Coupling of an Acyl Migration Prodrug Strategy with Bio-activation to Improve Oral Delivery of the HIV-1 Protease Inhibitor Atazanavir

Article abstract of DOI:10.1021/acs.jmedchem.8b00277

HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, 1), remain important medicines to treat HIV-1 infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer, which results in additional drug-drug interactions that are sometimes difficult to manage. We investigated a chemo-activated, acyl migration-based prodrug design approach to improve the pharmacokinetic profile of 1 but failed to obtain improved oral bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1) yielded substantially better oral drug delivery of 1 when compared to the parent itself, the simple acyl migration-based prodrug, and the corresponding simple l-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo, and (3) offered the benefit of mitigating the pH-dependent absorption associated with 1, thereby potentially reducing the risk of decreased bioavailability with concurrent use of stomach-acid-reducing drugs.

Full text of DOI:10.1021/acs.jmedchem.8b00277

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Coupling of an Acyl Migration Prodrug Strategy with Bio-activation
To Improve Oral Delivery of the HIV‑1 Protease Inhibitor Atazanavir
Murugaiah A. M. Subbaiah,*,† Nicholas A. Meanwell,‡ John F. Kadow,‡,⊗ Lakshumanan Subramani,†
Mathiazhagan Annadurai,† Thangeswaran Ramar,† Salil D. Desai,§ Sarmistha Sinha,⊥
Murali Subramanian,⊥ Sandhya Mandlekar,⊥ Srikanth Sridhar,§ Shweta Padmanabhan,⊥
Priyadeep Bhutani,⊥ Rambabu Arla,⊥ Susan M. Jenkins,#,⊗ Mark R. Krystal,∥,⊗ Chunfu X. Wang,∥,⊗
and Ramakanth Sarabu†
†
§
⊥
Departments of Medicinal Chemistry, Biopharmaceutics, and Pharmaceutical Candidate Optimization, Biocon-Bristol Myers
Squibb R&D Centre, Biocon Park, Bommasandra IV Phase, Jigani Link Road, Bangalore 560099, India
‡
∥
#
Departments of Discovery Chemistry and Molecular Technologies, Virology, and Pharmaceutical Candidate Optimization,
Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06924, United States
S
* Supporting Information
ABSTRACT: HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, 1), remain important medicines to treat HIV-1
infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer,
which results in additional drug−drug interactions that are sometimes difficult to manage. We investigated a chemo-activated,
acyl migration-based prodrug design approach to improve the pharmacokinetic profile of 1 but failed to obtain improved oral
bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed
to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1)
yielded substantially better oral drug delivery of 1 when compared to the parent itself, the simple acyl migration-based prodrug,
and the corresponding simple L-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo,
and (3) offered the benefit of mitigating the pH-dependent absorption associated with 1, thereby potentially reducing the risk of
decreased bioavailability with concurrent use of stomach-acid-reducing drugs.
potential for the development of fixed-dose drug combinations
(Figure 1).
INTRODUCTION
■
Combination antiretroviral therapy (cART), which is presently
the most effective treatment for AIDS, has made it possible for
HIV-1-infected patients to enjoy a high quality of life with a
significant extension of life expectancy.1,2 HIV-1 protease
inhibitors (PIs) enabled cART and became a key component of
drug combinations because of their robust efficacy, durability in
Atazanavir (ATV, 1), an azapeptide-based PI, was the first
once-daily PI to be approved and has been included in the
WHO’s list of essential medicines.5,6 1 is marketed alone or in
combination with the PK enhancers ritonavir or cobicistat.7 1 is
characterized by sub-optimal oral bioavailability, which can be
attributed to poor solubility at higher pH, high efflux,8 and
extensive first-pass metabolism. As a consequence of the
solubility limitation, co-administration of 1 + ritonavir with the
proton pump inhibitor (PPI) omeprazole leads to a 75%
reduction in the exposure of 1, indicative of pH-dependent
absorption.9,10 1 requires clinical co-administration with a PK
̈
treatment-naive patients, and high genetic barrier to resistance
development.3,4 As a class, PIs are known for sub-optimal
physicochemical and ADME properties, which contribute to
poor oral bioavailability. This frequently demands high doses of
the active drug and in most cases requires co-dosing with a
pharmacokinetic (PK) enhancer in order to effectively deliver
PIs. The resultant drug−drug interactions and related toxicities
contribute to reduced patient compliance and limit the
Received: February 21, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.8b00277
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design approach was found to be essential because simple
prodrugs like the L-Val derivative 3 (Scheme 1) that were based
a
Scheme 1. Synthesis of Direct L-Val Ester 3
Figure 1. Reduced patient compliance as an outcome of poor oral
bioavailability of PIs. Adapted with permission from ref 11.
enhancer that inhibits CYP3A metabolism, affording higher oral
bioavailability, a longer plasma t1/2, and a higher Ctrough than
that of unboosted 1.
Attempts have been made to develop next-generation PIs
that would exhibit improved pharmacokinetic, resistance, and/
or safety profiles.3,4 As an example, a deuterated version of 1,
D24-ATV, has been investigated in an effort to enhance
metabolic stability by taking advantage of the kinetic isotope
effect to slow metabolism.12 However, D24-ATV exhibited only
a modest improvement in plasma exposure in rats compared to
1. Moreover, this approach does not address the pH-dependent
absorption observed with 1. There has been a growing interest
in the exploration of prodrugs across all therapeutic classes, and
a well-designed prodrug of 1 has the potential to address both
its pharmaceutical and pharmacokinetic limitations.13,14 Differ-
ent prodrug approaches have been investigated in the literature
to improve the ADME properties of the PIs, although none
have been applied to 1.11,15,16 Phosphate prodrugs of the PIs
lopinavir, ritonavir, and amprenavir have been explored in an
effort to address dissolution-limited absorption and/or high
load of solubilizing excipients, which led to the discovery of the
marketed agent, fosamprenavir.17,18 Amino acid and dipeptide
prodrugs have been shown to improve absorptive permeability
by the combined effect of active transport and decreased efflux
and to reduce susceptibility to first-pass metabolism.19−21
Peptide-based prodrugs of an analogue of darunavir that are
designed to be activated by the ubiquitous dipeptidyl peptidase-
IV enzyme have also been reported.22 In addition to proton-
coupled peptide transporter 1 (PepT1)-mediated transport of
amino acid-based prodrugs, other prodrug strategies have been
designed to take advantage of carrier-mediated transport and
include, for example, ascorbic acid-conjugated prodrugs for
targeting the sodium-dependent vitamin C transporter
(SVCT),23 biotin-derived prodrugs for targeting the sodium-
dependent multivitamin transporter (SMVT),24 and mono-
carboxylate ester prodrugs to target the fatty acid transport
proteins (FATPs)25 for placental delivery. Acyl migration
prodrugs, which were designed to undergo pH-dependent
activation and improve aqueous solubility, have also been
explored.26,27
a
Reagents and conditions: (a) N-Boc-L-Val, DCC, DMAP, DCM, RT,
12 h, 74.1%; (b) 4 N HCl in diethyl ether, 0 °C, 1 h, 85%.
on appending solubilizing elements to the secondary alcohol of
1 were unsuccessful at improving oral bioavailability, with the
predominant circulation of prodrug itself.11 This result
prompted an examination of a design approach based on an
O-to-N acyl migration concept that potentially offers the
advantage of facile conversion of prodrug and higher atom
economy which arises from the rearrangement of the prodrug
to the parent without the release of a promoiety (Scheme 2).26
This traceless prodrug strategy eliminates the release of a
promoiety byproduct in vivo, reducing the potential for the
introduction of additional toxicities.
The acyl migration prodrug 4 was retrosynthetically designed
to release 1 by the chemical process depicted in Scheme 2.
Prodrug 4 was synthesized in two steps from 5, an intermediate
in the synthesis of 1,28 as delineated in Scheme 3. O-Acylation
of 5 with N-(methoxycarbonyl)-tert-L-leucine 6 followed by
deprotection of the N-Boc group using 4 N HCl in dioxane
afforded the amine 4 as the HCl salt. The intermediate 6 was
prepared in one step from L-tert-leucine on treatment with
methyl chloroformate following the literature protocol.29
Prodrug 4 showed decreasing solution stability with increasing
pH, with excellent stability at pH 1 and 4 (t1/2 > 334 h) and
lower stability at pH 6.5 (t1/2 = 6.6 h); data are compiled in
Table 1. Cognizant of solubility considerations associated with
1, stability studies were performed in 30% aqueous acetonitrile.
Consequently, the stability of 4 in a 100% aqueous system is
expected to be lower than the t1/2 of 6.6 h at pH 6.5 determined
in the mixed solvent system. The low stability of 4 at pH 6.5
was anticipated based on the design strategy which relies upon
rearrangement to 1 after amine deprotonation. Prodrug 4
RESULTS AND DISCUSSION
■
We report herein the design of a novel double prodrug of 1 that
relies upon sequential enzyme-mediated release of an amine
that is designed to undergo an acyl migration to deliver the
parent drug that (i) enables superior exposure based on
improved pharmaceutical properties and a sustained release in
vivo and (ii) attenuates the pH-dependent absorption of 1. This
B
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Article
a
Scheme 2. Chemoactivable Prodrug Design Based on an O-
to-N Acyl Migration
Table 1. Stability and Solubility for 1 and Prodrugs
a
stability (t1/2, h) at 37 °C solubility (mg/mL) at 25 °C
parent/prodrug pH 1.0 pH 4.0 pH 6.5 pH 1.0 pH 4.0
pH 6.5
1
>500
376.4
>500
>500
>500
>500
>500
334.3
>500
334.3
>500
>500
>500
6.6
1.69
>0.9
>1.8
>1.8
>1.1
>0.5
<0.001
>0.4
<0.001
ND
4
9
120.4
6.6
<0.001
>1.5
<0.001
ND
b
11
14
16
107.5
214.9
0.12
0.01
>1.43
0.027
a
Stability studies used 30% acetonitrile in buffer. Buffers used were 0.1
N aqueous HCl (pH 1.0), acetate buffer (pH 4.0), and phosphate
buffer (pH 6.5) at a concentration of 50 mM. ND = not determined.
b
Stability t1/2 = 1.8 h at pH 7.4.
suggests that the prodrug will be adequately stable in the
stomach but will begin to release the parent after reaching the
intestine. The prodrug was evaluated in Sprague−Dawley (SD)
rats following oral administration at a dose of 3 mg/kg using a
precipitation-resistant formulation to avoid any potential
impact of dissolution on the exposure. Disappointingly, the
exposure of 1 in this experiment was lower (relative F = 1%)
when compared to the exposure obtained from a solution
formulation of 1 (F = 20%) (Table 2 and Figure 2b). The
trough concentration of the parent, obtained following
administration of the prodrug, was also below the limit of
quantification (BLQ).
a
Retrosynthetic design of a traceless acyl migration-based prodrug
involving the transfer of the tert-leucine unit to the proximal secondary
alcohol moiety.
