Taxumarienes A-G, seven new α-glucosidase inhibitory taxane-diterpenoids from the leaves of Taxus mairei.

Seven new taxane diterpenoids taxumarienes A-G (1-7) were isolated from the leaves of Taxus mairei, along with seven known analogous (8-14). The structures of the new compounds were elucidated based on the analysis of NMR and MS spectroscopy. All isolates were evaluated for their α-glucosidase inhibitory activities. Among them, taxumarienes A (1) and F (6) showed potent effect with IC50 values of 5.9 ±â€¯1.30 µM and 3.7 ±â€¯0.75 µM, respectively.

Isolation, Purification, and Identification of Taxol and Related Taxanes from Taxol-Producing Fungus Aspergillus niger subsp. taxi.

The content of taxol in the bark of yews is very low, and this is not affordable from the environmental point of view. Thus, it is a necessity to look for alternative sources of taxol production to solve its supply. Currently, a large portion of the taxol in the market comes from chemical semi-synthesis, but the semi-synthetic precursors such as baccatin III and 10-deacetyl-baccatin III are extracted from needles and twigs of yew trees. Taxol-producing fungi as a renewable resource is a very promising way to increase the scale of taxol production. Our group has obtained a taxol-producing endophytic fungus, Aspergillus niger subsp. taxi HD86-9, to examine if A. niger can produce the taxanes. Six compounds from the fermentation broth of strain HD86-9 were isolated and identified by 1H NMR, 13C NMR, and ESI-MS. The results showed that the six compounds included four taxane diterpenoids (taxol, cephalomannine, baccatin III, and 10-deacetyl-baccatin III) and two non-taxane compounds (ß-sitosterol and flavonoid isovitexin). The study verified that the taxanes can be produced by the A. niger, which is very important to taxol production via chemical semi-synthesis. Additionally, the finding is potentially very significant to solve the taxol semi-synthetic precursors extracted from needles and twigs of yew trees, and the precursor production can be easily increased through the culture condition optimization, genetic breeding, and metabolic engineering of the A. niger.

Taxanes from Taxus wallichiana var. mairei cultivated in the southern area of the Yangtze River in China.

A new and 25 known taxane diterpenoids were isolated from the dried whole plants of Taxus wallichiana var. mairei cultivated in the southern area of the Yangtze River in China. The chemical structure of new compound 1, named taxiwallinine, was established by means of MS and NMR analyses. Among the 25 known taxane compounds 2-26, compounds 10, 12, 14, 15, 17 and 21-23 were first isolated from this plant. Taxiwallinine and taxol were screened for the cytotoxic effect on human breast cancer cell line MCF-7 with IC50 values of 20.898 µg/mL and 0.008 µg/mL, respectively. Taxiwallinine showed moderate cytotoxicity to this cell line. These results demonstrate that T. wallichiana var. mairei cultivated in the southern area of the Yangtze River in China could be a renewable resource for extracting the taxane derivatives and the new compound, taxiwallinine, could be a potential anti-tumour product.

α-Glucosidase Inhibitory and Cytotoxic Taxane Diterpenoids from the Stem Bark of Taxus wallichiana.

From a CH2Cl2 extract of the bark of Taxus wallichiana, six new taxoids, wallitaxanes A-F (1-6), were isolated, together with 29 known compounds. The structures of the new compounds were elucidated on the basis of spectroscopic data interpretation. Wallitaxane D (4) was identified as an opened oxetane-type taxoid having the first naturally occurring C(H)-20 acetal group, while wallitaxanes E (5) and F (6) are representative of the rare abeo-taxoid class. The isolated compounds were evaluated for their α-glucosidase inhibitory activity and for cytotoxicity against the HeLa human cervical cancer cell line. In the present work, taxanes were found to exhibit α-glucosidase inhibitory activity for the first time, and wallitaxane A (1) showed the most potent effect, with an IC50 value of 3.6 µM. In turn, 7-epi-taxol (16) and 7-epi-10-deacetyltaxol (17) showed IC50 values of 0.05 and 0.085 nM, respectively, against HeLa cells.

