Inhibitors of HIV-1 attachment. Part 9: An assessment of oral prodrug approaches to improve the plasma exposure of a tetrazole-containing derivative

Article abstract of DOI:10.1016/j.bmcl.2012.10.125

7-(2H-Tetrazol-5-yl)-1H-indole 3 was found to be a potent inhibitor of HIV-1 attachment but the compound lacked oral bioavailability in rats. The cause of the low exposure was believed to be poor absorption attributed to the acidic nature of the tetrazole moiety and, in an effort to address this liability, three more lipohilic tetrazole analogs, N-acetoxymethyl 4, N-pivaloyloxymethyl 5, and N-methyl 6, were evaluated as potential oral prodrugs in rats. Prodrug 5 was ineffective in improving the plasma concentration of 3 in vivo but compound 4 provided a 15-fold enhancement of the plasma concentration of 3. Most interestingly, oral dosing of analog 6 afforded a substantial increase in the plasma concentration of the parent in rats when compared to dosing of parent. This represents a novel example of a methyl tetrazole that acts as a prodrug for a free NH tetrazole-containing compound.

Full text of DOI:10.1016/j.bmcl.2012.10.125

Bioorganic & Medicinal Chemistry Letters 23 (2013) 209–212
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitors of HIV-1 attachment. Part 9: An assessment of oral prodrug
approaches to improve the plasma exposure of a tetrazole-containing derivative
⇑
Kap-Sun Yeung , Zhilei Qiu, Zheng Yang, Lisa Zadjura, Celia J. D’Arienzo, Marc R. Browning,
Steven Hansel, Xiaohua Stella Huang, Betsy J. Eggers, Keith Riccardi, Ping-Fang Lin, Nicholas A. Meanwell,
John F. Kadow
Bristol-Myers Squibb Research & Development, 5 Research Parkway, PO Box 5100, Wallingford, CT 06492, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 7 November 2012
7-(2H-Tetrazol-5-yl)-1H-indole 3 was found to be a potent inhibitor of HIV-1 attachment but the com-
pound lacked oral bioavailability in rats. The cause of the low exposure was believed to be poor absorp-
tion attributed to the acidic nature of the tetrazole moiety and, in an effort to address this liability, three
more lipohilic tetrazole analogs, N-acetoxymethyl 4, N-pivaloyloxymethyl 5, and N-methyl 6, were eval-
uated as potential oral prodrugs in rats. Prodrug 5 was ineffective in improving the plasma concentration
of 3 in vivo but compound 4 provided a 15-fold enhancement of the plasma concentration of 3. Most
interestingly, oral dosing of analog 6 afforded a substantial increase in the plasma concentration of the
parent in rats when compared to dosing of parent. This represents a novel example of a methyl tetrazole
that acts as a prodrug for a free NH tetrazole-containing compound.
Keywords:
Antivirals
HIV-1 attachment inhibitors
Indole glyoxamide
Tetrazole
Produrgs
Ó 2012 Elsevier Ltd. All rights reserved.
Highly active anti-retroviral therapy (HAART) has significantly
reduced the incidence of mortality associated with human immu-
nodeficiency virus-1 (HIV-1) infection/AIDS, and transformed the
disease from an acute to a chronic one.1 However, the emergence
and persistence of drug-resistant HIV-1 mutant viruses, HAART-re-
lated toxicities, patient non-compliance and the appearance of co-
morbidities associated with long-term therapy remain significant
medical challenges.2 Inhibitors of new viral targets thus continue
to be evaluated for the treatment of HIV-1 infection in order to
help overcome these major problems.3
A key first step during the HIV-1 entry into the host cells is the
attachment of its envelope glycoprotein gp120 to the host cell
receptor CD4. A subsequent conformational change in gp120 leads
to the binding of this binary complex to a co-receptor, either the
chemokine receptor CCR5 in the early stages of infection or CXCR4
in more advanced infection. A further conformational change in
gp120 induced by co-receptor binding triggers activation of the
transmembrane protein gp41, which inserts its hydrophobic
N-terminal into the host cell membrane as a prelude to fusion be-
tween the viral and host cell membranes, resulting in the release of
the viral genetic material into the host.4 Indole- or azaindole-3-
glyoxamide-derived small molecules that interfere with the
attachment of gp120 to CD4 by binding to the CD4 binding site
in gp120 were previously reported from these laboratories.5–7
BMS-488043 (1, Fig. 1) was advanced into clinical studies and
found to be safe and well tolerated in healthy human subjects.
Proof-of-concept for this novel antiviral mechanism has been
achieved with this compound in HIV-1 infected patients.7e,8 After
extensive optimization, this compound was supplanted in clinical
development by a phosphonooxymethyl prodrug (BMS-663068)
of an analog (BMS-626529) that showed an improved pre-clinical
profile.9,10
During the optimization of the early lead compound 2, an in-
dole-containing HIV-1 attachment inhibitor, the C-7-tetrazole ana-
log 3 (Fig. 2) was found to possess greatly enhanced potency
toward a HIV-1 JRFL pseudotype virus in a single cycle infectivity
assay. Further studies showed that 3 possessed nanomolar to
sub-nanomolar potency against both M- and T-tropic virus strains
in cell culture.7h Despite the fact that an NH tetrazole is a classical
carboxylic acid isostere that usually results in compounds in which
their tetrazolates are approximately 10 times more lipophilic than
the corresponding carboxylates,11 and certain tetrazole-containing
drugs, for example the angiotensin II receptor antagonist, losartan,
is well absorbed and orally bioavailable, 3 showed negligible oral
exposure (F = 2.3%) when administered orally to rats.12 This was
attributed to its low membrane permeability as a result of its ion-
ized state at pH 6.5 (calculated pKa of the tetrazole-NH of 3 = 4.2)
and predicted by a 17 nm/sec Caco-2 permeability value measured
at pH 6.5. As discussed in Part 8 of this series of reports,7h the oral
bioavailability of the C7-heteroaryl substituted analogs of 2 ap-
peared to correlate with membrane permeability, and a Caco-2
Pc of >100 ensured good bioavailability. Since 3 exhibited a
⇑
Corresponding author.
E-mail address: kapsun.yeung@bms.com (K.-S. Yeung).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.125

