CN112745268B

CN112745268B - Crystal form of benzimidazole derivative and preparation method thereof

Info

Publication number: CN112745268B
Application number: CN202011156500.6A
Authority: CN
Inventors: 叶景泉; 周先强; 邵启云; 冯君; 贺峰
Original assignee: Jiangsu Hengrui Medicine Co Ltd
Current assignee: Shandong Shengdi Pharmaceutical Co ltd; Jiangsu Hengrui Medicine Co Ltd
Priority date: 2019-10-31
Filing date: 2020-10-26
Publication date: 2022-09-16
Anticipated expiration: 2040-10-26
Also published as: CN112745268A

Abstract

The present disclosure relates to crystalline forms of benzimidazole derivativesAnd a preparation method. In particular, the disclosure relates to form a of the compound of formula I and methods of making the same. The A crystal form of the compound of the formula I has advantages in stability and hygroscopicity, is more suitable for drug development, can meet the medicinal requirements of production, transportation and storage, has stable production process, can be repeatedly controlled, and can be suitable for industrial production.

Description

Crystal form of benzimidazole derivative and preparation method thereof

Technical Field

The disclosure belongs to the field of pharmaceutical chemistry, and relates to a crystal form of a benzimidazole derivative, in particular to a crystal form A of a compound shown in a formula I and a preparation method thereof.

Background

Nuclear receptors are ligand-regulated transcription factors that regulate development, immunity, and cellular metabolism, and are one of the major drug target classes of human diseases. The retinoid-related orphan receptor gamma (ROR γ) protein is a member of the NR1 subfamily of nuclear receptors and has a typical nuclear receptor domain structure consisting of a DNA binding domain, a ligand binding domain, a hinge domain, and an activation function 2 domain (Benoit G, et al, pharmaceutical Reviews, 58 (4): 798-836, 2006; Zhang, Y., et al, Acta pharmaceutical Sinica Sinica, 36: 71-87,2015). In contrast to most other nuclear receptors that bind as dimers, ROR γ recognizes and binds as a monomer. It binds to a specific DNA sequence, usually consisting of TAAA/TNTAGGTCA, and is called ROR response element (RORE).

There are two subtypes of ROR γ, ROR γ 1 and ROR γ 2 (also known as ROR γ t) which are produced from the same RORC gene, possibly by selection of other promoters (Villey I et al, eur.j.immunol., 29 (12): 4072-80, 1999). Because both subtypes of ROR γ (ROR γ 1 and ROR γ t) are produced from the same mRNA, with the same ligand binding domain, they differ only in their protein N-termini (Jetten, a.m., 2009; Ivanov, i.i.et al, 2006). The small molecule inhibitor is combined in a ligand binding domain to inhibit the function of a receptor, so that the two subtypes of ROR gamma cannot achieve selectivity, and the small molecule inhibitors (or regulators) of ROR gamma are called as ROR gamma small molecule inhibitors (or regulators) and do not divide into subtypes.

The two subtypes, rory, are very different in tissue expression distribution. ROR γ t is expressed mainly in thymus and several immune cells, while ROR γ 1 is expressed in many tissues, such as thymus, liver, muscle, testis, pancreas, prostate, heart, etc. (Jetten, a.m., 2009; Zhang, y.et al, 2015). One of the functions of ROR γ 1 is reported in the literature to be the regulation of the human biological clock, involved in the regulation of the circadian rhythm (Jetten, a.m., 2009). Type 17 helper immunoregulatory T cells (TH17) are the major source of autoimmune disease (Ivanov, i.i.et al, 2006), both subtypes of ROR γ are expressed in TH17 cells, regulating type 17 helper immunoregulatory T cell differentiation and inducing gene transcription (Ruan, q.e., et al, 2011). Cytokines IL-6 and TGF- β induce differentiation of undifferentiated CD 4T helper cells into Th17 cells, Th17 cells express high levels of ROR γ T, induce transcription of IL-23 receptor genes in undifferentiated CD 4T helper cells, IL23 receptors in turn promote and stabilize production of Th17 cells, forming part of a positive feedback loop (Ivanov, i.i.et al, 2006; Jetten, a.m., 2009). Meanwhile, ROR gamma t can induce the gene transcription of proinflammatory effect cytokines such as cytokines IL-17A, IL-17F, IL-21, IL-22 and the like, and the inflammatory process is enhanced. Similar to ROR γ T, ROR γ 1 is also expressed in Th17 cells, and can also regulate differentiation of 17-type helper immunoregulatory T cells (Th17) and induce gene transcription (Ruan, q., et al., 2011). Pharmacological antagonism of ROR γ has therapeutic potential for autoimmune diseases, making it an attractive target for small molecule inhibitors.

