Thiotetrazole alkynylacetanilides as potent and bioavailable non-nucleoside inhibitors of the HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases

Article abstract of DOI:10.1016/j.bmcl.2007.06.012

A series of aryl thiotetrazolylacetanilides were synthesized and found to be potent inhibitors of the HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases. The incorporation of an alkynyl fragment on the aniline provided inhibitors with ex

Full text of DOI:10.1016/j.bmcl.2007.06.012

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4437–4441
Thiotetrazole alkynylacetanilides as potent and bioavailable
non-nucleoside inhibitors of the HIV-1 wild type
and K103N/Y181C double mutant reverse transcriptases
*
´
Alexandre Gagnon, Ma’an H. Amad, Pierre R. Bonneau, Rene Coulombe,
Patrick L. DeRoy, Louise Doyon, Jianmin Duan, Michel Garneau,
Ingrid Guse, Araz Jakalian, Eric Jolicoeur, Serge Landry,
Eric Malenfant, Bruno Simoneau and Christiane Yoakim
Boehringer Ingelheim (Canada) Ltd., Research and Development, 2100 Cunard Street, Laval, Que., Canada, H7S 2G5
Received 5 April 2007; revised 31 May 2007; accepted 4 June 2007
Available online 8 June 2007
Abstract—A series of aryl thiotetrazolylacetanilides were synthesized and found to be potent inhibitors of the HIV-1 wild type and
K103N/Y181C double mutant reverse transcriptases. The incorporation of an alkynyl fragment on the aniline provided inhibitors
with excellent cellular activity and extensive SAR led to the identification of one inhibitor having good oral bioavailability in rats.
Ó 2007 Elsevier Ltd. All rights reserved.
In 2006, 4.3 million people became newly infected
with HIV, bringing the total number of people living
with AIDS throughout the world to an unprecedented
level of 39.5 million.1 Although the introduction of
highly active antiretroviral therapy (HAART) in
1996 led to a drastic reduction of AIDS related
mortality, 2.9 million deaths were nonetheless
recorded in the past year as a consequence of this
disease. Despite the recent availability of simplified
regimens, many patients still undergo treatment
changes due to complex dosing schedules, metabolic
mutations rapidly occur and cross resistance within the
class is then generally observed.7 Thus, the next
generation NNRTI must be potent against mutant
strains that confer resistance to current NNRTIs.
Moreover, excellent pharmacokinetics should be
targeted in order to achieve a once a day dosing.8
With these goals in mind, we initiated a high throughput
screening (HTS) campaign aimed at identifying a potent
and structurally novel NNRTI. Using an enzymatic
assay,9 we screened for inhibition against the clinically
relevant K103N/Y181C HIV-1 double mutant reverse
transcriptase (RT) since this mutation leads to broad
resistance against all the available NNRTIs.10 Follow-
ing this exercise, we discovered that compound 1 is a
potent inhibitor of the HIV-1 K103N/Y181C RT
(Fig. 1). We subsequently found that compound 1 is also
a low nanomolar inhibitor of the wild type (WT) RT.11
complications
or
overwhelming
side
effects.2
Moreover, treatment failure following appearance of
drug resistance,3 transmission of resistant viruses,4
and superinfection5 are serious concerns and new
therapeutic drugs are therefore urgently needed.6
Non-nucleoside
reverse
transcriptase
inhibitors
(NNRTIs) are important components of the first line
HAART regimens. However, selection of resistance
We recently reported the SAR study on the aryl
tetrazole and the anilide moiety and demonstrated that
the nitro group can be replaced by a chlorine atom with
approximately twofold loss in activity against the
double mutant.12 Additionally, optimization of the aryl
tetrazole led to the introduction of the 2-chloro-4-tert-
butyl scaffold shown in compound 2. This potent
inhibitor, which has an IC50 value below 100 nM against
Keywords: Thiotetrazoles; Alkynes; Non-nucleoside reverse transcrip-
tase inhibitor (NNRTI); SAR; Pharmacokinetics; Molecular modeling;
Double mutant reverse transcriptase.
*
Corresponding author. Tel.: +1 450 682 4640; fax: +1 514 682
4189; e-mail: agagnon@lav.boehringer-ingelheim.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.012

