TW202016091A - A pharmaceutically acceptable salt and crystal form of otr inhibitor and preparation method thereof - Google Patents

Abstract

The disclosure provides a pharmaceutically acceptable salt and crystal form of OTR inhibitor and preparation method thereof. Specifically, the disclosure provides a pharmaceutically acceptable salt and crystal form of OTR inhibitor 5-(3-(3-(6-Fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4H-1,2,4-triazol-4-yl)-2-methoxypyridin and preparation method thereof. This pharmaceutically acceptable salt improves the dissolution of free alkali, physical or chemical stability of OTR inhibitor, and is of great significance for the development of medicines suitable for industrial production and with good biological activity.

Description

Pharmaceutically acceptable salt, crystal form of OTR inhibitor and preparation method

The present disclosure provides the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1, 2,4-triazol-4-yl)-2-methoxypyridine pharmaceutically acceptable salts, crystal forms and preparation methods thereof.

PCT/CN2017/117421 (application date 2017.12.20) describes a compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxy Methyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine, as a free base, exhibits highly selective OTR inhibition and has good brain permeability , Can effectively block the downstream function of oxytocin receptor mediated by oxytocin.

Nearly half of the drug molecules are in the form of salts. At the same time, salt formation can improve some undesirable physical, chemical or biological properties of drugs. Developed relative to 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2, 4-Triazol-4-yl)-2-methoxypyridine, a salt having more excellent physical and chemical properties or pharmaceutical properties is of great significance.

At the same time, in view of the importance of the solid drug crystal form and its stability in clinical treatment, the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidine-1- Group)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine polymorphic form of pharmaceutically acceptable salt, suitable for industrial development It is of great significance to produce drugs with good biological activity.

The present disclosure provides the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1, Pharmaceutically acceptable salt of 2,4-triazol-4-yl)-2-methoxypyridine, wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, mesylate, phosphate, citric acid Salt, benzoate or fumarate.

Preferably, the chemical ratio of the compound to the acid molecule is about 1:2~2:1, and may be about 1:2, 1:1, 2:1.

In an alternative embodiment, the chemical ratio of the compound to hydrogen chloride is about 1:1.

In an alternative embodiment, the chemical ratio of the compound to sulfuric acid is about 1:1 or 2:1.

In an alternative embodiment, the chemical ratio of the compound to phosphoric acid is about 1:1, 2:1.

In an alternative embodiment, the chemical ratio of the compound to methanesulfonic acid is about 1:1.

The present disclosure also provides a method for preparing the aforementioned pharmaceutically acceptable salts, including: compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-( methoxymethyl) -4 H -1,2,4- triazol-4-yl) -2-methoxy-pyridine in step with an acid into a salt.

The solvent used for salt formation in the present disclosure is at least one selected from water, alcohols, halogenated hydrocarbons, ethers, nitriles, alcohols, esters or ethers, preferably selected from isopropanol, acetone, methyl alcohol Based on at least one of tertiary butyl ether, acetonitrile, ethanol, ethyl acetate, and water.

In an alternative embodiment, the method for preparing the aforementioned pharmaceutically acceptable salt further includes the steps of volatile solvent or crystallizing with stirring and filtering.

The present disclosure also provides a pharmaceutical composition containing a pharmaceutically acceptable salt of the aforementioned compound and optionally a pharmaceutical adjuvant selected from pharmaceutically acceptable carriers, diluents, or excipients.

The present disclosure also provides the use of the aforementioned pharmaceutically acceptable salts in the preparation of a medicament for the treatment or prevention of a disease or condition known or shown to inhibit oxytocin to produce a beneficial effect, the disease or condition being selected from sexual dysfunction, sexual desire Hypothyroidism, sexual arousal disorder, orgasm disorder, intercourse pain disorder, premature ejaculation, preterm delivery, complication of delivery, appetite and eating disorders, benign prostatic hyperplasia, premature delivery, dysmenorrhea, congestive heart failure, arterial hypertension, cirrhosis , Renal hypertension, high intraocular pressure, obsessive-compulsive and behavioral disorders and neuropsychiatric diseases, preferably selected from sexual dysfunction, sexual arousal disorder, orgasm disorder, sexual intercourse pain disorder and premature ejaculation.

The present disclosure also provides the use of the aforementioned pharmaceutically acceptable salts in the preparation of a medicament for antagonizing oxytocin.

The present disclosure provides the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1, Form A of 2,4-triazol-4-yl)-2-methoxypyridine hydrochloride, X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, at 6.92, 12.54, 15.23, 16.32, 22.70 , 27.44, 28.10 have characteristic peaks.

In an alternative embodiment, the crystal form A has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, which has characteristic peaks at 6.92, 12.54, 15.23, 16.32, 18.89, 19.52, 20.90, 22.70, 27.44, 28.10 .

In some embodiments, the Form A crystal has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 6.92, 12.54, 15.23, 16.32, 16.63, 18.15, 18.89, 19.52, 20.90, 22.70, 24.93, 25.80, There are characteristic peaks at 27.44 and 28.10.

In other embodiments, the crystal form A has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ as shown in Figure 1.

Preparation of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- The method of triazole-4-yl)-2-methoxypyridine hydrochloride crystal form A includes: (a) Compound 5-(3-(3-(6-fluoronaphthalen-1-yl)aza Cyclobutan-1-yl)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (I) and stirred Dissolve or dissolve by heating, (b) add hydrochloric acid dropwise, stir and crystallize; the volume (ml) of the solvent (I) used in this method is 1 to 50 times the weight (g) of the compound, which can be 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 times; the solvent (I) is selected From isopropyl alcohol/water, ethyl acetate.

The present disclosure provides the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1, Form B of 2,4-triazol-4-yl)-2-methoxypyridine hydrochloride, X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, at 14.62, 15.65, 19.21, 23.66, 24.15 , 25.92, 27.19 have characteristic peaks.

In an alternative embodiment, the Form B crystal has a characteristic peak at 11.76, 14.62, 15.65, 19.21, 23.66, 24.15, 25.92, 27.19, 28.01, 29.54 with an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ .

In some embodiments, the Form B crystal has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 11.76, 14.62, 15.65, 16.43, 18.70, 19.21, 23.66, 24.15, 24.56, 25.60, 25.92, 27.19, There are characteristic peaks at 28.01 and 29.54.

In other embodiments, the X-ray powder diffraction pattern of the crystal form B expressed as the diffraction angle 2θ is shown in FIG. 2.

Preparation of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- The method of triazole-4-yl)-2-methoxypyridine hydrochloride crystal form B includes: (a) Compound 5-(3-(3-(6-fluoronaphthalen-1-yl)aza Cyclobutan-1-yl)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (II) and stirred Dissolve or dissolve by heating, (b) add hydrochloric acid dropwise, stir and crystallize, and beat; the volume (ml) of the solvent (II) described in this method is 1 to 50 times the weight (g) of the compound, which can be 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 times; the solvent (II ) Is selected from ethanol, acetone, acetonitrile.

Also provided in this disclosure is the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1 , 2,4-triazol-4-yl)-2-methoxypyridine hydrochloride form C, X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, at 6.94, 21.51, 22.71, 24.99, There are characteristic peaks at 25.80, 27.45 and 28.14.

In an alternative embodiment, the crystal form C has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, with characteristic peaks at 6.94, 12.54, 16.30, 20.89, 21.51, 22.71, 24.99, 25.80, 27.45, 28.14 .

In some embodiments, the C form, the X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, is at 6.94, 12.54, 16.30, 18.15, 18.88, 19.50, 20.89, 21.51, 22.71, 24.78, 24.99, 25.80, There are characteristic peaks at 27.45 and 28.14.

