Intermediate for synthesis of paclitaxel

Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T. chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T. chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures.[2]

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Uptake in the presence of paclitaxel and other taxanes [2]
To determine the influence of other taxanes on the uptake of Flutax-2®, aggregated suspension cell cultures were used. Suspension cell cultures were incubated with Flutax-2® in the presence of other taxanes (paclitaxel, baccatin III, cephalomannine, and 10-deacetyltaxol). These taxanes are structurally similar to paclitaxel, with several differences: baccatin III lacks the side-chain derived from phenylalanine; cephalomannine has a linear carbon chain at the third carbon position of the side-chain instead of a phenyl group; 10-deacetyltaxol lacks the acetyl group at position C-10 (Figure 3). Only paclitaxel was found to significantly (p < 0.05) inhibit the cellular uptake of Flutax-2® (Figure 4A). These results clearly demonstrate the specificity of paclitaxel uptake. Additionally, the inhibition of Flutax-2® was shown to depend linearly on the amount of unlabeled paclitaxel present (Figure 4B). At a constant concentration of Flutax-2®, the addition of paclitaxel is able to competitively inhibit the cellular association of the fluorescently labeled ligand. The competitive inhibition indicates that the two compounds are being transported by a specific mechanism.

Acetic acid-induced writhing [3]
All compounds [e.g. 10-Deacetylbaccatin-III (10-DAB)] revealed significant analgesic activity, especially tasumatrol B which reduced the acetic acid-induced writhing by 72.23% at 40 mg/kg dose, in comparison to the group treated only with saline (Table 1).

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Hot-plate test [3]
According to the results of hot-plate test, statistically no significant difference (Table 2) was observed between the animals treated with taxoids and the animals treated with saline.

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Carrageenan-induced oedema [3]
Isolated compounds [e.g. 10-Deacetylbaccatin-III (10-DAB)]significantly reduced the oedema induced by carrageenan. However, tasumatrol B was found to be the most active compound showing significant (p < 0.05) and highly significant (p < 0.01) activity at test doses of 20 and 40 mg/kg.

In vitro lipoxygenase inhibition assay [3]
Enzyme inhibition assays were performed by using different concentrations of the isolated compound Sieboldogenin. Lipoxygenase inhibitory activity was measured by slightly modifying the spectrometric method as developed by Tappel (1962). Lipoxygenase (EC 1.13.11.12) type I-B (Soybean) and linoleic acid were used without further purification. 160 μL of sodium phosphate buffer, 0.1 mm (pH 7.0), 10 mL of the sample solutions (test compounds) and 20 μL of lipoxygenase solution were mixed and incubated for 5 min at 258°C. The reaction was initiated by the addition of 10 μL linoleic acid substrate solution and the absorption change with the formation of (9Z,11E)-13S)-13-hydroperoxyoctadeca-9,11-dienoate was followed for 10 min. The test sample and the control were dissolved in 50% ethanol. All the reactions were performed in triplicate. Baicalein was used as positive control for lipoxygenase inhibition (Khan et al., 2009). The inhibitory concentration (IC50) values were calculated using the EZFit Enzyme Kinetics program.

Acetic acid-induced writhing [3]
The acetic acid abdominal constriction test (Koster et al., 1959) was used with modification according to Nisar et al. (2008b). Mice were divided into various groups of five mice each and starved for 18 h. The negative control group received saline 10 mL/kg, p.o., while test groups received various doses of the test compounds via oral route. Meanwhile, the positive control group received acetylsalicylic acid (ASA); 100 mg/kg, p.o. After half an hour, all mice received a 0.7% aqueous solution of acetic acid 10 mg/kg, i.p. and writhings were counted for 10 min after acetic acid injection.

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Hot-plate test [3]
A hot-plate latency test was performed according to the method described by Zhang et al. (2009). Animals were habituated twice to the hot-plate in advance. For testing, mice were placed on a hot-plate maintained at 55 ± 0.5°C. The time that elapsed until the incidence of either a hind paw licking or a jump off the surface was recorded as the hot-plate latency. Mice with baseline latencies of < 5 s or > 30 s were eliminated from the study. After the determination of baseline response latencies, hot-plate latencies were redetermined at 30, 60 and 120 min after oral administration of test drugs (aspirin as the reference drug).