Following poor in vivo exposure of 1 from 4, attention was
focused on masking the amine of the acyl migration prodrug 4
through conjugation with an enzyme-releasable promoiety. The
objectives were to improve solution stability at pH 6.5 and
enable absorption of the intact prodrug, with enzyme-mediated
cleavage of the prodrug occurring post-absorption. To the best
of our knowledge, this approach does not have precedent in the
literature. The criteria for choosing the promoiety were that it
should (a) retain a polar, solubility-enhancing element and (b)
also facilitate intestinal permeability through either passive
diffusion or active transport. Contrary to the traceless nature of
the acyl migration prodrug 4, the conjugative acyl migration
prodrugs are expected to release byproducts in vivo. Hence, the
design elements should incorporate safety considerations that
the released promoiety or byproduct should be associated with
an acceptable toxicological profile. This design principle would
deliver a prodrug to circulation that would be dependent upon
enzyme-mediated cleavage to liberate 4, which was anticipated
to rearrange to 1 at physiological pH based on the in vitro
analysis.
a
Scheme 3. Synthesis of Acyl Migration Prodrug 4
As the initial step in this direction, the conjugation of 4 with
a non-basic but polar dioxolenone promoiety was explored,
incorporated via a carbamate linker. This promoiety has found
clinical application in the angiotensin II receptor antagonists,
olmesartan medoxomil30 and azilsartan medoxomil,31 and the
antibacterial agents, prulifloxacin32 and ceftobiprole medocar-
il.33 The dioxolenone prodrug 9 was derived in a single step
upon treatment of 4 with the activated carbonate of 4-
(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (8),34 as depicted
in Scheme 4. This prodrug moiety was expected to undergo
bioconversion in intestinal mucosa, the liver, and plasma by the
action of multiple enzymes (especially carboxymethylene-
butenolidase homologue and paraoxonase 1) to release the
active parent 1 via 4 in addition to the byproducts carbon
dioxide and diacetyl.35−37 Prodrug 9 showed acceptable
stability across a range of pH values, with an 18-fold
a
Reagents and conditions: (a) intermediate 6, DCC, DMAP, DCM,
RT, 12 h, 52.5%; (b) 4 N HCl in dioxane, 0 °C, 1 h, 87%.
exhibited good aqueous solubility at pH 1 and 4 (>0.4 mg/
mL), but solubility was not determined at pH 6.5 due to the
short half-life of the prodrug at this pH. The in vitro profile of 4
C
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Table 2. Pharmacokinetic Data Obtained after Oral Administration of 1 at 3 mg/kg and Prodrugs at an Equivalent Dose of 3
a
mg/kg of Parent in SD Rats
compound dosed
analyte
Cmax (nM)
Tmax (h)
AUClast (nM·h)
%F
Ctrough at 24 h (nM)
prodrug/parent ratio
1
3
1
266
10
0.5
1.7
0.5
0.4
0.3
1.3
0.3
0.4
0.3
1.7
1.7
0.4
2.3
0.3
384
42
20
2.2
NA
0.25
BLQ
1.09
BLQ
BLQ
0.3
NA
112
1
3
1529
8
4731
16
b
4
1
0.83
NA
4.9
0.1
NA
3.4
4
68
79
b
9
1
180
42
194
19
10.1
NA
9
BLQ
BLQ
BLQ
4.5
b
11
14
1
56
83
4.3
11
1
4
1
NA
b
310
124
2160
37
1446
579
4306
167
8434
75.3
NA
NA
4
2.0
14
1
10.0
0.3
b
16
8.7
50.5
16
2209
NA
49.7
a
Values shown are mean SD (n = 3). NA = not available, BLQ = below limit of quantification. The formulation used for these experiments was
b
10% v/v DMAc, 40% v/v PEG-400, 50% v/v of 30% w/v HPβCD in pH 4.0 citrate buffer (50 mM). Relative F (oral bioavailability relative to that
of 1).
Figure 2. Exposure−time profiles obtained following oral administration of 1 and the prodrugs in SD rats.
improvement in solution stability at pH 6.5 when compared to
4. Compound 9 exhibited pH-dependent solubility which was
optimal only at acidic pH. Administration of 9 to rats resulted
in the 12-fold improved exposure of 1 when compared to
administration of 4, but this was still lower than that of the
directly dosed parent (Table 2 and Figure 2c). Plasma
concentrations of intermediate 4 were not monitored in this
study. Encouragingly, dosing of 9 resulted in a measurable
Ctrough concentration of 1 of 0.3 nM at 24 h post-dose, a
contrast to the results observed with prodrug 4. While this
improvement was modest, it provided some confidence that
improved intestinal stability would positively impact exposure.
Inspired by this result, we examined optimization of the
promoiety in an effort to further improve oral bioavailability.
The conjugation of 4 with amino acid esters was investigated
next. Across therapeutic areas and drug classes, amino acid-
based prodrugs have been reported to improve both solubility
and absorption, some by relying on active transport, and to
mitigate metabolic clearance of parent compounds.39 The
introduction of the amino acid side chain was accomplished
through a carbamate-based methylene linker. Accordingly, 4
was treated with chloromethyl chloroformate to afford the less-
stable chloromethyl carbamate derivative, which was subjected
to O-alkylation with N-Boc-L-Val to afford the carbamate
methylene N-Boc-L-Val derivative 10. Deprotection of 10 by
D
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a
Scheme 4. Carbamate-Based Conjugation of 4
a
Reagents and conditions: (a) (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate (8), DIPEA, DMAP, dioxane, 100 °C, 24 h, 10.1%;
(b) (i) chloromethyl chloroformate, pyridine, DCM, 0 °C, 3 h, (ii) N-Boc-L-Val, NaI, DIPEA, DMF, 50 °C, 14 h, 26.5% for two steps; (c) 4 N HCl
in dioxane, 0 °C, 1 h, 76%; (d) 2-chloro-2-methylpropyl chloroformate,38 pyridine, DCM, 0 °C, 3 h, ∼90%; (e) N-Boc-L-Val, NaI, DIPEA, DMF, 50
°C, 14 h, 29.7%; (f) 4 N HCl in dioxane, 0 °C, 1 h, 32%; (g) N-Boc-D-Val, NaI, DIPEA, DMF, 60 °C, 8 h, 31.9%; (h) 4 N HCl in dioxane, 0 °C, 1 h,
71.5%.
exposure to 4 N HCl in dioxane afforded the corresponding L-
Val amino acid prodrug 11. Assuming initial enzymatic cleavage
of L-Val amino acid ester of this prodrug and subsequent
spontaneous decomposition of the resultant intermediate 4 to
release the parent, the prodrug can be expected to release L-Val,
formaldehyde, and carbon dioxide as potential byproducts in
vivo in the initial enzymatic step. Natural amino acids like L-Val,
which serve as the building blocks of proteins, are ubiquitous in
the human body, and hence the release of L-Val as a promoiety
should not pose a significant safety concern.39 L-Val was
primarily selected as a prodrug moiety because of the clinical
precedent of Val-based ester prodrugs in, for example,
valacyclovir and valgancyclovir.40 The potential for toxicity
associated with the release of formaldehyde from a prodrug has
been reviewed in context of the estimated daily exposure to this
molecule in humans, where it is generated from a variety of
endogenous and exogenous substrates.41 Prodrug 11 displayed
acceptable stability at pH 1 and 4 but significantly reduced
E
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Article
stability at the higher pH values relevant for absorption. At
lower pH values, the amino acid ester prodrugs exist in the salt
form, which confers good solubility. However, with increasing
pH, the amine salt converts into the free amine, which may
negatively impact solubility. Prodrug 11 showed excellent
solubility at the tested pH range (pH 1−4) and exhibited 5-fold
higher plasma exposure of 1 in rats compared to that delivered
by 4 following oral administration (Table 2 and Figure 2d).
However, this value was still lower than that obtained by dosing
1 directly. The trough concentration of 1, which was released
from 11, was also BLQ. Intermediate 4 was not monitored for
its systemic circulation in this study.
Scheme 5. Potential in Vivo Degradation Pathway of the
Conjugative Acyl Migration Prodrug 14 To Release the
Parent Drug 1
The poor stability of 11 at higher pH values prompted an
investigation of the effect of installing alkyl substitution on the
methylene bridge, assuming that such steric encumbrance
around the carbamate and ester functionalities of the promoiety
would reduce the hydrolytic lability.42 The enhanced stability
was expected to minimize the intestinal degradation of the
prodrug and facilitate greater absorption of the prodrug itself.
Accordingly, the carbamate-based isobutyl L-Val prodrug 14
was synthesized from 4 in three steps. Compound 4 was treated
with 1-chloro-2-methylpropyl chloroformate38 to afford crude
product 12 which, due to modest stability, was immediately
subjected to O-alkylation with N-Boc-L-Val to afford the
carbamate isobutylene N-Boc-L-Val derivative 13. Deprotection
of 13 using 4 N HCl in dioxane afforded the corresponding L-
Val amino acid prodrug 14. Chiral HPLC analysis of 13
indicated the stereoisomeric purity of at least 95%, suggesting
the possibility of a stereoselective O-alkylation.43 The
introduction of the isopropyl substituent resulted in an 16-
fold increase in the chemical stability of 14 at pH 6.5 compared
to the unsubstituted L-Val prodrug 11 (Table 1). Oral
administration of 14 to SD rats resulted in a significant
increase in the plasma levels of 1, delivering a 90-fold higher
exposure of 1 than prodrug 4 and a ∼4-fold higher exposure
and 18-fold higher Ctrough concentration of 1 than those
following direct dosing of the parent drug (Table 2 and Figure
2e). Combining the exposure of circulating 14, 4, and 1, the
total exposure was 16-fold higher than that from the direct
dosing of 1. These results suggested that the prodrug not only
was efficiently absorbed, but also was subject to reduced
metabolic degradation by CYP enzymes.
Based on our design hypothesis, prodrug 14 is expected to
undergo enzymatic hydrolysis as the first step to release
intermediate 4 and equimolar amounts of L-Val, isobutyr-
aldehyde, and carbon dioxide (Scheme 5). The intermediate 4
subsequently undergoes acyl migration-based degradation to
release the parent. As a naturally occurring substance,
isobutyraldehyde is encountered in several food items and
metabolized to isobutyric acid, an intermediate in L-Val
metabolism.44 In addition, isobutyraldehyde is a food additive
permitted for human consumption as a synthetic flavoring
substance and adjuvant by the Federal Drug Administration
(FDA). Clinical precedent for the release of isobutyraldehyde
as a byproduct of prodrug activation is illustrated by the
marketed drug fosinopril,45 a prodrug of the phosphinic acid-
based angiotensin-converting enzyme inhibitor fosinoprilat, and
arbaclofen placarbil,46 a prodrug of the gamma amino butyric
acid type B receptor agonist arbaclofen.
in plasma supports a stepwise activation process involving
cleavage of conjugate 14 to release 4, which then undergoes
acyl migration to afford 1. The lower AUC of 4 compared to 1
and 14 reflects the different rates of activation of 4 and 14, with
the enzyme-mediated cleavage as the rate-determining step.
