A Rapamycin Derivative, and a Preparation Method, Pharmaceutical Composition and Use Thereof

Abstract

The present invention pertains to the field of pharmaceutical chemicals, and relates to a rapamycin derivative of Formula I, and a preparation method, pharmaceutical composition and use thereof. The compounds of the present invention overcome the defects of rapamycin in terms of water solubility and metabolic properties, and some of the compounds have an in vitro anti-tumor activity superior to rapamycin, have less toxicity to normal cells than rapamycin, and have very good druggability.

##STR00001##

Claims

1. A compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof: ##STR00051## wherein, R.sub.1 and R.sub.2 are independently selected from H, A and B, and R.sub.1, R.sub.2 cannot be H simultaneously; ##STR00052## wherein, in Formula A or Formula B, arrows refer to sites where A or B links to mother ring of Formula I; n independently is 1, 2, 3, 4, 5, 6 or 7; R.sub.4 is independently selected from fluorine, chlorine, bromine, iodine, nitro, and cyano; X.sub.1, X.sub.2, X.sub.3, X.sub.4, Y.sub.1, Y.sub.2, Y.sub.3, Y.sub.4, and Y.sub.5 are independently selected from C, S, O, N and Se atoms; X.sub.1˜X.sub.2, X.sub.2˜X.sub.3, X.sub.3˜X.sub.4, Y.sub.1˜Y.sub.2, Y.sub.2˜Y.sub.3, Y.sub.3˜Y.sub.4, Y.sub.4˜Y.sub.5 are independently single bond or double bond; Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8, Z.sub.9 are independently selected from hydrogen atom, hydroxy, aldehyde group, carboxyl, amino, cyano, halogen, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylthiol, C.sub.3-C.sub.10 cycloalkoxy, C.sub.1-C.sub.6 alkenyl, eneynylheterocyclic ring, heterocycloalkyl, substituted heterocycloalkyl, aromatic ring, aromatic heterocyclic ring, benzo-aromatic heterocyclic ring, wherein the C.sub.1-C.sub.6 alkyl, aromatic ring, aromatic heterocyclic ring, benzo-aromatic heterocyclic ring are not substituted or substituted with 1, 2, 3, 4 or 5 substituents independently selected from the following groups: —F, —Cl, —Br, —I, nitro, hydroxy, amino, cyano, C.sub.1-C.sub.6 alkylthiol, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl, C.sub.1-C.sub.6 alkynyl and C.sub.1-C.sub.6 alkoxy.

2. The compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof according to claim 1, wherein: Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8, Z.sub.9 are independently selected from hydrogen atom, hydroxy, aldehyde group, carboxyl, amino, cyano, halogen, C.sub.1-C.sub.3 alkyl, C.sub.3-C.sub.6 cycloalkyl, C.sub.1-C.sub.3 alkoxy, C.sub.1-C.sub.3 alkylthiol, C.sub.3-C.sub.6 cycloalkoxy and C.sub.1-C.sub.3 alkyl-enyl.

3. The compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof according to claim 1, wherein in Formula A, N atom together with X.sub.1, X.sub.2, X.sub.3, X.sub.4 form a thiazole ring.

4. The compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof according to claim 1, wherein: R.sub.1 and R.sub.2 are independently selected from H, carbonylmethyl-(4-methyl-thiazole R.sub.4 salt-3-yl), carbonylmethyl-(4, 5-dimethyl-thiazole R.sub.4 salt-3-yl), carbonylmethyl-(pyridine R.sub.4 salt-1-yl), carbonylmethyl-(3-hydroxy-pyridine R.sub.4 salt-1-yl), carbonylmethyl-(3-methyl-pyridine R.sub.4 salt-1-yl) and carbonylmethyl-(4-methyl-pyridine R.sub.4 salt-1-yl), wherein R.sub.4 is independently selected from fluorine, chlorine, bromine, iodine, nitro and cyano; and R.sub.1, R.sub.2 cannot be H simultaneously.

5. The compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof according to claim 1, wherein the compound is selected from the following compounds: 31,42-O-carbonylmethyl-(4-methyl-thiazole bromide salt-3-yl)-rapamycin, 42-O-carbonylmethyl-(4-methyl-thiazole bromide salt-3-yl)-rapamycin, 31-O-carbonylmethyl-(4-methyl-thiazole bromide salt-3-yl)-rapamycin, 31,42-O-carbonylmethyl-(4,5-dimethyl-thiazole bromide salt-3-yl)-rapamycin, 42-O-carbonylmethyl-(4,5-dimethyl-thiazole bromide salt-3-yl)-rapamycin, 31-O-carbonylmethyl-(4,5-dimethyl-thiazole bromide salt-3-yl)-rapamycin, 31,42-O-carbonylmethyl-(pyridine bromide salt-1-yl)-rapamycin, 31,42-O-carbonylmethyl-(3-hydroxy-pyridine bromide salt-1-yl)-rapamycin, 31,42-O-carbonylmethyl-(3-methyl-pyridine bromide salt-1-yl)-rapamycin, 42-O-carbonylmethyl-(pyridine bromide salt-1-yl)-rapamycin, 42-O-carbonylmethyl-(3-methyl-pyridine bromide salt-1-yl)-rapamycin, 42-O-carbonylmethyl-(3-hydroxy-pyridine bromide salt-1-yl)-rapamycin, 42-O-carbonylmethyl-(4-methyl-pyridine bromide salt-1-yl)-rapamycin, 31,42-O-carbonylmethyl-(4-methyl-pyridine bromide salt-1-yl)-rapamycin, 31-O-carbonylmethyl-(3-methyl-pyridine bromide salt-1-yl)-rapamycin, 31-O-carbonylmethyl-(pyridine bromide salt-1-yl)-rapamycin, and 31-O-carbonylmethyl-(4-methyl-pyridine bromide salt-1-yl)-rapamycin.

