Synthesis and antitumor activity of novel 10-substituted camptothecin analogues

In an attempt to improve the antitumor activity and decrease the cytotoxicity of camptothecin, 18 new 10-substituted camptothecin derivatives were prepared. The cytotoxicity in vitro on cancer cell lines and antitumor activity in vivo, and inhibitory properties of topoisomerase I of these derivatives were evaluated. Most of these derivatives possessed lower cytotoxicities than CPT, and the compounds 13, 21, 22, 23, and 24 showed similar topoisomerase I inhibitory activity to CPT. Analogues 13 exhibited the best antitumor activity in vivo among all derivatives we prepared.     

Synthesis and antitumor activity of 20-O-linked nitrogen-based camptothecin ester derivatives

A series of nitrogen-based 20S-hydroxyl camptothecin ester derivatives were prepared. 3-Aminopropionate of camptothecin was found more cytotoxic in vitro on several human tumor cell lines than 3-amidopropionate of camptothecin. Ester 16 showed best antitumor activity in vivo and in vitro in all esters we prepared.     

Synthesis and biological evaluation of 7-alkenyl homocamptothecins as potent topoisomerase I inhibitors

Homocamptothecin (hCPT) is a camptothecin (CPT) derivative with a seven-membered β-hydroxylactone E ring, which shows higher lactone stability and improves topoisomerase I (Topo I) inhibition activity. In an attempt to improve the antitumor activity of homocamptothecins, a series of 7-alkenyl-homocamptothecin derivatives was designed and synthesized based on a semisynthetic route starting from CPT. Most of the synthesized compounds exhibit higher cytotoxic activities on the A-549 tumor cell line than topotecan (TPT). Some compounds such as 2a and 2o show a broad in vitro antitumor spectrum and exhibit superior Topo I-inhibition activity.     

In vitro cytotoxicity and in vivo distribution after direct delivery of PEG-camptothecin conjugates to the rat brain

Low water solubility and rapid elimination from the brain inhibits local delivery via implants and other delivery systems of most therapeutic drugs to the brain. We have conjugated the chemotherapy drug, camptothecin (CPT), to poly(ethylene glycol) (PEG) of molecular weight 3400 using previously established protocols. These new conjugates are very water-soluble and hydrolyze at a pH-dependent rate to release the active parent drug. We have studied the uptake of these conjugates by cells in vitro and quantified their cytotoxicity toward gliosarcoma cells. These conjugates were loaded into biodegradable polymeric controlled-release implants, and their release characteristics were studied in vitro. We implanted similar polymeric disks into rat brains and used a novel sectioning scheme to determine the concentration profile of CPT in comparison to conjugated CPT in the brain after 1, 7, 14, and 28 days. We have found that PEGylation greatly increases the maximum achievable drug concentration and greatly enhances the distribution properties of CPT, compared to corelease of CPT with PEG. Although only one percent of CPT in the conjugate system was found in the hydrolyzed, active form, drug concentrations were still significantly above cytotoxic levels over a greater distance for the conjugate system. On the basis of these results, we believe that PEGylation shows great promise toward increasing drug distribution after direct, local delivery in the brain for enhanced efficacy in drug treatment.     

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.     

Design,synthesis, and cytotoxic activity of novel 7-substituted camptothecin derivatives incorporating piperazinyl-sulfonylamidine moieties

In our continuing search for camptothecin (CPT)-derived antitumor drugs, novel 7-substituted CPT derivatives incorporating piperazinyl-sulfonylamidine moieties were designed, synthesized and evaluated for cytotoxicity against five tumor cell lines (A-549, MDA-MB-231, MCF-7, KB, and KB-VIN). All of the derivatives showed promising in vitro cytotoxic activity against the tested tumor cell lines, and were more potent than irinotecan. Remarkably, most of the compounds exhibited comparable cytotoxicity against the multidrug-resistant (MDR) KB-VIN and parental KB tumor cell lines, while irinotecan lost activity completely against KB-VIN. Especially, compounds 13r and 13p (IC₅₀ 0.38 and 0.85 μM, respectively) displayed the greatest cytotoxicity against the MDR KB-VIN cell line and merit further development into preclinical and clinical drug candidates for treating cancer, including MDR phenotype.     

Topoisomerase I‐Mediated Antiproliferative Activity of 10‐Substituted and 12‐Substituted Homocamptothecins

Homocamptothecin (hCPT) is an E‐ring modified camptothecin (CPT) analogue, which showed pronounced inhibitory activity of topoisomerase I. In search of novel hCPT‐type anticancer agents, two series of hCPT derivatives were synthesized and evaluated in vitro against three human tumor cell lines. The results indicated that the 10‐substituted hCPT derivatives had a considerably higher cytotoxic activity than the 12‐substituted ones. Among the 10‐substituted compounds, 8a, 8b, 9b , and 9i showed an equivalent or even more potent activity than the positive control drug topotecan against the lung cancer cell line A‐549. Moreover, the hCPT analogues 8a and 8b exhibited a higher topoisomerase I inhibitory activity than CPT at a concentration of 100 μM .     

