Inhibitors of human immunodeficiency virus type 1 (HIV-1) attachment 13. Synthesis and profiling of a novel amminium prodrug of the HIV-1 attachment inhibitor BMS-585248

Article abstract of DOI:10.1021/jm301638a

In vitro studies suggested that the ammonium salt 2 could be a viable prodrug of the HIV-1 attachment inhibitor 1. Increased systemic exposure of the parent drug 1 following oral administration of the amminium salt 2 when compared to similar studies using

Full text of DOI:10.1021/jm301638a

Article
pubs.acs.org/jmc
Inhibitors of Human Immunodeficiency Virus Type 1 (HIV-1)
Attachment 13. Synthesis and Profiling of a Novel Amminium
Prodrug of the HIV‑1 Attachment Inhibitor BMS-585248
,
†
‡
†
‡
,‡
‡
Alicia Regueiro-Ren,* Jean Simmermacher-Mayer, Michael Sinz,* Kim A. Johnson,
‡
‡
‡
‡
Xiaohua Stella Huang, Susan Jenkins, Dawn Parker, Sandhya Rahematpura, Ming Zheng,
Nicholas A. Meanwell, and John F. Kadow
†
†
‡
Departments of Medicinal Chemistry and Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and
Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States
ABSTRACT: In vitro studies suggested that the ammonium salt
2
could be a viable prodrug of the HIV-1 attachment inhibitor 1.
Increased systemic exposure of the parent drug 1 following oral
administration of the amminium salt 2 when compared to similar
studies using solution dosing of the parent compound was
observed in the in vivo studies in both rats and dogs. At high
doses, the improvement in oral exposure of the parent drug was
even more evident, indicating that the increased solubility of the
amminium salt 2 can overcome dissolution-limited absorption
and demonstrating the potential utility of this compound as a
prodrug of 1.
INTRODUCTION
■
BMS-585248 (1), a potent, third-generation HIV-1 attachment
inhibitor (AI) with a promising initial in vitro and in vivo
pharmacokinetic profile, was selected for preclinical evaluation in
higher species and eventually as a candidate for clinical
1
development. However, at high doses, 1 displayed poor oral
exposure which was attributed to poor solubility (7 μg/mL) and,
as a result, its development presented a challenge. Several
strategies were explored in order to improve the exposure of 1.
Among them were alternative formulations and the use of
classical prodrugs such as phosphate-based approaches which
2
had proved successful with previous compounds in this class. In
Figure 1. BMS-585248 (1) and its amminium prodrug (2).
a parallel approach, we focused on introducing polar or ionizable
substituents onto 1 in an effort to increase solubility and thus
overcome the dissolution-limited absorption. Installation of an
amino moiety in the pyridyl group was considered as both a
means of introducing a charge into the structure as well as a
handle for further elaboration. However, when 1 was submitted
to amination conditions, the amino group was introduced to the
triazole moiety rather than the pyridine to afford the novel N-
hypothesized that the N−N bond in 2 could be susceptible to a
similar cleavage, resulting in the release of the parent drug 1.
Preliminary in vitro experiments demonstrated that conversion
to the parent was possible. Subsequently, increased systemic
exposure of the parent drug 1 following oral administration of the
amminium salt 2 when compared to similar studies using
solution dosing of the parent compound was observed in both
rats and dogs. At high doses, the improvement in oral exposure of
parent drug was even more evident, indicating that the increased
solubility of the amminium salt 2 can overcome the dissolution-
limited absorption and demonstrating the potential utility of this
compound as a prodrug. Several other experiments helped
determine the mechanism by which the parent drug is released as
3
,4
amino-triazolium (amminium) salt 2 (Figure 1).
Amminium salt 2 displayed improved aqueous solubility (1.76
mg/mL) but poor antiviral potency (EC50 = 1.76 nM) and
considerably decreased membrane permeability when compared
with 1 (15 nm/s vs 150 nm/s in the Caco-2 assay). On the basis
of this profile, 2 did not appear to have potential to be a useful
antiviral agent and we were unaware of examples in the literature
where amminium salts served as viable prodrugs. However, it is
known that N-oxides can act as prodrugs by releasing the parent
Received: November 6, 2012
Published: February 1, 2013
5
,6
drug in vivo via reductive cleavage of the N−O bond. We
©
2013 American Chemical Society
1670
dx.doi.org/10.1021/jm301638a | J. Med. Chem. 2013, 56, 1670−1676

Journal of Medicinal Chemistry
Article
Figure 2. Nitrogen chemical shift assignment for 2. The 15N chemical shifts were referenced to NH (liq) at −380.2 ppm..
3
Figure 3. Rat plasma concentration−time profiles of 1 and 2 after intravenous (1 mg/kg) and oral (5 mg/kg) dosing of 2 (mean ± SD).
