Glaucocalyxin a derivative, pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof and uses thereof in preparation of drugs for treating psoriasis

Abstract

A glaucocalyxin A derivative, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof and uses thereof in the preparation of drugs for treating psoriasis.

Claims

1. A method for treating psoriasis, comprising administrating a therapeutically effective amount of a glaucocalyxin A derivative, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the glaucocalyxin A derivative or the pharmaceutically acceptable salt thereof to a subject in need thereof, wherein the glaucocalyxin A derivative is a compound of formula I: ##STR00015## wherein R.sub.1 and R.sub.2 are independently hydrogen, amino, cyclohexyl, alkyl, alkenyl, aryl, or heteroaryl.

2. The method according to claim 1, wherein R.sub.1 and R.sub.2 are independently hydrogen, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, n-heptyl, n-octyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, n-hexyl, allyl, cyclohexyl, phenyl, benzyl, p-methoxyphenyl, o-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl, o-chlorophenyl, N-3,4-dichlorophenyl, N-3,5-dichlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 2,3,4-trifluorophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethyl phenyl, 2-chloro-5-trifluoromethyl phenyl, 2-bromo-5-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl, 2,4,6-trimethylphenyl, 4-propylphenyl, N-p-tolyl, N-methyl-N-phenyl, acetylthioamino, N-acetyl, 1-naphthyl, 2-phenylethyl, 3,5-dimethyl-1-pyrazolyl, 2-isopropylphenyl, 3-chloro-4-methylphenyl, 2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl, p-isopropylphenyl, 2-methoxyethyl, 2-morpholinylethyl, 2-furanylmethyl, 2,4,5-triphenyl, 2,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, pyridine-3-methyl, 3-morpholinylpropyl, 5-fluoro-2-methylphenyl, 4-iodophenyl, 2-piperidinylethyl, 3-bromophenyl, p-methoxybenzyl, 2,3-dichlorophenyl, 4-trifluoromethoxyphenyl, p-methylbenzyl, 3-methoxybenzyl, 4-phenoxyphenyl, 5-chloro-2-methylphenyl, 2-ethoxyphenyl, 4-hydroxyphenyl, 2,4-dimethoxyphenyl, 2-ethylphenyl, 4-ethylphenyl, p-dimethylaminophenyl, N-decyl, 2,6-diethylphenyl, 4-bromophenyl, 2,4,6-trichlorophenyl, 3-hydroxyphenyl, 3-methoxyphenyl, 2,5-dichlorophenyl, 2-bromophenyl, 2,6-dimethylphenyl, 5-chloro-2-methoxyphenyl, 2,6-dichlorophenyl, 2,5-dimethoxyphenyl, pentafluorophenyl, 4-ethoxyphenyl, 3-phenylpropyl, 2,5-difluorophenyl, 2-methyl-3-chlorophenyl, 3,4-dimethylphenyl, 3-methylphenyl, or 2,5-dimethylphenyl.

3. The method according to claim 1, wherein R.sub.1 and R.sub.2 are independently hydrogen, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, n-heptyl, n-octyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, n-hexyl, allyl, cyclohexyl, phenyl, or benzyl.

4. The method according to claim 1, wherein R.sub.1 and R.sub.2 are independently hydrogen, amino, methyl, ethyl, n-propyl, isopropyl, n-butyl, n-hexyl, allyl, cyclohexyl, phenyl, or benzyl.

5. The method according to claim 1, wherein the glaucocalyxin A derivative is any one of the following Formulas Ia to Ip, or a combination of at least two thereof: ##STR00016## ##STR00017## ##STR00018##

6. The method according to claim 1, wherein the pharmaceutically acceptable salt is an organic or inorganic acid salt of the glaucocalyxin A derivative.

7. The method according to claim 6, wherein the organic acid salt is any one selected from the group consisting of tartrate, stearate, oxalate, citrate, lactate, sorbate, fumarate, formate, acetate, benzoate, besylate, ethanesulfonate, resinate, trifluoroacetate, maleate, methanesulfonate, fumarate, amino acid salt, and nicotinate, or a combination of at least two selected therefrom.

8. The method according to claim 6, wherein the inorganic acid salt is any one selected from the group consisting of iodate, phosphate, sulfate, hydroiodide, hydrobromide, nitrate, bromate, and hydrochloride, or a combination of at least two selected therefrom.

