Discovery of a novel 5-carbonyl-1H-imidazole-4-carboxamide class of inhibitors of the HIV-1 integrase-LEDGF/p75 interaction

Article abstract of DOI:10.1016/j.bmc.2013.07.047

Though much progress has been made in the inhibition of HIV-1 integrase catalysis, clinical resistance mutations have limited the promise of long-term drug prescription. Consequently, allosteric inhibition of integrase activity has emerged as a promising approach to antiretroviral discovery and development. Specifically, inhibitors of the interaction between HIV-1 integrase and cellular cofactor LEDGF/p75 have been validated to diminish proviral integration in cells and deliver a potent reduction in viral replicative capacity. Here, we have contributed to the development of novel allosteric integrase inhibitors with a high-throughput AlphaScreen-based random screening approach, with which we have identified novel 5-carbonyl-1H-imidazole-4-carboxamides capable of inhibiting the HIV-1 integrase-LEDGF/p75 interaction in vitro. Following a structure-activity relationship analysis of the initial 1H-imidazole-4,5- dicarbonyl core, we optimized the compound's structure through an industrial database search, and we went further to synthesize a selective and non-cytotoxic panel of inhibitors with enhanced potency.

Full text of DOI:10.1016/j.bmc.2013.07.047

Bioorganic & Medicinal Chemistry 21 (2013) 5963–5972
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a novel 5-carbonyl-1H-imidazole-4-carboxamide class
of inhibitors of the HIV-1 integrase–LEDGF/p75 interaction q
Erik Serrao a, ,à, Zhong-Liang Xu b, , Bikash Debnath a,§, Frauke Christ c, Zeger Debyser c, Ya-Qiu Long b,
,
⇑
Nouri Neamati a,
⇑
,§
a Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA
b CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
c Laboratory for Molecular Virology and Gene Therapy, Division of Molecular Medicine, Katholieke Universiteit Leuven (KULeuven), Kapucijnenvoer 33,
B-3000 Leuven, Flanders, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Though much progress has been made in the inhibition of HIV-1 integrase catalysis, clinical resistance
mutations have limited the promise of long-term drug prescription. Consequently, allosteric inhibition
of integrase activity has emerged as a promising approach to antiretroviral discovery and development.
Specifically, inhibitors of the interaction between HIV-1 integrase and cellular cofactor LEDGF/p75 have
been validated to diminish proviral integration in cells and deliver a potent reduction in viral replicative
capacity. Here, we have contributed to the development of novel allosteric integrase inhibitors with a
high-throughput AlphaScreen-based random screening approach, with which we have identified novel
5-carbonyl-1H-imidazole-4-carboxamides capable of inhibiting the HIV-1 integrase–LEDGF/p75 interac-
tion in vitro. Following a structure–activity relationship analysis of the initial 1H-imidazole-4,5-dicar-
bonyl core, we optimized the compound’s structure through an industrial database search, and we
went further to synthesize a selective and non-cytotoxic panel of inhibitors with enhanced potency.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Received 21 April 2013
Revised 17 July 2013
Accepted 26 July 2013
Available online 2 August 2013
Keywords:
HIV-1
Integrase
LEDGF/p75
5-carbonyl-1H-Imidazole-4-carboxamide
Protein–protein interaction
Antiviral
PDB ID code2B4J
1. Introduction
the active site.4 Though raltegravir was shown to inhibit viral repli-
cation in the low nanomolar range and exhibit a relatively low de-
Traditionally, HIV-1 drug development research has focused on
inhibiting the active sites of viral enzymes.1–3 The first HIV-1 integr-
ase (IN) inhibitor (raltegravir) approved by the FDA in October of
2007 was believed to function by chelating the metal cofactor in
gree of cellular toxicity,5 an assortment of viral resistance
mutations have limited the plausibility of its long-term prescrip-
tion.6 A significant effort has been assigned to the generation of
me-too analogues of raltegravir in recent years in the hopes of cir-
cumventing viral resistance, but escape mutants have steadily
developed in the clinic.7–9 Indeed, all currently prescribed active
site-directed HIV-1 inhibitors have led to the generation of po-
tency-limiting resistance.10 Combination of potent antiretrovirals
into drug cocktails has transformed the management of treat-
ment-experienced patients by delivering a durable knockdown of
viral load, viral transmission, and chronic host immune activa-
tion,11–13 but viral eradication looks to require novel modes of enzy-
matic inactivation.
Allosteric inhibition of IN catalytic activity has emerged as an
interesting trend in current antiretroviral drug design and discov-
ery.14 The rationale is that targeting a non-active-site region on IN
that is still essential for its catalysis, such as a cofactor binding site
or dimerization hotspot, could yield the same inhibitory potency as
active site inhibitors, yet evoke a divergent panel of viable
resistance mutations. Hence, addition of an allosteric IN inhibitor
Abbreviations: IN, HIV-1 integrase; LEDGF/p75, lens epithelium-derived growth
factor/p75; HBD, hydrogen bond donor; HBA, hydrogen bond acceptor; IC50, 50%
inhibitory concentration; EC50, 50% effective concentration; CC50, 50% cytotoxic
concentration; CCID50, cell culture infective dose.
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding authors. Address: University of Michigan College of Pharmacy,
North Campus Research Complex, 2800 Plymouth Road, Bldg 520, Room 1364, Ann
Arbor, MI 48109-2800, USA. Tel.: +1 323 442 2341; fax: +1 323 442 1390 (N.N.).
E-mail addresses: yqlong@mail.shcnc.ac.cn (Y.-Q. Long), Neamati@umich.edu
(N. Neamati).
These authors equally contributed to this work.
Address: Department of Cancer Immunology and AIDS, Dana-Farber Cancer
à
Institute, Harvard Medical School, Boston, MA 02215, USA.
§
Address: Department of Medicinal Chemistry, College of Pharmacy, North
Campus Research Complex, University of Michigan, Ann Arbor, MI 48109, USA.
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.047

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E. Serrao et al. / Bioorg. Med. Chem. 21 (2013) 5963–5972
to antiretroviral therapy regimens should deliver a sharp initial
reduction in viral copies/mL. By far the most studied cellular cofac-
tor of HIV-1 integration to date is lens epithelium-derived growth
factor/p75 (LEDGF/p75). This human protein was originally found
to colocalize with IN and stimulate its activity15 and later found
to exert this stimulation by tethering IN to host cell chromatin.16
In a proof-of-concept study that validated the LEDGF/p75–IN inter-
action as an antiviral target, the IN-binding LEDGF/p75 domain
was overexpressed in human cells and was proven to compete
for IN binding with the endogenous full-length cofactor, thereby
inhibiting HIV replication.17
Since these initial discoveries, the IN-LEDGF/p75 interface has
been identified,18,19 and an initial panel of small-molecules has
been rationally developed to bind to this interface and potently in-
hibit the IN–LEDGF/p75 interaction.20–22 These molecules are on
their way to clinical development, but a diversity of structural clas-
ses of IN-LEDGF/p75 inhibitors is essential for the advancement of
the field. Here, we have contributed to this field with the identifi-
cation of a novel class of 5-carbonyl-1H-imidazole-4-carboxamide
inhibitors of the IN–LEDGF/p75 interaction, which have achieved
in vitro inhibition of the IN–LEDGF/p75 interaction in the nanomo-
lar range. Rational synthesis of optimized analogues increased the
potency of our inhibitors, and all compounds developed herein
were non-cytotoxic in MT-4 cells.
an additional saponification was required to remove the acetate
protecting group on the 4-hydroxy group.
