WO2000063212A1

WO2000063212A1 - Derivatives of triptolide, and preparation and uses thereof

Info

Publication number: WO2000063212A1
Application number: PCT/CN1999/000123
Authority: WO
Inventors: Dayuan Wang; Xiaoping Gao; Wenwu Li; Bogang Li
Original assignee: Chengdu Diao Pharmaceutical Group Co Ltd; W & K International Inc
Current assignee: Chengdu Diao Pharmaceutical Group Co Ltd; W & K International Inc
Priority date: 1999-04-16
Filing date: 1999-08-20
Publication date: 2000-10-26
Anticipated expiration: 2001-10-16
Also published as: AU5368699A

Abstract

The present invention provides a compound having Formula (1) wherein R1 is H, an alkyl containing 1-4 carbon atoms, -Ac, -C(=O)(CH2)nCO2, or an amino acid, wherein n is an integer of 1-4; R2 is -SCN, -NCS. The compounds significantly inhibit tumor cell growth both in vitro and in vivo.

Description

Derivatives of Triptolide, and Preparation and Uses Thereof

Technical Field of the Invention The present invention relates to derivatives of triptolide, the method for producing the same and their uses in treatment of cancer diseases. The present invention also relates to the pharmaceutical compositions containing the derivatives of the invention.

Background of the Invention Lei Gong Teng (Tripterygium wilfordii Hook F) is an herb with a variety of bioactivities, which can be used in traditional Chinese medicine. Several laboratories have demonstrated its anti-cancer activity. Kopachan et al. (Kopachan, SM, William AC, Richard GD et al., J. Am. Chem. Soc, 1972:94(2): 7194) isolated from Lei Gong Ten a diterpine containing epoxy (Triptolide) and demonstrated that 0.2 and 0.25 mg/kg of triptolide (intrapertoneal injection) can prolong the life time of L₆₁₅ leukemia mice. 1 ng/ml of triptolide can inhibit in vitro proliferation of KB cell of nasopharyngeal carcinoma (Zhang Qinmu et al, Zhong Guo Yao Li Xue Bao. 1981 ; 2:128). Triptolide inhibit colony formation of breast or stomach cancer cells to a similar extend as that of human leukemia HL-60 cells, and the IC₅₀ is 0.504 -1.22μg/L. The experiment on effect of triptolide on cytokinetic of Hela cells indicates that triptolide has cytocidal effects on phase-synchronized cells, and cells at the S phase are most sensitive to triptolide. It has been demonstrated that triptolide inhibits synthesis of RNA and protein, selectively inactivates_sulfhydryl of phosphofructokinase, inhibits synthesis of liver glycogen, interferes with DNA replication (Xu Jianhua et al, Zhong Guo Yao Li Xue Bao. 1989; 10:550). Owing to epoxy diterpine isolated from Lei Gong Teng have multiple bioactivities, it has drawn great attention among scientists. The chemical synthesis, structure modification and bioactivities of it have been extensively investigated (Berchtold GA et al, J. Am. Chem. Soc. 1980; 102:1200). Synthesized triptolide was achieved in the early 1980s, but it involves numerous reaction steps and strict reaction conditions.

Yu Dequan et al (Yu Dequan et al, Yao Xue Xue Bao. 1992; 27:11) made structure modification of triptolide and obtained a number of valuable analogs. Because the effective bioactive dosage of triptolide is closed to its LD₅₀, it can not be applied to clinical practice. Due to unique immunomodulation mechanism of triptolide compared with other natural agents, currently most of researches are focused on its immunosuppressing effects, and few of them relate to its anti-cancer effects. The advancement in the field of molecular oncology allows the use of oncogene as a novel target to which an anticancer drug directs. Bcl-2 is an oncogenic protein that inhibits programmed cell death. It involves the physiologic and pathological processes in organisms. Bcl-2 was also found to suppress the activation of caspases and became an important target for the therapy of tumors.