9. The method according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable auxiliary.

10. The method according to claim 9, wherein the pharmaceutical composition is in the form of an oral preparation, a parenteral preparation, or an external preparation.

11. The method according to claim 1, wherein the psoriasis is selected from the group consisting of psoriasis vulgaris, arthritic psoriasis, pustular psoriasis, and erythrodermic psoriasis.

12. A glaucocalyxin A derivative having the structure of formula I′: ##STR00019## wherein R.sub.1 and R.sub.2 are independently amino, cyclohexyl, alkyl, alkenyl, aryl, or heteroaryl.

13. The glaucocalyxin A derivative according to claim 12, wherein R.sub.1 and R.sub.2 are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, n-heptyl, n-octyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, n-hexyl, allyl, cyclohexyl, phenyl, or benzyl.

14. The glaucocalyxin A derivative according to claim 12, which has any one of the following Formulas If, In, Io, or Ip: ##STR00020##

15. A pharmaceutically acceptable salt of the glaucocalyxin A derivative according to claim 12, wherein the pharmaceutically acceptable salt is an organic or inorganic acid salt of the glaucocalyxin A derivative.

16. The pharmaceutically acceptable salt of glaucocalyxin A derivative according to claim 15, wherein the organic acid salt is any one selected from the group consisting of tartrate, stearate, oxalate, citrate, lactate, sorbate, fumarate, formate, acetate, benzoate, besylate, ethane sulfonate, resinate, trifluoroacetate, maleate, methanesulfonate, fumarate, amino acid salt, and nicotinate.

17. The pharmaceutically acceptable salt of glaucocalyxin A derivative according to claim 15, wherein the inorganic acid salt is any one selected from the group consisting of iodate, phosphate, sulfate, hydroiodide, hydrobromide, nitrate, bromate, and hydrochloride.

18. A method for preparing the glaucocalyxin A derivative according to claim 12, comprising reacting 2-bromoglaucocalyxin A with N,N-dialkylthiourea to provide a glaucocalyxin A derivative of formula I′.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the time- and dose-dependent effect of the hydrobromide of the compound of Formula If (Compound 23) on the proliferation of human keratinocytes;

(2) FIG. 2 is a graph showing the inhibitory effect of the hydrobromide of the compound of Formula If (Compound 23) on xylene-induced auricular swelling in mice; wherein the abscissa represents the grouping, comprising blank control group, dexamethasone (30 mg/kg) treatment group, Compound 23 (10 mg/kg) treatment group, and Compound 23 (50 mg/kg) treatment group sequentially from left to right.

DETAILED DESCRIPTION

(3) The technical solutions of the present application are further described below by way of illustrating specific embodiments. It should be understood by those skilled in the art that the examples are only described to help understand the present application and should not be considered as a limitation of the present application.

Example 1 (Preparation of a Hydrobromide Salt of the Compound of Formula If)

Step 1: Preparation of 2-bromoglaucocalyxin A

(4) ##STR00008##

(5) PyBr.sub.3 (96.22 mg, 0.30 mmol) was added to a solution of glaucocalyxin A (100 mg, 0.30 mmol) in DMF (4 ml) at room temperature. The reaction mixture was stirred at room temperature for 5 h, then poured into water, suction-filtered, and dried to give a mixture of two epimers as a white solid (106.33 mg, yield 86.1%), which was directly used for the next reaction without separation. MS ESI-MS m/z 412 [H.sup.+].

Step 2: Preparation of a Hydrobromide Salt of the Compound of Formula If

(6) ##STR00009##

(7) N,N-Dimethylthiourea (12.66 mg, 0.12 mmol) was' added to a solution of 2-bromoglaucocalyxin A (50 mg, 0.12 mmol) in ethanol (4 ml) at room temperature. The reaction mixture was heated under reflux for 3 h. The mixture was slowly cooled down, and a solid was precipitated, suction-filtered, and dried to give a white solid powder (Compound 23, 40.54 mg, yield 67.91%). MS ESI-MS nm/z 417 [H.sup.+].

