WO2016037552A1 - 结晶型ARB-NEPi复合物及其制备方法和应用 - Google Patents

结晶型ARB-NEPi复合物及其制备方法和应用 Download PDF

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WO2016037552A1
WO2016037552A1 PCT/CN2015/089051 CN2015089051W WO2016037552A1 WO 2016037552 A1 WO2016037552 A1 WO 2016037552A1 CN 2015089051 W CN2015089051 W CN 2015089051W WO 2016037552 A1 WO2016037552 A1 WO 2016037552A1
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biphenyl
ylmethyl
methyl
tetrazol
amino
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PCT/CN2015/089051
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English (en)
French (fr)
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李响
申志祥
张磊
徐耀昌
吕爱锋
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上海翰森生物医药科技有限公司
江苏豪森药业股份有限公司
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Priority to CN201580030123.2A priority Critical patent/CN106414416B/zh
Publication of WO2016037552A1 publication Critical patent/WO2016037552A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/51—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D257/04—Five-membered rings

Definitions

  • the invention belongs to the technical field of medicines, in particular, the invention relates to a dual-effect ARB-NEPi complex new crystal form and a preparation method and application thereof.
  • Neutral endopeptidase (EC 3.4.24.11; enkephalinase; peptidase; NEP) is a zinc-containing metalloproteinase that cleaves various peptide substrates at the amino terminus of an aromatic amino acid.
  • Substrates of such enzymes include, but are not limited to, atrial natriuretic peptide (ANF, also known as ANP), brain natriuretic peptide (BNP), met and leu enkephalin, bradykinin, neurokinin A, and substance P.
  • ANF is a family of vasodilatation, diuretic and antihypertensive peptides, one form - ANF 99-126 is a circulating peptide hormone released by the heart during heart expansion.
  • the function of ANF is to maintain the homeostasis of salt and water and regulate blood pressure.
  • ANF is rapidly inactivated by at least two processes in the circulation: receptor-mediated clearance and enzyme inactivation at NEP.
  • NEP inhibitors enhance hypotension, diuresis, natriuresis, and plasma ANF response in experimental animals following pharmacological ANF injection.
  • the enhancement of ANF by two specific NEP inhibitors is generally disclosed in U.S. Patent No. 4,749, 688, which is incorporated herein by reference. In the same year, U.S.
  • Patent 4,740,499 discloses that thiorphan and ceratophene can also be used to enhance the application of atrial peptides.
  • NEP inhibitors can lower blood pressure and exert ANF-like effects such as diuresis in some forms of experimental hypertension and increase in the excretion of cyclic guanosine 3', 5'-monophosphate (cGMP). Since the antibodies to ANF will counteract the decrease in blood pressure, the antihypertensive effect of NEP inhibitors is mediated through ANF. Long-term and uncontrolled hypertensive vascular disease will eventually lead to various pathological changes in target organs such as heart and kidney. Sustained high blood pressure can also increase the incidence of stroke. Therefore, there is a strong need to evaluate the efficacy of antihypertensive treatment by examining other cardiovascular endpoints other than blood pressure reduction to further discover the benefits of combination therapy.
  • Angiotensin II is a hormone that causes vasoconstriction, which in turn causes hypertension and heart strain. Angiotensin II is known to interact with receptors on the surface of target cells. Two receptor subtypes of angiotensin II have been identified, known as AT1 and AT2. Recently, great efforts have been made to identify substances that bind to the AT1 receptor. It is now known that angiotensin receptor blockers (ARBs, angiotensin II antagonists) are capable of lowering blood pressure by preventing angiotensin II from binding to its receptor on the vessel wall. Because of their ability to inhibit the AT1 receptor, such antagonists can be used to combat hypertension, or to treat congestive heart failure and other indications.
  • ARBs angiotensin receptor blockers
  • Chinese patent CN101098689 discloses a dual action complex (particularly a supramolecular complex) of an angiotensin antagonist (ARB) and a neutral endopeptidase inhibitor (NEPi), having the following general formula Description: [ARB(NEPi)]Na 1-3 ⁇ xH 2 O, wherein x is 0-3, preferably 2.5, specifically refers to [3-((1S,3R)-1-biphenyl-4-ylmethyl-) 3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4 '-Methylmethyl ⁇ amino)butyric acid]Na 3 ⁇ 2.5H 2 O (LCZ696), the simplified structure is as follows:
  • the single crystal structure is determined to obtain the unit cell constant and the atomic position, and the single crystal diffraction pattern can be corresponding to the corresponding solid by a computer-aided calculation method.
  • the program used is the Powder Pattern in the application software Materials Studio (Accelrys).
  • the patent also discloses crystalline [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- Melting initial temperature of 3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ 2.5H 2 O
  • the (onset temperature) and the maximum peak temperature (Peak temperature) were 139 ° C and 145 ° C, respectively.
  • Such complexes can also be characterized by infrared absorption spectroscopy obtained by Fourier transform attenuated total reflection infrared spectroscopy (ATR-FTIR) spectrometer (Nicolet Magna-IR 560) using the important bands described below (with wavelength data) Reciprocal (cm -1 )) Qualitative: 2956 (w), 1711 (st), 1637 (st), 1597 (st), 1488 (w), 1459 (m), 1401 (st), 1357 (w) , 1295 (m), 1266 (m), 1176 (w), 1085 (m), 1010 (w), 1942 (w), 907 (w), 862 (w), 763 (st), 742 (m) , 698 (m), 533 (st).
  • ATR-FTIR Fourier transform attenuated total reflection infrared spectroscopy
  • the complex is characterized in particular by the following peaks: 1711 (st), 1637 (st), 1597 (st) and 1401 (st).
  • the error of all absorption bands of ATR-IR is ⁇ 2 cm -1 .
  • Such complexes can also be characterized by Raman chromatography as determined by Raman chromatography with a 785 nm laser excitation source (Kaiser Optical Systems, Inc.) with the following important bands (reciprocal of wavelength data (cm -1) ) indicates: 3061 (m), 2930 (m, width), 1612 (st), 1523 (m), 1461 (w), 1427 (w), 1287 (st), 1195 (w), 1108 (w), 11053(w), 1041(w), 1011(w), 997(m), 866(w), 850(w), 822(w), 808(w), 735(w), 715(w), 669 (w), 643 (w), 631 (w), 618 (w), 602 (w), 557 (w), 522 (w), 453 (w), 410 (w), 328 (w).
  • the error of all Raman bands is ⁇ 2 cm -1 .
  • the centerless spacer group P2 1 determined by the single crystal X-ray structure is a spacer group of conventional enantiomerically pure molecules. There are two general positions in the spacer group, which means that for 12 structural units in the unit cell, there must be 18 sodium ions and 15 water in the asymmetric unit.
  • Trisodium hemipentahydrate can be referred to as a sodium supramolecular complex and can be coordinated by an oxygen ligand. These oxygens are derived from the 12 carboxylate groups and 18 carbonyl groups of the above section and 13 of the 15 water molecules. This crystallization is an infinite three-dimensional network of these sodium complexes.
  • Chinese patent CN101098689 discloses a dual action complex of angiotensin antagonist (ARB) and neutral endopeptidase inhibitor (NEPi), specifically [3-((1S,3R)-1-biphenyl- 4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazole-5- Base) biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ 2.5H 2 O (LCZ696).
  • ARB angiotensin antagonist
  • NEPi neutral endopeptidase inhibitor
  • LCZ696 The physical and chemical properties of LCZ696 have obvious disadvantages, that is, they are easily halogenated at higher humidity (solid adsorption of moisture) Post-liquefaction).
  • solid halogenation of compounds has a very negative impact on drug development and products.
  • the chemical properties of the compound after liquefaction are not stable enough to affect the efficacy. Therefore, the liquefaction of the compound has an adverse effect on all aspects of the drug product.
  • New and more stable LCZ696 new crystal form or new hydrate to improve or overcome the halogenation problem. Hydrate and crystalline state, and the absorbent CN101098689 reported on three hemipentahydrate (LCZ696) compared to significant progress and improvement.
  • the inventors have proposed a new crystal form of ARB-NEPi complex and its preparation method and application in the in-depth study technology, and the ARB-NEPi complex refers to [3-((1S, 3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-methyl-2'-(pentanoyl ⁇ 2 "-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O, the new crystalline form and hydrate obtained by the invention have better physical and chemical properties and improve
  • the halogenation property of the crystal form in the prior art is more favorable to the pharmaceutically developable crystal form, easy to preserve, stable and reliable in quality, and the preparation method thereof can be industrially applied, and has broad medicinal prospects.
  • the invention provides a crystalline form of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid-( S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O,
  • the powder X-ray diffraction pattern is located at: 4.1 ⁇ 0.2 °, 4.9 ⁇ 0.2 °, 5.2 ⁇ 0.2 °, 9.6 ⁇ 0.2 °, 12.5 ⁇ 0.2 °, 16.9 ⁇ 0.2 °, 18.1 ⁇ 0.2 °, 19.4 ⁇ 0.2 ° and 27.1 ⁇
  • the powder X-ray diffraction pattern preferably further comprises 8.3 ⁇ 0.2°, 15.4 ⁇ 0.2°, 17.4 ⁇ 0.2°, 18.8 ⁇ 0.2°, 20.0 ⁇ 0.2°, 21.3 ⁇ 0.2°, 22.6 ⁇ 0.2°. , 22.9 ⁇ 0.2 °, 24.7 ⁇ 0.2 °, 25.2 ⁇ 0.2 °, 25.7 ⁇ 0.2 °, 27.8 ⁇ 0.2 °, and a peak at a diffraction angle (2 ⁇ ) of 29.9 ⁇ 0.2 °.
  • the powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2 ⁇ ) shown in Fig. 1, and the X-ray powder diffraction data is as shown in Table 1:
  • the powder X-ray diffraction pattern measured by a Burker D8 powder diffractometer comprises the following lattice plane spacing: 18.2 ⁇ 0.1, 16.9 ⁇ 0.1, 10.7 ⁇ 0.1, 7.1 ⁇ 0.1, 5.2 ⁇ 0.1, 4.9 ⁇ 0.1, 4.6 ⁇ 0.1, 4.2 ⁇ 0.1, 3.9 ⁇ 0.1, 3.5 ⁇ 0.1 and 3.0 ⁇ 0.1.
  • the powder X-ray diffraction pattern preferably further comprises: 7.9 ⁇ 0.2°, 14.7 ⁇ 0.2°, 15.5 ⁇ 0.2°, 16.1 ⁇ 0.2°, 20.2 ⁇ 0.2°, 20.8 ⁇ 0.2°, 25.5 ⁇ 0.2 °, a peak at a diffraction angle (2 ⁇ ) of 27.7 ⁇ 0.2°, 27.9 ⁇ 0.2°, and 29.2 ⁇ 0.2°; preferably, the powder X-ray diffraction pattern and the peak at the diffraction angle (2 ⁇ ) shown in Fig. 5 are basically Same on the same.
  • the powder X-ray diffraction pattern preferably further comprises diffraction angles at: 8.9 ⁇ 0.2°, 14.5 ⁇ 0.2°, 15.2 ⁇ 0.2°, 16.0 ⁇ 0.2°, 25.8 ⁇ 0.2°, and 27.9 ⁇ 0.2° ( The peak at 2 ⁇ ); preferably, its powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2 ⁇ ) shown in FIG.
  • the powder X-ray diffraction pattern preferably further comprises: 14.7 ⁇ 0.2°, 15.1 ⁇ 0.2°, 17.6 ⁇ 0.2°, 18.7 ⁇ 0.2°, 22.6 ⁇ 0.2°, 23.2 ⁇ 0.2°, 24.5 ⁇ 0.2. And a peak at a diffraction angle (2 ⁇ ) of 29.1 ⁇ 0.2°; preferably, the powder X-ray diffraction pattern thereof is substantially the same as the peak at the diffraction angle (2 ⁇ ) shown in FIG.
  • the powder X-ray diffraction pattern preferably further comprises: 6.3 ⁇ 0.2°, 13.6 ⁇ 0.2°, 14.2 ⁇ 0.2°, 14.8 ⁇ 0.2°, 15.2 ⁇ 0.2°, 17.6 ⁇ 0.2°, 18.7 ⁇ 0.2 °, peaks at diffraction angles (2 ⁇ ) of 22.6 ⁇ 0.2°, 23.3 ⁇ 0.2°, 24.6 ⁇ 0.2° and 29.2 ⁇ 0.2°; preferably, the powder X-ray diffraction pattern and the diffraction angle (2 ⁇ shown in FIG. 21) The peaks at ) are basically the same.
  • substantially identical as used herein with respect to the position of the X-ray diffraction peak means to consider typical peak position and intensity variability. For example, those skilled in the art will appreciate that the peak position (2 theta) will vary depending on the XRPD instrument, sometimes varying up to as much as 0.2 degrees. In addition, those skilled in the art will appreciate that XRPD sample preparation methods, XRPD instruments, sample crystallinity, sample usage, and crystal orientation will result in changes in relative peak intensities in the XRPD diffraction pattern of the sample.
  • the X is selected from the group consisting of 2.0 to 9.0, preferably from 2.5 to 6.5, more preferably from 2.5 to 3.5, still more preferably from 2.5 to 3.5, most preferably from 2.5 to 3.0 ⁇ 0.2.
  • the present invention provides a crystalline form of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl) Propionate-(S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ XH 2 O, the crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S) -3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O is a supramolecular structure
  • each asymmetric unit contains 6 ARBs, 6 NEPis, 18 sodium atoms, and 15-39 water molecules.
