Synthesis and cytostatic evaluation of 4-N-alkanoyl and 4-N-alkyl gemcitabine analogues

Article abstract of DOI:10.1021/jm401586a

The coupling of gemcitabine with functionalized carboxylic acids (C9-C13) or reactions of 4-N-tosylgemcitabine with the corresponding alkyl amines afforded 4-N-alkanoyl and 4-N-alkyl gemcitabine derivatives. The analogues with a terminal hydroxyl group on the alkyl chain were efficiently fluorinated under conditions that are compatible with protocols for ¹⁸F labeling. The 4-N-alkanoylgemcitabines showed potent cytostatic activities in the low nanomolar range against a panel of tumor cell lines, whereas cytotoxicity of the 4-N-alkylgemcitabines were in the low micromolar range. The cytotoxicity for the 4-N-alkanoylgemcitabine analogues was reduced approximately by 2 orders of magnitude in the 2′-deoxycytidine kinase (dCK)-deficient CEM/dCK ^- cell line, whereas cytotoxicity of the 4-N-alkylgemcitabines was only 2-5 times lower. None of the compounds acted as efficient substrates for cytosolic dCK; therefore, the 4-N-alkanoyl analogues need to be converted first to gemcitabine to display a significant cytostatic potential, whereas 4-N-alkyl derivatives attain modest activity without measurable conversion to gemcitabine.

Full text of DOI:10.1021/jm401586a

Article
pubs.acs.org/jmc
Synthesis and Cytostatic Evaluation of 4‑N‑Alkanoyl and 4-N-Alkyl
Gemcitabine Analogues
Jesse Pulido,† Adam J. Sobczak,† Jan Balzarini,§ and Stanislaw F. Wnuk*,†,‡
‡
†Department of Chemistry and Biochemistry, Department of Environmental and Occupational Health, Florida International
University, Miami, Florida 33199, United States
§Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
S
* Supporting Information
ABSTRACT: The coupling of gemcitabine with functionalized carboxylic acids
(C9−C13) or reactions of 4-N-tosylgemcitabine with the corresponding alkyl
amines afforded 4-N-alkanoyl and 4-N-alkyl gemcitabine derivatives. The
analogues with a terminal hydroxyl group on the alkyl chain were efficiently
fluorinated under conditions that are compatible with protocols for 18F labeling.
The 4-N-alkanoylgemcitabines showed potent cytostatic activities in the low
nanomolar range against a panel of tumor cell lines, whereas cytotoxicity of the 4-
N-alkylgemcitabines were in the low micromolar range. The cytotoxicity for the
4-N-alkanoylgemcitabine analogues was reduced approximately by 2 orders of
magnitude in the 2′-deoxycytidine kinase (dCK)-deficient CEM/dCK− cell line,
whereas cytotoxicity of the 4-N-alkylgemcitabines was only 2−5 times lower.
None of the compounds acted as efficient substrates for cytosolic dCK; therefore, the 4-N-alkanoyl analogues need to be
converted first to gemcitabine to display a significant cytostatic potential, whereas 4-N-alkyl derivatives attain modest activity
without measurable conversion to gemcitabine.
either the exocyclic 4-N-amine or 5′-hydroxyl group.24 The
INTRODUCTION
■
hydrolyzable amide modifications facilitate a slow release of
Gemcitabine (2′,2′-difluoro-2′-deoxycytidine, dFdC) is a
gemcitabine, increasing its bioavailability and uptake while also
chemotherapeutic nucleoside analogue used in the treatment
providing resistance to enzymatic deamination.25−32 In 2004,
of solid tumors in various cancers.1,2 Synthesized first in 1988
Immordono et al. reported the increased anticancer activity of
by Hertel et al.,3 gemcitabine represents first-line therapy for
4-N-stearoyl gemcitabine, which was stable in plasma and
pancreatic and nonsmall cell lung cancers.4−6 Gemcitabine is
showed an improved resistance to deamination.28 Couvreur
hydrophilic by nature, and its cellular uptake is primarily
and Cattel developed the 4-N-squalenoyl gemcitabine prodrug
facilitated by human equilibrative nucleoside transport protein
1 (hENT1).7 Gemcitabine is activated via phosphorylation to
(SQgem) as a chemotherapeutic nanoassembly that accumu-
lates in the cell membranes prior to releasing gemcitabine26
its 5′-monophosphate (dFdCMP) by deoxycytidine kinase
(dCK).8,9 dFdCMP then undergoes subsequent phosphoryla-
(Figure 1). SQgem overcomes the low efficacy of gemcitabine
in chemoresistant pancreatic cell lines and is currently
tion by intracellular kinases to its diphosphate (dFdCDP) and
undergoing preclinical development.31 The orally active 4-N-
triphosphate (dFdCTP) forms.10,11 dFdCTP can incorporate
into DNA and inhibit DNA polymerase by chain termination
valproylgemcitabine prodrug 1 (LY2334737), currently under-
going phase I clinical trials,30,32 was designed to be resistant to
during DNA replication and repair processes, invariably
triggering apoptosis.10−12 It can also participate in self-
deamination by hydrolysis under acidic conditions similar to
those found in the human digestive system25,29 while
potentiation by inhibiting CTP synthetase and depleting CTP
systematically releasing gemcitabine upon action by carbox-
pools available to compete with dFdCTP for incorporation into
RNA.12,13 Moreover, dFdCD(T)P inhibits both R1 and R2
ylesterase 2. Recently, the gemcitabine prodrug with a Hoechst
subunits of ribonucleotide reductase (RNR),14−20 depleting the
conjugate attached to the 4-amino group targeting extracellular
DNA has been reported with low toxicity but high tumor
deoxyribonucleotide pool available to compete with dFdCTP
efficacy.27 In addition, the lipophilic gemcitabine prodrug CP-
4126 with the 5′-OH group esterified with an elaidic fatty acid
has shown antitumor activity in various xenograft models.33 It
remains active when orally administered;33 however, it has not
yet met criteria for advancement to phase II clinical trials.34
for incorporation into DNA.15,16 Gemcitabine is therapeutically
restricted by high toxicity to normal cells and rapid intracellular
deamination into inactive 2′,2′-difluorouridine (dFdU) by
cytidine deaminase (CDA).21
Because clinical studies have indicated that prolonged
infusion times with lower doses of gemcitabine can be effective
while reducing toxicity to normal cells,22,23 various prodrug
strategies have been developed featuring acyl modifications on
Received: October 10, 2013
Published: December 16, 2013
© 2013 American Chemical Society
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Figure 1. The acylated gemcitabine prodrugs.
a
Scheme 1. Synthesis of Lipophilic 4-N-Alkanoyl Gemcitabine Derivatives
a
Reagents and conditions: (a) R′(CH2)nCOOH/NMM/HOBt/EDCI/DMF/DMSO/60 °C/overnight; (b) (i) TMSCl/Pyr/CH3CN, 0 °C/2.5 h,
(ii) BrCH2(CH2)9COOH/CDI/CH3CN/65 °C/overnight; (c) BrCH2(CH2)9COOH/ClCOOEt/Et3N/DMF (d) (Boc)2O/KOH/1,4-dioxane; (e)
BrCH2(CH2)9COCl/NaHCO3/CH2Cl2; (f) DAST/CH2Cl2; (g) TFA
Very recently, the 5′-acylated gemcitabine prodrugs with a
coumarin or boron-dipyrromethene/biotin conjugate have been
reported for monitoring drug delivery at subcellular levels by
fluorescence imaging.35,36
(HOBt)/N-methylmorpholine (NMM)) in DMF/DMSO
(3:1) at 60 °C afforded 4-N-undecanoylgemcitabine 3 (50%,
Scheme 1). Analogous coupling of 2a with 8-nonenoic acid, 10-
undecenoic acid, 12-tridecenoic acid, 11-fluoroundecanoic acid
(S4; see the Supporting Information), or 11-hydroxyundeca-
noic acid afforded 4-N-acyl analogues 4−8 in 40−66% yield
after silica gel purification. It is noteworthy that these couplings
in the presence of HOBt typically progressed to >90%
completion (TLC), whereas the 1,1′-carbonyldiimidazole-
(CDI)-mediated coupling44 of 2a with the corresponding
carboxylic acids in CH3CN and pyridine without HOBt
proceeded less efficiently.
Unexpectedly, the HOBt-promoted coupling of 2a with 11-
bromoundecanoic acid led to the formation of 4-N-[11-(1H-
benzotriazol-1-yloxy)undecanoyl derivative 9 in a 53% yield
rather than the expected 4-N-(11-bromoundecanoyl) derivative
12. Other attempts to synthesize the bromo derivative either by
transiently protecting 2a with a trimethylsilyl group45 followed
by treatment with 11-bromoundecanoic acid/CDI or by
employing a mixed anhydride procedure26 (11-bromoundeca-
noic acid/ethyl chloroformate/TEA) gave chloro derivative 10
instead. The labile nature of the terminal bromide necessitated
an alternative approach for the preparation of 12. We found
that condensation of 3′,5′-di-O-Boc-protected gemcitabine46 2b
with 11-bromoundecanoyl chloride (S5; see the Supporting
Information) provided bromo derivative 11 in a 33% isolated
yield. The deprotection of 11 with TFA gave desired 12 (86%).
1-(2′-Deoxy-2′-18F-fluoro-β-D-arabinofuranosyl)cytosine
([18F]-FAC) was developed by Radu et al.37 as a PET tracer
possessing a substrate affinity for dCK and CDA comparable to
gemcitabine. Determination of [18F]-FAC uptake and pretreat-
ment levels of dCK serve as a noninvasive prognosticator for
gemcitabine chemotherapy response.37−40 dCK-specific PET
tracers, such as 1-(2′-deoxy-2′-18F-fluoro-β-L-arabinofuranosyl)-
cytosine ([18F]-L-FAC) and 1-(2′-deoxy-2′-18F-fluoro-β-L-arabi-
nofuranosyl)-5-methylcytosine (L-18F-FMAC), which possess
no substrate affinity to CDA, were also developed to study
uptake of [18F]-FAC and serve as additional predictive tools for
gemcitabine treatment response.41,42
Herein, we report synthesis and cytostatic activity of a series
of gemcitabine analogues with 4-N-alkanoyl or 4-N-alkyl chains
modified with a terminal hydroxyl, halide, or alkene groups.
The 4-N-alkyl analogues stable toward deamination were
designed to examine their anticancer activities and also to
explore their compatibility with radiofluorination protocols.
CHEMISTRY
■
Condensation of gemcitabine (2a, dFdC) with undecanoic acid
under peptide coupling conditions25,43 (N-dimethylaminoprop-
yl)-N′-ethyl-carbodiimide (EDCI)/1-hydroxybenzotriazole
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Scheme 2. Synthesis of Fluorinated 4-N-Alkanoyl Gemcitabine Derivatives by the Addition of HF to the Olefin
a
Scheme 3. Synthesis of 4-N-Alkyl Gemcitabine Derivatives
a
Reagents and conditions: (a) TsCl/Et3N/1,4-dioxane; (b) CH2CH(CH2)9NH2 or HOCH2CH2(CH2)9NH2; (c) TFA
a
Scheme 4. Synthesis of 4-N-Fluoroalkyl Gemcitabine Derivatives
a
Reagents and conditions: (a) (i) TMSCl/Pyr, (ii) TsCl, (iii) MeOH/NH3; (b) BnO(CH2)11NH2/Et3N/1,4-dioxane; (c) 2,6-lutidine/DMAP/
BzCl/CH2Cl2; (d) CAN/CH3CN; (e) DAST/CH2Cl2; (f) MeOH/NH3/rt; (g) MsCl/Et3N/CH2Cl2/0 °C; (h) KF/K2CO3/K222/CH3CN/110 °C,
(ii) MeONa/MeOH/100 °C
The coupling of 2b with 11-hydroxyundecanoic acid yielded
protected 4-N-(11-hydroxyundecanoyl) derivative 13 (37%),
bearing a hydroxyl group at the alkyl chain suitable for further
chemical modifications. Thus, fluorination of 13 with DAST
afforded 4-N-(11-fluoroundecanoyl) derivative 14 (40%).
