Synthesis and biological activity evaluation of cytidine-5′-deoxy-5- fluoro-N-[(alkoxy/aryloxy)] carbonyl-cyclic 2′,3′-carbonates

Article abstract of DOI:10.1016/j.ejmech.2012.06.023

Capecitabine, an oral prodrug of 5-FU was developed to improve the tumor selectivity and tolerability. To enhance the efficacy of capacitabine, a series of 5′-deoxy-5-fluorocytidine derivatives 5a-e were synthesized. In the present study, we investigated antitumor activity of 5′-deoxy-5- fluorocytidine derivatives both in vivo and in vitro methods. Title compounds were non-mutagenic to Salmonella typhimurium tester strain in Ames test. Compounds 5d and 5e are potent to inhibit the proliferation of NCI-69, PZ-HPV-7, MCF-7 and HeLa cells in MTT assay. In particular, 5d and 5e showed potent antitumor activities against L1210 leukemia cell line. Collectively, these findings suggest that 5d and 5e are more potent anti-cancer compounds than capecitabine.

Full text of DOI:10.1016/j.ejmech.2012.06.023

European Journal of Medicinal Chemistry 54 (2012) 690e696
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity evaluation of cytidine-50-deoxy-5-fluoro-N-
[(alkoxy/aryloxy)] carbonyl-cyclic 20,30-carbonates
,
V. Jhansi Rani a f, A. Raghavendra b, P. Kishore c, Y. Nanda Kumar d, K. Hema Kumar e,
,
K. Jagadeeswarareddy f
*
a Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, India
b Department of Biotechnology and Environmental Sciences, Thapar University, Patiala, Punjab 147004, India
c Department of Pharmacology, Toxicology, and Therapeutics, The University of Kansas Medical Center Kansas City, KS 66160, USA
d Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati 517 502, India
e Aptus Biosciences Pvt. Ltd., SVS Medical College campus, Mahabubnagar 509 002, India
f Sugen Life Sciences Pvt Ltd, Tirupati 517 505, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Capecitabine, an oral prodrug of 5-FU was developed to improve the tumor selectivity and tolerability. To
enhance the efficacy of capacitabine, a series of 50-deoxy-5-fluorocytidine derivatives 5aee were
synthesized. In the present study, we investigated antitumor activity of 50-deoxy-5-fluorocytidine
derivatives both in vivo and in vitro methods. Title compounds were non-mutagenic to Salmonella
typhimurium tester strain in Ames test. Compounds 5d and 5e are potent to inhibit the proliferation of
NCI-69, PZ-HPV-7, MCF-7 and HeLa cells in MTT assay. In particular, 5d and 5e showed potent antitumor
activities against L1210 leukemia cell line. Collectively, these findings suggest that 5d and 5e are more
potent anti-cancer compounds than capecitabine.
Received 28 January 2012
Received in revised form
11 June 2012
Accepted 12 June 2012
Available online 2 July 2012
Keywords:
Capecitabine
50-Deoxy-5-fluorocytidine
5-Fluorouracil
Toxicity
Published by Elsevier Masson SAS.
Antitumor activity
1. Introduction
toxicity of the drugs. The indiscriminate nature of current cancer
chemotherapy and drug resistance has initiated a search for more
Cancer is a common health problem worldwide, although
cancer types vary considerably between affluent and less affluent
countries [1]. Cancer is responsible for 1 in 8 deaths worldwide. In
fact, cancer causes more deaths than AIDS, tuberculosis and malaria
combined [2]. Cancer incidence and mortality rate are expected to
rise steeply and will overtake heart disease as the number one killer
of people around the world, according to a report by the Interna-
tional Agency for Research on Cancer (IARC) [2].
In spite of the use of surgical treatment and irradiation,
chemotherapy still remains an important option for the treatment
of solid cancers. Regardless of extensive research efforts, the ther-
apeutic treatment of tumor patients is still not satisfying. Chemo-
therapeutic drugs are designed in such way that they preferentially
target tumor cells without harming normal cells or tissues. Unfor-
tunately, it often affects parts of the body not directly affected by
the cancer itself in most cases, with harsh side effects due to the
selective approaches.
Most antitumor agents are cytotoxic and are associated with
severe side effects, including myleo-suppression, skin toxicity and
intestinal toxicity. There have been several attempts to reduce such
side effects by means of prodrug strategies for the tumor-selective
delivery of cytotoxic agents [3]. The present study describes the
tumor-selective delivery of 5-fluorouracil (5-FU) by sequential
conversion of the new 5-fluoropyrimidine carbamate (5aee) by
endogenous enzymes preferentially localized in human liver and
tumor tissue.
5-Fluorouracil (5-FU) is a pyrimidine analog and a standard anti-
tumor agent acting as thymidylate synthase inhibitor belonging to
antimetabolite family [4] and rapidly metabolized by enzyme dihy-
dropyrimidine dehydrogenase (DPD) [5]. However, 5-FU is poorly
tumor-selective, however, and its therapy causes highly incidences of
toxicity in the bone marrow, central nervous system, gastrointestinal
tract and skin. Thus, many compounds have been developed for
advanced selectivity and to reduce toxicity such as UFT, 5-ethynyluracil,
S-1(tegafur þ 5-Chloro-2,4-dihydroxypyridine þ Oxonic acid), tegafur,
doxifluridine (50-DFUR), carmofur and capecitabine [6]. Capecitabine
* Corresponding author. Tel.: þ91 949 3660267; fax: D91 877 2276118.
