Synthesis and metabolic studies of host-directed inhibitors for antiviral therapy

Article abstract of DOI:10.1021/ml400166b

Targeting host cell factors required for virus replication provides an alternative to targeting pathogen components and represents a promising approach to develop broad-spectrum antiviral therapeutics. High-throughput screening (HTS) identified two classes of inhibitors (2 and 3) with broad-spectrum antiviral activity against ortho- and paramyxoviruses including influenza A virus (IAV), measles virus (MeV), respiratory syncytial virus (RSV), and human parainfluenza virus type 3 (HPIV3). Hit-to-lead optimization delivered inhibitor 28a, with EC₅₀ values of 0.88 and 0.81 μM against IAV strain WSN and MeV strain Edmonston, respectively. It was also found that compound 28a delivers good stability in human liver S9 fractions with a half-life of 165 min. These data establish 28a as a promising lead for antiviral therapy through a host-directed mechanism.

Full text of DOI:10.1021/ml400166b

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Metabolic Studies of Host-Directed Inhibitors for
Antiviral Therapy
Terry W. Moore,† Kasinath Sana,† Dan Yan,‡ Stefanie A. Krumm,‡ Pahk Thepchatri,† James P. Snyder,†,§
Jose Marengo,† Richard F. Arrendale,† Andrew J. Prussia,† Michael G. Natchus,† Dennis C. Liotta,†,§
́
Richard K. Plemper,‡,∥ and Aiming Sun*,†
†Emory Institute for Drug Development, Yerkes National Primate Research Center, Emory University, 954 Gatewood Road, NE,
Atlanta, Georgia 30329, United States
‡University Center for Inflammation, Immunity & Infection, Georgia State University, Atlanta, Georgia 30303, United States
§Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
∥Department of Pediatrics, Emory University School of Medicine, 2015 Uppergate Drive, Atlanta, Georgia 30322, United States
S
* Supporting Information
ABSTRACT: Targeting host cell factors required for virus
replication provides an alternative to targeting pathogen
components and represents a promising approach to develop
broad-spectrum antiviral therapeutics. High-throughput screening
(HTS) identified two classes of inhibitors (2 and 3) with broad-
spectrum antiviral activity against ortho- and paramyxoviruses
including influenza A virus (IAV), measles virus (MeV),
respiratory syncytial virus (RSV), and human parainfluenza virus type 3 (HPIV3). Hit-to-lead optimization delivered inhibitor
28a, with EC50 values of 0.88 and 0.81 μM against IAV strain WSN and MeV strain Edmonston, respectively. It was also found
that compound 28a delivers good stability in human liver S9 fractions with a half-life of 165 min. These data establish 28a as a
promising lead for antiviral therapy through a host-directed mechanism.
KEYWORDS: Myxovirus inhibitor, host-directed, influenza A, respiratory syncytial virus, benzimidazole, metabolic stability
he orthomyxovirus and paramyxovirus families comprise
Tmany pathogens that cause common respiratory illnesses;
among them are measles virus, mumps virus, respiratory
syncytial virus, human parainfluenza virus (paramyxoviruses),
and influenza viruses A and B (orthomyxoviruses). Together,
the acute respiratory illnesses caused by these viruses represent
a major medical need.1 In particular, the likelihood of a
pandemic from influenza A makes developing new and broadly
active therapies a pressing global need.2 Our research group has
undertaken a program of discovering and developing antivirals
that target host factors implicated in virus reproduction.
Although this runs counter to the prevailing virus-targeted
mechanistic approach to discovering antivirals, the potential
benefits of such a strategy include decreased incidence of
resistance because host factors mutate less readily than viral
factors and a broadened spectrum of antiviral activity because
many of the same host factors are co-opted by different
viruses.3,4 A potential drawback to this approach is the
nontrivial effort required to define mechanism of action since
we use a whole-cell phenotypic screen to identify these
antivirals.5 To date, our most successful compound series has
been exemplified by the broadly active benzimidazole 1 (JMN3-
003), which has low nanomolar activity against a number of
different viruses in cell culture.6,7 In our efforts to develop a
backup to 1, we have re-examined two different hits (scaffolds 2
and 3, Figure 1) that were described in the same high-
throughput screening paper in which we first disclosed
benzimidazole 1.5 This report details our attempts to
simultaneously improve potency against both an orthomyx-
ovirus (influenza A) and paramyxovirus (measles) representa-
tive and to understand the metabolic profile in liver S9 fractions
for 2 and 3.
