INOSITOL DERIVATIVE AND USE THEREOF

Abstract

Involved are an inositol derivative and a use thereof. Specifically, provided is a compound, said compound includes two or many parts, as represented by formula (D), connected via a common central linker L2, where formula (D) is as shown below.

##STR00001##

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer, rotamer or tautomer thereof, ##STR00084## formed by connecting, via a common linker L.sup.2, two or more moieties of formula (D) shown below: ##STR00085## wherein, ##STR00086##  o and q are each independently selected from the group consisting of 0 to 2, the value of o+q is 0, 1 or 2, and L.sup.1 represents —O—, —NH—, —C(═O)—, —OC(═O)—, —NHC(═O)—, —S— or a single bond; at least one X is each independently selected from the R.sup.1 being selected from or comprising polyethylene glycol of formula R.sup.4—(OCH.sub.2CH.sub.2).sub.gO— or R.sup.4—(OCH.sub.2CH.sub.2).sub.g— or polyglycerol of formula R.sup.4—O—(CH.sub.2—CHOR.sup.5—CH.sub.2O).sub.g—, the remaining X are each independently selected from the group consisting of hydrogen, —OH, OPO.sub.3.sup.2−, OPSO.sub.2.sup.2— or OSO.sub.3.sup.− and CO.sub.2.sup.−, and each formula (D) is identical or different, wherein the polyethylene glycol or the polyglycerol is optionally substituted with one or more groups selected from the group consisting of hydroxy, deuterium, halogen, a nitrile group, nitro, amino, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, —OC(O)R.sup.4, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene; L.sup.2 is a common central linker connected to formula (D), comprises C.sub.2-20 linear or branched alkylene, C.sub.2-20 linear or branched alkyleneoxy, C.sub.2-20 linear or branched alkyleneamino, C.sub.2-20 linear or branched alkylenemercapto, C.sub.2-20 linear or branched alkenylene, C.sub.2-20 linear or branched alkenyleneoxy, C.sub.2-20 cycloalkylene, —(OCH.sub.2CH.sub.2).sub.eO—, —(OCH.sub.2CH.sub.2).sub.e— or —O—(CH.sub.2—CHOR.sup.2—CH.sub.2O).sub.e—, and optionally comprises a moiety A, wherein the moiety A is selected from the group consisting of an oxygen atom, a nitrogen atom, a sulfur atom, C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, and 5-membered or 6-membered heterocyclyl or aryl ring group or heteroaryl ring group, and the alkylene, alkyleneoxy, alkyleneamino, alkylenemercapto, alkenylene, alkenyleneoxy, cycloalkylene, —(OCH.sub.2CH.sub.2).sub.eO—, —(OCH.sub.2CH.sub.2).sub.e— or —O—(CH.sub.2—CHOR.sup.2—CH.sub.2O).sub.e— is optionally substituted with one or more groups selected from the group consisting of hydroxy, halogen, deuterium, amino, a nitrile group, nitro, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene, the C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyl or C.sub.3-7 cycloalkylene being optionally substituted with halogen, hydroxy, deuterium, amino, a nitrile group or nitro; when the moiety A is selected from the group consisting of C.sub.1-6 alkyl and a nitrogen atom, A can be optionally substituted with R.sup.3, wherein R.sup.3 is selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR′, NR′(R″), COOR′ and CONR′(R″), the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl being optionally substituted with one or more groups selected from the group consisting of alkyl, alkoxy, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl, heteroaryl, nitro, a nitrile group, hydroxy, halogen, haloalkyl, haloalkoxy, halocycloalkyl, haloheterocyclyl, haloaryl and haloheteroaryl; when the moiety A is selected from a sulfur atom, A can be optionally substituted with oxygen; R.sup.4 is selected from the group consisting of hydrogen, C.sub.1-6 alkyl and C.sub.3-7 cycloalkyl, the C.sub.1-6 alkyl or C.sub.3-7 cycloalkyl being optionally substituted with one or more groups selected from the group consisting of deuterium, a nitrile group, nitro, amino, hydroxy and halogen; R.sup.2 or R.sup.5 is each independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl and a glycerol chain; R′ or R″ is independently selected from the group consisting of hydrogen, hydroxy, alkyl, alkoxy, alkenyl, acyl, aryl and heteroaryl, the alkyl, alkoxy, aryl or heteroaryl being optionally substituted with one or more groups selected from the group consisting of halogen, alkyl, cycloalkyl, alkoxy, alkenyl, alkynyl, oxy, hydroxy, nitro, a nitrile group and —R.sup.a; and R.sup.a is selected from the group consisting of aryl and heteroaryl, the aryl or heteroaryl being optionally substituted with one or more groups selected from the group consisting of halogen, alkyl, cycloalkyl, alkoxy, alkenyl, alkynyl, oxy, hydroxy, aryl, heteroaryl, nitro, a nitrile group, haloalkyl, haloalkoxy, halocycloalkyl, haloheterocyclyl, haloaryl and haloheteroaryl; g is 1-200; n is selected from the group consisting of 2 to 8, e.g., 2, 3, 4, 6, 7 and 8; e is an integer of 1-20.

2. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, being: ##STR00087##

3. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, comprising a moiety of formula (D) shown as formula (Da) or (Db) ##STR00088## wherein L.sup.1 and X are as defined in claim 1.

4. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, being: ##STR00089## including ##STR00090##

5. (canceled)

6. (canceled)

7. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein L.sup.2 is selected from the group consisting of —(OCH.sub.2CH.sub.2).sub.eO—, —(OCH.sub.2CH.sub.2).sub.e— and —O—(CH.sub.2—CHOR.sup.2—CH.sub.2O).sub.e—, wherein e is an integer of 1-20, e.g., 2, 3, 4, 5, 6, 7 or 8, and the —(OCH.sub.2CH.sub.2).sub.eO—, —(OCH.sub.2CH.sub.2).sub.e— or —O—(CH.sub.2—CHOR.sup.2—CH.sub.2O).sub.e— is optionally substituted with one or more groups selected from the group consisting of hydroxy, halogen, deuterium, amino, a nitrile group, nitro, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene.

