Efficient syntheses of clofarabine and gemcitabine from 2-deoxyribonolactone

Article abstract of DOI:10.1080/15257771003597758

The development of a new methodology to achieve electrophilic fluorination of triisopropylsilyl-protected 2-deoxyribonolactone has been employed to synthesize clofarabine and gemcitabine with improved synthetic efficiency versus prior synthetic methods. These studies highlight the versatility of this new methodology to obtain medically relevant 2′-fluoronucleosides.

Full text of DOI:10.1080/15257771003597758

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Efficient Syntheses of Clofarabine and
Gemcitabine From 2-Deoxyribonolactone
Yana Cen a & Anthony A. Sauve a
a Department of Pharmacology, Weill Medical College of Cornell
University, New York, New York, USA
Version of record first published: 24 Feb 2010.
To cite this article: Yana Cen & Anthony A. Sauve (2010): Efficient Syntheses of Clofarabine and
Gemcitabine From 2-Deoxyribonolactone, Nucleosides, Nucleotides and Nucleic Acids, 29:2, 113-122
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Nucleosides, Nucleotides and Nucleic Acids, 29:113–122, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257771003597758
EFFICIENT SYNTHESES OF CLOFARABINE AND GEMCITABINE
FROM 2-DEOXYRIBONOLACTONE
Yana Cen and Anthony A. Sauve
Department of Pharmacology, Weill Medical College of Cornell University, New York,
New York, USA
The development of a new methodology to achieve electrophilic fluorination of triisopropylsilyl-
2
protected 2-deoxyribonolactone has been employed to synthesize clofarabine and gemcitabine with
improved synthetic efficiency versus prior synthetic methods. These studies highlight the versatility of
this new methodology to obtain medically relevant 2ꢁ-fluoronucleosides.
Keywords Diastereoselective; electrophilic fluorination; 2-deoxyribonolactone
INTRODUCTION
Nucleosides bearing fluorine or fluorinated substituents within the car-
bohydrate moiety are of value in biochemical research and therapeutic
treatment.[1–4] The presence of fluorine functional groups alters the phys-
ical and chemical properties of nucleosides with respect to their nonfluo-
rinated counterparts and can cause increased resistance to metabolic de-
composition.[3] Some best selling prescription drugs are fluorinated nucle-
osides,[1] such as the prodrug clofarabine (1, 2-chloro-2ꢁ-deoxy-2ꢁ-arabino-
fluoroadenosine, Figure 1), a purine nucleoside antimetabolite used clini-
cally to treat pediatric patients with relapsed or refractory acute lymphocytic
leukemia.[5] Clofarabine metabolites deplete 2-deoxynucleotide pools within
cells by inhibiting ribonucleotide reductase. This, in turn, restricts DNA syn-
thesis, although it exhibits toxicity by other mechanisms as well.[5] Another
well-known drug, Gemcitabine (2, Gemzar, 2ꢁ-deoxy-2ꢁ,2ꢁ-difluorocytidine,
Figure 1; Eli Lilly, Indianapolis, IN, USA), is a gem-difluoronucleoside, that
is widely used in the clinical setting to treat ovarian,[6] pancreatic,[7] and
breast cancers[8] with annual sales well in excess of 1 billion dollars a year.[9]
Gemcitabine metabolites interfere directly with DNA replication[10,11] and
Received 2 September 2009; accepted 4 December 2009.
Address correspondence to Anthony A. Sauve, Department of Pharmacology, Weill Medical College
of Cornell University, 1300 York Avenue, New York, NY 10065, USA. E-mail: aas2004@med.cornell.edu
113

