STEROL DERIVATIVES AND PREPARATION METHOD AND USES THEREOF

Abstract

The present invention pertains to pharmaceutical chemical field, and relates to a sterol derivative as well as preparation method and uses thereof. Specifically, the present invention relates to a compound of Formula I, or a pharmaceutically acceptable salt, ester or ether thereof, wherein, R.sub.1 is selected from the group consisting of —OH, ═O(carbonyl), H, and C.sub.1-C.sub.3 alkyl; R.sub.2 is selected from the group consisting of —OH, H, and C.sub.1-C.sub.3 alkyl; R.sub.3 is selected from the group consisting of —OH, ═O, H, and C.sub.1-C.sub.3 alkyl; R.sub.4 is selected from the group consisting of —OH, H, and C.sub.1-C.sub.3 alkyl; and none, one, two, three or four of R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are —OH. The compound of the present invention can inhibit HMG-CoA reductase, tumor cells and lipase effectively, and is a potential drug for reducing blood-fat, antitumors, or for losing weight.

##STR00001##

Claims

1. A method for effecting weight loss in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt, ester or ether thereof, ##STR00012## wherein, R.sub.1 is selected from the group consisting of —OH, ═O, H, and C.sub.1-C.sub.3 alkyl; R.sub.2 is selected from the group consisting of —OH, H, and C.sub.1-C.sub.3 alkyl; R.sub.3 is selected from the group consisting of —OH, ═O, H, and C.sub.1-C.sub.3 alkyl; and R.sub.4 is selected from the group consisting of —OH, H, and C.sub.1-C.sub.3 alkyl.

2. The method of claim 1, wherein the compound is: ##STR00013##

3. The method of claim 1, wherein the subject has hyperlipidemia, atherosclerosis, coronary heart disease, diabetes, or a combination thereof.

4. The method of claim 1, wherein the compound is an ester of Formula (II): ##STR00014## wherein the ester is selected from the group consisting of formate, acetate, propionate, p-tolylsulfonate and trifluoromethylsulfonate; said ether is tert-butyl-dimethyl silyl ether.

5. The method of claim 1, wherein the compound is tetraacetate of compound of Formula II, tetra-p-tolylsulfonate of compound of Formula II, tetra(trifluoromethylsulfonate) of compound of Formula II, or tetra(tert-butyl-dimethyl-silyl ether) of compound of Formula II.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0187] FIG. 1: inhibition curve of a compound of Formula II on growth of HCT116 cells.

[0188] FIG. 2: inhibition curve of a compound of Formula II on growth of H22 cells.

[0189] FIG. 3: inhibition curve of a compound of Formula II on growth of HepG2 cells.

[0190] FIG. 4: inhibition curve of a compound of Formula II on growth of S180 cells.

[0191] FIG. 5: inhibition curve of a compound of Formula II on growth of YAC-1 cells.

[0192] FIG. 6: inhibition curve of a compound of Formula II on growth of THP1 cells.

[0193] FIG. 7: inhibition curve of a compound of Formula II on growth of U937 cells.

[0194] FIG. 8: inhibition curve of a compound of Formula II on growth of B16-F10 cells.

[0195] FIG. 9: absorbance working curve of oleic acid.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0196] The present invention is further illustrated with the following examples, but those skilled in the art would understand the following examples are merely used to illustrate the present invention, rather than to limit the protection scope of the present invention. For those technologies or conditions not specifically described in the examples, they were performed according to the technologies or conditions as described in the documents in the art or according to the product specifications. For those reagents and instruments whose manufacturers were not given, they were all conventional products commercially available in markets.

Example 1: Preparation of Compound of Formula II (1)

[0197] Operation Steps:

[0198] 1) About 1 kg of dry powder of content of Xuezhikang capsules (produced by Beijing Peking University WBL Biotechnology Co., Ltd.) was provided, ultrasonic extraction was performed for 3 times with dichloromethane in 2-6 times by volume as solvent, 20-40 minutes for each time, all extracted solutions were combined, concentrated and subjected to solvent recovery under reduced pressure, to obtain 91 g of dichloromethane refined extract.

[0199] 2) 50 g of the dichloromethane refined extract was loaded on silica gel column for chromatography separation, and eluted in gradient manner with petroleum ether and ethyl acetate. The volume ratio of petroleum ether-ethyl acetate was 75:25, 50:50, 25:75, 0:100 in sequence.

