Synthesis of new pyrimidine nucleoside derivatives with nitric oxide donors for antiviral activity

Article abstract of DOI:10.1016/j.cclet.2011.01.010

New pyrimidine nucleoside derivatives with nitric oxide (NO) donor were systematically synthesized. The antivirus activities of these nucleoside analogues against vesicular stomatitis virus (VSV) in Wish cell were evaluated. It was demonstrated that most of compounds had stronger antiviral acitivity than acyclovir, while their toxicities were similar or lower to acyclovir.

Full text of DOI:10.1016/j.cclet.2011.01.010

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 899–902
www.elsevier.com/locate/cclet
Synthesis of new pyrimidine nucleoside derivatives
with nitric oxide donors for antiviral activity
Jing Bo Shi a,b, Song Xu a, Ya Ping Wang c, Jing Jing Li a, Qi Zheng Yao a,
*
a School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
b School of Pharmacy, Anhui Medical University, Hefei 230032, China
c Anhui Zhaoke Pharmaceutical Co., Ltd., Hefei 230088, China
Received 3 November 2010
Available online 18 May 2011
Abstract
New pyrimidine nucleoside derivatives with nitric oxide (NO) donor were systematically synthesized. The antivirus activities of
these nucleoside analogues against vesicular stomatitis virus (VSV) in Wish cell were evaluated. It was demonstrated that most of
compounds had stronger antiviral acitivity than acyclovir, while their toxicities were similar or lower to acyclovir.
# 2011 Qi Zheng Yao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Pyrimidine nucleoside; NO-donor; Antivirus; Furoxans; Oximes; Synthesis
Structural analogues of nucleosides, nucleoside analogues (NA), together with nucleobases and nucleotide
analogues, are commonly used in the treatment of viral infection and cancer. In both cases, they act as antimetabolite
agents and interfere with the synthesis of cellular or viral nucleic acids [1,2]. Most of these agents are hydrophilic
molecules and therefore require specialized transporter proteins to enter cells. Once inside, they are activated by
intracellular metabolic steps to triphosphate derivatives. Active derivatives of nucleoside analogues can exert
cytotoxic activity by being incorporated into and altering the DNA and RNA macromolecules or by interfering with
various enzymes involved in synthesis of nucleic acids, such as DNA polymerases and ribonucleotide reductase. These
actions result in inhibition of DNA synthesis and apoptotic cell death [3,4].
NAs such as didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), zidovudine (AZT), stavudine (d4T) and
abacavir (ABC) are used in antiviral treatment. These compounds belong to the class of nucleoside reverse
transcriptase (RT) inhibitors (NRTI) [2]. Major problems in the treatment of viral and cancer diseases with NAs are
acquirement of resistance and side effects such as delayed cytotoxicity. Accordingly, its therapeutic efficacy is limited,
the development of new NAs with potent anti-virus and anti-tumor activity should be of great significance.
Nitric oxide (NO) is one of the most important signaling molecules within physiology and pathophysiology
[5]. NO is produced by a group of enzymes called nitric oxide synthases (NOS). It is generally accepted that there
are three main isoforms of the NOS enzyme, endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible
(iNOS) [6].
* Corresponding author.
E-mail address: qz_yao@yahoo.com.cn (Q.Z. Yao).
1001-8417/$ – see front matter # 2011 Qi Zheng Yao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.01.010