114
Y. Cen and A. A. Sauve
NH2
N
N
HO
TIPSO
TIPSO
F
N
F
O
?
O
O
O
O
Clofarabine
N
NFSi, LiHMDS
THF, -78oC
(72%)
Cl
TIPSO
OH
TIPSO
NFSi, LiHMDS
THF, -78oC
(71%)
NH2
N
TIPSO
HO
F
F
F
N
O
O
O
?
O
Gemcitabine
TIPSO
F
HO
SCHEME 1 General strategy for synthesis of gemcitabine and clofarabine.
inhibit ribonucleotide reductase.[10,11] Despite their importance as cancer
drugs current procedures to synthesize clofarabine and gemcitabine are still
of limited efficiency.
We recently developed highly efficient syntheses of silyl-protected
2-deoxy-2-fluoro (arabino and gem-difluoro)-furanoses from 2-deoxyri-
bonolactone (Scheme 1) using a diastereoselective electrophilic fluorina-
tion methodology.[12] It is important to note that the sugars are precur-
sors for synthesis of 2ꢁ-deoxy-2ꢁ-fluoronucleosides. We wondered if we could
successfully synthesize clofarabine and gemcitabine (Figure 1) by use of
these methods as shown in Scheme 1. We herein describe new methods
to synthesize clofarabine and gemcitabine via routes utilizing electrophilic
fluorination of protected 2-deoxyribonolactone. These syntheses provide
substantially improved yields versus other reported methods. These studies
demonstrate the efficiency of electrophilic fluorination methodologies to
construct commercially important 2ꢁ-fluoro-substituted nucleosides.
RESULTS AND DISCUSSION
Previous Syntheses of Clofarabine
Clofarabine was first synthesized by Montgomery and coworkers (Scheme
2).[13] The synthetic route depends on a protected arabino-fluororiboside
NH2
NH2
N
N
HO
HO
N
F
F
F
N
N
O
O
N
O
Cl
HO
OH
1 (Clofarabine)
FIGURE 1 Structures of clofarabine and gemcitabine.
2 (Gemcitabine)

Syntheses of Clofarabine and Gencitabine
115
BzO
BzO
BzO
BzO
F
OAc
KHF2
2,3-butanediol
O
O
O
O
SO2Cl2, DMF
imidazole
HBr
CH2Cl2, 0oC
(81%)
(54% after 2 steps)
OAc
OAc
OAc
OAc OBz
OAc
OAc OH
OAc
OSO2N
N
HBr/CH2Cl2
NH2
N
Cl
N
N
HO
BzO
BzO
2,6-dichloropurine
4Å MS
dichloroethane, reflux
(32%)
N
NH3/ethanol
r.t., 3 days
then LiOH
(42%)
F
F
F
N
N
O
O
O
N
N
Br
Cl
Cl
OH
OAc
OAc
1
SCHEME 2 Montgomery’s synthesis of clofarabine.
precursor that is coupled to 2,6-dichloropurine. Installation of the 6-
amino group occurred concomittantly with removal of ester protect-
ing groups by ethanolic ammonia and base. The key protected arabino-
fluororibofuranose was obtained by conversion of a 2-ribo-hydroxyl group
to a 2-arabino-fluoro group by nucleophilic fluoride. The use of nucleophilic
fluoride to obtain 2-arabino-fluororibose and 2ꢁ-arabino-fluoronucleosides
has been the conventional method of synthesis for these compounds for
several decades.[14] Clofarabine was prepared in 6 steps in 6% overall
yield.
Recently, ILEX Products Inc. (San Antonio, TX, USA) reported an
improved synthesis of clofarabine (Scheme 3).[15] The newer method uti-
lizes an appropriate stereochemically pure (α-anomer) 2-deoxy-2-fluoro-1-
bromosugar (Scheme 3) obtained by nucleophilic fluorination chemistry
from 1-O-acetyl-2,3,5-tri-O-benzoyl-α-D-ribofuranose.[15,16] The base conju-
gation step to form the nucleoside is achieved via a preformed 2-chloro-6-
amino purine and proceeds with excellent stereoselectivity (β/α = 21/1).
Deprotection completed the synthesis in 6 steps 14% overall yield starting
from a fully protected ribose starting material.[15]
Bz O
Bz O
1) HBr , CH Cl , 0oC
B zO
SO Cl , DM F
imidaz ole
( 85%)
BzO
F
OA c
K HF
2
O
O
O
O
2,3-butanediol
( 63%)
2
2
2
2
2) H
O
2
OBz
OBz
OSO
OBz
(81%)
OB z OB z
O Bz
OBz
OBz OH
N
2
N
HBr/HOA C
(95~98%)
NH
N
NH
2
2
N
N
HO
B zO
B zO
N
F
F
F
2-chloroadenine
N
N
NaOMe/M eO H
O
O
O
t
N
r .t.
( 64%)
N
K OBu , CH CN
3
Cl
Cl
tA mOH, DCE , 55oC
B r
(50%)
OH
OB z
OBz
1
SCHEME 3 ILEX’s synthesis of clofarabine.

