U.S. patent application number 10/598973 was filed with the patent office on 2008-11-06 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 Caroline Mary Cook, Michael Dennis Dowle, Christopher David Edlin, Martin Redpath Johnson, Paul Spencer Jones, Mika Kristian Lindvall, Alison Judith Redgrave, Naimisha Trivedi.
Application Number | 20080275078 10/598973 |
Document ID | / |
Family ID | 34962723 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080275078 |
Kind Code |
A1 |
Cook; Caroline Mary ; et
al. |
November 6, 2008 |
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: R.sup.1 is C.sub.1-3alkyl,
C.sub.1-3fluoroalkyl, or --CH.sub.2CH.sub.2OH; R.sup.2 is hydrogen,
methyl or C.sub.1fluoroalkyl; R.sup.3 is of sub-formula (aa) or
(bb): ##STR00002## wherein Y is NCONH.sub.2 and n.sup.1 is 0 or 1;
R.sup.4 is H; and R.sup.5 is a group of the sub-formula (x), (y),
(y1) or (z): ##STR00003## These compounds are PDE4 inhibitors.
Inventors: |
Cook; Caroline Mary;
(Hertfordshire, GB) ; Dowle; Michael Dennis;
(Hertfordshire, GB) ; Edlin; Christopher David;
(Hertfordshire, GB) ; Johnson; Martin Redpath;
(Hertfordshire, GB) ; Jones; Paul Spencer;
(Hertfordshire, GB) ; Lindvall; Mika Kristian;
(Emeryville, CA) ; Trivedi; Naimisha;
(Hertfordshire, GB) ; Redgrave; Alison Judith;
(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
Greenford, Middlesex
GB
|
Family ID: |
34962723 |
Appl. No.: |
10/598973 |
Filed: |
March 15, 2005 |
PCT Filed: |
March 15, 2005 |
PCT NO: |
PCT/GB2005/000987 |
371 Date: |
May 5, 2008 |
Current U.S.
Class: |
514/303 ;
546/120 |
Current CPC
Class: |
A61P 11/02 20180101;
A61P 17/00 20180101; A61P 17/06 20180101; A61P 25/28 20180101; A61P
11/00 20180101; A61P 27/16 20180101; A61P 1/04 20180101; A61P 25/24
20180101; A61P 11/06 20180101; A61P 19/02 20180101; A61P 13/12
20180101; A61P 29/00 20180101; A61P 27/02 20180101; A61P 31/04
20180101; A61P 9/10 20180101; A61P 35/00 20180101; A61P 37/08
20180101; A61P 25/00 20180101; A61P 37/00 20180101; A61P 43/00
20180101; C07D 471/04 20130101 |
Class at
Publication: |
514/303 ;
546/120 |
International
Class: |
A61K 31/437 20060101
A61K031/437; C07D 487/04 20060101 C07D487/04; A61P 11/06 20060101
A61P011/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 16, 2004 |
GB |
0405893.9 |
Mar 14, 2005 |
GB |
0505214.7 |
Claims
1.-32. (canceled)
33. A compound of formula (I) or a salt thereof: ##STR00174##
wherein: R.sup.1 is C.sub.1-3alkyl, C.sub.1-3fluoroalkyl, or
--CH.sub.2CH.sub.2OH; R.sup.2 is hydrogen, methyl or
C.sub.1fluoroalkyl; R.sup.3 is a 4-(aminocarbonyl)cyclohexyl group
of sub-formula (aa), or an N-aminocarbonyl-piperidinyl or
-pyrrolidinyl group of sub-formula (bb): ##STR00175## wherein Y is
NCONH.sub.2 and n1 is 0 or 1; R.sup.4 is hydrogen; R.sup.5 is
sub-formula (x), (y), (y1) or (z): ##STR00176## wherein in
sub-formula (x), n=0, 1 or 2; in sub-formula (y) and (y1), m=1 or
2; and in sub-formula (z), r=0, 1 or 2; wherein in sub-formula (x)
and (y) and (y1), none, one or two of A, B, D, E and F are
nitrogen; and the remaining of A, B, D, E and F are independently
CH or CR.sup.6; wherein each R.sup.6 independently is halo;
C.sub.1-6alkyl; C.sub.1-4fluoroalkyl; C.sub.1-4alkoxy;
C.sub.1-2fluoroalkoxy; C.sub.3-6cycloalkyloxy; --C(O)R.sup.16a;
--C(O)OR.sup.30; --S(O).sub.2--R.sup.16a;
R.sup.16a--S(O).sub.2--NR.sup.15a--; R.sup.7R.sup.8N--S(O).sub.2--;
C.sub.1-2alkyl-C(O)--R.sup.15aN--S(O).sub.2--;
C.sub.1-4alkyl-S(O)--, Ph-S(O)--, R.sup.7R.sup.8N--CO--;
--NR.sup.15--C(O)R.sup.16a; R.sup.7R.sup.8N; OH;
C.sub.1-4alkoxymethyl; C.sub.1-4alkoxyethyl;
C.sub.1-2alkyl-S(O).sub.2--CH.sub.2--;
R.sup.7R.sup.8N--S(O).sub.2--CH.sub.2--;
C.sub.1-2alkyl-S(O).sub.2--NR.sup.15a--CH.sub.2--; --CH.sub.2--OH;
--CH.sub.2CH.sub.2--OH; --CH.sub.2--NR.sup.7R.sup.8;
--CH.sub.2--CH.sub.2--NR.sup.7R.sup.8; --CH.sub.2--C(O)OR.sup.30;
--CH.sub.2--C(O)--NR.sup.7R.sup.8;
--CH.sub.2--NR.sup.15a--C(O)--C.sub.1-3alkyl;
--(CH.sub.2).sub.n.sup.14-Het.sup.1 where n.sup.14 is 0 or 1; cyano
(CN); Ar.sup.5b; or phenyl, pyridinyl or pyrimidinyl wherein the
phenyl, pyridinyl or pyrimidinyl independently are optionally
substituted by one or two of fluoro, chloro, C.sub.1-2alkyl,
C.sub.1 fluoroalkyl, C.sub.1-2alkoxy or C.sub.1 fluoroalkoxy; or
where two adjacent R.sup.6 taken together are --O--(CMe.sub.2)--O--
or --O--(CH.sub.2).sub.n.sup.14a--O-- where n.sup.14a is 1 or 2;
wherein sub-formula (y) and (y1), independently, are optionally
substituted by oxo at a ring carbon adjacent the 6-membered
aromatic ring; wherein in sub-formula (z), G is O or S or NR.sup.9
wherein R.sup.9 is hydrogen, C.sub.1-4alkyl or
C.sub.1-4fluoroalkyl; none, one, two or three of J, L, M and Q are
nitrogen; and the remaining of J, L, M and Q are independently CH
or CR.sup.6 where R.sup.6, independently of any other R.sup.6; and
wherein: R.sup.7 and R.sup.8 are independently hydrogen;
C.sub.1-4alkyl; C.sub.3-6cycloalkyl; or phenyl optionally
substituted by one or two substituents independently selected from
the group consisting of: fluoro, chloro, C.sub.1-2alkyl,
C.sub.1fluoroalkyl, C.sub.1-2alkoxy and C.sub.1fluoroalkoxy; or
R.sup.7 and R.sup.8 together are --(CH.sub.2).sub.n6-- or
--C(O)--(CH.sub.2).sub.n7-- or --C(O)--(CH.sub.2).sub.n10--C(O)--
or --(CH.sub.2).sub.n.sup.8--X.sup.7--(CH.sub.2).sub.n.sup.9-- or
--C(O)--X.sup.7--(CH.sub.2).sub.n10-- in which: n.sup.6 is 3, 4, 5
or 6, n.sup.7 is 2, 3, 4, or 5, n.sup.8 and n.sup.9 and n.sup.10
independently are 2 or 3, and X.sup.7 is O or NR.sup.14; R.sup.7a
is hydrogen or C.sub.1-4alkyl; R.sup.8a is hydrogen or methyl;
R.sup.14, independent of other R.sup.14, is hydrogen;
C.sub.1-4alkyl; C.sub.1-2fluoroalkyl; cyclopropyl;
--C(O)--C.sub.1-4alkyl; --C(O)NR.sup.7aR.sup.8a; or
--S(O).sub.2--C.sub.1-4alkyl; R.sup.15, independent of other
R.sup.15, is hydrogen; C.sub.1-4alkyl; C.sub.3-6cycloalkyl; or
phenyl optionally substituted by one or two substituents selected
from the group consisting of: halo, C.sub.1-2alkyl,
C.sub.1fluoroalkyl, C.sub.1-2alkoxy and C.sub.1fluoroalkoxy;
R.sup.15a, independent of other R.sup.15a, is hydrogen or
C.sub.1-14alkyl; R.sup.16a is: C.sub.1-6alkyl; C.sub.3-6cycloalkyl
optionally substituted by one oxo, OH; C.sub.1-2alkyl;
C.sub.3-6cycloalkyl-CH.sub.2--; pyridinyl optionally substituted on
a ring carbon atom by one of: halo, C.sub.1-2alkyl,
C.sub.1fluoroalkyl, C.sub.1-2alkoxy or C.sub.1fluoroalkoxy;
Ar.sup.5c; phenyl optionally substituted by one or two substituents
independently selected from the group consisting of: halo,
C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy and
C.sub.1-2fluoroalkoxy; benzyl optionally substituted on its ring by
one or two substituents independently selected from the group
consisting of halo, C.sub.1-2alkyl, C.sub.1fluoroalkyl,
C.sub.1-2alkoxy and C.sub.1fluoroalkoxy; or a 4-, 5-, 6- or
7-membered saturated heterocyclic ring connected at a ring-carbon
and containing one or two ring-hetero-atoms independently selected
from the group consisting of O, S, and N; wherein any ring-nitrogen
which is present is NR.sup.27 where R.sup.27 is H, C.sub.1-2alkyl
or --C(O)Me; and wherein the ring is optionally substituted at
carbon by one C.sub.1-2alkyl or oxo substituent, provided that any
oxo substituent is substituted at a ring-carbon atom bonded to a
ring-nitrogen; R.sup.30, independent of other R.sup.30, is
hydrogen, C.sub.1-4alkyl or C.sub.3-6cycloalkyl; Ar.sup.5b and
Ar.sup.5c independently are a 5-membered aromatic heterocyclic ring
containing one O, S or NR.sup.15a, wherein the ring can optionally
additionally contain one or two N atoms, and wherein the
heterocyclic ring is optionally substituted on a ring carbon atom
by a substituent which is halo, C.sub.1-2alkyl, C.sub.1fluoroalkyl,
--CH.sub.2OH, --CH.sub.2--OC.sub.1-2alkyl, OH or
--CH.sub.2--NR.sup.28R.sup.29 wherein R.sup.28 and R.sup.29
independently are H or methyl; and Het.sup.1 is a 4-, 5-, 6- or
7-membered saturated heterocyclic ring connected at a ring-carbon
and containing one or two ring-hetero-atoms independently selected
from the group consisting of O, S, and N; wherein any ring-nitrogen
which is present is present as NR.sup.31 where R.sup.31 is H,
C.sub.1-2alkyl or --C(O)Me; and wherein the ring is optionally
substituted at carbon by one group which is C.sub.1-2alkyl or oxo,
provided that any oxo group is substituted at a ring-carbon atom
bonded to a ring-nitrogen.
34. A compound or salt as claimed in claim 33 wherein R.sup.1 is
C.sub.2-3alkyl, C.sub.2fluoroalkyl or --CH.sub.2CH.sub.2OH.
35. A compound or salt as claimed in claim 34 wherein R.sup.1
ethyl.
36. A compound or salt as claimed in claim 33 wherein R.sup.2 is
hydrogen.
37. A compound or salt as claimed in claim 33 wherein: R.sup.3 is
##STR00177## NHR.sup.3 is NHR.sup.3 is a
cis-[4-(aminocarbonyl)cyclohexan-1-yl]amino group or NHR.sup.3 is a
racemic [4-(aminocarbonyl)cyclohexan-1-yl]amino group.
38. A compound or salt as claimed in claim 37 wherein: R.sup.3 is
##STR00178## NHR.sup.3 is a
cis-[4-(aminocarbonyl)cyclohexan-1-yl]amino group.
39. A compound or salt as claimed in claim 37 wherein R.sup.3 is:
##STR00179##
40. A compound or salt as claimed in claim 33 wherein R.sup.7 and
R.sup.8 independently are hydrogen or C.sub.1-2alkyl; or R.sup.7
and R.sup.8 together are --(CH.sub.2).sub.n.sup.6-- or
--(CH.sub.2).sub.n.sup.8--X.sup.7--(CH.sub.2).sub.n.sup.9--wherein
X.sup.7 is NR.sup.14 or 0, n.sup.6 is 4 or 5, and n.sup.8 and
n.sup.9 are both 2; R.sup.14 is H, C.sub.1-2alkyl, or --C(O)Me;
R.sup.15, independent of other R.sup.15, is hydrogen or
C.sub.1-12alkyl; R.sup.15a is hydrogen or C.sub.1-2alkyl; and
R.sup.16a is C.sub.1-4alkyl, and
41. A compound or salt as claimed in claim 33 wherein R.sup.5 is
sub-formula (x) or (z).
42. A compound or salt as claimed in claim 33 wherein when R.sup.5
is sub-formula (z); none, one or two of J, L, M and Q are nitrogen;
one or none of J, L, M or Q is CR.sup.6; R.sup.9 is hydrogen or
methyl, r is 1, and, for sub-formula (z), R.sup.6 is independently
OH, C.sub.1-2alkyl or C.sub.1fluoroalkyl.
43. A compound or salt as claimed in claim 33 wherein R.sup.5 is
sub-formula (x), and in sub-formula (x) none or one of A, B, D, E
and F are nitrogen; none, one or two of A, B, D, E and F are
CR.sup.6; and the remaining of A, B, D, E and F are CH.
44. A compound or salt as claimed in claim 43 wherein R.sup.5 is
sub-formula (x), n is 1 and each of A, B, D, E and F is
independently CH or CR.sup.6.
45. A compound or salt as claimed in claim 43 wherein R.sup.5 is
benzyl optionally substituted on the phenyl ring with one or two
R.sup.6 substituents.
46. A compound or salt as claimed in claim 33 wherein, in
sub-formula (x), (y), (y1) and (z), each R.sup.6, independently of
any other R.sup.6, is fluoro, chloro, bromo, iodo, methyl, ethyl,
n-propyl, isopropyl, isobutyl, trifluoromethyl, --CH.sub.2OH,
methoxy, ethoxy, C.sub.1fluoroalkoxy, C.sub.1-3alkylS(O).sub.2--,
C.sub.1-3alkylS(O).sub.2--NH--, Me.sub.2N--S(O).sub.2--,
H.sub.2N--S(O).sub.2--, --CONH.sub.2, or C.sub.1-3
alkylS(O).sub.2--CH.sub.2--.
47. A compound or salt as claimed in claim 33 wherein, in
sub-formula (x), (y), (y1) and (z), each R.sup.6, independently of
any other R.sup.6, is fluoro, chloro, bromo, methyl, ethyl,
n-propyl, isopropyl, trifluoromethyl, --CH.sub.2OH, methoxy,
difluoromethoxy, methylsulphonyl, methyl-SO.sub.2--NH-- or
methyl-SO.sub.2--CH.sub.2--.
48. A compound or salt as claimed in claim 33 wherein R.sup.5 is
benzyl, (monoalkyl-phenyl)methyl, [mono(fluoroalkyl)-phenyl]methyl,
(monohalo-phenyl)methyl, (monoalkoxy-phenyl)methyl,
[mono(fluoroalkoxy)-phenyl]methyl,
[mono(N,N-dimethylamino)-phenyl]methyl,
[mono(methyl-SO.sub.2--NH--)-phenyl]methyl,
[mono(methyl-SO.sub.2--)-phenyl]methyl, (dialkyl-phenyl)methyl,
(monoalkyl-monohalo-phenyl)methyl,
[mono(fluoroalkyl)-monohalo-phenyl]methyl, (dihalo-phenyl)methyl,
(dihalo-monoalkyl-phenyl)methyl,
[dihalo-mono(hydroxymethyl)-phenyl]methyl, or
(dialkoxy-phenyl)methyl.
49. A compound or salt as claimed in claim 48 wherein R.sup.5 is
(monoC.sub.1-3 alkyl-phenyl)methyl; (monoC.sub.1
fluoroalkyl-phenyl)methyl; (monoC.sub.1-12alkoxy-phenyl)methyl;
[mono(C.sub.1 fluoroalkoxy)-phenyl]methyl;
(diC.sub.1-2alkyl-phenyl)methyl;
(monoC.sub.1-2alkyl-monohalo-phenyl)methyl; (dihalo-phenyl)methyl;
(dihalo-monoC.sub.1-2alkyl-phenyl)methyl; or
[dihalo-mono(hydroxymethyl)-phenyl]methyl.
50. A compound or salt as claimed in claim 48 wherein R.sup.5 is:
benzyl optionally substituted on the phenyl ring with one or two
R.sup.6 substituents; wherein one of the R.sup.6 is: Ar.sup.5b, or
phenyl or pyridinyl wherein the phenyl or pyridinyl independently
are optionally substituted by one of fluoro, chloro,
C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C.sub.1fluoroalkoxy; and wherein Ar.sup.5b is a 5-membered aromatic
heterocyclic ring containing one O, S or NR.sup.15a wherein the
ring can optionally additionally contain one N atom, and wherein
the heterocyclic ring is optionally substituted on a ring carbon
atom by a group which is C.sub.1-2alkyl or C.sub.1 fluoroalkyl.
51. A compound or salt as claimed in claim 50 wherein: in
sub-formula (x), A is CR.sup.6, wherein the R.sup.6 at position A
is Ar.sup.5b or the optionally substituted phenyl or pyridinyl, and
when the R.sup.6 at position A is optionally substituted phenyl or
pyridinyl, it is phenyl or pyridinyl independently optionally
substituted at the 3- or 4-position by one of fluoro, chloro,
C.sub.1-12alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C.sub.1fluoroalkoxy; and when the R.sup.6 at position A is
Ar.sup.5b then it is ##STR00180##
52. A compound or salt of formula (I) as claimed in claim 33
excluding
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide or a salt
thereof.
53. A compound or salt of formula (I) according to claim 33 which
is:
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-(methyloxy)pheny-
l]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,4-difluorophenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-(trifluoromethyl-
)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(4-hydroxyphenyl)me-
thyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-(phenylmethyl)-1H-py-
razolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(methylsulfonyl)-
phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(3-hydroxyphenyl)me-
thyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,3-dichlorophenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,3-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(2-biphenylylmethyl)-1-ethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chloro-2-fluorophenyl)me-
thyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-({4-[(difluoromethyl)oxy]phe-
nyl}methyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chloro-2-methylphenyl)me-
thyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4'-chloro-4-biphenylyl)met-
hyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-(methylsulfonyl)-
phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(trifluoromethy-
l)-2-biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chlorophenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(4-biphenylylmethyl)-1-ethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(ethyloxy)-4-bi-
phenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(3-biphenylylmethyl)-1-ethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(trifluoromethy-
l)-4-biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(3'-methyl-2-biphen-
ylyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[3'-(methyloxy)-2-b-
iphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3'-chloro-2-biphenylyl)met-
hyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4'-chloro-2-biphenylyl)met-
hyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(4'-methyl-2-biphen-
ylyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(methyloxy)-2-b-
iphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-furanyl)pheny-
l]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-({2-[6-(methyloxy)-3-
-pyridinyl]phenyl}methyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-thienyl)pheny-
l]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(4-methyl-3-thie-
nyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(5-methyl-2-bipheny-
lyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-methyl-2-(3-thie-
nyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-furanyl)-4-me-
thylphenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3'-chloro-5-methyl-2-biphe-
nylyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methy-
l]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-(methyloxy)pheny-
l]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-(methylsulfonyl)phen-
yl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,3-dimethylphenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(2-biphenylylmethyl)-1-ethyl-1H--
pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chloro-2-fluorophenyl)methyl-
]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,3-dichlorophenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[2-(methylsulfonyl)phen-
yl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chlorophenyl)methyl]-1-ethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-(methyloxy)phenyl]me-
thyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(4-biphenylylmethyl)-1-ethyl-1H--
pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-[(4-hydroxyphenyl)methyl-
]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-(phenylmethyl)-1H-pyrazo-
lo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-difluorophenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-({4-[(difluoromethyl)oxy]phenyl}-
methyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(3-biphenylylmethyl)-1-ethyl-1H--
pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-[(4-fluorophenyl)methyl]-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chloro-2-methylphenyl)methyl-
]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4'-chloro-4-biphenylyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-{[2,3-dichloro-6-(hydroxymethyl)-
phenyl]methyl}-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-{[3,4-bis(methyloxy)phenyl]methy-
l}-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(2,4-dimethylphenyl)m-
ethyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, or
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(3,4-dimethylphenyl)m-
ethyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide.
54. A compound of formula (IC) or a salt thereof: ##STR00181##
wherein: R.sup.1c is ethyl or C.sub.2fluoroalkyl; R.sup.2c is
hydrogen or methyl; NHR.sup.3c is
[1-(aminocarbonyl)-4-piperidinyl]amino,
cis-[4-(aminocarbonyl)cyclohexan-1-yl]amino group, or a racemic
[4-(aminocarbonyl)cyclohexan-1-yl]amino group; and NHR.sup.5c is
sub-formula (xx1), (xx2), (xx3) or (xx4): ##STR00182## wherein
n.sup.X1 and n.sup.X2 independently are 0 or 1, and X.sub.1,
X.sub.2, X.sub.3 and X.sub.4 independently are CH.sub.2 or O,
provided that one or both of X.sub.1 and X.sub.2 are CH.sub.2, and
provided that one or both of X.sub.3 and X.sub.4 are CH.sub.2.
55. A compound or salt as claimed in claim 54 wherein R.sup.1c is
ethyl; R.sup.2c is hydrogen; R.sup.3c is ##STR00183## NHR.sup.5c is
sub-formula (xx1) or (xx2).
56. A compound or salt as claimed in claim 55 which is:
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(2,3-dihydro-1-benzofuran-5--
ylmethyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-(5,6,7,8-tetrahydro-1-na-
phthalenylmethyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, or
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(2,3-dihydro-1-benzofuran-5-ylme-
thyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide.
57. A pharmaceutical composition comprising a compound of formula
(I) or a pharmaceutically acceptable salt thereof, as defined in
claim 33 and one or more pharmaceutically acceptable carriers.
58. A method for treating a respiratory disease which is asthma,
chronic obstructive pulmonary disease (COPD) or allergic rhinitis;
or a topical disease which is atopic dermatitis which method
comprises administering a therapeutically effective amount of a
compound or salt of formula (I) according to claim 33, alone or
mixed with a pharmaceutically carrier.
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, rheumatoid
arthritis, allergic rhinitis, psoriasis or atopic dermatitis.
BACKGROUND TO THE INVENTION
[0002] U.S. Pat. No. 3,979,399, U.S. Pat. No. 3,840,546, and U.S.
Pat. No. 3,966,746 (E.R. Squibb & Sons) disclose 4-amino
derivatives of pyrazolo[3,4-b]pyridine-5-carboxamides wherein the
4-amino group NR.sub.3R.sub.4 can be an acyclic amino group wherein
R.sub.3 and R.sub.4 may each be hydrogen, lower alkyl (e.g. butyl),
phenyl, etc.; NR.sub.3R.sub.4 can alternatively be a 3-6-membered
heterocyclic group such as pyrrolidino, piperidino and piperazino.
The compounds are disclosed as central nervous system depressants
useful as ataractic, analgesic and hypotensive agents.
[0003] U.S. Pat. No. 3,925,388, U.S. Pat. No. 3,856,799, U.S. Pat.
No. 3,833,594 and U.S. Pat. No. 3,755,340 (E.R. Squibb & Sons)
disclose 4-amino derivatives of
pyrazolo[3,4-b]pyridine-5-carboxylic acids and esters. The 4-amino
group NR.sub.3R.sub.4 can be an acyclic amino group wherein R.sub.3
and R.sub.4 may each be hydrogen, lower alkyl (e.g. butyl), phenyl,
etc.; NR.sub.3R.sub.4 can alternatively be a 5-6-membered
heterocyclic group in which an additional nitrogen is present such
as pyrrolidino, piperidino, pyrazolyl, pyrimidinyl, pyridazinyl or
piperazinyl. The compounds are mentioned as being central nervous
system depressants useful as ataractic agents or tranquilisers, as
having antiinflammatory and analgesic properties. The compounds are
mentioned as increasing the intracellular concentration of
adenosine-3',5'-cyclic monophosphate and for alleviating the
symptoms of asthma.
[0004] H. Hoehn et al., J. Heterocycl. Chem., 1972, 9(2), 235-253
discloses a series of 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid
derivatives with 4-hydroxy, 4-chloro, 4-alkoxy, 4-hydrazino, and
4-amino substituents. Ethyl
4-(n-butylamino)-1-ethyl-1H-pyrazolo[3,4-b]-pyridine-5-carboxylate
is disclosed therein; this compound is cartazolate.
[0005] The compound tracazolate, ethyl
4-(n-butylamino)-1-ethyl-6-methyl-1H-pyrazolo[3,4-b]-pyridine-5-carboxyla-
te, is known as an anxiolytic agent (e.g. see J. B. Patel et al.,
Eur. J. Pharmacol., 1982, 78, 323). Other 1-substituted 4-(NH.sub.2
or NH-alkyl)-1H-pyrazolo[3,4-b]-pyridine-5-carboxylic acid esters
and amides are disclosed as potential anxiolytic agents in T. M.
Bare et al., J. Med. Chem., 1989, 32, 2561-2573. CA 1003419, CH 553
799 and T. Denzel, Archiv der Pharmazie, 1974, 307(3), 177-186
disclose 4,5-disubstituted 1H-pyrazolo[3,4-b]pyridines
unsubstituted at the 1-position.
[0006] Japanese laid-open patent application JP-2002-20386-A (Ono
Yakuhin Kogyo K K) published on 23 Jan. 2002 discloses
pyrazolopyridine compounds of the following formula:
##STR00004##
wherein R.sup.1 denotes 1) a group --OR.sup.6, 2) a group
--SR.sup.7, 3) a C2-8 alkynyl group, 4) a nitro group, 5) a cyano
group, 6) a C1-8 alkyl group substituted by a hydroxy group or a
C1-8 alkoxy group, 7) a phenyl group, 8) a group --C(O)R.sup.8, 9)
a group --SO.sub.2NR.sup.9R.sup.10, 10) a group
--NR.sup.11SO.sub.2R.sup.12, 11) a group --NR.sup.13C(O)R.sup.14 or
12) a group --CH.dbd.NR.sup.15. R.sup.6 and R.sup.7 denote i) a
hydrogen atom, ii) a C1-8 alkyl group, iii) a C1-8 alkyl group
substituted by a C1-8 alkoxy group, iv) a trihalomethyl group, v) a
C3-7 cycloalkyl group, vi) a C1-8 alkyl group substituted by a
phenyl group or vii) a 3-15 membered mono-, di- or tricyclic hetero
ring containing 1-4 nitrogen atoms, 1-3 oxygen atoms and/or 1-3
sulphur atoms. R.sup.2 denotes 1) a hydrogen atom or 2) a C1-8
alkoxy group. R.sup.3 denotes 1) a hydrogen atom or 2) a C1-8 alkyl
group. R.sup.4 denotes 1) a hydrogen atom, 2) a C1-8 alkyl group,
3) a C3-7 cycloalkyl group, 4) a C1-8 alkyl group substituted by a
C3-7 cycloalkyl group, 5) a phenyl group which may be substituted
by 1-3 halogen atoms or 6) a 3-15 membered mono-, di- or tricyclic
hetero ring containing 1-4 nitrogen atoms, 1-3 oxygen atoms and/or
1-3 sulphur atoms. R.sup.5 denotes 1) a hydrogen atom, 2) a C1-8
alkyl group, 3) a C3-7 cycloalkyl group, 4) a C1-8 alkyl group
substituted by a C3-7 cycloalkyl group or 5) a phenyl group which
may be substituted by 1-3 substituents. In group R.sup.3, a
hydrogen atom is preferred. In group R.sup.4, methyl, ethyl,
cyclopropyl, cyclobutyl or cyclopentyl are preferred. The compounds
of JP-2002-20386-A are stated as having PDE4 inhibitory activity
and as being useful in the prevention and/or treatment of
inflammatory diseases and many other diseases.
[0007] 1,3-Dimethyl-4-(arylamino)-pyrazolo[3,4-b]pyridines with a
5-C(O)NH.sub.2 substituent similar or identical to those in
JP-2002-20386-A were disclosed as orally active PDE4 inhibitors by
authors from Ono Pharmaceutical Co. in: H. Ochiai et al., Bioorg.