[0200] 3) 5.0 g of fraction of petroleum ether-ethyl acetate (25:75) was taken, separated with C18 reversed column chromatography, eluted with methanol-water (10:90-100:0) in gradient manner to obtain 4 fractions (methanol-water 10:90, 50:50, 75:25, 100:0), in which 1.3 g of the elution fraction of methanol-water (75:25) was purified with semi-preparative high performance liquid chromatography, acetonitrile-0.2% acetic acid aqueous solution (45:55) was used as mobile phase, flow rate was 4 mL/min, C18 semi-preparative chromatography column (10×250 mm, 5 μm) was stationary phase, detection wavelength of DAD detector was 270 nm, chromatographic peaks at 9.2 minute were collected, accumulated for many times, then concentrated, freeze-dried to obtain about 40 mg of the compound.

Example 2: Preparation of Compound of Formula II (2)

[0201] Operation Steps:

[0202] 1) 5 kg of dry powder of Monascus-fermented rice was provided, ultrasonic extraction was performed for 3 times with dichloromethane in 2-6 times by volume as solvent, 20-40 minutes for each time, all extracted solutions were combined, concentrated and subjected to solvent recovery under reduced pressure, to obtain 78 g of dichloromethane refined extract.

[0203] 2) 30 g of the dichloromethane refined extract was loaded on silica gel column for chromatography separation, and eluted in gradient manner with petroleum ether and ethyl acetate. The volume ratio of petroleum ether-ethyl acetate was 75:25, 50:50, 25:75, 0:100 in subsequence.

[0204] 3) 3.0 g of fraction of petroleum ether-ethyl acetate (25:75) was taken, separated with C18 reversed column chromatography, eluted with methanol-water (10:90-100:0) in gradient manner to obtain 4 fractions (methanol-water 10:90, 50:50, 75:25, 100:0), in which 0.8 g of the elution fraction of methanol-water (75:25) was purified with semi-preparative high performance liquid chromatography, acetonitrile-0.2% acetic acid aqueous solution (45:55) was used as mobile phase, flow rate was 4 mL/min, C18 semi-preparative chromatography column (10×250 mm, 5 μm) was stationary phase, detection wavelength of DAD detector was 270 nm, chromatographic peaks at 9.2 minute were collected, accumulated for many times, then concentrated, freeze-dried to obtain about 25 mg of the compound.

Example 3: Preparation of Compound of Formula II (3)

[0205] In a reaction flask, toluene (350 mL) was added, then Compound A (4.4 g, 9.2 mmol) and p-toluenesulfonic acid monohydrate (0.59 g, 3.1 mmol) were added, heated to refluxing and water was removed by azeotropism for 7 hours, cooled to room temperature after reaction, solvent was removed under reduced pressure, then silica gel column chromatograph was performed for purification, to obtain a sticky oily compound, i.e., compound of Formula II.

##STR00009##

Example 4: Preparation of Compound of Formula II (4)

[0206] In a reaction flask, pyridine (100 mL) was added, then Compound A (4.4 g, 9.2 mmol) and 2,4,6-triisopropylsulfonyl chloride (0.93 g, 3.1 mmol) were added, heated and reacted at 50° C. for 20 hours, solvent was removed under reduced pressure, then silica gel column chromatograph was performed for purification, to obtain a sticky oily compound, i.e., compound of Formula II.

##STR00010##

Example 5: Identification of Structure of Compounds

[0207] The samples used were compounds prepared in Examples 1, 2, 3, 4. The results confirmed that all compounds prepared in Examples 1-4 were the same, i.e., compound of Formula II.

[0208] 1. Physical and Chemical Data of Compounds White powder, optical rotation: [α].sup.25.sub.D -50.00 (c 0.118, CH.sub.2Cl.sub.2:MeOH=1:1);

[0209] UV spectrum showed 3 maximum absorption peaks, separately, λ.sub.max(CH.sub.2Cl.sub.2:MeOH)=271.6 nm, 282.2 nm, 293.8 nm.

[0210] FT-IR (KBr, cm.sup.−1) spectrum: 3392(—OH), 2969, 2933 (saturated hydrocarbon), 1652, 1647(C═C), 1456, 1378 (gem-dimethyl).