3. Results
3.1. High-throughput screening generated a hit 4-
(phenylcarbamoyl)-1H-imidazole-5-carboxylic acid compound
Toward the discovery of novel inhibitors of the IN–LEDGF/p75
interaction, we initiated a high-throughput screening effort using
our in-house library of over 50,000 unique compounds. To acquire
this library, we previously built a drug-like model considering
approximately, 3200 FDA-approved small-molecules and drugs in
clinical trials,25,26 as well as approximately, 11,000 toxic and car-
cinogenic compounds,27–30 using machine learning technique
(Decision forest) within Pipeline Pilot (Accelyrs, Inc.). The whole
dataset was divided into training and test sets randomly consisting
of both good (drug) and bad (non-drug) molecules. The training set
was used to generate the model, and the test set was used for the
purpose of validation of this model. A decision forest of 25 decision
trees was used to generate the drug-like model, where decision
trees were built using a recursive portioning method. In the test
set, out of 1638 drugs, 1335 drugs are predicted as drugs and out
of 5666 toxic compounds, 5031 are predicted as toxics, which
proves the robustness of the model. We have used the drug-like
model to filter eight million commercially available compounds
from Asinex, Enamine, Vitas M Laboratory, etc.,31–33 which resulted
in an approximate output of five million drug-like structures. We
went further to cluster these compounds using 2D fingerprinting
(EFCP-6) and the Tanimoto coefficient to derive a final in-house li-
brary of 50,000 diverse compounds representing the chemical
space of 8 million. We have procured these compounds in 1 mg
scale from these commercial vendors and have demonstrated the
utility of this library in generating unique and potent inhibitors
of HIV-1 IN catalysis.34–36
2. Chemistry
Based on the hit (compound 1) that was identified by
high-throughput screening, SAR study and pharmacophore explo-
ration were conducted with respect to the optimal substituents
at the 4,5-dicarboxyl sites. As outlined in Scheme 1, a series of
5-carbonyl-1H-imidazole-4-carboxamides (2–16) were designed
and synthesized by incorporating different aromatic rings into po-
sition 4 and different aliphatic amino moieties into position 5.
The 5-formyl-1H-imidazole-4-carboxamide analogues were
synthesized according to the literature-reported methodology23,24
with our modification. As depicted in Scheme 2, the imidazole-
4,5-dicarboxylic acid was readily converted to the 1H-imidazole-
4,5-dicarbonyl dichloride in 89% yield by treating with SOCl2 in
toluene with catalytic DMF, followed by esterification with phenol
to afford the key intermediate 17 in 94% yield. Nucleophilic
addition–elimination of the lactam dimer 17 by various amines
produced amide-ester 18a–c. On one hand direct saponification
of 17 immediately after aminolysis yielded the acid-amides 2, 3,
5, 6 and 7 in good isolated yields (55–72% for two steps). On the
other hand, the nucleophilic attack of the ester-amide 18a or 18b
by hydrazine or various amines under heating or microwave
irradiation generated the asymmetric 4,5-dicarboxamide 8–10,
and 14–16 in an overall yield of 62–87%, Further treatment with
20% Pd(OH)2/C to remove the benzyl protection group
afforded 18c-derived compounds 11–13. For the synthesis of
4-hydroxypiperidinyl carboxamide containing compounds (9, 12),
Using an AlphaScreenÒ assay previously validated for sensitive
and specific detection of inhibition of the IN–LEDGF/p75 interac-
tion,20,22 we randomly screened 10,000 unique compounds from
our in-house library. Of multiple chemical classes of inhibitors
identified, we selected 4-(phenylcarbamoyl)-1H-imidazole-5-car-
boxylic acid (compound 1, Fig. 1) for further development, based
on the simplicity of its structure and potency in vitro (IC50 = 6 4 -
lM). This particular compound was non-cytotoxic in MTT assay
and exhibited specificity for inhibition of IN-LEDGF/p75, as it was
inactive in our quench counter-screen assay (Table 1). We have
previously described the details of this assay.37 Interestingly, com-
pound 1 was ineffective at inhibiting IN enzymatic activity in
terms of 30-processing and strand transfer (Table 1), differing from
what has been recently observed with separate classes of
IN-LEDGF/p75 inhibitors.38
Compound 1 was docked into the LEDGF/p75 binding site of
HIV-1 IN (PDB: 2B4J) for exploration of predicted binding mode,
OH
N
or R2:
NH
R2
R2
NH
NH
OH
Identification
of optimal R1
NH
Identification
of optimal R2
∗
3
SAR Study
N
N
O
O
O
O
4
N
O
O
N
O
2
1
N
H
N
H
N
H
Pharmacophore
exploration
O
N
H
5
OH
R1
2-7
R1
8-16
Hit, 1
R1 = OH, NH-alkyl, NHNH2, N-dialkyl
R2 = aryl, heteroaryl
Scheme 1. The medicinal chemistry approach for the SAR study and structural optimization based on the HTS hit and molecular docking.

E. Serrao et al. / Bioorg. Med. Chem. 21 (2013) 5963–5972
5965
O
R2
O
OPh
NH
OH
OH
OH
2
R2 = p-F-Ph
N
c, d
N
O
O
N
a, b
3 R2 = p-F-Benzyl
5 R2 = Py
O
O
N
N
N
N
H
N
H
6
R2 = p-NO2-Ph
PhO
O
7 R2 = p-NH2-Ph
O
17
c
OH
R2
R2
NH
NH
NH
N
O
O
e, f or e, d, f
N
N
O
O
O
O
e
N
H
N
H
N
H
R1
R1
OPh
11 R1 = Morpholine
12
8 R2 = Py, R1 = Morpholine
18a R2 = Py
18b
R1 = 4-OH-Piperidine
13 R1 = Piperazine
9
R2 = Py, R1 = 4-OH-piperidine
R2 = p-Me-Ph
18c R2 = 2-(Benzyloxy)phenyl
10 R2 = Py, R1 = Piperazine
14 R2 = p-Me-Ph, R1 = NHNH2
15
R2 = Py, R1 = NHNH2
16 R2 = Py, R1 = MeNH
Scheme 2. Reagents and conditions: (a) SOCl2, toluene, cat. DMF, 50 °C; (b) phenol, pyridine (Py), DCM, 0 °C to rt; (c) R2NH2, THF; (d) LiOH, THF/H2O, rt; (e) R1NH2, THF, 50 °C
or R1NH2, DMF, MW, 130 °C (for the heterocyclic amide containing compounds 8–13); (f) 20% Pd(OH)2/C, MeOH, rt.