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Carrageenan-induced oedema [3]
The method of Winter et al. (1962) was utilized to assess the antiinflammatory potential of the test sample via testing its ability to inhibit the carrageenan-induced hind paw oedema, as reported earlier (Khan et al., 2009). Test samples and the control samples were administered orally in groups to rats. After 1 hour, acute inflammation at the desired site was induced by subplantar injection of 1% suspension of carrageenan (0.1 mL) using 2% gum acacia as a suspending agent in normal saline, in the right hind paw of the rats. The paw volume was measured plethysmometrically (Ugo Basile, Italy) at ‘0’ and 3 h after the carrageenan injection. Indomethacin 5 mg/kg, p.o. suspended in 2% gum acacia was used as the positive control. Percentage inhibition of the inflammation was determined by applying statistics on raw data followed by the calculation of percentage inhibition for each group by comparing with the control group. The formula used for comparison was: %I = 1 − (dt/dc) × 100, where dt is the difference in paw volume in the drug-treated group, dc is the difference in paw volume in control group and I stands for inhibition of inflammation.

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Cotton-pellet oedema model [3]
In order to evaluate the antiinflammatory effect of the isolated compounds on the cotton-pellet oedema model, male rats were divided into four groups of five animals. To each animal, two cotton wool pellets weighing 18 ± 1 mg were implanted subcutaneously, one on each side of the abdomen. Animals were kept under light ether anesthesia according to the method reported by Swingle and Shideman (1972). Each compound was administered once daily throughout the experimental period of 7 days. On the day 8 after implantation, rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). After induction of anesthesia, pellets were dissected, dried at 55°C for 15 h, and weighed after cooling. The mean granuloma weights as well as the percentage granuloma inhibition of the test compounds were calculated.

Acute toxicity[3]
All compounds (e.g., 10-deacetylbaccine-III (10-DAB)) were confirmed to be safe 48 hours after administration. Statistical analysis showed no significant difference in mortality and morbidity between the negative control group and other treatment groups.

[1]. Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis. Plant Biol (Stuttg). 2013 Jan;15(1):19-26.

[2]. Paclitaxel uptake and transport in Taxus cell suspension cultures. Biochem Eng J. 2012 Apr 15;63:50-56.

[3]. Analgesic and antiinflammatory activities of taxoids from Taxus wallichiana Zucc. Phytother Res. 2012 Apr;26(4):552-6.

[4]. Taxol biosynthesis: Molecular cloning of a benzoyl- CoA:taxane 2α-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli. PNAS, 2000 , 97(25)：13591-13596.

10-Deacetylated baccatin III is a tetracyclic diterpenoid compound belonging to the secondary α-hydroxy ketone class. Its function is related to baccatin III. It has been reported that 10-deacetylated baccatin III exists in Taxus sumatrana, Taxus cuspidata, and other organisms with relevant data. This study investigated the transport of paclitaxel in Taxus canadensis suspension cultures using a paclitaxel fluorescent analog (Flutax-2®) combined with flow cytometry. Experiments were performed using isolated protoplasts and aggregated suspension cell cultures. Results showed that Flutax-2® maintained stability exceeding 90% in Taxus canadensis suspension cultures within the required incubation time (24 hours). Unlabeled paclitaxel was shown to inhibit cellular uptake of Flutax-2®, while structurally similar taxanes, such as cefadroxil, baccatin III, and 10-deacetylbaccatin III, did not inhibit Flutax-2® uptake. Flutax-2® uptake kinetics were saturated. These results suggest the existence of a specific transport system for paclitaxel. Suspension cells induced by methyl jasmonate accumulated 60% more Flutax-2® than uninduced cells, likely due to enhanced cell storage capacity after methyl jasmonate induction. The existence of a specific transport mechanism for paclitaxel is an important preliminary result that will lay the foundation for further research and the development of targeting strategies to increase the secretion of paclitaxel into extracellular mediators. [2]