E-mail address: kanalajreddy@gmail.com (K. Jagadeeswarareddy).
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2012.06.023

V. Jhansi Rani et al. / European Journal of Medicinal Chemistry 54 (2012) 690e696
691
(N4-pentyloxycarbonyl-50-deoxy-5-fluorocytidine) is designed as
a ‘pro-drug’ to the cytotoxic agent 5-FU and to be administered
orally [7]. To improve the efficacy and safety profiles of the oral
Characteristic IR stretching absorptions were observed in the
regions 3101e3235 cmꢀ1 and 3402e3488 cmꢀ1 for NeH [8], OeH
[9], respectively. The ester carbonyl stretching frequency was
observed in the range 1722e1746 cmꢀ1. In the 1H NMR spectra of
title compounds 4d, 4e, 5d and 5e, the chemical shifts of aromatic
hydrogens of the phenyl ring appeared as doublets in the region
fluoropyrimidine 50-DFUR, capecitabine,
a new orally available
5-fluoropyrimidine carbamate, were synthesized so that it passes intact
through the intestinal mucosa and then selectively delivers 5-FU to
tumor tissues following its enzymatic conversion: first to 50-deoxy-5-
fluorocytidine (50-DFCR) by cytidine deaminase, and finally to 5-FU
by thymidine phosphorylase. The latter two enzymes are preferen-
tially localized in tumor tissues. The catabolic pathway of 5-FU
comprises dihydro-5-fluorouracil (FUH2, via dihydropyrimidine dehy-
d
6.42e7.88 [9,10]. The NeH hydrogen resonated as a broad singlet
in the region
d 10.65e11.25 [10]. The OH protons were observed as
a singlet in the region 8.52e11.34 ppm 13C NMR chemical shifts for
title compounds were observed in their expected regions. The
carbamide carbon (eCONH) gave signal at
d 153.6e155.6 [8]
drogenase, DPD), 5-fluoro-ureidopropionic acid (FUPA) and fluoro-
b-
(Scheme 1).
alanine (FBAL) [5]. To enhance the efficacy of capecitabine, but not its
toxicity, we designed a series of 50-deoxy-5-fluorocytidine derivatives,
cytidine-50-deoxy-5-fluoro-N-[(alkoxy/aryloxy)] carbonyl-cyclic 20,30-
carbonates as prodrugs of known DPD inhibitor, 5-FU derivatives
selectively in tumors, similar to the design of capecitabine [10]. These
novel derivatives showed higher antitumor activity than capecitabine
in vivo.
3. Pharmacology
The bacterial reverse mutation assay was used to evaluate the
mutagenic potential of 50-deoxy-5-fluorocytidine derivatives, to
induce gene mutations in the bacterial reverse mutation assay in
comparison to solvent control according to the plate incorporation
test (Trial I) and the pre-incubation test (Trial II) using histidine
dependent auxotrophic mutants of Salmonella typhimurium (strains
TA1535, TA1537, TA98, TA100 and TA102). Cytotoxicity of the 50-
deoxy-5-fluorocytidine derivatives 4bee and 5aee was examined
by using cells of human lung (NCI-69), prostrate (PZ-HPV-7), breast
(MCF-7), and cervical (HeLa) cancer cell lines and expressed in
terms of inhibitory activity (IC50, mg/mL) obtained by the MTT
method. The results are summarized in Table 1. The in vivo anti-
tumor activity of compounds 5d and 5e was investigated with
murine leukemia L1210 and the results are listed in Fig. 1 [11]. The
activity was evaluated in terms of mean survival time (MST). L1210
cell line for implantation was maintained at 37 ꢁC under an
atmosphere of 5% CO2 in a 75 cm2 culture flask and subcultured
once or twice per week in RPMI 1640 medium containing 10% fetal
2. Chemistry
The synthesis of series of 50-deoxy-5-fluorocytidine derivatives,
cytidine-50-deoxy-5-fluoro-N [(alkoxy/aryloxy)] carbonyl-cyclic
20,30-carbonates, was carried out by the following way. In the first
step 20,30-di-O-acetyl-50-deoxy-5-fluorocytidine was treated with
various chloroformates in the presence of K2CO3 in acetone to
obtain the intermediates 4aee. They were further reacted with
carbonyl diimidazole (CDI) in methylene dichloride (CH2Cl2) to
afford the title compounds 5aee.
The chemical structures of all the title compounds 4aee and
5aee were characterized by IR, 1H, 13C NMR and APCI-MS studies
and their data are presented in the experimental section.
Scheme 1. Synthetic route.

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V. Jhansi Rani et al. / European Journal of Medicinal Chemistry 54 (2012) 690e696
Table 1
Cytotoxicities of new 50-deoxy-5-fluorocytidine derivatives against tumor cells.
