N-acetyl amino acid ESTER derivatives of betulin and preparation method thereof

Abstract

The present invention disclosed N-acetyl amino acid ester derivatives of betulin and the preparation method thereof, the method comprising the steps that in the presence of an alkaline substance, a catalyst and a racemization-inhibitor, the carboxyl group of N-acetyl amino acid is activated by a coupling agent; and then the activated N-acetyl amino acid is reacted with betulin via esterification reaction to obtain the N-acetyl amino acid ester derivative of betulin. The present invention provided a simple synthesis method to synthesize the N-acetyl amino acid ester derivatives of betulin by using betulin as a precursor compound and modifying the molecular structure of betulin. Such structural modification of betulin significantly enhances the anti-tumor activity of the betulin derivatives and therefore has important values.

Claims

1. An N-acetyl amino acid ester derivative of betulin, wherein the derivative has the following structural formula I: ##STR00018## wherein R is selected from one of ##STR00019##

2. A method for preparing the N-acetyl amino acid ester derivative of betulin of claim 1, comprising the steps that in the presence of an alkaline substance, a catalyst and a racemization-inhibitor, the carboxyl group of N-acetyl amino acid is activated by a coupling agent; and then the activated N-acetyl amino acid is reacted with betulin via esterification reaction to obtain the N-acetyl amino acid ester derivative of betulin.

3. The method for preparing the N-acetyl amino acid ester derivative of betulin according to claim 2, further comprising the steps that a N-acetyl amino acid, an alkaline substance, a catalyst and a racemization-inhibitor are added into an organic solvent, and a coupling agent is added after adequate stirring, to react for 0.5 to 4 hours at 2050 C. under nitrogen; and then, betulin is added to react for 8 to 24 hours at 2050 C., thereby to obtain the N-acetyl amino acid ester derivative of betulin.

4. The method for preparing the N-acetyl amino acid ester derivative of betulin according to claim 2, further comprising the purification step of the acetyl amino acid ester derivative of betulin, including: after completion of the reaction, the reaction solution is added dropwise into the distilled water to form a solution with precipitate; the solution is filtered under suction to give a solid precipitate, and then the solid precipitate is purified via silica gel column chromatography after drying, thereby to obtain the purified N-acetyl amino acid ester derivative of betulin.

5. The method for preparing the N-acetyl amino acid ester derivative of betulin according to claim 3, wherein the organic solvent is selected from N, N-dimethylacetamide.

6. The method for preparing the N-acetyl amino acid ester derivative of betulin according to claim 2, wherein the alkaline substance is selected from N-ethyldiisopropylamine.

7. The method for preparing the N-acetyl amino acid ester derivative of betulin according to claim 2, wherein the catalyst is selected from 4-dimethylaminopyridine.

8. The method for preparing the N-acetyl amino acid ester derivative of betulin according to claim 2, wherein the racemization-inhibitor is selected from 1-hydroxybenzotriazole.

9. The method for preparing the N-acetyl amino acid ester derivative of betulin according to claim 2, wherein the coupling agent is selected from 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride.

10. The method for preparing the N-acetyl amino acid ester derivative of betulin according to claim 2, wherein the N-acetyl amino acid is selected from one of N-acetyl-glycine, N-acetyl-alanine, N-acetyl-valine, N-acetyl-proline, N-acetyl-leucine and N-acetyl-isoleucine.

Description

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(1) While this invention may be embodied in many different forms, there are described in detail herein specific embodiments of the invention. This description is an exemplification of the principles of the invention and is not intended to limit the invention to the particular embodiments illustrated.

(2) For the purposes of this disclosure, like reference numerals in the figures shall refer to like features unless otherwise indicated.

(3) The present invention is further described in combination of the following examples in details.

(4) The present invention provides an N-acetyl amino acid ester derivative of betulin, with the following structural formula:

(5) ##STR00004##
Wherein, R is selected from one of

(6) ##STR00005##

(7) The specific derivatives synthesized by the present invention are shown in Table 1.

