ES2357110T3 - IMMUNOMODULATION HETEROCYCLIC COMPOUND. - Google Patents

Abstract

A compound of formula (I) or a salt, hydrate or solvate acceptable for pharmaceutical or veterinary use of this: ** Formula ** wherein R1 and R3 independently represent an H; F; Cl; Br; -NO2; -CN; C1-C6 alkyl optionally substituted by F or Cl; or C1-C6 alkoxy optionally substituted by F; R4 represents a carboxylic acid group (-COOH) or an ester thereof, or -C (= O) NR6R7, -NR7C (= O) R6, -NR7C (= O) OR6, -NHC (= O) NR7R6 or -NHC (= S) NR7R6 where R6 represents an H, or a radical of formula - (Alk) mQ where m is 0 or 1 Alk is a divalent linear or branched C1-C12 alkylene radical optionally substituted, or C2 alkenylene C12, or C2-C12 alkynylene or a divalent C3-C12 carbocyclic radical; any of the radicals that may contain one or more bonds -O-, -S- or -N (R8) - wherein R8 represents an H or C1-C4 alkyl, C3-C4 alkenyl, C3-C4 alkynyl, or C3 cycloalkyl -C6, and Q represents an H; -NR9R10 wherein R9 and R10 independently represent an H; C1-C4 alkyl; C3-C4 alkenyl; C3-C4 alkynyl; C3-C6 cycloalkyl; an ester group; an optionally substituted carbocyclic or heterocyclic group; or R9 and R10 form a ring when taken together with the nitrogen to which they are attached, which ring is optionally substituted; and R7 represents an H or C1-C6 alkyl; or when taken together with the atom or atoms to which R6 and R7 bind form an optionally substituted monocyclic heterocyclic ring having 5, 6 or 7 ring atoms; and X represents a bond or a divalent radical of the formula - (Z) n- (Alk) - or - (Alk) - (Z) n- where Z represents -O-, -S- or - NH-, Alk is as defined in relation to R6 and n is 0 or 1, where unless otherwise specified, in the context in which this occurs, the term "substituted" as applied to any fraction in this document means substituted with at least one substituent, selected from (C1-C6) alkyl, (C1-C6) alkenyl, (C2-C6) alkynyl, fluorinated-substituted (C1-C6) alkyl, fluorinated-substituted (C1-C6) alkenyl, fluorinated-substituted (C1-C6) alkynyl, (C1-C6) alkoxy and fluorinated-substituted (C1-C6) alkoxy (including the special case where a ring is substituted on the C atoms of the adjacent ring by an alkylenedioxy or ethylenedioxy) , alkylthio (C1-C6), phenyl, benzyl, phenoxy, benzyloxy, hydroxy, mercapto, amino, fluorine, chlorine, bromine, cyano, nitro, oxo, -COOH, -SO2OH, -CONH2, -SO2NH2, -COR A, -COOR A, -SO2OR A, -NHCOR A, -NHSO2R A, -CONHR A, - SO2NHR A, -NHR A, -NR ARB, -CONR ARB or -SO2NR ARB in which RA and RB are independently a (C1-C6) alkyl or (C2-C6) alkoxy or a monocyclic 5- or carbocyclic heterocyclic group 7 ring members, or RA and RB form a ring, when taken together with the nitrogen to which they are attached; or wherein "substituted" means substituted by a phenyl, benzyl, phenoxy, or benzyloxy, the phenyl ring thereof may itself be substituted with any of the foregoing, with the exception of phenyl, benzyl, phenoxy, or benzyloxy.

Description

The present invention relates to novel heterocyclic compounds, to the methods for their preparation, to the compositions containing them, and to the methods and use for the clinical treatment of medical conditions that can benefit from immunomodulation, for example autoimmune disease. , rheumatoid arthritis, multiple sclerosis, diabetes, asthma, transplants, systemic lupus erythematosus and psoriasis. More particularly, the present invention relates to novel heterocyclic compounds, which are antagonists of CD80, capable of inhibiting interactions between CD80 and CD28, useful for immuno-inhibition.

Background of the Invention

The immune system has the ability to control homeostasis between lymphocyte activation and inactivation through different regulatory mechanisms during and after an immune response. Among these are the mechanisms that specifically inhibit and / or deactivate an immune response. Thus, when an antigen is present through the MHC molecules with the T-cell receptor, the T-cells become properly activated only in the presence of additional co-stimulatory signals. In the absence of these accessory signals there is no lymphocyte activation and either a state of functional inactivation called anergy or tolerance is induced, or the T-cell is specifically suppressed by apoptosis.

Said co-stimulating signal involves the interaction of CD80 in the presenting cells of the specialized antigen with CD28 on the T-cells, and it has been shown that this signal is essential for the activation of the complete T-cell. (Lenschow et al. (1996) Annu. Rev. Immunol., 14, 233-258). Therefore it would be desirable to provide compounds that inhibit this interaction of CD80 / CD28. twenty

WO 03/004495 describes the substituted 3-oxo-dihydropyrazolo [4,3-c] quinolin-2-yl-phenyl derivatives, which are CD80 antagonists, capable of inhibiting interactions between CD80 and CD28 and has a pharmaceutical composition which comprises said compound, a method and the use of said compounds for the clinical treatment of medical conditions that may benefit from immunomodulation, for example rheumatoid arthritis, multiple sclerosis, diabetes, asthma, transplants, systemic lupus erythematosus and psoriasis. 25

US 4,591,589 discloses the 2-aryl-pyrazolo [4,3-c] cinnolin-2-yl derivatives which have anxiolytic properties, in this document they are also known as 2-aryl-2,5-dihydro-3-pyrazolo [4, 3-c] cinnolin-3-ones. In the compounds herein, the aryl group is optionally substituted by an alkyl, alkoxy, halogen, halomethyl or aralkyloxy.

Detailed description of the invention

In accordance with the present invention there is provided a compound of formula (I) or one of its salts, hydrates or solvates acceptable for veterinary or pharmaceutical use:

where

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; 35

R4 represents a carboxylic acid group (-COOH) or an ester thereof, or -C (= O) NR6R7, -NR7C (= O) R6, -NR7C (= O) OR6, -NHC (= O) NR7R6 or - NHC (= S) NR7R6 where

R6 represents an H, or a radical of formula - (Alk) m-Q where

m is 0 or 1

Alk is an optionally substituted divalent linear or branched C1-C12 alkylene radical, or C2-C12 alkenylene or C2-C12 alkynylene or a divalent C3-C12 carbocyclic radical, any of the radicals that may contain one or more -O- bonds, -S- or -N (R8) -, wherein R8 represents an H or C1-C4 alkyl, C3-C4 alkenyl, C3-C4 alkynyl, or C3-C6 cycloalkyl, and

Q represents an H; -NR9R10 wherein R9 and R10 independently represents an H; C1-C4 alkyl; C3-C4 alkenyl; C3-C4 alkynyl; C3-C6 cycloalkyl; an ester group; an optionally substituted carbocyclic or heterocyclic group; or R9 and R10 form a ring when taken together with the nitrogen to which they are attached, which ring is optionally substituted; and 10

R7 represents an H or C1-C6 alkyl; or when taken together with the atom or atoms to which R6 and R7 bind form an optionally substituted monocyclic heterocyclic ring having 5, 6 or 7 ring atoms; Y

