TWI684449B - Arb-nepi crystallization complex, the preparation and use thereof - Google Patents

Abstract

The present invention discloses ARB-NEPi crystallization complex, the preparation and use thereof. The X-ray power diffraction pattern of ARB-NEPi complex which is [3-((1S,3R)-1-biphenyl-4-yl-methyl-3-ethoxycarbonyl-1-butyl-carbamoyl) propanoic acid-(S)-3’-methyl-2’-(pentanoyl {2’-(tetrazol-5-yl) biphenyl-4’-yl methyl} amino) butyrate] Na3‧XH₂O is defined as the description. The present invention also discloses drug combinations containing the effect dosage of [3-((1S,3R)-1-biphenyl-4-yl-methyl-3-ethoxycarbonyl-1-butyl-carbamoyl) propanoic acid-(S)-3’-methyl-2’-(pentanoyl{2’-(tetrazol-5-yl) biphenyl-4’-yl methyl} amino) butyrate] Na3‧XH₂O and their use of treating or preventing diseases related to neutral endopeptidase, cardiovascular, hypertension, acute and chronic heart failure, congestive heart failure, left ventricular dysfunction, hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter or harmful vascular remodeling with broad application prospect.

Description

Crystalline ARB-NEPi composite and its preparation method and application

The present invention belongs to the technical field of medicine. Specifically, the present invention relates to a double-acting new crystal form of ARB-NEPi complex and its preparation method and application.

Neutral endopeptidase (EC3.4.24.11; enkephalinase; bipeptidase; NEP) is a zinc-containing metalloprotease that can cleave various peptide substrates at the amino terminus of aromatic amino acids. The substrates of this enzyme include, without limitation, atrial natriuretic peptide (ANF, also known as ANP), brain natriuretic peptide (BNP), met and leu enkephalin, bradykinin, neurokinin A and P substances.

ANF is a vasodilator, diuretic and antihypertensive peptide. One type, ANF99-126, is a circulating peptide hormone released by the heart in the case of heart dilation. The function of ANF is to maintain the balance of salt and water in the body and regulate blood pressure. ANF is rapidly deactivated in the circulation by at least two processes: receptor-mediated clearance and enzyme deactivation at NEP. NEP inhibitors enhance hypotension, diuresis, urinary sodium excretion, and plasma ANF response after pharmacological ANF injection in experimental animals. Enhancement of ANF by two specific NEP inhibitors. In 1988, US Patent No. 4,749,688 generally disclosed that NEP can be used to enhance ANF. In the same year, the US patent US4740499 It is disclosed in the application that thiorphan and keratophan can also be used to enhance atrial peptides. In addition, NEP inhibitors can lower blood pressure and exert ANF-like effects such as diuresis in some forms of experimental hypertension and the effect of increasing the excretion of cyclic guanosine 3',5'-monophosphate (cGMP). Because ANF antibodies will counteract the decrease in blood pressure, the antihypertensive effect of NEP inhibitors is mediated by ANF. Long-term and uncontrolled hypertensive vascular disease will eventually lead to various pathological changes in target organs such as heart and kidney. Persistent hypertension can also lead to an increased incidence of stroke. Therefore, there is a strong need to evaluate the efficacy of antihypertensive therapy, that is, to examine other cardiovascular end points in addition to blood pressure reduction to further discover the benefits of combination therapy.

In 1993, Gary Ksander et al. disclosed a class of biaryl-substituted 4-aminobutyric acid derivatives in US Patent No. 5,217,996. Such compounds were found to have significant NEP inhibitory activity. The most representative one is N -(3-Carboxy-1-oxopropyl)-(4S)-p-phenylphenylmethyl)-4-amino-2R-methylbutyric acid ethyl ester (also known as AHU-377).

Vasopressin II is a hormone that causes vasoconstriction, which in turn causes high blood pressure and heart strain. It is known that vasopressin II can interact with receptors on the surface of target cells. Has been identified vascular plus The two receptor subtypes of vasopressin II are called AT1 and AT2. Recently, people have made great efforts to identify substances that can bind to the AT1 receptor. It is now known that vasopressin receptor blockers (ARBs, vasopressin II antagonists) can lower blood pressure by preventing vasopressin II from binding to its receptor on the vessel wall. Because of their ability to inhibit AT1 receptors, such antagonists can be used to combat hypertension or to treat congestive heart failure and other indications.

In 2008, Chinese patent CN101098689 disclosed a double-acting complex (especially supramolecular complex) of vasopressin antagonist (ARB) and neutral endopeptidase inhibitor (NEPi), which has the following general formula: Description: [ARB(NEPi)]Na _1-3 ‧xH ₂ O, where x is 0-3, preferably 2.5, specifically [3-((1S,3R)-1-biphenyl-4-ylmethyl Yl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl) Biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧2.5H ₂ O (LCZ696), the simplified structure is as follows:

Also disclosed is the crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3 '-Methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧2.5H ₂ O(LCZ696) The data of crystalline powder diffraction of is:

For a given substance, a good method to verify the average lattice spacing and intensity of the lattice obtained by the X-ray diffraction real detection method is to check whether these values are consistent with the analytical calculation data of the complex single crystal structure.

The unit cell constant and atomic position can be obtained by measuring the structure of the single crystal, and the single crystal diffraction pattern can be corresponding to the corresponding solid through the computer-aided calculation method. The program used is the one in the application software Materials Studio (Accelrys) Powder Pattern. The following table shows the comparison of these data obtained by XRPD measurement and calculation of single crystal data, namely [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butan Carbamoylamino) propionic acid-(S)-3'-methyl-2'-(pentylamino{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino ) Butyric acid] Trisodium hemipentahydrate important data plane spacing and strength XRPD measured value and single crystal calculated value of the data comparison.

The patent also discloses crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S) -3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧2.5H ₂ O The melting initial temperature (onset temperature) and the maximum peak temperature (Peak temperature) were 139°C and 145°C, respectively.

Such compounds can also be qualitatively obtained by using Fourier transform attenuated total reflection infrared spectroscopy (ATR-FTIR) spectrometer (Nicolet Magna-IR 560) spectrometer (Nicolet Magna-IR 560), using the important bands described below (inverse of the wavelength data (cm ^-1 ) said) 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). The complex is particularly characterized by the following peaks: 1711 (st), 1637 (st), 1597 (st) and 1401 (st). The error of all absorption bands of ATR-IR is ±2cm ^-1 . The intensity of the absorption band is expressed as follows: (w) = weak; (m) = medium; and (st) = strong.

Such complexes can also be characterized by Raman chromatography measured by a Raman chromatograph with a 785 nm laser excitation source (Kaiser Optical Systems, Inc.), with the following important bands (in reciprocal of wavelength data (cm ^-1 ) Means: 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 ± 2cm ^-1 . The intensity of the absorption band is expressed as follows: (w) = weak; (m) = medium; and (st) = strong.

The centerless spacer group P21 determined by the single crystal X-ray structure is a conventional spacer group of pure molecules of mirror image isomers. There are two general positions in the spacer group, which means that for the 12 structural units in the unit cell, there must be 18 sodium ions and 15 water in the asymmetric unit.

Based on the single crystal structure analysis, [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)- 3'-methyl-2'-(pentyl acetyl {2"-(Tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid] trisodium hemipentahydrate supramolecular asymmetric unit each contains 6 ARB and NEPi moieties , 18 sodium atoms and 15 water molecules. [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate) propionic acid -(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]trisodium penta Hydrates can be called sodium supramolecular complexes and can be coordinated by oxygen ligands. These oxygens come from the 12 carboxylate groups and 18 carbonyl groups in the above part and 13 of the 15 water molecules. The crystal is an infinite three-dimensional network structure of these sodium complexes.

Chinese patent CN101098689 discloses a dual-acting complex of vasopressin antagonist (ARB) and neutral endopeptidase inhibitor (NEPi), specifically [3-((1S,3R)-1-biphenyl- 4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazole- 5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧2.5H ₂ O (LCZ696). The physical and chemical properties of LCZ696 have obvious shortcomings, that is, they are easily halogenated at higher humidity ( Solids absorb water and liquefy.) In pharmacy, the solid halogenation of compounds has a very adverse effect on drug development and products. For example, it is difficult to make solid oral preparations after hygroscopic liquefaction, and the drug content is difficult to accurately quantify. During the tabletting process, sticky punches and plaques are easily generated in the tablet, and the quality of the hard formulation is also generated. In addition, the chemical properties of the compound after liquefaction are not stable enough to affect the therapeutic effect. Therefore, the liquefaction of the compound has an adverse effect on all aspects of the pharmaceutical product. Therefore, there is an urgent need to find a new and more stable new crystal form or new hydrate of LCZ696 to improve or overcome the halogenation problem. In the present invention, the new hydrate and its crystalline state are found, which are the same as the three reported in CN101098689 in terms of hygroscopicity. Compared with sodium hemipentahydrate (LCZ696), it has made significant progress and improvement.

