Synthesis, biological evaluation and in silico modeling of novel pan-genotypic NS5A inhibitors

B i o o r g an i c &M e d i c i n a l C h e m i s tr y 2 8 (2 020 )1 15716

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

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

Synthesis, biological evaluation and in silico modeling of novel pan-

genotypic NS5A inhibitors

⁎

Andrey A. Ivashchenko^a,b,c, Yan A. Ivanenkov^b,e,, Vladimir A. Aladinskiy^c, Ruben N. Karapetian^a,

Angela G. Koryakova^a, Alexey A. Ryakhovskiy^a, Oleg D. Mitkin^a, Dmitry V. Kravchenko^a,

Nikolai P. Savchuk^a,d, Bogdan A. Zagribelnyy^c, Alexander V. Ivashchenko^a,b,d

^aChemical Diversity Research Institute, Rabochaya St. 2a, Khimki, Moscow Region 141401, Russia

^bChemDiv, 6605 Nancy Ridge Drive San Diego, CA 92121, United States

^cMoscow Institute of Physics and Technology (State University), 9 Institutskiy lane, Dolgoprudny City, Moscow Region 141700, Russia

^dAvisa Pharmaceuticals LLC, 1835 E. Hallandale Beach Blvd, #442, Hallandale Beach, Fl 33009, United States

^eInstitute of Biochemistry and Genetics Russian Academy of Science (IBG RAS) Ufa Scientiﬁc Centre, Ufa, Russia

A R T I C L E I N F O

A B S T R A C T

Keywords:

A series of novel small-molecule pan-genotypic hepatitis C virus (HCV) NS5A inhibitors with picomolar activity

NS5A inhibitors

HCV

containing

2-[(2S)-pyrrolidin-2-yl]-5-[4-(4-{2-[(2S)-pyrrolidin-2-yl]-1H-imidazol-5-yl}buta-1,3-diyn-1-yl)

phenyl]-1H-imidazole core was designed based on molecular modeling study and SAR analysis. The constructed

in silico model and docking study provide a deep insight into the binding mode of this type of NS5A inhibitors.

Based on the predicted binding interface we have prioritized the most crucial diversity points responsible for

improving antiviral activity. The synthesized molecules were tested in a cell-based assay, and compound 1.12

showed an EC₅₀value in the range of 2.9–34 pM against six genotypes of NS5A HCV, including gT3a, and

demonstrated favorable pharmacokinetic proﬁle in rats. This lead compound can be considered as an attractive

candidate for further clinical evaluation.

Medicinal chemistry

In silico modeling

Combinatorial synthesis

1. Introduction

chronic HCV infection, the risk of cirrhosis ranges between 15% and

30% within 20 years.⁴

Hepatitis C infection caused by hepatitis C virus (HCV)^1,2is one of

the most common liver diseases and is widespread throughout the

world. HCV infection is of growing international concern due to its

substantial eﬀect on morbidity and mortality. A leading cause of cir-

rhosis, hepatocellular carcinoma (HCC), liver transplantation, and liver-

related death worldwide, the HCV-related disease burden continues to

increase as the infected population advances to late stage liver disease.

The disease inﬂicts an immense health and economic burden on

countries due to the infection's hepatic and extrahepatic eﬀects. The

2015 global prevalence estimate of 71.1 million people have hepatitis C

infection.³In 2016, approximately most 399 000 people died of hepa-

titis C, mostly from cirrhosis and hepatocellular carcinoma (primary

liver cancer). HCV causes both acute and chronic infection. New HCV

infections are usually asymptomatic. Some persons get acute hepatitis

which does not lead to a life-threatening disease. Around 30%

(15–45%) of infected persons spontaneously clear the virus within

6 months of infection without any treatment. The remaining 70%

(55–85%) of persons will develop chronic HCV infection. Of those with

Until recently, HCV was classiﬁed into 8 distinct genotypes that

diﬀered by > 30% at the nucleotide level. Genotypes are further di-

vided into subtypes with a sequence divergence of > 15%.⁴To date, 86

conﬁrmed HCV subtypes have been described.⁵Genotypes 1, 2 and 3

are distributed worldwide, genotype 1 being the predominant one,

speciﬁcally subtypes 1a and 1b.³Genotype 1 is the best established

treatment.⁶Because of the large genetic diversity of genotype 2, often

joining recombinants with other genotypes, there are some strains of

genotype 2, more resistant to therapy.⁷The most diﬃcult in the treat-

ment of genotype 3.^8,9Within the six major clinical HCV genotypes,

genotype 3 represents 22–30% of all infection and is described as a

unique entity with higher rates of steatosis, faster progression to cir-

rhosis, and higher rates of hepatocellular carcinoma.⁹

In recent years, unconditional success has been achieved in the

combinatorial therapy of hepatitis C.^6–11One of the main components

of the combinatorial therapy of HCV infection are nonstructural protein

5A (NS5A) inhibitors which displayed much higher potency in con-

trolling HCV infection than other drugs. This is because NS5A exerts

^⁎Corresponding author at: ChemDiv, 6605 Nancy Ridge Drive San Diego, CA 92121, United States.

E-mail address: yai@chemdiv.com (Y.A. Ivanenkov).

https://doi.org/10.1016/j.bmc.2020.115716

Received 11 February 2020; Received in revised form 7 August 2020; Accepted 16 August 2020

A v a i l a b l e o n l i n e 2 0 A u gu s t 2 020

A.A. Ivashchenko, et al.

Bioorganic&MedicinalChemistry28(2020)115716

Abbreviations

uronium

DIPEA

MeCN

EtOAc

Val

Diisopropylethylamine

Acetonitrile

HCV

NS5A

SAR

EC₅₀

gT3a

HCC

FDA

gT1b

SM

Hepatitic C Virus

Nonstructural protein 5A (HCV)

Structure-Activity Relationship

Half-maximal eﬀective concentration

Genotype 3a (HCV)

Hepatocellular carcinoma

Food and Drug Administration

Genotype 1b (HCV)

Structural modiﬁcations

Domain I (NS5A)

Ethyl acetate

Valine

Ph

Phenyl

Gly

Glycine

gT1a

gT4a

gT5a

Huh7

PK

Genotype 1a (HCV)

Genotype 4a (HCV)

Genotype 5a (HCV)

Hepatoma cell line

Pharmacokinetics

Per-oral route of administration

Area under curve

D -I

MD

RMSF

RMSD

Trp

PPI

Molecular dynamics

Root mean square ﬂuctuation

Root mean square deviation

Tryptophan

PO

AUC

AUC_INFArea under curve (inﬁnite time)

AUC_lastArea under curve (the last point)

Protein-Protein Interaction

Nonstructural protein 4B (HCV)

Resistance Assosiated Variant

Arginine (except R¹and R²– radicals)

Alanine

C_max

T_max

T_1/2

Maximal plasma concentration

The time take to reach Cmax

Half-life time

NS4B

RAV

R

MRT_INFMean residence time (inﬁnite approximation)

MRT_lastMean residence time (the last time point)

A

Y

Tyrosine

F_abs

Absolute bioavailability

Nuclear magnetic resonance

High-performance liquid chromatography

Ultra Violet irradiation

Q

Glutamine

NMR

HPLC

UV

S

Serine

EC₅₀

T

Half-maximal eﬃcacy concentration

Threonine

HRMS

ESI

High Resolution Mass Spectrometry

Electrospray Ionization

P

Proline

Moc

gT2a

C

Methoxycarbonyl group

Genotype 2a (HCV)

Cysteine

TEA

DCM

Triethylamine

Dichloromethane

LC/MS Liquid chromatography/ mass-spectroscopy

DMSO‑d₆Dimethyl sulfoxide (fully deuterated)

BB

Building block

BDD

boc

THF

DMF

Et₃N

MeOH

Buta-1,3-diyne-1,4-diyl group

tert-Butyloxycarbonyl group

Tetrahydrofurane

ESIHRMS High resolution electrospray ionization mass spectrometry

FBS

Fetal Bovine Serum

DMEM

CC₅₀

EDTA

IV

Dulbecco's modiﬁed Eagle's medium

Half-maximal cytotoxic concentration

Ethylenediaminetetraacetic Acid

Intravenous route of administration

Mass-spectroscopy

N,N-dimethylformamide

Triethylamine

Methanol

Pd(PPh₃)₄Tetrakis(triphenylphosphine)palladium (0)

MS

HATU

Hexaﬂuorophosphate

azabenzotriazole

tetramethyl

UPLC

Ultra High-Performance Liquid Chromatography

functionally essential eﬀects in regulation of viral replication, assembly

and egress, it has been considered a drug target for antiviral therapeutic

intervention.¹²The ﬁrst among pan-genotypic NS5A inhibitors was

Daclatasvir (BMS-790052),^13–15approved of FDA July 24, 2015. It was

followed by Elbasvir, (MK-8742),¹⁶Ledipasvir (GS-5885),¹⁷AV-4025,¹⁸

Ombitasvir (ABT-267),¹⁹Samatasvir,^20,21Ruzasvir (MK-8408)²²and

others. The large and ever-growing diversity of HCV genotypes and

their subtypes dictates the search for new pan-genetic inhibitors of

NS5A HCV with improved characteristics, which is currently associated

primarily with the structural modiﬁcations (SM) of known core struc-

tures. The powerful picomolar pan-genotype inhibitors MK-6169^23,24

and Pibrentasvir (ABT-530)²⁵(Fig. 1) are examples of this SM. They

were obtained as a result of a SM of Elbasvir and Ombitasvir, respec-

tively.

conﬁguration²⁶(Fig. 1).

2.1. In Silico modelling

Considering the last trends in the title area, side-chain modiﬁcations

in the linker area may lead to an improved pan-genotypic covering and

higher activity. For instance, Elbasvir by Merck & Co. (Fig. 1) was

launched in 2016 for the oral treatment of chronic HCV infection in

combination with grazoprevir. The compound showed picomolar in-

hibition potency against many HCV genotypes, including 3a. Although

the predicted binding mode of Elbasvir was presented,²⁷the detailed

docking studies have not been published yet. This side-chain mod-

iﬁcation motivated us to develop a special in silico model of dimeric 3D

target structure with an attached N-terminal α-helices of NS5A using

homology morphing. The main focus was placed on genotype 3a due to

its prevalence within the Russian Federation in contrast to other geo-

graphic areas. In order to reconstruct the N-terminal region of NS5A

gt3a, we exploited the binding mode-II model of gt1b published by

Nettles et al. as a template structure. The authors reported two distinct

asymmetric binding modes of Daclatasvir. Both models were validated

experimentally with a library of structural analogs of Daclatasvir and

are consistent with the studies dedicated to investigating the me-

chanism of action of NS5A inhibitors. Moreover, in the recent bioin-

formatic analysis of gt1a and gt1b HCV protein sequences, it was stated

In this paper, we present the rational design of novel AV-4025¹⁸

derivatives with an improved inhibition potency against HCV geno-

types, especially gT3a. Particularly, we have developed in silico model

for prediction the target activity mainly focusing on substituents at the

phenyl ring in the linker fragment (Fig. 1).

2. Results and discussion

The structure of the vast majority of small-molecule NS5A inhibitors

contains a linker and two terminal “End Caps” with pre-deﬁned stereo-

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Fig. 1. Common topological pharmacophore model for NS5A inhibitors.

that the structural insights derived from the binding mode-II model

agree with data on clinically relevant mutations of HCV.²⁸In our pre-

vious work, we demonstrated that morphing Caps leads to inequivalent

changes in antiviral potency.¹⁸This fact also supports the hypothesis

regarding asymmetric binding modes of NS5A inhibitors.