210
K.-S. Yeung et al. / Bioorg. Med. Chem. Lett. 23 (2013) 209–212
O
parent 3 over a 5 min interval in HLMs, thus indicating that the
cleavage of the carboxymethyl group in 4 and 5 was not mediated
by CYP 450 enzymes. The acetoxymethyl analog 4 was tested in the
in vitro Caco-2 (pH 6.5) assay to assess its potential for absorption.
Low recovery (<70%) of the compound was observed however, rais-
ing concern that 4 might be either chemically unstable in the assay
medium or prematurely hydrolyzed in vivo by the esterases pres-
ent in the enterocytes and gut lumen. When dosed orally to rats,
the acetoxymethyl analog 4 nevertheless demonstrated a satisfy-
ing 15-fold increase in the concentration of parent tetrazole 3 in
rat plasma at both 30 and 120 min post dosing when compared
to levels of 3 obtained after dosing the parent compound (Table 1).
However, the pivaloylxymethyl analog 5 was an ineffective pro-
drug and did not increase the exposure of parent tetrazole 3
in vivo. This compound likely possessed low intestinal solubility
since a Caco-2 permeability value could not be determined for 5
because it was too insoluble in the medium used to conduct the
Caco-2 assay. The rat plasma concentration of 3 increased threefold
at the first 30 min after dosing with 5 but dropped to a concentra-
tion twofold less than that from dosing of 3 at the 120 min time
point. The higher concentration at the earlier time point may indi-
cate that the intact prodrug has improved absorption compared to
the parent drug. The low solubility of the compound or the rapid
conversion to parent prior to absorption could explain the observa-
tion of limited exposure at the later time point.
Interestingly, and somewhat unexpectedly, the plasma concen-
tration of the parent tetrazole 3 obtained after dosing of the meth-
yltetrazole 6 increased almost proportionally over time, and was
4- and 8-fold higher than after dosing with 3 at 30 and 120 min
post-dosing, respectively. Methyltetrazole 6 was not metabolically
stable in NADPH-supplemented HLMs, showing a half-life of
12 min, which compares to >200 min obtained for the parent tetra-
zole 3. CYP 2B6-mediated N-demethylation of an N-methyl-5-
phenyltetrazole-containing bradykinin B1 receptor antagonist
was previously found to be the major pathway of metabolism in
human liver microsomes and hepatocytes.20 CYP 450-mediated
activation of prodrugs has been demonstrated as a viable prodrug
release mechanism,21 so it is plausible that the parent tetrazole 3
was released via CYP 450-mediated oxidative N-demethylation of
6 in the liver. Based on the plasma concentrations of 3 present at
the two time points, it appears possible that the acetoxymethyl
prodrug 4 underwent fast absorption while the methyltetrazole 6
was absorbed more slowly. However, the differences in the plasma
exposure of 3 could have also resulted from differences in the rate
of enzymatic conversion of 4 and 6 to the parent in vivo.
OMe
O
N
N
N
O
N
H
MeO
BMS-488043 (1)
Figure 1. HIV-1 attachment inhibitor BMS-488043.