Multiple shoot cultures of Ophiorrhiza rugosa var. decumbens Deb and Mondal – A viable renewable source for the continuous production of bioactive Camptotheca alkaloids apart from stems of the parent plant of Nothapodytes foetida (Wight) Sleumer

Camptotheca alkaloids were isolated from multiple shoot cultures of O. decumbens (0.056% dry weight) and stems of N. foetida. The cytotoxicity of the extracts and products were tested in a panel of five cell lines. Crude extract from O. decumbens (Cr-Od) and N. foetida (Cr-Nf) showed more potent cytotoxic activity as compared to the isolated camptothecin from O. decumbens (CPT-Od) and N. foetida (CPT-Nf). CPT isolated from shoot cultures contained biological activity suggesting the possibility of using this system of O. decumbens as a renewable source for the production of camptotheca alkaloids. 9-Methoxy camptothecin (9-mCPT), isolated from N. foetida, was a very effective cytotoxic agent as compared to Cr-Nf or CPT-Nf. The IC₅₀ of 9-mCPT was 0.84, 0.32, and 0.35 μg/ml for A549, MCF7 and Jurkat cell lines and >3 μg/ml for U937. Viability assays using MTT dye were further confirmed by assessing extent of apoptosis in these cells. These findings suggest that shoot cultures of O. decumbens offer a rich alternative plant source for the anticancer compound, CPT and 9-mCPT is a more potent compound in N. foetida as compared to CPT.     

Synthesis and pharmacological evaluation of novel non-lactone analogues of camptothecin

Ten novel camptothecin (CPT) derivatives devoid of the lactone function in the E-ring were synthesized and evaluated as anticancer agents. Several of these CPT analogues bearing a five-membered E-ring are potent inhibitors of the DNA relaxation and cleavage reactions catalyzed by topoisomerase I and exhibit promising cytotoxic activities with IC(50) values in the nM range. This is the first successful design of lactone-free CPT, providing thus a new avenue to the development of topoisomerase I targeted antitumor agents.     

On the role of E-ring oxygen atoms in the binding of camptothecin to the topoisomerase I-DNA covalent binary complex

A recent X-ray crystallographic analysis of the binding of a water soluble camptothecin analogue to the human topoisomerase I-DNA covalent binary complex has suggested the existence of some novel features in the way that camptothecin is bound to the binary complex. Four additional models based on chemical and biochemical data have also been proposed. Presently we describe S-containing analogues of camptothecin prepared on the basis of these models, and report their ability to form stable ternary complexes with human topoisomerase I, and to mediate cytotoxicity at the locus of topoisomerase I. The results indicate that replacement of the 20-OH group of CPT with a SH functionality results in diminution of the potency of CPT as a topoisomerase I poison, while replacement of the O atoms at positions 20 and 21 with S atoms results in essentially complete loss of topoisomerase I inhibitory activity.     

Synthesis and Biological Evaluation of 7‐Alkenyl Homocamptothecins as Potent Topoisomerase I Inhibitors

Homocamptothecin (hCPT) is a camptothecin (CPT) derivative with a seven‐membered β‐hydroxylactone E ring, which shows higher lactone stability and improves topoisomerase I (Topo I) inhibition activity. In an attempt to improve the antitumor activity of homocamptothecins, a series of 7‐alkenyl‐homocamptothecin derivatives was designed and synthesized based on a semisynthetic route starting from CPT. Most of the synthesized compounds exhibit higher cytotoxic activities on the A‐549 tumor cell line than topotecan (TPT). Some compounds such as 2a and 2o show a broad in vitro antitumor spectrum and exhibit superior Topo I‐inhibition activity.     

Anti-proliferative and pro-apoptotic effect of CPT13, a novel camptothecin analog, on human colon cancer HCT8 cell line

10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) is a novel semi-synthetic analogue of camptothecin, our previous report had shown that it possessed higher in vitro cytoxicity activity towards human colon cancer HCT8 cell line than topotecan. In this study, the anti-proliferative effect of CPT13 on HCT8 cell line in vitro was analyzed. In order to further explore the underlying mechanism of cell growth inhibition of CPT13 towards HCT8 cell line, the cell cycle distribution, apoptosis proportion, the nuclei morphological changes and caspase-8 and caspase-3 activities were measured. Additionally the changes of mitochondrial morphology and membrane potential (DeltaPsim) were analyzed by atomic force microscopy (AFM) and flow cytometry, respectively. The results showed that CPT13 inhibited HCT8 cell growth by causing cell cycle arrest at G2/M transition and induced apoptosis, as evidenced by the typical apoptotic morphology such as condensation and fragmentation of nuclei and formation of apoptotic bodies. The changes of mitochondrial morphology, dose-dependently decrease in DeltaPsim and the enhancement of caspase-8 and caspase-3 activities were observed in different concentrations of drug treatment group. Our results suggest that CPT13 induces apoptosis by alternations of mitochondrial transmembrane depolarization, activation of caspase-8 and caspase-3. Therefore, CPT13 appears to be a potent drug against human colon cancer via induction of apoptosis and may be used as an alternative drug to therapy cancer.     

