Investigation of the alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors as potential cAMP phosphodiesterase-4B2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.12.015

The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number of ADAM analog

Full text of DOI:10.1016/j.bmcl.2007.12.015

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 1530–1533
Investigation of the alkenyldiarylmethane non-nucleoside
reverse transcriptase inhibitors as potential
cAMP phosphodiesterase-4B2 inhibitors
Matthew D. Cullen,a York-Fong Cheung,b Miles D. Houslay,b Tracy L. Hartman,c
Karen M. Watson,c Robert W. Buckheit, Jr.,c Christophe Pannecouque,d
Erik De Clercqd and Mark Cushmana,*
aDepartment of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences,
and the Purdue Cancer Center, Purdue University, West Lafayette, IN 47907, USA
bMolecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical Life Science,
University of Glasgow, Glasgow G12 8QQ, Scotland, UK
cImQuest Biosciences, Inc., 7340 Executive Way, Suite R, Frederick, MD 21704, USA
dRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 25 October 2007; revised 2 December 2007; accepted 6 December 2007
Available online 14 December 2007
Abstract—The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase
inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number
of ADAM analogues have been identified that protect HIV-infected cells from the cytopathic effects of the virus by an unknown,
HIV-1 RT-independent mechanism. Since the phosphodiesterase 4 family is required for HIV infection, the effect of various
ADAMs on the activity of PDE4B2 was investigated in an effort to determine if the ADAMs could possibly be targeting phospho-
diesterases. Six compounds representative of the ADAM class were tested for inhibition of cAMP hydrolysis by PDE4B2 enzymatic
activity. Four ADAMs were found to be weak inhibitors of PDE4B2 and two of them were inactive. The experimental results are
consistent with an antiviral mechanism that does not include inhibition of PDE4 isoforms.
Ó 2007 Elsevier Ltd. All rights reserved.
Acquired immune deficiency syndrome (AIDS) is esti-
mated to have claimed more than 25 million lives since
it was first described in 1981, making it one of the most
deadly epidemics in history.1 Increasing appreciation of
the complex biology involved with human immunodefi-
ciency virus (HIV) infection has led to the successful
development of antiviral agents that are used clinically
to combat the progression of AIDS. However, a cure
for AIDS does not appear to be on the horizon, and
HIV infection continues to spread on a pandemic scale.1
It is quite clear that finding a solution to the problem of
HIV infection will be one of this century’s greatest chal-
lenges in medical science.
Until a cure is discovered, clinicians will have to rely on
the various therapeutic agents that have been developed
to combat HIV infection and replication. Unfortu-
nately, the low polymerase fidelity of HIV reverse trans-
criptase allows the virus to rapidly mutate and develop
resistance to the existing spectrum of anti-HIV
agents.2–4 In fact, it has been reported that when antivi-
ral-naive patients begin highly active antiretroviral ther-
apy (HAART), it is possible to detect drug-resistant
strains of HIV in the patients’ circulation as early as
two months after initial treatment.5–7 HIV’s rapid muta-
bility has recently led to the emergence of multi-drug-
resistant viral strains, and thus the latest challenge has
been to develop antiviral agents that are active against
both the wild type form of the virus as well as the most
common drug-resistant strains.
Keywords: PDE4B2; Phosphodiesterase; HIV-1; Non-nucleoside reve-
rse transcriptase inhibitor; NNRTI; Alkenyldiarylmethane; ADAM.
The alkenyldiarylmethane (ADAM) non-nucleoside re-
verse transcriptase inhibitors (NNRTIs) inhibit HIV-1
reverse transcriptase (RT) by an allosteric mecha-
*
Corresponding author. Tel.: +1 765 494 1465; fax: +1 765 494
6790; e-mail: cushman@pharmacy.purdue.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.015

