Novel cyclopropyl-indole derivatives as HIV non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1021/ml3000462

The HIV pandemic represents one of the most serious diseases to face mankind in both a social and economic context, with many developing nations being the worst afflicted. Due to ongoing resistance issues associated with the disease, the design and synthesis of anti-HIV agents presents a constant challenge for medicinal chemists. Utilizing molecular modeling, we have designed a series of novel cyclopropyl indole derivatives as HIV non-nucleoside reverse transcriptase inhibitors and carried out their preparation. These compounds facilitate a double hydrogen bonding interaction to Lys101 and efficiently occupy the hydrophobic pockets in the regions of Tyr181/188 and Val179. Several of these compounds inhibited HIV replication as effectively as nevirapine when tested in a phenotypic assay.

Full text of DOI:10.1021/ml3000462

Letter
pubs.acs.org/acsmedchemlett
Novel Cyclopropyl-Indole Derivatives as HIV Non-Nucleoside Reverse
Transcriptase Inhibitors
Mohammad Hassam,† Adriaan E. Basson,‡ Dennis C. Liotta,§ Lynn Morris,‡ Willem A. L. van Otterlo,†
and Stephen C. Pelly*,†
†Department of Chemistry and Polymer Science, Stellenbosch University, Western Cape, South Africa
‡National Institute for Communicable Diseases, Johannesburg, South Africa
§Department of Chemistry, Emory University, Atlanta, Georgia, United States
S
* Supporting Information
ABSTRACT: The HIV pandemic represents one of the most serious diseases to
face mankind in both a social and economic context, with many developing nations
being the worst afflicted. Due to ongoing resistance issues associated with the
disease, the design and synthesis of anti-HIV agents presents a constant challenge
for medicinal chemists. Utilizing molecular modeling, we have designed a series of
novel cyclopropyl indole derivatives as HIV non-nucleoside reverse transcriptase
inhibitors and carried out their preparation. These compounds facilitate a double
hydrogen bonding interaction to Lys101 and efficiently occupy the hydrophobic
pockets in the regions of Tyr181/188 and Val179. Several of these compounds
inhibited HIV replication as effectively as nevirapine when tested in a phenotypic
assay.
KEYWORDS: NNRTI, indole, HIV, cyclopropyl, rational design
ith more than thirty years of the acquired immunode-
ficiency syndrome (AIDS) plaguing the human race, a
(BBB).4 This is particularly important to tackle viral reservoirs
located across the BBB, not only to reduce viral levels overall
but importantly to prevent complications such as the onset of
AIDS dementia complex.5 Furthermore, unlike the drugs in the
other therapeutic categories, the structural variance in known
NNRTIs is quite remarkable. Currently there are five licensed
NNRTIs (Figure 1), as well as many compounds which have
been developed as inhibitors for HIV RT.1−3,6−9 This structural
variation presents a wonderful opportunity in creative
exploration for the development of new NNRTIs.
In light of the need for new anti-HIV agents and the benefits
described above of the NNRTIs specifically, we embarked upon
a study to examine the receptor−ligand crystal structures of the
licensed NNRTIs to better understand their binding in the
allosteric pocket.10−13 Not surprisingly, given the general
hydrophobic nature of this binding pocket, very few electro-
static interactions were observed between the ligand and the
receptor. Nevirapine, for instance, makes no direct electrostatic
interactions with the protein, although there is a hydrogen
bonding interaction between the amide carbonyl and Leu234
via a bridging water molecule. In the case of efavirenz, however,
of particular interest to us was the double hydrogen bond
interaction made between the amide portion of the carbamate
and Lys101.
W
cure for the disease continues to remain elusive. Currently it is
estimated that over 33 million people are living with HIV,1 the
majority of which are in sub-Saharan Africa.2 Fortunately,
significant progress has been made in developing therapeutic
agents to control viral replication within HIV infected
individuals, resulting in delaying the onset of AIDS. However,
the high mutation rate of the virus and the resulting emergence
of resistance means that researchers are running a never ending
marathon to keep developing new drugs to control the viral
levels in HIV sufferers.
