Design and synthesis of imidazole N-H substituted amide prodrugs as inhibitors of hepatitis C virus replication

Article abstract of DOI:10.1016/j.bmcl.2015.06.006

Abstract Twenty-five novel imidazole N-H substituted Daclatasvir (BMS-790052, DCV) analogues (8a-8y) were designed and synthesized as potential prodrugs. Structure modifications were performed in order to improve potency and pharmacokinetic (PK) properties. All target compounds were evaluated in a hepatitis C virus (HCV) genotype 1b replicon, and the 2-oxoethyl acetate substituted compound 8t showed similar anti-HCV activity (EC₅₀ = 0.08 nM) to that of the lead compound Daclatasvir. Moreover, the utility of prodrug 8t was demonstrated through similar exposure of the parent compound when the prodrugs were dosed in vivo. PK studies showed that prodrug 8t was an ideal candidate for a slower and sustained release form of Daclatasvir.

Full text of DOI:10.1016/j.bmcl.2015.06.006

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of imidazole N–H substituted amide prodrugs
as inhibitors of hepatitis C virus replication
a
b
b
b
c
b,d,
Xi Zong a, Jin Cai a, , Junqing Chen , Peng Wang , Gaoxin Zhou , Bo Chen , Wei Li , Min Ji
⇑
⇑
a School of Chemistry & Chemical Engineering, Southeast University, Nanjing 210096, China
b School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China
c Department of Medicinal Chemistry and the Institute for Therapeutics Discovery and Development, University of Minnesota, 717 Delaware Street SE, Minneapolis, MN 55414, USA
d Suzhou Key Laboratory of Biomaterials and Technologies & Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou 215123, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-five novel imidazole N–H substituted Daclatasvir (BMS-790052, DCV) analogues (8a–8y) were
designed and synthesized as potential prodrugs. Structure modifications were performed in order to
improve potency and pharmacokinetic (PK) properties. All target compounds were evaluated in a hepati-
tis C virus (HCV) genotype 1b replicon, and the 2-oxoethyl acetate substituted compound 8t showed sim-
ilar anti-HCV activity (EC50 = 0.08 nM) to that of the lead compound Daclatasvir. Moreover, the utility of
prodrug 8t was demonstrated through similar exposure of the parent compound when the prodrugs were
dosed in vivo. PK studies showed that prodrug 8t was an ideal candidate for a slower and sustained
release form of Daclatasvir.
Received 19 January 2015
Revised 27 April 2015
Accepted 1 June 2015
Available online xxxx
Keywords:
Hepatitis C virus
NS5A
Ó 2015 Elsevier Ltd. All rights reserved.
Prodrug
Daclatasvir
Pharmacokinetics
Hepatitis C virus (HCV) infection is a global epidemic with an
associated high risk for serious liver disease. An estimated 200 mil-
lion people have been infected by HCV worldwide.1 HCV usually
progresses to a chronic state that persists for decades, but eventu-
ally becomes responsible for the development of chronic liver dis-
eases, such as liver cirrhosis and hepatocellular carcinoma.2,3 The
traditional standard of care for HCV infection through treatment
with pegylated-interferon and ribavirin has significant toxicity,
and its sustained virological response rate is less than 50% in those
infected by the genotype-1 (GT-1) virus.4,5
HCV was identified as a positive strand RNA virus with highly
genetically diverse species that were classified into six genotypes
(1–6) with more than 90 different subtypes.6 The viral proteins
can be divided into structural and nonstructural (NS) precursor
regions. Previous studies have reported that the NS proteins NS2,
NS3, NS4A, NS4B, NS5A, and NS5B are important for the replication
of HCV virus; thus, the development of direct-acting antiviral
agents against HCV infection has focused predominantly on inhibi-
tors of the viral NS proteins.7,8 Recently, several NS3/4A, NS5A, and
NS5B inhibitors have been approved for the treatment of HCV GT-1
infection and have attracted considerable interest.
and assembly of viral particles.9–11 Several NS5A inhibitors exhibit
robust potency profiles in HCV replicon assays in vitro and sup-
pression of HCV replication in clinical trials (Fig. 1). This family
of compounds includes some of the most active antiviral com-
pounds known, with low picomolar median effective concentra-
tions (EC50) in HCV replicon assays.
