C14-hydroxyl esterified amino acid derivative of triptolide, and preparation method and use thereof

Abstract

The present invention belongs to the field of natural medicines and medicinal chemistry, and relates to novel esterified amino acid derivatives of triptolide of general formula I and general formula II or pharmaceutically acceptable adducts, complexes, salts, and catabolites and metabolites thereof, preparation methods of these compounds, pharmaceutical compositions comprising the compound, and uses thereof in preparing drugs against tumors, immune diseases, or diseases related to abnormal expression of XPB or Pol II or oncogene c-myc. ##STR00001##

Claims

1. A C14-hydroxyl esterified amino acid derivative of triptolide selected from the following compounds: ##STR00018## ##STR00019##

2. A pharmaceutical composition, comprising the C14-hydroxyl esterified amino acid derivative of triptolide according to claim 1, and an optional pharmaceutically acceptable excipient.

3. A pharmaceutically acceptable salt of the C14-hydroxyl esterified amino acid derivative of triptolide according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the results of the immunoblot analysis of XPB and Pol II protein levels in tumor cells and normal cells.

(2) FIG. 2 shows the results of the immunoblot analysis of the inhibitory effects of 14-D-Valine-TPL on the activities of XPB and Pol II in human THP-1 leukemia cells.

(3) FIG. 3 shows the results of the immunoblot analysis of the inhibitory effects of 14-D-Valine-TPL on the activity of c-myc in human K562 leukemia cells.

DETAILED DESCRIPTION OF THE INVENTION

(4) The chemical raw materials used in the following examples are all commercially available or obtained by the synthesis methods well known in the art.

EXAMPLE 1

Synthesis and Identification of Compound 14-D-Valine-TPL (14-Dextro-Valine-Triptolide)

(5) ##STR00014##

(6) In the scheme, TPL: triptolide; D-Boc-Valine: Dextro-(N-Boc)-Valine; DIC: N,N-diisopropylcarbodiimide; DMAP: 4-dimethylaminopyridine; DCM: dichloromethane; TFA: trifluoroacetic acid.

(7) Step 1: Triptolide TPL (500 mg, 1.39 mmol, 1.0 eq.) and Dextro-(N-Boc)-Valine (1500 mg, 6.91 mmol, 5 eq.) were dissolved in dichloromethane (20 mL), and cooled to 0 C. N,N-diisopropylcarbodiimide (4 mL, 13 mmol, 9.35 eq.) and 4-dimethylaminopyridine (130 mg, 1.07 mmol, 0.8 eq.) were added into the mixed solution at 0 C., and reacted for 24 hours at 25 C. The reaction solution was diluted with ethyl acetate and washed with water, and then washed with saturated ammonium chloride, dried and concentrated to give a crude product. The crude product was purified on a preparative silica gel column to produce D-Boc-Valine-TPL (14-Dextro-Boc-Valine-Triptolide) as a white solid (250 mg), with a yield of 32.2%.

(8) Step 2: The product D-Boc-Valine-TPL (14-D-Boc-Valine-Triptolide) (200 mg) in the previous step was dissolved in dichloromethane (15 mL), and 3 mL of trifluoroacetic acid was added dropwise. After the addition, the reaction solution was reacted at 25 C. for 3.5 hours. The reaction solution was diluted with dichloromethane and washed with an aqueous sodium bicarbonate solution, and then dried and concentrated to give a crude product. The crude product was purified by preparative high-performance liquid chromatography to produce D-Valine-TPL (14-Detxtro-Valine-Triptolide) as a white solid (120 mg), with a yield of 73%.

(9) LC-MS: Retention time: 0.83 min (UV220: 97.8%), m/z: 460.28 (M+H). 1H NMR (400 MHz, CDCl3) 5.11 (s, 1H), 4.78-4.62 (m, 2H), 4.01 (s, 1H), 3.86 (d, J=2.7 Hz, 1H), 3.58 (d, J=2.4 Hz, 1H), 3.52 (d, J=4.8 Hz, 1H), 2.72 (d, J=11.9 Hz, 1H), 2.47 (s, 1H), 2.33 (d, J=16.3 Hz, 1H), 2.26-2.08 (m, 2H), 1.94 (dd, J=36.2, 22.1 Hz, 2H), 1.58 (dd, J=12.0, 4.5 Hz, 1H), 1.23 (d, J=5.6 Hz, 4H), 1.16 (d, J=4.8 Hz, 3H), 1.03 (s, 3H), 0.93 (d, J=6.4 Hz, 3H), 0.81 (d, J=6.5 Hz, 3H).

EXAMPLE 2

Synthesis and Identification of Compound 14-L-Valine-D-Valine-TPL (14-Levo-Valine-Dextro-Valine-Triptolide)

(10) ##STR00015##

(11) In the scheme, D-Valine-TPL: 14-Dextro-Valine-Triptolide; L-Boc-Valine: Levo-(N-Boc)-Valine; HATU: 2-(7-aza-benzotriazole)-N,N, N,N-tetramethyluronium hexafluorophosphate; DIPEA: N,N-diisopropylethylamine; DCM: dichloromethane; TFA: trifluoroacetic acid.

(12) Step 1: D-Valine-TPL (14-Dextro-Valine-Triptolide) (110 mg, Example 1) and Levo-(N-Boc)-Valine (1.2 eq.) were dissolved in dichloromethane (10 mL), and HATU (2-(7-aza-benzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate, 1.5 eq.) and DIPEA (N,N-diisopropylethylamine, 2 eq.) were added into the mixed solution, and reacted at 25 C. for half an hour. The reaction solution was washed with a 1M potassium carbonate solution, and dried and concentrated to give a crude product. The crude product was purified on a preparative silica gel column to produce 14-L-Boc-Valine-D-Valine-TPL (14-Levo-(N-Boc)-Valine-Dextro-Valine-Triptolide) as a white solid (85 mg).

