Discovery of dimeric inhibitors by extension into the entrance channel of HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.bmcl.2011.12.132

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with computational guidance. Extension of azine-containing inhibitors into the entrance channel between Lys103 and Glu138 has led to the discovery of potent and structurally novel derivatives including dimeric inhibitors in an NNRTI-linker-NNRTI motif.

Full text of DOI:10.1016/j.bmcl.2011.12.132

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1565–1568
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of dimeric inhibitors by extension into the entrance channel
of HIV-1 reverse transcriptase
Anil R. Ekkati a, Mariela Bollini a, Robert A. Domaoal b, Krasimir A. Spasov b, Karen S. Anderson b,
,
⇑
William L. Jorgensen a,
⇑
a Department of Chemistry, Yale University, New Haven, CT 06520, USA
b Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase is being pursued with computational
guidance. Extension of azine-containing inhibitors into the entrance channel between Lys103 and Glu138
has led to the discovery of potent and structurally novel derivatives including dimeric inhibitors in an
NNRTI-linker-NNRTI motif.
Received 7 December 2011
Revised 27 December 2011
Accepted 29 December 2011
Available online 5 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Anti-HIV agents
Non-nucleoside inhibitors of HIV reverse
transcriptase
Computer-aided drug design
NNRTI
Dimeric inhibitors
O
Non-nucleoside inhibitors of HIV-1 reverse transcriptase
(NNRTIs) are a mainstay of combination therapies for the treat-
ment of HIV infection.1 They bind to an allosteric site, which leads
to deactivating conformational changes at the proximal polymer-
ase active site.2,3 However, the clinical utility of NNRTIs is chal-
lenged by rapid emergence of drug-resistant, variant strains of
the virus.4 Thus, much effort has been put into the development
of new NNRTIs with improved resistance profiles with simulta-
neous concern for diminished side-effects and ease of administra-
tion.5 The work has mostly featured classical medicinal chemistry
with extensive analoging of multiple core structures, which have
typically arisen from high-throughput screening.3 As an alterna-
tive, our group has emphasized computer-aided structure-based
design in an attempt to reduce the number of compounds that
need to be synthesized and assayed.6 Significant success has been
achieved in several series; for example, 1–3 have been reported to
inhibit replication of wild-type HIV-1 (IIIB) in infected human T-
cells with EC50 values of 2, 11, and 0.3 nM.7–9 These structures
illustrate the diversity of NNRTIs; however, crystallographic10
and modeling studies reveal common features with the side chains
for inhibitors like 2–4 fitting into two channels in the NNRTI
binding site.
N
OCH3
N
O
O
CN
F
F
O
NH
N
N
H
N
1
2
F
CN
O
O
O
I
O
S
N
O
N
H
O
N
O
H
F
4
3
As illustrated for 4 in Figure 1, the benzyl or phenoxy groups of
2–4 reside in the tunnel lined by Tyr181, Tyr188, Trp229, and
Phe227, which leads towards the polymerase active site, while the
heteroaryl containing side chains occupy the groove lined by
⇑
Corresponding authors.
E-mail address: william.jorgensen@yale.edu (W.L. Jorgensen).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.132

