Salt of tetrahydroisoquinoline derivatives, preparation method thereof and medical application thereof

Abstract

The present invention relates to a pharmaceutically acceptable salt of (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, a preparation method thereof, a pharmaceutical composition comprising the pharmaceutically acceptable salt, and the use of the pharmaceutically acceptable salt as a therapeutic agent, in particular as an angiotensin II type 2 receptor antagonist.

Description

Technical Field

[0001] The present invention relates to a pharmaceutically acceptable salt form of (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, a method for preparing the same, a pharmaceutical composition comprising the same, and the use of the same as a therapeutic agent, in particular as an angiotensin II type 2 receptor antagonist. Background Art

[0002] Neuropathic pain is a chronic pain disease caused by primary damage or dysfunction of the nervous system. It can be divided into peripheral neuropathic pain and central neuropathic pain according to the location of the lesion. Trauma, inflammation, infection or compression can cause neuropathic pain, such as diabetic neuropathy (DNP), postherpetic neuralgia (PHN), primary neuropathy, secondary neuropathy, peripheral neuropathy, and neuropathy caused by mechanical nerve damage or biochemical nerve damage. At present, the drugs used in clinical treatment of neuropathic pain mainly include antiepileptic drugs, antidepressants and narcotic analgesics, such as gabapentin, pregabalin, tricyclic antidepressants, etc. However, these drugs are not targeted, the therapeutic effect is very limited, and there are serious side effects, including cognitive changes, sedation, nausea, tolerance and dependence, which are far from meeting the clinical drug needs. Therefore, it is necessary to study the pathogenesis of neuropathic pain, find clear targets for drug action, and develop new drugs that can effectively treat neuropathic pain with fewer adverse reactions.

[0003] Angiotensin II receptor is a G protein-coupled receptor with angiotensin II as its ligand. It is an important component of the renin-angiotensin system. The main subtypes of angiotensin II receptor include type 1 receptor (AT1R) and type 2 receptor (AT2R). AT1R and AT2R have only about 30% of the same amino acid sequence, but angiotensin II, as its main ligand, has similar affinity with both.

[0004] AT1R is the most well-studied angiotensin receptor. Activation of AT1R receptor can cause smooth muscle contraction, secretion of aldosterone and vasopressin, increased sodium reabsorption by renal tubules, activation of central and peripheral sympathetic nerves, and myocardial hypertrophy. Therefore, antagonizing angiotensin II at the receptor level has become a research hotspot in the search for new antihypertensive drugs, and a series of sartan antihypertensive drugs have been born.

[0005] AT2R is expressed in large quantities in various embryonic tissues and less distributed in normal adult tissues, but its expression increases after tissue damage. AT2R is related to blood pressure regulation, nerve growth, pain control and myocardial regeneration. Drugs targeting AT2R can improve cardiovascular function, relieve neuropathic pain, etc. The compound olodanrigan (EMA401) developed by Spinifex, an Australian company, is a highly selective AT2R antagonist. It is currently in Phase II clinical trials. This candidate drug has a good therapeutic effect on neuropathic pain such as diabetic neuropathy and postherpetic neuralgia. At the same time, Spinifex is also developing the AT2R antagonist EMA-400. Olodanrigan and EMA-400 are both prepared by WO 93 / 23378, and the structure is as follows:

[0006]

[0007] A series of patent applications for AT2R antagonists have been published, including WO2016113668, WO2015003223 and WO2013110135. The research and application of AT2R antagonists have made certain progress, but there is still huge room for improvement. It is still necessary to continue researching and developing new AT2R antagonists. Summary of the invention

[0008] The present invention provides a pharmaceutically acceptable salt of (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (1m), a preparation method thereof, a pharmaceutical composition comprising the pharmaceutically acceptable salt, and the use thereof as a therapeutic agent, especially as an angiotensin II type 2 receptor antagonist. The pharmaceutically acceptable salt is an alkali metal salt, calcium salt or magnesium salt formed by (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and an organic base or an inorganic base.