Deprotection of 13 or 14 with TFA gave 7 (87%) or 8
(82%), respectively.
Recently, Haufe et al. established the methodology for
radiofluorination of terminal olefins employing the [18F]-HF/
pyridine reagent.47 We performed the model fluorination under
this condition by employing regular, nonradioactive HF/
pyridine with olefin 5. Thus, treatment of 5 with Olah’s
reagent (70% HF in pyridine) in an HDPE vessel at 0 °C for 2
h gave a regioisomeric mixture of 10-fluoro, 9-fluoro, and 8-
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Table 1. In Vitro Cytostatic Activity of Representative 4-N-Modified Analogues on the Tumor Cell Lines L1210, CEM/0, CEM/
dCK−, HeLa, and MCF-7
IC50 (μM)
compound
L1210
CEM/0
CEM/dCK−
HeLa
MCF-7
a
1
1.1 0.7
5.2 2.3
161
8
0.76 0.30
0.55 0.49
2a
3
0.013 0.001
0.014 0.002
0.024 0.017
0.018 0.016
0.021 0.018
0.023 0.003
0.053 0.040
7.0 3.0
0.069 0.002
0.060 0.012
0.14 0.00
7.6 0.5
5.8 0.5
0.0099 0.0041
0.0089 0.0024
0.042 0.005
0.012 0.007
0.013 0.007
0.049 0.030
0.011 0.004
3.4 0.0
0.0072 0.0002
0.0053 0.0023
0.0079 0.0002
0.0062 0.0029
0.0079 0.0012
0.0081 0.0005
0.0077 0.0006
28 14
4
20
12
2
9
5
0.071 0.015
0.069 0.002
0.24 0.19
6
6.8 1.8
19
7
6
8
0.059 0.009
7.2 0.8
60 15
19
21
27
13
86 10
28
6
29 11
140 28
134 18
22
17
4
4
27
26
3
7
28
2
4
a
In free-base form.
fluoro derivatives 15−17 with an isomeric ratio of 75:20:5
(91%, Scheme 2). The 1H and 19F NMR spectra were
diagnostic for the regioisomeric composition [19F δ −179.79
(m, 0.05 F), −178.83 (m, 0.2 F), −170.27 (m, 0.75 F)].
Fluorination involved Markovnikov addition of HF to the
double bond, whereas generation of minor isomers 16 and 17 is
attributed to migration of the carbocation along the chain
during the addition reaction, as has been observed before.48
Because addition of HF to 5 gave multiple products and
radiolabeling with [18F]-HF was reported to proceed with low
radiochemical efficiencies,47 whereas other commonly used
fluorination protocols required conditions49 under which the 4-
N-alkanoyl linkage might be cleaved, we turned our attention to
developing 4-N-alkyl gemicitabine analogues.
Synthesis of 4-N-alkyl gemcitabine derivatives, which, to the
best of our knowledge, are limited to short 4-N-alkyl
modifications and their anticancer activities have not been
studied in depth.50 The 4-N-alkyl analogues are expected to be
resistant to chemical hydrolysis as well as CDA-catalyzed
deamination.51 From the available methods for the N-alkylation
of cytosine nucleosides,52−55 we found that the alkylation of the
4-exocyclic amine group in gemcitabine could be achieved
efficiently by displacement of a 4-N-tosylamine group54 with an
aliphatic alkyl amine. Thus, reaction of 2b with TsCl in the
presence of Et3N in 1,4-dioxane afforded protected 4-N-
tosylgemcitabine 18 (45%, Scheme 3). Treatment of 18 with
10-undecenyl amine effected simultaneous displacement of the
p-toluenesulfonamido group from the C4 position of the
cytosine ring and deprotection to give 4-N-(10-undecenyl)
derivative 19. Analogous reaction of 18 with 11-amino-
undecanol (S7; see the Supporting Information) gave 5′-
monoprotected 11-hydroxyundecanyl analogue 20 (47%) as
the major product in addition to fully deprotected 21 (24%).
Deprotection of 20 with TFA provided 21 with 38% overall
yield from 18.
Information) proceeded efficiently to give 4-N-(11-
benzyloxyundecanyl)gemcitabine 23 in 61% isolated yield.
Subsequent treatment of 23 with BzCl yielded fully protected
analogue 24 (60%). The lengthy treatment of 24 with ceric
ammonium nitrate (CAN) effected the selective removal of the
benzyl group to give 3′,5′-di-O-benzoyl-protected 11-hydrox-
yundecanyl analogue 25 (70%). It is noteworthy that attempted
hydrogenolysis of 24 (H2/Pd−C/EtOH/24 h) produced 25
with inconsistent yields of 5−50% in addition to substantial
quantities of other byproducts including the 5,6-dihydro
reduced derivative.
Fluorination of 25 with DAST afforded 4-N-(11-fluorounde-
canyl) derivative 26, and subsequent deprotection with
methanolic ammonia at room temperature gave 4-N-fluoroalkyl
analogue 27 (43% overall from 25). We also examined
fluorination of 25 under conditions that are compatible with
general radiosynthetic protocols for 18F labeling.46,56 Thus,
reaction of 25 with MsCl/Et3N gave mesylate precursor 28
(90%). Fluorination of the latter with KF/K2CO3/Kryptofix
2.2.2 in CH3CN at 110 °C for 18 min yielded protected fluoro
analogue 26, and subsequent debenzoylation with 0.5 M
MeONa/MeOH at 100 °C for 8 min and purification by HPLC
afforded desired 4-N-fluoroalkyl gemcitabine 27 (overall 62%
from 28, in a total of 50 min). This fluorination protocol meets
the criteria for working with 18-fluorine isotope, which has
limited availability and a short half-life (110 min) and as such is
applicable for labeling studies.
CYTOSTATIC ACTIVITY
■
The growth-inhibitory activities of the 4-N-acyl (3−8) and 4-N-
alkyl (19, 21, and 27) gemcitabine analogues were assessed on
a panel of murine and human tumor cell lines (Table 1). All 4-
N-alkanoyl 3−8 analogues demonstrated potent antiprolifer-
ative activities with IC50 values in the low nanomolar range,
similar to gemcitabine 2a, probably acting as prodrugs as
established before.25 However, 4-N-alkylgemcitabine derivatives
19, 21, and 27 showed cytostatic activities at IC50 values in the
low to modest micromolar range. It appears that the cytostatic
activity only varies slightly between compounds with different
chain lengths or functional groups. The activity for the 4-N-acyl
gemcitabine derivatives was drastically diminished (by almost 2
orders of magnitude) in the dCK-deficient CEM/dCK− cell
line, again implying the role for dCK in the metabolism of these
compounds.24 Interestingly, cytotoxicity of 4-N-alkylgemcita-
bines 19, 21, and 27 in dCK-deficient CEM/dCK− cells was
Owing to instability of the Boc protection group during
displacement of the p-toluenesulfonamido group, we explored
other protection strategies that would lead to gemcitabine
derivatives suitable for the selective modification of the primary
hydroxyl group on the 4-N-alkyl chain. Thus, transient
protection of 2a with TMSCl and subsequent treatment with
TsCl followed by methanolic ammonia afforded 4-N-
tosylgemcitabine 22 in 96% yield (Scheme 4). Displacement
of the p-toluenesulfonamido group from 22 with O-benzyl-
protected 11-aminoundecanol (S11; see the Supporting
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Figure 2. Time-point evaluation of the stability and resistance to deamination for gemcitabine (A), 4-N-alkanoylgemcitabine 7 (B), and 4-N-
alkylgemcitabine 21 (C) in 50% human serum in PBS.
only 2−5 times lower. It is noteworthy that 4-N-valproylgem-
citabine 1 in its free-base form showed cytostatic activity in the
low micromolar range, which is more comparable to our 4-N-
alkyl analogues than the 4-N-acyl counterparts. The inhibitory
activities for 1 are in agreement with the cytotoxicity described
by Pratt et al. on the NCI-60 panel, who reported IC50 values of
1 that were 80-fold greater than the value for gemcitabine on
most cell lines.30
The selected compounds were also investigated for their
interaction with dCK or mitochondrial thymidine kinase (TK-
2), which also has 2′-deoxycytidine kinase activity. None of the
prodrug derivatives showed significant inhibition of the
phosphorylation of dCyd or dThd by dCK and TK-2,
respectively. When directly evaluated as a potential substrate
for dCK, 4-N-alkanoyl derivatives 6 and 8 displayed very poor
substrate activity (<1%), whereas 4-N-alkyl analogues 11 and
12 displayed no measurable substrate activity under exper-
imental conditions that converted gemcitabine to its 5′-
monophosphate by at least 15%. Taken together, these findings
suggest that the 4-N-alkanoyl analogues first need to be
converted to gemcitabine before acting as an efficient substrate
for dCK. This release of gemcitabine from the 4-N-alkanoyl
prodrugs seems to occur quite efficiently given their
pronounced cytostatic potential in the cell cultures and the
marked loss of cytostatic activity in the dCK-deficient CEM
tumor cell cultures. Instead, the rather modest antiproliferative
activities of the 4-N-alkyl analogues may be attributed, at least
to a certain extent, to a poor cellular uptake and/or to an
inefficient conversion to the parental gemcitabine that may fall
outside the assay’s detection limits (<1%). Moreover, the fact
that the cytostatic activity of the 4-N-alkyl analogues are only
moderately decreased in dCK-deficient CEM tumor cell
cultures may not only confirm a poor, if any, intracellular
conversion to gemcitabine but also point to a potentially
different mechanism of cytostatic activity of these prodrugs. To
gain insight into the metabolism of the 4-N-alkylgemcitabine
derivatives, the stabilities of representative 4-N-alkanoyl 7 and
4-N-alkyl 21 analogues toward hydrolysis and resistance to
enzymatic deamination were evaluated in parallel with
gemcitabine in human serum and in murine liver extract.
Figure 2 shows that gemcitabine was deaminated to its inactive
uracil derivative, dFdU, as a function of time (panel A), whereas
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Figure 3. Time-point evaluation of the stability of 4-N-alkanoylgemcitabine 7 and 4-N-alkylgemcitabine 21 in murine liver extract in PBS.
commercially available except for 11-fluoroundecanoic acid (S4), 11-
bromoundecanoyl chloride (S5), 11-aminoundecanol (S7), and 11-
benzyloxyundecan-1-amine (S11), which were synthesized as
described in the Supporting Information. The purity of the synthesized
compounds was determined to be ≥95% by elemental analysis (C, H,
N) and/or HPLC on Phenomenex Gemini RP-C18 with an isocratic
mobile phase (50% CH3CN/H2O) and a flow rate of 5 mL/min.
Representative HPLC chromatograms are included in the Supporting
Information.
