Isolation, synthesis, and characterization of impurities and degradants from the clofarabine process

Article abstract of DOI:10.1021/op800182x

The identification of clofarabine process impurities and their subsequent isolation, synthesis, and characterization is described. Two isomeric process impurities resulting from N₆-attachment of a fluoroarabinose to clofarabine were found. Clofarabine's base degradation products, which were different from the process impurities, were also synthesized and characterized. These compounds resulted from modifications to the sugar moiety, the purine ring, or both. A mechanistic rationale for the formation of the various process impurities and degradation products is provided.

Full text of DOI:10.1021/op800182x

Organic Process Research & Development 2008, 12, 1229–1237
Isolation, Synthesis, and Characterization of Impurities and Degradants from the
Clofarabine Process
Bruce G. Anderson, William E. Bauta, William R. Cantrell, Jr.,* Tracy Engles, and Dennis P. Lovett
Genzyme Corporation, Quality and Technical Operations, 14805 Omicron DriVe, San Antonio, Texas 78245, U.S.A.
Scheme 1. Clofarabine manufacturing processa
Abstract:
The identification of clofarabine process impurities and their
subsequent isolation, synthesis, and characterization is described.
Two isomeric process impurities resulting from N6-attachment of
a fluoroarabinose to clofarabine were found. Clofarabine’s base
degradation products, which were different from the process
impurities, were also synthesized and characterized. These com-
pounds resulted from modifications to the sugar moiety, the purine
ring, or both. A mechanistic rationale for the formation of the
various process impurities and degradation products is provided.
a Reagents, conditions, and yields: a) HBr/HOAc, CH2Cl2, rt, 19 h, 89% yield;
b) KOt-Bu, MeCN, ClCH2CH2Cl, tert-amyl-OH, 50 °C, 19 h, 50% yield; c)
MeOH, NaOMe, rt, 5 h; d) recrystallization, MeOH, 64 °C to rt, 64% yield (2
steps).
Introduction
Clofarabine (1) is the active ingredient in the recently
approved pediatric antileukemia drug Clolar. The process for
manufacturing clofarabine1 resulted in some impurities at
g0.10% (HPLC area). Consequently, it was a regulatory
requirement to isolate and characterize these substances. Six
major degradation products resulted from heating clofarabine
in aqueous sodium hydroxide. This report describes the
identification, synthesis (or isolation), and characterization of
the clofarabine impurities and degradants.
Process Impurities. The clofarabine process is shown in
Scheme 1.1 Fluoroarabinose 2 is converted to the corresponding
bromosugar 3 using HBr/HOAc in dichloromethane. Bromo-
sugar 3 is next condensed with 2-chloroadenine (4) using KOt-
Bu. The resulting nucleoside 5 is precipitated from n-butyl
acetate using heptane, then purified by slurrying in hot methanol.
Nucleoside 5 is deprotected using catalytic sodium methoxide
in methanol, followed by recrystallization from methanol to give
clofarabine (1).
Purine Impurities. Six potential and observed nucleoside
impurities were prepared. Some of these were anticipated to
arise from related impurities in the starting materials. For
example, purines 10 and 11 were observed as impurities in
2-chloroadenine and would react to form nucleosides 13 and
14, respectively. Nucleoside 15 was detected as a minor
component in the API, resulting from displacement of the
2-chloro substituent of 4 by bromide during the condensation
step. Nucleoside 16 was also detected in a manufacturing batch
and determined to arise from the 2-chlorosugar impurity 8. The
syntheses of these impurities are shown in Scheme 2 and eq 1.
Although this process is more efficient than earlier ones,2 it
resulted in a number of process impurities routinely observed
in the API in levels greater than 0.10% (HPLC area). Accord-
ingly,3 these impurities as well as several potential impurities,
were identified and characterized.
The chloroarabinose 8 was synthesized by conversion of 6
to the triflate 7 followed by treatment with LiCl.4 Treatment of
8 with HBr/HOAc gave the bromosugar 9. Purine 10 was made
by reacting dimethylamine with 2,6-dichloropurine.5 Purine 11
is commercially available. Diazotization of 2,6-diaminopurine
in the presence of antimony tribromide afforded purine 12.6
The coupling reactions to produce the nucleosides 13-16
employed conditions similar to those of the coupling step of
the clofarabine process. Nucleoside 17 was thought to have
originated from the formal SNAr reaction of sodium methoxide
* Author to whom correspondence should be addressed. E-mail:
bill.cantrell@genzyme.com.
(1) Bauta, W. E.; Schulmeier, B. E.; Burke, B.; Puente, J. F.; Cantrell,
W. R., Jr.; Lovett, D.; Goebel, J.; Anderson, B.; Ionescu, D.; Gou, R.
Org. Process Res. DeV. 2004, 8, 889–896.
(2) Montgomery, J. A.; Shortnacy-Fowler, A. T.; Clayton, S. D.; Riordan,
J. M.; Secrist, J. A., III. J. Med. Chem. 1992, 35, 399–401.
Montgomery, J. A.; Fowler, A. T.; Secrist, J. A., III. Patent WO 01/
60383 A, 2001.
(3) The following are the ICH documents and FDA Guidelines which
cover the topic of impurities and degradants for control of drugs during
development to ensure patient safety: ICH guidelines Q6A, Q3A, Q3B,
Q2A, Q2B, Q1A(R2), Q1E; FDA Guidelines: “Analytical Procedures
and Methods Validation”, “INDs - Approaches to Complying with
CGMP During Phase 1”, “INDs for Phase 2 and Phase 3 Studies
Chemistry, Manufacturing, and Controls Information”.
(4) Tann, C. H.; Brodfuehrer, P. R.; Brundidge, S. P.; Sapino, C., Jr.;
Howell, H. G. J. Org. Chem. 1985, 50, 3644–3647.
(5) Montgomery, J. A.; Holum, L. B. J. Am. Chem. Soc. 1958, 80, 404–
408.
(6) Sampath, U.-S.; Bartlett, L. U.S. Patent 6,252,061 B1, 2001.
10.1021/op800182x CCC: $40.75
Published on Web 11/21/2008
2008 American Chemical Society
Vol. 12, No. 6, 2008 / Organic Process Research & Development
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1229

Scheme 2. Synthesis of purine impuritiesa
a Reagents and conditions: a) Tf2O, CH2Cl2, -30 to -4 °C, 3 h; b) LiCl, NMP, rt, 18 h; c) HBr/HOAc, CH2Cl2, rt, 17 h; d) KOt-Bu, CaH2, MeCN, ClCH2CH2Cl,
50 °C, 1 to 16 h; e) MeOH, NaOMe, rt, 2 to 28 h.
Scheme 3. Possible pathway to the bis-sugar impurities
of anomeric stereochemistry at C1′. Indeed, this is supported
by observed NOEs between H8 and H3′ in both 19a and 19b.
Compound 19a has an additional NOE between H8 and H3′′,
which supports an assignment where these substituents are on
the same face of the sugar ring. Interestingly, the H1′′ resonanace
in all the bis-sugar compounds appears broad, while H1′ is a
with clofarabine in the deprotection step and was formed under
the deprotection conditions (eq 1).
Bis-sugar Nucleosides. Two isomeric process impurities
were detected by LC/MS with a mass of 437. The structures
19a,b were proposed for these compounds. They were thought
to arise via deprotonation of the exocyclic purine nitrogen and
sharp doublet of doublets.
subsequent condensation with 3 to afford 18a,b, which then
Degradants. ICH guidelines require that drug substances
gave rise to 19a,b upon deprotection (Scheme 3).7
and drug products be stressed to aid in the development of
Attempts to form 18a,b by addition of excess KOt-Bu and
excess bromosugars 3 to 5 failed to significantly increase the
amount of 18a,b in the mixture, even when zinc chloride was
used as a promoter.6 It was found that the best conditions for
formation of 18 were simultaneous addition of 3 and KOt-Bu
to a 65-70 °C solution of protected clofarabine 5 in MeCN,
whereby the isomers constituted up to 34% of the crude mixture
by HPLC and were isolable by silica chromatography. However,
when the individual purified isomers (18a or 18b) were exposed
to the debenzoylation conditions (NaOMe, MeOH), rapid
isomerization to a 55:45 mixture of the unprotected bis-sugar
nucleosides (19a,b) occurred. The resulting isomeric mixture
could then be separated by preparative HPLC. The mechanism
for this isomerization is unclear, but may involve deprotonation
of the exocyclic N6 to reversibly afford the imine 19c (eq 2).
NMR (1H, 13C, COSY, NOESY) data for 19a and 19b
support the assigned structures. The stereochemical assignment
at the anomeric carbons (C1′ and C1′′) is somewhat more tenuous.