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A. Gagnon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4437–4441
NO2
Cl
N N
N N
H
H
N
N
N
N
S
S
N
N
O
O
Cl
1
2
IC50 (Wild type) = 9.5 nM
IC50 (Wild type) = 7.8 nM
IC50 (K103N/Y181C) = 766 nM
IC50 (K103N/Y181C) = 94 nM
Figure 1. Hit and lead compounds identified following high throughput screening and hit to lead campaigns.
the double mutant RT, was used as a starting point for
our NNRTI lead optimization program.
metabolically unstable and was therefore replaced by a
cyclopropyl group.15 While the impact of this change
on the IC50 values is considerable in the case of the
carboxylic acid 4, the effect is insignificant in the case
of the sulfonamide 6, validating this modification.
In the tetrazolyl series, we12,13 and others11 have
demonstrated that the introduction of substituents at
the para position of the anilide leads to substantial
improvement in potency. Interestingly, this beneficial
effect has also been observed in a closely related triazolyl
series.14 Being well aware of this fact and having secured
the left and the right sides of the inhibitor, we embarked
on finding a novel appendage that would allow
modulation of the overall physico-chemical properties
of the molecule while retaining excellent potency against
the double mutant RT.
The introduction of a phenyl acetic acid moiety
(compound 8) gave an acceptable IC50 value for the wild
type and the double mutant RT. However, since it
presents the disadvantage of increasing the molecular
weight of the inhibitor, we hypothesized that this
extraneous phenyl group could be replaced by a simpler
linear fragment such as a saturated carbon chain. In the
event, application of this hypothesis led to poor activity
against the double mutant, as indicated by compound 9.
However, the assumption proved to be successful when
an alkynyl moiety was introduced (compound 10).
Moreover, a threefold improvement in potency was
observed by replacing the propiolic acid fragment by a
homopropargylic alcohol group (compound 11).
Gratifyingly, no erosion of potency was observed when
going to the cyclopropyl analogue 12. In addition, the
presence of the alcohol at the terminal position should
allow the introduction of diverse functional groups.
As shown in Table 1, the introduction of a carboxylic
acid, a sulfonamide or a methylsulfone at the para
position of the anilide gave inhibitors with low nanomo-
lar potencies against the wild type and the K103N/
Y181C double mutant RT (compounds 3, 5, and 7).
Although the presence of the bulky tert-butyl group
ensured good potency, this fragment was found to be
Table 1. SAR on the right hand side anilide
A molecular model of compound 12 with the HIV-1 WT
RT was generated in the allosteric site of the enzyme
(Fig. 2). As illustrated, the tetrazole is orthogonal to
the cyclopropyl-chlorophenyl, whereas the amide is in
a trans conformation, bringing the two chlorine atoms
of both aryl groups in close proximity. The carbonyl
of the inhibitor is involved in a H-bond interaction with
Cl
N N
H
N
N
S
N
O
Cl
R
R'
IC50 (nM)9
a
Compound R0
R
WT K103N/Y181C
3
4
5
6
7
t-Bu CO2H
c-Pr CO2H
5.0
16
66
151
36
t-Bu SO2NH2
c-Pr SO2NH2
t-Bu SO2Me
5.8
5.0
3.1
45
26
t-Bu
8
10
33
CO2H
9
t-Bu CH2CH2CO2H
t-Bu C„CCO2H
t-Bu C„CCH2CH2OH
c-Pr C„CCH2CH2OH
10
150
38
10
11
12
2.9
5.5
2.0
12
8.9
Figure 2. Molecular model of 12 in the allosteric site of HIV-1 WT
RT.
a IC50 values are means of at least two independent experiments.

