Host-directed inhibitors of myxoviruses: Synthesis and in vitro biochemical evaluation

Article abstract of DOI:10.1021/ml200125r

Drugs targeted to viral proteins are highly vulnerable to the development of viral resistance. One little explored approach to the treatment of viral diseases is the development of agents that target host factors required for virus replication. Myxoviruses are predominantly associated with acute disease and, thus, ideally suited for this approach since the necessary treatment time is anticipated to be limited. High-throughput screening previously identified benzimidazole 22407448 with broad antiviral activity against different influenza virus and paramyxovirus strains. Hit to lead chemistry has generated 6p (JMN3-003) with potent antiviral activity against a panel of myxovirus family members exhibiting EC₅₀ values in the low nanomolar range.

Full text of DOI:10.1021/ml200125r

LETTER
pubs.acs.org/acsmedchemlett
Host-Directed Inhibitors of Myxoviruses: Synthesis and in Vitro
Biochemical Evaluation
Aiming Sun,*,† J. Maina Ndungu,† Stefanie A. Krumm,‡,§ Jeong-Joong Yoon,‡,§ Pahk Thepchatri,†
Michael Natchus,† Richard K. Plemper,‡,§,|| and James P. Snyder†,^
†Emory Institute for Drug Discovery, Emory University, Atlanta, Georgia 30322, United States
‡Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, United States
§Children's Healthcare of Atlanta, Atlanta, Georgia 30322, United States
Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, United States
^Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States
S Supporting Information
b
ABSTRACT: Drugs targeted to viral proteins are highly
vulnerable to the development of viral resistance. One little
explored approach to the treatment of viral diseases is the
development of agents that target host factors required for virus
replication. Myxoviruses are predominantly associated with
acute disease and, thus, ideally suited for this approach since
the necessary treatment time is anticipated to be limited. High-
throughput screening previously identified benzimidazole
22407448 with broad antiviral activity against different influenza virus and paramyxovirus strains. Hit to lead chemistry has
generated 6p (JMN3-003) with potent antiviral activity against a panel of myxovirus family members exhibiting EC50 values in the
low nanomolar range.
KEYWORDS: Host-directed, non-nucleoside, small molecule inhibitor, influenza virus, myxovirus, benzimidazole
yxoviruses are responsible for the majority of human
morbidity and mortality cases due to viral respiratory
likely target host cells.11 This approach yielded several benzi-
midazoles of the same class [22407448 (BM-1),12 22404943,
and 22407466] as well-behaved inhibitors of MeV. Potencies
(EC50) against MeV are 0.2, 0.7 and 2.1 μM, respectively
(Figure 1). Hit BM-1 in particular is unusual since it shows
broad-spectrum antiviral action against various paramyxo-
viruses in the low micromolar to nanomolar range.11
To confirm the activity, BM-1 was resynthesized and
reassayed. For hit confirmation, doseÀresponse curves were
generated. Synthesized BM-1 revealed behavior identical to
the original library member against CDV, HPIV3, and MeV. In
parallel, MTT assays were employed to determine compound-
induced cytotoxicity in the absence of viral infection. Synthesis
was initiated by coupling of 1-fluoro-2-nitrobenzene 1 with p-
anisidine 2 in the presence of potassium carbonate to provide
N-(4-methoxyphenyl)-2-nitroaniline 3. Reduction of 3 gave
diamine 4, which was treated with 1,1-thiocarbonyldiimidazole
in dichloromethane to afford 1-(4-methoxyphenyl)-1H-benzimi-
dazole-2-thiol 5. The 2-thio-imidazole 5 was transformed to its
potassium salt and coupled with 2-bromo-N-(3,5-dichloropyri-
din-2-yl)propanamide to give BM-1. The approach has been
M
illness globally.1 Influenza virus is the leading cause of these
events in North America, although vaccine prophylaxis is widely
available. Despite extensive research, no vaccines currently exist
for several major pathogens of the paramyxovirus family such as
respiratory syncytial virus (RSV) or different human parain-
fluenza viruses (HPIVs). Ribavirin (RBV) is a synthetic nucleo-
side analogue with broad-spectrum antiviral activity. Although
RBV is approved for the treatment of hepatitis C virus, RSV, and
Lassa fever virus infections, its efficacy is limited, and the drug is
compromised by several side effects.2À5 Previously, we utilized
high-throughput screening (HTS) to identify small molecule
inhibitors against measles virus (MeV) RNA-dependent RNA
polymerase (RdRp) activity.6À8 However, viral adaptation has
demonstrated that robust resistance to inhibition by these com-
pounds can originate from single point mutations in the targeted
viral protein.9,10 In an attempt to counteract viral escape from
inhibition, we have explored targeting host factors required for
viral replication rather than viral proteins directly. Anticipated
advantages of this strategy include a decreased frequency of viral
escape from inhibition and a broadened pathogen target spec-
trum. HTS in combination with counterscreening for a broa-
dened viral target spectrum that extends to other pathogens of
the myxovirus families has identified several antiviral hits that
Received: May 24, 2011
Accepted: August 23, 2011
Published: August 23, 2011
r
2011 American Chemical Society
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Figure 1. Anti-MeV hits identified by high-throughput screening.