Figure 4. Dog plasma concentration−time profiles of 1 and 2 after intravenous (1 mg/kg) and oral (5 mg/kg) dosing of 2 (mean ± SD).
8
well as illustrate the potential to apply this methodology to other
unrelated chemotypes.
methods, i.e., hydroxylamine-O-sulfonic acid; O-(2,4-dinitro-
9
phenyl) hydroxylamine rendered no desired product, which
seemed most likely due to the poor solubility of 1 in the reaction
media. The resulting amminium tosylate salt was converted into
the amminium chloride salt by chromatography using an ion
exchange column. All in vitro and in vivo experiments were
performed using the chloride salt. The connection between the
CHEMISTRY
■
The conversion of 1 into amino salt 2 was accomplished in 25%
yield by regioselective amination of 1 using a methylene chloride
7
solution of NH OTs at ambient temperature. Other amination
2
1
671
dx.doi.org/10.1021/jm301638a | J. Med. Chem. 2013, 56, 1670−1676

Journal of Medicinal Chemistry
Article
Figure 5. Rat dose escalation study after oral dosing of 2 (prodrug) and 1.
amino group (NH ) and the triazole N12 nitrogen was
Table 1. Pharmacokinetic Parameters from Rat Dose
Escalation Study
2
confirmed by the observation of the H−N long-range correlation
between the amino proton H15 (9.06 ppm) and the N12
nitrogen (265.1 ppm) (Figure 2). This conclusion is further
supported by the NOE observed between the N15 proton and
H13 (9.01 ppm).
parameter
2
1
dose (mg/kg)
5
25
12
72
200
77
15
6.6
42
75
200
11
C
max (μM)
1.3
8
8.4
115
AUCtot (μM·h)
996
145
RESULTS AND DISCUSSION
■
In Vivo Conversion of 2 in Rats and Dogs. Conversion of
the prodrug 2 to the parent 1 was observed following both
intravenous (IV) and oral (PO) administration in rats. The
plasma profiles obtained from the pharmacokinetic experiments
are shown in Figure 3. Following IV administration of 2 to male
rats, the prodrug had a moderate clearance (23 mL/min/kg), a
low volume of distribution (0.29 L/kg), and a short half-life (0.3
h). The conversion of prodrug was rapid after IV dosing with 1
appearing at the earliest time point (5 min). Following IV
administration of 2, 1 had 2-fold higher AUC than 2 (3220 vs
In Vitro Conversion of 2. With in vivo rat and dog
pharmacokinetic studies showing markedly increased systemic
exposure of the parent after administration of the amminium
prodrug, further in vitro and in vivo experiments were conducted
in order to investigate the mechanism by which the prodrug is
converted to parent drug in intact animals. Despite rapid and
extensive conversion in vivo, 2 was found to be stable in Tris-HCl
buffer (pH 7.4), rat blood, rat plasma, rat liver cytosol,
microsomes, and hepatocytes when the compound was
incubated under conventional aerobic conditions. Typically
microsomal enzymes oxidize compounds, however, they also
have the ability to reduce compounds and this reduction can be
enhanced under anaerobic incubation conditions. Low
amounts of conversion of prodrug to parent were observed in
rat and human liver microsomes when incubated under
anaerobic conditions (∼10% and 4%, respectively). Additional
incubations using recombinant CYPs under anaerobic conditions
indicated CYP3A4 and CYP2D6 have the ability to convert
prodrug to parent, however, the amounts formed were too low to
accurately quantify.
Contribution of Gut Metabolism in the Conversion of
2. Because the cleavage occurred under reducing conditions in
vitro, additional in vivo experiments were conducted to
determine if the reductive metabolism was mediated by the
liver or microflora present in the gastrointestinal tract. To assess
the effect of intestinal microflora on metabolism, an antibiotic-
treated rat model was used. Figure 6 shows the pharmacokinetic
profiles of both prodrug 2 and parent 1 after oral administration
of 2. The exposure (AUC and Cmax) of 1 following administration
of 2 to antibiotic-treated rats decreased 3.5-fold and 5.6-fold,
respectively, when compared to vehicle-treated animals (Table
2). However, pretreatment of rats with antibiotics did not alter
the systemic exposure of 2 following an oral dose of 2. These data
indicate that removal of the anearobic bacteria responsible for gut
reductive metabolism has an impact on the formation of 1
(significantly lower parent drug), yet has only a minor effect on
1
553 nM·h, respectively). Following oral administration of 2,
very low exposure of the prodrug was observed, with a Cmax of 23
nM while relatively high exposure of 1 was observed, with a Cmax
of 3121 nM 6 h postdose. The exposure of 1 was much greater
than 2 after oral dosing (28098 vs 20.8 nM·h, respectively),
indicating substantial conversion of the parent drug from the
prodrug. Similar increases in systemic exposure of the parent
drug following administration of prodrug were also observed in
male beagle dogs (Figure 4). Following IV administration of 2 at
1
0
1
mg/kg, moderate clearance (13.8 mL/min/kg), a low volume
of distribution (0.3 L/kg), and a short half-life of 2 (0.9 h) were
observed. Conversion to 1 after IV adminstration occurred with
an AUC 1/AUC 2 of ∼16%. Following oral administration of 2 in
dogs, low exposure of the prodrug was observed (C : 139 nM);
max
however, the exposure of the parent was ∼8-fold higher. The
exposure of 1 was again much greater than 2 after oral dosing
(
8064 vs 217 nM·h, respectively). These data in rats and dogs
indicate that the prodrug 2 is rapidly and extensively converted to
the parent drug 1 in vivo.
