Investigation on the role of the tetrazole in the binding of thiotetrazolylacetanilides with HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases

Article abstract of DOI:10.1016/j.bmcl.2008.12.074

The role of the tetrazole moiety in the binding of aryl thiotetrazolylacetanilides with HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases was explored. Different acyclic, cyclic and heterocyclic replacements were investigated in order t

Full text of DOI:10.1016/j.bmcl.2008.12.074

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1199–1205
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Investigation on the role of the tetrazole in the binding
of thiotetrazolylacetanilides with HIV-1 wild type and K103N/Y181C
double mutant reverse transcriptases
*
Alexandre Gagnon , Serge Landry, René Coulombe, Araz Jakalian, Ingrid Guse, Bounkham Thavonekham,
Pierre R. Bonneau, Christiane Yoakim, Bruno Simoneau
Boehringer Ingelheim (Canada) Ltd., Research and Development, 2100 Cunard Street, Laval, Que., Canada H7S 2G5
a r t i c l e i n f o
a b s t r a c t
Article history:
The role of the tetrazole moiety in the binding of aryl thiotetrazolylacetanilides with HIV-1 wild type and
K103N/Y181C double mutant reverse transcriptases was explored. Different acyclic, cyclic and heterocy-
clic replacements were investigated in order to evaluate the conformational and electronic contribution
of the tetrazole ring to the binding of the inhibitors in the NNRTI pocket. The replacement of the tetrazole
by a pyrazolyl group led to reversal of selectivity, providing inhibitors with excellent potency against the
double mutant reverse transcriptase.
Received 18 November 2008
Revised 17 December 2008
Accepted 17 December 2008
Available online 24 December 2008
Keywords:
Non-nucleoside reverse transcriptase
inhibitors (NNRTIs)
Ó 2008 Elsevier Ltd. All rights reserved.
Thiotetrazoles
Electrostatic potential
Molecular modeling
Double mutant reverse transcriptase
NO2
Cl
According to the world health organization, every day, more
than 6800 people become infected with human immunodeficiency
virus (HIV) and over 5700 people die from acquired immunodefi-
ciency syndrome (AIDS) throughout the world.1 With an estimated
number of 33.2 million people living with AIDS in 2007, an ade-
quate access to HIV treatment remains critical. Twelve years after
the introduction of highly active antiretroviral therapy (HAART) for
the treatment of HIV, the development of potent and well tolerated
new drugs remains an active field of research.2 The HIV reverse
transcriptase (RT) enzyme plays an essential role in the replication
of the HIV virus by performing the retrotranscription of the viral
RNA into viral DNA. Therefore, the HIV-RT constitutes a major tar-
get for antiretroviral drug development.3 The non-nucleoside re-
verse transcriptase inhibitors (NNRTIs) interact in an allosteric
site located approximately 10 Å away from the catalytic site, caus-
ing enzyme inhibition.4 Although NNRTIs are commonly used in
HAART regimens, the low genetic barrier to mutation associated
with first line NNRTIs creates a need for new drugs that exhibit
activity against the most prevalent mutants.5
N
N
N
N N
H
H
N
N
N
S
S
N
N
O
O
O
Cl
OH
1
2
IC50 (WT) = 9.5 nM
IC50 (K103N/Y181C) = 766 nM
IC50 (WT) = 3.7 nM
IC50 (K103N/Y181C) = 28 nM
Figure 1. Hit compound 1 and optimized inhibitor 2 obtained following uHTS
campaign and lead optimization program.
which was identified in the course of a ultra high-throughput
screening (uHTS) campaign, also showed submicromolar potency
against the clinically relevant K103N/Y181C double mutant (DM)
RT.8 Excellent potency against this mutant is highly desirable for
second generation drugs since it plays a central role in the develop-
ment of resistance in patients exposed to nevirapine, efavirenz and
delavirdine.9 An intensive lead optimization program subsequently
led to the design of compound 2 which possesses key features that
provide optimal binding to the reverse transcriptase, such as a 2,4-
disubstituted left hand side aryl group and a 2-chloro-4-alkynyl
anilide right hand side fragment.10 We recently disclosed the de-
tailed SAR studies of the alkynyl series11 and showed that com-
pound 2 gives good cellular potency against the WT and the
K103N/Y181C DM RT in combination with good pharmacokinetic
parameters in rats.