[0211] 2. Determination of molecular formula

[0212] HR-ESI-MS gave m/z 483.3104 [M+Na].sup.+(calcd. 483.3081, err 2.3), so that the molecular weight of the compound was deduced as 460.32. .sup.1H-NMR and .sup.13C-NMR showed that there were 40 hydrogen signals and 28 carbon signals. DEPT showed there were 6 quaternary carbons, 10 CH, 6 CH.sub.2 and 6 CH.sub.3. In .sup.13C-NMR, 4 olefinic carbon signals were at 117.2 ppm, 119.6 ppm, 139.1 ppm and 140.6 ppm. The analysis of combining HSQC and .sup.13C-NMR showed there were 6 oxygenated carbons linked to oxygen atoms at 70.5 ppm, 72.4 ppm, 74.5 ppm, 80.4 ppm, 83.8 ppm and 84.4 ppm. In view of molecular weight with .sup.1H, .sup.13C-NMR and DEPT spectra in combination, if the compound had 6 oxygen atoms, its molecular weight would exceed 460.32, thus it could be deduced that the compound should have 5 oxygen atoms and 4 hydrogen atoms that did not present signals in .sup.1H-NMR spectrum. Thus, among the above 5 oxygen atoms, 4 oxygen atoms belonged to 4 hydroxyls, while the 5.sup.th oxygen existed in form of ether. According to the above analysis, it could be determined that the molecule had 28 carbon atoms, 44 hydrogen atoms, and 5 oxygen atoms, and the molecular formula was C.sub.28H.sub.44O.sub.5.

[0213] 3. Determination of the Chemical Structure

[0214] By analyzing the .sup.13C-NMR and DEPT spectra of the compound, there were 28 carbon atoms, in which 6 carbon atoms were of methyl forms. Since 3 maximum absorption peaks at 271.6 nm, 282.2 nm and 293.8 nm in UV spectrum were substantively identical to those of ergosterol, it was primarily determined that the compound had framework of ergosterol. Its unsaturation degree was 7 based on calculation of molecular formula. Thus, it could be deduced that, besides 6 degrees of unsaturation including 2 double bonds and 4 rings of ergosterol framework, the compound further had one degree of unsaturation and it must be a ring, so that this ring could be determined as an epoxy formed with the 5.sup.th oxygen atom as center. The HSQC and HMBC spectra were analyzed in combination and showed that the 6 carbon atoms linked to oxygen atoms were separately assigned to 4 carbon atoms linked to 4 hydroxyls (70.5, 72.4, 74.5, 80.4 ppm) and 2 carbon atoms linked to the residual one oxygen atom (83.8, 84.4 ppm). Deep analysis of HMBC spectra showed that the 5.sup.th ring system was a 5-membered ring consisting of C-16 (83.8 ppm), C-17 (66.9 ppm), C-20 (80.4 ppm), C-22 (84.4 ppm) and oxygen atom. This satisfied degree of unsaturation and number of carbon atoms linked to oxygen atoms. Further analysis of mass spectrum gave molecular weight and molecular formula, which confirmed the above analysis. In sum, the above analysis indicated that the new compound was: 16,22-epoxy-ergosta-5,7-dien-3,20,23,25-tetraol. The chemical structure was shown in the following Formula II:

##STR00011##

[0215] 4. NMR Data of Compound of Formula II

[0216] The data were shown in Table 1.