(8) TABLE-US-00001 TABLE 1 No. of the derivatives R YWZ-001 YWZ-002 YWZ-003 YWZ-004 YWZ-005 0 YWZ-006

(9) The present invention also provides a method for preparing N-acetyl amino acid ester derivative of betulin, comprising the following steps: in the presence of an alkaline substance, a catalyst and a racemization-inhibitor, the carboxyl group of N-acetyl amino acid is activated by a coupling agent; and then the activated N-acetyl amino acid is reacted with betulin via esterification reaction to obtain the N-acetyl amino acid ester derivative of betulin.

(10) Specifically, the method for preparing the N-acetyl amino acid ester derivative of betulin particularly comprises that a N-acetyl amino acid, an alkaline substance, a catalyst and a racemization-inhibitor are added into an organic solvent, and a coupling agent is added after adequate stirring, to react for 0.5 to 4 hours at 2050 C. under nitrogen; and then betulin is added to react for 8 to 24 hours at 2050 C., thereby to obtain the N-acetyl amino acid ester derivative of betulin.

(11) In an embodiment of the invention, the method further comprises the purification step of the N-acetyl amino acid ester derivative of betulin, including: after completion of the reaction, the reaction solution is added dropwise into distilled water to form a solution with precipitate; the solution is filtered under suction to give a solid precipitate, and then the solid precipitate is purified via silica gel column chromatography after drying, thereby to obtain the purified N-acetyl amino acid ester derivative of betulin.

(12) In another embodiment of the invention, the organic solvent is selected from N, N-dimethylacetamide.

(13) In another embodiment of the invention, the alkaline substance is selected from N-ethyldiisopropylamine.

(14) In another embodiment of the invention, the catalyst is selected from 4-dimethylaminopyridine.

(15) In another embodiment of the invention, the racemization-inhibitor is selected from 1-hydroxybenzotriazole.

(16) In another embodiment of the invention, the coupling agent is selected from 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride.

(17) In another embodiment of the invention, the N-acetyl amino acid is selected from one of N-acetyl-glycine, N-acetyl-alanine, N-acetyl-valine, N-acetyl-proline, N-acetyl-leucine and N-acetyl-isoleucine.

EXAMPLES

Example 1

Preparation of the Derivative YWZ-001

(18) ##STR00012##

(19) 1 mmol of N-acetyl-glycine, 1.2 mmol of 1-hydroxybenzotriazole (HOBT), 0.1 mmol of 4-dimethylaminopyridine (DMAP) and 3 mmol N-ethyldiisopropylamine (DIPEA) were added into 5 ml of N,N-dimethylacetamide (DMAC) under magnetic stirring, and then 1 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) was added under nitrogen to react for 1 hr at 25 C. for activating the carboxyl group of N-acetyl-glycine.

(20) Then, 1.0 mmol of betulin was added thereinto and reacted for 12 hrs at 25 C. After completion of the reaction, the reaction solution was added dropwise into 250 ml of distilled water to form a solution with precipitation and the solution was filtered under suction to give a solid precipitate, and then the solid precipitate was dried in a blast oven at 60 C. and purified via silica gel column chromatography (petroleum ether:ethyl acetate=2:1 (volume)) to give 0.35 g of white solid of N-acetyl amino acid ester derivative of betulin YWZ-1, with a yield of 65%. Melting point (mp): 216217 C.; Mass spectrometry ESI-MS: m/z 541 [MH].sup..

(21) .sup.1H NMR (400 MHz, CDCl.sub.3): 4.68 (s, 1H, H-29a), 4.59 (s, 1H, H-29b), 4.37 (d, J=10.9 Hz, 1H, H-28.sub.a), 4.07 (d, J=5.0 Hz, 2H, H-2), 3.92 (d, J=10.9 Hz, 1H, H-28b), 3.18 (dd, J.sub.1=4.8 Hz, J.sub.2=4.8 Hz, 1H, H-3), 2.43 (dt, J.sub.1=5.9 Hz, J.sub.2=5.7 Hz, J.sub.3=5.8 Hz, 1H, H-19), 2.05 (s, 3H, H-4), 1.67 (s, 3H, H-30), 1.02 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.81 (s, 3H), 0.75 (s, 3H).

