Discovery and Characterization of Potent Pan-Genotypic HCV NS5A Inhibitors Containing Novel Tricyclic Central Core Leading to Clinical Candidate

Article abstract of DOI:10.1021/acs.jmedchem.9b01562

The identification of a novel class of potent pan-genotypic NS5A inhibitors with good pharmacokinetic profile suitable for potential use in treating HCV infections is disclosed here. The present series of compounds are with less complex tricyclic central core, identified through a systematic SAR study carried out on biphenyl moiety. The SAR outcome has confirmed the requirement of near planar and linear conformation of the molecule to achieve the best pan-genotypic activity. In addition, SAR with substituted imidazoles on improvement of antiviral activity is disclosed. The newly identified compounds 12, 16, 19-21 have shown desirable pharmacokinetic profiles with a favorable uptake of compounds in liver and maintained a significant concentration for up to 8 h in the liver. In addition, compounds 20 and 21 have shown superior pan-genotypic anti-HCV activity compared to ledipasvir and daclatasvir. Additional characterization and preliminary safety assessment resulted in the identification of compound 20 as a potential clinical candidate.

Full text of DOI:10.1021/acs.jmedchem.9b01562

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Drug Annotation
Discovery and Characterization of Potent Pan-Genotypic HCV NS5A Inhibitors
Containing Novel Tricyclic Central Core Leading to Clinical Candidate
Vidya Ramdas, Rashmi Talwar, Moloy Banerjee, Advait Arun Joshi, Amit Kumar Das,
Deepak Sahebrao Walke, Prashant Borhade, Usha Dhayagude, Rajesh Loriya, Ganesh
Gote, Apparao Bommakanti, Aruna Sivaram, Gautam Agarwal, Arnab Goswami, Prashant
Nigade, Maneesh Mehta, Vinod Patil, Dipak Modi, Hemant Kumar, Sadanand Mallurwar,
Amruta Dash, Falguni Modi, Sandip Kuldharan, Pratima Srivastava, Minakshi Singh, Lakshmi
Narasimham, Jayasagar Gundu, Sharad Sharma, Rajender Kumar Kamboj, and Venkata P. Palle
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01562 • Publication Date (Web): 11 Nov 2019
Downloaded from pubs.acs.org on November 12, 2019
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Discovery and Characterization of Potent Pan-Genotypic
HCV NS5A Inhibitors Containing Novel Tricyclic Central
Core Leading to Clinical Candidate
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Vidya Ramdas,* Rashmi Talwar, Moloy Banerjee, Advait Arun Joshi, Amit Kumar
Das, Deepak Sahebrao Walke, Prashant Borhade, Usha Dhayagude, Rajesh Loriya,
Ganesh Gote, Apparao Bommakanti, Aruna Sivaram, Gautam Agarwal, Arnab
Goswami, Prashant Nigade, Maneesh Mehta, Vinod Patil, Dipak Modi, Hemant
Kumar, Sadanand Mallurwar, Amruta Dash, Falguni Modi, Sandip Kuldharan,
Pratima Srivastava, Minakshi Singh,Lakshmi Narasimham, Jayasagar Gundu, Sharad
Sharma, Rajender Kumar Kamboj, Venkata P. Palle
Novel Drug Discovery & Development, Lupin Ltd., Lupin Research Park, Survey No. 46 A /47 A,
Village Nande, Taluka Mulshi, Pune 412115, India
ABSTRACT: Identification of a novel class of potent pan-genotypic NS5A inhibitors with good
pharmacokinetic profile suitable for potential use in treating HCV infections are disclosed here. Present
series of compounds are with less complex tricyclic central core, identified through a systematic SAR study
carried out on biphenyl moiety. The SAR outcome has confirmed the requirement of near planar and linear
conformation of the molecule to achieve the best pan-genotypic activity. In addition, SAR with substituted
imidazoles on improvement of anti-viral activity is disclosed. The newly identified compounds 12, 16, 19-
2
1 have shown desirable pharmacokinetic profile with a favorable uptake of compounds in liver and
maintained a significant concentration for up to 8h in the liver. In addition, compounds 20 and 21 have
shown superior pan-genotypic anti-HCV activity compared to ledipasvir and daclatasvir. Additional
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characterization and preliminary safety assessment resulted in identification of compound 20 as a
potential clinical candidate.
INTRODUCTION
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Hepatitis C is a liver disease caused by the Hepatitis C Virus (HCV) and is a chronic blood-borne disease
with high persistence rates (80%) and affects an estimated 71 million people globally based on a 2018
1
report from the World Health Organization (WHO). It is often referred to as “silent disease” because the
infection is asymptomatic making it very difficult for detection at early stage and consequently making it
difficult for early treatment. Unless successfully treated, chronic HCV infection can result in liver cirrhosis,
2
an increased incidence of hepatocellular carcinoma, and ultimately liver failure. Importantly, humans are
the only reservoir for HCV infection and most commonly spread by direct contact with infected
blood/blood products. According to data from WHO, approximately 399000 people die each year from
hepatitis C, mostly from cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine to prevent
HCV (in contrast to HAV and HBV).1
Hepatitis C Virus is a member of the flaviviridae family and there are 11 different genotypes known (e.g.
1
,2, 3 ,4 etc.) with 30% heterogeneity in their nucleotide sequence. ~100 different subtypes exist termed
quasispecies” (a, b, c etc.). The distribution, transmission and disease progression varies amongst the
genotypes. Worldwide 6 genotypes are most commonly present. Genotype 1 is the commonest in US
75%) followed by 2 & 3 (10% each). Genotype 3 is more prevalent in Asian populations and genotype 4 is
“
(
present (difficult-to-treat segment) in Middle East patient population.3
The treatment of Hepatitis C virus infection (HCV) has seen a revolution over the past decade from a
poorly tolerated early standard of care (SOC) involving pegylated interferon-α (IFN) and ribavirin with low
sustained virologic response (SVR) to the treatment regimen involving highly effective direct-acting
4
antivirals (DAAs) with SVR rates over 90%. In spite of this transformation in the treatment paradigms in
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HCV therapy, there were challenges existed in the treatment of HCV due to the presence of geographical
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and ethnic diversity present in the virus along with resistance associated variants (RAV). To deal with this
diversity along with the suppression of emergence of RAV arising from a single mechanism of action, it
was logical that the treatment paradigm involve administration of a combination of direct acting antiviral
agents which target different key steps of the HCV life cycle. Based on this strategy, three classes of DAA
have been developed which target the NS3/4A protease, the NS5A protein and the NS5B RNA-dependent
RNA polymerase. Combination therapy of DAAs with different mechanism of actions reduced the drug
resistance and increased the effectiveness of the anti-virals leading to very high SVR rates and reduced
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the treatment durations to 12 weeks of therapy. Recently, USFDA has even approved an eight week
regimen of combination therapy for treatment of HCV GT1-6.
The HCV NS5A protein has no known enzymatic activity, but it is essential for HCV RNA replication and
virus assembly. It has potential to simultaneously inhibit multiple steps in viral replication and/or viral
assembly and the exact mechanism of action is unclear. There is no known mammalian homologue of
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NS5A protein. NS5A inhibitor is a core component of all oral IFN-free/RBV-free DAA regimen due to its
extreme potency coupled with relatively high barrier to resistance and broad spectrum coverage of major
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HCV genotypes. The NS5A inhibitors produce the most rapid reduction in viral load with low picomolar
activity and they have exhibited additive or synergistic effects with protease and polymerase inhibitors.
