Computationally-guided optimization of a docking hit to yield catechol diethers as potent anti-HIV agents

Article abstract of DOI:10.1021/jm201134m

A 5-μM docking hit has been optimized to an extraordinarily potent (55 pM) non-nucleoside inhibitor of HIV reverse transcriptase. Use of free energy perturbation (FEP) calculations to predict relative free energies of binding aided the optimizations by identifying optimal substitution patterns for phenyl rings and a linker. The most potent resultant catechol diethers feature terminal uracil and cyanovinylphenyl groups. A halogen bond with Pro95 likely contributes to the extreme potency of compound 42. In addition, several examples are provided illustrating failures of attempted grafting of a substructure from a very active compound onto a seemingly related scaffold to improve its activity. (Figure presented)

Full text of DOI:10.1021/jm201134m

Article
pubs.acs.org/jmc
Computationally-Guided Optimization of a Docking Hit to Yield
Catechol Diethers as Potent Anti-HIV Agents
Mariela Bollini,† Robert A. Domaoal,‡ Vinay V. Thakur,† Ricardo Gallardo-Macias,† Krasimir A. Spasov,‡
Karen S. Anderson,*,‡ and William L. Jorgensen*,†
†Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, United States
‡Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, United States
S
* Supporting Information
ABSTRACT: A 5-μM docking hit has been optimized to an extraordinarily potent (55 pM)
non-nucleoside inhibitor of HIV reverse transcriptase. Use of free energy perturbation (FEP)
calculations to predict relative free energies of binding aided the optimizations by iden-
tifying optimal substitution patterns for phenyl rings and a linker. The most potent
resultant catechol diethers feature terminal uracil and cyanovinylphenyl groups. A halogen
bond with Pro95 likely contributes to the extreme potency of compound 42. In addition,
several examples are provided illustrating failures of attempted grafting of a substructure
from a very active compound onto a seemingly related scaffold to improve its activity.
progressed to phase III clinical trials.9 Various attributes of 1
are appealing including the fact that it is a diphenylmethane
INTRODUCTION
Though inhibition of multiple HIV proteins is therapeutically
■
viable, HIV reverse transcriptase (RT) has been the key target.1
derivative with a novel terminal uracil group, that it likely has
diminished metabolic liabilities compared to those of 3, and
Nucleoside RT inhibitors (NRTIs) including AZT are incor-
better computed aqueous solubility according to QikProp,10
porated into the product DNA causing premature strand termi-
and refinement of substituents in the phenyl rings can be
nation, while the non-nucleoside RT inhibitors (NNRTIs) bind
expected to be productive. Thus, optimization of 1 was initiated
to an allosteric site ca. 10-Å away from the polymerase active
site.2 Our efforts at discovery of new NNRTIs are intended to
using a computationally driven approach, primarily guided by
results of free-energy perturbation (FEP) calculations for
address continuing issues concerning the possible emergence of
new viral strains, improved dosing, long-term tolerability, and
complexes of the inhibitors with HIV-RT.4
safety.3 Numerous compounds in multiple series have been
prepared that are both potent against the wild-type (WT) virus
and that have auspicious computed pharmacological proper-
ties.4,5 Improvement in the performance of our compounds
against clinically relevant viral variants is still desired. To address
resistance from the outset, docking was done on multiple RT
structures to seek consensus high-scoring hits. More than two
million compounds from the ZINC library were screened with
Glide using a conventional WT structure (1rt4), one with an
alternative “down” conformation for Tyr181 (2be2) and a
structure that incorporated the troublesome Tyr181Cys muta-
tion (1jla).6 Though only nine compounds were purchased,
three showed 5−12 μM activity against one or both viral strains
in infected T-cell assays.
As described here, among the three actives, we have most
pursued lead-optimization for compound 1, which showed
4.8 μM potency toward WT HIV-1.6 Compound 1 bears some
structural similarity to the ligands, TNK-651 (2) and R221239
(3), from the 1jla and 2be2 crystal structures, respectively.7,8
Their roots can be traced back further to thymine analogues in
the HEPT class including emivirine (MKC-442, 4), which
EXPERIMENTAL AND COMPUTATIONAL
METHODS
■
Ultimately, the synthetic efforts focused on the preparation of diphe-
nylmethanes (Scheme 1) and catechol diethers (Schemes 2−4). The
o-benzylphenols in Scheme 1 arose from Friedel−Crafts reactions of
arylmethyl halides or alcohols with phenols.11,12 The catechol ether
intermediates were prepared from substituted phenols and the aryl
fluorides using SNAr reactions followed by treatment with boron
tribromide or lithium chloride (Schemes 2 and 3). The final
Received: August 24, 2011
Published: November 14, 2011
© 2011 American Chemical Society
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a
Scheme 1. Synthesis of Compounds 5−15
a
Reagents: (a) DIAD, bromoethanol, Ph3P, THF, rt, overnight; (b) 180 °C, 1.5 h.
