Crystallographic study of a novel subnanomolar inhibitor provides insight on the binding interactions of alkenyldiarylmethanes with human immunodeficiency virus-1 reverse transcriptase

Article abstract of DOI:10.1021/jm901167t

Two crystal structures have been solved for separate complexes of alkenyldiarylmethane (ADAM) nonnucleoside reverse transcriptase inhibitors (NNRTI) 3 and 4 with HIV-1 reverse transcriptase (RT). The structures reveal inhibitor binding is exclusively hydr

Full text of DOI:10.1021/jm901167t

J. Med. Chem. 2009, 52, 6467–6473 6467
DOI: 10.1021/jm901167t
Crystallographic Study of a Novel Subnanomolar Inhibitor Provides Insight on the Binding Interactions
of Alkenyldiarylmethanes with Human Immunodeficiency Virus-1 Reverse Transcriptase†
Matthew D. Cullen,#,‡ William C. Ho,#,§ Joseph D. Bauman,§ Kalyan Das,§ Eddy Arnold,§ Tracy L. Hartman,
Karen M. Watson, Robert W. Buckheit Jr., Christophe Pannecouque,^ Erik De Clercq,^ and Mark Cushman*,‡
‡Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and Purdue University
Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, §Department of Chemistry and Chemical Biology, Center for
Advanced Biotechnology and Medicine, Rutgers University, 679 Hoes Lane West, Piscataway, New Jersey 08854, ImQuest Biosciences, Inc.,
7340 Executive Way, Suite R, Frederick, Maryland 21704, and ^Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000
Leuven, Belgium. # Contributed equally to this research.
Received August 5, 2009
Two crystal structures have been solved for separate complexes of alkenyldiarylmethane (ADAM)
nonnucleoside reverse transcriptase inhibitors (NNRTI) 3 and 4 with HIV-1 reverse transcriptase (RT).
The structures reveal inhibitor binding is exclusively hydrophobic in nature and the shape of the
inhibitor-bound NNRTI binding pocket is unique among other reported inhibitor-RT crystal
structures. Primarily, ADAMs 3 and 4 protrude from a large gap in the back side of the binding pocket,
placing portions of the inhibitors unusually close to the polymerase active site and allowing 3 to form a
weak hydrogen bond with Lys223. The lack of additional stabilizing interactions, beyond the observed
hydrophobic surface contacts, between 4 and RT is quite perplexing given the extreme potency of the
compound (IC50 e 1 nM). ADAM 4 was designed to be hydrolytically stable in blood plasma, and an
investigation of its hydrolysis in rat plasma demonstrated it has a significantly prolonged half-life in
comparison to ADAM lead compounds 1 and 2.
Introduction
capable of grasping genomic material for enzymatic proces-
sing.3
The human immunodeficiency virus (HIV), the causative
agent of the acquired immune deficiency syndrome (AIDS), is
a retrovirus that relies on a myriad of viral proteins to help
infect CD4þ cells and enable its replication. One of these
proteins, reverse transcriptase (RT), facilitates viral replica-
tion by transcribing HIV’s single-stranded, RNA-based gen-
ome to a double-stranded DNA equivalent that is compatible
with the host cell’s replication machinery. The genomic
transcription abilities of RT are made possible by both the
DNA polymerase and RNase H activities, which are abso-
lutely required for the virulence and replication of HIV.1 As
such, RT is an attractive target for antiviral therapy, with an
array of drugs currently in use for the treatment of HIV
infection through inhibition of RT enzymatic function.
Structurally, RT is a heterodimeric protein composed of 66
and 51 kDa subunits (known as p66 and p51, respectively),
each of which consists of connection, thumb, palm, and finger
domains. The four domains of the p66 and p51 subunits are
identical in amino acid sequence from their N-termini for 440
residues.2 However, the heavier p66 subunit bears an addi-
tional 120 residues at its C-terminus, which form the RNase H
domain. Despite their nearly identical sequences, the p66 and
p51 subunits have surprisingly different ternary structures,
which result in RT assuming the shape of a hand that is
As previously mentioned, several antiretroviral drugs have
been developed to inhibit RT, and they are classified by their
mechanism of action.4-7 The nucleoside/nucleotide reverse
transcriptase inhibitors (NRTIs/NtRTIs)a halt RT’s polymer-
ase activity by acting as dNTP mimics that, when incorpo-
rated into viral DNA, terminate synthesis of the viral DNA
chain.8,9 The other category of HIV-1 RT inhibitors, non-
nucleoside reverse transcriptase inhibitors (NNRTIs), bind to
˚
a hydrophobic, allosteric site located ∼10 A from the poly-
merase active site. When NNRTIs, such as nevirapine10 and
efavirenz,11 bind to this hydrophobic site, the native arrange-
ment of amino acid side chains is forced to change such that
several of the residues point toward or intrude upon the
polymerase active site.3,12 In fact, crystallographic studies
on various HIV-1 RT-NNRTI complexes have shown that
binding of the inhibitors causes significant conformational
changes within RT, primarily through the displacement of
sheets β12-14 in the DNA primer grip, resulting in the
enzyme binding DNA in a nonproductive manner.3,12 Be-
cause of their usually tolerable toxicities, NNRTIs have
enjoyed increased use in highly active antiretroviral therapy
(HAART) in recent years. However, the clinical efficacy of
these inhibitors has been attenuated by the emergence of drug-
resistant HIV-1 strains that bear mutant forms of RT.6,13
†PDB code for 3 with HIV-1 RT, 3IS9; PDB code for 4 with HIV-1
RT, 3IRX.
*To whom correspondence should be addressed. E-mail: cushman@
pharmacy.purdue.edu. Phone: 765-494-1465. Fax: 765-494-6790.
a Abbreviations: ADAM, alkenyldiarylmethane; HAART, highly
active antiretroviral therapy; NNRTI, nonnucleoside reverse transcrip-
tase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; NtRTI,
nucleotide reverse transcriptase inhibitor; TI, therapeutic index.
r
2009 American Chemical Society
Published on Web 09/23/2009
pubs.acs.org/jmc

6468 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Cullen et al.