In other embodiments, the crystal form C, the X-ray powder diffraction pattern represented by the diffraction angle 2θ is shown in FIG. 4.

Preparation of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- The method of the crystal form C of triazol-4-yl)-2-methoxypyridine hydrochloride includes: (a) the compound 5-(3-(3-(6-fluoronaphthalen-1-yl) nitrogen Heterocyclobutan-1-yl)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (III), Stir to dissolve or heat to dissolve, (b) add hydrochloric acid dropwise and stir to crystallize; the volume (ml) of the solvent (III) used in this method is 1 to 50 times the weight (g) of the compound, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 times, the solvent (III) It is selected from methyl tertiary butyl ether.

Also provided in this disclosure is the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1 , 2,4-triazol-4-yl)-2-methoxypyridine sulfate form A, X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, at 12.66, 18.50, 19.90, 21.64, 23.61 , 24.22, 26.34 have characteristic peaks.

In an alternative embodiment, the crystalline form A of the sulfate salt has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 12.66, 14.65, 18.50, 19.90, 21.64, 22.05, 23.61, 24.22, 24.75, 26.34 There are characteristic peaks.

In some embodiments, the crystalline form A of the sulfate salt has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 7.28, 12.66, 14.04, 14.65, 17.60, 18.50, 19.90, 21.64, 22.05, 23.61, 24.22 , 24.75, 26.34, 26.70 have characteristic peaks.

In other embodiments, the crystalline form A of the sulfate salt has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ as shown in FIG. 7.

Preparation of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- The method of Form A of triazol-4-yl)-2-methoxypyridine sulfate includes:

(a) The compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2 ,4-Triazol-4-yl)-2-methoxypyridine is added to the solvent (IV), dissolved by stirring or dissolved by heating

(b) Add sulfuric acid dropwise and stir to crystallize; the volume (ml) of the solvent (IV) used in this method is 1 to 40 times the weight (g) of the compound, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 times; the solvent (IV) is selected from acetonitrile, ethanol, Acetone, ethyl acetate.

Also provided in this disclosure is the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1 , 2,4-triazol-4-yl)-2-methoxypyridine sulfate form B, X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, at 11.87, 13.22, 14.62, 15.29, 18.49 , 22.66, 23.61 have characteristic peaks.

In an alternative embodiment, the crystalline form B of the sulfate salt of the compound has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 11.87, 13.22, 14.62, 15.29, 18.49, 19.89, 21.65, 22.66, 23.61, There is a characteristic peak at 24.18.

In some embodiments, the crystalline form B of the sulfate salt of the compound has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 7.78, 11.87, 12.66, 13.22, 14.62, 15.29, 18.49, 19.89, 21.65, 22.66, There are characteristic peaks at 23.61, 24.18, 24.62 and 26.36.

In other embodiments, the crystalline form B of the sulfate salt of the compound has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ as shown in FIG. 9.

Preparation of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- The method of Form B of triazol-4-yl)-2-methoxypyridine sulfate includes:

(a) The compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2 ,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (V), dissolved by stirring or dissolved by heating

(b) Add sulfuric acid dropwise and stir to crystallize; the volume (ml) of the solvent (V) used in this method is 1 to 40 times the weight (g) of the compound, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 times; the solvent (V) is selected from methyl tert-butyl ether.

Also provided in this disclosure is the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1 ,2,4-triazol-4-yl)-2-methoxypyridine methanesulfonate, Form A, X-ray powder diffraction pattern expressed at a diffraction angle of 2θ, at 6.66, 16.81, 16.90, 18.92 , 19.19, 22.92 and 24.88 have characteristic peaks.

In an alternative embodiment, the crystalline form A of the compound mesylate salt has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 6.66, 11.00, 16.81, 16.90, 18.92, 19.19, 21.06, 22.92, 23.96 There is a characteristic peak at 24.88.

In some embodiments, the form A of the compound mesylate salt has an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 6.66, 11.00, 14.87, 16.81, 16.90, 18.92, 19.19, 20.27, 21.06, There are characteristic peaks at 22.92, 23.96, 24.88, 27.85 and 29.50.

In other embodiments, the crystalline form A of the mesylate salt of the compound has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ as shown in FIG. 14.

Preparation of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- The method of Form A of triazol-4-yl)-2-methoxypyridine mesylate includes:

(a) The compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2 ,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (VI), dissolved by stirring or dissolved by heating

(b) Add methanesulfonic acid dropwise and stir to crystallize; the volume (ml) of the solvent (VI) used in this method is 1 to 40 times the weight (g) of the compound, which can be 1, 2, 3, 4, 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 times; the solvent (VI) is selected from ethyl acetate Ester, methyl tertiary butyl ether.

The present disclosure also provides a pharmaceutical composition containing the aforementioned crystalline form of a pharmaceutically acceptable salt and optionally a pharmaceutical excipient selected from pharmaceutically acceptable carriers, diluents or excipients.

The present disclosure also provides a pharmaceutical composition prepared from the aforementioned crystal form.

The present disclosure also provides the use of the aforementioned crystalline forms of pharmaceutically acceptable salts in the preparation of a medicament for the treatment or prevention of diseases or conditions known to be or which may be shown to inhibit beneficial effects of oxytocin, the diseases or conditions being selected from Dysfunction, hyposexual disorder, sexual arousal disorder, orgasm disorder, painful intercourse, premature ejaculation, pre-term delivery, complication of delivery, appetite and eating disorders, benign prostatic hyperplasia, premature delivery, dysmenorrhea, congestive heart failure, arterial hypertension Blood pressure, cirrhosis, renal hypertension, high intraocular pressure, obsessive-compulsive and behavioral disorders and neuropsychiatric disorders are preferably selected from sexual dysfunction, sexual arousal disorder, orgasm disorder, painful intercourse pain disorder and premature ejaculation.

The disclosure also provides the use of the aforementioned crystalline form of a pharmaceutically acceptable salt in the preparation of a medicine for antagonizing oxytocin.

According to the description of the hygroscopicity characteristics and the definition of hygroscopicity gain in "Guidelines for the Moisture Absorption of 9103 Drugs" in the four parts of the Chinese Pharmacopoeia 2015 Edition, Deliquescence: Absorb enough water to form a liquid; very hygroscopic: the weight gain of moisture is not less than 15%; hygroscopic: the weight gain of moisture is less than 15% but not less than 2%; slightly hygroscopic: moisture Weight gain is less than 2% but not less than 0.2%; no or almost no hygroscopicity: wet weight gain is less than 0.2%.

5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2 , 4-Triazol-4-yl)-2-methoxypyridine hydrochloride form A under 20.0% RH-80.0% RH condition, the moisture gain increased 0.769%, slightly hygroscopic.

5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2 , 4-triazol-4-yl)-2-methoxypyridine hydrochloride B crystal form 20.0% RH-80.0% RH, wet weight gain 0.0641%, no or almost no wettability.

5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2 , 4-triazol-4-yl)-2-methoxypyridine methanesulfonate salt A crystal form 20.0%RH-80.0%RH, wet weight gain 0.1061%, no or almost no wettability.

The "X-ray powder diffraction pattern" described in this disclosure is measured using Cu-Kα radiation.

The "X-ray powder diffraction pattern or XRPD" mentioned in this disclosure refers to the Bragg formula 2d sinθ=nλ (where λ is the wavelength of X-ray, λ=1.54056Å, and the order of diffraction n is any positive integer , Generally take the first-order diffraction peak, n=1), when X-rays enter the crystal or part of the crystal sample with a d-lattice plane spacing at a grazing angle θ (coincidence of the incident angle, also called Bragg angle) On the surface, the Bragg equation can be satisfied, so that this group of X-ray powder diffraction patterns are measured.