[0009] The salt form has excellent activity in treating primary neuropathy, secondary neuropathy, peripheral neuropathy, neuropathy caused by mechanical nerve damage or biochemical nerve damage, postherpetic neuralgia, diabetic neuropathy or related neurological diseases, significantly improved solubility, good pharmacokinetic properties in animals, low toxicity, and is suitable for preparing preparations for treating neurological diseases.

[0010]

[0011] The alkali metal salt of the present invention is selected from sodium salt, potassium salt or lithium salt; preferably sodium salt.

[0012] Typical pharmaceutically acceptable salts of the compound of formula (1m) of the present invention include, but are not limited to:

[0013]

[0014] The present invention also relates to a method for preparing a pharmaceutically acceptable salt of a compound of formula (1m), which comprises reacting (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid with a metal hydroxide, an alkali metal carbonate, an alkali metal bicarbonate or an alkali metal alkoxide, wherein the metal hydroxide is selected from sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, preferably sodium hydroxide; the alkali metal carbonate is selected from sodium carbonate or potassium carbonate; the alkali metal bicarbonate is selected from sodium bicarbonate or potassium bicarbonate; the alkali metal alkoxide is selected from sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium methoxide, potassium ethoxide or potassium tert-butoxide, preferably sodium methoxide or sodium ethoxide.

[0015] Generally, the above preparation process can be carried out under cooling, room temperature or heating conditions. It is worth noting that the choice of reaction temperature has a certain influence on different salt-forming reactions, which is also well known to those skilled in the art. The salt-forming reaction temperature of the present invention is from room temperature to the boiling point of the solvent used. Those skilled in the art can easily determine the most preferred reaction temperature for a specific salt-forming reaction by conventional technical means in the art.

[0016] The present invention relates to a pharmaceutical composition, which contains a therapeutically effective dose of a pharmaceutically acceptable salt of a compound of formula (1m) and a pharmaceutically acceptable carrier. The present invention also relates to the use of the pharmaceutical composition in the preparation of drugs for treating primary neuropathy, secondary neuropathy, peripheral neuropathy, neuropathy caused by mechanical nerve damage or biochemical nerve damage, postherpetic neuralgia, diabetic neuropathy or related neurological diseases.

[0017] The present invention relates to the use of a pharmaceutically acceptable salt of a compound of formula (1m) or a pharmaceutical composition thereof in the preparation of a medicament for treating primary neuropathy, secondary neuropathy, peripheral neuropathy, neuropathy caused by mechanical nerve damage or biochemical nerve damage, postherpetic neuralgia, diabetic neuropathy or related neurological diseases.

[0018] The present invention relates to the use of a pharmaceutically acceptable salt of a compound of formula (1m) or a pharmaceutical composition thereof in the preparation of angiotensin II type 2 receptor antagonists.

[0019] The present invention relates to a method for treating primary neuropathy, secondary neuropathy, peripheral neuropathy, neuropathy caused by mechanical nerve damage or biochemical nerve damage, postherpetic neuralgia, diabetic neuropathy or related neurological diseases, which comprises administering an effective therapeutic amount of a pharmaceutically acceptable salt of a compound of formula (1m), or a pharmaceutical composition thereof, to a patient in need of treatment.

[0020] The present invention relates to a method for inhibiting angiotensin II type 2 receptor, which comprises contacting angiotensin II type 2 receptor with a pharmaceutically acceptable salt of a compound of formula (1m) or a pharmaceutical composition thereof.

[0021] The present invention relates to a pharmaceutically acceptable salt of a compound of formula (1m), or a pharmaceutical composition thereof, for use as a drug for treating primary neuropathy, secondary neuropathy, peripheral neuropathy, neuropathy caused by mechanical nerve damage or biochemical nerve damage, postherpetic neuralgia, diabetic neuropathy or related neurological diseases.

[0022] The present invention relates to a pharmaceutically acceptable salt of a compound of formula (1m), or a pharmaceutical composition thereof as a drug for inhibiting angiotensin II type 2 receptor.

