Pyrazoloquinolines with immunomodulating activity The present invention relates to novel heterocyclic compounds, to methods for their preparation, to composi- tions containing them, and to methods and use for clini- cal treatment of medical conditions which may benefit from immunomodulation, including rheumatoid arthritis, multiple sclerosis, diabetes, asthma, transplantation, systemic lupus erythematosis and psoriasis. More parti- cularly the present invention relates to novel hetero- cyclic compounds, which are CD80 antagonists capable of inhibiting the interactions between CD80. and CD28.

Background of the invention The immune system possesses the ability to control the homeostasis between the activation and inactivation of lymphocytes through various regulatory mechanisms du- ring and after an immune response. Among these are. mecha- nisms that specifically inhibit and/or turn off an immune response. Thus, when an antigen is presented by MHC mole- cules to the T-cell receptor, the T-cells become properly activated only in the presence of additional co-stimula- tory signals. In the absence of accessory signals there is no lymphocyte activation and either a state of func- tional inactivation termed anergy or tolerance is indu- ced, 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) A paper by Erbe et al, in J. Biol. Chem. Vol. 277, No. 9, pp 73. 63-7368 (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:

A B DESCRIPTION OF THE INVENTION According to the present invention there is provided a compound of formula (I) or a pharmaceutically or vete- rinarily acceptable salt thereof: wherein R1 and R3 independently represent H; F; Cl ; Br ; -NO2 ; -CN ; C1-C6 alkyl optionally substituted by F or Cl ; or C1- C6 alkoxy optionally substituted by F; R2 represents H, or optionally substituted C1-C6 alkyl, C3-C7 cycloalkyl or optionally substituted phenyl; Y represents-O-,-S-, N-oxide, or-N (R5)-wherein Rs represents H or C1-C6 alkyl ; X represents a bond or a divalent C1-C6 alkylene radical; R4 represents-C (=O) NR6R7, -NR7C (=O) R6, -NR7C (=O) OR6, -NHC (=O) NHR6, or-NHC (=S) NHR6 wherein R6 represents H, or a radical of formula- (Alk) b-Q wherein b is 0 or 1, and

Alk is an optionally substituted divalent straight chain or branched Cl-Cl2 alkylene, C2-Ci2 alkenylene or C2- C12 alkynylene radical which may be interrupted by one or more non-adjacent-O-,-S-or-N (R8)- radicals wherein R8 represents H or Cl-C4 alkyl, C3-C4 alkenyl, C3-C4 alkynyl, or C3-C6 cycloalkyl, and Q represents H;-CF3 ; -OH;-SH ;-NR8R8 wherein each R8 may be the same or different; an ester group; or an optionally substituted phenyl, C3-C7 cycloalkyl, C5-C7 cycloalkenyl or heterocyclic ring having from 5 to 8 ring atoms; and R7 represents H or CL-CE, alkyl ; or when taken to- gether with the atom or atoms to which they are attached R6 and R7 form an optionally substituted heterocyclic ring having from 5 to 8 ring atoms.

Compounds of general formula (I) are CD80 antago- nists. They inhibit the interaction between CD80 and CD28 and thus the activation of T cells, thereby modulating the immune response.

Accordingly the invention also includes : (i) a compound of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof for use in the treatment of conditions which benefit from immunomodula- tion.

(ii) the use of a compound of formula (I) or a phar- maceutically or veterinarily acceptable salt thereof in the manufacture of a medicament for the treatment of con- ditions which benefit from immunomodulation,.

(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.

(iv) a pharmaceutical or veterinary composition com- prising a compound of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof together with a

pharmaceutically or veterinarily acceptable excipient or carrier.