Compound
R
IC50 (mg/mL)a
NCI-69
PZ-HPV-7
MCF-7
HeLa
0.04 ꢃ 0.002
5-FU
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
e
0.28 ꢃ 0.02
0.30 ꢃ 0.015
0.37 ꢃ 0.01
0.17 ꢃ 0.018
0.16 ꢃ 0.019
0.25 ꢃ 0.012
0.25 ꢃ 0.037
0.09 ꢃ 0.001
0.07 ꢃ 0.02
0.69 ꢃ 0.04
0.71 ꢃ 0.053
0.12 ꢃ 0.01
Pentyl
0.05 ꢃ 0.012
0.09 ꢃ 0.0023
0.09 ꢃ 0.0015
0.01 ꢃ 0.005
0.03 ꢃ 0.0011
0.08 ꢃ 0.0018
0.18 ꢃ 0.007
0.13 ꢃ 0.0021
0.03 ꢃ 0.0019
0.006 ꢃ 0.0009
2-Methyl-1-butyl
3-Methyl-1-butyl
Nitrophenyl
Chlorophenyl
Pentyl
2-Methyl-1-butyl
3-Methyl-1-butyl
Nitrophenyl
Chlorophenyl
0.37 ꢃ 0.021
0.10 ꢃ 0.005
0.09 ꢃ 0.01
0.12 ꢃ 0.004
0.18 ꢃ 0.02
0.10 ꢃ 0.02
0.18 ꢃ 0.002
0.33 ꢃ 0.01
0.14 ꢃ 0.002
0.20 ꢃ 0.01
0.08 ꢃ 0.0034
0.07 ꢃ 0.0025
0.10 ꢃ 0.005
0.006 ꢃ 0.00051
0.030 ꢃ 0.005
0.31 ꢃ 0.018
0.030 ꢃ 0.008
0.003 ꢃ 0.0012
0.35 ꢃ 0.012
0.006 ꢃ 0.0012
0.003 ꢃ 0.0015
a
Activities against tumor cells (NCI-69, human lung; PZ-HPV-7, human prostrate; MCF-7, human breast; HeLa, human cervical) were measured by MTT assay after 3 days of
incubation.
bovine serum. The tumor cells were resuspended in PBS to
the presence and absence (10% v/v S9 mix) of metabolic activation,
is non-mutagenic to S. typhimurium tester strains viz., TA1537,
TA1535, TA98, TA100 and TA102 when tested under the specified
conditions. The reference mutagens showed a distinct increase in
induced revertant colonies in all the tester strains both in the
presence as well as in the absence of metabolic activation, without
showing cytotoxicity.
a concentration of 1 ꢂ 107/mL. Similarly, L1210 cells (1 ꢂ 106/mL)
were inoculated intraperitoneally (ip) into male BDF-1 mice on day
0. The test compounds 5d and 5e and the reference compound,
capecitabine, were orally administered (po) 20 times for 4 weeks
and the maximum doses were decided by considering acute
toxicity.
As shown in Table 1, all the new 50-deoxy-5-fluorocytidine
derivatives 4 and 5 were potent to inhibit the proliferation of
NCI-69, PZ-HPV-7, MCF-7 and HeLa cells. Among all obtained
compounds, 5d and 5e showed excellent inhibitory activity on the
above cell lines, with IC50 values in the range of 0.003e0.030 mg/
mL. It is evident that the in vitro potency of those bearing
aromatic substituents (5d, R ¼ p-nitrophenyl; 5e, R ¼ p-chlor-
ophenyl) is better than that of those bearing alkyl and isoalkyl
groups (5b, R ¼ pentyl; 4b and 5b, R ¼ 2-methyl-1-butyl, 4c and 5c,
R ¼ 3-methyl-1-butyl).
4. Results and discussion
All the new 50-deoxy-5-fluorocytidine derivatives 4 and 5 were
assessed for its potential to induce gene mutations according to the
plate incorporation test (experiment I) and the pre-incubation test
(experiment II) using Ames tester strains TA1535, TA1537, TA98,
TA100 and TA102 along with the different positive controls for
different strains. The assay was performed with and without liver
microsomal activation based on the results of the pre-experiment.
Each concentration and the controls were tested in triplicate. The
title compounds 4aee and 5aee were tested at the following
concentrations 0.312, 0.625, 1.25, 2.5, 5 mg/plate, both in the
presence of metabolic activation (þS9) and in the absence of
metabolic activation (ꢀS9). The results revealed that there was no
positive mutagenic effect in strains TA1537, TA1535, TA98, TA100
and TA102 both in the presence (10% v/v S9 mix) and in the absence
of metabolic activation at any of the tested dose level when
compared with the concurrent vehicle control.
We initially evaluated that the in vivo activity of the new 50-
deoxy-5-fluorocytidine derivative 5d and 5e and the reference
compounds capecitabine. The toxicity (LD50) of 5d and 5e by oral
administration was 2000 mg/kg for single dose and 200 mg/kg/day
daily for 28 days in mice [13,14]. This value for the single dose is
high compared to 115 mg/kg of 5-FU [15]. As shown in Fig. 1, the
new 50-deoxy-5-fluorocytidine derivatives 5d and 5e gave good
antitumor activity over a broad dose range compared to capecita-
bine [16]. The compound 5d and 5e showed MST ranging from 23.4
to 23.7 at 0.16-mmol/kg/day to 27.1 and 26.3 days at 0.080 mmol/
kg/day. Reference compound capecitabine show MST of 19.6 and
20.3 days at 0.16 and 0.08 mmol/kg/day dose, respectively. The
above results clearly showed that compound 5d increased the MST
of the mice by 3.8 and 6 days at 0.16 and 0.08 mmol/kg/day dose,
respectively, when compared with capecitabine treated mice.
