SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity

Article abstract of DOI:10.1016/j.bmcl.2012.05.035

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Cross

Full text of DOI:10.1016/j.bmcl.2012.05.035

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4263–4272
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
SAR analysis of a series of acylthiourea derivatives possessing
broad-spectrum antiviral activity
James R. Burgeson, Amy L. Moore, Jordan K. Boutilier, Natasha R. Cerruti, Dima N. Gharaibeh,
Candace E. Lovejoy, Sean M. Amberg, Dennis E. Hruby, Shanthakumar R. Tyavanagimatt,
⇑
Robert D. Allen III, Dongcheng Dai
SIGA Technologies, Inc., 4575 SW Research Way, Suite 230, Corvallis, OR 97333, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum
antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the
family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen,
Received 19 March 2012
Revised 3 May 2012
Accepted 8 May 2012
Available online 17 May 2012
compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC50 = 0.25
lM) and
La Crosse virus (EC50 = 0.27 M) in cytopathic effect (CPE) assays. SAR analysis was performed to further
l
improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we
identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La
Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of
antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity
Keywords:
Broad-spectrum inhibitor
Acylthiourea
Antiviral
against
a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus
SAR
(Arenaviridae), and dengue virus (Flaviviridae).
Ó 2012 Elsevier Ltd. All rights reserved.
We live in a world surrounded by a myriad of diverse viruses
which are found in every ecosystem. A majority of these are harm-
less to humans but an extensive list of highly infectious viral fam-
ilies capable of generating high morbidity and mortality are
included under the umbrella of NIAID Category A, B, and C patho-
gens.1 Many of these viruses pose threats through weaponization
as well as being endemic in areas where military personnel may
be deployed. These pathogens include viral families such as Poxvir-
idae (variola virus), Filoviridae (Ebola and Marburg viruses), and
Bunyaviridae (Rift Valley fever and La Crosse viruses). Given the
rather large number of these more virulent pathogens from such
diverse viral families, an approach targeting broad-spectrum anti-
viral activity would be preferable to targeting each individual
virus. Currently, there are limited options of efficacious broad-
spectrum antivirals on the market. Ribavirin is active against a
number of RNA and DNA viruses including influenzas, flaviviruses,
and agents of many viral hemorrhagic fevers. This purine nucleo-
side analog likely functions through lethal mutagenesis, although
multiple direct and indirect mechanisms have been proposed.2
Ribavirin co-administered with PEGylated interferon-alpha (INF-
side effects including hemolytic anemia.4,5 This treatment is also
quite costly, making it unfeasible for widespread use. Additionally,
ribavirin exhibits teratogenicity in some animal species.6,7
In order to uncover molecular scaffolds possessing potential
broad-spectrum antiviral activity, high-throughput screening
(HTS) was performed on a library of 206,000 small molecules.
HTS assayed activity against Rift Valley fever virus (RVFV) and La
Crosse virus (LACV), which represent two distinct genera in the
genetically diverse Bunyaviridae family. Molecules which displayed
inhibitory activity against either of these viruses at a fixed concen-
tration (5
sequently tested against both RVFV and LACV across a range of
concentrations (0.009–25 M) to characterize activity. During this
lM) and contained chemically viable scaffolds were sub-
l
screening process, two hits were identified, compounds 1 and 2
(Fig. 1), which shared an acylthiourea moiety. Acylthiourea deriv-
atives have been reported in the literature for possessing a broad
O
O
S
H
N
O
S
a
) is used as a treatment for hepatitis C virus infections.3 However,
N
H
N
H
O
Cl
Cl
N
H
N
H
this combination treatment has inherent issues such as serious
2
1
⇑
Corresponding author. Tel.: +1 541 766 3758; fax: +1 541 753 9999.
E-mail address: ddai@siga.com (D. Dai).
Figure 1. Broad-spectrum antiviral hits discovered via high-throughput screening.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.035

4264
J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
C
VV (1, EC50 = 0.25
similar (1, EC50 = 0.27
l
M; 2, EC50 = 0.5
l
M), while LACV activity was
D
l
M; 2, EC50 = 0.28
l
M).13 Analogs were also
O
H
N
B
S
tested for cytotoxicity to make certain the observed antiviral activ-
ity was not due to cellular toxicity. Beyond the initial probing of
the scaffold, more advanced SAR studies focused on the design of
compounds with more drug-like properties by improving the var-
ious ADMET characteristics of the molecules, while retaining
broad-spectrum antiviral potency.
A
O
O
Cl
N
H
N
H
1
The overall SAR strategy involved probing one particular seg-
ment of each HTS scaffold at a time, while essentially keeping
the other segments fixed. The majority of the segment A analogs
were based on the scaffold of 1 since this compound was about
twofold more potent than 2 against VV. The general synthetic
pathway utilized to prepare analogs 9a–p is outlined in Scheme
1. The chemistry was straightforward with an amide coupling fol-
lowed by reduction of the nitro group to provide intermediate 6.
This intermediate was then reacted with thioisocyanate 8, which
was formed in situ, to afford the final products.
Concerning segment B chemistry, sulfur alkylated compounds
10a–d were prepared using the general procedure outlined in
Scheme 2. As will be discussed, the intended chemistry was alkyl-
ation of the amide nitrogen of segment B, as opposed to the ob-
served sulfur alkylation. The alkylation procedures involved the
use of an alkyl halide and sodium hexamethyldisilazane (NaHMDS)
or potassium carbonate which resulted in the same S-alkyl prod-
ucts. For 1, alkylation beyond S-ethyl was explored with alkyl
groups containing amino and ester functionalities.
