GLAUCOCALYXIN A DERIVATIVE AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

Provided is a glaucocalyxin A derivative, or salt thereof, as represented by the formula (I), a method for preparation of said glaucocalyxin A derivative, and a use for said glaucocalyxin A derivative in preparing pharmaceuticals for fighting autoimmune diseases and tumors, e.g. difficult-to-treat diseases such as systemic lupus erythematosus, psoriasis and triple-negative breast cancer

##STR00001##

Claims

1. Glaucocalyxin A derivative represented by formula (I): ##STR00032## wherein R is any one selected from the group consisting of dimethylamino, diethylamino, piperidine-1-yl, piperazine-1-yl, hexamethyleneiminyl, morpholine-1-yl, N-phenyl-N-(3-oxocyclohexanyl)amino, N-p-chlorophenyl-N-(2-oxo-butanyl)amino, N-2-chlorophenyl-N-(2-oxo-butanyl)amino, benzylamino, purinyl-9-yl, 2-amino-6-hydroxypurine-9-yl, 4-methylpiperazine-1-yl, N-phenyl-N-methyl-amino, dibenzyl-amino, imidazole-1-yl, 2-methyl imidazole-1-yl, N-phenyl-N-(3-oxo-butanyl)amino, pyrrole-1-yl, 2-hydroxyacylpyrrole-1-yl, 2-methylpyrrolidine-1-yl, 3-methylpyrrole-1-yl, 2-oxo-pyrrole-1-yl, 3-aminoacylphenylamino, p-aminoacylphenylamino, amino acid substitutional; or a salt thereof.

2. A method for preparation of glaucocalyxin A derivative according to claim 1, which comprises subjecting glaucocalyxin A and a R group donor compound to an addition reaction in the presence of a catalyst to afford the product.

3. The method according to claim 2, wherein the catalyst is any one or more selected from the group consisting of sodium methoxide, sodium ethoxide, pyridine, sodium carbonate and potassium carbonate.

4. The method according to claim 2, wherein the mole ratio of the R group to the glaucocalyxin A is (1 to 10): 1.

5.-10. (canceled)

11. The method according to claim 2, wherein the mole ratio of the catalyst to the glaucocalyxin A is (1 to 10): 1.

12. The method according to claim 2, wherein the reaction is carried out at a temperature between 30 C. and 60 C.

13. The method according to claim 2, wherein the reaction is carried out in a solvent which comprises any one or more selected from the group consisting of alcohol, ketine, ether, ester and haloalkane.

14. The method according to claim 13, wherein the alcohol comprises any one or more selected from the group consisting of methanol, ethanol, isopropanol, isobutanol and tert-butanol; the ketone comprises any one or more selected from the group consisting of acetone and 2-butanone; the ether comprises any one or more selected from the group consisting of ethyl ether, dioxane, isopropyl ether, methyl tert-butyl ether and tetrahydrofuran; the ester comprises any one or more selected from the group consisting of methyl acetate, ethyl acetate and butyl acetate; the haloalkane comprises dichloromethane and trichloromethane;

15. The method according to claim 13, further comprising a step of evaporating the solvent and/or detecting with TLC and/or HPLC after the reaction.

16. A method for preparation of the salt of glaucocalyxin A derivative according to claim 1, comprising: dissolving the glaucocalyxin A derivative into an organic solvent to form a solution, then subjecting the solution and an acid to a salt formation reaction, while controlling the pH of the solution, to give the salt of glaucocalyxin A derivative.

17. The method according to claim 16, wherein the acid includes organic acid and inorganic acid.

18. The method according to claim 17, wherein the inorganic acid comprises any one selected from the group consisting of hypoiodous acid, hypochlorous acid, hypobromous acid, iodic acid, perchloric acid, peroxydisulfuric acid, peroxydicarbonic acid, peroxycarbonic acid, pyrophosphoric acid, pyrosulfuric acid, pyrosulfurous acid, tetrathioic acid, phosphoric acid, thiosulfuric acid, sulfuric acid, chloric acid, metaphosphoric acid, hydroiodic acid, hydronitric acid, hydrofluoric acid, hydrogen sulfide, hydrochloric acid, hydrobromic acid, tetraboric acid, carbonic acid, nitric acid, bromic acid, sulfurous acid, phosphorous acid, chlorous acid, hydrochloric acid, nitrous acid, orthophosphoric acid, orthosulfuric acid and orthocarbonic acid; and the organic acid comprises any one selected from the group consisting of tartaric acid, oxalic acid, malic acid, citric acid, ascorbic acid, benzoic acid, salicylic acid, caffeic acid, lactic acid, sorbic acid, fumaric acid, formic acid, acetic acid, benzoic acid, ethanedioic acid, succinic acid, pyruvic acid, -keto-succinic acid, benzenesulfonic acid, ethanesulfonic acid, resin acid, trifluoroacetic acid, maleic acid, tetrasulfonic acid, methanesulfonic acid, fumaric acid and amino acid.