Table 1. Antiviral Activity and Hydrolysis Half-Lives of ADAMs 1-4
EC50 (μM)b
CC50 (μM)c
CEM-SS
13
5.1
17
3.0
compd
IC50 (μM)a
HIV-1RF
HIV-1IIIB
HIV-2ROD
MT-4
half-life t1/2 ( SD (min)d
1g
2h
3i
4
0.30
0.02
0.1
0.3
NAe
NAe
NAe
NAe
91
17
6.2 ( 0.4
1.3 ( 0.09
12.4 ( 0.7f
238 ( 8.5
0.03
0.7
0.09
0.8
0.38
<0.001
>100
0.07
0.18
3.9
a Inhibitory activity versus HIV-1 RT with poly(rC) oligo(dG) as the template/primer. b EC50 is the 50% inhibitory concentration for inhibition of the
3
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 Metabolic half-life of the compound when it was incubated with rat plasma;
determined from a minimum of two replicates. e Not active. f Previously reported in ref 18. g Reference 17. h Reference 19. i Reference 18.
There is great need for the development of NNRTIs that are
efficacious against both wild-type and drug-resistant forms of
HIV-1 RT.
For several years, our group has been developing a series of
alkenyldiarylmethane-based NNRTIs that exhibit antiviral
activity against a number of the common drug-resistant strains
of HIV-1 that bear RT mutations.14-20 Previous hypothetical
binding models developed for potent inhibitors 1 and 2 showed
the alkenyldiarylmethanes (ADAMs) oriented in accordance
which was elaborated with iodide 920 through the Stille
reaction to afford ADAM 4.
Evaluation of RT Inhibition, Antiviral Activity, and Hydro-
lytic Stability. As is standard in our investigations, ADAM 4
was evaluated in a functional enzyme assay for inhibition of
HIV-1 RT. Additionally, the cytotoxicity and ability of
ADAM 4 to protect cells from the cytopathic effects of
HIV-1RF, HIV-1IIIB, or HIV-2ROD were evaluated. The meta-
bolic half-life of 4 in rat plasma was also determined in a
metabolic stability assay. The results of the RT inhibitory,
antiviral, metabolic, and cytotoxicity analyses for ADAM 4
are presented in Table 1. Associated data for compounds 1-3
are included in Table 1 for comparison.17-19
Concerning inhibition of RT enzymatic activity, ADAM 4
is the most potent analogue among the four compounds under
discussion, exhibiting an impressive IC50 of less than or equal
to 1 nM. The detection limits of our functional enzyme assay
precluded a more exact IC50 determination for 4. Despite
ADAM 4’s remarkable potency in the in vitro assay, the
manifestation of this analogue’s cytoprotective efficacy in
whole cells is surprisingly lower than expected and compar-
able to that of ADAM 1 (IC50 = 0.3 μM). The discrepancy
between ADAM 4’s enzyme inhibitory potency and its cyto-
protective activity could be the result of many factors, includ-
ing the fact that the enzymatic assay, in contrast to the cellular
.
with the “butterfly” model, as described by Schafer, which
reflects the binding orientation of NNRTIs like nevirapine,
TIBO, and R-APA.16,19,21 However, these models are not
sufficiently general for the ADAM class of inhibitors, nor do
they correlate with the RT SAR. It is well-known that the
NNRTI binding pocket is plastic in nature and will conform to
the structure of the specific inhibitor that is bound, thus
making molecular modeling studies problematic. Recent crys-
tallographic studies involving new structural classes of
NNRTIs have revealed compounds that are highly flexible
(large number of rotatable bonds) and similar in structure and
can each bind to RT in different orientations.22,23 Further-
more, potent inhibitors can even have more than one binding
orientation if the structure is flexible enough to allow for
“wiggling” and “jiggling” inside the binding pocket.22,23 Yet,
the malleable nature of the NNRTI binding pocket obviates
attempts to generate an accurate binding model via de novo
methods.
Given that current ADAM-RT interaction models lack
correlation with SAR data, our desire to identify key
protein-ligand interactions for this class of NNRTIs
necessitated the acquisition of ADAM-RT crystal struc-
tures. Herein we reveal the first X-ray crystal structures of
HIV-1 RT in complex with an ADAM (compounds 3 and
4). The present report also discloses the synthesis and
antiviral evaluation of the incredibly potent NNRTI 4
(IC50 e 1 nM). ADAM 4 was designed as part of an
investigative effort to replace the enzymatically labile
methyl esters in lead compound 1 with bioisosteres that
would confer hydrolytic stability in blood plasma while
maintaining the desired pharmacological activity.
system, uses the synthetic template/primer poly(rC) oligo-
3
(dG). Significant cytotoxicity is observed for each of the
analogues with ADAM 4 unfortunately presenting the nar-
rowest therapeutic index (TI = 32). Because the current goal
of our research is to improve on the metabolic stability of the
ADAMs throughisostericreplacement ofenzymatically labile
methylesters, ADAM4 was evaluatedinour ratblood plasma
stability assay, and the results were compared to previous
leads (ADAM 1). ADAM 4 was found to be at least 40-fold
more stable (t1/2 = 238 min) than 1, indicating the fused
oxazolidinone and oxadiazole isosteres rendered 4 less prone
to enzymatic degradation.