Scheme 1. Synthesis of Screening Hit BM-1a
Figure 2. (a) StructureÀactivity modification strategy for hit com-
pound BM-1; (b) modification of A and C sectors of BM-1.
The p-ethoxy analogue is the exception with reduced toxicity
at 20 μM (entry 6c). Encouragingly, 6p (JMN3-003) with a
substituted phenyl in sector C demonstrated strong antiviral
activity (EC50 = 170 nM) against MeV and cell cytotoxicity
over 75 μM (entry 6p, Table 1). As outlined in the context of
the recently reported detailed molecular characterization of
JMN3-003, antiviral activities were evaluated using actively
dividing cells, while CC50 values were determined for con-
fluent cell populations, thus reflecting acute toxicity. Inde-
pendent assessment of cell proliferation revealed a cytostatic
effect for JMN3-003. This was found, however, not to be the
basis for the antiviral effect.13 To understand the relationship
between chirality and potency, R- and S-isomers of 6p were
isolated by chiral HPLC. Both isomers were subjected to the
MeV inhibition assay. Potency of the S-isomer was essentially
identical to the racemic mixture 6p, while the antiviral
capacity of the R-isomer was slightly reduced (Table 1). A
newly developed methodology for the asymmetric synthesis
of both isomers will be reported elsewhere.
For part A of hit modification, we initially tried to replace
the fused benzene of the benzoimidazole with pyridine and
functionalize the benzene moiety by substitution with Me, Br
or COOEt. Unfortunately, most of the analogues experienced
either significant reduction or complete loss of activity. Thus, we
did not pursue this series further.
For replacement of the 2-thioacetamide linker, five different
variations of the central tether with an equivalent number of
chain atoms were prepared. Compound designations 11À15
(Schemes 2À4) and the corresponding activities are recorded
in Table 2, illustrating a significant reduction or loss of activity
relative to BM-1. Systematic structural modification revealed
that substitution of benzyl for phenyl at the benzimidazole
N1 position delivers analogues with similar anti-MeV viral
activity (Figure 2, sector A). Benzyl derivatives 20 and 21
deliver fairly good potency with EC50 at 0.5 and 0.4 μM,
respectively (Table 2). Thus, both derivatives 11 and 13 bear a
benzyl group instead of the p-methoxy phenyl group as shown
a Reagents: (a) K2CO3, 160 ꢀC, 5 h. (b) Pd/C (10%), H2 (50 psi),
MeOH, 2 h. (c) 1,1-Thiocarbonyldiimidazole, CH2Cl2, room tempera-
ture, 4 h. (d) KOH, EtOH, reflux, 2 h, and then 2-bromo-N-(3,5-
dichloropyridin-2-yl)propanamide 7.
used for the synthesis of a set of BM-1 derivatives, which are
described below (Scheme 1).
A structureÀactivity profile has begun to emerge by examina-
tion of the three molecular fragments circumscribed in Figure 2,
namely, the benzimidazole (A), the α-thio-amide linker (B) and
the substituted pyridine ring (C).
The first stage of hit optimization focused on introducing a
variety of aromatic rings as pyridine replacements, since
modification of the C sector is achieved in a straightforward
fashion by employing the same synthetic methodology uti-
lized for the preparation of BM-1 (Scheme 1). A small and
compact library of 30À50 analogues was obtained by utilizing
different α-halide amides for the final coupling step. Substi-
tuted pyridines, pyrazoles, triazines, thiazoles and other func-
tionalities were used instead of 7. A second group of analogues
was prepared by employing alternative anilines in sector A.
The para-methoxy group was replaced by hydrogen, ethoxy,
fluoro and hydroxyl, among others. Sector C analogues with
chloro, methyl and trifluoromethyl substituents show activ-
ities very similar to BM-1 (6a and 6d, Table 1). The corre-
sponding ethoxy analog delivers slightly better activity (entry
6c), while pyrazole and isoxazole replacements furnish 2-fold
reduced potency (entries 6j and 6n). The fluoro-analogue is
virtually equipotent to BM-1 (entry 6b). The thiazole func-
tionality reduces activity by 10-fold (entry 6m), while triazines
are significantly weaker still (entries 6i and 6k). However,
most of the active compounds listed in Table 1 are toxic in the
Trypan blue exclusion assay with CC50 values of 1À10 μM.