Evidently, the in vivo antitumor activity of 5d and 5e is better than
that of capacitabine (Fig. 1). We also evaluated that the in vivo
activity of the 50-deoxy-5-fluorocytidine derivative 5a, and the
reference compounds capecitabine. Because of systemic toxicity we
administered 5a through po daily for two weeks, where as
compounds 5d and 5e were administered 5 days a week for 3
weeks. As shown in Fig. 2, the antitumor activity of 5a against L1210
leukemia in mice showed the best mean survival time value at high
dose range, 17.3 days at 1.5 mmol/kg/day, after po administration
compared to MST of capecitabine (12.8 days) [12].
Trial II results have shown a negative effect in strain TA1537,
TA1535, TA98, TA 100 and TA102 both in the presence (10% v/v
S9 mix) and in the absence of metabolic activation at any of the
tested dose level when compared with the concurrent vehicle
control. From the results of this study, it is concluded that the new
50-deoxy-5-fluorocytidine derivatives in all the concentrations in
Finally the correlation models generated among IC50 values and
predicted log P values showed non linear graphs indicating that
their correlation is not well significant. The values of R (coefficient
of determination) and R2 (correlation coefficient) of the four
correlation models are shown in Table 2 and the correlation models
are shown in Fig. 3. The models are explaining that the two
Fig. 1. MST time was measured after treating the L1210 cells implanted BDF-1 male
mice with different concentrations of capecitabine, 5d and 5e compounds, 5 days
a week for 3 weeks. 5d, 5e increases the mean survival time of mice compared with
either with untreated control (18.2 days) or capecitabine treated group.

V. Jhansi Rani et al. / European Journal of Medicinal Chemistry 54 (2012) 690e696
693
a solution of triphosgene (5 g, 0.0167 mol) at 0 ꢁC in the presence of
TEA (1.688 g, 0.0167 mol) over a period of 15 min. The reaction
mixture was further stirred at room temperature 6 h, progress of the
reaction was monitored by TLC (ethyl acetate: hexane 1:1). After
completion of the reaction, it was filtered and washed with 2 volumes
(10 mL) of THF to obtain the liquid compound 2-methyl-1-butyl
chloroformate.
6.1.2. Preparation of 50-deoxy-5-fluoro-N4-(2-methyl-1-butyloxycarbonyl)
cytidine [10]
To a solution of K2CO3 (0.626g, 0.0453 mol)inacetone(80 mL)was
added 20,30-di-O-acetyl-50-deoxy-5-fluorocytidine (14 g, 0.0425 mol)
in a 500 mL 4-necked RB flask fitted with magnetic bar, thermo
pocket, guard tube and nitrogen inlet and stirred for 10e15 min 2-
Methyl-1-butyl chloroformate first half (2.279 g, 0.0151 mol) was
added through additional funnel over a period of 15e20 min at room
temperature and continued to stirred for 3 h. After completion of 3 h,
second half of 3-methyl-1-butyl chloroformate (1.129 g, 0.0075 mol)
added at 25e30 ꢁC, progress of the reaction was monitored by TLC
(ethylacetate: hexane 1:1). After completion of the reaction, the
mixture was filtered and washed with 2 volumes (50 mL) of acetone.
To the acetone layer 4 volumes of 10% NaOH solution added at ꢀ10
to ꢀ15 ꢁC over 2 h. After completion of the reaction indicated by TLC,
the pH of the reaction mixture adjusted to 6.0 by using concentrated
HCl. The acetonewasdistilled off completely by using rotaevaporator.
Thus obtained aqueous layer extracted with ethyl acetate 5 volumes
(50 mL) and ethyl acetate layer reduced to 1 volume stirred for 30 min
then 5 volumes of tertiary methyl butyl ether (TBME) (50 mL) was
added, the obtained precipitatewasstirred for 1 h at 20e25ꢁC and 2 h
at 0e5 ꢁC filtered off washed with 9:1 volumes of TBME and ethyl
acetate and suck dried to obtained the crude product and it was
further purified by column chromatography (1:1 hexane/EtOAc) to
get the title compound.
Fig. 2. MST time was measured after treating the L1210 cells implanted BDF-1 male
mice with different concentrations of capecitabine, 5a daily for two weeks. 5a
increases the mean survival time of mice compared with either with untreated control
(8.9 days) or capecitabine treated group.
variables are not showing linear relationship and their cytotoxicity
levels are independent among all the compounds.
5. Conclusion
In conclusion, we have synthesized the new 50-deoxy-5-
fluorocytidine derivatives 4 and 5 which show negative mutagenic
activity in the bacterial reverse mutation test without inducing gene
mutations by base pair changes or frame shifts in the genome of the
strains used and show better antitumor activity than that of 5-FU or
capecitabine. In particular, 5d and 5e show potent antitumor activ-
ities against L1210 leukemia cell line. The results provided a foun-
dation for future design and development of antitumor compounds
and also more potent drugs for cancer treatment.