satisfactory terminal half-life (67 min) and a low total clearance
(3.1 mL/min/kg) after IV dosing to rats, and given its superior
in vitro antiviral profile compared to 2, a prodrug approach
designed to improve the oral exposure of 3 was considered as a
means of establishing its potential to advance into further studies.
Although examples of prodrugs of carboxylic acids to improve
absorption are abundant in the literature,13 examples of prodrugs
of tetrazoles are rare.14 However, the precedent provided by the
few literature examples of tetrazole prodrugs suggested that this
could potentially be a viable approach. For example, a model tetra-
zole prodrug, 2-pivaloyloxymethyl-5-phenyltetrazole, underwent
rapid hydrolysis to the parent compound in rat plasma.15 Hydroly-
sis of a pivaloyloxymethyl-5-phenyltetrazole prodrug of an angio-
tensin II receptor antagonist to the parent was demonstrated in rat
small intestinal homogenates, liver homogenates and plasma, with
half-lives of 22.7, 9.80 and 3.94 min, respectively. These data were
interpreted by the authors of the study to suggest that much of the
intact prodrug had the potential to be absorbed through the gut
wall and into the circulation without extensive hydrolysis, with
facile hydrolysis of the prodrug by esterases in the liver and plas-
ma. When the prodrug was actually dosed intravenously to rats,
a time-dependent decrease in the AII-induced pressor response
in the animals was observed.16 In another published study, the 2-
methyl-1-(2H-tetrazol-2-yl)propyl pivalate prodrug of the angio-
tensin II receptor antagonist BMS-183920 increased the oral bio-
availability of the parent BMS-183920 in rats from 11% to 39%
when dosed orally.17–19
Three potential prodrugs of the parent tetrazole 3, N-acetoxy-
methyl derivative 4, the N-pivaloyloxymethyl homolog 5 and the
simple N-methyl tetrazole 6 were prepared, and their anti-viral po-
tency and the extent of oral absorption and conversion of prodrug
to parent in rats were evaluated. As shown in Table 1, compounds 4
and 6 exhibited a similar level of potency to the parent tetrazole 3
against the JRFL pseudotyped virus, while 5 was about sixfold less
potent. The prodrugs retained the desirable low cytotoxicity exhib-
ited by 3. Both 4 and 5 were unstable in human liver microsomes
(HLMs) in the absence of the CYP 450 enzyme co-factor, NADPH.
Under these conditions, both compounds completely released the
In conclusion, three tetrazole analogs, N-acetoxymethyl 4, N-
pivaloyloxymethyl 5 and N-methyl 6, were evaluated as prodrugs
of the potent HIV attachment inhibitor 3 in an effort to improve
O
F
O
N
N
O
O
N
H
3
F
N
N
O
N
N
pseudotyped-JRFL
EC50 = 0.034 nM
CC50 = 245
N
N
H
M
µ
O
N
M-tropic Bal EC50 = 0.37 nM
T-tropic BRU EC50 = 6.38 nM;
NL4-3 EC50 = 9.41 nM
H
2
pseudotyped-JRFL
EC50 = 1.24 nM
CC50 = >300
CacoPc (pH 6.5) = 17 nm/s Rat %F = 2.3
M
µ
HLM t1/2 = >200 min
IV t1/2 = 67 min
CL total = 3.1 ml/min/kg
Figure 2. HIV-1 attachment inhibitor lead compound 2 and the C7-tetrazole analog 3.