X represents a bond or a divalent radical of the formula - (Z) n- (Alk) - or - (Alk) - (Z) n- where Z represents a -O-, -S- or -NH-, Alk is as defined in relation to R6 and n is 0 or 1-,

wherein unless otherwise specified, in the context in which this occurs, the term "substituted" 15 as applied to any fraction herein means substituted with at least one substituent, selected from alkyl (C1 -C6), (C1-C6) alkenyl, (C2-C6) alkynyl, fluorinated-substituted (C1-C6) alkyl, fluorinated-substituted (C1-C6) alkenyl, fluorinated-substituted (C1-C6) alkynyl, alkoxy ( C1-C6) and fluoro-substituted (C1-C6) alkoxy (including the special case where a ring is substituted in C atoms of the adjacent ring by an alkylenedioxy or ethylenedioxy), alkylthio (C1-C6), phenyl, benzyl, phenoxy , benzyloxy, hydroxy, mercapto, amino, fluorine, chlorine, bromine, cyano, nitro, oxo, -20 COOH, -SO2OH, -CONH2, -SO2NH2, -CORA, -COORA, -SO2ORA, -NHCORA, -NHSO2RA, - CONHRA, -SO2NHRA, -NHRA, -NRARB, -CONRARB or -SO2NRARB where RA and RB are independently an alkyl group (C1-C6) or alkoxy (C2-C6) or a monocyclic carbocyclic or heterocyclic group of 5-7 members Ring ros, or RA and RB, form a ring when taken together with the nitrogen to which they bind;

or 25

wherein "substituted" means substituted by a phenyl, benzyl, phenoxy, or benzyloxy, the phenyl ring thereof may itself be substituted with any of the foregoing, with the exception of phenyl, benzyl, phenoxy, or benzyloxy.

Compounds (I) may exist in the form of tautomers, such as (I1) and (I2):

From now on, the compounds of the invention may be represented and referred to, in any tautomeric form (I), and it should be understood that any and all tautomeric forms of structure (I), in particular (I1) and (I2 ), are included in the invention.

The compounds of general formula (I) are antagonists of CD80. They show the interaction between CD80 and CD28 and thus the activation of T cells, therefore the modulation of the immune response. 5

Consequently the invention also includes:

(i) a compound of formula (I) or one of its acceptable salts for pharmaceutical or veterinary use for use in the treatment of conditions that can be treated with immunomodulation, and in particular by immuno-inhibition.

(ii) the use of a compound of formula (I) or one of its salts acceptable for pharmaceutical or veterinary use in the manufacture of a medicament for the treatment of conditions that can be treated with immunomodulation, and 10 in particular for immunoinhibition .

(iii) a method of immunomodulation, and in particular of immuno-inhibition, in mammals, including humans, comprising administration to a mammal in need of such treatment, of an effective immunomodulatory dose of a compound of formula (I) or a of its salts acceptable for pharmaceutical or veterinary use. fifteen

(iv) a pharmaceutical or veterinary composition comprising a compound of formula (I) or a salt thereof acceptable for pharmaceutical or veterinary use together with an excipient or carrier acceptable for pharmaceutical or veterinary use.

Conditions that can be treated with immunomodulation include:

Acute disseminated encephalomyelitis 20

Adrenal insufficiency

Allergic angitis and granulomatosis

Amyloidosis

Ankylosing spondylitis

Asthma 25

Autoimmune Addison's Disease

Autoimmune alopecia

Chronic autoimmune active hepatitis

Autoimmune hemolytic anemia

Autoimmune Neutrogena 30

Autoimmune thrombocytopenic purpura

Behçet's disease

Cerebellar degeneration

Chronic active hepatitis

Chronic inflammatory demyelinating polyradiculoneuropathy 35

Chronic neuropathy with monoclonal gammopathy

Classic polyarteritis nodosa

Congenital adrenal hyperplasia

Cryopathies

Dermatitis herpetiformis

Diabetes

Eaton-Lambert Myasthenic Syndrome 5

Encephalomyelitis

Bullous Epidermolysis Acquired

Erythema nodosa

Enteropathy due to gluten sensitivity

Goodpasture 10 syndrome

Guillain Barre syndrome

Hashimoto's thyroiditis

Hyperthyroidism

Idiopathic hemachromatosis

Idiopathic membranous glomerulonephritis 15

Vasculitis isolated from the central nervous system

Kawasaki disease

Minimal changes kidney disease

Various vasculitis

Mixed connective tissue disease 20

Multifocal motor neuropathy with conduction block

Multiple sclerosis

Myasthenia gravis

Opsoclono-myoclonus syndrome

Pemphigoid 25

Pemphigus

Pernicious anemia

Polymyositis / dermatomyositis

Post-infectious arthritis

Primary biliary sclerosis 30

Psoriasis

Reactive arthritis

Reiter's disease

Retinopathy

Rheumatoid arthritis

Sclerosing cholangitis 5

Sjogren's syndrome

Rigid Person Syndrome

Subacute thyroiditis

Systemic lupus erythematosus

Systemic necrotizing vasculitis 10

Systemic sclerosis (scleroderma)

Takayasu arteritis

Temporal arteritis

Thromboangeitis obliterans

Autoimmune polyglandular syndrome type I and type II 15

Ulcerative colitis

Uveitis

Wegener granulomatosis

As used herein, the term "ester" refers to a group of the form -COOR, where R is a radical derived as the notion of ROH alcohol. Examples of the ester groups include physiologically hydrolyzable esters such as methyl, ethyl, n- and iso-propyl, n-, sec- and tert-butyl, and benzyl esters.

As used herein, the term "alkylene" refers to a linear or branched alkyl chain having two unmet valencies, for example -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH (CH3) CH2-, -CH (CH2CH3) CH2CH2CH2-, and -C (CH3) 3.

As used herein, the term "alkenylene" refers to a linear or branched alkenyl chain that has two unmet valencies, for example -CH = CH-, -CH2CH = CH-, -C (CH3) = CH- , and -CH (CH2CH3) CH = CHCH2-.

As used herein, the term "alkynylene" refers to a linear or branched alkynyl chain having two unmet valencies, for example -CC-, -CH2CC-, and -CH (CH2CH3) CCCH2 -.

Unless otherwise specified, in the context in which this occurs, the term "substituted" as applied to any fraction in this document means substituted with at least one substituent, selected from 30 of, for example, (C1-C6) alkyl, (C1-C6) alkenyl, (C2-C6) alkynyl, fluorinated-substituted (C1-C6) alkyl, fluorinated-substituted (C1-C6) alkenyl, fluorinated-substituted (C1-C6) alkynyl , (C1-C6) alkoxy and fluoro-substituted (C1-C6) alkoxy (including the special case where a ring is substituted on the C atoms of the adjacent ring by an alkylenedioxy such as methylenedioxy or ethylenedioxy), (C1-C6) alkylthio ), phenyl, benzyl, phenoxy, benzyloxy, hydroxy, mercapto, amino, fluorine, chlorine, bromine, cyano, nitro, oxo, -COOH, -SO2OH, -CONH2, -SO2NH2, -CORA, -COORA, -SO2ORA, - NHCORA, -NHSO2RA, -35 CONHRA, -SO2NHRA, -NHRA, -NRARB, -CONRARB or -SO2NRARB where RA and RB are independently an alkyl (C1-C6) or alkoxy (C2-C6) group or a carbocyclic group or I have 5-7-membered monocyclic ring, or RA and RB form a ring when taken together with the nitrogen to which they bind. In the case where "substituted" means substituted by a phenyl, benzyl, phenoxy, or benzyloxy, the phenyl ring thereof may itself be substituted with any of the foregoing, with the exception of phenyl, benzyl, phenoxy, or benzyloxy. 40

As used herein, the term "aryl" refers to a mono-, bi- or tri-cyclic carbocyclic aromatic radical, and to two such covalently bonded radicals illustrative of such radicals are phenyl, biphenyl and naphthyl.