Hence, this class of inhibitors has seen a significant focus since the disclosure of BMS-790052 in 2010.9
Some of the first generation inhibitors showed a low resistance barrier as well as weaker activity against
genotypes GT2/3. With significant progress made over the past few years in the development of NS5A
inhibitors, so far there are six NS5A inhibitors, daclatasvir,10 ledipasvir,11 velpatasvir,12 ombitasvir,13
14
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pibrentasvir and elbasvir have been approved for the treatment of chronic HCV in various combination
with other DAAs, Figure 1.
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Our goal was to identify and develop a novel, pan-genotypic, oral NS5A inhibitor for utility in treatments
for HCV infections. Many of the reported structures of NS5A inhibitors from Merck, Abbvie and Gilead are
with 5-7 chiral centers.12-15 One of the major structural modification carried out by different groups is
around the biphenyl spacer between the two peptidic moieties.11-19 Some of the approved drugs possess
more complex tetracyclic or pentacyclic spacer as a central core, Figure 1. Herein, we report design,
synthesis and anti-viral activity of some of the novel NS5A inhibitors having simplified tricyclic core with
varying degree of conformational flexibility.
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RESULTS AND DISCUSSION
Figure 1. FDA Approved HCV NS5A Inhibitors with Different Type of Central Core
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Our objective was to identify a novel, potent pan-genotypic NS5A inhibitor with good pharmacokinetic
profile. To meet this prime objective, our plan was to develop systematic structure-activity relationship
(
SAR) around biphenyl central core with the aim of determining the impact of molecular conformation on
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pan-genotypic anti-HCV activity. Towards this goal we have designed some of the novel modification on
biphenyl core of first-in-class NS5A inhibitor BMS-790052 (daclatasvir) and tested their inhibitory activity
against both GT1b and GT2a replicons, which was used as primary cell based assay to evaluate all the
synthesized compounds. To begin with, we tried some of the heterocyclic or carbocyclic ring spacer
between the two phenyl rings as shown in compounds 1-4. Compounds 1-3 have shown sub-nanomolar
(
100-300 pM) activity for GT1b, however their activity against GT2a were in low nM range, Table 1.
20
21
Although compounds 1 and 2 were reported earlier with GT1b activity, their activity against GT2a were
not known. Compound 4 has shown activity in nM range for both GT1b and 2a. When we compare the
genotypic activity (GT1b and GT2a) of these compounds with in-house generated anti-HCV activity of
daclatasvir, the SAR indicates that both length and conformation of the molecule have impact on potency
22
of anti-HCV activity and the effect due to conformational change is very significant with respect to GT1b
potency.
Table 1. Anti-viral Activity of NS5A Inhibitors with Ring Spacer between Two Phenyl Rings
GT1b IC50
nM
GT2a IC50
nM
Compound
Ring spacer
Daclatasvir
BMS-790052)
Absent
0.023±0.012* 0.009±0.003*
(
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0.312±0.059
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*
In-house data
Then we tried some of the novel tetracyclic system with different out-of-plane conformation as central
core as shown in compounds 5-7. Compounds 5 & 6 have significantly lost the potency for both the
genotypes. However, compound 7 has shown 0.08 nM for GT1b and 27 nM for GT2a, Table 2, in similar
lines with compounds 1-3. Again, SAR of these modifications confirmed the near planar conformational
requirement of the molecule for potent pan-genotypic activity and out-of-plane conformation was not
tolerated.20, 22 Compounds 5-7 were tested as diastereomeric mixture due to challenges in isolation of
pure isomers.
Table 2. Anti-viral Activity of NS5A Inhibitors with Tetracyclic Core as a Spacer between Peptidic
Moieties
GT1b IC50
nM
GT2a IC50
nM
Compound Tetracyclic core
5
79% inhibition @ 1 μM 13% inhibition @ 1 μM
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75% inhibition @ 0.1 μM 11% inhibition @ 1 μM
0.08
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Based on these outcome, we have decided to keep our focus on studying the degree of conformational
flexibility of biphenyl which is tolerated for pan-genotypic activity by bridging the biphenyl to a
synthetically less complex, achiral tricyclic systems and by which to identify a novel and potent pan-
genotypic compounds with good PK profile across the species. Examination of known SAR around biphenyl
core indicated that in one of the early first generation NS5A inhibitors, ledipasvir, Fig.1, Gilead has tried
to fuse the biphenyl core to a substituted fluorene system as a spacer and the compound was found to
be around thousand fold less potent than daclatasvir for GT2a (21 nM) and GT3a (41 nM).11b This data
intrigued us and we have synthesized compounds 8-12 by tying the biphenyl system to a larger tricyclic
core with a bridge of varying length and tested their activity against GT1b and GT2a, since our initial
optimization goal is to identify a compound with low pM activity in both GT1b and 2a replicon assay.
21
Compound 8 with [6,6,6] system has shown low pM activity for both GT1b and GT2a. The larger ring
systems which will provide more flexibility to conformation of biphenyl as shown in compounds 9 and 10
were tried to check the effect of ring enlargement on activity. Compound 9 has shown around 300 pM
activity for both GT1b and GT2a, about twenty to thirty fold decrease in potency compared to compound
8. Further ring enlargement as shown in compound 10 has shifted the activity to nM range for GT2a (1.3
nM), Table 3. Then, gem diMe and spiro cyclopropyl substitutions were introduced on compound 9 and
checked the potency of resultant compounds 11 and 12. Both the substitutions have improved the
potency by 2-4 fold for GT1b, however different tendency was observed for GT2a. For further
improvement of potency of compounds 9 and 12, we have explored substitution on imidazoles by keeping
the central tricyclic core constant. When imidazoles were substituted with halogens like chlorine,
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compounds 13-16 have shown significant improvement in potency for GT1b compared to the
corresponding unsubstituted parent compounds 9 and 12. When both the imidazoles were substituted
with chlorine as in compounds 14 and 16, the improvement in potency for GT1b is remarkable (>10 fold)
compared to when only one of the imidazole is substituted with chlorine. However, alkyl groups such as
ethyl or cyclopropyl substitution on imidazoles as shown in compounds 17 and 18 reduced the potency,
in particular they were not tolerated for GT2a. We are not sure whether chloro substitution on imidazole
is bringing a favorable active conformation to these type of molecules, because chloro substitution on
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both the imidazole of daclatasvir did not alter its potency. To our knowledge, this kind of SAR on bis-
imidazole moiety is observed for the first time as we did not come across any reports wherein drastic
change in replicon activity with respect to substitution on imidazoles is noted.
Table 3. Anti-viral Activity of NS5A Inhibitors with Rigid to Flexible Tricyclic Core as a Spacer
GT1b IC50
pM
GT2a IC50
pM
Compound
821
R
R’
Tricyclic core
-H
-H
15
9
9
-H
-H
-H
-H
288±24
314±47
1
0
194
1300
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11
-H
-H
-H
-H
135
1800
1
2
69±19
176±79
1
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1
1
1
3
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5
-Cl
-Cl
-Cl
-H
-Cl
-H
51±0.3
19±1.4
21
53±3
31±20
671
1
6
-Cl
-Cp
-Et
-Cl
4±1
322±196
6800
17
-Cp
-Et
115±36
219±63
1
8
80000
As our goal was to further improve the potency for GT2a, we have focused our attention on [6,6,6] tricyclic
system which has shown the best potency for GT 1b and GT2a. We were interested to try bioisosteric
replacement of one of the phenyl ring on central tricyclic core. With that interest, compounds 19-23 were
synthesized with different type of bridge between phenyl and thiophene rings. To our delight, all the
compounds have shown low pM activity for GT1b. However, it is interesting to note that Shi J et al have
reported earlier that bioisosteric replacement of one of the phenyl to thiophene ring on daclatasvir led to
2
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significant decrease in potency for GT1b (90.6 nM). This huge difference in potency can’t be even
attributed to assay variation. It is probably the near planar conformation of the central core in compounds
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9-23 along with linearity of the molecules resulted in significant improvement of potency. In addition,
we have again observed a strong SAR correlation for GT2a, Table 4a. Compounds 19-21 with 2 atom bridge
have shown low pM activity for GT2a, which is a remarkable improvement in potency for GT2a compared
to Ledipasvir containing [6,5,6] tricyclic system.11a However, compounds 22 and 23 with 3 atom bridge
have shown either high pM or low nM activity. Overall, the SAR clearly indicates that there is a near planar
conformational requirement for the central core for pan-genotypic anti-HCV activity and the two atom
bridge was optimum to provide best potency for both GT1b and 2a out of two to six atom bridges used to
tie the biphenyl core. It is important to note that in the case of ledipasvir one of the imidazole ring is
replaced by benzimidazole. Compound 24, wherein thiophene ring was replaced with thiazole ring, did
not show any effect upto 1 M for GT2a. This is probably due to non-linear conformation of the molecule.