  • each asymmetric unit contains 6 ARBs, 6 NEPis, 18 sodium atoms and 15 water molecules or 18 ⁇ 0.5 water molecules.
  • each asymmetric unit contains 6 ARBs, 6 NEPis, 18 sodium atoms, and 17.5 water molecules, and its structure is as follows:
  • each of the asymmetric units comprises 15 coordination water molecules and 2.5 network space water molecules.
  • the single crystal size of the supramolecular structure is 0.36 x 0.34 x 0.10 mm, unit cell volume
  • the specific cultivation method of the single crystal is as follows: weigh about 510 mg of the sample into a 20 mL glass bottle, add 17 mL of 98% acetone/water, dissolve the sample at 65 ° C, and filter it on average to 17 new 4 mL glass bottles. The sample was placed at a constant temperature of 45 ° C to obtain the above single crystal. Its single crystal diffraction and analysis are as follows:
  • the crystal structure was analyzed by the direct method (sir2011) on a microcomputer, all non-hydrogen atom positions were obtained from the E map, and the structural parameters and discriminant atom types were corrected using the least squares method (SHELXL-2014/7), and geometric calculations and differences were used.
  • the minimum stoichiometric formula was finally determined to be C 288 H 365 N 36 Na 18 O 65.50 , the molecular weight of the single molecule was calculated to be 5792.93, and the calculated crystal density was 1.240 g/cm 3 .
  • the crystal structure is a sodium salt complex formed by the coordination of valsartan and AHU-377. These coordinating oxygen atoms are derived from the oxygen of 12 carboxy groups of valsartan and AHU377 molecules, 18 carbonyl groups, and oxygen in 15 water molecules. These oxygen atoms coordinate with 18 sodium ions to form a coordinated network structure. There are 2.5 water molecules in the interstices between the networks. There are hydrogen bonds between the molecules.
  • the network structure in the crystalline state has hydrogen bonding force and van der Waals force.
  • each asymmetric unit contains 6 ARBs, 6 NEPis, 18 sodium atoms, and 17.5 water molecules, and its structure is as follows:
  • each of the asymmetric units has 16 coordination water molecules and 2.5 network space water molecules.
  • the single crystal size of the supramolecular structure is 0.42 ⁇ 0.36 ⁇ 0.18 mm, and the unit cell volume
  • step 2 Heat the sample obtained in step 1 into 5 4-mL glass vials and cap tightly.
  • the sample obtained in the step 2 was placed in a circulating water tank for a program cooling test (program: equilibration at 60 ° C for 1 hour, and reduction to 20 ° C for 24 hours) to obtain the above single crystal. Its single crystal diffraction and analysis are as follows:
  • ⁇ scanning is used, the maximum 2 ⁇ angle is 56°, the scanning range is 0-180°, the swing angle is 0.5°, the interval is 0.5°, the scanning speed is 10s/°, and each screen is scanned once.
  • the crystal structure was analyzed by direct method (sir-2011) on a microcomputer, all non-hydrogen atom positions were obtained from E map, structural parameters were determined by least squares method, and atomic species were discriminated, and all hydrogen was obtained by geometric calculation method and difference Fourier method.
  • the minimum stoichiometric formula was determined to be C 288 H 365 N 36 Na 18 O 66.50 , the molecular weight of the single molecule was calculated to be 5181.99, and the calculated crystal density was 1.263 Mg/m 3 .
  • the molecular arrangement in the crystalline state belongs to the P2 1 space group, and the absolute configuration of the molecule can be determined by the absolute configuration of the Flack factor and the known hand-type organic molecule.
  • the main skeleton of the compound was the organic molecule valsartan and AHU377, and the Na salt formed.
  • the asymmetric unit there were six valsartan, six AHU-377 and 18 Na ions, and these 18 Na ions.
  • the oxygen of the carboxyl group of the six AHU-377, the oxygen of the amide group forms a coordination network structure.
  • the network gap is composed of 2.5 waters, and there are hydrogen bonds between the molecules.
  • the network structure of the lower coordination maintains its stable arrangement in space by hydrogen bonding force and van der Waals force.
  • the 5-form)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O structure has a value of about 3.1 in the formula.
  • Another aspect of the invention also provides a crystalline form of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid- (S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O
  • the preparation method comprises the following steps:
  • the addition method may be directly added or the basic sodium compound is dissolved in a suitable solvent;
  • the step 2) may be mixed with the basic sodium compound, or the basic sodium compound may be directly added to the system of the step 1) or the basic sodium compound may be dissolved in a suitable solvent and then added to the step 1) system. in.
  • the organic solvent in the step (1) includes but is not limited to the following solvents: methanol, ethanol, n-propanol, isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane Ring, N,N-dimethylformamide, dimethyl sulfoxide, isopropyl acetate, ethyl acetate or a mixture thereof.
  • the basic sodium compound in the step (2) is selected from the group consisting of sodium hydroxide, sodium carbonate, sodium hydrogencarbonate, sodium acetate, sodium methoxide, sodium formate, sodium propionate, sodium acrylate, sodium benzoate or a mixture thereof.
  • Preferred is sodium hydroxide, sodium hydrogencarbonate or a mixture thereof.
  • the method for creating supersaturation conditions described in the step (3) includes cooling crystallization, cooling-ultrasonic coupling crystallization, evaporation solvent crystallization, addition of anti-solvent or sol-gel replacement.
  • the anti-solvent refers to a solvent having a low solubility of the target solid.
  • the anti-solvent replacement means that a part of the original solvent is removed, a part of the anti-solvent is added, and after balancing, a part of the original solvent is removed until it is substantially replaced with an anti-solvent.
  • the humidity of the dry environment of step 5) is controlled to be 45% to 70%; preferably, the humidity of the dry environment is controlled to be 50% to 65%; more preferably, the humidity of the dry environment is controlled to be 55% to 65%.
  • the drying method described in the step (5) may be vacuum drying, evaporation, nitrogen drying or the like.
  • the water content in the solid obtained in the step (5) is optionally 4.6 to 11.6%, preferably 4.6 to 6.6%.
  • a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethyl) Oxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-yl Methyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O and a pharmaceutically acceptable carrier or excipient.
  • said X is selected from the group consisting of 2.5, 3, 6.5; most preferably 3.
  • Another aspect of the present invention provides the aforementioned crystalline [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid- (S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O or The use of the aforementioned pharmaceutical composition for the preparation of a medicament for treating or preventing a disease associated with a neutral endopeptidase, cardiovascular, antihypertensive.
  • Another aspect of the present invention provides the aforementioned crystalline [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid- (S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O or
  • the aforementioned pharmaceutical composition is prepared for treating or preventing acute and chronic heart failure such as congestive heart failure, left ventricular dysfunction, hypertrophic cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmia, atrial fibrillation, atrium Use in flutter or harmful vascular remodeling drugs.
  • the aforementioned crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-( S)-3'-methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O or the foregoing
  • the pharmaceutical composition is prepared for treating or preventing myocardial infarction and its sequelae, atherosclerosis, angina pectoris, diabetic or non-diabetic renal insufficiency, secondary aldosteronism, primary or secondary pulmonary hypertension, Diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary nephropathy, renal vascular hypertension, diabetic retinopathy, migraine, peripheral pulmonary hypertension,
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof with other chemical components, or other components such as physiological/pharmaceutical Accepted carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • Figure 1 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 1.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid ]Na 3 ⁇ 2.5H 2 O powder X-ray diffraction Figure; the ordinate is the intensity and the abscissa is 2 ⁇ (°).
  • Figure 2 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 1.
  • Figure 3 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 4.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid Na 3 ⁇ 6.5H 2 O powder X-ray diffraction Figure; the ordinate is the intensity and the abscissa is 2 ⁇ (°).
  • Figure 4 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid-(S)- in Example 4.
  • Figure 5 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 5.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid Na 3 ⁇ 3.1H 2 O powder X-ray diffraction Figure; the ordinate is the intensity and the abscissa is 2 ⁇ (°).
  • Figure 6 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 5.
  • Figure 7 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 2.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid Na 3 ⁇ 2.5H 2 O powder X-ray diffraction Figure; the ordinate is the intensity and the abscissa is 2 ⁇ (°).
  • Figure 8 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 2.
  • Figure 9 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 7.
  • the ordinate is the intensity and the abscissa is 2 ⁇ (°).
  • Figure 10 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 7.
  • Figure 11 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 3.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid Na 3 ⁇ 2.5H 2 O powder X-ray diffraction Figure; the ordinate is the intensity and the abscissa is 2 ⁇ (°).
  • Figure 12 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 3.
  • Figure 13 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 8.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid ]Na 3 ⁇ 2.9H 2 O powder X-ray diffraction Figure; the ordinate is the intensity and the abscissa is 2 ⁇ (°).
  • Figure 14 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 8.
  • Figure 15 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 5.
  • Figure 16 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 5.
  • Figure 17 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 6.
  • Figure 18 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 6.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid 6 Na 18 ⁇ 18.5H 2 O powder thermogravi Analysis (TGA) chart; the ordinate is the percent change in weight (%) and the abscissa is the temperature (°C).
  • Figure 19 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 8.
  • Figure 20 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 8.
  • Figure 21 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 9.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid 6 Na 18 ⁇ 17.5H 2 O powder X-ray Diffraction pattern; the ordinate is the intensity and the abscissa is 2 ⁇ (°).
  • Figure 22 is a graph of [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- in Example 9.
  • 3'-Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid 6 Na 18 ⁇ 17.5H 2 O powder thermogravi Analysis (TGA) chart; the ordinate is the percent change in weight (%) and the abscissa is the temperature (°C).
  • Figure 23 Changes in animal body weight during model induction; ordinate is animal body weight (grams) and abscissa is time (weeks).
  • Figure 24 shows changes in the systolic blood pressure of the rat tail artery during model induction; the ordinate is the animal systolic pressure (mm Hg) and the abscissa is the time (weeks).
  • Figure 25 Changes in the diastolic pressure of the rat tail artery during model induction; the ordinate is the animal diastolic pressure (mm Hg) and the abscissa is time (weeks).
  • Figure 26 shows the change in mean pressure of the rat tail artery during model induction; the ordinate is mean arterial pressure (mm Hg) and the abscissa is time (weeks).
  • Figure 27 Changes in systolic blood pressure of the rat tail artery after administration of drug A and drug B; the ordinate is the animal systolic pressure (mm Hg) and the abscissa is time (weeks).
  • Figure 28 shows changes in diastolic blood pressure of the rat tail artery after administration of the drug A and drug B; the ordinate is the diastolic blood pressure of the animal (mmH) and the abscissa is the time (week).
  • Figure 29 Changes in mean pressure of the tail artery of rats after administration of drug A and drug B; the ordinate is mean arterial pressure (mm Hg) and the abscissa is time (weeks).
  • Figure 30 Changes in animal heart rate during drug C and drug D administration.
  • Figure 31 Changes in systolic blood pressure of animals during drug C and drug D administration.
  • FIG. 32 Changes in diastolic blood pressure during administration of Drug C and Drug D.
  • Figure 33 Changes in mean arterial pressure of animals during drug C and drug D administration.
  • Crystalline [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-methyl -2'-(Pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ X H 2 O is characterized by their X-ray powder diffraction pattern.
  • the X-ray powder diffraction pattern of the salt was taken on a Bruker D8 Discover X-ray powder diffractometer of GADDS (General Area Diffraction Detector System) CS operating in a reflective mode.
  • the tube voltage and current quantities were set to 40kV and 40mA acquisition scans, respectively.
  • the sample was scanned for a period of 60 seconds in the range of 2 ⁇ from 3.0° to 40°.
  • the diffractometer was calibrated using a corundum standard for the peak position indicated by 2 ⁇ . All analyses were performed at room temperature, typically between 20 ° C and 30 ° C. Data was acquired and integrated using GADDS for the 4.1.14T version of WNT software.
  • the diffraction pattern was analyzed using the DiffracPlus software released in 2003 with version 9.0.0.2 Eva.
  • the XRPD sample is prepared by passing the sample onto a single crystal silicon wafer and pressing the sample powder with a glass slide or equivalent to ensure that the surface of the sample is flat and of a suitable height.
  • the sample holder was then placed in a Bruker XRPD instrument and a powder X-ray diffraction pattern was acquired using the instrument parameters described above.
  • Measurement differences associated with such X-ray powder diffraction analysis results are produced by a variety of factors including: (a) errors in sample preparation (eg, sample height), (b) instrument error, (c) calibration differences, ( d) operator error (including those that occur when determining peak position), and (e) properties of the substance (eg, preferred orientation error). Calibration errors and sample height errors often result in displacement of all peaks in the same direction. In general, this calibration factor will align the measured peak position to the expected peak position and may be in the range of the expected 2 ⁇ value ⁇ 0.2°.
  • AHU-377 free acid 200 mg was weighed, 212 mg of valsartan and 58.3 mg of sodium hydroxide were placed in a 20-mL glass vial, and then 10 mL of isopropyl acetate was added and sealed. The mixture was placed in an ultrasonic water of 0 ° C at a power of 100 W, 35 KHz, and reacted for 4 hours to obtain a suspension containing a white solid. The suspension was centrifuged (8000 rpm) for 10 minutes, the supernatant was discarded, and the remaining solid was dried by nitrogen purge for 4 hours, and a nitrogen flow rate of about 5 ml/min gave a powdery solid.
