CN106632349B

CN106632349B - The hydrate and its preparation and application of the salt of pyran derivate or its salt

Info

Publication number: CN106632349B
Application number: CN201610954824.1A
Authority: CN
Inventors: 范江; 陈清平; 汪成涛
Original assignee: Haisike Pharmaceutical Group Ltd By Share Ltd
Current assignee: Haisike Pharmaceutical Group Ltd By Share Ltd
Priority date: 2015-10-29
Filing date: 2016-10-27
Publication date: 2019-05-28
Anticipated expiration: 2036-10-27
Also published as: CN106632349A

Abstract

The present invention relates to the hydrates and its preparation and application of the salt of pyran derivate or its salt, more particularly to dipeptides kinases-IV inhibitor pharmaceutically acceptable salt or the hydrate of its salt, and its preparation method and application, furthermore it is related to salt or hydrate of its salt of compound shown in formula (I) and its preparation method and application:

Description

The hydrate and its preparation and application of the salt of pyran derivate or its salt

Technical field

The present invention relates to a kind of pyran derivate pharmaceutically acceptable salt or the hydrates and its crystal form knot of its salt Structure, preparation method or its pharmaceutical composition and they preparing the purposes on dipeptides kinases-IV (DPP-IV) inhibitor medicaments.

Background technique

Dipeptidyl peptidase-IV (Dipeptidyl Peptidase, DPP-IV, EC3.4.14.5) is a serine stretch protein Enzyme, from the polypeptide N-terminal penultimate hydrolyzing N end dipeptides containing L-PROLINE and l-Alanine.By enhancing incretin Activation plays effect, belongs to non-insulin therapeutic agent.DPP-IV inhibitor is without the adverse reactions such as weight gain and oedema.

PCT/CN2015/080570 patent application discloses new pyran derivate (2R, 3S, 5R) -2- (2,5- difluorobenzene Base) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) pyrrolo- [3,4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro - 2H- pyranose -3- amine, structural formula such as formula (A).The structure, which is shown, has good inhibiting effect to DPP-IV, has for pre- Anti- and/or treatment type-2 diabetes mellitus potential.

Summary of the invention

Present invention is primarily aimed at provide pyran derivate: (2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- first Base -2- (1- methyl-1 H- tetrazolium -5- base) pyrrolo- [3,4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyranose -3- The hydrate and its crystal form of the salt of amine or its salt.In the preparation of drug of the present invention or combinations thereof object, the hydration of salt or its salt Object and its crystal form specific ionization alkali cpd have better stability, and high temperature resistant, high humidity and intense light irradiation make it be more suitable for drug agent The preparation of type.

The present invention relates to the salt of compound shown in a kind of formula (I) or the hydrates of its salt:

The preferred solution of the invention, the salt of compound shown in formula (I) or the hydrate of its salt are formula (II) structure:

Wherein,

HA be pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, malonic acid, oxalic acid, adipic acid, It is trifluoroacetic acid, succinic acid, salicylic acid, benzoic acid, phthalic acid, lactic acid, tartaric acid, malic acid, benzene sulfonic acid, methanesulfonic acid, right Toluenesulfonic acid, citric acid or fumaric acid；

N is selected from 1 or 2；

M is selected from 0~3.

The preferred solution of the invention, HA are selected from hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, malonic acid, oxalic acid, adipic acid, trifluoro Acetic acid, succinic acid, salicylic acid, benzoic acid, phthalic acid, lactic acid, tartaric acid, malic acid, benzene sulfonic acid, methanesulfonic acid, to toluene Sulfonic acid, citric acid or fumaric acid.

The preferred solution of the invention, when HA is selected from hydrochloric acid, phosphoric acid or p-methyl benzenesulfonic acid, n is selected from 1 or 2；When HA is selected from benzene first When acid, citric acid or fumaric acid, n is selected from 1.

The preferred solution of the invention, a kind of formula (III) compound represented:

Wherein, m is selected from 1~3.

The preferred solution of the invention, formula (III) compound represented are crystal form I, are radiated using Cu-K α, X-ray powder Diffracting spectrum the position following 2 θ have characteristic diffraction peak: 6.7 ° ± 0.2 °, 13.8 ° ± 0.2 °, 14.5 ° ± 0.2 °, 24.2 ° ± 0.2 ° and 27.9 ° ± 0.2 °.

The preferred solution of the invention, formula (III) compound represented are crystal form I, are radiated using Cu-K α, X-ray powder Diffracting spectrum also the position following 2 θ have characteristic diffraction peak: 14.0 ° ± 0.2 °, 16.5 ° ± 0.2 °, 17.8 ° ± 0.2 °, 19.3 ° ± 0.2 °, 20.3 ° ± 0.2 ° and 21.0 ° ± 0.2 °.

The preferred solution of the invention, formula (III) compound represented are crystal form I, are radiated using Cu-K α, X-ray powder Diffracting spectrum also the position following 2 θ have characteristic diffraction peak: 13.4 ° ± 0.2 °, 15.4 ° ± 0.2 °, 22.7 ° ± 0.2 °, 24.5 ° ± 0.2 ° and 25.0 ° ± 0.2 °.

The preferred solution of the invention, the X-ray powder diffraction collection of the crystal form I of formula (III) compound represented is substantially as schemed Shown in 1.

The preferred solution of the invention, the differential scanning calorimetry thermogram of formula (III) compound represented are basic such as Fig. 2 institute Show.

The thermal gravimetric analysis curve of the preferred solution of the invention, formula (III) compound represented is substantially as shown in Figure 3.

The preferred solution of the invention, formula (III) compound represented, m=3.

The present invention also provides a kind of pharmaceutical compositions, and it includes the hydrations of the salt or its salt as described above of therapeutically effective amount Object and pharmaceutically acceptable carrier or excipient.

The present invention also provides the hydrate or logical formula (II) of the salt of logical formula (I) compound represented or its salt or general formulas (III) compound, or pharmaceutical composition as described above are being prepared for preventing and/or treating diabetes and/or glycosuria Application in the drug of sick complication.

Preferably, the diabetic complication includes diabetic retinopathy, diabetic neuropathy and diabetic keratopathy One of nephrosis is a variety of.

Preferably, the diabetes include type-2 diabetes mellitus.

The present invention also provides a kind of method for treating metabolic disease, arbitrarily described the method includes the administration present invention The hydrate or foregoing pharmaceutical composition of salt or its salt.

The synthetic method of the compounds of this invention

In order to complete the purpose of the present invention, the compounds of this invention can be prepared by following scheme:

Formula (IV) compound reacts to obtain formula (II) with HA；

Or

Formula (V) compound reacts to obtain formula (II) with HA；

Wherein,

HA is selected from hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, malonic acid, oxalic acid, adipic acid, trifluoroacetic acid, succinic acid, water Poplar acid, benzoic acid, phthalic acid, lactic acid, tartaric acid, malic acid, benzene sulfonic acid, methanesulfonic acid, p-methyl benzenesulfonic acid, citric acid or richness Horse acid；

N is selected from 1 or 2；

M is selected from 0~3；

P is amino protecting group.

Preferably, HA is selected from hydrochloric acid, phosphoric acid, benzoic acid, p-methyl benzenesulfonic acid, citric acid or fumaric acid.

It is highly preferred that HA is the hydrochloric acid that mass fraction is 15%~37%.

After logical formula (IV) compound or logical formula (V) compound can be dissolved with solvent, HA acid is added and is reacted, in which: Solvent, which can be all, can dissolve the solvent of logical formula (IV) compound or logical formula (V) compound, such as ethyl alcohol；Reaction is 0 ~90 DEG C can be with.