We6 and others7 have recently reported the discovery of aryl-
thiotetrazolyl acetanilide 1 (Fig. 1) as a low nanomolar inhibitor
of the wild type (WT) HIV-1 RT. More importantly, compound 1,
* Corresponding author. Tel.: +1 450 682 4640; fax: +1 514 682 4189.
E-mail address: alexandre.gagnon@boehringer-ingelheim.com (A. Gagnon).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.074

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A. Gagnon et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1199–1205
Table 1
Previous studies conducted by us on the tetrazolyl inhibitors
Evaluation of cyclic and acyclic linkers
bound in the WT NNRTI pocket suggested that the tetrazolyl group
is likely involved in relatively weak associations with the side
chains of Y181 and/or K103.12 Consequently, we proposed at that
time that this heterocycle is more than simply a scaffold that ori-
ents the pharmacophores in the optimal geometry. In order to have
a better understanding of the role of this group in the binding of
the inhibitors with the RT enzyme, we initiated SAR studies cen-
tered around this area of the molecule. As a first step toward this
goal, we recently reported that amides, carbamates, ureas and thio-
amides 3 efficiently behave as tetrazole replacements (Fig. 2).12
Additionally, others have reported that analogs of 4 provide excel-
lent potency against the WT and the K103N/Y181C RT, showing
that the triazole is also a good tetrazole surrogate.13 Recently, thi-
adiazoles 5, which we first reported in 2005,10 were also shown to
be adequate tetrazole replacements.14
We would like to report herein our studies in the evaluation of
the electronic and conformational contribution of the tetrazolyl
group to the binding of the inhibitors with the HIV-1 RT. Taking
lessons from series 3, 4 and 5 we set for ourselves the objective
of finding a rigidified linker that would provide the optimal bind-
ing conformation in conjunction with the required electronic fin-
gerprint (Fig. 3). Knowing from previous modeling studies that
the tetrazole is in the vicinity of residue 103,11,12 we utilized the
clinically relevant K103N/Y181C double mutant RT as a probe to
gain insights into the nature and strength of the interactions be-
tween the linker and the protein.
Cl
H
N
X
Cl
O
A
IC50 (nM)15
K103N/Y181C
a
Compound
Linker-X
A
WT
1.9
N N
N
N
6
OCH2CO2H
22
S
7
8
CH2CO2H
2.4
15
48
OCH2CO2H
785
9
OCH2CO2H
CH2CO2H
272
18
3272
592
10
S
We initiated our studies by exploring the conformational role of
the tetrazolyl group (Table 1) through the use of simple all-carbon
cyclic and acyclic linkers. In the event, replacing the tetrazolyl
group in 6 with a Z alkene (compound 8) resulted in a 7-fold reduc-
tion of potency against the WT-RT, showing that a simple double
bond is capable of partially retaining the potency against the
WT-RT. However, a drastic decrease of activity against the DM-
RT was observed. Surprisingly, the E alkene 9 retained submicrom-
olar potency against the WT-RT, but led to poor activity against the
DM-RT. These isomeric alkenes clearly demonstrate a preference
for a cis geometry, supporting the hypothesis that the tetrazole
partially acts as an orienting scaffold. Using compound 7 as a ref-
11
CH2CO2H
177
1390
O
a
IC50 values are the mean of at least two independent experiments.
erence, we then evaluated the effect of introducing a phenyl linker
and observed a good IC50 against the WT for thiophenyl 10. How-
ever, phenylether 11 suffered from significant loss of potency, indi-
cating a preference for a sulfur over an oxygen as the connecting
atom. The erosion of potency against the WT-RT and the drastic
loss of activity against the DM-RT observed for all of these linkers
suggest that an important electronic contribution is provided by
the tetrazole which might additionally be interacting with the sur-
rounding residues in the binding pocket.