  • the obtained hydrate was 2.5 hydrate.
  • the TGA analysis chart is shown in Figure 8.
  • the weight loss on the TGA map is due to water loss in the crystal and can be used to determine the moisture content in the crystal.
  • the KF moisture method was used to determine the moisture content in the crystal as a reference. The results obtained are shown in the table below:
  • the obtained hydrate was 2.5 hydrate.
  • the TGA analysis chart is shown in Figure 12.
  • the weight loss on the TGA map is due to water loss in the crystal and can be used to determine the moisture content in the crystal.
  • the KF moisture method was used to determine the moisture content in the crystal as a reference. The results obtained are shown in the table below:
  • the obtained hydrate was 2.5 hydrate.
  • AHU-377 free acid 200 mg was weighed, 212 mg of valsartan and 58.3 mg of sodium hydroxide were placed in a 20-mL glass vial, and then 10 mL of ethyl acetate was added thereto, and sealed.
  • the mixture was placed at 0 ° C for ultrasound In the water of the wave, the power was 100 W, 35 KHz, and the reaction was carried out for 4 hours to obtain a suspension containing a white solid.
  • the suspension was centrifuged (8000 rpm) for ten minutes, the supernatant was discarded, and the remaining solid was dried by nitrogen purge for 4 hours, and a nitrogen flow rate of about 5 ml/min gave a powdery solid.
  • the obtained hydrate was 6.5 hydrate.
  • AHU-377 free acid 200 mg was weighed, 212 mg of valsartan and 58.3 mg of sodium hydroxide were placed in a 20-mL glass vial, and then 10 mL of acetone was added thereto, and sealed.
  • the mixture was placed in an ultrasonic water of 0 ° C at a power of 100 W, 35 KHz, and reacted for 4 hours to obtain a suspension containing a white solid.
  • the suspension was centrifuged (8000 rpm) for ten minutes, the supernatant was discarded, and the remaining solid was dried by nitrogen purge for 4 hours, and a nitrogen flow rate of about 5 ml/min gave a powdery solid.
  • the obtained hydrate was 3.1 hydrate.
  • the sample obtained in the step 2 was placed in a circulating water tank for a program cooling test (program: equilibration at 60 ° C for 1 hour, and reduction to 20 ° C for 24 hours) to obtain the above single crystal.
  • the cell of the supramolecular complex is shown in Figure 15, which comprises two asymmetric units whose single crystal diffraction and resolution are as follows:
  • the single crystal is in the form of colorless flakes.
  • the crystal structure was analyzed by direct method (sir-2011) on a microcomputer, all non-hydrogen atom positions were obtained from E map, structural parameters were determined by least squares method, and atomic species were discriminated, and all hydrogen was obtained by geometric calculation method and difference Fourier method.
  • the minimum stoichiometric formula was determined to be C 288 H 367 N 36 Na 18 O 66.50.
  • the molecular weight of the single molecule was calculated to be 5180.93, and the calculated crystal density was 1.263 Mg/m 3 .
  • the molecular arrangement in the crystalline state belongs to the P 21 space group, and the absolute configuration of the molecule can be determined by the absolute configuration of the Flack factor and the known chiral organic molecule.
  • the asymmetric unit there were six valsartan, six AHU377 and 18 Na ions. These 18 Na The ions are coordinated with six valsartan, oxygen in the carboxyl group of six AHU377, and the oxygen of the amide group to form a coordination network structure.
  • the network gap is composed of 2.5 waters, and there are hydrogen bonds between the molecules.
  • the coordination network structure maintains its stable arrangement in space by hydrogen bonding force and van der Waals force.
  • the crystal diffraction pattern of the product powder obtained in Example 5 was highly consistent with the calculated value of the single crystal.
  • a small amount of peak is missing, which is caused by the preferred orientation of the powder crystal. This phenomenon is particularly noticeable for sheet crystals.
  • the crystal form of the powder crystal is highly consistent with the single crystal calculation crystal pattern.
  • the single crystal structure was consistent with the powder crystal structure.
  • the resulting single crystal TGA analysis is shown in Figure 16.
  • the weight loss on the TGA map is due to water loss in the crystal and can be used to determine the moisture content in the crystal.
  • the KF moisture method was used to determine the moisture content in the crystal as a reference. The results obtained are shown in the table below:
  • the obtained single crystal was 3.1 hydrate, which was consistent with the results of single crystal analysis.
  • Example 6 From the comparison of the above experimental data, the crystal powder diffraction pattern of the product powder obtained in Example 6 was also highly consistent with the calculated value of the single crystal of Example 5, showing a high degree of uniformity of the crystal form. Example 6 was also consistent with the powder crystal measurements in Example 5.
  • the TGA analysis chart is shown in Figure 18.
  • the weight loss on the TGA map is due to water loss in the crystal and can be used to determine the moisture content in the crystal.
  • the KF moisture method was used to determine the moisture content in the crystal as a reference. The results obtained are shown in the table below:
  • the TGA analysis chart is shown in Figure 10.
  • the weight loss on the TGA map is due to water loss in the crystal and can be used to determine the moisture content in the crystal.
  • the KF moisture method was used to determine the moisture content in the crystal as a reference. The results obtained are shown in the table below:
  • the obtained hydrate was a trihydrate.
  • the TGA analysis is shown in Figure 14.
  • the weight loss on the TGA pattern is due to the crystal.
  • the water content caused by water loss can be used to determine the moisture content in the crystal.
  • the KF moisture method is used to determine the moisture content in the crystal. The results are shown in the following table:
  • the obtained single crystal was 2.9 hydrate.
  • Example 8 The 510 mg sample prepared in Example 8 was placed in a 20 mL glass vial, 17 mL of 98% acetone/water was added, and the sample was dissolved at 65 ° C, and filtered on average to 17 new 4 mL glass bottle caps and placed. The above single crystal was obtained by constant temperature at 45 ° C.
  • Figure 19 The cell of the supramolecular complex is shown in Figure 19, which comprises two asymmetric units whose single crystal diffraction and resolution are as follows:
  • the crystal is colorless, and the crystal size used for diffraction analysis is 0.36x 0.34x 0.10mm. It belongs to monoclinic system and the space group is P2 1 .
  • the crystal structure was analyzed by the direct method (sir2011) on a microcomputer, all non-hydrogen atom positions were obtained from the E map, and the structural parameters and discriminant atom types were corrected using the least squares method (SHELXL-2014/7), and geometric calculations and differences were used.
  • the minimum stoichiometric formula was determined to be C 288 H 365 N 36 Na 18 O 65.50 , the molecular weight of the individual molecule was calculated to be 5792.93, and the calculated crystal density was 1.240 g/cm 3 .
  • the chemical name is: [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'- Methyl-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid] 6 Na 18 ⁇ 17.5H 2 O; the chemical formula is: [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-methyl-2 '-(Pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ 2.9H 2 O
  • the crystal diffraction pattern of the product powder obtained in Example 8 was highly consistent with the calculated value of the corresponding single crystal.
  • a small amount of peak is missing, which is caused by the preferred orientation of the powder crystal. This phenomenon is particularly noticeable for sheet crystals.
  • the crystal form of the powder crystal is highly consistent with the single crystal calculation crystal pattern.
  • the single crystal structure was consistent with the powder crystal structure.
  • the resulting single crystal TGA analysis chart is shown in Fig. 20.
  • the weight loss on the TGA map is due to water loss in the crystal and can be used to determine the moisture content in the crystal.
  • the KF moisture method was used to determine the moisture content in the crystal as a reference. The results obtained are shown in the table below:
  • the obtained single crystal was 2.9 hydrate, which was consistent with the single crystal analysis result.
  • Example 9 From the comparison of the above experimental data, the crystal diffraction pattern of the product powder obtained in Example 9 was highly consistent with the calculated value of the single crystal of Example 8, showing a high degree of uniformity of the crystal form. Example 9 was also consistent with the powder crystal measurements in Example 8.
  • the TGA analysis is shown in Figure 22.
  • the weight loss on the TGA map is due to water loss in the crystal and can be used to determine the moisture content in the crystal.
  • the KF moisture method was used to determine the moisture content in the crystal as a reference. The results obtained are shown in the table below:
  • the obtained single crystal was 2.9 hydrate.
  • Drug A [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-A Base-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ 3.1H 2 O was obtained as described in Example 6.
  • Drug B Valsartan is a commercially available drug substance.
  • Rat tail blood pressure measuring instrument BP-2010AUL.CN10120479
  • Rat gavage 16#80mm diameter, round head
  • mice Male Sprague Dawley (SD) rats, weighing 180-200 grams, were supplied by Vital River Laboratories, Beijing, China. Animals are housed in the SPF barrier system of the Animal Center and follow international standard temperature, humidity, and light control systems. The experimental animal operating protocol was approved by the ICATC Committee of the Platform Animal Center.
  • the abdominal wall muscle tissue was sutured with a 3-0 surgical suture thread, and then the skin was sutured with a 2-0 surgical suture.
  • the animals were placed on a 37-degree electric blanket to keep the animals awake, and it was confirmed that the animals were able to return to the cages for normal feeding after free feeding and drinking.
  • Animals were observed daily for the first week after surgery to confirm that there were no postoperative wound infections and splitting. Changes in animal body weight and heart rate in the awake state were measured weekly from the first week after surgery to monitor changes in blood pressure and heart rate in the animals.
  • Animals were tested for tail vein pressure for 4-5 weeks for a continuous observation of the trend of elevated blood pressure in the animals. Animals with systolic blood pressure exceeding 160 mmHg were selected for pharmacodynamic test
  • Drug A [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid-(S)-3'-A -2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ 3.1H 2 O
  • drug B valsartan
  • a single oral administration test was started, and administration was started 9-10am early, and the next day was 10:00AM.
  • the blood pressure changes of 2-4HR, 6-8HR and 24HR animals were observed within 24 hours.
  • -1 is the administration group
  • group-2 is the blank control group (only for the drug carrier)
  • the rat tail artery pressure was monitored once a week after modeling. Animals began to develop blood pressure from the first week of modeling. The rat tail artery pressure monitoring for 4-5 weeks showed that the blood pressure of the animals tends to maintain a steady increase from 3 weeks after modeling (Fig. 24-26).
  • a and B showed a slight decrease in blood pressure, including systolic blood pressure, diastolic blood pressure, and mean arterial pressure.
  • the optimal antihypertensive effect was achieved in 6-8 hours; a slight rebound occurred in 24 hours (see Figure 27). -29).
  • the antihypertensive effect of drug A is superior to that of drug B.
  • Drug C [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-A Base-2'-(pentanoyl ⁇ 2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl ⁇ amino)butyric acid]Na 3 ⁇ 2.9H 2 O was obtained according to the preparation of Example 9.
  • Drug D Valsartan is a commercially available drug substance.
  • Rat tail blood pressure measuring instrument BP-2010AUL.CN10120479
  • Rat gavage 16#80mm diameter, round head
  • mice Male Sprague Dawley (SD) rats, weighing 180-200 grams, were supplied by Vital River Laboratories, Beijing, China. Animals are housed in the SPF barrier system of the Animal Center and follow international standard temperature, humidity, and light control systems. The experimental animal operating protocol was approved by the ICATC Committee of the Platform Animal Center.
  • the abdominal wall muscle tissue was sutured with a 3-0 surgical suture thread, and then the skin was sutured with a 2-0 surgical suture.
  • the animals were placed on a 37-degree electric blanket to keep the animals awake, and it was confirmed that the animals were able to return to the cages for normal feeding after free feeding and drinking.
  • Animals were observed daily for the first week after surgery to confirm that there were no postoperative wound infections and splitting. Changes in animal body weight and heart rate in the awake state were measured weekly from the first week after surgery to monitor changes in blood pressure and heart rate in the animals.
  • Animals were tested for tail vein pressure for 4-5 weeks for a continuous observation of the trend of elevated blood pressure in the animals. Animals with systolic blood pressure exceeding 160 mmHg were selected for pharmacodynamic test
  • 64 animals were selected, SBP>160mmHg; the experiment was divided into 4 groups, sham operation group (group-1), hypertension model group (group-2), control group (drug D) (group-3), drug group C (group-4).
  • the drug was administered orally once a day at a drug volume of 10 mL/kg for one week, once every three days, and blood pressure changes of 6-8 hr and 24 hr after administration of the animals were examined.
  • Animals were started 9-10 am daily. After the administration, the physiological changes after the administration of the animals were closely observed, mainly for clinical characterization, and if abnormal phenomena were recorded and analyzed in time; 6-8 hr (15:30-17:30 P.M) and 24 hr after administration, respectively.
  • the blood pressure and heart rate of the rat tail artery were measured at 09:30-11:30 the next day. The experiment lasted for one week.
  • Heart rate observations of the sham-operated group (Group-1) for one week showed that the average heart rate fluctuated between 330 beats/min to 350 beats/min.
  • the average heart rate of hypertensive animals before administration was about 380 beats/min.
  • the animal heart rate did not show significant acceleration or slowdown during one week of continuous administration.
  • the heart rate of the animals administered in the drug group C (group 4) for one week showed a steady slow slowdown, which was consistent with the heart rate of the sham-operated animals 3 days after the administration. After a week of continuous administration, the heart rate of the administered animals showed a sustained steady slowdown (see Figure 30).
  • the sham-operated group (Group-1) of the test animals maintained their blood pressure in the normal range for one week of continuous tests, including systolic blood pressure, diastolic blood pressure and mean arterial pressure.