Conditions which benefit from immunomodulation in- clude: Adrenal insufficiency Allergic angiitis and granulomatosis Amylodosis Ankylosing spondylitis Asthma Autoimmune Addison's disease Autoimmune alopecia Autoimmune chronic active hepatitis Autoimmune hemolytic anemia Autoimmune neutropenia Autoimmune thrombocytopenic purpura Autoimmune vasculitides Behçet's disease Cerebellar degeneration Chronic active hepatitis Chronic inflammatory demyelinating polyradiculoneuropathy Dermatitis herpetiformis Diabetes Eaton-Lambert myasthenic syndrome Encephalomyelitis Epidermolysis bullosa Erythema nodosa Gluten-sensitive enteropathy Goodpasture's syndrome Graft versus host disease Guillain-Barre syndrome Hashimoto's thyroiditis Hyperthyrodism Idiopathic hemachromatosis Idiopathic membranous glomerulonephritis Minimal change renal disease Mixed connective tissue disease Multifocal motor neuropathy

Multiple sclerosis Myasthenia gravis Opsoclonus-myoclonus syndrome Pemphigoid Pemphigus Pernicious anemia Polyarteritis nodosa Polymyositis/dermatomyositis Post-infective arthritides Primary biliary sclerosis Psoriasis Reactive arthritides Reiter's disease Retinopathy Rheumatoid arthritis Sclerosing cholangitis Sjogren's syndrome Stiff-man syndrome Subacute thyroiditis Systemic lupus erythematosis Systemic sclerosis (scleroderma) Temporal arteritis Thromboangiitis obliterans Transplantation rejection Type I and type II autoimmune polyglandular syndrome Ulcerative colitis Uveitis Wegener's granulomatosis As used herein the term"alkylene"refers to a straight or branched alkyl chain having two unsatisfied valencies, for example-CH2-,-CH2CH2-,-CH2CH2CH2-, - CH (CH3) CH2-,-CH (CH2CH3) CH2CH2CH3, and-C (CH3) 3.

As used herein the term"heteroaryl"refers to a 5- or 6-membered aromatic ring containing one or more he- teroatoms. Illustrative of such groups are thienyl, fu- ryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thia-

diazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimi- dinyl, pyrazinyl, triazinyl.

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 hetero- atoms selected from S, N and O, including for example, pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyra- zolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzofuranyl, pyranyl, isoxazolyl, quinuclidinyl, aza-bicyclo [3.2. 1] octanyl, benzimidazolyl, methylenedioxyphenyl, maleimido and suc- cinimido groups.

Unless otherwise specified in the context in which it occurs, the term"substituted"as applied to any moie- ty herein means substituted with one or more of the fol- lowing substituents, namely (Cl-C6) alkyl, trifluoromethyl, (Cl-C6) alkoxy (including the special case where a ring is substituted on adjacent ring C atoms by methylenedioxy or ethylenedioxy), trifluoromethoxy, (Cl-C6) alkylthio, phe- nyl, benzyl, phenoxy, (C3-C8) cycloalkyl, hydroxy, mercap- to, amino, fluoro, chloro, bromo, cyano, nitro, oxo, <BR> <BR> <BR> - COOH,-SO2OH,-CONH2,-SO2NH2,-CORA,-COORA,-SO2ORA,<BR> <BR> <BR> <BR> <BR> <BR> - NHCORA,-NHS02RA,-CONHRA,-SO2NHRA,-NHRA,-NRARB, -CONRARB or-SO2NRARB wherein RA and R3 are independently a (Cl-C6) alkyl group. In the case where"substituted"means substituted by (C3-C8) cycloalkyl, phenyl, benzyl or phe- noxy, the ring thereof may itself be substituted with any of the foregoing, except (C3-C8) cycloalkyl phenyl, benzyl or phenoxy.

As used herein the unqualified term"carbocyclyl"or "carbocyclic"refers to a 5-8 membered ring whose ring atoms are all carbon.

Some compounds of the invention contain one or more chiral centres because of the presence of asymmetric car- bon atoms. The presence of asymmetric carbon atoms gives

rise to stereoisomers or diastereoisomers with R or S stereochemistry at each chiral centre. The invention in- cludes all such stereoisomers and diastereoisomers and mixtures thereof.

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.

In the compounds of the invention the following are examples of the several structural variables: R1 may be, for example, H, F, C1, methyl, methoxy, or methylenedioxy. Currently it is preferred that R1 is H, Cl or especially F; R2 may be, for example H, methyl, methoxy, cyclopro- pyl, phenyl, or fluoro-, chloro-, methyl, or methoxy- substituted phenyl. H or cyclopropyl is presently prefer- red; R3 may be, for example, H, F, C1, methyl, methoxy, or methylenedioxy. Currently it is preferred that R3 is F or Cl, and it is most preferred that R3 be H; Y may be, for example,-O-,-S-, or-N (Rs)- wherein R5 represents H or methyl. -NH-or-S-is presently preferred.

