U.S. patent application number 10/717519 was filed with the patent office on 2005-09-15 for immunomodulatory compounds.
This patent application is currently assigned to ACTIVE BIOTECH AB. Invention is credited to Brennan, Chris James, Coulter, Thomas Stephen, Ghiron, Chiara, Goran Pettersson, Lars Olof, Huxley, Philip, Matthews, Ian Richard, Thrige, Dorthe da Graca, Uddin, Muhammed Kamal.
Application Number | 20050203118 10/717519 |
Document ID | / |
Family ID | 32398347 |
Filed Date | 2005-09-15 |
United States Patent
Application |
20050203118 |
Kind Code |
A9 |
Matthews, Ian Richard ; et
al. |
September 15, 2005 |
Immunomodulatory compounds
Abstract
The present invention relates to novel heterocyclic compounds,
to methods for their preparation, to compositions containing them,
and to methods and use for clinical treatment of medical conditions
which may benefit from immunomodulation, including rheumatoid
arthritis, multiple sclerosis, diabetes, asthma, transplantation,
systemic lupus erythematosis and psoriasis. More particularly the
present invention relates to novel heterocyclic compounds, which
are CD80 antagonists capable of inhibiting the interactions between
CD80 and CD28.
Inventors: |
Matthews, Ian Richard;
(Abingdon, GB) ; Coulter, Thomas Stephen;
(Abingdon, GB) ; Ghiron, Chiara; (Abingdon,
GB) ; Brennan, Chris James; (Abingdon, GB) ;
Uddin, Muhammed Kamal; (Abingdon, GB) ; Goran
Pettersson, Lars Olof; (Lund, SE) ; Thrige, Dorthe da
Graca; (Lund, SE) ; Huxley, Philip; (Abingdon,
GB) |
Correspondence
Address: |
BUCHANAN INGERSOLL PC
(INCLUDING BURNS, DOANE, SWECKER & MATHIS)
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
ACTIVE BIOTECH AB
Lund
SE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0116461 A1 |
June 17, 2004 |
|
|
Family ID: |
32398347 |
Appl. No.: |
10/717519 |
Filed: |
November 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60428240 |
Nov 22, 2002 |
|
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60482122 |
Jun 25, 2003 |
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Current U.S.
Class: |
514/292 ;
514/405; 546/82; 548/359.5 |
Current CPC
Class: |
A61P 27/02 20180101;
A61P 13/12 20180101; A61P 17/06 20180101; A61P 1/16 20180101; A61P
7/02 20180101; A61P 29/00 20180101; C07D 491/04 20130101; A61P
19/02 20180101; C07D 471/04 20130101; A61P 1/00 20180101; C07D
519/00 20130101; A61P 3/10 20180101; A61P 21/04 20180101; A61P
17/00 20180101; A61P 11/06 20180101; A61P 19/00 20180101; A61P
37/02 20180101; A61P 7/00 20180101; A61P 35/00 20180101; A61P 25/00
20180101; A61P 21/00 20180101; A61P 5/14 20180101; C07D 495/04
20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/292 ;
514/405; 546/082; 548/359.5 |
International
Class: |
A61K 031/4745; A61K
031/4162 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 22, 2002 |
SE |
0203471-8 |
May 6, 2003 |
SE |
0301299-4 |
Jun 25, 2003 |
SE |
0301851-2 |
Claims
1. A compound of formula (I) or a pharmaceutically veterinarily
acceptable salt thereof: 143wherein R.sub.1 and R.sub.3
independently represent H; F; Cl; Br; -NO.sub.2; --CN;
C.sub.1-C.sub.6 alkyl optionally substituted by F or Cl; or
C.sub.1-C.sub.6 alkoxy optionally substituted by F; R.sub.2
represents H, or optionally substituted C.sub.1-C.sub.6 alkyl,
C.sub.3-C.sub.7 cycloalkyl or optionally substituted phenyl; Y
represents --O--, --S--, N-oxide, or --N(R.sub.5)-- wherein R.sub.5
represents H or C.sub.1-C.sub.6 alkyl; X represents a bond or a
divalent C.sub.1-C.sub.6 alkylene radical; R.sub.4 represents
--C(.dbd.O)NR.sub.6R.sub.7, --NR.sub.7C(.dbd.O)R.sub.6,
--NR.sub.7C(.dbd.O)OR.sub.6, --NHC(.dbd.O)NHR.sub.6 or
--NHC(.dbd.S)NHR.sub.6 wherein R.sub.6 represents H, or a radical
of formula -(Alk).sub.b-Q wherein b is 0 or 1 and Alk is an
optionally substituted divalent straight chain or branched
C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene or
C.sub.2-C.sub.12 alkynylene radical which may be interrupted by one
or more non-adjacent --O--, --S-- or --N(R.sub.8)-- radicals
wherein R.sub.8 represents H or C.sub.1-C.sub.4 alkyl,
C.sub.3-C.sub.4 alkenyl, C.sub.3-C.sub.4 alkynyl, or
C.sub.3-C.sub.6 cycloalkyl, and Q represents H; --CF.sub.3; --OH;
--SH; --NR.sub.8R.sub.8 wherein each R.sub.8 may be the same or
different; an ester group; or an optionally substituted phenyl,
C.sub.3-C.sub.7 cycloalkyl, C.sub.5-C.sub.7 cycloalkenyl or
heterocyclic ring having from 5 to 8 ring atoms; and R.sub.7
represents H or C.sub.1-C.sub.6 alkyl; or when taken together with
the atom or atoms to which they are attached R.sub.6 and R.sub.7
form an optionally substituted heterocyclic ring having from 5 to 8
ring atoms.
2. A compound as claimed in claim 1 wherein R.sub.1 is H, F, Cl,
methyl or methoxy.
3. A compound as claimed in claim 1 or claim 2 wherein R.sub.2 is
H, methyl, methoxy, cyclopropyl, phenyl, or fluoro-, chloro-,
methyl, or methoxy-substituted phenyl.
4. A compound as claimed in any of the preceding claims wherein
R.sub.3 is H, F, Cl, methyl, methoxy, or methylenedioxy.
5. A compound as claimed in any of the preceding claims wherein Y
is --O--, --S--, or --N(R.sub.5)-- wherein R.sub.5 represents H or
methyl.
6. A compound as claimed in any of the preceding claims wherein X
is a bond, or a --CH.sub.2-- or --CH.sub.2CH.sub.2-- radical.
7. A compound as claimed in any of the preceding claims wherein
R.sub.4 represents --C(.dbd.O)NHR.sub.6,
--NR.sub.7C(.dbd.O)R.sub.6, --NR.sub.7C(.dbd.O)OR.sub.6,
--NHC(.dbd.O)NHR.sub.6 or --NHC(.dbd.S)NHR.sub.6 and in these
R.sub.6 is H or a radical of formula -Alk.sub.b-Q wherein b is 0 or
1 and Alk is a --(CH.sub.2).sub.n--,
--CH((CH.sub.2).sub.mCH.sub.3)(CH.sub.2).sub.n--,
--CH((CH.sub.2).sub.mCH-
.sub.3)((CH.sub.2).sub.pCH.sub.3)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.n--O--(CH.sub.2).sub.m--, or
--(CH.sub.2).sub.n--O--(CH.- sub.2).sub.nO--(CH.sub.2).sub.m--,
radical where n is 1, 2, 3 or 4 and m and p are independently 0, 1,
2, 3 or 4, and Q represents H, --OH, --COOCH.sub.3 phenyl,
cyclopropyl, cyclopentyl, cyclohexyl, pyridyl, furyl, thienyl, or
oxazolyl. and R.sub.7 is H, or when taken together with the
nitrogen atom to which they are attached R.sub.6 and R.sub.7 form a
pyrrolidine-2-one or pyrrolidine-2,5-dione ring.
