In vitro activity of novel derivatives of 1,3-oxazole-4-carboxylate and 1,3-oxazole-4-carbonitrile against human cytomegalovirus

Article abstract of DOI:10.1007/s00044-019-02365-x

Ten 5-functionalized derivatives of 1,3-oxazole-4-carboxylate and 1,3-oxazole-4-carbonitrile were synthesized and their antiviral activities against the human cytomegalovirus (HCMV) were evaluated in vitro. Bioassays showed that seven compounds exhibited

Full text of DOI:10.1007/s00044-019-02365-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02365-x
ORIGINAL RESEARCH
In vitro activity of novel derivatives of 1,3-oxazole-4-carboxylate and
1,3-oxazole-4-carbonitrile against human cytomegalovirus
Maryna V. Kachaeva1 Stepan G. Pilyo1 Caroll B. Hartline2 Emma A. Harden2 Mark N. Prichard2
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Victor V. Zhirnov1 Volodymyr S. Brovarets
1
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Received: 9 January 2019 / Accepted: 21 May 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Ten 5-functionalized derivatives of 1,3-oxazole-4-carboxylate and 1,3-oxazole-4-carbonitrile were synthesized and their
antiviral activities against the human cytomegalovirus (HCMV) were evaluated in vitro. Bioassays showed that seven
compounds exhibited considerably higher antiviral activity (EC50: < 0.05 μM) against a normal laboratory HCMV strain
(AD-169) in human foreskin fibroblast cells than Ganciclovir (EC50 = 0.32 μM), an anti-HCMV agent in clinical use.
Additionally, the HCMV-resistant isolate (GDGr K17) was tested for sensitivity to 1,3-oxazole derivatives with most
antiviral potency against the strain AD169. A one of them (5-((2-hydroxyethyl)(methyl)amino)-2-(4-methylphenyl)-1,3-
oxazole-4-carbonitrile) showed very high potency (EC50: < 0.05; CC50: >150 µM, and SI50 = 3125) towards the resistant
isolate compared to standard drugs Cidofovir (EC50 = 0.10 µM, CC50: >30 µM and SI50: <4). But, in contrast to the primary
assays, the antiviral activity of these compounds against both the normal strain and the resistant isolate of HCMV were
considerably less than one of Cidofovir in secondary assay. These results provided evidence that derivatives of 1,3-oxazole
could be useful for developing new anti-HCMV drugs.
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Keywords Antiviral discovery 1 3-oxazole derivatives Human cytomegalovirus HFF cells
Introduction
against HCMV. Therefore, anti-HCMV drugs and immu-
notherapy have been approved for the treatment and pro-
phylaxis of HCMV diseases (Ahmed 2011; Tan 2014;
Hanson and Swaminathan 2015). HCMV is a double-
stranded DNA virus that belongs to the β-subfamily in large
family Herpesviridae. It is among the largest of the known
human herpes virus. The genome of HCMV has been
completely sequenced and encodes more than 200 proteins
(Chee et al. 1990). Not all of the proteins have been iden-
tified and the functions of all identified proteins have not
been characterized. However, three of the proteins are cri-
tical to the activity of antiviral agents. The first protein is
HCMV DNA polymerase. This protein is the common,
ultimate target for antiviral drugs currently approved for the
treatment of HCMV infections. At present, anti-HCMV
drugs that inhibit HCMV DNA polymerase are the
nucleoside analogs Ganciclovir, and its oral prodrug Val-
ganciclovir, Cidofovir, and the pyrophosphate analog fos-
carnet (Britt and Prichard 2018). All these substances
inhibit HCMV replication. However, these drugs have poor
oral bioavailability and many shortcomings, including
associated toxicities, and the emergence of drug-resistant
viruses (Ahmed 2011). The second, the UL97 protein
Human cytomegalovirus (HCMV) is a ubiquitous pathogen
that severely affects individuals with impaired or immature
immune-defence functions (Collins-McMillen et al. 2018;
Li et al. 2018; Yong et al. 2018). Since HCMV infects
~60% of people in the developed world and over 99% in
developing countries, the risk of morbidity due to HCMV
disease is a significant problem for public health (Griffiths
et al. 2015). So far, there is no widely available vaccine
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02365-x) contains supplementary
material, which is available to authorized users.
