Semisynthesis of DB-67 and other silatecans from camptothecin by thiol-promoted addition of silyl radicals

Thiol- or acid-promoted additions of silyl radicals to camptothecin are reported. At 105 degrees C, mixtures of 7-silyl (favored) and 12-silyl camptothecins are formed alongside substantial amounts of recovered camptothecin. At 160 degrees C, 12-silyl isomers are formed preferentially, but the total mass balance is substantially reduced. The silyl radical addition is featured in short semi-syntheses of DB-67 (7-tert-butyldimethylsilyl-10-hydroxy camptothecin) from both camptothecin and 10-hydroxycamptothecin.     

Evaluation of two models for human topoisomerase I interaction with dsDNA and camptothecin derivatives

Human topoisomerase I (Top1) relaxes supercoiled DNA during cell division. Camptothecin stabilizes Top1/dsDNA covalent complexes which ultimately results in cell death, and this makes Top1 an anti-cancer target. There are two current models for how camptothecin and derivatives bind to Top1/dsDNA covalent complexes (Staker, et al., 2002, Proc Natl Acad Sci USA 99: 15387-15392; and Laco, et al., 2004, Bioorg Med Chem 12: 5225-5235). The interaction energies between bound camptothecin, and derivatives, and Top1/dsDNA in the two models were calculated. The published structure-activity-relationships for camptothecin and derivatives correlated with the interaction energies for camptothecin and derivatives in the Laco et al. model, however, this was not the case for several camptothecin derivatives in the Stacker et al. model. By defining the binding orientation of camptothecin and derivatives in the Top1/dsDNA active-site these results allow for the rational design of potentially more efficacious camptothecin derivatives.     

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.     

Potentiation of cell killing by low-dose-rate radiation by camptothecin is related to an increase in the level of DNA double-strand breaks

We investigated the ability of camptothecin to potentiate cell killing by low-dose-rate irradiation and whether this potentiation was associated with an increase in the level of residual DNA double-strand breaks (DSBs). Human melanoma (Sk-Mel-3) cells, grown to the confluent phase, were treated with low-dose-rate radiation (0.88 cGy/min) alone, camptothecin alone, or concurrent camptothecin and low-dose-rate radiation. Cell survival was determined using a clonogenic assay. The interactions between camptothecin and low-dose-rate radiation were analyzed further using isobolograms. DNA DSBs were determined using the neutral comet assay. We found that 10 and 25 microM camptothecin, but not 1 microM, camptothecin potentiated cell killing significantly relative to that seen with low-dose-rate radiation alone. Unexpectedly, the potentiation of the effects of low-dose-rate radiation by camptothecin was accompanied by large increases in the alpha parameter of the linear-quadratic fit rather than in the beta parameter. This suggests a modification of intrinsic radiosensitivity rather than of repair of sublethal damage. From isobologram analysis, low-dose-rate radiation interacted either additively or supra-additively with 25 or 10 microM camptothecin. Conversely, the interaction of low-dose-rate radiation with 1 microM camptothecin was subadditive. Finally, there were strong correlations (correlation coefficients >0.9) between surviving fraction and either comet tail length or comet tail moment after concurrent treatment with 25 microM camptothecin and low-dose-rate radiation. This suggests that the level of residual DNA DSBs was a good indicator of cell killing after treatment with low-dose-rate radiation plus 25 microM camptothecin.     

An endophytic Neurospora sp. from Nothapodytes foetida producing camptothecin

The medicinal plant, Nothapodytes foetida contains a number of important alkaloids like camptothecin (an anticancer drug molecule) but its concentration is less to meet the existing demand of this important molecule, so in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as potential source of anticancer drug lead compound i.e. camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of precursor anticancer drug molecule in future at large scale.     

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.     

Studies on the effects of the antitumor agent camptothecin and derivatives on DNA. Camptothecin potentiated cleavage of DNA by bleomycin in vitro

Addition of sodium camptothecin (2a, Fig. 1) in comparable low concentrations to the glycopeptide antitumor antibiotic bleomycin (BLM) leads to enhanced rates of single-strand scission of PM2-covalently closed circular DNA, whereas sodium camptothecin alone has no effect. A similar enhancement of DNA scission by sodium camptothecin is produced with the 1 : 1 bleomycin-iron complex alone or in conjunction with NADPH as an additional reductant. The interpretation that camptothecin may substitute for the reducing requirement of the antibiotic is supported by its oxidation at 37°C by the 1 : 1 bleomycin iron complex, by iron salts or more efficiently by hydrogen peroxide to the known hemiacetal (3, Fig. 1).Electrochemical studies of 2a, its analogues and selected model compounds established that the α-pyridone ring D is most susceptible to a one-electron reduction at a reversible potential of ?0.95 ± 0.01 V. The reduced camptothecin is a transient species readily capable of donating an electron. This process may by compatible with a coupled reduction of the sequestered Fe(III) in the glycopeptide antibiotic necessary for the expression of antibiotic and antitumor properties. The results may provide a mechanistic rationale for the observed potentiation of the antitumor activity of bleomycin by camptothecin in vivo.     

