Large-scale purification of 13-dehydroxybaccatin III and 10-deacetylpaclitaxel, semi-synthetic precursors of paclitaxel, from cell cultures of Taxus chinensis

Journal of Chromatography A, 1123 (2006) 15–21

Large-scale purification of 13-dehydroxybaccatin III and 10-deacetylpaclitaxel, semi-synthetic precursors of paclitaxel, from cell cultures of Taxus chinensis Sang-Hyun Pyo a,b , Ho-Joon Choi a,∗ , Byung-Hee Han b,∗ a

Samyang Genex Food & Bio Research Center, 63-2 Hwaam-Dong, Yuseong-Gu, Daejeon 305-717, South Korea b Department of Chemistry, Chungnam National University, 220 Gung-Dong, Yuseong-Gu, Daejeon 305-764, South Korea

Received 21 January 2006; received in revised form 4 April 2006; accepted 12 April 2006 Available online 23 May 2006

Abstract The taxane derivatives 13-dehydroxybaccatin III (13-DHB III) and 10-deacetylpaclitaxel (10-DAP) can be used as semi-synthetic precursors of paclitaxel. These precursors were isolated in a simple and economical procedure during purification of paclitaxel from plant cell culture extracts. No additional costs for cell culture or extraction by silica-gel low-pressure chromatography were incurred. Precipitation from dichloromethane/n-hexane followed by HPLC on ODS and silica-gel resins resulted in paclitaxel of 99.5% purity with 80% overall yield. ODS low-pressure chromatography and THF/n-hexane precipitation yielded 13-dehydroxybaccatin III at >99% purity with 87.1% overall yield, and ODS low-pressure chromatography alone yielded 10-deacetylpaclitaxel at >90% purity with 93.4% overall yield. These compounds are of sufficient purity for use in semi-synthesis of paclitaxel. Both compounds were obtained in high yield at >99.5% purity using ODS-HPLC. The procedures described allow the simultaneous purification of 13-dehydroxybaccatin III, 10-deacetylpaclitaxel, and paclitaxel with only minimal additional expense for reagents or equipment. © 2006 Elsevier B.V. All rights reserved. Keywords: 13-Dehydroxybaccatin III; 10-Deacetylpaclitaxel; Paclitaxel; Purification; Chromatography; Semi-synthesis

1. Introduction Paclitaxel (Genexol, Taxol), a compound originally isolated from the bark of the Pacific yew tree Taxus brevifolia in 1971 [1], has been one of the most important anticancer agents of recent decades [2,3]. Due to its novel anticancer activity, paclitaxel has been approved by the U.S. Food and Drug Administration as a treatment for refractory ovarian, breast, and other cancers [2–4]. However, use of T. brevifolia as the primary source of paclitaxel for research and clinical purposes is not environmentally sustainable because the yield of paclitaxel from bark is only about 0.01% (w/w) [5,6], and bark stripping leads to the destruction of scarce plant material.

∗

Corresponding authors. Tel.: +82 42 821 6547; fax: +82 42 823 1360. E-mail addresses: [email protected] (H.-J. Choi), [email protected] (B.-H. Han). 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.04.093

Numerous attempts to develop an economical and reliable alternative method for paclitaxel production have been made, including semi-synthesis, total-synthesis, and plant and tissue cell culture [7–13]. Cell culture methodologies offer the advantage of renewable source material. Plant cell cultures are also a potential source of several useful starting materials for semisynthesis, including 13-dehydroxybaccatin III (13-DHB III) and 10-deacetylpaclitaxel (10-DAP). Commercial semi-synthetic methods have also been developed for production of paclitaxel from baccatin III and 10-deacetylbaccatine III [7,8]. 13-DHB III and 10-DAP have not been historically favored as starting materials for semi-synthesis because their concentrations in plant materials are much lower than that of 10-deacetylbaccatin III [14], which is used for commercial production of paclitaxel. As a result, procedures for purification of 13-DHB III and 10DAP have not been well defined. HPLC has been used to isolate taxanes including 10-deacetylbaccatin III, baccatin III, cephalomannine, and 10-DAP from plant materials on an analytical scale [15–17], but not on a preparative scale.

