Macharistol, a new cytotoxic cinnamylphenol from the stems of Machaerium aristulatum.

A new cinnamylphenol, macharistol (1), along with a known pterocarpan, (+)-medicarpin (2), were isolated as cytotoxic constituents from the stems of Machaerium aristulatum. In addition, a known pterocarpan, (+)-maackiain (3), and a known isoflavone, formononetin (4), were identified as inactive constituents. Compound 1 was evaluated in the in vivo hollow fiber assay with KB, Col-2, and hTERT-RPE1 cells and found to be inactive at the highest dose (25 mg/kg body weight) tested.

Isolation of symlandine from the roots of common comfrey (Symphytum officinale) using countercurrent chromatography.

Three pyrrolizidine alkaloids, symlandine, symphytine, and echimidine (1-3), were isolated from the roots of Symphytum officinale using a one-step countercurrent chromatography procedure. The structures of 1-3 were confirmed by several spectroscopic techniques including 2D NMR methods. This is the first description of the separation of symlandine (1) from its stereoisomer, symphytine (2).

Bioactive constituents of the roots of Licania intrapetiolaris.

Fractionation of a methanol extract of the roots of Licania intrapetiolaris, as directed by activity against the KB assay, has led to the isolation of two novel clerodane diterpenoids, intrapetacins A (1) and B (2), and the known triterpenoid cucurbitacin B (3). The structures of 1 and 2 were deduced from one- and two-dimensional NMR experiments, including relative stereochemical assignments based on NOESY correlations and COSY coupling constants. Compound 3 was the most potent against the KB assay, but both 1 and 2 displayed moderate cytotoxicity. When evaluated against an antifungal assay using Aspergillus niger, 2 caused a significant zone of inhibition of fungal growth, while 1 was completely inactive. To the best of our knowledge, this is the first report of the isolation of bioactive compounds from the genus Licania.

Selective drug resistant human osteosarcoma cell lines.

Chemotherapy in combination with surgery has been shown to be effective for the control of osteosarcoma. Development of resistance to chemotherapeutic agents is a recurring clinical problem. To investigate this phenomena, human osteosarcoma cells, TE-85, were exposed to increasing doses of Taxol or Taxotere during a 9-month period. Highly resistant subclones (TE-85TXL; TE-85TXR, respectively) were developed. Chemosensitivities are presented for TE-85 cell line and these new lines to Taxol, Taxotere, doxorubicin, cisplatin, and topotecan. Drug concentrations that inhibited cell growth by 50% compared with untreated cells were determined. The TE-85TXL cells showed resistance greater than 1,000-fold to Taxol and Taxotere and 60-fold to doxorubicin. The TE-85TXR cells showed resistance greater than 1,000-fold to Taxol, 800-fold to Taxotere, and 90-fold to doxorubicin. There was little cross resistance to topotecan and enhanced sensitivity to cisplatin. The role of P-170 glycoprotein in Taxol and Taxotere resistance was explored. Coincubation with verapamil, to block the actions of P-170 glycoprotein, partly reversed resistance to Taxol, Taxotere, and doxorubicin in both cell lines. Anti-P-170 glycoprotein antibodies revealed positive staining in TE-85TXL and TE-85TXR cell lines. Flow cytometry revealed reduced accumulation of doxorubicin in resistant cells. These data indicate that a human osteosarcoma cell line will develop resistance to Taxol and Taxotere, which is mediated in part by the P-170 glycoprotein.

New bioactive aromatic compounds from Vismia guianensis.

Five benzophenones, vismiaguianones A-E, and two benzocoumarins, vismiaguianins A and B were isolated from the CHCl3 extract of the roots of Vismia guianensis by bioassay-directed fractionation using the DNA strand-scission assay and KB cell line. Of the isolates obtained, vismiaguianone B exhibited DNA strand-scission activity, whereas vismiaguianones D and E and vismiaguianin A were found to be significantly cytotoxic.

Camptothecin analogues with enhanced antitumor activity at acidic pH.