The role of polyamine architecture on the pharmacological activity of open lactone camptothecin-polyamine conjugates

A series of camptothecin open-ring lactone tripartate conjugates were synthesized, in which polyamine side chains with different architecture (ethane-1,2-diamine, spermidine, homospermidine, spermine, and 4,8,13,17-tetraza-icosane-1,20-diamine) are linked to the 21-carboxylic function through an amidic bond, while the 17-CH(2)OH is acetylated. The rationale for the synthesis of these compounds was to explore the influence of the polyamine architecture on the activity of these CPT conjugates into cells, since the positively charged ammonium cations would favor interaction through electrostatic binding to the negatively charged DNA backbone. Topoisomerase I-mediated DNA cleavage assay was used to investigate the ability of these compounds to stimulate the DNA damage. The cleavage pattern was found to be similar to that of SN38 for all the new CPTs. The CPT tripartates were tested for growth inhibition ability against the human non-small-cell lung cancer carcinoma NCI-H460 cell line. Although these compounds were less potent than topotecan, SN38, and CPT after 1 h of treatment, the antiproliferative effects greatly increased after 72 h of exposure. The growth inhibition potency during long-term exposure is correlated with the number of charges of the 21-amide substituent. Both cleavage assay and in vitro effects support the interpretation that the compounds may have inhibitory activity also in the open-ring form. The architecture of the polyamine moiety is important for antiproliferative activity, and a balance between the hydrophilic and lipophilic properties of the polyamine is critical for CPT potency.     

Synthesis and biological relationships of 3',6-substituted 2-phenyl-4-quinolone-3-carboxylic acid derivatives as antimitotic agents

As part of a continuing search for potential anticancer drug candidates in the 2-phenyl-4-quinolone series, 3',6-substituted 2-phenyl-4-quinolone-3-carboxylic acid derivatives and their salts were synthesized and evaluated. Preliminary screening showed that carboxylic acid analogs containing a m-fluoro substituted 2-phenyl group displayed the highest in vitro anticancer activity. Activity decreased significantly if a chlorine or methoxy group replaced the fluorine atom. 3'-Fluoro-6-methoxy-2-phenyl-4-quinolone-3-carboxylic acid (68) had the highest in vitro cytotoxic activity among all tested carboxylic acid derivatives and their salts. The mechanism of action may be similar, but not identical, to that of tubulin binding drugs, such as navelbine and taxol. Compound 68 merits further investigation as a novel hydrophilic antimitotic agent.     

Maleimide-oligo(ethylene glycol) derivatives of camptothecin as albumin-binding prodrugs: synthesis and antitumor efficacy

In situ binding of thiol-reactive prodrugs to the cysteine-34 position of circulating albumin is a new approach in drug delivery. Therefore, five maleimide-bearing derivatives of the anticancer drug camptothecin (CPT) were developed as albumin-binding prodrugs. These compounds were synthesized by reacting heterobifunctional cross-linkers based on oligo(ethylene glycols) [3-6 (O-CH(2)-CH(2)) units] bearing a maleimide group on one end and a carboxylic acid group on the other with camptothecin 20-O-glycinate. Incorporating oligo(ethylene glycol) chains into the prodrugs enhanced their water-solubility when compared to the parent compound (up to 27-fold). HPLC studies showed that the prodrugs react almost quantitatively with the cysteine-34 position of endogenous albumin within a few minutes after incubation of the CPT derivatives with human blood plasma. The therapeutic potential of two of the prodrugs was assessed in nude mice bearing a colon xenograft (HT-29). Both albumin-binding derivatives of camptothecin were well-tolerated and showed enhanced antitumor efficacy when compared to CPT.     

Novel cytotoxic 7-iminomethyl and 7-aminomethyl derivatives of camptothecin

A series of new 7-iminomethyl derivatives of camptothecin were obtained from camptothecin-7-aldehyde and aromatic, alicyclic and aliphatic amines. Their hydrogenation led to the corresponding amines. All the imines and the less polar amines showed a marked increase of the cytotoxic activity against H460 non-small lung carcinoma cell line, with respect to topotecan. The lipophilicity of the substituent in position 7 of camptothecin seems to play an important role for cytotoxic potency. The 7-phenyliminomethyl derivative showed efficacy comparable to topotecan in vivo against NSCLC H460 xenografted in athymic nude mice.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

Camptothecin binds to a synthetic peptide identified by a T7 phage display screen

An analysis of non-biotinylated camptothecin (CPT) binding to the C-20-biotinylated CPT binding peptide NSSQSARR was carried out using two methods, quartz-crystal microbalance (QCM) and surface plasmon resonance (SPR). The peptide was immobilized peptide on a sensor chip and showed a dissociation constant (KD) of approximately 0.1 microM against CPT in QCM and SPR experiments.     

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.