116
Y. Cen and A. A. Sauve
A New Efficient Synthesis of Clofarabine From
2-Deoxyribonolactone
Improved access to silyl-protected 2-deoxy-2-arabino-fluorofuranose[12]
led us to wonder if we could construct clofarabine in a succinct man-
ner. Starting from TIPS-protected 2-deoxyribonolactone 3, diastereoselec-
tive electrophilic fluorination of 3 with LiHMDS with the presence of NFSi
at −78◦C produced only the arabino-isomer 4 in 72% isolated yield as re-
ported (Scheme 4).[12] Reduction of 4 with DIBAL-H provided lactol 5
in 91% yield.[12] 5 was activated with methanesulfonylchloride and triethy-
lamine, which formed only the α-chloro sugar 6 in quantitative yield.[12]
6
was coupled with 2,6-dichloropurine[17] by analogy to the method of Mont-
gomery.[13] The coupling provided both β and α isomers with a 3.5 to 1 ratio
favoring the desired β-isomer. The mixture of protected nucleosides 7 (α
and β) was then submitted to the next step without purification. Amination
of 7 with ammonia in isopropanol in a sealed tube at 105◦C afforded the
desired 6-amino-2-chloropurine nucleoside 8. At this point the β-anomer
and α-anomer were separately purified by silica gel chromatography. Yield
of the stereochemically pure silyl-protected β clofarabine after coupling and
amination steps was 65% whereas the yield of the α isomer was 16%, which
was fully characterized, but we did not deprotect it. Removal of the protect-
ing groups from the β isomer occurred in 90% yield to provide clofarabine
as an off-white solid.[13,15] The synthesis of clofarabine from TIPS-protected
lactone was 6 steps with overall yield of 38% (Scheme 4). Table 1 shows
our method requires the same number of steps as prior methods, but has a
significant improvement in overall yield (14% vs. 38%).
Previous Syntheses of Gemcitabine
Synthetic procedures to produce gemcitabine were developed by Chou
and coworkers for commercial production of the nucleoside and are adapted
from Hertel’s method.[18] The method of Chou involves coupling of ethyl
bromodifluoroacetate and isopropylidene glyceraldehyde to obtain the im-
portant precursor a protected gem-difluorolactone that can be used for nu-
cleoside synthesis.[19] The route has the pitfall that it is not diastereoselective
TABLE 1 Comparison of three different clofarabine syntheses
Method
Steps
Total yield (%)
Montgomery
ILEX
Sauve
6a
6a
6b
6
14
38
aFrom acyl-protected ribofuranose.
bFrom silyl-protected 2-deoxyribonolactone.

Syntheses of Clofarabine and Gencitabine
117
O
BzO
O
O
O
F
F
O
O
+
O
Br
OEt
H
CO2Et
F
F
F
BzO
F
OR
O
NH2
OTMS
recrystallization
N
N
N
O
N
N
F
BzO
O
BzO
O
HO
O
TM SO
OH
OMs
F
F
F
BzO
BzO
HO
F
F
2
SCHEME 4 Eli Lilly’ synthesis of gemcitabine.
and depends on crystallization to obtain the preferred isomer (Scheme 5).
Shen’s group reported a slightly improved method for synthesis of gemc-
itabine which was quite similar to the Chou method, differing mostly in the
use of a different protection scheme.[20] The Shen method requires 10 steps
and still requires crystallization step for isomer purification, with an overall
yield of 10% (starting from isopropylidene glyceraldehyde, Scheme 6).
A New Efficient Synthesis of Gemcitabine
We recently reported that arabino-fluorolactone 4 could be fluorinated
with NFSi at −78◦C to access the corresponding gem-difluorolactone.[12] 2-
deoxy-2,2-difluororibonolactone 9 was obtained in an overall yield of 51%
yield from the initial TIPS-protected 2-deoxy-ribonolactone (Schemes 4
and 7).[12] Lactol 10 was obtained via DIBAL-H reduction of 9 in
91% yield. We then prepared 1-mesylate 11.[12] Coupling of 11 with
bis(trimethylsilyl)cytosine yielded nucleoside 12 in both α and β configu-
rations (Scheme 7). Integration of 1ꢁ-hydrogen on NMR spectrum indicated
that the stereochemistry ratio of coupling was α/β = 1:1, similar to that
reported by other workers for this step.[19,20] Subsequent deprotection and
HO
O
RO
O
RO
O
O
O
stereospecific
crystallization
O
F
F
F
HO
RO
RO
RO
F
F
F
O
R =
NHAc
N
Ph
O
N
RO
RO
O
O
2
OTs
F
F
RO
F
F
SCHEME 5 Shen’s synthesis of gemcitabine.