To confirm the biological activity of series 2 and 3, these two
hits along with a small set of analogues were synthesized and
tested in reporter gene assays. In the case of IAV assays, a firefly
luciferase minireplicon reporter was driven by superinfection of
cells with influenza A/sw/Texas/2009 or influenza A/Aichi/
1968. EC50 (50% inhibitory concentrations) were calculated by
means of four-parameter nonlinear regression-fitting; values in
parentheses represent 95% confidence intervals. Syntheses of 2
and closely related analogues were initiated by treatment of
commercially available benzimidazolylacetonitrile 4 with
sodium nitrite in acetic acid. The resulting cyano oxime 5
was combined with hydroxylamine and cyclized to afford
furazan 6. The latter was further coupled with acyl chlorides 7
to deliver 8, followed by alkylation to give compound 2 and its
analogues (Scheme 1). Analogues with a thiazole, pyrazole, or
Received: May 2, 2013
Accepted: June 9, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400166b | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 1. Structures of host-directed antivirals and their biological profiles.
pyrazine instead of the furazan moiety were also synthesized
(Schemes S2−5, Supporting Information).
To obtain differentially substituted analogues, the acetophe-
nones 15 were brominated 16, coupled with methylamine, and
acidified to give α-ammonium ketones 17.8 These analogues
were cyclized using sodium thiocyanate to give the
thioimidazoles 18.8,11 Alkylation delivered the thioethers 19,
and bromination gave the 5-bromoimidazoles 20.8,12 mCPBA
oxidation provided the sulfone 21, and Suzuki coupling yielded
the tetra-substituted imidazoles 22 (Scheme 3). The synthesis
of 28a and analogues is shown in Scheme 4 starting with
treatment of 1-methyl-4,5-diphenylimidazole 29 with n-
butyllithium and DMF to afford 2-formyl imidazole 30.13
Reductive amination of 30 with different aromatic amines
delivered amines 31, which underwent either a second
reductive amination with formaldehyde or benzaldehyde or
LiHMDS-mediated alkylation to furnish 28a−d, respectively.
In exploring the SAR around 3, we found that replacing the
propargyl group with a hydrogen atom (13a) or a benzyl group
(13e) led to a complete loss of potency against both IAV and
MeV (13a), replacement with an ethyl (13d) or allyl group
(13f) led to reduced potency, and replacement with a methyl
group (13b) yielded analogues with comparable potency to
unmodified 3 against influenza, particularly, indicating a
preference for small alkyl substituents at the 1-position. Tris-
fluoro analogue 13c was synthesized and found to have reduced
potency against IAV and MeV as compared to 3. In an attempt
to increase potency, solubility, and metabolic stability, two of
the three phenyl rings were independently replaced with
pyridine (13g and 13h), and both compounds completely lost
activity.
Having demonstrated promising activity within this series, we
turned our attention to the metabolic stability of the
compounds. We assayed a subset of the compounds for
metabolic stability in human and hamster liver S9 fractions, the
latter species chosen for its likely use in a potential efficacy
study. We selected a panel of compounds for this study to
probe likely metabolic transformations: nucleophilic addition
into the heterocyclic sulfone and subsequent elimination of the
sulfinate, oxidation of the aromatic rings, oxidation of the N-
alkyl group, or oxidation of the methylene group α to the
sulfonyl. While rapidly metabolized in pooled hamster liver S9
fractions (T1/2 < 2.5 min), compounds 3, 13a, and 13b showed
a slightly prolonged half-life in the pooled human liver S9
a
Scheme 1
a
Reagents and conditions: (a) NaNO2, AcOH−H2O. (b) NH2OH−
i
HCl/KOH, CH3(CH2OCH2)3CH3−H2O. (c) 7, Pr2NEt/THF, then
KOH−CH3OH. (d) Ethyl bromoacetate, Pr2NEt/THF.
i
Compound 2 and analogues were retested against both IAV
and MeV. Replacement of the ethyl ester with a 4-aminophenyl
moiety (2b) led to a slight loss of activity, while replacement
with hydrogen caused a complete loss of potency against both
viruses (2a). All series 2 analogues featuring an altered central
ring system lost bioactivity (2c−g, Table 1). Active compounds
2 and 2b were subjected to further chemical and metabolic
stability testing against human and hamster liver S9 fractions
(T1/2 < 5 min in the presence or absence of cofactors).
Although the potency of compound 2 is good, we speculate
that the biological activity of this compound might arise from
its metabolic or chemical instability, which ultimately led us to
drop pursuit of this series in favor of alternative scaffold 3.
