Efficient synthesis of gemcitabine 5′-O-triphosphate using gemcitabine 5′-O-phosphoramidate as an intermediate

Article abstract of DOI:10.1055/s-0034-1378353

A new efficient approach for the synthesis of gemcitabine triphosphate has been developed. The method is based on the ring-opening reaction of 2-cyanoethoxy-2-oxo-1,3,2-oxathiaphospholane with protected gemcitabine in the presence of DBU. Subsequent treatment of gemcitabine monophosphate with DCC in the presence of ammonia provides gemcitabine 5′-O-phosphoramidate. Finally, this compound, on reaction with pyrophosphate, furnishes gemcitabine 5′-triphosphate in 50% yield. Georg Thieme Verlag Stuttgart. New York.

Full text of DOI:10.1055/s-0034-1378353

LETTER
▌1851
letter
Efficient Synthesis of Gemcitabine 5′-O-Triphosphate Using Gemcitabine
5′-O-Phosphoramidate as an Intermediate
Synthesis of Gemcitabine Triphosphate
Renata Kaczmarek,a Ewa Radzikowska,a Janina Baraniak*a,b
a
Department of Bioorganic Chemistry, Center of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112,
90-363 Łódź, Poland
Fax +48(42)6815483; E-mail: baraniak@cbmm.lodz.pl
b
Institute of Chemistry, Environmental Protection and Biotechnology, Jan Długosz University, Armii Krajowej 13/15, 42-201 Częstochowa,
Poland
Received: 15.04.2014; Accepted after revision: 28.05.2014
for assessing the uptake and retention time of gemcitabine
in tumors and potentially identifying tumors sensitive to
the drug.10
Abstract: A new efficient approach for the synthesis of gem-
citabine triphosphate has been developed. The method is based on
the ring-opening reaction of 2-cyanoethoxy-2-oxo-1,3,2-oxathia-
phospholane with protected gemcitabine in the presence of DBU.
Subsequent treatment of gemcitabine monophosphate with DCC in
the presence of ammonia provides gemcitabine 5′-O-phosphorami-
date. Finally, this compound, on reaction with pyrophosphate, fur-
nishes gemcitabine 5′-triphosphate in 50% yield.
Only one synthetic method for the preparation of gem-
citabine 5′-O-triphosphate has been reported in the litera-
ture, in which activated gemcitabine 5′-O-phosphate was
used as a key intermediate.11 This intermediate was ob-
tained by phosphorylation of the 5′-hydroxyl function of
gemcitabine with POCl3 in trimethyl phosphate, followed
by transformation to a 1-methylimidazolium derivative.
Further treatment with an acetonitrile solution of tris(tet-
rabutylammonium)hydrogen pyrophosphate gave
dFdCTP in 17% yield.
Key words: oxathiaphosphorylation, gemcitabine 5′-O-phosphate,
gemcitabine 5′-O-phosphoramidate, gemcitabine 5′-O-triphosphate
Gemcitabine (difluorodeoxycytidine, dFdC, Gemzar®) is
a deoxycytidine analogue with two fluorine atoms substi-
tuted for the two hydrogen atoms in the 2′-position of the
deoxyribose moiety. dFdC is registered as an anticancer
drug for the treatment of a number of different solid tu-
mors including nonsmall cell lung (NSCL), pancreatic,
ovary, bladder, and breast cancer.1–3 Gemcitabine is also
active against lymphomas.4 Studies on the metabolism of
gemcitabine have demonstrated that this compound is a
prodrug, which is subsequently converted intracellularly
by deoxycytidine kinases into the corresponding 5′-mono-
phosphate (dFdCMP), 5′-diphosphate (dFdCDP), and, fi-
nally, to its active 5′-triphosphate form (dFdCTP).5 These
metabolites inhibit two cellular processes required for
DNA biosynthesis. dFdCDP is a very potent inhibitor of
ribonucleotide reductase6 (RNR), while dFdCTP, after be-
ing incorporated into the C sites of the DNA during repli-
cation, inhibits DNA polymerases.6,7 Furthermore, 2′,2′-
difluoro-2′-deoxycytidine triphosphate is also inserted
into RNA, at a similar level as in DNA.8
Due to our long-term interest in the synthesis of nucleo-
side phosphates and polyphosphates12 we wished to elab-
orate a procedure for the selective preparation of
gemcitabine monophosphate and its further transforma-
tion into gemcitabine triphosphate.