Apoptosis of tumor cells can be induced by triptolide (Tengchaicri T et al, Cancer Lett. 1998; 133:169). However, the mechanism involves the processes that have not yet been identified. The present invention provides a new anticancer agent that is an analog of the compound triptolide. The compound shows excellent cytocidal activity against cancer cells of renal, ovarian, melanoma, breast or colon origin, especially the cells overexpressing bcl-2 oncogene. The present invention also provides a method of producing the compound, and a new anti-tumor therapeutic strategy based on the use of the compound in combination with an antisense oligonucleotide that is an effective inhibitor of human cancer cell growth in vitro and in vivo. It is an object of the present invention to provide a triptolide derivative with low toxicity and useful in clinical application.

It is another object of the present invention to provide the uses of triptolide derivatives in the preparation of pharmaceutical compositions used in the treatment of cancers. It is still another of the present invention to provide a method for producing the triptolide derivatives.

It is yet another object of the present invention to provide a pharmaceutical composition comprising the derivative of the present invention.

Detailed Description of the Invention

The present invention provides a triptolide derivative with the following formula:

wherein R is H, alkyl containing 1-4 carbon atom(s), -Ac, -C (=O) (CH₂)n CO₂, or an amino acid, wherein n is an integer of 1-4; R₂ is -SCN, or -NCS. Due to rearrangement of atoms within the molecule, -SCN and ^" HCS is interchangeable.

Preferably, R₂ is -SCN and R, is H.

Yu et al. opened the epoxide at C₁₂ and C₁₃ by using different agents and obtained a series of triptolide derivatives including tripchlororide and tripbromide. The halogen at C₁₂ position may be removed in vivo under enzymatic catalysis, and the oxygen ring at C₁₂ and C₁₃ may be formed again and toxicity of triptolide may be recovered. The present invention uses ammonium thiocyanide to react with triptolide under mild condition to open epoxide at C₁₂ and C₁₃, and obtains a compound, 12- -thiocyano- 13- -hydroxy triptolide (hereinafter referred to as T12) with high anti-cancer activity and low toxicity. The compound thus obtained has the following formula:

wherein R₁ is H, alkyl containing 1-4 carbon atom(s), -Ac, -C (=O) (CH₂)n CO₂, or an amino acid, wherein n is an integer of 1-4. R can be added with knowtimethod.

The present invention also provides a method for producing the triptolide derivatives, comprising the step of reacting triptolide represented by the following formula with ammonium thiocyanide under heating conditions.

wherein R^ is H, alkyl containing 1-4 carbon atom(s), -Ac, -C (=O) (CH₂)n CO₂, or an amino acid, wherein n is an integer of 1-4. Preferably, the reaction is performed in an organic solvent at a temperature of 65-

.0.

87^"C. More preferably, the organic solvent is t-butyl alcohol.

The present invention also provides the uses of the compound of the present invention in the preparation of pharmaceutical compositions for treatment of cancers. In the above uses, the cancers to be treated may be cancers of breast, lung, renal, melanoma, colon or ovarian origin. Preferably, the cancers are of melanoma, renal, colon, ovarian and breast origin.

The present invention further provides a pharmaceutical composition comprising an effective amount of the derivatives of the invention for treatment of cancers, and pharmaceutical acceptable carriers or vehicles.

The cancers to be treated in the composition may be of breast, lung, renal, melanoma, colon or ovarian origin. Preferably, the cancers are of melanoma, renal, ovarian, colon and breast origin.

The present invention still provides a composition comprising the derivatives of the present invention and bcl-2 antisense oligonucleotides in a synergistic effective amount for treatment of cancers, and pharmaceutically acceptable carriers or vehicles.

Brief Description of the Drawings:

Figure 1 shows DNA degradation of T12-induced cell apoptosis;

Figure 2 shows T12-induced Bcl-2 cleavage in HL-60 cells;

Figure 3 shows comparison of Bcl-2 cleavage induced by T12 and other 3 compounds;

Figure 4 shows synergistic effect of T12 and bcl-2 antisense oligonucleotides on Col- 06 cells.

The following experiments were made in accomplishing this invention.