ROR γ has been identified as a key mediator in the pathogenesis of several diseases, such as rheumatoid arthritis, psoriasis, multiple sclerosis, inflammatory bowel disease, crohn's disease, sjogren's syndrome, asthma, and the like. (Louten et al, J.Allergy Clin.Immunol., 123: 1004-. Some other diseases, such as chronic dry eye, Kawasaki disease, mucosal leishmaniasis, and Hashimoto's thyroiditis, are characterized by increased Th17 ratios and/or increased levels of Th17 marker cytokines, such as IL-17, IL-22, and IL-23. (Chen, Y. et al, Mucosal. Immunol., 7 (1): 38-45,2014; Jia, S., et al, Clin. exp. Immunol., 162: 131. cona 137, 2010; Boaventura, VS et al, Eur. J. Immunol., 40: 2830. cona 2836, 2010; Figueroa-Vega, N. et al, J. Clin. Endocrinol. Metab., 95: 953. cona 62, 2010). In each of the above examples, the inhibitory effect can be enhanced by simultaneously inhibiting ROR α. ROR γ t inhibitors are currently being developed for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. See Jun r.huh and Dan r.littman, eur.j.immunol., 42 (9): 2232, 2237(2012), WO2012/027965, WO2013/029338 and US 2015/291607.

Compounds of formula I are provided in application PCT/US 19/30526:

the entire contents of this application are incorporated herein.

The crystal structure of the medicinal active ingredient often affects the chemical stability of the medicament, and the difference of crystallization conditions and storage conditions can cause the change of the crystal structure of the compound and sometimes bring about other forms of crystal forms. Generally, amorphous drug products do not have regular crystal structures and often have other defects, such as poor product stability, fine crystallization, difficult filtration, easy agglomeration, poor flowability and the like. Therefore, it is necessary to improve various properties of the above products, and intensive research is needed to find a crystal form with high purity and good physicochemical stability.

Disclosure of Invention

The present disclosure provides crystalline form a of the compound of formula I.

The present disclosure provides a crystalline form a of the compound of formula I having an X-ray powder diffraction pattern with characteristic peaks at diffraction angles 2 θ of 11.827, 13.183, 14.339, 15.441, 16.495, 18.100, 18.365, 19.858.

Furthermore, the X-ray powder diffraction pattern of the crystal form A has characteristic peaks at diffraction angles 2 theta of 11.827, 13.183, 14.339, 15.441, 16.495, 18.100, 18.365, 19.858, 20.958, 21.204, 22.465, 23.721 and 24.536.

Furthermore, the X-ray powder diffraction pattern of the crystal form A has characteristic peaks at diffraction angles 2 theta of 11.827, 13.183, 14.339, 15.441, 16.495, 17.461, 18.100, 18.365, 19.270, 19.858, 20.958, 21.204, 22.003, 22.465, 22.968, 23.721, 24.536, 25.978, 26.653, 28.387, 28.818, 29.641, 30.986 and 32.785.

The disclosure also relates to a preparation method of the crystal form A of the compound shown in the formula I, which comprises the step of taking one or more of hydrocarbon solvent, ether solvent, alcohol solvent, ester solvent, ketone solvent, nitrile solvent, halogenated hydrocarbon solvent, nitrogen-containing solvent, water and dimethyl sulfoxide as a solvent to precipitate crystals of the compound shown in the formula I.

The present disclosure also relates to a process for preparing form a of the compound of formula I, comprising: taking a certain amount of the compound of the formula I, adding a proper amount of solvent, separating out solid, filtering and drying to obtain the crystal form A of the compound of the formula I. In certain embodiments, the solvent used in the preparation of the form a of the compound of formula I according to the present disclosure is selected from one or more of hydrocarbon solvents, ether solvents, alcohol solvents, ester solvents, ketone solvents, nitrile solvents, halogenated hydrocarbon solvents, nitrogen-containing solvents, water, and dimethyl sulfoxide.

The hydrocarbon solvents include but are not limited to n-hexane, n-heptane, p-xylene; the ether solvent includes, but is not limited to, diethyl ether, propylene glycol methyl ether, methyl tert-butyl ether, isopropyl ether or 1, 4-dioxane; the alcoholic solution includes but is not limited to methanol, ethanol, isopropanol, n-propanol, isoamyl alcohol or trifluoroethanol; the ester solvent includes, but is not limited to, ethyl acetate, isopropyl acetate or butyl acetate; the ketone solvents include, but are not limited to, acetone, acetophenone, 4-methyl-2-pentanone; the nitrile solvents include, but are not limited to, acetonitrile, propionitrile; the halogenated hydrocarbon solvents include, but are not limited to, methyl chloride, methylene chloride, 1, 2-dichloroethane, chloroform or carbon tetrachloride; the nitrogen-containing solvent includes, but is not limited to, nitromethane, N-dimethylformamide, N-dimethylacetamide.

In some embodiments, the solvent used in the preparation of form a is selected from an isopropyl alcohol/isopropyl ether mixed solvent, an isopropyl acetate/n-hexane mixed solvent, an isopropyl alcohol/water mixed solvent, a methyl tert-butyl ether/isopropyl alcohol mixed solvent, acetonitrile, an isopropyl ether/ethanol mixed solvent, a methyl tert-butyl ether, toluene, an isopropyl alcohol/n-hexane mixed solvent, or an ethyl acetate/n-hexane mixed solvent.

The crystallization mode of the crystal form A of the compound shown in the formula I is selected from room temperature crystallization, cooling crystallization, volatilization crystallization or crystal seed addition induced crystallization.

