U.S. patent application number 12/375685 was filed with the patent office on 2009-12-31 for pyrazolo[3,4-b]pyridine compounds, and their use as pde4 inhibitors.
This patent application is currently assigned to Glaxo Group Limited. Invention is credited to David George Allen, Nicola Mary Aston, Rodger Phillip Barnett, Reshma Manesh Chudasama, Caroline Jane Day, Christopher David Edlin, Leanda Jane Kindon, Naimisha Trivedi.
Application Number | 20090325952 12/375685 |
Document ID | / |
Family ID | 38996907 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090325952 |
Kind Code |
A1 |
Allen; David George ; et
al. |
December 31, 2009 |
PYRAZOLO[3,4-B]PYRIDINE COMPOUNDS, AND THEIR USE AS PDE4
INHIBITORS
Abstract
The invention provides a compound of formula (I) or a salt
thereof: ##STR00001## wherein Ar has the sub-formula (x):
##STR00002## as defined herein, and the use of the compounds as
inhibitors of PDE4.
Inventors: |
Allen; David George;
(Hertfordshire, GB) ; Aston; Nicola Mary;
(Hertfordshire, GB) ; Barnett; Rodger Phillip;
(Hertfordshire, GB) ; Chudasama; Reshma Manesh;
(Hertfordshire, GB) ; Day; Caroline Jane;
(Hertfordshire, GB) ; Edlin; Christopher David;
(Hertfordshire, GB) ; Kindon; Leanda Jane;
(Hertfordshire, GB) ; Trivedi; Naimisha;
(Hertfordshire, GB) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION;CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Assignee: |
Glaxo Group Limited
|
Family ID: |
38996907 |
Appl. No.: |
12/375685 |
Filed: |
July 31, 2007 |
PCT Filed: |
July 31, 2007 |
PCT NO: |
PCT/GB07/02908 |
371 Date: |
January 30, 2009 |
Current U.S.
Class: |
514/234.2 ;
514/303; 544/127; 546/119 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 9/00 20180101; C07D 471/04 20130101; A61P 19/02 20180101; A61P
11/08 20180101; A61P 11/02 20180101; A61P 17/00 20180101; A61P
19/00 20180101; A61P 37/08 20180101; A61P 11/00 20180101; A61P
11/06 20180101; A61P 13/12 20180101; A61P 27/02 20180101; A61P
17/06 20180101; A61P 1/04 20180101; A61P 29/00 20180101; A61P 35/00
20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/234.2 ;
546/119; 544/127; 514/303 |
International
Class: |
A61K 31/437 20060101
A61K031/437; C07D 471/04 20060101 C07D471/04; A61K 31/5377 20060101
A61K031/5377; A61P 29/00 20060101 A61P029/00; A61P 37/08 20060101
A61P037/08; A61P 11/00 20060101 A61P011/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 1, 2006 |
GB |
0615286.2 |
Apr 5, 2007 |
GB |
0706790.3 |
Jul 30, 2007 |
GB |
0714815.8 |
Claims
1-64. (canceled)
65. A compound of formula (I) or a salt thereof: ##STR00303##
wherein Ar has the sub-formula (x): ##STR00304## wherein: when
Q.sup.1 is NH or NMe, then Q.sup.2 is --C(O)--, --S(O).sub.2--,
--C(O)NH-- or --C(O)NMe--; or when Q.sup.1 is a bond or --O--, then
Q.sup.2 is a bond; or when Q.sup.1 is --C(O)--, then Q.sup.2 is NH
or NMe; or when Q.sup.1 is --S(O).sub.2--, then Q.sup.2 is NH, NMe
or a bond; L is (CH.sub.2).sub.n wherein n is 4, 5, 6, 7, 8, 9, 10,
11, 12 or 13; or L is
--(CH.sub.2).sub.m.sup.1--O--(CH.sub.2).sub.m.sup.2--, wherein
--(CH.sub.2).sub.m.sup.2-- is bonded to the NR.sup.5R.sup.6 group,
and wherein m.sup.1 is 1, 2, 3, 4, 5, 6, 7, 8 or 9, and m.sup.2 is
2, 3, 4, 5, 6, 7, 8 or 9; provided that m.sup.1+m.sup.2 is 3, 4, 5,
6, 7, 8, 9, 10, 11 or 12, and provided that when m1 is 1 then
Q.sup.2 is --C(O)--; R.sup.5 is hydrogen, methyl, ethyl, n-propyl,
isopropyl, --CH.sub.2CH.sub.2OH, --CH.sub.2CH(Me)OH,
--CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OMe, or
--CH.sub.2CH.sub.2CH.sub.2OMe; R.sup.6 is C.sub.1-4alkyl, or
R.sup.6 is C.sub.1-4alkyl substituted by one OH or OC.sub.1-3 alkyl
substituent which is not substituted at the alkyl carbon atom which
is bonded to the nitrogen atom of the NR.sup.5R.sup.6 group; or
R.sup.5 and R.sup.6 taken together are
--(CH.sub.2).sub.2--X--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--X--(CH.sub.2).sub.3--,
--(CH.sub.2).sub.p.sup.1--, --CHR.sup.7a--(CH.sub.2).sub.p.sup.2--,
or --(CH.sub.2).sub.p.sup.3--CHR.sup.7b-(CH.sub.2).sub.p.sup.4--;
in which X is O or NR.sup.8 wherein R.sup.8 is a hydrogen atom (H)
or C.sub.1-3alkyl; R.sup.7a is --CH.sub.2OH, --CH.sub.2OC.sub.1-3
alkyl, --CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OC.sub.1-3 alkyl, or
C.sub.1-3alkyl; R.sup.7b is OH, OC.sub.1-3alkyl, --CH.sub.2OH,
--CH.sub.2OC.sub.1-3alkyl, --CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2OC.sub.1-3alkyl, or C.sub.1-3alkyl; p.sup.1 is 4,
5 or 6; p.sup.2 is 3, 4 or 5; p.sup.3 is 1 or 2 and p.sup.4 is 2, 3
or 4, provided that p.sup.3+p.sup.4 is 3, 4 or 5; R.sup.1 is
C.sub.1-3alkyl, --CH.sub.2--C.sub.1-2fluoroalkyl, or
--CH.sub.2CH.sub.2OH; R.sup.2 is a hydrogen atom (H), methyl,
ethyl, n-propyl, isopropyl, n-butyl, C.sub.1-2fluoroalkyl,
cyclopropyl, cyclobutyl, or (cyclopropyl)methyl-; R.sup.4 is
hydrogen, methyl or ethyl; R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl, or optionally substituted
mono-unsaturated-C.sub.5-7cycloalkenyl, or an optionally
substituted heterocyclic group of sub-formula (aa), (bb) or (cc),
or a bicyclic group of sub-formula (ee); ##STR00305## in which
n.sup.1 and n.sup.2 independently are 1 or 2; and in which Y is O,
S, SO.sub.2, or NR.sup.10; where R.sup.10 is hydrogen, methyl,
C(O)NH.sub.2, C(O)-methyl, or C(O)--C.sub.1fluoroalkyl; and
wherein, when R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl, then R.sup.3 is C.sub.4-7cycloalkyl optionally
substituted on a ring carbon(s) with one or two substituents
independently being: oxo (.dbd.O); OH; methoxy; C.sub.1
fluoroalkoxy; NH.sub.2; C.sub.1-2alkyl; C.sub.1fluoroalkyl;
--CH.sub.2OH; --CH(Me)OH; --CH.sub.2CH.sub.2OH; --CH.sub.2NH.sub.2;
--C(O)OH; --C(O)NHR.sup.24 wherein R.sup.24 is H or methyl;
--C(O)R.sup.25 wherein R.sup.25 is methyl; fluoro; hydroxyimino
(.dbd.N--OH); or (C.sub.1-2alkoxy)imino (.dbd.N--OR.sup.26 where
R.sup.26 is C.sub.1-2alkyl); and wherein any OH, methoxy,
fluoroalkoxy or NH.sub.2 substituent is not bonded to the R.sup.3
ring carbon bonded to the --NH-- group of formula (I); and wherein,
when R.sup.3 is the optionally substituted heterocyclic group of
sub-formula (aa), (bb) or (cc), then R.sup.3 is the heterocyclic
group of sub-formula (aa), (bb) or (cc) optionally substituted on a
ring carbon(s) with one or two oxo (.dbd.O) substituents; and
wherein, when R.sup.3 is optionally substituted
mono-unsaturated-C.sub.5-7cycloalkenyl, then the cycloalkenyl is
optionally substituted on a ring carbon with one substituent being
fluoro or methyl, and the R.sup.3 ring carbon bonded to the --NH--
group of formula (I) does not partake in the cycloalkenyl double
bond; and wherein: when R.sup.3 is the heterocyclic group of
sub-formula (aa) and Y is NR.sup.10, then R.sup.10 is not
C(O)-methyl, or C(O)--C.sub.1 fluoroalkyl; and when R.sup.3 is the
heterocyclic group of sub-formula (bb) and Y is NR.sup.10, then
R.sup.10 is not methyl; and when R.sup.3 is the heterocyclic group
of sub-formula (cc), then Y is O, S, SO.sub.2 or NR.sup.10 wherein
R.sup.10 is H or methyl; and wherein: when R.sup.3 is optionally
substituted C.sub.4-7cycloalkyl, then any --C(O)NHR.sup.24 or
--C(O)R.sup.25 substituent on a ring carbon is: at the 3-position
of a R.sup.3 cyclobutyl ring; or at the 3- or 4-position of a
R.sup.3 cyclopentyl ring; or at the 4-position of a R.sup.3
cyclohexyl ring; or at the 3-, 4-, 5- or 6-position of a R.sup.3
cycloheptyl ring, wherein, in this connection, the 1-position of
the R.sup.3 cycloalkyl ring is deemed to be the connection point to
the --NH-- in formula (I), that is the ring atom connecting to the
--NH-- in formula (I)); and wherein: when R.sup.3 is optionally
substituted C.sub.4-7cycloalkyl, then any OH, methoxy,
fluoroalkoxy, --CH.sub.2OH, --CH(Me)OH, --CH.sub.2CH.sub.2OH,
--CH.sub.2NH.sub.2, or --C(O)OH substituent on a ring carbon is: at
the 3-position of a R.sup.3 cyclobutyl ring; or at the 3- or
4-position of a R.sup.3 cyclopentyl ring; or at the 3-, 4- or
5-position of a R.sup.3 cyclohexyl ring; or at the 3-, 4-, 5- or
6-position of a R.sup.3 cycloheptyl ring.
66. A compound or salt as claimed in claim 65, wherein R.sup.1 is
ethyl.
67. A compound or salt as claimed in claim 65, wherein R.sup.2 is
ethyl.
68. A compound or salt as claimed in claim 65, wherein, when
R.sup.3 is the heterocyclic group of sub-formula (aa), (bb) or
(cc), then R.sup.3 is a heterocyclic group of sub-formula (bb) and
n.sup.1 is 1.
69. A compound or salt as claimed in claim 65, wherein NHR.sup.3 is
a sub-formula (c), (h), (k2), (k3), (n), (o), (o2), (p9) or (p13).
##STR00306##
70. A compound or a salt thereof as claimed in claim 65, wherein
R.sup.4 is hydrogen.
71. A compound or salt thereof as claimed in claim 65, wherein Ar
is sub-formula (x1): ##STR00307##
72. A compound or salt thereof as claimed in claim 65, wherein
Q.sup.1 is NH or NMe, in which case Q.sup.2 is --C(O)--; or when
Q.sup.1 is a bond then Q.sup.2 is a bond; or when Q.sup.1 is
--C(O)-- then Q.sup.2 is NH or NMe.
73. A compound or salt thereof as claimed in claim 65, wherein L is
(CH.sub.2).sub.n.
74. A compound or salt thereof as claimed in claim 73, wherein n is
6, 7, 8 or 9.
75. A compound or salt thereof as claimed in claim 65, wherein:
R.sup.5 is a hydrogen atom (H), methyl, ethyl, n-propyl, isopropyl,
or --CH.sub.2CH.sub.2OH; R.sup.6 is --CH.sub.2CH.sub.2OH,
--CH.sub.2CH(Me)OH, --CH.sub.2CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OMe, or
--CH.sub.2CH.sub.2CH.sub.2OMe; or R.sup.5 and R.sup.6 are taken
together and are --(CH.sub.2).sub.2--X--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.p.sup.1--, or
--CHR.sup.7a--(CH.sub.2).sub.p.sup.2--.
76. A compound or a salt thereof according to claim 65 which is:
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide,
4-[(1,6-diethyl-5-{[({4-[(8-{(2R)-2-[(methyloxy)methyl]-1-pyrrolidiny-
l}octanoyl)amino]phenyl}carbonyl)amino]methyl}-1H-pyrazolo[3,4-b]pyridin-4-
-yl)amino]-1-piperidinecarboxamide,
4-[(1,6-diethyl-5-{[({4-[(8-{(2S)-2-[(methyloxy)methyl]-1-pyrrolidinyl}oc-
tanoyl)amino]phenyl}carbonyl)amino]methyl}-1H-pyrazolo[3,4-b]pyridin-4-yl)-
amino]-1-piperidinecarboxamide,
4-({1,6-diethyl-5-[({[4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}-amin-
o)phenyl]carbonyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-pi-
peridinecarboxamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-[(8-{(2R)-2-[(methyloxy)methyl]-1-pyrrolidinyl}
octanoyl)-amino]benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-[(8-{(2S)-2-[(methyloxy)methyl]-1-pyrrolidinyl}octanoyl)-
-amino]benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-{[8-(4-morpholinyl)octanoyl]amino}benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-{[8-(1-pyrrolidinyl)octanoyl]amino}benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-[4-({4-[(2-hydroxyethyl)(methyl)amino]butyl}oxy)butyl]be-
nzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
-b]pyridin-5-yl]methyl}-4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-(4-{[4-(1-pyrrolidinyl)butyl]oxy}butyl)benzamide,
4-[(1,6-diethyl-5-{[({4-[4-({4-[(2-hydroxyethyl)(methyl)amino]butyl}-oxy)-
butyl]phenyl}carbonyl)amino]methyl}-1H-pyrazolo[3,4-b]pyridin-4-yl)amino]--
1-piperidinecarboxamide,
4-({1,6-diethyl-5-[({[4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)phenyl]-car-
bonyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarb-
oxamide,
4-({1,6-diethyl-5-[({[4-(4-{[4-(1-pyrrolidinyl)butyl]oxy}butyl)ph-
enyl]-carbonyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piper-
idinecarboxamide,
N-{[1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]-
pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)-amino]octanoyl}amino)-
benzamide,
N-{[1-ethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3-
,4-b]pyridin-5-yl]methyl}-4-[8-(4-morpholinyl)octyl]benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-{8-[(2-hydroxyethyl)(methyl)amino]octyl}benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-3-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]p-
yridin-5-yl]methyl}-2-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)be-
nzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
-b]pyridin-5-yl]methyl}-4-({6-[(2-hydroxyethyl)(methyl)amino]hexanoyl}amin-
o)benzamide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({7-[(2-hydroxyethyl)(methyl)amino]heptanoyl}amino)benza-
mide,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]-
pyridin-5-yl]methyl}-4-({10-[(2-hydroxyethyl)(methyl)amino]decanoyl}amino)-
benzamide, or
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({11-[(2-hydroxyethyl)(methyl)amino]undecanoyl}amino)ben-
zamide.
77. A method of treatment and/or prophylaxis of an inflammatory
and/or allergic disease in a patient in need thereof, which method
comprises administering to the patient a therapeutically effective
amount of a compound of formula (I) as defined in claim 65 or a
pharmaceutically acceptable salt thereof.
78. The method as claimed in claim 78, wherein the disease is
chronic obstructive pulmonary disease (COPD), asthma, rhinitis,
atopic dermatitis or psoriasis.
79. A pharmaceutical composition comprising a compound of formula
(I) or a pharmaceutically acceptable salt thereof according to
claim 65 and a pharmaceutically acceptable excipient.
Description
[0001] The present invention relates to pyrazolo[3,4-b]pyridine
compounds or salts thereof, processes for their preparation,
intermediates usable in these processes, and pharmaceutical
compositions containing the compounds or salts. The invention also
relates to the use of the pyrazolo[3,4-b]pyridine compounds or
salts thereof in therapy, for example as inhibitors of
phosphodiesterase type IV (PDE4) and/or for the treatment and/or
prophylaxis of inflammatory and/or allergic diseases such as
chronic obstructive pulmonary disease (COPD), asthma, or rhinitis
(e.g. allergic and/or non-allergic rhinitis).
BACKGROUND TO THE INVENTION
[0002] Phosphodiesterase 4 inhibitors (PDE4 inhibitors) may be
useful in the treatment and/or prophylaxis of a variety of
diseases/conditions, especially inflammatory and/or allergic
diseases, in mammals such as humans, for example: chronic
obstructive pulmonary disease (COPD) (e.g. chronic bronchitis
and/or emphysema), asthma, rhinitis (e.g. allergic or non-allergic
rhinitis), rheumatoid arthritis, atopic dermatitis, psoriasis,
urticaria, allergic conjunctivitis, vernal conjunctivitis,
eosinophilic granuloma, septic shock, inflammatory bowel disease
(e.g. ulcerative colitis and/or Crohn's disease), reperfusion
injury of the myocardium and/or brain, chronic glomerulonephritis,
endotoxic shock, or adult respiratory distress syndrome, in mammals
such as humans.
[0003] Chronic obstructive pulmonary disease (COPD) is often
characterised by the presence of airflow obstruction due to chronic
bronchitis and/or emphysema (e.g., see S. L. Wolda, Emerging Drugs,
2000, 5(3), 309-319).
[0004] PDE4 inhibitors, for example cilomilast and roflumilast, are
thought to be effective in the treatment of COPD. For example, see
S. L. Wolda, Emerging Drugs, 2000, 5(3), 309-319; Z. Huang et al.,
Current Opinion in Chemical Biology, 2001, 5: 432-438; H. J. Dyke
et al., Expert Opinion on Investigational Drugs, January 2002,
11(1), 1-13; C. Burnouf et al., Current Pharmaceutical Design,
2002, 8(14), 1255-1296; A. M. Doherty, Current Opinion Chem. Biol.,
1999, 3(4), 466-473; A. M. Vignola, Respiratory Medicine, 2004, 98,
495-503; D. Spina, Drugs, 2003, 63(23), 2575-2594; and references
cited in the aforementioned publications; G. Krishna et al., Expert
Opinion on Investigational Drugs, 2004, 13(3), 255-267 (see
especially pp. 259-261 and refs. 102-111 and 201 therein); and B.
J. Lipworth, The Lancet, 2005, 365, 167-175.
[0005] The PDE4 inhibitor cilomilast (Ariflo.TM.) at 15 mg orally
twice daily appears to improve forced expiratory volume in 1 s
(FEV.sub.1) in COPD patients (C. H. Compton et al., The Lancet,
2001, vol. 358, 265-270), and appears to have antiinflammatory
effects in COPD patients (E. Gamble et al., Am. J. Respir. Crit.
Care Med., 2003, 168, 976-982). On cilomilast, see also R. D.
Border et al., Chest, 2003, vol. 124 Suppl. 4, p. 170S (abstract)
and J. D. Eddleston et al., Am. J. Respir. Crit. Care Med., 2001,
163, A277 (abstract). The PDE4 inhibitor roflumilast appears to
show small improvements in FEV.sub.1 in COPD patients (see B. J.
Lipworth, The Lancet, 2005, 365, 167-175, and refs 49-50
therein).
[0006] PDE4 inhibitors are thought to be effective in the treatment
and/or prophylaxis of asthma (e.g. see M. A. Giembycz, Drugs,
February 2000, 59(2), 193-212; Z. Huang et al., Current Opinion in
Chemical Biology, 2001, 5: 432-438; H. J. Dyke et al., Expert
Opinion on Investigational Drugs, January 2002, 11(1), 1-13; C.
Burnouf et al., Current Pharmaceutical Design, 2002, 8(14),
1255-1296; A. M. Doherty, Current Opinion Chem. Biol., 1999, 3(4),
466-473; P. J. Barnes, Nature Reviews--Drug Discovery, October
2004, 831-844; B. J. Lipworth, The Lancet, 2005, 365, 167-175; and
references cited in the aforementioned publications).
[0007] The PDE4 inhibitor roflumilast, given orally at 500 ug once
daily for 9 days, is reported to be effective in improving rhinal
airflow during the treatment period (compared to placebo), in
humans with histories of allergic rhinitis but asymptomatic at
screening, and who were challenged with intranasal allergen
provocation (pollen extracts) daily beginning the third day of
treatment and each time approx. 2 hours after study drug
administration (B. M. Schmidt et al., J. Allergy & Clinical
Immunology, 108(4), 2001, 530-536).
[0008] PDE4 inhibitors may be effective in the treatment of
rheumatoid arthritis (e.g. see H. J. Dyke et al., Expert Opinion on
Investigational Drugs, January 2002, 11(1), 1-13; C. Burnouf et
al., Current Pharmaceutical Design, 2002, 8(14), 1255-1296; and A.
M. Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473; and
references cited in these publications).
[0009] PDE4 inhibition has been suggested for the treatment of
inflammatory bowel disease (e.g. ulcerative colitis and/or Crohn's
disease), see K. H. Banner and M. A. Trevethick, Trends Pharmacol.
Sci., August 2004, 25(8), 430-436.
[0010] For the use of PDE4 inhibitors in atopic dermatitis, see for
example: [0011] J. M. Hanifin et al., "Type 4 phosphodiesterase
inhibitors have clinical and in vitro anti-inflammatory effects in
atopic dermatitis", J. Invest Dermatol., 1996, 107(1), 51-56; which
reports reductions of inflammatory parameters in atopic dermatitis
patients treated with PDE4 inhibitor CP80,633 (0.5% ointment, twice
daily topical application); [0012] C. E. M. Griffiths et al.,
"Randomized comparison of the type 4 phosphodiesterase inhibitor
cipamfylline cream, cream vehicle and hydrocortisone 17-butyrate
cream for the treatment of atopic dermatitis", Br. J. DermatoL,
2002, 147(2), 299-307, which reports that cipamfylline (0.15%)
cream is significantly more effective than vehicle, but
significantly less effective than hydrocortisone 17-butyrate (0.1%)
cream, in the treatment of atopic dermatitis patients; [0013] T. C.
Roos et al., "Recent advances in treatment strategies for atopic
dermatitis", Drugs, 2004, 64(23), 2639-2666 (see e.g. page 2657 and
refs. 201-209 therein); [0014] A. M. Doherty, Current Opinion Chem.
Biol., 1999, 3(4), 466-473 (e.g. see p. 470); and [0015] H. J. Dyke
et al., Expert Opinion Invest Drugs, 2002, 11(1), 1-13 (e.g. see p.
7 and refs. 74, 75 and 76 cited therein); and references cited in
the above references.
[0016] For the use of the PDE4 inhibitors SB 207499 (cilomilast)
and AWD 12-281 in mouse models of the allergic type of dermatitis,
see W. Baumer et al., Eur. J. Pharmacol., 2002, 446, 195-200 and W.
Baumer et al., J. Pharmacy Pharmacol., 2003, 55, 1107-1114.
[0017] WO 2004/056823 A1 (PCT/EP2003/014867, Glaxo Group Limited)
discloses certain pyrazolo[3,4-b]pyridine compounds or salts
thereof; and their use as PDE4 inhibitors.
[0018] WO 2004/024728 A2 (PCT/EP2003/011814, Glaxo Group Limited)
discloses pyrazolo[3,4-b]pyridine compounds or salts thereof with a
4-NHR.sup.3 group and a 5-C(O)--X group, according to the following
formula, wherein X is NR.sup.4R.sup.5 or OR.sup.5a, and R.sup.2 is
a hydrogen atom (H), methyl or C.sub.1fluoroalkyl. These
pyrazolo[3,4-b]pyridine compounds and salts are disclosed as being
inhibitors of phosphodiesterase type IV (PDE4):
##STR00003##
[0019] WO 2004/024728 has been reviewed, and WO 2004/056823
mentioned, in Expert Opin. Ther. Patents, 2005 (January edition),
15(1), 111-114.
[0020] Further pyrazolo[3,4-b]pyridine compounds or salts thereof,
and their use as PDE4 inhibitors, are disclosed in patent
publications WO 2005/058892 A1 (PCT/EP2004/014490), WO 2005/090348
A1 (PCT/GB2005/000983), WO 2005/090352 A1 (PCT/EP2005/003038), WO
2005/090353 A1 (PCT/GB2005/000976), WO 2005/090354 A1
(PCT/GB2005/000987) (all Glaxo Group Limited). Of these, WO
2005/090348 A1 discloses pyrazolo[3,4-b]pyridine compounds or salts
thereof with a 4--NHR.sup.3 group and a 5-C(O)--NH--W group,
according to the following formula, wherein W is Ar,
--CR.sup.4R.sup.5Ar or a group (y) or (yl), and R.sup.2 is
C.sub.2-6alkyl, C.sub.3-6cycloalkyl or
--(CH.sub.2).sub.n.sup.4C.sub.3-6cycloalkyl:
##STR00004##
[0021] Copending patent application PCT/GB2006/003626, published on
5 Apr. 2007 as WO 2007/036733 A1, discloses pyrazolo[3,4-b]pyridine
compounds of the following formula or salts thereof, and their use
as PDE4 inhibitors:
##STR00005##
[0022] Copending patent application PCT/GB2006/003627, published on
5 Apr. 2007 as WO 2007/036734 A1, discloses
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-3-methyl-5-isoxazolecarboxamide or a salt thereof,
and its use as PDE4 inhibitor.
THE INVENTION
[0023] We have now found new pyrazolo[3,4-b]pyridine compounds
having a long side-chain containing a basic nitrogen atom, which
compounds inhibit phosphodiesterase type IV (PDE4).
[0024] The present invention therefore provides a compound of
formula (I) or a salt thereof (in particular, a pharmaceutically
acceptable salt thereof):
##STR00006##
wherein Ar has the sub-formula (x):
##STR00007##
wherein: Q.sup.1 is NH or NMe, in which case Q.sup.2 is --C(O)--,
--S(O).sub.2--, --C(O)NH-- or --C(O)NMe-; or Q.sup.1 is a bond or
--O--, in which case Q.sup.2 is a bond; or Q.sup.1 is --C(O)--, in
which case Q.sup.2 is NH or NMe; or Q.sup.1 is --S(O).sub.2--, in
which case Q.sup.2 is NH, NMe or a bond; and L is (CH.sub.2).sub.n
wherein n is 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; or L is
--(CH.sub.2).sub.m.sup.1--O--(CH.sub.2).sub.m.sup.2--, wherein it
is the --(CH.sub.2).sub.m.sup.2-- which is bonded to the
NR.sup.5R.sup.6 group, and wherein m.sup.1 is 1, 2, 3, 4, 5, 6, 7,
8 or 9, and m.sup.2 is 2, 3, 4, 5, 6, 7, 8 or 9; provided that
m.sup.1+m.sup.2 is 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and provided
that when m.sup.1 is 1 then Q.sup.2 is --C(O)--; and R.sup.5 is a
hydrogen atom (H), methyl, ethyl, n-propyl, isopropyl,
--CH.sub.2CH.sub.2OH, --CH.sub.2CH(Me)OH,
--CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OMe, or
--CH.sub.2CH.sub.2CH.sub.2OMe; and R.sup.6 is C.sub.1-4alkyl, or
R.sup.6 is C.sub.1-4alkyl substituted by one OH or OC.sub.1-3alkyl
(e.g. OMe) substituent which is not substituted at the alkyl carbon
atom which is bonded to the nitrogen atom of the NR.sup.5R.sup.6
group (for example R.sup.6 can be --CH.sub.2CH.sub.2OH,
--CH.sub.2CH(Me)OH, --CH.sub.2CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OMe, or
--CH.sub.2CH.sub.2CH.sub.2OMe); or R.sup.5 and R.sup.6 taken
together are --(CH.sub.2).sub.2--X--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2--X--(CH.sub.2).sub.3--,
--(CH.sub.2).sub.p.sup.1--, --CHR.sup.7a--(CH.sub.2).sub.p.sup.2--,
or --(CH.sub.2).sub.p.sup.3--CHR.sup.7b--(CH.sub.2).sub.p.sup.4--;
in which: X is O or NR.sup.8 wherein R.sup.8 is a hydrogen atom (H)
or C.sub.1-3alkyl (e.g. H or methyl); R.sup.7a is --CH.sub.2OH,
--CH.sub.2OC.sub.1-3alkyl (e.g. --CH.sub.2OMe),
--CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OC.sub.1-3alkyl (e.g.
--CH.sub.2CH.sub.2OMe), or C.sub.1-3alkyl such as methyl (e.g.
R.sup.7a can be --CH.sub.2OH or preferably --CH.sub.2OMe); R.sup.7b
is OH, OC.sub.1-3alkyl (e.g. OMe), --CH.sub.2OH,
--CH.sub.2OC.sub.1-3alkyl (e.g. --CH.sub.2OMe),
--CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OC.sub.1-3alkyl (e.g.
--CH.sub.2CH.sub.2OMe), or C.sub.1-3alkyl such as methyl (e.g.
R.sup.7b can be OH or OMe); p.sup.1 is 4, 5 or 6 (e.g. 4 or 5),
p.sup.2 is 3, 4 or 5 (e.g. 3 or 4), p.sup.3 is 1 or 2 and p.sup.4
is 2, 3 or 4 (e.g. 2 or 3), provided that p.sup.3+p.sup.4 is 3, 4
or 5 (e.g. 3 or 4); and wherein: R.sup.1 is C.sub.1-3alkyl,
--CH.sub.2--C.sub.1-2-fluoroalkyl, or --CH.sub.2CH.sub.2OH; R.sup.2
is a hydrogen atom (H), methyl, ethyl, n-propyl, isopropyl,
n-butyl, C.sub.1-2fluoroalkyl, cyclopropyl, cyclobutyl, or
(cyclopropyl)methyl-; R.sup.4 is a hydrogen atom (H), methyl or
ethyl; and R.sup.3 is optionally substituted C.sub.4-7cycloalkyl,
or optionally substituted mono-unsaturated-C.sub.5-7cycloalkenyl,
or an optionally substituted heterocyclic group of sub-formula
(aa), (bb) or (cc), or a bicyclic group of sub-formula (ee);
##STR00008##
in which n.sup.1 and n.sup.2 independently are 1 or 2; and in which
Y is O, S, SO.sub.2, or NR.sup.10; where R.sup.10 is a hydrogen
atom (H), methyl, C(O)NH.sub.2, C(O)-methyl, or
C(O)--C.sub.1fluoroalkyl; and wherein, when R.sup.3 is optionally
substituted C.sub.4-7cycloalkyl, then R.sup.3 is
C.sub.4-7cycloalkyl optionally substituted on a ring carbon(s) with
one or two substituents independently being: oxo (.dbd.O); OH;
methoxy; C.sub.1fluoroalkoxy; NH.sub.2; C.sub.1-2alkyl;
C.sub.1fluoroalkyl; --CH.sub.2OH; --CH(Me)OH; --CH.sub.2CH.sub.2OH;
--CH.sub.2NH.sub.2; --C(O)OH; --C(O)NHR.sup.24 wherein R.sup.24 is
H or methyl; --C(O)R.sup.25 wherein R.sup.25 is methyl; fluoro;
hydroxyimino (.dbd.N--OH); or (C.sub.1-2alkoxy)imino
(.dbd.N--OR.sup.26 where R.sup.26 is C.sub.1-2alkyl); and wherein
any OH, methoxy, fluoroalkoxy or NH.sub.2 substituent is not bonded
to the R.sup.3 ring carbon bonded to the --NH--group of formula
(I); and wherein, when R.sup.3 is the optionally substituted
heterocyclic group of sub-formula (aa), (bb) or (cc), then the
heterocyclic group of sub-formula (aa), (bb) or (cc) is optionally
substituted on a ring carbon(s) with one or two oxo (.dbd.O)
substituents; and wherein, when R.sup.3 is optionally substituted
mono-unsaturated-C.sub.5-7cycloalkenyl, then the cycloalkenyl is
optionally substituted on a ring carbon with one substituent being
fluoro or methyl, and the R.sup.3 ring carbon bonded to the --NH--
group of formula (I) does not partake in the cycloalkenyl double
bond; and wherein: when R.sup.3 is the heterocyclic group of
sub-formula (aa) and Y is NR.sup.10, then R.sup.10 is not
C(O)-methyl, or C(O)--C.sub.1fluoroalkyl; and when R.sup.3 is the
heterocyclic group of sub-formula (bb) and Y is NR.sup.10, then
R.sup.10 is not methyl; and when R.sup.3 is the heterocyclic group
of sub-formula (cc), then Y is O, S, SO.sub.2 or NR.sup.10 wherein
R.sup.10 is H or methyl; and wherein: when R.sup.3 is optionally
substituted C.sub.4-7cycloalkyl, then any --C(O)NHR.sup.24 or
--C(O)R.sup.25 substituent on a ring carbon is: at the 3-position
of a R.sup.3 cyclobutyl ring; or at the 3- or 4-position of a
R.sup.3 cyclopentyl ring; or at the 4-position of a R.sup.3
cyclohexyl ring; or at the 3-, 4-, 5- or 6-position of a R.sup.3
cycloheptyl ring (wherein, in this connection, the 1-position of
the R.sup.3 cycloalkyl ring is deemed to be the connection point to
the --NH-- in formula (I), that is the ring atom connecting to the
--NH-- in formula (I)); and wherein: when R.sup.3 is optionally
substituted C.sub.4-7cycloalkyl, then any OH, methoxy,
fluoroalkoxy, --CH.sub.2OH, --CH(Me)OH, --CH.sub.2CH.sub.2OH,
--CH.sub.2NH.sub.2, or --C(O)OH substituent on a ring carbon is: at
the 3-position of a R.sup.3 cyclobutyl ring; or at the 3- or
4-position of a R.sup.3 cyclopentyl ring; or at the 3-, 4- or
5-position of a R.sup.3 cyclohexyl ring; or at the 3-, 4-, 5- or
6-position of a R.sup.3 cycloheptyl ring.
[0025] In compounds, for example in the compounds of formula (I),
an "alkyl" group or moiety may be straight-chain or branched. Alkyl
groups, for example C.sub.1-8alkyl or C.sub.1-6alkyl or
C.sub.1-4alkyl or C.sub.1-3alkyl or C.sub.1-2alkyl, which may be
employed include C.sub.1-6alkyl or C.sub.1-4alkyl or C.sub.1-3alkyl
or C.sub.1-2alkyl such as methyl, ethyl, n-propyl, n-butyl,
n-pentyl, or n-hexyl or any branched isomers thereof such as
isopropyl, t-butyl, sec-butyl, isobutyl, 3-methylbutan-2-yl,
2-ethylbutan-1-yl, or the like.
[0026] A corresponding meaning is intended for "alkoxy",
"alkylene", and like terms derived from alkyl. For example,
"alkoxy" such as C.sub.1-6alkoxy or C.sub.1-4alkoxy or
C.sub.1-2alkoxy includes methoxy, ethoxy, propyloxy, and oxy
derivatives of the alkyls listed above. "Alkylsulfonyl" such as
C.sub.1-4alkylsulfonyl includes methylsulfonyl (methanesulfonyl),
ethylsulfonyl, and others derived from the alkyls listed above.
"Alkylsulfonyloxy" such as C.sub.1-4alkylsulfonyloxy includes
methanesulfonyloxy (methylsulfonyloxy), ethanesulfonyloxy, et
al.
[0027] "Cycloalkyl", for example C.sub.3-8cycloalkyl (e.g.
C.sub.4-7cycloalkyl), includes cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
Suitably, a C.sub.3-8cycloalkyl group can be C.sub.3-6cycloalkyl or
C.sub.5-6cycloalkyl or C.sub.4-7cycloalkyl or C.sub.6-7cycloalkyl,
that is contains a 3-6 membered or 5-6 membered or 4-7 membered or
6-7 membered carbocyclic ring.
[0028] "Fluoroalkyl" includes alkyl groups with one, two, three,
four, five or more fluorine substituents, for example
C.sub.1-4fluoroalkyl or C.sub.1-3fluoroalkyl or
C.sub.1-2fluoroalkyl such as monofluoromethyl, difluoromethyl,
trifluoromethyl, pentafluoroethyl, 2,2,2-trifluoroethyl
(CF.sub.3CH.sub.2--), 2,2-difluoroethyl (CHF.sub.2CH.sub.2--),
2-fluoroethyl (CH.sub.2FCH.sub.2--), etc. "Fluoroalkoxy" includes
C.sub.1-4fluoroalkoxy or C.sub.1-2fluoroalkoxy such as
trifluoromethoxy, pentafluoroethoxy, monofluoromethoxy,
difluoromethoxy, etc.
[0029] A halogen atom ("halo") present in compounds, for example in
the compounds of formula (I), means a fluorine, chlorine, bromine
or iodine atom ("fluoro", "chloro", "bromo" or "iodo"), for example
fluoro, chloro or bromo.
[0030] When the specification states that atom or moiety A is
"bonded" or "attached" to atom or moiety B, it means that
atom/moiety A is directly bonded to atom/moiety B usually by means
of a covalent bond or a double covalent bond, and excludes A being
indirectly attached to B via one or more intermediate
atoms/moieties (e.g. excludes A-C-B); unless it is clear from the
context that another meaning is intended.
[0031] In the invention, Ar has the sub-formula (x).
[0032] Ar can be of sub-formula (x1), (x2) or (x3):
##STR00009##
[0033] Ar can for example be of sub-formula (x1) or (x3).
Preferably, Ar is of sub-formula (x1).
[0034] In one embodiment,
Q.sup.1 is NH or NMe (e.g. NH), in which case Q.sup.2 is --C(O)--,
--S(O).sub.2-- or --C(O)NH-- (e.g. --C(O)-- or --S(O).sub.2--); or
Q.sup.1 is a bond or --O--, in which case Q.sup.2 is a bond; or
Q.sup.1 is --C(O)--, in which case Q.sup.2 is NH or NMe (e.g. NH);
or Q.sup.1 is --S(O).sub.2--, in which case Q.sup.2 is NH or NMe or
a bond (e.g. NH or NMe such as NH).
[0035] In one embodiment, suitably, Q.sup.1 is NH or NMe (e.g. NH),
in which case Q.sup.2 is --C(O)--;
or Q.sup.1 is a bond, in which case Q.sup.2 is a bond; or Q.sup.1
is --C(O)--, in which case Q.sup.2 is NH or NMe (e.g. NH).
[0036] Suitably, Q.sup.1 is NH or NMe (e.g. NH), in which case
Q.sup.2 is --C(O)--;
or Q.sup.1 is a bond, in which case Q.sup.2 is a bond.
[0037] L is (CH.sub.2).sub.n or L is
--(CH.sub.2).sub.m.sup.1--O--(CH.sub.2).sub.m.sup.2--.
[0038] Typically, L is (CH.sub.2).sub.n.
[0039] Suitably, n is 5, 6, 7, 8, 9, 10 or 11, such as 5, 6, 7, 8,
9 or 10.
[0040] Preferably, n is 6, 7, 8 or 9, such as 7.
[0041] m.sup.1 can for example be 2, 3, 4, 5, 6, 7, 8 or 9. In
particular, m.sup.1 is 2, 3, 4, 5 or 6.
[0042] Suitably, m.sup.1 is 3, 4 or 5. Preferably, m.sup.1 is
4.
[0043] In particular, m.sup.2 is 2, 3, 4, 5 or 6. Suitably, m.sup.2
is 3, 4 or 5. Preferably, m.sup.2 is 4.
[0044] m.sup.1+m.sup.2 can for example be 4, 5, 6, 7, 8, 9, 10, 11
or 12. In particular, m.sup.1+m.sup.2 is 4, 5, 6, 7, 8, 9 or
10.
[0045] Suitably, m.sup.1+m.sup.2 is 6, 7, 8, 9 or 10. Preferably,
m.sup.1+m.sup.2 is 8.
[0046] In one embodiment, when L is (CH.sub.2).sub.n, then:
Q.sup.1 is NH or NMe (e.g. NH), in which case Q.sup.2 is --C(O)--,
--S(O).sub.2--, --C(O)NH-- or --C(O)NMe- (e.g. --C(O)-- or
--S(O).sub.2--); or Q.sup.1 is a bond or --O-- (e.g. a bond), in
which case Q.sup.2 is a bond; or Q.sup.1 is --C(O)--, in which case
Q.sup.2 is NH or NMe (e.g. NH); or Q.sup.1 is --S(O).sub.2--, in
which case Q.sup.2 is NH or NMe or a bond (e.g. NH or NMe such as
NH); and when L is
--(CH.sub.2).sub.m.sup.1--O--(CH.sub.2).sub.m.sup.2--, then:
Q.sup.1 is NH or NMe (e.g. NH), in which case Q.sup.2 is --C(O)--;
or Q.sup.1 is a bond or --O-- (e.g. a bond), in which case Q.sup.2
is a bond; or Q.sup.1 is --C(O)--, in which case Q.sup.2 is NH or
NMe (e.g. NH).
[0047] In one embodiment, when L is (CH.sub.2).sub.n, then:
Q.sup.1 is NH or NMe (e.g. NH), in which case Q.sup.2 is --C(O)--,
--S(O).sub.2--, --C(O)NH-- or --C(O)NMe- (e.g. --C(O)-- or
--S(O).sub.2--); or Q.sup.1 is a bond or --O-- (e.g. a bond), in
which case Q.sup.2 is a bond; or Q.sup.1 is --C(O)--, in which case
Q.sup.2 is NH or NMe (e.g. NH); or Q.sup.1 is --S(O).sub.2--, in
which case Q.sup.2 is NH or NMe or a bond (e.g. NH or NMe such as
NH); and when L is
--(CH.sub.2).sub.m.sup.1--O--(CH.sub.2).sub.m.sup.2--, then:
Q.sup.1 is a bond or --O-- (e.g. a bond), in which case Q.sup.2 is
a bond.
[0048] In one embodiment, suitably, when L is (CH.sub.2).sub.n,
then:
[0049] Q.sup.1 is NH or NMe (e.g. NH), in which case Q.sup.2 is
--C(O)--;
or Q.sup.1 is a bond, in which case Q.sup.2 is a bond; or Q.sup.1
is --C(O)--, in which case Q.sup.2 is NH or NMe (e.g. NH); and when
L is --(CH.sub.2).sub.m.sup.1--O--(CH.sub.2).sub.m.sup.2--, then
Q.sup.1 is a bond, and Q.sup.2 is a bond.
[0050] Suitably, when L is (CH.sub.2).sub.n, then:
Q.sup.1 is NH, in which case Q.sup.2 is --C(O)--; or Q.sup.1 is a
bond, in which case Q.sup.2 is a bond; and when L is
--(CH.sub.2).sub.m.sup.1--O--(CH.sub.2).sub.m.sup.2--, then Q.sup.1
is a bond, and Q.sup.2 is a bond.
[0051] Suitably, (when R.sup.5 and R.sup.6 are not taken together),
R.sup.5 is a hydrogen atom (H), methyl, ethyl, n-propyl, isopropyl,
or --CH.sub.2CH.sub.2OH. Preferably, R.sup.5 is methyl, ethyl,
n-propyl or isopropyl, such as methyl.
[0052] Suitably, (when R.sup.5 and R.sup.6 are not taken together),
R.sup.6 is --CH.sub.2CH.sub.2OH, --CH.sub.2CH(Me)OH,
--CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2OMe, or --CH.sub.2CH.sub.2CH.sub.2OMe.
Preferably, R.sup.6 is --CH.sub.2CH.sub.2OH.
[0053] In one embodiment, when R.sup.5 and R.sup.6 are taken
together, then R.sup.5 and R.sup.6 taken together can be
--(CH.sub.2).sub.2--X--(CH.sub.2).sub.2--,
--(CH.sub.2).sub.p.sup.1--, --CHR.sup.7a--(CH.sub.2).sub.p.sup.2--,
or
--(CH.sub.2).sub.p.sup.3--CHR.sup.7b--(CH.sub.2).sub.p.sup.4--.
[0054] Suitably, when R.sup.5 and R.sup.6 are taken together, then
R.sup.5 and R.sup.6 taken together are
--(CH.sub.2).sub.2--X--(CH.sub.2).sub.2--, --(CH.sub.2).sub.p1--,
or --CHR.sup.7a--(CH.sub.2).sub.p.sup.2--.
[0055] R.sup.8 can be for example a hydrogen atom (H) or methyl,
such as methyl.
[0056] X is suitably O.
[0057] R.sup.7a can for example be --CH.sub.2OH, --CH.sub.2OMe,
--CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2OMe, or C.sub.1-3alkyl such
as methyl.
[0058] R.sup.7a is suitably --CH.sub.2OH or preferably
--CH.sub.2OMe.
[0059] R.sup.7b can for example be OH, OMe, --CH.sub.2OH, or
--CH.sub.2OMe or methyl.
[0060] R.sup.7b can for example be OH or OMe.
[0061] p.sup.1 can suitably be 4 or 5, such as 4.
[0062] p.sup.2 can suitably be 3 or 4, such as 3.
[0063] p.sup.4 can suitably be 2 or 3, provided that
p.sup.3+p.sup.4 is 3, 4 or 5.
[0064] p.sup.3+p.sup.4 can suitably be 3 or 4.
[0065] When R.sup.1 is C.sub.1-3alkyl or
--CH.sub.2--C.sub.1-2fluoroalkyl, it can be straight-chained or
branched. When R.sup.1 is C.sub.1-3alkyl then it can be methyl,
ethyl, n-propyl, or isopropyl. When R.sup.1 is
--CH.sub.2--C.sub.1-2fluoroalkyl: then R.sup.1 can for example be
--CH.sub.2--C.sub.1fluoroalkyl such as 2,2,2-trifluoroethyl
(CF.sub.3CH.sub.2--), 2,2-difluoroethyl (CHF.sub.2CH.sub.2--), or
2-fluoroethyl (CH.sub.2FCH.sub.2--).
[0066] In the invention, R.sup.1 is C.sub.1-3alkyl (e.g. methyl,
ethyl or n-propyl), --CH.sub.2--C.sub.1-2fluoroalkyl or
--CH.sub.2CH.sub.2OH. R.sup.1 for example can be C.sub.1-3alkyl,
--CH.sub.2--C.sub.1fluoroalkyl, or --CH.sub.2CH.sub.2OH.
[0067] Suitably, R.sup.1 is C.sub.2-3alkyl (e.g. ethyl or
n-propyl), --CH.sub.2--C.sub.1fluoroalkyl (e.g.
CF.sub.3--CH.sub.2--) or --CH.sub.2CH.sub.2OH.
[0068] Preferably, R.sup.1 is ethyl, n-propyl or
--CH.sub.2CH.sub.2OH.
[0069] Most preferably, R.sup.1 is ethyl.
[0070] R.sup.2 can for example be a hydrogen atom (H), methyl,
ethyl, n-propyl, isopropyl, C.sub.1fluoroalkyl (such as CF.sub.3 or
CHF.sub.2 or CH.sub.2F), C.sub.2fluoroalkyl such as C.sub.2F.sub.5
or C.sub.1fluoroalkyl-CH.sub.2-- [e.g. 2,2,2-trifluoroethyl
(CF.sub.3CH.sub.2--), 2,2-difluoroethyl (CHF.sub.2CH.sub.2--) or
2-fluoroethyl (CH.sub.2FCH.sub.2--)], or cyclopropyl.
[0071] R.sup.2 can for example be methyl, ethyl, C.sub.1fluoroalkyl
(such as CF.sub.3 or CHF.sub.2 or CH.sub.2F), or C.sub.2fluoroalkyl
such as C.sub.2F.sub.5 or C.sub.1fluoroalkyl-CH.sub.2-- [e.g.
2,2,2-trifluoroethyl (CF.sub.3CH.sub.2--), 2,2-difluoroethyl
(CHF.sub.2CH.sub.2--) or 2-fluoroethyl (CH.sub.2FCH.sub.2--)].
[0072] R.sup.2 can for example be a hydrogen atom (H), methyl,
ethyl or C.sub.1fluoroalkyl (such as CF.sub.3 or CHF.sub.2 or
CH.sub.2F). Suitably, R.sup.2 is a hydrogen atom (H), methyl or
ethyl. Alternatively, suitably, R.sup.2 is methyl, ethyl or
C.sub.1fluoroalkyl (such as CF.sub.3 or CHF.sub.2 or
CH.sub.2F).
[0073] Preferably, R.sup.2 is methyl or ethyl.
[0074] Most preferably, R.sup.2 is ethyl.
[0075] R.sup.4 can for example be a hydrogen atom (H) or
methyl.
[0076] Preferably, R.sup.4 is a hydrogen atom (H).
[0077] Preferably, in R.sup.3 there is one substituent on a ring
carbon or no substituent on a ring carbon.
[0078] Preferably, R.sup.3 is the optionally substituted
C.sub.4-7cycloalkyl or the optionally substituted heterocyclic
group of sub-formula (aa), (bb) or (cc).
[0079] Optionally, when R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl, it is not unsubstituted cyclopentyl. In this
case, suitably, R.sup.3 is optionally substituted
C.sub.6-7cycloalkyl or optionally substituted cyclobutyl.
[0080] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl,
it is suitably optionally substituted C.sub.6-7cycloalkyl or
optionally substituted cyclobutyl, preferably optionally
substituted C.sub.6cycloalkyl (i.e. optionally substituted
cyclohexyl).
[0081] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl,
then R.sup.3 is C.sub.4-7cycloalkyl (e.g. C.sub.6-7cycloalkyl or
cyclobutyl) optionally substituted on a ring carbon(s) with one or
two substituents independently being (e.g. one substituent being):
oxo (.dbd.O); OH; methoxy; C.sub.1fluoroalkoxy (e.g.
trifluoromethoxy or difluoromethoxy); NH.sub.2; C.sub.1-2alkyl such
as methyl; C.sub.1fluoroalkyl such as --CH.sub.2F or --CHF.sub.2;
--CH.sub.2OH; --CH(Me)OH; --CH.sub.2CH.sub.2OH; --CH.sub.2NH.sub.2;
--C(O)OH; --C(O)NHR.sup.24 wherein R.sup.24 is H or methyl
(preferably H); --C(O)R.sup.25 wherein R.sup.25 is methyl; fluoro;
hydroxyimino (.dbd.N--OH); or (C.sub.1-12alkoxy)imino
(.dbd.N--OR.sup.26 where R.sup.26 is C.sub.1-2alkyl); and wherein
any OH, methoxy, fluoroalkoxy or NH.sub.2 substituent is not bonded
to the R.sup.3 ring carbon bonded to the --NH-- group of formula
(I).
[0082] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl,
then R.sup.3 can suitably be C.sub.4-7cycloalkyl (e.g.
C.sub.6-7cycloalkyl or cyclobutyl) optionally substituted on a ring
carbon(s) with one or two substituents independently being (e.g.
one substituent being): oxo (.dbd.O); OH; NH.sub.2; C.sub.1-2alkyl
such as methyl; C.sub.1fluoroalkyl such as --CH.sub.2F or
--CHF.sub.2; --CH.sub.2OH; --CH(Me)OH; --C(O)NHR.sup.24 wherein
R.sup.24 is H or methyl (preferably H); --C(O)R.sup.25 wherein
R.sup.25 is methyl; fluoro; hydroxyimino (.dbd.N--OH); or
(C.sub.1-2alkoxy)imino (.dbd.N--OR.sup.26 where R.sup.26 is
C.sub.1-2alkyl).
[0083] Preferably, when R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl, then R.sup.3 is C.sub.4-7cycloalkyl (e.g.
C.sub.6-7cycloalkyl or cyclobutyl) optionally substituted on a ring
carbon(s) with one or two substituents independently being (e.g.
one substituent being): oxo (.dbd.O); OH; methyl; --CH.sub.2F;
--CHF.sub.2; --CH.sub.2OH; --C(O)NHR.sup.24 wherein R.sup.24 is H;
fluoro; hydroxyimino (.dbd.N--OH); or methoxyimino
(.dbd.N--OR.sup.26 where R.sup.26 is methyl).
[0084] More preferably, when R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl, then R.sup.3 is C.sub.4-7cycloalkyl (e.g.
C.sub.6-7cycloalkyl or cyclobutyl) optionally substituted on a ring
carbon(s) with one or two substituents independently being (e.g.
one substituent being): oxo (.dbd.O); OH; methyl; --C(O)NHR.sup.24
wherein R.sup.24 is H; fluoro; or hydroxyimino (.dbd.N--OH).
[0085] Still more preferably, when R.sup.3 is optionally
substituted C.sub.4-7cycloalkyl, then R.sup.3 is
C.sub.4-7cycloalkyl (e.g. C.sub.6-7cycloalkyl or cyclobutyl)
optionally substituted on a ring carbon(s) with one or two
substituents independently being (e.g. one substituent being): oxo
(.dbd.O); OH; --C(O)NHR.sup.24 wherein R.sup.24 is H; or
hydroxyimino (.dbd.N--OH).
[0086] In one optional embodiment, in R.sup.3, the
C.sub.4-7cycloalkyl can be unsubstituted.
[0087] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl
or optionally substituted C.sub.5-7cycloalkenyl, e.g. optionally
substituted (C.sub.6-7cycloalkyl or cyclobutyl or
C.sub.5-7cycloalkenyl), such as optionally substituted
C.sub.6cycloalkyl (optionally substituted cyclohexyl) or optionally
substituted cyclohexenyl, the one or two optional substituents on a
ring carbon(s) if present suitably can comprise a substituent (for
example is or are substituent(s)) at the 3-, 4- and/or
5-position(s), e.g. at the 3- and/or 4-position(s), of the R.sup.3
cycloalkyl or cycloalkenyl ring.
[0088] (In this connection and generally herein, the 1-position of
the R.sup.3 ring, e.g. of the R.sup.3 cycloalkyl or cycloalkenyl
ring, is deemed to be the connection point to the --NH-- in formula
(I), that is the ring atom connecting to the --NH-- in formula
(I)).
[0089] Suitably, for R.sup.3, and in particular when R.sup.3 is
optionally substituted C.sub.4-7cycloalkyl or optionally
substituted C.sub.5-7cycloalkenyl, R.sup.3 is not substituted
(other than optionally by alkyl or fluoroalkyl) at the ring atom
connecting to the --NH-- in formula (I), and R.sup.3 is not
substituted (other than optionally by alkyl, fluoroalkyl or
NHR.sup.21) at the two ring atoms either side of (bonded to) the
connecting atom. For example, suitably, for R.sup.3, and in
particular when R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl or optionally substituted
C.sub.5-7cycloalkenyl, R.sup.3 is not substituted at the ring atom
connecting to the --NH-- in formula (I), and R.sup.3 is not
substituted at the two ring atoms either side of (bonded to) the
connecting atom.
[0090] Suitably, for R.sup.3, and in particular when R.sup.3 is
optionally substituted C.sub.4-7cycloalkyl or optionally
substituted C.sub.5-7cycloalkenyl, the one or two optional R.sup.3
ring-carbon substituents if present can comprise a substituent (for
example is or are substituent(s)):
(a) at the 3-position of a R.sup.3 cyclobutyl ring, or (b) at the
3- and/or 4-position(s) of a R.sup.3 cyclopentyl or cyclopentenyl
ring, or (c) at the 3-, 4- and/or 5-position(s) of a R.sup.3
cyclohexyl or cyclohexenyl ring, or (d) at the 3-, 4-, 5- and/or
6-position(s) of a R.sup.3 cycloheptyl or cycloheptenyl ring,
and/or (f) at the 1-, 2- and/or highest-numbered-position(s) of a
R.sup.3 cycloalkyl or cycloalkenyl ring, for alkyl or fluoroalkyl
substituent(s), and/or (g) at the 2- and/or
highest-numbered-position(s) of a R.sup.3 cycloalkyl or
cycloalkenyl ring, for NH.sub.2 or fluoro substituent(s).
[0091] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl,
then any OH, methoxy, fluoroalkoxy, --CH.sub.2OH, --CH(Me)OH,
--CH.sub.2CH.sub.2OH, --CH.sub.2NH.sub.2, or --C(O)OH substituent
on a ring carbon is: at the 3-position of a R.sup.3 cyclobutyl
ring; or at the 3- or 4-position of a R.sup.3 cyclopentyl ring; or
at the 3-, 4- or 5-position of a R.sup.3 cyclohexyl ring (such as
at the 3- or 5-position of a R.sup.3 cyclohexyl ring especially for
any OH substituent); or at the 3-, 4-, 5- or 6-position (e.g. 4- or
5-position) of a R.sup.3 cycloheptyl ring. Suitably, when R.sup.3
is optionally substituted C.sub.4-7cycloalkyl, then any OH,
methoxy, fluoroalkoxy, --CH.sub.2OH, --CH(Me)OH,
--CH.sub.2CH.sub.2OH or --CH.sub.2NH.sub.2, or --C(O)OH substituent
(or any OH substituent) on a ring carbon is at the 3- or 4-position
of a R.sup.3 cyclopentyl ring; or more suitably at the 3-, 4- or
5-position, such as at the 3- or 5-position, of a R.sup.3
cyclohexyl ring.
[0092] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl,
then any --C(O)NHR.sup.24 or --C(O)R.sup.25 substituent on a ring
carbon is: at the 3-position of a R.sup.3 cyclobutyl ring; or at
the 3- or 4-position of a R.sup.3 cyclopentyl ring; or at the
4-position of a R.sup.3 cyclohexyl ring; or at the 3-, 4-, 5- or
6-position (e.g. 4- or 5-position) of a R.sup.3 cycloheptyl ring.
When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl, then
any --C(O)NHR.sup.24 or --C(O)R.sup.25 substituent, or any
--C(O)NHR.sup.24 substituent, on a ring carbon is suitably at the
3-position of a R.sup.3 cyclobutyl ring or at the 4-position of a
R.sup.3 cyclohexyl ring. When R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl, it is preferable for any --C(O)NHR.sup.24
substituent to be at the 4-position of a R.sup.3 cyclohexyl
ring.
[0093] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl,
any NH.sub.2 substituent on a ring carbon is at any position other
than the 1-position (the ring atom connecting to the --NH-- in
formula (I)), e.g. at the 2-, 3-, 4-, 5-, 6- or 7-position.
Suitably, any NH.sub.2 substituent is at the 2-, 3-, 4-, 5- or
6-position, for example at the 3-, 4- or 5-position or at the 3- or
5-position, of a R.sup.3 cyclohexyl ring.
[0094] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl
or optionally substituted C.sub.5-7cycloalkenyl, any alkyl or
fluoroalkyl substituent on a ring carbon can for example be at the
1-, 2-, 3-, 4-, 5-, 6- or 7-position, for example at the 1-, 2-,
3-, 5- or 6-position, e.g. the 1-position, of the R.sup.3 ring.
Preferably, any such alkyl or fluoroalkyl substituent on a ring
carbon is at the 1-, 2-, 3-, 5- or 6-position, or more preferably
at the 1-, 3- or 5-position, of a R.sup.3 cyclohexyl or
cyclohexenyl ring.
[0095] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl
or optionally substituted C.sub.5-7cycloalkenyl, any fluoro
substituent on a ring carbon can for example be at the 2-, 3-, 4-,
5-, 6- or 7-position, for example at the 2-, 3-, 4-, 5- or
6-position, such as at the 3- or 4-position, of the R.sup.3 ring.
Suitably, any fluoro substituent on a ring carbon is at the 3-, 4-
or 5-position, in particular at the 4-position, of a R.sup.3
cyclohexyl or cyclohexenyl ring.
[0096] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl,
any oxo (.dbd.O), hydroxyimino (.dbd.N--OH); or
(C.sub.1-2alkoxy)imino (.dbd.N--OR.sup.26) substituent on a ring
carbon can for example be at the 3-, 4- or 5-position, e.g. at the
4-position, of the R.sup.3 cycloalkyl (e.g. C.sub.6-7cycloalkyl
e.g. cyclohexyl, or cyclobutyl) ring. Any such substituent can for
example be at the 3-position of a R.sup.3 cyclobutyl ring or at the
4-position of a R.sup.3 cyclohexyl ring. Preferably, any such
substituent is at the 4-position of a R.sup.3 cyclohexyl ring.
[0097] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl
(e.g. C.sub.6-7cycloalkyl or cyclobutyl, optionally substituted),
then R.sup.3 is suitably cyclohexyl (i.e. unsubstituted); or
cycloheptyl
[0098] (i.e. unsubstituted); or cyclohexyl substituted on a ring
carbon by one substituent being oxo (.dbd.O), OH, NH.sub.2,
C.sub.1-2alkyl, C.sub.1fluoroalkyl such as --CH.sub.2F or
--CHF.sub.2, --CH.sub.2OH, --CH(Me)OH, --C(O)NHR.sup.24 wherein
R.sup.24 is H or methyl (preferably H), --C(O)R.sup.25, fluoro,
hydroxyimino (.dbd.N--OH), or (C.sub.1-2alkoxy)imino
(.dbd.N--OR.sup.26 wherein R.sup.26 is C.sub.1-2alkyl); or
cyclohexyl substituted by two fluoro substituents; or cyclobutyl
(i.e. unsubstituted); or cyclobutyl substituted on a ring carbon
with one substituent being oxo (.dbd.O), OH, methyl, --CH.sub.2F,
--CHF.sub.2, --CH.sub.2OH, --C(O)NHR.sup.24 wherein R.sup.24 is H
or methyl (preferably H), fluoro, hydroxyimino (.dbd.N--OH), or
methoxyimino (.dbd.N--OR.sup.26 where R.sup.26 is methyl).
Preferably, when R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl (e.g. C.sub.6-7cycloalkyl or cyclobutyl,
optionally substituted), then R.sup.3 is cyclohexyl (i.e.
unsubstituted); or cycloheptyl (i.e. unsubstituted); or cyclohexyl
substituted on a ring carbon by one substituent being oxo (.dbd.O),
OH, NH.sub.2, C.sub.1-2alkyl, C.sub.1fluoroalkyl such as
--CH.sub.2F or --CHF.sub.2, --CH.sub.2OH, --C(O)NHR.sup.24 wherein
R.sup.24 is H, fluoro, hydroxyimino (.dbd.N--OH), or (methoxy)imino
(.dbd.N--OR.sup.26 wherein R.sup.26 is methyl); or cyclohexyl
substituted by two fluoro substituents; or cyclobutyl (i.e.
unsubstituted); or cyclobutyl substituted on a ring carbon with one
substituent being oxo (.dbd.O), OH, methyl, --CH.sub.2F,
--CHF.sub.2, --CH.sub.2OH, --C(O)NHR.sup.24 wherein R.sup.24 is H,
fluoro, hydroxyimino (.dbd.N--OH), or methoxyimino
(.dbd.N--OR.sup.26 where R.sup.26 is methyl). More preferably, when
R.sup.3 is optionally substituted C.sub.4-7cycloalkyl (e.g.
C.sub.6-7cycloalkyl or cyclobutyl, optionally substituted), then
R.sup.3 is cyclohexyl (i.e. unsubstituted); or cyclohexyl
substituted on a ring carbon by one oxo (.dbd.O), hydroxyimino
(.dbd.N--OH), --C(O)NH.sub.2, methyl or OH substituent; or
cyclobutyl substituted on a ring carbon by one --C(O)NHR.sup.24
substituent wherein R.sup.24 is H. The optional substituent can for
example be at the 3- or 4-position of the R.sup.3 cyclohexyl ring.
Preferably, when R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl (e.g. C.sub.6-7cycloalkyl or cyclobutyl,
optionally substituted), then any OH substituent on a ring carbon
is preferably at the 3-position of a R.sup.3 cyclohexyl ring,
and/or any oxo (.dbd.O), hydroxyimino (.dbd.N--OH), or
(C.sub.1-2alkoxy)imino (.dbd.N--OR.sup.26) substituent on a ring
carbon is preferably at the 4-position of a R.sup.3 cyclohexyl ring
or at the 3-position of a R.sup.3 cyclobutyl ring, and/or any alkyl
or fluoroalkyl substituent is preferably at the 1-, 3- or
5-position of a R.sup.3 cyclohexyl ring.
[0099] When R.sup.3 is optionally substituted cyclobutyl, then
R.sup.3 can preferably be cyclobutyl (i.e. unsubstituted) or more
preferably 3-(aminocarbonyl)cyclobutyl (i.e.
3-(aminocarbonyl)cyclobutan-1-yl) (e.g. in a cis or trans
configuration, preferably cis).
[0100] When R.sup.3 is optionally substituted cyclopentyl, R.sup.3
can for example be cyclopentyl (i.e. unsubstituted) or more
suitably 3-hydroxy-cyclopentyl.
[0101] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl
(e.g. optionally substituted C.sub.6-7cycloalkyl or optionally
substituted cyclobutyl), R.sup.3 can for example be cyclobutyl
(i.e. unsubstituted), 4-hydroxy-cyclohexyl (i.e.
4-hydroxycyclohexan-1-yl) (e.g. racemic or in a cis or trans
configuration), 4-methylcyclohexyl (e.g. racemic),
2-aminocyclohexyl (e.g. racemic or in a cis or trans configuration,
preferably trans), 4-aminocyclohexyl (e.g. racemic or in a cis or
trans configuration, preferably racemic or cis), 3-oxocyclohexyl,
4-acetylcyclohexyl (e.g. racemic or in a cis or trans
configuration, preferably racemic or cis),
4-(1-hydroxyethyl)cyclohexyl (e.g. racemic or in a cis or trans
configuration with respect to the ring, preferably racemic or cis),
or 3-(hydroxymethyl)cyclohexyl (e.g. racemic or in a cis or trans
configuration).
[0102] However, when R.sup.3 is optionally substituted
C.sub.4-7cycloalkyl (e.g. optionally substituted
C.sub.6-7cycloalkyl or optionally substituted cyclobutyl), R.sup.3
is more preferably cyclohexyl (i.e. unsubstituted), cycloheptyl
(i.e. unsubstituted), 3-hydroxy-cyclohexyl (i.e.
3-hydroxycyclohexan-1-yl) (e.g. racemic or in a cis or trans
configuration, preferably racemic or cis), 4-oxo-cyclohexyl (i.e.
4-oxocyclohexan-1-yl), 4-(hydroxyimino)cyclohexyl (i.e.
4-(hydroxyimino)cyclohexan-1-yl),
4-(C.sub.1-2alkoxyimino)cyclohexyl, 4-(aminocarbonyl)cyclohexyl
(i.e. 4-(aminocarbonyl)cyclohexan-1-yl) (e.g. racemic or in a cis
or trans configuration, preferably racemic or cis),
1-methylcyclohexyl (e.g. racemic), 3-methylcyclohexyl (e.g.
racemic), 4,4-(difluoro)cyclohexyl, 3-aminocyclohexyl (e.g. racemic
or in a cis or trans configuration), 4,4-(hydroxymethyl)cyclohexyl
(e.g. racemic or in a cis or trans configuration), or
3-(aminocarbonyl)cyclobutyl (i.e. 3-(aminocarbonyl)cyclobutan-1-yl)
(e.g. racemic or in a cis or trans configuration, preferably
cis).
[0103] A "cis configuration" in general includes mixtures of
configurations wherein the cis configuration is the major
component.
[0104] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl
(e.g. optionally substituted C.sub.6-7cycloalkyl or optionally
substituted cyclobutyl), R.sup.3 is still more preferably
cyclohexyl (i.e. unsubstituted), 3-hydroxy-cyclohexyl (i.e.
3-hydroxycyclohexan-1-yl) (preferably racemic or in a cis
configuration), 4-oxo-cyclohexyl (i.e. 4-oxocyclohexan-1-yl),
4-(hydroxyimino)cyclohexyl (i.e. 4-(hydroxyimino)cyclohexan-1-yl),
4-(aminocarbonyl)cyclohexyl (i.e. 4-(aminocarbonyl)cyclohexan-1-yl)
(preferably racemic or in a cis configuration), or
3-(aminocarbonyl)cyclobutyl (i.e. 3-(aminocarbonyl)cyclobutan-1-yl)
(preferably racemic or in a cis configuration).
[0105] When R.sup.3 is optionally substituted
mono-unsaturated-C.sub.5-7cycloalkenyl, suitably it is optionally
substituted mono-unsaturated-C.sub.5-6cycloalkenyl, preferably
optionally substituted mono-unsaturated-C.sub.6cycloalkenyl (i.e.
optionally substituted mono-unsaturated-cyclohexenyl=optionally
substituted cyclohexenyl). For example, the R.sup.3 cyclohexenyl
can be optionally substituted cyclohex-3-en-1-yl.
[0106] When R.sup.3 is optionally substituted
mono-unsaturated-C.sub.5-7cycloalkenyl, suitably the R.sup.3
cycloalkenyl (e.g. cyclohexenyl) is substituted on a ring carbon
with one fluoro substituent or is unsubstituted. For example, the
R.sup.3 optionally substituted cycloalkenyl can be
cyclohex-3-en-1-yl (i.e. unsubstituted) or
4-fluoro-cyclohex-3-en-1-yl.
[0107] For R.sup.3 cycloalkenyl, the optional substituent(s) on a
ring carbon can for example be at the 1-, 2-, 3-, 4-, 5- or
6-position(s) of the cycloalkenyl ring.
[0108] When R.sup.3 is the heterocyclic group of sub-formula (aa),
(bb) or (cc), then Y is suitably O or NR.sup.10. When R.sup.3 is
the heterocyclic group of sub-formula (aa) or (bb), then Y is
preferably O or N--C(O)--NH.sub.2.
[0109] R.sup.10 can for example be a hydrogen atom (H), methyl,
C(O)NH.sub.2, C(O)-methyl or C(O)--C.sub.1fluoroalkyl.
[0110] Suitably, R.sup.10 is not methyl.
[0111] Suitably, R.sup.10 is a hydrogen atom (H), C(O)NH.sub.2,
C(O)-methyl or C(O)--C.sub.1fluoroalkyl (e.g. C(O)--CF.sub.3). More
suitably, R.sup.10 is H, C(O)NH.sub.2 or C(O)-methyl; in particular
C(O)NH.sub.2.
[0112] When R.sup.3 is the heterocyclic group of sub-formula (aa),
(bb) or (cc), then it is preferable that R.sup.3 is the
heterocyclic group of sub-formula (aa) or (bb), more preferably of
sub-formula (bb).
[0113] In sub-formula (bb), n.sup.1 is preferably 1. In sub-formula
(cc), n.sup.2 is preferably 1. That is, six-membered rings are
preferred in the R.sup.3 heterocyclic group.
[0114] In the invention, the R.sup.3 heterocyclic group of
sub-formula (aa), (bb) or (cc) is optionally substituted on a ring
carbon with one or two (e.g. one) substituents being oxo
(.dbd.O).
[0115] In the R.sup.3 heterocyclic group of sub-formula (aa), (bb)
or (cc), any oxo (.dbd.O) substituent on a ring carbon is suitably
on a carbon atom bonded (adjacent) to Y. In one embodiment, any oxo
(.dbd.O) substituent on a ring carbon can be on a carbon atom
bonded (adjacent) to Y only when Y is O or NR.sup.10, for example
only when Y is NR.sup.10.
[0116] In the R.sup.3 heterocyclic group of sub-formula (aa), (bb)
or (cc), any oxo (.dbd.O) substituent on a ring carbon can suitably
be at the 2-, 3-, 4-, 5- or 6-position of the R.sup.3 heterocyclic
ring. For example any ring-carbon oxo (.dbd.O) substituent(s) can
be: at the 2-, 4- or 5-position(s) (e.g. 2-position or 4-position,
or two oxo substituents at 2- and 4-positions) of a R.sup.3
heterocyclic group of sub-formula (aa), at the 2-, 4-, 5- or
6-position(s) (e.g. 4-position) of a six-membered R.sup.3
heterocyclic group of sub-formula (cc) wherein n.sup.2 is 1, at the
2-, 3-, 5-, 6- or 7-position(s) (e.g. 5-position) of a
seven-membered R.sup.3 heterocyclic group of sub-formula (bb)
wherein n.sup.1 is 2, or at the 2-, 4-, 5-, 6- or 7-position(s)
(e.g. 2-position) of a seven-membered R.sup.3 heterocyclic group of
sub-formula (cc) wherein n.sup.2 is 2.