* Volodymyr S. Brovarets
brovarets@bpci.kiev.ua
1
Department of Chemistry of Bioactive Nitrogen-Containing
Heterocyclic Bases, V.P. Kukhar Institute of Bioorganic
Chemistry and Petrochemistry, NAS of Ukraine, 1, Murmanskaya
str, Kyiv 02094, Ukraine
2
Department of Pediatrics, University of Alabama at Birmingham,
Birmingham, AL 35233, USA

Medicinal Chemistry Research
kinase of HCMV (HCMV phosphotransferase) is respon-
sible for phosphorylating antiviral drugs to their active
forms (Prichard 2009). The HCMV UL97 kinase inhibitor
maribavir is new perspective drug because of oral bioa-
vailability and lack of cross-resistance with currently used
drugs (Frange and Leruez-Ville 2018). The third, the sub-
unit UL56 of the HCMV terminase enzyme complex is
responsible for the terminal phase of the virus life cycle.
Letermovir inhibits the terminal phase of the virus life cycle
by targeting the subunit UL56 (Chou 2015). It is currently
the most active molecule against HCMV and a preserved
activity against viruses resistant to currently available
molecules (Lischka et al. 2010; Frange and Leruez-Ville
2018). Combination of anti-HCMV agents that involve
drugs with different mechanisms of action, which could
reduce the incidence of drug resistance, is not yet available
because of limited number of anti-HCMV agents and the
lack of clinical trials (Cai et al. 2014).
Specificity against virus replication is a key issue in
chemotherapy. Because of the close interaction between
viral reproduction and normal cellular metabolism, it was
originally thought difficult to interrupt the viral life cycle
without adversely affecting the host cell metabolism.
However, it is now clear that several events associated with
viral replicative amplification either do not occur in normal
uninfected cells or are controlled by virus-specified
enzymes that differ structurally and functionally from the
corresponding host cell enzymes. For example, Cidofovir is
an acyclic nucleoside phosphonate, which nonspecifically
blocks viral replication because of more efficient conversion
of the prodrug to the active precursor in virus-infected cells
or by selective inhibition of a viral DNA polymerase
(Magee et al. 2005). It also inhibits human polymerases but
this action is 60–80 times weaker than its actions on viral
DNA polymerases (Upadhyayula and Michaels 2013).
Until recent years, there were rather few chemotherapies for
HCMV infections. The five antiviral drugs approved by the
Food and Drug Administration have shown adverse reactions
and the antiviral drug resistance were reported (Michel and
Mertens 2006; Chou 2008). Hence, this warrants the need for
urgent development of novel antiviral drugs. 1,3-Oxazole
compounds containing nitrogen and oxygen atoms in the five-
membered aromatic ring are readily able to bind with a variety
of biological structures via different non-covalent interactions,
and thus display various biological activities (Joshi et al.
2017). 1,3-Oxazole derivatives are among the most useful
heterocyclic compounds from both synthetic and medicinal
chemistry aspects (Zhang et al. 2018). A series of novel 1,3-
oxazole derivatives were synthesized and their antiviral
activities against the hepatitis C virus (HCV), the coxsackie
and HIV-1 viral strains were evaluated in vitro (see rev.
Swellmeen 2016). Some 1,3-oxazole derivatives showed
strong activity against the HCV and HIV-1 at low con-
centrations (EC50 < 2.0 μM and 14 nM, respectively) and had
low cytotoxicity. Recently, we have synthesized novel multi-
substituted 1,3-oxazole derivatives with different moieties,
such as piperazine, piperidine, sulfonamide, and carbonitrile
for evaluation of their antiviral activities (Kachaeva et al.
