Prodrug Strategies to Improve the Solubility of the HCV NS5A Inhibitor Pibrentasvir (ABT-530)

Article abstract of DOI:10.1021/acs.jmedchem.0c00956

A research program to discover solubilizing prodrugs of the HCV NS5A inhibitor pibrentasvir (PIB) identified phosphomethyl analog 2 and trimethyl-lock (TML) prodrug 9. The prodrug moiety is attached to a benzimidazole nitrogen atom via an oxymethyl linkage to allow for rapid and complete release of the drug for absorption following phosphate removal by intestinal alkaline phosphatase. These prodrugs have good hydrolytic stability properties and improved solubility compared to PIB, both in aqueous buffer (pH 7) and FESSIF (pH 5). TML prodrug 9 provided superior in vivo performance, delivering high plasma concentrations of PIB in PK studies conducted in mice, dogs, and monkeys. The improved dissolution properties of these phosphate prodrugs provide them the potential to simplify drug dosage forms for PIB-containing HCV therapy.

Full text of DOI:10.1021/acs.jmedchem.0c00956

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Article
Prodrug Strategies to Improve the Solubility
of HCV NS5A Inhibitor Pibrentasvir (ABT-530)
John T. Randolph, Eric A. Voight, Stephen N. Greszler, Brice Uno, James Newton,
Kenneth Gleason, DeAnne Stolarik, Cecilia Van Handel, Daniel Bow, and David A. DeGoey
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00956 • Publication Date (Web): 03 Sep 2020
Downloaded from pubs.acs.org on September 6, 2020
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Prodrug Strategies to Improve the Solubility of HCV NS5A Inhibitor
Pibrentasvir (ABT-530)
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9
John T. Randolph*, Eric A. Voight, Stephen N. Greszler, Brice E. Uno, James N. Newton, Kenneth
M. Gleason, DeAnne Stolarik, Cecilia Van Handel, Daniel A. J. Bow, David A. DeGoey
Abbvie Incorporated, Global Pharmaceutical Research and Development
1 North Waukegan Road, North Chicago, IL 60064
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KEYWORDS: hepatitis C, antiviral, NS5A, pibrentasvir, ABT-530, prodrug
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Abstract
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A research program to discover solubilizing prodrugs of HCV NS5A inhibitor pibrentasvir
(PIB) identified phosphomethyl analog 2 and trimethyl-lock (TML) prodrug 9. The prodrug moiety
is attached to a benzimidazole nitrogen via an oxymethyl linkage to allow for rapid and complete
release of the drug for absorption following phosphate removal by intestinal alkaline
phosphatase. These prodrugs have good hydrolytic stability properties and improved solubility
compared to PIB, both in aqueous buffer (pH 7) and FESSIF (pH 5). TML prodrug 9 provided
superior in vivo performance, delivering high plasma concentrations of PIB in mouse, dog and
monkey PK studies. The improved dissolution properties of these phosphate prodrugs give them
potential to simplify drug dosage forms for PIB-containing HCV therapy.
9
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F
O
O
O
HO
P
O
HO
O
O
N
N
9
NH
F
F
HN
O
O
N
N
N
HN
O
P
N
N
O
O
O
O
OH
OH
R
O
2
F
F
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Introduction
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Pibrentasvir (ABT-530) is a second-generation HCV inhibitor that targets the NS5A
protein, which is used in combination with the HCV NS3 protease inhibitor glecaprevir (ABT-493)
for the treatment of HCV genotypes 1-6.1-6 Pibrentasvir (PIB) is a single-digit picomolar inhibitor
of wild-type replicons for all major HCV genotypes that maintains potency (<10-fold resistance)
against variant forms of HCV that are highly resistant to other NS5A inhibitors.7 Selection
experiments in replicon cells for genotypes 1-6 found no significant resistance to PIB.8 Treatment
response in patients with chronic HCV infection is strongly associated with the presence of
baseline resistance-associated substitutions (RASs) to HCV direct-acting antivirals (DAAs). This is
particularly true in the case of NS5A inhibitor-containing regimens, since NS5A inhibitor-resistant
viruses have better fitness and persist much longer than viruses that are resistant to NS5B and
NS3/4a inhibitors. The presence of baseline RASs had minimal impact on SVR rates for GT 1-6
HCV patients treated with glecaprevir/pibrentasvir (G/P) in registration trials.9 This is consistent
with findings that PIB treatment-emergent NS5A variants have multiple resistance-conferring
mutations (no single-mutation resistant variants observed), and replicons engineered to contain
the PIB-resistant mutations have poor replicative fitness.10
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Mavyret is a fixed dose combination of 300 mg glecaprevir (GLE) and 120 mg PIB that is
administered as three pills, once a day, with food.11 A key determinant in the daily dose of PIB
is the effect of the intestinal P-gp transporter on drug absorption. Human PK studies found a
greater than linear increase in plasma drug concentrations with oral doses up to 120 mg, which
is the dose required to saturate transporter effects.12 Furthermore, co-dosing with 300 mg of
GLE, a P-gp inhibitor, boosts human plasma drug concentrations of PIB by 3-fold.13 Each one-
gram Mavyret capsule contains 100 mg of GLE and 40 mg of PIB, as well as 860 mg of excipients
to help stabilize the drugs in the amorphous form, improving solubility and drug delivery. While
both GLE and PIB are large drug molecules with physicochemical properties that present
challenges for dosing, PIB presents the far greater challenge, as evidenced by the comparison of
properties in Figure 1. PIB has a much higher MW and LogD and is essentially insoluble in water
at neutral pH. PIB is also much more highly bound to human plasma proteins and has poor in
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vitro permeability, with no measurable passive diffusion in PAMPA and apparent permeability
(Papp) <1 X 10 6 cm/s in canine kidney epithelial (MDCK) cells.
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Figure 1. Physicochemical properties of glecaprevir and pibrentasvir.
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F
F
F
O
O
N
N
O
N
N
O
NH
O
O
F
F
O
O
S
N
N
O
N
HN
O
HN
NH
NH
HN
O
NH
O
O
N
N
O
O
O
F
O
F
F
F
Glecaprevir (GLE)
MW = 839
LogD = 2.5
Aq sol. (pH 7) = 0.3 mg/mL
Aq sol. (pH 2) < 0.1 mg/mL
Pibrentasvir (PIB)
MW = 1113
LogD = 5.7
Aq sol. (pH 7) < 0.0001 mg/mL
Aq sol. (pH 1) = 0.42 mg/mL
FeSSIF sol. (pH 5) = 0.17 mg/mL
PAMPA = 0.0 x 10-6 cm/s
MDCK < 1 x 10-6 cm/s
FeSSIF sol. (pH 5) = 0.04 mg/mL
PAMPA = 5.2 x 10-6 cm/s
MDCK = 1.4 x 10-6 cm/s
Human PPB = 97.5%
Human PPB > 99.9%
PIB is the largest drug molecule approved for oral administration since cyclosporine A
received FDA approval in 1983, and as such PIB presents unique and perhaps unprecedented
problems for achieving a convenient oral dosing formulation.14-16 Indeed, the poor solubility
properties of PIB necessitate a high excipient/drug ratio to provide good drug absorption, and
this is the primary reason that the G/P drug combination is delivered in a three-pill, once-daily
regimen. With the goal of providing a treatment option for HCV patients with a reduced pill
burden, we investigated solubilizing prodrugs of PIB that could enable a single pill G/P
formulation with bioequivalence to Mavyret. Our studies primarily focused on PIB prodrug
designs that employed a phosphate solubilizing group. Phosphates provide good water
solubility and chemical stability properties, and a variety of phosphate prodrugs are described
in the literature that are designed to rapidly bioconvert to release the parent drug by the action
of phosphatases that are abundant in the brush border of enterocytes.17 Phosphate prodrugs
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have been successfully applied in research to identify new antiretroviral agents that reduce pill
burden by improving dissolution of HIV protease inhibitors and lowering the excipient/drug
ratio.18-21
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Figure 2. Design strategies for solubilizing prodrugs of PIB.