8. A compound as claimed in claim 1 wherein R.sub.1 is H, F, or Cl;
R.sub.2 is H; R.sub.3 is H, F, or Cl; Y is --NH--; X is a bond; and
R.sub.4 represents --C(.dbd.O)NHR.sub.6,
--NR.sub.7C(.dbd.O)R.sub.6, --NR.sub.7C(.dbd.O)OR.sub.6 or
--NHC(.dbd.O)NHR.sub.6 wherein: R.sub.6 is H or a radical of
formula -Alk.sub.b-Q wherein b is 0 or 1 and Alk is a
--(CH.sub.2).sub.n--,
--CH((CH.sub.2).sub.mCH.sub.3)(CH.sub.2).sub.n--,
--CH((CH.sub.2).sub.mCH.sub.3)((CH.sub.2).sub.pCH.sub.3)(CH.sub.2).sub.n--
-, --(CH.sub.2).sub.n--O--(CH.sub.2).sub.m--, or
--(CH.sub.2).sub.n--O--(C- H.sub.2).sub.n--O--(CH.sub.2).sub.m--,
radical where n is 1, 2, 3 or 4 and m and p are independently 0, 1,
2, 3 or 4, and Q represents H, --OH, --COOCH.sub.3 phenyl,
cyclopropyl, cyclopentyl, cyclohexyl, pyridyl, furyl, thienyl, or
oxazolyl. and R.sub.7 is H, or when taken together with the
nitrogen atom to which they are attached R.sub.6 and R.sub.7 form a
pyrrolidine-2-one or pyrrolidine-2,5-dione ring.
9. A compound as claimed in claim 1 wherein R.sub.1 is F, R.sub.2
is H or cyclopropyl, R.sub.3 is H, X is a bond, and R.sub.4 is
--C(.dbd.O)NHR.sub.6, --NRHC(.dbd.O)R.sub.6, or
--NHC(.dbd.O)NHR.sub.6.
10. N-(3-Dimethylamino
propyl)-4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo-
[4,3-c]quinolin-2-yl]-benzamide, or pharmaceutically or
veterinarily acceptable salt thereof.
11. A compound as claimed in any of claims 1 to 10 for use in the
treatment of conditions which benefit from immunomodulation.
12. The use of a compound as claimed in any of claims 1 to 10 in
the manufacture of a medicament for the treatment of conditions
which benefit from immunomodulation.
13. A method of immunomodulation in humans and non-human primates,
comprising administration to a subject in need of such treatment an
immunomodulatory effective dose of a compound as claimed in any of
claims 1 to 10.
14. A pharmaceutical or veterinary composition comprising a
compound as claimed in any of claims 1 to 10 together with a
pharmaceutically or veterinarily acceptable excipient or carrier.
Description
[0001] The present invention relates to novel heterocyclic
compounds, to methods for their preparation, to compositions
containing them, and to methods and use for clinical treatment of
medical conditions which may benefit from immunomodulation,
including rheumatoid arthritis, multiple sclerosis, diabetes,
asthma, transplantation, systemic lupus erythematosis and
psoriasis. More particularly the present invention relates to novel
heterocyclic compounds, which are CD80 antagonists capable of
inhibiting the interactions between CD80 and CD28.
BACKGROUND OF THE INVENTION
[0002] The immune system possesses the ability to control the
homeostasis between the activation and inactivation of lymphocytes
through various regulatory mechanisms during and after an immune
response. Among these are mechanisms that specifically inhibit
and/or turn off an immune response. Thus, when an antigen is
presented by MHC molecules to the T-cell receptor, the T-cells
become properly activated only in the presence of additional
co-stimulatory signals. In the absence of accessory signals there
is no lymphocyte activation and either a state of functional
inactivation termed anergy or tolerance is induced, or the T-cell
is specifically deleted by apoptosis. One such co-stimulatory
signal involves interaction of CD80 on specialised
antigen-presenting cells with CD28 on T-cells, which has been
demonstrated to be essential for full T-cell activation. (Lenschow
et al. (1996) Annu. Rev. Immunol., 14, 233-258)
[0003] A paper by Erbe et al, in J. Biol. Chem. Vol. 277, No. 9, pp
7363-7368 (2002), describes three small molecule ligands which bind
to CD80, and inhibit binding of CD80 to CD28 and CTLA4. Two of the
disclosed ligands are fused pyrazolones of structures A and B:
1
DESCRIPTION OF THE INVENTION
[0004] According to the present invention there is provided a
compound of formula (I) or a pharmaceutically or veterinarily
acceptable salt thereof: 2
[0005] wherein
[0006] R.sub.1 and R.sub.3 independently represent H; F; Cl; Br;
--NO.sub.2; --CN; C.sub.1-C.sub.6 alkyl optionally substituted by F
or Cl; or C.sub.1-C.sub.6 alkoxy optionally substituted by F;
[0007] R.sub.2 represents H, or optionally substituted
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.7 cycloalkyl or optionally
substituted phenyl;
[0008] Y represents --O--, --S--, N-oxide, or --N(R.sub.5)--
wherein R.sub.5 represents H or C.sub.1-C.sub.6 alkyl;
[0009] X represents a bond or a divalent C.sub.1-C.sub.6 alkylene
radical;
[0010] R.sub.4 represents --C(.dbd.O)NR.sub.6R.sub.7,
--NR.sub.7C(.dbd.O)R.sub.6, --NR.sub.7C(.dbd.O)OR.sub.6,
--NHC(.dbd.O)NHR.sub.6, or --NHC(.dbd.S)NHR.sub.6 wherein
[0011] R.sub.6 represents H, or a radical of formula -(Alk)b-Q
wherein b is 0 or 1, and
[0012] Alk is an optionally substituted divalent straight chain or
branched C.sub.1-C.sub.12 alkylene, C.sub.2-C.sub.12 alkenylene or
C.sub.2-C.sub.12 alkynylene radical which may be interrupted by one
or more non-adjacent --O--, --S-- or --N(R.sub.8)-- radicals
wherein R.sub.8 represents H or C.sub.1-C.sub.4 alkyl,
C.sub.3-C.sub.4 alkenyl, C.sub.3-C.sub.4 alkynyl, or
C.sub.3-C.sub.6 cycloalkyl, and
[0013] Q represents H; --CF.sub.3; --OH; --SH; --NR.sub.8R.sub.8
wherein each R.sub.8 may be the same or different; an ester group;
or an optionally substituted phenyl, C.sub.3-C.sub.7 cycloalkyl,
C.sub.5-C.sub.7 cycloalkenyl or heterocyclic ring having from 5 to
8 ring atoms; and
[0014] R.sub.7 represents H or C.sub.1-C.sub.6 alkyl; or when taken
together with the atom or atoms to which they are attached R.sub.6
and R.sub.7 form an optionally substituted heterocyclic ring having
from 5 to 8 ring atoms.
[0015] Compounds of general formula (I) are CD80 antagonists. They
inhibit the interaction between CD80 and CD28 and thus the
activation of T cells, thereby modulating the immune response.
[0016] Accordingly the invention also includes:
[0017] (i) a compound of formula (I) or a pharmaceutically or
veterinarily acceptable salt thereof for use in the treatment of
conditions which benefit from immunomodulation.
[0018] (ii) the use of a compound of formula (I) or a
pharmaceutically or veterinarily acceptable salt thereof in the
manufacture of a medicament for the treatment of conditions which
benefit from immunomodulation,.
[0019] (iii) a method of immunomodulation in humans and non-human
primates, comprising administration to a subject in need of such
treatment an immunomodulatory effective dose of a compound of
formula (I) or a pharmaceutically or veterinarily acceptable salt
thereof.
[0020] (iv) a pharmaceutical or veterinary composition comprising a
compound of formula (I) or a pharmaceutically or veterinarily
acceptable salt thereof together with a pharmaceutically or
veterinarily acceptable excipient or carrier.
[0021] Conditions which benefit from immunomodulation include:
[0022] Adrenal insufficiency
[0023] Allergic angiitis and granulomatosis
[0024] Amylodosis
[0025] Ankylosing spondylitis
[0026] Asthma
[0027] Autoimmune Addison's disease
[0028] Autoimmune alopecia
[0029] Autoimmune chronic active hepatitis
[0030] Autoimmune hemolytic anemia
[0031] Autoimmune neutropenia
[0032] Autoimmune thrombocytopenic purpura
[0033] Autoimmune vasculitides
[0034] Behcet's disease
[0035] Cerebellar degeneration
[0036] Chronic active hepatitis
[0037] Chronic inflammatory demyelinating
polyradiculoneuropathy
[0038] Dermatitis herpetiformis
[0039] Diabetes
[0040] Eaton-Lambert myasthenic syndrome
[0041] Encephalomyelitis
[0042] Epidermolysis bullosa
[0043] Erythema nodosa
[0044] Gluten-sensitive enteropathy
[0045] Goodpasture's syndrome
[0046] Graft versus host disease
[0047] Guillain-Barre syndrome
[0048] Hashimoto's thyroiditis
[0049] Hyperthyrodism
[0050] Idiopathic hemachromatosis
[0051] Idiopathic membranous glomerulonephritis
[0052] Minimal change renal disease
[0053] Mixed connective tissue disease
[0054] Multifocal motor neuropathy
[0055] Multiple sclerosis
[0056] Myasthenia gravis
[0057] Opsoclonus-myoclonus syndrome
[0058] Pemphigoid
[0059] Pemphigus
[0060] Pernicious anemia
[0061] Polyarteritis nodosa
[0062] Polymyositis/dermatomyositis
[0063] Post-infective arthritides
[0064] Primary biliary sclerosis
[0065] Psoriasis
[0066] Reactive arthritides
[0067] Reiter's disease
[0068] Retinopathy
[0069] Rheumatoid arthritis
[0070] Sclerosing cholangitis
[0071] Sjogren's syndrome
[0072] Stiff-man syndrome
[0073] Subacute thyroiditis
[0074] Systemic lupus erythematosis
[0075] Systemic sclerosis (scleroderma)
[0076] Temporal arteritis
[0077] Thromboangiitis obliterans
[0078] Transplantation rejection
[0079] Type I and type II autoimmune polyglandular syndrome
[0080] Ulcerative colitis
[0081] Uveitis
[0082] Wegener's granulomatosis
[0083] As used herein the term "alkylene" refers to a straight or
branched alkyl chain having two unsatisfied valencies, for example
--CH.sub.2--, --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH(CH.sub.3)CH.sub.2--,
--CH(CH.sub.2CH.sub.3)CH.sub.2CH.sub.2CH.sub.3, and
--C(CH.sub.3).sub.3.
