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 attachment inhibitor 2-(4-benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1 H -pyrrolo[2,3- c ]pyridin-3-yl)-2-oxoethanone (BMS-488043)

Article abstract of DOI:10.1021/jm201218m

BMS-663749, a phosphonooxymethyl prodrug 4 of the HIV-1 attachment inhibitor 2-(4-benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin- 3-yl)-2-oxoethanone (BMS-488043) (2) was prepared and profiled in a variety of preclinical in vitro and in vivo models designed to assess its ability to deliver parent drug following oral administration. The data showed that prodrug 4 had excellent potential to significantly reduce dissolution rate-limited absorption following oral dosing in humans. Clinical studies in normal healthy subjects confirmed the potential of 4, revealing that the prodrug significantly increased both the AUC and C_max of 2 compared to a solid capsule formulation containing the parent drug upon dose escalation. These data provided guidance for further efforts to obtain an effective HIV-1 attachment inhibitor.

Full text of DOI:10.1021/jm201218m

Article
pubs.acs.org/jmc
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
Attachment Inhibitor 2-(4-Benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-
1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanone (BMS-488043)
John F. Kadow,*,† Yasutsugu Ueda,† Nicholas A. Meanwell,† Timothy P. Connolly,† Tao Wang,†
Chung-Pin Chen,† Kap-Sun Yeung,† Juliang Zhu,† John A. Bender,† Zhong Yang,† Dawn Parker,†
Pin-Fang Lin,† Richard J. Colonno,† Marina Mathew,† Daniel Morgan,† Ming Zheng,† Caly Chien,‡
and Dennis Grasela‡
†Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States
‡Bristol-Myers Squibb Research and Development, P.O. Box 5400, Hopewell, New Jersey 08543-5400, United States
S
* Supporting Information
ABSTRACT: BMS-663749, a phosphonooxymethyl prodrug 4 of the HIV-1 attachment
inhibitor 2-(4-benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-ox-
oethanone (BMS-488043) (2) was prepared and profiled in a variety of preclinical in vitro
and in vivo models designed to assess its ability to deliver parent drug following oral
administration. The data showed that prodrug 4 had excellent potential to significantly
reduce dissolution rate-limited absorption following oral dosing in humans. Clinical studies
in normal healthy subjects confirmed the potential of 4, revealing that the prodrug
significantly increased both the AUC and Cmax of 2 compared to a solid capsule formulation
containing the parent drug upon dose escalation. These data provided guidance for further
efforts to obtain an effective HIV-1 attachment inhibitor.
INTRODUCTION
■
A significant number of approved single antiviral agents and
fixed-dose combinations that span six mechanistic classes are
now available for use in the treatment of HIV-1 infection.1
Despite the considerable advances made in effectively
controlling viremia in HIV-1-infected patients using combina-
tion antiretroviral therapy, a cure for this disease is not
imminent and there is an expanding population of HIV-1
patients relying on existing therapies whose medical needs are
Figure 1.
not completely satisfied.2 The increasing occurrence of drug-
resistant strains and the emergence of comorbidities associated
with long-term highly active combination antiretroviral therapy
(cART) strongly suggests that the need for mechanistically
distinct, non-cross-resistant drugs will continue to be an
imperative.3 Members of a previously disclosed class of orally
bioavailable, small molecule HIV-1 attachment inhibitors (AIs)
have been shown to bind to and induce conformational changes
within the HIV-1 viral envelope gp120 protein that interfere
with its interaction with the cellular CD4 receptor, the initial
point of host cell engagement by the virus.4−9 Potent and
selective inhibition has been observed in vitro against
macrophage-, T-, and dual-tropic HIV-1 strains7 by BMS-
37880610 (1) and BMS-48804311 (2), AIs that have been
advanced into clinical studies (Figure 1).10−15
1 was the first AI advanced into clinical studies, but the
plasma exposure in normal healthy human volunteers was
inadequate to progress this compound into proof-of-concept
trials in HIV-1 infected subjects.11,12,14 AI 2 represented an
advance from 1 that allowed evaluation of the antiviral activity,
safety, and tolerability in a multiple ascending dose study
conducted in HIV-1-infected adults. In a placebo-controlled
trial, 800 and 1800 mg doses of 2 were administered orally
twice daily for 8 days with daily viral load (VL) monitoring up
to 14 days.13,15 In subjects receiving an 1800 mg dose of 2, 67%
(8/12) experienced a VL decline of >1.0 log10, with 42% (5/
Received: September 14, 2011
Published: February 22, 2012
© 2012 American Chemical Society
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12) having a VL decline >1.5 log10. This contrasted with the
placebo-treated subjects, all of whom experienced a VL
reduction of <0.4 log10. Inhibitor 2 was well tolerated, and
there were no serious adverse events and no discontinuations
from the study. This proof-of-concept study validated the AIs as
a novel class of small molecules with clinically relevant antiviral
activity in HIV-1-infected subjects and has stimulated further
efforts to identify a drug candidate in this class.
