Fluorocyclopentenyl-cytosine with broad spectrum and potent antitumor activity

Article abstract of DOI:10.1021/jm3004009

On the basis of the potent biological activity of cyclopentenyl- pyrimidines, fluorocyclopentenyl-pyrimidines were designed and synthesized from d-ribose. Among these, the cytosine derivative 5a showed highly potent antigrowth effects in a broad range of

Full text of DOI:10.1021/jm3004009

Brief Article
pubs.acs.org/jmc
Fluorocyclopentenyl-cytosine with Broad Spectrum and Potent
Antitumor Activity†
Won Jun Choi,‡,§ Hwa-Jin Chung,∥ Girish Chandra,‡ Varughese Alexander,‡ Long Xuan Zhao,‡,⊥
Hyuk Woo Lee,‡ Akshata Nayak,‡ Mahesh S. Majik,‡ Hea Ok Kim,‡ Jin-Hee Kim,‡ Young B. Lee,#
Chang H. Ahn,# Sang Kook Lee,*,∥ and Lak Shin Jeong*,‡
‡Department of Bioinspired Science and College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
§College of Pharmacy, Dongguk University, Kyungki-do 410-774, Korea
∥College of Pharmacy, Seoul National University, Seoul 151-742, Korea
⊥College of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116-029, China
#Rexan Pharmaceuticals, Inc., Bethesda, Maryland 20850, United States
S
* Supporting Information
ABSTRACT: On the basis of the potent biological activity of cyclopentenyl-pyrimidines, fluorocyclopentenyl-pyrimidines were
designed and synthesized from D-ribose. Among these, the cytosine derivative 5a showed highly potent antigrowth effects in a
broad range of tumor cell lines and very potent antitumor activity in a nude mouse tumor xenograft model implanted with A549
human lung cancer cells. However, its 2′-deoxycytidine derivative 5b did not show any antigrowth effects, indicating that 2′-
hydroxyl group is essential for the biological activity.
hydrolase and to exhibit potent antiviral activity against
vesicular stomatitis virus (VSV).5 Compound 2 was shown to
be a novel dual mechanism-based inhibitor of SAH hydrolase.5
On the other hand, the cyclopentenyl-cytosine analogue 3
showed potent antitumor and antiviral activities by reducing
cytidine-5′-triphosphate (CTP) pools.6 Another 5-fluorocyto-
sine analogue 4 also exhibited potent antiviral activity against
the West Nile virus although the compound is rather cytotoxic.7
Thus, on the basis of the potent biological activity of 3 and 4,
there is interest in designing and synthesizing the fluorocyclo-
pentenyl-cytosine derivative 5a (R = OH) and measuring its
biological activity. Additionally, the synthesis of its 2′-deoxy
analogue 5b (R = H) will help determine if the 2′-hydroxyl
group is critical for the biological activity. Moreover, it is also
interesting to study the biological target involved in the
antitumor activity of 5a. Herein, we report the synthesis of the
fluorocyclopentenyl-cytosine derivatives 5a and 5b, the potent
antitumor activity of 5a, both in in vitro and in vivo studies, and
the mechanism of action of 5a.
INTRODUCTION
■
Neplanocin A (1, Figure 1),1,2 a naturally occurring nucleoside,
shows potent antiviral and antitumor activities by inhibiting S-
RESULTS AND DISCUSSION
■
For the synthesis of the desired nucleosides, the key
intermediate 138,9 was first synthesized with a benzyl protecting
group, as shown in Scheme 1, because other protecting groups
such as trityl, TBS, and TBDPS were not successful in the
electrophilic vinyl fluorination.
