3-Aryl-1,2,4-oxadiazole Derivatives Active Against Human Rhinovirus

Article abstract of DOI:10.1021/acsmedchemlett.8b00134

The human rhinovirus (hRV) is the causative agent of the common cold that often aggravates respiratory complications in patients with asthma or chronic obstructive pulmonary disease. The high rate of mutations and variety of serotypes are limiting the development of anti-hRV drugs, which emphasizes the need for the discovery of novel lead compounds. Previously, we identified antiviral compound 1 that we used here as the starting material for developing a novel compound series with high efficacy against hRV-A and -B. Improved metabolic stability was achieved by substituting an ester moiety with a 1,2,4-oxadiazole group. Specifically, compound 3k exhibited a high efficacy against hRV-B14, hRV-A21, and hRV-A71, with EC₅₀ values of 66.0, 22.0, and 3.7 nM, respectively, and a relevant hepatic stability (59.6 and 40.7% compound remaining after 30 min in rat and human liver microsomes, respectively). An in vivo study demonstrated that 3k possessed a desirable pharmacokinetic profile with low systemic clearance (0.158 L·h^-1·kg^-1) and modest oral bioavailability (27.8%). Hence, 3k appears to be an interesting candidate for the development of antiviral lead compounds.

Full text of DOI:10.1021/acsmedchemlett.8b00134

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3-Aryl-1,2,4-Oxadiazole Derivatives Active Against Human Rhinovirus
Jinwoo Kim, Jin Soo Shin, Sunjoo Ahn, Soo Bong Han, and Young-Sik Jung
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00134 • Publication Date (Web): 13 Apr 2018
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ACS Medicinal Chemistry Letters
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3ꢀArylꢀ1,2,4ꢀOxadiazole Derivatives Active Against Human Rhinovirus
Jinwoo Kim,† Jin Soo Shin,† Sunjoo Ahn,† Soo Bong Han,†, ‡ and Young-Sik Jung†, ‡,*
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†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
KEYWORDS Human rhinovirus, Capsid-binding, Antiviral compound, Small molecule inhibitor, Oxadiazole.
ABSTRACT: The human rhinovirus (hRV) is the causative agent of the common cold that often aggravates respiratory complica-
tions in patients with asthma or chronic obstructive pulmonary disease. The high rate of mutations and variety of serotypes are lim-
iting the development of anti-hRV drugs, which emphasizes the need for the discovery of novel lead compounds. Previously, we
identified antiviral compound 1 that we used here as the starting material for developing a novel compound series with high effica-
cy against hRV-A and B. Improved metabolic stability was achieved by substituting an ester moiety with a 1,2,4-oxadiazole group.
Specifically, compound 3k exhibited a high efficacy against hRV-B14, hRV-A21, and hRV-A71, with EC50 values of 66.0, 22.0,
and 3.7 nM, respectively, and a relevant hepatic stability (59.6 and 40.7% compound remaining after 30 min in rat and human liver
microsomes, respectively). An in vivo study demonstrated that 3k possessed a desirable pharmacokinetic profile with low systemic
clearance (0.158 L·h-1·kg-1) and modest oral bioavailability (27.8%). Hence, 3k appears to be an interesting candidate for the devel-
opment of antiviral lead compounds.
The human rhinovirus (hRV), a member of the Enterovirus
genus in the Picornaviradae family, is a persistent threat to
public health. It is known to cause ~60% of upper respiratory
tract symptoms such as the common cold. Moreover, recent
studies conducted with improved detection methods suggest
that hRV infections can aggravate inflammatory illnesses such
as asthma, chronic obstructive pulmonary disease, and otitis
media.1-6 Analyses of viral specimens from pediatric patients
with asthma exacerbations identified a high prevalence of
hRV.7-9 A recent study also revealed that early life hRV
wheezing illnesses increase the risk of asthma development at
adolescence.10
cluding rearrangements of the endoplasmic reticulum and
Golgi secretory apparatus, although the specific steps vary
among subspecies.21-23
Several drug candidates have been developed to combat
hRV infections.24,25 Capsid-binding inhibitors include plecon-
aril and vapendavir that associate with the canyon to stabilize
the capsid structure, thereby preventing viral genome intru-
sion.26,27 Inhibitors of 3C protease, such as rupintrivir and V-
7404, block the maturation of viral proteins,28,29 whereas envi-
roxime, an inhibitor of viral protein 3A, prevents viral replica-
tion.30 However, there is a need for the discovery of novel
anti-hRV agent candidates because current antiviral drugs are
not approved for hRV treatment due to high treatment failure
rates and significant side effects.