Interestingly, the plasma exposure profiles of 4 and 1 differ
depending on whether 4 or 14 is dosed (Table 2). Dosing of 4
provides a 5-fold higher plasma exposure of this prodrug than
for 1. However, after dosing 14, the ratio of 4 to 1 is inverted
and 2.5-fold in favor of the parent drug. Because the generation
of 1 from 14 without the intervention of 4 is limited, a potential
explanation may include a nonlinear increase in 1 from the
higher concentration of 4 in plasma or a more rapid rate of
conversion of 4 to 1 when 4 is enzymatically released from 14.
Because prodrugs 4 and 14 mask the transition-state-
mimicking, pharmacophoric secondary alcohol moiety of 1,
they would not be expected to be effective HIV-1 PIs. However,
prodrug 14 demonstrated antiviral activity in an HIV-1
infection assay (EC50 = 5.3 nM), suggesting that in cell culture
the prodrug can undergo bioactivation and conversion to
generate 1, which mediates the antiviral activity. Similarly, the
intermediate prodrug 4 showed high functional potency (EC50
The parallel PK profiles of 14 and 4 depicted in Figure 2e
suggest that 14 acts as a depot, steadily releasing 4 which then
converts into 1 in the systemic circulation, reflecting a
formation rate-mediated clearance of 1. The observation of 4
F
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= 1.8 nM). However, both sets of data reflect some limitation
on parent drug release in this setting since the EC50 value for 1
was 0.7 nM.
Prodrug 16, a diastereomer of 14, was synthesized next by
replacing L-Val with D-Val in order to understand the impact of
absolute configuration on stability, solubility, and the PK
profile. Accordingly, 16 was synthesized by treating the 1-
chloro-2-methylpropyl carbamate derivative 12 with N-Boc-D-
Val to afford the carbamate isobutylene N-Boc-D-Val ester 15
and subsequent deprotection of 15 using HCl. Analogous to
the observation for 13, chiral HPLC analysis of 15 indicated a
chiral purity of at least 95%, suggestive of a stereoselective
reaction. The D-Val prodrug 16 showed higher stability at pH
6.5 and higher solubility at pH 4 and 6.5 than the L-Val prodrug
14. In SD rats, oral dosing of 16 produced a 9-fold lower
exposure of 1 than 14 but a 2-fold higher exposure of the
circulating prodrug than 14 (Table 2 and Figure 2f). The
prodrug-to-parent ratio observed in the case of 16 was 15-fold
higher when compared to that of 14. This is consistent with a
lower rate of activation of the D-Val prodrug 16 than the L-Val-
based 14. This can be rationalized by slower cleavage of the
unnatural D-Val amino acid by hydrolytic enzymes compared to
the prodrug based on natural L-Val. Combining the exposure of
16 and 1, the total AUC was 22-fold higher than that from
direct dosing of 1, suggesting more efficient absorption and
lower metabolic clearance of this prodrug than the parent. As in
the case of 14, the in vivo cleavage of 16 to generate 1
presumably results from an initial enzymatic step that involves
the release of acyl migration intermediate 4 and equimolar
amounts of D-Val, isobutyraldehyde, and carbon dioxide.
This double prodrug approach was essential because the
simple L-Val ester 3, which may be preferred for practical
reasons associated with ease of synthesis, performed poorly
following oral administration to rats. Dosing of 3 gave 9-fold
lower exposure of 1 than direct administration of the drug itself
and 34-fold lower exposure than the exposure of the parent 1
released following administration of the acyl migration L-Val
prodrug 14 (Figure 4).
Figure 4. Comparative chart, indicating superior oral delivery of 1
from acyl migration L-Val prodrug 14 compared to simple direct L-Val
ester 3. For prodrug 14, the combined AUC of 14 and its intermediate
4 [4306 + 579 nM·h] is included.
Table 3. Bidirectional Caco-2 Permeability and in Vitro
Metabolic Stability Data
Papp (nm/s)
Caco-2 Caco-2
t1/2 (min)
parent/
prodrug
Caco-2
efflux ratio
rat
rat whole
blood
a
b
A-B
B-A
hepatocytes
1
<15
23
470
348
ND
ND
114
73
>31
15
28
79
<5
<5
56
53
>120
>120
<5
4
9
ND
ND
<15
<15
ND
ND
>8
11
14
16
20
>120
>120
>5
a
b
Apical-to-basolateral direction. Basolateral-to-apical direction.
not exhibit higher absorptive permeability in heterogeneous
human epithelial colorectal adenocarcinoma (Caco-2) cells
than the parent or acyl migration prodrug 4, they have shown
significantly lower efflux which could, in part, contribute to the
improved exposure. These results suggest that conjugation of 4
as carbamate isobutylene Val derivatives contributed to reduced
affinity for efflux transporters. Prodrugs 14 and 16 were
incubated in rat hepatocytes, considered to be better predictors
of metabolism in vivo than liver microsomal preparations
because they express all hepatic metabolizing enzymes.47 Both
14 and 16 exhibited improved half-lives compared to 1,
suggesting that the prodrugs were less prone to metabolic
degradation than the parent drug. The rat whole blood stability
profile of 14 and 16 indicates high stability, which is consistent
with the observed slow rate of cleavage in vivo, which is
presumably mediated by esterases or peptidases, leading to a
circulating depot of 14 and 16 that slowly releases 1 in vivo.
With the promising PK data in hand, the effect of gastric pH
on delivery of 1 by 14 was examined using a suspension
formulation evaluated in a rat model where gastric pH was
modulated by treatment with either pentagastrin or famoti-
dine.9,10,48 1 is known to depend upon an acidic gastric
environment for adequate dissolution and subsequent
absorption in vivo.9,10 Concomitant administration of PPIs
and 1 is currently not recommended because as acid-
suppressing agents, PPIs are known to reduce the exposure
of 1, thereby increasing the risk of sub-optimal therapy and
resistance development.49 Following oral administration of 14
to male SD rats, there was no significant difference in the
systemic exposure of 1 and 14 by pre-treatment with either
pentagastrin to enhance stomach acidity or famotidine to
reduce acidity, as depicted in Figure 5. In contrast, a 492-fold
The improved combined exposure of 1 and the prodrug
following oral dosing of 14 and 16 (Figure 3) can be attributed
to several factors, including improved aqueous solubility (Table
1), reduced efflux, and reduced susceptibility to CYP 450-
mediated metabolism (Table 3). Although these conjugates do
Figure 3. Comparative chart of exposures of prodrug and parent,
indicating the superior performance of prodrugs 14 and 16 when
compared to unconjugated acyl migration prodrug 4 or its other
conjugates. For prodrug 14, the combined AUC of 14 and its
intermediate 4 [4306 + 579 nM·h] is included.
G
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Article
conversion to 1 that was responsible for the activity. The
reduced metabolism of 14 and 16, as determined from in vitro
studies, and the improved exposure suggest that the prodrugs
mitigate, in part, the major limitation of CYP3A metabolism.
This discovery opens opportunities for the development of
metabolism-mitigating, depot-acting prodrugs. However, this
approach will require (1) refinement of prodrug cleavage in
vivo, especially, for achieving dose-proportional exposure, (2)
understanding the performance of the prodrug in vivo in higher
species and the potential for cross-species differences in
enzymatic cleavage of the prodrug moiety, and (3) assessment
of the potential safety and other liabilities [for example,
profiling for hERG and CYP2D6 inhibition due to the presence
of an ionizable amine in the promoiety and for hepatic uridine
diphosphate glucuronosyltransferase 1A1 (UGT1A1) inhibition
to assess hyperbilirubinemia potential because of the known
inhibition of UGT1A1 by the parent drug] associated with
developing a prodrug with high and sustained oral exposure.
Figure 5. AUC profiles of 1, obtained following oral administration of
the prodrug 14 in a rat pH model study using a suspension
formulation of 0.5% methyl cellulose and 99.5% water.
difference in systemic exposure was observed between the
pentagastrin and famotidine arms following direct adminis-
tration of 1 (Figure 6).48 These data suggest that 14 is able to
EXPERIMENTAL SECTION
■
Materials and General Methods. All anhydrous reactions were
performed under an atmosphere of N2 or Ar gas with magnetic
stirring. All reagents and solvents were purchased from commercial
suppliers (e.g., Aldrich, Alfa, Acros, Spectrochem, and Sonia) and were
used without further purification, unless otherwise stated. Reactions
were monitored by thin layer chromatography (TLC) or LC/MS.
TLC was performed using E. Merck pre-coated silica plates (60F-254)
with 0.25 mm thickness and visualized using short-wave UV light or
developing agents (KMnO4, phosphomolybdic acid, or p-anisalde-
hyde). For LC/MS monitoring, samples were run on a Waters Aquity
system coupled with a Waters Micromass SQ Mass Spectrometer
(buffer, 10 mM ammonium acetate or 0.01% trifluoroacetic acid; run
time, 3 min; flow rate, 0.7 mL/min). Crude compounds were purified
by either normal phase column chromatography using a Teledyne
ISCO CombiFlash system or reverse phase chromatography using
preparative HPLC instruments (Agilent 1200 series) equipped with a
high-pressure quaternary pump, a diode array detector, and a universal
loop injector with different C18 columns, for example, Sunfire,
XBridge phenyl, and Kinetex.
Figure 6. Comparison of AUC0−24 h profiles of 1, obtained following
oral administration of 14 and 1 itself in the rat pH model. Data for the
direct dosing of 1 taken from ref 48.
Analytical purity of the compounds was determined by HPLC
analysis to be ≥95%, unless otherwise noted. The following HPLC
methods were used to determine the analytical purity of the
compounds.
substantially mitigate pH-dependent absorption in rats and
hence may offer the benefit of reduced drug−drug interaction
with PPIs, thereby potentially improving the profile of the
agent. The lack of the pH effect on absorption of 14 may be
attributed to higher solubility and/or a salt form that can drive
dissolution and kinetic solubility.
(i) Analytical HPLC method A: column = Sunfire C18 [150 × 4.6
mm] 3.5 μm; buffer = 0.05% trifluoroacetic acid in water;
mobile phase A = buffer:MeCN [95:5]; mobile phase B =
MeCN:buffer [95:5]; 10% B to 100% B; run time = 23 min;
flow rate = 1.0 mL/min).
(ii) Analytical HPLC method B: column = XBridge phenyl C18
[150 × 4.6 mm] 3.5 μm; buffer = 0.05% trifluoroacetic acid in
water; mobile phase A = buffer: MeCN [95:5]; mobile phase B
= MeCN: buffer [95:5]; 10% B to 100% B; run time = 23 min;
flow rate = 1.0 mL/min).
