Bioorganic & Medicinal Chemistry Letters 25 (2015) 210–215

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

A prodrug strategy for the oral delivery of a poorly soluble HCV NS5B
thumb pocket 1 polymerase inhibitor using self-emulsifying drug
delivery systems (SEDDS)
Pierre L. Beaulieu a,⇑, Josie De Marte b, Michel Garneau b, Laibin Luo b, Timothy Stammers a, Chitra Telang c,
Dominik Wernic a, George Kukolj b, Jianmin Duan b
a
Medicinal Chemistry Department, Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
b
Biology Department, Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
c
Boehringer Ingelhein Pharmaceuticals Inc., 175 Briar Ridge Road, Ridgefield, CT 06877, United States

a r t i c l e i n f o a b s t r a c t

Article history: A prodrug approach was developed to address the low oral bioavailability of a poorly soluble (<0.1 lg/mL
Received 8 October 2014 in pH 6.8 buffer) but highly permeable thumb pocket 1 HCV NS5B polymerase inhibitor. Bioconversion
Revised 23 November 2014 rates of structurally diverse prodrug derivatives were evaluated in a panel of in vitro assays using
Accepted 26 November 2014
microsomes, from either liver or intestinal tissues, simulated intestinal fluids, simulated gastric fluids
Available online 4 December 2014
or plasma. In vivo bioconversion of promising candidates was evaluated following oral administration
to rats. The most successful strategy involved modification of the parent drug carboxylic acid moiety
Keywords:
to glycolic amide esters which improved solubility in lipid-based self-emulsifying drug delivery systems
Hepatitis C virus
Prodrug
(SEDDS). Crystalline prodrug analog 36 (mp 161 °C) showed good solubility in individual SEDDS compo-
NS5B polymerase nents (up to 80 mg/mL) compared to parent 2 (<3 mg/mL; mp 267 °C) and cross-species bioconversions
Antiviral which correlated with in vitro stability in liver microsomes.
Inhibitor Ó 2014 Elsevier Ltd. All rights reserved.

Considerable progress has been realized in the last twenty years
toward the treatment of chronic hepatitis C virus (HCV) infection.
From initial 20% sustained viral response rates (SVR) for HCV
genotype 1 (GT1; the predominant HCV variant in North America,
Europe and Japan) using interferon-a (IFN) immunotherapy, a com-
bination of pegylated-IFN and a broad spectrum antiviral agent,
ribavirin (RBV) rapidly became the standard of care (SoC), providing
improved response rates (45% SVR) in the GT1 patient popula-
tion.1 However, severe side-effects and contraindications associ-
ated with IFN/RBV-based regimens have stimulated the search for
Direct Acting Antivirals (DAAs) with the potential to enhance effi-
cacy and tolerability. Recently, a second generation DAA targeting
the virally encoded NS3/4A protease and a nucleotide analog inhib-
itor of the NS5B polymerase were approved for use in combination
with SoC and are providing patients with shorter treatment
durations and improved tolerance (SVR from 80 to >90% in GT1).2
Despite these advances, the new regimens still necessitate
co-treatment with PegIFN/RBV or other DAAs to prevent emergence
of DAA-resistant virus. Consequently, recent drug-discovery efforts
have focused on the development of IFN-free regimens, combining
⇑ Corresponding author. Tel.: +1 (450) 682 4640; fax: +1 (450) 682 8434.
E-mail address: resgeneral.lav@boehringer-ingelheim.com (P.L. Beaulieu).
DAAs with complementary modes of actions with the potential to
further improve cure rates (SVR >90% in GT1) and tolerability.3
The U.S. FDA recently approved Gilead’s HarvoniÒ (sofosbuvir
NS5B nucleotide/ledipasvir NS5A combination), the first once-daily
single tablet regimen for the treatment (SVR12 rates of 94–99%) of
GT1 chronic HCV infection.3h Toward a similar end, we have been
pursuing allosteric inhibitors of the HCV NS5B polymerase as part-
ners in IFN-free combination therapy with the protease inhibitor
faldaprevir.3a,b
The HCV NS5B RNA-dependent RNA polymerase is a virally-
encoded enzyme that does not have a mammalian equivalent and
is therefore a target of choice for the development of antiviral
agents devoid of mechanism-based toxicity. This enzyme plays a
crucial role in the virus life cycle and is responsible for the synthesis
and replication of HCV RNA.4 NS5B is susceptible to inhibition by
nucleotide analogs that act as nonobligate RNA chain-terminators.5
In addition, the enzyme is sensitive to small molecules that bind to
one of four distinct allosteric sites and interfere with conforma-
tional changes that are necessary for function.6
We have described over the last years the discovery and pro-
gression of a class of benzimidazole and indole-based NS5B inhib-
itors that bind to the thumb pocket 1 allosteric site and prevent
inter-domain interactions between the thumb and finger regions

http://dx.doi.org/10.1016/j.bmcl.2014.11.071
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
 P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 210–215 211

