CN111484505B - Hydrochloride crystal form of bicyclic ROR gamma inhibitor - Google Patents

Abstract

The present invention provides a crystalline hydrochloride form of a bicyclic ROR gamma inhibitor, more specifically (S) -N- ((5- (ethylsulfonyl) pyridin-2-yl) methyl) -6-isopropyl-5- (((1r, 4s) -4- (trifluoromethyl) cyclohexyl) methyl) -5, 6-dihydro-4H-thieno [2, 3-c)]A crystalline form of pyrrole-2-carboxamide hydrochloride, a process for its preparation, a pharmaceutical composition and its use. The compound of the formula I has high crystal stability and small hygroscopicity, has advantages in physical property, safety and metabolic stability, and has high patent medicine value.

Description

Hydrochloride crystal form of bicyclic ROR gamma inhibitor

Cross Reference to Related Applications

The present invention claims priority and benefit of patent application No. 201910082311.X, filed on 28.1.2019 to the national intellectual property office, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The invention belongs to the field of medicinal chemistry, and relates to a hydrochloride crystal form of a bicyclic ROR gamma inhibitor, in particular to a crystal form of (S) -N- ((5- (ethylsulfonyl) pyridine-2-yl) methyl) -6-isopropyl-5- (((1R, 4S) -4- (trifluoromethyl) cyclohexyl) methyl) -5, 6-dihydro-4H-thieno [2,3-c ] pyrrole-2-formamide hydrochloride, and a preparation method, a pharmaceutical composition and application thereof.

Background

Retinoic acid-related orphan nuclear receptors (RORs) are members of the nuclear receptor family, which are capable of modulating a variety of physiological and biochemical processes. The ROR family includes three types ROR α, ROR β, and ROR γ. Three different RORs can be expressed in different tissues and regulate different physiological processes. ROR α is distributed mainly in liver, skeletal muscle, skin, lung, adipose tissue, kidney, thymus and brain; ROR β acts primarily on the central nervous system; ROR γ can be expressed in many tissues, including liver, animal fat and skeletal muscle.

ROR γ has two subtypes: ROR γ 1 and ROR γ t (ROR γ 2). ROR γ 1 is found in many tissues, such as: thymus, muscle, kidney and liver, while ROR γ t is expressed only in immune cells. ROR γ T is thought to regulate T cell helper T17 (Th 17) differentiation. Th17 is a type of helper T cell that produces interleukin 17 (IL-17) and other cytokines. Th17 cells are implicated in the pathology of numerous autoimmune and inflammatory diseases including, but not limited to, psoriasis, multiple sclerosis, rheumatoid arthritis, crohn's disease, asthma, chronic obstructive pulmonary disease, behcet's disease, and irritable bowel syndrome.

Patent applications of Vitae Pharmaceuticals Inc in the prior art, such as WO2014179564, WO2015116904, WO2016061160, etc.; and patent applications of glatiramer company, such as WO2013045431, WO2013160418, WO2013160419 and the like, all disclose a series of compounds which can be used as ROR gamma inhibitors. In view of the great potential value of ROR gamma inhibitors, it is necessary to further search for compounds having ROR gamma inhibitory function and to study the chemical stability, solid state stability, in vivo metabolic properties, shelf life and other drug-forming factors.

Disclosure of Invention

The present invention provides a compound of formula I in crystalline form,

in some embodiments, the present invention provides a compound of formula I in a crystalline form, which is form a.

In some embodiments, the form a crystal of the compound of formula I is characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 Θ, at about 14.9 °, 20.6 °, 21.4 °, 23.0 °, and 24.9 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks expressed in terms of 2 Θ at about 8.0 °, 11.9 °, 13.7 °, 14.9 °, 18.3 °, 19.4 °, 20.6 °, and 21.4 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks expressed in terms of 2 Θ at about 8.0 °, 11.9 °, 13.7 °, 14.9 °, 18.3 °, 19.4 °, 20.6 °, 21.4 °, 21.9 °, 23.0 °, and 24.9 °; typically, the X-ray powder diffraction spectrum has diffraction peaks expressed in terms of 2 Θ at about 8.0 °, 9.7 °, 11.9 °, 13.7 °, 14.9 °, 15.7 °, 16.0 °, 18.3 °, 19.4 °, 20.1 °, 20.6 °, 21.1 °, 21.4 °, 21.9 °, 22.5 °, 23.0 °, 24.1 °, 24.9 °, 26.8 °, and 30.0 °; typically, the X-ray powder diffraction spectrum has diffraction peaks expressed in terms of 2 θ at about 8.0 °, 9.5 °, 9.7 °, 10.4 °, 11.9 °, 13.7 °, 14.9 °, 15.7 °, 16.0 °, 18.3 °, 19.4 °, 20.1 °, 20.6 °, 21.1 °, 21.4 °, 21.9 °, 22.5 °, 23.0 °, 23.7 °, 24.1 °, 24.9 °, 26.8 °, 27.6 °, 29.3 °, 30.0 °, 30.8 °, 31.2 °, 33.5 °, and 34.2 °.

In some embodiments, form a crystals of the compound of formula I characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 Θ, at about 14.89 °, 20.57 °, 21.40 °, 22.95 °, and 24.87 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks expressed in terms of 2 Θ at about 7.97 °, 11.89 °, 13.69 °, 14.89 °, 18.28 °, 19.41 °, 20.57 °, and 21.40 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks expressed in terms of 2 θ at about 7.97 °, 11.89 °, 13.69 °, 14.89 °, 18.28 °, 19.41 °, 20.57 °, 21.40 °, 21.89 °, 22.95 °, and 24.87 °; typically, the X-ray powder diffraction spectrum has diffraction peaks expressed in terms of 2 θ at about 7.97 °, 9.68 °, 11.89 °, 13.69 °, 14.89 °, 15.68 °, 15.98 °, 18.28 °, 19.41 °, 20.14 °, 20.57 °, 21.12 °, 21.40 °, 21.89 °, 22.45 °, 22.95 °, 24.10 °, 24.87 °, 26.82 ° and 30.04 °; typically, the X-ray powder diffraction spectrum has diffraction peaks expressed in terms of 2 θ at about 7.97 °, 9.45 °, 9.68 °, 10.41 °, 11.89 °, 13.69 °, 14.89 °, 15.68 °, 15.98 °, 18.28 °, 19.41 °, 20.14 °, 20.57 °, 21.12 °, 21.40 °, 21.89 °, 22.45 °, 22.95 °, 23.71 °, 24.10 °, 24.87 °, 26.82 °, 27.59 °, 29.27 °, 30.04 °, 30.83 °, 31.18 °, 33.45 °, and 34.20 °.

As an embodiment of the present invention, the peak position and intensity of the characteristic peak of the X-ray powder diffraction spectrum of the form a crystal of the compound of formula I are shown in table 1:

table 1: XRPD pattern characterization data for form a crystals

In one embodiment of the present invention, the form A crystal of the compound of formula I has an X-ray powder diffraction pattern as shown in figure 1.

In one embodiment of the invention, the form a crystals of the compound of formula I have a Differential Scanning Calorimetry (DSC) measurement in which the onset of the absorption peak is at about 208 ℃.

In one embodiment of the present invention, the form A crystals of the compound of formula I have a Differential Scanning Calorimetry (DSC) measurement in which the peak of the absorption peak is at about 222 ℃.

In one embodiment of the present invention, the Differential Scanning Calorimetry (DSC) measurement of the form A crystals of the compound of formula I is shown in FIG. 2.

In one embodiment of the present invention, the thermogravimetric analysis (TGA) pattern of the form a crystal of the compound of formula I is shown in figure 3.

In one embodiment of the present invention, the dynamic gas sorption (DVS) pattern of form a crystals of the compound of formula I is shown in figure 4.

The A-type crystal of the compound of the formula I can exist in a non-solvate crystal form or a solvate crystal form, wherein the solvate refers to a solvate formed by an organic solvent and/or water and the compound of the formula I, and preferably exists in a non-solvate crystal form.

In some embodiments, the present invention provides a compound of formula I in a crystalline form, which is form B.

In some embodiments, form B crystals of the compound of formula I are characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 Θ, at about 13.9 °, 17.1 °, 19.2 °, 19.6 °, 21.9 °, and 25.1 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks expressed in terms of 2 Θ at about 7.4 °, 8.5 °, 13.9 °, 15.5 °, 16.3 °, 17.1 °, 19.2 °, 19.6 °, and 20.0 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks, expressed in terms of 2 Θ, at about 7.4 °, 8.5 °, 13.9 °, 15.5 °, 16.3 °, 17.1 °, 19.2 °, 19.6 °, 20.0 °, 21.9 °, 24.0 °, 25.1 °, 25.5 °, 26.0 °, and 29.0 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks expressed in terms of 2 θ at about 7.4 °, 8.5 °, 11.0 °, 11.9 °, 13.9 °, 15.5 °, 16.3 °, 17.1 °, 18.3 °, 19.2 °, 19.6 °, 20.0 °, 20.5 °, 21.2 °, 21.6 °, 21.9 °, 23.1 °, 24.0 °, 25.1 °, 25.5 °, 26.0 °, 26.7 °, 27.9 °, 29.0 ° and 29.2 °; typically, the X-ray powder diffraction spectrum has diffraction peaks expressed in terms of 2 θ at about 7.4 °, 8.5 °, 9.7 °, 11.0 °, 11.9 °, 13.9 °, 14.7 °, 15.5 °, 16.3 °, 17.1 °, 18.3 °, 19.2 °, 19.6 °, 20.0 °, 20.5 °, 21.2 °, 21.6 °, 21.9 °, 22.7 °, 23.1 °, 24.0 °, 25.1 °, 25.5 °, 26.0 °, 26.7 °, 27.9 °, 29.0 °, 29.2 °, 30.0 °, and 30.3 °.