a
Scheme 2. Synthesis of Compounds 20−27, 36, and 44
a
Reagents: (a) K2CO3, DMSO, 110 °C, 2−5 h; (b) BBr3, CH2Cl2, −78 °C, overnight; (c) DIAD, bromoethanol, Ph3P, THF, rt, overnight; (d) 2,4-
bis((trimethylsilyl)oxy)pyrimidine, 180 °C, 1.5 h, or 3-benzoylpyrimidine-2,4(1H,3H)-dione, K2CO3, DMF, overnight, rt then MeOH, NH4OH, 2 h, rt;
o
(e) Fe, NH4Cl, EtOH, H2O, 75 °C; (f) NaNO2, conc HCl, KI, 80 °C, 2 h; (g) acrylonitrile, PdCl2(PPh3)2, Et3N, DMF, 140 C, 2 h.
compounds were prepared in a two-step sequence via the Mitsunobu reac-
tion to install the bromoethoxy linker, followed by 2,4-bis(trimethylsiloxy)-
The principal computations were conjugate-gradient energy
minimizations and Monte Carlo/FEP (MC/FEP) calculations, which
yield relative free energies of binding. The calculations were performed
with the MCPRO program18 and followed standard protocols.4,5
Coordinates of HIV-RT complexes were mostly constructed from the
2be2 crystal structure8 using the BOMB program.4 The model
included the 175 amino acid residues nearest the ligand. Short
conjugate-gradient minimizations were carried out on the initial
structures for all complexes to relieve any unfavorable contacts.
Coordinates for the free ligands were obtained by extraction from the
complexes. The unbound ligands and complexes were solvated in
TIP4P water spheres (“caps”) with a 25-Å radius; after the removal of
water molecules in too close contact with solute atoms, ca. 2000 and
1250 water molecules remained for the unbound and bound MC
simulations. The FEP calculations utilized 11 windows of simple
overlap sampling.19 Each window covered 10−15 million (M) con-
figurations of equilibration and 10−30 M configurations of averaging
at 25 °C. The ligand and side chains with any atom within ca. 10 Å
of the ligand were fully flexible, while the protein backbone was kept
fixed during the MC runs. The energetics were evaluated with the
OPLS-AA force field for the protein,20 OPLS/CM1A for the ligands,21
and TIP4P for water.22
pyrimidine alkylation (5−15 and 20−32).13
However, when substituent X was (E)-cyanovinyl (CV), methox-
yethoxy (MOEO), or 3-hydroxypropan-1-oxy (HOPO), the last step
did not yield the desired product. To circumvent this, the N-benzoyl
uracil derivatives14 were made followed by alkylation with the alkyl bro-
mide and cleavage of the benzoyl moiety to afford the target compounds
in good yields (35−49).15
In particular, when 3-cyanovinyl-substituted catechol ethers were re-
quired, Heck coupling of aryl iodides with acrylonitrile using PdCl2(PPh3)2
as catalyst was effective. This reaction afforded separable mixtures of E:Z
(70:30) stereoisomers in 50−70% yield.
Finally, compounds 33 and 34 were obtained via the Mitsunobu re-
action with the corresponding alcohols and catechol ethers (Scheme 4).
For compound 35, the aliphatic linker was added via the alkylation of
66 with 4-bromomethylpyridine. The identity of all assayed com-
pounds was confirmed by 1H and 13C NMR and high-resolution mass
spectrometry; purity was >95% as judged by high-performance liquid
chromatography. Small molecule crystal structures were obtained by
direct methods on data collected using a Rigaku Mercury2 CCD area
detector with graphite monochromated Mo−Kα radiation.
For the biology, activities against the IIIB and variant strains of
HIV-1 were measured using MT-2 human T-cells; EC50 values are ob-
tained as the dose required to achieve 50% protection of the infected
cells by the MTT colorimetric method. CC50 values for the inhibi-
tion of MT-2 cell growth by 50% are obtained simultaneously.5,16,17
The antiviral and toxicity curves used triplicate samples at each
concentration.
RESULTS AND DISCUSSION
■
A typical structure for the complexes is illustrated in Figure 1.