(13) Step 2: The product 14-L-Boc-Valine-D-Valine-TPL (14-Levo-(N-Boc)-Valine-Dextro-Valine-Triptolide) (85 mg) in the previous step was dissolved in dichloromethane (5 mL), and 1 mL of trifluoroacetic acid was added dropwise. After the addition, the reaction solution was reacted at 25 C. for 3.5 hours. The reaction solution was diluted with dichloromethane and washed with an aqueous sodium bicarbonate solution, and then dried and concentrated to give a crude product. The crude product was purified by preparative high-performance liquid chromatography to produce 14-L-Valine-D-Valine-TPL (14-Levo-Valine-Dextro-Valine-Triptolide) as a white solid (51.5 mg).

(14) LC-MS: Retention time: 0.96 min (UV220: 97.7%), m/z: 559.44 (M+H). 1H NMR (400 MHz, CDCl3) 7.52 (d, J=7.8 Hz, 1H), 5.60 (b, 3H), 5.06 (s, 1H), 4.80-4.65 (m, 2H), 4.58 (dd, J=7.8, 4.0 Hz, 1H), 4.20 (s, 1H), 3.88 (d, J=2.7 Hz, 1H), 3.57 (d, J=2.2 Hz, 1H), 3.50 (d, J=5.3 Hz, 1H), 2.72 (d, J=11.7 Hz, 1H), 2.47-2.09 (m, 5H), 1.92 (dd, J=17.9, 10.2 Hz, 2H), 1.57 (dd, J=11.5, 5.1 Hz, 1H), 1.25 (dd, J=12.2, 5.5 Hz, 2H), 1.13-1.04 (m, 11H), 1.02 (s, 3H), 0.92 (t, J=8.4 Hz, 3H), 0.83 (d, J=6.7 Hz, 3H).

EXAMPLE 3

Synthesis and Identification of Compound 14-L-Valine-TPL (14-Levo-Valine-Triptolide)

(15) The compound 14-L-Valine-TPL (14-Levo-Valine-Triptolide) was synthesized following the method in Example 1, while replacing D-Boc-Valine-TPL (Dextro-N-Boc-Valine) with L-Boc-Valine (Levo-N-Boc-Valine).

(16) LC-MS: Retention time: 0.96 min (UV220, 100%), m/z: 460.28 (M+H). 1H NMR (300 MHz, CDCl3) 5.10 (d, J=5.4 Hz, 1H), 4.73-4.60 (m, 2H), 3.99 (s, 1H), 3.91-3.79 (m, 1H), 3.63-3.44 (m, 2H), 2.67 (s, 1H), 2.55-1.79 (m, 7H), 1.65-1.47 (m, 1H), 1.31-1.09 (m, 7H), 1.01 (s, 3H), 0.97-0.86 (m, 3H), 0.85-0.72 (m, 3H).

EXAMPLE 4

Synthesis and Identification of Compound 14-L-Valine-L-Valine-TPL (14-Levo-Valine-Levo-Valine-Triptolide)

(17) The compound 14-L-Valine-L-Valine-TPL (14-Levo-Valine-Levo-Valine-Triptolide) was synthesized following the method in Example 2, while replacing 14-D-Valine-TPL (14-Dextro-Valine-Triptolide, Example 1) with 14-L-Valine-TPL (14-Levo-Valine-Triptolide, Example 3), and replacing D-Boc-Valine-TPL (Dextro-N-Boc-Valine) with L-Boc-Valine (Levo-N-Boc-Valine).

(18) LC-MS: Retention time: 1.78 min (ELSD: 99.6%), m/z: 559.50 (M+H). 1H NMR (400 MHz, CDCl3) 7.83 (s, 1H), 5.08 (d, J=7.5 Hz, 1H), 4.75-4.65 (m, 2H), 4.57 (dd, J=8.7, 4.7 Hz, 1H), 3.84 (dd, J=5.8, 3.2 Hz, 1H), 3.53 (ddd, J=17.4, 11.7, 4.1 Hz, 2H), 2.70 (s, 1H), 2.43-2.27 (m, 3H), 2.23-2.12 (m, 2H), 2.04-1.88 (m, 2H), 1.67-1.54 (m, 1H), 1.30-1.20 (m, 1H), 1.12-0.99 (m, 10H), 0.95 (t, J=7.5 Hz, 5H), 0.90-0.77 (m, 3H).

EXAMPLE 5

Synthesis and Identification of Compound 14-D-Valine-D-Valine-TPL (14-Dextro-Valine-Dextro-Valine-Triptolide)

(19) The compound 14-D-Valine-D-Valine-TPL (14-Dextro-Valine-Dextro-Valine-Triptolide) was synthesized following the method in Example 2, while replacing L-Boc-Valine-TPL (Levo-N-Boc-Valine) with D-Boc-Valine (Dextro-N-Boc-Valine).

(20) LC-MS: Retention time: 1.95 min (ELSD: 98.0%), m/z: 559.50 (M+H). 1H NMR (400 MHz, CDCl3) 7.81 (d, J=8.8 Hz, 1H), 5.08 (s, 1H), 4.69 (s, 2H), 4.61 (dd, J=8.8, 4.5 Hz, 1H), 3.84 (d, J=3.2 Hz, 1H), 3.57 (d, J=2.7 Hz, 1H), 3.49 (d, J=5.8 Hz, 2H), 2.74-2.68 (m, 1H), 2.39-2.27 (m, 3H), 2.22-2.10 (m, 2H), 1.94 (ddd, J=25.7, 16.2, 10.6 Hz, 2H), 1.59 (dd, J=12.4, 4.6 Hz, 1H), 1.28-1.20 (m, 1H), 1.06 (dt, J=12.6, 6.1 Hz, 12H), 0.95 (dd, J=12.7, 6.9 Hz, 6H), 0.85 (d, J=6.9 Hz, 3H).