It is high to react obtained product purity, it is alternatively that, obtained product can also be recrystallized further.When recrystallization, Product can choose ionized water and the mixed liquor of ethyl alcohol dissolves, and after heating for dissolving, product is precipitated with suitable crystallization solvent, described Crystallization solvent, such as isopropyl acetate, ethyl acetate, isopropyl ether or methyl tertiary butyl ether(MTBE).

Reaction of the invention tracks extent of reaction according to the common method of the those skilled in the art such as TLC or HPLC.

In dihydrochloride lanthanum chloride hydrate of the present invention, the molar ratio of hydrochloric acid and free alkali compound is 3:1 to 10:1.

In the present invention participate in reaction acid generally 0 DEG C~be added drop-wise to reaction system under room temperature, can be at 0 DEG C or less It is added dropwise.

Unless there are opposite statement, term used in the present invention has following meanings.

Carbon, hydrogen, oxygen, sulphur, nitrogen or halogen involved in group and compound of the present invention include their same position Carbon, hydrogen, oxygen, sulphur, nitrogen or halogen involved in element and group of the present invention and compound are optionally further by one or more Their a corresponding isotopes are substituted, and wherein the isotope of carbon includes¹²C、¹³C and C¹⁴, the isotope of hydrogen includes protium (H), deuterium (D, also known as heavy hydrogen), tritium (T, also known as superheavy hydrogen), the isotope of oxygen include¹⁶O、¹⁷O and¹⁸The isotope of O, sulphur includes³²S 、³³S、³⁴S and³⁶The isotope of S, nitrogen includes¹⁴N and¹⁵N, the isotope of fluorine¹⁹The isotope of F, chlorine includes³⁵Cl and³⁷Cl, bromine it is same Position element include⁷⁹Br and⁸¹Br。

Compound described herein can be containing one or more asymmetric centers, and it is possible thereby to racemate, outer Racemic mixture, single enantiomter, non-enantiomer mixture and single diastereoisomer exist.

The amount for the compound that " effective dose " has guided tissue, system or subject physiologic or medicine to translate, this amount is institute Seek, the one or more of symptoms for being enough to prevent treated illness or illness with subject when including applying occur Or mitigate it to the amount of compound to a certain degree."IC₅₀" refer to half-inhibitory concentration, refer to when reaching maximum suppression effect half Concentration.Numberical range a~b is indicated from several a to the arbitrary number of several b, for example, " m is selected from 1~3 " indicates that m can be selected from 1 to 3 The numerical value of meaning.Various analytical technology analyses known to persons of ordinary skill in the art can be used in crystalline structure of the present invention, including But it is not limited to, X-ray powder diffraction (XRD), differential scanning calorimetry (DSC) and/or thermogravimetry (TG).Thermogravimetric analysis (Thermogravimetric Analysis, TGA), is called thermogravimetry (Thermogravimetry, TG).

Detailed description of the invention

Fig. 1, which is compound 1, uses the alpha-emitting X-ray powder diffraction of Cu-K in 80 DEG C of dry 16 hours resulting crystal form I Map.

Fig. 2 is differential scanning calorimetry (DSC) thermogram of compound 1 in 80 DEG C of dry 16 hours resulting crystal form I.

Fig. 3 is thermogravimetric (TG) curve-micro- entropy thermogravimetric analysis of the compound 1 in 80 DEG C of dry 16 hours resulting crystal form I (DTG) curve.

Fig. 4, which is compound 1, uses the alpha-emitting X-ray powder diffraction figure of Cu-K in 80 DEG C of dry 8 hours resulting crystal form I Spectrum.

Fig. 5, which is compound 1, uses the alpha-emitting X-ray powder diffraction of Cu-K in 60 DEG C of dry 10 hours resulting crystal form I Map.

Fig. 6, which is compound 1, uses the alpha-emitting X-ray powder diffraction figure of Cu-K in 30 DEG C of dry 8 hours resulting crystal form I Spectrum.

Fig. 7 A is molecule ball-and-stick model in the monocrystalline of the compound 1 determined by single crystal diffraction；Fig. 7 B is corresponding with Fig. 7 A The absolute configuration determined by monocrystalline；Fig. 7 C is hydrogen bond schematic diagram in monocrystalline (O2~O4 indicates hydrone in figure).

Fig. 8 is that the crystal form I in embodiment 9 before the recrystallization of compound 1 uses the alpha-emitting X-ray powder diffraction figure of Cu-K Spectrum.

Fig. 9 is that the crystal form in embodiment 9 after the recrystallization of compound 1 uses the alpha-emitting X-ray powder diffraction collection of Cu-K.

Specific embodiment

Below by way of the beneficial effect of the specific embodiment implementation process that the present invention will be described in detail and generation, it is intended to which help is read Reader more fully understands essence and feature of the invention, does not limit the scope of the present invention.

The structure of compound is by nuclear magnetic resonance (NMR) and/or mass spectrum (MS) come what is determined.The measurement of NMR is to use (Bruker ADVANCE III 400) nuclear magnetic resonance spectrometer, measurement solvent are deuterated dimethyl sulfoxide (DMSO-d₆), deuterated chloroform (CDCl₃), deuterated methanol (CD₃OD), inside it is designated as tetramethylsilane (TMS).(Agilent 6120B (ESI)) is used in the measurement of MS. The measurement of HPLC uses Agilent 1260DAD high pressure liquid chromatograph (100 × 4.6mm of Zorba × SB-C18).Thin-layer chromatography Silica gel plate uses Yantai Huanghai Sea HSGF254 or Qingdao GF254 silica gel plate, what the silica gel plate that thin-layered chromatography (TLC) uses used Specification is 0.15mm~0.20mm, and the specification that thin-layer chromatography isolates and purifies product use is 0.4mm~0.5mm.Column chromatography is general It the use of 200~300 mesh silica gel of Yantai Huanghai Sea silica gel is carrier.Without specified otherwise, methyl tertiary butyl ether(MTBE), tetrabutylammonium bromide, hydrogen Change sodium, trifluoroacetic acid purchase in Chengdu Ke Long chemical reagent factory；Two dimethyl dicarbonate butyl esters, N, N'- dicarbapentaborane diimidazole, N, The purchase of O- dimethyl hydroxylamine hydrochloride is in special (Chengdu) the medical science Co., Ltd of Ace；Cesium carbonate, N- hydroxysuccinimide, The purchase of diphenyl methylene glycine ethyl ester is in the resistance to Jilin Chemical of peace；The purchase of 2,5- difluoro bromobenzene is in the silent limited public affairs of medical sci-tech of Shanghai moral Department；Isopropylmagnesium chloride/lithium chloride tetrahydrofuran solution purchase is in lark prestige Science and Technology Ltd.；Propargyl benzene sulfonate, four Butyl ammonium fluoride, three (acetoxyl group) sodium borohydrides, the purchase of tetrabutyl hexafluorophosphoric acid amine are in this Reagent Company of Adama；Ring penta 2 Bis- (triphenylphosphine) ruthenic chloride (II) purchases of alkenyl are in ACROS orgainics；Borane dimethylsulf iotade purchase is in splendid remote chemical section Skill (Shanghai) Co., Ltd.；Sodium perborate purchase recovers fine chemistry industry research institute in Tianjin；Chlorine { [(1R, 2R)-(-) -2- amino - 1,2- diphenyl-ethyl] (phenyl-pentafluoride sulphonyl) amino (p-cymene) ruthenium (II) purchase in Strem chemical；Iodomethane purchase It buys in Chinese medicines group medicine company limited liability company.Nitrogen atmosphere refers to that reaction flask connects the nitrogen balloon of an about 1L volume.Hydrogen Atmosphere refers to that reaction flask connects the hydrogen balloon of an about 2L volume.Hydrogenation usually vacuumizes, and is filled with hydrogen, operates 3 repeatedly It is secondary.Without specified otherwise in embodiment, solution refers to aqueous solution.Without specified otherwise in embodiment, the temperature of reaction is room temperature.Room temperature Temperature is 10 DEG C~30 DEG C.