6. A method for preparing the compound of Formula I according to claim 1, which comprises steps of any one of the following processes (1) to (3): Process (1): Preparation of compounds of Formula I with position 31,42 bis-substituted ##STR00053## Process (2): Preparation of compounds of Formula I with position 42 mono-substituted ##STR00054## ##STR00055## Process (3): Preparation of compounds of Formula I with position 31 mono-substituted ##STR00056## ##STR00057## wherein in the above methods (1) to (3), R.sub.3 is independently selected from F, Cl, Br and I; the other symbols are independently as defined in claim 1.

7. The method according to claim 6, wherein in the last reaction step of Process (1), Process (2) or Process (3), a reagent is added, and the reagent is independently selected from 5-membered heterocyclic compound, and 6-membered heterocyclic compound.

8. A pharmaceutical composition, which comprises the compound or a pharmaceutically acceptable salt or hydrate thereof of claim 1.

9. (canceled)

10. A coronary stent, comprising a drug coating comprising the compound or a pharmaceutically acceptable salt or hydrate thereof according to claim 1.

11. A method for suppressing immune, inhibiting mTOR, inhibiting mTORC1, inhibiting PI3K-Akt-mTOR signal pathway, inhibiting T lymphocyte proliferation, combating tumors, promoting tumor cell apoptosis, allowing cell cycle arrest at G1, lowering arterial embolism, combating aging, combating Alzheimer's disease, preventing organ rejection, combating inflammation or combating bacteria in vivo or in vitro, comprising a step of administering to a subject or a cell in need thereof an effective amount of the compound or a pharmaceutically acceptable salt or hydrate thereof according to claim 1.

12.-13. (canceled)

14. A method for treatment and/or prophylaxis of kidney cancer, lymphoma, lung cancer, liver cancer, breast cancer, neuroendocrine cancer, uterine sarcoma or gastric cancer, comprising a step of administering to a subject in need thereof, an effective amount of the compound or a pharmaceutically acceptable salt or hydrate thereof according to claim 1.

15. The compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof according to claim 1, wherein in Formula B, N atom together with Y.sub.1, Y.sub.2, Y.sub.3, Y.sub.4, Y.sub.5 form a pyridine ring.

16. The compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof according to claim 1, wherein Z.sub.1, Z.sub.2, Z.sub.3, Z.sub.4, Z.sub.5, Z.sub.6, Z.sub.7, Z.sub.8, Z.sub.9 are independently selected from hydrogen atom, hydroxy, and methyl.

17. The compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof according to claim 1, wherein R.sub.1 and R.sub.2 are independently selected from H, carbonylmethyl-(4-methyl-thiazole bromide salt-3-yl), carbonylmethyl-(4, 5-dimethyl-thiazole bromide salt-3-yl), carbonylmethyl-(pyridine bromide salt-1-yl), carbonylmethyl-(3-hydroxy-pyridine bromide salt-1-yl), carbonylmethyl-(3-methyl-pyridine bromide salt-1-yl) and carbonylmethyl-(4-methyl-pyridine bromide salt-1-yl), and R.sub.1, R.sub.2 are not H simultaneously.

18. The method according to claim 7, wherein the reagent is independently selected from thiazole, pyridine, thiazole substituted with one or more C.sub.1-C.sub.6 alkyl, pyridine substituted with one or more C.sub.1-C.sub.6 alkyl, thiazole substituted with one or more halogen atoms, and pyridine substituted with one or more halogen atoms.

19. The method according to claim 7, wherein the reagent is independently selected from 4-methylthiazole, 4,5-dimethylthiazole, pyridine, 3-hydroxypyridine, 3-methylpyridine and 4-methylpyridine.

20. The pharmaceutical composition of claim 8 further comprising a pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIG. 1: Inhibition effects of compounds on phosphorylation of Thr389 of S6K1 and Ser473 of Aktin tumor cell A549; A, compounds of Examples No. 1-8; B, Compounds of Examples No. 9-17.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0080] The embodiments of the present invention are illustrated in details in conjunction with examples. However, those skilled in the art would understand the following examples are merely to illustrate the present invention, rather than to limit the scope of the present invention. When specific conditions are not given in the examples, conventional conditions or conditions recommended by manufacturers are applied. The reagents or instruments which manufactures are not given are all conventional products commercially available in markets.

[0081] Melting points of compounds are measured by SRY-1 type melting point instrument, in which temperatures are not calibrated. 1H-NMR spectra are measured by VARIAN INOVA 600 type NMR spectrometer. Mass spectra are measured by API-150EX LC/MS high resolution magnetic mass spectrometer.