In a rat dose escalation study, 2 and 1 were dosed orally at 5,
5, and 200 mg/kg and 5, 15, and 200 mg/kg, respectively. Dose-
2
limiting absorption was observed after administration of 1 at
higher doses of 75 and 200 doses mg/kg, as would be expected
for a poorly soluble compound. However, dose-proportional
increases in Cmax and greater than dose-proportional increases in
AUC of parent drug 1 were observed following administration of
the prodrug up to 200 mg/kg (Figure 5, Table 1).
1
672
dx.doi.org/10.1021/jm301638a | J. Med. Chem. 2013, 56, 1670−1676

Journal of Medicinal Chemistry
Article
Figure 6. Exposure profiles of 1 and 2 after oral dosing of 2 (5 mg/kg) in antibiotic treated animals. Each data point is expressed as mean ± SD.
Table 2. Pharmacokinetic Parameters from Rats Pretreated
with an Antibiotic
and the parent in the portal vein (prehepatic) were similar to the
respective profiles in the jugular vein (posthepatic) samples.
Overall, very low levels of 2 (74 nM and 35 nM·h, Cmax and AUC
parameter
treatment
2
1
(
portal vein), respectively) were observed in both portal and
+ antibiotics − antibiotics + antibiotics − antibiotics
jugular vein samples and the majority of drug-related material
was parent drug (2137 nM and 23873 nM·h, Cmax and AUC
(portal vein), respectively) (jugular vein data not shown). These
data suggest that the majority of the prodrug conversion occurs at
the level of the intestine
To further evaluate the metabolite disposition of the prodrug,
a biotransformation study was conducted using bile duct
cannulated rats (Scheme 1). Relative percentages of 2, an N-
acetyl conjugate 3, and 1 in plasma were 68%, 15.7%, and 16%,
respectively (pooled plasma from 0 to 2 h post dose). The major
drug-related components observed in bile were the N-acetyl
conjugate 3 and 2, with a metabolite-to-prodrug ratio of 9:1,
Cmax (nM)
79
70
18
7
AUClast (nM·h)
148
112
13761
47535
the exposure of the prodrug 2 (minor increases in the exposure of
).
Because both gut microflora and hepatic enzymes (CYP2D6
and CYP3A4) appeared to mediate the reductive metabolism of
, the relative contribution of hepatic vs gut metabolism in the
2
2
conversion of 2 was evaluated using dual cannulated portal vein
and jugular vein rats. Following oral administration of the
prodrug 2, the concentration vs time profiles of both the prodrug
Scheme 1. Metabolites of 2 Observed in Bile Duct Cannulated Rats
1
673
dx.doi.org/10.1021/jm301638a | J. Med. Chem. 2013, 56, 1670−1676

Journal of Medicinal Chemistry
Article
while 1 was not observed in bile. In addition to 2, both the N-
acetyl conjugates 3 and 1 were detected in urine at relative
percentages of 80%, 16.5% ,and 3.5%, respectively.
High-Throughput in Vitro Method for the Screening of
Amminium Prodrug. To investigate the utility of this prodrug
with other N-amino compounds, as well as to develop a higher
throughput method to assess the participation of intestinal
microflora in the metabolism of this type of prodrug, 2 was
incubated in fresh rat fecal homogenate. After a 2 h incubation, a
pressure liquid chromatography (HPLC) and LC-MS analyses were
conducted using Shimadzu LC-10AS pumps and a SPD-10AV UV−vis
detector set at 254 nm with the MS detection performed with a
Micromass Platform LC spectrometer for LC in electrospray mode.
NMR ( H, C, and N) spectra were recorded on a Bruker 500 MHz
spectrometer equipped with a 5 mm TCI cryo probe in DMSO. The
proton and carbon chemical shift were referenced to TMS at 0.00 and
are reported in ppm. The nitrogen chemical shift was referenced to
1
13
15
liquid NH at −380.2 ppm. High-resolution mass spectra (HMRS) were
3
recorded on an LTQ-Orbitrap.