Experiment 1 : Preparation of 12- -thiocyano-13- -hydroxy triptolide

Triptolide (3.6g, 0.01 mole) was added to 400ml t-butyl alcohol under stirring at

50 C. Upon the triptolide was completely dissolved, 11.4g ammonium thiocyanide (0.15 mol) was added to t-butyl alcohol solution of triptolide in parts. Then the reaction mixture was stirred at 80 C for 24 hours. 400ml ethyl acetate was added after the reaction was completed. The organic phase was washed for three times with the saturated NaCI solution and dried with anhydrous MgSO₄. The filtered solution was evaporated at 60 C on a rotary evaporator to remove the solvent and the residue was dissolved with acetone. The acetone solution was checked by TLC, developed in CHCI₃/CH₃OH (95:5) solution. The 12- -thiocyano-13- -hydroxy triptolide was the major product on TLC with traces of triptolide and other by-products. 10g 70-140 μ silica gel was added to the acetone solution with stirring, and then the acetone was evaporated. Triptolide was first removed by silica gel (40-70 μ) with CHCI₃, and then eluted with CHCI_3/CH₃OH solution (95:5). Fractions of T12 were collected under TLC-monitoring. Combine the T12 fractions, and the solvent was evaporated under vacuum. The residue was re-crystallized with acetone to obtain the crystal of 12- -thiocyano-13- -hydroxy triptolide (3.77g, 90% yield), mp: 265-266°C, Rf: 0.15 (CHCI₃/CH₃OH, 95:5), purple-red color in Kedd's reagent.

Elemental analysis: C₂₁H₂₅NSO₆ , calculated value (%): C, 60.14. H, 6.01 , N, 3.34,

tested value (%): C, 59.85. H, 5.93. N, 3.26 [α]² _D ⁵ - 114.06°C (acetone, 4.17 mg/ml);

IR (KBr) crrr¹ : 3450, 3000, 2152, 1739, 1674, 1441 , 1032, 980. MS m/z

..CH 2

(%): 419 (M , 13), 404 (-CH ₃, 14.5), 361 ( ^~C-^CH , 13), 343 (-H₂O, 13), 271 (14), 241 (23), 151 (48), 137 (89), 72 (91), 61 (100).

¹HNMR, ppm: 0.8 (3H, d, J=6.77Hz, 16-CH₃ ), 0.95 (3H, s, 20-CH₃ ), 0.99 (3H, d, J=6.88 Hz, 17-CH₃), 1.28 (1 H, m, 1- H). 1.45 (1H, m, 1- H), 1.85

(1 H, t, J=14.17 Hz, 6- H), 2.0 (1H, m, 2-H), 2.18 (1H, m, 2-H), 2.20 (1H, m, 6- H), 2.25 (1H, m, 15-H), 2.67 (1H, m, 5-H), 3.0 (1 H, d, J=4.4Hz, 14-H), 3.35 (1 H, m, 7-H), 3.75 (1H, d, J=5.61 Hz, 11-H), 3.90 (1H, d, J=5.87Hz, 12-H), 4.85(2H, m, 19-H), 5.0 (1 H, s, 3-OH), 5.45 (1H, d, J=4.2 Hz, 14- OH);

³CNMR, ppm: 14 (16-C), 15.5 (20-C), 16 (17-C), 17 (2-C), 22.5 (6-C), 29 (15-C), 30 (1-C), 35 (10-C), 39 (5-C), 51 (11-C), 57.5 (12-C), 60.5 (8-C), 61.5 (7-C), 67 (9-C), 70.5 (19-C), 74 (14-C), 76.5 (13 -C), 115 (SCN), 123 (3-C), 162 (4-C), 173(18-C). Experiment 2: Inhibition of T12 on the growth of 6 human cancer cell lines in vitro

A detailed description of standard procedures and the method of data analyses performed by NCI have been published (Skehan P. et al., J. Nalt. Cancer Inst., 1990; 82:1107). The assay involves plating the cells, preincubating for 24 hours, followed by a 48 hours continuous drug exposure at appropriate concentration against 6 cell lines originated from 6 tumors (breast, colon, lung, melanoma, ovarian and renal). The cytotoxicity was assessed with the sulforhodamine B (SRB) protein assay. An evaluation parameter was used: The drug concentration which inhibits growth by 50% (Gl₅₀). The growth inhibitory activity of the present invention (T12) was examined and compared with two other compound: Triptolide (T, leading compound of T12) and T8 (a derivative of T, see Jung, M. J., 1/1999 PCT/US98/08562). A known chemotherapeutic agent, taxol was also tested in the experiment, so as to compare the anticancer activity of known compounds and that of the compound in the present invention.