The present disclosure also relates to a process for preparing form a of the compound of formula I, comprising: taking a certain amount of the compound of the formula I, adding a proper amount of an isopropanol/isopropyl ether mixed solvent, an isopropyl acetate/n-hexane mixed solvent, an isopropanol/water mixed solvent, a methyl tert-butyl ether/isopropanol mixed solvent, acetonitrile, an isopropyl ether/ethanol mixed solvent, a methyl tert-butyl ether, toluene, an isopropanol/n-hexane mixed solvent or an ethyl acetate/n-hexane mixed solvent, separating out a solid, filtering and drying to obtain the crystal form A of the compound of the formula I.

The present disclosure also relates to a process for preparing form a of the compound of formula I, comprising: taking a certain amount of the compound of the formula I, adding a proper amount of methyl tert-butyl ether, toluene, an isopropanol/n-hexane mixed solvent or an ethyl acetate/n-hexane mixed solvent or an isopropanol/water mixed solvent, pulping at room temperature (preferably 48h), filtering and drying to obtain the crystal form A of the compound of the formula I.

The present disclosure also relates to a process for preparing form a of the compound of formula I, comprising: taking a certain amount of the compound of the formula I, adding a proper amount of an isopropanol/isopropyl ether mixed solvent, an isopropyl acetate/n-hexane mixed solvent, an isopropanol/water mixed solvent, a methyl tert-butyl ether/isopropanol mixed solvent and acetonitrile or an isopropyl ether/ethanol mixed solvent, heating, stirring, dissolving, stirring at room temperature for 12-24h, preferably 16-20h, separating out a solid, filtering and drying to obtain the crystal form A of the compound of the formula I.

The present disclosure also relates to pharmaceutical compositions comprising the crystalline form a of compound I and optionally one or more pharmaceutically acceptable carriers and/or diluents. The pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form. For example, the pharmaceutical preparation containing the crystal form a of the compound I of the present disclosure may be formulated as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (the preparation is prepared by using the crystal form a of the compound I of the present disclosure or the injections themselves contain the crystal form a of the compound I of the present disclosure, including injections, sterile powders for injection and concentrated solutions for injection), suppositories, inhalants or sprays.

In addition, the pharmaceutical compositions of the present disclosure may also be administered to a patient or subject in need of such treatment by any suitable mode of administration, e.g., oral, parenteral, rectal, pulmonary, or topical administration. When used for oral administration, the pharmaceutical composition can be prepared into oral preparations, for example, oral solid preparations such as tablets, capsules, pills, granules and the like; or oral liquid preparations such as oral solution, oral suspension, syrup, etc. When formulated into oral preparations, the pharmaceutical preparations may further contain suitable fillers, binders, disintegrants, lubricants and the like. When used for parenteral administration, the pharmaceutical preparation can be prepared into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When prepared into injections, the pharmaceutical composition may be manufactured by a conventional method in the existing pharmaceutical field. When preparing injection, the pharmaceutical preparation can be added with no additive, or added with proper additive according to the nature of the medicine. When used for rectal administration, the pharmaceutical preparation may be formulated into suppositories and the like. For pulmonary administration, the pharmaceutical formulation may be formulated as an inhalant or a spray. In certain embodiments, the form a of the compounds of formula I of the present disclosure is present in a pharmaceutical composition or medicament in a therapeutically and/or prophylactically effective amount. In certain embodiments, the form a of the compound of formula I described in the present disclosure is present in a pharmaceutical composition or medicament in the form of a unit dose.

The present disclosure further relates to a pharmaceutical composition prepared from form a of the compound of formula I and at least one pharmaceutically acceptable carrier, diluent or excipient.

The present disclosure further relates to a process for preparing a pharmaceutical composition comprising mixing the crystalline form a of the compound of formula I of the present disclosure with at least one pharmaceutically acceptable carrier, diluent or excipient.

The present disclosure further relates to the use of said form a of the compound of formula I for the manufacture of a medicament for the treatment of a rory mediated disease or condition. Such ROR γ mediated diseases or conditions include, but are not limited to, inflammatory and autoimmune diseases and cancer, wherein inflammatory and autoimmune diseases include, but are not limited to, arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, osteoarthritis, focal purulent, ulcerative colitis, ankylosing spondylitis, autoimmune diabetes, type I diabetes, autoimmune ocular disease, autoimmune thyroid disease, type I immune hypersecretion syndrome, type II autoimmune polyendocrine syndrome, multiple sclerosis, inflammatory bowel disease, inflammatory bowel syndrome, juvenile idiopathic arthritis, chronic inflammatory bowel disease, chronic inflammatory bowel disease,

syndrome, crohn's disease, asthma, kawasaki disease, hashimoto's thyroiditis, infectious disease, ankylosing spondylitis, Chronic Obstructive Pulmonary Disease (COPD), lung disease, glomerulonephritis, myocarditis, thyroiditis, dry eye, uveitis, Behcet's disease, asthma, atopic dermatitis, contact dermatitis, allograft rejection, polymyositis, GVHD, acne, ulcerative colitis, systemic lupus erythematosus, scleroderma, bronchitis, dermatomyositis, and allergic rhinitis; wherein the cancer includes but is not limited to non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, synovial sarcoma, breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, renal cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, melanin pigment, and cell membrane, and can be used in combination with other therapeutic agentsTumors, solid tumors, gliomas, glioblastomas, hepatocellular carcinomas, papillary renal tumors, head and neck tumors, leukemias, lymphomas, myelomas, and non-small cell lung cancers.