96% conversion of prodrug 2 to parent 1 was observed. This
1-Amino-3-{3-[2-(4-benzoyl-piperazin-1-yl)-2-oxo-acetyl]-4-
fluoro-1H-pyrrolo[2,3-c]pyridin-7-yl}-3H-[1,2,3]triazol-1-ium. A
same in vitro method was used with several other N-aminated
heterocycles (4 and 5) (Figure 7), each of which showed
conversion to parent compound (4, 88%, and 5, 35%
conversion).
cloudy semisolution of 1-(4-benzoyl-piperazin-1-yl)-2-(4-fluoro-7-
1
[
(
1,2,3]triazol-1-yl-1H-pyrrolo[2,3-c]pyridin-3-yl)-ethane-1,2-dione
160 mg, 0.35 mmol) in CH Cl was treated with 5 mL of a CH Cl
2
2
2
2
solution of H NOTs (prepared as indicated below) and stirred at rt for 3
2
h, at which point the reaction had become homogeneous. The solvent
was removed in vacuo, and the residue was dissolved in MeOH and
purified using a medium pressure reverse phase column (YMC C18)
and 10% MeCN/99% H O/1% HCl to 50% MeCN/49% H O/1% HCl
2
2
as the mobile phase to afford the chloride salt of compound 2 as a pale-
yellow solid (40 mg, 23%). Analytical RP-HPLC t = 6.70 min (Sunfire
R
C18 3.5 μm, 3.0 mm × 150 mm, 15 min gradient, 1 mL/ming, 9.5%
MeCN/H O/0.1%TFA to 95% MeCN/H O/0.1%TFA, 254 nm, purity
2
2
Figure 7. Model N-aminated heterocycles 4 and 5.
+
9
9.1%). HRMS m/z: (M ) calcd for C H O N F, 463.1637; found,
22 20 3 8
1
4
63.1623. H NMR (500 MHz, DMSO-d ) δ 13.63 (br s, 1H), 9.55 (br
6
s, 1H), 9.06 (br s, 2H), 9.01 (br s, 1H), 8.65 (s, 1H), 8.44 (s, 1H), 7.45
(br s, 5H), 3.69 (br s, 4H), 3.49 (br s, 4H). 13C NMR (126 MHz,
CONCLUSION
■
DMSO-d ) δ 184.0, 169.3, 165.4, 153.6 (d, J = 263.4 Hz), 142.5, 135.5,
An amminium salt 2 of the 1,2,3-triazole-containing HIV-1
attachment inhibitor 1 was found to behave as a prodrug,
showing good systemic exposure of the parent 1 following oral
administration to both rats and dogs. The improved solubility of
the amminium prodrug allowed for higher doses to be
administered and overcame the solubility-limited absorption
associated with the parent drug. In principle, any compound with
a nitrogen-containing heterocycle can be aminated and the
amminium salt, providing that it is chemically stable, could be
used as a potential prodrug. This approach provides a potentially
useful method to help with the progression in the clinic and
6
1
26.5 (d, J = 29.1 Hz), 126.7, 122.2 (d, J = 20.9 Hz), 113.4, 45.3, 40.8.
The trifluoroacetate salt of compound 2 was obtained by purification
of the crude reaction productusing reverse phase HPLC (YMC C18 S5
0 × 50 mm column) and 10% MeOH/90% H O/0.1% TFA to 90%
2
2
MeOH/10% H O/0.1% TFA as the mobile phase.
2
1
-Amino-2-phenyl-6,7-dihydro-5H-pyrrolo[1,2-a]imidazol-1-
ium 4-methylbenzenesulfonate (4). Prepared from 2-phenyl-6,7-
1
4
dihydro-5H-pyrrolo[1,2-a]imidazole following the procedure de-
scribed above (white solid, 62% yield). Analytical RP-HPLC tR
=
1
1
0.38 min (Xbridge Phenyl 3.5 μm, 3.0 mm × 150 mm, 15 min gradient,
mL/min, 9.5% MeCN/H O/0.1%TFA to 95% MeCN/H O/0.1%
2
2
+
+
1
1
1,12
TFA, 254 nm, purity 95.4%). MS: (ES ) m/z (M) = 200.14. H NMR
further development of BCS class 2 compounds,
where this
(
(
(
500 MHz, DMSO-d ) δ 7.86 (s, 1H), 7.79−7.69 (m, 2H), 7.59−7.50
6
structural modification can be introduced. The in vitro and in
vivo experiments indicate that the amminium salt is predom-
inately cleaved presystemically by the gut microflora to release
the parent compound 1, which is highly permeable and readily
absorbed. An in vitro assay using rat feces, which are rich in gut
microflora, was developed and used to find that the amminium
salt of pyridines and imidazoles are converted efficiently to the
parent compound, demonstrating broader applicability of the
prodrug technology. Although gut physiology and diet will
influence the microflora between animals and humans in rate and
extent of reduction, the reductive cleavage reaction should
nonetheless occur to some extent across all species. Some
variation in the gut microflora across the human population is
also anticipated; however, the differences are not expected to be
dramatic. However, gut microflora can be sensitive to antibiotics
and patients undergoing treatment with an antibiotic may have
significantly different levels/types of intestinal flora. This can
precipitate a drug−drug interaction in which the antibiotic
therapy would reduce the efficiency of the prodrug release from
an amminium species. This is an aspect of this prodrug
technology that requires further consideration and evaluation.