[()TD$FIG]
900
J.B. Shi et al. / Chinese Chemical Letters 22 (2011) 899–902
O
R
OH
R
O
1
1
OH
R
OH
1
a
b
O
O
O
c
N
N
N
N
O
O
1a R =H
2a R =H
2b R =CH
3a R =H
1
3b R =CH
1 3
1
1
1
1b R =CH
1
3
3
HO
OH
OH
O
C
N
4a R =CH , n=0
3
3
3
3
5a R =CH , n=0
3
3
3
3
3
4b R =CH , n=2
3
5b R =CH , n=2
C
H
4c R =p-Cl-Ph, n=0
R
R
3
3
5c R =p-Cl-Ph, n=0
H
n
2
2
n
Scheme 1. General method for the synthesis of NO donors 3a–b, 5a–c. Reagents and conditions: (a) NaNO2, AcOH, rt. 3 h; (b) succinic anhydride,
ACN, Py, rt. 24 h; (c) NH2OH HCl (aq), KOH, EtOH, reflux, 3 h.
Indeed, previous studies have shown that synthesized NO-releasing nucleoside analogues have strong cytotoxicity
against human carcinoma cells or have strong antiviral activity in vitro [7–9]. Furoxans and alkyl- or aryloximes are
thermally stable compounds and represent an important class of NO-donors, which can produce high levels of NO in
vitro, and inhibit the growth of tumors in vivo [7,10].
In order to get more potent antiviral and/or antitumor NAs, a series of new stavudine and b-thymidine derivatives
combined NO donors, namely furoxans or aryloximes, were designed and synthesized.
The synthetic routes of substituted furoxan or aryloxime NO donors 3a–b, 5a–c are outlined in Scheme 1. The
substituted furoxans were prepared in a three-step sequence. The starting material cinnamyl alcohol derivates 1a, 1b
were converted to 2a, 2b by treatment with NaNO2 and glacial acetic acid in 40–90% yield [11]. 2a, 2b were acylated
by succinic anhydride to generate succinic acid mono ester 3a, 3b. The substituted oxime NO donors were synthesized
from the ketone derivates 4a–c, treated with NH2OHꢀHCl and KOH to produce the oxime derivates 5a–c in 50–66%
yield [12].
The target compounds 8a–b, 10a–d, 11a–b, 12a–d, 13, 15a–c, so called NO donor/NA hybrids, were synthesized as
outlined in Scheme 2. To prepare compounds 8a–b, b-thymidine was treated with triphenylmethyl chloride (TrCl) in
dry pyridine to yield 6, then reacted with 3a or 3b in the presence of N0,N0-dicyclohexylcarbodiimide (DCC) and 4-
(dimethylamino)pyridine (DMAP) in CH2Cl2 to lead the compounds 7a–b. Finally, the protected groups of 7a–b were
removed with CF3COOH to give the compounds 8a–b [13]. The compounds 10a–d, 12a–d were preparated by using
uridine or 5-iodouridine as starting material. It was respectively converted to 20,30-O-isopropylideneuridine 9a–b by
treatment with 4-methylbenzenesulfonic acid, triethoxymethane and acetone at room temperature in 60–70% yield
[14]. The esterification of compounds 3a or 3b with 9a or 9b in the above-mentioned reaction conditions gave the
compounds 10a–d with yields in the range of 40–76%. Subsequently, treatment of 10a–d with CF3COOH cleaved the
20,30-O-isopropylidene-protected group to give the compounds 12a–d [13]. b-Thymine or 20-deoxy-5-fluorouridine
was treated with two equilibrium of 3a in the presence of DCC and DMAP in CH2Cl2 to generate the compounds 11a–
b in yield of 50–69%, respectively. Stavudine was reacted with 3b to form the compound 13 [13] in 42.1% yield. In
order to get 15a–c, stavudine was reacted with succinic anhydride in pyridine to bring the corresponding ester 14. The
esterification of compounds 5a, 5b or 5c with 14 in the presence of DCC and DMAP in CH2Cl2 lead to the compounds
15a–c, respectively [13].
The antiviral activity of synthesized NO releasing nucleosides against vesicular stomatitis virus(VSV) in Wish cell
was evaluated by previously reported method [15]. Acyclovir and stavudine were selected as positive control
compounds. The TC0 (maximal atoxic concentration) values of individual compounds for Wish cell and ED50 values
against VSV are presented in Table 1.
As shown in Table 1, ten of prepared nucleosides showed potent anti-VSVactivities which were stronger than that
of acyclovir, while most of their toxicities were similar to or lower than that of acyclovir, except 10b, 10d, 13.
Although stavudine is used as a clinical antiviral agent, anti-VSV activities of stavudine derivatives 13 and 15b were
not higher than other thymidine derivatives. It showed that the combined NO donors at 50-position of sugar may play an
important role in enhancing their antiviral activities.

[()TD$FIG]
J.B. Shi et al. / Chinese Chemical Letters 22 (2011) 899–902
901
2
O
N
O
O
N
R
R
2
2
HN
R
HN
2
HN
O
O
N
O
N
HO
N
O
O
N
N
TrO
O
O
O
TrO
O
c
O
b
O
O
O
O
R
O
1
OH
O
R
1
O
O
6 R =CH
2
3
7a R =H, R =CH
3
8a R =H, R =CH
3
8b R =CH , R =CH
1
2
1
1
2
7b R =CH , R =CH
1
3
2
O
3
3
2
3
a
O
N
O
N
R
R
2
R
2
2
HN
HN
HN
O
O
N
O
O
HO
HO
R
O
N
1
d
O
O
O
O
e
O
OHR
4
O
O
O
O
O
N
O
R =H, I, CH , F
4
2
3
10c R =CH , R =H
10a R =H, R =H
9a R =H
1
3
2
1
2
R =H, OH
2
10d R =CH , R =I
10b R =H, R =I
9b R =I
2
3
2
2
2
2
O
O
g
f
R
R
2
2
HN
HN
O
O
O
N
O
N
O
N
N
R
O
R
O
N
O
O
1
1
O
O
O
O
O
O
N
O
O
O
R
1
O
HO
OH
N
N
O
O
O
11a R =H, R =CH
11b R =H, R =F
12c R =CH , R =H
1 3 2
12d R =CH , R =I
2 3 2
12a R =H, R =H
1
1
2
2
3
1
2
2
12b R =H, R =I
2
O
N
O
N
CH
3
CH
3
HN
O
HN
O
O
O
H C
O
O
3
h
O
HO
O
N
O
13
N
O
Stavudine
i
O
N
O
N
CH
3
CH
HN
O
3
HN
O
O
O
O
O
O
HO
j
O
O
N
HO
O
15a R =CH , n=0
3
3
O
C
R
15b R =CH , n=2
3
3
3
H
2
n
15c R =p-Cl-Ph, n=0
14
3
Scheme 2. General method for the synthesis of 8a–b, 10a–d, 11a–b, 12a–d, 13, 15a–c. Reagents and conditions: (a) TrCl, Py, reflux, 45 min; (b) 3a
or 3b, DCC, DMAP, CH2Cl2, rt. 24 h; (c) CF3COOH, rt. 1 h; (d) uridine, 4-methylbenzenesulfonic acid, triethoxymethane, acetone, rt. 24 h; (e) 3a or
3b, DCC, DMAP, CH2Cl2, rt. 24 h; (f) CF3COOH, rt. 1 h; (g) 3a, DCC, DMAP, CH2Cl2, rt. 24 h; (h) 3b, DCC, DMAP, CH2Cl2, rt. 24 h; (i) succinic
anhydride, Py, reflux, 3 h; (j) 5a, 5b or 5c, DCC, DMAP, CH2Cl2, rt. 24 h.

902
J.B. Shi et al. / Chinese Chemical Letters 22 (2011) 899–902
Table 1
Toxicity for Wish cell and anti-VSV effect of control compounds and NO-NA derivatives 8a–b, 10a–d, 11a–b, 12a–d, 13, 15a–c.
Compound
Acyclovir
Stavudine
8a
8b
10a
10b
10c
10d
11a
(TC0) (mmol/L)
ED50 (mmol/L)
2775
2788
N.D.
1210
N.D.
589
560
4566
546
8949
765
830.8
180.2
229.7
172.1
186.9
284.1
282.0
Compound
11b
12a
12b
12c
13
15a
15b
15c
(TC0) (mmol/L)
ED50 (mmol/L)
762
603
485
1174
460.3
6098
1366
N.D.
1287
70
301.0
N.D.
N.D.
238.6
278.7
N.D.
Note: N.D.: not determined.
Further NO-release and anti-tumor studies of these new nucleosides in correlated cells are currently in progress.
Additional results will be reported in due course.
Acknowledgments
We are grateful to the National Natural Science Foundation of China (No. 30572240).
References
[1] H. Hillaireau, T. Le Doan, P. Couvreur, J. Nanosci. Nanotechnol. 6 (2006) 2608.
[2] A.R. Van Rompay, M. Johansson, A. Karlsson, Pharmacol. Ther. 100 (2003) 119.
[3] C.M. Galmarini, J.R. Mackey, C. Dumontet, Lancet Oncol. 3 (2002) 415.
[4] C.M. Galmarini, J.R. Mackey, C. Dumontet, Leukemia 15 (2001) 875.
[5] R. Schulz, T. Rassaf, P.B. Massion, et al. Pharmacol. Ther. 108 (2005) 225.
[6] J.A. Coulter, H.O. McCarthy, J. Xiang, et al. Nitric Oxide 19 (2008) 192.
[7] S. Moharram, A. Zhou, L.I. Wiebe, et al. J. Med. Chem. 47 (2004) 1840.
[8] J.B. Shi, J. Gao, Q.Z. Yao, et al. Chin. Chem. Lett. 20 (2009) 404.
[9] S. Wang, J. Chang, S. Pan, et al. Helv. Chim. Acta 87 (2004) 327.
[10] P.G. Wang, M. Xian, X. Tang, et al. Chem. Rev. 102 (2002) 1091.
[11] A.M. Gasco, R. Fruttero, G. Sorba, et al. Liebigs Ann. Chem. 1991 (1991) 1211.
[12] G. Massolini, M.L. Carmellino, A. Baruffini, Farmaco Sci. 42 (1987) 117.
[13] The data of selected compounds. 8a: white foam; mp 72–74 8C; EMS-MS: 539[M+Na]+, 517[M+H]+, 515[Mꢁ1]+, C23H24N4O10(Mr: 516.46);
1H NMR (300 MHz, CDCl3): d 9.1(s, 1H, NH), 7.52–7.71 (m, 6H, ArH, 6-CH), 6.20 (q, 1H, 10-CH), 5.37 (t, 1H, 40-CH), 5.19 (s, 2H, O-CH2),
4.10 (q, 1H, 30-CH), 3.89 (d, 2H, J = 3 Hz, 50-CH2), 2.68 (s, 5H, 2ꢂCH2, OH), 2.34–2.45 (m, 2H, 20-CH2), 1.91 (s, 3H, CH3); Anal. Calcd. for.
C
23H24N4O10 + 0.2CH3COOCH2CH3(534.08): C 53.52, H 4.83, N 10.49; found: C 53.76, H 4.82, N 10.21. 12a: white foam; mp 68–70 8C;
EMS-MS: 541[M+Na]+, 519[M+H]+, C22H22N4O11 (Mr: 518.43); 1H NMR (300 MHz, CDCl3): d 9.78 (s, 1H, NH), 7.52–7.70 (m, 6H, ArH, 6-
CH), 5.75–5.79 (m, 2H, 5-CH, 10-CH), 5.16 (s, 2H, O-CH2), 4.19–4.38 (m, 5H, 20-CH, 30-CH, 40-CH, 50-CH2), 2.9 (s, 2H, 2ꢂOH), 2.67 (s, 4H,
2ꢂCH2); Anal. Calcd. for C22H22N4O11 + 0.25CH3COOCH2CH3(540.45): C 51.11, H 4.48, N 10.37; found: C 50.61, H 4.70, N 9.91. 13: white
foam; mp 69–72 8C; EMS-MS: 535[M+Na]+, 513[M+H]+, C24H24N4O9 (Mr: 512.47); 1H NMR (300 MHz, CDCl3): d 8.66 (s, 1H, NH), 7.53–
7.72 (m, 5H, ArH), 7.21 (d, 1H, 10-CH), 6.98 (d, 1H, J = 3 Hz, 40-CH), 6.24 (q, 1H, O-CH), 5.89 (d, 2H, O-CH2), 5.01 (s, 1H, 6-CH), 4.41–4.21
(m, 2H, –CH CH–), 2.60–2.61 (m, 4H, 2ꢂCH), 1.90 (d, 3H, CH3), 1.62 (s, 3H, CH3); Anal. Calcd. for C24H24N4O9 + CH3COOCH2CH3
(600.58): C 55.90, H 5.52, N 9.31; found: C 55.85, H 5.07, N 9.48. 15a: white foam; mp 78–80 8C; EMS-MS: 480[M+Na]+, 456[Mꢁ1]+,
C22H23N3O8(Mr: 457.43); 1H NMR (300 MHz, CDCl3): d 8.43 (s, 1H, NH), 7.59 (d, 2H, ArH), 7.28 (d, 1H, 10-CH), 6.96 (d, 1H, 40-CH), 6.85
(d, 2H, ArH), 5.91–6.3 (dd, 2H, 50-CH2), 5.05 (s, 1H, 6-CH), 4.22–4.52 (m, 2H, –C C–), 2.67–2.94 (m, 4H, 2ꢂCH2), 2.33 (s, 3H, CH3), 1.93
(s, 3H, CH3); Anal. Calcd. for C22H23N3O8 + 1.75H2O (488.93): C 54.04, H 5.46, N 8.59; found: C 53.99, H 5.01, N 8.34.
[14] N.C. Yung, J.J. Fox, J. Am. Chem. Soc. 83 (1961) 3060.
[15] Y.J. Deng, J.B. Shi, Q.Z. Yao, et al. Acta Chim. Sin. 64 (2006) 1911.