Optimization of benzyloxazoles as non-nucleoside inhibitors of HIV-1 reverse transcriptase to enhance Y181C potency

Article abstract of DOI:10.1016/j.bmcl.2012.11.115

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved activity towards Tyr181Cys containing variants was pursued with the assistance of free energy perturbation (FEP) calculations. Optimization of the 4-R substituent in 1 led to ethyl and isopropyl analogs 1e and 1f with 1-7 nM potency towards both the wild-type virus and a Tyr181C variant.

Full text of DOI:10.1016/j.bmcl.2012.11.115

Accepted Manuscript
Optimization of benzyloxazoles as non-nucleoside inhibitors of HIV-1 reverse
transcriptase to enhance Y181C potency
Mariela Bollini, Ricardo Gallardo-Macias, Krasimir A. Spasov, Julian Tirado-
Rives, Karen S. Anderson, William L. Jorgensen
PII:
S0960-894X(12)01567-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2012.11.115
BMCL 19879
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Accepted Date:
31 October 2012
27 November 2012
Please cite this article as: Bollini, M., Gallardo-Macias, R., Spasov, K.A., Tirado-Rives, J., Anderson, K.S.,
Jorgensen, W.L., Optimization of benzyloxazoles as non-nucleoside inhibitors of HIV-1 reverse transcriptase to
enhance Y181C potency, Bioorganic & Medicinal Chemistry Letters (2012), doi: http://dx.doi.org/10.1016/j.bmcl.
2012.11.115
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Optimization of benzyloxazoles as non-nucleoside inhibitors of
HIV-1 reverse transcriptase to enhance Y181C potency
Mariela Bollinia, Ricardo Gallardo-Maciasa, Krasimir A. Spasovb, Julian Tirado-Rivesa,
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
Received October XX, 2012; Accepted Month XX, 2010
Abstract— Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved activity towards Tyr181Cys containing
variants was pursued with the assistance of free energy perturbation (FEP) calculations. Optimization of the 4-R substituent in 1 led to
ethyl and isopropyl analogs 1e and 1f with 1 – 7 nM potency towards both the wild-type virus and a Tyr181C variant. ©2012 Elsevier
Science Ltd. All rights reserved.
Non-nucleoside inhibitors of HIV-1 reverse
transcriptase (NNRTIs) are an important component of
combination therapies for treatment of HIV infection.1,2
Their further improvement is important to seek safer
alternatives that can be given in low dosages and that
may be needed to combat newly emerging viral
variants. Compounds that can penetrate the blood-brain
barrier well might provide an additional advance for
enhance interactions in a distal region of the NNRTI
binding site that might yield general benefits for
activity.7c Specifically, 1a (R = X = H) has an EC50 of
13 nM towards wild-type HIV-1, but shows no activity
towards a Y181C-containing strain. The X = Cl analog
1b fares only a little better with EC50s of 6 nM for the
addressing latent viral reservoirs.3
A continuing
challenge for developing NNRTIs is achievement of
activity against clinically relevant viral variants that
incorporate single and multiple mutations in the reverse
WT virus and 420 nM for the Y181C variant.7b
A
remedy was sought by extending the inhibitors to the
‘east’ to occupy a channel between Phe227 and Pro236.
Though it was possible to replace the cyano group of 1
with novel alternatives, the best that was found for
activity was 2a (R = H) with EC50s of 31 nM and 3 µM,
respectively.7b
As described here, a new effort for the oxazoles 1
has been made by analyzing the potential for productive
modifications of the 4-R group. The strategy was to
seek improved Y181C activity, even if there is some
loss of WT activity. Structural model building with the
BOMB program and OPLS force fields10 suggested that
some elaboration of R might be possible, but that
substituents much larger than methyl might lead to
steric clashes with the WT protein. However, as
illustrated for the case with R = ethyl in Figure 1, it was
expected that a group such as ethyl or propyl might
constructively occupy the space vacated by the Tyr181
transcriptase enzyme (HIV-RT).
A
particularly
troublesome mutation has been Tyr181Cys (Y181C),
which often arises quickly in patients who begin
NNRTI therapy.4 The first generation drugs, nevirapine
and delavirdine, are inactive towards HIV-1 strains with
this mutation, and the second-generation efavirenz is
debilitated by Y181C when combined with
Lys103Asn.1,4 In contrast, the most recent introductions,
etravirine and rilpivirine, show sub-10 nM potency in
cell assays towards these variants and many others.5
In our own work, several new classes of NNRTIs
have been explored.6-9 The Y181C variant has always
been problematic and it has required deliberate efforts
to overcome. One solution
involved reduction of
contact of the inhibitors with Tyr181,8 while another
took advantage of a crystal structure with an alternative
orientation of Tyr181.9 A third approach was to