Being now aware of the apparent electronic contribution of the
tetrazole in the binding of the inhibitors with the RT, we then
turned our attention to different five-membered heterocyclic link-
ers. The synthetic route that we designed to access these inhibitors
is based on a previously reported sequence utilized for the prepa-
ration of the tetrazolyl inhibitors.11 As illustrated in Scheme 1, two
approaches efficiently lead to the desired compounds. The first ap-
proach involves the preparation of a bromoacetanilide fragment 13
from the corresponding aniline 12. This compound is then coupled
with a thio or hydroxy aryl heterocycle 14, directly giving the de-
sired compound 15. Alternatively, the acetate can be installed first
onto 14, leading to the tert-butyl acetate 16. Removal of the tert-
butyl group under standard conditions followed by amide bond
formation then provides the final product 15.
Cl
O
H
N
N
X
Cl
O
CO2H
3
X = CH2, O, NH, S
N
N
X
H
N
N
S
H
N
N
S
N
R'
S
O
O
Y
R
4
R
5
Figure
thiadiazoles
double mutant reverse transcriptases.
2. Amides, carbamates, ureas, thiocarbamates 3, triazoles
4 and
5
as potent inhibitors of the HIV-1 wild type and K103N/Y181C
The synthesis of compound 15 where the linker is a triazole
(compound 28), a pyrazole (compounds 29, 30, 31, 32, 33, and
40), an imidazole (compounds 35 and 36), a thiadiazole (com-
pound 37) or a thiazole (compound 38) has been described in an
earlier report.10 For the introduction of a pyrrolyl linker into frag-
ment 17, the synthesis commences with the Stille coupling be-
tween stannyl pyrrole 18 and 3-chloro-4-iodotoluene, furnishing
the 2-arylpyrrole 19 (Scheme 2). Bromination of 19 followed by
lithium-halogen exchange and trapping with TIPS-disulfide then
provides the protected thiopyrrole 20. Removal of the protecting
Rigidified
linker
Y
Z
H
X
N
A
R'
O
R
Figure 3. Design of a conformationally constrained linker acting as a tetrazole
replacement.

A. Gagnon et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1199–1205
1201
Cl
O
I
H2N
Cl
Cl
a, b
R
12
a
22
23
c, d
Cl
Cl
H
N
H
N
XH
Br
X
Cl
O
Cl
O
O
13
R
R
OH
N
OMe
O
S
S
b
15
O
Cl
Cl
O
e, f, g
R
R
14
e, f
c
25
24
Scheme 3. Synthetic sequence to fragment 25: (a) Butyl vinyl ether (1.2 equiv),
triethylamine (1.2 equiv), Pd(OAc)2 (0.2 equiv), dppp (0.2 equiv), DMF, 80 °C, 5 h;
O
OH
X
X
Cl
O
Cl
O
(b) HCl (4 N in dioxane), dioxane/water (1:1), rt, 1 h (52%,
2 steps); (c) Br2
d
(1.1 equiv), dioxane, rt, 1 h (73%); (d) methyl thioglycolate (1.1 equiv), triethyl-
amine (1.1 equiv), CH2Cl2, rt, 1 h (99%); (e) Br2 (1.0 equiv), AcOH, rt, 0.5 h; (f)
formamide (20 equiv), 150 °C, 1 h (39%, 2 steps, unoptimized); (g) 1.0 N aq. NaOH
(1.5 equiv), DMSO, rt, 1 h (97%).
16
17
R
R
Scheme 1. Synthetic routes to compound 15: (a) aniline 12, bromoacetyl chloride
(1.1 equiv), triethylamine (1.1 equiv), CH2Cl2, rt, o.n. (80–100%); (b) fragment 14,
bromoacetamide 13 (1.0 equiv), K2CO3 (1.2 equiv), DMF, rt, 2 h (80–100%); (c)
fragment 14, tert-butyl bromoacetate (1.1 equiv), K2CO3 (1.5 equiv), DMF, rt, o.n.
