Novel C2-C3' N-peptide linked macrocyclic taxoids. Part 2: synthesis and biological activities of docetaxel analogues with a peptide side chain at C2 and their macrocyclic derivatives

The synthesis of a series of novel docetaxel analogues possessing a peptide side chain at the C2 position as well as peptide macrocyclic taxoids is described. These compounds were designed to mimic a region of the alpha-tubulin loop equivalent to the paclitaxel binding pocket of beta-tubulin. Fifteen new peptide taxoids were obtained and evaluated as inhibitors of microtubule disassembly as well as cell proliferation. The relationships between these new taxoids and the tau protein motif interacting with microtubules are discussed.     

Taxoids profiling of suspension Taxus chinensis var. mairei cells in response to shear stress

To develop an optimal bioprocess for paclitaxel (Taxols) supply, taxoid biosynthetic pathway regulation must be better understood. The main taxoid metabolites (paclitaxel, baccatin III, taxol C, etc.) in Taxus cell culture showed great difference under shear stress. However, the regulating mechanism of taxoids metabolism under shear stress remained elusive. Here an efficient metabolic profiling approach combined with multivariate analysis was employed to profile taxoids changes of Taxus cells under laminar shear stress. A total of 21 taxoids were identified and quantified by ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry. The result showed the contents of paclitaxel and baccatin III were reduced by shear stress, indicating the inhibitory effect of shear stress on paclitaxel biosynthesis. The levels of other taxoids uninvolved in paclitaxel biosynthesis were decreased except several metabolites. Further analysis of mapping measured taxoids concentrations onto paclitaxel biosynthesis pathway illustrating proposed intermediates and “off-pathway” metabolites revealed shear stress might disrupt the appropriate cyclization process of geranylgeranyl-pyrophosphate, aggravate the inappropriate order of hydroxylations and acylations, and not be good for functional group oxetane formation. These findings revealed the possible mechanism for shear stress limiting paclitaxel production and might have important biotechnological applications to increase the yields of paclitaxel and relevant precursors.     

Design, synthesis and biological activity of novel C2-C3' N-Linked macrocyclic taxoids

A series of novel macrocyclic taxoids was designed and synthesized by connecting the C-2 and C-3' N positions of the taxoid framework with various tethers. Cytotoxicity of these macrocyclic taxoids was evaluated against a human breast cancer cell line LCC6-WT, and a couple of the taxoids exhibited 0.09-0.3 microM IC(50) values.     

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.     

联合调控对中国红豆杉细胞关键酶基因表达的影响

红豆杉悬浮培养细胞可以持续提供抗癌药物紫杉醇及一些紫杉烷类。在中国红豆杉悬浮培养细胞中,云南紫杉烷C(Tc)是主要的紫杉烷。为了更理性地调控紫杉醇或有用紫杉烷的生产,有必要深入了解其生物合成过程。采用实时定量PCR(Real-time Quantitative PCR,即RQPCR)技术考察经调控后紫杉醇及紫杉烷代谢中关键酶基因—TASY,T5αH,TDAT,T10βH,TαH,T14βH表达水平的变化。在细胞培养的第7天和12天,分别以100μmol/L2,3-二羟丙基茉莉酸(DHPJA)诱导,同时在细胞培养第7天进行20g/L蔗糖饲喂、100g/LXAD-7HP的原位吸附。该联合调控处理使得细胞培养第30天时,Tc产量高达1517±37mg/L,是对照处理的11.1倍,是DHPJA重复诱导联合蔗糖饲喂处理的1.7倍。RQ-PCR结果显示:DHPJA的加入可使6个基因表达水平显著提高,但在12小时后快速下降,需补充DHPJA以再次提高基因表达水平。吸附剂同时引入会延缓基因表达水平的提高速度,但却能维持基因表达处于一个较高的水平,表现为在细胞培养中后期,基因表达水平将显著高于无吸附剂的调控体系。与13α-羟化相对应的TαH基因有所不同,吸附剂的存在更显著地抑制其表达,但仍有维持表达的功能。     

Syntheses and biological activity of C-3'-difluoromethyl-taxoids

A series of new taxoids bearing difluoromethyl group at the C-3' position and modifications at the C-10 and C-14 positions has been synthesized and their biological activities studied. The in vitro cytotoxicity assay results indicate that these newly developed taxoids exhibit comparable to several times better activity against drug-sensitive cell line LCC6-WT, and 40-70 times better activity against the corresponding drug-resistant cancer cell line LCC6-MDR as compared to that of paclitaxel. Apoptosis analysis has revealed the exceptional activity of SB-T-12843 (1e) in inducing apoptosis in both MDR-bearing and MDR-negative cancer cells.     

Synthesis and biological evaluation of new taxoids derived from 2-deacetoxytaxinine J

A small library of 2-deacetoxytaxinine J (DAT-J) 1 derivatives was synthesised and tested in vitro for their reversal activity in human mammary carcinoma MDR cell line MCF7-R. One of the new taxoids showed to be active at 0.1 microM when tested in combination with paclitaxel.     