Example 1: Preparation of 31,42-O-carbonylmethyl-(4-methyl-thiazole bromide salt-3-yl-rapamycin (Compound 1)

[0082] Step 1: 1 g (1.09 mmol) of rapamycin was dissolved in 10 ml of dry dichloromethane, cooled to −10° C., then added with 0.87 g (1 mmol) of dry pyridine. To the reaction solution, 2.2 g (10 mmol) of bromoacetyl bromide in 5 ml of dichloromethane solution was slowly added dropwise, the addition was finished within 30 min, and the reaction was continued for 10 min then stopped. The reaction solution was adjusted to neutral pH with 1 mol/L hydrochloric acid, washed with distilled water 20 ml×3, then purified with column chromatography, to obtain 0.73 g of Intermediate 1 in form of white foam (n=1, R.sub.3=Br, R.sub.4=Br).

[0083] Step 2: 0.73 g (0.63 mmol) of Intermediate 1 (n=1, R.sub.3=Br, R.sub.4=Br) was dissolved in 20 ml of acetone, added with 0.94 g (9.45 mmol) of 4-methylthiazole, reacted at 60° C. for 5 h, the product was purified by column to obtain 0.7 g of Compound 1 in form of light yellow granules.

[0084] M.p. 142-145° C.; MS: 1193 [M-2Br-H]+, 597[(M-2Br)/2]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.18 (d, 2H), 8.07 (s, 2H), 6.46 (s, 1H), 6.38 (m, 1H), 6.24 (m, 1H), 6.22 (m, 1H), 5.71 (s, 1H), 5.59 (s, 2H), 5.46 (m, 1H), 5.33 (s, 1H), 4.91 (m, 3H), 4.53 (s, 2H).

Example 2: Preparation of 42-O-carbonylmethyl-(4-methyl-thiazole bromide salt-3-yl)-rapamycin (Compound 2)

[0085] Step 1: 2 g (2.19 mmol) of rapamycin was dissolved in 25 ml of dry ethyl acetate, cooled to 0-5° C. To the reaction solution, 1.50 g (22 mmol) imidazole was added, 1.2 g (11 mmol) of trimethylchlorosilane in 5 ml of ethyl acetate solution was slowly added dropwise within 30 min, until the conversion to Intermediate 2 was completed. To the reaction solution, 10 ml of 0.5 mol/L H.sub.2SO.sub.4 solution was slowly added dropwise within 3 h, until the conversion of Intermediate 2 was completed, and then the product was purified with column chromatography, to obtain 1.3 g of Intermediate 3 in form of white foam.

[0086] Step 2: 1.3 g (1.3 mmol) of Intermediate 3 was dissolved in 15 ml of dichloromethane, cooled to −10° C., then added with 1.03 g (13 mmol) of dry pyridine. To the reaction solution, 1.31 g (6.5 mmol) of bromoacetyl bromide in 5 ml solution was slowly added dropwise, and the addition was completed within 30 min, the reaction was continued for 10 min then stopped. The reaction solution was adjusted to neutral pH with 1 mol/L hydrochloric acid, washed with distilled water 20 ml×3, then purified with column chromatography, to obtain 1.2 g of Intermediate 4 (n=1, R.sub.3=Br, R.sub.4=Br) in form of white foam.

[0087] Step 3: 1.2 g (1.1 mmol) of Intermediate 4 (n=1, R.sub.3=Br, R.sub.4=Br) was dissolved in 15 ml of acetone, added dropwise with 0.7 ml of 1 mol/LH.sub.2SO.sub.4 aqueous solution, the addition was completed within 1 h, the reaction was continued for 30 min, and the conversion of the reactant was completed. By column separation and purification, 1.05 g of Intermediate 5 (n=1, R.sub.3=Br, R.sub.4=Br) in form of white foam was obtained.

[0088] Step 4: 1.05 g (0.95 mmol) of Intermediate 5 (n=1, R.sub.3=Br, R.sub.4=Br) was dissolved in 20 ml of acetone, added with 0.94 g (9.5 mmol) of 4-methylthiazole, reacted at 60° C. for 5 h, the product was purified by column to obtain 1.02 g of Compound 2 in form of light yellow granules.

[0089] M.p. 130-133° C.; MS: 1053.8 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.16 (d, 1H), 8.06 (s, 1H), 6.45 (s, 1H), 6.38 (m, 1H), 6.22 (m, 1H), 6.15 (m, 1H), 5.62 (s, 2H), 5.46 (M, 1H), 5.24 (s, 1H), 5.08 (d, 1H), 4.97 (s, 1H), 4.93 (s, 1H), 4.68 (m, 1H), 4.00 (s, 2H), 3.97 (s, 1H).

Example 3: Preparation of 31-O-carbonylmethyl-(4-methyl-thiazole bromide salt-3-yl)-rapamycin (Compound 3)

[0090] Step 1: 2 g (2 mmol) of Intermediate 3 was dissolved in 20 ml of ethyl acetate, cooled to 0-5° C. To the reaction solution, 1.36 g (20 mmol) of imidazole was added, 1.5 g (10 mmol) of tert-butyldimethylchlorosilane in 5 ml of ethyl acetate solution was slowly added dropwise with within 30 min, the reaction was continued for 30 min; by column separation, 1.3 g of Intermediate 6 was obtained. To the reaction solution, 0.5 ml of 0.5 mol/LH.sub.2SO.sub.4 solution was added within 30 min, and the addition was finished within 1 h, until the conversion of reactant was completed. By column separation and purification, 1.05 g of Intermediate 7 in form of white foam was obtained.