More than 160 hRV serotypes have been identified and
grouped into three species, hRV-A, B, and C, that are each
divided into various subspecies.11 Like other picornaviruses,
hRV has a positive-sense, single-stranded RNA genome pack-
aged in an icosahedral capsid composed of four viral proteins
(VP1 to VP4).12 The capsid features canyons around its five-
fold symmetry axes that contain binding sites for host recep-
tors such as the intracellular adhesion molecule 1 (ICAM-1),
which recognizes most of the hRV subspecies.13-15 The neu-
tralizing epitopes, which are hypervariable among the hRV
subspecies, are also located along the canyons.16-18 In addition,
the canyons of several hRV subspecies harbor small molecules,
the “pocket factors,” that are probably recovered from the host
to facilitate receptor recognition by stabilizing the capsid
structure.19 Receptor binding induces conformational changes
in the capsid to promote its decomposition and enable the in-
jection of the genome into the host cell.20 Viral RNA transla-
tion produces a single polyprotein, which is processed into its
various parts such as 2A and 3C viral proteases that are crucial
for maturation of viral proteins. Subsequent viral replication is
accompanied by alterations in the host cell architecture, in-
Recently, we revealed a novel series of small-molecule cap-
sid-binding inhibitors with high effectiveness against replica-
tion of hRV-A and B (Figure 1).31 An ester moiety and the
high hydrophobicity of the inhibitors represented targets for
optimization that may lead to improvements in metabolic sta-
bility and pharmacokinetics. Here, we hypothesized that these
improvements can be achieved by substituting the ester with
an oxadiazole moiety. This study describes new 3-aryl-1,2,4-
oxadiazole derivatives that exhibit strong activity against
hRV-B14, A21, and A71, along with significant metabolic
stability and hydrophilicity.
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aReagents and conditions: (a) DMF, SOCl2, 80 °C, 4 h, then
NH3, H2O, THF, 0 °C to 25 °C, 16 h; (b) TFAA, pyridine,
ClCH2CH2Cl, 25 °C, 1.5 h; (c) NH2OH, H2O, EtOH, 90 °C, 48 h;
(d) ROCl, pyridine, 0 °C to 120 °C, 16 h; (e) BBr3, CH2Cl2, -78
°C to 25 °C, 16 h; (f) 11, neat, 150 °C, 60 h; (g) DMF, SOCl2,
45 °C to 75 °C, 72 h, then CH3NH2, H2O, THF, 0 °C to 25 °C, 4 h.
1
2
3
4
5
6
7
8
9
Although compound 2 derivatives possess a lower hydro-
phobicity than compound 1 because of the oxadiazole substitu-
tion, the new derivatives are extended in length, and additional
alkyl length variations of two or more carbon atoms on the
oxadiazole ring could significantly interfere in the interaction
with one end of the viral capsid canyon. We assumed that
changing the benzo[b]thiophene core in 2 to phenyl in 3 would
not only further increase the hydrophilicity but also provide
space for additional variations on the core fragment and the
oxadiazole ring. Although the orientation of the oxadiazole
ring became distorted, the anti-hRV activities were unlikely to
be diminished because, in our previous study, the naphthyl
analogs of 1 had also retained activity against hRV-A and B
species.
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Figure 1. Previously discovered anti-hRV compound (1) and new
derivatives (2 and 3). aLiver microsomal stability: amount remain-
ing after 30 min (in percent).