6. Experimental protocols
6.1. Chemistry
6.1.3. Preparation of 50-deoxy-5-fluoro-N4-(2-methyl-1-butyloxycarbonyl)
cytidine-20,30-carbonate
Chemicals were purchased from SigmaeAldrich, Merck and
Lancaster, used as such without further purification. We procured
the compound 3 (20,30-di-O-acetyl-50-deoxy-5-fluorocytidine) from
Hefei NodMed Pharmacy Co., Ltd. China. All solvents used for
spectroscopy and other physical studies were reagent grade and
were further purified by literature methods. Melting points (m.p.)
were determined using a calibrated thermometer by Guna Digital
Melting Point apparatus. Infrared spectra (IR) were recorded on
a Nicolet 380 FT-IR spectrophotometer. Samples were recorded as
potassium bromide (KBr) discs. 1H and 13C NMR spectra were
recorded in DMSO-d6 on a Bruker AMX 300 MHz spectrometer
operating at 300 MHz for 1H, 75 MHz for 13C NMR and the chemical
shifts values are expressed in parts per million (ppm) with refer-
ence to tetramethylsilane (TMS). LC mass spectra were recorded on
a Jeol SX 102 DA/600 mass spectrometer.
Methylene dichloride (120 mL) was taken in a 500 mL 4-necked RB
flask fittedwithmagneticbar, thermopocket, guard tube and nitrogen
inlet. 50-Deoxy-5-fluoro-N4-(2-methyl-1-butyloxycarbonyl)-cytidine
(4 g, 0.0114 mol) was added in one lot, once the clear solution was
formed, CDI (2.257 g, 0.01393 mol) was added in one lot under
nitrogen atmosphere and the reaction mixture was stirred over
10e12 h. After the completion of the reaction (TLC), the reaction
mixturewas filteredandwashedwith2volumes(20mL)ofmethylene
dichloride. Thus obtained crude product was further purified by
column chromatography (1:1 hexane/EtOAc) to get the title
compound. The desired product was isolated and concentrated in rota
evaporator. Finally it is dried under vacuum at 50 ꢁC to get the pure
product.
The same experimental procedure was adopted for the prepa-
ration of the remaining title compounds.
6.1.1. Preparation of 2-methyl-1-butyl chloroformate
To the stirred solution of 2-methyl-1-butanol (1.474 g, 0.0167 mol)
in dry tetrahydrofuran (20 mL) at nitrogen atmosphere, was added
6.1.3.1. Synthesis of 50-deoxy-5-fluoro-N4-(n-pentyloxycarbonyl) cyti-
dine (4a). Yield: 90%; m.p. 119e121 ꢁC; IR (KBr) 3488, 3210,
2975e2886, 1728, 1686 cmꢀ1 1H NMR (300 MHz, DMSO-d6)
; d:
Table 2
Correlation analysis.
0.91e0.98 (m, 6H), 1.12e1.32 (m, 4H), 1.44e1.64 (m, 2H), 3.65 (q,
J ¼ 6.0 Hz,1H), 3.89e4.05 (m, 4H), 5.06 (d, J ¼ 6.0 Hz,1H), 5.42 (s,1H),
5.66 (d, J ¼ 3.0 Hz, 1H), 8.06 (br s, 1H), 10.55 (br, s, 1H). 13C NMR
Tumor cell typea
Rb
R2c
NCI-69
PZ-HPV-7
MCF-7
HeLa
0.1166
0.0774
0.3029
0.3792
0.0136
0.0060
0.0917
0.1438
(75 MHz, DMSO-d6) d: 155.6, 129.8, 93.7, 81.0, 75.9, 71.8, 65.9, 38.5,
35.5, 26.9, 26.0, 22.9,18.5,16.6,11.6. LCMS m/z (%) 360 [M þ H]þ; Anal.
Calcd. for C15H22FN3O6: C, 50.14; H, 6.17; N,11.69%. Found: C, 50.08; H,
6.10; N, 11.63%.
a
The type of tumor cells for which their respective IC50 values were taken for the
construction of correlation models.
6.1.3.2. Synthesis of 50-deoxy-5-fluoro-N4-(2-methyl-1-butyloxycarbonyl)
cytidine (4b). Yield: 85%; m.p. 119e120 ꢁC; IR (KBr) 3502, 3226,
b
Coefficient of determination.
Correlation coefficients calculated from correlation analysis.
c

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V. Jhansi Rani et al. / European Journal of Medicinal Chemistry 54 (2012) 690e696
Fig. 3. Correlation plots generated for IC50 and log P values of the compounds. A. NCI-69, B. PZ-HPV-7, C. MCF-7 and D. HeLa cell types.
2966e2880, 1720, 1683 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6)
DMSO-d6)d: 155.8,155.3,152.8,152.0,142.1,140.3,131.8,128.1,121.5,125.3,
d: 0.80e0.87 (m, 6H), 1.04e1.46 (m, 5H), 1.61e1.71 (m, 1H), 3.69
92.8, 83.2, 82.4, 76.5, 18.0. LCMS m/z (%) 411 [M þ H]þ; Anal. Calcd. for
C16H15FN4O8: C, 46.84; H, 3.68; N, 13.65%. Found: C, 46.86; H, 3.65; N,
13.62%.
