Enterovirus inhibitory activity of C-8-tert-butyl substituted 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones

Article abstract of DOI:10.1016/j.bmcl.2017.05.030

Members of a series of 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones (1, Fig. 2) were prepared and tested against representative enteroviruses including Human Coxsackievirus B1 (Cox B1), Human Coxsackievirus B3 (Cox B3), human Poliovirus 3 (PV3), human Rhinovirus 14 (HRV14), human Rhinovirus 21 (HRV 21) and human Rhinovirus 71 (HRV 71). The C-8-tert-butyl group on the tetrahydrobenzene ring in these substances was found to be crucial for their enterovirus activity. One member of this group, 1e, showed single digit micromolar activities (1.6–8.85?μM) against a spectrum of viruses screened, and the highest selectivity index (SI) values for Cox B1 (>11.2), for Cox B3 (>11.5), and for PV3 (>51.2), respectively. In contrast, 1p, was the most active analog against the selected HRVs (1.8–2.6?μM), and showed the highest selectivity indices among the group of compounds tested. The SI values for 1p were 11.5 for HRV14, 8.4 for HRV21, and 12.1 for HRV71, respectively.

Full text of DOI:10.1016/j.bmcl.2017.05.030

Accepted Manuscript
Enterovirus Inhibitory Activity of C-8-tert-Butyl Substituted 4-Aryl-6,7,8,9-
tetrahydrobenzo[4,5]thieno[3,2- e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones
Bishyajit Kumar Biswas, Yashwardhan R. Malpani, Neul Ha, Do-Hyun Kwon,
Jin Soo Shin, Hae-Soo Kim, Chonsaeng Kim, Soo Bong Han, Chong-Kyo Lee,
Young-Sik Jung
PII:
S0960-894X(17)30511-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.030
BMCL 24975
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 April 2017
6 May 2017
10 May 2017
Please cite this article as: Kumar Biswas, B., Malpani, Y.R., Ha, N., Kwon, D-H., Soo Shin, J., Kim, H-S., Kim,
C., Bong Han, S., Lee, C-K., Jung, Y-S., Enterovirus Inhibitory Activity of C-8-tert-Butyl Substituted 4-
Aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2- e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones, Bioorganic
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.05.030
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Bioorganic & Medicinal Chemistry Letters
Enterovirus Inhibitory Activity of C-8-tert-Butyl Substituted 4-Aryl-6,7,8,9-
tetrahydrobenzo[4,5]thieno[3,2- e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones
Bishyajit Kumar Biswas,a,†,‡ Yashwardhan R. Malpani,a,†,‡ Neul Ha,†,‡ Do-Hyun Kwon,† Jin Soo Shin,†
Hae-Soo Kim,† Chonsaeng Kim,† Soo Bong Han†,‡ Chong-Kyo Lee,† and Young-Sik Jung†,‡,
†Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, 141 Gajeongro, Yuseong, Daejeon 34114, Republic of Korea
‡Department of Medicinal Chemistry and Pharmacology, University of Science and Technology, 217 Gajeongro, Yuseong, Daejeon 34113, Republic of Korea.
a,These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Members of a series of 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-
a]pyrimidin-5(4H)-ones (1, Fig 2) were prepared and tested against representative enteroviruses
including Human Coxsackievirus B1 (Cox B1), Human Coxsackievirus B3 (Cox B3), human
Poliovirus 3 (PV3), human Rhinovirus 14 (HRV14), human Rhinovirus 21 (HRV 21) and
human Rhinovirus 71 (HRV 71). The C-8-tert-butyl group on the tetrahydrobenzene ring in
these substances was found to be crucial for their enterovirus activity. One member of this
group, 1e, showed single digit micromolar activities (1.6-8.85 μM) against a spectrum of viruses
screened, and the highest selectivity index (SI) values for Cox B1 (>11.2), for Cox B3 (>11.5),
and for PV3 (>51.2), respectively. In contrast, 1p, was the most active analog against the
selected HRVs (1.8-2.6 μM), and showed the highest selectivity indices among the group of
compounds tested. The SI values for 1p were 11.5 for HRV14, 8.4 for HRV21, and 12.1 for
HRV71, respectively.
Revised
Accepted
Available online
Keywords:
4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-
e]triazolo[4,3-a]pyrimidin-5(4H)-one
Poliovirus, Rhinovirus
Enterovirus,
Antiviral
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +82-42-8607135; fax: +82-42-8607160; e-mail: ysjung@krict.re.kr