(iii) Analytical HPLC method C: column = Kinetex Evo C18 [100
× 4.6 mm] 2.6 μm; buffer = 0.05% trifluoroacetic acid in water;
mobile phase A = buffer: MeCN [95:5]; mobile phase B =
MeCN: buffer [95:5]; 10% B to 100% B; run time = 23 min;
flow rate = 1.0 mL/min).
(iv) Analytical HPLC method D: column = Kinetex Biphenyl C18
[100 × 4.6 mm] 2.6 μm; buffer = 0.05% trifluoroacetic acid in
water; mobile phase A = buffer: MeCN [95:5]; mobile phase B
= MeCN: buffer [95:5]; 10% B to 100% B; run time = 23 min;
flow rate = 1.0 mL/min).
CONCLUSIONS
■
In conclusion, the reported results suggest that a conjugative
acyl migration prodrug design principle can be useful as a drug
delivery approach to significantly improve the oral bioavail-
ability of 1, with the advantage that a circulating prodrug results
in an extended release of parent drug that is reflected in a
higher Ctrough concentration. Such a novel drug delivery is
warranted in the light of the drug delivery challenges, reported
with the direct ester prodrugs of PIs, including the direct L-Val
ester of 1. This conjugation, engineered to undergo a sequential
bio- and chemo-activation process, was found to play a critical
role in the enhanced exposure of 1, since the simple acyl
migration prodrug 4 failed to deliver a significant amount of the
parent in vivo. Additionally, the lead prodrug 14 demonstrated
potential to mitigate the drug−drug interaction observed
between the parent drug and PPIs, as evident from the pH
effect study. The prodrug demonstrated potent antiviral
activity, which suggested efficient cellular penetration and
Chiral purity was determined by the following methods on a Agilent
HPLC SFC hybrid instrument using CHIRALPAK columns:
H
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Journal of Medicinal Chemistry
Article
6.96 min and 98.6%. HRMS (ESI/Orbitrap) m/z: [M+H]+ calcd for
C43H62N7O8 804.4654; found 804.4649.
(i) Chiral HPLC method A: column = CHIRALPAK IC [250 ×
4.6 mm] 5.0 μm; co-solvent: 0.2% ammonia in CH3CN:MeOH
(1:1 mixture); run time = 15 min; flow rate = 4 mL/min).
(ii) Chiral HPLC method B: column = CHIRALPAK ID [250 ×
4.6 mm] 5.0 μm; co-solvent: 0.2% ammonia in CH3CN:MeOH
(1:1 mixture); run time = 15 min; flow rate = 4 mL/min).
(2S,3S)-3-Amino-1-(2-((S)-2-((methoxycarbonyl)amino)-3,3-
dimethylbutanoyl)-1-(4-(pyridin-2-yl)benzyl)hydrazinyl)-4-phenyl-
butan-2-yl (S)-2-((Methoxycarbonyl)amino)-3,3-dimethylbutanoate
Dihydrochloride (4). To the solid of (5S,10S,11S)-11-benzyl-5-(tert-
butyl)-15,15-dimethyl-3,6,13-trioxo-8-(4-(pyridin-2-yl)benzyl)-2,14-
dioxa-4,7,8,12-tetraazahexadecan-10-yl (S)-2-((methoxycarbonyl)-
amino)-3,3-dimethylbutanoate (7) (800 mg, 0.994 mmol) was added
4 N HCl in dioxane (4.75 mL, 19.88 mmol) at 0 °C under nitrogen
atmosphere. After being stirred at 0 °C for 1 h, the reaction mixture
was concentrated in vacuo at 30 °C. The residue was stirred with ether.
The solvent was carefully decanted. The resultant solid was dried
under vacuum to get crude product as an off-white solid. The solid was
dissolved in a solvent mixture of acetonitrile and water. The resulting
mixture was frozen and lyophilized for 12.0 h to afford the title
Known compounds were characterized by comparing their 1H
NMR spectra to the previously reported data. New compounds were
characterized by 1H NMR and MS. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded on Bruker 300 and 400 MHz
instruments. Chemical shifts are reported in ppm (δ) relative to the
internal standard tetramethylsilane (TMS, δ 0.00 ppm). NMR data are
reported as follows: chemical shift (multiplicity [singlet (s), doublet
(d), triplet (t), quartet (q), pentet (p), multiplet (m), and broad
singlet (br s)], coupling constant [Hz], and integration). For LC/MS
characterization of the compounds, reverse phase analytical HPLC/
MS experiments were performed on Agilent 1200 Series system
coupled with a single quadrupole instrument (ESI) or Waters Aquity
system coupled with a Waters Micromass SQ Mass Spectrometer.
HRMS data were obtained on an Agilent HPLC/Q-TOF G6540A
mass spectrometer using APPI or APCI methods in positive or
negative ion detection modes.
Experimental Procedures. (2S,3S)-3-((S)-2-((Methoxycarbonyl)-
amino)-3,3-dimethylbutanamido)-1-(2-((S)-2-((methoxycarbonyl)-
amino)-3,3-dimethylbutanoyl)-1-(4-(pyridin-2-yl)benzyl)-
hydrazinyl)-4-phenylbutan-2-yl (tert-Butoxycarbonyl)-L-valinate
(2). To a solution of atazanavir (0.200 g, 0.284 mmol) in anhydrous
DCM (5.0 mL) were sequentially added (tert-butoxycarbonyl)-L-valine
(0.92 g, 0.426 mmol), DCC (0.117 g, 0.567 mmol), and DMAP (34.8
mg, 0.284 mmol). The reaction mixture was stirred at room
temperature for 12 h and filtered through a Celite bed. The Celite
bed was washed with ethyl acetate. The filtrate was concentrated in
vacuo. The resulting crude compound was purified by preparative
HPLC (preparative HPLC conditions: column: XBridge phenyl C18
(19 × 250 mm; 5 μm); mobile phase A: 10 mM ammonium acetate in
water; mobile phase B: acetonitrile; flow rate: 20 mL/min; gradient: 0/
50, 11/80). The pooled HPLC fraction was concentrated under
reduced pressure at 30 °C. The residue was then dissolved in a solvent
mixture of acetonitrile and water, and the solution was lyophilized for
12 h to obtain the title product as an off-white solid (190 mg, 74.1%).
1H NMR (400 MHz, DMSO-d6) δ = 8.86 (br s, 1H), 8.65 (d, J = 4.5
1
product (700 mg; 87%) as a colorless solid. H NMR (400 MHz,
DMSO-d6) δ 9.65−9.45 (m, 1H), 8.78−8.69 (m, 1H), 8.67−8.55 (m,
2H), 8.16−8.03 (m, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.74−7.63 (m,
1H), 7.57−7.49 (m, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 7.5 Hz,
3H), 7.20 (s, 2H), 7.02−6.80 (m, 1H), 5.06−4.88 (m, 1H), 4.14−3.86
(m, 4H), 3.70−3.65 (m, 3H), 3.41 (s, 4H), 3.24−3.08 (m, 2H), 3.05−
2.93 (m, 1H), 2.91−2.77 (m, 1H), 0.98 (s, 9H), 0.62 (s, 9H); MS
(ES): m/z 705.4 [M+H]+. Analytical HPLC purity and retention time:
method A = 95.61% and 6.498 min; method B = 98.81% and 7.913
min. HRMS (ESI/Orbitrap) m/z: [M+H]+ calcd for C38H53N6O7
705.3970; found 705.3966.
(S)-2-((Methoxycarbonyl)amino)-3,3-dimethylbutanoic Acid
(6).29 To a stirred solution of (S)-2-amino-3,3-dimethylbutanoic acid
(10.0 g, 76 mmol) in anhydrous 1,4-dioxane (50 mL) were
sequentially added a solution of 2 M sodium hydroxide (114 mL,
229 mmol) and methyl chloroformate (11.78 mL, 152 mmol) at 0 °C
dropwise. The reaction mixture was stirred at 60 °C for 18 h. The
mixture was subsequently cooled to ambient temperature and
extracted with DCM. The aqueous layer was acidified with 1.5 N
HCl. The resulting solution was extracted with ethyl acetate. The
organic layer was washed with brine solution, dried over anhydrous
sodium sulfate, filtered, and concentrated under vacuum to get the
crude product as a colorless oil, which was stirred in hexane. The
precipitated solid was filtered and dried to get the title product (12.0 g,
63.4 mmol, 83%) as a colorless solid. 1H NMR (300 MHz, DMSO-d6)
δ 12.48 (br s, 1H), 7.26 (d, J = 9.1 Hz, 1H), 3.79 (d, J = 9.1 Hz, 1H),
3.54 (s, 3H), 0.94 (s, 9H).
(5S,10S,11S)-11-Benzyl-5-(tert-butyl)-15,15-dimethyl-3,6,13-tri-
oxo-8-(4-(pyridin-2-yl)benzyl)-2,14-dioxa-4,7,8,12-tetraazahexa-
decan-10-yl (S)-2-((Methoxycarbonyl)amino)-3,3-dimethylbuta-
noate (7). To a solution of methyl ((S)-1-(2-((2S,3S)-3-((tert-
butoxycarbonyl)amino)-2-hydroxy-4-phenylbutyl)-2-(4-(pyridin-2-yl)-
benzyl)hydrazinyl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (5) (3 g,
4.73 mmol) in DCM (20 mL) were added (S)-2-((methoxycarbonyl)-
amino)-3,3-dimethylbutanoic acid 6 (1.343 g, 7.10 mmol), dicyclo-
hexylcarbodiimide (1.953 g, 9.47 mmol), and 4-dimethylamino-
pyridine (0.578 g, 4.73 mmol). The reaction mixture was stirred at
room temperature for 12 h and filtered through a Celite bed, which
was subsequently washed with ethyl acetate. The combined filtrate was
concentrated in vacuo at 30 °C. The crude product was purified by RP
HPLC (preparative HPLC conditions: Sunfire C18 (250 × 30 mm; 5
μm), mobile phase A: 10 mM ammonium acetate in water at pH 4.5,
mobile phase B: acetonitrile, flow rate: 27 mL/min, gradient: 0/50, 7/
85). The pooled HPLC fraction was concentrated in vacuo at 30 °C.
The residue was dissolved in a solvent mixture of acetonitrile and
water. The resultant mixture was lyophilized for 12 h to get the title
product as a white solid (2 g; 52.5%). 1H NMR (400 MHz, MeOH-d4)
δ 8.65−8.56 (m, 1H), 7.94−7.78 (m, 4H), 7.48 (d, J = 8.0 Hz, 3H),
7.36 (ddd, J = 7.4, 4.9, 1.3 Hz, 1H), 7.30−7.14 (m, 5H), 6.63−6.46
(m, 1H), 5.21−5.05 (m, 1H), 4.38−4.26 (m, 1H), 4.22−4.01 (m, 3H),
3.78−3.65 (m, 4H), 3.54 (s, 3H), 3.30−3.22 (m, 1H), 3.15−3.03 (m,
1H), 2.80 (br s, 1H), 2.73−2.58 (m, 1H), 2.17 (s, 1H), 1.33 (s, 7H),
1.20 (s, 2H), 1.09 (s, 9H), 0.82 (s, 9H); MS (ES): m/z 805.4 [M+H]+.