F
O
HO
P
O
HO
O
R
O
O
N
N
R
O
unstable
F
F
A
F
F
N
N
N
N
C
N
N
N
H
H
B
O
O
N
O
O
O
O
O
O
PIB presents several unique chemistry challenges to identify soluble prodrugs that
efficiently deliver the drug molecule in vivo and have stability properties suitable for
development. Not the least of these is the absence of a hydroxyl or amine group on the drug
molecule to serve as a convenient handle for prodrug substitution. Available groups for
attaching a prodrug moiety include the benzimidazole nitrogen atoms and the carbamate
nitrogens at the dipeptide endcap termini. Success was highly dependent on identifying linkers
(promoieties) that would provide good hydrolytic stability while also being rapidly cleaved by
enzymes in the intestine to release PIB at the site of absorption. Suitable linkers were limited
to alkyl substituents due to the hydrolytic instability of acylimidazole and imide functionalities
(ie., carbamate linking groups). An oxymethyl linking group provided a viable strategy for
introducing solubilizing groups on the imidazole rings at positions A or B. This approach
provided prodrugs with good stability properties that efficiently deliver PIB following enzymatic
removal of the solubilizing group due to hydrolytic instability of the hydroxymethyl
intermediate. The simplest application of this strategy was to introduce a phosphomethyl
substituent at position A or B, and this was the initial focus of the medicinal chemistry team.
Alternatively, a solubilizing group could be introduced via an ester linkage on the oxymethyl
substituent at positions A or B. These strategies could not be applied to attach prodrugs at
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position C, since the hydroxymethyl intermediate was found to be hydrolytically stable, thus
undermining efficient drug release.
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Because the benzimidazole nitrogen atoms are not chemically equivalent, introducing
solubilizing groups at positions A and B resulted in the formation of mixtures of isomeric
prodrugs that had to be separated. Solubility and stability properties of the individual
benzimidazole-linked prodrug isomers were not equivalent. Finally, the C2-symmetrical
structure of PIB presented challenges in minimizing the production of bis-substituted analogs
having solubilizing groups on both benzimidazoles. A single solubilizing prodrug group was
preferred to avoid problems with efficient drug release that could result from having two
prodrug groups. While prodrugging position C would potentially simplify the chemoselectivity
problem (two identical carbamate groups), modifying the terminal carbamate presented
different synthetic challenges, and limited efforts will not be described here.
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Chemistry
Phosphomethyl prodrugs were synthesized by reacting PIB with sodium hydride in
anhydrous DMF at 0 oC, followed by the addition of 1.0 equivalents of di-tert-butyl
(chloromethyl)phosphate (Scheme 1). The crude alkylation product was reacted with HCl in
dioxane, and the product mixture was separated by C18 HPLC to give the mono-phosphomethyl
products 1 and 2 in >30% overall yield. Bis-phosphomethyl prodrugs were prepared in a similar
manner by reacting with 3 equivalents of the chloromethylphosphate alkylating reagent, and
were obtained in a 1:2:1 ratio of “up,up”, “up,down”, and “down,down” isomers by HPLC
purification.
Chloromethyl ester alkylating reagents were used to prepare prodrugs having oxymethyl
ester linkages to the benzimidazole group. The chemistry to install the prodrug could be
directed to a single benzimidazole group by blocking the other benzimidazole with a Boc
protecting group. Mono-Boc analog 3 (Scheme 1) could be obtained in approximately 50%
yield by reacting PIB with 0.8 equivalents of di-tert-butyl decarbonate and catalytic DMAP in
tetrahydrofuran, followed by separation of the product mixture by column chromatography on
silica gel. This process favored formation of the “down” Boc isomer depicted in the scheme,
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but both isomers were present in the product. Chloromethyl ester reagents, prepared from the
corresponding carboxylic acids by reaction with chloromethyl chlorosulfate under phase-
transfer conditions, were reacted with 3 to give the desired mono-alkylated benzimidazole
products as a mixture that favored the “up” isomer, usually by at least 3 to 1. This approach
allowed for the use of excess alkylating reagent to drive the reaction to completion. Removal
of the Boc protecting group with TFA gave prodrugs 4, which were separated from the minor
“down” isomer by HPLC.
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F
Scheme 1
Ar
F
N
F
OPO3H2
Ar
F
F
Ar
F
F
N
N
N
N
N
a
N
N
+
PIB
N
N
N
O
N
N
O
N
O
N
O
H
H
H
H
H
H
N
O
N
N
O
OPO3H2
O
N
O
O
O
O
O
O
1
2
O
O
O
b
R
O
O
Ar
F
F
Ar
F
F
HN
N
N
N
N
N
c, d
N
N
N
N
N
N
O
N
N
H
H
H
H
H
N
O
N
O
N
O
O
O
O
O
N
O
O
O
O
(major)
O
4
3
O
O
O
O
O
+ "up" isomer
+ minor "down" isomer
o
Reagent conditions: a.
b.
i. sodium hydride, di-tert-butyl (chloromethyl)phosphate, DMF, 0 C; ii. 4N HCl in dioxane;
c. R
d.
CO2CH2Cl, Cs2CO3, DMF; TFA, CH2Cl2.
di-tert-butyl dicarbonate, DMAP, THF;
An alternative approach to prodrugs 4 was to avoid Boc intermediate 3 and alkylate PIB
with the chloromethyl ester reagents. This approach required the use of less than 1 equivalent
of alkylating reagent to provide the desired mono-alkylated benzimidazole product as the major
reaction component. Purification of the final products required removal of minor bis-alkylated
products and unreacted PIB by HPLC. While the Boc-protection approach via intermediate 3
eliminated bis-alkylation products and simplified the final HPLC purification, the overall yield of
prodrug was similar due to the inefficient protection step (conversion of PIB to 3). This is
illustrated in Scheme 2, which shows a synthesis of prodrugs using both approaches. The tert-
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butyl phosphate derivative of methyl 4-hydroxy-phenylacetate was prepared using the 2-step
sequence of phosphite formation followed by peroxide oxidation. Ester hydrolysis was
followed by chloromethylation using phase-transfer conditions to give chloromethyl ester 5.
This reagent was used to alkylate Boc-protected PIB intermediate 3 using cesium carbonate as
the base to give a 51% yield of isomeric products that favored the “up” isomer (substitution at
position A in Figure 2). Removal of the t-butyl and Boc protecting groups using TFA in
dichloromethane, followed by HPLC purification to remove the minor “down” isomer provided
prodrug 6 in 31% overall yield (based on consumed 3). In another example, carboxylic acid 7,
prepared using the published procedure,22 was converted to the chloromethyl ester alkylating
reagent 8, which reacted with PIB using potassium tert-butoxide as the base to give a 39% yield
of mono-alkylated isomeric products that strongly favored the “up” isomer (position A in Figure
2). Debenzylation and HPLC purification provided prodrug 9 in 24% overall yield (based on
consumed PIB).
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H2O3PO
F
Scheme 2
Ar
F
N
F
O
O
N
Ar
F
F
O
Cl
O
O P
O
N
CO2Me
a, b
c, d
N
O
HO
O
N
N
N
O
N
O
H
H
H
31%
N
O
N
O
O
O
5
6
O
O
OPO3H2
F
Cl
O
O
O
CO2H
BnO
BnO
O
O
Ar
F
e, f
b
O
P
BnO
BnO
P
N
N
N
O
O
24%
N
N
8
N
N
7
H
H
H
N
O
N
O
O
O
O
9
O
O
O
Reagent conditions: a.
i. 1H-tetrazole, di-tert-butyl diisopropylphosphoramidite, DMA, ii. 50% aq. H2O2, iii. LiOH, aq. THF;
c. d.
b.
3
Cs2CO3, , DMF; TFA,
NaHCO3, chloromethyl chlorosulfate, tetrabutylammonium hydrogen sulfate, water, CH2Cl2;
e.