[0084] As used herein the term "heteroaryl" refers to a 5- or
6-membered aromatic ring containing one or more heteroatoms.
Illustrative of such groups are thienyl, furyl, pyrrolyl,
imidazolyl, benzimidazolyl, thiazolyl, pyrazolyl, isoxazolyl,
isothiazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl,
pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl.
[0085] As used herein the unqualified term "heterocyclyl" or
"heterocyclic" includes "heteroaryl" as defined above, and in
particular means a 5-8 membered aromatic or non-aromatic
heterocyclic ring containing one or more heteroatoms selected from
S, N and O, including for example, pyrrolyl, furanyl, thienyl,
piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl,
thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl,
morpholinyl, piperazinyl, indolyl, morpholinyl, benzofuranyl,
pyranyl, isoxazolyl, quinuclidinyl, aza-bicyclo[3.2.1]octanyl,
benzimidazolyl, methylenedioxyphenyl, maleimido and succinimido
groups.
[0086] Unless otherwise specified in the context in which it
occurs, the term "substituted" as applied to any moiety herein
means substituted with one or more of the following substituents,
namely (C.sub.1-C.sub.6)alkyl, trifluoromethyl,
(C.sub.1-C.sub.6)alkoxy (including the special case where a ring is
substituted on adjacent ring C atoms by methylenedioxy or
ethylenedioxy), trifluoromethoxy, (C.sub.1-C.sub.6)alkylthio,
phenyl, benzyl, phenoxy, (C.sub.3-C.sub.8)cycloalkyl, hydroxy,
mercapto, amino, fluoro, chloro, bromo, cyano, nitro, oxo, --COOH,
--SO.sub.2OH, --CONH.sub.2, --SO.sub.2NH.sub.2, --COR.sup.A,
--COOR.sup.A, --SO.sub.2OR.sup.A, --NHCOR.sup.A,
--NHSO.sub.2R.sup.A, --CONHR.sup.A, --SO.sub.2NHR.sup.A,
--NHR.sup.A, --NR.sup.AR.sup.B, --CONR.sup.AR.sup.B or
--SO.sub.2NR.sup.AR.sup.B wherein R.sup.A and R.sup.B are
independently a (C.sub.1-C.sub.6)alkyl group. In the case where
"substituted" means substituted by (C.sub.3-C.sub.8)cycloalkyl,
phenyl, benzyl or phenoxy, the ring thereof may itself be
substituted with any of the foregoing, except
(C.sub.3-C.sub.8)cycloalkyl phenyl, benzyl or phenoxy.
[0087] As used herein the unqualified term "carbocyclyl" or
"carbocyclic" refers to a 5-8 membered ring whose ring atoms are
all carbon.
[0088] Some compounds of the invention contain one or more chiral
centres because of the presence of asymmetric carbon atoms. The
presence of asymmetric carbon atoms gives rise to stereoisomers or
diastereoisomers with R or S stereochemistry at each chiral centre.
The invention includes all such stereoisomers and diastereoisomers
and mixtures thereof.
[0089] Salts of salt forming compounds of the invention include
physiologically acceptable acid addition salts for example
hydrochlorides, hydrobromides, sulphates, methane sulphonates,
p-toluenesulphonates, phosphates, acetates, citrates, succinates,
lactates, tartrates, fumarates and maleates; and base addition
salts, for example sodium, potassium, magnesium, and calcium salts.
Where the compound contains an amino group, quaternary amino salts
are also feasable, and are included in the invention.
[0090] In the compounds of the invention the following are examples
of the several structural variables:
[0091] R.sub.1 may be, for example, H, F, Cl, methyl, methoxy, or
methylenedioxy. Currently it is preferred that R.sub.1 is H, Cl or
especially F;
[0092] R.sub.2 may be, for example H, methyl, methoxy, cyclopropyl,
phenyl, or fluoro-, chloro-, methyl, or methoxy-substituted phenyl.
H or cyclopropyl is presently preferred;
[0093] R.sub.3 may be, for example, H, F, Cl, methyl, methoxy, or
methylenedioxy. Currently it is preferred that R.sub.3 is F or Cl,
and it is most preferred that R.sub.3 be H;
[0094] Y may be, for example, --O--, --S--, or --N(R.sub.5)--
wherein R.sub.5 represents H or methyl. --NH-- or --S-- is
presently preferred.
[0095] X may be, for example a bond, or a --CH.sub.2-- or
--CH.sub.2CH.sub.2-- radical. A bond is presently preferred.
[0096] R.sub.4 represents --C(.dbd.O)NR.sub.6R.sub.7,
--NR.sub.7C(.dbd.O)R.sub.6, --NR.sub.7C(.dbd.O)OR.sub.6,
--NHC(.dbd.O)NHR.sub.6, or --NHC(.dbd.S)NHR.sub.6. Of these
--NR.sub.7C(.dbd.O)R.sub.6, and especially
--C(.dbd.O)NR.sub.6R.sub.7 and --NHC(.dbd.O)NHR.sub.6 are curently
preferred. R.sub.7 is preferably H, but a wide range of R.sub.6
substituents have given rise to highly active compounds of the
invention. Many exemplary R.sub.6 substituents appear in the
compounds of the Examples below.
[0097] R.sub.6 may be, for example, H or a radical of formula
-Alk.sub.b-Q wherein b is 0 or 1 and
[0098] Alk may be, for example a --(CH.sub.2).sub.n--,
--CH((CH.sub.2).sub.mCH.sub.3)(CH.sub.2).sub.n--,
--C((CH.sub.2).sub.mCH.- sub.3)((CH.sub.2).sub.pCH.sub.3)
(CH.sub.2).sub.n--, --(CH.sub.2).sub.n--O--(CH.sub.2).sub.m--,
--(CH.sub.2).sub.n--NH--(CH.su- b.2).sub.m--, or
--(CH.sub.2).sub.n--NH--(CH.sub.2).sub.m--NH--(CH.sub.2).- sub.p--
radical where n is 1, 2, 3 or 4 and m and p are independently 0, 1,
2, 3 or 4, and
[0099] Q may represent H, --OH, --COOCH.sub.3, phenyl, cyclopropyl,
cyclopentyl, cyclohexyl, pyridyl, furyl, thienyl, or oxazolyl;
and
[0100] R.sub.7 may be, for example, H, or when taken together with
the atom or atoms to which they are attached R.sub.6 and R.sub.7
may form a heterocyclic ring of 5, 6 or 7 members.
[0101] Specific examples of R.sub.4 groups include those present in
the compounds of the Examples herein.
[0102] Compounds of the invention may be prepared by synthetic
methods known in the literature, from compounds which are
commercially available or are accessible from commercially
available compounds. For example, compounds of formula (I) wherein
R.sub.4 is a group --NR.sub.7C(.dbd.O)R.sub.6 may be prepared by
acylation of an amine of formula (II) with an acid chloride of
formula (III): 3
[0103] Compounds of the invention wherein R.sub.4 is a group
--NHC(.dbd.O)NHR.sub.6 may be prepared by reaction of an amine of
formula (IIA) with an isocyanate of formula (IIIA) 4
[0104] Compounds of the invention wherein R.sub.4 is a group
--C(.dbd.O)NHR.sub.6 may be prepared by reaction of an acid
chloride of formula (IIB) with an amine NHR.sub.6R.sub.7: 5
[0105] Compounds of the invention wherein R.sub.4 is a group
--NR.sub.7C(.dbd.O)OR.sub.6 may be prepared by reaction of an amine
of formula (II) with a chloroformate ClC(.dbd.O)OR.sub.6.