Detailed Description

In the description and claims of this application, unless otherwise indicated, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. However, for a better understanding of the present disclosure, definitions and explanations of some of the relevant terms are provided below. In addition, where the definitions and explanations of terms provided herein are inconsistent with the meanings that would normally be understood by those skilled in the art, the definitions and explanations of terms provided herein shall control.

"ethereal solvents" as described in this disclosure include, but are not limited to: tetrahydrofuran, diethyl ether, propylene glycol methyl ether, methyl tert-butyl ether, isopropyl ether or 1, 4-dioxane.

Specific examples of "alcoholic solvents" described in the present disclosure include, but are not limited to: methanol, ethanol, isopropanol, n-propanol, isoamyl alcohol or trifluoroethanol.

The "ester solvents" described in this disclosure include, but are not limited to: ethyl acetate, isopropyl acetate or butyl acetate.

Specific examples of "ketone solvents" described in the present disclosure include, but are not limited to: acetone, acetophenone and 4-methyl-2-pentanone.

Specific examples of "nitrile solvents" described in the present disclosure include, but are not limited to: acetonitrile or propionitrile.

Specific examples of "halogenated hydrocarbon solvents" described in the present disclosure include, but are not limited to: methyl chloride, dichloromethane, chloroform or carbon tetrachloride.

The "X-ray powder diffraction pattern or XRPD" described in this disclosure is obtained by Cu-ka diffraction.

The differential scanning calorimetry or DSC in the present disclosure refers to measuring the temperature difference and heat flow difference between a sample and a reference substance during the temperature rise or constant temperature process of the sample to characterize all the physical changes and chemical changes related to the thermal effect and obtain the phase change information of the sample.

The "2 theta or 2 theta angle" referred to in the present disclosure refers to the diffraction angle, theta is the bragg angle in degrees or degrees, and the error range of 2 theta may be ± 0.3, ± 0.2 or ± 0.1.

Advantageous effects of the invention

The compound of formula I provided by the disclosure has advantages in the aspects of crystal form A stability and hygroscopicity, is more suitable for drug development, can meet the medicinal requirements of production, transportation and storage, has a stable production process, can be repeatedly controlled, and can be suitable for industrial production.

Drawings

FIG. 1 is an XRPD pattern for an amorphous form of a compound of formula I;

figure 2 is an XRPD pattern of form a of compound of formula I;

FIG. 3 is a DSC of form A of compound of formula I;

figure 4 is a TGA profile of compound a crystalline form of formula I;

FIG. 5 is a DVS moisture absorption spectrum of form A of compound of formula I;

figure 6 is a comparison of XRPD patterns before and after DVS detection of form a of compound of formula I;

Detailed Description

The present disclosure will be explained in more detail with reference to examples, which are merely illustrative of the technical solutions of the present disclosure, and the spirit and scope of the present disclosure is not limited thereto.

The present disclosure will be explained in more detail with reference to examples, which are provided only for illustrating the technical solutions of the present disclosure and do not limit the spirit and scope of the present disclosure.

Test conditions of the apparatus used for the experiment:

the structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shift (. delta.) at 10 ^-6 The units in (ppm) are given. NMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD) and Tetramethylsilane (TMS) as an internal standard.

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

HPLC was carried out using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18150X 4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18150X 4.6mm column).

XRPD was X-ray powder diffraction detection: the measurement is carried out by using a BRUKER D8 type X-ray diffractometer, and the specific information is acquired: cu anode (40kV, 40mA), Cu-Ka ray

The scanning mode is as follows: : θ/2 θ, scan range: 10-48 degrees.

DSC is differential scanning calorimetry: the measurement adopts a METTLER TOLEDO DSC 3+ differential scanning calorimeter, the temperature rise rate is 10 ℃/min, the specific temperature range refers to a corresponding map (mostly 25-300 or 25-350 ℃), and the nitrogen purging speed is 50 mL/min.

TGA is thermogravimetric analysis: the detection adopts a METTLER TOLEDO TGA 2 type thermogravimetric analyzer, the heating rate is 10 ℃/min, the specific temperature range refers to a corresponding graph (mostly 25-300 ℃), and the nitrogen purging speed is 20 mL/min.

DVS is dynamic moisture adsorption: adopting Surface Measurement Systems adaptation 2, at 25 ℃, starting from 50% of humidity, examining the humidity range to be 0% -95%, stepping to be 10%, judging the standard that each gradient mass change dM/dT is less than 0.002, TMAX is less than 360min, and circulating for two circles.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), a developing solvent used for the reaction, a system of eluents for column chromatography used for purifying compounds and a developing solvent system for thin layer chromatography including: a: the volume ratio of the n-hexane/ethyl acetate system is adjusted according to the different polarities of the compounds, and a small amount of basic or acidic reagents such as triethylamine, acetic acid and the like can be added for adjustment.