BACKGROUND: Camptothecin (CPT) is a specific inhibitor of the nuclear enzyme topoisomerase I, which is involved in cellular DNA replication and transcription. Topoisomerase I is therefore an attractive target for anticancer drug development, and two analogues of CPT, topotecan (TPT) and irinotecan (CPT-11), have demonstrated significant antitumor activity in the clinic. This activity is limited, however, by lability of the CPT E ring lactone, which forms the inactive hydroxy acid at physiological pH. The reaction is reversible at acidic pH, which provides a rationale for selectivity, because many solid tumors create an acidic extracellular environment while maintaining a normal intracellular pH. PURPOSE: To exploit the tumor-selective pH gradient to improve the efficacy of CPT-based chemotherapy. METHODS: CPT analogues were evaluated by growth inhibition assay in three human breast cancer cell lines that had been adapted to in vitro culture at acidic pH versus the respective cells cultured at physiological pH. The MCF-7, MDA-MB-231, and MCF-7/hc cell lines represent the hormone-dependent and hormone-independent stages of the disease, and a MCF-7 variant that is resistant to the alkylating agent 4-hydroperoxycyclophosphamide (4-HC), respectively. Antiproliferative activity of SN-38 (the active metabolite of CPT-11), and TPT was compared to that of CPT and two CPT analogues, 10,11-methylenedioxy-CPT (MDC), and the alkylating derivative, 7-chloromethyl-10,11-MDC (CMMDC). RESULTS: In general, MDC was the most potent and TPT or CPT the least potent analogue, regardless of pH. However, if the comparison was based on magnitude of potentiation by pH, a different rank order emerged. CPT was modulated 4-fold; MDC, SN-38, and TPT were each modulated 5- to 6-fold, while the activity of CMMDC was increased 10- to 11-fold by acidic pH in MCF-7 lines, and 65-fold in MDA-MB-231 cells. Thus MDC was the superior CPT analogue based on potency, but CMMDC was the best candidate for pH modulation. Drug specificity was also observed. While the alkylating agent, 4-HC, was 2- to 3-fold more active at acidic pH, modulation was not observed for 5-fluorouracil, doxorubicin, or paclitaxel. Preliminary mechanism studies indicated that pH modulation of CPT analogues was directly correlated to intracellular levels of glutathione. In addition, protein-associated DNA strand breaks were more rapidly induced at acidic pH. CONCLUSION: These results suggest that CPT-based drug development and resulting chemotherapy could benefit from evaluation of differential activity at acidic versus physiological pH. Analogues have been identified that could have improved therapeutic indices based on the pH gradient that selectively exists in human tumors.

7- and 10-substituted camptothecins: dependence of topoisomerase I-DNA cleavable complex formation and stability on the 7- and 10-substituents.

7-Alkyl, 7-alkyl-10-hydroxy, 7-alkyl-10-methoxy, and 7-alkyl-10, 11-methylenedioxy analogs of camptothecin have been synthesized and evaluated for their ability to trap human DNA topoisomerase I in cleavable complexes. The 7-alkyl chain lengths varied linearly from methyl to butyl. The concentration required to produce cleavable complexes with purified topoisomerase I in 50% of the plasmid DNA (EC(50)) was reduced by 1 order of magnitude by the introduction of a 10-methoxy or 7-alkyl group compared with camptothecin. The EC(50) values were reduced by 2 orders of magnitude with a 10-hydroxy or 10, 11-methylenedioxy moiety compared with camptothecin. The steady-state EC(50) concentrations for all of the analogs tested were slightly dependent on substitution at the 7-position, but this dependence was least with the 10-methoxy series. The kinetics of the reversibility of the complexes formed with all analogs was only slightly influenced by the length of the 7-substitution, with the trend that ethyl or greater lengths led to slightly reduced rate constants for cleavable complex reversal. These results were also observed for DNA-protein cross-link formation by the analogs in isolated CEM cell nuclei. Our data indicate that in vitro cleavable complex stability, as determined by the apparent rate constants for complex dissociation, does not reflect the in vitro biological activity of these camptothecin analogs. However, complex stability in vivo may be important for the antitumor activity of the compounds.

New biphenyl compounds with DNA strand-scission activity from Clusia paralicola.

Three new biphenyl derivatives, clusiparalicolines A (1), B (2), and C (3), were isolated from the roots of Clusia paralicola by bioassay-directed fractionation using the DNA strand-scission and the KB human cancer cell line cytotoxicity assays. Compounds 1 and 2 were found to be active in the DNA strand-scission assay, whereas all three compounds exhibited modest cytotoxicity against the KB cell line. The structures of 1-3 were elucidated by spectroscopic methods including 1D and 2D NMR techniques.

