Synthesis of Ester-Linked Paclitaxel-Glycoside Conjugate as a Water-Soluble Paclitaxel Derivative—Maltoside Modification of Paclitaxel through Ester-Linker (Ester-Spacer)

Abstract

To synthesize a water-soluble paclitaxel derivative, the anomers of diols of allyl 2,3,4-tri-O-benzyl-6-O-tritylglycoside (maltoside) were prepared, which can be separated by chromatographic procedure. One anomer was converted into α-glycosyloxyacetic acid (maltosyloxyacetic acid) by oxidative cleavage of the diol and subsequent oxidation. Ester-linked paclitaxel-glycoside conjugate, 7-glycolylpaclitaxel 2″-O-α-maltoside, was provided by condensation of 2′-TES paclitaxel with α-glycosyloxyacetic acid (maltosyloxyacetic acid) followed by deprotection of hydroxy groups.

Paclitaxel is one of the most important anticancer agents used in the treatment of breast and ovarian cancers. ¹ It presents disadvantages such as low water solubility and toxicity toward normal tissues. To date many efforts have been made to modify paclitaxel chemically in order to create a more soluble and more easily delivered drug. ² Paclitaxel prodrugs, that incorporate acids, have attracted much attention, because an ester linkage improves the solubility of paclitaxel and can be hydrolyzed by hydrolytic enzymes to release paclitaxel. ³

Glycosylation of bioactive compounds has been the subject of increasing attention, since it can enhance their water solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve their bio- and pharmacological properties.^4-6 So far, little attention has been paid to the synthesis of ester-linked paclitaxel-glycoside conjugates as water-soluble paclitaxel prodrugs, which employ enzymatic cleavage by hydrolytic enzymes including esterases and glycosidases as their mode of activation. On the other hand, convenient drug delivery system (DDS) using liposomes and bionanocapsules has attracted pharmacological attention. ⁷ However, the DDS system is not effective for delivering paclitaxel, because the present nano-particles can incorporate only soluble drugs.

On continuing the study to develop the technology for delivering paclitaxel, we investigated the synthesis of ester-linked paclitaxel-sugar conjugate, that is, 7-glycolylpaclitaxel 2″-O-α-maltoside.

The ester-linked paclitaxel-sugar conjugate, 7-glycolylpaclitaxel 2″-O-α-maltoside, was totally synthesized by chemical procedures. Allyl alcohol was linked to maltose by ether binding using triallyl orthoformate and acetyl chloride to give the product 1. DMAP (4-dimethylaminopyridine), which is a derivative of pyridine, is more basic than pyridine. Its basicity is caused by resonance stabilization from the NMe₂ substituent. DMAP is an important nucleophilic catalyst for tritylations. After neutralization of the reaction mixture with pyridine, the product 1 was tritylated using trityl chloride in the presence of catalyst DMAP. The evaporation, extraction, neutralization with saturated sodium hydrogen carbonate solution, dryness with magnesium sulfate, and concentration successfully prepared allyl 2,3,4-tri-O-benzyl-6-O-tritylmaltoside. After purification process by silica gel column chromatography, benzylation of allyl 2,3,4-tri-O-benzyl-6-O-tritylmaltoside was carried out with BnBr and NaH using dibenzo-18-crown-6 as a catalyst to give allyl 2,3,4-tri-O-benzyl-6-O-tritylmaltoside. Dihydroxylation of allyl 2,3,4-tri-O-benzyl-6-O-tritylmaltoside was proceeded in the presence of OsO₄ as an oxidant to the diastereomers of diols. It was found that anomers were separable by silica gel column chromatography to prepare diols of α-anomer 2. Oxydation of diols of α-anomer 2 by NaIO₄ to give carboxymethyl-2,3,4-tri-O-benzyl-6-O-tritylmaltoside (3). Further oxidation was carried out with NaClO₂ to ensure the oxidation of aldehydes.

The hydroxy group of paclitaxel was regioselectively triethylsilated by chlorotriethylsilane. After neutralization, dryness, concentration, and purification by column chromatography, 2′-TES ester of paclitaxel was prepared successfully. The condensation of 2′-TES ester of paclitaxel with carboxymethyl-2,3,4-tri-O-benzyl-6-O-tritylmaltoside (3) was carried out in the presence of EDCI/DMAP. After extraction, concentration, and purification, the product 4 was prepared. The hydroxy groups of 4 were deprotected by Pd black in HOAc-H₂O to give 7-glycolylpaclitaxel 2″-O-α-maltoside (5) (see Figure 1).

Figure 1.

Synthesis of 7-glycolylpaclitaxel 2″-O-α-maltosicde. (a) HC(OCH₂CH = CH₂)₃/allyl alcohol (10 eq)/AcCl (1.2 eq), 50 °C, 24 h; (b) Ph₃CCl (1.1 eq)/Py/DMAP (0.05 eq), rt, 24 h; (c) BnBr/NaH/dibenzo-18-crown-6/DMF/THF, rt, 12 h; (d) OsO₄/50% NMO/dioxane, rt, 24 h, quant.; (e) NaIO₄/dioxane, rt, 12 h; (f) NaClO₂/t-BuOH/dioxane, rt, 3 h, quant.; (g) EDCI/DMAP/CH₂Cl₂, rt, 12 h, quant.; (h) H₂/Pd black, rt, 24 h, quant.