Example 2 (Preparation of a Hydrobromide Salt of the Compound of Formula In)

(8) ##STR00010##

(9) N,N-Diethylthiourea (15.87 mg, 0.12 mmol) was added to a solution of 2-bromoglaucocalyxin A (50 mg, 0.12 mmol) in ethanol (4 ml) at room temperature. The reaction mixture was heated under reflux for 3 h. The mixture was slowly cooled down, and a solid was precipitated, suction-filtered, and dried to give a white solid powder (38.23 mg, yield 60.62%). MS ESI-MS m/z 445 [H.sup.+].

Example 3 (Preparation of a Hydrobromide-Salt of the Compound of Formula Io)

(10) ##STR00011##

(11) N,N-Dipropylthiourea (19.23 mg, 0.12 mmol) was added to a solution of 2-bromoglaucocalyxin A (50 mg, 0.12 mmol) in ethanol (4 ml) at room temperature. The reaction mixture was heated under reflux for 3 h. The mixture was slowly cooled down, and a solid was precipitated, suction-filtered, and dried to give a white solid powder (35.48 mg, yield 53.41%). MS ESI-MS m/z 473 [H.sup.+].

Example 4 (Preparation of a Hydrobromide Salt of the Compound of Formula Ip)

(12) ##STR00012##

(13) N,N-Diisopropylthiourea (19.23 mg, 0.12 mmol) was added to a solution of 2-bromoglaucocalyxin A (50 mg, 0.12 mmol) in ethanol (4 ml) at room temperature. The reaction mixture was heated under reflux for 3 h. The mixture was slowly cooled down, and a solid was precipitated, suction-filtered, and dried to give a white solid powder (30.17 mg, yield 45.41%). MS ESI-MS m/z 473 [H.sup.+].

Example 5

(14) In this example, the glaucocalyxin A derivatives of the present application, or pharmaceutically acceptable salts thereof, were examined for the effect on the proliferation of human keratinocytes. In this example, the glaucocalyxin A derivatives as shown in Table 1 were examined by using the following procedure.

(15) A cell suspension was prepared and inoculated into three 96-well plates at 8000 cells in a volume of 150 μl per well, which were then cultured overnight in an incubator at 37° C., 5% CO.sub.2. On the next day, 50 μl of test compound solution at different concentrations was added to each well. Triplicate wells were set up for each concentration. A vehicle control group (without compound), a cell control group (without compound and vehicle), and a blank medium control group (without cells, a well for null adjustment) were also set up, and cultured for an additional 48 hours. CCK-8 staining working solution was added at 110 μL per well, and cultured for additional 1-4 h. At the end of the culture, the absorbances were measured at 450 nm (OD450 nm) on a Bio-Tek enzyme-linked immunosorbent assay. The results were expressed as the mean OD450 nm value of triplicates±standard deviation (x±sd). The Origin 7.0 was used for plotting and analysis. A fitting curve was obtained by plotting the OD value of each group against the logarithmic concentration of the test compound, and then the IC.sub.50 value was determined.

(16) It can be seen from the IC.sub.50 values in Table 1 that within the range of concentrations tested, the glaucocalyxin A derivatives and salts thereof significantly affected the growth and survival of the cultured human keratinocytes (HaCaT) after 48 hours of treatment, suggesting that the glaucocalyxin A derivatives had a significant dose-dependent inhibitory effect on the proliferation of human epidermal keratinocytes. The IC.sub.50 value of each compound was shown in Table 1. It can be seen that the glaucocalyxin A derivatives (Formula I) had an anti-psoriatic activity with an IC.sub.50 of about 1-10 μM, indicating a low effective dose. It can be seen from the data in Table 1 that when both R.sub.1 and R.sub.2 in Formula I were substituted, the pharmacological activity of the compound was generally enhanced, and superior to the glaucocalyxin A derivative reported in CN104356090A which was mono-substituted or unsubstituted on the nitrogen atom. Among them, Compound 23 was the most effective one.