Merck has earlier reported fused tricyclic system containing different heterocycle as NS5A inhibitors and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
24
observed similar SAR trend for GT2a. From the current SAR, compounds 19-21 were found to be the best
compounds in terms of potency for both GT1b and GT2a and hence we have decided to profile them in
detail.
Table 4a. Anti-viral Activity of NS5A Inhibitors with Rigid Thiophene Containing Tricyclic Core as a Spacer
GT1b IC50 GT2a IC50
Compound
19
R
R’
Tricyclic core
pM
pM
-H
-H
21±2
21±17
2
0
-H
-H
12±4
5±2
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2
2
1
2
3
4
5
-H
-H
-H
-H
-Cl
-Cl
-H
20±5
21
30±15
801
-H
-H
-H
-Cl
-Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
22±2
73
10600
0% @ 1μM
52±34
1300
20±15
8
26
Table 4b. Anti-viral Activity of NS5A Inhibitors with Pyrrolidine Modification
GT1b
GT2a
Compound
Structure
IC50 pM IC50 pM
2
2
7
8
13
21
11
337
Based on our earlier SAR learning, we have prepared chloro substituted analogs of compounds 22 and 23
to improve the potency. These analogs 25-26 have shown improvement in potency, in particular, about
1
0 fold improvement for GT2a, however they were inferior compared to compounds 19-21, Table 4a. We
have also tried amino acid modification on compound 20 with the aim of improving the potency while
conserving the central core structure. When one of the pyrrolidine was replaced with bridged pyrrolidine
ring, compound 27 had retained the potency for GT1b and it has shown two fold decrease in potency for
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
GT2a. But, when both the pyrrolidine rings were replaced with bridged pyrrolidine rings as shown in
compound 28, significant decrease in potency was observed for GT2a, Table 4b.
Table 5. Metabolic Rate, CYP Inhibition and Cytotoxicity Profile of Selected Compounds
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cytotoxicity
Inhibition @ 3, 10µM
Metabolic Rate
nmol/min/mg)
% CYP Inhibition
(@ 10 µM)
%
(
Cmpd.
RLM
0.01
0.0
HLM
0.02
0.0
2D6
2C9
0
3A4
Huh7
4, NT
25, NT
2, NT
0, 14
HepG2
8, 35
23, 27
0, 6
HEK
17, 33
44, 63
2, 28
1
1
2
6
17
6
0
29
35
42
41
6
19
0.01
0.01
0.01
0.01
0.04
0.02
36
12
13
56
12
22
2
2
0
1
3, 22
0, 16
0, 36
3, NT
14, 22
Table 6. Pharmacokinetic Profile and Liver Exposure of Selected Compounds in Rats
IV (1 mpk)
PO (10 mpk)d
LE (μM) PE (μM) L/P
(@ 4h) (@ 4h) Ratio
(4 h)
Cmpd.
CL
AUClast
T1/2
(h)
Vss
AUClast
Cmax
μM)
(ml/m
in/kg)
%F
(
(
h*μM)
(l/kg)
(h*μM)
2.1±
0.2
2.3
0.7
۪
±
1.9±
0.8
a1
2
47
56
30
30
7.6
7.1
7.4
8.5
0.62
0.64
1.25
0.60
13
11
1.1±0.2
17±3
5.2±1.8
2.4±1.1
6±2
b
1
0.43± 3.8±
0.5±
0.1
3
7±12 10±3
6
9
0
0.1
0.1
b
1
1.5±
0.3
1.1± 1.6±
0.1
2
2
±0.2
10±1
6
0.4
b
2
2±
0.4
2±
0.6
±0.6
11±4
1±0.9
6±6
14.5
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9
c
1.9±
.1
1.5±
0.6
2
±0.3
10±2
2±0.3
6±2
30
7.9
1.39
5.5
2
1
0
Vehicle: aIV: 0.9% Normal Saline (100); bIV: DMA:PluronicF86:Saline (10:10:80); cIV: PEG-300:PG:Saline (20:15:65); dPO:
Tween:0.5% w/v CMC in MQ water (1:99)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compounds 12, 16, 19-21 were chosen for detailed ADME profiling based on their in-vitro potency. We
were also interested in understanding the influence of chloride substitution on physchem and ADME
properties compared to the parent unsubstituted imidazole analog. Hence, compound 16 was chosen
over compound 14, though compound 16 was inferior to 14 for GT2a potency. All the compounds were
found to be metabolically stable and have shown <50% inhibition at 10 M for major CYP isoforms such
as CYP2D6, 2C9 and 3A4, except for compound 19 which has shown 56% inhibition at 10 μM for CYP3A4,
Table 5. In vitro cytotoxicity potential of the selected compounds were tested in Huh7, HepG2 and HEK
cells up to a maximum concentration of 10 M. Except for compound 16 in HEK cell line, significant
cytotoxicity was not observed with any of the other compounds, Table 5. Compounds 16, 19 and 20 were
found to have >99% plasma protein binding in both rat and human. Main draw back with many NS5A
inhibitors reported in the literature was poor oral bio-availability and sub-optimal t1/2 suitable for once
daily dosing.12-14,25 Merck has extensively worked on chiral tetracyclic system as a spacer between the
peptidic moieties. Detailed SAR studies led to identification of many clinical candidates from this core
modification. However, most of these compounds with complex tetracyclic core have shown either poor
2
5
or reasonable PK profile across species. Whereas, all the selected compounds 12, 16, 19-21 containing
simple and novel tricyclic core from current SAR study have shown good rat PK profile with AUC >2μM.h,
t
1/2 about 2-4 hours and oral bioavailability of ≥30%. Also, we did not see any significant difference in
overall profile of compound 12 vs 16 except for decrease in kinetic solubility of 16 over 12 (data not
shown). More importantly, to our delight all these compounds have shown favorable liver uptake with
sustained exposure in rats for up to 8h and a ratio of liver to plasma concentration at 4h ranging from 5.5-
14.5 : 1, Table 6.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Based on overall profile, three compounds from [6,6,5] system, 19-21 were chosen for dose escalating PK
study in rats and dog PK studies. All three compounds have shown dose dependent increase in exposure
in rats, Table 7. Also, the compounds have shown very good exposure, t1/2 and oral bioavailability in dogs,
Table 8 and dose related increase in exposure was observed with compound 20, Table 7.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 8. Pharmacokinetic Profile of Compounds 19, 20 and 21 in Dog
IV ( 1 mpk)
CL
PO (10 mpk)
Cmpd. Species
AUClast
T1/2
(h)
Vss
AUClast
Cmax
(μM)
%F
(
ml/min/kg)
(
h*μM)
(l/kg)
(h*μM)
a
100, 83,
Dog
4±2
4±1
5±2
1±0.6
6±4
93,25,11*
1
9
0
3
7*
b
c
1
Dog
Dog
3±1
3±1
4±0.4
3±0.6
6±1
7±4
2±0.5
2±0.1
5±2
3±1
49±11
40±17
100
2
100
2
a
b
Vehicle: IV: DMA:PluronicF68:Saline (10:10:80); PO: 0.5% CMC:Tween80 (1:99); IV : DMA:Pluronic F68:20%Captisol in Saline
(10:10:80); PO: 0.5%w/vCMC with 1% Tween; IV: DMA:PEG300:Saline (10:20:70); PO: Gelucire 44/14:Labrasol:PG:Tween-80:H2O
c
(
15:15:10:1:59); *2/3 dogs shown emesis
Table 7. Single Dose Rising PK Profile of Compounds 19, 20 and 21 in Rat
PO
Specie
Cmpd.