Med. Chem. Lett., 5 Jan. 2004 issue, vol. 14(1), pp. 29-32
(available on or before 4 Dec. 2003 from the Web version of the
journal: "articles in press"). Full papers on these and similar
compounds as orally active PDE4 inhibitors are: H. Ochiai et al.,
Bioorg. Med. Chem., 2004, 12(15), 4089-4100 (stated to have been
available online 20 June 2004), and H. Ochiai et al., Chem. Pharm.
Bull., 2004, 52(9), 1098-1104 (stated to have been published online
15 Jun. 2004).
[0008] EP 0 076 035 A1 (ICI Americas) discloses
pyrazolo[3,4-b]pyridine derivatives as central nervous system
depressants useful as tranquilisers or ataractic agents for the
relief of anxiety and tension states.
[0009] J. W. Daly et al., Med. Chem. Res., 1994, 4, 293-306 and D.
Shi et al., Drug Development Research, 1997, 42, 41-56 disclose a
series of 4-(amino)substituted
1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid derivatives, including
ethyl
4-cyclopentylamino-1-methyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate,
and their affinities and antagonist activities at A.sub.1- and
A.sub.2A-adenosine receptors, and the latter paper discloses their
affinities at various binding sites of the GABA.sub.A-receptor
channel. S. Schenone et al., Bioorg. Med. Chem. Lett., 2001, 11,
2529-2531, and F. Bondavalli et al., J. Med. Chem., 2002, vol. 45
(Issue 22, 24 October 2002, allegedly published on Web Sep. 24,
2002), pp. 4875-4887 disclose a series of
4-amino-1-(2-chloro-2-phenylethyl)-1H-pyrazolo[3,4-b]pyridine-5-
-carboxylic acid ethyl esters as A.sub.1-adenosine receptor
ligands.
[0010] WO 02/060900 A2 appears to disclose, as MCP-1 antagonists
for treatment of allergic, inflammatory or autoimmune disorders or
diseases, a series of bicyclic heterocyclic compounds with a
--C(O)--NR.sup.4--C(O)--NR.sup.5R.sup.6 substituent, including
isoxazolo[5,4-b]pyridines and 1H-pyrazolo[3,4-b]pyridines (named as
pyrazolo[5,4-b]pyridines) with the
--C(O)--NR.sup.4--C(O)--NR.sup.5R.sup.6 group as the 5-substituent
and optionally substituted at the 1-, 3-, 4-, and/or 6-positions.
Bicyclic heterocyclic compounds with a --C(O)NH.sub.2 substituent
instead of the --C(O)--NR.sup.4--C(O)--NR.sup.5R.sup.6 substituent
are alleged to be disclosed in WO 02/060900 as intermediates in the
synthesis of the --C(O)--NR.sup.4--C(O)--NR.sup.5R.sup.6
substituted compounds. See also WO 02/081463 A1 for similar MCP-1
antagonists.
[0011] WO 00/15222 (Bristol-Myers Squibb) discloses inter alia
pyrazolo[3,4-b]pyridines having inter alia a C(O)--X.sub.1 group at
the 5-position and a group E.sub.1 at the 4-position of the ring
system. Amongst other things, X.sub.1 can for example be
--OR.sub.9, --N(R.sub.9)(R.sub.10) or
--N(R.sub.5)(-A.sub.2-R.sub.2), and E.sub.1 can for example be
--NH-A.sub.1-cycloalkyl, --NH-A.sub.1-substituted cycloalkyl, or
--NH-A.sub.1-heterocyclo; wherein A.sub.1 is an alkylene or
substituted alkylene bridge of 1 to 10 carbons and A.sub.2 can for
example be a direct bond or an alkylene or substituted alkylene
bridge of 1 to 10 carbons. The compounds are disclosed as being
useful as inhibitors of cGMP phosphodiesterase, especially PDE type
V, and in the treatment of various cGMP-associated conditions such
as erectile dysfunction. Compounds with a cycloalkyl or heterocyclo
group directly attached to --NH-- at the 4-position of the
pyrazolo[3,4-b]pyridine ring system and/or having PDE4 inhibitory
activity do not appear to be disclosed in WO 00/15222.
[0012] G. Yu et. al., J. Med. Chem., 2001, 44, 1025-1027 discloses
some
4-[(3-chloro-4-methoxybenzyl)amino]-pyrazolopyridine-5-carboxamides
as selective PDE5 inhibitors.
[0013] H. de Mello, A. Echevarria, et al., J. Med. Chem., 2004,
47(22), 5427-5432, believed to have been published online on or
before 21 Sep. 2004, discloses 3-methyl or 3-phenyl
4-anilino-1H-pyrazolo[3,4-b]pyridine 5-carboxylic esters as
potential anti-Leishmania drugs.
[0014] Copending patent application PCT/EP2003/014867, filed on 19
Dec. 2003 in the name of Glaxo Group Limited, published on 8 Jul.
2004 as WO 2004/056823 A1, discloses and claims
pyrazolo[3,4-b]pyridine compounds or salts thereof with a
4-NR.sup.3R.sup.3a group (R.sup.3a is preferably H) and with a
group Het at the 5-position of the pyrazolo[3,4-b]pyridine, wherein
Het is usually a 5-membered optionally substituted heteroaryl
group. PCT/EP2003/014867 (WO 2004/056823 A1) also discloses the use
of these compounds as PDE4 inhibitors and for the treatment and/or
prophylaxis of inter alia COPD, asthma or allergic rhinitis.
[0015] Copending patent application PCT/EP03/11814, filed on 12
Sep. 2003 in the name of Glaxo Group Limited, published on 25 Mar.
2004 as WO 2004/024728 A2, 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 this formula (I):
##STR00005##
wherein: R.sup.1 is C.sub.1-4alkyl, C.sub.1-3fluoroalkyl,
--CH.sub.2CH.sub.2OH or --CH.sub.2CH.sub.2CO.sub.2C.sub.1-2alkyl;
R.sup.2 is a hydrogen atom (H), methyl or C.sub.1fluoroalkyl;
R.sup.3 is optionally substituted C.sub.3-8cycloalkyl or optionally
substituted mono-unsaturated-C.sub.5-7cycloalkenyl or an optionally
substituted heterocyclic group of sub-formula (aa), (bb) or
(cc);
##STR00006##
in which n1 and n2 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),
C.sub.1-4alkyl, C.sub.1-2fluoroalkyl, CH.sub.2C(O)NH.sub.2,
C(O)NH.sub.2, C(O)--C.sub.1-2alkyl, C(O)--C.sub.1fluoroalkyl or
--C(O)--CH.sub.2O--C.sub.1-2alkyl; or R.sup.3 is a bicyclic group
(dd) or (ee):
##STR00007##
and wherein X is NR.sup.4R.sup.5 or OR.sup.5a.
[0016] In PCT/EP03/11814 (WO 2004/024728 A2), R.sup.4 is a hydrogen
atom (H); C.sub.1-6alkyl; C.sub.1-3fluoroalkyl; or C.sub.2-6alkyl
substituted by one substituent R.sup.11.
[0017] In PCT/EP03/11814 (WO 2004/024728 A2), R.sup.5 can be: a
hydrogen atom (H); C.sub.1-8alkyl; C.sub.1-8 fluoroalkyl;
C.sub.3-8cycloalkyl optionally substituted by a C.sub.1-2alkyl
group; --(CH.sub.2).sub.n.sup.4--C.sub.3-8cycloalkyl optionally
substituted, in the --(CH.sub.2).sub.n.sup.4-- moiety or in the
C.sub.3-8cycloalkyl moiety, by a C.sub.1-2alkyl group, wherein
n.sup.4 is 1, 2 or 3; C.sub.2-6alkyl substituted by one or two
independent substituents R.sup.11;
--(CH.sub.2).sub.n.sup.11--C(O)R.sup.16;
--(CH.sub.2).sub.n.sup.12--C(O)NR.sup.12R.sup.13;
--CHR.sup.19--C(O)NR.sup.12R.sup.13;
--(CH.sub.2).sub.n.sup.12--C(O)OR.sup.16;
--(CH.sub.2).sub.n.sup.12--C(O)OH; --CHR.sup.19--C(O)OR.sup.16;
--CHR.sup.19--C(O)OH;
--(CH.sub.2).sub.n.sup.12--SO.sub.2--NR.sup.12R.sup.13;
--(CH.sub.2).sub.n.sup.12--SO.sub.2R.sup.16; or
--(CH.sub.2).sub.n.sup.12--CN; --(CH.sub.2).sub.n.sup.13-Het; or
optionally substituted phenyl.
[0018] Alternatively, in PCT/EP03/11814 (WO 2004/024728 A2),
R.sup.5 can have the sub-formula (x), (y), (y1) or (z):
##STR00008##
wherein in sub-formula (x), n=0, 1 or 2; in sub-formula (y) and
(y1), m=1 or 2; and in sub-formula (z), r=0, 1 or 2; and wherein in
sub-formula (x) and (y) and (y1), none, one or two of A, B, D, E
and F are independently nitrogen or nitrogen-oxide
(N.sup.+--O.sup.-) provided that no more than one of A, B, D, E and
F is nitrogen-oxide, and the remaining of A, B, D, E and F are
independently CH or CR.sup.6; and provided that when n is 0 in
sub-formula (x) then one or two of A, B, D, E and F are
independently nitrogen or nitrogen-oxide (N.sup.+--O.sup.-) and no
more than one of A, B, D, E and F is nitrogen-oxide;
[0019] In PCT/EP03/11814 (WO 2004/024728 A2), each R.sup.6,
independently of any other R.sup.6 present, is: a halogen atom;
C.sub.1-6alkyl; C.sub.1-4fluoroalkyl; C.sub.1-4alkoxy;
C.sub.1-2fluoroalkoxy; C.sub.3-6cycloalkyloxy; --C(O)R.sup.16a;
--C(O)OR.sup.30; --S(O).sub.2--R.sup.16a;
R.sup.16a--S(O).sub.2--NR.sup.15a--; R.sup.7R.sup.8N--S(O).sub.2--;
C.sub.1-2alkyl-C(O)--R.sup.15aN--S(O).sub.2--;
C.sub.1-4alkyl-S(O)--; Ph-S(O)--; R.sup.7R.sup.8N--CO--;
--NR.sup.15--C(O)R.sub.1-6; R.sup.7R.sup.8N; OH;
C.sub.1-4alkoxymethyl; C.sub.1-4alkoxyethyl;
C.sub.1-2alkyl-S(O).sub.2--CH.sub.2--;
R.sup.7R.sup.8N--S(O).sub.2--CH.sub.2--;
C.sub.1-2alkyl-S(O).sub.2--NR.sup.15a--CH.sub.2--; --CH.sub.2--OH;
--CH.sub.2CH.sub.2--OH; --CH.sub.2--NR.sup.7R.sup.8;
--CH.sub.2--CH.sub.2--NR.sup.7R.sup.8; --CH.sub.2--C(O)OR.sup.30;
--CH.sub.2--C(O)--NR.sup.7R.sup.8;
--CH.sub.2--NR.sup.15a--C(O)--C.sub.1-3alkyl;
--(CH.sub.2).sub.n.sup.14-Het.sup.1 where n.sup.14 is 0 or 1; cyano
(CN); Ar.sup.5b; or phenyl, pyridinyl or pyrimidinyl wherein the
phenyl, pyridinyl or pyrimidinyl independently are optionally
substituted by one or two of fluoro, chloro, C.sub.1-2alkyl,
C.sub.1fluoroalkyl, C.sub.1-12alkoxy or C.sub.1 fluoroalkoxy;
or two adjacent R.sup.6 taken together can be --O--(CMe.sub.2)--O--
or --O--(CH.sub.2).sub.n.sup.14a--O-- where n.sup.14a is 1 or
2.
[0020] The pyrazolo[3,4-b]pyridine compounds of formula (I) and
salts thereof disclosed in PCT/EP03/11814 (WO 2004/024728 A2) are
disclosed as being inhibitors of phosphodiesterase type IV (PDE4),
and as being useful for the treatment and/or prophylaxis of a
variety of diseases/conditions, especially inflammatory and/or
allergic diseases in mammals such as humans, for example: asthma,
chronic obstructive pulmonary disease (COPD) (e.g. chronic
bronchitis and/or emphysema), atopic dermatitis, urticaria,
allergic rhinitis, allergic conjunctivitis, vernal conjunctivitis,
eosinophilic granuloma, psoriasis, rheumatoid arthritis, septic
shock, ulcerative colitis, Crohn's disease, reperfusion injury of
the myocardium and brain, chronic glomerulonephritis, endotoxic
shock, adult respiratory distress syndrome, multiple sclerosis,
cognitive impairment (e.g. in a neurological disorder), depression,
or pain. PCT/EP03/11814 (WO 2004/024728 A2) states that the
compounds of formula (I) and/or their pharmaceutical compositions
may be administered by oral, parenteral, inhaled (topical to the
lung), or nasal administration. However, the use of the
pyrazolo[3,4-b]pyridine compounds by external topical
administration is not disclosed.
[0021] PCT/EP03/11814 (WO 2004/024728 A2) does not disclose any
specific pyrazolo[3,4-b]pyridine compounds having a 4-position
group NHR.sup.3 in which R.sup.3 is an optionally substituted
heterocyclic group of sub-formula (aa), (bb) or (cc) and in which Y
is NR.sup.10 wherein R.sup.10 is C(O)NH.sub.2. Also, WO 2004/024728
A2 does not disclose any specific pyrazolo[3,4-b]pyridine compounds
having a 4-position group NHR.sup.3 in which R.sup.3 is a
4-(aminocarbonyl)cyclohexyl group.
[0022] WO 2004/024728 has been reviewed, and WO 2004/056823
mentioned, in Expert Opin. Ther. Patents, 2005 (January edition),
15(1), 111-114.
The Invention
[0023] We have now found new pyrazolo[3,4-b]pyridine compounds,
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):
##STR00009##
wherein: R.sup.1 is C.sub.1-3alkyl, C.sub.1-3fluoroalkyl, or
--CH.sub.2CH.sub.2OH; R.sup.2 is a hydrogen atom (H), methyl or
C.sub.1 fluoroalkyl; R.sup.3 is a 4-(aminocarbonyl)cyclohexyl (i.e.
4-(aminocarbonyl)cyclohexan-1-yl) group of sub-formula (aa), or an
N-aminocarbonyl-piperidinyl or -pyrrolidinyl group of sub-formula
(bb);
##STR00010##
wherein Y is NCONH.sub.2 and n.sup.1 is 0 or 1; and wherein the
cyclohexyl group of sub-formula (aa) or the piperidinyl or
pyrrolidinyl groups of sub-formula (bb) are not further substituted
on any ring carbon; R.sup.4 is a hydrogen atom (H);
[0025] R.sup.5 is a group of the sub-formula (x), (y), (y1) or
(z):
##STR00011##
wherein in sub-formula (x), n=0, 1 or 2; in sub-formula (y) and
(y1), m=1 or 2; and in sub-formula (z), r=0, 1 or 2; wherein in
sub-formula (x) and (y) and (y1), none, one or two of A, B, D, E
and F are nitrogen; and the remaining of A, B, D, E and F are
independently CH or CR.sup.6; wherein, each R.sup.6, independently
of any other R.sup.6 present, is: a halogen atom; C.sub.1-6alkyl
(e.g. C.sub.1-4alkyl or C.sub.1-2alkyl); C.sub.1-4fluoroalkyl (e.g.
C.sub.1-2fluoroalkyl); C.sub.1-4alkoxy (e.g. C.sub.1-2alkoxy);
C.sub.1-2fluoroalkoxy; C.sub.3-6cycloalkyloxy; --C(O)R.sup.16a;
--C(O)OR.sup.30; --S(O).sub.2--R.sup.16a (e.g.
C.sub.1-2alkylsulphonyl, that is C.sub.1-2alkyl-SO.sub.2--);
R.sup.16a--S(O).sub.2--NR.sup.15a (e.g.
C.sub.1-2alkyl-SO.sub.2--NH--); R.sup.7R.sup.8N--S(O).sub.2--;
C.sub.1-2alkyl-C(O)--R.sup.15aN--S(O).sub.2--;
C.sub.1-4alkyl-S(O)--, Ph-S(O)--, R.sup.7R.sup.8N--CO--;
--NR.sup.15--C(O)R.sup.16a; R.sup.7R.sup.8N; OH;
C.sub.1-4alkoxymethyl; C.sub.1-4alkoxyethyl;
C.sub.1-2alkyl-S(O).sub.2--CH.sub.2--;
R.sup.7R.sup.8N--S(O).sub.2--CH.sub.2--;
C.sub.1-2alkyl-S(O).sub.2--NR.sup.15a--CH.sub.2--; --CH.sub.2--OH;
--CH.sub.2CH.sub.2--OH; --CH.sub.2--NR.sup.7R.sup.8;
--CH.sub.2--CH.sub.2--NR.sup.7R.sup.8; --CH.sub.2--C(O)OR.sup.30;
--CH.sub.2--C(O)--NR.sup.7R.sup.8;
--CH.sub.2--NR.sup.15a--C(O)--C.sub.1-3alkyl;
--(CH.sub.2).sub.n.sup.14-Het.sup.1 where n.sup.14 is 0 or 1; cyano
(CN); Ar.sup.5b; or phenyl, pyridinyl or pyrimidinyl wherein the
phenyl, pyridinyl or pyrimidinyl independently are optionally
substituted by one or two of fluoro, chloro, C.sub.1-2alkyl,
C.sub.1fluoroalkyl, C.sub.1-2alkoxy or C.sub.1fluoroalkoxy; or
where two adjacent R.sup.6 taken together are --O--(CMe.sub.2)--O--
or --O--(CH.sub.2).sub.n.sup.14--O-- where n.sup.14 is 1 or 2;
wherein sub-formula (y) and (y1), independently, are optionally
substituted by oxo (.dbd.O) at a ring carbon adjacent the
6-membered aromatic ring (for example, sub-formula (y) can
optionally be
##STR00012##
or sub-formula (y1) can optionally be
##STR00013##
wherein in sub-formula (z), G is O or S or NR.sup.9 wherein R.sup.9
is a hydrogen atom (H), C.sub.1-4alkyl or C.sub.1-4fluoroalkyl;
none, one, two or three of J, L, M and Q are nitrogen; and the
remaining of J, L, M and Q are independently CH or CR.sup.6 where
R.sup.6, independently of any other R.sup.6 present, is as defined
herein; and wherein: R.sup.7 and R.sup.8 are independently a
hydrogen atom (H); C.sub.1-4alkyl (e.g. C.sub.1-2alkyl such as
methyl); C.sub.3-6cycloalkyl; or phenyl optionally substituted by
one or two substituents independently being: fluoro, chloro,
C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C.sub.1fluoroalkoxy; or R.sup.7 and R.sup.8 together are
--(CH.sub.2).sub.n.sup.6-- or --C(O)--(CH.sub.2).sub.n.sup.7-- or
--C(O)--(CH.sub.2)n.sup.10--C(O)-- or
--(CH.sub.2).sub.n.sup.8--X.sup.7--(CH.sub.2).sub.n.sup.9-- or
--C(O)--X.sup.7--(CH.sub.2)n.sup.10-- in which: n.sup.6 is 3, 4, 5
or 6 (suitably n.sup.6 is 4 or 5), n.sup.7 is 2, 3, 4, or 5
(suitably n.sup.7 is 3 or 4), n.sup.8 and n.sup.9 and n.sup.10
independently are 2 or 3 (suitably independently 2), and X.sup.7 is
O or NR.sup.14; R.sup.7a is a hydrogen atom (H) or C.sub.1-4alkyl
(suitably H or C.sub.1-2alkyl, more suitably H or methyl); R.sup.8a
is a hydrogen atom (H) or methyl (suitably H); R.sup.14,
independent of other R.sup.14, is a hydrogen atom (H);
C.sub.1-4alkyl (e.g. C.sub.1-2alkyl); C.sub.1-2fluoroalkyl (e.g.
CF.sub.3); cyclopropyl; --C(O)--C.sub.1-4alkyl (e.g. --C(O)Me);
--C(O)NR.sup.7aR.sup.8a (e.g. --C(O)NH.sub.2); or
--S(O).sub.2--C.sub.1-4alkyl (e.g. --S(O).sub.2Me) (preferably,
R.sup.14 is: H, C.sub.1-2alkyl, or --C(O)Me); R.sup.15, independent
of other R.sup.15, is a hydrogen atom (H); C.sub.1-4alkyl (e.g.
.sup.tBu or C.sub.1-2alkyl e.g. methyl); C.sub.3-6cycloalkyl; or
phenyl optionally substituted by one or two of: a halogen atom,
C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C.sub.1fluoroalkoxy (preferably R.sup.15 is H or C.sub.1-2alkyl,
more preferably H); R.sup.15a, independent of other R.sup.15a, is a
hydrogen atom (H) or C.sub.1-4alkyl (e.g. H, .sup.tBu or
C.sub.1-2alkyl such as methyl; preferably R.sup.15a is H or
C.sub.1-2alkyl, more preferably H);
R.sup.16a is:
[0026] C.sub.1-6alkyl (e.g. C.sub.1-4alkyl or C.sub.1-2alkyl);
C.sub.3-6cycloalkyl (e.g. C.sub.5-6cycloalkyl) optionally
substituted by one oxo (.dbd.O), OH or C.sub.1-2alkyl substituent
(e.g. optionally substituted at the 3- or 4-position of a
C.sub.5-6cycloalkyl ring; and/or preferably unsubstituted
C.sub.3-6cycloalkyl); C.sub.3-6cycloalkyl-CH.sub.2-- (e.g.
C.sub.5-6cycloalkyl-CH.sub.2--); pyridinyl (e.g. pyridin-2-yl)
optionally substituted on a ring carbon atom by one of: a halogen
atom, C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C.sub.1fluoroalkoxy; Ar.sup.5c; phenyl optionally substituted by
one or two substituents independently being: a halogen atom,
C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C.sub.1fluoroalkoxy; benzyl optionally substituted on its ring by
one or two substituents independently being: a halogen atom,
C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C.sub.1fluoroalkoxy; or a 4-, 5-, 6- or 7-membered saturated
heterocyclic ring connected at a ring-carbon and containing one or
two ring-hetero-atoms independently selected from O, S, and N;
wherein any ring-nitrogens which are present are present as
NR.sup.27 where R.sup.27 is H, C.sub.1-2alkyl or --C(O)Me; and
wherein the ring is optionally substituted at carbon by one
C.sub.1-2alkyl or oxo (.dbd.O) substituent, provided that any oxo
(.dbd.O) substituent is substituted at a ring-carbon atom bonded to
a ring-nitrogen; R.sup.30, independent of other R.sup.30, is a
hydrogen atom (H), C.sub.1-4alkyl or C.sub.3-6cycloalkyl; Ar.sup.5b
and Ar.sup.5c independently is/are a 5-membered aromatic
heterocyclic ring containing one O, S or NR.sup.15a in the
5-membered ring, wherein the 5-membered ring can optionally
additionally contain one or two N atoms, and wherein the
heterocyclic ring is optionally substituted on a ring carbon atom
by one of: a halogen atom, C.sub.1-2alkyl, C.sub.1fluoroalkyl,
--CH.sub.2OH, --CH.sub.2--OC.sub.1-2alkyl, OH (including the keto
tautomer thereof) or --CH.sub.2--NR.sup.28R.sup.29 wherein R.sup.28
and R.sup.29 independently are H or methyl; and Het.sup.1 is a 4-,
5-, 6- or 7-membered saturated heterocyclic ring connected at a
ring-carbon and containing one or two ring-hetero-atoms
independently selected from O, S, and N; wherein any ring-nitrogens
which are present are present as NR.sup.31 where R.sup.31 is H,
C.sub.1-2alkyl or --C(O)Me; and wherein the ring is optionally
substituted at carbon by one C.sub.1-2alkyl or oxo (.dbd.O)
substituent, provided that any oxo (.dbd.O) substituent is
substituted at a ring-carbon atom bonded to a ring-nitrogen.
[0027] 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.
[0028] 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.
[0029] "Cycloalkyl", for example C.sub.3-8cycloalkyl, includes
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl, and the like. Preferably, a C.sub.3-8cycloalkyl group
is C.sub.3-6cycloalkyl or C.sub.5-6cycloalkyl, that is contains a
3-6 membered or 5-6 membered carbocyclic ring.
[0030] "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--),
2fluoroethyl (CH.sub.2FCH.sub.2--), etc. "Fluoroalkoxy" includes
C.sub.1-4fluoroalkoxy or C.sub.1-2fluoroalkoxy such as
trifluoromethoxy, pentafluoroethoxy, monofluoromethoxy,
difluoromethoxy, etc. "Fluoroalkylsulfonyl" such as
C.sub.1-4fluoroalkylsulfonyl includes trifluoromethanesulfonyl,
pentafluoroethylsulfonyl, etc.
[0031] 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.
[0032] 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.
[0033] When R.sup.1 is C.sub.1-3alkyl or C.sub.1-3fluoroalkyl, it
can be straight-chained or branched. Where R.sup.1 is
C.sub.1-3alkyl then it can for example be methyl, ethyl, n-propyl,
or isopropyl. When R.sup.1 is C.sub.1-3fluoroalkyl, then R.sup.1
can for example be C.sub.1fluoroalkyl such as monofluoromethyl,
difluoromethyl, trifluoromethyl; or R.sup.1 can be
C.sub.2fluoroalkyl such as pentafluoroethyl or more preferably
C.sub.1fluoroalkyl-CH.sub.2-- such as 2,2,2-trifluoroethyl
(CF.sub.3 CH.sub.2--), 2,2-difluoroethyl (CHF.sub.2CH.sub.2--), or
2fluoroethyl (CH.sub.2FCH.sub.2--).
[0034] R.sup.1 is C.sub.1-3alkyl (e.g. methyl, ethyl or n-propyl),
C.sub.1-3fluoroalkyl or --CH.sub.2CH.sub.2OH. R.sup.1 can be
C.sub.1-3alkyl, C.sub.1-2fluoroalkyl, or --CH.sub.2CH.sub.2OH.
Preferably, R.sup.1 is C.sub.2-3alkyl (e.g. ethyl or n-propyl),
C.sub.2fluoroalkyl (e.g. C.sub.1fluoroalkyl-CH.sub.2-- such as
CF.sub.3--CH.sub.2--) or --CH.sub.2CH.sub.2OH; in particular ethyl,
n-propyl or --CH.sub.2CH.sub.2OH. More preferably, R.sup.1 is
C.sub.2alkyl or C2fluoroalkyl. R.sup.1 is most preferably
ethyl.
[0035] Preferably, R.sup.2 is a hydrogen atom (H) or methyl, for
example a hydrogen atom (H).
[0036] In the group of sub-formula (bb), n.sup.1 is preferably 1.
Therefore, preferably, the group of sub-formula (bb), or more
preferably R.sup.3, is:
##STR00014##
[0037] When R.sup.3 is an N-aminocarbonyl-pyrrolidinyl group of
sub-formula (bb), (i.e. wherein n.sup.1 is 0), then preferably
NHR.sup.3 is:
##STR00015##
(wherein the --NH-- connection point is underlined), and including
mixtures of configurations wherein the illustrated configuration is
the major component.
[0038] In R.sup.3, the piperidinyl or pyrrolidinyl group of
sub-formula (bb) is not substituted on a ring carbon.
[0039] In R.sup.3, the cyclohexyl group of sub-formula (aa) has no
further optional substituents (beyond 4-CONH.sub.2).
[0040] It will be appreciated that when R.sup.3 is the substituted
cyclohexyl group of sub-formula (aa), then the substituent can be
in the cis or trans configuration with respect to the --NH-- group
of formula (I) to which R.sup.3 is attached (bonded). The present
invention covers each configuration as well as mixtures of
configurations, in particular wherein the stated configuration is
the major component. Therefore, the --C(O)NH.sub.2 substituent on
the cyclohexyl group of sub-formula (aa) can for example be 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.
[0041] Preferably, the aminocarbonyl substituent on the cyclohexyl
group of sub-formula (aa) is in the cis configuration with respect
to the --NH-- group of formula (I) to which R.sup.3 is attached
(bonded), i.e. preferably NHR.sup.3 is a
cis-[4-(aminocarbonyl)cyclohexan-1-yl]amino group (including
mixtures of configurations wherein the stated cis configuration is
the major component).
[0042] Alternatively, NHR.sup.3 can be a racemic
[4-(aminocarbonyl)cyclohexan-1-yl]amino group.
[0043] Preferably, R.sup.7 and/or R.sup.8 are independently a
hydrogen atom (H); C.sub.1-2alkyl such as methyl;
C.sub.3-6cycloalkyl; or phenyl optionally substituted by one of:
fluoro, chloro, C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy
or C fluoroalkoxy; or R.sup.7 and R.sup.8 together are
--(CH.sub.2).sub.n.sup.6- or
--(CH.sub.2).sub.n.sup.8--X.sup.7--(CH.sub.2).sub.n.sup.9-- wherein
X.sup.7 is NR.sup.14 or preferably O.
[0044] When R.sup.7 is cycloalkyl or optionally substituted phenyl,
then preferably R.sup.8 is neither cycloalkyl nor optionally
substituted phenyl.