Thus, paclitaxel (water solubility, ca. 0.4 μg/mL) was converted into hydrophilic paclitaxel derivative, that is, 7-glycolylpaclitaxel 2″-O-α-maltoside, which was much more water soluble than paclitaxel, by organic synthetic procedures. In this study, we investigated the convenient methods using diols of allyl 2,3,4-tri-O-benzyl-6-O-tritylmaltoside, which can be separated by column chromatography. Further studies on the DDS using the paclitaxel derivative, 7-glycolylpaclitaxel 2′′-O-α-maltoside, are now in progress in our laboratory.

Experimental

General

In the synthesis of ester-linked paclitaxel-glycoside conjugate, 7-glycolylpaclitaxel 2″-O-α-maltoside, commercially available chemical reagents such as paclitaxel were used. The nuclear magnetic resonance (NMR) spectra were recorded in CD₃OD.

Synthesis of 7-Glycolylpaclitaxel 2″-O-α-Maltoside

To a mixture of triallyl orthoformate and acetyl chloride in allyl alcohol was added maltose. The mixture was stirred at 50 °C for a day. The mixture was neutralized by addition of pyridine, and the solvent was removed by evaporation. The condensed product 1 was treated with trityl chloride in pyridine with DMAP at room temperature for a day. The mixture was evaporated and the product triol was dissolved in 1 M HCl followed by extraction with toluene. The toluene layer was washed with saturated sodium hydrogen carbonate solution, dried with magnesium sulfate, and concentrated in vacuo. The crude products were purified by silica gel column chromatography. The purified triol was benzylated with BnBr and NaH in the presence of catalyst dibenzo-18-crown-6 to allyl 2,3,4-tri-O-benzyl-6-O-tritylmaltoside. Allyl 2,3,4-tri-O-benzyl-6-O-tritylmaltoside was dihydroxylated with OsO₄ to give diols. Purification of anomers of diols prepared diols of α-anomer 2. Diols of α-anomer 2 were transformed by oxidative cleavage with NaIO₄ to the corresponding carboxylic acid, carboxymethyl-2,3,4-tri-O-benzyl-6-O-tritylmaltoside (3). Aldehydes were oxidized with NaClO₂.

To a solution of paclitaxel and imidazole in DMF was added chlorotriethylsilane dropwise at room temperature. The mixture was stirred at room temperature for 2 h and diluted with ethyl acetate. The mixture was washed with water and brine, dried over magnesium sulfate, and concentrated in vacuo. Column chromatography of the residue on silica gel gave 2′-TES ester of paclitaxel. To a mixture of 2′-TES ester of paclitaxel in the presence of EDCI/DMAP in CH₂Cl₂ was added carboxymethyl-2,3,4-tri-O-benzyl-6-O-tritylmaltoside (3). The mixture was stirred at room temperature for 12 h. The reaction mixture was extracted with ethyl acetate. The organic layer was concentrated in vacuo and purified by column chromatography on silica gel to give the product 4. To a solution of Pd black in HOAc-H₂O was added the product. The suspension was stirred under hydrogen atmosphere at room temperature for 24 h. Extraction of the reaction mixture with n-butanol followed by column chromatography on silica gel yielded 7-glycolylpaclitaxel 2″-O-α-maltoside (5).

Product Identification

Spectral data of 7-glycolylpaclitaxel 2″-O-α-maltoside are as follows.

¹H NMR (400 MHz, CD₃OD, δ in ppm): δ 1.11 (s, H-16), 1.16 (s, H-17), 1.81 (s, H-19), 1.87 (m, H-6β), 2.00 (m, H-18, H-14α), 2.16 (s, CH₃ in 10Ac), 2.21 (m, H-14β), 2.37 (s, CH₃ in 4Ac), 2.58 (m, H-6α), 3.26 to 3.87 (m, H-2″, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, 6b), 3.88 (m, H-3), 4.20 (m, H-7, 20), 4.75 (d, J = 4.4 Hz, H-2′), 4.83 (m, H-1b), 5.00 (m, H-5), 5.16 (d, J = 2.8 Hz, H-1a), 5.64 (m, H-2, 3′), 6.15 (m, H-13), 6.21 (s, H-10), 7.29 (t, J = 3.8 Hz, p-H in Ph), 7.39 to 7.57 (m, m-H in NBz, p-H in NBz, m-H in OBz, o-H in Ph, m-H in Ph), 7.65 (m, p-H in OBz), 7.85 (d, J = 5.6 Hz, o-H in NBz), 8.10 (d, J = 6.0 Hz, o-H in OBz).