2017). Bioassays showed that the synthesized compounds 1-
((2,5-diphenyl-1,3-oxazole-4-yl)sulfonyl)-3-methylpiperidine,
2,5-diphenyl-1,3-oxazole-4-sulfonamide,
1-((2,5-diphenyl-
1,3-oxazole-4-yl)sulfonyl)piperidine, and 4-cyano-2-phenyl-
1,3-oxazole-5-sulfonamide exhibited potent antiviral activity
against low-risk HPV-11 (IC50: 1.7–9.6 μM) in a transient
DNA replication assay and exhibited low cytotoxicity in
HEK293 cells compared to Cidofovir, an antiviral agent in
clinical use. These data indicate that 1,3-oxazole derivatives
are promising compounds in the search for design of new anti-
HCMV drugs. The number of existing drug as antiviral is not
enough and development of new antiviral drugs is still in
requirement because of the viral resistance. There are no
reports of anti-HCMV activity of 1,3-oxazole derivatives in
available literature.
The present study is an exploratory investigation of anti-
HMСV activity by novel 1,3-oxazole derivatives designed
and synthesized in Kyiv, and evaluated with in vitro anti-
viral assays against HCMV.
Material and methods
Chemistry
1,3-Oxazole-5-sulfonyl amides 1–4 and 7–10 (Table 1)
have been synthesized from sulfonyl chlorides (Kornienko
et al. 2012) by refluxing with appropriate amine and excess
of triethylamine (Kachaeva et al. 2018)
By the reaction of 2-acylamino-3,3-dichloroacrylonitrile
(Drach et al. 1974) with appropriate amine using excess of
triethylamine (Drach et al. 1974) 5-amino-1,3-oxazoles 5
and 6 (Table 1) have been prepared.
The structure of synthesized 1,3-oxazole derivatives are
confirmed by NMR (1H and 13C NMR), IR spectroscopy,
chromato-mass, and elemental analysis. All CH-proton
1
signals of compound 10 are visible in the H NMR spec-
trum. Signal of the OH group of compound 7 is seen at
3.94, of compound 6—at 4.92 ppm. NH2 group (4) is
1
observed in the H NMR spectrum at 7.58 ppm, in IR—at
3424 cm–1. The intensive absorption bands of SO2-group of
compounds 4, 7, and 10 are observed in the IR spectra at
1154–1164 and 1339–1395 сm−1. Also the broad intensive
bands at 1741 cm−1 corresponded to ester C=O bond of
compound 4 and intensive bands at 2198–2253 cm–1 cor-
responded to CN group of 6, 7, and 10 were observed.

Medicinal Chemistry Research
Table 1 Chemical structures of compounds
Compound
Structure
Name
1
methyl 5-((5-amino-3-phenyl-1H-
pyrazol-1-yl)sulfonyl)-2-phenyl-1,3-
oxazole-4-carboxylate
O
OMe
N
N
N
O
S
O
O
NH2
O
2
3
methyl 5-((5-amino-3-methyl-1H-
pyrazole-1-yl)sulfonyl)-2-phenyl-1,3-
oxazole-4-carboxylate
OMe
N
Me
N
N
O
S
O
O
NH2
methyl 5-((5-amino-3-phenyl-1H-
pyrazole-1-yl)sulfonyl)-2-(4-
methylphenyl)-1,3-oxazole-4-
carboxylate
O
OMe
N
N
N
O
S
O
O
O
Me
NH2
4
methyl 5-((5-amino-1H-1,2,4-triazole-
1-yl)sulfonyl)-2-phenyl-1,3-oxazole-
4-carboxylate
OMe
N
N
N
O
N
S
O
O
NH2
N
5
6
5-((2-hydroxyethyl)amino)-2-(4-
methylphenyl)-1,3-oxazole-4-
carbonitrile
N
N
OH
N
H
O
Me
N
5-((2-hydroxyethyl)