NH
N
O
O
N
H
OH
Figure 1. Compound 1—our initial hit inhibiting the HIV-1 IN–LEDGF/p75 interaction. High-throughput random screening utilizing an AlphaScreen-based luminescence
assay resulted in discovery of a novel 5-formyl-1H-imidazole-4-carboxamide class of allosteric IN inhibitors.
Table 1
Biological activities of 5-carbonyl-1H-imidazole-4-carboxamide derivatives
Quench%inhibitiond
MTT/MT-4
M) EC50 (lM)
a
b
c
Compound
IN-LEDGF/p75, IC50
(l
M)
IN 30-P, IC50
(l
M)
IN ST, IC50
(lM)
e
f
CC50
(l
1
2
3
4
5
6
7
8
6
10
4
2
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
<50
<50
<50
<50
<50
<50
<50
<50
<50
<50
<50
<50
<50
<50
<50
<50
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
13
5.3 0.6
1.0 0.3
>20
>20
>20
>20
>20
3
9
10
11
12
13
14
15
16
>20
16 10
0.4 0.1
>20
a
b
c
d
e
f
Concentration required to inhibit the in vitro protein–protein interaction by 50%.
Concentration required to inhibit IN 30-processing catalysis by 50%.
Concentration required to inhibit IN strand transfer catalysis by 50%.
Percent inhibition exerted by compounds in quench counter-screen when tested at 20
Cytotoxic concentration reducing MT-4 cell viability by 50%.
l
M.
Effective concentration required to reduce HIV-1 induced cytopathic effect by 50% in MT-4 cells.
and this mode of binding provided vital information for a struc-
ture-guided optimization strategy. The carboxylic moiety of 1
was shown to form two hydrogen bond (H-bond) interactions with
the backbone NHs of His171 and Glu170 (Fig. 2). The phenyl ring of
compound 1 packed into the hydrophobic pocket formed by IN res-
idues Thr125, Ala128, Trp131, Trp132, and Gln168.
3.2. Initial synthesized analogues demonstrate R1 and R2
structural requirements
Based on our observations above, we began to design and
synthesize analogues with substitutions at each terminal region
of the 5-formyl-1H-imidazole-4-carboxamide hit scaffold (Fig. 3).

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E. Serrao et al. / Bioorg. Med. Chem. 21 (2013) 5963–5972
LEDGF/p75 inhibition, with an IC50 value of 10
2 lM (Table 1).
We found that an additional carbon extension of the fluorophenyl
group, in our synthesized 4-((4-fluorobenzyl)carbamoyl)-1H-imid-
azole-5-carboxylic acid (compound 3), significantly increased IC50
in the AlphaScreen assay above our upper measurement threshold
of 20 lM (Table 1). Similarly, maintaining the original fluorophenyl
R2 substituent but replacing the R1 hydroxyl with a methoxy in
methyl 4-((4-fluorophenyl)carbamoyl)-1H-imidazole-5-carboxyl-
ate (compound 4), abolished inhibitory potency to IC50 >20 lM
(Table 1). Multiple inactive analogues that also bear a methylated
R1 moiety are displayed in Supplementary Figure 1. Compound 4
was an analogue from our in-house database and was not synthe-
sized. These three compounds led to the conclusion that a HBA is
necessary at the R1 position, while a phenyl substituent with com-
pact modifications is preferred at the R2 position.
Enhanced potency was achieved with synthesized analogues
containing diverse compact R2 phenyl modifications. Inclusion
of an ortho nitrogen in the R2 phenyl, generating 4-(pyridine-2-
ylcarbamoyl)-1H-imidazole-5-carboxylic acid (compound 5),
Figure 2. Compound 1 was docked onto the LEDGF/p75 binding site of IN protein in
PDB 2B4J. Predicted ligand–receptor interactions included two H-bonds between
the carboxylic moiety of 1 and backbone NHs of E170 and H171. Between the
imidazole NH of 1 and the side chain OH of IN residue T174. Meanwhile, the phenyl
ring of the ligand packed into the hydrophobic pocket occupied by LEDGF/p75
residue I365.
showed acceptable activity at IC50 = 13
3 lM (Table 1). Alterna-
tively, a nitro- or amino-substituted phenyl ring is preferred for
the R2 structure, that is, 4-((4-nitrophenyl)carbamoyl)-1H-imidaz-
ole-5-carboxylic
1H-imidazole-5-carboxylic acid (compounds 6 and 7) showed an
improved potency at IC50 = 5.3 0.6 M and 1.0 0.3 M, respec-
acid
and
4-((4-aminophenyl)carbamoyl)-
l
l
We designated two variable regions, with R1 describing the original
position of the terminal carboxylate hydroxyl moiety, and R2
describing the phenyl substituent at the opposite terminus of the
molecule. We first synthesized 4-((4-fluorophenyl)carbamoyl)-
1H-imidazole-5-carboxylic acid (compound 2) with a fluorophenyl
R2 substitution, and we found similar in vitro activity for IN-
tively (Table 1). Similar to hit compound 1, compounds 2–7 were
nontoxic, inactive in our quench counter-screen at the maximum
dose of 20 lM, and inactive against IN catalytic activity (Table 1).
The best compound 7 was docked onto the LEDGF/p75 binding
site of IN protein (PDB: 2B4J) for exploration of binding mode
(Fig. 4). It displayed nearly similar binding interactions with IN
as compound 1. Specifically, the carboxylic oxygens formed H-
bonds with the backbone NHs of Glu170 and His171 on IN. The
imidazole NH adjacent to the carboxylic group formed an H-bond
with the side chain oxygen of Thr174. The aniline group packed
into the hydrophobic pocket formed by IN residues Thr125,
Ala128, Trp131, Trp132, and Gln168. Figure 4 shows compound 7
docked onto IN at the LEDGF/p75 binding interface.
R2
NH
N
O
O
N
H
R1
Compound
Structure
R2
R1
F
OH
2
3
F
OH
F
OCH3
4
5
N
OH
OH
O2N
H2N
6
7
OH
Figure 4. Compound 7 is docked onto IN protein at the IN–LEDGF/p75 interaction
interface. Specific interactions included the compound’s carboxylate oxygens
forming hydrogen bonds with the backbone NHs of IN E170 and H171. Another
hydrogen bond formed between the imidazole NH of 7 and the side chain OH of IN
residue T174. The aniline group of compound 7 packed into the hydrophobic pocket
formed by IN residues Thr125, Ala128, Trp131, Trp132, and Gln168.
