Discovery of a synthetic dual inhibitor of HIV and HCV infection based on a tetrabutoxy-calix[4]arene scaffold

Article abstract of DOI:10.1016/j.bmcl.2010.02.043

A potential anti-HIV and HCV drug candidate is highly desirable as coinfection has become a worldwide public health challenge. A potent compound based on a tetrabutoxy-calix[4]arene scaffold that possesses dual inhibition for both HIV and HCV is described. Structural activity relationship studies demonstrate the effects of lower-rim alkylation in maintaining cone conformation and upper-rim interacting head groups on the calix[4]arene play key roles for its potent dual antiviral activities.

Full text of DOI:10.1016/j.bmcl.2010.02.043

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2137–2139
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a synthetic dual inhibitor of HIV and HCV infection based on a
tetrabutoxy-calix[4]arene scaffold
Lun K. Tsou a, Ginger E. Dutschman b, Elizabeth A. Gullen b, Maria Telpoukhovskaia a,
a,
Yung-Chi Cheng b, , Andrew D. Hamilton
*
*
a Department of Chemistry, Yale University, New Haven, CT 06520, United States
b Department of Pharmacology, School of Medicine, Yale University, New Haven, CT 06520, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A potential anti-HIV and HCV drug candidate is highly desirable as coinfection has become a worldwide
public health challenge. A potent compound based on a tetrabutoxy-calix[4]arene scaffold that possesses
dual inhibition for both HIV and HCV is described. Structural activity relationship studies demonstrate
the effects of lower-rim alkylation in maintaining cone conformation and upper-rim interacting head
groups on the calix[4]arene play key roles for its potent dual antiviral activities.
Received 8 January 2010
Accepted 10 February 2010
Available online 13 February 2010
Keywords:
Protein surface recognition
Calixarenes
Ó 2010 Elsevier Ltd. All rights reserved.
HIV–HCV coinfection inhibitors
Coinfection with both human immunodeficiency virus 1 (HIV)
and hepatitis C virus (HCV) has become a public health challenge,
afflicting more than 10 million people worldwide.1,2 Complicating
the challenge is the realization that with the advancement in
highly active antiretroviral therapy (HAART), liver disease has
emerged as a leading cause of death among HIV-infected patients.3
Treatment of coinfected patients with HAART has resulted in more
liver toxicity,4 creating a major health problem as there is currently
no specific anti-HCV drug available. Therefore, the discovery of a
dual compound as a potential drug candidate that can block HIV
and HCV infection would be desirable. Herein, we report the first
synthetic dual inhibitor of HIV and HCV infection in vitro based
on a new class of compounds derived from a tetrabutoxy-calix[4]-
arene scaffold.
The secretion of vascular endothelial growth factor (VEGF) is
elevated in HIV infected T-cells and is a major factor in the devel-
opment of Kaposi’s sarcoma.5 In addition, platelet derived growth
factor receptor (PDGFR) is expressed by uninfected T-cell lines, in
which secretion of PDGF is also observed.6 Although the impact
of these processes is known for the development of Kaposi’s sar-
coma, the effect of these growth factors and receptors on HIV rep-
lication itself is still under investigation. The discovery of
calix[4]arene derivatives that block VEGF and PDGF with their
respective receptors7–10 provided an opportunity to investigate
the effect of this compound series on the replication of HIV. More-
over, a number of studies have shown that certain calixarene deriv-
atives have interesting activities against viruses, enveloped viruses,
bacteria, fungi, and cancerous cells.11 In these reports,12,13 the mac-
rocyclic scaffolds are based on pyrogallol calixarenes, calixarenes
and resorcarenes of different ring sizes (4, 6, and 8 units) without
functionality at the lower rim and with sulfite, phosphite or simple
carboxylic acid groups on the top rim. These studies suggested that
the antiviral activity was derived from the compounds binding to
the viral envelope and inhibiting its interaction with cells.
In this study, we utilized the series of calix[4]arene derivatives
that were previously shown to inhibit the binding of VEGF, PDGF to
their respective receptors.9 The three initial compounds tested for
antiviral activity and cytotoxicity 1–3 contained a lower ring n-bu-
tyl group and an upper ring carboxamide-linked isophthalic group
with a benzyl ester, free carboxylic acid and cyclohexylamide arm,
respectively, on one of the carboxylic acids. Compound 2 showed
the most promising anti-HIV activity (Table 1) with an EC50 value
of 0.3 lM and an ID50 value of 90 lM in an MT-2 cellular assay.
Compounds 1 and 3 showed little to no antiviral activity. Notably,
these results showed no correlation between antiviral activity and
the ability of the compounds to inhibit the interaction of VEGF and
PDGF with their receptors. Compound 2 was equally potent against
three different laboratory HIV strains: HIV-IIIB, NL-43, and
LaiM184 V. Moreover, antiviral activity was determined in differ-
ent cell lines including MT-2, which are human T-cells with
HTLV-1 virus, CEMx174, which is a human T cell line, and TZM-
bl, which is a HeLa cell line with a firefly luciferase, that is, con-
trolled by an HIV promoter. These cell lines are different in their
acquired viruses, extracellular proteins, and cytoplasm contents.
* Corresponding authors. Tel./fax: +1 203 214 1089.
E-mail addresses: ktsou@rockefeller.edu (L.K. Tsou), yccheng@yale.edu (Y.-C.
Cheng), andrew.hamilton@yale.edu (A.D. Hamilton).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.043