Comparative example 1 preparation of Compound of formula I (preparation of example 152,153 of application No. PCT/US 19/30526)

Preparation of 3- (6-chloro-5- (2- (difluoromethoxy) phenyl) -1H-benzo [ d ] imidazol-2-yl) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) propanamide

First step preparation of 6-chloro-2 '- (difluoromethoxy) - [1, 1' -diphenyl ] -3, 4-diamine

A mixture of 4-bromo-5-chlorobenzene-1, 2-diamine (1.5g, 6.78mmol), 2- (2- (difluoromethoxy) phenyl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborocyclopentane (2.2g, 8.15mmol), tris (dibenzylideneacetone) dipalladium (620mg), tri-tert-butylphosphonium tetrafluoroborate (393mg), sodium carbonate (1.7g, 13.7mmol), 1, 4-dioxane (50mL) and water (10mL) was deoxygenated, heated to 90 ℃ and stirred for 3 hours. The reaction solution was concentrated under reduced pressure, and the residue was directly loaded on an ISCO solid column and eluted with a mixed solvent of n-hexane/ethyl acetate to give a white solid product (1.0g, yield 51.9%) MS (+) ES:285(M + H) +.

Second step preparation of ethyl 4- ((4-amino-6-chloro-2 '- (dichloromethoxy) - [1, 1' diphenyl ] -3-yl) amino) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) -4-oxobutanoate

Preparation of ethyl 4- ((5-amino-2-chloro-2 '- (dichloromethoxy) - [1, 1' diphenyl ] -4-yl) amino) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) -4-oxobutanoate

To a solution of 6-chloro-2 '- (difluoromethoxy) - [1, 1' -diphenyl ] -3, 4-diamine (543mg, 1.9mmol), 2- (4- ((cyclopropylmethyl) sulfonyl) phenyl) -4-ethoxy-4-oxobutanoic acid (500mg, 1.47mmol) and DMF (5mL) were added EDCl (560mg, 2.93mmol), HOBT (447mg, 2.93mmol) and DIPEA (380mg, 2.94mmol), and the reaction solution was stirred warm for 2 hours. The reaction was adsorbed on 5g silica gel, loaded onto a silica gel column and eluted with 45% ethyl acetate in n-hexane to give a mixture of ethyl 4- ((4-amino-6-chloro-2 '- (dichloromethoxy) - [1, 1' diphenyl ] -3-yl) amino) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) -4-oxobutanoate and ethyl 4- ((5-amino-2-chloro-2 '- (dichloromethoxy) - [1, 1' diphenyl ] -4-yl) amino) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) -4-oxobutanoate as a white solid (600mg, yield: 62.3%) ms (esi):607(M + H) +.

Third step preparation of ethyl 3- (6-chloro-5- (2- (difluoromethoxy) phenyl) -1H-benzo [ d ] imidazol-2-yl) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) propionate

4- ((4-amino-6-chloro-2 '- (dichloromethoxy) - [1, 1' diphenyl)]-3-yl) amino) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) -4-oxobutanoic acid ethyl ester and 4- ((5-amino-2-chloro-2 '- (dichloromethoxy) - [1, 1' diphenyl)]A solution of a mixture of ethyl (4-yl) amino) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) -4-oxobutanoate (800mg) in acetic acid (15mL) was heated to 80 ℃ and the reaction stirred for 2 hours. Concentrating the reaction solution under reduced pressure, purifying the obtained residue by column chromatography, eluting with 60% ethyl acetate in n-hexane solution to obtain 3- (6-chloro-5- (2- (dichloromethoxy) phenyl) -1H-benzo [ d ]]Imidazol-2-yl) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) propionic acid ethyl ester was an off-white solid (600mg, yield: 77.3%). MS (ESI) 589(M + H) ⁺ .

Fourth step preparation of (S) -3- (6-chloro-5- (2- (difluoromethoxy) phenyl) -1H-benzo [ d ] imidazol-2-yl) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) propanamide

Preparation of (R) -3- (6-chloro-5- (2- (difluoromethoxy) phenyl) -1H-benzo [ d ] imidazol-2-yl) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) propanamide

To a solution of ethyl 3- (6-chloro-5- (2- (difluoromethoxy) phenyl) -1H-benzo [ d ] imidazol-2-yl) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) propanoate (400mg, 0.68mmol) in methanol (5mL) was added a 7N methanolic ammonia solution (4.8mL, 33.9mmol), and the reaction was warmed to 60 ℃ and stirred for 12 hours. The reaction solution was concentrated under reduced pressure and the crude product was purified by column chromatography using n-hexane and ethyl acetate as eluent to give 3- (6-chloro-5- (2- (difluoromethoxy) phenyl) -1H-benzo [ d ] imidazol-2-yl) -3- (4- ((cyclopropylmethyl) sulfonyl) phenyl) propanamide (177mg, compound of formula I).