Tumor Cell and Enzyme Sources. Murine leukemia L1210,
human lymphocyte CEM, and human cervical carcinoma HeLa cell
lines were obtained from ATCC (Rockville, MD). Human breast
carcinoma MCF-7 cells were a kind gift from G. Peters (Amsterdam,
The Netherlands). The dCK-deficient CEM cell line was obtained
upon selection in the presence of araC and was found to be deficient
in cytosolic dCK activity.
4-N-alkanoylgemcitabine prodrug 7 was slowly converted to
gemcitabine, which was then gradually deaminated to dFdU
(panel B). However, 4-N-alkylgemcitabine derivative 21 was
neither deaminated nor was there any measurable conversion to
gemcitabine observed (panel C). When 7 and 21 were exposed
to the murine liver extract, 7 was rapidly converted to
gemcitabine (and dFdU), whereas 21 was fully stable for at
least 2 h (Figure 3). These findings again support the
assumption that 7 is enzymatically efficiently converted to
gemcitabine, whereas 21 is not, explaining the differences in the
cytostatic activity of both compounds. Although the cellular
target for the antiproliferative activity of the 4-N-alkyl analogues
is currently unclear, it may be different from the inhibition of
DNA synthesis.
In conclusion, we have demonstrated that the coupling of
gemcitabine with various carboxylic acids or reaction of 3′,5′-di-
O-Boc-protected gemcitabine with acyl halides gives 4-N-
alkanoylgemcitabine analogues with a hydroxyl, fluoro, chloro,
bromo, or alkene functional group on the alkyl chain.
Displacement of the p-toluenesulfonamido group from 4-N-
tosylgemcitabine with alkyl amines provided 4-N-alkylgemcita-
bine analogues suitable for further chemical modifications,
including fluorination compatible with synthetic protocols for
18F labeling. The 4-N-alkanoylgemcitabine analogues showed
potent antiproliferative activities against the L1210, CEM,
HeLa, and MCF-7 cell lines, with IC50 values in the low
nanomolar range, whereas the cytostatic activity of the 4-N-
alkylgemcitabine derivatives was in the low to modest
micromolar range. The 4-N-alkanoyl derivatives display
significant cytostatic activity, acting as efficient prodrugs,
whereas the 4-N-alkyl analogues appear to attain their modest
activity without measurable conversion to gemcitabine. The
cytostatic activity appears to be independent of the length of
the alkyl chain and varies slightly for the different functional
groups present on the molecule.
General Synthetic Procedure for Preparation of the 4-N-Acyl
Gemcitabine Derivatives (3−9). Procedure A. N-Methylmorpho-
line (1.1 equiv), 1-hydroxybenzotriazole (1.1 equiv), the appropriate
carboxylic acid (1.1 equiv), and 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (1.3 equiv) were sequentially added to a stirred solution
of gemcitabine hydrochloride (2a, 1.0 equiv) in DMF/DMSO (3:1, 2
mL) at ambient temperature under Argon. The reaction mixture was
then gradually heated to 65 °C (oil-bath) and was kept stirring
overnight. After the reaction was completed (TLC), the reaction
mixture was cooled to 15 °C and partitioned between a small amount
of brine and EtOAc. The organic phase was separated, and the
aqueous layer was extracted with fresh portions of EtOAc (3 × 30
mL). The combined organic layers were then sequentially washed with
20% LiCl/H2O, saturated NaHCO3/H2O, and brine, dried over
Na2SO4, and evaporated under reduced pressure to give crude
products 3−9.
4-N-(Undecanoyl)-2′-deoxy-2′,2′-difluorocytidine (3). Treat-
ment of 2a (34 mg, 0.11 mmol) with commercially available
undecanoic acid (23.3 mg, 0.120 mmol) by procedure A gave 45.7
mg of the crude product, which was then column chromatographed
1
(5% MeOH/EtOAc) to give 3 (23.8 mg, 50%) as a white solid. H
NMR (CD3OD) δ 0.90 (t, J = 6.9 Hz, 3H, CH3), 1.27−1.39 (m, 14H,
7× × CH2), 1.63−1.70 (m, 2H, CH2), 2.45 (t, J = 7.4 Hz, 2H, CH2),
3.79−3.83 (m, 1H, H5′), 3.94−3.99 (m, 2H, H5′, H4′), 4.31 (dt, J =
20.8, 10.5 Hz, 1H, H3′), 6.24−6.28 (m, 1H, H1′), 7.50 (d, J = 7.6 Hz,
1H, H5), 8.34 (d, J = 7.6 Hz, 1H, H6). 13C NMR (CD3OD) δ 14.41,
23.69, 25.93, 30.15, 30.40, 30.40, 30.56, 30.64, 33.03, 38.16, 60.29
(C5′), 70.21 (t, J = 23.1 Hz, C3′), 82.86 (d, J = 8.6 Hz, C4′), 86.44
(dd, J = 26.6, 38.3 Hz, C1′), 98.26 (C5′), 123.90 (t, J = 259.3 Hz,
C2′), 145.94 (C6), 157.65 (C2), 164.80 (C4), 175.97. 19F NMR
(CD3OD) δ −120.09 (br d, J = 240.9 Hz, 1F), −119.14 (dd, J = 11.3,
240.9 Hz, 1F). HRMS (ESI+) m/z calcd for C20H31F2N3NaO5 [M +
Na]+, 454.2124; found, 454.2136.
EXPERIMENTAL SECTION
■
The 1H (400 MHz), 13C (100 MHz), or 19F (376 MHz) NMR spectra
were recorded at ambient temperature in solutions of CDCl3, MeOH-
d4, or DMSO-d6, as noted. The reactions were followed by TLC with
Merck Kieselgel 60-F254 sheets, and products were detected with a 254
nm light or with Hanessian’s stain. Column chromatography was
performed using Merck Kieselgel 60 (230−400 mesh). Reagent-grade
chemicals were used, and solvents were dried by reflux distillation over
CaH2 under nitrogen gas unless otherwise specified, and reactions
were carried out under an Ar atmosphere. The carboxylic acid and
amine derivatives used for the coupling with gemcitabine were
4-N-(8-Nonenoyl)-2′-deoxy-2′,2′-difluorocytidine (4). Treat-
ment of 2a (34 mg, 0.110 mmol) with commercially available 8-
nonenoic acid (21 μL, 19.5 mg, 0.120 mmol) by procedure A gave
196
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Journal of Medicinal Chemistry
Article
29.0 mg of the crude product, which was then column chromato-
63.01, 70.23 (t, J = 23.0 Hz, C3′), 82.88 (d, J = 9.0 Hz, C4′), 86.47
(dd, J = 27.0, 37.6 Hz, C1′), 98.25 (C5), 123.91 (t, J = 258.9 Hz, C2′),
145.95 (C6), 157.67 (C2), 164.82 (C4), 176.00. 19F NMR (CD3OD)
δ −120.16 (br d, J = 239.0 Hz, 1F), −119.21 (dd, J = 10.5, 242.6 Hz,
1F). HRMS (ESI+) m/z calcd for C20H31F2N3NaO6 [M + Na]+,
470.2073; found, 470.2073.
graphed (70 → 100% EtOAc/hexane) to give 4 (20 mg, 45%) as a
1
white solid. H NMR (CD3OD) δ 1.32−1.46 (br s, 6H, 3 × CH2),
1.65−1.69 (m, 2H, CH2), 2.03−2.07 (m, 2H, CH2), 2.45 (t, J = 7.4
Hz, 2H, CH2), 3.81 (dd, J = 12.3, 2.8 Hz, 1H, H5′), 3.96−3.99 (m,
2H, H5″, H4′), 4.30 (td, J = 12.2, 8.6 Hz, 1H, H3′), 4.90−5.01 (m,
2H, CH2), 5.81 (ddt, J = 16.9, 10.0, 3.4 Hz, 1H, CH), 6.24−6.28 (m,
1H, H1′), 7.50 (d, J = 7.6 Hz, 1H, H5), 8.34 (d, J = 7.6 Hz, 1H, H6).
13C NMR (CD3OD) δ 25.90, 29.87, 29.90, 30.00, 34.79, 38.15, 60.31
(C5′), 70.23 (dd, J = 21.9, 23.4 Hz, C3′), 82.86 (C4′), 86.14 (d, J =
20.1 Hz, C1′), 98.28 (C5), 114.83, 123.94 (t, J = 259.2 Hz, C2′),
140.03, 145.97 (C6), 157.37 (C2), 164.84 (C4), 175.97. 19F NMR
(CD3OD) δ −120.13 (br d, J = 242.5 Hz, 1F), −119.21 (dd, J = 11.4,
240.0 Hz, 1F). HRMS (ESI+) m/z calcd for C18H25F2N3NaO5 [M +
Na]+, 424.1654; found, 424.1656.
4-N-(11-Fluoroundecanoyl)-2′-deoxy-2′,2′-difluorocytidine
(8). Treatment of 2a (69.8 mg, 0.233 mmol) with 11-fluoroundecanoic
acid (S4, 52 mg, 0.256 mmol) by procedure A gave 82.7 mg of the
crude product, which was then column chromatographed (70%
1
EtOAc/hexane) to give 8 (42.1 mg, 41%) as a white solid. H NMR
(CD3OD) δ 1.35 (br s, 12H, 6 × CH2), 1.62−1.74 (m, 4H, 2 × CH2),
2.47 (t, J = 7.4 Hz, 2H, CH2), 3.83 (dd, J = 3.0, 12.8 Hz, 1H, H5′),
3.96−4.02 (m, 2H, H5″, H4′), 4.32 (dt, J = 8.6, 12.2 Hz, 1H, H3′),
4.42 (dt, J = 6.1, 47.5 Hz, 2H, CH2), 6.28 (t, J = 7.2 Hz, 1H, H1′), 7.51
(d, J = 7.6 Hz, 1H, H5), 8.35 (d, J = 7.6 Hz, 1H, H6). 13C NMR
(CD3OD) δ 25.95, 26.35, 30.16, 30.38, 30.48, 30.59, 31.50, 31.69,
38.17, 60.29(C5′), 70.20 (t, J = 23.0 Hz, C3′), 82.85 (dd, J = 2.3, 3.6
Hz, C4′), 84.89 (d, J = 163.8 Hz, CH2F), 86.47 (dd, J = 29.6, 34.7 Hz,
C1), 98.29 (C5), 123.94 (t, J = 259.2 Hz, C2′), 145.96 (C6), 157.69
(C2), 164.83 (C4), 176.01. 19F NMR (CD3OD) δ −219.87 (tt, J =
24.7, 47.5 Hz, 1F), −120.09 (br d, J = 239.0 Hz, 1F), −119.17 (br dd, J
= 10.2, 239.0 Hz, 1F). MS (ESI) m/z 450 (100, [M + H]+). HRMS
(ESI+) m/z calcd for C20H30F3N3NaO5 [M + Na]+, 472.2023; found,
472.2011.