Figure 1. The rationale for the design of fluorocyclopentenyl-cytosine
derivatives 5a and 5b.
adenosylhomocysteine (SAH) hydrolase, which catalyzes the
interconversion of S-adenosylhomocysteine into adenosine and
L-homocysteine.3 Compound 1 causes the depletion of the
cofactor NAD+ by inhibiting SAH hydrolase. This inhibition is
reversed by the addition of excess cofactor NAD+.4 Its 6′-fluoro
analogue 2 was also reported to be a potent inhibitor of SAH
We previously reported that the Grignard reaction to the
ketone 8 in the case of the benzyl protecting group produced
desired β-hydroxydiene 11 as the minor isomer and α-
Received: March 22, 2012
Published: April 23, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Synthesis of the Key Intermediate 13 with Benzyl
Protecting Group
Scheme 3. Synthesis of the 2′-Deoxy Derivatives 5b and 22
a
a
Reagents and conditions: (a) c-H2SO4, acetone, then TBDPSCl,
imidazole; (b) Ph3PCH3Br, KOt-Bu, THF; (c) (COCl)2, DMSO,
CH2Cl2, −78 °C, 1 h, then Et3N, rt; (d) CH2CHMgBr, THF, −78
°C; (e) n-Bu4NF, THF; (f) (i) Bu2Sn(O), toluene, 15 h, (ii) TBAI,
BnBr, 50 °C, 16 h; (g) Grubbs catalyst, toluene, 80 °C; (h) PDC,
molecular sieves, DMF, rt, 18 h.
Scheme 2. Synthesis of the 2′-Hydroxyl Derivatives 5a and
a
19
a
Reagents and conditions: (a) TIDPSCl2, pyridine, rt; (b) PhOC(S)-
Cl, pyridine, then n-Bu3SnH, AIBN, toluene, 120 °C; (c) TBAF, THF,
rt; (d) (i) Ac2O, pyridine, (ii. POCl3, Et3N, 1,2,4-triazole, (iii)
NH4OH, 1,4-dioxane, (iv) NH3/MeOH.
of 6 followed by Swern oxidation produced ketone 8. A
Grignard reaction of 8 with vinylmagnesium bromide yielded
the diene 9 as a single stereoisomer. Removal of the TBDPS
group in 9, followed by selective benzylation of the resulting
diol 1010 using organotin chemistry, produced the benzylate 11.
A ring-closing metathesis (RCM) reaction of 11 using the
second-generation Grubbs catalyst gave the β-hydroxycyclo-
pentene 12. Oxidative rearrangement of 12 with PDC in DMF
yielded the benzylated cyclopentenone 136 with concomitant
minor formation of its benzoylated derivative, resulting from
the benzylic oxidation (see page S4 in Supporting Information).
The key intermediate 13 was first converted to known
glycosyl donor 175 and then to the final cytosine derivative 5a
(Scheme 2). Conventional iodination of 13 with iodine and
pyridine in CCl4 was slow, but when the solvent was changed
to THF, iodocyclopentenone 14 was obtained at a very good
yield. Reduction of 14 with sodium borohydride, followed by
TBDPS protection of the resulting 15, produced TBDPS ether
16. Electrophilic vinyl fluorination was successfully achieved by
adding n-BuLi to a solution of 16 and N-fluorobenzenesulfo-
nimide (NFSI) at −78 °C, giving 17 after desilylation.5
Condensation of 17 with N3-benzoyluracil under the standard
Mitsunobu conditions yielded the uracil derivative 18. Treat-
ment of 18 with methanolic ammonia, followed by treatment
with boron tribromide, produced the uracil derivative 19. The
uracil derivative 19 was converted to the cytosine derivative 5a
in three steps. Treatment of 19 with acetic anhydride gave the
triacetate, which was converted to the triazole derivative by
a
Reagents and conditions: (a) I2, pyridine, THF; (b) NaBH4, CeCl3-
7H2O, MeOH; (c) TBDPSCl, imidazole, DMF; (d) NFSI, n-BuLi,
THF, −78 °C; (e) n-Bu4NF, THF; (f) N3-benzoyluracil, DEAD, Ph3P,
THF; (g) NH3/MeOH; (h) BBr3, CH2Cl2, −78 °C ; (i) (i) Ac2O,
pyridine, (ii) POCl3, Et3N, 1,2,4-triazole, (iii) NH4OH, 1,4-dioxane;
(iv) NH3/MeOH.