[0045] Most preferably, R.sup.7 and/or R.sup.8 independently are a
hydrogen atom (H) or C.sub.1-2alkyl. It is preferable that R.sup.7
is a hydrogen atom (H).
[0046] Preferably n.sup.6 is 4 or 5. Preferably n.sup.7 is 2, 3 or
4. Preferably, n.sup.8, n.sup.9 and/or n.sup.10 is/are
independently 2.
[0047] Preferably, R.sup.7 and R.sup.8 independently are a hydrogen
atom (H) or C.sub.1-2alkyl; or R.sup.7 and R.sup.8 together are
--(CH.sub.2).sub.n.sup.6-- or
--(CH.sub.2).sub.n.sup.8--X.sup.7--(CH.sub.2).sub.n.sup.9-- wherein
X.sup.7 is NR.sup.14 or preferably O, n.sup.6 is 4 or 5, and
n.sup.8 and n.sup.9 are both 2;
[0048] Preferably, R.sup.14 is H, C.sub.1-2alkyl, or --C(O)Me.
[0049] Preferably, R.sup.15, independent of other R.sup.15, is a
hydrogen atom (H) or C.sub.1-2alkyl.
[0050] Preferably, R.sup.15a is a hydrogen atom (H) or
C.sub.1-2alkyl.
[0051] Preferably, R.sup.16a is C.sub.1-4alkyl, e.g. C.sub.1-2alkyl
such as methyl.
[0052] In one embodiment, R.sup.5 is a group of sub-formula (x) or
(z). Preferably, R.sup.5 is a group of sub-formula (x).
[0053] In sub-formula (x), (y) and (y1), in particular in
sub-formula (x), it is preferred that none, one or two of A, B, D,
E and F are nitrogen; none, one, two or three of A, B, D, E and F
are CR.sup.6; and the remaining of A, B, D, E and F are CH. More
preferably, none, one or two of A, B, D, E and F are nitrogen;
none, one or two of A, B, D, E and F are CR.sup.6; and the
remaining of A, B, D, E and F are CH. Yet more preferably, none or
one of A, B, D, E and F are nitrogen, and/or preferably none, one
or two of A, B, D, E and F are CR.sup.6.
[0054] Preferably, sub-formula (x) is: benzyl; optionally
substituted on the phenyl ring with one or two R.sup.6
substituents.
[0055] In one embodiment, sub-formula (y1) is:
##STR00016##
wherein R.sup.6a is or independently are either R.sup.6 as defined
herein or preferably hydrogen.
[0056] Preferably, in sub-formula (z), none, one or two of J, L, M
and Q are nitrogen.
[0057] In sub-formula (x), (y), (y1) and/or (z), preferably, each
R.sup.6, independently of any other R.sup.6 present, is a fluorine,
chlorine, bromine or iodine atom, methyl, ethyl, n-propyl,
isopropyl, C.sub.4alkyl, trifluoromethyl, --CH.sub.2OH, methoxy,
ethoxy, C.sub.1fluoroalkoxy (e.g. trifluoromethoxy or
difluoromethoxy), OH, C.sub.1-3alkylS(O).sub.2-- (such as
methylsulphonyl which is MeS(O).sub.2--),
C.sub.1-3alkylS(O).sub.2--NH-- such as methyl-SO.sub.2--NH--,
Me.sub.2N--S(O).sub.2--, H.sub.2N--S(O).sub.2--, --CONH.sub.2,
--CONHMe, --CO.sub.2H, cyano (CN), NMe.sub.2, t-butoxymethyl, or
C.sub.1-3alkylS(O).sub.2--CH.sub.2-- such as
methyl-SO.sub.2--CH.sub.2--. More preferably, each R.sup.6,
independently of any other R.sup.6 present, is a fluorine,
chlorine, bromine or iodine atom, methyl, ethyl, n-propyl,
isopropyl, isobutyl, trifluoromethyl, --CH.sub.2OH, methoxy,
ethoxy, --C.sub.1fluoroalkoxy (e.g. trifluoromethoxy or
difluoromethoxy), C.sub.1-3alkylS(O).sub.2-- such as
methylsulphonyl, C.sub.1-3alkylS(O).sub.2--NH-- such as
methyl-SO.sub.2--NH--, Me.sub.2N--S(O).sub.2--,
H.sub.2N--S(O).sub.2--, --CONH.sub.2, or
C.sub.1-3alkylS(O).sub.2--CH.sub.2-- such as
methyl-SO.sub.2--CH.sub.2. Still more preferably, each R.sup.6,
independently of any other R.sup.6 present, is a fluorine, chlorine
or bromine atom, methyl, ethyl, n-propyl, isopropyl,
trifluoromethyl, --CH.sub.2OH, methoxy, difluoromethoxy,
methylsulphonyl, methyl-SO.sub.2--NH-- or
methyl-SO.sub.2--CH.sub.2--.
[0058] In sub-formula (x), preferably, one, two or three R.sup.6
substituents are present in B, D and/or E; so that for example in
sub-formula (x), one, two or three R.sup.6 substituents are present
in the ortho- (2- and/or 6-) and/or meta- (3- and/or 5-) and/or
para- (4-) positions with respect to the --(CH.sub.2).sub.n--
side-chain.
[0059] Preferably, R.sup.5 has the sub-formula (x), n is 1 and each
of A, B, D, E and F is independently CH or CR.sup.6; that is
R.sup.5 has the sub-formula (x) and is optionally substituted
benzyl.
[0060] In one preferable embodiment, R.sup.5 has the sub-formula
(x) and is: benzyl, (monoalkyl-phenyl)methyl,
[mono(fluoroalkyl)-phenyl]methyl, (monohalo-phenyl)methyl,
(monoalkoxy-phenyl)methyl, [mono(fluoroalkoxy)-phenyl]methyl,
[mono(N,N-dimethylamino)-phenyl]methyl,
[mono(methyl-SO.sub.2--NH--)-phenyl]methyl,
[mono(methyl-SO.sub.2--)-phenyl]methyl, (dialkyl-phenyl)methyl,
(monoalkyl-monohalo-phenyl)methyl,
[mono(fluoroalkyl)-monohalo-phenyl]methyl, (dihalo-phenyl)methyl,
(dihalo-monoalkyl-phenyl)methyl,
[dihalo-mono(hydroxymethyl)-phenyl]methyl, or
(dialkoxy-phenyl)methyl such as (3,4-dimethoxy-phenyl)methyl. The
substituents can preferably be further defined, as defined in
preferable embodiments herein. In one preferable embodiment,
R.sup.5 is of sub-formula (x) and is: (monoalkyl-phenyl)methyl,
[mono(fluoroalkyl)-phenyl]methyl, (monohalo-phenyl)methyl,
(monoalkoxy-phenyl)methyl, [mono(fluoroalkoxy)-phenyl]methyl,
[mono(N,N-dimethylamino)-phenyl]methyl, (dialkyl-phenyl)methyl,
(monoalkyl-monohalo-phenyl)methyl, (dihalo-phenyl)methyl or
(dihalo-monoalkyl-phenyl)methyl or
[dihalo-mono(hydroxymethyl)-phenyl]methyl. More preferably, in this
embodiment, R.sup.5 is: [0061] (monoC.sub.1-3alkyl-phenyl)methyl
such as (4-C.sub.1-3alkyl-phenyl)methyl; [0062] (monoC.sub.1
fluoroalkyl-phenyl)methyl such as (4-C.sub.1
fluoroalkyl-phenyl)methyl; [0063]
(monoC.sub.1-2alkoxy-phenyl)methyl such as
(4-C.sub.1-2alkoxy-phenyl)methyl; [0064]
[mono(C1fluoroalkoxy)-phenyl]methyl such as
(4-C1fluoroalkoxy-phenyl)methyl; [0065]
(diC.sub.1-2alkyl-phenyl)methyl or (dimethyl-phenyl)methyl such as
(3,4-dimethyl-phenyl)methyl, (2,4-dimethyl-phenyl)methyl,
(3,5-dimethyl-phenyl)methyl, (2,3-dimethyl-phenyl)methyl or
(2,5-dimethyl-phenyl)methyl; more preferably
(3,4-dimethyl-phenyl)methyl or (2,4-dimethyl-phenyl)methyl; [0066]
(monoC.sub.1-2alkyl-monohalo-phenyl)methyl or
(monoC.sub.1-2alkyl-monochloro-phenyl)methyl such as
(4-methyl-3-chloro-phenyl)methyl, (3-methyl-4-chloro-phenyl)methyl,
(2-methyl-4-chloro-phenyl)methyl; [0067] (dihalo-phenyl)methyl such
as (2-chloro-4-fluorophenyl)methyl or (2,4-difluoro-phenyl)methyl
or (4-bromo-2-fluorophenyl)methyl or preferably
(4-chloro-2-fluorophenyl)methyl; for example
(dichloro-phenyl)methyl such as (3,4-dichloro-phenyl)methyl or
(2,4-dichloro-phenyl)methyl or (2,6-dichloro-phenyl)methyl or
preferably (2,3-dichloro-phenyl)methyl; [0068]
(dihalo-monoC.sub.1-2alkyl-phenyl)methyl e.g.
(2,4-dichloro-6-methyl-phenyl)methyl; or [0069]
[dihalo-mono(hydroxymethyl)-phenyl]methyl such as
[2,3-dichloro-6-(hydroxymethyl)-phenyl]methyl.
[0070] In an alternative preferable "biaryl" embodiment, R.sup.5 is
of sub-formula (x) and is: benzyl optionally substituted on the
phenyl ring with one or two (e.g. one) R.sup.6 substituents;
wherein one of the R.sup.6 is: Ar.sup.5b, or phenyl, pyridinyl or
pyrimidinyl wherein the phenyl, pyridinyl or pyrimidinyl
independently are optionally substituted by one or two of fluoro,
chloro, C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C1fluoroalkoxy; and wherein Ar.sup.5b is a 5-membered aromatic
heterocyclic ring containing one O, S or NR.sup.15a in the
5-membered ring, wherein the 5-membered ring can optionally
additionally contain one or two N atoms, and wherein the
heterocyclic ring is optionally substituted on a ring carbon atom
by one of: a halogen atom, C.sub.1-2alkyl, C.sub.1fluoroalkyl,
--CH.sub.2OH, --CH.sub.2--OC.sub.1-2alkyl, OH (including the keto
tautomer thereof) or --CH.sub.2--NR.sup.28R.sup.29 wherein R.sup.28
and R.sup.29 independently are H or methyl.
[0071] In this "biaryl" embodiment, more preferably, R.sup.5 is of
sub-formula (x) and is: benzyl optionally substituted on the phenyl
ring with one or two (e.g. one) R.sup.6 substituents; wherein one
of the R.sup.6 is: Ar.sup.5b, or phenyl or pyridinyl wherein the
phenyl or pyridinyl independently are optionally substituted by one
of fluoro, chloro, C.sub.1-2alkyl, C.sub.1fluoroalkyl,
C.sub.1-2alkoxy or C1fluoroalkoxy; and wherein Ar.sup.5b is a
5-membered aromatic heterocyclic ring containing one O, S or
NR.sup.15a in the 5-membered ring, wherein the 5-membered ring can
optionally additionally contain one N atom, and wherein the
heterocyclic ring is optionally substituted on a ring carbon atom
by one of: C.sub.1-2alkyl or C.sub.1fluoroalkyl.
[0072] In this "biaryl" embodiment, when one R.sup.6 is optionally
substituted phenyl or pyridinyl, it can in particular be phenyl or
pyridinyl independently optionally substituted at the 3- or
4-position (e.g. 3-position) by one of fluoro, chloro,
C.sub.1-2alkyl, C.sub.1fluoroalkyl, C.sub.1-2alkoxy or
C1fluoroalkoxy (for example by one of chloro, C.sub.1alkyl,
C.sub.1fluoroalkyl or C.sub.1 alkoxy). When one R.sup.6 is
Ar.sup.5b then it can for example be
##STR00017##
[0073] In this "biaryl" embodiment, preferably, in sub-formula (x),
A is CR.sup.6, wherein the R.sup.6 at position A is: Ar.sup.5b, or
phenyl, pyridinyl or pyrimidinyl wherein the phenyl, pyridinyl or
pyrimidinyl independently are optionally substituted by one or two
of fluoro, chloro, C.sub.1-2alkyl, C.sub.1fluoroalkyl,
C.sub.1-2alkoxy or C.sub.1 fluoroalkoxy.
[0074] In this "biaryl" embodiment, NR.sup.4R.sup.5 can for example
be:
##STR00018## ##STR00019##
(connecting nitrogen underlined).
[0075] In another alternative embodiment, R.sup.5 has the
sub-formula (z), and one or preferably none of J, L, M or Q is
CR.sup.6, and/or R.sup.9 is a hydrogen atom (H) or methyl.
Preferably r is 1. Preferably, for (z), R.sup.6 is independently OH
(including any keto tautomer thereof), or more preferably
C.sub.1-2alkyl (e.g. methyl) or C.sub.1fluoroalkyl.
[0076] In a preferred embodiment, the compound of formula (I) or
the salt thereof is: [0077]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0078]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0079]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-(methyloxy)pheny-
l]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0080]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0081]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0082]
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0083]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,4-difluorophenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0084]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-(trifluoromethyl-
)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0085]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1
ethyl-N-[(4-hydroxyphenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamid-
e, [0086]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-(phenylmeth-
yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0087]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(methylsulfonyl)-
phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0088]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(3-hydroxyphenyl)me-
thyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0089]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,3-dichlorophenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0090]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,3-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0091]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(2-biphenylylmethyl)-1-ethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0092]
4-{1-[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chloro-2-fluorophenyl)-
methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0093]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-({4-[(difluoromethyl)oxy]phe-
nyl}methyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
[0094]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chloro-2-methylphenyl)me-
thyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0095]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4'-chloro-4-biphenylyl)met-
hyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0096]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-(methylsulfonyl)-
phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0097]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(trifluoromethy-
l)-2-biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
[0098]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chlorophenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0099]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(4-biphenylylmethyl)-1-ethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0100]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(ethyloxy)-4-bi-
phenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0101]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(3-biphenylylmethyl)-1-ethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0102]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(trifluoromethy-
l)-4-biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
[0103]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(3'-methyl-2-biphen-
ylyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0104]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[3'-(methyloxy)-2-b-
iphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0105]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3'-chloro-2-biphenylyl)met-
hyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0106]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4'-chloro-2-biphenylyl)met-
hyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0107]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(4'-methyl-2-biphen-
ylyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0108]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(methyloxy)-2-b-
iphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0109]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-furanyl)pheny-
l]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0110]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-({2-[6-(methyloxy)-3-
-pyridinyl]phenyl}methyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
[0111]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-thienyl)pheny-
l]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0112]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(4-methyl-3-thie-
nyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0113]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(5-methyl-2-bipheny-
lyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0114]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-methyl-2-(3-thie-
nyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0115]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-furanyl)-4-me-
thylphenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0116]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3'-chloro-5-methyl-2-biphe-
nylyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
[0117]
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0118]
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methy-
l]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0119]
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-(methyloxy)pheny-
l]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0120]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-(methylsulfonyl)phen-
yl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0121]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,3-dimethylphenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0122]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(2-biphenylylmethyl)-1-ethyl-1H--
pyrazolo[3,4-b]pyridine-5-carboxamide, [0123]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chloro-2-fluorophenyl)methyl-
]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0124]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,3-dichlorophenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0125]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[2-(methylsulfonyl)phen-
yl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0126]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chlorophenyl)methyl]-1-ethyl-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0127]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-(methyloxy)phenyl]me-
thyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0128]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(4-biphenylylmethyl)-1-ethyl-1H--
pyrazolo[3,4-b]pyridine-5-carboxamide, [0129]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-[(4-hydroxyphenyl)methyl-
]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0130]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-(phenylmethyl)-1H-pyrazo-
lo[3,4-b]pyridine-5-carboxamide, [0131]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-difluorophenyl)methyl]-1-e-
thyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0132]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-({4-[(difluoromethyl)oxy]phenyl}-
methyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0133]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(3-biphenylylmethyl)-1-ethyl-1H--
pyrazolo[3,4-b]pyridine-5-carboxamide, [0134]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-[(4-fluorophenyl)methyl]-
-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0135]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chloro-2-methylphenyl)methyl-
]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0136]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4'-chloro-4-biphenylyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0137]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-{[2,3-dichloro-6-(hydroxymethyl)-
phenyl]methyl}-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide,
[0138]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-{[3,4-bis(methyloxy)phenyl]methy-
l}-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0139]
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(2,4-dimethylphenyl)m-
ethyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, or [0140]
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(3,4-dimethylphenyl)m-
ethyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide; or a salt
thereof, e.g. a pharmaceutically acceptable salt thereof.
[0141] According to one embodiment of the invention, the compound
of formula (I) or the salt thereof is not
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide or a salt
thereof.
[0142] A further aspect of the invention provides a compound of
formula (IC) or a salt thereof (in particular, a pharmaceutically
acceptable salt thereof):
##STR00020##
wherein: R.sup.1c is ethyl or C2fluoroalkyl (preferably ethyl);
R.sup.2c is a hydrogen atom (H) or methyl (e.g. H); NHR.sup.3c is a
[1-(aminocarbonyl)-4-piperidinyl]amino group, a
cis-[4-(aminocarbonyl)cyclohexan-1-yl]amino group (including
mixtures of configurations wherein the stated cis configuration is
the major component), or a racemic
[4-(aminocarbonyl)cyclohexan-1-yl]amino group; and NHR.sup.5c is of
sub-formula (xx1), (xx2), (xx3) or (xx4):
##STR00021##
wherein n.sup.X1 and n.sup.X2 independently are 0 or 1, and
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 independently are CH.sub.2 or
O, provided that one or both of X.sub.1 and X.sub.2 are CH.sub.2,
and provided that one or both of X.sub.3 and X.sub.4 are
CH.sub.2.
[0143] NHR.sup.5c can for example be of sub-formula (xx1) or
(xx2).
[0144] In one embodiment, in sub-formula (xx1), both of X.sub.1 and
X.sub.2 are CH.sub.2. In one embodiment, in sub-formula (xx2), both
of X.sub.3 and X.sub.4 are CH.sub.2. NHR.sup.5c can for example
be:
##STR00022##
[0145] R.sup.3c is preferably
##STR00023##
That is, preferably NHR.sup.3c is a
[1-(aminocarbonyl)-4-piperidinyl]amino group.
[0146] The compound of formula (IC) or the salt thereof can be for
example: [0147]
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(2,3-dihydro-1-benzofuran-5--
ylmethyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, [0148]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-(5,6,7,8-tetrahydro-1-na-
phthalenylmethyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, or
[0149]
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(2,3-dihydro-1-benzofuran-5-ylme-
thyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide; or a salt
thereof, for example a pharmaceutically acceptable salt
thereof.
Salts, Solvates, Isomers, Tautomeric Forms, Molecular Weights,
Etc.
[0150] Because of their potential use in medicine, the salts of the
compounds of formula (I) are preferably pharmaceutically
acceptable. Suitable pharmaceutically acceptable salts may include
acid or (where acidic groups are present within formula (I)) base
addition salts.
[0151] A pharmaceutically acceptable acid addition salt is
optionally formed by combination of a compound of formula (I) with
a suitable inorganic or organic acid (such as hydrobromic,
hydrochloric, sulfuric, nitric, phosphoric, succinic, maleic,
formic, acetic, propionic, fumaric, citric, tartaric, lactic,
benzoic, salicylic, glutamaic, aspartic, p-toluenesulfonic,
benzenesulfonic, methanesulfonic, ethanesulfonic,
naphthalenesulfonic such as 2-naphthalenesulfonic, 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.
[0152] A pharmaceutically acceptable acid addition salt of a
compound of formula (I) optionally comprises or is for example a
hydrobromide, hydrochloride, sulfate, nitrate, phosphate,
succinate, maleate, formate, acetate, propionate, fumarate,
citrate, tartrate, lactate, benzoate, salicylate, glutamate,
aspartate, p-toluenesulfonate, benzenesulfonate, methanesulfonate,
ethanesulfonate, naphthalenesulfonate (e.g. 2-naphthalenesulfonate)
or hexanoate salt. A pharmaceutically acceptable acid addition salt
of a compound of formula (I) can in particular comprise or be a
hydrobromide, hydrochloride, sulfate, nitrate, phosphate,
p-toluenesulfonate, benzenesulfonate, methanesulfonate,
ethanesulfonate or naphthalenesulfonate (e.g.
2-naphthalenesulfonate) salt.
[0153] Where acidic groups are present within formula (I), a
pharmaceutically acceptable base addition salt can be 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.
[0154] Where acidic groups are present within formula (I), other
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).
[0155] Other non-pharmaceutically acceptable salts, eg. oxalates,
are in one less preferable embodiment used, for example in the
isolation of compounds of the invention, and are included within
the scope of this invention.
[0156] The invention includes within its scope all possible
stoichiometric and non-stoichiometric forms of the salts of the
compounds of formula (I).
[0157] Also included within the scope of the invention are all
solvates, hydrates and complexes of compounds and salts of the
invention.
[0158] Certain groups, substituents, 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.
[0159] 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.
[0160] 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.
[0161] Especially when intended for oral or external topical
medicinal use, compound(s) of formula (I) can have a molecular
weight of 1000 or less, for example 800 or less, in particular 650
or less or 600 or less. Molecular weight here refers to that of the
unsolvated "free base" compound, that is excluding any molecular
weight contributed by any addition salts, solvent (e.g. water)
molecules, etc.
Synthetic Process Routes
[0162] The following processes can be used to make the compounds of
formula (I) as hereinbefore defined. Most of the following
synthetic processes are exemplified for compounds of Formula (I)
wherein R.sup.2 is a hydrogen atom (H). However, some or all of
these processes can also be used with appropriate modification,
e.g. of starting materials and reagents, for making compounds of
Formula (I) wherein R.sup.2 is methyl.
Process A
[0163] To form a compound of formula (I), a carboxylic acid of
formula (II) can be converted into an activated compound of formula
(III) wherein X.sup.1=a leaving group substitutable by an amine (as
defined below) and subsequently the activated compound can be
reacted with an amine of formula NHR.sup.4R.sup.5:
##STR00024##
[0164] For example, the activated compound (the compound of formula
(III)) can be the acid chloride. This can be formed from the
carboxylic acid (II) e.g. by reaction with thionyl chloride, either
in an organic solvent such as chloroform or without solvent.
Alternatively, the activated compound (the compound of formula
(III)) can be an activated ester wherein the leaving group X.sup.1
is
##STR00025##
[0165] The latter activated compound of formula (III) can be formed
from the carboxylic acid (II) either: [0166] (a) by reaction of the
carboxylic acid with a carbodiimide such as EDC
(1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide), or a salt thereof
e.g. hydrochloride salt, preferably followed by reaction of the
resulting product with 1-hydroxybenzotriazole (HOBT); reaction (a)
being carried out usually in the presence of a solvent (e.g.
anhydrous) such as dimethyl formamide (DMF) or acetonitrile, and/or
usually in the presence of a base, e.g. tertiary organic amine
base, such as diisopropylethylamine (.sup.iPr.sub.2NEt=DIPEA),
and/or usually at room temperature (e.g. about 20 to about
25.degree. C.), and/or for example under anhydrous conditions; or:
[0167] (b) by reaction with
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU) or
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU), in the presence of a base, e.g.
tertiary organic amine base, such as diisopropylethylamine
(.sup.iPr.sub.2NEt=DIPEA), and usually in the presence of a solvent
such as dimethyl formamide (DMF) or acetonitrile and/or for example
under anhydrous conditions and/or usually at room temperature (e.g.
about 20 to about 25.degree. C.). Compounds of formula (II) can be
prepared by hydrolysis of an ester of formula (IV), wherein R.sup.7
is alkyl such as C.sub.1-4alkyl e.g. methyl or ethyl. This
procedure can for example involve reaction of (IV) with either:
[0168] (a) a base, e.g. alkali metal hydroxide such as sodium
hydroxide, potassium hydroxide or lithium hydroxide, in a solvent
e.g. an aqueous solvent (e.g. aqueous solvent comprising a mixture
of water and a water-miscible organic solvent) such as aqueous
ethanol or aqueous dioxane or [0169] (b) an acid such as
hydrochloric acid in a solvent e.g. an aqueous solvent such as
aqueous dioxane:
##STR00026##
[0170] The invention also provides in one aspect a compound of
formula (II), and in another aspect a compound of formula (IV), as
defined herein.
[0171] When R.sup.3 is the N-aminocarbonyl-piperidinyl or
-pyrrolidinyl group of sub-formula (bb), the compound of formula
(IV) can be prepared by reacting a compound of formula (IVa) or a
salt thereof (e.g. 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 (IVa) into a
[(1-aminocarbonyl)-4-piperidinyl]amino group or
[(1-aminocarbonyl)-3-pyrrolidinyl]amino group respectively:
##STR00027##
[0172] The urea-forming reagent may be benzyl isocyanate (followed
later by debenzylation e.g. reductive debenzylation), or preferably
the urea-forming reagent is tri(C.sub.1-4alkyl)silyl isocyanate
such as a tri(C.sub.1-2alkyl)silyl isocyanate, preferably
trimethylsilyl isocyanate. The reaction of the compound (IVa) or
salt thereof to the compound (IV) can be carried out in the
presence of a base such as N,N-diisopropylethylamine
(.sup.iPr.sub.2NEt=DIPEA), for example in more than one mole
equivalent compared to the number of moles of (IVa) or salt. The
reaction is optionally carried out at room temperature or by
heating to reflux.
[0173] The reaction of the compound (IVa) or salt thereof to
compound (IV) can be carried out in an organic solvent, the solvent
preferably not being an aqueous-organic solvent system or mixture.
The organic solvent can optionally be tetrahydrofuran (THF).
However, although THF appears at first sight to work satisfactorily
on an about 4 g scale (see Intermediate 4, n.sup.1=1,
R.sup.7=ethyl, HCl salt of (IVa)), it appears at first sight that
the yield might decrease when the reaction scaled up e.g. to about
33.5 g (see first half of Intermediate 4A, n.sup.1=1,
R.sup.7=ethyl, HCl salt of (IVa)). It seems that the solubility of
the HCl salt of (IVa) where n.sup.1=1, R.sup.7=ethyl in THF is
limited. Therefore, preferably the reaction of compound (IVa) or
salt to compound (IV) is carried out in a solubilising organic
solvent comprising (e.g. consisting essentially of or being) an
organic solvent capable of dissolving the compound of formula (IVa)
or salt thereof (whichever is used) to a substantially greater
extent than THF. For example, the solubilising organic solvent can
be dichloromethane or (probably) chloroform. Dichloromethane
appears to work satisfactorily on a scale of about 33.5 g, for
n.sup.1=1, R.sup.7=ethyl and using the HCl salt of compound (IVa)
(see e.g. second half of Intermediate 4A). (See also Intermediate
4B). In the reaction, the compound (IVa) or salt thereof is
preferably substantially wholly in solution, rather than being at
least partly in suspension, in the organic solvent.
[0174] Compound (IVa) or the salt thereof can be prepared from
compound (IVb), wherein Prot is a nitrogen protecting group such as
(tert-butyloxy)carbonyl (Boc), by deprotection of the nitrogen
protecting group. Boc removal can be effected by suitable acidic
conditions, such as hydrogen chloride (e.g. 4M) in 1,4-dioxane:
##STR00028##
[0175] Compound (IVb), wherein R.sup.7 is ethyl and Prot is Boc,
can be prepared according to a method, for example as described in
Intermediate 2 or 2A herein, by reaction of a compound of formula
(V) (illustrated below wherein R.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 (DIPEA), and/or in an organic solvent
such as acetonitrile. The reaction may require heating e.g. to ca.
60-100.degree. C. (e.g. ca. 80-90.degree. C.), for example for
about 16-18 hours:
##STR00029##
[0176] For one preparation of the compound of formula (V) wherein
R.sup.7 is ethyl, see e.g. Intermediate 1 herein, and/or see Scheme
1 and compound 12 in G. Yu et. al., J. Med. Chem., 2001, 44,
1025-1027. Therefore, a compound of formula (V) can be prepared by
reaction of a compound of formula (VI) with, for example,
diethylethoxymethylene malonate (where R.sup.7=Et) with heating,
followed by reaction with phosphorous oxychloride, again with
heating (see for example Intermediate 1 hereinafter):
##STR00030##
[0177] Where the desired amino pyrazole of formula (VI) is not
commercially available, preparation can be achieved using methods
described by Dorgan et. al. in J. Chem. Soc., Perkin Trans. 1, (4),
938-42; 1980, by reaction of cyanoethylhydrazine with a suitable
aldehyde of formula R.sup.40 CHO in a solvent such as ethanol, with
heating, followed by reduction with, for example sodium in a
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.