4-N-[11-(1H-Benzotriazol-1-yloxy)-undecanoyl]-2′-deoxy-
2′,2′-difluorocytidine (9). Treatment of 2a (50 mg, 0.167 mmol)
with commercially available 11-bromoundecanoic acid (48.7 mg, 0.184
mmol) by procedure A gave 85.5 mg of the crude product, which was
then column chromatographed (5% MeOH/EtOAc) to give 9 (50 mg,
53%) as a white solid. 1H NMR (DMSO-d6) δ 1.28 (br s, 10H, CH2),
1.45−1.57 (m, 4H, CH2), 1.73−1.80 (m, 2H, CH2), 2.40 (t, J = 7.3
Hz, 2H, CH2), 3.66 (br d, J = 13.6 Hz, 1H, H5′), 3.80 (br d, J = 13.6
Hz, 1H, H5″), 3.89 (dt, J = 2.7, 8.4 Hz, 1H, H4′), 4.20 (br dt, J = 9.1,
12.6 Hz, 1H, H3′), 4.55 (t, J = 6.5 Hz, 2H, CH2), 5.35 (br t, J = 4.6
Hz, 1H, OH), 6.17 (t, J = 7.5 Hz, 1H, H1′), 6.39 (br s, 1H, OH), 7.28
(d, J = 7.6 Hz, 1H, H5), 7.48 (t, J = 7.6 Hz, 1H, Ar), 7.64 (t, J = 7.6
Hz, 1H, Ar), 7.82 (d, J = 8.4 Hz, 1H, Ar), 8.07 (d, J = 8.4 Hz, 1H, Ar),
8.25 (d, J = 7.6 Hz, 1H, H6), 10.99 (br s, 1H, NH). 13C NMR
(CD3OD) δ 25.90, 26.64, 29.12, 30.06, 30.24, 30.28, 30.35, 30.40,
38.14, 58.32, 60.30, 70.23 (t, J = 23.1 Hz, C3′), 82.32, 82.89 (m, C4′),
98.25 (C5), 110.16, 120.50, 123.92, 126.38, 128.72, 129.55, 144.49,
145.95, 157.66, 164.81, 175.99. 19F NMR (CD3OD) δ −120.09 (br d,
J = 239.0 Hz, 1F), −119.14 (dd, J = 243.7, 12.3 Hz, 1F). HRMS
(ESI+) m/z calcd for C26H34F3N6NaO6 [M + Na]+, 587.2406; found,
587.2442.
4-N-(10-Undecenoyl)-2′-deoxy-2′,2′-difluorocytidine (5).
Treatment of 2a (40 mg, 0.134 mmol) with commercially available
undecylenic acid (31 μL, 28 mg, 0.148 mmol) by procedure A gave
114 mg of the crude product, which was then column chromato-
graphed (80 → 100% EtOAc/hexane) to give 5 (38 mg, 66%) as a
white solid. UV (CH3OH) λmax 252 nm (ε 15 150), 286 nm (ε 8950),
1
λmin 228 nm (ε 5900), 275 nm (ε 8650). H NMR (DMSO-d6) δ
1.23−1.29 (br s, 8H, 4 × CH2), 1.30−1.39 (m, 2H, CH2), 1.50−1.57
(m, 2H, CH2), 2.01 (q, J = 7.0 Hz, 2H, CH2), 2.40 (t, J = 7.3 Hz, 2H,
CH2), 3.66 (br d, J = 12.4 Hz, 1H, H5″), 3.81 (br d, J = 12.4 Hz, 1H,
H5′), 3.89 (dt, J = 8.5, 2.7 Hz, 1H, H4′), 4.19 (q, J = 10.6 Hz, 1H,
H3′), 4.93 (d quin, J = 10.1, 1.0 Hz, 1H, CH), 4.99 (d quin, J = 17.2,
1.7 Hz, 1H, CH), 5.33 (br t, J = 5.0 Hz, 1H, OH), 5.79 (tdd, J = 6.6,
10.3, 17.1 Hz, 1H, CH), 6.17 (t, J = 7.5 Hz, 1H, H1′), 6.35 (br s, 1H,
OH), 7.29 (d, J = 7.6 Hz, 1H, H5), 8.24 (d, J = 7.6 Hz, 1H, H6), 10.98
(br s, 1, NH). 13C NMR (CD3OD) δ 25.95, 30.08, 30.15 (2 × CH2),
30.37, 30.39, 34.88, 38.18, 60.32 (C5′), 70.24 (dd, J = 21.9, 23.4 Hz,
C3′), 82.89 (dd, J = 2.7, 5.2 Hz, C4′), 86.48 (dd, J = 25.8, 38.2 Hz,
C1′), 98.28 (C5), 114.73, 123.93 (t, J = 259.2 Hz, C2′), 140.13,
145.97 (C6), 157.69 (C2), 164.83 (C4), 176.00. 19F NMR (CD3OD)
δ −120.09 (br d, J = 239.6 Hz, 1F), −119.16 (dd, J = 10.9, 239.9 Hz,
1F). MS (ESI+) m/z 430 (100, [M + H]+). HRMS (ESI+) m/z calcd
for C20H29F2N3NaO5 [M + Na]+, 452.1967; found, 452.1982. Anal.
Calcd for C20H29F2N3O5·0.5H2O (438.47): C, 54.79; H, 6.90; N, 9.58.
Found: C, 54.48; H, 6.53; N, 9.21.
4-N-(12-Tridecenoyl)-2′-deoxy-2′,2′-difluorocytidine (6).
Treatment of 2a (30 mg, 0.1 mmol) with commercially available 12-
tridecenoic acid (23 mg, 0.11 mmol) by procedure A gave 43.1 mg of
the crude product, which was then column chromatographed (70 →
1
80% EtOAc/hexane) to give 6 (20.1 mg, 44%) as a white solid. H
NMR (CD3OD) δ 1.27−1.38 (m, 14H, 7 × CH2), 1.66 (quin, J = 6.9
Hz, 2H, CH2), 2.04 (dd, J = 14.3, 6.7 Hz, 2H, CH2), 2.45 (t, J = 7.4
Hz, 2H, CH2), 3.81 (dd, J = 12.4, 2.8 Hz, 1H, H5′), 4.07−3.88 (m,
2H, H5″, H4′), 4.31 (dt, J = 20.8, 10.4 Hz, 1H, H3′), 4.89−5.00 (m,
2H, CH2), 5.80 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H, CH), 6.26 (t, J = 7.2
Hz, 1H, H1′), 7.50 (d, J = 7.6 Hz, 1H, H5), 8.34 (d, J = 7.6 Hz, 1H,
H6). 13C NMR (CD3OD) δ 25.94, 30.11, 30.15, 30.20, 30.39, 30.53 (2
× CH2), 30.62, 34.87, 38.16, 60.30, 70.24 (t, J = 23.1 Hz, C3′), 82.83
(C4′), 86.46 (t, J = 32.2 Hz, C1′), 98.26 (C5), 114.67, 123.1 (t, J =
260.1 Hz, C2′), 140.14, 145.95 (C6), 157.68 (C2), 164.82 (C4),
176.0. 19F NMR (CD3OD) δ −120.13 (br d, J = 239.4 Hz, 1F),
−119.21 (dd, J = 9.3, 239.3 Hz, 1F). HRMS (ESI+) m/z calcd for
C22H33F2N3NaO5 [M + Na]+, 480.2280; found, 480.2289.
4-N-(11-Chloroundecanoyl)-2′-deoxy-2′,2′-difluorocytidine
(10). Method A. TMSCl (79 μL, 68 mg, 0.630 mmol) was added to a
suspension of 2a (150 mg, 0.500 mmol) in Pyr/MeCN (3:1, 2 mL) at
0 °C under Ar and stirred for 2.5 h, resulting in a clear solution. In a
separate vessel, carbonyldiimidazole (CDI, 22.5 mg, 0.138 mmol) was
added to a solution of 11-bromoundecanoic acid (36.5 mg, 0.138
mmol) in MeCN (1 mL) portionwise, and the mixure was stirred at
ambient temperature. After 30 min, the latter solution was combined
with the previously prepared solution of transiently protected
nucleoside, and the new reaction mixture was stirred at 65 °C
overnight. After 19 h, EtOH (2 mL) was added to the mixture
followed by H2O (4 mL), and the solution was stirred at 65 °C for 20
min. The volatiles were then evaporated under reduced pressure, the
residue was partitioned between EtOAc and H2O, the pH was adjusted
to 2.0 with phosphoric acid, and the aqueous layer was extracted with
EtOAc. The combined organic layer was washed with saturated
NaHCO3/H2O and brine, dried over Na2SO4, and evaporated under
reduced pressure, and the resulting residue (47.2 mg) was column
chromatographed (70% EtOAc/hexane) to give 10 (11 mg, 5%) as a
4-N-(11-Hydroxyundecanoyl)-2′-deoxy-2′,2′-difluorocyti-
dine (7). Treatment of 2a (58 mg, 0.194 mmol) with commercially
available 11-hydroxyundecanoic acid (43 mg, 0.213 mmol) by
procedure A gave 75.5 mg of the crude product, which was then
column chromatographed (7.5% MeOH/CHCl3) to give 7 (35 mg,
1
40%) as a white solid. H NMR (CD3OD) δ 1.33 (br s, 12H, 6 ×
CH2), 1.49−1.54 (m, 2H, CH2), 1.66 (quin, J = 7.2 Hz, 2H, CH2),
2.45 (t, J = 7.4 Hz, 2H, CH2), 3.53 (t, J = 6.6 Hz, 2H, CH2), 3.81 (dd,
J = 3.1, 12.8 Hz, 1H, H5′), 3.94−3.99 (m, 2H, H4′, H5′), 4.26−4.34
(m, 1H, H3′), 6.26 (t, J = 7.3 Hz, 1H, H1′), 7.49 (d, J = 7.6 Hz, 1H,
H5), 8.33 (d, J = 7.6 Hz, 1H, H6). 13C NMR (CD3OD) δ 25.93,
26.94, 30.13, 30.37, 30.48, 30.53, 30.64, 33.65, 38.17, 60.30 (C5′),
1
white solid. H NMR (CD3OD) δ 1.34 (br s, 10H, 2 × CH2), 1.41−
1.49 (m, 2H, CH2), 1.66−1.71 (m, 2H, CH2), 1.73−1.82 (m, 2H,
CH2), 2.47 (t, J = 7.5 Hz, 2H, CH2), 3.56 (t, J = 6.7 Hz, 2H, CH2),
3.83 (dd, J = 12.7, 3.1 Hz, 1H, H5′), 3.96−4.03 (m, 2H, H5″, H4′),
4.27−4.37 (m, 1H, H3′), 6.28 (t, J = 7.2 Hz, 1H, H1′), 7.51 (d, J = 7.6
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Hz, 1H, H5), 8.36 (d, J = 7.6 Hz, 1H, H6). 13C NMR (CD3OD) δ
25.94, 27.93, 29.94, 30.13, 30.35, 30.43, 30.51, 33.83, 38.15, 45.74,
60.31 (C5′), 70.25 (C3′), 82.89 (C4′), 86.81 (C1′), 98.26 (C5),
123.93 (t, J = 258.0 Hz, C2′), 145.97 (C6), 157.71 (C2), 164.86 (C4),
176.02 (CO). 19F NMR (CD3OD) δ −120.13 (br d, J = 240.2 Hz, 1F),
−119.2 (br dd, J = 10.9, 240.2 Hz, 1F). MS (ESI+) m/z 466 (100, [M
+ H]+ for 35Cl), 468 (100, [M + H]+ for 37Cl). HRMS (ESI+) m/z
calcd for C20H3035ClF2N3NaO5 [M + Na]+, 488.1734; found,
488.1742.