  • the blood pressure of the drug D (group-3) showed a slow fluctuation in the course of administration for one week, which showed that the antihypertensive effect was most obvious 6-8 hours after each administration. It rose slightly after 24 hours.
  • the drug group C (group-4) the blood pressure of the animals showed consistency and slow fluctuation in the administration test for one week, which showed that the antihypertensive effect was most obvious 6-8 hours after each administration, and mild after 24 hours.
  • the rebound, including systolic blood pressure, diastolic blood pressure and mean arterial pressure see Figure 31-33).

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Abstract

公开了结晶型ARB-NEPi复合物及其制备方法和应用，ARB-NEPi复合物是指[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3'-甲基-2'-(戊酰基{2"-(四唑-5-基)联苯-4'-基甲基}氨基)丁酸]Na 3·X H 2O，并公开了含有效量结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3'-甲基-2'-(戊酰基{2"-(四唑-5-基)联苯-4'-基甲基}氨基)丁酸]Na 3·X H 2O药物组合物及其在制备治疗或预防与中性内肽酶有关的疾病、心血管、抗高血压、急慢性心衰如、充血性心衰、左心室机能障碍、肥厚性心肌病、糖尿病性心肌病、室上性和室性心律不齐、心房纤维颤动、心房扑动或有害的血管重构等药物中的用途。

Description

结晶型ARB-NEPi复合物及其制备方法和应用 技术领域
本发明属于药物技术领域，具体地，本发明涉及双重作用的ARB-NEPi复合物新晶型及其制备方法和应用。
背景技术
中性内肽酶(EC 3.4.24.11；脑啡肽酶；必肽酶；NEP)是一种可以在芳族氨基酸的氨基末端上裂解各种肽底物的包含锌的金属蛋白酶。这种酶的底物非限制性地包括心钠素(ANF，也被称为ANP)、脑钠肽(BNP)、met和leu脑啡肽、缓激肽、神经激肽A和P物质。
ANF是一族血管舒张、利尿和抗高血压的肽类，一种形式——ANF 99-126是在心脏扩张情况中由心脏释放的循环肽激素。ANF的功能是维持盐和水的体内平衡以及调节血压。ANF在循环中被至少两个过程迅速灭活：受体-介导的清除和在NEP进行的酶灭活。NEP抑制剂增强了实验动物进行药理学ANF注射后的低血压、利尿、促尿钠排泄和血浆ANF响应。通过两种特定的NEP抑制剂进行的ANF的增强，1988年，美国专利US4749688中一般性地公开了可以用NEP来增强ANF。同年，美国专利US4740499中公开了噻奥芬(thiorphan)和凯拉托芬也可以用于增强心房肽的应用。此外，NEP抑制剂可降低血压和发挥ANF-样作用如在一些形式的实验性高血压中的利尿和增加环鸟苷3’，5’-单磷酸(cGMP)排泄的作用。因为ANF的抗体将抵消血压的降低，所以NEP抑制剂的抗高血压作用是通过ANF介导的。长期和不进行控制的高血压血管疾病最终将导致靶器官如心和肾的各种病理学改变。持续的高血压也能导致中风的发生率增加。因此，强烈需要对抗高血压治疗的功效进行评估，即对除了血压降低以外的其它心血管终点事件进行检查以进一步发现联合治疗的益处。
1993年，Gary Ksander等在美国专利US5217996中公开了一类联芳基取代的4-氨基丁酸衍生物，该类化合物被发现具有明显的NEP抑制活性，其中最具代表性的一个化合物为N-(3-羧基-1-氧代丙基)-(4S)-对-苯基苯基甲基)-4-氨基-2R-甲基丁酸乙酯(又称AHU-377)。
Figure PCTCN2015089051-appb-000001
血管紧张素II是引起血管收缩的激素，而血管收缩又导致高血压和心脏劳损。人们已经知道血管紧张素II能够与靶细胞表面上的受体相互作用。目前已经鉴别出血管紧张素II的两种受体亚型，称为AT1和AT2。最近一段时间，人们付出了巨大的努力鉴定能够与AT1受体结合的物质。现在已经知道，血管紧张素受体阻断剂(ARBs，血管紧张素II拮抗剂)能够通过阻止血管紧张素II与其在血管壁上的受体结合，从而导致血压降低。由于能够抑制AT1受体，所以此类拮抗剂可以用于抗高血压，或者用于治疗充血性心衰以及其它适应症。
2008年，中国专利CN101098689公开了一种血管紧张素剂拮抗剂(ARB)和中性内肽酶抑制剂(NEPi)双重作用的复合物(特别是超分子络合物)，具有如下通式所述：[ARB(NEPi)]Na1-3·xH2O，其中x为0-3，优选2.5，具体指[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O(LCZ696)，简化结构如下所示：
Figure PCTCN2015089051-appb-000002
还公开了结晶形式[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O(LCZ696)的晶型粉末衍射的数据为：
Figure PCTCN2015089051-appb-000003
对于一种给定的物质，验证X-射线衍射实检测方法获得的晶格平均晶面间距和强度的好的方法是检测这些值是否与复杂的单晶结构解析计算的数据相一致。
单晶结构测定得到晶胞常数和原子位置，通过计算机辅助计算的方法可以将单晶衍射图能够对应于相应固体。采用的程序为应用软件Materials Studio(Accelrys)中的Powder Pattern。下表显示了这些通过XRPD测定和计算单晶数据得到的数据比较，即[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]三钠半五水合物的重要的晶格平面间隔和强度的XRPD测定值和单晶计算值的数据比较。
Figure PCTCN2015089051-appb-000004
该专利还公开了结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O的熔化初始温度(onset temperature)和最大峰温度(Peak temperature)分别为139℃和145℃。
此类复合物也可以通过采用傅里叶变换衰减全反射红外光谱法(ATR-FTIR)光谱仪(Nicolet Magna-IR 560)而得到的红外吸收光谱定性，采用下面所述的重要波段(以波长数据的倒数(cm-1)表示)定性：2956(w)，1711(st)，1637(st)，1597(st)，1488(w)，1459(m)，1401(st)，1357(w)，1295(m)，1266(m)，1176(w)，1085(m)，1010(w)，1942(w)，907(w)，862(w)，763(st)，742(m)，698(m)，533(st)。络合物的特征特别在于下列峰：1711(st)，1637(st)，1597(st)和1401(st)。ATR-IR的所有吸收波段的误差为±2cm-1。吸收波段的强度表示如下：(w)＝弱；(m)＝中；和(st)＝强。
此类复合物也可以通过由拉曼色谱仪测定的拉曼色谱定性，该色谱仪具有785nm激光激发源(Kaiser Optical Systems，Inc.)，以下列重要波段(以波长数据的倒数(cm-1)表示：3061(m)，2930(m，宽)，1612(st)，1523(m)，1461(w)，1427(w)，1287(st)，1195(w)，1108(w)，11053(w)，1041(w)，1011(w)，997(m)，866(w)，850(w)，822(w)，808(w)，735(w)，715(w)，669(w)，643(w)，631(w)， 618(w)，602(w)，557(w)，522(w)，453(w)，410(w)，328(w)。所有的拉曼波段的误差±2cm-1。吸收波段的强度表示如下：(w)＝弱；(m)＝中；和(st)＝强。
单晶X-射线结构测定的无中心间隔群P21为常规的对映体纯的分子的间隔群。该间隔群中有两个一般位置(general position)，这意味着对于单位晶胞中的12个结构单位，在不对称单元中一定有18个钠离子和15个水。
基于单晶结构解析，[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]三钠半五水合物超分子的不对称单元每一个均含有6个ARB和NEPi部分、18个钠原子和15个水分子。[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]三钠半五水合物可以称为钠超分子络合物，可以被氧配基配位。这些氧来自上述部分的12个羧酸盐基团和18个羰基基团以及15个水分子中的13个。该结晶是这些钠络合物的无限三维空间网状结构。
中国专利CN101098689公开了一种血管紧张素剂拮抗剂(ARB)和中性内肽酶抑制剂(NEPi)双重作用的复合物，具体指[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O(LCZ696)。LCZ696理化性质具有明显的缺点，即在较高的湿度下，容易卤化(固体吸附水分后液化)。在药学上，化合物固体卤化对药物开发和产品等各方面带来十分不利的影响。比如，吸湿液化后难以制成固体口服制剂，药物含量也难以准确定量。在压片过程中，容易发生粘冲和片剂产生花斑，硬性制剂质量。另外，化合物液化后化学性质不够稳定，影响疗效。因此，化合物的液化对药物产品的方方面都有不利的影响。因此急需要找到新的更加稳定的LCZ696新晶型或者新的水合物，来改善或克服卤化问题。本发明中发现新的水合物及其结晶态，在吸湿性上与CN101098689中报道的三钠半五水合物(LCZ696)相比，有明显进步和提高。
发明内容
为了解决现有技术存在的缺陷，发明人在深入研究的技术上提出了一种ARB-NEPi复合物新晶型及其制备方法和应用，ARB-NEPi复合物是指[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，本发明得到的新晶型和水合物具有更好的理化性质，改善了现有技术中晶型的卤化性质，更利于药学上可开发的晶型，易于保存，质量稳定可靠，其制备方法，可工业应用，具有广阔的药用前景。
本发明一方面提供了一种结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其粉末X射线衍射图包括位于:4.1±0.2°，4.9±0.2°，5.2±0.2°，9.6±0.2°，12.5±0.2°，16.9±0.2°，18.1±0.2°，19.4±0.2°和27.1±0.2°的衍射角(2θ)处的峰。
作为进一步优选的方案，其粉末X射线衍射图优选还包括位于8.3±0.2°，15.4±0.2°，17.4±0.2°，18.8±0.2°，20.0±0.2°，21.3±0.2°，22.6±0.2°，22.9±0.2°，24.7±0.2°，25.2±0.2°，25.7±0.2°，27.8±0.2°,和29.9±0.2°的衍射角(2θ)处的峰。
最优选，其粉末X射线衍射图与图1中显示的衍射角(2θ)处的峰基本上相同，其X射线粉末衍射数据如表1所示：
表1
Figure PCTCN2015089051-appb-000005
作为进一步优选的方案，Burker D8粉末衍射仪测定的粉末X射线衍射图包括下列晶格平面间隔：
Figure PCTCN2015089051-appb-000006
18.2±0.1，16.9±0.1，10.7±0.1，7.1±0.1，5.2±0.1，4.9±0.1，4.6±0.1，4.2±0.1，3.9±0.1，3.5±0.1和3.0±0.1。
作为进一步优选的方案，其粉末X射线衍射图优选还包括位于：7.9±0.2°，14.7±0.2°，15.5±0.2°，16.1±0.2°，20.2±0.2°，20.8±0.2°，25.5±0.2°，27.7±0.2°，27.9±0.2°和29.2±0.2°的衍射角(2θ)处的峰；优选的，其粉末X射线衍射图与图5中显示的衍射角(2θ)处的峰基本上相同。
作为进一步优选的方案，其粉末X射线衍射图优选还包括位于：8.9±0.2°，14.5±0.2°，15.2±0.2°，16.0±0.2°，25.8±0.2°和27.9±0.2°的衍射角(2θ)处的峰；优选的，其粉末X射线衍射图与图17中显示的衍射角(2θ)处的峰基本上相同。
作为进一步优选的方案，其粉末X射线衍射图优选还包括位于：14.7±0.2°，15.1±0.2°，17.6±0.2°，18.7±0.2°，22.6±0.2°，23.2±0.2°，24.5±0.2°和29.1±0.2°的衍射角(2θ)处的峰；优选的，其粉末X射线衍射图与图13中显示的衍射角(2θ)处的峰基本上相同。
作为进一步优选的方案，其粉末X射线衍射图优选还包括位于：6.3±0.2°，13.6±0.2°，14.2±0.2°，14.8±0.2°，15.2±0.2°，17.6±0.2°，18.7±0.2°，22.6±0.2°，23.3±0.2°，24.6±0.2°和29.2±0.2°的衍射角(2θ)处的峰；优选的，其粉末X射线衍射图与图21中显示的衍射角(2θ)处的峰基本上相同。
本文所使用的关于X射线衍射峰位置的术语“基本上相同的”意指考虑典型的峰位置和强度可变性。例如，本领域技术人员将理解，峰位置(2θ)将由于XRPD仪器不同，而造成测量值有所变化，有时这种变化达有时多达0.2°。此外，本领域技术人员将理解，XRPD样品制样方法，XRPD仪器，样品结晶度，样品用量以及晶体择优取向等因素将导致样品XRPD衍射图中相对峰强度的改变。
作为进一步优选的方案，所述X选自2.0～9.0，优选自2.5～6.5，更优选自2.5～3.5，更优选自2.5～3.5，最优选自2.5、3.0±0.2。
作为更进一步优选的方案，本发明提供了一种结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，所述结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O是超分子结构的结构简式，其包含不对称单位，每个不对称单元均含有6个ARB、6个NEPi、18个钠原子及12-54个水分子，其中所述的ARB为(S)-N-戊酰基-N-{[2’-(1H-四唑-5-基)联苯-4-基]甲基}-缬氨酸，所述的NEPi为(2R，4S)-5-联苯-4-基-4-(3-羧基-丙酰基氨基)-2-甲基-戊酸乙基酯。
优选的，每个不对称单元均含有6个ARB、6个NEPi、18个钠原子及15-39个水分子。
更优选的，每个不对称单元均含有6个ARB、6个NEPi、18个钠原子及15个水分子或18±0.5个水分子。
更优选的，每个不对称单元均含有6个ARB、6个NEPi、18个钠原子及17.5个水分子，其结构如下：
Figure PCTCN2015089051-appb-000007
更优选的，所述每个不对称单元中包含15个配位水分子，2.5个网络空间水分子。
更优选的，所述超分子结构的单晶呈无色片状，单斜晶系，空间群为P21,晶胞参数：
Figure PCTCN2015089051-appb-000008