118
Y. Cen and A. A. Sauve
TABLE 2 Comparison of three different gemcitabine syntheses
Method
Steps
Total yield (%)
Chou
Shen
Sauve
8a
10a
6b
5.8
10
17
aFrom isopropylidene glyceraldehyde.
bFrom silyl-protected 2-deoxyribonolactone.
TIPSO
TIPSO
HO
F
NFSi, LiHMDS
THF, -78oC
TIPSCl
imidazole
O
O
O
O
O
O
DMF, r.t.
(92%)
(72%)
TIPSO
TIPSO
OH
4
3
DIBAL-H
toluene, -78oC
(91%)
Cl
N
TIPSO
TIPSO
TIPSO
MsCl, Et3N
CH2Cl2
(quantitative)
N
2,6-dichloropurine
4Å MS
F
F
F
O
N
O
O
N
OH
Cl
dichloroethane, reflux
Cl
TIPSO
TIPSO
TIPSO
7
6
5
NH3/isopropanol
sealed tube 105oC
(65% after 2 steps)
NH2
N
NH2
N
N
HO
TIPSO
N
Me4NF, AcOH
F
F
N
N
O
O
DMF
N
N
Cl
(90%)
Cl
HO
TIPSO
8
1
SCHEME 6 Stereoselective synthesis of clofarabine.
TIPSO
TIPSO
TIPSO
F
F
F
F
O
NFSi, LiHMDS
THF, -78oC
(71%)
DIBAL-H, toluene
-78oC
O
O
O
O
OH
(91%)
TIPSO
TIPSO
TIPSO
F
10
4
9
MsCl, Et3N
CH2Cl2
(quantitative)
NH2
N
NH2
N
O
HO
TIPSO
TIPSO
F
F
F
bis(trimethylsilyl)cytosine
TMSOTf
1,2-dichloroethane
N
N
Me4NF, AcOH
DMF
O
O
O
O
OMs
(36% after two steps)
TIPSO
F
HO
F
TIPSO
12 (
F
2
= 1/1)
11
SCHEME 7 Stereoselective synthesis of gemcitabine.