(methyl)amino)-2-(4-methylphenyl)-
1,3-oxazole-4-carbonitrile
OH
N
O
Me
Me
N
7
4-cyano-N-(2-hydroxyethyl)-2-(4-
methylphenyl)-1,3-oxazole-5-
sulfonamide
N
H
N
S
O
O
O
O
OH
Me
N
8
9
4-cyano-N-(2-hydroxyethyl)-N-
methyl-2-(4-methylphenyl)-1,3-
oxazole-5-sulfonamide
N
Me
N
S
O
N
O
OH
Me
N-(2-(4-chlorophenyl)-2-(1-
piperidinyl)ethyl)-4-cyano-2-phenyl-
1,3-oxazole-5-sulfonamide
N
N
H
N
S
O
O
O
Cl
N
S
10
N-(2-(4-chlorophenyl)-2-(1-
piperidinyl)ethyl)-4-cyano-2-(4-
methylphenyl)-1,3-oxazole-5-
sulfonamide
N
N
H
N
O
O
O
Me
Cl

Medicinal Chemistry Research
General chemistry methods
Methyl 5-((5-amino-1H-1,2,4-triazol-1-yl)sulfonyl)-2-
phenyl-1,3-oxazole-4-carboxylate (4)
Melting points were determined on a Fisher–Johns appa-
White solid (75%); mp (acetonitrile) 179–181 °C; IR
(KBr) νmax/cm−1 1049, 1162 (SO2), 1339 (SO2), 1418, 1741
(C=O), 3424 (NH2). 1H NMR (500 MHz, (CD3)2SO) δ
3.89 (3H, s, CH3O), 7.58 (s, 2H, NH2), 7.61–7.64 (2H, m,
ArH), 7.68–7.74 (2H, m, ArH), 8.00–8.03 (2H, m, ArH).
13C NMR (125 MHz, CDCl3) δ 53.8, 124.6, 127.7, 130.1,
133.7, 136.1, 145.8, 154.0, 158.2, 159.3, 162.9. LCMS, m/z
350 [M + 1]+. Anal. calcd. for C13H11N5O5S: C, 44.70%;
H, 3.17%; N, 20.05%; S, 9.18%. Found: C, 44.71%; H,
3.15%; N, 20.12%; S, 9.26%.
ratus. IR spectra were recorded on a Vertex-70 spectrometer
1
from KBr pellets. H and 13C NMR spectra were recorded
on Varian Mercury 400 (400 MHz) and Bruker Avance
DRX 500 (500 and 125 MHz, respectively) spectrometers in
(CD3)2SO or CDCl3 taking its residual protons signal as a
standard. LCMS analysis was performed on an Agilent
1200 Series system equipped with a diode array and a
G6130A mass-spectrometer (atmospheric pressure electro-
spray ionization). Combustion elemental analysis was per-
formed in the V.P. Kukhar Institute of Bioorganic
Chemistry and Petrochemistry of the NAS of Ukraine
analytical laboratory.
5-(2-Hydroxyethylamino)-2-(4-methylphenyl)-1,3-
oxazole-4-carbonitrile (5) have been described in Koza-
chenko et al. (2012).
Chemicals and reagents were purchased from commer-
cially available sourses. 2-Methylaminoethanol, 2-
aminoethanol and 1H-1,2,4-triazole-5-amine were pur-
chased from Aldrich. 2-(4-Chlorophenyl)-2-piperidin-1-
ylethanamine was synthesized by the previously described
method (Briner et al. 2006).
5-(2-Hydroxyethylmethylamino)-2-(4-methylphenyl)-
1,3-oxazole-4-carbonitrile (6) White solid (70%); mp
(ethanol) 104–106 °C; IR (KBr) νmax/cm−1 1048, 1634,
1
2198 (CN), 3446 (OH). H NMR (400 MHz, (CD3)2SO) δ
2.34 (3H, s, CH3), 3.21 (3H, s, NCH3), 3.54–3.57 (2H, m,
CH2), 3.62–3.65 (2H, m, CH2), 4.92 (1H, t, J = 5.2 Hz,
OH), 7.30 (2H, d, J = 8.0 Hz, ArH), 7.72 (2H, d,
J = 8.0 Hz, ArH). 13C NMR (125 MHz, CDCl3) δ 21.5,
38.0, 54.0, 59.9, 84.8, 116.8, 123.1, 125.4, 129.5, 140.5,
150.9, 160.5. LCMS, m/z 258 [M + 1]+. Anal. calcd. for
C14H15N3O2: C, 65.36; H, 5.88; N, 16.33. Found: C, 65.28;
H, 5.86; N, 16.35.