AUClast
Dose
mpk)
Tmax
(h)
Cmax
(μM)
s
(
(
h*μM)
1
0
0.25-2
1-4
6±1
14±2
43±16
0.9±0.4
3±2
24±7
Rat
DRF
1
9a
30
00
77±6
468±164
4±2
1
2-4
1
0
4-6
Rat
DRF
20b
30
4-6
24±17
127±49
49±11
100
10
4-6
15±5
5±2
2
0a
1±0.3
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3
0
4±0.6
3±0
2-4
10±4
17±4
0.3±0.2
2±1
86±33
179±60
2±0.9
Dog
DRF
1
00
1
0
21b
Rat DRF
30
4-6
28±12
76±31
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
00
2-6
7±1
a
Vehicle: 0.5% w/v CMC with 1% Tween;
Tween 80:Gelucire:Labrasol:PG:Milli Q (1:15:15:10:59)
b
The best two compounds 20 and 21, on the basis of their pM potency and good pharmacokinetic profile
across the species, were chosen for testing the anti-viral activity against other genotypes GT3a, 4a and 6a.
10
Compound 20 has shown 3 fold better potency for GT3a compared to daclatasvir and several hundred
fold better over ledipasvir,11b Table 9. Compound 21 was found to be relatively inferior compared to
compound 20. We have tested both these compounds in predominant HCV GT1a & GT1b NS5A variants
such as L31V, Y93H, and GT1a Y93N, Q30E and some of the double mutants which are known to cause
resistance. Though the compounds have shown less shift in potency for some of the variants, the overall
26
profile with respect to fold resistance relative to wild type was similar in line with daclatasvir. Compound
20 has been selected for further development studies based on its overall superior profile.
Table 9. Anti-viral Activity of Compounds 20 & 21 for GT3a/4a/6a
Parameters
20
12
5
21
20
30
31
75
9
Daclatasvir10
GT1b IC (pM)
9
71
50
146
12
54
50
GT2a IC (pM)
50
GT1a IC (pM)
2
4
7
7
50
GT3a IC (pM)
50
GT4a IC (pM)
3
50
GT6a IC (pM)
2
8
55
50
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CONCLUSION
In conclusion, we have identified compounds 20 and 21 having a less complex central tricyclic core as
novel and potent pan-genotypic NS5A inhibitors with good pharmacokinetic profile. A systematic SAR
study by bridging the biphenyl central core with a linker of varying length from two - six atom to examine
the role of degree of conformational flexibility of biphenyl central core on pan-genotypic anti-HCV activity
led to an understanding that 2 atom bridge is optimal to obtain molecules with desired pan-genotypic
activity. Also, the observed SAR confirmed that near planar and linear conformation is required to achieve
the best potency across various genotypes. As aimed, we could achieve a remarkable improvement in
potency against GT2a and 3a replicons with compounds 20 and 21 compared to ledipasvir and daclatasvir.
Overall, compound 20 has shown a superior pan-genotypic profile and a good pharmacokinetic profile
coupled with a favorable liver uptake and maintained significant level of concentration for up to 8h in the
liver. Additional characterization and preliminary safety assessment resulted in identification of
compound 20 as a potential clinical candidate.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CHEMISTRY
Synthesis of C2 symmetric compounds 9-12 containing dibenzooxaheterocycle as a central core is
delineated in Scheme 1. A base mediated dialkylation of the phenolic hydroxyl groups of 1,1’-(2,2’-
dihydroxy-[1,1’-biphenyl]-4,4’-diyl)-diethanone (29) with 1,3-dibromopropane or 1,4-dibromobutane or
2,2-dimethylpropane-1,3-diyl-bis-(4-methylbenzenesulfonate) or
a
Mitsunobu reaction with
cyclopropane-1,1’-diyldimethanol gave the annulated products with different ring size (9 and 10) and
substitution pattern. The diacetyl compounds (30a-d) thus obtained, underwent alpha halogenation
reaction with bromine to give the corresponding bis-phenacyl bromides (31a-d). The bromination reaction
was not selective in producing only the α-brominated products, but some tri and tetra-brominated
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9
/
products were also obtained resulting from α, α -dibromination of the acetyl group. This heterogenous
mixture of polybrominated products were debrominated to the required bis-phenacyl bromides by careful
27
reduction with diethyl phosphite in presence of triethylamine. O-Alkylation of these dibromides with N-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Boc-L-Proline gave the ketoesters (32a-d), which underwent cyclization to the imidazole moieties (33a-d)
28
in presence of excess ammonium acetate in refluxing toluene. Cleavage of the Boc protecting groups in
presence of trifluoroacetic acid and bis amidation of the resultant pyrrolidines with Moc-Valine under
standard HATU/DIEA condition provided the final product, compounds 9-12.
Scheme 1. Preparation of Compounds 9-12
Reagents and conditions: (a) (i) 1,3-dibromopropane or 1,4-dibromobutane or 2,2-dimethylpropane-1,3-
o
diyl bis(4-methylbenzenesulfonate), K
2
CO
3
, DMF, 90 C, 16-18h, 61-72%; or cyclopropane-1,1-
, DCM-Toluene-THF (2:1:1), 0 C to RT, 18h, 37%; (b) (i) Br
N, THF, 0 C to RT, 1-2h, 53-91%; (c) N-Boc-L-proline, EtN(i-Pr)
o
o
diyldimethanol, DIAD, PPh
3
2
, DCM, 0 C to RT, 1-
o
2
h; (ii) HP(O)(OEt)
2
, Et
3
2
, MeCN, RT, 3-4h, 48-
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
9
3%; (d) NH
4
OAc, Toluene, 115 C, 18h, 41-59%; (e) (i) TFA, DCM, RT, 2h; (ii) (methoxycarbonyl)-L-valine,
o
HATU, EtN(i-Pr)
2
, DMF, 0 C to RT, 18h, 15-40%
In order to investigate the influence of substituted imidazoles on the potency of compounds 9 & 12 for
GT1b and GT2a, mono and di chlorination and alkylation of the imidazole rings were planned, Scheme 2.