We further investigated the amino-acid variants in the N-terminal α-

helices and D-I of NS5A from HCV gT1b and gT3a to determine those

which can be associated with the resistance to AV-4025 treatment

(Fig. 3). To assess the signiﬁcance of these variants, we aligned the

known model of gt1b in complex with daclatasvir and our homology

model of gt3a (Fig. 4). Among the ﬁrst 100 residues, we highlighted the

following key diﬀerences: (i) a side-chain of A-R30 was shown to pro-

vide strong hydrogen bond with carbamate moiety,²⁷thus R30A variant

results in loss of this interaction and can be considered as one of the

vital RAV (Fig. 4A); (ii) T56R substitution in chain B may lead to steric

clashes between a core structure and a bulky guanidinium moiety of B-

R56 which is able to prevent tight ﬁtting the cleft between D-I and AH

consequently not allowing to appropriately place a peptide-like cap for

H-bonding with B-Y93 (Fig. 4B); (iii) in our model we observe that the

side chain of B-R6 is directed at the Cap binding site oppositely to gt1b

(Fig. 4C), such orientation likely caused by two amino-acid replace-

ments A-T56R and B-S3D. Indeed, the proximity of positively charged

A-R56 may lead to the displacement of the B-R6 functional group while

B-D3 stabilizes this orientation through electrostatic interactions and H-

bond.

We used the homology modeling tool implemented in Maestro

Schrödinger Software²⁹to produce ten homology models. In order to

evaluate the obtained 3D structures, we docked Elbasvir into all ten

models and selected the model with the lowest energy score (Emodel of

−115) for further steps. The Elbasvir predicted binding mode is re-

presented in Fig. 2. The linker tightly occupied the cleft between D-I of

the chain B and the AH of the chain A. B-R56 supposedly provided pi-

cation interaction with indole of the tetracyclic scaﬀold. Interestingly,

in our model, imidazole formed H-bond with a backbone carbonyl of

G92 instead of the bridge with T95 as it was predicted for Daclatasvir,

GSK-2336805, and GS-5885 in the model of gt1b.²⁷The phenyl func-

tion was oriented towards the membrane and provided additional

contacts with the α-helix. The model suggests that one of the peptide-

like caps binds in the region of the ﬂexible linker of chain B (Cap

binding site I), forming a hydrogen bond between NH of A30 and

carbonyl, while valine-carbamate moiety contacts P29, A30 and L31.

Another End Cap occupied the binding site II, where both carbonyls of

Pro-Val-carbamate were predicted to bind to two arginines A-R6 and B-

R6 through H-bonds. Noteworthy, that NH of this Cap was directed at

the hydroxyl of A-Y93 and possibly able to provide H-bond bridging.

Interestingly, previously published models³⁰have predicted, that

Q54 of each monomer in gT1b forms a relatively strong H-bond with

imidazole or pyrrolidino-carbonyl, hence Q54S substitution may at-

tenuate activity. However, due to signiﬁcant structural diﬀerences, the

binding mode-II model by Nettles et al., as well as the gt3a model

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Fig. 2. The predicted binding mode of Elbasvir (orange) to NS5A gt3a. The protein structure is depicted as green. Compound and key residues are represented as a

thick tube model. Orange arrows indicate the putative binding sites of End Caps.

described here, do not suggest such an interaction. Noteworthy, the

presence of two arginine groups in the proximity of a binding site may

cause electrostatic repulsion results in reducing the binding aﬃnity.

This hypothesis was comprehensively discussed in the study concerned

with the structural insights into the binding modes of NS5A in-

hibitors.³⁰The authors have suggested that resistance of gT1a Q30R

mutant is likely explained by repulsion between R30 and R56. In ac-

cordance with our model of NS5A gT3a, the binding site contains four

arginine residues A-R6, B-R6, A-R56, and B-R56. We also suggested that

tight binding with NS5A can be aﬀected by the proximity of these re-

sidues.

substitutions introduced in the linker may occupy either the pocket in

D-I formed by side chains of B-T95 and B-R56 or space between A-L31

and A-D10 and consequently increase interaction surface area. Another

opportunity to enhance the binding aﬃnity is to ﬁll the room in the cap-

binding site II more tightly by replacing isopropyl with a more bulky

function. Additionally, as mentioned above, R30A substitution results

in loss of H-bonding with carbamate moiety. Thus, another chance to

improve activity is to realize more tight contact with a ﬂexible linker by

modifying the isopropyl group for a more large fragment.

Following our molecular modeling eﬀorts, we have designed a series

of AV-4025 structural analogs – a small-sized virtual library contained

synthetically accessible compounds. Phenyl fragment of the spacer was

substituted with aryl functions, cycloalkyl, and alkyl moieties, readily

available in our proprietary collection, at positions R¹or/and R²(see

Table 1). In addition, we also replaced a canonical isopropyl moiety at

the Cap region by S- and R-phenyl fragment. All the proposed structures

were then docked using the developed model and ranked by virtual

scoring values as well as synthesized and evaluated in a replicon-based

assay against six diﬀerent HCV genotypes in the absence of human

plasma.

The built model was exploited to provide structural insight into the

binding mode of AV-4025 in order to indicate the opportunities for

improving the aﬃnity towards NS5A gT3a. The putative binding mode

of AV-4025 is depicted in Fig. 5. The diynyl-phenyl core was predicted

to ﬁt the groove formed by A-G33, A-P35, B-Y93, A-D10, and A-R-6.

Imidazole ring distal to phenyl of the spacer was accommodated by side

chains of A-R6, A-W9, B-Y93 and provided the Cap’s orientation sui-

table for forming H-bond between hydroxyphenyl of B-Y93 and NH of

carbamate. The isopropyl function was directed at B-R6 and A-Y93. The

End Cap occupying the binding site I and corresponding imidazole in-

teracted with a ﬂexible region of chain A, particularly with A-A30, A-

G33, A-L34. Furthermore, the model predicted that Val-carbamate

moiety was oriented towards D-I of chain B, thereby providing H-bonds

with NH of B-Y161 and a side chain of B-E92. Based on the analysis of

AV-4025 binding mode, we have suggested that well-tuned

Modeling suggested that the linker substitutions of 1.1–1.4 are

surrounded by residues B-T95, B-R56, A-L31, and A-D10 (Fig. 6). Ca-

tion-pi interactions with the guanidinium function of B-R56 were pre-

dicted for phenyl, naphthyl, biphenyl of 1.1, 1.2, and 1.4, respectively.

The orientation of 4-tert-butylphenyl introduced in the linker of 1.3 was

not suitable for providing cation-pi interaction, but it more tightly

Fig. 3. The alignment of amino-acid sequences for N-terminal α-helix, linker region and D-I for NS5A gT1b (ANQ34308) and gT3a (AFP27699).

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Fig. 4. The superposition of two structural models for the N-terminal region of NS5A: gt1b (green) in complex with daclatasvir and gt3a (magenta) described in the

present article. Daclatasvir is represented by grey color and a thick tube model. (A) Cap binding site I: A-R30 is represented by a thick tube model, the side chain of A-

A30 is represented by a space-ﬁlling model. (B) The cleft occupied by a linker of an inhibitor: B-T56 and the side chain of B-R56 are represented by a thick tube and a

space-ﬁlling model, respectively. The orange arrow indicates the possible steric collision between the biphenyl linker of daclatasvir and the guanidinium function of

B-R56. (C) Cap binding site II: A-R6 and B-D3 are represented by a thick tube model, the guanidinium group of B-R6 is depicted as a space-ﬁlling model. The orange

curved arrow indicates the predicted replacement of the B-R6 side chain observing in the model of gt3a. The orange arrow indicates the possible steric clash between

the B-R6 side chain and End Cap of daclatasvir.

Fig. 5. The predicted binding mode of AV-4025 (orange) to NS5A gT3a. Key residues are represented by a ball-and-stick model, while the inhibitor is depicted as a

tube model.

occupied the space between side chains of A-L31 and A-D10. Methyl

and ﬂuorine at the R²position of 1.5 and 1.6 demonstrated contacts

with B-Y93 and pyrrolidine of A-P35, meanwhile phenyl introduced in

the spacer was placed in a similar manner as for 1.1.

unsubstituted phenyl ring (compound 1.1) led to two-fold increase in

activity against gT3a in contrast to the parent compound. The sub-

stitution with 4-tert-butylphenyl (1.3) resulted in the same potency

towards gT3a (EC₅₀= 1286 pM), however the activity against gT1a

signiﬁcantly dropped. Compound 1.2 containing 2-naphthyl substituent

According to the results presented in Table 1, the introduction of the

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Fig. 6. The predicted binding modes of 1.1 (orange), 1.2 (grey), 1.3 (cyan) and 1.4 (magenta) to NS5A gT3a. Inhibitors and key binding residues are depicted as a

thick tube model.

demonstrated picomolar activity against gT3a (EC₅₀= 864 pM). A

decrease in the potency of 1.4 can be explained by the torsional angle

between phenyl rings in biphenyl-3-yl moiety, and their relative spatial

orientations resulted in unfavorable steric clashes with protein struc-

ture, while 2-naphthyl is coplanar with the unsubstituted phenyl. For

compounds 1.5 and 1.6 containing methyl and ﬂuorine substituents at

position R², respectively, we have obtained higher scorings in contrast

to 1.1. These molecules were predicted to form more contacts along

with a protein interface, speciﬁcally with the pyrrolidine ring of A-P35

and the side chain of B-Y93. Indeed, both molecules demonstrated an

improved activity against all the HCV isoforms evaluated vs. compound

1.1.

fragments in the Cap-binding sites I and II for 1.7 and 1.9, respectively,

oppositely to their stereoisomers.

Biological testing showed that the replacement of N-Moc-^L-valine

with N-Moc-(S)-phenylglycine in 1.8 and 1.10 leads to a 2-fold re-

duction in the target activity towards gT1b as well as against other

genotypes (Table 1). Then, we have demonstrated that the opposite

stereo-conﬁguration at this chiral point improves gT3a activity, and this

In our previous SAR study we demonstrated that stereo-conﬁgura-

tion of Caps dramatically aﬀects antiviral activity towards gT1b.¹⁸De

facto, AV-4025 contains four (S)-chiral centers, and it showed the

highest antiviral eﬃciency among combinatorial partners. Herein, we

investigated the impact of the replacement of N-Moc-valine group with

N-Moc-phenylglycine in both Caps and exploited molecular docking to

assess the diﬀerences in putative binding modes (Fig. 7A). N-Moc-(R)-

phenylglycine moiety of 1.7 occupied the binding site I, phenyl frag-

ment of the Cap provided contacts with B-Y161 and B-R160 side chains.

Methyl group of carbamate was predicted to interact with A-L34 and A-

G33 of the ﬂexible region, as well as NH of the same Cap formed H-

bond with B-E92 of D-I. The (S)-phenylglycine of 1.8 interacted with A-

G33 and A-A30, while carbamate was placed near B-Y161 and B-R160

interacting with their side chains, however, without H-bond B-E92. The

pyrrolidine ring attached to this Cap was oriented to contact with B-

T95. Other binding mode was predicted for compound 1.9 during

docking studies (Fig. 7B). Cap-associated phenyl ring was positioned

deeply within site II thereby occupying the pocket formed by B-I37, B-

F36, and A-Y93 and providing hydrophobic interactions with them.

Carbamate of this Cap interacted with A-F36 and A-D3 side chains by

methyl function. Noteworthy, NH formed H-bond with hydroxyphenyl

of B-Y93. Hydrogen bonding between A-R6 and amide of pyrrolidine

moiety was observed as well. In contrast, (S)-phenyl group of 1.10 was

directed towards B-Y93 as well as interacted with the side chains of

both R6, A-D3, and B-Y93. Carbamate function was predicted to pro-

vide contacts with B-R6, A-Y93, B-P35, B-F36, and B-G60. Finally,

compounds 1.7 and 1.9 exhibited dramatically lower docking scores

rather than 1.8 and 1.10 (Table 1). This fact can be explained by the

presence of H-bonds and more appropriate orientations of phenyl

Fig. 7. (A) Putative binding modes of 1.7 (magenta) and 1.8 (cyan) with phe-

nylglycine instead of ^L-Val in the Cap proximal to phenyl fragment of a linker.

(B) Putative binding modes of 1.9 (cyan) and 1.10 (magenta) with phenylgly-

cine instead of ^L-Val in the Cap distal to phenyl fragment of a linker.

Compounds and key binding residues are represented as a thick tube model.