[0106] The following Examples illustrate the preparation of
compounds of the invention:
Preparation of Intermediate 1
2-(4-Nitrophenyl)-6-fluoro-2,5-dihydropyrazolo[4,3-c]-quinolin-3-one
[0107] 6
[0108] 4-Nitrophenylhydrazine (2.28 g, 0.014 mol) was added in one
portion to a stirred solution of
4-chloro-8-fluoro-quinoline-3-carboxylic acid ethyl ester (3.58 g,
0.014 mol) in anhydrous n-butyl alcohol (50 ml) at room
temperature. The mixture was refluxed for 16 h under nitrogen,
cooled to room temperature and then filtered to leave an orange
solid. The solid was purified by washing sequentially with ethyl
acetate (20 ml) and heptane (20 ml) and then finally dried under
suction to give the pyrazolone (3.93 g, 87%) as a dark orange
solid, LCMS m/z 325.24 [M+H].sup.+ @ R.sub.T 1.47 min.
Preparation of Intermediate 2
2-(4-Aminophenyl)-6-fluoro-2,5-dihydropyrazolo[4,3-c]-quinolin-3-one
[0109] 7
[0110] Tin (II) chloride dihydrate (12.5 g, 0.055 mol) was added in
one portion to a stirred solution of
2-(4-nitro-phenyl)-6-fluoro-2,5-dihydro--
pyrazolo[4,3-c]quinolin-3-one (intermediate 1) (3.59 g, 0.011 mol)
in ethyl alcohol (110 ml) at room temperature. The mixture was then
heated to 80.degree. C. for 8 h, cooled to room temperature and
filtered to leave a yellow solid. The solid was suspended in a
bi-phasic solution of ethyl acetate (1L), a saturated solution of
Rochelles salt (500 ml) and a saturated solution of sodium
bicarbonate (500 ml) and stirred at room temperature for 2 h. The
mixture was filtered and the remaining solid was washed with water
and dried under vacuum to afford the title compound (3.39 g, 99%)
as a bright yellow solid, LCMS m/z 295.30 [M+H].sup.+ @ R.sub.T
0.84 min.
EXAMPLE 1
N-[4-(6-Fluoro-3-oxo-3,5-dihydropyrazolo[4,3-c]quinolin-2-yl)-phenyl]-2-me-
thyl-butyramide
[0111] 8
[0112] (.+-.)-2-Methylbutyryl chloride (13.6 .mu.l, 0.11 mmol) was
added dropwise over 30 sec to a stirred solution of
2-(4-amino-phenyl)-6-fluoro-
-2,5-dihydro-pyrazolo[4,3-c]quinolin-3-one (Intermediate 2) (30 mg,
0.10 mmol), triethylamine (14 .mu.l, 0.11 mmol) and
4-dimethylaminopyridine (2.4 mg, 0.02 mmol) in dichloromethane (1
ml) at room temperature. The mixture was stirred at room
temperature for 16 h. The yellow solid was then filtered and
purified by washing sequentially with a saturated solution of
sodium bicarbonate (1 ml), ethyl acetate (1 ml) and ethyl alcohol
(0.5 ml) and finally dried under suction to give the title compound
(10 mg, 26%) as a bright yellow solid, LCMS m/z 379.36 [M+H].sup.+
@ R.sub.T 1.18 min. .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 9.89
(1H, s), 8.52 (1H, s), 8.15 (2H, d J 9.0 Hz), 8.01 (1H, d J 7.0
Hz), 7.69 (2H, d J 9.0 Hz) 7.57-7.46 (2H, m), 2.46-2.39 (1H, m),
1.69-1.36 (2H, m), 1.11 (3H, d J 6.8 Hz), 0.91(3H, t J 7.3 Hz).
[0113] The title compound, and compounds of subsequent Examples,
were tested in the assay described below in the Assay Section, to
determine their activities as inhibitors of the CD80-CD28
interaction. The present title compound had an activity rating of
***.
EXAMPLES 2-49
[0114] The following compounds were synthesized by the route
described in Example 1, substituting the appropriate acid chloride
for (.+-.)-2-methylbutyryl chloride:
EXAMPLE 2
2-Methyl-pentanoic acid
[4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quin-
olin-2-yl)-phenyl]-amide
[0115] 9
[0116] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 9.92 (1H, s),
8.53 (1H, s) 8.12 (2H, d J 9.2 Hz), 8.05 (1H, d J 7.6 Hz), 7.70
(2H, d J 9.2 Hz), 7.63-7.53 2H, m), 1.68-1.58 (1H, m), 1.38-1.28
(3H, m), 1.11 (3H, d J 6.6 Hz), 0.91 (3H, t J 7.1 Hz).
[0117] Activity ***
EXAMPLE 3
1-Methyl-1H-pyrrole-2-carboxylic acid
[4-(6-fluoro-3-oxo-3,5-dihydro-pyraz-
olo[4,3-c]quinolin-2-yl)-phenyl]-amide
[0118] 10
[0119] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 9.76 (1H, s),
8.50 (1H, s), 8.26 (2H, d 9.0 Hz), 7.97-7.94 (1H, m), 7.73 (2H, d J
9.0 Hz), 7.39-7.28 (2H, m), 7.07-7.01 (2H, m), 3.91 (3H, s)
[0120] Activity *
EXAMPLE 4
N-[4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-3-m-
ethyl-butyramide
[0121] 11
[0122] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 9.92 (1H, s),
8.52 (1H, s), 8.14 (2H, d J 9.2 Hz), 8.01 (1H, d J 7.3 Hz), 7.67
(2H, d J 9.2 Hz), 7.57-7.47 (2H, m), 2.21 (2H, d J 6.8 Hz),
2.14-2.07 (1H, m), 0.96 (6H, d J 6.6 Hz).
[0123] Activity **
EXAMPLE 5
2-Propyl-pentanoic acid
[4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quin-
olin-2-yl)-phenyl]-amide
[0124] 12
[0125] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 9.93 (1H, s),
8.53 (1H, s), 8.11 (2H, d J 9.0 Hz), 8.05 (1H, d J 7.8 Hz), 7.70
(2H, d J 9.0 Hz), 7.59-7.46 (2H, m), 2.46-2.35 (1H, m), 1.63-1.27
(4H, m), 0.90(6H, t J 7.1 Hz).