[0117] (In this connection and generally herein, the 1-position of
the R.sup.3 heterocyclic ring is deemed to be the connection point
to the --NH-- in formula (I), that is the ring atom connecting to
the --NH-- in formula (I), and the remaining positions of the ring
are then numbered so that the ring heteroatom takes the lowest
possible number).
[0118] In the R.sup.3 heterocyclic group of sub-formula (aa), (bb)
or (cc), no substitution is allowed at the 1-position ring-carbon
of the R.sup.3 heterocyclic ring.
[0119] However, it is generally preferable that, in R.sup.3, the
heterocyclic group of sub-formula (aa), (bb) or (cc) is not
substituted on a ring carbon (i.e. is not substituted on any ring
carbon).
[0120] When R.sup.3 is the heterocyclic group of sub-formula (aa)
and Y is NR.sup.10, then R.sup.10 is not C(O)-methyl, or
C(O)--C.sub.1fluoroalkyl.
[0121] In one preferable embodiment, when R.sup.3 is the
heterocyclic group of sub-formula (aa), then Y is O, S, SO.sub.2,
NH or NC(O)NH.sub.2 (in particular Y can be O, S, NH or
NC(O)NH.sub.2, such as NC(O)NH.sub.2).
[0122] When R.sup.3 is the heterocyclic group of sub-formula (bb)
and Y is NR.sup.10 (e.g. when NHR.sup.3 is
##STR00010##
wherein the --NH-- connection point of the NHR.sup.3 group to the
4-position of the pyrazolopyridine of formula (I) is underlined),
then R.sup.10 is not methyl.
[0123] Therefore, when R.sup.3 is the heterocyclic group of
sub-formula (bb), then Y is O, S, SO.sub.2 or NR.sup.10 wherein
R.sup.10 is H, C(O)NH.sub.2, C(O)-methyl or
C(O)--C.sub.1fluoroalkyl (e.g. C(O)--CF.sub.3).
[0124] When R.sup.3 is the heterocyclic group of sub-formula (bb),
then R.sup.10 is preferably H, C(O)NH.sub.2 or C(O)-methyl, for
example C(O)NH.sub.2 or C(O)-methyl, more preferably
C(O)NH.sub.2.
[0125] When R.sup.3 is the heterocyclic group of sub-formula (cc),
then Y is O, S, SO.sub.2 or NR.sup.10 wherein R.sup.10 is H or
methyl.
[0126] Suitably, when R.sup.3 is the heterocyclic group of
sub-formula (cc), then: either Y is O, S, SO.sub.2 or NR.sup.10
wherein R.sup.10 is H, or NHR.sup.3 is of sub-formula (m4):
##STR00011##
wherein the --NH-- connection point of the NHR.sup.3 group to the
4-position of the pyrazolopyridine of formula (I) is
underlined.
[0127] Suitably, when R.sup.3 is the heterocyclic group of
sub-formula (cc), then Y is O, S, SO.sub.2 or NR.sup.10 wherein
R.sup.10 is H, or Y is O or NR.sup.10 wherein R.sup.10 is H.
[0128] In one embodiment, for sub-formula (bb) and/or for
sub-formula (cc), Y is O or NR.sup.10.
[0129] When R.sup.3 is optionally substituted C.sub.4-7cycloalkyl
(e.g. optionally substituted C.sub.6-7cycloalkyl or optionally
substituted cyclobutyl) or optionally substituted
mono-unsaturated-C.sub.5-7cycloalkenyl or an optionally substituted
heterocyclic group of sub-formula (aa), (bb) or (cc), then a
substituent on a ring carbon can be racemic or in the cis or trans
configuration with respect to the --NH-- group of formula (I) to
which R.sup.3 is attached (bonded). A cis or trans configuration
includes mixtures of configurations wherein the stated
configuration is the major component. In this context, "racemic"
refers to a mixture of isomers containing substantially equal
amounts of the cis and trans configurations with respect to a
substituent and the --NH-- group on the R.sup.3 ring, and in this
context "racemic" does not refer to isomerism at atoms other than
R.sup.3 ring carbon atoms. For example, an OH or --C(O)NHR.sup.24
substituent on C.sub.6-7cycloalkyl or cyclobutyl can for example be
in the cis configuration and/or a NH.sub.2 substituent on
C.sub.6-7cycloalkyl can for example be racemic or in the cis or
trans configuration, with respect to the --NH--group of formula (I)
to which R.sup.3 is attached (bonded), including mixtures of
configurations wherein the stated configuration is the major
component.
[0130] When R.sup.3 is a bicyclic group of sub-formula (ee), then
NHR.sup.3 can be of sub-formula (c6) or (c7):
##STR00012##
wherein the --NH-- connection point of the NHR.sup.3 group to the
4-position of the pyrazolopyridine of formula (I) is
underlined.
[0131] Preferably, NHR.sup.3 is of sub-formula (al), (b), (c), (c
1), (c 2), (c 3), (c 4), (c 5), (c 6), (c 7), (d), (e), (f), (g),
(g2), (g4), (h), (i), (j), (k), (k1), (k2), (k3), (L), (m), (m1),
(m3), (m4), (n), (o), (o1), (o2), (o3), (p), (p1), (p2), (p3),
(p5), (p6), (p9), (p10), (p12), (p13), (p14), (p15), or (q):
##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017##
[0132] In the sub-formulae (al) to (q) etc above, the --NH--
connection point of the NHR.sup.3 group to the 4-position of the
pyrazolopyridine of formula (I) is underlined.
[0133] Preferably, NHR.sup.3 is of sub-formula (c), (c1), (c 2), (c
3), (c 4), (c 5), (c 6), (c 7), (d), (e), (f), (g4), (h), (i), (j),
(k), (k1), (k2), (k3), (L), (m), (m1), (m3), (m4), (n), (o), (o1),
(o2), (o3), (p), (p2), (p5), (p6), (p9), (p10), (p12), (p13),
(p14), (p15) or (q); or preferably NHR.sup.3 is of sub-formula
(a1), (c), (c1), (c 2), (c 3), (c 4), (c 5), (c 6), (c 7), (d),
(e), (f), (g4), (h), (i), (j), (k), (k1), (k2), (k3), (L), (m),
(m1), (m3), (m4), (n), (o), (o1), (o2), (o3), (p), (p1), (p2),
(p5), (p6), (p9), (p10), (p12), (p13), (p14), (p15) or (q).
[0134] More preferably, NHR.sup.3 is of sub-formula (c), (c1), (c
4), (c 5), (h), (i), (k), (k2), (k3), (m1), (n), (o), (o2), (o3),
(p2), (p5), (p6), (p9), (p10), (p13) or (p15).
[0135] NHR.sup.3 is more preferably of sub-formula (c), (h), (k),
(k2), (k3), (n), (o), (o2), (p9) or (p13); still more preferably
NHR.sup.3 is (c), (h), (k2), (k3), (n), (o), (o2), (p9) or
(p13).
[0136] Most preferably, R.sup.3 is tetrahydro-2H-pyran-4-yl or
1-(aminocarbonyl)-4-piperidinyl; that is NHR.sup.3 is most
preferably of sub-formula (h) or (k2), as shown above, in
particular of sub-formula (h).
[0137] When NHR.sup.3 is of sub-formula (n), then it can be in the
trans configuration. But preferably it is in the cis configuration,
i.e. preferably it is a cis-(3-hydroxycyclohexan-1-yl)amino group
(including mixtures of configurations wherein the cis configuration
is the major component), or it is racemic.
[0138] When NHR.sup.3 is of sub-formula (p9), then it can be in the
trans configuration. But preferably it is in the cis configuration,
i.e. preferably it is a cis-[4-(aminocarbonyl)cyclohexan-1-yl]amino
group (including mixtures of configurations wherein the cis
configuration is the major component), or it is racemic.
[0139] When NHR.sup.3 is of sub-formula (p12), then it can be in
the trans configuration. But, preferably, it is in the cis
configuration, i.e. preferably NHR.sup.3 is a
cis-[4-acetylcyclohexan-1-yl]amino group (including mixtures of
configurations wherein the cis configuration is the major
component), or it is racemic.
[0140] When NHR.sup.3 is of sub-formula (p13), then it can be in
the trans configuration. But, preferably, it is in the cis
configuration, i.e. preferably NHR.sup.3 is a
cis-[3-(aminocarbonyl)cyclobutan-1-yl]amino group (including
mixtures of configurations wherein the cis configuration is the
major component), or it is racemic.
[0141] The NHR.sup.3 group of sub-formula (p10), (p14) or (p15),
independently, can for example be racemic; or it can be in the cis
configuration with respect to the ring (including mixtures of
configurations wherein the cis configuration is the major
component).
[0142] Preferably, the compound of formula (I) or the salt thereof
is: [0143]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4--
b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino-
)benzamide, [0144]
4-[(1,6-diethyl-5-{[({4-[(8-{(2R)-2-[(methyloxy)methyl]-1-pyrrolidinyl}oc-
tanoyl)amino]phenyl}carbonyl)amino]methyl}-1H-pyrazolo[3,4-b]pyridin-4-yl)-
amino]-1-piperidinecarboxamide, [0145]
4-[(1,6-diethyl-5-{[({4-[(8-{(2S)-2-[(methyloxy)methyl]-1-pyrrolidinyl}oc-
tanoyl)amino]phenyl}carbonyl)amino]methyl}-1H-pyrazolo[3,4-b]pyridin-4-yl)-
amino]-1-piperidinecarboxamide, [0146]
4-({1,6-diethyl-5-[({[4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino-
)phenyl]carbonyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-pip-
eridinecarboxamide, [0147]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-[(8-{(2R)-2-[(methyloxy)methyl]-1-pyrrolidinyl}octanoyl)-
amino]benzamide, [0148]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-[(8-{(2S)-2-[(methyloxy)methyl]-1-pyrrolidinyl}octanoyl)-
amino]benzamide, [0149]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-{[8-(4-morpholinyl)octanoyl]amino}benzamide,
[0150]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-{[8-(1-pyrrolidinyl)octanoyl]amino}benzamide,
[0151]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-[4-({4-[(2-hydroxyethyl)(methyl)amino]butyl}oxy)butyl]be-
nzamide, [0152]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)benzamide,
[0153]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-(4-{[4-(1-pyrrolidinyl)butyl]oxy}butyl)benzamide,
[0154]
4-[(1,6-diethyl-5-{[({4-[4-({4-[(2-hydroxyethyl)(methyl)amino]butyl}oxy)b-
utyl]phenyl}carbonyl)amino]methyl}-1H-pyrazolo[3,4-b]pyridin-4-yl)amino]-1-
-piperidinecarboxamide, [0155]
4-({1,6-diethyl-5-[({[4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)phenyl]carb-
onyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarbo-
xamide, [0156]
4-({1,6-diethyl-5-[({[4-(4-{[4-(1-pyrrolidinyl)butyl]oxy}butyl)phenyl]car-
bonyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarb-
oxamide, [0157]
N-{[1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]-
pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)b-
enzamide, [0158]
N-{[1-ethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-
-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide,
[0159]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4--
b]pyridin-5-yl]methyl}-4-[8-(4-morpholinyl)octyl]benzamide, [0160]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-{8-[(2-hydroxyethyl)(methyl)amino]octyl}benzamide,
[0161]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4--
b]pyridin-5-yl]methyl}-3-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino-
)benzamide, [0162]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-2-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide, [0163]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({6-[(2-hydroxyethyl)(methyl)amino]hexanoyl}amino)benzam-
ide, [0164]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({7-[(2-hydroxyethyl)(methyl)amino]heptanoyl}amino)benza-
mide, [0165]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({10-[(2-hydroxyethyl)(methyl)amino]decanoyl}amino)benza-
mide, or [0166]
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({11-[(2-hydroxyethyl)(methyl)amino]undecanoyl}amino)ben-
zamide; or a salt thereof such as a pharmaceutically acceptable
salt thereof.
[0167] The structures of the above-listed specific compounds or
salts, or embodiments (e.g. specific salts) thereof, are given in
the Examples hereinafter.
[0168] In the most preferred embodiment of the invention, the
compound of formula (I) or the salt thereof is:
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide, whose formula is
##STR00018##
or a salt thereof (e.g. the compound or a pharmaceutically
acceptable salt thereof).
[0169] Therefore, another preferred aspect of the invention
provides
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide, whose formula is
##STR00019##
or a salt thereof (e.g. the compound or a pharmaceutically
acceptable salt thereof.
[0170] See for example Example 1A1, 1A2, 1A3, 1B, 1C, 1D, 1E, 1F,
1G, 1H1, 1H2, 1J1, or 1J2 disclosed herein. This above-illustrated
compound or a salt thereof can be for inhaled or intranasal
administration e.g. to a mammal such as a human, monkey, rodent
(e.g. rat or mouse) or dog, in particular to a human.
[0171] This compound
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide, encompassed by the presently invented compounds of formula
(I), or a salt thereof (e.g. the monohydrochloride salt thereof,
may be suitable for use via an inhaled route of administration, in
particular as a PDE4 inhibitor. Preliminary tests appear to
indicate that this specific compound or a salt thereof may exhibit
a reasonable level of antiinflammatory efficacy and/or duration of
action (2 or more hours duration of action after i.t.
administration to the rat as an aqueous solution, and 4 hours
duration of action after i.t. administration to the rat as a dry
powder), when measured using an intratracheal (i.t.) rat
LPS-induced neutrophilia model (see In Vivo Assay 1 hereinbelow),
wherein LPS is lipopolysaccharide.
[0172] In one embodiment of the invention, the compound or salt can
be
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a hydrochloride (e.g. monohydrochloride or dihydrochloride),
hydrobromide (e.g. monohydrobromide or dihydrobromide), phosphate,
succinate (e.g. hemisuccinate), fumarate (e.g. hemifumarate),
1-naphthoate, 1-hydroxy-2-naphthoate ("xinafoate") (e.g.
mono-1-hydroxy-2-naphthoate), L-glutamate, camphorsulfonate (e.g.
(+)-camphorsulfonate and/or 10-camphorsulfonate and/or
mono-camphorsulfonate, e.g. mono-(+)-10-camphorsulfonate), or
1,5-naphthalenedisulfonate (e.g. hemi-1,5-naphthalenedisulfonate)
salt thereof. This compound or the salt thereof can e.g. be for
inhaled or intranasal administration e.g. to a mammal such as a
human.
[0173] In particular, the compound or salt can be
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a hydrochloride (e.g. monohydrochloride or dihydrochloride),
hydrobromide (e.g. monohydrobromide or dihydrobromide), or
succinate (e.g. hemisuccinate) salt thereof. This compound or the
salt thereof can be for inhaled or intranasal administration e.g.
to a mammal such as a human.
[0174] Preferably, the compound or salt is a hydrochloride (e.g.
monohydrochloride or dihydrochloride) salt of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide, in particular the monohydrochloride salt.
[0175] It is currently thought that there are at least two solid
forms of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride. The two currently known solid forms are:
[0176] a first, preferred, solid form (herein named "Form 1") which
is thought to be a substantially-anhydrous solid form ("anhydrate")
and which (e.g. from its XRPD spectrum) is thought to be
crystalline, and [0177] a second, less-preferred, solid form
(herein named "Form 2") which is thought to be a hydrated solid
form ("hydrate") and which (e.g. from its XRPD spectrum) is thought
to be crystalline.
[0178] Therefore, one preferable aspect of the invention provides
crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate.
[0179] Suitably, the crystalline monohydrochloride Form 1 anhydrate
is substantially free of (e.g. <30% w/w or <25% w/w or
<20% w/w or <10% w/w or <5% w/w is present as) the
crystalline monohydrochloride Form 2 hydrate. For example,
suitably, the crystalline monohydrochloride Form 1 anhydrate is
substantially free of (e.g. <30% w/w or <25% w/w or <20%
w/w or <10% w/w or <5% w/w is present as) any other form of
the monohydrochloride or dihydrochloride salt.
[0180] Suitably, the purity of the crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate is such that less than 30%
w/w (e.g. less than 25% w/w, or less than 20% w/w, or less than 10%
w/w, or less than 5% w/w) of the
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide hydrochloride salt is present as the crystalline
monohydrochloride Form 2 hydrate.
[0181] Preferably, 70% w/w or more, or 75% w/w or more, or 80% w/w
or more, or 90% w/w or more, or 95% w/w or more, of the
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide hydrochloride present is present as the
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate.
[0182] The crystalline monohydrochloride Form 2 hydrate is thought
to have a greater propensity to take up water than the crystalline
monohydrochloride Form 1 anhydrate, e.g. as the relative humidity
(RH) increases. In addition, water is believed to be readily lost
from the Form 2 hydrate (a) on heating to only slightly above room
temperature and/or (b) on reduction in the relative humidity
(R.sup.H) (e.g. water appears to be lost from the Form 2 hydrate
when the R.sup.H is reduced to 0% R.sup.H).
[0183] Differential scanning calorimetry (DSC) data for the
monohydrochloride Form 2 hydrate generally shows an endotherm,
interpreted as water loss from the Form 2 hydrate, at temperatures
only slightly above room temperature. In a DSC thermogram of one
sample of the crystalline monohydrochloride Form 2 hydrate,
measured using increasing temperature starting from about room
temperature, amongst other thermal features, the following
endotherms (interpreted to represent water loss events) were
seen:
(i) a first endotherm/water loss event having an onset of very
approximately 49.degree. C. (a "broad" endotherm, i.e. seen at a
broad temperature range starting from about 49.degree. C. and
ending at a higher temperature, possibly representing gradual water
loss e.g. of unbound water), and (ii) a second endotherm/water loss
event having an onset of very approximately 97.degree. C. (a
"sharper" endotherm, possibly representing loss of bound water),
although it is noted that the end of the first water loss event
often merges into the start of the second water loss event.
[0184] These low-temperature water losses may pose developability
risks for the crystalline monohydrochloride Form 2 hydrate, in
that, for example, it is likely to be less suitable than the Form 1
anhydrate for micronisation (which may warm the drug substance),
which micronisation may for example be carried out before
formulation of the drug substance into an inhalable pharmaceutical
composition.
[0185] In comparison, crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate typically shows a melt onset
temperature in its DSC thermogram of very approximately
190-210.degree. C. or above (depending on a variety of factors such
as the impurity profile, sample preparation, prior micronisation,
etc.), and generally has no significant thermal events (i.e. no or
only minor thermal events), at lower temperatures.
[0186] In gravimetric vapour sorption studies conducted at
25.degree. C., one sample of the crystalline monohydrochloride Form
1 anhydrate showed a moisture uptake as follows, measured as %
weight gain over a selected relative humidity (RH) range as RH
increases: about 0.5% weight gain when RH increased from 30% to
75%, and about 1.0% to about 1.1% weight gain when RH increased
from 30% to 90%. This appears to show that the crystalline
monohydrochloride Form 1 anhydrate has a non-negligible but small
propensity to take up moisture as the R.sup.H increases, at least
at 25.degree. C.
[0187] Overall, the crystalline monohydrochloride Form 1 anhydrate
is thought to be more suitable than the crystalline
monohydrochloride Form 2 hydrate, e.g. for micronisation and/or for
post-micronisation formulation into an inhalable pharmaceutical
composition.
[0188] The crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate can be characterised for
example by its X-ray powder diffraction (XRPD) spectrum. For
example, the solid monohydrochloride salt prepared in Example 1A2
(synthesis first-mentioned herein) is thought to be crystalline
monohydrochloride Form 1 anhydrate, and its detailed XRPD spectrum
is disclosed at the end of Example 1A2 (first synthesis)
herein.
[0189] The following four XRPD peaks of the crystalline
monohydrochloride Form 1 anhydrate are thought to particularly
distinguish the Form 1 anhydrate from the Form 2 hydrate:
5.1.+-.0.1.degree. 2.theta. (d-spacing ca. 17.3 .ANG.),
10.7.+-.0.1.degree. 2.theta. (d-spacing ca. 8.3 .ANG.),
23.1.+-.0.1.degree. 2.theta. (d-spacing ca. 3.9 .ANG.), and
23.5.+-.0.1.degree. 2.theta. (d-spacing ca. 3.8 .ANG.), the stated
degrees two-theta values being when the XRPD spectrum is measured
with a X-ray powder diffractometer using Cu K.alpha. (copper
K-alpha) radiation, a step size of 0.0167.degree. 2.theta. or less,
and a time per step of 31.75 seconds or more. The following
additional two XRPD peaks of the crystalline monohydrochloride Form
1 anhydrate may also partially distinguish this Form 1 anhydrate
from the Form 2 hydrate: 10.3.+-.0.1.degree. 2.theta. (d-spacing
ca. 8.6 .ANG.) and 17.5.+-.0.1.degree. 2.theta. (d-spacing ca. 5.1
.ANG.), the stated degrees two-theta values being when the XRPD
spectrum is measured with a X-ray powder diffractometer using Cu
K.alpha. (copper K-alpha) radiation, a step size of 0.0167.degree.
2.theta. or less, and a time per step of 31.75 seconds or more;
however, there are thought to be shoulders or low intensity peaks
of another form in fairly close proximity that make these
latter-mentioned two peaks slightly less distinguishing than the
first-mentioned four XRPD peaks of the crystalline
monohydrochloride Form 1 anhydrate.
[0190] In contrast, one sample of the less-preferred crystalline
monohydrochloride Form 2 hydrate had an XRPD spectrum comprising
peaks at substantially the following degrees 2.theta. (degrees
2-theta) values and d-spacings: 3.9.+-.0.1.degree. 2.theta.
(d-spacing ca. 22.4 .ANG.), 7.8.+-.0.1.degree. 2.theta. (d-spacing
ca. 11.4 .ANG.), 8.9.+-.0.1.degree. 2.theta. (d-spacing ca. 10.0
.ANG.), 11.7.+-.0.1.degree. 2.theta. (d-spacing ca. 7.6 .ANG.),
13.5.+-.0.1.degree. 2.theta. (d-spacing ca. 6.6 .ANG.),
13.9.+-.0.1.degree. 2.theta. (d-spacing ca. 6.4 .ANG.),
15.6.+-.0.1.degree. 2.theta. (d-spacing ca. 5.7 .ANG.),
16.0.+-.0.1.degree. 2.theta. (d-spacing ca. 5.5 .ANG.),
18.1.+-.0.1.degree. 2.theta. (d-spacing ca. 4.9 .ANG.),
19.5.+-.0.1.degree. 2.theta. (d-spacing ca. 4.6 .ANG.),
20.7.+-.0.1.degree. 2.theta. (d-spacing ca. 4.3 .ANG.),
21.4.+-.0.1.degree. 2.theta. (d-spacing ca. 4.2 .ANG.),
22.5.+-.0.1.degree. 2.theta. (d-spacing ca. 3.9 .ANG.),
22.9.+-.0.1.degree. 2.theta. (d-spacing ca. 3.9 .ANG.),
24.4.+-.0.1.degree. 2.theta. (d-spacing ca. 3.6 .ANG.), and
25.4.+-.0.1.degree. 2.theta. (d-spacing ca. 3.5 .ANG.), the stated
degrees two-theta values being when the XRPD spectrum is measured
with a X-ray powder diffractometer using Cu K.alpha. (copper
K-alpha) radiation, a step size of 0.0167.degree. 2.theta. or less,
and a time per step of 31.75 seconds or more. Of these Form 2
hydrate peaks, the following three XRPD peaks are thought to
distinguish the crystalline monohydrochloride Form 2 hydrate from
the Form 1 anhydrate 3.9.+-.0.1.degree. 2.theta.,
8.9.+-.0.1.degree. 2.theta., and 11.7.+-.0.1.degree. 2.theta., the
stated degrees two-theta values being when the XRPD spectrum is
measured with a X-ray powder diffractometer using Cu K.alpha.
(copper K-alpha) radiation, a step size of 0.0167.degree. 2.theta.
or less, and a time per step of 31.75 seconds or more.
[0191] Therefore, in particular, the crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate can be characterised by
having an X-ray powder diffraction (XRPD) spectrum comprising two,
three or all (e.g. three or all, e.g. all) of the following peaks
at substantially the following degrees 20 (degrees two-theta)
values:
5.1.+-.0.1.degree., 10.7.+-.0.1.degree., 23.1.+-.0.1.degree.,
23.5.+-.0.1.degree., wherein the XRPD spectrum is measured with a
X-ray powder diffractometer using Cu K.alpha. (copper K-alpha)
radiation, a step size of 0.0167.degree. 2.theta. or less, and a
time per step of 31.75 seconds or more (and preferably using a
sample mounted on a silicon wafer plate, e.g. a silicon wafer zero
background plate, and/or e.g. as a layer of powder).
[0192] Alternatively, the crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate can be characterised by
having an X-ray powder diffraction (XRPD) spectrum comprising four,
five or all (e.g. five or all, e.g. all) of the following peaks at
substantially the following degrees 2.theta. (degrees two-theta)
values:
5.1.+-.0.1.degree., 10.3.+-.0.1.degree., 10.7.+-.0.1.degree.,
17.5.+-.0.1.degree., 23.1.+-.0.1.degree., 23.5.+-.0.1.degree.,
[0193] wherein the XRPD spectrum is measured with a X-ray powder
diffractometer using Cu K.alpha. (copper K-alpha) radiation, a step
size of 0.0167.degree. 2.theta. or less, and a time per step of
31.75 seconds or more (and preferably using a sample mounted on a
silicon wafer plate, e.g. a silicon wafer zero background plate,
and/or e.g. as a layer of powder).
[0194] Alternatively, the crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate can be characterised by
having an X-ray powder diffraction (XRPD) spectrum comprising 6 or
more, or 9 or more, or 12 or more, or 15 or more, or all, of the
following peaks at substantially the following degrees 20 (degrees
two-theta) values:
5.1.+-.0.1.degree., 7.8.+-.0.1.degree., 10.3.+-.0.1.degree.,
10.7.+-.0.1.degree., 14.3.+-.0.1.degree., 15.2.+-.0.1.degree.,
17.5.+-.0.1.degree., 18.6.+-.0.1.degree., 18.8.+-.0.1.degree.,
19.7.+-.0.1.degree., 20.3.+-.0.1.degree., 21.4.+-.0.1.degree.,
21.9.+-.0.1.degree., 22.4.+-.0.1.degree., 23.1.+-.0.1.degree.,
23.5.+-.0.1.degree., 24.0.+-.0.1.degree., and wherein the XRPD
spectrum also comprises two, three or all (e.g. three or all) of
the following peaks at substantially the following degrees 2.theta.
(degrees two-theta) values: 5.1.+-.0.1.degree.,
10.7.+-.0.1.degree., 23.1.+-.0.1.degree., 23.5.+-.0.1.degree.,
wherein the XRPD spectrum is measured with a X-ray powder
diffractometer using Cu K.alpha. (copper K-alpha) radiation, a step
size of 0.0167.degree. 2.theta. or less, and a time per step of
31.75 seconds or more (and preferably using a sample mounted on a
silicon wafer plate, e.g. a silicon wafer zero background plate,
and/or e.g. as a layer of powder).
[0195] Alternatively or additionally, the crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate can be characterised for
example by its solid-form infrared (IR) spectrum.
[0196] The monohydrochloride Form 1 anhydrate can for example be
characterised by a solid-form IR spectrum (e.g. as measured using
an FT-IR spectrometer such as a Nicolet Avatar 360 FT-IR
spectrometer, and/or e.g. as measured at 4 cm.sup.-1 or 2 cm.sup.-1
resolution) comprising 7 or more, or 10 or more, or 15 or more, or
18 or more, or all, of the following peaks:
3252, 2938, 2857, 1688, 1639, 1598, 1571, 1526, 1503, 1437, 1407,
1355, 1304, 1254, 1185, 1162, 1139, 1123, 1086, 1035, 1014, 988,
964, 860, 820 and 770 cm.sup.-1, with a variation allowed for each
peak of .+-.2 cm.sup.-1 such as .+-.1 cm.sup.-1, provided that the
IR spectrum comprises a peak at 1639 (.+-.2 or .+-.1)
cm.sup.-1.
[0197] The solid-form IR spectrum of the monohydrochloride Form 1
anhydrate includes a band at 1639 (.+-.2 or .+-.1) cm.sup.-1 which
is thought to be capable of distinguishing from the IR spectrum of
the solid-from monohydrochloride Form 2 hydrate. The
monohydrochloride Form 2 hydrate includes an
apparently-characteristic band at 1623 (.+-.2 or .+-.1) cm.sup.-1
which is not present in the Form 1 anhydrate IR spectrum. For
example, representative solid-form IR spectrum peaks of a sample of
the monohydrochloride Form 2 hydrate include: 3244, 2940, 2858,
1677, 1623, 1602, 1569, 1526, 1510, 1444, 1407, 1353, 1309, 1257,
1227, 1165, 1137, 1085, 1048, 1011, 995, 961 and 860 cm.sup.-1
(with each peak being .+-.2 or .+-.1 cm.sup.-1).
[0198] Alternatively or additionally, the crystalline
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate can be characterised for
example by its solid-form Raman spectrum.
[0199] The monohydrochloride Form 1 anhydrate can for example be
characterised by a solid-form Raman spectrum comprising 5 or more,
or 7 or more, or 10 or more, or all, of the following peaks: 2937,
1671, 1643, 1608, 1563, 1502, 1439, 1343, 1304, 1253, 1191, 1123,
1015, 864 and 826 cm.sup.-1, with a variation allowed for each peak
of .+-.2 cm.sup.-1 such as .+-.1 cm.sup.-1,
[0200] provided that the Raman spectrum comprises a peak at 1643
(.+-.2 or .+-.1) cm.sup.-1.
[0201] In comparison, representative solid-form Raman spectrum
peaks of a sample of the monohydrochloride Form 2 hydrate include:
2937, 1684, 1607, 1572, ca. 1435 (broad), 1353, 1312, 1255, 1227,
1191, 1126, 1081, 1011, 886, 824, 634, 515 and 332 cm.sup.-1 (with
each peak being .+-.2 or .+-.1 cm.sup.-1).
[0202] In an alternative embodiment of the invention, the compound
of formula (I) or the salt thereof is for example a phosphate,
fumarate (e.g. hemifumarate or monofumarate, such as hemifumarate),
1-hydroxy-2-naphthoate ("xinafoate") (e.g. mono- or
di-1-hydroxy-2-naphthoate, such as
mono-1-hydroxy-2-naphthoate=mono-xinafoate), or
(+)-camphorsulfonate [e.g. (+)-10-camphorsulfonate, or mono- or
di-(+)-camphorsulfonate, e.g. mono- or di-(+)-10-camphorsulfonate]
salt of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide. This salt can e.g. be for inhaled or intranasal administration
e.g. to a mammal such as a human.
[0203] In an alternative separate embodiment of the invention, the
compound of formula (I) or the salt thereof does not include,
and/or is not,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]-
pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)b-
enzamide, whose formula is
##STR00020##
or a salt thereof.
[0204] In one preferred embodiment of the invention, the compound
of formula (I) or the salt thereof is:
4-({1,6-diethyl-5-[({[4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)phenyl]carb-
onyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarbo-
xamide, whose formula is
##STR00021##
or a salt thereof (e.g. the compound or a pharmaceutically
acceptable salt thereof).
[0205] In an alternative separate embodiment of the invention, the
compound of formula (I) or the salt thereof does not include,
and/or is not,
4-({1,6-diethyl-5-[({[4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)phenyl-
]carbonyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidine-
carboxamide, whose formula is
##STR00022##
or a salt thereof.
Salts, Solvates, Isomers, Tautomeric Forms, Molecular Weights,
etc.
[0206] Because of their potential use in medicine, the salts of the
compounds of formula (I) are preferably pharmaceutically
acceptable. Suitable pharmaceutically acceptable salts can include
acid addition salts, or less commonly (e.g. if a C(O)OH group is
present in the compound) base addition salts.
[0207] In one embodiment, a pharmaceutically acceptable acid
addition salt is optionally formed by mixing of a compound of
formula (I) with a pharmaceutically acceptable inorganic or organic
acid (such as hydrobromic, hydrochloric, sulfuric, nitric,
phosphoric, succinic, maleic, formic, acetic, propionic, fumaric,
citric, tartaric, lactic, benzoic, 1-naphthoic,
1-hydroxy-2-naphthoic ("xinafoic"), salicylic, glutamic such as
L-glutamic, aspartic, para-toluenesulfonic, benzenesulfonic,
methanesulfonic, ethanesulfonic, naphthalenesulfonic such as
2-naphthalenesulfonic, naphthalenedisulfonic such as
1,5-naphthalenedisulfonic, camphorsulfonic (e.g.
10-camphorsulfonic), 1,2,4-benzenetricarboxylic,
hydroxyethylidene-1,1-diphosphonic, or hexanoic acid), optionally
in a suitable solvent such as an organic solvent, to give the salt
which is usually isolated for example by crystallisation and
filtration. The organic solvent used in the pharmaceutically
acceptable acid addition salt formation process may e.g. be
anhydrous or water-containing. Depending on the properties of
and/or suitabilities for use with the compound of formula (I), the
organic solvent may be for example selected from methanol, ethanol
(e.g. anhydrous), n-propanol (propan-1-ol, e.g. anhydrous),
isopropanol (IPA, propan-2-ol, e.g. substantially anhydrous IPA or
IPA:water mixtures such as IPA containing about 1-2% water),
butanol, pentan-1-ol (e.g. substantially anhydrous), ethyl acetate,
methyl isobutyl ketone (MIBK), chloroform, dichloromethane,
toluene, or mixtures thereof. A pharmaceutically acceptable
inorganic or organic acid can for example be: hydrobromic,
hydrochloric, sulfuric, nitric, phosphoric, succinic, maleic,
1-naphthoic, 1-hydroxy-2-naphthoic ("xinafoic"), glutamic such as
L-glutamic, para-toluenesulfonic, benzenesulfonic, methanesulfonic,
ethanesulfonic, naphthalenesulfonic such as 2-naphthalenesulfonic,
naphthalenedisulfonic such as 1,5-naphthalenedisulfonic,
camphorsulfonic (e.g. 10-camphorsulfonic and/or
(+)-camphorsulfonic), 1,2,4-benzenetricarboxylic, or
hydroxyethylidene-1,1-diphosphonic acid.
[0208] A pharmaceutically acceptable acid addition salt of a
compound of formula (I) can comprise or be for example a
hydrobromide (e.g. monohydrobromide or dihydrobromide),
hydrochloride (e.g. monohydrochloride or dihydrochloride), sulfate,
nitrate, phosphate, succinate (e.g. hemisuccinate), maleate,
formate, acetate, propionate, fumarate (e.g. hemifumarate),
citrate, tartrate, lactate, benzoate, 1-naphthoate,
1-hydroxy-2-naphthoate ("xinafoate") (e.g.
mono-1-hydroxy-2-naphthoate), salicylate, glutamate such as
L-glutamate, aspartate, para-toluenesulfonate (e.g. mono- or
di-para-toluenesulfonate), benzenesulfonate, methanesulfonate (e.g.
mono- or di-methanesulfonate), ethanesulfonate,
naphthalenesulfonate (e.g. 2-naphthalenesulfonate),
naphthalenedisulfonate (e.g. 1,5-naphthalenedisulfonate),
camphorsulfonate (e.g. 10-camphorsulfonate and/or
(+)-camphorsulfonate, e.g. mono-(+)-10-camphorsulfonate),
1,2,4-benzenetricarboxylate, hydroxyethylidene-1,1-diphosphonate,
or hexanoate salt.
[0209] In one embodiment, a pharmaceutically acceptable base
addition salt is optionally formed by reaction of a compound of
formula (I) with a suitable inorganic or organic base (e.g.
triethylamine, ethanolamine, triethanolamine, choline, arginine,
lysine or histidine), optionally in a suitable solvent such as an
organic solvent, to give the base addition salt which is usually
isolated for example by crystallisation and filtration.
[0210] Other suitable pharmaceutically acceptable salts include
pharmaceutically acceptable metal salts, for example
pharmaceutically acceptable alkali-metal or alkaline-earth-metal
salts such as sodium, potassium, calcium or magnesium salts; in
particular pharmaceutically acceptable metal salts of one or more
carboxylic acid moieties that may be present in the compound of
formula (I).
[0211] Other non-pharmaceutically acceptable salts, e.g. oxalates
or trifluoroacetates, may be used, for example in the isolation of
compounds of the invention, and are included within the scope of
this invention.
[0212] The invention includes within its scope all possible
stoichiometric and non-stoichiometric forms of the salts of the
compounds of formula (I).
[0213] Also included within the scope of the invention are all
solvates, hydrates and complexes of compounds and salts of the
invention.
[0214] Certain compounds or salts included in the present invention
may be present as isomers. The present invention includes within
its scope all such isomers, including racemates, enantiomers and
mixtures thereof.
[0215] In the compounds or salts, pharmaceutical compositions,
uses, methods of treatment/prophylaxis, methods of preparing, etc.
according to the present invention, where a defined isomeric
configuration e.g. stereochemical configuration is described or
claimed, the invention includes a mixture comprising (a) a major
component of the compound or salt which is in the described or
claimed configuration, together with (b) one or more minor
components of the compound or salt which is/are not in the
described or claimed configuration. Preferably, in such a mixture,
the major component of the compound or salt which is in the
described or claimed configuration represents 70% or more, or 75%
or more, more preferably 85% or more, still more preferably 90% or
more, yet more preferably 95% or more, yet more preferably 98% or
more, of the total amount of compound or salt present in the
mixture on a molarity basis.
[0216] The percentage of one isomeric/stereochemical component in a
mixture of different isomeric/stereochemical components, and if
appropriate enantiomeric and/or diastereomeric excesses, can be
measured using techniques known in the art. Such methods include
the following:
[0217] (1) Measurement using NMR (e.g. .sup.1H NMR) spectroscopy in
the presence of chiral agent. One can measure a nuclear magnetic
resonance (NMR) spectrum (preferably a .sup.1H NMR spectrum, and/or
a solution-phase NMR spectrum e.g. in CDCl.sub.3 or D6-DMSO
solvent) of the compound/salt mixture in the presence of a suitable
chiral agent which "splits" the NMR peaks of a given atom in
different isomers into different peak positions. The chiral agent
can be: i) an optically pure reagent which reacts with the
compound/salt e.g. to form a mixture of diastereomers, ii) a chiral
solvent, iii) a chiral molecule which forms a transient species
(e.g. diastereomeric species) with the compound/salt, or iv) a
chiral shift reagent. See e.g. J. March, "Advanced Organic
Chemistry", 4th edn., 1992, pages 125-126 and refs. 138-146 cited
therein. A chiral shift reagent can be a chiral lanthanide shift
reagent such as tris[3-trifluoroacetyl-d-camphorato]europium-(III)
or others as described in Morrill, "Lanthanide Shift Reagents in
Stereochemical Analysis", VCH, New York, 1986. Whatever the chiral
agent is that is used, usually, the relative integrals
(intensities) for the NMR peaks of a given atom or group in
different isomers can provide a measurement of the relative amounts
of each isomer present.
[0218] (2) Measurement using chiral chromatography, especially on
an analytical scale. A suitable chiral column which separates the
different isomeric components can be used to effect separation,
e.g. using gas or liquid chromatography such as HPLC, and/or e.g.
on an analytical scale. The peaks for each isomer can be integrated
(area under each peak); and a comparison or ratio of the integrals
for the different isomers present can give a measurement of the
percentage of each isomeric component present. See for example:
"Chiral Chromatography", Separation Science Series Author: T. E.
Beesley and R. P. W. Scott, John Wiley & Sons, Ltd.,
Chichester, UK, 1998, electronic Book ISBN: 0585352690, Book ISBN:
0471974277.
[0219] (3) Separation of pre-existing diastereomeric mixtures which
are compounds/salts of the invention can be achieved (usually
directly, without derivatisation) using separation techniques such
as gas or liquid chromatography. Diastereomeric ratios and/or
excesses can thereby be derived e.g. from the relative peak areas
or relative separated masses.
[0220] (4) Conversion with a chiral/optically-active agent and
subsequent separation of the resulting isomers, e.g. diastereomers.
Conversion can be via derivatisation of a derivatisable group (e.g.
--OH, --NHR) on the compound/salt with an optically-active
derivatising group (e.g. optically active acid chloride or acid
anhydride); or can be via formation of an acid or base addition
salt of the compound by treatment of the compound with an
optically-active acid or base, such as + or - di-para-toluoyl
tartaric acid. After derivatisation, separation of the resulting
isomers e.g. diastereomers, can be using gas or liquid
chromatography (usually non-chiral); or (especially with isomeric
salts) can be by selective crystallisation of a single isomeric
e.g. diastereoisomeric salt. Determination of isomeric ratios
and/or excesses can be using chromatography peak areas or
measurement of mass of each separated isomer.
[0221] See e.g. J. March, "Advanced Organic Chemistry", 4th edn.,
1992, pages 120-121 and 126, and refs. 105-115 and 147-149 cited
therein.
[0222] (5) Measurement of optical activity [alpha] of mixture and
comparison with optical activity of pure isomer [alpha]max if
available (e.g. see J. March, "Advanced Organic Chemistry", 4th
edn., 1992, page 125 and refs. 138-139 cited therein). This assumes
a substantially linear relationship between [alpha] and
concentration.
[0223] Certain of the groups, e.g. heteroaromatic ring systems,
included in compounds of formula (I) or their salts may exist in
one or more tautomeric forms. The present invention includes within
its scope all such tautomeric forms, including mixtures.
Synthetic Process Routes
[0224] The following non-limiting processes can generally be used
to prepare the compounds of formula (I):
##STR00023##
wherein Ar has the sub-formula (x):
##STR00024##
[0225] Some of the following synthetic processes may be exemplified
for compounds of Formula (I) with particular Ar groups (in
particular embodiments within the Ar sub-formula (x)) and/or with
particular substitution patterns e.g. particular values of Q.sup.1
and Q.sup.2 and L. However, at least some of these processes are
likely to be able to be usable with appropriate modification(s),
e.g. modification(s) of starting materials and reagents, which
modification(s) may be extensive, for making other compounds of
Formula (I).
Process 1A
[0226] (For example, the compounds or salts whose names and/or
chemical structures are given in and which are the products of
Examples 1A1, 1A2, 1A3, 1B, 1C, 1D, 1E, 1F, 1G, 1H1, 1H2, 1J1, 1J2,
3, 4, 9, 11, 12, 17, 18, 25, 26 and 29 to 34, may optionally be
prepared by this Process 1A).
[0227] Compounds of formula (II), which are compounds of formula
(I) wherein Ar has sub-formula (x), Q.sup.1 is NH, Q.sup.2 is
--C(O)--, and L is (CH.sub.2).sub.n, wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, and n are as defined herein, or
salts thereof, can be prepared by reaction (substitution) of a
compound of formula (III), wherein X.sup.1 is a suitable leaving
group such as mesylate (methanesulfonate), tosylate
(p-toluenesulfonate) or a halogen atom, in particular a halogen
atom such as preferably a bromine atom, and wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4 and n are as defined herein, with an
amine of formula (IV), wherein R.sup.5 and R.sup.6 are as defined
herein. Suitable conditions for the reaction include heating in a
suitable solvent such as N,N-dimethylformamide, in the presence of
a suitable base (e.g. a base having a low nucleophilicity such that
it does not substantially displace the X.sup.1 leaving group) such
as N,N-diisopropylethylamine, e.g. heating at a suitable
temperature such as about 70-90.degree. C. e.g. about 80-85.degree.
C. Alternative conditions include heating under microwave
irradiation in a suitable solvent such as N,N-dimethylformamide,
e.g. at a suitable temperature such as about 140.degree. C.
##STR00025##
[0228] In general, the amines of formula (IV) are either
commercially available (for example in some cases from Aldrich),
known in the literature, or may be prepared e.g. by conventional
means.
[0229] Compounds of formula (III), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1, and n are as defined herein (in
particular where X.sup.1=Br), typically may be prepared from
compounds of formula (V), wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are as defined herein, by reaction with a suitable
acylating agent (VI), wherein X.sup.1 and n are as defined herein
(in particular where X.sup.1=Br) and X.sup.2 represents a halogen
atom such as a chlorine or bromine atom, preferably a chlorine
atom. Suitable conditions include carrying out the reaction in a
suitable anhydrous solvent such as dichloromethane or chloroform,
in the presence of a suitable base such as
N,N-diisopropylethylamine or triethylamine, at a suitable
temperature such as from 0.degree. C. to room temperature, and
suitably under nitrogen.
##STR00026##
[0230] Compounds of formula (VI), wherein X.sup.1, X.sup.2 and n
are as defined herein, typically can be prepared from compounds of
formula (VII), wherein X.sup.1 and n are as defined herein (in
particular where X.sup.1.dbd.Br), by reaction with for example
thionyl chloride (for X.sup.2.dbd.Cl), phosphoryl chloride (for
X.sup.2.dbd.Cl), or phosphoryl bromide (for X.sup.2.dbd.Br)
(preferably thionyl chloride for X.sup.2.dbd.Cl), or other suitable
reagents e.g. as described in R. C. Larock, Comprehensive Organic
Transformations, 2.sup.nd Ed, Wiley, 1999. Suitable conditions for
X.sup.2.dbd.Cl include the use of thionyl chloride, e.g. at room
temperature, optionally in a suitable non-aqueous (e.g. anhydrous)
aprotic organic solvent such as toluene.
##STR00027##
[0231] Compounds of formula (V), wherein R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are as defined herein, can generally be prepared from
compounds of formula (VIII), wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are as defined herein, and X.sup.3 is a protecting group,
by deprotection.
##STR00028##
[0232] X.sup.3 can be a nitrogen protecting group e.g. as described
in T. W. Greene and P. G. M Wuts Protecting Groups in Organic
Synthesis, Wiley 1999. Suitably, X.sup.3 is tert-butoxycarbonyl
(`BOC`). When X.sup.3 is the BOC protecting group, standard methods
(e.g. as described in Greene) can be used for the deprotection
reaction (VIII) to (V), for example treatment with a solution of
hydrogen chloride in 1,4-dioxane such as 4M HCl in 1,4-dioxane,
e.g. at room temperature.
[0233] Compounds of formula (VIII), wherein R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are as defined herein, and X.sup.3 is a
protecting group such as BOC, may typically be prepared by reaction
between compounds of formula (IX), wherein R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are as defined herein, and compounds of formula
(X) wherein X.sup.3 is as defined herein such as BOC and X.sup.4 is
a suitable leaving group.
##STR00029##
[0234] The activated compound (the compound of formula (X)) can for
example be an activated ester and X.sup.4 the leaving group
thereof. For example the leaving group X.sup.4 can be of
sub-formula (bt):
##STR00030##
[0235] The activated compound of formula (X), e.g. wherein X.sup.4
is of sub-formula (bt), can be formed from the carboxylic acid of
formula (Xa), wherein X.sup.3 is as defined herein such as BOC:
##STR00031##
[0236] In one embodiment the activated compound of formula (X)
wherein X.sup.4 is of sub-formula (bt) is formed from the
carboxylic acid of formula (Xa) by the following reaction (a). In
this reaction (a), the carboxylic acid (Xa) is reacted with a
suitable organic di-substituted carbodiimide, such as
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide [also named
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide] or a salt thereof
such as the hydrochloride salt thereof (EDC), or such as
N,N'-dicyclohexylcarbodiimide (DCC), followed by reaction of the
resulting product with 1-hydroxybenzotriazole (when X.sub.5 is CH)
or 1-hydroxy-7-azabenzotriazole (when X.sub.5 is N). In one
embodiment, this reaction (a), to form (X) wherein X.sup.4 is of
sub-formula (bt) from (Xa), is carried out in a suitable organic
solvent e.g. an aprotic organic solvent (preferably anhydrous) such
as N,N-dimethylformamide or acetonitrile, e.g. under anhydrous
conditions and/or e.g. at a suitable temperature such as room
temperature (e.g. about 18 to about 25.degree. C.). In one optional
embodiment, this reaction (a) is carried out in the presence of a
tertiary amine base such as N,N-diisopropylethylamine
(.sup.iPr.sub.2NEt=DIPEA).
[0237] In another embodiment, the activated compound of formula (X)
wherein X.sup.4 is of sub-formula (bt) is formed from the
carboxylic acid of formula (Xa) by the following reaction (b). In
reaction (b), the carboxylic acid (Xa) is reacted with a suitable
1-hydroxybenzotriazole-based or 1-hydroxy-7-azabenzotriazole-based
coupling agent, such as (i), (ii), (iii) or (iv): [0238] (i)
O-(benzotriazol-1-yl)-N,N,N'N'-tetramethyluronium
hexafluorophosphate (also called HBTU; when X.sub.2 is CH), or
[0239] (ii) 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU; when X.sub.2 is CH), or [0240] (iii)
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU, when X.sub.2 is N), or [0241] (iv)
(benzotriazol-1-yloxy)-trispyrrolidinophosphonium
hexafluorophosphate (PyBOP; when X.sub.2 is CH)).
[0242] In one optional embodiment, this reaction (b), to form (X)
wherein X.sup.4 is of sub-formula (bt) from (Xa), is carried out in
the presence of a tertiary amine base such as
N,N-diisopropylethylamine (.sup.iPr.sub.2NEt=DIPEA). In one
embodiment, this reaction (b) is usually carried out in the
presence of a solvent such as an aprotic organic solvent (e.g.
anhydrous solvent) such as N,N-dimethylformamide or acetonitrile,
e.g. under anhydrous conditions and/or at a suitable temperature
such as room temperature (e.g. about 18 to about 25.degree.
C.).
[0243] In one alternative embodiment, an activated compound of
formula (X) is the product (adduct) formed from the reaction of the
carboxylic acid of formula (Xa) with a suitable organic
di-substituted carbodiimide (e.g. R'--N.dbd.C.dbd.N--R') [wherein
the carbodiimide can e.g. be
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a salt thereof
such as the hydrochloride salt thereof (EDC), or
dicyclohexylcarbodiimide (DCC)]. In this case, in the activated
compound of formula (X) being the carboxylic acid-carbodiimide
adduct, X.sup.4 is --O--C(NHR').dbd.N--R'' or
--O--C(NHR'').dbd.N--R'. This is the situation e.g. when the
carbodiimide is reacted with the carboxylic acid (Xa) without
1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole being
present. In one embodiment, this reaction is carried out in a
suitable organic solvent e.g. an aprotic organic solvent
(preferably anhydrous) such as N,N-dimethylformamide or
acetonitrile, e.g. under anhydrous conditions and/or e.g. at a
suitable temperature such as room temperature (e.g. about 18 to
about 25.degree. C.). In one optional embodiment, this reaction (a)
is carried out in the presence of a tertiary amine base such as
N,N-diisopropylethylamine (.sup.iPr.sub.2NEt=DIPEA).
[0244] In another alternative embodiment, the activated compound of
formula (X) is for example the acid chloride (wherein X.sup.4 is
Cl). In one embodiment, this acid chloride is for example formed
from the corresponding carboxylic acid (Xa) either (a) by reaction
with thionyl chloride, either in an organic solvent such as
chloroform or without solvent, or (b) by reaction with oxalyl
chloride in N,N-dimethyloformamide (e.g. catalytic DMF in
dichloromethane or in DMF solvent). When an acid chloride within
formula (X) is used to prepare the compound of formula (VIII), the
reaction with amine (IX) is usually carried out in the presence of
a tertiary amine base such as N,N-diisopropylethylamine
(.sup.iPr.sub.2NEt=DIPEA) and/or in a suitable aprotic organic
solvent (e.g. anhydrous solvent) such as acetonitrile or
dichloromethane, for example at room temperature (e.g. about 18 to
about 25.degree. C.). Note: this acid chloride method may not be
ideal for Boc-protected compounds.
[0245] Compounds of formula (IX), wherein R.sup.1, R.sup.2 and
R.sup.3 are as defined herein and R.sup.4 represents hydrogen, can
be prepared by hydrogenation of an azide compound of formula (XI),
wherein R.sup.1, R.sup.2 and R.sup.3 are as defined herein, in the
presence of a suitable catalyst such as a palladium catalyst, e.g.
palladium on carbon, in a suitable solvent such as ethanol, e.g. at
a suitable temperature such as room temperature:
##STR00032##
[0246] Compounds of formula (XI), wherein R.sup.1, R.sup.2 and
R.sup.3 are as defined herein, may be prepared from compounds of
formula (XII), wherein R.sup.1, R.sup.2 and R.sup.3 are as defined
herein and wherein X.sup.6 is a leaving group such as a halogen
atom, mesylate (methanesulfonate), tosylate (p-toluenesulfonate),
or triflate (trifluoromethanesulfonate) (suitably a halogen atom
such as a chlorine atom).
[0247] For example the compounds of formula (XII), e.g. wherein
X.sup.6 is Cl, can be reacted with an azide salt such as sodium,
lithium or potassium azide, in a suitable solvent such as
dimethylsulfoxide such as dry DMSO, e.g. at a suitable temperature
such as room temperature, to give compounds of formula (XI).
##STR00033##
[0248] Compounds of formula (XII) or acid addition salts thereof
(e.g. sulfonate such as benzenesulfonate or methanesulfonate salt
thereof), wherein R.sup.1, R.sup.2 and R.sup.3 and X.sup.6 are as
defined herein (e.g. wherein X.sup.6 is Cl, mesylate, tosylate or
triflate), can be prepared by reaction of compounds of formula
(XIII), wherein R.sup.1, R.sup.2 and R.sup.3 are as defined herein,
with a suitable reagent such as thionyl chloride (for when X.sup.6
is Cl), oxalyl chloride (for when X.sup.6 is Cl), methanesulfonyl
chloride or methanesulfonic anhydride (for when X.sup.6 is
mesylate), or para-toluenesulfonyl chloride or para-toluenesulfonic
anhydride (for when X.sup.6 is tosylate), preferably thionyl
chloride. Suitable conditions, for when X.sup.6 is Cl, include
reacting with thionyl chloride in a suitable non-aqueous (e.g.
anhydrous) aprotic organic solvent such as toluene or anisole
(methoxybenzene), e.g. with heating to ca. 60-90.degree. C. such as
to ca. 85.degree. C. (e.g. with toluene), or e.g. at
20.+-.5.degree. C. (e.g. with anisole), and optionally also in the
presence of an anhydrous aryl (e.g. phenyl), alkyl (e.g. methyl) or
trifluoromethyl sulfonic acid, such as benzenesulfonic acid or
methanesulfonic acid (e.g. to prepare the sulfonate such as
benzenesulfonate salt of (XII), e.g. when X.sup.6 is Cl, for
example to try to improve the stability of the "benzylic"
chloride). Alternative conditions include reacting compounds of
formula (XIII) with thionyl chloride and methanesulfonic acid or
benzenesulfonic acid in a suitable non-aqueous (e.g. anhydrous)
aprotic organic solvent such as dichloromethane, e.g. at a suitable
temperature such as room temperature.
##STR00034##
[0249] Preferably, in the process, the compound of formula (XII) or
the acid addition salt thereof (e.g. sulfonate such as
benzenesulfonate or methanesulfonate salt thereof, and/or e.g. when
X.sup.6 is Cl) is not isolated. Rather, it is preferably left in
solution, optionally with partial or full exchange of solvents. The
solution of the compound of formula (XII) or the acid addition salt
thereof (e.g. when X.sup.6 is Cl) can be reacted directly in the
next step.
[0250] Alternatively, compounds of formula (XI) wherein R.sup.1,
R.sup.2 and R.sup.3 are as defined herein can be prepared directly
from compounds of formula (XIII) wherein R.sup.1, R.sup.2 and
R.sup.3 are as defined herein.
[0251] For example, compounds of formula (XI) may be prepared by
reacting compounds of formula (XIII) with an azide salt, e.g.
sodium azide, in the presence of a halogenating agent such as
carbon tetrabromide and a phosphine such as triphenylphosphine
under suitable conditions, such as N,N-dimethylformamide, e.g. at a
suitable temperature such as between 0.degree. C. and room
temperature (see e.g. Toyota et. al. Journal of Organic Chemistry,
2000, 65(21), 7110-7113).
##STR00035##
[0252] This route, (XIII) to (XI) directly, may be suitable for
where R.sup.3 is a urea-containing group [such as a
N-aminocarbonyl-piperidinyl or N-aminocarbonyl-pyrrolidinyl group
within sub-formula (bb) or (aa), that is wherein NHR.sup.3 is of
sub-formula (k2) or (k3)], because it is noted that these R.sup.3
urea-containing groups may not be tolerant of thionyl chloride
which may be used in converting (XIII) to (XII) wherein X.sup.6 is
Cl and thence to (XI).
[0253] In another alternative embodiment of particular interest, an
amine compound of formula (IX) or a salt thereof (e.g. HCl salt
thereof), wherein R.sup.1, R.sup.2 and R.sup.3 are as defined
herein and R.sup.4 is as defined herein (in particular where
R.sup.4 is a hydrogen atom), may usually be prepared directly from
a compound of formula (XII) or an acid addition salt thereof,
wherein R.sup.1, R.sup.2 and R.sup.3 and X.sup.6 are as defined
herein, without first converting to an azide compound of formula
(XI). For example, in compound (XII) or the acid addition salt
thereof, X.sup.6 can in particular be a chlorine atom. When X.sup.6
is a chlorine atom, a suitable acid addition salt such as an
arylsulfonate, alkylsulfonate or trifluoromethylsulfonate salt (in
particular a benzenesulfonate or methanesulfonate salt) of the
compound of formula (XII) can for example be used, in particular
when R.sup.1 and R.sup.2 are ethyl and when R.sup.3 is of the
sub-formula (h) that is when R.sup.3 is
tetrahydro-2H-pyran-4-yl.
##STR00036##
[0254] This reaction, converting the compound (XII) or the acid
addition salt thereof (e.g. sulfonate such as benzenesulfonate
salt) to the amine compound (IX) or the salt thereof (e.g. HCl
salt), may for example be carried out under suitable conditions,
for example by reaction of a compound of formula (XII) or an acid
addition salt thereof with an aminating agent. When R.sup.4
represents a hydrogen atom, and optionally for example when X.sup.6
is a chlorine atom, a suitable aminating agent may be used, e.g. an
alkali-metal hexamethyldisilazide such as lithium
hexamethyldisilazide, sodium hexamethyldisilazide or potassium
hexamethyldisilazide (in particular lithium hexamethyldisilazide),
in a suitable non-aqueous non-alcohol (aprotic) organic solvent
(e.g. anhydrous solvent) such as tetrahydrofuran (THF), for example
at a suitable temperature such as about 25 to about 50.degree. C.,
for example ca. 30-45.degree. C. or ca. 30-40.degree. C. The
compound (XII) or the salt thereof is suitably added slowly to a
mixture or solution of the alkali-metal hexamethyldisilazide (e.g.
lithium hexamethyldisilazide) in the aprotic organic solvent (e.g.
THF), to try to minimise alkylation of any in-situ deprotected
amine (IX) by the compound (XII) or the salt thereof. The reaction
with the suitable aminating agent (e.g. with the alkali-metal
hexamethyldisilazide) is suitably followed by treatment with an
aqueous acid such as aqueous hydrochloric acid (e.g. 2-10M, e.g.
about 5M), for example at a suitable temperature such as from
0.degree. C. to room temperature, for example at 5-15.degree. C. or
ca. 10.degree. C. In one particular embodiment, a solution of the
produced amine (IX) or the salt thereof in an organic solvent
(suitably in an organic solvent having a low water miscibility,
such as comprising 2-methyl-THF or a mixture of sec-butanol and
isopropanol) is extracted with aqueous base, such as concentrated
(e.g. ca. 32% w/w) NaOH solution, to form the amine compound (IX)
as the "free base". Optionally, a mono-acid-addition salt, e.g.
monohydrochloride, of the amine (IX) can be formed by mixing the
"free base" amine compound (IX) (e.g. in an organic solvent such as
comprising 2-methyl-THF or a mixture of sec-butanol and
isopropanol) with about 1 equivalent (e.g. 1.03 equiv.) of a
suitable acid such as HCl (e.g. aqueous hydrochloric acid such as
concentrated aq. HCl e.g. ca. 36% w/w aq. HCl), preferably under
conditions such that the mono-acid-addition salt, e.g.
monohydrochloride, of the amine (IX) crystallises from a or the
organic solvent present.
[0255] In one particular simplified embodiment of the conversion of
compound (XII) or a salt thereof to amine compound (IX) or a salt
thereof, when X.sup.6 is a chlorine atom in the compound of formula
(XII) and when R.sup.4 is a hydrogen atom in the compound of
formula (IX), the precursor alcohol compound of formula (XIII) or a
salt thereof is converted into the amine of formula (IX) or a salt
thereof, via the compound of formula (XII) or a salt thereof,
without substantially purifying and/or without substantially
isolating the compound of formula (XII) or the salt thereof wherein
X.sup.6 is a chlorine atom (Cl). In this embodiment, the compound
of formula (XII) or the salt thereof wherein X.sup.6 is a chlorine
atom can for example be in the form of the benzenesulfonate salt,
in particular when R.sup.1 and R.sup.2 are ethyl and when R.sup.3
is of the sub-formula (h) that is when R.sup.3 is
tetrahydro-2H-pyran-4-yl. In the first step, conversion of compound
(XIII) to compound (XI) or a salt thereof wherein X.sup.6 is
C.sup.1 is optionally carried out using thionyl chloride in a
suitable non-aqueous (e.g. anhydrous) aprotic organic solvent such
as anisole or toluene, e.g. at 20.+-.5.degree. C. or with heating
to ca. 60-90.degree. C., and optionally in the presence of an
organic sulfonic acid such as benzenesulfonic acid to prepare the
sulfonate e.g. benzenesulfonate salt of (XII), wherein X.sup.6 is
Cl. The second step, the compound (XII) or the salt thereof
(wherein X.sup.6 is Cl), dissolved and/or suspended in anisole
and/or toluene, is optionally converted to compound (IX) or a salt
thereof wherein R.sup.4 is H, e.g. as described elsewhere
hereinabove or hereinbelow. See Intermediate 7A herein for a
specific example of this two-step reaction:
##STR00037##
[0256] Compounds of formula (XIII), wherein R.sup.1, R.sup.2, and
R.sup.3 are as defined herein, can be prepared by reaction of
compounds of formula (XIV), wherein R.sup.1, R.sup.2 and R.sup.3
are as defined herein, and wherein X.sup.7 is an alkyl group such
as a C.sub.1-6 or C.sub.1-4 alkyl (e.g. straight-chain alkyl) group
e.g. in particular ethyl, with a suitable reducing agent in a
suitable solvent, e.g. at a suitable temperature.
[0257] One suitable reducing agent is lithium borohydride, in which
case: [0258] a suitable solvent can for example comprise or be an
aprotic organic solvent (e.g. anhydrous) such as tetrahydrofuran
(THF, e.g. dry), optionally mixed with toluene (e.g. dry), or THF
alone; [0259] and (for lithium borohydride), preferably, dry
methanol (e.g. 3 to 15 equivalents, e.g. about 9 equivalents, but
preferably not in very large excess) can be used, in particular in
admixture with the suitable aprotic organic solvent such as THF, to
speed up the reduction reaction; [0260] and/or (for lithium
borohydride) a suitable reaction temperature can be from room
temperature to the reflux temperature, e.g. about 50 to about
75.degree. C., e.g. about 60 to about 70.degree. C., e.g.
63-69.degree. C. or 64-68.degree. C.
[0261] Another reducing agent is di-iso-butylaluminium hydride
(e.g. solution in toluene), in which case: a suitable solvent is
dichloromethane and/or toluene, and/or a suitable reaction
temperature can be about 0.degree. C.
##STR00038##
[0262] Compounds of formula (XIV), wherein R.sup.1, R.sup.2 and
R.sup.3 and X.sup.7 are as defined herein, may be prepared by
reaction of a compound of formula (XV) with an amine of formula
R.sup.3NH.sub.2, for example generally according to the method
described by Yu et. al. in J. Med. Chem., 2001, 44, 1025-1027. The
reaction is preferably carried out in the presence of a base such
as triethylamine or N,N-diisopropylethylamine, and/or in an organic
solvent such as ethanol, dioxane, 1-methyl-2-pyrrolidinone (NMP) or
acetonitrile. The reaction may require heating e.g. to ca.
60-180.degree. C., for example at 115.degree. C.:
##STR00039##
[0263] When R.sup.3 is the N-aminocarbonyl-piperidinyl or
N-aminocarbonyl-pyrrolidinyl group within sub-formula (bb) or (aa),
that is wherein NHR.sup.3 is of sub-formula (k2) or (k3), the
compound of formula (XIV) can be prepared by reacting a compound of
formula (XIVa), wherein R.sup.1, R.sup.2 and X.sup.7 are as defined
herein and n.sup.3=0 or 1, or a salt thereof (e.g. a hydrochloride
salt thereof) with a urea-forming reagent capable of converting the
(4-piperidinyl)amino or (3-pyrrolidinyl)amino group in the compound
of formula (XIVa) into a [(1-aminocarbonyl)-4-piperidinyl]amino
group or [(1-aminocarbonyl)-3-pyrrolidinyl]amino group as in the
below-illustrated embodiment of formula (XIV) respectively:
##STR00040##
[0264] The urea-forming reagent may be benzyl isocyanate (followed
later by debenzylation e.g. reductive debenzylation), or preferably
the urea-forming reagent is a tri(C.sub.1-4alkyl)silyl isocyanate
such as a tri(C.sub.1-2alkyl)silyl isocyanate, preferably
trimethylsilyl isocyanate. The conversion of the compound (XIVa) or
salt thereof to the compound (XIV) is in one embodiment carried out
in the presence of a suitable base such as
N,N-diisopropylethylamine, in a suitable solvent such as
dichloromethane or chloroform, at a suitable temperature such as at
room temperature or at the reflux temperature of the solvent.
[0265] Compound (XIVa), wherein R.sup.1, R.sup.2, X.sup.7 and
n.sup.3 are as defined herein, or a salt thereof, can be prepared
from compound (XlVb), wherein R.sup.1, R.sup.2, X.sup.7 and n.sup.3
are as defined herein and Prot is a nitrogen protecting group such
as (tert-butyloxy)carbonyl, by removal of the nitrogen protecting
group. For example, removal of the (tert-butyloxy)carbonyl group
can be effected under suitable acidic conditions, such as with
hydrogen chloride (e.g. 4M) in a suitable solvent such as
1,4-dioxane:
##STR00041##
[0266] Compound (XIVb), wherein R.sup.1, R.sup.2, and n.sup.3 are
as defined herein, X.sup.7 is ethyl and Prot is
(tert-butyloxy)carbonyl, can be prepared by reaction of a compound
of formula (XV), wherein R.sup.1 and R.sup.2 are as defined herein
and X.sup.7=ethyl, with 1,1-dimethylethyl
4-amino-1-piperidinecarboxylate (e.g. commercially available from
AstaTech, Philadelphia, USA) or 1,1-dimethylethyl
3-amino-1-pyrrolidinecarboxylate (e.g. commercially available from
Aldrich). The reaction is optionally carried out in the presence of
a base such as triethylamine or N,N-diisopropylethylamine,
optionally in a suitable organic solvent such as acetonitrile, at a
suitable temperature such as 60-100.degree. C. (e.g. 80-90.degree.
C.):
##STR00042##
[0267] Compounds of formula (XV), wherein R.sup.1, R.sup.2, and
X.sup.7 are as defined herein can be prepared by reaction of
compounds of formula (XVI), wherein R.sup.1 is as defined herein,
with a dialkyl (1-chloroalkylidene)propanedioate of formula (XVII)
(which is (R.sup.2)(Cl)C.dbd.C(CO.sub.2X.sup.7).sub.2, wherein
R.sup.2 and X.sup.7 are as defined herein; for example a diethyl
(1-chloroalkylidene)propanedioate for when X.sup.7 is Et; and e.g.
R.sup.2 can be Et), followed by reaction with phosphorous
oxychloride (POCl.sub.3). Suitable conditions for reaction of
compounds of formula (XVI) with a dialkyl
(1-chloroalkylidene)propanedioate of formula (XVII) include
heating, for example in a suitable solvent such as toluene, and for
example in the presence of a suitable base such as triethylamine,
e.g. at a suitable temperature such as the reflux temperature of
the solvent. Suitable conditions for the reaction of the
intermediate [formed from (XVI) and the dialkyl
(1-chloroalkylidene)propanedioate (XVIII)] with phosphorous
oxychloride (POCl.sub.3) can include heating, e.g. heating at the
reflux temperature of phosphorous oxychloride.
##STR00043##
[0268] In one embodiment, a compound of formula (XVII), wherein
R.sup.2 and X.sup.7 are as defined herein, is prepared by reaction
of a compound of formula (XVIII), wherein R.sup.2 and X.sup.7 are
as defined herein, with phosphorus oxychloride (POCl.sub.3) in the
presence of a suitable base such as tributylamine, at a suitable
temperature such as ca. 80-130.degree. C., for example ca.
100-120.degree. C.
##STR00044##
[0269] In one embodiment, a compound of formula (XVIII), wherein
R.sup.2 and X.sup.7 are as defined herein, is prepared by reaction
of a dialkyl malonate of formula (XIX), wherein X.sup.7 is as
defined herein, with magnesium chloride (suitably anhydrous) and a
suitable non-aqueous base such as triethylamine, in a suitable
solvent (e.g. anhydrous solvent) such as acetonitrile, at a
suitable temperature such as ca. 5-10.degree. C., followed by
addition of an acid chloride of formula (XX), for example propanoyl
chloride when R.sup.2 is ethyl, at a suitable temperature such as
between 10.degree. C. and room temperature. In one embodiment, the
reaction is for example carried out under anhydrous conditions:
##STR00045##
[0270] Generally, many of the compounds of formulae (XIX) and (XX)
are either known compounds or may be prepared e.g. by conventional
means. For example, the compound of formula (XIX) where X.sup.7 is
ethyl, and the compound of formula (XX) where R.sup.2 represents
methyl or ethyl, are commercially available, e.g. from Aldrich or
elsewhere.
[0271] For examples of syntheses of compounds (XVIII) and compounds
(XVII), in which R.sup.2 is cyclopropyl, n-propyl, ethyl,
cyclobutyl and (cyclopropyl)methyl-, see for example Intermediates
1, 3 to 6 and 7 to 11 on pages 60-62 of WO 2005/090348 A1 (Glaxo
Group Limited).
[0272] In an alternative embodiment, a compound of formula (XV),
wherein R.sup.1, R.sup.2 and X.sup.7 are as defined herein, is
prepared by reaction of a compound of formula (XVI), wherein
R.sup.1 is as defined herein, with a compound of formula (XXI),
wherein R.sup.2 and X.sup.7 are as defined herein, with heating,
followed by reaction with phosphorous oxychloride, again with
heating (e.g. see Yu et. al. in J. Med. Chem., 2001, 44,
1025-1027). Compounds of formula (XXI) can for example be diethyl
[(ethyloxy)methylidene]propanedioate (wherein R.sup.2 is H and
X.sup.7 is Et, available from Aldrich) or diethyl
[1-(ethyloxy)ethylidene]propanedioate (wherein R.sup.2 is Me and
X.sup.7 is Et, see Eur. Pat. Appl. (1991), EP 413918 A2).