α＝90°，β＝119.278(9)°，γ＝90°。
更优选的，所述超分子结构的单晶大小为0.36x 0.34x 0.10mm，晶胞体积
Figure PCTCN2015089051-appb-000009
Figure PCTCN2015089051-appb-000010
晶胞内分子数Z＝2，单位晶格的独立区域中有两个单元。
其单晶的具体培养方法为：称取约510mg样品至20mL玻璃瓶中，加入17mL98％丙酮/水，在65℃条件下将样品溶解，并平均趁热过滤至17个新的4mL玻璃瓶，放置45℃条件下，恒温放置，得到上述单晶。其单晶衍射及解析如下：
1、单晶衍射试验：
在Rigaku XtaLAB P200衍射仪上，-100℃时收集衍射强度数据，CuKα辐射，人工多层膜聚焦镜，准直管φ＝0.30mm，晶体与Pilatus200K探测器距离为35mm，管压40kV,管流30mA，ω扫描，最大2θ角为146.9°，扫描范围为0-180°，回摆角度为1°，间隔为1°，扫描速度为5-10s/°,每个画面扫描一次，总计摄取3579幅图像，总衍射点为251362个，独立衍射点为59848个
(Rint＝0.0769)，可观察点(F2≧2σF2)为48100个，θ角到67.687°时数据完整度为99.3％。
2、单晶解析方法：
在微机上用直接法(sir2011)解析晶体结构，从E图上获得全部非氢原子位置，使用最小二乘法(SHELXL-2014/7)修正结构参数和判别原子种类，使用几何计算法和差值Fourier法获得氢原子位置，最终可靠因子R1＝0.0700，wR2＝0.1691，S＝1.070，Flack因子为0.07(2)。最终确定最小化学计量式为C288H365N36Na18O65.50，计算单个分子的分子量为5792.93，计算晶体密度为1.240g/cm3。
Figure PCTCN2015089051-appb-000011
3、解析结果：
单晶解析结果表明：每个超分子AHU-377-缬沙坦-钠单晶的不对称单元中包含6个缬沙坦、6个AHU-377、18个钠离子和17.5个水分子。该晶体结构是由缬沙坦和AHU-377配位形成的钠盐复合物。这些配位氧原子来自缬沙坦和AHU377分子中12个羧基的氧、18个羰基以及15个水分子中的氧。这些氧原子与18个钠离子配位形成一个配位的网络结构，网络空隙间有2.5个水分子，分子间存在氢键联系，晶态下配位的网络结构以氢键作用力和范德华力维系其在空间的稳定排列。形成一个钠盐复合物的无限的三维网络结构。晶体各个组成部分之间的摩尔比为AHU-377:缬沙坦:钠离子:水分子＝1:1:3:2.9，即所述结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O结构简式中X得取值约为2.9。
更优选的，每个不对称单元均含有6个ARB、6个NEPi、18个钠原子及17.5个水分子，其结构如下：
Figure PCTCN2015089051-appb-000012
更优选的，所述每个不对称单元中为16个配位水分子，2.5个网络空间水分子。
更优选的，所述超分子结构的单晶呈无色片状，单斜晶系，空间群为P21,晶胞参数：
Figure PCTCN2015089051-appb-000013
α＝90°，β＝119.561(2)°，γ＝90°。
更优选的，所述超分子结构的单晶大小为0.42×0.36×0.18mm，晶胞体积
Figure PCTCN2015089051-appb-000014
晶胞内分子数Z＝2，单位晶格的独立区域中有一个分子。
其单晶的具体培养方法：
1.称取约100mg实施例5得到的白色固体，至20-mL玻璃瓶中，加入5mL乙腈，超声将样品充分分散，再加入100uL H2O，60℃水浴加热，将样品溶解至清。
2.趁热将步骤1中得到的样品过滤至5个4-mL的玻璃瓶中，盖紧瓶盖。将步骤2中得到的样品放置循环水槽进行程序降温实验(程序：60℃平衡1小时，用24小时降至20℃)得到上述单晶。其单晶衍射及解析如下：
1、单晶衍射：
用Rigaku Saturn70CCD面探测仪收集衍射强度数据，MoKα辐射，人工多层膜聚焦镜，准直管φ＝0.30mm，晶体与CCD距离为45mm，管压50kV,管流16mA，在φ分别为0，90，180°时采用ω扫描，最大2θ角为56°，扫描范围为0-180°，回摆角度为0.5°，间隔为0.5°，扫描速度为10s/°,每个画面扫描一次，总计摄取1080幅图像，总衍射点为130648个，独立衍射点为68235个(Rint＝0.0479)，可观察点(F2≧2σF2)为53754个，数据完整度为98.9％。
2、单晶解析：
在微机上用直接法(sir-2011)解析晶体结构，从E图上获得全部非氢原子位置，使用最小二乘法修正结构参数和判别原子种类，使用几何计算法和差值Fourier法获得全部氢原子位置，最终可靠因子R1＝0.0651，wR2＝0.1447，S＝1.046，Flack因子为-0.01(10)。最终确定最小化学计量式为C288H365N36Na18O66.50,计算单个分子的分子量为5811.99，计算晶体密度为1.263Mg/m3
晶态下分子排列属P21空间群，分子的绝对构型可以通过Flack因子和已知手型有机分子的绝对构型确定。
Figure PCTCN2015089051-appb-000015
3、解析结果：
结果表明：该化合物分子主要骨架是有机分子缬沙坦和AHU377，形成的Na盐，在不对称单元中有六个缬沙坦，六个AHU-377和18个Na离子，这18个Na离子分别和六个缬沙坦，六个AHU-377中羧基的氧，酰胺基的氧配位，形成一个配位的网络结构，网络空隙间由2.5个水，分子间存在氢键联系，晶态下配 位的网络结构以氢键作用力和范德华力维系其在空间的稳定排列。晶体各个组成部分之间的摩尔比为AHU-377:缬沙坦:钠离子:水分子＝1:1:3:3.1，即所述结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O结构简式中X得取值约为3.1。
本发明另一方面还提供一种结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O的制备方法，包括如下步骤：
(1)将(S)-N-戊酰基-N-{[2′-(1H-四唑-5-基)-联苯-4-基]-甲基}-缬氨酸和(2R，4S)-5-联苯-4-基-4-(3-羧基-丙酰基氨基)-2-甲基-戊酸乙酯溶于适当的有机溶剂；
(2)与碱性钠化合物相混合,加入方式可以直接加入或者将碱性钠化合物溶于适当的溶剂中加入；
(3)自然产生沉淀、降温、加入晶种诱导析晶或通过创造过饱和条件来产生固体沉淀物；
(4)固液分离；
(5)控制干燥环境的湿度烘干步骤(4)分离得到的固体。
作为进一步优选的方案，步骤2)与碱性钠化合物相混合的方式可以在步骤1)体系中直接加入碱性钠化合物或者将碱性钠化合物溶于适当的溶剂中然后再加入步骤1)体系中。
作为进一步优选的方案，步骤(1)中所述有机溶剂包括但不限定于以下溶剂：甲醇、乙醇、正丙醇、异丙醇、正丁醇、乙腈、丙酮、甲乙酮、四氢呋喃、二氧六环、N，N-二甲基甲酰胺、二甲基亚砜、醋酸异丙酯、乙酸乙酯或其混合物。
作为进一步优选的方案，步骤(2)中所述碱性钠化合物选自氢氧化钠、碳酸钠、碳酸氢钠、醋酸钠、甲醇钠、甲酸钠、丙酸钠、丙烯酸钠、苯甲酸钠或其混合物；优选氢氧化钠、碳酸氢钠或其混合物。
作为进一步优选的方案，步骤(3)中所述的创造过饱和条件的方法包括降温析晶、降温-超声耦合析晶、蒸发溶剂析晶、加入反溶剂逼晶或反溶剂替换等方式。反溶剂是指目标固体溶解度较低的溶剂。反溶剂替换方式是指，除去一部分原有溶剂，加入一部分反溶剂，平衡后再除去一部分原有溶剂，直至基本上更换为反溶剂。
作为进一步优选的方案，步骤5)的干燥环境的湿度控制在45％～70％；优选干燥环境的湿度控制在50％～65％；更优选干燥环境的湿度控制在55％～65％。
作为进一步优选的方案，步骤(5)中所述的烘干方法可以采用真空干燥、蒸发、氮气干燥等。
作为更进一步优选的方案，任选的，步骤(5)中得到的固体中水含量为4.6～11.6％，优选4.6～6.6％。
本发明另一方面提供了一种药物组合物，所述药物组合物包含治疗有效剂量的前述结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O以及药学上可接受的载体或赋形剂。
作为更进一步优选的方案，所述的X选自2.5、3、6.5；最优选3。
本发明的另一方面提供了前述结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O或前述其药物组合物在制备治疗或预防与中性内肽酶有关的疾病、心血管、抗高血压的药物中的用途。
通过改善功效和具有更高的相应率而产生了更有效的抗高血压治疗，不论是对于恶性高血压、原发性高血压、肾血管性高血压、糖尿病性高血压、单纯收缩期高血压还是对于其它继发性高血压而言都是如此。
本发明的另一方面提供了前述结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O或前述其药物组合物在制备治疗或预防急慢性心衰如、充血性心衰、左心室机能障碍、肥厚性心肌病、糖尿病性心肌病、室上性和室性心律不齐、心房纤维颤动、心房扑动或有害的血管重构药物中的用途。
本发明的在一方面提供前述结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O或前述其药物组合物在制备治疗或预防心肌梗塞及其后遗症、动脉粥样硬化、心绞痛、糖尿病性或非糖尿病性肾机能不全、继发性醛固酮增多症、原发性或继发性肺高血压、糖尿病性肾病、肾小球肾炎、硬皮病、肾小球硬化、原发性肾病的蛋白尿、肾血管高血压、糖尿病性视网膜病、偏头痛、外周血管病、雷诺氏病、腔的增生、认知机能障碍、青光眼或中风药物中的用途。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/药学上可接受的盐或前体药物与其他化学组分的混合物，以或其他组分例如生理学/药学上可接受的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
与现有技术相比，本发明的优点在于：
本发明制备得到的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基) 丁酸]Na3·X H2O的新晶型X取值为2.5、2.9、3.0、3.1的水合物，较之前公开的2.5水合物，具有更高的配位水分子，实验结果显示具有更好的抗吸湿性，从而明显改善了药物的理化性质，有利于药物的应用与开发。大大降低了该药物制剂、化学稳定性、包装与存储等方面的风险。
附图说明
图1为实施例1中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图2为实施例1中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图3为实施例4中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·6.5H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图4为实施例4中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·6.5H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图5为实施例5中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·3.1H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图6为实施例5中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·3.1H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图7为实施例2中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图8为实施例2中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图9为实施例7中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·3H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图10为实施例7中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·3H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图11为实施例3中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图12为实施例3中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.5H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图13为实施例8中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.9H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图14为实施例8中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.9H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图15为实施例5中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·18.5H2O超分子络合物的晶胞(cell)图示，它包括两个不对称单元。
图16为实施例5中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·18.5H2O超分子络合物单晶的热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图17为实施例6中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·18.5H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图18为实施例6中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·18.5H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图19为实施例8中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·17.5H2O超分子络合物的晶胞(cell)图示，它包括两个不对称单元。
图20为实施例8中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6 Na18·17.5H2O超分子络合物单晶的热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图21为实施例9中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·17.5H2O粉末X射线衍射图；纵坐标为强度，横坐标为2θ(°)。
图22为实施例9中[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·17.5H2O粉末热重分析(TGA)图；纵坐标为重量变化百分比(％)，横坐标为温度(℃)。
图23模型诱导过程中动物体重的变化；纵坐标为动物体重(克)，横坐标为时间(周数)。