General procedure for the synthesis of compounds
1–4 and 7–10
To a solution of appropriate 1,3-oxazole-5-sulfonyl chloride
(0.01 mol) and amine (0.011 mol) in THF (30 ml), Et3N
(0.011 mol) was added. The mixture was refluxed for 2 h
and kept at 20–25 °C for 12 h. The precipitate was filtered
off the solvent was removed in a vacuum. The residue was
treated with water, filtered off, dried at 70–80 °C and
recrystallized from ethanol or acetonitrile.
4-Cyano-N-(2-hydroxyethyl)-2-(4-methylphenyl)-1,3-
oxazole-5-sulfonamide (7) White solid (74%); mp (etha-
nol) 132–134 °C; IR (KBr) νmax/cm−1 1054, 1154 (SO2),
1
1351 (SO2), 1499, 1554, 2253 (CN), 3513 (OH). H NMR
(400 MHz, (CD3)2SO) δ 2.41 (1H, s, CH3), 3.17 (2H, t, J =
4.8 Hz, CH2), 3.43–3.44 (2H, m, CH2), 3.94 (1H, br s, OH),
7.44 (2H, d, J = 7.6 Hz, ArH), 7.92 (2H, d, J = 7.6 Hz,
ArH), 8.92 (1H, s, NH). 13C NMR (125 MHz, CDCl3) δ
21.7, 46.0, 60.1, 111.5, 116.1, 122.1, 127.6, 130.5, 143.9,
154.1, 163.1. LCMS, m/z 308 [M + 1]+. Anal. calcd for
C13H13N3O4S: C, 50.81%; H, 4.26%; N, 13.67%; S,
10.43%. Found: C, 50.78%; H, 4.24%; N, 13.60%; S,
10.55%.
4-Cyano-N-(2-hydroxyethyl)-N-methyl-2-(4-methyl-
phenyl)-1,3-oxazole-5-sulfonamide (8) have been descri-
bed in Kachaeva et al. (2018).
N-(2-(4-Chlorophenyl)-2-piperidin-1-ylethyl)-4-
cyano-2-phenyl-1,3-oxazole-5-sulfonamide (9) have been
described in Kachaeva et al. (2018).
General procedure for the synthesis of compounds 5
and 6
To a solution of N-(2,2-dichloro-1-cyanovinyl)-4-methyl-
benzamide (0.01 mol) in 30 ml of THF, triethylamine
(0.022 mol) and an appropriate amine (2-methylami-
noethanol or 2-aminoethanol (0.011 mol) were added. The
mixture was stirred at room temperature during 12 h. The
residue was triturated with water to give a crude product
which was separated, dried at 70–80 °C, and recrystallized
from ethanol.
Methyl 5-((5-amino-3-phenyl-1H-pyrazol-1-yl)sulfo-
nyl)-2-phenyl-1,3-oxazole-4-carboxylate (1) have been
described in Kachaeva et al. (2018).
Methyl 5-((5-amino-3-methyl-1H-pyrazol-1-yl)sulfo-
nyl)-2-phenyl-1,3-oxazole-4-carboxylate (2) have been
described in Kachaeva et al. (2018).