m, 3H), 7.47 (d, J = 7.9 Hz, 2H), 7.11 (d, J = 7.9 Hz), 6.60 (s, 2H), 4.27
t, J = 7.3 Hz, 2H), 3.21 (t, J = 7.6 Hz, 2H), 2.66 (quin, J = 7.5 Hz, 2H),
2
.29 (s, 3H). 13C NMR (126 MHz, DMSO-d ) δ 153.1, 145.7, 137.7,
6
137.6, 129.5, 128.8, 128.5, 128.1, 126.2, 125.5, 113.9, 48.7, 24.8, 23.2,
20.8. 15N NMR (DMSO-d ) δ 182.3, 175.5, 71.6.
6
7
Preparation of H
NOTs. Tosyl chloride (2.1 g, 11.0 mmol) was
2
added in portions to a solution of ethyl N-hydroxyacetimidate (1.2 g,
1.6 mmol) and triethylamine (8.88 mL, 63.7 mmol) in anhydrous
1
DMF (20 mL) at 0 °C. After addition was completed, the mixture was
placed at room temperature and stirred for 1 h. The reaction mixture was
poured over ice−water (100 mL) and stirred for 10 min. A white-
yellowish solid was formed and collected by filtration. The solid was
washed with water (3 × 50 mL) (1.3 g of the solid was collected). LC/
13
+
+
MS: (ES ) m/z (M + 1) = 258. t = 1.52 min. This solid was treated
R
with 70% HClO (5 mL) and stirred at rt for 10 min, then in a water bath
4
at 60 °C for 1 min and again at room temperature for 20 min. H
O (100
2
mL) was added, and the reaction was extracted with CH Cl (50 mL).
2
2
The organic layer was washed with H O (50 mL), dried over sodium
2
sulfate, filtered, and used as-is.
Metabolics and Pharmacokinetics. Human and rat liver
microsomes, rat liver cytosol, human recombinant CYP’s, and rat
CYP450 reductase were purchased from BD Gentest (Woburn, MA).
Rat hepatocytes were isolated and prepared at Bristol-Myers Squibb
1
5
(
Berry et al. ). Solvents were purchased from Mallinckrodt Baker
EXPERIMENTAL SECTION
■
(Phillipsburg, NJ), and chemicals, including PEG-400, NADPH,
neomycin, tetracycline HCl, and bacitracin, were obtained from
Sigma-Aldrich (St. Louis, MO). LB Broth was obtained from
LifeTechnologies (Grand Island, NY). All other chemicals used were
Chemistry. All commercial reagents and anhydrous solvents were
purchased from commercial sources and were used without further
purification or distillation, unless otherwise noted. Analytical high
1
674
dx.doi.org/10.1021/jm301638a | J. Med. Chem. 2013, 56, 1670−1676

Journal of Medicinal Chemistry
Article
from commercial suppliers. Dual cannulated portal vein/jugular vein
rats and bile duct cannulated Spraque−Dawley rats were purchased
from Hilltop Lab Animals, Inc. (Scottdale, PA). Beagle dogs were
purchased from Marshall Farms (North Rose, NY).
In Vivo Pharmacokinetics in Rat and Dog. Compound 2
trifluoroacetate salt was administered to groups of three male rats by
either IV bolus (1 mg/kg; all doses were 1 equivalent) or oral gavage (5
mg/kg, fasted overnight prior to the dosing). The dosing solution was
prepared in PEG-400/ethanol (90/10, v/v) at 1 mg/mL (dosing
solution concentration was 1 equivalent) for both IV and oral
administration. Plasma samples were collected at intervals up to 24 h
and were stored at −20 °C until analysis.
In male beagle dogs (n = 3), the IV and PO studies of 2 were
conducted in a crossover fashion. The IV and PO legs were separated by
a two-week washout period. In the IV study, 2 hydrochloride salt was
infused at 1 mg/kg (equivalent to 0.88 mg/kg 1) as a solution in water
over 5 min. In the oral study, the dogs were fasted overnight before
dosing. Prodrug 2 hydrochloride salt was dissolved in water and
administered by oral gavage at 5 mg/kg. Plasma samples were collected
at intervals up to 24 h and were stored at −20 °C until analysis.