(17) TABLE-US-00001 TABLE 1 Comp. IC50 No. General formula R group Molecular formula MW (μM, 48 h) 01 a hydrochloride salt R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•HCl 424.98 3.21 of Formula I both H 02 an acetate salt of R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•C.sub.2H.sub.4O.sub.2 448.57 3.52 Formula I both H 03 a maleic acid salt R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•C.sub.4H.sub.4O.sub.4 504.59 4.02 of Formula I both H 04 a fumarate salt of R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•C.sub.4H.sub.4O.sub.4 504.59 4.10 Formula I both H 05 a sulfate salt of R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•H.sub.2SO.sub.4 486.60 3.45 Formula I both H 06 a phosphate salt of R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•H.sub.3PO.sub.4 486.52 2.91 Formula I both H 07 a nitrate salt of R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•HNO.sub.3 451.52 3.28 Formula I both H 08 a hydrobromide salt R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•HBr 469.50 2.85 of Formula I both H 09 a citrate salt of R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•C.sub.6H.sub.8O.sub.7 580.66 2.42 Formula I both H 10 a tartrate salt of R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•C.sub.4H.sub.6O.sub.6 538.61 3.86 Formula I both H 11 a methanesulfonate R.sub.1 and R.sub.2 are C.sub.21H.sub.28N.sub.2O.sub.3S•CH.sub.4O.sub.3S 484.62 3.00 salt of Formula I both H 12 a hydrobromide R.sub.1 is amino and C.sub.21H.sub.29N.sub.3O.sub.3S•HBr 484.45 1.21 salt of Formula I R.sub.2 is H 13 a hydrobromide R.sub.1 is 4-pyridyl C.sub.26H.sub.31N.sub.3O.sub.3S•HBr 546.52 3.62 salt of Formula I and R.sub.2 is H 14 a hydrobromide R.sub.1 is n-propyl C.sub.24H.sub.34N.sub.2O.sub.3S•HBr 511.52 7.00 salt of Formula I and R.sub.2 is H 15 a hydrobromide R.sub.1 is isopropyl C.sub.24H.sub.34N.sub.2O.sub.3S•HBr 511.52 6.95 salt of Formula I and R.sub.2 is H 16 a hydrobromide R.sub.1 is n-butyl and C.sub.25H.sub.36N.sub.2O.sub.3S•HBr 525.55 8.88 salt of Formula I R.sub.2 is H 17 a hydrobromide R.sub.1 is n-hexyl and C.sub.27H.sub.40N.sub.2O.sub.3S•HBr 553.60 4.29 salt of Formula I R.sub.2 is H 18 a hydrobromide R.sub.1 is allyl and R.sub.2 C.sub.24H.sub.32N.sub.2O.sub.3S•HBr 509.50 7.24 salt of Formula I is H 19 a hydrobromide R.sub.1 is cyclohexyl C.sub.27H.sub.38N.sub.2O.sub.3S•HBr 551.58 8.16 salt of Formula I and R.sub.2 is H 20 a hydrobromide R.sub.1 is Phenyl and C.sub.27H.sub.32N.sub.2O.sub.3S•HBr 545.54 9.31 salt of Formula I R.sub.2 is H 21 a hydrobromide R.sub.1 is Benzyl and C.sub.28H.sub.34N.sub.2O.sub.3S•HBr 559.56 9.20 salt of Formula I R.sub.2 is H 22 Formula If R.sub.1 is methyl and C.sub.23H.sub.32N.sub.2O.sub.3S•HBr 416.58 0.95 R.sub.2 is methyl 23 a hydrobromide salt R.sub.1 is methyl and C.sub.23H.sub.33BrN.sub.2O.sub.3S•HBr 497.49 0.93 of Formula If R.sub.2 is methyl 24 a hydrobromide salt R.sub.1 is methyl and C.sub.22H.sub.30N.sub.2O.sub.3S•HBr 483.47 3.17 of Formula I R.sub.2 is H 25 a hydrobromide salt R.sub.1 is ethyl and C.sub.23H.sub.32N.sub.2O.sub.3S•HBr 497.49 5.49 of Formula I R.sub.2 is H 26 a hydrobromide salt R.sub.1 is propyl and C.sub.24H.sub.34N.sub.2O.sub.3S•HBr 511.52 7.86 of Formula I R.sub.2 is H 27 a hydrobromide salt R.sub.1 is isopropyl and C.sub.24H.sub.34N.sub.2O.sub.3S•HBr 511.52 6.92 of Formula I R.sub.2 is H 28 a hydrobromide salt R.sub.1 is ethyl and R.sub.2 C.sub.25H.sub.36N.sub.2O.sub.3S•HBr 525.55 1.26 of Formula In is ethyl 29 a hydrobromide salt R.sub.1 is propyl and R.sub.2 C.sub.27H.sub.40N.sub.2O.sub.3S•HBr 553.60 1.85 of Formula Io is propyl 30 a hydrobromide salt R.sub.1 is isopropyl and C.sub.27H.sub.40N.sub.2O.sub.3S•HBr 553.60 2.37 of Formula Ip R.sub.2 is isopropyl 31 glaucocalyxin A C.sub.20H.sub.28O.sub.4 332.43 30.46 32 dimethylamino glaucocalyxin A C.sub.22H.sub.36ClNO.sub.4 413.98 18.74 hydrochloride as disclosed in CN104761460A

