Synthesis, antitumor activity evaluation of 2-selenocyano-3-selenocyanoalkyloxyestradiols with a bisselenocyanate structure.

The combination of steroid structure and selenocyano group offers high potential for the design and synthesis of new potential anti-tumor drugs. Beginning with estradiol, a series of 2-selenocyano-3-selenocyanoalkyloxyestradiol derivatives with remarkable antiproliferative activity was synthesized. Additionally, a 2,4-bisselenocyanoestradiol was synthesized by directly selenocyanating estradiol diacetate. It was found that the cytotoxicity of 2-selenocyano-3-selenocyanoalkyloxyestradiol derivatives was significantly increased in comparison to the corresponding monoselenocyanate precursor, whereas the cytotoxicity of the 2, 4-bisselenocyanoestradiol derivative was significantly reduced compared to the respective monosubstituted precursor. The introduction of the second selenocyano group at different locations of estradiol shows a various impact on the cytotoxicity of the compounds. Among them, compound 3e showed the best cytotoxicity, with an IC50 value of less than 5 µM against the tested tumor cells, and strong inhibitory activities against HeLa and MCF-7 cell xenograft tumors in zebrafish, suppressing tumor cell migration and neovascularization. Notably, compound 3e was more effective at inhibiting neovascularization of MCF-7 cell xenograft tumors than the positive control 2-methoxyestradiol. Furthermore, compound 3e showed excellent anti-oxidative stress effect in zebrafish. Therefore, these estrogen bisselenocyanate compounds may be promising anti-tumor agents, warranting further investigation.

Discovery and biological evaluation of pregnenolone selenocyanoamides with potential anticancer and antimicrobial activities.

Starting with pregnenolone, a 20-carbonyl group was converted into an amino group through a series of chemical reactions. This amino group was further converted into selenocyanoalkylamide, leading to the synthesis of six pregnenolone selenocyanoalkylamide derivatives. These compounds were then screened for antitumor activity in vitro, yielding promising results. Compounds 4b-4f show higher inhibitory activity than the positive control abiraterone and 2-methoxyestradiol, with IC50 values lower than 10 µmol/L against breast, ovarian, and cervical cancer cell lines that closely related to human hormone expression levels. The Annexin V assay of compound 4f revealed that compounds inhibited tumor cell proliferation primarily through the induction of programmed apoptosis. The zebrafish test results indicated that compound 4d had significant inhibitory activity against MCF-7 cell xenografts in vivo. Moreover, the antibacterial test indicated that compounds 4a and 4d-4e had better inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) than the positive controls vancomycin and ampicillin. These results suggest that these compounds may hold promise as novel antitumor agents or antimicrobial agents for further study.

Normalized TSH strategy can improve the initial assessment of thyroid nodules.

BACKGROUND: Serum thyrotropin (TSH) has been recommended for the initial assessment of patients with thyroid nodules to exclude functional thyroid nodules (FTN). However, the sensitivity of TSH is very low. The increased level of thyroid peroxidase antibody (TPOAb) is considered to be one of the reasons. OBJECTIVE: To investigate whether normalized TSH (nTSH) can improve diagnostic efficiency by removing TPOAb interference in the first evaluation of thyroid nodules compared with traditional TSH strategy. METHODS: Thyroid nodules were retrospectively analysed in 90 patients with FTN and 1038 patients with non-functioning thyroid nodules (non-FTN). The regression coefficient (ß) of TPOAb affecting the TSH levels was assessed in patients with thyroid nodules, and then, the nTSH level was calculated based on the following formula: nTSH = TSH-ß*TPOAb. We used nTSH levels to initially evaluate the thyroid nodules instead of the traditional TSH values and finally compared the results of the two strategies. RESULTS: The sensitivity, specificity, accuracy, positive prediction rate (PPV) and negative prediction rate (NPV) of nTSH for accessing FTN were 50.00%, 87.70%, 84.67%, 26.01% and 95.29%, respectively, which were better than the values of 48.90%, 78.70%, 76.33%, 16.60% and 94.67% associated with TSH, respectively (p < 0.001). CONCLUSION: Serum TPOAb testing is recommended for the first assessment of thyroid nodules. Normalized TSH levels can improve assessment efficiency compared to traditional TSH assessment, increase the specificity and reduce an unnecessary 99mTc-TS test.