Separation and purification of two taxanes and one xylosyl-containing taxane from Taxus wallichiana Zucc.: A comparison between high-speed countercurrent chromatography and reversed-phase flash chromatography.

10-Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10-deacetylbaccatin III (1), baccatin III (2), and 7ß-xylosyl-10-deacetyltaxol (3) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high-speed countercurrent chromatography or reversed-phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high-performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n-hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high-speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed-phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high-speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed-phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.

Comparison of Pharmacokinetics and Biodistribution of 10-Deacetylbaccatin III after Oral Administration as Pure Compound or in Taxus chinensis Extract: A Pilot Study.

Taxanes are a class of bioactive compounds isolated from the Taxus species. 10-Deacetylbaccatin III is one of the popular taxane compounds with antitumor activity, but the pharmacokinetic profile of this compound remains elusive. Previously, we prepared the taxane fractions from the twigs and leaves of Taxus chinensis var. mairei containing 20.4 % 10-deacetylbaccatin III. This study aimed to investigate the pharmacokinetics of 10-deacetylbaccatin III and biodistribution, and explore the potential changes when it was administered in the form of taxane extracts. A simple, sensitive, and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitative determination of 10-deacetylbaccatin III in biosamples. The results showed that 10-deacetylbaccatin III, after oral dosing, displayed a quick absorption into the blood and distribution into major organs. Oral administration of 10-deacetylbaccatin III in the form of taxane mixtures led to a 16-fold increase in the systemic exposure of pure 10-deacetylbaccatin III, with the AUC0-U in the plasma increasing from 25.75 ± 11.34 to 231.36 ± 70.12 µg h/L (p < 0.0001). Moreover, the concentrations of 10-deacetylbaccatin III in major tissues were significantly enhanced when given in taxane extracts. These findings revealed pharmacokinetic interactions in the taxane components from T. chinensis var. mairei, which contributed to an enhanced systemic exposure of pharmacologically active taxanes.

[Extraction of 10-Deacetyl Baccatin by Supercritical CO2 from Taxus yunnanensis Branches and Leaves].

OBJECTIVE: To study the supercritical CO2 fluids extraction (SFE) method to extract the components from Taxus yunnanensis. METHODS: Medicinal meterials were extracted by supercritical CO2, and then purified by industrial chromatography. Using the extraction yield of 10-DAB as the index,single factor test was carried out to investigate the effect of co-solvent, extraction time, extraction pressure, extraction temperature, pressure and temperature of separation kettle I. Then orthogonal experiment was used to optimize the best extraction condition. RESULTS: The suitable extraction condition was as follows: the ratio of co-solvent (80% ethanol) amount and the madicinal materials was 3: 1, Separation kettle I pressure was 14 MPa, separation kettle I temperature was 40 °C, extraction pressure was 25 MPa, extraction temperature was 60 T and extraction time was 90 min. The extract was separated by industrial chromatographic and then crystallized. CONCLUSION: The supercritical CO2 extraction and purification process of 10-DAB were simple and feasible.

Infrared and Raman spectroscopic methods for characterization of Taxus baccata L.--Improved taxane isolation by accelerated quality control and process surveillance.

Different yew species contain poisonous taxane alkaloids which serve as resources for semi-synthesis of anticancer drugs. The highly variable amounts of taxanes demand new methods for fast characterization of the raw plant material and the isolation of the target structures during phyto extraction. For that purpose, applicability of different vibrational spectroscopy methods in goods receipt of raw plant material and in process control was investigated and demonstrated in online tracking isolation and purification of the target taxane 10-deacetylbaccatin III (10-DAB) during solvent extraction. Applying near (NIRS) and mid infrared spectroscopy (IRS) the amount of botanical impurities in mixed samples of two different yew species (R(2)=0.993), the leave-to-wood ratio for Taxus baccata material (R(2)=0.94) and moisture in dried yew needles (R(2)=0.997) can be quantified. By partial least square analysis (PCA) needles of different Coniferales species were successfully discriminated by Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy (ATR-FT-IR). The analytical potential of ATR-FT-IR and Fourier Transform-Raman Spectroscopy (FT-RS) in process control of extraction and purification of taxanes is demonstrated for determination of the water content in methanolic yew extracts (R(2)=0.999) and for quantification of 10-DAB (R(2)=0.98) on a highly sophisticated level. The decrease of 10-DAB in the plant tissue during extraction was successfully visualized by FT-IR imaging of thin cross sections providing new perspectives for process control and design.