(18) The glaucocalyxin A denoted as Compound 31 had the structure of

(19) ##STR00013##
which is disclosed, for example, as the “wangzaozi ne B” in CN1541643A. The dimethylamino glaucocalyxin A hydrochloride as disclosed in CN104761460A denoted as Compound 32 had the structure of

(20) ##STR00014##

Example 6

(21) In this example, the glaucocalyxin A derivatives of the present application and pharmaceutically acceptable salts thereof were examined for the relationship between the inhibitory rate thereof on the proliferation of human keratinocytes and its concentration. Compound 23 as shown in Table 1 was used as an exemplary compound to be examined by using the following procedure.

(22) A cell suspension was prepared and inoculated into three 96-well plates at 8000 cells in a volume of 150 μl per well, which were then cultured overnight in an incubator at 37° C., 5% CO.sub.2. On the next day, 50 μl of test compound solution at different concentrations was added to each well. Triplicate wells were set up for each concentration. A vehicle control group (without compound), a cell control group (without compound and without vehicle), and a blank medium control group (without cells, a well for null adjustment) were also set up, and cultured for additional 24, 48, and 72 hours. CCK-8 staining working solution was added at 110 μL per well, and cultured for additional 1-4 h. At the end of the culture, the absorbances were measured at 450 nm (OD450 nm) on a Bio-Tek enzyme-linked immunosorbent assay. The results were expressed as the mean OD450 nm value of the triplicates±standard deviation (x±sd). Prism Graph was used for plotting and analysis. The cell inhibitory rate of each group was calculated according to the mean value of the treatment group with each compound at each concentration and the mean value of the vehicle control group, that is, the cell inhibitory rate (%)=[1−(the mean value of the treatment group−the blank control group)/(the mean value of the vehicle control group−the blank control group)]×100%. A fitting curve was obtained by plotting the cell inhibitory rate of each group against the logarithmic concentration (Log C) of the test compound, and then the IC.sub.50 value was determined.

(23) The measurement results of Compound 23 were shown in FIG. 1, from which it can be seen that within the range of concentrations tested, Compound 23 significantly affected the growth and survival of the cultured HaCaT keratinocytes after 24, 48, and 72 hours of treatment. Compound 23 inhibited the proliferation of HaCaT cells in a dose- and time-effect relationship, suggesting that Compound 23 had a significant dose-dependent inhibitory effect on the proliferation of human epidermal keratinocytes, wherein the inhibitory effect was enhanced as the drug action time prolonged. It can be seen from FIG. 1 that the IC.sub.50 values of Compound 23 at 24 h, 48 h and 72 h were 1.935, 0.933, and 0.787 μM, respectively.

Example 7

(24) In this example, the glaucocalyxin A derivatives and pharmaceutically acceptable salts thereof were detected for the inhibitory effect thereof on xylene-induced auricular swelling in mice. Compound 23 was used as an exemplary compound, and the detection method was as follows.

(25) 20 mice were randomly divided into 4 groups with 5 mice in each group, which were blank control group, 30 mg/kg dexamethasone treatment group, Compound 23 (10 mg/kg) treatment group, and Compound 23 (50 mg/kg) treatment group, respectively. 0.25-0.3 mL depending on the body weight was intragastrically administrated for 7 consecutive days, and an equal volume of 5% glucose injection was administrated to the blank group. On the 8th day, xylene was used to produce a model of auricular swelling in the right auricle of mice. 15 μl of xylene, an inflammatory inducer, was applied to both sides of the right auricle of each mouse, and the left auricle was left un-treated as a normal auricle control. After 1 hour, the mice were sacrificed by cervical dislocation. Both auricles were cut off along the auricle baseline, and then punched at the same position by using a puncher with a diameter of 8 mm to give auricle pieces, which were then weighed on an electronic analytical balance. The auricular swelling rate and the auricular swelling inhibitory rate were calculated according to the following formula. Swelling rate (%)=(weight of the auricle piece from the inflammatory side−weight of the auricle piece from the non-inflammatory side)/weight of the auricle piece from the non-inflammatory side×100%; swelling inhibitory rate (%)=(average swelling degree of the model group−average swelling degree of the administration group)/average swelling degree of the model group×100%.