Synthesis and antitumor activity of some cholesterol-based selenocyanate compounds.

The introduction of selenium-containing functional groups into steroids to study the biological activities of related derivatives is rarely reported in the literature. In the present study, using cholesterol as raw material, four cholesterol-3-selenocyanoates and eight B-norcholesterol selenocyanate derivatives were synthesized, respectively. The structures of the compounds were characterized by NMR and MS. The results of the in vitro antiproliferative activity test showed that the cholesterol-3-selenocyanoate derivatives did not exhibit obvious inhibitory on the tested tumor cell lines. However, the B-norcholesterol selenocyanate derivatives obtained by structural modification of cholesterol showed good inhibitory activity against the proliferation of tumor cell. Among them, compounds 9b-c, 9f and 12 showed similar inhibitory activity against tested tumor cells as positive control 2-methoxyestradiol, and better than Abiraterone. At the same time, these B-norcholesterol selenocyanate derivatives displayed a strong selective inhibitory against Sk-Ov-3 cell line. Except for compound 9g, the IC50 value of all B-norcholesterol selenocyanate compounds against Sk-Ov-3 cells was less than 10 µM, and compound 9d was 3.4 µM. In addition, Annexin V-FITC/PI double staining was used to analyze the cell death mechanism. The results showed that compound 9c could induce Sk-Ov-3 cells to enter programmed apoptosis in a dose-dependent manner. Furthermore, the in vivo antitumor experiments of compound 9f against zebrafish xenograft tumor showed that 9f displayed obvious inhibitory effect on the growth of human cervical cancer (HeLa) xenograft tumor in zebrafish. Our results provide new thinking for the study of such compounds as new antitumor drugs.

Synthesis and Antiproliferative Activity Evaluation of B-norcholesterol-6- amide Compounds.

BACKGROUND: The structure modification of steroids is commonly used to change the biological activity of steroids in medicinal chemistry. In recent years, it has been found that some derivatives derived from the structural modification of cholesterol display good inhibitory activity against tumor cell proliferation in vitro. METHODS: Using cholesterol as the starting material, different types of B-norcholesterol-6-amide derivatives were synthesized by the reaction of 6-carboxyl-B-norcholesterol with different alkyl amines or 6-amino-B-norcholesterol with different acyl chlorides. The inhibitory activity of compounds on the proliferation of tumor cell lines was investigated by the MTT method. RESULTS: The results showed that the B-norcholesterol-6-amide compounds displayed distinct cytotoxicity against Sk-Ov-3 cells but caused no obvious damage against HEK-293T cells. Additionally, the steroidal amide derivatives formed from 6-amino-B-norcholesterol showed stronger cytotoxicity than those produced from 6-carboxyl-B-norcholesterol. Specially, compounds with chloroalkyl structure displayed significant inhibitory activity against all tumor cells tested. Among them, compounds 19-21 showed cytotoxicity like 2-methoxyestradiol as a positive control, and the IC₅₀ value of compound 20 on HeLa cells was 3.9 µM. CONCLUSION: After introducing chloroalkyl acyl groups into 6-position of 6-amino-B-norcholesterol, the cytotoxicity of resulting B-norcholesterol-6-amide compounds can be greatly enhanced.

Straightforward synthesis of steroidal selenocyanates through oxidative umpolung selenocyanation of steroids and their antitumor activity.

Straightforward access to steroidal selenocyanates in a single assembly step from steroids remains a significant challenge. However, the development of novel method for the synthesis of steroidal selenocyanates and further investigation of their bioactivities have largely lagged behind. In this work, selenocyano groups were directly introduced into the 17- or 21-position of pregnenolone, the 2-position of estradiol, and the 16-position of estrone. A total of 16 estrogen selenocyanate derivatives with diverse structures were synthesized, and the tumor cell lines closely related to the expression level of estrogen were used to investigate the inhibitory activity of the target products on tumor cell proliferation in vitro. The results revealed that the 17-selenocyano-substituted pregnenolone selenocyanate derivatives 1b-3b exhibit obvious inhibitory activity against the tested tumor cell lines. Additionally, the 2-selenocyano-substituted estradiol derivatives and 16-selenocyano-substituted estrone derivatives exhibit selective inhibitory on HeLa cell lines. Among them, 2-selenocyano-3-methoxyestradiol-17-benzoate (7e) displayed an IC50 value of 4.1 µM against HeLa cells and induced programmed apoptosis in HeLa cancer cells. Furthermore, compound 7e could significantly inhibit the growth of human cervical cancer xenografts in zebrafish in vivo. This approach provides new insights for future steroid antitumor drug design.