X may be, for example a bond, or a-CH2-or-CH2CH2- radical. A bond is presently preferred.

R4 represents-C (=O) NR6R-7,-NR-7C (=O) R6,-NR, C (=0) OR6, -NHC (=O) NHR6, or-NHC (=S) NHR6. Of these-NR, C (=O) R6, and especially-C (=O) NR6R and-NHC (=O) NHR6 are curently preferred. R7 is preferably H, but a wide range of R6 substituents have given rise to highly active compounds

of the invention. Many exemplary R6 substituents appear in the compounds of the Examples below.

R6 may be, for example, H or a radical of formula - Alkb-Q wherein b is 0 or 1 and Alk may be, for example a- (CH2) n-, - CH ( (CH2) mCH3) (CH2) n-,- ( (CH2) MCH3) ( (CH2) pCH3) (CH2) - (CH2) n-0- (CH2) m-,- (CH2) n-NH- (CH2) m-, or - (CH2) n-NH- (CH2) m-NH- (CH2) p- radical where n is 1,2, 3 or 4 and m and p are independently 0,1, 2,3 or 4, and Q may represent H, -OH,-COOCH3, phenyl, cyclopro- pyl, cyclopentyl, cyclohexyl, pyridyl, furyl, thienyl, or oxazolyl; and R7 may be, for example, H, or when taken together with the atom or atoms to which they are attached R6 and R7 may form a heterocyclic ring of 5, 6 or 7 members.

Specific examples of R4 groups include those present in the compounds of the Examples herein.

Compounds of the invention may be prepared by syn- thetic 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 R4 is a group-NR7C (=O) R6 may be prepared by acylation of an amine of formula (II) with an acid chloride of formula (III): Compounds of the invention wherein R4 is a group -NHC (=O) NHR6 may be prepared by reaction of an amine of formula (IIA) with an isocyanate of formula (IIIA)

Compounds of the invention wherein R4 is a group -C (=O) NHR6 may be prepared by reaction of an acid chloride of formula (IIB) with an amine NHR6R7 :

Compounds of the invention wherein R4 is a group -NR7C (=O) OR6 may be prepared by reaction of an amine of formula (II) with a chloroformate ClC (=O) OR6

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 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 nit- rogen, 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] + @ RT 1.47 min.

Preparation of Intermediate 2 2- (4-Aminophenyl)-6-fluoro-2, 5-dihydropyrazolo [4, 3-c] - quinolin-3-one 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 °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 solu- tion of Rochelles salt (500 ml) and a saturated solution of sodium bicarbonate (500 ml) and stirred at room tem- perature for 2h. The mixture was filtered and the re- maining solid was washed with water and dried under va- cuum to afford the title compound (3.39 g, 99 %) as a bright yellow solid, LCMS m/z 295.30 [M+H] + @ RT 0.84 min.

Example 1 N- [4- (6-Fluoro-3-oxo-3,5-dihydropyrazolo [4,3-c] quinolin- 2-yl)-phenyl]-2-methyl-butyramide ()-2-Methylbutyryl chloride (13.6 µl, 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 il, 0.11 mmol) and 4-dimethylaminopyri- dine (2.4 mg, 0.02 mmol) in dichloromethane (1 ml) at room temperature. The mixture was stirred at room tempe- rature for 16 h. The yellow solid was then filtered and purified by washing sequentially with a saturated solu- tion of sodium bicarbonate (1 ml), ethyl acetate (1 ml) and ethyl alcohol (0.5 ml) and finally dried under suc- tion to give the title compound (10 mg, 26 %) as a bright yellow solid, LCMS m/z 379.36 [M+H] + @ RT 1.18 min. 8H (400 MHz, (CD3) 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).

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 The following compounds were synthesized by the route described in Example 1, substituting the appro- priate acid chloride for ()-2-methylbutyryl chloride: Example 2 2-Methyl-pentanoic acid [4- (6-fluoro-3-oxo-3, 5-dihydro- pyrazolo [4, 3-c] quinolin-2-yl)-phenyl]-amide bH (400 MHz, (CD3) 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).