(26) The inhibitory rate of Compound 23 against xylene-induced auricular swelling in mice was shown in FIG. 2. It can be seen from FIG. 2 that both 10 mg/kg of Compound 23 and 50 mg/kg of Compound 23 were effective for inhibiting the auricular swelling in mice, indicating that compound 23 had an acute anti-inflammatory effect.

Example 8

(27) In this example, the glaucocalyxin A derivatives and pharmaceutically acceptable salts thereof were detected for the effect thereof on ConA-induced proliferation of mouse spleen lymphocytes. Compound 23 was used as an exemplary compound, and the detection method was as follows

(28) 2×10.sup.7/mL cell suspension was prepared and a 5 μg/μL ConA stock solution was added thereto at a final concentration of 5 μg/mL. The resulting mixture was inoculated into a 96-well culture plate at 0.1 mL per well. Then 0.1 mL of test compound at different concentrations was added to each well. Triplicate or quadruplicate wells were set up for each group, and a vehicle control group (without compound), a stimulant control group (with ConA, without compound, and without vehicle), a cell control group (without ConA, without compound, and without vehicle), and a blank medium control group (without cells, a well for null adjustment) were also set up, and cultured at 37° C., 5% CO.sub.2 for 48 h. MTT solution at 5 mg/mL was added at 20 μl per well, and cultured for an additional 4 hours. The culture plate was taken out and centrifuged at 1000 rpm for 10 min. The supernatant of each well was aspirated, and 100 μL of SDS cell lysis solution was added to each well, and cultured overnight. On the next day, the absorbance at 550 nm (OD.sub.550nm) was measured on an enzyme-linked immunoassay analyzer. The results were expressed as the mean OD.sub.550nm value of the triplicates or quadruplicates±standard deviation (x±sd). SPSS software version 17.0 was used for statistical analysis of the mean values, that is, the vehicle group was compared with the stimulant group, and the test group of each compound concentration was compared with the corresponding vehicle control group. The cell survival rate of each group was calculated according to the mean value for the compound treatment group and the mean value for the vehicle control group, that is, the cell survival rate (%)=the mean value for the compound treatment group/the mean value for the vehicle control group×100%. A linear regression equation was obtained by plotting the cell survival rate of each group against the concentration of the test compound, statistical test of the correlation coefficient was conducted, and then the IC.sub.50 value was determined.

(29) The results of the effect of treatment with Compound 23 for 48 hours on 5 μg/mL ConA-induced proliferation of mouse spleen lymphocytes were shown in Table 2. The results of the effect of treatment with cyclosporin A for 48 hours on 5 μg/mL ConA-induced proliferation of mouse spleen lymphocytes were shown in Table 3. The results of the effect of treatment with triptolide for 48 hours on 5 μg/mL ConA-induced proliferation of mouse spleen lymphocytes were shown in Table 4. As can be seen from the data in Tables 2-4, within the range of concentrations tested, Compound 23 had a significant effect on 5 μg/mL ConA-induced proliferation of mouse spleen lymphocytes after 48 hours of treatment, and had an IC.sub.50 value of about 9.41 μM. The control drugs cyclosporine A and triptolide also showed varying degrees of inhibition at different tested concentrations.

(30) TABLE-US-00002 TABLE 2 Number of OD.sub.550nm Survival Linear regression Concentration samples value rate equation and (μg/mL) (n) (x ± sd) (%) IC.sub.50 (μg/mL) 100 4 0.02 ± 0.02**.sup.## 1 y = −48.524 Logx + 50 4 0.02 ± 0.01**.sup.## 2 79.226 25 4 0.02 ± 0.01**.sup.## 2 r = 0.8554 12.5 4 0.07 ± 0.02**.sup.## 6 n = 7, p.sub.2 < 0.05 6.25 4 0.29 ± 0.03**.sup.## 25 IC.sub.50 = 4.00 3.125 4 0.52 ± 0.04**.sup.## 45 (9.41 μM) 1.5625 4 1.18 ± 0.16 101 0.78125 4 1.14 ± 0.11 98 DMSO 4 1.16 ± 0.02 — vehicle group (5.sub.  ) Stimulant 4 1.14 ± 0.07 — control group Cell control 4 0.54 ± 0.03 — group Note: (1) Compared with the stimulant control group, *<0.05; **<0.01 (2) Compared with the vehicle group, .sup.#<0.05; .sup.##<0.01