[0023] Detailed description of the invention

[0024] Unless stated otherwise, the terms in the specification and claims have the following meanings.

[0025] "Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0026] Synthesis method of the compound of the present invention

[0027] In order to achieve the purpose of the present invention, the present invention adopts the following technical solution:

[0028] (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid base addition salt is prepared by the following method:

[0029] (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is reacted with a metal hydroxide, an alkali metal carbonate, an alkali metal bicarbonate or an alkali metal alkoxide in a water-miscible organic solvent, wherein:

[0030] The metal hydroxide is selected from sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, and magnesium hydroxide, preferably sodium hydroxide;

[0031] The alkali metal carbonate is selected from sodium carbonate or potassium carbonate;

[0032] The alkali metal bicarbonate is selected from sodium bicarbonate or potassium bicarbonate;

[0033] The alkali metal alkoxide is selected from sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium methoxide, potassium ethoxide or potassium tert-butoxide, preferably sodium methoxide or sodium ethoxide. DETAILED DESCRIPTION

[0034] The following examples are used to further describe the present invention, but these examples are not intended to limit the scope and spirit of the present invention.

[0035] Example

[0036] The examples provide the preparation of representative compounds represented by formula (1m) and related structural identification data. It must be noted that the following examples are used to illustrate the present invention rather than to limit the present invention. 1 H NMR spectra were measured using a Bruker instrument (400 MHz), and chemical shifts were expressed in ppm using tetramethylsilane as an internal standard (0.00 ppm). 1 H NMR notation: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of a doublet, dt = doublet of a triplet. Coupling constants, when given, are given in Hz.

[0037] The mass spectrum is obtained by LC / MS, and the ionization method can be ESI or APCI.

[0038] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.15mm-0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm-0.5mm.

[0039] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0040] In the following examples, unless otherwise indicated, all temperatures are degrees Celsius. Unless otherwise indicated, various starting materials and reagents are commercially available or synthesized according to known methods. Commercially available materials and reagents are used directly without further purification. Unless otherwise indicated, commercial manufacturers include but are not limited to Aldrich Chemical Company, ABCR GmbH & Co. KG, Acros Organics, Guangzan Chemical Technology Co., Ltd. and Jingyan Chemical Technology Co., Ltd.

[0041] CDCl3: deuterated chloroform.

[0042] DMSO-d6: deuterated dimethyl sulfoxide.

[0043] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0044] The compound is purified using a silica gel column chromatography eluent system and thin layer chromatography, wherein the eluent system is selected from: A: petroleum ether and ethyl acetate system; B: dichloromethane and methanol system; C: dichloromethane and ethyl acetate; wherein the volume ratio of the solvent varies according to the polarity of the compound, and a small amount of acidic or alkaline reagents, such as acetic acid or triethylamine, may also be added for adjustment.

[0045] Example 1

[0046] Sodium (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

[0047]

[0048] first step

[0049] Methyl (Z)-3-(2-(benzyloxy)-3-methoxyphenyl)-2-((tert-butoxycarbonyl)amino)acrylate

[0050] Dissolve (±)-BOC-A-phosphonoglycine trimethyl ester 1b (9.8 g, 33 mmol) and tetramethylguanidine (4.0 g, 34.4 mmol) in 100 mL of tetrahydrofuran. Cool the reaction solution to 0°C and add a tetrahydrofuran solution (5 mL) of 2-(benzyloxy)-3-methoxybenzaldehyde 1a (7.0 g, 28.7 mmol) and react at room temperature overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. Ethyl acetate (40 mL) was added to dissolve the residue, which was washed with 10% citric acid solution (30 mL) and saturated brine (30 mL) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: System A) to obtain (Z)-methyl 3-(2-(benzyloxy)-3-methoxyphenyl)-2-((tert-butoxycarbonyl)amino)acrylate 1c (9.5 g, white solid). Yield: 80% MS m / z (ESI): 314.0 [M-100]

[0051] Step 2

[0052] (S)-3-(2-(Benzyloxy)-3-methoxyphenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid methyl ester