Figure 3. Initial 5-formyl-1H-imidazole-4-carboxamide derivatives. Two variable
regions describe substitutions at the original position of the terminal carboxylate
hydroxyl moiety and the phenyl substituent at the opposite terminus of the
molecule.

E. Serrao et al. / Bioorg. Med. Chem. 21 (2013) 5963–5972
5967
3.3. Piperidine-based R1 position substitutions generated
inactive analogues
molecular conformation imparted by the imidazole is essential
for proper in vitro activity.
To explore the tolerance for extension of the HBD/HBA at the R1
position, a panel of analogues containing piperidine-based substit-
uents in place of the original R1 hydroxyl group were synthesized
(Fig. 5). Specifically, we prepared compounds containing morpho-
line, piperidin-4-ol, and piperazine R1 moieties with either pyri-
dine (compounds 8–10, respectively) or phenol R2 substituents
(compounds 11–13, respectively). Unfortunately, all of the above
compounds proved inactive in our AlphaScreen assay, and were
also nontoxic and inactive in our quench counter-screen and IN
enzymatic assay (Table 1). Molecular docking analysis of the bind-
ing mode of these compounds may explain this lack of activity. In
docking inactive compounds 8, 9, 11, and 12, we found a consistent
absence of an H-bond between the compound and IN residue
His171. All active compounds in this report that we studied with
molecular docking exhibited this particular hydrogen bond. Con-
versely, inactive compounds 8, 9, 11, and 12 lacked this interaction
but still were predicted to form H-bonds with the backbone NH of
Glu170 and packed a substituent into the hydrophobic pocket cre-
ated by IN residues Thr125, Ala128, Trp131, Trp132, and Gln168. A
separate panel of inactive analogues discovered during high-
throughput screening of in-house analogues of compound 1 is
shown in Supplementary Figure 2. These compounds did not
contain piperidine-based R1 substituents, but rather exhibited a
central pyrazine in place of the original 1H-imidazole component.
This analogue panel clearly demonstrated that the overall
3.4. Inclusion of R1 hydrazine yielded an IN-LEDGF/p75 inhibitor
with significantly increased potency
A concluding synthesis of analogues including an alternate
hydrazine HBD at the R1 position yielded encouraging results
(Fig. 6). Maintaining a simple toluene as the R2 substituent (com-
pound 14) gave 5-(hydrazinecarbonyl)-N-(p-tolyl)-1H-imidazole-
4-carboxamide, which exhibited a modest IC50 of 16 10 lM
against IN-LEDGF/p75 in vitro (Table 1). However, when reverting
to a pyridine R2 component, a substantial increase in potency was
observed, as 5-(hydrazinecarbonyl)-N-(pyridin-2-yl)-1H-imidaz-
ole-4-carboxamide (compound 15) exhibited an IC50 value of
0.4 0.1
tion of the hydrazine with a methylamine in N5-methyl-N4-(pyri-
dine-2-yl)-1H-imidazole-4,5-dicarboxamide (compound 16)
lM (Table 1). In line with our prior observations, substitu-
abolished in vitro activity entirely (Table 1). Molecular docking
analysis revealed that top compound 15 formed maximum interac-
tions with IN (Fig. 7). Specifically, hydrogen bonds were formed be-
tween side chain and backbone NHs of His171 and both the oxygen
and terminal NH at the R1 position of compound 15. The com-
pound’s R1 position oxygen and carbonyl oxygen from the amide
linker both formed hydrogen bonds with the backbone NH of Glu
170. While compound 15’s pyridine moiety packed into the hydro-
phobic pocket formed by IN residues Thr125, Ala128, Trp131,
Trp132, and Gln168, an additional hydrogen bond was formed be-
tween the compound’s amide NH and the backbone oxygen of
Gln168. The array of interaction points between compound 15
and IN at its interface with LEDGF/p75 may explain the com-
pound’s exceptional potency. Figure 7 shows compound 15 docked
onto IN at the LEDGF/p75 binding interface. Compounds 14–16 all
followed the trend of non-cytotoxicity in MTT assay and inactivity
in our quench counter-screen or IN enzymatic assay. Unfortu-
nately, none of the compounds detailed in this report exhibited
inhibition of HIV-induced cytopathic effect in MT-4 cell culture.
However, the lack of toxicity even at high doses gives us confi-
dence that further structural optimization may hold potential for
generating compounds that not only inhibit IN-LEDGF/p75
in vitro, but also during viral replication.
R2
NH
N
O
O
N
H
R1
R1
Compound
8
Structure
R2
N
N
O
N
N
N
9
R2
OH
N
NH
N
O
10
11
O
N
H
N
H
R1
R1
Compound
14
Structure
OH
OH
N
R2
O
N
NHNH2
12
13
N
OH
N
15
16
NHNH2
MeNH
OH
N
N
H
Figure 5. 5-Formyl-1H-imidazole-4-carboxamide analogues containing piperidine-
Figure 6. 5-Formyl-1H-imidazole-4-carboxamide analogues containing a hydra-
based substituents in place of the original R1 hydroxyl group.
zine HBD at the R1 position.

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E. Serrao et al. / Bioorg. Med. Chem. 21 (2013) 5963–5972
containing a benzophenoneamide at the R2 position, exhibited 16
and 17
M activity against 30-processing and strand transfer,
l
respectively. This compound contained considerably more bulk at
the R2 position than our top leads, which may have lent additional
binding specificity to the IN active site over the IN-LEDGF/p75
interfacial hotspot. On the other hand, this particular molecule
may not be binding to the IN catalytic site at all, but rather allos-
terically inhibiting enzymatic activity. Investigation of the binding
mode of this and other analogous compounds with IN is ongoing in
our laboratory.
Aside from targeting IN, analogous compounds to our
5-carbonyl-1H-imidazole-4-carboxamides have been described as
modulators of JAK-247 and characterized for their antiproliferative
and antituberculosis activity.48,49 The fact that such closely related
compounds can exhibit broad activity profiles underscores the
importance of selectivity monitoring during the development pro-
cess. Though none of our reported analogues exhibited cytotoxicity
in MTT assay, the potential for off-target effects with this class of
compounds is apparent. However, the formulations of these
5-carbonyl-1H-imidazole-4-carboxamides are optimal for clinical
development, and distinctive functional groups on our core
scaffold can be further optimized. Structure–activity analysis
yielded strict structural requirements for in vitro potency that
included a HBA/HBD presence at the R1 position, a central imidaz-
ole substituent, and a more lax phenyl-based R2 requirement. Our
future synthetic strategy will exploit these findings with the hope
of enhancing the antiviral potency of these inhibitors while still
maintaining their nontoxic profile.