Figure 2. Segmentation of the acylthiourea scaffold—segments A, B, C, and D.
range of biological activities including antibacterial,8 antifungal,9
anticancer,10 antiviral,11 and antithyroid12 activities. Compound 1
was further tested against other viral families and showed sub-
micromolar antiviral potency against vaccinia virus (Poxviridae),
Ebola virus (Filoviridae), influenza virus (Orthomyxoviridae), human
immunodeficiency virus type 1 (Retroviridae), Tacaribe virus and
lymphocytic choriomeningitis virus (Arenaviridae), encephalomyo-
carditis virus (Picornaviridae), Sindbis virus (Togaviridae), dengue
virus (Flaviviridae), Andes virus (Bunyaviridae), and respiratory syn-
cytial virus (Paramyxoviridae).
Concerning the SAR analysis of this acylthiourea scaffold shared
by 1 and 2, a strategy was employed involving the segmentation of
the molecule as shown in Figure 2. As illustrated, 2 is very similar
to 1 with the major difference being the addition of the chloro-
benzamide portion (segment D). The majority of the efforts were
concentrated on changing one segment at a time. Initially, changes
were made in order to probe the chemical space of the scaffold and
determine the effect on antiviral potency. This included incorpo-
rating groups of various size, electron donating/accepting capabil-
ity, as well as polar and ionizable groups. This study also made it
possible to uncover which moieties of the HTS hits might be crucial
for activity. To ensure subsequent analogs displayed broad-spec-
trum activity, they were screened against both vaccinia virus
(VV) and LACV. Compound 1 displayed superior activity against
Additional segment B analogs were prepared which incorpo-
rated an amidine moiety as shown in Scheme 3. The synthesis in-
volved separate treatment of thiocyanate compounds 11 and 12
with 4-tert-butylbenzamidine and NaHMDS to yield 13a and 13b.
For 13a, there was enough material synthesized to prepare the cor-
responding HCl salt form.
Continuing with segment B, the thiourea group was replaced
with a guanidine moiety for compound 16 as shown in Scheme
O
O
O
O
H
N
H
N
NH2
a
b
Cl
-O
+
-O
O
Cl
N+
O
O
Cl
N+
O
H2N
Cl
6
3
5
4
O
O
O
c
Cl
H
R
NCS
R
N
O
S
7
8
O
Cl
d
N
H
N
R
H
9a-p
Scheme 1. Reagents and conditions: (a) TEA, 4:1 DCM/THF, 0 °C to rt; (b) SnCl2, MeOH, 65 °C; (c) NH4SCN, acetone; (d) DCM, rt.
O
S
O
S
N
H
N
H
N
H
N
H
Cl
Cl
10a
a
2
O
O
H
N
H
N
R
O
S
O
S
O
Cl
O
Cl
N
H
N
H
N
H
N
H
b
. R = Et
c. R = CH2CH2NMe2
. R = CH2COOt-Bu
10b-d
1
d
Scheme 2. Reagents and conditions: (a) RX, 1 M NaHMDS in THF, THF, ꢀ60 °C to rt or RX, K2CO3, DMF, rt to 50 °C.

J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
4265
HCl
group or reduction of a nitro moiety. For 30j–l and 30o, the corre-
sponding HCl salt forms were prepared.
N
S
N
H
N
H
The chemistry outlined in Scheme 9 was used to isolate 32,
which involved reductive alkylation of 29 with 2-chlorobenzalde-
hyde. Amidine derivative 35 was synthesized following Scheme
10. Imido thioester 34 was generated first, via alkylation of the cor-
responding thioamide, and then reacted with intermediate 29 to
produce the final product.
Some of the analogs which contained structural changes in mul-
tiple segments included the amino-linked pyridyl analogs 39a–c
(Scheme 11). The synthesis of these compounds involved a palla-
dium-catalyzed amination reaction, followed by reduction of the
nitro moiety with tin chloride to form 38. These intermediates
were reacted with thioisocyanate derivative 8 followed by HCl salt
formation to generate 39a–c.
Cl
SCN
O
a
13a
11
Cl
O
H
N
N
S
H
N
O
Cl
N
H
N
H
12
Cl
O
SCN
13b
Scheme 3. Reagents and conditions: (a) 4-tert-butylbenzamidine, NaHMDS, THF,
60 °C, 4 h.
4. The synthesis involved a three-step procedure initiated by react-
ing compound 6 with Boc-protected methylisothiourea 14 in the
presence of mercuric chloride to produce 15. Following deprotec-
tion and acylation with 4-tert-butylbenzoylchloride, compound
16 was isolated.
We took advantage of the ease of sulfur alkylation in segment B
to install a cyanoguanidine moiety in place of the thiourea as illus-
trated in Scheme 5. After synthesizing the corresponding thioether
(17) of compound 1, a microwave-mediated reaction with sodium
hydrogen cyanamide afforded compound 18.
The majority of the segment C analogs were based on com-
pound 2 due to ease of preparation and a general synthetic path-
way is illustrated in Scheme 6.14 These analogs were mostly
synthesized by coupling compound 8 with the appropriate aniline
derivatives (19). In some cases, an additional modification was re-
quired, such as deprotection, to obtain the final product.
