Antitumor agents 216. Synthesis and evaluation of paclitaxel-camptothecin conjugates as novel cytotoxic agents

Five conjugates (16-20) composed of a paclitaxel and a camptothecin derivative joined by an imine linkage were synthesized and evaluated as cytotoxic agents and as inhibitors of DNA topoisomerase I. All of the conjugates were potent inhibitors of tumor cell replication with improved activity relative to camptothecin. Significantly, compounds 16-18 were more active than paclitaxel and camptothecin against HCT-8 (colon adenocarcinoma) cell replication, and the spectrum of activity was different from a simple mixture of paclitaxel and camptothecin. All of the conjugates were significantly less potent than camptothecin as inhibitors of human topoisomerase I in vitro with 16, 18, and 19 showing only marginal activity at 50 microM. Based on activity against drug-resistant cell line replication, one could conclude that the conjugates are simply acting as 'weak taxanes', but the spectrum of activity, particularly against MCF-7 and HCT-8, strongly suggests that a novel mechanism of action has been achieved through conjugation.     

Synthesis and antitumor activity of 7-ethyl-9-alkyl derivatives of camptothecin

A series of new camptothecin derivatives, as topoisomerase I inhibitor, were synthesized to identify potent antitumor agents. The synthesis method was based on the Claisen rearrangement of 10-allyloxy-7-ethylcamptothecin. All of the compounds were assayed for cytotoxicity against two human tumor cell lines, Bel7402, HCT116, and showed good potency in vitro. Compounds 2, 4, 9, were assessed for the stability of lactone in human plasma. And then compound 2 was tested for antitumor activity in vitro against mouse tumor sarcoma-180. The results suggested that the small alkyl groups in the both 7- and 9-positions of camptothecin could promote liposolubility, antitumor activity in vitro and vivo, though did not bring much increase of the stability of lactone.     

Analysis of stereoelectronic properties of camptothecin analogues in relation to biological activity

Camptothecin and four of its 10,11-methylenedioxy analogues were examined for their activity against the pathogenic protozoan Leishmania donovani in vitro. The methylenedioxy analogues were 36- to 180-fold more potent than the parent camptothecin, possessing IC50 values ranging from 160 to 32 nM against the parasite. Our finding that the methylenedioxy camptothecins possess greater activity than camptothecin, which is also the case for other cell types and for the generation of cleavable complex in the presence of DNA and purified mammalian topoisomerase I, prompted us to examine the molecular features of camptothecin and methylenedioxy camptothecin analogues. A delocalization of positive potential was observed in the methylenedioxy camptothecin analogues, which could increase the affinity of these molecules for DNA. In addition, geometrical and electronic differences between the E ring of camptothecin and its methylenedioxy analogues were noted. One or both of these factors may contribute to the superior biological activity of the methylenedioxy camptothecin analogues.     

Suzuki coupling based synthesis and in vitro cytotoxic evaluation of 7-heteroaryl-substituted camptothecin analogs

In an effort to discover potent antitumor agents, a series of novel C-7-heteroaryl-substituted camptothecin derivatives were designed and synthesized via microwave-promoted Suzuki coupling reaction. These analogs were then assessed for cytotoxicity against three human tumor cell lines, A549, HCT116, HT-29, and inhibitory effects on topoisomerase I. All of the new compounds showed potent inhibition of human tumor cell growth, among which compound 10a showed higher cytotoxic activity than that of SN-38. Furthermore, this series of compounds retained or enhanced Topo I inhibition.     

Design,synthesis and biological evaluation of lapachol derivatives possessing indole scaffolds as topoisomerase I inhibitors

A series of novel lapachol derivatives possessing indole scaffolds was designed and synthesized. The in vitro anti-proliferative activity of these novel compounds was evaluated in Eca109 and Hela cell lines. Almost all the tested compounds showed manifested potent inhibitory activity against the two tested cancer cell lines. Topo I-mediated DNA relaxation activity indicated that these novel compounds have potent Topoisomerase I inhibition activity. The most potent compounds 4n and 4k demonstrated more cytotoxicity than camptothecin and was comparable to camptothecin in inhibitory activities on Topoisomerase I in our biological assay. In addition, the Hoechst 33342 staining method also showed that the complex can induce Hela cell apoptosis.     

Synthesis and antitumor activity of camptothecin derivatives bearing five-membered heterocycle containing 10-substituents

A series of new camptothecin derivatives bearing five-membered ring heterocycle containing substituents in the 10-position were synthesized and evaluated for in vitro cytotoxic activity. Camptothecin derivatives bearing a pyrrole or a thiophene ring were significantly more potent than camptothecin, however those bearing furan were less potent than camptothecin.     

Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I

Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex.     