Figure 7. Compound 15 is docked onto IN protein at the IN–LEDGF/p75 interaction
interface. Specific interactions included the compound forming hydrogen bonds
between His171 and both the R1 position oxygen and terminal NH, as well as
between backbone NH of E170 and the oxygens of R1 position and linker amide. The
pyridine moiety of compound 15 formed
a hydrophobic interaction with IN
residues Thr125, Ala128, Trp131, Trp132, and Gln168, an additional hydrogen bond
formed between the compound’s amide NH and the backbone oxygen of Gln168.
4. Discussion
IN has to date been shown to distinctly interact with a variety of
host proteins, and many of these interactions have been demon-
strated to be essential for viral replication.39,40 The majority of re-
ported efforts to disrupt these essential interactions via rational
small-molecule development have revolved around the IN-
LEDGF/p75 interaction. A benzoic acid derivative known as D7741
5. Conclusions
From an initial high-throughput screening program, we identi-
fied 4-(phenylcarbamoyl)-1H-imidazole-5-carboxylic acid as a po-
tent and selective inhibitor of the in vitro interaction between HIV-
1 IN and LEDGF/p75. Molecular docking guided a structure-based
chemical synthesis that ultimately yielded a class of nontoxic
5-carbonyl-1H-imidazole-4-carboxamide inhibitors of the above
interaction with submicromolar IC50 values. Though our top com-
pounds did not exhibit antiviral activity in MT-4 cells, favorable
interactions with IN residues essential for the LEDGF/p75 interac-
tion were predicted. Work is ongoing in our laboratory to optimize
the chemical properties of our top compounds toward more potent
inhibition of HIV-1 replication in cell culture.
and
300320 were first reported to inhibit IN-LEDGF/p75 binding with
low micromolar activity. Since these discoveries series of
a LEDGF/p75 structure-based compound named CHIBA-
a
2-(quinolin-3-yl)acetic acid-based molecules has been rationally
designed and developed to inhibit IN-LEDGF/p75 binding with
reported IC50 values as low as 0.046 l
M.42 Though these com-
pounds exhibited potent antiviral activity, they were shown to also
evoke IN resistance mutations, namely the A128T substitution.22
However, recent reports demonstrate that the promotion of IN
multimerization by these so-called LEDGINs or ALLINIs plays a
significant, if not more important, role in ultimately inhibiting viral
replication.43–46 All of this work has revealed not only that inhibi-
tors of IN-LEDGF/p75 can be successful at allosteric IN inhibition
leading to antiviral activity, but also that there is a necessity for
discovering novel inhibitory structural scaffolds.
6. Experimental section
6.1. Expression and purification of recombinant HIV-1 integrase
and LEDGF/p75 proteins
Here we present a novel panel of 5-carbonyl-1H-imidazole-
4-carboxamide inhibitors of the IN–LEDGF/p75 interaction with
our best compound exhibiting submicromolar activity. This class
of compounds has not been previously developed in antiviral re-
search, and the structural scaffold significantly differs from all
prior IN-LEDGF/p75 inhibitors. Thus, we anticipate a divergent vir-
al resistance profile to emerge in response to in vivo drug pressure.
All of the compounds reported in this study were non-toxic in a
human cell line and lacked allosteric inhibition of IN enzymatic
activity. Though the compounds reported herein did not achieve
antiviral effects in vitro, our work here illustrates the power of ra-
tional compound optimization following high-throughput random
screening, and lends credence to the belief that such advances can
be accomplished in the academic realm. Our active compounds
have not been previously reported, though the central scaffold
has been described by our group for targeting the HIV-1 IN cata-
lytic site.35 Specifically, 4-((2-benzoyl-4-bromophenyl)carbam-
oyl)-1H-imidazole-5-carboxylic acid (Supplementary Fig. 1, S15),
The plasmids for His-IN, GST-IN, and LEDGF/p75 were ex-
pressed in Escherichia coli BL21(De3) pLysS expression strain (Invit-
rogen) after induction by IPTG (1 mM) at 0.75 optical density
absorbance (595 nm). The culture was grown for an additional
3–4 h at 37 °C, followed by centrifugation at 3000 rpm in a bucket
rotor centrifuge (Beckman) for 20 min. Cell lysis, Ni-affinity chroma-
tography purification, and dialysis were performed as detailed in.50
6.2. IN-LEDGF/p75 AlphaScreen assay and emission quench
AlphaScreen assay
The AlphaScreen assay was performed according to the manu-
facturer’s protocol (PerkinElmer, Benelux). Reactions were per-
formed in a 25 ll final volume in 384-well Optiwell™ microtiter
plates (PerkinElmer). The reaction buffer contained 25 mM Tris–
HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.01% (v/v) Tween-20

E. Serrao et al. / Bioorg. Med. Chem. 21 (2013) 5963–5972
5969
and 0.1% (w/v) bovine serum albumin. His6-tagged integrase
(300 nM final concentration) was incubated with compound at a
final concentration of 20 lM for 30 min at 4 °C. FLAG-tagged
LEDGF/p75 protein was then added at 300 nM final concentration,
and the reaction was incubated for an additional 60 min at 4 °C.
ported in parts per million relative to TMS and referenced to the
solvent in which they were run. ESI-MS spectra were recorded on
a Finnigan LCQ Deca mass spectrometer. Liquid chromatography–
mass spectrometry (LC–MS) was carried out on Waters Micro-
mass ZQ2000.
Subsequently, 5
FLAG antibody coated acceptor beads were added to a final con-
centration of 20 g/mL of both beads. Proteins and beads were
lL Ni-chelate-coated donor beads and 5 lL anti-
6.5.1. Diphenyl 5,10-dioxo-5,10-dihydrodiimidazo[1,5-a:10,50-
d]pyrazine-1,6-dicarboxylate (17)
l
incubated for 1 h at 30 °C in order to allow association to occur.
Exposure of the reaction to direct light was omitted as much as
possible and the emission of light from the acceptor beads was
measured in the EnVision plate reader (PerkinElmer) and analyzed
using the EnVision manager software. The emission quench
AlphaScreen assay was carried out identically to the IN-LEDGF/
p75 AlphaScreen assay, except instead of the IN and LEDGF/p75
proteins, a FLAG-tagged GST construct was added at a final con-
centration of 500 nM.
To a solution of imidazole-4,5-dicarboxylic acid (400 mg,
2.56 mmol) in 5 mL of toluene was added 1.12 mL of thionyl chlo-
ride (15.38 mmol) and 0.1 mL of DMF at rt. The reaction mixture
was stirred at 90 °C for 16 h. Then 5 mL of DCM was added to
the suspension and the solvent was removed by evaporation. This
process was repeated twice. The resulting acid chloride was dis-
solved in 10 mL of DCM and cooled to 0 °C. Phenol (2.82 mmol)
in 5 mL of DCM was added and the resulting solution was stirred
at 0 °C for 1 h. Then the solid product was collected by vacuum fil-
tration, washed with 10 mL of DCM twice, and dried under vacuum
to yield 0.632 g of crude product 17 (83.6% over two steps) as a tan
solid. 1H NMR (300 Hz, DMSO) d 9.12 (s, 2H), 7.52–7.56 (m, 4H),
7.34–7.40 (m, 6H); ESI-MS: 429 [M+1]+.