Method B. Et3N (28 μL, 0.200 mmol) was added to a mixture of
11-bromoundecanoic acid (26.6 mg, 0.100 mmol) in THF (1 mL),
and the mixture was stirred at ambient temperature under Ar. The
reaction mixture was then cooled to −15 °C followed by the dropwise
addition of a solution of ClCO2Et (19 μL, 0.200 mmol) in THF (0.5
mL) with continued stirring. After 15 min, a solution of 2a (30 mg,
0.100 mmol) in DMF/DMSO (2.5 mL, 1.5:1) was added dropwise,
and the reaction mixture was allowed to warm to ambient temperature
and was kept stirring overnight. After 24 h, the reaction was treated
with NaHCO3 and extracted with EtOAc (3×). The combined organic
layer was washed with brine, dried over Na2SO4, and evaporated under
reduced pressure, and the residue was column chromatographed (70%
EtOAc/hexane) to give 10 (7 mg, 15%) with data as above.
4-N-(11-Bromoundecanoyl)-3′,5′-di-O-(tert-butoxycarbon-
yl)-2′-deoxy-2′,2′-difluorocytidine (11). A solution of 2b46 (35.5
mg, 0.077 mmol) and NaHCO3 (400 mg, 4.76 mmol) in CH2Cl2 (0.5
mL) was added to a stirred solution of 11-bromoundecanoyl chloride
(S5, 0.1 mL, 122 mg, 0.43 mmol) in CH2Cl2 (1 mL) at 0 °C under Ar.
After 15 min, the reaction mixture was allowed to warm to ambient
temperature and was kept stirring for 3 h. The reaction mixture was
quenched by addition of saturated NaHCO3/H2O, the mixture was
partitioned with water, and the aqueous layer was extracted with
CH2Cl2 (2 × 10 mL). The combined organic layer was washed with
brine, dried over Na2SO4, and evaporated under reduced pressure, and
the resulting residue (141.0 mg) was chromatographed (25% EtOAc/
hexane) to give 11 (18 mg, 33%) as a colorless oil. 1H NMR (CDCl3)
δ 1.30 (br s, 10H, 5 × CH2), 1.40−1.45 (m, 2H, CH2), 1.53 (s, 18H, 6
× CH3), 1.68 (quin, J = 7.3 Hz, 2H, CH2), 1.86 (quin, J = 7.3 Hz, 2H,
CH2), 2.48 (t, J = 7.5 Hz, 2H, CH2), 3.42 (t, J = 6.9 Hz, 2H, CH2),
4.37−4.50 (m, 3H, H4′, H5′,5″), 5.14 (dt, J = 4.5, 11.2 Hz, 1H, H3′),
6.46 (dd, J = 7.3, 9.5 Hz, 1H, H1′), 7.51 (d, J = 7.6 Hz, 1H, H5), 7.85
(d, J = 7.6 Hz, 1H, H6), 9.05 (br s, 1H, NH). 13C NMR (CDCl3) δ
24.77, 27.54, 27.70, 28.14, 28.72, 28.96, 29.22, 29.26, 29.33, 32.82,
34.07, 37.58, 63.87 (C5′), 72.64 (dd, J = 17.2, 34.0 Hz, C3′), 77.79
(C4′), 83.37, 84.21 (m, C1′), 84.83, 97.02 (C5), 120.40 (dd, J = 260.7,
267.3 Hz, C2′), 145.27 (C6), 151.42, 152.91, 153.94 (C2), 163.40
(C4), 174.17. 19F NMR (CDCl3) δ −120.00 (br d, J = 246.9 Hz, 1F),
−115.57 (dt, J = 11.4, 246.9 Hz, 1F). MS (ESI+) m/z 710 (100, [M +
H]+ for 79Br), 712 (100, [M + H]+ for 81Br).
4-N-(11-Hydroxyundecanoyl)-3′,5′-di-O-(tert-butoxycarbon-
yl)-2′-deoxy-2′,2′-difluorocytidine (13). Treatment of 2b (39 mg,
0.084 mmol) with 11-hydroxyundecanoic acid (29 mg, 0.14 mmol) by
procedure A gave 102 mg of the crude product, which was then
column chromatographed (55 → 65% EtOAc/hexane) to give 13 (20
mg, 37%) as a colorless oil. 1H NMR (CDCl3) δ 1.30 (br s, 12H, 6 ×
CH2), 1.53 (s, 18H, 6 × CH3), 1.58 (quin, J = 6.9 Hz, 2H, CH2), 1.69
(quin, J = 7.4 Hz, 2H, CH2), 2.47 (t, J = 7.5 Hz, 2H, CH2), 3.65 (t, J =
6.6 Hz, 2H, CH2), 4.37−4.50 (m, 3H, H4′, H5′,5″), 5.14 (″dt, J = 4.8,
11.1 Hz, 1H, H3′), 6.46 (dd, J = 7.2, 9.5 Hz, 1H, H1′), 7.51 (d, J = 7.6
Hz, 1H, H5), 7.85 (d, J = 7.6 Hz, 1H, H6), 9.08 (br s, 1H, NH). 13C
NMR (CDCl3) δ 24.75, 25.65, 27.53, 27.69, 28.88, 29.11, 29.16, 29.27,
29.38, 32.75, 37.78, 62.99, 63.88 (C5′), 72.67 (dd, J = 17.0, 33.8 Hz,
C3′), 77.73 (C4′), 83.33, 84.16 (dd, J = 18.2, 37.9 Hz, C1′), 84.77,
97.08 (C5), 120.42 (t, J = 263.8 Hz, C2′), 144.78 (C6), 151.44,
152.93, 154.67 (C2), 162.93 (C4), 173.46. 19F NMR (CDCl3) δ
−120.00 (br d, J = 246.7 Hz, 1F), −115.58 (dt, J = 11.4, 246.7 Hz,
1F); MS (ESI+) m/z 648 (100, [M + H]+).
Treatment of 13 (4.0 mg, 0.008 mmol) with TFA as descibed for 12
gave 7 (3.1 mg, 87%) with data as reported above.
4-N-(11-Fluoroundecanoyl)-3′,5′-di-O-(tert-butoxycarbon-
yl)-2′-deoxy-2′,2′-difluorocytidine (14). A chilled (−78 °C)
solution of DAST (6.2 μL, 7.6 mg, 0.048 mmol) in CH2Cl2 (500
μL) was added to a stirred solution of 13 (9.8 mg, 0.016 mmol) in
CH2Cl2 (1.5 mL) at −78 °C. After 30 min, the reaction mixture was
allowed to warm to ambient temperature and was kept stirring. After 2
h, the reaction mixture was then poured into a separatory funnel
containing a chilled solution of NaHCO3/H2O (10 mL, pH 8) and
was then extracted with CHCl3 (3 × 10 mL). The combined organic
layer was washed with brine, dried over MgSO4, and evaporated under
reduced pressure, and the resulting residue (14 mg) was column
chromatographed (5% MeOH/CHCl3) to give 14 (4.2 mg, 40%) as a
colorless oil. 1H NMR (CDCl3) δ 1.28 (br s, 12H, 6 × CH2), 1.51 (s,
9H, t-Bu), 1.52 (s, 9H, t-Bu), 1.60−1.78 (m, 4H, 2 × CH2), 2.45 (t, J =
7.4 Hz, 2H, CH2), 4.38−4.47 (m, 3H, H4′, H5′, H5″), 4.44 (dt, J =
6.2, 47.3 Hz, 2H, CH2), 5.12−5.15 (m, 1H, H3′), 6.43 (t, J = 7.3 Hz,
1H, H1′), 7.51−7.54 (m, 1H, H5), 7.87 (d, J = 7.0 Hz, 1H, H6). 19F
NMR (CDCl3) δ −217.97 (dt, J = 25.0, 47.4 Hz, 2F), −120.27 (br d, J
= 240.7 Hz, 1F), −115.77 (dt, J = 10.9, 247.4 Hz, 1F). HRMS (ESI+)
m/z calcd for C30H46F3N3NaO9 [M + Na]+, 672.3078; found,
672.3096.
Treatment of 14 (4.0 mg, 0.008 mmol) with TFA as descibed for 12
gave 8 (2.9 mg, 82%) with data as reported above.
4-N-(10-Fluoroundecanoyl)-2′-deoxy-2′,2′-difluorocytidine
(15). Chilled hydrogen fluoride/pyridine (70%, 1.0 mL) was added to
5 (20 mg, 0.044 mmol) in an HDPE vessel at 0 °C and stirred. After 2
h, the reaction mixture was treated with saturated NaHCO3/H2O (10
mL) and extracted with EtOAc (3 × 10 mL). The combined organic
layer was washed with brine, dried over Na2SO4, and evaporated under
reduced pressure, and the resulting residue (24.6 mg) was then
column chromatographed (70% EtOAc/hexane) to give 15 (19 mg,
91%; isomeric mixture of 15/16/17 in a 75:20:5 ratio) as a white solid.
UV (CH3OH) λmax 250 nm (ε 13 250), 298 nm (ε 5350), λmin 226 nm
(ε 4650), 279 nm (ε 4700). Major isomer 15 had: 1H NMR (DMSO-
d6) δ 1.22 (br d, J = 6.1 Hz, 2H, CH2), 1.27 (br s, 8H, 4 × CH2), 1.29
(br s, 2H, CH2), 1.44−1.62 (m, 5H, CH2, CH3), 2.40 (t, J = 7.3 Hz,
2H, CH2), 3.66 (dt, J = 12.5, 4.3 Hz, 1H, H5″), 3.81 (br d, J = 12.0 Hz,
1H, H5′), 3.89 (br d, J = 8.5 Hz, 1H, H4′), 4.19 (sep, J = 6.4 Hz, 1H,
H3′), 4.64 (dsex, J = 49.0, 6.0 Hz, 1H, CH), 5.31 (t, J = 5.1 Hz, 1H,
OH), 6.17 (t, J = 7.5 Hz, 1H, H1′), 6.33 (d, J = 5.8 Hz, 1H, OH), 7.29
(d, J = 7.6 Hz, 1H, H5), 8.24 (d, J = 7.6 Hz, 1H, H6), 10.98 (s, 1H,
NH). 13C NMR (DMSO-d6) δ 24.30, 24.42, 24.46, 28.37, 28.57, 28.71,
28.74, 36.13, 36.35, 58.78 (C5′), 68.37 (t, J = 22.5 Hz, C3′), 81.01 (t, J
= 3.9 Hz, C4′), 84.50 (d, J = 82.2 Hz, C1′), 90.53 (d, J = 162.9 Hz),
95.87 (C5), 124.18 (d, J = 260.1 Hz, C2′), 144.68 (C6), 154.17 (C2),
162.85 (C4), 174.06. 19F NMR (DMSO-d6) δ −170.27 (symmetric m,
0.75F), δ −116.91 (br s, 2F). MS (ESI+) m/z 450 (100, [M + H]+).