Enteroviruses are positive-sense non-enveloped ssRNA viruses.
Various viruses, such as coxsackievirus (Cox A),
from ChemBank at KRICT was conducted using in vitro
phenotypic (cytopathogenic) assays on Cox B3 and PV3 in HeLa
cells. This study led to our discovery that 8-(tert-butyl)-4-phenyl-
6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[4,3-
a]pyrimidin-5(4H)-one (1) displays a good antiviral activity
A
coxsackievirus B (Cox B), poliovirus (PV), human rhinovirus
(HRV) and enterovirus (EV), belong to this genus. Owing to
frequent recombination and low replication fidelity, these
viruses, like other RNA viruses, have high mutation rates.1
Consequently various serotypes are present in each of the
enteroviurses. For example, 29 serotypes of Cox A and B, and
more than 160 serotypes of HRV with genetic variations are
known.2 Also PV can be divided to three serotypes (PV1, PV2
and PV3).2 These enteroviruses cause many different types of
disease such as myocarditis (Cox B), encephalitis (Cox A and B),
aseptic meningitis (Cox A and B), poliomyelitis (PV), hand, foot
and mouth disease (Cox A or EV71), and acute hemorrhagic
conjunctivitis (EV70, Cox A24).3 In addition, HRVs cause
common colds4 and they are responsible for the exacerbation of
asthma5 and Chronic Obstructive Pulmonary Disease (COPD).5c,6
Even mild and generally self-limiting infections caused by HRV
costs billions of dollars in terms of missed work days, medical
visits and treatment.7
profile (Figure 2). This substance
1 has 50% effective
concentrations (EC50 values) of 8.65 μM for Cox B3 and 1.95 μM
for PV3.
Figure 2. Initial hit compound 1 from HTS and its EC50 values
against Cox B3 and PV3
In our efforts to optimize the anti-enteroviral activity of this
substance, we modified cyclohexyl ring A, aromatic ring B and
triazole ring C moieties (Figure 2). The cyclohexyl ring A was
modified by incorporation of various alkyl groups. The thiophene
amino ester intermediates 3a-h were prepared using Gewald’s
reaction13 starting with appropriate substituted cyclohexanones
A few small molecules have been observed to inhibit the
replication of these viruses.8 For instance, Pleconaril and
Vapendavir are known to bind to capsid,8-9 which prevent their
entry into the host cells. Rupintrivir inhibits 3C protease and,
thus, interfers with viral polyprotein processing.10 Enviroxime
inhibits viral RNA replication by targeting 3A and related
proteins.11 However, because of insufficient efficacies or side
effects observed during their clinical studies, none of these
substances have been developed into drugs approved for
treatment of these viral infections.12 Therefore, a new small
molecule inhibitor that shows broad-spectrum inhibition of the
replication of these enteroviruses is highly desired.
2a-h (Scheme 1). These intermediates
3 reacted with
isothiocyanates to produce intermediates 4 (path I, Scheme 2).
Alternatively the same intermediates 4 were obtained through
path II (Scheme 2), where the thiophene amino esters 3 was
converted to stable isothiocyanate derivatives 6, which were
treated with aniline to form thiourea derivatives 7. Cyclization of
7 in mild ethanolic NaOH solution generated intermediates 4.
Intermediates 4 were converted to hydrazine containing analogs 5
using hydrazine hydrate. The resulting hydrazines 5 were reacted
with formic acid/trialkyl orthoformate to form the triazole ring
containing
4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-
e]triazolo[4,3-a]pyrimidin-5(4H)-ones 1a-h possessing different
R1 substituents. Modifications of the aromatic ring B, conducted
using the same sequence with different substituted
isothiocyanates (path I) or various substituted anilines (path II),
produced the desired products 1i-r with different R2 substitutions
(Scheme 2). Substances containing R3 substituted triazole rings
(C, Figure 2) including Me (1s), Ph (1t), NH2 (1u) and thiol (1v)
were also prepared (Scheme 2). In addition, 1s and 1t were
obtained by treatment of 5e with acetic acid and benzoyl
chloride. Finally, 1u and 1v were generated by reaction of 5e
with CNBr and CS2.14
Figure 1. Known virus inhibitors
In the preliminary phase of this effort, High-Throughput
Screening (HTS) of a chemical library of 100,000 compounds