Synthesis of estrone selenocyanate Compounds, anti-tumor activity evaluation and Structure-activity relationship analysis.

Introducing different functional groups into steroid can bring unexpected changes in biological activity of the steroid. Using estrone as a raw material, through the functional group conversion and modification of the 17-carbonyl, the structural fragments with selenocyano groups were instilled in the form of amide, ester, and oxime ester, respectively, and various 17-substituted estrone selenocyanate derivatives were synthesized. In addition, different 3-substituted estrone selenocyanate derivatives were synthesized by introducing different selenocyanoalkoxy fragments into the 3-position of estrone in the form of alkyl ether. Furthermore, the selenocyano-containing moieties were embedded into the 2-position of estrone by means of amide, affording diverse 2-selenocyanoamide-estrone derivatives. The antiproliferative activities of the target compounds were screened by selecting tumor cell lines related to the expression of human hormones. The results showed that the introduction of selenocyano group into estrone could endow estrone with significant biological activity of inhibiting the proliferation of tumor cells. Structure-activity relationship research showed that the cytotoxicity of 3-selenocyanoalkoxy-estrone was further increased with the extension of alkyl carbon-chain within 8 carbon chain lengths. In addition, the cytotoxicity of the products with selenocyano via the form of amide was stronger than that of ester or ether. Selenocyano moiety instilled at the 2-position of estrone in the form of amide was more cytotoxic than that of 17- or 3-position. Among them, compound 21a has better inhibitory activity on tested tumor cells than positive controls Abiraterone and 2-methoxyestradiol. Research showed that the compound 21c induced programmed apoptosis in Sk-Ov-3 cancer cells, and compound 17d inhibited significantly the growth of human cervical cancer zebrafish xenografts in vivo, offering useful insights into the synthesis of steroid antitumor drugs.

Rh(III)-Catalyzed Synthesis of Amino-isocoumarins with N-Functionalized Cyclic Carbonates via C-H/O-H Annulation.

A practical method to access amino-isocoumarins catalyzed by a Rh(III) complex through redox-neutral C-H/O-H annulation has been disclosed. The use of N-functionalized cyclic carbonates is crucial to facilitate the catalytic turnover, and a broad spectrum of amino-isocoumarin derivatives were prepared with satisfactory yields. Amino-isocoumarin estrone conjugated with a selenocyano functionality was identified to be nearly four times as active as the marketed drug abiraterone against T47D cancer cells.

Identification of estrogen receptor down-regulators for endocrine resistant breast cancer.

Resistance to endocrine therapies remains an impediment for the treatment of estrogen receptor (ER) positive breast cancer. ER down regulator Fulvestrant has showed great activity to overcome the endocrine resistance. However, Fulvestrant has poor bioavailability due to the hydrophobicity. Identification of novel ER down regulator is still important. Compounds 172 and 183 are two steroidal compounds with androgen scaffold but significantly down regulated ER in multiple breast cancer cell lines. RT-PCR results indicated that both compounds did not affect ER gene expression. Proteasome inhibitor MG132 could attenuate ER down regulation effect of the compounds, suggesting that the ER down regulation was via ubiquitin-proteasomal pathway. Furthermore, compounds 172 and 183 could downregulate ER in endocrine resistant breast cancer cell model long term estrogen deprivation (LTED) MCF-7 cells. Hydrophobicity of compounds 172 and 183 were determined and showed improved solubility compared to Fulvestrant. All these results suggested that compounds 172 and 183 could be potential lead compounds for drug development for the treatment of endocrine resistance breast cancer.

Synthesis and antiproliferative evaluation of some novel estradiol selenocyanates.