HRMS (ESI+) m/z calcd for C20H30F3N3NaO5 [M + Na]+, 472.2030;
found, 472.2048. Anal. Calcd for C20H30F3N3O4·H2O·0.33CH3CN
4-N-(11-Bromoundecanoyl)-2′-deoxy-2′,2′-difluorocytidine
(12). Compound 11 (32 mg, 0.045 mmol) was dissolved in TFA (1.0
mL), and the mixture was stirred at 20 °C. After 4 h, the reaction
mixture was diluted with toluene, the volatiles were evaporated, and
the residue was coevaporated with a fresh portion of toluene. The
resulting residue (32 mg) was column chromatographed (80 → 100%
1
EtOAc/hexane) to give 12 (19.9 mg, 86%) as a colorless solid. H
NMR (CD3OD) δ 1.31−1.41 (m, 10H, 5 × CH2), 1.41−1.52 (m, 2H,
CH2), 1.63−1.73 (m, 2H, CH2), 1.81−1.89 (m, 2H, CH2), 2.47 (t, J =
7.4 Hz, 2H, CH2), 3.45 (t, J = 6.7 Hz, 2H, CH2), 3.75−3.89 (m, 1H,
H5″), 3.93−4.05 (m, 2H, H4′, H5″), 4.32 (dt, J = 8.5, 12.2 Hz, 1H,
H3′), 6.28 (t, J = 7.3 Hz, 1H, H1′), 7.51 (d, J = 7.6 Hz, 1H, H5), 8.35
(d, J = 7.6 Hz, 1H, H6). 13C NMR (CD3OD) δ 25.94, 29.17, 29.80,
30.13, 30.34, 30.43, 30.48, 34.01, 34.42, 38.17, 60.32 (C5′), 70.25 (dd,
J = 22.2, 23.6 Hz, C3′), 82.88 (d, J = 8.6 Hz, C4′), 86.48 (dd, J = 26.6,
37.6 Hz, C1), 98.29 (C5), 123.93 (t, J = 259.9 Hz, C2′), 145.98 (C6),
157.69 (C2), 164.84 (C4), 176.03. 19F NMR (CD3OD) δ −120.10 (br
d, J = 240.0 Hz, 1F), −119.17 (ddd, J = 3.9, 12.1, 240.0 Hz, 1F). MS
(ESI+) m/z 510 (100, [M + H]+ for 79Br), 512 (100, [M + H]+ for
81Br). HRMS (ESI+) m/z calcd for C20H3079BrF2N3NaO5 [M + Na]+,
532.1229; found, 532.1239.
198
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Journal of Medicinal Chemistry
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(481.03): C, 51.59; H, 6.91; N, 9.70. Found: C, 51.36; H, 6.89; N,
9.97.
0.018 mmol) was dissolved in TFA (1.0 mL), and the reaction mixture
was stirred at 18 °C. After 5 h, the reaction mixture was diluted with
toluene (2 mL), the volatiles were evaporated, and the residue was
coevaporated with a toluene (2 × 1 mL). The resulting residue (17
mg) was then column chromatographed (1% MeOH/EtOAc) to give
21 (2.2 mg, 29% from 20; 38% overall from 18) as a colorless oil. 1H
NMR (CD3OD) δ 1.30−1.41 (m, 14H, 7 × CH2), 1.50−1.57 (m, 2H,
CH2), 1.58−1.64 (m, 2H, CH2), 3.39 (t, J = 7.1 Hz, 2H, CH2), 3.55 (t,
J = 6.6 Hz, 2H, CH2), 3.80 (dd, J = 3.3, 12.6 Hz, 1H, H5′), 3.89 (td, J
= 2.8, 8.3 Hz, 1H, H4′), 3.95 (br dd, J = 2.0, 12.6, 1H, H5″), 4.26 (dt, J
= 8.3, 12.1 Hz, 1H, H3′), 5.87 (d, J = 7.6 Hz, 1H, H5), 6.23 (t, J = 8.0
Hz, 1H, H1′), 7.74 (d, J = 7.6 Hz, 1H, H6). 13C NMR (CD3OD) δ
26.94, 28.01, 29.97, 30.42, 30.58, 30.63, 30.66, 30.71, 33.67, 41.74,
60.56 (C5′), 63.03, 70.63 (dd, J = 22.0, 24.8 Hz, C3′), 82.23 (dd, J =
3.8, 5.0 Hz, C4′), 85.82 (C1), 97.32 (C5), 124.04 (t, J = 259.8 Hz,
C2′), 140.77 (C6), 158.29 (C2), 165.37 (C4). 19F NMR (CD3OD) δ
−119.90 (br d, J = 239.2 Hz, 1F), −118.83 (dd, J = 11.6, 239.2 Hz,
1F). MS (ESI+) m/z 434 (100, [M + H]+). HRMS (ESI+) m/z calcd
for C20H33F2N3NaO5 [M + Na]+, 456.2280; found, 456.2287.
4-N-(p-Toluenosulfonyl)-2′-deoxy-2′,2′-difluorocytidine
(22). TMSCl (5.1 mL) was added to a suspension of 2a (600 mg, 2.0
mmol) in anhydrous pyridine (10 mL), and the mixture was stirred at
ambient temperature under Ar. After 2 h, TsCl (3.8 g, 20.027 mmol)
was added, and the reaction mixture was gradually heated to 60 °C
(oil-bath) and kept stirring. After 20 h, volatiles were evaporated under
reduced pressure, and the resulting residue was treated with MeOH/
NH3 (10 mL) and stirred at ambient temperature overnight. After 24
h, volatiles were evaporated under reduced pressure, and the resulting
residue was column chromatographed (90% EtOAc/hexane) to give
22 (808 mg, 96%) as a white solid. 1H NMR (CD3OD) δ 2.42 (s, 3H,
CH3), 3.78 (dd, J = 3.4, 12.8 Hz, 1H, H5′), 3.90−3.95 (m, 2H, H4′,
H5″), 4.28 (dt, J = 8.4, 12.0 Hz, 1H, H3′), 6.13 (dd, J = 5.3, 9.5 Hz,
1H, H1′), 6.65 (d, J = 8.2 Hz, 1H, H5), 7.36 (d, J = 8.0 Hz, 2H, Ar),
7.79 (d, J = 8.3 Hz, 2H, Ar), 7.99 (d, J = 8.1 Hz, 1H, H6). 13C NMR
(CD3OD) δ 21.43, 60.34 (C5′), 70.21 (dd, J = 18.8, 27.2 Hz, C3′),
82.99 (d, J = 8.4, C4′), 85.46 (dd, J = 23.9, 41.3 Hz, C1′), 98.46 (C5),
123.84 (t, J = 258.7 Hz, C2′), 127.58 (Ar), 130.52 (Ar), 140.71 (Ar),
142.62 (C6), 144.66 (Ar), 150.21 (C2), 160.54 (C4). 19F NMR
(CD3OD) δ −120.17 (br s, 1F), −119.41 (dd, J = 4.1, 12.7 Hz, 1F).
MS (ESI+) m/z 440 (100, [M + Na]+). HRMS (ESI+) m/z calcd for
C16H17F2N3NaO6S [M + Na]+, 440.0698; found, 440.0711.
4-N-(11-Benzyloxyundecanyl)-2′-deoxy-2′,2′-difluorocyti-
dine (23). In a tightly sealed container, a solution of 22 (158 mg,
0.383 mmol), 11-(benzyloxy)undecanyl amine (S11; 945 mg, 3.41
mmol), and TEA (2 mL) in 1,4-dioxane was stirred at 75 °C. After 96
h, the volatiles were evaporated under reduced pressure, and the
resulting residue was column chromatographed (1% MeOH/EtOAc)
to give 23 (122 mg, 61%). 1H NMR (CD3OD) δ 1.28 (br s, 10H, 5 ×
CH2), 1.32 (br s, 4H, 2 × CH2), 1.54−1.61 (m, 4H, 2 × CH2), 3.36 (t,
J = 7.1 Hz, 2H, CH2), 3.47 (t, J = 6.6 Hz, 2H, CH2), 3.79 (dd, J = 3.3,
12.6, 1H, H5′), 3.88−3.96 (m, 2H, H4′, H5″), 4.25 (dt, J = 8.3, 12.0
Hz, 1H, H3′), 4.48 (s, 2H, CH2), 5.89 (d, J = 7.6 Hz, 1H, H5), 6.21 (t,
J = 8.0 Hz, 1H, H1’), 7.32 (br s, 5H, Ar), 7.70 (d, J = 7.6 Hz, 1H, H6).
13C NMR (CD3OD) δ 27.23, 28.00, 29.96, 30.42, 30.48, 30.60, 30.63,
30.65, 30.71, 41.74, 60.54, 68.14 (C3′), 71.44, 73.86, 82.24 (t, J = 2.95
Hz, C4′), 85.92 (dd, J = 26.7, 37.7 Hz, C1′), 97.31 (C5), 124.04 (t, J =
258.7 Hz, C2′), 128.62, 128.84, 129.35, 139.87, 140.75, 158.27,
165.34. 19F NMR (CD3OD) δ −119.47 (br d, J = 236.7 Hz, 1F),
−118.42 (dd, J = 8.6, 236.7 Hz, 1F). HRMS (ESI+) m/z calcd for
C27H39F2N3NaO5 [M + Na]+, 546.2750; found, 546.2774.
Minor isomers 16 [4-N-(9-fluoroundecanoyl)] and 17 [4-N-(8-
1
fluoroundecanoyl)] had the following distinguishable peaks. H NMR
(DMSO-d6) δ 4.41 (d quin, J = 49.6, 5.8 Hz, 0.25, CHF). 19F NMR
(DMSO-d6) δ −179.79 (symmetric m, 0.20F), −178.83 (m, 0.05F),
−116.91 (br s, 2F).
4-N-(p-Toluenosulfonyl)-3′,5′-di-O-(tert-butoxycarbonyl)-2′-
deoxy-2′,2′-difluorocytidine (18). Et3N (1.45 mL, 10.5 mmol) and
TsCl (997 mg, 5.2 mmol) were added to a solution of 2b (242 mg,
0.52 mmol) in dry 1,4-dioxane (4.0 mL) and stirred at ambient
temperature under Ar. The tightly sealed reaction mixture was then
gradually heated to 65 °C and kept stirring. After 24 h, the reaction
mixture was diluted with EtOAc and partitioned with a saturated
NaHCO3/H2O solution, and the aqueous layer was then extracted
with EtOAc (2×). The combined organic layer was washed with brine,
dried over Na2SO4, and evaporated under reduced pressure, and the
resulting residue (403 mg) was then column chromatographed (35%
EtOAc/hexane) to give 18 (146 mg, 45%) as a colorless, solidifying
oil. 1H NMR δ 1.49 (s, 9H, 3 × CH3), 1.52 (s, 9H, 3 × CH3), 2.43 (s,
3H, CH3), 4.46−4.32 (m, 3H, H4′, H5′, 5″), 5.11 (ddd, J = 4.0, 5.3,
12.8 Hz, 1H, H3′), 5.80 (br s, 1H, H5), 6.24 (dd, J = 6.6, 10.6 Hz, 1H,
H1′), 7.31 (d, J = 8.1 Hz, 2H, Ar), 7.48 (dd, J = 1.9, 8.1, Hz, 1H, H6),
7.84 (d, J = 8.3 Hz, 2H, Ar), 10.96 (br s, 1H, NH). 13C NMR δ 21.54,
27.51, 27.65, 63.80 (C5′), 72.40 (dd, J = 16.9, 33.8 Hz, C3′), 78.02
(dd, J = 2.2, 4.7 Hz, C4′), 83.31 (dd, J = 20.6, 38.7 Hz, C1′), 83.41,
84.99, 98.41 (C5), 120.38 (dd, J = 260.2, 266.5 Hz, C2′), 126.71 (Ar),
129.58 (Ar), 138.26 (d, J = 3.4 Hz, Ar), 139.88 (d, J = 2.2 Hz, C6),
143.74 (Ar), 147.16 (C2), 151.35, 152.82, 154.82 (C4). 19F NMR δ
−120.59 (br d, J = 247.6 Hz, 1F), −115.80 (br d, J = 247.6 Hz, 1F).
MS (ESI+) m/z 618 (100, [M + H]+). HRMS (ESI+) m/z calcd for
C26H33F2N3NaO10S [M + Na]+, 640.1747; found, 640.1754.