O
H
N N
N meable prodrugs would require cleavage of the pro-moiety subse-
H quent to absorption (e.g., in plasma or in the liver target organ) to
O
N
COOH
BILB 1941 (1)
1a/1b replicon EC50 = 153 / 84 nM
O using HATU, followed by deprotection if necessary, to produce a
H
N N N
N OEt
Cl H In order to validate the prodrug concept, the solubility of the
O
N
2 COOH
1a/1b replicon EC50 = 29 / 11 nM
Figure 1. HCV NS5B polymerase thumb pocket 1 leads.
of the protein, resulting in a replication-deficient complex. This
effort culminated with the discovery of BILB 1941 (1, Fig. 1), the
first NS5B thumb pocket 1 inhibitor to demonstrate antiviral activ-
ity in HCV GT1 infected patients (up to 2.5 log10 reduction in
viremia).7
Unfortunately, GI intolerance observed in man at the highest
dose (450 mg, q8h) precluded further advancement of 1 and efforts
were subsequently focused on the design of backup compounds
with improved potency and tolerability. Compound 2 is a develop-
ment candidate that displayed 5–7-fold better potency in subge-
nomic replicon assays as a result of reduced ionization potential
and increased cell membrane permeability (pKa = 6.5 versus 4.5
for 2 and 1, respectively) and an improved preclinical PK profile.8
Inhibitor 2 was classified as a BCS 2 compound with high perme-
ability (Caco-2 apical ? basolateral permeability = 13  10 6 cm/s
at 10 lM nominal concentration) but very low aqueous solubility
at pH 6.8 (crystalline free form <0.1 lg/mL, mp = 267 °C) that pre-
sented a significant development challenge for conventional oral
delivery. Several approaches were explored to identify formula-
tions that would be suitable for delivery of the predicted effica-
cious doses of this compound (400 mg twice daily).8,9 While
to direct administration of parent drug, while gut-stable and per-
deliver parent drug.11
The synthesis of prodrugs of 2 exploits the carboxylic acid func-
tionality and is described in Scheme 1. It involves alkylation of 28
with various alkyl halides or condensation with alcohols or amines
variety of ester and amide-linked proanalogs.12
crystalline forms of 2 and its methyl ester 3 in individual SEDDS
components, and the extent of in vivo conversion of prodrug 3 to
parent 2 were determined. Consistent with a 5-fold increase in
c log P (from 5.3 to 6.1) and a lower melting point (215 °C vs
267 °C), methyl ester 3 showed improved solubility in lipids
(10–14 mg/mL vs <3 mg/mL). Following oral administration of the
methyl ester 3 to rats in a Tween/Methocell + 1% NMP suspension
(solvent A) at a dose of 10 mg/kg (Table 3) the plasma exposure of
the released parent was limited compared to much higher plasma
concentrations observed following the same dose of parent 2
administered in the same formulation. Based on the solubility of 3
in individual SEDDS components and in vivo bioconversion/plasma
exposures of 2 and 3 following oral administration in rats (data not
shown), a screening formulation consisting of 10.6% PEG400, 5.4%
Cremophor, 2.6% Labrasol and 1.4% Capmul in water (solvent B)
was selected for screening prodrugs. The oral PK profile following
administration of crystalline 3 to rats using solvent B is shown
graphically in Figure 2 and compared to direct administration of
crystalline parent (2). Using the screening SEDDS formulation (sol-
vent B), plasma concentrations of parent drug derived from biocon-
version of the methyl ester prodrug 3 were significantly improved
O
H
N N N OEt
N
Cl H
O
N
2 COOH