The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2θ is ±0.30, which can be -0.30, -0.29, -0.28 , -0.27, -0.26, -0.25, -0.24, -0.23, -0.22, -0.21, -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12,- 0.11, -0.010, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, preferably ±0.20.

The "crystal plane spacing or crystal plane spacing (d value)" mentioned in this disclosure means that the spatial lattice selects three non-parallel unit vectors a, b, c connecting two adjacent lattice points, which will The lattice is divided into juxtaposed parallelepiped units, called interplanar spacing. The spatial lattice is divided according to the determined parallel hexahedral unit connection to obtain a set of linear grids, called spatial grids or lattices. The lattice and lattice reflect the periodicity of the crystal structure with geometric dots and lines, respectively. Different crystal planes have different surface spacings (ie, the distance between two adjacent parallel crystal planes); the unit is Å Or Egypt.

The "differential scanning calorimetry or DSC" mentioned in this disclosure refers to the measurement of the temperature difference and heat flow difference between the sample and the reference during the sample heating or constant temperature process to characterize all physical changes related to thermal effects and Chemical change, get the phase change information of the sample.

The drying temperature in the present disclosure is generally 25°C to 100°C, preferably 40°C to 70°C, and can be dried under normal pressure or under reduced pressure. Preferably, the drying is done under reduced pressure.

The chemical reagents or biological reagents used in this disclosure can be purchased from commercial sources.

Test conditions for the instruments used in the experiments in this disclosure:

1. Differential Scanning Calorimeter (DSC)

Instrument model: Mettler Toledo DSC 3 ⁺ STAR ^e System

Purge gas: nitrogen

Heating rate: 10.0℃/min

Temperature range: 25-250℃

2. X-ray Powder Diffraction (XRPD)

(1) Instrument model: Bruker D8 Discover A25 X-ray powder diffractometer

Ray: Monochromatic Cu-Kα rays (λ=1.5406)

Scanning method: θ/2θ, scanning range: 10-48°

Voltage: 40KV, current: 40mA

3. Thermogravimetric Analysis (TGA)

Instrument model: Mettler Toledo TGA2

Purge gas: nitrogen

Heating rate: 10.0℃/min

Temperature range: 25-250℃

4. DVS is dynamic moisture adsorption

The detection adopts SMS DVA Advantage, at 25℃, the humidity is from 0-90%, the step is 10%, the humidity is from 90-95%, the step is 5%, the judgment standard is that each gradient mass change dM/dT is less than 0.002, T _{MAX is} less than 360min, and the cycle is two cycles.

5. Ion Chromatography (HPIC): Instrument

American DionexICS-5000 ion chromatograph; separation column: IonPac AS14A, detection method: conductivity; eluent: NaHCO ₃ 0.0010M+Na ₂ CO ₃ 0.0035M; flow rate 1.0mL/min

The structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). The NMR shift (δ) is given in units of 10 ^-6 (ppm). The measurement of NMR was performed by Bruker AVANCE-400 nuclear magnetic instrument. The solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD), and the internal standard was four. Methyl silane (TMS).

Figure 1: XRPD pattern of crystal form A of hydrochloride.

Figure 2: XRPD pattern of Form B of the hydrochloride salt.

Figure 3: TGA pattern of crystalline form B of hydrochloride.

Figure 4: XRPD pattern of crystal form C of hydrochloride.

Figure 5: DSC pattern of the C form of the hydrochloride salt.

Figure 6: TGA pattern of the C form of hydrochloride.

Figure 7: XRPD pattern of sulfate A crystal form.

Figure 8: TGA pattern of sulfate A crystal form.

Figure 9: XRPD pattern of Form B sulfate.

Figure 10: TGA pattern of Form B of sulfate.

Figure 11: XRPD pattern of Form A of formate.

Figure 12: DSC pattern of Form A of formate.

Figure 13: TGA pattern of Form A of formate.

Figure 14: DVS spectrum of crystal form B of hydrochloride.

The disclosure will be explained in more detail below in conjunction with examples or experimental examples. The examples or experimental examples in this disclosure are only used to illustrate the technical solutions in this disclosure, and do not limit the essence and scope of this disclosure.

The reaction progress in the examples is monitored by thin layer chromatography, the developing agent used in the reaction, the eluent system of column chromatography used for purifying the compound, and the developing agent system package of thin layer chromatography Including: A: dichloromethane/methanol system, B: petroleum ether/ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine and acetic acid can also be added Make adjustments.

Example 1: Compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H- 1,2 , 4-triazol-4-yl)-2-methoxypyridine

The first step: 3-(6-fluoro-3,4-dihydronaphthalen-1-yl)azetidine-1-carboxylic acid third butyl ester 1c

The third butyl 3-iodoazetidine-1-carboxylate 1b (1134.58mg, 4.01mmol, prepared by the well-known method " Organic Letters, 2014 , 16(23), 6160-6163"), Iodine (39.12mg, 0.15mmol), zinc (604.65mg, 9.25mmol) were added to the reaction flask, protected by argon, the reaction was 0.5 hours, bis (dibenzylideneacetone) palladium (141.15mg, 0.15mmol), 2- Dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (73.48mg, 0.15mmol) and 4-bromo-7-fluoro-1,2-dihydronaphthalene 1a (700mg, 3.08mmol, It was prepared by a well-known method " Chemistry-A European Journal, 2015 , 21(14), 5561-5583"). The above reaction solution was added. After the addition, the reaction was heated and stirred at 50°C for 3 hours. After cooling to room temperature, the reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain the title product 1c (700 mg), yield: 74.8%.

MS m/z (ESI): 304.1 [M+1].

Step 2: 3-(6-fluoronaphthalen-1-yl)azetidine-1-carboxylic acid third butyl ester 1d

Dissolve 2,3-dichloro-5,6-dicyano-p-benzoquinone (336.72 mg, 1.48 mmol) and 1c (300 mg, 0.99 mmol) in a 30 mL toluene solution. After addition, the reaction was carried out at 80°C for 12 hours. It was cooled to room temperature, distilled under reduced pressure, the solvent was removed, and the resulting residue was purified by thin-layer chromatography with the developer system B to obtain the title product 1d (180 mg), yield: 60.4%.

MS m/z (ESI): 302.2 [M+1].

Step 3: 3-(6-fluoronaphthalen-1-yl)azetidine hydrochloride 1e

1d (180 mg, 0.60 mmol) and 0.5 mL of 4M hydrogen chloride in 1,4-dioxane were dissolved in 30 mL of dichloromethane, and the reaction was completed for 2 hours. The above reaction solution was concentrated under reduced pressure to obtain the crude title product 1e (120 mg, brown solid). The product was directly subjected to the next reaction without purification.

MS m/z (ESI): 202.1 [M+1].

Step 4: 3-(6-fluoronaphthalen-1-yl)-N-(6-methoxypyridin-3-yl)azetidine-1-thioamide 1g

Crude products 1e (120mg, 0.6) and 1f (99.11mg, 0.60mmol, prepared by the well-known method " Bioorganic & Medicinal Chemistry Letters, 2010 , 20(2), 516-520") were added to a 50mL tetrahydrofuran solution, After completion, the reaction solution was stirred for 2 hours. The reaction solution containing 1 g of the title product was used directly in the next step without purification.

MS m/z (ESI): 368.1 [M+1].

