Synthesis and antiviral evaluation of novel N-6 substituted adenosine analogues

Article abstract of DOI:10.1016/j.tetlet.2016.11.059

A series of adenosine analogues were synthesized and their biological evaluation was tested against Coxsackie virus B3 (CVB3) and Herpes simplex virus type 1(HSV-1) in HEp-2 cells. The hydrophobic constant, acute toxicity, carcinogenicity and mutagenicity were calculated. Analogues with piperazine derivatives 8b showed promising activities against CVB3 with a lower IC₅₀value and higher selectivity index, their efficacy was better than that of the commercialized medicine, Ribavirin. These described adenosine analogues exhibit potent antiviral activities against several viruses, and offer new leads for further development.

Full text of DOI:10.1016/j.tetlet.2016.11.059

Accepted Manuscript
Digest paper
Synthesis and antiviral evaluation of novel N-6 substituted adenosine analogues
Youwu Hao, Gaohui Zhou, Wei Wu, Yang Zhang, Liming Tao, Jianhua Yao,
Wenping Xu
PII:
S0040-4039(16)31527-1
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.11.059
TETL 48345
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 September 2016
12 November 2016
14 November 2016
Please cite this article as: Hao, Y., Zhou, G., Wu, W., Zhang, Y., Tao, L., Yao, J., Xu, W., Synthesis and antiviral
evaluation of novel N-6 substituted adenosine analogues, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/
j.tetlet.2016.11.059
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tetrahedron Letters
Synthesis and antiviral evaluation of novel N-6 substituted adenosine analogues
Youwu Haoa , Gaohui Zhoua,Wei Wua, Yang Zhanga, Liming Taoa, Jianhua Yaob, Wenping Xua, 
aShanghai Key Laboratory of Chemical Biology, School of Pharmacy,East China University of Science and Technology, Shanghai 200237, China
bShanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of adenosine analogues were synthesized and their biological evaluation was tested
against Coxsackie virus B3 (CVB3) and Herpes simplex virus type 1(HSV-1) in HEp-2 cells.
The hydrophobic constant, acute toxicity, carcinogenicity and mutagenicity were calculated.
Analogues with piperazine derivatives 8b showed promising activities against CVB3 with a
lower IC50value and higher selectivity index, their efficacy was better than that of the
commercialized medicine, Ribavirin. These described adenosineanalogues exhibit potent
antiviral activities against several viruses, and offer new leads for further development.
Available online
Keywords:
Adenosine analogues
antiviral activities
2016 Elsevier Ltd. All rights reserved.
Coxsackie virus B3
Herpes simplex virus type 1
higher selectivity index
———
Corresponding author. Tel.: +86 2 164 253 756; fax: +86 2 159 883 730; e-mail: xuwp@ecust.edu.cn

2
Tetrahedron
1. Introduction
2. Results and discussion
Nucleoside analogues are recognized as an important class of
2.1. Chemistry
biologically active compounds.1-5 Nucleotides and their
derivatives are involved in many cellular processes, including
cell growth and division, and they have been successfully used in
the treatment of various viral infections and as antitumor agents
Scheme 1 shows the synthetic routes to the target compounds,
designed by computer simulation,and their yields and conditions.
Following previously reported procedures,22,23we obtained
adenosine with an adjacent protected hydroxyl group (compound
2) to improve solubility in organic solvents and for convenience
in later reactions. We employed the Sandmeyer reaction to obtain
compound 3 from compound 2.24 To improve the conversion rate,
we used both tert-butyl nitrite (t-BuONO) and TMCS in this step.
Because they are easily decomposed in the light and moist
atmosphere, we performed the reaction in dark and dry
conditions in an argon-filled reactor. Furthermore, we used the
best mole ratio (compound 2: TMCS: t-BUNO = 1:7:8.4) to
determine the highest yield. We dripped a solution of compound
3 in acetonitrile into a solution of piperazine and K2CO3 in
acetonitrile, and stirred for 6 h at room temperature. We obtained
a 90% yield of compound 4 after chromatography purification.
From compound 4 to compound 7, the reaction conditions were
slightly different due to the different substituent groups. When
there are electron-withdrawing groups in the aromatic ring,
increasing compound 6 and a longer reaction time will enhance
the reaction. To a methanol solution of compounds 6a-h, we
added trimethylamine, and stirred this mixture at room
temperature; we then added a solution of compound 4 in
methanol and refluxed these solutions for 6-8 h. We obtained
compounds 7a-h after evaporation and washing. After de-
protecting the hydroxyl groups of compounds7a-h, we obtained
the final compounds 8a-h by column chromatography and
identified the structures using1H nuclear magnetic resonance
(NMR), 13C NMR and high-resolution mass spectrometry
(HRMS).
7
in clinical settings.6, For example, didanosine, approved by
FDA, is well known for the treatment area of HIV-I infection.8
Lamivudine, has been widely used in the treatment of HBV since
being approved by the FDA in 1998.9Dicarmide, has been
reported to inhibit CCR5, which is the major co-receptor for
HIV-1 entry.10 An adenosine nucleoside analogue, NITD008,
which has been reported to be an antiviral reagent that
specifically inhibits flaviviruses, has been reported to effectively
suppress the propagation of different strains of EV71 in RD,
293T and Vero cells with a relatively high selectivity index.11
However, a number of side effects of traditional antiviral drugs
have been observed in clinical treatment, including mitochondrial
damage, myopathy, kidney injury, and lactic acidosis.12In
addition to these adverse effects, drug resistances has been
frequently reported.13-15Tenofovir (TFV), which became
commercially available in 2001, and is well known for its use in
the treatment of HIV-I infection, may cause treatment failure and
is limited in its clinical use due to the rapid emergence of drug
resistance.16
Fig. 1Adenosineanalogues of biologically active compounds
Based on the previous structure of adenosine analoguesand
their antiviral activities, with a novel scaffold of adenosine and a
heterocyclic ring in mind, we designed numerous compounds
using an intermediate derivatization approach. To reduce the
number of compounds and the workload, and increase the ratio of
the active compounds, we first calculated the hydrophobic
constant, acute toxicity, carcinogenic toxicity and mutagenic
toxicity of each of the designed compounds and their
representative structures. We then excluded compounds that did
not obey the “Lipinski’s rule of five”17 or exhibited potential
toxicity. Our antileukemic and antiviral activity prediction results
prompted us to investigate adenosine substituted with
a
piperazine-derivatives-based core structure to discover novel
adenosine derivatives with potential antitumor or antiviral
activities. N-6 substituted with piperazine derivatives can
increase the drug-likeness of target compounds, because many
piperazine designer drugs have already been clinically used in
recent decades, and in recent years many newpiperazine active
compounds are continually being reported.18-21 Here, we report
the synthesis, calculation, and antiviral evaluation of a new series
of adenosine analogues.

8h
399.0±2.5
>60
—
Scheme 1.Reagents and reaction conditions:(a) acetone, MeOH, rt, 4 h, 95%;
(b) TMCS, tert-Butyl nitrite, DCM, rt, overnight, 60%; (c) K2CO3, acetonitrile, rt,
8 h, 90%; (d) chloroacetyl chloride, Et3N, DCM, 0℃，1-6h; 80%-95% (e) 6a-i,
MeOH, refluxing, 6-18 h, 50%-70%; (f) HCOOH:H2O(1:1), 50℃, 4-8 h, 39%-
61%.
Ribavirin
1069.9±356.9
36.8±9.6
29.1
a: 4 repeats with 3 independent experiments, mean±SD
In vitro virus yield reduction assay.We infected the pre-
cultured HEp-2 cells with either CVB3 or HSV-1, washed them
with a phosphate-buffered saline (PBS), then added the three
compounds (8b, 8f, and 8g) and Ribavirin at concentration of 32
mg L-1. We harvested the infected cells after further incubation
for 48 h. We obtained the supematants after they were frozen and
thawed three times. We used the Reed-Muench method29to
calculate the infective virus titer. As shown in Fig.2, the
compounds could obviously decrease the titer of both CVB3 and
HSV-1, and compound 8f, in particular, performed better than
Ribavirin.
2.2. calculation
Table 1 lists ourcalculations of the hydrophobic constant (log
P, predicted by CISOC-log P25), acute toxicity (predicted by
CISOC-PSAT26), carcinogenicity (predicted by CISOC-PSCT27),
and mutagenicity (predicted by CISOC-PSMT28) of some
representative and structurally diverse compounds. The
calculated log P values were less than 5 and met the log P
criteria, and the acute toxicity levels were all within 50≤ LD50 <
500 mg kg−1, with no carcinogenic and mutagenic toxicity risk.
Table 1.Calculation results of log P, for acute, carcinogenic and
CVB3
HSV-1
mutagenic toxicities
9
8
7
6
5
4
3
2
1
0
Acute
tox.a
Car.
Tox.b
Mutagenic
tox.c
Compd
Log P
8a
8b
8c
8d
8e
8f
0.6
3.64
3.75
3.74
3.8
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
0.8
0.14
0.51
0.8
3.75
3.73
3.68
3.77
1.01
1.15
‐ 0.98
8g
8h
Virus control
8f
8b
8g
Ribavirin
a: Rat, oral, less than 2, LD50 < 1 mg kg−1; equal or more than 2 and less
than 3, 1 ≤ LD50 < 50 mg kg−1; equal or more than 3 and less than 4, 50≤
LD50 < 500 mg kg−1; equal or more than 4 and less than 5, 500 ≤ LD50 <
5000 mg kg−1; equal or more than 5, LD50 ≥ 5000 mg kg−1.
b: Rat, oral, P: probability of non carcinogenic is low; N: probability of non
carcinogenic is high.
Fig. 2 The titer of CVB3 and HSV-1 treated with compounds
3. Conclusions
In summary, we synthesized a series of novel adenosine
analogues as antiviral agents and calculated the hydrophobic
constant, acute toxicity, carcinogenicity and mutagenicity of each.
Compared with Ribavirin, some of the compounds showed potent
inhibitory activities against CVB3 along with low cytotoxicities.
Compound 8b, N-6 substituted with piperazine derivatives,
exhibited better inhibitory activities against CVB3 and a better
selectivity index than Ribavirin. Compounds 8a-g had lower IC50
values than the compound 8h, which indicates that the phenyl
substitution of piperazine moiety may more effectively enhance
antiviral activities than pyridine substitution in this series of
compounds. This might be related to the lonepair of electrons in
the aromatic ring. Furthermore, as shown in the in vitro virus
yield reduction assay, compounds 8f and 8g can reduce the
infective virus titer; better than the compound 8b and Ribavirin,
which might be related to the electron-withdrawing group
(trifluoromethyl) or the steric hindrance effects of the CF3 group.
These results indicate that these kinds of adenosine analogues
could be developed as potent antiviral agents in future.
c: Salmonella typhimurium. P: positive; N: negative.
2.3. Biological evaluation
In vitro cytotoxicity and antiviral activity assay. Using the
commercialized drugRibavirinas a reference compound, we
evaluated these new adenosine analogues by MTT assay to
determine their cytotoxicities and antiviral activities in HEp-2
cells infected with CVB3 strains (Table 2). Table 2 shows the
results, which are expressed as TC50 (50% cytotoxic
concentration), IC50 (50% CVB3 cytoprotective concentration
against CVB3 induced cytopathic effect) and TI (selectivity
index represented by the TC50/IC50 ratio). Seven analogues
exhibited promising antiviral activity against CVB3, especially
against compound 8b, for which the IC50value was 5.1 mg L-1
and the TI value was 41, the values obtained for this compound
were better than those obtained for Ribavirin, for which the IC50
value was 36.8 mg L-1 and TI value was 29.1.
Table2. Anti-CVB3 activities and cytotoxicity of compounds
in HEp-2 cellsa
4. Acknowledgements
Compound
TC50 / mg L-1
103.3±13.0
208.2±18.7
240.5±25.0
239.5±25.9
141.0±12.1
116.2±9.6
IC50 / mg L-1
19.0±3.1
5.1±2.3
TI
This work was financially supported by National Key
Technologies R&D Program (2011BAE06B04). We are grateful
to the colleges of Wuhan University, School of Basic Medical
Science, Institute of Medical Virology, for their helpful works in
the antiviral assays.
8a
8b
8c
8d
8e
8f
5.5
41.0
13.3
8.2
18.1±1.4
29.1±3.9
17.4±1.0
10.0±0.7
10.6±2.0
8.1
11.6
14.0
8g
148.9±10.1