As our initial interest in studying the ADAM scaffold
was spurred by the ability of initial leads to retain antiviral
activity against several common NNRTI-resistant HIV-1
strains, the antiviral activity of 4 was evaluated against
NNRTI-resistant HIV strains. Specifically, CEM-SS cells
infected with HIV-1 strain NL4-3 which bore either the
K103N or Y181C RT mutant were treated with ADAM
4 to determine the residual cytoprotective activity of the
inhibitor. The results of the experiment are presented in
Table 2, with data for ADAM 1 and nevirapine included
for comparison. Unlike that of ADAM 1, the cytoprotec-
tive activity of 4 was completely abolished (EC50 >100
μM) by two of the most common NNRTI resistance
mutations, K103N and Y181C. A priori one would have
expected ADAM 4 to exhibit moderate antiviral activity,
Results and Discussion
Chemistry. Several routes for the synthesis of ADAM
analogues have been reported.17,24,25 The synthesis of
ADAM 3 has been previously described.26 The synthesis of
compound 4 centers on a series of palladium-catalyzed cross-
coupling reactions recently utilized to prepare a set of
hydrolytically stable ADAM analogues.20 Briefly, the synth-
esis of ADAM 4 began with the coupling of known com-
pounds 5 and 6 under standard Sonogashira conditions
(Scheme 1). Cross-coupling product 7 was treated with
tributyltin hydride and Pd(PPh3)4 to obtain stannane 8,20

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6469
Scheme 1a
Table 2. Antiviral Activity of ADAMs 1 and 4 against NNRTI-
Resistant HIV-1 Strains
EC50 (μM)a
compd
WTb
K103Nc
Y181Cd
1f
4
0.1
0.7
NTe
0.42
>100
0.6
6.1
>100
0.8
nevirapine
a EC50 is the 50% inhibitory concentration for inhibition of the
cytopathic effect of HIV-1RF in CEM-SS cells. b Cytoprotective activity
against HIV-1RF bearing wild-type reverse transcriptase. c Cytoprotec-
tive activity against HIV-1 bearing reverse transcriptase containing
the K103N mutation. d Cytoprotective activity against HIV-1 bearing
reverse transcriptase containing the Y181C mutation. e Not tested.
f Data were taken from ref 16.
ADAM-RT Complex Structure and Analysis. The
ADAM-RT structures were determined at final resolutions
of 2.6 and 2.8 A for compounds 3 and 4, respectively, with
˚
associated diffraction and refinement data presented in
Table 3. The overall conformation of RT in the crystal
structures is typical of most NNRTI-RT complexes
(Figure 1) in which the p66 thumb region is hyperextended.
The side chains of Tyr181, Tyr188, and Trp229 are rotated or
shifted toward the polymerase active site, and the β12-14
˚
sheet is displaced by approximately 2 A relative to the
structure of apo-RT.
In general, both 3 and 4 occupy nearly the entire volume of
the NNRTI-binding pocket, making very few, if any, polar
contacts with the protein (Figure 2). The latter observation is
surprising but not unprecedented given the recent crystal
structure of the highly potent NNRTI, TMC278 (10), which
showed that drastic improvements in potency were achieved
by the addition of an increased number of hydrophobic
interactions.27 Although the NNRTI-binding pocket is
known to be predominantly hydrophobic in nature and
known nonnucleoside inhibitors have significant hydropho-
bic character, most NNRTI-RT complexes, including 10,
exhibit one or more hydrogen bonds between the inhibitor
and protein. Our past SAR studies indicated the ester
functionalities are a crucial component of the ADAM phar-
macophore and were suspected of participating in one or
more hydrogen bonds with RT.
In comparing the NNRTI-binding pockets containing 3
and 4 to those of other published NNRTI-RT crystal
structures, it was observed that the shape of the ADAM-
binding pocket resembles that of the bis(heteroaryl)-
piperazine (BHAP) series, to which delavirdine belongs.
For NNRTI binding, it is commonly accepted that inhibitors
bind to one of two NNRTI-binding pocket “shapes”, one of
which is represented by nevirapine (PDB code 1VRT) and
the other, delavirdine (PDB code 1KLM). The nevirapine-
binding pocket is characterized as small and encapsulating,
where no surface gaps are observed in the pocket. In the case
of delavirdine, the inhibitor is quite large and the NNRTI-
binding pocket expands to accommodate its volume. The
binding pocket acquires additional space by breaking the
hydrogen bond between the carbonyl of Pro236 and main
chain amide of Lys101, which creates a large gap in the
pocket near the surface of the protein. Additionally, the
β12-14 sheets are displaced further than in the nevirapine
pocket, which provides supplementary volume and results in
the opening of another gap toward the back of the pocket.
The aforementioned hole in the back of the binding pocket is
significantly larger in the two ADAM structures, relative to
a Reagents and conditions: (a) cat. PdCl2(PPh3)2, cat. CuI, Et3N,
THF; (b) cat. Pd(PPh3)4, Bu3SnH, THF, 0 ꢀC; (c) cat. Pd(PPh3)4, cat.
CuI, CsF, DMF, 60 ꢀC.
even in the presence of the two mutations, on the basis of
the point mutation data for ADAM 1. Such a drastic
change in activity suggests that ADAM 4 is incapable of
adapting conformationally to the structural changes in-
duced in the NNRTI binding pocket by the K103N and
Y181C mutations, whereas the slightly more flexible
ADAM 1 can adopt an alternative binding conformation
or orientation.

6470 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Cullen et al.
Table 3. Crystallographic Data Collection and Structural Refinement
Statistics
RT/ADAM 3
RT/ADAM 4
Crystallographic Data
space group
˚
a, b, c (A)
C2 C2
162.82, 73.73, 108.69 164.64, 75.39, 110.66
β (deg)
99.55
100.08
˚
resolution range (A)
total no. of reflections
no. of unique reflections 41 074
50-2.55
486 221
50-2.8
362 418
28 872
a,b
Rmerge
I/σa
0.086 (0.512)
18.3 (3.0)
98.7 (100.0)
0.091 (0.368)
10.1 (1.8)
87.2 (92.6)
completeness (%)a
Refinement
1 677
no. of reflections in
free set
1 414
R
Rwork
c
25.34
28.27
0.008
1.281
82
23.62
28.86
0.010
1.325
91
d
Rfree
˚
rmsd bond lengths (A)
rmsd angles (deg)
av B factor of protein
Figure 1. An overlay of the backbone tube structures of unliganded
apo-RT (1HMV, yellow), the RT/3 complex (red), and the RT/4
complex (green). The crystal structures obtained for the RT/3 and
RT/4 complexes show hyperextension of the thumb region and
displacement of the fingers, relative to the structure of unliganded
apo-RT. In areas of very close overlap, the red and green tubes
appear as either red or green.