As expected from the crystal structures for HEPT analogues
and 3, the terminal phenyl ring resides in the π-box formed by
Tyr181, Tyr188, Phe227, and Trp229, and the uracilylethoxy
side chain projects into the channel lined by Pro225, Phe227,
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a
Scheme 3. Synthesis of Compounds 28−32 and 40−47
a
Reagents: (a) K2CO3, DMSO, 110 °C 2−5 h; (b) Fe, NH4Cl, EtOH, H2O, 75 °C; (c) NaNO2, conc HCl, CuCl, 80 °C, 1 h; (d) CH3B(OH)2,
Cu(OAc)2, pyr, dioxane, reflux; (f) acrylonitrile, PdCl2(PPh3)2, Et3N, DMF, 140 o C, 2 h; (g) BBr3, CH2Cl2, −78 °C, overnight or LiCl, DMF 160 °C,
overnight; (h) DIAD, bromoethanol, Ph3P, THF, rt, overnight; (i) 3-benzoylpyrimidine-2,4(1H,3H)-dione, K2CO3, DMF, rt, overnight then NH4OH,
MeOH, rt.
a
Scheme 4. Synthesis of Compounds 33−35
a
Reagents: (a) DIAD, bromoethanol, Ph3P, THF, rt, overnight; (b) NaBH4, MeOH, 0 °C to rt, 3 h; (c) NaH, DMF, rt, 2 h.
Pro236, and Tyr318. The computations further indicate that
alternative conformations are possible for the side chain with
the two illustrated in Figure 1 as the most probable. The
gauche-anti-anti (gaa) conformer on the left is close to that
found for the methoxymethyl fragment in the crystal structures
for HEPT analogues,7 while the aag alternative allows the
formation of a hydrogen bond between the carbonyl group of
Lys103 and the uracilyl NH. It should also be noted that the
“down” orientation of the phenolic unit of Tyr181 follows from
the 2be2 structure.8 It is possible that for some of the inhibitors
considered here that the more parallel “up” orientation with
Tyr181 and Tyr188, as in the HEPT structures,7 is preferred.
Diphenylmethanes. The MC/FEP calculations began
with a chlorine scan for the terminal phenyl ring in 1. With
numbering the ring such that the chlorines in Figure 1 are at
the 2- and 5-positions, replacement of hydrogen by chlorine
was predicted to be favorable (more negative free energy of
binding, ΔGb) by 1.4, 1.9, and 3.8 kcal/mol at C2, C5, and C6
and unfavorable by 1.4 and 0.7 kcal/mol at C3 and C4. The
computed uncertainties are ±0.2 kcal/mol. However, since
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Figure 1. Computed structures of a catechol diether (31) bound to HIV-RT starting from the 2be2 crystal structure. Two possible conformations of
the uracilylethoxy side chain, gaa (left) and aag (right), are illustrated. Carbon atoms of the inhibitor are colored gold.
double substitution was envisioned and the results might not
be additive, MC/FEP calculations were executed for all 10
unique dichloro possibilities, as summarized in Table 1. Such
Table 2. Anti-HIV-1 Activity (EC50) and Cytotoxicity (CC50)
(μM) of Diphenylmethane Derivatives
Table 1. MC/FEP Results for the Change in Free Energy of
Binding upon Introducing Two Chlorines in the Terminal
a
Phenyl Ring
compd
R1
X
Y
R2
EC50
CC50
1
4-Me
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
5-Cl
H
H
H
H
H
H
H
H
H
H
Me
F
4.8
72
23
28
11
88
10
15
49
22
27
21
12
5
H
H
1.2
6
2-Cl
3-Cl
2-Cl
3-Cl
2-Cl
2-Cl
H
H
0.62
1.5
7
H
8
4-Cl
5-Cl
5-Cl
6-Cl
H
2.9
H to Cl
ΔΔGb
σ
9
1.3
C2/C3
C2/C4
C2/C5
C2/C6
C3/C4
C3/C5
C3/C6
C4/C5
C4/C6
C5/C6
2.00
2.43
0.18
0.20
0.22
0.25
0.23
0.19
0.24
0.23
0.21
0.20
10
11
12
13
14
15
0.38
0.31
2.4
−2.12
−3.69
6.27
H
H
2.5
H
H
Cl
H
2.2
−1.13
3.63
2-Cl
H
0.41
1.79
more potent against the WT virus with an EC50 of 1.2 μM. For
substitution in the terminal phenyl ring, 2-Cl substitution (6)
was found to be more favorable than the 3-Cl alternative (7),
consistent with the computed results for C2(C6) and C3(C5).