图24模型诱导过程中鼠尾动脉收缩压的变化；纵坐标为动物收缩压(毫米汞柱)，横坐标为时间(周数)。
图25模型诱导过程中鼠尾动脉舒张压的变化；纵坐标为动物舒张压(毫米汞柱)，横坐标为时间(周数)。
图26模型诱导过程中鼠尾动脉平均压的变化；纵坐标为平均动脉压(毫米汞柱)，横坐标为时间(周数)。
图27药物A和药物B给药后鼠尾动脉收缩压的变化；纵坐标为动物收缩压(毫米汞柱)，横坐标为时间(周数)。
图28模药物A和药物B给药后鼠尾动脉舒张压的变化；纵坐标为动物舒张压(毫米汞柱)，横坐标为时间(周数)。
图29药物A和药物B给药后鼠尾动脉平均压的变化；纵坐标为平均动脉压(毫米汞柱)，横坐标为时间(周数)。
图30药物C和药物D给药期间动物心率的变化。
图31药物C和药物D给药期间动物收缩压的变化。
图32药物C和药物D给药期间动物舒张压的变化。
图33药物C和药物D给药期间动物平均动脉压的变化。
具体实施方式
以下提供的具体实施例以及制备方法例将进一步举例说明本发明实施方案的特定方面。下列实施例的范围将不以任何方式限制本发明的范围。
方法和材料
结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O由它们的X射线粉末衍射图表征。因此，在具有使用Cu Kα辐射
Figure PCTCN2015089051-appb-000016
以反射方 式操作的GADDS(一般面积衍射检测器系统)CS的Bruker D8 Discover X射线粉末衍射仪上采集所述盐的X射线粉末衍射图。管电压和电流量分别设置为40kV和40mA采集扫描。在3.0°至40°的2θ范围内扫描样品60秒的时期。针对2θ表示的峰位置，使用刚玉标准品校准衍射仪。在通常是20℃-30℃的室温下实施所有分析。使用用于4.1.14T版WNT软件的GADDS，采集和积分数据。使用2003年发行的具有9.0.0.2版Eva的DiffracPlus软件，分析衍射图。XRPD样品的制备，通过是将样品至于单晶硅片上，用玻璃片或等效物压样品粉末以确保样品的表面平坦并有适当的高度。然后将样品支架放入Bruker XRPD仪器，并使用上文描述的仪器参数采集粉末X射线衍射图。由包括以下的多种因素产生与这类X射线粉末衍射分析结果相关的测量差异：(a)样品制备物(例如样品高度)中的误差，(b)仪器误差，(c)校准差异，(d)操作人员误差(包括在测定峰位置时出现的那些误差)，和(e)物质的性质(例如优选的定向误差)。校准误差和样品高度误差经常导致所有峰在相同方向中的位移。一般地说，这个校准因子将使测量的峰位置与预期的峰位置一致并且可以在预期的2θ值±0.2°的范围中。
所得到的[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]三钠水合物晶型用热重分析仪(TGA)进行热分析，所用TGA仪器型号为TA Q500(Thermo Analysis)。TGA分析方法参数如下：温度范围为室温至300摄氏度，扫描速率为10摄氏度每分钟，保护气体为氮气(流速25毫升/分钟)。
所得到的[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O晶型水分含量用卡尔费休库伦水分分析仪进行分析，所用水分分析仪器型号为C20(梅特勒)。操作方法：首先通过开始分析对话框中的开始进行预滴定，待持续测定的漂移值降低至规定值以下，进入样品测定模式。点击按键开始测试样品，从样品中快速取出约20-100mg样品，置于滴定杯中。按下确认键，分析自动开始。被测样品的总重包括皮重已经预先称量得到，再次测量加样后样品的质量包括皮重。总重量减去加样后的重量即为水分测定样品的实际加入量。滴定结束后显示结果对话框，将实际加入量输入，自动得到含水量的结果。
实施例1
称取200mg AHU-377游离酸(油状)，212mg缬沙坦和58.3mg氢氧化钠置于20-mL玻璃瓶中，然后加入10mL醋酸异丙酯，密闭。将该混合物置于0℃的超声波水域中，功率100W，35KHz，反应4小时，得到含白色固体混悬液。将混悬液离心(8000转/分钟)10十分钟，弃去上清液，氮气吹扫干燥剩余固体4小时，氮气流量约5毫升/分钟，得到粉末状固体。其粉末X射线衍射图(XRPD)如图1所示。TGA分析图见图2。TGA图上的失重是由于晶体内水分失水造成的，可以 用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000017
根据水分测定结果可知，所得水合物为2.5水合物。
实施例2
称取151.2mg AHU-377游离酸(油状)，160mg缬沙坦置于50-mL茄形瓶中，加入15.3mL丙酮，再加入2.8mL 15.8mg/mL NaOH，旋转蒸发至干燥，再加入8mL丙酮，室温磁力搅拌24小时，得到含白色固体混悬液。将混悬液离心(8000转/分钟)10十分钟，弃去上清液，氮气吹扫干燥剩余固体4小时，氮气流量约5毫升/分钟，得到粉末状固体。其粉末X射线衍射图(XRPD)如图7所示。TGA分析图见图8。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000018
根据水分测定结果可知，所得水合物为2.5水合物。
实施例3
称取151.2mg AHU-377游离酸(油状)，160mg缬沙坦置于50-mL茄形瓶中，加入15.3mL丙酮，再加入2.8mL 15.8mg/mL NaOH，旋转蒸发至干燥，再加入8mL乙腈，室温磁力搅拌24小时，得到含白色固体混悬液。将混悬液离心(8000转/分钟)十分钟，弃去上清液，氮气吹扫干燥剩余固体4小时，氮气流量约5毫升/分钟，得到粉末状固体。其粉末X射线衍射图(XRPD)如图11所示。TGA分析图见图12。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000019
根据水分测定结果可知，所得水合物为2.5水合物。
实施例4
称取200mg AHU-377游离酸(油状)，212mg缬沙坦和58.3mg氢氧化钠置于20-mL玻璃瓶中，然后加入10mL乙酸乙酯，密闭。将该混合物置于0℃的超声 波水域中，功率100W，35KHz，反应4小时，得到含白色固体混悬液。将混悬液离心(8000转/分钟)十分钟，弃去上清液，氮气吹扫干燥剩余固体4小时，氮气流量约5毫升/分钟，得到粉末状固体。其粉末X射线衍射图(XRPD)如图3所示。TGA分析图见图4。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000020
根据水分测定结果可知，所得水合物为6.5水合物。
实施例5
称取200mg AHU-377游离酸(油状)，212mg缬沙坦和58.3mg氢氧化钠置于20-mL玻璃瓶中，然后加入10mL丙酮，密闭。将该混合物置于0℃的超声波水域中，功率100W，35KHz，反应4小时，得到含白色固体混悬液。将混悬液离心(8000转/分钟)，十分钟，弃去上清液，氮气吹扫干燥剩余固体4小时，氮气流量约5毫升/分钟，得到粉末状固体。其粉末X射线衍射图(XRPD)如图5所示。TGA分析图见图6。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
根据水分测定结果可知，所得水合物为3.1水合物。
其对应单晶的具体培养方法：
1.称取约100mg实施例5得到的白色固体，至20-mL玻璃瓶中，加入5mL乙腈，超声将样品充分分散，再加入100uL H2O，60℃水浴加热，将样品溶解至清。
2.趁热将步骤1中得到的样品过滤至5个4-mL的玻璃瓶中，盖紧瓶盖。
3.将步骤2中得到的样品放置循环水槽进行程序降温实验(程序：60℃平衡1小时，用24小时降至20℃)得到上述单晶。
其超分子络合物的晶胞(cell)图15示，它包括两个不对称单元，其单晶衍射及解析如下：
单晶晶体呈无色片状，衍射分析所用晶体大小为0.42×0.36×0.18mm,属于单斜晶系，空间群为P21,晶胞参数：
Figure PCTCN2015089051-appb-000022
Figure PCTCN2015089051-appb-000023
α＝90°，β＝119.561(2)°，γ＝90°，晶胞体积
Figure PCTCN2015089051-appb-000024
Figure PCTCN2015089051-appb-000025
晶胞内分子数Z＝2，单位晶格的独立区域中有一个分子。
用Rigaku Saturn70 CCD面探测仪收集衍射强度数据，MoKα辐射，人工多层膜聚焦镜，准直管φ＝0.30mm，晶体与CCD距离为45mm，管压50kV,管流16mA，在φ分别为0，90，180°时采用ω扫描，最大2θ角为56°，扫描范围为0-180°，回摆角度为0.5°，间隔为0.5°，扫描速度为10s/°,每个画面扫描一次，总计摄取1080幅图像，总衍射点为130648个，独立衍射点为68235个(Rint＝0.0479)，可观察点(F2≧2σF2)为53754个，数据完整度为98.9％。
在微机上用直接法(sir-2011)解析晶体结构，从E图上获得全部非氢原子位置，使用最小二乘法修正结构参数和判别原子种类，使用几何计算法和差值Fourier法获得全部氢原子位置，最终可靠因子R1＝0.0651，wR2＝0.1447，S＝1.046，Flack因子为-0.01(10)。最终确定最小化学计量式为C288H367N36Na18O66.50,计算单个分子的分子量为5810.93，计算晶体密度为1.263Mg/m3，化学名称为：[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·18.5H2O；化学简式为：[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·3.1H2O
晶态下分子排列属P21空间群，分子的绝对构型可以通过Flack因子和已知手性有机分子的绝对构型确定。
Figure PCTCN2015089051-appb-000026
单晶解析结果表明：该化合物分子主要骨架是有机分子缬沙坦和AHU377，形成的Na盐，在不对称单元中有六个缬沙坦，六个AHU377和18个Na离子，这18个Na离子分别和六个缬沙坦，六个AHU377中羧基的氧，酰胺基的氧配位，形成一个配位的网络结构，网络空隙间由2.5个水，分子间存在氢键联系，晶态下配位的网络结构以氢键作用力和范德华力维系其在空间的稳定排列。
实施例5所得粉晶粉末XRPD测定值与单晶计算值比较如下表所示：
Figure PCTCN2015089051-appb-000027
Figure PCTCN2015089051-appb-000028
从上述数据对比来看，实施例5中所得产品粉末晶体衍射图与单晶计算值高度一致。粉末晶体衍射图中，有少量的峰缺失，这是粉末晶体的择优取向造成的。这一现象对于片状晶体来说尤为明显。但通过现有峰位置对比，显示粉末晶体的晶型与单晶计算晶型图的高度一致性。从而证明单晶结构与粉末晶体结构一致。
所得单晶TGA分析图见图16。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000029
根据TGA和KF测量值可知，所得单晶为3.1水合物，与单晶解析的结果一致。
实施例6
精密称取284g AHU377，300g缬沙坦于50L玻璃反应釜中，加入29L乙腈，机械搅拌溶解化合物得到澄清溶液。精密称取NaOH 82.8g于240mL水溶解。将该NaOH水溶液缓慢加入到玻璃反应釜内，并用机械搅拌快速搅拌。当NaOH水溶液滴加完毕,约4小时，继续搅拌30min后，加入实施例5中得到的2.9水合物的单晶约50mg。继续搅拌直至晶体析出，搅拌过夜后，离心过滤得到白色晶体。所得白色固体在50摄氏度下真空干燥过夜后，置于55％相对湿度下，平衡72小时，得到产品。
其粉末X射线衍射图(XRPD)如图17所示。实施例6所得粉晶粉末XRPD测定值与单晶计算值比较如下表所示：
Figure PCTCN2015089051-appb-000030
Figure PCTCN2015089051-appb-000031
从上述实验数据对比来看，实施例6中所得产品粉末晶体衍射图与实施例5单晶计算值也高度一致，显示晶型的高度一致性。实施例6也与实施例5中粉末晶体测量值也一致。
TGA分析图见图18。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000032
实施例7
称取151.2mg AHU-377游离酸(油状)，160mg缬沙坦置于50-mL茄形瓶中，加入15.3mL丙酮，再加入2.8mL 15.8mg/mL NaOH，旋转蒸发至干燥，再加入8mL乙酸乙酯，室温磁力搅拌24小时，得到含白色固体混悬液。将混悬液离心(8000转/分钟)十分钟，弃去上清液，氮气吹扫干燥剩余固体4小时，氮气流量约5毫升/分钟，得到粉末状固体。其粉末X射线衍射图(XRPD)如图9所示。TGA分析图见图10。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000033
根据水分测定结果可知，所得水合物为3水合物。
实施例8
精密称取283.5mg AHU377，300mg缬沙坦于50-mL茄形瓶中，加入27mL丙酮，加入磁力搅拌子，溶解化合物得到澄清溶液。精密称取NaOH 82.8mg于2-mL HPLC小瓶中，加入240uL水溶解。将该NaOH溶液全部转移到丙酮溶 液中，磁力搅拌。室温下继续磁力搅拌混悬液过夜。室温下，旋转蒸发去大约一半的丙酮溶剂，然后在加入15mL的醋酸异丙酯，继续旋转蒸发去一半体积，再加入15mL醋酸异丙酯，磁力搅拌过夜。氮气保护下抽滤，待滤液滤干后，加入约10mL醋酸异丙酯重新洗涤，氮气保护下抽滤，至干约2小时。
收集干燥滤饼，50℃真空干燥过夜。然后将干燥后的固体转移至60％±5％的湿度，室温下过夜，发生再水合过程，得到稳定的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.9H2O。其粉末X射线衍射图(XRPD)如图13所示。TGA分析图见图14。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000034
根据TGA和KF测量值可知，所得单晶为2.9水合物。
其对应单晶的具体培养方法：
实施例8中制备的510mg样品至20mL玻璃瓶中，加入17mL 98％丙酮/水，在65℃条件下将样品溶解，并平均趁热过滤至17个新的4mL玻璃瓶盖紧瓶盖，放置45℃条件下，恒温放置，得到上述单晶。其超分子络合物的晶胞(cell)图19示，它包括两个不对称单元，其单晶衍射及解析如下：
晶体呈无色片状，衍射分析所用晶体大小为0.36x 0.34x 0.10mm,属于单斜晶系，空间群为P21,晶胞参数：
Figure PCTCN2015089051-appb-000035
Figure PCTCN2015089051-appb-000036
α＝90°，β＝119.278(9)°，γ＝90°，晶胞体积
Figure PCTCN2015089051-appb-000037
晶胞内分子数Z＝2，单位晶格的独立区域中有两个单元。
在Rigaku XtaLAB P200衍射仪上，-100℃时收集衍射强度数据，CuKα辐射，人工多层膜聚焦镜，准直管φ＝0.30mm，晶体与Pilatus200K探测器距离为35mm，管压40kV,管流30mA，ω扫描，最大2θ角为146.9°，扫描范围为0-180°，回摆角度为1°，间隔为1°，扫描速度为5-10s/°,每个画面扫描一次，总计摄取3579幅图像，总衍射点为251362个，独立衍射点为59848个(Rint＝0.0769)，可观察点(F2≧2σF2)为48100个，θ角到67.687°时数据完整度为99.3％。