6 cell lines were grown in RPMI 1649 containing 5% fetal bovine serum and 5mM L-glutamine. T, T8 and T12 were tested for their effect on cell growth. Cells were harvested from exponential-phase maintenance culture, counted by trypan blue exclusion method, and dispensed on 96-well culture plates in 100 μl volumes. Following a 24 hr incubation at 37°C, 5% CO₂, 100% relative humidity, 100 μl of culture medium, the culture medium containing drug or the culture medium containing drug vehicle was dispensed within appropriated wells. Plates were then incubated for 48 hours. The growth inhibitory activity of T, T8, T12 were evaluated with SRB assay (Skehan P., 1990). Cell culture were fixed in site by adding 50 μl of cold 50% (wt/vol) trichloroacetic acid (TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded and the plates were washed five times with deionized water and dried. 100 μl of SRB solution was added to each well, and the cultures were incubated for 10 minutes at room temperature. Unbound SRB was removed by washing four times with 1% acetic acid and the plates were air-dried. Bound dyes was solved with Tris buffer, and the optical densities were read on an automated spectrophotometric plate reader at a single wavelength of 490nm. Gl₅₀ were determined using NCI system. The results expressed in terms of Gl₅₀ are shown in Table 1. Table 1. Effects of T12, T and Taxol on GI50 ( μg/ml) of 6 cell lines

Tumor cell lines T12 T T8 Taxol

Bre-01 (breast) 0.055 0.04 0.3 0.06

Col-06 (colon) 0.03 0.02 0.1 0.007

Lu-06 (lung) 0.6 0.09 0.5 0.075

Mel-08 (melanoma) 0.09 0.02 0.25 0.07

Ov-01 (ovarian) 0.02 0.006 0.06 0.05

Re-01 (renal) 0.06 0.03 0.25 1

From Table 1 , it can be seen that the compound of the present invention has a marked inhibition on all 6 cell lines, and the inhibition effects of T12 in renal cancer cells was significantly higher than that of taxol.

Example 3: Inhibitory effect of T12 on colony formation of renal cancer cell line

Clonogenic assays have the advantage of selecting tumor cells in a mixed population, since only the latter have the capacity to grow at low density. The assay has been widely used to assess the effects of tumor chemotherapy.

Re-01 cells were harvested from exponential-phase maintenance culture, counted by trypan blue exclusion, and dispensed in 6-well culture plates (50 cells/ well) in 0.5 ml volumes. Following a 24 hours incubation at 37°C, 5% CO₂, 100% relative humidity, 0.5 ml of culture medium, the culture medium containing drug or the culture medium containing drug vehicle was dispensed within appropriated plates. Culture plates were then incubated for 14 days. Cell colonies were fixed in site followed by Giemsa staining and counted under microscopy. After using a colony forming assay with renal cancer cells and continuous drug exposure for 21 days, T12 showed significant inhibition effects (50% of growth inhibition) in renal cancer cell lines at 0.005 μg/ml of drug concentration.

Experiment 4: Inhibition of T12 on the growth of human melanoma in nude mice L1210 and P388 leukemia were developed in 1945 and 1955 respectively, and played an important role in the screening of candidates of anticancer agents. However, whether P388 or L1210 is a poor predictor for solid tumor active drugs has yet to be investigated. A number of drugs, that are active against L1210 or P388, are inactive against experimental solid tumors. A nude mouse, deficient T-cells, cannot reject foreign tissues. If the nude mice are kept in a germ-free environment, the lifetime can nearly be increased to the length of a normal mouse, and the xenografts can produce many biological characters similar to those of human tumor tissue.