The disubstituted compounds also mostly followed the com-
puted trends with the most potent being the 2,6-analogue (11)
at 310 nM, which was followed by the 2,5- and 3,5-analogues,
10 and 9, at 380 nM and 1.3 μM. The 2,4-dichloro analogue 8
was less active than anticipated, and the range of activities was
compressed from what might have been expected from the
computed ΔΔGb values. The compression is normal and may
result from comparing free energies of binding with results of
cell-based assays and force-field inadequacies.4,5 Overall, a ca.
15-fold gain in potency was achieved in going from 1 to 10 or 11.
It is notable that the FEP calculations pointed out the
viability of 2,5- and 2,6-disubstitution, whereas the literature on
more potent HEPT and emivirine analogues is dominated by
3,5-disubstituted cases.23 The same is true for the compounds
in the 3-series.24 In the absence of the FEP results, even with
−2.26
2.52
a
ΔΔGb is the computed change in free energy of binding (kcal/mol)
for introducing the two chlorines; ± σ is the computed uncertainty.
C6 is proximal to Tyr181 in the binding site (see Figure 1).
exhaustive double substituent scans should find general utility
in ligand design. The results continued to support the viability
of substitutions at C2, C5, and C6 with the most favorable
di-substitutions being C2/C5, C2/C6, and C4/C6, which
correspond to the 2,5-, 2,6-, and 2,4-dichloro analogues. The
3,5-dichloro analogue is also predicted to be better bound than
the parent compound. A term that could be added for the 3,5-
and 2,6-analogues is a factor of 2 symmetry benefit (−0.4 kcal/
mol) over the unsymmetrical 2,5- or 2,4-analogues, since only
one conformer is favorable in the latter cases (C2/C5 and
C4/C6).
Related experimental results are presented in Table 2. The
methyl group in 1 was replaced by chlorine to give 5, which is
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display of the optimized structures, the preferences are not
obvious. Most of the disubstituted possibilities look reasonable
with the possible exception of X or Y = 4-Cl, which appears to
yield a steric conflict with Trp229.
Table 3. MC/FEP Results for the Change in Free Energy of
Binding (kcal/mol) upon Introducing Two Chlorines in a
Catechol Diether
Several additional diphenylmethane derivatives were prepared.
The results for 12−14 in comparison with those for 5 demonstrate
that substitution at the 5-position in the uracil ring is not beneficial,
while the results for 15 vs 6 show that a 5-Cl substituent in the
central ring is somewhat preferred to the 4-Cl isomer.
Catechol Diethers. The next consideration was the
position of the oxygen in the linkers, especially given the alter-
natives suggested by 2 and 3. Thus, MC/FEP calculations were
executed for perturbing structure 16 with X = Y = Z = CH2
to the three compounds in which X, Y, or Z are individually
oxygen. The resultant ΔΔGb values were −5.95, 0.64, and
−2.07 kcal/mol, respectively, with uncertainties of ±0.3 kcal/
mol. Thus, the phenoxy substructure as in 3 was predicted to be
much favored. This likely reflects conformational preferences.25
It is easier for a diphenyl ether fragment than a diphenyl-
methane one to achieve the near perpendicular arrangement,
illustrated in Figure 1, which is most complementary to the
positioning of Tyr188 and Trp229.
H to Cl
ΔΔGb
σ
C2/C3
C2/C4
C2/C5
C2/C6
C3/C4
C3/C5
C3/C6
C4/C5
C4/C6
C5/C6
−3.87
1.88
−5.56
1.44
0.14
0.20
0.18
0.21
0.18
0.15
0.24
0.17
0.30
0.19
2.82
−3.28
6.26
2.16
6.42
4.48
As recorded in Table 4, headway was rapidly made. The
results for 20−25 show that the 2,5-, 3,5-, and 2,6-substitution
Table 4. Anti-HIV-1 Activity (EC50) and Cytotoxicity (CC50)
(μM) of Catechol Diether Derivatives
a
compd
R1
4-Cl
X
Y
R2
EC50
0.14
CC50
20
21
22
23
24
25
26
27
28
29
30
31
32
40
41
42
43
44
45
46
47
48
49
3-CN
2-Cl
5-CN
6-CN
5-CN
5-CN
5-CN
5-CN
5-CN
5-CN
5-CN
5-CN
H
H
H
H
H
H
H
F
Cl
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
80
4-Cl
4-Cl
5-Cl
5-Cl
0.12
50
33
60
>100
>100
17
31
>100
94
>100
71
>100
>100
19
3-Cl
2-Cl
0.090
0.100
0.046
0.014
0.013
0.017
6.0
3-CN
3-Cl
5-Cl
In fact, when a gas-phase conformational search is performed
for the parent o-methoxy derivatives of diphenylmethane and
diphenyl ether with the OPLS-AA force field, conformers 17
and 18 are the global minima. For 17, there is only this mini-
mum, while there are 5 unique minima for 18. Conformer 19 is
the second minimum, only 0.37 kcal/mol higher in energy than
18. Thus, catechol diethers like 18 are well preorganized to
bind to HIV-RT in the desired manner (Figure 1).