Intermediate 1:((2R, 3S) -2- (2,5- difluorophenyl) -5- carbonyl tetrahydro -2H- pyrans -3- base) carbamic acid uncle Butyl ester (intermediate 1)

tert-butyl((2R,3S)-2-(2,5-difluorophenyl)-5-oxotetrahydro-2H-pyran-3- yl)carbamate

Step 1: 2- Aminopentyl -4- ynoic acid ethyl esters (1B)

ethyl 2-aminopent-4-ynoate

At room temperature, 1A (50g, 0.187mol) is dissolved in methyl tertiary butyl ether(MTBE) (300mL), propargyl benzene sulfonate is added (44g, 0.224mol) and tetrabutylammonium bromide (6.1g, 0.019mol), is warming up to 50 DEG C, be added cesium carbonate (121.8g, 0.374mol), it is reacted overnight at a temperature of 50 DEG C.Reaction solution is filtered, filter cake is washed with methyl tertiary butyl ether(MTBE) (40mL × 2), Merge organic phase, concentrated by rotary evaporation to half volume is added hydrochloric acid solution (3mol/L, 100mL), stirs 1 hour at room temperature, stands Liquid separation, water phase are extracted with methyl tertiary butyl ether(MTBE) (70mL × 2), are collected water phase, are concentrated to get 1B.

Step 2: 2- ((tertbutyloxycarbonyl) amino) -4- alkynes valeric acid (1C)

2-((tert-butoxycarbonyl)amino)pent-4-ynoic acid

Sodium hydroxide (33.7g, 0.842mol) is dissolved in water (100mL), is added dropwise to 1B (26.4g, 0.187mol) dropwise In, it stirs 2 hours at room temperature, the methyl tertiary butyl ether(MTBE) (125mL) that two dimethyl dicarbonate butyl esters (45g, 0.206mol) are added dropwise is molten Liquid stirs 4 hours at room temperature.Liquid separation is stood, water phase is washed with methyl tertiary butyl ether(MTBE) (80mL × 2), the salt of water phase 3mol/L Acid solution adjusts pH about 3, is extracted with methyl tertiary butyl ether(MTBE) (100mL × 2), merges organic phase, organic phase saturated sodium chloride water Solution (30mL × 2) washing, anhydrous magnesium sulfate are dried, filtered, are spin-dried for, and obtain yellow oily liquid 1C (33g, yield 83%).

Ms m/z(ESI):212.0[M-1]。

Step 3: (1- (methoxyl group (methyl) amino) -1- carbonyl amyl -4- alkynes -2- base) t-butyl carbamate (1D)

tert-butyl(1-(methoxy(methyl)amino)-1-oxopent-4-yn-2-yl)carbamate

1C (33g, 0.155mol) is dissolved in n,N-Dimethylformamide (200mL), temperature is controlled less than 10 DEG C, is added N, N'- carbonyl dimidazoles (32.58g, 0.201mol) react 1 hour at 0 DEG C, and N, O- dimethyl hydroxylamine hydrochloride is added (19.6g, 0.186mol), is stirred overnight at room temperature.It is added dropwise water (150mL), stirs 1 hour, with acetic acid second into reaction solution Ester (100mL × 2) extraction, merges organic phase, and organic phase successively uses saturated sodium bicarbonate solution (60mL × 3), saturated sodium-chloride Solution (60mL × 3) washing, anhydrous magnesium sulfate dries, filters, and is concentrated, residue with silica gel column chromatography separating-purifying (petroleum ether/ Ethyl acetate (v/v)=10:1), obtain white solid 1D (35g, yield 88.2%).

Ms m/z(ESI):156.9[M-100]。

Step 4: (1- (2,5- difluorophenyl) -1- carbonyl amyl -4- alkynes -2- base) t-butyl carbamate (1E)

tert-butyl(1-(2,5-difluorophenyl)-1-oxopent-4-yn-2-yl)carbamate

Under nitrogen protection, 2,5- difluoro bromobenzene (15.05g, 78mmol) is dissolved in dry toluene (50mL), ice salt bath drop Temperature to -10 DEG C hereinafter, isopropylmagnesium chloride/lithium chloride tetrahydrofuran solution (66mL, 1.3mol/L) is added dropwise, be maintained at - 10 DEG C or so are stirred 1 hour, and dry tetrahydrofuran (100mL) solution of 1D (10g, 39mmol) is added dropwise, and are kept for -10 DEG C of temperature, It finishes, reacts 4 hours at room temperature, cool the temperature to -10 DEG C or so, be added dropwise saturated ammonium chloride solution (40mL), stir 10 minutes, pH 5~6 is adjusted with the hydrochloric acid solution of 3mol/L, stands liquid separation, water phase is extracted with methyl tertiary butyl ether(MTBE) (50mL × 2) It takes, merges organic phase, organic phase is washed with saturated sodium chloride solution (30mL × 2), and anhydrous sodium sulfate dries, filters, concentration, residual Object silica gel column chromatography separating-purifying (petrol ether/ethyl acetate (v/v)=50:1~8:1) is stayed, faint yellow solid 1E is obtained (10.1g, yield 83.5%).

Ms m/z(ESI):210.1[M-100]。

Step 5: ((1R, 2S) -1- (2,5- difluorophenyl) -1- Hydroxy pentyl -4- alkynes -2- base) t-butyl carbamate (1F)

tert-butyl((1R,2S)-1-(2,5-difluorophenyl)-1-hydroxypent-4-yn-2-yl) carbamate

1E (16.07g, 52mmol) is dissolved in tetrahydrofuran (100mL), is added triethylene diamine (17.39g, 155mmol) (p-cymene) ruthenium (II) is (i.e. with chlorine { [(1R, 2R)-(-) -2- amino -1,2- diphenyl-ethyl] (phenyl-pentafluoride sulphonyl) amino } RuCl (p-cymene) (R, R)-FSDPEN) (0.37g, 0.52mmol), formic acid (14.27g, 310mmol) is added dropwise, adds Finish, overnight in 40 DEG C of reactions.Reaction solution is concentrated and removes tetrahydrofuran and formic acid, water (60mL) and hydrochloric acid are added into residue (3mol/L, 10mL) is extracted with methyl tertiary butyl ether(MTBE) (90mL × 3), is merged organic phase, organic phase saturated sodium bicarbonate solution (35mL × 2) washing, anhydrous magnesium sulfate dry, filter, and are concentrated, residue silica gel column chromatography separating-purifying (petroleum ether/acetic acid Ethyl ester (v/v)=60:1~10:1), obtain faint yellow jelly 1F (15.37g, yield 95%).