In the clinical studies with 2 in HIV-1 infected patients, both
a high dose (800 or 1800 mg BID) and concomitant
administration of a high fat meal were required in order to
achieve adequate exposure and antiviral activity. A single 200
mg dose administered as a solution had 2-fold greater exposure
than that obtained from a single dose of a clinical capsule
without a concomitant high fat meal (capsule containing wet
milled crystalline active pharmaceutical ingredient (API) (95%
<23 μm, SA = 0.8 m2/g), data summarized in Figure 2. The
leading to its characterization as a BCS class 2 molecule.16 On
the basis of this appreciation, a number of alternate delivery
strategies were explored that were designed to improve the
exposure of 2. Nanosizing and amorphous formulation
approaches were examined with some success, providing
approximately 5- and 9-fold enhancements in exposure in
dogs, respectively, compared to the formulation used in the
clinical capsule.17 These data, along with the data from solution
dosing, provided evidence that addressing dissolution-limited
absorption had the potential to improve plasma exposure of 2
following oral administration.
Phosphate prodrugs, either directly attached to a molecule or
incorporated via linkers, have been used to successfully enhance
the solubility and exposure of a range of compounds
administered by either an intravenous (IV) or oral route.18−33
The physicochemical properties predictive of success with an
oral phosphate prodrug approach have been studied, but due to
the number of variables, the potential for utility must be
assessed experimentally.30,31 Previous experience with phos-
phate-based prodrugs of the antifungal agent ravuconazole and
taxane derivatives suggested that a phosphonooxymethyl
prodrug moiety might be readily attached to the indole
nitrogen of 2. It was anticipated that this moiety would provide
parent drug via the intermediacy of a short-lived hydroxymethyl
intermediate 3 after phosphatase cleavage of prodrug 4 (BMS-
663749)34 and would offer considerable potential to provide a
functional solution to the formulation problem (Scheme
1).28,33,35,36 The purpose of this work was to prepare prodrug
4 and assess its potential to remedy the high dose and solubility
limited absorption that were two significant reasons parent 2
was not a viable HIV-1 attachment inhibitor for use in
combination therapy to treat HIV-1 infection.
Figure 2. AUC (INF) vs dose after single oral administration of 2 as a
solution or capsule formulation under fasting or fed (high fat meal)
conditions.
Synthesis. The synthesis and identification of a stable form
of 4, ideally a crystalline salt, which could be reproducibly
prepared and used to perform definitive profiling, was achieved
in the following manner The chemistry initially used to prepare
prodrug 4 from parent 2 was an adaption of the process used to
prepare a phosphonooxymethyl prodrug of ravuconazole and is
summarized in Scheme 2.28
evidence available suggested that the absorption of 2 was
significantly limited by poor dissolution and/or poor solubility,
with exposure in humans ostensibly reaching a plateau at a dose
of 800 mg in the absence of a high fat meal. Both the high fat
meal and the excessive pill burden necessitated by these dose
regimens were considered to be liabilities that ultimately
precluded further development of the parent compound.
The performance of 2 in vivo in which the area under the
concentration−time curve (AUC) plateaus at a dose of 800 mg
with fasting is consistent with its experimentally measured low
solubility (Table 1) and high intrinsic membrane permeability,
The route shown in Scheme 2 comprises of four steps, the
last two of which can be accomplished without isolation of the
penultimate free acid intermediate 4. To ensure reproducible
reactivity, di-tert-butyl chloromethyl phosphate (6) was freshly
prepared from 5 and chloroiodomethane for each individual
preparative experiment. An initial survey of conditions revealed
that deprotonation of 2 was most effectively performed with 3
equiv of NaH and that the iodine-catalyzed alkylation
proceeded most efficiently using 5 equiv of 6. This reaction
was typically performed on batches of 8−10 g of 2 based on the
preparative reproducibility of the alkylating agent. Silica gel
chromatography provided the diester 7 in purity sufficient to
allow the subsequent deprotection, salt formation, and
crystallization steps to produce material of consistently high
purity. A crystallization of a final salt form was found to be the
most expeditious method to produce material of sufficient
purity for toxicology studies. TFA in dichloromethane was
initially used to deprotect diester 7, followed by isolation of
crude material and then generation of the desired salt, typically
in an acetone/water solvent system in the case of lysine.