MS(+)ES:560(M+H) ⁺

¹ H NMR(400MHz,CD ₃ OD)δppm 7.91(d,2H)7.64(d,3H)7.38-7.52(m,2H)7.19-7.36(m,3H)6.64(s,1H)4.92-5.00(m,1H)3.38(d,1H)3.07-3.21(m,3H)0.83-1.01(m,1H)0.37-0.63(m,2H),0.11(q,2H)

The resulting product was identified as an amorphous form of the compound of formula I, with the XRPD pattern shown in figure 1.

Test example 1.LanthaScreen TR-FRET ROR gamma-LBD and Biochemical experiments with coactivating peptides

Materials and reagents

1.RORγLBD-GST tagged(Cat No.RORC-114H，Creative Biomart)

2.Fluorescein-D22 coactivator，(Cat No.PV4386，Invitrogen)

3.LanthaScreen ^TM Tb anti-GST anti body (Cat. No. PV3550, Invitrogen)

4.TR-FRET coregulatory buffer D(Cat No，PV4420，Invitrogen)

5.DTT(Cat No.P2325，Fisher)

6.384 well assay plate (Cat No.6008280, Perkin Elmer)

Tecan Infinite M1000 plate reader (Tecan)

Experimental procedure

Complete TR-FRET Coreglator Buffer D was prepared by adding 1M DTT to TR-FRET Coreglator Buffer D to a final concentration of 5mM DTT. Compound dilutions were performed in Complete TR-FRET Coregator buffer D. The highest dose was 3 μ M, 7-fold dilution, for a total of 7 doses. To each well of the 384-well plate, 10. mu.L was added. For negative and positive controls, 10. mu.L completetR-FRET Coregulator Buffer D was added.

The ROR γ LBD solution was prepared using Complete TR-FRET Coregator buffer D. The final concentration of ROR γ LBD solution at each reaction was 25 ng. In addition to the addition of 5 u L Complete TR-FRET Coreglator Buffer D negative hole, 5 u L ROR gamma LBD solution is added to 384 hole determination plate in all holes.

Complete TR-FRET Coreglator Buffer D was used to prepare a solution containing 0.6. mu.M Fluorescein-D22 and 8nM Tbanti-GST antibody, and 5. mu.L of each of the prepared solutions was added to all wells of the 384 well assay plate.

The 384 well plate was briefly mixed gently on a plate shaker and reacted at room temperature for 1 hour with exclusion of light. The 384 well plates were sealed with plastic film to avoid evaporation.

The plates were read on a Tecan Infinite M1000 plate reader at wavelengths of 520nm and 495 nm. Make itIC was calculated using GraphPad Prism by plotting the logarithm of compound concentration versus the percent inhibition ₅₀ The value is obtained. IC's of the compounds of formula I are shown in Table 1 ₅₀ The value is obtained.

Test example 2. assay for inhibiting IL-17A cytokine production in human peripheral blood mononuclear cells

Experimental materials and instruments

1.Human PBMC(Stemcell,Cat No.70025.1)

2.Lymphocyte medium(Zenbio,Cat No.LYMPH-1)

3.TexMACS(Miltenyi Biotec,Cat No.130-097-196)

4.Human Cytostim(Miltenyi Biotec,Cat No.130-092-173)

Human IL-17ELISA, Human IL-17 enzyme linked immunosorbent assay kit (R & D Systems, D1700)

6.96 well cell culture plate (Fisher Scientific, Cat No.07-200-80)

Tecan SPARK plate reader (Tecan)

Experimental procedure

Frozen human Peripheral Blood Mononuclear Cells (PBMC) were rapidly thawed in pre-warmed lymphocyte medium, centrifuged at 1000rpm for 10min, the cell culture supernatant was removed, the cells were gently suspended in TexMACS medium, and the cells were counted. T cell activating reagent cytostim (10. mu.L/mL) was added to the cell suspension in proportion, and then the cells were seeded in 96-well cell culture plates at a density of 1X 105 peripheral blood mononuclear cells/well. Test compounds were diluted in gradient using TexMACS medium and added to each experimental well, 2-3 parallel wells per group. Negative control wells containing cells alone without cytostim were prepared to obtain background readings. The cell culture plates were incubated in a 5% carbon dioxide incubator at 37 ℃ for 3 days. Cell culture supernatant was collected after 3 days of drug treatment and centrifuged to remove suspended matter. IL-17A in the supernatant was then quantified using an IL-17A enzyme linked immunosorbent assay kit. The IC of compound inhibition was calculated using the log (inhibitor) vs. response- -Variable slope (four parameters) algorithm of GraphPad Prism 6.0 ₅₀ The value is obtained. The inhibition rate calculation formula is as follows:

in the calculation formula, Inhibition rate is Inhibition percent; OD (NC) is the reading of the negative control with cells without cytostim and without compound; OD (PC) is the reading of the positive control with cytostim added to the cells but no compound added; OD (compound) is the reading for cells plus cytostim and plus compound.