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Paclitaxel has been purified from yew tree and plant cell cultures on a large scale using methods such as liquid chromatography (LC), recrystallization, and simulated moving-bed chromatography [18–22]. In this report, we describe modifications of the paclitaxel purification procedure that allow efficient and low-cost purification of 13-DHB III and 10-DAP from plant cell cultures concurrently with purification of paclitaxel. 13DHB III and 10-DAP were readily isolated using low-pressure LC (LPLC). This method is suitable for large-scale production of these precursors for the purpose of semi-synthetic production of paclitaxel. 2. Experimental 2.1. Plant materials and culture conditions Taxus chinensis cells were maintained in suspension culture at 24 ◦ C with shaking at 150 rpm in the dark. The cells were cultured [23] in modified Gamborg’s B5 medium supplemented with 3% (w/v) sucrose, 10 ␮M naphthalene acetic acid, 0.2 ␮M 6-benzylamino purine, 0.1% (w/v) casein hydrolysate, and 0.1% (w/v) of 2-(N-morpholino) ethanesulfonic acid. Cell cultures were transferred to fresh medium every 2 weeks. In production cultures, maltose was added to the culture medium on days 7 and 21 at 1 and 2% (w/v), respectively. To initiate production of paclitaxel, the culture was supplemented with AgNO3 at 4 ␮M [23]. Biomass was recovered using a CA150 clarifying decanter (Westfalia) and an Alfa Laval high-speed centrifuge (model no. BTOX 205GD-35CDEFP). Paclitaxel in the culture medium was recovered by adsorption with DIAION HP20 resin (Mitsubishi Chemical, Japan) for 24 h, followed by filtration.

2.3. Extraction of 13-DHB III, paclitaxel, and 10-DAP from suspension-cultured cells The biomass recovered from cell cultures was added to methanol at a preferred ratio of 1:1 (w/v), stirred at room temperature for 30 min, and filtered to yield a methanol extract. The biomass was extracted in this manner at least 4 times. The methanol extracts were pooled and concentrated under reduced pressure at 20–40 ◦ C until the volume was reduced by 70–80%. To eliminate polar impurities, the concentrated methanol extract was re-extracted with dichloromethane (CH2 Cl2 ). Dichloromethane was added to the methanol extract to dilute the methanol to 28% (v/v), the solution was stirred at room temperature for 30 min, and the phases were separated. Extraction was performed at least 3 times. The extracted methanol phases were then pooled and dried under reduced pressure at room temperature. The residue was dissolved in 10 ml dichloromethane per gram of dried extract and filtered using Sylopute (Fuji Silysia, Japan) as a filter aid. The filtrate was washed 5 times with dichloromethane. The resulting solution was then applied to a 16 mm × 900mm column that was packed with silica-gel 60N (Timely, Japan) and equilibrated with 1.5% (v/v) methanol in dichloromethane. The column was eluted isocratically with the same solution to yield first 13-DHB III and then paclitaxel. After paclitaxel eluted, the concentration of methanol was increased to 5.0% to elute 10-DAP. For each compound, the corresponding fractions were pooled and dried by rotary evaporation. The resulting dried compounds were used in further experiments to optimize the purification process. 2.4. Purification of 13-DHB III using ODS-LPLC and precipitation from THF/n-hexane

2.2. Quantitative analysis Quantitative analysis of intermediates and products was performed using an HP1090 HPLC system (Hewlett-Packard, USA) fitted with a Curosil PFP column (Phenomenex, 4.6 mm × 250 mm, pore diameter 5 ␮m). The column was eluted with a water–acetonitrile gradient from 65:35 (v/v) to 35:65 (v/v) over 30 min at a flow rate of 1.0 ml/min. Elution was monitored at 227 nm (paclitaxel) or 255 nm (internal standard) with the builtin photodiode-array detector. Recovered paclitaxel was dried and re-dissolved in methanol for quantitative analysis. Paclitaxel and n-propyl paraben were purchased from Sigma and used as standards. Paclitaxel purity was determined using an internal standard assay with reference paclitaxel as the standard. Purity was calculated by comparing the response ratio determined for the test sample with that obtained from the reference paclitaxel. Paclitaxel concentrations were estimated by reference to the paclitaxel peak area. The purity of the recovered 13-DHB III and 10-DAP was determined using reference samples of 13-DHB III and 10-DAP as external standards. Standard curves obtained from HPLC analysis of the reference samples were compared to curves obtained for the test samples.