However, the yield and purity of both the crude material and
final salt was not always reproducible as scales increased to tens
of grams and additional manipulation was required to get the
crude phosphate product ready for salt formation and
Table 1. Physicochemical Properties of 4b vs 2
4b phosphate
prodrug
2 parent compound
∼0.04 at pH 4−8
aqueous solubility at room
temperature (mg/mL)
0.22 at pH 1.4
>12 at pH 5.4
>12 at pH 8.9
0.9 at pH 1.5
0.25 at pH 10
pKa
2.2
6.1
2.6
9.3
lipophilicity
<−1.0
1.5
(log D at pH
6.5)
(log P at pH 6.5)
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Scheme 1
Scheme 2. Synthesis of 4b
crystallization. A more preferable procedure was developed
which avoided the use of highly acidic TFA and the need to
isolate the crude phosphate free acid. Thus, deprotection of 7
was accomplished by heating at 40 °C in a mixture of acetone
and water. The use of acetone/water as solvent allowed for
efficient deprotection of the phosphate group, subsequent in
situ salt formation, and crystallization. The discovery of these
conditions provided an advance over alternative conditions to
produce and isolate the prodrug in higher yields and purity on
large scales. The phosphate prodrug 4 could be isolated as
either the free acid or as a range salts. A survey of organic and
inorganic salts identified the mono (S)-(+)-lysine salt mono
hydrate as the most suitable candidate for advancing into key in
vivo studies with a crystalline form that could be reproducibly
prepared and crystallized to meet purity and stability
requirements. The overall yield of the lysine salt mono hydrate
starting from 2 was approximately 56%.
Preclinical Profiling of 4b. The physical and pharmaceut-
ical characteristics of the lysine salt of 4 confirmed that this
prodrug possessed significantly improved solubility and
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sufficient chemical stability in both the solid state and solution
to support oral dosing studies (Table 1). Two pKa values of 2.2
and 6.1 were measured by potentiometric titration of the diacid
4. Lysine salt 4b crystallized as an off-white powder containing
a small amount of amorphous material, and the solubility at
room temperature increased from 0.22 mg/mL at pH 1.4 to
>12 mg/mL at pH 5.4 and pH 8.9. Concentrations of >100
mg/mL have been achieved in water for in vivo toxicology
studies. In comparison, the aqueous solubility at room
temperature of the parent compound 2 was determined to be
approximately 0.04 mg/mL in the pH range of 4−8, 0.9 mg/
mL at pH 1.5 and 0.25 mg/mL at pH 10.0, as crystalline
material.
Table 2. Summary of the Pharmacokinetic Parameters of
Prodrug 4 and Parent 2 Following IV and PO
Administration of Prodrug Lysine Salt 4b in Various
a
Species
parameters
IV dosing
rat
dog
monkey
n = 3
n = 3
n = 3
b
dose (mg/kg)
Prodrug 4
1.4 (1.1)
1.2 (0.95)
1.3 (1.1)
Cl (mL/min/kg)
Vss (L/kg)
T1/2 (h)
14 4.2
70 16
4.4 0.18
0.048 0.017
1.0 0.78
0.18 0.059
0.70
0.12 0.019
0.16 0.052
0.14 0.020
0.30 0.094
0.15 0.010
0.070 0.0057
0.80
MRT (h)
IV AUC conversion 1.5
Prodrug 4 is hydrolyzed by alkaline phosphatase (ALP) to
form 2.30,37,38 Because multiple ALP isoforms are widely
distributed in various tissues, quantitative in vitro to in vivo
correlations were not attempted.39−43 Therefore, the studies
were limited to a qualitative assessment of ALP-dependent
hydrolysis in different tissues. As shown in Scheme 1, the
hydroxymethyl indole adduct 3 arises from enzyme-mediated
dephosphorylation of the phosphonooxymethyl prodrug 4.