Table 1
MC/FEP Results for Relative Free Energies of Binding (kcal/mol).
Wild-Type
∆∆Gb
Tyr181Cys
∆∆Gb
a
a
σ
R
H
F
σ
0.0
0.03
0.0
0.07
0.08
0.09
0.11
0.19
0.24
0.26
0.34
0.34
0.35
0.30
0.30
0.0
-0.14
-2.33
-3.09
-4.72
-7.62
-9.18
-10.17
-8.25
-7.59
-8.87
-4.73
-6.07
0.0
0.05
0.07
0.08
0.11
0.18
0.25
0.23
0.34
0.29
0.30
0.28
0.30
Cl
-2.38
-3.33
-4.11
-5.71
-4.34
-4.93
-3.46
-2.55
-3.90
-4.11
-3.14
Clxb
Me
Et
Pr
i-Pr
(R)-s-Bu
(S)-s-Bu
t-Bu
OMe
OEt
σ is the computed uncertainty in ΔΔGb.b Includes an extra point charge
a
on Cl to enable halogen bonding; see ref. 14.
resultant relative free energies of binding are
summarized in Table 1.
For the WT protein, the prediction is that binding
should be enhanced by replacing R = H by a methyl or
ethyl group, but further enlargement would be
detrimental. Halogen and ether substituents are also
predicted to be less effective than small alkyl groups.
Then, the results for the Y181C variant are striking with
the prediction that there would be significantly greater
gain than for the WT enzyme and that peak binding
should occur for R = isopropyl. It might be noted that
by visualization, the R = s-Bu analogs look very nice
with the two carbon branch extending into the Tyr181
cavity (Figure 2). Again, visualization is inadequate.
Thus, the expectation was that for R = Et and i-Pr, the
WT activity should be good relative to R = H, but there
should be significantly greater gains in potency towards
the Y181C-bearing virus.
The synthesis of analogs of 1 then proceeded
featuring the previously utilized cyclization of α-
azidoketones and phenyl isothiocyanates (Scheme 1).7
The azides were prepared from substituted 2,6-
difluorophenylacetic acids via the α-bromo analogs, as
previously described (Scheme 2).7 In turn, the desired
acids were prepared starting with aldehydes, which
were reduced to alcohols using NaBH4. The alcohols
were converted to the bromomethyl derivatives with
CBr4 and PPh3, and then treated with TMSCN using
K2CO3 as an additive in MeCN to provide the
corresponding nitriles in good yield. The nitriles were
finally hydrolyzed to the requisite carboxylic acids
under acidic or basic conditions (Scheme 2).
Figure 1. Snapshots of 1e bound to the NNRTI site for wild-type (top)
and Tyr181Cys HIV-RT from MC/FEP simulations. Carbon atoms of 1e
are in yellow. Some residues are omitted for clarity.
to Cys181 change. The problem with such structural
visualization is that too many complexes look good.
One cannot see if contacts are, in fact, too close, and
one cannot visualize potential entropic losses owing to
restriction of translational, rotational, or torsional
degrees of freedom. Thus, as in the past, we turned to
free-energy perturbation (FEP) calculations with
configurational sampling at 25 ºC using Monte Carlo
(MC) simulations to obtain quantitative predictions.6-10
The MC/FEP calculations followed the same
protocols as previously described.7,8 Initial coordinates
of the complexes were constructed from the 1S9E PDB
file11 using the MCPRO12 and BOMB programs.10 The
Y181C variant was generated manually from the WT
structure and all complexes were relaxed with short
conjugate gradient minimizations. The model included
the 178 amino acid residues nearest the ligand. The
unbound ligands and complexes were solvated in 25-Å
caps with 2000 and 1250 TIP4P water molecules. The
FEP calculations utilized 11 windows of simple overlap
sampling. Each window covered 10-15 million (M)
configurations of equilibration and 20-30
M
Installation of the 4-R group in the benzyl ring of
the aldehydes was generally straightforward starting
with the 4-bromo, hydroxyl, or hydroxymethyl
precursor.15-20 The one problematic case was for R = t-
Bu (Scheme 3). 4-t-Butylbenzoate methyl ester
configurations of averaging. The energetics were
evaluated with the OPLS-AA force field for the protein,
OPLS/CM1A for the ligands, and TIP4P for water.13
The MC/FEP calculations were carried out for both the
WT and Y181C variant of HIV-RT with MCPRO; the