In the oral dose escalation study in rats, 2 trifluoroacetate salt was
administered to groups of three male rats by oral gavage at 5, 25, and 200
mg/kg and 1 was given orally at 15, 75, and 200 mg/kg. The dosing
solutions were prepared in water with 0.5% methylcellulose.
buffer (0.1 M phosphate buffer and 10 mM MgCl ) was purged with
2
nitrogen for at least 1 h prior to the start of the incubation and anaerobic
incubations were performed under a blanket of humidified nitrogen
added directly to sample tubes. Aliquots of the cytosol (2 mg/mL),
microsomes (1 mg/mL), recombinant CYPs (made up to 1 mg/mL
with the vector control), and P450 reductase (1 mg/mL) were
incubated with 2 and 1 mM NADPH for 60 min in a shaking water bath
at 37 °C. Finally, prodrugs 2, 4, and 5 (40 uM) were incubated in rat
feces in LB media (3 g of fresh rat feces suspended in 25 mL of LB
1
6
media) for 2 h at 37 °C in a shaking water bath. Aliquots were removed
and quenched with an equal volume of ice-cold acetonitrile. Following a
10 min centrifugation at ∼1500g, the supernatant was collected and
prodrugs and metabolites were detected using LC-UV/MS/MS.
For all in vitro incubations, final substrate (2) concentration was 10
μM. At each time point, aliquots of the mixture were removed and
immediately quenched with an equal volume of ice-cold acetonitrile.
Following a 10 min centrifugation at ∼1500g, the supernatant was
collected and 2 and its metabolite (1) were quantitated using LC/MS/
MS.
LC/MS-MS Analysis of 1 and 2. For quantitation of 1 and 2,
following protein precipitation, supernatants were injected into an
API4000 (Applied Biosystems, Foster City, CA) triple quadropole mass
spectrometer equipped with a Turboionspray ionization interface
operating in the positive ionization mode. The source temperature
was 600 °C. The HPLC system used was an Agilent 1200 series (Agilent
Technologies, Santa Clara, CA) and a HTC PAL autosampler (Leap
Technologies, Cary, NC) linked to a Phenomenex Luna 2.5 um C18-
HST analytical column (2.0 mm × 50 mm, 2.5 um; Torrance, CA). The
mobile phase, which consisted of 0.1% formic acid in water (A) and 0.1%
formic acid in acetonitrile (B), was delivered at a flow rate of 0.6 mL/
min. Detection of each analyte was achieved through selected reaction
Pharmacokinetics in Antibiotic-Treated Rats. Two groups of
male rats (n = 3, each group) were dosed for 2 days with an antibiotic
mixture or saline (control animals). Bacitracin (400 mg/kg BID),
neomycin sulfate (400 mg/kg BID), and tetracycline (200 mg/kg) were
16
included in the antibiotic mixture (Watanabe et al. ). After antibiotic or
saline pretreatment, animals received a 5 mg/kg oral dose of 2. Plasma
samples were collected at intervals up to 24 h and were stored at −20 °C
until analysis.
+
monitoring. Ions representing the precursor (M + H) species for 2 and
Pharmacokinetics in Dual Cannulated JVC/PVC Rats. Dual
cannulated jugular vein cannulated/portal vein cannulated (JVC/PVC)
male rats (n = 3) received a 5 mg/kg oral dose of 2. Plasma samples were
collected from the portal vein and the jugular vein up to 24 h and were
stored at −20 °C until analysis.
Rat Bile Duct Cannulated Study. Bile duct cannulated/jugular
vein cannulated (BDC/JVC) male rats (n = 3) received a 1 mg/kg IV
dose of 2. Plasma, bile, and urine samples were collected up to 6 h.
Biotransformation of 2 was evaluated in bile and urine samples. For
biotransformation analysis, chromatographic separations were carried
out with an HPLC system that consisted of an Accela UPLC
1 were selected in quadrupole 1 and collisionally dissociated with
nitrogen to generate specific product ions, which were subsequently
monitored by quadrupole 3. Standard curves defining the dynamic range
of the bioanalytical method were prepared in the respective biological
matrix and processed in the same fashion as the test samples. Transitions
monitored for 2 and 1 were 463.2 → 245.1 and 448.2 → 202.1,
respectively. The analysis of 1 and 2 was conducted against a standard
curve ranging from 5 to 5000 nM. The standard curve was fitted with a
linear regression weighted by the square of the reciprocal concentration
2
(
1/x ).