##STR00031##
[0178] According to one alternative optional embodiment of Process
A, a compound of formula (IV), wherein R.sup.7 is alkyl such as
C.sub.1-4alkyl e.g. methyl or ethyl, is optionally prepared
according to a method, for example as described in Scheme 1 of Yu
et. al., J. Med Chem., 2001, 44, 1025-1027, by reaction of a
compound of formula (V) with an amine of formula R.sup.3NH.sub.2.
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 or acetonitrile. The
reaction may require heating e.g. to ca. 60-100.degree. C., for
example ca. 80-90.degree. C.:
##STR00032##
[0179] In another alternative embodiment of Process A, the 4-chloro
substituent in the compound of formula (V) can be replaced by a
bromine or iodine 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 an alkoxy group --OR.sub.35 such as
--OC.sub.1-4alkyl (in particular--OEt) or a group
--O--S(O).sub.2--R.sup.37, wherein R.sup.37 is C.sub.1-8alkyl (e.g.
C.sub.1-4alkyl or C.sub.1-2alkyl such as methyl),
C.sub.1-6fluoroalkyl (e.g. C.sub.1-4fluoroalkyl or
C.sub.1-2fluoroalkyl such as CF.sub.3 or C.sub.4F.sub.9), or phenyl
wherein the phenyl is optionally substituted by one or two of
independently C.sub.1-2alkyl, halogen or C.sub.1-2alkoxy (such as
phenyl or 4-methyl-phenyl). The reaction may be carried out with or
without solvent and may require heating.
Process B
[0180] Compounds of formula (I) can be prepared by reaction of a
compound of formula (VII) with an amine of formula R.sup.3NH.sub.2
or a salt (e.g. HCl salt) thereof. The reaction is preferably
carried out in the presence of a base, e.g. tertiary organic amine
base, such as triethylamine or N,N-diisopropylethylamine (DIPEA),
and/or in an organic solvent such as ethanol, tetrahydrofuran
(THF), dioxane or acetonitrile. The reaction may require heating,
e.g. to ca. 60-100.degree. C. or ca. 80-90.degree. C., for example
for 8-72 or 12-48 or 24-48 hours:
##STR00033##
[0181] For an example of a preparation of HCl salts of some amines
of formula R.sup.3NH.sub.2, see e.g. Intermediates 12, 15, 19 and
20 herein.
[0182] Compounds of formula (VII) can be prepared in a two step
procedure (e.g. see Bare et. al. in J. Med. Chem. 1989, 32,
2561-2573). This process involves, first, reaction of a compound of
formula (VIII) with thionyl chloride (or another agent suitable for
forming an acid chloride from a carboxylic acid), either in an
organic solvent such as chloroform or THF, or as a neat solution,
preferably under substantially anhydrous conditions (e.g. under a
nitrogen or argon atmosphere). This reaction may require heating
(e.g. to reflux) and the thus-formed acid chloride intermediate may
or may not be isolated. Step two involves reaction of the resulting
acid chloride intermediate with an amine of formula
R.sup.4R.sup.5NH, in an organic solvent such as THF or chloroform
and may also involve the use of a base such as triethylamine or
diisopropylethylamine (DIPEA), See, for example, Intermediate 7
(first step) and Intermediates 8, 9, and 10 (second step)
herein:
##STR00034##
[0183] Compounds of formula (VIII) can be prepared by hydrolysis of
an ester of formula (V) according to the method described by Yu et.
al. in J Med. Chem., 2001, 44, 1025-1027. This procedure preferably
involves reaction with a base such as sodium hydroxide or potassium
hydroxide in a solvent e.g. an aqueous solvent such as aqueous
ethanol or aqueous dioxane:
##STR00035##
[0184] In an alternative embodiment of Process B, the 4-chloro
substituent in the compound of formula (VII) can be replaced by a
bromine or iodine atom.
Process C: Conversion of One Compound of Formula (I) or Salt
Thereof into Another Compound of Formula (I) or Salt Thereof.
[0185] One compound of formula (I) or salt thereof can be converted
into another compound of formula (I) or salt thereof. This
conversion can for example comprise or be one or more of the
following processes C1 to C6 or C7:
C1. 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. C2. A reduction process, for example reduction of
a ketone or a carboxylic acid to an alcohol. C3. Alkylation, for
example alkylation of an amine or of a hydroxy group. C4.
Hydrolysis, e.g. hydrolysis of an ester to the corresponding
carboxylic acid or salt thereof. C5. Deprotection, e.g.
deprotection (e.g. deacylation or t-butyloxycarbonyl (BOC) removal)
of an amine group. C6. Formation of an ester or amide, for example
from the corresponding carboxylic acid. C7. A carbon-carbon bond
coupling process, for example conversion of compounds of formula
(Ib) to compounds of formula (Ia), for example as follows.
Compounds of formula (I) which are of formula (Ia) are optionally
prepared by reaction of a compound of formula (Ib), where Hal.sup.2
is a chlorine, bromine or iodine atom (preferably a bromine or
iodine atom), with an aryl boronic acid (IX) or the corresponding
boronate ester. The reaction can for example use a catalyst such as
palladium tetrakis(triphenylphosphine) and a base such as sodium
carbonate, in one or more solvents such as a mixture of
dimethylformamide and water. The reaction may require heating, e.g.
to about 150.degree. C., and the rate of reaction may perhaps be
further enhanced by the use of a microwave reactor.
##STR00036##
[0186] In this reaction of (Ib) to (Ia), Ar.sup.B is typically one
R.sup.6 which is: Ar.sup.5b, or phenyl or pyridinyl wherein the
phenyl or pyridinyl independently are optionally substituted by one
of fluoro, chloro, C.sub.1-2alkyl, C.sub.1fluoroalkyl,
C.sub.1-2alkoxy or C.sub.1fluoroalkoxy; and wherein Ar.sup.5b is a
5-membered aromatic heterocyclic ring containing one O, S or
NR.sup.15a in the 5-membered ring, wherein the 5-membered ring can
optionally additionally contain one N atom, and wherein the
heterocyclic ring is optionally substituted on a ring carbon atom
by one of: C.sub.1-2alkyl or C.sub.1fluoroalkyl.
[0187] The present invention therefore also provides a method of
preparing a compound of formula (I) or a salt thereof:
##STR00037##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as
defined herein, the method comprising: (a) converting a compound of
formula (IIA) into an activated compound of formula (IIIA) wherein
X.sup.1=a leaving group substitutable by an amine:
##STR00038##
and subsequent reaction of the activated compound of formula (IIIA)
with an amine of formula R.sup.4R.sup.5NH; or (b) reacting a
compound of formula (VIIA):
##STR00039##
wherein Hal is a chlorine, bromine or iodine atom (such as a
bromine atom or preferably a chlorine atom), with an amine of
formula R.sup.3NH.sub.2 or a salt (e.g. HCl salt) thereof, or (c)
converting one compound of formula (I) or salt thereof into another
compound of formula (I) or salt thereof (e.g. by a carbon-carbon
bond coupling process, e.g. as described herein); and, in the case
of (a), (b) or (c), optionally converting the compound of formula
(I) into a salt thereof e.g. a pharmaceutically acceptable salt
thereof.
[0188] The optional or preferable features of steps (a), (b), (c)
can be according to Processes A, B or C mentioned above.
[0189] The present invention also provides: (g) a method of
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.
[0190] The present invention also provides a compound of formula
(I) or a salt thereof, prepared by a method as defined herein.
Medical Uses
[0191] 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; or e.g. for use in the
treatment and/or prophylaxis of cognitive impairment or depression
in a mammal such as a human) and/or can be for use as a
phosphodiesterase 4 (PDE4) inhibitor. "Therapy" may include
treatment and/or prophylaxis.
[0192] 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.
[0193] 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, or e.g. for the treatment and/or prophylaxis of
cognitive impairment or depression in a mammal.
[0194] 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, cognitive
impairment or depression 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.
[0195] Phosphodiesterase 4 inhibitors are thought to 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: asthma, chronic
obstructive pulmonary disease (COPD) (e.g. chronic bronchitis
and/or emphysema), atopic dermatitis, urticaria, allergic rhinitis,
allergic conjunctivitis, vernal conjunctivitis, eosinophilic
granuloma, psoriasis, rheumatoid arthritis, septic shock,
ulcerative colitis, Crohn's disease, reperfusion injury of the
myocardium and brain, chronic glomerulonephritis, endotoxic shock,
adult respiratory distress syndrome, multiple sclerosis, cognitive
impairment (e.g. in a neurological disorder such as Alzheimer's
disease), depression, or pain (e.g. inflammatory pain). Ulcerative
colitis and/or Crohn's disease are collectively often referred to
as inflammatory bowel disease.
[0196] In the treatment and/or prophylaxis, the inflammatory and/or
allergic disease is preferably chronic obstructive pulmonary
disease (COPD), asthma, rheumatoid arthritis, allergic rhinitis,
psoriasis or atopic dermatitis in a mammal (e.g. human). More
preferably, the treatment and/or prophylaxis is of COPD, asthma,
psoriasis or atopic dermatitis in a mammal (e.g. human).
[0197] Most preferably, 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) is preferably used.
For treatment and/or prophylaxis of atopic dermatitis, inhaled
administration is usually not suitable.
[0198] "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).
[0199] "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.
[0200] "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.
[0201] For the use of PDE4 inhibitors in atopic dermatitis, see for
example: [0202] 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); [0203] 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; [0204] 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); [0205] A. M.
Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473 (e.g. see
p. 470); and [0206] 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.
[0207] 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.
[0208] PDE4 inhibitors 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; and 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).
[0209] 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).
[0210] 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).
[0211] PDE4 inhibitors are thought to be effective in the treatment
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; and
references cited in the aforementioned publications).
[0212] PDE4 inhibitors are thought to be effective in the treatment
of allergic rhinitis (e.g. see B. M. Schmidt et al., J. Allergy
& Clinical Immunology, 108(4), 2001, 530-536).
[0213] PDE4 inhibitors are thought to be effective in the treatment
of rheumatoid arthritis and multiple sclerosis (e.g. see H. J. Dyke
et al., Expert Opinion on Investigational Drugs, January 2002,
11(1), 1-13; C. Bumouf 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).
[0214] PDE4 inhibitors have been suggested as having analgesic
properties and thus being effective in the treatment of pain (A.
Kumar et al., Indian J. Exp. Biol., 2000, 38(1), 26-30).
[0215] In the invention, the treatment and/or prophylaxis can be of
cognitive impairment e.g. cognitive impairment in a neurological
disorder such as Alzheimer's disease. For example, the treatment
and/or prophylaxis can comprise cognitive enhancement e.g. in a
neurological disorder. See for example: H. T. Zhang et al. in:
Psychopharmacology, June 2000, 150(3), 311-316 and
Neuropsychopharmacology, 2000, 23(2), 198-204; and T. Egawa et al.,
Japanese J. Pharmacol., 1997, 75(3), 275-81.
[0216] PDE4 inhibitors such as rolipram have been suggested as
having antidepressant properties (e.g. J. Zhu et al., CNS Drug
Reviews, 2001, 7(4), 387-398; O'Donnell, Expert Opinion on
Investigational Drugs, 2000, 9(3), 621-625; and H. T. Zhang et al.,
Neuropsychopharmacology, October 2002, 27(4), 587-595; J. M.
O'Donnell and H.-T. Zhang, Trends Pharmacol. Sci., March 2004,
25(3), 158-163; and T. E. Renau, Curr. Opinion Invest. Drugs, 2004,
5(1), 34-39).
[0217] 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.
Pharmaceutical Compositions, Routes of Administration, and
Dosing
[0218] For use in medicine, the compounds or salts of the present
invention are usually administered as a pharmaceutical
composition.
[0219] 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.
[0220] The pharmaceutical composition can be for use in the
treatment and/or prophylaxis of any of the conditions described
herein, in particular atopic dermatitis in a mammal such as a
human.
[0221] 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,
the method comprising mixing the compound or salt with the one or
more pharmaceutically acceptable carriers and/or excipients.
[0222] The invention also provides a pharmaceutical composition
prepared by said method.
[0223] The compounds of formula (I) and/or the pharmaceutical
composition may be administered, for example, by external topical
(e.g. skin topical), oral, parenteral (e.g. intravenous,
subcutaneous, or intramuscular), inhaled or nasal
administration.
[0224] Accordingly, the pharmaceutical composition can be suitable
for (e.g. adapted for) external topical (e.g. skin topical), oral,
parenteral (e.g. intravenous, subcutaneous, or intramuscular),
inhaled or nasal administration. The pharmaceutical composition is
preferably suitable for inhaled administration or more preferably
is suitable for external topical (e.g. skin topical)
administration, e.g. to a mammal such as a human. Inhaled
administration involves topical administration to the lung e.g. by
aerosol or dry powder composition.
[0225] Although the compound of formula (I) or salt thereof may be
administered orally, oral administration is not presently thought
to be a preferred route of administration, at least for the
compound
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide ("free base"
form) (e.g. Example 2, 2A or 2B). This is because, without
intending to be bound by this data, preliminary tests appear to
indicate a low systemic exposure after oral administration of
Example 2 or 2A ("free base" form) to rat(s), at a dose level of
about 1 mg of the compound per kg bodyweight, when formulated in
approximately [10% DMSO and 90% PEG200/water (70:30 PEG200:water
ratio)].
[0226] 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) (less
preferred) a tablet or capsule for oral administration e.g. for
oral administration to a human.
[0227] 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 External Topical
Administration
[0228] The pharmaceutical composition of the invention is
preferably suitable for (e.g. adapted for) external topical (e.g.
skin topical) administration, for example to a mammal such as a
human. More preferably, the pharmaceutical composition suitable for
external topical administration is for the treatment and/or
prophylaxis of atopic dermatitis in a mammal such as a human.
[0229] "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.
[0230] 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. The pharmaceutical composition can alternatively be
a DMSO-containing solution such as a DMSO/acetone solution or
DMSO/water solution (DMSO=dimethyl sulfoxide); a DMSO-containing
solution can be used for experimental animal tests, but is not
usually desirable for use in humans.
[0231] 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 is suitably present in 0.05% to 10%, preferably 0.1% to 5%,
more preferably 0.1% to 3%, still more preferably 0.2% to 3% (e.g.
about 0.5% or about 2.5%), yet more preferably 0.2% to 1.5% (e.g.
about 0.5%), by weight of the composition (w/w).
[0232] 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 below, within the Inhalable pharmaceutical
compositions section, for more details.
[0233] Aqueous solubility: A preliminary screen aims to estimate
roughly the aqueous solubility of compounds by (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 present
in this solubility screen "filtrate", the results from this
preliminary screen for the compound
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide ("free base"
form) (e.g. Example 2 or 2A) (about 60 micrograms of the
compound/ml) appear to suggest generally that this particular
Example 2 or 2A compound (in the "free base" form) has a generally
moderate aqueous solubility at about room temperature.
[0234] Lipophilicity: The compound
4-{1-[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methy-
l]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide ("free base"
form) (e.g. Example 2 or 2A) is thought to have a clogP (calculated
log of the octanol/water partition coeficient (P)) of approximately
2.5, suggesting moderate lipophilicity. This particular compound
(as the free base) is believed to have a measured logD
(D=distribution coefficient, wherein log D is generally log P
corrected for ionization) of approximately 3.4 at pH=7.4, again
suggesting modest/moderate lipophilicity.
[0235] Therefore, preferably, in a pharmaceutical composition
suitable for external topical (e.g. skin topical) administration,
the compound of formula (I) or salt thereof is
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide or a
pharmaceutically acceptable salt thereof, in particular
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide ("free base"
form).
[0236] Solubilising and/or skin-penetration-enhancing agents: An
external-topical pharmaceutical composition, e.g. an ointment or an
oil-in-water cream or water-in-oil cream, preferably 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.), preferably propylene glycol. The solubiliser
and/or skin-penetration enhancer suitably does not comprise DMSO.
The solubiliser and/or skin-penetration enhancer is preferably both
a solubiliser and skin-penetration enhancer, and/or is suitably
present in 0.5% to 50%, preferably 5% to 50%, more preferably 7% to
30%, still more preferably 7% to 25%, yet more preferably about 10%
to about 20% (e.g. about 10% or about 20%), by weight of the
composition (w/w). 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 compatible with the active
pharmaceutical ingredient. Note that the agent preferably functions
both as a solubilising agent and a skin-penetration-enhancing
agent.
[0237] Surfactants: An external-topical pharmaceutical composition,
e.g. an ointment or an oil-in-water cream or water-in-oil cream,
preferably includes 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 one or more of the following: a polyoxyl
C.sub.12-22alkyl ether (e.g. a polyoxyl C.sub.14-20alkyl ether such
as polyoxyl cetyl ether or polyoxyl stearyl 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),
cetyl alcohol and/or stearyl alcohol (e.g. wherein the total of any
cetyl alcohol and any stearyl alcohol present is 0.1% to 15% w/w,
e.g. 1% to 10% w/w such as about 2% to about 5% w/w), and sodium
dodecyl sulphate (SDS) (e.g. present at 0.3% to 2% w/w such as
about 1% w/w). Polyoxyl stearyl ether (steareth) can e.g. be
polyoxyl 2 stearyl ether (steareth 2) or polyoxyl 21 stearyl ether
(steareth 21).
[0238] DMSO-containing solutions: One possible external-topical
pharmaceutical composition is a solution of the compound of formula
(I) or the pharmaceutically acceptable salt thereof present at ca.
0.5% to ca. 2.5% w/w in a DMSO-containing solvent such as in
DMSO/acetone or in DMSO/water; for example a solution of the
compound or salt present at ca. 0.5% to ca. 2.5% w/w in
DMSO/acetone (1:1). DMSO-containing solutions, often being capable
of high skin penetration, are often good experimental pre-clinical
formulations for use in animals, but their likely skin irritancy
generally make them less suitable for use in humans such as
patients, e.g. atopic dermatitis patients.
[0239] 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, preferably of a
consistency suitable for skin-spreadability.
[0240] Preferably, 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.
[0241] 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.
[0242] The oil phase (oily ointment base) can for example be
present at 25 to 85% w/w (e.g. 50 to 80% w/w) in an ointment (e.g.
emulsion or homogeneous single phase), at 25 to 85% w/w (e.g. 35 to
70% w/w) in an water-in-oil cream (e.g. emulsion), or at 8 to 55%
w/w (e.g. 10 to 45% w/w) in an water-in-oil cream (e.g.
emulsion).
[0243] Exemplary ointments: An exemplary external-topical
pharmaceutical composition is an ointment comprising: [0244] the
compound of formula (I) or pharmaceutically acceptable salt thereof
present at 0.1% to 5% w/w (e.g. 0.1% to 3% w/w or 0.2% to 1.5%
w/w); [0245] an oil phase (oily ointment base) present at 25% to
99% w/w or 25% to 85% w/w or 50% to 80% w/w (for example, the oil
phase can comprise white petrolatum present at 25 to 75% w/w or 45
to 75% w/w, and optionally also comprising mineral oil (e.g. as
solubiliser and emollient) present at 2.5% to 15% w/w such as 4% to
12% w/w); [0246] one or more surfactants (e.g. polyoxyl stearyl
ether) present in total at 0.5% to 10% w/w or 3% to 10% w/w; and
one or more agents acting as a skin-penetration enhancer
(preferably acting as both a solubiliser and skin-penetration
enhancer and/or preferably 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 [0247] 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 [0248] 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).
[0249] The above exemplary composition, including the oil "phase"
and the penetration enhancer, can optionally be a homogeneous
single phase. However, in one embodiment of the above exemplary
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.
[0250] 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 preferably 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, preferably
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., preferably 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.
[0251] Optionally, an ointment can comprise a polyethylene glycol
base, e.g. present at 25 to 98% w/w such as 50 to 95% w/w, instead
of or as well as an oily ointment base.
[0252] 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.
[0253] Water-in-oil creams: These usually have an increased aqueous
content compared to ointments. Preferably, the water-in-oil cream
is a water-in-oil cream emulsion. That is, preferably, in the
water-in-oil cream, an oil phase and an aqueous phase have been
emulsified to form a water-in-oil cream emulsion.
[0254] An exemplary external-topical pharmaceutical composition is
a water-in-oil cream (e.g. cream emulsion) comprising: [0255] the
compound of formula (I) or pharmaceutically acceptable salt thereof
present at 0.1% to 5% w/w (e.g. 0.1% to 3% w/w or 0.2% to 1.5%
w/w); [0256] 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, and optionally also
comprising mineral oil (e.g. as solubiliser and emollient) present
at 2.5% to 15% w/w or 4% to 12% w/w); [0257] water present in 2% to
30% w/w, e.g. 5% to 25% or 10% to 22% w/w; [0258] 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 [0259] one or more agents
acting as a skin-penetration enhancer (preferably acting as both a
solubiliser and skin-penetration enhancer and/or preferably
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 [0260]
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 [0261] 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).
[0262] Oil-in-water creams: These usually have an increased aqueous
content compared to ointments and water-in-oil creams. Preferably,
the oil-in-water cream is a 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 a oil-in-water cream
emulsion.
[0263] Preferable 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.
[0264] Preferably, the oil-in-water cream contains 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.
[0265] An exemplary external-topical pharmaceutical composition is
a oil-in-water cream (e.g. cream emulsion) comprising: [0266] the
compound of formula (I) or pharmaceutically acceptable salt thereof
present at 0.1% to 5% w/w (e.g. 0.1% to 3% w/w or 0.2% to 1.5%
w/w); [0267] an oil phase (oily ointment base) containing one or
more ingredients capable of acting as emollients, the oil phase
being present at 5% to 60% w/w or preferably 20% to 60% w/w or 30%
to 60% w/w or more preferably 30% to 55% w/w; [0268] water present
in 12% to 75% w/w or 15% to 75% w/w or 15% to 60% w/w, preferably
15% to 50% w/w or 20% to 40% w/w; [0269] one or more surfactants
present in total at 0.5% to 12% w/w, e.g. 3% to 10% w/w; and [0270]
one or more agents acting as a skin-penetration enhancer
(preferably acting as both a solubiliser and skin-penetration
enhancer and/or preferably 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 [0271] 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 [0272] 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.
[0273] In the above exemplary oil-in-water cream composition, the
oil phase preferably comprises mineral oil (e.g. as emollient and
solubiliser) present at 15% to 50% w/w or 20% to 45% w/w, 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.
[0274] In the above exemplary oil-in-water cream composition, the
one or more surfactants preferably 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.
[0275] 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 preferably 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, preferably 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., preferably 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.
[0276] 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.
Inhalable and Intranasal Pharmaceutical Compositions, and Particle
Size Reduction
[0277] Compositions suitable for (e.g. adapted for) nasal or
inhaled administration may conveniently be formulated as aerosols,
drops, gels or dry powders.
[0278] Aerosol formulations, e.g. for inhaled administration, can
comprise a solution or fine suspension of the active substance in a
pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol
formulations can be presented 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) which is
intended for disposal once the contents of the container have been
exhausted.
[0279] Where the dosage form comprises an aerosol dispenser, it
preferably contains a suitable propellant under pressure such as
compressed air, carbon dioxide, or an organic propellant such as a
chlorofluorocarbon (CFC) or more preferably hydrofluorocarbon
(HFC). Suitable CFC propellants include dichlorodifluoromethane,
trichlorofluoromethane and dichlorotetrafluoroethane. Suitable HFC
propellants include 1,1,1,2,3,3,3-heptafluoropropane and
1,1,1,2-tetrafluoroethane. The aerosol dosage forms can also take
the form of a pump-atomiser.
[0280] Particle size reduction: For pharmaceutical compositions
suitable for (e.g. adapted for) inhaled administration, it is
preferred that the compound or salt of formula (I) is 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 preferable particle size of the size-reduced (e.g. micronised)
compound or salt is defined by a D50 value of about 0.5 to about 10
microns, e.g. about 1 to about 7 microns (e.g. as measured using
laser diffraction). For example, it is preferable for the compound
or salt of formula (I) to have a particle size defined by: a D10 of
about 0.3 to about 3 microns (e.g. about 0.5 to about 2 microns, or
about 1 micron), and/or a D50 of about 0.5 to about 10 microns or
about 1 to about 7 microns (e.g. about 2 to about 5 microns or
about 2 to about 4 microns), and/or a D90 of about 1 to about 30
microns or about 2 to about 20 microns or about 3 to about 15
microns (e.g. about 5 to about 15 microns or about 5 to about 10
microns); for example as measured using laser diffraction.
[0281] 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.
[0282] 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 (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 (preferably all
of) the following: a Malvern Mastersizer longbed version, a
dispersing medium of 0.1% Tween 80 in water, a stir rate 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.
[0283] For a small-scale non-limiting example of micronisation, see
the Micronisation Example hereinafter.
[0284] Dry powder inhalable compositions: For pharmaceutical
compositions suitable (e.g. adapted for) inhaled administration,
the pharmaceutical composition may for example be a dry powder
inhalable composition. Such a composition can comprise a powder
base such as lactose or starch, the compound of formula (I) or salt
thereof (preferably in particle-size-reduced form, e.g. in
micronised form), and optionally a ternary agent such as L-leucine,
mannitol, trehalose, magnesium stearate and/or cellobiose
octaacetate (e.g. alpha-D-isomer of cellobiose octaacetate, e.g.
available from Aldrich). For cellobiose octaacetate and storage
stability, see WO 03/088943.
[0285] Preferably, the dry powder inhalable composition comprises a
dry powder blend of lactose and the compound of formula (I) or salt
thereof. The lactose is preferably lactose hydrate e.g. lactose
monohydrate and/or is preferably inhalation-grade and/or fine-grade
lactose. Preferably, the particle size of the lactose is 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. More preferably, the particle size of the lactose is
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
is 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. It is suitable
that about 3 to about 30% (e.g. about 10%) (by weight or by volume)
of the particles are less than 50 microns or less than 20 microns
in diameter. For example, without limitation, a suitable
inhalation-grade lactose is E9334 lactose (10% fines) (Borculo Domo
Ingredients, Hanzeplein 25, 8017 JD Zwolle, Netherlands).
[0286] 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 1% to about 50%, e.g. about 5% to
about 40%, e.g. about 20 to about 30%) by weight of the
composition.
[0287] An illustrative non-limiting example of a dry powder
inhalable composition is given in the Composition Examples
below.
[0288] Dry powder inhalation devices: 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 is 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 is usually 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.
Pharmaceutical Compositions for Oral or Parenteral
Administration
[0289] 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
parenterally acceptable aqueous carrier (e.g. sterile water) or
parenterally acceptable oil. Alternatively, the solution can be
lyophilised. 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.
[0290] A pharmaceutical composition 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.
[0291] A liquid formulation 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, ethanol or 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.
[0292] The composition can optionally be in unit dose form such as
a tablet or capsule for oral administration, e.g. for oral
administration to a human.
[0293] A pharmaceutical composition 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 (polyvinylpyrollidone), 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
polyvinylpyrollidone). 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.
[0294] A pharmaceutical composition 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.
Dosing Regimens
[0295] 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 0.05% to 10%, preferably 0.1% to 5%, more preferably 0.1% to 3%,
still more preferably 0.2% to 3% (e.g. about 0.5% or about 2.5%),
yet more preferably 0.2% to 1.5% (e.g. about 0.5%/0), by weight of
the composition. 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.
[0296] A pharmaceutical composition can optionally be in unit dose
form. In the pharmaceutical composition, a or each dosage unit for
oral or parenteral administration can for example contain from 0.01
to 3000 mg, for example 0.5 to 1000 mg, of a compound of the
formula (I) or a pharmaceutically acceptable salt thereof,
calculated as the free base. A or each dosage unit for nasal or
inhaled administration can for example contains from 0.001 to 50
mg, e.g. 0.01 to 5 mg, of a compound of the formula (I) or a
pharmaceutically acceptable salt thereof, calculated as the free
base.
[0297] When a parenteral or oral composition is used,
pharmaceutically acceptable compound or salt of the invention can
optionally be administered to a mammal (e.g. human) in a daily oral
or parenteral dose of 0.001 mg to 50 mg per kg body weight per day
(mg/kg/day), for example 0.01 to 20 mg/kg/day or 0.03 to 10
mg/kg/day or 0.1 to 2 mg/kg/day, of the compound of the formula (I)
or a pharmaceutically acceptable salt thereof, calculated as the
free base.
[0298] When an inhaled or nasal composition is used, a
pharmaceutically acceptable compound or salt of the invention can
optionally be administered to a mammal (e.g. human) in a daily
nasal or inhaled dose of: 0.0001 to 5 mg/kg/day or 0.0001 to 1
mg/kg/day, e.g. 0.001 to 1 mg/kg/day or 0.001 to 0.3 mg/kg/day or
0.001 to 0.1 mg/kg/day or 0.005 to 0.3 mg/kg/day, of the compound
of the formula (I) or a pharmaceutically acceptable salt thereof,
calculated as the free base.