In order to solve the defects in the existing technology, the inventor proposed a new crystal form of ARB-NEPi complex and its preparation method and application in the technology of in-depth research. ARB-NEPi complex refers to [3-((1S,3R) -1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl-2'-(pentylacetate{2 "-(Tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O, the new crystal form and hydrate obtained by the present invention have better physical and chemical properties, The halogenation property of the crystal form in the prior art is improved, which is more conducive to the pharmacologically developable crystal form, easy to preserve, stable and reliable quality, and its preparation method can be industrially applied and has broad medical prospects.

One aspect of the present invention provides a crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-( S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O , Its powder X-ray diffraction pattern includes: 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 The peak at a diffraction angle (2θ) of 27.1±0.2°.

As a further preferred solution, the powder X-ray diffraction pattern preferably further includes 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± Peaks at diffraction angles (2θ) of 0.2°, 22.9±0.2°, 24.7±0.2°, 25.2±0.2°, 25.7±0.2°, 27.8±0.2°, and 29.9±0.2°.

Most preferably, the powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in Figure 1, and the X-ray powder diffraction data are shown in Table 1 :

As a further preferred solution, the powder X-ray diffraction pattern measured by the Burker D8 powder diffractometer includes the following lattice plane spacing: D(Å) 21.6±0.1, 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.

As a further preferred solution, its powder X-ray diffraction pattern is also preferred Including: 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° and 29.2± The peak at the diffraction angle (2θ) of 0.2°; preferably, the powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in FIG. 5.

As a further preferred solution, the powder X-ray diffraction pattern preferably further includes diffraction 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 the angle (2θ); preferably, the powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in Figure 17. As a further preferred solution, the powder X-ray diffraction pattern preferably further includes: 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 Peaks at the diffraction angle (2θ) of ±0.2° and 29.1±0.2°; preferably, the powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in FIG. 13.

As a further preferred solution, the powder X-ray diffraction pattern preferably further includes: 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 Peaks at diffraction angles (2θ) of ±0.2°, 22.6±0.2°, 23.3±0.2°, 24.6±0.2°, and 29.2±0.2°; preferably, the powder X-ray diffraction pattern shown in Figure 21 The peaks at the diffraction angle (2θ) are basically the same.

The term "substantially the same" as used herein with respect to X-ray diffraction peak positions means that typical peak positions and intensity variability are considered. For example, those skilled in the art will understand that the peak position (2θ) will vary due to different XRPD instruments, and sometimes this variation may be as much as 0.2°. In addition, those skilled in the art will understand that the XRPD sample preparation method, Factors such as XRPD instrument, sample crystallinity, sample dosage, and preferred crystal orientation will cause changes in the relative peak intensity in the XRPD diffraction pattern of the sample.

As a further preferred solution, X is selected from 2.0 to 9.0, preferably from 2.5 to 6.5, more preferably from 2.5 to 3.5, more preferably from 2.5 to 3.5, and most preferably from 2.5, 3.0±0.2.

As a further preferred solution, the present invention provides a crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate Group) Propionic acid-(S)-3'-methyl-2'-(pentylacetate{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-butylcarbamate) propionic acid -(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O is a simple structural formula of supramolecular structure, which contains asymmetric units, each asymmetric unit contains 6 ARB, 6 NEPi, 18 sodium atoms and 12-54 water molecules, wherein the ARB is (S)-N-pentyl-N-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-valine, the NEPi is (2R, 4S)-5-Biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoic acid ethyl ester.

Preferably, each asymmetric unit contains 6 ARBs, 6 NEPis, 18 sodium atoms and 15-39 water molecules.

More preferably, each asymmetric unit contains 6 ARBs, 6 NEPis, 18 sodium atoms, and 15 water molecules or 18±0.5 water molecules.

More preferably, each asymmetric unit contains 6 ARBs, 6 NEPis, 18 sodium atoms and 17.5 water molecules. Its structure is as follows:

More preferably, each asymmetric unit contains 15 coordination water molecules and 2.5 network water molecules.

More preferably, the single crystal of the supramolecular structure is colorless plate-like, monoclinic crystal system, space group is P 2 ₁ , unit cell parameters: a=20.567(8)Å, b=42.166(13)Å,c =20.503(7)Å, α=90°, β=119.278(9)°, γ=90°.

More preferably, the size of the single crystal of the supramolecular structure is 0.36×0.34×0.10 mm, the unit cell volume V=15509(9)Å ³ , the number of molecules in the unit cell Z=2, and there are independent regions in the unit lattice Two units.

The specific cultivation method of the single crystal is: Weigh about 510mg sample into a 20mL glass bottle, add 17mL 98% acetone/water, dissolve the sample at 65℃, and filter to 17 new 4mL glass bottles while hot , Placed under constant temperature at 45 ℃ to obtain the above single crystal. The single crystal diffraction and analysis are as follows:

=0.30mm，晶體與Pilatus 200K探測器距離為35mm，管壓 40kV，管流30mA，ω掃描，最大2θ角為146.9°，掃描範圍為0-180°，回擺角度為1°，間隔為1°，掃描速度為5-10s/°，每個畫面掃描一次，總計攝取3579幅圖像，總衍射點為251362個，獨立衍射點為59848個(Rint=0.0769)，可觀察點(F²≧2 σ F²)為48100個，θ角到67.687°時資料完整度為99.3%。 1. Single crystal diffraction test: on the Rigaku XtaLAB P200 diffractometer, collect the diffraction intensity data at -100°C, CuK α radiation, artificial multilayer film focusing mirror, collimating tube

=0.30mm, the distance between the crystal and Pilatus 200K detector is 35mm, the tube pressure is 40kV, the tube current is 30mA, ω scan, the maximum 2θ angle is 146.9°, the scan range is 0-180°, the swing angle is 1°, the interval is 1 °, scanning speed is 5-10s/°, each screen is scanned once, a total of 3579 images are taken, the total diffraction point is 251362, the independent diffraction point is 59848 (Rint=0.0769), the observable point (F ² ≧ 2 σ F ² ) is 48100, and the data completeness is 99.3% when the angle θ reaches 67.687°.

2. Single crystal analysis method: use the direct method (sir2011) to analyze the crystal structure on the microcomputer, obtain all non-hydrogen atom positions from the E map, use the least square method (SHELXL-2014/7) to modify the structural parameters and identify the type of atom, Using geometric calculation method and difference Fourier method to obtain the hydrogen atom position, the final reliability factor R1=0.0700, wR2=0.1691, S=1.070, Flack factor is 0.07(2). Finally, the minimum stoichiometric formula was determined to be C ₂₈₈ H ₃₆₅ N ₃₆ Na ₁₈ O _65.50 , the molecular weight of a single molecule was calculated to be 5792.93, and the crystal density was calculated to be 1.240 g/cm ³ .

3. Analysis results: The single crystal analysis results show that: each supermolecular AHU-377-valsartan-sodium single crystal asymmetric unit contains 6 valsartan, 6 AHU-377, 18 sodium ions and 17.5 Water molecules. The crystal structure is a sodium salt complex formed by the coordination of valsartan and AHU-377. These coordinated oxygen atoms come from the oxygen of 12 carboxyl groups, 18 carbonyl groups and 15 water molecules of valsartan and AHU377 molecules. These oxygen atoms coordinate with 18 sodium ions to form a network structure of coordination elements. There are 2.5 water molecules between the network voids. There are hydrogen bonds between the molecules. The network structure of the coordination elements in the crystalline state is hydrogen bonds The force and van der Waals force maintain its stable arrangement in space. An infinite three-dimensional network structure of sodium salt complexes is formed. The molar ratio between the various components of the crystal is AHU-377: valsartan: sodium ion: water molecule = 1: 1: 3: 2.9, that is, the crystalline form [3-((1S,3R)-1-lian Benzene-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate) propionic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(four (Oxazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O in the structural formula X has a value of about 2.9.

More preferably, each asymmetric unit contains 6 ARBs, 6 NEPis, 18 sodium atoms and 17.5 water molecules. Its structure is as follows:

More preferably, there are 16 coordination water molecules and 2.5 network space water molecules in each asymmetric unit.

More preferably, the single crystal of the supramolecular structure is in the form of colorless flakes, monoclinic crystal system, space group is P 2 ₁ , unit cell parameters: a=20.6864(10)Å, b=41.7011 (14)Å,c =20.8069(10)Å, α=90°, β=119.561(2)°, γ=90°.

More preferably, the size of the single crystal of the supramolecular structure is 0.42×0.36×0.18 mm, the unit cell volume V=15612.6(12)Å ³ , the number of molecules in the unit cell Z=2, and there are independent regions in the unit lattice A molecule.

The specific cultivation method of its single crystal: Weigh about 100mg of the white solid obtained in Example 5 into a 20-mL glass bottle, add 5mL of acetonitrile, fully disperse the sample by ultrasonic wave, then add 100uLH ₂ O, and heat at 60°C in a water bath. The sample dissolved until clear.

While hot, filter the sample obtained in step 1 into 5 4-mL glass bottles and close the cap tightly.