EXAMPLE 6

Synthesis and Identification of Compound 14-L-Phenylalanyl-L-Tyrosine-TPL (14-Levo-Phenylalanyl-Levo-Tyrosine-Triptolide)

(21) ##STR00016## ##STR00017##

(22) In the scheme, TPL: triptolide; L-Boc-O-TBDMS-Tyrosine: Levo-(N-Boc-O-t-butyldimethyllsilyl)-Tyrosine; DIC: N,N-diisopropylcarbodiimide; DMAP: 4-dimethylaminopyridine; DCM: dichloromethane; TFA: trifluoroacetic acid. TBAF: tetrabutylammonium fluoride;

(23) D-Valine-TPL: 14-Dextro-Valine-Triptolide; L-Boc-Phenylalanine: Levo-(N-Boc)-Phenylalanine; HATU: 2-(7-aza-benzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate; DIPEA: N,N-diisopropylethylamine;

(24) Compound L-Tyrosine-TPL (14-Levo-Tyrosine-Triptolide) was synthesized following the method in Example 1, while replacing D-Boc-Valine-TPL (Dextro-N-Boc-Valine) with L-Boc-O-TBDMS-Tyrosine: Levo-(N-Boc-O-t-butyldimethyllsilyl)-Tyrosine.

(25) The compound 14-L-Phenylalanyl-L-tyrosine-TPL (14-Levo-Phenylalanyl-Levo-tyrosine-Triptolide) was synthesized following the method in Example 2, while replacing 14-D-Valine-TPL (14-Dextro-Valine-Triptolide, Example 1) with the compound L-Tyrosine-TPL (14-Levo-Tyrosine-Triptolide), and replacing D-Boc-Valine-TPL (Dextro-N-Boc-Valine) with L-Boc-Phenylalanine (Levo-(N-Boc)-Phenylalanine).

(26) LC-MS: Retention time: 1.06 min (UV220: 97.8.0%), m/z: 671.56 (M+H). 1H NMR (400 MHz, DMSO) 9.34 (s, 0H), 9.17 (s, 0H), 8.07 (d, J=15.1 Hz, 1H), 7.42-7.14 (m, 2H), 7.13-7.03 (m, 1H), 6.89 (d, J=7.6 Hz, 0H), 6.74-6.62 (m, 1H), 5.01 (d, J=6.3 Hz, 0H), 4.82 (d, J=17.7 Hz, 1H), 4.64-4.58 (m, 0H), 4.03 (d, J=3.2 Hz, 1H), 3.94 (d, J=3.2 Hz, 0H), 3.25 (d, J=14.2 Hz, 0H), 3.15 (s, 0H), 3.05 (dd, J=13.9, 6.2 Hz, 0H), 2.94-2.77 (m, 1H), 2.64 (s, 1H), 2.31-2.07 (m, 1H), 1.92 (d, J=24.5 Hz, 1H), 1.87-1.72 (m, 1H), 1.66-1.53 (m, 1H), 1.38-1.17 (m, 1H), 1.04 (d, J=6.8 Hz, 0H), 0.97-0.88 (m, 2H), 0.80 (dd, J=6.7, 3.8 Hz, 1H), 0.65 (d, J=6.8 Hz, 3H).

EXAMPLE 7

Comparison of Therapeutic Windows Between the Compound 14-D-Valine-TPL of the Invention and Triptolide (TPL)

(27) (1) Experimental Materials

(28) Leukemia cell strains: Sup-B15 (Ph+acute lymphoblastic leukemia), CEM (acute lymphoblastic leukemia, ALL), and Molt-4 (acute lymphoblastic leukemia, ALL). Normal blood cell samples: peripheral blood from healthy volunteers. Primary reagent: 14-D-Valine-TPL of the invention. Primary instruments: cell incubator and microplate reader.
(2) Experimental Method

(29) 5000 well-grown leukemia cells or normal human blood cells were taken and seeded into wells of a 96-well cell culture plate. The medium was 1640 cell medium containing 10% fetal bovine serum. 14-D-Valine-TPL with different concentrations was added, and after being uniformly mixed, the plate was placed in a carbon dioxide (5% CO2) cell incubator, and incubated at 37 C. for 72 hours. The concentration of live cells was then determined using the MTT assay. In this experiment, the cell viability in the control group (without the compound treatment) was set to be 100%, and the cell viability (%) after the action of the compound the half maximal growth inhibitory concentration of leukemia cells at 72 hours (IC50 value at 72 hours) and the therapeutic window index were calculated. Therapeutic window index=IC50 value of normal blood cells/IC50 value of leukemia cells. The therapeutic window index of < or =1 indicates that there is no therapeutic window, and the compound is non-selective with the same toxicity to normal cells and tumor cells. The therapeutic window index of >1 indicates that there is a therapeutic window, and the compound is more toxic to tumor cells than to normal cells. A larger therapeutic window indicates a better selectivity of the compound for tumor cells.