T12 was evaluated in nude mice against human melanoma xenografts. Female Bab/c nude mice, 6-8 weeks old, were obtained from Shanghai Institute of Pharmaceutical Industry, and randomly assigned to experimental groups of n = 6 nude mice. The tumor cells Mel-08 used were implanted subcutaneously into the auxiliary region ( 1X10⁷ cells/0.2 mi/mouse). T12 treatment was initiated 24 hours after the implantation of the tumor cells. T12 (formulated in 0.5% of CMC-Na solution) were injected intraperitoneally for 14 days (4mg/kg body weight/day), and the tumor were removed at the indicated times. Dacarbazine (DTIC, 20 mg/kg/day) and 0.5% of CMC-Na solution (0.5 ml/mouse) were injected intraperitoneally as positive and negative control, respectively. Tumor inhibition (%) were calculated as follows:

[(C-T)/ C]' 100

wherein C and T represent the mean tumor weight for control and treated groups, respectively.

DTIC and the compound of the present invention showed potent inhibitory effects (54.92% and 50.17%) on the growth of melanoma in nude mice, whereas no effects on tumor growth were observed for control group.

Experiment 5: Detection of fragmentation of genomic DNA by agrose gel electrophoresis

Apoptotic cell death involves the activation of a preprogrammed cascade of molecular events that culminate in DNA degradation, nuclear disintegration and packaging of the cell's remnants into membrane-enclosed "apoptosis bodies" which are subsequently removed by phagocytosis. Generally, agents who directly or indirectly cause DNA damage induce apoptosis. The formation of distinct DNA fragments of oligonucleosomal size (180-200 bp lengths) is a biochemical hallmark of apoptosis in many cells. The DNA is cleaved between the nucleosomes resulting in a "ladder" of DNA fragments of multiples of 180-200 bp. The presence of extensive DNA breaks in apoptotic cells has provided the basis for the development of flow cytometric techniques to identify apoptotic cells and to determine their position in the cell cycle. The knowledge of the cell cycle phase specificity of antitumor drugs, therefore, is helpful in developing clinical protocols, especially involving drug combinations. Attempts have been made to classify the drugs according to whether they show selectivity toward noncycling or cycling cells or to particular phases of the cell cycle.

Following incubations with the designated concentrations and schedules of T12, 3 x 10⁶ HL-60 cells were washed in PBS, and then lysed in 100 μl lysis solution (50mM Tris, pH 8.0; 10 mM EDTA and 0.25% NP-40) containing 3 μl proteinase K (1 mg/ml). The lysates were incubated at 37 °C for 30 minutes. Supernatants (20μl) were collected as described for agarose gel electrophoresis (Frank T, 1993).

FIG 1 shows that an exposure to 1 μg/ml T12 for 4, 12 and 24 hours (lanes 3-5) causes intemucleosomal DNA fragmentation of the genomic DNA of HL-60 cells, and no intemucleosomal DNA fragmentation of the genomic DNA for the control and 2 hours with T12 (lanes 1 and 2).

Experiment 6: Flow cytometry analysis of apoptosis and cell cycle

Apoptotic cells were detected by flow cytometry using P1 (Nicoletti, 1991). HL-

60 lines, Re-01 cell lines with or without T12 were incubated at 37 C for 4-72 hours (2, 4, 8 and 24 hours for HL-60 cell lines; 12, 24, 48 and 72 hours for Re-01 cell lines). After centrifugation (700 g at room temperature for 5 minutes), the pellets were washed for two times and then resuspended in 50 μl of PBS. Aliquots containing 10⁶ cells/ 50 μl were fixed in 70% ethanol at -20^°C overnight. Cells were washed and resuspended in PBS (0.5 ml) containing 0.25 mg/ml RNase and 0.1 mg/ml PI. Samples were kept in dark for 30 minutes. Cytometry was performed using the Epics Elite flow cytometer (COULTER, USA) equipped with a 488-nm argon-ion laser. Each of the analysis was performed with the Elite 4.0 and DNA Multicycle software. The percentages of G0/G1 , S and G2/M cells were also calculated by cycle analysis on the gated apoptotic population. Table 2 and 3 demonstrate respectively the induction of apoptosis in HL-60 and Re-01 cells following incubation with 1 μg/ml T12. The low molecular weight DNA from cells undergoing apoptosis following suspension in the phosphate-citrate buffer gave rise to a sub-G1 population on DNA content frequency histograms.