19. The method according to claim 16, wherein the organic solvent comprises any one or more selected from the group consisting of alcohol, ketone, ether, ester and haloalkane.

20. The method according to claim 19, wherein the alcohol comprises any one or more selected from the group consisting of methanol, ethanol, isopropanol, isobutanol and tert-butanol; the ketone comprises any one or more selected from the group consisting of acetone and 2-butanone; the ether comprises any one or more selected from the group consisting of ethyl ether, dioxane, isopropyl ether, methyl tert-butyl ether and tetrahydrofuran; the ester comprises any one or more selected from the group consisting of methyl acetate, ethyl acetate and butyl acetate; the haloalkane comprises dichloromethane and trichloromethane.

21. The method according to claim 16, wherein the pH of the solution is controlled by hydrogen chloride solution.

22. The method according to claim 21 wherein the hydrogen chloride solution comprises any one selected from the group consisting of aqueous solution of hydrogen chloride, methanol solution of hydrogen chloride, ethanol solution of hydrogen chloride, isopropanol solution of hydrogen chloride, n-propanol solution of hydrogen chloride, isobutanol solution of hydrogen chloride, ethyl acetate solution of hydrogen chloride, acetone solution of hydrogen chloride, ethyl ether solution of hydrogen chloride and dioxane solution of hydrogen chloride;

23. The method according to claim 21, wherein the pH of the solution is controlled between 6.0 and 8.0.

24. The method according to claim 16, wherein the reaction is carried out at a temperature between 30 C. and 60 C.

25. A method for treating an autoimmune disease and/or a cancer in a subject in need thereof, comprising administration of a medicament comprising the glaucocalyxin A derivative or the salt thereof according to claim 1 to the subject.

26. The method according to claim 25, wherein the autoimmune disease is systemic lupus erythematosus or psoriasis; and the cancer comprises any one or more of the following group consisting of triple-negative breast cancer, glioma, cervical cancer, esophageal cancer, lung cancer, liver cancer, choriocarcinoma, oral epidermoid carcinoma, prostate cancer, rectal cancer.

Description

DESCRIPTION OF THE DRAWINGS

[0038] The drawings to be used in the description of the examples will be briefly described below, in order to illustrate the technical solutions of the embodiments of the present invention more clearly. Apparently, the drawings in the following description are only embodiments of the invention, and other drawings may be obtained by those skilled in the art without creative work.

[0039] FIG. 1 shows a .sup.1H NMR spectrum of a glaucocalyxin A derivative of the present invention.

[0040] FIG. 2 shows a .sup.13C NMR spectrum of a glaucocalyxin A derivative of the present invention.

[0041] FIG. 3 shows a LC-MS spectrum of a salt of glaucocalyxin A derivative of the present invention.

[0042] FIG. 4 shows a graph depicting IC.sub.50 values (i.e., half effective inhibitory concentration) of a glaucocalyxin A derivative of the present invention for the inhibition of proliferation of five types of cell lines.

[0043] FIG. 5 shows a graph depicting IC.sub.50 values of GLA for the inhibition of proliferation of five types of cell lines.

[0044] FIG. 6 shows the comparison of the IC.sub.50 of a glaucocalyxin A derivative of the present invention for the inhibition of proliferation of various cell lines with that IC.sub.50 of GLA.

[0045] FIG. 7 shows a X-Y scatter plot of the concentration versus inhibitory rate of a glaucocalyxin A derivative of the present invention.

[0046] FIG. 8 shows a X-Y scatter plot of the concentration versus inhibitory rate of GLA.

DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention will be described, clearly and completely, with the combination of the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the present invention rather than all. All other embodiments obtained by those general skilled in the art under the premise of no creative work, on the basis of embodiments of the present invention, are within the scope of the present invention.

Example 1

[0048] This example discloses a method for the preparation of a glaucocalyxin A derivative, comprising: glaucocalyxin A nitrogenous derivative which was obtained by modification of glaucocalyxin A, was reacted with an acid to give a salt of glaucocalyxin A nitrogenous derivative with good solubility in water.