[0053] Methyl (Z)-3-(2-(benzyloxy)-3-methoxyphenyl)-2-((tert-butoxycarbonyl)amino)acrylate 1c (5.0 g, 12.0 mmol), (R)-N-diphenylphosphino-N-methyl-(S)-2-(diphenylphosphino)ferrocenylethylamine (90 mg, 0.06 mmol) and rhodium (I) di(1,5-cyclooctadiene)tetrafluoroborate (100 mg, 0.024 mmol) were dissolved in 50 mL of methanol, hydrogen was replaced three times, and a hydrogen balloon was inserted, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: System A) to obtain methyl (S)-3-(2-(benzyloxy)-3-methoxyphenyl)-2-((tert-butoxycarbonyl)amino)propanoate 1d (3.2 g, colorless oil) with a yield of 64%.

[0054] MS m / z(ESI):316.0[M-100]

[0055] 1H NMR (400MHz, DMSO-d6) δ7.48-7.32(m,5H),7.23(d,J=8.0Hz,1H),6.98-6.96(m,2H),6.78(dd,J=6.4,2.0Hz,1H),4.96(q,J=10.4Hz, 2H), 4.20(td,J=8.8,5.2Hz,1H),3.82(s,3H),3.55(s,3H),3.05(dd,J=13.4,5.0Hz,1H),2.71(dd,J=13.2,10.0Hz,1H),1.30(s,9H).

[0056] Step 3

[0057] (S)-2-Amino-3-(2-(benzyloxy)-3-methoxyphenyl)propanoic acid methyl ester hydrochloride

[0058] Methyl (S)-3-(2-(benzyloxy)-3-methoxyphenyl)-2-((tert-butoxycarbonyl)amino)propanoate 1d (3.2 g, 7.7 mmol) was dissolved in 10 mL of 1,4-dioxane, and a solution of hydrogen chloride in 1,4-dioxane (9.6 mL, 38.5 mmol, 4 M) was added, and the mixture was reacted at room temperature for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain methyl (S)-2-amino-3-(2-(benzyloxy)-3-methoxyphenyl)propanoate hydrochloride 1e (2.7 g, white solid), with a yield of 100%.

[0059] MS m / z(ESI):316.0[M+1]

[0060] 1 H NMR(400MHz,DMSO-d6)δ8.62(s,3H),7.47-7.32(m,5H),7.03-7.02(m,2H),6.81–6.78(m,1H), 4.94(q,J=11.2Hz,2H),4.04(t,J=7.2Hz,1H),3.83(s,3H),3.50(s,2H),3.05(d,J=7.2Hz,2H).

[0061] Step 4

[0062] (S)-5-(Benzyloxy)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester hydrochloride

[0063] (S)-2-amino-3-(2-(benzyloxy)-3-methoxyphenyl)propionic acid methyl ester hydrochloride 1e (1.3 g, 3.7 mmol) was dissolved in 2N dilute hydrochloric acid (26 mL), argon was replaced three times, and stirred at room temperature for 30 minutes. Formaldehyde aqueous solution (2.8 mL, 37 mmol, 37 wt.%) and tetrahydrofuran (5 mL) were added in sequence, and argon was replaced three times. The reaction was allowed to react overnight at room temperature. After the reaction was completed, acetonitrile was added to the reaction solution, and the reaction was concentrated under reduced pressure. The reaction was repeated several times to obtain (S)-5-(benzyloxy)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester hydrochloride 1f (400 mg, white solid) with a yield of 30%.

[0064] MS m / z(ESI):328.0[M+1]

[0065] 1 H NMR (400MHz, DMSO-d6) δ10.12(s,2H),7.45–7.33(m,5H),7.05(d,J=8.8Hz,1H),7.00(d,J=8.4Hz,1H),4.96(d,J=2.0Hz,2H),4.41(d d,J=10.8,5.2Hz,1H),4.22(q,J=15.6Hz,2H),3.82(s,3H),3.77(s,3H),3.21(dd,J=17.2,5.2Hz,1H),2.92(dd,J=17.6,11.2Hz,1H).