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correlates with the results of our docking simulations. Indeed, 1.7 and

1.9 showed EC₅₀values of 329 and 629 pM, respectively. This ob-

servation is consistent with the data reported earlier by the group from

Bristol-Myers Squibb.³¹Interestingly, modiﬁcation of the Cap that is

distal to the phenyl fragment in the linker has more impact on the

antiviral proﬁle as compared to the proximal one. These results are in

agreement with our data published previously.¹⁸Compound with (R)-

chiral point at the proximal Cap was 23-fold less active than AV-4025,

while R-conﬁguration at the distal Cap resulted in a 120-fold reduction

in the desired activity. These SAR observations indicate that Caps bind

within diﬀerent sites. Moreover, during the modeling, we have found

that the distal Cap interacts with Y93, a key amino acid residue asso-

ciated with resistance.

slightly improved potency as compared to 1.12. Summarizing, com-

pounds 1.12 and 1.15 demonstrated an enhanced virologic proﬁle in

contrast to AV-4025 and inhibited the replication of six HCV genotypes

with an activity in low picomolar range.

2.2. Chemistry

The key step for the preparation of compounds 1.1–1.24 was

Sonogashira cross-coupling reaction between 2-[(2S)-1-acylpyrrolidin-

2-yl]-5-(4-buta-1,3-diyn-1-ylphenyl)-1H-imidazole and 2-[(2S)-1-acet-

ylpyrrolidin-2-yl]-5-iodo-1H-imidazole or between 2-[(2S)-1-acet-

ylpyrrolidin-2-yl]-5-(4-iodophenyl)-1H-imidazole and 2-[(2S)-1-acet-

ylpyrrolidin-2-yl]-4-buta-1,3-diyn-1-yl-1H-imidazole with a subsequent

modiﬁcation of pyrrolidine fragment to get the ﬁnal Cap-containing

molecules with the desired stereo-conﬁguration. Building blocks (BBs)

were radially synthesized as described previously¹⁸or obtained from

commercial sources. The target compounds were synthesized starting

from the reaction between BB (II) and tert-butyl (2S)-2-(4-iodo-1H-

imidazol-2-yl)pyrrolidine-1-carboxylate (III) after the elimination of

trimethylsilyl group from BB (I) (Scheme 1). Products (IV) were ob-

tained in good yield and used for further versatile transformation. Thus,

intermediate (IV) was deprotected (−Boc) upon alkaline conditions

resulted in compound (V). Acylation of (V) with Moc-^L-Valine, N-Moc-

(R)-phenylglycine or N-Moc-(S)-phenylglycine led to the desired pro-

duct 1.1–1.24 (∙2HCl) in moderate-to-high yields (see exp. section). The

ﬁnal products were obtained as the optically pure stereomers con-

taining four chiral centers. The structure of substituents R¹and R²as

well as Caps attached to (V) via acylation can readily be identiﬁed in

Table 1.

Taking into account the results of molecular docking and SAR stu-

dies, compound 1.9 was selected as the most promising starting point

for subsequent morphing. As exempliﬁed in Fig. 8, compounds were

predicted to provide beneﬁt H-bond with B-Y93 as well as tightly oc-

cupied the interface formed by N-terminal α-helix, protein linker region

and D-I. Surprisingly, the modeling was not resulted in the highly

scored binding modes for compounds 1.20 and 1.21 with substitutions

in both R¹and R²positions. The values of Emodel scoring function for

other compounds are lower than −95. Inhibitors 1.12–1.24 exhibited

ﬁrm picomolar activity against gT3a, excepting methylpiperazine

analog 1.11 demonstrated EC₅₀value > 10 nM despite quite high

docking score (Emodel = −100.9). It likely indicates that aryl sub-

stitutions introduced at the linker part are more preferable than satu-

rated cycles. However, this observation cannot be explained in the

scope of the present model and requires further computational eﬀorts.

Replacing the distal Cap with N-Moc-(R)-phenylglycine and the addi-

tion of phenyl ring at position R¹resulted in compound 1.12 with an

EC₅₀value of 34 pM. The addition of tert-butyl group at phenyl frag-

ment reduced activity (1.13 and 1.14). However, compound 1.16

bearing dimethylamino phenyl moiety inhibited HCV replication with a

similar to 1.12 manner, except gT1a and gT2a isoforms. Introducing

2,3-diﬂuorophenyl fragment (compound 1.15) led to enhanced activity,

particularly against gT3a isoform (EC₅₀= 16 pM, two-fold more active

than 1.12). Compounds 1.17–1.19 bearing more bulky functionalities

as well as more ﬂexible ones (1.22–1.24) did not demonstrate sig-

niﬁcant improvements. Considering the results obtained for compounds

1.5 and 1.6, we investigated the impact of methyl- or ﬂuoro-substituent

at position R²of compound 1.12 on gT3a replication. These mod-

iﬁcations resulted in compounds 1.20 and 1.21, respectively, with

3. Biological evaluation

3.1. Cell-based assay

Antiviral activity of the synthesized compounds was evaluated

against six distinct genotypes of NS5A HCV (gT1b, gT1a, gT2a, gT3a,

gT4a, and gT5a). The standard human hepatoma cell line Huh7 har-

boring the HCV replicon was used as a test system. The obtained results

are summarized in Table 1. As shown in Table 1, inhibitors 1.7 and 1.9

containing phenyl in the End Caps at R-conﬁguration are 13- and 6.8-

times more active with respect to gT3a than AV-4025, respectively. At

the same time, compounds 1.8 and 1.10 bearing phenyl in the end caps

Fig. 8. Predicted binding mode for compound 1.15 (orange) to NS5A gT3a that illustrates the impact of Cap and linker modiﬁcations on ligand-protein interactions.

Compound and key residues are represented as a thick tube model.

12

A.A. Ivashchenko, et al.

Bioorganic&MedicinalChemistry28(2020)115716

R²

N

a

N

b

N

boc

BDD SiMe₃

I

BDD

H

+

N

H

R¹

boc

H

I

II

III

R²

N

c

N

d,e

N

H

boc

1.1-1.24 (*2HCl)

BDD

H

boc

NH

N

H

R¹

V

IV

Scheme 1. The common route for the preparation of compounds 1.1–1.24 (∙2HCl): BDD – buta-1,3-diyne-1,4-diyl, R¹and R²are listed in Table 1 below. Reagents and

conditions: (a) K₂CO₃, THF, MeOH; (b) Pd(PPh₃)₄, CuI, THF (or DMF), Et₃N, 40–45 °C; (c) HCl, dioxane; (d) for Moc-L-Val-OH: HATU, DMF, DIPEA, 4 °C, while for

Moc-(R)-PhGly-OH or Moc-(S)-PhGly-OH: HATU, MeCN, DIPEA, −20 °C; (e) HCl, dioxane (or in EtOAc).

at S-conﬁguration are 2.6- and 1.1-times less active against gT3a than

the parent molecule. These data correlate well with the results obtained

during the molecular docking study (Fig. 9). In general, inhibitors

containing bulky substituents simultaneously in the linker (R¹) and in

the Cap fragment are signiﬁcantly more active with respect to NS5A

HCV in contrast to AV-4025. For instance, salt compositions of com-

pounds 1.12, 1.15, 1.16, 1.20, 1.21 and 1.23 inhibit gT1b isoform

with EC₅₀value in the range of 1.5–2.9 pM, gT1a with

EC₅₀= 28.0–260.0 pM, gT2a with EC₅₀= 1.0–14.0 pM, gT3a with

EC₅₀= 16.0–91.0 pM, gT4a with EC₅₀= 5.7–12.0 pM, and gT5a with

EC₅₀= 19.0–52.0 pM. In this series of compounds, the most attractive

are compounds 1.12 and 1.15, which are the most active against gT3a

(EC₅₀= 34.0 pM and EC₅₀= 16.0 pM, respectively). The weakest

activity was shown for compound 1.11·4HCl containing 4-methyl pi-

perazine substituent.

their antiviral activity and estimate the prediction power of the de-

veloped in silico model. The biological evaluation conﬁrmed the high

predictive ability of the model, and we observed consistency between

scoring values and activities (Fig. 9). The biological testing has revealed

compound 1.12 as the most promising lead candidate. The compound

eﬀectively inhibited HCV replication in human hepatoma cell line

Huh7, demonstrated pan-speciﬁc picomolar activity against six HCV

isoforms, and did not show any cytotoxicity. In particular, it blocked

the replication of HCV gT3a with an EC₅₀value of 34 pM, whereas the

parent compound (AV-4025) was 126-times less active. PK study per-

formed for lead compound 1.12 has provided impressive results making

this molecule very attractive for subsequent clinical evaluation.

5. Experemental section

5.1. Chemistry

3.2. Pharmacokinetics in rats

Starting materials, as a rule, are commercially available products.

All chemicals and solvents were used as received from suppliers without

further puriﬁcation. The crude reaction mixtures were concentrated

under reduced pressure by removing organic solvents. Several starting

compounds and BBs were synthesized in according to the reported

synthetic procedures: tert-butyl (2S)-2-(4-iodo-1H-imidazol-2-yl)-pyr-

rolidine-1-carboxylate,³²(2S)-2-(5-{4-[4-(trimethylsilyl)buta-1,3-diyn-

Pharmacokinetic proﬁle (PK) for compounds 1.12∙2HCl, 1.15 and

1.15∙2HCl was determined in rats (for details see exp. section below).

The obtained data is summarized in Table 2. As shown in Table 2,

compound 1.12∙2HCl signiﬁcantly exceeds its analogs in its PK prop-

erties and can be reasonably regarded as the most attractive candidate

for further clinical evaluation. For instance, upon PO administration,

1.12∙2HCl demonstrated 43-times more favorable AUC value in contrast

to 1.15∙2HCl and 13-times higher bioavailability.

1-yl]phenyl}-1H-imidazol-2-yl)pyrrolidine-1-carboxylate,¹⁸

methyl

[(1S)-1-{[(2S)-2-(4-iodo-1H-imidazol-2-yl)pyrrolidin-1-yl]carbonyl}-2-

4. Conclusion

Recently, we have described the synthesis and extensive biological

evaluation of small-molecule HCV NS5A inhibitor AV-4025.¹⁸In the

present paper, in order to improve antiviral activity, we have performed

a rational design of novel pan-genotypic HCV NS5A inhibitors (AV-

4025 analogs) containing modiﬁed linker and Caps. Docking study with

AV-4025 has provided a valuable insight into the potential binding

mode of the parent compound and allowed us to ﬁrmly indicate key

diversity points for enhancing antiviral eﬃciency. Especially, the sub-

stitution of phenyl moiety in the spacer by relatively bulky aromatic

fragments was considered as the most promising strategy to achieve the

greater activity. Furthermore, we predicted that the substitution of

isopropyl moiety in the Cap regions by phenyl fragment with inverse

stereo-conﬁguration might also lead to reliable improvements. Based on

the obtained results, we designed a small-sized virtual library of

structures bearing appropriate substituents attached to the predicted

diversity points and carried out molecular docking study. Taking into

account the obtained scoring values and synthetic capabilities, we have

synthesized 24 compounds using combinatorial schemes to evaluate

Fig. 9. The plot represents the relationship between docking score values

(Emodel) and potency against HCV gt3a (pEC₅₀). Parent compound AV-4025 is

highlighted in cyan, compounds 1.1–1.6 are highlighted in orange, compounds

1.7–1.10 are highlighted in green, compounds 1.12–1.24 are highlighted in

yellow. Compound 1.11 was excluded from the plot due to its exact EC₅₀value

is unknown, 1.20 and 1.21 were excluded due to the absence of adequate

binding modes.

13

A.A. Ivashchenko, et al.

Bioorganic&MedicinalChemistry28(2020)115716

Table 2

PK proﬁle for compounds 1.12∙2HCl, 1.15 and 1.15∙2HCl.