[0126] Activity *
EXAMPLE 6
5-[4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)phenylcarbam-
oyl]-pentanoic acid methyl ester
[0127] 13
[0128] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 9.85 (1H, s),
8.47 (1H, s), 8.25 (2H, d J 9.0 Hz), 7.91-7.90 (1H, m), 7.59 (2H, d
J 9.0 Hz), 7.29-7.20 (2H, m), 3.61 (3H, s), 2.38-2.28 (4H, m),
1.64-1.50 (4H, m)
[0129] Activity ***
EXAMPLE 7
N-[4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-2,2-
-dimethyl-propionamide
[0130] 14
[0131] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 9.26 (1H, S),
8.52 (1H, s), 8.15 (2H, d J 9.2 Hz), 8.03 (1H, d J 8.8 Hz), 7.71
(2H, d J 9.2 Hz), 7.56-7.47 (2H, m), 1.26 (9H, s)
[0132] Activity **
[0133] Examples 8 to 28 were also prepared by the method of Example
1 using the appropriate acid chloride:
1 15 M.S. Example X R (MH+) Activity 8 6-F 16 443.4 ** 9 6-F
--CH.sub.2Cl 371.31 ** 10 6-F 17 389.34 * 11 6-F 18 485.45 * 12 6-F
CO.sub.2Me 381.34 ** 13 6-F OEt 367.18 14 6-F 19 507.43 * 15 6-F 20
466.41 ** 16 6-F Me 337.36 ** 17 6-F
CH(Et)CH.sub.2CH.sub.2CH.sub.2Me 421.46 * 18 6-F CH(Et).sub.2
393.41 *** 19 6-F 21 405.41 ** 20 6-F 22 448.44 ** 21 6-F 23 481.35
** 22 6-F 24 423.42 *** 23 6-F (CH.sub.2).sub.8CO.sub.2Me 493.51 **
24 6-F iPr 365.36 *** 25 6-F CH.sub.2OCH.sub.2CH.sub.2OMe 411.4 **
26 6-F CH(Me) (nPr) 393.42 *** 27 6-F CH.sub.2OMe 367.24 ** 28 6-F
25 390.33 ** 29 6-F CH.sub.2CH.sub.2CH.sub.2N.sup.+(Me).sub.3 422.1
(M+) *** 30 6-F CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 408.3 *** 31
6-F CH.sub.2NHCH.sub.2CH.sub.2CH.sub.2N(Me) (Ph) 499.3 * 32 6-F 26
485.3 * 33 6-F 27 505.1 *** 34 6-F 28 517.2 *** 35 6-F 29 477.1 ***
36 6-F 30 457.1 ** 37 6-F 31 463.1 ** 38 6-F 32 438.3 ** 39 6-F 33
463.2 *** 40 6-F 34 460.4 ** 41 6-F
CH.sub.2NHCH.sub.2CH.sub.2N(iPr).sub.2 479.4 ** 42 6-F 35 420.2 **
43 H CH(NH.sub.2)CH.sub.3 348.3 ** 44 H CH(Me)nPr 375.3 * 45 H iPr
347.3 ** 46 6-F CH(NH.sub.2)CH.sub.3 366.3 *** 47 H CH(Me)Et 361.3
** 48 6-F 36 529.1 ** 49 6-F CH.sub.2N(Me)CH.sub.2Ph 456.4 **
Preparation of Intermediate 3
3-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic
acid
[0134] 37
[0135] 3-Hydrazinobenzoic acid (1.91 g, 0.013 mol) was added in one
portion to a stirred solution of
4-chloro-8-fluoro-quinoline-3-carboxylic acid ethyl ester (2.93 g,
0.011 mol) in n-butanol (60 ml) at room temperature. The solution
was heated to reflux for 16 h, cooled to room temperature and the
resulting yellow solid filtered, washed with tert-butyl methyl
ether and then dried. The solid was redissolved in a solution of
tetrahydrofuran:water (2:1; 21 ml) and lithium hydroxide (1.27 g,
0.031 mol) was then added. After stirring at room temperature for
16 h, concentrated hydrochloric acid (3 ml) was added dropwise to
the mixture to precipitate a yellow solid which was filtered and
dried under vacuum to give the title compound (intermediate 3)
(2.32 g, 63%) as a bright yellow solid.
Preparation of Intermediate 4
3-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoyl
chloride
[0136] 38
[0137] Oxalyl chloride (20 ml, 0.2 mol) was added dropwise over 2
min to a stirred solution of
3-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-
-2-yl)-benzoic acid (intermediate 3) (2.0 g, 6.1 mmol) in
dichloromethane (10 ml) at room temperature. N,N-Dimethylformamide
(50 .mu.l) was then added and the resulting mixture heated to
50.degree. C. for 1 h. The solution was then cooled to room
temperature and then concentrated in vacuo to leave the title
compound (intermediate 4) (2.0 g, 96%) as a beige solid.
EXAMPLE 50
3-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-N-(3-methoxy-p-
ropyl)-benzamide
[0138] 39
[0139] 3-Methoxypropylamine (0.026 g, 0.29 mmol) was added to a
stirred solution of
3-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-b-
enzoyl chloride (intermediate 4) (26 mg 0.29 mmol) in
tetrahydrofuran (2 ml) and the mixture stirred at room temperature
for 15 min. Triethylamine (0.2 ml, 1.4 mmol) was then added and the
resulting mixture stirred overnight. 1 M Hydrochloric acid (3-4 ml)
was added dropwise to precipitate a yellow solid which was filtered
and dried under suction to give the amide (79 mg, 0.20 mmol) as a
yellow solid, LCMS m/z 395.25 [M+H].sup.+ @ R.sub.T 1.04 min;
.delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 8.59 (1H, m), 8.57 (1H,
s), 8.39 (1H, app d J 9.3 Hz), 8.08 (1H, app d J 7.3 Hz), 7.66-7.53
(5H, m), 3.37-3.33 (4H, m), 3.27 (3H, s), 1.83-1.77 (2H, m).
[0140] Activity **
EXAMPLE 51
N-Ethyl-3-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]-quinolin-2-yl)-benza-
mide
[0141] 40
[0142] Prepared by the method of Example 53 substituting ethylamine
for 3-methoxypropylamine.
[0143] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) major rotomer
quoted; 8.56 (1H, br s), 8.47 (1H, m), 8.21 (2H, d J 8.5 Hz), 7.94
(2H, d J 8.5 Hz), 3.96 (3H, s), 3.31 (2H, q J 7.3 Hz), 2.58 (3H,
s), 1.15 (3H, t J 7.4 Hz).
[0144] Activity **
EXAMPLE 52
N-Benzyl-3-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]-quinolin-2-yl)-benz-
amide
[0145] 41
[0146] Prepared by the method of Example 53 substituting
benzylamine for 3-methoxypropylamine.
[0147] LCMS m/z 427.16 [M+H].sup.+ @ R.sub.T 1.28 min.
[0148] Activity *
[0149] Examples 53 to 64 were prepared by the method of example 50,
using the appropriate amine.
2 42 M.S. Example X R R' (MH+) Activity 53 6-F
CH.sub.2CH.sub.2CH.sub.2N(Me).sub- .2 Me 422.5 * 54 6-F
CH.sub.2CH.sub.2l CH.sub.2N(Me).sub.2 H 408.4 ** 55 6-F 43 H 420.4
* 56 6-F 44 H 434.4 * 57 6-F 45 H 448.4 ** 58 6-F
CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 H 422.4 ** 59 6-F
CH.sub.2CH.sub.2OMe H 381.3 ** 60 6-F Et Et 379.3 * 61 6-F
CH.sub.2CO.sub.2Me H 395.2 * 62 6-F CH.sub.2CCH H 361.3 ** 63 6-F
CH.sub.2Ph Me 427.2 ** 64 6-F 46 463.3 *
EXAMPLE 65
N-(3-Dimethylamino
propyl)-4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-
-c)quinolin-2-yl]-benzamide
Step 1
2-cyclopropyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid ethyl
ester
[0150] 47
[0151] A solution of 3-cyclopropyl-3-oxo-propionic acid methyl
ester (6.2 g, 0.038 mols), 2-amino benzoic acid ethyl ester (4.95
g, 0.03 mols) and p-toluene sulfonic acid (0.04 g, 0.2 mmols) in
toluene (25 ml) was heated at 125.degree. C. for 2 h; 15 ml of
solvent was then distilled. To the residual orange solution was
added sodium ethoxide (2 M, 15 ml) in ethanol (reaction mixture
turns red). This red mixture was stirred at 120.degree. C. for 2 h;
15 ml of solvent was again distilled. The reaction mixture was left
to cool to room temperature, diluted with ethyl acetate (1 litre),
extracted with HCl 0.1 M and water. The combined organic extracts
were dried over sodium sulfate and concentrated in vacuo to leave
an orange residue which was washed once with cold ethyl acetate to
yield 2-cyclo-propyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid
ethyl ester (3.87 g, 53%) as an off-white solid. LCMS m/z 244.14
[M+H].sup.+ @ R.sub.T 0.78 min, 89%, m/z 230.11 [Acid+H].sup.+ @
R.sub.T 1.27, 11%.
[0152] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 11.04 (1 H, s),
8.06 (1 H, dd, J.sub.1 1.1, J.sub.2 8.1), 7.76-7.66 (2 H, m), 7.36
(1 H, td, J.sub.1 1.1, J.sub.2 7.5), 3.89 (3 H, s), 2.16 (1 H, m),
1.18 (4 H, d, J 7.0).
Step 2
4-Chloro-2-cyclopropyl-quinoline-3-carboxylic acid ethyl ester
[0153] 48
[0154] Phosphorus oxychloride (0.77 ml, 0.082 mols) was added in
one portion to a suspension of
2-cyclopropyl-4-oxo-1,4-dihydro-quinoline-3-ca- rboxylic acid ethyl
ester (1.0 g, 0.041 mols) in acetonitrile and the mixture was
heated at 75.degree. C. for 90 minutes (becomes a clear solution
above 65.degree. C.). The resulting light brown solution was poured
into saturated sodium bicarbonate (100 ml); the suspension was
extracted with ethyl acetate and the combined organic extracts were
dried and concentrated in vacuo to leave
4-Chloro-2-cyclopropyl-quinoline-3-car- boxylic acid ethyl ester
(1.15 g, 106%) as an off-white solid. R.sub.f(AcOEt)=0.73.