Selenocyano fragments with different structural characteristics have been successfully installed into the 3- and 17-position of estradiol through the etherification and esterification of its 3 or 17-hydroxyl group respectively. A total of 12 new estradiol selenocyanates were synthesized and their structures were characterized by NMR and HRMS. The tumor cell lines related to the expression of human hormones were selected as the screening objects, and the antiproliferative activity of the target compounds was further investigated. The results showed that the introduction of selenocyano group in estradiol could endue estradiol with the activity of inhibiting tumor cell proliferation, and 3-selenocyanoalkyl estradiol ethers had stronger cytotoxicity than their 17-selenocyanocarboxylates counterpart. Among them, IC50 value of compound 3e on HeLa cells was 5.69 µM. The information obtained from the studies may be useful for the design and development of novel chemotherapeutic drugs.

Apoptosis inducing properties of 3-biotinylate-6-benzimidazole B-nor-cholesterol analogues.

In this work, a series of Biotin-substituted B-nor-cholesteryl benzimidazole compounds were synthesized. The antiproliferativeactivities of these compounds were evaluated in vitro using a series of human cancer cell lines, including HeLa (cervical cancer), SKOV3 (ovarian cancer), T-47D (thymus gland cancer), MCF-7 (human breast cancer) and HEK293T (normal renal epithelial) cells. These compounds displayed distinct antiproliferative activities against the currently tested cancer cells. The apoptotic properties induced by compound 6d were further investigated. Our results showed that compound 6d could induce the apoptosis of SKOV3 cells, blocking the cell growth in S-phase. Western blotting analyses revealed that compound 6d can induce cell apoptosis via the mitochondria-dependent pathway.

Studies on apoptosis induced by B-norcholesteryl benzimidazole compounds in HeLa cells.

Certain B-norcholesteryl benzimidazole compounds were found to mediate marked anti-tumor proliferative effects in vitro in our earlier study. Here, the mechanism of action of these anti-tumor effects was evaluated using HeLa human cervical cancer cells. Methods for detecting cell invasion and migration, Annexin V-PI double staining, cell cycle status, and mitochondrial membrane potential Δψm were employed. These compounds were confirmed to significantly inhibit the proliferation of HeLa cells in vitro. Compound 1 induced apoptosis in S phase, compound 2induced apoptosis in the G0/G1 phase and compound 3 induced late apoptosis in the G2/M phase. These compounds induced HeLa cell apoptosis through depolarization of mitochondrial membrane potential Δψm in a dose-dependent manner. B-norcholesteryl benzimidazole compounds induced morphological changes in HeLa cells and inhibited proliferation, invasion and metastasis. Apoptosis was promoted by mechanisms involving p21 and p53 in this cervical cancer cell line.

Untargeted metabolomics study and pro-apoptotic properties of B-norcholesteryl benzimidazole compounds in ovarian cancer SKOV3 cells.

The current study aims to evaluate the antiproliferative activity of B-norcholesteryl benzimidazole compounds in human ovarian cancer cells (SKOV3). Our experimental data indicates that the tested compounds can induce apoptosis in SKOV3 cells, block S-phase growth, and decrease mitochondrial membrane potential. Western blot results showed that B-norcholesteryl benzimidazole compounds (1 and 2) induced apoptosis in SKOV3 cells via activation of the mitochondrial signaling pathway. Following SKOV3 cells treatment with compounds 1 and 2, the cell metabolism was assessed using the UHPLC-QE-MS (Ultra High Performance Liquid Chromatography-Q Exactive Orbitrap- Mass Spectrometry) non-target metabolomics analysis method. The results showed 10 metabolic pathways that mediated the effects of compound 1, including arginine and proline metabolism; alanine, aspartate, and glutamate metabolism; histidine metabolism; D-glutamine and D-glutamine and D-glutamate metabolism; cysteine and methionine metabolism; aminoacyl-tRNA biosynthesis; purine metabolism; Glutathione metabolism; D-Arginine and D-ornithine metabolism; and Nitrogen metabolism. From the perspective of metabolomics, compound 1 inhibits intracellular metabolism, protein synthesis, and slows down energy metabolism in SKOV3 cells. These changes result in the inhibition of proliferation and signal transduction, abrogate invasive and metastatic properties, and induce apoptosis, thus, exerting anti-tumor effects. Application of compound 2 altered activation of metabolic pathways in SKOV3 cells. The main metabolic pathways involved were glycerophospholipid metabolism; arginine and proline metabolism; purine metabolism; glycine, serine, and threonine metabolism; and ether lipid metabolism. The metabolic pathway with the greatest impact and the deepest enrichment was the glycerophospholipid metabolism. In conclusion, compound 2 inhibits proliferation of SKOV3 cells by interfering with glycerate metabolism, which plays a major role in regulation of cell membrane structure and function. Additionally, compound 2 can inhibit the invasion and metastasis of SKOV3 cells and induce apoptosis via interfering with the metabolism of arginine and proline.