SCN
R
F
PPh , CH Cl ,
3 2 2
O
+
NH
rt, overnight
N
X
F
F
R
F
X
Y
O
Y
1, 2, 3
N
3
X= H, Cl
Y= CN, 3-PyO
Scheme 1. Synthesis of 2-anilinyl-5-benzyloxazoles.
Figure 2. Snapshot of the (R)-s-butyl analog 1i bound to the NNRTI site
for Tyr181Cys HIV-RT from MC/FEP simulations. The s-butyl group is
likely too constrained to yield improved potency over the i-Pr analog.
Scheme 2. Synthesis of α-azidoacetophenones.
Table 2
Anti-HIV-1 Activity (EC50) and cytotoxicity (CC50), μMa
EC50
K103N/
Y181C
Compound
R
X
H
Cl
Cl
WT
Y181C
NA
CC50
8
10
7
31
10
5
65
29
40
15
15
16
5
1a
1b
1c
1d
1e
1f
1g
H
H
F
0.013
0.0062
0.0091
0.011
0.0013
0.0052
0.024
0.068
0.120
0.028
0.0036
0.031
0.005
1.3
NA
NA
NA
7.2
0.21
0.12
10.7
15.0
2.8
0.75
6.3
4.5
0.420
0.910
0.210
0.0069
0.0072
1.5
Scheme 3. Synthesis of a 4-t-butylbenzyl alcohol.
Me Cl
Et Cl
i-Pr Cl
underwent selective dinitration at the positions meta to
the t-Bu group. Both nitro groups were reduced, but
various conditions for a Balz-Schiemann reaction all
failed to effect replacement of the diazonium groups by
fluorine. However, it was possible to prepare the 2,6-
dichloro derivative from the diamine via a Sandmeyer
reaction, which was followed by reduction of the ester
to the alcohol and conversion to the carboxylic acid via
steps b-d in Scheme 2.
c-Pr Cl
CH2c-Pr Cl
s-Bu Cl
OEt Cl
CH2OMe Cl
H
1h
1.2
0.15
1ib
1j
1k
2a
0.048
0.690
3.2
0.060
NA
2b
Et
0.80
NA
3c
t-Bu Cl
6
nevirapine
efavirenz
etravirine
rilpivirine
0.11
0.002
0.001
NA
0.010
0.008
NA
>100
15
11
8
0.030
0.005
0.002
The identities of all assayed compounds were
0.00067 0.00065
1
confirmed by H and 13C NMR and high-resolution
a Results using human MT-2 cells. Antiviral and toxicity curves used
triplicate samples at each concentration. NA = not active. b Racemic. c
The 4-t-butyl-2,6-dichlorobenzyl analog of 1b.
mass spectrometry; purity was >95% as judged by high-
performance liquid chromatography. Activities against
the IIIB and variant strains 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. CC50
values for inhibition of MT-2 cell growth by 50% are
obtained simultaneously.6-9,21,22 The results are
summarized in Table 2.
For the WT virus, the ethyl analog 1e did turn out to
be the most potent with an EC50 of 1 nM. The gain of a
factor of 5 over 1b is less than what might have been
expected from Table 1. However, computed free
energies of binding are being compared to activities in a
cell-based assay and a compression of the computed
results is normal.6-10,23 Additional elaboration of the R
group was unproductive, as expected, and the 4-t-butyl-
2,6-dichloro analog 3 is only a 1 µM NNRTI. For the
Y181C HIV-1 variant, it was gratifying that the
qualitative predictions from the MC/FEP calculations
were confirmed. Indeed, there is pronounced
improvement in the activity towards the Y181C-
containing virus peaking with the ethyl and isopropyl
analogs 1e and 1f with EC50s of 7 nM. As from the
computations, the s-Bu analog 1i is significantly less
active. For 1f, the potency towards the WT and Y181C
variant form of HIV-1 are essentially the same, in sharp
contrast to the case for the parent 1b. There is also
improvement in potency towards the challenging
K103N/Y181C double mutant. Though 1f has about the
same potency towards HIV-1 bearing the two mutations
as the drug nevirapine has towards the WT virus, the
potencies of etravirine and rilpivirine towards the
variant are 24- and 60-fold greater still.
Another interesting comparison is for the isopropyl
and cyclopropyl analogs 1f and 1g. The cyclopropyl