4-N-(10-Undecenyl)-2′-deoxy-2′,2′-difluorocytidine (19). In a
tightly sealed vessel, a mixture of 18 (40 mg, 0.065 mmol) and 1-
amino-10-undecene (0.50 mL, 404 mg, 2.4 mmol) was stirred at 60
°C. After 30 h, the volatiles were evaporated, and the resulting residue
was column chromatographed (8% MeOH/EtOAc) to give 19 (9.5
mg, 36%) as colorless viscous oil. UV (CH3OH) λmax 268 nm (ε 11
1
600), λmin 228 nm (ε 7800). H NMR (CD3OD) δ 1.43−1.30 (m,
12H, 6 × CH2), 1.65−1.56 (m, 2H, CH2), 2.03−2.09 (m, 2H, CH2),
3.39 (t, J = 7.1 Hz, 2H, CH2), 3.80 (dd, J = 3.3, 12.6 Hz, 1H, H5′),
3.89 (td, J = 2.8, 8.3 Hz, 1H, H4′), 3.95 (d, J = 12.6 Hz, 1H, H5″),
4.26 (dt, J = 8.3, 12.1 Hz, 1H, H3′), 4.91−5.02 (m, 2H, CH2), 5.82
(tdd, J = 6.7, 10.3, 17.0 Hz, 1H, CH), 5.87 (d, J = 7.6 Hz, 1H, H5),
6.23 (t, J = 8.0 Hz, 1H, H1′), 7.74 (d, J = 7.6 Hz, 1H, H6). 13C NMR
(CD3OD) δ 28.01, 29.98, 30.12, 30.19, 30.42, 30.51, 30.63, 34.88,
41.75, 60.56 (C5′), 70.67 (dd, J = 22.4, 23.8 Hz, C3′), 82.26 (dd, J =
3.6, 5.0 Hz, C4′), 85.94 (dd, J = 26.0, 38.0 Hz, C1), 97.33 (C5),
114.68, 124.05 (t, J = 258.4 Hz, C2′), 140.16, 140.77 (C6), 158.30
(C2), 165.37 (C4). 19F NMR (CD3OD) δ −119.89 (br d, J = 240.1
Hz, 1F), −118.80 (br d, J = 240.1 Hz, 1F). MS (ESI+) m/z 416 (100,
[M + H]+). HRMS (ESI+) m/z calcd for C20H31F2N3NaO4 [M +
Na]+, 438.2175; found, 438.2178. Anal. Calcd for C20H31F2N3O4·
0.5H2O·0.5CH3CN (445.01): C, 56.68; H, 7.59; N, 11.02. Found: C,
56.93; H, 7.77; N, 10.76.
4-N-(11-Hydroxyundecanyl)-2′-deoxy-2′,2′-difluorocytidine
(21). 11-Amino-1-undecanol (S7; 88 mg, 0.47 mmol) and Et3N (0.5
mL) were added to a solution of 18 (23.2 mg, 0.038 mmol) in 1,4-
dioxane (0.5 mL) and stirred at ambient temperature under Ar. The
reaction mixture was then gradually heated to 65 °C (oil bath) and
kept stirring overnight. After 40 h, the volatiles were evaporated, and
the residue (97 mg) was column chromatographed (1 → 3% MeOH/
4-N-(11-Benzyloxyundecanyl)-3′,5′-di-O-benzoyl-2′-deoxy-
2′,2′-difluorocytidine (24). BzCl (50 μL, 0.49 mmol) was added to
a solution of 23 (117 mg, 0.22 mmol), 2,6-lutidine (64 μL, 0.89
mmol), and 4-dimethylaminopyridine (27 mg, 0.22 mmol) in CH2Cl2
(10 mL), and the mixture was stirred at 35 °C under Argon. After 20
h, the reaction mixture was diluted with CH2Cl2 (40 mL) and
partitioned with H2O, and the aqueous layer was extracted with
CH2Cl2 (2 × 20 mL). The combined organic layers were sequentially
washed with 1 M HCl (20 mL), saturated NaHCO3/H2O (20 mL),
and brine (20 mL), dried over Na2SO4, and evaporated under reduced
1
EtOAc) to give monoprotected product 20 (9.5 mg, 47%). H NMR
(CD3OD) δ 1.32 (br s, 12H, 6 × CH2), 1.49 (s, 9H, t-Bu), 1.49−1.61
(m, 4H, 2 × CH2), 3.37 (t, J = 7.1 Hz, 2H, CH2), 3.49−3.62 (m, 2H,
CH2), 4.17 (dt, J = 9.9, 19.4 Hz, 1H, H4′), 4.02−4.09 (m, 1H, H3′),
4.48 (dd, J = 2.6, 12.4 Hz, 1H, H5′), 4.33 (dd, J = 4.3, 12.4 Hz, 1H,
H5″), 5.86 (d, J = 7.6 Hz, 2H, H5), 6.25 (t, J = 8.2 Hz, 2H, H1′), 7.51
(d, J = 7.6 Hz, 2H, C6). MS (ESI+) m/z 534 (100, [M + H]+)]
followed by 21 (4 mg, 24%) of 90% purity. Compound 20 (9.5 mg,
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pressure, and the resulting residue (157 mg) was column chromato-
24.9, 47.3 Hz, 1F), −120.62 (br d, J = 203.1, 1F), −115.40 (dt, J =
14.1, 247.3 Hz, 1F). MS (ESI+) m/z 644 (100, [M + H]+). HRMS
(ESI-TOF+) m/z calcd for C34H40F3N3NaO6 [M + Na]+, 666.2761;
found, 666.2763.
graphed (1% MeOH/CHCl3) to give 24 (50.6 mg, 60%) as a mixture
1
of rotamers (80:20). The major rotamer had the following peaks. H
NMR (CD3OD) δ 1.24 (br s, 12H, 6 × CH2), 1.51−1.62 (m, 4H, 2 ×
CH2), 3.20 (t, J = 7.1 Hz, 2H, CH2), 3.39 (t, J = 12.3 Hz, 2H, CH2),
4.48 (s, 2H, CH2), 4.49−4.53 (m, 1H, H5′), 4.63−4.67 (m, 1H, H5′),
4.73−4.79 (m, 1H, H4′), 5.54 (d, J = 7.6 Hz, 1H, H5), 5.57−5.61 (m,
1H, H3′), 6.60−6.65 (m, 1H, H1′), 7.26−7.33 (m, 5H, Ar), 7.41−7.49
(m, 4H, Ar), 7.55−7.64 (m, 2H, Ar), 8.02−8.08 (m, 4H, Ar). 19F
NMR (CD3OD) δ −120.48 (br d, J = 246.7 Hz, 1F), −115.34 (dt, J =
13.6, 246.7 Hz, 1F). MS (ESI+) m/z 754 (100, [M + Na]+). HRMS
(ESI+) m/z calcd for C41H47F2N3NaO7 [M + Na]+, 754.3274; found,
754.3303.
The minor rotamer had the following distinguishable peaks. 1H
NMR (400 MHz, CDCl3) δ 3.20−3.21 (m, 2H, CH2N), 5.71 (d, J =
7.8 Hz, 1H, H5). 19F NMR δ −115.98 (dt, J = 12.9, 247.5 Hz, 1F)
4-N-(11-Fluoroundecanyl)-2′-deoxy-2′,2′-difluorocytidine
(27). Method A. Compound 26 (10.6 mg, 0.017 mmol) was dissolved
in methanolic ammonia (2 mL) and stirred at ambient temperature.
After 2 h, volatiles were evaporated under reduced pressure, and the
resulting residue was chromatographed (5% MeOH/EtOAc) to give
27 (6.5 mg, 90%) as a clear oil. 1H NMR (CD3OD) δ 1.32 (brs, 14H,
7 × CH2), 1.55−1.73 (m, 4H, 2 × CH2), 3.37 (t, J = 7.1 Hz, 2H,
CH2), 3.78 (dd, J = 3.3, 12.6 Hz, 1H, H5′), 3,87 (dt, J = 3.0, 8.28 Hz,
1H, H3′), 3.93 (d, J = 13.3 Hz, 1H, H5″), 4.20−4.28 (m, 1H, H4′),
4.40 (dt, J = 6.1, 47.6 Hz, 1H, CH2), 5.85 (d, J = 7.6 Hz, 1H, H5), 6.21
(t, J = 7.96 Hz, H1′), 7.73 (d, J = 7.6 Hz, 1H, H6). 19F NMR δ
−219.94 (tt, J = 25.5, 47.3 Hz, 1F, CH2F), −119.60 (br s, 1F),
−119.14 (br s, 1F). MS (ESI) m/z 436 (100, [M + H]+). HRMS
(ESI+) m/z calcd for C20H32F3N3NaO4 [M + Na]+, 458.2237; found,
458.2256.
The minor rotamer had the following distinguishable peaks. 1H
NMR (CD3OD): δ 5.70 (d, J = 7.8, 1H, H5). 19F NMR (CD3OD): δ
−115.82 (dt, J = 13.4, 246.7, 1F).
4-N-(11-Hydroxyundecanyl)-3′,5′-di-O-benzoyl-2′-deoxy-
2′,2′-difluorocytidine (25). Ammonium cerium(IV) nitrate (63 mg,
0.115 mmol) was added to a solution of 24 (106 mg, 0.145 mmol) in
CH3CN/H2O (9:1, 5 mL), and the mixture was stirred at ambient
temperature overnight. Additional portions of CAN (240 mg) were
added to the reaction mixture every 24 h until no starting material
could be detected by TLC. After 72 h, the reaction was quenched by
the addition of saturated NaHSO3 (20 mL), the volatiles were
evaporated under reduced pressure, and the resulting aqueous residue
was extracted with EtOAc (3 × 20 mL). The combined organic layers
were washed with brine (20 mL), dried over Na2SO4, and evaporated
under reduced pressure, and the resulting residue (103.5 mg) was then
column chromatographed (1% MeOH/EtOAc) to give 25 (66 mg,
70%) as a mixture of rotamers (72:28). The major rotamer had the
Method B. In a tightly sealed cylindrical pressure vessel with screw
cap, a solution of KF (1.6 mg, 0.028 mmol), K2CO3 (3.8 mg, 0.028
mmol), Kryptofix 2.2.2 (10.5 mg, 0.028 mmol), and 28 (5.0 mg, 0.007
mmol) in CH3CN (1 mL) was stirred at 110 °C. After 18 min, the
reaction mixture was quickly cooled in a water bath and vacuum
filtered into another pressure vessel. The effluent containing crude 26
was concentrated under reduced pressure, and the resulting residue
was treated with 0.5 CH3ONa/MeOH (1 mL), stirred, and heated at
100 °C. After 8 min, the reaction mixture was neutralized with 1 N
HCl and evaporated under reduced pressure to dryness. The crude
sample was then dissolved in 45% CH3CN/H2O to a total volume of
4.5 mL, passed through a 0.2 μm PTFE syringe filter, and then injected
into a semipreparative HPLC column (Phenomenex Gemini RP-C18
column; 5 μ, 25 cm × 1 cm) via a 5 mL loop. The HPLC column was
eluted with an isocratic mobile phase mixture of 45% CH3CN/H2O at
a flow rate of 5 mL/min to give 27 (1.9 mg, 62% overall yield from 28,
tR = 13.1 min) with spectral properties as above.