Our first attempt towards the synthesis of gemcitabine 5′-
O-phosphate (1) was based on phosphoramidite chemis-
try, which was also used by Desmaële for the synthesis of
squalenoyl gemcitabine monophosphate.13 N4,O3′-dibenz-
oylgemcitabine (2) was phosphitylated by means of 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite (3,
Scheme 1) to yield 4, which, after hydrolysis in the pres-
ence of 1-H-tetrazole, gave the expected gemcytabine H-
phosphonate derivative 5 isolated by silica gel column
chromatography in 54% yield and characterized by 31P
NMR [δ (CD3CN) = 9.33 and 8.74 ppm] and MS–FAB
{m/z = 587 [M – 1]}. Compound 5 was oxidized with an
iodine–base–water mixture to yield 6, which was then de-
benzoylated with concentrated aqueous ammonia. Unfor-
tunately, the desired gemcitabine 5′-O-phosphate (1) was
isolated by ion-exchange column chromatography in only
3% yield (characterized by 31P NMR and MS–FAB). One
might presume that the retro-Michael deprotection of the
cyanoethyl group occurring under ammonia treatment is
responsible for this extremely low yield.
Compared with ara-C, gemcitabine is phosphorylated
more efficiently and is eliminated more slowly, thus of-
fers a longer retention time of the active forms in tumor
cells.9 Moreover, gemcitabine cytotoxicity is enhanced by
a number of unique self-potentiating mechanisms that
maintain high intracellular concentrations of the active
metabolites. It is worth mentioning that dFdCTP is used as
the standard in imaging studies using a radiolabeled probe
However, 31P NMR inspection of the reaction mixture ob-
tained after oxidizing of 5 showed, alongside the signal
corresponding to 6 (δ = –1.32 ppm; ca. 20%), unidentified
signals at δ = 14.80 and 11.77 ppm.
SYNLETT 2014, 25, 1851–1854
Advanced online publication: 09.07.2014
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DOI: 10.1055/s-0034-1378353; Art ID: st-2014-d0319-l
© Georg Thieme Verlag Stuttgart · New York

1852
R. Kaczmarek et al.
LETTER
NHBz
NHBz
NHBz
N
N
N
N
N
O
P
H
(i-Pr)2N
N(i-Pr)2
N
O
O
O
b
a
HO
P
O
CEO
O
O
O
O
+
Cl
P
CEO
F
OCE
F
F
F
F
BzO
F
BzO
BzO
2
3
4
5
OCE = OCH2CH2CN
c
NH2
NHBz
N
N
N
O–
O
d
N
O
O
–O
P
O
CEO
P
O
O
O
O
–O
F
F
F
BzO
HO
F
1
6
Scheme 1 Synthetic approach to the gemcitabine 5′-O-phosphate (1) based on phosphoramidite-type of chemistry. Reagents and conditions:
(a) DIPEA, CH2Cl2; (b) 1-H-tetrazole, MeCN–H2O; (c) I2–H2O–pyridine, Et3N; (d) NH4OH.
In the light of the above results we turned our attention to To obtain this reagent 3-hydroxypropionitrile (10) was re-
the oxathiaphospholane methodology, originally devel- acted with 2-chloro-1,3,2-oxathiaphospholane (7)18 in the
oped by Stec for the stereocontrolled synthesis of P-chiral presence of elemental sulfur in pyridine (Scheme 3).
analogues of oligonucleotides.14 This methodology has
been also used for thiophosphorylation and phosphoryla-
S
S
S
tion of various alcohols, including nucleosides.12a,15 In this
approach, 5′-O-(2-thiono-1,3,2-oxathiaphospholane) de-
rivatives of nucleosides, on reacting with 3-hydroxypro-
pionitrile in the presence of DBU, were transformed into
the corresponding nucleoside-5′-O-(O-2-cyanoethyl-
phosphorothioate)s. After removing the 2-cyanoethyl
moiety and benzoyl protecting groups with concentrated
aqueous ammonia, the desired phosphorothioate monoes-
ters were isolated in good to reasonable yields. The phos-
phorothioate compounds could then be oxidized to the
corresponding phosphates upon treatment with PhIO2.16
CN
a
CN
P
Cl
P
O
+
HO
O
O
7
10
9
Scheme 3 Synthesis of 2-cyanoethoxy-2-thiono-1,3,2-oxathiaphos-
pholane (9). Reagents and conditions: (a) S8, pyridine.