Activity *** Example 3 l-Methyl-lH-pyrrole-2-carboxylic acid [4- (6-fluoro-3-oxo- 3,5-dihydro-pyrazolo [4, 3-c] quinolin-2-yl)-phenyl]-amide

8H (400 MHz, (CD3) 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).

Activity * Example 4 N- [4- (6-Fluoro-3-oxo-3,5-dihydro-pyrazolo [4, 3-c] quinolin- 2-yl)-phenyll-3-methyl-butyramide 5H (400 MHz, (CD3) 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).

Activity ** Example 5 2-Propyl-pentanoic acid [4- (6-fluoro-3-oxo-3, 5-dihydro- pyrazolo [4, 3-c] quinolin-2-yl)-phenyl]-amide AH (400 MHz, (CD3) 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).

Activity * Example 6 5- [4- (6-Fluoro-3-oxo-3, 5-dihydro-pyrazolo [4, 3-c] quinolin- 2-yl) phenylcarbamoyl]-pentanoic acid methyl ester 8H (400 MHz, (CD3) 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).

Activity *** Example 7 N- [4- (6-Fluoro-3-oxo-3, 5-dihydro-pyrazolo [4, 3-c] quinolin- 2-yl)-phenyl]-2, 2-dimethyl-propionamide

AH (400 MHz, (CD3) 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).

Activity ** Examples 8 to 28 were also prepared by the method of Example 1 using the appropriate acid chloride :

Example X R M. S. Activity (MH+) 8 6-F X-o 443. 4 i 9 6-F-CH2C1 371. 31 ** 10 6-F O A 389. 34 * 11 6-F N 485. 45 N\--/0 12 6-F C°2Me 381. 34 ** 13 6-F OEt 367. 18 14 6-F 507. 43 nor nPr 15 6-F-jV 466. 41 ** N N_ < 16 6-F Me 337. 36 ** 17 6-F CH (Et) CHZCH2CHZMe 421. 46 * 18 6-F CH (Et) 2 393. 41 * _

Preparation of Intermediate 3 3- (6-Fluoro-3-oxo-3, 5-dihydro-pyrazolo [4, 3-c] quinolin-2- yl)-benzoic acid

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 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 (501) was then added and the resulting mixture heated to 50 °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-propyl)-benzamide

3-Methoxypropylamine (0.026g, 0. 29mmol) was added to a stirred solution of 3- (6-fluoro-3-oxo-3, 5-dihydro- pyrazolo [4, 3-c] quinolin-2-yl)-benzoyl chloride (interme- diate 4) (26 mg 0. 29mmol) in tetrahydrofuran (2 ml) and the mixture stirred at room temperature for 15 min. Tri- ethylamin (0.2 ml, 1.4 mmol) was then added and the re- sulting mixture stirred overnight. 1M 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]+ @ PT 1.04 min; 6H (400 MHz, (CD3) 2SO) B-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).

Activity ** Example 51 N-Ethyl-3-(6-fluoro-3-oxo-3, 5-dihydro-pyrazolo [4,3-c]- quinolin-2-yl)-benzamide Prepared by the method of Example 53 substituting ethylamine for 3-methoxypropylamine.

#H(400 MHz, (CD3) 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).

Activity ** Example 52 N-Benzyl-3- (6-fluoro-3-oxo-3, 5-dihydro-pyrazolo [4,3-c]- quinolin-2-yl)-benzamide

Prepared by the method of Example 53 substituting benzylamine for 3-methoxypropylamine.

LCMS m/z 427.16 [M+H] + @ RT 1.28 min.

Activity * Examples 53 to 64 were prepared by the method of example 50, using the appropriate amine. Example X R R'M. S. Activity (MH+) 53 6-F CH2CH2CH2N (Me) 2 Me 422. 5 * 54 6-F CH2CH2CH2N (Me) 2 H 408. 4 ** 55 6-F H 420. 4 * ..')-NH, .. _ 56 6-F H 434. 4 . _ _ 57 6-F H 448. 4 58 6-F cH2cH2cH2cH2N (Me) 2 H 422. 4 ** 59 6-F CH2CH2OMe H 381. 3 _ 60 6-F Et Et 379. 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

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°C for 2h; 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°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 HC1 0.1 M and water. The combined organic extracts were dried over sodium sulfate and con- centrated 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]+ @ RT 0.78 min, 89%, m/z 230.11 [Acid+H] @ RT 1.27, 11%.