5-Cl
5-Cl
3-Cl
3-Cl
3-Cl
3-Cl
5-CN
5-CN
5-CN
5-CN
5-CN
5-CN
5-Cl
5.3
0.27
2-Cl
2-Cl
3-Cl
5-Cl
5-Cl
H
0.043
0.020
0.015
0.005
0.000055
0.0032
0.00032
0.020
0.16
3-CV
3-CV
3-CV
3-CV
3-CV
3-CV
2-Cl
H
Given these results, a complete dichlorine FEP scan was per-
formed for the model inhibitor in Table 3. The results favor 2,3-,
2,5-, and 3,5-substitution, with the 2,5-pattern, illustrated in Figure 1,
the most favored. A monochlorine scan for the catechol ring was
also performed for the corresponding 3,5-dichlorophenyl analog.
This yielded ΔΔGb values of −1.2, −2.6, −3.1, and −2.8 kcal/mol
(±0.2 kcal/mol) for introducing a chlorine at the 3′, 4′, 5′, and 6′
positions. Consequently, the synthetic focus turned to catechol
diethers with the favored substitution patterns in both rings. How-
ever, since SNAr chemistry was envisioned for the synthesis of the
core (Schemes 2 and 3), an activating group was needed in one of
the rings; cyano groups were chosen as they are the most similar
sterically to chlorine, and model-building indicated that they should
be viable.
5-Cl
5-Cl
5-F
5-F
5-Cl
H
10
5-Cl
5-F
48
45
30
>100
13
5-NHMe
3-F,5-CN
3-Cl,5-Cl
5-CN
5-CN
2-Cl
H
0.83
3-MOEO
3-HOPO
H
H
0.54
1.8
>100
>100
a
CV = (E)-cyanovinyl; MOEO = methoxyethoxy; HOPO = 3-hydroxy-
propan-1-oxy.
patterns for the phenoxy ring all provide active compounds, and
a chlorine is indeed preferred at the 5′ position in the catechol
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ring over the 4′-alternative. Compound 25 is a potent NNRTI
with an EC50 of 14 nM, and it has a large safety margin since no
cytotoxicity was observed to the limit of the tested con-
centration range, 100 μM. The results for 26 and 27 then
reconfirm that small substituents at C5 in the uracil ring have
little effect. However, potency is dramatically lost by replace-
ment of the 5′-Cl by cyano in 28 and 29. Modeling indicates
that the 5′-nitrile nitrogen in the complexes is ca. 3.1 Å from
the carbonyl oxygen of Lys101. In addition, if a 5-cyano group
in the phenoxy ring is positioned over the catechol ring, there
would be dipole−dipole repulsion between the two cyano
groups. The situation is strikingly relieved by replacing the
phenoxy cyano group with chlorine in progressing to 32,
which brings the anti-HIV activity back to 20 nM. The good
potency, 43 nM, for the 2,5-dichloro isomer 31 (Figure 1) is
also consistent with the expectations from the FEP results, and the
benefit of the second chlorine is apparent in comparison to the
results for 30. The difference in performance of the 3-chloro,5-
cyanophenoxy substituent depending on the substitution of the
catechol ring is notable (22, 25, and 28). The spectroscopic
characterization of the structures for some of the compounds
was also confirmed by crystallography, e.g., for 20 in Figure 2.
Thus, deletion of the uracil, which might be suggested by
considering emivirine (4), is detrimental.
By this juncture, some results on the activity of the more
potent compounds toward variant strains of HIV-1 containing
the Tyr181Cys mutation and the challenging Lys103Asn/
Tyr181Cys double mutation in HIV-RT had been obtained
(Table 5). Optimism might be justified from the general
Table 5. Anti-HIV Activity (EC50) and Average Cytotoxicity
(CC50) (μM) of Catechol Diether Derivatives and Reference
NNRTIs for WT and Viral Variants
EC50
compd
20
WT
0.14
Y181C
K103N/Y181C
CC50
Figure 2. Small molecule crystal structure of 20. Details are in the
Supporting Information.
21
2.2
77
22
0.090
0.014
6.7
1.2
32
It is noted that the conformation of 20 in the crystal lattice is
identical to that for the aag conformer of 31 in Figure 1.