Total and semisynthesis and in vitro studies of both enantiomers of 20-fluorocamptothecin

Both enantiomers of 20-fluorocamptothecin and the racemate have been prepared by total synthesis. The (R)-enantiomer is essentially inactive in a topoisomerase-I/DNA assay, while the (S)-enantiomer is much less active than (20S)-camptothecin. The lactone ring of 20-fluorocamptothecin hydrolyzes more rapidly than that of camptothecin in PBS. The results provide insight into the role of the 20-hydroxy group in the binding of camptothecin to topoisomerase-I and DNA.     

Induction of cleavage in topoisomerase I c-DNA by topoisomerase I enzymes from calf thymus and wheat germ in the presence and absence of camptothecin.

In this study, we further examined the sequence selectivity of camptothecin in mammalian topoisomerase I cDNA from human and Chinese hamster. In the absence of camptothecin, almost all the bases at the 3'-terminus of cleavage sites are T for calf thymus and wheat germ topoisomerase I. In addition, wheat germ topoisomerase I exhibits preference for C (or not T) at -3 and for T at -2 position. As for camptothecin-stimulated cleavage with topoisomerase I, G (or not T) at +1 is an additional strong preference. This sequence selectivity of camptothecin is similar to that previously found in SV40 DNA, suggesting that camptothecin preferentially interacts with topoisomerase I-mediated cleavage sites where G is the base at the 5'-terminus. These results support the stacking model of camptothecin (Jaxel et al. (1991) J. Biol. Chem. 266, 20418-20423). Comparison of calf thymus and wheat germ topoisomerase I-mediated cleavage sites in the presence of camptothecin shows that many major cleavage sites are similar. However, the relative intensities are often different. One of the differences was attributable to a bias at position -3 where calf thymus topoisomerase I prefers G and wheat germ topoisomerase I prefers C. This difference may explain the unique patterns of cleavage sites induced by the two enzymes. Sequencing analysis of camptothecin-stimulated cleavage sites in the surrounding regions of point mutations in topoisomerase I cDNA, which were found in camptothecin-resistant cell lines, reveals no direct relationship between DNA cleavage sites in vitro and mutation sites.     

Enhanced in vitro multiplication of Nothapodytes nimmoniana Graham using semisolid and liquid cultures and estimation of camptothecin in the regenerated plants

Nothapodytes nimmoniana (Icacinaceae) yields camptothecin (isoquinoline alkaloid) which is a potent anti-cancer drug. The major objectives of the present study were to develop an efficient protocol for mass propagation of N. nimmoniana using liquid medium and to compare regeneration with semisolid cultures; as also to quantify the amount of camptothecin in regenerated plants. Adventitious shoots were induced from the callus derived from nodal explants on semisolid and liquid Murashige and Skoog (MS) medium supplemented with 1.0, 2.0, 5.0 and 10.0???M 6-benzylaminopurine or kinetin or 2-isopentenyl adenine (2-iP). The highest number of adventitious shoots was regenerated on medium supplemented with 2.0???M BAP. Compared to semisolid medium (41.9 shoots per explant), liquid medium (165.9 shoots per explant) was found suitable for shoot induction and shoot multiplication. Shoots were rooted on MS semisolid medium of one-fourth strength containing IBA (2.4???M) and IAA (5.7???M). The plantlets were acclimatized in a growth chamber at 25°C, 60% relative humidity, with 16-h photoperiod (40???mol?m^?2?s^?1). The camptothecin content was determined in ex vitro plants using HPLC. The analysis revealed that the leaves and stems of ex vitro plants had a considerable amount of camptothecin and these plants could be used as a raw material for camptothecin extraction.     

Investigation into the functional impact of the vancomycin C-ring aryl chloride

A vancomycin aglycon analogue that possesses a reduced C-ring and an intact E-ring chloride was prepared and its antimicrobial activity towards Staphylococcus aureus and binding affinity to model cell wall ligands were established. Comparison of the derivative with a series of vancomycin aglycon analogues that possess and lack the chloro substituents on the aryl C- and E-rings defines the impact and further refines the role the C-ring chloride plays in promoting both target binding affinity and binding selectivity for d-Ala-d-Ala and its impact on antimicrobial activity.     

Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes

Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism. Keywords: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation     

Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives

SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k') and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.     