The oxadiazole moieties of the new derivatives were pre-
pared from corresponding nitrile groups through cyclization of
an N-hydroxyimidamide intermediate (Scheme 1 and 2). The
benzo[b]thiophenyl oxadiazole compounds 2 were synthesized
according to the synthetic route outlined in Scheme 1. A refer-
ence procedure was employed to synthesize ben-
zo[b]thiophene core fragment 4, which was converted into
amide 5 via the acid chloride intermediate using SOCl2. Am-
ide 5 was further cyanated with trifluoroacetic anhydride
(TFAA) and pyridine using chlorinating solvents. Addition of
NH2OH in H2O-EtOH medium yielded the N’-
hydroxyimidamide intermediate 7. Oxadiazole moieties with
diverse alkyl chains were obtained by the cyclization using
corresponding acid chlorides. Demethylation of the 6-methoxy
mask with BBr3 exposed the phenolic hydroxyl group for sub-
sequent coupling (9). The coupling partner, 4-chloro-N-
methylpicolinamide (11), was generated from picolinic acid 10
by over-chlorination in SOCl2 and DMF followed by ami-
dation. Coupling of 9 and 11 under neat conditions at 150 °C
yielded compounds 2 with one exception. The trifluoromethyl
compound 2e was obtained by creating a pre-coupled amide
from 11 and 6-hydroxy-3-methylbenzo[b]thiophene-2-
carboxamide because of its instability under BBr3 demethyla-
tion condition (see Supporting Information for details).
The synthetic route to phenyl oxadiazole compounds 3 is
presented in Scheme 2. N’-Hydroxybenzimidamides 15 were
obtained from corresponding 4- or 3-hydroxybenzonitriles 14
using NH2OH. Commercially unavailable benzonitriles were
synthesized from hydroxybenzoic acids 12 via the amide in-
termediates 13. To obtain 14, cyanation of 13 was performed
using TFAA and pyridine in THF because using the reagents
in dichloroethane gave lower yields. Continuing the synthetic
route by cyclization of 15 with TFAA or acyl chlorides yield-
ed
phenyl
oxadiazole
cores
16.
4-Chloro-N,N-
dimethylpicolinamide 17 was prepared by methylating 11
using MeI and NaH. The phenolic group of 16 was coupled
with 11 or 17 under neat conditions to obtain the desired com-
pounds 3. Because of side reactions during oxadiazole ring
formation, the pyridine analog 3s was synthesized using a
synthetic
route
like
in
Scheme
1.
The
6-
(oxadiazolyl)naphthalen-2-ol counterpart 3t, however, was
synthesized like the phenyl oxadiazole derivatives (See Sup-
porting Information for the details).
Scheme 1. General synthetic route to benzo[b]thiophene
Scheme 2. General synthetic route to phenyl compounds.a
compounds.a
N
OH
c
R
S
S
7
NH2
O
O
4
5
R = CO2H
a
b
R = CONH2
d
6 R = CN
N
O
N
HO
S
N
Y
R
8
9
Y = OMe
Y = OH
O
10
e
g
f
N
N
N
H
N
O
N
H
Cl
N
S
O
R
O
11
2
O
R = Me, Et, cPr, iPr
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aReagents and conditions: (a) DMF, SOCl2, 70 °C, 5 h, then
Specifically, an improved anti-hRV21 activity was observed
for 3j, exhibiting EC50 values in the range between 2.5 and
73.0 nM against the three hRV species (Entry 16). Further
improvement was obtained by adding a methyl group in the
ortho position towards oxadiazole, which significantly re-
duced the cytotoxicity (Entry 17). Compound 3k exhibited
EC50 values of 66, 22, and 3.7 nM against hRV-B14, A21, and
A71, respectively. Although the dimethyl derivative 3l still
exhibited high efficacy against hRV-A strains, the activity
against hRV-B14 was strongly reduced along with a strong
increase in cytotoxicity (Entry 18). The N,N-
dimethylpicolinamide analogs 3m–o also exhibited high cyto-
toxicity associated with CC50 values that were no longer above
the EC50 values against hRV-A strains (Entries 19–21). Vari-
ants with meta-substituted phenyl core 3p–r did not exhibit
useful antiviral activity profiles, presumably, because these
inhibitor molecules are bent, which may prevent the critical
hydrophobic interaction between the oxadiazole moiety and
the viral pocket (Entries 22–24). In addition, the pyridine de-
rivative 3s with low cytotoxicity showed only moderate antivi-
ral activity (Entry 25), whereas the naphthalene derivative 3t
was effective against hRV-A71, but its cytotoxicity was too
high for its other modest activities against hRV-B14 and A-21
(Entry 26).