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Table 1. Antiviral Activity and Cytotoxicity of Various Substituted Anilides 6
a 50% inhibitory concentrations 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 experiments ( SEMs (standard errors of the mean); highest concentration
assessed, 75 μM. b CC50 values represent range of two experiments; highest concentration assessed, 75 μM. c CC50 not determined (ND) when EC50
1.0 μM.
>
in Table 2. Compound 11, incorporating NH in the central
linker, introduces additional hydrogen bond capacity and a
new pKa center, while 12, 13, 14 and 15 (CH2, O, SO and
SO2, respectively) sustain somewhat different linker geome-
tries by comparison with the sulfur-containing tether of BM-
1. The importance of sulfur versus other atoms was also
observed during discovery of the HIV reverse transcriptase
inhibitor RDEA-806,14,15 which shares the central 2-thio-
acetamide linker with BM-1 and has achieved success in
clinical trials (Figure 3). Obviously, a sulfur atom in the
linker is essential. However, its precise role at the binding site
remains to be fully defined. Comparative conformational
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Scheme 2. Synthesis of 2-Aminobenzimidazolea
a Reagents and conditions: (a) p-Methoxybenzyl bromide, K2CO3, CH3CN, reflux. (b) DL-Alanine methyl ester, K2CO3, CH3CN, 165 ꢀC, microwave,
1 h. (c) HCl (conc.)/H2O, reflux, 2 h. (d) Oxalyl chloride (2.0 equiv of 2.0 M in CH2Cl2), CH2Cl2, DMF (cat.). (e) 2-Chloro-4-methylaniline,
4-(dimethylamino)pyridine, (DMAP) (cat.), pyridine.
Scheme 3. Synthesis of the Carbon Analogue of 6pa
a Reagents and conditions: (a) CH2Cl2, 40 ꢀC. (b) HCl (4 N), MeOH, 100 ꢀC. (c) LiOH, THF/H2O. (d)iPrOCOCl, 4-methylmorpholine, 2-chloro-4-
methylaniline, CH2Cl2/DMF.
searches for otherwise identical analogues with O, NH and
CH2 replacements for S suggest that sulfur is possibly uni-
que in providing an energetically accessible bioactive
conformation.16
Analogue 11 was prepared as outlined in Scheme 2.
2-Chloro-benzimidazole was treated with p-methoxybenzyl
bromide in the presence of potassium carbonate to provide
chloro-benzimidazole 8. The latter was coupled with race-
mic alanine methyl ester in the microwave at 165 ꢀC for
1 h to afford 2-aminoimidazole 9, which was hydrolyzed to
the corresponding acid 10. The latter was treated with oxalyl
chloride followed by coupling with 2-chloro-4-methylani-
line to furnish the final product 11 (Scheme 2).
The synthesis of carbon analogue 12 proceeded from reaction
of diamine 4 with succinic anhydride to give the benzimidazole
16. Hydrolysis of 16 under basic conditions with lithium hydro-
xide (LiOH) in a mixture of THF and water delivered acid 17,
which was further coupled with 2-chloro-4-methylaniline in the
presence of isopropyl chloroformate and 4-methylmorpholine to
afford 12 (Scheme 3).
The synthesis of the oxygen analogue 13 commenced with the
protection of benzyl-L-lactate as a silyl ether, which on hydrogeno-
lysis furnished acid 18. BOP-mediated coupling of 18 with 2-chloro-
4-methylaniline followed by cleavage of the silyl group furnished
alcohol 19. Treatment of 19 with NaH facilitated coupling with
1-benzyl-2-chloro-1H-benzimidazole to afford 13 (Scheme 4).