Results of the experiment

The biochemical experiment results of LanthaScreen TR-FRET ROR gamma-LBD and coactivated peptide show that the combination of the compound of formula I and ROR gamma-LBD causes the conformational change, which leads to the weakening of the affinity of ROR gamma-LBD to coactivated peptide, and inhibits the activity of ROR gamma, IC ₅₀ Was 10nM (Table 1). In addition, ROR γ inhibitors may inhibit the activity of immune helper T cell type 17 (Th17) and immune toxic T cell type 17 (Tc17), including the production of IL-17A cytokines, to reduce the overactivity of the immune system. To determine the functional activity of ROR γ inhibitors in cells, T cells were activated with artificial superantigens (Cytostim) in human PBMC cells, promoting T cell differentiation, and thereby secreting cytokines, including IL-17. The experimental results show that the compound of the formula I can inhibit the production of IL-17A cytokine and IC in human peripheral blood mononuclear cells ₅₀ Was 21nM (Table 1). In summary, compounds of formula I can reduce IL-17A cytokine production by inhibiting the activity of ROR γ.

TABLE 1 inhibition of ROR gamma binding and IL-17 production in human peripheral blood mononuclear cells by compounds of formula I

Example 1 preparation of Compound A Crystal form of formula I

The compound of formula I (1.5g, 2.67mmol) was added to 35mL of a mixed solvent of isopropanol and isopropyl ether (V: V ═ 13:22), heated to 70 ℃, stirred to dissolve, slowly cooled to room temperature, stirred at room temperature for 18 hours, filtered, the filter cake was collected, and dried under vacuum to give the product (1.37g, yield: 91.3%). The product is defined as crystal form A by X-ray powder diffraction detection, and the spectrogram is shown in figure 2. The DSC spectrum is shown in figure 3, and the first endothermic peak is 251.58 ℃, and the second endothermic peak is 285.55 ℃; the TGA spectrum is shown in figure 4.

DVS testing showed that under normal storage conditions (i.e., 25 ℃, 60% RH), the sample had a moisture absorption weight gain of about 0.37%; under accelerated test conditions (i.e., 70% RH), the moisture pick-up weight is about 0.42%; under extreme conditions (90% RH), the hygroscopic weight gain was about 0.54%. The desorption process of this sample coincides with the adsorption process during the 0% -95% RH humidity change. And (4) after DVS detection, re-testing the crystal form, wherein the crystal form is not transformed. The DVS spectrum is shown in FIG. 5, and the X-ray powder diffraction contrast spectrum before and after DVS detection is shown in FIG. 6.

TABLE 2 characteristic peaks of the form A of the compound of formula I

Example 2 preparation of Compound A Crystal form of formula I

The compound of formula I (50mg, 89.3 μmol) was added to a mixed solution of 1mL isopropyl acetate and n-hexane (V: V ═ 1:1), heated to 70 ℃, stirred to dissolve, slowly cooled to room temperature, stirred at room temperature for 16 hours to precipitate a white solid, filtered, the cake was collected, and dried under vacuum to give the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 3 preparation of Compound A Crystal form of formula I

The compound of formula I (50mg, 89.3 μmol) was added to a mixed solution of 1mL isopropanol and water (V: V ═ 4:1), heated to 70 ℃, stirred to dissolve, slowly cooled to room temperature, stirred at room temperature for 16 hours to precipitate a white solid, filtered, the cake collected, and dried under vacuum to give the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 4 preparation of Compound A Crystal form of formula I

The compound of formula I (50mg, 89.3 μmol) was added to a mixed solution of 1.5mL methyl t-butyl ether and isopropanol (V: V ═ 2:1), heated to 70 ℃, stirred to dissolve, slowly cooled to room temperature, stirred at room temperature for 16 hours to precipitate a white solid, filtered, collected as a cake, and dried under vacuum to give the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 5 preparation of Compound A Crystal form of formula I

The compound of formula I (50mg, 89.3. mu. mol) was added to 1mL acetonitrile, heated to 70 ℃ and stirred to dissolve, slowly cooled to room temperature, stirred at room temperature for 16 hours to precipitate a white solid, which was filtered, the filter cake was collected and dried in vacuo to give the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 6 preparation of Compound A Crystal form of formula I

The compound of formula I (50mg, 89.3 μmol) was added to a mixed solution of 1.25mL isopropyl ether and ethanol (V: V ═ 4:1), heated to 70 ℃, stirred to dissolve, slowly cooled to room temperature, stirred at room temperature for 16 hours to precipitate a white solid, filtered, the cake was collected, and dried under vacuum to give the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 7 preparation of Crystal form A of Compound I

The compound of formula I (50mg, 89.3. mu. mol) was added to 1mL of methyl tert-butyl ether as a suspension, slurried at room temperature under stirring for 48 hours, filtered, the filter cake collected, and dried in vacuo to give the title product (40mg, yield: 80%).

The product is of A crystal form through X-ray powder diffraction detection.