Comparison of in vitro activities of camptothecin and nitidine derivatives against fungal and cancer cells.

The activities of a series of camptothecin and nitidine derivatives that might interact with topoisomerase I were compared against yeast and cancer cell lines. Our findings reveal that structural modifications to camptothecin derivatives have profound effects on the topoisomerase I-drug poison complex in cells. Although the water-soluble anticancer agents topotecan and irinotecan are less active than the original structure, camptothecin, other derivatives or analogs with substitutions that increase compound solubility have also increased antifungal activities. In fact, a water-soluble prodrug appears to penetrate into the cell and release its active form; the resulting effect in complex with Cryptococcus neoformans topoisomerase I is a fungicidal response and also potent antitumor activity. Some of the compounds that are not toxic to wild-type yeast cells are extremely toxic to the yeast cells when the C. neoformans topoisomerase I target is overexpressed. With the known antifungal mechanism of a camptothecin-topoisomerase I complex as a cellular poison, these findings indicate that drug entry may be extremely important for antifungal activity. Nitidine chloride exhibits antifungal activity against yeast cells through a mechanism(s) other than topoisomerase I and appears to be less active than camptothecin analogs against tumor cells. Finally, some camptothecin analogs exhibit synergistic antifungal activity against yeast cells in combination with amphotericin B in vitro. Our results suggest that camptothecin and/or nitidine derivatives can exhibit potent antifungal activity and that the activities of camptothecin derivatives with existing antifungal drugs may be synergistic against pathogenic fungi. These new compounds, which exhibit potent antitumor activities, will likely require further structural changes to find more selective activity against fungal versus mammalian cells to hold promise as a new class of antifungal agents.

Pre-clinical evaluation of SN-38 and novel camptothecin analogs against human chronic B-cell lymphocytic leukemia lymphocytes.

The topoisomerase I inhibitor camptothecin and its analogs have potent activity against a wide range of solid tumors and several hematologic malignancies. Previous studies with these compounds using the MTT metabolic inhibition assay have shown significant cytotoxicity against lymphocytes from patients with chronic B-cell lymphocytic leukemia (B-CLL). Yet the water soluble analogue, topotecan, which was inhibitory at > 1 microM in vitro, had no clinical activity in vivo. In the present study, we evaluated the in vitro cytotoxicities of SN-38, the active form of irinotecan, and two newer water soluble camptothecin derivatives 10,11-methylenedioxy-20(S)-camptothecin glycinate (MDCG) and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin glycinate (CMMDCG). These two glycinate esters are prodrugs for 10,11-methylenedioxy-20(S)-camptothecin (MDC) and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin (CMMDC), respectively. Effects on cellular metabolism, induction of apoptosis, and overall cell survival were used to evaluate chemosensitivity. We report that the relative cytotoxic potency for these compounds is MDC > or = CMMDC > or = SN-38 >> TPT > CPT-11, where MDC, CMMDC, and SN-38 were over an order of magnitude more cytotoxic than TPT and CPT-11. We also investigated potential mechanisms underlying the unexpected cytotoxicity of these camptothecin derivatives in B-CLL cells that are known to be arrested in G0/G1 of the cell cycle, and found that this class of compounds inhibited [3H]uridine incorporation. We therefore postulate that the inhibition of RNA rather than DNA synthesis may be responsible for the observed cytotoxicity in non-cycling B-CLL cells.

Cytotoxic constituents from the roots of Tovomita brevistaminea.

Two known xanthones, trapezifolixanthone and manglexanthone were isolated as cytotoxic constituents from the CHCl3 extract of the roots of Tovomita brevistaminea by bioassay-guided fractionation using the KB cell line. In addition, a new compound, tovophenone C, and two known compounds, tovophenones A and B which are benzophenones, were found to be inactive constituents in this investigation. The structure of the new isolate was determined by detailed analysis of spectroscopic parameters including its 1D and 2D NMR spectroscopy data.

Human DNA topoisomerase I: quantitative analysis of the effects of camptothecin analogs and the benzophenanthridine alkaloids nitidine and 6-ethoxydihydronitidine on DNA topoisomerase I-induced DNA strand breakage.

