Synthesis and in vitro antiviral evaluation of 4-substituted 3,4-dihydropyrimidinones

Article abstract of DOI:10.1016/j.bmcl.2016.12.010

A series of 4-substituted 3,4-dihydropyrimidine-2-ones (DHPM) was synthesized, characterized by IR,¹H NMR,¹³C NMR and HRMS spectra. The compounds were evaluated in vitro for their antiviral activity against a broad range of DNA and R

Full text of DOI:10.1016/j.bmcl.2016.12.010

Accepted Manuscript
Synthesis and in vitro antiviral evaluation of 4-substituted 3,4-dihydropyrimi-
dinones
Dhanabal Kumarasamy, Biswajit Gopal Roy, Joana Rocha-Pereira, Johan
Neyts, Satheeshkumar Nanjappan, Subhasis Maity, Musfiqua Mookerjee, Lieve
Naesens
PII:
S0960-894X(16)31277-X
http://dx.doi.org/10.1016/j.bmcl.2016.12.010
BMCL 24499
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
22 May 2016
8 November 2016
2 December 2016
Please cite this article as: Kumarasamy, D., Roy, B.G., Rocha-Pereira, J., Neyts, J., Nanjappan, S., Maity, S.,
Mookerjee, M., Naesens, L., Synthesis and in vitro antiviral evaluation of 4-substituted 3,4-dihydropyrimidinones,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.010
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Synthesis and in vitro antiviral evaluation of 4-substituted 3,4-
dihydropyrimidinones
a,
b
c
c
Dhanabal Kumarasamy *, Biswajit Gopal Roy , Joana Rocha-Pereira , Johan Neyts ,
d
a
a
c
Satheeshkumar Nanjappan , Subhasis Maity , Musfiqua Mookerjee , and Lieve Naesens
a
Department of Pharmaceutical Chemistry, NSHM College of Pharmaceutical Technology, 124, B.L Saha
Road, Kolkata 700053, India
b
th
Department of Chemistry, School of Physical Sciences, Sikkim University, 6 Mile, Tadong, Gangtok, Sikkim
7
c
37102, India
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven,
Minderbroedersstraat 10, Leuven 3000, Belgium
d
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research
[NIPER-H], Balanagar, Hyderabad 500037, India
Keywords: Dihydropyrimidinones, DHPM, Antiviral activity, Cytotoxicity, Bunyavirus
*
Corresponding author, Tel.: +91-33-24032300; fax: +91-33-24032301; E-mail: dhanabalrx@gmail.com
1

Abstract
A series of 4-substituted 3,4-dihydropyrimidine-2-ones (DHPM) was synthesized,
1
13
characterized by IR, H NMR, C NMR and HRMS spectra. The compounds were evaluated
in vitro for their antiviral activity against a broad range of DNA and RNA viruses, along with
assessment for potential cytotoxicity in diverse mammalian cell lines. Compound 4m, which
possesses a long lipophilic side chain, was found to be a potent and selective inhibitor of
Punta Toro virus, a member of the Bunyaviridae. For Rift Valley fever virus, which is
another Bunyavirus, the activity of 4m was negligible. DHPMs with a C-4 aryl moiety
bearing halogen substitution (4b, 4c and 4d) were found to be cytotoxic in MT4 cells.
2