For further purification of 13-DHB III, the dried compound obtained from silica-gel chromatography was dissolved in 10 ml of 70% (v/v) methanol in water and subjected to LPLC on a 16 mm × 900-mm column packed with C18 ODS (100-␮m particle size; Timely, Japan). The sample was injected onto the column at a linear velocity of 3–5 cm/min and eluted with a 62% (v/v) solution of methanol in water. Elution was monitored with a UV detector at 227 nm, and fractions containing at least 90% 13-DHB III were pooled and dried under reduced pressure. The dried 13-DHB III was then dissolved in tetrahydrofuran (THF), dripped into n-hexane to form a final THF:n-hexane ratio of 1:5–1:6. This mixture was allowed to stand at −20 ◦ C for 2 days to allow precipitation of 13-DHB III and filtered. The resulting 13-DHB III, which was greater than 99% pure, was dried at 30 ◦ C under reduced pressure. To optimize this step of the purification, various amounts of 13-DHB III (0.5–2.5 g) were applied to the column, and the purity and yield were assessed. Reverse-phase HPLC was used to obtain 13-DHB III of even greater purity (>99.5%), using previously described conditions [16]. The 90%-pure pooled ODS-LPLC fractions dissolved in methanol were applied to a 50 mm × 50-mm C18 ODS column (Shiseido, Japan) at a linear velocity of 3–5 cm/min. The column

S.-H. Pyo et al. / J. Chromatogr. A 1123 (2006) 15–21

was eluted with a 65% (v/v) solution of methanol in water, with monitoring at 227 nm. The active fractions containing 13-DHB III were pooled and dried under reduced pressure. 2.5. Purification of 10-DAP using ODS-LPLC The dried, crude 10-DAP obtained from silica-gel chromatography was subjected to ODS-LPLC using the same procedure as described above for 13-DHB III. The active fractions containing partially purified 10-DAP of >90% purity were pooled and dried under reduced pressure. Further purification was achieved using ODS-HPLC as described above for 13-DHB III. The resulting 10-DAP was >99.5% pure. 2.6. Purification of paclitaxel using dichloromethane/n-hexane precipitation, ODS-HPLC, and silica-gel HPLC Paclitaxel of >99.5% purity was obtained using a previously published procedure [16] consisting of dichloromethane/nhexane precipitation, ODS-HPLC, and silica-gel HPLC. Dried, crude paclitaxel was dissolved in dichloromethane at 100 mg/ml and dripped into nine volumes of n-hexane. The resulting paclitaxel precipitate was recovered by filtration and dried at 30 ◦ C under reduced pressure. The dried paclitaxel was dissolved in methanol and injected onto a 50 mm × 500-mm C18 ODS HPLC column at a linear velocity of 3–5 cm/min. The column was eluted with a 65% solution of methanol in water with monitoring at 227 nm. Fractions containing paclitaxel were pooled and dried under vacuum. The residue was then dissolved in dichloromethane and injected onto a 50 mm × 500-mm silica-gel HPLC column at a linear velocity of 3–5 cm/min. The column was eluted with a 1.5% solution of methanol in dichloromethane with monitoring at 227 nm. Frac-

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tions containing paclitaxel were pooled and dried under reduced pressure. 3. Results and discussion 3.1. Preparation of crude 13-DHB III, paclitaxel, and 10-DAP from suspension-cultured cells Crude 13-DHB III, paclitaxel, and 10-DAP were isolated from cell cultures using solvent extractions and silica-gel LPLC, as shown in Fig. 1. Methanol extraction followed by dichloromethane extraction yielded a crude extract containing paclitaxel, 13-DHB III, and 10-DAP of 5.8, 2.7, and 4.2% purity, respectively. After the crude extract was dissolved in dichloromethane and filtered, the resulting solution was loaded into a silica-gel column equilibrated with 1.5% (v/v) methanol in dichloromethane. As shown in Figs. 2 and 3, 13-DHB III and paclitaxel were eluted sequentially with 1.5% (v/v) methanol in dichloromethane. The methanol concentration was then increased to 5.0% to rapidly elute 10-DAP. The column was regenerated with 50% (v/v) methanol in dichloromethane. As shown in Fig. 2, the peaks for each compound were sufficiently resolved to achieve >95% yield on a large scale. Therefore, this procedure was optimal for simultaneous extraction of the three compounds from crude extract. The fractionated compounds were concentrated and dried to yield crude 13-DHB III, paclitaxel, and 10-DAP that were 21.5, 28.7, and 25.3% pure, respectively. 13-DHB III and 10-DAP, two compounds useful as semisynthetic precursors of paclitaxel, were efficiently separated from plant cell cultures by this process. In this procedure, 13DHB III and 10-DAP were isolated as by-products during largescale purification of paclitaxel, producing high yields of these

Fig. 1. Schematic diagram of purification process for 13-DHB III, paclitaxel, and 10-DAP from plant cell cultures: (a) L/L (liquid/liquid) extraction; (b) LPLC (low-pressure liquid chromatography).