Compound ID

1.12∙2HCl

1.15

1.15∙2HCl

Parameter

IV,

PO,

IV,

PO,

IV,

PO,

2 mg/kg

10 mg/kg

2 mg/kg

10 mg/kg

2 mg/kg

10 mg/kg

AUC_INF, h*ng/ml

AUC_last, h*ng/ml

8327

8256

7763

0.083

1.2

13,098

12,591

2980

2.0

285

279

114

0.083

4.6

4.7

4.2

–

61.7

58.7

38.3

1.0

2734

2724

1155

0.083

3.2

328.7

270.8

15.2

0.7

C

_max, ng/ml

T_max, h

T

_1/2, h

1.6

0.6

2.5

MRT_INF_obs, h

MRT_last, h

1.3

3.1

1.5

3.3

0.4

1.2

2.8

1.4

3.2

0.3

F

_abs, %

–

31.0

4.3

–

2.4

methylpropyl]carbamate,³³(S)-3-methyl-1-oxo-1-((S)-2-(5-((4-(2-((S)-

pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl)buta-1,3-diynyl)-1H-imi-

dazol-2-yl)pyrrolidin-1-yl)butan-2-ylcarbamate trihydrochloride,¹⁸2-

[(2S)-1-(1-tert-butoxyethenyl)pyrrolidin-2-yl]-5-ethynyl-1H-imida-

zole.³⁴Nuclear magnetic resonance (NMR) spectra were recorded using

Bruker DPX-400 spectrometer at room temperature (rt) with tetra-

methyl silane as an internal standard. The chemical shifts (δ) are re-

ported in parts per million (ppm), and the signals are reported as s

(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s

(broad singlet). The purities of the ﬁnal compounds were determined by

HPLC and were > 98%. The HPLC conditions for assessing purity were

as follows: Shimadzu HPLC, XBridge C18, 4.6 mm × 250 mm (3.5 μm);

gradient of 0.1% TFA in 5% acetonitrile/water (A) and 0.1% TFA

acetonitrile (B); ﬂow rate, 0.5 ml/min; acquisition time, 20 min; de-

tection wavelengths, UV 214 and 254 nm. The preparative HPLC system

included two sets of Shimadzu LC-8A pumps, a Shimadzu SCL 10Avp

controller, and a Shimadzu SPD 10Avp detector. A Reprosil-Pur C18-AQ

10 μm, 250 mm × 20 mm column was used. The mobile phase was a

gradient of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B). LC/

MS analysis was carried out using PE Sciex API 165 system equipped

with electrospray in positive ion mode, [M+H]⁺, and Shimadzu HPLC

in 3.5 ml of dioxane 4 M HCl solution in dioxane (3.5 ml) was added.

The mixture was stirred for 15 h, then rotovapped to obtain 2-[(2S)-

pyrrolidin-2-yl]-5-[4-(4-{2-[(2S)-pyrrolidin-2-yl]-1H-imidazol-5-yl}

buta-1,3-diyn-1-yl)phenyl]-1H-imidazole (V). Intermediate (V) was

then dissolved in DMF (5 ml) or MeCN (5 ml) and Caps were added as

follows: N-Moc-^L-valine (0.51–0.61 mmol), HATU (0.24–0.72 mmol)

and DIPEA (2.3–2.4 mmol) or N-Moc-R-(or S-)phenylglycine

(0.21 mmol or 0.34 mmol) and HATU (0.34 mmol) and DIPEA (1 mmol

or 1.7 mmol, after cooling to −20 °C). The resulting mixture was stirred

in a fridge for 2–4 h, then was diluted with DCM, washed with 5% citric

acid solution (for phenylglycine derivative with 5% Na₂CO₃solution),

dried over Na₂SO₄, rotovapped and subjected to HPLC to give the de-

sired products 1.1–1.4 with 61–65% yield, 1.5 (35%), 1.6 (31%), 1.7

and 1.8 (82%), 1.9 and 1.10 (56%), 1.11 (69%), 1.12–1.24 (28–77%).

The corresponding dihydrochlorides or tetrahydrochloride (for com-

pound 1.17) were obtained by the addition of 4 M HCl (an excess) so-

lution in dioxane (or in EtOAc) to the ﬁnal compound dissolved in

acetone (~4 ml). The resulting mixture was diluted with 4 ml of ether,

the precipitate was ﬁltered, washed with ether and dried in vacuum to

aﬀord the desired salts.

Methyl [(S)-1-((S)-2-{5-[(5-{2-[(S)-1-((S)-2-methoxycarbonylamino-

3-methylbutyryl)pyrrolidin-2-yl]-1H-imidazol-5-yl}biphenyl-2-yl)buta-1,3-

diynyl]-1H-imidazol-2-yl}pyrrolidine-1-carbonyl)-2-methylpropyl]-carba-

mate dihydrochloride (1.1·2HCl): LC/MS 787 (M+H)⁺. ¹H NMR

(DMSO‑d₆, 400 MHz) δ 15.41 (brs, 0.4H), 14.90 (brs, 0.7H), 8.30 (s,

0.1H), 8.28 (s, 0.9H), 8.04 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.93 (brs,

2H), 7.89 (d, J = 8.0 Hz, 1H), 7.66 (m, 2H), 7.51 (m, 3H), 7.27 (m,

1.77H), 7.18 (m, 0.12H), 6.85 (m, 0.11H), 5.74 (m, 0.05H), 5.45 (m,

0.05H), 5.18 (d, J = 6.8 Hz, 0.95H) 5.03 (dd, J₁= 8.0 Hz, J₂= 6.0 Hz,

0.95H), 4.12 (t, J = 7.6 Hz, 1H), 4.06 (t, J = 8.0 Hz, 1H), 3.98 (m, 1H),

3.81 (m, 3H), 3.53 (2 s, 5.45H), 3.40 (s, 0.35H), 3.29 (s, 0.2H), 2.37 (m,

1H), 2.26 (m, 1H), 2.18 (m, 2H), 2.10 (m, 2H), 2.00 (m, 4H), 0.78 (m,

12H). ESIHRMS m/z calcd for C₄₄H₅₀N₈O₆[M+H]⁺787.3926; found

787.3908.

system equipped with

a Waters XBridge C18 3.5 μm column

(4.6 mm × 150 mm). High-resolution mass spectrometry (HRMS) data

was obtained using Bruker microTOF II instrument with electrospray

ionization (ESI) in the Department of Structural Studies of Zelinsky

Institute of Organic Chemistry, Moscow. The measurements were per-

formed in positive ion mode (interface capillary voltage – 4500 V) or in

negative ion mode (3200 V). The analyzed mass range was from m/z 50

to m/z 3000 Da, and external or internal calibration was performed

with Electrospray Calibrant Solution (Fluka). Syringe injection was

used for solutions in acetonitrile, methanol, or water (ﬂow rate 3 ml/

min). Nitrogen was used as the dry gas; the interface temperature was

set at 180 °C.

Methyl [(S)-1-((S)-2-{5-[(4-{2-[(S)-1-((S)-2-methoxycarbonylamino-

3-methylbutyryl)pyrrolidin-2-yl]-1H-imidazol-5-yl}-2-[naphthalen-2-yl]

phenyl)buta-1,3-diynyl]-1H-imidazol-2-yl}pyrrolidine-1-carbonyl)-2-me-

thylpropyl]-carbamate dihydrochloride (1.2·2HCl): LC/MS molecular ion

peak was at 837 (M+H)⁺, ¹H NMR (DMSO‑d₆, 400 MHz) δ 15.46 (brs,

0.4H), 14.90 (brs, 0.7H), 8.32 (s, 0.1H), 8.30 (s, 0.9H), 8.20 (m, 2H),

8.07 (d, J = 8.8 Hz, 1H), 8.02 (m, 3H), 7.94 (m, 1H), 7.88 (brs, 2H),

7.82 (m, 1H), 7.60 (m, 2H), 7.27 (m, 1.8H), 7.15 (m, 0.1H), 6.84 (m,

0.1H), 5.75 (m, 0.05H), 5.42 (m, 0.05H), 5.18 (d, J = 6.8 Hz, 0.95H),

5.01 (dd, J₁= 7.6 Hz, J₂= 5.6 Hz, 0.95H), 4.12 (t, J = 7.6 Hz, 1H),

4.05 (t, J = 8.0 Hz, 1H), 3.99 (m, 1H), 3.80 (m, 3H), 3.53, 3.54 (2 s,

5.5H), 3.35 (s, 0.3H), 3.30 (s, 0.2H), 2.38 (m, 1H), 2.21 (m, 3H), 2.09

(m, 2H), 1.97 (m, 4H), 0.80 (m, 12H). ESIHRMS m/z calcd for

5.1.1. General procedure for compounds 1.1–1.24

To a solution of tert-butyl (2S)-2-(5-{4-[4-(trimethylsilyl)buta-1,3-

diyn-1-yl]phenyl}-1H-imidazol-2-yl)pyrrolidine-1-carboxylate

(I,

0.75–2 mmol) in THF (5 ml) and methanol (5 ml) K₂CO₃

(2.25–3.39 mmol) was added and the resulting mixture was stirred

under argon atmosphere for 2 h, then ﬁltered and rotovapped. To a

solution of the obtained tert-butyl (2S)-2-[5-(4-buta-1,3-diyn-1-yl-

phenyl)-1H-imidazol-2-yl]pyrrolidine-1-carboxylate (II) in THF (6 ml)

or DMF (4 ml) tert-butyl (S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-

carboxylate (III) (0.5–0.85 mmol), TEA (2 ml), Pd(PPh₃)₄

(0.025–0.043 mmol), and CuI (0.05–0.085 mmol) were added. The

resulting mixture was then stirred under argon at 40–45 °C for 15–48 h.

Then, the mixture was diluted with DCM, ﬁltered, washed with satu-

rated NH₄Cl solution, rotovapped and subjected to HPLC to give tert-

butyl (2S)-2-{5-[4-(4-{2-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-

1H-imidazol-5-yl}buta-1,3-diyn-1-yl)phenyl]-1H-imidazol-2-yl}pyrroli-

dine-1-carboxylate IV in 28–35% yield. To a solution of IV (0.24 mmol)

C

₄₈H₅₂N₈O₆[M+H]⁺837.4082; found 837.4069.

Methyl [(S)-1-((S)-2-{5-[4-(3′-tert-butyl-5-{2-[(S)-1-((S)-2-methox-

ycarbonylamino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-bi-

phenyl-2-yl)-buta-1,3-diynyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-

14

A.A. Ivashchenko, et al.

Bioorganic&MedicinalChemistry28(2020)115716

2-methyl-propyl]-carbamate dihydrochloride (1.3·2HCl): LC/MS 844 (M

+H)⁺. ¹H NMR (DMSO‑d₆, 400 MHz) δ 15.39 (brs, 0.5H), 14.84 (brs,

0.6H), 8.29 (s, 0.1H), 8.26 (s, 0.9H), 8.05 (s, 1H), 7.94 (m, 1H), 7.88

(m, 1H), 7.83 (brs, 0.85H), 7.74 (s, 1H), 7.48 (m, 3H), 7.28 (m, 1.7H),

7.13 (m, 0.03H), 6.86 (m, 0.05H), 5.73 (m, 0.03H), 5.39 (m, 0.04H),

5.17 (t, J = 6.8 Hz, 0.97H), 5.02 (dd, J₁= 7.6 Hz, J₂= 5.2 Hz, 0.96H),

4.12 (t, J = 7.6 Hz, 1H), 4.05 (t, J = 8.0 Hz, 1H), 3.98 (m, 1H), 3.81

(m, 3H), 3.53 (2 s, 5.5H), 3.40 (s, 0.3H), 3.30 (s, 0.2H), 2.37 (m, 1H),

2.06–2.27 (m, 5H), 1.98 (m, 4H), 1.35 (s, 9H), 0.75–0.93 (m, 12H).

ESIHRMS m/z calcd for C₄₈H₅₈N₈O₆[M+H]⁺843.4552; found

843.4541.

400 MHz) δ 15.17 (brs, 0.4H), 14.71 (brs, 0.7H), 8.26 (s, 0.1H), 8.17 (s,

0.9H), 7.99 (m, 0.35H), 7.93 (m, 3.6H), 7.78 (m, 2H), 7.71 (m, 0.05H),

7.43 (m, 0.25H), 7.38 (m, 0.5H), 7.30 (m, 5.3H), 7.16 (m, 0.05H), 5.57 (d,

J = 8.8 Hz, 0.9H), 5.43 (m, 0.1H), 5.32 (m, 0.1H), 5.22 (m, 0.9H), 5.05

(m, 1H), 4.07 (t, J = 7.6 Hz, 1H), 3.78 (m, 4H), 3.54, 3.55 (2 s, 5.4H), 3.44

(s, 0.3H), 3.39 (s, 0.3H), 2.36 (m, 1H), 2.26 (m, 1H), 2.06 (m, 7H), 0.88 (t,

J = 6.8 Hz, 0.6H), 0.79, 0.82 (2d, J = 6.8 Hz, 5.4H). ESIHRMS m/z calcd

for C₄₁H₄₄N₈O₆[M+H]⁺745.3456; found 745.3445.