The sample obtained in step 2 was placed in a circulating water tank to perform a program cooling experiment (program: equilibrate at 60°C for 1 hour, and reduce to 20°C in 24 hours) to obtain the above single crystal. The single crystal diffraction and analysis are as follows:

1. Single crystal diffraction: use Rigaku Saturn70 CCD surface detector to collect diffraction intensity data, MoK α radiation, artificial multilayer focusing lens, collimating tube Φ=0.30mm, distance between crystal and CCD is 45mm, tube pressure 50kV, tube current 16mA , Use ω scanning when Φ is 0, 90, 180° respectively, the maximum 2θ 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/°, Scanning each screen once, a total of 1080 images are taken, the total diffraction point is 130648, the independent diffraction point is 68235 (Rint=0.0479), the observable point (F2≧2 σ F2) is 53754, and the data integrity is 98.9%.

2. Single crystal analysis: use the direct method (sir-2011) to analyze the crystal structure on the microcomputer, obtain all the non-hydrogen atom positions from the E map, use the least square method to modify the structural parameters and identify the type of atoms, use the geometric calculation method and the difference The value Fourier method obtains all hydrogen atom positions. The final reliability factor is R1=0.0651, wR2=0.1447, S=1.046, and the Flack factor is -0.01(10). Finally, the minimum stoichiometric formula is C ₂₈₈ H ₃₆₅ N ₃₆ Na ₁₈ O _66.50 , the molecular weight of a single molecule is calculated to be 5811.99, and the crystal density is calculated to be 1.263Mg/m ^3. The molecular arrangement in the crystalline state belongs to the P 2 ₁ space group. The configuration can be determined by the Flack factor and the absolute configuration of known palmitic organic molecules.

3. Analysis results: The results show that the main skeleton of the compound molecule is the organic molecules valsartan and AHU377, the Na salt formed, there are six valsartan in the asymmetric unit, six AHU-377 and 18 Na ions, These 18 Na ions are coordinated with six valsartan, six carboxyl oxygens in AHU-377, and amide amine oxygens to form a network structure of coordination elements. There is hydrogen bonding between the molecules, and the network structure of the coordination element in the crystalline state maintains its stable arrangement in space by the hydrogen bonding force and van der Waals force. The molar ratio between the various components of the crystal is AHU-377: valsartan: sodium ion: water molecule = 1:1: 3: 3.1, that is, the crystalline form [3-((1S,3R)-1-lian Benzene-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate) propionic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(four (Oxazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧XH ₂ O in the structural formula X gets a value of about 3.1

Another aspect of the present invention also provides a crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid- (S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ The preparation method of O includes the following steps: (1) The (S)-N-pentyl-N-{[2'-(1H-tetrazol-5-yl)-biphenyl-4-yl]-methyl }-Valine and (2R,4S)-5-biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-valeric acid ethyl ester dissolved in a suitable organic Solvent; (2) Mixed with basic sodium compound, the way of addition can be directly added or the basic sodium compound is dissolved in a suitable solvent; (3) Precipitation occurs naturally, cooling, adding seed crystal to induce crystallization or by creating Supersaturated conditions to produce solid precipitate; (4) solid-liquid separation; (5) control the humidity of the drying environment drying step (4) separated solids.

As a further preferred solution, the method of mixing the basic sodium compound in step 2) can be directly added to the basic sodium compound in the system of step 1) or the basic sodium compound is dissolved in an appropriate solvent and then added in step 1) System.

As a further preferred solution, the organic solvent in step (1) includes, but is not limited to, the following solvents: methanol, ethanol, n-propanol, isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dioxygen Six rings, N, N- Dimethylformamide, dimethyl sulfoxide, isopropyl acetate, ethyl acetate or mixtures thereof.

As a further preferred solution, the basic sodium compound in step (2) is selected from sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate, sodium methoxide, sodium formate, sodium propionate, sodium acrylate, sodium benzoate or Mixture; preferably sodium hydroxide, sodium bicarbonate or mixtures thereof.

As a further preferred solution, the method for creating supersaturated conditions described in step (3) includes cooling crystallization, cooling-ultrasonic coupled crystallization, evaporating solvent crystallization, adding antisolvent crystallization or antisolvent replacement. Anti-solvent refers to a solvent with low solubility of the target solid. The anti-solvent replacement method is to remove a part of the original solvent, add a part of the anti-solvent, and then remove a part of the original solvent after equilibration until it is basically replaced with the anti-solvent.

As a further preferred solution, the humidity of the drying environment in step 5) is controlled at 45% to 70%; the humidity of the preferred drying environment is controlled at 50% to 65%; and the humidity of the better drying environment is controlled at 55% to 65% .

As a further preferred solution, the drying method described in step (5) may use vacuum drying, evaporation, nitrogen drying, and the like.

As a further preferred solution, optionally, the water content in the solid obtained in step (5) is 4.6% to 11.6%, preferably 4.6% to 6.6%.

Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of the aforementioned crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxy Carbonylcarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'- Methylmethyl}amino)butyric acid]Na ₃ ‧XH ₂ O and a pharmaceutically acceptable carrier or excipient.

As a still further preferred solution, said X is selected from 2.5, 3, 6.5; optimal 3.

Another aspect of the present invention provides the aforementioned crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid -(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O or the use of the aforementioned pharmaceutical composition in the preparation of a medicament for the treatment or prevention of diseases related to neutral endopeptidase, cardiovascular and antihypertensive.

By improving efficacy and having a higher corresponding rate, more effective antihypertensive treatment has been produced, whether it is for malignant hypertension, primary hypertension, renal vascular hypertension, diabetic hypertension, simple systolic hypertension The same is true for other secondary hypertension.

Another aspect of the present invention provides the aforementioned crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid -(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O or the aforementioned pharmaceutical composition is used in the treatment or prevention of acute and chronic heart failure, congestive heart failure, left ventricular dysfunction, hypertrophic cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, Use in atrial flutter or harmful vascular remodeling drugs.

An aspect of the present invention provides the aforementioned crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-( S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O Or the aforementioned pharmaceutical composition in the preparation or treatment of myocardial infarction and its sequelae, atherosclerosis, angina pectoris, diabetic or non-diabetic renal insufficiency, secondary aldosteronism, primary or secondary pulmonary hypertension Blood pressure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, proteinuria of primary nephropathy, renal vascular hypertension, diabetic retinopathy, migraine, peripheral vascular disease, Raynaud's disease, cavity Use in medicine for hyperplasia, cognitive dysfunction, glaucoma or stroke.

"Pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiological/pharmaceutically acceptable salt or prodrug thereof with other chemical components, other compositions such as physiological/pharmaceutically acceptable 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 thus exerts the biological activity.

Compared with the prior art, the advantage of the present invention is that: the crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butan Carbamate)-propionic acid-(S)-3'-methyl-2'-(pentylamino{2"-(tetrazol-5-yl)biphenyl-4'ylmethyl}amino) Butyric acid] Na ₃ ‧XH ₂ O The new crystalline form X takes the values of 2.5, 2.9, 3.0 and 3.1 hydrates. Compared with the previously disclosed 2.5 hydrates, it has higher coordination water molecules. The experimental results show that it has more Good anti-hygroscopicity, which obviously improves the physical and chemical properties of the drug, is conducive to the application and development of the drug. It greatly reduces the risks of the drug preparation, chemical stability, packaging and storage.

The first figure is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧2.5H ₂ O Powder X-ray diffraction pattern; the ordinate is intensity and the abscissa is 2θ(°).

Figure 2 is [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ .2.5H ₂ O Powder thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 3 shows [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧6.5H ₂ O Powder X-ray diffraction pattern; the ordinate is intensity and the abscissa is 2θ(°).

Figure 4 is [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧6.5H ₂ O Powder thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 5 is [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'pentanyl{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧3.1H ₂ O powder X Ray diffraction pattern; the ordinate is intensity, and the abscissa is 2θ(°).

Figure 6 is the example [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧3.1H ₂ O Powder thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 7 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧2.5H ₂ O Powder X-ray diffraction pattern; the ordinate is intensity and the abscissa is 2θ(°).

Figure 8 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧2.5H ₂ O Powder thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 9 is [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧3H ₂ O powder X-ray diffraction pattern; the ordinate is intensity, and the abscissa is 2θ(°).

Figure 10 is [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧3H ₂ O powder Thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 11 is [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧2.5H ₂ O Powder X-ray diffraction pattern; the ordinate is intensity and the abscissa is 2θ(°).

Figure 12 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S )-3'-methyl-2'pentanyl{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧2.5H ₂ O powder heat Gravimetric analysis (TGA) graph; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 13 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'methyl-2'pentanyl{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧2.9H ₂ O powder X-ray Diffraction pattern; ordinate is intensity, and abscissa is 2θ(°).