[0066] Step 5

[0067] (S)-5-(Benzyloxy)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester (S)-5-(Benzyloxy)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester hydrochloride 1f (80 mg, 0.22 mmol), 2-chloro-6-fluorobenzo[d]oxazole 1g (37 mg, 0.22 mmol) and triethylamine (91 μL, 0.66 mmol) were dissolved in 2 mL of tetrahydrofuran and reacted at 50-60°C for 5 hours. After the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by thin layer chromatography (developing solvent: System A) to give (S)-5-(benzyloxy)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester 1h (60 mg). Yield: 59%.

[0068] MS m / z(ESI):462.9[M+1]

[0069] 1H NMR (400MHz, CDCl3) δ7.49-7.29(m,6H),7.06(dd,J=7.8,2.2Hz,1H),6.96-6.86(m,3H),5.19(dd,J=6.4,2.4Hz,1H),5.05(d,J=10.8Hz,1 H),4.95(d,J=11.2Hz,1H),4.90(d,J=15.6Hz,1H),4.76(d,J=15.2Hz,1H),3.89(s,3H),3.66-3.61(m,4H),2.94(dd,J=16.4,6.4Hz,1H).

[0070] Step 6

[0071] (S)-2-(6-Fluorobenzo[d]oxazol-2-yl)-5-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester

[0072] (S)-5-(Benzyloxy)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester 1h (600 mg, 1.3 mmol) and 10% palladium on carbon (300 mg, 50% w) were dissolved in 10 mL of methanol, and a hydrogen balloon was inserted to replace the hydrogen 4 times, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, and the diatomaceous earth was washed with a mixed solvent of ethyl acetate and methanol (V:V=1:1) (100 mL×3) and dichloromethane (100 mL×3) in turn, and the filtrate was concentrated under reduced pressure to obtain crude (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-5-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester 1i (500 mg), with a yield of 100%. MS m / z(ESI):372.9[M+1]

[0073] 1 H NMR(400MHz, DMSO-d6)δ8.86(br,1H),7.50(dd,J=8.8,2.4Hz,1H),7.35(dd,J=8 .6,5.4Hz,1H),7.10-7.04(m,1H),6.88(d,J=8.4Hz,1H),6.74(d,J=8.4Hz,1H),5 .25(dd,J=6.4,2.4Hz,1H),4.81(d,J=15.2Hz,1H),4.63(d,J=15.6Hz,1H),3.79 (s,3H),3.59(s,3H),3.46(dd,J=16.2,1.8Hz,1H),3.04(dd,J=16.8,6.4Hz,1H).

[0074] Step 7

[0075] (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester

[0076] 2-(Chloromethyl)-5-methoxypyridine hydrochloride 1j (113 mg, 0.72 mmol), potassium carbonate (149 mg, 1.08 mmol) and (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-5-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester 1i (100 mg, 0.27 mmol) were dissolved in 6 mL of N,N-dimethylformamide in sequence and reacted at 70°C for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, 100 mL of ethyl acetate and 50 mL of water were added, the mixture was separated, the organic phase was collected, the aqueous phase was extracted with ethyl acetate (50 mL × 2), the organic phases were combined, washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester 1k (133 mg), yield: 100%. The crude product can be further separated and purified by column chromatography (eluent: B system) for characterization.

[0077] MS m / z(ESI):493.9[M+1]

[0078] 1 H NMR (400MHz, CDCl3) δ8.30(d,J=2.4Hz,1H),7.59(d,J=8.8Hz,1H),7.32-7.29(m,2H),7. 07(dd,J=7.8,2.2Hz,1H),6.96-6.86(m,3H),5.21(dd,J=6.4,2.4Hz,1H),5.12(d,J=12.0 Hz,1H),5.05(d,J=11.6Hz,1H),4.91(d,J=15.6Hz,1H),4.77(d,J=15.6Hz,1H),3.89(s,3 H),3.87(s,3H),3.67(dd,J=16.4,2.4Hz,1H),3.63(s,3H),3.02(dd,J=16.4,6.4Hz,1H).