[0091] Step 2: 1.05 g (1.02 mmol) Intermediate 7 was dissolved in 10 ml of dichloromethane, cooled to −10° C., then added with 0.81 g (10.2 mmol) of dry pyridine. To the reaction solution, 1.03 g (5.1 mmol) of bromoacetyl bromide in 5 ml solution was slowly added dropwise, the addition was completed within 30 min. The reaction was continued for 10 min and then stopped. The reaction solution was adjusted to neutral pH with 1 mol/L hydrochloric acid, washed with distilled water 20 ml×3, then purification was carried out by using column chromatography method, to obtain 0.73 g of Intermediate 8 (n=1, R.sub.3=Br, R.sub.4=Br) in form of white foam.

[0092] Step 3: 0.73 g (0.64 mmol) of Intermediate 8 (n=1, R.sub.3=Br, R.sub.4=Br) was dissolved in 10 ml of acetone, added dropwise with 0.5 ml of 2 mol/L H.sub.2SO.sub.4 solution, the addition was completed within 30 min, the reaction was continued for 30 min, until the conversion of reactant was completed. By column separation and purification, 0.4 g of Intermediate 9 (n=1, R.sub.3=Br, R.sub.4=Br) in form of white foam was obtained.

[0093] Step 4: 0.4 g (0.39 mmol) of Intermediate 9 (n=1, R.sub.3=Br, R.sub.4=Br) was dissolved in 20 ml of acetone, added with 0.38 g (3.90 mmol) of 4-methylthiazole, reacted at 60° C. for 5 h; by column separation, 0.27 g (0.24 mmol) of Compound 3 in form of light yellow granules was obtained.

[0094] M.p. 130-132° C.; MS: 1054 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.16 (d, 1H), 8.05 (s, 1H), 6.45 (s, 1H), 6.37 (m, 1H), 6.25 (m, 1H), 6.18 (m, 2H), 5.70 (s, 2H), 5.46 (m, 1H), 5.35 (s, 1H), 4.55 (d, 2H), 4.04 (s, 1H).

Example 4: Preparation of 31,42-O-carbonylmethyl-(4,5-dimethyl-thiazole bromide salt-3-yl)-rapamycin (Compound 4)

[0095] Preparation method was referred to Example 1 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=4, 5-dimethylthiazole).

[0096] m.p. 135-139° C.; MS: 1220.9 [M-2Br-H]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.03 (d, 2H), 6.46 (s, 1H), 6.37 (m, 1H), 6.24 (m, 1H), 6.18 (m, 1H), 6.12 (m, 1H), 5.76 (m, 3H), 5.62 (s, 2H), 5.46 (m, 1H), 5.32 (s, 1H), 5.32 (s, 1H), 4.93 (s, 1H), 4.83 (m, 2H), 4.61 (s, 1H), 4.55 (s, 1H), 4.52 (s, 1H).

Example 5: Preparation of 42-O-carbonylmethyl-(4,5-dimethyl-thiazole bromide salt-3-yl)-rapamycin (Compound 5)

[0097] Preparation method was referred to Example 2 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.3=4, 5-dimethylthiazole).

[0098] m.p. 135-138° C.; MS: 1067.9 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.03 (d, 1H), 6.49 (s, 1H), 6.37 (m, 1H), 6.21 (m, 1H), 6.14 (m, 2H), 5.62 (m, 2H), 5.46 (m, 1H), 5.23 (s, 1H), 5.08 (s, 1H), 4.97 (s, 1H), 4.93 (s, 1H), 4.68 (s, 1H), 4.06 (s, 2H), 4.01 (s, 1H).

Example 6: Preparation of 31-O-carbonylethyl-(4, 5-dimethyl-thiazole bromide salt-3-yl)-rapamycin (Compound 6)

[0099] Preparation method was referred to Example 3 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=4, 5-dimethylthiazole).

[0100] m.p. 125-127° C.; MS: 1067.7 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 10.02 (d, 1H), 6.45 (s, 1H), 6.37 (m, 1H), 6.25 (m, 1H), 6.20 (m, 1H), 6.15 (m, 1H), 5.70 (m, 2H), 5.45 (m, 1H), 5.33 (s, 1H), 5.08 (s, 1H), 4.93 (s, 1H), 4.81 (m, 1H), 4.56 (m, 3H), 4.03 (m, 1H).

Example 7: Preparation of 31,42-O-carbonylmethyl-(pyridine bromide salt-1-yl)-rapamycin (Compound 7)

[0101] Preparation method was referred to Example 1 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=pyridine).

[0102] m.p. 140-142° C.; MS: 1153 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.96 (d, 4H), 8.72 (m, 2H), 8.22 (m, 4H), 6.44 (s, 1H), 6.40 (m, 1H), 6.24 (m, 1H), 6.17 (m, 2H), 5.87 (m, 1H), 5.79 (m, 1H), 5.48 (m, 1H), 5.29 (s, 1H), 4.95 (m, 2H), 4.81 (s, 1H), 4.52 (m, 3H).

Example 8: Preparation of 31, 42-O-carbonylmethyl-(3-hydroxy-pyridine bromide salt-1-yl)-rapamycin (Compound 8)

[0103] Synthesis method was referred to Example 1 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=3-hydroxypyridine).

[0104] m.p. 120-124° C.; MS; 1185.4 [M-2Br-H]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 12.04 (s, 2H), 8.60 (m, 4H), 8.00 (m, 4H), 6.43 (m, 2H), 6.12 (m, 3H), 5.61 (m, 3H), 5.56 (m, 1H).