Methyl {(R)-1-[(S)-2-(5-{[4-(2-{(S)-1-[(S)-2-(methoxycarbonylamino)-2-

phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)phenyl]buta-1,3-diynyl}-1H-

imidazol-2-yl)pyrrolidine-1-yl]-3-methyl-1-oxobutan-2-yl}-carbamate

dihy-

Methyl [(S)-1-((S)-2-{5-[4-(5-{2-[(S)-1-((S)-2-methoxycarbonylamino-3-

methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-[1,1′;3′,1′']terphenyl-2-yl)-

buta-1,3-diynyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-

carbamate dihydrochloride (1.4·2HCl): LC/MS 864 (M+H)⁺. ¹H NMR

(DMSO‑d₆, 400 MHz) δ 15.49 (brs, 0.65H), 14.91 (brs, 0.85H), 8.32 (s,

0.1H), 8.30 (s, 0.9H), 8.19 (s, 1H), 8.00, 8.02 (2 s, 1H), 7.92 (m, 2.8H),

7.81 (m, 2H), 7.77 (m, 1H), 7.70 (m, 1H), 7.63 (m, 1H), 7.49 (m, 2H), 7.39

(m, 1H), 7.27 (m, 1.75H), 7.17 (m, 0.13H), 6.86 (m, 0.12H), 5.76 (m,

0.05H), 5.44 (m, 0.08H), 5.18 (t, J = 7.0 Hz, 0.95H), 5.03 (dd,

J₁= 7.6 Hz, J₂= 5.6 Hz, 0.92H), 4.11 (t, J = 7.8 Hz, 1H), 4.05 (t,

J = 8.0 Hz, 1H), 4.00 (m, 1H), 3.81 (m, 3H), 3.53 (s, 5.5H), 3.37 (s, 0.3H),

3.29 (s, 0.2H), 2.37 (m, 1H), 1.92–2.29 (m, 9H), 0.74–0.93 (m, 12H).

ESIHRMS m/z calcd for C₅₀H₅₄N₈O₆[M+H]⁺863.4239; found 863.4238.

Methyl {(S)-1-[(S)-2-(5-{[5-(2-{(S)-1-[(S)-2-(methoxycarbonylamino)-3-

methylbutyryl]pyrrolidin-2-yl}-1H-imidazol-5-yl)-3-methylbiphenyl-2-yl]buta-

1,3-diynyl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-methylpropyl}-carba-

mate (1.5·2HCl): LC/MS 801 (M+H)⁺, ¹H NMR (DMSO‑d₆, 400 MHz) δ

15.12 (brs, 0.6H), 14.72 (brs, 0.7H), 8.22 (s, 1H), 7.90 (s, 1H), 7.79 (m,

2H), 7.61 (m, 2H), 7.53 (m, 2H), 7.47 (m, 1H), 7.27 (d, J = 8.4 Hz, 1.8H),

7.07 (m, 0.1H), 6.86 (m, 0.1H), 5.66 (m, 0.05H), 5.32 (m, 0.08H), 5.15 (t,

J = 6.8 Hz, 0.95H), 5.01 (dd, J₁= 7.6 Hz, J₂= 4.8 Hz, 0.92H), 4.11 (t,

J = 7.8 Hz, 1H), 4.04 (t, J = 8.0 Hz, 1H), 3.93 (m, 1H), 3.83 (m, 1H), 3.77

(m, 2H), 3.53, 3.54 (2 s, 5.5H), 3.41 (s, 0.3H), 3.30 (s, 0.2H), 2.54 (s, 3H),

2.37 (m, 1H), 1.91–2.24 (m, 9H), 0.76–0.92 (m, 12H). ESIHRMS m/z calcd

for C₄₅H₅₂N₈O₆[M+H]⁺801.44082; found 801.4090.

drochloride (1.9∙2HCl): LC/MS 745 (M+H)⁺. ¹H NMR (DMSO‑d₆,

400 MHz) δ 15.34 (brs, 0.6H), 14.82 (brs, 0.8H), 8.20 (s, 1H), 7.96 (m, 3H),

7.85 (d, J = 8.0 Hz, 0.7H), 7.77 (m, 2H), 7.66 (m, 0.3H), 7.31 (m, 6H),

5.74 (m, 0.1H), 5.51 (m, 0.8H), 5.17 (t, J = 6.8 Hz, 0.9H), 5.09 (m, 0.8H),

5.00 (d, J = 7.6 Hz, 0.2H), 4.91 (m, 0.2H), 4.12 (t, J = 7.6 Hz, 1H), 3.98

(m, 1H), 3.83 (m, 1H), 3.70 (m, 1H), 3.54, 3.55 (2 s, 5.5H), 3.46 (s, 0.3H),

3.41 (m, 1H), 3.31 (s, 0.2H), 2.37 (m, 1H), 2.20 (m, 3H), 2.02 (m, 5H),

0.91 (m, 0.4H), 0.76, 0.82 (2d, J = 6.4 Hz, 5.6H). ESIHRMS m/z calcd for

C₄₁H₄₄N₈O₆[M+H]⁺745.3456; found 745.3443.

Methyl {(S)-1-[(S)-2-(5-{[4-(2-{(S)-1-[(S)-2-(methoxycarbonylamino)-2-

phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)phenyl]buta-1,3-diynyl}-1H-

imidazol-2-yl)pyrrolidine-1-yl]-3-methyl-1-oxobutan-2-yl}-carbamate

dihy-

drochloride (1.10∙2HCl): LC/MS 745 (M+H)⁺. ¹H NMR (DMSO‑d₆,

400 MHz) δ 15.35 (brs, 0.5H), 14.80 (brs, 0.6H), 8.20 (s, 1H), 7.98 (m, 3H),

7.78 (m, 2.2H), 7.68 (m, 0.7H), 7.60 (s, 0.1H), 7.32 (m, 5.3H), 7.16 (m,

0.5H), 6.96 (m, 0.2H), 5.73 (m, 0.05H), 5.56 (m, 0.15H), 5.49 (m, 0.8H),

5.32 (m, 0.15H), 5.17 (m, 1.05H), 5.09 (m, 0.8H), 4.12 (t, J = 7.6 Hz, 1H),

3.97 (m, 1H), 3.86 (m, 2H), 3.53, 3.54 (2 s, 5.9H), 3.31 (s, 0.1H), 3.11 (m,

1H), 2.37 (m, 1H), 2.06 (m, 7H), 1.84 (m, 1H), 0.91 (m, 0.3H), 0.76, 0.82

(2d, J = 6.8 Hz, 5.7H). ESIHRMS m/z calcd for C₄₁H₄₄N₈O₆[M+H]⁺

745.3456; found 745.3448.

Methyl {(S)-1-[(S)-2-(5-{4-[(2-{(S)-1-[(R)-2-(methoxycarbonylamino)-2-

phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-1,3-diynyl]-3-(4-methylpi-

perazin-1-yl)phenyl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-methyl-

propyl}-carbamate tetrahydrochloride (1.11∙4HCl): LC/MS: 822 (M+H)^{+ 1}

H

Methyl

{(S)-1-[(S)-2-(5-{[3-ﬂuoro-5-(2-{(S)-1-[(S)-2-(methox-

NMR (DMSO‑d₆, 400 MHz) δ 15.64 (brs, 0.6H), 14.90 (brs, 0.8H), 11.25

(brs, 1H), 8.29 (s, 0.05H), 8.26 (s, 0.95H), 7.99 (s, 0.8H), 7.86 (m, 0.2H),

7.69 (m, 2.8H), 7.58 (m, 1.2H), 7.34 (m, 4.6H), 7.26 (d, J = 8.0 Hz, 0.8H),

7.16 (m, 0.4H), 6.97 (m, 0.2H), 5.76 (m, 0.04H), 5.55 (m, 0.13H), 5.49 (d,

J = 8.0 Hz, 0.77H), 5.30 (d, J = 8.0 Hz, 0.12H) 5.19 (t, J = 7.2 Hz, 1H),

5.10 (m, 0.94H), 4.12 (t, J = 7.6 Hz, 1H), 4.02 (m, 1H), 3.89 (m, 1H), 3.80

(m, 3H), 3.54 (m, 2H), 3.53, 3.54 (2 s, 5.8H), 3.36 (m, 2.2H), 3.23 (m, 2H),

3.10 (m, 1H), 2.85 (d, J = 4.0 Hz, 3H), 2.37 (m, 1H), 1.91–2.24 (m, 6H),

1.84 (m, 1H), 0.93 (d, J = 6.8 Hz, 0.2H), 0.90 (d, J = 6.8 Hz, 0.2H), 0.83

(d, J = 6.8 Hz, 2.8H), 0.76 (d, J = 6.8 Hz, 2.8H). ESIHRMS m/z calcd for

C₄₆H₅₄N₁₀O₆[M+H]⁺843.4300; found 745.3457.

ycarbonylamino)-3-methylbutyryl]pyrrolidin-2-yl}-1H-imidazol-5-yl)bi-

phenyl-2-yl]buta-1,3-diynyl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-

methylpropyl}-carbamate dihydrochloride (1.6·2HCl): LC/MS 805 (M

+H)⁺. ¹H NMR (DMSO‑d₆, 400 MHz) δ 14.86 (brs, 0.6H), 8.34 (s,

0.1H), 8.32 (s, 0.9H), 8.04 (m, 1H), 7.92 (s, 2H), 7.68 (m, 2H), 7.53 (m,

3H), 7.26 (t, J = 8.0 Hz, 1.77H), 7.15 (m, 0.15H), 6.85 (m, 0.08H),

5.69 (m, 0.05H), 5.40 (m, 0.08H), 5.16 (t, J = 6.8 Hz, 0.95H), 5.02 (dd,

J₁= 7.6 Hz, J₂= 5.2 Hz, 0.92H), 4.11 (t, J = 7.6 Hz, 1H), 4.05 (t,

J = 8.0 Hz, 1H), 3.97 (m, 1H), 3.80 (m, 3H), 3.53, 3.54 (2 s, 5.5H),

3.40 (s, 0.3H), 3.31 (s, 0.2H), 2.36 (m, 1H), 2.05–2.30 (m, 5H), 1.98

(m, 4H), 1.35 (s, 9H), 0.75–0.92 (m, 12H). ESIHRMS m/z calcd for

Methyl {(S)-1-[(S)-2-(5-{6-[(2-{(S)-1-[(R)-2-(methoxycarbonylamino)-2-

phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-1,3-diynyl]biphenyl-3-yl}-

C

₄₄H₄₉FN₈O₆[M+H]⁺805.3832; found 805.3828.

Methyl {(R)-1-[(S)-2-(5-{[4-(2-{(S)-1-[(S)-2-(methoxycarbonylamino)-3-

1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-methylpropyl}-carbamate

dihy-

1

methylbutanoyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)phenyl]buta-1,3-diynyl}

drochloride (1.12∙2HCl, 66%): LC/MS 821 (M+H)⁺. H NMR (DMSO‑d₆,

400 MHz) δ 15.35 (brs, 0.6H), 14.84 (brs, 0.6H), 8.30 (s, 0.1H), 8.28 (s,

0.9H), 8.02 (m, 1H), 7.96 (m, 1.8H), 7.90 (m, 1H), 7.82 (m, 0.2H), 7.67 (m,

2.8H), 7.55 (m, 2H), 7.49 (m, 1H), 7.26–7.37 (m, 5.5H), 7.13 (m, 0.45H),

6.95 (m, 0.25H), 5.71 (m, 0.05H), 5.55 (m, 0.15H), 5.47 (d, J = 7.6 Hz,

0.8H), 5.30 (d, J = 7.6 Hz, 0.15H), 5.17 (t, J = 6.8 Hz, 1H), 5.07 (m,

0.85H), 4.12 (t, J = 7.6 Hz, 1H), 3.96 (m, 1H), 3.85 (m, 2H), 3.53, 3.54

(2 s, 5.9H), 3.30 (s, 0.1H), 3.11 (m, 1H), 1.90–2.21 (m, 5H), 1.83 (m, 1H),

0.92 (d, J = 6.8 Hz, 0.15H), 0.89 (d, J = 6.8 Hz, 0.15H), 0.83 (d,

J = 6.8 Hz, 2.85H), 0.76 (d, J = 6.8 Hz, 2.85H). ESIHRMS m/z calcd for

C₄₇H₄₈N₈O₆[M+H]⁺821.3769; found 821.3761.