The chlorination of compounds 9 & 12 was done in the presence of NCS and the corresponding mono and
dichlorinated products 13, 14 & 15, 16, respectively were separated by preparative HPLC. The ethyl and
cyclopropyl substituted analogs were obtained by bromination of the imidazole rings of Boc-protected
pyrrolidine intermediate 33d in presence of phenyl trimethyl ammonium tribromide, subsequent Suzuki
coupling with the corresponding boronic acids and conversion to the final compounds 17 & 18 by Boc-
deprotection and amide coupling.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Preparation of Compounds 13-18
o
Reagents and conditions: (a) NCS, DMF, 45 C, 1.5h, 7-36%; (b) Phenyltrimethylammonium tribromide,
o
o
THF, 0 C to RT, 2h, 57% ; (c) R
3
B(OH)
2
, K
3
PO
4
, PCy
3
, Pd(OAc)
2
, Toluene-H O (5:1), 110 C, 45 min, MW, 41-
2
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2
3
4
5
6
7
8
9
o
4
1
2%; (d) (i) TFA, DCM, RT, 2h; (ii) (methoxycarbonyl)-L-valine, HATU, EtN(i-Pr)
0%
2
, DMF, 0 C to RT, 15-18h, 8-
Scheme 3 depicts the reaction sequence followed for the construction of rigid tricyclic core containing
thiophene ring and its subsequent conversion to the peptide capped final compounds. The starting
materials for the oxygen containing tricycle were 7-bromochroman-4-one (36a) and 8-bromo-3,4-
dihydrobenzo[b]oxepin-5(2H)-one (36b) while for the six and seven membered carbocycle annulated
tricycles, α-tetralone (36c) and 6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-one (36d), respectively served
as the starting points. The α-methylene ketones were converted to the corresponding β-chloro vinyl
aldehydes (37a-d) by the Vilsmeier-Haack reaction, which after a base mediated cyclisation with
mercaptoacetone provided the thiophene fused tricycles (38a-d) with an acetyl group adjacent to the
sulfur atom.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Preparation of Compounds 19, 21-23
o
o
Reagents and conditions: (a) DMF, POCl
MeOH, 0 C to RT, 3-4h, 77-89% (c) (i) Acetyl Chloride, AlCl
3
, CHCl
3
, 0 C to 60 C, 16h, 50-93%; (b) Mercaptoacetone, NaOMe,
, DCM, 0 C to RT, 48h, 80-85% (when R
, Dioxane, 90 C, 18h; 4N HCl, RT, 1 h, 67-68% (when R
o
o
3
1
= H)
o
(ii) Tributyl(1-ethoxyvinyl)stannane, PdCl
2 3
(PPh )
2
1
=
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
o
Br); (d) (i) Br
2
, DCM, 0 C to RT, 1h; (ii) HP(O)(OEt)
2
, Et
3
N, THF, 0 C, 1-2h, 88-96%; (e) N-Boc-L-proline, EtN(i-
o
Pr) , MeCN, RT, 3-4h, 66-90%; (f) NH
(methoxycarbonyl)-L-valine, HATU, EtN(i-Pr)
2
4
OAc, Toluene, 115 C, 15-18h, 51-70%; (g) (i) TFA, DCM, RT, 2h; (ii)
o
2
, DMF, 0 C to RT, 15-18h, 24-89%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The other acetyl group on the phenyl ring was either installed by a Stille reaction with Tributyl(1-
ethoxyvinyl)stannane and subsequent acid hydrolysis of the enol ether intermediate (for 39a-b) or a
Friedel-Crafts reaction with acetyl chloride in presence of AlCl (for 39c-d). Further elaboration of the bis-
3
acetyl tricycle core to the final compounds (19, 21-23, 25 & 26) followed the same reaction sequence,
methods and conditions as discussed in Schemes 1 and 2.
Scheme 4. Preparation of Compounds 20 & 28
o
Reagents and conditions: (a) DDQ, Toluene, 110 C, 60h, 55%; (b) (i) Br
THF, 0 C, 2h, 97%; (c) (1R,3S,4S)-2-(tert-butoxycarbonyl)-2-azabicyclo[2.2.1]heptane-3-carboxylic acid or
N-Boc-L-proline, EtN(i-Pr)
DCM, RT, 2h; (ii) (methoxycarbonyl)-L-valine, HATU, EtN(i-Pr)
2
, DCM, RT, 2h; (ii) HP(O)(OEt)
2
, Et N,
3
o
o
o
2
, MeCN, 50 C, 3h, 80%; (d) NH
4
OAc, Toluene, 115 C, 16-18h, 66-74%; (e) (i) TFA,
o
2
, DMF, 0 C to RT, 18h, 54-68%
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5
6
7
8
9
For the synthesis of compound 20 with completely planar [6,6,5] fused tricyclic central core, intermediate
3
9c was aromatized into naphtho[1,2-b]thiophene core 43 in presence of DDQ and the acetyl groups on
both ends were further elaborated to the final compounds 20 & 28 with proline-valine and a [2.2.1]
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
bicyclic proline-valine, respectively as the peptide cap according to methods discussed in Schemes 1 and
2
. In order to have an unsymmetrical peptidic cap at both ends of the rigid tricyclic core, the synthetic
sequence as shown in Scheme 5 was adopted for compound 27. The key intermediate identified for this
unsymmetrically capped compound was the tricyclic core 50, which was synthesized from 6-Bromo-1-
tetralone 47 in three steps. The acetyl group in 50 underwent an α-tosyloxylation reaction in presence of
29
[
hydroxy(tosyloxy)iodo]benzene (HTIB, Koser’s reagent). O-alkylation of the tosylate 51 with (1R,3S,4S)-
-(tert-butoxycarbonyl)-2-azabicyclo[2.2.1]heptane-3-carboxylic acid gave the ketoester 52 which was
2
cyclized to the imidazole derivative 53. With the bicyclic proline added at one end of the core, the bromo
was converted to the boronate 54 via Miyaura boration. The proline at the other end of the core was
added via a Suzuki coupling of the boronate with tert-butyl (S)-2-(5-iodo-1H-imidazol-2-yl)pyrrolidine-1-
carboxylate to give intermediate 55 which upon Boc deprotection and bis amidation of the resultant
pyrrolidines with Moc-Valine gave the final compound 27.
Scheme 5. Preparation of Compound 27
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
o
Reagents and conditions: (a) DMF, POCl
3
, CHCl
3
, 0 C to 60 C, 6h, 83%; (b) Mercaptoacetone, NaOMe,
o
o
MeOH, 0 C to RT, 4h, 91%; (c) MnO
2
, Toluene, 100 C, 26h, 85%; (d) hydroxytosyloxyiodobenzene, MeCN,
o
7
Pr)
PdCl
5 C, 8h, 82%; (e) (1R,3S,4S)-2-(tert-butoxycarbonyl)-2-azabicyclo[2.2.1]heptane-3-carboxylic acid, EtN(i-
o
o
2
, MeCN, 50 C, 4h, 70%; (f) NH
4
OAc, Toluene, 115 C, 18h, 62%; (g) bis(pinacolato)diboron, KOAc, PCy ,
3
o
2
(dppf)-DCM, Dioxane, 95 C, 8h, 92%; (h) tert-butyl (S)-2-(5-iodo-1H-imidazol-2-yl)pyrrolidine-1-
o
carboxylate, K
3
PO
4
, Pd(PPh
3
)
4
, PCy
3
, Toluene-H
2
O (3:1), 100 C, 24h, 45%; (i) (i) TFA, DCM, RT, 2h; (ii)
, DMF, 0 C to RT, 18h, 9%
o
(methoxycarbonyl)-L-valine, HATU, EtN(i-Pr)
2
Compound 24 containing the thiazole fused 6,6,5-tricyclic core was synthesized starting from 6-Bromo-1-
tetralone 47, which upon cyclisation with thiourea using Hantzch thiazole synthesis conditions gave the
tricyclic thiazole amine 56, Scheme 6. The amine was converted to bromo under diazotization conditions
and the dibromo compound 57 was converted to the diacetyl compound 58 by a Stille reaction at both
ends. The diacetyl compound thus obtained underwent alpha halogenation reaction with bromine to give
the corresponding bis-phenacyl bromide 59 with simultaneous aromatization of the tricyclic core. Further
elaboration of 59 to the final compound 24 followed the same protocols as discussed in Scheme 1.