##STR00046##
[0273] Where the desired amino pyrazole of formula (XVI) is not
commercially available, in one alternative embodiment, a method of
preparation of (XVI) comprises reaction of
3-hydrazinopropanenitrile (available from Lancaster Synthesis) with
a suitable aldehyde of formula R.sup.40CHO in a suitable solvent
such as ethanol, with heating, followed by reduction with, for
example sodium in a suitable solvent such as t-butanol. R.sup.40
should be chosen so as to contain one less carbon atom than
R.sup.1, for example R.sup.40=methyl will afford R.sup.1=ethyl. See
also the method(s) described by Dorgan et. al. in J. Chem. Soc.,
Perkin Trans. 1, 1980, (4), 938-942:
##STR00047##
[0274] In an alternative less-preferred embodiment of Process 1A,
the 4-chloro substituent in the compound of formula (XV) is
replaced by another halogen atom, such as a bromine atom, or by
another suitable leaving group which is displaceable by an amine of
formula R.sup.3NH.sub.2. The leaving group can, for example, be a
straight-chain alkoxy group --OR.sup.35 being --OMe, --OEt or
--OPr.sup.n, or a group --O--S(O).sub.2--R.sup.37, wherein R.sup.37
is methyl, CF.sub.3, or phenyl or 4-methyl-phenyl. The reaction is
optionally carried out with or without solvent. The reaction may
require heating.
[0275] In one embodiment, a compound of formula (XI), wherein
R.sup.1 and R.sup.2 are as defined herein and R.sup.3 represents
the N-aminocarbonyl-piperidinyl or N-aminocarbonyl-pyrrolidinyl
group of sub-formula (bb) or (aa), e.g. wherein NHR.sup.3 is of
sub-formula (k2) or (k3), is alternatively prepared from a compound
of formula (XXXVIII), wherein R.sup.1 and R.sup.2 are as defined
herein, n.sup.3 is 0 or 1, and Prot represents a suitable nitrogen
protecting group such as tert-butoxycarbonyl. Conditions can
include: [0276] suitable acidic conditions (for N-deprotection)
such as hydrogen chloride in a suitable solvent such as 1,4-dioxane
at a suitable temperature such as room temperature, followed by
[0277] reacting the resultant N-deprotected piperidine/pyrrolidine
with a urea-forming reagent (e.g. trimethylsilyl isocyanate) so as
to convert the (4-piperidinyl)amino or (3-pyrrolidinyl)amino group
in the compound into a [(1-aminocarbonyl)-4-piperidinyl]amino group
or [(1-aminocarbonyl)-3-pyrrolidinyl]amino group in the
below-illustrated embodiment of formula (XI) respectively [optional
urea-forming conditions can include: carrying out the reaction in
the presence of a suitable base such as N,N-diisopropylethylamine,
in a suitable solvent such as dichloromethane or chloroform, at a
suitable temperature such as at room temperature or at the reflux
temperature of the solvent.].
##STR00048##
[0278] In one embodiment, a compound of formula (XXXVIII), wherein
R.sup.1 and R.sup.2, n.sup.3 and Prot are as defined herein, is
prepared from a compound of formula (XXXIX), wherein R.sup.1 and
R.sup.2, n.sup.3 and Prot are as defined herein. Conditions can
include reaction of a compound of formula (XXXIX) with an azide
such as sodium azide and a halogenating agent such as carbon
tetrabromide, in the presence of a suitable phosphine such as
triphenylphosphine, in a suitable solvent such as
N,N,-dimethylformamide, at a suitable temperature such as between
0.degree. C. and room temperature.
##STR00049##
[0279] In one embodiment, a compound of formula (XXXIX), wherein
R.sup.1 and R.sup.2, n.sup.3 and Prot are as defined herein, is
prepared from a compound of formula (XL), wherein R.sup.1 and
R.sup.2, n.sup.3 Prot and X.sup.7 are as defined herein, by
reduction with a suitable reducing agent such as lithium
borohydride, in a suitable solvent such as a mixture of
tetrahydrofuran and methanol, at a suitable temperature such as at
the reflux temperature of the solvent.
##STR00050##
[0280] In one embodiment, a compound of formula (XL), wherein
R.sup.1 and R.sup.2, n.sup.3 Prot and X.sup.7 are as defined
herein, is prepared from compounds of formula (XV), wherein
R.sup.1, R.sup.2, and X.sup.7 are as defined herein, by reaction of
a compound of formula (XV) with an amine of formula (XLI), wherein
Prot and n.sup.3 are as defined herein. The reaction is optionally
carried out in the presence of a base such as triethylamine or
N,N-diisopropylethylamine, and/or in an organic solvent such as
ethanol, dioxane, 1-methyl-2-pyrrolidinone (NMP) or acetonitrile.
The reaction may require heating e.g. to ca. 60-180.degree. C., for
example at 120.degree. C.:
##STR00051##
Process 1A--Alternative Embodiment 1A1
[0281] (For example, Examples 1A1, 1A2, 1B, 1C, 3, 4, 9, 11, 12,
17, 18, 25, 26 and 29 to 34 may optionally be prepared by Process
1A alternative embodiment 1A1).
[0282] As an alternative embodiment within Process 1A, as the
penultimate step, compounds of formula (III), wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4, n and X.sup.1 are as defined herein, may
also be prepared by reaction between compounds of formula (IX),
wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined
herein, and compounds of formula (XXVI), wherein X.sup.1 and n are
as described herein and X.sup.9 is a suitable leaving group,
preferably a halogen atom such as a chlorine atom; suitable
conditions include stirring in a suitable anhydrous solvent such as
dichloromethane, in the presence of a base such as triethylamine,
at a suitable temperature such as between 0.degree. C. and room
temperature, for example between 0.degree. C. and 10.degree. C.
##STR00052##
[0283] Compounds of formula (XXVI), wherein X.sup.1, n and X.sup.9
are as described herein, may be prepared from carboxylic acids of
formula (XXVII), wherein X.sup.1 and n are as described herein, by
treatment with a suitable activating agent such as thionyl
chloride, phosphoryl chloride or other reagents as described in R.
C. Larock, Comprehensive Organic Transformations, Wiley, 2.sup.nd
Ed., 1999. For example when X.sup.9 is chlorine compounds of
formula (XXVI) may be prepared from compounds of formula (XXVII) by
heating with thionyl chloride at a suitable temperature such as
100.degree. C.
##STR00053##
[0284] Compounds of formula (XXVII), wherein X.sup.1 and n are as
described herein, may be prepared by hydrolysis of compounds of
formula (XXV), wherein X.sup.1, n and X.sup.8 are as described
herein. Preferably X.sup.8 is tert-butyl and the formation of
compounds of formula (XXVII) from compounds of formula (XXV) can be
achieved by treatment with a suitable acid such as 4M hydrogen
chloride in a suitable solvent such as 1,4-dioxane, at a suitable
temperature such as room temperature.
##STR00054##
[0285] Compounds of formula (XXV) may be prepared as described
herein.
Process 1B
[0286] (For example, the compounds or salts whose names and/or
chemical structures are given in and which are the products of
Examples 1A1, 1A2, 1A3, 1B, 1C, 1D, 1E, 1F, 1G, 1H1, 1H2, 1J1, 1J2,
3, 4, 9, 11, 12, 17, 18, 25, 26 and 29 to 34, may optionally be
prepared by Process 1B. Process 1B is a process of particular
interest for the preparation of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a salt thereof e.g. this compound or a hydrochloride salt
thereof, e.g. see Example 1B first preparation and/or Example 1A2
alternative preparation no. 1).
[0287] In the embodiment which is "Process 1B", a compound of
formula (II) or a salt thereof, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6 and n are as defined herein (in
particular when R.sup.4 is a hydrogen atom), is prepared by
reaction of a compound of formula (IX) or a salt thereof (e.g. a
HCl salt thereof, e.g. monohydrochloride salt thereof), wherein
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined herein (in
particular when R.sup.4 is a hydrogen atom), with a compound of
formula (XXII) or a salt thereof, wherein R.sup.5, R.sup.6 and n
are as defined herein and X.sup.4 is a suitable leaving group (e.g.
wherein X.sup.4 is as described hereinbelow). [As described above,
a compound of formula (II) is a compound of formula (I) wherein Ar
has sub-formula (x), Q.sup.1 is NH, Q.sup.2 is --C(O)--, and L is
(CH.sub.2).sub.n.]
##STR00055##
[0288] For various processes for the preparation of an amine
compound of formula (IX) or a salt thereof (e.g. a HCl salt
thereof, in particular for when R.sup.4 is a hydrogen atom, and for
processes for the preparation of the precursor intermediates usable
to prepare the amine (IX) or a salt thereof, see the processes
already extensively disclosed under the heading "Process 1A"
hereinabove.
[0289] The compound of formula (XXII) or salt thereof can for
example be a suitable activated carboxylic acid derivative wherein
the leaving group X.sup.4 is the leaving group of said activated
carboxylic acid derivative. In the case of this activated
carboxylic acid derivative, the leaving group X.sup.4 can for
example be of sub-formula (bt):
##STR00056##
[0290] In the case of this activated carboxylic acid derivative,
the leaving group X.sup.4 can alternatively be for example
--O--C(NHR').dbd.N--R'' or --O--C(NHR'').dbd.N--R', wherein R' and
R'' are the substituents (which may be the same or different) of an
organic (di-substituted) carbodiimide reagent
R'--N.dbd.C.dbd.N--R'' which may have been used to prepare (XXII)
or a salt thereof from the corresponding carboxylic acid.
[0291] The leaving group X.sup.4 can alternatively be, for example,
a chlorine atom (Cl), a bromine atom (Br), 1-imidazolyl, or
t-Bu-C(O)--O--.
[0292] Preferably, the compound of formula (XXII) or the salt
thereof (for example the suitable activated carboxylic acid
derivative) is not isolated. Preferably, it is reacted with the
amine of formula (IX) or the salt thereof to prepare the compound
of formula (II) or the salt thereof, directly after formation of
the compound of formula (XXII) or the salt thereof and without
isolation of said compound (XXII) or salt.
[0293] A compound of formula (XXII), wherein R.sup.5, R.sup.6, n
and X.sup.4 are as defined herein, or a salt thereof can be formed
from a carboxylic acid of formula (XXIII) or a salt (e.g. acid
addition salt such as a HCl salt) thereof, wherein R.sup.5,
R.sup.6, and n are as defined herein.
##STR00057##
[0294] In one embodiment, a compound of formula (XXII) or a salt
thereof, wherein the leaving group X.sup.4 is of sub-formula (bt),
is prepared from the corresponding carboxylic acid of formula
(XXIII) or a salt thereof by the following reaction (a). In this
reaction (a), the carboxylic acid (XXIII) or salt is reacted with a
suitable organic carbodiimide e.g. organic di-substituted
carbodiimide, such as
1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide [also named
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide] or a salt thereof
such as a hydrochloride salt thereof (EDC), or such as
N,N'-dicyclohexylcarbodiimide (DCC), followed by reaction of the
resulting product with 1-hydroxybenzotriazole (when X.sub.5 is CH)
or 1-hydroxy-7-azabenzotriazole (when X.sub.5 is N).
[0295] In an alternative embodiment, a compound of formula (XXII)
or a salt thereof, wherein the leaving group X.sup.4 is of
sub-formula (bt), is prepared from the corresponding carboxylic
acid of formula (XXIII) or a salt thereof by the following reaction
(b). In this reaction (b), the carboxylic acid (XXIII) or salt is
reacted with a suitable 1-hydroxybenzotriazole-based or
1-hydroxy-7-azabenzotriazole-based coupling agent, such as (i),
(ii), (iii) or (iv): [0296] (i)
O-(benzotriazol-1-yl)-N,N,N'N'-tetramethyluronium
hexafluorophosphate (also called HBTU, when X.sub.2 is CH), or
[0297] (ii) 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU, when X.sub.2 is CH), or [0298] (iii)
O-(7-azabenzotriazol-1-yl)-N,N,N' N'-tetramethyluronium
hexafluorophosphate (HATU, when X.sub.2 is N), or [0299] (iv)
(benzotriazol-1-yloxy)-trispyrrolidinophosphonium
hexafluorophosphate (PyBOP, when X.sub.2 is CH).
[0300] In one embodiment, this reaction (b) is carried out in the
presence of a base e.g. a tertiary amine base such as
N,N-diisopropylethylamine (.sup.iPr.sub.2NEt=DIPEA), and/or usually
in the presence of a solvent such as an aprotic organic solvent
(e.g. anhydrous solvent) such as N,N-dimethylformamide or
acetonitrile, e.g. under anhydrous conditions and/or at a suitable
temperature such as room temperature (e.g. about 18 to about
25.degree. C.).
[0301] Reaction (a) of the carboxylic acid (XXIII) or a salt
thereof with the suitable organic carbodiimide (e.g. DCC; or more
preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a salt
thereof such as a hydrochloride salt thereof to prepare compound
(XXII) or a salt thereof, in particular in the presence of
1-hydroxybenzotriazole, followed by reaction of the resulting
compound (XXII) or the salt thereof with the amine of formula (IX)
or the salt thereof, can be carried out as follows: [0302] in the
presence of a suitable organic solvent (preferably anhydrous or at
least substantially dry), such as one or more C.sub.1-7alkyl
alcohols (in particular one or more C.sub.4-6alkyl alcohols, such
as pentanol, e.g. pentan-1-ol or pentan-2-ol, or butanol, e.g.
butan-2-ol or butan-1-ol, or hexanol e.g. hexan-1-ol),
N,N-dimethylformamide (DMF), N,N-dimethylacetamide,
1-methyl-2-pyrrolidinone (NMP), acetonitrile, tetrahydrofuran (e.g.
anhydrous), dichloromethane, or a mixture of any of these (e.g.
N,N-dimethylacetamide and pentan-1-ol, or NMP and pentan-1-ol);
and/or [0303] at a suitable temperature such as about room
temperature (e.g. about 15 to about 25.degree. C., or about 18 to
about 25.degree. C.); and/or [0304] under an inert atmosphere such
as nitrogen; and/or [0305] in the presence of a non-aqueous base
such as solid potassium carbonate (e.g. having about 325 mesh
particle size), solid sodium carbonate, or a non-aqueous tertiary
amine base such as N,N-diisopropylethylamine
(.sup.iPr.sub.2NEt=DIPEA) or triethylamine; and/or [0306] using an
aqueous alkaline (e.g. concentrated aqueous alkaline) extraction of
a or the organic phase (e.g. using conc. aq. NaOH or conc. aq.
KOH), after the reaction which has prepared compound (II) or a salt
thereof [this extraction may help to decrease the amount of certain
possible reaction byproducts (e.g. ester byproducts, e.g. when the
solvent comprises an alcohol and/or when the starting material
compound (XXII) or (XXIII) or a salt thereof contains an alcoholic
OH group in the R.sup.5 and/or R.sup.6 group) that might perhaps be
formed during preparation of the product (II) or the salt
thereof].
[0307] In particular where the compound of formula (II) or the salt
thereof is
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a salt thereof, reaction (a) of the carboxylic acid (XXIII)
or a salt thereof with the suitable organic carbodiimide (e.g. DCC;
or more preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or
a salt thereof such as a hydrochloride salt thereof) to prepare the
compound (XXII) or a salt thereof, in particular in the presence of
1-hydroxybenzotriazole, followed by reaction of the resulting
compound (XXII) or the salt thereof with the amine of formula (IX)
or the salt thereof, can be in particular carried out: [0308] in
the presence of a hydrochloride salt (e.g. monohydrochloride) (e.g.
solid e.g. crystalline) of an amine compound of formula (IX);
and/or [0309] in the presence of a suitable organic solvent
(preferably anhydrous or at least substantially dry) being or
comprising an organic solvent with low water-miscibility such as
one or more C.sub.4-6alkyl alcohols (such as pentanol, e.g.
pentan-1-ol or pentan-2-ol, or butanol, e.g. butan-2-ol or
butan-1-ol, or hexanol e.g. hexan-1-ol) (e.g. to allow aqueous
extractions more easily, e.g. see below); and/or [0310] at a
suitable temperature such as about room temperature (e.g. about 15
to about 25.degree. C., or about 18 to about 25.degree. C.); and/or
[0311] under an inert atmosphere such as nitrogen; and/or [0312] in
the presence of a non-aqueous inorganic base such as solid
potassium carbonate (e.g. having about 325 mesh particle size) or
solid sodium carbonate, and for a solid base preferably with rapid
stirring (e.g. about 160 rpm); and/or [0313] using an aqueous
alkaline (e.g. concentrated aqueous alkaline) extraction of a or
the organic phase (e.g. using conc. aq. NaOH or conc. aq. KOH),
after the reaction which has prepared compound (II) or a salt
thereof [this extraction may help to decrease the amount of certain
possible reaction byproducts (e.g. ester byproducts, e.g. when the
solvent comprises an alcohol and/or due to the starting material
compound (XXII) or (XXIII) or the salt thereof generally containing
a 2-hydroxyethyl group) that might perhaps be formed during
preparation of the
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or the salt thereof]; and/or [0314] using an aqueous acidic
extraction of a or the organic phase (e.g. using aq. HCl) after the
reaction to prepare (II) or a salt thereof, and optionally after an
aqueous alkaline extraction which may be present, with the amount
of aqueous acid used in the acid extraction being such that the pH
of the aqueous phase is adjusted to pH 5.75.+-.0.25 [e.g. to
decrease the amount of certain possible basic byproducts and/or
basic impurities that might perhaps be present, for example after
preparation of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide].
[0315] See e.g. Example 1A2 (alternative preparation no. 1, plant
method), for one example of the above reaction conditions to
prepare
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide before conversion to the monohydrochloride salt thereof.
[0316] Where a salt of the compound of formula (II) (or (I)) is
prepared, in one embodiment this is salt optionally prepared after
the preparation of the compound (1I) or (I) and without isolation
of said compound, e.g. by crystallisation of the salt directly from
essentially the same or similar organic solvent as that used in the
reaction to prepare the compound (1I) or (I) (e.g. the organic
solvent can be one or more C.sub.4-6alkyl alcohols, such as
pentanol e.g. pentan-1-ol), e.g. by salt crystallisation from the
reaction mixture. In one embodiment, this salt crystallisation is
optionally done:
(i) by conversion if necessary of the compound (II) or (I) and/or
the salt thereof, prepared from the reaction and/or extraction
thereof, into substantially wholly the "free base" form, e.g. by
aqueous alkaline extraction of the non-extracted or
previously-extracted reaction mixture; and/or (ii) by optional
reduction of the volume of and/or heating of the reaction solvent,
if necessary or desirable, e.g. after preparation of compound (II)
or (I) and after aqueous extraction of the reaction mixture, and/or
(iii) by mixing an appropriate amount of an appropriate acid such
as HCl with the reaction mixture (e.g. ca. 1 mole equivalent of HCl
to prepare a monohydrochloride salt, e.g. using 2-10M aq. HCl, e.g.
4-6 M aq. HCl); and/or (iv) by optional mixing with the reaction
solvent of an organic "antisolvent" capable of crystallising the
salt of the compound (1I) or (I) from the organic
solvent-antisolvent mixture, typically followed by cooling of the
mixture and/or isolation eg by filtration of the crystallised salt.
In particular, depending on the solubility properties of the salt
of the compound, tert-butyl methyl ether (TBME) is optionally used
as such an antisolvent, especially for preparing a salt of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide (e.g. the HCl salt e.g. monohydrochloride salt thereof).
[0317] See e.g. Example 1A2 (alternative preparation no. 1, plant
method), for one specific example of the above salt preparation
conditions, in the preparation of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride.
[0318] In one alternative embodiment of when the compound of
formula (XXII) or salt is a suitable activated carboxylic acid
derivative, the compound of formula (XXII) or a salt thereof is the
product (adduct) formed from the reaction of the carboxylic acid of
formula (XXII) or salt thereof with a suitable organic carbodiimide
e.g. organic di-substituted carbodiimide (e.g.
R'--N.dbd.C.dbd.N--R') [e.g. wherein the carbodiimide can be
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a salt thereof
such as a hydrochloride salt thereof (EDC), or
N,N'-dicyclohexylcarbodiimide (DCC)]. In this case, in the compound
of formula (XXII) or salt which is the carboxylic acid-carbodiimide
adduct, the leaving group X.sup.4 is --O--C(NHR').dbd.N--R'' or
--O--C(NHR'').dbd.N--R', wherein R' and R'' are the substituents
(which may be the same or different) of the organic
(di-substituted) carbodiimide reagent used. This is the situation
for example when the carbodiimide is reacted with the carboxylic
acid (XXIII) or a salt thereof without 1-hydroxybenzotriazole or
1-hydroxy-7-azabenzotriazole being present. In one embodiment, this
reaction is carried out in a suitable organic solvent e.g. an
aprotic organic solvent (preferably anhydrous) such as
N,N-dimethylformamide or acetonitrile, e.g. under anhydrous
conditions and/or e.g. at a suitable temperature such as room
temperature (e.g. about 18 to about 25.degree. C.). In one optional
embodiment, this reaction is carried out in the presence of a
tertiary amine base such as N,N-diisopropylethylamine
(.sup.iPr.sub.2NEt=DIPEA).
[0319] In another alternative embodiment, the compound of formula
(XXII) or salt thereof is for example the acid chloride (in which
case, the leaving group X.sup.4 is a chlorine atom (Cl)).
[0320] In one embodiment, this acid chloride is for example
prepared from the corresponding carboxylic acid (XXIII) or a salt
thereof by reaction with oxalyl chloride, usually in a suitable
organic solvent (e.g. anhydrous) such as DMF or dichloromethane
(e.g. dichloromethane or another solvent together with a catalytic
amount of DMF), and/or e.g. under anhydrous conditions, and/or e.g.
at room temperature or at or below 20.degree. C. When an acid
chloride of formula (XXII) wherein X.sup.4 is Cl, or a salt
thereof, is used to prepare the compound of formula (II) or (I),
the reaction with amine (IX) or a salt thereof is usually carried
out in the presence of a tertiary amine base such as
N,N'-diisopropylethylamine (iPr.sub.2NEt=DIPEA) and/or in a
suitable aprotic organic solvent (e.g. anhydrous solvent) such as
acetonitrile or dichloromethane, for example at room temperature
(e.g. about 18 to about 25.degree. C.).
[0321] In the compound (XXII) or a salt thereof, further
alternative values of the leaving group X.sup.4 include a bromine
atom (Br), 1-imidazolyl (e.g. as prepared from the acid (XXIII) or
salt using carbonyl diimidazole), and t-Bu-C(O)--O-- (e.g. as
prepared from the acid (XXIII) or salt using t-Bu-C(O)--CI).
[0322] Compounds of formula (XXIII), wherein R.sup.5, R.sup.6 and n
are as defined herein, can for example typically be formed by
hydrolysis of esters of formula (XXIV), wherein R.sup.5, R.sup.6,
and n are as defined herein and X.sup.8 is an alkyl group, such as
ethyl or preferably tert-butyl. The hydrolysis may be achieved
under suitable conditions, such as by reaction with 4M hydrogen
chloride in 1,4-dioxane (e.g. for when X.sup.8 is tert-butyl but
usually not when X.sup.8 is ethyl), at a suitable temperature such
as room temperature, optionally in dry dichloromethane.
##STR00058##
[0323] Compounds of formula (XXIV), wherein R.sup.5, R.sup.6 n and
X.sup.8 are as defined herein, can for example typically be formed
by substitution of compounds of formula (XXV), wherein X.sup.8 and
n are as defined herein and X.sup.1 is a leaving group as defined
herein (e.g. for X.sup.1 as defined in Process 1A hereinabove, e.g.
X.sup.1 can be a bromine atom), with an amine of formula (IV),
wherein R.sup.5 and R.sup.6 are as defined herein, optionally in
the presence of a suitable base such as N,N-diisopropylethylamine,
in a suitable solvent such as N,N-dimethylformamide, at a suitable
temperature such as between room temperature and 100.degree. C.,
for example at about 60.degree. C.
##STR00059##
[0324] Compounds of formula (XXV), wherein X.sup.8 and X.sup.1 and
n are as defined herein, can for example typically be formed from
compounds of formula (XXVI), wherein X.sup.8 is as defined herein,
by reaction with a suitable acylating agent (VI), wherein X.sup.1,
X.sup.2 and n are as defined herein e.g. hereinabove (e.g. for
X.sup.1 and X.sup.2 as defined in Process 1A hereinabove, e.g.
X.sup.1 can be a bromine atom and/or X.sup.2 can be a chlorine
atom). Suitable conditions include carrying out the reaction in a
suitable anhydrous solvent such as dichloromethane or chloroform,
in the presence of a suitable non-aqueous base e.g. a tertiary
amine base such as N,N-diisopropylethylamine or triethylamine, at a
suitable temperature such as between 0.degree. C. and room
temperature, e.g. under anhydrous conditions.
##STR00060##
[0325] Compounds of formula (XXVI) are generally either
commercially available, known in the literature or may be prepared
e.g. by conventional means. For example ethyl 4-aminobenzoate
(X.sup.8 is ethyl) is commercially available, for example from
Aldrich. Preferably X.sup.8 is tert-butyl; for example tert-butyl
4-aminobenzoate and tert-butyl 3-aminobenzoate are commercially
available, for example from Fluka.
Process 1C
[0326] (For example, the compounds or salts prepared/preparable in
Examples 1A1, 1A2, 1A3, 1B, 1C, 1D, 1E, 1F, 1G, 1H1, 1H2, 1J1, 1J2,
3, 4, 9, 11, 12, 17, 18, 25, 26 and 29 to 34 may optionally be
prepared by Process 1C. Process 1C is a process which is of
interest for the preparation of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a salt thereof, e.g. this compound or a hydrochloride salt
thereof such as a monohydrochloride).
[0327] In Process 1C, a compound of formula (XLII), wherein
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and n are as defined herein (in
particular when R.sup.4 is a hydrogen atom), and wherein R.sup.5'
is a hydrogen atom (H), methyl, ethyl, n-propyl, or isopropyl (e.g.
methyl), and wherein R.sup.6 is C.sub.1-4alkyl substituted by one
OH substituent (e.g. wherein R.sup.6' is --CH.sub.2CH.sub.2OH,
--CH.sub.2CH(Me)OH, --CH.sub.2CH.sub.2CH.sub.2OH, or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, in particular wherein
R.sup.6' is --CH.sub.2CH.sub.2OH), which is one embodiment of a
compound of formula (I), or a salt thereof, is prepared by
deprotection of a compound of formula (XLIII) or a salt thereof,
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and n are as defined
herein (in particular when R.sup.4 is a hydrogen atom), wherein
R.sup.5' is a hydrogen atom (H), methyl, ethyl, n-propyl, or
isopropyl (e.g. methyl), and wherein R.sup.6-prot is C.sub.1-4alkyl
substituted by one protected OH substituent O-Prot' (e.g. wherein
R.sup.6-prot is --CH.sub.2CH.sub.2--O-Prot',
--CH.sub.2CH(Me)-O-Prot', --CH.sub.2CH.sub.2CH.sub.2--O-Prot', or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--O-Prot', in particular wherein
R.sup.6-prot is --CH.sub.2CH.sub.2--O-Prot').
##STR00061##
[0328] For example, O-Prot' can be any protected OH group which is
deprotectable by treatment with acid or base or fluoride ions. In
particular, O-Prot' can be --OC(O)--C.sub.1-6alkyl such as OAc
(acetate), OC(O)--CF.sub.3, --OC(O)aryl such as --OC(O)-phenyl, or
--O-(tri-organo)silyl such as O-trialkylsilyl such as O-TBDMS
(tert-butyldimethylsilyloxy) or O-TMS (trimethylsilyloxy).
[0329] Deprotection of the compound of formula (XLIII) or the salt
thereof can be via standard OH-deprotection conditions, e.g. using
acid or more preferably base in particular strong base such as
strong inorganic base (e.g. NaOH or KOH, e.g. aqueous and/or
ethanolic NaOH or KOH, in particular concentrated (e.g. 5-12M or
10-11M) aqueous NaOH or KOH) for deprotection where O-Prot' is
--OC(O)--C.sub.1-6alkyl such as OAc (acetate) or --OC(O)--CF.sub.3
or --OC(O)aryl. Deprotection mediated by base, e.g. where O-Prot'
is --OC(O)--C.sub.1-6alkyl or --OC(O)--CF.sub.3 or --OC(O)aryl, can
in particular be carried out under heating (e.g. at ca.
40-60.degree. C. such as ca. 50.degree. C.) and/or in the presence
of an organic solvent such as a C.sub.1-7alcohol for example a
C.sub.4-6alcohol e.g. pentanol such as pentan-1-ol. Where O-Prot'
is O-trialkylsilyl such as O-TBDMS or O-TMS, this can be
deprotected sometimes by acid or more usually by fluoride ions such
as by use of tetra-n-butylammonium fluoride. For other
OH-protecting groups and their chemistry, see Theadora Green,
"Protective Groups in Organic Synthesis".
[0330] Compounds of formula (XLIII) or salts thereof can typically
be formed by the following reaction of an amide of formula (XLV) or
a salt thereof, wherein R.sup.4 and n are as defined herein (in
particular wherein R.sup.4 is a hydrogen atom), R.sup.5' is a
hydrogen atom (H), methyl, ethyl, n-propyl, or isopropyl (e.g.
methyl), and R.sup.6-prot is as defined herein, with a compound of
formula (XLIV) or an acid addition salt thereof (e.g. a sulfonate
salt thereof, e.g. methanesulfonate or benzenesulfonate salt of
(XLIV)), wherein X.sup.11 is a suitable leaving group such as
mesylate (methanesulfonate), tosylate (p-toluenenesulfonate),
triflate (trifluoromethanesulfonate), or a chlorine, bromine or
iodine atom (in particular a chlorine atom). Compounds of formula
(XLIV), where X.sup.11 is mesylate, tosylate, triflate, or a
chlorine, bromine or iodine atom, are generally thought to be
potent electrophiles.
##STR00062##
[0331] Conditions can include heating compound (XLIV) or the acid
addition salt thereof (e.g. a sulfonate salt thereof, e.g.
methanesulfonate or benzenesulfonate salt of (XLIV)) and compound
(XLV) or a salt thereof together, e.g. at ca. 50-100.degree. C. or
ca. 60-80.degree. C., e.g. at ca. 60.degree. C., for example in a
suitable solvent, such as N-methyl-2-pyrrolidinone (that is
1-methyl-2-pyrrolidinone, also called NMP), dimethyl sulfoxide,
ethyl acetate, n-propyl acetate, chloroform, diethylene glycol
dimethyl ether ["diglyme", (CH.sub.3OCH.sub.2CH.sub.2).sub.2O],
N,N-dimethylformamide (DMF), tetrahydrofuran (THF), or mixtures
thereof. Suitably, the solvent can be NMP, dimethyl sulfoxide,
ethyl acetate, n-propyl acetate, chloroform, diethylene glycol
dimethyl ether, or mixtures thereof. Preferably, the solvent is
1-methyl-2-pyrrolidinone (NMP).
[0332] Due to its reactivity, the compound of formula (XLIV) or the
acid addition salt thereof (e.g. sulfonate salt thereof, e.g.
methanesulfonate or benzenesulfonate salt of (XLIV)), wherein
X.sup.11 is a suitable leaving group such as mesylate
(methanesulfonate), tosylate (p-toluenenesulfonate), triflate
(trifluoromethanesulfonate), or a chlorine, bromine or iodine atom
(in particular a chlorine atom), is preferably not isolated; e.g.
it can be prepared in solution and used directly in solution (with
or without partial or full solvent replacement), e.g. by being used
in THF and/or NMP solution, in the next step (i.e. reaction with
the amide of formula (XLV) or the salt thereof).
[0333] The compound of formula (XLIV) or the acid addition salt
thereof can for example be prepared as described in Process 1A
herein. However, when X.sup.11 is Cl, see the detailed scheme below
marked "Scheme--Process 1C (to Example 1A2)", an embodiment
intended e.g. for the preparation of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a salt thereof such as the monohydrochloride thereof, for
some modified processes/reagents/reaction conditions leading to the
relevant acid addition salt (e.g. methanesulfonate salt) of the
compound of formula (XLIV) wherein R.sup.1 and R.sup.2 are both
ethyl, R.sup.3 is tetrahydro-2H-pyran-4-yl, and
[0334] X.sup.11 is Cl. See in particular Stages 1c, 1d, 2a, and 2b
within that Scheme for alternative reagents and/or reaction
conditions. For specific examples of these modified
processes/reagents/reaction conditions, see Intermediate 2B (for
Stage 1c preparation), Intermediate 2C (for Stage 1d preparation),
Intermediate 3 (alternative preparation) (for Stage 2a
preparation), and Intermediate 4 (alternative preparation) (for
Stage 2b preparation) as disclosed herein.
[0335] One preferred specific route within Process 1C, e.g. for the
preparation of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a salt thereof, in particular the monohydrochloride salt
thereof, is illustrated in "Scheme--Process 1C (to Example 1A2)" as
follows:
##STR00063## ##STR00064##
[0336] The final deprotection step in the specific route
["Scheme--Process 1C (to Example 1A2)"] shown above can preferably
involve:
(i) extracting the acetyl-O-protected product within formula
(XLIII) into n-pentanol (pentan-1-ol); (ii) adding aqueous NaOH
solution (e.g. ca. 2M to ca. 10M such as ca. 10M), optionally with
heating e.g. at ca. 40-60.degree. C. such as ca. 50.degree. C., to
cleave the acetate protecting group (whereupon the deprotected OH
product (the "free base" compound within formula (XLII) and within
formula (I)) stays in the n-pentanol phase); and (iii) removing the
aqueous phase (optionally after addition of water); and (iv)
optionally (if a monoHCl salt is required) adding ca. 1 equivalent
of aqueous HCl (eg ca. 5M aq. HCl) to produce the product,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,-
4-b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}ami-
no)benzamide monohydrochloride.
[0337] As part of the general preparation of compounds of formula
(XLII) or salts thereof, a compound of formula (XLV) or a salt
thereof can for example be prepared by the following generalised
route:
##STR00065##
[0338] Typical conditions and/or reagents for the protection of the
OH group within R.sup.6' of compound (XLVI) ro a salt thereof, to
form (XLV) or a salt thereof, can include conditions suitable for
OH-protection with the OH-protecting group used. For example, where
O-Prot' (within R.sup.6-prot of compound (XLV)) is OAc (acetate),
acetic anhydride can be used, e.g. in a suitable solvent (e.g.
anhydrous) such as acetonitrile, and/or in the presence of an
organic base such as triethylamine, and/or e.g. with heating such
as at ca. 60-90.degree. C., for example heating at ca.
81-82.degree. C. e.g. with acetonitrile solvent. Alternatively,
acetyl chloride can be used for acetyl OH-protection. For other
OH-protecting groups and their chemistry, see Theadora Green,
"Protective Groups in Organic Synthesis".
[0339] Conditions and/or reagents for the coupling of the compound
(XLVII) or a salt thereof with the amine R.sup.5'R.sup.6'NH or a
salt thereof to prepare compound (XLVI) or a salt thereof can
include: [0340] using at least 2 equivalents (e.g. 3-10
equivalents, preferably 5-10 equivalents, such as 6-9 equivalents,
e.g. ca. 7 equivalents) of the amine R.sup.5'R.sup.6'NH [or if not
then using another non-nucleophilic organic or inorganic base to
remove the HBr formed in the reaction, such as pyridine,
2,6-dimethylpyridine, tri-n-butylamine, N,N-diisopropylethylamine
(iPr.sub.2NEt=DIPEA), or DABCO, or K.sub.2CO.sub.3 or
Na.sub.2CO.sub.3], and/or [0341] using a suitable dipolar aprotic
solvent such as dimethylsulfoxide, NMP, DMF, or dichloromethane (in
particular dimethylsulfoxide), and/or [0342] either heating e.g. at
ca. 40-80.degree. C. or ca. 50-80.degree. C. e.g. at ca. 60.degree.
C., or carrying out the reaction at room temperature.
[0343] Conditions and/or reagents for the coupling of the
aminobenzamide or N-alkyl-(amino)benzamide compound (XLVIII) or a
salt thereof with the specific acid chloride compound within
formula (VI) will normally include generally anhydrous/dry
conditions. and/or can optionally include: [0344] using a suitable
organic or inorganic non-nucleophilic base such as pyridine,
2,6-dimethylpyridine, tri-n-butylamine, N,N-diisopropylethylamine
(iPr.sub.2NEt=DIPEA), or DABCO, or K.sub.2CO.sub.3 or
Na.sub.2CO.sub.3 (in particular tri-n-butylamine or suitably
pyridine), and/or [0345] using a suitable solvent (e.g. generally
dry) such as a C.sub.4-6ketone, a C.sub.2-4alkyl acetate,
C.sub.1-3alkyl-CN, dichloromethane, chloroform, tetrahydrofuran or
toluene; in particular 2-butanone (methyl ethyl ketone, MEK),
pentanone such as 3-pentanone, n-propyl acetate, n-butyl acetate,
acetonitrile, propionitrile (CH.sub.3CH.sub.2--CN), butyronitrile
(CH.sub.3CH.sub.2CH.sub.2--CN), dichloromethane, tetrahydrofuran or
toluene; preferably 2-butanone or butyronitrile
(CH.sub.3CH.sub.2CH.sub.2--CN).
[0346] The compounds of formula (VI) wherein X.sup.1 is Br and
X.sup.2 is Cl can be obtained by the skilled man, for example using
process(es) as described elsewhere herein.
Process 1D
[0347] In Process 1 D, in order to prepare a compound of formula
(XLIIa), wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and n are as
defined herein (in particular when R.sup.4 is a hydrogen atom), and
wherein R.sup.5'' and R.sup.6'' are as defined herein for R.sup.5
and R.sup.6 respectively provided that there is no OH group in
R.sup.5'' or R.sup.6'' or [R.sup.5'' and R.sup.6'' taken together]
and provided that R.sup.8 is not H, which is one embodiment of a
compound of formula (I), or a salt thereof, an amide of formula
(XLVa) or a salt thereof, wherein R.sup.4 and n are as defined
herein (in particular when R.sup.4 is a hydrogen atom), and wherein
R.sup.5'' and R.sup.6'' are as defined herein for R.sup.5 and
R.sup.6 respectively provided that there is no OH group in
R.sup.5'' or R.sup.6'' or [R.sup.5'' and R.sup.6'' taken together]
and provided that R.sup.8 is not H, can be reacted with a compound
of formula (XLIV) or an acid addition salt thereof (e.g. a
sulfonate salt thereof e.g. methanesulfonate or benzenesulfonate
salt thereof, wherein X.sup.11 is a suitable leaving group such as
mesylate (methanesulfonate), tosylate (p-toluenenesulfonate),
triflate (trifluoromethanesulfonate), or a chlorine, bromine or
iodine atom (in particular a chlorine atom):
##STR00066##
[0348] Compounds of formula (XLVa) are optionally prepared by a
process analogous to that of compound (XLV) or (XLVI) in Process 1C
(e.g. see above), but without the OH-protection step.
Process 3
[0349] In general terms, the Process 3 can be as follows: A
compound of formula (XXXa), wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4R.sup.5, R.sup.6, m.sup.1, and m.sup.2 are as defined herein
(suitably, m.sup.1 and m.sup.2 are both 4), or a salt thereof, may
typically be prepared by reaction by substitution of a compound of
formula (XXXIa), or a salt thereof, wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1, m.sup.1, and m.sup.2 are as defined
herein, with an amine of formula (IV), or a salt thereof, wherein
R.sup.5 and R.sup.6 are as defined herein. At least about 2
equivalents of the amine of formula (IV) or the salt thereof can be
used. Suitable conditions include heating in a suitable solvent
such as N,N-dimethylformamide, suitably in the presence of a
suitable base e.g. organic tertiary amine base such as
N,N-diisopropylethylamine, e.g. at a suitable temperature such as
ca. 60-100.degree. C. e.g. ca. 85.degree. C. Alternative conditions
include heating under microwave irradiation in a suitable solvent
such as N,N-dimethylformamide, e.g. at a suitable temperature such
as ca. 140-150.degree. C.
##STR00067##
[0350] For the suitable embodiment wherein m.sup.1 and m.sup.2 are
both 4: Compounds of formula (XXX), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4 R.sup.5, and R.sup.6 are as defined herein, or
salts thereof, may typically be prepared by reaction by
substitution of a compound of formula (XXXI), or a salt thereof,
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and X.sup.1 are as
defined herein, with an amine of formula (IV), or a salt thereof
wherein R.sup.5 and R.sup.6 are as defined herein. At least about 2
equivalents of the amine of formula (IV) or the salt thereof can be
used. Suitable conditions include heating in a suitable solvent
such as N,N-dimethylformamide, suitably in the presence of a
suitable base e.g. organic tertiary amine base such as
N,N-diisopropylethylamine, e.g. at a suitable temperature such as
ca. 60-100.degree. C. e.g. ca. 85.degree. C. Alternative conditions
include heating under microwave irradiation in a suitable solvent
such as N,N-dimethylformamide, e.g. at a suitable temperature such
as ca. 140-150.degree. C.
##STR00068##
[0351] Compounds of formula (XXXI), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4 and X.sup.1 are as defined herein, may typically
be prepared by reaction between compounds of formula (IX), wherein
R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined above, and
compounds of formula (XXXIII) wherein X.sup.1 and X.sup.4 are as
defined herein (e.g. X.sup.1 can in particular be Br and/or X.sup.4
can in particular be Cl).
##STR00069##
[0352] Compounds of formula (XXXIII) wherein X.sup.4 is a chlorine
atom (CI) (and optionally wherein X.sup.1 is Br) can e.g. be
prepared as illustrated in Intermediates 26, 25, 24, 23, and/or 22
herein.
[0353] Alternatively, the activated compound (the compound of
formula (XXXIII)) can for example be an activated carboxylic acid
derivative wherein the leaving group X.sup.4 is
##STR00070##
[0354] The latter activated compound of formula (XXXIII) can be
formed from the carboxylic acid of formula (XXXIV), wherein X.sup.1
is as defined above:
##STR00071##
(a) by reaction of the carboxylic acid (XXXIV) with a suitable
carbodiimide such as 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide
[also named 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide], or a
salt thereof e.g. hydrochloride salt, preferably followed by
reaction of the resulting product with 1-hydroxybenzotriazole, in a
suitable solvent (preferably anhydrous) such as
N,N-dimethylformamide or acetonitrile, e.g. at a suitable
temperature such as room temperature (e.g. about 18 to about
25.degree. C.); or: (b) by reaction of the carboxylic acid (XXXIV)
with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate or
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, e.g. in the presence of a base such as
N,N-diisopropylethylamine, and/or usually in the presence of a
solvent such as N,N-dimethylformamide or acetonitrile e.g. at a
suitable temperature such as room temperature (e.g. about 18 to
about 25.degree. C.).
[0355] Compounds of formula (XXXIV), wherein X.sup.1 is as defined
herein, may for example be prepared by hydrolysis of an ester of
formula (XXXV), wherein X.sup.1 and X.sup.8 are as defined herein.
Conditions can include reaction with a base such as sodium
hydroxide or potassium hydroxide in a suitable solvent such as
aqueous ethanol or aqueous dioxane, or if X.sup.8 is tert-butyl
then 4M hydrogen chloride in dioxane can be used.
##STR00072##
[0356] Compound of formula (XXXV), wherein X.sup.1 and X.sup.8 are
as defined herein, may for example be prepared by reacting
compounds of formula (XXXVI), wherein X.sup.8 is as defined herein
(e.g. where X.sup.8=tert-butyl, can optionally be prepared
according to Tetsuo Miwa et. al. J. Org. Chem. 1993, 58(7), 1696)
with a suitable alkylating agent of formula (XXXVII), wherein
X.sup.1 is as defined herein.
##STR00073##
Process 4
[0357] Compounds of formula (XLII), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are as defined herein, may
typically be prepared by reaction by substitution of compounds of
formula (XLIII) or a salt thereof, wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4 and X.sup.1 are as defined herein, with an amine
of formula (IV) or a salt thereof, wherein R.sup.5 and R.sup.6 are
as defined herein. Suitable conditions include heating in a
suitable solvent such as N,N-dimethylformamide, suitably in the
presence of a suitable base e.g. an organic tertiary amine base
such as N,N-diisopropylethylamine, e.g. at a suitable temperature
such as ca. 60-100.degree. C. e.g. ca. 85.degree. C. Alternative
conditions include heating under microwave irradiation in a
suitable solvent such as N,N-dimethylformamide, e.g. at a suitable
temperature such as ca. 140-150.degree. C.
##STR00074##
[0358] Compounds of formula (XLIII), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4 and X.sup.1 are as defined above, may for example
be prepared from compounds of formula (XLIV), wherein R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are as defined above. For example,
when X.sup.1 represents a halogen atom such as a bromine atom,
compounds of formula (XLIII) may be prepared from compounds of
formula (XLIV) and a suitable halogenating agent such as carbon
tetrabromide, in the presence of a suitable phosphine such as
triphenylphosphine, in a suitable solvent such as dichloromethane,
at a suitable temperature for example between 0.degree. C. and room
temperature.
##STR00075##
[0359] Compounds of formula (XLIV), wherein R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 are as defined above, may for example be
prepared from compounds of formula (IX), wherein R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 are as defined above, and the compound of
formula (XLV), in the presence of a suitable amide coupling reagent
such as
(1H-1,2,3-benzotriazol-1-yloxy)(tri-1-pyrrolidinyl)phosphonium
hexafluorophosphate (available from Aldrich) and a suitable base
such as N,N-diisopropylethylamine, in a suitable solvent such as
anhydrous N,N-dimethylformamide, at a suitable temperature such as
room temperature.
##STR00076##
[0360] The compound of formula (XLV) is a known compound (e.g. can
optionally be prepared according to K. Fukai JP11174621).
Process 5
[0361] Compounds of formula (XLVI), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6 and n are as defined herein, may
typically be prepared by reaction by substitution of compounds of
formula (XLVII) or a salt thereof, wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1 and n are as defined herein, with an
amine of formula (IV) or a salt thereof, wherein R.sup.5 and
R.sup.6 are as defined herein. Suitable conditions include heating
in a suitable solvent such as N,N-dimethylformamide, suitably in
the presence of a suitable base e.g. an organic tertiary amine base
such as N,N-diisopropylethylamine, e.g. at a suitable temperature
such as ca. 60-100.degree. C., e.g. ca. 85.degree. C. Alternative
conditions include heating under microwave irradiation in a
suitable solvent such as N,N-dimethylformamide, e.g. at a suitable
temperature such as ca. 140-150.degree. C.
##STR00077##
[0362] Compounds of formula (XLVII), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1 and n are as defined herein, may e.g. be
prepared according to the following scheme:
##STR00078##
Process 6
[0363] Compounds of formula (XLVIII), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6 and n are as defined herein, may
typically be prepared by reaction by substitution of compounds of
formula (XLIX) or a salt thereof, wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1 and n are as defined herein, with an
amine of formula (IV) or a salt thereof, wherein R.sup.5 and
R.sup.6 are as defined herein. Suitable conditions include heating
in a suitable solvent such as N,N-dimethylformamide, suitably in
the presence of a suitable base e.g. an organic tertiary amine base
such as N,N-diisopropylethylamine, e.g. at a suitable temperature
such as ca. 60-100.degree. C. e.g. ca. 85.degree. C. Alternative
conditions include heating under microwave irradiation in a
suitable solvent such as N,N-dimethylformamide, e.g. at a suitable
temperature such as ca. 140-150.degree. C.
##STR00079##
[0364] Compounds of formula (XLIX), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1 and n are as defined above, may for
example be prepared according to the following scheme:
##STR00080##
Process 7
[0365] Compounds of formula (L), wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6 and n are as defined herein, may
typically be prepared by reaction by substitution of a compound of
formula (LI) or a salt thereof, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, X.sup.1 and n are as defined herein, with an amine of
formula (IV) or a salt thereof, wherein R.sup.5 and R.sup.6 are as
defined herein. Suitable conditions include heating in a suitable
solvent such as N,N-dimethylformamide, suitably in the presence of
a suitable base e.g. an organic tertiary amine base such as
N,N-diisopropylethylamine, e.g. at a suitable temperature such as
ca. 60-100.degree. C. e.g. ca. 85.degree. C. Alternative conditions
include heating under microwave irradiation in a suitable solvent
such as N,N-dimethylformamide, e.g. at a suitable temperature such
as ca. 140-150.degree. C.
##STR00081##
[0366] Compounds of formula (LI), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1 and n are as defined above, may e.g.
typically be prepared according to the following scheme:
##STR00082##
[0367] Compounds of formula (V) may e.g. typically be prepared as
described above.
Process 8
[0368] Compounds of formula (LII), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6 and n are as defined herein, may
typically be prepared by reaction by substitution of a compound of
formula (LII) or a salt thereof, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, X.sup.1 and n are as defined herein, with an amine of
formula (IV) or a salt thereof, wherein R.sup.5 and R.sup.6 are as
defined herein. Suitable conditions include heating in a suitable
solvent such as N,N-dimethylformamide, suitably in the presence of
a suitable base e.g. an organic tertiary amine base such as
N,N-diisopropylethylamine, e.g. at a suitable temperature such as
ca. 60-100.degree. C. e.g. ca. 85.degree. C. Alternative conditions
include heating under microwave irradiation in a suitable solvent
such as N,N-dimethylformamide, e.g. at a suitable temperature such
as ca. 140-150.degree. C.
##STR00083##
[0369] Compounds of formula (LIII), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1 and n are as defined above, may e.g.
typically be prepared according to the following scheme:
##STR00084##
Process 9
[0370] Compounds of formula (LIV), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6 and n are as defined herein, may
typically be prepared by reaction by substitution of a compound of
formula (LV) or a salt thereof, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, X.sup.1 and n are as defined herein, with an amine of
formula (IV) or a salt thereof, wherein R.sup.5 and R.sup.6 are as
defined herein. Suitable conditions include heating in a suitable
solvent such as N,N-dimethylformamide, suitably in the presence of
a suitable base e.g. an organic tertiary amine base such as
N,N-diisopropylethylamine, e.g. at a suitable temperature such as
ca. 60-100.degree. C. e.g. ca. 85.degree. C. Alternative conditions
include heating under microwave irradiation in a suitable solvent
such as N,N-dimethylformamide, e.g. at a suitable temperature such
as ca. 140-150.degree. C.
##STR00085##
[0371] Compounds of formula (LV) wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4, X.sup.1 and n are as defined above, may e.g. typically be
prepared according to the following scheme:
##STR00086##
[0372] Compound of formula (IX) may e.g. typically be prepared as
described above.
Process 10
[0373] Compounds of formula (LVI), wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6 and n are as defined herein, may
e.g. typically be prepared by reaction by substitution of a
compound of formula (LVII) or a salt thereof, wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4, X.sup.1 and n are as defined herein,
with an amine of formula (IV) or a salt thereof, wherein R.sup.5
and R.sup.6 are as defined herein. Suitable conditions include
heating in a suitable solvent such as N.sub.7N-dimethylformamide,
suitably in the presence of a suitable base e.g. an organic
tertiary amine base such as N,N-diisopropylethylamine, e.g. at a
suitable temperature such as ca. 60-100.degree. C. e.g. ca.
85.degree. C. Alternative conditions include heating under
microwave irradiation in a suitable solvent such as
N,N-dimethylformamide, e.g. at a suitable temperature such as ca.
140-150.degree. C.
##STR00087##
[0374] Compounds of formula (LVII) wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, X.sup.1 and n are as defined above, may e.g.
typically be prepared according to the following scheme:
##STR00088##
[0375] Compounds of formula (IX) may e.g. be prepared as described
above.
Process 11
[0376] Compounds of formula (XIV) wherein R.sup.2 represents
fluoroalkyl (for example trifluoromethyl) may e.g. typically be
prepared according to the following scheme, followed by appropriate
subsequent steps, e.g. as described in Process 1A or 1B or 1C:
##STR00089##
Process 12
[0377] Compounds of formula (IX) wherein R.sup.4 represents methyl
or ethyl may e.g. typically be prepared according to the following
scheme, wherein R.sup.4-C1 represents H when R.sup.4 is methyl and
R.sup.4-C1 represents methyl when R.sup.4 is ethyl; followed by
appropriate subsequent steps, e.g. as described in Process 1A or 1B
or 1C:
##STR00090##
Process F: Conversion of One Compound of Formula (I), or a Salt
Thereof into Another Compound of Formula (I) or a Salt Thereof.
[0378] In optional Process F, one compound of formula (I) or salt
thereof (or a protected version thereof, such as an N-protected
version thereof e.g. BOC--N-protected or
benzyloxycarbonyl-N-protected version; or a derivative thereof such
as an ester derivative thereof) is optionally converted into
another compound of formula (I) or a salt thereof. This conversion
reaction can for example occur within the R.sup.3 group. This
conversion optionally comprises or is one or more of the following
processes F1 to F10:
F1. Conversion of a ketone into the corresponding oxime. F2. An
oxidation process. For example, the oxidation process can comprise
or be oxidation of an alcohol to a ketone (e.g. using Jones
reagent) or oxidation of an alcohol or a ketone to a carboxylic
acid. F3. A reduction process, for example reduction of a ketone or
a carboxylic acid to an alcohol. F4. Acylation, for example
acylation of an amine (e.g. see Examples 329-349 and Example 353 of
WO 2004/024728 A2 for suitable process details), or acylation of a
hydroxy group. F5. Alkylation, for example alkylation of an amine
or of a hydroxy group. F6. Hydrolysis, e.g. hydrolysis of an ester
to the corresponding carboxylic acid or salt thereof (e.g. see
Examples 351, 488, 489, 650, 651 of WO 2004/024728 A2 for suitable
process details). F7. Deprotection, e.g. deprotection of (e.g.
deacylation of, or t-butyloxycarbonyl (BOC) removal from, or
benzyloxycarbonyl removal from) an amine group. BOC deprotection is
usually carried out under acidic conditions e.g. using hydrogen
chloride in an organic solvent such as dioxan. Benzyloxycarbonyl
deprotection is optionally carried out by hydrogenation. F8.
Formation of an ester or amide, for example from the corresponding
carboxylic acid. and/or F9. Beckmann rearrangement of one compound
of formula (I) into another compound of formula (I), for example
using cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) together
with a formamide such as DMF, e.g. at room temperature (see L. D.
Luca, J. Org. Chem., 2002, 67, 6272-6274). The Beckmann
rearrangement can for example comprise conversion of a compound of
formula (I) wherein NHR.sup.3 is of sub-formula (o2)
##STR00091##
into a compound of formula (I) wherein NHR.sup.3 is of
sub-formula
##STR00092##
and process details can be for example as illustrated in Examples
658 and 659 of WO 2004/024728 A2.
Synthetic Process Summary
[0379] The present invention therefore also provides a process for
preparing a compound of formula (I) or a salt (e.g.
pharmaceutically acceptable salt) thereof:
##STR00093##
wherein Ar has the sub-formula (x):
##STR00094##
and wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
L, Q.sup.1 and Q.sup.2 are as defined herein, the process
comprising: (1A) to prepare a compound of formula (II), which are
compounds of formula (I) wherein Ar has sub-formula (x), Q.sup.1 is
NH, Q.sup.2 is --C(O)--, and L is (CH.sub.2).sub.n, wherein
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and n are as
defined herein, or a salt thereof, [0380] reaction (substitution)
of a compound of formula (III), wherein X.sup.1 is a suitable
leaving group such as mesylate (methanesulfonate), tosylate
(p-toluenesulfonate) or a halogen atom (suitably a halogen atom, in
particular a bromine atom), and R.sup.1, R.sup.2, R.sup.3, R.sup.4
and n are as defined herein, with an amine of formula (IV), wherein
R.sup.5 and R.sup.6 are as defined herein:
##STR00095##
[0380] or (1B) to prepare a compound of formula (II) or a salt
thereof, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6 and n are as defined herein (in particular when R.sup.4 is
a hydrogen atom), reaction of a compound of formula (IX) or a salt
thereof (e.g. a HCl salt thereof, e.g. monohydrochloride salt
thereof, wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as
defined herein (in particular when R.sup.4 is a hydrogen atom),
with a compound of formula (XXII) or a salt thereof, wherein
R.sup.5, R.sup.6 and n are as defined herein and X.sup.4 is a
suitable leaving group (e.g. wherein X.sup.4 is as described
herein):
##STR00096##
or (1C) to prepare a compound of formula (XLII), wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4 and n are as defined herein (in
particular when R.sup.4 is a hydrogen atom), and wherein R.sup.5'
is a hydrogen atom (H), methyl, ethyl, n-propyl, or isopropyl (e.g.
methyl), and wherein R.sup.6' is C.sub.1-4alkyl substituted by one
OH substituent (in particular wherein R.sup.6' is
--CH.sub.2CH.sub.2OH), which is one embodiment of a compound of
formula (I), or a salt thereof, by carrying out steps (1C)(i) and
(1C)(ii):
[0381] (IC)(i) formation of a compound of formula (XLIII) or a salt
thereof, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and n are as
defined herein (in particular when R.sup.4 is a hydrogen atom),
wherein R.sup.5' is a hydrogen atom (H), methyl, ethyl, n-propyl,
or isopropyl (e.g. methyl), and wherein R.sup.6-prot is
C.sub.1-4alkyl substituted by one protected OH substituent O-Prot'
(in particular wherein R.sup.6-prot is
--CH.sub.2CH.sub.2--O-Prot'), wherein O-Prot' is any protected OH
group which is deprotectable by treatment with acid or base or
fluoride ions [in particular, --OC(O)--C.sub.1-6alkyl such as OAc
(acetate), OC(O)--CF.sub.3, --OC(O)aryl such as --OC(O)-phenyl, or
--O-(tri-organo)silyl such as O-trialkylsilyl such as O-TBDMS
(tert-butyldimethylsilyloxy) or O-TMS (trimethylsilyloxy)], by
reaction of an amide of formula (XLV) or a salt thereof, wherein
R.sup.4 and n are as defined herein (in particular wherein R.sup.4
is a hydrogen atom), R.sup.5' is a hydrogen atom (H), methyl,
ethyl, n-propyl, or isopropyl (e.g. methyl), and R.sup.6-prot is as
defined herein, with a compound of formula (XLIV) or an acid
addition salt thereof (e.g. a sulfonate salt thereof e.g.
methanesulfonate or benzenesulfonate salt thereof), wherein
X.sup.11 is a suitable leaving group such as mesylate
(methanesulfonate), tosylate (p-toluenenesulfonate), triflate
(trifluoromethanesulfonate), or a chlorine, bromine or iodine atom
(in particular a chlorine atom):
##STR00097##
and then (1C)(ii) deprotection of the compound of formula (XLIII)
or the salt thereof to prepare the prepare a compound of formula
(XLII) or a salt thereof:
##STR00098##
or (1D) to prepare a compound of formula (XLIIa), wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4 and n are as defined herein (in
particular when R.sup.4 is a hydrogen atom), and wherein R.sup.5''
and R.sup.6'' are as defined herein for R.sup.5 and R.sup.6
respectively provided that there is no OH group in R.sup.5'' or
R.sup.6'' or [R.sup.5'' and R.sup.6'' taken together] and provided
that R.sup.8 is not H, which is one embodiment of a compound of
formula (I), or a salt thereof, reaction of an amide of formula
(XLVa) or a salt thereof, wherein R.sup.4 and n are as defined
herein (in particular when R.sup.4 is a hydrogen atom), and wherein
R.sup.5'' and R.sup.6'' are as defined herein for R.sup.5 and
R.sup.6 respectively provided that there is no OH group in
R.sup.5'' or R.sup.6'' or [R.sup.5'' and R.sup.6'' taken together]
and provided that R.sup.8 is not H, with a compound of formula
(XLIV) or an acid addition salt thereof (e.g. a sulfonate salt
thereof e.g. methanesulfonate or benzenesulfonate salt thereof),
wherein X.sup.11 is a suitable leaving group such as mesylate
(methanesulfonate), tosylate (p-toluenenesulfonate), triflate
(trifluoromethanesulfonate), or a chlorine, bromine or iodine atom
(in particular a chlorine atom):
##STR00099##
or (3) to prepare a compound of formula (XXXa), wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4R.sup.5, R.sup.6, m.sup.1, and m.sup.2 are
as defined herein (suitably, m.sup.1 and m.sup.2 can both be 4), or
a salt thereof, reaction by substitution of a compound of formula
(XXXIa), or a salt thereof,
[0382] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, X.sup.1,
m.sup.1, and m.sup.2 are as defined herein, with an amine of
formula (IV), or a salt thereof, wherein R.sup.5 and R.sup.6 are as
defined herein:
##STR00100##
or (4 to 10) a process according to the final step in the synthesis
of the compound of formula (I) as described in Processes 4, 5, 6,
7, 8, 9, or 10 (as described herein, e.g. as described
hereinabove); or (F) converting one compound of formula (I) or a
salt thereof, or a protected version thereof or a derivative (e.g.
ester derivative) thereof, into another compound of formula (I) or
a salt thereof (e.g. as described in Process F hereinabove); and,
in the case of any of Process 1A, 1B, 1C, 1 D, 3, or 4 to 10, or
(F), optionally converting the compound of formula (I) into a salt
thereof such as a pharmaceutically acceptable salt thereof; or (G)
to prepare a salt of a compound of formula (I), converting a
compound of formula (I) into a salt thereof such as a
pharmaceutically acceptable salt thereof.
[0383] Preferred, suitable or optional features of the steps in any
of Processes 1A, 1B, 1C, 1D, 2A, or 3 to 10, or (F), independently
of each other, can be as described above for such
correspondingly-numbered and/or -lettered Processes, with all
necessary changes being made.
[0384] The present invention also provides: (G) a process for
preparing a pharmaceutically acceptable salt of a compound of
formula (I) comprising conversion of the compound of formula (I) or
a salt thereof into the desired pharmaceutically acceptable salt
thereof. Such processes can for example be as described herein,
e.g. as described in the Salts section above. For example, a
pharmaceutically acceptable salt can be an acid addition salt, or
less commonly (e.g. if a C(O)OH group is present in the compound) a
base addition salt. In one embodiment, a pharmaceutically
acceptable acid addition salt is optionally prepared by reaction of
a compound of formula (I) with a suitable inorganic or organic acid
(e.g. as described hereinabove).
[0385] The present invention also provides a compound of formula
(I) or a salt thereof, prepared by (or obtainable by) a method as
defined herein.
MEDICAL USES
[0386] The present invention also provides a compound of formula
(I) or a pharmaceutically acceptable salt thereof for use as an
active therapeutic substance in a mammal such as a human. The
compound or salt can be for use in the treatment and/or prophylaxis
of any of the diseases/conditions described herein (e.g. for use in
the treatment and/or prophylaxis of an inflammatory and/or allergic
disease in a mammal such as a human, monkey, rodent (e.g. rat or
mouse) or dog, in particular in a human) and/or can be for use as a
phosphodiesterase 4 (PDE4) inhibitor. "Therapy" may include
treatment and/or prophylaxis.
[0387] Also provided is the use of a compound of formula (I) or a
pharmaceutically acceptable salt thereof in the manufacture of a
medicament (e.g. pharmaceutical composition) for the treatment
and/or prophylaxis of any of the diseases/conditions described
herein in a mammal such as a human, e.g. for the treatment and/or
prophylaxis of an inflammatory and/or allergic disease in a mammal
such as a human.
[0388] Also provided is a method of treatment and/or prophylaxis of
any of the diseases/conditions described herein in a mammal (e.g.
human) in need thereof, e.g. a method of treatment and/or
prophylaxis of an inflammatory and/or allergic disease in a mammal
(e.g. human) in need thereof, which method comprises administering
to the mammal (e.g. human) a therapeutically effective amount of a
compound of formula (I) as herein defined or a pharmaceutically
acceptable salt thereof.
[0389] Phosphodiesterase 4 inhibitors may be useful in the
treatment and/or prophylaxis of a variety of diseases/conditions,
especially inflammatory and/or allergic diseases, in a mammal such
as a human, for example: chronic obstructive pulmonary disease
(COPD) (e.g. chronic bronchitis and/or emphysema), asthma, rhinitis
(e.g. allergic and/or non-allergic rhinitis), rheumatoid arthritis,
atopic dermatitis, psoriasis, urticaria, allergic conjunctivitis,
vernal conjunctivitis, eosinophilic granuloma, septic shock,
inflammatory bowel disease (e.g. ulcerative colitis and/or Crohn's
disease), reperfusion injury of the myocardium and/or brain,
chronic glomerulonephritis, endotoxic shock, or adult respiratory
distress syndrome, in a mammal such as a human; in particular:
chronic obstructive pulmonary disease (COPD) (e.g. chronic
bronchitis and/or emphysema), asthma, rhinitis (e.g. allergic
and/or non-allergic rhinitis), atopic dermatitis, psoriasis,
urticaria, allergic conjunctivitis, vernal conjunctivitis,
eosinophilic granuloma, inflammatory bowel disease (e.g. ulcerative
colitis and/or Crohn's disease), or adult respiratory distress
syndrome, in a mammal such as a human.
[0390] In the treatment and/or prophylaxis using the compound of
formula (I) or the pharmaceutically acceptable salt thereof, the
inflammatory and/or allergic disease can for example be chronic
obstructive pulmonary disease (COPD), asthma, rhinitis (e.g.
allergic and/or non-allergic rhinitis), atopic dermatitis or
psoriasis, in a mammal (e.g. human). Preferably, the compound of
formula (I) or the pharmaceutically acceptable salt thereof is for
the treatment and/or prophylaxis of COPD, asthma or rhinitis (e.g.
allergic and/or non-allergic rhinitis), in a mammal (e.g.
human).
[0391] PDE4 inhibitors, for example cilomilast and roflumilast, are
thought to be effective in the treatment of COPD. For example, see
S. L. Wolda, Emerging Drugs, 2000, 5(3), 309-319; Z. Huang et al.,
Current Opinion in Chemical Biology, 2001, 5:432-438; H. J. Dyke et
al., Expert Opinion on Investigational Drugs, January 2002, 11(1),
1-13; C. Burnouf et al., Current Pharmaceutical Design, 2002,
8(14), 1255-1296; A. M. Doherty, Current Opinion Chem. Biol., 1999,
3(4), 466-473; A. M. Vignola, Respiratory Medicine, 2004, 98,
495-503; D. Spina, Drugs, 2003, 63(23), 2575-2594; and references
cited in the aforementioned publications; G. Krishna et al., Expert
Opinion on Investigational Drugs, 2004, 13(3), 255-267 (see
especially pp. 259-261 and refs. 102-111 and 201 therein); and B.
J. Lipworth, The Lancet, 2005, 365, 167-175.
[0392] The PDE4 inhibitor cilomilast (Ariflo.TM.) at 15 mg orally
twice daily appears to improve forced expiratory volume in Is
(FEV.sub.1) in COPD patients (C. H. Compton et al., The Lancet,
2001, vol. 358, 265-270), and appears to have antiinflammatory
effects in COPD patients (E. Gamble et al., Am. J. Respir. Crit.
Care Med., 2003, 168, 976-982). On cilomilast, see also R. D.
Border et al., Chest, 2003, vol. 124 Suppl. 4, p. 170S (abstract)
and J. D. Eddleston et al., Am. J. Respir. Crit. Care Med., 2001,
163, A277 (abstract). The PDE4 inhibitor roflumilast appears to
show small improvements in FEV.sub.1 in COPD patients (see B. J.
Lipworth, The Lancet, 2005, 365, 167-175, and refs 49-50
therein).
[0393] COPD is often characterised by the presence of airflow
obstruction due to chronic bronchitis and/or emphysema (e.g., see
S. L. Wolda, Emerging Drugs, 2000, 5(3), 309-319).
[0394] For treatment and/or prophylaxis of COPD or asthma in a
mammal (e.g. human), inhaled or parenteral administration to the
mammal of the compound of formula (I) or a pharmaceutically
acceptable salt thereof can be used, preferably inhaled
administration.
[0395] PDE4 inhibitors are thought to be effective in the treatment
and/or prophylaxis of asthma (e.g. see M. A. Giembycz, Drugs,
February 2000, 59(2), 193-212; Z. Huang et al., Current Opinion in
Chemical Biology, 2001, 5: 432-438; H. J. Dyke et al., Expert
Opinion on Investigational Drugs, January 2002, 11(1), 1-13; C.
Burnouf et al., Current Pharmaceutical Design, 2002, 8(14),
1255-1296; A. M. Doherty, Current Opinion Chem. Biol., 1999, 3(4),
466-473; P. J. Barnes, Nature Reviews--Drug Discovery, October
2004, 831-844; B. J. Lipworth, The Lancet, 2005, 365, 167-175; and
references cited in the aforementioned publications).
[0396] In one preferred embodiment, the compound of formula (I) or
the pharmaceutically acceptable salt thereof is for the treatment
and/or prophylaxis of rhinitis, such as allergic rhinitis (e.g.
seasonal allergic rhinitis or perennial allergic rhinitis) and/or
non-allergic rhinitis (e.g. vasomotor rhinitis or cold air
rhinitis), in a mammal such as a human. One embodiment involves the
treatment and/or prophylaxis of mixed allergic/non-allergic
rhinitis in a mammal such as a human. By way of example, for
rhinitis such as allergic and/or non-allergic rhinitis, intranasal
or parenteral administration of the compound of formula (I) or a
pharmaceutically acceptable salt thereof is optionally used.
[0397] The PDE4 inhibitor roflumilast, given orally at 500 ug once
daily for 9 days, is reported to be effective in improving rhinal
airflow during the treatment period (compared to placebo), in
humans with histories of allergic rhinitis but asymptomatic at
screening, and who were challenged with intranasal allergen
provocation (pollen extracts) daily beginning the third day of
treatment and each time approx. 2 hours after study drug
administration (B. M. Schmidt et al., J. Allergy & Clinical
Immunology, 108(4), 2001, 530-536).
[0398] PDE4 inhibitors may be effective in the treatment of
rheumatoid arthritis (e.g. see H. J. Dyke et al., Expert Opinion on
Investigational Drugs, January 2002, 11(1), 1-13; C. Burnouf et
al., Current Pharmaceutical Design, 2002, 8(14), 1255-1296; and A.
M. Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473; and
references cited in these publications). For rheumatoid arthritis,
parenteral administration is optionally used.
[0399] PDE4 inhibition has been suggested for the treatment of
inflammatory bowel disease (e.g. ulcerative colitis and/or Crohn's
disease), see K. H. Banner and M. A. Trevethick, Trends Pharmacol.
Sci., August 2004, 25(8), 430-436.
[0400] In one embodiment, the compound or salt can for example be
for use in the treatment and/or prophylaxis of an inflammatory
and/or allergic skin disease, such as atopic dermatitis or
psoriasis, in a mammal such as a human.
[0401] In one optional embodiment, the treatment and/or prophylaxis
is of atopic dermatitis in a mammal such as a human or pig,
preferably in a human, in particular in a human aged 21 years or
less, e.g. 18 years or less. For treatment and/or prophylaxis of
atopic dermatitis in a mammal, external topical administration to
the mammal of the compound of formula (I) or a pharmaceutically
acceptable salt thereof (e.g. topical administration to the skin
e.g. to skin affected by the atopic dermatitis) can be used, though
alternatively oral or parenteral administration can be used. For
treatment and/or prophylaxis of atopic dermatitis, inhaled
administration is usually not suitable.
[0402] "Atopic dermatitis" has been proposed to include two general
sub-classes: (1) an "allergic (extrinsic)" type of atopic
dermatitis which generally occurs in the context of sensitization
to environmental allergens and/or which is generally accompanied by
elevated serum IgE levels; and (2) an "non-allergic (intrinsic)"
type of atopic dermatitis generally with little or no detectable
sensitization and/or generally with normal or low serum IgE levels
(N. Novak et al., J. Allergy Clin. Immunol., 2003, 112, 252-262;
and T. C. Roos et al., Drugs, 2004, 64(23), 2639-2666, see e.g.
pages 2640-2641). The compound of formula (I) or the
pharmaceutically acceptable salt thereof can therefore be for the
treatment and/or prophylaxis of allergic (extrinsic) atopic
dermatitis and/or non-allergic (intrinsic) atopic dermatitis in a
mammal (e.g. human or pig, preferably human).
[0403] "External topical" administration means topical
administration to an external body part (i.e. excluding, for
example, the lung or mouth, but including the lips), preferably
excluding the eye.