2
˚
atoms (A )
av B factor of inhibitor 84
74
2
atoms (A )
˚
no. of protein atoms
no. of inhibitor atoms
no. of water atoms
7933
37
7941
36
126
0
a Numbers in parentheses denote statistics for last resolution shell.
P P
P P
b Rmerge
=
l|Ih,j - Ih|/
h,i, where Ih is the mean intensity of
h
symmetry-related reflections. chRwork
=
and Fc are the observed and calculated structure factor amplitudes,
||Fo| - |Fc||/ |Fo|, where Fo
P
P
P
P
respectively. d Rfree
=
||Fo| - |Fc||/ |Fo| for reflections in the test set.
the delavirdine structure, and allows both inhibitors to
protrude from the pocket, placing them unusually close to
the RT polymerase site (Figure 3). In fact, the 50 nitrile
˚
functionality on ADAM 3 is held only 6 A, with an un-
obstructed “line-of-site”, from the catalytically active resi-
due Asp186 in the polymerase active site! The previous
observation can only be made for the crystal structure
of aryloxypyridinone R165481 (11) (PDB code 2B5J), which
˚
shows the nitrile of the ligand lays only 4.9 A from aspartate
186.28 In comparison to the nevirapine and delavirdine struc-
tures, the shortest paths from each inhibitor to Asp186 are
˚
8.6 and 8.9 A in length, respectively, where the paths are
obstructed by Y181 and Y188.
A more detailed analysis of the structures revealed that
ADAM 4 creates a smaller pocket and binds slightly deeper
relative to 3. Yet in both crystals the alkenyl side chains of the
ADAMs display significant edge-to-face interactions with
the aromatic rings of Tyr181, Tyr188, and Trp229, in addi-
tion to general hydrophobic interactions with the side chains
of Leu100 and Leu234. Most notably, the vinyl hydrogen on
each ligand is nearly buried in the π cloud of Trp229’s indole
ring. The Trp229 residue is highly conserved among HIV
strains and is part of the DNA/RNA primer grip, thus
disrupting the alignment of this residue that can significantly
impair RT polymerase activity.29-31 The aryl rings situated
trans to the side chain in both ligands are buried deep in the
pocket and occupy a region normally blocked by residues
Phe227, Trp229, and Tyr188. With 3 and 4 bound so deeply
in the pocket, the side chain of Phe227 becomes pinned
against the peptide backbone, forcing a chain rotation of
the local residues (219-228). This chain displacement opens
a large hole at the back of the binding pocket and, in the case
Figure 2. (Top) Depiction of the NNRTI-binding pocket obtained
from the RT/ADAM 3 crystal structure. (Bottom) Depiction of the
NNRTI-binding pocket obtained from the RT/ADAM 4 crystal
structure. The view for each panel is from the “front side” of the
binding pocket, from the point of view of the putative solvent
channel through which NNRTIs are believed to enter. The stereo-
view is programmed for wall-eyed viewing.
of ADAM 3, brings the side chain of Lys223 in close
proximity to the “trans” aryl ring’s ester. The proximity of
Lys223 hints at a potential hydrogen bond, but the intera-
˚
tomic distance of 3.6 A from the side chain amine to the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6471
Figure 3. Overlay of the crystal structures of RT bound to ADAM 4 (red), nevirapine (blue), and delavirdine (green). The NNRTI binding
pocket is more compact when nevirapine is bound in contrast to a more open pocket when delavirdine or ADAM 4 are present; β12, β13, and
β14 are rearranged to make room for the larger inhibitors.
nearest ether-type oxygen of 3 is at the outer limits of
hydrogen-bonding distance. In comparison, electron density
for residues 218-223 in the RT/ADAM 4 crystal structure is
completely absent, suggesting a large degree of disorder in
this region of the protein. Such an observation precludes the
assignment of a hydrogen bond between 4 and Lys223 in the
crystal structure; however, such an association may theore-
tically arise under physiological conditions.
Waals contacts between the inhibitor and Tyr181 residue, it
is unsurprising that mutation of this residue significantly
impacts the antiviral activity of 4. However, the crystal
structure shows no specific interactions between 4 and
Lys103, suggesting conformational changes in the binding
pocket residues lead to attenuation of 4’s antiviral activity.
If one were to assume ADAM 1 binds to RT in a manner
similar to 3, then ADAM 1 would exhibit many of the same
binding interactions with RT as are observed for ADAM 4.
Yet, the point mutation data indicate that modulating the
shape of the NNRTI binding pocket via replacement of the
Tyr181 and Lys103 residues results in small to moderate
changes in the affinity of 1 for HIV-1 RT. This observation
can be rationalized in two ways: (1) ADAM 1 binds to RT in
an orientation vastly different from 3 and 4, making analyses
based on the two ADAM-RT crystal structures impossible;
(2) because 1 possesses more conformational flexibility
relative to 4, the former analogue is capable of adopting a
favorable, alternative binding conformation. In the absence
of additional crystallographic or accurate modeling data for
ADAM 1, validation of either scenario is problematic.
Conclusion. Described herein are the first crystal structures
of HIV-1 RT in complex with two ADAM NNRTIs. Of the
two inhibitors discussed, ADAM 4 has been identified as a
subnanomolar inhibitor of HIV-1 RT enzymatic activity in
vitro, and the synthesis of this compound has been outlined.