Example 9: Preparation of 31,42-O-carbonylmethyl-(3-methyl-pyridine bromide salt-1-yl)-rapamycin (Compound 9)

[0105] Preparation method was referred to Example 1 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=3-methylpyridine).

[0106] m.p. 159-162° C.; MS: 1181.2 [M-2Br-H]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 9.00 (s, 1H), 8.89 (m, 2H), 8.81 (m, 1H), 8.57 (s, 2H), 8.14 (m, 2H), 6.46 (s, 1H), 6.38 (m, 1H), 6.23 (m, 1H), 6.14 (m, 2H), 5.80 (m, 5H), 5.46 (m, 1H), 5.29 (m, 1H).

Example 10: Preparation of 42-O-carbonylmethyl-(pyridine bromide salt-1-yl)-rapamycin (Compound 10)

[0107] Preparation method was referred to Example 2 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=pyridine).

[0108] m.p. 135-137° C.; MS: 1034.1 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 9.07 (d, 1H), 9.02 (m, 1H), 8.71 (m, 1H), 8.24 (m, 2H), 8.14 (m, 2H), 6.45 (s, 1H), 6.41 (m, 1H), 6.22 (m, 1H), 6.14 (m, 1H), 5.80 (m, 5H), 5.67 (m, 2H), 5.44 (m, 1H), 5.25 (s, 1H), 5.08 (m, 1H).

Example 11: Preparation of 42-O-carbonylmethyl-(3-methyl-pyridine bromide salt-1-yl)-rapamycin (Compound 11)

[0109] Preparation method was referred to Example 2 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=3-methylpyridine).

[0110] m.p. 138-140° C.; MS: 1048 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 9.07 (d, 1H), 9.02 (m, 1H), 8.71 (m, 1H), 8.24 (m, 2H), 8.14 (m, 2H), 6.45 (s, 1H), 6.41 (m, 1H), 6.22 (m, 1H), 6.14 (m, 1H), 5.80 (m, 5H), 5.67 (m, 2H), 5.44 (m, 1H), 5.25 (s, 1H), 5.08 (m, 1H).

Example 12: Preparation of 42-O-carbonylmethyl-(3-hydroxy-pyridine bromide salt-1-yl)-rapamycin (Compound 12)

[0111] Preparation method was referred to Example 2 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=3-hydroxypyridine).

[0112] m.p. 146-148° C.; MS: 1050 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 12.08 (s, 1H), 8.60 (m, 1H), 8.53 (m, 1H), 8.02 (m, 2H), 6.45 (m, 2H), 6.38 (s, 1H), 6.22 (m, 1H), 6.12 (m, 2H), 5.60 (m, 2H), 5.48 (m, 1H), 5.25 (m, 1H), 5.08 (m, 1H).

Example 13: Preparation of 42-O-carbonylmethyl-(4-methyl-pyridine bromide salt-1-yl)-rapamycin (Compound 13)

[0113] Preparation method was referred to Example 2 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=4-methylpyridine).

[0114] m.p. 157-160° C.; MS: 1048.1 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.89 (d, 1H), 8.05 (m, 2H), 6.45 (s, 1H), 6.41 (m, 1H), 6.22 (m, 1H), 6.12 (m, 2H), 5.61 (m, 3H), 5.46 (m, 1H), 5.25 (m, 1H), 5.08 (d, 1H), 4.93 (m, 2H).

Example 14: Preparation of 31,42-O-carbonylmethyl-(4-methyl-pyridine bromide salt-1-yl)-rapamycin (Compound 14)

[0115] Preparation method was referred to Example 1 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=4-methylpyridine).

[0116] m.p. 181-183° C.; MS: 1181.2 [M-2Br-H]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.90 (d, 2H), 8.78 (d, 2H), 8.06 (m, 4H), 6.46 (d, 1H), 6.38 (m, 1H), 6.20 (m, 1H), 6.14 (m, 2H), 5.80 (d, 1H), 5.62 (m, 4H), 5.47 (m, 1H), 5.29 (s, 1H).

Example 15: Preparation of 31-O-carbonylmethyl-(3-methyl-pyridine bromide salt-1-yl)-rapamycin (Compound 15)

[0117] Preparation method was referred to Example 3 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=3-methylpyridine).

[0118] m.p. 138-140° C.; MS: 1048.1 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.88 (d, 1H), 8.80 (d, 1H), 8.57 (m, 1H), 8.12 (m, 1H), 6.45 (m, 1H), 6.40 (m, 1H), 6.23 (m, 1H), 6.17 (m, 2H), 5.81 (d, 1H), 5.74 (m, 2H), 5.47 (m, 1H) 5.28 (s, 1H).

Example 16: Preparation of 31-O-carbonylmethyl-(pyridine bromide salt-1-yl)-rapamycin (Compound 16)

[0119] Preparation method was referred to Example 3 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=pyridine).

[0120] m.p. 140-142° C.; MS: 1034 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.96 (d, 2H), 8.72 (m, 1H), 8.22 (m, 2H), 6.44 (s, 1H), 6.40 (m, 1H), 6.24 (m, 1H), 6.17 (m, 2H), 5.87 (m, 1H), 5.79 (m, 1H), 5.48 (m, 1H), 5.29 (s, 1H), 4.95 (m, 2H), 4.81 (s, 1H), 4.52 (m, 3H).

Example 17: Preparation of 31-O-carbonylmethyl-(4-methyl-pyridine bromide salt-1-yl)-rapamycin (Compound 17)

[0121] Preparation method was referred to Example 3 (n=1, R.sub.3=Br, R.sub.4=Br, R.sub.5=4-methylpyridine).