TABLE-US-00001 TABLE 1 NMR data of compound of Formula II (600 MHz, CDC13, J in Hz) .sup.1H-NMR, .sup.13C-NMR, HMBC No. (ppm) (ppm) DEPT (H.fwdarw.C) 1 1.28 (1H, m) 38.4 CH.sub.2 — 1.90 (1H, m) 2 1.48 (1H, m) 32.1 CH.sub.2 — 1.89 (1H, m) 3 3.62 (1H, m) 70.5 CH — 4 2.39 (1H, m) 40.9 CH.sub.2 — 2.46 (1H, m) 5 — 140.6 C — 6 5.56 (1H, m) 119.6 CH 4, 7 7 5.38 (1H, m) 117.2 CH — 8 — 139.1 C — 9 1.95 (1H, m) 46.0 CH — 10 — 37.3 C — 11 1.60 (1H, m) 20.8 CH.sub.2 — 1.77 (1H, m) 12 1.25 (1H, m) 39.3 CH.sub.2 — 2.08 (1H, m) 13 — 43.0 C 14 1.87 (1H, m) 54.0 CH 13 15 1.75 (1H, m) 34.2 CH.sub.2 14 2.23 (1H, m) 16 4.66 (1H, m) 83.8 CH 13, 14, 15 17 1.90 (1H, m) 66.9 CH 20, 22 18 1.12 (3H, s) 14.6 CH.sub.3 27, 12, 13, 14 19 0.96 (3H, s) 16.6 CH.sub.3 9, 10, 1 20 — 80.4 C — 21 1.43 (3H, s) 27.8 CH.sub.3 20, 22, 24 22 4.19 (1H, brs) 84.4 CH 21, 24, 23, 20 23 3.66 (1H, brd, J = 72.4 CH 28, 24, 25, 20 9.0 Hz) 24 1.75 (1H, m) 46.4 CH 27, 28 25 — 74.5 C — 26 1.20 (3H, s) 30.3 CH.sub.3 25, 24 27 1.23 (3H, s) 24.0 CH.sub.3 25, 24 28 0.82 (3H, d, J = 7.2 14.0 CH.sub.3 23, 25, 24 Hz) Notation: “—” represents concern signal does not exist.

Example 6: Preparation of Tetraacetate of Compound of Formula II

[0217] The mixture of Compound A (4.23 g, 9.2 mmol) and 2 drops of concentrated sulfuric acid in 20 mL of acetic anhydride was heated with water-bath to 50° C., reacted for 2 hours, cooled, added with saturated NaHCO.sub.3 aqueous solution, extracted with toluene for 3 times, the organic layer was evaporated under rotation to obtain a product, and further purified by silica gel column chromatography to obtain tetraacetate of compound of Formula II via silica gel column chromatography. Pyridine catalysis could be used for prevention of configuration reversion.

Example 7: Preparation of Tetra-p-Tolylsulfonate of Compound of Formula II

[0218] In a dry 25 mL flask, compound of Formula II (4.23 g, 9.2 mmol) and 50 mL of dry dichloromethane were added, cooled to 0° C., p-tolylsulfonyl chloride (10.56 g, 55.3 mmol) was added, triethylamine (7.46 g, 73.6 mmol) was added dropwise under stirring. After end of dropwise adding, the reaction mixture solution was stirred at 20° C. for 1 hour, washed with water (50 mL×3), dried with anhydrous sodium sulfate, filtrated, dried out solvent by evaporation to obtain a crude product, and subjected to rapid column chromatography to obtain tetra-p-tolylsulfonate of compound of Formula II.

Example 8: Preparation of Tetra(Trifluoromethylsulfonate) of Compound of Formula II

[0219] Compound of Formula II (4.23 g, 9.2 mmol) and pyridine (4.36 g, 55.2 mmol) were added in sequence into a flask containing 50 mL of dry dichloromethane under stirring, then trifluoromethylsulfonate (12.46 g, 44.2 mmol) was slowly added dropwise at room temperature (25° C.) within about 20 minutes, reacted for 2 hours, concentrated filtrate, the residue was subjected to separation with silica gel column chromatography to obtain tetra(trifluoromethylsulfonate) of compound of Formula II.

Example 9: Preparation of Tetra(Tert-Butyl-Dimethyl-Silyl Ether) of Compound of Formula II

[0220] In a 100 mL round bottom flask, 20 mL of THF and 1.26 g of NaH (content: 70%, 36.8 mmol) wrapped with mineral oil were added, stirred to sufficiently disperse NaH, added dropwise with compound of Formula II (4.23 g, 9.2 mmol) in THF (10 mL), reacted at room temperature under vigorous agitation for 1 hour. TBDMS-C1 (tert-butyldimethylchlorosilane) (5.53 g, 36.8 mmol) was dissolved in 10 mL THF, added dropwise to the above reaction solution under stirring, the speed of dropwise adding was controlled so that the reaction temperature was not extremely high, after end of dropwise adding, the reaction was continued at room temperature under stirring for 1.5 hours. The reaction solution was poured in water, extracted with dichloromethane; the organic phase was washed with water to neutrality, washed with saturated brine, dried with anhydrous sodium sulfate. The filtrate was concentrated, separated with column chromatography, to obtain tetra (tert-butyl-dimethyl-silyl ether) of compound of Formula II.