CH2Cl2;
potassium tert-butoxide, PIB, DMF; f. Pd(OH)2, H2, THF.
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Results and Discussion
Table 1. Properties of phosphomethyl PIB prodrugs.
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F
F
A
N
F
Ar F
A'
N
F
N
N
Ar =
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N
N
N
N
H
H
N
B
B'
N
O
O
O
O
O
O
O
O
Aq. Solubility
(µg/mL)
Prodrug structure
Stabilitya
Compd
ID
A
B
X
A’
X
B’
pH 1
422
pH 7.4
<0.1
pH 1
pH 7.4
PIB
1
H
H
H
H
>4000
37.3
>4000
6.4
100%
100%
100%
100%
100%
2
X
X
O
P
10
11
12
134.7
566.6
>1000
122.8
>1000
>1000
OH
O
OH
X
X
X
X
dog PK (40 mg p.o.)b
mouse PK (3 mg/kg p.o.)b
Compd
ID
PIB Prodrug
PIB Prodrug
Cmax
Cmax
Cmax
-
Cmax
AUC
2730
3860
1470
429
AUC
AUC
-
AUC
-
PIBc
PIB
1
239
339
152
55
-
-
-
-
-
280
17
5170
278
-
2.2
0
-
12.6
0
20
0
93
0
2
141
396
2660
6070
10
40
355
11
66
611
20
353
12
275
2580
28
477
a Percent prodrug remaining after 24 h at 37 oC in 0.1 N aq HCl (pH 1) or phosphate buffer (pH 7.4); b Doses
c
adjusted for MW of the prodrug to give an equivalent dose to PIB; 40 mg PIB phase 2 tablet; Vehicle = 1:99 (v/v)
DMSO:0.2% copovidone in FASSIF; Cmax (ng/mL); AUC (ng.h/mL).
Phosphomethyl prodrugs (Table 1) had good hydrolytic stability, with no degradation
observed by HPLC following 24-hour incubation at 37 oC in either 0.1 N aq HCl or neutral pH
phosphate buffer. Solubility and PK properties differed based on the position of the prodrug
substituent. Prodrug 1, which has the phosphomethyl substituent in position B, was much
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more soluble than prodrug 2, which is substituted in position A. The reason for the large
difference in solubility is not understood but may be due to a difference in structural
conformation resulting in a disparity in solvent access for the phosphate groups. The role
played by the benzo-ring substituents (fluorine and pyrrolidine) on the relative solubility of
these 2 regioisomeric prodrugs is not known. Solubility of the 3 isomeric bis-phosphomethyl
prodrugs 10, 11, and 12 was not improved in comparison to prodrug 1.
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The ability of phosphomethyl prodrugs to release the drug PIB was assessed in PK
studies in dog and mouse. Oral doses were adjusted for MW of the prodrug to deliver an
equivalent dose of PIB. Dog studies provided a comparison between dosing the prodrug and
dosing the 40 mg tablet (amorphous drug in a polymer support) that was used in Phase 2
clinical studies (crushed to facilitate dosing in dog). The more soluble prodrug 1 gave better
plasma drug concentrations in dog than prodrug 2, but plasma drug concentrations in mouse
were much better for prodrug 2. Low plasma concentrations of the prodrug were observed in
both dog and mouse for prodrug 1, but not for prodrug 2. The bis-phosphomethyl prodrugs
also showed species variability in drug delivery, with the unsymmetrically substituted prodrug
12 giving the best performance in dog and prodrug 10 giving the best performance in mouse.
All phosphomethyl prodrugs gave poor PK results in at least one species in comparison to
dosing with PIB. Very importantly, PK results in mouse were not predictive of results in dog,
indicating that screening prodrugs in mouse would not be a dependable strategy.
The observed variability and suboptimal performance in PK studies raised concerns that
simple phosphomethyl prodrug designs could be poor substrates for alkaline phosphatase
enzymes, resulting in inefficient drug release in the gut. Thus, alternative prodrug designs
having a variety of promoiety linking groups between the solubilizing group and the large drug
molecule were explored (Table 2). Carbamate-linked phosphate prodrug 13 showed good
solubility in neutral pH phosphate buffer, and much improved solubility in fed-state simulated
intestinal fluid (FESSIF) in comparison to PIB. Not surprisingly, the acylimidazole linkage
resulted in some loss in solubility at low pH. It was also not surprising that acyl-linked prodrug
13 had poor hydrolytic stability, with less than 20% prodrug remaining after 3 hours in neutral
phosphate buffer. The poor hydrolytic stability of prodrug 13 was also observed with a few
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other carbamate-linked designs that were investigated but not included in Table 2, and the
combined stability data for these prodrugs indicated that prodrug attachment to the
benzimidazole would require an alkyl linkage to provide hydrolytic stability suitable for
development.
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Valine ester prodrugs have been extensively applied in antiviral and anticancer drug
research due to their ability to improve absorption of nucleoside analogs through active uptake
by peptide transporters.23-25 Our investigation of valine ester prodrugs of PIB was based in part
on the possibility for solubility improvement by simple structural desymmetrization.
Unfortunately, valine ester prodrugs 14 and 15 had poor hydrolytic stability and did not
improve solubility in phosphate buffer in comparison to PIB. Although prodrug 14 provided
good plasma concentrations of PIB in PK studies, unwanted concentrations of the prodrug were
also observed in dog plasma. Bis-valine ester prodrugs having a valine ester substituent
attached to both benzimidazole groups were also investigated, but bis-valine ester prodrugs
were found to have solubility and hydrolytic stability properties that were no better than
mono-ester prodrugs 14 and 15.
Prodrugs with better hydrolytic stability were achieved by acylating the valine ester
beta-amino group. Phosphoglycolate 16, fumarate 17, and phosphomethylcarbamates 18 and
19, were stable at pH 1. However, the half-life in phosphate buffer at 37 oC was < 24 hours for
these valine ester-linked prodrugs. Phosphate prodrugs 16 and 18 had surprisingly poor
solubility at neutral pH, providing little or no improvement relative to PIB. The excellent
solubility for prodrug 19 in comparison to 18 again demonstrated the superior solubility
properties for prodrugs having a solubilizing substituent in position B on the benzimidazole ring.
Unfortunately, the chemistry to make these oxymethyl ester prodrugs was selective for the A-
substituent regioisomer (product ratio ~5:1 A:B under standard alkylation conditions using
cesium carbonate and the Boc-protected valine chloromethylester alkylating agent). Both
phosphomethylcarbamate prodrugs 18 and 19 gave high plasma concentrations of PIB in mouse
PK studies. However, 18 performed poorly in dog, and 19 was not tested in dog because of its
undesirable hydrolytic stability profile, as well as the inefficient synthetic route.
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Prodrugs having carboxylic acid solubilizing groups were also investigated, such as
benzoic acid prodrug 20, which had poor solubility properties. Prodrug 20 was tested as a
mixture of regioisomers due to difficulty isolating the individual prodrug isomers in pure form.
Mouse PK found that 20 provided low concentrations of PIB and significant concentrations of
the prodrug in plasma samples, indicating undesired absorption of carboxylic acid prodrugs
(also observed for fumaric acid prodrug 17). The phosphate-substituted phenylacetate prodrug
6 was more soluble than benzoic acid 20 in neutral pH phosphate buffer and provided good
plasma concentrations of PIB in the PK studies. Benzoate-linked prodrugs 21 and 22 had better
hydrolytic stability and improved solubility compared to phenylacetate-linked prodrug 6, but
prodrugs 21 and 22 did not perform well in PK studies.
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13
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16
17
18
19
20
21
22
23
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Table 2. Properties of ester-linked oxymethyl benzimidazole PIB prodrugs.