Pilot studies on scale-up biocatalysis of 7-ß-xylosyl-10-deacetyltaxol and its analogues by an engineered yeast.

Paclitaxel content in yew tree is extremely low, causing a worldwide shortage of this important anticancer drug. Yew tree can also produce abundant 7-ß-xylosyl-10-deacetyltaxol that can be bio-converted into 10-deacetyltaxol for semi-synthesis of paclitaxel. However, the bio-conversion by the screened natural microorganisms was inefficient. We have constructed the recombinant yeast with a glycoside hydrolase gene from Lentinula edodes and explored the bioconversion. Based on previously established reaction conditions, the bioconversion of 7-ß-xylosyl-10-deacetyltaxol or its extract was further optimized and scaled up with the engineered yeast harvested from 200-L scale high-cell-density fermentation. The optimization included the freeze-dried cell amount, dimethyl sulfoxide concentration, addition of 0.5% antifoam supplement, and substrate concentration. A 93-95% bioconversion and 83% bioconversion of 10 and 15 g/L 7-ß-xylosyltaxanes in 10 L reaction volume were achieved, respectively. The yield of 10-deacetyltaxol reached 10.58 g/L in 1 L volume with 15 g/L 7-ß-xylosyl-10-deacetyltaxol. The conversion efficiencies were not only much higher than those of other reports and our previous work, but also realized in 10 L reaction volume. A pilot-scale product purification was also established. Our study bridges the gap between the basic research and commercial utilization of 7-ß-xylosyl-10-deacetyltaxol for the industrial production of semi-synthetic paclitaxel.

Isolation and cytotoxicity evaluation of taxanes from the barks of Taxus wallichiana var. mairei.

Fifteen taxanes (1-15) including a new taxane glucoside, 7ß,9α,10ß-triacetoxy-13α-hydroxy-5α-O-(ß-d-glucopyranosyl)taxa-4(20),11-diene (1), were isolated from the barks of Taxus wallichiana var. mairei. Compounds 1-15 representing three sub-types of 6/8/6-taxane were evaluated in vitro for anti-proliferative activity against a panel of parental and drug-resistant cancer cells. Potent compounds were found while several exhibited selective cytotoxicity. Especially, 3, 8, and 10 showed selective inhibition to breast carcinoma cell line MCF-7, while 13 selectively inhibited taxol resistant human ovarian carcinoma cell line A2780/TAX (IC50=0.19µM), being more potent than the clinical drugs taxol (IC50=4.4µM) and docetaxol (IC50=0.42µM), and less cytotoxic to mouse embryonic fibroblast cell line NIH-3T3, a cell line close to normal cell line. The possible P-glycoprotein evasion mechanism of 13 against A2780/TAX and the preliminary structure-activity relationships (SARs) of this group of compounds were also discussed.

Preparative isolation of paclitaxel and related taxanes from cell cultures of Taxus chinensis using reversed-phase flash chromatography.

In this study, paclitaxel, baccatin III, taxuyunnanine C and sinenxane C were successfully separated by reversed-phase flash chromatography on a manually packed C18 column from Taxus chinensis cell culture extract. The crude cell culture extract was first treated with Al2O3 column chromatography and then divided into two parts: fraction 1 and fraction 2. Ten milligrams of baccatin III and 19 mg of paclitaxel were obtained from 100 mg dried fraction 1. Fifty-two milligrams of taxuyunnanine C and 11 mg sinenxane C were obtained from 100 mg dried fraction 2. The purities of the four compounds were 98.02%, 98.53%, 98.93% and 98.76%, respectively. Their structures were characterised by using UV, MS and NMR. These results indicate that paclitaxel and related taxanes including baccatin III can be obtained from cell culture in a highly pure state using reversed-phase flash chromatography.