On the basis of the mechanism, one would not anticipate loss
stability-indicating analytical methods.8 Clofarabine is a rela-
tively stable compound; very little degradation was observed
upon exposure to acid, peroxide, and light. However, several
degradants were observed when clofarabine was dissolved in
aqueous 1 M NaOH and heated to 80 °C for 1 h. The reaction
mixture was sampled, and the resulting HPLC chromatogram
revealed six major degradants. The degradants were labeled A
through F according to their relative retention times (RRT).
Table 1 shows the results of the UV and mass spectral analysis.
The UV spectra of degradants A, B, and C are noticeably
different from that of clofarabine as well as different from each
other. Assuming that there is no contribution by the sugar
moiety to the UV spectra, we thought that degradants A, B,
and C had undergone some modification of the purine.
Comparison of the UV spectra of degradant C and guanosine
showed a remarkable similarity. The UV spectra of degradants
D, E, and F are very similar to clofarabine, suggesting that little
or no modification of the purine had occurred in these
(8) Reynolds, D. W.; Facchine, K. L.; Mullaney, J. F.; Alsante, K. M.;
(7) Related structures have been reported: Jain, P.; Anand, N. Indian
J. Chem. 1968, 6, 616–618.
Hatajik, T. D.; Motto, M. G. Pharm. Technol. 2002, (February), 48–
56.
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Table 1. Results from LC/MS analysis of the degradant
mixture
Scheme 4. Independent synthesis of degradant C (23)a
MS isotopic
RRT UVmax, nm % area mass Cl pattern?
cmpd
degradant A (29) 0.322 219, 277
degradant B (20) 0.489 248, 292
degradant C (23) 0.572 252
degradant D (28) 0.872 264
degradant E (24) 0.920 264
2.64 265
5.34 285
6.04 285
5.02 283
6.15 283
53.58 303
6.87 570
no
no
no
yes
yes
yes
yes
clofarabine (1)
1.000 264
degradant F (31) 1.450 264
a Reagents and conditions: a) KOt-Bu, CaH2, MeCN, ClCH2CH2Cl, tert-amyl-
OH, rt, 20 h; b) NaOH, H2O, rt, 72 h.
degradants. Degradants A, B, and C also showed loss of the
signature isotopic chlorine pattern in the mass spectrum.
Assuming replacement of chloride by hydroxyl via formal SNAr
reaction, then the expected mass of the isomeric guanidines
would be 285. Both degradants B and C have a mass of 285.
Degradants D and E had a molecular weight of 283, which
could result from a base-induced HF elimination. Degradant A
has a mass of 265 which could result from the loss of HF in
addition to replacement of chlorine by hydroxyl. NMR data
later confirmed the loss of fluorine by lack of distinctive
coupling patterns in both the proton and carbon spectra of
degradants A, D, and E. Degradant F has a mass of 570, which
was assumed to be a dimer of clofarabine minus HCl.
The alkaline degradation was repeated under various condi-
tions via design of experiments (DoE) in an attempt to optimize
the production of individual degradants for isolation. Unfortu-
nately, the enhancements in individual degradant levels was
insufficient for preparative purposes in most cases. The best
approach to characterizing the degradants was to synthesize
them. The degradants with a mass of 285 were our first synthetic
targets. It is known that peroxide anion is more nucleophilic
than hydroxide.9 Therefore, clofarabine was reacted with
hydrogen peroxide in aqueous lithium hydroxide to cleanly give
degradant B (20).10 After purification and characterization, 20
was assigned the structure shown in eq 3.
Degradant E was successfully synthesized and isolated by
using nonaqueous degradation conditions (eq 4). When clo-
farabine was heated with solid NaOH in DMSO, clean
formation of degradant E resulted along with the starting
material. Unfortunately, further heating resulted in decomposi-
tion of the desired degradant. Isolation of degradant E (24) was
accomplished by chromatography, and sufficient material was
acquired for characterization. Extensive NMR experiments
indicated that the structure was 24 as shown in eq 4. Proton
NMR showed seven resonances attributed to the sugar moiety
(compared to eight for clofarabine). The D2O exchange experi-
ment showed the peak at δ 5.97 as the only peak that was
diminishing, which indicated that only one hydroxyl group
remained in the molecule. DEPT and HETCOR indicated one
methylene and four methine carbon resonances on the sugar
moiety. As stated previously, both proton and carbon spectra
showed the absence of fluorine. The COSY spectrum indicated
that the hydroxyl group was in the 2′ position. The other salient
feature of the proton spectrum was separation of the 2 H5′
resonances by 0.78 ppm. The spectra of most of the other
nucleosides we studied showed the H5′ protons to be indistin-
guishable from each other. One explanation for this phenom-
enon is restricted rotation about the C4′-C5′ bond, which could
be accomplished by the oxatane ring found in the structure.
We assumed that the other degradant with a mass of 285
was derived by a base-induced rearrangement as was reported
in the hydroxide degradation of cladribine.11 We reasoned that
a nucleoside such as 22 would react with hydroxide to give the
isomeric degradant C (23). Synthesis of the isomeric nucleoside
22 was accomplished under clofarabine coupling conditions,
and the subsequent SNAr reaction with hydroxide cleanly
produced 23 (see Scheme 4).
The formation of 24 might have occurred via intramolecular
attack of the 5′ alkoxide to C4′ of a C3′-C4′ epoxide. These
nucleoside epoxides are known12 and are usually formed by
base-induced internal nucleophilic displacement of halide with
alkoxide.
Coincidently, a nucleoside epoxide of structure 28 fit the
data that was obtained for degradant D (Scheme 5). We set out
to independently synthesize degradant D from the corresponding
2′-bromonucleoside 27. The triflate 7 was treated with KBr to
give the bromide 25. Compound 25 was converted to the 1′,2′-
dibromo sugar 26 using the clofarabine process conditions. The
subsequent coupling reaction with 2-chloroadenine (4) gave the
(9) Smith, M. B.; March, J. March’s AdVanced Organic Chemistry, 5th
ed.; Wiley Inter-Science: New York, 2001; p 445.
(10) (a) Pewkupec, S.; Svedruzˇic´-Gazivoda, T.; Mrvosˇ-Sermek, D.; Nagl,
A.; Grdisˇa, M.; Pavelic´, K.; Balzarini, J.; De Clerq, E.; Folkers, G.;
Scapozza, L.; Mintas, M.; Raic´-Malic´, S. J. Med. Chem. 2003, 46,
5763–5772. (b) Cantrell, W. R., Jr.; Bauta, W. E.; Engles, T.
Tetrahedron Lett. 2006, 47, 4249.
(11) Andrzejewska, M.; Dzierzgowska-Szmidt, A.; Kazimierczuk, Z. Phar-
mazie 2003, 58, 122–124.
(12) Robles, R.; Rodr´ıguez, C.; Izquierdo, I.; Plaza, M. T.; Mota, A.; de
Cienfuegos, L. A. Tetrahedron: Asymmetry 2000, 11, 3069–3077.
Vol. 12, No. 6, 2008 / Organic Process Research & Development
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Scheme 5. Independent synthesis of degradant D (28a
Experimental Section
a Reagents and conditions: a) LiBr, NMP, rt, 20 h; b) HBr/HOAc, CH2Cl2,
rt, 23 h; c) KOt-Bu, CaH2, MeCN, ClCH2CH2Cl, tert-amyl-OH, rt, 18 h; d)
MeOH, NaOMe, rt, 16 days.
Reactions were run under nitrogen. 1H, 13C, and 19F NMR
spectra were obtained at 400, 100, and 376 MHz, respectively.
IR spectra were obtained as KBr pellets. UV spectra were
obtained as solutions in H2O/MeCN or H2O/MeOH. HPLC data
were collected using photodiode array detectors on dual pump
systems. Conditions for HPLC are given in the Supporting
Information.
Scheme 6. Synthetic pathways towards degradant A (29)a
Arabinose Triflate 7. Triflic anhydride (2.5 mL, 14.9 mmol)
was added to a solution of 6 (5.63 g, 12.2 mmol), CH2Cl2 (65
mL), and pyridine (4.9 mL, 60.6 mmol) over 2 min at -30 °C
during which time the temperature rose to -25 °C. The reaction
was allowed to warm to -4 °C over 2.75 h. Reaction progress
was judged complete by TLC (silica gel GHLF, 50% EtOAc/
50% hexanes, UV254, Rf of 6 ) 0.50, Rf of 7 ) 0.62). A solution
of NaHCO3 (50 mL, 5 wt %) was added (off-gassing). The
layers were separated, and the aqueous layer was extracted with
CH2Cl2 (25 mL). The organic portions were dried (MgSO4)
and concentrated. Heptane was added to the residue, and the
mixture was concentrated. The residue was dissolved in tert-
butyl methyl ether (TBME, 100 mL), and the solution was
filtered through silica gel. The silica was washed with TBME
(2 × 100 mL), and the combined filtrates were concentrated to
a Reagents and conditions: a) H2O, 100 °C, 5 h; b) H2O2, LiOH, H20, 60 °C,
4 h, trace.
protected nucleoside 27. Treatment with a slight excess of
sodium methoxide cleanly produced degradant D (28).