(30) (3) Experimental Results

(31) The experimental results are shown in Table 1, which indicates that the compound 14-D-Valine-TPL of the invention has a better therapeutic window, while triptolide has substantially no therapeutic window. Table 1 shows that triptolide (TPL) had a small therapeutic window (2.0) only for Molt-4 cells, while its therapeutic windows for other 3 leukemia cells, Jurkat, CEM and Sup-B15, were all <1, which were 0.64, 0.93 and 0.94, respectively. These indicates that triptolide has substantially no selectivity for normal cells and tumor cells, which is consistent with the result reported in the literatures that TPL has substantially no therapeutic window. In contrast, 14-D-Valine-TPL of the invention has therapeutic window indices of 5.54-18.58 for 4 different types of human leukemia cells that have been assayed, indicting that 14-D-Valine-TPL has a larger therapeutic window for all 4 types of leukemia, with a therapeutic window index for Molt-4 cells being up to 18.58.

(32) TABLE-US-00002 TABLE 1 Comparison of the therapeutic windows between 14-D-Valine-TPL and triptolide (TPL) 14-D-Valine-TPL TPL Therapeutic Therapeutic IC50 (nM/L) window IC50 window Leukemia Cell Line Jurkat 36.301 5.54 33.829 0.64 CEM 25.488 7.89 23.286 0.93 Molt-4 10.824 18.58 10.718 2.03 Sup-B15 20.674 9.73 23.153 0.94 Normal Blood Cell No1 202.706 29.289 No2 198.826 20.772 No3 205.784 9.383 No4 199.835 8.968 No5 213.719 20.072 No6 189.872 20.445 No7 203.546 19.515 No8 184.509 9.710 No9 210.835 57.443 Overall 201.07 21.733

EXAMPLE 8

Determination of in vitro Activity of the Compound 14-D-Valine-TPL of the Invention Against Leukemia

(33) (1) Experimental Materials

(34) Leukemia cell strains: human KG-1a (acute myelogenous leukemia, AML-MO), THP-1 (acute myelogenous leukemia, AML-M5), NB4 (acute promyelocytic leukemia, AML), Kasumi-1 (acute myelogenous leukemia M2 type, AML-M2), KG-1 (acute myelogenous leukemia, AML), Jurkat (acute lymphoblastic leukemia, ALL), and H9 (acute lymphoblastic leukemia, ALL). Primary reagent: 14-D-Valine-TPL of the invention. Primary instruments: cell incubator and microplate reader.
(2) Experimental Method

(35) 5000 well-grown leukemia cells were taken and seeded into wells of a 96-well cell culture plate. The medium was 1640 cell medium containing 10% fetal bovine serum. 14-D-Valine-TPL with different concentrations was added, and after being uniformly mixed, the plate was placed in a carbon dioxide (5% CO.sub.2) cell incubator, and incubated at 37 C. for 72 hours. The concentration of live cells was then determined using the MTT assay. In this experiment, the cell viability in the control group (without the compound treatment) was set to be 100%, and the cell viability (%) after the action of the compound and the half maximal growth inhibitory concentration of leukemia cells at 72 hours (IC.sub.50 value at 72 hours) were calculated.

(36) (3) Experimental Results

(37) Experimental results are shown in Table 2. Table 2 shows that 14-D-Valine-TPL of the invention can induce the death of human acute myelogenous leukemia cells and acute lymphoblastic leukemia cells, and inhibit the growth of these leukemia cells.

(38) TABLE-US-00003 TABLE 2 Inhibitory effects of the derivatives of the invention on the growth of different types of leukemia cells 14-L-Val-14-L-Val- 14-D-Val-TPL 14-L-Val-TPL TPL Cell Line IC50 (nM/L) IC50 (nM/L) IC50 (nM/L) KG-1 49.0 8.86 KG-1a 31.0 20 THP-1 16.0 5.0 HL-60 65.8 U937 60.0 Jurkat 36.301 CEM 25.488 Molt-4 10.824 Sup-B15 20.674

EXAMPLE 9

Determination of in vivo Activity of the Compound 14-D-Valine-TPL of the Invention Against Human Leukemia in Mice

(39) (1) Experimental Materials

(40) Experiment animal: NOD/SCID mice obtained from the Shanghai Animal Center of the Chinese Academy of Sciences (CAS). Leukemia cell strains: human KG-1a (acute myelogenous leukemia, AML-MO), and THP-1 (acute monocytic leukemia, AML-M5), obtained from ATCC Library. Reagent: 14-D-Valine-TPL trifluoroacetate of the invention, dissolved with sterile PBS to a concentration of 10 mg/ml, and then diluted with sterile deionized water to a required working concentration. Primary instruments: cell incubator and microplate reader.
(2) Experimental Method

(41) 7-week old female NOD/SCID mice were taken and inoculated subcutaneously with 110.sup.7 cells (0.2 ml) per mouse in axilla, with human acute myelogenous leukemia KG-1a cells on the left and human acute monocytic leukemia THP-1 cells on the right. When tumors grew to a tumor size of around 0.5 cm, the mice were randomized into groups with 3 mice each. An equivalent amount of PBS was used for the control group, and the 14-D-Valine-TPL group was divided into two groups with 0.2 mg/kg body weight and 0.4 mg/kg body weight: intragastric administration 2 times a day (one at 8 a.m. and one at 4 p.m.) for consecutive 14 days of treatment. An observation was made for total 30 days, during which the conditions of the mice such as tumor size, weight, activity, diet, and the like were measured. At the end of the experiment, the mice were sacrificed, tumor tissues were taken and the tumors were weighed, and major organs and tissues of the mice such as liver, spleen, heart, lung, large intestine, small intestine, and the like were examined.

(42) (3) Experimental Results

(43) Experimental results are shown in Table 3 and Table 4.

