Tetrazole thioacetanilides: Potent non-nucleoside inhibitors of WT HIV reverse transcriptase and its K103N mutant

Article abstract of DOI:10.1016/j.bmcl.2006.02.024

A series of aryltetrazolylacetanilides was synthesized and evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors on wild-type virus and on the clinically relevant K103N mutant strain. Extensive SAR investigation led to potent compounds, with nanomolar activity on K103N, and orally bioavailable in rats.

Full text of DOI:10.1016/j.bmcl.2006.02.024

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2748–2752
Tetrazole thioacetanilides: Potent non-nucleoside inhibitors of
WT HIV reverse transcriptase and its K103N mutant
Ester Muraglia,a,* Olaf D. Kinzel,a Ralph Laufer,b Michael D. Miller,c Gregory Moyer,c
Vandna Munshi,c Federica Orvieto,a Maria Cecilia Palumbi,a Giovanna Pescatore,a
Michael Rowley,a Peter D. Williamsd and Vincenzo Summaa
aDepartment of Medicinal Chemistry, IRBM/MRL Rome, Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy
bDepartment of Pharmacology, IRBM/MRL Rome, Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy
cDepartment of Antiviral Research, WP/MRL, Sumneytown Pike PO Box 4, West Point, PA 19486, USA
dDepartment of Medicinal Chemistry, WP/MRL, Sumneytown Pike PO Box 4, West Point, PA 19486, USA
Received 19 January 2006; revised 3 February 2006; accepted 3 February 2006
Available online 28 February 2006
Abstract—A series of aryltetrazolylacetanilides was synthesized and evaluated as HIV-1 non-nucleoside reverse transcriptase inhib-
itors on wild-type virus and on the clinically relevant K103N mutant strain. Extensive SAR investigation led to potent compounds,
with nanomolar activity on K103N, and orally bioavailable in rats.
Ó 2006 Elsevier Ltd. All rights reserved.
Non-nucleoside reverse transcriptase inhibitors (NNR-
TIs)1 have found widespread use in HIV therapy in mul-
tidrug regimens and in the so-called highly active
antiretroviral therapy (HAART), a combination of
nucleoside HIV reverse transcriptase inhibitors
(NRTIs), NNRTIs and protease inhibitors (PIs).2 How-
ever, the main concern in the use of NNRTIs in the clin-
ic still remains the rapid emergence of drug resistance,
due to mutation of the HIV-RT enzyme.3
From high-throughput screening (HTS) of our com-
pound collection, two interesting compounds (1–2) were
identified as potent low nanomolar inhibitors of HIV-1
RT polymerase, with submicromolar activity in a cell as-
say6 and significant in vitro activity on the K103N mu-
tant strain (Fig. 1).
The two lead compounds proved not to be orally bio-
available and were rapidly metabolized in rat plasma
to the corresponding carboxylic acids.
The most frequent HIV mutant strain observed in pa-
tients failing therapy with NNRTIs is K103N.4 This
mutation is observed in patients treated with each of
the only three currently marketed NNRTI drugs: nevira-
pine, delavirdine and efavirenz,4,5 thus creating cross-re-
sistance and preventing therapeutic efficacy of any
further treatment with available NNRTIs.
This prompted us to undertake an extensive medicinal
chemistry effort aimed at improving the pharmacokinet-
Cl
NO2
N
N
N
N N
H
H
N
N
N
S
S
In this respect, the identification of novel NNRTIs,
characterized by high potency on both the wild-type
HIV-1 and the clinically relevant HIV-1 RT K103N mu-
tant strain, is a constant goal for drug development.
N
N
O
O
1
2
WT RT Pol (IC50) 5 nM
K103N shift 3x
WT RT Pol (IC50) 5 nM
K103N shift 4x
Keywords: HIV; Non-nucleoside reverse transcriptase inhibitors; Tet-
razole thioacetanilides.
*
Figure 1. Lead tetrazole thioacetanilide inhibitors of HIV-RT, iden-
tified by HTS.
Corresponding author. Tel.: +39 06 91093276; fax: +39 06
91093225; e-mail: Ester_Muraglia@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.024