Emerging and re-emerging viruses are capable of rapidly overcoming their natural
geographical boundaries and spreading among the human population. Most of these viral
1
,2
outbreaks are due to zoonotic viruses, some of which were previously unknown. With an
increasing risk for vector-borne outbreaks, the Bunyaviridae present a particular and global
threat to public health. Members of this virus family are the cause of Hantavirus
cardiopulmonary syndrome in the Americas and Schmallenberg virus-induced congenital
malformations in ruminants, which emerged in Europe in 2011. Severe fever with
thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS
virus. In addition, some well-known Bunyaviruses like Rift Valley fever virus (RVFV) and
Crimean-Congo haemorrhagic fever virus continue to emerge in new geographical
3
,4
locations. RVFV is transmitted by a wide variety of mosquitoes and its geographic
distribution is expanding, with the potential of reaching Europe in the near future. This virus
causes devastating epidemics in Africa and Arabia, killing hundreds of humans and
thousands of wild and livestock animals. The transmission of Bunyaviruses, and their cell
entry pathways and cell receptors remain poorly characterized, partly due to the fact that
experiments with wild-type RVFV are limited to enhanced biosafety level-4 (BSL-4)
5
facilities, which are very few in number. The lack of vaccines and antiviral medications
makes the potential spread of these Bunyaviruses a global concern. Recently, in an effort to
repurpose FDA-approved drugs for RVFV treatment, the anticancer drug sorafenib was found
6
to be an effective inhibitor of RVFV replication. Another agent that is able to suppress
4
,7
RVFV replication in cell culture is the polyanionic drug suramin.
Biginelli condensation is a well-known reaction that is more than 120 years old. It is
an exceptionally simple and straightforward acid-catalyzed one-pot cyclocondensation
reaction of ethylacetoacetate (1), aldehyde (2) and urea (3) that affords 4-substituted 3,4-
8
dihydropyrimidine-2-ones (DHPM) or Biginelli compounds. A number of variations to give
3

higher yields and expand the scope versus the traditional three-component Biginelli
condensation, resulted in many simple derivatives that emerged as promising therapeutic
agents. Monastrol was discovered as a prototype anti-cancer drug that inhibits the mitotic
9
kinesin-5 protein. Methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-
dihydropyrimidine-5-carboxylate (HAP-1) and its derivatives were developed as anti-
1
0
hepatitis B virus (HBV) agents targeting viral capsid assembly. Moreover, marine alkaloids
Crambescidins and Crambescins containing an DHPM core and isolated from the sponges
Crambe crambe and Batzella sp. were demonstrated to possess antiviral and cytotoxic
1
1
properties. Batzelladines A and B with DHPM core were identified as inhibitors of HIV gp-
1
2
1
20 binding to CD4 receptors. However, the majority of studies exclude or limit the
aliphatic aldehydes within their scope due to their troublesome behavior and unacceptable
1
3,14
yield in this multi-component reaction.
employing aliphatic aldehydes.
It is still desirable to optimize this reaction while
4

Figure 1. Biologically active DHPMs
The present work was aimed to synthesize 4-substituted 3,4-dihydropyrimidin-2(1H)-
ones using various aromatic and aliphatic aldehydes and evaluate their antiviral activity
against a broad range of DNA and RNA viruses, along with their cytotoxicity assessment in
diverse mammalian cell lines.
The synthesis of desired compounds is depicted in Scheme 1. 3,4-dihydropyrimidine-
2
-one bearing C-4 aryl (4a-g; 4i) and styryl (4h) moiety were prepared in excellent yields
5

1
5
(
(
83-95%) via neat condition without solvent and catalyst at ~98 ˚C. Whereas, reaction
Scheme 2) employing salicylic aldehyde (2a) in neat conditions, a tricyclic compound i.e.
oxygen bridged pyrimidinone (5) was obtained in good yield (76%). However, utilizing
aliphatic aldehydes in neat conditions lead to negligible yield of the desired compounds (data
not shown). C-4 unsubstituted DHPM (4j) and Dihydropyrimidine-2-one derivatives with C-
4
aliphatic group (4k-4m) were achieved by using excess urea under reflux conditions
employing acetic acid as a mild acid catalyst in ethanol. The yields ranged from 53-68%. All
1
13
the synthesized compounds were characterized by IR, H NMR, C NMR and HR-MS.
(
Supplementary Data)
Scheme 1. Synthesis of DHPMs (a) Compounds 4a-i, neat reaction condition at ~98 ˚C; (b)
Compounds 4j-m CH COOH, ethanol, reflux
3
6