Pharmacokinetics and Statistical Analysis. Noncompartmental
pharmacokinetic analysis of 1 and 2 were performed using Kinetica
Thermo Fisher Scientific, Waltham, MA) from the plasma concen-
(
ThermoFisher) and a 3.0 μm Waters Sunfire C-18 (2.1 mm × 150
mm) column maintained at 40 °C. The detectors were an Accela
ThermoFisher) photodiode array detector and an LTQ-Orbitrap
(
tration−time data. Statistical analyses (mean and standard deviation)
were performed using Microsoft Excel (Seattle, WA).
(
Discovery (ThermoFisher) mass spectrometer. The mobile phase,
which consisted of 0.1% formic acid in water (A) and acetonitrile (B),
was delivered at a flow rate of 0.35 mL/min. A gradient elution program
was used for chromatographic separation. The initial mobile phase
consisted of 90% solvent A and 10% solvent B, which was held for 2 min,
followed by a linear increase to 40% solvent B over 17 min. A step
increase to 90% solvent B was employed to wash the column and held
for 5 min before returning to the initial conditions. The column was
equilibrated for at least 5 min prior to the next injection. The mass
spectrometer was operated using an electrospray source in the positive
ion mode. Both full scan MS and data dependent MS/MS scanning were
used for detection.
In Vitro Incubations. Compound 2 was incubated in Tris-HCl
buffer (pH 7.4), rat blood, and rat plasma for 60 min at 37 °C.
Incubations were also conducted with commercially available
preparations of rat liver cytosol and microsomes as well as human
liver microsomes. Aliquots of the cytosol (2 mg/mL) and microsomes
AUTHOR INFORMATION
Corresponding Author
For A.R.R.: phone, 203-677-7416; fax, 203-677-7702; E-mail,
■
*
alicia.regueiroren@bms.com. For M.S.: phone, 203-677-6646;
fax, 203-677-6193; E-mail, michael.sinz@bms.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Pharmaceutical Candidate Optimization Group
(Biocon-Bristol Myers Squibb Research Center, Bangalore,
India) for conducting the antibiotic treated rat experiment.
(
1 mg/mL) were incubated with 2 and 1 mM NADPH for up to 2 h in a
ABBREVIATIONS USED
HIV-1, human immunodeficiency virus type 1; AI, attachment
inhibitor; BCS, Biopharmaceutics Classification System; BID
bis in die), twice a day; Caco-2, human colorectal
adenocarcinoma-2; ppm, parts per million; AUC, area under
1
5
■
shaking water bath at 37 °C. Rat hepatocytes were isolated in-house
and were incubated with 2 for 2 h at 37 °C in a humidified incubator with
9
5/5% air/CO2.
(
For anaerobic incubations, rat liver cytosol and microsomes, rat P450
reductase, human liver microsomes, and recombinant human CYP1A2,
2
B6, 2D6, 2C8, 2C9, 2C19, and 3A4 were used. Briefly, the incubation
the plasma concentration−time curve; CYP, cytochrome P450;
1
675
dx.doi.org/10.1021/jm301638a | J. Med. Chem. 2013, 56, 1670−1676

Journal of Medicinal Chemistry
Article
DMSO, dimethylsulfoxide; PEG, polyethylene glycol; LB Media,
(13) Sousa, T.; Paterson, R.; Moore, V.; Carlsson, A.; Abrahamsson, B.;
Basit, A. The gastrointestinal microbiota as a site for the
biotransformation of drugs. Int. J. Pharm. 2008, 363, 1−25.
Luria−Bertani media; Cmax, maximum plasma concentration; Cl,
clearance; V , volume of distribution; NADPH, β-nicotinamide
ss
(
14) Bonjouklian, R.; Dally, R. D.; De Dios, A.; Del Prado C., Miriam
adenine dinucleotide phosphate reduced tetrasodium salt; BDC,
bile-duct cannulated; IV, intravenous; PO, oral; JVC, jugular vein
cannulated; PVC, portal vein cannulated; HPLC, high pressure
liquid chromatography; LC-MS, liquid chromatography mass
spectroscopy; NMR, nuclear magnetic resonance; TMS,
trimethylsilane; HRMS, high resolution mass spectrometry;
MeOH, methanol; MeCN, acetonitrile; RP-HPLC, reverse phase
high pressure liquid chromatography; TFA, trifluoroacetic acid;
DMF, dimethylformamide; Rt, retention time; rt, room temper-
ature; min, minutes; h, hours
F.; Dominguez-Fernandez, C.; Jaramillo Aguado, C.; Lopez de Uralde-
Garmendia, B.; Montero Salgado, C.; Shepherd, T. A. Kinase inhibitors.
PCT WO 2005080380, 2005.
(15) Berry, M. N., Edwards, A. M., Barritt, G. J. Laboratory Techniques
in Biochemistry and Molecular Biology: Isolated Hepatocytes Preparation,
Properties and Applications; Elsevier: Amsterdam, 1991.