Step 3
4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic
acid
[0155] 49
[0156] 4-Chloro-2-cyclopropyl-quinoline-3-carboxylic acid ethyl
ester (1.15 g, 0.0041 mols) and 4-hydrazino-benzoic acid (1.0 g,
0.0068 mols) were stirred in ethanol (30 ml) at reflux for 16 h.
The bright yellow suspension was diluted with heptane, filtered,
washed with cold t-butylmethyl ether and left to dry under suction
to yield crude solid containing hydrazine. This solid was suspended
in 1 M HCl, filtered, washed with water and then dried in vacuo to
yield
4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic
acid (1.135 g, 80%) as a yellow solid, LCMS m/z 346.20 [M+H].sup.+
@ R.sub.T 1.05 min: 96% purity.
[0157] .delta..sub.H(400 MHz, (CD.sub.3).sub.2SO) 11.4 (1 H, s),
8.43 (2 H, d, J 8.1), 8.21 (1 H, dd, J.sub.1 1.2, J.sub.2 8.1),
8.07 (2 H, d, J 8.1), 7.92 (1 H, d, J 8.1), 7.67 (1 H, t, J 6.6),
7.52 (1 H, t, J 6.5), 3.43 (1 H, m), 1.59 (2 H, m), 1.43 (2 H,
m).
Step 4
4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]-quinolin-2-yl)-benzoyl
chloride
[0158] 50
[0159] To a suspension of finely ground
4-(4-cyclopropyl-3-oxo-3,5-dihydro-
-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid (0.19 g. 0.55 mmol) in
dichloromethane (4 ml) was added oxalyl chloride (1.6 ml, 0.01 mol)
followed by a drop of dimethyl formamide. The mixture was stirred
under nitrogen at 45.degree. C. for 8 h. The solvent was removed in
vacuo to yield
4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-be-
nzoyl chloride as a pale yellow solid, LCMS m/z [M+MeOH--Cl].sup.+
@ R.sub.T 1.46 min: 95% purity. Used without further
purification.
Step 5
N-(3-Dimethylamino
propyl)-4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,3-
-c]quinolin-2-yl]-benzamide
[0160] 51
[0161] To a partial solution of
4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazol-
o[4,3-c]quinolin-2-yl)-benzoyl chloride (0.1 g, 0.28 mmol) in
tetrahydrofurane (6 ml) under nitrogen was added a solution of
3-dimethylamino-propyl amine (0.03 g, 0.3 mmol) in tetrahydrofurane
(3 ml). The mixture was stirred at R.sub.T for 3 h. The solvent was
removed under reduced pressure and the yellow solid was washed with
a little saturated sodium bicarbonate, water and dried under vacuo
to yield N-(3-Dimethylamino
propyl)-4-(4-cyclopropyl-3-oxo-3,5-dihydro-pyrazolo[4,-
3-c]-quinolin-2-yl]-benzamide (57 mg, 47%) as a yellow solid. LCMS
m/z 430.11 [M+H].sup.+ @ R.sub.T 0.99 min: 100% purity.
[0162] Activity ***
Preparation of Intermediate 5
4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benzoyl
chloride
[0163] 52
[0164] To a suspension of finely ground
4-(6-Fluoro-3-oxo-3,5-dihydro-pyra-
zolo[4,3-c]quinolin-2-yl]-benzoic acid (1.1 g. 3.4 mmol) in
dichloromethane (6 ml) was added oxalyl chloride (2.4 ml, 29 mmol)
followed by a drop of dimethyl formamide. The mixture was stirred
under nitrogen at 45.degree. C. for 3 h. The solvent was removed in
vacuum to yield 4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,
3-c]quinolin-2-yl]-benzoy- l chloride (1.15 g, quantitative) as a
pale yellow solid that was used without further purification.
EXAMPLE 66
N-(3-Dimethylamino
propyl)-4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]qu-
inolin-2-yl]-benzamide hydrochloride
[0165] 53
[0166] To a partial solution of
4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-
-c]quinolin-2-yl]-benzoyl chloride (0.1 g, 0.3 mmol) in
tetrahydrofurane (5 ml) under nitrogen was added a solution of
3-dimethylamino-propyl amine (0.03 g, 0.3 mmol) in
tetrahydrofurane. The mixture was stirred at rt for 90 minutes. The
solvent was removed under reduced pressure and the yellow solid was
purified via FCC silica gel (gradient elution, MeOH:H.sub.2O, Fluka
C.sub.18 reverse phase) to yield N-(3-Dimethylamino
propyl)-4-(6-fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl]-benza-
mide hydrochloride (70 mg, 53%) as a yellow solid.
[0167] LCMS m/z 408.39 [M+H].sup.+ @ R.sub.T 0.89 min: 90%
purity.
[0168] Activity ***
EXMAPLES 67-141
Were Prepared Analogously From the Appropriate Benzoyl Chloride and
the Appropriate Amine
[0169]
3 54 M.S. Example X Z W R R' (MH+) Activity 67 6-F H H
--CH.sub.2CH.sub.2CH.sub.- 2CH.sub.2CH.sub.2-- 391.3 ** 68 6-F H H
--CH.sub.2Phenyl H 413.2 *** 69 6-F H H --CH.sub.2Phenyl Me 427.3
** 70 6-F H H --CH.sub.2CH.sub.2OMe H 381.2 *** 71 6-F H H
--CH.sub.2CH.sub.2N(Me).sub.2 H 394.3 *** 72 6-F H H
--CH.sub.2CO.sub.2Me H 395.3 *** 73 6-F H H
--CH.sub.2CH.sub.2CH.sub.2OMe H 395.2 *** 74 6-F H H
--CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 H 408.3 *** 75 6-F H H 55 H
431.3 ** 76 6-F H H 56 H 419.2 ** 77 6-F H H Et H 351.2 *** 78 6-F
H H Et Et 379.3 ** 79 6-F H H 57 H 420.4 *** 80 6-F H H
--CH.sub.2CH.sub.2CH.sub.2N(Me).sub.- 2 Me 422.4 *** 81 6-F H H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Me).s- ub.2 H 422.4 *** 82 6-F
H H 58 H 448.5 *** 83 6-F H H 59 H 434.4 *** 84 6-F H H 60 H 525.3
*** 85 6-F H H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Me).sub.- 2 H 450.3 ***
86 H H H --CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 H 390.2 *** 87 H H H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Me)- .sub.2 H 432.1 **
88 H H H --CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Et).- sub.2 H 432.2 **
89 H H H --CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 Me 404.2 ** 90 6-F H
2--Cl --CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 H 442.1 ** 91 H H H 61
H 416.1 ** 92 H H H 62 H 573.0 ** 93 H H H 63 H 445.1 ** 94 H H H
64 H 507.1 ** 95 6-F H H 65 H 591.0 *** 96 H 66 H
--CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 H 430.1 *** 97 6-F H H 67 H
464.1 *** 98 6-F H H 68 H 463.1 *** 99 6-F H 3--Cl 69 H 482.1 **
100 6-F H 2--Cl 70 H 497.1 ** 102 6-F H 2--Cl
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Et).sub.2 H 484.1 ** 103 6-F H
3--Cl --CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 H 442.1 ** 104 H 71 H
72 H 470.4 *** 105 6-F H H 73 516.3 * 106 6-F H H 74 H 470.3 ***
107 6-F H H --CH.sub.2CH.sub.2N(iPr).sub.2 H 451.4 *** 108 6-F H
2--Cl 75 H 496.2 ** 109 6-F H H 76 H 456.1 *** 110 6-F H 2--Cl
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(M- e).sub.2 H 456.1 ** 111 6-F
H H 77 406.2 ** 112 6-F H H 78 H 462.1 *** 113 6-F H H 79 H 436.1
*** 114 6-F H H 80 H 434.4 *** 115 6-F H H 81 H 476.1 *** 116 6-F H
H 82 H 496.1 *** 117 6-F H H 83 H 436.3 *** 118 6-F H H 84 H 462.3
*** 119 6-F H H 85 H 428.1 ** 120 6-F H H --CH.sub.2CH.sub.2SEt H
411.3 *** 121 6-F H H 86 H 448.3 ** 122 6-F H H 87 H 431.3 *** 123
6-F H H 88 H 434.3 ** 124 6-F H H
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Et).sub.2 H 450.4 *** 125 6-F
89 H 90 H 536.1 *** 126 6-F 91 H 92 H 516.2 *** 127 6-F H H 93 H
428.3 * 128 6-F H H --CH.sub.2CH.sub.2CH.sub.2SMe H 411.3 ** 129 H
94 H 95 H 498.5 *** 130 6-F 96 H 97 H 488.4 *** 131 6-F H H 98 H
446.3 *** 132 6-F 99 H --CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 H
448.2 *** 133 6-F 100 H 101 H 502.3 *** 134 6-F 102 H 103 H 486.3
*** 135 6-F 104 H --CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Et).sub.2 H
490.3 *** 136 6-F 105 H 106 H 546.2 ** 137 6-F 107 H 108 H 631.2
*** 138 6-F 109 H 110 H 468.2 ** 139 6-F 111 H 112 H 468.2 * 140
6-F 113 H 114 H 476.2 *** 141 6-F 115 H 116 H 474.3 ***
EXAMPLE 142
{3-[4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-ur-
eido}acetic acid ethyl ester
[0170] 117
[0171] Ethyl cyanatoacetate (31 mg, 0.24 mmol) was added in one
portion to a stirred solution of
2-(4-aminophenyl)-6-fluoro-2,5-dihydropyrazolo[4,3--
c]quinolin-3-one (intermediate 2) (50 mg, 0.17 mmol) in
N,N-dimethylformamide (2 ml) and the mixture stirred at room
temperature for 16 h. Water (1 ml) was then added to the mixture to
precipitate a solid, which was filtered, washed with water (1 ml)
and then ethyl acetate (1 ml) and finally dried by suction to leave
the urea as a yellow solid, LCMS m/z 424.40 [M+H].sup.+ @ R.sub.T
1.06 min.
