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 the compounds [e.g. 10-Deacetylbaccatin-III (10-DAB)] were found safe after 48 h of administration. Statistically, no considerable difference was observed between the negative control and other treatment groups both in terms of mortality and morbidity.

[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-deacetylbaccatin III is a tetracyclic diterpenoid and a secondary alpha-hydroxy ketone. It is functionally related to a baccatin III.
10-Deacetylbaccatin III has been reported in Taxus sumatrana, Taxus cuspidata, and other organisms with data available.

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The transport of paclitaxel in Taxus canadensis suspension cultures was studied with a fluorescence analogue of paclitaxel (Flutax-2(®)) in combination with flow cytometry detection. Experiments were carried out using both isolated protoplasts and aggregated suspension cell cultures. Flutax-2(®) was shown to be greater than 90% stable in Taxus suspension cultures over the required incubation time (24 hours). Unlabeled paclitaxel was shown to inhibit the cellular uptake of Flutax-2(®), although structurally similar taxanes such as cephalomannine, baccatin III, and 10-deacetylbaccatin III did not inhibit Flutax-2(®) uptake. Saturation kinetics of Flutax-2(®) uptake was demonstrated. These results indicate the presence of a specific transport system for paclitaxel. Suspension cells elicited with methyl jasmonate accumulated 60% more Flutax-2(®) than unelicited cells, possibly due to an increased cellular storage capacity following methyl jasmonate elicitation. The presence of a specific mechanism for paclitaxel transport is an important first result that will provide the basis of more detailed studies as well as the development of targeted strategies for increased paclitaxel secretion to the extracellular medium.[2]

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A study was conducted to identify constituents that might be responsible for analgesic and antiinflammatory conditions. 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. All the compounds were assessed for analgesic and antiinflammatory activities using an acetic acid-induced writhing model, a hot-plate test, a carrageenan-induced paw oedema model, a cotton-pellet oedema model and in vitro lipoxygenase inhibitory assay. All the compounds, especially tasumatrol B, revealed significant analgesic activity in comparison to a saline group based on an acetic acid-induced model. Similarly all of the test compounds, particularly tasumatrol B, showed significant antiinflammatory activity. However, all the compounds failed to exhibit any considerable activity in of the hot-plate test and the in vitro lipoxygenase inhibitory assay. This study has highlighted the potential of tasumatrol B to be further explored as a new lead compound for the management of pain and inflammation, one that has been discovered by scientific validation of the traditional medicinal use of T. wallichiana Zucc.[3]

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A cDNA clone encoding a taxane 2α-O-benzoyltransferase has been isolated from Taxus cuspidata. The recombinant enzyme catalyzes the conversion of 2-debenzoyl-7,13-diacetylbaccatin III, a semisynthetic substrate, to 7,13-diacetylbaccatin III, and thus appears to function in a late-stage acylation step of the Taxol biosynthetic pathway. By employing a homology-based PCR cloning strategy for generating acyltransferase oligodeoxynucleotide probes, several gene fragments were amplified and used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which several full-length acyltransferases were obtained and individually expressed in Escherichia coli. The functionally expressed benzoyltransferase was confirmed by radio-HPLC, 1H-NMR, and combined HPLC-MS verification of the product, 7,13-diacetylbaccatin III, derived from 2-debenzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as cosubstrates in the corresponding cell-free extract. The full-length cDNA has an open reading frame of 1,320 base pairs and encodes a protein of 440 residues with a molecular weight of 50,089. The recombinant benzoyltransferase has a pH optimum of 8.0, Km values of 0.64 mM and 0.30 mM for the taxoid substrate and benzoyl-CoA, respectively, and is apparently regiospecific for acylation of the 2α-hydroxyl group of the functionalized taxane nucleus. This enzyme may be used to improve the production yields of Taxol and for the semisynthesis of drug analogs bearing modified aroyl 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.)