[0404] "External topical" administration preferably is topical
administration to the skin, for example to the skin of an arm,
hand, leg, foot, head (e.g. face), neck and/or torso of a mammal
such as a human. External topical administration can for example be
to those parts of a mammal's skin affected by or susceptible to
atopic dermatitis.
[0405] For the use of PDE4 inhibitors in atopic dermatitis, see for
example: [0406] J. M. Hanifin et al., "Type 4 phosphodiesterase
inhibitors have clinical and in vitro anti-inflammatory effects in
atopic dermatitis", J. Invest. Dermatol., 1996, 107(1), 51-56;
which reports reductions of inflammatory parameters in atopic
dermatitis patients treated with PDE4 inhibitor CP80,633 (0.5%
ointment, twice daily topical application); [0407] C. E. M.
Griffiths et al., "Randomized comparison of the type 4
phosphodiesterase inhibitor cipamfylline cream, cream vehicle and
hydrocortisone 17-butyrate cream for the treatment of atopic
dermatitis", Br. J. Dermatol., 2002, 147(2), 299-307, which reports
that cipamfylline (0.15%) cream is significantly more effective
than vehicle, but significantly less effective than hydrocortisone
17-butyrate (0.1%) cream, in the treatment of atopic dermatitis
patients; [0408] T. C. Roos et al., "Recent advances in treatment
strategies for atopic dermatitis", Drugs, 2004, 64(23), 2639-2666
(see e.g. page 2657 and refs. 201-209 therein); [0409] A. M.
Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473 (e.g. see
p. 470); and
[0410] H. J. Dyke et al., Expert Opinion Invest. Drugs, 2002,
11(1), 1-13 (e.g. see p. 7 and refs. 74, 75 and 76 cited
therein);
and references cited in the above references.
[0411] For the use of the PDE4 inhibitors SB 207499 (cilomilast)
and AWD 12-281 in mouse models of the allergic type of dermatitis,
see W. Baumer et al., Eur. J. Pharmacol., 2002, 446, 195-200 and W.
Baumer et al., J. Pharmacy Pharmacol., 2003, 55, 1107-1114.
Pharmaceutical Compositions, Routes of Administration, and Dosage
Regimens
[0412] For use in medicine, the compounds or salts of the present
invention are usually administered as a pharmaceutical
composition.
[0413] The present invention therefore provides in a further aspect
a pharmaceutical composition comprising a compound of formula (I)
or a pharmaceutically acceptable salt thereof and one or more
pharmaceutically acceptable carriers and/or excipients.
[0414] The pharmaceutical composition can be for use in the
treatment and/or prophylaxis of any of the conditions described
herein, for example chronic obstructive pulmonary disease (COPD),
asthma, rhinitis (e.g. allergic rhinitis), atopic dermatitis or
psoriasis in a mammal (e.g. human).
[0415] The invention also provides a method of preparing a
pharmaceutical composition comprising a compound of formula (I), as
herein defined, or a pharmaceutically acceptable salt thereof, and
one or more pharmaceutically acceptable carriers and/or excipients,
[0416] the method comprising mixing the compound or salt with the
one or more pharmaceutically acceptable carriers and/or
excipients.
[0417] The invention also provides a pharmaceutical composition
prepared by said method.
[0418] The compounds of formula (I) or salts thereof and/or the
pharmaceutical composition may be administered, for example, by
inhaled, intranasal, external topical (e.g. skin topical),
parenteral (e.g. intravenous, subcutaneous, or intramuscular), or
oral administration, for example to a mammal such as a human.
Inhaled administration involves topical administration to the lung
e.g. by aerosol or dry powder composition.
[0419] Accordingly, the pharmaceutical composition can be suitable
for (e.g. adapted for) inhaled, intranasal, external topical (e.g.
skin topical), parenteral (e.g. intravenous, subcutaneous, or
intramuscular), or oral administration, e.g. to a mammal such as a
human. The pharmaceutical composition can for example be suitable
for inhaled, intranasal or external topical (e.g. skin topical)
administration, e.g. to a mammal such as a human.
[0420] Inhaled or intranasal administration, in particular inhaled
administration, is generally a preferred route of administration.
In particular, inhaled or intranasal administration is preferred
for
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a salt thereof such as the monohydrochloride salt thereof
[e.g. which can be as prepared in Example 1A2 (monohydrochloride
salt) or Example 1B ("free base")], and is thought to be preferred
for
4-({1,6-diethyl-5-[({[4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)phenyl]carb-
onyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarbo-
xamide or a salt thereof [e.g. which can be as prepared in Example
23 (formate salt) or Example 23A ("free base")].
[0421] Oral administration is generally not a preferred route of
administration.
[0422] The pharmaceutical composition can optionally be in unit
dose form. The unit dose form can for example be:
(a) a rupturable or peel-openable sealed dose container containing
a dry powder inhalable pharmaceutical composition (e.g. a plurality
of which are usually disposed inside a suitable inhalation device);
(b) a vial, ampoule or filled syringe for parenteral administration
e.g. comprising a solution or suspension of the compound or
pharmaceutically acceptable salt in a suitable carrier such as an
aqueous carrier or e.g. containing a lyophilised parenteral
pharmaceutical composition (the vial or ampoule can optionally be
manufactured using a blow-fill-seal process); or (c) a tablet or
capsule for oral administration e.g. for oral administration to a
human.
[0423] Alternatively, the composition can be in a form adapted for
the administration of varying amounts of composition as desired by
the user, such as a spreadable or sprayable external topical
composition such as a cream, an ointment, a gel, or a liquid.
Pharmaceutical Compositions Suitable for Inhalable or Intranasal
Administration, and Particle-Size Reduction
[0424] Pharmaceutical compositions suitable for (e.g. adapted for)
intranasal or inhaled administration may conveniently be formulated
as aerosols, solutions, suspensions, drops, gels or dry powders.
Pharmaceutical compositions suitable for (e.g. adapted for)
intranasal or inhaled administration may optionally be formulated
with one or more pharmaceutically acceptable carriers and/or
excipients such as an aqueous or non-aqueous vehicle, a suspending
agent, a thickening agent, a wetting agent, an isotonicity
adjusting agent, an antioxidant and/or a preservative.
[0425] For compositions suitable for intranasal or inhaled
administration, the compound of formula (I) or a pharmaceutically
acceptable salt thereof may typically be in a particle-size-reduced
form, which may be prepared by conventional techniques, for
example, micronisation and milling. Generally, the size-reduced
(e.g. micronised) compound of formula (I) or a pharmaceutically
acceptable salt thereof can be defined by a D.sub.50 value of about
0.5 to 10 microns, such as of about 2 to 4 microns (for example as
measured using laser diffraction).
Pharmaceutical Compositions Suitable for Intranasal
Administration
[0426] In one embodiment, compositions comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof are
suitable for intranasal administration. In the composition for
intranasal administration, a liquid vehicle such as an aqueous
(liquid) vehicle is typically used. In the composition for
intranasal administration, the compound of formula (I) or the salt
thereof can be present, for example, as a suspension in a liquid
vehicle (e.g. as a suspension in an aqueous (liquid) vehicle)
and/or as a solution for example as an aqueous solution.
[0427] Intranasal compositions comprising a compound of formula (I)
or a pharmaceutically acceptable salt thereof may permit the
compound(s) to be delivered to all areas of the nasal cavities (the
target tissue) and further, may permit the compound(s) to remain in
contact with the target tissue for longer periods of time. One
possible dosing regime for intranasal compositions would be for the
patient to inhale slowly through the nose subsequent to the nasal
cavity being cleared. During inhalation the composition would be
administered to one nostril while the other is manually compressed.
This procedure would then be repeated for the other nostril.
Typically, one or two sprays per nostril would be administered by
the above procedure up to two or three times each day, ideally once
daily. Intranasal compositions can be for once daily
administration.
[0428] Intranasal compositions may optionally contain one or more
suspending and/or thickening agents, one or more preservatives, one
or more wetting and/or solublising agents and/or one or more
isotonicity adjusting agents as desired. Compositions suitable for
intranasal administration may optionally further contain other
excipients, such as antioxidants (for example sodium
metabisulphite), taste-masking agents (such as menthol) and/or
sweetening agents (for example dextrose, glycerol, saccharin and/or
sorbitol).
[0429] A suspending and/or thickening agent, if included, will
typically be present in the intranasal composition in an amount of
from about 0.05 to about 5% w/w or from about 0.1 to about 5% w/w,
such as from about 0.05 to about 1% w/w (e.g. about 0.1 to about
0.5% w/w, e.g. about 0.2% w/w, e.g. for xanthan gum) or from about
1% to about 3% w/w, based on the total weight of the composition. A
suspending and/or thickening agent can include xanthan gum,
microcrystalline cellulose (e.g. Avicel.RTM.),
carboxymethylcellulose (e.g. carboxymethylcellulose sodium),
veegum, tragacanth, bentonite, methylcellulose and polyethylene
glycols; in particular xanthan gum, microcrystalline cellulose or
carboxy methylcellulose sodium. A suspending and/or thickening
agent of particular interest is xanthan gum, especially when the
compound of formula (I) or the salt thereof is
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a pharmaceutically acceptable salt thereof.
[0430] Suspending agents may also be included in compositions
suitable for inhaled or oral liquid administration, as
appropriate.
[0431] For stability purposes, intranasal compositions comprising a
compound of formula (I) or a pharmaceutically acceptable salt
thereof may be protected from microbial or fungal contamination and
growth by inclusion of a preservative. Examples of pharmaceutically
acceptable anti-microbial agents or preservatives may include
quaternary ammonium compounds (e.g. benzalkonium chloride,
benzethonium chloride, cetrimide and cetylpyridinium chloride),
mercurial agents (e.g. phenylmercuric nitrate, phenylmercuric
acetate and thimerosal), alcoholic agents (e.g. chlorobutanol,
phenylethyl alcohol and benzyl alcohol), antibacterial esters (e.g.
esters of para-hydroxybenzoic acid), chelating agents such as
disodium ethylenediaminetetraacetate (EDTA, also called disodium
edetate), and other anti-microbial agents such as chlorhexidine,
chlorocresol, sorbic acid and its salts (such as potassium
sorbate), polymyxin and sodium benzoate. In particular, the
pharmaceutically acceptable anti-fungal agent or preservative can
for example include potassium sorbate (e.g. 0.1% to 1% w/w, e.g.
about 0.3% w/w) and/or disodium ethylenediaminetetraacetate (EDTA,
also called disodium edetate) (e.g. 0.005% to 0.5% w/w, e.g. about
0.015% w/w). The preservative, if included, may be present in an
amount of between about 0.001 and 1% (w/w), such as about 0.015% to
0.3% (w/w), based on the total weight of the composition.
Preservatives may be included in compositions suitable for other
routes of administration as appropriate.
[0432] Compositions which contain a suspended medicament may
include a pharmaceutically acceptable wetting agent which functions
to wet the particles of medicament to facilitate dispersion thereof
in a or the aqueous phase of the composition. Typically, the amount
of wetting agent used will not cause foaming of the dispersion
during mixing. Examples of wetting agents include fatty alcohols,
esters and ethers, such as polysorbate e.g. a polyoxyethylene
sorbitan monooleate such as polyoxyethylene 20 sorbitan monooleate
(polysorbate 80). The wetting agent may be present in intranasal
compositions in an amount of between about 0.001 and 0.05% (w/w),
for example about 0.025% (w/w), based on the total weight of the
composition. Wetting agents may be included in compositions
suitable for other routes of administration, e.g. for inhaled
administration, as appropriate.
[0433] Compositions can alternatively or additionally contain a
solublising agent, which generally functions to maintain the
compound of formula (I) or salt in solution (e.g. aqueous solution)
and/or to reduce the amount of precipitation of said compound or
salt from solution (e.g. aqueous solution) and/or to decrease the
rate of precipitation of said compound or salt from solution (e.g.
aqueous solution). Such a solublising agent can include a
polysorbate, for example a polyoxyethylene sorbitan monooleate such
as polyoxyethylene 20 sorbitan monooleate (polysorbate 80) or
polyoxyethylene 5 sorbitan monooleate (polysorbate 81), or
polyoxyethylene 20 sorbitan monolaurate (polysorbate 20), or
polyoxyethylene 20 sorbitan monopalmitate (polysorbate 40), or
polyoxyethylene 20 sorbitan monostearate (polysorbate 60). [See The
Handbook of Pharmaceutical Excipients 4th edition 2003 pp. 479-483
for polysorbates, and for an explanation of polyoxyethylene 20
sorbitan monooleate meaning it is the prepared at least in part by
copolymerisation with about 20 moles of ethylene oxide for each
mole of sorbitol and/or sorbitol anhydrides used.] The solubilising
agent can be present in intranasal compositions in an amount of
from about 0.25% to about 10% (w/w) such as about 1% to about 10%
(w/w), for example about 1% to about 5% (w/w), based on the total
weight of the composition.
[0434] An isotonicity adjusting agent may be included to achieve
isotonicity with body fluids e.g. fluids of the nasal cavity,
resulting in reduced levels of irritancy. The isotonicity adjusting
agent can include sodium chloride, dextrose, xylitol or calcium
chloride, in particular xylitol or dextrose. An isotonicity
adjusting agent may be included in intranasal compositions in an
amount of from about 0.1 to about 10% w/w, such as from about 1% to
about 10% w/w or from about 2% to about 10% w/w, e.g. about 4 to
about 5% (w/w), based on the total weight of the composition.
Isotonicity adjusting agents may also be included in compositions
suitable for other routes of administration, for example in
compositions suitable for inhaled, oral liquid or parenteral
administration, as appropriate. A isotonicity adjusting agent of
particular interest is xylitol, especially when the compound of
formula (I) or the salt thereof is
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a pharmaceutically acceptable salt thereof.
[0435] Further, the intranasal compositions comprising a compound
of formula (I) or a pharmaceutically acceptable salt thereof may be
buffered by the addition of suitable buffering agents such as
sodium citrate, citric acid, phosphates such as disodium phosphate
(for example the dodecahydrate, heptahydrate, dihydrate and
anhydrous forms) or sodium phosphate, or mixtures thereof, in
particular sodium citrate and/or citric acid. Buffering agents may
also be included in compositions suitable for other routes of
administration as appropriate.
[0436] Compositions for administration topically to the nose or
lung for example, for the treatment of rhinitis, include
pressurised aerosol compositions (e.g. for inhaled
administration/topical to the lung administration) and aqueous
compositions for delivery to the nasal cavities by pressurised
pump. Compositions which are non-pressurised and adapted to be
administered topically to the nasal cavity are of particular
interest. Suitable compositions contain water as the diluent or
carrier for this purpose. Aqueous compositions for administration
to the lung or nose may be provided with excipients such as
buffering agents, tonicity modifying agents and the like. Aqueous
compositions may also be administered to the nose by
nebulisation.
[0437] A fluid dispenser may typically be used to deliver a fluid
composition to the nasal cavities. The fluid composition may be
aqueous or non-aqueous, but typically aqueous. Such a fluid
dispenser may have a dispensing nozzle or dispensing orifice
through which a metered dose of the fluid composition is dispensed
upon the application of a user-applied force to a pump mechanism of
the fluid dispenser. Such fluid dispensers are generally provided
with a reservoir of multiple metered doses of the fluid
composition, the doses being dispensable upon sequential pump
actuations. The dispensing nozzle or orifice may be configured for
insertion into the nostrils of the user for spray dispensing of the
fluid composition into the nasal cavity. A fluid dispenser of the
aforementioned type is described and illustrated in WO05/044354.
The dispenser has a housing which houses a fluid discharge device
having a compression pump mounted on a container for containing a
fluid composition. The housing has at least one finger-operable
side lever which is movable inwardly with respect to the housing to
cam the container upwardly in the housing to cause the pump to
compress and pump a metered dose of the composition out of a pump
stem through a nasal nozzle of the housing. In one embodiment, the
fluid dispenser is of the general type illustrated in FIGS. 30-40
of WO05/044354.
Particle Size Reduction of Compound of Formula (I) or Salt
Thereof.
[0438] In, for example, pharmaceutical compositions suitable for
(e.g. adapted for) inhaled administration, the compound or salt of
formula (I) can be in a particle-size-reduced form. The
size-reduced form can for example be obtained or obtainable by
micronisation. Micronisation usually involves subjecting the
compound/salt to collisional and/or abrasional forces in a
fast-flowing circular or spiral/vortex-shaped airstream often
including a cyclone component. The particle size of the
size-reduced (e.g. micronised) compound or salt can be defined by a
D50 value of about 0.5 to about 10 microns, e.g. about 1 to about 7
microns or about 1 to about 5 microns (e.g. as measured using laser
diffraction). For example, the compound or salt of formula (I) can
have a particle size defined by: a D10 of about 0.3 to about 3
microns (e.g. about 0.4 to about 2 microns, or about 0.5 to about 1
microns), and/or a D50 of about 0.5 to about 10 microns or about 1
to about 7 microns or (e.g. about 1 to about 5 microns or about 1.5
to about 5 microns or about 1.5 to about 4 microns), and/or a D90
of about 1 to about 30 microns or about 2 to about 20 microns or
about 2 to about 15 microns or about 3 to about 15 microns (e.g.
about 2 to about 10 microns or about 4 to about 10 microns); for
example as measured using laser diffraction.
[0439] In particle size measurements, D90, D50 and D10 respectively
mean that 90%, 50% and 10% of the material is less than the micron
size specified. D50 is the median particle size. DV90, DV50 and
DV10 respectively mean that 90%, 50% and 10% by volume of the
material is less than the micron size specified. DM90, DM50 and
DM10 respectively mean that 90%, 50% and 10% by weight of the
material is less than the micron size specified.
[0440] Laser diffraction measurement of particle size can use a dry
method (wherein a suspension of the compound/salt in an airflow
crosses the laser beam) or a wet method [wherein a suspension of
the compound/salt in a liquid dispersing medium, such as isooctane
or ca. 0.05% lecithin in isooctane or (e.g. if compound is soluble
in isooctane) 0.1% Tween 80 in water, crosses the laser beam]. With
laser diffraction, particle size is preferably calculated using the
Fraunhofer calculation; and/or preferably a Malvern Mastersizer or
Sympatec apparatus is used for measurement. For example, particle
size measurement and/or analysis by laser diffraction can use any
or all of (e.g. all of) the following apparatus and/or conditions:
a Malvern Mastersizer 2000 version apparatus, a dispersing medium
of isooctane or ca. 0.05% lecithin in isooctane or ca. 0.1% Tween
80 in water, a stirring speed of ca. 1500-2500 rpm, ca. 30 seconds
to ca. 3 mins (e.g. ca. 30 seconds) sonification prior to final
dispersion and analysis, a 300 RF (Reverse Fourier) lens, and/or
the Fraunhofer calculation with Malvern software. In another
example, particle size measurement and/or analysis by laser
diffraction can use any or all of (e.g. all of) the following
apparatus and/or conditions: a Malvern Mastersizer longbed version
apparatus, a dispersing medium of ca. 0.1% Tween 80 in water, a
stirring speed of ca. 1500 rpm, ca. 3 mins sonification prior to
final dispersion and analysis, a 300 RF (Reverse Fourier) lens,
and/or the Fraunhofer calculation with Malvern software.
[0441] An illustrative non-limiting example of a small-scale
micronisation process is now given:
[0442] Micronisation Example: Micronisation of a compound or salt
of one of the Examples [0443] Purpose: To micronise a compound or
salt of one of the Examples (described hereinafter), e.g. in an
amount of approximately 300-3000 mg (e.g. about 300-1000 mg)
thereof, using a Jetpharma MC1 micronizer. [0444] The parent
(unmicronised) and micronised materials are analyzed for particle
size by laser diffraction and crystallinity by PXRD. Micronisation
Example: General Equipment and material
TABLE-US-00001 [0444] Equipment/material Description and
specification Jetpharma MC1 Nitrogen supply: Air tank with pressure
resistant Micronizer reinforced tubing (e.g. with 275 psi rate
tubing) Analytical balance can e.g. be Sartorius Analytical Top
loader balance can e.g. be Mettler PM400 or Sartorius L420P Digital
Caliper can e.g. be VWR Electronic caliper Materials to be a
compound or salt of one of the Examples micronised
[0445] The Jetpharma MC1 Micronizer comprises a horizontal
disc-shaped milling housing having: a tubular compound inlet (e.g.
angled at ca. 30 degrees to the horizontal) for entry of a
suspension of unmicronised compound of formula (I) or salt in a
gasflow, a separate gas inlet for entry of gases, a gas outlet for
exit of gases, and a collection vessel (micronizer container) for
collecting micronised material. The milling housing has two
chambers: (a) an outer annular chamber in gaseous connection with
the gas inlet, the chamber being for receiving pressurised gas
(e.g. air or nitrogen), and (b) a disc-shaped inner milling chamber
within and coaxial with the outer chamber for micronising the input
compound/salt, the two chambers being separated by an annular wall.
The annular wall (ring R) has a plurality of narrow-bored holes
connecting the inner and outer chambers and
circumferentially-spaced-apart around the annular wall. The holes
opening into the inner chamber are directed at an angle (directed
part-way between radially and tangentially), and in use act as
nozzles directing pressurised gas at high velocity from the outer
chamber into the inner chamber and in an inwardly-spiral path
(vortex) around the inner chamber (cyclone). The compound inlet is
in gaseous communication with the inner chamber via a nozzle
directed tangentially to the inner chamber, within and near to the
annular wall/ring R. Upper and lower broad-diameter exit vents in
the central axis of the inner milling chamber connect to (a) (lower
exit) the collection vessel which has no air outlet, and (b) (upper
exit) the gas outlet. Inside and coaxial with the tubular compound
inlet and longitudinally-movable within it is positioned a venturi
inlet (V) for entry of gases. The compound inlet also has a
bifurcation connecting to an upwardly-directed material inlet port
for inputting material.
[0446] In use, the narrow head of the venturi inlet (V) is
preferably positioned below and slightly forward of the material
inlet port, so that when the venturi delivers pressurised gas (e.g.
air or nitrogen) the feed material is sucked from the material
inlet port into the gas stream through the compound inlet and is
accelerated into the inner milling chamber tangentially at a
subsonic speed. Inside the milling chamber the material is further
accelerated to a supersonic speed by the hole/nozzle system around
the ring (R) (annular wall) of the milling chamber. The nozzles are
slightly angled so that the acceleration pattern of the material is
in the form of an inwardly-directed vortex or cyclone. The material
inside the milling chamber circulates rapidly and particle
collisions occur during the process, causing larger particles to
fracture into smaller ones. "Centrifugal" acceleration in the
vortex causes the larger particles to remain at the periphery of
the inner chamber while progressively smaller particles move closer
to the centre until they exit the milling chamber, generally
through the lower exit, at low pressure and low velocity. The
particles that exit the milling chamber are heavier than air and
generally settle downward thorugh the lower exit into the
collection vessel (micronizer container), while the exhaust gas
rises (together with a minority of small particles of micronised
material) and escapes into the atmosphere at low pressure and low
velocity. A filter sock can optionally be placed in the upper gas
outlet to catch fine micronised material.
MICRONISATION EXAMPLE
General Procedure
[0447] The micronizer is assembled. The narrow head of the venturi
inlet is positioned below and slightly forward of the material
inlet port and is measured with a micro-caliper to make sure that
it is inserted correctly. The grind (ring) (R) and venturi (V)
pressures are adjusted according to the values specified in the
experimental design (refer to experimental section below) by
adjusting the valves on the pressure gauges on the micronizer. The
setup is checked for leakage by observing if there is any
fluctuation in the reading of the pressure gauges.
[0448] Note that the venturi (V) pressure is kept at least about 2
bars greater than the grind (ring) (R) pressure to prevent
regurgitation of material, e.g. outwardly from the material inlet
port.
[0449] Balance performance can be checked with calibration weights.
Specified amount of the parent material (input material) is fed
manually, generally quite slowly, into the input container of the
micronizer using a spatula. The input container plus material is
weighed. The equipment pressure is monitored during the
micronization process.
[0450] [Note: As an optional variation of these procedures, input
material may be passed through a sieve/screen (e.g. a 600 micron
screen) prior to micronisation in order to de-aggregate the input
material, if appropriate or desirable.]
[0451] Upon completion of the micronising run, the nitrogen supply
is shut off and the micronised material is allowed to settle into
the micronizer container. The Venturi pressure valve and the grind
(ring) pressure valve can be closed. The micronised powder in the
micronizer container (collection vessel) and the cyclone (above the
recovery vessel) are collected together into a pre-weighed and
labelled collection vial. Any fine micronised material which may
have collected in any filter sock, which may have been optionally
placed in the upper gas outlet of the microniser, can optionally be
tapped down into the collection vessel or cyclone. The weight of
the micronised material is recorded. The input container is
generally re-weighed in order to calculate the amount of input
material by difference. The micronizer can be disassembled and
residual PDE4 compound on the micronizer inner surface can be
rinsed e.g. with 70/30 isopropyl alcohol/water and collected into a
flask. The micronizer can then be thoroughly cleaned, e.g. in a
Lancer washing machine, and can be dried before subsequent runs are
performed.
[0452] In one embodiment, one example of suitable micronisation
conditions is: Material input amount about 300 mg to about 1000 mg;
Venturi Pressure (V) about 4 to about 10 bar (e.g. about 4-6 bar,
e.g. about 4-5 bar); Grind (Ring) Pressure (R) about 2 to about 6
bar (e.g. about 2 bar). Material feed rate can optionally be from
about 70 to about 200 mg/min.
% yield=[(Material from collection vessel+Material from
cyclone+optionally material from gas outlet)/Material input
amount].times.100.
Micronisation Examples Carried Out
Micronisation Example 1
[0453]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide monohydrochloride, substantially in the form of the Form
1 anhydrate thereof (e.g. which can be preparable in Example 1A2),
was micronised, by manual feeding into the microniser venturi,
using a general procedure and/or general apparatus generally
similar or analogous to those described above, and generally using
substantially the experimental equipment and parameters described
below. The parent (unmicronised) material was analyzed for particle
size by scanning electron microscopy (SEM).
Micronisation Example 1
Equipment and Process Details
TABLE-US-00002 [0454] Equipment/material Description and
specification Jetpharma MC1 Microniser Nitrogen supply: Nitrogen
cylinder with pressure resistant reinforced tubing Analytical
balance e.g. Sartorius Analytical Top loader balance e.g. Sartorius
L420P Pre-micronisation screening of input The input material is
thought not to have been material passed through a screen before
micronisation; but see below for screening performed during the
process of micronisation. Venturi Material Pressure (V) Particle
Size Particle Size Recovery input & Grind (ring) Data (microns)
Data (microns) yield of Example amount Pressure (R) (unmicronised
(micronised micronised no. (g) (bar) material), SEM material), SEM
material* 1 502 mg V = ca. 5 bar Agglomerates Primary equant About
56%. (see below). of equant particles 282 mg of R = 2 bar primary
generally of micronised particles. The about 2-4 material primary
particle microns or less collected size is particle size generally
about (e.g. generally 5 microns or about 2-3 less microns or less).
Some of the primary particles are present as loose agglomerates
[0455] Process notes for Micronisation Example 1: The Venturi
Pressure V was initially 5 bar (with R=2 bar). In response to an
apparently blocked venturi with material appearing to emit from the
venturi, the process was stopped after 3 minutes micronisation,
resumed again for a further 2 minutes and then stopped again. The
process was restarted with V increased to 6 bar, but there was a
blockage again with powder emitting from the venturi, so the
process was stopped again after 1 minute; it appeared that there
was a large lump of material which blew back. Apparently, the
presence of aggregates (agglomerates) was blocking the venturi
feed. Hence, the material was passed through a 355 micron screen
(sieve), to attempt to break up significant agglomerates, and then
micronisation was resumed, using V=5 bar and R=2 bar and feeding
the cohesive screened material into the venturi using a brush
rather than a spatula; there was no evidence of blow-back as the
screened material was fed into the venturi. This final stage,
micronising the screened material, lasted 7 minutes. The collected
micronised material (282 g) appeared to be cohesive in nature,
based on evidence of a residue on the venturi. There was some
evidence of material present in the outlet; there was no major
presence of material around the micronising ring.
Micronisation Example 2
[0456]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide hydrobromide (e.g. monohydrobromide, and/or e.g. which
can be as prepared in Example 1D), fed manually into the venturi,
was micronised, by manual feeding into the microniser venturi,
using a general procedure and/or general apparatus generally
similar or analogous to those described above, and generally using
substantially the experimental equipment and parameters described
below. The micronisation duration was about 5 minutes. The parent
(unmicronised) material was analyzed for particle size by scanning
electron microscopy (SEM).
Micronisation Example 2
Equipment and Process Details
TABLE-US-00003 [0457] Equipment/material Description and
specification Jetpharma MC1 Microniser Nitrogen supply: Nitrogen
cylinder with pressure resistant reinforced tubing Analytical
balance e.g. Sartorius Analytical Top loader balance e.g. Sartorius
L420P Pre-micronisation screening of input material Passed through
600 micron screen before micronisation, to try to de-aggregate
visible lumps Venturi Material Pressure (V) Particle Size Particle
Size Recovery input & Grind (ring) Data (microns) Data
(microns) yield of Example amount Pressure (R) (unmicronised
(micronised micronised no. (g) (bar) material), SEM material), SEM
material* 2 414.1 mg V = 5 bar Agglomerates Agglomerates 59.5% of
quite R = 2 bar of equant of equant based on cohesive primary
primary input pre- screened particles. The particles. screening;
material primary particle Primary 71.8% (weight size is generally:
particle size is based on micronised about 5 generally: input into
after microns or about 1-5 microniser. screening less, or about
microns, or 297.5 mg of 500 mg of 2-3 microns or about 2-3
micronised raw input less microns or less material material)
collected.
Micronisation Example 3
[0458]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide succinate (e.g. hemisuccinate and/or e.g. which can be as
prepared in Example 1E) was micronised, by manual feeding into the
microniser venturi, using a general procedure and/or general
apparatus generally similar or analogous to those described above,
and generally using substantially the experimental equipment and
parameters described below. The parent (unmicronised) material was
analyzed for particle size by scanning electron microscopy
(SEM).
Micronisation Example 3
Equipment and Process Details
TABLE-US-00004 [0459] Equipment/material Description and
specification Jetpharma MC1 Microniser Nitrogen supply: Nitrogen
cylinder with pressure resistant reinforced tubing Analytical
balance e.g. Sartorius Analytical Top loader balance e.g. Sartorius
L420P Pre-micronisation screening of material Not performed Venturi
Material Pressure (V) Particle Size Particle Size Recovery input
& Grind (ring) Data (microns) Data (microns) yield of Example
amount Pressure (R) (unmicronised (micronised micronised no. (g)
(bar) material), SEM material), SEM material* 3 500 mg V = ca. 4-5
bar Agglomerates Agglomerates about 60% (see of equant (some of
yield. Either below) primary reduced size) about 300 mg R = 2 bar
particles. The made up of or about primary particle equant primary
380 mg of size is particles. micronised generally about Primary
material 5 microns or particle size is recovered. less generally
about 4-5 microns or less
[0460] Process notes for Micronisation Example 3: The input
material appeared to be reasonably free-flowing and not excessively
aggregated or lumpy and so was not screened. The grind pressure (R)
was 2 bar and the initial venturi pressure (V) was 4 bar, and the
input material was fed manually to the microniser quickly, in an
attempt to reduce the risk of rapid and excessive micronisation.
After 5 minutes of the micronisation process, a change in noise was
heard, and it was observed that there was a build-up of input
material in the feed inlet funnel. The venturi pressure was
increased to 5 bar, which cleared the powder in the feed funnel.
There was no further recurrence of build-up/blockage in the feed
funnel. At the end of the process, some fine material was noted in
the filter outlet port. Micronised material was collected from the
collecting pot and the outlet funnel. Micronised material did not
appear to be excessively cohesive and was readily recovered.
Micronisation process time=10 minutes.
Micronisation Example 4
[0461]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide (the "free base", e.g. which can be as prepared in
Example I B) was micronised, by manual feeding into the microniser
venturi, using a general procedure and/or general apparatus
generally similar or analogous to those described above, and
generally using substantially the experimental equipment and
parameters described below. The parent (unmicronised) material was
analyzed for particle size by scanning electron microscopy
(SEM).
Micronisation Example 4
Equipment and Process Details
TABLE-US-00005 [0462] Equipment/material Description and
specification Jetpharma MC1 Micronizer Nitrogen supply: Nitrogen
cylinder with pressure resistant reinforced tubing Analytical
balance e.g. Sartorius Analytical Top loader balance e.g. Sartorius
L420P Pre-micronisation screening of input Passed through 600
micron screen before material micronisation, to try to break up
hard aggregates Venturi Material Pressure (V) Particle Size
Particle Size Recovery input & Grind (ring) Data (microns) Data
(microns) yield of Example amount Pressure (R) (unmicronised
(micronised micronised no. (g) (bar) material), SEM material), SEM
material* 4 451 mg V = 5 bar Irregular Equant about 57%, (weight R
= 2 bar particles of up particles of up based on available to about
200 to about 10 material for microns microns input to micronising
particle size particle size microniser. after 257.6 mg of screening
micronised 509.5 mg material raw input recovered. material)
Micronisation Example 5
[0463]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide monohydrochloride, substantially in the form of the Form
1 anhydrate thereof (e.g. which can be as prepared in Example 1A2
such as Example 1A2 alternative preparation no. 1, plant method),
was micronised on a larger scale micronisation equipment, a 4 inch
APTM microniser (available from APTM, Switzerland, "4 inch"
referring to the diameter of the micronisation ring). Experimental
parameters and procedures are substantially as described in the
table and text below or similar, including automatic feeding of the
input material into the microniser venturi. A detailed apparatus
and method description is not described herein. The parent
(unmicronised) material and the micronised material were analysed
for particle size by a laser diffraction method; particle size data
are thought to be substantially as given below.
TABLE-US-00006 Venturi Particle Size Particle Size Material
Pressure (V) Data (microns) Data (microns) Recovery input &
Grinding (unmicronised (micronised yield of amount pressure (G)
material), laser material), laser micronised (g) (bar) diffraction
diffraction material 800 g V = ca. 7 bar D50 = about D50 = about
721 g total (divided G = ca. 5 bar 77.8 microns 2.48 microns net
weight into pre-sampling separate (about 90% 443 g and yield) 357 g
lots)
[0464] For Micronisation Example 5, the feed rate (i.e. the rate of
feeding of the input material via a rotating screw feed into the
venturi inlet of the microniser), was 14.+-.2.5 g per minute. An
automatic (not manual) feeder was used.
[0465] Inhaled administration involves topical administration to
the lung, such as by aerosol or dry powder composition.
Aerosol Inhalable Compositions
[0466] An aerosol formulation (aerosol composition), e.g. for
inhaled administration, can be either a suspension or a
solution.
[0467] Aerosol formulations (aerosol compositions), e.g. for
inhaled administration, can optionally comprise a solution or fine
suspension of the compound of formula (I) or the pharmaceutically
acceptable salt thereof (active substance) in a pharmaceutically
acceptable aqueous or non-aqueous liquid (e.g. solvent).
[0468] An aerosol formulation (aerosol composition), e.g. for
inhaled administration, generally can contain a compound of formula
(I) or a pharmaceutically acceptable salt thereof and a suitable
propellant (e.g. under pressure) such a suitable organic propellant
such as a fluorocarbon or hydrofluorocarbon (HFC) or
hydrogen-containing chlorofluorocarbon (HCFC) or a mixture thereof.
A hydrofluorocarbon (HFC) propellant can comprise a
hydrofluoroalkane, such as 1,1,1,2-tetrafluoroethane or
1,1,1,2,3,3,3-heptafluoropropane or a mixture thereof. Other
propellants include chlorofluorocarbons (CFCs) such as
dichlorodifluoromethane, trichlorofluoromethane or
dichlorotetrafluoroethane.
[0469] An aerosol composition may optionally contain additional
formulation excipient(s) such as a surfactant and/or a cosolvent.
The surfactant can include, but is not limited to, oleic acid,
lecithin, an oligolactic acid or derivative e.g. as described in
WO94/21229 and/or WO98/34596. The cosolvent can for example
comprise ethanol.
[0470] Aerosol formulations, e.g. for inhaled administration, can
be presented e.g. as a dosage form, e.g. in single or multidose
quantities in sterile form in a sealed container, which can take
the form of a cartridge or refill for use with an atomising device
or inhaler. Alternatively the sealed container may be a unitary
dispensing device such as a single dose nasal inhaler or an aerosol
dispenser fitted with a metering valve (metered dose inhaler, or
MDI) which is intended for disposal once the contents of the
container have been exhausted. The aerosol formulation dosage form
can alternatively take the form of a pump-atomiser.
Dry Powder Inhalable Composiftions
[0471] For pharmaceutical compositions suitable (e.g. adapted for)
inhaled administration, the pharmaceutical composition may for
example be a dry powder inhalable composition.
[0472] Dry powder inhalable compositions may take the form of
capsules and cartridges of, for example, gelatine, or blisters of,
for example, laminated aluminium foil, for use in an inhaler or
insufflator. Dry powder inhalable compositions may be formulated
comprising a powder mix of a compound of formula (I) or a
pharmaceutically acceptable salt thereof and a suitable powder base
such as lactose or starch.
[0473] A dry powder inhalable pharmaceutical composition can
comprise a powder base such as lactose or starch, the compound of
formula (I) or salt thereof (suitably in particle-size-reduced
form, e.g. in micronised form), and optionally a ternary agent. The
ternary agent can for example comprise L-leucine, mannitol,
trehalose, magnesium stearate, calcium stearate and/or cellobiose
octaacetate (e.g. alpha-D-isomer of cellobiose octaacetate, e.g.
available from Aldrich). The ternary agent can in particular be
magnesium stearate, e.g. magnesium stearate present in an amount of
from about 0.05% to about 2% w/w or from about 0.1% to about 1% w/w
or from about 0.1% to about 0.5% w/w, such as from about 0.1% to
about 0.25% w/w or from about 0.25% to about 1% w/w, for example
about 0.5% w/w magnesium stearate. For the use of magnesium
stearate in dry powder inhalable compositions, see WO 00/28979 A1
(Skyepharma) and the corresponding publications U.S. Pat. No.
6,645,466 B1 and US 2004/0202616 A1; and/or see WO 00/53157 A1. For
cellobiose octaacetate and storage stability, see WO 03/088943.
[0474] The dry powder inhalable composition can comprise a dry
powder blend of lactose and the compound of formula (I) or salt
thereof. The lactose can be lactose hydrate e.g. lactose
monohydrate and/or can be inhalation-grade and/or fine-grade
lactose. The particle size of the lactose can for example be
defined by 90% or more (by weight or by volume) of the lactose
particles being less than 1000 microns (micrometres) (e.g. 10-1000
microns e.g. 30-1000 microns) in diameter, and/or 50% or more of
the lactose particles being less than 500 microns (e.g. 10-500
microns) in diameter. The particle size of the lactose can for
example be defined by 90% or more of the lactose particles being
less than 300 microns (e.g. 10-300 microns e.g. 50-300 microns) in
diameter, and/or 50% or more of the lactose particles being less
than 100 microns in diameter. Optionally, the particle size of the
lactose can be defined by 90% or more of the lactose particles
being less than 100-200 microns in diameter, and/or 50% or more of
the lactose particles being less than 40-70 microns in diameter. In
particular, the particle size of the lactose can be about 2% to
about 30% or about 3% to about 30% (e.g. 2% to 15%, or 7% to 11%,
e.g. about 10%) (by weight or by volume) of the particles are less
than 50 microns, or are less than 20 microns ("fines"), or are less
than 15 microns ("fines"), in diameter. For example, without
limitation, one suitable inhalation-grade lactose is lactose (e.g.
10% fines (e.g. E9334), or 6% fines, or 7% fines, or 11% fines),
obtainable from Friesland Foods Domo (formerly Borculo Domo
Ingredients), Hanzeplein 25, 8017 JD Zwolle, Netherlands.
[0475] In the dry powder inhalable composition the compound of
formula (I) or salt thereof can for example be present in about
0.1% to about 70% (e.g. about 0.1% to about 50%, about 0.1% to
about 30%, or about 0.1% to about 20%, or about 0.1% to about 10%,
such as about 0.4% to about 10%, e.g. about 1% to about 10%, e.g.
about 1% to about 5%) by weight of the composition.
[0476] Illustrative non-limiting examples of dry powder inhalable
compositions follow:
Dry Powder Composition Example 1
Dry Powder Lactose Blend Preparation
[0477] Using a size-reduced e.g. micronised form of the compound of
formula (I) or salt thereof (e.g. as prepared in a Micronisation
Example herein), a dry powder blend can, for example, be prepared
by mixing the required amount of the compound/salt (e.g. 10 mg, 1%
w/w) with inhalation-grade lactose containing 10% fines (e.g. 990
mg, 99% w/w) in a Teflon.TM. (polytetrafluoroethene) pot in a
Mikro-dismembrator ball-mill (but without a ball bearing) at %
speed (ca. 2000-2500 rpm) for about 4 hours at each blend
concentration. The Mikro-dismembrator (available from B. Braun
Biotech International, Schwarzenberger Weg 73-79, D-34212
Melsungen, Germany; www.bbraunbiotech.com) comprises a base with an
upwardly-projecting and sidewardly-vibratable arm to which is
attached the Teflon.TM. pot. The vibration of the arm achieves
blending.
[0478] Other blends can include: 10% w/w compound/salt (50 mg)+90%
w/w lactose (450 mg, inhalation-grade lactose containing 10%
fines).
[0479] Serial dilution of the 1% w/w blend can achieve e.g. 0.1%
and 0.3% w/w blends.
Dry Powder Composition Example 2
Dry Powder Lactose Blend Preparations
[0480] Using a micronised form of the compound of formula (I) or
salt thereof (e.g. as prepared in a Micronisation Example herein),
the dry powder blend can, for example, be prepared by mixing
(blending), in a high shear blender (such as an Aeromatic-Fielder
Turbo Rapid Volume blender), the required amount of the
compound/salt with inhalation-grade lactose, e.g. lactose (such as
lactose monohydrate) containing from 2% to 15% w/w fines, such as
7% to 11% w/w fines.
[0481] Examples of the contents of these dry powder inhalable
compositons are those which are in a dosage unit containing 12.5 mg
total amount of dry powder inhalable composition, which can have
different constituents as follows:
Dry Powder Composition Example 2(A)
[0482] (i) 10 micrograms of the micronised compound of formula (I)
or salt thereof (0.08% w/w of the formulation), plus
[0483] (ii) 12.49 mg of inhalation-grade lactose, containing from
7% to 11% lactose fines.
Dry Powder Composition Example 2(B)
[0484] (i) 50 micrograms of the micronised compound of formula (I)
or salt thereof (0.4% w/w of the formulation), plus
[0485] (ii) 12.45 mg of inhalation-grade lactose, containing from
7% to 11% lactose fines.
Dry Powder Composition Example 2(C)
[0486] (i) 100 micrograms of the micronised compound of formula (I)
or salt thereof (0.8% w/w of the formulation), plus
[0487] (ii) 12.4 mg of inhalation-grade lactose, containing from 7%
to 11% lactose fines.
Dry Powder Composition Example 2(D)
[0488] (i) 500 micrograms of the micronised compound of formula (I)
or salt thereof (4% w/w of the formulation), plus
[0489] (ii) 12.0 mg of inhalation-grade lactose, containing from 7%
to 11% lactose fines.
Dry Powder Composition Example 2(E)
[0490] (i) 1000 micrograms of the micronised compound of formula
(I) or salt thereof (8% w/w of the formulation), plus
[0491] (ii) 11.5 mg of inhalation-grade lactose, containing from 7%
to 11% lactose fines.
Dry Powder Composition Example 2(F) with Magnesium Stearate Ternary
Agent
[0492] (i) 50 micrograms of the micronised compound of formula (I)
or salt thereof (0.4% w/w of the formulation), plus
[0493] (ii) 62.5 micrograms of magnesium stearate (0.5% w/w of the
formulation), plus
[0494] (iii) 12.3875 mg of inhalation-grade lactose, containing
from 7% to 11% lactose fines.
Dry Powder Composition Example 2(G) with Magnesium Stearate Ternary
Agent
[0495] (i) 500 micrograms of the micronised compound of formula (I)
or salt thereof (4% w/w of the formulation), plus
[0496] (ii) 62.5 micrograms of magnesium stearate (0.5% w/w of the
formulation), plus
[0497] (iii) 11.9375 mg of inhalation-grade lactose, containing
from 7% to 11% lactose fines.
Dry Powder Composition Example 2(H) with Magnesium Stearate Ternary
Agent
[0498] (i) 1000 micrograms of the micronised compound of formula
(I) or salt thereof (8% w/w of the formulation), plus
[0499] (ii) 62.5 micrograms of magnesium stearate (0.5% w/w of the
formulation), plus
[0500] (iii) 11.4375 mg of inhalation-grade lactose, containing
from 7% to 11% lactose fines.
[0501] In the above dry powder inhalable compositons (Dry Powder
Composition Example 2(A) to 2(H)), the micronised compound of
formula (I) or salt thereof is preferably
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide or a salt thereof, such as the monohydrochloride salt thereof,
e.g. the monohydrochloride Form 1 anhydrate thereof.
[0502] The above dry powder inhalable compositons (Dry Powder
Composition Example 2(A) to 2(H)) can optionally be formed into a
dosage unit as follows:
[0503] (a) encapsulated in a rupturable or openable capsule, or
[0504] (b) contained within sealed, rupturable or peel-openable,
dose containers which are mounted longitudinally in a strip or
ribbon which can be inside a suitable inhalation device (e.g. the
DISKUS.TM. inhalation device, e.g. which can optionally be
substantially as described in GB 2,242,134 A, see below).
Dry Powder Inhalation Devices
[0505] Optionally, in particular for dry powder inhalable
compositions, a pharmaceutical composition for inhaled
administration can be incorporated into a plurality of sealed dose
containers (e.g. containing the dry powder composition) mounted
longitudinally in a strip or ribbon inside a suitable inhalation
device. The container can be rupturable or peel-openable on demand
and the dose, e.g. of the dry powder composition, can be
administered by inhalation via a device such as the DISKUS.TM.
device, marketed by GlaxoSmithKline. The DISKUS.TM. inhalation
device can e.g. be substantially as described in GB 2,242,134 A. In
such device at least one container for the pharmaceutical
composition in powder form (the at least one container preferably
being a plurality of sealed dose containers mounted longitudinally
in a strip or ribbon) is defined between two members peelably
secured to one another; the device comprises: means defining an
opening station for the said at least one container; means for
peeling the members apart at the opening station to open the
container; and an outlet, communicating with the opened container,
through which a user can inhale the pharmaceutical composition in
powder form from the opened container.
[0506] See also WO 03/061743 A1 which discloses a medicament
dispenser for use with plural elongate form medicament carriers,
each having multiple distinct medicament dose portions carried
thereby (e.g. as described in the claims thereof e.g. claim 1). See
also WO 2007/012871 A1 which discloses a medicament dispenser for
use with at least one medicament carrier carrying multiple distinct
medicament portions (e.g. as described in the claims thereof e.g.
claim 1).
Pharmaceutical Compositions Suitable for External Topical
Administration
[0507] A pharmaceutical composition of the invention can for
example be suitable for (e.g. adapted for) external topical (e.g.
skin topical) administration, for example to a mammal such as a
human. The pharmaceutical composition suitable for external topical
administration can suitably be for the treatment and/or prophylaxis
of atopic dermatitis in a mammal such as a human.
[0508] "External topical administration" is defined above under the
"medical uses" section. External topical administration can for
example be to those parts of the skin affected by or susceptible to
the disease or condition e.g. atopic dermatitis, in particular in a
mammal (e.g. human) suffering from or susceptible to atopic
dermatitis.
[0509] An external-topical pharmaceutical composition, e.g. skin
topical pharmaceutical composition, can for example be an ointment,
a cream (usually an oil-in-water or water-in-oil pharmaceutical
composition, usually an emulsion), an aqueous gel, or a
microemulsion.
[0510] In the external-topical pharmaceutical composition, e.g. an
ointment or an oil-in-water or water-in-oil composition, the
compound of formula (I) or the pharmaceutically acceptable salt
thereof can be present in 0.1% to 10%, such as 0.2% to 5%, or 0.5%
to 5%, or 1% to 5%, or 0.5% to 3% (e.g. about 1% or about 2%), by
weight of the composition (w/w).
[0511] In one optional embodiment, the compound of formula (I) or
the pharmaceutically acceptable salt thereof can optionally be in a
particle-size-reduced form, for example obtained or obtainable by
micronisation. This can be, for example, for use in a
pharmaceutical composition suitable for (e.g. adapted for) external
topical (e.g. skin topical) administration. See the Particle size
reduction sub-section herein, within the Inhalable pharmaceutical
compositions section, for more details.
Aqueous solubility: A preliminary screen, which can aim to estimate
roughly the aqueous solubility of a compound or salt of the
invention, can include (as an approximate summary): (i) creating a
ca. 10 mM solution of the compound in DMSO, (ii) diluting a portion
of this DMSO solution by mixing about 19 parts by volume of pH 7.4
aqueous phosphate buffered saline (PBS) buffer with 1 part by
volume of the ca. 10 mM DMSO solution, (iii) "filtering" the
mixture with the aid of centrifugation, and then (iv) measuring the
concentration of the dissolved compound in the "filtrate". Although
some DMSO (about 5% by volume) is usually present in this
solubility screen "filtrate", the results can be a very approximate
estimate of aqueous solubility, e.g. at room temperature.
[0512] Lipophilicity: The clogP (calculated log of the
octanol/water partition coefficient (P)) of a particular compound
or salt of the invention can estimate the lipophilicity of the
compound or salt.
[0513] Solubilising and/or skin-penetration-enhancing agents: In
one embodiment, an external-topical pharmaceutical composition,
e.g. an ointment or an oil-in-water cream or water-in-oil cream,
suitably includes an agent which acts as a skin-penetration
enhancer for and/or a solubiliser of the compound of formula (I) or
the salt thereof. The skin-penetration-enhancing- and/or
solubilising-agent can for example be propylene glycol, diethylene
glycol monoethyl ether (e.g. TRANSCUTOL.TM.) and/or caprylocaproyl
macrogolglycerides (e.g. LABRASOL.TM.), such as propylene glycol.
The solubiliser and/or skin-penetration enhancer suitably does not
comprise DMSO. The solubiliser and/or skin-penetration enhancer can
be both a solubiliser and skin-penetration enhancer, and/or can for
example be present in 0.5% to 50%, suitably 5% to 50%, more
suitably 7% to 30%, for example 7% to 25%, such as about 10% to
about 20% (e.g. about 10% or about 20%), by weight of the
composition (w/w).
[0514] The skin-penetration enhancer is for delivery of the
compound of formula (I) or salt thereof ("active agent" or "drug")
through the skin. Solubilization of the drug also helps. The
solubilising and/or skin-penetration-enhancing agents should
ideally (a) be safe and/or tolerable, (b) have as low a potential
for skin irritancy as possible consistent with being an effective
skin penetration enhancer, and (c) be compatibile with the active
pharmaceutical ingredient. Note that the agent can e.g. function
both as a solubilising agent and a skin-penetration-enhancing
agent.
[0515] Surfactants: An external-topical pharmaceutical composition,
e.g. an ointment or in particular an oil-in-water cream or
water-in-oil cream, can include a surfactant (e.g. as an
emulsifier), for example for achieving emulsification of
compositions having two or more phases. The total surfactant
content can for example be 0.3% to 20%, e.g. 0.5% to 15% or 0.5% to
12% or 0.5% to 10% or 1% to 12% or 3% to 10%, by weight of the
composition (w/w). The surfactant can for example comprise a
nonionic surfactant such as one or more of the following: a
polyoxyl C.sub.12-22alkyl ether (e.g. a polyoxyl C.sub.12-18alkyl
ether such as polyoxyl cetyl ether or polyoxyl stearyl ether or
polyoxyl lauryl ether) (e.g. present at 0.5% to 10% w/w, e.g. 2.5%
to 10% w/w such as about 5% to about 8% w/w), glycerol monostearate
(e.g. Arlacel 165.TM.) (e.g. present at 0.5% to 10% w/w, e.g. about
2% w/w), sorbitan monostearate (e.g. Span 60.TM.) (e.g. present at
0.05% to 10% w/w, e.g. about 1% w/w), and cetyl alcohol and/or
stearyl alcohol (e.g. cetostearyl alcohol, e.g. wherein the total
of any cetyl alcohol and any stearyl alcohol present is 0.5% to 15%
w/w, e.g. 1% to 10% w/w such as 2% to 10% w/w or 5% to 10% w/w).
Polyoxyl stearyl ether (steareth) can e.g. be a polyoxyl 2-21
stearyl ether, such as polyoxyl 2 stearyl ether (steareth-2),
polyoxyl 10 stearyl ether (steareth-10), polyoxyl 20 stearyl ether
(steareth-20) or polyoxyl 21 stearyl ether (steareth-21). Polyoxyl
cetyl ether (ceteth) can e.g. be a polyoxyl 2-20 cetyl ether such
as ceteth-2, ceteth-10 or ceteth-20. Polyoxyl alkyl ethers are also
named polyoxyethylene alkyl ethers. Alternatively or additionally,
the surfactant can for example comprise an ionic surfactant such as
sodium dodecyl sulfate (SDS)=sodium lauryl sulfate (e.g. SDS
present at 0.3% to 2% w/w such as 0.5% to 1.5% w/w).
[0516] Ointments and creams (and oil phase): An external-topical
pharmaceutical composition can be an ointment or an oil-in-water
cream or water-in-oil cream. The ointment or cream typically
contains an oil phase (oily ointment base). The oil phase (ointment
base) typically comprises an oil and/or a fat, suitably of a
consistency suitable for skin-spreadability.
[0517] In particular, an oil comprising or being white soft
paraffin (white petrolatum) and/or a mineral oil (such as liquid
paraffin) can be used. (Mineral oil can also be used as a
solubiliser and/or emollient). The white soft paraffin (white
petrolatum) can be of various grades, for example (for Penreco
supplier) Penreco Regent White grade, Penreco Snow White grade, or
Penreco Ultima White grade, in particular high melting point white
soft paraffin (e.g. of Penreco Ultima White grade).
Microcrystalline wax or beeswax or beeswax substitute can be used
as an oil/fat in the oil phase.
[0518] Alternatively or additionally, one or more fats like
straight or branched chain mono- or di-alkyl esters such as
isopropyl myristate, isopropyl palmitate, diisopropyl adipate,
isocetyl stearate, isostearyl isostearate, decyl oleate, butyl
stearate, 2-ethylhexyl palmitate, propylene glycol diester of
coconut fatty acids, or a mixed ester of 2-ethyl hexanoic acid with
a blend of cetyl or stearyl alcohols (e.g. known as Crodamol CAP),
may be used in the oil phase (some of these are also solubilisers
and/or surfactants). These may be used singly or in combination
depending on the properties required.
[0519] The oil phase (oily ointment base) can for example be
present at: [0520] 30% to 99.8% w/w (e.g. 50% to 99.5% w/w, e.g.
50% to 95% w/w, e.g. 60% to 95% w/w, e.g. 60% to 90% w/w) in an
ointment (e.g. emulsion or homogeneous single phase); [0521] 25% to
85% w/w (e.g. 35% to 70% w/w) in a water-in-oil cream (e.g.
emulsion); or [0522] 5% to 60% w/w or 8% to 55% w/w (e.g. 10% to
45% w/w or 12% to 30% w/w) in an oil-in-water cream (e.g.
emulsion).
[0523] Note that the w/w percentages for the oil phase (oily
ointment base), mentioned above or in the example formulations
below or generally herein, exclude the amount of any surfactant(s)
present (except for compound(s) listed herein as fats which also
have surfactant properties), and exclude the amount of any non-oil
non-fat solubilising and/or skin-penetration-enhancing agents
present.
[0524] Example ointments: As an example, an external-topical
pharmaceutical composition can be an ointment comprising: [0525]
the compound of formula (I) or pharmaceutically acceptable salt
thereof present at 0.1% to 10% w/w (e.g. 0.2% to 5% w/w, or 0.5% to
5% w/w, or 0.5% to 3% w/w); and [0526] an oil phase (oily ointment
base) present at 30% to 99.8% w/w or 50% to 99.5% w/w or 50% to 95%
w/w or 60% to 95% w/w or 60% to 90% w/w (i.e. by weight of the
composition).
[0527] For example, in the above example ointment, the oil phase or
composition can suitably comprise white petrolatum present at 25%
to 99.5% w/w or 45% to 99% w/w or 55% to 85% w/w (i.e. by weight of
the composition). Optionally, additionally or alternatively, the
oil phase or composition can comprise mineral oil (e.g. as
solubiliser and emollient) present at 2.5% to 25% w/w such as 4% to
20% w/w (i.e. by weight of the composition)].
[0528] In the above example ointment, the ointment can optionally
comprise one or more surfactants (e.g. polyoxyl stearyl ether,
polyoxyl cetyl ether or cetostearyl alcohol) present in total at
0.5% to 10% w/w or 3% to 10% w/w.
[0529] In the above example ointment, the ointment can optionally
comprise one or more agents acting as a skin-penetration enhancer
(in particular acting as both a solubiliser and skin-penetration
enhancer and/or in particular hydrophilic such as propylene glycol)
present in total at 0.5% to 50% w/w, such as 5% to 50% w/w or 7% to
30% w/w.
[0530] In the above example ointment, the ointment can optionally
comprise (a) one or more antioxidants (e.g. butylated
hydroxyanisole), e.g. present in total at 0.001% to 2% w/w such as
0.02% to 2% w/w; and/or (b) one or more preservatives, e.g. present
in total at 0.01% to 4% w/w such as 0.05% to 1% w/w (e.g.
methylparaben present at 0.05% to 2% w/w and/or propylparaben
present at 0.01% to 2% w/w).
[0531] The above example ointment composition, including the oil
"phase" and an optional penetration enhancer, can optionally be a
homogeneous single phase. However, in one embodiment of the above
example ointment composition, e.g. when using propylene glycol or
another hydrophilic solubiliser and penetration enhancer, the oil
phase (oily ointment base) and a hydrophilic phase containing the
hydrophilic solubiliser and penetration enhancer (e.g.
propylene-glycol-containing phase) have been emulsified to form an
ointment emulsion.
[0532] Ointment compositions having two phases can optionally be
prepared using an emulsification process whereby the hydrophilic
phase (e.g. propylene-glycol-containing phase) and oil phase are
first prepared in separate vessels. The hydrophilic phase can
optionally contain a penetration enhancer such as propylene glycol,
and optionally some or all of the compound of formula (I) or salt
thereof. The oil phase can optionally contain a surfactant.
Temperatures of both phases are maintained at elevated
temperatures, such as about 55-90.degree. C. or in particular from
above 70 to 90.degree. C., the oil phase temperature being
sufficiently high (e.g. from above 70 to 90.degree. C.) to melt the
oil phase. While hot, one phase is added to another while mixing,
e.g. using a high shear mixer, to effect emulsification, e.g.
keeping the temperature above 70.degree. C. such as from above 70
to 90.degree. C. The resulting ointment emulsion is allowed to
cool, e.g. to about 15-35.degree. C. such as to about 18-30.degree.
C., in particular while the agitation continues e.g. at lower
speeds. The ointment emulsion can then optionally be dispensed from
the manufacturing vessel and filled into primary packaging, for
example tubes or sachets.
[0533] Optionally, an ointment can comprise a polyethylene glycol
base, e.g. present at 25% to 99% w/w such as 50% to 98% w/w,
instead of or as well as an oily ointment base.
[0534] Creams: An external-topical pharmaceutical composition can
be a cream, e.g. a water-in-oil cream or an oil-in-water cream.
Creams can sometimes be more fluid than ointments, can sometimes
provide more moisture, and hence may in principle in certain cases
allow for improved and/or good efficacy in patients with atopic
dermatitis.
[0535] Water-in-oil creams: These usually have an increased aqueous
content compared to ointments. In particular the water-in-oil cream
can be a water-in-oil cream emulsion. That is, in particular, in
the water-in-oil cream, an oil phase and an aqueous phase can have
been emulsified to form a water-in-oil cream emulsion.
[0536] As an example, an external-topical pharmaceutical
composition can be a water-in-oil cream (e.g. cream emulsion)
comprising: [0537] the compound of formula (I) or pharmaceutically
acceptable salt thereof present at 0.1% to 10% w/w (e.g. 0.2% to 5%
w/w, or 0.5% to 5% w/w, or 0.5% to 3% w/w); [0538] an oil phase
(oily ointment base) present at 25% to 85% w/w or 35% to 70% w/w
[for example: comprising white petrolatum present at 25% to 75% w/w
or 30% to 65% w/w (i.e. by weight of the composition), and/or
comprising mineral oil (e.g. as solubiliser and emollient) present
at 2.5% to 20% w/w or 4% to 15% w/w (i.e. by weight of the
composition)]; [0539] water present in 2% to 30% w/w, e.g. 5% to
25% or 10% to 22% w/w; [0540] one or more surfactants (e.g.
polyoxyl stearyl ether) present in total at 0.5% to 12% w/w, such
as 3% to 10% w/w; and [0541] in particular, one or more agents
acting as a skin-penetration enhancer (e.g. acting as both a
solubiliser and skin-penetration enhancer and/or e.g. hydrophilic
such as propylene glycol) present in total at 0.5% to 50% w/w, such
as 5% to 50% w/w or 7% to 30% w/w; and [0542] optionally, one or
more antioxidants (e.g. butylated hydroxyanisole), e.g. present in
total at 0.001 to 2% w/w such as 0.02 to 2% w/w; and [0543]
optionally, one or more preservatives, e.g. present in total at
0.01 to 4% w/w such as 0.05 to 1% w/w (e.g. methylparaben present
at 0.05 to 2% w/w and/or propylparaben present at 0.01 to 2%
w/w).
[0544] Oil-in-water creams: These usually have an increased aqueous
content compared to ointments and water-in-oil creams. In
particular, the oil-in-water cream can be an oil-in-water cream
emulsion. That is, preferably, in the oil-in-water cream, an oil
phase and an aqueous phase have been emulsified to form an
oil-in-water cream emulsion.
[0545] Examples of oil-in-water creams are high-occlusion creams,
wherein, after topical administration to the skin, moisture loss
from the skin and/or from the cream is reduced or limited by means
of sufficiently high coverage of the skin and/or by providing a
sufficient barrier at the site of application.
[0546] In particular, the oil-in-water cream can contain one or
more emollients (hydrating agents), such as silicones (e.g.
dimethicone, e.g. dimethicone 360 or dimethicone 20), a
high-viscosity wax such as microcrystalline wax, and/or mineral
oil. A sufficiently high water content is also preferred, for
example wherein the water is present in 15% to 60% w/w, 20% to 50%
w/w, or 25% to 40% w/w.
[0547] As an example, an external-topical pharmaceutical
composition can be an oil-in-water cream (e.g. cream emulsion)
comprising: [0548] the compound of formula (I) or pharmaceutically
acceptable salt thereof present at 0.1% to 10% w/w (e.g. 0.2% to 5%
w/w, or 0.5% to 5% w/w, or 0.5% to 3% w/w); [0549] an oil phase
(oily ointment base), in particular containing one or more
ingredients capable of acting as emollients, the oil phase being
present at 5% to 60% w/w or 8% to 55% w/w or in particular 10% to
45% w/w or 12% to 30% w/w; [0550] water present in 7% to 75% w/w or
7% to 60% w/w or 10% to 60% w/w, in particular 15% to 50% w/w or
20% to 40% w/w; [0551] one or more surfactants present in total at
0.5% to 20% w/w, e.g. 3% to 15% w/w or 3% to 10% w/w; and [0552]
preferably, one or more agents acting as a skin-penetration
enhancer (e.g. acting as both a solubiliser and skin-penetration
enhancer and/or e.g. hydrophilic such as propylene glycol) present
in total at 0.5% to 50% w/w, preferably 5% to 50% w/w or 7% to 25%
w/w; and [0553] optionally, one or more solubilisers (e.g.
isopropyl myristate), e.g. present at 0.5% to 20% w/w, e.g. 3 to
12% w/w; and [0554] optionally, one or more buffers (e.g. citric
acid and/or dibasic sodium phosphate), e.g. present in total at
0.05 to 5% w/w.
[0555] In the above example oil-in-water cream composition, the oil
phase in particular can comprise mineral oil (e.g. as emollient and
solubiliser) present at 15% to 50% w/w or 20% to 45% w/w (i.e. by
weight of the composition), and/or comprises a high-viscosity wax
such as microcrystalline wax (e.g. as emollient) present at 5% to
25% w/w such as 8% to 15% w/w, and/or comprises a silicone (such as
dimethicone e.g. dimethicone 360 or dimethicone 20, e.g. as
emollient) present at 0.5% to 20% such as 0.5% to 10% or 1% to 5%
w/w.
[0556] In the above example oil-in-water cream composition, the one
or more surfactants can in particular comprise: glycerol
monostearate present at 0.5% to 10% w/w, and/or sorbitan
monostearate present at 0.05% to 10% w/w, and/or [cetyl alcohol
and/or stearyl alcohol] present in total at 0.1% to 15% or 1 to 10%
w/w.
[0557] Cream emulsions, e.g. water-in-oil or oil-in-water cream
emulsions, can be prepared by a process in which an aqueous phase
is prepared, e.g. prepared before emulsification. The aqueous phase
usually contains water and a solubiliser and/or skin-penetration
enhancer such as propylene glycol, and optionally contains some or
all of the compound of formula (I) or salt thereof, and/or
optionally contains surfactant. The oil phase, e.g. containing
white petrolatum and/or mineral oil, and/or optionally containing
surfactant, can be prepared in a separate vessel. Temperatures of
both phases are maintained at elevated temperatures, such as about
55-90.degree. C. or in particular from above 70 to 90.degree. C.,
the oil phase temperature being sufficiently high (e.g. from above
70 to 90.degree. C.) to melt the oil phase. While hot, one phase is
added to another while mixing, e.g. using a high shear mixer, to
effect emulsification, in particular keeping the temperature above
70.degree. C. such as from above 70 to 90.degree. C. The resulting
emulsion is allowed to cool, e.g. to about 15-35.degree. C. such as
to about 18-30.degree. C., in particular while the agitation
continues e.g. at lower speeds. The cream emulsion can then
optionally be dispensed from the manufacturing vessel and filled
into primary packaging, for example tubes or sachets.
[0558] Typically, a pharmaceutical composition of the invention
suitable for external topical administration can be administered
once daily, twice daily or more than twice daily, to external body
part(s), e.g. on the skin such as at a site of diseased skin, e.g.
skin suffering from atopic dermatitis.
Pharmaceutical Compositions Suitable for Parenteral or Oral
Administration
[0559] A pharmaceutical composition suitable for (e.g. adapted for)
parenteral (e.g. intravenous, subcutaneous, or intramuscular)
administration can comprise a solution or suspension of the
compound or pharmaceutically acceptable salt in a sterile
pharmaceutically and parenterally acceptable aqueous liquid carrier
(e.g. sterile water or a sterile aqueous solution) or in a
parenterally acceptable oil. Alternatively, an aqueous solution can
be lyophilised to prepare the parenteral composition. A lyophilised
pharmaceutical composition suitable for (e.g. adapted for)
parenteral administration may, in use, optionally be reconstituted
with a suitable solvent, e.g. sterile water or a sterile
parenterally acceptable aqueous solution, just prior to
administration. A pharmaceutical composition suitable for (e.g.
adapted for) parenteral administration may optionally comprise a
preservative.
[0560] As mentioned above, oral administration is generally not
thought to be a preferred route of administration. However, in the
event that oral administration is to be used, a pharmaceutical
composition suitable for oral administration can be liquid or
solid; for example it can be a syrup, suspension or emulsion, a
tablet, a capsule or a lozenge.
[0561] A liquid formulation (e.g. oral) can generally consist of a
suspension or solution of the compound or pharmaceutically
acceptable salt in a suitable pharmaceutically acceptable liquid
carrier(s), for example an aqueous solvent such as water, aqueous
ethanol or aqueous glycerine, or a non-aqueous solvent, such as
polyethylene glycol or an oil. The formulation may also contain a
suspending agent, preservative, flavouring and/or colouring
agent.
[0562] In one embodiment, the pharmaceutical composition is in unit
dose form, such as a tablet or capsule for oral administration,
e.g. for oral administration to a human.
[0563] A pharmaceutical composition suitable for oral
administration being a tablet can comprise one or more
pharmaceutically acceptable carriers and/or excipients suitable for
preparing tablet formulations. The carrier can for example be or
include lactose, cellulose (for example microcrystalline
cellulose), or mannitol. The tablet can also or instead contain one
or more pharmaceutically acceptable excipients, for example a
binding agent such as hydroxypropylmethylcellulose or povidone
(polyvinylpyrrolidone), a lubricant e.g. an alkaline earth metal
stearate such as magnesium stearate, and/or a tablet disintegrant
such as sodium starch glycollate, croscarmellose sodium, or
crospovidone (cross-linked polyvinylpyrrolidone). The
pharmaceutical composition being a tablet can be prepared by a
method comprising the steps of: (i) mixing the compound of formula
(I), as herein defined, or a pharmaceutically acceptable salt
thereof, with the one or more pharmaceutically acceptable carriers
and/or excipients, (ii) compressing the resulting mixture (which is
usually in powder form) into tablets, and (iii) optionally coating
the tablet with a tablet film-coating material.
[0564] A pharmaceutical composition suitable for oral
administration being a capsule can be prepared using encapsulation
procedures. For example, pellets or powder containing the active
ingredient can be prepared using a suitable pharmaceutically
acceptable carrier and then filled into a hard gelatin capsule.
Alternatively, a dispersion or suspension can be prepared using any
suitable pharmaceutically acceptable carrier, for example an
aqueous gum or an oil and the dispersion or suspension then filled
into a soft gelatin capsule.
Dosage Regimens
[0565] The pharmaceutical composition can optionally be in unit
dose form. The unit dose form can for example be:
(a) a rupturable or peel-openable sealed dose container containing
a dry powder inhalable pharmaceutical composition (e.g. a plurality
of which are usually disposed inside a suitable inhalation device);
(b) a vial, ampoule or filled syringe for parenteral administration
e.g. comprising a solution or suspension of the compound or
pharmaceutically acceptable salt in a suitable carrier such as an
aqueous carrier or e.g. containing a lyophilised parenteral
pharmaceutical composition (the vial or ampoule can optionally be
manufactured using a blow-fill-seal process); or (c) a tablet or
capsule for oral administration e.g. for oral administration to a
human.
[0566] In the pharmaceutical composition of the invention, a or
each dosage unit for inhaled or intranasal administration can for
example contain from 0.005 to 10 mg, such as 0.005 to 7.5 mg (e.g.
0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg or 1 mg), for example 0.02 to 2 mg
(e.g. 0.05 mg, 0.1 mg, 0.25 mg, 0.4 mg, 0.5 mg, 0.875 mg or 1 mg)
or 0.05 to 2 mg (e.g. 0.05 mg, 0.1 mg, 0.25 mg, 0.4 mg, 0.5 mg,
0.875 mg or 1 mg), of a compound (e.g. of formula (I)) or a
pharmaceutically acceptable salt thereof, calculated as the free
base. A or each dosage unit for oral or parenteral administration
can for example contain from 0.02 to 1000 mg, such as 0.2 to 350
mg, of a compound (e.g. of formula (I)) or a pharmaceutically
acceptable salt thereof, calculated as the free base.
[0567] When an inhalable or intranasal composition is used, a
pharmaceutically acceptable compound or salt of the invention can
for example be administered to a mammal (e.g. human) in a daily
inhaled or intranasal dose of: 0.0003 to 0.6 mg/kg body weight/day
or 0.0007 to 0.6 mg/kg body weight/day or 0.0007 to 0.1 mg/kg body
weight/day, e.g. 0.0007 to 0.015 mg/kg/day or 0.004 to 0.1
mg/kg/day, of the compound (e.g. of formula (I)) or a
pharmaceutically acceptable salt thereof, calculated as the free
base.