NH3, H2O, THF, 0 °C to 25 °C, 16 h; (b) TFAA, pyridine, THF,
0 °C to 25 °C, 16 h; (c) NH2OH, H2O, EtOH, 90 °C, 16 h; (d)
RCOCl or TFAA, pyridine, 0 °C to 120 °C, 16 h; (e) 11 or 17,
neat, 150 °C, 90 h; (f) DMF, SOCl2, 45 °C to 75 °C, 72 h, then
CH3NH2, H2O, THF, 0 °C to 25 °C, 4 h; (g) MeI, NaH, DMF, 0 °C
to 25 °C, 3 h.
1
2
3
4
5
6
7
8
The antiviral activities of oxadiazole compounds 2–3
against hRV-B14, A21, and A71 were assessed in H1HeLa
cells using the MTT assay with pleconaril as a reference (Ta-
ble 1). 5-Methyl-1,2,4-oxadiazole derivative 2a, a compound
with a molecular volume similar to 1, exhibited activity
against the three hRV strains at a nanomolar concentration
range (Entry 2). Longer and bulkier alkyl substitutions on po-
sition 5 of the oxadiazole ring resulted in significantly higher
cytotoxicity and slightly improved anti-hRV activity (Entries
3–5). Comparing the activities between ethyl-substituted 2b
and cyclopropyl-substituted 2c, their activities were similar
against hRV-B14, but 2c was much stronger against hRV-A21
than 2b, and approximately 50% less active against hRV-A71
(Entries 3, 4). Assessing the differences between 2b and iso-
propyl-substituted 2d, the activity of 2d was lower against
hRV-B14, higher against hRV-A21, and not different towards
hRV-A71 (Entry 5). 5-Trifluoromethyl derivative 2e had low
cytotoxicity but its activity towards the three hRV strains was
the lowest among the compound 2 variants, implying that elec-
tron-richness on the oxadiazole ring is critical for the anti-hRV
activities (Entry 6).
9
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11
12
13
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17
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The Phase I metabolic stability of selected compounds (2d,
3j, 3k, and 3l) was investigated in rat and human liver micro-
somes (Table 2). The compounds showed higher stability in rat
liver microsome than compound 1. Interestingly, 2d and 3l are
more stable than 1 in human liver microsome. The most active
and least cytotoxic compound 3k displayed improved Phase I
metabolic stability to compound 1. To elucidate the pharmaco-
kinetics of 3k, male Sprague-Dawley rats received doses of 5
and 10 mg·kg-1 via the intravenous and oral route, respectively.
The 3k plasma concentration was determined using LC-
MSMS after sample deproteinization in acetonitrile. The
plasma concentration-time data were analyzed by applying the
non-compartmental method using Phoenix WinNonlin (v6.4;
Pharsight Corp., Mountain View, CA, USA). After intravenous
dosing, the AUCt value at 5 mg·kg-1 was 31.3 µg·h·mL-1 along
with a low systemic clearance rate of 0.158 L·h-1·kg-1. After
the oral administration, 3k was slowly absorbed and reached a
Cmax of 3.9 µg·mL-1 . Then, the 3k plasma concentrations de-
clined with a terminal T1/2 of 3.2 h. The systemic exposure
(AUCt) following an oral dose was 17.4 µg·h·mL-1 and the oral
bioavailability was 27.8%.