(22) .sup.13C NMR (75 MHz, CDCl.sub.3): 170.66, 170.32, 150.01, 110.07, 78.97, 64.01, 55.34, 50.39, 48.82, 47.76, 46.52, 42.76, 41.50, 40.93, 38.93, 38.78, 37.70, 37.20, 34.52, 34.23, 29.69, 29.56, 28.07, 27.44, 27.08, 25.21, 23.06, 20.82, 19.18, 18.34, 16.18, 16.07, 15.47, 14.84.

Example 2

Preparation of the Derivative YWZ-002

(23) ##STR00013##

(24) 1 mmol of N-acetyl-alanine, 2 mmol of 1-hydroxybenzotriazole (HOBT), 0.2 mmol of 4-dimethylaminopyridine (DMAP) and 2 mmol N-ethyldiisopropylamine (DIPEA) were added into 8 ml of N,N-dimethylacetamide (DMAC) under magnetic stirring, and then 1.5 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) was added under nitrogen to react for 1.5 hrs at 20 C. for activating the carboxyl group of N-acetyl-alanine.

(25) Then, 1.1 mmol of betulin was added thereinto and reacted for 10 hrs at 20 C. After completion of the reaction, the reaction solution was added dropwise into 280 ml of distilled water to form a solution with precipitation and the solution was filtered under suction to give a solid precipitate, and then the solid precipitate was dried in a blast oven at 40 C. and purified via silica gel column chromatography (petroleum ether:ethyl acetate=5:1 (volume)) to give white solid of N-acetyl amino acid ester derivative of betulin YWZ-002, with a yield of 70%. Melting point (mp): 213214 C.; Mass spectrometry ESI-MS: m/z 555 [MH].sup..

(26) .sup.1H NMR (400 MHz, CDCl.sub.3): 4.68 (s, 1H, H-29a), 4.62 (m, 1H, H-2), 4.59 (s, 1H, H-29b), 4.43 (d, J=11.0 Hz, 1H, H-28a), 3.85 (d, J=10.8 Hz, 1H, H-28b), 3.18 (m, 1H, H-3), 2.42 (dt, 71=5.9 Hz, 72=5.6 Hz, 73=5.8 Hz, 1H, H-19), 2.02 (s, 3H, H-4), 1.67 (s, 3H, H-30), 1.42 (d, J=7.1 Hz, 3H, H-5), 1.03 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.82 (s, 3H), 0.75 (s, 3H).

(27) .sup.13C NMR (75 MHz, CDCl.sub.3): 173.76, 169.63, 150.07, 110.10, 79.05, 63.96, 55.39, 50.44, 48.91, 48.32, 47.82, 46.68, 42.82, 40.97, 38.98, 38.81, 37.75, 37.26, 34.57, 34.26, 29.80, 29.62, 28.11, 27.50, 27.10, 25.27, 23.34, 20.87, 19.25, 18.92, 18.38, 16.22, 16.10, 15.50, 14.90.

Example 3

Preparation of the Derivative YWZ-003

(28) ##STR00014##

(29) 1 mmol of N-acetyl-valine, 2 mmol of 1-hydroxybenzotriazole (HOBT), 0.3 mmol of 4-dimethylaminopyridine (DMAP) and 2.5 mmol N-ethyldiisopropylamine (DIPEA) were added into 4 ml of N,N-dimethylacetamide (DMAC) under magnetic stirring, and then 2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) was added under nitrogen to react for 4 hrs at 40 C. for activating the carboxyl group of N-acetyl-valine.