8.-15. (canceled)

16. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, being: ##STR00091## wherein X, L.sup.1 and L.sup.2 are as defined in claim 1; and R.sup.4 is selected from the group consisting of hydrogen, methyl, ethyl, trifluoromethyl and cyclopropyl, the polyethylene glycol being optionally substituted with one or more groups selected from the group consisting of hydroxy, halogen, deuterium, C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene; ##STR00092## wherein the moiety A is selected from the group consisting of C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, and 5-membered or 6-membered heterocyclyl or aryl ring group or heteroaryl ring group, the C.sub.3-7 cycloalkyl or 5-membered or 6-membered heterocyclyl or aryl ring group or heteroaryl ring group being optionally substituted with halogen, hydroxy, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, SR′, NR′(R″), COOR′ or CONR′(R″); r, s, t, j, k, l and v are each independently selected from the group consisting of integers of 1 to 6, e.g., 1, 2 or 3.

17. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein at least two X are selected from R.sup.1 and on the same formula (D), the R.sup.1 being selected from or comprising polyethylene glycol of formula R.sup.4—(OCH.sub.2CH.sub.2).sub.gO— or R.sup.4—(OCH.sub.2CH.sub.2).sub.g—, wherein R.sup.4 is selected from the group consisting of hydrogen, methyl, ethyl, trifluoromethyl and cyclopropyl; and the remaining X are each independently selected from the group consisting of hydrogen, —OH, OPO.sub.3.sup.2−, OPSO.sub.2.sup.2− or OSO.sub.3.sup.− and CO.sub.2.sup.−, wherein the polyethylene glycol is optionally substituted with one or more groups selected from the group consisting of hydroxy, halogen, deuterium, C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene.

18. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein n is 5 or 6; further, L.sup.2 comprises C.sub.2-20 linear or branched alkylene, —(OCH.sub.2CH.sub.2).sub.eO—, —(OCH.sub.2CH.sub.2).sub.e— or —O—(CH.sub.2—CHOR.sup.2—CH.sub.2O).sub.e—, and comprises a moiety A, wherein the moiety A is selected from the group consisting of C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, and 5-membered or 6-membered heterocyclyl or aryl ring group or heteroaryl ring group, and the alkylene, —(OCH.sub.2CH.sub.2).sub.eO—, —(OCH.sub.2CH.sub.2).sub.e— or —O—(CH.sub.2—CHOR.sup.2—CH.sub.2O).sub.e— is optionally substituted with one or more groups selected from the group consisting of hydroxyl, halogen, deuterium, C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene.

19. (canceled)

20. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein R.sup.3 is selected from the group consisting of hydrogen, hydroxy, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyl, and 5-membered or 6-membered heterocyclyl or aryl ring group or heteroaryl ring group.

21. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein L.sup.1 represents —O—, —C(═O)—, —OC(═O)— or a single bond.

22. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein R.sup.2 or R.sup.5 is selected from the group consisting of hydrogen, methyl and ethyl.

23.-26. (canceled)

27. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein the polyglycerol or polyethylene glycol has a molar mass selected from the group consisting of 100 g/mol to 3000 g/mol.

28. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein g is 2-100.

29. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein L.sup.2 is selected from the group consisting of: ##STR00093## and the L.sup.2 is further optionally substituted with one or more groups selected from the group consisting of hydroxy, halogen, deuterium, amino, a nitrile group, nitro, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene.

30.-34. (canceled)

35. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein g is 2 to 100, 2 to 50, 2 to 20, or 2 to 10.

36. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein L.sup.2 is a common central linker connected to formula (D) and comprises C.sub.2-20 linear or branched alkylene, C.sub.2-20 linear or branched alkyleneoxy, —(OCH.sub.2CH.sub.2).sub.eO— or —(OCH.sub.2CH.sub.2).sub.e—, wherein the alkylene, alkyleneoxy, —(OCH.sub.2CH.sub.2).sub.eO— or —(OCH.sub.2CH.sub.2).sub.e— is optionally substituted with one or more groups selected from the group consisting of hydroxy, deuterium, amino, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene, and e is as defined in claim 1.

37. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, wherein L.sup.2 is a common central linker connected to formula (D) and comprises C.sub.2-10 linear or branched alkylene, —(OCH.sub.2CH.sub.2).sub.eO— or —(OCH.sub.2CH.sub.2).sub.e—, wherein the alkylene, —(OCH.sub.2CH.sub.2).sub.eO— or —(OCH.sub.2CH.sub.2).sub.e— is optionally substituted with one or more groups selected from the group consisting of hydroxy, deuterium, amino, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.3-7 cycloalkyl and C.sub.3-7 cycloalkylene, and e is as defined in claim 1; L.sup.1 represents —O— or a single bond.

38. (canceled)

39. The compound or the pharmaceutically acceptable salt thereof, or the stereoisomer, rotamer or tautomer thereof according to claim 1, being: ##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##

40. A pharmaceutical composition, comprising the compound or the pharmaceutically acceptable salt thereof or the stereoisomer, rotamer or tautomer thereof according to claim 1 and a pharmaceutically acceptable auxiliary material optionally selected from at least one of pharmaceutical excipients.

41. A method for treating or preventing a calcium disorder in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof of the stereoisomer, rotamer, or tautomer thereof according to claim 1, wherein the calcium disorder is selected from the group consisting of kidney stone, cardiovascular calcification, cardiovascular diseases, osteoporosis, bone cancer, podagra, calcific tendonitis, calcinosis cutis, rheumatoid arthritis, bone mineral diseases, osteomalacia, adynamic bone, calciphylaxis and cardiovascular diseases.