Table 4 and 5 show the distribution of HL-60 and Re-01 cells in various phases of the cell cycle after treatment with T12. Exponentially growing cells were treated with and without 1 μg/ml T12 for 4 hours. T12 increases the proportion of cells in the G0/G1 phases with 1 μg/ml for 2 hours and 4 hours, and decreases the population of cells in S/G2/M phases, indicating that apoptosis is selective to S phase cells.

Table 2. DNA content in apoptotic HL-60 cells following incubation with T12

Treatment time (h) 0 2 4 8 24

Sub-G1 9.1 28.9 74.8 79.8 79.8

Table 3. DNA content in apoptotic Re-01 cells following incubation with T12

Treatment time (h) 0 12 24 48 72

Sub-G1 5.1 10.3 27.4 54.0 90.0

Table 4. Cell cycle Distribution in HL-60 cells treated with T12

Treatment time G0/G1 S G2/M

Control 32.0 53.7 14.3

2 29.5 59.2 11.3

4 71.8 25.3 2.9 Table 5. Cell cycle Distribution in Re-01 cells treated with T12

Treatment time G0/G1 S G2/M

Control 28.4 60.6 11.0

24 38.1 36.9 25.0

48 40.7 43.3 16.0

Experiment 7: Western blot detection of Bcl-2 proteins expression

Apoptosis has become a basic tool in developing and establishing new strategies of cancer therapy. However, the molecular mechanism of T12-induced apoptosis is not well understood. The Bcl-2 protein plays an essential role in preventing cell death and is one of the major factors causing tumor formation. During the study of the molecular mechanisms of T12 action in Bcl-2-overexpressing cells, we found that T12 can induce the cleavage and inactivation of Bcl-2 in a caspase-dependent manner. Both biochemical and genetic evidence indicates that Bcl-2 can regulate cell death induced by caspases. Recent studies has shown that caspase-3 (CPP32) which can cleave poly(ADP-ribose) polymerase (PARP) and play an important role in triptolide- induced T lymphocyte apoptosis (Yang Yili et. al. Immunopharmacology 40: 139- 149,1998).

Three cell lines (human leukemia HL-60 line, human breast cancer Bre-01 line and lymphoma Ly-01 line) were cultured at 1 x 10⁶/ml and treated with 1 μg/ml T12 for 24, 48 and 72 hours. Untreated cell lines were taken as control. Cells were washed and resuspended in lysis buffer containing 20 mM Tris.HCI (pH 8.0), 4% sodium dodecyl sulfate (SDS). Cell lysates were migrated on 12% SDS-polyacrylamide gel and transferred onto PVDF membrane. Membrane was blocked overnight with PBS containing 5% nonfat milk powder and incubated for 2 hours with anti-human Bcl-2 monoclonal antibody (Santa Cruz), followed by peroxidase-conjugated anti-lg antibody. Detection was carried out with ECL solution.

As shown in Figure 2, treatment of HL-60 cells with T12 for 24 and 48 hours (lanes 4 and 5) lead to a cleavage of the 26kDa Bcl-2 into the 20 kDa product. Similar result was observed in Bre-01 cells ( 48 hours, lane 7). Under the same conditions, lymphoma cells did not produce a 20 kDa fragment (24 hours, lane 1), even when cultured for 48 hours (lane 2). All treatment cell lines without T12 (lane 3 for lymphoma, lane 6 for HL-60, and 9 for Bre-01) were not changed for Bcl-2 expression.