6.3. Drug susceptibility assays
The inhibitory effect of antiviral drugs on the HIV-induced cyto-
pathic effect in MT-4 cell culture was determined by the MTT assay
as previously described.22
6.5.2. General procedure for the preparation of amide-ester
18a–c. Method A
6.4. Molecular docking
To a solution of 17 (100 mg, 0.233 mmol) in 30 mL of dry THF
was added 2-aminopyridine (30.8 mg, 0.328 mmol) in 2 mL of
THF dropwise over 2 min at 0 °C. The ice bath was removed after
10 min, and the solution was stirred at rt for 18 h. The solvent
was removed under vacuum, and the residue was purified by col-
umn chromatography on silica gel to give a white solid 18a
(84.9 mg, 84% yield). 1H NMR (400 MHz, CDCl3) d 12.37 (s, 1H),
11.52 (s, 1H), 8.40 (d, J = 3.9 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.95
(s, 1H), 7.80–7.72 (m, 1H), 7.46 (t, J = 7.9 Hz, 2H), 7.33 (d,
J = 9.2 Hz, 2H), 7.29 (s, 1H), 7.11 (dd, J = 7.1, 5.0 Hz, 1H). ESI-MS:
308 [M+1]+.
Select compounds were docked in the LEDGF binding site of
HIV-1 IN (PDB ID: 2B4J) using GOLD (Genetic Optimization for Li-
gand Docking) software package, version 4.0 (Cambridge Crystallo-
graphic Data Centre, Cambridge, UK). Prior to docking, compounds
were energy-minimized, and all possible combinations of stereo-
isomers and conformers were generated with LigPrep from Schro-
dinger using the OPLS-2005 force field.51 Within LigPrep, Epik
module was used to generate all possible ionization forms of li-
gands at pH 7.0 0.2. GOLD uses a genetic algorithm to explore
the conformational space of a compound inside the binding site
of a protein.52,53 Docking studies were performed using the stan-
dard default settings with 100 genetic algorithm (GA) runs on each
molecule. For each of the 100 independent GA runs, a maximum of
100,000 operations was performed on a set of five groups with a
population of 100 individuals. With respect to ligand flexibility,
special care was taken by including options such as flipping of ring
corners, amides, pyramidal nitrogens, secondary and tertiary
amines, and rotation of carboxylate groups, as well as torsion angle
distribution and post-process rotatable bonds as default. The
annealing parameters were used as default cutoff values of 3.0 Å
for hydrogen bonds and 4.0 Å for van der Waals interactions.
Hydrophobic fitting points were calculated to facilitate the correct
starting orientation of the compound for docking by placing the
hydrophobic atoms appropriately in the corresponding areas of
the active site. When the top three solutions attained root-mean-
square deviation (rmsd) values within 1.5 Å, docking was termi-
nated. GOLD-Score, a scoring function within the software, is a
dimensionless fitness value that takes into account the intra- and
intermolecular hydrogen bonding interaction energy, van der
Waals energy, and ligand torsion energy.52,53
6.5.3. Phenyl 4-(p-tolylcarbamoyl)-1H-imidazole-5-carboxylate
(18b)
The title compound was obtained from 17 (100 mg,
0.233 mmol) and p-toluidine (49.9 mg, 0.466 mmol) as described
above in the method A. Pale yellow solid, yield 78%. 1H NMR
(300 MHz, CDCl3) d 11.62 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.64
(d, J = 6.3, 2H), 7.46–7.23 (m, 7H), 2.32 (s, 3H). EI-MS:
321.3 [M]+.
6.5.4. Phenyl 4-((2-(benzyloxy)phenyl)carbamoyl)-1H-
imidazole-5-carboxylate (18c)
The title compound was obtained from 17 (100 mg, 0.233 mmol)
and 2-(benzyloxy)aniline (92.7 mg, 0.466 mmol) as described
above in the method A. Pale yellow solid, yield 81%. 1H NMR
(300 MHz, CDCl3) d 11.88 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.88
(s, 1H), 7.46–7.38 (m, 2H), 7.37–7.27 (m, 3H), 7.24–7.13 (m, 5H),
7.12–7.08 (m, 1H), 7.04–6.97 (m, 2H), 5.09 (s, 2H). EI-MS: 413.2
[M]+.
6.5.5. General procedure for the preparation of acid-amide 2, 3,
5–7. Method B. 4-((4-Fluorophenyl)carbamoyl)-1H-imidazole-5-
carboxylic acid (2)
6.5. General synthetic methods
To a solution of 17 (100 mg, 0.233 mmol) in 30 mL of dry THF
was added p-fluoroaniline (51.7 mg, 0.466 mmol) in 2 mL THF
dropwise over 2 min at 0 °C. The ice bath was removed after
10 min, and the solution was stirred at rt for 18 h. Solvent was re-
moved by concentrating under vacuum. The residue was dis-
solved in 5 mL of methanol and the potassium hydroxide
(200 mg, 3.57 mmol) was added to this suspension at 0 °C. The
ice bath was removed after 10 min, and the solution was
Starting materials, reagents and solvents were purchased from
commercial suppliers and were used without further purification.
THF and CH2Cl2 were dried by anhydrous sodium sulfate before
used. Analytical thin-layer chromatography (TLC) was performed
on silica gel (GF-254) plates. Visualization was effected with
ultraviolet light or iodine vapor. NMR spectra were recorded on
a Varian Mercury 300 or 400 MHz spectrometer. The data are re-

5970
E. Serrao et al. / Bioorg. Med. Chem. 21 (2013) 5963–5972
continuously stirred at rt for 8 h. Then the crude product was pre-
cipitated out when the pH was adjusted to 5 with 3 N HCl. The
solid was filtered and subsequently recrystallized from methanol
and ethyl acetate to give pale yellow solid 2, in an overall yield of
68% for two steps. 1H NMR (300 MHz, DMSO-d6) d 8.223 (s, 1H),
7.808 (d, J = 9.0, 2H), 7.256 (d, J = 9.0, 2H); ESI-MS: 250 [M+1]+;
HRESI-MS: m/z calcd for C11H9FN3O3 ([M+H]+) 250.1979; found
250.1983.