[0172] Activity ***
EXAMPLES 143 and 144
[0173]
4 118 Example 143 LCMS m/z 438.41 [M + H.sup.]+@ RT 1.13 min.
Activity ** 119 Example 144 LCMS m/z 514.46 [M + H.sup.]+@ RT 1.35
min. Activity *
[0174] The following compounds were synthesised by the method of
Example 142, substituting the appropriate isocyanate,
isothiocyanate or chloroformate for ethyl cyanatoacetate.
5 120 M.S. Example X Z Y R A (MH +) Activity 144 6-F H O iPr NH
380.3 *** 145 6-F H O nPr NH 380.3 *** 146 6-F H O tBu NH 394.4 ***
147 6-F H O Ph NH 414.3 ** 148 6-F H S 121 NH 394.3 ** 149 6-F H S
122 NH 436.4 * 150 6-F H O tBu O 395.3 *** 151 6-F H O Et O 367.2
** 152 6-F H O CH.sub.2CH.sub.2N(Me).sub.2 O 410.2 *** 153 H 123 O
Me O 375.3 ** 154 6-F H O CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 O
424.1 *** 155 6-F H O 124 O 512.3 ** 156 6-F H S nPentyl NH 424.4
** 157 6-F H S CH(CH.sub.3)CH(CH.sub.3)CH.sub.3 NH 424.4 ** 158 6-F
H O CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Et).sub.2 NH 465.4 *** 159 H
H O nPr NH 362.3 *** 160 H H S 125 NH 376.1 ** 161 6-F H O
CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2 NH 423.3 *** 162 H H O 126 NH
434.5 *** 163 6-F H O CH.sub.2CH.sub.2CH.sub.2CH.sub.2N(Me).sub.2
NH 437.2 *** 164 6-F H O 127 NH 463.5 ***
Intermediate 6: Preparation of methyl
4-oxothiochromane-3-carboxylate
[0175] 128
[0176] Dry tetrahydrofuran (60 ml) was cooled under nitrogen
atmosphere to -50 to -60.degree. C. 1M Lithium
bis(trimethylsily)amide solution in hexane (56 ml, 56 mmol) was
added. The temperature was kept at -50 to -60.degree. C. and
thiochroman-4-one was added dropwise over 20 min. Stirring was
continued at low temperature for 60 min. Methyl cyanoformate (4.84
ml, 60.9 mmol) was added dropwise over 5 min to the reaction
mixture. The obtained suspension was stirred at -50 to -60.degree.
C. for 80 min and then allowed to warm up to room temperature.
Saturated ammonium chloride solution (100 ml) was added. The phases
were separated, the aqueous phase extracted with ethyl acetate
(2.times.100 ml). The combined organic phases were washed with
water (50 ml), dried over magnesium sulphate, filtered and
concentrated under vacuum. An orange oil was obtained and purified
by column chromatography. The title compound was isolated as a
yellow solid (4.70 g, 21.1 mmol, 42%). LCMS: m/z 221
[M-H].sup.+.
Intermediate 7: Preparation of
4-(3-Oxo-3a,4-dihydro-3H-thiochromeno[4,3-c- ]pyrazol-2-yl)-benzoic
acid
[0177] 129
[0178] 4-Oxothiochromane-3-carboxylate (0.50 g, 2.25 mmol) and
hydrazinobenzoic acid (0.377 g, 2.48 mmol) were mixed in acetic
acid (6 ml). The mixture was heated to reflux for 30 min. Excess
acetic acid was distilled off to give a brown oil. Diethylether was
added, a precipitate formed which was collected by filtration and
dried under vacuum. The crude product was isolated as a red/brown
solid (797 mg). LCMS: m/z 325 [M+H].sup.+. No purification was
carried out.
Intermediate 8: Preparation of
4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl- )benzoic acid
[0179] 130
[0180] Crude
4-(3-Oxo-3a,4-dihydro-3H-thiochromeno[4,3-c]pyrazol-2-yl)-ben- zoic
acid (250 mg, 0.77 mmol) was dissolved in dimethyl sulphoxide (6
ml). O-Chloranil (189 mg, 0.77 mmol) was added and the mixture was
stirred at room temperature overnight. Water (20 ml) was added and
the solids were collected by filtration and washed with water. The
filter cake was triturated with toluene, filtered and dried under
vacuum. The title compound was isolated as a dark brown solid (230
mg, 0.71 mmol, 92%). LCMS: m/z 323 [M+H].sup.+
[0181] Alternatively crude
4-(3-Oxo-3a,4-dihydro-3H-thiochromeno[4,3-c]pyr- azol-2-yl)-benzoic
acid can be stirred in dimethyl sulphoxide under exposure to air.
It was found that air oxidation provides clean product, however the
reaction is much slower.
EXAMPLE 165
Preparation of
N-[3-(dimethylamino)propyl]-4-(3-oxothiochromeno[4,3-c]pyra-
zol-2(3H)-yl)benzamide
[0182] 131
[0183] 4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzoic acid (55
mg, 0.17 mmol) was suspended in anhydrous dimethyl acetamide (1
ml). Diisopropyl-ethyl amine (46.5 mg, 0.36 mmol, 62.mu.l) was
added followed by 3-dimethylaminopropylamine (17.5 mg, 0.17 mmol)
and
[(benzotriazol-1-yloxy)-dimethylamino-methylene]-dimethyl-ammonium
hexafluoro phosphate (65 mg, 0.17 mmol). The mixture was stirred at
room temperature for 4 h and was purified by preparative HPLC. The
title compound was isolated as a brown solid. LCMS: m/z 407
[M+H].sup.+
[0184] Activity **
EXAMPLE 166
Preparation of
N-[(cyclohexylamino)propyl]-4-(3-oxothiochromeno[4,3-c]pyra-
zol-2(3H)-yl)benzamide
[0185] 132
[0186] The reaction was carried out as described above. LCMS: m/z
461 [M+H].sup.+
[0187] Activity ***
EXAMPLE 167
Preparation of
N-(pyrrolidin-1-yl-butyl)-4-(3-oxothiochromeno[4,3-c]pyrazo-
l-2(3H)-yl)benzamide
[0188] 133
[0189] The reaction was carried out as described above. LCMS: m/z
447 [M+H].sup.+
[0190] Activity *
EXAMPLE 168
Preparation of
4-(3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)-N-1,2,2,6,6-pe-
ntamethylpiperidin-4-ylbenzamide
[0191] 134
[0192] The reaction was carried out as described above. LCMS: m/z
475 [M+H].sup.+
[0193] Activity **
Intermediate 9: Preparation of
3-[(2-fluorophenyl)sulfanyl]propanoic acid
[0194] 135
[0195] 2-Fluorothiophenol (5.0 g, 39 mmol) was dissolved in
tetrahydrofuran (50 ml) under a nitrogen atmosphere. Triethylamine
(3.94 g, 5.33 ml, 85.8 mmol) was added. Acrylic acid (2.81 g, 2.67
ml, 39 mmol) was dissolved in tetrahydrofuran and added dropwise to
the reaction solution over 2 h at room temperature. The mixture was
stirred at room temperature overnight. 1M Hydrochloric acid (50 ml)
was added and the phases were separated. The aqueous phase was
washed with ethyl acetate (2.times.50 ml). The combined organic
phases were dried over magnesium sulphate, filtered and
concentrated under vacuum. A yellow oil was obtained which
solidified upon storage at room temperature. The solid was
triturated with hexane, filtered and dried under vacuum. The title
compound was isolated as an off-white solid (4.19 g, 20.9 mmol,
54%).