Human DNA topoisomerase I (topo I) has been purified from normal placenta and from a recombinant baculovirus expression system. A new radiolabeled plasmid DNA assay has been used to quantitate the activity of the purified enzymes and to compare the ability of several types of topo I-targeted drugs to induce topo I-mediated DNA strand breaks. The 100-kDa recombinant enzyme form isolated from the baculovirus expression system is able to relax 2564 ng of supercoiled M-13 mp19 plasmid per minute per nanogram of enzyme. The addition of camptothecin (1 microM) to the reaction lowers the rate to 1282 ng per minute per nanogram of enzyme. The 100-kDa topo I from human placenta is able to relax 1092 ng of supercoiled plasmid per minute per nanogram of enzyme and the 68-kDa topo I form from placenta is able to relax 2069 ng of supercoiled plasmid per minute per nanogram of enzyme. Camptothecin (1 microM) decreases the relaxation rate of the placental enzymes about 50%. In the presence of several different types of topo I-targeted drugs, both the recombinant and placental enzymes are induced to cleave plasmid DNA. Quantitative DNA cleavage assays with radioactive plasmid DNA and 9-aminocamptothecin, topotecan, SN-38, 10, 11-methylenedioxycamptothecin, 7-ethyl-10, 11-methylenedioxycamptothecin, 7-chloromethyl-10, 11-methylenedioxycamptothecin, nitidine, and 6-ethoxy-5, 6-dihydronitidine indicate that the order of potency in inducing topo I-mediated DNA breakage is methylenedioxycamptothecin analogs > SN-38 > 9-aminocamptothecin > topotecan and camptothecin > nitidine compounds. The order of potency correlates with the half-lives of the topo I-DNA drug complex determined with radiolabeled DNA in 0.45 M NaCl at 30 degrees C. The half-life of the complex formed with 7-chloromethyl-10,11-methylenedioxycamptothecin is greater than 90 min whereas the half-life of the topo I-DNA complex with 6-ethoxy-5, 6-dihydronitidine is less than 15 s. The other drugs tested were found to have drug complex half-lives which fall between these two extremes.

Water soluble 20(S)-glycinate esters of 10,11-methylenedioxycamptothecins are highly active against human breast cancer xenografts.

Water-soluble 20(S)-glycinate esters of two highly potent 10,11-methylenedioxy analogues of camptothecin (CPT) have been synthesized and evaluated for their ability to eradicate human breast cancer tumor xenografts. The glycinate ester moiety increases the water solubility of the 10,11-methylenedioxy analogues 4-16-fold. However, in contrast to CPT-11, a water-soluble CPT analogue that was recently approved for second line treatment of colorectal cancer, the 20(S)-glycinate esters do not require carboxylesterase for conversion to their active forms. The glycinate esters are hydrolyzed to their parent, free 20(S)-hydroxyl active analogues in phosphate buffer (pH 7.5) and in mouse and human plasma. The glycinate esters are also 20-40-fold less potent than CPT-11 in inhibiting human acetylcholinesterase. In vivo, we examined 20(S)-glycinate-10,11-methylenedioxycamptothecin, 20(S)-glycinate-7-chloromethyl-10,11-methylenedioxycamptothecin, and CPT-11. We found that the two 10,11-methylenedioxy analogues had antitumor activity against breast cancer xenografts that was comparable to that of CPT-11. Our results indicate that water-soluble 20(S)-glycinate esters of highly potent CPT analogues provide compounds that maintain biological activity, do not require interactions with carboxylesterases, and do not inhibit human acetylcholinesterase.

New compounds with DNA strand-scission activity from the combined leaf and stem of Uvaria hamiltonii.

Two flavanones, hamiltones A (1) and B (2), an aurone, hamiltrone (3), a chalcone, hamilcone (4), and a tetrahydroxanthene, hamilxanthene (5), were isolated from Uvaria hamiltonii extracts guided initially by fractionation based on DNA strand-scission and/or 9KB cytotoxicity assays. Compounds 2-5 have not been reported previously, while 1 is new as a natural product. Structural assignments were made based on extensive spectroscopic measurements. Compounds 1-3 were inactive in the 9KB cytotoxicity assay, with compounds 4 and 5 having weak activity. In the DNA strand-scission assay, 3 was the most active compound found in the DNA strand-scission assay, being 10 times more potent than 1 or 2. Compound 4 was only weakly active, and 5 was inactive.

