Design, synthesis and biological evaluation of caffeoyl benzanilides as dual inhibitors of HIV integrase and CCR5

Article abstract of DOI:10.1039/c6md00311g

Novel series of caffeoyl benzanilide compounds as dual inhibitors of HIV-1 CCR5/IN were designed and synthesized. The biological results indicated that the acetylated compounds with double bonds were reduced, especially compound 9a, which showed potential activity against HIV-1 CCR5 tropic viruses with an EC₅₀ value of 4.85 μM, as well as binding affinity with IN (K_D 2.4 μM). Molecular modeling studies also suggested the possible binding mode of 9a with CCR5 and IN. These results indicated that 9a has the possibility of being a dual inhibitor of HIV-1.

Full text of DOI:10.1039/c6md00311g

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ARTICLE
Design, synthesis and biological evaluation of caffeoyl -
benzanilides as dual inhibitors of HIV integrase and CCR5†
Xuefeng Sun,a Ningning Fan,a Weisi Xu,c Yixing Sun,a Xiexin,a Ying Guo,a Liying Ma,c Junyi Liu,a,b *
and Xiaowei Wang,a *
Received 00th January 20xx,
Accepted 00th January 20xx
Novel series of caffeoyl-benzanilide compounds as dual inhibitors of HIV-1 CCR5/IN were designed and synthesized. The
biological results indicated that the acetylated compounds with double bond reduced, especially compound 9a, showed
potential activity against HIV-1 CCR5 tropic viruses with EC50 value (4.85µM) as well as binding affinities with IN (KD 2.4µM).
Molecular modeling studies also suggested the possible binding mode of 9a with CCR5 and IN respectively. These results
indicated that 9a has the possibility of being dual inhibitor of HIV-1.
DOI: 10.1039/x0xx00000x
www.rsc.org/
development of antiretroviral drugs.3 Thus, we focused our
attention on designing dual inhibitors of HIV-1 integrase and
Introduction
CCR5.
Human immunodeficiency virus-1 (HIV-1) is the causative
agent of the acquired immunodeficiency syndrome (AIDS).
Because of the variation and the persistence of viral, the
clinical management of AIDS is very complicated. Currently,
highly active antiretroviral therapy (HAART), constituted by
inhibitors of reverse transcriptase (RT) and protease (PR) or
integrase (IN) ones, successfully suppressed HIV-1 viral load to
an undetectable level.1 However, the efficiency of this therapy
is compromised by complicated dosing and intolerable
toxicities. Therefore, development of HIV-1 dual inhibitors,
which two scaffolds merged into one entity and act on
different sites of viral life cycle, would be an innovative
approach to reduce drug toxicity as well as improving patient
compliance.2
Caffeic acid phenethyl ester (CAPE) and flavonoid
analogues have been reported to have potential activity
against HIV-14,5 and considered safe with no mortality
observed in experimental mice. The SAR study indicated that
the dihydroxy styryl is a pharmacophore for anti-HIV activity.6
And pharmaco-kinetic studies of CAPE indicated that the ester
linkage is dramatically degraded by esterase and would also
limit its oral bioavailability. Therefore, according to the
principle of bioisostere, we decide to introduce the ketone
group instead of the unstable ester group.
According to the virus’ life cycle, the chemokine receptor
CCR5 was identified as an essential coreceptor for the
attachment of HIV-1 to the surface of CD4+ macrophages and
T-cells in entry level. Especially, CCR5 is an extracellular target
on the host cell surface.3 Therefore, inhibit the activity of CCR5
would be an effective strategy against HIV-1 virus.4 In addition
，the integrase, which integrated the proviral dsDNA into the
host cell chromosome, could be an attractive target for the
a.Department of Chemical Biology, School of Pharmaceutical Sciences, Peking
University, Beijing 100191, China
b.State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing
100191, China
c. State Key Laboratory for Infectious Disease Prevention and Control, Collaborative
Innovation Center for Diagnosis and Treatment of Infectious Diseases, National
Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control
and Prevention, Beijing, China
Figure1. Structures of target compounds
As we know, Kazmierski’s research group has exploited
7
†The authors declare no competing interests.
Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/x0xx00000x
and reported anilides as CCR5 antagonists comprehensively
.
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CONCISE ARTICLE
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Especially Smithkline Beecham Corp. disclosed various
benzanilides as antagonists of receptor. The docking study
indicated that the benzanilides would provide more
interactions with the binding pocket.8 Herein, we have the
caffeoyl moiety and benzanilides moiety merged into one
entity and propose to design novel caffeoyl - benzanilides
analogues as dual inhibitors of HIV-1 integrase and CCR5
(Figure 1).