Experimental Example 1: Test of Activity of Inhibiting HMG-CoA Reductase

[0221] 1. Test Materials

[0222] 1.1 Drugs

[0223] Compound of Formula II—Prepared in Examples 1-4.

[0224] Standard sample of lovastatin—purchased from Sigma.

[0225] 1.2 Enzymes

[0226] Rat liver microsomes (HMG-CoA reductase)—, could be commercially available or prepared by the following preparation method: taking out liver of male rat, washing with KESD buffer solution, centrifuging under 1200 g for 15 minutes, collecting supernatant, then centrifuging under 105,000 g twice, 90 minutes for each time, and collecting centrifugation deposit. The centrifugation deposit was added with 8.3% of glycerol, heated with 37° C. bath for 1 hour. The crude product of rat liver microsomes was purified with saturated ammonium sulfate, and 35-50% of purified fraction was collected. The purified fraction obtained could be stored in −80° C. refrigerator.

[0227] 1.3 Reagents

[0228] Potassium chloride, potassium dihydrogen phosphate, ethylenediamine tetraacetic acid, dithiothreitol—purchased from Beijing Chemical Reagents Co., Ltd.;

[0229] Nicotinamide adenine dinucleotide (NADPH)—purchased from Merk; 3-hydroxy-3-methylglutary coenzyme A (HMG-CoA)—purchased from Sigma.

[0230] 2. Test Methods

[0231] Compound of Formula II was dissolved in 75% ethanol solution, with initial concentration of 8.0 mg/mL, and diluted in gradient manner, to reach 4.0 mg/mL, 2.0 mg/mL, 1.0 mg/mL; lovastatin was used as positive control, dissolved with 75% ethanol solution to reach concentration of 2.0 mg/mL; in the test system, total volume was 250 μL, concentrations of ingredients were separately: potassium chloride 200 mM, potassium dihydrogen phosphate 160 mM, ethylenediamine tetraacetic acid 4 mM, dithiothreitol 10 mM, nicotinamide adenine dinucleotide and 3-hydroxy-3-methylglutary coenzyme A as two substrates separately had concentrations of: 200 μM and 50 μM, pH6.8, 30 μL of enzyme was added, 4 test groups were separately added with 10 μL of new compound solutions with different concentrations, the positive control group was added with 10 μL of lovastatin solution, the blank control group was added with 10 μL of 75% ethanol solution, and dynamitic changes of OD.sub.340 were detected with Versamax ELISA at 37° C. condition. The rate of decline of OD.sub.340 (expressed in slope value) as detected within 5 minutes was used to evaluate the activity of HMG-CoA reductase, and then to evaluate the activity of enzyme inhibitor and the results were shown in Table 2.

[0232] 3. Test Results

[0233] The results were shown in Table 2.

TABLE-US-00002 TABLE 2 Test results of activity of enzyme inhibitors Final Inhibitor concentration Inhibition concentration Inhibitor in system rate Sample name (mg/mL) volume (μL) (μg/mL) Slope (%) Blank control — — — 16.0 — Lovastatin 2.0 10 80 7.3 54.4 Compound of 8.0 10 320 7.5 53.1 Formula II 4.0 10 160 8.7 45.6 2.0 10 80 11.3 29.4 1.0 10 40 13.7 14.4 * Blank control was solvent; Lovastatin was positive control.

[0234] The test results showed that the compound of the present invention, especially, the compound of Formula II, had inhibition effect on activity of HMG-CoA reductase, and presented concentration-effect relationship. Its IC.sub.50 value was about 250 μg/mL, which indicated that the compound has good inhibition effects on activity of HMG-CoA reductase.

[0235] Further studying showed that the ester derivatives or ether derivatives of compound of Formula II, for example, “tetraacetate of compound of Formula II”, “tetra-p-tolylsulfonate of compound of Formula II”, “tetra(trifluoromethylsulfonate) of compound of Formula II” and “tetra(tert-butyl-dimethyl-silyl ether) of compound of Formula II” as prepared in Examples 6-9 also had similar activity of inhibiting HMG-CoA reductase.

Experimental Example 2: Test of Inhibiting Cancer Cells with Compound of Formula II

[0236] 1 Test materials

[0237] 1.1 Strains of cells

[0238] HCT116 and H22, purchased from Korean cell line bank, Seoul, Korea;

[0239] S180, HepG-2, YAC-1, Thp1, U937 and B16-F10, purchased from the Cell Bank of the Committee on Type Culture Collection of Chinese Academy of Sciences.

[0240] 1.2 Drugs

[0241] New compound (compound of Formula II) prepared in Examples 1-4.

[0242] 1.3 Reagents

[0243] MTT was purchased from Amresco; RPMI1640 and double antibody were purchased from Sigma; fetal bovine serum (FBS) was purchased from Gibco in USA; all other reagents were of analytical purity and made in China.

[0244] 2 Test Methods

[0245] Cancer cells of exponential growth phase were inoculated on 96-well plate, 2×10.sup.4 cells/well, added with drug until final drug concentrations were: 500, 250, 125, 62.5, 31.25, 15.625 and 7.8125 μg/mL, cultured at 37° C. in 5% CO.sub.2 cell culture incubator for 72 hours, then added with MTT, 10 μL/well, incubated in dark at 37° C. for 4 hours, culture medium was removed, 150 μL of DMSO or acidified isopropanol was added, vibrated for 5 minutes, and then OD values were measured under wavelength of 570 nm. This was repeated for 3 times, and a blank control was set. These cell strains used the same culture medium, i.e., RPMI1640 culture medium containing 10% fetal bovine serum and 1% double antibody (penicillin and steptomycin).

[0246] Calculation Formula:

Cell survival rate=(OD value of test group/OD value of control group)×100%.

[0247] The test steps could also refer to YANG Xiuwei, et al., “Screening for antitumor activities of strychnos alkaloids in vitro”, Journal of Modern Chinese Medicine, 2006, 8(9): 11-13.

[0248] 3 Test Results

[0249] 3.1 Activity of compound of Formula II against cancer (colon cancer) in vitro

[0250] The results were shown in FIG. 1. The results showed that the compound of Formula II had inhibition effect on growth of human colon cancer cell strain HCT116, and exhibited concentration-effect relationship. It could be seen that this compound had potency for prophylaxis and treatment of colon cancer.

[0251] 3.2 Activity of compound of Formula II against cancer (liver cancer) in vitro

[0252] 3.2.1 As showing in FIG. 2, the compound of Formula II had inhibition effect on growth of mice hepatoma cell line H22, and exhibited a concentration-effect relationship. Its IC.sub.50 value was about 50 μg/mL, which suggested that this compound had good inhibition effect on proliferation of mice hepatoma cells.

[0253] 3.2.2 As showing in FIG. 3, the compound of Formula II had inhibition effect on growth of human hepatoma cell line Hep22, and exhibited a concentration-effect relationship. Its IC.sub.50 value was about 200 μg/mL, which suggested that this compound had good inhibition effect on proliferation of human hepatoma cells.

[0254] 3.2.3 As showing in FIG. 4, the compound of Formula II had inhibition effect on growth of mice sarcoma cell line S180, and exhibited a concentration-effect relationship.

[0255] It could be seen that this compound had potency for prophylaxis and treatment of liver cancer.

[0256] 3.3 Activity of compound of Formula II against cancer (lymphoma) in vitro

[0257] 3.3.1 As showing in FIG. 5, the compound of Formula II had inhibition effect on growth of mice lymphoma cell YAC-1, and exhibited a concentration-effect relationship. Its IC.sub.50 value was about 62.5 μg/mL, which suggested that this compound had good inhibition effect on proliferation of mice lymphoma cells.

[0258] 3.3.2 As showing in FIG. 6, the compound of Formula II had inhibition effect on growth of human mononuclear lymphoma cell THP1, and exhibited a concentration-effect relationship. Its IC.sub.50 value was about 250 μg/mL, which suggested that this compound had good inhibition effect on proliferation of human mononuclear lymphoma cells. FIG. 6.

[0259] 3.3.3 As showing in FIG. 7, the compound of Formula II had inhibition effect on growth of human tissue lymphoma cell U937, and exhibited a concentration-effect relationship. Its IC.sub.50 value was about 50 μg/mL, which suggested that this compound had good inhibition effect on proliferation of human tissue lymphoma cells.

[0260] It could be seen that this compound had potency for prophylaxis and treatment of lymph cancer (lymphoma).

[0261] 3.4 Activity of compound of Formula II against cancer (melanoma) in vitro

[0262] The results were shown in FIG. 8. The results showed that the compound of Formula II had inhibition effect on growth of mice melanoma cell B16-F10, and exhibited concentration-effect relationship. Its IC.sub.50 value was about 62.5 μg/mL, which suggested that this compound had potency for prophylaxis and treatment of melanoma.

[0263] In sum, the compound of the present invention, especially compound of Formula II, had effective inhibition effects on many kinds of tumor cells, and was a potential drug for prophylaxis and/or treatment and/or adjunctive treatment of cancers.

[0264] Further studying showed that the ester derivatives or ether derivatives of compound of Formula II, for example, “tetraacetate of compound of Formula II”, “tetra-p-tolylsulfonate of compound of Formula II”, “tetra(trifluoromethyl sulfonate) of compound of Formula II” and “tetra(tert-butyl-dimethyl-silyl ether) of compound of Formula II” as prepared in Examples 6-9 also had similar activity of inhibiting tumor cells, and were potential drugs for prophylaxis and/or treatment and/or adjunctive treatment of cancers.

Experimental Example 3: Test of Activity of Inhibiting Lipase

[0265] 1 Test materials

[0266] 1.1 Drugs

[0267] Compound of Formula II.

[0268] Orlistat—purchased from Golden Elephant Pharmacy.

[0269] 1.2 Enzymes

[0270] Porcine pancreatic lipase—purchased from Sigma.

[0271] 1.3 Reagents

[0272] Oleic acid—purchased from Sigma.

[0273] Toluene, olive oil, pyridine, copper acetate, NaH.sub.2PO.sub.4, K.sub.2HPO.sub.4 were all of analytic purity and made in China.

[0274] 2 Test methods

[0275] The steps were shown as follows, and could also refer to JIANG Huifang, WANG Yaqin, LIU Chunguo, Comparison and improvement of three methods for measurement of lipase activity, Journal of Chemistry & Bioengineering, 2007, 24(8):72-75; and ZHU Xiaoqing, LU Jingci, HUO Shixin, et al., Inhibitory Effect on Lipase of Alkaloid Derived from Lotus Leaf, Journal of Shanghai University (Natural Science), 2007, 13(1):85-87.

[0276] 2.1 Preparation of light absorbance working curve for fatty acid: a series of oleic acid-toluene solutions with different concentrations (0-3.5 mmol/L, separately being 0, 0.225, 0.45, 0.675, 0.9, 1.125, 1.35, 1.8, 2.25, 2.7, and 3.5 mmol/L), 4 mL of each was taken and placed in conical flask, added with 1 mL of color developing agent, stirred with magnetic force for 3 minutes, oleic acid molecules and copper ions formed a green complex compound, after centrifugation, organic phase of upper layer was taken and measured at 714 nm to determine light absorbance.

[0277] 2.2 Enzyme solution was 0.5 mg/mL pancreatic lipase solution; buffer solution was 0.07M NaH.sub.2PO.sub.4—K.sub.2HPO.sub.4 phosphate buffer solution (pH 7.0); color developing agent was 5% copper acetate solution, which was regulated with pyridine to have pH 6.1.

[0278] 2.3 Measurement of Lipase Activity:

[0279] 3 mL of 0.07M phosphate buffer solution and 1 mL of olive oil were pre-heated at a constant temperature of 37° C. for 5 minutes in water bath under agitation with a magnetic stirrer, added with 1.3 mL of enzyme solution (negative control was that without enzyme inhibitor; positive control is that added with 100 μL of 0.1 mg/mL orlistat solution; 0.6, 0.9, 1.1, 1.3, 1.8 mg/mL compound of Formula II solutions were added, each 100 μL), stirred with magnetic force for 10 minutes, immediately added with 8 mL of toluene, stirred continuously for 2 minutes, the reaction was terminated, the generated oleic acid was extracted. The solutions were transferred to centrifuge tube, centrifuged under 4000 rpm for 10 minutes, and the organic phase and water phase were layered and clarified. 4 mL of the upper layer organic phase was taken and placed in a small conical flask, added with 1 mL of color developing agent, stirred with magnetic stirrer for 3 minutes, the generated oleic acid reacted with copper ions to form green complex compound. After centrifugation under 4000 rpm for 10 minutes, the upper layer of toluene solution containing copper oleate was taken, and measured with a spectrophotometer at 714 nm to determine its light absorbance. A blank solution without lipase was prepared as control by the same method, and concentration of fatty acid could be determined by referring to light absorbance working curve of oleic acid.