To confirm Bcl-2 were also cleaved by other compounds (extract from Triptergium, triptolide and T8) to produce a 20 kDa fragment, we examined the Bcl-2 expression and compared the difference of Bcl-2 cleavage among the compounds in HL-60 cells. Figure 3 shows the cleavage of Bcl-2 during apoptosis by four compounds (lane 1-5: control, extract, T, T8 and T12). T12 (lane 5) displayed a more significant activity to induce Bcl-2 cleavage than other compounds.

Experiment 8: Synergistic effect of T12-combinated with bcl-2 antisense oligonucleotides

It was reported that bcl-2 antisense oligonucleotides are effective inhibitors of human tumor cell growth in vitro and in vivo. This antitumor activity was associated with an early down-regulation of bcl-2 expression (mRNA), followed by inhibition of cell growth and induction of apoptosis. It has been demonstrated that the combination of certain chemotherapeutic agents and bcl-2 antisense oligonucleotides shows a synergistic inhibitory effect on melanoma cells in vitro and in vivo (Campbell M J et al, British Journal of Cancer. 1998; 77:739-744). However, no studies have been reported on the efficacy of the combination of bcl-2 antisense and T12 that are directed to the same molecular target (protein of bcl-2).

Colon cancer Col-06 cells (10⁶/mL) overexpressing bcl-2 cultured in RPMI-1640 medium supplemented with bovine serum, L-glutamine, and penicillin-streptomycin (100 μg/mL each) were treated with bcl-2 antisense (5μM). After 24 hours, T12 were added. The cytotoxicity of the antisense oligonucleotide or the antisense oligonucleotide + T12 in colon cancer cells was determined by SRB assay.

FIG 4 illustrates the combination of T12 and bcl-2 antisense oligonucleotide exerts significantly greater antitumor effects than either agent used alone on human colon and melanoma cell lines in vitro.

Experiment 9: Acute toxicity of T12

Kunming strain mouse (6 weeks old) was chosen for evaluating the acute toxicity of the compound of the present invention. Groups of mice (ten of each sex) were injected intraperitoneally with T12 at doses of 40.96, 51.2, 64, 80, and 100mg/kg in 0.5% of CMC-Na solution. The mice were observed for two weeks for lethality. The LD_JO (dose required to produce 50% lethality) were estimated to be approximately 74.42mg/kg using Bliss analysis. The LD₅₀ of triptolide was reported to be 0.85mg/kg (Folkman J, Nature med 1995, 1 :27). The result shows that the toxicity of T12 is significantly lower than that of T.

Claims

We claim:

1. A compound which is a derivative of triptolide having the following formula:

wherein, R- is H, an alkyl containing 1 -4 carbon atoms, -Ac, -C(=O)(CH ) CO , or an

2 n 2 amino acid, n is an integer of 1-4; R₂ is -SCN, or -NCS.

2. The compound of claim 1 , wherein R₂ is SCN, R, is H.

3. A method for preparation of the compound of claim 1 , comprising the step of reacting the triptolide represented by the following formula with ammonium thiocyanide under heating condition,

wherein, R. is H, an alkyl containing 1 -4 carbon atoms, -Ac, -C(=O)(CH ) CO , or an amino acid, n is an integer of 1-4.

4. The method of claim 3, wherein the reaction is carried out in an organic solvent at 65-87°C.

5. The method of claim 3, wherein the organic solvent is t-butyl alcohol.

6. Uses of the compound of claim 1 or 2 in the preparation of pharmaceutical composition for treatment of cancers.

7. The uses of claim 6, wherein said cancers are of ovarian, breast, lung, renal, melanoma, or colon origin.

8. A pharmaceutical composition, comprising the compound of claims 1 or 2 in an effective amount for treatment of cancers and a pharmaceutically acceptable carrier or vehicle.

9. The composition of claim 8, wherein said cancers are of breast, lung, renal, melanoma, colon or ovarian origin.

10. A pharmaceutical composition, comprising the compound of claim 1 or 2 and bcl-2 antisense oligonucleotide in a synergistic effect amount for treatment of cancers, and a pharmaceutically acceptable carrier or vehicle.

11. The composition of claim 10, wherein said cancers are of breast, lung, renal, melanoma, colon or ovarian origin.