Discovery of Novel Highly Potent Hepatitis C Virus NS5A Inhibitor

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Discovery of Novel Highly Potent Hepatitis C Virus NS5A Inhibitor (AV4025) Alexandre V. Ivachtchenko, Oleg D. Mitkin, Pavel M. Yamanushkin, Irina V. Kuznetsova, Elena A. Bulanova, Natalia A. Shevkun, Angela G. Koryakova, Ruben N. Karapetian, Vadim V. Bichko, Andrey S. Trifelenkov, Dmitry V. Kravchenko, Natalia V. Vostokova, Mark S. Veselov, Nina V. Chufarova, and Yan Andreevich Ivanenkov J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500951r • Publication Date (Web): 22 Aug 2014 Downloaded from http://pubs.acs.org on August 25, 2014

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Journal of Medicinal Chemistry

Discovery of Novel Highly Potent Hepatitis C Virus NS5A Inhibitor (AV4025)

Alexandre V. Ivachtchenko,a–c Oleg D. Mitkin,b Pavel M. Yamanushkin,b Irina V. Kuznetsova,b Elena A. Bulanova,b Natalia A. Shevkun,b Angela G. Koryakova,b Ruben N. Karapetian,b Vadim V. Bichko,a,c Andrey S. Trifelenkov,b Dmitry V. Kravchenko,b Natalia V. Vostokova,b Mark S. Veselov,d Nina V. Chufarovad and Yan A. Ivanenkov*,b-d

a

Alla Chem LLC, Hallandale Beach Blvd, 442, Fl 33009, USA

b

Chemical Diversity Research Institute, 141401 Khimki, Moscow Reg., Rabochaya St. 2a,

Russia c

ChemDiv, 6605 Nancy Ridge Drive San Diego, California 92121, USA

d

Moscow Institute of Physics and Technology (State University), 9 Institutskiy lane,

Dolgoprudny city, Moscow Reg., 141700, Russia

ABSTRACT: A series of next in class small-molecule Hepatitis C Virus (HCV) NS5A inhibitors with picomolar potency containing 2-pyrrolidin-2-yl-5-{4-[4-(2-pyrrolidin-2-yl-1Himidazol-5-yl)buta-1,3-diynyl]phenyl}-1H-imidazole cores was designed based on the SAR studies available for the reported NS5A inhibitors. Compound 13a (AV4025), with (S,S,S,S)stereochemistry (EC50 = 3.4 ± 0.2 pM, HCV replicon genotype 1b), was dramatically more active than were the compounds with two (S)- and two (R)-chiral centers. Human serum did not significantly reduce the antiviral activity (< 4-fold). Relatively favorable pharmacokinetic features and good oral bioavailability were observed during animal studies. Compound 13a was well tolerated in rodents (in mice, LD50 = 2326 mg/kg or higher), providing a relatively high therapeutic index. During safety, pharmacology and sub-chronic toxicity studies in rats and dogs, it was not associated with any significant pathological or clinical findings. This compound is currently being evaluated in Phase I/II clinical trials for the treatment of HCV infection.

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INTRODUCTION Hepatitis C infection caused by HCV is among the most common liver diseases and is widespread throughout the world. Based on annual World Health Organization (WHO) reports, more than 130–150 million people are infected and more than 350–500K individuals die from HCV-related liver pathologies.1 In accordance with the Centers for Disease Control and Prevention statistical estimation, approx. 3.2 million people are chronically infected in the USA.2 Acute disease states are frequently observed after long-term and asymptomatic periods. Approximately 75–85% of newly infected persons become chronically infected. Among these patients, 60–70% will suffer chronic liver disease. In 5–20% of cases, cirrhosis or liver cancer is diagnosed, resulting in 1–5% lethal outcomes. It is not surprising that HCV is the leading indication for liver transplantation.3

Recent years have seen a significant change in HCV treatment. Much of the challenge in current antiviral chemotherapy should address the development of novel direct-acting agents with a clear mechanism of action. Novel biological targets with high therapeutic value, including nonstructural HCV protein NS5A,4 have attracted increasing attention. NS5A is one of the key components in the HCV replication complex essential for virus RNA replication in host cells. It has successfully been validated in clinics as an anti-hepatitis C drug target with a high therapeutic potential.5,6 A number of NS5A inhibitors generally bearing a peptidomimetic core have recently been developed and show nano- and picomolar activity in vitro.7-14 Their structures can be roughly generalized by the simplified topological pharmacophore hypothesis (Figure 1). Their backbones consist of two amino-acid or peptide-based fragments (Capi, Capj) connected with linkers of different types and lengths (L). Capi

L

Capj

Figure 1. The common topological pharmacophore model for symmetric and pseudo-symmetric NS5A inhibitors. ACS Paragon Plus Environment

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Representative examples of reported NS5A inhibitors (1-12) fitting this pharmacophore are listed in Table 1.