(44) TABLE-US-00004 TABLE 3 Effects of 14-D-Valine-TPL (DV) on the growth of human acute myelogenous leukemia KG-1a cells in mice in vivo KG-1a Tumor weight (g) Body weight (g) Mouse Control DV DV Control DV DV No. group 0.2 0.4 group 0.2 0.4 1 4.06 0.5 0 23.0 (15.11) 20.4 19.1 2 4.14 0.42 0 22.3 (15.38) 18.5 20.4 3 3.06 1.49 0 23.0 (15.97) 20.2 21.0 4 0.06 0 19.9 21.1 5 0.98 0 18.1 21.4 6 0.2 0 19.0 20.8

(45) TABLE-US-00005 TABLE 4 Effects of 14-D-Valine-TPL (DV) on the growth of human acute myelogenous leukemia THP-1 cells in mice in vivo THP-1 Tumor weight (g) Body weight (g) Mouse Control DV DV Control DV DV No. group 0.2 0.4 group 0.2 0.4 1 3.83 0.28 0 23.0 (19.17) 20.4 19.1 2 2.78 0 0 22.3 (19.52) 18.5 20.4 3 3.97 0 0 23.0 (19.03) 20.2 21.0 0.21 0 19.9 21.1 0.06 0 18.1 21.4 0 0 19.0 20.8 Note: DV 0.2: 0.2 mg/kg; DV 0.4: 0.4 mg/kg.

(46) Table 3 and Table 4 show that 14-D-Valine-TPL of the invention can significantly inhibit the growth of two different types of human acute myelogenous leukemia cell lines KG-1a and THP-1 in mice in vivo, and exhibits a significant dose-dependent effect. 14-D-Valine-TPL with a dose of 0.4 mg per kilogram of the body weight enables a complete regression of KG-1a and THP-1 xenograft tumors, with no significant weight losses in the mice. Further, no significant abnormalities were seen in major organs such as heart, lung, liver, spleen, large intestine, small intestine and the like in anatomical examination of the mice. These results indicate that 14-D-Valine-TPL of the invention has a significant activity against different types of human acute myelogenous leukemia, and shows no significant toxic reaction.

EXAMPLE 10

Determination of in vivo Activity of 14-L-Valine-L-Valine-TPL (14-LLV-TPL) Against Human Leukemia in Mice

(47) (1) Experimental Materials

(48) Experiment animal: NOD/SCID mice obtained from the Shanghai Animal Center of the Chinese Academy of Sciences (CAS). Leukemia cell strain: human KG-1a (acute myelogenous leukemia, AML-M0), obtained from ATCC Library. Reagent: 14-LLV-TPL trifluoroacetate of the invention, dissolved with sterile PBS to a concentration of 10 mg/ml, and then diluted with sterile deionized water to a required working concentration. Primary instruments: cell incubator and microplate reader.
(2) Experimental Method

(49) 7-week old female NOD/SCID mice were taken and inoculated subcutaneously with 110.sup.7 cells (0.2 ml) per mouse in axilla, with human acute myelogenous leukemia KG-1a cells on the left. When tumors grew to a tumor size of around 50-100 mm.sup.3, the mice were randomized into groups with 3 mice each. An equivalent amount of PBS was used for the control group, and the TPL-LLV group was divided into two groups with 0.23 mg/kg body weight and 0.46 mg/kg body weight: intragastric administration, 2 times a day (one at 8 a.m. and one at 4 p.m.) for consecutive 14 days of treatment. An observation was made for total 30 days, during which the conditions of the mice such as tumor size, weight, activity, diet, and the like were measured. At the end of the experiment, the mice were sacrificed, tumor tissues were taken and the tumors were weighed, and major organs and tissues of the mice such as liver, spleen, heart, lung, large intestine, small intestine, and the like were examined.

(50) (3) Experimental Results

(51) Experimental results are shown in Table 5. Table 5 shows that 14-LLV-TPL of the invention can significantly inhibit the growth of human acute myelogenous leukemia cell line KG-1a in mice in vivo, and exhibits a significant dose-dependent effect.

(52) TABLE-US-00006 TABLE 5 Effects of 14-LLV-TPL on the growth of human acute myelogenous leukemia KG-1a cells in mice in vivo THP-1 Tumor volume (mm.sup.3) Body weight (g) Mouse Control LLV Control LLV No. group 0.23 LLV0.46 group 0.23 LLV0.46 1 1251.54 1105.64 317.61 19.8 20.5 18.6 2 1196.78 804.56 383.11 18.8 19.4 20.0 3 2628.39 644.07 356.82 19.1 19.7 18.9 Overall 1692.24 851.42 352.51 19.2 19.9 19.2 811.2 243.33 32.96 0.51 0.57 0.74

EXAMPLE 11

Determination of in vivo Activity of 14-L-Valine-D-Valine-TPL (14-DLV-TPL) Against Human Leukemia in Mice

(53) (1) Experimental Materials

(54) Experiment animal: NOD/SCID mice obtained from the Shanghai Animal Center of the Chinese Academy of Sciences (CAS). Leukemia cell strain: human KG-1a (acute myelogenous leukemia, AML-M0), obtained from ATCC Library. Reagent: 14-DLV-TPL trifluoroacetate of the invention, dissolved with sterile PBS to a concentration of 10 mg/ml, and then diluted with sterile deionized water to a required working concentration. Primary instruments: cell incubator and microplate reader.
(2) Experimental Method

(55) 7-week old female NOD/SCID mice were taken and inoculated subcutaneously with 110.sup.7 cells (0.2 ml) per mouse in axilla, with human acute myelogenous leukemia KG-1a cells on the left. When tumors grew to a tumor size of around 50-100 mm.sup.3, the mice were randomized into groups with 3 mice each. An equivalent amount of PBS was used for the control group, and the TPL-DLV group was divided into two groups with 0.23 mg/kg body weight and 0.46 mg/kg body weight: intragastric administration, 2 times a day (one at 8 a.m. and one at 4 p.m.) for consecutive 14 days of treatment. An observation was made for total 30 days, during which the conditions of the mice such as tumor size, weight, activity, diet, and the like were measured. At the end of the experiment, the mice were sacrificed, tumor tissues were taken and the tumors were weighed, and major organs and tissues of the mice such as liver, spleen, heart, lung, large intestine, small intestine, and the like were examined.