[0079] Step 8

[0080] (S)-2-(6-Fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

[0081] (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid methyl ester 1k (133 mg, 0.27 mmol) was dissolved in 4 mL of tetrahydrofuran, 3 mL of a mixed solution of calcium chloride (481.74 mg, 4.34 mmol) in isopropanol and water (V:V=2:1) ​​was added, and then 3 mL of sodium hydroxide solution (56 mg, 1.4 mmol) was added, and the reaction was allowed to react at room temperature overnight. After the reaction, 80 mL of ethyl acetate and 100 mL of water were added, and the pH value of the reaction solution was adjusted to pH = 5-6 with 1 M dilute hydrochloric acid. The organic phase was collected and the aqueous phase was extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was separated and purified by preparative column to obtain (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 1m (15 mg), with a yield of 12%.

[0082] MS m / z(ESI):479.9[M+1]

[0083] 1 H NMR(400MHz,DMSO-d6)δ8.30(d,J=2.8Hz,1H),7.58(d,J=8.4Hz,1H),7.52-7.48(m,2H ),7.34(dd,J=8.6,5.0Hz,1H),7.09-7.01(m,3H),5.07(dd,J=6.2,2.6Hz,1H),5.00(d, J=11.2Hz,1H),4.93(d,J=11.2Hz,1H),4.79(d,J=15.6Hz,1H),4.66(d,J=15.2Hz,1H) ,3.87(s,3H),3.83(s,3H),3.51(dd,J=16.0,2.4Hz,1H),3.00(dd,J=16.2,6.6Hz,1H).

[0084] Step 9

[0085] Sodium (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate

[0086] (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid 1m (0.40 g, 0.84 mmol) was added to 6 mL of ethyl acetate, and NaOH / ethanol solution (0.05 g / mL, 0.668 mL) was added under stirring in an ice-water bath to adjust the pH to 7-8. The solution was concentrated to dryness to obtain sodium (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate 1 (0.42 g) with a yield of 100%.

[0087] MS m / z(ESI):480.2[M+1]

[0088] 1 H NMR (400MHz, DMSO-d6) δ8.26(s,1H),7.62(d,J=8.5Hz,1H),7.46(d,J=8.6Hz,1H),7.38(d,J=8.4Hz,1H),7.24(dd,J=8.2,4.9Hz,1H),7.01-6 .90(m,3H),4.93(s,2H),4.71(s,2H),4.66(d,J=5.5Hz,1H),3.84(s,3H),3.79(s,3H),3.67(d,J=16.1Hz,1H),2.78(dd,J=16.1,6.4Hz,1H).

[0089] Test example:

[0090] Solubility experiment

[0091] According to the conventional solubility determination method, the solubility of the compound of the present invention in four different systems was tested: phosphate buffer PBS (pH 7.4), methanol, 0.1% HCl and water. The results are shown in the following table:

[0092]

[0093]

[0094] Conclusion: The solubility of the sodium salt of the compound of the present invention (Compound 1) is significantly improved compared with its free acid (Compound 1m).

[0095] Biological evaluation

[0096] Test Example 1 Test of the antagonistic activity of the compounds of the present invention on human AT2R ligand binding

[0097] Angiotensin II Type 2 Receptor (AT2R) is involved in neuronal differentiation and regeneration, cell proliferation and angiogenesis, and the maintenance of bone mass. AT2R inhibitors can be used to treat pain and abnormal neuroregenerative diseases, inhibit tumor cell proliferation, and increase bone mass. The following method studies the degree of antagonism of the compounds of the present invention to AT2R through an AT2 ligand binding test.

[0098] 1. Reagents and consumables

[0099]

[0100]

[0101] 2. Reagent preparation

[0102] (1) 10 mM Angiotensin II human: Dissolve 10 mg Angiotensin II human (purity 99.09%) in 0.947 mL deionized water, aliquot and store at -80°C;

[0103] (2) Preparation of compound stock solution

[0104] All compounds were dissolved in dimethyl sulfoxide and prepared as 10 mM stock solutions according to standard protocols.