conventional nano-milling, solid dispersion with free acid or saltif- PRO-X / Cs2CO3 (X = Cl, Br, I)
ication of the acid did not provide a suitable path for the parent or
drug, the development of an amorphous solid dispersion utilizing PRO-OH or PRO-NH2 / HATU / Et 3N
the potassium salt form was pursued with some success. More-
O
over, 2 (calcd log P = 5.3; measured log D7.4 = 4.1) did not have suf- H
OEt
N N N
ficient solubility in excipients (<3 mg/mL) for delivery of the Cl
N
H
O
projected doses of parent drug using generally-acceptable lipid for- N
mulations (e.g., self-emulsifying drug delivery systems or SEDDS: Y = O, NH or NR
Y
PRO
glyceryl acetate, glycerine, miglyol, Cremophor, Labrasol, solutol, O
PEG400, Capmul MCM, PG, Captex 300).10 Knowing the risks asso- Polar / ionizable prodrugs
ciated with the development of metastable amorphous solids, a 4 L-Malic ester 12 O-(CH2)2-N+Me3Br -
5 L-Asp α-amide 13 NH-(CH2)2-OH
parallel investigation explored whether prodrugs of 2 may provide 6 L-Glu α-amide 14 NMe-(CH2)2OH
an alternative to delivering the required dose of parent drug in 7 L-Arg α-amide 15 O-CH2COOH
human. 8 O-(CH2)2-NMe2
16 O-CH2CO-(4-methylpiperazino)
9 O-(CH2)2-morpholino
Consistent with its high permeability and low solubility, the 10 O-(CH2)2-pyrrolidino
17 O-CH2-OP(O)(Otert-Bu)OH
18 O-CH2-OP(O)OHO-
animal pharmacokinetic profile following oral dosing suggested 11 O-(CH2)2-(4-methylpiperazino)
that absorption of compound 2 was solubility-limited. The overall Lipophilic / non-ionizable prodrugs
properties of 2 suggested that a prodrug strategy may be applied to 19 OEt 31 O-CH2COOBu
improve oral delivery with a novel formulation. Since 2 displayed 20 OPr 32 O-CH2COOBn
21 Oisoamyl
good intrinsic absorption, two approaches were considered: (i) 22 Ohexyl
33 O-CH2COO(4-Cl-Bn)
34 O-CH2CONH2
polar or ionizable pro-moieties expected to improve aqueous solu- 23 O-CH2CCl3
24 OBn 35 O-CH2CONHMe
bility in conventional delivery systems (aqueous solubility objec- 25 O-(CH2)2OPh 36 O-CH2CONMe2
tive: >100 lg/mL) that would be cleaved in the stomach prior to 26 O-(CH2)2-O-(CH2)2OMe 37 O-CH2CONMePr
absorption and (ii) a less conventional prodrug approach that 27 O-(CH2)2-N-pyrrolidone 38 O-CH2CONMeiPr
28 O-CH2-OCOtertBu 39 O-CH2CO-pyrrolidino
aimed to increase solubility in SEDDS (lipid solubility objective: 40 O-CH2CO-morpholino
29 O-CH2COOMe
>100 mg/mL). Prodrugs that can be cleaved either chemically or 30 O-CH2COOEt
enzymatically in the gut or stomach to release highly permeable
parent drug should provide systemic exposure to 2 comparable Scheme 1. Synthesis and structure of investigated prodrugs.
212 P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 210–215

Figure 2. Rat PK profile of crystalline 2 and its methyl ester prodrug 3 following an oral dose of 10 mg/kg in SEDDS solvent B.

compared to direct administration of parent drug in the same sol-
vent B and comparable to delivery of 2 using solvent A (Table 3).
The relatively low levels of circulating 3 (Cmax = 0.84 lM) and
time-plasma concentration profile of the prodrug (Fig. 2) suggested
a rapid initial bioconversion of the prodrug, followed by a slow
decay t1/2 in plasma. These encouraging results prompted a thor-
ough investigation of prodrugs to identify candidates with
improved properties including formulability in SEDDS (ideally
>100 mg/mL) and in vivo bioconversion rates (rapid conversion
and minimal circulating prodrug).
The list of investigated prodrugs is provided in Scheme 1. Their
in vitro bioconversion rates were assessed by measuring t1/2 fol-
lowing incubation with simulated gastric fluids (SGF), simulated
intestinal fluids (SIF), human and rat intestinal microsomes (HIM,
RIM), human and rat liver microsomes (HLM, RLM), and human
and rat plasma (HPL, RPL).13 The data are presented in Table 1
for polar/ionizable (4–17) and neutral/lipophilic (3, 18–40)
prodrugs. Using the profile of methyl ester 3 as a reference
(RLM t1/2 = 98 min), in vitro half-lives <75 min in human-derived
media (e.g., HIM, HLM, HPL etc.) or SIF/SGF were considered to