As used herein, the term "unclassified carbocyclyl" or "carbocyclic" includes aryl, cycloalkyl and cycloalkenyl and refers to a ring system (monocyclic, bicyclic, tricyclic or bond) whose ring 5 atoms are all carbon.

As used herein, the term "unqualified cycloalkyl" refers to a carbocyclic ring system that contains only single bonds between the ring carbons.

As used herein, the term "unqualified cycloalkenyl" refers to a carbocyclic ring system that contains at least one double bond between a pair of ring carbons. 10

As used herein, the term "heteroaryl" refers to a mono-, bi- or tri-cyclic aromatic radical, which contains one or more heteroatoms selected from S, N and O. Illustrative of such radicals are thienyl, benztienil, furyl, benzfuril, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisotiazolil, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolil, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl. fifteen

As used herein, the term "heterocyclyl" or "unclassified heterocyclic" includes "heteroaryl" as defined above, and in particular means a mono-, bi- or tri-cyclic radical or non-aromatic bond, which contains one or more heteroatoms selected from S, N and O, and with groups consisting of a monocyclic non-aromatic radical containing one or more of said heteroatoms that covalently bind to another said radical or with a monocyclic carbocyclic radical. Illustrative of such radicals are pyrrolyl, furanyl, tetrahydrofuranyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzylpropylhydranyl, quinfolinyl, tetraphyl, trichlorohydranyl, quinolinyl, tetraphyl, cranyl, citropyl, citropyl, cranyl; , isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido.

Some compounds of the invention contain one or more chiral centers due to the presence of asymmetric carbon atoms. The presence of asymmetric carbon atoms results in the stereoisomers or diastereomers with stereochemistry R or S in each chiral center. The invention includes all stereoisomers and diastereoisomers and mixture thereof.

Salts of compounds of the invention that form salts include base salts and physiologically acceptable acid addition salts. Appropriate acid addition salts are formed from acids that form non-toxic salts. Examples include the salts of acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisilate, esylate, format, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsilate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, sucrate, stearate, succinate, tartrate, tosylate and trifluoroacetate. Appropriate base salts are formed from bases that form non-toxic salts. Examples include the salts of aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc.

Methods

The compounds of the invention, wherein R4 represents an amide group -C (= O) NR6R7, can be prepared by reacting the appropriate HNR6R7 amine, with a compound of formula (II) to amide the carboxylic acid group:

the symbols being R1, R3, X, R6 and R7, as defined in relation to the previous formula (I).

The compounds (II) (i.e. the compounds (I) of the invention wherein R4 is a carboxylic acid group) can be prepared, by reacting a compound of formula (III) with a hydrazine of formula (IV):

This reaction can lead to the preparation of a mixture of the position isomers (IIA) and (IIB):

 5

from which the desired isomer (IIA) can be separated.

Compounds (I) wherein R 4 is an ester or amide group can also be prepared from an intermediate (III) by reaction with the appropriate hydrazine (VAT)

wherein R4 is an ester or amide group. Again the reaction can give rise to a mixture of the analogs of the ester or the amide of the carboxylic acids (IIA) and (IIB), from which the isomer (I) of the desired ester or amide can be pull apart. Alternatively, the carboxylic acid compound (II) can simply be esterified, or amidated.

Compounds (I) wherein R4 is a group -NR7C (= O) R6 "reverse amide", can be prepared by reordering Curtius (see Ninomiya, K .; Shioiri, T .; Yamada, S. Tetrahedron (1974 ), 30 (14), 2151-7) of carboxylic acid (II) to isocyanate (V)

followed by hydrolysis of the isocyanate group to an amino group and the acylation of the amino group with, for example, the Cl-C (= O) R6 chloride acid. In cases where R7 is not hydrogen, the R7 substituent can be introduced after the isocyanate reduction step or after the acylation step.

In an alternate route for the compounds of the invention "reverse amide" (R4 = -NR7C (= O) R6), a compound of structure (V) in which the isocyanate fraction is replaced by a nitro group can be reduced to corresponding amine 5, which can then be acylated to form the desired reverse amide.

Compounds (I), wherein R4 is a urea group -NHC (= O) NHR6 or thiourea group -NHC (= O) NHR6, can also be prepared from the isocyanate (V) or the corresponding isothiocyanate by reaction with the appropriate amine H2NR6.

Compounds (I), wherein R4 is a carbamate -NR7C (= O) OR6 group, can be prepared by reacting the isocyanate with an appropriate R6OH alcohol.

Other details of the synthetic methods for the preparation of the compounds (I) of the invention, and intermediates such as (III), can be found in the examples herein.

In the compounds of the invention:

The radical R4X- is preferably in position 4 of the phenyl ring. fifteen

X can be, for example, a bond, or a radical -CH2- or -CH2CH2-. A link is preferred at the moment.

R3 can be, for example, H, F, Cl, methyl, methoxy, or methylenedioxy. It is preferred that R3 is H.

R1 can be, for example, H, F, Cl, methyl, methoxy, or methylenedioxy. It is preferred herein that R1 is hydrogen or fluorine, particularly at position 6 of the 3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl ring system. twenty

R4 represents a carboxylic acid group (-COOH) or an ester thereof, or -C (= O) NR6R7, -NR7C (= O) R6, -NR7C (= O) OR6 or -NHC (= O) NHR6, all as defined above.

When R4 is an ester group, examples include those of the formula -COOR wherein R is methyl, ethyl n- or isopropyl, n-, sec- or tert-butyl, or benzyl ester.

R6, when present, represents an H, or a radical of formula - (Alk) m-Q where m, Alk and Q are as defined above.

When m is 1, Alk can be, for example, a linear or branched C1-C6 alkylene radical, such as -CH2-, -CH2CH2-, -CH2CH2CH2-, and -CH2CH (CH3) CH2-. Alk can also be, for example, a divalent cyclopropylene, cyclopentylene or cyclohexylene radical. The Alk radical may be optionally substituted by, for example, OH, oxo, CF3, methoxy or ethoxy. The Alk radical may optionally contain a hetero atom, for example in the form of an ether, thioether or amino bond.

The group Q may represent, for example, hydrogen; -NR9R10 wherein R9 and R10 may be the same or different and selected from hydrogen, methyl, ethyl, n- or isopropyl or tert-butyl; an ester group for example a methyl, ethyl or benzyl ester; or an optionally substituted aryl, aryloxy, cycloalkyl, cycloalkenyl or heterocyclic group, for example a phenyl, phenoxy, cyclopentyl, cyclohexyl, furyl, thienyl, quinuclidinyl, piperidyl, or piperazinyl group. 35

R7 when present, represents an H or C1-C6 alkyl, for example methyl, ethyl n- or iso-propyl, n-, sec- or tert-butyl;

or when taken together with the atom or atoms to which R6 and R7 bind form a monocyclic heterocyclic ring having 5, 6 or 7 ring atoms.