Hz, 1H), 7.97−7.82 (m, 4H), 7.79−7.68 (m, 1H), 7.42−7.28 (m, 4H),
7.20−7.10 (m, 5H), 6.80−6.59 (m, 2H), 4.94 (br s, 1H), 4.72−4.51
(m, 1H), 4.02−3.84 (m, 4H), 3.66 (d, J = 9.1 Hz, 1H), 3.55 (s, 3H),
3.41 (s, 3H), 3.00 (br s, 2H), 2.64 (d, J = 7.2 Hz, 2H), 2.19−2.02 (m,
1H), 1.38 (s, 9H), 0.94 (d, J = 6.8 Hz, 6H), 0.84 (s, 9H), 0.74 (s, 9H);
MS (ES): m/z 905.4 [M+H]+. Analytical HPLC-RT and purity:
method C = 4.168 min and 99.95%; method D = 5.973 min and
99.94%. HRMS (ESI/Orbitrap) m/z: [M+H]+ calcd for C48H70N7O10
904.5179; found 904.5161.
(2S,3S)-3-((S)-2-((Methoxycarbonyl)amino)-3,3-dimethylbutan-
amido)-1-(2-((S)-2-((methoxycarbonyl)amino)-3,3-dimethyl-
butanoyl)-1-(4-(pyridin-2-yl)benzyl)hydrazinyl)-4-phenylbutan-2-yl
L-Valinate Dihydrochloride (3). In a 5 mL round-bottom flask,
(2S,3S)-3-((S)-2-((methoxycarbonyl)amino)-3,3-dimethylbutan-
amido)-1-(2-((S)-2-((methoxycarbonyl)amino)-3,3-dimethyl-
butanoyl)-1-(4-(pyridin-2-yl)benzyl)hydrazinyl)-4-phenylbutan-2-yl
(tert-butoxycarbonyl)-L-valinate (2) (0.065 g, 0.072 mmol) was taken
and cooled to 0 °C. A solution of 4 M HCl in diethyl ether (1.80 mL,
7.19 mmol) was added, and the reaction mixture was stirred at 0 °C
for 1 h. The mixture was then concentrated under vacuum. The
residue was dissolved in a mixture of acetonitrile and water. The
resultant mixture was frozen and lyophilized for 12 h to get the title
1
product as an off-white solid (55 mg; 85%). H NMR (300 MHz,
D2O) δ 8.71−8.65 (m, 1H), 8.60−8.51 (m, 1H), 8.25−8.18 (m, 1H),
7.97−7.88 (m, 1H), 7.83−7.72 (m, 2H), 7.64−7.57 (m, 2H), 7.29−
7.11 (m, 5H),5.22−5.10 (m, 1H), 4.11−3.71 (m, 5H), 3.63−3.36 (m,
7H), 3.04−2.87 (m, 3H), 2.68−2.39 (m, 2H), 1.13−0.96 (m, 6H),
0.71 (s, 9H), 0.67 (s, 9H); (ES): m/z 804.9 [M+H]+. Analytical
HPLC-RT and purity: method A = 6.17 min and 97.8%; method B =
I
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Article
HRMS (ESI/Orbitrap) m/z: [M+H]+ calcd for C43H61N6O9
805.4495; found 805.4465.
The organic layer was washed with water and brine, dried over
anhydrous sodium sulfate, and concentrated under vacuum to get the
crude product as a light brownish oil, which was purified by RP HPLC
(preparative HPLC conditions: X Bridge phenyl 250 × 19 mm; mobile
phase A: 10 mM ammonium acetate in water at pH 4.5; mobile phase
B: acetonitrile; flow rate: 17 mL/min.; isocratic mobile phase A:B =
40:60 with a run time of 30 min). The HPLC fraction was
concentrated under reduced pressure at ∼30 °C. The residue was
dissolved in a solvent mixture of acetonitrile and water, and frozen
solution was lyophilized for 12 h to get the title product as an off-white
solid (100 mg; 26.5% for two steps).1H NMR (400 MHz, MeOH-d4)
δ 8.66−8.55 (m, 1H), 7.93−7.80 (m, 4H), 7.51−7.48 (m, 2H), 7.40−
7.34 (m, 1H), 7.30−7.15 (m, 5H), 5.73−5.60 (m, 2H), 5.12−5.03 (m,
1H), 4.56−4.47 (m, 1H), 4.20−4.09 (m, 2H), 4.06−3.95 (m, 2H),
3.70 (s, 4H), 3.53 (s, 3H), 3.28−3.19 (m, 1H), 3.12−3.02 (m, 1H),
2.91−2.78 (m, 1H), 2.76−2.66 (m, 1H), 2.10−1.99 (m, 1H), 1.43 (s,
9H), 1.07 (s, 9H), 0.93−0.84 (m, 6H), 0.80 (s, 9H); MS (ES): m/z
978.4 [M+H]+. Analytical HPLC-RT and purity: method A = 10.349
min and 97.63%; method B = 10.861 min and 99.57%. HRMS (ESI/
Orbitrap) m/z: [M+H]+ calcd for C50H72N7O13 978.5183; found
978.5149.
(5S,10S,11S,18S)-18-Amino-11-benzyl-5-(tert-butyl)-19-methyl-
3,6,13,17-tetraoxo-8-(4-(pyridin-2-yl)benzyl)-2,14,16-trioxa-
4,7,8,12-tetraazaicosan-10-yl (S)-2-((methoxycarbonyl)amino)-3,3-
dimethylbutanoate Dihydrochloride (11). To the solid of
(5S,10S,11S,18S)-11-benzyl-5-(tert-butyl)-18-isopropyl-22,22-dimeth-
yl-3,6,13,17,20-pentaoxo-8-(4-(pyridin-2-yl)benzyl)-2,14,16,21-tet-
raoxa-4,7,8,12,19-pentaazatricosan-10-yl (S)-2-((methoxycarbonyl)-
amino)-3,3-dimethylbutanoate (10) (90 mg, 0.092 mmol) was
added 4 N HCl in dioxane (0.5 mL, 2.00 mmol) at 0 °C under
nitrogen atmosphere. After being stirred at 0 °C for 1 h, the reaction
mixture was concentrated in vacuo at 30 °C. The residue was stirred
with ether. The solvent was carefully decanted. The resultant solid was
dried under vacuum to get the crude product as an off white solid. The
solid was dissolved in a mixture of acetonitrile and water. The resulting
mixture was frozen and lyophilized for 12.0 h to afford the title
product as a white solid (67.33 mg; 76%). 1H NMR (400 MHz,
MeOH-d4) δ 8.82 (d, J = 5.02 Hz, 1H), 8.58 (t, J = 7.65 Hz, 1H),
8.25−8.38 (m, 1H), 7.83−8.01 (m, 3H), 7.71 (d, J = 8.28 Hz, 2H),
7.54 (d, J = 9.54 Hz, 1H), 7.12−7.35 (m, 5H), 5.72−5.86 (m, 2H),
5.08 (br s, 1H), 4.60 (br s, 1H), 4.04−4.25 (m, 3H), 3.81−3.95 (m,
1H), 3.71 (s, 4H), 3.47−3.58 (m, 3H), 3.25 (dd, J = 7.28, 13.05 Hz,
1H), 3.06−3.18 (m, 1H), 2.89 (dd, J = 4.77, 13.80 Hz, 1H), 2.65−2.80
(m, 1H), 2.20 (qd, J = 6.90, 11.42 Hz, 1H), 1.08 (s, 9H), 0.93−1.03
(m, 6H), 0.81 (s, 9H); MS (ES): m/z 878.4 [M+H]+. Analytical
HPLC-RT and purity: method A = 6.415 min and 98.33%, method B
= 7.754 min and 99.16%. HRMS (ESI/Orbitrap) m/z: [M+H]+ calcd
for C45H64N7O11 878.4658; found 878.4648.
(5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl (4-Nitrophenyl) Carbo-
nate (8).34 A stirred solution of 4-(hydroxymethyl)-5-methyl-1,3-
dioxol-2-one (1.4 g, 10.76 mmol) in DCM (15 mL) was cooled to 0
°C. Pyridine (8.70 mL, 108 mmol) was added, and the reaction
mixture was stirred for 5 min. Then 4-nitrophenyl chloroformate (8.68
g, 43.0 mmol) was added, and the reaction mixture was stirred at room
temperature for 24 h. Water was added. The reaction mixture was
extracted with DCM (3 × 50 mL). The combined organic layer was
washed with 1.5 N HCl and brine, dried over anhydrous sodium
sulfate, filtered, and concentrated to get a brownish gum. The crude
product was purified by CombiFlash (silica gel 60−120 mesh; 50%
ethyl acetate in hexane as an eluent) to get the title product (1.8 g,
5.98 mmol, 55.5%) as a white solid. 1H NMR (300 MHz, CHCl3-d) δ
8.35−8.26 (m, 2H), 7.44−7.37 (m, 2H), 5.04 (s, 2H), 2.23 (s, 3H).
MS (ES): The desired mass peak was not obtained.
(5S,10S,11S)-11-Benzyl-5-(tert-butyl)-15-(5-methyl-2-oxo-1,3-di-
oxol-4-yl)-3,6,13-trioxo-8-(4-(pyridin-2-yl)benzyl)-2,14-dioxa-
4,7,8,12-tetraazapentadecan-10-yl (S)-2-((Methoxycarbonyl)-
amino)-3,3-dimethylbutanoate (9). To a stirred solution of
(2S,3S)-3-amino-1-(2-((S)-2-((methoxycarbonyl)amino)-3,3-
dimethylbutanoyl)-1-(4-(pyridin-2-yl)benzyl)hydrazinyl)-4-phenyl-
butan-2-yl (S)-2-((methoxycarbonyl)amino)-3,3-dimethylbutanoate
dihydrochloride (4) (0.3 g, 0.386 mmol) in dioxane (10 mL) were
added DIPEA (0.202 mL, 1.157 mmol), DMAP (0.024 g, 0.193
mmol), and (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (4-nitrophenyl)
carbonate 8 (0.228 g, 0.771 mmol). The reaction mixture was heated
at 100 °C for 24 h and cooled to ambient temperature. The reaction
mixture was concentrated under reduced pressure to remove the
solvent. The residue was partitioned between water and ethyl acetate.