Example 8 preparation of Crystal form A of Compound I

The compound of formula I (50mg, 89.3. mu. mol) was added to 1mL of toluene as a suspension, slurried at room temperature and stirred for 48 hours, filtered, the cake collected and dried in vacuo to give the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 9 preparation of Compound A Crystal form of formula I

The compound of formula I (50mg, 89.3 μmol) was added to a mixed solution of 1mL of isopropanol and n-hexane (V: V ═ 1:1) to give a suspension, and the suspension was slurried at room temperature with stirring for 48 hours, filtered, the cake was collected and dried in vacuo to give the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 10 preparation of Compound A Crystal form of formula I

The compound of formula I (50mg, 89.3 μmol) was added to a mixed solution of 1mL of ethyl acetate and n-hexane (V: V ═ 1:1) to give a suspension, and the suspension was slurried at room temperature with stirring for 48 hours, filtered, the cake was collected and dried in vacuo to give the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 11 preparation of Compound A Crystal form of formula I

The compound of formula I (50mg, 89.3 μmol) was added to a mixed solution of 1mL isopropyl alcohol and water (V: V ═ 1:1) to give a suspension, and slurried at room temperature for 48 hours, followed by filtration, collection of a cake and vacuum drying to obtain the product (40mg, yield: 80%). The product is of A crystal form through X-ray powder diffraction detection.

Example 12 Crystal form influence factor experiment of Compound I

A crystal form A sample of the compound A in the formula I is placed open and spread, the stability of the sample under the conditions of heating (40 ℃, 60 ℃), illumination (4500Lux) and high humidity (RH 75 percent and RH 90 percent) is inspected, and the sampling inspection period is 30 days.

The experimental results are as follows:

TABLE 3 influence factor experiment results of compound A crystal form of formula I

And (4) experimental conclusion:

the influence factors of table 3 show that: the crystal form A of the compound of the formula I has good physical and chemical stability after being placed for 30 days under the conditions of illumination, high temperature of 40 ℃, high temperature of 60 ℃, high humidity of 75 percent and high humidity of 90 percent.

Example 13 Long-term accelerated stability test of Compound A form of formula I

Samples of form A of compound of formula 1 were tested for long-term (25 deg.C, 60% RH), accelerated (40 deg.C, 75% RH) stability for 3 months.

Table 4, results of long term accelerated stability experiments on samples of form a of compound of formula I:

the long term accelerated stability test results from table 4 show that: the physicochemical stability of the crystal form A sample of the compound of the formula I is good after being placed for 3 months under the conditions of long-term (25 ℃, 60% RH) and accelerated (40 ℃, 75% RH) stability.

Claims

1. The crystal form A of the compound of the formula I has an X-ray powder diffraction pattern, and has characteristic peaks at diffraction angles 2 theta of 11.827, 13.183, 14.339, 15.441, 16.495, 18.100, 18.365 and 19.858,

2. the crystalline form a of the compound of formula I according to claim 1, having an X-ray powder diffraction pattern with characteristic peaks at diffraction angles 2 Θ of 11.827, 13.183, 14.339, 15.441, 16.495, 18.100, 18.365, 19.858, 20.958, 21.204, 22.465, 23.721, 24.536.

3. A crystalline form a of the compound of formula I according to claim 1 having an X-ray powder diffraction pattern with characteristic peaks at diffraction angles 2 Θ of 11.827, 13.183, 14.339, 15.441, 16.495, 17.461, 18.100, 18.365, 19.270, 19.858, 20.958, 21.204, 22.003, 22.465, 22.968, 23.721, 24.536, 25.978, 26.653, 28.387, 28.818, 29.641, 30.986, 32.785.

4. The crystalline form a according to any one of claims 1 to 3, having a 2 Θ value error within ± 0.2.

5. The process for the preparation of the form A of the compound of formula I according to any one of claims 1 to 4, comprising the step of precipitating crystals of the compound of formula I with one or more solvents selected from the group consisting of hydrocarbon solvents, ether solvents, alcohol solvents, ester solvents, nitrile solvents, and water,

the hydrocarbon solvent is selected from n-hexane, n-heptane and p-xylene;

the ether solvent is selected from diethyl ether, propylene glycol methyl ether, methyl tert-butyl ether, isopropyl ether or 1, 4-dioxane;

the alcohol solution is selected from methanol, ethanol, isopropanol, n-propanol, isoamylol or trifluoroethanol;

the ester solvent is selected from ethyl acetate, isopropyl acetate or butyl acetate;

the nitrile solvent is selected from acetonitrile and propionitrile.

6. The process for preparing crystalline form a according to claim 5, wherein the solvent is selected from the group consisting of an isopropyl alcohol/isopropyl ether mixed solvent, an isopropyl acetate/n-hexane mixed solvent, an isopropyl alcohol/water mixed solvent, a methyl tert-butyl ether/isopropyl alcohol mixed solvent, acetonitrile, an isopropyl ether/ethanol mixed solvent, a methyl tert-butyl ether, toluene, an isopropyl alcohol/n-hexane mixed solvent and an ethyl acetate/n-hexane mixed solvent.

7. A pharmaceutical composition comprising the crystalline form a according to any one of claims 1 to 4, further comprising one or more pharmaceutically acceptable carriers, diluents or excipients.

8. A pharmaceutical composition prepared from the crystalline form a according to any one of claims 1-4 and at least one pharmaceutically acceptable carrier, diluent or excipient.

9. A process for preparing a pharmaceutical composition comprising admixing the form a of any one of claims 1-4 and at least one pharmaceutically acceptable carrier, diluent, or excipient.

10. Use of the crystalline form a of any one of claims 1-4, or the pharmaceutical composition of claim 7 or 8, in the manufacture of a medicament for treating a rory-mediated disease or disorder.