[0568] A compound, e.g. of formula (I), or a pharmaceutically
acceptable salt thereof of the invention can, for example, be
administered to a human in a total daily inhaled or intranasal dose
of: 0.005 to 10 mg per day, or 0.02 to 7.5 mg per day, or 0.05 to
7.5 mg per day, or 0.05 to 4 mg per day (e.g. 1 mg per day), or
0.25 to 2 mg per day, of the compound (e.g. of formula (I)) or a
pharmaceutically acceptable salt thereof, calculated as the free
base. These total daily doses can be administered as a single dose
once daily, or can represent the summation of two or more separate
doses administered at different times of the day (e.g. two doses
per day administered every ca. 12 hours). These total daily doses
can e.g. be for administration to an adult human e.g. of 50-120 kg
or 60-100 kg body weight.
[0569] For example, a human inhaled or intranasal dosage regimen of
0.05 to 2 mg (e.g. 0.05 mg, 0.1 mg, 0.4 mg, 0.5 mg, 0.875 mg or 1
mg) of the compound or the salt thereof once or twice per day, or
0.2 to 2 mg (e.g. 0.4 mg, 0.5 mg, 0.875 mg or 1 mg) once or twice
per day, calculated as the free base, can optionally be
administered to a human, for example in the treatment and/or
prophylaxis of COPD, asthma or rhinitis (e.g. allergic or
non-allergic rhinitis) in the human.
[0570] When a parenteral or oral composition is used, a
pharmaceutically acceptable compound or salt of the invention is
optionally, for example, administered to a mammal (e.g. human) in a
daily parenteral or oral dose of 0.0003 mg to 15 mg per kg body
weight per day (mg/kg/day), for example 0.003 to 5 mg/kg/day, of
the compound (e.g. of formula (I)) or a pharmaceutically acceptable
salt thereof, calculated as the free base.
[0571] A compound, e.g. of formula (I), or pharmaceutically
acceptable salt thereof of the invention is optionally, for
example, administered to a human (e.g. adult human) in a total
daily parenteral or oral dose of 0.02 mg to 1000 mg per day or 0.2
to 350 mg per day of the compound (e.g. of formula (I)) or a
pharmaceutically acceptable salt thereof, calculated as the free
base.
[0572] In a pharmaceutical composition suitable for (e.g. adapted
for) external topical administration, e.g. an ointment or an
oil-in-water or water-in-oil composition, the compound of formula
(I) or the pharmaceutically acceptable salt thereof can be present
in 0.1% to 10%, such as 0.2% to 5%, or 0.5% to 5%, or 0.5% to 3%,
by weight of the composition (w/w). Typically, an external-topical
pharmaceutical composition can be administered once daily, twice
daily or more than twice daily, to external body part(s), e.g. to
the skin such as at a site of diseased skin. The amount
administered is usually such as substantially to cover the site(s)
of diseased skin.
Combinations
[0573] The compounds, salts and/or pharmaceutical compositions
according to the invention may also be used in combination with
another therapeutically active agent, for example, a .beta..sub.2
adrenoreceptor agonist, an anticholinergic compound (e.g.
muscarinic (M) receptor antagonist), an anti-histamine, an
anti-allergic, an anti-inflammatory agent, an antiinfective agent
or an immunosuppressant.
[0574] The invention thus provides, in a further aspect, a
combination comprising a compound of formula (I) or a
pharmaceutically acceptable salt thereof together with another
therapeutically active agent, for example, a muscarinic (M)
receptor antagonist, a .beta..sub.2-adrenoreceptor agonist (beta-2
adrenoreceptor agonist), an anti-histamine, an anti-allergic, an
anti-inflammatory agent, an antiinfective agent or an
immunosuppressant.
[0575] The invention also provides, in a further preferred aspect,
a combination comprising a compound of formula (I) or a
pharmaceutically acceptable salt thereof together with a muscarinic
(M) receptor antagonist.
[0576] The muscarinic (M) receptor antagonist can be an M.sub.1,
M.sub.2, M.sub.1/M.sub.2, or M.sub.3 receptor antagonist, such as a
M.sub.3 receptor antagonist, in particular a M.sub.3 receptor
antagonist which selectively antagonises (e.g. antagonises 10 times
or more strongly) the M.sub.3 receptor over the M.sub.1 and/or
M.sub.2 receptor.
[0577] For combinations of a muscarinic (M) receptor antagonist
with PDE4 inhibitors, see for example WO 03/011274 A2 and WO
02/069945 A2/US 2002/0193393 A1 and US 2002/052312 A1, and some or
all of these publications give examples of anticholinergic
compounds/muscarinic (M) receptor antagonists which may be used
with the compounds of formula (I) or salts, and/or suitable
pharmaceutical compositions. For example, the muscarinic receptor
antagonist can comprise or be an ipratropium salt (e.g. ipratropium
bromide), an oxitropium salt (e.g. oxitropium bromide), or more
preferably a tiotropium salt (e.g. tiotropium bromide); see e.g. EP
418 716 A1 for tiotropium.
[0578] Muscarinic antagonists which can be optionally used in the
combination of the present invention include a compound (including
a pharmaceutically acceptable salt thereof) defined by claim 1, 2,
3 or 4 of WO 2005/037280 A1. These compounds are stated or implied
as being muscarinic (e.g. M.sub.3) acetylcholine receptor
antagonists.
[0579] Muscarinic antagonists which can be optionally used in the
combination of the present invention include those disclosed in WO
2005/037280, WO 2005/046586 and WO 2005/104745. The muscarinic
antagonist can be: [0580]
(3-endo)-3-(2,2-di-2-thienylethenyl)-8,8-dimethyl-8-azoniabicyclo[3.2.]oc-
tane iodide; [0581]
(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1-
]octane bromide; [0582]
4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicycl-
o[2.2.2]octane bromide; or [0583]
(1R,5S)-3-(2-cyano-2,2-diphenylethyl)-8-methyl-8-{2-[(phenylmethyl)oxy]et-
hyl}8-azoniabicyclo[3.2.1]octane bromide; or a salt of any of the
foregoing.
[0584] The muscarinic (M) receptor antagonist, e.g. M.sub.3
receptor antagonist, is preferably for inhaled administration, more
preferably in particle-size-reduced form e.g. as defined herein.
More preferably, both the muscarinic (M) receptor antagonist and
the compound of formula (I) or the pharmaceutically acceptable salt
thereof are for inhaled administration. Suitably, the muscarinic
receptor antagonist and the compound of formula (I) or salt are for
simultaneous administration. The muscarinic receptor antagonist
combination is preferably for treatment and/or prophylaxis of
COPD.
[0585] In one embodiment, the combination includes a
.beta..sub.2-adrenoreceptor agonist (beta-2 adrenoreceptor agonist)
being salmeterol (e.g. as racemate or a single enantiomer such as
the R-enantiomer), salbutamol, formoterol, salmefamol, fenoterol,
carmoterol, indacaterol or terbutaline, or a salt thereof (e.g.
pharmaceutically acceptable salt thereof), for example the
xinafoate salt of salnieterol, the sulphate salt or free base of
salbutamol or the fumarate salt of formoterol. Long-acting
.beta.2-adrenoreceptor agonists are preferred, especially those
having a therapeutic effect over a 12-24 hour period such as
salmeterol or formoterol. Preferably, the .beta.2-adrenoreceptor
agonist is for inhaled administration, e.g. once per day and/or for
simultaneous inhaled administration; and more preferably the
.beta.2-adrenoreceptor agonist is in particle-size-reduced form
e.g. as defined herein. Preferably, the .beta..sub.2-adrenoreceptor
agonist combination is for treatment and/or prophylaxis of COPD or
asthma. Salmeterol or a pharmaceutically acceptable salt thereof,
e.g. salmeterol xinofoate, can be administered to humans at an
inhaled dose of 25 to 50 micrograms twice per day (measured as the
free base).
[0586] Examples of .beta..sub.2-adrenoreceptor agonists e.g. long
acting .beta..sub.2-adrenoreceptor agonists for use in the
combination include those described in WO 02/066422A, WO 03/024439,
WO 02/070490, WO 02/076933, WO 03/072539, WO 03/091204, WO
2004/016578, WO2004/022547, WO 2004/037807, WO 2004/037773, WO
2004/037768, WO 2004/039762, and WO 2004/039766.
[0587] In particular, long-acting .beta..sub.2-adrenoreceptor
agonists (beta-2 adrenoreceptor agonists) can include compounds of
formula (XX) (described in WO 02/066422);
##STR00101##
or a salt or solvate thereof, wherein in formula (XX): m.sup.X is
an integer of from 2 to 8; n.sup.X is an integer of from 3 to 11,
with the proviso that m.sup.X+n.sup.X is 5 to 19, R.sup.11X is
--XSO.sub.2NR.sup.16XR.sup.17X wherein X is
--(CH.sub.2).sub.p.sup.X-- or C.sub.2-6 alkenylene; R.sup.16X and
R.sup.17X are independently selected from hydrogen, C.sub.1-6alkyl,
C.sub.3-7cycloalkyl, C(O)NR.sup.18XR.sup.19X, phenyl, and phenyl
(C.sub.1-4alkyl)-, or R.sup.16X and R.sup.17X, together with the
nitrogen to which they are bonded, form a 5-, 6-, or 7-membered
nitrogen containing ring, and R.sup.16X and R.sup.17X are each
optionally substituted by one or two groups selected from halo,
C.sub.1-6alkyl, C.sub.1-6haloalkyl, C.sub.1-6alkoxy,
hydroxy-substituted C.sub.1-6alkoxy, --CO.sub.2R.sup.18X,
--SO.sub.2NR.sup.18XR.sup.19X, --CONR.sup.18XR.sup.19X,
--NR.sup.1XC(O)R.sup.19X, or a 5-, 6- or 7-membered heterocylic
ring; R.sup.18X and R.sup.19X are independently selected from
hydrogen, C.sub.1-8alkyl, C.sub.3-6cycloalkyl, phenyl, and phenyl
(C.sub.1-4alkyl)-; and p.sup.X is an integer of from 0 to 6,
preferably from 0 to 4; R.sup.12X and R.sup.13X are independently
selected from hydrogen, C.sub.1-6alkyl, C.sub.1-6alkoxy, halo,
phenyl, and C.sub.1-6haloalkyl; and R.sup.14X and R.sup.15X are
independently selected from hydrogen and C.sub.1-4alkyl with the
proviso that the total number of carbon atoms in R.sup.14X and
R.sup.15X is not more than 4.
[0588] Suitable .beta..sub.2-adrenoreceptor agonists disclosed in
WO 02/066422 include: [0589]
3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}ami-
no)hexyl]oxy}butyl)benzenesulfonamide, or [0590]
3-(3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-hydroxymethyl)phenyl]ethyl}amino-
)heptyl]oxy}propyl)benzenesulfonamide; or a salt thereof.
[0591] A preferred .beta..sub.2-adrenoreceptor agonist disclosed in
WO 03/024439 is: [0592]
4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyet-
hyl}2-(hydroxymethyl)phenol or a salt thereof. Suitably, this can
be for inhaled administration.
[0593] Another example of .beta.2-adrenoreceptor agonist disclosed
in WO 2004/037773 is: [0594]
4-{(1R)-2-[(6-{4-[3-(cyclopentylsulfonyl)phenyl]butoxy}hexyl)amino]-1-hyd-
roxyethyl}-2-(hydroxymethyl)phenol or a salt thereof.
[0595] An anti-histamine usable in a combination of a compound of
formula (I) or salt can for example be for oral administration
(e.g. this can be as a separately-administrable tablet), and can be
for treatment and/or prophylaxis of allergic rhinitis. Examples of
anti-histamines for oral administration include methapyrilene, or
H.sub.1 antagonists such as cetirizine, loratadine (e.g.
Clarityn.TM.), desloratadine (e.g. Clarinex.TM.) or fexofenadine
(e.g. Allegra.TM.).
[0596] An anti-histamine usable in a combination of a compound of
formula (I) or salt can for example be for intranasal
administration. An anti-histamine for intranasal administration can
e.g. be azelastine or a salt thereof (e.g. azelastine
hydrochloride, e.g. 0.1% w/v aqueous solution), or levocabastine or
a salt thereof (e.g. levocabastine hydrochloride). The
anti-histamine olopatadine (e.g. as olopatadine HCl) can be used
e.g. as eye drops.
[0597] Other possible combinations include, for example, a
combination comprising a compound of formula (I) or a
pharmaceutically acceptable salt thereof together with another
anti-inflammatory agent such as an anti-inflammatory
corticosteroid; or a non-steroidal anti-inflammatory drug (NSAID)
such as a leukotriene antagonist (e.g. montelukast), an iNOS
inhibitor, a tryptase inhibitor, a elastase inhibitor, a beta-2
integrin antagonist, a adenosine 2a agonist, or a 5-lipoxogenase
inhibitor; or an antiinfective agent (e.g. an antibiotic or an
antiviral). An iNOS inhibitor is optionally for oral
administration. Examples of iNOS inhibitors (inducible nitric oxide
synthase inhibitors) include those disclosed in WO 93/13055, WO
98/30537, WO 02/50021, WO 95/34534 and WO 99/62875.
[0598] In a combination comprising a compound of formula (I) or a
pharmaceutically acceptable salt thereof together with an
anti-inflammatory corticosteroid (which can for example be for
treatment and/or prophylaxis of asthma, COPD, rhinitis e.g.
allergic rhinitis, psoriasis or atopic dermatitis), then the
anti-inflammatory corticosteroid can be fluticasone propionate
(e.g. see U.S. Pat. No. 4,335,121), beclomethasone 17-propionate
ester, beclomethasone 17,21-dipropionate ester, dexamethasone or an
ester thereof, mometasone or an ester thereof (e.g. mometasone
furoate), betamethasone valerate (for external topical
administration), clobetasol propionate (for external topical
administration), ciclesonide, budesonide, flunisolide, or a
compound as described in WO 02/12266 A1 (e.g. as claimed in any of
claims 1 to 22 therein), or a pharmaceutically acceptable salt of
any of the above. If the anti-inflammatory corticosteroid is a
compound as described in WO 02/12266 A1, then it can be Example 1
therein {which is
6.alpha.,9.alpha.-difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hy-
droxy-16.alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic
acid S-fluoromethyl ester} or Example 41 therein {which is
6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.-methyl-17.alpha.-[(-
4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17.beta.-ca-
rbothioic acid S-fluoromethyl ester}, or a pharmaceutically
acceptable salt thereof. The anti-inflammatory corticosteroid can
for example be for inhaled, intranasal or external topical
administration. Fluticasone propionate can be used and is
preferably for inhaled administration to a human either (a) at a
dose of 250 micrograms once per day or (b) at a dose of 50 to 250
micrograms twice per day. In a combination comprising betamethasone
valerate for external topical administration, the betamethasone
valerate can be present at from about 0.025% to about 0.1% w/w in
an externally-topically-administrable composition such as a cream
or ointment. In a combination comprising clobetasol propionate for
external topical administration, the clobetasol propionate can be
present at about 0.0525% w/w or about 0.05% w/w in an
externally-topically-administrable composition such as a cream or
ointment.
[0599] Also provided is a combination comprising a compound of
formula (I) or a pharmaceutically acceptable salt thereof together
with .beta..sub.2-adrenoreceptor agonist and an anti-inflammatory
corticosteroid, for example as described in WO 03/030939 A1.
Preferably this combination is for treatment and/or prophylaxis of
asthma, COPD or allergic rhinitis. The .beta..sub.2-adrenoreceptor
agonist and/or the anti-inflammatory corticosteroid can be as
described above and/or as described in WO 03/030939 A1. In this
"triple" combination, the .beta.2-adrenoreceptor agonist can for
example be salmeterol or a pharmaceutically acceptable salt thereof
(e.g. salmeterol xinafoate), and the anti-inflammatory
corticosteroid can for example be fluticasone propionate.
[0600] Other examples of combinations, in particular for external
topical administration (e.g. versus atopic dermatitis), include,
for example, a combination comprising a compound of formula (I) or
a pharmaceutically acceptable salt thereof together with an
immunosuppressant, e.g. a calcineurin inhibitor such as
pimecrolimus or tacrolimus. The immunosuppressant can in particular
be an externally-topically administrable immunosuppressant such as
pimecrolimus (e.g. pimecrolimus at ca. 1% w/w concentration in a
topical composition such as a cream, and/or e.g. Elidel.TM.) or
tacrolimus (e.g. tacrolimus at from about 0.03% to about 0.1% w/w
concentration in a topical composition such as an ointment, and/or
e.g. Protopic M). The externally-topically administrable
immunosuppressant can be administered or administrable in a
external-topical composition separately from the compound or salt
of the invention, or it can be contained with the compound of
formula (I) or pharmaceutically acceptable salt in a combined
externally-topically-administrable composition.
[0601] For external topical administration, e.g. versus an
inflammatory and/or allergic skin disease such as atopic dermatitis
or psoriasis, in a combination of the compound or salt of the
invention together with an anti-infective agent, the anti-infective
agent can include (e.g. be) an externally-topically-administrable
antibacterial, such as mupiricin or a salt thereof (e.g. mupirocin
calcium salt) (e.g. Bactroban.TM.) or such as an
externally-topically-administrable pleuromutilin antibacterial
(e.g. retapamulin or a salt thereof, which can be present in about
1% w/w by weight of an externally-topically-administrable
pharmaceutical composition, such as an ointment). Alternatively or
additionally, for external topical administration, the
anti-infective agent can include an
externally-topically-administrable antifungal such as clotrimazole
(e.g. at about 1% to about 10% w/w or at about 1% to about 2% w/w
in a topical composition), or ketoconazole, or terbinafine (e.g. as
HCl salt and/or at about 1% w/w).
[0602] For external topical administration, e.g. versus atopic
dermatitis, a combination with an anti-itch compound may optionally
be used.
[0603] The combinations referred to above may be presented for use
in the form of a pharmaceutical composition and thus a
pharmaceutical composition comprising a combination as defined
above together with one or more pharmaceutically acceptable
carriers and/or excipients represent a further aspect of the
invention.
[0604] The individual compounds of such combinations may be
administered either sequentially or simultaneously in separate or
combined pharmaceutical composition.
[0605] In one embodiment, the combination as defined herein can be
for simultaneous inhaled administration and is disposed in a
combination inhalation device. Such a combination inhalation device
is another aspect of the invention. Such a combination inhalation
device can comprise a combined pharmaceutical composition for
simultaneous inhaled administration (e.g. dry powder composition),
the composition comprising all the individual compounds of the
combination, and the composition being incorporated into a
plurality of sealed dose containers mounted longitudinally in a
strip or ribbon inside the inhalation device, the containers being
rupturable or peel-openable on demand; for example such inhalation
device can be substantially as described in GB 2,242,134 A
(DISKUS.TM.) and/or as described above. Alternatively, the
combination inhalation device can be such that the individual
compounds of the combination are administrable simultaneously but
are stored separately (or wholly or partly stored separately for
triple combinations), e.g. in separate pharmaceutical compositions,
for example as described in PCT/EP03/00598 filed on 22 Jan. 2003,
published as WO 03/061743 A1 (which discloses a medicament
dispenser for use with plural elongate form medicament carriers,
each having multiple distinct medicament dose portions carried
thereby, e.g. as described in the claims thereof e.g. claim 1)
and/or WO 2007/012871 A1 (which discloses a medicament dispenser
for use with at least one medicament carrier carrying multiple
distinct medicament portions, e.g. as described in the claims
thereof e.g. claim 1).
[0606] The invention also provides a method of preparing a
combination as defined herein, [0607] the method comprising either
[0608] (a) preparing a separate pharmaceutical composition for
administration of the individual compounds of the combination
either sequentially or simultaneously, or [0609] (b) preparing a
combined pharmaceutical composition for administration of the
individual compounds of the combination simultaneously, [0610]
wherein the pharmaceutical composition comprises the combination
together with one or more pharmaceutically acceptable carriers
and/or excipients.
[0611] The invention also provides a combination as defined herein,
prepared by a method as defined herein.
Biological Test Methods
PDE 3, PDE 4B, PDE 4D, PDE 5, PDE 6 in Vitro Assay Methods
[0612] The biological activity of the compounds or salts of the
invention can be measured in the assay methods shown below, or in
generally similar or generally analogous assay methods.
[0613] Some of the Examples disclosed herein and encompassed within
the invention are selective PDE4 inhibitors, i.e. they inhibit PDE4
(e.g. PDE4B) more strongly than they inhibit PDE3 and/or more
strongly than they inhibit PDE5 and/or more strongly than they
inhibit PDE6. It is to be recognised that such selectivity is not
essential to the invention.
Possible PDE Enzyme Sources and Literature References
[0614] Human recombinant PDE4B, in particular the 2B splice variant
thereof (HSPDE4B2B), is disclosed in WO 94/20079 and also M. M.
McLaughlin et al., "A low Km, rolipram-sensitive, cAMP-specific
phosphodiesterase from human brain: cloning and expression of cDNA,
biochemical characterisation of recombinant protein, and tissue
distribution of mRNA", J. Biol. Chem., 1993, 268, 6470-6476. For
example, in Example 1 of WO 94/20079, human recombinant PDE4B is
described as being expressed in the PDE-deficient yeast
Saccharomyces cerevisiae strain GL62, e.g. after induction by
addition of 150 uM CuSO.sub.4, and 100,000.times.g supernatant
fractions of yeast cell lysates are described for use in the
harvesting of PDE4B enzyme.
[0615] Human recombinant PDE4D (HSPDE4D3A) is disclosed in P. A.
Baecker et al., "Isolation of a cDNA encoding a human
rolipram-sensitive cyclic AMP phoshodiesterase (PDE IVD)", Gene,
1994, 138, 253-256.
[0616] Human recombinant PDE5 is disclosed in K. Loughney et al.,
"Isolation and characterisation of cDNAs encoding PDE5A, a human
cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide
phosphodiesterase", Gene, 1998, 216, 139-147.
[0617] PDE3 can be purified from bovine aorta as described by H.
Coste and P. Grondin, "Characterisation of a novel potent and
specific inhibitor of type V phosphodiesterase", Biochem.
Pharmacol., 1995, 50, 1577-1585.
[0618] PDE6 can be purified from bovine retina as described by: P.
Catty and P. Deterre, "Activation and solubilization of the retinal
cGMP-specific phosphodiesterase by limited proteolysis", Eur. J.
Biochem., 1991, 199, 263-269; A. Tar et al. "Purification of bovine
retinal cGMP phosphodiesterase", Methods in Enzymology, 1994, 238,
3-12; and/or D. Srivastava et al. "Effects of magnesium on cyclic
GMP hydrolysis by the bovine retinal rod cyclic GMP
phosphodiesterase", Biochem. J, 1995, 308, 653-658.
Inhibition of PDE 3, PDE 4B, PDE 4D, PDE 5 or PDE 6 Activity:
Radioactive Scintillation Proximity Assay (SPA)
[0619] The ability of compounds to inhibit catalytic activity at
PDE4B or 4D (human recombinant), PDE3 (from bovine aorta), PDE5
(human recombinant) or PDE6 (from bovine retina) can optionally be
determined by Scintillation Proximity Assay (SPA) in a 96-well
format.
[0620] Test compounds (as a solution in DMSO, suitably about 2
microlitre (ul) volume of DMSO solution) are preincubated at
ambient temperature (room temperature, e.g. 19-23.degree. C.) in
Wallac Isoplates (code 1450-514) with PDE enzyme in 50 mM Tris-HCl
buffer pH 7.5, 8.3 mM MgCl.sub.2, 1.7 mM EGTA, 0.05% (w/v) bovine
serum albumin for 10-30 minutes (usually 30 minutes). The enzyme
concentration is adjusted so that no more than 20% hydrolysis of
the substrate defined below occurs in control wells without
compound, during the incubation. For the PDE3, PDE4B and PDE4D
assays, [5',8-.sup.3H]Adenosine 3',5'-cyclic phosphate (Amersham
Pharmacia Biotech, code TRK.559; or Amersham Biosciences UK Ltd,
Pollards Wood, Chalfont St Giles, Buckinghamshire HP8 4SP, UK) is
added to give 0.05uCi per well and about 10 nM final concentration.
For the PDE5 and PDE6 assays, [8.sup.-3H]Guanosine 3',5'-cyclic
phosphate (Amersham Pharmacia Biotech, code TRK.392) is added to
give 0.05uCi per well and about 36 nM final concentration. Plates
containing assay mixture, suitably approx. 100 ul volume of assay
mixture, are mixed on an orbital shaker for 5 minutes and incubated
at ambient temperature for 1 hour. Phosphodiesterase SPA beads
(Amersham Pharmacia Biotech, code RPNQ 0150) are added (about 1 mg
per well) to terminate the assay. Plates are sealed and shaken and
allowed to stand at ambient temperature for 35 minutes to 1 hour
(suitably 35 minutes) to allow the beads to settle. Bound
radioactive product is measured using a WALLAC TRILUX 1450
Microbeta scintillation counter. For inhibition curves, 10
concentrations (e.g. 1.5 nM-30 uM) of each compound are assayed.
Curves are analysed using ActivityBase and XLfit (ID Business
Solutions Limited, 2 Ocean Court, Surrey Research Park, Guildford,
Surrey GU2 7QB, United Kingdom) Results are expressed as pIC.sub.50
values.
[0621] In an alternative to the above radioactive SPA assay, PDE4B
or PDE4D inhibition can be measured in the following Fluorescence
Polarisation (FP) assay:
Inhibition of PDE4B or PDE4D activity: Fluorescence Polarisation
(FP) assay
[0622] The ability of compounds to inhibit catalytic activity at
PDE4B (human recombinant) or PDE4D (human recombinant) can
optionally be determined by IMAP Fluorescence Polarisation (FP)
assay (IMAP Explorer kit, available from Molecular Devices
Corporation, Sunnydale, Calif., USA; Molecular Devices code: R8062)
in 384-well format.
[0623] The IMAP FP assay is able to measure PDE activity in an
homogenous, non-radioactive assay format. The FP assay uses the
ability of immobilised trivalent metal cations, coated onto
nanoparticles (tiny beads), to bind the phosphate group of Fl-AMP
that is produced on the hydrolysis of fluorescein-labelled (Fl)
cyclic adenosine mono-phosphate (Fl-cAMP) to the non-cyclic Fl-AMP
form. Fl-cAMP substantially does not bind. Binding of Fl-AMP
product to the beads (coated with the immobilised trivalent
cations) slows the rotation of the bound Fl-AMP and leads to an
increase in the fluorescence polarisation ratio of parallel to
perpendicular light. Inhibition of the PDE reduces/inhibits this
signal increase.
[0624] Test compounds (small volume, e.g. ca. 0.5 to 1 microlitres
(ul), suitably ca. 0.5 ul, of solution in DMSO) are preincubated at
ambient temperature (room temperature, e.g. 19-23.degree. C.) in
black 384-well microtitre plates (supplier: NUNC, code 262260) with
PDE enzyme in 10 mM Tris-HCl buffer pH 7.2, 10 mM MgCl.sub.2, 0.1%
(w/v) bovine serum albumin, and 0.05% NaN.sub.3 for 10-30 minutes.
The enzyme level is set by experimentation so that reaction is
linear throughout the incubation. Fluorescein adenosine
3',5'-cyclic phosphate (from Molecular Devices Corporation,
Molecular Devices code: R7091) is added to give about 40 nM final
concentration (final assay volume usually ca. 20-40 ul, suitably
ca. 20 ul). Plates are mixed on an orbital shaker for 10 seconds
and incubated at ambient temperature for 40 minutes. IMAP binding
reagent (as described above, from Molecular Devices Corporation,
Molecular Devices code: R7207) is added (60 ul of a 1 in 400
dilution in binding buffer of the kit stock solution) to terminate
the assay. Plates are allowed to stand at ambient temperature for 1
hour. The Fluorescence Polarisation (FP) ratio of parallel to
perpendicular light is measured using an Analyst.TM. plate reader
(from Molecular Devices Corporation). For inhibition curves, 10
concentrations (e.g. 1.5 nM-30 uM) of each compound are assayed.
Curves are analysed using ActivityBase and XLfit (ID Business
Solutions Limited, 2 Ocean Court, Surrey Research Park, Guildford,
Surrey GU2 7QB, United Kingdom). Results are expressed as
pIC.sub.50 values.
[0625] In the FP assay, reagents can be dispensed using
Multidrop.TM. (available from Thermo Labsystems Oy, Ratastie 2, PO
Box 100, Vantaa 01620, Finland).
[0626] For a given PDE4 inhibitor, the PDE4B (or PDE4D) inhibition
values measured using the SPA and FP assays can differ
slightly.
[0627] Biological Data obtained for some of the Examples (PDE4B
inhibitory activity, either as one reading (n=1) or as an average
of 2 or more readings (n=2 or more) are generally as follows, based
on measurements only, generally using SPA and/or FP assay(s)
generally as described above or generally similar or generally
analogous to those described above. In each of the SPA and FP
assays, absolute accuracy of measurement is not possible, and the
pIC.sub.50 readings given are generally thought to be accurate only
up to very approximately .+-.0.5 of a log unit, depending on the
number of readings made and averaged:
TABLE-US-00007 PDE4B pIC.sub.50 Example (generally in FP assay)
numbers (.+-.about 0.5) Example 1A1 about 10.3 (n = 8) Example 1B
about 10.2 (n = 2) Example 1C about 10.3 (n = 1) Example 9 about
10.3 (n = 1) Example 11 about 10.3 (n = 1) Example 12 about 10.5 (n
= 1) Example 17 about 9.8 (n = 1) Example 19 about 10.4 (n = 2)
Example 20 about 10.0 (n = 1) Example 21 about 10.2 (n = 2) Example
23 about 10.1 (n = 1) Example 23A about 10.0 (n = 1) Example 26
about 9.6 (n = 2) Example 31 about 10.1 (n = 1) Example 34 about
9.0 (n = 1)
[0628] A majority of the Examples, namely Examples 1A1,1B, 1C, 3,
4, 9, 11, 12, 17, 18, 19, 20, 21, 22, 23, 23A, 24, 25, 26, 30, 31,
32, 33 and 34, have been tested for PDE4B inhibition, mostly or all
using the FP assay generally as described above or a generally
similar or generally analogous assay. This tested majority of the
Examples generally have PDE4B inhibitory activities in the range of
pIC.sub.50=about 9.0 (about 0.5) to about 10.6 (about 0.5),
generally as measured using the FP assay generally as described
above or a generally similar or generally analogous assay.
[0629] Some or all of the PDE4B-tested Examples have also been
tested, for PDE3 and/or PDE5 inhibition using the above-described
assays or generally similar or generally analogous assays or other
assays (for example FP or SPA assays). The compound of Example 1B
and the salt of Example 1A1 each exhibit a larger PDE4B pIC.sub.50
value than any of the PDE3, PDE5 and PDE6 pIC.sub.50 values for the
same compound or salt. That is, the compound of Example 1B and the
salt of Example 1A1 each inhibit PDE4B more strongly than they
inhibit PDE3, PDE5 and PDE6 (as measured in whatever assays, e.g.
FP or SPA assays, happen to have been used).
[0630] The PDE4D pIC.sub.50 values of some compounds or salts of
the invention are as follows, as measured using an FP assay or a
generally similar assay: [0631] the salt of Example 1A1: PDE4D
pIC.sub.50=about 10.7 (about 0.5 or more) (n=8). [0632] the
compound of Example 1B: PDE4D pIC.sub.50=about 10.4 (about 0.5 or
more) (n=2). [0633] the salt of Example 9: PDE4D
pIC.sub.50>about 11.0 (.+-. about 0.5 or more) (n=1). [0634] the
salt of Example 19: PDE4D pIC.sub.50=about 10.7 (.+-.about 0.5 or
more) (n=2). [0635] the salt of Example 26: PDE4D pIC.sub.50=about
9.4 (.+-.about 0.5 or more) (n=2). [0636] the compound of Example
23A: PDE4D pIC.sub.50=about 10.5 (.+-. about 0.5 or more) (n=1).
Emesis: Some known PDE4 inhibitors can cause emesis and/or nausea
to greater or lesser extents, especially after systemic exposure
e.g. after oral administration (e.g. see Z. Huang et al., Current
Opinion in Chemical Biology, 2001, 5: 432-438, see especially pages
433-434 and refs cited therein). Therefore, it would be preferable,
but not essential, if a PDE4 inhibitory compound or salt of the
invention were to cause only limited or manageable emetic
side-effects, e.g. after inhaled or parenteral or external-topical
administration. Emetic side-effects can for example be measured by
the emetogenic potential of the compound or salt when administered
to ferrets or monkeys; for example the time to onset, extent,
frequency and/or duration of vomiting, retching and/or writhing in
ferrets or monkeys is optionally measured, after intratracheal or
parenteral or intraperitoneal (or oral) administration of the PDE4
inhibitor compound or salt. See for example A. Robichaud et al.,
"Emesis induced by inhibitors of [PDE IV] in the ferret",
Neuropharmacology, 1999, 38, 289-297, erratum Neuropharmacology,
2001, 40, 465-465.
[0637] Other side effects: Some known PDE4 inhibitors can cause
other side effects such as headache and/or other central nervous
system (CNS--) mediated side effects; and/or gastrointestinal (GI)
tract disturbances. Therefore, it would be preferable but not
essential if a particular PDE4 inhibitory compound or salt of the
invention were to cause only limited or manageable side-effects in
one or more of these side-effect categories.
Other In Vitro Assays:
[0638] Other assays which may also be optionally used to further
profile compounds or salts of the invention include the
following.
Inhibition of TNF-.alpha. (TNF-Alpha) Production in Human PBMC
(Peripheral Blood Mononuclear Cell) Assay (MSD technology)
[0639] This is an optional supplementary test, e.g. for potentially
inhalably-administrable PDE4 inhibitors.
[0640] A 96-well plate (96 MicroWell.TM. Plates
Nunclon.TM..DELTA.-High Flange Design, Fisher Scientific UK, Bishop
Meadow Road, Loughborough LE 11 5 RG, Leicestershire, UK) is
prepared by initially adding to column 1 ca. 10 mM of test compound
dissolved in DMSO. For a more potent compound, a more diluted
solution in DMSO may be used. The compound is further diluted with
DMSO into columns 2 to 9 by 8 successive 3-fold dilutions using the
Biomek.RTM. FX Laboratory Automation Workstation (Beckman Coulter,
Inc., 4300 N. Harbor Boulevard, P.O. Box 3100, Fullerton, Calif.
92834-3100, USA). Column 10 is used as a DMSO negative control
(High Signal, 0% response), whilst column 11, which contains 10 mM
of the PDE4 inhibitor roflumilast, is used as a positive control
(Low Signal, 100% response). About 1 .mu.l (about 1 ul) of compound
is transferred to the compound plate using the Biomek.RTM. FX.
[0641] PBMC cells (peripheral blood mononuclear cells) are prepared
from heparinised human blood (using 1% v/v Heparin Sodium 10001
U/ml Endotoxin Free, Leo Laboratories Ltd., Cashel Road, Dublin 12.
Ireland, Cat No: PL0043/0149) from normal volunteers using the
Accuspin.TM. System-Histopaque.RTM.-1077 essentially (Sigma-Aldrich
Company Ltd., The Old Brickyard New Rd, Gillingham, Dorset SP8 4XT,
UK). About 20 ml of blood is overlaid onto 15 ml Histopaque.RTM. in
Accuspin.TM. tubes. The tube is then centrifuged at about 800 g for
ca. 20 minutes. The cells are collected from the interface, washed
by centrifugation (ca. 1300 g, ca. 10 minutes) and resuspended in
RPMI1640 medium (Low endotoxin RPMI1640 medium, Cat No: 31870-025,
Invitrogen Corporation Invitrogen Ltd, 3 Fountain Drive, Inchinnan
Business Park, Paisley PA4 9RF, UK) containing 10% foetal calf
serum, 1% L-glutamine (Invitrogen Corporation, Cat No: 25030024)
and 1% penicillin/streptomycin (Invitrogen Corporation, Cat No:
15140-122). Viable cells are counted by trypan blue staining and
diluted to 1.times.10.sup.6 viable cells/ml. About 50 .mu.l (about
50 ul) of diluted cells and about 75 .mu.l (about 75 ul) of LPS
(ca. 1 ng/ml final; Sigma Cat No: L-6386) are added to the compound
plate, which is then incubated at 37.degree. C., 5% CO.sub.2, for
20 hours.
[0642] The supernatant is removed and the concentrations of
TNF-.alpha. are determined by electrochemiluminescence assay using
the Meso Scale Discovery (MSD) technology (Meso Scale Discovery,
9238 Gaither Road, Gaithersburg, Md. 20877, USA). See the
"TNF-.alpha. (TNF-alpha) MSD Assay" described below for typical
details.
[0643] Results can be expressed as pIC50 values for inhibition of
TNF-.alpha. (TNF-alpha) production in PBMCs, and it should be
appreciated that these results can be subject to a possibly-large
variability or error.
Inhibition of TNF-.alpha.(TNF-alpha) Production in Human PBMC
(Peripheral Blood Mononuclear Cell) Assay (IGEN Technology)
[0644] This is an optional supplementary test, e.g. for potentially
inhalably-administrable PDE4 inhibitors.
[0645] Test compounds are prepared as a ca. 10 mM stock solution in
DMSO and a dilution series prepared in DMSO with 8 successive
3-fold dilutions, either directly from the 10 mM stock solution or
from a more dilute solution in DMSO. The compound is added to assay
plates using a Biomek Fx liquid handling robot.
[0646] PBMC cells (peripheral blood mononuclear cells) are prepared
from heparinised human blood from normal volunteers by
centrifugation on histopaque at ca. 1000 g for ca. 30 minutes. The
cells are collected from the interface, washed by centrifugation
(ca. 1300 g, ca. 10 minutes) and resuspended in assay buffer (RPMI
1640 containing 10% foetal calf serum, 1% L-glutamine and 1%
penicillin/streptomycin) at 1.times.10.sup.6 cells/ml. Ca. 50 .mu.l
(ca. 50 ul) of cells are added to microtitre wells containing ca.
0.5 or ca. 1.0 .mu.l (ul) of an appropriately diluted compound
solution. Ca. 75 .mu.l (ul) of LPS (lipopolysaccharide) (ca. 1
ng/ml final) is added and the samples are incubated at 37.degree.
C., 5% CO.sub.2, for 20 hours. The supernatant is removed and the
concentrations of TNF-.alpha. are determined by
electrochemiluminescence assay using the IGEN technology or by
ELISA (see below).
[0647] Results can be expressed as pIC50 values for inhibition of
TNF-.alpha. (TNF-alpha) production in PBMCs, and it should be
appreciated that these results can be subject to a possibly-large
variability or error.
PBMC Assay Results:
[0648] For the following Examples, which are compounds of formula
(I) or salts thereof, and generally when using the one of the above
assays, or a generally similar or generally analogous assay, the
measured and/or mean pIC50 values for inhibition of TNF-.alpha.
(TNF-alpha) production in PBMCs are generally as follows (subject
to a possibly-large variability or error):
TABLE-US-00008 PBMC pIC.sub.50 Example (MSD assay) numbers (n = no.
of measurements) 1A1 about 9.3 (n = 85) 1B about 9.2 (n = 29) 9
about 8.4 (n = 4) 19 about 9.6 (n = 12) 23 about 9.2 (n = 4) 23A
about 9.1 (n = 8) 26 about 7.8 (n = 8)
Inhibition of TNF-.alpha. (TNF-alpha) Production in Human Whole
Blood
[0649] This is an optional supplementary test. For example, as the
assay may measure the effect of PDE4 inhibitors after loss by
protein binding, it might possibly be relevant to
externally-topically-administrable PDE4 inhibitors as
protein-binding-loss of compound is possible during transport
through the skin.
[0650] Test compounds are prepared as a ca. 10 mM stock solution in
DMSO and a dilution series prepared in DMSO with 8 successive
3-fold dilutions, either directly from the 10 mM stock solution or
from a more dilute solution in DMSO. The compound is added to assay
plates using a Biomek Fx liquid handling robot.
[0651] Heparinised blood drawn from normal volunteers is dispensed
(ca. 100 .mu.l=ca. 100 ul) into microtitre plate wells containing
ca. 0.5 or ca. 1.0 .mu.l (ul, microlitres) of an appropriately
diluted test compound solution. After ca. 1 hr incubation at ca.
37.degree. C., 5% CO.sub.2, ca. 25 .mu.l (ca. 25 ul) of LPS
(lipopolysaccharide) solution (S. typhosa) in RPMI 1640 (containing
1% L-glutamine and 1% Penicillin/streptomycin) is added (ca. 50
ng/ml final). The samples are incubated at ca. 37.degree. C., 5%
CO.sub.2, for ca. 20 hours, and ca. 100 .mu.l (ca. 100 ul)
physiological saline (0.138% NaCl) is added, and diluted plasma is
collected using a Platemate or Biomek FX liquid handling robot
after centrifugation at ca. 1300 g for ca. 10 min. Plasma
TNF-.alpha. content is determined by electrochemiluminescence assay
using the MSD technology (see below), the IGEN technology (see
below) or by enzyme linked immunosorbant assay (ELISA) (see
below).
[0652] Results can be expressed as plC50 values for inhibition of
TNF-.alpha. (TNF-alpha) production in Human Whole Blood, and it
should be appreciated that these results can be subject to a
possibly-large variability or error.
TNF-.alpha. (TNF-alpha) MSD Assay
[0653] Using the Biomek FX, 25 .mu.l (25 ul) of MSD Human Serum
Cytokine Assay Diluent (Meso Scale Discovery, 9238 Gaither Road,
Gaithersburg, Md. 20877) is added to a 96-well High-Bind MSD plate
pre-coated with anti-hTNF alpha capture antibody (MA6000) and then
incubated for 24 hours at 4.degree. C. to prevent non-specific
binding. About 20 .mu.l (ul) of supernatant from the PBMC plate or
about 40 .mu.l (ul) of supernatant from the whole blood (WB) plate
are then transferred from columns 1-11 to columns 1-11 of the MSD
plate using the Biomek FX. About 20 .mu.l (ul) of TNF-.alpha.
standard (Cat No. 210-TA; R&D Systems Inc., 614 McKinley Place
NE, Minneapolis, Minn. 55413, USA) are added to column 12 of the
MSD plate to generate a standard calibration curve (about 0 to
30000 pg/ml final).
[0654] For the Whole Blood assay, plates are washed after 2 hours
shaking with a Skanwasher 300 version B (Skatron Instruments AS. PO
Box 8, N-3401 Lier, Norway). About 40 .mu.l (ul) of diluted
sulfo-TAG antibody (ca. 1 pg/ml final) is added, the plates are
shaken at room temperature for 1 hours, and the plates washed again
as above. About 150 .mu.l (ul) of Read Buffer T (2.times.) is added
to the plates, which are then read on a MSD Sector 6000. For the
PBMC assay, about 20 .mu.l (ul) of diluted sulfo-TAG antibody (ca.
1 pg/ml final) is added to each well, and the plates/wells are
shaken at room temperature for 2 hours. Finally, about 90 .mu.l
(ul) of MSD Read Buffer P (diluted to 2.5 times with distilled
water) is added and the plates are read on a MSD Sector 6000.
Data Analysis
[0655] Data analysis is performed with ActivityBase/XC50 module (ID
Business Solutions Ltd., 2 Occam Court, Surrey Research Park,
Guildford, Surrey, GU2 7QB, UK). Data are normalized and expressed
as % inhibition using the formula
100*((U-C.sub.1)/(C.sub.2-C.sub.1)) where U is the unknown value,
C.sup.1 is the average of the high signal (0%) control wells
(column 10), and C2 is the average of the low signal (100%) control
wells (column 11). Curve fitting is performed with the following
equation: y=A+((B-A)/(1+(10 x/10 C) D)), where A is the minimum
response, B is the maximum response, C is the log10(IC50), and D is
the Hill slope. The results for each compound are recorded as pIC50
values (--C in the above equation).
TNF-.alpha. (TNF-alpha) IGEN Assay
[0656] Ca. 50 .mu.l supernatant from either whole blood or PBMC
assay plates is transferred to a 96 well polypropylene plate. Each
plate also contains a TNF-.alpha. standard curve (ca. 0 to 30000
pg/ml: R+D Systems, 210-TA). Ca. 50 .mu.l (ul) of
streptavidin/biotinylated anti-TNF-.alpha. antibody mix, ca. 25
.mu.l ruthenium tagged anti-TNF-.alpha. monoclonal and ca. 100pI
PBS containing 0.1% bovine serum albumin are added to each well and
the plates are sealed and shaken for ca. 2 hours before being read
on an IGEN instrument.
TNF-.alpha. (TNF-alpha) ELISA Assay (Enzyme Linked Immunosorbant
Assay)
[0657] Human TNF-.alpha. can be assayed using a commercial ELISA
assay kit (AMS Biotechnology, 211-90-164-40) according to the
manufacturers' instructions but with TNF-.alpha. calibration curves
prepared using Pharmingen TNF-.alpha. (cat. No. 555212).
In Vivo Biological Assays
[0658] The in vitro enzymatic PDE4B inhibition assay(s) described
above or generally similar or analogous assays should be regarded
as being the primary test(s) of biological activity. However, some
additional in vivo biological tests, which are optional only, and
which are not an essential measure of activity, efficacy or
side-effects, and which have not necessarily been carried out, are
described below.
In Vivo Assay 1. LPS Induced Pulmonary Neutrophilia in Rats: Effect
of Intratracheally Administered PDE4 Inhibitors
[0659] This assay is an animal model of acute inflammation in the
lung--specifically neutrophilia induced by lipopolysaccharide
(LPS)--and allows the study of putative inhibition of such
neutrophilia (anti-inflammatory effect) by an intratracheally
(i.t.) administered PDE4 inhibitor ("drug"). The PDE4 inhibitors
are suitably in dry powder, aqueous solution or aqueous suspension
form. I.t. administration is one model of inhaled administration,
allowing topical delivery to the lung.
[0660] Animals: Male CD (Sprague Dawley Derived) rats supplied by
Charles River, United Kingdom are housed in groups of 5 rats per
cage, acclimatised after delivery for at least 7 days with
bedding/nesting material regularly changed, fed on SDS diet R1
pelleted food given ad lib, and supplied with daily-changed
pasteurised animal grade drinking water.
[0661] Device for dry powder administration: For these studies, a
Penn Century DP-4 dry powder intratracheal (i.t.) delivery device
(insufflator) is used for administration of a dry powder blend of
vehicle (inhalation grade lactose) and the PDE4 inhibitor compound
or salt of the invention ("drug"). Each device is numbered and the
sample loading chamber is unscrewed from the main device and
weighed. The i.t. delivery needle is also weighed. The drug+lactose
blend or inhalation grade lactose (vehicle control) is then added
to the sample chamber and this is then re-weighed. The sample
chamber is fitted back on to the main device to prevent any drug
being lost. This procedure is repeated for all of the Penn century
devices to be used in the study (one separate device for each
animal). After dosing to the animals, the sample chambers and
needles are re-weighed in order to determine the amount of sample
that was expelled from the device.
[0662] Device for aqueous solution administration: A blunt dosing
needle, whose forward end is slightly angled to the needle axis, is
used, with a flexible plastic portex canula inserted into the
needle. The portex cannula is inserted into the lumen of the blunt
stainless steel dosing needle and this is carefully inserted into
the back of the animal's throat and into the trachea via the
larynx. A known volume (200 microlitres) of the vehicle (0.2% Tween
80.TM. in saline) or of the solution (or suspension) drug
composition is administered into the trachea using a plastic
syringe that is attached to the portex cannula and dosing needle. A
new internal cannula is used for each different drug group. After
dosing, the needle is removed from the trachea and the animals are
removed from the table and continually observed until they recover
from the effects of anaesthesia. The animals are then returned to
the holding cages and given free access to food and water, and are
observed and any unusual behavioural changes noted. (Aqueous
suspensions can be handled generally similarly.)
[0663] Drugs and Materials: Lipopolysaccharide (LPS)
(Serotype:0127:B8, e.g. from Sigma, UK; e.g. L3129 prepared by
phenol extraction) is dissolved in phosphate-buffered saline
(PBS).
[0664] For dry powder, aqueous solution or aqueous suspension
administration, PDE4 inhibitors are preferably used in size-reduced
(e.g. micronised) form, for example according to the Micronisation
Example(s) disclosed herein.
[0665] For dry powder administration of the drug, the PDE4
inhibitor compound or salt of the invention (drug) is administered
as a dry powder blended with inhalation-grade (respiratory-grade)
lactose, i.e. as a dry powder composition. The target quantity of
dry powder composition administered i.t. to each animal is 2 mg.
This remains constant throughout the studies. To achieve different
doses, blends of the drug dry powder and lactose, containing
different concentrations of the PDE4 inhibitor compound or salt of
the invention (drug), are prepared. The following dry powder
blends, or generally similar blends with generally analogous but
nonidentical concentrations, are generally used for these studies:
[0666] Blend 1: 0.1667% w/w of drug in lactose, which can e.g. be
used to dose rats at about 10 .mu.g/kg (about 10 ug/kg), depending
on the bodyweight of the rats used (e.g. ca. 300-350 g bodyweight);
[0667] Blend 2: 0.5% w/w of drug in lactose, which can e.g. be used
to dose rats at about 30 .mu.g/kg (about 30 ug/kg), depending on
the bodyweight of the rats used (e.g. ca. 300-350 g bodyweight);
[0668] Blend 3: 1.667% w/w of drug in lactose, which can e.g. be
used to dose rats at about 100 .mu.g/kg (about 100 ug/kg),
depending on the bodyweight of the rats used (e.g. ca. 300-350 g
bodyweight); [0669] Blend 4: 5% w/w of drug in lactose, which can
e.g. be used to dose rats at about 300 .mu.g/kg (about 300 ug/kg),
depending on the bodyweight of the rats used (e.g. ca. 300-350 g
bodyweight).
[0670] For dry powder administration of the drug, one suitable
inhalation-grade lactose that is typically used is: lactose
monohydrate; and/or lactose having about 10% fines (i.e. has about
10% of material under 15 um (15 micron) particle size, measured by
Malvern particle size). Other inhalation-grade lactoses might have
e.g. from about 7% to about 11% fines.
[0671] For aqueous solution compositions of the drug, a composition
of 0.2% Tween 80.TM. (e.g. available from Sigma, e.g. 073K 00643)
in phosphate buffered saline (PBS) is prepared and used as the
vehicle. The Sigma catalogue 2004-2005 and The Handbook of
Pharmaceutical Excipients (HPE) 4th edition 2003 pp. 479-483 define
Tween 80.TM. as a brand of polysorbate 80 (according to HPE,
polysorbate 80 is also named polyoxyethylene 20 sorbitan
monooleate, i.e. having been copolymerised with about 20 moles of
ethylene oxide for each mole of sorbitol and/or sorbitol anhydrides
used). Solution compositions are prepared by adding the required
volume of 0.2% Tween 80.TM. in PBS to the pre-weighed PDE4
inhibitor compound or salt of the invention (drug). The average
weight of the rats generally used in these studies is approximately
350 g; the i.t. solution dose volume is 200 microlitres. Thus, for
a target dose of 100 .mu.g/kg (100 ug/kg), a 0.175 mg/ml solution
of the drug is prepared. Lower concentrations are achieved by
preparing serial dilutions from the 0.175 mg/ml stock solution
concentration. All solutions were prepared immediately prior to
dosing. The formulation is usually sonicated, e.g. for
approximately 2-15 minutes e.g. ca. 2 minutes, prior to use and is
observed to be a solution (if it is a solution) by visual
examination.
[0672] Similar to aqueous solutions, aqueous suspensions of a drug
can be prepared by adding the required volume of vehicle to the
pre-weighed drug; the vehicle used can for example be saline alone
or preferably a mixture of saline/Tween.TM. (e.g. 0.2% Tween 80.TM.
in phosphate buffered saline). The aqueous suspension is usually
sonicated, e.g. for about 10-15 minutes, prior to use.
[0673] Preparation, and dosing with PDE 4 inhibitor: Rats are
anaesthetised by placing the animals in a sealed Perspex chamber
and exposing them to a gaseous mixture of isoflourane (4.5%),
nitrous oxide (3 litres/minute) and oxygen (1 litre/minute). Once
anaesthetised, the animals are placed onto a stainless steel i.t.
dosing support table. They are positioned on their back at
approximately a 35.degree. angle. A light is angled against the
outside of the throat to highlight the trachea. The mouth is opened
and the opening of the upper airway visualised. The procedure
varies for aqueous solution and dry powder administration of PDE4
inhibitors as follows:
[0674] Dosing with an aqueous solution: A portex cannula is
introduced via a blunt metal dosing needle that has been carefully
inserted into the rat trachea. The animals are intratracheally
dosed with vehicle or PDE4 inhibitor via the dosing needle with a
new internal canula used for each different drug group. The
formulation is slowly (ca. 5 seconds) dosed into the trachea using
a syringe attached to the dosing needle. The procedure with an
aqueous suspension is generally similar.
[0675] Dosing with a Dry Powder: The intratracheal dosing device (a
Penn Century dry powder insufflator, DP-4) is inserted into the rat
trachea up to a pre-determined point established to be located
approximately 1 cm above the primary bifurcation. Two.times.3 ml of
air is delivered using the Penn Century dry powder insufflator
device by depressing a plastic syringe (ideally coinciding with the
animal inspiring), aiming to expel the entire drug quantity from
the tap. After dosing, the device is removed from the airway, and
then is stored in an upright postion in a sealed plastic bag. At a
later stage, the device is re-weighed to determine the quantity of
dry powder delivered.
[0676] After dosing with either aqueous solution (or wet
suspension) or dry powder, the animals are removed from the table
and observed constantly until they have recovered from the effects
of anaesthesia. The animals are returned to the holding cages and
given free access to food and water; they are observed and any
unusual behavioural changes noted.
[0677] Exposure to LPS: About 2 hours after i.t. dosing with
vehicle control or the PDE4 inhibitor, the rats are placed into
sealed Perspex containers and exposed to an aerosol of LPS
(nebuliser concentration ca. 150 .mu.gml.sup.-1=ca. 150 ug/ml) for
ca. 15 minutes. Aerosols of LPS are generated by a nebuliser
(DeVilbiss, USA) and this is directed into the Perspex exposure
chamber. Following the 15-minute LPS-exposure period, the animals
are returned to the holding cages and allowed free access to both
food and water.
[0678] In an alternative embodiment, the rats can be exposed to LPS
less than 2 hours (e.g. about 30 minutes) after i.t. dosing.
[0679] In another alternative embodiment, the rats can be exposed
to LPS more than 2 hours (e.g. ca. 4 hours to ca. 36 hours, such as
4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours or 36
hours, in particular 4 hours or 12 hours) after i.t. dosing by
vehicle or PDE4 inhibitor, to test whether or not the PDE4
inhibitor has a long duration of action (which is preferable but
not essential).
[0680] Bronchoalveolar lavage: About 4 hours after LPS exposure the
animals are killed by overdose of sodium pentobarbitone (i.p.). The
trachea is cannulated with polypropylene tubing and the lungs are
lavaged (washed out) with 3.times.5 ml of heparinised (25 units/ml)
phosphate buffered saline (PBS).
[0681] Neutrophil cell counts: The Bronchoalveolar lavage (BAL)
samples are centrifuged at ca. 1300 rpm for ca. 7 minutes. The
supernatant is removed and the resulting cell pellet resuspended in
ca. 1 ml PBS. A cell slide of the resuspension fluid is prepared by
placing ca. 100 .mu.l (ca. 100 ul) of resuspended BAL fluid into
cytospin holders and then is spun at ca. 5000 rpm for ca. 5
minutes. The slides are allowed to air dry and then stained with
Leishmans stain (ca. 20 minutes) to allow differential cell
counting. The total cells are also counted from the resuspension.
From these two counts, the total numbers of neutrophils in the BAL
are determined. For a measure of PDE4-inhibitor-induced inhibition
of neutrophilia, a comparison of the neutrophil count in rats
treated with vehicle and rats treated with PDE4 inhibitors is
conducted.
[0682] By varying the dose of the PDE4 inhibitor used in the dosing
step (e.g. 0.3 or 0.1 mg of PDE4 inhibitor per kg of body weight,
down to e.g. 0.01 mg/kg), a dose-response curve can be generated,
and thence an ED.sub.50 value. The ED.sub.50 value is typically the
dose required to achieve 50% of the maximum level of inhibition
achievable for that particular PDE4 inhibitor (drug) in the
particular composition used in that particular study. The ED.sub.50
value is not usually the dose required for 50% inhibition of
neutrophilia.
[0683] Results: In an assay(s), generally involving LPS-induced
pulmonary neutrophilia in rats and the effect of intratracheally
administered PDE4 inhibitors to those rats, which assay(s) is or
are generally similar to or generally analogous to the
above-mentioned assay, the following results, given below, were
measured. It is noted that these results might be subject to a
possibly significant margin of error; and generally results may
vary as the assay or measurement method varies:
[0684] (1)
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3-
,4-b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}am-
ino)benzamide dihydrochloride (e.g. which can be as prepared in
Example 1A1), in micronised form, showed an estimated ED.sub.50 of
very approximately 12.3 micrograms/kg body weight, for inhibition
of LPS-induced pulmonary neutrophilia in rats, when administered to
rats intratracheally (i.t.) as an aqueous solution in 0.2% Tween
80.TM. in phosphate buffered saline, the administration to the rats
being about 2 hours prior to the LPS challenge. The individual
values of percentage mean inhibition of LPS-induced pulmonary
neutrophilia in rats at different doses, which led to this
estimated ED.sub.50 value of very approximately 12.3 micrograms/kg,
were as follows (n=6-8 for each of the following four dosing
groups):
[0685] about 33% mean inhibition (p>0.05) after dosing at about
10 micrograms/kg body weight;
[0686] about 56% mean inhibition (p<0.01) after dosing at about
30 micrograms/kg body weight;
[0687] about 76% mean inhibition (p<0.01) after dosing at about
100 micrograms/kg body weight; and about 74% mean inhibition
(p<0.01) after dosing at about 300 micrograms/kg body
weight.
[0688] (2)
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3-
,4-b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}am-
ino)benzamide (e.g. which can be as prepared in Example 1B), in
micronised form, showed an estimated ED.sub.50 of very
approximately 10.1 micrograms/kg body weight, for inhibition of
LPS-induced pulmonary neutrophilia in rats, when administered to
rats intratracheally (i.t.) as an aqueous solution in 0.2% Tween
80.TM. in phosphate buffered saline, the administration to the rats
being about 2 hours prior to the LPS challenge. The individual
values of percentage inhibition (arithmetic mean) of LPS-induced
pulmonary neutrophilia in rats at different doses, which led to
this estimated ED.sub.50 value of very approximately 10.1
micrograms/kg, were as follows (using: p values are vs.
LPS+Tween/PBS vehicle control; ANOVA followed by Dunnet's post
test; and n=5-6 for each of the following three dosing groups):
[0689] about 29% arithmetic mean inhibition (p>0.05) after
dosing at about 10 micrograms/kg body weight;
[0690] about 75% arithmetic mean inhibition (p<0.001) after
dosing at about 30 micrograms/kg body weight; and about 59%
arithmetic mean inhibition (p<0.05) after dosing at about 100
micrograms/kg body weight.
[0691] (3)
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3-
,4-b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}am-
ino)benzamide monohydrochloride (e.g. which can be as prepared in
Example 1A2), in micronised form, gave the following arithmetic
mean percentage inhibitions of LPS-induced pulmonary neutrophilia
in rats, when administered to rats intratracheally (i.t.) as a dry
powder composition blended in inhalation grade lactose (with about
10% fines), the administration to the rats being about 2 hours
prior to the LPS challenge (using: p values are vs. LPS+vehicle
control; ANOVA followed by Dunnet's post test; note these results
are summations (means) derived from about 4 separate experiments
each using some but not all of the following four dosing groups,
and with, for each experiment, n=4 or 6 for each of the dosing
groups tested):
[0692] about 29% arithmetic mean inhibition (p>0.05) after
dosing at about 10 micrograms/kg body weight;
[0693] about 44% arithmetic mean inhibition (p<0.05) after
dosing at about 30 micrograms/kg body weight;
[0694] about 39% arithmetic mean inhibition (p>0.05) after
dosing at about 100 micrograms/kg body weight; and
[0695] about 55% arithmetic mean inhibition (p<0.05) after
dosing at about 300 micrograms/kg body weight.
[0696] (4)
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3-
,4-b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}am-
ino)benzamide monohydrochloride (e.g. which can be as prepared in
Example 1A2), in micronised form, gave about 60% inhibition
(p<0.05) of LPS-induced pulmonary neutrophilia in rats, when
about 100 micrograms of the drug/kg body weight was administered to
the rats intratracheally (i.t.) as a dry powder composition blended
in inhalation grade lactose (with about 10% fines), the
administration to the rats being about 4 hours prior to the LPS
challenge.
In Vivo Assay 2. LPS Induced Nasal Neutrophilia in Rats: Effect of
Intranasally Administered PDE4 Inhibitors
[0697] This assay is an animal model of acute inflammation in the
nose--specifically neutrophilia induced by lipopolysaccharide
(LPS)-- and allows the study of putative inhibition of such
neutrophilia (anti-inflammatory effect) by an intranasally (i.n.)
administered PDE4 inhibitor ("drug"). The PDE4 inhibitor is
preferably in the form of an aqueous suspension or aqueous
solution. Bolus, i.n. administration allows topical delivery to the
nose.
Animals: Male CD rats (Sprague Dawley derived), weighing between
300-350 g are supplied by Charles River, UK and transported to the
test site. On arrival at the test site, the animals are housed in
groups of 4 or 5 per cage and complete a minimum acclimatisation
period of 7 days. The animals are housed in solid bottomed cages
using grade 8 sawdust. The bedding and nesting materials are
changed 3 times per week. Diet comprises SDS diet R1 pelleted food
given ad lib. Pasteurised animal grade drinking water is supplied
and changed daily.
Drugs and Materials
[0698] Lipopolysaccharide (Serotype: 0127:B8, e.g. available from
Sigma, UK; e.g. L3129 prepared by phenol extraction) is used for
all studies and is dissolved in phosphate-buffered saline
(PBS).
[0699] Tween 80.TM. (e.g. available from Sigma e.g. 023K 01575). A
mixture of 0.2% Tween 80.TM. in phosphate buffered saline (PBS) is
prepared as vehicle for solution compositions. The Sigma catalogue
2004-2005 and The Handbook of Pharmaceutical Excipients (HPE) 4th
edition 2003 pp. 479-483 define Tween 80.TM. as a brand of
polysorbate 80 (according to HPE, polysorbate 80 is also named
polyoxyethylene 20 sorbitan monooleate, i.e. having been
copolymerised with about 20 moles of ethylene oxide for each mole
of sorbitol and/or sorbitol anhydrides used).
[0700] The PDE4 inhibitor compound or salt of the invention
("drug") is preferably used in size-reduced (e.g. micronised) form,
for example according to the Micronisation Example(s) disclosed
herein.
[0701] A solution of compound (0.7 mg/ml) is generally prepared by
adding 0.2% Tween 80.TM. in phosphate buffered saline (PBS) to a
pre-weighed micronised sample of the drug. The solution composition
is sonicated, e.g. for approximately 2 min, and is observed to be a
solution (if it is a solution) by visual assessment. A fixed volume
of 50 microlitres (25 microlitres into each nostril) of either the
drug-containing solution composition or a saline/Tween 80.TM.
vehicle is administered to all animals. Each animal weighs, on
average, 350 g. To achieve a dose of 100 .mu.g/kg (100 ug/kg), each
animal receives a total of 35 .mu.g (35 ug) of the drug. The dose
volume is 50 microlitres (25 microlitres to each nostril), and
therefore a concentration of 35 .mu.g (ug) per 50 microlitres (0.7
mg/ml) is prepared. To achieve lower dose concentrations, serial
dilutions are prepared in saline/Tween 80.TM.
[0702] Intranasal dosing with the PDE4 inhibitor (drug): Rats are
anaesthetised by placing the animals in a sealed Perspex chamber
and exposing them to a gaseous mixture of isofluorane (5%), nitrous
oxide (2 L/min) and oxygen (1 L/min). Once anaesthetised, the
animals are i.n. dosed with vehicle or PDE4 inhibitor aqueous
compositions using a 10-100-microlitre-range Gilson pipette and
plastic disposable tip. A volume of 25 microlitres is administered
into each nostril by carefully inserting the pipette tip into the
nostril and dispensing the required volume. When the dosing
procedure is completed, the animal is returned to the cage and
observed at regular intervals until it had recovered from the
effects of the anaesthetic.
[0703] Exposure to LPS: Thirty minutes after i.n. dosing with
vehicle or PDE4 inhibitor, the rats are anaesthetised as previously
described and a 25 microlitres of a solution of LPS (175
.mu.g/kg=175 ug/kg) is administered into each nostril using the
same technique described for dosing of drug or vehicle. When the
dosing procedure is completed, the animal is returned to the cage
and observed at regular intervals until it had recovered from the
effects of the anaesthetic.
[0704] Nasal Lavage: Four hours after LPS exposure, the animals
areeuthanased by overdose of sodium pentobarbitone (i.p.). The
trachea is exposed and a small incision made approximately 1 cm
below the larynx. A polypropylene cannula is inserted into the
trachea towards the larynx and into the upper airway. The nasal
cavity is then lavaged with 15 ml of heparinised (10 units/ml)
phosphate buffered saline. The lavage fluid is collected in a 20 ml
plastic sterilin tube which is positioned against the nostrils.
[0705] Cell Counts: The nasal lavage samples are centrifuged at
1300 rpm for 7 minutes. The supernatant is removed and the
resulting cell pellet is resuspended in 0.5 ml PBS. A cell slide of
the resuspension fluid is prepared by placing 75 microlitres of
resuspended nasal lavage fluid into cytospin holders and then spun
at 500 rpm for 5 min. The slides are allowed to air dry and then
stained with Leishmans stain (20 minutes) to allow differential
cell counting. The total cells are also counted from the
resuspension. From these two counts, the total numbers of
neutrophils in the nasal lavage fluid is determined. For a measure
of PDE4-inhibitor-induced inhibition of neutrophilia, a comparison
of the neutrophil count in rats treated with vehicle and rats
treated with the PDE4 inhibitor is conducted. By varying the dose
of the PDE4 inhibitor used in the dosing step (e.g. 0.3 or 0.1 mg
of PDE4 inhibitor per kg of body weight, down to e.g. 0.01 mg/kg),
a dose-response curve can be generated, and thence an ED.sub.50
value.
[0706] Results: In an assay(s), generally involving LPS-induced
nasal neutrophilia in rats and the effect of intranasally
administered PDE4 inhibitors to those rats, which assay(s) is or
are generally similar to or generally analogous to the
above-mentioned assay, the following results were measured:
[0707]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide monohydrochloride (e.g. which can be as prepared in
Example 1A2), in micronised form, showed an estimated ED50 of very
approximately 4.6 micrograms/kg body weight, for inhibition of
LPS-induced nasal neutrophilia in rat, when administered to the rat
intranasally (i.n.) as an aqueous solution in 0.2% Tween 80.TM. in
phosphate buffered saline, the administration to the rat being
about 30 minutes prior to the LPS challenge. The individual values
of percentage inhibition (arithmetic mean) of LPS-induced nasal
neutrophilia in rats at different doses, which led to this
estimated ED.sub.50 value of very approximately 4.6 micrograms/kg,
were as follows (using: p values are vs. LPS+Tween/PBS vehicle
control; ANOVA followed by Dunnet's post test; note: these data
represent a summation from 2 or more separate experiments):
[0708] essentially no inhibition after dosing at about 1
micrograms/kg body weight;
[0709] small and non-statistically-significant inhibition after
dosing at about 3 micrograms/kg body weight;
[0710] about 62% arithmetic mean inhibition (p<0.01) after
dosing at about 10 micrograms/kg body weight (for 10 ug/kg
experiment, assumed rat bodyweight was ca. 350 g at time of test,
rats (n=6) weighed ca. 225-300 g at delivery one month before
test); and
[0711] about 79% arithmetic mean inhibition (p<0.01) after
dosing at about 100 micrograms/kg body weight (N.B. the 100 ug/kg
dosing was a separate experiment with rats (n=6) weighing ca.
175-225 g at delivery nine days before test).
In Vivo Assay A:
Activity of Topically-Applied Compounds in a Pig Model of Atopic
Dermatitis: Effect of Compounds, Applied by Skin Topical
Administration, on the Dinitrofluorobenzene (DNFB)-Induced Delayed
Type Hypersensitivity (DTH) Response in Pigs
General Study Design:
[0712] The pig DTH (delayed type hypersensitivity) model of contact
hypersensitivity utilizes the Th2-mediated inflammatory response in
pig skin to mimic the pathology of atopic dermatitis in humans. The
model measures the potential anti-inflammatory effect of compounds,
topically-applied to the skin, on the acute DTH (delayed type
hypersensitivity) response in castrated male Yorkshire pigs.
[0713] In general in the assay, pigs (domestic Yorkshire pigs,
15-18 kg at time of sensitization, castrated males) are first
sensitized by topical application of ca. 10% (w/v)
dinitrofluorobenzene (DNFB) dissolved in DMSO:acetone:olive oil
(ca. 1:5:3) (ca. 40 mg DNFB, 400 microlitre solution total) to the
ears (outer) and groin (inner). The pigs are then challenged 12
days later with ca. 0.6% (w/v) DNFB applied to randomized sites on
the shaved back of the pigs (ca. 90 micrograms/site; sites are
identified and numbered by grid made with marking pen).
[0714] On the day of challenge, the treatments are performed at the
challenge sites at about 2 hours prior to and about 6 hours after
challenge (for DMSO/acetone solutions/suspensions containing the
PDE4 inhibitor, to maximize exposure to drug), or at about 30
minutes after and about 6 hours after challenge (for topical
ointments or creams containing the PDE4 inhibitor, representing a
more clinically relevant treatment protocol).
[0715] One day (about 24 hrs) after challenge, and optionally again
at ca. 48 hrs post challenge, test sites are visually evaluated for
intensity and extent of erythema by measuring the diameter of the
reaction at its widest point and assigning scores of 0 to 4 for
each of erythema intensity and erythema extent. Induration (a
measure of swelling) is also scored 0 to 4. Scores for erythema
intensity, erythema extent and induration are assigned according to
the following criteria: Intensity of Erythema: 0=normal, 1=minimal,
barely visible, 2=mild, 3=moderate, 4=severe. Extent of Erythema
(not raised): 0=no edema, 1=macules of pin head size, 2=lentil
sized macules, 3=confluent macules, 4=diffuse over entire site.
Induration (palpable): 0=normal, 1=nodules of pin head size,
2=doughy lentil sized nodules, 3=confluent firm nodules, 4=diffuse
hard lesion. The summed visual score at ca. 24 hours includes the
individual scores for erythema intensity, erythema extent, and
induration; so the maximal summed score for each site would be 12.
High summed scores can generally indicate a high inflammatory
response. Visual scores are subject to some inaccuracy/error.
[0716] Differences in the summed score between adjacent control
(placebo) and treatment sites on the grids are calculated. This
difference value is then used to determine the percent inhibition
compared to the summed score for the control (placebo) sites. The
more negative the difference value, the greater the calculated
inhibition. Percent inhibition of (percent inhibition compared to)
the mean summed score can be calculated.
[0717] About 24 hours after challenge, treatment sites can
optionally also be visually evaluated for lesion area.
EXAMPLES
[0718] The various aspects of the invention will now be described
by reference to the following examples. These examples are merely
illustrative and are not to be construed as a limitation of the
scope of the present invention.
[0719] In this section, "Intermediates" can represent syntheses of
intermediate compounds intended for use in the synthesis of one or
more of the "Examples", and/or "Intermediates" can represent
syntheses of intermediate compounds which can possibly be used in
the synthesis of compounds of formula (I) or salts thereof.