The reaction of crude 9 with protected gemcitabine (2)
was carried out in the presence of DBU in acetonitrile
(Scheme 4). The diester 11 so obtained was converted into
the desired phosphorothioate 12 by overnight treatment
with concentrated aqueous ammonia at room temperature,
and 12 was subsequently isolated from the reaction mix-
ture by means of ion-exchange chromatography on
DEAE-Sephadex A-25 in 67% yield. Its structure was
confirmed by 31P NMR spectroscopy [δ (D2O) = 43.90
and 43.55 ppm] and MS–FAB {m/z = 358 [M – 1]} anal-
yses. Unexpectedly, compound 12 upon treatment with
PhIO2 furnished mainly (40% yield) a gemcitabine disul-
fide derivative 13 {31P NMR: δ (D2O) = 16.80 ppm; MS–
FAB: m/z = 715 [M – 1]} while desired 1 was obtained in
only 2% yield.
NHBz
NHBz
N
N
N
N
S
P
O
S
O
S
O
O
a
O
HO
O
O
P
Cl
+
F
F
F
F
BzO
BzO
2
7
8
Scheme 2 Oxathiaphosphorylation of the protected gemcitabine (2).
Reagents and conditions: (a) S8, pyridine.
To avoid the use of an oxidizing reagent with 12 we mod-
ified our approach and oxidized 9 with selenium dioxide
in acetonitrile.15c The 31P NMR spectrum of the reaction
mixture recorded after ten minutes revealed a signal at
around δ = 45 ppm, indicating the formation of 2-cyano-
ethoxy-2-oxo-1,3,2-oxathiaphospholane (14), which was
reacted with protected gemcitabine (2) for four hours in
the presence of DBU in acetonitrile (Scheme 5). In this
case the desired gemcitabine 5′-O-phosphate (1) was
Unfortunately, direct oxathiaphosphorylation of the pro-
tected gemcitabine (2) by means of 2-chloro-1,3,2-oxathia-
phospholane (7) in the presence of elemental sulfur gave
the expected compound 8 in only 15% yield (Scheme 2).
Hence,
2-cyanoethoxy-2-thiono-1,3,2-oxathiaphos-
pholane (9) was synthesized as an alternative phosphoro-
thioylating reagent.17
Synlett 2014, 25, 1851–1854
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis of Gemcitabine Triphosphate
1853
NHBz
NH2
NHBz
N
N
N
N
N
S–
S–
P
S
P
S
O
–O
P
O
N
O
O
b
HO
CEO
O
a
O
O
O
+
OCE
O
O
O
F
F
F
F
F
BzO
HO
F
BzO
2
9
11
12
c
NH2
NH2
N
–O
O–
P
F
OH
N
O–
P
N
O
O
O
P
S
S
O
N
O
–O
O
F
O
+
O
O
O
F
O
O
N
F
HO
F
HO
F
N
1
13
NH2
Scheme 4 Reaction of protected gemcitabine (2) with 2-cyanoethoxy-2-thiono-1,3,2-oxathiaphospholane (9). Reagents and conditions: (a)
DBU, MeCN; (b) NH4OH; (c) PhIO2, H2O.
formed in 67% overall yield. Its structure was confirmed using ion-exchange chromatography on DEAE-Sephadex
by 31P NMR spectroscopy [δ (D2O) = 3.69 ppm] and MS– A-25. Its structure was confirmed by 31P NMR spectros-
FAB analysis {m/z = 342 [M – 1]}.19
copy [δ (D2O) = 9.41 ppm] and MS–FAB analysis {m/z =
341 [M – 1]}.21
NHBz
N
NHBz
NH2
NH2
N
N
O–
N
N
O
P
S
O–
P
N
O
+
O
O
O–
P
HO
CEO
P
a
O
O
OCE
a
–O
O
N
O
N
O
O
H2N
O
O
O
O
F
F
F
O
O
BzO
F
F
BzO
F
2
14
6
HO
F
HO
F
b
1
16
NH2
b
NH2
N
O–
P
N
O
O
O
N
O
–O
O
O
–O
P
O–
O
P
O–
O
P
O–
O
N
O
O
O
F
F
HO
F
F
1
HO
15
Scheme 5 Synthetic approach to the gemcitabine 5′-O-phosphate (1)
based on using 2-cyanoethoxy-2-oxo-1,3,2-oxathiaphospholane as a
phosphorylating reagent. Reagents and conditions: (a) DBU, MeCN;
(b) NH4OH.
Scheme 6 Synthesis of gemcitabine 5′-O-triphosphate (15) using
gemcitabine 5′-O-phosphoramidate (16) as an intermediate. Reagents
and conditions: (a) DCC, NH4OH; (b) bis-(tri-n-butyl)ammonium py-
rophosphate (17), DMF.