(100%); (d) CF3CO2H (10 equiv), CH2Cl2, rt, o.n. (90–100%); (e) ClCOCOCl (1.1 equiv),
DMF (0.1 equiv), CH2Cl2, rt, 1.5 h; (f) aniline 12 (1.1 equiv), pyridine (2.0 equiv),
CH2Cl2, rt, 4 h, 70–80% (2 steps).
modification indicates that substitution para to the aniline pro-
vides superior potency against the DM-RT. In addition, the potency
against the DM-RT could be enhanced by replacing the methyl
group in 7 by a larger tert-butyl fragment, as exemplified by com-
pound 27. To evaluate the importance of the nitrogen atoms at po-
sition 3 and 4 in compound 7, we then prepared triazole 28 and
pyrazole 29. Interestingly, removing the nitrogen atom at position
4 (tetrazole numbering) had no detectable impact on the IC50
against the WT-RT (triazole 28). However, proceeding further with
the removal of N3 (tetrazole numbering) led to a 4-fold reduction
of potency against the WT-RT (pyrazole 29). In both cases, a drastic
loss of potency against the DM-RT was observed.16 Substitution at
position 4 was clearly not tolerated, as illustrated by compound 30,
which was completely inactive against both RTs. The pyrazolyloxy
31 was found to be slightly less potent than the thio analogue 29,
but yet tolerated by the WT-RT. This is in clear contrast to the dras-
tic shift that we observed when going from the thioaryl 10 to the
phenoxy 11. The two isomeric pyrazoles 32 and 33 demonstrate
that substitution is only tolerated at the position adjacent to the
aryl group. Additionally, pyrrole 34 was more than 500-times less
potent than the structurally similar pyrazole 32, indicating a pref-
erence for a nitrogen at position 5 (pyrrole numbering). The above
modifications allow us to conclude that a nitrogen atom is pre-
ferred over a CH at position 3 and 4 of the heterocyclic linker. Sec-
ondly, it appears that substitution is permited exclusively at
position 2.
N
Cl
a
SnMe3
N
18
19
b, c
OH
N
N
S
STIPS
O
d, e
Cl
Cl
21
20
Scheme 2. Synthetic sequence to fragment 21: (a) 3-chloro-4-iodotoluene
(0.9 equiv), Cl2Pd(PPh3)2 (0.05 equiv), toluene, 110 °C, o.n. (22%, unoptimized); (b)
PBr3 (0.1 equiv), NBS (1 equiv), THF, À78 °C, 1 h then rt, 3 h (83%); (c) n-BuLi
(1.0 equiv), TMEDA (1.0 equiv), THF, À78 °C, 1 h then TIPS-S-S-TIPS (1.0 equiv),
À78 °C to rt, 2 h (35%, unoptimized); (d) ethyl bromoacetate (2.5 equiv), toluene/
DMF (1:1), 0 °C then TBAF (2.5 equiv in toluene), 0.5 h (88%); (e) 1.0 N aq. LiOH
(1.5 equiv), THF/MeOH (3:1), 0 °C (100%).
Being now educated on the tolerance of the molecule towards
substitution and deletion of nitrogen atoms, we next prepared
the imidazole 35 in accordance with the above observations. In
the event, excellent potency against the WT-RT was observed for
this inhibitor, associated with modest activity against the DM-RT.
The 4-methylimidazole 36 was found to be completely inactive
against the DM-RT, confirming the observation that substitution
at position 3 of the heterocycle is not tolerated. Thiadiazole 37,
which possesses heteroatoms at positions 2, 3 and 4 (tetrazole
numbering) proved to be the most potent tetrazole replacement,
with a very good IC50 against the WT-RT and only a 2-fold loss of
activity against the DM-RT. Unfortunately, the low metabolic sta-
bility associated with this compound hampered further efforts
involving this heterocyclic linker. Replacing one nitrogen atom
with carbon in 37 led to thiazole 38 which suffered from modest
potency against both RTs, indicating once again a preference for
a nitrogen atom at position 3 (tetrazole numbering). The negative
group in the presence of ethyl bromoacetate followed by saponifi-
cation gives fragment 21. This pyrrolyl intermediate is finally con-
verted into inhibitor 34 according to Scheme 1.