[0299] The pharmaceutically acceptable compounds or salts of the
invention can optionally be administered to a human in a daily dose
(for an adult patient) of, for example, an oral or parenteral dose
of 0.01 mg to 3000 mg per day or 0.5 to 1000 mg per day e.g. 2 to
500 mg per day, or a nasal or inhaled dose of 0.001 to 300 mg per
day or 0.001 to 50 mg per day or 0.01 to 30 mg per day or 0.01 to 5
mg per day or 0.02 to 2 mg per day, of the compound of the formula
(I) or a pharmaceutically acceptable salt thereof, calculated as
the free base.
Combinations
[0300] 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.2
adrenoreceptor agonist, an anti-histamine, an anti-allergic, an
anti-inflammatory agent, an antiinfective agent or an
immunosuppressant.
[0301] 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
.beta..sub.2-adrenoreceptor agonist, an anti-histamine, an
anti-allergic, an anti-inflammatory agent, an antiinfective agent
or an immunosuppressant.
[0302] Preferably, the .beta..sub.2-adrenoreceptor agonist is
salmeterol (e.g. as racemate or a single enantiomer such as the
R-enantiomer), salbutamol, formoterol, salmefamol, fenoterol or
terbutaline, or a salt thereof (e.g. pharmaceutically acceptable
salt thereof), for example the xinafoate salt of salmeterol, the
sulphate salt or free base of salbutamol or the fumarate salt of
formoterol. Long-acting .beta..sub.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..sub.2-adrenoreceptor agonist is for inhaled administration,
e.g. once per day and/or for simultaneous inhaled administration;
and more preferably the .beta..sub.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, is preferably administered to humans at an inhaled dose
of 25 to 50 micrograms twice per day (measured as the free
base).
[0303] Preferred long acting .beta..sub.2-adrenoreceptor agonists
include those described in WO 02/066422A, WO 03/024439, WO
02/070490 and WO 02/076933.
[0304] Especially preferred long-acting .beta..sub.2-adrenoreceptor
agonists include compounds of formula (XX) (described in WO
02/066422):
##STR00040##
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.sub.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.7X, 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.1XR.sup.19X,
--NR.sup.18XC(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.1-6alkyl, 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
[0305] 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.
[0306] Preferred .beta..sub.2-adrenoreceptor agonists disclosed in
WO 02/066422 include:
3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}ami-
no)hexyl]oxy}butyl)benzenesulfonamide and
3-(3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-hydroxymethyl)phenyl]ethyl}-amin-
o)heptyl]oxy}propyl)benzenesulfonamide.
[0307] A preferred .beta..sub.2-adrenoreceptor agonist disclosed in
WO 03/024439 is: [0308]
4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyet-
hyl}-2-(hydroxymethyl)phenol.
[0309] An anti-histamine usable in a combination of a compound of
formula (I) or salt can for example be for oral administration
(e.g. as a combined composition such as a combined tablet), and can
be for treatment and/or prophylaxis of allergic rhinitis. Examples
of anti-histamines include methapyrilene, or H1 antagonists such as
cetirizine, loratadine (e.g. Clarityn.TM.), desloratadine (e.g.
Clarinex.TM.) or fexofenadine (e.g. Allegra.TM.).
[0310] The invention also provides, in a further aspect, a
combination comprising a compound of formula (I) or a
pharmaceutically acceptable salt thereof together with an
anticholinergic compound, e.g. a muscarinic (M) receptor antagonist
in particular an M.sub.1, M.sub.2, M.sub.1/M.sub.2, or M.sub.3
receptor antagonist, more preferably a M.sub.3 receptor antagonist,
still more preferably 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. For combinations of anticholinergic compounds/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.
[0311] The anticholinergic compound or 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. Preferably, the anticholinergic compound or
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.
[0312] 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 preferably for oral
administration. Suitable 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.
[0313] Exemplary 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.TM.). 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.
[0314] For external topical administration, e.g. versus atopic
dermatitis, a combination of the compound or salt of the invention
together with an anti-infective agent can include an
externally-topically-administrable antibacterial such as mupiricin
or a salt (e.g. calcium salt) thereof (e.g. Bactroban.TM.), or an
externally-topically-administrable pleuromutilin antibacterial.
Alternatively or additionally, for external topical administration
an externally-topically-administrable antifungal such as
clortrimazole, clotrimazole or ketoconazole can be used.
[0315] For external topical administration, e.g. versus atopic
dermatitis, a combination with an anti-itch compound may optionally
be used.
[0316] 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 or allergic rhinitis),
then preferably the anti-inflammatory corticosteroid is 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), 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 preferably it is
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 external topical, intranasal or inhaled
administration. Fluticasone propionate is preferred 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.
[0317] 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. Most
preferably, in this "triple" combination, the
.beta..sub.2-adrenoreceptor agonist is salmeterol or a
pharmaceutically acceptable salt thereof (e.g. salmeterol
xinafoate) and the anti-inflammatory corticosteroid is fluticasone
propionate.
[0318] 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.
[0319] The individual compounds of such combinations may be
administered either sequentially or simultaneously in separate or
combined pharmaceutical composition.
[0320] 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 (e.g. as described in the claims thereof
e.g. claim 1).
[0321] The invention also provides a method of preparing a
combination as defined herein, the method comprising either [0322]
(a) preparing a separate pharmaceutical composition for
administration of the individual compounds of the combination
either sequentially or simultaneously, or [0323] (b) preparing a
combined pharmaceutical composition for administration of the
individual compounds of the combination simultaneously, [0324]
wherein the pharmaceutical composition comprises the combination
together with one or more pharmaceutically acceptable carriers
and/or excipients.
[0325] 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 Primary Assay Methods
[0326] The activity of the compounds or salts of the invention can
be measured in the assay methods shown below. Preferred compounds
of the invention are selective PDE4 inhibitors, i.e. they inhibit
PDE4 (e.g. PDE4B and/or PDE4D, preferably PDE4B) more strongly than
they inhibit PDE3 and/or more strongly than they inhibit PDE5
and/or more strongly than they inhibit PDE6 (though such
selectivity is not essential to the invention).
Possible PDE Enzyme Sources and Literature References
[0327] 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.
[0328] 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 IV.sub.D)",
Gene, 1994, 138, 253-256.
[0329] 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.
[0330] PDE3 can be purified from bovine aorta, e.g. 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.
[0331] PDE6 can be purified from bovine retina, e.g. 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)
[0332] 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. [0333] Test compounds (as a solution in DMSO, preferably
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 0.05 uCi 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 0.05 uCi per well and about 36 nM final
concentration. Plates containing assay mixture, preferably 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 (preferably 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.
[0334] 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
[0335] 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 a 384-well format.
[0336] 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.
[0337] Test compounds (small volume, e.g. ca. 0.5 to 1 microlitres
(ul), preferably 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, preferably
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.
[0338] In the FP assay, all reagents are generally dispensed using
Multidrop.TM. (available from Thermo Labsystems Oy, Ratastie 2, PO
Box 100, Vantaa 01620, Finland).
[0339] For a given PDE4 inhibitor, the PDE4B (or PDE4D) inhibition
values measured using the SPA and FP assays can differ slightly.
However, in a regression analysis of 100 test compounds (not
necessarily compounds of the invention), the pIC.sub.50 inhibition
values measured using SPA and FP assays have been found generally
to agree within about 0.5 log units, for each of PDE4B and PDE4D
(linear regression coefficient 0.966 for PDE4B and 0.971 for PDE4D;
David R. Mobbs et al., "Comparison of the IMAP Fluorescence
Polarisation Assay with the Scintillation Proximity Assay for
Phosphodiesterase Activity", poster presented at 2003 Molecular
Devices UK & Europe User Meeting, 2 Oct. 2003, Down Hall,
Harlow, Essex, United Kingdom).
[0340] 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 as follows, based on
current measurements only, and using the above or similar or
analogous assay methods. In each of the SPA and FP assays, absolute
accuracy of measurement is not possible, and the readings given are
generally accurate only up to about .+-.0.5 of a log unit,
depending on the number of readings made and averaged:
TABLE-US-00001 PDE4B pIC.sub.50 Example number (.+-.about 0.5) 1, 2
9.1 to 9.2 3, 4, 5, 8 8.5 to 9.0 9, 46 6.7 to 6.8 10 to 22, 24, 26
8.6 to 9.9 23, 25, 27 to 32, 34 to 44 8.2 to 9.6 45, 47 8.3 to 8.9
48 to 68 7.9 to 8.95 69, 70 about 8.0 71, 72 7.0 to 7.5
Emesis: Some known PDE4 inhibitors can cause emesis and/or nausea
to greater or lesser extents, e.g. after systemic exposure (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 external
topical, oral or parenteral administration. Emetic side-effects can
for example be measured by the emetogenic potential of the compound
or salt when administered to ferrets; for example one can measure
the time to onset, extent, frequency and/or duration of vomiting,
retching and/or writhing in ferrets after oral or parenteral
administration of the compound or salt. See for example in vivo
Assay 4 hereinafter for a measurement method for anti-inflammatory
effect, emetic side-effects and therapeutic index (TI) in the
ferret. See also 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. However,
optionally, emetic side-effects and therapeutic index (TI) after
oral administration in rats can be conveniently measured by
monitoring the pica feeding behaviour of rats after administration
of the compound or salt of the invention (see In Vivo Assay 2
below).
[0341] Other side effects: Some known PDE4 inhibitors can cause
other side effects such as headache and 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.
In Vivo Biological Assays
[0342] The in vitro enzymatic PDE4B inhibition assay(s) described
above or generally similar assays should be regarded as being the
primary test(s) of biological activity. However, some additional in
vivo biological tests, which are optional, 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 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:
[0343] 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.
[0344] 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).
[0345] 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).
[0346] 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.
[0347] 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.
[0348] About 24 hours after challenge, treatment sites can
optionally also be visually evaluated for lesion area.
Specific Study Design and Results:
[0349] The anti-inflammatory effect of the compound
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide ("Example 2 or 2A
compound"), applied topically to the skin, on the acute DTH
response in castrated male Yorkshire pigs is compared to that of
another PDE4 inhibitor cipamfylline (BRL-61063) and the topical
immunomodulator pimecrolimus.
[0350] In the above assay, the Example 2 or 2A compound is
topically administered: either (A) at ca. 2.5% (w/v) concentration
in a solution of ca. 10% DMSO/90% acetone (ca. 50 microlitres or
ca. 1.5 mg/site) at about 2 hours prior to and about 6 hours after
the DNFB challenge; or (B) at ca. 0.5% (w/w) concentration in an
ointment containing propylene glycol (PG)* (ca. 25 mg of
formulation/site) at about 30 minutes after and about 6 hours after
the DNFB challenge. (* The PG ointment used or usable with the
Example 2 or 2A compound generally contains inter alia
approximately the following constituents [in % (w/w)]: ca. 69-70%
white petrolatum, ca. 5% mineral oil, ca. 5% polyoxyl stearyl ether
(e.g. Volpo S2) and ca. 20% propylene glycol. See for example
Composition Example C1 hereinafter for a suitable ointment
formulation.)
[0351] The Example 2 or 2A compound at ca. 0.5% (w/w) in PG
ointment inhibits the mean summed score by about 13-14% compared to
the placebo PG ointment (p<0.05 by ANOVA). The Example 2 or 2A
compound administered at ca. 2.5% (w/v) in DMSO/acetone solution
gives a reduction of about 21% in the mean summed score compared to
vehicle alone (p<0.05 by ANOVA). In comparison, cipamfylline at
ca. 2.5% (w/v) in DMSO/acetone and cipamfylline at ca. 0.25% (w/w)
in ointment reduces the summed scores by about 8% and about 6-7%,
respectively (effects that are not statistically significant),
whereas pimecrolimus applied in a 1% (w/w) cream formulation
inhibits the summed scores by about 29% (p<0.05 by ANOVA).
[0352] Further, the skin administration of the Example 2 or 2A
compound appears to decrease the lesion area when added in
DMSO/acetone or PG ointment (results subject to some
inaccuracy/error).
[0353] These results appear to demonstrate the potential
anti-inflammatory activity of the Example 2 or 2A compound ("free
base" form) in an acute pig DTH (delayed type hypersensitivity)
model when the compound is topically administered to the skin in a
suitable formulation.
In Vivo Assay 1. LPS-Induced Pulmonary Neutrophilia in Rats: Effect
of Orally Administered PDE4 Inhibitors
[0354] Pulmonary neutrophil influx is thought to be a significant
component to the family of pulmonary diseases like chronic
obstructive pulmonary disease (COPD) which can involve chronic
bronchitis and/or emphysema (G. F. Filley, Chest. 2000; 117(5);
251s-260s). The purpose of this neutrophilia model is to study the
potentially anti-inflammatory effects in vivo of orally
administered PDE4 inhibitors on neutrophilia induced by inhalation
of aerosolized lipopolysaccharide (LPS), modelling the neutrophil
inflammatory component(s) of COPD. See the literature section below
for scientific background.
[0355] Male Lewis rats (Charles River, Raleigh, N.C., USA) weighing
approximately 300-400 grams are pretreated with either (a) test
compound, for example suspended in about 0.5% methylcellulose
(obtainable from Sigma-Aldrich, St Louis, Mo., USA) in water or (b)
vehicle only, delivered orally in a dose volume of ca. 10 ml/kg.
Generally, dose response curves can for example be generated using
the following approx. doses of PDE4 inhibitors: 2.0, 0.4, 0.08,
0.016 and 0.0032 mg/kg. About thirty minutes following
pretreatment, the rats are exposed to aerosolized LPS (Serotype E.
Coli 026:B6 prepared by trichloroacetic acid extraction, obtainable
from Sigma-Aldrich, St Louis, Mo., USA), generated from a nebulizer
containing a ca. 100 .mu.g/ml LPS solution (ca. 100 ug/ml). Rats
are exposed to the LPS aerosol at a rate of ca. 4 L/min for ca. 20
minutes. LPS exposure is carried out in a closed chamber with
internal dimensions of roughly 45 cm length.times.24 cm
width.times.20 cm height. The nebulizer and exposure chamber are
contained in a certified fume hood. At about 4 hours-post LPS
exposure the rats are euthanized by overdose with pentobarbital at
ca. 90 mg/kg, administered intraperitoneally. Bronchoalveolar
lavage (BAL) is performed through a 14 gauge blunt needle into the
exposed trachea. Five, 5 ml washes are performed to collect a total
of 25 ml of BAL fluid. Total cell counts and leukocyte
differentials are performed on BAL fluid in order to calculate
neutrophil influx into the lung. Percent neutrophil inhibition at
each dose (cf. vehicle) is calculated and a variable slope,
sigmoidal dose-response curve is generated, usually using Prism
Graph-Pad. The dose-response curve is used to calculate an ED50
value (in mg per kg of body weight) for inhibition by the PDE4
inhibitor of the LPS-induced neutrophilia. [0356] Alternative
method: In an alternative simpler embodiment of the procedure, a
single oral dose of 10 mg/kg or more usually 1.0 mg/kg or 0.3 mg/kg
of the PDE4 inhibitor (or vehicle) is administered to the rats, and
percent neutrophil inhibition is calculated and reported for that
specific dose.
[0357] Literature: [0358] Filley G. F. Comparison of the structural
and inflammatory features of COPD and asthma. Chest. 2000; 117(5)
251s-260s. [0359] Howell R E, Jenkins L P, Fielding L E, and Grimes
D. Inhibition of antigen-induced pulmonary eosinophilia and
neutrophilia by selective inhibitors of phosphodiesterase types 3
and 4 in brown Norway rats. Pulmonary Pharmacology. 1995; 8: 83-89.
[0360] Spond J, Chapman R, Fine J, Jones H, Kreutner W, Kung T T,
Minnicozzi M. Comparison of PDE 4 inhibitors, Rolipram and SB
207499 (Ariflo.TM.), in a rat model of pulmonary neutrophilia.
Pulmonary Pharmacology and Therapeutics. 2001; 14:157-164. [0361]
Underwood D C, Osborn R R, Bochnowicz S, Webb E F, Rieman D J, Lee
J C, Romanic A M, Adams J L, Hay D W P, and Griswold D E. SB
239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory
cytokines, MMP-9, and fibrosis in lung. Am J Physiol Lung Cell Mol.
Physiol. 2000; 279: L895-L902.
In Vivo Assay 2. Rat Pica Model of Emesis
[0362] Background: Selective PDE4 inhibitors are thought to inhibit
inflammation in various in vitro and in vivo models by increasing
intracellular levels of cAMP of many immune cells (e.g.
lymphocytes, monocytes). However, a side effect of some PDE4
inhibitors in some species is emesis. Because many rat models of
inflammation are well characterized, they can be used in procedures
(see e.g. In Vivo Assay 1 above) to show beneficial
anti-inflammatory effects of PDE 4 inhibitors. However rats have no
emetic response (they have no vomit reflex), so that the
relationship between beneficial anti-inflammatory effects of PDE 4
inhibitors and emesis is difficult to study directly in rats.
[0363] However, in 1991, Takeda et al. (see Literature section
below) demonstrated that the pica feeding response is analogous to
emesis in rats. Pica feeding is a behavioural response to illness
in rats wherein rats eat non-nutritive substances such as earth or
in particular clay (e.g. kaolin) which may help to absorb toxins.
Pica feeding can be induced by motion and chemicals (especially
chemicals which are emetic in humans), and can be inhibited
pharmacologically with drugs that inhibit emesis in humans. The Rat
Pica Model, In Vivo Assay 2, can determine the level of pica
response of rats to PDE 4 inhibition at pharmacologically relevant
doses in parallel to in vivo anti-inflammatory Assays in (a
separate set of) rats (e.g. In Vivo Assay 1 above).
[0364] Anti-inflammatory and pica assays in the same species
together can provide data on the "therapeutic index" (TI) in the
rat of the compounds/salts of the invention. The Rat TI can for
example be calculated as the ratio of a) the potentially-emetic
Pica Response ED50 dose from Assay 2 to b) the rat
anti-inflammatory ED50 dose (e.g. measured by rat
neutrophilia-inhibition in eg In Vivo Assay 1), with larger TI
ratios possibly indicating lower emesis at many anti-inflammatory
doses. This might allow a choice of a non-emetic or low-emetic
pharmaceutical dose of the compounds or salts of the invention
which has an anti-inflammatory effect. It is recognised however
that achieving a low-emetic PDE4 inhibitory compound is not
essential to the invention.
[0365] Procedure: On the first day of the experiment, the rats are
housed individually in cages without bedding or "enrichment". The
rats are kept off of the cage floor by a wire screen. Pre-weighed
food cups containing standard rat chow and clay pellets are placed
in the cage. The clay pellets, obtainable from Languna Clay Co,
City of Industry, Calif., USA, are the same size and shape as the
food pellets. The rats are acclimated to the clay for 72 hours,
during which time the cups and food and clay debris from the cage
are weighed daily on an electronic balance capable of measuring to
the nearest 0.1 grams. By the end of the 72 hour acclimation period
the rats generally show no interest in the clay pellets.
[0366] At the end of 72 hours the rats are placed in clean cages
and the food cups weighed. Rats that are still consuming clay
regularly are removed from the study. Immediately prior to the dark
cycle (the time when the animals are active and should be eating)
the animals are split into treatment groups and dosed orally with a
dose of a compound or salt of the invention (different doses for
different treatment groups) or with vehicle alone, at a dose volume
of ca. 2 ml/kg. In this oral dosing, the compound/salt can for
example be in the form of a suspension in about 0.5%
methylcellulose (obtainable Sigma-Aldrich, St. Louis, Mo., USA) in
water. The food and clay cups and cage debris are weighed the
following day and the total clay and food consumed that night by
each individual animal is calculated.
[0367] A dose response is calculated by first converting the data
into quantal response, where animals are either positive or
negative for the pica response. A rat is "pica positive" if it
consumes greater than or equal to 0.3 grams of clay over the mean
of its control group. The D50 value is usually calculated using
logistic regression performed by the Statistica software
statistical package. A Pica Response ED50 value in mg per kg of
body weight can then be calculated.
[0368] The Pica Response ED50 value can be compared to the
neutrophilia-inhibition ED50 values for the same compound
administered orally to the rat (measurable by In Vivo Assay 1
above), so that a Therapeutic Index (TI) in rats can be calculated
thus:
Rat Therapeutic index ( T I ) ( 50 / 50 ) = Pica Response ED 50
value rat neutrophilia - inhibition ED 50 value ##EQU00001##
[0369] In general, the Therapeutic Index (TI) calculated this way
is often substantially different to, and for example can often be
substantially higher than, the TI (D20/D50) calculated in the
ferret (see In vivo Assay 4 below).
[0370] Alternatively, e.g. for a simpler test, the In Vivo Assay 2
(pica) can use only a single oral dose of the test compound (e.g.
10 mg/kg orally).
[0371] Literature: [0372] Beavo J A, Contini, M., Heaslip, R. J.
Multiple cyclic nucleotide phosphodiesterases. Mol. Pharmacol.
1994; 46:399-405. [0373] Spond J, Chapman R, Fine J, Jones H,
Kreutner W, Kung T T, Minnicozzi M. Comparison of PDE 4 inhibitors,
Rolipram and SB 207499 (Ariflo.TM.), in a rat model of pulmonary
neutrophilia. Pulmonary Pharmacology and Therapeudtics. 2001;
14:157-164. [0374] Takeda N, Hasegawa S, Morita M, and Matsunaga T.
Pica in rats is analogous to emesis: an animal model in emesis
research. Pharmacology, Biochemistry and Behavior. 1991;
45:817-821. [0375] Takeda N, Hasegawa S, Morita M, Horii A, Uno A,
Yamatodani A and Matsunaga T. Neuropharmacological mechanisms of
emesis. I. Effects of antiemetic drugs on motion- and
apomorphine-induced pica in rats. Meth Find Exp Clin Pharmacol.
1995; 17(9) 589-596. [0376] Takeda N, Hasegawa S, Morita M, Horii
A, Uno A, Yamatodani A and Matsunaga T. Neuropharmacological
mechanisms of emesis. II. Effects of antiemetic drugs on
cisplatin-induced pica in rats. Meth Find Exp Clin Pharmacol. 1995;
17(9) 647-652.
In Vivo Assay 3. LPS Induced Pulmonary Neutrophilia in Rats: Effect
of Intratracheally Administered PDE4 Inhibitors
[0377] This assay is an animal model of inflammation in the
lung--specifically neutrophilia induced by lipopolysaccharide
(LPS)--and allows the study of putative inhibition of such
neutrophilia (anti-inflammatory effect) by intratracheally (i.t.)
administered PDE4 inhibitors. The PDE4 inhibitors are preferably in
dry powder or wet suspension form. I.t. administration is one model
of inhaled administration, allowing topical delivery to the
lung.
[0378] Animals: Male CD (Sprague Dawley Derived) rats supplied by
Charles River, Raleigh, N.C., USA or Charles River, United Kingdom
are housed in groups of 5 rats per cage, acclimatised after
delivery for at least 5 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.
[0379] Device for dry powder administration: Disposable 3-way tap
between dosing needle and syringe. The intratracheal dosing device
(a 3-way sterile tap, Vycon 876.00; or Penn Century dry powder
insufflator, DP-4) is weighed, the drug blend or inhalation grade
lactose (vehicle control) is then added to the tap, the tap is
closed to prevent loss of drug, and the tap is re-weighed to
determine the weight of drug in the tap. After dosing, the tap is
weighed again to determine the weight of drug that had left the
tap. The needle, a Sigma Z21934-7 syringe needle 19-gauge 152 mm (6
inches) long with luer hub, is cut by engineering to approximately
132 mm (5.2 inches), a blunt end is made to prevent them damaging
the rat's trachea, and the needle is weighed prior to and after
drug delivery to confirm that no drug is retained in the needles
after dosing.
[0380] Device for wet suspension administration: This is similar to
the above but 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.
[0381] Drugs and Materials: Lipopolysaccharide (LPS)
(Serotype:0127:B8) is dissolved in phosphate-buffered saline (PBS).
PDE4 inhibitors are preferably used in size-reduced (e.g.
micronised) form, for example according to the Micronisation
Example given herein.
[0382] For dry powder administration of the drug, the Dry Powder
Formulation Example given herein, comprising drug and
inhalation-grade lactose, can optionally be used. One suitable
inhalation-grade lactose that can be used has 10% fines (10% of
material under 15 um (15 micron) particle size measured by Malvern
particle size).
[0383] Wet suspensions of the drug (aqueous) can be prepared by
adding the required volume of vehicle to the drug; the vehicle used
can for example be saline alone or a mixture of saline/tween (e.g.
0.2% tween 80). The wet suspension is usually sonicated for ca. 10
minutes prior to use.
[0384] 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.sup.-1) and oxygen (1
litre.minute.sup.-1). 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 wet suspension and dry powder
administration of PDE4 inhibitors as follows:
[0385] Dosing with a Wet suspension: 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. 10 seconds) dosed into the trachea using a syringe
attached to the dosing needle.
[0386] Dosing with a Dry Powder: The intratracheal dosing device (a
three-way sterile tap device, Vycon 876.00; or Penn Century dry
powder insufflator, DP-4) and needle are inserted into the rat
trachea up to a pre-determined point established to be located
approximately 1 cm above the primary bifurcation. Another operator
holds the needle at the specified position whilst 2.times.4 ml of
air (using 3-way tap device) is delivered through the three-way tap
by depressing the syringes (ideally coinciding with the animal
inspiring), aiming to expel the entire drug quantity from the tap.
(Alternatively, 2.times.3 ml of air is delivered using Penn Century
dry powder insufflator device.). After dosing, the needle and tap
or device are removed from the airway and the tap closed off to
prevent any retained drug leaving the tap.
[0387] After dosing with either wet suspension or dry powder, the
animals are then 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.
[0388] 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.g.ml.sup.-1=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.
[0389] [In an alternative embodiment, the rats can be exposed to
LPS less than 2 hours (e.g. about 30 minutes) after i.t. dosing. In
another alternative embodiment, the rats can be exposed to LPS more
than 2 hours (e.g. ca. 4 to ca. 24 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 not
essential).]
[0390] 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
ravaged (washed out) with 3.times.5 mls of heparinised (25
units.ml.sup.-1) phosphate buffered saline (PBS).
[0391] 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.
[0392] By varying the dose of the PDE4 inhibitor used in the dosing
step (e.g. 0.2 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.
In Vivo Assay 4. Evaluation of Therapeutic Index of
Orally-Administered PDE 4 Inhibitors in the Conscious Ferret
1.1 Materials
[0393] The following materials can be used for these studies:
[0394] PDE4 inhibitors are prepared for oral (p.o.) administration
by dissolving in a fixed volume (ca. 1 ml) of acetone and then
adding cremophor to ca. 20% of the final volume. Acetone is
evaporated by directing a flow of nitrogen gas onto the solution.
Once the acetone is removed, the solution is made up to final
volume with distilled water. LPS is dissolved in phosphate buffered
saline.
1.2 Animals
[0395] Male ferrets (Mustela Pulorius Furo, weighing 1-2 kg) are
transported and allowed to acclimatise for not less than 7 days.
The diet comprises SDS diet C pelleted food given ad lib with
Whiskers.TM. cat food given 3 times per week. The animals are
supplied with pasteurised animal grade drinking water changed
daily.
1.3 Experimental Protocol(s)
[0396] 1.3.1 Dosing with PDE4 Inhibitors
[0397] PDE4 inhibitors are administered orally (p.o.), using a dose
volume of ca. 1 ml/g. Ferrets are fasted overnight but allowed free
access to water. The animals are orally dosed with vehicle or PDE 4
inhibitor using a ca. 15 cm dosing needle that is passed down the
back of the throat into the oesophagus. After dosing, the animals
are returned to holding cages fitted with perspex doors to allow
observation, and given free access to water. The animals are
constantly observed and any emetic episodes (retching and vomiting)
or behavioural changes are recorded. The animals are allowed access
to food ca. 60-90 minutes after p.o. dosing.
1.3.2 Exposure to LPS
[0398] About thirty minutes after oral dosing with compound or
vehicle control, the ferrets are placed into sealed perspex
containers and exposed to an aerosol of LPS (ca. 30 .mu.g/ml ca. 30
ug/ml) for ca. 10 minutes. Aerosols of LPS are generated by a
nebuliser (DeVilbiss, USA) and this is directed into the perspex
exposure chamber. Following a 10-minute exposure period, the
animals are returned to the holding cages and allowed free access
to water, and at a later stage, food. General observation of the
animals continues for a period of at least 2.5 hours post oral
dosing. All emetic episodes and behavioural changes are
recorded.
1.3.3 Bronchoalveolar Lavage and Cell Counts
[0399] About six hours after LPS exposure the animals are killed by
overdose of sodium pentobarbitone administered intraperitoneally.
The trachea is then cannulated with polypropylene tubing and the
lungs lavaged twice with ca. 20 ml heparinised (10 units/ml)
phosphate buffered saline (PBS). The bronchoalveolar lavage (BAL)
samples are centrifuged at ca. 1300 rpm for ca. 7 minutes. The
supernatant is removed and the resulting cell pellet re-suspended
in ca. 1 ml PBS. A cell smear of re-suspended fluid is prepared and
stained with Leishmans stain to allow differential cell counting. A
total cell count is made using the remaining re-suspended sample.