Novel 7-alkyl methylenedioxy-camptothecin derivatives exhibit increased cytotoxicity and induce persistent cleavable complexes both with purified mammalian topoisomerase I and in human colon carcinoma SW620 cells.

An alkylating camptothecin (CPT) derivative, 7-chloromethyl-10,11-methylenedioxy-camptothecin (7-CM-MDO-CPT) was recently shown to produce irreversible topoisomerase I (top1) cleavage complexes by binding to the +1 base of the scissile strand of a top1 cleavage site. We demonstrate that 7-CM-EDO-CPT (7-chloromethyl-10,11-ethylenedioxy-camptothecin) also induces irreversible top1-DNA complexes. 7-CM-MDO-CPT, 7-CM-EDO-CPT, and the nonalkylating derivative 7-ethyl-10,11-methylenedioxy-camptothecin (7-E-MDO-CPT) also induced reversible top1 cleavable complexes, which were markedly more stable to salt-induced reversal than those induced by 7-ethyl-10-hyroxy-CPT, the active metabolite of CPT-11. This greater stability of the top1 cleavable complexes was contributed by the 7-alkyl and the 10,11-methylene- (or ethylene-) dioxy substitutions. Studies in SW620 cells showed that 7-E-MDO-CPT, 7-CM-MDO-CPT, and 7-CM-EDO-CPT are more potent inducers of cleavable complexes and more cytotoxic than CPT. The reversal of the cleavable complexes induced by 7-E-MDO-CPT, 7-CM-MDO-CPT, and 7-CM-EDO-CPT was markedly slower after drug removal than that for CPT, which is consistent with the data with purified top1. By contrast to CPT, 7-E-MDO-CPT, 7-CM-MDO-CPT, and 7-CM-EDO-CPT were cytotoxic irrespective of the presence of 10 microM aphidicolin. These results suggest that 7-E-MDO-CPT, 7-CM-MDO-CPT, and 7-CM-EDO-CPT are more potent top1 poisons than CPT and produce long lasting top1 cleavable complexes and greater cytotoxicity than CPT in cells.

Comparative molecular field analysis and molecular modeling studies of 20-(S)-camptothecin analogs as inhibitors of DNA topoisomerase I and anticancer/antitumor agents.

Conformational studies and comparative molecular field analysis (CoMFA) were undertaken for a series of camptothecin (CPT) analogs to correlate topoisomerase I inhibition with the steric and electrostatic properties of 32 known compounds. The resulting CoMFA models have been used to make predictions on novel CPT derivatives. Using the newly derived MM3 parameters, a molecular database of the 32 CPT analogs was created. Various point atomic charges were generated and assigned to the MM3 minimized structures, which were used in partial least-squares analyses. Overall, CoMFA models with the greatest predictive validity were obtained when both the R- and S-isomers were included in the data set, and semiempirical charges were calculated for MM3 minimized low-energy lactone structures. A cross-validated R2 of 0.758 and a non-cross-validated R2 of 0.916 were obtained for MM3 minimized structures with PM3 ESP charges for the 32 CPT analogs. The derived QSAR equations were used to assign topoisomerase I inhibition values for compounds in this study and compounds not included in the original data set. Prior to its appearance in the literature, an IC50 of 103 nM was predicted for the 10,11-oxazole derivative. This CoMFA predicted value compared favorably with the recently reported value of 150 nM. The CoMFA model was also evaluated by predicting the activities of recently reported 11-aza CPT and trione derivatives. The predicted activity (IC50 = 249 nM) for 11-aza CPT compared well with the reported value of 383 nM.

Triterpenes from the combined leaf and stem of Lithospermum caroliniense.

An investigation of the combined leaf and stem of Lithospermum caroliniense afforded two new pentacyclic triterpenoids based on the olean-12-ene and taraxast-12-ene skeletal types. The structures of these compounds were elucidated on the basis of spectral analysis as 1 alpha,3 beta,23-trihydroxyolean-12-ene-28-oic acid and 3 alpha,19 beta,21 alpha,23-tetrahydroxytaraxast-12-ene-28-oic acid.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.