Syntheses of Clofarabine and Gencitabine
119
HPLC purification provided both isomers of gemcitabine (2) with the β
isomer in 36% yield and the α isomer in 42% from the mesylate. The synthesis
of β-gemcitabine was completed in 6 steps with 17% overall yield (starting
from fully protected 2-deoxyribonolactone). This new synthesis exceeds the
methods of Chou and Shen for selectivity, brevity and yield (Table 2).
CONCLUSION
TIPS-protected 2-deoxyribonolactone is a versatile precursor to 2-deoxy-
2-fluororibonolactones and furanoses via electrophilic fluorination.[12]
Here we demonstrated that electrophilic fluorination of TIPS-protected 2-
deoxyribonolactone can be used to synthesize the commercially important
fluorinated nucleosides gemcitabine and clofarabine with greater efficiency
as compared with prior published methods. The TIPS groups do not ap-
pear to present an impediment to nucleoside coupling steps, which still
occur with desirable stereochemical yield. Furthermore, the TIPS groups
are readily removed after nucleoside formation, to obtain the desired nu-
cleosides in high yield. These results demonstrate improved synthetic access
to 2ꢁ-fluoronucleosides by methods integrating electrophilic fluorination of
TIPS-protected 2-deoxyribonolactone.
EXPERIMENTAL
General
1H and 13C NMR spectra were obtained using either a Bruker 400 MHz
AMX or a Bruker Avance DMX 500 MHz spectrometer (Bruker Biospin,
Rhinstetten, Germany). Mass spectra were recorded either on a PerSeptive
(PerSpective Biosystems, Inc., Framingham, MA, USA) Voyager DE STR
MALDI-TOF mass spectrometer (PerSpective Biosystems, Inc., Framingham,
MA, USA) or Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies,
Inc., Wilmington, DE, USA). HPLC analyses were performed on a Hitachi
elite lachrom system equipped with Diode array detector using C18 columns
(Hitachi High Technologies, Tokyo, Japan). Compounds 3 to 6, 9 to 11 were
synthesized as described before.
2,6-dichloro-9-(2-deoxy-2-fluoro-3,5-di-O-(triisopropylsilyl)-D-arabino
furanosyl)-9H-purine (7). To a solution of 6 (36 mg, 0.075 mmol) in 1.1
˚
mL of 1,2-dichloroethane were added 10 mg of 4 A molecular sieves and
2,6-dichloropurine (21.5 mg, 0.113 mmol). The reaction mixture was
refluxed at 100◦C overnight. Solvent was then removed in vacuo, the residue
was redissolved in CHCl3 and filtered, the filtrate was concentrated under
reduced pressure. NMR of reaction mixture indicates the formation of both
α and β isomers with a ratio of 1/3.5 (α/β). The crude product was used
for the next step without further purification.

120
Y. Cen and A. A. Sauve
6-amino-2-chloro-9-(2-deoxy-2-fluoro-3,5-di-O-(triisopropylsilyl)-β-D-
arabinofuranosyl)-9H-purine (8). 7 (75 mg, 0.118 mmol) was dissolved in 4
mL of ammonia in isopropanol solution (2 M), the reaction was carried out
in a sealed tube at 105◦C overnight. The reaction mixture was then cooled
to room temperature and concentrated under reduced pressure. Column
chromatography (hexanes: ethyl acetate 3:1) afforded 8 (47 mg, 0.076
mmol, 65%) as a colorless oil, and its α-anomer (11.5 mg, 0.019 mmol, 16%)
as white solid. 8 1H NMR (CDCl3, 500 MHz), δ ppm: 0.96 (stack, 42H), 3.95
(m, 2H), 4.00 (q, J = 4.5 Hz, 1H), 4.75 (dd, J = 3.1, 18 Hz, 1H), 5.00 (dd,
J = 2.8, 52 Hz, 1H), 6.08 (s, broad, 2H), 6.42 (dd, J = 3.5, 19.8 Hz, 1H),
8.00 (d, J = 2.8 Hz, 1H). 13C NMR (125 MHz, CDCl3), δ ppm: 11.9, 12.1,
17.89, 17.91, 62.0, 75.0, 75.2, 82.7, 82.8, 85.7, 95.0, 96.6, 117.9, 140.6, 150.8,
154.3, 156.0. HRMS (ESI): calcd. for C28H51ClFN5O3Si2: 615.3203; Found:
615.3203. α-anomer 1H NMR (CDCl3, 500 MHz), δ ppm: 1.01 (stack, 42H),
3.80 (dd, J = 1.1, 7.4 Hz, 1H), 3.87 (ddd, J = 2.1, 5.0, 10.5 Hz, 1H), 4.48 (t,
J = 5.2 Hz, 1H), 4.71 (d, J = 15.3 Hz, 1H), 5.35 (d, J = 49.5 Hz, 1H), 5.77
(s, broad, 2H), 6.34 (d, J = 15.9 Hz, 1H), 8.06 (s, 1H). 13C NMR (125 MHz,
CDCl3), δ ppm: 11.88, 11.91, 17.7, 17.8, 17.88, 17.90, 62.6, 75.6, 75.8, 88.2,
88.5, 99.2, 100.7, 118.6, 139.6, 150.6, 154.3, 155.8. HRMS (ESI): calcd. for
C28H51ClFN5O3Si2: 615.3203; Found: 615.3204.
6-amino-2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-9H-purine
(Clofarabine, 1). 8 (18.5 mg, 0.03 mmol) was dissolved in 1 mL of DMF,
to this solution were added acetic acid (7.2 mg, 0.12 mmol) and tetram-
ethylammonium fluoride (11 mg, 0.12 mmol). The reaction was allowed
to stir at room temperature overnight. Solvent was removed in vacuo and
column chromatography (ethyl acetate:methanol 20:1) afforded 1 (8.2 mg,
1
0.027 mmol, 90%) as a pale yellow foam. H NMR (DMSO, 500 MHz), δ
ppm: 3.63 (m, 2H), 3.84 (q, J = 4.9 Hz, 1H), 4.42 (dt, J = 4.9, 19 Hz,
1H), 5.23 (dt, J = 4.3, 53 Hz, 1H), 6.31 (dd, J = 4.6, 13.7 Hz, 1H), 7.88
(s, broad, 2H), 8.27 (d, J = 1.8 Hz, 1H). 13C NMR (125 MHz, DMSO), δ
ppm: 60.3, 72.3, 72.5, 81.3, 81.5, 83.4, 83.5, 84.5, 96.1, 117.3, 140.0, 150.2,
153.3, 156.8. HRMS (ESI): calcd. for C11H11ClFN5O3: 303.0534; Found:
303.0540.
2ꢁ-deoxy-2ꢁ,2ꢁ-difluoro-3ꢁ,5ꢁ-di-O-(triisopropylsilyl)cytidine (12). Freshly
prepared bis(trimethylsilyl)cytosine (75 mg, 0.03 mmol) was dissolved in
1 mL of 1,2-dichloroethane, to this solution was added TMSOTf (6.7 mg,
0.03 mmol). The mixture was stirred at room temperature for 30 mins before
11 (10 mg, 0.018 mmol) was added, and the reaction was refluxed overnight.
The reaction mixture was cooled to room temperature and solvent was re-
moved under reduced pressure. NMR spectrum of reaction mixture indicates
the formation of both α and β isomers with a ratio of 1/1. The crude product
12 was used for the next step without further purification.
2ꢁ-deoxy-2ꢁ,2ꢁ-difluorocytidine (Gemcitabine, 2). To a solution of 12
(10 mg, 0.017 mmol) in 1 mL of DMF were added acetic acid (5.2 mg,