1
following peaks. H NMR (CD3OD) δ 1.24 (br s, 14H, 7 × CH2),
1.51−1.64 (m, 4H, 2 × CH2), 3.20 (t, J = 7.0 Hz, 2H, CH2), 3.63 (t, J
= 6.6 Hz, 2H, CH2), 4.50−4.58 (m, 1H, H4′), 4.64−4.71 (m, 1H,
H5′), 4.75−4.82 (m, 1H, H5″), 5.57−5.62 (m, 1H, H3′) 5.60 (d, J =
7.6 Hz, 1H, H5), 6.58−6.61 (m, 1H, H1′), 7.31 (d, J = 7.6 Hz, 1H,
H6), 7.41−7.65 (m, 6H, Ar), 8.02−8.11 (m, 4H, Ar). 13C NMR
(CDCl3) δ 25.79, 26.93, 29.20, 29.29, 29.44, 29.47, 29.53, 29.57, 32.78,
41.14, 62.90, 63.01, 71.86 (m, C3′), 77.74 (C4′), 83.51 (br s, C1′),
96.55 (C5), 120.98 (t, J = 256.3 Hz, C2′), 128.10, 128.70, 128.81,
129.39, 129.81, 130.27, 133.60, 134.28, 139.86, 155.75, 163.59, 165.05,
166.09. 19F NMR δ −115.36 (dt, J = 13.7, 246.3 Hz, 1F), −120.50 (br
d, 1F). MS (ESI+) m/z 664 (100, [M + Na]+). HRMS (ESI+) m/z
calcd for C34H41F2N3NaO7 [M + Na]+, 664.2805; found, 664.2837.
The minor rotamer had the following distinguishable peaks. 1H
NMR (400 MHz, CDCl3) δ 3.40−3.41 (m, 2H, CH2), 5.71 (d, J = 7.8
Hz, 1H, H5). 19F NMR δ −115.85 (dt, J = 12.4, 246.0 Hz, 1F).
4-N-(11-Fluoroundecanyl)-3′,5′-di-O-benzoyl-2′-deoxy-
2′,2′-difluorocytidine (26). A chilled (−78 °C) solution of DAST
(14 μL, 17.2 mg, 0.107 mmol) in CH2Cl2 (250 μL) was added to a
stirred solution of 25 (21.7 mg, 0.034 mmol) in CH2Cl2 (1 mL) at
−78 °C. After 30 min, the reaction mixture was allowed to warm to
ambient temperature and was kept stirring. After 3 h, the reaction
mixture was poured into a separatory funnel containing an ice-cold
solution of Na2HCO3 in H2O (10 mL, pH 8) and was extracted with
CHCl3 (3 × 10 mL). The combined organic layers were washed with
brine, dried over MgSO4, and evaporated under reduced pressure, and
the resulting oily residue (20.5 mg) was then column chromato-
graphed (40% EtOAc/hexane) to give 26 (10.6 mg, 48%) as a mixture
4-N-[11-(Methanesulfoxy)undecanyl]-3′,5′-di-O-benzoyl-2′-
deoxy-2′,2′-difluorocytidine (28). Et3N (3.8 μL, 2.7 mg, 0.027
mmol) and MsCl (1.5 μL, 2.3 mg, 0.020 mmol) were sequentially
added to a stirred solution of 25 (11.6 mg, 0.018 mmol) in CH2Cl2 at
0 °C. After 5 min, the reaction mixture was allowed to warm to
ambient temperature and was kept stirring. After 3 h, the reaction
mixture was then partitioned between H2O and CH2Cl2, and the
aqueous layer was extracted with CH2Cl2 (2 × 20 mL). The combined
organic layers were washed with brine, dried over MgSO4, and
evaporated under reduced pressure, and the resulting residue (12.1
mg) was column chromatogramphed (50% EtOAc/hexane) to give 28
(11.7 mg, 90%) as a mixture of rotamers (71:29). The major rotamer
1
had the following peaks. H NMR (CDCl3) δ 1.25 (brs, 14H, 7 ×
CH2), 1.55−1.77 (m, 4H, 2 × CH2), 2.99 (s, 3H, CH3), 3.46−3.52 (m,
2H, CH2), 4.21 (t, J = 6.6 Hz, 2H, CH2), 4.51−4.58 (m, 1H, H4′),
4.64−4.81 (m, 2H, H5′, H5″), 5.55 (d, J = 7.6 Hz, 1H, H5), 5.59−5.63
(m, 1H, H3′), 6.55−6.67 (m, 1H, H1′), 7.32 (dd, J = 1.6, 7.5 Hz, 1H,
H6), 7.43−7.51 (m, 4H, Ar), 7.57−7.66 (m, 2H, Ar), 8.03−8.10 (m,
4H, Ar). 13C NMR (CDCl3) δ 21.15, 25.47, 26.94, 29.00, 29.22, 29.30,
29.39, 29.46, 29.55, 37.50, 41.18, 63.02 (C5′), 70.39, 71.96 (dd, J =
17.3, 35.8 Hz, C3′), 77.36 (C4′), 84.00 (br s, C1′), 96.22 (C5), 120.93
(t, J = 262.8 Hz, C2′), 128.13, 128.71, 128.82, 129.41, 129.82, 130.28,
133.61, 134.28, 139.97 (C6), 155.66, 163.55, 165.05, 166.08. 19F NMR
δ −120.61 (brd, J = 261.9 Hz, 1F), −115.38 (dt, J = 14.1, 246.7 Hz,
1F). MS (ESI) m/z 720 (100, [M + H]+). HRMS (ESI+) m/z calcd for
C35H43F2N3NaO9S [M + Na]+, 742.2580; found, 742.2603.
1
of rotamers (76:24). The major rotamer had the following peaks. H
NMR (CDCl3) δ 1.27 (br s, 14H, 7 × CH2), 1.55−1.69 (m, 4H, 2 ×
CH2), 3.47−3.52 (m, 2H, CH2), 4.43 (dt, J = 6.2, 47.4 Hz, 2H, CH2),
4.51−4.55 (m, 1H, H4′), 4.67 (dd, J = 4.5, 12.3 Hz, 1H, H5′), 4.79
(dd, J = 3.2, 12.3 Hz, 1H, H5″), 5.54 (d, J = 7.6 Hz, 1H, H5), 5.58−
5.63 (m, 1H, H3′), 6.61 (brs, 1H, H1′), 7.32 (d, J = 7.5 Hz, 1H, H6),
7.42−7.66 (m, 6H, Ar), 8.03−8.16 (m, 4H, Ar). 13C NMR (CDCl3) δ
25.30, 27.01, 29.24, 29.32, 29.49, 29.56, 29.83, 30.44, 30.63, 41.28,
63.02 (C5′), 71.87 (br s, C3′), 79.16 (br s, C4′), 83.56 (br s, C1′),
84.37 (d, J = 164.0 Hz′), 96.17 (C5), 121.88 (t, J = 255.7 Hz, C2′),
128.14, 128.73, 128.84, 129.43, 129.84, 130.31, 133.59, 134.29, 140.10,
155.46, 163.39, 165.06, 166.10. 19F NMR (CDCl3) δ −217.94 (tt, J =
The minor rotamer had the following distinguishable peaks. 1H
NMR (CDCl3) δ 3.23−3.25 (m, CH2N), 5.77 (d, J = 7.9 Hz, H5). 19
NMR δ −115.98 (dt, J = 13.1, 234.1 Hz, 1F).
F
Cytostatic Activity Assays.33 The compounds tested were added
to murine leukemia L1210, human T-lymphocyte CEM, human
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cervical carcinoma HeLa, and human breast carcinoma MCF-7 cell
cultures in 96-well microtiter plates. After 2 (L1210), 3 (CEM), or 4
(HeLa, MCF-7) days of incubation at 37 °C, the number of living cells
was determined by a Coulter counter. The 50% inhibitory
concentration (IC50) was defined as the compound concentration
required to inhibit cell proliferation by 50%.
N′-ethyl-carbodiimide; HOBt, 1-hydroxybenzotriazole; NMM,
N-methylmorpholine; CDI, 1,1′-carbonyldiimidazole; TEA,
triethylamine; DAST, (diethylamino)sulfur trifluoride; HDPE,
high-density polyethylene; TK-2, thymidine kinase
REFERENCES
dCK and TK-2 Activity Assays. The activity of recombinant
mitochondrial thymidine kinase (TK-2) and cytosolic 2′-deoxycytidine
kinase (dCK) and the 50% inhibitory concentration of the test
compounds were assayed in a 50 μL reaction mixture containing 50
mM Tris/HCl, pH 8.0, 2.5 mM MgCl2, 10 mM dithiothreitol, 0.5 mM
CHAPS, 3 mg/mL bovine serum albumin, 2.5 mM ATP, 1 μM
[5-3H]dCyd or [CH3-3H]dThd, and enzyme. The samples were
incubated at 37 °C for 30 min in the presence or absence of different
concentrations (5-fold dilutions) of the test compounds. Aliquots of
45 μL of the reaction mixtures were spotted on Whatman DE-81 filter-
paper disks. The filters were washed three times for 5 min each in 1
mM ammonium formate, once for 1 min in water, and once for 5 min
in ethanol. The radioactivity retained on the filter discs was
determined by a scintillation counter. To evaluate substrate activity
against TK-2 and dCK, the tested compounds were added to the
enzyme reaction mixture at 100 μM, and conversion to their 5′-
monophosphates was monitored by HPLC on an anion-exchange
Partisil Sax column.
Human Serum and Murine Liver Extract Stability Assays.
The compounds tested were exposed to 50% human serum in
phosphate buffered saline (PBS) or murine liver extract in PBS at 100
μM concentrations and incubated for 0, 60, and 240 min (human
serum) or 0, 30, and 120 min (murine liver extract) at 37 °C. At each
time point (0, 60, and 240 min), an aliquot was withdrawn and
subjected to HPLC analysis on a reverse-phase RP-18 column (mobile
phase: acetonitrile/H2O). Elution times were 13.2 and 16.4 min for
dFdU and gemcitabine, respectively, and 22.8 and 22.7 min for
compounds 7 and 21, respectively.
■
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, representative HPLC chromato-
grams, and characterization data for 11-fluoroundecanoic, 11-
bromoundecanoyl, 11-aminoundecanol, and 11-(benzyloxy)-
undecan-1-amine. This material is available free of charge via
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AUTHOR INFORMATION
Corresponding Author
*Telephone 305-348-6195; E-mail wnuk@fiu.edu.
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This investigation was partially supported by award
SC1CA138176 from NIGMS and NCI. J.P. is grateful to the
MBRS RISE program (NIGMS; R25 GM61347) for his
fellowship. The research of J.B. was supported by KU Leuven
(GOA 10/014). We are grateful to Mrs. Lizette van Berckelaer
and Mrs. Ria Van Berwaer for excellent technical assistance.
ABBREVIATIONS USED
■
dFdC, 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine); hENT1,
human equilibrative nucleoside transport protein 1; dCK,
deoxycytidine kinase; RNR, ribonucleotide reductase; CTP,
cytidine triphosphate; dFdU, 2′,2′-difluorouridine; CDA,
cytidine deaminase; SQgem, 4-N-squalenoylgemcitabine;
[18F]-FAC, 1-(2′-deoxy-2′-18F-fluoro-β-D-arabinofuranosyl)-
cytosine); L-18F-FMAC, 1-(2′-deoxy-2′-18F-fluoro-β-L-arabino-
furanosyl)-5-methylcytosine; EDCI, N-dimethylaminopropyl)-
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