E. Muraglia et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2748–2752
2749
ic properties together with the intrinsic activity on the
K103N mutant.
This observation proved to be extremely important
when we discovered that anilides substituted in the 4-po-
sition with sulfonamide (Table 2, compound 12), car-
boxamide, or aliphatic chains bearing a tertiary amine,
were stable in rat plasma.
A SAR study at the anilide moiety of over 300 com-
pounds (a selection displayed in Table 1) revealed that
2-substitution is the only allowed monosubstitution,
nitro and halogen being the substituents of choice
(compounds 1–5). Monosubstitutions at the 3- or 4-
positions are generally not well tolerated (compounds
6–7), and 2,6-disubstitution completely abolishes activ-
ity (compound 8). The preferred aromatic disubstitu-
tion pattern is 2,4 (compounds 9–11), and, in this
latter case, the preferred 2-substituents are halogens,
as already seen for 2-monosubstitution, and also
methyl (as in compound 11). Introduction of a 2-
methyl gained significant activity from inactive 4-
substituted compounds (compare 11 with 7), though
to a lesser extent compared to chlorine (compare 10
with 7).
By introducing chlorine or methyl in the 2-position, we
were able to obtain stability in rat plasma, conferred by
the 4-substituent, and very high intrinsic potency on
both the WT and K103N mutant enzymes and in spread
cell based assays, conferred by the 2-substituent.
A simultaneous SAR study on the aryl linked to the tet-
razole core (Table 3) provided a further opportunity to
identify potent compounds. The pattern of aromatic
substitution is more tolerant compared to the anilide,
accepting most of the disubstitutions, except 3,4-disub-
stitution (compound 22) and the preferred substituents
are again halogens and methyl.
Table 1. SAR at anilide moiety
N
N
NHR
O
N
S
N
a
WT RT Pol IC50 (nM)
c
Compound
R
K103N shiftb
3
CIC95 (nM) 10% FBS/50% NHS
11/90
NO2
Br
I
3
3
4
5
7
5
3
100/810
31
312/1250
NO2
6
7
8
200
nad
nad
ndd
ndd
ndd
nad
nad
nad
NO2
Cl
Cl
NO2
Cl
9
10
11
4
22
3
5
3
31/400
Cl
78/1250
1250/nad
NO2
NO2
165
a IC50 values are means of at least two independent experiments.
b Ratio IC50 K103N RT Pol mutant/IC50 WT RT Pol.
c The two values of CIC95 refer to cell based assays in the presence of 10% FBS (fetal bovine serum) and 50% NHS (normal human serum). See Ref. 6.
d na: not active (>5 lM), nd: not determined.

2750
E. Muraglia et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2748–2752
Table 2. Anilide substitutions that confer stability in plasma
N
N
NHR
N
S
N
O
a
WT RT Pol IC50 (nM)
c
CIC95 (nM) 10% FBS/50% NHS
Compound
R
K103N shiftb
ndd
12
1252
1250/nad
50/580
SO2NH2
SO2NH2
13
54
1
Cl
14
15
16
4
55
3
3
1
2
16/62
31/62
8/16
SO2NH2
CONH2
Cl
Cl
Cl
CONH2
H
N
17
18
5
2
5
8/16
N
O
O
13
62/62
N
a IC50 values are means of at least two independent experiments.
b Ratio IC50 K103N RT Pol mutant/IC50 WT RT Pol.
c The two values of CIC95 refer to cell based assays in the presence of 10% FBS (fetal bovine serum) and 50% NHS (normal human serum). See Ref. 6.
d na: not active (>5 lM), nd: not determined.
More interestingly, the aryl SAR study gave compounds
with low or no shift (K103N shift = 1) on the K103N
mutant, which could be identified by appropriate substi-
tutions on the aryl rings.
total absence of oral exposure and extremely high values
of plasma clearance, the latter being due, at least in part,
to the already discussed hydrolytic metabolism.
The substitution of the labile 2-nitroanilide with more
stable anilides, such as in 13–14, not only significantly
decreased the plasma clearance, but produced orally
bioavailable compounds.
With the above SAR studies in hand, we envisaged the
possibility to optimize the activity on both the wild-type
RT enzyme and the K103N mutant strain by combining
some of the most active anilides with some of the best
aryls. The most relevant results are reported in Table 4.
One of the best compounds tested was 29, which exhib-
ited acceptable oral bioavailability and plasma
clearance.
We were delighted to see that by suitable combinations
of the anilides and aryls it is possible to obtain very po-
tent compounds on the WT RT, retaining the same low
nanomolar enzymatic activity on the relevant mutant
K103N.
In summary, an extensive SAR demonstrated that aryltet-
razolyl thioacetanilides are potent inhibitors of HIV-1
RT, with low nanomolar intrinsic activity on the enzyme
and submicromolar antiviral activity in HIV infected
cells. With a suitable combination of substitution pat-
terns both on the aryl linked to the tetrazole core and
the anilide aryl, it is possible to identify compounds which
maintain the same intrinsic activity on the wild-type HIV-
1 enzyme and the clinically relevant K103N mutant.
The possibility to combine the best aryls with the ani-
lides that confer the highest activity, but also the re-
quired metabolic stability, allowed also the
optimization of the pharmacokinetic properties of the
series (Table 5).
The 2-nitrophenylanilide 3 had the same pharmacoki-
netic profile as the lead compound 1, characterized by
The tetrazole derivatives were synthesized as outlined in
Scheme 1, starting from the commercially available