Step 5: (E)-3-(6-fluoronaphthalen-1-yl)-N-(6-methoxypyridin-3-yl)azetidine-1-thioimidic acid methyl ester 1h

1 g (200 mg, 0.54 mmol) of the crude product was dissolved in a 50 mL tetrahydrofuran solution, cooled to 0° C., and potassium tert-butoxide (183.23 mg, 1.63 mmol) was dissolved in the above solution, and the reaction was completed for 1 hour. Methyl 4-methylbenzenesulfonate (101.37 mg, 0.54 mmol) was added to the above reaction solution, and stirred at room temperature for 12 hours. 50 mL of ethyl acetate was added to the reaction solution, washed with water (20 mL×3), the organic phases were combined, the organic phase was distilled under reduced pressure, the solvent was removed, and the resulting residue was purified by thin-layer chromatography with the developer system B to obtain the title product 1h (100mg), yield: 48.2%.

MS m/z (ESI): 382.1 [M+1].

Sixth step: 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2, 4-triazol-4-yl)-2-methoxypyridine 1

Dissolve 1h (100mg, 0.26mmol), trifluoroacetic acid (0.1mL, 0.13mmol) and 2-methoxyacetyl hydrazide (27.29mg, 0.26mmol) in 50mL of tetrahydrofuran. Time. After cooling to room temperature, the solvent was removed under reduced pressure, and the resulting residue was purified and purified by thin layer chromatography using developer system A to obtain the title product 1 (30 mg), yield: 26.7%.

MS m/z (ESI): 420.1 [M+1].

¹ H NMR (400MHz, CD ₃ OD) δ 8.32 (s, 1H), 7.75-7.84 (m, 3H), 7.51-7.55 (m, 2H), 7.43 (d, 1H), 7.25-7.32 (m, 1H) ), 7.00 (d, 1H), 4.51-4.66 (m, 1H), 4.35 (t, 4H), 4.10 (t, 2H), 4.00 (s, 3H), 3.26 (s, 3H).

Test Example 1: Determination of human OTR inhibitory activity

1. Experimental materials and instruments

1. Fluo-4 NW calcium analysis kit (F36206, invitrogen)

2. MEM (Hyclone, SH30024.01B)

3. G418 sulfate (Enzo, ALX-380-013-G005)

4. Fetal bovine serum (GIBCO, 10099)

5. Sodium pyruvate solution (sigma, S8636-100ML)

6. MEM non-essential amino acid solution (100×) (sigma, M7145-100ML)

7. Flexstation 3 Multifunctional Microplate Reader (Molecular Devices)

8. Poly-D-lysine 96-well plate, black/clean (356692, BD)

9. Oxytocin (synthesized by Jill Biochemical Co., Ltd.)

10. pcDNA3.1 (invitrogen, V79020)

11. pcDNA3.1-hOTR (NM-000706) (synthesized and constructed into pcDNA3.1 plasmid by Jinweizhi Biotechnology Co., Ltd.)

12. HEK293 cells (Cat. No. GNHu18, Chinese Academy of Sciences Cell Bank)

2. Experimental procedure

The pcDNA3.1-hOTR plasmid was transfected into HEK293 cells with Lipofectamine® 3000 transfection reagent; G418 screening was started the next day, and simple strain cell lines were selected.

One day in advance, HEK293/human OTR stable transfectant cells were planted in 96-well plates at a density of 25,000 cells/well. The next day, first prepare the loading buffer containing Fluo-4 dye using the reagents in the Fluo-4 NW calcium analysis kit, then remove the medium, add 100 μl of loading buffer containing Fluo-4 dye to each well, 37°C And incubate for 30 minutes. When the time is up, the plate is moved to room temperature and equilibrated for 10 minutes. The compound was formulated into 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , and 10 ¹ nM, 1 μl was added to each well, and incubated at room temperature for 10 minutes. The flexstation 3 microplate reader was used for detection, and 50 μl of 3nM oxytocin polypeptide was automatically added by the machine, and the value was immediately read at 494/516nM. IC ₅₀ values for compounds at different concentrations corresponding to the values of fluorescence can be calculated by Graphpad Prism obtain IC ₅₀ = 2nM, demonstrate that the compounds having significant inhibitory effect on the activity of human OTR.

Test Example 2: Determination of human V1aR inhibitory activity

1. Experimental materials and instruments

1. Fluo-4 NW calcium analysis kit (F36206, invitrogen)

2. MEM (Hyclone, SH30024.01B)

3. G418 sulfate (Enzo, ALX-380-013-G005)

4. Fetal bovine serum (GIBCO, 10099)

5. Sodium pyruvate solution (sigma, S8636-100ML)

6. MEM non-essential amino acid solution (100×) (sigma, M7145-100ML)

7. Flexstation 3 Multifunctional Microplate Reader (Molecular Devices)

8. Poly-D-lysine 96-well plate, black/clean (356692, BD)

9. Vasopressin (Tocris, 2935)

10. pcDNA3.1 (invitrogen, V79020)

11. pcDNA3.1-V1aR (NM-000706) (synthesized and constructed into pcDNA3.1 plasmid by Jinweizhi Biotechnology Co., Ltd.)

12. HEK293 cells (Cat. No. GNHu18, Chinese Academy of Sciences Cell Bank)

2. Experimental procedure

The pcDNA3.1-V1aR plasmid was transfected into HEK293 cells with Lipofectamine® 3000 transfection reagent; G418 screening was started the next day, and simple cell lines were selected.

One day in advance, HEK293/human V1aR stable transfectant cells were seeded in 96-well plates at a density of 25,000 cells/well. The next day, first prepare the loading buffer containing Fluo-4 dye using the reagents in the Fluo-4 NW calcium analysis kit, then remove the medium, add 100 μl of loading buffer containing Fluo-4 dye to each well, 37°C And incubate for 30 minutes. When the time is up, the plate is moved to room temperature and equilibrated for 10 minutes. The compound was formulated into 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , and 10 ¹ nM, 1 μl was added to each well, and incubated at room temperature for 10 minutes. The detection was performed with a flexstation 3 microplate reader, and 50 μl of 3 nM vasopressin polypeptide was automatically added by the machine, and the value was immediately read at 494/516 nM. IC ₅₀ values for compounds at different concentrations corresponding to the values of fluorescence can be calculated by Graphpad Prism obtain IC ₅₀ = 4.5nM, compounds described human V1aR weak inhibiting activity, selective for OTR described inhibitory activity.

Test Example 3: Determination of the inhibitory activity of compounds on human V1bR

The inhibitory effect of the compounds in this disclosure on the activity of human-derived V1bR protein expressed in HEK293/human-derived V1bR cells was determined by the following experimental method:

1. Experimental materials and instruments

1. Fluo-4 NW calcium analysis kit (F36206, invitrogen)

2. MEM (Hyclone, SH30024.01B)

3. G418 sulfate (Enzo, ALX-380-013-G005)

4. Fetal bovine serum (GIBCO, 10099)

5. Sodium pyruvate solution (sigma, S8636-100ML)

6. MEM non-essential amino acid solution (100×) (sigma, M7145-100ML)

7. Flexstation 3 Multifunctional Microplate Reader (Molecular Devices)

8. Poly-D-lysine 96-well plate, black/clean (356692, BD)

9. Vasopressin (Tocris, 2935)

10. pcDNA3.1 (invitrogen, V79020)

11. pcDNA3.1-V1bR (NM-000706) (synthesized and constructed into pcDNA3.1 plasmid by Jinweizhi Biotechnology Co., Ltd.)

12. HEK293 cells (Cat. No. GNHu18, Chinese Academy of Sciences Cell Bank)

2. Experimental procedure

The pcDNA3.1-V1bR plasmid was transfected into HEK293 cells with Lipofectamine® 3000 transfection reagent; G418 was added the next day to obtain HEK293/human-derived V1bR pool cell line.