Scheme 2. Synthesis of oxygen bridged pyrimidinone (5)
Antiviral activity and cytotoxicity were determined by previously described methods.
1
6,17,18
The compounds plus viruses were added to 96-well plates containing subconfluent cell
cultures. After 3 to 5 days incubation, the virus-induced cytopathic effect (CPE) was scored
which, depending on the virus, consisted of cell lysis, plaque formation, or giant cell
formation. Scores of 1, 2, 3 or 4 were given to conditions in which the virus affected 25, 50,
7
5 or 100% of the cell monolayer, respectively. The virus panel included: (i) evaluated in
African green monkey kidney Vero cells: Parainfluenza-3 virus; Reovirus-1; Sindbis virus;
Coxsackie virus B4; Punta Toro virus; Rift Valley fever virus (MP-12 strain); and Yellow
fever virus; (ii) evaluated in human embryonic lung (HEL) fibroblast cells: herpes simplex
virus types 1 and 2; varicella-zoster virus; cytomegalovirus; vaccinia virus; and adenovirus;
(
iii) evaluated in human cervix carcinoma HeLa cells: vesicular stomatitis virus; Coxsackie
virus B4; and respiratory syncytial virus; (iv) evaluated in Crandell-Rees feline kidney
CRFK) cells: feline herpesvirus and feline coronavirus; (v) evaluated in Madin-Darby canine
(
kidney (MDCK) cells: influenza A and B virus; (vi) evaluated in human MT-4 T-lymphoblast
cells: HIV types 1 and 2. The antiviral activity was assessed based on CPE reduction, as
determined by microscopic scoring of the CPE or virus plaque formation. For HIV-1 and -2,
7

the CPE was measured with the colorimetric formazan-based MTS [3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium] assay.
Cytotoxicity of the compounds was assessed by microscopic examination, yielding
the minimal cytotoxic concentration (MCC). Alternatively, the colorimetric formazan-based
MTS assay was used to determine the 50% cytotoxic concentration (CC50) value.
The antiviral activities and cytotoxicity of the synthesized compounds (4a-m and 5)
were determined in CPE reduction assays with a broad and diverse panel of DNA and RNA
viruses and using relevant mammalian cell lines. The results of the evaluation in Vero cell
cultures are presented in Table 1. Compound 4m was found to be a selective and potent
inhibitor of Punta Toro virus (PTV), a member of the family Bunyaviridae and the genus
Phlebovirus. 4m was approximately 7 times more potent than the viral entry inhibitor dextran
sulphate (DS-10000) and broad-spectrum acting mycophenolic acid (an inhibitor of the
cellular IMP dehydrogenase). Compared to the broad antiviral agent ribavirin, 4m proved 17
times more potent with superior activity (EC50 value of 3 µM) and a selectivity index (ratio of
5
0% cytotoxic concentration to 50% effective antiviral concentration) of ≥33. The anti-PTV
effect of 4m was dose-dependent achieving 81% protection against virus-induced CPE at 20
1
9
µM of compound. Since PTV is related to the most clinically relevant Phlebovirus RVFV,
we next evaluated the effect of 4m against RVFV. This virus is classified as a BSL-4 virus,
2
0,21
and we therefore used the low-pathogenic MP-12 strain. Although about 40% reduction
in RVFV-induced CPE was seen at a compound concentration of 100 µM, 4m was unable to
achieve 50% inhibition at this high concentration (data not shown). Also, when evaluated
against PTV in HeLa cells, 4m was found to be inactive, which contrasts to the marked effect
seen in Vero cells (see above). Hence, the antiviral effect of 4m appears to be cell-dependent,
alike what was described for the anti-Ebola virus activity of brincidofovir, an antiviral drug
2
2
with a lipophilic moiety that is structurally similar to that of 4m.
8