Description

DETAILED DESCRIPTION

[0171] The present disclosure is further described below with reference to examples, which are not intended to limit the scope of the present disclosure.

[0172] Experimental procedures without conditions specified in the examples of the present disclosure are generally conducted according to conventional conditions, or according to conditions recommended by the manufacturer of the starting materials or commercial products. Reagents without specific origins indicated are commercially available conventional reagents.

Example 1

[0173] ##STR00051##

[0174] To a 250 mL single-neck flask were added compound 1-1 (synthesized according to the method in CN108367080A, 3.04 g, 10 mmol) and DMF (30 mL), and the mixture was cooled in an ice bath, followed by the addition of NaH (0.96 g, 24 mmol). Compound 1-2 (synthesized according to the method described in U.S. Pat. No. 6,645,951, 6.03 g) was dissolved in DMF (30 mL), and the solution was added dropwise to the above reaction system. The mixture was stirred at room temperature until the reaction was substantially completed, and extracted with MTBE. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography to give compound 1-3 (2.15 g).

[0175] Ms (ESI): m/z 509 [M+H].sup.+.

[0176] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ 5.51 (s, 1H), 4.43 (s, 1H), 4.25-4.20 (m, 3H), 4.12-4.11 (m, 2H), 3.75-3.50 (m, 16H), 3.37 (s, 6H), 0.93 (s, 9H), 0.13 (s, 6H).

##STR00052##

[0177] To a 100 mL plastic flask were added compound 1-3 (1.52 g, 2.99 mmol) and THF (15 mL), and the mixture was cooled in an ice bath, followed by the addition of pyridine hydrogen fluoride (15 mL, containing 65-70% HF). The resulting mixture was reacted at room temperature for 12 h. The reaction solution was added to a saturated aqueous sodium bicarbonate solution to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 1-4 (1.1 g). Ms (ESI): m/z 395 [M+H].sup.+.

##STR00053##

[0178] To a 100 mL single-neck flask were added compound 1-4 (1.379 g, 3.5 mmol) and DMF (14 mL), and the mixture was cooled in an ice bath, followed by the addition of NaH (120 mg). After the addition, compound 1-5 (synthesized according to the method described in U.S. Pat. No. 4,001,279, 1.75 g) was dissolved in DMF (20 mL), and the solution was added dropwise to the above reaction solution until the reaction was substantially completed. The reaction solution was diluted with water (70 mL), and extracted with MTBE (35 mL×2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography to give compound 1-6 (1.43 g).

[0179] Ms (ESI): m/z 637 [M+H].sup.+.

[0180] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ 7.810-7.791 (d, 2H), 7.356-7.337 (d, 2H), 5.48 (s, 1H), 4.47-4.16 (m, 6H), 3.82-3.53 (m, 24H), 3.37 (s, 6H), 2.45 (s, 3H).

##STR00054##

[0181] To a 50 mL single-neck flask were added compound 1-4 (0.788 g, 2 mmol) and DMF (8 mL), and the mixture was cooled in an ice bath, followed by the addition of NaH (120 mg). After the addition, compound 1-6 (1.271 g) was dissolved in DMF (12 mL), and the solution was added dropwise to the above mixture. The resulting mixture was heated to 40° C. until the reaction was substantially completed. The reaction solution was diluted with water (40 mL), and extracted with methyl tert-butyl ether. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give compound 1-7 (1.08 g).

[0182] Ms (ESI): m/z 859 [M+H].sup.+.

[0183] .sup.1H-NMR (400 MHz, CDCl.sub.3): 5.50 (s, 2H), 4.50-4.45 (m, 2H), 4.40-4.32 (m, 4H), 4.30-4.25 (m, 4H), 3.83-3.54 (m, 42H), 3.38 (s, 12H).

[0184] To a 100 mL single-neck flask were added compound 1-7 (1.08 g, 1.26 mmol), MeOH (29 mL) and 0.1 N HCl (29 mL), and the mixture was heated to slight reflux. The reaction solution was directly concentrated to give the crude product (1.06 g). Ms (ESI): m/z 839 [M+H].sup.+.

##STR00055##

[0185] To a 50 mL single-neck flask were added the product of the previous step (600 mg, 0.7161 mmol) and tetrazole (451 mg, 6.444 mmol), and the mixture was dissolved in dichloromethane/acetonitrile (5 mL/18 mL), followed by the dropwise addition of compound 1-8 (2.223 g). After the addition, the resulting mixture was stirred at room temperature for 18 h. The reaction solution was cooled to −50° C., and m-chloroperoxybenzoic acid (1.312 g) was added. The mixture was heated to room temperature, and stirred until the reaction was substantially completed. The reaction was quenched with water (50 mL), and the reaction solution was extracted with methyl tert-butyl ether. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product. The crude product was purified by silica gel column chromatography to give compound 1-9 (453 mg, HPLC purity >98%).

[0186] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ 7.30-7.26 (m, 60H), 5.05-5.00 (m, 24H), 4.27 (s, 2H), 4.15-3.90 (m, 6H), 3.81-3.74 (m, 16H), 3.54-3.28 (m, 28H), 3.16 (s, 12H).

##STR00056##

[0187] To a 25 mL single-neck flask were added compound 1-9 (162 mg, 0.06 mmol), Pd(OH).sub.2/C (50 mg, wet), EtOH/H.sub.2O (5 mL/5 mL), and sodium bicarbonate (34 mg). The mixture was reacted for 8 h under hydrogen atmosphere, and filtered. The filtrate was concentrated to remove most of organic solvent, and lyophilized to give the target product 1a (87 mg, yield: 100%).

[0188] .sup.1H-NMR (400 MHz, D.sub.2O): δ 4.21 (s, 2H), 3.99-3.69 (m, 50H), 3.34 (s, 12H).