Table 1
Prodrug in vitro half-lives (t1/2 in min)a,13

HLM HIM RLM RIM SIF SGF HPL RPL cLogPb

3 764 544 98 109 412 150 >300 >300 6.1
4 334 475 4.2
5 4 >300 3.6
6 254 32 4.0
7 28 4.0
8 90 >300 19 88 396 109 >300 84 6.0
9 55 910 35 210 257 20 >300 96 5.6
10 85 23 6.5
11 111 13 5.6
12 >300 104 25 4.7
13 98 89 4.9
14 78 135 5.0
15 186 70 5.0
16 21 9 5.1
17 89 8 6.2
18 10 28 9 5 78 4.4
19 >300 >300 184 >300 >300 20 >300 491 6.5
20 >300 >300 21 >300 >300 134 >300 915 7.5
21 >300 >300 85 >300 >300 86 371 1081 8.0
22 1073 764 53 8.6
23 >300 >300 14 >300 >300 105 >300 78 7.9
24 >300 >300 130 >300 >300 110 >300 134 7.8
25 >300 989 7.8
26 51 64 5.7
27 41 47 5.7
28 247 336 <2 7.0
29 126 125 5.8
30 96 147 6.2
31 359 947 7.2
32 1569 >300 132 >300 98 182 >300 396 7.5
33 337 682 8.1
34 59 23 36 4.8
35 34 30 5.3
36 64 256 38 123 417 53 >300 86 5.4
37 19 19 5.8
38 39 16 6.2
39 99 88 6.3
40 48 205 44 76 190 51 >300 110 5.0
a
Half-lives are color-coded as follows: green for t1/2 <75 min; red for t1/2 >75 min.
b
c Log P values calculated using JChem 5.0.0 (http://www.chemaxon.com).
 P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 210–215 213

provide sufficient bioconversion potential and those derivatives Table 3

were prioritized for further investigation. Rat oral PK parameters for parent 2 and prodrugs (10 mg/kg)