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Fig. 2. Chromatogram from silica-gel LPLC of plant cell culture extracts.

useful compounds without incurring any additional cost for culture or extraction. 3.2. Purification of 13-DHB III using ODS-LPLC and THF/n-hexane precipitation The further purification of crude 13-DHB III needs to be a useful starting material for semi-synthesis of paclitaxel. Therefore, the crude sample (21.5% pure) was applied to an ODS column (100-␮m particle size) that was equilibrated with a solution of 61–63% (v/v) methanol in water. Isocratic elution yielded 13-DHB III that was 81% pure with a 95.8% yield (Fig. 4). Further purification was achieved by precipitation of the 81%-pure 13-DHB III from THF/n-hexane to yield 99.5%-pure 13-DHB III. The sample was stored at −20 ◦ C for 2 days. Purities and yields for several different sets of precipitation conditions were compared to determine optimal conditions (Table 1). For sets 1–3, the ratio of THF:n-hexane was varied. At the highest THF ratio (set 1), the purity was greatest and the yield was lowest. As the proportion of n-hexane increased, purity decreased and yield increased. Based on these results, a THF:n-hexane ratio of 10:55 (v/v) was selected. Following this, the amounts of starting material were varied (sets 4–6). The yield of 13-DHB III increased proportionally to the amount of starting material, but the purity decreased. A starting amount of 1.0–1.5 g in 10 ml THF was concluded to be optimal (set 2). The resulting purity and yield were 99% (Fig. 3) and 93.2%, respectively. Even greater purity was achieved using ODS-HPLC fractionation of the 81%-pure ODS-LPLC sample, which yielded 13-DHB III that was >99.5 pure, with a 79.6% yield (Table 2). Table 1 Effect of precipitation of 13-DHB III under various sets of conditions Set

Fig. 3. Reverse-phase HPLC analysis of fractions from silica-gel LPLC of plant cell culture extracts: (A) crude extract; (B) chromatogram of 13-DHB III fraction; (C) chromatogram of paclitaxel fraction; (D) chromatogram of 10-DAP fraction. Arrows indicate peaks of 13-DHB III, paclitaxel, and 10-DAP, respectively.

1 2 3 4 5 6

Starting material

THF

n-Hexane

Precipitate

Purity (%)

Amount (g)

(ml)

(ml)

Purity (%)

Yield (%)

81 81 81 81 81 81

1.0 1.0 1.0 0.5 1.5 2.5

10 10 10 10 10 10

45 55 65 55 55 55

99.1 99.0 97.0 99.2 98.3 94.5

87.0 92.8 93.0 90.2 93.5 94.6

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Fig. 5. Chromatograms from 10-DAP purification: ODS-LPLC (A); reversephase HPLC chromatograms of 10-DAP fractions from ODS-LPLC (B); and ODS-HPLC (C).

3.3. Purification of 10-DAP using ODS-LPLC

Fig. 4. Chromatograms from 13-DHB III purification: ODS-LPLC (A); reversephase HPLC chromatograms of 13-DHB III fraction from ODS-LPLC (B); THF/n-hexane precipitate (C); and 13-DHB III fraction from ODS-HPLC (D).

Table 2 Summary of purification of 13-DHB III from plant cell cultures Purification process

13-DHB III (g)

Purity (%)

Step yield (%)

Overall yield (%)

Crude extract Silica-LPLC ODS-LPLC THF/n-Hexane (ODS-HPLC)b

8.1 7.9 1.03a 0.96a (0.82)a

2.7 21.5 81.0 99.0 (99.5)

100 97.5 95.8 93.2 (79.6)

97.5 93.4 87.1 (74.4)

a Amount of 13-DHB III obtained from 1.075 g 13-DHB III (crude sample 5 g) fractionated by silica-gel LPLC. b The ODS-HPLC was carried out using ODS-LPLC sample without THF/nHexane precipitation. The yield was calculated from ODS-LPLC step.