One day in advance, HEK293/human V1bR pool cells were seeded in 96-well plates at a density of 25,000 cells/well. The next day, first prepare the loading buffer containing Fluo-4 dye using the reagents in the Fluo-4 NW calcium analysis kit, then remove the medium, add 100 μl of loading buffer containing Fluo-4 dye to each well, 37°C And incubate for 30 minutes. When the time is up, the plate is moved to room temperature and equilibrated for 10 minutes. The compound was formulated into 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , and 10 ¹ nM, 1 μl was added to each well, and incubated at room temperature for 10 minutes. The detection was performed with a flexstation 3 microplate reader, and 50 μl of 3 nM vasopressin polypeptide was automatically added by the machine, and the value was immediately read at 494/516 nM. The IC ₅₀ value of the compound can be the fluorescence value corresponding to different concentrations. The IC ₅₀ =26 μM calculated by Graphpad Prism software, indicating that the compound has no significant inhibitory effect on human V1bR activity, indicating that it has selective inhibitory effect on OTR activity.

Test Example 4: Determination of the inhibitory activity of compounds on human V2R

The inhibitory effect of the compounds in this disclosure on the activity of human-derived V2R protein expressed in HEK293/human-derived V2R cells was determined by the following experimental method:

1. Experimental materials and instruments

1.cAMP dynamic 2 kit-1,000 experiments (62AM4PEB, Cisbio)

2.MEM (Hyclone, SH30024.01B)

3. G418 sulfate (Enzo, ALX-380-013-G005)

4. Fetal bovine serum (GIBCO, 10099)

5. Sodium pyruvate solution (sigma, S8636-100ML)

6. MEM non-essential amino acid solution (100×) (sigma, M7145-100ML)

7.PheraStar Multi-function Microplate Reader (BMG)

8. Corning/Costar 384-well non-adsorption microplate-black NBS plate (4514, Corning)

9. Cell dissociation solution, without enzyme, PBS (13151014-100ml, Thermo Fisher Scientific)

10. HBSS, calcium, magnesium, phenol red free (14025-092, Invitrogen)

11. HEPES, 1M buffer solution (15630-080, GIBCO)

12.BSA (0219989725, MP Biomedicals)

13. IBMX (I7018-250MG, sigma)

14. Vasopressin (Tocris, 2935)

15.pcDNA3.1 (invitrogen, V79020)

16.pcDNA3.1-V2R(NM-000054) (synthesized and constructed into pcDNA3.1 plasmid by Jinweizhi Biotechnology Co., Ltd.)

17. HEK293 cells (Cat. No. GNHu18, Chinese Academy of Sciences Cell Bank)

2. Experimental procedure

The pcDNA3.1-V2R plasmid was transfected into HEK293 cells with Lipofectamine® 3000 transfection reagent; G418 was added the next day to obtain HEK293/human-derived V2R pool cell line.

1) Dissociate cells:

Use cell dissociation solution to dissociate HEK293/human-derived V2R pool cells without cell dissociation from the cell culture dish, dissociate the cells into a single, pipette evenly after termination, centrifuge, remove the supernatant with experimental buffer 1 (1x HBSS+20mM HEPES+ 0.1% BSA) Resuspend the cells and count, adjust the cell density to 1250 cells/5μl, that is 2.5*10 ⁵ /ml.

2) Dispensing

Compounds were formulated with pure DMSO to a series of concentrations of 20 mM, 6.67 mM, 2.22 mM, 0.74 mM, 0.25 mM, 0.08 mM, 27.4 μM, 9.14 μM, 3.05 μM, 1.02 μM, 0.34 μM and 0 μM (DMSO). Then the compound was made up to a 4-fold use concentration using Experiment Buffer 2 (Experiment Buffer 1+1 mM IBMX).

Agonist: Use 460 μM vasopressin mother solution, first make up 2 μM with DMSO, and then dilute with experiment buffer 2 to 0.5 nM concentration.

Standard: The first point is a stock solution of 20 μl (2848 nM). From the second point, dilute it in 4 times with experiment buffer 1 in total, 11 concentrations in total.

3) Medicine incubation:

1. Add the mixed cells to a 384-well plate, 5μl/well, without changing the tip.

2. Add 2.5μl/well of the compound to be tested and the positive compound, and the tip needs to be replaced.

3. Centrifuge at 1000 for 1 min, shake for 30 sec to mix, and incubate at room temperature for 30 min.

4. Standard curve wells need to add 5μl/well of experiment buffer 2.

5. Add 2.5μl per well of the prepared agonist. Need to change the tip, centrifuge at 1000rpm for 1min, shake for 30sec to mix, and incubate at room temperature for 30min.

6. Prepare cAMP-d2 (component in cAMP dynamic 2 kit) and Anti-cAMP-Eu-Cryptate (component in cAMP dynamic 2 kit) in the dark, according to the ratio of 1:4 with cAMP lysate ( cAMP Dynamic 2 kit components) and mix well. Add 5μl/well of the prepared cAMP-d2 liquid to each well, then add 5μl/well of Anti-cAMP-Eu-Cryptate, mix by shaking for 30sec, and incubate at room temperature in the dark for 1h.

4) Plate reading: PheraStar multi-function microplate reader reads HTRF signals.

5) Data processing

The data in this experiment was processed with data processing software Graphpad Prism to obtain IC ₅₀ =4.9 μM, indicating that the compound had no significant inhibitory effect on human V2R activity, indicating that it had selective inhibitory effect on OTR activity.

Test Example 5: Determination of rat brain permeability activity

The permeation activity of compounds in this disclosure on rat brain is determined by the following experimental method:

1. Experimental materials and instruments

1. RED device insertion (Device Inserts) (Thermo Scientific, QL21291110)

2. API 4000 Q-trap Linear Ion Trap Mass Spectrometer (Applied Biosystems)

3.LC-30A Ultra High Pressure Liquid Chromatography System (Shimadzu)

4. pH7.4 PBS (100mM, stored in refrigerator at 4℃)

5. SD rats, provided by Jie Jie Jie Experimental Animal Co., Ltd., animal production license number SCXK (Shanghai) 2013-0006.

2. Experimental animal operation

Four SD rats, half male and one female, light/dark adjustment for 12/12 hours, constant temperature 24±3℃, humidity 50-60%, free to eat and drink. After fasting overnight, they were administered by intragastric administration. The dosage is 10mg/kg, and the drug group is sacrificed after blood collection 0.5h~2h after administration (blood collection volume 0.5ml). The blood sample is placed in a heparinized test tube and centrifuged at 3500rpm for 10min to separate the plasma, which is recorded as plasma 1, at 20℃ Preservation; after the animal was sacrificed, the head was decapitated, the brain tissue was taken, and the remaining blood was blotted with filter paper, which was recorded as brain tissue 1, and stored at 0°C after 10 minutes. Another 3 animals were used to take blank plasma and brain tissue 2 and the treatment method was the same as the administration group.

3. Plasma protein binding balance dialysis process

3.1 Sample preparation

0.2%)，用於該濃度血漿蛋白結合率的測定。移取上述配好的50μl血漿樣品，記為T₀，置於-80℃冰箱保存待測。 Dilute the drug compound to 50 mM with DMSO to obtain the stock solution I; transfer the appropriate amount of the stock solution I and dilute with methanol to obtain a 200 μM diluted stock solution II; transfer 10 μl of the stock solution II to a 1.5 ml Eppendorf tube, add 990 μl of blank plasma and mix well 2μM plasma sample 2 (DMSO content was obtained

0.2%) for the determination of plasma protein binding rate at this concentration. Pipette 50μl above with a good plasma sample, referred to as T _0, -80 ℃ refrigerator disposed under test.

3.2 Experimental process

Insert the RED device into the balanced dialysis tube and insert it into the 96-well bottom plate. Take 300 μl of the above-prepared plasma sample 2 containing the analyte and the corresponding blank plasma sample, and place them in the red marked well (plasma chamber). Take 500 μl of pH 7.4 phosphate buffered saline solution and place it in another well (buffer chamber) marked side by side in red. According to the above steps, the concentration of each compound is 2-3 samples. When finished, the 96-well bottom plate was covered with sealing tape, and the whole bottom plate was placed in the thermal mixer, equilibrated at 37°C for 4 h at 400 rpm. After the incubation, remove the 96-well bottom plate device from the thermal mixer to complete the equilibrium dialysis. Take 50μl of equilibrated plasma sample or dialysate sample, add 50μl of corresponding unbalanced drug-free blank phosphate buffer or drug-free blank plasma, add internal standard (acetonitrile preparation) 300μl, vortex to mix for 5min , Centrifuge for 10min (4000rpm), and take the supernatant for LC/MS/MS analysis. Without incubation, the T ₀ sample was directly measured by the LC/MS/MS method established above to determine the total drug (plasma chamber) and free drug (buffer chamber) and internal standard chromatographic peak area ratio, and calculate the free percentage (f _{u plasma} % ).

4. Brain tissue protein binding balance dialysis process

Brain tissue protein binding balance dialysis process: blank brain tissue 2 was made into blank brain homogenate with pH7.4 PBS at a ratio of dilution factor=11, compound was added to make 2 μM brain homogenate, and the rest was combined with plasma protein The operation was the same. The established LC/MS/MS method was used to determine the ratio of chromatographic peak area of total drug (brain homo chamber) and free drug (buffer chamber) to internal standard, and calculate the free percentage ( _{fu brain hom} %).

5. Brain permeability test method

1) The established LC/MS/MS method was used to determine the drug concentration in plasma 1 and brain tissue 1 0.5h after administration, which is the total concentration (C _{total, p} and C _{total, b} ); 2) Using the RED Device Inserts device, the protein binding rate of the compound in rat plasma and brain tissue was measured by balanced dialysis method to calculate the free percentage (f _{u plasma} %, f _{u brain} %); plasma free percentage (f _{u plasma%} %)=C _buffer /C _plasma ×100%; free percentage of brain homogenate (f _{u brain hom} %)=C _buffer /C _{brain hom} ×100%; free percentage of brain tissue (f _{u brain} %)=f _{u brain hom} /(Df-(Df-1)*f _{u brain hom} )×100%; here Df=11

3) Use the following formula to calculate the blood-brain permeability index Kp-unbound.

6. Test results and discussion

Compound Brain Permeability Index

Conclusion: The compounds in this disclosure have good brain permeability.

Test Example 6: Pharmacokinetic test of the compound

1. Summary

SD male rats were used as test animals, and the compounds of Example 2, Example 17, Compound 34, Example 37, and Example 38 were administered to rats by intragastric administration using LC/MS/MS. The concentration of the drug in the plasma at different times after the compound of Example 39, the compound of Example 42 and the compound of Example 43. Study the pharmacokinetic behavior of the compounds in this disclosure in rats and evaluate their pharmacokinetic characteristics.

2. Test plan

2.1 Test drug

The compound of Example 2, the compound of Example 17, the compound of Example 34, the compound of Example 37, the compound of Example 38, the compound of Example 39, the compound of Example 42 and the compound of Example 43.

2.2 Test animals

There were 24 healthy adult SD rats, males were divided into 8 groups on average, and 3 rats in each group were purchased from Shanghai Jie Si Jie Experimental Animal Co., Ltd. Animal production license number: SCXK (Shanghai) 2013-0006.

2.3 Drug preparation

Weigh a certain amount of drug, add 2.5% volume of DMSO and 97.5% volume of 10% solutol HS-15 to make 0.2mg/mL colorless clear transparent liquid.

2.4 Administration

The SD rats were given by intragastric administration after fasting overnight. The dosage was 30.0 mg/kg and the volume was 10.0 mL/kg.

3. Operation

The compound of Example 2, the compound of Example 17, the compound of Example 34, the compound of Example 37, the compound of Example 38, the compound of Example 39, the compound of Example 42 and the compound of Example 43 were administered to rats by intragastric administration before administration And 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 hours after administration, 0.2mL of blood was collected from the orbit, placed in a heparinized test tube, centrifuged at 4℃, 3500 rpm for 10 minutes to separate the plasma, at -20℃ Store and eat 2 hours after administration.

Determination of test compound content in rat plasma after intragastric administration of different concentrations of drugs: 50μL of rat plasma at each time after administration, adding internal standard solution camptothecin 50μL (100ng/mL), acetonitrile 150μL, vortex Spin-mix for 5 minutes, centrifuge for 10 minutes (4000 rpm), and take 3 μL of the supernatant of the plasma sample for LC/MS/MS analysis.

4. Results of pharmacokinetic parameters

結論：本披露中化合物的藥物代謝吸收較好，具有藥物代謝動力學優勢。 The pharmacokinetic parameters of the compounds in this disclosure are as follows:

Conclusion: The compounds in this disclosure have better drug metabolism and absorption, and have the advantages of pharmacokinetics.

Example 2: 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H- 1,2, Form A of 4-triazol-4-yl)-2-methoxypyridine hydrochloride

250 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was added to 95% isopropanol/water 3ml, stirred to dissolve, hydrochloric acid (2mol/L) was added dropwise according to 1:1.1eq (equivalent), and stirring was continued, Filter and dry in vacuo to give the product.

The ion chromatography (HPIC) test result of the obtained product: the chloride ion content is 7.86%, indicating that the molar ratio of the compound in the salt to hydrochloric acid is about 1:1.

The XRPD pattern of the crystalline sample is shown in Figure 1. The melting peak point is near 183.41℃, and the characteristic peak position is shown in Table 1 below:

Example 3: Hydrochloride A crystal form

20 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was dissolved in 400ul of ethyl acetate, hydrochloric acid (2mol/L) was added dropwise according to 1:1.1eq, stirring was continued, filtration and vacuum drying were carried out to obtain the product.

Example 4: 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H- 1,2, Form B of 4-triazol-4-yl)-2-methoxypyridine hydrochloride

250 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was dissolved in 3 ml of acetone, hydrochloric acid (2 mol/L) was added dropwise according to 1:1.1 eq, stirring was continued, filtered and dried in vacuum to obtain the product.

The ion chromatography (HPIC) detection result of the obtained product: the chloride ion content is 7.83%, indicating that the molar ratio of the compound in the salt to hydrochloric acid is about 1:1.

The XRPD pattern of the crystalline sample is shown in Figure 2, and the TGA pattern is shown in Figure 3. The melting peak point is near 182.73°C, and the characteristic peak position is shown in Table 2 below:

Example 5: Hydrochloride Form B

20 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was dissolved in 400ul of ethanol, hydrochloric acid (2mol/L) was added dropwise according to 1:1.1eq, stirring was continued, filtration and vacuum drying were performed to obtain the product.

Example 6: Hydrochloride Form B

250 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was dissolved in 3ml of acetonitrile, hydrochloric acid (2mol/L) was added dropwise according to 1:1.1eq, stirring was continued, filtered and dried in vacuo to obtain the product.

Example 7: 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H- 1,2, Form C of 4-triazol-4-yl)-2-methoxypyridine hydrochloride

250 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine is added to 5ml of methyl tert-butyl ether, hydrochloric acid (2mol/L) is added dropwise according to 1:1.1eq, and the temperature is raised and lowered within the range of 10℃-50℃ The procedure is stirred, filtered and dried under vacuum to obtain the product.