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.     

Synthesis and biological evaluation of novel taxoids designed for targeted delivery to tumors

The use of drug-antibody conjugates affords a method for the targeted delivery of anticancer drugs specifically to cancer cells. Monoclonal antibodies alone usually do not possess high therapeutic efficacy, however, they are capable of targeting tumor markers selectively. We have prepared taxoids with significantly higher cytotoxicity than paclitaxel and docetaxel. These taxoids now meet the high potency required for use in a targeted-delivery approach using monoclonal antibodies. The synthesis and biological evaluation of these taxoids are reported.     

Synthesis and structure-activity relationships of new second-generation taxoids

A series of second-generation taxoids bearing a substitutent on the C-2-benzoyl group and modifications at C-3′/C-10 positions was synthesized. These taxoids exhibited 2–3 orders of magnitude higher potency than that of paclitaxel against drug-resistant human breast cancer cell lines. It is also noteworthy that three taxoids showed almost no difference in activity against drug-resistant and drug-sensitive cell lines, which are categorized as “advanced second generation taxoids“.     

Cyclostreptin (FR182877), an antitumor tubulin-polymerizing agent deficient in enhancing tubulin assembly despite its high affinity for the taxoid site

Cyclostreptin (FR182877), a bacterial natural product, was reported to have weak paclitaxel-like activity with tubulin but antitumor activity in vivo. We used synthetic cyclostreptin in studies of its mechanism of action. Although less potent than paclitaxel in several human cancer cell lines, cyclostreptin was active against cells resistant to paclitaxel and epothilone A. At equitoxic concentrations with paclitaxel, cyclostreptin was more effective in arresting MCF-7 cells in mitosis and equivalent in bundling microtubules in PtK(2) cells. Tubulin assembly with paclitaxel occurs at low temperatures and in the absence of GTP or microtubule-associated proteins (MAPs). Brisk assembly with cyclostreptin required MAPs, GTP, and higher reaction temperatures. On the basis of turbidimetry, cyclostreptin-induced microtubules were more stable in the cold than the paclitaxel-induced polymer. Moreover, at 37 degrees C cyclostreptin was a strong competitive inhibitor of the binding of radiolabeled paclitaxel to tubulin polymer, with an apparent K(i) value of 88 nM. Competition studies versus a fluorescent taxoid across a temperature range, in comparison with paclitaxel and docetaxel, showed that only the binding of cyclostreptin to microtubules was markedly reduced at 4 degrees C versus temperatures over 30 degrees C. The binding of cyclostreptin to microtubules was characterized by a relatively greater endothermic and entropic profile as compared with those of the taxoid binding reactions, which are characterized more by exothermic and enthalpic interactions. Molecular modeling showed that cyclostreptin formed a pharmacophore with taxoids but formed hydrogen bonds only with the S9-S10 and M loops in the taxoid site. Initial studies also indicate that, relative to paclitaxel, cyclostreptin is more deficient in nucleation than elongation of polymer.     

Seasonal changes in the concentrations of four taxoids in Taxus baccata L. during the autumn-spring period.

The concentrations of four common taxoids: baccatin III, paclitaxel, cephalomannine and 10-deacetylbaccatin III (10-DAB III) were measured in fresh needles and stems of Taxus baccata L. during the late autumn-spring period (November'96-April'97) which has not been investigated to date in this species. Baccatin III, paclitaxel and 10-DAB III were present on the surface of the twigs in concentrations of 8-26 micrograms/1000 g (fresh weight). Changes in the levels of baccatin III and paclitaxel inside the needles and stems showed similar trends over the investigated period. From November to March the total level of taxoids differed between the needles and stems, and were the same only in April. Total levels in fresh needles were stable from December to March. The highest concentrations of 10-DAB III in the whole analysed period in fresh stems were measured, as well as in the fresh needles except for samples collected in November and December when the levels of cephalomannine were higher. The concentrations of paclitaxel were usually the lowest. These results confirm that epigenetic factors--date of collection (and thus phyllogenesis) and kind of plant tissue--determine taxoid levels during the late autumn-spring period in T. baccata. The opposite patterns of changes for 10-DAB III and cephalomannine, especially in the fresh needles, suggest a possible role for 10-DAB III in the biosynthetic pathway to cephalomannine, a less polar taxoid with a side-chain at position C-13. As well, owing to the thermolability of taxoids, the influence of low temperatures in December and January could explain the highest observed concentrations of 10-DAB III in the fresh stems and needles, respectively.     