A few segment C analogs were based on the extended scaffold of
compound 1 including 26, for which the synthetic pathway is out-
lined in Scheme 7. Following the initial amide coupling with the
Boc-protected aniline derivative (23), the nitro group was reduced
using iron powder and ammonium chloride to afford compound
25. This material was reacted with thioisocyanate derivative 8,
deprotected with TFA and treated with HCl to afford 26. As will
be discussed, 26 turned out to be our most potent compound
against both VV and LACV.
For segment D analogs 30a–o, Scheme 8 outlines the chemistry
used to prepare these targets. Various reaction conditions were uti-
lized for coupling the corresponding carboxylic acid starting mate-
rial since the amide couplings proved to be very problematic. In
some cases, the corresponding acyl chloride starting material was
utilized, as opposed to the carboxylic acid, in order to isolate the
compounds in a reasonable yield. For some of the analogs (30h–l
and 30o), following the amide bond formation it was necessary
to deprotect the amino moiety either through removal of a Boc
A systematic substitution strategy was employed to probe
segment A using a variety of groups aimed at replacement of the
tert-butyl moiety in an attempt to reduce overall lipophilicity of
the molecule. Substitutions were made at all three positions, ortho,
meta and para, using hydrogen, chloro, nitro, methyl, methoxy, and
amino for compounds 9a–p (Fig. 3). These groups were chosen to
determine the effects of size and electronic character on this seg-
ment. Unfortunately, all substitutions resulted in a complete loss
of activity against both VV and LACV. Another series of molecules
included substitution of the 4-tert-butylphenyl ring with a 2-, 3-,
or 4-pyridyl ring, which would allow for ionization and a potential
increase in solubility. All of these compounds were inactive as well.
Two analogs were also tested in which a saturated ring was substi-
tuted in place of the phenyl ring, both with and without the 4-tert-
butyl substituent. Interestingly, with the tert-butyl group present
there was no antiviral potency observed, while removal of this
moiety resulted in an analog with a single digit micromolar VV
EC50 value. A possible explanation may involve disfavored posi-
tioning of this bulky tert-butyl substituent due to the preferred
conformation of the saturated ring. From this study of segment
A, it was ascertained that a bulky lipophilic group was necessary
to retain antiviral potency attained by the HTS hits. There was an
analog (Fig. 4, R = 4-CH3) which contained a methyl group in
segment A and displayed submicromolar activity against both VV
(EC50 = 0.77 lM) and LACV (EC50 = 0.44 l
M).13 In this instance,
the bulky, lipophilic tert-butyl group was instead present in seg-
ment D. It was hypothesized that there could be an inversion of
the scaffold at the location of biological interaction, thus filling
the presumed hydrophobic pocket. Interestingly, subsequent ana-
logs possessing this scaffold inversion did not show any antiviral
activity and therefore further work was not pursued.
At the outset, changes to segment B, the acylthiourea portion,
were not attempted as we believed this portion was crucial for
O
O
H
N
H
N
O
H
BocHN
S
O
NH
a
N
NBoc
b, c
+
O
Cl
O
Cl
NBoc
14
N
H
N
O
Cl
BocHN
N
H
H
H2N
16
6
15
Scheme 4. Reagents and conditions: (a) HgCl2, DIEA, DCM, rt, 18 h; (b) TFA, DCM, rt, 18 h; (c) 4-tert-butylbenzoylchloride, DIEA, DCM, rt, 18 h.
N
O
O
O
H
N
N
N
O
S
O
Cl
O
Cl
a
b
N
N
N
H
N
H
1
17
18
Scheme 5. Reagents and conditions: (a) MeI, K2CO3, DMF, rt, 4 h; (b) NaNHCN, iPrOH,
x (300 W), 80 °C, 10 min.

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J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
O
O
S
R
a
NCS
N
H
N
H
R
+
H2N
8
19
20a-u, 21a-p, 22a-i
Scheme 6. Reagents and conditions: (a) acetone, rt, 2 h.
NHBoc
Cl
O
H
N
H2N
NO2
b
a
Cl
+
O
Cl
O2N
BocHN
4
23
24
HCl
NH2
O
NHBoc
H
H
N
N
NCS
O
S
c, d
+
O
Cl
O
Cl
N
H
N
H
H2N
8
25
26
Scheme 7. Reagents and conditions: (a) DIEA, THF, rt, 3 h; (b) iron powder, NH4Cl (aq), THF, rt, 3 days; (c) acetone, rt, 2 h; (d) TFA/DCM (1:1), DCM, 0 °C, then 2 M HCl in
diethyl ether, rt, 3 h.
O
O
O
NHBoc
O
S
NHBoc
b
NCS
a
N
H
N
H
+
H2N
O
28
27
8
O
H
N
R
NH2
O
S
c
O
S
O
N
N
N
H
N
H
H
H
29
30a-o
Scheme 8. Reagents and conditions: (a) acetone, rt, 2 h; (b) TFA/DCM, rt, 2 h; (c) RCOOH, Ghosez reagent, pyridine, DCM, 0 °C to rt, 6 h or RCOOH, PyBOP, N-
methylmorpholine, DMF, rt, 18 h or RCOOH, HATU, DMF, rt, 5 h or RCOCl, TEA or pyridine, DCM, rt.