In some embodiments, form B crystals of the compound of formula I characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 Θ, at about 13.88 °, 17.12 °, 19.23 °, 19.64 °, 21.93 °, and 25.07 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks expressed in terms of 2 Θ at about 7.39 °, 8.46 °, 13.88 °, 15.52 °, 16.26 °, 17.12 °, 19.23 °, 19.64 °, and 20.03 °; typically, the X-ray powder diffraction spectrum exhibits diffraction peaks expressed in terms of 2 θ at about 7.39 °, 8.46 °, 13.88 °, 15.52 °, 16.26 °, 17.12 °, 19.23 °, 19.64 °, 20.03 °, 21.93 °, 23.96 °, 25.07 °, 25.54 °, 26.04 °, and 28.95 °; typically, the X-ray powder diffraction spectrum has diffraction peaks expressed in terms of 2 θ at about 7.39 °, 8.46 °, 10.96 °, 11.87 °, 13.88 °, 15.52 °, 16.26 °, 17.12 °, 18.26 °, 19.23 °, 19.64 °, 20.03 °, 20.45 °, 21.22 °, 21.58 °, 21.93 °, 23.11 °, 23.96 °, 25.07 °, 25.54 °, 26.04 °, 26.69 °, 27.87 °, 28.95 °, and 29.24 °; typically, the X-ray powder diffraction spectrum has diffraction peaks expressed in terms of 2 θ at about 7.39 °, 8.46 °, 9.72 °, 10.96 °, 11.87 °, 13.88 °, 14.70 °, 15.52 °, 16.26 °, 17.12 °, 18.26 °, 19.23 °, 19.64 °, 20.03 °, 20.45 °, 21.22 °, 21.58 °, 21.93 °, 22.74 °, 23.11 °, 23.96 °, 25.07 °, 25.54 °, 26.04 °, 26.69 °, 27.87 °, 28.95 °, 29.24 °, 30.02 °, and 30.27 °.

As an embodiment of the present invention, the peak position and intensity of the characteristic peak of the X-ray powder diffraction spectrum of the B-type crystal of the compound of formula I are shown in Table 2:

table 2: XRPD pattern characterization data for form B crystals

Numbering	Diffraction angle 2 theta (°)	Relative Strength (%)	Numbering	Diffraction angle	2 theta (°)	Relative Strength (%)
							1	7.39	28.8	16	21.22	13.0
2	8.46	41.3	17	21.58	17.5
						3	9.72	8.2	18	21.93	48.3
4	10.96	12.3	19	22.74	8.4
						5	11.87	10.5	20	23.11	13.1
6	13.88	52.4	21	23.96	21.4
						7	14.70	9.2	22	25.07	58.1
8	15.52	32.2	23	25.54	23.7
						9	16.26	28.9	24	26.04	21.1
10	17.12	76.4	25	26.69	13.3
						11	18.26	19.1	26	27.87	10.0
12	19.23	100.0	27	28.95	27.9
						13	19.64	69.5	28	29.24	15.5
14	20.03	41.8	29	30.02	8.3
						15	20.45	14.1	30	30.27	6.8

In one embodiment of the present invention, the form B crystal of the compound of formula I has an X-ray powder diffraction pattern as shown in FIG. 5.

In one embodiment of the present invention, the Differential Scanning Calorimetry (DSC) measurement of the form B crystals of the compound of formula I is shown in FIG. 6.

In one embodiment of the present invention, the thermogravimetric analysis (TGA) pattern of form B crystals of the compound of formula I is shown in figure 7.

The B-type crystal of the compound of the formula I can exist in a non-solvate crystal form or a solvate crystal form, and the solvate refers to a solvate formed by an organic solvent and/or water and the compound of the formula I.

In another aspect, the present invention provides a method for preparing form a crystal of compound I above, comprising:

(1) Dissolving a free base of a compound of formula I in a solvent;

(2) Adding hydrochloric acid, stirring for crystallization, filtering and drying to obtain the compound A type crystal of the formula I.

In some embodiments, the solvent in step (1) above is selected from acetone or tetrahydrofuran.

In some embodiments, the molar volume ratio of free base of the compound of formula I to solvent in step (1) above is 1mmol: 5-20 mL; preferably 1mmol: 10-20 mL.

In some embodiments, the compound of formula I in step (2) above has a free base to hydrochloric acid molar ratio of 1:1 to 1.2, preferably 1:1 to 1.1, more preferably 1:1 to 1.05.

In some embodiments, the stirring temperature of step (2) above is 30 to 60 ℃, preferably 40 to 60 ℃, more preferably 40 to 50 ℃; the stirring time is 2 to 20 hours, preferably 5 to 10 hours.

In some embodiments, the hydrochloric acid in step (2) may be HCl gas or a solution of HCl gas dissolved in a solvent, or may be commercially available concentrated hydrochloric acid or diluted hydrochloric acid obtained by diluting concentrated hydrochloric acid with a solvent, and the solvent is preferably tetrahydrofuran; the molar concentration of the dilute hydrochloric acid is 0.5 to 6mol/L, preferably 1 to 1.5mol/L, and more preferably 1 to 1.2mol/L.

In another aspect, the present invention provides a process for preparing form B crystals of compound of formula I, comprising:

(1) Crystallizing compound A in a solvent to obtain suspension or solution;

(2) And (3) placing the suspension or the solution in a constant-temperature mixing instrument, shaking, centrifuging and drying to obtain the B-type crystal of the compound in the formula I.

In some embodiments, the solvent in the step (1) is selected from methanol, ethanol, acetonitrile or a mixed solvent of methanol and water.

In some embodiments, the solvent in the step (1) is selected from a mixed solvent of methanol and water, wherein the volume ratio of methanol to water is 1:0.5 to 2, preferably 1:0.5 to 1.5, more preferably 1:0.5 to 1.

In some embodiments, the molar volume ratio of the compound of formula I to the solvent in step (1) is 1mmol: 7-20 mL, preferably 1mmol:7 to 15mL, more preferably 1mmol:10 to 12mL.

In some embodiments, the constant temperature in step (2) is 30 to 60 ℃, preferably 40 to 60 ℃, and more preferably 40 to 50 ℃.

In some embodiments, the shaking time in step (2) is from 12 to 72 hours, preferably from 24 to 72 hours, more preferably from 48 to 72 hours.

In another aspect, the present invention provides a crystalline composition of form A crystals of compound I, wherein the form A crystals of compound I account for more than 50%, preferably more than 75%, more preferably more than 90%, and most preferably more than 95% of the weight of the crystalline composition. Other crystalline or amorphous forms of the compound of formula I, including but not limited to the B-form crystal of the compound of formula I, may also be present in the crystalline composition in small amounts.

In another aspect, the present invention provides a crystalline composition of form B crystals of a compound of formula I, wherein the form B crystals of said compound of formula I comprise more than 50%, preferably more than 75%, more preferably more than 90%, and most preferably more than 95% by weight of said crystalline composition. Other crystalline or amorphous forms of the compound of formula I, including but not limited to form a crystals of the compound of formula I, may also be present in the crystalline composition in minor amounts.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of the compound of formula I above, or a crystalline composition thereof; the pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier or other excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of form a crystal of a compound of formula I above, or a composition of form a crystals of a compound of formula I above; the pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier or other excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of form B crystals of a compound of formula I as described above, or a composition of form B crystals of a compound of formula I as described above; the pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier or other excipient.

In another aspect, the present invention provides a crystalline form of a compound of formula I including form a crystals of a compound of formula I as described above or form B crystals of a compound of formula I as described above, the use of a crystalline composition as described above or a pharmaceutical composition as described above in the manufacture of a medicament for the treatment of a ROR γ receptor mediated disease.

In another aspect, the present invention provides a method of treating a ROR γ receptor mediated disease, comprising administering to a mammal in need thereof a therapeutically effective amount of a crystalline form of a compound of formula I, including form a crystals of a compound of formula I as described above or form B crystals of a compound of formula I as described above, a crystalline composition as described above or a pharmaceutical composition as described above.

In another aspect, the present invention provides crystalline forms of the compound of formula I for use in the treatment of ROR γ receptor mediated diseases comprising form a crystals of the compound of formula I as described above or form B crystals of the compound of formula I as described above, a crystalline composition as described above or a pharmaceutical composition as described above.

In some embodiments of the invention, the mammal is a human.

In some embodiments of the invention, the ROR γ receptor mediated disease includes, but is not limited to, psoriasis. In the present invention, the pharmaceutical composition can be formulated into a certain dosage form, and the administration route is preferably oral administration, parenteral administration (including subcutaneous, intramuscular, and intravenous), rectal administration, and the like. For example, dosage forms suitable for oral administration include tablets, capsules, granules, powders, pills, powders, lozenges, syrups or suspensions; formulations suitable for parenteral administration include aqueous or non-aqueous solutions or emulsions for injection; dosage forms suitable for rectal administration include suppositories with hydrophilic or hydrophobic carriers. The dosage forms may also be formulated as desired to provide rapid, delayed or modified release of the active ingredient.