The organic layer was washed with brine, dried over anhydrous
sodium sulfate, filtered, and concentrated to get a brownish gum. The
crude product was purified using RP-HPLC (preparative HPLC
conditions: Sunfire C18 (150 × 19 mm; 5 μm), mobile phase A: 10
mM ammonium acetate in water at 4.5 pH, mobile phase B:
acetonitrile, flow rate: 17 mL/min, gradient: 0/30, 7/65). The fraction
was concentrated using high vacuum at 30 °C. The residue was
dissolved in a mixture of acetonitrile and water. The mixture was
frozen and lyophilized for 12 h to get the title product (0.035 g, 0.039
mmol, 10.09% yield) as a white solid. 1H NMR (400 MHz, MeOH-d4)
δ 8.61 (td, J = 2.5, 1.0 Hz, 1H), 7.94−7.82 (m, 4H), 7.51 (d, J = 8.0
Hz, 2H), 7.36 (ddd, J = 7.3, 5.0, 1.3 Hz, 1H), 7.30−7.12 (m, 5H), 5.12
(br s, 1H), 4.81 (d, J = 14.1 Hz, 1H), 4.62 (d, J = 14.1 Hz, 1H), 4.42
(br s, 1H), 4.18−4.00 (m, 3H), 3.74 (s, 1H), 3.70 (s, 3H), 3.54 (s,
3H), 3.28−3.19 (m, 1H), 3.18−3.04 (m, 1H), 2.84 (dd, J = 13.6, 5.0
Hz, 1H), 2.67−2.55 (m, 1H), 2.03 (s, 3H), 1.07 (s, 9H), 0.84 (s, 9H);
LCMS (ES): m/z 861.4 [M+H]+. Analytical HPLC RT and purity:
method A = 7.85 min and 95.78%; method B = 9.32 min and 95.88%.
HRMS (ESI/Orbitrap) m/z: [M+H]+ calcd for C44H57N6O12
861.4029; found 861.3986.
(5S,10S,11S)-11-Benzyl-5-(tert-butyl)-15-chloro-16-methyl-
3,6,13-trioxo-8-(4-(pyridin-2-yl)benzyl)-2,14-dioxa-4,7,8,12-
tetraazaheptadecan-10-yl (2S)-2-((Methoxycarbonyl)amino)-3,3-
dimethylbutanoate (12). To a solution of (2S,3S)-3-amino-1-(2-
((S)-2-((methoxycarbonyl)amino)-3,3-dimethylbutanoyl)-1-(4-(pyri-
din-2-yl)benzyl)hydrazinyl)-4-phenylbutan-2-yl (S)-2-((methoxy-
carbonyl)amino)-3,3-dimethylbutanoate dihydrochloride (4) (300
mg, 0.426 mmol) in anhydrous DCM (2 mL) were added pyridine
(0.172 mL, 2.128 mmol) and 1-chloro-2-methylpropyl chloroformate
(0.124 mL, 0.851 mmol) under nitrogen atmosphere. After being
stirred at 0 °C for 3 h, the reaction mixture was partitioned between
1.5 N HCl and DCM. The organic layer was washed with water and
brine, dried over anhydrous sodium sulfate, and concentrated under
vacuum to get the crude product as a brown solid (320 mg, ∼90%).
The crude product was found to be unstable for purification
(CombiFlash/Prepartive HPLC) and hence was used as such in the
next reaction. MS (ES): m/z 839.4 [M+H]+
(5S,10S,11S,18S)-11-Benzyl-5-(tert-butyl)-18-isopropyl-22,22-di-
methyl-3,6,13,17,20-pentaoxo-8-(4-(pyridin-2-yl)benzyl)-2,14,16,21-
tetraoxa-4,7,8,12,19-pentaazatricosan-10-yl (S)-2-((Methoxy-
carbonyl)amino)-3,3-dimethylbutanoate (10). To a solution of
(2S,3S)-3-amino-1-(2-((S)-2-((methoxycarbonyl)amino)-3,3-
dimethylbutanoyl)-1-(4-(pyridin-2-yl)benzyl)hydrazinyl)-4-phenyl-
butan-2-yl (S)-2-((methoxycarbonyl)amino)-3,3-dimethylbutanoate
dihydrochloride (4) (300 mg, 0.426 mmol) in DCM (2 mL) were
added pyridine (0.172 mL, 2.128 mmol) and chloromethyl chloro-
formate (0.076 mL, 0.851 mmol) under nitrogen atmosphere. After
being stirred at 0 °C for 3 h, the reaction mixture was partitioned
between 1.5 N HCl and DCM. The organic layer was washed with
water and brine, dried over anhydrous sodium sulfate, and
concentrated under vacuum to get the crude product as an off-white
solid (300 mg). MS (ES): m/z 797.4 [M+H]+. To the solution of this
crude product in DMF (1 mL) were added N-Boc-L-Val (123 mg,
0.564 mmol), DIPEA (0.329 mL, 1.881 mmol), and sodium iodide
(56.4 mg, 0.376 mmol). The reaction mixture was heated at 50 °C for
14 h. The reaction mixture was partitioned between water and EtOAc.
(5S,10S,11S,18S)-11-Benzyl-5-(tert-butyl)-15,18-diisopropyl-
22,22-dimethyl-3,6,13,17,20-pentaoxo-8-(4-(pyridin-2-yl)benzyl)-
2,14,16,21-tetraoxa-4,7,8,12,19-pentaazatricosan-10-yl (2S)-2-
((Methoxycarbonyl)amino)-3,3-dimethylbutanoate (13). To a sol-
ution of (5S,10S,11S)-11-benzyl-5-(tert-butyl)-15-chloro-16-methyl-
3,6,13-trioxo-8-(4-(pyridin-2-yl)benzyl)-2,14-dioxa-4,7,8,12-tetraaza-
J
DOI: 10.1021/acs.jmedchem.8b00277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
heptadecan-10-yl (2S)-2-((methoxycarbonyl)amino)-3,3-dimethyl-
butanoate (12) (252 mg, 0.300 mmol) in DMF (1.0 mL) were
added N-Boc-L-Val (98 mg, 0.450 mmol), DIPEA (0.262 mL, 1.501
mmol), and sodium iodide (45.0 mg, 0.300 mmol). The reaction
mixture was heated at 50 °C for 14 h. The reaction mixture was diluted
with water and extracted with EtOAc. The organic layer was washed
with water and brine, dried over anhydrous sodium sulfate, and
concentrated under vacuum to give the crude product. The crude
product was purified by RP HPLC (Kinetex C18 (250 × 30 mm);
mobile phase A: 10 mM ammonium acetate in water; mobile phase B:
acetonitrile; flow rate: 20 mL/min.; gradient: 0/30, 10/70) to afford
the title product (100 mg; 29.7%).1H NMR (400 MHz, MeOH-d4) δ
8.64−8.58 (m, 1H), 7.88 (d, J = 8.5 Hz, 4H), 7.58−7.50 (m, 2H),
7.40−7.33 (m, 1H), 7.30−7.14 (m, 5H), 6.52−6.39 (m, 1H), 5.12−
5.04 (m, 1H), 4.58−4.46 (m, 1H), 4.23−4.03 (m, 3H), 4.02−3.89 (m,
1H), 3.71 (s, 4H), 3.52 (s, 3H), 3.30−3.23 (m, 1H), 3.16−3.00 (m,
1H), 2.89−2.81 (m, 1H), 2.78−2.63 (m, 1H), 2.10−1.90 (m, 2H),
1.42 (s, 9H), 1.08 (s, 9H), 0.96−0.85 (m, 12H), 0.79 (s, 9H); MS
(ES): m/z 1020.4 [M+H]+. Analytical HPLC-RT and purity: method
B = 19.092 min and 98.1%; chiral HPLC-RT and purity: method A =
1.85 min and 95.1%, method B = 2.97 min and 100%. HRMS (ESI/
Orbitrap) m/z: [M+H]+ calcd for C53H78N7O13 1020.5652; found
1020.5614.
7.33 (m, 1H), 7.30−7.14 (m, 5H), 6.45 (d, J = 5.0 Hz, 1H), 5.10 (br s,
1H), 4.58 (br s, 1H), 4.23−4.13 (m, 2H), 4.10−3.95 (m, 2H), 3.71 (s,
3H), 3.66 (d, J = 2.5 Hz, 1H), 3.51 (s, 3H), 3.29−3.18 (m, 1H), 3.18−
3.03 (m, 1H), 2.90−2.80 (m, 1H), 2.76−2.63 (m, 1H), 2.19−2.05 (m,
1H), 1.92 (dd, J = 12.5, 7.0 Hz, 1H), 1.50−1.37 (m, 9H), 1.08 (s, 9H),
0.98−0.85 (m, 12H), 0.81 (s, 9H); LCMS (ES): m/z 1020.4 [M+H]+.
Analytical HPLC-RT and purity: method B = 11.57 min and 99.34%;
chiral HPLC-RT and purity: method A = 1.87 min and 97.1%, method
B = 2.76 min and 97.3%. HRMS (ESI/Orbitrap) m/z: [M+H]+ calcd
for C53H78N7O13 1020.5652; found 1020.5715.
(5S,10S,11S,18R)-18-Amino-11-benzyl-5-(tert-butyl)-15-isoprop-
yl-19-methyl-3,6,13,17-tetraoxo-8-(4-(pyridin-2-yl)benzyl)-2,14,16-
trioxa-4,7,8,12-tetraazaicosan-10-yl (2S)-2-((Methoxycarbonyl)-
amino)-3,3-dimethylbutanoate Dihydrochloride (16). In a 5 mL
round-bottom flask, (5S,10S,11S,18R)-11-benzyl-5-(tert-butyl)-15,18-
diisopropyl-22,22-dimethyl-3,6,13,17,20-pentaoxo-8-(4-(pyridin-2-yl)-
benzyl)-2,14,16,21-tetraoxa-4,7,8,12,19-pentaazatricosan-10-yl (2S)-2-
((methoxycarbonyl)amino)-3,3-dimethylbutanoate (15) (0.04 g, 0.039
mmol) was taken and cooled to 0 °C. A solution of 4 M HCl in
dioxane (2.47 mL, 9.87 mmol) was added, and the reaction mixture
was stirred at 0 °C for 1 h. The mixture was then concentrated under
vacuum. The residue was then triturated with ether, and the ether layer
was decanted. The residue was dissolved in a mixture of acetonitrile
and water. The resultant mixture was frozen and lyophilized for 12 h to
get the title product (29.44 mg, 0.028 mmol, 71.5% yield) as a white
(5S,10S,11S,18S)-18-Amino-11-benzyl-5-(tert-butyl)-15-isoprop-
yl-19-methyl-3,6,13,17-tetraoxo-8-(4-(pyridin-2-yl)benzyl)-2,14,16-
trioxa-4,7,8,12-tetraazaicosan-10-yl (2S)-2-((methoxycarbonyl)-
amino)-3,3-dimethylbutanoate Dihydrochloride (14). To the solid
of (5S,10S,11S,18S)-11-benzyl-5-(tert-butyl)-15,18-diisopropyl-22,22-
dimethyl-3,6,13,17,20-pentaoxo-8-(4-(pyridin-2-yl)benzyl)-2,14,16,21-
tetraoxa-4,7,8,12,19-pentaazatricosan-10-yl (2S)-2-((methoxy-
carbonyl)amino)-3,3-dimethylbutanoate (13) (90 mg, 0.088 mmol)
was added 4 N HCl in dioxane (4.1 mL, 16.46 mmol) at 0 °C under
nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 1 h
and subsequently concentrated in vacuo at 30 °C. The residue was
stirred with ether, and the ether layer was decanted. The resultant solid
was dried in vacuo to get crude product as an off-white solid. The solid
was dissolved in a mixture of solvent acetonitrile and water. The
resulting mixture was frozen and lyophilized for 12 h to afford the title
1
solid. H NMR (400 MHz, MeOH-d4) δ 8.81 (d, J = 5.0 Hz, 1H),
8.61−8.51 (m, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.98−7.85 (m, 3H), 7.72
(d, J = 8.5 Hz, 2H), 7.35−7.17 (m, 5H), 6.46 (d, J = 4.5 Hz, 1H), 5.14
(d, J = 4.5 Hz, 1H), 4.52 (br s, 1H), 4.15 (s, 3H), 3.92 (d, J = 4.0 Hz,
1H), 3.70 (s, 3H), 3.59 (br s, 1H), 3.51 (s, 4H), 3.30−3.24 (m, 1H),
3.15 (br s, 1H), 2.91 (dd, J = 13.6, 4.5 Hz, 1H), 2.76−2.67 (m, 1H),
2.37−2.26 (m, 1H), 2.01 (d, J = 4.5 Hz, 1H), 1.15−1.03 (m, 15H),
0.99−0.90 (m, 6H), 0.81 (s, 9H); LCMS (ES): m/z 920.4 [M+H]+.