Currently there are six therapeutic targets utilized for anti-
HIV treatment, and within this group, the enzyme reverse
transcriptase (RT), which is crucial for the conversion of single
stranded viral RNA into double stranded DNA, provides two
opportunities for therapeutic intervention. The first of these is
at the actual catalytic site utilizing DNA base mimics which act
as chain terminators, and in fact the very first drug to be used in
the treatment of HIV (AZT) operates in this manner.3
However, located just 10 Å from the catalytic site is a
hydrophobic and relatively small cleft known as the non-
nucleoside binding pocket, and the resulting allosteric effect of
specific compounds binding in this pocket significantly inhibits
the normal functioning of the enzyme. These non-nucleoside
reverse transcriptase inhibitors (NNRTIs), being small and
somewhat nonpolar molecules, are the only anti-HIV drugs
which are suitable candidates to cross the blood brain barrier
Received: February 21, 2012
Accepted: May 2, 2012
Published: May 2, 2012
© 2012 American Chemical Society
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Figure 1. Currently licensed NNRTIs.
We envisaged that an indole-2-carboxamide based structure
would also facilitate the same interactions as efavirenz, and
indeed, a survey of the literature revealed that we were not the
first to consider this scaffold. In 1993 Williams et al. reported
that phenylsulfinyl-indole 1 showed potent activity against HIV
RT (Figure 2).14 Following this, several other groups became
Figure 3. Our envisaged indole-cyclopropyl NNRTI 5 docked within
the NNRTI allosteric site (A), and its corresponding schematic
representation (B), as well as that of the carboxylate 6 (C).
the cyclopropyl moiety snugly occupied the small hydrophobic
pocket in the region of Val179. Furthermore, the two important
hydrogen bonding interactions between the ligand and Lys101
were maintained as well as additional favorable interactions in
the form of π stacking between the phenyl ring and Tyr181, as
well as a σ−π interaction between the para-phenyl proton and
the indole side chain of Trp229. The amide −NH2 group was
directed out toward the entrance of the site hydrogen bonding
to HOH454, which facilitated a bridging connection to Glu138
(Figure 3B). Our concern, however, was that although the
nonpolar cyclopropyl moiety was well suited to the Val179
pocket, overall the compound may prove to be too hydro-
phobic, resulting in poor efficacy in our assays. Therefore, we
decided to initially synthesize the corresponding carboxylate 6
(Figure 3C) as a proof of concept compound, as the
carboxylate group could facilitate the same hydrogen bond
acceptor connection to Lys101 while maintaining the electro-
static connections to the water molecules at the site entrance.
At this point we returned to the issue of the cyclopropyl
group as a replacement moiety for the sulfonamide. Indeed,
although this group clearly fulfilled its role in terms of spatial
characteristics, we sought to find more conclusive evidence that
its positioning would be effective in binding to RT. Of the five
licensed NNRTIs used in HIV therapy, two structures contain a
cyclopropyl group, and fortuitously, crystal structures exist for
both compounds bound within HIV RT.12,13 Therefore, an
alignment and superimposition of the two structures bound
within their respective receptors was performed with our
docked structure in 2RF2, and the positioning of the
cyclopropyl ring was examined for each ligand. In the case of
efavirenz, the ring was found to occupy the Tyr181/188
binding pocket, bearing no resemblance to our envisaged
positioning. However, in the case of nevirapine, the cyclopropyl
ring was found to occupy a nearly identical position in the
region of the small Val179 pocket (Figure 4).
Figure 2. Indole based NNRTIs.
interested in this class of indole-based NNRTIs, and the
molecules evolved from sulfinyls 1 to sulfonyls 2 and eventually
to sulfonamide compounds 3, which were very potent
inhibitors of HIV RT.15−18 Work in this area continues, and
recently another group has published a variant on these
molecules containing phosphorus instead of sulfur 4.19 In fact,
the phosphoindole derivative IDX-899 is currently under
clinical trial evaluation.20,21
In light of the fact that these indole derivatives exhibited
excellent potency against HIV RT, we wished to retain this
structural scaffold and investigate novel functional groups
decorating the 2, 3, and 5 positions of the indole. Of particular
importance to us was to retain the hydrogen bonding
interactions to Lys101 facilitated by the indole NH (as
donor) and a suitable hydrogen bond acceptor moiety at the
2-position of the indole. A study of the X-ray crystal structure
of 3 bound within HIV RT revealed that although the
sulfonamide group adequately filled the Val179 binding
pocket,22 no electrostatic interactions were observed with the
receptor residues. We envisaged that a cyclopropyl group may
be well accommodated in this position and indeed could be
more suitable given the hydrophobic nature of the pocket.