Daclatasvir (BMS-790052, DCV) is an oral, once-daily, highly
selective NS5A inhibitor developed by Bristol–Myers Squibb. As
the first-in-class inhibitor targeting NS5A, the EC50 values of DCV
are 9 and 28 pM against GT-1b and GT-2a replicons, respectively.12
DCV is active against multiple HCV genotypes. Lemm et al.
reported that DCV shows the highest in vitro potency among all
known anti-HCV compounds, with a picomolar range of EC50 val-
ues against HCV replicons from various genotypes. In addition,
DCV has been marketed and launched successfully to treat GT-1
HCV-infected patients.13
Preparation of a prodrug is an efficient approach to enhance the
biopharmaceutical, physicochemical, or pharmacokinetic (PK)
properties of pharmacologically potent compounds, thereby
improving the development and utility of a potential drug.14
Previous reports on DCV have demonstrated that the imidazole
N–H is essential to the anti-HCV activity, as internal hydrogen
bonding interactions may exist between the valine C@O and imida-
zole N–H on both sides of the dimer to control the configurations of
DCV.15 Amides and esters are two of the most frequently used
moieties in designing small-molecule prodrugs because hydrolases
The NS5A protein was speculated to play a critical role in the
replication of HCV RNA, the modulation of the host cell responses,
⇑
Corresponding authors. Tel.: +86 0512 62729923; fax: +86 025 83272349.
E-mail addresses: caijin@seu.edu.cn (J. Cai), jimin@seu.edu.cn (M. Ji).
http://dx.doi.org/10.1016/j.bmcl.2015.06.006
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zong, X.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.006

2
X. Zong et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
F
F
H
N
N
N
N
H3CO2CHN
N
CO2CH3
O
H3CO2C NH
HN
N
NH
O
NH
O
N
NH
NHCO2CH3
N
N
GS-5885 ledipasvir
BMS-790052 Daclatasvir DCV
NHCO2CH3
NHCO2CH3
O
N
NH
O
O
N
N
N
N
O
Ph
N
NHCO2CH3
O
O
N
HN
HN
NH
H3CO2CHN
O
N
MK-8742 elbasvir
ABT-267 ombitasvir
S
S
N
O
H3CO2CHN
H3CO2CHN
HN
NH
O
O
N
N
NHCO2CH3
N
O
N
N
NH
NHCO2CH3
NH
N
N
O
GSK2336805
IDX-719 samatasvir
O
Figure 1. Chemical structure of NS5A inhibitors.
are abundant in the body.16 To discover novel prodrugs of NS5A
inhibitors with improved cellular potency, PK properties, and min-
imized side effects, we designed and synthesized 25 prodrugs of
DCV (Fig. 2) and tested their anti-HCV activities.
The symmetrical structure of the target compounds was syn-
thesized according to general Scheme 1. 1,10-([1,10-Biphenyl]-
4,40-diyl)diethanone 1 was first converted to 1,10-([1,10-biphe-
nyl]-4,40-diyl)bis(2-bromoethanone) 2 with Br2 in CH2Cl2, which
was then coupled with N-Boc protected proline in the presence
of N,N-diisopropylethylamine (DIPEA). Refluxing the compound 3
with ammonium acetate in toluene formed the imidazole ring
and led to the versatile Boc-protected intermediate 4. After the
removal of Boc protection with 6 N HCl, the resulting amine was
N
N
H3CO2CHN
O
N
HN
NH
O
NHCO2CH3
coupled with N-(methoxycarbonyl)-L-valine 6 by using o-(7-az-
N
abenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
rophosphate in the presence of DIPEA to provide
hexafluo-
(DCV).
7
BMS-790052 Daclatasvir DCV
Acylation of 7 with corresponding acyl chlorides gave 8a–8y in fair
to excellent overall yields. All the target compounds were new
chemical entities and are shown in Table 1.