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8
9
10
11
12
13
14
Plasma PIB concentrationsb
Mouse Dog
AUC (PD) AUC (PD)
Prodrug structure
Solubility (µg/mL)
pH 1 pH 7.4 FeSSIF
Stabilitya
15
Compd
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
ID
A
B
X
pH 1
pH 7.4
Cmax
408
Cmax
339
PIB
13
H
422
23
<0.1
631
169
6720
456 (0)
2340 (0)c
3860
O
X
X
>1000
>1000
54%
78%
<1%
5%
26
196
379
2140 (0)
3940 (568)
O
OPO3H2
O
14
15
>1000
>1000
<0.1
<0.2
129c
NH2
O
O
O
H
N
16
17
X
186
203
0.7
97%
97%
31%
67%
216c
15
3710 (0)c
231 (354)
112
1210 (0)
O
O
OPO3H2
O
H
N
CO2H
X
X
<0.2
542
0
0 (0)
O
O
O
18
19
114
271
<0.1
>1000
>1000
95%
95%
38%
28%
498
513
8840 (0)
8930 (0)
16
122 (0)
H
N
O
OPO3H2
CO2H
O
X
X
>1000
O
40 20
41
X
232
<0.2
122
77%
59%
59
1100 (413)
O
42
OPO3H2
O
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
X
X
45
135
827
>1000
>1000
74%
96%
71%
905
96
16100 (0)
1510 (0)
155
0
1920 (0)
0 (0)
O
O
21
397
100%
OPO3H2
O
22
23
24
X
X
>1000
146
>1000
49
91%
82%
76%
100%
76%
80%
46
819 (0)
5570 (0)
12600 (0)
X
X
>1000
>1000
336
784
462
288
4510 (0)
2940 (0)
O
O
OPO3H2
OPO3H2
O
609
874
O
25
2
<0.2
>1000
15%
28%
584
9560 (0)
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OPO3H2
26
27
28
29
X
X
99
<0.2
92
>1000
>1000
>1000
>1000
99%
98%
92%
92%
89%
84%
92%
90%
622
132
380
567
10700 (0)
2420 (0)
6490 (0)
9750 (0)
115
297
1150 (0)
3020 (0)
O
O
X
X
543
>1000
>1000
>1000
>1000
O
N
O
O
N
OPO3H2
OPO3H2
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O
9
X
572
0.5
794
87%
100%
1210
20600 (0)
249
3210 (0)
a Percent prodrug remaining after 24 h at 37 oC in 0.1 N aq HCl (pH 1) or phosphate buffer (pH 7.4); b Prodrugs dosed p.o. in
dog (equiv. to 40 mg PIB) or mouse (equiv. to 6 mg/kg PIB) in 0.2% HPMC (doses adjusted for MW of the prodrug); c Prodrug
14 and 16 mouse dose equiv. to 3 mg/kg PIB; AUC (ng.h/mL); prodrug (PD) AUC in parenthesis.
A variety of self-immolative prodrugs were investigated that were designed to release
PIB through an intramolecular lactonization following dephosphorylation. A self-releasing
mechanism was expected to contribute to better PK performance across species, since drug-
release would not be dependent on esterase expression levels. In general, self-immolative
prodrugs that were investigated all provided good plasma PIB concentrations in both mouse
and dog PK studies. Phosphobutyrate prodrugs 23 and 24 provided further evidence for the
differences in solubility properties that favor prodrug substituents on position B of the
benzimidazole (Figure 2). Phenylacetate-linked ortho-phosphate prodrug 25 had poor
hydrolytic stability, whereas benzoate-linked ortho-phosphomethyl prodrugs 26 and 27 had
better stability and improved solubility. However, prodrug 26 had poor solubility at neutral pH,
and the more soluble isomer 27 provided only modest plasma concentrations of PIB in a mouse
PK study. Phosphomethylpyridyl carbamate prodrugs 28 and 29, which employ phosphate
versions of a prodrug design used in a marketed antifungal agent,26 had excellent solubility and
hydrolytic stability properties and performed well in PK studies. Unlike other prodrugs where
both benzimidazole regioisomers were tested, these pyridylcarbamate-linked prodrugs had
excellent solubility properties regardless of whether the solubilizing substituent was attached at
the A or B position on the benzimidazole ring.
The trimethyl-lock (TML) prodrug strategy has been extensively explored as an effective
method for increasing solubility and enhancing delivery of a wide variety of drug molecules.27
Following dephosphorylation, a rapid lactonization of the ortho-hydroxyphenyl-
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dimethylpropionate promoiety results in efficient drug release.22 TML prodrug 9 provided high
plasma concentrations of PIB in mouse and dog; 4-fold higher in mouse in comparison to dosing
with PIB alone. Prodrug 9 had excellent hydrolytic stability, with no significant degradation
observed after 24 hours at 37 oC in neutral pH buffer, making it one of the most stable ester-
linked prodrugs that we investigated. Solubility of 9 in neutral phosphate buffer was low, but
measurable (unlike PIB which has no measurable solubility above pH 2). The good solubility in
FESSIF for 9 would indicate adequate dissolution in the GI, which is consistent with the
excellent PK performance of this prodrug.
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Prodrugs were selected for PK studies in monkey based on overall properties of
solubility and stability, as well as the ability to efficiently deliver PIB in PK studies in mouse and
dog. Select prodrugs were dosed as a suspension in 0.5% HPMC (dose adjusted for MW of the
prodrug to deliver 2 mg/kg PIB equivalent), and multiple plasma samples were collected at
regular time intervals up to 12 hours post dosing to determine concentrations of both PIB and
the prodrug that was dosed. Prodrugs were not detected in any of the plasma samples, with
the exception of prodrug 2, which was present in low concentrations in samples collected up to
6 hours post dosing (AUC PIB/AUC 2 = 40). Several prodrugs tested in monkey provided equal
or better AUC/dose in comparison to dosing with PIB (Figure 3), including phosphomethyl
prodrug 2, valine ester prodrug 14, phosphophenylacetate prodrugs 6 and 25, and trimethyl-
lock prodrug 9. Disappointing results for pyridylcarbamate prodrug 28 were confirmed in a
repeat study. The most likely explanation for the poor performance of prodrugs 26 and 28 is
inefficient drug release by intestinal ALP in monkey, but we have no evidence to support this
theory and PK studies in monkey with similar prodrugs have not been reported.
Of the phosphate prodrugs that performed well in monkey, 2, 6, and 9 were selected as
the most promising prospects for development based on an overall profile of improved
solubility relative to PIB, hydrolytic stability properties deemed adequate to initiate salt-
selection studies (half-life > 24 h in stability assays), and efficient delivery of PIB in PK studies in
multiple preclinical species (Table 3).
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Figure 3. Monkey PK for PIB prodrugs.
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Table 3. Properties of leading PIB prodrug development candidates.
Property
PIB
< 0.1
7.6
2
6
9
Aq. Sol. mg/mL (pH 7.4)
6.5 [>65x PIB]
135
0.5 [>5x PIB]
Calc. LogD (pH 7.4)
FeSSIF Sol. g/mL (pH 5)
Stability (t1/2 @ pH 7.4)
3.8
--
5.5
7.5
169
>1000
794 [5x PIB]
> 3 days
> 3 days
> 1 day
16,100
--
Mouse AUC (ng.h/mL)
6,720
2,660*
429
20,600
3,210
240
PIB equiv. dose = 6 mg/kg
Dog AUC (ng.h/mL)
PIB equiv. dose = 40 mg
Monkey AUC (ng.h/mL)
PIB equiv. dose = 2 mg/kg
3,860
166
1,920
197
160
*dosed at 3 mg/kg PIB equiv. dose
Conclusion
A research program to identify solubilizing prodrugs of PIB identified three phosphate
analogs, each having important differences in properties as determined by their promoiety
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linking group. Not surprisingly, hydrolytic stability favored non-ester containing
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phosphomethyl prodrug 2, while prodrug efficiency determined by performance in PK studies
favored the ester-linked prodrugs, particularly TML prodrug 9. The relative PK performance,
which was not related to observed solubility differences, was likely due to the drug releasing
efficiency of the promoiety group. Differences in the rate of dephosphorylation by intestinal
alkaline phosphatase may account for the relative performance of prodrugs 2 and 9, while
prodrug 6 required esterase activity to release the drug.28 The observed differences in
solubility for PIB phosphate prodrugs are not well understood. Properties of the promoiety
matter, as evidenced by the poor solubility of the highly lipophilic TML prodrug 9, which has a
calculated LogD very similar to PIB. Phosphate solubilization for these prodrugs may be
affected by overall conformation in solution, which could be an explanation for the solubility
differences observed for isomeric prodrugs having substituents in positions A or B of the
benzimidazole. Solid forms of all prodrugs evaluated were amorphous, consistent with
chromatographic isolation. However, obtaining a crystalline solid form is necessary to assess
stability and hygroscopicity prior to initiating formulation studies. The three prodrugs identified
were advanced to salt selection experiments, with the goal of identifying a crystalline solid
form. Hydrolytic stability for prodrug 6 was not adequate to obtain a stable salt form,
prohibiting its advancement. Stable salt forms of 2 and 9 were identified and are being
investigated.