Figure 14 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propanoic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧2.9H ₂ O Powder thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 15 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid] ₆ Na ₁₈ ‧18.5H ₂ The cell diagram of the supramolecular complex of O, which includes two asymmetric units. Figure 16 shows the [3-((1S,3R)-1-biphenyl-4-ylmethyl group in Example 5 Yl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl) Biphenyl-4'-ylmethyl}amino)butyric acid] ₆ Na ₁₈ ‧18.5H ₂ O supramolecular complex single crystal thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), The abscissa is temperature (°C).

FIG. 17 is [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid] ₆ Na ₁₈ ‧18.5H ₂ O powder X-ray diffraction pattern; the ordinate is intensity, and the abscissa is 2θ (°).

Figure 18 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid] ₆ Na ₁₈ ‧18.5H ₂ O powder thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 19 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid] ₆ Na ₁₈ ‧17.5H ₂ The cell diagram of the O supramolecular complex, which includes two asymmetric units.

Figure 20 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid] ₆ Na ₁₈ ‧17.5H ₂ Thermogravimetric analysis (TGA) diagram of O supramolecular complex single crystal; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 21 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid] ₆ Na ₁₈ ‧17.5H ₂ O powder X-ray diffraction pattern; the ordinate is intensity, and the abscissa is 2θ (°).

Figure 22 is [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S )-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid] ₆ Na ₁₈ ‧17.5H ₂ O powder thermogravimetric analysis (TGA) diagram; the ordinate is the weight change percentage (%), and the abscissa is the temperature (°C).

Figure 23 shows the changes in animal body weight during model induction; the ordinate is the animal body weight (g), and the abscissa is time (weeks).

Figure 24 shows the changes in the systolic pressure of the rat tail artery during the induction of the model; the ordinate is the animal systolic pressure (mm Hg), and the abscissa is the time (weeks).

Figure 25 shows the changes in rat tail artery diastolic pressure during model induction; the ordinate is the animal diastolic pressure (millimeters of mercury), and the abscissa is time (weeks).

Figure 26 shows the change in the mean pressure of the rat tail artery during the induction of the model; the ordinate is the mean arterial pressure (mm Hg), and the abscissa is the time (weeks).

Figure 27 shows the changes in the systolic blood pressure of the caudal arteries of rats after the administration of drugs A and B;

Figure 28 shows the changes in the diastolic pressure of the tail artery of the rat after administration of drug A and drug B; the ordinate is the animal diastolic pressure (millimeters of mercury), and the abscissa is the time (weeks).

Figure 29 shows the change in the mean pressure of the tail artery of the rat after administration of drug A and drug B; the ordinate is the mean arterial pressure (mm Hg), and the abscissa is the time (weeks).

Figure 30 shows the changes in animal heart rate during the administration of Drug C and Drug D.

Figure 31 shows the changes in animal systolic blood pressure during the administration of Drug C and Drug D.

Figure 32 shows the changes in animal diastolic blood pressure during the administration of Drug C and Drug D.

Figure 33 shows the changes in the mean arterial pressure of animals during the administration of Drug C and Drug D.

The specific examples and preparation methods provided below will further illustrate specific aspects of the embodiments of the present invention. The scope of the following examples will not limit the scope of the present invention in any way.

Methods and materials

Crystalline (3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl Yl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ .XH ₂ O is diffracted by their X-ray powder Figure characterization. Therefore, the X-ray powder diffraction pattern of the salt was collected on a Bruker D8 Discover X-ray powder diffractometer with GADDS (General Area Diffraction Detector System) CS operated in reflection using CuK α radiation (1.54Å) The tube voltage and current are set to 40kV and 40mA acquisition scan respectively. The sample is scanned within a range of 3.0° to 40° 2θ for a period of 60 seconds. For the peak position represented by 2θ, the corundum standard is used to calibrate the diffractometer. All analyses were carried out at room temperature from 20°C to 30°C. GADDS for WNT software version 4.1.14T was used to collect and integrate data. Diffac Plus software with Eva version 9.0.0. 2 released in 2003 was used to analyze the diffraction pattern. XRPD sample preparation, by placing the sample on a single crystal silicon wafer, pressing the sample powder with a glass sheet or equivalent to ensure that the surface of the sample is flat and has an appropriate height. Then put the sample holder into the Bruker XRPD instrument and use The powder X-ray diffraction pattern is collected by the instrument parameters described above. The measurement differences associated with such X-ray powder diffraction analysis results from a variety of factors including: (a) error in the sample preparation (eg, sample height), ( b) instrument error, (c) calibration difference, (d) operator error (including those errors that occur when determining the peak position), and (e) the nature of the substance (such as better orientation errors). Calibration errors and samples Height errors often cause all peaks to shift in the same direction. Generally speaking, this calibration factor will make the measured peak position consistent with the expected peak position and can be within the expected range of 2θ ± 0.2°.

The resulting [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'- Thermogravimetric analyzer for methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]trisodium hydrate crystal form( TGA) for thermal analysis, the model of the TGA instrument used is TAQ500 (Thermo Analysis). The parameters of the TGA analysis method are as follows: the temperature range is from room temperature to 300 degrees Celsius, the scan rate is 10 degrees Celsius per minute, and the protective gas is nitrogen (flow rate 25 ml/min) ).

The resulting [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'- Methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧XH ₂ O The Fischer Coulometer moisture analyzer is used for analysis. The model of the moisture analyzer used is C20 (Mettler). Operation method: First, start pre-titration through the start analysis dialog box, and the drift value to be continuously measured falls below the specified value. Enter the sample measurement mode. Click the button to start testing the sample, quickly remove about 20-100 mg of the sample from the sample and place it in the titration cup. Press the confirm key, the analysis starts automatically. The total weight of the tested sample including the tare weight has been weighed in advance Obtain and measure the quality of the sample after adding the sample again including the tare weight. The total weight minus the weight after the sample is the actual amount of the sample for moisture determination. After the titration, the result dialog box is displayed, and the actual amount of input is entered to automatically obtain The result of the amount of water.

Example 1

根據水分測定結果可知，所得水合物為2.5水合物。 Weigh 200 mg of AHU-377 free acid (oily), 212 mg of valsartan and 58.3 mg of sodium hydroxide in a 20-mL glass bottle, then add 10 mL of isopropyl acetate and seal. The mixture was placed in an ultrasonic water area at 0°C, power 100W, 35KHz, and reacted for 4 hours to obtain a white solid-containing suspension. The suspension was centrifuged (8000 rpm) for 10 minutes, the supernatant was discarded, the remaining solid was dried by nitrogen purge for 4 hours, and the nitrogen flow was about 5 ml/min to obtain a powdery solid. The powder X-ray diffraction pattern (XRPD) is shown in Figure 1. See Figure 2 for the TGA analysis chart. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the results of moisture measurement, the obtained hydrate is 2.5 hydrate.

Example 2

根據水分測定結果可知，所得水合物為2.5水合物。 Weigh 151.2 mg of AHU-377 free acid (oily), 160 mg of valsartan in a 50-mL concentrated bottle, add 15.3 mL of acetone, then add 2.8 mL of 15.8 mg/mL NaOH, rotate to dryness, then add 8 mL Acetone, magnetically stirred at room temperature for 24 hours to obtain a white solid-containing suspension. The suspension was centrifuged (8000 rpm) for 10 minutes, the supernatant was discarded, the remaining solid was dried by nitrogen purge for 4 hours, and the nitrogen flow was about 5 ml/min to obtain a powdery solid. The powder X-ray diffraction pattern (XRPD) is shown in Figure 7. See Figure 8 for the TGA analysis chart. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the results of moisture measurement, the obtained hydrate is 2.5 hydrate.

Example 3

根據水分測定結果可知，所得水合物為2.5水合物。 Weigh 151.2 mg of AHU-377 free acid (oily), 160 mg of valsartan in a 50-mL concentrated bottle, add 15.3 mL of acetone, then add 2.8 mL of 15.8 mg/mL NaOH, rotate to dryness, then add 8 mL Acetonitrile was stirred magnetically at room temperature for 24 hours to obtain a white solid suspension. The suspension was centrifuged (8000 rpm) for ten minutes, the supernatant was discarded, the remaining solid was dried by nitrogen purge for 4 hours, and the nitrogen flow rate was about 5 ml/min to obtain a powdery solid. The powder X-ray diffraction pattern (XRPD) is shown in Figure 11. See Figure 12 for the TGA analysis chart. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the results of moisture measurement, the obtained hydrate is 2.5 hydrate.

Example 4

根據水分測定結果可知，所得水合物為6.5水合物。 Weigh 200 mg of AHU-377 free acid (oily), 212 mg of valsartan and 58.3 mg of sodium hydroxide in a 20-mL glass bottle, then add 10 mL of ethyl acetate and seal. The mixture was placed in an ultrasonic water area at 0°C, power 100W, 35KHz, and reacted for 4 hours to obtain a white solid-containing suspension. The suspension was centrifuged (8000 rpm) for ten minutes, the supernatant was discarded, the remaining solid was dried by nitrogen purge for 4 hours, and the nitrogen flow rate was about 5 ml/min to obtain a powdery solid. The powder X-ray diffraction pattern (XRPD) is shown in Figure 3. See Figure 4 for TGA analysis chart. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the result of moisture measurement, the obtained hydrate is 6.5 hydrate.