At this point, consideration was given to possible replacement of
the uracil group and/or variation of the linking chain. Though
multiple options were tried, none emerged as competitive with the
uracilylethoxy substituent. For example, 33, the 4-pyridinyl analogue
of 31, was synthesized and found to worsen the EC50 value 18-
fold to 0.76 μM. The isomer 34 with the methylenoxymethyl
linker was significantly less potent still (12 μM), and shorten-
ing the linker to OCH2 in 35 also did not improve the activity
(1.8 μM). The 3-pyridinyl-, 2,4-pyrimidinyl-, 5-pyrazolyl-, 5-oxa-
zolyl-, and 4-pyridinyl-N-oxide-analogues of 34 also showed no
activity below micromolar levels.
Thus, the transfer of parts of a potent inhibitor to a less
potent one, e.g., the methyleneoxymethyl linker of 2 to 33 or
35, is an undependable strategy.26 Conformational differences
are affected by the rest of the structures. For the present cate-
chol derivatives, in conformational searches using OPLS-AA for
2-ethoxyanisole, the aa and ga conformers for the ethoxy group
are the lowest in energy; they correspond to the conformers in
Figure 1. For 2-methoxymethylanisole, the corresponding ga
conformer is not an energy minimum; it collapses to aa. Finally,
the importance of the uracilylmethyl group was clearly demon-
strated through the preparation of 36, whose EC50 of 9.9 μM
can be compared to the 14 nM for 25. The benefits of addi-
tional van der Waals interactions and the hydrogen bond
between Lys103 and the uracilyl NH are likely contributors.
25
0.52
1.7
>100
13
26
0.013
0.62
1.7
27
0.017
0.24
0.57
2.8
21
31
0.043
0.92
59
32
0.020
0.80
>43
8.0
>61
>90
22
40
0.015
0.98
41
0.005
0.37
1.6
42
0.000055
0.0032
0.00032
0.020
0.049
0.15
0.22
0.90
0.085
ND
10
43
48
44
0.016
2.8
45
45
30
nevirapine
nevirapine
efavirenz
0.11
>100
>10
>100
>10
0.030
0.037
0.005
0.004
0.002
0.001
>100
a
0.081
0.002
0.010
0.002
0.008
0.007
0.00065
0.0013
15
11
a
efavirenz
0.0014
0.001
etravirine
a
etravirine
0.001
rilpivirine
rilpivirine
0.00067
0.0004
>1
8
a
a
Literature data; see ref 28.
topological similarities of 3 and the present catechol diethers
and the fact that 3 is reported to show 1−10 nM activity toward
WT HIV-1 and many variants including Y181C and K103N/
Y181C.8 However, the catechol diethers through compound
32 are not as potent against WT HIV-1 as 3 (2 nM), and the
performance against the Y181C variant and double mutant was
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also found to be diminished (Table 5). The best compound
at this point was 27, which has EC50 values of 17, 240, and
570 nM toward WT, Y181C, and the K103N/Y181C variants.
The 14- and 34-fold ratios between the variant and WT activities
are better than typical,24 so it seemed that a prerequisite for
further improvement was to drive down the WT EC50 by
another factor of 10 or more.
Cyanovinyl Analogues. To this end, introduction of a
cyanovinyl group in the terminal phenyl ring was considered. This
had proved profitable in another series, which included 37,27 and
it also finds precedent in R165481 (38)8 and rilpivirine (39).28 All
three of these cyanovinyl-containing NNRTIs have activities below
5 nM for both WT HIV-1 and the Y181C-containing variant.
considered with the cyanovinyl group at the 3-position. Energy
minimizations for the complexes of the three alternatives
indicated a preference for substitution at C5 or C6 based on the
protein−ligand interaction energies of −70.0, −73.9, and −73.1
kcal/mol. The interaction energy for the reference compound
41 is −71.4 kcal/mol. The 4-chlorine leads to a steric clash with
Trp229, while the 5-chlorine projects into a pocket formed by
Pro95, Pro97, Leu100, and Tyr181, and the 6-chlorine nuzzles
between Leu100 and Tyr181. However, for the C5 and C6
options, evaluation of the conformational energetics suggests
more ligand strain for the C6 isomer owing to the placement
of the 6-Cl over the catechol ring. In view of the interaction
energy improvement for the addition of the 5-chlorine and the
anticipated benefits of burial of more hydrophobic surface area,
a significant activity boost was expected.