Isolation and identification of a 10-deacetyl baccatin-III-producing endophyte from Taxus wallichiana.

Endophytic fungi of inner root bark of Taxus wallichiana var. mairei were investigated in order to find endophytes producing 10-DABIII (10-deacetyl baccatin III). Purified colonies were cultured in potato dextrose broth (PDB), and then the organic extracts from fungi were analyzed with HPLC, LC-MS, and (1)H NMR. Of 102 fungal endophytes isolated from the inner root bark, only one strain named IRB54 can yield 10-DABIII but no taxol and baccatin III. In PDB culture medium, its productivity was 187.564 ug/l. Based on its morphological characteristics and molecular data, the IRB54 strain was identified as Trichoderma sp. The isolation of the fungus IRB54 yielding 10-DABIII will provide an alternative resource to manufacture taxol/taxotere via semi-synthesis and some useful clues for improving the understanding of taxane synthesis evolution.

NMR spectroscopic search module for Spektraris, an online resource for plant natural product identification--Taxane diterpenoids from Taxus × media cell suspension cultures as a case study.

Development and testing of Spektraris-NMR, an online spectral resource, is reported for the NMR-based structural identification of plant natural products (PNPs). Spektraris-NMR allows users to search with multiple spectra at once and returns a table with a list of hits arranged according to the goodness of fit between query data and database entries. For each hit, a link to a tabulated alignment of (1)H NMR and (13)C NMR spectroscopic peaks (query versus database entry) is provided. Furthermore, full spectroscopic records and experimental meta information about each database entry can be accessed online. To test the utility of Spektraris-NMR for PNP identification, the database was populated with NMR data (total of 466 spectra) for ∼ 250 taxanes, which are structurally complex diterpenoids (including the anticancer drug taxol) commonly found in the genus Taxus. NMR data generated with metabolites purified from Taxus cell suspension cultures were then used to search Spektraris-NMR, and enabled the identification of eight taxanes with high confidence. A ninth isolated metabolite could be assigned, based on spectral searches, to a taxane skeletal class, but no high confidence hit was produced. Using various spectroscopic methods, this metabolite was characterized as 2-deacetylbaccatin IV, a novel taxane. These results indicate that Spektraris-NMR is a valuable resource for rapid and reliable identification of known metabolites and has the potential to contribute to de-replication efforts in novel PNP discovery.

[Chemical constituents of bark of Taxus chinensis var. mairei].

OBJECTIVE: To study the chemical constituents in the bark of Taxus chinensis var. mairei collected from southeast of China. METHODS: Chemical constituents were isolated and purified by column chromatography, Prep-TLC, and preparative HPLC. The structures were identified on the basis of 1D-and 2D-NMR spectral analysis. RESULTS: Twelve taxane diterpenoids were isolated from the bark of Taxus chinensis var. mairei grown in southeast of China. They were identified as: taxagifine (1), decinnamoyltaxagifine (2), 19-debenzoyl-19-acetyltaxinine M(3), 9-dihydro-13-acetyl-baccatin III (4), 7, 9-dideacetylbaccatin IV (5), 1,3-dihydro-taxinine (6), taxumairol C (7), taxezopidine J (8), 7-xylosyl-10-deacetyl-taxol A (9),10-deacetyltaxol (10), taxicin II (11), and 2alpha, 7beta, 10beta-triacetoxy-5alpha, 13alpha-dihydroxy-2 (3 --> 20) abeotaxa-4 (20), 11-dien-9-one (12). CONCLUSION: Compounds 1, 2, 4 - 6, 8, 9, 11 and 12 are obtained from this plant for the first time. Compound 7 is obtained from the bark of Taxus chinensis var. mairei for the first time.

A new taxane diterpenoid from Taxus chinensis var. mairei.

Taxadiene (3), a new taxane diterpenoid with an unusual hydroxy substituting at C-17, and six known compounds including two taxane diterpenoids (1 and 2) and four flavonoids (4-7) were isolated from the whole seedling of the Taxus chinensis var. mairei. Among them, compound 7 was isolated from T. chinensis var. mairei for the first time. Structures of these compounds were elucidated on the basis of spectroscopic data and by comparison with reported literature data.