"Examples" are generally examples of compounds or salts of the
invention, for example compounds of formula (I) or salts
thereof.
Abbreviations used herein: [0720] AcOH acetic acid [0721] Ac.sub.2O
acetic anhydride [0722] BEMP
2-t-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphazine
[0723] BOC.sub.2O di tert-butyl carbonate [0724] DMSO dimethyl
sulfoxide [0725] DCC N,N'-dicyclohexylcarbodiimide [0726] DCM
dichloromethane [0727] DMF N,N-dimethylformamide [0728] DIPEA
N,N-diisopropylethyl amine (iPr.sub.2NEt) [0729] EDC
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride [0730]
EtOAc ethyl acetate [0731] Et.sub.2O diethyl ether [0732] Et.sub.3N
triethylamine [0733] EtOH ethanol [0734] HATU
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate; also named
0-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate [0735] HBTU
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [0736] HOBT
hydroxybenzotriazole=1-hydroxybenzotriazole [0737] IPA isopropanol
(isopropyl alcohol) [0738] Lawesson's reagent
2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulphide
[0739] LPS lipopolysaccharide [0740] MeCN acetonitrile [0741] MeOH
methanol [0742] MDAP mass directed autoprep HPLC [0743] NMP
1-methyl-2-pyrrolidinone (also named 1-methyl-2-pyrrolidone or
N-methyl-2-pyrrolidinone) [0744] PyBOP
(Benzotriazol-1-yloxy)-trispyrrolidinophosphonium
hexafluorophosphate [0745] TBME tert-butyl methyl ether [0746] TMS
tetramethylsilane [0747] THF Tetrahydrofuran [0748] TFA
Trifluoroacetic acid [0749] barg generally, this represents the
pressure relative to the prevailing atmospheric pressure (so that
e.g. if the atmospheric pressure at the time is 1 atmosphere then
-0.9 barg is 0.9 atmospheres below the prevailing external 1
atmosphere=0.1 atmosphere absolute pressure). [0750] HPLC high
performance liquid chromatography [0751] h hours [0752] min minutes
[0753] LCMS or LC/MS liquid chromatography/mass spectroscopy [0754]
NMR nuclear magnetic resonance (in which: s=singlet, d=doublet,
t=triplet, q=quartet, dd=doublet of doublets, m=multiplet, H=no. of
protons) [0755] SPE solid phase extraction. Unless otherwise
specified, the solid phase is usually silica gel. Aminopropyl SPE,
if used, typically refers to a silica SPE column with aminopropyl
residues immobilised on the solid phase (eg. IST Isolute.TM.
columns). It is thought that compounds isolated by SPE are usually
isolated as the free base. [0756] SCX solid phase extraction (SPE)
column with benzene sulfonic acid residues immobilised on the solid
phase (eg. IST Isolute.TM. columns). When eluting with
ammonia/methanol, it is thought that compounds isolated by SCX are
usually in the free base form. [0757] TLC thin layer chromatography
[0758] T.sub.RET or R.sub.T retention time (e.g. from LCMS) [0759]
T.sub.int internal temperature of the reaction mixture [0760]
T.sub.max maximum internal temperature, e.g. during an addition
[0761] Room temperature (ambient temperature): this is usually in
the range of about 18 to about 25.degree. C.
General Experimental Details
Machine Methods Used Herein:
LCMS (Liquid Chromatography/Mass Spectroscopy)
[0762] Waters ZQ mass spectrometer operating in positive ion
electrospray mode, mass range 100-1000 amu.
UV wavelength: 215-330 nm Column: 3.3 cm.times.4.6 mm ID (internal
diameter), 3 .mu.m (3 micrometres) ABZ+PLUS Flow Rate: 3 ml/min
Injection Volume: 5 .mu.l (5 microlitres) Solvent A: 0.05% v/v
solution of formic acid in a mixture of [95% acetonitrile and 5%
water] Solvent B: aqueous solution of [0.1% v/v formic acid+10
mMolar ammonium acetate] Gradient: Mixtures of Solvent A and
Solvent B are used according to the following gradient profiles
(expressed as % Solvent A in the mixture): 0% A/0.7 min, 0-100%
A/3.5 min, 100% A/1.1 min, 100-0% A/0.2 min.
[0763] It should be noted that retention times (T.sub.RET) quoted
herein are inherently variable (e.g. the variability can be about
+/-0.2 min or more.). Variability can arise e.g. when samples are
run on different Waters machines, or on the same Waters machine at
different times of day or under slightly different conditions, even
when the same type of column and identical flow rates, injection
volumes, solvents and gradients are used.
Mass Directed Automated Preparative HPLC Column, Conditions and
Eluent Method A (Eluent Containing Formic Acid)
[0764] The preparative HPLC column generally used is a Supelcosil
ABZplus (10 cm.times.2.12 cm internal diameter; particle size 5
.mu.m=5 micrometres). A mass spectrometer attached to the end of
the column can detect peaks arising from eluted compounds.
UV detection wavelength: 200-320 nm Flow rate: 20 ml/min
Injection Volume: 0.5 to 1.0 ml
[0765] Solvent A: 0.1% v/v aqueous formic acid solution Solvent B:
0.05% v/v solution of formic acid in a mixture of [95% acetonitrile
and 5% water] Gradient systems: mixtures of Solvent A and Solvent B
are used according to a choice of 5 generic gradient profiles
(expressed as % Solvent B in the mixture), ranging from a start of
0 to 50% Solvent B, with all finishing at 100% Solvent B to ensure
total elution. It is thought that compounds isolated by this method
are usually isolated as formate salts.
Mass Directed Automated Preparative HPLC Column, Conditions and
Eluent Method B (Eluent Containing Trifluoroacetic Acid)
[0766] The preparative HPLC column generally used is a C18 column
(10 cm.times.2.12 cm internal diameter; particle size 5 .mu.m=5
micrometres). A mass spectrometer attached to the end of the column
can detect peaks arising from eluted compounds.
UV detection wavelength: 200-320 nm Flow rate: 20 ml/min
Injection Volume: 0.5 to 1.0 ml
[0767] Solvent A: 0.1% v/v aqueous trifluoroacetic acid solution
Solvent B: solution of 0.1% v/v trifluoroacetic acid in
acetonitrile Gradient systems: mixtures of Solvent A and Solvent B
are used according to a choice of 5 generic gradient profiles
(expressed as % Solvent B in the mixture), ranging from a start of
0 to 50% Solvent B, with all finishing at 100% Solvent B to ensure
total elution.
[0768] It is thought that compounds isolated by this method are
usually isolated as trifluoroacetate salts.
Mass Directed Automated Preparative HPLC Column, Conditions and
Eluent
Method C (Eluent Containing Trifluoroacetic Acid)
[0769] The preparative HPLC column generally used is a C18 column
(10 cm.times.2.12 cm internal diameter; particle size 5 .mu.m=5
micrometres). A mass spectrometer attached to the end of the column
can detect peaks arising from eluted compounds.
UV detection wavelength: 210-254 nm Flow rate: 20 ml/min
Injection Volume: 0.5 to 1.0 ml
[0770] Solvent A: 0.1% v/v aqueous trifluoroacetic acid solution
Solvent B: solution of 0.1% v/v trifluoroacetic acid in
acetonitrile Gradient systems: mixtures of Solvent A and Solvent B
are used according to a choice of 5 generic gradient profiles
(expressed as % Solvent B in the mixture), ranging from a start of
0 to 50% Solvent B, with all finishing at 100% Solvent B to ensure
total elution.
[0771] It is thought that compounds isolated by this method are
usually isolated as trifluoroacetate salts.
`Hydrophobic Frit`
[0772] This generally refers to a Whatman PTFE filter medium
(frit), pore size 5.0 .mu.m (5.0 micrometres), housed in a
polypropylene tube.
Evaporation of Product Fractions after Purification
[0773] Reference to column chromatography, SPE and preparative HPLC
purification includes evaporation of the product containing
fractions to dryness by an appropriate method.
Celite
[0774] References to celite generally refer to the filter agent
Celite 545 (e.g. available from Aldrich).
Intermediates and Examples
[0775] Reagents not detailed in the text below are usually
commercially available from chemicals suppliers, e.g. established
suppliers such as Sigma-Aldrich. The addresses and/or contact
details of the suppliers for some of the starting materials
mentioned in the Intermediates and Examples below or the Assays
above, or suppliers of miscellaneous chemicals in general, are as
follows: [0776] AB Chem, Inc., 547 Davignon, Dollard-des-Ormeaux,
Quebec, H9B 1Y4, Canada [0777] ABCRGmbH & CO. KG, P.O. Box 21
01 35, 76151 Karlsruhe, Germany [0778] ACB Blocks Ltd; Kolokolnikov
Per, 9/10 Building 2, Moscow, 103045, Russia [0779] Aceto Color
Intermediates (catalogue name), Aceto Corporation, One Hollow Lane,
Lake Success, N.Y., 11042-1215, USA [0780] Acros Organics, A
Division of Fisher Scientific Company, 500 American Road, Morris
Plains, N.J. 07950, USA [0781] Aldrich (catalogue name),
Sigma-Aldrich Company Ltd., Dorset, United Kingdom, telephone: +44
1202 733114; Fax: +44 1202 715460; ukcustsv@eurnotes.sial.com; or
[0782] Aldrich (catalogue name), Sigma-Aldrich Corp., P.O. Box
14508, St. Louis, Mo. 63178-9916, USA; telephone: +1-314-771-5765;
fax: +1-314-771-5757; custserv@sial.com; or [0783] Aldrich
(catalogue name), Sigma-Aldrich Chemie GmbH, Munich, Germany;
telephone: +49 89 6513 0; Fax: +49 89 6513 1169;
deorders@eurnotes.sial.com. [0784] Alfa Aesar, A Johnson Matthey
Company, 30 Bond Street, Ward Hill, Mass. 01835-8099, USA [0785]
Amersham Biosciences UK Ltd, Pollards Wood, Chalfont St Giles,
Buckinghamshire HP8 4SP, United Kingdom [0786] Apin Chemicals Ltd.,
82 C Milton Park, Abingdon, Oxon OX14 4RY, United Kingdom [0787]
Apollo Scientific Ltd., Unit 1A, Bingswood Industrial Estate,
Whaley Bridge, Derbyshire SK23 7LY, United Kingdom [0788] Arch
Corporation, 100 Jersey Avenue, Building D, New Brunswick, N.J.
08901, USA [0789] Array Biopharma Inc., 1885 33rd Street, Boulder,
Colo. 80301, USA [0790] Asinex-Reag. [0791] AstaTech, Inc., 8301
Torresdale Ave., 19C, Philadelphia, Pa. 19136, USA [0792] Austin
Chemical Company, Inc., 1565 Barclay Blvd., Buffalo Grove, Ill.
60089, USA [0793] Avocado Research, Shore Road, Port of Heysham
Industrial Park, Heysham, Lancashire LA3 2XY, United Kingdom [0794]
Bayer AG, Business Group Basic and Fine Chemicals, D-51368
Leverkusen, Germany [0795] Berk Univar plc, Berk House, P.O.Box 56,
Basing View, Basingstoke, Hants RG21 2E6, United Kingdom [0796]
Bionet Research Ltd; Highfield Industrial Estate, Camelford,
Cornwall PL32 9QZ UK [0797] Butt Park Ltd., Braysdown Works,
Peasedown St. John, Bath BA2 8LL, United Kingdom [0798] Chemical
Building Blocks (catalogue name), Ambinter, 46 quai Louis Bleriot,
Paris, F-75016, France [0799] ChemBridge Europe, 4 Clark's Hill
Rise, Hampton Wood, Evesham, Worcestershire WR11 6FW, United
Kingdom [0800] ChemService Inc., P.O.Box 3108, West Chester, Pa.
19381, USA [0801] CiventiChem, PO Box 12041, Research Triangle
Park, N.C. 27709, USA [0802] Combi-Blocks Inc., 7949 Silverton
Avenue, Suite 915, San Diego, Calif. 92126, USA [0803] Dynamit
Nobel GmbH, Germany; also available from: Saville Whittle Ltd (UK
agents of Dynamit Nobel), Vickers Street, Manchester M40 8EF,
United Kingdom [0804] E. Merck, Germany; or E. Merck (Merck Ltd),
Hunter Boulevard, Magna Park, Lutterworth, Leicestershire LE17 4XN,
United Kingdom [0805] Esprit Chemical Company, Esprit Plaza, 7680
Matoaka Road, Sarasota, Fla. 34243, USA [0806] Exploratory Library
(catalogue name), Ambinter, 46 quai Louis Bleriot, Paris, F-75016,
France [0807] Fluka Chemie AG, Industriestrasse 25, P.O. Box 260,
CH-9471 Buchs, Switzerland [0808] Fluorochem Ltd., Wesley Street,
Old Glossop, Derbyshire SK13 7RY, United Kingdom [0809]
Heterocyclic Compounds Catalog (Florida Center for Heterocyclic
Compounds, University of Florida, PO Box 117200, Gainsville, Fla.
32611-7200 USA [0810] ICN Biomedicals, Inc., 3300 Hyland Avenue,
Costa Mesa, Calif. 92626, USA [0811] Interchim Intermediates
(catalogue name), Interchim, 213 Avenue Kennedy, BP 1140,
Montlucon, Cedex, 03103, France [0812] Key Organics Ltd., 3,
Highfield Indusrial Estate, Camelford, Cornwall PL32 9QZ, United
Kingdom [0813] Lancaster Synthesis Ltd., Newgate, White Lund,
Morecambe, Lancashire LA3 3DY, United Kingdom [0814] Manchester
Organics Ltd., Unit 2, Ashville Industrial Estate, Sutton Weaver,
Runcorn, Cheshire WA7 3 PF, United Kingdom [0815] Matrix
Scientific, P.O. Box 25067, Columbia, S.C. 29224-5067, USA [0816]
Maybridge Chemical Company Ltd., Trevillett, Tintagel, Cornwall
PL34 OHW, United Kingdom [0817] Maybridge Combichem (catalogue
name), Maybridge Chemical Company Ltd., Trevillett, Tintagel,
Cornwall PL34 OHW, United Kingdom [0818] Maybridge Reactive
Intermediates (catalogue name), Maybridge Chemical Company Ltd.,
Trevillett, Tintagel, Cornwall PL34 OHW, United Kingdom [0819]
MicroChemistry Building Blocks (catalogue name),
MicroChemistry-RadaPharma, Shosse Entusiastov 56, Moscow, 111123,
Russia [0820] Miteni S. p. A., Via Mecenate 90, Milano, 20138,
Italy [0821] Molecular Devices Corporation, Sunnydale, Calif., USA
[0822] N.D. Zelinsky Institute, Organic Chemistry, Leninsky
prospect 47, 117913 Moscow B-334, Russia [0823] Oakwood Products
Inc., 1741, Old Dunbar Road, West Columbia, S.C., 29172, USA [0824]
OmegaChem. Inc., 8800, Boulevard de la Rive Sud, Levis, PQ, G6V
9H1, Canada [0825] Optimer Building Block (catalogue name), Array
BioPharma, 3200 Walnut Street, Boulder, Colo. 80301, USA [0826]
Peakdale Molecular Ltd., Peakdale Science Park, Sheffield Road,
Chapel-en-1e-Frith, High Peak SK23 OPG, United Kingdom [0827]
Pfaltz & Bauer, Inc., 172 East Aurora Street, Waterbury, Conn.
06708, USA [0828] Qualigens Fine Chemicals [0829] Rare Chemicals
(catalogue name), Rare Chemicals GmbH, Schulstrasse 6, 24214
Gettorf, Germany [0830] Rieke [0831] SALOR (catalogue name) (Sigma
Aldrich Library of Rare Chemicals), Aldrich Chemical Company Inc,
1001 West Saint Paul Avenue, Milwaukee, Wis. 53233, USA [0832]
Sigma (catalogue name), Sigma-Aldrich Corp., P.O. Box 14508, St.
Louis, Mo. 63178-9916, USA; see "Aldrich" above for other non-US
addresses and other contact details [0833] SIGMA-RBI, One
Strathmore Road, Natick, Mass. 01760-1312, USA [0834] Spectrochem
[0835] Synchem OHG Heinrich-Plett-Strasse 40, Kassel, D-34132,
Germany [0836] Syngene International Pvt Ltd, Hebbagodi, Hosur
Road, Bangalore, India. [0837] TCI America, 9211 North Harborgate
Street, Portland, Oreg. 97203, USA [0838] TimTec Building Blocks A
or B, TimTec, Inc., P 0 Box 8941, Newark, Del. 19714-8941, USA
[0839] TimTec Overseas Stock, TimTec Inc., 100 Interchange Blvd.
Newark, Del. 19711, USA [0840] TimTec Stock Library, TimTec, Inc.,
P 0 Box 8941, Newark, Del. 19714-8941, USA [0841] Trans World
Chemicals, Inc., 14674 Southlawn Lane, Rockville, Md. 20850, USA
[0842] Tyger [0843] Ubichem PLC, Mayflower Close, Chandlers Ford
Industrial Estate, Eastleigh, Hampshire SO53 4AR, United Kingdom
[0844] Ultrafine (UFC Ltd.), Synergy House, Guildhall Close,
Manchester Science Park, Manchester M15 6SY, United Kingdom
TABLE-US-00009 [0844] TABLE 1 Intermediates Intermediate No. Name 1
1-ethyl-1H-pyrazol-5-amine 2 diethyl
(1-chloropropylidene)propanedioate 2A diethyl
(1-chloro-1-propen-1-yl)propanedioate and/or diethyl
(1-chloropropylidene)propanedioate 2B diethyl
{1-[(1-ethyl-1H-pyrazol-5-yl)amino]propylidene}propanedioate 2C
ethyl
1,6-diethyl-4-hydroxy-1H-pyrazolo[3,4-b]pyridine-5-carboxylate 3
ethyl 4-chloro-1,6-diethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
4 ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridine-5-carboxylate 5
[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methanol 6
5-(azidomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazolo[3,4-b]pyridin-4-amine 7
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazolo[3,4-b]pyridin-4-amine 7A
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazolo[3,4-b]pyridin-4-amine monohydrochloride 8
1,1-dimethylethyl{4-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-
1H-pyrazolo[3,4-b]pyridin-5-
yl]methyl}amino)carbonyl]phenyl}carbamate 9
4-amino-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide hydrochloride 10
8-bromooctanoyl chloride 11
4-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-
ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 13 ethyl
4-[(1-{[(1,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]-1,6-
- diethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate 14
1,1-dimethylethyl
4-{[1,6-diethyl-5-(hydroxymethyl)-1H-pyrazolo[3,4-
b]pyridin-4-yl]amino}-1-piperidinecarboxylate 15 1,1-dimethylethyl
4-{[5-(azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-
b]pyridin-4-yl]amino}-1-piperidinecarboxylate 16
4-{[5-(azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-
1-piperidinecarboxamide 17
4-{[5-(aminomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-
1-piperidinecarboxamide 19 1,1-dimethylethyl
[4-({[(4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1,6-
diethyl-1H-pyrazolo[3,4-b]pyridin-5-
yl)methyl]amino}carbonyl)phenyl]carbamate 20
4-{[5-({[(4-aminophenyl)carbonyl]amino}methyl)-1,6-diethyl-1H-
pyrazolo[3,4-b]pyridin-4-yl]amino}-1-piperidinecarboxamide
hydrochloride 21
4-[(5-{[({4-[(8-bromooctanoyl)amino]phenyl}carbonyl)amino]methyl}-
1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)amino]-1-
piperidinecarboxamide 22 1,1-dimethylethyl
4-(4-hydroxy-1-butyn-1-yl)benzoate 23 1,1-dimethylethyl
4-(4-hydroxybutyl)benzoate 24 1,1-dimethylethyl
4-{4-[(4-bromobutyl)oxy]butyl}benzoate 25
4-{4-[(4-bromobutyl)oxy]butyl}benzoic acid 26
4-{4-[(4-bromobutyl)oxy]butyl}benzoyl chloride 27
4-{4-[(4-bromobutyl)oxy]butyl}-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-
4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 28
4-{[5-({[(4-{4-[(4-bromobutyl)oxy]butyl}phenyl)carbonyl]amino}methyl)-
1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-
piperidinecarboxamide 29 diethyl (1-chloroethylidene)propanedioate
30 ethyl 4-chloro-1-ethyl-6-methyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate 31 ethyl
1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate 32
[1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methanol 33
5-(azidomethyl)-1-ethyl-6-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazolo[3,4-b]pyridin-4-amine 34
5-(aminomethyl)-1-ethyl-6-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazolo[3,4-b]pyridin-4-amine 35 1,1-dimethylethyl
4-[(8-bromooctanoyl)amino]benzoate 36
4-[(8-bromooctanoyl)amino]benzoic acid 37
4-[(8-bromooctanoyl)amino]-N-{[1-ethyl-6-methyl-4-(tetrahydro-2H-
pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 38
1,1-dimethylethyl 4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzoate 39
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzoic acid
39A 4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzoic
acid hydrogen chloride 40
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-(8-hydroxyoctyl)benzamide 41
4-(8-bromooctyl)-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-
1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 42
1,1-dimethylethyl
{2-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-
1H-pyrazolo[3,4-b]pyridin-5-
yl]methyl}amino)carbonyl]phenyl}carbamate 43 1,1-dimethylethyl
{3-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-
1H-pyrazolo[3,4-b]pyridin-5-
yl]methyl}amino)carbonyl]phenyl}carbamate 44
2-amino-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 45
3-amino-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 46
2-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-
ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 47
3-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-
ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 48
5-(aminomethyl)-1-ethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-
b]pyridin-4-amine hydrochloride 49
4-[(6-bromohexanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-
ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 50
4-[(7-bromoheptanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-
ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 51
4-[(10-bromodecanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-
4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 52
4-[(11-bromoundecanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-
pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide 53
4-[(8-bromooctanoyl)amino]benzamide 54
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide 55
2-[(8-{[4-(aminocarbonyl)phenyl]amino}-8-
oxooctyl)(methyl)amino]ethyl acetate
Intermediate 1: 1-Ethyl-1H-pyrazol-5-amine
##STR00102##
[0846] Acrylonitrile (536 g, 10.11 moles, commercially available
e.g. from E. Merck) is added dropwise to hydrazine hydrate (511 g,
10.22 moles, commercially available e.g. from Qualigens Fine Chem.)
maintaining the reaction temperature between 30 and 35.degree. C.
(a little exothermicity can be observed). After completion of the
addition the reaction mixture is stirred at the same temperature
for an additional hour. Water from the mixture is removed at
45-50.degree. C. under reduced pressure to give the Michael
addition product (2-cyanoethylhydrazine, e.g. this can be a pale
yellow oil), which can be used in the next step without further
purification.
[0847] A solution of acetaldehyde (561 ml, 9.99 moles, commercially
available e.g. from Sigma-Aldrich) in ethanol (1008 ml) is added
dropwise to a stirred solution of 2-cyanoethylhydrazine (835 g,
9.82 moles) in ethanol (700 ml) at such a rate that the temperature
does not rise above 35.degree. C. The batch is heated under reflux
for 1 hour and ethanol is removed under reduced pressure to afford
the Schiff's base (e.g. this can be a pale yellow oil), which can
be directly used in the subsequent step.
[0848] The Schiff's base is added to a stirred mixture of
sodium-tert-butoxide (908 g, 9.46 mol) in t-butanol and the
reaction mixture is heated under reflux for 12 hours. The cooled
reaction mixture is then poured into water (23 L), stirred for 0.5
hours and extracted with diethyl ether (1.times.10 L and 3.times.6
L). The combined organic layer is dried (Na.sub.2SO.sub.4) and the
solution is evaporated to dryness. The residue is dissolved in
dichloromethane (1.5 L) to remove impurities and the solvent is
evaporated to dryness to give the title compound (e.g. this can be
a red oil), which can optionally be used without further
purification.
Intermediate 2: Diethyl (1-chloropropylidene)propanedioate
##STR00103##
[0850] To a mixture of diethyl malonate (704 g, e.g. available from
Spectrochem) and acetonitrile (3.8 L) was added anhydrous magnesium
chloride immediately (419 g, e.g. available from Lancaster).
Triethylamine (1222 ml) was added dropwise, maintaining the
temperature at 5-10.degree. C., followed by the dropwise addition
of propionyl chloride (406 g), maintaining the temperature at or
below 30.degree. C. The reaction mixture was kept at 10-15.degree.
C. for 1 hour, and then the mixture was kept overnight at room
temperature. The next day, aqueous hydrochloric acid (1 M) was
added to the reaction mixture until the pH of the mixture was about
2.0 (approx. 4.8 L was required). The mixture was extracted with
diethyl ether (3.times.800 ml). The combined ethereal extracts were
washed with aqueous hydrochloric acid (1M, 2.times.1000 ml),
followed by water (2.times.1000 mL) and finally with brine
(2.times.1000 mL). Evaporation of the solvent from the ethereal
extracts under reduced pressure afforded diethyl
propanoylpropanedioate (845 g) as a yellow oil.
[0851] Tri-n-butylamine (316 ml) was added dropwise to a mixture of
the above keto-diester derivative (300 g) in phosphorus oxychloride
(POCl.sub.3, 3.1 L) at room temperature. The reaction mixture was
then heated under reflux for 7 hours. After cooling to room
temperature, excess phosphorus oxychloride was removed by
distillation under reduced pressure. The reaction mixture was then
cooled to room temperature and extracted with a 1:2 mixture of
hexane and diethyl ether (3.times.1.2 L). The combined organic
extracts were washed with aqueous hydrochloric acid (1 M, 1.times.1
L), aqueous sodium hydroxide solution (0.1 M, 2.times.500 ml),
brine (2.times.500 ml) and dried. Evaporation of the solvent under
reduced pressure afforded the title compound (245 g) as a red oil
which was not further purified.
Intermediate 2A: Diethyl (1-chloro-1-propen-1-yl)propanedioate
and/or diethyl (1-chloropropylidene)propanedioate
[0852] There are two stages to this reaction, as follows:
"Stage 1a": Preparation of diethyl propanoylpropanedioate
##STR00104##
[0853] The following reaction was conducted under a nitrogen
atmosphere. The volumes and equivalents stated are relative to the
diethyl malonate.
[0854] Diethyl malonate (25 g, 23.7 ml, 0.156 mol, 1.0 equivalent)
was charged to a clean dry reaction vessel followed by acetonitrile
(125 ml, 5 volumes). The solution was cooled to 0-5.degree. C.
Anhydrous magnesium chloride (MgCl.sub.2, 15.04 g, 0.158 mol, 1.01
equivalents) was added in 6 portions over 15 minutes, ensuring the
internal temperature (T.sub.int) was less than 20.degree. C. (the
maximum internal temperature during the addition (T.sub.max)
actually was 15.degree. C.). The slurry was cooled to 0-5.degree.
C. and triethylamine (43.3 ml, 31.59 g, 0.312 mol, 2.0 equivalents)
was slowly charged to the vessel from a dropping funnel over 27
minutes, maintaining T.sub.int<20.degree. C. (T.sub.max actually
was 9.degree. C.). A solution of propionyl chloride (13.7 ml, 14.59
g, 0.158 mol, 1.01 equivalents) in acetonitrile (10 ml) was added
slowly via a dropping funnel to the reaction mixture over 20
minutes, maintaining T.sub.int<20.degree. C. (T.sub.max actually
was 15.degree. C.). The reaction mixture was stirred at
10-15.degree. C. for about 54-60 minutes, and then allowed to warm
to 20-25.degree. C. (cooling removed) and stirred at 20-25.degree.
C. for about 2 hours. The reaction mixture was cooled. Toluene (50
ml) was then added in one portion, followed by 1 M aqueous
hydrochloric acid (115 ml) which was added over 9 minutes via a
dropping funnel (during which time an ice bath was used, with
T.sub.max being 19.degree. C.). The reaction mixture was stirred
for 35 minutes, and then transferred to a separating funnel after
standing for 5 minutes. Some grey solids were observed in the
organic layer. The pH of the aqueous layer was about 6. Aqueous
hydrochloric acid (2 M, 23 ml) was added, after which the pH of the
aqueous layer was about 0-1. The two layers were then separated and
the aqueous layer was back-extracted with toluene (50 ml). The back
extraction was repeated. The combined organic layers were then
washed with 1 M citric acid (50 ml) (whereupon the organic layer
became milky), followed by 2 M aqueous hydrochloric acid (40 ml),
water (40 ml), 15 wt % brine solution (50 ml) and a further 15 wt %
brine solution (50 ml). The resulting slightly translucent
opalescent organic layer was concentrated using a rotary evaporator
to a volume of 100 ml. The yield of the reaction using a .sup.1H
NMR assay was calculated as about 32.3 g, about 0.149 moles, about
95.8% theory yield. This mixture of diethyl propanoylpropanedioate
in toluene was used directly in the Stage 1b reaction below.
"Stage 1b": Preparation of diethyl
(1-chloro-1-propen-1-yl)propanedioate
##STR00105##
[0855] The following reaction was conducted under a nitrogen
atmosphere.
[0856] Triethylamine (19.17 ml, 16.59 g, 0.1639 moles, 1.1
equivalents) was added in four portions to a reaction vessel
containing the 100 ml toluene solution of diethyl
propanoylpropanedioate [calculated by .sup.1H NMR to contain about
32.3 g (about 0.149 moles, 1 equivalent) of diethyl
propanoylpropanedioate] which was prepared in the Intermediate 2
(Stage la) process disclosed hereinabove. The reaction mixture was
heated to 70.degree. C. A mixture of phosphorus oxychloride
(POCl.sub.3, 20.83 ml, 34.27 g, 0.2235 moles, 1.5 equivalents) in
toluene (60 ml) was slowly added to the rapidly-stirred reaction
vessel, maintaining T.sub.int<85.degree. C. The contents were
then stirred at 85.degree. C. for about 21 hours. The reaction
mixture was then cooled using an ice bath to approximately
5.degree. C., and water (90 ml) was added over 1 hour (with the
first 70 ml added very slowly), with the maximum internal
temperature during the addition (T.sub.max) being 18.degree. C. The
biphase was vigorously stirred at 5-10.degree. C. for 15 minutes,
and then the two layers were allowed to separate. The aqueous layer
was separated and discarded. To the organic layer was added water
(90 ml) in one portion and the mixture stirred for 15 mins and then
allowed to separate. Due to slow separation and/or partial
emulsification at the interface, most of the aqueous layer was
removed, and then brine (20 ml) was added in an attempt to aid the
separation. After 20 mins, the aqueous layer, including some
emulsion and solid/scum from the interface, was separated and
discarded. 1 M Sodium bicarbonate solution (90 ml) was added to the
organic layer and the biphase was stirred for 30 minutes. After the
two layers were separated, extra toluene (60 ml) was added to the
organic layer. The organic layer was concentrated to 100 ml volume
in vacuo. The yield of the reaction using a .sup.1H NMR assay was
calculated as about 28.2 g, about 0.120 moles, about 80.7% theory
yield.
[0857] Note: .sup.1H NMR analysis, of a mini-worked-up sample of
the crude reaction mixture and/or of the final toluene product
solution, appears to suggest that the major product of the reaction
is the unconjugated vinyl chloride, diethyl
(1-chloro-1-propen-1-yl)propanedioate, as illustrated above.
Intermediate 2B:
Diethyl{1-[(1-ethyl-1H-pyrazol-5-yl)amino]propylidene}propanedioate
("Stage 1c" preparation)
##STR00106##
[0859] The following reaction was conducted under a nitrogen
atmosphere.
[0860] 5-Amino-1-ethylpyrazole (5.85 g, 0.0526 moles, 1.06
equivalents) was added in one portion to 31.35 g of a solution at
room temperature, wherein the solution is of diethyl
(1-chloro-1-propen-1-yl)propanedioate and/or diethyl
(1-chloropropylidene)propanedioate (about 11.65 g, 0.0496 moles,
1.0 equivalent) in toluene (about 19.70 g). Triethylamine (14 ml,
10.05 g, 0.0993 moles, 2.0 equivalents) was added slowly to the
reaction mixture ensuring T.sub.int<30.degree. C. by use of an
ice bath. The reaction mixture was then heated at 90.degree. C. for
approximately 15.5 hours and then was allowed to cool to room
temperature. The reaction mixture was filtered, the filter cake was
washed with toluene (23 ml), and then the filter cake was pulled
dry. Toluene (12 ml) was added to the filtrate, and the solution
was concentrated in vacuo to about 55 ml volume. Toluene (12 ml)
was added to the solution which was then concentrated down to about
55 ml, and this toluene (12 ml) addition and evaporation was
repeated. The toluene solution (about 55 ml) was washed with 2M
aqueous sodium hydroxide solution (55 ml), and the two layers were
separated. The organic layer was concentrated to dryness to yield
diethyl
{1-[(1-ethyl-1H-pyrazol-5-yl)amino]propylidene}propanedioate as a
brown liquid (11.11 g, about 72% theory yield). HPLC (5 min generic
solvent gradient): T.sub.RET about 3.05. Mass Spectrum: Found:
MH.sup.+ 310.1, (M-H).sup.- 308.1.
Intermediate 2C: Ethyl
1,6-diethyl-4-hydroxy-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
("Stage 1d" preparation)
##STR00107##
[0862] The following reaction was conducted under a nitrogen
atmosphere.
[0863] Diethyl
{1-[(1-ethyl-1H-pyrazol-5-yl)amino]propylidene}propanedioate (1.00
g, 3.23 mmol, e.g. which can be as prepared in Intermediate 2B) was
dissolved in toluene (8 ml). To the resulting dark orange solution,
1,8-diazabicyclo[5.4.0]undec-7-ene ("DBU", 0.97 ml, 0.98 g, 6.46
mmol, 2.0 equivalents) was added, and the reaction mixture was
heated at reflux for 17 hours. Further toluene (8 ml) was added to
the reaction mixture, after a reduction in volume after heating had
been noted. The reaction mixture was allowed to cool, and was
washed with water (8 ml). The two layers were separated, and the
organic layer was discarded after HPLC analysis suggested that the
majority of the product was in the aqueous layer. The aqueous
layer, whose pH was 12, was acidified to pH 1 using concentrated
aqueous hydrochloric acid (0.6 ml), whereupon gas was seen to
evolve and solid precipitated spontaneously from solution. This
solid was filtered and washed with water. The resulting beige solid
(0.55 g) was dried in a vacuum oven at 60.degree. C. overnight. The
title compound was obtained as a beige solid (0.46 g, about 54%
theory yield). HPLC (5 min generic solvent gradient): T.sub.RET
about 2.33. Mass Spectrum: Found: MH.sup.+ 264.1, (M-H).sup.-
262.1.
Intermediate 3: Ethyl
4-chloro-1,6-diethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
##STR00108##
[0865] Triethylamine (230 ml) was added dropwise to a mixture of
diethyl (1-chloropropylidene)propanedioate (208 g, e.g. which can
optionally be as prepared in Intermediate 2) and
1-ethyl-1H-pyrazol-5-amine (101 g, e.g. which can optionally be as
prepared in Intermediate 1) in toluene (2.65 L). The mixture was
heated under reflux for 16 hours. The reaction mixture was then
cooled to room temperature, and filtered through a sintered glass
funnel to remove the solid present. The filtrate was evaporated
under reduced pressure. The residue was then treated with
phosphorus oxychloride (POCl.sub.3, 2.65 L) and the mixture was
heated under reflux for 16 hours. Excess phosphorus oxychloride was
removed by distillation (e.g. under reduced pressure). The reaction
mixture was then cooled and poured onto a mixture of saturated
aqueous sodium hydrogen carbonate solution (4 L) and ethyl acetate
(1.5 L), maintaining the temperature at 5.degree. C. The organic
layer was separated and the aqueous layer further extracted with
ethyl acetate (2.times.1 L). The combined organic extracts were
washed with aqueous sodium hydrogen carbonate solution (1.times.2
L, e.g. optionally a saturated solution thereof) and were dried
(Na.sub.2SO.sub.4). Evaporation of solvent under reduced pressure
afforded the crude product (202 g). The crude product was purified
by chromatography (silica gel 60-120 mesh, 3.5 kg) eluting with 3%
ethyl acetate in hexane. Fractions containing the product were
pooled and evaporated to give the title compound (133 g) (which in
one embodiment can be e.g. a pale yellow thick liquid or oil which
may solidify on standing). .sup.1H NMR (200 MHz, chloroform-d)
.delta. (delta) ppm 1.37 (3H, t, J=7.1 Hz), 1.45 (3H, t, J=7.1 Hz),
1.57 (3H, t, J=7.2 Hz), 2.91 (2H, q, J=7.1 Hz), 4.48 (2H, q, J=7.2
Hz), 4.57 (2H, q, J=7.1 Hz) 8.05 (1H, s).
Intermediate 3 (alternative preparation): Ethyl
4-chloro-1,6-diethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
("Stage 2a" preparation)
##STR00109##
[0867] The following reaction was conducted under a nitrogen
atmosphere.
[0868] Ethyl
1,6-diethyl-4-hydroxy-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
(0.10 g, 0.38 mmol, 1 equivalent, e.g. which can be as prepared in
Intermediate 2C) was dissolved in dichloromethane (1.5 ml). Oxalyl
chloride (0.07 ml, 0.10 g, 0.76 mmol, 2.0 equivalents) was added in
one portion to the stirred solution. The reaction mixture, a
solution, was stirred at room temperature for 2 hours, then was
heated to 39.degree. C. and stirred for 22.5 hours at this
temperature, and then allowed to cool. The reaction mixture was
diluted with toluene and then the solvent was removed in vacuo to
give a brown oil which solidified on standing at room temperature
to yield the title compound as a brown solid (0.086 g, about 80%
theory yield). HPLC (5 min generic solvent gradient): T.sub.RET
about 3.43. Mass Spectrum: Found: MH.sup.+ 282.1.
Intermediate 4: Ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate
##STR00110##
[0870] To a solution of ethyl
4-chloro-1,6-diethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (36.0
g, 127.8 mmol, 1 equivalent, e.g. which can optionally be as
prepared in Intermediate 3) in N-methyl-2-pyrrolidinone (NMP, 300
ml) was added N,N-diisopropylethylamine (44.51 ml, 33.03 g, 255.6
mmol, 2.0 equivalents), resulting in a colour change from yellow to
orange. Tetrahydro-2H-pyran-4-amine (15.51 g, 153.3 mmol, 1.2
equivalents, commercially available e.g. from Peakdale or
Combi-Blocks) was added, and the reaction mixture was heated at
115.degree. C. with stirring overnight (for about 21.5 hours). The
reaction mixture was cooled to room temperature and poured into
water (1200 ml), forming an oily orange mixture. This mixture was
extracted with ethyl acetate (4.times.250 ml). The organic extracts
were combined, were washed with water (50 ml) and 5% aqueous
lithium chloride solution (50 ml), and were dried
(brine/MgSO.sub.4), filtered and evaporated to give an orange oil
(46.25 g) as the residue. This residue was purified by silica gel
(1 kg) chromatography, eluting with 2:1 cyclohexane:ethyl acetate
(6000 ml) followed by 1:1 cyclohexane:ethyl acetate (3000 ml). The
fractions containing the product only (whose R.sub.F is about 0.40
by TLC in 2:1 cyclohexane:ethyl acetate) were pooled and evaporated
to give the title compound as a mobile yellow oil (33.90 g, ca.
76.6% yield) which solidified to a yellow solid on standing. LCMS
m/z 347 [MH.sup.+]; T.sub.RET=3.01 min.
Intermediate 4 (Alternative Preparation): Ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate ("Stage 2b" preparation)
##STR00111##
[0872] The following reaction was conducted under a nitrogen
atmosphere.
[0873] Ethyl
4-chloro-1,6-diethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate [0.75
g, 2.66 mmol, 1.0 equivalent] was dissolved in
1-methyl-2-pyrrolidinone (NMP, 4 ml). 4-Amino-tetrahydro-2H-pyran
acetate (0.51 g, 3.19 mmol, 1.2 equivalents) was added in one
portion to the stirred solution at room temperature. The reaction
mixture was stirred for 15 minutes and then potassium carbonate
(1.10 g, 7.99 mmol, 3.0 equivalents) was added in one portion. The
reaction mixture was heated to and at 100.degree. C. for 16 hours,
and then cooled before partitioning between ethyl acetate (25 ml)
and water (25 ml) whereupon an emulsion formed. To this biphase
mixture/emulsion was added brine, followed by water (20 ml) and
then ethyl acetate (20 ml), to aid separation. The two layers were
separated and the aqueous layer was washed with ethyl acetate (20
ml). The organic layers were combined and washed with 1M aqueous
hydrochloric acid (3.times.25 ml). To the combined aqueous layers
was added ethyl acetate (25 ml); the resulting biphasic mixture was
stirred vigorously, and then 10M aqueous sodium hydroxide solution
(14.5 ml) was added to this stirred mixture until the pH of the
now-warm mixture was measured to be about 13. The mixture was
allowed to separate. The separated aqueous layer was washed with
ethyl acetate (20 ml). The combined organic layers were washed with
water (20 ml) followed by brine (20 ml). The organic layer was
concentrated to dryness to give an orange oil which visually
appeared to contain sodium chloride or similar crystals. This oil
was dissolved in ethyl acetate and washed with a minimal amount of
water; separation was poor. The organic layer was separated and
concentrated to dryness, to give the title compound as an orange
oil (still containing some crystals) which solidified after
standing overnight at room temperature (0.58 g, about 63%
yield).
Intermediate 5:
[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol
##STR00112##
[0875] A solution of ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate (17.94 g, 51.85 mmol, e.g. which can optionally be as
prepared in Intermediate 4) in dichloromethane (150 ml) was cooled
to 0.degree. C. under an atmosphere of nitrogen, and was treated
with di-iso-butylaluminum hydride (100 ml of a 1.5 M solution in
toluene, 150 mmol) added dropwise over 30 minutes, keeping the
temperature steady at 0.degree. C. The reaction was stirred at
0.degree. C. for a further 30 minutes after completion of the
addition. The reaction was quenched by the dropwise addition of
saturated aqueous potassium sodium tartrate solution (120 ml),
keeping the temperature below 5.degree. C. The resulting mixture
was diluted with ethyl acetate (300 ml) and the whole filtered
through a pad of Celite, to form two layers. During this filtration
the sinter funnel imploded. Material spilt on the fumehood floor
was soaked up and recovered as much as possible. The organic layer
was separated and dried (MgSO.sub.4), the drying agent was filtered
off, and the filtrate evaporated to give a first batch of product.
The aqueous layer and the celite were extracted with a larger
amount of ethyl acetate to give a second batch of product after
evaporation. The two batches of product were combined to give the
title compound (14 g) as an off-white waxy solid.
Intermediate 5 (Alternative preparation no. 1):
[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol
##STR00113##
[0877] To a mixture of ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate (60.43 g, 174 mmol, 1 equivalent, e.g. which can
optionally be as prepared in Intermediate 4) in dry tetrahydrofuran
(300 ml) was added dry methanol (28.3 ml, 22.3 g, 698 mmol, 4
equivalents), followed by slow addition of lithium borohydride
(LiBH.sub.4, 2 M solution in tetrahydrofuran, 262 ml, 523 mmol, 3
equivalents) over 30 minutes. The mixture was heated as the lithium
borohydride was added, reaching reflux after about 20 minutes.
Further aliquots of methanol (14.1 ml, 2 equivalents, each time)
were added after 1 hour and again after 1.5 hours, and the heating
was continued for a further 30 minutes (for 2 hours total). The
reaction mixture was cooled (ice/water bath) and was carefully
treated with methanol (100 ml, non-dried) followed by cautious
addition of water (200 ml) which resulted in cloudiness and then a
precipitate. Addition of more water (800 ml) produced a more
homogenous suspension which was stirred for 1 hour and which was
then extracted with dichloromethane (about 1.5 L total volume). The
organic extracts were combined, were washed with water and then
brine, and were dried and evaporated to give the title compound as
a white solid (49.84 g). LCMS m/z 305 [MH.sup.+]; T.sub.RET=1.79
and 1.83 min (double peak).
Intermediate 5 (Alternative Preparation no. 2, Plant Method):
[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol
##STR00114##
[0878] Summary of Plant Method
[0879] All weights, volumes (vol., vols) and equivalents are
relative to the ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridine-5-carboxylate.
[0880] The reaction is conducted under a nitrogen atmosphere, in a
plant, and the organic reaction solvents are usually dry or
moderately dry.
[0881] To a stirred solution of lithium borohydride (LiBH.sub.4, 3
eq, 4.34vol, 2M solution in tetrahydrofuran (THF)) and dry toluene
(4 vols) at 64-68.degree. C., is added a solution of ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate (1 wt, e.g. which can optionally be as prepared in
Intermediate 4) in dry THF (1 vol.) and dry methanol (9 eq,
1.05vol.) over at least 2.5 hours. The reaction is monitored by
HPLC and is typically complete about 60 minutes (50-70 mins) after
the end of the addition. The reaction mixture is then cooled down
to 20.+-.3.degree. C., and water (3 vol.) is added dropwise over at
least 30mins followed by aqueous sodium hydroxide solution (10.8M,
about 32% w/w, 6.0 vols, 8.1 wt) (dropwise over about 30-40 mins),
ensuring the temperature remains below 40.degree. C. The
temperature of the reaction is then adjusted to 37.+-.3.degree. C.
and stirred vigorously for at least 90 minutes. The organic layer
and aqueous layer are separated and the aqueous layer is treated
with sulphuric acid (11 vol., 13.2 wt) to remove any remaining
lithium borohydride. Water (2.5 vols) is added to the organic layer
over at least 30 mins followed by aqueous sodium hydroxide solution
(10.8M, about 32% w/w, 2 vols, 2.7 wt) over about 30-40 mins, and
the biphase is stirred vigorously at 37.+-.3.degree. C. for at
least 30 minutes. The organic layer is again separated and the
organic solution is concentrated in vacuo to 3 vols, which
precipitates the product, usually as yellow solid. Toluene (3 vol.)
is then added and the slurry is reconcentrated in vacuo to 3 vol.
The suspension is then cooled to 10.+-.3.degree. C. and aged for at
least 30 min. The solid is then filtered and washed with toluene (3
vol.). The product
[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol is then dried in vacuo at about 45.degree. C. to
constant temperature.
[0882] Step-Wise Methodology Used in One Pilot Plant Batch
[0883] All weights, volumes (vol., vols) and equivalents are
relative to the ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridine-5-carboxylate.
[0884] The reaction was conducted under a nitrogen atmosphere, in a
pilot plant.
[0885] 1. Making up solution of ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate:
Dry THF (1 vol., 11 litres) was charged to a glass lined reactor
followed by dry methanol (1.0 vol., 11 litres), the mixture was
stirred at 20.+-.5.degree. C. for about 10 min at 75 rev./min, and
ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate (1 wt, 11.0 kg) was added. The walls of the charge
hole (for the solid ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate) were washed clean with dry methanol (0.05 vol., 0.55
litres) which went into the reactor. The contents of the glass
lined reactor were stirred at 20.+-.5.degree. C. for about 1 hr 11
minutes at 75 rev./min.
[0886] 2. Dry toluene (4 vol., 44 litres) was charged to a separate
glass lined reactor and the contents were stirred.
[0887] 3. A 2M solution of lithium borohydride in THF (3.89 wt,
42.8 kg) was charged to the stirred toluene-containing reactor over
about 1 hour from a cylinder using a pyrophoric dispensing
manifold. The contents of the reactor were heated to
66.+-.3.degree. C. (target range 64-68.degree. C.) over about 50
mins.
[0888] 4. The solution made up in step 1 was then charged to the
stirred toluene and LiBH.sub.4 solution via a peristaltic pump over
2 hrs 34 minutes, maintaining the reactor contents at a temperature
of 66.+-.3.degree. C. (target range 64-68.degree. C.) whilst
maintaining an even paced addition rate.
[0889] 5. After the addition of the ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate, the reaction contents were stirred at
66.+-.3.degree. C. (target range 64-68.degree. C.) (contents
temperature) at 75 rev./min for 65 minutes, and the reaction was
sampled for HPLC analysis. HPLC indicated<6% a/a (about 4.7%) of
the ethyl
1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine--
5-carboxylate remaining, and so the reaction was considered
complete.
[0890] 6. Straight after the sample has been taken, the contents
were cooled to 20.+-.3.degree. C. over about 30 mins with
stirring.
[0891] 7. With the stirrer speed of the reactor increased to 125
rev./min, water (3 wt, 33.00 kg, 33 litres) was added to the
reaction via a peristaltic pump over 33 minutes, during which the
contents temperature was kept below 40.degree. C. (T.sub.max of the
contents was 32.degree. C.). During this water addition the product
usually precipitates forming a thick suspension.
[0892] 8. After the water addition was complete the reactor
contents were cooled to 20.+-.5.degree. C.
[0893] 9. Aqueous sodium hydroxide solution (about 32% w/w, 8.1 wt,
89.10 kg) was added to the reactor via a peristaltic pump over 35
minutes.
[0894] 10. After the addition of the sodium hydroxide solution, the
contents of the reactor were stirred at 37.+-.3.degree. C. for 90
minutes.
[0895] 11. The agitator was stopped and the layers were allowed to
separate for at least 15 min.
[0896] 12. The lower aqueous layer was separated and transferred to
another vessel.
[0897] 13. The upper organic layer was cooled to 20.+-.3.degree.
C., and was held for about 9.5 hours.
[0898] 13A. With the stirrer speed of the reactor increased to 125
rev./min, water (2.5 wt, 27.50 kg) was added to the organic layer
via a peristaltic pump over 30 minutes, during which time the
contents temperature was kept below 40.degree. C.
[0899] 14. The reactor contents were cooled to 20.+-.5.degree. C.,
and aqueous sodium hydroxide solution (about 32% ww, 2.7 wt, 29.70
kg) was added via a peristaltic pump over 35 minutes.
[0900] 15. The contents of the reactor were stirred at
37.+-.3.degree. C. for 33 minutes.
[0901] 16. The stirrer was stopped and the layers were allowed to
separate for 15 minutes.
[0902] 17. The lower aqueous layer was separated and transferred to
another vessel.
[0903] 18. The remaining reactor contents (organic layer) was
stirred and cooled to 5.+-.3.degree. C. and then medium vacuum was
applied (about -0.8 to -0.85 barg vacuum) to degas the reactor
contents (to remove the hydrogen formed in the reaction).
[0904] 19. The reactor contents were adjusted to 5.degree. C. with
stirring, and the reactor contents were heated under a vacuum of
about -0.83 barg. Initially the reactor contents were heated to
about 26.degree. C. and eventually to about 45.degree. C., to
remove solvent by vacuum distillation until the batch was
concentrated to about 33 litres (about 3 vols). The distillation is
complete after 4.5 hours of heating, leaving a slurry in the
reactor.
[0905] 20. The vacuum was released and toluene (3 vols, 33 litres)
was charged to the reactor.
[0906] 21. The reactor contents were adjusted to 5.degree. C. with
stirring.
[0907] 22. The reactor contents were heated, under a vacuum ranging
from about -0.84 barg to about -0.91 barg. The reactor contents
were heated to about 40.degree. C. (at about -0.84 barg) and then
to a maximum of 46.degree. C. (at about -0.88 barg) and then to
about 40.degree. C. (at about -0.91 barg), to remove solvent by
vacuum distillation until the batch was concentrated to about 33
litres (about 3 vols). The distillation was complete after about 4
hours 18 minutes of heating.
[0908] 23. The vacuum was released and the contents were cooled to
10.+-.3.degree. C. and stirred at this temperature for about 6.25
hours at 75 rev./min.
[0909] 24. The resulting slurry was then transferred to a 20 micron
filter cloth, and filtered under nitrogen pressure (0.5-1
barg).
[0910] 25. The filter cake was washed with toluene (3 vol., 33
litres) under nitrogen pressure (0.5-1 barg), and then solvent was
blown from the filter cake using nitrogen pressure over 11
minutes.
[0911] 26. The damp filter cake was transferred to polythene lined
trays, covered with muslin bags, placed in a vacuum tray dryer, and
was dried by heating to 47.+-.3.degree. C. under vacuum (using a 5
L/min nitrogen bleed to the dryer, to minimise build-up of
condensed solvents in the oven), until a constant temperature of
the solid was measured for at least 4 hours. The batch was cooled
to below 30.degree. C.
[0912] 27. The product
[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol was obtained as a solid (8.55 kg, 88.5% theory
yield).
[0913] * Note: The remaining additional steps, below, concern the
acidic quenching of the aqueous layers and mother liquors which is
done for safety reasons to remove any borohydride residues left in
these solutions on plant. These stages are waste management steps,
separate to the core production of product, and so are not
essential and may or may not be performed as is desired.
[0914] 28. The aqueous layers from step 12 & 17 are stirred and
cooled to 5.+-.3.degree. C.
[0915] 29. 5M sulphuric acid (13.2 wt, 145.2 kg) is charged to the
aqueous layers via peristaltic pump over 3 hrs 52 minutes
maintaining a contents temperature below 40.degree. C.
[0916] 30. Post the acid addition, the contents are stirred at
5.+-.5.degree. C. for 2 hrs 18 minutes. The pH is checked, if the
pH<3 the quench is considered complete.
[0917] 31. The reactor is degassed using vacuum and purging with
nitrogen before the aqueous solution is discharged and labelled as
waste.
[0918] 32. The mother liquors produced during the filtration steps
(24 & 25) are transferred to a reactor, stirred and cooled to
5.+-.3.degree. C.
[0919] 33. Water (5 litres) is charged to the liquors.
[0920] 34. 5M sulphuric acid (1 kg) is charged to the aqueous
layers via peristaltic pump over 1 hr maintaining a contents
temperature below 40.degree. C.
[0921] 35. After the acid addition, the contents are stirred at
5.+-.5.degree. C. for 2 hrs 18 minutes. The pH is checked; if the
pH<3 the quench is considered complete.
[0922] 36. The reactor is degassed using vacuum and purging with
nitrogen before the biphasic solution is discharged and labelled as
waste.
Intermediate 6:
5-(Azidomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine
##STR00115##
[0924] Thionyl chloride (SOCl.sub.2, 50.37 ml, 82.11 g, 69 mmol)
was added dropwise over 15 minutes to a suspension of
[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol (13.98 g, 46 mmol, e.g. which can optionally be as
described in Intermediate 5) in toluene (140 ml) at room
temperature. The mixture was then heated at reflux at about
85.degree. C. for 4 hours, was allowed to cool, and then was
evaporated to dryness and azeotroped with three further portions of
toluene. The residue obtained was dissolved in dry
dimethylsulfoxide (100 ml) and treated with sodium azide (4.49 g,
69 mmol), and the mixture was stirred at room temperature for 18
hours. The mixture was poured into saturated aqueous sodium
bicarbonate solution and was extracted with 3 portions of ethyl
acetate. The combined organic extracts were washed with saturated
brine and water, and were then dried (Na.sub.2SO.sub.4), the drying
agent was filtered off, and the filtrate was evaporated to dryness.
The residue obtained was purified by flash chromatography, eluting
with 1:1 cyclohexane:ethyl acetate. Fractions containing the
product without substantial amounts of impurities were evaporated
to give the title compound (5.6 g+4.75 g=10.35 g).
Intermediate 7:
5-(Aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine
##STR00116##
[0926] A suspension of
5-(azidomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine (10.3 g, 34 mmol, e.g. which can be as prepared
in Intermediate 6) in ethanol (200 ml) was added to 10% palladium
on carbon (1 g) and stirred under an atmosphere of hydrogen for 3
hours at room temperature. The catalyst was removed by filtration
under nitrogen and was washed well with ethanol. The filtrate was
evaporated to dryness to give a dark oil. Ether was added and then
evaporated to give the title compound as a foamy semi-solid (9.35
g).
Intermediate 7 (Alternative Preparation no. 1):
5-(Aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine
[0927] In a hydrogenation flask, ethanol (300 ml) was added to 10%
palladium on carbon (9.9 g, 0.2 wt. equivalents, 50% wet), followed
by
5-(azidomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine (49.62 g, 151 mmol, 1 equivalent, e.g. which can
be as prepared in Intermediate 6) in ethanol (900 ml). The mixture
was hydrogenated overnight at room temperature and room pressure
(using the Wright valve); during which the flask was vented and
refilled once. The catalyst was removed by filtration and the
filtrate was evaporated to give a grey solid. The residue was
purified by column chromatography, using a relatively small amount
of silica (1500 ml of 9385 silica), eluting initially with 5%
methanol in dichloromethane (to elute some fast-moving impurities),
followed by 10% and finally 15% methanol in dichloromethane to give
the title compound as an almost white solid (41.25 g). LCMS m/z 304
[MH.sup.+]; T.sub.RET=1.65 and 1.69 min (split peak). NMR (d6-DMSO)
showed a trace of methanol present in the product.
Intermediate 7A:
5-(Aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine monohydrochloride (Plant Method)
##STR00117##
[0928] Intermediate 7A Process Scheme
##STR00118##
[0929] Intermediate 7A Process Summary
[0930] All weights and volumes are relative to
[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol starting material, unless otherwise stated.
[0931] The reactions are conducted under an atmosphere of nitrogen
in plant, and the organic reaction solvents are usually dry or
moderately dry.
[0932]
[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]py-
ridin-5-yl]methanol (1 wt) (e.g. preferably substantially as
prepared in Intermediate 5, alternative preparation no. 2, plant
method) is suspended in dry anisole (5.97 wt, 6 vol) and treated
with solid benzensulfonic acid (0.52 wt, about 1 equiv.). This
suspension is aged at 20.+-.5.degree. C. for at least 30 minutes.
Thionyl chloride (0.45 wt, about 1.15 equiv.) is added at
20.+-.5.degree. C. over at least 20 minutes, and stirred for
between 20 and 30 minutes. The reaction is then sampled for HPLC.
The resulting solution of
5-(chloromethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-
-b]pyridin-4-amine benzenesulfonate is treated with dry toluene (2
vol) and then placed under medium vacuum (about -0.85 barg, e.g.
about 100-150 mbar) to remove hydrogen chloride and sulfur dioxide,
and then the vacuum is increased (about -0.95 barg, e.g. about 50
mbar) to azeotrope off excess thionyl chloride and toluene.
[0933] This solution/slurry of
5-(chloromethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-
-b]pyridin-4-amine benzenesulfonate (ca. 7.5 vol) is added to a
solution of lithium hexamethyldisilazide (LiHMDS) in
tetrahydrofuran (1.3M in THF, 5.73 wt, 6.51 vol, about 2.5 equiv.)
at 35.+-.5.degree. C. over 60-70 minutes and then stirred for a
further 15 to 30 minutes.
[0934] The mixture is cooled to 10.+-.5.degree. C. and 5M
hydrochloric acid (3.78 wt, 3.6 vol) is added. The phases are
separated and the lower aqueous layer is transferred back into the
vessel, and the organic phase is discarded. 2-Me-THF
(2-methyl-tetrahydrofuran) (8 vol) is added and then the biphasic
mixture is adjusted to pH>13 with 32% w/w aqueous sodium
hydroxide solution (2.25 wt) and then warmed to 30.+-.5.degree. C.
The layers are separated and the lower aqueous layer is back
extracted with further 2-Me-THF (2 vol). The combined organic
phases are washed with 15% w/w aqueous sodium chloride solution
(2.2 wt).
[0935] The amount of
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine in solution is quantified via yieldaliser (uses a
solution-based assay, using a comparator standard solution of known
concentration), and an appropriate amount of 36% w/w aqueous
hydrochloric acid (1.03 equivalents based on the calculated and/or
measured amount of the product amine) is added at 55.+-.5.degree.
C. The suspension is held for 2 hours at 55.+-.5.degree. C., and
then cooled to 5.+-.5.degree. C. over 3 hours and held at
5.+-.5.degree. C. for at least 3 hours. The slurry is filtered and
the separated solid is washed with 2-Me-THF (2.times.2vol) and
dried in vacuo at 60.degree. C. to constant weight or batch
temperature to give the product
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine monohydrochloride (Intermediate 7A).
Intermediate 7A: Detailed Step-wise Methodology
[0936] All weights and volumes are relative to
[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol starting material, unless otherwise stated.
[0937] The reactions are conducted under an atmosphere of nitrogen
in plant.
Chlorination Reaction
[0938] 1. Anisole (5.97 wt, 71.6 kg) is charged to the reactor.
[0939] 2.
1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]-
pyridin-5-yl]methanol (lwt, 12.0 kg) (e.g. preferably substantially
as prepared in Intermediate 5, alternative preparation no. 2, plant
method) is charged to the reactor.
[0940] 3. Benzenesulfonic acid (0.52 wt, 6.24 kg) is charged to the
reactor and the contents are stirred at 20.+-.5.degree. C. for 34
minutes.
[0941] 4. Thionyl chloride (0.45 wt, 5.4 kg) is charged to the
reactor via peristaltic pump over 28 minutes, maintaining the
reactor contents at 20.+-.5.degree. C.
[0942] 5. The reactor contents are stirred at 20.+-.5.degree. C.
for 28 minutes and then the contents are sampled for HPLC analysis.
The volume of the reaction is noted.
[0943] 6. Toluene (2 vols, 24 litres) is then charged to the
stirred reactor.
[0944] 7. The reactor contents are placed under vacuum (ca -0.85
barg) and stirred at 20.+-.5.degree. C. for 1 hr 6 minutes.
[0945] 8. The reactor contents are then cooled to 10.degree. C.
[0946] 9. The reactor is then subjected to full vacuum for the
distillation (-0.95 to -0.97 barg).
[0947] 10. The batch is then heated, with an initial internal
temperature of 18.degree. C. rising to 40.degree. C. by the end of
the distillation. The end point of the distillation is the volume
noted in step 5. This can be reached e.g. after 2 hrs 43 minutes of
heating.
[0948] 11. The vacuum is released and the contents are stirred at
20.+-.5.degree. C. This is the anisole slurry of
5-(chloromethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-
-b]pyridin-4-amine benzenesulfonate. In this example an extra
charge of 15 litres of anisole is made to this slurry.
[0949] Amination Reaction
[0950] 12. To a different reactor, lithium hexamethyldisilazide
(LiHMDS) solution (1.3M in THF, 5.73 wt, 68.8 kg) is charged and
then the contents are stirred and heated to 35.+-.5.degree. C.
[0951] 13. The slurry of
5-(chloromethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-
-b]pyridin-4-amine benzenesulfonate (from step 11) is added to the
LiHMDS solution in portions via a saunders valve over 46 minutes.
The reactor contents temperature is maintained at 35.+-.5.degree.
C.
[0952] 14. In this example several rinses are conducted to clean
the lines. A line wash of the Kammer valve line (which can become
blocked) is conducted to clear residual solid using anisole (2 kg),
and a vessel rinse using anisole (0.25 wt, 3.0 kg) is conducted to
rinse further
5-(chloromethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-
-b]pyridin-4-amine benzenesulfonate into the LiHMDS solution.
[0953] 15. After the residual solid is rinsed in, the amination
reaction is stirred at 35.+-.5.degree. C. for 20 minutes.
[0954] 16. The amination reaction is sampled for completion by HPLC
analysis.
[0955] 17. The reactor contents are then stirred and cooled to
10.+-.5.degree. C.
[0956] 18. 5M aqueous hydrochloric acid (3.78 wt, 45.4 kg) is
charged to the reactor via a peristaltic pump over 52 minutes,
maintaining an internal temperature of 10.+-.5.degree. C.
[0957] 19. The resultant biphase is stirred at 10.+-.5.degree. C.
for at least 15 minutes.
[0958] 20. The stirrer is stopped and the 2 layers are allowed to
separate. The lower aqueous layer is transferred to a drum and is
an aqueous solution of
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine hydrochloride. The pH is checked (expect
pH<5).
[0959] 21. The upper organic layer is discharged as waste.
[0960] 22. Further line and vessel rinses are conducted running
from the ex-thionyl chloride reactor vessel through the Kammer
valve to the ex-amination reactor. The rinses are THF (30 litres)
and water (60 litres).
[0961] 23. The aqueous solution of
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine hydrochloride (from step 20) is charged to a
reactor and stirred.
[0962] 24. 2-Methyl-tetrahydrofuran (2-Me-THF) (8 vol, 96 litres)
is charged to the aqueous solution.
[0963] 25. Aqueous sodium hydroxide solution (32% ww, 2.25 wt, 27
kg) is charged to the reactor via a peristaltic pump over 30
minutes. Reactor contents are maintained at 10.+-.5.degree. C.
[0964] 26. The reactor contents are heated to 30.+-.5.degree. C.
and stirred for at least 15 minutes.
[0965] 27. The stirrer is stopped and the two layers are allowed to
separate. The lower aqueous layer is transferred to a drum and is
an aqueous solution of the product amine. Its pH is checked (expect
pH>13).
[0966] 28. The upper layer is also transferred to a drum and is a
2-methyl-tetrahydrofuran solution of the product amine.
[0967] 29. The aqueous solution of the product amine (from step 27)
is charged to the reactor and 2-methyl-tetrahydrofuran (2-Me-THF)
(2 vols, 24 litres) is added to back extract the aqueous layer
further. The biphase is stirred at 30.+-.5.degree. C. for at least
15 minutes.
[0968] 30. The stirrer is stopped and the two layers are allowed to
separate. The lower aqueous layer is discharged as waste, and the
upper organic layer is retained in the reactor.
[0969] 31. The 2-methyl-tetrahydrofuran solution of the product
amine (from step 28) is charged back into the reactor and the
combined organic layers are stirred.
[0970] 32. 15 wt % aqueous sodium chloride solution (2.2 wt, 26.4
kg) is charged to the reactor.
[0971] 33. The biphase is heated to 30.+-.5.degree. C. and stirred
for at least 15 minutes.
[0972] 34. The stirrer is stopped and the two layers are allowed to
separate. The lower aqueous layer is discharged as waste, the upper
organic layer is retained in the reactor, and the volume of the
solution is noted.
[0973] 35. The reactor contents are sampled for HPLC analysis. An
approximate yield of the product
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine is obtained from this analysis.
Formation and Isolation of Hydrochloride Salt of Amine
[0974] 36. The contents of the reactor are heated to 55.+-.5
DC.
[0975] 37. Concentrated aqueous hydrochloric acid (1.03 eq.
relative to yield of
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyra-
zolo[3,4-b]pyridin-4-amine calculated in step 35, e.g. in this
example this can be about 2.37 kg) is added to the reactor via a
peristaltic pump over 20 minutes, maintaining the reactor contents
at 55.+-.5.degree. C.
[0976] 38. The reactor contents are stirred at 55.+-.5.degree. C.
for 2 hrs 6 minutes.
[0977] 39. The reactor contents are gradually cooled to
5.+-.5.degree. C. over 3 hours.
[0978] 40. The slurry is then stirred at 5.+-.5.degree. C. for at
least 3 hours. In this example, the reactor contents are stirred at
5.+-.5.degree. C. overnight for a stir time of 19 hrs 53
minutes.
[0979] 41. The slurry is transferred to a 20 micron filter cloth,
is allowed to settle for 10 mins, and is filtered under nitrogen
pressure (0.5 to 1 barg).
[0980] 42. The filter cake is washed with 2-methyl-tetrahydrofuran
(2 vols, 24 litres) under nitrogen pressure (0.5 to 1 barg).
[0981] 43. The filter cake is washed with further
2-methyl-tetrahydrofuran (2 vols, 24 litres) under nitrogen
pressure (0.5 to 1 barg). Further solvent is removed from the cake
by blowing under nitrogen pressure over 1 hr 40 minutes.
[0982] 44. The damp filter cake is transferred to polythene lined
trays, covered with muslin bags, and placed in a vacuum tray dryer.
The vacuum tray dryer is put under vacuum and is heated to
60.+-.5.degree. C. to dry the product, using a 5 L/min nitrogen
bleed to the dryer (to minimise build-up of condensed solvents in
the oven). The drying is considered complete when the product
temperature probe reads a constant temperature for at least 4
hours.
[0983] 45. The product
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine monohydrochloride (Intermediate 7A) is collected
as a solid, usually as a white solid.
Intermediate 8: 1,1-Dimethylethyl
{4-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]py-
ridin-5-yl]methyl}amino)carbonyl]phenyl}carbamate
##STR00119##
[0985] A solution of
4-({[(1,1-dimethylethyl)oxy]carbonyl}amino)benzoic acid (0.96 g,
4.05 mmol, e.g. available from Fluka) in N,N-dimethylformamide (20
ml) was treated with
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU, 1.7 g, 4.45 mmol, 1.1 equiv.) and then
N,N-diisopropylethylamine (1.77 ml, 10.1 mmol, 2.5 equiv.). The
resulting solution was allowed to stand at room temperature for 10
minutes.
5-(Aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine (1.24 g, 4.05 mmol, which can e.g. be as prepared
in Intermediate 7) was added to this solution and the resultant
mixture was allowed to stand at room temperature for 2 hours. The
N,N-dimethylformamide solvent was removed by evaporation and the
residue was partitioned between saturated aqueous sodium
bicarbonate solution (100 ml) and dichloromethane (150 ml). The
organic layer was separated and collected through a first
hydrophobic frit. Meanwhile, there was some solid which did not
dissolve in either the organic or aqueous layer. This solid, which
contained some product, was removed by filtration, was partitioned
between dichloromethane and water, and the organic layer was
isolated through a second hydrophobic frit. The combined organic
layers from the first and second hydrophobic frits were evaporated
to dryness. The residue from the organic layers was dissolved in
dichloromethane and methanol and was purified by passing through
two 50 g silica SPE cartridges, eluting with a cyclohexane:ethyl
acetate gradient (some product eluted with 100% ethyl acetate)
followed by 9:1 ethyl acetate: methanol (in which more product
eluted). Fractions containing the product were combined and
evaporated to dryness. Trituration with diethyl ether, and
filtration and drying in vacuo of the solid gave the title compound
as a cream-coloured solid (1.75 g). LCMS showed MH.sup.+=523;
T.sub.RET=2.75 min.
Intermediate 9:
4-Amino-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
-b]pyridin-5-yl]methyl}benzamide hydrochloride
##STR00120##
[0987] To 1,1-dimethylethyl
{4-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]py-
ridin-5-yl]methyl}amino)carbonyl]phenyl}carbamate (1.75 g, 3.35
mmol, which can e.g. be as prepared in Intermediate 8) was added a
4M solution of HCl in 1,4-dioxane (15 ml) resulting in a
precipitate. This mixture was allowed to stand at room temperature
overnight. The solvent was removed by evaporation; co-evaporation
with diethyl ether (3.times.30 ml) gave the title compound as a
pale yellow/brown foam (2.12 g, >100%, NMR showed unknown
impurity present). LCMS showed MH.sup.+=423; T.sub.RET=2.23
min.
Intermediate 10: 8-Bromooctanoyl chloride
##STR00121##
[0989] To 8-bromooctanoic acid (2.5 g, 11.2 mmol, commercially
available e.g. from Aldrich) in toluene (50 ml) was added thionyl
chloride (2 ml), and the mixture was stirred at room temperature
overnight. The solvent was evaporated and the residue was
co-evaporated with dichloromethane (2.times.25 ml) to give a brown
oil which solidified on standing (found to be starting acid by
NMR). This material was redissolved in toluene (25 ml), thionyl
chloride (2 ml) was added, and the mixture was stirred at room
temperature for 8 hours and then left standing overnight. The
reaction mixture was evaporated to dryness and the residue was
co-evaporated with dichloromethane (2.times.30 ml) to give the
title compound as a brown oil (2.4 g), as a 3:2 mixture of product:
starting acid (by NMR). .sup.1H NMR (400 MHz, chloroform-d) .delta.
(delta) ppm 1.37 (4H, m), 1.45 (2H, m), 1.67 (2H, m), 1.86 (2H, m),
2.90 (2H, t, J=7 Hz), 3.42 (2H, t, J=7 Hz).