Medaer, B.; De Knaep, F.; Bohets, H.; De Clerck, F.;
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compound is less active against the WT virus by a
factor of 5 and 200-fold less active towards the Y181C
variant. In the MC/FEP simulations, the isopropyl group
is oriented much as in Figure 1 (bottom) with significant
projection of one methyl group towards the Tyr181
cavity. Though FEP calculations were not done for the
cyclopropyl analog, model building does show the
expected reduced projection owing to the ca. 60º
internal CCC angles. A cyclopropyl group is decidedly
more compact than an isopropyl group, which is
disadvantageous in the present case. A cyclopropyl
group is also somewhat less lipophilic as apparent in
observed octanol/water log P values of 3.3 for
cyclopropylbenzene and 3.7 for isopropylbenzene.24
Finally, to integrate the investigation of the ‘eastern
extensions’7b with the present work, 2b was
synthesized. In comparison to 2a, addition of the ethyl
group provides a 5-fold activity boost against the WT
virus and a 50-fold gain against the Y181C-bearing
variant. The fold gains are almost the same as for the 1b
and 1e pair.
In summary, further study of oxazoles 1 as NNRTIs
has focused on optimization of the 4-R group to yield
greater potency against strains of HIV-1 containing the
Tyr181Cys mutation in the reverse transcriptase
enzyme. Results of MC/FEP calculations were highly
provocative in predicting that substantial differential
gains would be obtained for the viral variant over the
wild-type virus for R = ethyl and isopropyl. This was
confirmed experimentally through synthesis and
assaying of the analogs in Table 2. 1e and 1f emerged as
NNRTIs with sub-10 nM potency towards both viral
strains.
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Acknowledgements
Gratitude is expressed to the National Institutes of
Health (AI44616, GM32136, GM49551) for support.
Receipt of reagents through the NIH AIDS Research
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Optimization of benzyloxazoles as non-nucleoside inhibitors of HIV-1
reverse transcriptase to enhance Y181C potency
Mariela Bollini, Ricardo Gallardo-Macias, Krasimir A. Spasov, Julian Tirado-Rives,
Karen S. Anderson,* and William L. Jorgensen*
Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved
activity towards Tyr181Cys containing variants was pursued with the assistance of free
energy perturbation (FEP) calculations. Optimization of the 4-substituent in the benzyl
ring of 1 led to ethyl and isopropyl analogs with 1 – 7 nM potency towards both the wild-
type virus and a Tyr181C variant