Initial docking studies on cyclopropyl derivative 5 (Figure 3A)
revealed that it adopted a pose very similar to that of 3 and that
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an HIV-1 retroviral vector system for the production of virus-
like particles (VLPs). Compounds were incubated with 293T
cells and VLP for 48 h, after which the inhibition of HIV was
quantified by luminescence measurement.24,25 Disappointingly,
in this assay 6 proved to be a rather poor inhibitor with an IC50
value of 28.5 μM, significantly less effective than nevirapine,
which had an IC50 value of 0.087 μM under these conditions
(Table 1). After some consideration it was surmised that, in
Table 1. Phenotypic Assay Values (IC50/μM) of Cyclopropyl
Indole Derivatives Evaluated against Wild Type HIV as Well
as the Corresponding Toxicity Values (CC50/μM)
Figure 4. Overlay of nevirapine (purple) cocrystallized in HIV RT
(PDB 1VRT) and our docked indole derivative 6 (orange) showing
the similar placement of the cyclopropyl moiety.
With a promising candidate in mind, we set about the
synthesis of 6, starting from commercially available ethyl 5-
chloroindole-2-carboxylate 7 (Scheme 1). Installation of the
required phenyl functionality was readily achieved by Friedel−
Crafts acylation using benzoyl chloride to furnish 9. The indole
−NH was then protected as the Boc carbamate 13 in
preparation for the Wittig reaction to follow. To this end, 13
was subjected to typical Wittig conditions employing the
methyl triphenylphosponium ylide, which converted the ketone
to the desired alkene 17 yet also resulted in loss of the Boc
protecting group to some extent, thereby additionally
producing 21. This unplanned event turned out to be
inconsequential, as either compound could be converted
directly to the desired cyclopropyl indole derivative 25 in the
presence of diethyl zinc, diiodomethane, and trifluoroacetic
acid.23 We also attempted to shorten the synthesis by
completely avoiding the installation of the Boc group, but
unfortunately in our hands, this led to very poor yields being
obtained in the Wittig step. Finally, to complete our synthesis,
the ethyl ester 25 was hydrolyzed to the corresponding
carboxylic acid 29 and after purification, the carboxylate was
recrystallized as the triethyl ammonium salt 6. With the proof
of concept compound 6 in hand, we were finally in a position to
test its efficacy as an anti-HIV agent. To this end, we utilized an
in vitro single-cycle, nonreplicative phenotypic assay employing
light of the fact that our evaluation method is a whole cell assay,
the paltry potency may indeed not be as a result of the
a
Scheme 1. Synthesis of Cyclopropyl Indoles
a
Reagents and conditions: (a) benzoyl chloride, 2-thiophenecarbonyl chloride, or acetyl chloride, AlCl3, DCE; (b) Boc2O, DMAP, THF; (c)
MePPh3Br, n-BuLi, THF; (d) Et2Zn, CH2I2, TFA, DCM; (e) KOH, EtOH, then HCl; (f) Et3N, Et2O.
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a
compound ineffectively binding to the HIV NNRTI binding
pocket but may simply be as a result of poor membrane
permeability given that 6 contains the negatively charged
carboxylate group, which would remain ionized at physiological
pH. To investigate this possibility, the preceding ester 25 was
subjected to the same phenotypic assay and confirmed our
suspicions, as it inhibited HIV replication as effectively as
nevirapine, with an IC50 value of 0.085 μM and comparatively
low cytotoxicity (CC50).26 Modeling studies indicate that the
ester analogue 25 could be well accommodated within the
binding site, with the ethyl group directed out the site opening
(Figure 5). Work is ongoing in this area, and we are in the
process of synthesizing several ester analogues with hydrophilic
end groups which we hope will exhibit improved potency.
Scheme 2. Conversion of Indole-2-carboxylates to Amides
a
Reagents and conditions: (a) PyBOP, HOBt, NH3, DIPEA.
Scheme 3. Reduction of the Ethyl Indole-2-carboxylate to
a
the Alcohol
a
Reagents and conditions: (a) LiAlH4, Et2O.
effect of replacing the sp2 hybridized carbonyl oxygen hydrogen
bond acceptor with an sp3 hybridized alcohol acceptor. In
principal, one could envisage that the oxygen could still fulfill its
role as an acceptor for the amide proton of Lys101. However,
in our docking studies the most favored pose always positioned
the oxygen in such a manner that hydrogen bonding could not
occur, even in rigorous docking exercises where numerous
starting conformations were considered to ensure adequate
sampling of the conformational space for these compounds.