From this, the total number of neutrophils in the BAL sample is
determined.
1.3.4 Pharmacodynamic Readouts
[0400] The following parameters are recorded:
a) % inhibition of LPS-induced pulmonary neutrophilia to determine
the dose of PDE4 inhibitor which gives 50% inhibition (D50). b)
Emetic episodes--the number of vomits and retches are counted to
determine the dose of PDE4 inhibitor that gives a 20% incidence of
emesis (D20). c) A therapeutic index (TI), using this assay, is
then calculated for each PDE4 inhibitor using the following
equation:
Ferret Therapeutic index ( T I ) ( D 20 / D 50 ) = D 20 incidence
of emesis in ferret D 50 inhibition of neutrophilia in ferret
##EQU00002##
[0401] It is noted that the Ferret Therapeutic index (TI) (D20/D50)
calculated using this in vivo Assay 4 is often substantially
different to, and for example is often substantially lower than,
the Rat TI (50/50) calculated using the rat oral inflammation and
pica feeding Assays 1+2.
[0402] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
EXAMPLES
[0403] 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. In this section, "Intermediates"
represent syntheses of intermediate compounds intended for use in
the synthesis of the "Examples". "Examples" are generally exemplary
compounds or salts of the invention, for example a compound of
formula (I) or a salt thereof. The "Composition Examples" are
non-limiting illustrations of the pharmaceutical compositions of
the invention.
Abbreviations Used Herein:
[0404] DCM dichloromethane DIPEA diisopropylethyl amine
(.sup.iPr.sub.2NEt) DMF dimethyl formamide DMSO dimethyl sulfoxide
EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
EtOAc ethyl acetate EtOH ethanol h hours HATU
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate HCl hydrogen chloride or hydrochloric acid HOBT
hydroxybenzotriazole=1-hydroxybenzotriazole M molarity, or moles
per litre MeCN acetonitrile MeOH methanol NaHCO.sub.3 sodium
bicarbonate NaOH sodium hydroxide Na.sub.2SO.sub.4 sodium sulfate
KOH potassium hydroxide THF tetrahydrofuran HPLC high performance
liquid chromatography SPE solid phase extraction NMR nuclear
magnetic resonance (in which: s=singlet, d=doublet, t=triplet,
q=quartet, dd=doublet of doublets, m=multiplet, H=no. of protons)
LCMS liquid chromatography/mass spectroscopy TLC thin layer
chromatography h hours .sup.TRET retention time (generally from
LCMS)
[0405] Room (ambient) temperature this is usually in the range of
about 20 to about 25.degree. C.
General Experimental Details
Machine Methods Generally Used Herein:
LCMS (Liquid Chromatography/Mass Spectroscopy)
[0406] Waters ZQ mass spectrometer operating in positive ion
electrospray mode, mass range 100-1000 amu.
[0407] UV wavelength: 215-330 nM
[0408] Column: 3.3 cm.times.4.6 mm ID, 3 .mu.m ABZ+PLUS
[0409] Flow Rate: 3 ml/min
[0410] Injection Volume: 5 .mu.l
[0411] Solvent A: 95% acetonitrile+0.05% formic acid
[0412] Solvent B: 0.1% formic acid+10 mMolar ammonium acetate
[0413] Gradient: 0% A/0.7 min, 0-100% A/3.5 min, 100% A/1.1 min,
100-0% A/0.2 min
[0414] It should be noted that retention times (T.sub.RET) quoted
herein may vary slightly (+/-0.1 min.) when samples are run on
different Waters machines, even when the same type of column and
identical flow rates, injection volumes, solvents and gradients are
used.
LCMS (Liquid Chromatography/Mass Spectroscopy)
(For Intermediates 2a, 3a, 4a and 5a, and Example 2B Only)
[0415] Agilent 1100 mass spectrometer operating in positive ion
electrospray mode, mass range 100-700 amu.
[0416] UV wavelength: 214-254 nM
[0417] Column: 2.1 cm.times.50 mm ID, 5 .mu.m Zorbax
[0418] Flow Rate: 1 ml/min
[0419] Injection Volume: 1 .mu.l
[0420] Solvent A: water+0.02% trifluoroacetic acid
[0421] Solvent B: acetonitrile+0.018% trifluoroacetic acid
[0422] Gradient: 10-80% A/3.0 min, 80% A/1.2 min, 80-10% A/1.0
min
Mass Directed Autoprep HPLC
[0423] Prep column: a Supelcosil ABZplus (10 cm.times.2.12 cm)
(usually 10 cm.times.2.12 cm.times.5 .mu.m).
[0424] UV wavelength: 200-320 nM
[0425] Flow: 20 ml/min
[0426] Injection Volume: 1 ml; or more preferably 0.5 ml
[0427] Solvent A: 0.1% formic acid
[0428] Solvent B: 95% acetonitrile+5% formic acid; or more usually
99.95% acetonitrile+0.05% formic acid
[0429] Gradient: 100% A/1 min, 100-80% A/9 min, 80-1% A/3.5 min, 1%
A/1.4 min, 1-100% A/0.1 min
Autoprep Gilson Reverse-Phase HPLC (for Example 2B)
[0430] Prep column: YMC ODS-A (50 mm.times.50 mm)
[0431] UV wavelength: 215-254 nM
[0432] Flow: 70 ml/min
[0433] Injection Volume: 3 ml
[0434] Solvent A: water
[0435] Solvent B: acetonitrile
[0436] Gradient: 35-95% B/5 min
Chiral Columns for Chromatographic Purification
[0437] ChiralPak AS columns can be obtained from:
[0438] Chiral Technologies Europe Sarl, Illkirch, France
(Telephone: 0033(0)388795200; (cte@chiral.fr; www.chiral.fr).
Intermediates and Examples
[0439] 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 chemicals in general, are as follows: [0440]
Aldrich (catalogue name), Sigma-Aldrich Company Ltd., Dorset,
United Kingdom, telephone: +44 1202 733114; Fax: +44 1202 715460;
ukcustsv@eurnotes.sial.com; or [0441] Aldrich (catalogue name),
Sigma-Aldrich Corp., P.O. Box 14508, St. Louis, Mo. 63178-9916,
USA; telephone: 314-771-5765; fax: 314-771-5757; custserv@sial.com;
or [0442] Aldrich (catalogue name), Sigma-Aldrich Chemie GmbH,
Munich, Germany; telephone: +49 89 6513 0; Fax: +49 89 6513 1169;
deorders@eurnotes.sial.com. [0443] Apin Chemicals Ltd; 3D Milton
Park, Abingdon, Oxon, OX14 4RU, United Kingdom [0444] AstaTech
Inc., 8301 Torresdale Ave., 19C, Philadelphia, Pa. 19136, USA
[0445] Fluka Chemie AG, Industriestrasse 25, P.O. Box 260, CH-9471
Buchs, Switzerland [0446] Interchim Intermediates (catalogue name),
Interchim, 213 Avenue Kennedy, BP 1140, Montlucon, Cedex, 03103,
France [0447] Lancaster Synthesis Ltd., Newgate, White Lund,
Morecambe, Lancashire LA3 3DY, United Kingdom [0448] Matrix
Scientific, P.O. Box 25067, Columbia, S.C. 29224-5067, USA [0449]
Maybridge Chemical Company Ltd., Trevillett, Tintagel, Cornwall
PL34 OHW, United Kingdom [0450] Trans World Chemicals, Inc., 14674
Southlawn Lane, Rockville, Md. 20850, USA
TABLE-US-00002 [0450] Table of Intermediates Int No Name 1 Ethyl
4-chloro-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate 2 Ethyl
4-[(1-{[(1,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]-1-
ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate 3 Ethyl
1-ethyl-4-(4-piperidinylamino)-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate hydrochloride 4 Ethyl
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-
- b]pyridine-5-carboxylate 5
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid 6
4-chloro-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid 7
4-chloro-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carbonyl chloride 8
4-chloro-N-[(2,4-dimethylphenyl)methyl]-1-ethyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide 9
4-chloro-N-[(3,4-dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide 10
4-chloro-1-ethyl-N-{[4-(methyloxy)phenyl]methyl}-1H-pyrazolo[3,4-
b]pyridine-5-carboxamide 11 1,1-dimethylethyl
[1-(aminocarbonyl)-4-piperidinyl]carbamate 12
4-amino-1-piperidinecarboxamide hydrochloride 13 1,1-dimethylethyl
[4-(aminocarbonyl)cyclohexyl]carbamate 15
4-aminocyclohexanecarboxamide hydrochloride 17 1,1-dimethylethyl
[cis-4-(aminocarbonyl)cyclohexyl]-carbamate 18 1,1-dimethylethyl
[trans-4-(aminocarbonyl)cyclohexyl]-carbamate 19
cis-4-aminocyclohexanecarboxamide hydrochloride 20
trans-4-aminocyclohexanecarboxamide hydrochloride 21 ethyl
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate 22 ethyl
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate 23
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid 24
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylic acid 25 ethyl
4-[((3S)-1-{[(1,1-dimethylethyl)oxy]carbonyl}-3-pyrrolidinyl)amin-
o]- 1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate 26 ethyl
4-[((3R)-1-{[(1,1-dimethylethyl)oxy]carbonyl}-3-pyrrolidinyl)amin-
o]- 1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate 27 ethyl
1-ethyl-4-[(3S)-3-pyrrolidinylamino]-1H-pyrazolo[3,4-b]pyridine-5-
- carboxylate hydrochloride 28 ethyl
1-ethyl-4-[(3R)-3-pyrrolidinylamino]-1H-pyrazolo[3,4-b]pyridine-5-
- carboxylate hydrochloride 29 ethyl
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-1-ethyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate 30 ethyl
4-{[(3R)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-1-ethyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate 31
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-1-ethyl-1H-pyrazolo[3,-
4- b]pyridine-5-carboxylic acid 32
4-{[(3R)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-1-ethyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylic acid 33
4-[(1-{[(1,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]-1-ethyl-1-
H- pyrazolo[3,4-b]pyridine-5-carboxylic acid 34
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2-bromophenyl)methyl]-
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 35
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2-bromo-4-
methylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
Intermediate 1
Ethyl 4-chloro-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
[0451] Intermediate 1 is thought to be capable of being prepared
from commercially available 5-amino-1-ethyl pyrazole as described
by G. Yu et. al. in J. Med. Chem., 2001, 44, 1025-1027:
##STR00041##
[0452] One specific method of preparing Intermediate 1 is as
follows:
Intermediate 1
Ethyl 4-chloro-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
##STR00042##
[0454] A mixture of 5-amino-1-ethyl pyrazole (806 g) (e.g.
commercially available from Aldrich) and diethyl
ethoxymethylenemalonate (1621 ml) (e.g. commercially available from
Aldrich) was stirred and heated at 160.degree. C. under nitrogen,
in a 5 litre flask fitted with a Dean-Stark apparatus, for 1.5 h.
Ethanol that distilled out of the reaction mixture (320 ml) was
collected in the Dean-Stark apparatus. The reaction mixture was
stirred and heated at 160.degree. C., under nitrogen, for a further
6 h, cooled to room temperature and divided into two batches (1200
ml+1000 ml: "Batch 1" and "Batch 2"). The first (1200 ml) batch
("Batch 1") was divided into two roughly equal portions. Phosphorus
oxychloride (1.85 litres) was added to each portion. The reaction
mixtures were then heated at reflux in two 51 flasks for 13 h.
Excess phosphorus oxychloride was distilled from both flasks under
reduced pressure. The residues were cooled to room temperature,
then the contents of both flasks were poured slowly onto one
portion (10 kg) of crushed ice. The mixture was stirred for 15 min
and then extracted with diethyl ether (3.times.2.5 litres). The
combined organics were washed with water (2 litres) and brine
(2.times.2 litres), then dried over Na.sub.2SO.sub.4. Evaporation
of the solvent afforded the crude Intermediate 1 as a brown oil
(865 g) which solidified immediately on cooling. An identical
procedure was used to prepare a further 710 g of crude Intermediate
1 as a solid from "Batch 2" using 3.1 litres of phosphorus
oxychloride; i.e. a total of 1575 g of crude Intermediate 1 was
isolated as a solid. This solid (430 g) was dissolved in hexane
(4.3 litres, i.e. 10 vols.) by heating to 50.degree. C. with
stirring. Activated charcoal (64.5 g) was added. The mixture was
stirred at 50.degree. C. for 1.0 h, then filtered through a celite
bed. The celite bed was washed with hexane (2.times.430 ml). The
combined filtrate and the washings were concentrated to about 950
ml and left to stand at 10-15.degree. C. overnight. The resultant
suspension was filtered. The residual solid was washed with chilled
hexane (3.times.215 ml slurry wash, plus 2.times.400 ml
displacement wash) and dried to give Intermediate 1 (280 g) as a
pale yellow solid. The combined mother liquor and the washings were
concentrated to about 300 ml, then cooled and left to stand at
10-15.degree. C. overnight to afford an additional 30 g of
Intermediate 1. LCMS showed MH.sup.+=254; T.sub.RET=3.09 min.
Intermediate 2
Ethyl
4-[(1-{[(1,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]-1-ethy-
l-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
##STR00043##
[0456] A solution of Intermediate 1 (0.5 g, 2 mmol),
1,1-dimethylethyl 4-amino-1-piperidinecarboxylate (0.59 g, 2.9
mmol) (e.g. available from AstaTech) and DIPEA (0.87 ml, 5 mmol,
2.5 equivalents) in MeCN (15 ml) was heated at reflux for 18 h. The
reaction mixture was cooled. The solvent was removed under reduced
pressure and the residue was partitioned between DCM (50 ml) and
saturated NaHCO.sub.3 solution (40 ml). The organic fraction was
collected through a hydrophobic frit and concentrated under reduced
pressure. The residue was purified by passing through a 100 g
silica cartridge, using a gradient of 0% to 100% EtOAc in
cyclohexane as the eluent, and the fractions containing the product
were concentrated under reduced pressure to yield Intermediate 2 as
a solid (0.74 g). LCMS showed MH.sup.+=418; T.sub.RET=3.43 min.
[0457] Alternative optional synthesis: A solution of Intermediate 1
(2.3 g) and 1,1-dimethylethyl 4-amino-1-piperidinecarboxylate (2 g)
in MeCN (50 ml) and DIPEA (8.6 ml) (optionally also with about
1.5-2 ml EtOH) is heated, for example at 85.degree. C. or
90.degree. C., for 16 h. The solvents are removed under reduced
pressure and the residue is partitioned between DCM (e.g. about 65
ml) and water (e.g. about 30 ml). The organic fraction is collected
through a hydrophobic frit and the solvents are removed under
reduced pressure to yield Intermediate 2.
Intermediate 2A
Ethyl
4-[(1-{[(1,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]-1-ethy-
l-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
##STR00044##
[0459] A solution of Intermediate 1 (25 g) in MeCN (565 ml) was
treated with solid 1,1-dimethylethyl
4-amino-1-piperidinecarboxylate (21.7 g) and DIPEA (93.4 ml, 69.3
g). The reaction mixture was heated at 90.degree. C. for 16 h.
After cooling the reaction mixture, the solvents were removed under
reduced pressure and the residue was partitioned between DCM (1100
ml) and water (800 ml). The organic fraction was dried
(MgSO.sub.4), was filtered and the solvents were removed under
reduced pressure. The residue was subject to flash column
chromatography (3:1 hexane/EtOAc) to yield Intermediate 2A as a
yellow solid (39.54 g). LCMS showed MH.sup.+=418; T.sub.RET=3.13
min.
[0460] .sup.1H NMR (400 MHz, chloroform-d) .delta. ppm 9.44 (d,
J=7.83 Hz, 1H), 8.89 (s, 1H), 7.94 (s, 1H), 4.49 (q, J=7.33 Hz,
2H), 4.35 (q, J=7.16 Hz, 2H), 3.95-4.11 (m, 3H), 3.18 (t, J=10.86
Hz, 2H), 2.10-2.20 (br d, 2H), 1.62-1.73 (m, 2H), 1.47-1.53 (m,
12H), 1.41 (t, J=7.07 Hz, 3H).
Intermediate 3
Ethyl
1-ethyl-4-(4-piperidinylamino)-1H-pyrazolo[3,4-b]pyridine-5-carboxyl-
ate hydrochloride
##STR00045##
[0462] Intermediate 2 (4.1 g) was treated with 4.0M hydrogen
chloride in 1,4-dioxane (30 ml) and the reaction mixture was
stirred at 22.degree. C. for 1 h. The solvents were removed to give
Intermediate 3 as a white solid (4.0 g). LCMS showed MH.sup.+=318;
T.sub.RET=2.1 min.
Intermediate 3A
Ethyl
1-ethyl-4-(4-piperidinylamino)-1H-pyrazolo[3,4-b]pyridine-5-carboxyl-
ate hydrochloride
##STR00046##
[0464] Intermediate 2A (39.54 g) was treated with 4M HCl in
1,4-dioxane (300 ml) and the reaction mixture was stirred at room
temperature for 1 h. The solvents were removed and the residue
subject to vacuum overnight, to give Intermediate 3A as a white
solid (34.21 g, some dioxane still present; from NMR theoretically
33.5 g present excluding dioxane). LCMS showed MH.sup.+=318;
T.sub.RET=1.90 min.
[0465] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 9.33 (d,
J=8.08 Hz, 1H), 9.21 (br s, 1H), 9.10 (br s, 1H), 8.74 (s, 1H),
8.44 (s, 1H), 4.43 (q, J=7.20 Hz, 2H), 4.32 (q, J=7.09 Hz, 2H),
3.20-3.35 (m, 4H), 2.23 (d, J=11.62 Hz, 2H), 1.76-1.87 (m, 2H),
1.38 (q, J=7.20 Hz, 3H), 1.34 (q, J=7.09 Hz, 3H).
Intermediate 4
Ethyl
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-b-
]pyridine-5-carboxylate
##STR00047##
[0467] A suspension of Intermediate 3 (4 g) in THF (100 ml) was
treated with DIPEA (2.6 ml) followed by trimethylsilyl isocyanate
(1.99 ml, 1.7 g) and the solution was stirred at 22.degree. C. for
2 h. The volatile solvents were removed under reduced pressure and
the residue was partitioned between DCM (e.g. 50 ml) and water
(e.g. 25 ml). The organic and aqueous layers were separated. The
aqueous phase was extracted with DCM (e.g. 50 ml). The organic
layers were combined, separated from water by passing through a
hydrophobic frit and concentrated under reduced pressure to yield
Intermediate 4 as a solid (4 g). LCMS showed MH.sup.+=361;
T.sub.RET=2.45 min.
Intermediate 4A
Ethyl
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-b-
]pyridine-5-carboxylate
##STR00048##
[0469] A suspension of Intermediate 3A (theoretical 33.5 g
excluding dioxane present) in THF (880 ml) was treated with
trimethylsilyl isocyanate (17.5 ml, 14.9 g) followed by DIPEA (22.6
ml, 16.8 g) and the solution was stirred at room temperature for 5
h. LCMS indicated formation of only a small amount of product, so
more DIPEA (22.6 ml) was added and the mixture was stirred for an
additional 24 h. The THF was removed under reduced pressure. The
residue was dissolved/diluted in DCM (1000 ml), was washed with
brine (200 ml), was dried (MgSO.sub.4) and was evaporated under
reduced pressure to give a residue which from LCMS appeared to
contain a small amount of product.
[0470] The residue was dissolved in DCM (1000 ml), was treated with
trimethylsilyl isocyanate (17.5 ml) followed by DIPEA (22.6 ml) and
the solution was stirred at room temperature for 48 h.
Trimethylsilyl isocyanate (17.5 ml, 14.9 g) followed by DIPEA (22.6
ml, 16.8 g) were again added and the solution was stirred at room
temperature for an additional 48 h. The volatile solvents were
removed under reduced pressure and the residue was dissolved in DCM
(600 ml) and washed with brine (2.times.200 ml). The organic layer
was dried (MgSO.sub.4), was filtered and was concentrated under
reduced pressure. The solid residue was stirred in diethyl ether
(1000 ml) for 2 h until pulverized and was collected by filtration
to yield Intermediate 4A as a light-yellow solid (24.56 g). LCMS
showed MH.sup.+=361; T.sub.RET=2.19 min. m.p.=126-127.degree.
C.
[0471] .sup.1H NMR (400 MHz, chloroform-d) .delta. ppm 9.48 (d,
J=7.83 Hz, 1H), 8.90 (s, 1H), 7.95 (s, 1H), 4.65 (s, 2H), 4.50 (q,
J=7.24 Hz, 2H), 4.35 (q, J=7.12 Hz, 2H), 4.1-4.2 (m, 1H), 3.87-3.93
(m, 2H), 3.26 (ddd, J=13.58, 10.17, 3.03 Hz, 2H), 2.16-2.23 (m,
2H), 1.70-1.80 (m, 2H), 1.51 (t, J=7.24 Hz, 3H), 1.41 (t, J=7.12
Hz, 3H).
Intermediate 4B
Ethyl
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-b-
]pyridine-5-carboxylate
##STR00049##
[0473] A suspension of ethyl
1-ethyl-4-(4-piperidinylamino)-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
hydrochloride (13 g, 36.77 mmol) (e.g. Intermediate 3) in DCM (300
ml) was treated with trimethylsilyl isocyanate (5 g) followed by
DIPEA (10 ml) and was stirred at 22.degree. C. for 3 h. The mixture
was diluted with water, the organic layer was separated from the
aqueous layer by passing through a hydrophobic frit, and the
solvents were removed from the organic layer to give Intermediate
4B (10 g). LCMS showed MH.sup.+=361; T.sub.RET=2.6 min.
Intermediate 5
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-b]pyrid-
ine-5-carboxylic acid
##STR00050##
[0475] A solution of Intermediate 4 (4 g) in EtOH (50 ml) was
treated with a solution of NaOH (1.77 g) in water (20 ml) and the
reaction mixture was heated at 60.degree. C. for 5 h. The solvents
were removed and the residue was dissolved in water (ca. 8 ml), the
pH was adjusted to 3 (2M HCl) and the resultant precipitate was
collected by filtration and dried at 60.degree. C. under vacuum.
LCMS indicated that partial hydrolysis of the urea portion had
occurred. Therefore, the precipitate from the reaction was
dissolved in EtOH (100 ml), the solution was treated with
trimethylsilyl isocyanate (3 ml) and DIPEA (10 ml) and then stirred
at room temperature overnight. The solvents were removed, water was
added to the residue, the pH was adjusted to 3 (2M HCl), the
mixture was cooled to 0.degree. C. for 30 minutes, and the
resultant precipitate was collected by filtration and dried to give
Intermediate 5 as a white solid (2.66 g). LCMS showed MH.sup.+=333;
T.sub.RET=2.0 min.
Intermediate 5A
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-b]pyrid-
ine-5-carboxylic acid
##STR00051##
[0477] A solution of Intermediate 4A (24.26 g) in EtOH (360 ml) and
water (120 ml) was treated with lithium hydroxide monohydrate (11
g) and the reaction mixture was stirred at room temperature
overnight. The EtOH was removed under reduced pressure. Aqueous 1N
HCl solution (300 ml) was added to the residue and the resultant
precipitate was cooled in an ice bath for 1 h, was collected by
filtration, washed with cold water, dried in a vacuum dessicator
overnight, and then further dried in a vacuum oven under reduced
pressure at 60.degree. C. overnight to give Intermediate 5A as a
white solid (22.4 g). LCMS showed MH.sup.+=333; T.sub.RET=1.23 min.
m.p.=204-206.degree. C.
[0478] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 9.50 (d,
J=8.08 Hz, 1H), 8.69 (s, 1H), 8.25 (s, 1H), 6.04 (br s, 2H), 4.37
(q, J=7.20 Hz, 2H), 4.14-4.24 (m, 1H), 3.80 (d, J=13.64 Hz, 2H),
3.15 (t, J=10.86 Hz, 2H), 1.96-2.03 (m, 2H), 1.39-1.47 (m, 2H),
1.37 (t, J=7.20 Hz, 3H).
Intermediate 6
4-chloro-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid
##STR00052##
[0480] A solution of Intermediate 1 (20 g, 78.8 mmol) in
1,4-dioxane (100 ml) was treated with a solution of KOH (18 g of
pellets) in water (30 ml) and the reaction mixture was stirred at
room temperature for 24 h. The solvent was evaporated and the
residue was acidified to pH3 (2M hydrochloric acid). The resultant
white precipitate was collected by filtration and dried under
vacuum overnight to give Intermediate 6 as a white solid (16.9 g).
LCMS showed MH.sup.+=226; T.sub.RET=2.61 min.
Intermediate 7
4-chloro-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carbonyl chloride
##STR00053##
[0482] A solution of Intermediate 6 (17.8 g, 78.8 mmol) in thionyl
chloride (SOCl.sub.2, 100 ml) was heated at reflux under nitrogen
for 3.5 h. The solution was allowed to cool to room temperature
overnight. The thionyl chloride was removed in vacuo, any remaining
thionyl chloride was removed in vacuo by azeotropic distillation
with toluene (ca. 30 ml), and this was repeated to remove the
thionyl chloride, to give Intermediate 7 as a beige solid (16.86
g). LCMS (in MeOH, hence methyl ester) showed MH.sup.+=240
(MH.sup.+ for methyl ester); T.sub.RET=2.88 min.
Intermediate 8
4-chloro-N-[(2,4-dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-
-5-carboxamide
##STR00054##
[0484] A solution of Intermediate 7 (6 g, 24.6 mmol) and DIPEA
(3.17 g, 24.6 mmol) in THF (60 ml) was stirred for 20 min. A
one-third aliquot (20 ml) of the resultant solution was added to
2,4-dimethyl-benzylamine (1.1 .mu.g, 8.2 mmol) (e.g. available from
Trans World Chemicals). The reaction mixture was stirred under
nitrogen at room temperature for 24 h. The solvents was removed in
vacuo and the residue was partitioned between DCM (50 ml) and 5%
citric acid solution (50 ml). The organic layer was separated using
a hydrophobic frit, washed with 0.5M NaHCO.sub.3 solution (50 ml),
dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo to
give Intermediate 8 as a white powder (1.61 g). LCMS showed
MH.sup.+=343; T.sub.RET=3.22 min.
[0485] The following Intermediate 10 can be prepared in a similar
manner from Intermediate 7 and the appropriate amine reagent:
TABLE-US-00003 ##STR00055## ##STR00056## PossibleSourceof
aminereagent MH.sup.+ion T.sub.RET(min) Intermediate10 ##STR00057##
Aldrich 345 2.9
Intermediate 9
4-chloro-N-[(3,4-dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-
-5-carboxamide
##STR00058##
[0487] A solution of Intermediate 7 (6 g) and DIPEA (3.17 g) in THF
(60 ml) was stirred for 20 min. A one-third aliquot (20 ml) of the
resultant solution was added to 3,4-dimethyl-benzylamine (1.11 g)
(e.g. available from Trans World Chemicals). The reaction mixture
was stirred under nitrogen at room temperature for 24 h, more THF
(20 ml) being added to aid dissolution of the reactants. The
solvent was removed in vacuo and the residue was partitioned
between DCM (50 ml) and 5% citric acid solution (50 ml). The
organic layer was separated using a hydrophobic frit, washed with
0.5M NaHCO.sub.3 solution (50 ml), dried (Na.sub.2SO.sub.4),
filtered and concentrated in vacuo to give Intermediate 8 as a
white powder (2.39 g). LCMS showed MH.sup.+=343; T.sub.RET=3.34
min.
Intermediate 11
1,1-dimethylethyl [1-(aminocarbonyl)-4-piperidinyl]carbamate
##STR00059##
[0489] A solution of 1,1-dimethylethyl 4-piperidinylcarbamate (0.35
g) (e.g. AstaTech and/or Aldrich) in DCM (10 ml) was treated with
trimethylsilyl isocyanate (1.1 ml, 0.86 g). The reaction mixture
was stirred at room temperature for 8 h and then left to stand at
room temperature over the weekend. The mixture was diluted with DCM
(10 ml) and washed with saturated NaHCO.sub.3 solution (20 ml). The
organic phase was separated and collected through a hydrophobic
frit. The aqueous phase was extracted with DCM. The organics were
combined and evaporated to dryness to give Intermediate 11 as a
white foam (0.29 g). .sup.1H NMR (400 MHz in CDCl.sub.3, 27.degree.
C., 8 ppm) 4.45 (br. s, 3H), 3.90 (d, 2H), 3.65 (br. m, 1H),
2.9-3.0 (dt, 2H), 1.95-2.0 (br. dd, 2H), 1.45 (s, 9H), 1.3-1.4 (dq,
2H).