N-(2-(4-Chlorophenyl)-2-(1-piperidinyl)ethyl)-4-
cyano-2-(4-methylphenyl)-1,3-oxazole-5-sulfonamide
(10) Yellow solid (69%); mp (ethanol) 148–150 °C; IR
(KBr) νmax/cm−1 1092, 1161 (SO2), 1395 (SO2), 1496,
1
Methyl 5-((5-amino-3-phenyl-1H-pyrazol-1-yl)sulfo-
1588, 1616, 2253 (CN), 3321 (NH). H NMR (500 MHz,
nyl)-2-(4-methylphenyl)-1,3-oxazole-4-carboxylate
(3)
(CD3)2SO) δ 1.18 (2H, br s, CH2), 1.35 (4H, br s, 2CH2),
have been described in Kachaeva et al. (2018).
2.12 (2H, br s, CH2), 2.26 (2H, br s, CH2), 2.41 (3H, s,

Medicinal Chemistry Research
Table 2 Antiviral activity against the normal strain as well as the
resistant isolate (GDGr K17) of HCMV, and cytotoxicity of the 1,3-
oxazole derivatives in HFF cell line. Compound concentrations are in µM
CH3), 3.41–3.48 (2H, m, CH2, CH), 3.59–3.66 (2H, m,
CH2), 7.18 (2H, d, J = 8.5 Hz, ArH), 7.27 (2H, d,
J = 8.5 Hz, ArH), 7.43 (2H, d, J = 8.0 Hz, ArH), 7.87 (2H,
d, J = 8.0 Hz, ArH). 13C NMR (125 MHz, (CD3)2SO) δ
21.71, 24.4, 26.0, 44.5, 50.6, 63.1, 67.9, 111.5, 115.8,
121.9, 127.6, 128.2, 130.6, 132.5, 135.4, 143.9, 154.2.
LCMS, m/z 485 [M + 1]+. Anal. calcd for C24H25ClN4O3S:
C, 59.44; H, 5.20; N, 11.55; S, 6.61. Found: C, 59.41; H,
5.22; N, 11.65; S, 6.72.
Compound
Strain AD169
GDGr K17 (resistant
isolate)
EC50
CC50 SI50
EC50
CC50
SI50
1
<0.05 17.0
<0.05 87.2
<0.05 16.0
>6.00 29.0
>356
>1.20 4.5
<4
2
>1818 >30
74.0
<2
3
>334
<5
<0.05 4.0
>83
4
Antiviral and cytotoxicity assays
5
<0.05 >150 >3125 >30
106.2
<4
6
<0.05 >150 >3125 <0.05 >150
>3125
<2
CPE assay
7
<0.05 88
>1833 >30
53.7
2.46
8
<0.05 16.4
>6.00 20.7
>6.00 12.6
>342
<3
0.05
46
For the primary assay, antiviral activity and cytotoxicity
were evaluated using CellTiter-Glo™ Assay (Promega
Corporation, USA) by methods reported recently (Hartline
et al. 2018). This is a simple, quantifiable method of
determining viral-induced cytopathic effects (CPE) and
compound toxicity in host cells. Briefly, CPE reduction
assays were performed in medium consisting of MEM with
Earle’s salts, 2% FBS and standard concentrations of L-
glutamine, penicillin, and gentamycin. Cells were seeded
into 384-well microtiter plates and subsequently incubated
at 37 °C in a humidified 5% CO2 incubator for 24 h to
allow the formation of confluent monolayers. Dilutions of
test compounds were then prepared directly in the plates in
a series of five-fold dilutions in duplicate wells to yield
final concentrations that range from 300 to 0.1 µM or from
10 to 0.003 µM. Monolayers were then infected at a mul-
tiplicity of infection of 0.005 PFU/cell with HCMV and
incubated further until 100% CPE was observed in the
virus control wells. Cytopathology was determined by the
addition of CellTiter-Glo reagent according to the manu-
facturer’s suggested protocol. Concentrations of test com-
pounds sufficient to reduce CPE by 50% (EC50) were
interpolated from the experimental data. Cytotoxicity was
also determined with CellTiter-Glo and concentrations of
the compounds that decreased cell viability by 50% (CC50)
were also calculated from the data and selective index (SI)
values were calculated as the CC50/EC50 as a measure of
antiviral activity.