Study on the interactions between B-norcholesteryl benzimidazole compounds with ct-DNA.

The study of molecule-DNA interaction is very important for designing an improved therapeutic agent. In previous studies, we synthesized some B-norcholesteryl benzimidazole compounds, and the tests on cancer cells showed that these compounds had good in vitro anti-cancer activities. In order to further investigate mechanism of their actions, three different B-norcholesteryl benzimidazole compounds were selected and interaction of these compounds with the calf thymus DNA (ct-DNA) was monitored by using various methods including UV-Vis and fluorescence spectroscopic techniques, viscosity measurement, and circular dichroism (CD). The results proved a hypochromic effect accompanied with a slight red-shift due to the interaction of the molecules with ct-DNA. According to the UV-Vis and fluorescence spectra, the mentioned compounds were bound to DNA, preferentially through partial intercalation into the DNA helix. Moreover, the ethidium bromide (EB) and Hoechst 33258 competitive binding experiments were also used to confirm the interaction mode of the compounds with ct-DNA. In the Hoechst 33258 displacement experiment, no significant change in the fluorescence intensity was observed. Additional assays such as iodide quenching, viscosity, and CD spectroscopy further confirmed that intercalation should be the major binding mode of the selected compounds with DNA. The cytotoxicity of these three compounds was also evaluated by MTT method, and the results confirmed that binding ability of these compounds to DNA was consistent with their cytotoxicity behavior. The experimental results indicated a higher binding affinity for compound 3 compared to the other compounds. This research provided a better understanding on the molecular mechanism of the interaction between B-norcholesteryl benzimidazole compounds and tumor cells, and offered a beneficial perspective to the designation of novel B-norsteroidal anticancer compounds.

Synthesis and antiproliferative evaluation of novel steroid-benzisoselenazolone hybrids.

The two different types of steroidal benzisoselenazolone hybrids were synthesized by incorporating benzisoselenazolone scaffold into dehydroepiandrosterone and B-norcholesterol. The antiproliferative activity of the synthesized compounds against some carcinoma cell lines were investigated. The results showed that some of these compounds have better inhibitory activity than abiraterone on the proliferation of tumor cells associated with human growth hormone, and have less cytotoxicity on normal human cells. In particular, the IC50 values of the compound 8a and 8f are 5.4 and 6.5 µmol/L against human ovarian carcinoma (SKOV3) cell line, and possess SI values of 13.9 and 10.5, respectively. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.

Synthesis, characterization, and biological evaluations of some steryl 2-methoxybenzoates as anticancer agents.

Using cholesterol, stigmasterol and sitosterol as starting materials, a series of 7-subsitituted-ster-3-yl 2-methoxybenzoate analogs were prepared through reacting with 2-methoxybenzoyl chloride and introducing some function groups, such as carbonyl, hydroxyl and various thiosemicarbazones, at 7-position of steroidal nucleus. The structures of these new compounds were characterized by IR, NMR and HRMS. Their antiproliferative activities were evaluated by using several types of cancer cells. Interestingly, the compounds displayed potent antiproliferative activity against CNE-2 (nasopharyngeal carcinoma cell lines), BEL-7402 (human liver cancer cell lines) and HepG2 (human liver cancer cell lines), suggesting that they have potential to be drug candidates for cancer treatment.

Antibacterial and Antitumor Potential of Actinomycetes Isolated from Mangrove Soil in the Maowei Sea of the Southern Coast of China.