This intermediate has not yet been observed in any in vitro or
in vivo studies and apparently collapses spontaneously to 2,
simultaneously liberating one molecule of formaldehyde per
molecule of 2 (Scheme 1). This is not surprising because the
measured pKa of the indole N−H of 2 is 9.3, and literature
precedent suggests that the hydroxymethyl indole intermediate
3 would be unstable when the pKa of the indole N−H is less
than 11.44 On the basis of the available toxicologic information
on orally administered formaldehyde, other pharmaceuticals,
and dietary sources, the risks associated with chronic low level
oral exposure to formaldehyde are minimal when compared to
the potential benefits of realizing a new efficacious agent for the
treatment of HIV.45 Clinical studies with salts of prodrug 4 or
similar prodrugs of closely related molecules have to date not
identified toxicities attributed to formaldehyde exposure.46 The
Caco-2 permeability coefficient (Pc) of the parent molecule 2 is
high, >100 nm/s at pH 6.5, which is comparable to metoprolol,
a highly membrane permeable marker that displays 100%
absorption in humans.11 Parent compound 2 was rapidly
formed following IV administration of 4b to rats, dogs and
monkeys. The IV AUC conversion ratios were 1.5 in rats, 0.80
in dogs, and 0.70 in monkeys, suggesting good conversion from
4b to 2 (The ratios were calculated from 2 AUC after prodrug
4 dosing/2 AUC after 2 dosing for IV dosing). No or very low
levels of 4 were detected in rat, dog, and monkey plasma after
oral administration of 4b. The absolute bioavailability of 2
following oral administration of 4b was determined to be 62%,
93% and 67% in rats, dogs, and monkeys, respectively (Table
2). These data are consistent with reports describing the high
levels of ALP expression in the gut that efficiently cleave the
prodrug presystemically.47,48
c
ratio
PO dosing
n = 3
7.9 (6.3)
n = 3
n = 3
b
dose (mg/kg)
6.6 (5.2)
7.1 (5.6)
Parent 2 Formed from Prodrug 4b
d
bioavailability (%)
Cmax (nM)
62
93
67
3943 983
0.83 0.29
13168 1452
1.5 0.24
14017 1261
0.25
16354 6381
0.83 0.14
34183 2668
4.8 1.3
Tmax (h)
AUCtot (nM·h)
T1/2 (h)
82844 16499
4.1 0.52
a
Data from IV and PO dosing of parent 2 in all three species has been
published.11 Free acid of 4b (equivalent dose of 2). The ratios were
b
c
calculated from ((2 AUC after prodrug 4 dosing)/(2 AUC after 2
dosing)) for IV dosing. The absolute oral bioavailability was
calculated from the dose-normalized AUC of 2 after oral dosing of
4b divided by the historical IV AUCtot data (3348 nM·h at 1 mg/kg)
of 2.1
d
As can be seen from the data, 4b, when dosed orally at ∼200
mg/kg equivalent of 2, provided ∼3 fold higher Cmax and ∼2-
fold higher AUC of 2 in rats when compared to the AUC from
2 administered as a suspension at the similar dose.
The findings from in vitro and in vivo studies in animals as
well as from in vitro human tissues predicted that 2 would be
formed after oral administration of 4b to human subjects. The
data from preclinical studies supported further development of
4b, and the compound was advanced sequentially into pre-IND
characterization and phase I studies in normal healthy
volunteers.35,49
Human Clinical Studies with 4b. The lysine salt 4b was
formulated as a solution from drug in bottle and evaluated in a
single ascending dosing study in normal healthy human
volunteers (N = 6 per dose cohort) at doses equivalent to
25, 50, 100, 200, 400, and 800 mg of parent 2. The results from
this study are compiled in Figures 4, 5, and 6 and Table 3 and
were compared with those from the historical studies in which
2 was dosed in a clinical capsule formulation and as a 200 mg
solution (data summarized in Figure 7).15 Following dosing of
4, parent drug 2 was observed in plasma with Tmax achieved
within 30 min, suggesting that the parent drug was both
released from the prodrug and rapidly absorbed. As can be seen
from Figures 4−6, and Table 3, overall exposure and both Cmax
and AUC increased at least proportionally with dose over the
range studied, 25−800 mg. The 800 mg dose equivalent of
prodrug 4 resulted in a significantly higher AUC of 2 but a
somewhat lower C12 than that obtained from 800 mg of 2
administered as solid in a capsule concomitantly with a high fat
meal (Table 3). Only relatively low levels of the prodrug 4 were
detectable in human plasma at the 800 mg dose of 4 in 4 out of
5 subjects up to 1.5 h postdose. The T1/2 of 2 measured from 2
to 12 h after an 800 mg dose of 4b was 1.5 0.2 h, significantly
In an oral dose escalation study conducted in rats
administered an aqueous solution of the mono lysine salt 4b,
the increase in AUC of 2 was approximately dose-proportional
over the doses of 16, 72, and 267 mg/kg (free acid).