In the next step gemcitabine 5′-O-phosphate (1) was con-
verted into gemcitabine 5′-O-triphosphate (15). The most
widely used method of synthesis of nucleoside 5′-O-tri-
phosphates involves a nucleophilic attack of a pyrophos-
phate anion on a DCC/N-activated nucleoside 5′-O-
phosphate.20 We took advantage of this type of activation
and activated gemcitabine 5′-monophosphate (1) with
DCC/ammonium hydroxide in DMF (Scheme 6). After
heating at 80 °C for eight hours, gemcitabine 5′-O-phos-
phoramidate (16) was formed and isolated in 79% yield
Finally, 16 was converted into 15 using the procedure em-
ployed by Tomasz for the preparation of 5′-O-triphos-
phate derivatives of 3′,5′-diribonucleoside phosphates.22
The reaction was carried out in the presence of bis-(tri-n-
butyl)ammonium pyrophosphate (17, Scheme 6) in DMF
solution at 65 °C for 13 hours and its progress was moni-
tored by 31P NMR. Gemcitabine 5′-O-triphosphate (15,
dFdCTP) was isolated as its tris(triethylammonium) salt
in 50% yield using HPLC and was characterized by 31P
© Georg Thieme Verlag Stuttgart · New York
Synlett 2014, 25, 1851–1854

1854
R. Kaczmarek et al.
LETTER
(13) Caron, J.; Elise Lepeltier, E.; Reddy, L. H.; Lepêtre-
Mouelhi, S.; Wack, S.; Bourgaux, C.; Couvreur, P.;
Desmaële, D. Eur. J. Org. Chem. 2011, 14, 2615.
(14) Stec, W. J.; Grajkowski, A.; Karwowski, B.; Kobylanska,
A.; Koziołkiewicz, M.; Misiura, K.; Okruszek, A.; Wilk, A.;
Guga, P.; Boczkowska, M. J. Am. Chem. Soc. 1995, 117,
12019.
(15) (a) Baraniak, J.; Korczyński, D.; Stec, W. J. J. Org. Chem.
1999, 64, 4533. (b) Baraniak, J.; Kaczmarek, R.; Stec, W. J.
Tetrahedron Lett. 2000, 41, 9139. (c) Baraniak, J.;
Kaczmarek, R.; Korczyński, D.; Wasilewska, E. J. Org.
Chem. 2002, 67, 7267.
(16) (a) Mielniczak, G.; Łopusiński, A. Synlett 2001, 505.
(b) Kulik, K.; Radzikowska, E.; Kaczmarek, R.; Baraniak,
J.; Stec, W. J.; De Clerck, E.; Balzarini, J.; Pannecouqe, C.
Antiviral Chem. Chemother. 2011, 21, 143.
NMR spectroscopy [δ (D2O) = –10.43 (d), –11.04 (d), and
–22.59 (t) ppm] and MS analysis {MALDI, m/z = 501.7
[M – 1]}.23
In conclusion, a new efficient route for the synthesis of
gemcitabine 5′-O-triphosphate (dFdCTP) has been devel-
oped using gemcitabine monophosphate (1) as an inter-
mediate. Whereas some strategies presented herein have
been used previously for the synthesis of diverse nucleo-
side monophosphates,12a,15,16b they could not be imple-
mented for the preparation of the desired compound 1.
dFdCMP (1) was eventually prepared by the ring-opening
reaction of 2-cyanoethoxy-2-oxo-1,3,2-oxathiaphos-
pholane (14) with protected gemcitabine (2) in the pres-
ence of DBU. Subsequent treatment of 1 with DCC in the
presence of ammonia provided gemcitabine 5′-O-phos-
phoramidate (16) in good yield, and this activated form of
monophosphate was reacted with the pyrophosphate an-
ion furnishing gemcitabine 5′-O-triphosphate (15) in rea-
sonable yield.
(17) Zmudzka, K.; Nawrot, B.; Chojnacki, T.; Stec, W. J. Org.
Lett. 2004, 6, 1385.
(18) Martynov, I. V.; Kruglyak, Y. L.; Leibovskaya, G. A.;
Khromova, Z. I.; Strukov, O. G. Zh. Obshch. Khim. 1969,
39, 996.
(19) Experimental Procedure for the Synthesis of Compound
1
To a mixture of compound 2 and DBU in MeCN, crude
oxathiaphospholane 14 was added. After stirring at r.t. for 4
h the mixture was evaporated in vacuo, and the residue was
dissolved in 20% aq NH3 and left for 24 h at r.t. The mixture
was then evaporated, and product 1 was isolated by ion-
exchange chromatography in 67% yield.