CK2-Mediated Hyperphosphorylation of Topoisomerase I Targets Serine 506, Enhances Topoisomerase I–DNA Binding,and Increases Cellular Camptothecin Sensitivity

Topoisomerase I is the target for a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. In this study we have identified a novel site of CK2-mediated topoisomerase I (topo I) phosphorylation at serine 506 (PS506) that is relevant to topo I function and to cellular responses to these topo I-targeted drugs. CK2 treatment induced hyperphosphorylation of recombinant topo I and expression of the PS506 epitope, and resulted in increased binding of topo I to supercoiled plasmid DNA. Hyperphosphorylated topo I was approximately three times more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but had similar DNA cleavage activity once bound to DNA. The PS506 epitope was expressed in cancer cell lines with elevated CK2 activity, hyperphosphorylated topo I, and increased sensitivity to camptothecin. In contrast, PS506 was not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we demonstrate a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. Our results show that the PS506 epitope is an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and may thus serve as a diagnostic or prognostic biomarker and predict tumor responsiveness to widely used topo I-targeted therapies.     

A novel DNA topoisomerase I inhibitor with different mechanism from camptothecin induces G2/M phase cell cycle arrest to K562 cells

DNA topoisomerase I (Top1) is an essential nuclear enzyme and a validated target for anticancer agent screening. In a previous study, we found that indolizinoquinoline-5,12-dione derivatives show significant biological activity against several human cancer cell lines. To understand their mechanism of inhibition of cancer cell growth, one indolizinoquinoline-5,12-dione derivative, CY13II, was further studied as lead. Our present results indicate that CY13II shows more potent antiproliferative activity against K562 cells than camptothecin. Additionally, K562 cells were arrested in G2/M, and their growth rate decreased after treatment with CY13II at micromolar concentration. Biochemical Top1 assays indicate that CY13II exhibits a different inhibitory mechanism from camptothecin. Unlike camptothecin, CY13II specifically inhibits the catalytic cleavage activity of Top1 instead of forming the drug-enzyme-DNA covalent ternary complex.     

Design,Synthesis and Biological Activity of (20S,21S)‐7‐Cyclohexyl‐21‐fluorocamptothecin Carbamates as Potential Antitumor Agents

(20S,21S)‐7‐Cyclohexyl‐21‐fluorocamptothecin was discovered by a fluorine drug design strategy with potent antitumor activity and increased metabolic stability. In continuous efforts to find novel antitumor agents derived from natural product camptothecin, 20‐carbamates of the active compound (20S,21S)‐7‐cyclohexyl‐21‐fluorocamptothecin have been designed and synthesized. Among them, one compound with the diethylamino group showed greater antiproliferative activity than the other 20‐carbamate derivatives. The following biological activity assays indicated that the above compound is a valuable lead compound with excellent Topo I inhibitory activity and solution stability.     

Camptothecin analogs with bulky, hydrophobic substituents at the 7-position via a Grignard reaction

By developing a new synthetic procedure for introduction of side chains onto the camptothecin ring system, we were able to achieve the preparation of a number of analogs bearing bulky, hydrophobic groups directly attached to the 7-position. These include 7-tert-butylcamptothecin, 7-benzylcamptothecin and the corresponding 10,11-methylenedioxycamptothecins. This method involves the reaction of an appropriate orthoaminobenzonitrile with various Grignard reagents to give the corresponding orthoaminoketones. Friedlander condensation of the latter with the key tricyclic ketone leads to 7-substituted camptothecin analogs. We report the activity of these compounds as topoisomerase I poisons and their ability to inhibit growth of selected tumor cell lines.     

Synthesis of new camptothecin analogs with improved antitumor activities

Novel hexacyclic camptothecin analogs containing cyclic amidine, urea, or thiourea moiety were designed and synthesized based on the proposed 3D-structure of the topoisomerase I (Topo I)/DNA/camptothecin ternary complex. The analogs were prepared from 9-nitrocamptothecin via 7,9-diaminocamptothecin derivatives as a key intermediate. Among them, 7c exhibited in vivo antitumor activities superior to CPT-11 in human cancer xenograft models in mice at their maximum tolerated doses though its in vitro antiproliferative activity was comparable to SN-38 against corresponding cell lines.     

Camptothecin: current perspectives

This review provides a detailed discussion of recent advances in the medicinal chemistry of camptothecin, a potent antitumor antibiotic. Two camptothecin analogues are presently approved for use in the clinic as antitumor agents and several others are in clinical trials. Camptothecin possesses a novel mechanism of action involving the inhibition of DNA relaxation by DNA topoisomerase I, and more specifically the stabilization of a covalent binary complex formed between topoisomerase I and DNA. This review summarizes the current status of studies of the mechanism of action of camptothecin, including topoisomerase I inhibition and additional cellular responses. Modern synthetic approaches to camptothecin and several of the semi-synthetic methods are also discussed. Finally, a systematic evaluation of novel and important analogues of camptothecin and their contribution to the current structure-activity profile are considered.     