Example 5

根據水分測定結果可知，所得水合物為3.1水合物。 Weigh 200 mg of AHU-377 free acid (oily), 212 mg of valsartan and 58.3 mg of sodium hydroxide in a 20-mL glass bottle, then add 10 mL of acetone and seal. The mixture was placed in an ultrasonic water area at 0°C, power 100W, 35KHz, and reacted for 4 hours to obtain a white solid-containing suspension. The suspension was centrifuged (8000 rpm) for ten minutes, the supernatant was discarded, the remaining solid was dried by nitrogen purge for 4 hours, and the nitrogen flow was about 5 ml/min to obtain a powdery solid. The powder X-ray diffraction pattern (XRPD) is shown in Figure 5. See Figure 6 for TGA analysis chart. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the results of moisture measurement, the obtained hydrate was 3.1 hydrate.

It corresponds to the specific cultivation method of single crystal: Weigh about 100 mg of the white solid obtained in Example 5 into a 20-mL glass bottle, add 5 mL of acetonitrile, fully disperse the sample by ultrasonic wave, then add 100 uL H ₂ O, and heat in a 60°C water bath , Dissolve the sample until clear.

While hot, filter the sample obtained in step 1 into 5 4-mL glass bottles and close the cap tightly.

The sample obtained in step 2 was placed in a circulating water tank to perform a program cooling experiment (program: equilibrate at 60°C for 1 hour, and reduce to 20°C in 24 hours) to obtain the above single crystal.

The cell of its supramolecular complex is shown in Figure 15. It includes two asymmetric units. Its single crystal diffraction and analysis are as follows: the single crystal is colorless plate-like, and the crystal size used for diffraction analysis is 0.42 ×0.36×0.18mm, belonging to monoclinic system, space group P21, unit cell parameters: a=20.6864(10)Å, b=41.7011(14)Å, c=20.8069(10)Å, α=90°, β=119.561(2)°, γ=90°, unit cell volume V=15612.6(12)Å ³ , the number of molecules in the unit cell Z=2, there is one molecule in the independent region of the unit lattice.

Collect the diffraction intensity data with Rigaku Saturn70 CCD surface detector, MoK α radiation, artificial multi-layer film focusing mirror, collimating tube Φ=0.30mm, the distance between the crystal and CCD is 45mm, the tube pressure is 50kV, the tube current is 16mA, and the Φ is 0 , 90, 180 ° using ω scan, the maximum 2θ angle is 56 °, the scan range is 0-180 °, the swing angle is 0.5 °, the interval is 0.5 °, the scanning speed is 10s/°, each screen scan once, A total of 1080 images were taken, with a total of 130648 diffraction points, 68235 independent diffraction points (Rint=0.0479), 53754 observable points (F2≧2 σ F2), and the data integrity was 98.9%.

Use the direct method (sir-2011) to analyze the crystal structure on the microcomputer, obtain all non-hydrogen atom positions from the E map, use the least square method to modify the structural parameters and identify the type of atoms, and use the geometric calculation method and the difference Fourier method to obtain all hydrogen For the atomic position, the final reliability factor is R1=0.0651, wR2=0.1447, S=1.046, and the Flack factor is -0.01(10). The final minimum stoichiometric formula is C ₂₈₈ H ₃₆₇ N ₃₆ Na ₁₈ O _66.50 , the molecular weight of a single molecule is calculated to be 5810.93, the crystal density is calculated to be 1.263Mg/m ³ , the chemical name is: [3-((1S,3R)- 1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate) propionic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(Tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid] ₆ Na ₁₈ ‧18.5H ₂ O; the chemical formula is: [3-((1S,3R)-1 -Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl-2'-(pentylacetate{2"- (Tetrazole-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧3.1H ₂ O The molecular arrangement in the crystalline state belongs to the P ₂₁ space group, the absolute configuration of the molecule can The factors and the absolute configuration of known palmitic organic molecules are determined.

The single crystal analysis results show that the main skeleton of the compound molecule is the organic molecules valsartan and AHU377, the Na salt formed, there are six valsartan in the asymmetric unit, six AHU377 and 18 Na ions, these 18 Na The ions coordinate with six valsartan, the oxygen of the carboxyl group in the six AHU377, and the oxygen of the amide group to form a network structure of coordination elements. There are 2.5 water between the network spaces, and there are hydrogen bonds between the molecules. In the crystalline state, the network structure of the coordination element maintains its stable arrangement in space by hydrogen bonding force and van der Waals force.

The XRPD measured value of the powder crystal powder obtained in Example 5 is compared with the calculated value of the single crystal as shown in the following table:

From the comparison of the above data, the powder crystal diffraction pattern of the product obtained in Example 5 is highly consistent with the calculated value of the single crystal. In the powder crystal diffraction pattern, a small amount of peaks are missing, which is caused by the preferred orientation of the powder crystals. This phenomenon is particularly obvious for plate crystals. However, the comparison of the existing peak positions shows that the crystal form of the powder crystal is highly consistent with the calculated crystal form of the single crystal. This proves that the single crystal structure is consistent with the powder crystal structure.

根據TGA和KF測量值可知，所得單晶為3.1水合物，與單晶解析的結果一致。 The obtained single crystal TGA analysis chart is shown in Figure 16. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the measured values of TGA and KF, the obtained single crystal was 3.1 hydrate, which was consistent with the results of single crystal analysis.

Example 6

Precisely weigh 284g of AHU377, 300g of valsartan in a 50L glass reactor, add 29L of acetonitrile, and dissolve the compound mechanically to obtain a clear solution. Precisely weigh 82.8g of NaOH and dissolve it in 240mL of water. The NaOH aqueous solution was slowly added to the glass reaction kettle, and rapidly stirred with mechanical stirring. When the NaOH aqueous solution was added dropwise, about 4 hours, after stirring for 30 min, about 50 mg of the single crystal of 2.9 hydrate obtained in Example 5 was added. Continue to stir Until crystals precipitated, after stirring overnight, centrifugal filtration gave white crystals. The resulting white solid was dried under vacuum at 50 degrees Celsius overnight, placed at 55% relative humidity, and equilibrated for 72 hours to obtain a product.

The powder X-ray diffraction pattern (XRPD) is shown in Figure 17. The XRPD measured value of the powder crystal powder obtained in Example 6 is compared with the calculated value of the single crystal as shown in the following table:

From the comparison of the above experimental data, the powder crystal diffraction pattern of the product obtained in Example 6 is also highly consistent with the calculated value of the single crystal of Example 5, showing a high degree of consistency of the crystal form. Example 6 is also consistent with the measurement of the powder crystal in Example 5.

See Figure 18 for the TGA analysis chart. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

Example 7

根據水分測定結果可知，所得水合物為3水合物。 Weigh 151.2 mg of AHU-377 free acid (oily), 160 mg of valsartan in a 50-mL concentrated bottle, add 15.3 mL of acetone, then add 2.8 mL of 15.8 mg/mL NaOH, rotate to dryness, then add 8 mL Ethyl acetate was stirred magnetically at room temperature for 24 hours to obtain a white solid suspension. The suspension was centrifuged (8000 rpm) for ten minutes, the supernatant was discarded, the remaining solid was dried by nitrogen purge for 4 hours, and the nitrogen flow rate was about 5 ml/min to obtain a powdery solid. The powder X-ray diffraction pattern (XRPD) is shown in Figure 9. The TGA analysis chart is shown in Figure 10. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the results of moisture measurement, the obtained hydrate is a trihydrate.

Example 8

Precisely weigh 283.5 mg of AHU377 and 300 mg of valsartan in a 50-mL concentration bottle, add 27 mL of acetone, add a magnetic stirrer, and dissolve the compound to obtain a clear solution. Precisely weigh 82.8 mg of NaOH into a 2-mL HPLC vial and add 240 uL of water to dissolve. The NaOH solution was transferred to the acetone solution and stirred magnetically. The suspension was magnetically stirred overnight at room temperature. At room temperature, about half of the acetone solvent was removed by rotary evaporation, then 15 mL of isopropyl acetate was added, half of the volume was continued by rotary evaporation, and then 15 mL of isopropyl acetate was added, and the magnetic stirring was performed overnight. Suction filtration under nitrogen protection. After the filtrate is filtered, add about 10 mL of isopropyl acetate to wash again, and filter under nitrogen protection to dry for about 2 hours.

根據TGA和KF測量值可知，所得單晶為2.9水合物。 The dried filter cake was collected and dried under vacuum at 50°C overnight. Then transfer the dried solid to a humidity of 60% ± 5%, overnight at room temperature, the rehydration process occurs, and a stable crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl Yl-3-ethoxycarbonyl-1-butylcarbamoyl)propanoic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl) Biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧2.9H ₂ O. Its powder X-ray diffraction pattern (XRPD) is shown in Figure 13. TGA analysis chart is shown in Figure 14. TGA chart The above weight loss is caused by the loss of water in the crystal, and can be used for accurate determination of the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal.