(q, J ¼ 6.0 Hz, 1H), 3.84e4.16 (m, 4H), 5.21 (d, J ¼ 3.6 Hz, 1H), 5.72
(d, J ¼ 3.2 Hz, 1H), 5.80 (s, 1H), 7.85 (br, s, 1H), 10.21 (br, s, 1H). 13C NMR
(75 MHz, DMSO-d6) d: 155.4, 129.7, 93.7, 81.0, 75.9, 71.8, 50.0, 48.3, 35.5,
35.3, 26.9, 22.19,19.6,16.7,11.6. LCMS m/z (%) 358 [M ꢀ H]ꢀ; Anal. Calcd.
for C15H22FN3O6: C, 50.14; H, 6.17; N, 11.69%. Found: C, 50.19; H, 6.13; N,
11.69%.
6.1.3.5. Synthesis of 50-deoxy-5-fluoro-N4-(4-chloro-phenyloxycarbonyl)
cytidine (4e). Yield: 86%; m.p. 153e155 ꢁC; IR (KBr) 3426, 3198,
2952, 1726, 1672 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6)
d
: 1.28e1.34 (m, 3H), 3.68 (q, J ¼ 6.0 Hz, 1H), 3.85e3.91 (m, 2H),
6.1.3.3. Synthesis of 50-deoxy-5-fluoro-N4-(3-methyl-1-butyloxycarbonyl)
cytidine (4c). Yield: 85%; m.p. 120e121 ꢁC; IR (KBr) 3508, 3229e3121,
5.26 (d, J ¼ 3.0 Hz, 1H), 5.72 (d, 1H), 5.80 (s, 1H), 6.02 (d, 2H,
J ¼ 8.0 Hz), 6.42 (d, 2H, J ¼ 8.0 Hz), 7.88 (br, s, 1H), 10.52 (br, s, 1H).
2965e2907, 1722, 1684 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6)
13C NMR (75 MHz, DMSO-d6)
d: 155.2, 154.3, 152.6, 152.1, 142.6,
d
: 0.85e0.91 (m, 6H), 1.12e1.43 (m, 5H), 1.61e1.71 (m, 1H), 3.69
140.4, 131.2, 126.8, 125.3, 121.4, 92.4, 83.4, 83.0, 75.2, 18.4. LCMS
m/z (%) 400 [M þ H]þ; Anal. Calcd. for C16H15ClFN3O6: C, 48.07; H,
3.78; N, 10.51%. Found: C, 48.01; H, 3.74; N, 10.47%.
(q, J ¼ 6.0 Hz, 1H), 3.66e3.99 (m, 4H), 5.25 (d, J ¼ 3.1 Hz, 1H), 5.72
(d, J ¼ 6.0 Hz, 1H), 5.80 (s, 1H), 7.84 (br, s, 1H), 9.32 (br, s, 1H). 13C NMR
(75 MHz, DMSO-d6) d: 155.6, 129.4, 93.9, 81.4, 75.2, 71.5, 51.5, 48.7, 35.8,
35.3, 26.5, 22.30, 19.8, 16.8, 11.9. LCMS m/z (%) 358 [M ꢀ H]ꢀ; Anal. Calcd.
for C15H22FN3O6: C, 50.14; H, 6.17; N, 11.69%. Found: C, 50.15; H, 6.14; N,
11.68%.
6.1.3.6. Synthesis of 50-deoxy-5-fluoro-N4-(n-pentyloxycarbonyl)cytidine-
20,30-carbonate (5a). Yield: 88%; m.p. 142e143 ꢁC; IR (KBr) 3103e3195,
2933e2958, 1803, 1749, 1630 cmꢀ1 1H NMR (300 MHz, DMSO-d6)
;
d
: 1.12 (t, J ¼ 6.0 Hz, 3H), 1.24e1.54 (m, 7H), 1.76 (t, J ¼ 6.0 Hz, 2H), 4.25
6.1.3.4. Synthesis of 50-deoxy-5-fluoro-N4-(4-nitro-phenyloxycarbonyl)
cytidine (4d). Yield: 87%; m.p. 147e148 ꢁC; IR (KBr) 3458, 3235, 2986,
(t, J ¼ 6.0 Hz, 2H), 4.51e4.58 (m, 1H), 5.23 (m, 1H), 5.73 (m, 1H), 6.02 (d,
1H), 8.47 (d, J ¼ 9.0 Hz, 1H), 11.12 (br, s, 1H). 13C NMR (75 MHz, DMSO-d6)
1728, 1684 cmꢀ1;1H NMR (300 MHz, DMSO-d6)
d:1.32e1.46(m,3H),3.70
d: 153.6, 153.1, 152.6, 151.0, 138.6, 135.4, 130.9, 92.7, 83.2, 82.1, 65.4, 27.8,
(q, J ¼ 6.2 Hz, 1H), 3.85e3.89 (m, 2H), 5.25 (d, J ¼ 3.1 Hz, 1H), 5.68
(d, J ¼ 3.0 Hz, 1H), 5.80 (s, 1H), 6.64 (d, 2H, J ¼ 8.4 Hz), 6.67
(d, 2H, J ¼ 8.0 Hz), 7.86 (br, s, 1H), 10.26 (br, s, 1H). 13C NMR (75 MHz,
27.3, 21.6, 18.8, 13.7. LCMS m/z (%) 384 [M ꢀ H]ꢀ; Anal. Calcd. for
C16H20FN3O7: C, 49.87; H, 5.23; N, 10.90%. Found: C, 49.86; H, 5.20; N,
10.92%.