(56) (3) Experimental Results

(57) Experimental results are shown in Table 6. Table 6 shows that 14-DLV-TPL of the invention can significantly inhibit the growth of human acute myelogenous leukemia cell line KG-1a in mice in vivo, and exhibits a significant dose-dependent effect.

(58) TABLE-US-00007 TABLE 6 Effects of 14-DLV-TPL on the growth of human acute myelogenous leukemia KG-1a cells in mice in vivo THP-1 Tumor volume (mm.sup.3) Body weight (g) Mouse Control DLV DLV Control DLV DLV No. group 0.23 0.46 group 0.23 0.46 1 1251.54 1038.41 50.35 19.8 19.6 18.8 2 1196.78 562.66 90.96 18.8 19.5 19.6 3 2628.39 505.81 265.32 19.1 17.8 18.5 Overall 1692.24 702.29 135.54 19.2 19.0 19.0 811.2 192.47 114.21 0.51 1.01 0.57

EXAMPLE 12

Determination of in vivo Activity of 14-L-Phenylalanyl-L-Tyrosine-TPL (14-LPT-TPL) Against Human Leukemia in Mice

(59) (1) Experimental Materials

(60) Experiment animal: NOD/SCID mice obtained from the Shanghai Animal Center of the Chinese Academy of Sciences (CAS). Leukemia cell strain: human KG-1a (acute myelogenous leukemia, AML-M0), obtained from ATCC Library. Reagent: 14-LPT-TPL trifluoroacetate of the invention, dissolved with sterile PBS to a concentration of 10 mg/ml, and then diluted with sterile deionized water to a required working concentration. Primary instruments: cell incubator and microplate reader.
(2) Experimental Method

(61) 7-week old female NOD/SCID mice were taken and inoculated subcutaneously with 110.sup.7 cells (0.2 ml) per mouse in axilla, with human acute myelogenous leukemia KG-1a cells on the left. When tumors grew to a tumor size of around 50-100 mm.sup.3, the mice were randomized into groups with 3 mice each. An equivalent amount of PBS was used for the control group, and the TPL-DLV group was divided into two groups with 0.32 mg/kg body weight and 0.64 mg/kg body weight: intragastric administration, 2 times a day (one at 8 a.m. and one at 4 p.m.) for consecutive 14 days of treatment. An observation was made for total 30 days, during which the conditions of the mice such as tumor size, weight, activity, diet, and the like were measured. At the end of the experiment, the mice were sacrificed, tumor tissues were taken and the tumors were weighed, and major organs and tissues of the mice such as liver, spleen, heart, lung, large intestine, small intestine, and the like were examined.

(62) (3) Experimental Results

(63) Experimental results are shown in Table 7. Table 7 shows that 14-LPT-TPL of the invention can significantly inhibit the growth of human acute myelogenous leukemia cell line KG-1a in mice in vivo, and exhibits a significant dose-dependent effect.

(64) TABLE-US-00008 TABLE 7 Effects of 14-LPT-TPL on the growth of human acute myelogenous leukemia KG-1a cells in mice in vivo THP-1 Tumor volume (mm.sup.3) Body weight (g) Mouse Control LPT LPT Control LPT LPT No. group 0.32 0.64 group 0.32 0.64 1 1251.54 131.17 0 19.8 21.4 17.4 2 1196.78 168.07 0 18.8 20 19.4 3 2628.39 93.12 0 19.1 20.4 16.1 Overall 1692.24 130.78 0 19.2 20.6 17.6 811.2 37.48 0.72 1.01 1.66

EXAMPLE 13

Determination of in vivo Activity of 14-D-Valine-D-Valine-TPL (14-DDV-TPL) Against Human Leukemia in Mice

(65) (1) Experimental Materials

(66) Experiment animal: NOD/SCID mice obtained from the Shanghai Animal Center of the Chinese Academy of Sciences (CAS). Leukemia cell strain: human K562/ADR (chronic myelogenous leukemia, CML), obtained from ATCC Library. Reagent: 14-DDV-TPL trifluoroacetate of the invention, dissolved with sterile PBS to a concentration of 10 mg/ml, and then diluted with sterile deionized water to a required working concentration. Primary instruments: cell incubator and microplate reader.

(67) (2) Experimental Method

(68) 7-week old female NOD/SCID mice were taken and inoculated subcutaneously with 110.sup.7 cells (0.2 ml) per mouse in axilla, with human K562/ADR cells on the left. When tumors grew to a tumor size of around 50-100 mm.sup.3, the mice were randomized into groups with 3 mice each. An equivalent amount of PBS was used for the control group, and the TPL-DDV group was divided into two groups with 0.23 mg/kg body weight and 0.46 mg/kg body weight: intragastric administration, 2 times a day (one at 8 a.m. and one at 4 p.m.) for consecutive 14 days of treatment. An observation was made for total 30 days, during which the conditions of the mice such as tumor size, weight, activity, diet, and the like were measured. At the end of the experiment, the mice were sacrificed, tumor tissues were taken and the tumors were weighed, and major organs and tissues of the mice such as liver, spleen, heart, lung, large intestine, small intestine, and the like were examined.