Purification of 28 was accomplished by recrystallization in
boiling water. Examination of the mother liquors by HPLC
revealed a significant amount of degradant A with a small
amount of degradant E (24). Therefore, degradant A (29) was
easily prepared when crude 28 was heated in water at 100 °C
(Scheme 6). Interestingly, traces of 29 were also observed in
the SNAr reaction of clofarabine with alkaline peroxide. Further
evidence for the assignment of structure 29 for degradant A is
the similarity to the proton NMR of 24.
1
a pale yellow oil which was used in the next step as is. H
Attempts made to prepare degradant F by reacting clofara-
bine (1) with the analogous difluoronucleoside 30 were suc-
cessful on small scale (eq 5). However, when this reaction was
scaled up, none of the desired dimers could be isolated.
Therefore, degradant F was isolated from a degradation mixture
using the DoE optimized conditions. The structure of 31 was
confirmed by comparing a proton spectrum with the corre-
sponding D2O wash spectrum. Three peaks were observed to
diminish upon addition of D2O: a doublet at 5.96 ppm and two
triplets at 5.15 and 5.09 ppm, respectively. These peaks
corresponded to one 3′-OH and two 5′-OH’s, which would give
the structure shown for compound 31 in eq 5. Peak assignments
were based mostly on the COSY data. The H8 and NH2 protons
were assigned by analogy to compounds 1 and 17. Surprisingly,
none of the isomer derived from attachment of 5′-OH was
detected by LC/MS in the degradation mixtures.
In summary, all significant impurities in the clofarabine
process over 0.1% were either synthesized or isolated, and were
characterized. In addition, all of the six major compounds from
the aqueous sodium hydroxide degradation of clofarabine were
isolated and characterized. Confirmation of structure by inde-
pendent syntheses was achieved for all compounds except for
compound 31.
NMR (CDCl3) δ 8.15-8.01 (m, 6H), 7.65-7.58 (m, 3H),
7.50-7.39 (m, 6H), 6.87 (d, 1H, J ) 4), 5.79 (dd, 1H, J ) 6,
3), 5.55 (dd, 1H, J ) 6, 4), 4.86 (q, 1H, J ) 3), 4.75 (dd, 1H,
J ) 12, 3), 4.63 (dd, 1H, J ) 12, 3). 13C NMR (CDCl3) 165.8,
165.5, 164.7, 134.0, 133.9, 133.5, 130.09, 130.0, 129.6, 129.1,
128.8, 128.6, 128.5, 128.4, 118.4 (q, JCF ) 320), 93.1, 82.0,
79.4, 69.9, 63.4 ppm. UV (H2O/MeCN) λmax1 230 nm, λmax2
274 nm.
Chloroarabinose 8. A suspension of 7 (7.24 g, 12.2 mmol,
assuming 100% yield), LiCl (2.58 g, 60.9 mmol), and N-
methylpyrrolidinone (NMP, 25 mL) were stirred at ambient
temperature for 17.5 h. The reaction was judged complete by
TLC (silica gel GHLF, 50% EtOAc/50% hexanes, UV254, Rf
of 7 ) 0.62, Rf of 8 ) 0.65). H2O (100 mL) was added, and
the mixture was extracted with TBME (3 × 75 mL). The
organic portions were washed with H2O (100 mL), dried
(MgSO4), and concentrated. Purification by chromatography
(silica gel, EtOAc/hexanes, 3/17) gave 8 as a clear oil (5.11 g,
98.1% purity, 86% yield). 1H NMR (CDCl3) δ 8.12-8.03 (m,
6H), 7.61-7.53 (m, 3H), 7.44-7.37 (m, 6H), 6.68 (s, 1H), 5.62
(d, 1H, J ) 3), 4.85-4.74 (m, 3H), 4.62 (s, 1H). 13C NMR
(CDCl3) 166.1, 165.2, 164.5, 133.8, 133.7, 133.1, 129.8, 129.7,
129.4, 129.0, 128.6, 128.5, 128.4, 128.3, 102.4, 84.3, 79.9, 63.9,
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61.8 ppm. IR (KBr) 3436, 3063, 1725, 1601, 1452, 1316, 1270,
1108, 1067, 1025, 937, 708 cm-1. UV (H2O/MeCN) λmax1
230 nm, λmax2 274 nm. MS m/z [M + Na]+ ) 503. Anal.
Calcd for C26H21ClO7: C, 64.90; H, 4.40; Cl, 7.37. Found: C,
64.47; H, 4.26; Cl, 7.45.
(DMSO-d6) δ 13.0 (br s, 1H), 8.11 (s, 1H), 7.64 (s, 2H). UV
(H2O/MeOH) λmax1 212 nm, λmax2 265 nm. MS m/z [M +
H]+ ) 214.
Nucleoside 13. A solution of 3 (7.71 g, 18.2 mmol) in 1,2-
dichloroethane (DCE, 6.5 mL) was added to a suspension of
10 (3.00 g, 15.2 mmol), CaH2 (0.64 g, 15.2 mmol), MeCN (5
mL), and KOt-Bu (15.9 mL, 1.0 M, 15.9 mmol) at 50 °C over
10 min. The reaction was stirred at 50 °C for 16 h. The mixture
was filtered, and the filtrate was concentrated to give a brown
tar (9.74 g). Purification by chromatography (silica gel, EtOAc/
heptane, 3/7) gave the protected nucleoside (4.83 g, 95.3%
purity, 56% yield). Mp ) 84-86 °C. 1H NMR (DMSO-d6) δ
8.20 (d, 1H, J ) 3), 8.10-8.08 (m, 2H), 7.99-7.97 (m, 2H),
7.74-7.70 (m, 1H), 7.67-7.63 (m, 1H), 7.60-7.52 (m, 2H),
7.50-7.48 (m, 2H), 6.57 (dd, 1H, J ) 19, 4), 5.89 (dm, 1H, J
) 19), 5.77 (dm, 1H, J ) 50), 4.78-4.67 (m, 3H), 3.68 (br s,
1H), 3.18 (br s, 1H). 13C NMR (DMSO-d6) 165.4, 164.8, 154.5,
152.9, 150.9, 138.7 (d, JCF ) 6), 133.9, 133.5, 129.7, 129.2,
128.7, 128.6, 117.7, 92.9 (d, JCF ) 193), 82.1 (d, JCF ) 18),
78.4, 76.3 (d, JCF ) 29), 63.7, 37.3 ppm. 19F NMR (DMSO-
d6) -198.3 (dt, J ) 51, 19) ppm. IR (KBr) 3446, 2933, 1726,
1602, 1452, 1314, 1271, 1109, 711 cm-1. UV (H2O/MeOH)
λmax1 223 nm, λmax2 276 nm. MS m/z [M + H]+ ) 540.
Anal. Calcd for C26H23ClFN5O5: C, 57.84; H, 4.29; Cl, 6.57;
F, 3.52; N, 12.97. Found: C, 58.44; H, 4.32; Cl, 6.34; F, 3.45;
N, 12.39. NaOMe (0.16 mL, 25 wt %, 0.70 mmol) was added
to a suspension of the protected nucleoside (1.91 g, 3.54 mmol)
in MeOH (25 mL). The mixture was stirred for 2 h and HOAc
(40 µL, 0.7 mmol) was added. The filtrate was concentrated
and the residue was purified by chromatography (silica gel,
EtOAc) to give 13 as a white solid (0.91 g, 95.9% purity, 75%
Chlorobromosugar 9. HBr/HOAc (3.7 mL, 33 wt %, 21.4
mmol) was added to a solution of 3 (4.88 g, 10.1 mmol) in
CH2Cl2 (25 mL). The mixture was stirred at ambient temper-
ature for 17 h. The reaction was judged complete by TLC (silica
gel GHLF, 15% EtOAc/85% hexanes, UV254, Rf of 8 ) 0.23,
Rf of 9 ) 0.40). Saturated NaHCO3 (100 mL) was added with
stirring (off-gassing). The mixture was extracted with CH2Cl2
(60 mL). The organics were washed with saturated NaHCO3
(75 mL), dried (MgSO4), and concentrated to give 9 as a yellow
oil (4.36 g, 98% recovery), which was used in the next step as
is. 1H NMR (CDCl3) δ 8.12-8.09 (m, 4H), 7.64-7.54 (m, 2H),
7.50-7.41 (m, 4H), 6.61 (s, 1H), 5.58 (d, 1H, J ) 4), 4.93 (s,
1H), 4.90-4.72 (m, 3H). 13C NMR (CDCl3) 166.1, 165.5,
133.9, 133.3, 130.0, 129.9, 129.4, 128.6, 128.5, 128.4, 90.4,
85.2, 79.6, 66.5, 62.6 ppm. IR (neat) 3063, 3033, 2955, 2926,
2870, 1726, 1602, 1451, 1266, 1098, 710 cm-1. MS m/z [M +
Cl]- ) 473.