Analytical HPLC RT and purity: method A = 8.32 min and 94.81%;
method B = 6.23 min and 94.53%. HRMS (ESI/Orbitrap) m/z: [M
+H]+ calcd for C48H70N7O11 920.5128; found 920.5099.
ASSOCIATED CONTENT
* Supporting Information
■
1
S
product as a white solid (27.89 mg; 31.8%). H NMR (400 MHz,
MeOH-d4) δ 8.78 (d, J = 5.02 Hz, 1H), 8.42−8.55 (m, 1H), 8.23 (d, J
= 8.53 Hz, 1H), 7.86 (d, J = 8.53 Hz, 3H), 7.68 (d, J = 8.53 Hz, 2H),
7.50−7.35 (br s, 1H), 7.14−7.32 (m, 5H), 6.50 (d, J = 5.02 Hz, 1H),
5.08 (br s, 1H), 4.55 (br s, 1H), 4.08−4.24 (m, 3H), 3.94 (d, J = 4.02
Hz, 1H), 3.61−3.72 (m, 4H), 3.43−3.52 (m, 3H), 3.02−3.26 (m, 2H),
2.63−2.95 (m, 2H), 1.96−1.99(m 2H), 0.76−1.08 (m, 29H); MS
(ES): m/z = 920.4 [M+H]+. Analytical HPLC-RT and purity: method
A = 7.21 min and 95.46%; method B = 8.16 min and 96.11%. HRMS
(ESI/Orbitrap) m/z: [M+H]+ calcd for C48H70N7O11 920.5128; found
920.5097.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b00277.
Details of in vitro and in vivo experiments (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: +91-(0)9731600213. E-mail: murugaiah.andappan@
syngeneintl.com or murugaiah.subbaiah123@gmail.com.
■
(5S,10S,11S,18R)-11-Benzyl-5-(tert-butyl)-15,18-diisopropyl-
22,22-dimethyl-3,6,13,17,20-pentaoxo-8-(4-(pyridin-2-yl)benzyl)-
2,14,16,21-tetraoxa-4,7,8,12,19-pentaazatricosan-10-yl (2S)-2-
((Methoxycarbonyl)amino)-3,3-dimethylbutanoate (15). To a sol-
ution of (5S,10S,11S)-11-benzyl-5-(tert-butyl)-15-chloro-16-methyl-
3,6,13-trioxo-8-(4-(pyridin-2-yl)benzyl)-2,14-dioxa-4,7,8,12-tetraaza-
heptadecan-10-yl (2S)-2-((methoxycarbonyl)amino)-3,3-dimethyl-
butanoate (12) (0.5 g, 0.453 mmol) in DMF (8.0 mL) was added
N-Boc-D-Val (0.197 g, 0.905 mmol) followed by DIPEA (0.395 mL,
2.263 mmol) and sodium iodide (0.068 g, 0.453 mmol). The reaction
mixture was heated at 60 °C for 8 h. The mixture was partitioned
between ethyl acetate and water. The organic layer was concentrated
under vacuum to give the residue, which was purified using RP- HPLC
(preparative HPLC conditions: Sunfire C18 (150 × 19 mm; 5 μm),
mobile phase A: 0.1% HCOOH in water, mobile phase B: acetonitrile,
flow: 18 mL/min, gradient: 0/30, 10/80). The fraction was
concentrated using high vacuum at 30 °C. The residue was dissolved
in a mixture of acetonitrile and water. The resultant mixture was frozen
and lyophilized for 12 h to get the title product (0.15 g, 0.144 mmol,
ORCID
Murugaiah A. M. Subbaiah: 0000-0001-5707-966X
Nicholas A. Meanwell: 0000-0002-8857-1515
Present Address
⊗For J.F.K., S.J., M.R.K., and C.W.: ViiV Healthcare, 36 East
Industrial Road, Branford, CT 06405, USA
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Arvind Mathur and Percy Carter for their
support and inspiration. We especially acknowledge Dr. Sridhar
Desikan for his guidance at the early stage of the program and
Dr. Michael Sinz for critical review of the manuscript. We thank
Drs. Punit Marathe, Michael Hageman, and Bruce Car for
productive discussions. We acknowledge Dr. Arun Kumar
1
31.9% yield) as a white solid. H NMR (400 MHz, MeOH-d4) δ 8.61
(d, J = 4.0 Hz, 1H), 7.95−7.80 (m, 4H), 7.57−7.47 (m, 2H), 7.40−
K
DOI: 10.1021/acs.jmedchem.8b00277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(15) Vierling, P.; Greiner, J. Prodrugs of HIV protease inhibitors.
Curr. Pharm. Des. 2003, 9, 1755−1770.
Gupta and team for an intermediate supply, the Discovery
Analytical Sciences team for analytical support, and Dr.
Sivaprasad Putlur for HRMS support.
(16) Calogeropoulou, T.; Detsi, A.; Lekkas, E.; Koufaki, M. Strategies
in the Design of Prodrugs of Anti-HIV Agents. Curr. Top. Med. Chem.
2003, 3, 1467−1495.
ABBREVIATIONS USED
■
(17) Ouyang, H. Fosamprenavir: a Prodrug of Amprenavir.
Biotechnol.: Pharm. Aspects 2007, 5, 541−549.
(18) DeGoey, D. A.; Grampovnik, D. J.; Flosi, W. J.; Marsh, K. C.;
Wang, X. C.; Klein, L. L.; McDaniel, K. F.; Liu, Y.; Long, M. A.; Kati,
W. M.; Molla, A.; Kempf, D. J. Water-Soluble Prodrugs of the Human
Immunodeficiency Virus Protease Inhibitors Lopinavir and Ritonavir.
J. Med. Chem. 2009, 52, 2964−2970.
(19) Patel, M.; Mandava, N.; Gokulgandhi, M.; Pal, D.; Mitra, A. K.
Amino Acid Prodrugs: An Approach to Improve the Absorption of
HIV-1 Protease Inhibitor, Lopinavir. Pharmaceuticals 2014, 7, 433−
452.
(20) Wang, Z.; Pal, D.; Mitra, A. K. Stereoselective Evasion of P-
Glycoprotein, Cytochrome P450 3A, and Hydrolases by Peptide
Prodrug Modification of Saquinavir. J. Pharm. Sci. 2012, 101, 3199−
3213.
(21) Gaucher, B.; Rouquayrol, M.; Roche, D.; Greiner, J.; Aubertin,
A. M.; Vierling, P. Prodrugs of HIV Protease Inhibitors-Saquinavir,
Indinavir and Nelfinavir-Derived from Diglycerides or Amino Acids:
Synthesis, Stability and Anti-HIV Activity. Org. Biomol. Chem. 2004, 2,
345−357.
(22) De Kock, H. A.; Wigerinck, P. T. B. P.; Balzarini, J. Preparation
of Peptidyl HIV Prodrugs Which are Cleavable by CD26. World
Patent Application 2004/099135, Nov 18, 2004.
ATV, atazanavir; BLQ, below limit of quantification;; cART,
combination antiretroviral therapy; DMAc, dimethylacetamide;
FATP, fatty acid transport protein; HPβCD, hydroxypropyl β-
cyclodextrin; PepT1, proton-coupled peptide transporter 1; PI,
protease inhibitor; PPI, proton pump inhibitor; SMVT,
sodium-dependent multivitamin transporter; SVCT, sodium-
dependent vitamin C transporter; UGT1A1, uridine diphos-
phate glucuronosyltransferase 1A1
REFERENCES
■
(1) Zhan, P.; Pannecouque, C.; De Clercq, E.; Liu, X. Anti-HIV Drug
Discovery and Development: Current Innovations and Future Trends.
J. Med. Chem. 2016, 59, 2849−2878.
(2) Samji, H.; Cescon, A.; Hogg, R. S.; Modur, S. P.; Althoff, K. N.;
Buchacz, K.; Burchell, A. N.; Cohen, M.; Gebo, K. A.; Gill, M. J.;
Justice, A.; Kirk, G.; Klein, M. B.; Korthuis, P. T.; Martin, J.;
Napravnik, S.; Rourke, S. B.; Sterling, T. R.; Silverberg, M. J.; Deeks,
S.; Jacobson, L. P.; Bosch, R. J.; Kitahata, M. M.; Goedert, J. J.; Moore,
R.; Gange, S. J. Closing the Gap: Increases in Life Expectancy Among
Treated HIV-Positive Individuals in the United States and Canada.
PLoS One 2013, 8, e81355.
(3) Ghosh, A. K.; Osswald, H. L.; Prato, G. Recent Progress in the
Development of HIV-1 Protease Inhibitors for the Treatment of HIV/
AIDS. J. Med. Chem. 2016, 59, 5172−5208.
(23) Luo, S.; Wang, Z.; Patel, M.; Khurana, V.; Zhu, X.; Pal, D.;
Mitra, A. K. Targeting SVCT for Enhanced Drug Absorption:
Synthesis and In Vitro Evaluation of a Novel Vitamin C Conjugated
Prodrug of Saquinavir. Int. J. Pharm. 2011, 414, 77−85.
(24) Luo, S.; Kansara, V. S.; Zhu, X.; Mandava, N. K.; Pal, D.; Mitra,
A. K. Functional Characterization of Sodium-Dependent Multivitamin
Transporter in MDCK-MDR1 Cells and its Utilization as a Target for
Drug Delivery. Mol. Pharmaceutics 2006, 3, 329−339.