8H (400 MHz, (CD3) 2SO) 11. 04 (1 H, s), 8. 06 (1 H, dd, J1 1.1, J2 8.1), 7.76-7. 66 (2 H, m), 7.36 (1 H, td, J1 1. 1, J2 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 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-carboxylic acid ethyl ester (1. O g, 0.041 mols) in acetonitrile and the mixture was heated at 75°C for 90 minutes (becomes a clear solution above 65°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-carboxylic acid ethyl ester (1.15 g, 106 %) as an off-white solid. Rf (AcOEt) = 0.73.

Step 3 4- (4-cyclopropyl-3-oxo-3, 5-dihydro-pyrazolo [4,3- c] quinolin-2-yl)-benzoic acid

4-Chloro-2-cyclopropyl-quinoline-3-carboxylic acid ethyl ester (1.15 g, 0.0041 mols) and 4-hydrazino-benzoic acid (l. Og, 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 HC1, filtered, washed with water and then dried in vacuo to yield 4- (4-cyclopropyl-3-oxo-3, 5-dihydro-pyrazolo [4, 3- cjquinolin-2-yl)-benzoic acid (1.135 g, 80 %) as a yellow solid, LCMS m/z 346.20 [M+H] + @ RT 1.05 min: 96% purity.

8H (400 MHz, (CD3) 2SO) 11.4 (1 H, s), 8.43 (2 H, d, J 8.1), 8.21 (1 H, dd, J1 1.2, J2 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

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 nit- rogen at 45 °C for 8 h. The solvent was removed in vacuo to yield 4- (4-cyclopropyl-3-oxo-3, 5-dihydro-pyrazolo [4, 3- cjquinolin-2-yl)-benzoyl chloride as a pale yellow solid, LCMS m/z [M+MeOH-Cl] + @ RT 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 To a partial solution of 4- (4-cyclopropyl-3-oxo-3, 5- dihydro-pyrazolo [4, 3-c] quinolin-2-yl) -benzoyl chloride (0. 1 g, 0.28 mmol) in tetrahydrofurane (6 ml) under nit- rogen was added a solution of 3-dimethylamino-propyl

amine (0.03 g, 0.3 mmol) in tetrahydrofurane (3 ml). The mixture was stirred at RT for 3 h. The solvent was remo- ved under reduced pressure and the yellow solid was wa- shed with a little saturated sodium bicarbonate, water and dried under vacuo to yield N- (3-Dimethylamino pro- <BR> <BR> <BR> pyl)-4- (4-cyclopropyl-3-oxo-3, 5-dihydro-pyrazolo [4, 3-cJ- guinoIin-2-y-U-jbenzamide (57 mg, 47 %) as a yellow solid.

LCMS m/z 430.11 [M+H] + @ RT 0.99 min: 100% purity.

Activity *** Preparation of Intermediate 5 4- (6-Fluoro-3-oxo-3, 5-dihydro-pyrazolo [4, 3-c] quinolin-2- yl]-benzoyl chloride To a suspension of finely ground 4- (6-Fluoro-3-oxo-3, 5- dihydro-pyrazolo [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 °C for 3 h. The solvent was removed in vacuum to yield 4- (6-Fluoro-3-oxo-3, S-dihydro-pyrazolo [4, 3-c] quinolin-2- yl]-benzoyl 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] quinolin-2-yl]-benzamide hydrochloride 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 : H2O, Fluka C18 reverse phase) to yield N- (3-Dimethylamino propyl)-4- (6- fluoro-3-oxo-3, 5-dihydro-pyrazolo [4, 3-c) quinolin-2-yl)- benzamide hydrochloride (70 mg, 53 %) as a yellow solid.

LCMS m/z 408.39 [M+H] + @ RT 0.89 min: 90% purity.