[0049] Wherein, the modification reaction equation from glaucocalyxin A to glaucocalyxin A nitrogenous derivative is shown as below:

##STR00005##

[0050] The glaucocalyxin A (GLA) was used as raw material, and the catalyst was used in an amount so that the molar ratio of the catalyst to the raw material glaucocalyxin A was 1 to 10, and the temperature was controlled between 30

[0051] The R in RH is a donor of nitrogen, and has a structure identical to that of R in glaucocalyxin A derivative. R is any one of the following groups: dimethylamino, diethylamino, piperidine, piperazine, hexamethyleneimine, morpholine, N-methy 1piperazine, methylaniline, dibenzylamine, imidazole, 2-methyl imidazole, 4-Phenylamino-butanone, 3-(phenyl amino) cyclohexanone, p-chlorophenyl-amino-butanone, o-chlorophenyl-amino-butanone, benzylamine, purine, 2-amino-6-hydroxypurine, Pyrrole, pyrrole-2-carboxylic acid, 2-methylpyrrolidine, 3-methylpyrrole, 3-aminobenzamide, p-aminobenzamide and various amino acid.

[0052] The obtained glaucocalyxin A derivative has a structural formula as shown below:

##STR00006##

[0053] The structure of R in the above structural formula may be any one of the structures of the group in Table 1:

TABLE-US-00001 TABLE 1 The name and structure of R group Name of the Structure of the Name of the Structure of the Group Group Group Group dimethylamino N(CH.sub.3).sub.2 N-methyl piperazine [00007] diethylamino N(CH.sub.2CH.sub.3).sub.2 methylaniline [00008] piperidine [00009] dibenzylamine [00010] piperazine [00011] imidazole [00012] hexamethyleneimine [00013] 2-methyl imidazole [00014] morpholine [00015] 4-phenylamino- butanone [00016] 3-(phenylamino) cyclohexanone [00017] pyrrole [00018] p-chlorophenylamino- butanone [00019] pyrrole-2- carboxylic acid [00020] o-chlorophenylamino- butanone [00021] 2-methylpyrrolidine [00022] benzylamine [00023] 3-methylpyrrole [00024] Purine [00025] 2-pyrrolidone [00026] 2-amino-6- hydroxypurine [00027] 3-aminobenzamide [00028] amino acid [00029] p-Aminobenzamide [00030]

[0054] The glaucocalyxin A derivative described as above was then reacted with an acid to give a salt of glaucocalyxin A derivative with good solubility in water. The acid which can react with glaucocalyxin A derivative includes those shown in Table 2.

TABLE-US-00002 TABLE 2 The acids which can react with glaucocalyxin A derivative Inorganic Acid Organic Acid hydrochloric hypoiodous acid oxalic acid benzenesulfonic acid acid hydrobromic hypochlorous acid tartaric acid ethanesulfonic acid acid sulfuric acid hypobromous acid p-toluenesulfonic formic acid acid nitric acid perchloric acid methanesulfonic naphthalenesulfonic acid acid phosphoric acid peroxydisulfuric fumaric acid maleic acid acid iodic acid peroxydicarbonic citric acid amino acid acid peroxycarbonic pyrophosphoric nicotinic acid lactic acid acid acid pyrosulfuric acid pyrosulfurous resin acid acetic acid acid tetrathioic acid thiosulfuric acid malic acid ascorbic acid chloric acid metaphosphoric benzoic acid salicylic acid acid hydroiodic acid hydronitric acid caffeic acid sorbic acid hydrofluoric acid hydrogen sulfide fumaric acid ethanedioic acid hydrochloric tetraboric acid succinic acid pyruvic acid acid carbonic acid bromic acid -keto-succinic trifluoroacetic acid acid sulfurous acid phosphorous acid tetrasulfonic acid chlorous acid nitrous acid orthophosphoric orthosulfuric acid acid orthocarbonic acid

Example 2

[0055] This example discloses a method for the preparation of dimethylamino glaucocalyxin A hydrochloride, which comprises mainly the following steps:

[0056] STEP ONE: The preparation of dimethylamino glaucocalyxin A. 1. Glaucocalyxin A (1.00 g) was mixed with an organic solvent (20 mL) under stirring at room temperature till the glaucocalyxin A was dissolved completely.