Ms m/z(ESI):334.2[M+23]。

Step 6: ((2R, 3S) -2- (2,5- difluorophenyl) -3,4- dihydro -2H- pyrans -3- base) t-butyl carbamate (1G)

tert-butyl((2R,3S)-2-(2,5-difluorophenyl)-3,4-dihydro-2H-pyran-3-yl) carbamate

It will be dissolved in n,N-Dimethylformamide (75mL) under 1F (15.37g, 49.4mmol) heating condition, the tetrabutyl be added Hexafluorophosphoric acid amine (2.49g, 6.42mmol), N- hydroxysuccinimide (2.84g, 24.75mmol), triphenylphosphine (0.86g, It 3.26mmol) replaces three times, vacuumizes 15 minutes with sodium bicarbonate (2.16g, 25.69mmol), nitrogen, cyclopentadienyl group is added Bis- (triphenylphosphine) ruthenic chloride (II) (i.e. CpRuCl (PPh₃)₂) (1.79g, 2.47mmol), nitrogen is replaced three times, and is vacuumized 15 minutes, under nitrogen protection, it is warming up to 85 DEG C of reactions overnight.Water (300mL) and methyl tertiary butyl ether(MTBE) are added into reaction solution (200mL), is filtered with silica gel, and liquid separation after filtrate stands, water phase is extracted with methyl tertiary butyl ether(MTBE) (90mL × 2), merges organic phase, Organic phase is washed with saturated sodium bicarbonate solution (60mL × 2), and anhydrous sodium sulfate dries, filters concentration, residue silicagel column Chromatographic purification (petrol ether/ethyl acetate (v/v)=80:1~30:1), obtains pale yellow powder solid 1G (8.9g, yield 57.9%).

Ms m/z(ESI):256.2[M-56]。

Step 7: ((2R, 3S) -2- (2,5- difluorophenyl) -5- hydroxy tetrahydro -2H- pyrans -3- base) tertiary fourth of carbamic acid Ester (1H)

tert-butyl((2R,3S)-2-(2,5-difluorophenyl)-5-hydroxytetrahydro-2H- pyran-3-yl)carbam ate

1G (8.9g, 28.6mmol) is dissolved in dry methyl tertiary butyl ether(MTBE) (90mL), is added dry toluene (9mL), Temperature is down to -10 DEG C, and borane dimethylsulf iotade tetrahydrofuran solution (2mol/L, 35.9mL) is added dropwise, reacts 3.5 at 0 DEG C Hour.It is slowly added to water (4mL), is added dropwise sodium hydroxide solution (1mol/L, 89mL), stirs 15 minutes, was added portionwise Boratex (13.2g, 85.8mmol), is stirred overnight at room temperature.Liquid separation is stood, water phase is extracted with methyl tertiary butyl ether(MTBE) (50mL × 2), Merge organic phase, organic phase wash with saturated sodium chloride solution (20mL × 2), and anhydrous sodium sulfate dries, filters, and is concentrated, to residual Addition toluene (50mL) in object is stayed, 90 DEG C of dissolutions is heated to, n-hexane (200mL) is added dropwise in reaction solution, it is solid that white is precipitated Body, filtering, n-hexane (30mL × 2) wash filter cake, and concentration removes solvent, obtains white solid powder 1H (7.9g, yield 84%).

Ms m/z(ESI):274.1[M-56]。

Step 8: ((2R, 3S) -2- (2,5- difluorophenyl) -5- carbonyl-tetrahydro -2H- pyrans -3- base) carbamic acid uncle Butyl ester (intermediate 1)

tert-butyl((2R,3S)-2-(2,5-difluorophenyl)-5-oxo-tetrahydro-2H-pyran- 3-yl)carbamate

1H (11.53g, 35.03mmol) is dissolved in methylene chloride (130mL), is cooled to 0 DEG C, this Martin oxidation will be worn Agent (29.72g, 70.06mmol) adds in reaction solution in batches, is warmed to room temperature reaction 4 hours naturally.It is cooled to 0 DEG C, by saturated carbon Sour hydrogen sodium solution (60mL) is added dropwise in reaction solution, stirs 20 minutes, and filtering, filtrate stands liquid separation, water phase methyl tertbutyl Ether (60mL × 3) extraction, merges organic phase, and organic phase is washed with saturated sodium bicarbonate solution (30mL × 2), and anhydrous sodium sulfate is dry Dry, filtering and concentrating, residue is obtained white with silica gel column chromatography separating-purifying (petrol ether/ethyl acetate (v/v)=10:1-4:1) Color crystalline powder intermediate 1 (10.85g, yield 94.7%).

Ms m/z(ESI):272.0[M-56]；

¹H NMR(400MHz,DMSO-d₆):δ7.29-7.13(m,4H),4.77–4.75(d,1H),4.22-4.12(d, 2H),4.08-4.02(m,1H),2.75-2.70(m,2H),1.23(s,9H)。

Embodiment 1

(2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) pyrrolo- [3,4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine dihydrochloride hydrate (compound 1)

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl-1H- tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- amine dihydrochloride hydrate

Step 1: 2- ethyoxyl -2- ethyl acetimidate hydrochloride (1b)

ethyl 2-ethoxy-2-iminoacetate hydrochloride

Cyanoformic ester 1a (9.9g, 0.1moL) is dissolved in dry ether (50mL), and is added dry anhydrous Ethyl alcohol (5.52g, 0.12moL) is passed through dry hydrogen chloride gas under ice bath stirring until saturation (system weight gain 5g) and continuing Stirring 2 hours, filters the white solid of precipitation, and filter cake is washed with anhydrous ether (20mL × 3), and it is solid to obtain white for vacuum drying Body 1b (14g, yield 77%).

Step 2: 5- tert-butyl 2- ethyl 3a, 4,6,6a- nafoxidine simultaneously [3,4-d] imidazoles -2,5 (1H)-dicarboxylic acid esters (1c)

5-tert-butyl 2-ethyl 3a,4,6,6a-tetrahydropyrrolo[3,4-d]imidazole-2,5 (1H)-dicarboxy-late

Bis- amido pyrrolidines -1- carboxylate hydrochloride (980mg, 4.126mmoL) of tert-butyl 3,4- is dissolved in hexafluoro isopropyl It in alcohol (10mL), is added with stirring 1b (886mg, 4.538mmoL), 50 DEG C are stirred 16 hours.Reaction solution concentration is spin-dried for, is added Saturated salt solution (50mL) adjusts pH value to 2~3 with dilute hydrochloric acid, is extracted with EtOAc (50mL × 2).Water phase is collected, is used in combination Saturated sodium bicarbonate solution tune pH value 7~8 is extracted with EtOAc (50mL × 4), merges organic phase, and anhydrous sodium sulfate is dry, Filtering, concentration, column chromatographic isolation and purification (methylene chloride/methanol (v/v)=20:1) obtain white solid 1c (700mg, yield 60%).

MS m/z(ESI):284.1[M+1]；

¹H NMR(400MHz,CDCl₃):δ4.64(S,2H),4.37-4.33(q,2H),3.72-3.69(d,2H),3.55- 3.50(dd,2H),1.44(s,9H),1.40-1.37(t,3H)。

Step 3: 5- tert-butyl 2- ethyl 4,6- pyrrolin simultaneously [3,4-d] imidazoles -2,5 (1H)-dicarboxylic acid esters (1d)

(2R,4R)-tert-butyl 4-(tert-butyldimethylsilyloxy)-2-(methoxymethyl) pyrrolidine-1-carboxylate

Oxalyl chloride (652.5mg, 5.14mmoL) is dissolved in dry methylene chloride (15mL), is cooled down with dry ice acetone bath To -78 DEG C, the lower dimethyl sulfoxide (803.96mg, 10.29mmoL) that drying is added dropwise is stirred, is stirred 30 minutes.Into reaction solution Methylene chloride (5mL) solution of 1c (970mg, 3.43mmoL) is added dropwise, continues stirring 20 minutes.Diisopropyl is added dropwise at -78 DEG C Ethamine (2.216g, 17.15mmoL) is raised to room temperature reaction 2 hours naturally.Saturated ammonium chloride solution is added into reaction solution (50mL), saturated sodium chloride solution (50mL) and methylene chloride (50mL), liquid separation, water phase are extracted with methylene chloride with (50mL × 3) It takes, merges organic phase, saturated sodium chloride solution (50mL × 3) washing, anhydrous sodium sulfate is dried, filtered, is concentrated, column chromatography for separation Purifying (petrol ether/ethyl acetate=1:1) obtains white solid 1d (520mg, yield 54%).