The influence of ultraviolet radiation on the content of pharmacologically active taxoids in yew tissues

The aim of the work was the investigation of the influence of UV radiation on the taxoids contents in yew tissues. The experiment was performed using Taxus baccata var. Aurea Corr. twigs irradiated with UV C (lambda = 254 nm) and UV A (lambda = 366 nm) in comparison to control samples. Multistep procedure of sample preparation was applied before the analysis of paclitaxel and 10-DAB III: SPE using alumina - for purification from the chlorophylles, waxes and polar balasts as well as zonal micropreparative TLC on silica - for isolation of partially separated fractions. The quantitation of some taxoids in the isolated fractions was performed using RP-HPLC procedure in system C18/acetonitrile + water. The experiments with UV-A and especially UV-C radiation showed changes in concentrations of paclitaxel and its precursor-10-deacetylbaccatin III. The results can be utilized to increase the yield of the taxoids isolated for medicinal and practical purposes.     

Selamectin Is the Avermectin with the Best Potential for Buruli Ulcer Treatment

A comprehensive analysis was done to evaluate the potential use of anti-parasitic macrocyclic lactones (including avermectins and milbemycins) for Buruli ulcer (BU) therapy. A panel containing nearly all macrocyclic lactones used in human or in veterinary medicine was analyzed for activity in vitro against clinical isolates of Mycobacterium ulcerans. Milbemycin oxime and selamectin were the most active drugs against M. ulcerans with MIC values from 2 to 8 μg/mL and 2 to 4 μg/mL, respectively. In contrast, ivermectin and moxidectin, which are both in clinical use, showed no significant activity (MIC> 32 μg/mL). Time-kill kinetic assays showed bactericidal activity of selamectin and in vitro pharmacodynamic studies demonstrated exposure-dependent activity. These data together with analyses of published pharmacokinetic information strongly suggest that selamectin is the most promising macrocyclic lactone for BU treatment.     

Design and synthesis of de novo cytotoxic alkaloids through mimicking taxoid skeleton

Based on a common pharmacophore model and the hypothesis that the baccatin core of taxoids is a scaffold securing the proper orientation of the side chains, a bicyclic alkaloid scaffold was designed as a baccatin surrogate. Using this scaffold, two novel macrocyclic and open-chain 'taxoid-mimicking' compounds were synthesized. Two of these 'taxoid-mimics', 2 and 3, were found to possess cytotoxicity with micromolar level IC50 values against human breast cancer cell lines.     

New macrocyclic musk compounds

The syntheses and olfactory evaluations of eight new macrocyclic musks with a 1,6-dioxa structure (1a-d and 1'a-d) as well as of twelve optically active 3-methyl macrolides (5a-c and 5'a-c) are reported. These macrocycles were synthesized via intramolecular metathesis mediated by the Grubbs catalyst. Despite the absence of a C=O function, the 1,6-dioxa compounds, both unsaturated (1) and saturated (1'), possess musky odors similar to those of macrocyclic ketones and lactones. Especially 16-membered rings were found to display an intense and pleasant musk character. However, in the case of optically active 3-methyl macrolides (5, 5'), only the (R)-configured 15- and 16-membered rings had intense and pleasant musk notes.     

MS/MS profiling of taxoids from the needles of Taxus wallichiana

Ammonium cationisation has been used for taxoid profiling of partially purified methanolic extracts of needles of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP Hills, Darjeeling, Sikkim and Arunachal Pradesh) by electrospray ionisation tandem mass spectrometry (MS/MS). The MS/MS spectra of the [M + NH4]+ or [M + H]+ ions gave structurally diagnostic fragment ions which revealed information about the taxane skeleton as well as the number and nature of the substituents. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl group with an acetoxy/benzoyloxy group from C-9. The identification of the taxoids was achieved by comparison of the MS/MS spectra with those of authentic taxoids or was based on biogenetic grounds. The results were corroborated by liquid chromatography-MS analysis. Out of the 50 taxoids identified, 21 belonged to the rearranged class. The presence of paclitaxel in the samples from four regions was confirmed: the study also revealed the occurrence of several basic taxoids in these samples. MS/MS profiling by electrospray ionisation was shown to be a fast and reliable technique for the analysis of taxoid samples.     

Novel C2-C3' N-peptide linked macrocyclic taxoids. Part 1: Synthesis and biological activities of docetaxel analogues with a peptide side chain at C3'

A series of novel docetaxel analogues possessing a peptide side chain at the C3'-N position was synthesized. These compounds were designed to mimic a region of the alpha-tubulin loop that is equivalent to the paclitaxel binding pocket in beta-tubulin. Eight new peptidic taxoids were obtained and evaluated as inhibitors of microtubule disassembly, as well as for their cytotoxicity.     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals

To synthesize new highly active taxoids, we designed and synthesized 9 beta-dihydro-9,10-acetal taxoids. In vitro study of these analogues clearly showed them to be more potent than docetaxel.     

Conformationally restricted paclitaxel analogues: macrocyclic mimics of the "hydrophobic collapse" conformation

Conformationally restricted macrocyclic analogues of paclitaxel were prepared, by connecting the 3'-phenyl group and the 2-benzoate moiety with two-atom tethers to mimic the "hydrophobic collapse" paclitaxel conformation. The analogues did not show activity in a tubulin assembly assay.