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

Article abstract of DOI:10.1016/j.bmcl.2008.03.063

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

Full text of DOI:10.1016/j.bmcl.2008.03.063

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2957–2958
Synthesis of gemcitabine triphosphate (dFdCTP)
as a tris(triethylammonium) salt
Prabhakar A. Risbood,a,* Charles T. Kane, Jr.,b Md. Tafazzal Hossain,b
Sudhakarrao Vadapallib and Satish K. Chaddab
aDrug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
bStarks Associates Inc., 1280 Niagara Street, Buffalo, NY 14213, USA
Received 4 March 2008; accepted 21 March 2008
Available online 25 March 2008
Abstract—First synthesis of gemcitabine triphosphate (dFdCTP) as a tris(triethylammonium) salt is reported.
Published by Elsevier Ltd.
Gemcitabine (20, 20-difluoro-20-deoxycytidine) is regis-
tered 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
Gemcitabine hydrochloride was phosphorylated by por-
tionwise addition to a mixture of phosphorus oxychlo-
ride (POCl3) in trimethyl phosphate at 5 ꢀC.5 The
reaction was warmed to room temperature and stirred
for 2 h. The progress of the reaction was followed by
HPLC (small aliquots were worked up by treatment
with aqueous NaHCO3). Workup of the reaction affor-
ded monophosphate 2 (dFdCMP), as a white solid
(105%). This material was used in the next step without
1
further purification. H NMR (500 MHz, CD3OD): d
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 d 3.57 (s) and 3.55 (s). MS: Electro-
spray (negative), Calcd for C9H12F2N3O7P = 343.
Found: (MꢀH)ꢀ 342 (100%). HPLC: Luna C-18 col-
umn (4.6 · 150 mm, 5 l), isocratic 10% MeOH/90%
20 mM NH4OAc, flow rate: 1 mL/min at 20 ꢀC, detec-
tion: UV at 254 nm, results: the compound elutes at
1.837 min with 92.77% area.
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 triphos-
phate (dFdCTP) in our hands. Our synthesis incorpo-
rates the method described by Bogachev,4 using a
modified purification procedure to give gemcitabine tri-
phosphate as the tris(triethylammonium) salt.
Monophosphate 2 (dFdCMP) was treated with trifluo-
roacetic anhydride in the presence of triethylamine and
N, N-dimethylaniline in acetonitrile at ꢀ5 ꢀC. The reac-
tion mixture was warmed to room temperature and stir-
red for 20 min until the formation of a clear solution
indicating the completion of the reaction. This reaction
mixture was then evaporated in vacuo to complete dry-
ness (TFA free) to yield crude 3, which was used in the
next step without further purification. Crude 3 was dis-
solved in acetonitrile containing 1-methylimidazole and
triethylamine. The mixture was stirred at 0 ꢀC for 5 min,
Keyword: Gemcitabine triphosphate (dFdCTP).
*
Corresponding author. Tel.: +1 301 435 9159; fax: +1 301 480
4817; e-mail: risboodp@mail.nih.gov
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.03.063

2958
P. A. Risbood et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2957–2958
NH2
N
NHCOCF3
N
NH2 HCl
N
N
O
N
O
O
N
O
O
H2O3PO
O P O
OH
F3C
HO
O
O
O
c
a, b
F
F
F
OH F
F3C
O
F
F
OH
O
1
2
3
NHCOCF3
N
NH2
N
N
O
O
P O
O
N
O
O
O
O
Me N
d
e, f
N
O
HO P O P O P
O
+
-
O
OH F
F
OH OH OH
F
O
F
F3C
3 Et3N
O
5
4
Scheme 1. Reagents and conditions: (a) POCl3, (MeO)3PO; (b) H2O; (c) (CF3CO)2O; (d) 1-methylimidazole; (e) (nBu4N)3HP2O7; (f) DEAE
Sephadex A25, Et3NHCO3 (pH 7.5).
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 min at room temper-
ature, the reaction mixture was quenched by the addi-
tion 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 trieth-
ylammonium 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 con-
firmed by repeating the reaction sequence several times.
Acknowledgments
We thank Linda J. Whited for her assistance with the
HPLC analysis. This project has been funded in whole
or in part with Federal funds from the National Cancer
Institute, National Institutes of Health, Department of
Health and Human Services, under Contract No. N02-
CM-52209.
Supplementary data
Detailed experimental procedures for the synthesis of
compounds 2, 3, 4, and 5 are included in the Supporting
Information section of this report. Supplementary data
associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2008.03.063.
1H NMR (500 MHz, D2O): d 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): d ꢀ10.35 to ꢀ10.54 (m, 1P); ꢀ10.94 to ꢀ11.03
(d, 1P); ꢀ22.79 (m, 1P). HRMS: TOF Electrospray
(negative): Calcd for MꢀH = C9H13F2N3O13P3 =
501.9629, Found: m/z (relative intensity) 501.9611
(100%), (MꢀH)ꢀ. HPLC: Bio Basic column and step
gradient elution using ammonium acetate buffer and
acetonitrile eluted 52 with a retention time of 4.17 min
showing 97.03% purity.
References and notes
1. Norenberg, J. P. U.S. Patent 20070025910, 2007; Chem.
Abstr. 2007, 146, 200935.
2. The following reference describes an assay of
dFdCTP: Veltkamp, S. A.; Hillebrand, M. J. X.;
Rosing, H.; Jansen, R. S.; Wickremsinha, E. R.;
Perkins, E. J.; Schellens, J. H. M.; Beijnen, J. H. J.
Mass Spectrom. 2006, 41, 1633, This reference
describes obtaining dFdCTP from Eli Lilly and
Company; however, the method of synthesis or
isolation was not indicated.
3. Burgess, K.; Cook, D. Chem. Rev. 2000, 100, 2047.
4. Bogachev, V. S. Russ. J. Bioorg. Chem. 1996, 22, 599.
5. Bonjouklian, R.; Grindey, G. B.; Hertel, L. W. European
Patent Application 0376518, 1990; Chem. Abstr. 1991, 114,
43489.
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).