2-Chloro-N,N-dimethyl-9H-purin-6-amine 10. Triethy-
lamine (4.6 mL, 33 mmol) was added to a suspension of 2,6-
dichloropurine (3.10 g, 16.4 mmol), dimethylamine hydrochlo-
ride (2.01 g, 24.6 mmol), and DMF (10 mL) over 1 min. A
solid mass formed with accompanying exotherm. More DMF
(4 mL) was added, and the mixture was stirred for 45 min.
Saturated NaHCO3 (50 mL) was added, and the mixture was
stirred for 10 min and filtered. The wet cake was dried (50 °C,
27 Torr) to give 10 as a white solid (3.00 g, 97.2% purity, 90%
yield). Mp ) 296-297 °C (dec). 1H NMR (DMSO-d6) δ 13.06
(br s, 1H), 8.09 (s, 1H), 3.41 (br s, 6H). 13C NMR (DMSO-d6)
154.4, 152.3, 152.0, 138.2, 117.9, 37.8 ppm. IR (KBr) 3099,
2955, 2826, 2700, 1593, 1359, 1316, 1277, 964 cm-1. UV
(H2O/MeOH) λmax1 223 nm, λmax2 276 nm. MS m/z [M +
H]+ ) 198. Anal. Calcd for C7H8ClN5: C, 42.54; H, 4.08; Cl,
17.94; N, 35.44. Found: C, 42.60; H, 3.89; Cl, 18.16; N, 35.14.
2-Bromo-9H-purin-6-amine 12. Dibromomethane (150
mL) and tert-butyl nitrite (90 wt %, 9.8 mL, 74.3 mmol) were
added to a suspension of 1,2-diaminopurine (10.08 g, 67.2
mmol), antimony(III) bromide (36.40 g, 101 mmol), and DMSO
(100 mL) over 20 min, and the mixture was stirred at ambient
temperature for 16 h. The reaction was neutralized with
saturated NaHCO3 until pH ) 7. The mixture was filtered, the
crude solid (60% purity) was dried (50 °C, 27 Torr) and
suspended in 1.0 M NaOH (100 mL). The mixture was filtered,
and the solids were washed with H2O (5 × 10 mL) and then
discarded. The filtrate was neutralized with 12 M HCl until the
pH ) 6. The resulting suspension was filtered, and the solid
was dried (50 °C, 27 Torr). The solid (8.68 g) was suspended
in H2O (100 mL), and the mixture was heated at 85 °C for 1 h.
The mixture was cooled to ambient temperature and filtered.
The solid was washed with H2O (3 × 10 mL) and dried (45
°C, 24 Torr) to give 12 as a brown solid (5.29 g, 68% purity).
The crude solid was used in the coupling step as is. 1H NMR
1
yield). Mp ) 169-170 °C. H NMR (DMSO-d6) δ 8.30 (d,
1H, J ) 2), 6.36 (dd, 1H, J ) 14, 5), 5.97 (d, 1H, J ) 5), 5.25
(dt, 1H, J ) 53, 4), 5.10 (t, 1H, J ) 6), 4.44 (ddd, 1H, J ) 19,
9, 5), 3.87 (dd, 1H, J ) 10, 5), 3.73-3.62 (m, 5H), 3.19 (br s,
3H). 13C NMR (DMSO-d6) 154.4, 152.7, 150.9, 138.7, 117.8,
95.2 (d, JCF ) 192), 83.5 (d, JCF ) 4.2), 81.5 (d, JCF ) 17),
72.5 (d, JCF ) 23), 60.3, 37.4 ppm. 19F NMR (DMSO-d6)
-198.9 (dt, J ) 53, 18) ppm. IR (KBr) 3374, 3132, 2925, 1723,
1606, 1354, 1311, 1035, 793 cm-1. UV (H2O/MeOH) λmax1
218 nm, λmax2 276 nm. MS m/z [M + H]+ ) 332. Anal. Calcd
for C12H15ClFN5O3: C, 43.45; H, 4.56; Cl, 10.69; F, 5.73; N,
21.11. Found: C, 43.50; H, 4.42; Cl, 10.66; F, 5.94; N, 21.13.
Nucleoside 14. A solution of 3 (2.48 g, 5.86 mmol) in DCE
(10 mL) was added to a suspension of 11 (0.82 g, 4.9 mmol),
CaH2 (0.21 g, 4.9 mmol), MeCN (10 mL), and KOt-Bu (5.4
mL, 1.0 M, 5.4 mmol) at 50 °C over 30 min. The reaction was
stirred at 50 °C for 16 h and filtered, and the filtrate was
concentrated to give a brown tar. Purification by chromatog-
raphy (silica gel, acetone/EtOAc, 3/7) gave the protected
nucleoside as a white solid (0.50 g, 97.6% purity, 20% yield).
1
Mp ) 127-128 °C. H NMR (DMSO-d6) δ (mixture of
anomers). 13C NMR (DMSO-d6) (mixture of anomers). 19F
NMR (DMSO-d6) minor anomer -190.5 (dt, J ) 50, 16) ppm,
major anomer -198.7 (dt, J ) 50, 20) ppm. IR (KBr) 3460,
3329, 3189, 1724, 1604, 1271, 1109, 711 cm-1. UV (H2O/
MeOH) λmax1 220 nm, λmax2 279 nm. MS m/z [M + H]+ )
493. Anal. Calcd for C24H21FN6O5: C, 58.53; H, 4.30; F, 3.86;
Vol. 12, No. 6, 2008 / Organic Process Research & Development
•
1233

N, 17.07. Found: C, 58.99; H, 4.31; F, 3.96; N, 16.19. NaOMe
(0.10 mL, 25 wt %, 0.44 mmol) was added to a suspension of
the protected nucleoside (1.09 g, 2.21 mmol) in MeOH (23 mL).
The mixture was stirred for 17 h and HOAc (0.2 mL) was
added. The filtrate was concentrated and triturated with heptane.
Purification by chromatography (silica gel, EtOAc/CH2Cl2, 2/3)
gave 14 as a white solid (0.12 g, 99.0% purity, 19% yield).
Mp ) 175-178 °C (dec). 1H NMR (DMSO-d6) δ 7.81 (d, 1H,
J ) 3), 6.81 (s, 2 H), 6.20 (dd, 1H, J ) 17, 4), 5.94 (d, 1H, J
) 4), 5.91 (s, 2H), 5.11 (dt, 1H, J ) 53, 4), 5.10 (br t, 1H, J
) 5), 4.43-4.36 (m, 1H), 3.83 (dd, 1H, J ) 10, 5), 3.68-3.59
(m, 2H). 13C NMR (DMSO-d6) 160.4, 156.2, 151.5, 136.0,
112.4, 95.3 (d, JCF ) 192), 83.5, 81.1 (d, JCF ) 17), 72.9 (d,
JCF ) 23), 60.6 ppm. 19F NMR (DMSO-d6) -198.5 (dt, J )
52, 17) ppm. IR (KBr) 3337, 3204, 2929, 1605, 1478, 1415,
1344, 1279, 1222, 1040, 792 cm-1. UV (H2O/MeOH) λmax1
215 nm, λmax2 255 nm, λmax3 279 nm. MS m/z [M + H]+ )
285. Anal. Calcd for C10H13FN6O3: C, 42.26; H, 4.61; F, 6.68;
N, 29.57. Found: C, 42.05; H, 4.88; F, 6.38 N, 25.90.