hydroxydiene as the major isomer, but the minor isomer 11
could only undergo oxidative rearrangement to give 13.8 Thus,
we first synthesized β-hydroxydiene 9 with the TBDPS
protecting group stereoselectively in five steps, according to a
previously published procedure,8 and then converted the
TBDPS group into the benzyl group. Treatment of D-ribose
with acetone and c-H2SO4, followed by TBDPS protection of
the resulting 2,3-acetonide, gave the lactol 6. A Wittig reaction
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Table 1. Broad Spectrum and Potent Antitumor Activity (IC50, μM) of Compound 5a in a Variety of Human Tumor Cell Lines
tumor cell lines
OVCAR-3
(ovary)
MCF-7
MDA-MB-231
(breast)
HeLa
(cervix)
PC-3
(prostate)
LNCap
(prostate)
HepG2
(liver)
A549
(lung)
UMRC2
(kidney)
(breast, hormone dependent)
IC50 (μM)
IC50 (μM)
0.80
0.34
0.18
1.35
0.63
2.67
0.79
0.50
0.83
tumor cell lines
NCIH226
(lung)
HT-29
HCT116
(colon)
SK-MEL-28 PANC-1
(melanoma)
1.38
U251
(brain)
MKN45
(stomach)
K562
(leukemia)
(colon)
(pancreas)
0.62
0.25
0.28
0.19
0.83
0.34
0.82
Figure 2. Inhibition of tumor growth by 5a using an A549 xenograft model. (A,B) A549 cells (3 × 106 cells) were injected subcutaneously into the
right flank of nude mice. When tumor volumes reached ca. 100 mm3, treatment was initiated. 5a (3 or 10 mg/kg) was administered intraperitoneally
three times a week for three weeks. Tumor volumes were measured with a caliper every 2−3 days. (C) Weight of the removed xenograft tumors on
day 38 was measured. *p < 0.05, **p < 0.01 indicates statistically significant differences from the control group. (D) Body weight changes of the
mice were monitored during the experiments.
Removal of the TIPDS group of 21 with tetra-n-butylammo-
nium fluoride produced the final 2′-deoxyuridine derivative 22.
The 2′-deoxyuridine derivative 22 was converted to the 2′-
deoxycytidine derivative 5b according to the same procedure
used in Scheme 2. All synthesized final nucleosides 5a, 5b, 19,
and 22 were tested for in vitro antigrowth effects in a variety of
human tumor cell lines using the sulforhodamine B (SRB)
Figure 3. Effect of 5a on the expression of DNMT-1 protein in MDA-
method. The cytotoxic effects were measured at a concen-
MB-231 cancer cells. Cells were treated without or with 5a at 1, 1.5, 2,
tration of 1 μM of the final nucleosides. As shown in Table 1,
or 5 μM for 24 h. The expression of DNMT-1 protein was determined
only the cytosine derivative 5a exhibited highly potent
by Western blot analysis. Actin was determined as a loading control.
antitumor activity against a broad range of tumor cell lines at
Representative data are shown from two independent cell culture
the above-mentioned concentration. Interestingly, compound
5a showed much weaker antileukemic activity than 3,6 a CTP-
synthetase inhibitor. This finding indicates that different
mechanism of action may be involved in the antitumor activity
of 5a. However, the corresponding 2′-deoxy analogue 5b did
not show any cytotoxic effect, indicating that the 2′- hydroxyl
group is essential for the biological activity of compound 5a.
Lung cancer is the leading cause of cancer-related death in
the world.11 Along with surgery and radiotherapy, chemo-
therapy is one of the most common treatments for lung cancer
therapy. Although the overall survival has been improved
significantly in advanced nonsmall cell lung cancer (NSCLC)
using platinum-based combinations with chemotherapeutic
agents such as vinorelbine, gemcitabine, and the taxanes during
experiment.
treating with POCl3 and 1,2,4-triazole in the presence of
triethylamine. The triazole derivative was converted to the
cytosine derivative by treating with ammonium hydroxide in
1,4-dioxane. Removal of the acetate group with methanolic
ammonia yielded the final cytosine derivative 5a.
To determine if a ribosyl template is essential for the
biological activity, the 2′-deoxy derivative 5b was synthesized as
shown in Scheme 3. Compounds 5a was treated with
TIPDSCl2 to give the 3′,5′-TIPDS-protected nucleoside 20.