O
O
H
N
NH2
a
O
S
O
S
H
+
Cl
N
H
N
H
N
H
N
H
O
Cl
32
31
29
Scheme 9. Reagents and conditions: (a) NaBH(OAc)3, AcOH, DCE, rt.
a
MeS
H2N
O
O
H
N
34
NH Cl
HCl
33
Cl
S
NH2
O
S
O
S
NH Cl
N
H
N
b
N
H
N
35
29
H
H
Scheme 10. Reagents and conditions: (a) MeI, acetone, reflux, 5 h; (b) DIEA, EtOH, reflux, 2 h.
activity due to the presence of this group in both HTS hits. During
formulation studies, it was determined that the amide of the acy-
lthiourea was labile to basic conditions and we observed subse-
quent hydrolysis. Therefore, molecules were designed to reduce
this liability including methylene or amidine replacement of the
carbonyl along with alkylation of the proximal amide-nitrogen to
make the region more sterically hindered (Table 1). When alkyl-
ation was performed as the final step in order to install steric bulk
to the amide-nitrogen of 1 and 2, the major products that were iso-
lated were in fact 10a and 10b, stemming from sulfur alkylation. It
was interesting to discover that these compounds had similar po-
tency to the parent compounds. We tried to exploit this feature of

J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
4267
H
N
X
H
O
S
Y
Z
NO2
37
H2N
X
N
X
a, b
c
HCl
Y
Z
Y
Z
+
N
H
N
H
36
Br
H2N
39a-c
38
X, Y, Z = CH or N
X, Y, Z = CH or N
X, Y, Z = CH or N
Scheme 11. Reagents and conditions: (a) Pd2(dba)2, BINAP, Cs2CO3, DMF, 80 °C, 18 h; (b) SnCl2, EtOAc, MeOH/H2O, reflux, 18 h; (c) 8, acetone, rt, 2 h then 2 M HCl in Et2O.
O
attachment. Compared to 2, compound 20b showed a fivefold de-
crease in activity, while compound 20c displayed a complete loss
of antiviral activity. The 2- and 3-pyridyl ring analogs (20s and
20t) were inactive while the synthesis of the 4-pyridyl analog
was unsuccessful. The inactivity of these pyridine ring compounds
is in contrast with the unsubstituted phenyl (20a) and cyclohexane
(20u) rings which displayed single digit micromolar activity.
As previously discussed, substitution of segment C in the ortho-
or meta-positions with chloro or methyl produced active analogs
(2, 20b, 20j and 20k). With this in mind, analogs were prepared
in which disubstitution of segment C was explored and the results
are shown in Table 3. A couple analogs were tested that incorpo-
rated a trifluoromethyl group (21c and 21d), as opposed to a sim-
ple methyl moiety, to reduce potential oxidative metabolism of
this ring and the alkyl group itself. Compounds containing a hydro-
xyl group (21o and 21p) were also included in this study as this
group may increase solubility through hydrogen bonding. Some
fairly potent analogs were identified and activity usually tracked
with the pattern of chloro substitution in the 2-position in combi-
nation with a bulky lipophilic group at the 3-, 4-, or 5-positions.
Unfortunately, none of these substitution patterns resulted in an
analog that surpassed the potency of the parent compound (2). It
should also be noted that the hydroxyl analogs (21o and 21p)
showed signs of cytotoxicity with single digit micromolar CC50
values.
Continuing with segment C, it was observed that an amino group
was well tolerated in the meta- and para-positions (20q and 20r) for
the scaffold of 2. In addition to antiviral potency against both VV and
LACV, these analogs offered the advantage of improved solubility, at
least in acidic conditions, due to the addition of this ionizable group.
It was interesting to note that the combination of these two primary
amine groups (22a), which independently display activity, did not
possess potency against the selected viruses. It was evident from
22b that the methoxy group of 1 may not have a dramatic effect
on activity since this compound displayed similar potency to 20r,
which lacked the methoxy moiety. When the 4-NH2 group was com-
bined with 2-chloro substitution (22c), the result was a dramatic in-
crease in antiviral potency when compared to 20r. This is in
agreement with the trend observed in Table 3 which illustrated
advantageous substitution patterns involving the 2-chloro moiety.
When we attempted to further improve solubility by installing an
aminomethyl group, as for compounds 22d and 22e, we observed
a dramatic increase in solubility, but with complete loss of antiviral
activities. Additional SAR studies led to monoalkylated (22f) and
dialkylated (22g) amino groups installed at the para-position which
possessed increased basicity, for ease of salt formation, along with
the potential to reduce in vivo oxidation. These groups were also
combined with 2-chloro substitution which led to an increase in po-
tency for 22h, but did not have much effect for compound 22i. In all
cases, the HCl salt forms of these compounds were synthesized in an
attempt to increase water solubility. All of these results are summa-
rized in Table 4.
H
N
O
S
O
Cl
N
H
N
H
R
9a-p
R = H, Cl, NO2, CH3, OCH3, NH2
Figure 3. Systematic SAR study of segment A.