For the introduction of an oxazole into structure 17, we started
with a Heck reaction between 3-chloro-4-iodotoluene 22 and butyl
vinyl ether to provide, after treatment under acidic conditions, ke-
tone 23 (Scheme 3). This ketone was then brominated and treated
with methyl thioglycolate in the presence of base to give 24. This
sulfide was then subjected to bromination prior to undergoing con-
densation with formamide, and saponification, affording the de-
sired thio-oxazolyl acetic acid 25. This fragment was finally
converted into inhibitor 39 under conditions reported in scheme 1.
To begin our SAR studies, we first prepared reference compound
26 where the eastern biphenyl aniline is replaced by a simple 2-
chloro aniline (Table 2). The loss of potency associated with this

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A. Gagnon et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1199–1205
Table 2
Evaluation of heterocyclic linkers
Cl
H
N
Het
X
Cl
O
A
R
IC50 (nM)15
a
Compound
Het-X
R
A
WT
2.3
K103N/Y181C
48
3
4
N N
7
Me
Ph-4-CH2CO2H
N
2
S
N
1
26
27
Me
t-Bu
H
8.8
7.7
809
28
Ph-4-CH2CO2H
1
5
N
28
Me
Ph-4-CH2CO2H
2.4
730
2
1
N
S
S
4
N
3
5
4
3
29
30
Me
Me
Ph-4-CH2CO2H
Ph-4-CH2CO2H
7.9
420
N
N
2
>10,000
>10,000
N
N
S
N
31
32
Me
Me
Ph-4-CH2CO2H
Ph-4-CH2CO2H
18
770
178
O
N
2
3
N
4
1.9
N
1
S
5
1
5
N
33
34
Me
Me
Ph-4-CH2CO2H
n.d.b
6480
2679
N
2
S
4
3
5
4
3
Ph-4-OCH2CO2H
>1000
N
S
S
1
2
3
4
N
1
2
35
36
Me
Me
Ph-4-CH2CO2H
9.5
549
5
N
3
4
N
1
2
H
>1000
>10,000
5
S
N
N
S
N
37
Me
Ph-4-CH2CO2H
2.0
107
S

A. Gagnon et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1199–1205
1203
Table 2 (continued)
IC50 (nM)15
a
Compound
Het-X
R
A
WT
200
K103N/Y181C
796
1
2
S
38
39
Me
Ph-4-CH2CO2H
3
N
4
S
5
O
N
Me
Ph-4-CH2CO2H
Ph-4-CH2CO2H
365
1458
22
S
H
1
2
N N
40
t-Bu
2089
3
O
5
4
a
IC50 values are the mean of at least two independent experiments.
n.d., not determined.
b
impact on the potency was further exacerbated with the replace-
ment of the sulfur in 38 by an oxygen (oxazole 39). All of the het-
erocyclic linkers explored thus far indicate that the tetrazole is not
simply acting as an orienting group, but is presumably also inter-
acting with the protein, as supported by the considerable loss of
potency observed in all cases with the DM-RT. However, this exer-
cise demonstrates that triazoles, pyrazoles, imidazoles and thi-
adiazoles are good tetrazole replacements, providing potent
inhibitors of the wild type reverse transcriptase.
Table 3
SAR studies on the oxypyrazole acetanilide series
H
1
2
Cl
N
N
H
3
N
R
O
5
4
O
Cl
A
We then became interested in the introduction of a H-bond do-
nor in the linker, resulting in the design of pyrazole 40. To our great
delight, an IC50 of 22 nM against the DM-RT was observed for this
inhibitor. More fascinating was the significant loss of activity ob-
served against the WT-RT. Intrigued by this reversal of selectivity,
we initiated a more detailed SAR study on this pyrazolyl scaffold
(Table 3).
To evaluate the importance of the methyl group at position 5 of
the pyrazole, we first prepared the des-methyl 41 and observed a
significant reduction in the potency against the DM-RT. However,
encouraged by the cellular potency obtained against the DM-RT,
we pursued our investigation on these NH-pyrazolyl inhibitors.