[0057] 2. After filtration, dimethylamine solution (33%, 0.68 mL) was added drop-wise with stirring, and the mixed solution was stirred for 12 h at room temperature and monitored by TLC. 3. After completion of the reaction, the reaction system was subjected to distillation under reduced pressure at 4050 dimethylamino glaucocalyxin A (1.05 g) as powder. The yield of dimethylamino glaucocalyxin A was 93%, and its purity was 99.2% by HPLC.

[0058] STEP TWO: The preparation of dimethylamino glaucocalyxin A hydrochloride.

[0059] 1. To an organic solvent (10 mL) was added dimethylamino glaucocalyxin A (1.00 g) under ice bath, and the mixture was completely stirred till the dimethylamino glaucocalyxin A was dissolved.

[0060] 2. After filtration, at a controlled temperature 0, hydrogen chloride solution was added to the mixture until pH 7 was reached, and the resultant was then stirred for 30 min to give a white solid.

[0061] 3. The precipitation was filtered to afford dimethylamino glaucocalyxin A hydrochloride (1.00 g) as a white-like solid, with a yield of 92% and a purity of 99.5% by HPLC.

[0062] In practice, the organic solvents used in the STEP ONE and STEP TWO described above may be one or more of the following solvents: alcohol, ketone, ether, ester and haloalkane. In the present example, the organic solvent in STEP ONE is methanol, and the organic solvent in STEP TWO is isopropyl alcohol.

[0063] In other examples, the alcohols described above may be any one or more selected from the group consisting of methanol, ethanol, isopropanol, isobutanol and tert-butanol; the ketone described above is acetone or 2-butanone; the ether described above may be any one or more selected from the group consisting of ethyl ether, dioxane, isopropyl ether, methyl tert-butyl ether and tetrahydrofuran; the ester described above may be any one or more selected from the group consisting of methyl acetate, ethyl acetate and butyl acetate; the haloalkane described above is dichloromethane or trichloromethane.

[0064] The hydrogen chloride solution used in STEP TWO may be any one selected from the group consisting of aqueous solution of hydrogen chloride, methanol solution of hydrogen chloride, ethanol solution of hydrogen chloride, isopropanol solution of hydrogen chloride, n-propanol solution of hydrogen chloride, isobutanol solution of hydrogen chloride, ethyl acetate solution of hydrogen chloride, acetone solution of hydrogen chloride, ethyl ether solution of hydrogen chloride and dioxane solution of hydrogen chloride.

[0065] In the present example, the hydrogen chloride solution described above is, preferably, isopropanol solution of hydrogen chloride.

[0066] In the present example, the dimethylamino glaucocalyxin A hydrochloride was characterized by H spectrum using NMR, and the result was shown as FIGS. 1-2.

[0067] As seen from FIG. 1, the molecular formula of dimethylamino glaucocalyxin A hydrochloride salt is C.sub.22H.sub.36ClNO.sub.4.HCl, .sup.1H NMR (DMSO) :0.98 (6H, d, 2CH.sub.3), 1.05 (3H, m, CH.sub.3), 1.74 (6H, m, 2CH.sub.3), 3.91 (1H, s, CH), 6.20 (1H, s, CH), 4.83 (1H, s, CH), 5.86 (1H, s, CH), 6.20 (1H, s, CH), 10.58 (1H, s, OH).

[0068] As seen from FIG. 2, the molecular formula of dimethylamino glaucocalyxin A hydrochloride salt is C.sub.22H.sub.36ClNO.sub.4.HCl, .sup.13C NMR (DMSO) :

##STR00031##

[0069] In the present example, the obtained dimethylamino glaucocalyxin A hydrochloride was subjected to LC-MS detection, and the detection result was shown as FIG. 3.

[0070] As seen from the spectrum of FIG. 3, the molecular formula of dimethylamino glaucocalyxin A hydrochloride salt is C.sub.22H.sub.36ClNO.sub.4.HCl, MS m/e: 378.6 (M+).

Example 3

[0071] The procedure of Example 3 was substantially identical to that of Example 2, except that the controlled temperature and pH in the preparation of dimethylamino glaucocalyxin A hydrochloride in STEP TWO were different. In the present example, the controlled temperature was 30

Example 4

[0072] The procedure of Example 4 was substantially identical to that of Example 2, except that the controlled temperature and pH in the preparation of dimethylamino glaucocalyxin A hydrochloride in STEP TWO were different. In the present example, the controlled temperature was 60

Example 5

[0073] Compared to Example 2, the procedure of Example 5 involves some differences in that the controlled temperature was 10 in the preparation of dimethylamino glaucocalyxin A hydrochloride in STEP TWO.