Interestingly, the crystal structures for the ADAM com-
plexes reveal that inhibitors 3 and 4, which differ in enzy-
matic inhibitory activity by at least 2 orders of magnitude,
exhibit nearly identical binding orientations and interac-
tions with the NNRTI-binding pocket. Moreover, the bind-
ing interactions observed in the protein-inhibitor comp-
lexes are exclusively hydrophobic in nature with additional
π-stacking attractions. The deep binding exhibited by 3
and 4 allows the inhibitors to protrude through the back of
the NNRTI binding pocket and into the polymerase active
site, making the ADAMs unique among other published
NNRTI-RT complexes. The former observation has led to
the hypothesis that it may be possible to tether an ADAM
to a traditional nucleoside/nucleotide inhibitor (e.g., AZT,
tenofovir), generating a dual inhibitor that may simultaneously
Further analysis of the crystal structures shows stacking
between the aromatic ring of Tyr181 and the terminal side
chain functionalities of the ligands in both structures. Ad-
ditionally, the methyl ester (ADAM 3) and oxazolone
(ADAM 4) moieties on the ADAM rings oriented trans to
the side chains show similar stacking interactions with the
aromatic side chain of Phe227. Given the observed stacking
interactions between the methyl ester of 3 and Phe227,
replacement of this functional group by the cyclic oxazolone
in 4 may have the benefit of rigidifying the atoms in the
proper planar orientation for π-stacking with Phe227. This
may be one reason for the increased potency of 4. It is
interesting to note that the IC50’s of ADAMs 2 and 4 differ
by over an order of magnitude with 4 being the more potent
of the two. The major difference between 2 and 4 is that 2
retains the methyl ester on the end of its alkenyl arm, while 4
has an oxadiazole in this place. If 2 and 4 bind to RT in a
similar fashion, the π-stacking with Tyr181 (which is also
observed in the cocrystal structure of 3) should be retained.
However, 4 has the benefit of a rigidified, planar heterocycle
for this stacking interaction while 2 retains the methyl ester.
The aromatic rings of 3 and 4 that are oriented cis to the
side chains are cradled by the hydrophobic residues Leu100,
Val106, Val179, and Leu234. The protein-ligand interac-
tions described for both structures are primarily hydropho-
bic in nature and help in explaining why the carboxylate
analogues, derived from hydrolysis of the ADAM esters, are
biologically inactive.
With regard to the point mutation data for ADAMs 1 and
4, the application of information gleaned from the two
ADAM-RT crystal structures leads to several interesting
observations and conclusions on the data. In the case of
ADAM 4, given the significant edge-to-face and van der

6472 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Cullen et al.
bind to the NNRTI and NtRTI and polymerase binding sites.
Such a compound would undoubtedly be an extremely potent
RT inhibitor and potential antiviral agent.
method by mixing 1 μL of protein-inhibitor complex and 1 μL
of mother liquor sealed over a 500 μL reservoir solution. The
best crystals appeared at 4 ꢀC using a mother liquor consisting of
50 mM imidazole, pH 6.4, 10% PEG 8000, 100 mM ammonium
sulfate, 10 mM spermine, and 30 mM magnesium sulfate.
Crystals were harvested and flash cooled in liquid nitrogen after
transferring to mother liquor supplemented with 25% ethylene
glycol. Data were collected at the Cornell High Energy Syn-
chrotron Source (CHESS) F1 beamline. The program
HKL200033 was used to index, scale, and merge reflection data.
The phase problem was solved by molecular replacement using
the program Molrep and the protein coordinates of the RT52A/
10 complex (PDB code 2ZD1) as the starting model.34 Upon
initial examination of the difference Fourier density, the pre-
sence of each respective inhibitor was clear. Refinement of
protein coordinates proceeded by iterative rounds of manual
fitting of electron density using the program COOT35 followed
by reciprocal space refinement using CNS.36 In the final stages
of model refinement, as the Rwork and Rfree converged, the
respective inhibitors were built into the electron density and
subjected to coordinate and B-factor minimization.
Experimental Section
1
All H NMR spectra were obtained at 300 MHz in CDCl3,
using the residual CHCl3 resonance as the internal standard for
chemical shifts. A Perkin-Elmer 1600 series FT-IR spectrometer
was used to record infrared spectra of all compounds. Preparative
TLC separations utilized Analtech Uniplates with glass-sup-
ported silica (20 ꢀ 20 cm, 2000 μm thickness) and UV indicator
(254 nm). The progress of reactions was monitored with Baker-
flex silica gel IB2-F plates (0.25 mm thickness). Unless specifically
mentioned, chemicals and solvents were of minimum reagent
grade and used as obtained from commercial sources without
further purification. Anhydrous tetrahydrofuran was prepared
by distillation from sodium-benzophenone ketal radical. The
hydrolytic stability assay utilized lyophilized rat plasma (lot no.
065K7555) from Sigma Chemical Co., St. Louis, MO. Elemental
analyses were performed at the Purdue University Microanalysis
Laboratory. All yields reported refer to isolated yields. Elemental
analysis established >95% purity of ADAM 4.