Especially preferred are cases where R4 represents -C (= O) NR6R7 or -NHC (= O) NR7R6 where R7 is a hydrogen and R6 represents a radical of formula - (Alk) mQ, where m is 1 and the radical divalent Alk contains 3 or 4 40 carbon atoms and is unsubstituted, and Q represents -NR9R10 where R9 and R10 independently represent an H; C1-C4 alkyl; C3-C4 alkenyl; C3-C4 alkynyl; C3-C6 cycloalkyl; an ester group; an optionally substituted carbocyclic or heterocyclic group; or form a ring when taken together with the nitrogen to which they are attached, a ring that is optionally substituted.

A specific preferred subset of the compounds of the invention has the formula (IC):

where X and R4 are as specified above. In this subset, the radical R4X- may be in position 4 of the phenyl ring. This subset includes in particular, the compounds where X is a bond and R4 is -C (= O) NR6R7 where R6 and R7 are as specified above. For example, in such compounds R6 can be a quinuclidinyl and R7 a hydrogen. 5

Specific compounds of the invention include those of the Examples herein.

A preferred compound of the invention is 4- (6-fluor-3-oxo-1,3-dihydro-pyrazolo [4,3-c] cinnolin-2-yl) -N- (2,2-difluoro-ethyl ) -benzamide, of formula (A)

or one of its salts, hydrates or solvates acceptable for veterinary or pharmaceutical use. 10

Another preferred compound of the invention is N- [3- (tert-butyl-methyl-amino) -butyl] -4- (6-fluor-3-oxo-1,3-dihydro-pyrazolo [4,3-c ] cinnolin-2-yl) -benzamide, of formula (B):

or one of its salts, hydrates or solvates acceptable for veterinary or pharmaceutical use.

Another object of the present invention is a compound of the structure, selected from one of the groups consisting of the following formulas:

or one of its salts, hydrates or solvates acceptable for veterinary or pharmaceutical use.

As mentioned above, the invention includes a pharmaceutical or veterinary composition comprising a compound of formula (I) or a salt thereof acceptable for pharmaceutical or veterinary use together with an excipient or carrier acceptable for pharmaceutical or veterinary use. In such compositions, it will be understood that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, route of administration. , excretion rate, combination of drugs and the cause and severity of the particular disease, under treatment. Optimum dose levels and dosage frequency will be determined by clinical trials.

The compounds that are the subject of the invention can be prepared for administration by any route consistent with their pharmacokinetic properties. The orally administrable compositions may be in the form of tablets, capsules, powders, granules, grajeas, liquid or gel preparations, such as suspensions or parenteral oral, topical, or sterile solutions. Tablets and capsules for administration may be in the form of unit dose presentation, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tablet compression lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, suspensions, solutions, emulsions, syrups or aqueous or oily elixirs, or they may be presented as a dry product for reconstitution with water or other appropriate vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, hydrogenated edible fats gelatin; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerin, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional coloring or flavoring agents.

For topical application on the skin, the drug can be manufactured in a cream, lotion or ointment. The cream or ointment formulations that can be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutical products such as the British Pharmacopoeia.

For topical application in the eyes, the drug can be manufactured in a solution or suspension in an appropriate sterile aqueous or non-aqueous vehicle. Additives, for example regulatory solutions such as sodium metabisulfite or disodium edetate; preservatives including bactericidal and fungicidal agents such as phenyl acetate

of mercury or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents such as hypromellose may also be included.

The active ingredient can also be administered parenterally in a sterile medium. Depending on the vehicle and the concentration used, the drug can be both suspended and dissolved in the vehicle. Advantageously, adjuvants such as a local anesthetic, preservatives and regulatory agents can be dissolved in the vehicle. 5

The embodiments of the invention are described in the following non-limiting Examples:

The following abbreviations are used in the experimental descriptions:

DMF Dimethyl Formamide

DMA Dimethyl Acetamide

DMSO Dimethylsulfoxide 10

HBTU O-Benzotriazol-1-yl-N, N, N ’, N’-tetramethyluronium hexafluorophosphate

HPLC High Resolution Liquid Chromatography

LCMS Liquid Chromatography / Mass Spectrometry

NMR Magnetic Resonance Spectroscopy

Example 1 15

Stage 1: Preparation of malonic acid (phenylhydrazone):

Sodium mesoxalate monohydrate (5.00 g, 27.8 mmol) was dissolved in 1 M hydrochloric acid (50 ml) to provide a colorless cloudy solution. Phenylhydrazine (3.00 g, 2.72 ml, 27.8 mmol) was added dropwise at room temperature to the stirred mixture. A yellow precipitate formed was collected by filtration after 20 90 min and washed with water (50 ml). The cake of the filtrate was triturated with ethyl acetate / hexane [1: 1], filtered and dried under vacuum. The title compound was isolated as a yellow powder (4.74 g, 22.7 mmol, 82%). LCMS: m / z 207 [M-H] +.

Alternatively, the product can be extracted from the aqueous phase with ethyl acetate (2 x 250 ml), the organic phase is dried over magnesium sulfate, filtered and the solvent is removed under vacuum. 25

Stage 2: Preparation of (phenylhydrazono) malonoyl dichloride:

Malonic acid (phenylhydrazone) (1.00 g, 4.80 mmol) was mixed under an inert atmosphere with dry chloroform (15 ml) to provide a yellow suspension. The mixture was stirred at room temperature and phosphorus pentachloride (2.19 g, 10.5 mmol) was added gradually. The reaction mixture was heated at reflux for 1.5 h, to provide a green solution. The mixture was cooled to room temperature and diluted with hexane (15

ml) A green precipitate formed, was collected by filtration and dried under vacuum. The title compound was isolated as a green powder (645 mg, 2.63 mmol, 53%).

Stage 3: Preparation of methyl 4-hydroxycinoline-3-carboxylate

The (phenylhydrazono) malonoyl dichloride (2.45 g, 0.01 mmol) was mixed under an inert atmosphere with 1,2-dichloroethane 5 (15 ml) to provide a yellow suspension. Titanium tetrachloride (1.89 g, 1.09 ml) was added dropwise to form a brown solution. The mixture was heated at reflux overnight, cooled to room temperature and quenched with methanol (15 ml). Stirring was continued for 30 min and the volatiles were removed under vacuum. Water (100 ml) was added and the suspension obtained was extracted with n-butanol (2 x 50 ml). The combined organic phases were washed with water (2 x 20 ml) and concentrated under vacuum. The title compound was isolated as a green solid (1.04 g, 5.10 mmol, 51%). LCMS: m / z 205 [M + H] +.

Stage 4: Preparation of methyl 4-chlorocinoline-3-carboxylate:

Thionyl chloride (8.15 g, 5 ml) was added dropwise under an inert atmosphere to methyl 4-hydroxyquinoline-3-carboxylate (0.50 g, 2.45 mmol). The mixture was heated at reflux for 1.5 h, cooled to room temperature and the excess thionyl chloride was removed under vacuum. Toluene (5 ml) was added to the residue. The mixture was stirred at room temperature overnight. The solids were collected by filtration and dried under vacuum. The title compound was isolated as a brown solid (248 mg, 1.11 mmol, 45%). LCMS: m / z 223 [M + H] +.