-1H-imidazol-2-yl)pyrrolidine-1-yl]-2-oxo-1-phenylethyl}-carbamate

dihy-

drochloride (1.7∙2HCl): LC/MS 745 (M+H)⁺. ¹H NMR (DMSO‑d₆,

400 MHz) δ 15.30 (brs, 0.3H), 14.69 (brs, 0.8H), 8.23 (s, 0.9H), 8.00 (d,

J = 8.4 Hz, 1.8H), 7.93 (s, 1H), 7.78 (m, 2.1H), 7.71 (d, J = 7.6 Hz, 1H),

7.33 (m, 5.5H), 7.16 (m, 0.17H), 7.07 (m, 0.23H), 6.99 (m, 0.3H), 5.83 (d,

J = 8.0 Hz, 0.1H), 5.58 (m, 0.05H), 5.51 (d, J = 8.0 Hz, 0.8H), 5.43 (m,

0.15H), 5.39 (d, J = 7.2 Hz, 0.1H), 5.25 (m, 0.9H), 5.05 (m, 0.9H), 4.12 (t,

J = 7.6 Hz, 1H), 3.97 (m, 1H), 3.82 (m, 2H), 3.51, 3.52 (2 s, 5.7H), 3.43 (s,

0.3H), 3.15 (m, 1H), 2.26 (m, 2H), 2.15 (m, 1H), 2.02 (m, 5H), 1.88 (m,

1H), 0.88 (t, J = 7.2 Hz, 0.7H), 0.79, 0.82 (2d, J = 6.8 Hz, 5.3H).

ESIHRMS m/z calcd for C₄₁H₄₄N₈O₆[M+H]⁺745.3456; found 745.3457.

Methyl {(S)-1-[(S)-2-(5-{[4-(2-{(S)-1-[(S)-2-(methoxycarbonylamino)-3-

methylbutanoyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)phenyl]buta-1,3-diynyl}

Methyl {(S)-1-[(S)-2-(5-{4′-tert-butyl-6-[(2-{(S)-1-[(R)-2-(methox-

ycarbonylamino)-2-phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-

1,3-diynyl]biphenyl-3-yl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-me-

thylpropyl}-carbamate dihydrochloride (1.13∙2HCl, 75%): LC/MS 877 (M

+H)⁺. ¹H NMR (DMSO‑d₆, 400 MHz) δ 15.34 (brs, 0.5H), 14.86 (brs,

-1H-imidazol-2-yl)pyrrolidine-1-yl]-2-oxo-1-phenylethyl}-carbamate

dihy-

drochloride (1.8∙2HCl): LC/MS 745 (M+H)⁺. ¹H NMR (DMSO‑d₆,

15

A.A. Ivashchenko, et al.

Bioorganic&MedicinalChemistry28(2020)115716

0.6H), 8.30 (s, 0.1H), 8.28 (s, 0.9H), 8.02 (s, 1H), 7.96 (m, 1.7H), 7.89

(m, 1H), 7.82 (d, J = 8.0 Hz, 0.2H), 7.67 (d, J = 8.0 Hz, 0.8H), 7.62 (d,

J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.36 (m, 4H), 7.29 (m, 1H),

7.12 (m, 0.4H), 6.95 (m, 0.2H), 5.73 (m, 0.04H), 5.55 (m, 0.12H), 5.47

(d, J = 7.6 Hz, 0.8H), 5.30 (d, J = 7.6 Hz, 0.14H) 5.17 (t, J = 6.8 Hz,

1H), 5.08 (m, 0.9H), 4.12 (t, J = 7.6 Hz, 1H), 3.97 (m, 1H), 3.85 (m,

2H), 3.52, 3.54 (2 s, 5.8H), 3.29 (s, 0.2H), 3.10 (m, 1H), 2.37 (m, 1H),

1.90–2.21 (m, 6H), 1.83 (m, 1H), 1.35 (s, 9H), 0.92 (d, J = 6.8 Hz,

0.2H), 0.89 (d, J = 6.8 Hz, 0.2H), 0.83 (d, J = 6.8 Hz, 2.8H), 0.76 (d,

J = 6.8 Hz, 2.8H). ESIHRMS m/z calcd for C₅₁H₅₆N₈O₆[M+H]⁺

877.4395; found 877.4377.

(m, 0.02H), 5.57 (m, 0.07H), 5.48 (d, J = 7.6 Hz, 0.8H), 5.30 (d,

J = 7.6 Hz, 0.11H) 5.19 (t, J = 7.2 Hz, 1H), 5.09 (m, 1H), 4.12 (t,

J = 7.6 Hz, 1H), 3.99 (m, 3H), 3.85 (m, 2H), 3.53, 3.54 (2 s, 5.8H), 3.50

(m, 2H), 3.30 (s, 0.2H), 3.17 (m, 5H), 2.82 (d, J = 4.4 Hz, 3H), 2.36 (m,

1H), 2.14 (m, 3H), 1.97 (m, 3H), 1.83 (m, 1H), 0.92 (d, J = 6.8 Hz, 0.2H),

0.89 (d, J = 6.8 Hz, 0.2H), 0.83 (d, J = 6.8 Hz, 2.8H), 0.76 (d, J = 6.8 Hz,

2.8H). ESIHRMS m/z calcd for C₅₂H₅₈N₁₀O₆[M+H]⁺919.4613; found

919.4603.

Methyl {(S)-1-[(S)-2-(5-{6-[(2-{(S)-1-[(R)-2-(methoxycarbonylamino)-2-

phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-1,3-diynyl]-1,1′:3′,1″-ter-

phenyl-3-yl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-methylpropyl}-carba-

mate dihydrochloride (1.18∙2HCl, 62%): LC/MS 897 (M+H)⁺. ¹H NMR

(DMSO‑d₆, 400 MHz) δ 15.42 (brs, 0.6H), 14.86 (brs, 0.8H), 8.32 (s, 0.1H),

8.30 (s, 0.9H), 8.17 (m, 1H), 7.99 (m, 1H), 7.94 (m, 2.9H), 7.80 (m, 3.4H),

7.67 (m, 3H), 7.50 (m, 2.3H), 7.37 (m, 5.6H), 7.26 (d, J = 8.4 Hz, 1H),

7.08 (m, 0.5H), 6.93 (m, 0.3H), 5.74 (m, 0.06H), 5.54 (m, 0.15H), 5.47 (d,

J = 7.6 Hz, 0.77H), 5.29 (d, J = 7.6 Hz, 0.16H) 5.18 (t, J = 7.2 Hz,

0.97H), 5.07 (m, 0.89H), 4.12 (t, J = 7.6 Hz, 1H), 3.99 (m, 1H), 3.84 (m,

2H), 3.51, 3.53 (2 s, 5.8H), 3.30 (s, 0.2H), 3.10 (m, 1H), 2.38 (m, 1H),

1.90–2.22 (m, 6H), 1.83 (m, 1H), 0.92 (d, J = 6.8 Hz, 0.2H), 0.89 (d,

J = 6.8 Hz, 0.2H), 0.81 (d, J = 6.8 Hz, 2.8H), 0.75 (d, J = 6.8 Hz, 2.8H).

ESIHRMS m/z calcd for C₅₃H₅₂N₈O₆[M+H]⁺897.4082; found 897.4083.

Methyl {(S)-1-[(S)-2-(5-{6-[(2-{(S)-1-[(R)-2-(methoxycarbonylamino)-2-

phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-1,3-diynyl]-1,1′:4′,1″-ter-

phenyl-3-yl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-methylpropyl}-carba-

mate dihydrochloride (1.19∙2HCl, 62%): LC/MS 897 (M+H)⁺. ¹H NMR

(DMSO‑d₆, 400 MHz) δ 15.38 (brs, 0.5H), 14.85 (brs, 0.6H), 8.29 (s, 1H),

8.09 (m, 1H), 7.99 (m, 1H), 7.92 (m, 1.6H), 7.86 (m, 1.9H), 7.79 (m, 3.7H),

7.65 (m, 0.7H), 7.51 (m, 2H), 7.35 (m, 5.5H), 7.08 (m, 0.38H), 6.92 (m,

0.22H), 5.73 (m, 0.06H), 5.52 (m, 0.15H), 5.46 (d, J = 8.0 Hz, 0.8H), 5.28

(d, J = 8.0 Hz, 0.2H) 5.18 (t, J = 6.8 Hz, 0.94H), 5.06 (m, 0.85H), 4.12 (t,

J = 7.6 Hz, 1H), 3.98 (m, 1H), 3.84 (m, 2H), 3.53, 3.54 (2 s, 3H), 3.49 (s,

2.75H), 3.31 (s, 0.25H), 3.09 (m, 1H), 2.38 (m, 1H), 2.18 (m, 2H), 2.09 (m,

1H), 2.03 (m, 1H), 1.93 (m, 2H), 1.81 (m, 1H), 0.92 (d, J = 6.8 Hz, 0.2H),

0.90 (d, J = 6.8 Hz, 0.2H), 0.83 (d, J = 6.8 Hz, 2.8H), 0.77 (d, J = 6.8 Hz,

2.8H). ESIHRMS m/z calcd for C₅₃H₅₂N₈O₆[M+H]⁺897.4082; found

897.4065.

Methyl {(S)-1-[(S)-2-(5-{3′-tert-butyl-6-[(2-{(S)-1-[(R)-2-(methox-

ycarbonylamino)-2-phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-

1,3-diynyl]biphenyl-3-yl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-me-

thylpropyl}-carbamate dihydrochloride (1.14∙2HCl, 56%): LC/MS 877 (M

+H)⁺. ¹H NMR (DMSO‑d₆, 400 MHz) δ 15.42 (brs, 0.4H), 14.84 (brs,

0.6H), 8.30 (s, 0.1H), 8.27 (s, 0.9H), 8.07 (m, 1H), 7.95 (m, 1H), 7.89

(m, 1.7H), 7.82 (d, J = 7.6 Hz, 0.2H), 7.73 (s, 1H), 7.66 (d, J = 7.6 Hz,

0.8H), 7.48 (m, 3.2H), 7.35 (m, 4.4H), 7.29 (m, 1H), 7.27 (d,

J = 8.8 Hz, 0.8H), 7.12 (m, 0.5H), 6.93 (m, 0.25H), 5.74 (m, 0.05H),

5.54 (m, 0.14H), 5.47 (d, J = 7.6 Hz, 0.77H), 5.30 (d, J = 7.6 Hz,

0.18H) 5.18 (t, J = 6.8 Hz, 1H), 5.07 (m, 0.9H), 4.12 (t, J = 7.6 Hz,

1H), 3.98 (m, 1H), 3.85 (m, 2H), 3.52, 3.54 (2 s, 5.8H), 3.30 (s, 0.2H),

3.10 (m, 1H), 2.38 (m, 1H), 2.18 (m, 2H), 2.00 (m, 1H), 1.97 (m, 3H),

1.83 (m, 1H), 1.36, 1.37 (2 s, 9H), 0.92 (d, J = 6.8 Hz, 0.2H), 0.89 (d,

J = 6.8 Hz, 0.2H), 0.83 (d, J = 6.8 Hz, 2.8H), 0.76 (d, J = 6.8 Hz,

2.8H). ESIHRMS m/z calcd for C₅₁H₅₆N₈O₆[M+H]⁺877.4395; found

877.4372.