In addition, to further explore the structure-activity
relationships (SAR), the unsaturated double bond was reduced
to single bond to enhance the flexibility and various
substituted groups on the amide side chain were designed. To
improve the permeability, the bisphenol acetylated
compounds were also studied in our report.9
Scheme 2. Synthesis of serious II target compounds. Reagents and conditions:
(
) 3,4-dihydroxy benzaldehyde, CH3SO3H, neat, rt, 5 min; ( ) acetic anhydride,
Results and Discussion
pyridine, rt, 15min; ( ) 10% Pd/C, H2, CH2Cl2, rt; ( ) R’COCl, CH2Cl2 , rt; ( ) HCl,
H2O, acetone, reflux.
The synthesis of serious I target compounds is outlined in
Schemes 1. (E)-4-(4-nitrophenyl)but-3-en-2-one (1), which was
prepared by condensation of 4-nitrobenzaldehyde with
acetone according to the reported procedure,10 was
Firstly, condensation reaction was carried out between
1
and 3,4-dihydroxy benzaldehyde with methane sulfonic acid as
coupling agent to furnish the important intermediate
hydrogenated using 10% Pd/C as the catalyst in CH2Cl2 to
11,12
afford the saturated compound
Subsequently, reaction of
2
in high yield
.
with various acid chlorides to
compound
was hydrogenated according to the methods mentioned above
to yield the saturated compound . Reaction of with the
6. Acetylation of 6 could afford compound 7 which
2
furnish compounds 3a-f, which condensed with 3,4-dihydroxy
benzaldehyde in the presence of pyrrolidine and acetic acid to
give the corresponding target compounds 4a-f. To improve
permeability, acetylation reaction was performed to obtain the
corresponding compounds 5a-f in high yields.
8
8
corresponding acid chlorides to afford the series II compounds
9a-f. Subsequently, deacetylation was performed to give the
bisphenyl target compounds 10a-f in high yields.
Figure 2 The RU value of target compounds at the concentration of 25µM.
Scheme 1 Synthesis of serious I target compounds. Reagents and conditions:(
10% Pd/C, H2, CH2Cl2, rt.; ( ) R’COCl, CH2Cl2 rt. ( ) 3,4-dihydroxy benzaldehyde,
pyrrolidine, acetic acid, THF, reflux; ( ) acetic anhydride, pyridine, rt, 5-15min.
)
Subsequently, the surface plasma resonance (SPR)-based
competitive assay12 was employed to evaluate the target
compounds’ binding affinity with IN by monitoring the alteration of
response unit (RU) with the addition of compounds at different
concentrations.13 Firstly, IN was covalently immobilized to the
sensor chip (CM5) surface by the standard primary amine coupling
method. Then compounds were screened at the concentration of
25µM with running buffer as negative control. All the sensor grams
Because of the low yield of preparing compounds 4a-f, it
would be difficult to get series II compounds (10a-f) by directly
reduction. Therefore, another synthetic pathway was designed
to obtain the corresponding saturated compounds 9a-f and
10a-f. (Scheme 2)
2 | J. Name., 2012, 00, 1-3
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CONCISE ARTICLE
were processed by automatic correction for non-specific bulk
refractive index effects. The primary results were illustrated in
Figure 2.
and cyclohexyl (6c 89.8µM) substituents would be beneficial
for binding.
Table 1 Equilibrium Dissociation Constants (KD) of some compounds
After the preliminary screen at one concentration, the seven
promising ones (4a, 4c, 4e, 4f, 9a, 10a, 10b) were selected to
perform in dose response assay with two models to determine the
equilibrium dissociation constants (KD) (Figure 3 and 4). The results
were summarized in Table 1.
O
RO
RO
O
N
R'
H
4a,4c,4e,4f
4c
compds 4a
4e
4f
9a
10a
10c
R
H
H
H
H
Ac
H
H
R’
KD (µM) 163.6 89.8 43.2
61.3
2.4
110.5 80.9
The ability of these new analogues to inhibit HIV-1 CCR5
was evaluated using a cell based assay, which carried out in
TZM-bl cells infected by HIV-1 Bal with Maraviroc as reference
compound. TZM-bl cells are used for determination of anti-HIV
activity due to easy and quick visualization of infected cells.
Known as R5 isolate, Bal was used in screening studies
.