Mangroves are the tidal forest existing in the intertidal zone and usually considered as the special marine ecosystem. In the present study, 452 actinomycetes were recovered from nine diferent sites at Maowei Sea Mangrove Reserve in Qinzhou (Guangxi province, China). Among them, Seventy-four strains were purified for 16s RNA gene sequencing and further characterization. The results indicated that the majority of isolates belonged to the genera Streptomyces, including 17 species. Streptomyces sanyensis was the dominant species (31.1%), followed by Streptomyces griseorubens (17.5%), Streptomyces viridobrunneus (10.8%) and other Streptomyces species. Only one rare actinomycete, Stenotrophomonas was discovered. The isolation of actinomycetes was obviously related to the type of soil and edaphic conditions. Rhizosphere-associated soils gave almost 62.2% actinomycete isolates, nearly twice as many as the non-rhizosphere-associated soils. In addition, 20 actinomycete strains (27%) presented varied antibacterial activities towards four tested organisms, including two drug-resistant clinical strains (MRSA and VRE), while some species of Streptomyces like S.sanyensis, S.viridobrunneus, S.tanashiensis, S.parvus, S.flavotricini, and S.parvulus exhibited remarkable cytotoxic activities. Further bioinformatical analysis of these 29 bioactive strains for secondary metabolites biosynthetic machineries revealed that nonribosomal peptide synthetase (NRPS) was detected in 20 isolates (68.9%), whereas type-I polyketide synthase (PKS-I) and type-II polyketide synthase (PKS-II) were detected in 16 and all of the 29 strains, respectively. Hence, our work demonstrated that actinomycetes from mangroves in Maowei Sea Mangrove Reservewere fascinating reservoirs for antibacterial and antitumor natural products discovery.

Synthesis and antiproliferative activity of 17-[1',2',3']-selenadiazolylpregnenolone compounds.

Using pregnenolone as a starting material, some 3-substituted 17-[1',2',3']-selenadiazolylpregnenolone derivatives were synthesized, and their structures were characterized by IR, NMR and HRMS. The in vitro antitumor activity of the compounds was assayed against PC-3、SKOV3、T47D、MCF-7 and HEK293T cell lines. The results show that some compounds display selective antiproliferative activity against PC-3 and SKOV3 cells lines and are almost inactive to normal kidney epithelial cells (HEK293T). The IC50 value are much better than that of abiraterone (positive control).

Preclinical Study of Novel Curcumin Analogue SSC-5 Using Orthotopic Tumor Xenograft Model for Esophageal Squamous Cell Carcinoma.

PURPOSE: Tumor xenograft model is an indispensable animal cancer model. In esophageal squamous cell carcinoma (ESCC) research, orthotopic tumor xenograft model establishes tumor xenograft in the animal esophagus, which allows the study of tumorigenesis in its native microenvironment. MATERIALS AND METHODS: In this study,we described two simple and reproducible methods to develop tumor xenograft at the cervical or the abdominal esophagus in nude mice by direct injection of ESCC cells in the esophageal wall. RESULTS: In comparing these two methods, the cervical one presented with more clinically relevant features, i.e., esophageal stricture, body weight loss and poor survival. In addition, the derived tumor xenografts accompanied a rapid growth rate and a high tendency to invade into the surrounding structures. This model was subsequently used to study the anti-tumor effect of curcumin, which is known for its potential therapeutic effects in various diseases including cancers, and its analogue SSC-5. SSC-5 was selected among the eight newly synthesized curcumin analogues based on its superior anti-tumor effect demonstrated in an MTT cell proliferation assay and its effects on apoptosis induction and cell cycle arrest in cultured ESCC cells. Treatment of orthotopic tumor-bearing mice with SSC-5 resulted in an inhibition in tumor growth and invasion. CONCLUSION: Taken together, we have established a clinically relevant orthotopic tumor xenograft model that can serve as a preclinical tool for screening new anti-tumor compounds, e.g., SSC-5, in ESCC.

Synthesis and Cytotoxic Evaluation of Steroidal Copper (Cu (II)) Complexes.

Using estrone and pregnenolone as starting materials, some steroidal copper complexes were synthesized by the condensation of steroidal ketones with thiosemicarbazide or diazanyl pyridine and then complexation of steroidal thiosemicarbazones or steroidal diazanyl pyridines with Cu (II). The complexes were characterized by IR, NMR, and HRMS. The synthesized compounds were screened for their cytotoxicity against HeLa, Bel-7404, and 293T cell lines in vitro. The results show that all steroidal copper (II) complexes display obvious antiproliferative activity against the tested cancer cells. The IC50 values of complexes 5 and 12 against Bel-7404 (human liver carcinoma) are 5.0 and 7.0 µM.