Step 5: Preparation of 4- (3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoic acid:

 twenty

4-Hydrazinobenzoic acid (68.4 mg, 0.45 mmol) was mixed at room temperature with ethanol (5 ml) to provide a cream-colored suspension. Methyl 4-chlorocinoline-3-carboxylate (100 mg, 0.45 mmol) was added and the mixture was heated between 45-50 ° C for 1 h. The reaction mixture was cooled to room temperature and the solvent was removed under vacuum. Ethyl acetate (10 ml) was added to the residue. The mixture was stirred at room temperature for 1 h. The solids were collected by filtration and dried under vacuum. The title compound was isolated as a brown powder (120 mg, 0.39 mmol, 86%). LCMS: m / z 307 [M + H] +. NMR [DMSO-d6]:  = 7.69-7.77 (m, 1 -Haril); 7.81-7.90 (m, 2 Haril); 8.05 (d, J = 8.85, 2 Haril); 8.20 (d, J = 7.92 Hz, 1 Haril); 8.33 (d, J = 8.85 Hz, 2 Haril); 14.64 (s, NH).

Alternatively the reaction can be carried out at room temperature. In this case, a longer reaction time of 2-3 h may be needed. 30

Example 2

Preparation of N - [(dimethylamino) propyl] -4- (3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzamide:

4- (3-Oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoic acid (25 mg, 0.08 mmol) was mixed with DMF (1 ml). Diisopropylethylamine (21 mg, 28 µl, 0.16 mmol) and 3-dimethylaminopropylamine (8.2 mg, 10.0 µl, 0.09 5 mmol) were added, followed by HBTU (30.3 mg, 0.08 mmol). The mixture was stirred at room temperature for 2 h. The product was purified by preparative HPLC. The title compound was isolated as a red solid (12.6 mg, 0.032 mmol, 40%). LCMS: m / z 391 [M + H] +.

Example 3

Preparation of N-benzyl-4- (3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzamide: 10

4- (3-Oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoic acid (52 mg, 0.17 mmol) was mixed with DMF (2 ml). Diisopropylethylamine (22 mg, 29 µl, 0.17 mmol) and benzylamine (18.2 mg, 18.6 µl, 0.17 mmol) were added, followed by HBTU (64.5 mg, 0.17 mmol). The mixture was stirred at room temperature for 4 h. The product was purified by preparative HPLC. The title compound was isolated as a red solid (6.6 mg, 0.02 mmol, 10%). LCMS: 15 m / z 396 [M + H] +.

Example 4

Stage 1: Preparation of 4- (3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoyl chloride:

Thionyl chloride (90 ml) was added to 4- (3-oxo-1,3-dihydro-2H-pyrazolo- [4,3-c] cinnolin-2-yl) benzoic acid (2.36 g, 7.70 mmol) . The mixture was heated at reflux for 2 h, under a nitrogen atmosphere. A dark red solution was obtained, cooled to room temperature and excess thionyl chloride was removed under vacuum. Toluene (30 ml) was added to the residues and the mixture was stirred at room temperature under a nitrogen atmosphere until precipitation was complete. The solids were collected by filtration and washed with toluene (2 x 30 ml). The title compound was isolated as a red solid (2.20 g, 6.77 mmol, 88%) LCMS: m / z 321 [M + H] + (methyl ester resulting from the sample formed in methanol).

Stage 2: Preparation of the N - [(cyclohexylamino) propyl] -4- (3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzamide:

 10

The 4- (3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoyl chloride (97 mg, 0.30 mmol) was dissolved in anhydrous DMA (2 ml). Diisopropylethylamine (39 mg, 53 µl, 0.60 mmol) was added followed by N-cyclohexyl-1,3-propanediamine (52 mg, 0.60 mmol). The mixture was stirred for 30 min. Water (5 ml) was added to provide a dark red suspension. The mixture was extracted with n-butanol (2 x 20 ml). The combined organic phases were washed with water and concentrated under vacuum, until a precipitation was observed. Hexane (20 ml) and ethyl acetate (10 ml) were added, the solids were collected by filtration and dried under vacuum. The product was isolated as a dark red powder (82 mg, 0.18 mmol, 62%). LCMS: m / z 445 [M + H] +.

Example 5:

Stage 1: Preparation of [[2-Fluorophenyl) Hydrazone] Malonic acid:

 twenty

Sodium mesoxalate monohydrate (2.21 g, 12.3 mmol) was dissolved in 1M hydrochloric acid (50 ml), to provide a colorless cloudy solution. 2-fluorophenylhydrazine hydrochloride (2.00 g, 12.3 mmol) was added gradually at room temperature to the stirred mixture. A yellow precipitate forms, the mixture is diluted with water (50 ml) and stirring is continued overnight. Ethyl acetate (150 ml) was added, the phases were mixed vigorously until the solids had dissolved. The phases were separated and the aqueous phase was washed with ethyl acetate (50 ml). The combined organic phases were dried over magnesium sulfate, filtered and the solvent removed under vacuum. The title compound was isolated as a yellow powder (2.55 g, 11.7 mmol, 92%). LCMS: m / z 227 [M-H] +.

Stage 2: Preparation of [(2-Fluorophenyl) Hydrazone] Malonoyl Dichloride:

The (2-Fluorophenylhydrazono) malonic acid (1.33 g, 5.88 mmol) was mixed under an inert atmosphere with dry chloroform (20 ml) to provide a yellow suspension. The mixture was stirred at room temperature and phosphorus pentachloride (2.69 g, 12.9 mmol) was added little by little. The reaction mixture was heated at reflux for 2 h, to provide a dark yellow solution. The mixture was cooled to room temperature and concentrated under vacuum until precipitation occurs. The solids were collected by filtration, washed with hexane (30 ml) and dried under vacuum. The title compound was isolated as a yellow powder (760 mg, 2.89 mmol, 49%).

Stage 3: Preparation of methyl 8-fluor-4-hydroxycinoline-3-carboxylate:

The (2-Fluorophenylhydrazono) malonoyl dichloride (19.4 g, 74 mmol) was mixed under an inert atmosphere with 1.2-10 dichloroethane (100 ml), to provide a yellow suspension. Titanium tetrachloride (13.9 g, 8.08 ml, 74 mmol) was added dropwise to form a brown solution. The mixture was heated at reflux overnight. In addition, titanium tetrachloride (13.9 g, 8.08 ml, 74 mmol) was added and heating is continued for 24 h. The reaction mixture was cooled to 0-5 ° C and quenched with methanol (50 ml). Stirring was continued for 1 h at room temperature and the volatiles were removed under vacuum. Water (300 ml) was added and the suspension obtained was extracted with ethyl acetate (3 x 100 ml). The combined organic phases were dried over magnesium sulfate, filtered and concentrated under vacuum. A yellow solid was obtained (12 g of crude product). LCMS: m / z 223 [M + H] +.

Stage 4: Preparation of 4- (6-fluor-3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoic acid:

 twenty

The crude 8-Fluoro-4-hydroxyquinoline-3-carboxylate from the previous step (1.00 g, 4.95 mmol), was dissolved in thionyl chloride (50 ml). The solution was heated at reflux for 2-3 h, until no other gas production was observed. The reaction mixture was cooled to room temperature and the excess thionyl chloride was removed under vacuum. The crude intermediate was formed as an azeotrope with toluene (3 x 25 ml). A dark brown solid was obtained, which was taken with ethanol (25 ml). 4-Hydrazinobenzoic acid (640 mg, 4.21 mmol) was added and the mixture was stirred at room temperature overnight. The solids were collected by filtration, suspended in 1M HCl (100 ml), filtered, washed with hexane (50 ml) and dried under vacuum. A brown solid was obtained (890 mg of crude product). LCMS: m / z [M + H] + 325.