(69) (3) Experimental Results

(70) Experimental results are shown in Table 8. Table 8 shows that 14-DDV-TPL of the invention can significantly inhibit the growth of human chronic myelogenous leukemia cell line K562/ADR cells in mice in vivo, and exhibits a significant dose-dependent effect.

(71) TABLE-US-00009 TABLE 8 Effects of 14-DDV-TPL on the growth of human chronic myelogenous leukemia K562/ADR cells in mice in vivo K562/ADR Tumor volume (mm.sup.3) Body weight (g) Mouse Control DDV DDV Control DDV DDV No. group 0.23 0.46 group 0.23 0.46 1 209.97 0 0 21 19.1 19.3 2 1059.16 0 0 20.3 19.7 20.5 3 0 0 0 16.3 21.2 23.2 Overall 423.04 0 0 19.2 20.0 21.0 560.81 2.54 1.08 2.0

EXAMPLE 14

Determination of in vivo Activity of 14-L-Valine-TPL (14-LV-TPL) Against Human Leukemia in Mice

(72) (1) Experimental Materials

(73) Experiment animal: NOD/SCID mice obtained from the Shanghai Animal Center of the Chinese Academy of Sciences (CAS). Leukemia cell strain: human K562/ADR (chronic myelogenous leukemia, CML), obtained from ATCC Library. Reagent: 14-LV-TPL trifluoroacetate of the invention, dissolved with sterile PBS to a concentration of 10 mg/ml, and then diluted with sterile deionized water to a required working concentration. Primary instruments: cell incubator and microplate reader.
(2) Experimental Method

(74) 7-week old female NOD/SCID mice were taken and inoculated subcutaneously with 110.sup.7 cells (0.2 ml) per mouse in axilla, with human K562/ADR cells on the left. When tumors grew to a tumor size of around 50-100 mm.sup.3, the mice were randomized into groups with 3 mice each. An equivalent amount of PBS was used for the control group, and the TPL-LV group was divided into two groups with 0.2 mg/kg body weight and 0.4 mg/kg body weight: intragastric administration, 2 times a day (one at 8 a.m. and one at 4 p.m.) for consecutive 14 days of treatment. An observation was made for total 30 days, during which the conditions of the mice such as tumor size, weight, activity, diet, and the like were measured. At the end of the experiment, the mice were sacrificed, tumor tissues were taken and the tumors were weighed, and major organs and tissues of the mice such as liver, spleen, heart, lung, large intestine, small intestine, and the like were examined.

(75) (3) Experimental Results

(76) Experimental results are shown in Table 9. Table 9 shows that 14-LV-TPL of the invention can significantly inhibit the growth of human chronic myelogenous leukemia cell line K562/ADR cells in mice in vivo, and exhibits a significant dose-dependent effect.

(77) TABLE-US-00010 TABLE 9 Effects of 14-LV-TPL on the growth of human chronic myelogenous leukemia K562/ADR cells in mice in vivo K562/ADR Tumor volume (mm.sup.3) Body weight (g) Mouse Control LV LV Control LV LV No. group 0.2 0.4 group 0.2 0.4 1 209.97 159.87 0 21 22 21.1 2 1059.16 73.63 30.71 20.3 18 21.2 3 0 0 96.79 16.3 22.2 20.8 423.04 77.83 42.50 19.2 20.7 21.0 560.81 80.02 49.46 2.54 2.37 0.21

EXAMPLE 15

Determination of the Activity of 14-D-Valine-TPL for Inhibiting T Lymphocytes and B Lymphocytes in vitro

(78) (1) Experimental Materials

(79) Leukemia cell strains: Jurkat (T lymphocyte), H9 (T lymphocyte), CEM (B lymphocyte), and Sup-B15 (B lymphocyte). Primary reagent: 14-D-Valine-TPL of the invention. Primary instruments: cell incubator and microplate reader.
(2) Experimental Method

(80) 5000 well-grown lymphocytes were taken and seeded into wells of a 96-well cell culture plate. The medium was 1640 cell medium containing 10% fetal bovine serum. 14-D-Valine-TPL with different concentrations was added, and after being uniformly mixed, the plate was placed in a carbon dioxide (5% CO.sub.2) cell incubator, and incubated at 37 C. for 72 hours. The concentration of live cells was then determined using the MTT assay. In this experiment, the cell viability in the control group (without the compound treatment) was set to be 100%, and the cell viability (%) after the action of the compound and the half maximal growth inhibitory concentration of leukemia cells at 72 hours (IC.sub.50 value at 72 hours) were calculated.

(81) (3) Experimental Results

(82) Experimental results are shown in Table 10. Table 10 shows that 14-D-Valine-TPL of the invention can significantly inhibit the growth of these T lymphocytes and B lymphocytes.

(83) TABLE-US-00011 TABLE 10 Inhibitory effects of 14-D-Valine-TPL on T/B lymphocytes proliferation 14-D-Valine-TPL Immune Cell Line IC50 (nM/L) Jurkat (T lymphocyte) 36.301 CEM (B lymphocyte) 25.488 Molt-4 (T lymphocyte) 10.824 Sup-B15 (B lymphocyte) 20.674

EXAMPLE 16

Expression of Transcription Factor TFIIH Subunit XPB and RNA Polymerase II (Pol II) in Different Types of Tumor Cells and Normal Cells

(84) It has been reported in some literatures that transcription factor TFIIH subunit XPB (xeroderma pigmentosum group B) and RNA polymerase II (Pol II) are the key target molecules for triptolide to exert its pharmacological effect [Titov D V, et al., XPB, a subunit of TFIIH, is a target of the natural product triptolide. Nature chemical biology. 2011, 7:182-188]. In order to know whether the expression levels of XPB and Pol II in tumor cells and normal cells are different, the present invention adopts the immunoblot technique to detect the protein levels of XPB and Pol II in different types of tumor cells and normal cells.