[0122] m.p. 145-148° C.; MS: 1048.1 [M-Br]+; 1H-NMR (600 MHz, DMSO-d6 δ ppm), 8.77 (d, 2H), 8.03 (d, 2H), 6.44 (s, 1H), 6.40 (m, 1H), 6.23 (m, 1H), 6.16 (m, 2H), 5.80 (d, 1H), 5.70 (d, 1H), 5.44 (m, 1H), 5.29 (m, 1H), 5.29 (s, 1H).

Example 18: Solubility Test

[0123] The Compounds 1-17 of the present invention were taken, and separately tested according to the Solubility Test Method of the Part II of Chinese Pharmacopoeia, 2010 Edition. The results are shown in Table 2:

TABLE-US-00002 TABLE 2 Water solubility of Compounds 1-17 Compound [00027] Water solubility Comparison to water solubility of No. R.sub.1 R.sub.2 (mg/ml) rapamycin Rapamycin H H 0.0026 1 1 [00028] [00029] 34.5 13269 2 [00030] H 0.028 11 3 H [00031] 10 3846 4 [00032] [00033] 100 38462 5 [00034] H 4.2 1615 6 H [00035] 23.3 8962 7 [00036] [00037] 85 32692 8 [00038] [00039] 0.1 38 9 [00040] [00041] 47 18077 10 [00042] H 0.025 10 11 [00043] H 6.7 2577 12 [00044] H 0.01 4 13 [00045] H 6 2308 14 [00046] [00047] 120 46154 15 H [00048] 26.7 10269 16 H [00049] 52.6 20231 17 H [00050] 62.5 24038

[0124] It can be seen from data of Table 2 that the compounds of the present invention have water solubility significantly higher than that of rapamycin, and even by 3 to 4 orders of magnitudes for some of these compounds.

Example 19: Experimental Evaluation of Toxicity on Rat Primary Hepatocytes

[0125] Experimental method: Liver lobes of SD rat were placed in sterilized plate, added with a suitable amount of liver cell cleaning solution (precooled at 4° C.), the liver was shredded and fibrous connective tissues were removed to form a liver cell suspension, screened with 200 mesh screen and placed in 50 ml centrifuge tube, centrifuged at 500 rpm for 1-2 min; the supernatant was removed, the precipitate was added with 20-30 ml of RPMI 1.640 (precooled at 4° C.) and washed 3 times; isovolumetric liver cell suspension was taken and suspended in Percoll separating solution I, mixed by turning upside down, centrifuged at 4° C. and 800 rpm for 10 min, the supernatant was discarded, the liver cells were washed with PBS once (800 rpm, 5 min). The liver cell precipitate was added into liver cell culture fluid, resuspended and diluted to 10 ml, and a suitable amount thereof was taken for counting and survival rate was determined by using 0.4% trypan blue. The liver cells with survival rate of 90% or more were inoculated in a density of 2×10.sup.3 on 96-well plate, 100 μl of RPMI 1640 culture medium was added to each well, culture was performed at 37° C., 5% CO.sub.2 for 24 h. The compounds were diluted with DMSO to desired concentrations and added to each well in an amount of 1 μl (final compound concentrations were usually initiated from 1000 μM, 10× gradient dilution, 6 gradients), and blank control was added with 1 μl of DMSO.

[0126] After the cells were cultured at 37° C., 5% CO.sub.2 for 24 h, 100 μl of ATP detection reagent was added, incubated under shock for 15 min. fluorescence values of wells were read by ELIASA. Cell survival rates for each example compounds were calculated, and then GIs values (concentrations of added compounds at which growth of 50% cells was inhibited) were calculated. The results were shown in Table 4. Obviously, the toxicities of the compounds were significantly less than that of rapamycin.

TABLE-US-00003 TABLE 4 GI.sub.50 values of some example compounds on rat primary hepatocytes Compound GI.sub.50 (μM) Rapamycin 24.822 Compound of Example 11 71.753 Compound of Example 13 44.936 Compound of Example 17 54.311

[0127] The data of Table 4 show that the compounds of the present invention have toxicities significantly less than rapamycin.

Example 20: Experiment of Inhibition Activities of the Compounds to mTORC1 and mTORC2

[0128] Molecular mechanism of action of the example compounds in tumor cell A549.

[0129] Experimental materials: DMEM high-glucose cell culture media (Hyclone Company), fetal calf serum (FBS) (Gibco Company), penicillin and streptomycin were purchased from North China Pharmaceutical Co., Ltd, phosphate buffer saline (PBS) was purchased from Gibco Company, pancreatic enzyme and dimethylsulfoxide (DMSO) were products of Sigma Company. A549 cell line (human lung adenocarcinoma cell line) was purchased from ATCC. Mouse anti-human p-p70S6K(T389) monoclonal antibody was purchased from Cell Signaling Technology Company, mouse anti-human p70S6K monoclonal antibody was purchased from Cell Signaling Technology Company, mouse anti-human p-AKT (S473) monoclonal antibody was purchased from Cell Signaling Technology Company, mouse anti-human AKTmonoclonal antibody was purchased from Cell Signaling Technology Company. Horseradish peroxidase-labeled goat anti-mouse, goat anti-mouse monoclonal antibodies were purchased from Cell Signaling Technology Company. Cell lysis solution was purchased from Beijing Solarbio Science and Technology Co., Ltd., loading buffer was purchased from Biyuntian Company, SDS electrophoretic buffer solution was purchased from Biyuntian Company, Towbin transfer buffer was purchased from Biyuntian Company, TBS was purchased from Biyuntian Company, TBST was purchased from Biyuntian Company. ECL chemiluminescence solution was purchased from Beijing Pulilai Genetic Technology Co., Ltd., developing solution and fixing solution were purchased from the Tenth Chemical Plant of Shijiazhuang, NC membrane was purchased from Whatman Company, photographic film was purchased from Carestream (Xiamen) Medical Equipment Co., Ltd., skimmed milk powder was purchased from Beijing Xikai Creative Technology Co., Ltd.