According to the measured values of TGA and KF, the obtained single crystal was 2.9 hydrate.

It corresponds to the specific cultivation method of single crystal: the 510 mg sample prepared in Example 8 is added to a 20 mL glass bottle, 17 mL of 98% acetone/water is added, the sample is dissolved under the condition of 65° C., and the average is filtered while hot to 17 new The 4mL glass bottle cap is tightly capped, placed at 45°C, and placed at a constant temperature to obtain the above single crystal. The cell of its supramolecular complex is shown in Figure 19. It includes two asymmetric units. Its single crystal diffraction and analysis are as follows: the crystal is colorless plate-like, and the size of the crystal used for diffraction analysis is 0.36×0.34 ×0.10mm, belongs to the monoclinic system, the space group is P 2 ₁ , unit cell parameters: a=20.567(8)Å, b=42.166(13)Å, c=20.503(7)Å, α=90°, β=119.278(9)°, γ=90°, unit cell volume V=15509(9)Å ³ , the number of molecules in the unit cell Z=2, there are two units in the independent region of the unit lattice.

=0.30mm，晶體與Pilatus 200K探測器距離為35mm，管壓40kV，管流30mA，ω掃描，最大2θ角為146.9°，掃描範 圍為0-180°，回擺角度為1°，間隔為1°，掃描速度為5-10s/°，每個畫面掃描一次，總計攝取3579幅圖像，總衍射點為251362個，獨立衍射點為59848個(Rint=0.0769)，可觀察點(F²≧2 σ F²)為48100個，θ角到67.687°時資料完整度為99.3%。 On the Rigaku XtaLAB P200 diffractometer, collect the diffraction intensity data at -100°C, CuK α radiation, artificial multilayer film focusing mirror, collimator tube

=0.30mm, the distance between the crystal and Pilatus 200K detector is 35mm, the tube pressure is 40kV, the tube current is 30mA, ω scan, the maximum 2θ angle is 146.9°, the scan range is 0-180°, the swing angle is 1°, the interval is 1 °, scanning speed is 5-10s/°, each screen is scanned once, a total of 3579 images are taken, the total diffraction point is 251362, the independent diffraction point is 59848 (Rint=0.0769), the observable point (F ² ≧ 2 σ F ² ) is 48100, and the data completeness is 99.3% when the angle θ reaches 67.687°.

Use the direct method (sir2011) to analyze the crystal structure on the microcomputer, obtain all non-hydrogen atom positions from the E map, use the least square method (SHELXL-2014/7) to modify the structural parameters and identify the type of atoms, use the geometric calculation method and the difference Fourier method obtains the position of hydrogen atom, the final reliability factor is R1=0.0700, wR2=0.1691, S=1.070, and Flack factor is 0.07(2). Finally, the minimum stoichiometric formula was determined to be C ₂₈₈ H ₃₆₅ N ₃₆ Na ₁₈ O _65.50 , the molecular weight of a single molecule was calculated to be 5792.93, and the crystal density was calculated to be 1.240g/cm3. The chemical name is: [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-Methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'ylmethyl}amino)butyric acid] ₆ Na ₁₈ ‧17.5H ₂ O; chemical formula Is: [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl Yl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧2.9H ₂ O

The single crystal analysis results show that the asymmetric unit of AHU377-valsartan-sodium single crystal contains 6 valsartan, 6 AHU377, 18 sodium ions and 17.5 water molecules. The crystal structure can be regarded as a sodium salt complex formed by the coordination of valsartan and AHU377. These coordinated oxygen atoms come from the oxygen of 12 carboxyl groups, 18 carbonyl groups and 15 water molecules of valsartan and AHU377 molecules. These oxygen atoms coordinate with 18 sodium ions to form a network structure of coordination elements. There are 2.5 water molecules between the network voids. There are hydrogen bonds between the molecules. The network structure of the coordination elements in the crystalline state is hydrogen bonds The force and van der Waals force maintain its stable arrangement in space. An infinite three-dimensional network structure of sodium salt complexes is formed. The molar ratio between the various components of the crystal is AHU377: valsartan: sodium ion: water molecule = 1: 1: 3: 2.9.

There is disorder in the single crystal. One of the 18 sodium is disordered and appears in two positions. The sodium occupancy rates of these two positions are 50%. At the same time, a valsartan ligand coordinated with disordered sodium (except for the portion of biphenyltetrazolium in the ligand) is disordered, with a occupancy rate of 50%. The aliphatic side chains on the other two valsartan ligands are disordered, and one benzene ring on the biphenyl on the AHU377 ligand is disordered. 0.5 of the 2.5 crystal waters are disordered in three positions. The XRPD measured value of the powder crystal powder obtained in Example 8 is compared with the calculated value of the single crystal as shown in the following table:

From the comparison of the above experimental data, the powder crystal diffraction pattern of the product obtained in Example 8 is highly consistent with the calculated value of its corresponding single crystal. In the powder crystal diffraction pattern, a small amount of peaks are missing, which is caused by the preferred orientation of the powder crystals. This phenomenon is particularly obvious for plate crystals. However, the comparison of the existing peak positions shows that the crystal form of the powder crystal is highly consistent with the calculated crystal form of the single crystal. This proves that the single crystal structure is consistent with the powder crystal structure.

The obtained single crystal TGA analysis chart is shown in Figure 20. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the measured values of TGA and KF, the obtained single crystal is 2.9 hydrate, which is consistent with the single crystal analysis result.

Example 9

Precisely weigh 284g of AHU377 and 300g of valsartan in a 50L glass reactor, add 27L of isopropanol, and dissolve the compound by mechanical stirring to obtain a clear solution. Precisely weigh 82.8g of NaOH in 240mL water to dissolve. The NaOH aqueous solution was slowly added to the glass reaction kettle, and rapidly stirred with mechanical stirring. When the dropwise addition of the NaOH aqueous solution was completed, about 6 hours, after stirring for 30 minutes, about 50 mg of the single crystal of 2.9 hydrate obtained in Example 8 was added. Continue stirring until crystals are precipitated. After stirring overnight, centrifugal filter to obtain white crystals. The powder X-ray diffraction pattern (XRPD) is shown in Figure 21. The comparison between the XRPD measured value of the powder crystal powder obtained in Example 9 and the calculated value of the single crystal is shown in the following table:

From the comparison of the above experimental data, the powder crystal diffraction pattern of the product obtained in Example 9 is highly consistent with the calculated value of the single crystal of Example 8, showing a high degree of consistency of the crystal form. Example 9 is also consistent with the measured value of the powder crystal in Example 8.

See Figure 22 for the TGA analysis chart. The weight loss on the TGA chart is caused by the loss of water in the crystal, which can be used to accurately determine the moisture content in the crystal. In addition, the KF moisture method is used as a reference to determine the moisture content in the crystal. The results obtained are shown in the table below:

According to the measured values of TGA and KF, the obtained single crystal was 2.9 hydrate.

Example 10

Compare the hygroscopicity of different hydrates. [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propionic acid-(S)-3'-methyl -2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧XH ₂ O various crystals are placed in HPLC vials, Place it open at 57% humidity (saturated sodium bromide solution) and observe the hygroscopicity and liquefaction time of the sample.

Example 11

[3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl- 2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O (drug A) and valsartan ( Drug B) Combination therapy in the rat hypertension model induced by renal artery stenosis

1 Experimental materials:

Drug A: [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'- Methyl-2'pentylamino{2"-(tetrazol-5-yl)biphenyl-4'ylmethyl}amino)butanoic acid]Na ₃ ‧3.1H ₂ O was prepared according to Example 6.

Drug B: Valsartan is a commercially available API.

2 Experimental equipment

Animal scales: JJ500, G&G, Jiangsu, China

Low temperature electric blanket: Jwilch, China

Surgical microscope: LuckbirdXTS-4A

Rat tail blood pressure measuring instrument: BP-2010AUL.CN10120479

Rat gastric lavage device: 16#80mm diameter, round head

3 Experimental methods 3.1 Unilateral renal artery narrowing induced rat renal hypertension model

Fifty male Sprague Dawley (SD) rats, weighing 180-200 grams, were provided by Vital River Laboratories, Beijing, China. Animals are housed in the SPF-level barrier system of the animal center, following the international standard temperature, humidity, and light control system. The experimental animal operation plan was approved by the IACUC committee of the platform animal center.