Treatment of 20 with phenyl chlorothionoformate in pyridine
gave the thiocarbonate, which was treated with n-Bu3SnH and
AIBN in refluxing toluene to give the 2′-deoxy derivative 21.
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the past decade, the one-year survival rates are typically 35%
and the two-year survival rates approach only 15−20% in
patients with advanced NSCLC.12−14 Therefore, the develop-
ment of novel approaches to prevent and treat lung cancer is an
important goal.
AUTHOR INFORMATION
■
Corresponding Author
*For L.S.J.: phone, 82-2-3277-3466; fax, 82-2-3277-2851; E-
mail, lakjeong@ewha.ac.kr. For S.K.L.: phone, 82-2-880-2475;
fax, 82-2-762-8322; E-mail, sklee61@snu.ac.kr.
Thus, we evaluated the antitumor activity of 5a in a nude
mouse tumor xenograft model implanted with A549 human
lung cancer cells to determine if in vitro antigrowth effects were
correlated with in vivo antitumor effects. A549 cells (3 × 106
cells/mouse) were injected subcutaneously into the right flank
region of male nu/nu mice. When the tumor reached to
approximately 100 mm3, compound 5a was administered three
times a week for three weeks by intraperitoneal injection (3 or
10 mg/kg). The tumor volume in the control group was
approximately 1400 mm3 on day 38 after the cells were
inoculated. Treatment of 5a significantly inhibited the tumor
growth, and the tumor volume (Figures 2A,B) and tumor
weight (Figure 2C) were decreased in a dose-dependent
manner. The percent inhibition of the tumor volume compared
with the vehicle-administered control group were 31.8% and
58.1% at 3 and 10 mg/kg doses of 5a, respectively. Under the
same experimental conditions, gemcitabine (10 mg/kg), a drug
clinically used to treat lung cancer, showed 76.7% inhibition.
No overt toxicity or body weight change was apparent in the
5a-treated group compared to the control group (Figure 2D).
These results suggest that compound 5a might be a promising
new chemotherapeutic candidate for the treatment of lung
cancer.
To determine the mechanism of action involved in potent
antitumor activity of 5a, we examined the effect of 5a against
DNA methyltransferase (DNMT-1) because of the structural
resemblance between 5a and 5-azacytidine,15 which is a potent
inhibitor of DNMT-1. When MDA-MB-231 (breast cancer)
cells were incubated with various concentrations of 5a, the
expression of DNMT-1 protein was inhibited in a dose-
dependent manner (Figure 3). This preliminary study indicates
that 5a shows different mechanism of action from 3,6 which is a
CTP-synthetase inhibitor.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the grant from the WCU project
(R31-2008-000-10010-0), National Research Foundation
(NRF), Seoul R&BD Program (ST100039), the GRRC
program of Gyeonggi province(GRRC-DONGGUK2011-
B02), and the Ewha Global Top5 Grant 2011 of Ewha
Womans University.
ADDITIONAL NOTE
■
†
Part of this work was presented in the 4th International
Symposium on Nucleic Acids Chemistry, Fukuoka, Japan,
September 20−22, 2005.
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CONCLUSIONS
■
On the basis of the potent biological activity of pyrimidine
nucleosides with a cyclopentene ring, we have synthesized
novel pyrimidine nucleosides with a fluorocyclopentene ring via
a stereoselective Grignard reaction and electrophilic vinyl
fluorination as key steps. Among the compounds tested,
cytosine derivative 5a demonstrated broad spectrum and potent
antitumor activity in a broad range of human tumor cell lines as
well as in a xenograft nude mouse model. These biological
activity data reported here suggest that compound 5a is a
promising, clinically useful anticancer agent that should be
further investigated. The detailed mechanism of action of 5a is
currently being studied and will be described in subsequent
reports.
ASSOCIATED CONTENT
■
S
* Supporting Information
Complete experimental procedures and characterization data
1
and H and 13C NMR, 19F copies of 19, 22, 5a, and 5b. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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