H
N
O
S
O
N
H
N
H
R
R = H, 4-CH3, 2-Cl, 2-NO2, 3,5-diCH3
Figure 4. Inversion of Scaffold.
sulfur alkylation and install polar groups that may improve the sol-
ubility, as for analogs 10c and 10d; however, these molecules did
not display any antiviral potency. Installation of an ethyl group
onto the other nitrogen atom of segment B was successful (40),
but this analog displayed a dramatic decrease in antiviral potency
against VV and was inactive against LACV. From the results of
methylene (41) and amidine replacement (13a and 13b) of the car-
bonyl moiety of segment B, it was apparent that this group may be
required due to the dramatic decrease in antiviral activity. Another
analog involved replacement of the thiourea moiety with a guani-
dine group (16) in an attempt to improve solubility characteristics
and reduce the liability of potential in vivo sulfur oxidation. Unfor-
tunately, this molecule was inactive against both viruses. Another
analog that was synthesized involved bioisosteric replacement of
the thiourea portion of 1 with a cyanoguanidine moiety (18). This
compound failed to display antiviral activity against either virus at
25
l
M, which was the highest concentration tested.
A large portion of the acylthiourea analogs were focused on
changes to segment C as this portion appeared to be the most ame-
nable to substitution based upon preliminary studies. Initially, a
systematic study was performed in an analogous manner to the
one used for segment A which involved phenyl ring substitutions
with hydrogen, chloro, nitro, carboxylic acid, methyl, methoxy,
and amino (Table 2). In addition, replacement of the phenyl ring
with a pyridine or cyclohexane ring was also explored. In this
study, the activity of 2 was used as a benchmark since these ana-
logs most closely resembled this HTS hit. While these substitutions
did not uncover analogs that offered superior antiviral potency, the
study did reveal which positions and types of functionalities may
be tolerated. Overall, substitution with electron donating groups,
such as methyl (20j–l), methoxy (20m–o), and amino (20p–r),
led to active analogs. Groups offering electron withdrawing capa-
bility through resonance (20d–i) were less tolerated, resulting in
many inactive analogs. It was interesting to note that chloro substi-
tution gave very different results based upon the position of
The extended scaffold of 1 was also used in segment C SAR stud-
ies involving replacement of the methoxy group (Table 5). One
such analog was derived by substituting the methoxy moiety with
an amino group (26) which offered improved solubility through
presence of an ionizable group and made salt formation possible.

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J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
While it was shown later that solubility was only modestly in-
creased, the antiviral activity was significantly improved against
both VV (EC50 = 0.06
ecule turned out to be the most potent analog identified.
lM) and LACV (EC50 = 0.05 lM) and this mol-
Table 1
Changes to segment B
Compound
Structure
VV EC50
1.91a
(l
M)
LACV EC50 (lM)
O
O
S
S
10a
0.51a
N
N
N
H
Cl
O
H
N
O
Cl
10b
0.23
0.30
N
H
N
O
O
H
N
S
10c
>25
>25
O
Cl
N
N
H
O
O
O
S
O
H
N
10d
>25
>25
O
Cl
N
N
H
O
H
N
O
S
O
Cl
40
10.1
>25
N
N
H
O
H
N
S
S
O
Cl
41
13a
13b
>25
>25
>25
3.01
N
N
H
H
NH
N
H
N
H
>25
Cl
O
H
N
NH
S
7.64b
O
Cl
N
H
N
H
O
H
N
O
NH
O
Cl
16
>25
nd
N
N
H
H

J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
4269
M)
Table 1 (continued)
Compound
Structure
VV EC50
(
lM)
LACV EC50 (l
N
O
N
N
O
18
>25
nd
O
Cl
N
N
nd = not determined.
a
Average value of multiple experiments.
One experiment displayed inactivity at highest concentration tested (25 lM).
b
Table 2
Table 3
Disubstitution study of segment C
Systematic SAR analysis of segment C
O
S
O
S
R
R'
R
N
H
N
H
N
H
N
H
20a-u
21a-p
Compound
R
VV EC50
(
lM)
LACV EC50 (lM)
Compound
R
R0
VV EC50
(lM)
LACV EC50
(l
M)
20a
2
Ph
2-ClPh
3-ClPh
4-ClPh
5.57a
0.35
1.58
>25
5.01b
0.28
4.16
>25
21a
21b
21c
21d
21e
21f
21g
21h
21i
21j
21k
21l
21m
21n
21o
21p
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-CH3
2-CH3
2-CH3
2-CH3
3-CH3
3-CH3
2-OH
2-OH
3-CH3
5-CH3
5-CF3
3-CF3
5-OCF3
6-CH3
5-Cl
1.83a
0.66a
1.00a
1.83
1.9
1.17
1.67b
0.6
20b
20c
20d
20e
20f
20g
20h
20i
0.85
>25
2-NO2Ph
3-NO2Ph
4-NO2Ph
2-CO2HPh
3-CO2HPh
4-CO2HPh
2-CH3Ph
3-CH3Ph
4-CH3Ph
2-OCH3Ph
3-OCH3Ph
4-OCH3Ph
2-NH2Ph
3-NH2Ph
4-NH2Ph
2-Pyridyl
3-Pyridyl
Cyclohexyl
3.64a
>25
3.70a
>25
2.49a
2.42b
>25
0.94a
2.45b
>25
4.08
>25c
>25
>25
>25
>25
>25
4-Cl
4-CH3
3-CH3
3-Cl
5-CH3
5-CH3
4-CH3
5-t-butyl
5-CH3
1.06a
1.83a
6.47b
2.86a
3.48a
3.93
0.98a
>25
0.88a
2.59a
0.919b
>25
>25
20j
20k
20l
0.83a
1.01a
1.91a
1.39
4.07
2.39
>25
0.63a
1.33a
4.86a
1.15
>25
4.43b
1.23
3.42
>25
20m
20n
20o
20p
20q
20r
20s
20t
20u
>25
>25
2.72a,b
4.41a,b
>25
>25
4.92
2.19a
2.27a
>25
a
Average value of multiple experiments.