M. D. Cullen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1530–1533
1531
nism.8–14 Early investigations revealed that a number of
the leading compounds, such as ADAM 2, retained anti-
viral activity against several common reverse transcrip-
tase mutants (for example Y188C and K103N)11 and
development of the ADAMs as potential AIDS thera-
peutics has therefore been pursued. During these stud-
ies, several ADAM analogues were identified that do
not inhibit the enzymatic activity of HIV-1 RT
in vitro, but do protect HIV-1-infected cells from the
cytopathic effect of the virus at micromolar and sub-
micromolar concentrations. Examples include ADAMs
3 and 4 (Table 1). Inhibition of HIV-1 RT is the
ADAMs’ usual mechanism of action, and the analogues
that exhibit RT-independent antiviral activity must exert
their antiviral effects by an alternative mechanism. Ef-
forts have therefore been made to elucidate this un-
known mechanism. A variety of alkenyldiarylmethanes
that are structurally related to those with anti-HIV
activity have been developed at Celgene Corp. as inhib-
itors of tubulin polymerization, inflammation, and
phosphodiesterase 4 enzymatic activity.15 Structural
similarities between the ADAM NNRTIs and Celgene’s
inhibitors suggested that some of the anti-HIV ADAMs
may exhibit additional pharmacological properties be-
sides inhibition of RT. This hypothesis led directly to
consideration of inhibition of phosphodiesterase 4 as a
potential antiviral mechanism for ADAM analogues
that exhibit RT-independent anti-HIV activity.
chemistry depicted in Route 2 was utilized for the syn-
theses of ADAMs 522 and 6.23 Sonogashira coupling
of aryl iodide 11 and terminal alkyne 12, followed by
hydrostannation, afforded stannane intermediate 13.
The stannane and aryl iodide 14 were coupled via the
Stille reaction to obtain the desired analogues 15.
CO2CH3 CO2CH3
H3CO
X
OCH3
X
R
X = Cl, R = CH2CH3
X = Br, R = CO2CH3
X = Br, R = Ph
1
2
3
4
X = Cl, R = SCH3
CO2CH3 CH3
CH3
CH3
H3CO
H3C
O
O
N
O
O
O
O
N
N
CH3
CH3
CH3
CO2CH3
CO2CH3
5
6
Studies have shown that infection of a T4 cell by HIV-1
requires the cell to be ‘activated’, and this immunologi-
cal response is highly regulated by intracellular levels of
cAMP.16–18 The phosphodiesterase family of hydrolases
is one group of enzymes that is responsible for regulat-
ing cellular cAMP levels.19 Expression of the phosphodi-
esterase 4 (PDE4) family is absolutely required for HIV
infection to occur, suggesting that inhibition of PDE4
isoforms by a small molecule is a potential therapeutic
strategy for the treatment of AIDS.16 Indeed, inhibitors
of PDE4 isoforms are capable of attenuating the viru-
lence of HIV and it has long been suggested that
PDE4 inhibitors be included in the HAART of AIDS.20
In light of this information, we decided to investigate the
inhibitory activities of various ADAMs on PDE4B2, a
member of the PDE4 family that has been implicated
as a key target for anti-inflammatory agents. The aim
of the presently reported investigation was to establish
if the ADAMs’ RT-independent antiviral mechanism in-
volves the inhibition of the PDE4 family. This commu-
nication details the PDE4B2 inhibitory and antiviral
activities of ADAMs 1–6.
The in vitro PDE4B2 inhibitory data24 for ADAMs 1–6
are presented in Table 1, along with the antiviral
data9,12,25–27 associated with the compounds. Nevirapine
is included for antiviral comparisons. Inhibition of
PDE4B2 enzymatic activity was observed for four of the
six compounds under investigation, suggesting that inhi-
bition of PDE4 isoforms by the ADAMs may be a general
phenomenon, given the strong similarities of PDE4 iso-
forms in sequence homology and structure.28 However,
despite the structural similarities with Celgene’s potent
PDE4 inhibitors, ADAMs 1–6 display either weak
PDE4B2 inhibitory activity or no activity, with the most
potent PDE4 inhibitor exhibiting 72% inhibition at a con-
centration of 100 lM.29–35 One of the main goals of the
present study was to determine if inhibition of PDE4
activity was integral to the RT-independent antiviral
mechanism of ADAMs such as 3 and 4. Little to no inhi-
bition of PDE4B2 was observed for compounds 3 and 4 at
a concentration 10- to 20-fold higher than their respective
cytoprotective efficacies (EC50 values). These data are
consistent with an RT-independent antiviral mechanism
that does not involve the inhibition of PDE4 isoforms.
Additionally, although other analogues in this study (2,
5, and 6) displayed significant inhibition of PDE4B2 at
a 100 lM concentration, PDE4 inhibition also does not
contribute to their antiviral efficacies, since inhibition of
cAMP hydrolysis would be negligible at the minimal con-
centrations required for ADAMs 2, 5, and 6 to protect
cells from the cytopathic effect of HIV-1. It is clear that
an alternative viral or cellular entity is being targeted
when the ADAMs display an RT-independent antiviral
mechanism.
The syntheses of ADAMs 1–6 have been previously
published, and thus a brief, general depiction of their
construction is outlined in Scheme 1. ADAMs 1–
48,11,21 were synthesized according to general synthetic
Route 1, in which a functionalized salicylic acid 7 was
condensed with formaldehyde to afford a diarylmethane
intermediate, which was subsequently alkylated and oxi-
dized to obtain benzophenone 8. Coupling of 8 with a
suitably functionalized aldehyde 9 under standard
McMurry conditions yielded the desired ADAM ana-
logues 10. The palladium-catalyzed cross-coupling