2138
L. K. Tsou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2137–2139
Table 1
acid di-tertbutyl ester and monobenzyl mono-tertbutyl 5-amin-
oisophthalate, respectively. The diacid functionalities were then
prepared by either benzyl ester deprotection with H2, Pd–C or tert-
butyl ester removal with TFA. The role of alkylation at the lower
rim was also studied through compounds 2, 9 and 11, allowing a
comparison of benzyl versus n-butyl group at the lower ring
hydroxyl.
The results (Table 2) display some interesting dependence of
antiviral activity on the shape and structure of the calix[4]arene.
Introduction of n-butyl groups at the lower rim has the effect of
locking the calix[4]arene scaffold into a cone conformation as the
bulky substitutions are unable to invert through the ring. Benzylat-
ed compound 11 also exists in a well-defined cone conformation as
indicated by the pair of doublets from the bridging methylene pro-
tons in the 1H NMR spectrum.14 Notably, compound 11 had an EC50
Correlation studies of anti-HIV activities to growth factor inhibition studies
a
a
Compds
HIV Inhibition
Cytotoxicity
VEGF IC50
M)
PDGF IC50
(lM)
a
a
IC50
(lM)
IC50
(l
M)
(
l
1
2
3
>50
0.36 ( 0.12)
>50
>50
>50
>50
0.48 ( 0.31)
4.96 ( 0.34)
>10
0.19 ( 0.06)
2.62 ( 0.5)
0.29 ( 0.08)
a
50 lM is the cutoff for the assay. Standard deviation is given in the parentheses.
However, antiviral activity was retained in all these cell lines,
which suggests that the anti-HIV activity of compound 2 is inde-
pendent of cell type. Compound 2 was further investigated against
other viruses and shown to have no effect on the replication of her-
pes simplex virus-1 (HSV-1), HSV-2, and hepatitis B virus (HBV).
However, an EC50 of 1.8
observed.
lM against hepatitis C virus (HCV) was
value of 0.3
l
M for anti-HIV and 1.4
l
M for anti-HCV activities,
M sug-
similar in potency to 2. However, the ID50 value was 18
l
These observations propelled us to prepare a small library of
compounds (Fig. 1) and screen for their antiviral activity against
HIV and HCV (Table 2). In particular, we investigated the effect of
a range of upper and lower rim modifications on the activity of
core scaffold 2. Calix[4]arenes with different diacid functionalities,
such as glutamate, aspartate and isophthalate were synthesized by
reacting the acyl chloride form of tetraacid derivative of tetrabut-
gesting toxicity at a significant lower concentration as it showed
toxicity at a lower concentration than that of compound 2. Tetra-
hydroxy calix[4]arene derivative 9, which contains an identical
upper periphery to that of 2 exhibited a drop in the EC50 value from
0.3 to 6.7 lM for HIV and from 1.8 to >50 lM for HCV. Computa-
tional studies suggest that a calix[4]arene with four hydroxyl
groups at the lower rim can stabilize the scaffold into a cone
oxy calix[4]arene with L-glutamic acid di-tertbutyl ester, L-aspartic
Figure 1. Synthetic routes for: (A) upper rim modifications, mono-t-butyl-5-aminoisophthalate. (B) Lower rim n-butyl modifications on compounds 1, 2 and 4–7. (C) Lower
rim benzylated compound 10 and tetrahydroxy calix[4]arene 8. D. Compounds 9 and 11.