V. Jhansi Rani et al. / European Journal of Medicinal Chemistry 54 (2012) 690e696
695
6.1.3.7. Synthesis of 50-deoxy-5-fluoro-N4-(2-methyl-1-butyloxycarbonyl)
cytidine-20,30-carbonate (5b). Yield: 84%; m.p. 143e145 ꢁC; IR (KBr) 3120,
mixed in a test tube and incubated at 37 ꢃ 2 ꢁC for 20 min. After
pre-incubation 2.0 mL overlay agar was added to each tube. The
mixture was poured on minimal agar plates.
2948, 1798, 1732, 1626 cmꢀ1; 1H NMR (300 MHz, DMSO-d6)
d: 0.56e0.76
(t, J ¼ 6.0 Hz, 3H), 1.00e1.23 (m, 7H), 1.34 (d, J ¼ 6.0 Hz, 2H), 4.16
(t, J ¼ 6.0 Hz, 2H), 4.83e4.87 (m, 1H), 5.36 (m, 1H), 5.64 (m, 1H), 6.87
(d, J ¼ 6.2 Hz, 1H), 8.06 (d, J ¼ 9.0 Hz, 1H), 10.65 (br, s, 1H). 13C NMR
After solidification the plates were incubated upside down for
48 h (approx) at 37 ꢃ 2 ꢁC in the dark.
(75 MHz, DMSO-d6)
d: 154.1, 153.2, 152.8, 150.1, 138.8, 135.6, 130.7, 94.9,
6.2.2. MTT assay
83.7, 80.2, 66.0, 29.3, 27.6, 21.9, 17.4, 12.8. LCMS m/z (%) 384 [M ꢀ H]ꢀ;
Anal. Calcd. for C16H20FN3O7: C, 49.87; H, 5.23; N,10.90%. Found: C, 49.88;
H, 5.22; N, 10.89%.
The HeLa (cervical carcinoma), MCF-7 (breast carcinoma), NCI-
69 (lung carcinoma), and PZ-HPV-7 (parostate carcinoma) cells
(obtained from American Type Culture Collection ATCC, Rockville,
MD, USA) were cultured as monolayers and maintained in Dul-
becco’s modified Eagle’s medium (DMEM) supplemented with 10%
6.1.3.8. Synthesis of 50-deoxy-5-fluoro-N4-(3-methyl-1-butyloxycarbonyl)
cytidine-20,30-carbonate (5c). Yield: 86%; m.p. 143e144 ꢁC; IR (KBr)
fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin
3124e3195, 2928e2955, 1812, 1758, 1628 cmꢀ1
;
1H NMR (300 MHz,
and 100 mg/mL streptomycin in a humidified atmosphere with 5%
CO2 at 37 ꢁC. The growth inhibition activity was assessed as
described previously, according to the slightly modified procedure
of the National Cancer Institute, Developmental Therapeutics
Program [21]. The cells were inoculated onto standard 96-well
micro titer plates on day 0. The cell concentrations were adjusted
according to the cell population doubling time (PDT): 1 ꢂ 104/mL
for HeLa, NCI-69 cell lines (PDT ¼ 20e24 h), 2 ꢂ 104/mL for MCF-7
cell line (PDT ¼ 33 h) and 3 ꢂ 104/mL for PZ-HPV-7 (PDT ¼ 47 h).
Test agents were then added in five dilutions (10ꢀ8e5 ꢂ 10ꢀ6 mol/l)
and incubated for a further 72 h. Working dilutions were freshly
prepared on the day of testing. After 72 h of incubation, the cell
growth rate was evaluated by performing the MTT assay, which
detects succinate dehydrogenase activity in viable cells. Each test
was performed in quadruplicate in three individual experiments.
The results are expressed as IC50, which is the concentration
necessary for 50% of inhibition.
DMSO-d6) d: 0.62e0.69 (m, 3H), 1.06e1.35 (m, 7H), 1.36e1.40 (d, 2H),
3.75e3.86 (t, J ¼ 6.0 Hz, 2H), 4.10e4.16 (m, 1H), 4.81e4.85 (m, 1H),
5.33e5.42 (m, 1H), 5.68e5.82 (m, 1H), 8.07 (d, J ¼ 6.0 Hz, 1H), 9.72 (br, s,
1H). 13C NMR (75 MHz, DMSO-d6)
d: 155.4, 152.9, 140.5, 137.2, 129.8, 93.7,
81.06, 75.9, 71.87, 65.9, 38.5, 26.9, 26.0, 23.7, 18.5, 11.6. LCMS m/z (%) 384
[M ꢀ H]ꢀ; Anal. Calcd. for C16H20FN3O7: C, 49.87; H, 5.23; N, 10.90%.
Found: C, 49.85; H, 5.21; N, 10.91%.