The syntheses of analogues of 3 were modified based on the
procedures of Laufer et al.8 Briefly, 4,5-diphenyl-2-thioimida-
zole was treated with alkyl bromides to give thioethers 10. The
thioether was either oxidized to the sulfone 11 first and
alkylated9 or, alternatively, alkylated and then oxidized to yield
13.10 Deprotonating the benzylic α-carbon and fluorinating
once or twice gave analogues 14a and 14b (Scheme 2).
B
dx.doi.org/10.1021/ml400166b | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 1. Analogues of Hit Series 2
a
EC50 values were calculated using the variable slope (four parameters) nonlinear regression-fitting algorithm embedded in the Prism 5 software
package (GraphPad Software). Values represent averages of four assessments; highest concentration assessed, 10 μM.
a
a
Scheme 2
Scheme 3
a
Reagents and conditions: (a) R′CH2Br, Cs2CO3, DMF. (b) R″X,
Cs2CO3, DMF. (c) H2O2/AcOH. (d) m-Chloroperoxybenzoic acid,
CH2Cl2. (e) KOtBu, N-fluorobenzenesulfonimide, THF. (f) LiN-
(SiMe3)2, N-fluorobenzenesulfonimide, THF.
a
Reagents and conditions: (a) Br2, AcOH. (b) NH2Me, CH2Cl2. (c)
HCl, MeOH. (d) NaSCN, DMF, 160 °C. (e) BrCH2R′, Cs2CO3,
DMF. (f) N-Bromosuccinimide, CCl4. (g) m-Chloroperoxybenzoic
acid, CH2Cl2. (h) R″B(OH)2, Na2CO3, Pd(PPh3)4.
fractions with half-lives of 19, 14, and 2−12 min, respectively
(Table 2). Surprisingly, the tris-fluorophenyl analogue 13c
showed rapid breakdown in human and hamster liver S9
fractions with both half-lives less than 2.5 min (Table 2).
Common to these four analogues is a sulfonylmethylene
moiety, which links the left-most imidazole and right-most aryl
rings of the molecule, presenting a potential metabolic
instability. We observed no metabolism in the absence of
cofactors, implying that the compounds were chemically stable
and that elimination of the sulfone as a sulfinate, were it to
occur, must be cofactor-mediated. Toward understanding the
role of the methylene carbon in the metabolism, we introduced
one or two fluorines into the molecule at the methylene carbon.
Although incorporation of one fluorine at that position (14a)
C
dx.doi.org/10.1021/ml400166b | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Scheme 4
a
n
Reagents and conditions: (a) BuLi/DMF, −78 °C. (b) Aniline/CH2Cl2, then NaBH(OAc)3,16 h. (c) HCHO (37% in H2O)/CH2Cl2, then
NaBH(OAc)3,16 h. (d) LiHMDS, RX, THF.
sulfonylmethylene represents a substantial metabolic liability
for this compound class (Table 2).
Table 2. Antiviral EC50 Values for Selected Sulfone
Analogues
We attempted to determine the identity of the metabolite(s)
using LC−MS/MS. After incubating 13b (100 μM) with
pooled human liver S9 fractions for 20 min, we observed a peak
(91% of total area) in the LC−MS that corresponded to a mass
with m/z of 576 (+187 Da) and 531 (+142 Da; data not
shown). Enhanced product ion (EPI) of the metabolite with m/
z 576 produced a fragment with m/z 298, which is consistent
with the mass of the 4,5-diphenylimidazole sulfone structure.
Taken together, these data are consistent with our hypothesis
of an unstable species, perhaps a reactive metabolite that arises
from hydrolysis of an α-hydroxysulfone intermediate or a
product of secondary metabolism.