Example 2

[0189] ##STR00057##

[0190] To a 250 mL single-neck flask were added compound 1-4 (1.8 g, 4.57 mmol) and DMF (20 mL), and the mixture was cooled in an ice bath, followed by the addition of NaH (220 mg). The resulting mixture was reacted at room temperature for half an hour. Compound 2-1 (3.35 g) was dissolved in DMF (40 mL), and the solution was added dropwise to the above mixture. The resulting mixture was heated to 40° C. and stirred for 18 h. The reaction solution was diluted with water (120 mL) to quench the reaction, and extracted with methyl tert-butyl ether (60 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give compound 2-2 (3 g). Ms (ESI): m/z 835[M+H].sup.+.

##STR00058##

[0191] To a 250 mL hydrogenation flask were added compound 2-2 (2.8 g, 3.36 mmol), Pd(OH).sub.2/C (1.4 g, wet) and THF (56 mL), and the mixture was heated to 40° C. and reacted for 12 h under hydrogen atmosphere (1 atm). The reaction solution was filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography to give compound 2-3 (1.675 g). Ms: 655[M+H].sup.+.

[0192] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ 5.50 (s, 2H), 4.54 (s, 3H), 4.47-4.24 (m, 10H), 3.93-3.53 (m, 25H), 3.38 (s, 6H).

[0193] The procedures of Example 1 were then referred to give the target product 2a.

[0194] .sup.1H-NMR (400 MHz, D.sub.2O): δ 4.43-3.63 (m, 36H), 3.37 (s, 6H).

Example 3

[0195] ##STR00059##

[0196] To a 250 mL single-neck flask were added compound 3-1 (synthesized according to the method described in Organic and Biomolecular Chemistry, 2014, 13, 866-875, 4.44 g, 12 mmol) and DMF (50 mL), and the mixture was cooled in an ice bath, followed by the addition of NaH (0.624 g). Compound 3-2 (5.98 g) was dissolved in DMF (60 mL), and the solution was added dropwise to the above mixture. The resulting mixture was stirred until the reaction was substantially completed. The reaction solution was diluted with water (240 mL) to quench the reaction, and extracted with methyl tert-butyl ether (30 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give compound 3-2 (3.36 g).

[0197] Ms (ESI): m/z 811 [M+H].sup.+.

[0198] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ 7.32-7.26 (m, 20H), 5.50 (s, 2H), 4.70-4.55 (m, 8H), 4.43 (s, 2H), 4.35-4.32 (m, 7H), 4.13-4.11 (m, 1H), 4.00-3.88 (m, 2H), 3.69-3.62 (m, 8H).

##STR00060##

[0199] To a 250 mL hydrogenation flask were added compound 3-2 (2.6 g, 3.21 mmol), Pd/C (1.3 g, wet) and THF (52 mL), and the mixture was heated to 40° C. and reacted for 3 h under hydrogen (1 atm) atmosphere. The reaction solution was filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography to give compound 3-3 (1.4 g, yield: 96.9%).

[0200] Ms (ESI): m/z 451 [M+H].sup.+.

[0201] .sup.1H-NMR (400 MHz, D.sub.2O): δ 5.50 (s, 2H), 4.50-4.44 (m, 4H), 4.40-4.30 (m, 4H), 4.25-4.24 (m, 2H), 4.00-3.90 (m, 2H), 3.82-3.80 (m, 4H), 3.73-3.71 (m, 4H).

##STR00061##

[0202] To a 250 mL single-neck flask were added compound 3-3 (1.4 g, 3.11 mmol), MeOH (30 mL), and 0.5 N HCl (30 mL), and the mixture was heated to 65° C. and reacted for 5 h. The reaction solution was directly concentrated to give compound 3-4 (1.14 g). Ms (ESI): m/z 431[M+H].sup.+.

[0203] The procedures of Example 1 were then referred to give the target product 3a.

[0204] .sup.1H-NMR (400 MHz, D.sub.2O): δ 4.50-4.40 (m, 4H), 4.20-3.95 (m, 12H), 3.62-3.60 (m, 4H).

Example 4

[0205] ##STR00062##

[0206] To a 50 mL single-neck flask were added compound 3-1 (740 mg, 2 mmol, synthesized according to the method described in Organic and Biomolecular Chemistry, 2014, 13, 866-875), compound 4-1 (1.377 g, 4 mmol, synthesized according to the method described in Tetrahedron Asymmetry, 1995, 5, 1097-1104) and DMSO (15 mL), and the mixture was cooled in an ice bath, followed by the addition of KOH (560 mg). The resulting mixture was stirred at 70° C. for 5 h. The reaction solution was diluted with water (30 mL) to quench the reaction, and extracted with methyl tert-butyl ether (40 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give compound 4-2 (1.02 g).

[0207] Ms (ESI): m/z 429 [M+H].sup.+.

##STR00063##

[0208] To a 100 mL single-neck flask were added compound 1-4 (1.64 g, 4.19 mmol) and DMF (5 mL), and the mixture was cooled in an ice bath, followed by the addition of NaH (252 mg). The resulting mixture was reacted at room temperature for half an hour. Compound 4-3 (1.93 g, synthesized according to the method described in Chemistry—A European Journal, 2009, 31, 7534-7538) was dissolved in DMF (17 mL), and the solution was added dropwise to the above mixture. The resulting mixture was heated to 45° C. and stirred for 5 h. The reaction solution was diluted with water (44 mL) to quench the reaction, and extracted with methyl tert-butyl ether (50 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give compound 4-4 (2.08 g).

[0209] Ms (ESI): m/z 529 [M+H].sup.+.

##STR00064##

[0210] To a 100 mL hydrogenation flask were added compound 4-4 (2.08 g, 3.94 mmol), Pd(OH).sub.2/C (1 g, wet) and THF (42 mL), and the mixture was heated to 45° C. and reacted for 5 h under hydrogen atmosphere (1 atm). The reaction solution was filtered, and the filtrate was concentrated to give compound 4-5 (1.632 g).