[0105] (3) Tag-lite angiotensin receptor red agonist: 8600 nM stock solution, stored at -80°C after aliquoting;

[0106] (4) 1X Tag-Lite Buffer (TLB): Dilute 5X TLB to 1X with deionized water.

[0107] 3. Experimental steps

[0108] (1) Prepare an appropriate amount of 1X TLB and mix well for later use;

[0109] (2) The test compound was diluted 5-fold, with a total of 10 concentration gradients;

[0110] (3) Transfer 160 nL / well of the diluted compound in step (2) to a working plate (3657, Corning) at 200 g, room temperature, for 1 min;

[0111] (4) Add 40 μl of 1X TLB to the above working plate, centrifuge at room temperature for 1 minute, 200 g (centrifugal force), shake on a shaker for 15 minutes to mix, and centrifuge at room temperature for 1 minute, 200 g (the working concentration of the compound is 4X);

[0112] (5) Dilute Tag-lite angiotensin receptor red agonist (8600 nM stock solution) to 12 nM using 1X TLB;

[0113] (6) Take 5 mL of 1X TLB in a 15 mL centrifuge tube;

[0114] (7) Thaw one tube of Tb-labeled-AT2R cells in a 37°C water bath until all the ice melts (1–2 min);

[0115] (8) Rapidly transfer the frozen-thawed cells to the 1X TLB in step (6), mix gently, and centrifuge at room temperature for 5 minutes at 1200g;

[0116] (9) Gently aspirate the supernatant, resuspend the cells in 1 mL of 1X TLB, add 1.7 mL of 1X TLB, mix well, and bring to room temperature for later use;

[0117] (10) Add 10 μL of cells to all test wells and centrifuge at room temperature for 3 seconds at 200 g; add 5 μL of the compound working solution 4X in step (4) to the corresponding wells; add 5 μL of the 4X Tag-lite angiotensin receptor red agonist diluted in step (5) to all test wells.

[0118] (11) The reaction plate was centrifuged at room temperature for 1 min at 200 g, allowed to stand at 25 °C for 1 h, and then centrifuged at room temperature for 1 min at 200 g. Data were collected using an Envision HTRF microplate reader, and the IC was calculated using a nonlinear fitting formula. 50 .

[0119] (12) Similarly, the same method was used, except that Tb-labeled-AT1R cells were used instead of Tb-labeled-AT2R cells to test the IC values ​​of the antagonistic activity of the compounds of the present invention against AT1R. 50 .

[0120] 4. Experimental results

[0121] IC of the compounds of the present invention for AT2R antagonist activity test 50 See the table below for values.

[0122] Compound No. <![CDATA[IC 50 (nM) / AT2R]]> <![CDATA[IC 50 (μM) / AT1R]]> Compound 1m 10 >10 Compound 1 6 >10

[0123] Conclusion: (1) The sodium salt of the compound of the present invention (Compound 1) and its free acid (Compound 1m) have significant antagonistic activity against AT2R;

[0124] (2) The IC of the sodium salt of the compound of the present invention (Compound 1) and its free acid (Compound 1m) for AT1R antagonism is 50 Values ​​> 10 μM, no antagonistic activity against AT1R;

[0125] Therefore, the compounds of the present invention have a highly selective antagonistic effect on AT2R.

[0126] Pharmacokinetic testing

[0127] 1. Purpose of the experiment

[0128] SD rats were used as test animals, and the pharmacokinetic characteristics of the compounds of the present invention in rats were studied by LC / MS / MS method, and the drug concentrations in plasma at different times were determined after intravenous injection or oral gavage.

[0129] 2. Experimental plan

[0130] 2.1 Experimental drugs and animals

[0131] Compound 1m and Compound 1

[0132] Six healthy adult Sprague Dawley (SD) male rats were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd.