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Sulfoxide 14 and sulfone 15 can be easily obtained by
oxidation of 6p with 1.0 and 2.0 equiv of 3-chloroperoxyben-
zoic acid (MCPBA), respectively. Benzene analogue 6p has
surfaced as the most promising candidate of this compound
series with superb antiviral potency and low cytotoxicity. The
compound shows potent activity against MeV and a selection
of clinically significant members of the para- and orthomyx-
ovirus families. We also compared the antimyxovirus activity
of 6p with the previously reported MeV RdRp inhibitor AS-
136a. The latter shows high selectivity against MeV, while 6p
exhibits a broad range of antimyxovirus activities with EC50
values ranging from 10 to 70 nM in virus yield reduction
assays depending on the target virus. Detailed biological
evaluation and target examination have been reported
elsewhere.13
counterscreening assays uncovered BM-1 as a well-behaved
inhibitor with the ability to block replication of a broad
range of myxovirus family members. By optimization of
BM-1, we have developed preliminary SAR within the three
pharmacophoric sectors highlighted in Figure 2. A variety
of structural modifications essentially abolish antiviral ac-
tivity or result in high cytotoxicity. Particularly influential
for the SAR of these agents are the structural constitu-
tions of the amide substituents and the sulfur atom of
the central tether. The most potent analogue 6p was
generated by replacing the pyridine ring in BM-1 with a
substituted phenyl ring. The compound shows activity
against MeV at 170 (viral CPE-reduction assay) and
30 nM (virus yield reduction assay) and does not display
any detectable acute cytotoxicity. Compound 6p was also
evaluated for its antiviral activity against a selection of
clinical-relevant paramyxovirus (RSV, MuV, and HPIV3)
and orthomyxovirus (influenza) family members. The com-
pound exhibits superb inhibitory activity against all of the
viruses tested with EC50 values ranging from 10 to 70 nM in
virus yield reduction assays.13 These results demonstrate
that 6p has great potential as a lead for development of host-
directed antiviral drugs. Pharmacokinetics evaluation,
in vivo efficacy, and expansion of 6p library are currently
in progress.
In summary, HTS has identified several hits in the benzi-
midazole class with potent anti-MeV activities. Follow-up
Scheme 4. Synthesis of the Oxygen Analogue of 6pa
a Reagents and conditions: (a) tert-Butyldimethylsilyl chloride, imida-
zole, DMF. (b) H2, Pd/C, MeOH. (c) Benzotriazole-1-yl-oxy-tris-
(dimethylamino)-phosphonium hexafluorophosphate (BOP), i-Pr2NEt,
2-chloro-4-methylaniline, CH2Cl2. (d) Tetrabutylammonium fluoride
(TBAF), THF. (e) NaH, 1-benzyl-2-chloro-1H-benzimidazole, DMF,
100 ꢀC.
Figure 3. Structural comparison of RDEA-806 and BM-1.
Table 2. Anti-MeV EC50 Values for S-Atom Replacements
ID
compd
EC50 (μM)a (MeV-Alaska)
CC50 (μM)b (Vero cells)
11
12
13
14
15
20
21
AS-228
>150
NDc
ND
ND
>75
ND
>75
>75
JMN3-006
JMN8-096
JMN3-004
JMN3-005
JMN5-010
JMN4-023
>150
>150
1.3 ( 0.06
>150
0.4 ( 0.06
0.7 ( 0.04
a 50% inhibitory concentrations 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 experiments ( SEMs; highest concentration assessed, 75
μM. b Values represent averages of two experiments; highest concentration assessed, 75 μM. c CC50 not determined (ND) when EC50 > 150 μM.
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’ ASSOCIATED CONTENT
(13) Krumm, S. A.; Ndungu, J. M.; Dochow, M.; Yoon, J.-J.; Sun, A.;
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.
S
Supporting Information. Experimental details for the
b
synthesis and characterization of 3À11, 12, 13, 15, 20 and 21
and experimental details for biology assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
(14) Moyle, G.; Boffito, M.; Shen, Z.; Manhard, K.; Sheedy, B.;
Hingorani, V.; Nguyen, M.; Nguyen, T.; Quart, B.; Yeh, L.; Ong, V.
RDEA806, a novel HIV non-nucleoside reverse transcriptase inhibitor,
shows positive outcome in treatment of n€aive HIV patients. 48 Annual
ICAAC/IDSA 46th Annual Meeting. Washington, DC, 2008.
(15) De La Rosa, M.; Kim, H. W.; Gunic, E.; Jenket, C.; Boyle, U.;
Koh, Y.; Korboukh, I.; Allan, M.; Zhang, W.; Chen, H.; Xu, W.; Nilar, S.;
Yao, N.; Hamatake, R.; Lang, S. A.; Hong, Z.; Zhang, Z.; Girardet, J.-L. Z.
Tri-substituted triazoles as potent non-nucleoside inhibitors of the HIV-
1 reverse transcriptase. Bioorg. Med. Chem. Lett. 2006, 16, 4444–4449.
(16) Conformational searches of the S, O, NH and CH2 analogues
were performed with the OPLS-2005 force field, and a putative binding
model was compared with the global minimum in each case.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 404-712-8680. Fax: 404-727-6689. E-mail: asun2@emory.edu.
Funding Sources
We are grateful to Dr. Dennis Liotta for generous financial
support and scientific discussions. This work was supported, in
part, by Public Health Service Grants AI071002 and AI085328
(to R.K.P.) from the NIH/NIAID and by Public Health Service
Grant HG003918-02 (to J.P.S.) from the NIH.
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paper for simplicity.
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