[0211] Ms (ESI): 439 [M+H].sup.+.

##STR00065##

[0212] To a 100 mL single-neck flask were added compound 4-5 (1.632 g, 3.73 mmol), triethylamine (756 mg, 7.46 mmol) and dichloromethane (32 mL), and the mixture was cooled in an ice bath and reacted at room temperature for half an hour, followed by the addition of TsCl (851 mg, 4.48 mmol) and DMAP (48 mg). The resulting mixture was reacted at room temperature for 18 h. The reaction solution was directly concentrated and purified by silica gel column chromatography to give compound 4-6 (2.08 g).

[0213] Ms (ESI): m/z 593[M+H].sup.+.

##STR00066##

[0214] To a 50 mL single-neck flask were added compound 4-2 (0.685 g, 1.74 mmol) and DMF (10 mL), and the mixture was cooled in an ice bath, followed by the addition of NaH (139 mg). The resulting mixture was reacted at room temperature for half an hour. Compound 4-6 (1.922 g) was dissolved in DMF (10 mL), and the solution was added dropwise to the above mixture. The resulting mixture was heated to 40° C. and stirred for 18 h. The reaction solution was diluted with water (40 mL) to quench the reaction, and extracted with methyl tert-butyl ether (40 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give compound 4-7 (1.627 g).

[0215] Ms (ESI): m/z 849 [M+H].sup.+.

[0216] .sup.1H-NMR (400 MHz, CDCl3) δ 7.29-7.26 (m, 10H), 5.49 (s, 2H), 4.68-4.24 (m, 10H), 3.93-3.51 (m, 25H), 3.38 (s, 6H), 1.18 (d, J=6.4 Hz, 6H).

##STR00067##

[0217] To a 100 mL hydrogenation flask were added compound 4-7 (1.627 g, 1.92 mmol), Pd(OH).sub.2/C (0.82 g, wet) and THF (33 mL), and the mixture was heated to 45° C. and reacted for 5 h under hydrogen atmosphere (1 atm). The reaction solution was filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography to give compound 4-8 (740 mg).

[0218] Ms (ESI): m/z 669 [M+H].sup.+.

[0219] .sup.1H-NMR (400 MHz, CDCl3) δ 5.50 (s, 2H), 4.55-4.24 (m, 11H), 3.88-3.53 (m, 26H), 3.38 (s, 6H), 1.18 (d, J=6.4 Hz, 6H).

##STR00068##

[0220] To a 50 mL single-neck flask were added compound 4-8 (740 mg, 1.11 mmol), MeOH (15 mL) and 0.5 N HCl (15 mL), and the mixture was heated to slight reflux and reacted for 2 h. The reaction solution was directly concentrated to give compound 4-9 (crude, 750 mg).

[0221] Ms (ESI): m/z 649[M+H].sup.+.

##STR00069##

[0222] To a 100 mL single-neck flask were added compound 4-9 (350 mg, 0.54 mmol) and tetrazole (910 mg, 12.96 mmol), and the mixture was dissolved in dichloromethane/acetonitrile (35 mL/15 mL) and reacted for 20 min, followed by the dropwise addition of compound 1-8 (2.24 g). After the addition, the resulting mixture was stirred at room temperature for 1 h. The reaction solution was cooled to about −50° C., and m-chloroperoxybenzoic acid (1.98 g) was added. The mixture was heated to room temperature and stirred for 2 h. The reaction was quenched with water (50 mL), and the reaction solution was extracted with methyl tert-butyl ether. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and directly concentrated to give a crude product. The crude product was purified by silica gel column chromatography to give compound 4-10 (700 mg, HPLC purity >98%).

[0223] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.40-7.12 (m, 80H), 5.04-4.92 (m, 34H), 4.52 (s, 1H), 4.40-4.02 (m, 7H), 3.81-3.72 (m, 9H), 3.53-3.15 (m, 22H), 1.07 (d, J=6 Hz, 3H).

##STR00070##

[0224] To a 50 mL single-neck flask were added compound 4-10 (537 mg, 0.197 mmol), 20% Pd(OH).sub.2/C (179 mg, wet), EtOH/H.sub.2O (16 mL/16 mL), and sodium bicarbonate (132 mg). The mixture was reacted for 3 h under hydrogen atmosphere, and filtered. The filtrate was concentrated to remove most of organic solvent, and lyophilized to give the target compound 4a (285 mg, yield: 100%).

[0225] .sup.1H-NMR (400 MHz, D.sub.2O) δ 4.54-4.49 (m, 2H), 4.25-3.63 (m, 42H), 3.42 (s, 6H), 1.35-1.31 (m, 3H).

Example 5

[0226] ##STR00071##

[0227] To a 100 mL single-neck flask were added compound 3-1 (0.9 g, 2.43 mmol) and DMAc (3.6 mL), and the mixture was cooled in an ice bath, followed by the addition of t-BuONa (0.47 g, 2.0 eq). After the addition, compound 3-2 (10 g, synthesized by referring to Organic and Biomolecular Chemistry, 2006, 4, 2082-2087) was dissolved in DMAc (27 mL), and the solution was added to the above mixture until the reaction was substantially completed. The reaction solution was diluted with water and filtered, and the filter cake was slurried and washed with MTBE. The organic phases were combined, concentrated and crystallized, and filtered to remove the filter cake, and the filtrate was concentrated. The crude product was purified by column chromatography to obtain compound 2-1 (1.1 g, 73.9%).

[0228] Ms (ESI): m/z 613 [M+H].sup.+.