MS m/z(ESI):282.1[M+1]；

¹H NMR(400MHz,CDCl₃):δ4.54(s,2H),4.47(s,2H),4.46-4.41(q,2H),1.51(s, 9H),1.44-1.41(t,3H)。

Step 4: 5- tert-butyl 2- ethyl 1- methyl -4,6- pyrrolin simultaneously [3,4-d] imidazoles -2,5 (1H)-dioctyl phthalate Ester (1e)

5-tert-butyl 2-ethyl 1-methyl-4,6-dihydropyrrolo[3,4-d]imidazole-2,5 (1H)-dicarboxylate

1d (5.4g, 19.2mmoL) is dissolved in n,N-Dimethylformamide (110mL), addition potassium carbonate (3.3g, 23.9mmoL), iodomethane (3.4g, 23.9mmoL) is added under ice bath, reacts at room temperature 2 hours, saturation chlorine is added into reaction solution Change ammonium salt solution (300mL) and saturated sodium chloride solution (200mL), extracted with ethyl acetate (200mL × 3), merges organic phase, It is washed with saturated sodium chloride solution (200mL × 3).It is dried, filtered with anhydrous sodium sulfate, filtrate decompression is concentrated, obtained pale yellow Color solid 1e (5.3g, yield 94%).

Step 5: tert-butyl 2- carbamyl -1- methyl -4,6- pyrrolin simultaneously [3,4-d] imidazoles -5 (1H)-formic acid esters (1f)

tert-butyl 2-carbamoyl-1-methyl-4,6-dihydropyrrolo[3,4-d]imidazole-5 (1H)-carboxy-late

By 1e (2.3g, 7.8mmol), it is dissolved in ammonia-methanol solution (40mL, 7mol/L, 280mmol), is warming up to 85 DEG C Tube sealing is stirred to react 20 hours.Reaction solution is cooled to room temperature, is filtered, filter cake is washed with ethyl acetate (10mL × 3), and filtrate is dense Merge after contracting with filter cake, obtains white solid 1f (1.914g, yield 93%).

Step 6: tert-butyl 2- cyano -1- methyl -4,6- pyrrolin simultaneously [3,4-d] imidazoles -5 (1H)-formic acid esters (1g)

tert-butyl 2-cyano-1-methyl-4,6-dihydropyrrolo[3,4-d]imidazole-5(1H)- carboxylate

It is dissolved in 1f (1.576g, 5.925mmol) in n,N-Dimethylformamide (45mL), phosphorus pentachloride is added (1.482g, 7.109mmol) is warming up to 50 DEG C and reacts 2 hours.Reaction solution is cooled to room temperature, is added ethyl acetate (50mL), Saturated sodium chloride solution (150mL), liquid separation, water phase are extracted with ethyl acetate (50mL × 3), merge organic phase, respectively with saturation Saline solution (50mL × 3), saturated sodium bicarbonate solution (50mL × 2) washing, anhydrous sodium sulfate dry, filter, filtrate are concentrated Obtain white solid 1g (1.25g, yield 85%).

Step 7: tert-butyl 1- methyl -2- (1H-TETRAZOLE -5- base) -4,6- pyrrolin simultaneously [3,4-d] imidazoles -5 (1H) - Formic acid esters (1h)

tert-butyl 1-methyl-2-(1H-tetrazol-5-yl)-4,6-dihydropyrrolo[3,4-d] imidazole-5(1H)-carboxylate

1g (1030mg, 4.153mmoL) is dissolved in n,N-Dimethylformamide (20mL), sodium azide is added (810mg, 12.459mmoL) and ammonium acetate (960mg, 12.459mmoL) is warming up to 120 DEG C of reactions overnight.1mol/L salt is added Acid solution adjusts pH value 1~2, adds water (200mL), filters, and filtrate is extracted with methylene chloride (50mL × 3).Merge organic phase, uses Anhydrous sodium sulfate dries, filters, and is spin-dried for, and drying obtains yellow solid 1h (1000mg, yield 83%).

MS m/z(ESI):292.1[M+1]。

Step 8: tert-butyl 1- methyl -2- (2- methyl -2H- tetrazolium -5- base) -4,6- pyrrolin simultaneously [3,4-d] miaow Azoles -5 (1H)-formic acid esters (1i-1)

tert-butyl 1-methyl-2-(2-methyl-2H-tetrazol-5-yl)-4,6-dihydropyrrolo [3,4-d]imidazole-5(1H)-carboxylate

Tert-butyl 1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) -4,6- pyrrolin simultaneously [3,4-d] imidazoles -5 (1H) - Formic acid esters (1i-2)

tert-butyl 1-methyl-2-(1-methyl-1H-tetrazol-5-yl)-4,6-dihydropyrrolo [3,4-d]imidazole-5(1H)-carboxylate

1h (1000mg, 3.436mmoL) is dissolved in n,N-Dimethylformamide (25mL), sodium hydride is added under ice bath Iodomethane (586mg, 4.124mmoL) is added after stirring 0.5 hour under ice bath, natural temperature reaction in (165mg, 4.124mmoL) Overnight.Reaction solution is slowly added in trash ice (50g), sodium chloride, which is added, is saturated solution, ethyl acetate (50mL × 4) extraction. Merge organic phase, washed with saturated sodium chloride solution (50mL × 3), dried, filtered, be spin-dried for anhydrous sodium sulfate, residue is used Silica gel column chromatography separating purification (petrol ether/ethyl acetate (v/v)=2:1~1:1), obtaining white solid 1i-1, (682mg is produced Rate 55%) and white solid 1i-2 (230mg, yield 18%).

MS m/z(ESI):306.1[M+1]。

1i-1:1H NMR (400MHz, DMSO): δ 4.58-4.54 (d, 2H), 4.39-4.37 (d, 2H), 4.33 (s, 3H), 3.99 (s, 3H), 1.47 (s, 9H).

1i-2:1H NMR (400MHz, DMSO): δ 4.54-4.50 (d, 2H), 4.44 (s, 3H), 4.34-4.31 (d, 2H), 3.92-3.91 (m, 3H), 1.47 (s, 9H).

Step 9: 1- methyl -2- (1- methyl-1 H- tetrazole radical -5- base) -1,4,5,6- nafoxidine simultaneously [3,4-d] imidazoles Diphenyl sulfonate (1j)

1-methyl-2-(1-methyl-1H-tetrazol-5-yl)-1,4,5,6-tetrahydropyrrolo[3,4- d]imidazole dibenzenesulfonate

By 1i-1 (682mg, 2.236mmoL), benzene sulfonic acid .1.5H₂O (827.3mg, 4.472mmoL) is dissolved in methylene chloride In (25mL), it is warming up to 40 DEG C of reactions overnight.Reaction solution concentration is spin-dried for, vacuum drying obtains 1j (1165mg), under directly using Single step reaction.