Unfortunately, having repaired the metabolic liability in 13b,
we found that fluorinating the metabolically labile methylene
resulted in complete loss of antiviral activity (14b and 15, Table
3). Understanding this key weakness within the molecule, we
replaced the sulfonylmethylene moiety altogether with a variety
of two-atom linkers. Replacing the sulfone with a thioether
moiety led to analogue 12, which exhibited not only reduced
antiviral activity against IAV, but also shortened compound
half-life in both hamster and human liver S9 fractions (12,
Table 3), a curious finding when one considers the difference in
electron density at the α-carbons of 13a and 12. Further
modification by replacing the sulfone with a carbonyl group led
to analogue 25, which showed much improved stability in
human liver S9 fractions (human S9 T1/2 > 90 min) but
diminished potency (Table 3). Replacing the sulfonylmethylene
with a sulfonamide 23 or a 1,2-disubstituted ethanol 26 caused
not only complete loss of activity but also failed to confer
metabolic stability. Compounds 24a and 24b, in which an
amide linker replaces the sulfonylmethylene, were about 100-
fold less potent than compound 3. A similar result was found
with replacement by an ethane linker (27, Table 3). Strikingly,
we found analogue 28a, with an aminomethyl linker, to return
good potency with EC50 values of 0.88 μM against IAV-WSN
and 0.81 μM against MeV-Edm. Compound 28a was also
found to be metabolically stable in pooled human liver S9
fractions with a half-life of 165 min. While the potency and
metabolic stability of 28a are very promising, the solubility of
28a is quite poor (<15 μg/mL at pH = 7.4). We attempted to
S9 stability
T1/2 (min)
a
EC50 (μM)
compd
R, R1, R2, R3
Influenza MeV
human
hamster
3
0.5
0.74
>10
4.0
19
<2.5
<2.5
13a
13b
R = H, R1 = R2 = R3 = H
>10
0.3
14
b
b
R = Me, R1 = R2 =
R3 = H
2−12
<2.5
b
b
13c
13d
13e
13f
R = Me, R1 = R2 =
R3 = p-F
>10
2.0
>10
1.0
<2.5
<2.5
c
c
R = Et, R1 = R2 =
R3 = H
ND
ND
ND
ND
ND
ND
ND
ND
c
c
R = Bn, R1 = R2 =
R3 = H
10
>10
4.6
ND
c
c
c
c
c
c
R = allyl, R1 = R2 = H,
R3 = p-F
4.2
ND
c
13g
13h
22a
22b
R = Me, R1 = R2 = H,
R3 = 4-py
>10
>10
>10
>10
>10
9
ND
c
R = Me, R1 = R2 = H,
R3 = 3-py
ND
c
R = Me, R1 = R3 = p-F,
R2 = 4-py
>10
>10
ND
c
R = Me, R1 = R3 = p-F,
R2 = 3-py
ND
a
EC50 were calculated using the variable slope (four parameters)
nonlinear regression-fitting algorithm embedded in the Prism 5
software package (GraphPad Software). Values represent averages of
b
four assessments; highest concentration assessed, 10 μM. Values
represent the range of two experiments carried out on different dates.
Where only one value is given, the half-lives were calculated to be the
c
same. Not determined (ND) when EC50 > 10 μM.
conferred no additional S9 stability, introduction of a second
fluorine (15) resulted in good stability in pooled hamster S9
fractions (T1/2 = 54 min) and excellent stability in the human
system (T1/2 > 90 min). These results suggest that the
D
dx.doi.org/10.1021/ml400166b | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 3. Analogues of 3 Containing Different Linker Moieties
a
b
Lacks 1-methyl group on imidazole ring. EC50 were calculated using the variable slope (four parameters) nonlinear regression-fitting algorithm
embedded in the Prism 5 software package (GraphPad Software). Values represent averages of four assessments; highest concentration
c
assessed,10 μM. Not determined when EC50 >10 μM.
address this issue by either generating salts of 22 or
incorporating hydrophilic groups in the right-hand portion of
the molecule. Compound 28b with a hydroxyl group, 28c with
a carboxyl group, and 28d with pyridine instead of a phenyl ring
were synthesized and subjected to solubility testing based on
nephelometry. Analogue 28c showed superb solubility of
greater than 300 μg/mL at pH = 7.4 (Table 3).
In conclusion, we have described two new series of analogues
that may have broad-spectrum antimyxovirus activity, albeit
with higher EC50 values than with our previously reported
scaffold 1.6,7,14 While the first of these series, based on furazan
2, delivered potent compounds, this series was chemically and
metabolically unstable. The second series, based on imidazole
3, yielded bioactive compounds that were compromised,
however, by metabolic liabilities likely arising from the benzylic
methylene group α to the sulfone. Replacing the sulfonyl-
methylene with a methyleneamine moiety leads to a bioactive
(EC50 value for 28a approximately 0.8−0.9 μM against different
myxovirus family members) and metabolically stable analogue
(human S9 T1/2 = 165 min). Once solubility is further
improved, the series would appear suitable for in vivo efficacy
testing studies.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the synthesis and characterization of 2,
2a-h, and 3−32. Experimental details for biology assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(A.S.) Phone: 404-727-4860. E-mail: asun2@emory.edu.