4
Tetrahedron
16. Iyidogan, P.; Anderson, K. S. Antiviral Chemistry &
Chemotherapy2014, 23, 217.
References and notes
17. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Advanced Drug Delivery Reviews2001, 46, 3.
18. YANG; Zhang, Y. H.; Yang, L. J.; Wang, L. X.; Xiao-Yu, W. U.;
Zhao, X. M.; Yu Chinese Journal of Organic Chemistry2008, 28,
2095.
19. Westwell, A. D. Drug Discovery Today2003, 8, 652.
20. Huang, L. H.; Xu, H. D.; Yang, Z. Y.; Zheng, Y. F.; Liu, H. M.
Steroids2014, 82, 1.
21. Fytas, C.; Zoidis, G.; Tsotinis, A.; Fytas, G.; Khan, M. A.; Akhtar,
S.; Rahman, K. M.; Thurston, D. E. European Journal of
Medicinal Chemistry2015, 93, 281.
22. Jing, L. I.; Gui-Zhen, A. O.; Chen, J. L.; Qiao, C. H. Chinese
Journal of Synthetic Chemistry2009.
23. Townsend, A. P.; Roth, S.; Williams, H. E.; Stylianou, E.;
Thomas, N. R. Organic Letters2009, 11, 2976.
24. Šála, M.; Palma, A. M. D.; Hřebabecký, H.; Dejmek, M.;
Dračínský, M.; Leyssen, P.; Neyts, J.; Mertlíková-Kaiserová, H.;
Nencka, R. Bioorganic & Medicinal Chemistry Letters2011, 21,
4271.
25. Liao, Q.; Yao, J.; Yuan, S. Molecular Diversity2006, 10, 301.
26. J. H. Yao, CISOC-PSAT, version 1.0, Shanghai Institute of
Organic Chemistry, Chinese Academy of Sciences, Shanghai,
2010.
1.
Périgaud, C.; Gosselin, G.; Imbach, J. L. Nucleosides &
Nucleotides1992, 11, 903.
Ichikawa, E.; Kato, K. Current Medicinal Chemistry2001, 8, 385.
Shimada, H.; Haraguchi, K.; Hotta, K.; Miyaike, T.; Kitagawa, Y.;
Tanaka, H.; Kaneda, R.; Abe, H.; Shuto, S.; Mori, K. Bioorganic
& Medicinal Chemistry2014, 22, 6174.
2.
3.
4.
5.
6.
Weiss, A. Chembiochem2013, 14, 2519.
Karran, P. British Medical Bulletin2006, 79-80, 153.
Tuncbilek, M.; Guven, E. B.; Onder, T.; Atalay, R. C. Journal of
Medicinal Chemistry2012, 55, 3058.
Bosch, L.; Harbers, E.; Heidelberger, C. Cancer Research1958,
18, 335.
Clercq, E. D. Future Virology2016, 3, 393.
Liu, Y.; Zhou, E.; Yu, K.; Zhu, J.; Zhang, Y.; Xie, X.; Li, J.; Jiang,
H. Molecules2008, 13, 2426.
7.
8.
9.
10. Schul, W.; Chen, Y. L.; Yin, Z.; Keller, T.; Shi, P. Y. Proceedings
of the National Academy of Sciences2009, 106, 20435.
11. Shang, L.; Wang, Y.; Jie, Q.; Bo, S.; Lin, C.; Lou, Z.; Peng, G.;
Sun, Y.; Zheng, Y. Antiviral Research2014, 112, 47.
12. HimaBindu, A.; Naga Anusha, P. Journal of Antivirals
&Antiretrovirals2011, 03, 88.
13. Bani-Sadr, F.; Carrat, F.; Pol, S.; Hor, R.; Rosenthal, E.; Goujard,
C.; Morand, P.; Lunel-Fabiani, F.; Salmon-Ceron, D.; Piroth, L.
Jaids Journal of Acquired Immune Deficiency Syndromes2005,
40, 47.
14. Kohler, J. J.; Lewis, W. Environmental & Molecular
Mutagenesis2007, 48, 166.
15. Qian-Qian, H. U.; Shi, L. L.; Tan, C. B.; Zhang, J.; Pan, X. F.;
Wei-Ren, X. U.; University, T. M. Chinese Journal of
Pharmacology & Toxicology2013, 27, 885.
27. Liao, Q.; Yao, J. H.; Li, F.; Yuan, S. G.; Doucet, J. P.; Panaye, A.;
Fan, B. T. Sar&Qsar in Environmental Research2004, 15, 217.
28. Liao, Q.; Yao, J.; Yuan, S. Molecular Diversity2007, 11, 59.
29. Jin, J.; Sun, S.; Jiang, C.; Ma, H.; Xu, L.; An, D.; Huang, X.;
Kong, W. Journal of Virological Methods2013, 187, 362.

Highlights:
1 A novel series of adenosine analogs were
synthesized as antiviral agents.
2 The phenyl substitution of piperazine moiety may
more effective.
3 Compounds 8b exhibited better inhibitory
activities against CVB3 than Ribavirin.
4 The target compounds were designed by
computer simulation.
5 These kinds of adenosine analogues might be
developed as potent antiviral agents.

6
Tetrahedron
Graphical Abstract
Synthesis and antiviral evaluation of novel
Leave this area blank for abstract info.
N-6 substituted adenosine analogues
Youwu Hao