The crystal form of the compound shown in the formula I comprises the A-type crystal of the compound shown in the formula I, and the A-type crystal has high stability, small hygroscopicity, better in-vivo metabolism level, longer half-life period, better inhibitory activity on retinoic acid related orphan nuclear receptor (ROR) gamma, better properties in the aspects of physical property, safety and metabolic stability, and higher value as a medicament.

In the present invention, the X-ray powder diffraction spectrum of the sample is measured under the following conditions: the instrument comprises: bruker D8 ADVANCE X-ray diffractometer; target: cu is K alpha; wavelength of light

2 θ angular range: 4-40 degrees; the scanning speed is 10 degrees/min; the rotating speed of the sample is 15rpm; cu target tube pressure and tube flow: 40KV and 40mA.

In the present invention, the DSC spectrum is measured under the following conditions: the instrument comprises the following steps: TA Q2000 differential scanning calorimeter; temperature range: 25 to 300 ℃; rate of temperature rise: 10 ℃/min.

In the present invention, TGA thermogravimetric analysis is determined under the following conditions: the instrument comprises the following steps: TA Q5000 thermogravimetric analyzer; temperature range: 25 to 300 ℃; rate of temperature rise: 10 ℃/min.

It is noted that in X-ray powder diffraction spectroscopy, the diffraction pattern obtained from a crystalline compound tends to be characteristic for a particular crystal, where the relative intensities of the bands (especially at low angles) may vary due to the dominant orientation effects resulting from differences in crystallization conditions, particle size, and other measurement conditions. Thus, the relative intensities of the diffraction peaks are not characteristic of the crystal in question, and it is judged whether, at the same time as the known crystalline phase, it is more important to note the relative positions of the peaks rather than their relative intensities. In addition, there may be slight errors in the position of the peaks for any given crystal, which are also well known in the crystallography art. For example, the position of the peak may shift due to a change in temperature when analyzing the sample, movement of the sample, calibration of the instrument, or the like, and the error in measurement of the 2 θ value is sometimes about ± 0.2 °. Therefore, this error should be taken into account when determining each crystalline structure. The peak position is usually expressed in the XRD pattern by 2 θ angle or plane distance d, with a simple conversion relationship between: d = λ/2sin θ, where d represents the face distance, λ represents the wavelength of the incident X-rays, and θ is the diffraction angle. For the same crystal of the same compound, the peak positions of the XRD spectrum have similarity as a whole, and the error of relative intensity may be large. It should also be noted that in the identification of mixtures, the absence of a portion of the diffraction lines may be due to, for example, a reduction in the content, in which case it is not necessary to rely on all the bands observed in a high-purity sample, and even one band may be characteristic for a given crystal.

DSC measures the transition temperature when a crystal absorbs or releases heat due to a change in its crystalline structure or melting of the crystal. For the same crystal of the same compound, the thermal transition temperature and melting point errors in successive analyses are typically within about 5 ℃, usually within about 3 ℃, or within about 2 ℃, which means that when we say a compound has a given DSC peak or melting point ± 5 ℃. DSC provides an auxiliary method to distinguish different crystals. Different crystalline morphologies can be identified by their different transition temperature characteristics. It is noted that the DSC peak or melting point for the mixture may vary over a larger range. Furthermore, the melting temperature is related to the rate of temperature rise due to decomposition that accompanies the process of melting the substance.

Definitions and explanations

As used in the description and claims of this invention, the following terms have the meanings indicated, unless specified to the contrary:

"mammal" includes humans and domestic animals such as laboratory mammals and domestic pets (e.g., cats, dogs, pigs, sheep, cows, sheep, goats, horses, rabbits), and non-domesticated mammals such as wild mammals and the like.

The term "pharmaceutical composition" refers to a formulation of a compound of the present invention with a vehicle generally accepted in the art for delivering biologically active compounds to a mammal, such as a human. The medium includes all pharmaceutically acceptable carriers for its use. The pharmaceutical composition facilitates administration of the compound to an organism.

The term "therapeutically effective amount" refers to a sufficient amount of a drug or pharmaceutical agent that is non-toxic but achieves the desired effect. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient and also on the particular active substance, and an appropriate effective amount in a case may be determined by a person skilled in the art in the light of routine tests.

In the present invention, "pharmaceutically acceptable carriers" are those which, when administered with the active ingredient, do not significantly irritate the organism and do not impair the biological activity and performance of the active compound.

In the present invention, "room temperature" means 20 to 25 ℃.

Drawings

FIG. 1 is an X-ray powder diffraction pattern of Compound I form A crystals prepared in example 2.

FIG. 2 is a Differential Scanning Calorimetry (DSC) measurement of form A crystals of compound I prepared in example 2.

FIG. 3 is a thermogravimetric analysis (TGA) profile of compound A form of compound I prepared in example 2.

FIG. 4 is a dynamic gas sorption (DVS) diagram of form A crystals of compound I prepared in example 2.

FIG. 5 is an X-ray powder diffraction pattern of form B crystals of compound I prepared in example 4.

FIG. 6 is a Differential Scanning Calorimetry (DSC) measurement of form B crystals of compound I prepared in example 4.

FIG. 7 is a thermogravimetric analysis (TGA) profile of compound B form of compound I prepared in example 4.

FIG. 8 is the clinical score results of in vivo efficacy studies in MOG35-55 induced mouse Experimental Autoimmune Encephalomyelitis (EAE).

FIG. 9 shows the results of the inhibition rate of in vivo efficacy studies in MOG35-55 induced mouse Experimental Autoimmune Encephalomyelitis (EAE).

FIG. 10 shows the results of the incidence of in vivo efficacy studies in MOG35-55 induced mouse Experimental Autoimmune Encephalomyelitis (EAE).

FIG. 11 shows the body weight changes of MOG35-55 induced in vivo efficacy studies in Experimental Autoimmune Encephalomyelitis (EAE) in mice.

Figure 12 is the clinical score results of an in vivo efficacy study in imiquimod-induced mouse experimental psoriasis (IMQ).

Figure 13 is the inhibition rate results of in vivo efficacy studies in imiquimod-induced mouse experimental psoriasis (IMQ).

Detailed description of the preferred embodiments

The following specific examples are included to provide those skilled in the art with a clear understanding of the invention and are included to provide a further understanding of the invention. They should not be considered as limiting the scope of the invention but merely as being exemplary illustrations and representative of the invention.

All operations involving easily oxidizable or hydrolyzable raw materials were carried out under nitrogen protection. Unless otherwise stated, the starting materials used in the present invention were all commercially available and used without further purification. The solvent used in the invention is directly purchased from the market and is directly used without special treatment. The compound is made by hand or

The software names, and the commercial compounds are under the supplier catalog name.

The invention employs the following abbreviations: TEA for triethylamine; EDCI represents 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride; HOBt represents 1-hydroxybenzotriazole; boc ₂ O represents di-tert-butyl dicarbonate; TLC for thin layer chromatography; etOAc for ethyl acetate; PE represents petroleum ether; DMF represents N, N-dimethylformamide; THF represents tetrahydrofuran.

Example 1: preparation of the free base of the Compound of formula I

Synthesis of Compound 1-2

To a solution of compound 1-1 (25 g) and N-methoxymethylamine hydrochloride (14.9 g) in 500mL of dichloromethane were added TEA (51.6 g,71.0 mL), EDCI (36.7 g) and HOBt (25.8 g), and the reaction solution was reacted at 10 to 20 ℃ for 16 hours, TLC (PE: etOAc = 1) showed completion of the reaction. The reaction solution was dispersed in 500mL of dichloromethane and 500mL of water, separated, and the organic phase was washed once with 500mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a crude product. Crude product was passed through flash column (220 g)

Flash column on silica gel, eluent ethyl acetate/petroleum ether, elution gradient: 0 to 100 percent of ethyl acetate/petroleum ether (V/V); flow rate: 100 mL/min) to obtain the compound 1-2.

¹ H NMR(400MHz,CHLOROFORM-d):δ＝3.71(s,3H),3.19(s,3H),2.62-2.74(m,1H),1.99-2.07(m, 3H),1.92(br d,J＝13.6Hz,2H),1.48-1.61(m,2H),1.32-1.44(m,2H)；

¹⁹ F NMR(400MHz,CHLOROFORM-d):δ＝-73.8。

Synthesis of Compounds 1-3

A solution of compound 1-2 (10 g) in 50mL of tetrahydrofuran was added dropwise at-65 ℃ to a 50mL tetrahydrofuran suspension of lithium aluminum hydride (1.90 g). After the dropwise addition is completed, the reaction solution reacts for 4 hours at a temperature of between 70 ℃ below zero and 65 ℃ below zero. TLC (PE: etOAc =5. The reaction was quenched with methanol (6.43 g) at-65 ℃. Heating to-10 deg.c, dropping 1M citric acid aqua to regulate pH to 4, and controlling the temperature to-10-5 deg.c. The resulting mixture was extracted with ethyl acetate (300mL. Times.3). The organic phases were combined, washed with 300mL of saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the crude compounds 1-3, which were used directly in the next step.