[0229] .sup.1H-NMR (400 MHz, CDCl3): δ 7.79-7.77 (d, J=8 Hz, 2H), 7.32-7.26 (m, 12H), 5.51 (s, 1H), 4.70-4.67 (d, J=12 Hz, 2H), 4.58-4.55 (d, J=12 Hz, 2H), 4.45 (s, 1H), 4.36-4.32 (m, 4H), 4.13-4.11 (m, 2H), 3.94 (s, 1H), 3.69-3.62 (m, 6H), 2.42 (s, 3H).

##STR00072##

[0230] To a 250 mL reaction flask were added compound 5-1 (0.50 g, 1.18 mmol), compound 2-1 (1.09 g, 1.78 mmol) and DMSO (4 mL), and the mixture was dissolved. Sodium tert-butoxide (0.17 g) was added at room temperature. After the addition, the mixture was reacted for 3 h, followed by the addition of methanol (0.2 mL). The resulting mixture was stirred for 0.5 h at room temperature. The reaction solution was added to a 20% ammonium chloride solution and methyl tert-butyl ether, followed by liquid separation. The aqueous phase was extracted with methyl tert-butyl ether, and the organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to give compound 5-2 (0.72 g, yield: 70.5%).

[0231] Ms (ESI): m/z 863 [M+H].sup.+.

[0232] .sup.1H-NMR (400 MHz, CDCl3) δ 7.29 (s, 10H), 5.50 (d, J=2.2 Hz, 2H), 4.68 (d, J=11.6 Hz, 2H), 4.58 (d, J=11.6 Hz, 2H), 4.49-4.42 (m, 2H), 4.35-4.34 (m, 6H), 4.30-4.28 (m, 2H), 3.97 (s, 1H), 3.82 (s, 1H), 3.76-3.60 (m, 18H), 3.41-3.27 (m, 10H), 1.14 (d, J=6.3 Hz, 6H).

[0233] The procedures of Example 4 were then referred to give the target product 5a. M+HM+H.sup.1H-NMR (400 MHz, D.sub.2O) δ 4.42 (d, J=9.4 Hz, 2H), 4.17-4.10 (m, 5H), 4.00-3.74 (m, 23H), 3.47-3.38 (m, 4H), 3.31 (s, 6H), 1.11 (d, J=6.4 Hz,

##STR00073##

[0234] To a 3 L three-neck flask were added compound a1 (1.01 g, 11.3 mmol) and THF (10 mL), and the mixture was cooled in an ice bath, followed by the dropwise addition of NaHMDS (6.19 mL, 1.1 eq). The resulting mixture was reacted in an ice bath for 2 h. Compound a2 (1.53 g) was added in portions to the reaction solution in an ice bath, and reacted for 2 h at room temperature until the reaction was substantially completed. Sulfuric acid and water were added to the reaction solution at room temperature and stirred for 16 h. The reaction solution was filtered, and the filtrate was concentrated. The crude product was distilled to give compound a3 (0.94 g, yield: 62.3%).

[0235] Ms (ESI): m/z 135 [M+H].sup.+.

[0236] .sup.1H-NMR (400 MHz, CDCl3): δ 3.72-3.64 (m, 4H), 3.59-3.52 (m, 1H), 3.40-3.32 (m, 5H), 2.68 (s, 1H), 1.13 (d, 3H, J=6.4 Hz).

##STR00074##

[0237] To a 1 L three-neck reaction flask were added compound a4 (0.72 g, 5.39 mmol), Et.sub.3N (0.654 g, 6.46 mmol), DMAP (0.066 g) and DCM (3.6 mL), and the mixture was cooled in an ice bath. Compound TsCl (1.13 g) was dissolved in DCM (3.6 mL), and the solution was added dropwise to the above mixture and reacted at room temperature for 1 h. After the addition, water was added, followed by liquid separation. The organic phase was washed sequentially with dilute hydrochloric acid and brine, dried, and filtered, and the filtrate was concentrated to give compound a5 (1.55 g, yield: 99.8%).

[0238] Ms (ESI): m/z 289 [M+H].sup.+.

[0239] .sup.1H-NMR (400 MHz, CDCl3): δ 7.80 (d, 2H, J=8.4 Hz), 7.33 (d, 2H, J=8.0 Hz), 4.14 (t, 2H, J=2.4 Hz), 3.71 (t, 2H, J=2.6 Hz), 3.60-3.56 (m, 1H), 3.35-3.25 (m, 5H), 2.44 (s, 3H), 1.08 (d, 3H, J=6.4 Hz).

##STR00075##

[0240] To a 1 L three-neck reaction flask were added compound 1-1 (0.30 g, 0.986 mmol), compound 1-2 (0.71 g, 2.46 mmol) and THF (4.5 mL), and a solution of NaHMDS in THF (1.7 mL, 3.45 mmol) was added dropwise and reacted until the reaction was substantially completed. The reaction solution was transferred into a 1 L Teflon flask, and cooled in an ice bath, followed by the addition of 65%-70% hydrogen fluoride pyridine solution (1.2 mL). The resulting mixture was reacted at room temperature. The reaction solution was washed with saturated sodium bicarbonate and extracted with dichloromethane. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by column chromatography to give compound 5-1 (0.165 g, yield: 40%).

[0241] Ms (ESI): m/z 423 [M+H].sup.+.

[0242] .sup.1H-NMR (400 MHz, CDCl3): δ 5.46 (s, 1H), 4.47-4.45 (m, 1H), 4.30-4.23 (m, 4H), 4.08 (s, 1H), 3.76-3.71 (m, 2H), 3.67-3.60 (m, 8H), 3.41-3.31 (m, 11H), 1.16-1.15 (m, 6H).

Example 6

[0243] ##STR00076## ##STR00077##

[0244] Compound 6-1 (0.85 g, 2.3 mmol) was dissolved in DMAc (3.5 mL), and the solution was cooled to 0° C., followed by the addition of t-BuONa (0.44 g, 4.6 mmol). A solution of compound 6-2 (7.7 g, 18.4 mmol, synthesized according to WO200923233A1) in DMAc (26 mL) was added dropwise and reacted until the reaction was substantially completed. The reaction solution was diluted with water (75 mL) and filtered, and the filter cake was slurried and washed with MTBE (80 mL×2). The organic phases were combined, concentrated and filtered, and the filtrate was concentrated. The crude product was purified by column chromatography to obtain compound 6-3 (1 g, 70%).