Anti-hRV activities and cytotoxicity of phenyl oxadiazole
derivatives 3a–t were also assessed. The para-substituted phe-
nyl variants 3a–d and 3g showed improved cytotoxicity but
exhibited mostly lower activities than their respective ben-
zo[b]thiophene counterparts (Entries 7–10, 13). Specifically,
3a had much lower anti-hRV activities than 2a. Although, the
tendencies observed for the activities of compounds 3b–d
were similar to their respective counterparts 2b–d, the activity
of 3c was lower against hRV-21 than against hRV-71, which
is the inverse activity pattern observed for 2c. Furthermore,
the extension of the 5-alkyl substitution to isobutyl or methox-
ymethyl significantly reduced anti-hRV activities, suggesting
that derivatives with 5-alkyl substitutions of more than two
carbon atoms cannot properly fit into the viral capsid canyon
(Entries 11, 12). Like 2e, the CF3 derivative 3g exhibited a
much lower antiviral activity than 3b–e (Entry 13).
Based on our previous study, we hypothesized that an addi-
tional alkyl substitution on the phenyl core of 3 could enhance
the interaction between the inhibitor and residue L25 of VP 3,
one of the viral capsid constituents.31 Addition of a methyl
group on the meta position towards oxadiazole preserved anti-
hRV activities but increased cytotoxicity (Entries 14–16).
Table 1. Inhibitory activities of compounds 2 and 3 against hRVs.
EC50 (µM)b
H1HeLa
Com-
pound
Entry
Core structure
R1
R2
CC50 (µM)a
hRV14
0.092
0.65
hRV21
0.073
0.059
0.077
0.06
hRV71
0.0094
0.061
0.014
0.031
0.013
1
2
3
4
5
pleconaril
19.7
>100
5.2
2a
2b
2c
2d
Me
Et
0.43
cPr
iPr
6.8
0.47
5.5
0.073
0.3
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6
7
2e
CF3
Me
Et
>100
2
0.99
8.7
0.3
1.6
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
>100
43.7
32.3
16.1
23.4
33.7
1.1
8
0.13
0.4
0.031
0.05
0.034
0.34
9
cPr
iPr
iBu
0.52
0.073
>23.4
10
11
12
0.38
2.2
CH2OM
e
63.4
8.3
2.1
1.7
9
13
14
15
16
17
18
19
20
21
3g
3h
3i
CF3
Et
H
35
25.2
20.0
8.9
30.5
7.4
9
9.3
0.39
0.34
0.073
0.066
0.3
>35.0
0.23
1.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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53
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60
2-methyl
2-methyl
2-methyl
3-methyl
2,6-dimethyl
H
0.0039
0.012
0.0025
0.0037
0.011
>9.0
cPr
iPr
iPr
iPr
iPr
iPr
iPr
0.4
3j
0.029
0.022
0.069
>9.0
>1.4
>1.8
3k
3l
3m
3n
3o
0.28
0.49
>1.8
2-methyl
2,6-dimethyl
1.4
1.8
>1.4
>1.8
22
23
24
3p
3q
3r
iPr
54.2
19.1
12.8
>19.1
>100
2.1
2.7
0.15
0.37
2
N
H
N
iBu
N
R1
O
CH2OM
e
40.3
O
N
O
>100
25
26
3s
iPr
H
>100
11.5
1.7
39.1
0.11
6.7
6.9
0.0084
aCC50: cytotoxic concentration (µM) that reduced cell viability by 50%; measured in H1HeLa cells using MTT assay. EC50: effective
concentration (µM) inhibited hRV replication by 50%; measured in H1HeLa cells using MTT assay.