¹ HNMR(400MHz,CHLOROFORM-d):δ＝9.65(s,1H),2.09-2.16(m,1H),2.03-2.05(m,4H), 1.92-1.99(m 1H),1.23-1.32(m,4H)。

Synthesis of Compounds 1 to 5

Compound 1-4 (50.0 g) was dissolved in N-methylpyrrolidone (500 mL), potassium carbonate (49.1 g) and sodium ethylmercaptide (31.0 g) were added to the reaction solution, and the mixture was stirred at 20 ℃ for 12 hours; TLC (PE: etOAc = 5) showed disappearance of starting material, poured the reaction into 1500 mL of water, and solid precipitated, filtered, and the filter cake was dried in vacuo to afford compound 1-5.

MS:m/z 165.1[M+H] ⁺ 。

Synthesis of Compounds 1-6

Dissolving the compounds 1-5 (58.0 g) in methanol (600 mL), dropwise adding a suspension of oxone complex salt (434.2 g) in 900mL of water to the reaction solution at 0 ℃, and then stirring the reaction solution at 0-20 ℃ for 16 hours; TLC (PE/EtOAc = 3) showed the starting material reaction was complete. The reaction solution was filtered, and excess oxone complex salt was quenched with sodium thiosulfate. After removing methanol under reduced pressure, extract with ethyl acetate (500 mL. Times.2), combine the organic phases, wash with saturated brine (300 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give the crude compounds 1-6. The crude product is purified by column chromatography (silica, 100-200 mesh silica gel, eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether: ethyl acetate (V: V) = 10) to obtain a compound 1-6.

MS:m/z 197.0[M+H] ⁺ ；

¹ H NMR(400MHz,CDCl ₃ -d):δ＝9.13(d,J＝1.6Hz,1H),8.38(dd,J＝8.4,2.4Hz,1H),7.94(d,J＝8.4 Hz,1H),3.21(Q,J＝7.2Hz,2H),1.35(d,J＝7.6Hz,3H)。

Synthesis of Compounds 1-7

Compounds 1-6 (10 g) were dissolved in dry methanol (100.00 mL), and dry palladium on carbon (10%) was added under nitrogen, and the mixture was replaced with nitrogen 3 times and then with hydrogen 3 times. Reaction solution in H ₂ (50 Psi) was stirred at 25 ℃ for 3 hours. When the LC-MS showed the reaction was complete, the reaction solution was filtered through Celite, and the filtrate was concentrated under reduced pressure to give compounds 1-7.

MS:m/z 201.0[M+H] ⁺ ；

¹ H NMR(400MHz,CDCl ₃ ):δ＝9.04(d,J＝2.0Hz,1H),8.15(dd,J＝2.4,8.4Hz,1H),7.56(d,J＝8.4 Hz,1H),4.12(s,2H),3.16(q,J＝7.6Hz,2H),1.32(t,J＝7.6Hz,3H)。

Synthesis of Compounds 1-10

To a suspension of compounds 1-8 (188 g) in water (188 mL) at 20 deg.C was added aqueous KOH (105.93 g, 85% purity) (188 mL). Stirring at 20 deg.C until compounds 1-8 are completely dissolved. The solution was cooled to 0-5 ℃ and under nitrogen, compound 1-9 (91.2g, 114.00mL) was added dropwise while maintaining the temperature. After the dropwise addition, the reaction solution was stirred at 0 to 5 ℃ for 3 hours. After the reaction was completed, the reaction solution was added with water (470 mL) and adjusted to pH =5 with concentrated hydrochloric acid (about 130 mL) by LC-MS. A large amount of solid is generated to generate a suspension, and the suspension is continuously stirred for 1 hour at the temperature of 0-5 ℃. The solid was filtered, washed with water (47 mL), collected and dried under vacuum at 60 ℃ to give compounds 1-10.

MS:m/z 170.9[M+H] ⁺ ；

¹ H NMR(400MHz,DEUTERIUM OXIDE):δ＝3.46(d,J＝4.0Hz,1H),3.32(br t,J＝6.8Hz,2H),2.92 (br t,J＝6.8Hz,2H),2.05-2.26(m,1H),0.94(br dd,J＝6.8,17.6Hz,6H)。

Synthesis of Compounds 1-11

Concentrated H to prepared absolute ethanol (450 mL) ₂ SO ₄ (150 mL) to the solution was added compound 1-10 (94 g). The reaction solution was stirred at 90 ℃ for 16 hours under nitrogen protection. After the reaction was completed, the reaction solution was poured into a saturated sodium bicarbonate solution until pH =8, and extracted three times with 300mL (100mL × 3) of dichloromethane. The combined organic phases were washed with 100mL (100mL. Times.1) of saturated brine, and the organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give crude products 1-11. The crude product was used directly in the next reaction without further purification.

¹ H NMR(400MHz,CHLOROFORM-d):δ＝3.74(s,3H),3.70(s,3H),2.90-3.01(m,2H),2.58-2.74(m, 2H),2.48-2.52(m,2H),1.91(qd,J＝6.8,13.2Hz,1H),0.94(dd,J＝2.0,6.8Hz,6H)。

Synthesis of Compounds 1-12

To a solution of compound 1-11 (96.0 g) in tetrahydrofuran (50 mL) was added 1M potassium tert-butoxide tetrahydrofuran (645 mL). The reaction mixture was stirred at 20 ℃ for 20 minutes under nitrogen. After the reaction is monitored by LC-MS, the reaction solution is concentrated and dried to obtain crude products, namely compounds 1-12. The crude product was used directly in the next reaction without further purification.

MS:m/z 186.0[M+H] ⁺ 。

Synthesis of Compounds 1-13

To a solution of compounds 1-12 (80.0 g) in ethanol (640 mL) was added concentrated hydrochloric acid (640 mL). The reaction mixture was stirred at 90 ℃ for 0.5 hour under nitrogen. After the reaction is monitored by LC-MS, the reaction solution is concentrated and dried to obtain crude product compound 1-13 hydrochloride. The crude product was used directly in the next reaction without further purification.

MS:m/z 127.9[M+H] ⁺ 。

Synthesis of Compounds 1-14

To a solution of compound 1-13 hydrochloride (70.0 g) in dichloromethane (500 mL) was added triethylamine (129.8 g, 178.62mL) and Boc ₂ O (112.0 g, 117.92mL). The reaction mixture was stirred at 20 ℃ for 2 hours under nitrogen. After completion of the reaction was monitored by LC-MS, water (300 mL) was added to the reaction solution, followed by extraction three times with 900mL (300mL. Times.3) of methylene chloride. The combined organic phases were washed with 200mL (200mLx 1) of saturated saline solution, and the organic phases were separatedDried over anhydrous sodium sulfate, filtered, concentrated and the crude product purified using a column chromatography (330 g)

Silica gel flash column, eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether/ethyl acetate (V/V) 0-30%; flow rate: 100 mL/min) to give compounds 1-14.

MS:m/z 171.9[M-56+H] ⁺ ；

¹ H NMR(400MHz,CHLOROFORM-d):δ＝3.93-4.10(m,1H),3.75-3.87(m,1H),3.56(br d,J＝8.8Hz, 1H),2.52-2.66(m,1H),2.40-2.51(m,1H),2.16-2.38(m,1H),1.50(s,9H),1.02(d,J＝6.8Hz,3H),0.96(d,J＝ 6.8Hz,3H)。

Synthesis of Compounds 1-15

POCl was added dropwise to DMF (14.3g, 15.00mL) at 0 deg.C ₃ (3.96g, 2.40mL), and the reaction mixture was stirred at 25 ℃ for 0.5 hour. Then, a solution of compounds 1 to 14 (3 g) in DMF (30 mL) was added dropwise to the reaction solution, and the reaction solution was stirred at 25 ℃ for 3.5 hours. After the completion of the reaction was monitored by LC-MS, eight reactions in parallel were combined, ice and a saturated aqueous solution of sodium acetate (800 mL) were added to the reaction solution at 0 ℃ and the reaction solution was stirred at 0 ℃ for 1 hour. The reaction solution was extracted three times with ethyl acetate (500 mL). The combined organic phases were washed once with brine (500 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and the crude product was purified by column chromatography (80 g)

Silica gel flash column, eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether/ethyl acetate (V/V) 0-30%; flow rate: 60 mL/min) to obtain the target compounds 1-15.

MS(ESI)m/z 217.8[M-56+H] ⁺ ；

¹ H NMR(400MHz,CHLOROFORM-d)：δ＝9.93-10.04(m,1H),4.62-4.83(m,1H),4.37-4.59(m,1H), 4.04-4.16(m,1H),2.25-2.48(m,1H),1.49(s,9H),1.02(br s,6H)。

Synthesis of Compounds 1-17

Triethylamine (10.54 g, 14.50 mL) was added to a dichloromethane (100 mL) solution of the compounds 1 to 15 (14.5 g) and the compounds 1 to 16 (7.02g, 6.mL), and the reaction solution was stirred at 25 ℃ for 9 hours and then at 50 ℃ for 3 hours. After the reaction was monitored by LC-MS, the reaction was concentrated, the residue was diluted with 100mL of water and the aqueous phase was extracted three times with ethyl acetate (100mL. Times.3). The combined organic phases were washed once with brine (100 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and the crude product was purified by column chromatography (80 g)

Silica gel flash column, eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether/ethyl acetate (V/V) 0-30%; flow rate: 60 mL/min) to give compounds 1-17.