1532
M. D. Cullen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1530–1533
Table 1. Antiviral and PDE4B2 inhibitory activities of ADAMs 1–6
% PDE4 inhibitiond
a
b
c
Compound
IC50 (lM)
EC50 (lM)
CC50 (lM)
CEM-SS
1RF
1IIIB
2ROD
MT-4
1
NTe
0.30
16
NTe
0.3
NTe
NAg
21
NTe
NAg
NAg
NAg
NTe
>29
13
NTe
91
NIf
40
2
0.001
13
5.3
3
>100
>100
0.02
2.6
>200
>20
5.1
>198
NTe
17
NIf
20
4
NTe
0.09
0.22
0.053
NTe
5
0.03
0.62
0.0015
NTe
72
6
0.5
0.08436
NTe
31
33
15
NTe
71
Nevirapine
Rolipram
NTe
NTe
NTe
100g
a Inhibitory activity versus HIV-1 RT with poly(rC).oligo(dG) as the template primer.
b EC50 is the concentration required to inhibit 50% of the cytopathic effect of HIV-1RF in CEM-SS cells, HIV-1IIIB in MT-4 cells, or HIV-2ROD in
MT-4 cells.
c CC50 is the cytotoxic concentration required to induce cell death for 50% of the mock-infected CEM-SS or MT-4 cells.
d The percent inhibition of PDE4B2 enzymatic activity observed when the compound was tested at a concentration of 100 lM.
e Not tested.
f No inhibition observed at 100 lM.
g The IC50 of rolipram is 105 8 nM under the assay conditions used in the present study.
CO2CH3 CO2CH3
General Route 1
H3CO
X
OCH3
CO2CH3 CO2CH3
OCH3
O
OCH3
H3CO
X
H
X
d
X
CO2CH3
a-c
+
X
O
R
7
9
8
R
10
X = Cl or Br
Ar1
General Route 2
Ar1
Ar2
Ar1
SnBu3
g
e,f
+
I
H3CO2C
Ar2
11
CO2CH3
13
CO2CH3
12
I
14
15
CH3
CO2CH3
OCH3
O
Ar1 and Ar2 =
O
or
N
CH3
CH3
Scheme 1. Reagents and conditions: (a) formaldehyde, methanol, 0 °C; (b) K2CO3, Me2SO4, acetone, reflux; (c) CrO3, methanol; (d) i—Zn dust,
TiCl4Æ2 THF, THF, reflux; ii—8, 9, THF, reflux; (e) 5 mol% PdCl2(PPh3)2, 10 mol% CuI, Et3N, THF; (f) 2 mol% Pd(PPh3)4, Bu3SnH, THF, 0 °C; (g)
10 mol% Pd(tBu3P)2, CsF, toluene, reflux.
In summary, the PDE4B2 inhibitory activities of select
ADAMs were determined. The weak to negligible inhi-
bition of PDE4B2 observed for ADAMs 3 and 4 demon-
strates that PDE4 isoforms are not integral to these
ADAMs’ RT-independent antiviral mechanism.
Facilities Improvement Program Grant, No. C06-
14499, also from the National Institutes of Health.
Work In MDH’s laboratory was supported by the Med-
ical Research Council (UK) Grants G8604010 and
G0400053; European Union Grant 037189 and Fonda-
tion Leducq 06 CVD 02.
Acknowledgments
References and notes
This investigation was made possible by funding from
the National Institutes of Health, DHHS through Grant
RO1-AI-43637 and research was conducted in a facility
constructed with the financial support of a Research
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