[0245] MS-ESI: m/z 617.2 [M+H].sup.+.

[0246] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.78 (d, J=8.3 Hz, 2H), 7.35-7.24 (m, 12H), 5.49 (s, 1H), 4.68 (d, J=11.6 Hz, 2H), 4.57 (d, J=11.6 Hz, 2H), 4.47-4.42 (m, 1H), 4.38-4.23 (m, 4H), 3.94 (d, J=1.3 Hz, 1H), 3.63 (d, J=24.6 Hz, 4H), 2.42 (s, 3H).

##STR00078##

[0247] Compounds 6-3 (1 g, 1.62 mmol) and 5-1 (0.57 g, 1.35 mmol) were dissolved in DMSO (6 mL), and sodium tert-butoxide (0.19 g, 2.0 mmol) was added and reacted at room temperature for 3 h. Methanol (0.2 mL) was added. The resulting mixture was stirred for 0.5 h at room temperature. The reaction solution was added to a 20% ammonium chloride solution (100 mL) and methyl tert-butyl ether (100 mL), followed by liquid separation. The aqueous phase was extracted with methyl tert-butyl ether (50 mL), and the organic phases were combined, washed with water (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography to give 6-4 (1.05 g, yield: 90%).

[0248] MS-ESI: m/z 867.4 [M+H].sup.+.

[0249] .sup.1H NMR (400 MHz, CDCl3) δ 7.29 (s, 10H), 5.50 (d, J=2.6 Hz, 2H), 4.68 (d, J=11.7 Hz, 2H), 4.58 (d, J=11.7 Hz, 2H), 4.48-4.42 (m, 2H), 4.41-4.25 (m, 8H), 3.96 (s, 1H), 3.81 (s, 1H), 3.76-3.54 (m, 14H), 3.41-3.28 (m, 9H), 3.22 (s, 1H), 1.14 (d, J=6.2 Hz, 6H).

[0250] The procedures of Example 4 were then referred to give the target product 6a.

[0251] .sup.1H NMR (400 MHz, D2O) δ 4.38 (dd, J=19.1, 9.6 Hz, 2H), 4.19-3.59 (m, 24H), 3.50-3.38 (m, 4H), 3.31 (s, 6H), 1.11 (d, J=6.4 Hz, 6H).

Example 7

[0252] ##STR00079##

[0253] The procedures of Example 4 were referred to give the target product 7a.

[0254] .sup.1H NMR (400 MHz, D2O) δ 4.35 (dd, J=19.0, 9.5 Hz, 2H), 4.10-3.67 (m, 28H), 3.44-3.36 (m, 4H), 3.29 (s, 6H), 1.09 (d, J=4.0 Hz, 6H).

Example 8

[0255] ##STR00080## ##STR00081##

[0256] Compound b1 (10.0 g, 11.1 mmol) was dissolved in THF (100 mL) and cooled to 0° C. NaHMDS (61 mL, 122.0 mmol) was added dropwise and reacted at 0° C. for 2 h. Compound a2 (15.2 g, 122.0 mmol) was added in portions and reacted at room temperature for 2 h. Sulfuric acid (61 mL) and water (61 mL) were added at room temperature and stirred for 16 h. NaOH was added to adjust pH to neutrality, and the reaction solution was filtered and concentrated. The residue was dissolved in methylene chloride (70 mL), and TEA (14.0 g) and DMAP (1.4 g) were sequentially added at room temperature, followed by the addition of a solution of TsCl (24.2 g) in methylene chloride (70 mL) in an ice bath. The resulting mixture was reacted at room temperature for 2 h. The reaction solution was washed sequentially with water and dilute hydrochloric acid. The organic phase was concentrated, and the residue was purified by column chromatography to give compound b3 (23 g, yield over two steps: 70%).

[0257] MS-ESI: m/z 289.1 [M+H].sup.+.

[0258] .sup.1H-NMR (400 MHz, CDCl3) δ 7.79 (d, 2H, J=8.4 Hz), 7.32 (d, 2H, J=8.4 Hz), 4.13 (t, 2H, J=5.2 Hz), 3.70 (t, 2H, J=5.2 Hz), 3.61-3.54 (m, 1H), 3.32-3.24 (m, 5H), 2.43 (s, 3H), 1.07 (d, 3H, J=6.4 Hz).

##STR00082##

[0259] Compound 8-1 (4.0 g, 11.6 mmol, synthesized according to Carbohydrate Research, 2002, 337, 2399-2410) and b3 (7.0 g, 24.4 mmol) were dissolved in DMSO (40 mL), and sodium tert-butoxide (2.8 g) was added at room temperature and reacted for 16 h. The reaction was extracted with water and EA, followed by liquid separation. The organic phase was concentrated, and the residue was purified by column chromatography to give compound 8-2 (3.8 g, yield: 56%).

[0260] MS-ESI: m/z 577.2 [M+H].sup.+.

[0261] .sup.1H-NMR (400 MHz, CDCl3) δ 7.86 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 2H), 5.46 (d, J=0.8 Hz, 1H), 5.04 (d, J=1.2 Hz, 1H), 4.45-4.43 (m, 1H), 4.27-4.21 (m, 4H), 3.68-3.58 (m, 10H), 3.41-3.30 (m, 10H), 2.44 (s, 3H), 1.15-1.13 (m, 6H).