MS(ESI)m/z 269.9[M-56+H] ⁺ ；

¹ H NMR(400MHz,CHLOROFORM-d)：δ＝7.50(d,J＝14.8Hz,1H),4.85-4.99(m,1H),4.44-4.61(m, 1H),4.29-4.37(m,1H),3.81(s,3H),2.29-2.60(m,1H),1.44(s,9H),0.99(t,J＝7.2Hz,3H),0.55(t,J＝7.2,8.4 Hz,3H)。

Synthesis of Compounds 1-18

To a solution of compounds 1 to 17 (13 g) in methanol (100 mL) was added a solution of sodium hydroxide (2.3 g) in water (10 mL), and the reaction mixture was stirred at 50 ℃ for 45 minutes. After the LC-MS detection reaction is finished, directly concentrating and spin-drying the reaction solution to obtain a crude product 1-18 sodium salt. The crude product was used directly in the next reaction without further purification.

MS(ESI)m/z 256[M-56+H] ⁺ 。

Synthesis of Compounds 1-19

To a suspension of the sodium salt of compound 1-18 (25 g) and compound 1-7 (9 g) in dichloromethane (100 mL) were added triethylamine (10.91g, 15 mL), EDCI (10 g) and HOBt (10 g). The reaction solution was stirred at 25 ℃ for 16 hours, and then at 50 ℃ for 3 hours. After the reaction was monitored by thin layer chromatography and LC-MS, the reaction was concentrated, the residue was diluted with 150mL of water, and the aqueous phase was extracted three times with ethyl acetate (100mLx 3). The combined organic phases were washed once with brine (100 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and the crude product was purified by column chromatography (40 g)

Silica gel flash column, eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether: 0 to 80 percent of ethyl acetate (V/V); flow rate: 40 mL/min) to give compounds 1-19.

MS(ESI)m/z 494.2[M+H] ⁺ ；

¹ H NMR(400MHz,CHLOROFORM-d)：δ＝8.99(d,J＝2.00Hz,1H),8.11(dd,J＝2.2,8.2Hz,1H),7.48 (d,J＝8.4Hz,1H),7.32(d,J＝9.60Hz,1H),4.86-5.00(m,1H),4.77(d,J＝4.0Hz,2H),4.53-4.65(m,1H), 4.34(dd,J＝4.0,13.80Hz,1H),3.06-3.13(m,3H),2.34-2.56(m,1H),1.44(s,9H),1.24-1.28(m,3H),1.00(t,J ＝7.04Hz,3H),0.56(t,J＝7.04Hz,3H)。

Synthesis of Compounds 1-20

To a solution of compounds 1 to 19 (6.3 g) in ethyl acetate (10 mL) was added a solution of hydrogen chloride in ethyl acetate (10 mL), and the reaction mixture was stirred at 25 ℃ for 0.5 hour. After the reaction is monitored by LC-MS, the reaction solution is directly concentrated and spin-dried to obtain crude product 1-20 hydrochloride. The crude product was used directly in the next reaction without further purification.

MS(ESI)m/z 394[M+H] ⁺ 。

Synthesis of the free base of the Compound of formula I

Triethylamine (2.04g, 2.8mL) was added to a suspension of the hydrochloride salt (5.5 g) of compound 1-20 in 1, 2-dichloroethane (80 mL), and the reaction mixture was stirred at 25 ℃ for 0.5 hour. Then, acetic acid (2.63g, 2.5 mL), compound 1-3 (2.6 g) and sodium borohydride acetate (5.50 g) were added to the reaction solution. After the addition was completed, the reaction mixture was stirred at 25 ℃ for 0.5 hour. After the reaction was monitored by thin layer chromatography and LC-MS, the reaction was concentrated, the residue was diluted with 150mL of water and the aqueous phase was extracted three times with ethyl acetate (100mL x 3). The combined organic phases were washed once with brine (100 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and the crude product was purified by column chromatography (40 g)

Silica gel flash column, eluent: petroleum ether/ethyl acetate; elution gradient: petroleum ether/ethyl acetate (V/V) 0-80%; flow rate: 40 mL/min) to yield a chemically pure product, which was passed through SFC (column: AD (250mm. X50mm, 10 um); mobile phase A: supercritical fluid carbon dioxide, B: neutral ethanol, wherein the volume ratio of B is 40%; the column temperature is 40 ℃; isolating the free base of the compound of formula I. The free base of the compound of formula I was analyzed by the SFC method using a SFC conditioned column AD-3. Sub.5CM _ETOH (DEA) _ 40. Sub.3ML _. Sub.5MIN _. T35. Mobile phase A is supercritical fluid carbon dioxide and B is ethanol containing 0.05% diethylamine, elution gradient: 0.00min; 5.00 min: A60%, B40%. Flow rate: 3.0mL/min column temperature: 35 ℃ RT =3.157min. ¹ H NMR(400MHz,CDCl ₃ )δ＝9.04(s, 1H),8.17–8.14(m,1H),7.55(d,J＝8.4Hz,1H),7.34(s,1H),7.18–7.16(m,1H),4.86–4.82(m,2H),4.20 (dd,J1＝3.6Hz,J2＝12.4Hz,1H),3.90–3.89(m,1H),3.49(dd,J1＝3.2Hz,J2＝12.4Hz,1H),3.16(q,J1＝ 11.2Hz,J2＝14.8Hz,2H),2.60–2.55(m,3H),2.00–1.90(m,5H),1.36–0.82(m,14H).

MS(ESI)m/z:558[M+H] ⁺ 。

The free base of the compound of formula I is purified using preparative high performance liquid chromatography (HCl system) to give the compound of formula I as amorphous.

Example 2: preparation of compound A crystal of formula I

The free base of the compound of formula I obtained in example 1 (112mg, 0.2mmol) was added to the reactor, followed by acetone (3 mL) to dissolve it, HCl/THF (100. Mu.L of commercially available concentrated HCl in 900. Mu.L of tetrahydrofuran, 173. Mu.L of the reaction mixture) was added slowly, after the addition, the reaction mixture was stirred overnight at 45 ℃ to find solid precipitated, cooled, filtered under reduced pressure, the solid was dispersed in a clean petri dish, and dried to constant weight in a vacuum oven (35 ℃ C.) to obtain the compound of formula I A crystals.

MS(ESI)m/z:558[M+H-HCl] ⁺ ；

¹ H NMR(400MHz,CDCl ₃ )δ＝11.23(s,1H),9.64(t,J＝5.6Hz,1H),8.96(s,1H),8.27-8.24(m,1H),7.88 (s,1H),7.64–7.60(m,1H),4.96–4.89(m,2H),4.67–4.63(m,2H),4.43–4.39(m,1H),3.52(s,2H),4.42– 3.36(m,2H),3.27–3.26(m,2H),2.48–1.88(m,7H),1.26–1.01(m,11H)。

Example 3: preparation of compound A crystal of formula I

The free base of the compound of formula I obtained in example 1 (112mg, 0.2mmol) was added to the reactor, followed by tetrahydrofuran (2 mL) to dissolve it, then the prepared HCl/THF (100. Mu.L of commercially available concentrated HCl in 900. Mu.L of tetrahydrofuran, 173. Mu.L of the reaction mixture was added) solution was slowly added, after the addition, the reaction mixture was stirred overnight at 45 ℃ to find solid precipitated, cooled, vacuum filtered, the solid was dispersed in a clean watch glass, and dried to constant weight in a vacuum drying oven (35 ℃) to obtain the compound of formula I A crystal.

Example 4: preparation of compound B form crystals of formula I

Compound I form A crystals obtained in example 2 (30 mg) were added to a reaction vessel, followed by addition of methanol (0.6 mL) to give a suspension, which was shaken in a homothermal (40 ℃) homogenizer for 48 hours, centrifuged, and the solid was dried overnight in a vacuum oven (30 ℃) to give compound I form B crystals.

Example 5: preparation of compound B form crystals of formula I

Compound I form A crystals obtained in example 2 (30 mg) were added to a reaction vessel, followed by addition of ethanol (0.6 mL) to give a suspension, which was shaken in a homomixer at constant temperature (40 ℃) for 48 hours, centrifuged, and the solid was dried in a vacuum oven (30 ℃) overnight to give compound I form B crystals.

Example 6: preparation of compound B form crystals of formula I

Compound I form A crystals (30 mg) obtained in example 2 were added to a reaction vessel, followed by addition of acetonitrile (0.4 mL) to give a suspension, which was shaken in a homomixer at a constant temperature (40 ℃ C.) for 48 hours, centrifuged, and the solid was dried overnight in a vacuum oven (30 ℃ C.) to give compound I form B crystals.

Example 7: preparation of compound B form crystals of formula I

Compound A form crystals of formula I obtained in example 2 (30 mg) were added to a reaction vessel, followed by addition of a mixed solvent of methanol (0.2 mL) and water (0.2 mL) to give a solution, which was shaken in a homomixer at a constant temperature (40 ℃ C.) for 48 hours, centrifuged, and the solid was dried in a vacuum oven (30 ℃ C.) overnight to give compound B form crystals of formula I.

Experimental example 1: stability test of Compound A form crystals of formula I

About 5mg of the type-A crystal of the compound of the formula I obtained in example 2 was accurately weighed, placed in a dry and clean glass bottle, spread out in a thin layer, and placed as a full-scale test sample in the condition of influencing factor test (60 ℃,92.5% RH) and in the acceleration condition (40 ℃/75% RH and 60 ℃/75% RH), which sample was fully exposed to loft, covered with an aluminum foil paper, and punctured with a small hole. Samples were taken for analysis on 5 days, 10 days. Samples placed under light (visible 1200000Lux, UV 200W) were fully exposed for lofting at room temperature. The stability results in table 3 show that the compound of formula I, form a, is highly stable and substantially non-hygroscopic.