Compound 15. A solution of 3 (6.61 g, 15.6 mmol) in DCE
(45 mL) was added to a suspension of 12 (67.5% purity, 4.50 g,
14.2 mmol), KOt-Bu (1.67 g, 14.9 mmol), CaH2 (0.60 g, 14.3
mmol), MeCN (25 mL), and tert-amyl alcohol (TAA, 25 mL)
at 50 °C over 38 min. The mixture was stirred at 50 °C for
16 h and filtered, and the filtrate was concentrated. Purification
by chromatography (silica gel, EtOAc/hexanes, 3/2) gave a solid
(3.00 g, 92% purity). Further purification by trituration in MeOH
(25 mL) gave the protected nucleoside (1.82 g, 98% purity,
23% yield). Mp ) 121-122 °C dec. 1H NMR (DMSO-d6) δ
8.19 (d, 1H, J ) 3), 8.19-7.98 (m, 4H), 7.76-7.50 (m, 6H),
6.57 (dd, 1H, J ) 18, 4), 5.94 (dq, 1H, J ) 19, 2), 5.80 (dq,
1H, J ) 51, 2), 4.82-4.68 (m, 3H). 13C NMR (DMSO-d6)
165.4, 164.8, 156.6, 150.0, 144.6, 139.8 (d, JCF ) 6), 133.9,
133.5, 129.7, 129.2, 128.7, 128.6, 118.0, 92.9 (d, JCF ) 189),
82.0 (d, JCF ) 17), 78.3, 76.3 (d, JCF ) 28), 63.7 ppm. 19F
NMR (DMSO-d6) -198.2 (dt, J ) 51, 18) ppm. IR (KBr) 3463,
3358, 3167, 2359, 1726, 1642, 1586, 1452, 1349, 1272, 1096,
711 cm-1. UV (H2O/MeCN) λmax1 214 nm, λmax2 231 nm,
λmax3 264 nm. MS m/z [M + H]+ ) 556. Anal. Calcd for
C24H19BrFN5O5: C, 51.81; H, 3.44; Br, 14.36; F, 3.41; N, 12.59.
Found: C, 53.01; H, 3.14; Br, 12.06; F, 3.55; N, 11.57. NaOMe
(0.14 mL, 25 wt %, 0.65 mmol) was added to a suspension of
the protected nucleoside (1.72 g, 3.09 mmol) in MeOH (35
mL),and the mixture was stirred at ambient temperature for
4.7 h. HOAc (0.5 mL) was added,and the reaction mixture was
washed with hexanes (3 × 30 mL). The bottom MeOH layer
was concentrated and the residue was triturated with IPA/hexane
(15 mL/2 mL). The suspension was filtered, and the solid was
washed with IPA/hexane (2 mL/2 mL). The solid was sus-
pended in acetone (50 mL) and filtered through silica gel. The
filtrate was concentrated to give 15 as a white solid (0.42 g,
98% purity, 39% yield). Mp ) 220-221 °C. 1H NMR (DMSO-
d6) δ 8.27 (s, 1H), 7.90 (s, 2H), 6.34 (dd, 1H, J ) 14, 4), 5.98
(br s, 1H), 5.25 (d, 1H, J ) 53), 5.10 (br s, 1H), 4.45 (d, 1H,
J ) 19), 3.87 (br s, 1H), 3.70-3.67 (m, 2H). 13C NMR (DMSO-
d6) 156.6, 150.0, 144.5, 139.9, 117.7, 95.3 (d, JCF ) 192), 83.5
(d, JCF ) 4), 81.4 (d, JCF ) 15), 72.6 (d, JCF ) 23), 60.4 ppm.
19F NMR (DMSO-d6) -198.8 (br dd, J ) 52, 15) ppm. IR
(KBr) 3323, 3160, 2927, 1666, 1594, 1426, 1355, 1298, 1178,
1035, 915, 804, 668, 547 cm-1. UV (H2O/MeOH) λmax1 213
nm, λmax2 263 nm. MS m/z [M + H]+ ) 348. Anal. Calcd for
C10H12BrFN5O3: C, 34.50; H, 3.18; Br, 22.95; F, 5.46; N, 20.12.
Found: C, 34.79; H, 2.88; Br, 23.54; F, 5.63; N, 19.91.
Nucleoside 16. A solution of 9 (4.35 g, 9.89 mmol) in DCE
(8 mL) was added to a suspension of 4 (1.53 g, 9.02 mmol),
KOt-Bu (1.06 g, 9.45 mmol), CaH2 (0.38 g, 9.03 mmol), MeCN
(8 mL), and TAA (8 mL) at 50 °C over 30 min, and the mixture
was stirred at 50 °C for 1 h. The reaction mixture was filtered,
the filtrate was concentrated, and the residue was purified by
slurrying in MeOH (3 × 44 mL) to give the protected
nucleoside as an off-white solid (1.78 g, 97% purity, 36% yield).
Mp ) 172-174 °C. 1H NMR (DMSO-d6) δ 8.38 (s, 1H), 7.98
(br s, 2H), 8.11-7.94, (m, 4H), 7.76-7.45 (m, 6H), 6.68 (d,
1H, J ) 6), 6.24 (t, 1H, J ) 6), 5.44 (t, 1H, J ) 6), 4.88-4.80
(m, 2H), 4.67 (q, 1H, J ) 6). 13C NMR (DMSO-d6) 165.4,
164.8, 156.8, 153.2, 149.8, 140.1, 133.9, 133.5, 129.6, 129.2,
128.8, 128.7, 128.6, 117.9, 83.6, 78.6, 77.8, 64.0, 60.3 ppm.
IR (KBr) 3452, 3331, 3177, 1724, 1639, 1593, 1452, 1307,
1271, 1094, 1027, 710 cm-1. UV (H2O/MeCN) λmax1 212 nm,
λmax2 231 nm, λmax3 264 nm. MS m/z [M + H]+ ) 528.
Anal. Calcd for C24H19Cl2N5O5: C, 54.56; H, 3.62; Cl, 13.42;
N, 13.26. Found: C, 54.26; H, 3.32; Cl, 13.39; N, 13.25. NaOMe
(0.14 mL, 25 wt %, 0.61 mmol) was added to a suspension of
the protected nucleoside (1.58 g, 2.99 mmol) in MeOH (31 mL)
and the mixture was stirred at ambient temperature for 28 h.
HOAc (0.4 mL) was added, the solvent was concentrated, and
the residue was triturated with hexanes (50 mL). The suspension
was filtered, and the crude solid was purified by crystallization
(acetone/MeOH) to give 16 as a white solid (0.46 g, 99% purity,
48% yield). Mp ) 237-241 °C (dec). 1H NMR (DMSO-d6) δ
8.41 (s, 1H), 7.88 (br s, 1H), 6.42 (d, 1H, J ) 6), 6.12 (br d,
1H, J ) 4), 5.22 (t, 1H, J ) 5), 4.80 (dd, 1H, J ) 8, 7), 4.45
(br q, 1H, J ) 4), 3.86-3.72 (m, 3H). 13C NMR (DMSO-d6)
156.8, 153.2, 150.3, 139.7, 117.5, 83.6, 82.6, 73.8, 63.6, 59.8
ppm. IR (KBr) 3389, 3216, 3113, 2906, 2360, 1649, 1600, 1461,
1305, 1083, 708 cm-1. UV (H2O/MeOH) λmax1 211 nm, λmax2
265 nm. MS m/z [M + H]+ ) 320. Anal. Calcd for
C10H11Cl2N5O3: C, 37.52; H, 3.46; Cl, 22.15; N, 21.88. Found:
C, 37.66; H, 3.23; Cl, 22.01; N, 21.84.
Compound 17. A suspension of clofarabine (1, 1.04 g, 3.42
mmol), MeOH (20 mL), and NaOMe (1.56 mL, 25 wt %, 6.84
mmol) was heated at 60 °C for 7 days. The reaction was
quenched with 5 M HCl until pH ) 6 and was filtered, and the
filtrate was concentrated. Purification by chromatography (silica
gel, MeOH/CH2Cl2, 1/9) gave 17 as a white solid (200 mg,
1
99% purity, 20% yield). Mp ) 216 °C. H NMR (CDCl3) δ
8.04 (s, 1H), 7.35 (s, 2H), 6.29 (dd, 1H, J ) 15, 4), 5.97 (s,
1H), 5.19 (dm, 1H, J ) 53), 5.07 (s br, 1H), 4.47 (d, 1H, J )
19), 3.83 (s, 4H), 3.70-3.62 (m, 2H). 13C NMR (DMSO-d6)
161.9, 156.7, 150.9, 138.2, 114.7, 95.3 (d, JCF ) 193), 83.4 (d,
JCF ) 3), 81.2 (d, JCF ) 17), 72.8 (d, JCF ) 24), 60.6, 54.0
ppm. IR (KBr) 3470, 3312, 2922, 1639, 1507, 1475, 1369, 1262,
1205, 1119, 1059, 953, 870, 703 cm-1. UV (H2O/MeOH)
λmax1 210 nm, λmax2 267 nm. MS m/z [M + Na]+ ) 322.
Anal. Calcd for C11H14FN5O4: C, 44.15; H, 4.72; F, 6.35; N,
23.40. Found: C, 43.74; H, 4.28; F, 6.09; N, 22.61.
1234
•
Vol. 12, No. 6, 2008 / Organic Process Research & Development

1
Protected Bis-sugars (18a,b). A suspension of 5 (11.38 g,
22.2 mmol), MeCN (80 mL), and CaH2 (0.479 g, 11.4 mmol)
was heated to 70 °C. Solutions of bromosugar 3 (2.35 g, 5.55
mmol in MeCN (5.6 mL)), and KOt-Bu (1.0 M, 5.6 mL, 5.6
mmol) were added simultaneously via syringe pump (0.2 mL/
min). Simultaneous addition was reiterated over 5 days until
no further reaction progress by HPLC was observed. Eight
additions were made, totaling 2 equiv each of 3 and KOt-Bu.