Example 6

Stage 1: Preparation of 4- (6-fluor-3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoic chloride: 30

The crude 4- (6-fluor-3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) -benzoic acid (1.45 g) from the previous step, is dissolved in thionyl chloride (50 ml). The mixture was heated at 70˚C for 2-3 h until no gas production was observed. The mixture was cooled to room temperature and the excess thionyl chloride was removed under vacuum. The residues were formed as azeotropes with toluene (2 x 20 ml), to provide a solid. Solid 5 was collected by filtration, washed with toluene and dried under vacuum. The product was isolated as a yellow powder (670 mg, 1.95 mmol). LCMS: m / z [M + H] + 339 (methyl ester resulting from a sample formed in methanol).

Stage 2: Preparation of 4- (6-fluor-3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) -N- (pyrrolid in-1-yl- butyl) benzamide: 10

The 4- (6-fluor-3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoyl chloride (100 mg, 0.29 mmol) was dissolved in anhydrous DMA (2 ml) Diisopropylethylamine (75 mg, 101 µl, 0.58 mmol) was added, followed by 1- (4- aminobutyl) pyrrolidine (41 mg). The mixture was stirred at room temperature overnight. Water (5 ml) and n-butanol (5 ml) were added. The phases were separated. The organic phase was washed with water (2 x 5 ml). Volatiles were removed under vacuum. The product was isolated as a brown powder (50 mg, 0.11 mmol, 37%). LCMS: m / z [M + H] + 463.

Example 7

Preparation of 4- (6-fluor-3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) -N- (1,2,2,6,6- pentamethylpiperidine-4-yl) benzamide:

The 4- (6-fluor-3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoyl chloride (100 mg, 0.29 mmol) was dissolved in anhydrous DMA (2 ml) Diisopropylethylamine (75 mg, 101 ml, 0.58 mmol) was added, followed by 4-amino-1,2,2,6,6-

pentamethylpiperidine (49 mg, 0.29 mmol). The mixture was stirred overnight. Water (5 ml) and n-butanol (5 ml) were added. The phases were separated. The organic phase was washed with water (2 x 5 ml) and the solution was concentrated under vacuum. The title compound was isolated as a red solid (50 mg, 0.105 mmol, 36%). LCMS: m / z [M + H] + 477.

Example 8 5

Stage 1: Preparation of 2- (4-nitrophenyl) -1,2-dihydro-3H-pyrazolo [4,3-c] cinnolin-3-one

Thionyl chloride (326 g, 200 ml), was added dropwise under an inert atmosphere to the methyl 4-hydroxyquinoline-3-carboxylate (10.0 g, 49 mmol). The mixture was heated at reflux for 2.5 h, cooled to room temperature and the excess thionyl chloride was removed under vacuum. Toluene (100 ml) was added to the residue and removed under vacuum. This procedure was repeated with more toluene (100 ml). A semi-solid brown material was obtained and carried with ethanol (200 ml). 4-Nitrophenylhydrazine (5.99 g, 39.2 mmol) was added little by little. The mixture was stirred at room temperature overnight. The mixture was heated at 40-45˚C for 1 h and cooled to room temperature. The solids were collected by filtration, triturated with ethanol (100 ml) and dried under vacuum. The title compound was isolated as a brown solid (8.42 g, 27.4 mmol, 70%). LCMS: m / z 308 [M + H] +. fifteen

Stage 2: Preparation of 2- (4-aminophenyl) -1,2-dihydro-3H-pyrazolo [4,3-c] cinnolin-3-one

2- (4-Nitrophenyl) -1,2-dihydro-3 H -pyrazolo [4,3-c] cinnolin-3-one (11.4 g, 37.2 mmol), was suspended in a mixture of ethanol (100 ml) and water (100 ml). Powdered iron (11.1 g, 200 mmol) and ammonium chloride (5.34 g, 100 mmol) were added. The mixture was heated at 80 ° C overnight, cooled to room temperature and basified with potassium carbonate at pH 9-10. The solids were removed by filtration through a pad of Celite®. The filtrate was extracted with n-butanol (2 x 200 ml). The combined organic phases were concentrated under vacuum to provide a red solid. The solid was triturated with methanol (100 ml), filtered and dried under vacuum. The title compound was isolated as a dark red powder (5.58g, 20.1 mmol, 57%). LCMS: m / z 278 [M + H] +. 25

Step 3: Preparation of the N- [3- (dimethylamino) propyl] -N '- [4- (3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) phenyl] urea

2- (4-Aminophenyl) -1,2-dihydro-3H-pyrazolo [4,3-c] cinnolin-3-one (44 mg, 0.16 mmol), was suspended in toluene under a nitrogen atmosphere (0.5 ml ) at 0-5˚C. DMA (0.5 ml) was added followed by N, N’-carbonyldiimidazole (26 mg, 0.16 mmol). The mixture was stirred for 1 h at 0-5 ° C, before being mixed with a solution of 3-dimethylaminopropylamine (18 mg, 0.18 mmol) in toluene (0.5 ml). Stirring was continued for 1 h and the product was purified by preparative HPLC. The title compound was isolated as a dark red powder (2.6 mg, 6 mmol, 4%). LCMS: m / z 406 [M + H] +.

Example 9: Preparation of 4- (3-oxo-1,3-dihydro-2H-pyrazolo [4,3-c] cinnolin-2-yl) benzoic ethyl ester.

The title compound was prepared by the method of Example 1 step 5, replacing 4-hydrazinobenzoic acid ethyl ester with the original acid. MS: MH + = 335.2

Results

The use of BIAcore biomolecular interaction analysis

Biotinylated human CD80 (hCD80-BT) is a recombinant soluble form of a membrane bound receptor molecule (CD80) that binds to CD28 to initiate T cell activation. The interaction between CD80 and CD28 has been extensively investigated. (Collins et al, 2002). Biotinylated human HLA-A2-tax is the recombinant soluble form of a membrane bound receptor molecule that has been used in this example as a control protein, and is not expected to interact with the compounds.

The BIAcore S51 ™ system was used to detect the compounds of Examples 1-4 above. A CM5 series S sensor chip was attached to the BIAcore S51 ™. Streptavidin was coupled to the carboxymethyl surface 20 using standard amine coupling. The surface of the chip was activated with EDC 0.2M / NHS 0.05M, followed by streptavidin binding (0.25 mg / ml in 10 mM sodium acetate pH 5.0) and saturation of unoccupied sites with 1M ethylenediamine.

The BIAcore S51 sensor chip has two separate sensor points for protein immobilization. hCD80-BT was immobilized on the streptavidin-covered surface of a sensor point until a response of approximately 3000 RU was observed. A protein to control the non-specific binding of the compound was immobilized at a second point of the sensor. The control protein used for these experiments was a soluble, biotinylated form of the human HLA protein.

The dilution series of the compounds (1000nM - 0.05nM) were prepared in a runner solution (10 mM, pH 7.4, 150 mM NaCl, 0.005% P20; 5% DMSO). 30

BIAcore S51 ™ was run at a flow rate of 30 µl / min using run regulator solution. Compounds and standard solutions of DMSO for the correction of data for solvent effects were injected. The data was recorded automatically and analyzed using the BIAcore S51 Evaluation software.

The interaction between CD80 and the endogenous protein ligand (CD28) is highly specific, but relatively weak, with a KD of 4750 nM, and a dissociation constant greater than 0.2 s-1. The compounds of Examples 2,3,4,6,7 have high affinity and longer residence time in CD80 than CD28, which has KDs of less than 100nM, and dissociation constants of 2x10-2, indicating that the cinolines will be capable of effectively competing with the endogenous ligand. The cinolines showed no detectable interaction with the control protein.