Single experiment displayed inactivity at highest concentration tested (25 lM).
b
>25
3.71
with an imidazole ring (30f and 30g) led to loss of activity. The
incorporation of amino (NH2) and methylamino (CH2NH2) substit-
uents was attempted for 30h–30l, which did show signs of VV
activity in most cases, but were also mostly inactive against LACV.
While acceptable antiviral potency was observed against VV for
30k and 30l, these compounds also displayed cytotoxicity with
single digit micromolar CC50 values. In addition, two analogs were
prepared that incorporated carboxylic acid group substitution onto
the phenyl ring of segment D (30m and 30n). Unfortunately, these
compounds did not show any activity against either virus tested at
a
b
c
Average value of multiple experiments.
Single experiment displayed inactivity at highest concentration tested (25
Single experiment showed activity of 2.29 M.
lM).
l
Interestingly, the hydroxyl analog (42), which could be a potential
metabolite of 1, displayed very similar potency to the parent. Over-
all, the data that was gathered regarding segment C suggested that
the extended scaffold of 1 may be involved in additional binding
interactions, when compared to compound 2, leading to an in-
crease in antiviral potency.
Analogs were also synthesized which probed the chemical
space around segment D with the hopes of improving drug-like
properties. Multiple attempts to incorporate an ionizable moiety
into segment D, for reducing lipophilicity and improving solubility,
are summarized in Table 6. One of these studies involved replace-
ment of the 2-chlorophenyl ring with aromatic heterocycles con-
taining one or multiple nitrogen atoms. It was observed that 3-
and 4-pyridyl ring containing compounds (30b and 30c) displayed
decent potency against VV, while the presence of a nitrogen atom
in the 2-position led to a dramatic decrease in activity (30a, 30d,
and 30e). It was also shown that replacement of the phenyl group
25 lM. The same was true for the benzimidazole derivative (30o).
For compounds 30a–o, we did not observe any activity against
LACV. The overall trend indicated that segment D was not amena-
ble to substitution with ionizable groups when broad-spectrum
activity is desired.
Attempts to replace the phenyl ring of segment D with an alkyl
chain linked to a terminal polar functionality [(NCH3)2, CO2CH3,
COOH] was shown to be unsuccessful at retaining antiviral potency
(43a–e). In addition, the dimethylamino analogs (43a and 43b)
showed signs of cytotoxicity with single digit micromolar CC50 val-
ues. In contrast, substitution with a simple N-acetyl group (43f)
displayed good potency against both viral targets. The potency
observed for a smaller, polar functionality in this region was not

4270
J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
Table 4
Amino group substitution in segment C
O
S
R
N
H
N
H
22a-i
Compound
R groups
VV EC50
(
lM)
LACV EC50
(l
M)
Solubility (
lg/mL)
H2O
SGFa
CTBb
c
22a
22b
22c
22d
22e
22f
22g
22h
22i
3,4-DiNH2
>25d
2.09e,f
0.62f
>25
>25
2.9
ND
0.1
121
258
1.9
1.0
0.2
ND
4.2
53
2.0
0.4
0.5
11
3-OCH3, 4-NH2
2-Cl, 4-NH2
2-CH2NH2
1.69e,f
0.75f
>25g
>25
6.4
261
248
190
36
3-CH2NH2
4-NHCH3
4-N(CH3)2
>25
24
1.16f
2.43
0.33f
3.26f
1.45
nd
0.3
ND
0.1
ND
2-Cl, 4-NHCH3
2-Cl, 4-N(CH3)2
0.24f
1.55f
3.2
ND
nd = not determined.
ND = not detectable.
a
SGF = simulated gastric fluid.
b
c
d
e
f
CTB = Caco-2 transport buffer.
Di-HCl salt was synthesized.
Single experiment showed activity of 7.78 lM.
Two experiments displayed inactivity at highest concentration tested (25
Average value of multiple experiments.
lM).
g
Single experiment showed activity of 6.93 lM.
Table 5
Table 6
Ionizable groups incorporated into segment D
Methoxy group substitution in segment C
R
O
H
N
H
N
Ar
O
S
O
S
O
Cl
O
N
H
N
H
N
H
N
H
R = OCH3, NH2 or OH
30a-o
Compound
R
VV EC50
(l
M)
LACV EC50
(
lM)
Compound
Ar
VV EC50
(lM)
LACV EC50 (lM)
1
26
42
OCH3
NH2
OH
0.25a
0.06a
0.23
0.27a
0.05a
0.21
30a
30b
30c
30d
30e
30f
30g
30h
30i
30j
30k
30l
30m
30n
2-Pyridyl
3-Pyridyl
4-Pyridyl
Pyrazyl
Pyrimidyl
2-Imidazole
4-Imidazole
2-Aminophenyl
3-Aminophenyl
4-Aminophenyl
3-Methylaminophenyl
4-Methylaminophenyl
3-Benzoic acid
4-Benzoic acid
N
>25
>25
nd
nd
1.72
2.03
>25
>25
>25
>25
2.61
>25
>25
>25
>25
>25
>25
>25
>25a
>25
>25
nd
a
Average value of multiple experiments.
repeated for the urea (43g) or guanidine (43h) analogs. All of these
results are summarized in Table 7.