13,16–23

As shown in Table 1, the first-in-class NS5A inhibitor, 1 (BMS-790052),

contains four (S)-chiral centers. Other known NS5A ligands share four (2-9), five (10 [GS-5885], 11 [MK-8742]) and six (12 [ABT-267]) chiral centers. A relatively rigid spacer of an optimal length is much more appropriate for good activity than a flexible one. Imidazole (in, e.g., 1 and 2) and its isosteric and bioisosteric analogues, including simple benzimidazole (e.g., 3 [IDX719]) or nontrivial carboxamide replacements (e.g., 12), can be reasonably regarded as privileged sub-structures. However, the spatial orientations of the substituents within the cap regions are crucial for tight binding. A detailed overview of possible binding modes predicted for compound 13a and other NS5A inhibitors using 3D-molecular docking studies is discussed below. The main drawback of the NS5A inhibitors currently being evaluated in clinical trials is in their relatively poor bioavailability and sub-optimal pharmacokinetic properties. For example, in 2009, Arrow Therapeutics discontinued the Phase II development of AZD-2836 and AZD7295 (standard oral formulation) against hepatitis C mainly because of their inappropriate pharmacokinetic profiles.15 On the other hand, compound 1 is an antiviral drug developed by Bristol-Myers Squibb that is currently undergoing registration in Japan for the treatment of chronic hepatitis C infection (genotype 1b) in combination with asunaprevir.

Table 1. Examples of known NS5A inhibitors with high activity against HCV replicon GT1b Capi-L-Capj

№ NHCO2 H3 C

1

O

H N

N

N

N N

N H

BMS-790052

O

Length[a], Å

0.009 ± 0.004[b]

Me

17.5 ± 0.5

Me

Me Me

EC50, nM

NHCO2 CH3

0.003 ± 0.001[c]



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2

NHCO2 H3 C

O

O

N

N H O

O

O NH

O

N

Me N

Me

NHCO2 CH3

0.0074 ± 0.0009[d]

17.5 ± 0.5

0.003[e]

20.5 ± 1.5

< 4.6[f]

14 ± 1

0.077[f]

16 ± 1

0.055[f]

16 ± 1

1.332[g]

14 ± 1

0.384[g]

21 ± 1

GSK2336805

S

NHCO 2H3 C

Me

N

Me

N

Me

3

H N

N

Me

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N H

NHCO 2 CH3

N

N

S

IDX-719 N (CH3 )2 O N

N

4

H N

O

N H

N

N

N O

N (H3 C )2

N(CH3 )2 O

5

H N

N

N

O

N H

N O

N

O N(CH3 )2

H N

6

NHCO 2 H3 C

O N

Me

NHCO 2 H3 C

7

NHCO2H3C

O

H N

N

N

O

Me O

Me N

N H

O

NHCO 2 CH3

H N

N

N

9

Me N H

O

Me Me

N

10

NHCO2CH3

Me Me

O

NHCO2CH3

N H

O N

0.026[h] 18.5 ± 1.5 0.009 ± 0.002[c]

F H N

N

N

Me Me

Me Me

O

N N

N

F NHCO2H3 C

Me

N N

H N NHCO2 H3 C

Me

N

Me Me

NHCO 2 CH3

N H

Me Me

8

N

Me

Me

N N

N

N H

O

0.004[h]

NHCO2CH3

GS-5885


17 ± 1

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NHCO2 H3 C

11

O O

N

N

Me Me

N

H N

N H

N

N

0.003 ± 0.001[i]

19 ± 1

0.0050 ± 0.0019[j]

18.5 ± 2.5

Me

O

MK-8742

Me

H3CO2 CHN

Me

CH3 O2 CHN

Me N

12

O O H N

HN

N

N O

Me Me

O

NHCO2 CH3

ABT-267 a

The distance between two key chiral points (arrow indicated, 1) was calculated for different minimized (in vacuo)

conformations using MOE Software for all the compounds presented.23 bIC50 value according to ref 16 and 17. c

Activity determined in this study (see the experimental section and conditions therein). dIC50 value according to ref

18. eIC50 value according to ref 19. fIC50 value according to ref 20. gIC50 value according to ref 13. hIC50 value according to ref 16. iIC50 value according to ref 21. jIC50 value according to ref 22.