L. K. Tsou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2137–2139
2139
Table 2
HIV and HCV antiviral activities and cytotoxicity assay results for compounds 1–11
a
a
a
a
Compds
HIV inhibition IC50
(
lM)
Cytotoxicity IC50
(
lM)
HCV inhibition IC50
(l
M)
CEM IC50 (lM)
1
2
3
4
5
6
7
8
9
>50
0.36 ( 0.12)
>50
2.9 ( 1.4)
3.0 ( 2.8)
2.4 ( 1.4)
<16
>50
>50
>50
13.0 ( 1.4)
>50
>50
16
>50
>50
25.6
1.8 ( 0.3)
4.1
12.5
50
>50
12.5
82
100
<12.5
>50
ND
30.1
1.5 ( 0.3)
4.1 ( 1.8)
5.7
>50
>50
>50
6.7 ( 2.9)
37.1 ( 6.1)
0.3
10
11
>50
18.0
>50
1.4
50
0.46
a
50 lM is the cutoff for assays. Standard deviation is given in parentheses.
conformation through intramolecular hydrogen bonding. However,
the cone conformation is in equilibrium with the inverted cone
conformation through a partial cone intermediate.14 Preorganiza-
tion of the scaffold into a cone conformation for projection of the
recognition groups appears to be important for both anti-HIV and
anti-HCV activities.
cytotoxicity. The molecular targets and mechanisms for anti-HIV
and anti-HCV activities with these calix[4]arene compounds are
under investigation and will provide valuable insight for future
attempts to improve potency.
Acknowledgment
While alkylation at the bottom rim was essential for activity,
the importance of projected diacid groups on the top rim was also
clear. Among the seven compounds with a butyl chain at the bot-
tom rim, compound 2 exhibited the most potent anti-HIV activity.
When the isophthalic derivative was replaced with aspartic acid in
compound 5, anti-HIV activity decreases about 10-fold but anti-
HCV activity slightly improved, while cytotoxicity remained the
same. This suggested that aromatic substitutions are superior to
aliphatic ones at the upper rim for HIV inhibition, while anti-
HCV activity appears not to be as sensitive to this change. Com-
pounds 1 and 3 contained benzyl ester and cyclohexylamide deriv-
atives of the isophthalate linkers, respectively, suggesting that
substituting both acid groups are required for potent antiviral
activity. Overall, these observations suggest that four charges on
the top rim are important for anti-HIV and anti-HCV activities.
The lack of antiviral activity in positively charged 7, confirms the
importance of the negative charges on the projected periphery of
the compounds.
In conclusion, the results demonstrate remarkable anti-HIV and
anti-HCV activities for a series of compounds based on the tetrabu-
tyl-calix[4]arene scaffold. We have shown that maintaining the
cone conformation of the scaffold is important for antiviral activity.
In addition, aromatic isophthalate spacers at the upper rim are
essential for anti-HIV activities and the diacid groups are also nec-
essary for the observed anti-HCV effects. Furthermore, we have
identified a potent compound that possesses dual inhibition for
both HIV and HCV in vitro. Moreover, it retains potency against dif-
ferent HIV strains in different cell lines while maintaining low
We thank the National Institutes of Health (GM 35208 to A.D.H)
and (AI 38204 to Y.-C.C.) for financial support of this work.
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