(E)-S-Methyl 5-(1-(3,7-Dimethyl-2-oxo-2,3-dihydrobenz-
[d]oxazol-5-yl)-5-(5-methyl-1,3,4-oxadiazol-2-yl)pent-1-enyl)-
2-methoxy-3-methylbenzothioate (4). An oven-dried flask was
charged with stannane 820 (202 mg, 0.319 mmol) and iodide 919
(140 mg, 0.533 mmol). Anhydrous DMF (3 mL) was added via a
syringe, and the resulting mixture was sparged with argon for
10 min. Cesium fluoride (195 mg, 1.28 mmol), Pd(PPh3)4 (38 mg,
0.033 mmol), and copper(I) iodide (16 mg, 0.084 mmol) were
quickly weighed and added to the flask in the order listed. The
reaction mixture was allowed to stir at 60 ꢀC, under an argon
atmosphere, for 14 h. The system was allowed to cool to room
temperature, and the reaction mixture was diluted with ethyl
acetate (5 mL), water (1 mL), and methanol (1 mL). The mixture
was sonicated at room temperature (30 s) and filtered through a
pad of silica (10 mL). The filter pad was washed with ethyl acetate
(50 mL), and the filtrate was washed with 1.0 M ethylenediami-
netetraacetic acid solution (pH = 10, 2 ꢀ 20 mL) and saturated
ammonium chloride solution (2 ꢀ 20 mL). The phases were
separated, and the organic phase was dried over magnesium
sulfate, filtered, and condensed in vacuo. The crude products
were purified by preparative thin-layer chromatography using
ethyl acetate as the eluant. The desired product was further
purified, two more times, by preparative thin-layer chromatog-
raphy for which 66% ethyl acetate-hexanes was used as the
developing solution for the first plate and 80% ethyl aceta-
te-toluene was used for the second. The desired product was
isolated as an opaque oil (6 mg, 4%). IR (neat) 2929, 2862, 1777,
1675, 1641, 1595, 1570, 1472, 1367, 1333, 1303, 1245, 1227, 1149,
1123, 1044, 1002 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.37 (d,
J = 2.1 Hz, 1 H), 7.08 (d, J = 2.1 Hz, 1 H), 6.74 (s, 1 H), 6.57 (s,
1 H), 5.97 (t, J = 7.5 Hz, 1 H), 3.88 (s, 3 H), 3.35 (s, 3 H), 2.82 (t,
J = 7.5 Hz, 2 H), 2.47 (s, 3 H), 2.45 (s, 3 H), 2.33 (s, 6 H), 2.25 (q,
J = 7.5 Hz, 2 H), 1.95 (p, J = 7.5 Hz, 2 H); ESIMS m/z (relative
intensity) 530 (MNaþ, 24), 508 (MHþ, 3), 478 (MHþ - SCH3 þ
H2O, 100). Anal. Calcd for C27H29N3O5S: C, 63.89; H, 5.76; N,
8.28. Found: C, 63.60; H, 5.96; N, 7.91.
RT Inhibition Assay. The ability of ADAM 4 to inhibit the
enzymatic activity of recombinant HIV-1 RT (p66/51 dimer)
was evaluated as previously described.24,37 Briefly, inhibition of
purified HIV-1 reverse transcriptase was determined by the
amount of 32P-labeled GTP incorporated into a nascent DNA
strand, with a poly(rC) oligo(dG) homopolymer primer, in the
3
presence of increasing concentrations of the target compounds.
In Vitro Antiviral Assays. The antiviral activity of ADAM 4
was determined for the HIV strains HIV-1RF, HIV-1IIIB, and
HIV-2ROD. The antiviral activity against HIV-1RF and NNRTI-
resistant strains (Y181C/K103N) was determined in infected
CEM-SS cells using the XTT cytoprotection assay, as previously
described.37 Evaluation of antiviral activity against the HIV-
1IIIB and HIV-2ROD strains was performed in infected MT-4
cells using the previously described MTT assay.18,38
In Vitro Hydrolytic Stability Assay Utilizing Rat Plasma.
ADAM 4 was tested for its hydrolytic stability in solutions of
reconstituted rat plasma using methods that have been previously
reported.18 The internal standard used was 1,1-diphenylethylene,
and rat plasma from Sigma was utilized for the experiments (lot no.
065K7555). The aliquot supernatants were analyzed using a
Waters binary HPLC system (Model 1525, 20 μL injection loop)
and a Waters dual wavelength absorbance UV detector (Model
2487) set for 254 nM. Data were collected and processed using the
Waters Breeze software (version 3.3) on a Dell Optiplex GX280
personal computer. The mobile phase consisted of 8:2 (v/v)
acetonitrile-water, and the Symmetry HPLC column (4.6 mm ꢀ
150 mm) was packed with C18 silica from Waters. The column was
maintained at room temperature during the analyses. The reported
half-life is an average calculated from two replicates. Half-lives for
the individual replicates were calculated from regression curves
fitted to plots of the compound concentration versus time.
Acknowledgment. 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 Facilities Improvement Program Grant, No. C06-
14499, also from the National Institutes of Health. E.A.’s
laboratory is supported by NIH Grant AI 27690 MERIT
Award and F32GM084726 (W.C.H.).
Crystallography. All crystallography experiments utilized an
RT construct (RT52A) specifically optimized to cocrystallize
with NNRTIs.32 RT was purified as described previously,
concentrated to 40 mg/mL, then flash frozen, and stored at
-80 ꢀC. Prior to use, RT was allowed to thaw on ice and then
diluted to a final concentration of 20 mg/mL in 10 mM Tris, pH
8.0, and 75 mM NaCl. ADAM inhibitors were added to RT at a
stoichiometry of 1:1.5 protein:inhibitor from stock solutions
previously dissolved in DMSO. Inhibitor and protein were
allowed to incubate at room temperature for 10 min and then
returned on ice. Crystals were grown using the vapor diffusion
References
(1) Cheng, Y. C.; Gao, W. Y.; Chen, C. H.; Vazquez-Padua, M.;
Starnes, M. C. DNA Polymerases Versus HIV Reverse Transcrip-
tase in AIDS Therapy. Ann. N.Y. Acad. Sci. 1990, 616, 217–223.
(2) Jacobo-Molina, A.; Ding, J.; Nanni, R. G.; Clark, A. D., Jr; Lu, X.;
Tantillo, C.; Williams, R. L.; Kamer, G.; Ferris, A. L.; Clark, P.;

Article
Hizi, A.; Hughes, S. H.; Arnold, E. Crystal Structure of Human
Immunodeficiency Virus Type I Reverse Transcriptase Complexed
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6473
Inhibitors of HIV-1 Reverse Transcriptase: Molecular Modeling and
X-ray Structure Investigations. J. Med. Chem. 1993, 36, 726–732.
(22) Das, K.; Clark, A. D.; Lewi, P. J.; Heeres, J.; de Jonge, M. R.;
Koymans, L. M. H.; Vinkers, H. M.; Daeyaert, F.; Ludovici,
D. W.; Kukla, M. J.; De Corte, B.; Kavash, R. W.; Ho, C. Y.;
Ye, H.; Lichtenstein, M. A.; Andries, K.; Pauwels, R.; de Bethune,
M. P.; Boyer, P. L.; Clark, P.; Hughes, S. H.; Janssen, P. A. J.;
Arnold, E. Roles of Conformational and Positional Adaptability in
Structure-Based Design of TMC125-R165335 (Etravirine) and
Related Non-nucleoside Reverse Transcriptase Inhibitors That
Are Highly Potent and Effective Against Wild-Type and Drug-
Resistant HIV-1 Variants. J. Med. Chem. 2004, 47, 2550–2560.