A. Gagnon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4437–4441
4439
the backbone NH of K103 and the left hand side aryl
group binds in a hydrophobic pocket formed by residues
W229, L234, Y188, and Y181. Detailed analysis shows
that the cyclopropyl group sits over W229, while the left
hand side phenyl is parallel to Y188. Interestingly, the
alkynyl group extends between P236 and V106, and
exposes its terminus to the solvent. Consequently, this
feature should be useful in the modulation of the
physico-chemical properties of the inhibitors.
double mutant, showing the necessity of having a substi-
tuted alkynyl group. Unfortunately, the propargylic
alcohol 20 and the acid 21 suffered either from poor met-
abolic stability or unacceptable cellular activity. To our
great pleasure, blocking the propargylic position with
methyl groups led to enhanced stability and potency, as
illustrated by compound 22. Additionally, this scaffold
was utilized as a handle for the introduction of basic
and acidic groups, leading to potent inhibitors 23 and
24, respectively. The dimethyl propargylic amine 25 gave
equivalent potency as the alcohol 22 against the double
mutant RT, but poorer half-life in rat liver microsomes.
Fortunately, good metabolic stability was obtained by
capping the amine with an acetic acid group (compound
26) or an amino acetate (compound 27). The homoprop-
argyl alcohol 28 and the acid 29 gave very low half-lives
in RLM. Happily, further elaboration leading to the
aminoacetic acid 30 and the dimethyl propionic acid
31, gave satisfactory potency and metabolic stability.
Hoping to further improve on the activity of compound
31, we transformed the acid into the corresponding acyl
sulfonamide 32, leading to a twofold improvement of the
EC50 value for the double mutant RT. However,
attempts to improve the metabolic stability of 31 by
replacing the methyl groups with a cyclopropane (com-
pound 33) led to a reduction in the half-life in RLM. Fur-
thermore, homologation of the acid (compound 34) led
to a minor improvement in cellular activity accompanied
by a drastic reduction in metabolic stability.
The synthetic route that we designed to access the alkynyl
inhibitors is expeditious and tolerates chemical diversity
(Scheme 1). As illustrated, 2-chloro-4-iodoaniline 13
was submitted to a standard Sonogashira coupling
reaction leading to alkynylanilines 14. The same starting
aniline 13 was protected as a tert-butyl carbamate
employing conditions developed for electronically
deactivated anilines.16 The installation of the cyclopropyl
unit was performed under Negishi conditions giving the
BOC-protected 2-chloro-4-cyclopropyl aniline 15. The
amine was then liberated under acidic conditions and
subsequently transformed into the corresponding
arylthiotetrazole 16 in a two-step sequence. Alkylation
with bromo acetic acid provided compound 17 which was
activated as the corresponding acid chloride and coupled
with anilines 14 to afford the desired inhibitors 18.
An extensive SAR study was then performed on the
terminal position of the alkyne. As shown in Table 2,
the enzymatic potency is given exclusively for the key
double mutant RT since all of the prepared inhibitors
showed IC50 values below 10 nM against the WT RT.
The cellular activity was measured for both the WT
and the K103N/Y181C double mutant RT viruses and
the half-lives in rat liver microsomes (RLM) were also
measured in order to select compounds for PK evalua-
tion. As illustrated, the introduction of a terminal alkyne
(compound 19) led to mediocre potency against the
Compounds having good cellular activity against the
WT and the double mutant RT in conjunction with
adequate half-life in RLM were selected for pharmaco-
kinetic evaluation in rats (Table 3). Based on these
criteria, compounds 27, 30, 31, and 32 were chosen for
PK profiling. Unfortunately, low exposure was achieved
following oral dosing with compounds 27, 30, and 32 at
5 mg/kg. However, to our delight, a Cmax of 5.0 lM and
N N
N
SH
Cl
NH2
NHBOC
d, e, f
N
H2N
Cl
Cl
Cl
a
b, c
14
R
I
13
15
N N
16
Cl
N N
H
OH
N
N
N
S
S
N
N
O
O
Cl
Cl
g
h, i
R
17
18
Scheme 1. Synthetic route to compound 18. Reagents and conditions (a) R-C„C-H (1.0 eq), CuI (0.1 eq), Pd(PPh3)4 (0.1 eq), Et2NH (3 eq), THF,
70 °C, o.n., 75–90%; (b) NaHMDS (2.1 eq), BOC2O (0.9 eq), THF, rt, o.n., 84%; (c) c-PrBr (3.0 eq), n-BuLi 2.5 M in hexanes (3.1 eq), ZnBr2 (3.6 eq),
Pd(PPh3)4 (0.1 eq), THF, reflux, 2 h, 72%; (d) HCl 4.0M in dioxane, rt, 5 h, 90%; (e) thiophosgene (1.1 eq), Et3N (3.3 eq), i-PrOH, rt, 4 h; (f) NaN3
(2.0 eq), acetonitrile, reflux, 48 h, 60% (2 steps); (g) BrCH2CO2H (1.1 eq), Et3N (2.5 eq), EtOH, rt, 2 h, 73%; (h) ClCOCOCl (1.1 eq), DMF (cat.),
CH2Cl2, rt, 1.5 h; (i) aniline 14 (1.1 eq), pyridine (2.0 eq), CH2Cl2, rt, 4 h, 55–75% (2 steps).

