HK1225379A1 - Chromene derivatives as inhibitors of tcr-nck interaction - Google Patents

Description

Chromene derivatives as inhibitors of the TCR-Nck interaction

Technical Field

The present invention relates to a group of compounds comprising a chromene nucleus and having the ability to inhibit lymphocyte proliferation by blocking TCR interaction with Nck, and thus such compounds are useful for the treatment of diseases or disorders where such interaction triggers a complication, such as transplant rejection, immune or autoimmune diseases or proliferative diseases.

Background

Autoimmune and inflammatory diseases, such as asthma, multiple sclerosis, allergy, rheumatoid arthritis, crohn's disease or psoriasis, are different types of diseases in which the adaptive immune system attacks the body's self-antigens, in particular by T-lymphocytes. It is generally accepted that T cells are central to all immunological mechanisms. T cells are capable of recognizing both foreign and self antigens and activating immune responses against these antigens. T cells recognize antigens via the T Cell Receptor (TCR), which is responsible for transmitting signals to the cytoplasm. Indeed, the fact that the haplotype Major Histocompatibility Complex (MHC) is the most important genetic risk factor for human autoimmune disease places T cells in the heart of all immunopathological events.

T cells recognize MHC-associated antigenic peptides (pMHC) via the T cell antigen receptor (TCR) and are able to translate small differences in the chemical composition of pMHC into different quantitative and qualitative results. Although there are a variety of control mechanisms that prevent activation of T cells bearing TCRs (which have significant affinity for self-peptide-bearing MHC), including suppression of potentially autoreactive T cells during maturation in the thymus, these mechanisms are somewhat inadequate in patients with autoimmune disease, and autoreactive T cells are activated and proliferate, overcoming homeostatic control.

Upon stimulation, the TCR is activated and undergoes conformational changes that result in the recruitment of different proteins that form the "TCR signal transductants" responsible for signal transduction and cell activation. This complex includes a cytoplasmic protein Nck that binds to the PRS motif (proline-rich sequence) present in the CD3 subunit of the T cell receptor. As a result, TCR conformation changes are stabilized and activation signals are efficiently transmitted.

Current therapies for immune diseases emerge as immunosuppressive strategies rather than as tolerogenic/immunomodulatory approaches. Azathioprine, methotrexate, mycophenolate mofetil, and cladribine are cytostatic agents. Other therapies promote depletion of T cells in lymph nodes (alemtuzumab, anti-CD 52) or their maintenance (fingolimod). Alternatively, indirect modulation of the immune system is also being used as a powerful strategy (BG-12). Therefore, although TCR signaling has a central role in activating T cells in autoimmune diseases, recent efforts to regulate activation of T cells have focused on modulating costimulatory signals, cytokine receptors, etc., with consequent lack of specificity and occurrence of a number of related side effects.

To develop specific immunomodulatory therapies, much effort has been focused on characterizing the role of Nck in T cell activation by many different research groups. Nck has been suggested to play an important role in the function of mature T cells through studies in knockout mice that lack Nck1 in all tissues and only conditionally lack Nck2 on T cells. In these models, the number of peripheral T cells expressing TCRs with low affinity for self-antigens is drastically reduced, and a general worsening of T cell activation by stimulation with weak antigens is observed. Furthermore, the importance of Nck was also emphasized by the generation of bone marrow chimeras, suggesting that the PRS motif (binding site for Nck in TCR) is important for activation of mature T cells by weak agonists rather than strong agonists. Likewise, mutations in the PRS sequence alter the ability of mice to activate adaptive immune responses in vivo. Furthermore, inhibitor peptides with high affinity for the SH3.1 domain of Nck alter the assembly of TCR signaling transductants, suggesting that recruitment of Nck is an early key step in TCR signaling, which represents a target for modulating immune responses.

Document WO2010/064707 describes a series of compounds derived from 2H-chromene for the prevention or treatment of diseases induced by undesired lymphocyte infiltration mediated by the sphingosine 1-phosphate receptor (S1P 1).

Document WO2012/042078 also describes chromene derivatives having the ability to inhibit TCR-Nck interactions in T cells and their use for the treatment of autoimmune diseases, inflammatory diseases or transplant rejection.

Therefore, it would be desirable to provide novel compounds that are capable of inhibiting TCR-Nck interactions in T lymphocytes and are good candidates. These compounds should show good activity in vivo pharmacological tests, good oral absorption upon oral administration and be metabolically stable and have a favourable pharmacokinetic profile. In addition, the compounds should be non-toxic and exhibit minimal side effects.

Disclosure of Invention

A first aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt, isomer, or solvate thereof, wherein:

R₁selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, -COR₅、-C(O)OR₅、-C(O)NR₅R₆、-CNR₅；

X is selected from-OH or-NR₂R₃；

R₂And R₃Independently selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COR₇、-C(O)OR₇、-C(O)NR₇R₈、-CNR₇、-OR₇、-NR₇R₈and-NR₇C(O)R₈；

Or, R₂And R₃Together with the nitrogen atom to which they are bound, form a substituted or unsubstituted heterocyclic ring;

R₄is halogen;

R₅、R₆、R₇and R₈Independently selected from hydrogen, C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl, and halogen.