MS m/z(ESI):206.1[M+1]。

Step 10: tert-butyl ((2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetra- Azoles -5- base) pyrrolo- [3,4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- base) carbamate (1k)

tert-butyl((2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl- 1H-tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- yl)carbamate

1j (1165mg, 2.236mmoL) and intermediate 1 (804.4mg, 2.459mmoL) are dissolved in N, N- dimethylacetamide In amine (10mL), at room temperature stir 0.5 hour, under ice bath be added three (acetoxyl group) sodium borohydrides (1279mg, 6.037mmoL), 0 DEG C stirring 0.5 hour after be warmed to room temperature naturally stirring 2 hours.Add intermediate 1 (400mg, 1.223mmoL) And three (acetoxyl group) sodium borohydrides (400mg, 1.887mmoL), it reacts at room temperature 2 hours.Unsaturated carbonate hydrogen is added in reaction solution It in sodium solution (100mL), stirs 0.5 hour, filtering, filter cake is molten with methylene chloride (100mL) after being washed with water (20mL × 3) Solution, dried, filtered, be spin-dried for anhydrous sodium sulfate, residue with silica gel column chromatography separating purification (methylene chloride/methanol (v/v)= 60:1), the crude product obtained is dissolved with ether (50mL), is added n-hexane (100mL), and filtering obtains yellow solid 1k (480mg, yield 42%).

MS m/z(ESI):517.1[M+1]。

Step 11: (2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- Base) pyrrolo- [3,4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine (1l)

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl-1H- tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- amine

1k (440mg, 0.853mmoL) is added in methylene chloride (5mL), trifluoroacetic acid (2mL) is added under ice bath, it is natural It is warmed to room temperature reaction 2 hours.Reaction solution is spin-dried for, saturated sodium bicarbonate solution (100mL) is added and adjusts pH value 7~8, water phase is used Methylene chloride (30mL × 4) extraction.Merge organic phase, is dried, filtered, be spin-dried for anhydrous sodium sulfate, residue silica gel column layer Analysis isolates and purifies (methylene chloride/methanol (v/v)=30:1, a small amount of ammonium hydroxide is added), obtains yellow solid 1l (134mg, yield 38%).

MS m/z(ESI):417.1[M+1]；

¹H NMR(400MHz,CD₃OD):δ7.23-7.06(m,3H),4.38(s,3H),4.32-4.26(m,2H),4.07- 4.05(t,2H),4.03(s,3H),3.95-3.94(t,2H),3.46-3.41(t,1H),3.17-3.09(m,1H),2.96- 2.90(m,1H),2.51-2.47(m,1H),1.58-1.49(q,1H)。

Step 12: (2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- Base) pyrrolo- [3,4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine dihydrochloride hydrate (compound 1)

1l (16g, 38.43mmol) and dehydrated alcohol (80mL) sequentially add in reaction flask, and concentrated hydrochloric acid (8mL) is added drop-wise to It in reaction solution, finishes and is stirred at room temperature 10 minutes, completely, it is solid that shallow white is precipitated in stirring after ten minutes for solid dissolution in reaction solution Body.Isopropyl acetate (56mL) is added drop-wise in reaction solution, room temperature continues stirring 1 hour.Reaction solution is filtered, filter cake acetic acid Isopropyl ester (30mL × 2) washing, is drained, and is dried in vacuo, is obtained pale solid compound 1 (14g, yield 85%).Fig. 2 is institute Compound 1 is obtained in differential scanning calorimetry (DSC) thermogram of 80 DEG C of dry 16 hours resulting crystal form I.Fig. 3 is compound 1 In micro- entropy thermogravimetric analysis (DTG) curve of thermogravimetric (TG) curve-of 80 DEG C of dry 16 hours resulting crystal form I.

It is 2 that compound 1, which measures HCl number contained by compound 1 by single crystal diffraction,.

Single crystal cultivation: taking about 5mg sample to be put in vial, and with 300 μ L EtOH Sonicate dissolved clarifications, aperture is waved at room temperature Hair, obtains small bulk crystals, adds in the alcohol saturated solution of sample using this crystal as crystal seed, and aperture volatilization obtains bulk Crystal.Single crystal diffraction instrument information and detection method parameter are as shown in table 1:

Table 1

Thermal station polarization light microscope information and detection method parameter are as shown in table 2:

Table 2

Gained cell parameter is as shown in table 3 below:

3 mono-crystalline structures information table of table

Fig. 7 A is molecule ball-and-stick model in the monocrystalline of the compound 1 determined by single crystal diffraction；Fig. 7 B is corresponding with Fig. 7 A The absolute configuration determined by monocrystalline；Fig. 7 C is hydrogen bond schematic diagram (in figure O2~O4 indicate hydrone) in monocrystalline, from Fig. 7 A~ 7C is it is found that an API molecule combines 3 hydrones, wherein the hydrone containing O2 is connect with API molecule by hydrogen bond, relatively surely It is fixed, it is the crystallization water；Two hydrones containing O3 and O4 are in free state, are easy to lose, but do not influence on structure cell character.

Embodiment 2

At room temperature, 1k (133g, 0.257mol) and ethyl alcohol (864.5mL) sequentially add in reaction flask, by concentrated hydrochloric acid (146.3mL) is added drop-wise in reaction solution, is finished and is reacted 3 hours in 60 DEG C~70 DEG C.Isopropyl acetate (296.2mL) is added drop-wise to In reaction solution, room temperature is naturally cooled to, shallow white solid is precipitated.Filtering, filter cake are washed with isopropyl acetate (300mL × 2), are taken out Dry, vacuum drying obtains pale solid compound 1 (102g, yield 80%).

Compound 1 is because of vacuum drying temperature, time difference in embodiment 1 or embodiment 2, contained by obtained hydrate Hydrone content is also different, that is, the hydrone number for including is different, and m value is different, is shown in Table 4.

Water content of 4 compound 1 of table after different temperatures and drying time are dry

Fig. 1 show the X- powder diagram of 80 DEG C of drying temperatures, dry 16 hours, Fig. 4 show 80 DEG C of drying temperatures, Dry 8 hours X- powder diagrams, Fig. 5 show the X- powder diagram of 60 DEG C of drying temperatures, dry 10 hours, Fig. 6 institute It is shown as the X- powder diagram of 30 DEG C of drying temperatures, dry 8 hours.Although it follows that the water in embodiment 1 and embodiment 2 Molecule content difference due to vacuum drying temperature and time is different, but the figure of its X- powder diffraction is almost the same, therefore this hair Bright (2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (the 1- methyl-1 H- tetra- thought containing 1~3 mole of water Azoles -5- base) pyrrolo- [3,4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine dihydrochloride all obtains I type knot It is brilliant.

Embodiment 3

(2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) pyrroles [3, 4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine-tosilate (compound 2)

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl-1H- tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- amine4-methylbenzenesulfonate

At room temperature, 1l (2.0g, 4.8mmol) is dissolved in ethyl acetate (60mL), reaction flask is added, by p-methyl benzenesulfonic acid Monohydrate (0.912g, 4.8mmol) is dissolved in ethyl acetate (20mL), is added drop-wise in above-mentioned reaction solution, room temperature is added dropwise Lower reaction 2 hours.Reaction solution has white solid precipitation, filters, filter cake ethyl acetate (10mL × 3), methyl tertiary butyl ether(MTBE) (10mL) washing, vacuum drying obtain white solid product compound 2 (2.5g, yield 88.6%, HPLC purity 99.5%).