TABLE 3 stability results for form A crystals of compound I

Relative retention time	0.55	0.74	0.86	0.98	1.11	1.12	1.25	1.37	1.38	1.39	1.55	1.71	1.95	TRS
															Starting sample	N/A	N/A	0.30	0.52	0.30	0.11	N/A	N/A	N/A	N/A	N/A	N/A	N/A	1.24
Light-shielding	N/A	N/A	0.31	0.52	0.31	0.11	N/A	N/A	N/A	N/A	N/A	N/A	N/A	1.24
															Illumination of light	0.05	N/A	0.32	0.54	0.30	0.11	N/A	N/A	N/A	N/A	N/A	N/A	N/A	1.32
60℃-5D	N/A	N/A	0.36	0.52	0.30	0.11	N/A	N/A	0.15	N/A	N/A	N/A	N/A	1.44
															60℃-10D	0.05	N/A	0.33	0.53	0.29	0.11	0.06	N/A	0.19	N/A	N/A	N/A	N/A	1.56
92.5％RH-5D	N/A	N/A	0.31	0.52	0.30	0.11	N/A	N/A	N/A	N/A	N/A	N/A	N/A	1.23
															92.5％RH-10D	N/A	N/A	0.31	0.52	0.30	0.11	0.05	N/A	N/A	N/A	N/A	N/A	N/A	1.29
40℃-75％-10D	N/A	N/A	0.33	0.52	0.28	0.11	N/A	N/A	N/A	N/A	N/A	N/A	N/A	1.23
															60℃-75％-10D	0.07	N/A	0.42	0.51	0.18	0.11	0.08	0.07	0.58	0.12	0.11	N/A	0.07	2.31
40℃-75％-1M	N/A	N/A	0.35	0.52	0.21	0.10	N/A	0.09	N/A	N/A	N/A	N/A	N/A	1.27
															60℃-75％-1M	0.10	0.09	0.49	0.49	0.11	N/A	0.10	0.19	0.14	0.25	0.09	0.07	0.13	3.25

Wherein N/A represents no detection, D represents days, and TRS represents total related substances.

Biological activity experiment 1-a: in vitro ROR γ inhibitory Activity assay

1. Test method

A reporter cell: the present experiment used reporter cells expressing the chimeric ROR γ receptor. The N-terminal DNA binding domain of the chimeric ROR γ receptor, i.e. the native ROR γ protein, was replaced with the DNA binding domain of the yeast Gal4 protein. The reporter luciferase is located downstream of the Gal4 activation sequence (UAS). As shown in table 4 below:

TABLE 4

Step 1: reporter cells were prepared as cell suspensions using INDIGO cell recovery medium (CRM, containing 5% charcoal-treated fetal bovine serum). The cell suspension was dispensed into a white 96-well plate at 100. Mu.l/well.

Step 2: 8 concentration gradients were set for each test compound, and each concentration was tested 2 times. Prior to the experiment, master stocks of test compounds were serially diluted in DMSO to prepare "1000X-concentration" solutions for each final test concentration. Subsequently, the compounds were further diluted with cell recovery medium (CRM, containing 5% charcoal treated fetal bovine serum) to prepare a "2X-concentration" of test working solution. Test solutions were added to test wells pre-loaded with reporter cells in a volume of 100 μ l/well, respectively, to obtain the desired final test concentration. The residual concentration of DMSO in all test wells was 0.1%. The test plate was incubated for 24 hours in a cell incubator, which set the conditions: temperature 37 ℃,5% CO ₂ And the humidity is 85 percent.

And step 3: after 24 hours of incubation, the plates were discarded, 100 μ l of luciferase assay reagent was added to each test well, and the fluorescence intensity in each well was read (Relative Luminescence Units, RLUs).

And (3) testing:

the test uses the reference compound ursolic acid as an internal standard of a positive compound to confirm the inhibition effect of the test compound on ROR gamma in a specific batch of report cells. The test of the reference compound and the test compound is performed simultaneously and thus exposed to the same test reagents and environment. The reference compound group containing solvent 0.1% DMSO was used as an internal positive compound standard to determine the effect of solvent DMSO on the test results and to calculate the percent ROR γ activity reduction.

2. Data processing

The test data was managed and archived by Microsoft Excel and the mean +/-Standard Deviation (SD), fold reduction, inhibition, coefficient of variation (% CV) and Z factor of RLU were calculated.

Coefficient of variation (% CV): 100 [ (SD/ave. Rlu) ];

fold reduction in inverse agonist: [ Ave.RLU ^Solvent /Ave.RLU ^{Test compounds} ]

Percent reduction in inverse agonist: 100 (1- [ Ave ^{Test compounds} /Ave.RLU ^Solvent ])；

The theoretical minimum reduction (0% reduction) is for the solvent control without compound;

factor Z: 1- [ (3. Multidot. SD) ^Solvent +SD ^{Test compounds} ])/(RLU ^Solvent _{_} RLU ^{Test compounds} )]。

3. Graphic data processing method

Dose Response Curves (DRC) for ROR γ tests for reference and test compounds were obtained by nonlinear fitting of the inhibition rate of ROR γ activity and the logarithmic value of compound concentration by GraphPad Prism software.

4. Test results

The results of the in vitro screening assays are detailed in table 5,

table 5: results of in vitro screening assays

Definition of biological activity: a: EC (EC) ₅₀ ≤100nM。

Biological Activity experiments 1-B: in vitro ROR γ inhibitory activity assay: TR-FRET screening

The compound can modulate (inhibit) the biological activity of a nuclear receptor ROR gamma, and the strength of the modulation (inhibition) effect can be evaluated by a TR-FRET (time resolved fluorescence energy resonance transfer) screening system. Nuclear receptor cofactors (coactivators and cosuppressors) can regulate transcription of a gene of interest by interacting with nuclear receptors. If the ligand (test compound) affects the interaction of the nuclear receptor and the cofactor, the ligand (test compound) can modulate the transcription of the corresponding gene.

The method adopts TR-FRET (time-resolved fluorescence resonance energy transfer) technology to determine the regulation capacity of the compound on the interaction of RORC2-LBD (restriction enzyme binding) of polypeptide indirectly bound by APC (phycocyanin) (namely, APC is indirectly bound with polypeptide through the binding of Streptavidin (Streptavidin) and biotin (biotin)) and connected with europium (Eu) -labeled anti-His antibody (Perkin Elmer # AD 0111). In the absence of the compound, RORC2 may bind to the APC-modified polypeptide. If the compound is an agonist, then its binding to RORC2 may enhance the interaction of RORC2 with APC-modified polypeptides; conversely, if the compound is an inverse agonist, its binding to RORC2 may inhibit the interaction of RORC2 with APC-modified polypeptides. The APC modified polypeptide and the Eu-labeled anti-His antibody-RORC 2-LBD complex approach each other to a certain distance to generate energy transfer and generate a TR-FRET signal.

1. The final concentration and reaction conditions of the reaction system of the present method are shown in the following table 6:

TABLE 6

SRC1 peptide: a polypeptide comprising LXLL-motif capable of binding to RORC 2-LBD; wherein LXLL-motif is a structural sequence comprising LXLL, L is leucine and X is any amino acid.

2. Experimental methods

2.1 preparation buffer:

a buffer solution consisting of 50mM Tris pH 7.0,50mM KCl,1mM Na-EDTA,0.01% BSA (0.1 mg/ml). 0.1mM DTT was freshly prepared before the experiment was performed.

2.2 preparation of working fluid without ROR γ:

working solutions containing 1.67nM Eu/His-resistant, 0.75. Mu.l/well SF9 cell lysate were freshly prepared prior to testing. Placed on ice prior to use.

2.3 preparation containing ROR γ working solution A working solution containing 1.67nM Eu/His-resistant, 0.75 μ l/well SF9 cell lysate was freshly prepared before testing. Placed on ice prior to use. 25.05nM ROR gamma was added to the working solution.

2.4 preparation of peptide-Streptomyces/APC mixture:

buffers containing 500nM polypeptide and 125nM streptavidin/APC were freshly prepared prior to testing. Placed on ice prior to use.