Cl
R
All synthesized DCV prodrugs were evaluated for inhibition of
HCV RNA replication in Huh7 cells containing a subgenomic HCV
1b replicon. When the R group was straight chain alkyl acyl, com-
pounds showed moderate anti-HCV activities, whereas longer alkyl
chains resulted in lowered potency, for example, 8a (EC50 = 12 nM)
> 8b (EC50 = 39 nM) > 8c (EC50 = 85 nM) > 8g (EC50 = 231 nM) > 8h
(EC50 = 324 nM) > 8i (EC50 = 542 nM). When the acyl alkyl terminal
was tertiary butyl (such as 8e, 8f, and 8j), anti-HCVactivity was com-
pletely lost. When the R group was acly annular alkane, ring exten-
sion (8l) or contraction (8m) resulted in a slight decrease in activity
when compared with 8k. Furthermore, some carbamate prodrugs of
DCV (8n–8s) were also synthesized. Surprisingly, the carbamate
prodrugs had no activity against HCV, except compound 8t, which
demonstrated anti-HCV activity with an EC50 of 0.08 nM, compara-
ble to the control compound DCV (EC50 = 0.009 nM). To discover
the prodrug with improved PK properties, we also designed aro-
matic prodrugs of DCV (8u–8y). Almost all of them possessed anti-
HCV activity with EC50 values ranging from 77 to 988 nM.
N
O
R
N
N
H3CO2CHN
O
N
N
R
NHCO2CH3
N
Target prodrug compounds
enzymatic or chemical
hydrolysis
N
N
H3CO2CHN
O
N
HN
NH
O
NHCO2CH3
N
BMS-790052 Daclatasvir DCV
Figure 2. A design for DCV prodrugs.
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X. Zong et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
O
O
O
O
CH2Br
O
O
a
b
N
N
Boc
O
BrH2C
O
O
O
Boc
2
3
1
O
HO
R
H
N
H
N
N
O
H
N
Boc
N
H
d
c
N
N
O
6
Boc
H
N
N
N
e
N
N
H
N
H
5
4
O
O
NH
NH
H
N
O
O
O
N
O
N
N
N
f
N
N
N
N
N
N
R
N
H
O
O
O
O
HN
HN
8a-8y
7 (BMS-790052)
O
O
Scheme 1. Synthetic route for the preparation of the target compounds 8a–8y. Reagents and conditions: (a) Br2, CH2Cl2, rt, overnight, 86%; (b) N-Boc-
L
-proline, MeCN, Et3N, rt,
2 h, 98%; (c) NH4OAc, toulene, 130 °C, 15 h, 85%; (d) 6 N HCl, MeOH, 50 °C, 4 h, 87%; (e) HATU, N-(methoxycarbonyl)-L-valine, DIPEA, rt, 14 h, 83%; (f) RCOCl, TEA, CH2Cl2, rt,
3 h, 64–87%.
Table 1
Table 1 (continued)
Chemical structure and anti-HCV activity of the target compounds 8a–8y
Compound
R
HCV replicona EC50(nM)
Compound
R
HCV replicona EC50(nM)
O
O
O
O
O
8q
8r
>100,000
8a
12
O
O
O
>100,000
8b
8c
39
85
O
8s
8t
>100,000
O
O
8d
8e
131
O
O
O
O
0.08
O
O
>100,000
O
O
O
O
O
8u
155
8f
8g
8h
8i
>100,000
231
8v
77
324
Cl
O
542
8w
8x
8y
231
687
988
8j
>100,000
O
O
O
O
8k
8l
110
124
BMS-790052
0.009
O
a
Data represent mean values of at least two experiments.
8m
195
O
O
O
8n
8o
8p
>100,000
>100,000
>100,000
Table 2
O
O
O
Conversion of Ester Prodrug 8t to Parent DCV in vitro
Compound
pH 2
pH 7
Blood
DCV
8t
Stable
Stable
Stable
Stable
Stable
Unstablea
a
t1/2 < 30 min.