Another aspect of the present invention relates to a compound of formula (II) or a pharmaceutically acceptable salt, isomer, or solvate thereof, wherein:

R₁selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, -COR₅、-C(O)OR₅、-C(O)NR₅R₆、-CNR₅；

R₂And R₃Independently selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COR₇、-C(O)OR₇、-C(O)NR₇R₈、-CNR₇、-OR₇、-NR₇R₈and-NR₇C(O)R₈；

Or, R₂And R₃Together with the nitrogen atom to which they are bound, form a substituted or unsubstituted heterocyclic ring;

R₄is halogen;

R₅、R₆、R₇and R₈Independently selected from hydrogen, C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl, and halogen.

The term "alkyl" in the present invention refers to a linear or branched hydrocarbon chain group having 1 to 6 carbon atoms (preferably 1 to 4 carbon atoms) bonded to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl and the like. The alkyl group may be optionally substituted with one or more substituents such as: halogen, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The term "cycloalkyl" refers in the present invention to a stable 3-to 6-membered (preferably 3-membered) monocyclic group, saturated or partially saturated, consisting solely of carbon and hydrogen atoms, for example cyclopropyl, cyclopentyl, cyclohexyl, and which may optionally be substituted by one or more groups such as: alkyl, halogen, hydroxy, alkoxy, carboxy, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The term "aryl" in the present invention means an aromatic carbocyclic chain having 6 to 18 carbon atoms, preferably having 6 to 14 carbon atoms, more preferably having 6 to 8 carbon atoms, and the aryl group may be constituted by a single or multiple rings, and in the case of being constituted by multiple rings, the rings are separate and/or fused rings. Non-limiting examples of aryl groups are phenyl, naphthyl, indenyl, and the like. Preferably the aryl group is phenyl or naphthyl. The aryl group may be optionally substituted with one or more substituents such as: alkyl, halogen, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The term "heteroaryl" is meant to comprise at least one group selected from: an aryl group of a heteroatom of nitrogen, oxygen or sulfur.

The term "heterocycle" in the present invention means a compound consisting of carbon atoms and at least one selected from the group consisting of: nitrogen, oxygen or sulfur heteroatoms constitute unsaturated, saturated or partially saturated stable 3-15 membered monocyclic or bicyclic groups. Preferably, the heterocyclic ring is a 4-8 membered heterocyclic ring having one or more heteroatoms, more preferably, the heterocyclic ring is a 5-6 membered heterocyclic ring having one or more heteroatoms. Examples of heteroaryl groups may be, but are not limited to: azepines, indoles, imidazoles, isothiazoles, thiadiazoles, furans, tetrahydrofurans, benzimidazoles, benzothiazoles, piperidines, pyrrolidines, piperazines, purines, quinolines. Preferably, the heterocyclic group is pyrrolidine or piperazine. Any position on the heterocyclic group may be substituted with one or more substituents such as: alkyl, halogen, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

"halogen" means fluorine, chlorine, bromine or iodine.

In a preferred embodiment, R₁Is substituted or unsubstituted C₁-C₄An alkyl group.

In a more preferred embodiment, R₁is-CH₃。

In another more preferred embodiment, R₁Is a quilt C₃-C₆Cycloalkyl-substituted C₁-C₄An alkyl group. In an even more preferred embodiment, R₁is-CH₂-a cyclopropyl group.

In another preferred embodiment, R₂Is H.

In another preferred embodiment, R₃Is substituted or unsubstituted C₁-C₄An alkyl group.

In a more preferred embodiment, R₃is-CH₂CH₃A group.

In another more preferred embodiment, R₃Is C substituted by a group-NR' R₁-C₄Alkyl, wherein R 'and R' are independently selected from H or C₁-C₄An alkyl group. In another even more preferred embodiment, R₃is-CH₂-CH₂-N(CH₃)₂A group.

In another preferred embodiment, R₂And R₃Forming a substituted or unsubstituted saturated 5-membered heterocyclic ring.

In another preferred embodiment, R₂And R₃Forming a substituted or unsubstituted saturated 6-membered heterocyclic ring.

In another more preferred embodiment, at least one position on the saturated heterocycle is substituted with C₁-C₄Alkyl substitution.

In another more preferred embodiment, the saturated 6-membered heterocyclic ring contains an insertion of a further unsubstituted or substituted C₁-C₄An alkyl-substituted N atom.

In another preferred embodiment, R₃To comprise additionallyInserted unsubstituted or by C₁-C₄An alkyl-substituted saturated 6-membered heterocyclic ring of N atoms.

In another preferred embodiment, R₄Is fluorine.