References 10

Collins AV et al. (2002) Immunity 17, 201-210 "The interaction properties of costimulatory molecules revisited"

Inhibition of the production of interlechin-2 (IL-2) by human Jurkat T cells.

Method

HumanRaji cells were dispensed at a concentration of 2x105 cells per well in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, 1% glutamine (RPMI medium) in a round bottom microtiter plate 96-well. The compounds under investigation (dissolved in 100% DMSO) were diluted to eight times the desired final concentration in RPMI medium and added to the desired final concentration for a total volume of 200 µl per well. After 20 minutes of incubation at 37˚C, Jurkat T cells were added at a concentration of 2x105 cells per well. The monoclonal antibody for CD3 (UCHT1, R&D Systems) was added to the cultures at a final concentration of 1 µg per ml, and where indicated, the monoclonal antibody for CD28 (CD28.2, BD-Pharmingen) was also added to a 2.5 µg concentration per ml. The cells were grown at 37˚C for 5 hours, after which the plates were centrifuged and the supernatants were cultured by ELISA IL-2 assay using the Eli-pair IL-2 kit (DIACLONE Research, Besancon, France) of According to the manufacturer's instructions.

By way of example, the compound of Example 2 (AV1142005) provided 65% inhibition at 30mM. 25

Homogeneous Late Resolution Fluorescence Test

The examples described above were tested in a Cell Free Homogeneous Late Resolution Fluorescence Test (HTRT) to determine their activity as inhibitors of the CD80-CD28 interaction.

In the assay, europium and allophycocyanin (APC) associated with CD28 and CD80 indirectly (through antibody linkers) to form a complex, which carries the europium and APC in close proximity to generate a signal. The complex comprises the following six proteins: fluorescent label 1, linker antibody 1, fusion protein CD28, fusion protein CD80, linker antibody 2, and fluorescent label 2. The table below describes these reagents in greater detail.

 Fluorescent label 1

 Anti-Rabbit IgG marked with Europium (1µg / ml)

 Linker Antibody 1

 Rabbit IgG specific for the mouse Fc fragment (3 µg / ml)

 CD28 fusion rotein

 Fc CD28-mouse fragment fusion protein (0.48µg / ml)

 CD80 fusion rotein

 Fusion CD80-mouse fragment fusion protein (C215) (1.9 µg / ml)

 binding body 2

 GαM-biotin: biotinylated goat IgG specific for mouse kappa chain (2 µg / ml)

 Fluorescent label 2

 SA-APC: streptavidin labeled with allophycocyanin (8 µg / ml)

In the formation of the complex, Europium and APC were carried in proximity and a signal is generated.

Non-specific interaction was measured by substituting a mouse Fab fragment (C215), for the fusion protein Fab fragment CD80 mouse (1.9 µg / ml). The test was carried out in black plates of 384 wells in a final volume of 30 µl. Test regulatory solution: 50mM Tris-HCl, 150mM NaCl pH7.8, containing 0.1% BSA (weight / vol.) Is added just before use.

The compounds were added to the above reagents in a series of concentrations ranging from 100 µM - 1.7nM. The reaction was incubated for 4 hours at room temperature. Double measurements were made using 40

a Wallac Victor 1420 Multilabel Accountant. First measurement: excitation 340nm, emission 665nm, delay 50µs, window time 200µs. second measurement: excitation 340nm, emission 615nm, delay 50µs, window time 200µs. The counts were automatically corrected for cross, off and background fluorescence. EC50 activities of the tested compounds were recorded as:

 5

The compounds of Examples 1-8, had the following activities in the HTRF assay described above:

Example 1 *

Example 2 ***

Example 3 ***

Example 4 *** 10

Example 5 *

Example 6 ***

Example 7 ***

Example 8 ***

Example 9 ** 15

Additional Examples

Other examples of the compounds of the invention were synthesized by methods analogous to those of Examples 1-8 cited above. The structures of the synthesized compounds are shown in the following Table, together with their activities in the HTRF assay described above.

Examples of the test results of the above compounds in the interlequine-2 (IL-2) production inhibition assay by human Jurkat T cells, described above, are as follows:

 Example No (see table)

 Compound Concentration (µM) Inhibition Percentage (relative to DMSO = 0%)

 478

 10 56.0

 376

 10 56.7

 353

 10 77.4

 429

 10 58.8

 349

 10 79.5

 68

 10 71.7

 235

 10 59.3

 288

 30 72

 162

 30 54.4

 350

 10 74.2

 381

 10 48.5

 442

 10 58.9

 482

 10 39.2

 472

 10 58.4

 453

 10 55.7

 53

 30 63.8

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is only for the convenience of the reader. It is not part of the European patent document. Even though great care has been taken in collecting references, errors or omissions cannot be excluded and the EPO disclaims all responsibility in this regard.

Patent documents cited in the description

• WO 03004495 A [0004] 5

• US 4591589 A [0005]

Non-patent literature cited in the description

• Lenschow et al. Annu Rev. Immunol., 1996, vol. 14, 233-258 [0003]

• Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron, 1974, vol. 30 (14), 2151-7 [0030]

• Collins AV et al. Immunity, 2002, vol. 17, 201-210 [0097] 10

Claims (30)