For candidates which exhibited in vitro t1/2 <75 min, solubility Prodruga Vehicleb [Prodrug]plasma [Parent 2]plasma
was determined in aqueous buffers or individual SEDDS compo- Cmax (lM) AUC (lM h) Cmax (lM) AUC (lM h)
nents and in vivo conversion to parent drug in rats was monitored
2 A n.a. n.a 7.4 25.5
following oral administration of amorphous, lyophilized solids. As 2 B n.a. n.a 2.1 7.5
seen from the color coding in Table 1 (t1/2 <75 min = green, t1/2 3 A 0.01 0.07 0.51 5.7
>75 min = red), liver microsomes (human and rat) as well as simu- 3 B 0.84 2.6 5.2 30.7
lated gastric fluids were most effective for converting prodrugs 8 B BLD BLD 1.6 10.1
9 B 0.07 0.2 7.2 35.2
into parent in vitro. Most compounds were relatively stable in
10 B BLD BLD 1.1 6.5
the presence of intestinal microsomes, simulated intestinal fluid 11 B BLD BLD 1.2 7.9
and plasma. 16 B BLD BLD 0.26 1.5
Polar/ionizable prodrugs were designed with a water-solubiliz- 17 A BLD BLD 0.08 0.49
18 A 0.05 0.09 4.2 14.5
ing entity (acid or base) attached to the carboxyl function of com-
22 B 0.40 2.2 0.2 3.2
pound 2 through an amide or ester linkage (compounds 4–17). 26 B 0.03 0.10 4.6 23.1
c log P values ranged from 3.6 to 6.5. Analogs that incorporated 27 B 0.04 0.06 6.1 25.6
acidic solubilizing groups (4–6) displayed good solubility at pH 28 B 0.04 0.30 1.7 19.6
6.8 (>0.5 mg/mL) and had potential for liberating parent drug in 34 B 0.04 0.13 4.4 16.7
36 B 0.02 BLD 9.2 38.5
SIF or SGF (5 and 6). However, neither the prodrugs nor parent
40 B BLD BLD 4.0 19.3
drug (2) were detected in plasma following oral administration
a
(10 mg/kg) of these derivatives to rats, most likely due to lack of Compounds 2 and 3 were crystalline solids. All other prodrugs were screened as
lyophilized powders and their solid state was not characterized.
absorption (Caco-2 permeability <0.2  10 6 cm/s).14 Zwitterionic b
Vehicle A: 0.5% Methyl cellulose, 0.3% Tween-80, 1% N-methylpyrrolidone in
arginine conjugate 7 had no significant solubility at pH 2–6.8 and water. Vehicle B: 10.6% PEG400, 5.4% Cremophor EL, 2.6% Labrasol, 1.4% Capmul
was not considered further. MCM in water (80%). n.a.: not applicable. BLD: below limit of detection.
Compounds 8–11 are esters of ethanolamine in which the pKa of
the amino group was modulated through substitution. These deriv-
atives displayed comparable and rapid bioconversion rates in the not meet criteria for further evaluation. Weakly ionizable glycolic
presence of RLM and pKa values ranging from 5.7 for 9 to 7.7–8.8 amide ester 16 (pKa = 6.6) displayed rapid in vitro bioconversion
for 8, 10 and 11. Although all four compounds had solubilities rates in both HLM and RLM and was evaluated in rats. While 16
<100 mg/mL in individual lipid vehicles (Table 2), in vivo biocon- remained undetectable, plasma levels of 2 were low (Table 3), sug-
version to 2 was evaluated following oral administration in rats gesting that the basic group restricted permeability. Derivatives 17
using the screening SEDDS formulation (solvent B) to verify and 18 belong to the methylenoxyphosphate class of prodrugs that
whether an in vitro/in vivo correlation (IVIVC) could be established has proven very successful for the delivery of poorly soluble drugs
in this species. Table 3 shows that the short RLM t1/2 values trans- in several therapeutic areas.11 Because of chemical instability in
lated into good in vivo bioconversion with parent drug plasma the free form both prodrugs were prepared as salts (lysine and
Cmax = 1.1–7.2 lM. On the other hand, the prodrugs themselves monosodium salts for 17 and 18, respectively). Since solubility
were not detected in plasma except for very low levels of 9 which was high at pH 6.8 (781 and 140 lg/mL, respectively), both com-
correlated with its relatively longer in vitro RLM t1/2 = 35 min. Pro- pounds were administered to rats using solvent A. As shown in
drug 9 with a lower pKa of 5.7 provided the best exposure of 2 Table 3, 17 did not provide significant exposure of parent 2 how-
(comparable to direct administration of crystalline 2, Table 3) ever, good in vivo conversion was observed for phosphate 18 (18
while 8, 10 and 11, which would be expected to ionize at physio- displayed short t1/2 in HIM, HLM, RIM, RLM and SGF, and it was
logical pH and thus lose permeability, provided lower plasma not possible to assess whether conversion to parent occurred
exposures to parent. Unfortunately, 9 did not fulfill lipid solubility through chemical cleavage due to sensitivity to acidic conditions
criteria (>100 mg/mL) that would allow delivery of the projected in the stomach prior to absorption or via biological processes med-
efficacious human dose of 400 mg BID using conventional 1 mL iated by phosphatases or esterases or both). The chemical instabil-
soft-gel capsules. Nevertheless, this data confirmed IVIVC between ity under both basic and acidic conditions of 18 was a potential
liver microsome stabilities and bioconversion in rats. issue for development and progression of this analog was stopped
Based on HLM conversion rates (t1/2 = 78 to >300 min, Table 1) as well.
choline salt 12, amides 13 and 14 and glycolic acid ester 15 did The remaining prodrugs (19–40) belong to the lipophilic, non-
ionizable class (Scheme 1) and displayed c log P = 4.8–8.6. Simple
Table 2
esters (19–25) were generally stable in vitro (HLM t1/2 >300 min)
Solubility of prodrugs in SEDDS componentsa
and were not pursued further except for analog 22 that showed
Prodrug Capmul Solutol Labrasol PEG400 some level of processing in the presence of SGF. However, prodrug
8 <100 <100 <100
22 provided only low plasma exposure to 2 and comparable circu-
9 <30 <60 <60
10 <60 <100 <60 lating concentrations of intact prodrug following oral administra-
11 <100 <100 tion to rats, not warranting further investigation.
16 <100 <100 <100 The HLM t1/2 values of glycolic ester 26 and N-(2-ethanol)pyr-
26 <60 <100 <30
27 <100 <100 <100
rolidone ester 27 prompted further evaluation in rats. Good bio-
34 <200 ~200 >200 >200 conversion was observed for both compounds (Table 3) and very
35 <200 <200 ~200 >200 low concentrations of un-cleaved prodrug were detected in
36 >100 >200 >200 >200 plasma. However, the lipid solubility of these compounds was
37 <200 <200 <200 <200
<200 <200 <200 >200
low (<100 mg/mL, Table 2) and precluded further advancement.
38
39 <200 ~200 ~200 >200 The tert-butylglyoxylate diester 28 is a prodrug that showed slow
40 <15 <15 <30 bioconversion in HLM/RLM microsomes but was rapidly processed
a
Solubility in mg/mL based on visual examination (green: in SGF due to its low chemical stability under acidic conditions. In
>100 mg/mL, yellow: 60–100 mg/mL, red: <60 mg/mL). vivo, 28 provided moderate plasma exposure to parent and low
214 P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 210–215