Oxidative stimuli-responsive nanoprodrug of camptothecin kills glioblastoma cells

The purpose of this study was to prepare and characterize nanometer-sized prodrug (nanoprodrug) of camptothecin. The camptothecin prodrug was synthesized using tetraethylene glycol spacer linked via carbonate bond to camptothecin and via ester bond to α-lipoic acid. The nanoprodrug was prepared through the spontaneous emulsification mechanism using the mixture of camptothecin prodrug and α-tocopherol which served as a structural matrix. The nanoprodrug was activated readily by porcine liver esterase and, with a much slower rate, by hydrolytic degradation. Upon longterm storage, the α-lipoic acid moiety of the camptothecin prodrug gradually oxidized without loss of structural integrity and therapeutic efficacy. Interestingly, the hydrolytic activation was negligible before the oxidation, but was significantly accelerated after the oxidation of the α-lipoic acid moiety, suggesting an oxidative stimuli-responsive activation of the prodrug. The camptothecin nanoprodrug was found to possess significant inhibitory effect on the proliferation of U87-MG glioma cells with an IC₅₀ of 20 nM.     

A novel method of eliminating non-neuronal proliferating cells from cultures of mouse dorsal root ganglia

1. We hypothesized that non-neuronal cells could be eliminated from primary dorsal root ganglion (DRG) cultures by including a DNA topoisomerase inhibitor (camptothecin) during culture.2. Exposure to 20 M camptothecin for 48 h, beginning at 3 days in vitro, reliably eliminates proliferating non-neuronal cells.3. Following camptothecin treatment, neurons survived and continued to extend neurites for several weeks without obvious defects in morphology or viability.4. Transient camptothecin exposure is therefore an efficient and fast-acting method to purify DRG neurons in culture.     

Simultaneous treatment with camptothecin and valproic acid suppresses induction of Bcl-XL and promotes apoptosis of MCF-7 breast cancer cells

Camptothecin derivatives have been widely used for chemotherapy in patients with various cancers, but intrinsic and acquired drug resistance is major drawback to be overcome. In the present study, we demonstrated that simultaneous treatment with camptothecin and valproic acid induced apoptosis of MCF-7 cells, whereas neither agent alone could efficiently induce apoptosis. This induction of apoptosis was associated with loss of the mitochondrial membrane potential and was caspase dependent. Further investigation showed that concurrent treatment modulated the expression of pro-apoptotic and anti-apoptotic genes. Bcl-X_L expression was induced in MCF-7 cells treated with camptothecin alone, but not in cells treated simultaneously with camptothecin and valproic acid. Ectopic overexpression of Bcl-X_L in MCF-7 cells completely suppressed the induction of apoptosis, even with simultaneous treatment. On the other hand, efficient induction of apoptosis was achieved by treatment with camptothecin and Bcl-X_L inactivation (using siRNA or BH3 mimetic). The cytotoxic effect of camptothecin combined with valproic acid was more than additive for MCF-7 cells. Taken together, our results suggest that simultaneous administration of camptothecin and valproic acid might be useful for anticancer therapy.     

An endophytic <Emphasis Type="Italic">Neurospora</Emphasis> sp. from <Emphasis Type="Italic">Nothapodytes foetida</Emphasis> producing camptothecin

The medicinal plant, Nothapodytes foetida, contains a number of important alkaloids like camptothecin (an anticancer drug molecule), but its concentration is less to meet the existing demand of this important molecule, in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as a potential source of anticancer drug lead compound, i.e., camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of an anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of a precursor anticancer drug molecule in the future at a large scale. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 225–231. The text was submitted by the authors in English.     

接种时期对丛枝菌根喜树幼苗喜树碱含量的影响

喜树(Camptotheca acuminata)是我国特有的多年生亚热带落叶阔叶树种, 因其次生代谢产物喜树碱具有良好的抗肿瘤活性而受到人们的广泛关注。通过温室盆栽接种试验, 观察了喜树幼苗不同生长时期接种蜜色无梗囊霉(Acaulospora mellea)和根内球囊霉(Glomus intraradices)对喜树幼苗喜树碱积累的影响。结果表明接种两种丛枝菌根真菌均促进了喜树幼苗喜树碱的积累, 表现为喜树碱产量(单株幼苗所含的喜树碱量, 喜树碱含量与幼苗生物量的乘积)的显著提高。进一步分析发现, 接种丛枝菌根真菌导致幼喜树苗喜树碱产量的提高, 早期(幼苗出土20天)接种主要是源于喜树碱含量的提高, 特别是叶片喜树碱含量的提高, 而晚期(幼苗出土60天)接种则主要是源于幼苗生物量的增加。     

胶束介导联合超声—微波辅助法提取喜树叶中喜树碱及羟基喜树碱

为了寻找一种新的方法从喜树叶中提取喜树碱和羟基喜树碱,充分利用喜树资源,以喜树叶粉末作为提取原料,采用超声—微波辅助提取技术提取喜树叶中活性成分喜树碱和羟基喜树碱。采用响应面优化方法设计,分别考察微波时间、料液比、微波功率、表面活性剂的浓度4个因素对总提取率的影响,优化得到最佳提取工艺条件。研究结果表明：当微波时间为10.5 min、料液比为1∶60、微波功率为850 W、表面活性剂浓度为 8.5 g·L^-1时总提取率达到最佳。最佳工艺下总提取率为0.056 4%。