The ion chromatography (HPIC) test result of the obtained product: the chloride ion content is 6.76%, indicating that the molar ratio of the compound in the salt to hydrochloric acid is about 1:1.

The XRPD pattern of the crystalline sample is shown in Figure 4, the DSC pattern is shown in Figure 5, the TGA pattern is shown in Figure 6, the melting peak point is around 170.63℃, and the characteristic peak position is shown in Table 3:

Example 8: 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H- 1,2, Form A of 4-triazol-4-yl)-2-methoxypyridine-sulfate

250 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine is added to acetonitrile, stirred to dissolve, sulfuric acid (2mol/L) is added according to 1:1.1eq, stirring is continued, filtered and dried in vacuum to obtain the product.

The ion chromatography (HPIC) detection result of the obtained product: the sulfate content is 32.56%, indicating that the molar ratio of the compound in the salt to sulfuric acid is about 1:2.

The XRPD pattern of the crystalline sample is shown in Figure 7 and the TGA pattern is shown in Figure 8. The characteristic peak positions are shown in Table 4 below:

Example 9: sulfate A crystal form

20 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was dissolved in 400ul of ethanol, sulfuric acid (2mol/L) was added dropwise according to 1:1.1eq, naturally volatilized, and filtered to obtain the product.

Example 10: Sulfate A crystal form

20 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was dissolved in 400ul of acetone, sulfuric acid (2mol/L) was added dropwise according to 1:1.1eq, stirring was continued, filtration and vacuum drying were carried out to obtain the product.

Example 11: Sulfate A crystal form

20 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was dissolved in 400ul of ethyl acetate, sulfuric acid (2mol/L) was added dropwise according to 1:1.1eq, stirring was continued, filtration and vacuum drying were carried out to obtain the product.

Example 12: 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H- 1,2, Form B of 4-triazol-4-yl)-2-methoxypyridine-sulfate

250 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4 -Triazol-4-yl)-2-methoxypyridine was dissolved in 5ml of methyl tert-butyl ether, and sulfuric acid (2mol/L) was added according to 1:1.1eq, in the range of 10℃-50℃ Stir, raise and lower the temperature program, filter, and vacuum dry to obtain the product.

The ion chromatography (HPIC) detection result of the obtained product: the sulfate content was 18.54%, indicating that the molar ratio of the compound in the salt to sulfuric acid was about 1:1.

The XRPD pattern of the crystalline sample is shown in Figure 9, and the TGA pattern is shown in Figure 10. The characteristic peak positions are shown in Table 5 below:

Example 13: 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H- 1,2, Form A of 4-triazol-4-yl)-2-methoxypyridine mesylate

250 mg of compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4H-1,2,4- Triazol-4-yl)-2-methoxypyridine was dissolved in 5ml of methyl tert-butyl ether, and methanesulfonic acid (2mol/L) was added according to 1:1.1eq, within the range of 10℃-50℃ Stir the temperature program, filter, and vacuum dry at 40°C to obtain the product.

The ion chromatography (HPIC) detection result of the obtained product: the mesylate content was 17.78%, indicating that the molar ratio of the compound in the salt to hydrochloric acid was about 1:1.

The XRPD pattern of the crystalline sample is shown in Figure 11, the DSC pattern is shown in Figure 12, the TGA pattern is shown in Figure 13, the melting peak point is near 207.46℃, and the characteristic peak position is shown in Table 6 below:

Example 14: Study on the hygroscopicity of the hydrochloride A form, the hydrochloride B form and the mesylate A form

Using Surface Measurement Systems advantage 2, at 25 ℃, the humidity starts from 50%, the humidity range is 0%-95%, the step is 10%, the judgment standard is that each gradient mass change dM/dT is less than 0.002, TMAX is less than 360min , Cycle two times.

Hydrochloride A crystalline sample is between 20.0% RH and 80.0% RH at 25°C. As the humidity increases, the amount of water absorption also increases, the weight change is 0.769%, and the humidity increase is less than 2%. Not less than 0.2%, the sample is slightly hygroscopic. Under normal storage conditions (ie, 60% humidity at 25°C), the water absorption is about It is 1.675%; under accelerated test conditions (that is, humidity 70%), the water absorption is about 1.738%; in extreme conditions (that is, humidity 90%), the water absorption is about 1.860%.

During the humidity change of 0%-95%, the desorption process of this sample basically coincided with the adsorption process; the X-ray powder diffraction comparison chart before and after DVS showed that the crystal form did not change before and after DVS.

Under the condition of 25℃, the hydrochloride B crystal sample is between 20.0%RH-80.0%RH, with the increase of humidity, the amount of water absorption also increases, the weight change is 0.0641%, and the weight gain is less than 0.2%. The sample has no or almost no hygroscopicity; under normal storage conditions (that is, 60% humidity at 25°C), the water absorption is about 0.0361%; under accelerated test conditions (that is, humidity 70%), the water absorption is about 0.0446%; in extreme conditions ( 90% humidity), the water absorption is about 0.0947%

During the humidity change of 0%-95%, the desorption process of this sample basically coincides with the adsorption process; the DVS spectrum is shown in Figure 14. The X-ray powder diffraction comparison chart before and after DVS shows that the crystal form has not changed before and after DVS.

The methanesulfonic acid crystalline sample is between 20.0% RH and 80.0% RH at 25°C. As the humidity increases, the amount of water absorption also increases, the weight change is 0.1061%, and the weight gain is less than 0.2%. The sample has no or almost no hygroscopicity; under normal storage conditions (ie, 60% humidity at 25°C), the water absorption is about 0.0993%; under accelerated test conditions (ie, humidity 70%), the water absorption is about 0.1154%; under extreme conditions ( That is 90% humidity), the water absorption is about 0.1755%.

During the humidity change of 0%-95%, the desorption process of this sample basically coincided with the adsorption process; the X-ray powder diffraction comparison chart before and after DVS showed that the crystal form did not change before and after DVS.

Example 15: Crystal form stability study

Place the salt crystals in an open flat position, and examine the stability of the samples under light (4500 Lux), high temperature (40℃, 60℃), and high humidity (RH 75%, RH 92.5%). The sampling period is 30 days.

結論：影響因素實驗表明：在光照、高溫40℃和60℃、高濕75%和92.5%條件下，鹽酸鹽的A和B及甲磺酸鹽的A晶型具有較好的物理、化學穩定性。

Conclusion: The experiment of influencing factors shows that: under the conditions of light, high temperature of 40 ℃ and 60 ℃, high humidity of 75% and 92.5%, the crystalline forms of hydrochloride A and B and mesylate A have better physical and chemical properties. stability.

Experimental Example 2: Long-term/accelerated stability

Form B (Example 4) was placed at 25 ℃, 60% RH and 40 ℃, 75% RH conditions to investigate its stability

The long-term/accelerated stability experiment shows that the hydrochloride crystal form B is placed under long-term accelerated stability conditions, its crystal form has not changed, and it has good physical stability. At the same time, there is no growth in related substances, and it has excellent chemistry stability.