Comparisons of the Cmax and AUC obtained in rats dosed
with either 2 or 4b, with doses plotted in parent drug
equivalents, are shown in Figure 3. The dosages of the free acid
of prodrug 4 studied correspond to molar equivalent dosages of
13, 57, and 211 mg/kg, respectively, of the parent 2 (Figure 3).
In all experiments, the AUC of the prodrug in plasma was
≤0.03% that of the parent.
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Figure 3. Comparison of Cmax and AUC of 2 in male rats administered either 2 or 4b. Data for 4b are from a single-dose study and data for 2 are
from day 1 of a 2 week exploratory oral dose toxicity study. Dosages of 4b are presented as molar equivalents of 2.
Figure 6. Cmax versus dose relationship in humans after single doses of
25−800 mg equivalents of 2 as prodrug 4b. The diagonal line depicts a
hypothetical linear relationship starting from the data at the lowest
Figure 4. Comparative mean profiles of 2 in humans after single oral
dose for comparison.
administration of 25−800 mg equivalents of 2 as prodrug (lysine salt)
4b.
parent when dosed as solution, exhibiting high bioavailability, a
very high Cmax, and a short T1/2, as summarized in Figure 7. The
oral solution data suggest complete conversion of 4b to 2 in
vivo after prodrug administration via the oral route.
In the human SAD study, a 400 mg dose of prodrug was
administered concomitantly with a high fat meal to a single
subject and the data compared to that obtained from the dosing
of the same dose of prodrug alone. The high fat meal appeared
to delay Tmax by about 0.75 h and decreased both the Cmax and
AUC by 57% and 32%, respectively, in this limited sample set.
DISCUSSION
■
The phosphonooxymethyl prodrug methodology succeeded in
overcoming the solubility- and dissolution-limited absorption
encountered with the solid formulation of the parent AI 2 when
dosed to human subjects in the initial clinical trials. Consistent
with its improved ability to deliver parent to plasma following
oral administration, the prodrug 4b achieved a higher AUC of
parent than the solid formulation at the highest dose studied
(800 mg) without the need for a high fat meal to boost
exposure. Indeed, the limited data obtained suggest that in
humans, coadministration with food is actually somewhat
detrimental to the exposure of the parent 2 from prodrug 4.
This suggests that the solubilization of the parent from the
prodrug is already highly optimized and food may actually
interfere with access of the prodrug to the gut wall. The dosing
of prodrug 4b at 200 mg equivalent of parent closely mimicked
that from dosing a solution of the parent, affording a T1/2 just
Figure 5. AUCt vs dose relationship in humans after single doses of
25−800 mg equivalents of 2 as prodrug 4b. The diagonal line depicts a
hypothetical linear relationship starting from the data at the lowest
dose for comparison.
shorter than the ∼10 h T1/2 observed for 2 following oral
dosing of parent 2, and approximately half of the 3 h measured
after dosing parent 2 as a solution. These data are consistent
with poor dissolution prolonging the absorption phase of the
parent drug and some precipitation occurring from solution.
Qualitatively, when comparing the data from dosing 2, either
as a solid in the clinical capsule or as a solution, with that of the
prodrug 4, dosing of the prodrug more closely mimicked that of
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a
Table 3. Human PK Parameters of Oral Dosing of 4b vs Oral Dosing of 800 mg 2 with a High Fat Meal
Cmax geometric mean (%CV)
Tmax median
AUC(INF) geometric mean (%CV) T1/2 mean C12 geometric mean (%CV)
PO treatment
800 mg 4b
(ng/mL)
(range) h
(ng/mL·h)
(SD) h
(ng/mL)
26762 (21)
12906 (24)
8419 (11)
3653 (18)
1371 (25)
784 (22)
1 (0.5−1)
64131 (13)
26168 (13)
14157 (12)
5727 (18)
2151 (17)
1162 (18)
21191 (20)
11 (5.4)
13 (15)
150(32)
59 (43)
33 (40)
12 (17)
400 mg 4b
200 mg 4b
100 mg 4b
50 mg 4b
25 mg 4b
0.63 (0.25−1)
0.5 (0.25−0.75)
0.5 (0.5−0.75)
0.5 (0.25−0.5)
0.5 (0.5−0.75)
4.0 (2.0−4.0)
6.7 (6.0)
1.9 (0.42)
1.4 (0.13)
1.0 (0.09)
14 (6.3)
b
<LLOQ
b
<LLOQ
800 mg 2 (capsule with
high fat meal)
4100 (13)
175 (88)
a
b
Analyte for all data is parent compound 2. LLOQ: lower level of quantitation.