levels of circulating prodrug. It is likely that most of the parent
drug detected in plasma resulted from acid-induced cleavage of
the pro-moiety in the stomach followed by absorption of highly
permeable 2. As in previous cases (e.g., 18) the low chemical stabil-
ity of 28 under acidic conditions was considered a barrier to fur-
ther progression. More chemically stable versions of 28
(compounds 29–33) were relatively stable in presence of HLM
(t1/2 = 96 to >300 min) and thus unsuitable for our purpose.
Analogs 34–40 are representatives of glycolic amide esters
which have been reported to be promising carboxylic acid prodrugs
due to their rapid cleavage in plasma.15 Interestingly, glycolic amide
esters of 2 were very stable in human plasma (e.g., 36 and 40). How-
ever, apart from pyrrolidine amide 39 (HLM t1/2 = 99 min), these
derivatives displayed good conversion rates in presence of human
and rat liver microsomes (t1/2 = 19–64 min, Table 1) and some ana-
logs (e.g., 34 and 36) also exhibited promising solubility in individ-
ual SEDDS components (Table 2).
Compounds 34, 36 and 40 were evaluated in rats and the three
analogs were efficiently converted to parent drug in vivo.
A retrospective analysis of a set of ester prodrugs that were effi-
ciently bio-converted to parent drug 2 in rats suggests that in most
cases, cleavage is likely mediated through the action of esterases
rather than CYP450-dependent metabolism (similar in vitro t1/2
were observed when incubations were performed with or without
NADPH), and occurs once the prodrugs have been absorbed into
systemic circulation. This hypothesis is supported by the rat IVIVC
with LM t1/2 and the lack of such a correlation with SGF stability as
depicted in Figure 3.
Of all analogs screened, 36 provided the highest plasma concen-
trations of 2 and only small amounts of the initial prodrug were
detectable in plasma following a single oral dose of 10 mg/kg.
Furthermore, this analog visually exhibited excellent solubility
(>100–200 mg/mL) in four lipid vehicles and consequently, 36
was selected for further profiling. The oral PK profile of 36 in rats,
monkeys and dogs using solvent B is shown in Table 4 and com-
pared to direct administration of parent 2.
As can be seen from the data in Table 4, the bioconversion rate
of the prodrug in LM is reasonably consistent with oral parent drug
delivery cross-species. Therefore, it is expected that 36 would also
bio-convert in human to release parent drug 2. Consistent with the
microsome data, 36 efficiently converted to 2 in rats and provided
a 2-fold improvement in oral exposure and bioavailability com-
pared to direct administration of parent. On the other hand expo-
sure and bioavailability in monkeys were reduced two-fold despite
increased conversion rates in liver microsomes (t1/2 = 19 min).
In both cases, uncleaved prodrug was not detected in plasma
samples. Consistent with the high metabolic stability of 36 in
dog microsomes (t1/2 = 588 min), exposure and bioavailability of
2 were significantly lower in this species when derived from pro-
drug compared to parent. The bioconversion in HLM however is
consistent with that observed in the rat and monkey (t1/2 =
64 min) suggesting that dog most likely represents an outlier
species. Rapid in vivo conversion is further supported by the
observation that similar profiles of parent drug 2 were obtained
following IV administration of either parent drug 2 or the prodrug
as shown for compound 36 in Table 5.
After screening several solvents and conditions 36 was success-
fully crystallized, providing a high degree of crystallinity as deter-
mined by XRPD and a significantly lower melting point (161 °C)
and heat of fusion (21 J/g) compared to crystalline 2 (267 °C,
120 J/g). The solubility of crystalline 36 was evaluated in 7 individ-
ual solvents that are acceptable for toxicological studies and clini-
cal use. Of these PEG400, Labrasol and Cremaphor EL gave the best
solubility results (73, 80 and 60 mg/mL, respectively), providing a
significant improvement over that of the crystalline forms of
parent 2 or the initial methyl ester prodrug 3.
SEDDS formulation optimization would be required to further
improve solubility and stability of the crystalline prodrug (e.g.,
combination of excipients). However, during development of the
Table 5
Rat IV PK parameters for 2 from administration of parent 2 or prodrug 36 (2 mg/kg)
iv AUC (lM h) MRT (h) t1/2 VSS (L/kg) Cltot (mL/min/kg)
2 20.1 2.5 2.4 0.38 2.6
36 10.5 2.8 2.1 0.73 4.4