tert-butyl group was incorporated at the C-8 position of the
tetrahydrobenzene ring in compounds selected for the further
SAR study.
The effects of the substituents on the N-4 phenyl ring (B, Figure
2) were determined next (Table 1 1i-r). The 2’-Me (1i) and 4’-
Me (1j) derivatives only showed moderate activity for inhibition
of Cox B1 and Cox B3. However, when R2 is more sterically
bulky than methyl (Me), the activities decrease (1k-r). However,
compared to those against Cox B1 and Cox B3, the inhibitory
activity against PV3 is less sensitive to the size of substituents. In
general, all derivatives except 1q and 1r possess moderate
activity against PV3. Thus, when R2 is H (1e) or a small group
like Me (1i and 1j), Cl (1k, 1l, and 1m), or OMe (1p), the
effective activity against PV3 is moderate (EC50 <10 µM).
However, when R2 is Br (1n and 1o), CF3 (1q), or p-Me-Ph (1r),
the PV3 activity is very low or absent (EC50 >10 µM). Even
though the antiviral activities against all three viruses are
dependent on phenyl ring at N-4substituents, the unsubstituted
compound (1e) shows the highest activity and the highest SI
values; >11.2 for Cox B1, >11.5 for Cox B3, and >51.2 for PV3.
Scheme 1. Generation of intermediates 3 through using Gewald’s
Reaction
The inhibitory activities of 1a-v were tested against three
representative strains of enteroviruses (Cox B1, Cox B3, and
PV3) (Table 1). The assays, involving measurements of the
reduction of virus-induced cytotoxicity (CPE) of HeLa cells,
employed the previously described method.15 For this purpose,
Pleconaril was utilized as a reference compound. In a SAR study,
the effects of substituents on the cyclohexyl ring (A, Figure 2) on
antiviral efficacy were evaluated (1a-h, Table 1). The data shows
that only the substance bearing a tert-butyl group at C-8 (1e)
displayed marginal effectiveness toward Cox B1, Cox B3 and
PV3 (EC50 = 8.85, 8.65 and 1.95 μM, respectively). However,
incorporation of other substituents such as methyl (1c), propyl
(1d) or phenyl (1f) at this position did not have any effect on
activity. Unsubstituted and differently substituted compounds (1a,
1b, 1g, and 1h) did not display any activity. Moreover, the
derivative 1e had a very low toxicity (CC50 >100). Therefore, the
To probe the effect of R3 substituents on the triazole ring (C,
Figure 2), Me (1s), Ph (1t), NH2 (1u), and SH (1v) were
introduced. However, none of these substances display enhanced
activities when compared to those of the unsubstituted compound
1e (Table 1).

Scheme 2. Synthetic Route to the analogs of 1
In addition to studies with the coxsackievirus and poliovirus, we
a percentage, is given in Table 2. Buspirone was used for
reference for this study. The results show that 1e is moderately
stable under these metabolic conditions.
also explored the anti-rhinovirus activity of the substances
against HRV14, HRV21 and HRV71 in H1 HeLa cells (Table 1)
(vide supra). In this effort, Pleconaril was used as a reference
compound. The hit compound, 1e, has good activities against all
three HRVs, but it displays a strong toxicity in H1 HeLa cells
(CC50 9.6 µM). Among all members of this series, the 4’-OMe
derivative 1p was found to be one of the most active against the
three selected HRVs, and to display the highest selectivity. The
SI values for 1p are 11.5 for HRV14, 8.4 for HRV21, and 12.1
for HRV71.
Table 2. In vitro microsomal metabolic stabilities of 1e in human
and rats.
The observations made in the study described above show that 1e
and 1p display properties that make them suitable lead
compounds in further exploratory efforts aimed at developing
broad spectrum anti-enteroviruse drugs. For this purpose, studies
focusing on optimization of the structure of these substances to
install drug-like activities and the mechanism of anti-virus action
are underway in our laboratory.
Even though the compounds investigated in this effort are at an
early stage of development, we assessed their microsomal
stabilities. In vitro metabolic stabilities of 1e were determined
using human and rat liver microsomal phase I. The amount of 1e
(calculated using quantitative mass spectrometry),16 expressed as
Table 1. Antiviral activities of 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-e]triazolo[4,3-a]pyrimidin-5(4H)-ones 1a-1v.