19.96a
1.63b
0.81b,c
>25
Substitutions of the amide group in segment D were explored
and summarized in Table 8. The carbonyl group was substituted
with a methylene for 32 and this analog had nearly the same
activity as the parent compound (1). This discovery provided
the potential for a backup series of compounds sharing this
same substitution if amide hydrolysis were to become an issue
for segment D. Replacement of the amide group with an ami-
dine (35) provided an ionizable moiety, while the thioamide
(44) derivative was synthesized to represent a closely related
surrogate with the potential to reduce possible hydrolysis at
this position. Both compounds resulted in a decrease in activity
when compared to 1.
>25
>25
30o
>25
>25
N
H
HCl
nd, not determined.
a
High variability for this analog with single digit micromolar potency observed
for one experiment.
b
Average value of multiple experiments.
One experiment displayed inactivity at highest concentration tested (25 lM).
c
Some of the alterations that were examined incorporated
changes to both segments C and D and are summarized in Table
9.15 For compounds purchased from commercial sources, it was
difficult to find molecules to probe only one specific part of the
acylthiourea scaffold. One set of analogs included rather substan-
tial changes including replacement of the entire segment D portion
with saturated heterocycles including piperidine, piperazine, and
morpholine (45a–e). The incorporation of these groups offered
the potential for improved solubility through both ionizable groups

J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
4271
Table 7
Table 9
Polar group appended alkyl chains in segment D
Significant changes to segments C and D
O
S
O
R
H
N
N
H
N
H
O
S
R
45a-n
N
H
N
H
43a-h
Compound
R
VV EC50
(lM)
LACV EC50
>25
(lM)
N
45a
>25
Compound
R
VV EC50
(l
M)
LACV EC50 (lM)
43a
43b
43c
43d
43e
43f
CO(CH2)2N(CH3)2
CO(CH2)4N(CH3)2
COCH2CO2CH3
COCH2CO2H
CO(CH2)2CO2H
COCH3
>25
>25
18.61
>25
>25
>25
>25
>25
>25
1.35
4.86
>25
N
45b
45c
>25
>25
>25
>25
>25
O
3.27a
>25
43g
43h
CONH2
C(@NH)NH2
N
O
>25
N
N
a
Average value of multiple experiments.
45d
>25
>25
Table 8
Amide group substitution of segment D
N
45e
45f
>25
>25
>25
>25
O
O
H
N
O
S
R
O
O
S
Cl
N
N
H
N
H
S
O
O
O
S
S
Compound
R
VV EC50
(lM)
LACV EC50 (lM)
N
N
45g
45h
>25
>25
>25
>25
H
1
32
35
44
C@O
CH2
C@NH
C@S
0.25a
0.34a
1.75a
0.99
0.27a
0.20a
2.06a
0.64
O
N
a
Average value of multiple experiments.
O
O
S
45i
N
>25
>25
H
and structural flexibility of the non-planar rings. However, these
molecules did not display any antiviral activity in our assays,
which may not be surprising given the structural disparity
compared to 1. In addition, they also appeared to be cytotoxic.
Another batch of compounds examined the replacement of the
amide linker of segment D with a sulfonamide linker (45f–i). The
sulfonamide substitution included linkage to a benzyl substituent
(45f), thiazole (45g), piperidine (45h) and cyclohexane (45i). In
all cases there was a complete loss of antiviral activity. A series
of compounds was also tested which included replacement of the
segment C phenyl ring with a thiazole ring (45j–l) and these were
inactive as well. It was apparent that the location of the amide por-
tion of segment D was very important given the inactivity of 45m
and 45n, with a large decrease in antiviral activity observed when
this group is moved away from the para-position. Overall, it ap-
peared that these relatively dramatic changes to segments C and
D were not tolerated even though segments A and B remained
unchanged.
Interestingly, we found that we were able to omit certain moi-
eties of 1 without having a substantial impact on activity (46a and
46b, Table 10).16 Compound 46a, which is lacking the chloro and
methoxy phenyl substituents, retained the broad-spectrum activ-
ity of the parent compound. The addition of 2-chloro substitution
(46b) did not appear to have much of an effect on antiviral po-
tency when compared to 46a. Compound 46c contained an amino
linker in segment D, as opposed to the amide linker of 1, and there
O
N+
O–
S
S
45j
>25
>25
>25
>25
N
N
O
O
45k
O
O
O
45l
S
>25
>25
N
O
O
Cl
Cl
45m
45n
>25
>25
>25
>25
N
H
N
H
was only a 2- to 3-fold decrease in activity. Structural modifica-
tions of 46c were performed and involved the addition of an

4272
J. R. Burgeson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4263–4272
Table 10
Key group probing of segment D
potency as well as drug-like properties of the acylthiourea scaf-
fold derived from the HTS hits.
R
Acknowledgements
O
S
N
H
N
H
We gratefully acknowledge the financial support from the
Department of Defense contract HDTRA1-10-C-0036. We thank
Andrew Wieczorek, Thuan Tran, Robert Jordan, Brita Hanson, Erin
VanderMay, Tracy Tan, Daniela Zima and Will Weimers for techni-
cal support and assay development. We also thank Ricerca Biosci-
ences for providing most of the compounds for this SAR study.