Like 13-DHB III, crude 10-DAP needs further purification for use in semi-synthesis of paclitaxel. Crude extracts were subjected to silica-gel LPLC, and the resulting crude 10DAP (25.3% pure) was further purified using ODS-LPLC, as described for 13-DHB III (Fig. 5). Isocratic elution yielded 10DAP that was 90.5% pure, with a 95.7% yield. Greater purity was achieved by ODS-HPLC fractionation of the 90.5%-pure ODS-LPLC sample. The final purity and yield were >99.5 and 88.4%, respectively (Table 3). Table 3 Summary of purification of 10-DAP from plant cell cultures Purification process

10-DAP (g)

Purity (%)

Crude extract Silica-LPLC ODS-LPLC ODS-HPLC

12.6 12.3 1.21a 1.07a

4.2 25.3 90.5 99.8

Step yield (%)

Overall yield (%)

97.6 95.7 88.4

100 97.6 93.4 82.6

a Amount of 10-DAP obtained from 1.265 g 10-DAP (crude sample 5 g) fractionated by silica-gel LPLC.

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Fig. 6. Proposed semi-synthesis of paclitaxel from 10-DAP and 13-DHB III.

3.4. Purification of crude paclitaxel using dichloromethane/n-hexane precipitation, ODS-HPLC, and silica-gel HPLC Using a previously published method [16], crude paclitaxel (28.7% pure) obtained by LPLC was further purified by precipitation from dichloromethane/n-hexane. The resulting purity and yield were 68.3 and 96%, respectively. ODS-HPLC then resulted in paclitaxel of 95.8% purity, with 92.5% yield, and subsequent silica-gel HPLC resulted in paclitaxel of >99.5% purity, with 95% yield. 3.5. Possible semi-synthesis of paclitaxel from 13-DHB III and 10-DAP Several reports have described semi-synthesis and totalsynthesis of paclitaxel [24–27]. As shown in Fig. 6, paclitaxel can be economically synthesized from 10-DAP using simple reactions, whereas semi-synthesis from 13-DHB III requires several reaction steps. Conversion of 10-DAP to paclitaxel could be carried out as a one-pot reaction in three steps: protection with a trimethylsilyl group, acetylation, and deprotection [24]. Alternatively, protected baccatin III could be synthesized from 13-DHB III by protection, oxidation, and reduction steps [9]; baccatin III could then be used to produce paclitaxel via several steps, including esterification with a side chain and deprotection [25–27]. Despite its increased complexity, semi-synthesis of paclitaxel from 13-DHB III is more cost-effective than purification of paclitaxel from yew tree bark, owing to the additional costs of its extraction and isolation from natural materials. Yew tree bark is of limited availability and sustainability, whereas the leaves of yew tree, which are renewable, include semi-synthetic precursors such as baccatin III and 10-DAP. Therefore, semi-synthesis of paclitaxel from 13-DHB III and 10-DAP isolated from cul-

tured cells offers an important alternative, renewable source of paclitaxel. 4. Summary: features and advantages of the optimized separation and purification process Silica-gel LPLC was carried out optimally using isocratic elution with 1.5% methanol in dichloromethane, which sequentially eluted 13-DHB III and paclitaxel, and a subsequent step elution with 5.0% methanol in dichloromethane, which eluted 10-DAP. Thus, three useful compounds were separated from crude extract in one chromatographic step. This method requires relatively simple equipment and operation, and features good resolution, a short operation time, and minimal use of solvent. An additional benefit is that the solvents used in this step (dichloromethane and methanol) are the same solvents used in the silica-HPLC step of paclitaxel purification. Use of the same solvents minimizes the amount of production equipment and allows reuse of the solvents after distillation in the same facility. During further purification of 13-DHB III and 10-DAP by ODS-LPLC, methanol and water were used as the solvents. These solvents are the same as those used in purification of paclitaxel by ODS-HPLC. Although their ratios were changed slightly (from 65 to 61–63% methanol) to obtain good peak separations, the same benefits of using the same solvents apply. In conclusion, paclitaxel, 13-DHB III, and 10-DAP can be simply and economically produced on an industrial scale, allowing recovery of important semi-synthetic precursors of paclitaxel while incurring minimal additional costs. References [1] M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon, A.T. McPhail, J. Am. Chem. Soc. 93 (1971) 2325.

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