(30) Then, 1.0 mmol of betulin was added thereinto and reacted for 24 hrs at 40 C. After completion of the reaction, the reaction solution was added dropwise into 240 ml of distilled water to form a solution with precipitation and the solution was filtered under suction to give a solid precipitate, and then the solid precipitate was dried in a blast oven at 60 C. and purified via silica gel column chromatography (petroleum ether:ethyl acetate=1:2 (volume)) to give white solid of N-acetyl amino acid derivative of betulin YWZ-003, with a yield of 51%. Melting point (mp): 184186 C.; Mass spectrometry ESI-MS: m/z 583 [MH].sup..

(31) .sup.1H NMR (400 MHz, CDCl.sub.3): 4.69 (s, 1H, H-29a), 4.62 (m, 1H, H-2), 4.59 (s, 1H, H-29b), 4.34 (m, 1H, H-28a), 3.86 (m, 1H, H-28b), 3.72 (m, 1H, H-5), 3.18 (m, 1H, H-3), 2.42 (dt, J.sub.1=5.4 Hz, J.sub.2=5.2 Hz, J.sub.3=5.5 Hz, 1H, H-19), 2.05 (s, 3H, H-4), 1.68 (s, 3H, H-30), 1.02 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.95 (d, J=8.7 Hz, 3H, H-6), 0.90 (d, J=6.8 Hz, 3H, H-7), 0.82 (s, 3H), 0.75 (s, 3H).

(32) .sup.13C NMR (75 MHz, CDCl.sub.3): 172.85, 170.09, 150.04, 110.06, 78.98, 63.94, 55.36, 50.42, 48.87, 48.83, 46.42, 42.77, 40.94, 38.94, 37.73, 37.21, 34.23, 31.56, 31.49, 29.58, 28.08, 27.46, 25.24, 23.40, 23.38, 20.84, 19.24, 19.10, 19.06, 18.33, 17.86, 16.19, 16.09, 16.04, 15.48, 14.89, 14.85.

Example 4

Preparation of the Derivative YWZ-004

(33) ##STR00015##

(34) 2 mmol of N-acetyl-proline, 2 mmol of 1-hydroxybenzotriazole (HOBT), 0.2 mmol of 4-dimethylaminopyridine (DMAP) and 5 mmol N-ethyldiisopropylamine (DIPEA) were added into 5 ml of N,N-dimethylacetamide (DMAC) under magnetic stirring, and then 2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) was added under nitrogen to react for 3.5 hrs at 50 C. for activating the carboxyl group of N-acetyl-proline.

(35) Then, 1.0 mmol of betulin was added thereinto and reacted for 16 hrs at 50 C. After completion of the reaction, the reaction solution was added dropwise into 260 ml of distilled water to form a solution with precipitate and the solution was filtered under suction to give a solid precipitate, then the solid precipitate was dried in a blast oven at 55 C. and purified via silica gel column chromatography (chloroform:methanol=10:1 (volume)) to give white solid of N-acetyl amino acid ester derivative of betulin YWZ-004, with a yield of 55%. Melting point (mp): 204206 C.; Mass spectrometry ESI-MS: m/z 581 [MH].sup..

(36) .sup.1H NMR (400 MHz, CDCl.sub.3): 4.67 (s, 1H, H-29a), 4.57 (s, 1H, H-29b), 4.50 (m, 1H, H-2), 4.39 (d, J=11.0 Hz, 1H, H-28a), 3.89 (d, J=10.8 Hz, 1H, H-28b), 3.64 (m, 1H, H-7a), 3.49 (m, 1H, H-7b), 3.17 (m, 1H, H-3), 2.42 (dt, J.sub.1=6.4 Hz, J.sub.2=5.7 Hz, J.sub.3=5.8 Hz, 1H, H-19), 2.19 (m, 1H, H-5a), 2.08 (s, 3H, H-4), 2.02 (m, 1H, H-5b), 1.97 (m, 1H, H-6a), 1.83 (m, 1H, H-6b), 1.67 (s, 3H, H-30), 1.02 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.81 (s, 3H), 0.75 (s, 3H).