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PIB presented unique challenges to the identification of solution-stable prodrugs with
improved aqueous solubility. Nitrogen atoms on the two unsymmetrically substituted
benzimidazole groups provided the only convenient handle for installing the solubilizing group,
giving rise to a difficult regioselectivity problem for synthesis. Promoiety designs that provided
suitable stability and good drug-releasing properties focused largely on the oxymethyl ester
linking strategy, however a convenient synthetic method using chloromethyl ester alkylating
reagents was selective for forming the regioisomer that was less desirable from a solubility
perspective. It is possible that the lessons learned in our work to identify solubilizing prodrugs
of PIB may be applicable to the unique structural features of this drug and might not inform
prodrug studies with other drug molecules. Nevertheless, strategies implemented here may
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provide a useful guide for approaches worth considering with other large and lipophilic
compounds where solubility-limited exposure prohibits advancement or requires over-
extensive enabling formulation technology.
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Experimental
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General Procedures. Reagents and solvents, including anhydrous solvents, were
obtained from commercial sources and used as supplied. Column chomatography was carried
out on silica gel. 1H NMR spectra were measured using a Varian 400-MR (400 MHz) or a Varian
Inova 500 (500 MHz) spectrometer. Chemical shifts are reported in ppm (δ) and referenced to
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1
an internal standard of tetramethylsilane (δ 0.00 ppm). H – 1H couplings are assumed to be
first-order, and peak multiplicities are reported in the usual manner. MS analysis was
conducted using a Finnigan SSQ7000 (ESI) mass spectrometer. Prodrugs were determined to be
>95% pure by analytical HPLC using an Agilent 1260 LC System (HPLC conditions: Agilent
poroshell 120 EC-C18 2.7 um, 4.6 x 150 mm; 5 uL injection, 1.0 mL/min flow rate, 25 oC column;
A: 0.1% H3PO4; B: ACN; 5% B for 1 min, ramp to 100% B at 18 min, hold 1 min).
Methods used to determine solubility of compounds. For equilibrium solubility,
sufficient solid was added to 3–4 mL liquid medium in triplicate such that excess solid remained
undissolved. The mixtures were equilibrated for 24–48 hours at 25 rpm on an end-over-end
tumbler model in a constant temperature water bath (25 ºC). After equilibration, the mixtures
were inspected to assure that excess solid remained. If not, additional solid was added, and the
mixtures were again equilibrated for 24–48 hours. The pH of aqueous suspensions was
determined after equilibration. The solid phase was separated from the liquid phase by
centrifugation at 4000 rpm at equilibration temperature 25 ºC. The supernatant was then
filtered through a 0.45 µm GHP membrane filter to discard the initial 2 mL (to saturate filter)
and capture the last 1 mL for analysis. The filtrate was diluted as needed for quantification and
injected to HPLC. The concentration was evaluated by linear regression against known
concentration standards.
Methods used to determine hydrolytic stability of compounds. A stock solution of
prodrug was prepared in DMSO (~1.25mg/mL). This DMSO stock was diluted into 0.1N HCl (pH
1) and 50 mM phosphate buffer (pH 7.4) for a final prodrug concentration of ~125ug/mL and
DMSO concentration of 10%. The solution was filtered through a 0.45 µm GHP membrane filter
and placed into an HPLC vial. The vial was inserted into a heated HPLC autosampler (37 oC) and
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injections were made at multiple time points over the course of 24hrs. Percent recovery based
on prodrug peak area was calculated relative to the time 0 injection.
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8
9
Methods for Pharmacokinetic evaluation of compounds. CD-1 mice were obtained
from Charles River Laboratories (Wilmington, MA). Beagle dogs (male/female) were obtained
from Marshall Farms (North Rose, NY). Cynomolgus monkeys (female) were obtained from
Covance (Denver, PA). Mice were allowed free access to food and water; dogs and monkeys
were fasted overnight prior to dosing with free access to water. Food was returned to dogs 12
hours after dosing or 4 hours after drug administration in monkeys. Compounds were dosed as
follows: in mouse by oral gavage as a solution or suspension in 0.2% HPMC at 10 mL/kg; in dog
in a gelatin capsule as a solution or suspension in 0.2% HPMC at 0.5 mL/kg; in monkey by
gastric intubation as a solution or suspension in 0.5% HPMC at 0.5 mL/kg. Blood samples,
collected into K2EDTA anticoagulant for plasma concentration analysis, were obtained from
each animal after dosing. Serial samples were obtained at 0.25, 0.5, 1.0, 3, 6, 9, 12 and 24
hours post dosing in mice and at 0.25, 0.5, 1.0, 1.5, 2, 3, 4, 6, 9, 12 and 24 hours post dosing in
dogs and monkeys. Plasma was separated by centrifugation (3200 x g, 5 minutes, ~4°C) and
stored frozen (<-15°C) until analysis.
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All animal studies were approved by the AbbVie Institutional Animal Care and Use
Committee (IACUC) and conducted in an AAALAC accredited facility to ensure high standards of
animal care and use.
Methyl ((2S,3R)-1-((S)-2-(6-((2R,5R)-1-(3,5-difluoro-4-(4-(4-fluorophenyl)piperidin-1-
yl)phenyl)-5-(5-fluoro-2-((S)-1-(N-(methoxycarbonyl)-O-methyl-L-threonyl)pyrrolidin-2-yl)-1H-
benzo[d]imidazol-6-yl)pyrrolidin-2-yl)-5-fluoro-1-((phosphonooxy)methyl)-1H-
benzo[d]imidazol-2-yl)pyrrolidin-1-yl)-3-methoxy-1-oxobutan-2-yl)carbamate (1). To a
solution of dimethyl ((2S,2'S,3R,3'R)-((2S,2'S)-2,2'-(6,6'-((2R,5R)-1-(3,5-difluoro-4-(4-(4-
fluorophenyl)piperidin-1-yl)phenyl)pyrrolidine-2,5-diyl)bis(5-fluoro-1H-benzo[d]imidazole-6,2-
diyl))bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-2,1-diyl))dicarbamate (PIB, 1.0 g, 0.90
mmol) in anhydrous N,N-dimethylformamide at 0 °C was added sodium hydride (60%
suspension in mineral oil, 54 mg, 1.35 mmol), and the resulting mixture was stirred at 0°C for 30
minutes. Di-tert-butyl (chloromethyl) phosphate (0.23 g, 0.90 mmol) was added, and the
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mixture was allowed to warm to ambient temperature and stirred for 16 hours. The mixture
was partitioned between 0.5 N aqueous HCl and ethyl acetate, and the organic layer was dried
over Na2SO4, filtered and concentrated in vacu. The crude product was dissolved in
dichloromethane (10 mL), and the solution was cooled in an ice bath while trifluoroacetic acid
(3 mL) was added dropwise. The resulting solution was allowed to warm to ambient
temperature and stirred for 3 hours. The solution was concentrated in vacuo, and the crude
product mixture was purified by C18 HPLC using a solvent gradient of 5-100% acetonitrile in
0.1% TFA aqueous buffer. Bis-phosphomethyl products eluted at 40-50% acetonitrile, followed
by 1, which was the first mono-alkyl product to elute. Lyophilization provided 1 as a colorless
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solid (0.22 g, 0.18 mmol, 20% yield): H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 10.2 Hz, 1H),
7.51 – 7.40 (m, 2H), 7.28 – 6.98 (m, 6H), 6.15 – 5.99 (m, 2H), 5.91 – 5.82 (m, 2H), 5.66 (d, J = 7.9
Hz, 1H), 5.55 (d, J = 8.3 Hz, 1H), 5.31 (dd, J = 7.3, 4.9 Hz, 1H), 5.13 (dd, J = 8.1, 4.8 Hz, 1H), 4.28 –
4.20 (m, 2H), 3.97 – 3.75 (m, 4H), 3.51 (s, 3H), 3.50 (s, 3H), 3.37 – 2.82 (m, 10H), 2.75 – 1.49 (m,
21H), 1.05 – 0.88 (m, 6H); MS (ESI) m/z 1223.3 [M+H]+.