Table 1. Antiviral activity and cytotoxicity in Vero cell cultures.
b
Antiviral EC50 (µM)
Cytotoxicity
MCC (µM)
Compound
a
Para-
influenza-
Reovirus-1
Sindbis
virus
Coxsackie Punta Toro
Yellow
fever virus
virus B4
virus
3
virus
4
a
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
112
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>250
1.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
20
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
58
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.0 ± 1.4
>100
20
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.8
4
b
4
c
4
d
4
e
4
f
4
4
g
h
100
4
4
i
j
>100
>100
>100
>100
≥100
>100
>100
>250
>100
4
k
4
l
e
4
m
5
c
DS-10,000
Ribavirin
>250
4
>250
>250
50
112
d
MPA
0.8
20
4
a
MCC: minimal cytotoxic concentration, or compound concentration producing minimal
alterations in cell morphology, as detected by microscopical inspection.
b
EC50: 50% effective concentration, or compound concentration giving 50% protection
against virus-induced cytopathic effect, as determined by microscopy.
c
DS-10,000: dextran sulphate of MW 10,000; these data are expressed in µg per ml.
d
MPA: mycophenolic acid.
e
Mean ± SD of two independent tests.
Among the DHPM series that we synthesized, the distinguished structural feature of
4
m is its very long lipophilic chain at position C-4 of the polar dihydropyrimidinone scaffold,
leading us to assume that the observed anti-PTV activity mainly depends on the undecyl
moiety which confers amphiphilic properties to 4m. Some amphiphilic molecules with a
large polar head group (inverted-cone-shaped) are known to target viral membranes and
9

2
3
display broad-spectrum antiviral activity. However, except for Punta Toro virus, 4m did not
have antiviral activity against all other viruses tested. This molecule displayed only modest
cytotoxicity (Table 2), with a minimal cytotoxic concentration (MCC) of 100 µM in Vero and
human embryonic lung (HEL) cells, and no toxicity at 100 µM compound concentration in
the other cell lines tested. Human MT4 T-lymphoblast cells were an exception with a 50%
cytotoxic concentration (CC50) of 50 µM. Of the other DHPM compounds tested, none had
noticeable antiviral activity. Compound 4h had marginal activity against varicella zoster virus
(
EC50 of 57 µM and MCC of 100 µM). Compounds 4b, 4c and 4d displayed no antiviral
activity yet some cytotoxicity in MT-4 cells.
Table 2. Cytotoxicity in diverse mammalian cell lines.
Cytotoxicity
a
b
Compound
MCC (µM)
CC50 (µM)
Vero
HeLa
MDCK
HEL
CRFK
MT-4
4
4
4
4
4
a
b
c
d
e
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
≥100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>125
96
76
64
>125
>125
>125
>125
>125
>125
>125
>125
50
4
f
4
4
g
h
100
4
4
i
j
>100
>100
>100
>100
≥100
>100
4
k
4
l
4
m
5
>100
>125
a
MCC: minimal cytotoxic concentration, or compound concentration producing minimal
alterations in cell morphology, as detected by microscopical inspection.
b
CC50: 50% cytotoxic concentration, determined by the colorimetric and formazan-based
MTS cell viability test.
1
0

The following cell lines were used: Vero, African green monkey kidney cells; HeLa, cervix
carcinoma cells; MDCK, Madin-Darby canine kidney cells; HEL, human embryonic lung
fibroblast cells; CRFK, Crandell-Rees feline kidney cells; MT-4, human T-lymphoblast cells.
In summary, an efficient reaction condition was developed for utilizing aliphatic
aldehydes in biginelli condensation. Ethyl 6-methyl-2-oxo-4-undecyl-1,2,3,4-tetrahydro-5-
pyrimidinecarboxylate (4m) displayed selective and potent antiviral activity against Punta
Toro virus. Based on these first results, further evaluation of 4m against other Bunyaviruses
(
such as Crimean-Congo haemorrhagic fever virus) is warranted. Also, mechanistic studies
are underway to understand the biochemical basis for its antiviral effect against Punta Toro
virus.
Acknowledgement
The authors acknowledge excellent technical assistance from Leentje Persoons, Frieda
De Meyer, Lies Van den Heurck, Kristien Erven and Jasper Rymenants. We thank Richard
Elliott (University of St Andrews, UK) for providing the RVFV MP-12 strain. This work was
supported by the Flemish Fonds voor Wetenschappelijk Onderzoek (FWO) via the Infect-
ERA EScential network.
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