(37) .sup.13C NMR (75 MHz, CDCl.sub.3): 172.86, 169.40, 150.26, 109.92, 79.02, 63.28, 58.85, 55.41, 50.47, 48.97, 47.85, 47.81, 46.75, 42.80, 40.97, 38.98, 38.82, 37.71, 37.25, 34.61, 34.24, 31.74, 29.91, 29.73, 28.12, 27.51, 27.13, 25.32, 24.96, 22.38, 20.87, 19.32, 18.38, 16.22, 16.12, 15.52, 14.90.

Example 5

Preparation of the Derivative YWZ-005

(38) ##STR00016##

(39) 1.5 mmol of N-acetyl-leucine, 1.5 mmol of 1-hydroxybenzotriazole (HOBT), 0.15 mmol of 4-dimethylaminopyridine (DMAP) and 4 mmol N-ethyldiisopropylamine (DIPEA) were added into 7 ml of N,N-dimethylacetamide (DMAC) under magnetic stirring, and then 2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) was added under nitrogen to react for 1.0 hr at 25 C. for activating the carboxyl group of N-acetyl-leucine.

(40) Then, 1.0 mmol of betulin was added thereinto and reacted for 5 hrs at 25 C. After completion of the reaction, the reaction solution was added dropwise into 258 ml of distilled water to form a solution with precipitate and the solution was filtered under suction to give a solid precipitate, and then the solid precipitate was dried in a blast oven at 45 C. and purified via silica gel column chromatography (chloroform:methanol=20:1 (volume)) to give white solid of N-acetyl amino acid ester derivative of betulin YWZ-005, with a yield of 40%. Melting point (mp): 142144 C.; Mass spectrometry ESI-MS: m/z 597 [MH].sup..

(41) .sup.1H NMR (400 MHz, CDCl.sub.3): 4.69 (s, 1H, H-29a), 4.67 (m, 1H, H-2), 4.59 (s, 1H, H-29b), 4.33 (m, 1H, H-28a), 3.87 (m, 1H, H-28b), 3.18 (m, 1H, H-3), 2.43 (m, 1H, H-19), 2.02 (s, 3H, H-4), 1.68 (s, 3H, H-30), 1.63 (m, 2H, H-5), 1.56 (m, 1H, H-6), 1.02 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.95 (d, J=5.5 Hz, 3H, H-7), 0.94 (d, J=4.0 Hz, 3H, H-8), 0.82 (s, 3H), 0.76 (s, 3H).

(42) .sup.13C NMR (75 MHz, CDCl.sub.3): 173.88, 169.86, 150.09, 110.05, 79.01, 63.93, 55.37, 50.94, 50.43, 48.87, 47.78, 46.53, 42.78, 42.09, 40.95, 38.95, 38.80, 37.72, 37.23, 34.62, 34.24, 29.62, 28.09, 27.48, 27.10, 25.26, 25.02, 23.33, 22.90, 22.26, 20.85, 19.24, 18.37, 16.20, 16.10, 15.49, 14.88, 14.87.

Example 6

Preparation of the Derivative YWZ-006

(43) ##STR00017##

(44) 1.8 mmol of N-acetyl-isoleucine, 1.8 mmol of 1-hydroxybenzotriazole (HOBT), 0.18 mmol of 4-dimethylaminopyridine (DMAP) and 5 mmol N-ethyldiisopropylamine (DIPEA) were added into 6.5 ml of N,N-dimethylacetamide (DMAC) under magnetic stirring, and then 1.8 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) was added under nitrogen to react for 3 hrs at 30 C. for activating the carboxyl group of N-acetyl-valine.

(45) Then, 1.0 mmol of betulin was added thereinto and reacted for 10 hrs at 30 C. After completion of the reaction, the reaction solution was added dropwise into 220 ml of distilled water to form a solution with precipitate and the solution was filtered under suction to give a solid precipitate, and then the solid precipitate was dried in a blast oven at 35 C. and purified via silica gel column chromatography (chloroform:methanol=8:1 (volume)) to give white solid of N-acetyl amino acid ester derivative of betulin YWZ-006, with a yield of 42%. Melting point (mp): 188190 C.; Mass spectrometry ESI-MS: m/z 597 [MH].sup..