Syntheses of Clofarabine and Gencitabine
121
0.087 mmol) and tetramethylammonnium fluoride (8 mg, 0.087 mmol). The
reaction was stirred at room temperature overnight, then was concentrated
and purified by HPLC (solvent was 20 mM ammonium acetate, compounds
were eluted at a flow rate of 2 mL/min) to afford 2 (β-isomer, tR = 16.4 min-
utes, 1.7 mg, 0.006 mmol, 36%) and α-isomer (tR = 13.2 minutes, 1.9 mg,
1
0.007 mmol, 42%). 2 H NMR (D2O, 500 MHz), δ ppm: 3.86 (dd, J = 5.0,
12.9 Hz, 1H), 4.01 (dd, J = 1.6, 12.9 Hz, 1H), 4.13 (m, 1H), 4.40 (m, 1H),
6.25 (t, J = 8.8 Hz, 1H), 6.30 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H). 13C
NMR (125 MHz, D2O), δ ppm: 59.0, 68.2, 68.4, 68.6, 80.4, 80.5, 83.4, 83.5,
83.6, 83.7, 94.5, 120.7, 123.0, 125.3, 140.8, 154.7, 165.6. HRMS (ESI): calcd.
for C9H11F2N3O4: 263.0718; Found:263.0719. α-anomer 1H NMR (D2O, 500
MHz), δ ppm: 3.82 (dd, J = 5.0, 12.9 Hz, 1H), 3.95 (dd, J = 1.6, 12.9 Hz, 1H),
4.44 (m, 1H), 4.57 (m, 1H), 6.29 (d, J = 8.0 Hz, 1H), 6.38 (dd, J = 5.9, 8.8
Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H). 13C NMR (125 MHz, D2O), δ ppm: 60.8,
70.4, 70.6, 70.8, 84.5, 84.7, 85.6, 85.7, 85.8, 85.9, 96.1, 123.4, 144.5, 149.5,
160.6. HRMS (ESI): calcd. for C9H11F2N3O4: 263.0718; Found:263.0721.
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