¹H NMR(400MHz,DMSO-d₆):δ8.01(s,3H),7.49(d,2H),7.38-7.33(m,3H),7.12(d, 2H),4.55(d,1H),4.34(s,3H),4.21(m,1H),4.06-3.99(m,4H),3.87(m,2H),3.60(m,1H), 3.41(m,2H),3.11(m,1H),2.50(m,1H),2.29(s,3H),1.72(m,1H)。

Embodiment 4

(2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) pyrroles [3, 4-d] imidazoles -5 (1H, 4H, 6H)-yl)-two tosilate (compound 3) of tetrahydro -2H- pyrans -3- amine

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl-1H- tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- amine bis(4-methylbenzenesulfonate)

At room temperature, 1l (2.0g, 4.8mmol) is dissolved in ethyl acetate (60mL), reaction flask is added, by p-methyl benzenesulfonic acid Monohydrate (1.824g, 9.6mmol) is dissolved in ethyl acetate (20mL), is added drop-wise to above-mentioned reaction solution, is added dropwise at room temperature Reaction 2 hours.Reaction solution has white solid precipitation, filters, filter cake ethyl acetate (10mL × 3), methyl tertiary butyl ether(MTBE) (10mL) washing, vacuum drying obtain white solid product compound 3 (3.1g, yield 85%, HPLC purity 99.63%).

¹H NMR(400MHz,DMSO-d₆):δ8.19(s,3H),7.49-7.47(m,4H),7.38-7.35(m,3H), 7.11(d,4H),4.75-4.52(m,5H),4.41-4.34(m,4H),4.03(m,4H),3.67-3.62(m,2H),2.70(m, 1H),2.27(s,6H),2.00(m,1H)。

Embodiment 5

(2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) pyrroles [3, 4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine-phosphate (compound 4)

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl-1H- tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- amine phosphate

At room temperature, 1l (2.5g, 6mmol) is dissolved in ethyl acetate (80mL), be added reaction flask, by phosphoric acid (0.693g, 6mmol, mass fraction 85%) it is dissolved in ethyl acetate (20mL), it is added drop-wise to above-mentioned reaction solution, is added dropwise and reacts at room temperature 2 hours.There is white solid precipitation in reaction solution, filters, filter cake ethyl acetate (10mL × 3), methyl tertiary butyl ether(MTBE) (10mL) Washing, vacuum drying, obtains white solid product compound 4 (1.6g, yield 52%, HPLC purity 99.53%).

¹H NMR(400MHz,DMSO-d₆):δ7.78(brs,5H),7.37-7.28(m,3H),4.50-4.37(m,1H), 4.34(s,3H),4.19-4.16(m,1H),4.04-3.98(m,5H),3.86(m,2H),3.41-3.36(m,2H),3.10- 3.05(m,1H),2.54(m,1H),1.70-1.67(m,1H)。

Embodiment 6

(2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) pyrroles [3, 4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine-benzoate (compound 5)

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl-1H- tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- amine benzoate

At room temperature, 1l (2.5g, 6mmol) is dissolved in ethyl acetate (80mL), reaction flask is added, by benzoic acid (0.733g, 6mmol) is dissolved in ethyl acetate (20mL), is added drop-wise to above-mentioned reaction solution, is added dropwise and is reacted 2 hours at room temperature. Reaction solution has white solid precipitation, filters, and filter cake ethyl acetate (10mL × 3), methyl tertiary butyl ether(MTBE) (10mL) wash, vacuum It is dry, obtain white solid product compound 5 (2.1g, yield 62%, HPLC purity 99.76%).

¹H NMR(400MHz,DMSO-d₆):δ7.95(d,2H),7.60(d,1H),7.49(t,2H),7.27-7.20(m, 3H),4.34(s,3H),4.19-4.14(m,2H),4.03-3.98(m,5H),3.84(m,2H),3.30(t,1H),3.01- 2.98(m,2H),2.35(d,1H),1.49-1.40(m,1H)。

Embodiment 7

(2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) pyrroles [3, 4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine-citrate (compound 6)

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl-1H- tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- amine2-hydroxypropane-1,2,3-tricarboxylate

At room temperature, 1l (2.5g, 6mmol) is dissolved in ethyl acetate (80mL), reaction flask is added, by citric acid (1.152g, 6mmol) is dissolved in ethyl alcohol (20mL), is added drop-wise to above-mentioned reaction solution, is added dropwise and is reacted 2 hours at room temperature.Reaction There is white solid precipitation in liquid, filter, filter cake ethyl acetate (10mL × 3), methyl tertiary butyl ether(MTBE) (10mL) wash, and vacuum is dry It is dry, obtain white solid product compound 6 (3.2g, yield 87.7%, HPLC purity 99.47%).

¹H NMR(400MHz,MeOD):δ7.36-7.20(m,3H),4.67-4.65(m,1H),4.39-4.35(m,4H), 4.15-4.09(m,2H),4.05-4.00(m,5H),3.59-3.54(m,2H),3.30-3.27(m,1H),2.86-2.72(m, 4H),2.72-2.65(m,1H),1.87-1.81(m,1H)。

Embodiment 8

(2R, 3S, 5R) -2- (2,5- difluorophenyl) -5- (1- methyl -2- (1- methyl-1 H- tetrazolium -5- base) pyrroles [3, 4-d] imidazoles -5 (1H, 4H, 6H)-yl) tetrahydro -2H- pyrans -3- amine-fumarate (compound 7)

(2R,3S,5R)-2-(2,5-difluorophenyl)-5-(1-methyl-2-(1-methyl-1H- tetrazol-5-yl)pyrrolo[3,4-d]imidazol-5(1H,4H,6H)-yl)tetrahydro-2H-pyran-3- amine fumarate

At room temperature, 1l (2.5g, 6mmol) is dissolved in ethyl acetate (80mL), reaction flask is added, by fumaric acid (0.696g, 6mmol) is dissolved in methanol (20mL), is added drop-wise to above-mentioned reaction solution, is added dropwise and is reacted 2 hours at room temperature.Reaction There is white solid precipitation in liquid, filter, filter cake ethyl acetate (10mL × 3), methyl tertiary butyl ether(MTBE) (10mL) wash, and vacuum is dry It is dry, obtain white solid product compound 7 (2.3g, yield 72%, HPLC purity 99.62%).

¹H NMR(400MHz,DMSO-d₆):δ7.31-7.23(m,3H),6.54(s,2H),4.34(m,4H),4.18- 4.15(m,1H),3.99-3.98(m,5H),3.85(m,2H),3.33(t,1H),3.17-3.12(m,1H),3.05-3.00(m, 1H),2.45(m,1H),1.60-1.52(m,1H)。

9 compound 1 of embodiment recrystallization

Compound 1 (53g, 0.108mol), deionized water (53mL) and dehydrated alcohol (106mL) are sequentially added into reaction flask In, it finishes and is warming up to 80 DEG C, completely to solid dissolution, be added active carbon (5.3g), finish, stir 30 minutes at 80 DEG C, while hot Filtering, filter cake are washed with hot dehydrated alcohol (26.5mL).Isopropyl acetate (344.5mL) is added drop-wise in filtrate by merging filtrate, White solid is precipitated in cooled to room temperature, continues stirring 3 hours, filtering, and filter cake is washed with isopropyl acetate (60mL × 2), It drains, is dried in vacuo (80 DEG C, 16 hours), the compound 1 (45g, yield 84.9%) after being refined.Compound after recrystallization No change has taken place for 1 crystal form, consistent with crystal form is compared by XRD spectrum after recrystallization before recrystallization.XRD spectrum is before recrystallizing Fig. 8.XRD spectrum is Fig. 9 before recrystallizing.

10 solubility studies of embodiment

Sample: compound 1 (being obtained by 1 condition of serial number in table 4), compound 1l.