Intermediate 10 (alternative synthesis): 8-Bromooctanoyl
chloride
[0990] 8-Bromooctanoic acid (2.0 g, 8.97 mmol, commercially
available e.g. from Aldrich) in thionyl chloride (2 ml) was stirred
at room temperature for 1 hour and then at 100.degree. C. for 2
hours. The thionyl chloride was removed by evaporation and the
residue was co-evaporated with dichloromethane (2.times.30 ml) to
give 8-bromooctanoyl chloride as a colourless mobile oil (2.05
g).
Intermediate 11:
4-[(8-Bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide
##STR00122##
[0992] To a stirred gummy mixture of
4-amino-N-{([1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,-
4-b]pyridin-5-yl]methyl}benzamide hydrochloride (2.0 g, 4.34 mmol,
which can e.g. be as prepared in Intermediate 9) in dichloromethane
(50 ml) cooled to 0.degree. C. under nitrogen was added
triethylamine (3.1 ml, 21.8 mmol, 5 equiv.) resulting in a white
suspension. To the suspension was added, by dropwise addition over
5 minutes, a solution of 8-bromooctanoyl chloride (2.1 g, 8.69
mmol, which can e.g. be as prepared in Intermediate 10) in
dichloromethane (10 ml). The mixture was then stirred at 0.degree.
C. for 1 hour, allowed to warm to room temperature and stirred for
a further 18 hours. The mixture was poured into ice, diluted with
dichloromethane, and the organic layer was separated and collected.
The aqueous layer was extracted with more dichloromethane (50 ml).
The organic layers were combined and evaporated to dryness to give
a brown gum, which was triturated with diethyl ether. The resulting
solid was filtered off and dried in vacuo to give the title
compound as a pale yellow solid (0.98 g) containing about 15% (by
LCMS) of unreacted starting material
(4-amino-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,-
4-b]pyridin-5-yl]methyl}benzamide hydrochloride).
[0993] LCMS of the product showed MH.sup.+=627/629 (bromine
isotopes); T.sub.RET=3.04 min. The pyrazolo[3,4-b]pyridine starting
material present showed MH.sup.+=423; T.sub.RET=2.25 min.
[0994] A white suspension was present in the aqueous phase from the
above extraction; this was extracted with ethyl acetate (3.times.70
ml) and the combined ethyl acetate layers were evaporated to
dryness to give a pale yellow/brown gum (1.91 g). This appeared by
LCMS to be very approximately 43%:57% starting material: product.
The gum was suspended in dry dichloromethane (50 ml), treated with
triethylamine (0.8 ml), and cooled to about 0.degree. C. using an
ice/water bath. To this mixture was added dropwise over 3 minutes
8-bromooctanoyl chloride (0.8 g, which can e.g. be as prepared in
Intermediate 10). After 10 mins at 0.degree. C., the mixture was
allowed to warm to room temperature, stirred for 5 hours and
allowed to stand at room temperature overnight. The mixture was
poured into ice, diluted with dichloromethane (30 ml) and the
layers separated. The aqueous phase was further extracted with
dichloromethane (20 ml), and the combined organic phases were
passed through a hydrophobic frit and evaporated to dryness. The
residue was triturated with diethyl ether, and the resulting solid
was filtered off and dried in vacuo to give the title compound as a
cream solid (0.66 g). LCMS showed MH.sup.+=629; T.sub.RET=3.04
min.
Intermediate 13: Ethyl
4-[(1-{[(1,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]-1,6-diethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
##STR00123##
[0996] A solution of ethyl
4-chloro-1,6-diethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (14.8
g, 52.6 mmol, e.g. which can be as prepared in Intermediate 3,
first or alternative preparation thereof) in
N-methyl-2-pyrrolidinone (140 ml) was treated with
N,N-diisopropylethylamine (22.9 ml, 131 mmol) followed by
1,1-dimethylethyl 4-amino-1-piperidinecarboxylate (11.6 g, 57.9
mmol, commercially available e.g. from AstaTech) and the mixture
was heated at 120.degree. C. for 24 hours. The reaction mixture was
cooled and poured into aqueous lithium chloride solution (5% LiCl,
1 L) and the aqueous phase was extracted with ethyl acetate
(3.times.250 ml). The combined organic extracts were dried
(Na.sub.2SO.sub.4) and the solvent was evaporated. The residue, an
oil, was purified by column chromatography using a column of silica
gel (ART9385, 500 ml) made up in 4:1 hexane; [0997] ethyl acetate,
eluting with 4:1 hexane:ethyl acetate (500 ml) and then 2:1
hexane:ethyl acetate until the product eluted. The fractions
containing the product were pooled and the solvent was evaporated
to give the title compound (18.55 g) as an oil which solidified on
standing. LCMS m/z 446 [MH.sup.+]; T.sub.RET=3.69 min.
Intermediate 14: 1,1-Dimethylethyl
4-{[1,6-diethyl-5-(hydroxymethyl)-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-
-piperidinecarboxylate
##STR00124##
[0999] A solution of ethyl
4-[(1-{[(1,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]-1,6-diethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (18.3 g, 41.1 mmol, e.g.
which can be as prepared in Intermediate 13) in tetrahydrofuran
(100 ml) was treated with lithium borohydride (LiBH.sub.4, 2M in
tetrahydrofuran, 40 ml, 80 mmol) followed by methanol (3.6 ml, 88.8
mmol), was heated under reflux for 3 hours, and then was cooled to
about room temperature. To the cooled mixture was added a further
portion of lithium borohydride (LiBH.sub.4, 2M in tetrahydrofuran,
40 ml, 80 mmol) followed by methanol (3.6 ml, 88.8 mmol). The
mixture was heated under reflux for 2 hours and allowed to cool to
room temperature. Solid lithium borohydride (1.6 g, 73.5 mmol) was
added followed by methanol (3.6 ml, 88.8 mmol), and the mixture was
heated under reflux for 2 hours and allowed to cool overnight. The
mixture was cooled in an ice/water bath, and treated with methanol
(20 ml). After stirring for 10 min, water (10 ml) was added. When
the effervescence ceased more water was added cautiously (500 ml
total volume) and the mixture was stirred for 45 minutes. The
mixture was partitioned between water and dichloromethane. The
aqueous phase was separated and extracted with dichloromethane
(.times.2). The combined organic extracts were washed with brine,
dried (Na.sub.2SO.sub.4) and the solvent was evaporated. The
residue was purified by column chromatography (800 ml of ART9385
silica gel, column made up using 1:1 hexane:ethyl acetate), using
chloroform to load the mixture and eluting with
hexane:ethyl:acetate (1:1, 800 ml), hexane:ethyl acetate (1:2,1800
ml) and finally neat ethyl acetate to complete elution of the
product. The fractions containing the product were combined and the
solvent was evaporated to give the title compound (12.4 g). LCMS
m/z 404 [MH.sup.+]; T.sub.RET=2.40 min.
Intermediate 15: 1,1-Dimethylethyl
4-{[5-(azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-p-
iperidinecarboxylate
##STR00125##
[1001] A suspension of 1,1-dimethylethyl
4-{[1,6-diethyl-5-(hydroxymethyl)-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-
-piperidinecarboxylate (14.0 g, 34.7 mmol, e.g. which can be as
prepared in Intermediate 14) in N,N-dimethylformamide (110 ml) was
treated with sodium azide (4.59 g, 70.6 mmol) and carbon
tetrabromide (23.1 g, 69.7 mmol). The stirred suspension was cooled
in an ice/water bath and a solution of triphenylphosphine (18.6 g,
70.9 mmol) in N,N-dimethylformamide (75 ml) was added dropwise over
30 minutes. The resulting yellow solution was allowed to warm to
room temperature and was stirred for a further 3.5 hours. A solid
separated during the stirring. The suspension was concentrated in
vacuo to about 1/3 the original volume and partitioned between
water (500 ml) and ethyl acetate (600 ml). The organic phase was
separated and washed with water (.times.2). The combined aqueous
phases were back-extracted with ethyl acetate. The combined organic
extracts were then washed with 5% aqueous lithium chloride solution
and were dried (Na.sub.2SO.sub.4), and the solvent was removed by
evaporation. The residue was purified by column chromatography (800
ml of ART9385 silica gel, column made up in 1:1 hexane:ethyl
acetate), loading the residue in dichloromethane, and eluting with
1:1 hexane:ethyl acetate. The fractions containing the product were
combined and the solvent was evaporated to give the title compound
as a white solid (12.6 g). LCMS m/z 429 [MH.sup.+]; T.sub.RET=2.92
min.
Intermediate 16:
4-{[5-(Azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-p-
iperidinecarboxamide
##STR00126##
[1003] A solution of 1,1-dimethylethyl
4-{[5-(azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-p-
iperidinecarboxylate (8.9 g, 20.8 mmol, e.g. which can be as
prepared in Intermediate 16) in 1,4-dioxane (50 ml) was treated
with 4M HCl in 1,4-dioxane (125 ml). After 2 hours the solvent was
evaporated and the residue was partitioned between dichloromethane
and saturated aqueous sodium bicarbonate solution. The organic
phase was collected and washed with saturated aqueous sodium
bicarbonate solution. The combined aqueous phases were
back-extracted with dichloromethane. The combined organic phases
were dried (Na.sub.2SO.sub.4) and the solvent was evaporated to
give an oil (LCMS showed MH.sup.+=329; T.sub.RET=1.75 min). The oil
was dissolved in dichloromethane (100 ml), and the solution was
treated with N,N-diisopropylethylamine (6.1 ml, 35.0 mmol) followed
by trimethylsilyl isocyanate (3.6 ml, 26.6 mmol, commercially
available e.g. trimethylsilyl isocyanate 85% from Aldrich). The
reaction mixture was stirred at room temperature overnight.
Additional N,N-diisopropylethylamine (6.1 ml, 35.0 mmol) and
trimethylsilyl isocyanate (3.6 ml, 26.6 mmol) was added. After
stirring for a further 7 hours, additional trimethylsilyl
isocyanate (3.6 ml) was added and stirring was continued over the
weekend (e.g. this can be about 62 hours). Water (100 ml) was added
and the mixture was stirred for 1 hour. The precipitated solid was
collected by filtration, washed with water followed by diethyl
ether, and then dried to give the title compound (2.21 g). LCMS m/z
372 [MH.sup.+]; T.sub.RET=2.08 min.
Alternative preparation of
4-{[5-(azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-p-
iperidinecarboxamide
[1004] 1,1-dimethylethyl
4-{[5-(azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-p-
iperidinecarboxylate (5.14 g) is treated with a 4M solution of
hydrogen chloride in dioxane (50 ml). The reaction mixture is left
to stand for 2 hours. The solvent is evaporated and co-evaporated
with dichloromethane (2.times.50 ml) then diethyl ether (2.times.50
ml) to give
5-(azidomethyl)-1,6-diethyl-N-4-piperidinyl-1H-pyrazolo[3,4-b]pyridin-4-a-
mine, hydrochloride salt. This product is dissolved in
dichloromethane (100 ml) and the solution is treated with DIPEA
(6.3 ml) and trimethylsilyl isocyanate (1.8 ml), and then is left
to stand at room temperature for 18 hours. Water (50 ml) is added
and the precipitated solid is collected by filtration, washed with
diethyl ether and dried to give
4-{[5-(azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino-
}-1-piperidinecarboxamide.
Intermediate 17:
4-{[5-(Aminomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-p-
iperidinecarboxamide
##STR00127##
[1006] 10% Palladium on carbon (0.65 g), in a flask flushed with
nitrogen, was treated with water (about 2 ml), ethanol (20 ml) and
a suspension of
4-{[5-(azidomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-p-
iperidinecarboxamide (4.1 g, 1 1.0 mmol, e.g. which can be as
prepared in Intermediate 16) in ethanol (100 ml). Additional
ethanol (200 ml) was used to wash the suspension into the flask.
The suspension was stirred under an atmosphere of hydrogen for 21
hours. The mixture was filtered through celite to remove catalyst,
and the filtrate was evaporated to dryness to give the title
compound as a grey foam (3.8 g). LCMS m/z 346 [MH.sup.+];
T.sub.RET=about 1.66 min.
Intermediate 19: 1,1-Dimethylethyl
[4-({[(4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1,6-diethyl-1H-pyrazolo-
[3,4-b]pyridin-5-yl)methyl]amino}carbonyl)phenyl]carbamate
##STR00128##
[1008] A solution of
4-({[(1,1-dimethylethyl)oxy]carbonyl}amino)benzoic acid (0.17 g,
e.g. available from Aldrich) in N,N-dimethylformamide (5 ml) was
treated with O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (0.27 g) followed by N,N-diisopropylethylamine
(0.31 ml) and the mixture allowed to stir at room temperature for
10 minutes. A suspension of intermediate 17 (0.25 g) in
N,N-dimethylformamide (2 ml) was added and the resultant solution
allowed to stand at room temperature for 16 h. The solvent was
removed by evaporation and the residue dissolved in dichloromethane
(5 ml) and applied directly to a 50 g silica SPE cartridge. Elution
with a gradient of 0-100% ethyl acetate: cyclohexane followed by a
gradient of 0-20% methanol: ethyl acetate gave the title compound
as a cream solid (0.41 g). LCMS showed MH.sup.+=565; T.sub.RET=2.53
min.
Intermediate 20:
4-{[5-({[(4-Aminophenyl)carbonyl]amino}methyl)-1,6-diethyl-1H-pyrazolo[3,-
4-b]pyridin-4-yl]amino}-1-piperidinecarboxamide hydrochloride
##STR00129##
[1010] A solution of Intermediate 19 (0.41 g) in methanol (8 ml)
was treated with 4M HCl in 1,4-dioxane (30 ml), resulting in a
yellow solution, and stirred at room temperature for 16 hours. The
solvent was evaporated to dryness, and dichloromethane (2.times.20
ml) added to the residue and evaporated to give the title compound
as a cream solid (0.49 g). LCMS showed MH.sup.+=465; T.sub.RET=2.03
min.
Intermediate 21:
4-[(5{[({4-[(8-Bromooctanoyl)amino]phenyl}carbonyl)amino]methyl}-1,6-diet-
hyl-1H-pyrazolo[3,4-b]pyridin-4-yl)amino]-1-piperidinecarboxamide
##STR00130##
[1012] A solution of Intermediate 20 (0.49 g) and triethylamine
(0.39 ml) in chloroform (20 ml) was cooled to 0.degree. C. and
treated dropwise with intermediate 10 (0.24 g). The mixture was
stirred at 0.degree. C. for 15 minutes, allowed to warm to room
temperature and stirred for a further 16 hours. Water (10 ml) was
added and the organic layer collected through a hydrophobic frit
and evaporated to dryness. The residue was purified by passing
through a 50 g silica SPE cartridge, eluting with a gradient of
0-50% methanol: dichloromethane, to give the title compound as a
white solid (0.3 g). LCMS showed MH+=669/671 (bromine isotope);
T.sub.RET=2.75 min.
Intermediate 22: 1,1-Dimethylethyl
4-(4-hydroxy-1-butyn-1-yl)benzoate
##STR00131##
[1014] A mixture of 1,1-dimethylethyl 4-iodobenzoate (1.1 g, 3.62
mmol, e.g. which can be preparable e.g. according to E. C. Taylor,
J. Org. Chem., 1995, 60(24), 7947), 3-butyn-1-ol (0.36 ml, 4.70
mmol, 1.3 equivalents), bis(triphenylphosphine)palladium(II)
dichloride [(PPh.sub.3).sub.2PdCl.sub.2] (0.13 g, 018 mmol, 5 mol
%) and copper (I)iodide (0.014 g, 0.07 mmol, 2 mol %) in
triethylamine (5 ml) was stirred at room temperature for 18 hours.
The mixture was diluted with dichloromethane (50 ml) and washed
with water (50 ml). The organic phase was collected by passing
through a hydrophobic frit and evaporated to give a dark brown oil
(1.3 g). This was purified by flash chromatography on silica gel
(100 g silica cartridge, FlashMaster II), eluting with a gradient
of 0 to 100% ethyl acetate in cyclohexane over 20 minutes.
Fractions containing product (obtained from 4:1 cyclohexane:ethyl
acetate eluant) were pooled and evaporated to dryness to give the
title compound as a brown gummy oil (0.9 g). 1H NMR (400 MHz,
chloroform-d) .delta. (delta) ppm 1.60 (9H, s), 1.85 (1H, t, J=6.27
Hz), 2.73 (2H, t, J=6.27 Hz), 3.85 (2H, q, J=6.27 Hz), 7.45 (2H, d,
J=8.53 Hz), 7.92 (2H, d, J=8.53 Hz).
Intermediate 23: 1,1-Dimethylethyl 4-(4-hydroxybutyl)benzoate
##STR00132##
[1016] A solution of 1,1-dimethylethyl
4-(4-hydroxy-1-butyn-1-yl)benzoate (0.9 g, e.g. which can be as
prepared in Intermediate 22) in a mixture of ethanol (50 ml) and
ethyl acetate (5 ml) was hydrogenated over platinum (IV) oxide
(PtO.sub.2, 0.09 g, 10% w/w) for 18 hours at room temperature. The
mixture was filtered through a bed of Celite, washing through with
ethanol. The solvents were evaporated to dryness to give the title
compound as a yellowy/brown oil (0.85 g). .sup.1H NMR (400 MHz,
chloroform-d) .delta. (delta) ppm 1.60 (14H, m), 1.70 (2H, m), 2.70
(2H, t, J=7.53 Hz & 7.78 Hz), 3.67 (2H, t, J=6.53 Hz & 6.27
Hz) 7.23 (2H, d, J=8.28 Hz), 7.91 (2H, d, J=8.03 Hz). Extra protons
at about 1.5-1.6 ppm may perhaps be due to residual H.sub.2O in
CDCl.sub.3. LCMS showed T.sub.RET=3.26 min with: MH.sup.+ not seen,
and [MH].sup.++18 adduct=268.
Intermediate 24: 1,1-Dimethylethyl
4-{4-[(4-bromobutyl)oxy]butyl}benzoate
##STR00133##
[1018] To 1,1-dimethylethyl 4-(4-hydroxybutyl)benzoate (0.85 g, 3.4
mmol, e.g. which can be as prepared in Intermediate 23) was added
1,4-dibromobutane (1.22 ml, 10.2 mmol, 3 equivalents, commercially
available e.g. from Aldrich), tetra-n-butylammonium bisulphate
(0.034 g, 0.1 mmol, 0.03 equivalents) and aqueous sodium hydroxide
solution (50% w/v NaOH, 5 ml). The resultant biphasic mixture was
stirred at room temperature over the weekend (this can be e.g.
about 64 hours), diluted with water (20 ml) and extracted with
diethyl ether (50 ml). The organic extract was washed with water
(20 ml), dried (Na.sub.2SO.sub.4) and evaporated to dryness. The
residue was purified by passing through a 50 g silica SPE cartridge
eluting with cyclohexane (100 ml), 9:1 cyclochexane:ethyl acetate
(100 ml), 4:1 cyclohexane:ethyl acetate (200 ml) and 1:1
cyclohexane:ethyl acetate (100 ml). The 4:1 cyclohexane:ethyl
acetate fraction containing the product was evaporated to give the
title compound (1.05 g). LCMS showed T.sub.RET=4.14 min with:
MH.sup.+=385 (trace), and [MH].sup.++18 adduct=402/404 (Bromine
isotope pattern).
Intermediate 25: 4-{4-[(4-Bromobutyl)oxy]butyl}benzoic acid
##STR00134##
[1020] 1,1-Dimethylethyl 4-{4-[(4-bromobutyl)oxy]butyl}benzoate
(1.05 g, 2.72 mmol, e.g. which can be as prepared in Intermediate
24) was treated with a 4M solution of HCl in 1,4-dioxane (10 ml, 40
mmol, 15 equivalents) and the mixture was allowed to stand at room
temperature overnight (about 16 hours). The solvent was evaporated
in vacuo followed by several co-evaporations with dichloromethane
and a trituration with diethyl ether (the material failed to
solidify). The diethyl ether was evaporated off to give the title
compound as greenish gum (0.91 g; product contained diethyl ether
by NMR). LCMS showed MH.sup.+=329/331 (Bromine isotope pattern);
T.sub.RET=3.60 min.
Intermediate 26: 4-{4-[(4-Bromobutyl)oxy]butyl}benzoyl chloride
##STR00135##
[1022] A suspension of 4-{4-[(4-bromobutyl)oxy]butyl}benzoic acid
(0.91 g, 2.77 mmol, e.g. which can be as prepared in Intermediate
25) in thionyl chloride (SOCl.sub.2, 2 ml) was stirred at room
temperature for 1 hour and then at 100.degree. C. for 2 hours. The
reaction mixture was cooled and the excess thionyl chloride was
removed by evaporation, followed by co-evaporation with
dichloromethane (2.times.30 ml) to give the title compound as a
brown oil (1.03 g). .sup.1H NMR (400 MHz, chloroform-d) .delta.
(delta) ppm 1.5-2.0 (10H, m); 2.75 (2H, m); 3.45 (4H, m); 7.35 (2H,
d); 8.05 (2H, d). The compound was not pure by .sup.1H NMR
(CDCl.sub.3), but it was not purified further.
Intermediate 27:
4-{4-[(4-Bromobutyl)oxy]butyl}-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-y-
lamino)-1H-pyrazolo[3,4-b]pyridin-6-yl]methyl}benzamide
##STR00136##
[1024] A solution of
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine (300 mg, 1 mmol, e.g. which can be as prepared in
Intermediate 7) and triethylamine (0.34 ml, 2.5 mmol, 2.5
equivalents) in dichloromethane (20 ml) was stirred and cooled in
an ice/water bath under an atmosphere of nitrogen.
4-{4-[(4-Bromobutyl)oxy]butyl}benzoyl chloride (520 mg, 1.5 mmol,
1.5 equivalents, e.g. which can be as prepared in Intermediate 26)
was added portion-wise over 2 minutes, and the mixture was stirred
at 0.degree. C. for 5 minutes and then at room temperature for 3
hours. Iced water was added to the reaction mixture and the organic
phase was separated and collected through a hydrophobic frit. The
aqueous phase was extracted with dichloromethane (10 ml), and the
organic layers were combined and evaporated to dryness to give a
brown oil. The residue was purified by flash chromatography (100 g
silica cartridge, FlashMaster II), eluting with a gradient of
0-100% ethyl acetate in cyclohexane over 40 minutes. The fractions
containing the product were combined and evaporated to dryness to
give the title compound as a glass (0.26 g). LCMS showed
MH.sup.+=614/616 (Bromine isotope pattern); T.sub.RET=3.06 min.
Intermediate 28:
4-{[5-({[(4-{4-[(4-Bromobutyl)oxy]butyl}phenyl)carbonyl]amino}methyl)-1,6-
-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-piperidinecarboxamide
##STR00137##
[1026] A solution of
4-{[5-(aminomethyl)-1,6-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-p-
iperidinecarboxamide (0.21 g, 0.6 mmol, e.g. which can be as
prepared in Intermediate 17) and triethylamine (0.21 ml, 1.52 mmol,
2.5 equivalents) in dichloromethane (20 ml) was stirred and cooled
in an ice/water bath under an atmosphere of nitrogen.
4-{4-[(4-Bromobutyl)oxy]butyl}benzoyl chloride (0.32 g, 0.91 mmol,
1.5 equivalents, e.g. which can be as prepared in Intermediate 26)
was added portion-wise over 2 minutes, and the mixture was stirred
at 0.degree. C. for 10 minutes and then at room temperature
overnight. Iced water was added to the reaction mixture and the
organic phase was separated and collected through a hydrophobic
frit. The aqueous phase was extracted with dichloromethane (50 ml),
and the combined organic extracts were evaporated to dryness to
give a whitish foam. The residue was purified by passing through a
20 g silica column, eluting with a gradient of cyclohexane/ethyl
acetate followed by a gradient of ethyl acetate/methanol. The
fractions containing the product (obtained from 9:1 ethyl
acetate/methanol) were pooled and evaporated to dryness to give the
title compound as a pale yellow foam (0.27 g). LCMS showed
MH.sup.+=656/658 (Bromine isotope pattern); T.sub.RET=2.88 min.
Intermediate 29: Diethyl (1-chloroethylidene)propanedioate
##STR00138##
[1028] To a cooled (10.degree. C.) suspension of diethyl malonate
(200 g, 1.41 mol) in dry acetonitrile (300 mL) was added anhydrous
magnesium chloride (119.37 g, 1.26 mol, e.g. available from
Lancaster) maintaining the temperature below 20.degree. C.
Triethylamine (348 ml) was added dropwise to the slurry followed by
the dropwise addition of a solution of acetyl chloride (98.12 g,
1.25 mol) in acetonitrile (100 ml) maintaining the temperature at
10-15.degree. C. Stirring was continued for 1 hour at 10-15.degree.
C. and the mixture allowed to warm to room temperature
overnight.
[1029] Hydrochloric acid (1 M) was added to the cooled reaction
mixture (10.degree. C.) until the pH of the mixture was about 1.0
(approx. 1.1 L was required). The mixture was extracted with
diethyl ether (2.times.800 ml). The combined ethereal extracts were
washed with hydrochloric acid (1 M, 1.times.600 ml) and brine.
Evaporation of the solvent under reduced pressure afforded diethyl
acetylpropanedioate as the product (233.28 g) as an orange oil.
[1030] To the above keto-diester derivative (233 g) in phosphorus
oxychloride (POCl.sub.3, 2.2L) was added tri-n-butylamine (250 ml)
dropwise and the solution then heated at 120.degree. C. for 7
hours. Excess phosphorus oxychloride was removed under reduced
pressure; and the cooled reaction mixture extracted with a 1:2
mixture of hexane and diethyl ether (3.times.1.2L). The combined
organic extracts were washed with hydrochloric acid (1 M, 2.times.1
L), NaOH solution (0.1M, 2.times.1 L), with water (2.times.1 L),
brine (2.times.1 L) and dried (Na.sub.2SO.sub.4). Evaporation of
the solvent under reduced pressure afforded the title compound (1
58 g) as a red oil which was used without further purification.
Intermediate 30: Ethyl
4-chloro-1-ethyl-6-methyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
##STR00139##
[1032] A mixture Intermediate 29 (158 g, 0.72 mol), Intermediate I
(79 g, 0.71 mol) and triethylamine (196 ml) in toluene (2L) was
heated under reflux for 16 hrs. The reaction mixture was then
cooled to room temperature, filtered and the solid residue washed
thoroughly with toluene. From the combined filtrate and the
washings, toluene was removed by evaporation under reduced
pressure. The residue treated with phosphorus oxychloride
(POCl.sub.3, 2L) and then heated under reflux for 16 hrs. Excess
phosphorus oxychloride was removed under reduced pressure. The
reaction mixture was diluted with ethyl acetate (1L) and cooled to
10.degree. C. Saturated aqueous sodium bicarbonate solution (800
ml) was added dropwise. The organic layer was separated, dried
(Na.sub.2SO.sub.4) and evaporated to give an oil.
[1033] The crude product was purified by chromatography (silica
gel, 60-120 mesh, 3 kg), eluting with 3% ethyl acetate in hexane.
The desired fractions were combined and evaporated to give the
title compound (76 g). .sup.1H NMR (200 MHz, chloroform-d) 8
(delta) ppm 1.42 (3H, t, J=7.2 Hz), 1.50 (3H, t, J=7.3 Hz), 2.68
(3H, s), 4.47 (2H, q, J=7.3 Hz), 4.56 (2H, q, J=7.2 Hz), 8.04 (1H,
s).
Alternative Preparation of ethyl
4-chloro-1-ethyl-6-methyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
[1034] A mixture of 5-amino-1-ethylpyrazole (1.614 g, 14.5 mmol)
and diethyl 2-(1-ethoxyethylidene)malonate [e.g. see J. Am. Chem.
Soc., 1931, 53, 1836] (3.68 g) is heated at 150.degree. C. under
Dean Stark conditions for 5 hours. Phosphorous oxychloride (25 ml)
is carefully added to the mixture and the resulting solution is
heated at 130.degree. C. under reflux for 18 hours. The mixture is
concentrated in vacuo and the residual oil is carefully added, with
cooling, to water (100 ml). The resulting mixture is extracted with
dichloromethane (3.times.100 ml) and the combined organic extracts
are dried over anhydrous sodium sulphate and concentrated in vacuo.
The residual oil is purified by Biotage chromatography (silica; 90
g) eluting with 5% ethyl acetate in petroleum ether. Fractions
containing the desired product are combined and concentrated in
vacuo to give ethyl
4-chloro-1-ethyl-6-methyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate.
Intermediate 31: Ethyl
1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyri-
dine-5-carboxylate
##STR00140##
[1036] Intermediate 30 (58 g, 217 mmol) in N-methyl-2-pyrrolidinone
(380 ml) was treated with N,N-diisopropylethylamine (75 ml, 433
mmol) and a solution of tetrahydro-2H-pyran-4-amine (26.2 g, 260
mmol, e.g. available from Peakdale or Combi-Blocks) in
N-methyl-2-pyrrolidinone (100 ml), and was then heated at 11
5.degree. C. overnight. The cooled mixture was poured into water
(2500 ml) and extracted with ethyl acetate (6.times.250 ml). The
combined organic extracts were washed with water and brine, dried
and evaporated. The residue was suspended in diethyl ether (about
150 ml) and the solid collected by filtration to give the title
compound (44.5 g) as a white solid. LCMS m/z 333 [MH.sup.+];
T.sub.RET=2.89 min.
[1037] The filtrate was evaporated, dissolved in ethyl acetate,
washed with water and brine, dried and evaporated. Treatment with
diethyl ether as described above gave a further quantity of the
title compound (9.6 g). A further repeat of this procedure on the
filtrate gave an additional quantity of the title compound (2.16
g).
Intermediate 32:
[1-Ethyl-6-methyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyr-
idin-5-yl]methanol
##STR00141##
[1039] Intermediate 31 (26.9 g, 80.9 mmol) in dry dichloromethane
(270 ml) under an atmosphere of nitrogen was cooled to 0.degree. C.
and treated with diisobutylaluminium hydride (162 ml of a 1.5M
solution in toluene, 243 mmol). The addition took 27 minutes. The
resulting pale yellow solution was stirred at 0.degree. C. for 35
minutes and quenched by careful addition of about 20% aqueous
potassium sodium tartrate solution (Rochelle's salt, 250 ml). This
was exothermic and produced effervescence (temperature reached a
maximum of 30.degree. C.). After stirring for 20 min the solids
were removed by filtration and washed with dichloromethane and
ethyl acetate and the aqueous phase of the filtrate extracted
firstly with dichloromethane then ethyl acetate. The combined
organics were washed with water then brine, dried and evaporated to
give 3 g of material. The aqueous phase was treated with more
potassium sodium tartrate solution, concentrated and exhaustively
extracted with ethyl acetate. The ethyl acetate extracts were
combined, dried and evaporated to give the title compound (17.9 g)
as a cream solid.
[1040] LCMS m/z 291 [MH.sup.+]; T.sub.RET=1.74, 1.81 min (split
peak).
Intermediate 33:
5-(Azidomethyl)-1-ethyl-6-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo-
[3,4-b]pyridin-4-amine
##STR00142##
[1042] A suspension of Intermediate 32 (9.7 g, 33.4 mmol) in
toluene (100 ml) was treated dropwise with thionyl chloride
(SOCl.sub.2, 36.6 ml, 500 mmol) over 10 minutes resulting in a
gummy lump. The mixture was heated to 80.degree. C. for 2.5 hours
by which time the lump had broken down to be a pale brown
suspension. The mixture was cooled, then evaporated and the residue
was azeotroped with more toluene (about 20 ml). The resultant pale
brown solid was suspended in dimethylsulphoxide (75 ml) and treated
with sodium azide (3.25 g, 50.1 mmol); the mixture became darker
and slightly more translucent. After 2 hours the mixture was
partitioned between saturated aqueous sodium bicarbonate solution
and ethyl acetate, the layers separated and the aqueous phase
extracted with more ethyl acetate. The combined organics were
washed with water then brine, dried and evaporated. The residue was
purified by silica gel chromatography (800 g) eluting with 1:1
cyclohexane:ethyl acetate. The fractions containing the product
were pooled and evaporated to give the title compound (7.6 g).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (delta) ppm 1.51 (3H, t),
1.68 (2H, m), 2.16 (2H, m), 2.64 (3H, s), 3.61 (2H, m), 4.05 (2H,
m), 4.13 (1H, m), 4.45 (2H, s), 4.47 (2H, q), 4.96 (1H, d), 7.88
(1H, s).
Intermediate 34:
5-(Aminomethyl)-1-ethyl-6-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo-
[3,4-b]pyridin-4-amine
##STR00143##
[1044] 10% palladium on carbon (50% wet, 2.1 g) was treated with
ethanol (20 ml) followed by a solution of Intermediate 33 (10.48 g)
in ethanol (180 ml), and was hydrogenated at room temperature and
pressure overnight. The catalyst was removed by filtration, washing
through with more ethanol, and the solvents were removed under
reduced pressure to give the title compound (9.0 g) as a grey foam:
.sup.1H NMR (400 MHz, d6 DMSO) .delta. (delta) ppm 1.36 (3H, t),
1.59 (2H, m), 2.0 (2H, m), 2.50 (3H, s), 3.37 (2H, br s), 3.62 (2H,
m), 3.89 (2H, s), 3.91 (2H, m), 4.14 (1H, m), 4.33 (2H, q), 7.62
(1H, d), 8.02 (1H, s).
Intermediate 35: 1,1-Dimethylethyl
4-[(8-bromooctanoyl)amino]benzoate
##STR00144##
[1046] A solution of 1,1-dimethylethyl 4-aminobenzoate (0.5 g, 2.6
mmol, commercially available e.g. from Fluka) in dichloromethane
(25 ml) was treated with triethylamine (1.8 ml, 12.9 mmol, 5
equivalents), and the mixture was stirred under an atmosphere of
nitrogen and cooled in an ice/water bath. The cold mixture was
treated with a solution of 8-bromooctanoyl chloride (0.75 g, 3.1
mmol, 1.2 equivalents, e.g. which can be as prepared in a or the
alternative synthesis of Intermediate 10) in dichloromethane (1 ml)
over 2 minutes. The resultant stirred mixture was allowed to warm
up from ca. 10.degree. C. to room temperature while stirring was
continued for an additional 1 8 hours. Iced water (10 ml) was
added, and the organic fraction was collected through a hydrophobic
frit. The aqueous fraction was extracted again with dichloromethane
(10 ml), and the organic phases were combined and evaporated to
dryness. The resulting gum was purified by passing through a 50 g
silica SPE cartridge, eluting with dichloromethane (400 ml)
followed by 10% ethyl acetate in dichloromethane. Product was
obtained from eluting with 10% ethyl acetate in dichloromethane to
give the title compound as a colourless gum (0.61 g). LCMS showed
MH.sup.+=398/400 (Br isotope pattern); T.sub.RET=3.82 min; with an
impurity at T.sub.RET=2.95 min. NMR (CDCl.sub.3) suggested that the
material was about 75% pure; this was not purified further.
Intermediate 36: 4-[(8-Bromooctanoyl)amino]benzoic acid
##STR00145##
[1048] To a solution of 1,1-dimethylethyl
4-[(8-bromooctanoyl)amino]benzoate (0.61 g, 1.5 mmol, as prepared
in Intermediate 35) in dichloromethane (2 ml) was added a 4M
solution of HCl in 1,4-dioxane (4 ml, about 10 equivalents), and
the mixture was left to stand at ambient temperature for 18 hours.
The mixture was evaporated to dryness and the residue was
co-evaporated with dichloromethane (20 ml) to give the title
compound as a white solid (0.51 g). LCMS showed MH.sup.+=342/344
(Br isotope pattern); T.sub.RET=3.34 min. NMR (d6-DMSO) suggested
that the material was again about 75% pure and that the impurity
from the previous reaction (Intermediate 35) was still present. The
mixture was used without further purification.
Intermediate 37:
4-[(8-Bromooctanoyl)amino]-N-{[1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4--
ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide
##STR00146##
[1050] A solution of 4-[(8-bromooctanoyl)amino]benzoic acid (0.25
g, 0.73 mmol, e.g. which can be as prepared in Intermediate 36) in
thionyl chloride (2 ml) was stirred under an atmosphere of nitrogen
for 1 hour, then heated at 100.degree. C. for 2 hours. The thionyl
chloride was evaporated and the residue co-evaporated with
dichloromethane (2.times.20 ml) to give a black gum. This was used
without further purification.
[1051] To a solution of
5-(aminomethyl)-1-ethyl-6-methyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo-
[3,4-b]pyridin-4-amine (0.14 g, 0.49 mmol, e.g. which can be as
prepared in Intermediate 34) in dichloromethane (10 ml) under an
atmosphere of nitrogen was added triethylamine (0.34 ml, 2.5 mmol)
and the mixture cooled in an ice/water bath. To this mixture was
added a solution of the acid chloride intermediate prepared above
in dichloromethane (10ml) dropwise over 3-5 minutes. The resultant
solution was stirred at 0-10.degree. C. for 2 hours. The mixture
was diluted with dichloromethane (10 ml), iced water (about 20 ml)
was added and the organic fraction collected through a hydrophobic
frit. The aqueous fraction was extracted with a further portion of
dichloromethane (1 0 ml), the organic layer collected through a
hydrophobic frit, and the combined organic extracts evaporated to
dryness. The resulting gum was purified flash chromatography on
silica gel, eluting with a gradient of 0-100% ethyl
acetate/cyclohexane followed by a gradient of 0-20% methanol/ethyl
acetate. Fractions containing the product were pooled and
evaporated to give the title compound as a yellow foam (0.1 g).
LCMS showed MH.sup.+=613/615 (Br isotope pattern); T.sub.RET=2.91
min. An impurity (ca. 15% by LC) was also seen with T.sub.RET 3.35
min, MH.sup.+=755/757--the material was used without further
purification.
Intermediate 38: 1,1-Dimethylethyl
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzoate
##STR00147##
[1053] To a solution of 2-(methylamino)ethanol (10.7 g, 11.5 ml,
144 mmol, 1.5 equivalents, commercially available e.g. from
Aldrich) in dry N,N-dimethylformamide (300 ml) was added
N,N-diisopropylethylamine (25 ml, 18.5 g, 144 mmol, 1.5
equivalents). To this mixture was added 1,1-dimethylethyl
4-[(8-bromooctanoyl)amino]benzoate (38.1 g, 96 mmol, 1 equivalent)
in dry N,N-dimethylformamide (250 ml) over 12 minutes. The mixture
was then heated to and at about 60.degree. C. for 4.25 hours. The
mixture was then cooled and the solvent was removed by evaporation
using high vacuum to give a golden oil. An attempt to partition
this oil between water and ethyl acetate gave a cloudy organic
phase and phase separation was poor; an oil separated out as a
third layer, some of which was collected and evaporated to give
crude product (16.69 g). The remaining emulsion was allowed to
stand overnight, giving a clear organic layer which was decanted
off, dried (MgSO.sub.4) and evaporated to give further crude
product as a golden oil (28.11 g). Attempts to further extract the
aqueous phase with ethyl acetate or dichloromethane gave an
emulsion again, from which further crude product (about 10 g) was
finally isolated. The combined crude product was purified by
chromatography on silica gel (about 1400 ml of 7734 silica),
eluting with 5% methanol in dichloromethane, then 8% methanol in
dichloromethane, then 10% methanol in dichloromethane, and finally
20% and 30% methanol in dichloromethane, to afford the title
compound as a white foam (28.4 g). LCMS showed MH.sup.+=393;
T.sub.RET=2.52 min.
Intermediate 39:
4-({8-[(2-Hydroxyethyl)(methyl)amino]octanoyl}amino)benzoic
acid
##STR00148##
[1055] To a mixture of 4-[(8-bromooctanoyl)amino]benzoic acid (50
mg, 0.15 mmol, as prepared in Intermediate 36) in
N,N-dimethylformamide (2 ml) was added 2-(methylamino)ethanol (24
microlitres, 0.3 mmol, 2 equivalents, commercially available e.g.
from Aldrich) followed by N,N-diisopropylethylamine (0.13 mL, 0.8
mmol, about 5 equivalents), and the resulting solution was heated
at 60.degree. C. for 16 hours. The solvent was removed by
evaporation, and the residue was purified by Mass Directed
Automated Preparative HPLC (Method A). Fractions containing product
were pooled and evaporated to dryness to give the title compound
(25.4 mg) as a pale brown solid. LCMS showed MH.sup.+=337;
T.sub.RET=2.03 min.
Intermediate 39A:
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzoic acid
hydrogen chloride
##STR00149##
[1057] To a solution of 1,1-dimethylethyl
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-benzoate (28.4
g, 72.4 mmol, e.g. which can be as prepared in Intermediate 38) in
dry dichloromethane (225 ml) was added a 4M hydrogen chloride
solution in 1,4-dioxane (145 ml, 579 mmol, 8 equivalents). The
mixture quickly became cloudy and was stirred overnight resulting
in a uniform white suspension. The solvents were evaporated off in
vacuo, using a high vacuum at the end, to give the title compound
(23.45 g) as a white solid. LCMS showed positive ion at m/z 336;
T.sub.RET=2.01 min. NMR (d6-DMSO) appeared to show a trace of
dichloromethane and 1,4-dioxane in the product.
Intermediate 40:
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-(8-hydroxyoctyl)benzamide
##STR00150##
[1059] A solution of Intermediate 7 (182 mg, 0.6 mmol),
4-(8-hydroxyoctyl)benzoic acid (100 mg, 0.4 mmol, e.g. preparable
according to K. Fukai JP11174621),
(1H-1,2,3-benzotriazol-1-yloxy)(tri-1-pyrrolidinyl)phosphonium
hexafluorophosphate (PyBOP, 208 mg, 0.4 mmol, e.g. available from
Aldrich) and N,N-diisopropylethylamine (0.276 ml, 1.6 mmol) in dry
N,N-dimethylformamide (3 ml) was allowed to stir at room
temperature for 4 hours. The solvent was evaporated and the residue
partitioned between chloroform (1 0 ml) and saturated aqueous
sodium bicarbonate solution (1 0 ml); the organic fraction was
collected by passing through a hydrophobic frit and evaporated. The
residue was dissolved in chloroform (20 ml), and the solution
passed through a 10 g aminopropyl SPE cartridge under gravity. The
cartridge was further eluted with 10% methanol in chloroform, and
product containing fractions evaporated to give the title compound
(330 mg). LCMS showed MH.sup.+=536; T.sub.RET=2.92 min.
Intermediate 41:
4-(8-Bromooctyl)-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyr-
azolo[3,4-b]pyridin-5-yl]methyl}benzamide
##STR00151##
[1061] Triphenylphosphine (197 mg, 0.75 mmol) was added dropwise in
dichloromethane (3 ml) to a mixture of Intermediate 40 (330 mg,
0.62 mmol) and carbon tetrabromide (247 mg, 0.74 mmol) in
dichloromethane (7 ml) at 0.degree. C. under an atmosphere of
nitrogen. The mixture was allowed to reach ambient temperature and
stirred for 3 hours, then allowed to stand for a further 16 hours.
The solvent was evaporated and the residue purified by flash
chromatography (20 g silica cartridge), eluting with a gradient of
0 to 100% ethyl acetate in cyclohexane over 30 minutes. Fractions
containing product were combined and evaporated to give the title
compound (60 mg) as a colourless oil. LCMS showed MH.sup.+=598/600
(bromine isotopes); T.sub.RET=3.42 min.
Intermediate 42: 1,1-Dimethylethyl
{2-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]py-
ridin-5-yl]methyl}amino)carbonyl]phenyl}carbamate
##STR00152##
[1063] A mixture of Intermediate 7 (250 mg, 0.83 mmol) and
2-({[(1,1-dimethylethyl)oxy]carbonyl}amino)benzoic acid (196 mg,
0.83 mmol, e.g. available from Fluka) in N,N-dimethylformamide (4
ml) was treated with N,N-diisopropylethylamine (0.569 ml, 3.3 mmol)
and (1H-1,2,3-benzotriazol-1-yloxy)(tri-1-pyrrolidinyl)phosphonium
hexafluorophosphate (472 mg, 0.91 mmol) and allowed to stir at room
temperature for 4 hours. An additional portion of
(1H-1,2,3-benzotriazol-1-yloxy)(tri-1-pyrrolidinyl)phosphonium
hexafluorophosphate (0.5 equiv., 0.41 mmol) was added and the
mixture stirred for an additional hour. The solvent was evaporated
and the residue dissolved in ethyl acetate (50 ml) and washed with
aqueous sodium bicarbonate solution (2.times.50 ml); the organic
fraction was dried (Na.sub.2SO.sub.4) and evaporated. The residue
was purified by chromatography on silica gel (50 g SPE cartridge),
eluting with a gradient of 0-100% ethyl acetate/cyclohexane
followed by a gradient of 0-20% methanol/ethyl acetate over 40
minutes. Fractions containing product were pooled and evaporated to
give the title compound (292 mg). LCMS showed MH.sup.+=523;
T.sub.RET=2.71 min.
Intermediate 43: 1,1-Dimethylethyl
{3-[({[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]py-
ridin-5-yl]methyl}amino)carbonyl]phenyl}carbamate
##STR00153##
[1065] A mixture of Intermediate 7 (250 mg, 0.83 mmol) and
3-({[(1,1-dimethylethyl)oxy]carbonyl}amino)benzoic acid (196 mg,
0.83 mmol, e.g. available from Fluka) in N,N-dimethylformamide (4
ml) was treated with N,N-diisopropylethylamine (0.569 ml, 3.3 mmol)
and (1H-1,2,3-benzotriazol-1-yloxy)(tri-1-pyrrolidinyl)phosphonium
hexafluorophosphate (472 mg, 0.91 mmol) and allowed to stir at room
temperature for 4 hours. An additional portion of
(1H-1,2,3-benzotriazol-1-yloxy)(tri-1-pyrrolidinyl)phosphonium
hexafluorophosphate (0.5 equivalents, 0.41 mmol) was added and the
mixture stirred for an additional hour. The solvent was evaporated
and the residue partitioned between ethyl acetate (50 ml) and
aqueous sodium bicarbonate solution (50 ml); saturated sodium
chloride solution was added and the layers allowed to separate
overnight. The aqueous layer was further extracted with ethyl
acetate (50 ml), and the organic fractions collected, dried
(Na.sub.2SO.sub.4) and evaporated; however the residue contained
very little product. A white solid was filtered off from the
aqueous layer and dried to give the title compound (193 mg). LCMS
showed MH.sup.+=523; T.sub.RET=2.99 min.
Intermediate 44:
2-Amino-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
-b]pyridin-5-yl]methyl}benzamide
##STR00154##
[1067] A solution of Intermediate 42 (292 mg, 0.56 mmol) in
methanol (8 ml) was treated with a 4M hydrogen chloride solution in
1,4-dioxane (24 ml) and allowed to stir at ambient temperature for
16 hours. The solvent was evaporated and co-evaporation with
dichloromethane (2.times.20ml) gave the title compound (182 mg) as
a gold powder. LCMS showed MH.sup.+=423; T.sub.RET=2.16 min.
Intermediate 45:
3-Amino-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
-b]pyridin-5-yl]methyl}benzamide
##STR00155##
[1069] A solution of Intermediate 43 (193 mg, 0.37 mmol)) in
methanol (8 ml) was treated with a 4M hydrogen chloride solution in
1,4-dioxane (24 ml) and allowed to stir at ambient temperature for
16 hours. The solvent was evaporated and co-evaporation with
dichloromethane (2.times.20 ml) gave the title compound (224 mg) as
a gold powder. LCMS showed MH.sup.+=423; T.sub.RET=2.36 min.
Intermediate 46:
2-[(8-Bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide
##STR00156##
[1071] To a solution of Intermediate 44 (182 mg, 0.43 mmol) in
dichloromethane (4 ml) at 0.degree. C. was added triethylamine
(0.282 ml) followed by dropwise addition of Intermediate 10 (191
mg, 0.79 mmol) over 5 minutes. The mixture was stirred for 1 hour
at 0.degree. C., allowed to warm to room temperature, and stirring
was continued for a further 18 hours. Dichloromethane (50 ml) was
added, the mixture poured into iced water and the organic fraction
collected. The aqueous layer was extracted with more
dichloromethane (50 ml), and the combined organic layers were
evaporated to dryness. The residue was purified by passing through
a 10 g silica SPE cartridge eluting with a gradient of 0-100% ethyl
acetate/cyclohexane over 40 minutes. Fractions containing product
were pooled and evaporated to give the title compound (68 mg) as a
colourless oil. LCMS showed MH.sup.+=627/629 (bromine isotopes);
T.sub.RET=3.14 min.
Intermediate 47:
3-[(8-Bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide
##STR00157##
[1073] To a solution of Intermediate 45 (224 mg, 0.53 mmol) in
dichloromethane (5 ml) at 0.degree. C. was added triethylamine
(0.347 ml) followed by dropwise addition of Intermediate 10 (235
mg, 0.97 mmol) over 5 minutes. The mixture was stirred for 1 hour
at 0.degree. C., allowed to warm to room temperature, and stirring
was continued for a further 18 hours. Dichloromethane (50 ml) was
added, the mixture poured into iced water, and the organic fraction
collected. The aqueous layer was extracted with more
dichloromethane (50 ml), and the combined organic layers were
evaporated to dryness. The residue was purified by passing through
a 10 g silica SPE cartridge, eluting with a gradient of 0-100%
ethyl acetate/cyclohexane over 40 minutes. Fractions containing
product were pooled and evaporated to give the title compound (42
mg) as a colourless oil. LCMS showed MH.sup.+=627/629 (bromine
isotopes); T.sub.RET=3.15 min.
Intermediate 48:
5-(Aminomethyl)-1-ethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]py-
ridin-4-amine hydrochloride
##STR00158##
[1075]
1-Ethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-
e-5-carboxamide (0.59 g, 2.04 mmol, e.g. preparable according to
WO2004/056823) was suspended in dry tetrahydrofuran (20 ml) and
borane-tetrahydrofuran complex (10.2 ml, 1 M soln in
tetrahydrofuran) was added. The reaction was heated under reflux
under nitrogen for 4 hours. The mixture was cooled, quenched with
methanol (20 ml), and allowed to stand at room temperature over the
weekend. The solvent was evaporated in vacuo and the residue
dissolved in methanol and applied to a 20 g SCX cartridge, eluting
with methanol followed by 2M ammonia/methanol. The ammonia/methanol
fractions were combined and evaporated to gave a yellow oil (0.38
g). The oil was dissolved in methanol, 2M hydrochoric acid (5 ml)
was added, and the mixture was heated at 70.degree. C. for 2 hours.
The mixture was cooled and solvent evaporated to give an orange oil
(0.4 g). Purification by flash chromatography, eluting with 0-30%
methanol and 1% triethylamine in dichloromethane over 20 minutes,
gave the title compound as an impure oil (0.32 g). LCMS showed
MH.sup.+=276; T.sub.RET=1.72 min.
[1076] The impure intermediate (0.31 g) was dissolved in methanol
(10 ml) and di-t-butyldicarbonate (0.25 g, 1.1 3 mmol) and
triethylamine (0.1 6 ml, 1.13 mmol) were added. The mixture was
left standing at room temperature for 16 hours. The solvent was
evaporated and the residue partitioned between dichloromethane (10
ml) and water (10 ml). The organic phase was collected by passing
through a hydrophobic frit and the solvent evaporated to give the
impure BOC-protected amine intermediate as an orange oil (0.26 g),
which was used without further purification. LC/MS showed
MH.sup.+=376; T.sub.RET=2.39 mins.
[1077] The BOC-protected intermediate (58 mg, including material
from another preparation) was treated with a 4M solution of HCl in
1,4-dioxane (1 ml) and the resulting mixture was left standing at
room temperature for 16 hours. The solvent was removed by
evaporation to give the title compound as a beige coloured solid
(55 mg). LCMS showed MH.sup.+=276; T.sub.RET=1.66 min.
Intermediates 49 to 52
##STR00159##
[1079] A solution of Intermediate 9 (0.10 g, 0.22 mmol) in
dichloromethane (5 ml) was treated with triethylamine (0.15 ml, 1.1
mmol) and the mixture was cooled to 0.degree. C. To the mixture was
added the appropriate acid chloride (see Table 2 below, 0.33 mmol).
After 5 minutes at 0.degree. C., the mixture was allowed to reach
room temperature and the reaction continued for a further 2
hours.
[1080] The mixture was treated with ice/water (2 ml). In the case
of Intermediate 49, this product precipitated out and was isolated
by filtration to give the title compound (74 mg; used without
further purification).
[1081] For isolation of Intermediates 50, 51 and 52, the organic
phase was separated by passing through a hydrophobic frit. The
aqueous phase was extracted further with dichloromethane (2.times.2
ml), and the organic phases were combined and evaporated to give
the crude product. Intermediate 51 was obtained as a brown oil (312
mg; generally used without further purification). Intermediate 52
was obtained as a brown oil (366 mg; generally used without further
purification). Intermediate 50 was purified by mass directed
preparative HPLC (Method A) to give the title compound as a white
solid (76 mg).
TABLE-US-00010 TABLE 2 ##STR00160## Inter- mediate number formed
Acid chloride reagent ##STR00161## One possible Source of, or one
possible publication mentioning, the acid chloride M.sup.+ ion
T.sub.RET (min) 49 n = 5 Aldrich 599, 2.72 601 50 n = 6 Advanced
Synthesis 613, 2.85 Technologies 615 51 n = 9 Chem & Pharm
Bull; 655, 3.22 31 (2), 454-65 657 52 n = 10 Advanced Synthesis 669
3.31 Technologies 671
Intermediate 53: 4-[(8-bromooctanoyl)amino]benzamide
##STR00162##
[1083] This preparation is an embodiment of the "Stage 4a"
preparation within Process IC described herein. The following
reaction was conducted under a nitrogen atmosphere. All volumes
("vol.") and equivalents are relative to the 4-aminobenzamide.
[1084] Pyridine (1.6 mL, 20 mmol, 1.1 equivalents) was added to a
slurry of 4-aminobenzamide (2.5 g, 18 mmol) in 2-butanone (40 mL,
16 vol.) with mechanical stirring. After about 2 hours, a solution
of 8-bromooctanoyl chloride* (3.7M, 5.5 mL, 20 mmol, 1.1
equivalents) in 2-butanone (10 mL, 4 vol.) was added dropwise over
18 min. The resulting mixture, a very viscous slurry, was stirred
at room temperature for 19 to 19.5 hours and then filtered through
two 42.5 mm diameter filter papers in a filter funnel. The reaction
vessel was rinsed with the filtrate and filtered through the same
filter funnel and filter papers. Water (25 mL, 10 vol.) was then
added to the filtration cake which was stirred well and filtered.
The washing process was repeated with more water (25 mL), and the
resultant filtration cake was then displacement washed with
methanol (10 mL, 4 vol.) and then tert-butyl methyl ether (TBME, 10
mL, 4 vol.). The residual solvents in the cake were removed under
suction, the damp solid was dried in vacuo at 50.degree. C.
overnight to yield the title compound as a white solid (5.99 g,
about 95-96% yield based on the amount of 4-aminobenzamide used).
HPLC (5 mins generic solvent gradient): T.sub.RET about 3.01 min.
Mass spectrum: Found: MH.sup.+ 341/343, (M-H).sup.- 339/341.
[1085] * 8-Bromooctanoyl chloride can optionally be prepared: (i)
substantially as described in this reference: Xue, F.; Seto, C. T.
"Structure-activity studies of cyclic ketone inhibitors of the
serine protease plasmin: design, synthesis, and biological
activity", Bioorganic & Medicinal Chemistry, 2006,14(24),
8467-8487; and/or (ii) as described in Intermediate 10 herein
(using the first or the alternative synthesis given).
Intermediate 53 (alternative preparation):
4-[(8-bromooctanoyl)amino]benzamide
##STR00163##
[1087] The following reaction is conducted under a nitrogen
atmosphere. Pyridine (1.6 mL, 1.1 equivalents) is added to a
mixture of 4-aminobenzamide (2.5 g, 18 mmol) and n-butyronitrile
(65 mL) at 20.degree. C. under an atmosphere of nitrogen. The
mixture is stirred for 15 minutes before a solution of
8-bromooctanoyl chloride (3.7M in toluene, 5.5 mL, 20 mmol, 1.1
equivalents) in n-butyronitrile (10 mL) is added over 20 minutes.
The mixture is then stirred at 20.degree. C. for 22 hours. Upon
complete reaction the mixture is filtered. The resulting product is
washed with methanol (10 mL), water (2.times.25 mL), methanol (10
mL) and tert-butyl methyl ether (TBME, 10 mL) before being dried in
vacuo to yield the title compound, generally as a solid.
Intermediate 54:
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide
##STR00164##
[1089] This preparation is an embodiment of the "Stage 4b"
preparation within Process 1C described herein. The following
reaction was conducted under a nitrogen atmosphere.
[1090] 2-(Methylamino)ethanol (4.07 mL, 50.6 mmol, 3 equivalents)
was added to a stirred slurry of
4-[(8-bromooctanoyl)amino]benzamide (5.76 g, 16.9 mmol, e.g. which
can be as prepared in Intermediate 53 (first or alternative
preparation thereof)) in dimethyl sulfoxide (30 mL). The mixture
was heated at 60.degree. C. for 2 hours, cooled to room
temperature, quenched with ice (150 g), stirred for about 30 mins
and filtered. Filtration, through a Whatman hardened filter paper,
was very slow, taking about 2 hours. The resultant wet white paste
was dissolved in methanol (about 130 mL) and filtered through a
SCX-2 SPE cartridge (70 g), eluting with methanol (about 500 mL)
and then 10% concentrated ammonia-methanol (about 700 mL).
Fractions from the ammonical methanol eluate containing UV active
component were pooled together and evaporated to dryness to give
the title compound as a white solid (5.15 g after drying at
60.degree. C. for 19 hours, about 91% yield based on the amount of
4-[(8-bromooctanoyl)amino]benzamide used). HPLC (5 mins generic
solvent gradient): T.sub.RET about 1.95 min. Mass spectrum: Found:
MH.sup.+ 336, (M-H).sup.- 334.
Intermediate 54 (Alternative Preparation):
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide
##STR00165##
[1092] The following reaction is conducted under a nitrogen
atmosphere. 2-(Methylamino)ethanol (9.5 g, 7 equivalents) is added
to a stirred mixture of 4-[(8-bromooctanoyl)amino]benzamide (5.76
g, 16.9 mmol, e.g. which can be as prepared in Intermediate 53
(first or alternative preparation thereof) and dimethyl sulfoxide
(30 mL). The mixture is stirred at 25.degree. C. until the reaction
is substantially complete and is then diluted with acetonitrile
(115 mL). After stirring for 1 hour the mixture is filtered, washed
with ethyl acetate (15 mL) and tert-butyl methyl ether (TBME, 15
mL) before being dried in vacuo to yield the title compound,
generally as a solid.
Intermediate 55:
2-[(8-{[4-(aminocarbonyl)phenyl]amino}-8-oxooctyl)(methyl)amino]ethyl
acetate
##STR00166##
[1094] This preparation is an embodiment of the "Stage 5"
preparation within Process 1C described herein. The following
reaction was conducted under a nitrogen atmosphere.
[1095] A mixture of
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide (6.05
g, 18 mmol, e.g. which can be as prepared in Intermediate 54),
acetic anhydride (4.25 mL, 45 mmol, 2.5 equivalents) and
triethylamine (7.5 mL, 54 mmol, 3 equivalents) in dry acetonitrile
(100 mL) was stirred at room temperature for 66 hours. Additional
acetic anhydride (3.40 mL, 36 mmol, 2 equivalents) was then added.
The mixture was heated under reflux for 2.5 hours and cooled to
room temperature with magnetic stirring. The resultant slurry was
filtered, washed with acetonitrile (12 mL then 6 mL) and the
residual solvents in the filter cake removed under suction. The
cake was then dried in vacuo at 50.degree. C. for 15 hours to
afford the title compound as a greyish brown solid (5.4 g, about
79% yield based on the amount of
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide
used). HPLC (5 mins generic solvent gradient): T.sub.RET about 2.05
min. Mass spectrum: Found: MH 378, (M-H).sup.- 376.
Optional Intermediates
[1096] In this Optional Intermediates section, "Intermediates"
generally represent syntheses of intermediate compounds which (in
some cases) might theoretically be usable in the synthesis of
compounds of formula (I) or salts thereof, but which have not
necessarily been used to prepare specific compounds of formula (I)
or salts thereof (and in most or all cases they have not been so
used).
Optional Intermediate 60
[1-ethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl-
]methanol
##STR00167##
[1098] A solution of 1 M diisobutylaluminium hydride in
dichloromethane (80 ml) is added dropwise to a stirred solution of
ethyl
1-ethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridine-5-ca-
rboxylate [e.g. see Intermediate 32 and/or Example 3 of WO
2004/024728 A2] (13.8 g) in dichloromethane (75 ml) at 0.degree. C.
under nitrogen. The reaction mixture is maintained below 5.degree.
C. during the addition, and is then stirred for 0.5 h at 0.degree.
C. The mixture is then quenched by addition of aqueous potassium
sodium tartrate (10% solution), diluted with water (150 ml) and the
organic phase separated. The aqueous phase is extracted with ethyl
acetate (2.times.250 ml) and the combined organics are dried
(magnesium sulphate) and evaporated. The residue is purified by
column chromatography on silica gel eluting with a gradient of
0-100% ethyl acetate in cyclohexane followed by 0-20% methanol in
ethyl acetate to give Intermediate 60.
Optional Intermediate 61
5-(chloromethyl)-1-ethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]p-
yridin-4-amine
##STR00168##
[1100] Intermediate 60 (80 mg) is treated with thionyl chloride (1
ml) and heated at 80.degree. C. for 1 h then allowed to cool. The
orange solution is evaporated to dryness and the residue azeotroped
with toluene (2.times.5 ml) to give Intermediate 61.
Optional Intermediate 62
5-(azidomethyl)-1-ethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]py-
ridin-4-amine
##STR00169##
[1102] A solution of Intermediate 61 (50 mg) in anhydrous
dimethylsulphoxide (0.2 ml) is treated with lithium azide (9 mg)
and the solution is stirred at room temperature for 20 h. A further
portion of lithium azide (1 5 mg) is then added, and after a
further day stirring at room temperature, water (0.25 ml) is added.
The solution is extracted with dichloromethane (2.times.5 ml) and
the combined organic extracts are passed through a hydrophobic frit
(6 ml) and then are blown to dryness. The residue is dissolved in
dichloromethane (0.5 ml) and applied to an SPE cartridge (silica; 1
g). The cartridge is eluted with 50% ethyl acetate in cyclohexane
and fractions containing the desired material are combined and
blown to dryness to give Intermediate 62.
Optional Intermediate 63
1-ethyl-N.sup.4-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridine-4,5-
-diamine
##STR00170##
[1104] A solution of Intermediate 62 (0.351 g) in ethanol (30 ml)
is added to palladium on carbon (5% wet, 0.050 g) and the mixture
is stirred at room temperature for 20 hours under an atmosphere of
hydrogen. The mixture is filtered through a glass fibre filter and
through celite, which is then washed with ethanol (50 ml). The
combined filtrates and washings are concentrated in vacuo to give
Intermediate 63.
TABLE-US-00011 One or two possible published Optional reference(s)
to (e.g. synthetic Intermediate reference to), or one possible
Number Structure of compound source of, the compound 100
##STR00171## Intermediate 8A of WO 2004/024728 A2 (Glaxo Group
Limited) 101 ##STR00172## Intermediate 6 of WO 2004/024728 A2
(Glaxo Group Limited), which refers to WO 00/42011 102 ##STR00173##
Intermediate 32 and/or Example 3 of WO 2004/024728 A2 (Glaxo Group
Limited); or Intermediate 4 of WO 2005/058892 A1 (Glaxo Group
Limited) 103 ##STR00174## Intermediate 5 of WO 2005/058892 A1
(Glaxo Group Limited) 104 ##STR00175## Example 207 of WO
2004/024728 A2 (Glaxo Group Limited); or Intermediate 6 of WO
2005/058892 A1 (Glaxo Group Limited) 105 ##STR00176## Example 204
of WO 2004/024728 A2 (Glaxo Group Limited) 106 ##STR00177## Example
205 of WO 2004/024728 A2 (Glaxo Group Limited) 107 ##STR00178##
Example 652 of WO 2004/024728 A2 (Glaxo Group Limited) 108
##STR00179## Intermediate 12 of WO 2005/058892 A1 (Glaxo Group
Limited) 109 ##STR00180## Intermediate 102 of WO 2005/058892 A1
(Glaxo Group Limited) 110 ##STR00181## Intermediate 103 of WO
2005/058892 A1 (Glaxo Group Limited) 111 ##STR00182## Intermediate
104 of WO 2005/058892 A1 (Glaxo Group Limited) 112 ##STR00183##
Intermediate 112 of WO 2005/058892 A1 (Glaxo Group Limited) 113
##STR00184## Intermediate 113 of WO 2005/058892 A1 (Glaxo Group
Limited) 114 ##STR00185## Intermediate 114 of WO 2005/058892 A1
(Glaxo Group Limited) 115 ##STR00186## Intermediate 115 of WO
2005/058892 A1 (Glaxo Group Limited) 116 ##STR00187## Intermediate
116 of WO 2005/058892 A1 (Glaxo Group Limited) 117 ##STR00188##
Intermediate 117 of WO 2005/058892 A1 (Glaxo Group Limited) 118
##STR00189## Intermediate 118 of WO 2005/058892 A1 (Glaxo Group
Limited) 119 ##STR00190## Intermediate 119 of WO 2005/058892 A1
(Glaxo Group Limited) 120 ##STR00191## Intermediate 146 of WO
2005/058892 A1 (Glaxo Group Limited) 121 ##STR00192## Intermediate
147 of WO 2005/058892 A1 (Glaxo Group Limited) 122 ##STR00193##
Intermediate 148 of WO 2005/058892 A1 (Glaxo Group Limited) 123
##STR00194## Intermediate 149 of WO 2005/058892 A1 (Glaxo Group
Limited) 124 ##STR00195## Intermediate 150 of WO 2005/058892 A1
(Glaxo Group Limited) 125 ##STR00196## Intermediate 151 of WO
2005/058892 A1 (Glaxo Group Limited) 126 ##STR00197## Intermediate
155 of WO 2005/058892 A1 (Glaxo Group Limited) 127 ##STR00198##
Intermediate 156 of WO 2005/058892 A1 (Glaxo Group Limited) 128
##STR00199## Intermediate 157 of WO 2005/058892 A1 (Glaxo Group
Limited) 129 ##STR00200## Intermediate 159 of WO 2005/058892 A1
(Glaxo Group Limited) 130 ##STR00201## Intermediate 160 of WO
2005/058892 A1 (Glaxo Group Limited) 131 ##STR00202## Intermediate
161 of WO 2005/058892 A1 (Glaxo Group Limited) 132 ##STR00203##
Example 185 of WO 2004/024728 A2 (Glaxo Group Limited); or
Intermediate 171 of WO 2005/058892 A1 (Glaxo Group Limited) 133
##STR00204## Example 20 of WO 2004/024728 A2 (Glaxo Group Limited)
134 ##STR00205## Example 186 of WO 2004/024728 A2 (Glaxo Group
Limited) 135 ##STR00206## Fluka Chemie AG, Germany (CAS
111769-27-8) 136 ##STR00207## E. Merck, Germany; or E. Merck (Merck
Ltd), Hunter Boulevard, Magna Park, Lutterworth, Leicestershire
LE17 4XN, United Kingdom (CAS 104530-80-5) 137 ##STR00208##
Intermediate 11 of WO 2004/024728 A2, and optionally reference
cited therein 138 ##STR00209## Intermediate 12 of WO 2004/024728
A2, and optionally references cited therein 139 ##STR00210## Sigma
Aldrich Library of Rare Chemicals (SALOR) (CAS-6338-70-1) 140
##STR00211## Intermediate 14 of WO 2004/024728 A2, and optionally
references cited therein 141 ##STR00212## Intermediate 3 of WO
2004/024728 A2 (Glaxo Group Limited) 142 ##STR00213## Intermediate
25 of WO 2004/024728 A2 (Glaxo Group Limited) 143 ##STR00214##
Example 8 of WO 2004/024728 A2 (Glaxo Group Limited) 144
##STR00215## Example 9 of WO 2004/024728 A2 (Glaxo Group Limited)
145 ##STR00216## Example 10 of WO 2004/024728 A2 (Glaxo Group
Limited) 146 ##STR00217## Example 11 of WO 2004/024728 A2 (Glaxo
Group Limited) 147 ##STR00218## Example 13 of WO 2004/024728 A2
(Glaxo Group Limited) 148 ##STR00219## Example 14 of WO 2004/024728
A2 (Glaxo Group Limited) 149 ##STR00220## Example 190 of WO
2004/024728 A2 (Glaxo Group Limited) 150 ##STR00221## Intermediate
54 of WO 2004/024728 A2 (Glaxo Group Limited) 151 ##STR00222##
Intermediate 58A of WO 2004/024728 A2 (Glaxo Group Limited) 152
##STR00223## Intermediate 62 of WO 2004/024728 A2 (Glaxo Group
Limited) 153 ##STR00224## Intermediate 63 of WO 2004/024728 A2
(Glaxo Group Limited) 154 ##STR00225## AB Chem, Inc., Canada
(mixture of cis and trans); or J. Chem. Soc., Perkin Trans. 1,
1994, 537 155 as Intermediate 154, but racemic cis- J. Chem. Soc.,
Perkin isomer, i.e. racemic cis-(3-hydroxy- Trans 1, 1994, 537
cyclohex-1-yl)-amine (discloses a 3.3:1 cis:trans mixture) 156
##STR00226## Aldrich; or TCI-America 157 ##STR00227## U.S. Pat. No.