Table 2 Inhibition of HIV-1 entry through CCR5 co-receptor as analyzed in TZM-
bl cells using R5 tropic virus isolates (Bal)
Compd
EC50
SIb
Compd
EC50
SIb
Figure 3 The curve fitting of six target compounds (4a, 4c, 4e, 4f, 10a and 10b)
with 1:1 binding model13
(µM)a
>50
(µM) a
4.85±0.57
4a
4b
4c
4d
4e
4f
< 1
< 1
< 3
< 1
7
9a
110
>50
9b
16.83±2.34 15
18.77±1.46 14
>50
9c
>50
9d
23.14±4.23
9
16.56±0.63
>50
9e
30.63±4.75 21
15.99±1.51 17
22.84±1.03 26
< 2
< 5
5
9f
5a
5b
5c
5d
5e
5f
>50
10a
10b
10c
10d
10e
10f
16.25±0.05
29.67±0.18
11.22±0.33
18.85±0.42
>50
42.46±0.31
6
5
18.34±0.82 10
2
36.30±1.15
>200
5
Figure 4 The curve fitting of compound 9a with steady state model14
12
< 1
>106
< 5
8
55.37±1.06
Maraviroc 0.001
As shown in Table 1, the saturated compounds could be
binding closely with IN than the corresponding ones with
double bond. Especially, the bisphenyl acetylated compound
9a exhibited excellently binding affinity with IN. The KD value
of 9a (2.4µM), which was determined from the steady state
model, was 46 folds and 68 folds lower than that of the
unacetylated 10a and 4a respectively. Considering the
different substitutions at amide moiety, thienyl (4f 61.3µM)
a Data represent the mean values of at least three experiments. b SI was
calculated based on the CC50 and EC50, respectively. Maraviroc was used as the
reference compounds
All synthesized compounds were tested for their
cytotoxicity before testing for cell based anti-HIV activity. The
anti-HIV activity and cytotoxicity of the compounds were
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illustrated in Table 2. Most of the acetylated compounds both of 9a and 10a. Therefore, compound 9a with more
exhibited better anti-HIV activity than that of the bisphenyl flexibility could fit well in the binding pocket of IN. This result
ones. One possible explanation for this result was the high coincidence quite well with our assumption.
permeability of acetylated compounds. Notably, compound 9a
once again exhibited significantly higher potency against R5
virus with low toxicity (EC50 = 4.85µM, SI = 110). These
biological results indicate that 9a would be the potent dual
inhibitor of HIV-1 CCR5/IN.
To explore the binding molds of our compounds with
CCR5 and IN, a representative inhibitor 9a was docked into IN
(PBD: 3OYA) and CCR5 (PBD: 4MBS) respectively using Glide of
Schrodinger suite 2015-1.
Figure 6. A: Binding mold of 9a (magenta) into the 3OYA of HIV-1 IN; B: Binding
Figure 5. Binding mold of 9a (magenta) into 4MBS of CCR5. Interactions are
mold of 10a (magenta) into the 3OYA of HIV-1 IN. Hydrogen bonds are shown as
yellow dashed line, green ball represent magnesium ion.
shown as yellow dashed line
Docking studies revealed that 9a could be flexibly docked
Conclusions
into the binding pocket of CCR5 and establish several ligand-
receptor interactions, which were characterized as: (i) Two
In conclusion, we have synthesized caffeoyl-benzanilide
hydrogen bonds would be an important factor for anti-HIV-1
analogues as dual inhibitors of cellular CCR5 and HIV-1 IN. The
activity. One is donated by the NH function of the amide on
biological evaluation data suggested that the acetylated
the side chain to the carbonyl oxygen of Glu283. Another one
compounds with double bond reduced could be active against
is the acetyl ester with Tyr 37 residue. (ii) Favorable π-π
R5 virus with low cytotoxicity. Interestingly, compound 9a
stacking interactions could be observed between the phenyl
inhibited the entry of CCR5 tropic viruses with EC50 values of
ring of 9a with the hydrophobic pocket defined by the side
4.85µM and also exhibited excellently binding affinity with IN
chains of Tyr108, Phe109, and Phe112. In addition, the
(KD 2.4µM). The biological and structural data provide more
aromatic ring of caffeoyl moiety interacted with TRP86 via π-π
information on caffeoyl- benzanilides as a valid scaffold for
stacking. (Figure 5)
dual inhibitors of HIV-1.
To further explore the binding mode of this analogue
Acknowledgements
with IN, compound 9a was docked into the catalytic core
domain (CCD) of IN (Figure 6A), and several interactions could
This research work was financially supported by the National
be observed. Firstly, the NH and the acyl of the amide formed
Natural Science Foundation of China (21172014, 20972011 and
two H-bonds with Gln186 and Tyr212 residues, respectively.
21042009) and Grants from the Ministry of Science and
Second, the carbonyl group of caffeoyl moiety coordinated
Technology of China (2009ZX09301-010).
with two Mg2+ metal ions (Figure 6A). Meanwhile, comparing
the docking results of compound 10a, we could find the
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Graphical Abstract
Novel serious of caffeoyl-benzanilides have been synthesized and evaluated
as dual inhibitors of HIV-1 CCR5/IN. Compound 9a exhibited the possibility of
being dual inhibitor of HIV-1.
9a EC50 = 4.8 µM
KD = 2.4 µM