(23) Das, K.; Lewi, P. L.; Hughes, S. H.; Arnold, E. Crystallography
and the Design of Anti-AIDS Drugs: Conformational Flexibility
and Positional Adaptability are Important in the Design of Non-
Nucleoside HIV-1 Reverse Transcriptase Inhibitors. Prog. Bio-
phys. Mol. Biol. 2005, 88, 209–231.
(24) Cushman, M.; Golebiewski, W. M.; Graham, L.; Turpin, J. A.;
Rice, W. G.; Fliakas-Boltz, V.; Buckheit, R. W., Jr. Synthesis and
Biological Evaluation of Certain Alkenyldiarylmethanes as Anti-
HIV-1 Agents Which Act as Non-Nucleoside Reverse Transcrip-
tase Inhibitors. J. Med. Chem. 1996, 39, 3217–3227.
(25) Xu, G.; Loftus, T. L.; Wargo, H.; Turpin, J. A.; Buckheit, R. W.;
Cushman, M. Solid-Phase Synthesis of the Alkenyldiarylmethane
(ADAM) Series of Non-Nucleoside Reverse Transcriptase Inhibi-
tors. J. Org. Chem. 2001, 66, 5958–5964.
(26) Silvestri, M. A.; Nagarajan, M.; De Clercq, E.; Pannecouque, C.;
Cushman, M. Design, Synthesis, Anti-HIV Activities, and Meta-
bolic Stabilities of Alkenyldiarylmethane (ADAM) Non-nucleo-
side Reverse Transcriptase Inhibitors. J. Med. Chem. 2004, 47,
3149–3162.
(27) Das, K.; Bauman, J. D.; Clark, A. D.; Frenkel, Y. V.; Lewi, P. J.;
Shatkin, A. J.; Hughes, S. H.; Arnold, E. High-Resolution Struc-
tures of HIV-1 Reverse Transcriptase/TMC278 Complexes: Stra-
tegic Flexibility Explains Potency against Resistance Mutations.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 1466–1471.
(28) Himmel, D. M.; Das, K.; Clark, A. D.; Hughes, S. H.; Benjahad,
A.; Oumouch, S.; Guillemont, J.; Coupa, S.; Poncelet, A.; Csoka,
I.; Meyer, C.; Andries, K.; Nguyen, C. H.; Grierson, D. S.; Arnold,
E. Crystal Structures for HIV-1 Reverse Transcriptase in Com-
plexes with Three Pyridinone Derivatives: A New Class of Non-
Nucleoside Inhibitors Effective against a Broad Range of Drug-
Resistant Strains. J. Med. Chem. 2005, 48, 7582–7591.
(29) Jacques, P. S.; Wohrl, B. M.; Ottmann, M.; Darlix, J. L.; Legrice,
S. F. J. Mutating the Primer Grip of P66 Hiv-1 Reverse-Tran-
scriptase Implicates Tryptophan-229 in Template-Primer Utiliza-
tion. J. Biol. Chem. 1994, 269, 26472–26478.
˚
with Double-Stranded DNA at 3.0 A Resolution Shows Bent
DNA. Proc. Nat. Acad. Sci. U.S.A. 1993, 90, 6320–6324.
(3) Rodgers, D. W.; Gamblin, S. J.; Harris, B. A.; Ray, S.; Culp, J. S.;
Hellmig, B.; Woolf, D. J.; Debouck, C.; Harrison, S. C. The
Structure of Unliganded Reverse Transcriptase from the Human
Immunodeficiency Virus Type 1. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 1222–1226.
(4) Gulick, R. M.; Staszewski, S. New Drugs for HIV Therapy. AIDS
2002, 16, S135–S144.
(5) De Clercq, E. New anti-HIV Agents and Targets. Med. Res. Rev.
2002, 22, 531–565.
(6) De Clercq, E. Non-Nucleoside Reverse Transcriptase Inhibitors
(NNRTIs): Past, Present, and Future. Chem. Biodiversity 2004, 1,
44–64.
(7) De Clercq, E. New Approaches toward Anti-HIV Chemotherapy.
J. Med. Chem. 2005, 48, 1297–1313.
(8) Vivet-Boudou, V.; Didierjean, J.; Isel, C.; Marquet, R. Nucleoside
and Nucleotide Inhibitors of HIV-1 Replication. Cell. Mol. Life
Sci. 2006, 63, 163–186.
(9) De Clercq, E. Nucleoside/Nucleotide Inhibitors of HIV Reverse
Transcriptase. Infect. Dis. Ther. 2003, 30, 485–504.
(10) Tabba, H. D.; Chang, R. S.; Smith, K. M. Isolation, Purification,
and Partial Characterization of Prunellin, an Anti-HIV Compo-
nent from Aqueous Extracts of Prunella vulgaris. Antiviral Res.
1989, 11, 263–274.
(11) Young, S. D.; Britcher, S. F.; Tran, L. O.; Payne, L. S.; Lumma,
W. C.; Lyle, T. A.; Huff, J. R.; Anderson, P. S.; Olsen, D. B.;
Carroll, S. S.; Pettibone, D. J.; O’Brien, J. A.; Ball, R. G.; Balani,
S. K.; Lin, J. H.; Chen, I.-W.; Schleif, W. A.; Sardana, V. V.; Long,
W. J.; Byrnes, V. W.; Emini, E. A. L-743,726 (DMP-266): a Novel,
Highly Potent Nonnucleoside Inhibitor of the Human Immunodi-
ficiency Virus Type 1 Reverse Transcriptase. Antimicrob. Agents
Chemother. 1995, 39, 2602–2605.