Methyl ((2S,3R)-1-((S)-2-(5-((2R,5R)-1-(3,5-difluoro-4-(4-(4-fluorophenyl)piperidin-1-
yl)phenyl)-5-(5-fluoro-2-((S)-1-(N-(methoxycarbonyl)-O-methyl-L-threonyl)pyrrolidin-2-yl)-1H-
benzo[d]imidazol-6-yl)pyrrolidin-2-yl)-6-fluoro-1-((phosphonooxy)methyl)-1H-
benzo[d]imidazol-2-yl)pyrrolidin-1-yl)-3-methoxy-1-oxobutan-2-yl)carbamate (2). Compound
2 was prepared using the methods described for synthesis of 1. It was the second of 2 mono-
alkylated products to elute during the HPLC purification step, and was obtained as a colorless
1
solid by lyophilization (0.16 g, 0.13 mmol, 14% yield): H NMR (501 MHz, DMSO-d6) δ 7.67 –
7.53 (m, 2H), 7.28 – 6.96 (m, 7H), 6.25 (t, J = 10.7 Hz, 1H), 6.02 (t, J = 11.0 Hz, 1H), 5.91 – 5.81
(m, 2H), 5.67 – 5.53 (m, 2H), 5.34 – 5.28 (m, 1H), 5.14 (dd, J = 8.2, 4.8 Hz, 1H), 4.26 (dd, J = 8.3,
6.1 Hz, 1H), 4.18 (dd, J = 8.2, 5.9 Hz, 1H), 3.88 – 3.70 (m, 4H), 3.52 (s, 3H), 3.50 (s, 3H), 3.34 –
2.79 (m, 10H), 2.71 – 1.52 (m, 21H), 1.08 – 0.69 (m, 6H); MS (ESI) m/z 1223.2 [M+H]+.
Methyl 2-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)acetate (30). To a stirring solution
of methyl 2-(4-hydroxyphenyl)acetate (2.49 g, 15.0 mmol) and 1H-tetrazole (83 mL, 37.5 mmol)
in N,N-dimethylacetamide (30.0 mL) was added di-tert-butyl diisopropylphosphoramidite (5.92
ml, 18.8 mmol), and the resulting mixture was stirred at ambient temperature for 4 hours. The
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mixture was then cooled to 0 °C, a solution of 50% aqueous hydrogen peroxide (2.30 ml, 37
mmol) was added, and the mixture was allowed to warm to ambient temperature and stirred
for 2 hours. The mixture was partitioned between saturated aqueous Na2CO3 and ethyl acetate
(3x), and the combined organic layers were dried over Na2SO4, filtered, and concentrated in
vacuo. The crude product was purified by column chromatography on silica gel, eluting with a
solvent gradient of 0-100% ethyl acetate in heptanes. Compound 30 was obtained as a
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colorless oil (5.2 g, 97%): H NMR (501 MHz, DMSO-d6) δ 7.31 – 7.22 (m, 4H), 7.15 – 7.06 (m,
2H), 3.66 (s, 2H), 3.61 (s, 3H), 1.44 (s, 18H).
Chloromethyl 2-(4-((di-tert-butoxyphosphoryl)oxy)phenyl)acetate (5). A solution of 30
(5.2 g, 14.51 mmol) in water (60.5 mL) and tetrahydrofuran (151 mL) was cooled to 0 °C, and a
solution of aqueous lithium hydroxide (0.3 M, 145 mL, 43.5 mmol) was added dropwise. The
reaction was stirred at 0 °C for 60 min, and 1.0 M aqueous HCl was added to acidify to pH 3.0.
The mixture was extracted with ethyl acetate, and the organic layer was washed with water and
brine. The solution was dried over Na2SO4, filtered, and concentrated in vacuo to give a
colorless oil, which was dissolved in dichloromethane (24 mL). To the solution was added water
(24 mL), NaHCO3 (4.78 g, 56.9 mmol) and tetrabutylammonium hydrogen sulfate (0.48 g, 1.42
mmol), and the resulting mixture was cooled to 0 °C and vigorously stirred while chloromethyl
chlorosulfate (2.59 ml, 28.5 mmol) was added dropwise. The stirred reaction mixture was
allowed to slowly warm to ambient temperature over 3 hours. The layers were separated, and
the organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude
product was purified by column chromatography on silica gel using a solvent gradient of 0-
100% ethyl acetate in heptanes. Compound 5 was obtained as a colorless oil (2.53 g, 6.44
1
mmol, 45% yield): H NMR (400 MHz, CDCL3) δ 7.25 – 7.16 (m, 4H), 5.70 (s, 2H), 3.66 (s, 2H),
1.51 (s, 18H).
Tert-butyl 6-((2R,5R)-1-(3,5-difluoro-4-(4-(4-fluorophenyl)piperidin-1-yl)phenyl)-5-(6-
fluoro-2-((S)-1-((2S,3R)-3-methoxy-2-((methoxycarbonyl)amino)butanoyl)pyrrolidin-2-yl)-1H-
benzo[d]imidazol-5-yl)pyrrolidin-2-yl)-5-fluoro-2-((S)-1-((2S,3R)-3-methoxy-2-
((methoxycarbonyl)amino)butanoyl)pyrrolidin-2-yl)-1H-benzo[d]imidazole-1-carboxylate (3).
To a stirred solution of dimethyl ((2S,2'S,3R,3'R)-((2S,2'S)-2,2'-(5,5'-((2R,5R)-1-(3,5-difluoro-4-(4-
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(4-fluorophenyl)piperidin-1-yl)phenyl)pyrrolidine-2,5-diyl)bis(6-fluoro-1H-benzo[d]imidazole-
5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-2,1-diyl))dicarbamate (PIB, 14.8 g,
13.3 mmol) in anhydrous tetrahydrofuran (111 mL) was added a solution of di-tert-butyl
dicarbonate (2.18 g, 10.0 mmol) and 4-dimethylaminopyridine (0.12 g, 1.0 mmol) in anhydrous
tetrahydrofuran (111 mL), dropwise over 60 minutes. The reaction mixture was poured into
water (250 mL) and extracted with ethyl acetate (2 x 200 mL), and the combined organic layers
were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude
product was purified by column chromatography on silica gel, eluting with a solvent gradient of
0-10% methanol in ethyl acetate. Compound 3, which is a mixture of mono-Boc benzimidazole
regioisomers, was obtained as an off-white solid (6.49 g, 54% yield).