2.5 10 concentration gradients were set for each test compound, and duplicate wells were set for each concentration. The test compounds were prepared as follows:

1) Mu.l of the sample was taken from a source plate containing 10mM compound in DMSO and added to the corning 3656 in columns 1 and 11

2) The compounds were diluted to 500. Mu.M by Bravo adding 38. Mu.l DMSO to columns 1 and 11

3) Transfer 10. Mu.l of 500. Mu.M compound solution to columns 1 and 11 of LDV plates by Bravo

4) Columns 2-10 and 12-22 of LDV plates by Bravo plus 7.5. Mu.l DMSO

5) Compounds were serially diluted by Bravo (4-fold, 10 doses, 2.5. Mu.l compound + 7.5. Mu.l DMSO)

6) Transfer of 250nL of compound solution from dose plate to test plate by Echo (Greiner 781076)

2.6 Experimental procedures:

1) To 22 columns of the 384-well plate tested, 15. Mu.l of a ROR γ -free working solution (Eu/anti-His final concentration of 1 nM) was added

2) Add 15. Mu.l of ROR γ -containing working solution (Eu/anti-His final concentration of 1nM, RORg final concentration of 15 nM) to columns 1-21 of the 384-well plate tested

3) Mu.l of the peptide-streptavidin/APC mixture (SA-APC final concentration of 50nM, SRC1 peptide final concentration of 200 nM) was added to the 384-well plate tested in columns 1-22

4) Fully shaking the plate on a plate shaker for 2 minutes

5) Test plates were incubated overnight at 4 deg.C

6) The test panels were left at room temperature for 1 hour for room temperature equilibration

7) Plates were centrifuged at 1000 rpm for 1 min

8) Reading the board on an Envision board reader

9) Data analysis using the ratio of the value of the emitted light at 665nm divided by the value of the emitted light at 615nm

3. Data analysis

The TR-FRET ratio F665/F615-compound concentration was plotted as a log curve using GraphPad Prism software and the EC50 value calculated, with a smaller value indicating a greater modulating (inhibiting) effect of the compound on the receptor ROR γ.

EC for ROR gamma action of the Compounds of formula I ₅₀ The values are given in table 7 below:

TABLE 7

Definition of biological activity: a: EC (EC) ₅₀ ≤100nM。

Biological activity experiment 2: pharmacokinetic evaluation

1. Experimental method

Balb/c mice (female, 15-30g, 7-9 weeks old, shanghai Ling Chang) were used to test the in vivo pharmacokinetics of the compounds, and the experimental method was as follows:

rodent pharmacokinetic characteristics after intravenous injection and oral administration of the compound are tested by a standard scheme, and the candidate compound is prepared into a clear solution in an experiment and is administered to mice by single intravenous injection and single oral administration. Intravenous Injection (IV) of the solvent 5% DMSO and 95% the mixed solvent of cremophor EL, oral (PO) of the solvent 1% tween80, 9% PEG400 and 90% water. Collecting whole blood sample within 48 hours, centrifuging at 4 ℃ for 15 minutes at 3000g, separating supernatant to obtain plasma sample, adding 20 times of acetonitrile solution containing internal standard to precipitate protein, centrifuging to obtain supernatant, adding equal times of water, centrifuging to obtain supernatant, sampling, quantitatively analyzing blood concentration by LC-MS/MS analysis method, and calculating drug parameters such as peak-reaching concentration, peak-reaching time, clearance rate, half-life, area under curve during drug administration, bioavailability, etc.

Wherein "10% of Cremophor EL" means a deionized water solution of Cremophor EL having a volume concentration of 10%, for example, taking 10ml of Cremophor EL as an example of preparation of 100ml, adding deionized water and stirring uniformly, and then adding deionized water to make the total volume 100ml. "5% DMSO/95% Cremophor EL mixed solvent", means a mixed solvent consisting of DMSO and 10% Cremophor EL, wherein DMSO represents 10% Cremophor EL, and 10% Cremophor EL represents 95% by volume of the mixed solvent. "1%" the mixed solvent of tween80, 9% PEG400 and 90% water "means that the volumes of tween80, PEG400 and water respectively account for 1%, 9% and 90% of the volume of the mixed solvent.

2. Test results

The test results are shown in Table 8.

Table 8: PK parameters in plasma of Compounds of formula I

"- -" means no test or no data obtained.

Biological activity experiment 3: inhibition assay for hERG potassium channel

1. Experimental method

(1) Cell preparation

CHO-hERG cells (from Shanghai pharmaceutical institute of Chinese academy of sciences) were cultured at 175cm ² In the culture flask, when the cell density grows to 60-80%, the culture solution is removed, washed once with 7mL PBS, and then digested by adding 3mL Detachin. After digestion is completed, 7mL of culture medium is added for neutralization, then centrifugation is carried out, supernatant is sucked off, and 5mL of culture medium is added for heavy suspension so as to ensure that the cell density is 2-5 × 106/mL.

(2) Preparation of intracellular and extracellular fluids

Table 9: composition of intracellular and extracellular fluids

(3) Electrophysiological recording procedure

The single cell high impedance sealing and the whole cell mode forming process are all automatically completed by a Q patch instrument, after a whole cell recording mode is obtained, the cell is clamped at minus 80 millivolts, before a depolarization stimulation of plus 40 millivolts for 5 seconds is given, a 50 millisecond-50 millivolt pre-voltage is given, then repolarization is carried out to minus 50 millivolts for maintaining for 5 seconds, and then the voltage returns to minus 80 millivolts. This voltage stimulus was applied every 15 seconds, and 2 minutes after recording extracellular fluid was administered for 5 minutes, and then the dosing process was started, with compound concentrations starting from the lowest test concentration, each test concentration being administered for 2.5 minutes, and after all concentrations were administered in succession, the positive control compound was administered at 3 μ M Cisapride. At least 3 cells (n.gtoreq.3) were tested per concentration.

(4) Preparation of Compounds

The compound stock solution at 20mM was diluted with the extracellular solution, and 5. Mu.L of the compound stock solution at 20mM was added to 2495. Mu.L of the extracellular solution, diluted 500-fold to 40. Mu.M, and then serially diluted 3-fold in the 0.2-fold% DMSO-containing extracellular solution in order to obtain the final concentration to be tested. The highest concentration tested was 40. Mu.M, which was in turn 40. Mu.M, 13.33. Mu.M, 4.44. Mu.M, 1.48. Mu.M, 0.49. Mu.M, 0.16. Mu.M for 6 concentrations. The final concentration of DMSO in the test concentration did not exceed 0.2%, and the concentration of DMSO had no effect on the hERG potassium channel.

(5) Data analysis

Experimental data were analyzed by XLFit software.

(6) Quality control

Environment: humidity is 20-50%, and temperature is 22-25 DEG C

Reagent: the experimental reagent is purchased from Sigma, and has purity of 98%

The experimental data in the report must meet the following criteria:

whole cell sealing impedance >100M omega

Tail current amplitude >400pA

Pharmacological parameters:

the inhibitory effect of multiple concentrations of Cisapride on the hERG channel was set as a positive control.

(7) Test results

hERG IC of compound of formula I ₅₀ The results are shown in Table 10.

Table 10: compound hERG IC of formula I ₅₀ Value result

Test sample	hERG IC 50 (μM)	Maximum concentration inhibition (%)
			A compound of formula I	>40	26.33
Cisapride	0.05	89.61

Biological activity experiment 4: in vivo efficacy studies in MOG35-55 induced mouse Experimental Autoimmune Encephalomyelitis (EAE)

The experimental method comprises the following steps:

preparation of MOG/CFA emulsion

Preparing CFA: weighing M.tuberculosis H37Ra, placing in an agate mortar, grinding into fine powder, and adding IFA to suspend into suspension of 4 mg/ml;

preparing MOG35-55 solution: weighing MOG35-55, dissolving in normal saline to prepare 2mg/ml solution, and preparing for use;

2mg/ml MOG35-55 and 4mg/ml CFA were mixed in equal volumes in a 40ml glass bottle and homogenized with a homogenizer for 1h (45 seconds for 15 seconds) to make a MOG/CFA emulsion which was placed on ice for use.

Preparation of PTX solution

PTX preparation: adding PBS (1 ml) into 50ug of PTX powder to prepare 50ug/ml mother solution, diluting to 1ug/ml with PBS before use, and using the solution at present;

induction of eae:

day of immunization was scored as day 0, with subsequent days noted in order. Isoflurane anesthetized mice were injected subcutaneously with 100 μ l of emulsion, three sites (middle of the shoulder and back, two sides of the tail near the groin), three sites each with 33 μ l of emulsion; 200ng (i.e. 200. Mu.l) of PTX solution was injected intraperitoneally 0 hours and 48 hours after the emulsion injection. The normal group of mice did not need to be immunized.

4. Administration and dose design

The immunized mice were randomly divided into 3 groups of 10 mice each by weight after the immunization from day 0, except for the normal group of mice, as detailed in table 11.FTY720 as a positive drug, the 0.05mg/ml dose is the effective dose commonly used in the EAE model.

The first group was normal mice, without any treatment; a second group was given Vehicle; FTY720 was administered in a third group at a dose of 0.5mg/kg once a day; the fourth group was administered the compound of formula I at a dose of 75mg/kg twice daily. For a total of 25 days. The volume of the drug for intragastric administration is 10ml/kg.

Table 11: grouping and dose design

*vehicle:DMSO/PEG400/H ₂ A mixed solvent of O; wherein DMSO/PEG400/H ₂ The volume ratio of O is 5.

* Vehicle (solvent): DDH ₂ O。

5. Disease index monitoring

Animal body weights were recorded three times a week from day 0-10 on day 0 of animal immunization, and were closely observed daily on day 11 with the scoring criteria shown in table 12 below:

table 12: clinical scoring criteria

Score value	Clinical symptoms
		0	Normal manifestation without obvious disease signs
1	A drooping tail and weakness of one side of the hind limb
		2	Drooping and weakness of the tail and stumbling gait of both hind limbs
3	Paralysis of unilateral hind limb weakness
		4	Paralysis of both hind limbs

6. Statistical treatment

Experimental data were expressed using Mean ± standard error (Mean ± SEM), clinical scores AUC were analyzed using One-way ANOVA, and p <0.05 was considered significantly different.