Thus, synthesis of the 3-cyanovinyl, 5-Cl analogue 42 was
carried out, and the activity results were gratifying. It is an
extraordinarily potent NNRTI. The initial WT assay yielded
an EC50 of 24 pM. To our knowledge, this is the lowest EC50
for an NNRTI that has been reported. Several pyrimidinone
“SDABO” analogues have been reported to have EC50 values
for WT HIV-1 (NL4-3) in the 70−440 pM range,29 and 39 is
another very potent NNRTI.28 Compound 42 was reassayed
side-by-side with 39 yielding EC50s of 55 and 670 pM, which
are the values reported in Table 5. A 5- or 6-substituent in the
phenoxy ring is also expected to help fill the space vacated upon
mutation of Tyr181 to cysteine. This notion and the benefits
of the improved activity for the cyanovinyl-containing inhib-
itors yielded EC50 values for 42 of 49 and 220 nM for the
variant HIV-1 strains containing the Y181C and K103N/Y181C
mutations (Table 5).
However, in view of the caveats above concerning the
methylenoxymethyl linker and uracil, modeling was carried
out, which did indicate that a m-cyanovinyl group could be
incorporated in the current series (Figure 3). It should make
Close analogues of 42 with replacement of the 5-Cl with
fluorine (43) or both chlorines with fluorines (44) were then
prepared. Compound 43 is significantly less active at 3.2 nM
for the WT virus, while 44 is still very potent at 320 pM.
Furthermore, 44 shows improved potency toward the viral
variants, 16 nM for Y181C and 85 nM for K103N/Y181C
(Table 5). The activity patterns for NNRTIs toward viral
variants are often not straightforward to interpret. For example,
the most potent S-DABO in one report has EC50 values of
100−400 pM toward WT HIV-1, but is no better than 870 nM
toward a K103N-bearing variant.29a However, it is generally
thought that smaller, flexible inhibitors have an advantage in
being able to better adjust to the structural changes that
accompany point mutations.28b This can help rationalize the
improved performance of the difluoro analogue 44 over 42
toward the mutant strains and the still better results with 39.
In particular, greater contact with Lys103 is expected for
the present compounds (Figure 3) than for rilpivirine. Thus, it
is not surprising that mutations of Lys103 may be more
detrimental for 42−44.
Table 5 includes data for four approved drugs with results
from both our measurements using infected MT2 cells and
those of Janssen et al. using MT4 cells;28 the directly com-
parable results are almost all within a factor of 2. In comparison
to the approved drugs in Table 5, 42 is by far the most potent
toward the wild-type virus. Compounds 42 and 44 also show
good potency toward the two mutant strains, though not at the
low-nanomolar levels of rilpivirine (39). The relatively low
cytotoxicity, CC50, toward human T-cells of many of the
present compounds in comparison to that of the most potent
drugs is also notable.
Figure 3. Computed structure of 42 (JLJ0494) bound to HIV-RT
illustrating the positioning of the cyanovinyl group between Tyr188
and Phe227. Carbon atoms of 42 are colored gold.
favorable van der Waals contact with Trp229 and be positioned
between Tyr188 and Phe227, as is observed in the crystal
structures for the complexes of 38 and 39.8,28 Since rilpivirine is
a marketed drug, potential liabilities of the cyanovinyl group as
a weak Michael acceptor seem acceptable.
Thus, continuing from 32 in Table 4, the 3-cyanovinyl
analogue 40 was prepared and showed improved activity at
15 nM. Switching to chlorine instead of cyano at the 5-position
in the catechol ring brought an expected further enhancement,
specifically, to 5 nM for 41. Then, for reintroduction of a
chlorine in the phenoxy ring, the 4-, 5-, and 6-Cl isomers were
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The computed structure for the complex of 42 (Figure 3)
indicates that 42 fills the space extremely well in the channels
running from Tyr181 past Trp229 and toward Phe227 and
Pro236. The hydrogen bond with the carbonyl group of Lys103
remains notable. Furthermore, there may also be a boost for a
halogen bond between the 5-Cl and the carbonyl oxygen of
Pro95.30 This is illustrated in Figure 4, though standard force
chlorine, bromine, and iodine enhanced the binding by factors
as large as 24, 49, and 74, respectively.32
A few additional compounds were synthesized. (1) The
analogue of 42 with a methylamino group at C5 in the catechol
ring, 45, is much less active (20 nM). Though it might benefit
from a favorable electrostatic interaction between the amino
group and the carbonyl of Lys101, the dehydration penalty
would be greater than that for 42. (2) Returning to 2 and 3,
one might be motivated to include a substituent at the 3-
position in the catechol ring, though the FEP results noted
above indicated that substitution at that site with chlorine
would be the least beneficial of the four possibilities. Addition
of the 3-fluoro group in 46 (0.16 μM) was found to provide
slight improvement over the results for 30 (0.27 μM), while the
3-chlorine in 47 (0.83 μM) was detrimental since the reference
compound would be expected to be more potent than 30. In
this case, the reason why the mapping from the precedents
fails is likely associated with the fact that 2 and 3 both form a
hydrogen bond with their NH groups and the carbonyl of
Lys101. As the 5-Cl or 5-CN in 46 or 47 aligns with the NH,
the present compounds are pushed away and rotated from
Lys101, which changes the positioning of the catechol ring
versus the central rings in 2 and 3. (3) As the region behind
Tyr188, Phe227, and Trp229 in Figure 3 opens into a solvent
Figure 4. Computed structure of 42 bound to HIV-RT illustrating
the possible halogen bond between the oxygen of Pro95 and the
5-chlorine in the terminal phenyl ring of the inhibitor. The O−Cl
distance is 3.4 Å.