9
10
<2
Ganciclovir
Cidofovir
0.32 >150 >463
17
>150.00 >8
>30.00 <4
0.10
Control and drug concentrations ranges are 0.048–150 μM. Vehicle is
DMSO. EC50 and CC50 are compound concentrations that reduce viral
replication and cell viability, respectively, by 50% in CellTiter-Glo
(cytopathic effect/toxicity) assay. Selectivity index (SI50) is calculated
as the CC50 value divided by the EC50 value
determined in tissue culture infectious dose assays per-
formed in HFF cells in 384-well plates.
Results and discussion
The effects of the 1,3-oxazole derivatives on antiviral
activity against a normal laboratory HCMV strain, AD-169,
and their cytotoxicity were first evaluated on HFF cells
using CellTiter-Glo (cytopathic effect/toxicity) assay (Table
2).
All compounds exhibited antiviral activity against the
normal HCMV strain AD169 and, with the exception of 4,
9, and 10, had EC50 values of <0.05 µM, and were con-
siderably more active than Ganciclovir (EC50 = 0.32 µM) in
this assay. The most selective compounds were 5 = 6 >>
7 ≈ 2 >> Ganciclovir > 1 ≈ 8 and 3.
Yield reduction assays
The HCMV-resistant isolate (GDGr K17) was tested for
sensitivity to the compounds with high antiviral activity
against the normal HCMV strain AD169 (Table 2). The 1,3-
oxazole derivatives 3 and 6 showed very high potency
(EC50: < 0.05 µM) towards the resistant isolate compared to
standard drug Cidofovir (EC50 = 0.10 µM). Compound 8
showed moderate activity and was still two times more
potent (EC50 = 0.05 µM) than Cidofovir. The most selective
compounds were 6 >> Cidofovir >> 3 > 8 > 1 = 5 > 2 = 7 >
Ganciclovir.
Selected compounds with good antiviral activity were
evaluated further with virus yield reduction (VYR) assays.
Briefly, monolayers of HFF cells in 96-well plates were
infected with HCMV a multiplicity of infection of 0.5 PFU/
cell in the presence of dilutions of the test compounds. At
4 days following infection, the plates containing mono-
layers of infected cells were frozen to lyse the cells, then the
contents were mixed and the titer of the progeny virus was

Medicinal Chemistry Research
Table 3 Anti-HCMV activity and cytotoxicity of 1,3-oxazole
derivatives in yield reduction assays in HFF cells. Compound
concentrations are in µM
important roles in the activation of viral IE gene expression.
Pharmacological inhibition of the HDM leads to severely
impaired viral IE gene expression and may block HCMV
lytic infection and reactivation (Gan et al. 2017). This may
suggest hypothetical mechanism of anti-HCMV action of
1,3-oxazoles derivatives here.
Compound
EC50
EC90
CC50
SI50
SI90
AD169
5
>20.00
>100.00
>20.00
4.65
>20.00
>100.00
>20.00
15.8
46.70
<2
1
<2
1
6
>100.00
73.30
Large numbers of compounds for their ability to inhibit
have been synthesized and tested in HFF cells in vitro but
only few have been developed (Fader et al. 2016; Britt and
Prichard 2018). Therefore, a problem has occurred with the
adequate of model cell line and/or experimental methods
that is used for antiviral screening of new compounds
(Griffiths 2002). HCMVs are highly restricted in their
tropism, so the human virus is challenging to study outside
of cultured cells. In fibroblast cell cultures HCMV repli-
cates produce CPE very slowly. This slow appearance of
CPE is misleading about the true nature of CMV replica-
tion. In vivo, in its natural human host, HCMV replicates
rapidly (Emery et al. 1999). This contrast between findings
in vivo and in vitro is only one of the differences between
replication of the virus in cell cultures and the human host.
Thus, we cannot exclude the possibility that these mole-
cules may demonstrate potent antiviral activity in animal
models.