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A. R. Ekkati et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1565–1568
Table 1
Anti-HIV-1 activity (EC50) and cytotoxicity (CC50), l
Ma
R
N
O
CN
N
N
R'O
N
H
5
Compound
R
OR0
EC50
CC50
5ab
OCH3
OCH3
OCH3
OCH3
OCH3
CH2CH3
CH2CH3
OCH3
OCH3
OCH3
H
OCH3
OCH2CH2OCH3
(OCH2CH2)2OCH3
(OCH2CH2)3OCH3
OCH2CH2OCH3
(OCH2CH2)2OCH3
OCH2CH2-4-MePip
OCH2CH2-2-THP
NH2
0.011
0.022
0.097
0.150
0.380
0.057
0.540
0.320
1.2
42
>100
8.6
13
4.2
2.1
9.8
7.0
4.2
5bb
5c
5d
5e
5f
5g
5h
5i
5jb
0.009
0.110
0.11
>100
Nevirapine
Figure 1. Rendering of the 2be2 crystal structure10 of 4 bound to HIV-RT. Carbon
atoms of 4 are in green. Some residues are omitted for clarity.
a
4-MePip = N-methylpiperazinyl; 2-THP = 2-tetrahydropyranyl (racemic).
Ref. 7b.
b
Phe227, Tyr318, Pro225, and Pro236. Another common feature is a
hydrogen bond between the amino groups of 1, 2, and 4 and the
carbonyl oxygen of Lys101 (not illustrated). And, for 1, the dimeth-
ylallyl group arches into the former channel interacting with
Tyr181, Tyr188, and Trp229, as in the 1rt4 crystal structure.11 In
addition to the tunnel and groove, the largely open region in front
of Lys103, Glu138, and Val179 in Figure 1 is considered to form
the entrance channel for the NNRTI binding site. This region appears
to have been previously unexplored in the development of NNRTIs.
Thus, we set out to consider growing substituents into this area
envisioning possible benefits for activity and modulation of molec-
ular properties. In the extreme, we are also interested in pursuing
development of chimeric NNRTIs. For example, an NNRTI1-linker-
NNRTI2 construct could be intriguing given different resistance
profiles for the two NNRTIs. The next step would be ‘keychain inhib-
itors’ in which multiple, different NNRTIs or other anti-HIV agents
are linked to a core, effectively providing combination therapy in
a single compound. Naturally, molecular weight/bioavailability
issues would likely become a concern at some point. The present
work just addresses growth of substituents into the entrance chan-
nel and the viability of dimeric NNRTIs with simultaneous occu-
pancy of the entrance channel and NNRTI binding site.
Figure 2. Computed structure of 5d bound to HIV-RT illustrating extension of the
polyether side chain into the entrance channel. Carbon atoms of 5d are in green.
Some residues are omitted for clarity.
We chose to begin with analogues of the triazene corresponding
to 1, which show similar antiviral activity, but diminished cytotox-
icity relative to the corresponding pyrimidines.7 We had also found
previously that the dimethoxy compound 5b was only two-fold less
potent than the monomethoxy 5a (Table 1). Model building was
then carried out for analogues in which the methoxy group near
the entrance channel was elaborated. The calculations consisted of
generation of structures of the complexes and conformational
searches with the BOMB program,6 followed by conjugate-gradient
optimizations using MCPRO12 and the OPLS/CM1A force field.13 The
protein coordinates were based on the 1s9e crystal structure for
which the ligand is a diaminotriazine derivative.14 The computa-
tions indicated that growth into the entrance channel was sterically
allowable using PEG-like extensions (Fig. 2).
R
N
O
R
N
O
CN
CN
N
N
O
N
N
NaH (60% oil)
Cl
N
H
R'O
N
H
R'OH, THF, rt, 48 h
5c-i
Al (i-Bu)3 (1 M in hexanes)
O
OH
O
°
Hexane, rt to 60 C, 5h, 51%
Scheme 1. Synthesis of substituted triazenes.
The desired compounds 5c–i were synthesized via SNAr reaction
between previously reported chlorotriazines7b and appropriate
alkoxides, as summarized in Scheme 1. The alcohol for 5i was pre-
pared by reductive rearrangement of 2-vinyloxytetrahydropyran.
Activities against the IIIB strain of HIV-1 were measured using
MT-2 human T-cells; EC50 values are obtained as the dose required
to achieve 50% protection of the infected cells by the MTT colori-
metric method. CC50 values for inhibition of MT-2 cell growth by
50% are obtained simultaneously.7,15,16 The identity of all assayed
compounds was confirmed by 1H and 13C NMR and high-resolution
mass spectrometry; purity was >95% as judged by high-perfor-
mance liquid chromatography.