The mixture was filtered, and the solvent was concentrated to
give a dark brown oil (27.6 g), which was dissolved in MeCN
(70 mL) and resubjected to the same reaction conditions. Seven
additions of 3 and KOt-Bu (0.25 equiv each per addition) were
performed over 3 days. HPLC analysis showed a level of 14%
and 19%, respectively, of 18a,b. The reaction mixture was
filtered and diluted with CH2Cl2 (700 mL). The solution was
washed with H2O (2 × 250 mL), dried (MgSO4), and
concentrated. Purification by chromatography (silica gel, hex-
anes/10-45% EtOAc) gave a yellow oil (0.530 g, 75% purity).
The oil was further purified by preparative scale HPLC (Nova-
Pak silica, hexanes/EtOAc, 69/31 w/0.2% NEt3) to give the
protected bis-sugars: 18a (101 mg, 94% purity). Mp ) 87-90
°C. 1H NMR (CDCl3) δ 8.11-8.07 (m, 10H), 7.41-7.69 (m,
12H), 6.78 (br m, 1H), 6.58 (br dd, 2H, J ) 22, 3), 5.75 (dd,
1H, J ) 18, 3), 5.64 (dd, 1 H, J ) 18, 3), 5.36 (dm, 1H, J )
52), 5.24 (dm, 1H, J ) 52), 4.80 (m, 2H), 4.66 (m, 2H), 4.53
(m, 2H). 13C NMR (CDCl3) 166.2, 166.1, 165.2, 165.2, 154.4,
154.0, 140.9 (d, JCF ) 8), 134.2, 133.9, 133.4, 133.1, 129.8
(m), 128.6 (m), 94.0 (d, JCF ) 188), 92.7 (d, JCF ) 192), 83.6,
83.4, 81.3, 79.5, 63.8, 63.3 ppm. IR (KBr) 3421, 3064, 1727,
1617, 1586, 1531, 1452, 1316, 1272, 1178, 1110, 1027, 918,
805, 710 cm-1. UV (H2O/MeOH) λmax1 218 nm, λmax2 230
nm, λmax3 266 nm. MS m/z [M + H]+ ) 854. Anal. Calcd for
C43H34ClF2N5O10: C, 60.46; H, 4.01; Cl, 4.15; F, 4.45; N, 8.20.
Found: C, 60.81; H, 4.03; Cl, 3.68; F, 4.38; N, 7.35. 18b (141
°C. H NMR (DMSO-d6) δ 8.41 (d, 1H, J ) 2), 8.18 (br s,
1H), 6.38 (dd, 1H, J ) 13, 5), 6.22 (br s, 1H), 5.98 (d, 1H, J
) 5), 5.73 (d, 1H, J ) 5), 5.26 (dt, 1H, J ) 52, 4), 5.12-4.90
(m, 3H), 4.44 (dm, 1H J ) 9), 4.30 (m, 1H), 3.87 (m, 1H),
3.72-3.63 (m, 3H), 3.55-3.51 (m, 2H). 13C NMR (DMSO-
d6) 154.1, 152.9, 150.4, 141.0, 95.8 (d, JCF ) 192), 95.3 (d, JCF
) 192), 83.5 (d, JCF ) 6), 82.8, 81.5 (d, JCF ) 17), 78.8 (br s),
73.1 (d, JCF ) 23), 72.4 (d, JCF ) 22), 61.4, 60.3 ppm. IR (KBr)
3401, 2935, 1622, 1536, 1467, 1426, 1343, 1238, 1038, 952
cm-1. UV (H2O/MeOH) λmax1 212 nm, λmax2 266 nm. MS
m/z [M + H]+ ) 438. Anal. Calcd for C15H18ClF2N5O6: C:
41.15; H, 4.14; Cl, 8.10; F, 8.68; N, 16.00. Found: C, 41.34;
H, 4.17; Cl, 8.30; F, 8.50; N, 15.75. 19b (50 mg, 99% purity).
Mp ) 134-135 °C. 1H NMR (DMSO-d6) δ 8.30 (d, 1H, J )
4), 8.19 (br s, 1H), 6.41 (dd, 1H, J ) 16, 4), 6.22 (br s, 1H),
5.15 (dm, 1H, J ) 52), 5.09 (dt, 1H, J ) 52, 3), 4.51 (dm, 1H,
J ) 18), 4.34 (dm, 1H, J ) 18), 4.11 (br q, 1H, J ) 3), 3.98
(q, 1H, J ) 3), 3.87-3.78 (m, 2H), 3.72-3.63 (m, 2H). 13C
NMR (DMSO-d6) 155.3, 142.5 (d, JCF ) 4), 100.9 (d, JCF
)
60), 96.8 (d, JCF ) 194), 85.6 (br d), 84.3 (d, JCF ) 17), 75.3
(d, JCF ) 24), 74.7 (d, JCF ) 25), 62.6, 62.1 ppm. IR (KBr)
3355, 2933, 1662, 1619, 1466, 1344, 1307, 1238, 1042, 680
cm-1. UV (H2O/MeOH) λmax1 210 nm, λmax2 268 nm. MS
m/z [M + H]+ ) 438. Anal. Calcd for C15H18ClF2N5O6: C:
41.15; H, 4.14; Cl, 8.10; F, 8.68; N, 16.00. Found: C, 40.55;
H, 4.39; Cl, 8.15; F, 7.67; N, 15.72.
Degradant B, Nucleoside 20. A solution of clofarabine (1,
0.352 g, 1.16 mmol), H2O (6 mL), LiOH (0.083 g, 3.47 mmol),
and H2O2 (0.24 mL, 30 wt %, 2.35 mmol) was stirred at 60 °C
for 24 h. HPLC analysis showed 88% conversion. A solution
of Na2S2O3 in H2O was added until the peroxide test (starch-
iodide paper) was negative. HOAc was added until the pH was
4-5, and the mixture was concentrated. Purification by chro-
matography (reverse phase, C-18, H2O/0-100% MeOH) gave
20 as a white solid (0.16 g, 99.3% purity, 48% yield). Mp )
1
mg, 85% purity). Mp ) 87-90 °C. H NMR (CDCl3) δ
1
8.11-8.08 (m, 9H), 7.66-7.41 (m, 12H), 6.70 (br s, 2H), 6.58
(dd, 1H, J ) 23, 3), 5.77-6.69 (m, 2H), 5.41 (d, 1H, J ) 49),
5.35 (dd, 1H, J ) 49, 2), 4.80-4.79 (m, 2H), 4.75-4.68 (m,
1H), 4.66-4.60 (m, 2H), 4.58-4.55 (m, 1H). 13C NMR
(CDCl3) 166.2, 166.1, 165.2, 165.1, 154.4, 153.7, 140.8 (d, JCF
) 5), 134.2, 134.0, 133.4, 133.2, 130.0, 129.83, 129.76, 129.6,
129.4, 128.7, 128.5, 128.4, 128.1, 118.5, 97.0 (d, JCF ) 188),
92.7 (d, JCF ) 193), 86.0 (br), 83.5 (d, JCF ) 17), 83.5, 81.2,
63.9, 63.3 ppm. 19F NMR (DMSO-d6) -189.1 (br d), -198.8
(dt, J ) 30, 19) ppm. IR (KBr) 3415, 3333, 3064, 1726, 1616,
1452, 1380, 1272, 1178, 1096, 1027, 805, 710 cm-1. UV (H2O/
MeOH) λmax1 218 nm, λmax2 230 nm, λmax3 268 nm. MS
m/z [M + H]+ ) 854. Anal. Calcd for C43H34ClF2N5O10: C,
60.46; H, 4.01; Cl, 4.15; F, 4.45; N, 8.20. Found: C, 60.74; H,
3.62; Cl, 3.81; F, 4.41; N, 7.96.