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(5-((2R,5R)-1-(3,5-Difluoro-4-(4-(4-fluorophenyl)piperidin-1-yl)phenyl)-5-(5-fluoro-2-
((S)-1-((2S,3R)-3-methoxy-2-((methoxycarbonyl)amino)butanoyl)pyrrolidin-2-yl)-1H-
benzo[d]imidazol-6-yl)pyrrolidin-2-yl)-6-fluoro-2-((S)-1-((2S,3R)-3-methoxy-2-
((methoxycarbonyl)amino)butanoyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl 2-(4-
(phosphonooxy)phenyl)acetate (6). To a solution of 3 (2.0 g, 1.65 mmol) in anhydrous N,N-
dimethylformamide (16.5 mL) was added cesium carbonate (0.81 g, 2.47 mmol), and the
resulting mixture was stirred at ambient temperature for 10 minutes. Compound 5 (1.30 g,
3.30 mmol) was added, and the reaction mixture was stirred at ambient temperature for 90
minutes. The reaction was quenched by the addition of saturated aqueous NH4Cl, and the
mixture was extracted with methyl tert-butyl ether (3 x 100 ml). The combined organic layers
were washed with water (2 x 100 mL) and brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The crude product was purified by column chromatography on silica gel using a
solvent gradient of 50-100% ethyl acetate in heptanes to give a mixture of mono-alkylated
benzimidazole regioisomeric products as a colorless solid (1.34 g, 51%). A portion of this solid
(0.35 g, 0.22 mmol) was dissolved in dichloromethane (3.72 mL) and the solution was cooled to
0 °C. Trifluoroacetic acid (0.74 mL) was added, and the solution was allowed to warm to
ambient temperature and stirred for 1 hour. The solution was concentrated in vacuo, and the
residue was dissolved in dichloromethane (5 ml) and concentrated in vacuo (2x). The crude
product was purified by C18 preparative HPLC, eluting with a solvent gradient of 20-95%
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acetonitrile in 0.1% aqueous TFA buffer. Compound 6 was isolated as a colorless solid by
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lyophilization (0.18 g, 0.13 mmol, 60% yield): H NMR (400 MHz, DMSO-d6) δ 7.67 (t, J = 10.1
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9
Hz, 2H), 7.33 – 7.20 (m, 5H), 7.16 (d, J = 6.8 Hz, 1H), 7.14 – 7.04 (m, 5H), 6.99 (d, J = 8.3 Hz, 1H),
6.39 (s, 2H), 5.91 (s, 1H), 5.88 (s, 1H), 5.70 – 5.64 (m, 1H), 5.58 (d, J = 6.1 Hz, 1H), 5.24 (dd, J =
7.6, 4.4 Hz, 1H), 5.18 (dd, J = 8.2, 5.1 Hz, 1H), 4.28 (dd, J = 8.5, 5.8 Hz, 5H), 4.20 (dd, J = 8.2, 6.0
Hz, 2H), 3.83 (m, 4H), 3.73 (m, 2H), 3.52 (d, J = 8.0 Hz, 6H), 3.42 – 3.37 (m, 1H), 3.32 (p, J = 5.7
Hz, 2H), 3.07 – 2.93 (m, 6H), 2.87 (m, 4H), 2.56 (m, 1H), 2.47 (m, 2H), 2.39 – 1.93 (m, 6H), 1.83
(d, J = 9.6 Hz, 2H), 1.74 – 1.57 (m, 4H), 0.95 (d, J = 6.2 Hz, 3H), 0.77 (d, J = 6.2 Hz, 3H); MS (ESI)
m/z 1357.3 [M+H]+.
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Chloromethyl 3-(2-((bis(benzyloxy)phosphoryl)oxy)-4,6-dimethylphenyl)-3-
methylbutanoate (8). A solution of 3-(2-((bis(benzyloxy)phosphoryl)oxy)-4,6-dimethylphenyl)-
3-methylbutanoic acid (7, 2.05 g, 4.25 mmol), prepared according to reference 22, in
dichloromethane (20 mL) was stirred at ambient temperature. Sodium bicarbonate (1.43 g,
16.99 mmol), tetrabutylammonium hydrogen sulfate (0.14 g, 0.43 mmol), and water (20 mL)
were added. Chloromethyl chlorosulfate (0.513 ml, 5.10 mmol) was added dropwise, and the
mixture was heated to 30-35 oC. After 15 minutes, the layers were separated, and the organic
layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was
purified by column chromatography on silica gel using a solvent gradient of 20-60% methyl tert-
butyl ether in heptanes. Compound 8 was obtained as an oil (2.02 g, 3.80 mmol, 90 % yield):
1H NMR (501 MHz, CDCl3) δ 7.36 – 7.27 (m, 10H), 7.09 (brs, 1H), 6.72 (brs, 1H), 5.45 (s, 2H), 5.15
– 5.07 (m, 4H), 2.95 (s, 2H), 2.52 (s, 3H), 2.16 (s, 3H), 1.58 (s, 6H).
(5-((2R,5R)-1-(3,5-Difluoro-4-(4-(4-fluorophenyl)piperidin-1-yl)phenyl)-5-(6-fluoro-2-
((S)-1-((2S,3R)-3-methoxy-2-((methoxycarbonyl)amino)butanoyl)pyrrolidin-2-yl)-1H-
benzo[d]imidazol-5-yl)pyrrolidin-2-yl)-6-fluoro-2-((S)-1-((2S,3R)-3-methoxy-2-
((methoxycarbonyl)amino)butanoyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl 3-(2-
((bis(benzyloxy)phosphoryl)oxy)-4,6-dimethylphenyl)-3-methylbutanoate (31). A solution of
dimethyl ((2S,2'S,3R,3'R)-((2S,2'S)-2,2'-(5,5'-((2R,5R)-1-(3,5-difluoro-4-(4-(4-
fluorophenyl)piperidin-1-yl)phenyl)pyrrolidine-2,5-diyl)bis(6-fluoro-1H-benzo[d]imidazole-5,2-
diyl))bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-2,1-diyl))dicarbamate (PIB, 8.43 g,
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7.57 mmol) in anhydrous N,N-dimethylformamide (42 mL) was cooled to 10 oC and potassium
tert-butoxide (1 M in tetrahydrofuran, 6.81 mL, 6.81 mmol) was added dropwise. After 5
minutes with cooling to 5 oC, a solution of 8 (2.01 g, 3.79 mmol) in N,N-dimethylformamide (1
ml) was added, and the mixture was allowed to warm to ambient temperature and stirred for
14 hours. To the reaction mixture was added aqueous 1 N HCl (45 mL) and ethyl acetate (90
mL), and the mixture was stirred for 15 min. The layers were separated, and the aqueous layer
was extracted with ethyl acetate (45 mL). The combined organic layers were washed with
aqueous 1N HCl (18 mL), brine (18 mL), saturated aqueous NaHCO3 (18 mL), and brine (18 mL).
The organic layer was dried over Na2SO4, filtered and concentrated, and the crude product was
purified by column chromatography on silica gel using a solvent gradient of 50-100% 19:1 ethyl
acetate:isopropanol in heptanes. Compound 31 was obtained as a colorless foam (2.37 g, 1.47
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mmol, 39 % yield): H NMR (501 MHz, DMSO-d6) δ 7.43 – 7.02 (m, 19H), 6.94 (br s, 1H), 6.78
(brs, 1H), 6.44 (br s, 1H), 6.30 (d, J = 11.7 Hz, 1H), 6.14 (d, J = 11.7 Hz, 1H), 5.84 (br s, 2H), 5.65-
5.45 (m, 2H), 5.18-5.09 (m, 2H), 5.06 (s, 2H), 5.05 (s, 2H), 4.26 (br s, 1H), 4.17 (t, J = 7.2 Hz, 1H),
3.81 (br s, 2 H), 3.74-3.65 (m, 1H), 2.81 (s, 3H), 3.51 (s, 3H), 3.52-2.77 (m, 18H), 2.57-2.32 (m,
5H), 2.25 (s, 3H), 2.03 (s, 3H), 1.47 (s, 3H), 1.44 (s, 3H), 2.27-1.38 (m, 9H), 1.08-0.88 (m, 4H),
0.73 (d, 3H); MS (ESI) m/z 1608 [M+H]+.