6.5.11. 5-(4-Hydroxypiperidine-1-carbonyl)-N-(pyridin-2-yl)-
1H-imidazole-4-carboxamide (9)
Piperidin-4-yl acetate (27.7 mg, 0.213 mmol) and 18a (60 mg,
0.194 mmol) in DMF (3.0 mL) were heated to 130 °C for 30 min
in a CEM microwave oven. The reaction mixture was allowed to
rt and the DMF was removed under vacuum. The residue was dis-
solved in 1 N LiOH (2 mL) and THF (2 mL). The solution was stirred
at rt for 6 h. After the reaction was completed, the pH of the aque-
ous phase was adjusted to 3 and was extracted with DCM
(10 mL ꢀ 2). The DCM was removed under vacuum, the residue
was purified by column chromatography on silica gel to give a
white solid 40.9 mg, (yield: 67% in two steps): 1H NMR
(300 MHz, CDCl3) d 13.44 (s, 1H), 12.91–12.25 (s, 1H), 8.37 (d,
J = 4.2 Hz, 1H), 8.22 (d, J = 8.3 Hz, 1H), 7.94 (s, 1H), 7.85 (t,
J = 7.8 Hz, 1H), 7.21–7.12 (m, 1H), 4.85–4.76 (m, 1H), 4.15–4.03
(m, 1H), 3.86–3.73 (m, 1H), 3.42–3.27 (m, 4H), 1.95–1.68 (m,
2H), 1.54–1.29 (m, 2H). ESI-MS: 316.2 [M+1]+; HRESI-MS: m/z
calcd for C15H18N5O3 ([M+H]+) 316.1410; found 316.1400.
6.5.6. 4-((4-Fluorobenzyl)carbamoyl)-1H-imidazole-5-
carboxylic acid (3)
The title compound was obtained from 17 (100 mg,
0.233 mmol) and p-fluorobenzylamine (58.3 mg, 0.466 mmol) as
described above in the method B. Pale yellow solid, yield 62%; 1H
NMR (300 MHz, DMSO-d6) d 8.060 (s, 1H), 7.363 (d, J = 7.8, 2H),
7.162 (d, J = 7.8, 2H), 4.494 (s, 2H); ESI-MS: 264 [M+1]+; HRESI-
MS: m/z calcd for C12H11FN3O3 ([M+H]+) 264.2245; found
264.2238.
6.5.12. 5-(Piperazine-1-carbonyl)-N-(pyridin-2-yl)-1H-
imidazole-4-carboxamide (10)
6.5.7. 4-(Pyridin-2-ylcarbamoyl)-1H-imidazole-5-carboxylic
acid (5)
The title compound was obtained from 18a (100 mg,
0.233 mmol) and piperazine (38.4 mg, 0.466 mmol) as described
above in the method C. white solid, yield 87%; 1H NMR
(300 MHz, CDCl3) d 8.37 (d, J = 3.9 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H),
7.94 (s, 1H), 7.85 (t, J = 7.7 Hz, 1H), 7.21–7.12 (m, 1H), 3.90–3.73
(m, 2H), 3.70–3.60 (m, 2H), 2.85–2.76 (m, 2H), 2.76–2.68 (m,
2H). ESI-MS: 301.2 [M+1]+; HRESI-MS: m/z calcd for C14H17N6O2
([M+H]+) 301.1413; found 301.1417.
The title compound was obtained from 17 (100 mg,
0.233 mmol) and 2-aminopyridine (84 mg, 0.466 mmol) as de-
scribed above in the method B. Pale yellow solid, yield 67%; 1H
NMR (300 MHz, DMSO-d6) d 8.281(s, 1H), 7.844(d, J = 11.7, 2H),
7.411(d, J = 11.7, 2H); 13C NMR (100 MHz, DMSO-d6) d 160.4,
150.7, 148.4, 138.5, 137.6, 137.5, 135.6, 129.8, 120.5, 114.4; ESI-
MS: 233 [M+1]+; HRESI-MS: m/z calcd for C10H9N4O3 ([M+H]+)
233.0675; found 233.0690.
6.5.13. N-(2-Hydroxyphenyl)-5-(morpholine-4-carbonyl)-1H-
imidazole-4-carboxamide (11)
6.5.8. 4-((4-Nitrophenyl)carbamoyl)-1H-imidazole-5-carboxylic
acid (6)
Morpholine (18.6 mg, 0.213 mmol) and 18c (80 mg, 0.194 mmol)
in DMF (3.0 mL) were heated to 130 °C for 30 min in a CEM micro-
wave oven. The reaction was allowed to cool and the DMF was re-
moved under vacuum. The residue was dissolved in EtOH (5 mL)
and 10 percent 20% Pd(OH)2/C was added thereto. The reaction
mixture was stirred at rt for 3 h under hydrogen atmosphere. After
completion of the reaction, the solution was filtered with a cellite
and the filtrate was distilled under a reduced pressure to afford the
title compound, which was purified by column chromatography on
silica gel to give a white solid 46.7 mg, (yield: 76.1% in two steps):
1H NMR (300 MHz, CDCl3) d 8.02–7.95 (m, 1H), 7.81 (s, 1H), 7.00–
6.95 (m, 1H), 6.86 (t, J = 1.4 Hz, 1H), 6.84–6.80 (m, 1H), 3.84–3.75
(m, 4H), 3.73–3.64 (m, 4H). ESI-MS: 317.2 [M+1]+; HRESI-MS: m/
z calcd for C15H17N4O4 ([M+H]+) 317.1250; found 317.1255.
The title compound was synthesized from 17 (100 mg,
0.233 mmol) and p-nitroaniline (64.4 mg, 0.466 mmol) as de-
scribed above in the method B. Pale yellow solid, yield 61%; 1H
NMR (300 MHz, DMSO-d6) d 8.298(s, 1H), 7.861(d, J = 9.0, 2H),
7.427(d, J = 9.0, 2H); ESI-MS: 277 [M+1]+; HRESI-MS: m/z calcd
for C11H9N4O5 ([M+H]+) 277.1331; found 277.1318.
6.5.9. 4-((4-Aminophenyl)carbamoyl)-1H-imidazole-5-
carboxylic acid (7)
The title compound was synthesized from 17 (100 mg,
0.233 mmol) and (9H-fluoren-9-yl)methyl (4-aminophenyl)carba-
mate (153.8 mg, 0.466 mmol) as described above in the method
B. Pale yellow solid, yield 72%; 1H NMR (300 MHz, DMSO-d6) d
8.362 (s, 1H), 8.267(d, J = 9.1, 1H), 7.804 (m, 2H), 7.106 (dd,
J = 5.4, 6.0, 1H); 13C NMR (100 MHz, DMSO-d6) d 161.22, 159.91,
137.68, 136.25, 133.12, 129.23, 127.93, 123.25, 122.44; ESI-MS:
247 [M+1]+; HRESI-MS: m/z calcd for C11H11N4O3 ([M+H]+)
247.0831; found 247.0815.