Intermediate 10: Preparation of
8-fluoro-2,3-dihydro-4H-thiochromen-4-one
[0196] 136
[0197] 3-[(2-Fluorophenyl)sulfanyl]propanoic acid (4.0 g, 20 mmol)
was mixed with concentrated sulphuric acid (20 ml) at 0-5.degree.
C. The reaction solution was stirred at 0 to 5.degree. C. for 3 h
then allowed to warm up to room temperature overnight. The mixture
was quenched dropwise into ice to give a white suspension. The
aqueous phase was extracted with ethyl acetate (1.times.200 ml,
1.times.100 ml). The combined organic phases were washed with
saturated sodium bicarbonate solution (1.times.50 ml), water
(1.times.50 ml), 1M hydrochloric acid (50 ml) and water (2.times.50
ml). The organic phase was dried over magnesium sulphate, filtered
and concentrated under vacuum. The title compound was isolated as a
yellow solid (2.10 g, 11.5 mmol, 58%).
Intermediate 11: Preparation of methyl
8-fluoro-4-oxothiochromane-3-carbox- ylate
[0198] 137
[0199] 1M Lithium hexamethyldisilazide solution in hexane (13.2 ml)
was dissolved in anhydrous tetrahydrofuran (20 ml) under nitrogen
atmosphere. The solution was cooled to -78.degree. C.
8-Fluoro-2,3-dihydro-4H-thiochr- omen-4-one (2.00 g, 11 mmol) was
dissolved in tetrahydrofuran (40 ml), the solution was transferred
to the dropping funnel and added dropwise over 30 min to the
reaction mixture maintaining the temperature below -60.degree. C.
An orange clear solution was obtained which was stirred at
-78.degree. C. to -65.degree. C. for 2 h. Methyl cyanoformate
(0.935 g, 0.87 ml) was dissolved in tetrahydrofuran (2 ml) and
added dropwise to the reaction solution. Stirring was continued at
low temperature for 1 h, the mixture was then allowed to warm to
room temperature. Saturated ammonium chloride solution (20 ml) and
water (10 ml) were added, the phases mixed for 5 min and separated.
The aqueous phase was washed with ethyl acetate (2.times.100 ml)
and the combined organic phases were dried over magnesium sulphate.
The mixture was filtered and the solvent removed under vacuum to
give an orange oil. The crude oil was purified by column
chromatography; mobile phase: hexanes, gradient to hexanes/ethyl
acetate [90:10]. The title compound was isolated as a yellow solid
(1.19 g, 4.95 mmol, 45%).
Intermediate 12: Preparation of
4-(6-fluoro-3-oxothiochromeno[4,3-c]pyrazo- l-2(3H)-yl)benzoic
acid
[0200] 138
[0201] Methyl 8-fluoro-4-oxothiochromane-3-carboxylate (1.19 g,
4.95 mmol) and 4-hydrazinobenzoic acid (755 mg, 4.95 mmol) were
mixed with glacial acetic acid (10 ml). The mixture was heated to
reflux for 4 h. Excess acetic acid was removed under vacuum to give
an orange oil. Ethyl acetate (10 ml) was added and the mixture
sonicated. Precipitation of an orange solid was observed. The
solids were collected by filtration and washed with ethyl acetate.
The filter cake was taken up in dimethyl suphoxide (10 ml) and
air-oxidised at room temperature for one week. Water (20 ml) was
added to the reaction mixture, the solids were collected by
filtration, slurried in ethyl acetate, filtered and dried under
vacuum. The title compound was isolated as an orange powder (175
mg, 0.51 mmol, 10%). LCMS: m/z 341.
EXAMPLE 169
Preparation of
N-[3-(dimethylamino)propyl]-4-(6-fluoro-3-oxothiochromeno[4-
,3-c]pyrazol-2(3H)-yl)benzamide
[0202] 139
[0203] 4-(6-Fluoro-3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)benzoic
acid (41 mg, 0.12 mmol) was dissolved in anhydrous
dimethyl-acetamide(1 ml). Diisopropyl-ethyl amine (46 mg, 0.36
mmol, 62 .mu.l) was added followed by
[(benzotriazol-1-yloxy)-dimethylamino-methylene]-dimethyl-ammonium
hexafluoro phosphate (65 mg, 0.17 mmol) and
3-dimethylaminopropylamine (12 mg, 0.12 mmol). The mixture was
stirred at room temperature overnight and purified by preparative
HPLC. The title compound was isolated as a brown solid. LCMS: m/z
425 [M+H].sup.+.
[0204] Activity **
EXAMPLE 170
Preparation of
N-[(cyclohexylamino)propyl]-4-(6-fluoro-3-oxothiochromeno[4-
,3-c]pyrazol-2(3H)-yl)benzamide
[0205] 140
[0206] The reaction was carried out as described above. LCMS: m/z
479 [M+H].sup.+.
[0207] Activity **
EXAMPLE 171
Preparation of
N-(pyrrolidin-1-yl-butyl)-4-(6-fluoro-3-oxothiochromeno[4,3-
-c]pyrazol-2(3H)-yl)benzamide
[0208] 141
[0209] The reaction was carried out as described above. LCMS: m/z
465 [M+H].sup.+.
[0210] Activity ***
EXAMPLE 173
Preparation of
4-(6-fluoro-3-oxothiochromeno[4,3-c]pyrazol-2(3H)-yl)-N-1,2-
,2,6,6-pentamethylpiperidin-4-ylbenzamide
[0211] 142
[0212] The reaction was carried out as described above. LCMS: m/z
493 [M+H].sup.+
[0213] Activity ***
Assay Section
[0214] The examples described above were tested in a cell free
Homogenous Time Resolved Fluorescence (HTRF) assay to determine
their activity as inhibitors of the CD80-CD28 interaction.
[0215] In the assay, europium and allophycocyanin (APC) are
associated with CD28 and CD80 indirectly (through antibody linkers)
to form a complex, which brings the europium and APC into close
proximity to generate a signal. The complex comprises the following
six proteins: fluorescent label 1, linker antibody 1, CD28 fusion
protein, CD80 fusion protein, linker antibody 2, and fluorescent
label 2. The table below describes these reagents in greater
detail.
6 Fluorescent Anti-Rabbit IgG labelled with Europium label 1 (1
.mu.g/ml) Linker Rabbit IgG specific for mouse Fc antibody 1
fragment (3 .mu.g/ml) CD28 fusion CD28 - mouse Fc fragment fusion
protein protein (0.48 .mu.g/ml) CD80 fusion CD80 mouse Fab fragment
(C215) fusion protein protein (1.9 .mu.g/ml) Linker
G.alpha.M.kappa.-biotin: biotinylated goat IgG antibody 2 specific
for mouse kappa chain (2 .mu.g/ml) Fluorescent SA-APC: streptavidin
labelled label 2 allophycocyanin (8 .mu.g/ml)
[0216] On formation of the complex, europium and APC are brought
into proximity and a signal is generated.
[0217] Non-specific interaction was measured by substituting a
mouse Fab fragment (C215) for the CD80 mouse Fab fragment fusion
protein (1.9 .mu.g/ml). The assay was carried out in black 384 well
plates in a final volume of 30.mu.l. Assay buffer: 50 mM Tris-HCl,
150 mM NaCl pH 7.8, containing 0.1% BSA (w/v) added just prior to
use.
[0218] Compounds were added to the above reagents in a
concentration series ranging between 100 .mu.M-1.7 nM. The reaction
was incubated for 4 hours at room temperature. Dual measurements
were made using a Wallac Victor 1420 Multilabel Counter. First
measurement: excitation 340 nm, emission 665 nm, delay 50 .mu.s,
window time 200 .mu.s. second measurement: excitation 340 nm,
emission 615 nm, delay 50 .mu.s, window time 200 .mu.s. Counts were
automatically corrected for fluorescence crossover, quenching and
background.
[0219] By way of illustration, the EC.sub.50 results for the
compounds of Examples 15, 21, 29, 35 and 83 were 8 .mu.M, 1.9
.mu.M, 950 nM, 148 nM and 90 nM respectively. For convenience, the
EC50 activities of compounds tested are recorded above in summary
form as:
EC50: *=>10 .mu.M, **=1-10 .mu.M, ***=<1 .mu.M.
* * * * *