Acknowledgment
These studies were supported financially by the Statutory Funds of
CMMS PAS.
31P NMR (202.45 MHz, D2O): δ = 3.69 ppm. 1H NMR (500
MHz, D2O): δ = 7.84–7.79 (d, 1 H), 6.17–6.12 (t, 1 H), 6.03–
6.00 (d, 1 H), 4.41–4.33 (m, 1 H), 4.17–4.11 (m, 1 H), 4.10–
4.05 (m, 1 H), 4.03–3.98 (m, 1 H) ppm. ESI-MS [M – 1]: m/z
= 342.
Supporting Information for this article, including detailed
experimental procedures for the syntheses of compounds 1, 2, 9, 14,
15 and 16 is available online at http://www.thieme-connect.com/
products/ejournals/journal/10.1055/s-00000083.SungIifoop
r
imaotrtnSupgIopi
n
foimarttr
n
(20) (a) Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1961,
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80, 3752. (d) Hoard, D. E.; Ott, D. G. J. Am. Chem. Soc.
1965, 87, 1785.
References and Notes
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Poore, G. A.; Todd, G. C.; Grindey, G. B. Cancer Res. 1990,
50, 4417.
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Transplant. 2010, 16, 1145.
(5) Gandhi, V.; Plunkett, W. Proc. Am. Assoc. Cancer Res.
1989, 30, 589.
(21) Experimental Procedure for the Synthesis of Compound
16
Compound 1 was dissolved in a mixture of 2 M NH4OH and
formamide, and to this solution DCC dissolved in t-BuOH
was added. The reaction mixture was heated at 80 °C for 10
h and then allowed to stand overnight at r.t. The mixture was
evaporated in vacuo, and the product was isolated by ion-
exchange chromatography in 78% yield.
31P NMR (202.45 MHz, D2O): δ = 9.41 ppm. 1H NMR (500
MHz, D2O): δ = 7.80–7.76 (d, 1 H), 6.19–6.13 (t, 1 H), 6.08–
6.02 (d, 1 H), 4.41–4.37 (m, 1 H), 4.14–4.10 (m, 1 H), 4.08–
4.01 (m, 1 H), 4.00–3.96 (m, 1 H) ppm. ESI-MS [M – 1]: m/z
= 341.
(6) Huang, P.; Plunkett, W. Semin. Oncol. 1995, 22, 19.
(7) Huang, P.; Chubb, S.; Hertel, L. W.; Grindey, G. B.;
Plunkett, W. Cancer Res. 1991, 51, 6110.
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Peters, G. J. Biochem. Pharmacol. 1993, 46, 762.
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S.; Wickremsinha, E. R.; Perkins, E. J.; Schellens, J. H. M.;
Beijnen, J. H. J. Mass Spectrom. 2006, 41, 1633.
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Chadda, S. K. Bioorg. Med. Chem. Lett. 2008, 18, 2957.
(12) (a) Olesiak, M.; Krajewska, D.; Wasilewska, E.;
Korczyński, D.; Baraniak, J.; Okruszek, A.; Stec, W. J.
Synlett 2002, 967. (b) Baraniak, J.; Wasilewska, E.;
Korczyński, D.; Stec, W. J. Tetrahedron Lett. 1999, 40,
8603. (c) Guranowski, A.; Starzyńska, E.; McLennan, A. G.;
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(22) Tomasz, J.; Simoncsits, A.; Kajtar, M.; Krug, R. M.;
Shatkin, A. J. Nucleic Acids Res. 1978, 5, 2945.
(23) (a) Experimental Procedure for the Synthesis of
Compound 15
Compound 16 was dissolved in anhydrous DMF, and bis-
(tri-n-butyl)ammonium pyrophosphate (17) in DMF was
added. After heating the homogeneous solution in a
stoppered flask at 65 °C for 13 h the mixture was evaporated,
and the product was isolated using HPLC in 50% yield.
31P NMR (202.45 MHz, D2O): δ = –10.43 (d), –11.04 (d),
–22.59 (t) ppm. 1H NMR (500 MHz, D2O): δ = 7.83–7.80 (d,
1 H), 6.14–6.09 (t, 1 H), 6.08–6.05 (d, 1 H), 4.46–4.38 (m, 1
H), 4.29–4.23 (m, 1 H), 4.18–4.13 (m, 1 H), 4.08–4.04 (m, 1
H) ppm. MALDI-MS [M – 1]: m/z = 501.7.
Synlett 2014, 25, 1851–1854
© Georg Thieme Verlag Stuttgart · New York

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