compound was not pursued in favor of candidates with
improved pharmacokinetic and antiviral properties.15,46,50
CONCLUSION
■
A phosphonooxymethyl prodrug of an azaindole-based HIV-1
attachment inhibitor was found to provide improved
biopharmaceutics properties, successfully overcoming the
dissolution- and solubility-limited absorption associated with
the parent drug and the requirement for concomitant dosing of
a high fat meal in order to achieve targeted plasma levels of
active drug. The parent drug was considered to be a good
candidate for prodrug methodology based on its high intrinsic
permeability, low solubility, and the projected need for a high
dose. Although the plasma levels achieved with parent drug 2
dosed with a high fat meal and the prodrug 4 dosed under
fasted conditions, were shown to be sufficient to demonstrate
clinical antiviral activity and establish proof-of-concept for this
drug class, the predicted dosing regimen of the drug fell short
of objectives. Administration of the prodrug 4 more fully
revealed inherent deficiencies in the pharmacokinetic properties
of 2, particularly the short T1/2, which may have been masked
by the slow absorption of parent drug, leading to a flip-flop
kinetic profile. Nevertheless, the clinical data established the
value of this prodrug methodology to overcome dissolution-
and solubility-limited absorption of a BCS class 2 molecule and
provided a platform for drug delivery via a prodrug that is
currently being used for a compound of this mechanistic class,
BMS-663068, that was formulated as an extended release tablet,
and demonstrated improved pharmacokinetic and antiviral
properties.15,46,50
Figure 7. Human plasma profiles of parent 2 from dosing of solution,
drug-in-capsule formulation, or prodrug 4b. The dose was 200 mg of
parent 2 or equivalent.
slightly longer than that from solution. The ∼10 h T1/2
observed after dosing of the parent drug was apparently
lengthened due to a major contribution from the effect of
extended and prolonged absorption, a function of the limited
solubility and slow dissolution in the gut. The solution dosing
also probably demonstrates some component of prolonged
absorption that was absent in the dosing of prodrug. A critical
contributor to the success of an oral phosphate approach is that
the rate of release of parent drug from the pro-moiety must be
commensurate with the rate of absorption and the data for this
prodrug suggest that this release is very rapid. In addition, the
parent drug generated from unmasking of the prodrug
presystemically must remain in solution long enough to be
absorbed and this also seems to be occurring with 2 in a near
optimal manner.
While the performance of the prodrug met expectations with
respect to improving the plasma delivery of 2, as demonstrated
by increased Cmax and AUC and reduced Tmax, the intrinsic
pharmacokinetic properties of 2 in humans are less than ideal,
with a short T1/2 in plasma. As a consequence, the C12 of the
drug in humans falls below the targeted protein binding-
adjusted EC50 measured in vitro that would give confidence
that this compound could be developed as a twice daily dosing
regimen. The C12 at the 800 mg dose of prodrug 4b was 149.57
32.32 ng/mL, which is less than the protein binding-adjusted
EC50 for 2, determined in vitro vs a panel of HIV subtype B
clinical virus isolates to be 156 ng/mL (370 nM). Because the
convenience of twice daily drug dosing was considered to be a
critical element to its future utility, further development of the
EXPERIMENTAL SECTION
■
Procedures for the preparation of 4b (mono-L-lysine salt): {3-[(4-
benzoylpiperazin-1-yl)(oxo)acetyl]-4,7-dimethoxy-1H-pyrrolo[2,3-c]-
pyridin-1-yl}methyl dihydrogen phosphate, L-lysine salt (1:1).
Preparation of 6: di-tert-butyl chloromethyl phosphate.28 The
tetrabutylammonium salt of di-tert butyl phosphate, (45.1 g, 0.1
mol) and chloroiodomethane (200 g, 1.14 mol) were combined in 100
mL of benzene and the mixture stirred at room temperature for 4 h.