K.-S. Yeung et al. / Bioorg. Med. Chem. Lett. 23 (2013) 209–212
211
Table 1
Antiviral activity, cytotoxicity and rat oral plasma exposure of 3 and its prodrugs 4, 5 and 6
O
F
O
N
N
O
N
N
N
H
N
N
R
Plasma [3] @30/120 min (ng/ml)a
22
22
Inhibitor
R
EC50 (nM)
CC50
(
lM)
3
4
5
6
H
34
60
190
20
245
36
228
19/25
261/378
60/13
CH2OCOMe
CH2OCOt-Bu
Me
>300
72/207
a
5 mg/kg P.O.; dosing vehicle 90% PEG 400/10% EtOH; each data was the average of those obtained from two rats.
upon the parent compound’s poor oral bioavailability in rats. After
oral dosing to rats, compound 5 had negligible effect on the plasma
concentration of 3 compared to dosing the parent compound;
however, analog 4 provided a 15-fold enhancement in exposure.
Surprisingly, N-methyltetrazole 6 also conferred a substantial in-
crease in the plasma concentration of the parent in rats over the
time course of the study. Thus analog 6 represents a novel example
of prodrug methodology for tetrazole-containing compounds.
These studies exemplify the feasibility of prodrug approaches to
improve the oral exposure of NH tetrazoles either through a hydro-
lytic or oxidative cleavage mechanism. These tactics, previously
examined in only very limited examples and seldomly explored
to date, are of potential application to discovery programs where
such compounds exhibit insufficient oral exposure due to poor
absorption.
The syntheses of 3 and analogs 4 to 6 are shown in Schemes 1
and 2. The 7-cyano-4-fluoroindole 8 was obtained from the cyana-
tion of 7-bromo-4-fluoroindole 7 using copper cyanide. Friedel–
Crafts acylation of 8 using oxalyl chloride provided acid chloride
9, which was coupled with N-benzoylpiperazine to form the inter-
mediate 10. Reaction of the cyano group of 10 with sodium azide
provided the parent tretrazole 3, which was methylated using trim-
ethylsilyldiazomethane to give the N-methyltetrazole 6. The prep-
aration of 4 and 5 relied on an alkylcarboxymethylation of the
tetrazole 11 with the corresponding halomethyl reagents. After Fri-
edel–Crafts acylation of 12, it was more convenient to hydrolyze the
intermediate acid chloride to the acid 13 since partial hydrolysis to
F
F
F
b
a
N
H
N
H
N
H
N
N
N
N
CN
8
N
N
N
NH
11
12
ROCO
O
OH
O
F
O
d
c
N
N
H
4, 5
+
HN
N
N
N
N
13
ROCO
Scheme 2. Synthesis of compounds 4 and 5. Reagents and conditions: (a) NaN3,
NH4Cl, DMF, 100 °C; 17 h; (b) BrCH2OCOMe (for 4), or ClCH2OCOt-Bu (for 5), K2CO3,
MeCN, rt, 22 h; (c) (i) (ClCO)2, CH2Cl2, rt, 21 h; (ii) i-Pr2EtN, THF, rt, 19 h; (d) EDC,
DMAP, DMF, rt, 20 r.23
the acid occurred during the coupling of the acid chloride with N-
benzoylpiperazine. The acid 13 was then coupled to N-benzoylpip-
erazine using carbodiimide to furnish the final products 4 and 5.
O
Cl
F
F
F
O
b
a
O
+
N
N
H
N
H
HN
N
H
Br
CN
8
CN
7
9
O
N
O
N
O
F
c
d
e
3
6
N
H
CN
10
Scheme 1. Synthesis of compounds 3 and 6. Reagents and conditions: (a) CuCN, DMF, 145 °C, 17 h; (b) (ClCO)2, CH2Cl2, reflux, 3 days; (c) i-Pr2EtN, THF, rt, 16 h, (d) NaN3,
NH4Cl, DMF, 85 °C; 12 h; (e) TMSCHN2, MeOH/PhH, rt, 90 min.23

212
K.-S. Yeung et al. / Bioorg. Med. Chem. Lett. 23 (2013) 209–212
10. Kadow, J. F.; Ueda, Y.; Connolly, T. P.; Wang, T.; Chen, C. P.; Yeung, K.-S.; Bender,
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23. The regiochemistry of alkylation was supported by NMR heteronuclear
multiple bond correlation (HMBC) studies on
6 and an N2-allyltetrazole
analog of 3 (prepared in the same manner as 4 and 5) in which the H-N long-
range correlation between the proton of the methyl group of 6 or allyl proton
and three nitrogen atoms of the tetrazole was observed.