Chemical studies on Taxus canadensis.

A series of new taxanes, 1-93, have been isolated, together with 37 known taxoids including Taxol(®) (paclitaxel) and cephalomannine, from the Canadian yew, Taxus canadensis (Taxaceae) in the past 30 years. These new taxoids possess various skeletons containing 5/7/6, 6/10/6, 6/5/5/6, 6/8/6, and 6/12 ring systems and six new taxanes with four novel skeletons, i.e., a taxane with a 6/6/8/6 ring system, a taxane with a [3.3.3] propellane skeleton, three taxanes with [3.3.3] [3.4.5] dipropellane sytems, as well as a novel taxane with a unique 5/5/4/6/6/6 hexacyclic skeleton, containing a unique [3.3.2] propellane, were isolated for the first time from natural sources. It should be emphasized that 13-acetyl-9-dihydrobaccatin III, a very useful starting material for the semisynthesis of Taxol(®) and Taxotere(®) , represents the most abundant taxane in the needles of this yew tree. These findings establish the above mentioned yew tree as significantly different from the remaining species. On the other hand, some chemical modifications on the taxanes isolated from this plant were carried out.

Synthesis, isolation, stereostructure and cytotoxicity of paclitaxel analogs from cephalomannine.

Four paclitaxel derivatives were afforded by preparative HPLC separation of two pairs of diastereoisomers, which were obtained by catalytic hydrogenation and epoxidation of the C-13 side-chain double bond of cephalomannine, a naturally occurring paclitaxel analog. The four paclitaxel derivatives were analyzed using NMR, CD spectroscopy, and side-chain hydrolysis in order to measure their optical rotations and GC characteristics. In this way, the stereoconfigurations of the products were determined. Evaluation of the compounds' activity indicated that they had differing cytotoxic activities: compound 5 had superior activity in BCG-823 tumor cells compared to paclitaxel, while compound 7 had superior activity in HCT-8 and A549 tumor cells compared to paclitaxel. These results indicate that the stereoconfiguration of the paclitaxel N-acyl side chain has a significant impact on its activity.

The development and clinical validation of a turbulent-flow liquid chromatography-tandem mass spectrometric method for the rapid quantitation of docetaxel in serum.

BACKGROUND: Docetaxel is a second generation taxane utilized as an anti-neoplastic agent in cancer chemotherapies. Traditional treatment regimens have resulted in significant adverse effects, resulting in the shift to more frequent drug administration at lower doses. As a result, it is important to monitor serum docetaxel concentrations to optimize efficacy and minimize adverse effects. METHODS: Serum containing docetaxel was combined with acetonitrile containing deuterated internal standard, and following protein precipitation, supernatant was diluted with water for on-line sample extraction. Following turbulent-flow chromatography (TFC), analytic separation was achieved on a Hypersil Gold C-18 (50×2.1mm) column and the eluent analyzed using a TSQ Vantage mass spectrometer with selected reaction monitoring. Matrix effects were characterized in addition to carryover, precision, linearity, recovery and functional sensitivity. RESULTS: The simple and complex precision for the assay at multiple concentrations was ≤6.2%. The assay has functional sensitivity of <3ng/ml, and is linear from 8.1 to 1978ng/ml. Method comparison studies with a reference HPLC-MS/MS method show a slope of 0.84 with a Spearman coefficient of 0.99. CONCLUSIONS: Based on the validation metrics, we have generated a sensitive and automated TFC-MS/MS method for docetaxel quantitation in serum.

A furantaxane with an unusual 6/8/6/5 ring system and potent tumor MDR reversal activity obtained via microbial transformation.

A furantaxane (4) with an unusual 6/8/6/5 ring system and two hydroxylated products (2, 3) were isolated following the biotransformation of a taxane (1) by Streptomyces griseus. The structures of the isolates were elucidated by spectroscopic analysis. The absolute configuration of 4, which exhibited potent reversal activity in the A549/taxol MDR tumor cell line, was unambiguously deduced by single-crystal X-ray diffraction.