(16) Watanabe, K.; Yamashita, S.; Furuno, K.; Kawasaki, H.; Gomita, Y.
Metabolism of omeprazole by gut flora in rats. J. Pharm. Sci. 1995, 84,
5
16−517.
REFERENCES
■
(
1) Regueiro-Ren, A.; Xue, Q. M.; Swidorski, J. J.; Gong, Y.-F.;
Mathew, M.; Parker, D. D.; Yang, Z.; Eggers, B.; D’Arienzo, C.; Sun, Y.;
Malinowski, J.; Gao, Q.; Wu, D.; Langley, D. R.; Colonno, R. J.; Chien,
C.; Grasela, D. M.; Zheng, M.; Lin, P.-F.; Meanwell, N. A.; Kadow, J. F.
Inhibitors of Human Immunodeficiency Virus Type 1 (HIV-1)
Attachment 12. Structure−Activity Relationships Associated with 4-
Fluoro-6-azaindole Derivatives Leading to the Identification of 1-(4-
Benzoyl-piperazin-1-yl)-2-(4-fluoro-7-[1,2,3]triazol-1-yl-1H-pyrrolo-
[
2
2,3-c]pyridin-3-yl)-ethane-1,2-dione (BMS-585248). J. Med. Chem.
013, DOI: 10.1021/jm3016377.
2) Kadow, J. F.; Ueda, Y.; Meanwell, N. A.; Connolly, T. P.; Wang, T.;
(
Chen, C.-P.; Yeung, K.-S.; Zhu, J.; Bender, J. A.; Yang, Z.; Parker, D.; Lin,
P.-F.; Colonno, R. J.; Mathew, M.; Morgan, D.; Zheng, M.; Chien, C.;
Grasela, D. Inhibitors of Human Immunodeficiency Virus Type 1 (HIV-
1) Attachment 6. Preclinical and Human Pharmacokinetic Profiling of
BMS-663749, a Phosphonooxymethyl Prodrug of the HIV-1 Attach-
ment Inhibitor 2-(4-Benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1H-
pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanone (BMS-488043). J. Med.
Chem. 2012, 55, 2048−2056.
(
3) Regueiro-Ren, A.; Jenkins, S. M.; Kim, S.; Mosure, K.;
Rahematpura, S.; Simmermacher, J., Sinz, M.; Zheng, M.; Johnson, K.;
Meanwell, N. A.; Kadow, J. F. Novel ammonium prodrug: synthesis,
pharmacokinetics, mechanism of release and scope. Abstracts of Papers,
2
2
40th ACS National Meeting and Exposition, Boston, MA, August 22−
6, 2010, MEDI-421.
(
4) Kadow, J. F.; Regueiro-Ren, A. Aminium salts of 1,2,3-triazoles as
prodrugs of drugs including antiviral agents. U.S. Patent US
0060142298, 2006.
5) Patterson, L H; McKeown, S R; Robson, T.; Gallagher, R.; Raleigh,
S M; Orr, S. Antitumor prodrug development using cytochrome P450
CYP) mediated activation. Anti-Cancer Drug Des. 1999, 14, 473−486.
6) Patterson, L. H. Rationale for the use of aliphatic N-oxides of
2
(
(
(
cytotoxic anthraquinones as prodrug DNA binding agents: a new class of
bioreductive agent. Cancer Metastasis Rev. 1993, 12, 119−134.
(
7) Bottaro, J. C. Stereospecific conversion of olefins into aziridines by
p-tolylsulfonylhydroxylamine. J. Chem. Soc., Chem. Commun. 1980,
60−561.
8) Raap, R. Amination of tetrazoles with hydroxylamine-O-sulfonic
acid: 1-and 2-aminotetrazoles. Can. J. Chem. 1969, 47, 3677−3681.
9) Bellettini, J. R.; Olson, E. R.; Teng, M.; Miller, M. J. In Encyclopedia
of Reagents for Organic Synthesis; Paquette, L. A., Ed.; Wiley: New York,
995; Vol 3, pp 2189−2190.
10) Goeptar, A.; Scheerens, H.; Vermeulen, N. Oxygen and
5
(
(
1
(
Xenobiotic Reductase Activities of Cytochromoe P450. Crit. Rev.
Toxicol. 1995, 25 (1), 25−65.
(
11) Dahan, A.; Miller, J. M.; Amidon, G. L. Prediction of solubility and
permeability class membership: provisional BCS classification of the
world’s top oral drugs. AAPS J. 2009, 11, 740−746.
(
12) Broccatelli, F.; Cruciani, G.; Benet, L. Z.; Oprea, T. I. BDDCS
Class Prediction for New Molecular Entities. Mol. Pharmaceutics 2012,
, 570−580.
9
1
676
dx.doi.org/10.1021/jm301638a | J. Med. Chem. 2013, 56, 1670−1676