[0280] 3 Definition and calculation formula of enzyme activity

[0281] Enzyme activity unit of lipase was defined as follows: an enzyme amount to release 1 μmol fatty acid under certain condition was defined as 1 unit of lipase activity (U).

[0282] The following formula was used to calculate enzyme activity: X=(cV)/(tV′) wherein: X represents enzyme activity of lipase, U/mL; c represents concentration of fatty acid, μmol/mL; V represents volume of fatty acid solution, mL; V′ represents amount of enzyme solution, mL; t represents action time, minute.

Inhibition rate=[(enzyme activity of lipase−enzyme activity of lipase after inhibition)/enzyme activity of lipase]×100%

[0283] 4 Test results

[0284] 4.1 Light absorbance working curve of oleic acid

[0285] As shown in FIG. 9.

[0286] 4.2 Test results of activity of enzyme inhibitors

[0287] As shown in following Table 3.

TABLE-US-00003 TABLE 3 Test results of activity of enzyme inhibitors Final concentration Concentration concentration of inhibitor in of generated Enzyme Inhibition of the inhibitor the system oleic acid activity rate Sample name (mg/mL) (pg/mL) Absorbance (mM) U/mL (%) Blank control — — 0.6680 2.75 1.69 — Orlistat 0.1 1.85 0.2042 0.81 0.50 69.84% Compound of 6 111 0.5665 2.32 1.43 15.38% Formula II 9 166 0.5035 2.06 1.27 24.85% 11 203 0.3998 1.63 1.00 40.83% 13 240 0.2743 1.10 0.68 59.76% 18 333 0.2611 1.04 0.64 62.13%

[0288] wherein the enzyme activity was calculated according to the calculation formula of the above section 3, wherein:

[0289] V =8 mL; V′=1.3 mL; t =10 minutes.

[0290] The test results showed that the compound of the present invention, especially the compound of Formula II, had good inhibition effect on activity of lipase, had a IC.sub.50 value of about 220 μg/mL, and exhibited concentration-effect relationship in inhibition effect. It could be seen that the compounds of the present invention were potential drug for prophylaxis and/or treatment and/or adjunctive treatment of obesity or diseases associated with obesity.

[0291] Further studying showed that the ester derivatives or ether derivatives of compound of Formula II, for example, “tetraacetate of compound of Formula II”, “tetra-p-tolylsulfonate of compound of Formula II”, “tetra(trifluoromethyl sulfonate) of compound of Formula II” and “tetra(tert-butyl-dimethyl-silyl ether) of compound of Formula II” as prepared in Examples 6-9 also had similar activity of inhibiting lipase, and were potential drugs for prophylaxis and/or treatment and/or adjunctive treatment of obesity or diseases associated with obesity.

[0292] Although the specific embodiments of the present invention have been described in details, those skilled in the art would understand these details can be modified or substituted according to the disclosed teachings, and all these changes are within the protection scope of the present invention. The whole scope of the present invention is defined by the appending claims.

[0293] This application is a divisional application of U.S. patent application Ser. No. 17/071,963, filed Oct. 15, 2020, which is a divisional application of U.S. patent application Ser. No. 14/368,494, filed Jun. 24, 2014, issued as U.S. Pat. No. 10,889,612, which is a 371 national stage application of International Patent Application No. PCT/CN2012/087360, filed Dec. 25, 2012, which claims the benefit of Chinese Patent Application No. 201110441993.2, filed Dec. 26, 2011, and Chinese Patent Application No. 201110442009.4, filed Dec. 26, 2011, and Chinese Patent Application No. 201210015475.9, filed Jan. 18, 2012, which applications are incorporated herein by reference in their entirety.