b
1000
I.V. 5 mg/kg
Table 2. In vitro liver microsomal phase I stability (% reꢀ
maining after 30 min).a
100
10
P.O. 10 mg/kg
Compound
Rat (%)
1.0 ± 0.1
Human (%)
42.7 ± 0.6
69.8 ± 8.9
9.9 ± 3.1
1
1
2d
99.2 ± 0.3
35.1 ± 6.3
59.6 ± 5.7
37.1 ± 2.6
0.1 ± 0.01
0.1
3j
3k
0.01
0.001
40.7 ± 1.0
61.1 ± 3.7
3.5 ± 0.5
3l
0
2
4
6
8
10 12 14 16 18 20 22 24
Buspirone
Time (h)
aEach value is presented as mean ± standard deviation of at
least three independent experiments.
Figure 2. Plasma concentration-time profiles of 3k in male rats (n
=3).
Table 3. Pharmacokinetics of 3k in male ratsa
I.V., 5 mg·kg-1
P.O., 10 mg·kg-1
2.7 ± 1.2
b
c
Parameter
Tmax (h)
NA
NA
Cmax
(
µg·mL-1
)
3.9 ± 2.4
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Tmax, time of maximum drug concentration; T1/2, terminal half-
T1/2 (h)
4.6 ± 1.1
3.2 ± 0.4
life; VP, viral capsid protein; VSS, steady-state volume of distribu-
tion
1
2
3
4
5
6
7
8
AUCt (µg·h·mL-1
)
31.3 ± 2.5
17.4 ± 5.4
( )
µg·h·mL-1
31.8 ± 2.8
17.4 ± 5.4
AUC∞
CL (L·h-1·kg-1
VSS
L·kg-1
Ft (%)
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0.158 ± 0.014
0.611 ± 0.029
NA
NA
NA
)
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(
)
27.8
aEach value is presented as mean ± standard deviation of at least
three independent experiments. bParameters from intravenous
administration. cParameters from oral administration.
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Conclusion
In this study, we used antiviral compound 1 as starting ma-
terial for developing two novel compound series, derivatives 2
and 3, that we examined for their inhibitory activity against
hRV-B14, A21, and A71. We showed that substituting the
ester moiety of compound 1 by a 1,2,4-oxadiazole group in 2
and 3 created molecules with antiviral activity and significant
metabolic stability. Several compounds were highly active
with EC50 values in the nanomolar range against the three hRV
species. Specifically, compound 3k displayed a high efficacy
against hRV-B14, hRV-A21, and hRV-A71, with EC50 values
of 66.0, 22.0, and 3.7 nM, respectively. In addition, the hepatic
stability of 3k was better than compound 1 in rat microsomes
and similar for both compounds in human microsomes. A
pharmacokinetics analysis of 3k in rats demonstrated that the
inhibitor had a low systemic clearance and a moderate oral
bioavailability. Hence, 3k represents an interesting candidate
for the development of novel antiviral lead compounds.
ASSOCIATED CONTENT
Supporting Information
Detailed synthetic procedures, UPLC purity and characterization
1
data for compounds 2–3, including the spectral copies of H and
13C NMR spectra (PDF). This material is available free of charge
on the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
* E-mail: ysjung@krict.re.kr
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This study was supported by Korea Research Institute of Chemi-
cal Technology (Grant No. KK1703-C00, KK1703-E00, and
KK1803-D00).
ABBREVIATIONS
AUCt, areas under the plasma concentration-time curve; AUC∞,
areas under the plasma concentration-time curve from time zero;
CL, total clearance from plasma; Cmax, maximum plasma concen-
tration; DMF, dimethylformamide; Ft, bioavailability; hRV, hu-
man rhinovirus; ICAM-1, intracellular adhesion molecule 1;
MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bro-
mide; TFAA, trifluoroacetic anhydride; THF, tetrahydrofuran;
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A Table of Contents Graphic (TOC)
3ꢀArylꢀ1,2,4ꢀOxadiazole Derivatives Active Against Human Rhinovirus
Jinwoo Kim, Jin Soo Shin, Sunjoo Ahn, Soo Bong Han, and Young-Sik Jung
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