Activity *** Exmaples 67-141 were prepared analogously from the appropriate benzoyl chloride and the appropriate amine Example X Z W R R'M. S. Activ (MH+) ity 67 6-F H H-CH2CH2CH2CH2CH2-391. 3 ** 68 6-F H H-CH2Phenyl H 413. 2 69 6-F H H-CH2Phenyl Me 427. 3 ** 70 6-F H H-CH2CH2OMe H 381. 2 *** 71 6-F H H-CH2CH2N (Me) 2 H 394. 3 72 6-F H H-CH2CO2Me H 395. 3 *** 73 6-F H H-CH2CH2CH2OMe H 395. 2 74 6-F H H-CH2CH2CH2N (Me) 2 H 408. 3 *** 75 6-F H H, H 431. 3 ** w 76 6-F H 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 ; H 420. 4 *** leb cNH2 80 6-F H H-CH2CH2CH2N (Me) 2 Me 422. 4 *** 81 6-F H H-CH2CH2CH2CH2N (Me) 2 H 422. 4 *** 82 6-F H H I N3 H 448. 5 *** *** 83 6-F H H H 434. 4 , 9 N 84 6-F H H H 525. 3 zu U 85 6-F H H-CH2CH2CH2CH2CH2N (Me) 2 H 450. 3 86 H H H-CH2CH2CH2N (Me) 2 H 390. 2 *** 87 H H H-CH2CH2CH2CH2CH2N (Me) 2 H 432. 1 ** Example 142 {3- [4- (6-Fluoro-3-oxo-3, 5-dihydro-pyrazolo [4, 3-c] quino- lin-2-yl)-phenyl]-ureido} acetic acid ethyl ester 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 (inter- mediate 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 preci- pitate 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] + @ RT 1.06 min.

Activity *** Examples 143 and 144 Example 143 LCMS m/z 438.41 [M+H1+ @ RT 1. 13 min.

Activity ** Example 144 LCMS m/z 514. 46 [M+H' @ RT 1.35 min.

Activity *

The following compounds were synthesised by the method of Example 142, substituting the appropriate isocyanate, isothiocyanate or chloroformate for ethyl cyanatoacetate. Example X Z Y R A M. S. Activ (MH+) ity 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 A NH 394. 3 ** 149 6-F H S 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 0 CH2CH2N (Me) 2 O 410. 2 *** 153 H 0 Me O 375. 3 ** w 154 6-F H O CH2CH2CH2N (Me) 2 O 424. 1 *** 155 6-F H Ouzo 512. 3 ** X- 156 6-F H S nPentyl NH 424. 4 ** 157 6-F H S CH (CH3) CH (CH3) CH3 NH 424. 4 158 6-F H O CH2CH2CH2CH2N (Et) 2 NH 465. 4 *** 159 H H O nPr NH 362. 3 *** 160 H H S A NH 376. 1 **

Intermediate 6: Preparation of methyl 4-oxothiochromane- 3-carboxylate

Dry tetrahydrofuran (60 ml) was cooled under nitrogen atmosphere to-50 to-60°C. 1M Lithium bis (trimethylsily) amide solution in hexane (56 ml, 56 mmol) was added. The temperature was kept at-50 to-60°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°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 x 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] +.

Intermediate 7: Preparation of 4- (3-Oxo-3a, 4-dihydro-3H- thiochromeno [4,3-c] pyrazol-2-yl)-benzoic acid 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] +. No purification was carried out.

Intermediate 8: Preparation of 4- (3-oxothiochromeno [4,3- c] pyrazol-2 (3H) -yl) benzoic acid

Crude 4- (3-Oxo-3a, 4-dihydro-3H-thiochromeno [4,3- c] pyrazol-2-yl)-benzoic 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] + Alternatively crude 4- (3-Oxo-3a, 4-dihydro-3H- thiochromeno [4, 3-c] pyrazol-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] pyrazol-2 (3H) -yl) benzamide

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, 62go) was added followed by 3- dimethylaminopropylamine (17.5 mg, 0.17 mmol) and [(benzotriazol-l-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] + Activity ** Example 166 Preparation of N- [ (cyclohexylamino) propyl]-4- (3- oxothiochromeno [4, 3-c] pyrazol-2 (3H) -yl) benzamide

The reaction was carried out as described above. LCMS: m/z 461 [M+H] + Activity *** Example 167 Preparation of N- (pyrrolidin-1-yl-butyl)-4- (3- oxothiochromeno [4, 3-c] pyrazol-2 (3H) -yl) benzamide The reaction was carried out as described above. LCMS: m/z 447 [M+H] + Activity * Example 168 Preparation of 4- (3-oxothiochromeno [4, 3-c] pyrazol-2 (3H) - yl)-N-1, 2,2, 6,6-pentamethylpiperidin-4-ylbenzamide