(31) TABLE-US-00003 TABLE 3 Number of OD.sub.550nm Survival Concentration samples value rate (μg/mL) (n) (x ± sd) (%) 10 4 0.40 ± 0.02**.sup.## 39 0.1 4 0.48 ± 0.03**.sup.## 47 Ethanol vehicle group 3 1.03 ± 0.11 — (1 ) Stimulant control group 4 0.90 ± 0.07 — Cell control group 4 0.53 ± 0.07 Note: (1) Compared with the stimulant control group, *<0.05; **<0.01 (2) Compared with the vehicle group, .sup.#<0.05; .sup.##<0.01

(32) TABLE-US-00004 TABLE 4 Number of OD.sub.550nm Survival Concentration samples value rate (μg/mL) (n) (x ± sd) (%) 0.1 4 0.42 ± 0.03**.sup.## 40 0.001 4 1.03 ± 0.10 98 DMSO vehicle group 3 1.05 ± 0.11 — (0.01 ) Stimulant control group 4 0.90 ± 0.07 — Cell control group 4 0.53 ± 0.07 — Note: (1) Compared with the stimulant control group, *<0.05; **<0.01 (2) Compared with the vehicle group, .sup.#<0.05; .sup.##<0.01

Example 9

(33) In this example, Compound 23 and Compound 24 were detected for their acute toxicities, and the detection method was as follows.

(34) Experimental procedure: 40 BALB/c mice, half male and half female, were randomly divided into groups after weight measurement. The weight of an individual should be within the range of the average body weight±20%. Animals should be allowed to acclimatize to the environment for at least 5 days before the test, and healthy (if female, it must be un-pregnant) mice were selected as test animals. The main inspections during the acclimatization period included: whether it met the quality indicators required at the time of ordering; general state check; and whether the weight reached the weight range required by the test. Unqualified abnormal animals were not included in this test. A single tail-vein injection was administrated to the mice at a low, medium, or high dose, which was 5 mg/kg, 10 mg/kg, or 40 mg/kg respectively according to a blank preparation pre-test. An equal volume of vehicle was intravenously injected to establish a control group.

(35) Observation method: (1) General state observation: Animals were observed for conditions including, but not limited to, appearance signs, administration sites (with or without bleeding, redness, purpura, induration, suppuration, or ulceration), hair coat, general behavior, mental state, gland secretion, color of skin and mucosa, respiratory state, fecal traits, genitals, and death, and other toxic symptoms. Mice were observed once respectively at 0 to 2 hours and 4 to 6 hours after each administration. If toxic symptoms occurred, the number of observations could be increased. (2) Gross anatomy observation: All the surviving mice in each group were dissected and observed on the 8th day of the experiment. The abnormal organs and tissues found in the observation of administration sites and in the gross anatomy observation that might be related to the test samples were photographed and recorded. (3) Disposal of dying animals: The state of the mouse and the observation time were recorded, and the body weight was measured. (4) Disposal of dead animals: The time of death or the time when the death was found was recorded, the body weight was measured, and then the dissection was quickly performed for gross observation to speculate the cause of death.

(36) Experimental results: No obvious toxic side effects were observed, and no significant weight loss and decreased diet were found in BALB/c mice treated with a single intravenous injection of Compound 23 (5 mg/kg, 10 mg/kg, or 40 mg/kg). All animals treated with a single intravenous injection of Compound 24 (10 mg/kg) died. The above results indicated that Compound 23 is safer than Compound 24.

(37) The applicant states that although the glaucocalyxin A derivatives, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the same according to the present application, and their use in the preparation of a medicament for treating psoriasis are illustrated herein through the above embodiments, the present application is not limited to the above embodiments, and it does not mean that the present application must rely on the above embodiments to implement. It should be apparent to those skilled in the art that, for any improvement of the present application, the equivalent replacement of the raw materials selected in the present application, the addition of auxiliary components, and the selection of specific modes, etc., will all fall within the protection scope and the disclosure scope of the present application.