4,219,660 158 ##STR00228## Aldrich 159 ##STR00229## Aldrich 160
##STR00230## Aldrich 161 ##STR00231## Pfaltz-Bauer 162 ##STR00232##
J. Org. Chem., 1985, 50 (11), 1859 163 ##STR00233## WO 99/12933 164
##STR00234## EP 1188744 165 ##STR00235## Sigma-Aldrich Company Ltd
166* ##STR00236## J. Med. Chem., 1994, 37 (17), 2360 167*
##STR00237## Aldrich 168* ##STR00238## Aldrich 169* ##STR00239##
Aldrich 170* ##STR00240## Peakdale Molecular Ltd 171 ##STR00241##
AstaTech 172 ##STR00242## 173 ##STR00243## Syngene or AstaTech 174
##STR00244## Fluka 175 ##STR00245## Aldrich 176 ##STR00246##
Aldrich
Examples
TABLE-US-00012 [1105] Table of Examples Example No. Name 1A1
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}-
amino)benzamide dihydrochloride 1A2
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}-
amino)benzamide monohydrochloride 1A3
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}-
amino)benzamide monohydrochloride (recrystallised) 1B
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide 1C
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide
trifluoroacetate (salt) 1D
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide
monohydrobromide 1E
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide hemisuccinate
1F
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide hemifumarate 1G
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide mono-xinafoate
(mono-1-hydroxy-2-naphthoate) 1H1 and
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
1H2 b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide phosphate 1J1
and
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
1J2 b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide
mono-(+)-camphorsulfonate 3
4-[(1,6-diethyl-5-{[({4-[(8-{(2R)-2-[(methyloxy)methyl]-1-
pyrrolidinyl}octanoyl)amino]phenyl}carbonyl)amino]methyl}-1H-
pyrazolo[3,4-b]pyridin-4-yl)amino]-1-piperidinecarboxamide
trifluoroacetate 4
4-[(1,6-diethyl-5-{[({4-[(8-{(2S)-2-[(methyloxy)methyl]-1-
pyrrolidinyl}octanoyl)amino]phenyl}carbonyl)amino]methyl}-1H-
pyrazolo[3,4-b]pyridin-4-yl)amino]-1-piperidinecarboxamide
trifluoroacetate 9 4-({1,6-diethyl-5-[({[4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)phenyl]carbonyl}amino)methyl]-
1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarboxamide
trifluoroacetate (salt) 11
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-[(8-{(2R)-2-[(methyloxy)methyl]-1-
pyrrolidinyl}octanoyl)amino]benzamide trifluoroacetate 12
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-[(8-{(2S)-2-[(methyloxy)methyl]-1-
pyrrolidinyl}octanoyl)amino]benzamide trifluoroacetate 17 formate
salt of N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}-4-{[8-(4-
morpholinyl)octanoyl]amino}benzamide 18 formate salt of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}-4-{[8-(1-
pyrrolidinyl)octanoyl]amino}benzamide 19 formate salt of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}-4-[4-({4-[(2-
hydroxyethyl)(methyl)amino]butyl}oxy)butyl]benzamide 20 formate
salt of N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}-4-(4-{[4-(4-
morpholinyl)butyl]oxy}butyl)benzamide 21 formate salt of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}-4-(4-{[4-(1-
pyrrolidinyl)butyl]oxy}butyl)benzamide 22 formate salt of
4-[(1,6-diethyl-5-{[({4-[4-({4-[(2-
hydroxyethyl)(methyl)amino]butyl}oxy)butyl]phenyl}carbonyl)amino]methyl}-
1H-pyrazolo[3,4-b]pyridin-4-yl)amino]-1-piperidinecarboxamide 23
formate salt of 4-({1,6-diethyl-5-[({[4-(4-{[4-(4-
morpholinyl)butyl]oxy}butyl)phenyl]carbonyl}amino)methyl]-1H-
pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarboxamide 23A
4-({1,6-diethyl-5-[({[4-(4-{[4-(4-
morpholinyl)butyl]oxy}butyl)phenyl]carbonyl}amino)methyl]-1H-
pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarboxamide 24
formate salt of 4-({1,6-diethyl-5-[({[4-(4-{[4-(1-
pyrrolidinyl)butyl]oxy}butyl)phenyl]carbonyl}amino)methyl]-1H-
pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarboxamide 25
formate salt of
N-{[1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4-ylamino)-
1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide 26 formate salt
of N-{[1-ethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-
pyrazolo[3,4-b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide 27
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-[8-(4-morpholinyl)octyl]benzamide 28
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-{8-[(2-
hydroxyethyl)(methyl)amino]octyl}benzamide 29
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-3-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide 30
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-2-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide 31
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({6-[(2-
hydroxyethyl)(methyl)amino]hexanoyl}amino)benzamide 32
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({7-[(2-
hydroxyethyl)(methyl)amino]heptanoyl}amino)benzamide 33
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({10-[(2-
hydroxyethyl)(methyl)amino]decanoyl}amino)benzamide 34
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-
b]pyridin-5-yl]methyl}-4-({11-[(2-
hydroxyethyl)(methyl)amino]undecanoyl}amino)benzamide
Example 1A1
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de dihydrochloride
##STR00247##
[1107] To a solution of
4-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (0.98 g, 1.56
mmol, e.g. which can be as prepared in Intermediate 11) in
N,N-dimethylformamide (50 ml) was added N,N-diisopropylethylamine
(0.54 ml, 3.12 mmol) followed by 2-(methylamino)ethanol (0.2 ml,
2.34 mmol, commercially available e.g. from Aldrich) and the
resulting solution was heated at 80.degree. C. for 16 hours. The
mixture was cooled, evaporated to dryness and the residue
partitioned between dichloromethane (30 ml) and saturated aqueous
sodium bicarbonate solution (30 ml). The aqueous portion was
extracted with more dichloromethane (30 ml) and the combined
organic extracts were passed through a hydrophobic frit and
evaporated to dryness to give an amber oil (1.3 g). The residue was
purified by passing through a 50 g silica SPE cartridge, loading
using dichloromethane, and eluting with dichloromethane (100 ml),
5% methanol/dichloromethane (100 ml), 10% methanol/dichloromethane
(200 ml), 20% methanol/dichloromethane (200 ml), 20% methanol/1%
triethylamine/dichloromethane (200 ml) and 25% methanol/1%
triethylamine/dichloromethane (400 ml). Fractions containing the
product (generally obtained from the 25% methanol/1%
triethylamine/dichloromethane elution) were combined and evaporated
to dryness. Co-evaporation with dichloromethane and diethyl ether
to remove excess triethylamine gave the product as a cream-coloured
foamy solid (0.54 g). Attempts to recrystallise the foam from
various solvents including ethyl acetate, ethyl acetate/diethyl
ether, isopropanol/water, and ethanol/diethyl ether, were
unsuccessful (material failed to crystallise). Hence the foam was
dissolved in dichloromethane, treated with a 4M solution of HCl in
1,4-dioxane (5 ml), and allowed to stand at room temperature
overnight. Evaporation of the solvent followed by co-evaporation
with diethyl ether and ethanol gave a foam. Attempts to
recrystallise this foam from various solvent combinations including
diethyl ether/ethyl acetate/ethanol were unsuccessful (material
failed to crystallise). The foam was triturated with diethyl ether,
filtered and dried to give the title compound as a cream solid
(0.45 g). LCMS showed MH.sup.+=622; T.sub.RET=2.24 min. Under the
microscope this material appeared to be partially crystalline.
The .sup.1H NMR spectrum for this or a similar sample of the title
compound was as follows: .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. (delta) ppm 13.90 (1H, br. s.), 10.27 (1H, s.), 9.63 (1H,
br. s.), 9.22 (1H, br. s.), 9.14 (1H, br. s.), 8.40 (1H, br. s.),
7.87 (2H, d, J=8.5 Hz), 7.71 (2H, d, J=8.5 Hz), 5.31 (1H, br. s.),
4.49-4.62 (4H, m), 4.24-4.39 (1H, m), 3.90 (2H, d, J=11.0 Hz), 3.72
(2H, t, J=5.0 Hz), 3.62 (2H, t, J=11.0 Hz), 2.91-3.28 (6H, m), 2.74
(3H, d, J=4.5 Hz), 2.34 (2H, t, J=7.0 Hz), 1.95 (2H, d, J=12.0 Hz),
1.54-1.77 (6H, m), 1.37 (3H, t, J=7.0 Hz), 1.25-1.33 (9H, m).
[1108] This compound is thought to be the dihydrochloride salt,
e.g. based on a comparison of its .sup.1H NMR spectrum with that of
Example 1A2 (e.g. that given below).
Example 1A2:
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride
##STR00248##
[1110]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide (the "free base", 20.3 g, e.g. which can be as prepared
in Example 1B) was charged to a 500 ml jacketed
temperature-controlled laboratory reactor and was suspended in
isopropanol (203 ml) under nitrogen. The slurry mixture was stirred
and was heated from 21.degree. C. to 65.degree. C. over 40 minutes
(the starting material dissolving at about 60.degree. C.), and when
at 65.degree. C. the mixture was treated with concentrated
hydrochloric acid (2.82 ml, 1.05 equivalents). This acid was rinsed
into the vessel with further isopropanol (5 ml). The temperature
was adjusted to 56.degree. C. and then seeds of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide hydrochloride (about 2 mg) (e.g. monohydrochloride or
dihydrochloride) were added. After cooling to 51.degree. C. over
1.25 h, the batch was held at 51.degree. C. for 5 minutes and then
cooled to 0-2.degree. C. over about 2 hours. Crystallisation
occurred during this cool.
[1111] The slurry was held at ca. 0-2.degree. C. for about one hour
and then heated to 45.degree. C. (Jacket temperature) over 2 hours,
and then cooled to 0.degree. C. over 2 hours. This `temperature
cycling` was repeated three further times, before holding at
0.degree. C. for very approximately half an hour to one hour. The
batch was filtered, and the solid was washed with chilled (ca.
5.degree. C.) isopropanol (2.times.30 ml) and dried in vacuo at
40-45.degree. C. to give the title compound as a solid (17.5
g).
[1112] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. (delta) ppm
10.31 (s, 1H), 10.01 (br. s., 1H), 8.86 (br. s., 1H), 8.02 (br. s.,
1H), 7.85 (d, J=8.5 Hz, 2H), 7.70 (d, J=8.5 Hz, 2H), 7.19 (br. s.,
1H), 5.30-5.35 (m, 1H), 4.53 (d, J=5.5 Hz, 2H), 4.30-4.39 (m, 2H),
4.12 (br. s., 1H), 3.85-3.90 (m, J=11.0 Hz, 2H), 3.71-3.78 (m,
J=4.9 Hz, 2H), 3.53-3.59 (m, J=11.0, 11.0 Hz, 2H), 2.91-3.24 (m,
6H), 2.73 (s, 3H), 2.32-2.37 (m, 2H), 1.89-1.94 (m, J=12.2 Hz, 2H),
1.48-1.73 (m, 6H), 1.25-1.34 (m, 12H).
[1113] This compound is thought to be the monohydrochloride salt,
e.g. based on a comparison of its .sup.1H NMR spectrum with that of
Example 1 .mu.l (e.g. that given above).
[1114] It is also thought that there are at least two solid (e.g.
crystalline) forms of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride, with a first solid (e.g. crystalline) form
(named "Form 1") being a preferred substantially-anhydrous solid
(e.g. crystalline) form (named "anhydrate") and with a second solid
(e.g. crystalline) form (named "Form 2") being a less-preferred
hydrated solid (e.g. crystalline) form (named "hydrate"). The solid
(e.g. crystalline) monohydrochloride salt prepared in the
above-described synthesis (Example 1A2) is thought to be
substantially the Form 1 anhydrate.
[1115] A sample of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate, prepared in the
above-described synthesis (Example 1A2), has substantially the
following X-ray powder diffraction (XRPD) characteristics. The
X-ray powder diffraction (XRPD) data were acquired on a PANalytical
X.Pert Pro powder diffractometer, model PW3040/60, serial number
DY1850 using an XCelerator detector. The acquisition conditions
were: radiation: Cu K.alpha. (copper K-alpha), generator tension:
40 kV, generator current: 45 mA, start angle: 2.0.degree. 2.theta.,
end angle: 40.0.degree. 2.theta., step size: 0.0167.degree.
2.theta., time per step: 31.75 seconds. The sample was prepared by
mounting a few milligrams of sample on Si wafer (zero background)
plate(s), resulting in a thin layer of powder. Characteristic XRPD
peak positions (angles) and calculated d-spacings for the
monohydrochloride Form 1 anhydrate, calculated from the raw data
using Highscore software, are shown in the following table, with a
margin of error of approximately .+-.0.1.degree. 2.theta. for each
of the peak assignments:
TABLE-US-00013 approximate degrees 2.theta. d-spacing in .ANG.
(2-theta) (Angstroms) 5.1 17.3 7.8 11.4 10.3 8.6 10.7 8.3 14.3 6.2
15.2 5.8 17.5 5.1 18.6 4.8 18.8 4.7 19.7 4.5 20.3 4.4 21.4 4.1 21.9
4.1 22.4 4.0 23.1 3.9 23.5 3.8 24.0 3.7
[1116] The following four XRPD peaks of the monohydrochloride Form
1 anhydrate, from the above list of XRPD peaks, are thought to
distinguish this Form 1 anhydrate from the Form 2 hydrate:
5.1.+-.0.1.degree. 2.theta. (d-spacing ca. 17.3 .ANG.),
10.7.+-.0.1.degree. 2.theta. (d-spacing ca. 8.3 .ANG.),
23.1.+-.0.1.degree. 2.theta. (d-spacing ca. 3.9 .ANG.),
23.5.+-.0.1.degree. 2.theta. (d-spacing ca. 3.8 .ANG.).
[1117] The following two XRPD peaks of the monohydrochloride Form 1
anhydrate are thought also partially to distinguish this Form 1
anhydrate from the Form 2 hydrate, but there are shoulders or low
intensity peaks of another form in fairly close proximity that make
these peaks slightly less distinguishing than the above four peaks:
10.3.+-.0.1.degree. 2.theta. (d-spacing ca. 8.6 .ANG.),
17.5.+-.0.1.degree. 2.theta. (d-spacing ca. 5.1 .ANG.).
[1118] The IR (infrared) spectrum of the solid product, the
monohydrochloride Form 1 anhydrate, was recorded using a Nicolet
Avatar 360 FT-IR spectrometer fitted with a diamond/ZnSe ATR
accessory. The FT-IR data were recorded over 64 scans at 4
cm.sup.-1 resolution. For the monohydrochloride Form 1 anhydrate
product, solid-form IR bands were observed at: 3252, 2938, 2857,
1688, 1639, 1598, 1571, 1526, 1503, 1437, 1407, 1355, 1304, 1254,
1185, 1162, 1139, 1123, 1086, 1035, 1014, 988, 964, 860, 820 and
770 cm.sup.-1 (with some variation or error possible for each peak,
e.g. of .+-.2 cm.sup.-1 or .+-.1 cm.sup.-1). It is thought that the
solid-form IR spectrum of the monohydrochloride Form 1 anhydrate
includes a characteristic band at 1639 (.+-.2 or .+-.1) cm.sup.-1.
In comparison, a solid-form sample (not prepared in this Example)
of the monohydrochloride Form 2 hydrate appears to include a
characteristic band at ca. 1623 (.+-.2 or .+-.1) cm.sup.-1.
Example 1A2
(Alternative Preparation No. 1, Plant Method):
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride
[1119] (n=1)
[1120] The monohydrochloride salt may be prepared directly from
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine monohydrochloride e.g. Intermediate 7A. For
example, the following process may be used, e.g. in plant:
##STR00249##
Summary of Plant Method
[1121] The following reaction is carried out under a nitrogen
atmosphere, in plant. All weights, volumes and equivalents are
relative to the
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine monohydrochloride.
[1122] To stirred n-pentanol (pentan-1-ol, 15 vol, 105 L) in a
glass-lined reactor at 18.degree. C. under nitrogen is added
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzoic acid
hydrogen chloride (0.95 wt, 6.65 kg) (e.g. which can be as prepared
in Intermediate 39A, or which can be specially ordered from
Sigma-Aldrich) and potassium carbonate
[1123] [325 mesh particle size (quite a fine particle size), 1.02
wt, 7.14 kg]. After stirring at 90 rpm for 10 minutes at
20.+-.5.degree. C., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC) (0.87 wt, 6.09 kg), 1-hydroxybenzotriazole
hydrate (HOBT.H.sub.2O) (0.19 wt, 1.33 kg) and
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine monohydrochloride (1 wt, 7.0 kg) (e.g. which is
preferably substantially as prepared in Intermediate 7A) are added
sequentially. The heterogeneous reaction mixture is warmed to
20.+-.5.degree. C., and is stirred at 90-160 rpm under nitrogen at
20.+-.5.degree. C. for 9 hours 20 minutes (90 minutes at 90 rpm, 90
minutes at 125 rpm, and 6 hours 20 minutes at 160 rpm). Water (10
vol, 70 L) is added and the mixture is stirred for 16 minutes while
maintaining the current batch temperature; the layers are allowed
to settle for 15 minutes and separated. To the upper organic layer
is added 32% w/w aqueous sodium hydroxide solution (2.5 wt, 17.5
kg) over a period of 37 minutes, while maintaining the reactor
contents at 20.+-.5.degree. C. The mixture is then heated to
45.+-.5.degree. C. and stirred at 75 rpm at 45.+-.5.degree. C. for
2 hours. The mixture is cooled to 20.+-.5.degree. C. and water (8
vol, 56 L) is added. The mixture is stirred for 15 minutes and
allowed to settle for 15 minutes. The lower aqueous layer is
removed and the remaining organic layer is held overnight without
stirring. 6.5% w/w aqueous sodium chloride solution (10.4 wt, 72.8
kg) is added to the organic layer. Aqueous hydrochloric acid (17%
w/w) is added over 83 minutes, while maintaining the reactor
contents at 20.+-.5.degree. C., with the amount of 17% w/w
hydrochloric acid used (e.g. about 7.0 kg to about 19.20 kg) being
such as to ensure that the pH is adjusted finally to 5.75.+-.0.25,
with a final pH of less than 5.5 being avoided to minimise loss of
the product (as hydrochloride) to the aqueous layer. The mixture is
stirred for 30 minutes and allowed to settle for 30 minutes. The
organic layer is separated, is stirred with 4% aqueous sodium
hydroxide solution (10.4 wt, 72.8 kg) for 15 minutes while
maintaining the current batch temperature, and is allowed to settle
for 30 minutes. The lower aqueous layer is removed. Water (10 vol,
70 L) is added to the organic layer and stirred for at least 15
minutes while maintaining the current batch temperature. The
agitator is then switched off. The reactor contents are heated to
25.+-.5.degree. C., allowed to settle for 30 minutes and then
cooled to 20.+-.5.degree. C. The lower aqueous layer is removed.
The organic layer is transferred from the reactor, the reactor is
washed with n-pentanol (pentan-1-ol, 2 vol, 14 L), and the combined
organic mixture is filtered to remove any insoluble matters while
being returned to the reactor. The organic mixture is allowed to
settle for about 1 hour, after which a lower aqueous layer forms
which is removed. The separated organic layer is allowed to settle
overnight, after which a lower aqueous layer forms which is
removed. A solution yield is obtained on the organic solution using
a calibrated HPLC system and 4.49M aqueous hydrochloric acid (1
equivalent, 3.80 kg) is added, maintaining the reactor contents at
20.+-.5.degree. C. The mixture is concentrated in vacuo to 10
volumes (70 L), by vacuum distillation at about -0.901 to -0.912
barg, at internal temperatures starting from 46.5.degree. C. and
rising to about 78.degree. C. (jacket temperature 60.degree. C.,
rising to 90.degree. C., and rising again to 100.degree. C.). The
mixture is then adjusted to 85.+-.5.degree. C. under an atmospheric
pressure of nitrogen. A slurry of seed crystals (0.007 kg, 0.001
wt) of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride (preferably the Form 1 anhydrate thereof) in
n-pentanol (pentan-1-ol, 0.07 L, 0.01 vol) is added. The mixture is
cooled to 60.+-.5.degree. C., stirred for 2 hours at
60.+-.5.degree. C., and then adjusted to 55.+-.5.degree. C.
Tert-butyl methyl ether (TBME) (4.3 vol. 30.1 L) is added over 5
minutes, maintaining the reactor contents at 50.+-.5.degree. C. The
resultant mixture is cooled to 20.+-.5.degree. C. over about 1
hour, and then is stirred for about 14 hours at 20.+-.5.degree. C.
The resultant slurry is filtered under 0.5 to 1 barg of nitrogen
pressure using a 20 micron filter cloth. The reactor and then the
filter cake is washed with n-pentanol (pentan-1-ol, 2.5 vol, 17.5
L), and the filter cake is then washed with TBME (2.5 vol, 17.5 L),
the filter cake washes being under 0.5 to 1 barg of nitrogen
pressure. Residual solvent in the filter cake is removed under
nitrogen pressure. Then the filtered solid is dried at
55.+-.5.degree. C. in vacuo in a vacuum tray dryer under a bleed of
5 L/min nitrogen until the product achieves a constant temperature
for at least 4 hours, and then is cooled to <30.degree. C., to
yield the desired product
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride.
[1124] The solid compound preparable by the above alternative
preparation no. 1 (Plant Method) is substantially in the form of
the crystalline monohydrochloride Form 1 anhydrate (see Example
1A2, first synthesis, for discussion of this form).
Example 1A2 alternative preparation no. 1
Plant Method--Detailed Step-Wise Methodology
[1125] This reaction is for carrying out in a 400 L glass lined
reactor; the reaction is carried out under a nitrogen
atmosphere.
[1126] All weights, volumes and equivalents are relative to
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine monohydrochloride.
[1127] 1. Purge the reactor with nitrogen.
[1128] 2. Add n-pentanol (pentan-1-ol, 15 vol, 105 L) to the 400L
glass lined reactor.
[1129] 3. Run the agitator at 90 rpm (revolutions per minute).
[1130] 4. Cool the contents to 18.degree. C.
[1131] 5. Charge the reactor with
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzoic acid
hydrogen chloride (0.95 wt, 6.65 kg) and then potassium carbonate
(325 mesh particle size (quite a fine particle size), 1.02 wt, 7.14
kg).
[1132] 6. Stir the contents at 20.+-.5.degree. C. for 10 minutes at
90 rpm.
[1133] 7. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC) (0.87 wt, 6.09 kg), 1-hydroxybenzotriazole
hydrate (HOBT.H.sub.2O) (0.19 wt, 1.33 kg) and
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine monohydrochloride (1 wt, 7.0 kg) to the
reactor.
[1134] 8. Heat the contents of the reactor to 20.+-.5.degree. C.,
and then stir the contents of the reactor at 20.+-.5.degree. C. for
90 minutes at 90 rpm.
[1135] 9. Adjust agitator speed to 125 rpm and stir the reactor
contents for a further 90 minutes.
[1136] 10. Adjust agitator speed to 160 rpm and stir the
heterogeneous reactor contents at 20.+-.5.degree. C. for a further
6 hours 20 minutes.
[1137] 11. Add water (10 vol, 70 L) to the reactor.
[1138] 12. Stir the contents of the reactor for 16 minutes,
maintaining the current batch temperature.
[1139] 13. Stop the agitator and allow the layers of the contents
to settle for 15 minutes.
[1140] 14. Remove the lower aqueous layer.
[1141] 15. Add 32% w/w aqueous sodium hydroxide solution (2.5 wt,
17.5 kg) to the reactor over a period of 37 minutes, maintaining
the reactor contents at 20.+-.5.degree. C.
[1142] 16. Heat the contents of the reactor to 45.+-.5.degree. C.
over a period of 12 minutes, and stir at 45.+-.5.degree. C. for 2
hours at 75 rpm.
[1143] 17. Cool the contents of the reactor to 20.+-.5.degree. C.
over a period of 18 minutes.
[1144] 18. Add water (8 vol, 56 L) to the reactor.
[1145] 19. Stir the contents of the reactor for 15 minutes,
maintaining the current batch temperature, before allowing the
layers to settle for 15 minutes.
[1146] 20. Remove the lower aqueous layer. The reactor contents are
held overnight with the agitator off.
[1147] 21. Charge the reactor with 6.5% w/w aqueous sodium chloride
solution (10.4 wt, 72.8 kg).
[1148] 22. Calibrate a pH probe using pH 7.0, 2.0 and 10.0
buffers.
[1149] 23. Add 17% w/w aqueous hydrochloric acid to the stirred
reactor over a period of 83 minutes (using a slower addition rate
towards the end of the addition), maintaining the reactor contents
at 20.+-.5.degree. C., with the amount of 17% w/w hydrochloric acid
used being such as to ensure that the pH of the contents is
adjusted to a final pH of 5.75.+-.0.25. A final pH of less than 5.5
is avoided to minimise loss of the product (as hydrochloride) to
the aqueous layer. [Note: If some alkaline aqueous phase is still
present in or together with the organic layer after step 20, e.g.
due to some emulsion formation, and if this alkaline aqueous phase
is not separated off before the aqueous sodium chloride solution
addition in step 21, then more than the theoretically-required
about 7.0 kg amount of 17% w/w hydrochloric acid will be required,
e.g. 19.20 kg may be required, to achieve a final pH of
5.75.+-.0.25.]
[1150] 24. Stir the contents of the reactor for 30 minutes,
maintaining the current batch temperature, before stopping the
agitator and allowing the layers to settle for 30 minutes.
[1151] 25. Remove the lower aqueous layer.
[1152] 26. Add 4% aqueous sodium hydroxide solution (10.4 wt, 72.8
kg) to the reactor.
[1153] 27. Stir the contents of the reactor for 15 minutes,
maintaining the current batch temperature, before stopping the
agitator and allowing the layers to settle for 30 minutes.
[1154] 28. Remove the lower aqueous layer.
[1155] 29. Add water (10 vol, 70 L) to the reactor.
[1156] 30. Stir the contents of the reactor for at least 15
minutes, maintaining the current batch temperature, and then stop
the agitator.
[1157] 31. Warm the contents of the reactor to 25.+-.5.degree. C.
(jacket temperature 40.+-.5.degree. C.).
[1158] 32. Allow the contents of the reactor to settle for 30
minutes.
[1159] 33. Cool the contents of the reactor to 20.+-.5.degree. C.
over 6 minutes.
[1160] 34. Remove the lower aqueous layer.
[1161] 35. Transfer the organic mixture from the reactor into
earthed polylined steel drums, add pentan-1-ol (2 vol, 14 L) to the
reactor, and discharge these pentan-1-ol washings into the drum
containing the organic mixture. Transfer the combined organic
mixture from the drum back into the reactor through a filter, in
order to filter the organic layer (to remove any insoluble
matters).
[1162] 36. Allow the organic mixture in the reactor to settle for
about 1 hour, to allow the organic mixture to separate into an
upper organic layer and a further lower aqueous layer. Remove the
lower aqueous layer.
[1163] 37. Allow the organic mixture to settle overnight, with the
agitator off, to allow further separation, and then remove the
lower aqueous layer.
[1164] 38. Determine the solution yield using a calibrated HPLC
system.
[1165] 39. Add 4.49M aqueous hydrochloric acid (3.80 kg, 1
equivalent) to the stirred reactor over 8 minutes, maintaining the
reactor contents at 20.+-.5.degree. C.
[1166] 40. Adjust the contents (about 140 litres) of the reactor to
35.degree. C. with stirring, and partly concentrate the reactor
contents via vacuum distillation (whereupon the batch temperature
can fall, e.g. to about 26.degree. C.). Release the vacuum and stop
distillation. Re-establish a vacuum with a jacket temperature of
about 60.degree. C. and an intended internal (batch) temperature of
about 45.degree. C., and then concentrate the reactor contents to
70 litres (10vol) via vacuum distillation at pressures ranging from
-0.901 to -0.912 barg, and with internal (batch) temperatures
starting from 46.5.degree. C. and rising to from 78.44.degree. C.
to 77.56.degree. C. (jacket temperature 60.degree. C., rising to
90.degree. C., and rising again to 100.degree. C.).
[1167] 41. Adjust the contents of the reactor to 85.+-.5.degree.
C.
[1168] 42. Add a slurry of seed crystals (0.007 kg, 0.001 wt) of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate in n-pentanol (pentan-1-ol,
0.07 L, 0.01 vol) to the contents of the reactor.
[1169] 43. Cool the contents of the reactor to 60.+-.5.degree.
C.
[1170] 44. Stir the contents of the reactor at 60.+-.5.degree. C.
for 2 hours at 75 rpm.
[1171] 45. Adjust the temperature of the reactor contents to
55.+-.5.degree. C.
[1172] 46. Add tert-butyl methyl ether (TBME) (4.3 vol, 30.1 L) to
the reactor over 5 minutes whilst keeping the temperature of the
reactor contents at 50.+-.5.degree. C.
[1173] 47. Cool the contents of the reactor to 201 5.degree. C.
over about 1 hour.
[1174] 48. Age the contents of the reactor at 20.+-.5.degree. C.
for approximately 14 hours with stirring at75 rpm.
[1175] 49. Transfer the reactor contents onto a 20 micron filter
cloth, allow the slurry to settle for about 10 minutes, and filter
off the solid under 0.5 to 1 barg nitrogen pressure.
[1176] 50. Wash the reactor with n-pentanol (pentan-1-ol, 2.5 vol,
17.5 L) with 75 rpm stirring at 20.+-.5.degree. C. for 13 minutes,
transfer the pentan-1-ol washings onto the filter cake, and wash
the filter cake under 0.5 to 1 barg nitrogen pressure.
[1177] 51. Wash the filter cake with tert-butyl methyl ether (TBME)
(2.5 vol, 17.5 L) under 0.5 to 1 barg nitrogen pressure, and blow
solvent from the cake under nitrogen pressure for 6 minutes.
[1178] 52. Transfer the damp filtered solid onto polythene lined
trays, cover with muslin bags, and place in a vacuum tray dryer.
Dry the solid in vacuo at 55.+-.5.degree. C. under a bleed of 5
L/min nitrogen until the temperature of a probe in the product
remains constant for at least 4 hours, and then cool the solid to
<30.degree. C. to give the product
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride, substantially in the form of the
monohydrochloride Form 1 anhydrate thereof.
[1179] When a method substantially as described in the above
detailed steps 1 to 52 was performed, a yield of 9.15 kg of solid
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride, substantially in the form of the
monohydrochloride Form 1 anhydrate, was obtained.
Example 1A2
(Alternative Preparation No. 2):
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride
##STR00250##
[1181] This preparation represent embodiment(s) of "Stage 6a
(chlorination), Stage 6b (coupling to form amide), and Stage 7
(acetyl deprotection)" preparations within Process 1C described
herein.
[1182] The following reactions were conducted under a nitrogen
atmosphere.
[1183] A solution of methanesulfonic acid (0.79 mL, 11 mmol, 1
equivalent) in dry dichloromethane (3.5 mL) was added dropwise over
3 minutes to a stirred solution of
[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin--
5-yl]methanol
[1184] [3.4 g, 11 mmol, 1 equivalent, e.g. which can be as prepared
in Intermediate 5 (alternative preparation no. 2, plant method)] in
dry dichloromethane (13.5 mL) under nitrogen. The mixture was
cooled in a water bath. A solution of thionyl chloride (1.1 mL, 15
mmol, 1.35 equivalents) in dry dichloromethane (3.5 mL) was added
dropwise whilst keeping the reaction temperature to <25.degree.
C. by addition of dry ice pellets into the water bath. After the
addition was complete, the reaction mixture was stirred for 40
mins. This mixture is thought to contain
5-(chloromethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyra-
zolo[3,4-b]pyridin-4-amine methanesulfonate salt, from LC analysis
during or at the end of the 40 min period which showed the
5-(methoxymethyl)-derivative derived from a methanol quench of the
5-(chloromethyl)-compound.
[1185] The reaction mixture was then concentrated to about half of
its original volume. Dry dichloromethane (15 mL) was added and the
mixture was then evaporated in vacuo to about half of its original
volume. Dry tetrahydrofuran (15 mL) was then added and the mixture
was evaporated in vacuo to about 10 mL.
2-[(8-{[4-(Aminocarbonyl)phenyl]amino}-8-oxooctyl)(methyl)amino]ethyl
acetate (4 g, 11 mmol, 1 equivalent, e.g. which can be as prepared
in Intermediate 55) was added to the resultant solution which was
evaporated, removing most of the solvent, to form a syrupy slurry.
Dry 1-methyl-2-pyrrolidinone (NMP, 30 mL) was added and the mixture
was stirred at 60.degree. C. for 3.75 hours (all the material
dissolved when the temperature reached about 50.degree. C.).
Additional
5-(chloromethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-
-b]pyridin-4-amine methanesulfonate salt (0.44 g, 1.06 mmol, e.g.
which can be as prepared in a generally analogous manner to that
described above) was added, and the reaction mixture was heated for
a further 1 hour at 60.degree. C. and was then allowed to stand at
room temperature for 17 hours. The reaction micture was quenched
with an aqueous potassium carbonate solution (5% w/v, 300 mL),
whereupon effervescence occurred and an exotherm was noted. The
reaction mixture was extracted with isopropyl acetate (2.times.100
mL). The combined organic extracts were washed with water (100 mL).
n-Pentanol (pentan-1-ol, 34 mL) was added. The organic mixture was
evaporated in vacuo to remove most of the isopropyl acetate. The
resultant solution was treated with aqueous sodium hydroxide
solution (10M, 7 mL), heated at 50.degree. C. for 1.5 hours and
allowed to cool to room temparature. Water (28 mL) was added, and
the mixture was stirred, allowed to settle and the layers were
separated. The resultant organic solution was washed with water (34
mL), the biphase mixture was allowed to settle for 1.5 hours and
the aqueous layer was separated. The concentration of the product,
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide (the "free base"), in the resultant organic solution was
determined quantitatively by HPLC to be about 133.6 to about 135.4
mg/mL, with a total volume of 43 mL, leading to an estimated yield
of the free base of about 5.78 g (estimated "solution yield" of
about 87.8%).
[1186] Aqueous hydrochloric acid (5M, 1.86 mL, amount estimated for
preparation of monohydrochloride salt) was then added. The mixture
was concentrated in vacuo at about 90.degree. C. (bath temperature)
to about 34 mL volume. (9.5 mL solvent was removed, including about
5.5 mL water as a lower layer). A microspatula of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride seed crystals (e.g. substantially in the form
of the Form 1 anhydrate thereof) was added. No crystallisation
occurred. Another 1.8 mL of solvent (including 0.6 ml of water as a
lower layer) was removed in vacuo at ca. 110.degree. C. (bath
temperature). The solution became hazy, and the mixture was cooled
to about 90.degree. C. (bath temperature). Another microspatula of
seed crystals was added. The hazy mixture was cooled to 60.degree.
C. and was aged for 1.5 hours. tert-Butyl methyl ether (TBME, 14.4
mL) was added. The mixture was allowed to cool to room temperature
and was stirred at room temperature, for 15.5 hours in total. The
resultant slurry was filtered, displacement washed with n-pentanol
(7 mL) and then TBME (7 mL). Residual solvents in the filter cake
were removed under suction for about 15 mins, and then the
resultant wet white filter cake (7.14 g) was dried in vacuo at
70.degree. C. for 22.25 hours to afford the title product,
substantially in the form of the monohydrochloride Form 1
anhydrate, as a white solid (4.75 g, molecular weight about 658.3,
so about 65-66% yield based on amount of
[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4--
b]pyridin-5-yl]methanol used). HPLC (5 mins generic solvent
gradient): T.sub.RET about 2.45 min. Mass spectrum: Found: MH.sup.+
622, (M-H).sup.- 620. A preliminary analysis suggests a purity of
about 97.7%.
Example 1A3
Recrystallisation of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de mono hydrochloride
[1187] Form 1 anhydrate (e.g. as prepared in Example 1A2 such as
alternative Plant preparation no. 1 thereof) is preferably
recrystallised from dry (e.g. anhydrous) n-propanol. An alternative
suitable recrystallisation solvent is anhydrous ethanol, though it
is thought this gives lower recrystallisation yields than anhydrous
n-propanol.
[1188] Generally, for the recrystallisation, it is considered
preferable not to conduct the recrystallisation in a
water-containing solvent, in order to reduce the risk of forming
the undesired monohydrochloride Form 2 hydrate.
[1189] One possible embodiment of Example 1A3 is as follows:
N-{([1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyri-
din-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benza-
mide monohydrochloride Form 1 anhydrate is dissolved in about 7
volumes of anhydrous n-propanol at about 85.degree. C., e.g. under
nitrogen. The temperature is adjusted to about 70.degree. C. The
mixture is seeded with a small amount of
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate product. The mixture is then
held at about 70.degree. C. for about two hours (e.g. with
stirring), cooled to 0.degree. C. over about 3 to 16 hours (e.g.
for about 12 hours) (e.g. with stirring), is aged at 0.degree. C.
for about 2 to about 8 hours (e.g. with stirring), and the
resulting solid product is separated by filtration. The filtered
solid product can optionally be dried in vacuo with heating e.g. at
55.+-.5.degree. C.
Example 1A3
(Alternative Preparation): Recrystallisation of
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate
[1190] n-Propanol (propan-1-ol, 7.8 L, 6 vol) was added to
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride Form 1 anhydrate (1.304 kg, 1 wt, which e.g.
can be as prepared in Example 1A2 (Alternative preparation no. 1,
Plant Method) or Example 1A2 (Alternative Preparation no. 2)), in a
first vessel, and the resulting suspension was heated under
nitrogen to 90.+-.3.degree. C. (e.g. 90-92.degree. C.) until
complete dissolution of the solid had been achieved. The resulting
solution (having reached 96.degree. C.) was then transferred over
about 20 minutes through a 5 micron filter into a second vessel
pre-heated to 85.+-.3.degree. C. under nitrogen. The first vessel
was washed with n-propanol (1.3 L, 1 vol) which was heated to
90.degree. C., and these hot n-propanol washings were then passed
through the previously-used lines and filter into the second vessel
over about 2 minutes. The resulting combined solution in the second
vessel was cooled to 70.+-.3.degree. C. and was seeded with a
slurry of micronised
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-benza-
mide monohydrochloride Form 1 anhydrate (about 1.3 g, 0.001 wt) in
n-propanol (10 ml). The resulting mixture was aged at
70.+-.3.degree. C. for 2 hours and then was cooled to
0.+-.3.degree. C. over 12 hours (approx. 0.1 degree/minute cooling
rate), all under nitrogen. The resulting slurry was then aged at
0.+-.3.degree. C. for 5 hours 10 minutes with stirring at 110 rpm.
The solid was collected by filtration under vacuum (using a ca. 24
cm PTFE mini filter fitted with Whatman No. 113 wet strengthened
filter paper with rough side up, and using a ca. 600 mbar vacuum
regulated by a Buchi vacuum controller). The filter cake was washed
twice with pre-filtered n-propanol (2.times.2.6 L, 2.times.2 vol)
under suction to free the product of most of the solvent. The
resulting filtered solid was transferred to polythene lined steel
trays, covered with mono filament cloth, and transported to an oven
in a sealed polythene bag. After removal of the bag once in the
oven, the solid product was dried in vacuo at 60.degree. C. to a
constant probe temperature and/or to a constant weight, using a ca.
70 mbar vacuum regulated by a Buchi vacuum controller, to give the
title product as the Form 1 anhydrate and as a white solid (1.096
kg; yield: 84% theory, 84% w/w).
Example 1B
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de
##STR00251##
[1192] A suspension of
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzoic acid
(7.38 g, 19.8 mmol, 1.2 equivalents, e.g. which can be as prepared
in Intermediate 39) in N,N-dimethylformamide (75 ml, about 10
volumes cf. the weight of the acid) was treated with
N,N-diisopropylethylamine (4.89 ml, 3.62 g, 28.1 mmol, 1.7
equivalents) resulting in a clear yellow solution.
N',N'-Dicyclohexylcarbodiimide (4.42 g, 21.45 mmol, 1.3
equivalents) and then 1-hydroxybenzotriazole hydrate (3.28 g, 21.45
mmol, 1.3 equivalents) were added, and the mixture was stirred at
room temperature for 15 minutes.
5-(Aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyra-
zolo[3,4-b]pyridin-4-amine (5 g, 16.5 mmol, 1 equivalent, e.g.
which can optionally be as prepared in Intermediate 7 including
alternative preparation no. 1 thereof was then added, resulting in
a cloudy yellow/orange solution which was allowed to stir at room
temperature overnight. As much N,N-dimethylformamide as possible
was evaporated off, the residue was partitioned between
dichloromethane and sodium bicarbonate solution, and then the
separated aqueous phase was extracted with more dichloromethane.
The combined organic extracts were washed with lithium chloride
solution, water and brine, were dried (MgSO.sub.4), and were
concentrated by evaporation, to give the crude product (15.5 g).
1.73 g of this crude product was removed. The remainder of the
crude product was purified by column chromatography on silica gel
(about 600 g), eluting with 10% methanol in dichloromethane,
followed by 20% methanol in dichloromethane, 20% methanol in
dichloromethane containing triethylamine, and finally 30% methanol
in dichloromethane containing triethylamine. The majority of the
fractions containing the product were combined and evaporated to
give the title compound (6.68 g) as a white foam. LCMS showed
MH.sup.+=622; T.sub.RET=2.21 min. The apparently-more-pure
fractions containing the product were combined and evaporated to
give more of the title compound (0.48 g).
Example 1B (Alternative preparation no. IA):
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide
##STR00252##
[1194] O-(7-Azabenzotriazol-1-yl)-N,N, N',N'-tetramethyluronium
hexafluorophosphate (HATU, 2.4 g, 6.3 mmol) was added to a solution
of 4-[(8-bromooctanoyl)amino]benzoic acid (2.1 g, 6.1 mmol) in
N,N-dimethylformamide (20 ml) followed by N,N-diisopropylethylamine
(2.5 g, 19 mmol). The solution was stirred at room temperature for
15 min, and
5-(aminomethyl)-1,6-diethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4--
b]pyridin-4-amine (1.7 g, 5.6 mmol. e.g. which can be substantially
as prepared in Intermediate 7 including alternative preparation no.
1 thereof was added as a solid. The solution was stirred at room
temperature for 3 hours, and then was added to aqueous sodium
bicarbonate solution (200 ml) and extracted with dichloromethane
(2.times.100 ml). The dried (sodium sulphate) organic extract was
evaporated gently to leave crude
4-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide mixed with
N,N-dimethylformamide (about 5 ml).
[1195] 2-(Methylamino)ethanol (3 ml, 35 mmol, commercially
available e.g. from Aldrich) was added to this crude mixture and
the solution was heated in a sealed microwave vessel (split between
three 10 ml vessels) at 150.degree. C. for 15 min. The resulting
mixture was evaporated and the residue purified on a 100 g silica
cartridge, eluting with a gradient starting with dichloromethane
and finishing with 30% methanol in dichloromethane containing 1%
triethylamine, to give a yellow gum (2.5 g) containing small
amounts of impurities. The gum was dissolved in 19:1 ethyl
acetate/methanol (100 ml) and washed with aqueous sodium carbonate
solution. The organic phase was evaporated onto silica and applied
to a column of silica (300 ml), eluting with 9:1 ethyl acetate: 2M
ammonia in methanol to give a gum (1.75 g). Trituration with
diethyl ether (30 ml) gave a white solid (1.5 g). LCMS showed
MH.sup.+=622; T.sub.RET=2.1 min. This product was used in the
preparation of Example 1B (Alternative preparation no. 1B), as
described below.
Example 1B
(Alternative Preparation No. 1B):
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide
##STR00253##
[1197] A mixture of
4-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (2.2 g, 3.5
mmol, e.g. which can be as prepared in Intermediate 11) and
2-(methylamino)ethanol (2 ml, 22 mmol, commercially available e.g.
from Aldrich,) in NAN-dimethylformamide (4 ml), was split between
two 10 ml sealed microwave vessels, and was heated at 140.degree.
C. under microwave irradiation for 15 minutes. The combined
reaction mixtures were poured into aqueous sodium carbonate
solution (Na.sub.2CO.sub.3, 100 ml) and extracted with ethyl
acetate (2.times.75 ml). The organic phase was washed with water
(100 ml) and evaporated. The residue was purified by column
chromatography on silica gel (700 ml), eluting with 19:1
dichloromethane: 2M ammonia in methanol, to give a gum (1.2 g). The
gum was triturated with diethyl ether (20 ml) to give a solid (1.1
g).
[1198] This 1.1 g of solid was combined with the white solid
material (1.5 g) prepared by Example 1B (Alternative preparation
no. 1A), see above for the method. The two batches of product were
blended by stirring in diethyl ether (50 ml) for 2 hours.
Filtration gave the title compound as a white powder (2.4 g). LCMS
showed MH.sup.+=622; T.sub.RET=2.14 min.
Example 1B (Alternative preparation no. 2):
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide
##STR00254##
[1200]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide monohydrochloride (20.0 g, 0.0304 mol, e.g. optionally
the Form 1 anhydrate thereof) was suspended in ethyl acetate (150
ml). Potassium carbonate (7.5 g in 50 ml water) was added dropwise,
and the mixture was stirred vigorously for 3 hours. The solid was
filtered off, washed with ethyl acetate (30 ml), and dried at
50.degree. C. Analysis indicates that this product contains about
1.5% of chloride. The product was suspended in isopropanol (160
ml), and Dowex 550A resin (10 g) was added. The suspension was
heated to 60.degree. C. for 1 hour, and was filtered while hot. The
filtrate was concentrated to leave a white foam. Dichloromethane
(100 ml) was added and the resulting mixture was concentrated to
dryness to give the title compound (the "free base"). Analysis
indicates about 0.3% of chloride remaining; NMR analysis indicates
about 0.3% of water present.
[1201] The product free base made by this "Example 1B (alternative
preparation no. 2)" procedure is thought, from a preliminary
analysis, to be present as a substantially amorphous form (i.e. a
substantially non-crystalline form), e.g. as ascertained by
substantially a lack of or minimal birefringence under microscopy
and/or as ascertained by X-ray powder diffraction.
Example 1B1
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de, or a Salt Thereof
[1202]
4-[(8-Bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-
-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (100 mg,
0.16 mmol, e.g. which can optionally be as prepared in Intermediate
11) was dissolved in N,N-dimethylformamide (4 ml). To one-quarter
of this solution, i.e. containing
4-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (25 mg, 0.04
mmol) in N,N-dimethylformamide (1 ml), was added
2-(methylamino)ethanol (0.0064 ml (6.4 microlitres), 0.08 mmol, 2
equivalents, commercially available e.g. from Aldrich) followed by
N,N-diisopropylethylamine (0.04 ml, about 6 equivalents). The
reaction mixture was heated at 85.degree. C. for 16 hours. The
solvent was evaporated and the residue was purified by eluting
through a 5 g silica SPE cartridge, with dichloromethane containing
20% methanol and 1% triethylamine as the eluent, to give the title
compound (18.7 mg), in the form of a cream foam, initially thought
to be in the form of the "free base". LCMS showed MH.sup.+=622;
T.sub.RET=2.20 min. However, later re-inspection of the 1H NMR
spectrum suggests that the product was isolated as a salt e.g. a
mono-salt (presumed to be a hydrobromide salt), e.g. from the
signals of protons adjacent to the (2-hydroxyethyl)(methyl)amino
group.
Example 1D
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de monohydrobromide
[1203] Isopropanol (15 ml) was added to
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide (the "free base", 1.5 g, e.g. which can be as prepared in
Example 1B) and the mixture was heated at 60.degree. C. for 1 hour
to dissolve the free base. 48% Aqueous hydrobromic acid (0.273 ml,
1 equiv.) was added, and the mixture was left to temperature cycle
between 0.degree. C. and 40.degree. C. over the weekend (about 3
days). The resulting solid was isolated by filtration, washed with
isopropanol (about 5 ml), left on the filter for a while, and then
dried under vacuum at 40.degree. C. in a vacuum oven overnight to
give the title compound (1.286 g). NMR appeared to show some
isopropanol present (e.g. very approx. 0.89% w/w). XRPD appeared to
suggest product was partly crystalline material. Ion chromatography
of the product appears to confirm that the stoichiometry of the
salt is that of a monohydrobromide.
Example 1E
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de hemisuccinate
[1204] Isopropanol (10 ml) was added to
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide (the "free base", 1 g, e.g. which can be as prepared in Example
I B) and the mixture was heated at 60.degree. C. for 0.5 hour to
dissolve the free base. A solution of succinic acid (199 mg, 1.05
equiv.) in isopropanol (2 ml) was added to the solution of the free
base, and the mixture was left to temperature cycle between
0.degree. C. and 40.degree. C. for about 3 days. The resulting
solid was isolated by filtration, washed with isopropanol (about 2
ml), left on the filter paper for a while, and dried under vacuum
at 40.degree. C. in an oven overnight to give the title compound
(0.833 g). NMR appeared to show approximately 1:0.471 stoichiometry
of free base: succinate, and some isopropanol present (e.g.
approximately 0.73% w/w). XRPD appeared to suggest the product was
mainly crystalline material.
Example 1F
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de hemifumarate
[1205]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide [the "free base", 100 mg, about 0.161 mmol, as a
substantially amorphous form thereof, as prepared in Example 1B
(alternative preparation no. 2)] was dissolved in isopropanol (1
ml) at room temperature. The resulting solution was added to
fumaric acid (18.7 mg.+-.1 mg, 1 equivalent) and heated to
40.degree. C. The reaction mixture was left to temperature cycle
between 0.degree. C. and 40.degree. C. overnight. The next day, the
reaction mixture was a slurry, and the solid from this was isolated
by filtration, washed with isopropanol (0.5 ml) and dried at
ambient temperature under vacuum overnight. 54.4 mg of dried solid
was obtained. NMR data appears to support a stoichiometry
consistent with the solid product being substantially a
hemifumarate salt.
Example 1G
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de mono-xinafoate (mono-1-hydroxy-2-naphthoate)
[1206]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide [the "free base", 100 mg, about 0.161 mmol, as a
substantially amorphous form thereof, as prepared in Example 1B
(alternative preparation no. 2)] was dissolved in isopropanol (1
ml) at room temperature. The resulting solution was added to
xinafoic acid (1-hydroxy-2-naphthoic acid) (30.3 mg.+-.1 mg, 1
equivalent) and heated to 40.degree. C. The reaction mixture was
left to temperature cycle between 0.degree. C. and 40.degree. C.
overnight. The next day, it remained a solution. Cyclohexane (0.5
ml) was added and the resulting solution was left to temperature
cycle between 0.degree. C. and 40.degree. C. overnight. The next
day, the reaction mixture was a slurry, and the solid from this was
isolated by filtration, washed with 1:1 isopropanol:cyclohexane (1
ml) and dried at ambient temperature under vacuum over the weekend.
58.6 mg of dried solid was obtained. NMR data appears to support a
stoichiometry consistent with the solid product being substantially
a mono-xinafoate (mono-1-hydroxy-2-naphthoate) salt.
Example 1H1
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de phosphate
[1207]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide [the "free base", 100 mg, about 0.161 mmol, as a
substantially amorphous form thereof, as prepared in Example 1B
(alternative preparation no. 2)] was suspended in methyl isobutyl
ketone (MIBK, 1 ml), producing a slightly turbid solution.
Phosphoric acid (H.sub.3PO.sub.4, 85 wt % solution in water
(commercially available e.g. from Aldrich), 7.9 microlitres, 1
equivalent) was added. Methyl isobutyl ketone (MIBK, 0.5 ml) was
added. A gum appeared at the bottom of the vial, which was left to
temperature cycle between 0.degree. C. and 40.degree. C. for about
2 days. A suspension had formed, and from this the resulting solid
was isolated by filtration, washed with isopropanol (0.5 ml) and
dried at 40.degree. C. under vacuum overnight. 74.4 mg of dried
solid was obtained.
Example 1H2
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de phosphate
[1208]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide [the "free base", 100 mg, about 0.161 mmol, as a
substantially amorphous form thereof, as prepared in Example 1B
(alternative preparation no. 2)] was dissolved in isopropanol (1
ml) at room temperature. Phosphoric acid (H.sub.3PO.sub.4, 85 wt %
solution in water (commercially available e.g. from Aldrich), 3.95
microlitres, 0.5 equivalents) was added. A gum formed, which
dissolved on heating to 60.degree. C. No precipitation occurred
after cooling to ambient temperature and about 30 minutes at
ambient temperature. Phosphoric acid (H.sub.3PO.sub.4, 85 wt %
solution in water (commercially available e.g. from Aldrich), 3.95
microlitres, 0.5 equivalents) was added. A gum formed, which did
not dissolve on heating to 60.degree. C. The reaction mixture was
left to temperature cycle between 0.degree. C. and 40.degree. C.
for about 2 days. A suspension had formed, and from this the
resulting solid was isolated by filtration, washed with isopropanol
(0.5 ml) and dried at 40.degree. C. under vacuum overnight. 68.3 mg
of dried solid was obtained.
Example 1J1
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de mono-(+)-camphorsulfonate
[1209]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide [the "free base", 100 mg, about 0.161 mmol, as a
substantially amorphous form thereof, as prepared in Example 1B
(alternative preparation no. 2)] and (+)-camphorsulfonic acid (37.4
mg.+-.1 mg, 1 equivalent) were suspended in methyl isobutyl ketone
(MIBK, 5 ml). The resulting solution/suspension was left to
temperature cycle between 0.degree. C. and 40.degree. C. overnight.
The next day, a suspension had formed, and from this the resulting
solid was isolated by filtration, washed with methyl isobutyl
ketone (MIBK, 1 ml) and dried at 40.degree. C. under vacuum
overnight. 111.4 mg of dried solid was obtained. NMR data appears
to support a stoichiometry consistent with the solid product being
substantially a mono-(+)-camphorsulfonate.
Example 1J2
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de mono-(+)-camphorsulfonate
[1210]
N-{[1,6-Diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b-
]pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)-
benzamide [the "free base", 100 mg, about 0.161 mmol, as a
substantially amorphous form thereof, as prepared in Example 1B
(alternative preparation no. 2)] and (+)-camphorsulfonic acid (37.4
mg.+-.1 mg, 1 equivalent) were suspended in ethyl acetate (6 ml).
The resulting suspension was left to temperature cycle between
0.degree. C. and 40.degree. C. overnight. The next day, a
suspension had formed, from which the resulting solid was isolated
by filtration, washed with ethyl acetate (1 ml) and dried at
40.degree. C. under vacuum overnight. 82.2 mg of dried solid was
obtained. NMR data appears to support a stoichiometry consistent
with the solid product being substantially a
mono-(+)-camphorsulfonate.
Examples 1C, 3, 4, 9, 11 and 12
[1211] 0.04 mmol of the appropriate
(8-bromooctanoyl)amino-alkylating agent (selected from those listed
below) in N,N-dimethylformamide (0.5 ml) was treated with a
solution of the appropriate amine (HNR.sup.5R.sup.6 wherein
NR.sup.5R.sup.6 is as defined in the table below; 0.08 mmol) in
N,N-dimethylformamide (0.5 ml) followed by
N,N-diisopropylethylamine (0.04 ml). The reaction mixture was
heated at 80.degree. C. for 16 hours and the solvent was removed in
vacuo. The residue was purified by mass directed preparative HPLC
(Method B) to give the appropriate title compound (with a structure
as shown in the table below) as a trifluoroacetate salt.
(8-Bromooctanoyl)amino-Alkylating Agents and Amounts Used:
[1212]
4-[(8-Bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-
-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (200.8
mg/8=25.10 mg, 0.04 mmol, e.g. which can be as prepared in
Intermediate II); and [1213]
4-[(5-{[({4-[(8-bromooctanoyl)amino]phenyl}carbonyl)amino]methyl}-1,6-die-
thyl-1H-pyrazolo[3,4-b]pyridin-4-yl)amino]-1-piperidinecarboxamide
(214.3 mg/8=26.79 mg, 0.04 mmol, e.q. which can be as prepared in
Intermediate 21).
TABLE-US-00014 ##STR00255## [1213] Starting NR.sup.5R.sup.6 One
material (wherein the possible Ex- can be connecting Source ample
as pre- nitrogen atom is of M - H T.sub.RET No. R.sup.3 Ar Q.sup.1,
Q.sup.2 pared in underlined) HNR.sup.5R.sup.6 ion (min) 1C
##STR00256## (x1a) Q.sup.1 is NH; Q.sup.2 is --C(O)-- Inter-
mediate 11 ##STR00257## Aldrich 620 2.24 3 ##STR00258## (x1a)
Q.sup.1 is NH; Q.sup.2 is --C(O)-- Inter- mediate 21 ##STR00259##
Aldrich 702 2.24 4 ##STR00260## (x1a) Q.sup.1 is NH; Q.sup.2 is
--C(O)-- Inter- mediate 21 ##STR00261## Aldrich 702 2.24 9
##STR00262## (x1a) Q.sup.1 is NH; Q.sup.2 is --C(O)-- Inter-
mediate 21 ##STR00263## Aldrich 662 2.15 11 ##STR00264## (x1a)
Q.sup.1 is NH; Q.sup.2 is --C(O)-- Inter- mediate 11 ##STR00265##
Aldrich 660 2.34 12 ##STR00266## (x1a) Q.sup.1 is NH; Q.sup.2 is
--C(O)-- Inter- mediate 11 ##STR00267## Aldrich 660 2.34
[1214] The salt prepared in Example 1C above is named
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide trifluoroacetate.
Examples 17, 18, 19, 20, 21, 22, 23 and 24
[1215] To a solution of the appropriate bromo alkylating agent
(0.04 mmol) (selected from those listed below) in
N,N-dimethylformamide (1 ml) were added the appropriate amine
HNR.sup.5R.sup.6 (wherein NR.sup.5R.sup.6 is as defined in the
table below) (0.08 mmol, 2 equivalents) followed by
N,N-diisopropylethylamine (0.04 ml, about 6 equivalents). The
reaction mixture was heated at 85.degree. C. for 16 hours. The
solvent was evaporated and the residue was purified by mass
directed preparative HPLC (Method A, using formic acid) to give the
title compound (with a structure as shown in the table below) as a
formate salt. In all cases, the product was present as a foam.
[1216] Bromo alkylating agents and amounts used: [1217]
4-[(8-Bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (100 mg/4=25
mg, 0.04 mmol, e.g. which can be as prepared in Intermediate 11);
[1218]
4-{4-[(4-Bromobutyl)oxy]butyl}-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-y-
lamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (98
mg/4=24.5 mg, 0.04 mmol, e.g. which can be as prepared in
Intermediate 27); and [1219]
4-{[5-({[(4-{4-[(4-Bromobutyl)oxy]butyl}phenyl)carbonyl]amino}methyl)-1,6-
-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-piperidinecarboxamide
(105 mg/4=26.25 mg, 0.04 mmol, e.g. which can be as prepared in
Intermediate 28).
TABLE-US-00015 ##STR00268## [1219] Starting NR.sup.5R.sup.6 One
material (wherein the possible Ex- can be connecting Source ample
as pre- nitrogen atom is of MH.sup.+ T.sub.RET No. R.sup.3 Q.sup.1,
Q.sup.2 L pared in underlined) HNR.sup.5R.sup.6 ion (min) 17
##STR00269## Q.sup.1 is NH; Q.sup.2 is --C(O)-- ##STR00270## Inter-
mediate 11 ##STR00271## Aldrich 634 2.20 18 ##STR00272## Q.sup.1 is
NH; Q.sup.2 is --C(O)-- ##STR00273## Inter- mediate 11 ##STR00274##
Aldrich 618 2.25 19 ##STR00275## Q.sup.1 and Q.sup.2 are a bond
##STR00276## Inter- mediate 27 ##STR00277## Aldrich 609 2.27 20
##STR00278## Q.sup.1 and Q.sup.2 are a bond ##STR00279## Inter-
mediate 27 ##STR00280## Aldrich 621 2.28 21 ##STR00281## Q.sup.1
and Q.sup.2 are a bond ##STR00282## Inter- mediate 27 ##STR00283##
Aldrich 605 2.28 22 ##STR00284## Q.sup.1 and Q.sup.2 are a bond
##STR00285## Inter- mediate 28 ##STR00286## Aldrich 651 2.12 23
##STR00287## Q.sup.1 and Q.sup.2 are a bond ##STR00288## Inter-
mediate 28 ##STR00289## Aldrich 663 2.14 24 ##STR00290## Q.sup.1
and Q.sup.2 are a bond ##STR00291## Inter- mediate 28 ##STR00292##
Aldrich 647 2.17
[1220] The specific procedure used for Example 23 was as
follows:
Example 23
Formate salt of
4-({1,6-diethyl-5-[({[4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)phenyl]carb-
onyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarbo-
xamide
##STR00293##
[1222] To a solution of
4-{[5-({[(4-{4-[(4-bromobutyl)oxy]butyl}phenyl)carbonyl]amino}methyl)-1,6-
-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-piperidinecarboxamide
(105 mg/4=26.25 mg, 0.04 mmol, e.g. which can be as prepared in
Intermediate 28) in N,N-dimethylformamide (1 ml) were added
morpholine (0.007 ml, 0.08 mmol, 2 equivalents) followed by
N,N-diisopropylethylamine (0.04 ml, about 6 equivalents). The
reaction mixture was heated at 85.degree. C. for 16 hours. The
solvent was evaporated and the residue was purified by mass
directed preparative HPLC (Method A, using formic acid) to give the
title compound as a cream foam (7.9 mg). LCMS showed MH.sup.+=663;
T.sub.RET=2.14 min.
Example 23A
4-({1,6-Diethyl-5-[({[4-(4-{[4-(4-morpholinyl)butyl]oxy}butyl)phenyl]carbo-
nyl}amino)methyl]-1H-pyrazolo[3,4-b]pyridin-4-yl}amino)-1-piperidinecarbox-
amide
##STR00294##
[1224] A mixture of
4-{[5-({[(4-{4-[(4-bromobutyl)oxy]butyl}phenyl)carbonyl]amino}methyl)-1,6-
-diethyl-1H-pyrazolo[3,4-b]pyridin-4-yl]amino}-1-piperidinecarboxamide
(1.1 g, 1.67 mmol, e.g. which can be as prepared in Intermediate
28) in N,N-dimethylformamide (20 ml) was treated with
N,N-diisopropylethylamine (0.567 ml) and morpholine (0.300 ml, 3.3
mmol) and heated at 65.degree. C. The solvent was removed under
vacuum and the residue was applied directly to a 100 g silica SPE
cartridge and eluted with a gradient of from 0% to 50% methanol in
dichloromethane over 40 mins. Fractions containing product were
pooled and evaporated to give a pale orange foam which appeared to
be about 85% pure by analytical HPLC. The residue was further
purified by mass directed preparative HPLC (Method C, using 0.1%
trifluoroacetic acid) in 50 mg portions, and, following evaporation
of the solvent from the appropriate fractions, the residual
material was dissolved in dichloromethane and washed with saturated
aqueous sodium bicarbonate solution (NaHCO.sub.3). The organic
fraction was collected by passing through a hydrophobic frit and
evaporated to give the title compound (0.300 g) as a white foam.
LCMS showed MH.sup.+=663; T.sub.RET=2.09 min.
Example 25
Formate salt of
N-{[1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]-
pyridin-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)b-
enzamide
##STR00295##
[1226] To a solution of
4-[(8-bromooctanoyl)amino]-N-{[1-ethyl-6-methyl-4-(tetrahydro-2H-pyran-4--
ylamino)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (50 mg,
0.08 mmol, e.g. which can be as prepared in Intermediate 37) in
N,N-dimethylformamide (1 ml) was added N,N-diisopropylethylamine
(0.085 ml, 0.49 mmol) and 2-(methylamino)ethanol (0.008 ml, 0.1
mmol, commercially available e.g. from Aldrich), and the resulting
solution was heated at 60.degree. C. for 2 hours. The solvent was
evaporated and the residue purified by mass directed preparative
HPLC (Method A). Evaporation of the solvent gave the title compound
as a cream foamy solid (22.2 mg). LCMS showed MH.sup.+=608;
T.sub.RET=2.16 min.
Example 26
Formate salt of
N-{[1-ethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-5-
-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzamide
##STR00296##
[1228] A solution of
4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzoic acid
(24 mg, 0.06 mmol, e.g. which can be as prepared in Intermediate
39) in N,N-dimethylformamide (1 ml) was treated with
5-(aminomethyl)-1-ethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]py-
ridin-4-amine hydrochloride (20 mg, 0.065 mmol, e.g. which can be
as prepared in Intermediate 48), N,N-diisopropylethylamine (0.036
ml, 0.21 mmol) and
0-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (25 mg, 0.065 mmol) and the resultant mixture
was allowed to stand at ambient temperature for 18 hours. The
solvent was evaporated and the residue partitioned between
dichloromethane (5 ml) and saturated aqueous sodium bicarbonate
solution (5 ml). The organic fraction was collected through a
hydrophobic frit, evaporated and purified by mass directed
preparative HPLC (Method A). Fractions containing the product were
pooled and evaporated to give the title compound (8.3 mg) as a pale
yellow solid. LCMS showed MH.sup.+=594; T.sub.RET=2.16 min.
Example 27
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-[8-(4-morpholinyl)octyl]benzamide
##STR00297##
[1230] A solution of
4-(8-bromooctyl)-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyr-
azolo[3,4-b]pyridin-5-yl]methyl}benzamide (36.3 mg, 0.06 mmol, e.g.
which can be as prepared in Intermediate 41) in
N,N-dimethylformamide (6 ml) was treated with morpholine (0.021 ml,
0.24 mmol) and N,N-diisopropylethylamine (0.042 ml, 0.24 mmol) and
heated at 60.degree. C. for 16 hours. The solvent was evaporated
and the residue purified by mass directed preparative HPLC (Method
A). Fractions containing product were pooled and evaporated to give
the title compound (11 mg). LCMS showed MH.sup.+=605;
T.sub.RET=2.39 min.
Example 28
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-{8-[(2-hydroxyethyl)(methyl)amino]octyl}benzamide
##STR00298##
[1232] A solution of
4-(8-bromooctyl)-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyr-
azolo[3,4-b]pyridin-5-yl]methyl}benzamide (36.3 mg, 0.06 mmol, e.g.
which can be as prepared in Intermediate 41) in
N,N-dimethylformamide (6 ml) was treated with
2-(methylamino)ethanol (0.0195 ml, 0.24 mmol, commercially
available e.g. from Aldrich) and N,N-diisopropylethylamine (0.042
ml, 0.24 mmol) and heated at 60.degree. C. for 16 hours. The
solvent was evaporated and the residue purified by mass directed
preparative HPLC (Method A). Fractions containing product were
pooled and evaporated to give the title compound (7 mg). LCMS
showed MH.sup.+=593; T.sub.RET=2.37 min.
Example 29
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-3-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de
##STR00299##
[1234] A solution of
3-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (42 mg, 0.067
mmol, e.g. which can be as prepared in Intermediate 47) in
N,N-dimethylformamide (3 ml) was treated with
2-(methylamino)ethanol (0.011 ml, 0.13 mmol, commercially available
e.g. from Aldrich) and N,N-diisopropylethylamine (0.0235 ml, 0.13
mmol) and heated at 60.degree. C. for 16 hours. The solvent was
evaporated and the residue purified by mass directed preparative
HPLC (Method A). Fractions containing product were pooled and
evaporated to give the title compound (33 mg). LCMS showed
MH.sup.+=622; T.sub.RET=2.21 min.
Example 30
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-2-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzami-
de
##STR00300##
[1236] A solution of
2-[(8-bromooctanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (68 mg, 0.11
mmol, e.g. which can be as prepared in Intermediate 46) in
N,N-dimethylformamide (5 ml) was treated with
2-(methylamino)ethanol (0.0175 ml, 0.22 mmol, commercially
available e.g. from Aldrich) and N,N-diisopropylethylamine (0.038
ml, 0.22 mmol) and heated at 60.degree. C. for 16 hours. The
solvent was evaporated and the residue purified by mass directed
preparative HPLC (Method A). Fractions containing product were
pooled and evaporated to give the title compound (5 mg). LCMS
showed MH.sup.+=622; T.sub.RET=2.15 min.
Examples 31 to 34
Example 31
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyridi-
n-5-yl]methyl}-4-({6-[(2-hydroxyethyl)(methyl)amino]hexanoyl}amino)benzami-
de
##STR00301##
[1238] To a solution of
4-[(6-bromohexanoyl)amino]-N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylami-
no)-1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}benzamide (74 mg, 0.12
mmol, e.g. which can be as prepared in Intermediate 49) in
N,N-dimethylformamide (3 ml) was added 2-(methylamino)ethanol
(0.019 ml, 0.24 mmol, commercially available e.g. from Aldrich) and
N,N-diisopropylethylamine (0.042 ml, 0.24 mmol), and the mixture
was heated at 85.degree. C. with stirring for 3 hours. On cooling
the solvent was removed by evaporation and the residue purified by
mass directed preparative HPLC (Method A) to give a yellow gum (50
mg). This gum was dissolved in dichloromethane (5 ml) and washed
with saturated aqueous sodium bicarbonate solution (3 ml), and the
organic phase was isolated by passing through a hydrophobic frit.
The aqueous phase was further extracted with dichloromethane (3
ml), and the combined organic phases evaporated to give the title
compound as a white residue (33 mg). LCMS showed MH.sup.+=594;
T.sub.RET=2.02 min.
[1239] Examples 32 to 34 were prepared in a generally similar
manner:
TABLE-US-00016 ##STR00302## Starting material can Example e.g. be
as number prepared in: n M.sup.+ ion T.sub.RET (min) 32
Intermediate 50 n = 6 608 2.12 33 Intermediate 51 n = 9 650 2.32 34
Intermediate 52 n = 10 664 2.40
Intranasal Composition Example
[1240] The following solution, a pharmaceutical composition
intended for intranasal administration to a mammal, such as a human
or dog or rat or monkey, (and/or for intranasal delivery of the
active drug substance), may be prepared.
[1241] A 1% w/w aqueous solution for intranasal administration is
as follows:
TABLE-US-00017 Quantity mg/g % w/w
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)- 10 mg 1
1H-pyrazolo[3,4-b]pyridin-5-yl]methyl}-4-({8-[(2-
hydroxyethyl)(methyl)amino]octanoyl}amino)- benzamide
monohydrochloride (e.g. this can e.g. be as prepared in Example
1A2, or in Example 1A2 alternative preparation no. 1), Micronized
Active Drug Substance Xylitol, EP and/or USP and/or JP (used as an
45 mg 4.5 isotonicity adjusting agent) Not Animal Origin Xanthan
Gum, EP and/or USP (used as thickening 2 mg 0.2 agent) Not Animal
Origin, Powder Potassium Sorbate, EP and/or USP 3 mg 0.3 Not Animal
Origin Edetate Disodium, Dihydrate (EDTA), 0.15 mg 0.015 EP and/or
USP And/or JP, Not Animal Origin, Crystalline Powder Sodium
Citrate, Dihydrate, USP 11.8 mg 1.18 Not Animal Origin, Crystalline
Powder Citric Acid Anhydrous, EP and/or USP 7.6 mg 0.76 Not Animal
Origin, Granular Powder Water for Injection BP, 100 Percent to 1 g
to 100 PL08828/0047, 1000 ml
Method of Manufacture
[1242] 1. To prepare the 4.5 pH Citrate Buffer solution: Dissolve
Sodium Citrate in the minimum amount of water, heat if necessary to
50-60.degree. C. to aid dissolution. Dissolve the Citric Acid in
the minimum amount of water, heat if necessary to 50-60.degree. C.
to aid dissolution. Pour the Sodium Citrate and Citric Acid into a
beaker and make up to 50% of the target mass of water required to
make the bulk solution.
[1243] 2. Divide the Citrate Buffer solution from step 1 into two
halves (pour equal quantities into two separate beakers).
[1244] 3. To one half of the prepared Citrate Buffer solution
(prepared in step 1) add the
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride micronized active drug substance, heat if
necessary to 50-60.degree. C. to aid dissolution, and dissolve with
the aid of a stirrer.
[1245] 4. In a separate beaker, add the Xylitol to approximately
20% of the target mass of water and mix until dissolved at 3000 rpm
using a Silverson mixer.
[1246] 5. Add the Xanthan Gum to the Xylitol solution and mix at
3000 rpm until dissolved.
[1247] 6. Dissolve the Edetate Disodium Dihydrate (EDTA) in the
minimum quantity of water. Heat to 50-60.degree. C. to aid
dissolution if required and add to the bulk, mixing at 3000
rpm.
[1248] 7. Gradually add the remaining Citrate Buffer solution
(prepared in step 1) to the Xylitol, Xanthan Gum, and EDTA
solution.
[1249] 8. Gradually incorporate the
N-{[1,6-diethyl-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[3,4-b]pyrid-
in-5-yl]methyl}-4-({8-[(2-hydroxyethyl)(methyl)amino]octanoyl}amino)benzam-
ide monohydrochloride previously dissolved in the Citrate Buffer
solution (prepared in step 3), to the solution (prepared in step 7)
and mix for a further 3mins.
[1250] 9. Dissolve the Potassium Sorbate in the minimum quantity of
water and add to the bulk, and mix for a further 3mins.
[1251] 10. Add water to make up to the target mass solution and mix
at 4200 rpm for 5 mins.
[1252] This formulation is preferably be stored under a nitrogen
headspace (and/or stored at about 5.degree. C.).
[1253] Alternative variations of this intranasal composition
example include the following:
[1254] 1. Add polysorbate 80 (polyoxyethylene 20 sorbitan
monooleate, e.g. about 1% to about 5% w/w) as a solubilising agent,
in order to reduce the amount of precipitation of, and/or to slow
down the rate of precipitation of, the drug substance from
solution.
and/or
[1255] 2. Vary the concentration of the active drug substance used
within the range of 0.05% w/w to 1% w/w, for example about 0.5%
w/w.
* * * * *
References