The reaction was carried out as described above. LCMS: m/z 475 [M+H] + Activity ** Intermediate 9: Preparation of 3- [ (2- fluorophenyl) sulfanyl] propanoic acid 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 x 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 3-[(2-Fluorophenyl) sulfanyl] propanoic acid (4.0 g, 20 mmol) was mixed with concentrated sulphuric acid (20 ml) at 0-5°C. The reaction solution was stirred at 0 to 5°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 x 200 ml, 1 x 100 ml). The combined organic phases were washed with saturated sodium bicarbonate solution (1 x 50 ml), water (1 x 50 ml), 1M hydrochloric acid (50 ml) and water (2 x 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-carboxylate

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°C. 8-Fluoro-2,3-dihydro-4H-thiochromen-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°C. An orange clear solution was obtained which was stirred at-78°C to-65°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 x 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] pyrazol-2 (3H)-yl) benzoic acid

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

4- (6-Fluoro-3-oxothiochromeno [4, 3-c] pyrazol-2 (3H)- yl) benzoic acid (41 mg, 0.12 mmol) was dissolved in anhydrous dimethyl-acetamided ml). Diisopropyl-ethyl amine (46 mg, 0.36 mmol, 62go) was added followed by [(benzotriazol-l-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] +.

Activity ** Example 170 Preparation of N-[(cyclohexylamino) propyl]-4-(6-fluoro-3- oxothiochromeno [4,3-c] pyrazol-2 (3H)-yl) benzamide

The reaction was carried out as described above. LCMS: m/z 479 [M+H] +.

Activity ** Example 171 Preparation of N- (pyrrolidin-1-yl-butyl)-4- (6-fluoro-3- oxothiochromeno [4, 3-c] pyrazol-2 (3H) -yl) benzamide The reaction was carried out as described above. LCMS: m/z 465 [M+H] +.

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

The reaction was carried out as described above. LCMS: m/z 493 [M+H] + Activity *** Assay Section 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.

In the assay, europium and allophycocyanin (APC) are associated with CD28 and CD80 indirectly (through anti- body linkers) to form a complex, which brings the euro- pium and APC into close proximity to generate a signal.

The complex comprises the following six proteins: fluo- rescent 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. Fluorescent Anti-Rabbit IgG labelled with Europium label 1 (lHg/ml) Linker Rabbit IgG specific for mouse Fc antibody 1 fragment (3yg/ml) CD28 fusion CD28-mouse Fc fragment fusion protein protein (0. 48pg/ml) CD80 fusion CD80 mouse Fab fragment (C215) fusion protein protein (l. 9pg/ml) Linker GaMK-biotin : biotinylated goat IgG antibody 2 specific for mouse kappa chain (2yg/ml) Fluorescent SA-APC: streptavidin labelled label 2 allophycocyanin (8yg/ml)

On formation of the complex, europium and APC are brought into proximity and a signal is generated.

Non-specific interaction was measured by substitu- ting a mouse Fab fragment (C215) for the CD80 mouse Fab fragment fusion protein (1. 9µg/ml). The assay was carried out in black 384 well plates in a final volume of 30Al.

Assay buffer: 50mM Tris-HCl, 150mM NaCl pH7. 8, containing 0. 1% BSA (w/v) added just prior to use.

Compounds were added to the above reagents in a concentration series ranging between 100µm - 1. 7nM. The reaction was incubated for 4 hours at room temperature.

Dual measurements were made using a Wallac Victor 1420 Multilabel Counter. First measurement: excitation 340nm, emission 665nm, delay 50As, window time 200µs. second measurement: excitation 340nm, emission 615nm, delay 50As, window time 200µs. Counts were automatically cor- rected for fluorescence crossover, quenching and back- ground.

By way of illustration, the EC50 results for the compounds of Examples 15,21, 29,35 and 83 were 8 µM, 1.9 M, 950 nM, 148nM and 90nM respectively. For convenience, the EC50 activities of compounds tested are recorded above in summary form as: EC50: * = >10 pM, ** = 1-10 pM, *** = <1 pM.