Intermediate 12
4-amino-1-piperidinecarboxamide hydrochloride
##STR00060##
[0491] Intermediate 11 (0.29 g) was treated with a 4M solution of
hydrogen chloride in 1,4-dioxane (5 ml), and was stirred at room
temperature for 4 h. The reaction mixture was evaporated to dryness
and co-evaporated with DCM to give a white foam. This was
triturated with diethyl ether and a small amount (a few drops) of
MeOH and the resulting white solid was filtered off and dried by
suction to give Intermediate 12 as a white solid (0.27 g,
impurities present). .sup.1H NMR (400 MHz in d.sub.6-DMSO,
27.degree. C., 6 ppm) 8.1 (br. s, 2H), 3.95 (d, 2H), 3.15 (m, 1H),
2.7 (dt, 2H), 1.85 (dd, 2H), 1.35 (m, 2H); impurities present.
Intermediate 13
1,1-dimethylethyl [4-(aminocarbonyl)cyclohexyl]carbamate
##STR00061##
[0493] A solution of
4-({[(1,1-dimethylethyl)oxy]carbonyl}amino)cyclohexanecarboxylic
acid (e.g. Fluka, 1 g) in DMF (30 ml) was treated with HATU (1.72
g) and DIPEA (5.4 ml). The reaction mixture was stirred at room
temperature for 10 min. A 0.5M solution of ammonia in 1,4-dioxane
(40 ml) was added and the reaction mixture was stirred at room
temperature for 72 h. The solvents were evaporated and the residue
was purified by loading the crude mixture onto a 50 g aminopropyl
SPE cartridge and eluting with EtOAc (100 ml), then MeOH (100 ml).
Intermediate 13 was isolated (by evaporation of the MeOH fraction)
as a yellow oil (0.99 g). LCMS showed MH.sup.+=242; T.sub.RET=2.2
min.
Intermediate 15
4-aminocyclohexanecarboxamide hydrochloride
##STR00062##
[0495] 4.0M Hydrogen chloride in 1,4-dioxane (14 ml) was added to
Intermediate 13 (0.99 g) and the reaction mixture was stirred at
room temperature for 30 min. The solvent was evaporated to give
Intermediate 15 as a yellow gum (1.03 g). .sup.1H NMR (400 MHz in
d.sub.6-DMSO, 27.degree. C., 6 ppm) 7.9 (br. S, 2H), 3.9 (br. S,
2H), 3.10 (m, 1H), 1.92 (m, 2H), 1.68 (m, 4H), 1.50 (m, 2H).
Intermediate 17
1,1-dimethylethyl [cis-4-(aminocarbonyl)cyclohexyl]-carbamate
##STR00063##
[0497] A solution of
cis-4-({[(1,1-dimethylethyl)oxy]carbonyl}amino)cyclohexane-carboxylic
acid (5.0 g) (e.g. available from Fluka), EDC (5.9 g) and HOBT
(4.17 g) was stirred for 20 min. Ammonia solution (Specific
Gravity=0.88; 8 ml) was added. The reaction mixture was stirred at
room temperature overnight, concentrated in vacuo and partitioned
between DCM and saturated sodium bicarbonate solution. The aqueous
phase was separated and washed with DCM. The combined organics were
dried over MgSO.sub.4 and concentrated in vacuo to give
Intermediate 17 (4.84 g) as a white solid. LCMS showed
MH.sup.+=243; T.sub.RET=2.3 min.
[0498] The following Intermediate 18 was prepared in a similar
manner from
trans-4-({[(1,1-dimethylethyl)oxy]carbonyl}amino)cyclohexanecarboxylic
acid (e.g. available from Fluka):
Intermediate 18
1,1-dimethylethyl
[trains-4-(aminocarbonyl)cyclohexyl]-carbamate
##STR00064##
[0500] LCMS showed MNH.sub.4.sup.+=260; T.sub.RET=2.24 min.
Intermediate 19
cis-4-aminocyclohexanecarboxamide hydrochloride
##STR00065##
[0502] 4.0M hydrogen chloride in dioxan (50 ml) was added to a
stirred solution of Intermediate 17 (4.84 g) in dioxan (100 ml).
The reaction mixture was stirred for 1 hour at room temperature and
then stood at 0.degree. C. for 3 days. The reaction mixture was
concentrated in vacuo to give Intermediate 19 (4.1 g) as a white
solid. LCMS showed MH.sup.+=143; T.sub.RET=0.31 min.
[0503] The following Intermediate 20 was prepared in a similar
manner from Intermediate 18:
Intermediate 20
trans-4-aminocyclohexanecarboxamide hydrochloride
##STR00066##
[0505] LCMS showed MH.sup.+=143; T.sub.RET=0.30 min.
Intermediate 21
ethyl
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-1H-pyrazolo[3,4-b-
]pyridine-5-carboxylate
##STR00067##
[0507] A solution of Intermediate 1 (2.0 g), Intermediate 19 (1.55
g) and DIPEA (6.9 ml) in EtOH (140 ml) was stirred and heated at
reflux overnight. More Intermediate 19 (420 mg) and DIPEA (3.5 ml)
were added. The reaction mixture was stirred and heated at reflux
overnight, cooled and concentrated in vacuo. The residue was
partitioned between DCM and saturated sodium bicarbonate solution.
The organic phase was concentrated in vacuo. The residue was
triturated in a mixture of DCM and cyclohexane to give a solid. The
solid was filtered off and dried to give Intermediate 21 (2.16 g)
as a yellow solid. LCMS showed MH.sup.+=360; T.sub.RET=2.56
min.
[0508] The following Intermediate 22 was prepared in a similar
manner from Intermediate 1 and Intermediate 20:
Intermediate 22
ethyl
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-1H-pyrazolo[3,4-
-b]pyridine-5-carboxylate
##STR00068##
[0510] LCMS showed MH.sup.+=360; T.sub.RET=2.84 min.
Intermediate 23
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-1H-pyrazolo[3,4-b]pyrid-
ine-5-carboxylic acid
##STR00069##
[0512] A mixture of Intermediate 21 (1.54 g) and sodium hydroxide
(0.68 g) in 95% aqueous EtOH (EtOH containing 5% water) (60 ml) was
stirred and heated at 50.degree. C. overnight. The solvent was
removed in vacuo. The residue was dissolved in water. The solution
was cooled to 0-5.degree. C., with stirring, and acidified with 2M
HCl. The resultant suspension was refrigerated for 3 days then
filtered under suction. The residue was dried in vacuo to give
Intermediate 23 (1.58 g) as a yellow solid. LCMS showed
MH.sup.+=332; T.sub.RET=2.06 min.
[0513] The following Intermediate 24 was prepared in an analogous
manner from Intermediate 1 and Intermediate 22:
Intermediate 24
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-1H-pyrazolo[3,4-b]pyr-
idine-5-carboxylic acid
##STR00070##
[0515] LCMS showed MH.sup.+=332; T.sub.RET=2.06 min.
Intermediate 25
ethyl
4-[((3S)-1-{[(1,1-dimethylethyl)oxy]carbonyl}-3-pyrrolidinyl)amino]--
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
##STR00071##
[0517] A solution of Intermediate 1 (680 mg), DIPEA (2.3 ml) and
1,1-dimethylethyl (35)-3-amino-1-pyrrolidinecarboxylate (500 mg)
(e.g. available from Aldrich) in MeCN (15 ml) was stirred and
heated at reflux for 16 h. The solvent was evaporated and the
residue was partitioned between DCM and water. The organic phase
was isolated by passage through a hydrophobic frit. The solvent was
evaporated and the residue was purified on a 100 g "flashmaster"
cartridge (e.g. available from Jones Chromatography Ltd., United
Kingdom), using a mixture of EtOAc and cyclohexane as the eluent,
to give Intermediate 25 (720 mg) as a solid. LCMS showed
MH.sup.+=404; T.sub.RET=3.20 min.
[0518] The following Intermediate 26 was prepared in a similar
manner from Intermediate 1 and 1,1-dimethylethyl
(3R)-3-amino-1-pyrrolidinecarboxylate (e.g. available from
Aldrich):
Intermediate 26
ethyl
4-[((3R)-1-{[(1,1-dimethylethyl)oxy]carbonyl}-3-pyrrolidinyl)amino]--
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
##STR00072##
[0520] LCMS showed MH.sup.+=404; T.sub.RET=3.20 min.
Intermediate 27
ethyl
1-ethyl-4-[(3S)-3-pyrrolidinylamino]-1H-pyrazolo[3,4-b]pyridine-5-ca-
rboxylate hydrochloride
##STR00073##
[0522] Intermediate 25 (720 mg) in a solution of 4.0M hydrogen
chloride in dioxan (30 ml) was stirred at room temperature for 3 h.
The solvent was evaporated to give Intermediate 27 (606 mg) as a
white solid. LCMS showed MH.sup.+=304; T.sub.RET=2.00 min.
[0523] The following Intermediate 28 was prepared in a similar
manner from Intermediate 26:
Intermediate 28
ethyl
1-ethyl-4-[(3R)-3-pyrrolidinylamino]-1H-pyrazolo[3,4-b]pyridine-5-ca-
rboxylate hydrochloride
##STR00074##
[0525] LCMS showed MH.sup.+=304; T.sub.RET=2.00 min.
Intermediate 29
ethyl
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-1-ethyl-1H-pyrazolo-
[3,4-b]pyridine-5-carboxylate
##STR00075##
[0527] A solution of Intermediate 27 (606 mg) in DCM (30 ml) was
stirred and treated with DIPEA (1.15 ml) followed by trimethylsilyl
isocyanate (1.03 ml). The reaction mixture was stirred at room
temperature for 2 h. The solution was washed with water. The
aqueous phase was extracted with dichloromethane. The combined
organics were passed through a hydrophobic frit and then
concentrated to give Intermediate 29 (660 mg) as a solid. LCMS
showed MH.sup.+=347; T.sub.RET=2.40 min.
[0528] The following Intermediate 30 was prepared in a similar
manner from Intermediate 28:
Intermediate 30
ethyl
4-{[(3R)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-1-ethyl-1H-pyrazolo-
[3,4-b]pyridine-5-carboxylate
##STR00076##
[0530] LCMS showed MH.sup.+=347; T.sub.RET=2.40 min.
Intermediate 31
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-b-
]pyridine-5-carboxylic acid
##STR00077##
[0532] A mixture of Intermediate 29 (660 mg) and sodium hydroxide
(300 mg) in EtOH (15 ml) and water (8 ml) was stirred and heated at
60.degree. C. for 2 h. The solvents were removed in vacuo. Water (8
ml) was added to the residue and the resultant solution was
acidified with 2M HCl. The resultant suspension was filtered under
suction. The residue was dried in vacuo to give Intermediate 31
(270 mg) as a solid. LCMS showed MH.sup.+=319; T.sub.RET=1.90
min.
[0533] The following Intermediate 32 was prepared in a similar
manner from Intermediate 30:
Intermediate 32
4-{[(3R)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-1-ethyl-1H-pyrazolo[3,4-b-
]pyridine-5-carboxylic acid
##STR00078##
[0535] LCMS showed MH.sup.+=319; T.sub.RET=1.90 min.
Intermediate 33
4-[(1-{[(1,1-dimethylethyl)oxy]carbonyl}-4-piperidinyl)amino]-1-ethyl-1H-p-
yrazolo[3,4-b]pyridine-5-carboxylic acid
##STR00079##
[0537] A mixture of Intermediate 2 (750 mg) and sodium hydroxide
(290 mg) in EtOH (20 ml) and water (5 ml) was stirred and heated at
50.degree. C. for 2.5h then cooled and concentrated under reduced
pressure. A solution of the residue in water (20 ml) was cooled to
0-5.degree. C., with stirring, and acidified to pH=5 with 2M HCl
solution. The resultant solid suspension was filtered. The solid
residue was washed with water and dried to give Intermediate 33
(575 mg) as a white solid. LCMS showed MH.sup.+=390; T.sub.RET=2.86
min.
Intermediate 34
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2-bromophenyl)methyl]-1-eth-
yl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00080##
[0539] A solution of Intermediate 5 (0.5 g), EDC (0.32 g), HOBT
(0.22 g) and DIPEA (0.65 ml) in DMF (10 ml) was stood at room
temperature for 10 min then added to 2-bromobenzylamine
hydrochloride (0.40 g) (e.g. Aldrich). The reaction mixture was
stood at room temperature for 20 h. The solvent was evaporated. The
residue was partitioned between DCM (50 ml) and saturated
NaHCO.sub.3 solution. The organic phase was separated by passing
through a hydrophobic frit and evaporated. The residual solid was
triturated with a mixture of methanol and ether. The suspension was
filtered and the residue was dried to give Intermediate 34 (0.52 g)
as a white solid. LCMS showed MH.sup.+=500+502; T.sub.RET=2.70
min.
[0540] The following Intermediate 35 was prepared in a similar
manner from Intermediate 5 and 2-bromo-4-methylbenzylamine:
Intermediate 35
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2-bromo-4-methylphenyl)meth-
yl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00081##
[0542] LCMS showed MH.sup.+=514+516; T.sub.RET=2.82 min.
Table of Examples
TABLE-US-00004 [0543] Example Number Example Name 1
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 2, 2A,
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 2B 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-5-{[(3,4-
dimethylphenyl)methyl]aminocarbonyl}-1-ethyl-1H-pyrazolo[3,4-
b]pyridine (same compound as Examples 2, 2A, alternative name) 2C
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-5-{[(3,4-
dimethylphenyl)methyl]aminocarbonyl}-1-ethyl-1H-pyrazolo[3,4-
b]pyridine hydrochloride 3
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-
(methyloxy)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
4
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 5
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methyl]-
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 8
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 9 4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 10 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,4-
difluorophenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
11 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-
(trifluoromethyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 12
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(4-
hydroxyphenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 13
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-(phenylmethyl)-
1H-pyrazolo[3,4-b]pyridine-5-carboxamide 14
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-
(methylsulfonyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 15
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(3-
hydroxyphenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 16
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,3-
dichlorophenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
17 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,3-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 18
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(2-biphenylylmethyl)-1-
ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 19
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chloro-2-
fluorophenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
20
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(2,3-dihydro-1-benzofuran-
5-ylmethyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 21
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-({4-
[(difluoromethyl)oxy]phenyl}methyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-
5-carboxamide 22
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chloro-2-
methylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
23 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4'-chloro-4-
biphenylyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
24 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-
(methylsulfonyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 25
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-
(trifluoromethyl)-2-biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 26
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4-chlorophenyl)methyl]-
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 27
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(4-biphenylylmethyl)-1-
ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 28
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(ethyloxy)-4-
biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 29
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(3-biphenylylmethyl)-1-
ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 30
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-
(trifluoromethyl)-4-biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 31
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(3'-methyl-2-
biphenylyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 32
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[3'-(methyloxy)-2-
- biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 33
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3'-chloro-2-
biphenylyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
34 4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(4'-chloro-2-
biphenylyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
35
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(4'-methyl-2-
biphenylyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 36
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4'-(methyloxy)-2-
- biphenylyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 37
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-
furanyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 38
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-({2-[6-
(methyloxy)-3-pyridinyl]phenyl}methyl)-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 39
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-
thienyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 40
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(4-methyl-3-
thienyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 41
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(5-methyl-2-
biphenylyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 42
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-methyl-2-(3-
thienyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 43
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[2-(3-furanyl)-4-
methylphenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 44
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3'-chloro-5-methyl-2-
biphenylyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
45 4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 46
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 47
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-
(methyloxy)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
48 4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-
(methylsulfonyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 49
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,3-dimethylphenyl)methyl]-
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 50
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(2-biphenylylmethyl)-1-ethyl-
1H-pyrazolo[3,4-b]pyridine-5-carboxamide 51
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chloro-2-
fluorophenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
52
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,3-dichlorophenyl)methyl]-
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 53
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[2-
(methylsulfonyl)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 54
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chlorophenyl)methyl]-1-
ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 55
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-
(methyloxy)phenyl]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
56
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(4-biphenylylmethyl)-1-ethyl-
1H-pyrazolo[3,4-b]pyridine-5-carboxamide 57
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-[(4-
hydroxyphenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 58
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-(phenylmethyl)-1H-
pyrazolo[3,4-b]pyridine-5-carboxamide 59
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-difluorophenyl)methyl]-
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 60
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-({4-
[(difluoromethyl)oxy]phenyl}methyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-
5-carboxamide 61
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(2,3-dihydro-1-benzofuran-5-
ylmethyl)-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 62
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-(3-biphenylylmethyl)-1-ethyl-
1H-pyrazolo[3,4-b]pyridine-5-carboxamide 63
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-[(4-
fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 64
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4-chloro-2-
methylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
65 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(4'-chloro-4-
biphenylyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
66 4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-{[2,3-dichloro-6-
(hydroxymethyl)phenyl]methyl}-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 67
4-{[4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-(5,6,7,8-tetrahydro-1-
naphthalenylmethyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxamide 68
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-{[3,4-
bis(methyloxy)phenyl]methyl}-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 69
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(2,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 70
4-{[(3S)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(3,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 71
4-{[(3R)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(2,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide 72
4-{[(3R)-1-(aminocarbonyl)-3-pyrrolidinyl]amino}-N-[(3,4-
dimethylphenyl)methyl]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxamide
Example 1
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(2,4-dimethylphenyl)methyl]--
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00082##
[0545] A solution of Intermediate 5 (0.066 mmol) in DMF (1 ml) was
mixed with EDC (0.066 mmol), HOBT (0.066 mmol) and DIPEA (0.151
mmol) followed by 2,4-dimethyl benzylamine (0.066 mmol) (e.g. Trans
World Chemicals Inc.). The reaction mixture was left to stand at
room temperature overnight (e.g. for 16 h). The DMF was evaporated
and the residue was partitioned between DCM (5 ml) and saturated
aqueous NaHCO.sub.3 solution (2 ml). The organic layer was
collected through a hydrophobic frit and evaporated. The residue
was purified by mass directed autoprep. HPLC to give the title
compound Example 1 as a gum (7.9 mg). LCMS showed MH.sup.+=450;
T.sub.RET=2.8 min.
Example 2
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]--
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00083##
[0547] A solution of Intermediate 5 (100 mg, 0.3 mmol) in dry DMF
(e.g. can be about 1 ml) was treated with EDC (63 mg, 0.33 mmol),
HOBT (45 mg, 0.33 mmol) and DIPEA (0.13 ml, 0.75 mmol). 10 minutes
later, 3,4-dimethyl-benzylamine (47 microlitres, 0.33 mmol) (e.g.
available from Trans World Chemicals Inc.) was added and the
resulting solution was left to stand at room temperature overnight.
The DMF was removed by evaporation and the residue was partitioned
between DCM and saturated aqueous NaHCO.sub.3 solution. The organic
layer was collected through a hydrophobic frit and was concentrated
in vacuo to dryness. The residue was purified by passing through a
20 g silica SPE cartridge, using firstly a gradient of EtOAc and
cyclohexane (increasing concentration of EtOAc) and then a step
gradient of EtOAc and methanol as the eluent. The product was
eluted in the fraction containing 4:1 EtOAc:MeOH. The solvents were
removed in vacuo to give a white solid (101 mg). NMR showed the
presence of EtOAc and DCM, so the solid was dried in vacuo at
40.degree. C. to give Example 2 (80 mg). LCMS showed MH.sup.+=450;
T.sub.RET=2.80 min.
[0548] .sup.1H NMR (400 MHz in d.sub.6-DMSO, 27.degree. C., .delta.
ppm) 9.9 (d, 1H), 8.93 (t, 1H), 8.61 (s, 1H), 8.19 (s, 1H), about
7.08 (s, 1H), 7.07 (d, 1H), 7.02 (d, 1H), 5.98 (s, 2H), 4.33-4.39
(m, 4H), 4.08-4.18 (br m, 1H), 3.75 (dt, 2H), 3.13 (td, 3H), 2.19
(s, 3H), 2.18 (s, 3H), 1.92-2.00 (m, 2H), 1.33-1.42 (m, 5H). Plus
some other peaks: possibly solvent.
[0549] A similar alternative method is: A solution of Intermediate
5 (0.066 mmol) in DMF (1 ml) is treated with EDC (0.066 mmol), HOBT
(0.066 mmol) and DIPEA (0.151 mmol) followed by
3,4-dimethylbenzylamine (0.066 mmol). The reaction mixture is left
to stand at 22.degree. C. for 16 h. The DMF is evaporated and the
residue is partitioned between DCM (5 ml) and saturated aqueous
NaHCO.sub.3 solution (2 ml). The organic layer is collected through
a hydrophobic frit and evaporated. The residue is purified by mass
directed autoprep. HPLC to give the title compound.
Example 2A
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]--
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00084##
[0551] A mixture of Intermediate 9 (27 mg, 0.08 mmol), Intermediate
12 (16 mg, 0.088 mmol) and DIPEA (35 microlitres, 0.2 mmol) in MeCN
(2 ml) was heated at reflux for 18 h. More of Intermediate 12 (0.5
mole equivalents, ca. 0.04 mmol, ca. 7 mg) was added. The reaction
mixture was heated at reflux for a further 24 h, cooled to room
temperature and the solvent removed in vacuo. The residue was
partitioned between DCM and water. The organic phase was collected
through a hydrophobic frit and evaporated to dryness. LCMS
indicated that there were two products of the same molecular
weight.
[0552] Therefore, the residue was purified by mass directed
autopreparative HPLC to give the title compound as Example 2A (4.4
mg); LCMS showed MH.sup.+=450 and T.sub.RET=2.79 min.
[0553] The other undesired product having the same molecular weight
as Example 2A was also isolated from the mass directed
autopreparative HPLC (0.6 mg); and for this compound LCMS showed
MH.sup.+=450 and T.sub.RET=2.69 min.
Example 2B
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-5-{[(3,4-dimethylphenyl)methyl]-
aminocarbonyl}-1-ethyl-1H-pyrazolo[3,4-b]pyridine
##STR00085##
[0555] A solution of Intermediate 5A (21.6 g) in DMF (300 ml) was
treated with 3,4-dimethyl benzylamine (9.71 ml, 9.23 g), HOBT (9.66
g) and DIPEA (25 ml, 18.5 g) followed by EDC (14.1 g). The reaction
mixture was stirred at room temperature overnight. The DMF was
evaporated under reduced pressure at 40.degree. C. and the residue
was partitioned between EtOAc (300 ml) and water (200 ml). The
organic layer was separated and the aqueous phase was extracted
with EtOAc (2.times.100 ml). The combined organic extracts were
washed with brine (200 ml), were dried (MgSO.sub.4), were filtered
and were evaporated under reduced pressure.
[0556] The residue was purified by flash column chromatography on
silica gel (1500 ml) using 95:5 DCM/MeOH as the eluting solvent.
The purest fractions were collected and evaporated under reduced
pressure. The residue was dissolved in EtOAc (500 ml), was washed
with 1 N NaOH solution (100 ml), was dried (MgSO.sub.4), was
filtered, was evaporated, and the residue was dried at 60.degree.
C. in a vacuum oven overnight to provide the title compound as a
pale yellow solid (12.5 g).
[0557] The remaining fractions from the flash column chromatography
were collected and purified by autoprep. HPLC (Gilson reverse-phase
HPLC, Solvent A water, Solvent B acetonitrile, see above for
details). The water-acetonitrile fractions containing the product
(UV detection) were combined, and the acetonitrile solvent was
removed under reduced pressure. The remaining water was decanted
off from the residue, and the residue was evaporated to dryness.
The solid was collected and washed with ether to give the title
compound as a pale yellow solid (6 g).
[0558] The two batches of product were combined, were dissolved in
MeOH and were evaporated under reduced pressure to provide the
title compound Example 2B as a pale yellow solid.
[0559] LCMS showed MH.sup.+=450; T.sub.RET=2.45 min.
m.p.=152-154.degree. C.
[0560] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 10.01 (d,
J=7.83 Hz, 1H), 8.97 (t, J=5.81 Hz, 1H), 8.63 (s, 1H), 8.21 (s,
1H), 7.02-7.10 (m, 3H), 6.01 (br s, 2H), 4.34-4.42 (m, 4H),
4.10-4.20 (m, 1H), 3.71-3.81 (m, 2H), 3.14 (t, J=10.74 Hz, 2H),
2.20 (s, 3H), 2.18 (s, 3H), 1.92-2.02 (m, 2H), 1.35-1.45 (m, 5H).
Plus peaks due to ether.
Example 2C
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-5-{[(3,4-dimethylphenyl)methyl]-
aminocarbonyl}-1-ethyl-1H-pyrazolo[3,4-b]pyridine hydrochloride
##STR00086##
[0562] A solution of Example 2B (15 mg) in MeOH (0.5 ml) was
treated with a solution of 1N hydrogen chloride in ether (10 ml).
The mixture was evaporated to provide the title compound (Example
2C) as a white solid (16 mg). m.p.=217-218.degree. C.
(decomposition).
[0563] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 10.84 (s,
1H), 9.48 (s, 1H), 8.77 (s, 1H), 8.46 (s, 1H), 7.05-7.14 (m, 3H),
about 6.0-6.6 (br s, 2H), 4.53 (q, J=7.12 Hz, 2H), 4.40 (d, J=5.56
Hz, 2H), 4.27 (br s, 1H), 3.76 (d, J=13.64 Hz, 2H), 3.13-3.23 (t,
J=10.95 Hz, 2H), 2.21 (s, 3H), 2.19 (s, 3H), 1.94-2.03 (m, 2H),
1.44-1.53 (m, 2H), 1.40 (t, J=7.17 Hz, 3H).
Example 3
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-{[4-(methyloxy)phenyl-
]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00087##
[0565] A mixture of Intermediate 10 (27 mg, 0.08 mmol),
Intermediate 12 (16 mg, 0.088 mmol) and DIPEA (35 microlitres, 0.2
mmol) in MeCN (2 ml) was heated at reflux for 18 h. More of
Intermediate 12 (0.5 mole equivalents, ca. 0.04 mmol, ca. 7 mg) was
added. The reaction mixture was heated at reflux for a further 24
h, cooled to room temperature and the solvent removed in vacuo. The
residue was partitioned between DCM and water. The organic phase
was collected through a hydrophobic frit and evaporated to dryness.
LCMS indicated that there were two products of the same molecular
weight.
[0566] Therefore, the residue was purified by mass directed
autopreparative HPLC to give the title compound as Example 3 (4.2
mg); LCMS showed MH.sup.+=452 and T.sub.RET=2.57 min.
[0567] The other product having the same molecular weight as
Example 3 was also isolated from the mass directed autopreparative
HPLC (1.4 mg); and for this compound LCMS showed MH.sup.+=452 and
T.sub.RET=2.37 min.
[0568] Alternative synthesis: A mixture of Intermediate 10 (27 mg)
and Intermediate 12 (16 mg) in MeCN (2 ml) is treated with DIPEA
(35 .mu.L=35 microlitres). The reaction mixture is heated at reflux
for 72 h. The solvent is evaporated and the residue is partitioned
between DCM (5 ml) and saturated aqueous NaHCO.sub.3 solution (2
ml). The organic layer is collected through a hydrophobic frit and
evaporated. The residue is purified by mass directed autoprep. HPLC
to give Example 3.
[0569] Examples 1 and 2 can also optionally be prepared using a
similar procedure (e.g. see Example 2A).
Example 4
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl]-1-et-
hyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00088##
[0571] A solution of Intermediate 9 (0.08 mmol) in MeCN (1 ml) was
reacted with Intermediate 15 (0.088 mmol) and DIPEA (0.2 mmol). The
reaction mixture was heated at reflux for 20 h. The solvents were
evaporated and the residue was partitioned between DCM (5 ml) and
water (2 ml). The organic phase was collected through a hydrophobic
frit and evaporated. The residue was purified by mass directed
autoprep. HPLC to give Example 4 as a white solid (23 mg). LCMS
showed MH.sup.+=449; T.sub.RET=2.8 min.
[0572] The following Example 5 was prepared from Intermediates 8
and 15 using a similar procedure:
TABLE-US-00005 ##STR00089## LC-MS Example Input aryl Input MH.sup.+
retention Number chloride cyclohexylamine Ion time 5 Intermediate 8
Intermediate 15 449 2.9
Alternative Preparation of Example 4 and Separation into Cis and
Trans Isomers (Examples 8 and 9)
4-{[4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl]-1-et-
hyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00090##
[0574] A solution of Intermediate 9 (75 mg) in MeCN (5 ml) was
reacted with Intermediate 15 (42 mg) and DIPEA (200 .mu.L). The
reaction mixture was heated at reflux for 16 h. The solvents were
evaporated and the residue was partitioned between DCM (10 ml) and
water (5 ml). The organic fraction was collected through a
hydrophobic frit and evaporated. The residue was separated into its
cis and trans isomers using a 25 cm Chiralpak AS column, eluting
with 40% EtOH: 60% heptane mixture to give the isomers.
Example 8
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl]--
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
[0575] First isomer to elute: isolated as a white solid (7.4 mg).
LCMS showed MH.sup.+=449; T.sub.RET=2.8 min. Confirmation of the
cis-relationship between the substituents on the cyclohexane ring
was demonstrated by NMR (1D TOCSY experiments).
Example 9
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-N-[(3,4-dimethylphenyl)methyl-
]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
[0576] Second isomer to elute: isolated as a white solid (7.8 mg).