Experimental Method

[0130] A549 cells were spread on a 96-well plate, the cells grew to density of 80%-90% and then cultured overnight under serum deprivation condition, co-incubated on the second day with 167 nM insulin and the compounds for 2 hours, then the cells were lysed and the phosphorylation levels for the example compounds at Thr389 site of S6K1 (corresponding to mTORC1) and Ser473 site of Akt (corresponding to mTORC2) were detected by Western Blot method, and which were used to semi-quantitatively reflect the inhibition levels of the compounds to mTORC1 and mTORC2 (in the bands of the results, absence of blot represented expression of corresponding protein was inhibited, while presence of blot represented the expression was not inhibited). Positive control compound was Rapamycin. The results of evaluation are shown in FIG. 1 (FIG. 1A and FIG. 1B). Specific steps were as follows:

[0131] 1. Treatment of Cells:

[0132] 1) the cells were inoculated on 6-well plate, and the cells spread 80%-90% after 24 h;

[0133] 2) the cells were washed with PBS once, then 2 mL serum-free culture media was used for replacement and the culture was performed overnight under serum deprivation condition;

[0134] 3) to the treated cells, 2 μL of cell culture fluid containing the compound to be tested (20 μM) and insulin (167 nM) was added and co-incubation was performed for 2 h.

[0135] 2. Lysis of Cells:

[0136] 1) the cells on the 6-well plate were washed off with 10 ml PBS, centrifuged at 1500 rpm for 5 min;

[0137] 2) the supernatant was discarded then 60 μl cell lysis buffer (containing protease inhibitor) was added, the cells were lysed on ice for 20-30 min, after 10 min, oscillated with oscillator for 2-3 s, and put back on ice;

[0138] 3) oscillation was performed again for 3 s before centrifugation, then centrifuged at 4° C., 13000 rpm, for 10 min;

[0139] 4) 2×loading buffer 60 μl was added, subjected to boiling water bath for 5 min, and cryopreservated at −20° C. or −80° C. for standby use;

[0140] 3. Western Blotting Analysis:

[0141] 1) SDS-PAGE, 80 V voltage was firstly used for running to spacer gel, then 120 V voltage was used for running separation gel;

[0142] 2) transfer, wet transfer, in ice-bath, 250 mA, 150 min;

[0143] 3) 5% milk was used for sealing at room temperature for 1-2 h (or overnight at 4° C.);

[0144] 4) primary antibody was added (diluted with TBS), stood overnight at 4° C.;

[0145] 5) TBST washing 3×10 min.

[0146] 6) secondary antibody was added (1: 2000 diluted with 3% skimmed milk powder in TBS), incubation was performed at room temperature at sealing film for 2 hours;

[0147] 7) TBST washing 3×10 min.

[0148] 8) ECL chemiluminescence developing solutions A, B, each in 500 μl, were mixed in a plate dish, and washed for 5 min;

[0149] 9) the film was placed in exposure clamp, exposed in darkroom for a certain time, then placed in developing solution for 2 min, in fixing solution for 5 min, the film was washed with tap water, and dried by baking;

[0150] 10) pictures were taken and kept.

[0151] The results showed that the compounds of the present invention could effectively inhibit mTORC1, and their inhibition effects are not inferior to rapamycin (rapamycin could only inhibit mTORC1, so that our compounds had activities not inferior to rapamycin).

Example 21: Experiment of Tumor Cell Inhibition

[0152] The activities of example compounds to tumor cell A549 were evaluated.

[0153] Experimental materials: DMEM high-glucose cell culture media (Hyclone Company), fetal calf serum (FBS) (Gibco Company), penicillin and streptomycin were purchased from North China Pharmaceutical Co., Ltd, phosphate buffer saline (PBS) was purchased from Gibco Company, Cell Titer-Glo® cell viability detection reagent was purchased from Promega Company, pancreatin and dimethylsulfoxide (DMSO) were products of Sigma Company. A549 cell line (human lung adenocarcinoma cell line) was purchased from ATCC.

Experimental Method

[0154] The cells were inoculated on a 96-well plate with white wall and penetrative bottom (Costar) in amount of 5000 cells per well, cultured at condition of 37° C., 5% CO.sub.2 for 24 h. The compounds to be tested were dissolved with DMSO and diluted to 100 mM, to obtain mother liquids of the compounds.

[0155] DMEM culture solution containing 2% FBS was used to diluted the compounds, in which concentration gradients were 3, and concentration range was 100 μM to 3 nM. The compounds in various dilution degrees were added to the cultured cells on the 96-well plate, 100 μl per well. Culture was performed at 37° C., CO.sub.2 for 72 h, supernatant was discarded, and cell viability detection test was performed.