Acknowledgement
This study was supported by Korea Research Institute of Chemical Technology (Grant No. KK1503-C00 and SI1507). We thank to
Korea Chemical Bank for providing the chemical library with which this work was conducted. We also thank the University of Science
and Technology for providing fellowships to the student authors.
References
1. S.F. Elena, R. Sanjuán, Journal of Virology, 79 (2005) 11555-11558.
2. http://www.picornaviridae.com/enterovirus/enterovirus.htm
3. (a) A.M. De Palma, et al., Medicinal Research Reviews, 28 (2008) 823-884; (b) P. Muir, Enteroviruses, Medicine, 2009, pp. 691-
694; (c) D.N. Irani, Neurologic Clinics, 26 (2008) 635-655; (d) P. Rice, Medicine, 41 (2013) 678-682; (e) H.A. Rotbart, Antiviral
Research, 53 (2002) 83-98; (f) T. Zaoutis, J.D. Klein, Pediatrics in Review, 19 (1998) 183.
4. (a) J.M. Rollinger, M. Schmidtke, Medicinal Research Reviews, 31 (2011) 42-92; (b) M.J. Mäkelä, et al., Journal of Clinical
Microbiology, 36 (1998) 539-542.
5. (a) J.E. Gern, Clinical & Experimental Allergy, 45 (2015) 32-42; (b) S.D. Message, et al., Proceedings of the National Academy of
Sciences, 105 (2008) 13562-13567; (c) D. Kurai, et al., Frontiers in Microbiology, 4 (2013).
6. H.-C. Kim, et al., Journal of Medical Virology, 88 (2016) 2092-2099.
7. (a) T. Heikkinen, A. Järvinen, The Lancet, 361 51-59; (b) T.A. Hicks, THE COMMON COLD, Marshall Cavendish Benchmark,
Tarrytown, New York 10591-90012006.
8. S.-R. Shih, et al., Medicinal Research Reviews, 24 (2004) 449-474.
9. A.K. Patick, Antiviral Research, 71 (2006) 391-396.
10. P.S. Dragovich, et al., Journal of Medicinal Chemistry, 42 (1999) 1213-1224.
11. (a) J.H. Wikel, et al., Journal of Medicinal Chemistry, 23 (1980) 368-372; (b) B.A. Heinz, L.M. Vance, Journal of Virology, 69
(1995) 4189-4197.
12. S.E. Jacobs, et al., Clinical Microbiology Reviews, 26 (2013) 135-162.
13. (a) L.D. Jennings, et al., Bioorganic & Medicinal Chemistry Letters, 15 (2005) 4731-4735; (b) K. Gewald, et al., Chemische
Berichte, 99 (1966) 94-100.
14. (a) Q.-h. Dai, et al., Journal of Heterocyclic Chemistry, 51 (2014) 1390-1394; (b) M.M. Ghorab, et al., Arzneimittelforschung, 56
(2006) 553-560; (c) H.A.H. El-Sherief, et al., Journal of Heterocyclic Chemistry, 45 (2008) 467-473; (d) S.N. Thore, et al., Journal
of Heterocyclic Chemistry, 52 (2015) 142-149.
15. H. Kang, et al., Antiviral Research, 124 (2015) 1-10.
16. See supplementary information.

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Enterovirus Inhibitory Activity of C-8-tert
butyl Substituted 4-Aryl-6,7,8,9-
tetrahydrobenzo[4,5]thieno[3,2-
e][1,2,4]triazolo[4,3-a]pyrimidin-5(4H)-ones
Bishyajit Kumar Biswas,a,†,‡ Yashwardhan R. Malpani,a,†,‡ Neul Ha,†,‡ Do-Hyun Kwon,† Jin Soo Shin,†
Hae-Soo Kim,† Chonsaeng Kim,† Soo Bong Han†,‡ Chong-Kyo Lee,† and Young-Sik Jung†,‡,
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