Example 6

[0074] Compared to Example 2, the procedure of Example 6 involves some differences in that the controlled temperature was 20 in the preparation of dimethylamino glaucocalyxin A hydrochloride in STEP TWO.

[0075] The organic solvents used in Examples 2-6 described above are those enumerated in the embodiments. The hydrogen chloride solution is most preferably isopropanol solution of hydrogen chloride or ethanol solution of hydrogen chloride, the reaction temperature in the STEP TWO is most preferably in the range of 1020 and the pH is most preferably 7.

[0076] As described in the Example below, the glaucocalyxin A derivative provided by the present invention was subjected to efficacy experiments. It can be known from the experiments that the glaucocalyxin A derivative can be used in the manufacture of a medicament against autoimmune diseases and cancers, such as triple-negative breast cancer, glioma, cervical cancer, esophageal cancer, lung cancer, liver cancer, choriocarcinoma, oral epidermoid carcinoma, prostate cancer, rectal cancer. Especially for the triple-negative breast cancer and glioma, the glaucocalyxin A derivative shows high targetability. The glaucocalyxin A derivative can be used in the medicament for the treatment of triple-negative breast cancer and fill the gaps in the treatment of triple-negative breast cancer. The autoimmune diseases comprise systemic lupus erythematosus or psoriasis.

Example 7

[0077] Cell Proliferation Assay (MTT):

[0078] The experiment assaying the inhibition of dimethylamino glaucocalyxin A hydrochloride salt (hereinafter referred to as GH02) and glaucocalyxin A (i.e. GLA) on cell proliferation of cancer cell lines was performed as follows: cells of cancer cell lines were adjusted to a density of 10.sup.4/100 L and were seeded into 96-well plates at 100 L per well and cultured overnight until the cells were completely adherent and grown up to 70-80% confluence. The mother liquid of GH02 and GLA (both of which concentrations were 100 mM) were diluted with complete medium, respectively, to final concentrations of 1, 3.125, 6.25, 12.5, 25, 50, 100 mol/L, and then such diluted solutions were added into wells of 96-well plates, with each concentration being performed in 4 replicates, and the group without cells acting as blank control, and then incubated in the incubator at 37.sub.2. After incubation for 48 h, MTT was added to each well to a final concentration of 0.5 mg/ml and further incubated in the incubator at 37.sub.2 for 4 h. Subsequently, the supernatant was removed, 150 L DMSO was added to each well and incubated with shaking for 10 min, and then the absorbance was measured at 490 nm on a microplate reader.

Inhibitory rate of the cell proliferation=[(absorbance of the control groupabsorbance of blank)(absorbance of the medication groupabsorbance of blank)]/(absorbance of the control groupabsorbance of blank)%

[0079] As shown in FIG. 4 to FIG. 6, MDA-MB-231 and MCF-7 are cell lines of breast cancer, SGC-7901 is a cell line of gastric cancer, U251 is a cell line of glioma, and A549 is a cell line of lung cancer.

[0080] FIG. 4 shows the IC.sub.50 values of GH02 against five cell lines described above, as seen from which, the GH02 displayed the lowest IC.sub.50 value against the MDA-MB-231 breast cancer cell line, i.e. GH02 exhibited the best efficacy against breast cancer.

[0081] FIG. 5 shows the IC.sub.50 values of GLA against five cell lines described above, as seen from which, the GLA displayed the lowest IC.sub.50 value against the MDA-MB-231 breast cancer cell line; and as seen from which, in combination with FIG. 6, GH02 and GLA exhibited the best efficacy against breast cancer.

[0082] As shown in FIG. 7, the inhibition of GH02 on each of the five cell lines described above increased with the increase of the concentration of GH02, i.e. it can inhibit the proliferation of cancer cells.

[0083] As shown in FIG. 8, the inhibition of GLA on each of the five cell lines described above increased with the increase of the concentration of GLA, i.e. it can inhibit the proliferation of cancer cells.

[0084] In addition, it can be understood that, as shown in FIG. 7 and FIG. 8, GH02 and GLA displayed the lowest IC.sub.50 values and the highest inhibitory rate against the MDA-MB-231 breast cancer cell line, and thus the glaucocalyxin A derivative of the present invention exhibited the best inhibition effect on breast cancer.

[0085] The foregoing description of the disclosed embodiments will enable one skilled in the art to achieve or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be embodied in other embodiments without departing from the spirit or scope of the invention. Accordingly, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.