Scheme 6. Preparation of Compound 24
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
t
o
Reagents and conditions: (a) Thiourea, I
h, 99%; (c) (i) Tributyl(1-ethoxyvinyl)stannane, PdCl
2
, Ethanol, 100 C, 4h, 80%; (b) BuONO, Cu(I)Br, MeCN, 50 C, 0.5
o
2
(PPh
3
) , Dioxane, 90 C, 18h; (ii) 4N HCl, RT, 1h, 73%;
2
(
d) Br
2
, DCM, RT, 18h, 99%; (e) (i) N-Boc-L-proline, EtN(i-Pr)
2
, CH
3
CN, RT, 4h; (ii) NH
4
OAc, Toluene, 18h,
, DMF, 0 C to RT, 18
o
o
115 C, 14%; (f) (i) TFA, DCM, RT, 2h; (ii) (methoxycarbonyl)-L-valine, HATU, EtN(i-Pr)
2
h, 13%
EXPERIMENTAL SECTION
Chemistry. All reagents were purchased from commercial suppliers and used without purification unless
otherwise noted. All anhydrous reactions were performed under a nitrogen atmosphere using dry
solvents. Silica gel column chromatography was carried out on ISCO CombiFlash Companion with
prepacked columns. Preparative HPLC were performed on a Gilson GX 281 series using a YMC ODS-A
column or YMC Triat column (50 mm X 250 mm, 10 µm) using either acid (0.1 % formic acid) or base (0.1%
1
NH
4
OH) modified MeCN-H
2
O gradients. Proton nuclear magnetic resonance ( H NMR) spectra were
recorded on a Bruker AvanceV-III 400 MHz spectrometer. All spectra were determined in the solvents
indicated, and chemical shifts are reported in δ units downfield from the internal standard
tetramethylsilane (TMS) with inter proton coupling constants reported in hertz (Hz). All NMR spectra were
analyzed using the MestReNova 10.0 program software. At ambient temperature, most of the final
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9
1
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1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
products exist as a mixture of rotamers. Liquid chromatography-mass spectrometry (LC-MS) analyses
were performed either on an Agilent 1200 Series LC instrument coupled to an Agilent Iontrap MS
instrument or a Thermo Scientific Accela LC instrument coupled to a Thermo Scientific MSQ Plus
instrument. HRMS spectrum were acquired by direct infusion of samples in positive ESI (Electro Spray
Ionization) mode on Thermo Q-Exactive HRMS at 1,40,000 resolution. Compound purity was determined
by reverse phase HPLC performed on an Agilent 1290 Infinity equipped with either a Zorbax Eclipse Plus
C18, 50 X 2.1 mm, 1.8 µm or an Acquity Beh C18, 50 X 2.1 mm, 1.7 µm column using either acid (0.1 %
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
formic acid) or base (0.1% NH
determined by HPLC and were greater than 95% for all the compounds except for compounds 19, 22 and
5. The synthetic procedures and the analytical data for the final compounds 3-7 along with the
intermediates thereof are provided in the supporting information.
4 2
OH) modified MeCN-H O gradients. Purity of the final compounds were
2
dimethyl
((2S,2'S)-((2S,2'S)-2,2'-(5,5'-(7,8-dihydro-6H-dibenzo[f,h][1,5]dioxonine-3,11-diyl)bis(1H-
imidazole-5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl))dicarbamate (9): To a
solution of 33a (0.09 g, 0.12 mmol) in DCM at 0 °C, trifluoroacetic acid (0.05 mL, 0.62 mmol) was added.
The reaction mixture was warmed to 25 °C and stirred for 2 h. The reaction mixture was concentrated
under reduced pressure. The content was taken up in DMF (2 mL) and cooled to 0 °C, after which DIPEA
(0.09 mL, 0.49 mmol) was added and the reaction mixture was stirred for 10 min. (S)-2-
((methoxycarbonyl)-amino)-3-methylbutanoic acid (0.04 g, 0.25 mmol) and HATU (0.09 g, 0.25 mmol)
were added to the reaction mixture at 0 °C. The reaction mixture was gradually warmed to 25 °C and
stirred for 18 h. Crushed ice was added to the reaction mixture and the precipitate was filtered.
1
Purification by preparative HPLC yielded an off-white solid (0.04 g, 40%). H NMR (400 MHz, CDCl
3
) δ 10.73
(brs, 1H), 10.36 (brs, 1H), 7.70 – 7.32 (m, 4H), 7.27 – 7.11 (m, 4H), 5.51 – 5.40 (m, 2H), 5.30 – 5.22 (m, 2H),
4
.48 – 4.43 (m, 2H), 4.40 – 4.28 (m, 2H), 3.93 – 3.81 (m, 2H), 3.77 – 3.70 (m, 6H), 3.64 (s, 2H), 3.19 – 2.98
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(
m, 2H), 2.48 – 2.28 (m, 2H), 2.28 – 2.18 (m, 2H), 2.09 – 1.97 (m, 4H), 1.14 – 0.89 (m, 12H). LCMS: m/z =
8
11.5 (M+H)+
dimethyl ((2S,2’S)-((2S,2’S)-2,2’-(5,5’-(6,7,8,9-tetrahydrodibenzo-[b,d]-[1,6]-dioxecine-3,12-diyl)-bis-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1H-imidazole-5,2))-bis-(pyrrolidine-2,1))-bis-(3-methyl-1-oxobutane-2,1-diyl))-dicarbamate
(10):
1
Synthesized from 33b by following an analogous procedure described for 9, (Yield = 15%). H NMR (400
MHz, CDCl
3
) δ 10.37 (s, 2H), 7.52 – 7.28 (m, 6H), 7.22 (s, 2H), 5.47 (d, J = 9.1 Hz, 2H), 5.34 – 5.26 (m, 2H),
.55 (s, 2H), 4.35 (t, J = 8.0 Hz, 4H), 3.85 (q, J = 8.2 Hz, 2H), 3.72 (s, 6H), 3.65 (d, J = 10.6 Hz, 2H), 3.09 (s,
H), 2.40 – 2.31 (m, 2H), 2.25 – 2.10 (m, 4H), 2.00 (d, J = 16.0 Hz, 4H), 1.08 – 0.80 (m, 12H); LCMS: m/z =
4
2
825.4 (M+H)+
dimethyl
((2S,2'S)-((2S,2'S)-2,2'-(5,5'-(6',8'-dihydrospiro[cyclopropane-1,7'-
dibenzo[f,h][1,5]dioxonine]-3',11'-diyl)bis(1H-imidazole-5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxobutane-2,1-diyl))dicarbamate (11): Synthesized from 33c by following an analogous
1
procedure described for 9, (Yield = 24%). H NMR (400 MHz, CD
3
OD): δ 7.53 – 7.46 (m, 2H), 7.45 – 7.40
(m, 2H), 7.38 – 7.32 (m, 2H), 7.26 – 7.22 (m, 2H), 5.18 (dd, J = 7.8, 5.3 Hz, 2H), 4.35 – 4.19 (m, 6H), 4.06 –
3
1
.99 (m, 2H), 3.95 – 3.88 (m, 2H), 3.67 (s, 6H), 2.43 – 2.28 (m, 4H), 2.28 – 2.21 (m, 2H), 2.10 – 2.01 (m, 4H),
.00 – 0.91 (m, 12H), 0.59 – 0.55 (m, 4H); LCMS: m/z = 837.5 (M+H)+.