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A study aimed to identify components that may have analgesic and anti-inflammatory effects. Tasumatrol B, 1,13-diacetyl-10-deacetylbaccatin III (10-DAD), and 4-deacetylbaccatin III (4-DAB) were isolated from the bark extract of Taxus wallichiana Zucc. The analgesic and anti-inflammatory activities of all compounds were evaluated using an acetic acid-induced writhing model, a hot plate test, a carrageenan-induced mouse paw edema model, a cotton ball edema model, and an in vitro lipoxygenase inhibition assay. In the acetic acid-induced mouse paw edema model, all compounds, especially tasumatrol B, showed significant analgesic activity, superior to the saline group. Similarly, all tested compounds, especially tasumatrol B, showed significant anti-inflammatory activity. However, none of the compounds showed significant activity in the hot plate test or the in vitro lipoxygenase inhibition assay. This study highlights the potential of tasumatrol B as a novel lead compound for the treatment of pain and inflammation, a discovery derived from the scientific validation of the traditional medicinal plant T. wallichiana Zucc. [3]

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A cDNA clone encoding taxane 2α-O-benzoyltransferase was isolated from Taxus cuspidata. This recombinase catalyzes the conversion of the semi-synthetic substrate 2-debenzoyl-7,13-diacetylbaccatin III to 7,13-diacetylbaccatin III, and therefore appears to play a role in the late acylation step of the paclitaxel biosynthesis pathway. Multiple gene fragments were amplified using a homology-based PCR cloning strategy to generate oligodeoxynucleotide probes for the acyltransferases. These probes were then used to screen cDNA libraries constructed from mRNA isolated from methyl jasmonate-induced Taxus cuspidata cells, from which multiple full-length acyltransferases were obtained and expressed in Escherichia coli. The functionally expressed benzoyltransferases were validated by radioactive HPLC, 1H-NMR, and HPLC-MS. The product of this enzyme is 7,13-diacetylbaccatin III, which is derived from the conversion of 2-debenzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as co-substrates in the corresponding cell-free extracts. The full-length cDNA has an open reading frame of 1320 base pairs, encoding a protein consisting of 440 amino acid residues with a molecular weight of 50089. The optimal pH of the recombinant benzoyltransferase is 8.0, and the Km values for taxane substrates and benzoyl-CoA are 0.64 mM and 0.30 mM, respectively, and it exhibits significant regioselectivity for the 2α-hydroxyacylation of functionalized taxane cores. This enzyme can be used to improve the production yield of paclitaxel and for the semi-synthesis of drug analogs with modified aromatic acyl groups at the C2 position. [4]

C29H36O10

544.59

544.23

C, 63.96; H, 6.66; O, 29.38

32981-86-5

154272

White to off-white solid powder

1.4±0.1 g/cm3

716.8±60.0 °C at 760 mmHg

231-236 °C

233.5±26.4 °C

0.0±2.4 mmHg at 25°C

1.624

3.51

4

10

5

39

1090

9

CC1=C2[C@H](C(=O)[C@@]3([C@H](C[C@@H]4[C@]([C@H]3[C@@H]([C@@](C2(C)C)(C[C@@H]1O)O)OC(=O)C5=CC=CC=C5)(CO4)OC(=O)C)O)C)O

YWLXLRUDGLRYDR-ZHPRIASZSA-N

InChI=1S/C29H36O10/c1-14-17(31)12-29(36)24(38-25(35)16-9-7-6-8-10-16)22-27(5,23(34)21(33)20(14)26(29,3)4)18(32)11-19-28(22,13-37-19)39-15(2)30/h6-10,17-19,21-22,24,31-33,36H,11-13H2,1-5H3/t17-,18-,19+,21+,22-,24-,27+,28-,29+/m0/s1

(2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-12b-acetoxy-4,6,9,11-tetrahydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxet-12-yl benzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)