Figure 3. IVIVC between rat plasma levels of 2 and SGF or RLM t1/2.

Table 4
Cross-species oral PK parameters for 2 from administration of amorphous parent 2 or prodrug 36 (10 mg/kg in solvent B)

LM t1/2 (min) Parent 2 from prodrug 36 36 From 2

Oral AUC (lM h) Oral F% Oral AUC (lM h) Oral AUC (lM h) Oral F%
Rat 38 38.5 38 BLD 19.3 19
Monkey 19 26 35 BLD 53 72
Dog 588 11.5 4 0.69 76 29
 P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 25 (2015) 210–215
parent drug 2, cross-species metabolite studies performed in vitro
and in vivo, identified small amounts of a genotoxic 4-amino-2-
ethoxycinnamic acid metabolite resulting from cleavage of the ani-
lide bond. This metabolite was also detected in animal plasma
samples from PK studies and upon incubation of 2 with simulated
gastric fluids (SGF). In human liver microsomes, the metabolite
was formed at a rate of 1 ppm/min in a NADPH-independent
manner. In light of the challenges associated with the progression
of such a compound, development of 2 and its prodrug 36 were
discontinued. Nevertheless, the feasibility of a prodrug approach
for delivery of the poorly soluble parent molecule using SEDDS
has been demonstrated.
Acknowledgments
We thank the following colleagues from Boehringer Ingelheim
(Canada) Ltd that participated in generating the data presented
in this Letter: Ma’an Amad, Martin Jutras, Hélène Montpetit, Franç-
ois Otis and Manon Rhéaume for ADME-PK support. Norman
Aubry, Colette Boucher and Serge Valois for analytical support.
We also wish to acknowledge contributions from Boehringer Ingel-
heim Pharmaceuticals Inc., Ridgefield, CT and Boehringer Ingel-
heim Pharma BIKG for their contributions to advanced preclinical
profiling of compound 36 and Professor Valentino Stella for helpful
discussions.
References and notes
1. (a) Fried, M. W.; Shiffman, M. L.; Reddy, K. R.; Smith, C.; Marinos, G.; Gonçales,
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treatment of genotype 1 chronic hepatitis C: http://www.gilead.com/news/
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11. (a) Beaumont, K.; Webster, R.; Gardner, I.; Dack, K. Cur. Drug Metabol. 2003, 4,
461; (b)Prodrugs, Challenges and Rewards Part 1 and Part 2; Stella, V., Borchardt,
R., Hageman, M., Oliyai, R., Maag, H., Tilley, J., Eds.; Springer: New York, 2007.
12. Prodrugs had >95% homogeneity as determined by reversed-phase HPLC
analysis and had 1H NMR and mass-spectral data consistent with their
structures. Inhibitors were found to be somewhat sensitive to light-induced
double bond isomerization and solids/solutions were stored protected from
light to avoid degradation.
13. Human liver microsomes were obtained from BD Gentest (Woburn, MA).
Human and rat intestinal microsomes were obtained from Xenotech (Lenexa,
KS). Human plasma was obtained from Biological Specialty Corporation
(Colmar, PA). Rat plasma was prepared in house by centrifugation of rat
whole-blood. Rat liver microsomes were prepared in-house as follows: male
Sprague-Dawley rats (350 g) were anesthetized with halothane, the liver was
removed and quickly rinsed in cold 1.15% KCl. The tissue was then blotted dry,
weighed, cut into small pieces and homogenized in 0.25 M sucrose (5 mL/g
tissue) using a polytron. Following homogenization, the tissue was further
ground with a Potter–Elvehjem tissue grinder (Wheaton). Homogenates were
centrifuged at 10,000g at 4 °C for 20 min, followed by centrifugation of the
supernatants for 60 min at 145,000g. The resulting pellets were centrifuged
once more at 145,000 g for 60 min and suspended in cold 0.066 M Tris–HCl