7
<4
>21
>31
<4
>6
>6
Ganciclovir
Cidofovir
>100.00
>20.00
0.63
2.9
GDGr K17 (resistant isolate)
5
>20.00
>100.00
>20.00
30.8
>20.00
>100.00
>20.00
98.7
46.70
<2
1
<2
1
6
>100.00
73.30
7
<4
>3
>20
<4
>1
>5
Ganciclovir
Cidofovir
>100.00
>20.00
0.97
3.4
Drug concentration ranges are 0.032–100 μM. Control concentration
ranges are 0.8–100 (Ganciclovir) and 0.16–20 μM (Cidofovir). Vehicle
is DMSO. EC50 and CC50—compound concentrations that reduce viral
replication and cell viability by 50% in yield reduction assay.
Selectivity index (SI50) is calculated as the CC50 value divided by
the EC50 value
Compounds 5, 6, and 7, which showed high activity against
the normal strain of HCMV in the primary in vitro antiviral
assays, were chosen for the secondary assays. The effects of
compounds 5, 6, and 7 on the viral replication and the cell
cytotoxicity in a HFF culture using yield assay were
investigated.
Conclusion
In contrast to the primary assay, the antiviral activity of
these compounds against both the susceptible and the
resistant isolate GDGr K17 of HCMV was not detectable
(Table 3). Differences in the antiviral activity measured by
CPE assay and the yield reduction assay may be related to
compound exposure is 14 days and 4 days, respectively.
In this assay, compounds 5 and 7 have shown the
identical antiviral activity against the normal strain AD169
and the resistant isolate GDGr K17, which is more than for
compound 6 but less than for control drugs.
Thus, only 5-amino- and 5-sulfonamide derivatives with
hydroxyethyl group (compounds 5 and 7) displayed the mild
biological activity (EC50: >20.0 μM) among the three analogs
of 1,3-oxazole-4-carbonitrile tested in the secondary assay.
Introducing into the 5-position of the 1,3-oxazole ring another
substituents led to a negative impact on HCMV inhibition.
All synthesized compounds belong to 2-phenyl-1,3-
oxazoles. It was shown that compounds of this type are
potent inhibitors of histone demethylases in vitro (Dulla
et al. 2013). As mentioned above, HCMV infects a broad
range of the population and establishes life-long latency in
the infected individuals. Periodically the latently infected
virus can reactivate and provoke morbidity in immuno-
compromised individuals. Upon reactivation, viral lytic
gene transcription is initiated by the expression of viral
immediate early (IE) genes, and histone demethylases play
Ten five-functionalized derivatives of 1,3-oxazole-4-car-
boxylate and 1,3-oxazole-4-carbonitrile have been synthe-
sized and evaluated for in vitro antiviral activity and
cytotoxicity. Bioassays showed that some of the synthesized
compounds exhibited considerably higher antiviral activity
(EC50: < 0.05 μM) against a normal laboratory HCMV
strain (AD-169), and the HCMV-resistant isolate (GDGr
K17) in HFF cells than Ganciclovir. In contrast to the pri-
mary assays, the antiviral activity of these compounds
against both the normal strain and the resistant isolate of
HCMV were less than one of cidofovir in secondary assay.
Additional research will be required to understand fully the
activities of these compounds against HCMV.
Disclaimer
This material should not be interpreted as representing the
viewpoint of the National Institute of Allergy and Infectious
Diseases (USA) and its Collaborative Antiviral Testing Group.
Acknowledgements We would like to thank Enamine Ltd. for the
material and technical support. These studies were funded in whole or
in part with Federal funds from the National Institutes of Allergy and
Infectious Diseases, National Institutes of Health, Department of Health
and Human Services, under contract HHSN272201100016I (MNP).

Medicinal Chemistry Research
Compliance with ethical standards
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Conflict of interest The authors declare that they have no conflict of
interest.
Hartline CB, Keith KA, Eagar J, Harden EA, Bowlin TL, Prichard MN
(2018) A standardized approach to the evaluation of antivirals
against DNA viruses: orthopox-, adeno-, and herpesviruses.
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Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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