All operations involved in this experiment were performed under the guidelines of KCI animal surgery SOP. The animals were anesthetized with pentobarbital sodium (50 mg/kg). After confirming that the animals were under anesthesia, the left abdomen was prepared with skin for strict surgical disinfection. A 2.0 cm long surgical incision was made layer by layer on the left abdominal kidney area and entered the abdominal cavity. Expose the left kidney. Carefully separate the left renal artery to completely separate it from the renal vein. 3-0 surgical sutures were used to perform left renal artery constriction (achieving 60% stenosis of the left renal artery). After the operation, it was confirmed that there was no intra-abdominal hemorrhage. The abdominal wall muscle layer tissue was sutured with 3-0 surgical suture thread, and then the skin was sutured with 2-0 surgical suture thread. After washing the skin wound, place the animal on a 37-degree electric blanket to keep it warm until the animal wakes up. After confirming that it can eat and drink water freely, return the animal to the rearing cage for normal rearing. Animals were observed daily during the first week after surgery to confirm that there were no postoperative wound infections and splits. From the first week after the operation, the changes in animal body weight and the caudal arterial pressure and heart rate were monitored weekly to monitor changes in animal blood pressure and heart rate.

3.2 Inclusion criteria for laboratory animals

Animals test the pressure of the tail artery of the rat for 4-5 weeks to observe the increase in blood pressure trend. Select animals with systolic blood pressure over 160mmHg to be enrolled for pharmacodynamic test

3.3 Experimental scheme

Drug A((3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'- Methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧3.1H ₂ O), Drug B( (Valsartan) were tested on a single oral administration, starting at 9-10AM early and ending at 10:00AM the next day. The blood pressure of animals 2-4HR, 6-8HR and 24HR after administration were observed within 24 hours Change. Group-1 is the drug administration group, Group-2 is the blank control group (only drug carrier)

3.4 Detection during administration

After administration, closely observe the physiological changes of all animals after oral administration, mainly including vomiting and diarrhea.

4 Experimental results 4.1 Model establishment of unilateral renal artery stenosis induced hypertension 4.1.1 Changes in animal weight

The weight of the animals was measured once a week for 4-5 weeks after the model surgery. The body weight of animals showed a gradual increase trend, and the average body weight of the animals reached about 400 grams by five weeks after surgery (see Figure 23).

4.1.2 Changes in animal blood pressure

After modeling, the caudal arterial pressure was monitored once awake every week. From the first week of modeling, the animals began to have increased blood pressure. The monitoring of rat tail arterial pressure for 4-5 weeks shows that the animal blood pressure tends to maintain a steady increase since 3 weeks after modeling (Figure 24-26).

4.2.1 Basic physiological observation of animals during administration

All animals did not show abnormal physiological changes during the administration, including gastrointestinal reactions such as vomiting, diarrhea, abnormal feeding, etc., and psychotic symptoms such as mental depression and abnormal movement.

4.2.2 Animal blood pressure changes during administration

Animals' blood pressure showed a slight decrease after 2-4 hours of administration of drugs A and B, including systolic blood pressure, diastolic blood pressure and mean arterial pressure. The optimal blood pressure lowering effect was achieved in 6-8 hours; a slight rebound in 24 hours (see page 27) -29 picture). The antihypertensive effect of drug A is better than that of drug B.

Example 12

[3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl- 2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧XH ₂ O combined with valsartan (Drug D) Experimental study on the therapeutic effect of a rat hypertension model induced by renal artery stenosis

1 Experimental materials

Drug C: [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'- Methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧2.9H ₂ O was prepared according to Example 9 obtain.

Drug D: Valsartan is a commercially available API.

2 Experimental equipment

Animal scales: JJ500, G&G, Jiangsu, China

Low temperature electric blanket: Jwilch, China

Surgical microscope: LuckbirdXTS-4A

Rat tail blood pressure measuring instrument: BP-2010AUL.CN10120479

Rat gastric lavage device: 16#80mm diameter, round head

3 Experimental methods 3.1 Unilateral renal artery narrowing induced rat renal hypertension model

Fifty male Sprague Dawley (SD) rats, weighing 180-200 grams, were provided by Vital River Laboratories, Beijing, China. Animals are housed in the SPF-level barrier system of the animal center, following the international standard temperature, humidity, and light control system. The experimental animal operation plan was approved by the IACUC committee of the platform animal center.

All operations involved in this experiment were performed under the guidelines of KCI animal surgery SOP. The animals were anesthetized with pentobarbital sodium (50 mg/kg). After confirming that the animals were under anesthesia, the left abdomen was prepared with skin for strict surgical disinfection. A 2.0 cm long surgical incision was made layer by layer on the left abdominal kidney area and entered the abdominal cavity. Expose the left kidney. Carefully separate the left renal artery to completely separate it from the renal vein. Use 3-0 surgical suture thread to perform left renal artery constriction (realize left renal artery 60% narrow). After the operation, it was confirmed that there was no intra-abdominal hemorrhage. The abdominal wall muscle layer tissue was sutured with 3-0 surgical suture thread, and then the skin was sutured with 2-0 surgical suture thread. After washing the skin wound, place the animal on a 37-degree electric blanket to keep it warm until the animal wakes up. After confirming that it can eat and drink water freely, return the animal to the rearing cage for normal rearing. Animals were observed daily during the first week after surgery to confirm that there were no postoperative wound infections and splits. From the first week after the operation, the changes in animal body weight and the caudal arterial pressure and heart rate were monitored weekly to monitor changes in animal blood pressure and heart rate.

3.2 Inclusion criteria for laboratory animals

The animals tested the pressure of the tail artery of the rats for 4-5 weeks to observe the increasing trend of the animals' blood pressure. Select animals with systolic blood pressure over 160mmHg to be enrolled for pharmacodynamic test

3.3 Experimental scheme

64 animals were selected with 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 administration group C (Group-4). Oral administration is performed once a day, the drug volume is 10 mL/kg, the administration is continued for a week, weighing is performed every three days, and the blood pressure changes of the animals after 6-8 hr and 24 hr are measured.

4During the administration period

Animals start dosing every morning at 9-10am. After administration, closely observe the physiological changes of animals after administration, mainly for clinical characterization, and record and analyze any abnormal phenomena in time; 6-8hr (15:30-17:30P.M) and 24hr (after administration) The next day 09:30-11:30) The blood pressure and heart rate of the tail artery of the rat were measured, and the experiment lasted for a week.

5 End of the test

After the last data acquisition was completed, all experimental animals were euthanized according to the KCI Animal Euthanasia SOP, and plasma (heparin anticoagulant) and tissues were collected: 2 blood samples, 1 mL each, 2 kidneys (right kidney), 2 adrenal glands One portion, the heart (two ventricles, two atria), all tissues were quick-frozen in liquid nitrogen and stored in a refrigerator at -80 degrees.

6 Experimental results 6.1 The model of unilateral renal artery stenosis-induced hypertension was successfully established, which is the same as the result of 4.1 in Example 11. 6.2 Basic physiological observation of animals during administration

All animals showed no abnormal physiological changes during the administration for a week, including gastrointestinal reactions such as vomiting, diarrhea, abnormal feeding, etc., and psychotic symptoms such as mental depression and abnormal movement.

6.3 Changes in animal heart rate during administration

The observation of animal heart rate in the sham operation group (Group-1) for one week showed that the average heart rate fluctuated from 330 beats/min to 350 beats/min. The average heart rate of hypertensive animals before administration was about 380 beats/min. In the model group (Group-2) and the drug D group, the animal heart rate did not show significant acceleration or slowdown during one week of continuous administration. Animal C group (Group 4) animals showed a steady and slow heart rate after one week of dosing, which was consistent with the heart rate of the sham-operated group after 3 days of dosing. After a week of continuous administration, the heart rate of the animals showed a sustained and steady slowdown (see Figure 30).

6.4 Changes in animal blood pressure during administration

The blood pressure of animals in the sham-operated group (Group-1) of the experiment was maintained in the normal range during the week-long experiment, including systolic pressure, diastolic pressure, and mean arterial pressure. Relative to the model group (Group-2), the blood pressure of the animals of Drug D (Group-3) showed a gradual decrease during the continuous one week of administration. The blood pressure reduction effect was the most obvious 6-8 hours after each administration , A slight recovery after 24 hours. In the drug group C (group-4), the blood pressure of the animals showed a consistent and slow decline in the continuous one-week dosing test, showing that the blood pressure lowering effect was the most obvious 6-8 hours after each administration, and mild after 24 hours Recovery, including systolic, diastolic and mean arterial pressure (see Figures 31-33).

[3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl- 2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧XH ₂ O (drug C) has the effect of slowing the heart rate of animals Function, fast onset, slowing heart rate to the level of animals in the sham-operated group for the first administration. Continuous administration for one week makes the heart rate of hypertensive animals stable at the average heart rate of animals in the sham-operated group.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solutions are modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, which should be covered within the scope of the patent application of the present invention.