Bis-sugars (19a,b). A mixture of protected bis-sugar isomers
(18a,b 834 mg, 0.976 mmol, 86% purity) was combined with
MeOH (8 mL) and NaOMe (90 µL, 25 wt %, 0.39 mmol) and
stirred at ambient temperature for 4.5 h. HOAc (30 µL) was
added, and the reaction mixture was concentrated. Purification
by preparative HPLC (Atlantis reverse-phase, 5 µm, 19 mm ×
100 mm, H2O/MeCN, 41/9, 14 mL/min) gave the bis-sugars
as white solids: 19a (127 mg, 95% purity). Mp ) 146-152
262-282 °C (dec). H NMR (DMSO-d6) δ 10.74 (br s, 1H),
7.84 (1H, d, J ) 2), 7.80 (br s, 2H), 6.13 (1H, dd, J ) 16, 4),
5.93 (1H, d, J ) 5), 5.09 (1H, dt, J ) 53, 4), 5.13 (1H, br s),
4.35 (1H, ddd, J ) 19, 8, 5), 3.79 (1H, q, J ) 5), 3.80-3.56
(2H, m). 13C NMR (DMSO-d6) 156.1, 151.8 (br), 137.7 (br
m), 104.4 (br), 95.4 (d, JCF ) 192), 83.5 (d, JCF ) 5), 81.0
(br), 72.8 (d, JCF ) 24), 60.4. IR (KBr) 3371, 3171, 1675, 1643,
1606, 1379, 1040 cm-1. UV (H2O/MeOH) λmax1 247 nm,
λmax2 292 nm. MS m/z [M + H]+ ) 285. Anal. Calcd for
C10H12FN5O4: C, 42.11; H, 4.24; F, 6.66; N, 24.55. Found: C,
41.96; H, 4.00; F, 6.43; N, 24.57.
Protected Nucleoside 22. A solution of 3 (13.83 g, 32.7
mmol) in DCE (25 mL) was added to a suspension of 21 (5.04
g, 29.7 mmol), TAA (28 mL), MeCN (25 mL), and KOt-Bu
(36 mL, 36 mmol) over 5 min. The reaction was stirred at
ambient temperature for 20 h. HOAc (0.3 mL, pH ) 6-7) was
added followed by CH2Cl2 (100 mL). The mixture was filtered,
and the flask and solids were washed with CH2Cl2 (2 × 50
mL). The mixture was concentrated, and the residue was
recrystallized from boiling MeOH to give 22 as a pale yellow
solid (7.72 g, 93% purity, 47% yield). A portion of this material
was purified by chromatography (silica gel, hexanes/0-100%
1
EtOAc) for characterization. Mp ) 87-89 °C. H NMR
Vol. 12, No. 6, 2008 / Organic Process Research & Development
•
1235

(DMSO-d6) δ 8.19 (d, 1H, J ) 3), 8.10 (dm, 2H, J ) 8), 8.01
(dm, 2H, J ) 8), 7.76-7.66 (m, 2H), 7.62-7.11 (m, 4H), 7.11
(s, 2H), 6.50 (dd, 1H, J ) 20, 4), 5.90 (dm, 1H, J ) 20), 5.77
(dm, J ) 52), 4.82-4.70 (m, 3H). 13C NMR (DMSO- d6) 165.5,
164.7, 160.0, 153.6, 149.8, 141.1 (d, JCF ) 6), 134.0, 133.5,
129.6, 129.2, 128.8, 128.7, 128.6, 92.8 (d, JCF ) 192), 82.0 (d,
JCF ) 15), 78.6, 76.7 (d, JCF ) 28), 63.7 ppm. 19F NMR
(DMSO-d6) -198.4 (dt, J ) 51, 19) ppm. IR (KBr) 3383, 1725,
1615, 1566, 1468, 1272, 1110, 908, 711 cm-1. UV (H2O/
MeOH) λmax1 223 nm, λmax2 308 nm. MS m/ z [M + H]+ )
512. Anal. Calcd for C24H19ClFN5O5: C, 56.31; H, 3.74; Cl,
6.93; F, 3.71; N, 13.68. Found: C, 56.10; H, 3.63; Cl, 6.81; F,
3.59; N, 13.44.
Degradant C, Nucleoside 23. A solution of 22 (3.48 g, 6.8
mmol), THF (45 mL), H2O (25 mL), and NaOH (0.85 g, 21.3
mmol) was stirred at ambient temperature for 7 h. HCl (12 M,
2 mL) was added, and THF was removed by rotary evaporation.
The remaining aqueous layer was washed with CH2Cl2 (2 ×
100 mL). The aqueous layer was stirred with NaOH (0.54 g,
13.6 mmol) and H2O2 (0.7 mL, 30 wt %, 6.8 mmol) at ambient
temperature for 30 min. A solution of Na2S2O3 in H2O was
added until the peroxide test (starch-iodide paper) was negative.
HC1 (12 M, 1.4 mL) was added until the pH was 5-6. The
suspension was filtered, and the flask and solids were washed
with THF (100 mL). Purification by chromatography (C-18,
H2O/5 to 100% MeOH) gave 23 as a white solid (0.36 g, 18%
yield). Mp ) 275-279 °C. 1H NMR (DMSO-d6) δ 10.68 (s,
1H), 7.79 (d, 1H, J ) 2), 6.54 (br s, 2H), 6.11 (dd, 1H, J ) 16,
4), 5.94 (d, 1H, J ) 5), 5.09 (dt, J ) 53, 4), 5.08 (t, 1H, J )
6), 4.35 (ddd, 1H, J ) 18, 8, 5), 3.79 (q, 1H, J ) 5), 3.60
(apparent octet 2H, J ) 6). 13C NMR (DMSO-d6) 157.2, 154.1,
151.4, 136.6, 116.1, 95.5 (d, JCF ) 192), 83.8 (d, JCF ) 5),
81.6 (d, JCF ) 17), 72.9 (d, JCF ) 23), 60.7 ppm. IR (KBr)
3396, 3132, 1691, 1535, 1376, 1045 cm-1.UV (H2O/MeOH)
λmax1 252 nm. MS m/z [M + H]+ ) 285. Anal. Calcd for
C10H12FN5O4: C, 42.11; H, 4.24; F, 6.66; N, 24.55. Found: C,
40.06; H, 4.23; F, 5.84; N, 21.73.
Degradant E compound 24. A suspension of clofarabine
(1, 1.59 g, 5.24 mmol), NaOH (1.26 g, 31.5 mmol), and DMSO
(110 mL) was heated to 54 °C over 30 min and held at that
temperature for 3 h. HPLC analysis showed formation of the
product (38% purity). The mixture was decanted, HOAc (2 mL)
was added, and the mixture was concentrated. The residue was
triturated with MeOH (200 mL), the suspension was filtered,
and the filtrate was concentrated. Purification by chromatog-
raphy (silica gel, acetone/EtOAc, 3/7) and crystallization from
a boiling mixture of IPA/H2O (8 mL/1.3 mL) gave 24 as a white
solid (0.38 g, 97.3% purity, 25% yield). Mp ) 239-242 °C.
1H NMR (DMSO-d6) δ 8.42 (s, 1H), 7.88 (br s, 2H), 6.27 (s,
1H), 5.98 (d, 1H, J ) 2), 5.24 (d, 1H, J ) 4), 5.17 (ddd, 1H,
J ) 6, 4, 2), 5.03 (s, 1H), 4.72 (dd, 1H, J ) 4, 8), 3.94 (dd,
1H, J ) 8, 2). 13C NMR (DMSO-d6) 156.8, 153.3, 150.9, 139.3,
117.7, 92.6, 90.6, 80.2, 77.0, 76.6 ppm. IR (KBr) 3412, 3331,
3218, 3126, 2887, 1652, 1597, 1576, 1460 cm-1. UV (H2O/
MeOH) λmax1 211 nm, λmax2 265 nm. MS m/z [M + H]+ )
284. Anal. Calcd for C10H10ClN5O3: C, 42.34; H, 3.55; Cl,
12.50; N, 24.69. Found: C, 42.28; H, 3.42; Cl, 12.65; N, 24.65.
Compound 25. LiBr (14.56 g, 168 mmol) was added to a
solution of 7 (19.94 g, 33.5 mmol) in NMP (50 mL), and the
mixture was stirred at ambient temperature for 20 h. H2O (250
mL) was added, and the mixture was extracted with TBME (2
× 250 mL). The combined organic portions were washed with
H2O (250 mL), dried (MgSO4), and concentrated to give 25 as
a pale yellow oil (17.12 g, 98.6% purity, 96% yield). 1H NMR
(DMSO-d6) δ 8.08-8.01 (m, 6H), 7.74-7.67 (m, 3H),
7.58-7.48 (m, 6H), 6.64 (s, 1H), 5.72 (d, 1H, J ) 3), 5.02 (s,
1H), 5.02-4.99 (m, 1H), 4.79 (dd, 1H, J ) 12, 4), 4.68 (dd,
1H, J ) 12, 6). 13C NMR (DMSO-d6) 165.4, 164.8, 163.9,
133.93, 133.87, 133.5, 129.5, 129.4, 129.2, 128.8, 128.7, 102.3,
83.4, 79.3, 63.8, 50.4 ppm. IR (KBr) 3434, 1725, 1270, 1095,
1067, 929, 709 cm-1. UV (H2O/MeOH) λmax1 229 nm, λmax2
274 nm. MS m/z [M + Na]+ ) 547. Anal. Calcd for
C26H21BrO7: C, 59.44; H, 4.03; Br, 15.21. Found: C, 59.62; H,
4.18; Br, 14.88.