(12) Hsiou, Y.; Ding, J.; Das, K.; Clark, A. D.; Hughes, S. H.; Arnold,
E. Structure of Unliganded HIV-1 Reverse Transcriptase at 2.7
Angstrom Resolution: Implications of Conformational Changes
for Polymerization and Inhibition Mechanisms. Structure 1996, 4,
853–860.
(13) Eshleman, S. H.; Jackson, J. B. Nevirapine Resistance After Single
Dose Prophylaxis. AIDS Rev. 2002, 4, 59–63.
(14) Cushman, M.; Golebiewski, M.; Buckheit, R. W., Jr.; Graham, L.;
Rice, W. G. Synthesis and Biological Evaluation of an Alkenyl-
diarylmethane (ADAM) which Acts as a Novel Non-nucleoside
HIV-1 Reverse Transcriptase Inhibitor. Bioorg. Med. Chem. Lett.
1995, 5, 2713–2716.
(30) Ghosh, M.; Jacques, P. S.; Rodgers, D. W.; Ottman, M.; Darlix,
J. L.; leGrice, S. F. J. Alterations to the Primer Grip of p66 HIV-1
Reverse Transcriptase and Their Consequences for Template-
Primer Utilization. Biochemistry 1996, 35, 8553–8562.
(31) Pelemans, H.; Esnouf, R.; De Clercq, E.; Balzarini, J. Mutational
Analysis of Trp-229 of Human Immunodeficiency Virus Type 1
Reverse Transcriptase (RT) Identifies this Amino Acid Residue as
a Prime Target for the Rational Design of New Non-Nucleoside
RT Inhibitors. Mol. Pharmacol. 2000, 57, 954–960.
(32) Bauman, J. D.; Das, K.; Ho, W. C.; Baweja, M.; Himmel, D. M.;
Clark, A. D.; Oren, D. A.; Boyer, P. L.; Hughes, S. H.; Shatkin,
A. J.; Arnold, E. Crystal Engineering of HIV-1 Reverse Transcrip-
tase for Structure-Based Drug Design. Nucleic Acids Res. 2008, 36,
5083–5092.
(33) Otwinowski, Z.; Minor, Z. Processing of X-Ray Diffraction
Data Collected in Oscillation Mode. Methods Enzymol. 1997,
276, 307–326.
(15) Cushman, M.; Casimiro-Garcia, A.; Williamson, K.; Rice, W. G.
Synthesis of a Non-Nucleoside Reverse Transcriptase Inhibitor
in the Alkenyldiarylmethane (ADAM) Series with Optimized
Potency and Therapeutic Index. Bioorg. Med. Chem. Lett. 1998,
8, 195–198.
(16) Casimiro-Garcia, A.; Micklatcher, M.; Turpin, J. A.; Stup, T. L.;
Watson, K.; Buckheit, R. W., Jr.; Cushman, M. Novel Modifica-
tions in the Alkenyldiarylmethane (ADAM) Series of Non-Nucleo-
side Reverse Transcriptase Inhibitors. J. Med. Chem. 1999, 42,
4861–4874.
(17) Xu, G.; Micklatcher, M.; Silvestri, M.; Hartman, T. L.; Burrier, J.;
Osterling, M. C.; Wargo, H.; Turpin, J. A.; Buckheit, R. W., Jr.;
Cushman, M. The Biological Effects of Structural Variation at the
Meta Position of the Aromatic Rings and at the End of the Alkenyl
Chain in the Alkenyldiarylmethane Series of Non-Nucleoside
Reverse Transcriptase Inhibitors. J. Med. Chem. 2001, 44, 4092–
4113.
(18) Silvestri, M. A.; Nagarajan, M.; De Clercq, E.; Pannecouque, C.;
Cushman, M. Design, Synthesis, Anti-HIV Activities, and Meta-
bolic Stabilities of Alkenyldiarylmethane (ADAM) Non-nucleo-
side Reverse Transcriptase Inhibitors. J. Med. Chem. 2004, 47,
3149–3162.
(19) Deng, B. L.; Cullen, M. D.; Zhou, Z. G.; Hartman, T. L.; Buckheit,
R. W.; Pannecouque, C.; De Clercq, E.; Fanwick, P. E.; Cushman,
M. Synthesis and Anti-HIV Activity of New Alkenyldiarylmethane
(ADAM) Non-nucleoside Reverse Transcriptase Inhibitors
(NNRTIs) Incorporating Benzoxazolone and Benzisoxazole
Rings. Bioorg. Med. Chem. 2006, 14, 2366–2374.
(34) Vagin, A.; Teplyakov, A. MOLREP: an Automated Program for
Molecular Replacement. J. Appl. Crystallogr. 1997, 30, 1022–1025.
(35) Emsley, P.; Cowtan, K. Coot: Model-Building Tools for Molecular
Graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60,
2126–2132.
(36) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Crystallography & NMR System: A New Software Suite for
Macromolecular Structure Determination. Acta Crystallogr., Sect.
D: Biol. Crystallogr. 1998, 54, 905–921.
(20) Cullen, M. D.; Deng, B. L.; Hartman, T. L.; Watson, K. M.;
Buckheit, R. W.; Pannecouque, C.; De Clercq, E.; Cushman, M.
Synthesis and Biological Evaluation of Alkenyldiarylmethane HIV-1
Non-Nucleoside Reverse Transcriptase Inhibitors that Possess
Increased Hydrolytic Stability. J. Med. Chem. 2007, 50, 4854–4867.
(37) Rice, W. G.; Bader, J. P. Discovery and In Vitro Development of
AIDS Antiviral Drugs as Biopharmaceuticals. Adv. Pharmacol.
(San Diego) 1995, 33, 389–438.
(38) Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.;
Herdewijn, P.; Desmyter, J.; De Clercq, E. Rapid and Automated
Tetrazolium-based Colorimetric Assay for Detection of Anti-HIV
Compounds. J. Virol. Methods 1988, 20, 309–321.
.
(21) Schafer, W.; Friebe, W.-G.; Leinert, W.; Mertens, A.; Poll, T.; von
der Saal, W.; Zilch, H.; Nuber, H.; Ziegler, M. L. Non-Nucleoside