In another preferred embodiment, the compound of formula (I) is selected from the following series:

- (4- (4-fluorophenyl) -6-methoxy-2H-chromen-3-yl) (pyrrolidin-1-yl) methanone,

-N-ethyl-4- (4-fluorophenyl) -6-methoxy-2H-chromene-3-carboxamide,

- (4- (4-fluorophenyl) -6-methoxy-2H-chromen-3-yl) (4-methylpiperazin-1-yl) methanone,

-N- (2- (dimethylamino) ethyl) -4- (4-fluorophenyl) -6-methoxy-2H-chromene-3-carboxamide,

- (6- (cyclopropylmethoxy) -4- (4-fluorophenyl) -2H-chromen-3-yl) (pyrrolidin-1-yl) methanone,

-6- (cyclopropylmethoxy) -N-ethyl-4- (4-fluorophenyl) -2H-chromene-3-carboxamide, and

- (6- (cyclopropylmethoxy) -4- (4-fluorophenyl) -2H-chromen-3-yl) (4-methylpiperazin-1-yl) methanone,

- (6- (cyclopropylmethoxy) -4- (4-fluorophenyl) -2H-chromen-3-yl) (pyrrolidin-1-yl) methanone,

-6- (cyclopropylmethoxy) -N-ethyl-4- (4-fluorophenyl) -2H-chromene-3-carboxamide,

- (6- (cyclopropylmethoxy) -4- (4-fluorophenyl) -2H-chromen-3-yl) (4-methylpiperazin-1-yl) methanone,

-6- (cyclopropylmethoxy) -N- (2- (dimethylamino) ethyl) -4- (4-fluorophenyl) -2H-chromene-3-carboxamide.

In another preferred embodiment, X is-OH.

In another preferred embodiment, the compound of formula (I) is 4- (4-fluorophenyl) -6-methoxy-2H-chromene-3-carboxylic acid.

Another aspect of the invention relates to the use of a compound of formula (I) as described above for the preparation of a medicament.

Another aspect of the invention relates to the use of a compound of formula (I) as described above for the preparation of a medicament for the treatment of a disease or condition mediated by TCR-Nck interactions in T lymphocytes.

Throughout this specification, the terms "treatment", "treating" or other grammatically related expressions of a disease refer to curative treatment as well as palliative or prophylactic treatment of such disease.

In a preferred embodiment, the disease or condition mediated by TCR-Nck interaction in T lymphocytes is selected from: transplant rejection, immunological, autoimmune and inflammatory diseases, neurodegenerative diseases, hematological diseases and proliferative diseases.

In a more preferred embodiment, the disease or condition mediated by TCR-Nck interactions in T lymphocytes is selected from: transplant rejection, rheumatoid arthritis, psoriatic arthritis, psoriasis, type I diabetes, complications associated with diabetes, multiple sclerosis, systemic lupus erythematosus, atopic dermatitis, mast cell mediated allergic reactions, leukemia, lymphoma, and thromboembolic and allergic complications associated with leukemia and lymphoma.

Another aspect of the invention relates to the use of a compound of formula (I) in the treatment of a disease or condition mediated by TCR-Nck interactions in T lymphocytes.

Another aspect of the invention relates to a pharmaceutical composition comprising a compound of formula (I) as described above and one or more pharmaceutically acceptable excipients.

The compounds, pharmaceutically acceptable salts and/or solvates thereof (e.g., pharmaceutical compositions containing them) described in the present invention may be used with other additional drugs to provide combination therapy. The additional agents may be part of the same pharmaceutical composition or may be provided in a separate composition for administration simultaneously or non-simultaneously with a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable isomer, solvate or salt thereof.

Unless otherwise indicated, the compounds of the present invention also include compounds that differ only in the presence of one or more isotopically enriched atoms. E.g. only H substituted by deuterium or tritium or carbon enriched¹³C or¹⁴C carbon or rich in¹⁵N-substituted compounds having this structure are within the scope of the present invention.

The compounds of formula (I) for therapeutic use are prepared in solid form or in the form of an aqueous suspension in a pharmaceutically acceptable diluent. These formulations may be administered by any suitable route of administration and for this purpose the formulations will be formulated in a manner suitable for the chosen route of administration. In a particular embodiment, the compounds of formula (I) provided herein are administered by oral, topical, rectal or parenteral (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous, and the like) routes. An overview of the drugs of administration in different drug forms and the excipients necessary to obtain such administration can be found, for example, in "treatyof galenic pharmacy (galenic treaty)" c.fauli illo,1993Luz a n5, saedicines, Madrid or other spanish, european and us pharmacopoeias or analogues.

For the therapeutic use of a compound of formula (I), an isomer, salt or solvate thereof, it will preferably be found in an acceptable or substantially pure pharmaceutical form, i.e. having a pharmaceutically acceptable level of purity apart from normal pharmaceutical additives such as diluents and carriers, and containing no substances which are considered toxic at normal dosage levels. The purity level of the active substance is preferably higher than 50%, more preferably higher than 70%, more preferably higher than 90%. In a preferred embodiment, the compound of formula (I) or an isomer, salt or solvate thereof is at a purity level of greater than 95%.