[0156] AfterCell Titer-Glo® reaction buffer solution was mixed with isometric substrate, the mixture was added to 96-well plate, 100 μl per well. Cell lysis was induced by horizontally shaking for 4 min. Reaction signal was stabilized by balancing at room temperature for 15 min. Chemiluminescence units in each well of the 96-well plate were detected by using chemiluminescence detector.

[0157] Inhibition rates at different dilution degrees of each compound were calculated according to chemiluminescence value of each well, and Origin 8.0 software was used for S-type curve fitting of each compound, and for calculating EC.sub.50 value. The results were shown in Table 5.

TABLE-US-00004 TABLE 5 EC.sub.50 values of concentration of some example compounds for tumor cell A549 Compound IC.sub.50 (μM) rapamycin 49.35 Compound of Example 1 17.39 Compound of Example 2 19.31 Compound of Example 3 3.04 Compound of Example 4 6.23 Compound of Example 5 7.40 Compound of Example 6 52.26 Compound of Example 7 46.68 Compound of Example 8 45.98 Compound of Example 9 25.12 Compound of Example 10 66.32 Compound of Example 11 52.80 Compound of Example 12 51.48 Compound of Example 13 44.80 Compound of Example 14 44.0 Compound of Example 15 52.70 Compound of Example 16 57.71 Compound of Example 17 40.80

[0158] The results showed that the compound of the present invention could effectively inhibit tumor cells, and some compounds had effects even superior to rapamycin, and thus are promising in the manufacture of anti-tumor drugs.

Example 22: Experiment of Inhibition to Drug-Resistant Tumor Cell Line

[0159] The activity of example compounds to multi-drug resistant tumor cell line MES-SA was evaluated.

[0160] Experimental materials: DMEM high-glucose cell culture media (Hyclone Company), fetal calf serum (FBS) (Gibco Company), penicillin and streptomycin were purchased from North China Pharmaceutical Co., Ltd, phosphate buffer saline (PBS) was purchased from Gibco Company, Cell Titer-Glo® cell viability detection reagent was purchased from Promega Company, pancreatin and dimethylsulfoxide (DMSO) were products of Sigma Company. MES-SA cell line (human uterine sarcoma cell line) was purchased from ATCC. MRC5 cell (human embryonic lung fibroblast) was purchased from ATCC.

Experimental Method

[0161] The cells were inoculated on a 96-well plate with white wall and penetrative bottom (Costar) in amount of 5000 cells per well, cultured at condition of 37° C., 5% CO.sub.2 for 24 h. The compounds to be tested were dissolved with DMSO and diluted to 100 mM, to obtain mother liquids of the compounds.

[0162] DMEM culture solution containing 2% FBS was used to diluted the compounds, in which concentration gradients were 3, and concentration range was 100 μM to 0.046 μM. The compounds in various dilution degrees were added to the cultured cells on the 96-well plate, 100 μl per well. Culture was performed at 37° C., CO.sub.2 for 72 h, supernatant was discarded, and cell viability detection test was performed.

[0163] AfterCell Titer-Glo® reaction buffer solution was mixed with isometric substrate, the mixture was added to 96-well plate, 100 μl per well. Cell lysis was induced by horizontally shaking for 4 min. Reaction signal was stabilized by balancing at room temperature for 15 min. Chemiluminescence units in each well of the 96-well plate were detected by using chemiluminescence detector.

[0164] Inhibition rates at different dilution degrees of each compound were calculated according to chemiluminescence value of each well, and Origin 8.0 software was used for S-type curve fitting of each compound, and for calculating EC.sub.50 value. The ratio of EC.sub.50 value of compound for MRC5 cell to EC.sub.50 value of compound for MES-SA cell was used as therapeutic index for the compound. The experimental results were shown in Table 6.

TABLE-US-00005 TABLE 6 EC.sub.50 values and therapeutic indexes of some example compounds for tumor cell MES-SA Compound IC.sub.50 (μM) Therapeutic index rapamycin <0.046 >635.09 Compound of Example 1 <0.046 >259.8 Compound of Example 2 <0.046 >1811.9 Compound of Example 3 <0.046 >1501.1 Compound of Example 4 <0.046 >787.2 Compound of Example 5 <0.046 >1371.1 Compound of Example 6 <0.046 >455.1 Compound of Example 7 <0.046 >442.7 Compound of Example 8 <0.046 >1441.6 Compound of Example 9 7.21 12.1 Compound of Example 10 10.93 3.4 Compound of Example 11 <0.046 >1888.7 Compound of Example 12 <0.046 >146.5 Compound of Example 13 <0.046 >1559.0 Compound of Example 14 <0.046 >2136.8 Compound of Example 15 <0.046 >331.5 Compound of Example 16 <0.046 >364.8 Compound of Example 17 <0.046 >317.7

[0165] The results showed that the compounds of the present invention could effectively inhibit proliferation of multi-drug resistant tumor cell line MES-SA, and their inhibition activities to normal cells were generally lower than their inhibition activities to MES-SA, so that they had good therapeutic indexes and good safety. Some compounds had effects superior to rapamycin, and were promising for making or acting as anti-tumor drugs.

[0166] Although specific models for carrying out the invention were described in details, those skilled in the art would understand that these details can be modified and changed according to the teachings of disclosures, and all these changes fall into the protection scope of the present invention. The whole protection scope of the present invention is given by the appended claims and any equivalents thereof.