(5-((2R,5R)-1-(3,5-difluoro-4-(4-(4-fluorophenyl)piperidin-1-yl)phenyl)-5-(6-fluoro-2-
((S)-1-((2S,3R)-3-methoxy-2-((methoxycarbonyl)amino)butanoyl)pyrrolidin-2-yl)-1H-
benzo[d]imidazol-5-yl)pyrrolidin-2-yl)-6-fluoro-2-((S)-1-((2S,3R)-3-methoxy-2-
((methoxycarbonyl)amino)butanoyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl 3-(2,4-
dimethyl-6-(phosphonooxy)phenyl)-3-methylbutanoate (9). To a solution of 31 (1.59 g, 0.99
mmol) in tetrahydrofuran (11 mL) was added to 20% Pd(OH)2/C, wet (163 mg, 0.59 mmol) in a
25 ml SS reactor. The reactor was purged with argon and the mixture was stirred under 50 psi
of hydrogen at 25 °C. After 16 hours, the mixture was filtered through a Celite packed column
and the catalyst was washed thoroughly with methanol. Acetonitrile (80 mL) was added, and
the mixture was sonicated and then stirred vigorously for 10 minutes to give the crude product
as a colorless solid. The crude product was purified by C18 preparative HPLC using a solvent
gradient of 40-95% acetonitrile in 0.1% aqueous TFA buffer. The clean fractions were pooled
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and concentrated in vacuo to ~10 mL to give a colorless slurry. The solid product was collected
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by filtration, washed with water, and dried under vacuum to give 9 as a colorless solid (0.86 g,
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0.60 mmol, 61% yield): H NMR (400 MHz, DMSO-d6) δ 7.39 (dd, J = 10.7, 7.8 Hz, 2H), 7.22 (dd,
J = 8.7, 5.5 Hz, 2H), 7.10 (dd, J = 7.2, 3.4 Hz, 2H), 7.03 (t, J = 8.8 Hz, 2H), 6.95 (s, 1H), 6.92 (d, J =
8.8 Hz, 1H), 6.29 (s, 1H), 6.25 (d, J = 11.6 Hz, 1H), 6.13 (d, J = 11.8 Hz, 1H), 5.82 (brd, J = 13.0 Hz,
2H), 5.55 (dd, J = 20.7, 6.0 Hz, 2H), 5.11 (ddd, J = 18.6, 7.6, 4.4 Hz, 2H), 4.22 (t, J = 7.4 Hz, 1H),
4.14 (dd, J = 8.3, 5.8 Hz, 1H), 3.85-2.73 (m, 21H), 3.04 (s, 3H), 2.94 (s, 2H), 2.79 (s, 2H), 2.31-1.69
(m, 10H), 2.18 (s, 4H), 2.05 (s, 3H), 1.69-1.50 (m, 4H), 1.46 (s, 3H), 1.42 (s, 3H), 1.04-0.89 (m,
3H), 0.70 (d, J = 6.2 Hz, 3H); MS (ESI) m/z 1428 [M+H]+.
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ASSOCIATED CONTENT
Supporting Information
Synthetic methods and analytical data for compounds 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29; analytical HPLC for 2, 6, and 9; molecular formula strings. This
material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*John T. Randolph - Phone: +1 847-937-7182. E-mail: john.randolph@abbvie.com.
ABBREVIATIONS USED:
HCV, hepatitis C virus; PIB, pibrentasvir; GLE, glecaprevir; GT, genotype; DAA, direct-acting
antiviral; SVR, sustained virologic response; NS, nonstructural; FESSIF, fed-state simulated
intestinal fluid; PK, pharmacokinetics; PAMPA, parallel artificial membrane permeability assay;
MDCK, Madin-Darby canine kidney, TFA, trifluoroacetic acid; DMF, N,N-dimethylformamide;
DMAP, 4-dimethylaminopyridine; Boc, tert-butoxy carbonyl.
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Disclosure: All authors are employees of AbbVie. The design, study conduct, and financial
support for this research were provided by AbbVie and Enanta. AbbVie and Enanta participated
in the interpretation of data, review, and approval of the publication.
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References
1. Lamb, Y. N. Glecaprevir/Pibrentasvir: First Global Approval Drugs 2017, 77, 1797-1804.
2. Forns, X.; Lee, S. S.; Valdes J.; Lens, S.; Ghalib, R.; Aguilar, H.; Felizarta, F.; Hassanein, T.;
Hinrichsen, H.; Rincon, D.; Morillas, R.; Zeuzem, S.; Horsmans, Y.; Nelson, D. R.; Yu, Y.;
Krishnan, P.; Lin C.-W.; Kort, J. J.; Mensa, F. J. Glecaprevir Plus pibrentasvir for Chronic
Hepatitis C Virus Genotype 1, 2, 4, 5, or 6 Infection in Adults with Compensated Cirrhosis
(EXPEDITION-1): A Single-Arm, Open-Label, Multicenter Phase 3 Trial Lancet 2017, 17, 1062-
1068.
3. Puoti, M.; Foster, G. R.; Wang, S.; Mutimer, D.; Gane, E.; Moreno, C.; Chang, T. T.; Lee, S.
S.; Marinho, R.; Dufour, J.-F.; Pol, S.; Hezode, C.; Gordon, S. C.; Strasser, S. I.; Thuluvath, R.
J.; Zhang, Z.; Lovell, S.; Pilot-Matias, T.; Mensa, F. J. High SVR12 with 8-Week and 12-Week
Glecaprevir/Pibrentasvir Therapy: An Integrated Analysis of HCV Genotype 1-6 Patients
without Cirrhosis J Hepatol. 2018, 69, 293-300.
4. Brown, R. S. Jr; Buti, M.; Rodrigues, L.; Chulanov, V.; Chuang, W.-L.; Aguilar, H.; Horvath,
G.; Zuckerman, E.; Carrion, B. R.; Rodriguez-Perez, F.; Urbanek, P.; Abergel, A.; Cohen, E.;
Lovell, S. S.; Schnell, G.; Lin, C.-W.; Zha, J.; Wang, S.; Trinh, R.; Mensa, F. J.; Burroughs, M.;
Felizarta, F. Glecaprevir/Pibrentasvir for 8 Weeks in Treatment-Naïve Patients with Chronic
HCV Genotypes 1-6 and Compensated Cirrhosis: The EXPEDITION-8 Trial J. Hepatol. 2020, 72,
441-449.
5. Mensa, F. J.; Lovell, S.; Pilot-Matias, T.; Liu, W. Glecaprevir/Pibrentasvir for the Treatment
of Chronic Hepatitis C Virus Infection Future Microbiol. 2018, 14, 89-110.
6. Huff, J.; Andersen, R. Glecaprevir/Pibrentasvir: The First 8-Week Pangenotypic HCV
Treatment Regimen for Patients 12 Years of Age and Older Annals of Pharmacother. 2020, 54,
262-276.
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7. Wagner, R.; Randolph, J. T.; Patel, S. V.; Nelson, L.; Matulenko, M. A.; Keddy, R.; Pratt, J.
K.; Liu, D.; Krueger, A. C.; Donner, P. L.; Hutchinson, D.; Flentge, C.; Betebenner, D.;
Rockway, T.; Maring, C. J.; Ng, T.; Krishnan, P.; Pilot-Matias, T.; Panchel, N.; Reisch, T.;
Dekhtyar, T.; Mondal, R.; Stolarik, D. F.; Gao, Y.; Gao, W.; Beno, D. W. A.; Kati, W. M.
Highlights of the Structure-Activity Relationships of Benzimidazole Linked Pyrrolidines Leading
to the Discovery of the Hepatitis C Virus NS5A Inhibitor Pibrentasvir (ABT-530) J. Med. Chem.
2018, 61, 4052-4066.
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