The introduction of our previously reported alkynyl aniline frag-
ment led to an improvement in the cellular potency against the
DM-RT, as exemplified by compound 42. Further advancement
was achieved with the introduction of the dimethyl butynoic acid
fragment (compound 43), giving our best EC50 against the DM-RT
for this class of compound.
a
a
Compound
R
A
IC50 (nM)15
EC50 (nM)17
WT
K103N
Y181C
WT
K103N
Y181C
41
42
H
>3300 122
>3330 24
4869
444
CO2H
OH
Me
Me
>5000 177
CO2H
43
2403
38
3884
73
a
IC50 and EC50 values are the mean of at least two independent experiments.
In order to evaluate the relation between the electrostatic prop-
erties of the linkers and the potency of the inhibitors, we next cal-
culated the electrostatic potential (ESP) of selected compounds
(Fig. 4). As illustrated, tetrazole 7, triazole 28 and thiadiazole 37,
which were all very potent against the WT-RT, all have a strong
negative potential localized around the edge of the heterocycle.
Conversely, pyrazoles 29 and thiazole 38, which had poorer po-
tency against the WT-RT, have a slightly positive ESP. In the case of
the pyrazole 40, which had poor potency against the WT-RT, a
strong positive potential localized on the NH is observed. Conse-
quently, it appears that a strong negative ESP near position 3 leads
to higher potency against the WT-RT, but not against the DM-RT.
To rationalize the unforeseen loss of potency against the WT-RT
observed with the NH-pyrazolyl inhibitors and to gain a deeper
understanding on the role of the linker in the binding of the tetraz-
olyl inhibitors, a molecular model of compound 27 and 40 in the
NNRTI binding pocket of the HIV-1 WT and K103N/Y181C DM
RTs was generated (Fig. 5).18 In accordance with our previous stud-
ies,11,12 the model shows the orthogonality between the tetrazole
and the directly attached aryl group. Additionally, the carbonyl of
the amide is at an optimal distance for a hydrogen bond with the
backbone of residue 103. The left hand side aryl group binds in a
hydrophobic pocket formed by residues 229, 188 and 181 while
the tetrazole rests in close proximity to residue 103. The erosion
of potency observed following substitution at position 3 and 4 (tet-
razole numbering) can be explained by a steric clash or an unfavor-
able interaction between the alkyl group and the side chain of
residue 103. Additionally, a steric repulsion between the CH2 group
of the acetamide and a 4-methyl group might result in a higher en-
ergy bioactive conformation.
In accordance with our initial hypothesis,12 our studies indicate
that the tetrazole can interact with residue 103. In fact, the model
suggests a possible (tetrazole)NÁÁÁÁH-Csp3 interaction between N3
and N4 of the tetrazole and the
c-H of the side chain of lysine-
103.19 Previous studies have established that the side chain of ly-
interaction.20 More-
sine is one of the best donors in the C–HÁÁÁÁ
p
over, this interaction is supported by the electrostatic potential
calculated for 7 which places the electronic density on the edge

1204
A. Gagnon et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1199–1205
against the DM-RT for many heterocyclic linkers would therefore
be explained by the lack of an adequate H-bond acceptor.
In conclusion, the role of the tetrazolyl group in the binding of
the thiotetrazole acetanilide inhibitors with the HIV-1 reverse
transcriptase has been studied through the design of different cyc-
lic and acyclic tetrazole replacements. We have demonstrated that
a simple Z alkene is capable of retaining most of the potency
against the WT-RT, supporting the hypothesis that the tetrazole
partially acts as an orienting scaffold. The exploration of numerous
heterocyclic linkers allowed the identification of potent inhibitors
of the WT-RT. A complete reversal of selectivity was observed for
the NH-pyrazolyl inhibitors, giving excellent potency against the
DM-RT associated with a complete loss of activity against the
WT-RT. The electrostatic potential study has demonstrated that
the tetrazole provides a strong negative potential located at the
edge of the heterocycle. Modeling studies of the tetrazolyl and
NH-pyrazolyl inhibitors suggested important interactions between
the heterocyclic linkers and residue 103, providing a rationale for
the potency observed against both RTs.
Acknowledgments
We are grateful to colleagues at Boehringer Ingelheim (Canada)
Ltd. for analytical support, biological evaluation and valuable help
during manuscript preparation.
References and notes
1. AIDS epidemic update, UNAIDS, World health organization, December 2007,
http://www.unaids.org.