E. Muraglia et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2748–2752
2751
Table 3. SAR at aryl moiety
NO2
N
N
H
N
N
S
N
R
O
a
WT RT Pol IC50 (nM)
c
Compound
R
Cl
Cl
F
K103N shiftb
1
CIC95 (nM) 10% FBS/50% NHS
31/200
Cl
19
4
20
21
23
10
1
1
78/1250
63/250
Cl
22
23
230
2
3
nad
Cl
Br
Cl
3
8/62
Cl
Cl
24
25
26
27
12
18
3
1
1
3
1
78/125
63/250
8/31
Cl
Cl
Cl
4
15/62
a IC50 values are means of at least two independent experiments.
b Ratio IC50 K103N RT Pol mutant/IC50 WT RT Pol.
c The two values of CIC95 refer to cell based assays in the presence of 10% FBS (fetal bovine serum) and 50% NHS (normal human serum). See Ref. 6.
d na: not active (>5 lM).
Table 4. Effect of combination of aryl and anilide SAR on potency and resilience on K103 mutant
Cl
N
N
H
N
N
S
N
R
O
SO2NH2
a
WT RT Pol IC50 (nM)
c
CIC95 (nM) 10% FBS/50% NHS
Compound
R
K103N shiftb
Cl
Cl
28
9
1
1
62/250
16/125
Cl
29
3
Cl
a IC50 values are means of at least two independent experiments.
b Ratio IC50 K103N RT Pol mutant/IC50 WT RT Pol.
c The two values of CIC95 refer to cell based assays in the presence of 10% FBS (fetal bovine serum) and 50% NHS (normal human serum). See Ref. 6.

2752
E. Muraglia et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2748–2752
Table 5. Rat PK profilesa
of PCl5 by a slightly modified literature procedure.7 All
the final compounds have been purified by RP-HPLC.
Compound
F (%)
Clp (ml/min/kg)
t1/2
3
14
13
29
0
33
60
33
272
125
85
0.23
0.83
1.1
Acknowledgments
37
We gratefully thank for D.M. and analytical studies Fa-
bio Bonelli, Annalise Di Marco, Fabrizio Fiore, Massi-
miliano Fonsi, Odalys Gonzales-Paz, Marina Taliani
and Maria Verdirame. This work was supported in part
by a grant from the MIUR.
a Oral and iv dosing at 3 mg/kg. PO formulation: suspension in
PEG400. iv formulation: solution in DMSO/PEG400/water
20:60:20.
N
N
N
N
N
N
a
b
References and notes
Ar NCS
S
CO2H
SH
N
N
Ar
Ar
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30
31
N
N
c
NR1R2
N
S
N
O
Ar
32
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aniline, toluene/CHCl3, rt 1–2 h then 70 °C 1–3 h. Purification by RP-
HPLC.
}
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isothiocyanates. Reaction with NaN3 produced the cor-
responding tetrazole-thiols 30, which were alkylated
with chloroacetic acid in aqueous conditions. The final
anilides were accessed by reaction of the thioacetic acids
31 with the required anilines in the presence of 1.2 equiv
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