6.1.3.9. Synthesis of 50-deoxy-5-fluoro-N4-(4-nitro-phenyloxycarbonyl)
cytidine-20,30-carbonate (5d). Yield: 86%; m.p. 167e169 ꢁC; IR (KBr) 3235,
2986, 1812, 1746, 1632 cmꢀ1; 1H NMR (300 MHz, DMSO-d6)
d: 1.32 (d,
J¼ 6.0 Hz, 3H), 4.09 (q, J¼ 6.0 Hz,1H), 4.78 (m, J¼ 6.0 Hz,1H), 5.31 (m,1H),
5.74 (d, J¼ 7.2 Hz,1H), 6.68 (d, 2H, J ¼ 8.2 Hz, Ar-H), 6.96 (d, 2H, J ¼ 8.2 Hz,
Ar-H), 7.68 (br, s, 1H), 11.25 (s, 1H). 13C NMR (75 MHz, DMSO-d6)
d: 156.2,
153.6,151.0,138.6,135.4,131.6,130.9,128.6, 92.7, 83.2, 83.0, 82.2, 65.4, 31.2,
29.7, 28.9,18.7. LCMS m/z(%) 435 [M ꢀ H]ꢀ; Anal. Calcd. for C17H13FN4O9:C,
46.80; H, 3.00; N, 12.81%. Found: C, 46.78; H, 2.97; N, 12.79%.
6.2.3. In vivo activities of new 50-deoxy-5-fluorocytidine derivatives
against murine leukemia L1210
6.1.3.10. Synthesis of 50-deoxy-5-fluoro-N4-(4-chloro-phenyloxycarbonyl)
cytidine-20,30-carbonate (5e). Yield: 86%; m.p. 157e159 ꢁC; IR (KBr)
L1210 cells were implanted intraperitoneally (ip) into BDF-1
male mice (6 weeks old) on day 0 and the mice were divided into
several groups (8 mice per group) on day 1. The test compounds
(capecitabine, 5d, 5e) were dissolved in saline and orally (po)
administrated 5 days a week for 3 weeks. Because of systemic
toxicity the test compound 5a was dissolved in saline and admin-
istrated per os (po) daily for 2 weeks.
3152, 2958, 1810, 1752, 1630 cmꢀ1; 1H NMR (300 MHz, DMSO-d6)
d:
1.35 (d, J ¼ 6.0 Hz, 3H), 4.12 (q, J ¼ 6.0 Hz,1H), 4.81 (q, J ¼ 6.0 Hz,1H),
5.32 (d, J ¼ 9.0 Hz,1H), 5.68 (br, s,1H), 6.02 (d, J ¼ 8.4 Hz, 2H), 6.54 (d,
J ¼ 8.4 Hz, 2H), 8.11 (d, J ¼ 9.0 Hz,1H),10.65 (s,1H).13C NMR (75 MHz,
DMSO-d6) d: 156.4,153.6,151.2,138.6,135.8,131.1,130.6,128.6,123.2,
94.1, 84.2, 83.2, 81.5, 65.4, 32.4, 29.4,18.5. LCMS m/z (%) 426 [M þ H]þ;
Anal. Calcd. for C17H13ClFN3O7: C, 47.96; H, 3.08; N, 9.87%. Found: C,
47.94; H, 3.05; N, 9.85%.
6.2.4. Construction of correlation models
Correlation models were constructed among IC50 values of the
compounds and their respective predicted log P (partition coeffi-
cient) values. The three dimensional models were constructed for all
the compounds (5-FU and 4ae5e) in Molecular Operating Envi-
ronment (MOE) working area [22]. The structures were energy
minimized in MMFF94x force filed with implicit solvated environ-
ment at an RMS gradient of 0.05. The stabilized conformations were
used to predict the log P values using the MOE descriptor calculator
module. The resultant values were used to construct the correlation
models taking IC50 values on X-axis and log P values on Y-axis.
6.2. Pharmacology
6.2.1. Bacterial reverse mutation assay
The assay was performed with and without liver microsomal
activation. Each concentration, including the negative, vehicle and
positive controls, were tested in triplicate. The test item was tested
at the following concentrations: 0.312, 0.625, 1.25, 2.5 and 5 mg/
plate, both in the presence of metabolic activation (þS9) and in the
absence of metabolic activation (ꢀS9) [14,17e19,20].
For each strain and dose level, including the controls three
plates were used. The following materials were mixed in a test tube
and poured onto the selective agar plates:
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100
or reference mutagen solution (positive control),
500 L S9 mix (for test with metabolic activation) or S9 mix
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100 L Bacterial suspension,
2000 L Overlay agar.
mL Test solution at each dose level, solvent, negative control
m
m
m
In the pre-incubation assay 100
m
L test solution, 500
mL S9 mix/
S9 mix substitution buffer and 100
mL bacterial suspensions were

696
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Glossary
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capecitabine at 2.00 mmol/kg/day) showed a small but significant decrease in
body weight relative to the group without tumor cells.
5-FU: 5-Fluorouracil
50-DFCR: 50-deoxy-5-fluorocytidine
50-DFUR: doxifluridine
APCI-MS: Atmospherics pressure chemical ionization-Mass spectrometry
CDI: Carbonyl diimidazole
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Amsterdam, 1977, pp. 1e17.
CDM: Methylene dichloride
DPD: dihydropyrimidine dehydrogenase
FUPA: 5-fluoro-ureidopropionic acid
MST: Mean survival time
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MTT: (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
TBME: tertiary methyl butyl ether
UFT: Tegafur-Uracil