2. For a review on recent developments in the treatment of AIDS, see: Armbruster,
C. Anti-Infective Agents Med. Chem. 2008, 7, 201.
Figure 4. Electrostatic potential (ESP) of selected inhibitors calculated at the HF/6-
31G level of theory. Red represents a negative potential; white represents a
neutral potential; blue represents a positive potential.
**
3. For an excellent review on the HIV-RT, see: Castro, H. C.; Loureiro, N. I. V.;
Pujol-Luz, M.; Souza, A. M. T.; Albuquerque, M. G.; Santos, D. O.; Cabral, L. M.;
Frugulhetti, I. C.; Rodrigues, C. R. Curr. Med. Chem. 2006, 13, 313.
4. For a recent reference on the mechanism of NNRTIs, see: Sluis-Cremer, N.;
Tachedjian, G. Virus Res. 2008, 134, 147.
5. For excellent reviews on the current status of NNRTIs, see: (a) Martins, S.;
Ramos, M. J.; Fernandes, P. A. Curr. Med. Chem. 2008, 15, 1083; (b) Sweeney, Z.
K.; Klumpp, K. Curr. Opin. Drug Discov. Dev. 2008, 11, 458; (c) Sahlberg, C.; Zhou,
X.-X. Anti-Infective Agents Med. Chem. 2008, 7, 101.
6. Simoneau, B.; Thavonekham, B.; Landry, S.; O’Meara, J.; Yoakim, C.; Faucher, A. -
M. 2004 WO 2004/050643.
7. (a) Muraglia, E.; Kinzel, O. D.; Laufer, R.; Miller, M. D.; Moyer, G.; Munshi, V.;
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G.; Muraglia, E.; Orvieto, F.; Williams, P. D. 2005 WO 2005/115147.
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Figure 5. Molecular model of 27 (yellow) and 40 (orange) in the allosteric site of
HIV-1 WT and K103N/Y181C RT.
of the heterocycle. In the case of the pyrazole 40, this interaction
would be replaced by an unfavorable (pyrazolyl)N–HÁÁ//ÁÁH-Csp
3
contact, resulting in a major loss of potency against the WT-RT.
In the case of the 103N mutant, the side chain of the asparagine
can accommodate a H-bond donor or H-bond acceptor by rotation
of the amide, resulting in a (tetrazole)NÁÁÁÁH2NCO103N for 27 or
(pyrazolyl)N–HÁÁÁÁO@C103N for 40. The poor potency observed
14. Zhan, P.; Liu, X.; Cao, Y.; Wang, Y.; Pannecouque, C.; De Clercq, E. Bioorg. Med.
Chem. Lett. 2008, 18, 5368.
15. IC50 values for WT and mutant RTs were obtained from
proximity assay using poly rC/biotin-dG15 and 3H-dGTP at 37ꢀC.
16. For compound 28, a 28-fold and 5-fold loss of potency was observed,
respectively, for K103N and Y181C single mutants, suggesting that the loss of
a scintillation
a

A. Gagnon et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1199–1205
1205
potency against the K103N/Y181C double mutant is mainly due to the 103
mutation.
minimization was performed on the binding site for each inhibitor/mutation
complex.
17. For EC50 determinations, C8166 cells were acutely infected (MOI 0.001) and
incubated with inhibitors for 3 days at 37 °C. Viral replication was assessed by
p24 ELISA of the culture supernatants.
19. For studies on related C–HÁÁÁÁO@C interactions, see: (a) Tóth, G.; Bowers, S.
G.; Truong, A. P.; Probst, G. Curr. Pharm. Design 2007, 13, 3476; (b) Panigrahi,
S. K.; Desiraju, G. R. Proteins: Structure, Function, and Bioinformatics 2007, 67,
18. Models of 27 and 40 were generated by modifying an unpublished in-house X-
128.
ray structure of
a
related compound. Mutations K103N and Y181C were
20. Brandl, M.; Weiss, M. S.; Jabs, A.; Sühnel, J.; Hilgenfeld, R. J. Mol. Biol. 2001, 307,
performed in silico based on pdb 1FKP and 1UWB, respectively. Energy
357.