4440
A. Gagnon et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4437–4441
Table 2. SAR on the alkynyl moiety
Table 2 (continued)
a
a
a
Cl
Compound
R
IC50
(nM)
EC50
(nM)17
t1/2
N N
H
(min)
RLM18
N
N
S
N
K103N/
Y181C
O
Cl
WT K103N/
Y181C
R
O
33
34
43
26
12
54
30
21
OH
OH
a
a
a
Compound
R
IC50
(nM)
EC50
(nM)17
t1/2
(min)
K103N/
Y181C
RLM18
5.8 36
O
WT K103N/
Y181C
a IC50, EC50, and t1/2 values are means of at least two independent
experiments.
19
20
21
22
23
24
H
44
20
47
17
80
23
82 505
5.4 31
66 355
2.4 17
3.4 22
6.2 29
4
7
Table 3. Rat PK profiles of best inhibitorsa
CH2OH
Compound
iv (2 mg/kg)
Oral (5 mg/kg)
CL
(mL/min kg)
t1/2 (h) Cmax
(lM)
AUC
(lM h)
F (%)
CO2H
20
84
15
29
27
30
25.6
41.9
10.8
17.5
1.6
1.5
3.5
0.72
0.28
0.26
5.0
1.0
0.84
9.5
1.7
21
29
66
22
OH
31
32b
0.63
a iv formulation: 70% PEG-400 (30% water). PO formulation: 0.5%
methocel and 0.3% Tween 80.
b Sodium salt.
O
N
O
O
an AUC of 9.5 lM h were observed for inhibitor 31.
Following intravenous administration at 2 mg/kg, an
acceptable clearance and half-life were obtained and
an oral bioavailability of 66% was calculated for this
inhibitor.
O
OH
NH2
25
26
38
30
2.7 25
1.9 29
31
70
In summary, we have demonstrated that the
structurally novel thiotetrazolyl acetanilides are potent
non-nucleoside inhibitors of the HIV-1 wild type and
K103N/Y181C double mutant reverse transcriptases.19
Introduction of an alkynyl moiety at the para position
of the anilide led to further improvement in potency
and allowed the modulation of physico-chemical
properties of the inhibitors. Extensive SAR studies
led to the identification of potent and metabolically
stable compounds. Following this exercise, rat PK
evaluation of selected inhibitors was conducted and
compound 31 was found to have the best PK profile.
H
N
OH
H
N
27
7.9
1.1 8.1
109
NH2
O
OH
28
29
17
32
8.2 51
9
O
11 139
19
OH
Acknowledgment
OH
N
H
30
31
32
23
28
19
1.6 13
7.1 58
4.2 27
79
90
82
O
O
We are grateful to colleagues at Boehringer Ingelheim
Canada for analytical support and biological
evaluation.
O
O
OH
O
References and notes
S
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H
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days at 37 °C. Viral replication was assessed by p24
ELISA of the culture supernatants.
18. t1/2 values for phase I oxidative metabolitic stability were
determined using rat liver microsomes at a concentration
of 2 lM for the compounds.
3
19. DeRoy, P.; Faucher, A.-M.; Gagnon, A.; Landry, S.;
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