Compound 26. HBr/HOAc (10.7 mL, 33 wt %, 62 mmol)
was added to a solution of 25 (16.71 g, 31.8 mmol) in CH2Cl2
(125 mL), and the mixture was stirred at ambient temperature
for 23 h. The mixture was poured into saturated NaHCO3 (300
mL) with stirring (off-gassing). The layers were separated, and
the aqueous layer was extracted with CH2Cl2 (100 mL). The
organic portions were washed with saturated NaHCO3 (100
mL), dried (MgSO4), and concentrated to give 26 as an oil
(14.80 g, 96% recovery). The crude material was used in the
next step as is.
Nucleoside 27. A solution of 26 (14.19 g, 29.3 mmol) in
DCE (22.5 mL) was added to a suspension of 4 (4.52 g, 26.7
mmol), TAA (25 mL), MeCN (22.5 mL), and KOt-Bu (32 mL,
32 mmol) over 8 min. After stirring at ambient temperature for
18 h, the reaction mixture was filtered, and the filtrate was
concentrated. MeOH (100 mL) was added, and the mixture was
heated to reflux. The suspension was cooled and filtered, and
the solid was dried (50 °C, 50-100 Torr). Purification by
chromatography (silica gel, EtOAc/hexanes, 3/2) gave 27 as a
white solid (2.85 g, 94.4% purity, 18% yield). Mp 172-175
°C. 1H NMR (DMSO-d6) δ 8.40 (s, 1H), 8.09 (d, 2H, J ) 7),
7.97 (br s, 1H), 7.93 (d, 2H, J ) 7), 7.74 (apparent t, 1H, J )
7), 7.66-7.58 (m, 3H), 7.45 (apparent t, 2H, J ) 8), 6.61 (d,
1H, J ) 8), 6.37 (t, 1H, J ) 7), 5.43 (t, 1H, J ) 7), 4.84 (d,
2H, J ) 5), 4.63 (q, 1H, J ) 6). 13C NMR (DMSO-d6) 165.4,
164.8, 156.9, 153.2, 149.7, 139.9, 134.0, 133.5, 129.6, 129.1,
128.8, 128.64, 128.57, 83.6, 79.0, 77.7, 64.1, 50.2 ppm. IR
(KBr) 3325, 3177, 1717, 1669, 1598, 1312, 1272, 1094, 710
cm-1. UV (H2O/MeOH) λmax1 213 nm, λmax2 231 nm, λmax3
264 nm. MS m/z [M + Na]+ ) 594. Anal. Calcd for
C24H19BrClN5O5: C, 50.32; H, 3.34; Br, 13.95; Cl, 6.19; N,
12.23. Found: C, 50.59; H, 3.09; Br, 13.56; Cl, 6.43, N, 11.98.
Degradant D, Compound 28. A suspension of 27 (1.33 g,
2.33 mmol), MeOH (3.6 mL), and NaOMe (0.59 mL, 25 wt
%, 2.6 mmol) was stirred at ambient temperature for 16 days.
HOAc (0.5 mL) was added, the mixture was concentrated, and
the residue was triturated with hexanes. Purification by chro-
matography (silica gel, hexanes/0-100% EtOH, 15 min) gave
28 as a white solid (0.347 g, 99.1% purity, 52% yield). Mp
207-209 °C. 1H NMR (DMSO-d6) δ 8.35 (s, 1H), 7.84 (br s,
2H), 6.13 (s, 1H), 5.03 (br t, 1H, J ) 5), 4.46 (d, 1H, J ) 3),
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4.20-4.18 (m, 2H), 3.58-3.51 (m, 2H). 13C NMR (DMSO-
d6) 156.8, 153.1, 150.2, 139.9, 117.8, 81.9, 81.3, 60.8, 58.5,
57.6 ppm. IR (KBr) 3414, 3328, 1644, 1315, 1065, 1018, 585
cm-1. UV (H2O/MeOH) λmax1 211 nm, λmax2 264 nm. MS
m/z [M + H]+ ) 284. Anal. Calcd for C10H10ClN5O3: C, 42.34;
H, 3.55; Cl, 12.50; N, 24.69. Found: C, 42.23; H, 3.44 Cl, 12.38,
N, 24.54.
white solid (74 mg, 99.0% purity, 0.8% yield). Mp ) 211-213
°C. 1H NMR (DMSO-d6) δ 8.33 (d, 1H, J ) 3), 8.12 (d, 1H,
J ) 3), 7.93 (br s, 2H), 7.53 (br s, 2H), 6.38 (dd, 1H, J ) 20,
4), 6.32 (dd, 1H, J ) 15, 5), 5.96 (d, 1H, J ) 5), 5.66 (dm, 1H,
J ) 17), 5.50 (dt, 1H, J ) 50, 2), 5.20 (dt, 1H, J ) 50, 4), 5.15
(t, 1H, J ) 6), 5.09 (t, 1H, J ) 6), 4.42 (ddd, 1H, J ) 14, 9,
5), 4.21 (dd, 1H, J ) 9, 5), 3.87-3.76 (m, 3H), 3.71-3.62 (m,
2H). 13C NMR (DMSO-d6) 159.9, 156.8, 153.4, 150.7, 150.2,
140.1 (d, JCF ) 6), 138.6 (d, JCF ) 4), 117.3, 115.1, 95.4 (d,
JCF ) 193), 93.0 (d, JCF ) 191), 83.5 (d, JCF ) 5), 82.9, 82.6
(d, JCF ) 17), 81.4 (d, JCF ) 17), 77.6 (d, JCF ) 28), 72.7 (d,
JCF ) 24), 60.8, 60.4 ppm. 19F NMR (DMSO-d6) -198.3 (dt,
J ) 53, 17 Hz), -199.0 (dt, J ) 48, 18 Hz) ppm. IR (KBr)
3429, 1649, 1595, 1475, 1346, 1215, 1044 cm-1. UV (H2O/
MeOH) λmax1 211 nm, λmax2 264 nm. MS m/z [M + H]+ )
571. Anal. Calcd for C20H21ClF2N10O6: C, 42.08; H, 3.71; Cl,
6.21; F, 6.66; N, 24.53. Found: C, 41.98; H, 3.78; Cl, 6.10; F,
6.63; N, 24.38.
Degradant A, Compound 29. A solution of 28 (0.393 g,
1.39 mmol) and H2O (11 mL) was heated at 100 °C for 6.2 h.
The volatiles were concentrated to give crude 29 (278 mg).
Purification by preparative HPLC (C-18, H2O/5-95% MeCN,
6 min) gave 29 as a white solid (0.107 g, 99% purity, 29%
1
yield). Mp ) 290 °C (dec). H NMR (DMSO-d6) δ 7.82 (s,
1H), 6.03 (s, 1H), 4.80 (d, 1H, J ) 3), 4.75 (s, 1H), 4.65
(apparent quintet, 1H, J ) 4), 3.48 (dd, 1H, J ) 12, 4), 3.24
(dd, 1H, J ) 11, 7). 13C NMR (DMSO-d6) 157.8, 153.1, 140.2,
132.8, 109.9, 86.3, 85.3, 74.0, 60.6, 56.1 ppm. IR (KBr) 3404,
3330, 3204, 1675, 1596, 1581, 1045, 844, 782 cm-1. UV (H2O/
MeOH) λmax1 220 nm, λmax2 239 nm, λmax3 276 nm. MS
m/z [M + H]+ ) 266. Anal. Calcd for C10H11N5O4: C, 45.28;
H, 4.18; N, 26.41. Found: C, 44.99; H, 4.18; N, 26.22.
Degradant F, Compound 31. A solution of 1 (9.73 g, 32.0
mmol), NaOH (2.88 g, 72 mmol), and H2O (145 mL) was
stirred at 80 °C for 1.9 h. HPLC analysis showed the reaction
mixture contained 11.9% 31. HOAc (2.5 mL) was added, and
the suspension was cooled to 5 °C and filtered. The solid was
triturated with H2O, and the mixture was filtered. The solid was
triturated with MeOH, and the mixture was filtered. Purification
by chromatography (2×, silica gel, hexanes/0-100% EtOH)
gave 31 (116 mg, 77% purity). Final purification by preparative
HPLC (C-18, 79% H2O/MeCN/MeOH, 79/14/7) gave 31 as a
Acknowledgment
We thank Dr. Phillip Bauer for helpful discussions concern-
ing the regulatory aspects of this work. We also thank Dr. Jim
Goebel for guidance regarding analytical chemistry issues.
Supporting Information Available
HPLC conditions and spectral data for all compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Received for review July 31, 2008.
OP800182X
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