buffer, pH 7.4, containing 250 mM sucrose and 5.4 mM tetrasodium EDTA
(1 mL/g of original liver weight). The total microsomal protein concentration
was determined by the method of Bradford with the Bio-Rad protein assay dye
reagent concentrate, using bovine serum albumin as a standard. Microsomes
were aliquoted and stored at –80 °C until needed. Simulated gastric fluid (SGF)
was prepared by adding 0.7 mL conc. HCl to 0.2% NaCl solution to obtain a final
pH of 1.2. Prior to use, porcine pepsin was added to an aliquot of solution
(3.2 mg/mL). Simulated intestinal fluid was prepared by dissolving 3.4 g of
KH2PO4 in water (350 mL), adding enough 0.2 N NaOH to obtain a final pH of
7.5 and completing to a total volume of 500 mL). On the day of the experiment,
porcine pancreatine was added to an aliquot of solution (10 mg/mL).
Incubations: subcellular fractions were incubated at 37 °C at a concentration
of 1 mg/mL in 0.066 M Tris buffer, pH 7.4 containing 2 lM compound.
Reactions were initiated by the addition of 2.5 mM NADPH and terminated
at the appropriate times (o, 10, 20 and 40 min) by quenching with an equal
volume of a 1:1 mix of acetonitrile/methanol. The collected samples were
centrifuged at 2000 g at 4 °C for 10 min and the resulting supernatants were
analyzed by HPLC. Plasma, simulated intestinal and simulated gastric fluid
were incubated with 2 lM compound at 37 °C. Samples were quenched 1:3
(plasma) or 1:2 (SIF, SGF) with a 1:1 mix of acetonitrile/methanol, followed by
centrifugation and analysis. HPLC analysis: Symmetry C-8, 2.1  150 mm
column heated to 40 °C; mobile phase: MeCN and 50 mM KH2PO4 (pH 3); PDA
detection at 200–400 nm. The concentration of parent compound was
measured at the wavelength representing the best signal to noise ratio. The
half-life (t1/2) was determined by the ratio of ln2 over the first order rate of
disappearance of the parent compound (0.693/-k).
14. All rat PK studies were performed at Boehringer Ingelheim (Canada) Ltd. PK
studies in dogs and monkeys were performed at LAB Pre-Clinical Research
International Inc., Laval, QC. All protocols involving animal experimentation
were reviewed and approved by the respective Animal Care and Use
Committee of each test facility. In-life procedures were in compliance with
the Guide for the Care and Use of Laboratory Animals from the Canadian
Council of Animal Care. All chemicals used were reagent grade or better.
Animals were fasted overnight prior to dosing. IV dose administration (2 mg/
kg) was performed using a 70% PEG400: 30% water dosing solution. Oral dose
administration (10 mg/kg) was performed using a suspension containing 0.3%
Tween-80 and 0.5% methylcellulose, with addition of 1% NMP as additional
solubilizer (n = 3 per dose and per route). Plasma samples were extracted by
solid phase extraction. Samples were analyzed by HPLC using either a UV diode
array detector between 200 and 400 nm with quantitative determination made
by peak height at the wavelength representing the best signal to noise ratio or
a LC/MS systems using the appropriate retention time and m/z+. Calibration
standards were prepared in blank plasma. The calibration curve was linear to
cover the time-concentration curve with a r2 values >0.99, and a lower limit of
quantification (LOQ) at 7–14 ng/mL depending on compounds. The temporal
profiles of drug concentrations in plasma were analyzed by noncompartmental
methods using WinNonlin (version 3.1; Scientific Consulting Inc, Cary, NC).
15. (a) Bundgaard, H.; Nielsen, N. M. Int. J. Pharm. 1988, 43, 101; (b) Nielsen, N. M.;
Bundgaard, H. J. Pharm. Sci. 1988, 77, 285.