Interestingly, these modeling results are consistent with our
assay results, which show more than an order of magnitude
decrease in potency for the IC50 values yet the CC50 values are
similar (Table 1), indicative that cell permeability is not the
issue and that these compounds certainly are poorer inhibitors
of HIV RT.
To investigate the effect of increasing the size of the halogen
at R3, the 5-bromoindole derivative 28 was synthesized. This
procedure, which started with ethyl 5-bromoindole-2-carbox-
ylate 8 (Scheme 1), resulted in the synthesis of 28 in five steps
and 15% overall yield. Analysis of the effectiveness of this
compound in our phenotypic assay showed a similar potency to
that of the corresponding 5-chloroindole analogue 25 (Table
1).
The final step in this work involved confirming that the
compounds were indeed inhibiting HIV replication by way of
binding to the allosteric pocket of HIV RT. To this end, our
most potent cyclopropyl-indole derivatives were tested against
eight RT single mutant strains of the virus. Of importance,
mutations at residues Lys103, Tyr181, and Tyr188, which
surround our cyclopropyl-indoles in their predicted binding
poses, caused significant changes in the inhibition values for
these compounds (Supporting Information Table 2). These
residues are also the site of action of current NNRTIs,
confirming that our novel compounds are indeed NNRTIs.
In conclusion, we have utilized molecular modeling to design
novel indole based HIV-1 non-nucleoside reverse transcriptase
inhibitors which are as potent as nevirapine. In addition, cellular
toxicity studies indicate promisingly low toxicity. We believe
that the compounds can be further optimized by more
appropriate substitution at the R1 position (Table 1), and
work is currently ongoing in this area.
Figure 5. Ester derivative 25 docked into the allosteric site of HIV RT
(PDB 2RF2) showing that it is well accommodated, with the ethyl
ester pointing out the opening of the site (nonpolar hydrogens have
been omitted for clarity).
Having obtained proof of concept for our indole-cyclopropyl
HIV NNRTIs, we set about investigating smaller groups to
occupy the Tyr181/188 pocket. Thiophene derivative 26
(Table 1), obtained from a Friedel−Crafts acylation with 2-
thiophenecarbonyl chloride (Scheme 1), showed a similar IC50
value to that of 25. Interestingly, we also synthesized the
methyl derivative 27, as modeling studies indicated that this
compound would bind significantly less effectively as a result of
inadequately filling the Tyr181/188 pocket. The IC50 result of
2.25 μM for this compound is consistent with our modeling
results. Moreover, analogous to our previous finding that the
carboxylate structure 6 performed poorly in the phenotypic
assay, the thiophene containing carboxylate 31 was also found
to be poorly effective against HIV in the phenotypic assay.
With the phenyl and thiophene esters (25 and 26) in hand,
we then decided to investigate several modifications to the
critical H-bond acceptor moiety at R1 (Table 1). To this end,
both compounds were converted to the corresponding amides.
In fact, direct conversion of the esters to the corresponding
amides in methanol saturated with ammonia did not proceed
even at reflux. Therefore, the esters were hydrolyzed to the
corresponding carboxylic acids (29 and 30) and installation of
the amides was achieved by way of a PyBOP mediated
coupling, thereby forming 5 and 32 (Scheme 2).27 The IC50
values for the amides 5 and 32 are similar to those obtained for
the corresponding esters, 25 and 26, respectively (Table 1).
The ester analogues 25 and 26 were also reduced to the
corresponding alcohols 33 and 34 (Scheme 3) to determine the
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures, spectral data, and modeling procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: scpelly@sun.ac.za. Telephone: +27-21-808-2180. Fax
+27-21-808-3360.
Funding
(14) Williams, T. M.; Ciccarone, T. M.; MacTough, S. C.; Rooney, C.
S.; Balani, S. K.; Condra, J. H.; Emini, E. A.; Goldman, M. E.;
Greenlee, W. J. 5-Chloro-3-(phenylsulfonyl)indole-2-carboxamide: a
novel, non-nucleoside inhibitor of HIV-1 reverse transcriptase. J. Med.
Chem. 1993, 36, 1291−1294.
This study was supported by the University of Stellenbosch,
NRF, NICD, and Emory University (S.C.).
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors thank the NRF for funding and the NICD for the
phenotypic assay components of the project. S.C. Pelly thanks
Emory University for funding a sabbatical related to this work
and the University of the Witwatersrand for sabbatical leave.
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