(85) (1) Experimental Materials

(86) Leukemia cell strains: Nalm6 (lymphoma), Jurkat (acute lymphoblastic leukemia), THP-1 (acute monocytic leukemia), KG-1a (acute myelogenous leukemia), HL-60 (acute myelogenous leukemia), NB4 (acute promyelocytic leukemia), and U937 (acute monocytic leukemia). Normal blood cell samples were obtained from volunteers.

(87) (2) Experimental Method: the Conventional Immunoblot Technique.

(88) Cellular proteins of leukemia cell and normal cell samples were extracted according to conventional methods, and separated through SDS-PAGE protein electrophoresis, and then transferred to NC membrane. Experimental procedures such as incubation with primary antibody and secondary antibody, development, exposure, and the like were performed according to conventional methods. GAPDH served as an internal reference protein. Results: As shown in FIG. 1, XPB and Pol II proteins were highly expressed in most tumor cells, and were expressed at low levels or not expressed in normal blood cells. This result indicates that the diseases related to abnormally high expression of XPB and Pol II may serve as the indications of triptolide and derivatives thereof.

(89) FIG. 1 shows the results of the expression levels of XPB and Pol II in different types of tumor cells and normal cells detected by adopting the immunoblot technique. 1: normal blood cells; 2: Nalm6; 3: Jurkat; 4: THP-1; 5: KG-1a; 6: HL-60; 7: NB4; 8: U937.

EXAMPLE 17

Inhibitory Effects of 14-D-Valine-TPL on the Activities of XPB and Pol II in Human THP-1 Leukemia Cells

(90) XPB and Pol II are known to be the key target molecules for triptolide to exert its pharmacological effect [Titov D V, et al., XPB, a subunit of TFIIH, is a target of the natural product triptolide. Nature chemical biology. 2011, 7:182-188]. In order to know whether the compounds of the invention have the activity to inhibit these two target molecules, the present invention adopts the cell culture technique and immunoblot technique to detect the effect of 14-D-Valine-TPL on the activities of XPB and Pol II in leukemia cells.

(91) (1) Experimental Materials

(92) Leukemia cell strain: human THP-1 leukemia cell strain (acute myelogenous leukemia-M5, AML-M5). Reagent: 14-D-Valine-TPL.
(2) Experimental Method

(93) Well-grown leukemia cells were taken and seeded into wells of a 6-well cell culture plate, with a density of 110.sup.6/ml. The medium was 1640 cell medium containing 10% fetal bovine serum. 14-D-Valine-TPL with different concentrations was added, and after being uniformly mixed, the plate was placed in a carbon dioxide (5% CO.sub.2) cell incubator, and incubated at 37 C. for 48 hours. Cellular proteins were then extracted, and the immunoblot technique was used to detect the expression levels of XPB and Pol II.

(94) (3) Experimental Results

(95) Experimental results are shown in FIG. 2. 14-D-Valine-TPL down-regulated the levels of XPB and Pol II in a dose-dependent manner. It can be seen that 14-D-Valine-TPL significantly inhibited the levels of XPB and Pol II in THP-1 cells at a concentration of 160 nM. These results indicate that the 14-D-Valine-TPL compound of the invention can inhibit the activities of XPB and Pol II, and is useful for the diseases related to abnormally high expressions of XPB and Pol II.

(96) FIG. 2 is the results of the inhibitory effects of 14-D-Valine-TPL on the activities of XPB and Pol II in human THP-1 leukemia cells detected by adopting the cell culture technique and the immunoblot technique.

EXAMPLE 18

Inhibitory Effects of 14-D-Valine-TPL on the Activity of c-Myc Oncogene in Human K562 Leukemia Cells

(97) Oncogene c-myc is known to also be one of the key target molecules for triptolide to exert its pharmacological effect [Stphan Visp, et al., Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA. Mol Cancer Ther 2009; 8:2780-2790]. In order to know whether the compounds of the invention have the activity to inhibit c-myc, the present invention adopts the cell culture technique and immunoblot technique to detect the effect of 14-D-Valine-TPL on the activity of c-myc in leukemia cells.

(98) (1) Experimental Materials

(99) Leukemia cell strain: human K562 leukemia cell strain (acute transformation of chronic myelogenous leukemia). Reagent: 14-D-Valine-TPL.
(2) Experimental Method

(100) Well-grown leukemia cells were taken and seeded into wells of a 6-well cell culture plate, with a density of 110.sup.6/ml. The medium was 1640 cell medium containing 10% fetal bovine serum. 14-D-Valine-TPL with different concentrations was added, and after being uniformly mixed, the plate was placed in a carbon dioxide (5% CO.sub.2) cell incubator, and incubated at 37 C. for 48 hours. Cellular proteins were then extracted, and the immunoblot technique was used to detect the expression level of c-myc.

(101) (3) Experimental Results

(102) Experimental results are shown in FIG. 3. 14-D-Valine-TPL down-regulated the level of c-myc protein in a dose-dependent manner. It can be seen that 14-D-Valine-TPL significantly inhibited the level of c-myc in K562 cells at a concentration of 80 nM. This result indicates that the 14-D-Valine-TPL compound of the invention can inhibit the activity of oncogene c-myc in tumor cells, and is useful for the diseases related to abnormally high expression of c-myc.