在微机上用直接法(sir2011)解析晶体结构，从E图上获得全部非氢原子位置，使用最小二乘法(SHELXL-2014/7)修正结构参数和判别原子种类，使用几何计算法和差值Fourier法获得氢原子位置，最终可靠因子R1＝0.0700，wR2＝0.1691，S＝1.070，Flack因子为0.07(2)。最终确定最小化学计量式为C288H365N36Na18O65.50，计算单个分子的分子量为5792.93，计算晶体密度为1.240 g/cm3。化学名称为：[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]6Na18·17.5H2O；化学简式为：[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.9H2O
Figure PCTCN2015089051-appb-000038
单晶解析结果表明：AHU377-缬沙坦-钠单晶的不对称单元中包含6个缬沙坦、6个AHU377、18个钠离子和17.5个水分子。该晶体结构可以看成是由缬沙坦和AHU377配位形成的钠盐复合物。这些配位氧原子来自缬沙坦和AHU377分子中12个羧基的氧、18个羰基以及15个水分子中的氧。这些氧原子与18个钠离子配位形成一个配位的网络结构，网络空隙间有2.5个水分子，分子间存在氢键联系，晶态下配位的网络结构以氢键作用力和范德华力维系其在空间的稳定排列。形成一个钠盐复合物的无限的三维网络结构。晶体各个组成部分之间的摩尔比为AHU377:缬沙坦:钠离子:水分子＝1:1:3:2.9。
单晶中存在无序，18个钠中有一个钠无序，出现在两个位置，这两个位置钠的占有率分别为50％。同时，和无序钠配位的一个缬沙坦配体(除了该配体中联苯四氮唑部分)无序，占有率分别为50％。另外两个缬沙坦配体上脂肪族侧链无序，以及一个AHU377配体上联苯的一个苯环无序，2.5个结晶水当中的0.5个结晶水无序，位于三个位置。
实施例8所得粉晶粉末XRPD测定值与单晶计算值比较如下表所示：
Figure PCTCN2015089051-appb-000039
Figure PCTCN2015089051-appb-000040
从上述实验数据对比来看，实施例8中所得产品粉末晶体衍射图与其对应单晶计算值高度一致。粉末晶体衍射图中，有少量的峰缺失，这是粉末晶体的择优取向造成的。这一现象对于片状晶体来说尤为明显。但通过现有峰位置对比，显示粉末晶体的晶型与单晶计算晶型图的高度一致性。从而证明单晶结构与粉末晶体结构一致。
所得单晶TGA分析图见图20。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000041
根据TGA和KF测量值可知，所得单晶为2.9水合物，与单晶解析结果一致。
实施例9
精密称取284g AHU377，300g缬沙坦于50L玻璃反应釜中，加入27L异丙醇，机械搅拌溶解化合物得到澄清溶液。精密称取NaOH 82.8g于240mL水溶解。将该NaOH水溶液缓慢加入到玻璃反应釜内，并用机械搅拌快速搅拌。当NaOH水溶液滴加完毕,约6小时，继续搅拌30min后，加入实施例8中得到的2.9水合物的单晶约50mg。继续搅拌直至晶体析出，搅拌过夜后，离心过滤得到白色晶体。
其粉末X射线衍射图(XRPD)如图21所示。实施例9所得粉晶粉末XRPD测定值与单晶计算值比较如下表所示：
Figure PCTCN2015089051-appb-000042
从上述实验数据对比来看，实施例9中所得产品粉末晶体衍射图与实施8单晶计算值高度一致，显示晶型的高度一致性。实施例9也与实施例8中粉末晶体测量值也一致。
TGA分析图见图22。TGA图上的失重是由于晶体内水分失水造成的，可以用于精确的晶体中的水分含量测定。此外，用KF水分法测定晶体内水分含量做参照。所得结果，见下表：
Figure PCTCN2015089051-appb-000043
根据TGA和KF测量值可知，所得单晶为2.9水合物。
实施例10
比较不同水合物的吸湿性。将[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O各种结晶放置在HPLC小瓶中，敞口放置在57％的湿度下(溴化钠饱和溶液)，观察吸湿情况及样品液化时间。
Figure PCTCN2015089051-appb-000044
实施例11
[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O
(药物A)与缬沙坦(药物B)组合治疗在肾动脉狭窄诱导的大鼠高血压模型的药效实验
1 实验材料：
药物A：[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·3.1H2O按照实施例6制备获得。
药物B：缬沙坦为市售原料药。
2 实验设备
动物体重秤：JJ500,G&G,Jiangsu,China
低温电热毯：Jwilch,China
手术显微镜：Luckbird XTS-4A
鼠尾血压测量仪：BP-2010AUL.CN10120479
大鼠灌胃器：16#80mm直径，圆头
3 实验方法
3.1 单侧肾动脉缩窄诱导大鼠肾性高血压模型建立
雄性Sprague Dawley(SD)大鼠50只，体重180-200克，由北京维通利华公司(Vital River Laboratories,Beijing,China)提供。动物饲养于动物中心SPF级屏障系统内，遵循国际标准温、湿、光控制系统。本实验动物操作方案经由该平台动物中心IACUC委员会审批确认。
在KCI动物手术SOP指导原则下实施本实验所涉及到的一切手术。动物经戊巴比妥钠腹腔麻醉(50mg/kg)，确认动物进入麻醉状态后，左侧腹部备皮，严格手术消毒，左侧腹部肾区逐层切开一2.0cm长手术切口，进入腹腔。暴露左侧肾脏。小心分离左肾动脉，使其与肾静脉彻底分离。采用3-0手术缝合丝线实行左肾动脉缩窄术(实现左肾动脉60％狭窄)。术后确认没有腹腔内出血，用3-0手术缝合丝线缝合腹壁肌层组织，然后用2-0手术缝合丝线缝合皮肤。清洗皮肤创面后将动物置于37度电热毯保温至动物苏醒，确认能够自由采食和饮水后将动物返回饲养笼正常饲养。术后第一周内每天观察动物，确认无术后伤口感染以及裂开。自术后第一周起每周测试动物体重变化以及清醒状态下鼠尾动脉压和心率以监测动物血压和心率的变化。
3.2 实验动物入组标准
动物连续4-5周测试鼠尾动脉压观察动物血压升高的趋势。选择收缩压超过160mmHg的动物入组进行药效学试验
3.3 实验方案
药物A([3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·3.1H2O)、药物B(缬沙坦)分别进行单次口服给药实验，早9-10am开始给药，次日10:00AM试验结束。24小时内分别观察给药后2-4HR、6-8HR和24HR动物的血压的变化。组-1为给药组，组-2为空白对照组(只给药物载体)
Figure PCTCN2015089051-appb-000045
3.4 给药期间检测
给药后密切观测所有动物口服给药后的生理变化，主要为包括呕吐和腹泻。
4 实验结果
4.1 单侧肾动脉狭窄诱导高血压的模型建立
4.1.1 动物体重变化
模型手术后连续4-5周每周测量一次动物体重。动物体重呈现逐渐增长趋势，至术后五周动物平均体重达到400克左右(见图23)。
4.1.2 动物血压变化
造模后每周监测一次清醒状态下鼠尾动脉压。造模第一周起动物开始出现血压升高。连续4-5周的鼠尾动脉压监测显示动物血压自造模后3周起趋于维持在稳定升高的状态(图24-26)。
4.2.1 给药期间动物基本生理观察
所有动物在给药期间并未出现异常生理变化，包括胃肠道反应诸如呕吐、腹泻、采食异常等，精神症状反应诸如精神萎靡、运动异常等。
4.2.2 给药期间动物血压变化
药物A和B给药2-4小时后动物血压呈现轻度下降，包括收缩压、舒张压以及平均动脉压，6-8小时达到最佳降压效果；24小时呈现轻度回升(见图27-29)。药物A的降压效果优于药物B的降压效果。
实施例12
[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O与缬沙坦(药物D)组合治疗在肾动脉狭窄诱导的大鼠高血压模型的药效实验
1 实验材料
药物C：[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·2.9H2O按照实施例9制备获得。
药物D：缬沙坦为市售原料药。
2 实验设备
动物体重秤：JJ500,G&G,Jiangsu,China
低温电热毯：Jwilch,China
手术显微镜：Luckbird XTS-4A
鼠尾血压测量仪：BP-2010AUL.CN10120479
大鼠灌胃器：16#80mm直径，圆头
3 实验方法
3.1 单侧肾动脉缩窄诱导大鼠肾性高血压模型建立
雄性Sprague Dawley(SD)大鼠50只，体重180-200克，由北京维通利华公司(Vital River Laboratories,Beijing,China)提供。动物饲养于动物中心SPF级屏障系统内，遵循国际标准温、湿、光控制系统。本实验动物操作方案经由该平台动物中心IACUC委员会审批确认。
在KCI动物手术SOP指导原则下实施本实验所涉及到的一切手术。动物经戊巴比妥钠腹腔麻醉(50mg/kg)，确认动物进入麻醉状态后，左侧腹部备皮，严格手术消毒，左侧腹部肾区逐层切开一2.0cm长手术切口，进入腹腔。暴露左侧肾脏。小心分离左肾动脉，使其与肾静脉彻底分离。采用3-0手术缝合丝线实行左肾动脉缩窄术(实现左肾动脉60％狭窄)。术后确认没有腹腔内出血，用3-0手术缝合丝线缝合腹壁肌层组织，然后用2-0手术缝合丝线缝合皮肤。清洗皮肤创面后将动物置于37度电热毯保温至动物苏醒，确认能够自由采食和饮水后将动物返回饲养笼正常饲养。术后第一周内每天观察动物，确认无术后伤口感染以及裂开。自术后第一周起每周测试动物体重变化以及清醒状态下鼠尾动脉压和心率以监测动物血压和心率的变化。
3.2 实验动物入组标准
动物连续4-5周测试鼠尾动脉压观察动物血压升高的趋势。选择收缩压超过160mmHg的动物入组进行药效学试验
3.3 实验方案
选取动物64只，SBP>160mmHg；实验分为4组，假手术组(组-1)、高血压模型组(组-2)、对照组(药物D)(组-3)、给药组药物C(组-4)。口服给药每天一次，药物体积10mL/kg，持续给药一周，每三天进行一次称重，并检测动物给药之后6-8hr和24hr的血压变化。
Figure PCTCN2015089051-appb-000046
4 给药期间检测
动物每天早9-10am开始给药。给药之后密切观察动物给药之后的生理变化，主要为临床表征，如有异常现象要及时记录和分析；分别于给药后6-8hr(15:30-17:30P.M)和24hr(次日09:30-11:30)检测大鼠尾动脉血压和心率，实验持续一周。
5 试验终了
试验最后一次数据采集终了后，按照KCI动物安乐死SOP对所有实验动物实行安乐死，并采集血浆(肝素抗凝剂)和组织：血样2份，每份1mL，肾脏(右肾)2份，肾上腺2份，心脏(心室两份、心房2份)，所有组织均液氮速冻，保存于-80度冰箱。
6 实验结果
6.1 单侧肾动脉狭窄诱导高血压的模型建立成功，与实施例11的4.1结果相同。
6.2 给药期间动物基本生理观察
所有动物在连续一周的给药期间并未出现异常生理变化，包括胃肠道反应诸如呕吐、腹泻、采食异常等，精神症状反应诸如精神萎靡、运动异常等。
6.3 给药期间动物心率变化
假手术组(组-1)连续一周的动物心率观察显示平均心率在330次/分到350次/分之间波动。高血压动物给药前平均心率在380次/分左右。模型组(组-2)和药物D组，动物心率在连续给药一周期间未表现出明显的加速或减慢。药物C组(组4)动物连续一周给药的动物心率显示平稳缓慢减缓，至给药3天后与假手术组动物心率一致。之后连续一周的给药动物心率呈现持续平稳的减缓(见图30)。
6.4 给药期间动物血压变化
试验的假手术组(组-1)动物血压在连续一周的试验中维持在正常范围，包括收缩压，舒张压和平均动脉压。相对模型组(组-2)而言，药物D(组-3)的动物血压在连续一周的给药过程中呈现波动性缓慢下降表现为每次给药后6-8小时降压效果最为明显，24小时后轻度回升。药物C组(组-4)，动物血压在连续一周的给药试验中呈现一致性、波动性缓慢下降，表现为每次给药后6-8小时降压效果最为明显，24小时后轻度回升，包括收缩压，舒张压和平均动脉压(见图31-33)。
[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O(药物C)具有减缓动物心率的功能，起效快，首次给药即减缓心率至假手术组动物的水平。连续一周的给药使得高血压动物的心率平稳在假手术组动物的平均心率水平。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非限制本发明，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围内。

Claims (29)

  1. 结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·XH2O，其粉末X射线衍射图包括位于:4.1±0.2°，4.9±0.2°，5.2±0.2°，9.6±0.2°，12.5±0.2°，16.9±0.2°，18.1±0.2°，19.4±0.2°和27.1±0.2°的衍射角(2θ)处的峰。
  2. 根据权利要求1所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，其粉末X射线衍射图还包括位于8.3±0.2°，15.4±0.2°，17.4±0.2°，18.8±0.2°，20.0±0.2°，21.3±0.2°，22.6±0.2°，22.9±0.2°，24.7±0.2°，25.2±0.2°，25.7±0.2°，27.8±0.2°,和29.9±0.2°的衍射角(2θ)处的峰；
    或者还包括位于7.9±0.2°，14.7±0.2°，15.5±0.2°，16.1±0.2°，20.2±0.2°，20.8±0.2°，25.5±0.2°，27.7±0.2°，27.9±0.2°和29.2±0.2°的衍射角(2θ)处的峰；
    或者还包括位于8.9±0.2°，14.5±0.2°，15.2±0.2°，16.0±0.2°，25.8±0.2°和27.9±0.2°的衍射角(2θ)处的峰；
    或者还包括位于14.7±0.2°，15.1±0.2°，17.6±0.2°，18.7±0.2°，22.6±0.2°，23.2±0.2°，24.5±0.2°和29.1±0.2°的衍射角(2θ)处的峰；
    或者还包括位于6.3±0.2°，13.6±0.2°，14.2±0.2°，14.8±0.2°，15.2±0.2°，17.6±0.2°，18.7±0.2°，22.6±0.2°，23.3±0.2°，24.6±0.2°和29.2±0.2°的衍射角(2θ)处的峰。
  3. 根据权利要求1所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，其粉末X射线衍射图与图1、图5、图17、图13、图21中显示的衍射角(2θ)处的峰基本上相同。
  4. 根据权利要求1所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基} 氨基)丁酸]Na3·X H2O，其特征在于，所述X选自2.0～9.0，优选自2.5～6.5，更优选自2.5～3.5，最优选自2.5、3.0±0.2。
  5. 根据权利要求1-4中任一项所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，所述结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O是超分子结构的结构简式，其包含不对称单位，每个不对称单位均含有6个ARB、6个NEPi、18个钠原子及12-54个水分子，其中所述的ARB为(S)-N-戊酰基-N-{[2’-(1H-四唑-5-基)联苯-4-基]甲基}-缬氨酸，所述的NEPi为(2R，4S)-5-联苯-4-基-4-(3-羧基-丙酰基氨基)-2-甲基-戊酸乙基酯；优选所述每个不对称单位含有15-39个水分子；更优选所述每个不对称单位含有15个水分子、39个水分子或18±0.5个水分子。