Claims (31)

A compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- A pharmaceutically acceptable salt of triazol-4-yl)-2-methoxypyridine selected from the group consisting of hydrochloride, sulfate, methanesulfonate, phosphate, citrate, and benzoate Or fumarate. The pharmaceutically acceptable salt as described in item 1 of the patent application scope, wherein the chemical ratio of the compound to the acid molecule is about 1:2~2:1. The pharmaceutically acceptable salt as described in item 2 of the patent application scope, wherein the chemical ratio of the compound to the acid molecule is about 1:2, 1:1, 2:1. A method for preparing a pharmaceutically acceptable salt according to any one of items 1 to 3 of the patent application scope, including: the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidine l-yl) -5- (methoxymethyl) -4 H -1,2,4- triazol-4-yl) -2-methoxy-pyridine in step with an acid into a salt. The method according to item 4 of the patent application scope, wherein the solvent used in the salt formation reaction is selected from at least one of isopropyl alcohol, acetone, methyl tertiary butyl ether, acetonitrile, ethanol, ethyl acetate, and water. A pharmaceutical composition comprising a pharmaceutically acceptable salt as described in item 1 of the patent application scope and optionally at least one pharmaceutical excipient selected from pharmaceutically acceptable carriers, diluents or excipients. The use of a pharmaceutically acceptable salt as described in item 1 of the patent application scope for the preparation of a medicament for the treatment or prevention of a disease or condition known or shown to inhibit beneficial effects of oxytocin, the disease or condition selected Self-sex dysfunction, hyposexual disorder, sexual arousal disorder, orgasm disorder, intercourse pain disorder, premature ejaculation, pre-birth delivery, childbirth complications, appetite and eating disorders, benign prostatic hyperplasia, premature delivery, dysmenorrhea, congestive heart failure, Arterial hypertension, cirrhosis, renal hypertension, high intraocular pressure, obsessive-compulsive disorder and behavioral disorders, and neuropsychiatric disorders. The use as described in item 7 of the patent application scope, wherein the disease or disorder is selected from sexual Dysfunction, sexual arousal disorder, orgasm disorder, sexual intercourse pain disorder and premature ejaculation. The use of a pharmaceutically acceptable salt as described in item 1 of the patent application scope is for preparing a medicine for antagonizing oxytocin. A compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- Form A of triazol-4-yl)-2-methoxypyridine hydrochloride is characterized by an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 6.92, 12.54, 15.23, 16.32, 22.70 , 27.44, 28.10 have characteristic peaks. A method for preparing crystal form A as described in item 10 of the patent application scope includes: (a) Compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidine-1- Group)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (I), stirred to dissolve or heated to dissolve, the The solvent (I) is selected from isopropanol/water, ethyl acetate, (b) hydrochloric acid is added dropwise, and crystallized by stirring. A compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- Form B of triazol-4-yl)-2-methoxypyridine hydrochloride is characterized by an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 14.62, 15.65, 19.21, 23.66, 24.15 , 25.92, 27.19 have characteristic peaks. The crystal form B as described in item 12 of the patent application range, in which the X-ray powder diffraction pattern expressed at a diffraction angle of 2θ is at 11.76, 14.62, 15.65, 19.21, 23.66, 24.15, 25.92, 27.19, 28.01, 29.54 There are characteristic peaks. The crystal form B according to item 12 or 13 of the patent application scope, wherein the X-ray powder diffraction pattern expressed at a diffraction angle of 2θ is at 11.76, 14.62, 15.65, 16.43, 18.70, 19.21, 23.66, 24.15, 24.56 , 25.60, 25.92, 27.19, 28.01, 29.54 have characteristic peaks. The crystal form B according to any one of items 12 to 14 of the patent application range, wherein the X-ray powder diffraction pattern expressed at a diffraction angle of 2θ is shown in FIG. 2. A method for preparing Form B according to any one of claims 12 to 15 of the patent application, including: (a) compound 5-(3-(3-(6-fluoronaphthalen-1-yl)aza Cyclobutan-1-yl)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (II) and stirred Dissolve or dissolve by heating. The solvent (II) is selected from ethanol, acetone and acetonitrile. (b) Hydrochloric acid is added dropwise, stirred for crystallization and beaten. A compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- The crystal form C of triazol-4-yl)-2-methoxypyridine hydrochloride is characterized by an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 6.94, 21.51, 22.71, 24.99, 25.80 , 27.45 and 28.14 have characteristic peaks. A method for preparing the crystal form C described in Item 17 of the patent application scope includes: (a) the compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidine-1- Group)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (III), stirred and dissolved by heating or the The solvent (III) is selected from methyl tert-butyl ether, (b) hydrochloric acid is added dropwise, and the crystal is stirred and crystallized. A compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- Form A of triazol-4-yl)-2-methoxypyridine sulfate is characterized by an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 12.66, 18.50, 19.90, 21.64, 23.61, There are characteristic peaks at 24.22 and 26.34. A method for preparing the crystalline form A described in item 19 of the patent application scope includes: (a) Compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidine-1- Group)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (IV), dissolved by stirring or heated to dissolve, The solvent (V) is selected from acetonitrile, ethanol, acetone and ethyl acetate. (b) Sulfuric acid is added dropwise and crystallized by stirring. A compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- Form B of triazol-4-yl)-2-methoxypyridine sulfate is characterized by an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 11.87, 13.22, 14.62, 15.29, 18.49, There are characteristic peaks at 22.66 and 23.61. A method for preparing Form B described in Item 21 of the patent application scope includes: (a) Compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidine-1- Group)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (V), dissolved by stirring or heated to dissolve, The solvent (VI) is selected from methyl tert-butyl ether, (b) sulfuric acid is added dropwise, and crystallized by stirring. A compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidin-1-yl)-5-(methoxymethyl)-4 H -1,2,4- Form A of triazol-4-yl)-2-methoxypyridine methanesulfonate is characterized by an X-ray powder diffraction pattern expressed at a diffraction angle of 2θ at 6.66, 16.81, 16.90, 18.92, There are characteristic peaks at 19.19, 22.92 and 24.88. A method for preparing crystal form A as described in item 23 of the patent application scope includes: (a) Compound 5-(3-(3-(6-fluoronaphthalen-1-yl)azetidine-1- Group)-5-(methoxymethyl)-4 H -1,2,4-triazol-4-yl)-2-methoxypyridine is added to the solvent (VI), dissolved by stirring or heated to dissolve, The solvent (VII) is selected from ethyl acetate and methyl tertiary butyl ether, (b) methanesulfonic acid is added dropwise, and crystallized by stirring. The crystalline form as described in any of items 10, 12 to 15, 17, 19, 21 or 23 of the patent application range, wherein the error range of the 2θ angle is ±0.30. The crystal form as described in item 25 of the patent application range, wherein the 2θ angle error range is ±0.20. A pharmaceutical composition containing the crystalline form described in any one of claims 10, 12, 17, 19, 21 or 23 and optionally selected from a pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition prepared from the crystal form described in any one of items 10, 12, 17, 19, 21 or 23 of the patent application. A use of the crystalline form described in any one of the patent application items 10, 12, 17, 19, 21 or 23, which is used in the preparation of a therapeutic or preventive known or may show a beneficial effect of inhibiting oxytocin A drug for a disease or disorder selected from sexual dysfunction, hyposexual disorder, sexual arousal disorder, orgasm disorder, painful sexual intercourse, premature ejaculation, pre-term delivery, complication of delivery, appetite and eating disorders, benign Prostatic hyperplasia, premature delivery, dysmenorrhea, congestive heart failure, arterial hypertension, cirrhosis, renal hypertension, high intraocular pressure, obsessive-compulsive and behavioral disorders, and neuropsychiatric disorders. The use as described in item 29 of the patent application scope, wherein the disease or disorder is selected from sexual dysfunction, sexual arousal disorder, orgasm disorder, sexual intercourse pain disorder and premature ejaculation. The use of the crystalline form described in any one of the patent application items 10, 12, 17, 19, 21 or 23 is used to prepare a medicine for antagonizing oxytocin.

Images