E
dx.doi.org/10.1021/ml400166b | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Funding
This work was supported, in part, by Public Health Service
Grants AI071002 and AI057157 (to R.K.P.) from the NIH/
NIAID.
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) World Health Organization. World Health Statistics, 2013.
(2) Why should influenza be a public health priority? Vaccine 2012,
30, 7418−7420.
(3) Watanabe, T.; Watanabe, S.; Kawaoka, Y. Cellular networks
involved in the influenza virus life cycle. Cell Host Microbe 2010, 7,
427−439.
(4) Konig, R.; Stertz, S.; Zhou, Y.; Inoue, A.; Hoffmann, H. H.;
Bhattacharyya, S.; Alamares, J. G.; Tscherne, D. M.; Ortigoza, M. B.;
Liang, Y. H.; Gao, Q. S.; Andrews, S. E.; Bandyopadhyay, S.; De Jesus,
P.; Tu, B. P.; Pache, L.; Shih, C.; Orth, A.; Bonamy, G.; Miraglia, L.;
Ideker, T.; Garcia-Sastre, A.; Young, J. A. T.; Palese, P.; Shaw, M. L.;
Chanda, S. K. Human host factors required for influenza virus
replication. Nature 2010, 463, 813−817.
(5) Yoon, J. J.; Chawla, D.; Paal, T.; Ndungu, M.; Du, Y. H.;
Kurtkaya, S.; Sun, A. M.; Snyder, J. P.; Plemper, R. K. High-throughput
screening-based identification of paramyxovirus inhibitors. J. Biomol.
Screening 2008, 13, 591−608.
(6) Moore, T. W.; Sana, K.; Yan, D.; Thepchatri, P.; Ndungu, J. M.;
Saindane, M. T.; Lockwood, M. A.; Natchus, M. G.; Liotta, D. C.;
Plemper, R. K.; Snyder, J. P.; Sun, A. M. Asymmetric synthesis of host-
directed inhibitors of myxoviruses. Beilstein J. Org. Chem. 2013, 9,
197−203.
(7) Sun, A. M.; Ndungu, J. M.; Krumm, S. A.; Yoon, J. J.; Thepchatri,
P.; Natchus, M.; Plemper, R. K.; Snyder, J. P. Host-directed inhibitors
of myxoviruses: Synthesis and in vitro biochemical evaluation. ACS
Med. Chem. Lett. 2011, 2, 798−803.
(8) Laufer, S. A.; Hauser, D. R. J.; Liedtke, A. J. Regiospecific and
highly flexible synthesis of 1,4,5-trisubstituted 2-sulfanylimidazoles
from structurally diverse ethanone precursors. Synth.-Stuttgart 2008,
253−266.
(9) Guravaiah, N.; Rao, V. R. Stereoselective synthesis of substituted
2-(Z-styrylsulfonyl)-imidazoles and benzothiazole. Synth. Commun.
2010, 40, 808−813.
(10) Sugimoto, H.; Nakamura, S.; Watanabe, Y.; Toru, T.
Enantioselective hydrogen atom transfer to alpha-sulfonyl radicals
controlled by selective coordination of a chiral Lewis acid to an
enantiotopic sulfonyl oxygen. Tetrahedron: Asymmetry 2003, 14,
3043−3055.
(11) Dodson, R. M.; Ross, F. The preparation of 2-alkylthioimida-
zoles. J. Am. Chem. Soc. 1950, 72, 1478−1480.
(12) Rao, K. V. P.; Sundaramurthy, V. Regioselective electrophilic
substitutions of 4h-imidazo[2,1-C][1,4]benzoxazine and 4h-imidazo-
[2,1-C][1,4]benzthiazine. J. Org. Chem. 1992, 57, 2737−2739.
(13) Zhou, Y. R.; Gong, Y. F. Asymmetric copper(II)-catalysed
nitroaldol (Henry) reactions utilizing a chiral C-1-symmetric
dinitrogen ligand. Eur. J. Org. Chem. 2011, 6092−6099.
(14) Krumm, S. A.; Ndungu, J. M.; Yoon, J. J.; Dochow, M.; Sun, A.
M.; Natchus, M.; Snyder, J. P.; Plemper, R. K. Potent host-directed
small-molecule inhibitors of myxovirus RNA-dependent RNA-
polymerases. PLoS One 2011, 6, e20069.
F
dx.doi.org/10.1021/ml400166b | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX