Extension into the entrance channel of HIV-1 reverse transcriptase - Crystallography and enhanced solubility

Article abstract of DOI:10.1016/j.bmcl.2013.06.093

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature extension into the entrance channel near Glu138. Complexes of the parent anilinylpyrimidine 1 and the morpholinoethoxy analog 2j with HIV-RT have received crystallographic characterization confirming the designs. Measurement of aqueous solubilities of 2j, 2k, the parent triazene 2a, and other NNRTIs demonstrate profound benefits for addition of the morpholinyl substituent.

Full text of DOI:10.1016/j.bmcl.2013.06.093

Accepted Manuscript
Extension into the entrance channel of HIV-1 reverse transcriptase – crystal‐
lography and enhanced solubility
Mariela Bollini, Kathleen M. Frey, José A. Cisneros, Krasimir A. Spasov,
Kalyan Das, Joseph D. Bauman, Eddy Arnold, Karen S. Anderson, William L.
Jorgensen
PII:
S0960-894X(13)00826-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.06.093
BMCL 20657
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Please cite this article as: Bollini, M., Frey, K.M., Cisneros, J.A., Spasov, K.A., Das, K., Bauman, J.D., Arnold, E.,
Anderson, K.S., Jorgensen, W.L., Extension into the entrance channel of HIV-1 reverse transcriptase –
crystallography and enhanced solubility, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/
10.1016/j.bmcl.2013.06.093
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Extension into the entrance channel of HIV-1 reverse
transcriptase – crystallography and enhanced solubility
Mariela Bollinia, Kathleen M. Freyb, José A. Cisnerosa, Krasimir A. Spasovb, Kalyan
Dasc, Joseph D. Baumanc, Eddy Arnoldc, Karen S. Andersonb*, and William L.
Jorgensena*
aDepartment of Chemistry, Yale University, New Haven, CT 06520-8107, USA
bDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066, USA
cDepartment of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
Received May 15, 2013; Accepted Month XX, 2013
Abstract— Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that feature extension into the entrance
channel near Glu138. Complexes of the parent anilinylpyrimidine 1 and the morpholinoethoxy analog 2j with HIV-RT have received
crystallographic characterization confirming the designs. Measurement of aqueous solubilities of 2j, 2k, the parent triazene 2a, and other
NNRTIs demonstrate profound benefits for addition of the morpholinyl substituent. ©2013 Elsevier Science Ltd. All rights reserved.
Non-nucleoside inhibitors of HIV-1 reverse
transcriptase (NNRTIs) are a central component of anti-
HIV therapy.1 There are five FDA-approved drugs in
the class: nevirapine, delavirdine, efavirenz, etravirine,
and rilpivirine. In view of the rapid mutation of the
virus, NNRTIs are given in combination therapies.
Though the class has demonstrated important utility,
further improvements are possible to address issues of
safety, administration, and virologic failure.2-4 There is
also need to be positioned to respond to the emergence
of pan-resistant viral variants and unknown effects of
long-term treatment.5
RT, the binding mode for 1 was confirmed by an X-ray
crystal structure. This structure, which was obtained by
the Rutgers group, is reported here and illustrated in
Figures 1 and 2.12
Consistent with the 1S9E structure and the
modeling, 1 resides in the NNRTI binding site with the
hydrophobic dimethylallyl group positioned in the π-
box formed by Tyr181, Tyr188, Phe227, and Trp229
(Figure1). There are also hydrogen bonds between the
backbone O and N of Lys101 and the amidine moiety of
1, which are clearer from the orthogonal view in Figure
2. This Figure also highlights a salt bridge between the
side chains of Glu138 and Lys101, a feature that is not
always found in NNRTI complexes, e.g., the 1S9E
structure.11 The X-ray results provide important
validation of the structure-activity patterns that were
obtained for 1 and its analogs.10 This includes
rationalization of the poor activity of these compounds
towards viral variants containing the clinically
important Tyr181Cys mutation owing to the substantial
contact of Tyr181 with the dimethylallyl group of 1.10,14
Our efforts at Yale to discover new series of
NNRTIs have combined computer-aided design,
synthesis, and biological assaying.6,7 For two picomolar
inhibitors, which feature
a
catechol diether
substructure,8 we also recently reported X-ray crystal
structures for their complexes with wild-type (WT)
HIV-RT.9 One of the earliest series that we had
investigated
was
anilinylazines
containing
a
dimethylallyloxy substituent. This work culminated in
the pyrimidine 1 and the corresponding 1,3,5-triazine
(2a), which yield EC50 values of 2 and 11 nM in a
standard assay using MT-2 cells infected with WT HIV-
1.10 Though results of molecular modeling, starting
from the 1S9E crystal structure of an anilinyltriazene,11
were reported for complexes of the inhibitors with HIV-

Figure 2. View from the right in Figure 1 of the crystal structure of 1
bound to WT HIV-RT. Some residues are omitted for clarity. Coordinates
have been deposited in the PDB as structure 4KO0.
Figure 1. Rendering of the 1.95-Å crystal structure of 1 with WT HIV-
RT. Carbon atoms of 1 are in yellow. Some residues are omitted for
clarity.
the parent 2a and dimethoxy analog 2b are 11- and 22-
nM inhibitors. It was even found that large, dimeric
NNRTIs could be accommodated, e.g., 3 gives an EC50
of 170 nM.16
Subsequent efforts included the probing of
relatively unexplored regions including the ‘eastern
channel’ or ‘groove’15 extending back and down from
the cyano group of 1 in Figure 1 and the ‘entrance
channel’ extending into the solvent-exposed region to
the left of Lys101 in Figure 2.16 The intentions were to
seek extensions that might (1) improve potency towards
both WT HIV-1 and variants containing common
NNRTI-induced point mutations, and/or (2) improve
physical properties of the NNRTIs. Extension into the
entrance channel for 1 and 2 would require attachment
of a substituent at C6 of the azine rings (Figure 1). It
was not clear that this would be successful owing to
possible unfavorable contacts, especially with Glu138
(Figure 2). However, the idea was pursued and it was
found that substantial substituents could be attached at
this position, though with some loss of potency.16 For
example, as summarized in Table 1, the methoxyethoxy
and methoxy-triethoxy containing 2c and 2e yield EC50
values of 97 and 380 nM in the MT-2 cell assay, while
The present report extends the investigations of the
entrance channel. Additional compounds were
synthesized and assayed, specifically, the amino-
terminating 2f and 2g and the morpholinoethoxy and
-propoxy analogs 2j – 2m. The latter compounds were
prepared as before via SNAr reactions of alkoxides with
the intermediate 4;16 however, an alternative route was
necessary for the amines (Scheme 1). The SNAr
reaction was performed earlier in the sequence to install
the side chain on dichloromethoxytriazene, and the
amino group was unmasked in the last step.
In the same manner as in previous reports,6-10,14-16
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 colorimetric method.
Simultaneously, CC50 values for inhibition of MT-2 cell
growth by 50% are obtained. The identity of all assayed
Table 1
Anti-HIV-1 Activity (EC50) and cytotoxicity (CC50), µMa
Compound
R
R’
H
OCH3
EC50
0.011
0.022
0.097
0.057
0.380
2.2
5.0
0.320
1.2
0.092
0.095
0.150
0.100
1.500
0.110
0.002
0.001
CC50
42
>100
8.6
2.1
4.2
23
2ab
OCH3
OCH3
OCH3
CH2CH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
CH2CH3
c-C3H5
OCH3
2bb
2cc
OCH2CH2OCH3
OCH2CH2OCH3
(OCH2CH2)3OCH3
(OCH2CH2)4NH2
(OCH2CH2)6NH2
OCH2CH2-4-MePiz
OCH2CH2-2-THP
OCH2CH2-Morph
OCH2CH2CH2-Morph
OCH2CH2-Morph
OCH2CH2-Morph
OCH2CH2CH2-4-Pip
2dc
1
compounds was confirmed by H and 13C NMR and
2ec
2f
high-resolution mass spectrometry; purity was >95% as
2g
18
2hc
7.0
4.2
2.5
3.6
0.8
0.9
5.6
>100
15
2ic
2j
2k
2l
2m
2n
nevirapine
efavirenz
rilpivirine
8
a
4-MePiz
= N-methylpiperazinyl; 2-THP = 2-tetrahydropyranyl
(racemic); Morph = N-morpholinyl; Pip = piperidinyl. b Ref. 10. c Ref. 16.

Table 2
Aqueous Solubility at pH 6.5 (S)
Compound
S, µg/mL
0.1
42.2
Compound
efavirenz
dapivirine
rilpivirine
S, µg/mL
68.0
2a
2j
2k
0.15
52.3
0.24a, 0.02b
a Ref. 18, pH 7.4. b Ref. 20, pH 7.
Scheme 1. Synthesis of 2f and 2g.
resolution, data were collected at the Brookhaven NSLS
on beam line X25, and the structure was solved by
molecular replacement using the 2ZD1 structure of
rilpivirine with HIV-RT25 as the search model. The
electron density for the non-nucleoside binding region is
ordered and provides clear characterization of the
interactions with the inhibitor (Figure 3). An alternative
rendering of the structure is provided in Figure 4.
judged by high-performance liquid chromatography.
The amines 2f and 2g were of interest to test the
effects of a positive charge at the end of the side chains
and increases in length beyond 2e. The results for 2f and
2g in Table 1 show that these modifications are not
beneficial for activity. The interest in the morpholine
containing compounds was particularly high since this
modification would likely improve the solubility of the
compounds. Poor solubility has often been an issue in
NNRTI development, stemming from the hydrophobic
character of the NNRTI binding site.1,16-19 Thus, it was
pleasing to find good activity for 2j (92 nM) and 2k (95
nM). This represents significant improvement over the
methylpiperazinyl and THP analogs 2h and 2i.
The positioning of 2j in the NNRTI binding site is
the same as for 1 including the hydrogen bonds with
Lys101 and the placement of the dimethylallyl group in
the π-box; however, the salt bridge between Glu138 and
Lys101 is no longer present (Figure 4). Overall, the co-
crystal structures reported here for 1 and 2j have an
rmsd of only 1.4 Å for an all-atom alignment. Since the
contact between Tyr181 and the dimethylallyl group is
retained, 2j does not show activity towards the Y181C-
containing viral variant. Most notably, the structure for
2j confirms the anticipated extension of the
morpholinoethoxy side chain into the entrance channel
towards Glu28. The carboxylate group of Glu138
contacts the carbon atoms of the ethoxy subunit with O-
C separations of 2.8 – 3.6 Å. The carboxylate oxygens
are also 3.8 and 4.4 Å from the morpholine nitrogen,
which is presumably protonated. In addition, the
morpholine oxygen is proximal to the carboxylate
carbon (4.1 Å) and oxygens (3.4 and 4.3 Å) of Glu28.
Though it might seem that replacement of the
oxygen of the morpholine with a positively charged
group would enhance binding, beneficial effects were
not apparent for the 4-methylpiperazinyl analog 2h
(Table 1). In this case, only one of the tertiary nitrogens
Furthermore, we quantified the aqueous solubilities
of 2a, 2j, 2k, dapivirine (TMC120), and efavirenz using
a shake-flask method with UV detection (Agilent
8453).21 The results in Table 2, which also includes
previous reports for rilpivirine, are striking. Addition of
the morpholinoalkoxy substituent to 2a to yield 2j or 2k
delivers a 400 to 500-fold improvement in solubility.
The results for 2j and 2k are similar to that for evafirenz
and place them within the normal range observed for
oral drugs.22 In contrast, the sub-µg/mL solubilities for
dapivirine and rilpivirine are well outside the normal
range. Though rilpivirine is an FDA-approved oral drug,
it likely has an unusual absorption mechanism involving
aggregates,23 while dapivirine is being pursued as a
vaginal microbicide.24 Thus, compounds 2j and 2k have
good aqueous solubility and potencies similar to that of
nevirapine. Though their activities in the WT cell assay
are 100-fold less than for rilpivirine or dapivirine, their
solubilities are 175 to 350-fold greater.
Finally, to confirm the expectation of the placement
of the morpholinoethoxy side chain in the entrance
channel and to elucidate its contacts, a co-crystal
structure of 2j and WT HIV-RT was pursued by the
investigators at Yale using similar methods as
previously reported.9 Crystals with the recombinant
RT52A enzyme were obtained that diffracted to 2.9-Å
Figure 3. Omit Fo-Fc electron density map at a contour level of 3.0 σ
showing 2j in the NNRTI binding site of HIV-1 RT with extension
towards Glu28 in the entrance channel.

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expressed, purified, and co-crystallized with 1, as
previously described.13 Data collection was performed
at the Cornell High Energy Synchrotron Source
(CHESS) F1 beamline. The diffraction data was
processed and scaled using HKL2000. The structure
was refined to 1.95-Å resolution to R and R-free of
0.237 and 0.275, respectively, using PHENIX. The
atomic coordinates and structure factors are deposited
in the PDB with ID 4KO0.
13. Bauman, J. D.; Das, K.; Ho, W. C.; Baweja, M.;
Himmel, D. M.; Clark, A. D. Jr.; Oren, D. A.; Boyer,
P. L.; Hughes, S. H.; Shatkin, A. J.; Arnold, E.
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Figure 4. Illustration of the crystal structure of 2j with HIV-1 RT; the
morpholinoethoxy side chain projects towards Glu28B. Coordinates have
been deposited in the PDB as structure 4KKO.
would be protonated near pH 7, since the pKa values
for 1,4-dimethylpiperazine are 8.4 and 3.8.26 Thus, a
favorable electrostatic interaction with either Glu138 or
Glu28 has to be sacrificed.
In summary, with compounds 2j and 2k it has been
demonstrated that it is possible to extend anilinylazine
NNRTIs into the entrance channel of the NNRTI
binding site to achieve profound improvements in
aqueous solubility. 2j and 2k are similar in anti-HIV
activity to the drug nevirapine, but they have 200 to
500-fold greater solubility than the parent compound 2a
and the diarylpyrimidines dapivirine and rilpivirine.
Analogous application of the present approach to the
latter series of compounds may be expected to yield
similar benefits for solubility, while hopefully retaining
their excellent activity towards viral variants.
Acknowledgements
Gratitude is expressed to the National Institutes of
Health (AI27690, AI44616, GM32136, GM49551) for
research support and fellowship support for K. M. F.
(AI104334). Receipt of reagents through the NIH AIDS
Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH is also greatly appreciated.
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A.; Anderson, K. S.; Jorgensen, W. L. Bioorg. Med.
Chem. Lett. 2012, 22, 1565.
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X-ray
HIV-RT