A series of enediynes as novel inhibitors of topoisomerase I.

A series of acyclic enediynes, 2-((6-substituted)-3-hexen-1,5-diynyl)benzonitriles (8--11), display potent inhibition against topoisomerase I without the formation of active biradical intermediates and show inhibitory activity against topoisomerase I at 10 microM, which is five times that of camptothecin from the results of agarose gel electrophoresis.     

Synthesis of nitrated indenoisoquinolines as topoisomerase I inhibitors

Indenoisoquinolines and dihydroindenoisoquinolines have been synthesized possessing a nitro-substituted isoquinoline ring in an effort to explore the effects of electron-withdrawing substituents on biological activity. The in vitro anticancer activities of these molecules have been tested in the National Cancer Institute's screen of 55 cell lines. The compounds have also been tested for topoisomerase I (top1) inhibition. The results indicate that these substances are a potent class of top1 inhibitors with sub-micromolar cytotoxicity mean graph midpoints (MGM) and top1 inhibition equal to camptothecin.     

Inhibition of DNA topoisomerase I by natural and synthetic mono- and dimeric protoberberine alkaloids

A series of natural (i.e., 1-7) and synthetic (i.e., 8-23) protoberberine alkaloids were evaluated for their inhibitory activities towards DNA topoisomerase I. Both the natural, monomeric protoberberine alkaloids and their mono-modified congeners showed only minor activities. In contrast, most of the dimeric protoberberine alkaloids, especially compounds 12-22, were highly active, with a similar cleavage efficiency as camptothecin (CPT), a well-known, potent topoisomerase-I inhibitor. Thus, these dimeric compounds are promising candidates to be further elaborated as anticancer leads. The mechanism of topoisomerase-I inhibition seems to be dependent on drug concentration for the dimeric protoberberines. At low concentration, they exhibit similar characteristics as CPT. At high concentration, this ability is mostly lost, and the dimers inhibit the relaxation activity of topoisomerase I. Thus, we suppose that the active, dimeric protoberberines strongly bind to plasmid DNA at elevated drug concentration. This most likely results in blocking the enzyme's access to plasmid DNA, thus inhibiting its relaxation.     

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.     

Efficient and chemoselective N-acylation of 10-amino-7-ethyl camptothecin with poly(ethylene glycol)

A new poly(ethylene glycol) (PEG) conjugate of 10-amino-7-ethyl camptothecin, a potent antitumor analogue of camptothecin, has been synthesized and preliminary in vivo tests have been performed. Successful chemoselective N-acylation of 10-amino-7-ethyl camptothecin was accomplished using phenyl dichlorophosphate, a coupling reagent used in esterification of alcohols, while other coupling methods failed, due to the low nucleophilicity of the amino group in position 10. The conjugate was tested against P388 murine leukemia cell lines and resulted equipotent to CPT-11, a camptothecin analogue already in clinical use.     

7-Silylcamptothecins (silatecans): A new family of camptothecin antitumor agents

The synthesis and biological evaluation of about one dozen 7-silylcamptothecin derivatives are described. Most new compounds show potencies comparable to or better than camptothecin itself. The best compound, 11-fluoro-10-amino-7-trimethylsilylcamptothecin, is more than 20 times more potent than camptothecin in cell assays.     

Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor

p14ARF (ARF) and topoisomerase I play central roles in cancer and have recently been shown to interact. The interaction activates topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, but the regulation of the interaction is poorly understood. We have used the H358 and H23 lung cancer cell lines and purified recombinant human topoisomerase I to demonstrate that the ARF/topoisomerase I interaction is regulated by topoisomerase I serine phosphorylation, a modification that regulates topoisomerase I activity. Both cell lines express wild-type ARF and topoisomerase I proteins at equivalent levels, but H23 topoisomerase I, unlike that of H358 cells, is largely devoid of serine phosphorylation, has low activity, and complexes poorly with ARF. The ability of H23 topoisomerase I to complex with ARF can be restored by treatment with the serine kinase, casein kinase II. Consistent with these observations, we show that the response of H23 cells to camptothecin treatment is unaffected by changes in intracellular levels of ARF. However, in H358 and PC-3 cells, which express a serine phosphorylated topoisomerase I that complexes with ARF, ectopic overexpression of ARF causes sensitization to camptothecin, and siRNA-mediated down-regulation of endogenous ARF causes desensitization to camptothecin. These biological responses correlate with increased and decreased levels, respectively, of ARF/topoisomerase I complex and DNA-bound topoisomerase I. Thus, ARF is a serine phosphorylation-dependent coregulator of topoisomerase I in vivo, and it regulates cellular sensitivity to camptothecin by interacting with topoisomerase I. Certain cancer associated defects affecting ARF/topoisomerase I complex formation could contribute to cellular resistance to camptothecin.