dimethyl
((2S,2'S)-((2S,2'S)-2,2'-(5,5'-(6',8'-dihydrospiro[cyclopropane-1,7'-
dibenzo[f,h][1,5]dioxonine]-3',11'-diyl)bis(1H-imidazole-5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-
methyl-1-oxobutane-2,1-diyl))dicarbamate dihydrochloride (12): Synthesized from 33d by following an
1
analogous procedure described for 9, (Yield = 32%). H NMR (400 MHz, CDCl
3
) δ 10.73 (brs, 1H), 10.36 (brs,
1
5
H), 7.60 – 7.52 (m, 2H), 7.52 – 7.43 (m, 2H), 7.26 – 7.22 (m, 2H), 7.20 – 7.10 (m, 2H), 5.53 – 5.39 (m, 2H),
.37 – 5.19 (m, 2H), 4.42 – 4.29 (m, 2H), 3.90 – 3.80 (m, 2H), 3.72 (s, 8H), 3.66 – 3.60 (m, 2H), 3.24 – 2.82
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1
1
1
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
m, 2H), 2.43 – 2.30 (m, 2H), 2.26 – 2.11 (m, 4H), 2.01 – 1.94 (m, 2H), 0.96 (s, 6H), 0.92 – 0.88 (m, 12H) ;
+
HRMS m/z calculated for C45
59
H N
8
O
8
[M + H ]: 839.4456, found 839.4445
methyl ((S)-1-((S)-2-(5-(11-(2-((S)-1-((S)-2-(methoxycarbonyl)amino-3-methylbutanoyl)pyrrolidin-2-yl)-
H-imidazol-5-yl)-7,8-dihydro-6H-dibenzo[f,h][1,5]dioxonin-3-yl)-4-chloro-1H-imidazol-2-yl)pyrrolidin-
1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (13) and dimethyl ((2S,2'S)-((2S,2'S)-2,2'-(5,5'-(7,8-dihydro-
H-dibenzo[f,h][1,5]dioxonine-3,11-diyl)bis(4-chloro-1H-imidazole-5,2-diyl))bis(pyrrolidine-2,1-
diyl))bis(3-methyl-1-oxobutane-2,1-diyl))dicarbamate (14): To a solution of 9, (0.30 g, 0.43 mmol) in DMF
3 mL) was added NCS (0.12 g, 0.86 mmol) and the mixture was heated at 45 ºC for 90 min., after which
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
(
the contents were added to water. The aqueous contents were extracted with EtOAc and the organic layer
was washed with brine. The organic layer was dried over sodium sulfate and purified by flash column
1
chromatography (30% EtOAc-hexane) to yield two compounds 13 (0.05 g, 18%). H NMR (400 MHz, CDCl
3
)
δ 10.80 (s, 1H), 10.37 (s, 1H), 7.64 – 7.58 (m, 1H), 7.52 – 7.40 (m, 2H), 7.36 – 7.30 (m, 2H), 7.26 – 7.17 (m,
2H), 5.49 – 5.38 (m, 2H), 5.31 – 5.21 (m, 2H), 4.50 – 4.38 (m, 4H), 3.94 – 3.79 (m, 2H), 3.79 – 3.68 (m, 8H),
3
.68 – 3.60 (m, 2H), 3.20 – 2.93 (m, 2H), 2.44 – 2.29 (m, 2H), 2.24 – 2.17 (m, 2H), 2.07 – 1.97 (m, 4H), 1.12
0.73 (m, 12H).; LCMS: m/z = 845.4 (M+H)+
–
1
and 14 (0.12 g, 36%) H NMR (400 MHz, DMSO-d
6
) δ 12.57 (s, 2H), 7.56 – 7.43 (m, 4H), 7.41 – 7.29 (m, 4H),
5
.05 – 4.93 (m, 2H), 4.45 – 4.32 (m, 4H), 4.05 (t, J = 8.4 Hz, 2H), 3.90 – 3.70 (m, 4H), 3.54 (s, 6H), 2.27 –
2.11 (m, 4H), 2.03 – 1.92 (m, 8H), 0.87 (dd, J = 15.6, 6.7 Hz, 12H); LCMS: m/z = 879.3 (M+H)+
methyl ((S)-1-((S)-2-(5-(11-(2-((S)-1-((S)-2-(methoxycarbonyl)amino-3-methylbutanoyl)pyrrolidin-2-yl)-
4
-chloro-1H-imidazol-5-yl)-7,7-dimethyl-7,8-dihydro-6H-dibenzo[f,h][1,5]dioxonin-3-yl)-1H-imidazol-2-
yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (15) and dimethyl ((2S,2'S)-((2S,2'S)-2,2'-(5,5'-
7,7-dimethyl-7,8-dihydro-6H-dibenzo[f,h][1,5]dioxonine-3,11-diyl)bis(4-chloro-1H-imidazole-5,2-
(
diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl))dicarbamate (16): Synthesized from
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7
8
9
1
1
2 by following an analogous procedure described for 13 and 14. (15), Yield = 7%; H NMR (400 MHz,
CDCl ): δ 10.80 (brs, 1H), 10.40 (brs, 1H), 7.60 – 7.52 (m, 1H), 7.49 – 7.43 (m, 1H), 7.37 – 7.28 (m, 3H), 7.26
7.19 (m, 2H), 5.48 – 5.38 (m, 2H), 5.25 (d, J = 8.1, 3.5 Hz, 2H), 4.39 – 4.30 (m, 2H), 4.26 – 4.15 (m, 2H),
3.93 – 3.82 (m, 2H), 3.72 (d, J = 1.3 Hz, 6H), 3.69 – 3.58 (m, 2H), 3.14 – 2.94 (m, 2H), 2.35 – 2.05 (m, 8H),
3
–
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.02 – 0.81 (m, 18H); LCMS: m/z = 873.4 (M+H)+
1
1
and (16), Yield = 21%; H NMR (400 MHz, CDCl
3
): δ 10.90 – 10.80 (brs, 2H), 7.48 – 7.41 (m, 2H), 7.34 (d, J
=
3
7.9 Hz, 4H), 5.42 (d, J = 9.1 Hz, 2H), 5.25 (dd, J = 8.1, 3.5 Hz, 2H), 4.43 – 4.08 (m, 2H), 3.91 – 3.77 (m, 2H),
.72 (s, 8H), 3.68 – 3.53 (m, 2H), 3.09 – 2.85 (m, 2H), 2.39 – 2.29 (m, 2H), 2.25 – 2.14 (m, 2H), 2.16 – 2.07
[M + H+]:
(m, 2H), 2.07 – 1.88 (m, 2H), 0.97 – 0.90 (m, 18H); HRMS m/z calculated for C45
H
57Cl
2 8
N O
8
9
07.3676, found 907.3679
dimethyl ((2S,2'S)-((2S,2'S)-((7,7-dimethyl-7,8-dihydro-6H-dibenzo[f,h][1,5]dioxonine-3,11-diyl)bis(4-
cyclopropyl-1H-imidazole-5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-
diyl))dicarbamate (17): Synthesized from 35a by following an analogous procedure described for 9, (Yield
1
= 8%). H NMR (400 MHz, CDCl
3
): δ 10.29 (br s, 1H), 10.00 (br s, 1H), 7.73 – 7.58 (m, 2H), 7.37 – 7.30 (m,
4
2
H), 5.43 (d, J = 9.1 Hz, 2H), 5.22 (s, 2H), 4.42 – 4.28 (m, 2H), 3.80 (d, J = 10.7 Hz, 2H), 3.71 (s, 8H), 3.61 (s,
H), 3.05 (d, J = 47.8 Hz, 2H), 2.42 – 1.91 (m, 10H), 0.97 (s, 14H), 0.93 – 0.89 (m, 12H) ; LCMS: m/z = 919.4
(
M+H)+
dimethyl ((2S,2'S)-((2S,2'S)-((7,7-dimethyl-7,8-dihydro-6H-dibenzo[f,h][1,5]dioxonine-3,11-diyl)bis(4-
ethyl-1H-imidazole-5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate
1
(
18): Synthesized from 35b by following an analogous procedure described for 9, (Yield = 10%). H NMR
(400 MHz, CDCl
3
): δ 10.24 (brs, 2H), 7.47 – 7.38 (m, 1H), 7.38 – 7.28 (m, 4H), 7.13 – 7.04 (m, 1H), 5.57 –
5
.40 (m, 2H), 5.32 – 5.16 (m, 2H), 4.47 – 3.98 (m, 4H), 3.93 – 3.76 (m, 2H), 3.72 (s, 6H), 3.69 – 3.45 (m, 2H),
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9
1
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1
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2
2
2
2
2
2
2
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3
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3
3
3
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4
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5
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5
5
5
5
5
5
5
6
3
0
.17 – 2.95 (m, 2H), 2.91 – 2.73 (m, 4H), 2.41 – 2.27 (m, 2H), 2.24 – 1.95 (m, 6H), 1.31 – 1.24 (m, 12H),
.96 – 0.83 (m, 12H) ; LCMS: m/z = 895.5 (M+H)+.