The compounds of the invention may be in crystalline form as free compounds or solvates and both forms are intended to be within the scope of the invention. As used herein, the term "solvate" includes two pharmaceutically acceptable solvates, i.e., solvates of the compounds of formula (I) that may be used in the preparation of a medicament, and solvates that may be useful as pharmaceutically unacceptable solvates in the preparation of pharmaceutically acceptable salts or solvates. The nature of the pharmaceutically acceptable solvate is not critical, provided that it is pharmaceutically acceptable. In a specific embodiment, the solvate is a hydrate. Solvates may be obtained by conventional solvation methods well known to those skilled in the art.

The compound of the present invention represented by formula (I), specifically, a specific compound belonging to the aforementioned general formula may include isomers depending on the presence of a plurality of bonds (e.g., Z, E), and may include optical isomers or enantiomers depending on the presence of a chiral center. Isomers, enantiomers or individual diastereomers and mixtures thereof are within the scope of the invention. The individual enantiomers or diastereomers, e.g., mixtures thereof, may be separated by conventional techniques.

Another aspect of the invention is a method of treating a disease or disorder mediated by TCR-Nck interactions in T cells, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I).

The term "therapeutically effective amount" as used herein refers to an amount of active compound sufficient to produce the desired effect in which the symptoms of the disease are reduced. The dose should not be used in an amount that causes undesirable side effects that are made undesirable by clinical evaluation and which render them therapeutically untreatable. In general, the dosage varies with the age, condition, sex and severity of the disease of the patient, as well as the route and frequency of administration, and will be specifically determined on a case-by-case basis.

Another aspect of the present invention relates to a process for obtaining a compound of formula (I) as described above, comprising the steps of:

a) reacting a compound of formula (II) with a compound of formula (III) and a compound of formula (IV),

wherein R is₁X and R₄Has the same meaning as in claim 1, and

b) converting a compound of formula (III) to another compound of formula (I) in one or more steps.

Throughout the description and claims the word "comprise" and variations of the word, do not exclude other technical features, additives, components or steps. Other objects, advantages and features of the invention will be in part apparent to those skilled in the art from the specification and practice of the invention. The following examples and figures are provided by way of illustration and are not intended to be limiting of the present invention.

Drawings

FIG. 1 shows the ability of each of the test compounds AX-105, AX-106, AX-107 and AX-108 of the present invention to inhibit T-lymphocyte proliferation.

FIG. 2 shows the ability of each of test compounds AX-129, AX-130, AX-131, AX-132 and AX-137 of the present invention to inhibit T lymphocyte proliferation.

Examples

The invention will be illustrated by tests performed by the inventors which show the effectiveness of the compounds of the invention.

Example 1: synthesis of Compounds of the invention

Synthesis schemes for AX-105 through AX-108

Synthesis of Compound AX-137: compound 1(3g, 1 eq.), AgNO₃A mixture of (3.5g, 2 equivalents) in ethanol (30ml) and NaOH (1.7g, 4 equivalents) dissolved in water (30ml) was refluxed at 85 ℃ and stirred at that temperature for 4 hours the reaction was monitored by TLC after completion, the mixture was acidified with 1MHCl and extracted with DCM (2 × 30ml) the combined organic layers were washed with water (20ml), brine solution (10ml) and dried with anhydrous sodium sulphate the organic layer was evaporated under reduced pressure yielding 1.5g of the desired product with a purity of 96.2% by HPLC.¹HNMR(CDCl₃)7.17-7.08(m, 4H), 6.89-6.87(d, 1H), 6.84-6.81(m, 1H), 6.21-6.20(d, 1H), 4.96(s, 2H), 3.61(s, 3H). For C₁₇H₁₃FO₄Theoretical value MS: 300.28, respectively; m⁺+1 actual value: 301.0.

synthesis of Compound AX-105A mixture of AX-137(0.2g, 1 eq), EDCl (0.14g, 1.1 eq), HOBt (0.09g, 1 eq), pyrrolidine (0.06g, 1.2 eq) and DIPEA (0.17g, 2 eq) in THF (5ml) was irradiated by microwaves for 10 minutes after which the THF was evaporated, the residue washed with saturated sodium bicarbonate solution and extracted with DCM (2 × 10 ml). the combined organic layers were washed with water (20ml), brine solution (10ml) and dried over anhydrous sodium sulfate and the organic layer evaporated under reduced pressure to yield 135mg of the desired product in 98% purity by HPLC.¹HNMR(CDCl₃)7.37-7.33(m，2H)，7.10-7.06(t，2H)，6.89-6.87(d，1H)，6.78-6.75(dd，1H)，6.46-6.45(d，1H)，4.82(s，2H)，3.66(s，3H)，3.31-3.27(t，2H)，3.02(b，2H)，1.68-1.63(m, 2H), 1.60-1.54(m, 2H). For C₂₁H₂₀FNO₃Theoretical value MS: 353.4, respectively; m⁺+1 actual value: 354.1.