LCMS showed MH.sup.+=449; T.sub.RET=2.8 min. Confirmation of the
trans-relationship between the substituents of the cyclohexane ring
was demonstrated by NMR (1D TOCSY experiments).
Examples 10 to 26
[0577] The following Examples 10 to 26 were prepared from
Intermediate 5 and the appropriate amine reagent
Ar--CH.sub.2NH.sub.2 using a procedure similar to that used in
Example 1 or Example 2:
TABLE-US-00006 ##STR00091## One Possible NHCH.sub.2Ar Source of
amine LC-MS Example (connecting nitrogen reagent MH.sup.+ retention
Number underlined) H.sub.2NCH.sub.2Ar Ion time 10 ##STR00092##
Aldrich 458 2.7 11 ##STR00093## Aldrich 490 2.9 12 ##STR00094##
Apin 438 2.4 13 ##STR00095## Aldrich 422 2.43 14 ##STR00096## WO
2002/030930A2 (2002) 500 2.49 15 ##STR00097## Apin 438 2.43 16
##STR00098## Aldrich 490 2.95 17 ##STR00099## Trans World 450 2.83
18 ##STR00100## Trans World 498 2.99 19 ##STR00101## Lancaster 474
2.85 20 ##STR00102## MaybridgeInternational 464 2.6 21 ##STR00103##
Aldrich 488 2.78 22 ##STR00104## Trans World 470 2.93 23
##STR00105## Interchim S.A. 532 3.26 24 ##STR00106## Matrix 500 2.4
25 ##STR00107## Not known 566 3.21 26 ##STR00108## Aldrich 456
2.81
Example 27
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-(4-biphenylylmethyl)-1-ethyl--
1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00109##
[0579] A solution of Intermediate 5 (25 mg), EDC (16 mg), HOBT (11
mg) and DIPEA (33 ul) in DMF (1 ml) was stood for 10 min then added
to (4-biphenylylmethyl)amine acetate (Aldrich, 22 mg). The reaction
mixture was left to stand at room temperature for 4 days. The
solvent was evaporated. The residue was partitioned between DCM (2
ml) and saturated NaHCO.sub.3 solution. The organic phase was
passed through a hydrophobic frit and concentrated. The residue was
purified by mass directed autoprep HPLC to give Example 27 (12 mg)
as a white solid. LCMS showed MH.sup.+=498; T.sub.RET=3.08 min.
Examples 28 to 30
[0580] The following Examples 28 to 30 were prepared from
Intermediate 5 and the appropriate amine reagent
Ar--CH.sub.2NH.sub.2 using the above procedure of Example 27 or a
similar procedure:
TABLE-US-00007 ##STR00110## One Possible NHCH.sub.2Ar Source of
amine LC-MS Example (connecting nitrogen reagent MH.sup.+ retention
Number underlined) H.sub.2NCH.sub.2Ar Ion time 28 ##STR00111## 542
3.20 29 ##STR00112## Trans World 498 3.06 30 ##STR00113## Interchim
S.A. 566 3.33
Example 31
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-1-ethyl-N-[(3'-methyl-2-bipheny-
lyl)methyl]-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00114##
[0582] A solution of Intermediate 34 (50 mg), (3-methylphenyl)
boronic acid (20 mg) (e.g. Aldrich), palladium
tetrakis(triphenylphosphine) (6 mg) and sodium carbonate (32 mg) in
a (3:1) mixture of DMF and water (1 ml) was heated at 1500 in a
sealed vessel in a microwave reactor for 10 min. The reaction
mixture was cooled to room temperature and the solvents were
evaporated. The residue was partitioned between DCM (2 ml) and
water (2 ml). The organic phase was separated by passing through a
hydrophobic frit to give a solution containing a mixture of Example
31 [piperidinyl --NCONH.sub.2] and the [piperidinyl --NH] adduct.
The solution was treated with DIPEA (26 ul) and trimethylsilyl
isocyanate (15 ul). The solution was left to stand at room
temperature overnight. The solvent was removed and the residue was
purified by mass directed autoprep HPLC to give Example 31 (35 mg)
as a white solid. LCMS showed MH.sup.+=512; T.sub.RET=3.02 min.
Examples 32 to 44
[0583] The following Examples 32 to 44 were prepared from
Intermediate 34 (Examples 32 to 40) or Intermediate 35 (Examples 41
to 44) and the appropriate boronic acid ArB(OH).sub.2, using the
above (Example 31) procedure or a similar procedure.
TABLE-US-00008 ##STR00115## Boronic Acid HNCH.sub.2Ar Reagent
ArB(OH).sub.2 LC-MS Example (connecting nitrogen (with one possible
MH.sup.+ retention Number underlined) source) Ion time 32
##STR00116## ##STR00117## 528 2.9 33 ##STR00118## ##STR00119## 532
3.04 34 ##STR00120## ##STR00121## 532 3.06 35 ##STR00122##
##STR00123## 512 3.03 36 ##STR00124## ##STR00125## 528 2.91 37
##STR00126## ##STR00127## 488 2.88 38 ##STR00128## ##STR00129## 529
2.82 39 ##STR00130## ##STR00131## 504 2.97 40 ##STR00132##
##STR00133## 518 2.91 41 ##STR00134## ##STR00135## 512 2.97 42
##STR00136## ##STR00137## 518 2.93 43 ##STR00138## ##STR00139## 502
2.85 44 ##STR00140## ##STR00141## 546 3.11
Example 45
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methyl]--
1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00142##
[0585] A solution of Intermediate 23 (30 mg), EDC (19 mg), HOBT
(13.5 mg) and DIPEA (39 ul) in DMF (1 ml) was stirred for 0.5 h
then added to 2,4-dimethylbenzylamine (e.g. Trans World Chemicals,
Inc., 16 mg). The reaction mixture was left to stand at room
temperature overnight. The solvent was removed and the residue was
partitioned between dichloromethane and 1.0M sodium bicarbonate
solution. The organic phase was dried and concentrated. The residue
was purified by mass directed autoprep. HPLC to give Example 45
(3.6 mg). LCMS showed MH.sup.+=449; T.sub.RET=2.78 min.
[0586] The following Examples 46 and 47 were prepared in a similar
manner from Intermediates 24 and 23 respectively:
Example 46
4-{[trans-4-(aminocarbonyl)cyclohexyl]amino}-N-[(2,4-dimethylphenyl)methyl-
]-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00143##
[0588] From Intermediate 24 and 2,4-dimethylbenzylamine (e.g. Trans
World Chemicals, Inc.). LCMS showed MH.sup.+=449; T.sub.RET=2.82
min.
Example 47
4-{[cis-4-(aminocarbonyl)cyclohexyl]amino}-1-ethyl-N-{[4-(methyloxy)phenyl-
]methyl}-1H-pyrazolo[3,4-b]pyridine-5-carboxamide
##STR00144##
[0590] From Intermediate 23 and 4-methoxybenzylamine (e.g.
Aldrich). LCMS showed MH.sup.+=451; T.sub.RET=2.55 min.
Examples 48 to 68
##STR00145##
[0592] General Procedure:
[0593] A mixture of Intermediate 23 (0.1 mmol), HATU (0.1 mmol) and
DIPEA (0.4 mmol) in DMF (0.4 ml) was shaken at room temperature for
10 min. A solution of the amine reagent ArCH.sub.2NH.sub.2 (0.1
mmol) in DMF (0.2 ml) was then added and the mixture was agitated
for several minutes to produce a solution. The solution was stored
at room temperature for 16-64 hours then concentrated in vacuo. The
residue was dissolved in chloroform (0.5 ml) and applied to a SPE
cartridge (aminopropyl, 0.5 g). The cartridge was eluted
successively with chloroform (1.5 ml), EtOAc (1.5 ml) and
EtOAc:MeOH (9:1, 1.5 ml). Fractions containing the desired product
were concentrated in vacuo and the residue purified by mass
directed autoprep HPLC.
[0594] The following Examples 48 to 68 were prepared from
Intermediate 23 and the appropriate amine reagent
ArCH.sub.2NH.sub.2 using this or a similar procedure. The Examples
were believed to be isolated as a mixture of cis and trans isomers
at the cyclohexane ring, with the cis isomer believed to be
predominating.
##STR00146##
(believed to be mixture of cis and trans isomers at the cyclohexane
ring, with the cis isomer believed to be predominating)
TABLE-US-00009 One Possible HNCH.sub.2Ar Source of amine LC-MS
Example (connecting nitrogen reagent MH.sup.+ retention Number
underlined) H.sub.2NCH.sub.2Ar Ion time 48 ##STR00147## Matrix 499
2.52 49 ##STR00148## Trans World 449 2.93 50 ##STR00149## Trans
World 497 3.1 51 ##STR00150## Lancaster 473 2.95 52 ##STR00151##
Aldrich 489 3.04 53 ##STR00152## WO 2002/030930A2 (2002) 499 2.61
54 ##STR00153## Aldrich 455 2.92 55 ##STR00154## Aldrich 451 2.71
56 ##STR00155## Aldrich 497 3.18 57 ##STR00156## Apin 437 2.5 58
##STR00157## Aldrich 421 2.71 59 ##STR00158## Aldrich 457 2.81 60
##STR00159## Aldrich 487 2.89 61 ##STR00160##
MaybridgeInternational 463 2.71 62 ##STR00161## Trans World 497
3.17 63 ##STR00162## Aldrich 439 2.77 64 ##STR00163## Trans World
469 3.03 65 ##STR00164## Interchim S A 531 3.38 66 ##STR00165## 519
2.88 67 ##STR00166## JP 05194108 A2(1993) 475 3.11 68 ##STR00167##
Aldrich 481 2.6
Examples 69 and 70
##STR00168##
[0596] General Procedure:
[0597] A mixture of Intermediate 31 (30 mg), HATU (120 mg) and
DIPEA (0.09 ml) in acetonitrile (2 ml) was added to the amine
ArCH.sub.2NH.sub.2 (0.09 mmol). The mixture was stood at room
temperature for 16 hours. The solvent was evaporated. The residue
was partitioned between DCM and saturated sodium bicarbonate
solution. The organic phase was separated and evaporated. The
residue was purified by mass directed autoprep. HPLC to obtain the
desired product.
Examples 71 and 72
##STR00169##
[0599] General Procedure:
[0600] A mixture of Intermediate 32 (30 mg), HATU (120 mg) and
DIPEA (0.09 ml) in acetonitrile (2 ml) was added to the amine
ArCH.sub.2NH.sub.2 (0.09 mmol). The mixture was stood at room
temperature for 16 hours. The solvent was evaporated. The residue
was partitioned between DCM and saturated sodium bicarbonate
solution. The organic phase was separated and evaporated. The
residue was purified by mass directed autoprep HPLC to obtain the
desired product.
[0601] The following Tabulated Examples 69 and 70 (prepared from
Intermediate 31 and appropriate amine reagent ArCH.sub.2NH.sub.2)
and Examples 71 and 72 (prepared from Intermediate 32 and
appropriate amine reagent ArCH.sub.2NH.sub.2) were synthesised
using the procedure stated above for the relevant example
number:
TABLE-US-00010 One Possible Exam- HNCH.sub.2Ar Source of LC-MS ple
(connecting nitrogen amine reagent MH.sup.+ retention Number
underlined) H.sub.2NCH.sub.2Ar Ion time 69 ##STR00170## Trans World
436 2.65 70 ##STR00171## Trans World 436 2.64 71 ##STR00172## Trans
World 436 2.6 72 ##STR00173## Trans World 436 2.66
Pharmaceutical Composition Examples and Micronisation Examples
Examples of Pharmaceutical Compositions Suitable for External
Topical Administration
[0602] In each of the Composition Examples C1, C1A, C1B, C2, C2A,
C3, C3A, C4, and C5 to C11 disclosed below, the compound of formula
(I) ("drug") in the composition for external topical administration
can for example be
4-{[1-(aminocarbonyl)-4-piperidinyl]amino}-N-[(3,4-dimethylphenyl)methyl]-
-1-ethyl-1H-pyrazolo[3,4-b]pyridine-5-carboxamide, for example the
compound of Example 2, 2A or 2B ("free base" form).
Composition Examples C1, C1A and C1B
Ointments
[0603] Exemplary pharmaceutical compositions being ointments
suitable for external topical administration are as follows:
TABLE-US-00011 Example C1 Example C1A Alternative Ingredients
Ingredient Ingredient Example C1B: (+preferable Concentration
Concentration Concentration specifications*) Function (% w/w) (%
w/w) range (% w/w) Compound of Active agent 0.5 0.5 0.1 to 3%
formula (I) ("drug") PDE4 (e.g. 0.2 to 1.5%) (as free base)
inhibitor White Petrolatum Wax/ 69.65 69.16 25 to 75% (white soft
paraffin) ointment (e.g. 45 to 75%) (e.g. USP) base Mineral Oil
(e.g. Solubilizer/ 4.975 5 2.5 to 15% USP or BP) emollient (e.g. 4
to 12%) Polyoxyl Stearyl Surfactant 4.975 5 0.5 to 10% Ether (e.g.
(e.g. 3 to 10%) Volpo S-2 .TM.) Propylene Glycol Solubilizer/ 19.90
20 0.5 to 50% (e.g. USP) Penetration (e.g. 5 to 50% or Enhancer 7
to 30%) Butylated Antioxidant 0 0.20 0%, Hydroxyanisole or 0.001 to
2% (e.g. NF or BP) (e.g. 0.02 to 2%) Methylparaben Preservative 0
0.11 0%, (e.g. NF) or 0.05 to 2% Propylparaben (e.g. Preservative 0
0.03 0%, NF) or 0.01 to 2% Total 100 100 *NF = National Formulary;
USP = US Pharmacopeia; BP = British Pharmacopeia
[0604] Composition Example C1 can be prepared by the following
method:
[0605] The white petrolatum (white soft paraffin) (140 g), the
mineral oil (10 g), and the polyoxyl stearyl ether (e.g. Volpo
S-2.TM.) (10 g) are mixed together and melted using a hot water
bath until all the ingredients are dissolved, to form an oil phase.
The oil phase is heated to ca. 65-70.degree. C. The propylene
glycol (40 g) is heated using a hot water bath to a temperature of
ca. 65-70.degree. C., and is then added slowly to the oil phase
under low homogenization stirring conditions (Polytron large
shaft). The homogenization is then increased and the mixture is
homogenized for 10 mins. The resulting formulation is then cooled
to room temperature, using a cold water bath to facilitate cooling,
to give an ointment formulation (ca. 200 g).
[0606] Approx. 5 g of the ointment formulation in a vial is heated
slowly in a hot water bath until the ointment reaches a temperature
of approximately 40-45.degree. C. The compound of formula (I)
("drug", in free base form, 25.3 mg) is added slowly into the vial
and then the resulting mixture is homogenized using a
microhomogenizer for approx. 10 minutes, to give an ointment
containing the drug (Composition Example C1). The drug can for
example be at least partly in suspension in the ointment.
[0607] Composition Example C1A (and/or alternative Composition
Example C1B) can be prepared by the following method:
[0608] The ointment formulation is manufactured by first preparing
the propylene glycol phase. The propylene glycol and the
appropriate amount of the drug are mixed to provide an initial
solution while stirring with a low shear propeller mixer. The
antioxidant and preservatives are also included in the propylene
glycol solution. The temperature of the propylene glycol solution
is maintained at about 55-65.degree. C. Concurrently, all of the
components of the oil phase (white petrolatum, polyoxyl stearyl
ether and mineral oil) are added into a separate container and
heated to about 75-85.degree. C. to melt and mix the
components.
[0609] The propylene glycol phase is then added into the oil phase
while maintaining the temperature at above 70.degree. C. (e.g. from
above 70.degree. C. to 90.degree. C., e.g. about 75-85.degree. C.)
and mixing with a high shear homogenizer for a minimum of 15
minutes (e.g. 15-60 minutes or 15-30 minutes). The emulsification
process can be carried out in conventional topical manufacturing
equipment, such as a Lee Kettle or Malt-Mat, which allows for
scraping of the materials from the sides of the vessel while the
phases are being emulsified.
[0610] Following the emulsification time of at least 15 minutes,
the product is cooled to about 30.degree. C., to form
(semi-solidify) the ointment emulsion. During this cooling time,
the homogenizer speed is reduced and low agitation is used. After
the product has cooled, and the ointment is produced (Composition
Example C1A or C1B), it is dispensed from the manufacturing vessel
into holding containers. It can then be packed into tubes, sachets
or other suitable packaging components, as necessary.
Composition Examples C2 and C2A
Water-in-Oil Cream Emulsion
[0611] An exemplary external-topical pharmaceutical composition
being a water-in-oil cream emulsion is as follows:
TABLE-US-00012 Ingredients Example C2: Alternative (+preferable
specifications: Ingredient Example C2A: NF = National Formulary;
Concentration Concentration USP = US Pharmacopeia, etc) Function (%
w/w) range (% w/w) Compound of formula (I) (as Active 0.5 0.1 to 3%
free base) ("drug") (e.g. 0.2 to 1.5%) White Petrolatum (e.g. USP)
Wax/ointment 41.34 25 to 75% base (e.g. 30 to 65%) Mineral Oil
(e.g. USP or BP) Solubilizer/ 10 2.5 to 15% emollient (e.g. 4 to
12%) Polyoxyl Stearyl Ether Surfactant 8 0.5 to 12% (e.g. 3 to 10%)
Propylene Glycol USP Solubilizer/ 20 0.5 to 50% Enhancer (e.g. 5 to
50%) Butylated Hydroxyanisole (e.g. Antioxidant 0.02 0.001 to 2% NF
or BP) (e.g. 0.02 to 2%) Methylparaben (e.g. NF) Preservative 0.11
0.05 to 2% Propylparaben (e.g. NF) Preservative 0.03 0.01 to 2%
Purified water (e.g. USP) Vehicle Qs 2 to 30% (20%) (e.g. 5 to 25%)
Total 100
[0612] Composition Example C2 (and/or alternative embodiment
Example C2A) can for example be prepared using a process similar to
that described for Composition Example C1.
[0613] For example, in one embodiment of the process, the water and
propylene glycol can be mixed together (optionally with the
surfactant, antioxidant and preservatives, and optionally with the
drug) to form an aqueous phase. The oil phase containing the white
petrolatum and mineral oil are prepared in a separate vessel.
Temperatures of both the aqueous and oil phases are maintained at
elevated temperatures, such as about 55-90.degree. C. or preferably
about [from above 70 to 90].degree. C., the oil phase temperature
being sufficiently high to melt the oil phase, and while hot, one
phase is added to another while mixing using a high shear mixer to
effect emulsification, preferably keeping the temperature above
70.degree. C. such as from above 70 to 90.degree. C. The final
emulsion is allowed to cool e.g. to about 18-35.degree. C. such as
about 30.degree. C., while the agitation continues at lower speeds.
The ointment can then be dispensed from the manufacturing vessel
and filled into the primary packaging, for example tubes or
sachets.
Composition Examples C3 and C3A
Oil-in-Water Cream Emulsion
[0614] An exemplary external-topical pharmaceutical composition
being a oil-in-water cream emulsion, and intended to be a high
occlusion composition, is as follows:
TABLE-US-00013 Example C3: Alternative Ingredient Example C3A:
Ingredients Concentration Concentration (+optional specifications)
Function (% w/w) range (% w/w) Compound of formula (I) (as free
Active 0.5 0.1 to 3% base) ("drug") (e.g. 0.2 to 1.5%) Mineral Oil
(e.g. USP) Solubilizer/ 32.5 15 to 50% emollient (e.g. 20 to 45%)
Dimethicone (Silicone Fluid Emollient 2.5 0.5 to 20% 360) (e.g. NF)
(e.g. 1 to 5%) Isopropyl Myristate (e.g. NF) Solubilizer 7.5 0.5 to
20% (e.g. 3 to 12%) Glycerol Monostearate (e.g. Surfactant 2 0.5 to
10% Arlacel 165 .TM.) Sorbitan Monostearate (e.g. Surfactant 1 0.05
to 10% Span 60 .TM.) Cetostearyl Alcohol (e.g. NF) Surfactant 2 0.1
to 15% (e.g. 1 to 10%) Microcrystalline Wax (Ross) Emollient/ 10 5
to 25% (e.g. NF) base (e.g. 8 to 15%) Propylene Glycol (e.g. USP)
Solubilizer/ 10 0.5% to 50% Enhancer (e.g. 7% to 25%) Citric Acid,
Hydrous Granular Buffer 0.05 0.05 to 5% (e.g. USP) Sodium
Phosphate, Dibasic (e.g. Buffer 0.06 0.05 to 5% USP) Imidurea
(Germall 115 .TM.) (e.g. Preservative 0.20 0.05 to 2% NF) Purified
Water (e.g. USP) vehicle 32 15 to 60% (e.g. 20 to 50%)
[0615] Composition Example C3 (and/or alternative embodiment
Example C3A) can for example be prepared using a process generally
analogous to that described in Composition Example C2 above.
Composition Example C4
Cream Emulsion
TABLE-US-00014 [0616] Concentration of Optional weight of
Ingredients ingredient (% w/w) ingredients (g) Oil Phase Mineral
oil 29.86 29.90 Steareth 2 2 2.05 Cetostearyl Alcohol 2 2.01
Arlacel 165 .TM. 2 2.10 (glycerol monostearate) Microcrystalline
wax 10 10.12 isopropyl myristate (IPM) 7.5 7.87 Dimethicone 2.5
2.65 Aqueous Phase Propylene glycol 22 22.09 Purified water 22
22.34 Methylparaben 0.11 0.1142 Propylparaben 0.03 0.0354 (Optional
Batch size: ca. 100 g).
Procedure
[0617] Cream base without drug: The combined ingredients of the oil
phase are melted in a hot water bath to a temperature of approx
60-70.degree. C. The combined ingredients of the aqueous phase are
also heated in a hot water bath to a temperature of approx.
60-70.degree. C. The aqueous phase is then added slowly to the oil
phase under low homogenization conditions and then is homogenized
at a higher speed for approx. 10 mins under low heat in the water
bath. With heating removed, the formulation is then stirred
manually with a spatula while being allowed to cool, until room
temperature is reached, giving a cream emulsion.
[0618] Cream containing drug: As a modification of the above
procedure, after the aqueous phase is heated to a temperature of
approx. 60-70.degree. C. and before addition to the oil phase, the
compound of formula (I) is added at 0.1% to 3% w/w or 0.2% to 1.5%
w/w (e.g. 0.5% w/w) to the pre-formed hot aqueous phase. Then, the
aqueous phase is added slowly to the oil phase under low
homogenization conditions and then is homogenized at a higher speed
for approx. 10 mins under low heat in the water bath. With heating
removed, the formulation is then stirred (e.g. manually with a
spatula) while being allowed to cool, until ca. 18-35.degree. C. or
ca. 18-30.degree. C. (e.g. 30.degree. C. or room temperature) is
reached, giving a cream emulsion (Composition Example C4).
Composition Examples C5, C6, C7, C8, C9, C10, and C11
Ointment Compositions
[0619] Exemplary ointments can be as follows:
TABLE-US-00015 Optional Ingedient ingredients Ingredients % w/w
weight (g) Example C5 Oil Phase White petrolatum 60 60.82 Mineral
oil 5 5.04 Steareth-2 5 5.09 Beeswax substitute 10 10.02
Hydrophilic phase Propylene glycol 20 20.32 Example C6 Oil Phase
White petrolatum 65 67.87 Mineral oil 5 5.12 Steareth-2 5 5.04
Beeswax substitute 5 5.05 Hydrophilic phase Propylene glycol 20
20.35 Example C7 Oil Phase White petrolatum 60 60.45 Mineral oil 10
10.04 Steareth-2 5 5.03 Beeswax substitute 5 5.07 Hydrophilic phase
Propylene glycol 20 20.28 Example C8 Oil Phase White petrolatum 60
60.22 Mineral oil 10 10.11 Cetostearyl alcohol 5 5.08 Beeswax
substitute 5 5.09 Hydrophilic phase Propylene glycol 20 20.29
Example C9 Oil Phase White petrolatum 55 55.70 Mineral oil 15 15.11
Cetostearyl alcohol 5 5.02 Beeswax substitute 5 5.07 Hydrophilic
phase Propylene glycol 20 20.08 Example C10 Oil Phase White
petrolatum 63 63.23 Mineral oil 10 10.09 Steareth-2 5 5.03 Beeswax
substitute 2 2.03 Hydrophilic phase Propylene glycol 20 20.18
Example C11 Oil Phase White petrolatum 58 58.50 Mineral oil 15
15.12 Cetostearyl alcohol 5 5.01 Beeswax substitute 2 2.00
Hydrophilic phase Propylene glycol 20 20.29 Optional Batch size ca.
100 g
[0620] Procedure for Composition Examples C5, C6, C7, C8, C9, C10,
and C11 (ointment base): The oil phase is melted in a hot water
bath to a temperature of approx. 60-70.degree. C. The hydrophilic
(propylene glycol) phase is also heated in a hot water bath to a
temperature of approx. 60-70.degree. C. The hydrophilic phase is
added slowly to the oil phase under low homogenization conditions
and is then homogenized at a higher speed for approx. 10 mins under
low heat in the water bath. With heating removed, the formulation
is then stirred manually with a spatula while being allowed to
cool, until room temperature is reached, giving an ointment.
[0621] Procedure for Composition Examples C5, C6, C7, C8, C9, C10,
and C11 (ointment containing drug): As a modification of the above
procedure, after the hydrophilic (propylene glycol) phase is heated
to a temperature of approx. 60-70.degree. C. and before addition to
the oil phase, the compound of formula (I) is added at 0.1% to 3%
w/w or 0.2% to 1.5% w/w (e.g. 0.5% w/w) to the pre-formed hot
hydrophilic phase. Then, the hydrophilic phase is added slowly to
the oil phase under low homogenization conditions and then is
homogenized at a higher speed for approx. 10 mins under low heat in
the water bath. With heating removed, the formulation is then
stirred (e.g. manually with a spatula) while being allowed to cool
to ca. 15-35.degree. C. or ca. 18-30.degree. C. (e.g. to ca.
30.degree. C. or room temperature), giving an ointment (Composition
Examples C5, C6, C7, C8, C9, C10, and C11).
Micronisation Example
[0622] Purpose: To micronize a compound of formula (I) or a
pharmaceutically acceptable salt thereof, e.g. in an amount of
approximately 600-1000 mg thereof, using a Jetpharma MC1
micronizer. [0623] The parent (unmicronised) and micronised
materials are analyzed for particle size by laser diffraction and
crystallinity by PXRD.
TABLE-US-00016 [0623] Equipment and material Equipment/material
Description and specification Jetpharma MC1 Micronizer Nitrogen
supply: Air tank with 275 psi rate tubing Analytical balance
Sartorius Analytical Top loader balance Mettler PM400 Digital
Caliper VWR Electronic caliper Materials to be micronised A
compound of formula (I)
[0624] 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.
[0625] 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
settle downward through 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.
[0626] Procedure:
[0627] 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 ring (R) and venturi (V) pressures
are adjusted according to the values specified in the experimental
design (e.g. 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.
[0628] Note that the venturi (V) pressure is kept at least 2 bars
greater than the ring (R) pressure to prevent regurgitation of
material, e.g. outwardly from the material inlet port.
[0629] Balance performance is checked with calibration weights.
Specified amount of the parent material is fed 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.
[0630] 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 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. The weight of the micronised material is recorded.
The input container is re-weighed in order to calculate the amount
of input material by difference. The micronizer is disassembled and
residual PDE4 compound on the micronizer inner surface is rinsed
with 70/30 isopropyl alcohol/water and collected into a flask. The
micronizer is then thoroughly cleaned in a Lancer washing machine
and dried before subsequent runs are performed.
TABLE-US-00017 Optional Experimental Parameters Parent
(unmicronised) material (Procedure 1): a compound of formula (I) or
a pharmaceutically acceptable salt thereof Balance(s): Sartorius
analytical Time Venturi needed to Material Pressure (V)/ feed
Procedure input ring (R) Intended material Actual feed-rate no.
amount (g) Pressure (bar) feed-rate (min+sec) (g/min) 1 ca. 0.9 g V
= 8 to 10 bar 180 to 200 mg/min procedure not R = 5.5 to 6 bar
carried out
[0631] The above optional parameters can be varied using the
skilled person's knowledge.
% yield=[(Material from vessel+Material from cyclone)/Material
input amount].times.100
Procedure 1 includes possible parameters and conditions and has not
been carried out.
Inhalable Composition Example
Dry Powder Formulation Example--Dry powder Lactose Blend
Preparation
[0632] Using a size-reduced e.g. micronised form of the compound of
formula (I) or salt thereof (e.g. optionally as prepared in the
Micronisation Example above), the dry powder blend is 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 34
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.
[0633] Other blends: 10% w/w compound/salt (50 mg)+90% w/w lactose
(450 mg, inhalation-grade lactose containing 10% fines).
[0634] Serial dilution of the 1% w/w blend can achieve e.g. 0.1%
and 0.3% w/w blends.
* * * * *
References