dimethyl
((2S,2'S)-((2S,2'S)-((4,5-dihydronaphtho[1,2-b]thiophene-2,7-diyl)bis(1H-imidazole-5,2-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate dihydrochloride salt
(19): Synthesized from 42c by following an analogous procedure described for 9, (Yield = 48%). The free
base was dissolved in MeOH and a 3 N HCl solution in MeOH was added to it and the resulting solution
was stirred for 30 min. The volume of MeOH was then reduced and acetone was added to precipitate out
a pale yellow solid, which was washed with diethyl ether and dried to obtain the dihydrochloride salt of
1
the title compound, Purity; 91.5% (HPLC). H NMR (400 MHz, CD
3
OD) δ 7.89 (s, 1H), 7.80 (s, 1H), 7.68 (d, J
=
1.7 Hz, 1H), 7.66 – 7.58 (m, 1H), 7.58 – 7.46 (m, 1H), 7.40 (s, 1H), 5.24 (dt, J = 12.2, 7.6 Hz, 2H), 4.24 (d,
J = 7.3 Hz, 2H), 4.13 (d, J = 10.3 Hz, 2H), 3.94 – 3.84 (m, 2H), 3.67 (s, 6H), 3.09 (t, J = 7.7 Hz, 2H), 3.01 – 2.89
(m, 2H), 2.62 – 2.52 (m, 2H), 2.33 – 2.06 (m, 8H), 0.96 – 0.88 (m, 12H); HRMS m/z calculated for
+
C
40
H
51
N
8
O
6
S [M + H ]: 771.3652, found 771.3634
dimethyl
((2S,2'S)-((2S,2'S)-(naphtho[1,2-b]thiophene-2,7-diylbis(1H-imidazole-5,2-
diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate dihydrochloride salt
(
20): Synthesized from 46a by following an analogous procedure described for 9 followed by conversion
1
to the dihydrochloride salt as in 19, (Yield = 68%). H-NMR (400 MHz, DMSO-d
6
): δ 8.61 (s, 1H), 8.35 – 8.19
(m, 2H), 8.14 – 8.10 (m, 1H), 8.02 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.42 – 7.19 (m, 2H), 5.27 –
5
2
1
.08 (m, 2H), 4.13 (q, J = 7.2 Hz, 2H), 4.05 – 3.91 (m, 2H), 3.91 – 3.78 (m, 2H), 3.55 (s, 6H), 2.45 – 2.31 (m,
13
H), 2.28 – 2.14 (m, 4H), 2.14 – 1.94 (m, 4H), 1.01 – 0.70 (m, 12H); C NMR (101 MHz, MeOD) δ 172.92,
58.13, 149.47, 149.15, 138.41, 136.62, 133.00, 131.16, 128.17, 128.08, 127.83, 126.17, 125.27, 124.24,
123.77, 123.65, 122.94, 115.57, 115.08, 58.57, 58.53, 53.72, 53.66, 51.47, 31.24, 29.77, 25.08, 18.63,
+
1
6.85; HRMS m/z calculated for C40
49
H N
8
O
6
S [M + H ]: 769.3496, found 769.3483
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dimethyl
((2S,2'S)-((2S,2'S)-((4H-thieno[3,2-c]chromene-2,7-diyl)bis(1H-imidazole-5,2-
diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (21): Synthesized from
1
4
2a by following an analogous procedure described for 9, (Yield = 89%). H-NMR (400 MHz, DMSO-d
6
): δ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12.06 (brs, 1H), 11.80 (brs, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.38 (d, J = 2.0 Hz, 1H), 7.31 (s, 1H), 7.25 (s, 1H),
7
4
.18 (d, J = 7.9 Hz, 1H), 7.03 (s, 1H), 5.29 – 5.21 (m, 2H), 5.10 – 4.96 (m, 2H), 4.08 – 4.02 (m, 2H), 3.79 (s,
H), 3.53 (s, 6H), 2.17 – 1.91 (m, 10H), 0.97 – 0.78 (m, 12H); HRMS m/z calculated for C39
49
H N
8
O S [M +
7
+
H ]: 773.3445, found 773.3432
dimethyl
((2S,2'S)-((2S,2'S)-((5,6-dihydro-4H-benzo[6,7]cyclohepta[1,2-b]thiophene-2,8-diyl)bis(1H-
imidazole-5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate
(22):
1
Synthesized from 42d by following an analogous procedure described for 9, (Yield = 85%). H-NMR (400
MHz, CD
3
OD): δ 7.62 – 7.40 (m, 2H), 7.35 – 7.22 (m, 2H), 7.24 – 7.15 (m, 1H), 7.15 – 7.07 (m, 1H), 5.23 –
5
.10 (m, 2H), 4.31 – 4.21 (m, 2H), 4.06 – 3.95 (m, 2H), 3.93 – 3.84 (m, 2H), 3.66 (d, J = 1.4 Hz, 6H), 2.76 –
2.68 (m, 4H), 2.41 – 2.27 (m, 4H), 2.25 – 2.17 (m, 4H), 2.12 – 2.03 (m, 4H), 0.98 – 0.91 (m, 12H); LCMS:
m/z = 785.5 (M+H)+
dimethyl ((2S,2'S)-((2S,2'S)-((4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2,8-diyl)bis(1H-imidazole-5,2-
diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (23): Synthesized from
1
4
2b by following an analogous procedure described for 9, (Yield = 24%). H NMR (400 MHz, DMSO-d
6
) δ
12.03 – 11.50 (m, 2H), 7.59 – 7.46 (m, 2H), 7.36 (d, J = 12.2 Hz, 3H), 7.05 (s, 1H), 5.12 – 4.96 (m, 2H), 4.35
–
4.18 (m, 2H), 4.09 – 4.00 (m, 2H), 3.84 – 3.75 (m, 4H), 3.53 (s, 6H), 3.22 – 3.07 (m, 4H), 2.23 – 1.92 (m,
H), 0.92 – 0.83 (m, 12H); LCMS: m/z = 394.3 (M+2H)+2
8
dimethyl ((2S,2'S)-((2S,2'S)-(naphtho[1,2-d]thiazole-2,7-diylbis(1H-imidazole-5,2-diyl))bis(pyrrolidine-
2
,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (24): Synthesized from 60 by following an
1
analogous procedure described for 9, (Yield = 13%). H-NMR (400 MHz, CD
3
OD): δ 8.74 (t, J = 9.4 Hz, 1H),
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