6.5.14. N-(2-Hydroxyphenyl)-5-(4-hydroxypiperidine-1-
carbonyl)-1H-imidazole-4-carboxamide (12)
Piperidin-4-yl acetate (27.7 mg, 0.213 mmol) and 18c (80 mg,
0.194 mmol) in DMF (3.0 mL) were heated to 130 °C for 30 min
in a CEM microwave oven. The reaction was allowed to cool and
the DMF was removed under vacuum. The residue was dissolved
in 1 N LiOH (2 mL) and THF (2 mL). The solution was stirred at rt.
After the reaction was completed, the pH of the aqueous phase
was adjusted to 3 and was extracted with DCM (10 mL ꢀ 2). The
combined organic phases were evaporated in vacuum, and the res-
idue was dissolved in EtOH (5 mL). 10 percent 20% Pd(OH)2/C was
added thereto, and the reaction mixture was stirred at rt for 3 h un-
der hydrogen atmosphere. After completion of the reaction, the
resulting solution was filtered with a cellite and distilled under a
reduced pressure to obtain the title compound, which was purified
by column chromatography on silica gel to give a white solid
36.2 mg, (yield: 56.7% in three steps): 1H NMR (300 MHz, CDCl3)
d 8.31–8.09 (m, 1H), 7.87 (s, 1H), 7.01–6.87 (m, 2H), 6.85–6.76
(m, 1H), 4.80 (s, 1H), 4.09 (s, 1H), 3.77 (s, 1H), 3.38–3.29 (m, 4H),
6.5.10. General procedure for the preparation of unsymmetric
4,5-dicarboxamide 8 and 10. Method C. 5-(Morpholine-4-
carbonyl)-N-(pyridin-2-yl)-1H-imidazole-4-carboxamide (8)
Morpholine (18.6 mg, 0.213 mmol) and 18a (60 mg,
0.194 mmol) in DMF (3.0 mL) were heated to 130 °C for 30 min
in a CEM microwave oven. The reaction was allowed to cool and
the DMF was removed under vacuum, the residue was purified
by column chromatography on silica gel to give a white solid
47.3 mg, (yield: 81%): 1H NMR (300 MHz, CDCl3) d 13.51 (s, 1H),
12.67 (s, 1H), 8.37 (d, J = 4.9 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.96
(s, 1H), 7.91–7.81 (m, 1H), 7.16 (dd, J = 6.8, 5.4 Hz, 1H), 3.82–3.68
(m, 4H), 3.68–3.59 (m, 2H), 3.40–3.28 (m, 2H). ESI-MS: 302.3
[M+1]+; HRESI-MS: m/z calcd for C14H16N5O3 ([M+H]+) 302.1253;
found 302.1264.

E. Serrao et al. / Bioorg. Med. Chem. 21 (2013) 5963–5972
5971
1.89–1.59 (m, 2H), 1.51–1.26 (m, 2H). ESI-MS: 331.1 [M+1]+; HRES-
Supplementary data
I-MS: m/z calcd for C16H18N4O4Na ([M+Na]+) 353.1226; found
353.1214.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.07.047.
6.5.15. N-(2-Hydroxyphenyl)-5-(piperazine-1-carbonyl)-1H-
imidazole-4-carboxamide (13)
References and notes
The title compound was prepared from 18c (80 mg,
0.194 mmol) and piperazine (18.3 mg, 0.213 mmol) with a proce-
dure similar to that for 11. White solid, yield 61%; 1H NMR
(300 MHz, CDCl3) d 8.18 (d, J = 7.5 Hz, 1H), 7.86 (s, 1H), 6.98–6.87
(m, 2H), 6.84–6.77 (m, 1H), 3.65–3.48 (m, 4H), 2.81–2.72 (m,
2H), 2.72–2.62 (m, 2H). ESI-MS: 316.1 [M+1]+; HRESI-MS: m/z
calcd for C15H18N5O3 ([M+H]+) 316.1410; found 316.1421.
1. Qian, K.; Morris-Natschke, S. L.; Lee, K. H. Med. Res. Rev. 2009, 29, 369.
2. El Safadi, Y.; Vivet-Boudou, V.; Marquet, R. Appl. Microbiol. Biotechnol. 2007, 75,
723.
3. Temesgen, Z.; Warnke, D.; Kasten, M. J. Expert Opin. Pharmacother. 2006, 7,
1541.
4. Nguyen, B. Y.; Isaacs, R. D.; Teppler, H.; Leavitt, R. Y.; Sklar, P.; Iwamoto, M.;
Wenning, L. A.; Miller, M. D.; Chen, J.; Kemp, R.; Xu, W.; Fromtling, R. A.; Vacca,
J. P.; Young, S. D.; Rowley, M.; Lower, M. W.; Gottesdiener, K. M.; Hazuda, D. J.
Ann. N.Y. Acad. Sci. 2011, 1222, 83.
5. Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi, M.; Ferrara, M.;
Fiore, F.; Gardelli, C.; Gonzalez Paz, O.; Hazuda, D. J.; Jones, P.; Kinzel, O.; Laufer,
R.; Monteagudo, E.; Muraglia, E.; Nizi, E.; Orvieto, F.; Pace, P.; Pescatore, G.;
Scarpelli, R.; Stillmock, K.; Witmer, M. V.; Rowley, M. J. Med. Chem. 2008, 51,
5843.
6.5.16. General procedure for the preparation of unsymmetric
4,5-dicarboxamide 14–16. Method D. 5-(Hydrazinecarbonyl)-N-
(p-tolyl)-1H-imidazole-4-carboxamide (14)
6. Ramkumar, K.; Neamati, N. Core Evid. 2010, 4, 131.
To a solution of 18b (100 mg, 0.164 mmol) in 30 mL of dry THF
was added 0.1 mL of hydrazine hydrate (85%) at 0 °C. The ice bath
was removed after 10 min, and the solution was stirred at 50 °C
overnight, the solid was filtered. The filtrate was concentrated un-
der vacuum to yield a solid that was crystallized and subsequently
recrystallized from methanol and ethyl acetate. The final product
was dried under vacuum to give 25 mg of 14 in an overall yield
of 64% for two steps; White solid; 1H NMR (300 MHz, DMSO-d6)
d 8.027 (s, 1H), 7.607 (d, J = 6.3, 2H), 7.206 (d, J = 6.3, 2H), 2.298
(s, 3H); 13C NMR (100 MHz, DMSO-d6) d 162.9, 156.3, 137.2,
136.4, 135.6, 134.2, 133.2, 130.0, 129.6, 121.7, 119.8, 21.0; ESI-
MS: 260 [M+1]+; HRESI-MS: m/z calcd for C12H14N5O2 ([M+H]+)
260.1069; found 260.1074.
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The title compound was prepared from 18a (80 mg,
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1H), 7.856 (dd, J = 4.8, 8.4, 1H), 7.170 (dd, J = 4.8, 8.4, 1H), 2.871
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Acknowledgments
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high-throughput screening. This work was supported by an NIH/
NIAID (R21 AI081610) grant, a research grant from the Campbell
foundation (N.N.), and a National Natural Science Foundation of
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