R'
Compound
R
R0
VV EC50
0.17a
(lM)
LACV EC50
0.15b
(lM)
H
N
46a
H
O
O
Supplementary data
H
N
46b
Cl
0.16
0.08
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
035. These data include MOL files and InChiKeys of the most
important compounds described in this article.
H
N
46c
H
H
0.82a
>25
1.7a
>25
H
N
References and notes
39ac
1. NIAID. NIAID Biodefense Research Agenda for Category
B and C Priority
N
Pathogens, Progress Report, June 2004. NIH Publication No. 04-5523, 2004.
2. Graci, J. D.; Cameron, C. E. Rev. Med. Virol. 2006, 16, 37.
3. Fried, M. W.; Shiffman, M. L.; Reddy, K. R.; Smith, C.; Marinos, G.; Gonçales, F. L.,
Jr.; Häussinger, D.; Diago, M.; Carosi, G.; Dhumeaux, D.; Craxi, A.; Lin, A.;
Hoffman, J.; Yu, J. N. Eng. J. Med. 2002, 347, 975.
4. Davis, G. L.; Esteban-Mur, R.; Rustgi, V.; Hoefs, J.; Gordon, S. C.; Trepo, C.;
Shiffman, M. L.; Zeuzem, S.; Craxi, A.; Ling, M. H.; Albrecht, J. N. Eng. J. Med.
1998, 339, 1493.
5. McHutchison, J. G.; Gordon, S. C.; Schiff, E. R.; Shiffman, M. L.; Lee, W. M.;
Rustgi, V. K.; Goodman, Z. D.; Ling, M. H.; Cort, S.; Albrecht, J. K. N. Eng. J. Med.
1998, 339, 1485.
6. Ferm, V. H.; Willhite, C.; Kilham, L. Teratology 1978, 17, 93. Teratology 1978, 17,
93–101.
H
N
39bc
39cc
H
H
24.5
>25
>25
7.7a
N
H
N
N
a
b
c
Average value of multiple experiments.
One experiment displayed inactivity at highest concentration tested (25
HCl salt form of compound.
lM).
7. Kochhar, D. M.; Penner, J. D.; Knudsen, T. B. Toxicol. Appl. Pharmacol. 1980, 52, 99.
8. Saeed, A.; Shaheen, U.; Hameed, A.; Haider Naqvi, S. Z. J. Fluorine Chem. 2009,
130, 1028.
9. del Campo, R.; Criado, J. J.; Gheorghe, R.; González, F. J.; Hermosa, M. R.; Sanz,
F.; Manzano, J. L.; Monte, E.; Rodríguez-Fernández, E. J. Inorg. Biochem. 2004, 98,
1307.
ionizable pyridyl ring to segment D in an attempt to improve sol-
ubility (39a–c). While solubility was increased, the analogs were
rendered inactive in our antiviral assays against both VV and
LACV. It was reasonable to expect at least modest activity against
VV based upon results from pyridyl ring incorporation into seg-
ment D, as observed for 30b and 30c. However, the absence of
the carbonyl linker appears to have greatly changed this
interaction.
10. Saeed, S.; Rashid, N.; Jones, P. G.; Ali, M.; Hussain, R. Eur. J. Med. Chem. 2010, 45,
1323.
11. (a) Sun, C.; Huang, H.; Feng, M.; Shi, X.; Zhang, X.; Zhou, P. Bioorg. Med. Chem.
Lett. 2006, 16, 162; (b) Wyles, D. L.; Kaihara, K. A.; Schooley, R. T. Antimicrob.
Agents Chemother. 2008, 52, 1862; (c) Severson, W. E.; McDowell, M.;
Ananthan, S.; Chung, D.-H.; Rasmussen, L.; Sosa, M. I.; White, E. L.; Noah, J.;
Jonsson, C. B. J. Biomol. Screen. 2008, 13, 879.
In summary, we have shown that the acylthiourea scaffold,
uncovered through HTS hits 1 and 2, displays broad-spectrum
activity against a variety of viral families. Through our SAR stud-
ies, it was evident that a bulky, lipophilic group (i.e., tert-butyl
moiety) in segment A and acylthiourea moiety in segment B were
required for broad-spectrum antiviral activity. As a result, it ap-
peared that the portions of the scaffold that were most amenable
to structural modifications were segments C and D. The activity
data suggested that the extended scaffold of 1 may be picking
up additional binding interactions, when compared to 2, leading
to an increase in potency. In the end, we were able to improve
12. Upadhyaya, J. S.; Srivastava, P. K. J. Indian Chem. Soc. 1982, 59, 767.
13. VV and LACV antiviral activity values were averages from multiple
experiments for compounds 1, 2 and the compound in Figure 4 (R = CH3).
14. Scheme 6 was used to prepare compounds 20a, 20b, 20f, 20m, 20n, 20p–r,
20u, 21a, 21b, 21d–f, 21i, 21j, 21l, 21m, 22a–i. Please note that for compounds
22a–i, additional modifications were required beyond those described in
Scheme 6 such as deprotection and reductive amination. The other compounds
contained in Tables 2 and 3 were purchased from commercial sources.
15. For the analogs in Table 9, all of these were purchased from commercial
sources except for 45m.
16. For Table 10, compounds 46a and 46c were purchased from commercial
sources.