The benzene was removed under vacuum, a portion of 500 mL of
Et2O added to the residue, and the insoluble solid filtered away.
Concentration of the filtrate in vacuo and removal of the volatiles on a
vacuum pump provided crude bis-tert-butyl chloromethyl phosphate 6
as a light-yellow or light-brown oil which was utilized in the next step
without further purification.
Preparation of 7: (3-(2-(4-benzoylpiperazin-1-yl)-2-oxoacetyl)-4,7-
dimethoxy-1H-pyrrolo[2,3-c]pyridin-1-yl)methyl di-tert-butyl phos-
phate. NaH (2.4 g, 60 mmol, 60% in oil) was added slowly to a
suspension of 2 (8.4 g, 20 mmol) in dry THF (120 mL) and the
mixture stirred for an hour at room temperature. Iodine (5 g, 20
mmol) dissolved in dry THF (10 mL) was added slowly and
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cautiously to the stirred solution at such a rate that foaming was kept
under control. Following completion of the addition, the mixture was
stirred at ambient temperature for an additional 15 min and 6 (bis-tert-
butyl chloromethyl phosphate ∼0.1 mol), obtained as described in
step one, was added. After stirring for 16 h, the reaction mixture was
poured into iced NH4OAc solution (30%, 120 mL) and extracted with
EtOAc (3 × 300 mL). The combined organic extracts were washed
with H2O (100 mL) and brine (100 mL), dried over Na2SO4, and
concentrated in vacuo to afford a residue which was purified by silica
gel chromatography. Elution with EtOAc/Et3N (100/1) followed by
EtOAc/MeOH (100/1) gave diester 7 (9.0−10.3 g, AP ∼75%) as a
light-yellow solid in yields of 70−80% over several runs.
0.77 mmol) was dissolved in a solution of H2O (3 mL) and acetone (3
mL). The mixture (pH: not determined) was stirred at 40 °C for 16 h
to allow the solvolysis to reach completion. To this mixture (pH 2.18;
prodrug 4−69 AP) was added 4 M aqueous lysine solution to adjust
the pH to 4.83 and the solution heated to 55 °C. Acetone (35 mL)
was added slowly into the reaction mixture over 30 min at 45−50 °C.
At 45 °C, and after adding about 15 mL of the acetone, the clear
solution was seeded with crystalline 4b and stirring at this temperature
was continued for 45 min. After complete addition of acetone, the
solution was cooled to room temperature over ∼4 h and stirred
overnight to complete the crystallization of 4b. The solid was collected
by filtration and dried via suction under N2 for 2 h. The white
crystalline solid was dried under house vacuum at 50−55 °C for 24 h
to afford 343 mg (93%) of 4b with purity and characterization data
similar to that obtained in the TFA procedure above.
In Vivo Studies. The studies described used the monolysine salt of
4b, unless stated otherwise. All in vivo PK studies in rats, dogs, and
monkeys were performed using aqueous solutions of prodrug for PO
and IV dosing.
Human Clinical Studies with 4b. The Lysine salt of 4b was
formulated as a solution from drug in bottle and evaluated in a single
ascending dosing study in healthy volunteers with cohorts of six
patients each at doses 25, 50, 100, 200, 400, and 800 mg of two parent
equivalents. The results from this study were compared to those from
the historical dosing of 2 in a clinical capsule formulation and from a
200 mg solution dose.15
1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 7.48 (s, 1H), 7.40 (b,
5H), 6.15 (d, 2H, J = 11.5 Hz), 4.05 (s, 3H), 3.90 (s, 3H), 3.90−3.30
(b, 8H), 1.39 (s, 18H); 13C NMR (125 MHz, CDCl3) 185.5, 170.7,
166.5, 146.9, 146.2, 139.6, 135.3, 130.2, 128.7, 128.4, 127.2, 124.5,
122.0, 120.8, 115.8, 83.8, 73.2, 57.3, 53.5, 46.1, 41.7, 29.8; MS m/z: (M
+ H)+ calcd for C31H42N4O9P, 645.27; found, 645.10.
Preparation of 4. A mixed solution of TFA (50 mL) and
dichloromethane (450 mL) was added to a round-bottom flask
containing 43.3 g of diester 7. After stirring at room temperature for
16 h, the reaction mixture was concentrated under vacuum to offer a
residue of crude 4 which was used in further steps without any
purification.