[0133] 2.2 Drug preparation and administration

[0134] (1) Drug configuration:

[0135] Weigh 76.32 mg of compound 1m, dissolve in 12.72 mL of 0.5% sodium carboxymethylcellulose (CMC-Na), vortex for 1 min until the compound is completely suspended, and the final concentration is 6 mg / mL;

[0136] 76.61 mg of compound 1 was weighed and dissolved in 12.768 mL of 0.5% sodium carboxymethylcellulose (CMC-Na). The mixture was vortexed for 1 minute until the compound was completely suspended. The final concentration was 6 mg / mL.

[0137] (2) Administration

[0138] Six healthy adult SD rats, half male and half female, were equally divided into two groups, each with 3 rats. After overnight fasting, the rats were intragastrically administered (po) with the compound 1m of the present invention and compound 1, both at a dose of 60 mg / kg.

[0139] 2.3 Sample collection

[0140] Before administration and 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours after administration, approximately 0.25 mL of blood was collected from the jugular vein and anticoagulated with sodium heparin. After blood sample collection, it was placed on ice and centrifuged to separate plasma (centrifugation conditions: 7000 rpm, 5 minutes). The collected plasma was stored below -70°C before analysis.

[0141] 2.4 Sample pretreatment

[0142] 300 μL of methanol (including internal standard working solution, loratadine 800 ng / mL) was added to 100 μL of plasma sample, vortexed for 5 minutes, centrifuged at 10,000 rpm for 10 minutes, and 1 μL of the mixed solution was injected into LC-MS / MS for analysis.

[0143] 3. Pharmacokinetic parameter results

[0144] The pharmacokinetic parameters of the compounds of the present invention and the positive control are shown in the following table.

[0145]

[0146] Conclusion: The sodium salt of the compound of the present invention (Compound 1) has significantly improved blood concentration and area under the curve compared with its free acid (Compound 1m).

Claims

1. A salt of (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, wherein the salt is selected from:

2. A method for preparing the salt according to claim 1, the method comprising: (S)-2-(6-fluorobenzo[d]oxazol-2-yl)-6-methoxy-5-((5-methoxypyridin-2-yl)methoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is reacted with a metal hydroxide, an alkali metal carbonate, an alkali metal bicarbonate or an alkali metal alkoxide.

3. The method according to claim 2, wherein: The metal hydroxide is selected from sodium hydroxide; the alkali metal carbonate is selected from sodium carbonate; the alkali metal bicarbonate is selected from sodium bicarbonate; the alkali metal alkoxide is selected from sodium methoxide, sodium ethoxide, and sodium tert-butoxide.

4. The method according to claim 3, wherein: The alkali metal alkoxide is selected from sodium methoxide or sodium ethoxide.

5. A pharmaceutical composition comprising a therapeutically effective dose of the salt according to claim 1 and a pharmaceutically acceptable carrier.

6. Use of the salt according to claim 1 or the pharmaceutical composition according to claim 5 in the preparation of angiotensin II type 2 receptor antagonist.

7. The use according to claim 6, wherein the angiotensin II type 2 receptor antagonist is a drug for treating or preventing neurological diseases.

8. The use according to claim 6, wherein the angiotensin II type 2 receptor antagonist is a drug for treating or preventing primary neuropathy, secondary neuropathy, peripheral neuropathy, neuropathy caused by mechanical nerve damage or biochemical nerve damage, postherpetic neuralgia, and diabetic neuropathy.

9. Use of the salt according to claim 1 or the pharmaceutical composition according to claim 5 in the preparation of a medicament for inhibiting angiotensin II type 2 receptor.

10. The use according to claim 9, wherein the preparation of a drug for inhibiting angiotensin II type 2 receptor is a drug for treating neurological diseases.

11. The use according to claim 9, wherein the preparation of the drug for inhibiting angiotensin II type 2 receptor is a drug for treating primary neuropathy, secondary neuropathy, peripheral neuropathy, neuropathy caused by mechanical nerve damage or biochemical nerve damage, postherpetic neuralgia, and diabetic neuropathy.

Images