synthesis of Compound AX-106A mixture of AX-137(0.2g, 1 eq), EDCl (0.14g, 1.1 eq), HOBt (0.09g, 1 eq), ethylamine (Etilamine). HCl (0.06g, 1.2 eq) and DIPEA (0.17g, 2 eq) in THF (5ml) was irradiated by microwaves for 10 minutes after which the THF was evaporated, the residue washed with saturated sodium bicarbonate solution and extracted with DCM (2 × 10 ml). the combined organic layers were washed with water (20ml), brine solution (10ml) and dried over anhydrous sodium sulfate and the crude product was purified by column chromatography (9% methanol in DCM) to give 100mg of the desired product by HPLC to a purity of 99.3%.¹HNMR(CDCl₃)7.29-7.27(m, 2H), 7.20-7.15(m, 2H), 6.89-6.86(d, 1H), 6.79-6.76(dd, 1H), 6.24-6.23(d, 1H), 3.63(s, 3H), 3.11-3.04(q, 2H), 0.78-0.74(t, 3H). For C₁₉H₁₈FNO₃Theoretical value MS: 327.35, respectively; m⁺+1 actual value: 328.1.

synthesis of Compound AX-107A mixture of AX-137(0.1g, 1 eq), EDCl (0.07g, 1.1 eq), HOBt (0.05g, 1 eq), N-methylpiperazine (0.04g, 1.2 eq) and DIPEA (0.09g, 2 eq) in THF (5ml) was irradiated by microwaves for 10 minutes after which the THF was evaporated, the residue was washed with a saturated solution of sodium bicarbonate and extracted with DCM (2 × 10 ml). the combined organic layers were washed with water (20ml), brine solution (10ml) and dried over anhydrous sodium sulfate and the organic layer was evaporated under reduced pressure to yield 68mg of the desired product, 94.9% pure by HPLC.¹HNMR(CDCl₃)7.31(b, 2H), 7.12-7.08(t, 2H), 6.89-6.87(d, 1H), 6.79-6.76(dd, 1H), 6.44(m, 1H), 4.95-4.91(d, 1H), 4.68-4.64(d, 1H), 3.66(s, 3H), 3.5-3.7(m, 3H), 3.04(b, 1H), 2.3-2.27(b, 1H), 2.13(b, 4H), 2.03(b, 1H), 1.49(b, 1H). For C₂₂H₂₃FN₂O₃Theoretical value MS: 382.4, respectively; m^-1 actual value: 383.0.

synthesis of Compound AX-108A mixture of AX-137(0.2g, 1 eq), EDCl (0.14g, 1.1 eq), HOBt (0.09g, 1 eq), N-dimethylethylenediamine (0.06g, 1.2 eq), and DIPEA (0.17g, 2 eq) in THF (5ml) was irradiated by microwaves for 10 minutes after which the THF was evaporated, the residue was washed with saturated sodium bicarbonate solution and extracted with DCM (2 × 10ml), the combined organic layers were washed with water (20ml), brine solution (10ml) and dried over anhydrous sodium sulfate and the crude product was purified by column chromatography (6% methanol in DCM) to give 80mg of the desired product with a purity of 97.7% by HPLC.¹HNMR(CDCl₃)7.28-7.25(m, 2H), 7.17-7.13(t, 2H), 6.88-6.86(d, 1H), 6.78-6.75(dd, 1H), 6.22-6.21(d, 1H), 5.80(b, 1H), 3.63(s, 3H), 3.13-3.09(q, 2H), 2.08-2.05(t, 2H), 1.97(s, 6H). For C₂₁H₂₃ClFN₂O₃Theoretical value MS: 370.42, respectively; actual value of M + + 1: 371.0.

synthesis schemes for AX-129 through AX-132

To a solution of compound 1(1.2g, 0.003mol) and 2-methyl-2-butene (3.92ml, 0.037mol) in t-butanol (25ml) at room temperature was added NaClO₂(1.07g, 0.003mol) and NaH₂PO₄(1.63g, 0.01mol) in water (2.5ml) and stirred at the same temperature until the SM was consumed. After about 30 minutes, the tert-butanol was evaporated off and the resulting solution was acidified with 2n hcl (pH 3-4). The resulting solid was filtered and dried in vacuo to give acid 2 as a pale yellow solid (1.1g, 88% yield), which was used for the preparation of amides AX-129 to AX-132 without further purification.¹HNMR(CDCl₃，400MHz)7.23(d，J＝6.8Hz，1H)，7.13-7.07(m，3H)，6.88-6.80(m，2H)，6.23(s，1H)，4.94(s，2H)，3.58(d，J＝6.8Hz，2H)，1.13(m，1H)，0.58(d，J＝7.6Hz，2H)，0.25(d，J＝4.4Hz，2H)。ES-MS[M-1]+：339.1。

