Synthesis of gemcitabine triphosphate (dFdCTP) as a tris(triethylammonium) salt

Abstract

First synthesis of gemcitabine triphosphate (dFdCTP) as a tris(triethylammonium) salt is reported.

Gemcitabine (2’, 2’-difluoro-2’-deoxycytidine) is registered as a clinical anti-cancer agent for the treatment of a number of solid tumor types including pancreatic, non-small cell lung (NSCL), ovary, bladder, and breast cancer.¹ Gemcitabine is metabolized intracellularly by nucleoside kinases to the active diphosphate (dFdCDP) and triphosphate (dFdCTP) nucleoside metabolites.¹ The NCI Cancer Imaging Program needed gemcitabine triphosphate (dFdCTP), to be used as a standard in imaging studies using a radiolabeled probe for assessing the uptake and retention of gemcitabine in tumors and potentially identifying tumors sensitive to the drug. To facilitate such biochemical, pharmacological and other detailed studies of the metabolism of gemcitabine, a steady supply of authentic gemcitabine triphosphate (dFdCTP) was essential.²

Here we wish to report the first synthesis of gemcitabine triphosphate (dFdCTP), as shown in Scheme 1. Although, several methods have been reported for the general synthesis of nucleoside triphosphates,³ many of these approaches did not produce the desired triphosphate (dFdCTP) in our hands. Our synthesis incorporates the method described by Bogachev,⁴ using a modified purification procedure to give gemcitabine triphosphate as the tris(triethylammonium) salt.

Scheme 1.

Reagents and conditions: (a) POCl₃, (MeO)₃PO; (b) H₂O; (c) (CF₃CO)₂O; (d) 1-methylimidazole; (e) (nBu₄N)₃HP₂O₇; (f) DEAE Sephadex A25 Et₃NHHCO₃ (pH 7.5).

Gemcitabine hydrochloride was phosphorylated by portion wise addition to a mixture of phosphorus oxychloride (POCl₃) in trimethyl phosphate at 5°C.⁵ The reaction was warmed to room temperature and stirred for two hours. The progress of the reaction was followed by HPLC (small aliquots were worked up by treatment with aq NaHC0₃). Work up of the reaction afforded the monophosphate 2 (dFdCMP), as a white solid (105%). This material was used in the next step without further purification. ¹H NMR (500 MHz, CD₃OD):δ 7.95-7.93 (d, 1H); 6.25-6.22 (m, 1H); 6.01 (m, 1H); 4.39-4.32 (m, 1H); 4.25-4.23 (m, 1H); 4.14-4.10 (m, 1H); 4.00-3.98 (m, 1H). An impurity related to trimethyl phosphate is seen at δ 3.57 (s) and 3.55 (s). MS: Electrospray (negative), Calc’d for C₉H₁₂F₂N₃O₇P = 343; Found: (M-H)^- 342 (100%),. HPLC: Luna C-18 column (4.6 × 150 mm, 5 µ), Isocratic 10% MeOH/90% 20mM NH₄OAc, Flow rate: 1 mL/min at 20°C, Detection: UV at 254 nm, Results: The compound elutes at 1.837 min with 92.77% area.

The monophosphate 2 (dFdCMP) was treated with trifluroacetic anhydride in the presence of triethylamine and N, N-dimethlyaniline in acetonitrile at −5°C. The reaction mixture was warmed to room temperature and stirred for 20 minutes until the formation of a clear solution indicating the completion of the reaction. This reaction mixture was then evaporated in vacuo to complete dryness (TFA free) to yield crude 3, which was used in the next step without further purification. Crude 3 was dissolved in acetonitrile containing 1-methylimidazole and triethylamine. The mixture was stirred at 0°C for 5 minutes, when TLC analysis of an aliquot showed no presence of 3. The reaction mixture containing 4 was allowed to warm to 20°C and then was treated with a solution of tris(tetrabutylammonium) hydrogen pyrophosphate in acetonitrile. After stirring for 7–10 minutes at room temperature, the reaction was quenched by the addition of water. Following an aqueous workup, the crude gemcitabine triphosphate (dFdCTP) was purified by chromatography on DEAE Sephadex A25 (1.6 × 20 cm bed volume) eluted with deionized water (one column volume), followed by elution with a linear gradient from deionized water (500 mL) to 0.4 M triethylammonium bicarbonate (pH 7.5, 500 mL). Fractions containing pure dFdCTP as a tris(triethylammonium) salt were combined and concentrated in vacuo to give the desired triphosphate 5 (dFdCTP)(140 mg, 17%). The reproducibility of the above procedure was confirmed by repeating the reaction sequence several times. ¹H NMR (500 MHz, D₂O):δ 8.17-8.15 (d, 1H); 6.39-6.38 (d, 1H); 6.28-6.26 (m, 1H); 4.66-4.57 (m, 1H); 4.44-4.42 (m, 1H); 4.32-4.25 (m, 2H); 3.23-3.19 (q, 18H); 1.30-1.27 (t, 27H); ³¹P NMR (202.456 MHz, D₂O):δ -10.35 - -10.54 (m, 1P); -10.94 - -11.03 (d, 1P); -22.79 (m, 1P). HRMS: TOF Electrospray (negative): Calc’d for C₉H₁₃F₂N₃O₁₃P₃ = 501.9629, Found: m/z (relative intensity) 501.9611 (100%), (M-H)^-. HPLC: Bio Basic column and gradient elution using ammonium acetate buffer and acetonitrile eluted 5 with a retention time of 4.17 min showing 97.03% purity.

Milligram quantities of this material (dFdCTP, NSC 746306) for the use as a standard can be obtained from the NCI (http://dtp.nci.nih.gov).