Methyl {(S)-1-[(S)-2-(5-{2′,3′-diﬂuoro-6-[(2-{(S)-1-[(R)-2-(methox-

ycarbonylamino)-2-phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-

1,3-diynyl]biphenyl-3-yl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-me-

thylpropyl}-carbamate dihydrochloride (1.15∙2HCl, 63%): LC/MS 857 (M

+H)⁺. ¹H NMR (DMSO‑d₆, 400 MHz) δ 15.31 (brs, 0.4H), 14.81 (brs,

0.7H), 8.26 (s, 0.95H), 8.07 (m, 1.95H), 7.93 (m, 1.74H), 7.81 (m,

0.25H), 7.61 (m, 1.92H), 7.35 (m, 7.28H), 7.11 (m, 0.58H), 6.93 (m,

0.33H), 5.70 (m, 0.08H), 5.52 (m, 0.13H), 5.47 (d, J = 8.0 Hz, 0.76H),

5.29 (m, 0.16H), 5.16 (t, J = 7.0 Hz, 1H), 5.06 (m, 0.87H), 4.11 (t,

J = 8.0 Hz, 1H), 3.96 (m, 1H), 3.84 (m, 2H), 3.53, 3.54 (2 s, 5.76H),

3.30 (s, 0.24H), 3.10 (m, 1H), 2.37 (m, 1H), 2.04 (m, 7H), 1.82 (m, 1H),

0.89, 0.91 (2d, J = 6.8 Hz, 0.55H), 0.76, 0.82 (2d, J = 6.8 Hz, 5.45H).

ESIHRMS m/z calcd for C₄₇H₄₆FN₈O₆[M+H]⁺857.3581; found

857.3584.

Methyl {(S)-1-[(S)-2-(5-{6-[(2-{(S)-1-[(R)-2-(methoxycarbonylamino)-2-

phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-1,3-diynyl]-5-methylbi-

phenyl-3-yl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-methylpropyl}-carba-

mate dihydrochloride (1.20∙2HCl, 77%): LC/MS 835 (M+H)⁺. ¹H NMR

(DMSO‑d₆, 400 MHz) δ 15.21 (brs, 0.6H), 14.78 (brs, 0.7H), 8.25 (s, 0.1H),

8.23 (s, 0.9H), 7.93 (m, 1.8H), 7.82 (m, 1.1H), 7.63 (m, 2.8H), 7.53 (m,

2.2H), 7.48 (m, 1H), 7.35 (m, 4.5H), 7.26 (d, J = 8.4 Hz, 0.9H), 7.12 (m,

0.5H), 6.94 (m, 0.2H), 5.69 (m, 0.05H), 5.54 (m, 0.1H), 5.47 (d,

J = 7.6 Hz, 0.8H), 5.29 (d, J = 7.6 Hz, 0.15H), 5.16 (t, J = 7.0 Hz, 1H),

5.07 (m, 0.9H), 4.11 (t, J = 7.6 Hz, 1H), 3.95 (m, 1H), 3.84 (m, 2H), 3.53,

3.54 (2 s, 5.8H), 3.30 (s, 0.2H), 3.10 (m, 1H), 2.55 (s, 3H), 2.37 (m, 1H),

1.89–2.22 (m, 6H), 1.83 (m, 1H), 0.91 (d, J = 6.8 Hz, 0.2H), 0.89 (d,

J = 6.8 Hz, 0.2H), 0.82 (d, J = 6.8 Hz, 2.8H), 0.76 (d, J = 6.8 Hz, 2.8H).

ESIHRMS m/z calcd for C₄₈H₅₀N₈O₆[M+H]⁺835.3926; found 835.3905.

Methyl {(1R)-2-[(2S)-2-{5-[4-(3-ﬂuoro-5-{2-[(2S)-1-{(2S)-2-[(meth-

oxycarbonyl)amino]-3-methylbutanoyl}pyrrolidin-2-yl]-1H-imidazol-5-yl}

biphenyl-2-yl)buta-1,3-diyn-1-yl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-2-oxo-

1-phenylethyl}carbamate (1.21∙2HCl, 71%): LC/MS 839 (M+H)⁺. ¹H

NMR (DMSO‑d₆, 400 MHz) δ 14.86 (brs, 0.6H), 8.35 (s, 0.1H), 8.32 (s,

0.9H), 8.05 (m, 1H), 7.97 (s, 0.8H), 7.93 (s, 1H), 7.68 (m, 2.8H), 7.55

(m, 3.2H), 7.35 (m, 4.5H), 7.25 (d, J = 8.8 Hz, 0.9H), 7.12 (m, 0.5H),

6.95 (m, 0.2H), 5.69 (m, 0.05H), 5.55 (m, 0.12H), 5.47 (d, J = 7.6 Hz,

0.8H), 5.29 (d, J = 7.6 Hz, 0.15H), 5.16 (t, J = 7.2 Hz, 0.96H), 5.07

(m, 0.92H), 4.12 (t, J = 7.6 Hz, 1H), 3.98 (m, 1H), 3.84 (m, 2H), 3.53,

3.54 (2 s, 5.8H), 3.31 (s, 0.2H), 3.11 (m, 1H), 2.36 (m, 1H), 2.17 (m,

2H), 2.08 (m, 1H), 1.97 (m, 3H), 1.83 (m, 1H), 0.92 (d, J = 6.8 Hz,

0.2H), 0.89 (d, J = 6.8 Hz, 0.2H), 0.83 (d, J = 6.8 Hz, 2.8H), 0.76 (d,

J = 6.8 Hz, 2.8H). ESIHRMS m/z calcd for C₄₇H₄₇FN₈O₆[M+H]⁺

839.3675; found 839.3665.

Methyl

{(S)-1-[(S)-2-(5-{4′-(dimethylamino)-6-[(2-{(S)-1-[(R)-2-

(methoxycarbonylamino)-2-phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-

yl)buta-1,3-diynyl]biphenyl-3-yl}-1H-imidazol-2-yl)pyrrolidine-1-car-

bonyl]-2-methylpropyl}-carbamate trihydrochloride (1.16∙2HCl, 74%):

LC/MS 864 (M+H)⁺. ¹H NMR (DMSO‑d₆, 400 MHz) δ 15.50 (brs,

0.6H), 14.93 (brs, 0.9H), 8.30 (s, 0.1H), 8.27 (s, 0.9H), 8.07 (s, 0.8H),

8.04 (s, 1H), 7.91 (m, 1H), 7.86 (m, 1H), 7.77 (m, 0.2H), 7.67 (m,

2.8H), 7.35 (m, 4.7H), 7.26 (d, J = 8.4 Hz, 1H), 7.14 (m, 2H), 6.99 (m,

0.3H), 5.77 (m, 0.1H), 5.64 (m, 0.15H), 5.48 (d, J = 7.6 Hz, 0.75H),

5.32 (d, J = 7.6 Hz, 0.15H) 5.19 (t, J = 6.8 Hz, 0.95H), 5.12 (m, 0.9H),

4.12 (t, J = 7.6 Hz, 1H), 4.00 (m, 1H), 3.85 (m, 2H), 3.52, 3.54 (2 s,

5.8H), 3.30 (s, 0.2H), 3.10 (m, 1H), 3.04 (s, 6H), 2.37 (m, 1H),

1.92–2.23 (m, 6H), 1.84 (m, 1H), 0.92 (d, J = 6.8 Hz, 0.2H), 0.90 (d,

J = 6.8 Hz, 0.2H), 0.83 (d, J = 6.8 Hz, 2.8H), 0.76 (d, J = 6.8 Hz,

2.8H). ESIHRMS m/z calcd for C₄₉H₅₃N₉O₆[M+H]⁺864.4191; found

864.4192.

Methyl {(S)-1-[(S)-2-(5-{6-[(2-{(S)-1-[(R)-2-(methoxycarbonylamino)-2-

phenylacetyl]pyrrolidin-2-yl}-1H-imidazol-5-yl)buta-1,3-diynyl]-4′-(4-methyl-

piperazin-1-yl)biphenyl-3-yl}-1H-imidazol-2-yl)pyrrolidine-1-carbonyl]-2-me-

thylpropyl}-carbamate tetrahydrochloride (1.17∙4HCl, 57%): LC/MS 919 (M

+H)⁺. ¹H NMR (DMSO‑d₆, 400 MHz) δ 15.52 (brs, 0.6H), 14.91 (brs,

0.7H), 11.19 (brs, 1H), 8.29 (s, 0.1H), 8.26 (s, 0.9H), 8.04 (m, 1.1H), 7.98

(s, 0.8H), 7.87 (m, 2.3H), 7.68 (d, J = 8.0 Hz, 0.75H), 7.62 (m, 2.2H), 7.34

(m, 4.7H), 7.26 (d, J = 8.4 Hz, 1H), 7.14 (m, 2.5H), 6.96 (m, 0.25H), 5.76

16

A.A. Ivashchenko, et al.

Bioorganic&MedicinalChemistry28(2020)115716

Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)-2-methoxycarbonylamino-2-

phenyl-acetyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-3-phenoxy-

phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-carbamate

dihydrochloride (1.22·2HCl, 28%): LC/MS 837 (M+H)⁺. ¹H NMR

(DMSO‑d₆, 400 MHz) δ 14.76 (brs, 0.6H), 8.19 (s, 1H), 7.95 (s, 1H), 7.86

(m, 1H), 7.79 (m, 1.4H), 7.66 (m, 0.8H), 7.59 (s, 0.8H), 7.59 (m, 0.2H),

7.42 (m, 2H), 7.34 (m, 4.4H), 7.24 (d, J = 8.0 Hz, 0.8H), 7.18 (t,

J = 6.8 Hz, 1H), 7.12 (m, 0.4H), 7.06 (d, J = 8.0 Hz, 2H), 6.94 (m, 0.2H),

5.64 (m, 0.01H), 5.54 (m, 0.08H), 5.47 (d, J = 8.4 Hz, 0.79H), 5.29 (d,

J = 7.6 Hz, 0.16H), 5.09 (m, 1.96H), 4.09 (t, J = 7.6 Hz, 1H), 3.92 (m,

1H), 3.83 (m, 3H), 3.52, 3.53 (2 s, 5.8H), 3.27 (s, 0.2H), 3.10 (m, 1H), 2.34

(m, 1H), 2.14 (m, 2H), 1.97 (m, 4H), 1.83 (m, 1H), 0.89 (d, J = 6.8 Hz,

0.2H), 0.86 (d, J = 6.8 Hz, 0.2H), 0.78 (d, J = 6.8 Hz, 2.8H), 0.73 (d,

J = 6.8 Hz, 2.8H). ESIHRMS m/z calcd for C₄₇H₄₈N₈O₇[M+H]⁺

837.33718; found 837.3710.

reﬁned as well. Finally, ten sets with ten structures in each one were

obtained. From each set, a model with the lowest potential energy was

selected for further assessment. Receptor Grid Generation tool was used

to generate a grid for the following docking procedures. Coordinates of

the center of the grid: X = 5.74, Y = 10.24, Z = 53.34. Box size: length

X = 17, box length Y = 10, box length Z = 17.

2D structures of ligands were preprocessed using the LigPrep tool at

default settings. Chiralities were speciﬁed prior to preprocessing and

inspected manually after conformers generation. Docking simulations

were performed with the Ligand Docking tool (Glide) at standard pre-

cision mode. Obtained conformers were sorted by the values of the

Glide Emodel score function and inspected manually.