Synthesis of Compound AX-129:

EDCl (164mg, 1.85mmol) and HOBT (85mg, 0.63mmol) were added to a solution of acid 2(194mg, 0.57mmol), pyrrolidine (32mg, 0.45mmol) and DIPEA (220mg, 1.71mmol) in THF (10ml) and the whole was irradiated under microwave (900W) for 4 min, the THF was concentrated to minimum volume, the reaction mass was diluted with ice cold water (20ml) and extracted with EtOAc (3 × 25 ml). the combined organic extracts were extracted with Na₂SO₄Drying and concentrating on a rotary evaporator to give a crude residue, which is FCC (SiO)₂Hexane-ethyl acetate mixture) to yield the amide AX-129 as an off-white solid (100mg, 45% yield).¹HNMR(DMSO-d₆)7.30-7.28(m，4H)，6.88-6.80(m，1H)，6.29(s，1H)，4.77(s，2H)，3.63(d，J＝6.8Hz，2H)，3.11(m，2H)，3.03(s，2H)，1.60-1.51(m，4H)，1.10(m，1H)，0.49(d，J＝8.0Hz，2H)，0.23(d，J＝4.4Hz，2H)。ES-MS[M+1]+：391.4。

Synthesis of Compound AX-130

AX-130: starting from acid 2(300mg, 0.882mmol) and substituting ethylamine for the pyrrolidine in the above step, AX-130 was prepared as a pale yellow viscous solid (130mg, 76% yield).¹HNMR(DMSO-d₆，400MHz):7.63(t，J＝4.8Hz，1H)，7.27-7.25(m，4H)，6.87-6.79(m，2H)，6.17(s，2H)，4.78(s，1H)，3.61(d，J＝5.1Hz，2H)，2.9(m，2H)，1.08(m，1H)，0.69(t，J＝7.2Hz，3H)，0.48(d，J＝8.0Hz，2H)，0.22(d，J＝4.4Hz，2H)。ES-MS[M+1]+：368.1。

Synthesis of Compound AX-131

AX-131: starting from acid 2(200mg, 0.588mmol) and substituting N-methylpiperazine for pyrrolidine in the above step, AX-131 was prepared as a light yellow viscous solid (120mg, 23% yield).¹HNMR(DMSO-d₆，400MHz)：7.32(m，4H)，6.91-6.84(m，2H)，6.27(s，1H)，4.81(s，2H)，4.30(m，1H)，3.89(m，1H)，3.63(d，J＝6.8Hz，2H)，3.18(m，1H)，2.75-2.56(m，4H)，1.09(m，1H)，0.49(d，J＝8.0Hz，2H)，0.23(d，J＝4.4Hz，2H)。ES-MS[M+1]+：423.1。

Synthesis of Compound AX-132:

in N₂Next, TBTU (514mg, 1.6mmol) was added to acid 2(218mg, 0.64mmol) in CH₂Cl₂(12ml), DMF (2.5ml) and DIEA (289mg, 2.244mmol) in a mixture; stirring was carried out at room temperature. After 1h, 4-amino-1-methylpiperidine (221mg, 1.923mmol) was added and the entire reaction mixture was stirred at room temperature for 6 h. By CH₂Cl₂The reaction mass was diluted (70ml) and washed with water (2 × 25 ml.) the organic layer was Na₂SO₄Drying and concentrating on a rotary evaporator to give a crude residue, which is FCC (SiO)₂：MeOH-CH₂Cl₂Mixture) to afford the light yellow oil amide AX-132(100mg, 39% yield).¹HNMR(DMSO-d₆，400MHz)：7.58(m，1H)，7.28-7.26(m，4H)，6.16(s，1H)，4.78(s，2H)，3.61(d，J＝6.4Hz，2H)，3.45(m，1H)，2.62(m，4H)，2.21-2.00(m，6H)，1.43(m，2H)，1.40-1.00(m，6H)，1.08(m，1H)，0.48(d，J＝7.6Hz，2H)，0.21(m，2H)。ES-MS[M+1]+：437.3。

Synthesis of Compound AX-137:

AX-137: NaOH (1.7g, 0.04mol) dissolved in water (30ml) was added to the stirred aldehyde 3(3.0g, 0.01mol), AgNO₃(3.5g, 0.02mol) in ethanol (30ml) and the reaction mass was refluxed at 85 ℃ for 4 hours. The reaction medium is acidified with 1MHCl (pH 2-3) and CH is added₂Cl₂(2 × 70ml) the combined organic extracts were washed with brine solution (50ml) followed by water (100ml) and anhydrous Na₂SO₄Drying and concentration gave AX-137(1.5g, 47% yield).¹HNMR(CDCl₃，400MHz)7.17-7.08(m，4H)，6.89-6.87(d，J＝8.8Hz，1H)，6.83(dd，J＝8.8，3.2Hz，1H)，6.21(d，J＝3.2Hz，1H)，4.96(s，2H)，3.61(s，3H)。ES-MS[M+1]+：301.0。