Experiment: it takes 2mL physiological saline and 2mL 0.5%CMC-Na solution to be separately added into BD centrifuge tube, is gradually added Sample, it is each that heating and strength shaking or ultrasound in 37 DEG C of water-baths is added after sample.Solution becomes after a certain amount of sample to be added Muddiness, is heated 30 minutes in 37 DEG C of water-baths and strength shakes or ultrasound can not make turbid phenomenon disappear.Experimental result is shown in Table 5。

5 solubility of table

Conclusion: compound 1 is significant relative to solubility of the compound 1l in physiological saline and 0.5%CMC-Na solution It improves.

11 stability study of embodiment

Sample: compound 1l, compound 1, compound 2, compound 3, compound 4, compound 5, compound 6 and compound 7。

Experiment: compound 1l, compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, chemical combination are taken Object 7 carries out under the conditions of high temperature (40 DEG C and 60 DEG C), high humidity (RH75% and RH92.50%), illumination (5500 ± 500lx) respectively Test, 0 day, 10 days, 12 days, 14 days, 20 days or 30 days HPLC detect purity (percentage expression), testing conditions and experiment knot Fruit is shown in Table 6~9.

HPLC detects purity conditions are as follows:

Chromatographic column: Xselect CSH C18 (4.6 × 150mm, 3.5um)；Column temperature: 35 DEG C；Detection wavelength: 275nm；Stream Dynamic phase: gradient elution is carried out by table 3；Flow velocity 1.0mL/min；Wherein A phase is 10mmol/L dipotassium hydrogen phosphate (pH=10.95), B It is mutually methanol.

Table 6HPLC mobile phase

T(min)	0.01	4.00	14.00	20.00	26.00	30.00	30.01	37.00
									A (%)	70	70	40	40	10	10	70	70
B (%)	30	30	60	60	90	90	30	30

The stability contrast of 7 compound 1 and compound 1l of table

The stability of 8 compound 1 of table

The stability of 9 compound 2-7 of table

Conclusion: compound 1 is no matter under high temperature, high humidity or illumination condition, and purity does not change substantially after 10 days, compares Its free alkali compound 1l, stability significantly improve.Meanwhile compound 1 is under high temperature, high humidity or illumination condition, 12 days or Purity does not change substantially after 30 days, has good stability.No matter compound 2, compound 3 are in high temperature, high humidity or illumination condition Under, purity does not change substantially after 14 days or 20 days, has good stability.

The external enzyme activity determination of embodiment 12DPP-IV

DPP-IV using the zymetology reaction assay the compounds of this invention of recombined human DPP-IV and H-Ala-Pro-AFC is external Enzyme activity.Buffer, to be measured is prepared according to DPP-IV Fluorescent ActivityAssay Kit (BPS Bioscience) Sample working solution, DPP-IV enzyme dilution and AFC substrate dilution.

Prepare 96 orifice plates, 80 μ L buffers are first added in every hole, and 5 μ L DPP-AFC- substrates are added later.It adds different dense 5 μ L buffers are added to test sample working solution, every 5 μ L of hole, blank group in degree.10 μ L DPP-IV finally are added in test group control 10 μ L buffers are added in blank control group in enzyme.Statistical analysis is carried out to data with 7.5 software of Origin, obtains each test The IC of compound₅₀Value, experimental result are shown in Table 10.

The external enzyme activity measurement result of table 10DPP-IV

Serial number	Compound number	IC₅₀(nM)
			1	Compound 1	1.37
2	Compound 2	2.02
			3	Compound 3	2.11
4	Compound 4	2.22
			5	Compound 5	1.80
6	Compound 6	1.57
			7	Compound 7	1.90

Conclusion: the compounds of this invention has the inhibitory activity of apparent DPP-IV enzyme.

Claims

1. a kind of salt of compound shown in formula (II) comprising formula (II) structure:

Wherein,

HA be selected from hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, malonic acid, oxalic acid, adipic acid, trifluoroacetic acid, succinic acid, salicylic acid, Benzoic acid, phthalic acid, lactic acid, tartaric acid, malic acid, benzene sulfonic acid, methanesulfonic acid, p-methyl benzenesulfonic acid, citric acid or fumaric acid；

N is selected from 1 or 2；

M is selected from 0.

2. salt according to claim 1, wherein

When HA is selected from hydrochloric acid, phosphoric acid or p-methyl benzenesulfonic acid, n is selected from 1 or 2；

When HA is selected from benzoic acid, citric acid or fumaric acid, n is selected from 1.

3. a kind of formula (III) compound represented:

Wherein, m is selected from 1~3.

4. compound according to claim 3, wherein compound shown in formula (III) is crystal form I, is radiated using Cu-K α, Its X-ray powder diffraction collection the position following 2 θ have characteristic diffraction peak: 6.7 ° ± 0.2 °, 13.8 ° ± 0.2 °, 14.5 ° ± 0.2 °, 24.2 ° ± 0.2 ° and 27.9 ° ± 0.2 °.

5. compound according to claim 4, wherein formula (III) compound represented is crystal form I, uses Cu-K α spoke Penetrate, X-ray powder diffraction collection also the position following 2 θ have characteristic diffraction peak: 14.0 ° ± 0.2 °, 16.5 ° ± 0.2 °, 17.8 ° ± 0.2 °, 19.3 ° ± 0.2 °, 20.3 ° ± 0.2 ° and 21.0 ° ± 0.2 °.

6. compound according to claim 5, wherein formula (III) compound represented is crystal form I, uses Cu-K α spoke Penetrate, X-ray powder diffraction collection also the position following 2 θ have characteristic diffraction peak: 13.4 ° ± 0.2 °, 15.4 ° ± 0.2 °, 22.7 ° ± 0.2 °, 24.5 ° ± 0.2 ° and 25.0 ° ± 0.2 °.

7. compound according to claim 6, wherein the X-ray powder diffraction collection of the crystal form I is basic such as Fig. 1 institute Show.

8. the compound according to any one of claim 3~7, wherein the differential scanning calorimetry heat analysis of the crystal form I Scheme substantially as shown in Figure 2.

9. the compound according to any one of claim 3~7, wherein the thermal gravimetric analysis curve of the crystal form I is substantially such as Shown in Fig. 3.

10. compound according to claim 3, wherein m=3.

11. a kind of prepare the method such as salt according to any one of claims 1 to 2, this method comprises:

Formula (IV) compound reacts to obtain formula (II) with HA；

Alternatively,

Formula (V) compound reacts to obtain formula (II) with HA；

Wherein,

N is selected from 1 or 2；

M is selected from 0；

P is amino protecting group.

12. according to the method for claim 11, wherein HA is selected from hydrochloric acid, phosphoric acid, benzoic acid, p-methyl benzenesulfonic acid, citric acid Or fumaric acid.

13. according to the method for claim 12, wherein HA is the hydrochloric acid that mass fraction is 15%~37%.

14. a kind of pharmaceutical composition, it includes the salt according to any one of claims 1 to 2 or right of therapeutically effective amount to want Compound described in asking any one of 3~10 and pharmaceutically acceptable carrier or excipient.

15. compound described in any one of salt according to any one of claims 1 to 2 or claim 3~10, or power Benefit require 14 described in pharmaceutical composition preparing the drug for preventing and/or treating diabetes and/or diabetic complication In application.

16. application according to claim 15, wherein the diabetic complication includes diabetic retinopathy, sugar Urinate one of characteristic of disease neuropathy and nephrosis or a variety of.

17. application according to claim 15, wherein the diabetes are type-2 diabetes mellitus.