Claims (31)

A crystalline form [3-((1S,3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'- Methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧XH ₂ O, its powder X-ray diffraction Figures include diffraction 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±0.2° The peak at the angle (2θ), wherein X is selected from 3.0±0.2 to 6.5. The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propane as described in item 1 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, where the powder X-ray diffraction pattern also includes 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 °, Peaks at the diffraction angles (2θ) of 27.7±0.2°, 27.9±0.2°, and 29.2±0.2°; or also include 8.9±0.2°, 14.5±0.2°, 15.2±0.2°, 16.0±0.2°, 25.8± Peaks at diffraction angles (2θ) of 0.2° and 27.9±0.2°; or also including 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 29.1±0.2° at the diffraction angle (2θ) peak; or also include 6.3±0.2°, 13.6±0.2°, 14.2±0.2°, 14.8±0.2°, 15.2±0.2°, 17.6 Peaks at diffraction angles (2θ) of ±0.2°, 18.7±0.2°, 22.6±0.2°, 23.3±0.2°, 24.6±0.2° and 29.2±0.2°. The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propane as described in item 1 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, wherein the powder X-ray diffraction pattern is substantially the same as the peak at the diffraction angle (2θ) shown in FIGS. 1, 5, 17, 13, and 21. The crystalline type [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butyl Carbamoyl) propionic acid-(S)-3'-methyl-2'-(pentylamino{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino) Butyric acid]Na ₃ .XH ₂ O, wherein the crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate Acetyl)propionic acid-(S)-3'-methyl-2'-(pentylacetyl{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid ]Na ₃ ‧XH ₂ O is a structural formula of supramolecular structure, which contains asymmetric units, each asymmetric unit contains 6 ARB, 6 NEPi, 18 sodium atoms and 12 to 54 water molecules, of which The ARB is (S)-N-pentyl-N-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-valine, the NEPi is (2R, 4S)-5-biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoic acid ethyl ester; each asymmetric unit contains 39 water molecules or 18±0.5 water molecules. The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propane as described in item 4 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, wherein each asymmetric unit contains 17.5 water molecules, and its structure is as follows:

The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propane as described in item 5 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, wherein each asymmetric unit contains 15 coordination water molecules and 2.5 network space water molecules. The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propane as described in item 5 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, wherein the single crystal of the supramolecular structure is a colorless plate, monoclinic system, space group is P 2 ₁ , unit cell parameters: a=20.567(8)Å, b=42.166(13)Å , C=20.503(7)Å, α=90°, β=119.278(9)°, γ=90°. The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propane as described in item 5 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, wherein the size of the single crystal of the supramolecular structure is 0.36×0.34×0.10 mm, the unit cell volume V=15509(9)Å ³ , the number of units in the unit cell Z=2, and the independent region of the unit lattice There are two units. The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamoyl)propylene as described in item 6 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, wherein each asymmetric unit contains 18.5 water molecules, and its structure is as follows:

The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamyl)propane as described in item 9 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, wherein each asymmetric unit contains 16 coordination water molecules and 2.5 network water molecules. The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamyl)propane as described in item 9 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, when X=3.1, wherein the single crystal of the supramolecular structure is colorless plate-like, monoclinic crystal system, space group is P 2 ₁ , unit cell parameter: a=20.6864(10)Å, b =41.7011(14)Å, c=20.8069(10)Å, α=90°, β=119.561(2)°, γ=90°. The crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamyl)propane as described in item 9 of the patent application Acid-(S)-3'-methyl-2'-(pentylacetate{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧ XH ₂ O, when X=3.1, wherein the size of the single crystal of the supramolecular structure is 0.42×0.36×0.18 mm, the unit cell volume V=15612.6(12)Å ³ , and the number of units in the unit cell Z=2, There are 2 cells in the independent area of the unit lattice. A crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butan according to any one of claims 1 to 12 Carbamoylamino) propionic acid-(S)-3'-methyl-2'-(pentylamino{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino )Butyric acid]Na _3. The preparation method of XH ₂ O includes the following steps: 1) Combine (S)-N-pentyl-N-{[2'-(1H-tetrazol-5-yl)- Phenyl-4-yl]-methyl}-valine and (2R,4S)-5-biphenyl-4-yl-4-(3-linker-propionylamino)-2-methyl- Ethyl valerate is dissolved in a suitable organic solvent; 2) mixed with basic sodium compound, the way of addition can be directly added or the basic sodium compound is dissolved in a suitable solvent; 3) natural precipitation occurs, and the addition of seed crystals induces Crystallization or creating solid precipitates by creating supersaturated conditions; 4) Solid-liquid separation; 5) Controlling the humidity of the drying environment Drying step 4) Separated solids. The preparation method as described in item 13 of the patent application scope, wherein the humidity of the drying environment in step 5) is controlled at 45% to 70%. The preparation method as described in item 14 of the patent application scope, wherein the humidity of the drying environment in step 5) is controlled at 50% to 65%. The preparation method as described in item 15 of the patent application scope, wherein the humidity of the drying environment in step 5) is controlled at 55% to 65%. The preparation method as described in item 13 of the patent application scope, wherein the method of mixing the basic sodium compound in step 2) can directly add the basic sodium compound in the system of step 1) or dissolve the basic sodium compound in an appropriate The solvent is then added to the system of step 1). The preparation method as described in item 13 of the patent application scope, wherein the organic solvent in step 1) is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, acetonitrile, acetone, methyl ethyl ketone, tetrahydrofuran, dihydrogen Oxyhexane, N,N-dimethylformamide, dimethyl sulfoxide, isopropyl acetate, ethyl acetate or mixtures thereof. The preparation method according to item 13 of the patent application scope, wherein the basic sodium compound in step 2) is selected from sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate, sodium methoxide, sodium formate, sodium propionate, Sodium acrylate, sodium benzoate or mixtures thereof. The preparation method as described in item 19 of the patent application scope, wherein the basic sodium compound in step 2) is selected from sodium hydroxide, sodium bicarbonate or a mixture thereof. The preparation method as described in item 13 of the patent application scope, wherein the method for creating supersaturated conditions described in step 3) is selected from cooling crystallization, cooling-ultrasonic coupling crystallization, evaporation of solvent crystallization, and addition of anti-solvent crystallization Or antisolvent replacement method. The preparation method according to item 13 of the patent application scope, wherein the drying method described in step 5) is selected from vacuum drying, evaporation, and nitrogen drying. The preparation method as described in item 13 of the patent application scope, wherein in step 5), the humidity is controlled to obtain the corresponding hydrate, and then further fully exposed to a specific humidity for hydration to obtain a hydrate with a target hydration degree. The preparation method as described in item 13 of the patent application scope, wherein the water content in the solid obtained in step 5) is 4.6% to 11.6%. The preparation method as described in item 24 of the patent application scope, wherein the water content in the solid obtained in step 5) is 4.6% to 6.6%. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises a therapeutically effective dose of the crystalline form [3-((1S,3R)-1-Lian according to any one of items 1 to 12 of the patent application Benzene-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate) propionic acid-(S)-3'-methyl-2'-(pentylacetate{2"-(four Oxazol-5-yl)biphenyl-4'-ylmethyl}amino)butyric acid]Na ₃ ‧XH ₂ O and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to item 26 of the patent application scope, wherein the pharmaceutical composition contains a therapeutically effective dose of the crystalline form [3-((1S, 3R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butylcarbamate)propionic acid-(S)-3'-methyl-2'-(pentylamino {2"-(Tetrazol-5-yl)biphenyl-4'-ylmethyl}amino)butanoic acid]Na ₃ ‧XH ₂ O and a pharmaceutically acceptable carrier or excipient. A crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butan as described in any one of claims 1 to 12 Carbamoylamino) propionic acid-(S)-3'-methyl-2'-(pentylamino{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino )Butyric acid]Na ₃ ‧XH ₂ O or the pharmaceutical composition of any one of the patent application items 26 to 27 in the preparation of treatment or prevention of diseases related to neutral endopeptidase, cardiovascular, anti-hypertensive Application of medicine. The application according to item 28 of the patent application scope, wherein the antihypertensive is selected from antimalignant hypertension, primary hypertension, renal vascular hypertension, diabetic hypertension, simple systolic hypertension or other secondary Sexual hypertension. A crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butan as described in any one of claims 1 to 12 Carbamoylamino) propionic acid-(S)-3'-methyl-2'-(pentylamino{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino )Butyric acid]Na ₃ ‧XH ₂ O or the pharmaceutical composition according to any one of the patent application items 26 to 27 in the preparation of treatment or prevention of acute and chronic heart failure, congestive heart failure, left ventricular dysfunction, hypertrophy The use of drugs for sexual cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter or harmful vascular remodeling. A crystalline form [3-((1S,3R)-1-biphenyl-4-ylmethyl-3-ethoxycarbonyl-1-butan as described in any one of claims 1 to 12 Carbamoylamino) propionic acid-(S)-3'-methyl-2'-(pentylamino{2"-(tetrazol-5-yl)biphenyl-4'-ylmethyl}amino )Butyric acid]Na ₃ ‧XH ₂ O or the pharmaceutical composition according to any one of patent application items 26 to 27 in the preparation of treatment or prevention of myocardial infarction and its sequelae, atherosclerosis, angina pectoris, diabetic or Non-diabetic renal insufficiency, secondary hyperaldosteronism, primary or secondary pulmonary hypertension, diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, proteinuria of primary nephropathy , Renal hypertension, diabetic retinopathy, migraine, peripheral vascular disease, Raynaud's disease, cognitive dysfunction, glaucoma or stroke.

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