Example 2: inhibition of T cell proliferation induced by TCR stimulation

The effect of compounds AX-105, AX-106, AX-107 and AX-108 on the ability of TCR to induce T lymphocyte proliferation was assessed using naive T lymphocytes (PBMC, peripheral blood mononuclear cells) obtained from blood of healthy human donors the PBMC of volunteers were isolated by centrifugation of venous blood with Ficoll-Paqueplus density gradient, the purified cells (NWT; NylonWoodT cells) were plated in triplicate in 96-well plates (0.5 × 10)⁵Per well) with 200 μ l of complete medium and stimulated with OKT3(10 μ g/ml) or OKT3(1 μ g/ml) + CD28 in the presence or absence of different compounds at concentrations of 1 and 10 μ M. Cultures were incubated for 3 days and 0.5uCi [3H ] was added]After TdR/well, the cultures were analyzed for the last 12 hours of culture. The radioactivity incorporated into the DNA was determined by liquid scintillation counting. As the cell divides, radioactivity is incorporated into the daughter cells which allows the degree of cell proliferation to be known. The inhibitory potency of the test compounds is shown in figure 1.

In addition, the effects of compounds AX-129, AX-130, AX-131, AX-132 and AX-137 were also analyzed on Peripheral Blood Mononuclear Cells (PBMC) obtained by centrifugation of venous blood using Ficoll-Paqueplus density gradients for purification of T cells, PBMC were passed through a Nylon wood column for cell division analysis, purified T cells were stained with carboxyfluorescein succinimidyl ester (SCFE), labeled cells (0.5 × 10)⁵Per well) were cultured in triplicate in 96-well plates with 200 μ l complete medium and stimulated with fixed OKT3(1 μ g/ml) for 6 days in the presence or absence of the aforementioned test compound, the percentage of proliferating cells (defined as CFSE low fluorescence) was determined by flow cytometry. The inhibitory potency of the test compounds is shown in figure 2.

These assays enable the evaluation and confirmation of the ability of compounds to inhibit T cell proliferation, and thus, help to demonstrate that such compounds are candidates for the development of new therapies for the treatment of T cell mediated autoimmune diseases.

Example 3: in vivo testing of type 1 diabetes models

The development of diabetes was monitored daily and recorded as positive in the case of glucosuria levels higher than 250mg/dl detected in two consecutive measurements taken daily for the RIP-MOVA model or biweekly for the NOD model. The effect of treatment on disease incidence and survival was evaluated in the RIP-MOVA model as used for insulitis. Histopathological evaluation of insulitis of sections H & E soaked in paraffin and fixed in formalin was assessed by blind testing using the following classification system: grade 0, no intrusion; grade 1, periinsulitis (periinsulitis); grade 2, intrusion > 25%; grade 3, intrusion > 75%; and stage 4, the remaining islets. In the second phase, the therapeutic effect of the compounds was evaluated by treatment of RIP-mOVA mice already suffering from diabetes. During this second phase, compounds were tested in NOD mouse models.

Claims (26)

1. A compound of formula (I) or a pharmaceutically acceptable salt, isomer, or solvate thereof, wherein:

R₁selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, -COR₅、-C(O)OR₅、-C(O)NR₅R₆、-CNR₅；

X is selected from-OH or-NR₂R₃；

R₂And R₃Independently selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COR₇、-C(O)OR₇、-C(O)NR₇R₈、-CNR₇、-OR₇、-NR₇R₈and-NR₇C(O)R₈；

Or, R₂And R₃Together with the nitrogen atom to which they are bound, form a substituted or unsubstituted heterocyclic ring;

R₄is halogen;

R₅、R₆、R₇and R₈Independently selected from hydrogen, C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl, and halogen.

2. A compound of formula (II) or a pharmaceutically acceptable salt, isomer, or solvate thereof, wherein:

R₁selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, -COR₅、-C(O)OR₅、-C(O)NR₅R₆、-CNR₅；

R₂And R₃Independently selected from hydrogen, substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -COR₇、-C(O)OR₇、-C(O)NR₇R₈、-CNR₇、-OR₇、-NR₇R₈and-NR₇C(O)R₈；

Or, R₂And R₃Together with the nitrogen atom to which they are bound, form a substituted or unsubstituted heterocyclic ring;

R₄is halogen;

R₅、R₆、R₇and R₈Independently selected from hydrogen, C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, aryl, heteroaryl, and halogen.