  6. 根据权利要求5所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，所述每个不对称单元含有17.5个水分子，其结构如下：
    Figure PCTCN2015089051-appb-100001
  7. 根据权利要求6所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，所述每个不对称单元中包含15个配位水分子，2.5个网络空间水分子。
  8. 根据权利要求6所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，所述超分子结构的单晶呈无色片状，单斜晶系，空间群为P21,晶胞参数：
    Figure PCTCN2015089051-appb-100002
    Figure PCTCN2015089051-appb-100003
    α＝90°，β＝119.278(9)°，γ＝90°。
  9. 根据权利要求6所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，所述超分子结构的单晶大小为0.36x0.34x0.10mm，晶胞体积
    Figure PCTCN2015089051-appb-100004
    晶胞内单元数Z＝2，单位晶格的独立区域中有两个单元。
  10. 根据权利要求5所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，所述每个不对称单元含有18.5个水分子，其结构如下：
    Figure PCTCN2015089051-appb-100005
  11. 根据权利要求10所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，其特征在于，所述每个不对称单元中包含16个配位水分子，2.5个网络空间水分子。
  12. 根据权利要求10所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，当X＝3.1时，其特征在于，所述超分子结构的单晶呈无色片状，单斜晶系，空间群为P21,晶胞参数：
    Figure PCTCN2015089051-appb-100006
    Figure PCTCN2015089051-appb-100007
    α＝90°，β＝119.561(2)°，γ＝90°。
  13. 根据权利要求10所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基
    羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O，当X＝3.1时，其特征在于，所述超分子结构的单晶大小为0.42×0.36×0.18mm，晶胞体积
    Figure PCTCN2015089051-appb-100008
    晶胞内单元数Z＝2，单位晶格的独立区域中有2个单元。
  14. 一种根据权利要求1-13中任一项所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O的制备方法，包括如下步骤：
    1)将(S)-N-戊酰基-N-{[2′-(1H-四唑-5-基)-联苯-4-基]-甲基}-缬氨酸和(2R，4S)-5-联苯-4-基-4-(3-羧基-丙酰基氨基)-2-甲基-戊酸乙酯溶于适当的有机溶剂；
    2)与碱性钠化合物相混合,加入方式可以直接加入或者将碱性钠化合物溶于适当的溶剂中加入；
    3)自然产生沉淀、加入晶种诱导析晶或通过创造过饱和条件来产生固体沉淀物；
    4)固液分离；
    5)控制干燥环境的湿度烘干步骤4)分离得到的固体。
  15. 根据权利要求14所述的制备方法，其特征在于，步骤5)的干燥环境的湿度控制在45％～70％；优选干燥环境的湿度控制在50％～65％；更优选干燥环境的湿度控制在55％～65％。
  16. 根据权利要求14所述的制备方法，其特征在于，步骤2)与碱性钠化合物相混合的方式可以在步骤1)体系中直接加入碱性钠化合物或者将碱性钠化合物溶于适当的溶剂中然后再加入步骤1)体系中。
  17. 根据权利要求14所述的制备方法，其特征在于，步骤1)中所述有机溶剂选自甲醇、乙醇、正丙醇、异丙醇、正丁醇、乙腈、丙酮、甲乙酮、四氢呋喃、二氧六环、N，N-二甲基甲酰胺、二甲基亚砜、醋酸异丙酯、乙酸乙酯或其混合物。
  18. 根据权利要求14所述的制备方法，其特征在于，步骤2)中所述碱性钠化合物选自氢氧化钠、碳酸钠、碳酸氢钠、醋酸钠、甲醇钠、甲酸钠、丙酸钠、丙烯酸钠、苯甲酸钠或其混合物；优选氢氧化钠、碳酸氢钠或其混合物。
  19. 根据权利要求14所述的制备方法，其特征在于，步骤3)中所述的创造过饱和条件的方法选自降温析晶、降温-超声耦合析晶、蒸发溶剂析晶、加入反溶剂逼晶或反溶剂替换方法。
  20. 根据权利要求14所述的制备方法，其特征在于，步骤5)中所述的烘干方法选自真空干燥、蒸发、氮气干燥。
  21. 根据权利要求14所述的制备方法，其特征在于，在步骤5)中控制湿度得到 相应水合物后进一步充分暴露在特定湿度下进行水合得到目标水合度的水合物。
  22. 根据权利要求14所述的制备方法，其特征在于，步骤5)中得到的固体中水含量为4.6～11.6％，优选4.6～6.6％。
  23. 药物组合物，其特征在于，所述药物组合物包含治疗有效剂量的根据权利要求1-13中任一项所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O以及药学上可接受的载体或赋形剂。
  24. 根据权利要求23所述的药物组合物，其特征在于，所述的X选自2.5、3、6.5；优选自3±0.2。
  25. 根据权利要求23所述的药物组合物，其特征在于，所述药物组合物包含治疗有效剂量的权利要求5-13任一项所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O以及药学上可接受的载体或赋形剂。
  26. 根据权利要求1-13中任一项所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O或权利要求23-25中任一项所述的药物组合物在制备治疗或预防与中性内肽酶有关的疾病、心血管、抗高血压的药物中的应用。
  27. 根据权利要求26所述的应用，其特征在于，抗高血压选自抗恶性高血压、原发性高血压、肾血管性高血压、糖尿病性高血压、单纯收缩期高血压或其它继发性高血压。
  28. 根据权利要求1-13中任一项所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O或权利要求23-25中任一项所述的药物组合物在制备治疗或预防急慢性心衰如、充血性心衰、左心室机能障碍、肥厚性心肌病、糖尿病性心肌病、室上性和室性心律不齐、心房纤维颤动、心房扑动或有害的血管重构药物中的应用。
  29. 根据权利要求1-13中任一项所述的结晶型[3-((1S，3R)-1-联苯-4-基甲基-3-乙氧基羰基-1-丁基氨甲酰基)丙酸-(S)-3’-甲基-2’-(戊酰基{2”-(四唑-5-基)联苯-4’-基甲基}氨基)丁酸]Na3·X H2O或权利要求23-25中任一项所述的药物组合 物在制备治疗或预防心肌梗塞及其后遗症、动脉粥样硬化、心绞痛、糖尿病性或非糖尿病性肾机能不全、继发性醛固酮增多症、原发性或继发性肺高血压、糖尿病性肾病、肾小球肾炎、硬皮病、肾小球硬化、原发性肾病的蛋白尿、肾血管高血压、糖尿病性视网膜病、偏头痛、外周血管病、雷诺氏病、腔的增生、认知机能障碍、青光眼或中风药物中的应用。
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WO2018069833A1 (en) 2016-10-10 2018-04-19 Laurus Labs Limited Stable amorphous form of sacubitril valsartan trisodium complex and processes for preparation thereof
WO2018178295A1 (en) 2017-03-31 2018-10-04 Alfred E. Tiefenbacher (Gmbh & Co. Kg) Stable hot-melt extrudate containing valsartan and sacubitril
WO2019073062A1 (en) 2017-10-13 2019-04-18 Alfred E. Tiefenbacher (Gmbh & Co. Kg) TABLET CONTAINING VALSARTAN AND SACUBITRIL
WO2019180735A1 (en) * 2018-03-19 2019-09-26 Natco Pharma Limited Stable pharmaceutical compositions comprising sacubitril-valsartan complex
CN110713465A (zh) * 2017-01-03 2020-01-21 上海博志研新药物技术有限公司 ARB-NEPi复合物、晶型、制备方法及应用
WO2020039386A1 (en) 2018-08-23 2020-02-27 Novartis Ag New pharmaceutical use for the treatment of heart failure
WO2020039394A1 (en) 2018-08-24 2020-02-27 Novartis Ag New drug combinations
US10668035B2 (en) 2018-02-07 2020-06-02 Novartis Ag Substituted bisphenyl butanoic ester derivatives as NEP inhibitors
CN111253330A (zh) * 2020-02-29 2020-06-09 广州白云山天心制药股份有限公司 沙坦类药物的新晶型及其制备方法和用途
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US11642329B2 (en) 2005-11-09 2023-05-09 Novartis Pharmaceuticals Corporation Amorphous solid form of compounds containing S—N-valeryl-N- {[2′-( 1 H-tetrazole-5-yl)-biphenyl-4-yl]-methyl}-valine and (2R,4S)-5-biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoic acid ethyl ester moieties and sodium cations
US11096918B2 (en) 2005-11-09 2021-08-24 Novartis Pharmaceuticals Corporation Amorphous solid form of compounds containing S—N-valeryl-N-{[2′-(1H-tetrazole-5-yl)-biphenyl-4-yl]-methyl}-valine and (2R,4S)-5-biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoic acid ethyl ester moieties and sodium cations
WO2017009784A1 (en) 2015-07-14 2017-01-19 Cadila Healthcare Limited Solid state forms of trisodium salt of valsartan/sacubitril complex and sacubitril
WO2018069833A1 (en) 2016-10-10 2018-04-19 Laurus Labs Limited Stable amorphous form of sacubitril valsartan trisodium complex and processes for preparation thereof
US11318116B2 (en) 2016-10-10 2022-05-03 Laurus Labs Limited Stable amorphous form of sacubitril valsartan trisodium complex and processes for preparation thereof
US10857132B2 (en) 2016-10-10 2020-12-08 Laurus Labs Limited Stable amorphous form of sacubitril valsartan trisodium complex and processes for preparation thereof
CN111592500A (zh) * 2017-01-03 2020-08-28 上海博志研新药物技术有限公司 ARB-NEPi复合物、晶型、制备方法及应用
CN110713465A (zh) * 2017-01-03 2020-01-21 上海博志研新药物技术有限公司 ARB-NEPi复合物、晶型、制备方法及应用
WO2018178295A1 (en) 2017-03-31 2018-10-04 Alfred E. Tiefenbacher (Gmbh & Co. Kg) Stable hot-melt extrudate containing valsartan and sacubitril
WO2019073062A1 (en) 2017-10-13 2019-04-18 Alfred E. Tiefenbacher (Gmbh & Co. Kg) TABLET CONTAINING VALSARTAN AND SACUBITRIL
US10668035B2 (en) 2018-02-07 2020-06-02 Novartis Ag Substituted bisphenyl butanoic ester derivatives as NEP inhibitors
US11426375B2 (en) 2018-02-07 2022-08-30 Novartis Ag Substituted bisphenyl butanoic ester derivatives as NEP inhibitors
WO2019180735A1 (en) * 2018-03-19 2019-09-26 Natco Pharma Limited Stable pharmaceutical compositions comprising sacubitril-valsartan complex
WO2020039386A1 (en) 2018-08-23 2020-02-27 Novartis Ag New pharmaceutical use for the treatment of heart failure
WO2020039394A1 (en) 2018-08-24 2020-02-27 Novartis Ag New drug combinations
CN111253330A (zh) * 2020-02-29 2020-06-09 广州白云山天心制药股份有限公司 沙坦类药物的新晶型及其制备方法和用途

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