A. R. Ekkati et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1565–1568
1567
It was encouraging that the methoxyethoxy analogue 5c re-
tained good potency at 97 nM and the penalties for additional eth-
yleneoxy groups were modest for 5d and 5e. Compound 5f, the
R = ethyl analogue of 5c, was also prepared and yielded improved
activity with an EC50 of 57 nM. Curiously, 5g, the ethyl analogue
of 5d, exhibited the opposite trend. To test if more branched alter-
natives could be tolerated, the N-methylpiperazinyl and 2-tetrahy-
dropyranyl derivatives, 5h and 5i, were considered; they were
20–40 lM. The dimeric constructs with longer linkers or amino
connectors (6e and f) did not show anti-viral activity below their
CC50 levels. As illustrated in Figure 3, 6b may fill the entrance
groove well. Longer linkers could cause the protruding NNRTI to
be pushed farther from the protein’s surface. The similar activities
for 5d and 6b indicate the addition of some favorable contacts be-
tween 6b and the protein to offset the increased loss of conforma-
tional freedom. The proof-of-concept success with 6a and 6b is
striking and provides a foundation for investigations of heterodi-
mers, NNRTI1-linker-NNRTI2.
found to be less active with EC50 values of 0.32 and 1.2 lM. Since
the MT-2 assay is cell-based, expectations for 5h were unclear in
view of the potential impact of protonation of the piperazine nitro-
gens on the cell permeability.
The stage is also set for construction of bifunctional inhibitors,
NNRTI-linker-Inh, where Inh is a member of a different class of
OCH3
N
O
OCH3
N
O
H3C
CN
CN
N
N
N
N
5i
O
N
N
O
O
N
N
5h
H
H
Prior results for the amino analogue 5j are also noted in Table 1. It
is a 9-nM NNRTI, and the corresponding methylamino analogue with
the cyano group replaced by chlorine was found to have good po-
tency, 31 nM, as well.7b Thus, amino connectors might also be viable.
Clearly, numerous additional model compounds could be explored
with alternative side chains including anionic ones, and further opti-
mization of the group R. Nevertheless, at this point, it was established
that substantial molecular growth was possible in the entrance chan-
nel with retention of antiviral activity at nanomolar levels.
Thus, attention turned to more ambitious Janus dimers, NNRTI-
linker-NNRTI. Computational modeling was performed to assess
the viability of dimers of analogues of 5a tethered as in 6. Use of
the BOMB program readily found low-energy structures with rela-
tively short linkers, for example, for 6b inFigure 3. The second copy
of the NNRTI can be accommodated in a cleft passing between
Lys32B and Lys172A. The illustrated conformer of 6b is well ex-
tended in the entrance channel. Six compounds were synthesized
from the chloromethoxytriazene,7b as summarized in Scheme 2.
The corresponding assay results are listed in Table 2.
anti-HIV agent, and the necessary attachment point to the NNRTI
is evident. Bifunctional inhibitors have previously been explored
with NNRTIs. Early work on NNRTI-(CH2)n-NRTI constructs pro-
vided compounds where the anti-viral activity seemed to arise so-
lely from the NNRTI component.17 The structural situation is
unclear, though it is possible that the NRTI resides in the entrance
channel. More recent efforts designed NNRTI-linker-NRTI inhibi-
tors such that the linker is in the tunnel passing Trp229; however,
there was no evidence that synergistic binding to the NNRTI and
NRTI sites was achieved.18 NNRTIs, particularly in the HEPT class,
have also been linked to a characteristic diketoacid fragment of
HIV integrase inhibitors.19 The linking to the NNRTI in these cases
was at the terminus that would reside in the Pro225–Pro236
groove (Fig. 1). Though these constructs retained strong inhibition
of HIV-RT, the inhibition of HIV integrase has only been in the
micromolar range.19 The present results open up the possibility
of exploring an alternative topology via connections to NNRTIs
such that the second inhibitor resides in the NNRTI-entrance chan-
nel. Aside from the present triazines and related pyrimidines (1)
such connections should be possible for the oxazole (2) and cate-
chol diether (3) series as well as for some other known NNRTIs
The diethers with the shortest linkers, 6a and 6b, do show good
activity with EC50 values of 390 and 170 nM, and low cytotoxicity,
O
OCH3
N
OCH3
N
O
NC
CN
N
N
L
N
H
N
O
O
N
N
H
6a-d
NaH, THF
60-76%
HO-L-OH
OCH3
N
O
CN
N
NaHCO3
H2N-L-NH2 THF
Cl
N
N
H
78-85%
O
OCH3
N
OCH3
N
O
NC
CN
N
N
L
N
H
N
N
N
H
N
N
H
H
6e-f
Figure 3. Computed structure of 6b bound to HIV-RT illustrating extension into the
entrance channel. Carbon atoms of the inhibitor are in green. Some residues are
omitted for clarity.
Scheme 2. Synthesis of dimeric inhibitors.

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A. R. Ekkati et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1565–1568
Table 2
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Ma
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6d
6e
6f
OCH2CH2O
OCH2CH2CH2O
OCH2CH2CH2CH2O
OCH2CH2OCH2CH2O
NHCH2CH2NH
0.390
0.170
NA
NA
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42
21
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HN
CN
N
N
N
H
7
Inh-LINKER
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Gratitude is expressed to the National Institutes of Health
(AI44616, GM32136, GM49551) for support. Receipt of reagents
through the NIH AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH is also greatly appreciated.