##STR00083##

[0262] Compound 8-3 (2.0 g, 3.47 mmol) was dissolved in methanol (20 mL), and a solution of 30% sodium methoxide in methanol (10 mL) was added. The mixture was heated to 70° C. and reacted for 3 h. The reaction solution was concentrated by rotary evaporation to remove methanol, and the residue was dissolved in water until it was clarified, extracted with dichloromethane, and concentrated to give compound 8-4 (1.3 g, yield: 88%).

[0263] MS-ESI: m/z 423.2 [M+H].sup.+.

[0264] The procedures of Example 4 were then referred to give the target product 8a.

[0265] .sup.1H NMR (400 MHz, D.sub.2O) δ 4.38 (dd, J=19.0, 9.5 Hz, 2H), 4.21-3.55 (m, 28H), 3.47-3.35 (m, 4H), 3.30 (s, 6H), 1.09 (d, J=6.4 Hz, 6H).

Test Example 1

[0266] The inhibition effect of the test compounds on the generation of the human plasma hydroxyapatite (HAP) was determined by the spectrophotometric pharmacodynamics (PD) assay.

Experimental Procedures

[0267] 1.1 Preparation of Reagents [0268] 1) Human plasma anticoagulated with EDTA-K2. [0269] 2) Mixed solution: disodium hydrogen phosphate 5 mM, calcium chloride 41.67 mM, adjusted to pH 7.4 and filtered through a 0.22 μm filter. [0270] 3) Sodium chloride solution: 0.15 M, adjusted to pH 7.4 and filtered through a 0.22 μm filter.

[0271] 2.1 Test Procedures [0272] 1) The test compounds were prepared using a 0.15 M sodium chloride solution. [0273] 2) The human plasma sample was reprocessed. A sufficient amount of plasma was taken and centrifuged at room temperature at 10000 g for 30 min for later use. [0274] 3) The plasma was added to a 96 well plate at 75 μL/well. [0275] 4) The candidate drug was added at 5 μL/well. [0276] 5) The mixed solution of disodium hydrogen phosphate and calcium chloride was added at 120 μL/well, with expected final concentrations of 1.5 mM and 12.5 mM, respectively. [0277] 6) The plate was incubated at room temperature (25° C.) on a shaker (750 rpm), and the absorbance at a wavelength of 550 nm was measured every 3 min. [0278] 7) The final reading was set as the slope of the change in absorbance between 6 min and 24 min.

[0279] 3 Calculation Formula

[00001]$\mathrm{Crystallization}\mathrm{inhibition}\mathrm{rate}\left(\%\right)=\frac{\begin{array}{c}\mathrm{Slope}\left(\mathrm{blank}\mathrm{control}\right)-\\ \mathrm{Slope}\left(\mathrm{blank}\mathrm{control}\right)\end{array}}{\mathrm{Slope}\left(\mathrm{blank}\mathrm{control}\right)}\times 100\%$

[0280] 4 Experimental Results

TABLE-US-00001 Sample IC50 (Mean ± SD, nM) INS 3001 1864 ± 142  1a (Example 1) 306 ± 104 2a (Example 2) 230 ± 163 3a (Example 3) 343 ± 154 Note: INS 3001 was prepared by referring to the method described in CN108367080

Test Example 2

1 Experimental Objective

[0281] In a Na.sub.2HPO.sub.4 and NaCl buffer system, the crystallization process of hydroxyapatite (HAP) is initiated by adding high-concentration CaCl.sub.2, and HAP crystals have a maximum absorption peak at a wavelength of 550 nm. The absorption peak intensity of the HAP crystals is positively correlated with the content of HAP in the system. The compound has the functions of binding to the HAP crystals and inhibiting the formation and growth of the HAP crystals. Therefore, the inhibitory activity of different series of compounds on the formation of the HAP crystals is evaluated by measuring the OD value of an inorganic salt solution system at 550 nm.

2 Experimental Procedures

[0282] 1) Preparation of an inorganic salt system: 25 mM CaCl.sub.2 solution; 55 mM Na.sub.2HPO.sub.4.12H.sub.2O solution; 1.5 M NaCl solution;
2) mixed solution: 55 mM Na.sub.2HPO.sub.4.Math.12H.sub.2O, 1.5 M NaCl and ddH.sub.2O were added according to the volume ratio of 1:1:6, and adjusted to pH 7.4;
3) 160 μL of the mixed solution was added to a 96-well plate, and 20 μL of each of the compound solutions at different concentrations (100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, and 0.01 μM);
4) 25 mM CaCl.sub.2 solution was added at 20 μL/well;
5) the plate was placed on a shaker (300 rpm) for uniformly mixing for 30 s; and
6) the absorbance at a wavelength of 550 nm was measured every 3 min at room temperature (the plate was placed on a shaker (300 rpm) for uniformly mixing for 30 s before measurement). The measurement time was 60 min.

3 Experimental Results

[0283] The formation of the HAP crystals was determined in a 96-well plate by monitoring absorbance. The absorbance at a wavelength of 550 nm was read every 3 min, and the plate was shaken continuously on a shaker for 1 h. Two linear equations were derived for the OD values during the reading of each experimental well: the first one represents a line of baseline absorbance with a slope approaching 0, and the second one represents the maximum slope of the increase of the baseline absorbance. The induction time of each sample for the information of HAP was calculated, and the intersection between the two lines (baseline absorbance and maximum slope of increase) represents the time required for the formation of the HAP crystals. The induction time was determined for each of the compounds at different concentrations and compared to the control induction time. IC50 was determined by nonlinear fitting of the time-concentration curve using GraphPad software.

TABLE-US-00002 TABLE 1 Compound IC50 (μM) 1a 1.04 ± 0.54 2a 4.04 ± 2.55 3a 5.19 ± 0.31 5a 1.13 ± 0.26 7a 1.35 ± 0.14 8a 1.73 ± 0.57

[0284] Reference: Mechanism of action of SNF472, a novel calcification inhibitor to treat vascular calcification and calciphylaxis [J]. British Journal of Pharmacology, 2020, 177(19).