Preparation of {3-[(4-benzoylpiperazin-1-yl)(oxo)acetyl]-4,7-dime-
thoxy-1H-pyrrolo[2,3-c]pyridin-1-yl}methyl dihydrogen phosphate, L-
lysine salt (1:1) (4b). First, 55 g of crude product 4 from the
preceding procedure was added to an aqueous solution of L-lysine
(1.36M, 70 mL) at room temperature. The resulting suspension (pH =
1.83) was added to a lysine solution (1.36 M, ∼40 mL) to reach pH
4.88. The resulting suspension was filtered through a pad of Celite.
The clear light-yellow filtrate (∼200 mL) was mixed with acetone (200
mL) and heated to 45 °C. Acetone (1400 mL) was added over 2 h at
45 °C. The clear solution was seeded and stirred at 45 °C for 2 h and
slowly cooled to room temperature (5 h) and the suspension stirred
overnight. The white solid was collected by filtration and dried under
house vacuum at 50 °C over 24 h to afford 41.2 g of 4b as an off-white
solid. The above solid was dissolved in 1:1 water−acetone (560 mL) at
45 °C. Acetone (700 mL) was added over a period of 1 h at 45 °C.
The clear solution was seeded and stirred at 45 °C for 2 h, slowly
cooled to room temperature (5 h), and the suspension stirred at room
temperature overnight. The white solid was collected by filtration and
dried under house vacuum at 50 °C over 36 h to afford 33 g of 4b as
an off-white solid. The material was >99% by LC/MS (method below)
and determined to be >98% pure based on the combustion analysis
and water determination.
4b was a 1.70 molar hydrate (4.20% w/w water) and 1.14 molar
lysine salt. Residual solvent analysis did not find any residual solvents.
1H NMR (500 MHz, D2O, 60 °C) δ 8.72 (s, 1H), 7.84 (m, 6H), 6.44
(d, 2H, J = 10 Hz), 4.41 (s, 3H), 4.27 (s, 3H), 4.3−3.7 (m, 8H), 4.10
(t, 1H, J = 5 Hz), 3.39 (t, 2H, J = 5 Hz), 2.30−1.80 (m, 6H). 13C
NMR (125 MHz, D2O, 27 °C) δ 186.7, 174.9, 173.2, 167.9, 147.7,
145.7, 142.6, 134.3, 131.1, 129.2, 127.1, 124.3, 122.4, 120.1, 113.8,
73.5, 57.1, 54.9, 54.4, 47.7, 47.1, 46.3, 45.7, 42.6, 42.1, 42.0, 41.5, 39.5,
30.2, 26.8, 21.8. HRMS m/z: (M − lysine + H)+ calcd for
C23H26N4O9P, 533.1437; found, 533.1425. Anal. Calcd for
C23H26N4O9P: C, 49.11; H 6.13; N, 12.05. Found: C, 48.93; H
6.26; N 12.07. Melting point 168−172 °C. HPLC/MS RT = 17.75
min. Method details: Waters 2695 HPLC with Finnigan UV 6000LP
PDA detection and LCQ Classic MS (electrospray probe); column,
Waters Atlantis dC18; 150 mm × 2.1 mm ID; 5 μm (at 30C); mobile
phase A, 0.05% TFA/water; mobile phase B, 0.05% TFA/acetonitrile;
flow, 0.30 mL/min 5−98%; B gradient, hold 5%B 0−2 min, 5−35%B
2−23 min, 35−98%B 23−35 min, hold 98%B 35−37 min, 98−5%B
37−37.3 min, hold 5%B 37.3−45 min; UV detection, UV at 260 nm
sampling: 2 Hz, bandwidth 7 nm, step 5 nm.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental procedures for assays, preclinical in vitro
and in vivo studies, and human clinical studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 203-677-6910. Email: john.kadow@bms.com.
ACKNOWLEDGMENTS
■
We thank Marc Browning for the bioanalytical support in
analyzing samples from animal PK studies and Jennifer Pizzano
for conducting the in-life animal PK studies. We also thank
Tong Li, MD, and co-workers at the former Bristol-Myers
Squibb Clinical Research Center at Hamilton Hospital,
Hamilton, NJ, for conducting the human clinical studies.
ABBREVIATIONS USED
■
API, active pharmaceutical ingredient; AUC, area under the
concentration−time curve; BCS, The Biopharmaceutics Clas-
sification System; cArt, combination antiretroviral therapy;
C
max, maximum concentration; INF, from time zero to infinity;
IV, intravenous; PO, oral
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