(46) .sup.1H NMR (400 MHz, CDCl.sub.3): 4.75 (m, 1H, H-2), 4.69 (s, 1H, H-29a), 4.59 (s, 1H, H-29b), 4.32 (m, 1H, H-28a), 3.86 (m, 1H, H-28b), 3.18 (m, 1H, H-3), 2.43 (m, 1H, H-19), 2.04 (s, 3H, H-4), 1.92 (m, 1H, H-5), 1.68 (s, 3H, H-30), 1.18 (m, 2H, H-6), 1.02 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H), 0.94 (m, 3H, H-7), 0.86 (d, J=6.7 Hz, 3H, H-8), 0.82 (s, 3H), 0.75 (s, 3H).

(47) .sup.13C NMR (75 MHz, CDCl.sub.3): 173.15, 170.15, 150.07, 110.06, 79.02, 63.97, 55.49, 55.37, 50.43, 48.85, 47.76, 46.47, 46.38, 42.79, 40.96, 38.96, 38.80, 38.06, 37.74, 37.23, 34.69, 34.25, 29.75, 29.61, 28.09, 27.48, 26.28, 25.28, 23.44, 20.86, 19.26, 18.37, 16.20, 16.10, 15.48, 14.87, 14.78, 11.91.

(48) The present invention also relates to the application of the N-acetyl amino acid ester derivatives of betulin for the preparation of anti-tumor drugs. By using MTT assay, experimental results of in vitro activity screening show that the N-acetyl acetyl amino acid ester derivatives of betulin provided by the present invention have significant anti-tumor effects and excellent dose-dependent manners.

(49) Human colon tumor cell lines HT29, pancreatic cancer cell lines MPC2 and ovarian cancer cell lines MDAH2774 were selected to determine the half inhibitory concentrations (IC50s) of the N-acetyl amino acid ester derivatives of betulin, betulinic acid and betulin, and the results are shown in Table 2.

(50) TABLE-US-00002 TABLE 2 The half inhibitory concentrations (IC50s) of the N-acetyl amino acid ester derivatives of betulin, betulinic acid and betulin against the three tumor cell lines. IC50 (mol/L) Compounds HT29 MPC2 MDAH2774 YWZ-002 6.58 0.24 4.24 0.23 10.56 0.62 YWZ-003 14.09 0.83 11.43 0.89 10.87 0.76 YWZ-004 8.88 0.62 5.32 0.21 2.13 0.05 YWZ-005 18.12 1.51 19.71 0.83 1.38 0.04 YWZ-006 15.03 1.31 10.17 0.69 7.26 0.10 Betulinic acid 32.66 0.62 38.58 2.91 39.54 2.19 Betulin >100 >100 >100

(51) As can be seen from Table 2, the N-acetyl amino acid ester derivatives of betulin provided by the present invention have very significant anti-tumor effects, especially for human colon cancer cell line HT29, pancreatic cancer cell lines MPC2 and ovarian cancer cell lines MDAH2774.

(52) Therefore, the present invention provided a simple synthesis method to synthesize the N-acetyl amino acid ester derivatives of betulin by using betulin as a precursor compound and modifying the molecular structure of betulin. Such structural modification of betulin significantly enhances the anti-tumor activity of the betulin derivatives and thereby has important value.

(53) The persons skilled in the art should understand that preferred embodiments of the invention are described for illustration purpose but not to limit the present invention, and any modification, replacement and change within the spirit and principles of the present invention should be fallen into the scope of the present invention.

(54) The above disclosure is intended to be illustrative and not exhaustive. This description will suggest many variations and alternatives to one of ordinary skill in this field of art. All these alternatives and variations are intended to be included within the scope of the claims where the term comprising means including, but not limited to. Those familiar with the art may recognize other equivalents to the specific embodiments described herein which equivalents are also intended to be encompassed by the claims.