1. A compound of formula (I) or a salt, hydrate or solvate acceptable for pharmaceutical or veterinary use thereof: where R1 and R3 independently represent an H; F; Cl; Br; -NO2; -CN; C1-C6 alkyl optionally substituted by F or 5 Cl; or C1-C6 alkoxy optionally substituted by F; R4 represents a carboxylic acid group (-COOH) or an ester thereof, or -C (= O) NR6R7, -NR7C (= O) R6, -NR7C (= O) OR6, -NHC (= O) NR7R6 or -NHC (= S) NR7R6 where R6 represents an H, or a radical of formula - (Alk) m-Q where m is 0 or 1 10 Alk is an optionally substituted divalent linear or branched C1-C12 alkylene radical, or C2-C12 alkenylene, or C2-C12 alkynylene or a divalent C3-C12 carbocyclic radical; any of the radicals that may contain one or more bonds -O-, -S- or -N (R8) - wherein R8 represents an H or C1-C4 alkyl, C3-C4 alkenyl, C3-C4 alkynyl, or C3 cycloalkyl -C6, and Q represents an H; -NR9R10 wherein R9 and R10 independently represent an H; C1-C4 alkyl; C3-C4 alkenyl; C3-C4 alkynyl; C3-C6 cycloalkyl; an ester group; an optionally substituted carbocyclic or heterocyclic group; or R9 and R10 form a ring when taken together with the nitrogen to which they are attached, which ring is optionally substituted; Y R7 represents an H or C1-C6 alkyl; or when taken together with the atom or atoms to which they bind R6 and R7 form an optionally substituted monocyclic heterocyclic ring having 5, 6 or 7 ring atoms; and 20 X represents a bond or a divalent radical of the formula - (Z) n- (Alk) - or - (Alk) - (Z) n- where Z represents -O-, -S- or -NH-, Alk is like is defined in relation to R6 and n is 0 or 1, wherein unless otherwise specified, in the context in which this occurs, the term "substituted" as applied to any fraction herein means substituted with at least one substituent, selected from (C1-) alkyl. C6), (C1-C6) alkenyl, (C2-C6) alkynyl, fluorinated-substituted (C1-C6) alkyl, fluorinated-substituted 25 (C1-C6) alkenyl, fluorinated-substituted (C1-C6) alkynyl, alkoxy ( C1-C6) and fluoro-substituted (C1-C6) alkoxy (including the special case where a ring is substituted on the C atoms of the adjacent ring by an alkylenedioxy or ethylenedioxy), alkylthio (C1-C6), phenyl, benzyl, phenoxy, benzyloxy, hydroxyl, mercapto, amino, fluorine, chlorine, bromine, cyano, nitro, oxo, -COOH, -SO2OH, -CONH2, -SO2NH2, -CORA, -COORA, -SO2ORA, -NHCORA, -NHSO2RA, - CONHRA, -SO2NHRA, -NHRA, -NRARB, -CONRARB or -SO2NRARB where RA and RB are independently a 30 (C1-C6) or C2-C6 alkoxy group or a 5-7 monocyclic carbocyclic or heterocyclic group me ring embras, or RA and RB form a ring, when taken together with the nitrogen to which they are attached; or wherein "substituted" means substituted by a phenyl, benzyl, phenoxy, or benzyloxy, the phenyl ring thereof may itself be substituted with any of the foregoing, with the exception of phenyl, benzyl, phenoxy, or benzyloxy. 35 2. A compound as claimed in claim 1, wherein the radical R4X- is at position 4 of the phenyl ring. 3. A compound as claimed in claim 1 or claim 2 wherein X is a bond. 4. A compound as claimed in any of the preceding claims, wherein R3 is a hydrogen. 5 5. A compound as claimed in any of the preceding claims, wherein R1 is a hydrogen or fluorine. 6. A compound as claimed in any preceding claim, wherein R4 represents a -C (= O) NR6R7 wherein R6 and R7 are as defined in claim 1. 7. A compound as claimed in any one of the preceding claims, wherein R4 represents a -10 NHC (= O) NR7R6 wherein R6 and R7 are as defined in claim 1. 8. A compound as claimed in claim 7, wherein R6 is a quinuclidinyl radical. 9. A compound as claimed in any preceding claim wherein R6 represents a radical of the formula - (Alk) mQ wherein m is 1 and the divalent radical Alk contains 3 or 4 carbon atoms and is unsubstituted, and Q represents a -NR9R10 where R9 and R10 independently represent an H; C1-C4 alkyl; C3-C4 alkenyl; C3-C4 alkynyl; C3-C6 cycloalkyl; an ester group; an optionally substituted carbocyclic or heterocyclic group; or forms a ring when taken together with the nitrogen to which they are attached, a ring that is optionally substituted. 10. A compound as claimed in any one of claims 6 to 8, wherein R7 is a hydrogen. 11. A compound as claimed in claim 1, wherein Q represents an H; -CF3; -OH; -SH; -NR8R8 20 where each R8 independently represents an H; C1-C4 alkyl; C3-C4 alkenyl; C3-C4 alkynyl; C3-C6 cycloalkyl; an ester group; an optionally substituted aryl, aryloxy, cycloalkyl, cycloalkenyl or heterocyclic group; or form a ring when taken together with the nitrogen to which they bind; Y R7 represents an H or C1-C6 alkyl; or when taken together with the atom or atoms to which R6 and R7 bind form a monocyclic heterocyclic ring having 5, 6 or 7 ring atoms. 25 12. A compound as claimed in claim 11, wherein R4 represents a carboxylic acid group (-COOH) or an ester group of the formula -COOR wherein R is methyl, ethyl, n- or iso-propyl, n- , sec- or tert-butyl or benzyl. 13. A compound as claimed in claim 11 or claim 12, wherein R6 represents a radical of formula - (Alk) mQ wherein m is 1, Alk is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH (CH3) CH2-, or a divalent cyclopropylene, cyclopentylene or cyclohexylene radical, optionally substituted by OH, oxo, CF3, methoxy or ethoxy, and Q represents a hydrogen; -NR8R8 wherein each R8 can be the same or different and selected from hydrogen, methyl, ethyl, n- or isopropyl or tert-butyl; a methyl, ethyl or benzyl ester; or an optionally substituted phenyl, phenoxy, cyclopentyl, cyclohexyl, furyl, thienyl, piperidyl, or piperazinyl group. 14. A compound as claimed in any one of claims 11 to 13 wherein R7 represents a methyl, ethyl, n- or iso-propyl, n-, sec- or tert-butyl ester; or when taken together with the atom or atoms to which R6 and R7 bind form a monocyclic heterocyclic ring having 5, 6 or 7 ring atoms. 15. A compound as claimed in any of claims 11 to 14 wherein R1 is H, F, Cl, methyl, methoxy, or methylenedioxy. 16. A compound as claimed in any one of claims 11 to 14 wherein R1 is F, at position 40 6 of the 3-oxo-1,3-dihydro-2H-pyrazolo ring system [4,3-c] cinnolin-2-il. 17. A compound as claimed in any one of claims 11 to 16, wherein R3 is H, F, Cl, methyl, methoxy, or methylenedioxy. 18. A compound as claimed in any one of claims 11 to 17, wherein X is a bond, or a radical -CH2- or -CH2CH2-. Four. Five 19. A compound as claimed in claim 1, wherein the compound has the structure of formula (IC) or one of its salts, hydrates or solvates acceptable for veterinary or pharmaceutical use: wherein X and R4 are as specified in any of claims 3, 7 to 10 or 11 to 18. 20. A compound as claimed in claim 18, wherein the radical R4X- is at position 4 of the phenyl ring. 21. A compound as claimed in claim 19 or claim 20, wherein X is a bond and R4 is -C (= O) NR6R7 wherein R6 and R7 are as specified in claim 11, or claim 13 or 14 respectively. 22. A compound as claimed in claim 1, wherein the compound has the structure 4- (6-fluoro-3-10 oxo-1,3-dihydro-pyrazolo [4,3-c] cinnolin-2-yl ) -N- (2,2-Difluoro-ethyl-yl) -benzamide, of formula (A) or one of its salts, hydrates or solvates acceptable for veterinary or pharmaceutical use. 23. A compound as claimed in claim 1, wherein the compound has the structure N- [3- (tert-butyl-methyl-amino) -butyl] -4- (6-fluor-3-oxo-1, 3-dihydro-pyrazolo [4,3-c] -cinnolin-2-yl) -benzamide, of formula (B): or one of its salts, hydrates or solvates acceptable for veterinary or pharmaceutical use. 24. A compound of the structure selected from one of the groups consisting of the following formulas: or one of its salts, hydrates or solvates acceptable for veterinary or pharmaceutical use. 25. A veterinary or pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 24 together with an excipient or carrier acceptable for pharmaceutical or veterinary use. 26. A compound as claimed in any one of claims 1 to 24, for use in the treatment of 5 conditions that can be treated with immunomodulation. 27. The use of a compound as claimed in any one of claims 1 to 24, in the manufacture of a medicament for the treatment of conditions that can be treated with immunomodulation. 28. A compound as claimed in any one of claims 1 to 24, for use in a method of immunomodulation in mammals, including humans, which comprises administration to a mammal, in need of such treatment, of an effective immunomodulatory dose of the compound. 29. A compound for use, as claimed in claim 26, the use as claimed in claim 27 or a method as claimed in claim 28, wherein the immunomodulation is immuno-inhibition. 30. A compound for use as claimed in claim 26, the use as claimed in claim 27 or a method as claimed in claim 28, wherein the condition is an autoimmune disease, rheumatoid arthritis, multiple sclerosis. , diabetes, asthma, transplant, systemic lupus erythematosus and psoriasis.