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X. Zong et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 3
Pharmacokinetic parametersa
Compound administered
Compound measured
Cmax (ug/mL)
Tmax (h)
T1/2 (h)
AUC(0–8) (ug ⁄ h/mL)
AUC(0–1) (ug ⁄ h/mL)
MRT (h)
DCV
8t
DCV
DCV
0.484 0.163
0.341 0.112
3.01 0.23
4.05 0.21
1.66 0.24
2.93 0.31
3.21 0.88
3.33 0.93
3.32 0.98
3.44 0.89
3.43 0.51
4.01 0.22
a
Each value represents the mean SD of 6 rats.
In the present study, we efficiently synthesized a series of pro-
drugs of DCV and evaluated their anti-HCV activity. Several target
compounds exhibited modest anti-HCV activity. Among the tested
molecules, compound 8t demonstrated potent inhibition of HCV
replication in a GT-1b replicon with EC50 = 80 pM. The utility of
prodrug 8t was demonstrated through similar exposure of the par-
ent compound when the prodrugs were dosed in vivo. Our PK stud-
ies of 8t demonstrated that this prodrug is an excellent candidate
for a slower and sustained release form of DCV. Finally, further
in vivo studies and structure optimizations are currently being
investigated in our laboratory and will be the subject of future
publications.
700
600
500
400
300
200
100
0
parent (DCV) dosing
Exposure of parent
from 8t
0
1
2
3
4
5
6
7
8
9
10
Time (h)
Figure 3. Comparison of in vivo exposure of parent (DCV) when rats are dosed with
parent (red) or prodrug (8t, green) at 5 mg/kg parent or parent equivalent.
Acknowledgments
This work is supported by The Fundamental Research Funds for
the Central Universities (2242014R30019), Technology Supporting
Program of Jiangsu province (BE2012657), and National Basin
Research Program of China (No. 2011CB933503).
Compounds with electron-withdrawing group at the para position
of benzene ring exhibited better potency than those with electron-
donating group, for example, 8v (IC50 = 77 nM) > 8u (IC50
=
155 nM) > 8w (IC50 = 231 nM). Among the 25 DCV derivatives tested
in HCV replicon cells, 16 compounds showed modest anti-HCV
activity. Moreover, the 2-oxoethyl acetate compound 8t seemed to
be the most effective.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.06.
006.
Previous reports have demonstrated that the imidazole N–H is
essential to the anti-HCV activity,15 the substituent at the N would
greatly decrease the activity. The synthesized prodrugs were
hydrolyzed to release the corresponding free DCV that would then
exert their anti-HCV activity (Fig. 2). We speculated that the most
potent anti-HCV activity achieved by 8t was due to its high-effi-
ciency hydrolysis to the corresponding parent DCV. In the stability
study, 8t was initially stable in both acidic (pH 2/water) and neu-
tral (pH 7.0/water) conditions (Table 2). Therefore, considering
anti-HCV activity and hydrolysis stability, the ester prodrug 8t
was selected for further PK studies to observe whether it is a good
candidate for a slower and sustained release form of DCV.
The selected compound 8t was evaluated in a rat PK study. As
shown in Figure 3, the systemic exposure of 8t was converted to
DCV after low oral doses (5 mg/kg). The drug exposure of 8t was
similar to that of the parent (DCV) itself at equivalent dose levels.
Although plasma concentrations of the DCV released from 8t were
lower than those of DCV during the first 4.1 h, they became higher
than that observed with DCV dosing and remained higher through-
out the remainder of the experiment. Table 3 summarizes the PK
parameters related to these curves. The areas under the curve from
time 0 to 8 h (AUC(0–8)) obtained from both curves were very sim-
ilar. However, the PK behavior of the drug released from the 8t and
the DCV itself was different. This result suggests that the prodrug
did not change the bioavailability of DCV, but was well-absorbed
and efficiently converted to DCV in vivo, prolonging its activity.
Therefore, DCV can be released from 8t in mice, and 8t and DCV
have similar anti-HCV activities, demonstrating that 8t can serve
as a prodrug of DCV.
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