fields including OPLS do not account for the benefits of
halogen bonding.31 The simple point-charge model with a
partial negative charge on chlorine is inadequate to describe
correctly the electrostatic field along the C−Cl bond axis.30
Nevertheless, the present, computed O−Cl distance of 3.43 Å
is near the ideal value of ca. 3.3 Å for such interactions.30,31 It
may be noted that when the complex is reoptimized with the
only change being replacement of the cyanovinyl group in 42
by chloro or cyano, the O−Cl distance increases to 3.62 and
3.72 Å, respectively. The shift toward Pro95 for 42 improves
the contact between the first methine unit of the vinyl group
and Trp229. Thus, the optimal halogen bonding likely requires
both the 3-chloro and 5-cyanovinyl groups. The significant
decrease in activity in going from 42 to 43 is also consistent
with loss of the halogen bond upon replacement of chlorine
with fluorine. Notably, a recent report on inhibition of cathep-
sin L also found large binding enhancements upon introduc-
tion of a halogen bond between a halophenyl group of the
inhibitors and a backbone carbonyl group.32 In comparison to
the unsubstituted case, introduction of a halogen bond with
filled channel heading toward the polymerase active site,
replacement of the (E)-cyanovinyl group with longer chains
that could extend into this region was explored. In comparison
to 40, 48 with a methoxyethoxy substituent was found to retain
significant activity (0.54 μM), while the more hydrophilic
hydroxypropoxy alternative in 49 diminishes the activity to
1.8 μM.
Other Diarylethers. While this work was in progress,
reports of other NNRTIs with diarylether substructures
appeared from Merck and Roche.33,34 The illustrated 50 and
51 appear to be the most promising compounds from these
efforts. They are clearly far more similar to each other than to
42−44.
Notably, the central benzene ring in 50 and 51 is 1,3-
disubstituted with the aryl appendages, while here it is 1,2-
disubstituted. The lengths of the linkers to the heteroaryl group
in the compounds are also different, the heterocycle is a
Scheme 5
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monocyle in 42−44, the cyanovinyl group is not present in 50
and 51, and they have a chlorine adjacent to the phenoxy
oxygen, which was not found to be desirable in the present
series (e.g., 47). Compound 50 is reported to have anti-HIV
activities in cell assays of 4.7, 13.8, and 141 nM for the WT,
Y181C, and K103N/Y181C variants,33 while the corresponding
values for 51 are 1, 1, and 4 nM.34a Thus, these compounds are
less potent toward wild-type HIV-1 than 42 or 44, while 51 has
the best results for the mutant strains.
potentials for liquid simulations; OPLS-AA, OPLS all-atom;
CM1A, charge model 1A; DIAD, diisopropyl azodicarboxylate
REFERENCES
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CONCLUSIONS
■
In summary, the present closely coupled experimental and
computational efforts began with virtual screening, which led
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ASSOCIATED CONTENT
* Supporting Information
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■
S
AUTHOR INFORMATION
Corresponding Author
*(K.S.A.) Phone: 203-785-4526. Fax: 203-785-7670. E-mail:
karen.anderson@yale.edu. (W.L.J.) Phone: 203-432-6278. Fax:
203-432-6299. E-mail: william.jorgensen@yale.edu.
■
ACKNOWLEDGMENTS
■
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. Gratitude is also expressed to Dr. Julian
Tirado-Rives for computational assistance and to
Dr. Christopher D. Incarvito for the small molecule X-ray
crystallography.
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ABBREVIATIONS USED
■
HIV, human immunodeficiency virus; HIV-RT, HIV reverse
transcriptase; NRTI, nucleoside inhibitor of HIV-RT; NNRTI,
non-nucleoside inhibitor of HIV-RT; OPLS, optimized
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