(25) Gerk, P. M.; Walsh, S. W.; Wang, M.; Landsberg, A. K. Prodrugs
of HIV Protease Inhibitors Utilizing a Transporter-Directed Uptake
Mechanism. U.S. Patent Appl. 20130267547, Oct 10, 2013.
(26) Skwarczynski, M.; Kiso, Y. Application of the O-N Intra-
molecular Acyl Migration Reaction in Medicinal Chemistry. Curr. Med.
Chem. 2007, 14, 2813−2823.
(27) Hamada, Y.; Matsumoto, H.; Yamaguchi, S.; Kimura, T.;
Hayashi, Y.; Kiso, Y. Water-Soluble Prodrugs of Dipeptide HIV
Protease Inhibitors Based on O–>N Intramolecular Acyl Migration:
Design, Synthesis and Kinetic Study. Bioorg. Med. Chem. 2004, 12,
159−170.
(28) Zhang, H.; Bonacorsi, S. J., Jr.; Chen, B.-C.; Leith, L. W.;
Rinehart, J. K.; Balasubramanian, B.; Barrish, J. C. A Facile and
Efficient Synthesis of d3-Labelled Reyataz. J. Labelled Compd.
Radiopharm. 2005, 48, 1041−1047.
(29) Reddy, B. P.; Krishna, B. V.; Sharma, V. M.; Reddy, K. R.;
Reddy, M. M. Preparation of Aza-Peptides Containing 2,2-
Disubstituted Cyclobutyl and/or Substituted Alkoxybenzyl Derivatives
as Antivirals. World Patent Application 2011/080562, July 7, 2011.
(30) Yanagisawa, H.; Koike, H.; Miura, S.-i. Olmesartan Medoxomil:
an Angiotensin II Receptor Blocker; John Wiley & Sons, Inc.: 2012; pp
45−66.
(31) Hjermitslev, M.; Grimm, D. G.; Wehland, M.; Simonsen, U.;
Krueger, M. Azilsartan Medoxomil, an Angiotensin II Receptor
Antagonist for the Treatment of Hypertension. Basic Clin. Pharmacol.
Toxicol. 2017, 121, 225−233.
(4) Midde, N. M.; Patters, B. J.; Rao, P.; Cory, T. J.; Kumar, S.
Investigational Protease Inhibitors as Antiretroviral Therapies. Expert
Opin. Invest. Drugs 2016, 25, 1189−1200.
(5) Farajallah, A.; Bunch, R. T.; Meanwell, N. A. Discovery and
Development of Atazanavir. In Antiviral Drugs; John Wiley & Sons,
Inc.: 2011; pp 1−17.
(6) 20th WHO Essential Medicines List (EML); World Health
Organization (WHO), Geneva, 2017; http://www.who.int/
medicines/publications/essentialmedicines/20th_EML2017_FINAL_
amendedAug2017.pdf?ua=1 (accessed March 23, 2018).
(7) Appendix B, Table 3: Characteristics of Protease Inhibitors;
https://aidsinfo.nih.gov/guidelines/htmltables/1/5482 (accessed
March 23, 2018).
(8) Kis, O.; Zastre, J. A.; Hoque, M. T.; Walmsley, S. L.; Bendayan, R.
Role of Drug Efflux and Uptake Transporters in Atazanavir Intestinal
Permeability and Drug-Drug Interactions. Pharm. Res. 2013, 30,
1050−1064.
(9) Klein, C. E.; Chiu, Y. L.; Cai, Y.; Beck, K.; King, K. R.;
Causemaker, S. J.; Doan, T.; Esslinger, H. U.; Podsadecki, T. J.;
Hanna, G. J. Effects of Acid-Reducing Agents on the Pharmacokinetics
of Lopinavir/Ritonavir and Ritonavir-Boosted Atazanavir. J. Clin.
Pharmacol. 2008, 48, 553−562.
(10) Zhu, L.; Persson, A.; Mahnke, L.; Eley, T.; Li, T.; Xu, X.;
Agarwala, S.; Dragone, J.; Bertz, R. Effect of Low-Dose Omeprazole
(20 mg daily) on the Pharmacokinetics of Multiple-Dose Atazanavir
with Ritonavir in Healthy Subjects. J. Clin. Pharmacol. 2011, 51, 368−
377.
(11) Subbaiah, M. A. M.; Meanwell, N. A.; Kadow, J. F. Design
Strategies in the Prodrugs of HIV-1 Protease Inhibitors to Improve the
Pharmaceutical Properties. Eur. J. Med. Chem. 2017, 139, 865−883.
(12) Harbeson, S. L.; Tung, R. D. Azapeptide Derivatives. U.S. Patent
8,158,805, April 17, 2012.
(32) Giannarini, G.; Tascini, C.; Selli, C. Prulifloxacin: Clinical
Studies of a Broad-Spectrum Quinolone Agent. Future Microbiol. 2009,
4, 13−24.
(33) Syed, Y. Y. Ceftobiprole Medocaril: A Review of Its Use in
Patients with Hospital- or Community-Acquired Pneumonia. Drugs
2014, 74, 1523−1542.
(13) Clas, S. D.; Sanchez, R. I.; Nofsinger, R. Chemistry-Enabled
Drug Delivery (Prodrugs): Recent Progress and Challenges. Drug
Discovery Today 2014, 19, 79−87.
(14) Rautio, J.; Karkkainen, J.; Sloan, K. B. Prodrugs - Recent
Approvals and a Glimpse of the Pipeline. Eur. J. Pharm. Sci. 2017, 109,
146−161.
L
DOI: 10.1021/acs.jmedchem.8b00277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(34) Alexander, J.; Bindra, D. S.; Glass, J. D.; Holahan, M. A.; Renyer,
M. L.; Rork, G. S.; Sitko, G. R.; Stranieri, M. T.; Stupienski, R. F.;
Veerapanane, H.; Cook, J. J. Investigation of (Oxodioxolenyl)methyl
Carbamates as Nonchiral Bioreversible Prodrug Moieties for Chiral
Amines. J. Med. Chem. 1996, 39, 480−486.
(35) Ishizuka, T.; Yoshigae, Y.; Murayama, N.; Izumi, T. Different
Hydrolases Involved in Bioactivation of Prodrug-Type Angiotensin
Receptor Blockers: Carboxymethylenebutenolidase and Carboxylester-
ase 1. Drug Metab. Dispos. 2013, 41, 1888−1895.
(36) Ishizuka, T.; Rozehnal, V.; Fischer, T.; Kato, A.; Endo, S.;
Yoshigae, Y.; Kurihara, A.; Izumi, T. Interindividual Variability of
Carboxymethylenebutenolidase Homolog, a Novel Olmesartan
Medoxomil Hydrolase, in the Human Liver and Intestine. Drug
Metab. Dispos. 2013, 41, 1156−1162.
(37) Ishizuka, T.; Fujimori, I.; Nishida, A.; Sakurai, H.; Yoshigae, Y.;
Nakahara, K.; Kurihara, A.; Ikeda, T.; Izumi, T. Paraoxonase 1 as a
Major Bioactivating Hydrolase for Olmesartan Medoxomil in Human
Blood Circulation: Molecular Identification and Contribution to
Plasma Metabolism. Drug Metab. Dispos. 2012, 40, 374−380.
(38) Poras, H.; Bonnard, E.; Dange, E.; Fournie-Zaluski, M.-C.;
Roques, B. P. New Orally Active Dual Enkephalinase Inhibitors
(DENKIs) for Central and Peripheral Pain Treatment. J. Med. Chem.
2014, 57, 5748−5763.
(39) Vig, B. S.; Huttunen, K. M.; Laine, K.; Rautio, J. Amino Acids as
Promoieties in Prodrug Design and Development. Adv. Drug Delivery
Rev. 2013, 65, 1370−1385.
(40) Krecmerova, M. Amino Acid Ester Prodrugs of Nucleoside and
Nucleotide Antivirals. Mini-Rev. Med. Chem. 2017, 17, 818−833.
(41) Dhareshwar, S. S.; Stella, V. J. Your Prodrug Releases
Formaldehyde: Should You be Concerned? No! J. Pharm. Sci. 2008,
97, 4184−4193.
(42) Hilfinger, J.; Shen, W. Preparation of Neuraminidase Inhibitor
Analogs and Prodrugs, Especially Zanamivir and Oseltamivir Acid
Analogs, with Enhanced Oral Bioavailability Useful in Treatment of
Viral Infections, Particularly Influenza. World Patent Application
2011/123856, Oct 6, 2011.
(43) Mechanistic studies of this serendipitous discovery of
diastereoselective O-alkylation are in progress.
(44) PubChem Website: Isobutyraldehyde; https://pubchem.ncbi.
nlm.nih.gov/compound/isobutyraldehyde (accessed March 23, 2018).
(45) Petrillo, E. W. Case Study: Fosinopril. Biotechnol.: Pharm.
Aspects 2007, 5, 551−558.
(46) Lal, R.; Sukbuntherng, J.; Tai, E. H. L.; Upadhyay, S.; Yao, F.;
Warren, M. S.; Luo, W.; Bu, L.; Nguyen, S.; Zamora, J.; Peng, G.; Dias,
T.; Bao, Y.; Ludwikow, M.; Phan, T.; Scheuerman, R. A.; Yan, H.; Gao,
M.; Wu, Q. Q.; Annamalai, T.; Raillard, S. P.; Koller, K.; Gallop, M. A.;
Cundy, K. C. Arbaclofen Placarbil, a Novel R-Baclofen Prodrug:
Improved Absorption, Distribution, Metabolism, and Elimination
Properties Compared with R-Baclofen. J. Pharmacol. Exp. Ther. 2009,
330, 911−921.
(47) Hoppe, E.; Hewitt, N. J.; Buchstaller, H. P.; Eggenweiler, H. M.;
Sirrenberg, C.; Zimmermann, A.; Marz, J.; Schwartz, H.; Saal, C.;
Meyring, M.; Hecht, S. A Novel Strategy for ADME Screening of
Prodrugs: Combined Use of Serum and Hepatocytes to Integrate
Bioactivation and Clearance, and Predict Exposure to Both Active and
Prodrug to the Systemic Circulation. J. Pharm. Sci. 2014, 103, 1504−
1514.
(48) Saxena, A.; Shah, D.; Padmanabhan, S.; Gautam, S. S.; Chowan,
G. S.; Mandlekar, S.; Desikan, S. Prediction of pH Dependent
Absorption Using In Vitro, In Silico, and In Vivo Rat Models: Early
liability Assessment During Lead Optimization. Eur. J. Pharm. Sci.
2015, 76, 173−180.
(49) Zhang, L.; Wu, F.; Lee, S. C.; Zhao, H.; Zhang, L. pH-
Dependent Drug-Drug Interactions for Weak Base Drugs: Potential
Implications for New Drug Development. Clin. Pharmacol. Ther. (N.
Y., NY, U. S.) 2014, 96, 266−277.
M
DOI: 10.1021/acs.jmedchem.8b00277
J. Med. Chem. XXXX, XXX, XXX−XXX