7. The experimental results are as follows:

(1) Clinical scoring

On day 13 post immunization, mice began to develop clinical symptoms of EAE. G2 The mean clinical score (of the solvent control group) gradually increased to reach 3.6 points by day 18, suggesting successful establishment of the model (see figure 8). G4 (compound of formula I) significantly reduced the clinical score of EAE mice in the experiment (see figure 8).

(2) Inhibition rate results

The inhibition rate of each of the remaining groups (G1, G3, G4) relative to G2 (solvent control group) was calculated by analyzing the clinical score curve for each animal in each group, calculating the area under the clinical score curve over time (i.e., "clinical score AUC" or "area under clinical score curve"), and calculating the average value of the AUC for the clinical scores between the groups, and the results are shown in fig. 9. The compound of formula I significantly reduced the clinical score AUC of EAE animals (p value <0.0001 compared to solvent control group) with an inhibition of 93.2%. The positive drug FTY720 can completely inhibit the clinical score of EAE animals, and the inhibition rate is 100%.

(3) Incidence results

See figure 10 for EAE incidence results. G4 The incidence of (compound of formula I) was 20% at day 19 and stabilized to the end of the experiment, showing good results. G2 (solvent control group) EAE incidence reached and was maintained at 100% at day 17 post immunization. G3 The incidence of (positive control FTY720 group) was consistently 0 (see fig. 10).

(4) Results of body weight changes

The body weight of mice in G2 (solvent control group) decreased on day 12, decreased greatly from day 12 to 16, and recovered slowly from day 17, compared to G1 (normal group). G3 The body weights (positive control FTY720 group) and G4 (compound of formula I) continued to increase continuously with the same trend, with a significant difference from the solvent control group. And G4 (compound of formula I) was always highest (fig. 11).

Biological activity experiment 5: in vivo efficacy study in efficacy and mechanism study of mouse psoriasis (IMQ) model

The experimental method comprises the following steps:

5.1 animal grouping and Imquimod-induced psoriasis model establishment in mice

C57BL/6 female mice of 6-8 weeks were taken, depilated on the backs, and sensitized after two days except for the sham operation group. The day of sensitization was randomized into 6 groups (8 per group): group I is a sham operation group; group II is vehicle control group, given PBS; group III is a positive control group, and 5mg/kg methotrexate is given; IV, V, and VI groups are ip, ig, and topocal treatment groups of compound of formula I, and 75mg/kg compound of formula I is administered by intraperitoneal injection, intragastric administration, and back smearing, respectively. Administration was started on the day of sensitization, once daily for group III and 2 times daily for groups IV-VI. On the day of sensitization, group II-VI mice were applied 60-80 mg of imiquimod cream (5%) to the dorsal skin for 4 consecutive days. (see Bouchaud, G., et al., epidermal IL-15Ralpha acts as an endogenesis antagnonist of pseudomorpha in motion and man.J.Exp.Med.,2013.210 (10): p.2105-17.)

Mice were weighed daily from the day of sensitization, observed for skin scaling, induration, erythema, and scored using a 4-grade scoring method: 0 point, no disease occurs; 1 point, slight; 2 points, medium; grade 3, severe; 4 points, very serious. (see Qin, S., et al, endogeneus n-3 complicated fatty acids detecting against infection-induced stress inflammation-like inflammation via the IL-17/IL-23 axis. Mol Med Rep,2014.9 (6): p.2097-104.)

5.2 study results: effect of compound of formula I on clinical score in imiquimod-induced psoriasis mice;

as shown in figure 12, the compound shown in the formula I can inhibit the phenomena of psoriasis model skin scale, induration and the like induced by imiquimod, wherein 75mg/kg of the compound shown in the formula I can be obviously (p is less than 0.01) when the compound is administrated by intraperitoneal injection, gastric lavage and back smearing, and the effect of reducing the clinical score is equivalent to that of 5mg/kg methotrexate.

As shown in figure 13, the results of inhibition rate for in vivo efficacy studies in imiquimod-induced mouse experimental psoriasis (IMQ) are shown by the relative ratio of the clinical scores AUC as described previously.

Table 13: effect on clinical Scoring of Imquimod-induced psoriasis mice after daily administration of the Compound of formula I (Mean + -SEM, n = 8)

Claims (19)

1. A crystal of the compound of formula I characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 Θ, at about 14.9 °, 20.6 °, 21.4 °, 23.0 °, and 24.9 °;

2. a crystal of the compound of formula I according to claim 1, characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 Θ, at about 8.0 °, 11.9 °, 13.7 °, 14.9 °, 18.3 °, 19.4 °, 20.6 °, 21.4 °, 21.9 °, 23.0 ° and 24.9 °.

3. A crystal of the compound of formula I according to claim 2, characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 Θ, at about 8.0 °, 9.7 °, 11.9 °, 13.7 °, 14.9 °, 15.7 °, 16.0 °, 18.3 °, 19.4 °, 20.1 °, 20.6 °, 21.1 °, 21.4 °, 21.9 °, 22.5 °, 23.0 °, 24.1 °, 24.9 °, 26.8 ° and 30.0 °.

4. A crystal of the compound of formula I according to claim 3, characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 θ, at about 8.0 °, 9.5 °, 9.7 °, 10.4 °, 11.9 °, 13.7 °, 14.9 °, 15.7 °, 16.0 °, 18.3 °, 19.4 °, 20.1 °, 20.6 °, 21.1 °, 21.4 °, 21.9 °, 22.5 °, 23.0 °, 23.7 °, 24.1 °, 24.9 °, 26.8 °, 27.6 °, 29.3 °, 30.0 °, 30.8 °, 31.2 °, 33.5 ° and 34.2 °.

5. A crystal of a compound of formula I as claimed in any one of claims 1 to 4, characterized by a Differential Scanning Calorimetry (DSC) measurement in which the onset of the absorption peak is at about 208 ℃.

6. A crystal of the compound of formula I characterized by an X-ray powder diffraction spectrum having diffraction peaks expressed in terms of 2 Θ at about 13.9 °, 17.1 °, 19.2 °, 19.6 °, 21.9 °, and 25.1 °;

7. a crystal of the compound of formula I according to claim 6, characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 θ, at about 7.4 °, 8.5 °, 13.9 °, 15.5 °, 16.3 °, 17.1 °, 19.2 °, 19.6 °, 20.0 °, 21.9 °, 24.0 °, 25.1 °, 25.5 °, 26.0 ° and 29.0 °.

8. A crystal of the compound of formula I as claimed in claim 7, characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 θ, at about 7.4 °, 8.5 °, 11.0 °, 11.9 °, 13.9 °, 15.5 °, 16.3 °, 17.1 °, 18.3 °, 19.2 °, 19.6 °, 20.0 °, 20.5 °, 21.2 °, 21.6 °, 21.9 °, 23.1 °, 24.0 °, 25.1 °, 25.5 °, 26.0 °, 26.7 °, 27.9 °, 29.0 ° and 29.2 °.

9. A crystal of the compound of formula I according to claim 8, characterized by an X-ray powder diffraction spectrum having diffraction peaks, expressed in terms of 2 θ, at about 7.4 °, 8.5 °, 9.7 °, 11.0 °, 11.9 °, 13.9 °, 14.7 °, 15.5 °, 16.3 °, 17.1 °, 18.3 °, 19.2 °, 19.6 °, 20.0 °, 20.5 °, 21.2 °, 21.6 °, 21.9 °, 22.7 °, 23.1 °, 24.0 °, 25.1 °, 25.5 °, 26.0 °, 26.7 °, 27.9 °, 29.0 °, 29.2 °, 30.0 ° and 30.3 °.

10. A crystalline composition, wherein crystals of a compound of formula I as defined in any one of claims 1 to 4 represent more than 50% by weight of the crystalline composition.

11. The crystalline composition as defined in claim 10, wherein the crystals of the compound of formula I as defined in any one of claims 1 to 4 represent more than 75% by weight of the crystalline composition.

12. The crystalline composition as defined in claim 11, wherein the crystals of the compound of formula I as defined in any one of claims 1 to 4 represent more than 90% by weight of the crystalline composition.

13. The crystalline composition as defined in claim 12, wherein the crystals of the compound of formula I as defined in any one of claims 1 to 4 represent more than 95% by weight of the crystalline composition.

14. A crystalline composition, wherein the crystals of the compound of formula I as defined in any one of claims 6 to 9 represent more than 50% by weight of the crystalline composition.

15. The crystalline composition as defined in claim 14, wherein the crystals of the compound of formula I as defined in any one of claims 6 to 9 represent more than 75% by weight of the crystalline composition.

16. The crystalline composition as defined in claim 15, wherein the crystals of the compound of formula I as defined in any one of claims 6 to 9 represent more than 90% by weight of the crystalline composition.

17. The crystalline composition as defined in claim 16, wherein the crystals of the compound of formula I as defined in any one of claims 6 to 9 represent more than 95% by weight of the crystalline composition.

18. A pharmaceutical composition comprising a crystalline form of a compound of formula I according to any one of claims 1 to 4 or a crystalline composition according to claim 10.

19. Use of a crystal of a compound of formula I according to any one of claims 1 to 4 or a crystal of a compound of formula I according to any one of claims 6 to 9, a crystalline composition according to claim 10 or 14 or a pharmaceutical composition according to claim 18 for the manufacture of a medicament for the treatment of a ROR γ receptor mediated disease.

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