5.3. Replicon antiviral assay

Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)-2-methoxycarbonylamino-2-

phenyl-acetyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-3-phensul-

phanyl-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-

carbamate dihydrochloride (1.23·2HCl, 36%): LC/MS 853 (M+H)⁺. ¹H

NMR (DMSO‑d₆, 400 MHz) δ 15.38 (brs, 0.3H), 14.91 (brs, 0.6H), 8.12 (s,

1H), 8.05 (s, 0.9H), 7.95 (m, 2H), 7.87 (m, 1H), 7.79 (m, 0.1H), 7.68 (d,

J = 7.6 Hz, 0.7H), 7.64 (m, 0.15H), 7.36 (m, 9.85H), 7.24 (d, J = 9.2 Hz,

0.8H), 7.14 (m, 0.35H), 6.98 (m, 0.15H), 5.69 (m, 0.05H), 5.60 (m, 0.1H),

5.48 (d, J = 7.6 Hz, 0.8H), 5.32 (d, J = 7.6 Hz, 0.15H), 5.13 (m, 1.9H),

4.10 (t, J = 7.6 Hz, 1H), 3.97 (m, 1H), 3.89 (m, 1H), 3.81 (m, 1H), 3.52,

3.53 (2 s, 5.85H), 3.27 (s, 0.15H), 3.11 (m, 1H), 2.34 (m, 1H), 2.16 (m,

2H), 2.06 (m, 1H), 1.97 (m, 3H), 1.84 (m, 1H), 0.90 (m, 0.4H), 0.79 (d,

J = 6.8 Hz, 2.8H), 0.73 (d, J = 6.8 Hz, 2.8H). ESIHRMS m/z calcd for

C₄₇H₄₈N₈O₆S [M+H]⁺853.3490; found 853.3477.

The human hepatoma cell line Huh7 harboring the HCV replicon

was used as the test line to perform the assay. Compounds were tested

for their inhibitory activities in the HCV gT1a, gT1b, gT2a, gT3a, gT4a,

gT5a subgenomic replicons assays with the addition of 10% FBS. Cells

were seeded at a density of 3 × 10³cells per well in 384-well plates

containing 40 μL of assay media (DMEM 1×, Cellgro, catalogue no. 10-

013-CV). After overnight incubation of the cells at 37 °C in a 5% CO₂

atmosphere compound treatment was initiated. A total of 10 serial

3.16-fold dilutions of the test compounds were prepared as 200 × stock

in DMSO. Then 5 × compounds solution was prepared fresh in the

assay medium and 10 μL of this 5 × solution was transferred to the

plates with cells. The ﬁnal concentration of the compounds was from

10 nM to 0.1 pM. The ﬁnal DMSO concentration was 0.5%. Test ex-

amples were then incubated for 3 days at 37 °C in a 5% CO₂atmo-

sphere. Bright-Glo reagent (25 ml) was added in each well of assay

plates after 72 h of incubation with compounds. Intensity of lumines-

cence were measured for each well on the Victor Light luminescence

counter after 3–5 min of incubation with Brigth-Glo reagent.

Methyl (S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)-2-methoxycarbonylamino-2-

phenyl-acetyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-3-(methyl-

phenyl-amino)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-

propyl]-carbamate trihydrochloride (1.24·3HCl, 37%): LC/MS 850 (M+H)⁺.

¹H NMR (DMSO‑d₆, 400 MHz) δ 15.21 (brs, 0.3H), 14.75 (brs, 0.6H), 8.22

(s, 1H), 7.89 (m, 0.8H), 7.85 (m, 1H), 7.76 (m, 2.1H), 7.65 (d, J = 7.6 Hz,

0.8H), 7.51 (m, 0.1H), 7.34 (m, 4.5H), 7.23 (m, 2.9H), 7.11 (m, 0.5H), 6.93

(m, 0.3H), 6.84 (t, J = 7.6 Hz, 1H), 6.77 (d, J = 8.0 Hz, 2H), 5.67 (m,

0.05H), 5.52 (m, 0.1H), 5.47 (d, J = 7.6 Hz, 0.8H), 5.29 (d, J = 7.6 Hz,

0.15H), 5.14 (t, J = 6.8 Hz, 1H), 5.06 (m, 0.9H), 4.10 (t, J = 7.6 Hz, 1H),

3.94 (m, 1H), 3.84 (m, 2H), 3.52, 3.53 (2 s, 5.8H), 3.35 (s, 3H), 3.31 (s,

0.2H), 3.10 (m, 1H), 2.35 (m, 1H), 1.89–2.20 (m, 6H), 1.82 (m, 1H), 0.90

(m, 0.4H), 0.81 (d, J = 6.8 Hz, 2.8H), 0.75 (d, J = 6.8 Hz, 2.8H).

ESIHRMS m/z calcd for C₄₈H₅₁N₉O₆[M+H]⁺850.4035; found 850.4033.

5.4. Cytotoxicity

Cytotoxicity of the tested compounds was evaluated in parallel

using the same human hepatoma cell line Huh7 in gT1b replicon by

analogy to the procedure described above. CellTiter-Glo reagent (10 ml)

was used instead of Bright-Glo reagent after 72 h of incubation with the

test compound. Intensity of luminescence was measured for each well

using Victor Light luminescence counter after 3–5 min of incubation.

All the compounds showed an CC₅₀value over 10 µM.

5.5. PK study in rats

5.2. Molecular modeling

Animal studies were carried out following protocols approved by

the Institutional Animal Care and Use Committees as well as institu-

tional guidelines for the proper, humane use of animals in research.

Male Sprague-Dawley rats (8–10 weeks) obtained from Charles River

Laboratories International, Inc., Germany were used for PK studies.

Animals were housed under controlled conditions (22–26 °C, relative

humidity 30–70%) with a natural light/dark cycle (12 h/12 h). Animals

were previously catheterized in the jugular vein and fasted for 12 h

before the study with free access to water. Compounds were dissolved

in 10% DMSO/10% Solutol/80% 0.05 M N-methylglucamine for IV

administration (5 ml/kg) and in 0.5% methyl cellulose for PO admin-

istration (10 ml/kg). Blood samples were taken in EDTA-containing

tubes 5, 15, 30 min, 1, 2, 4, 8 and 24 h after IV administration and 15,

30 min, 1, 2, 4, 8 and 24 h after oral administration. Plasma samples

(45 μL) were prepared by centrifugation of the blood, mixed with 5 μL

of acetonitrile:H₂O (1:1 v/v) and precipitated with 150 μL of acetoni-

trile containing the internal standard tolbutamide (50 ng/mL). Samples

were incubated at −20 °C for 15 min followed by centrifugation at

11,000 g for 10 min, and then the supernatants (130 μL) were trans-

ferred into a new plate for LC-MS/MS analysis. The analysis was

Ligands and protein preparation, homology modeling, docking si-

mulations were performed using the Maestro suite (release 2015-2).²⁹

The homodimeric structure of HCV NS5A gtT3a was constructed ex-

ploiting Structure Prediction Wizard implemented in Prime. The pro-

tein sequence of the N-terminal region (residues 1–198) was extracted

from the NCBI Protein database (access code AFP27699.1). The mode-II

structure of D-I attached to the terminal alpha-helices described by

Nettles et al.²⁷was applied as a template for homology modeling. Both

subunits were reconstructed simultaneously using a knowledge-based

method for a homo-multimer model type. Zn²⁺cations in the area of

zinc-ﬁngers and the ligand were included during the model construc-

tion. The obtained protein structure was preprocessed with the Protein

Preparation Wizard tool at the following settings: amino acid residue

state generation (Epik, at pH = 7.4

0) and restrained minimization

(converge heavy atoms to RMSD 0.8 Å). After preprocessing the ﬂexible

linker (residues 28–36) of chain A had been reﬁned, exploiting the

Reﬁne Loops tool implemented in Prime. The reﬁnement was per-

formed with an extended serial loop sampling method, recommended

for loops with 6–11 residues. Side chains were reﬁned within 4.5 Ȧ. It

resulted in ten models. For each of them, the same region of chain B was

17

A.A. Ivashchenko, et al.

Bioorganic&MedicinalChemistry28(2020)115716

performed using Agilent 1290 UPLC coupled with Qtrap5500 mass

spectrometer (ABSciex, USA). PK parameters were calculated using

noncompartmental analysis provided by the software WinNonlin

Professional 5.2 (Pharsight Corporation).

11. Spach DH, Kim HN, Treatment of HCV genotype 5 or 6. Hepatitis C online 2019, Sec.

5, Top. 5 or 6; 2019. https://www.hepatitisc.uw.edu/go/treatment-infection/

treatment-genotype-5-or-6/core-concept/all [accessed 16 January 2020].

12. Belda O, Targett-Adams P. Small molecule inhibitors of the hepatitis C virus-encoded

NS5A protein. Virus Res. 2012;170:1–14. https://doi.org/10.1016/j.virusres.2012.

09.007.

13. Nettles JH, Jenkins JL, Bender A, Deng Z, Davies JW, Glick M. Asymmetric binding to

NS5A by daclatasvir (BMS-790052) and analogs suggests two novel modes of HCV

inhibition. J Med Chem. 2006;49:6802–6810. https://doi.org/10.1021/jm501291c.

14. Belema M, Lopez OD, Bender JA, et al. Discovery and development of hepatitis C

virus NS5A replication complex inhibitors. J Med Chem. 2014;57:1643–1672.

https://doi.org/10.1021/jm401793m.

Author contributions

The manuscript was written through contributions of all authors. All

authors have given approval to the ﬁnal version of the manuscript.

15. Nitta S, Asahina Y, Matsuda M, et al. Eﬀects of resistance-associated NS5A mutations

in hepatitis C virus on viral production and susceptibility to antiviral reagents. Sci

Rep. 2016;6:34652. https://doi.org/10.1038/srep34652.

Funding

16. Coburn CA, Meinke PT, Chang W, et al. Discovery of MK-8742: an HCV NS5A in-

hibitor with broad genotype activity. Chem Med Chem. 2013;8:1930–1940. https://

doi.org/10.1002/cmdc.201300343.

The authors also acknowledge the ﬁnancial support of Russian

Science Foundation [grant number 17-74-30012], IBG RAS Ufa (in silico

study).

17. Link JO, Taylor JG, Xu L, et al. Discovery of ledipasvir (GS-5885): a potent, once-

daily oral NS5A inhibitor for the treatment of hepatitis C virus infection. J Med Chem.

2014;57:2033–2046. https://doi.org/10.1021/jm401499g.

Declaration of Competing Interest

18. Ivachtchenko AV, Mitkin OD, Yamanushkin PM, et al. Discovery of novel highly

potent hepatitis C virus NS5A inhibitor (AV4025). J Med Chem. 2014;57:7716–7730.

https://doi.org/10.1021/jm500951r.

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂu-

ence the work reported in this paper.

19. DeGoey DA, Randolph JT, Liu D, et al. Discovery of ABT-267, a pan-genotypic in-

hibitor of HCV NS5A. J Med Chem. 2014;57:2047–2057. https://doi.org/10.1021/

jm401398x.

20. Vince B, Hill JM, Lawitz EJ, et al. A randomized, double-blind, multiple-dose study of

the pan-genotypic NS5A inhibitor samatasvir in patients infected with hepatitis C

virus genotype 1, 2, 3 or 4. J Hepatol. 2014;60:920–927. https://doi.org/10.1016/j.

jhep.2014.01.003.

Acknowledgements

21. Bilello JP, Lallos LB, McCarville JF, et al. In vitro activity and resistance proﬁle of

samatasvir, a novel NS5A replication inhibitor of hepatitis C virus. Antimicrobobial

Agents Chemother. 2014;58:4431–4442. https://doi.org/10.1128/AAC.02777-13.

22. Tong L, Yu W, Chen L, et al. Discovery of ruzasvir (MK-8408): a potent, pan-genotype

HCV NS5A inhibitor with optimized activity against common resistance-associated

polymorphisms. J Med Chem. 2017;60:290–306. https://doi.org/10.1021/acs.

jmedchem.6b01310.

The authors gratefully thank Alexandre V. Ponomarev, Dr. Yulia A.

Pelogeykina, Ekaterina Yu. Volosova for excellent technical assistance.

Appendix A. Supplementary material

23. Yu W, Hu B, Zhong B, et al. Discovery of novel pan-genotypic HCV NS5A inhibitors

containing a novel tetracyclic core. Bioorg Med Chem Lett. 2019;29:700–706. https://

doi.org/10.1016/j.bmcl.2019.01.031.

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.bmc.2020.115716.

24. Yu W, Tong L, Selyutin O, et al. Discovery of MK-6169, a potent pan-genotype he-

patitis C virus NS5A inhibitor with optimized activity against common resistance-

associated substitutions. J Med Chem. 2018;61:3984–4003. https://doi.org/10.1021/

acs.jmedchem.7b01927.

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