3. The compound of any one of claims 1 or 2, wherein R₁Is substituted or unsubstituted C₁-C₄An alkyl group.

4. A compound according to claim 3, wherein R₁is-CH₃。

5. A compound according to claim 3, wherein R₁Is a quilt C₃-C₆Cycloalkyl-substituted C₁-C₄An alkyl group.

6. A compound of formula (I) according to claim 5, wherein R₁Is a group-CH₂-cyclopropyl.

7. A compound according to any one of claims 2 to 6, wherein R₂Is H.

8. A compound according to any one of claims 2 to 7, wherein R₃Is substituted or unsubstituted C₁-C₄An alkyl group.

9. The compound of claim 8, wherein R₃is-CH₂-CH₃A group.

10. The compound of claim 8, wherein R₃Is C substituted by a-NR' R "group₁-C₄Alkyl, wherein R 'and R' are independently selected from H or C₁-C₄An alkyl group.

11. The compound of claim 10, wherein R₃is-CH₂-CH₂-N(CH₃)₂A group.

12. A compound according to any one of claims 2 to 6, wherein R₂And R₃Forming a substituted or unsubstituted saturated five-membered heterocyclic ring.

13. A compound according to any one of claims 2 to 6, wherein R₂And R₃To form a substituted or unsubstituted saturated six-membered heterocyclic ring.

14. A compound according to any one of claims 12 or 13, wherein at least one position on the saturated heterocycle is substituted by C₁-C₄Alkyl substitution.

15. The compound of claim 13, wherein the saturated six-membered heterocyclic ring comprises an insertion of an additional unsubstituted or substituted C₁-C₄An alkyl-substituted N atom.

16. A compound according to any one of claims 2 to 7, wherein R₃To contain an insertion of a further unsubstituted or substituted C₁-C₄An alkyl-substituted N atom saturated six-membered heterocyclic ring.

17. The compound according to any one of claims 1 to 16, wherein R₄Is fluorine.

18. A compound of formula (I) according to claim 2, selected from the following series:

- (4- (4-fluorophenyl) -6-methoxy-2H-chromen-3-yl) (pyrrolidin-1-yl) methanone,

-N-ethyl-4- (4-fluorophenyl) -6-methoxy-2H-chromene-3-carboxamide,

- (4- (4-fluorophenyl) -6-methoxy-2H-chromen-3-yl) (4-methylpiperazin-1-yl) methanone,

-N- (2- (dimethylamino) ethyl) -4- (4-fluorophenyl) -6-methoxy-2H-chromene-3-carboxamide,

- (6- (cyclopropylmethoxy) -4- (4-fluorophenyl) -2H-chromen-3-yl) (pyrrolidin-1-yl) methanone,

-6- (cyclopropylmethoxy) -N-ethyl-4- (4-fluorophenyl) -2H-chromene-3-carboxamide, and

- (6- (cyclopropylmethoxy) -4- (4-fluorophenyl) -2H-chromen-3-yl) (4-methylpiperazin-1-yl) methanone,

- (6- (cyclopropylmethoxy) -4- (4-fluorophenyl) -2H-chromen-3-yl) (pyrrolidin-1-yl) methanone,

-6- (cyclopropylmethoxy) -N-ethyl-4- (4-fluorophenyl) -2H-chromene-3-carboxamide,

- (6- (cyclopropylmethoxy) -4- (4-fluorophenyl) -2H-chromen-3-yl) (4-methylpiperazin-1-yl) methanone,

-6- (cyclopropylmethoxy) -N- (2- (dimethylamino) ethyl) -4- (4-fluorophenyl) -2H-chromene-3-carboxamide.

19. The compound of claim 1, wherein X is-OH.

20. The compound of claim 19 which is 4- (4-fluorophenyl) -6-methoxy-2H-chromene-3-carboxylic acid.

21. Use of a compound of formula (I) according to any one of claims 1 to 20 for the preparation of a medicament.

22. Use of a compound of formula (I) according to any one of claims 1 to 20 for the preparation of a medicament for the treatment of a disease or condition mediated by TCR-Nck interactions in T lymphocytes.

23. The use according to claim 22, wherein the disease or condition mediated by TCR-Nck interactions in T lymphocytes is selected from: transplant rejection, immunological, autoimmune and inflammatory diseases, neurodegenerative diseases, hematological diseases and proliferative diseases.

24. The use of claim 23, wherein the disease or disorder mediated by TCR-Nck interaction in T lymphocytes is selected from: transplant rejection, rheumatoid arthritis, psoriatic arthritis, psoriasis, type I diabetes, complications associated with diabetes, multiple sclerosis, systemic lupus erythematosus, atopic dermatitis, mast cell mediated allergic reactions, leukemia, lymphoma, and thromboembolic and allergic complications associated with leukemia and lymphoma.

25. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 20 and one or more pharmaceutically acceptable excipients.

26. Process for obtaining a compound of formula (I) according to any one of claims 1 to 20, comprising the steps of:

a) reacting a compound of formula (III) with a compound of formula (IV) and a compound of formula (V),

wherein R is₁X and R₄Has the same meaning as in claim 1, and

b) converting a compound of formula (III) to another compound of formula (I) in one or more steps.