RESULTS AND DISCUSSION As shown in Table 1, two NS5A inhibitors (7 and 8) possessing diynyl-imidazole and diynyldiphenyl-imidazole linkers showed target activities of 1.332 nM and 0.384 nM, respectively, with spacer lengths of 14 ± 1 Å and 21 ± 1 Å. Interestingly, much more potent analogues, e.g., 11, 12 and 1, have linker lengths in the median range of 19 ± 2 Å. Based on these observations and related in silico modeling, we have tentatively suggested that diynyl-monophenyl-imidazole linkers (19-21 Å) can provide superior binding. To establish whether novel linkers with a predefined length could indeed serve as appropriate bioisosteric replacements for previously reported spacers, a relatively small pilot library of 17 small molecule potential α-helix mimetics (13a-k) was synthesized and evaluated against the HCV genotype 1b replicon in cell-based assays (Table 2). A detailed description of the performed biological trial is provided in the experimental section (vide infra). Based on the available structure-activity relationship (SAR) information and our in silico modeling, we have concluded that a rigid diynylbenzene linker of an optimal length could position the caps appropriately for good binding and exhibit an ACS Paragon Plus Environment


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appropriate pharmacokinetic (PK) profile because the obtained compounds have lower molecular weights (MW = 710.84, 13a) than do other reported NS5A inhibitors with similar geometries and structures, e.g., 1 (MW = 738.89) and 11 (MW = 881.05). With respect to lipophilicity and aqueous solubility, the following descriptors were calculated: 13a (LogP = 4.99), 1 (LogP = 6.33) and 11 (LogP = 8.61); 13a and 1 (PSA = 174.6 Å), 11 (PSA = 188.8 Å). Therefore, we predicted comparable or even more beneficial PK features for our compounds.

Table 2. Antiviral activity of compounds 13a-k against HCV genotype 1b replicon H N

N

N

N H

Cap1

Cap2

. 2HCl 13a-k

1

№

gT1b 10% FBS

gT1b 40%

EC50, nM

NHS EC50, nM

0.0034 ± 0.0002[a]

0.021 ± 0.002

17.3

46.97 ± 5.27

136.6 ± 8.3

297.9

24.34 ± 2.33

34.2 ± 3.1

52.6

66.39 ± 7.67

136.1 ± 9.4

48.2

221.2 ± 18.4

565.0 ± 27.3

68.9

2

Сap

Сap

CC50, µM

NHCO 2 Me

13a

i-Pr

O

(S)

(S)

N (S)

N (S)

i-Pr

O NHCO2Me

NHCO 2 Me

13b

i-Pr

O

(R)

(S)

N (R)

N

i-Pr

O (S)

NHCO2Me

NHCO 2Me (S)

13c

O

O

(S)

N

O

N (S)

O

(S)

NHCO2Me

NHCO 2Me (S,R)

13d

O

O

(S)

N

N (S)

O (S,R)

O

NHCO2Me

Me

13e

MeO

NHCO 2 Me (S,R)

O

(S)

N (S)

O

N

OMe (S,R)

Me NHCO2Me


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Me

NHCO 2 Me O

13f

(S)

N Me

Me N (S)

O

599 ± 35.3

1959.0 ± 89.6

52.2

0.079 ± 0.0077

0.243 ± 0.056

24.7

0.4089 ± 0.0549

0.737 ± 0.123

30.3

5.372 ± 1.2

36.1 ± 5.5

34.7

882.1 ± 84.5

6896 ± 521

59.7

993.7 ± 132

7437 ± 689

108.5

Me NHCO2Me

NHCO 2Me O

13g

i-Pr

(S)

(R)

N

N

(S) (S)

i-Pr

O NHCO2Me

NHCO 2 Me

13h

i-Pr

O

(S)

(S)

N (R)

N

i-Pr

O (S)

NHCO2Me

NHCO 2 Me

13i

i-Pr

O

(S)

(R)

N

N

(S)

i-Pr

O (S)

NHCO2Me

NHCO2Me O

13j

i-Pr

(R)

(S)

N

N

(S) (R)

i-Pr

O NHCO2Me

NHCO 2 Me

13k

i-Pr

O

(R)

(R)

N (R)

N

i-Pr

O (R)

NHCO2Me

a

three independent tests were performed

The

pseudo-symmetric

(S)-2-(5-{4-[4-((S)-2-pyrrolidin-2-yl-3H-imidazol-4-yl)-phenyl]-buta-

1,3-diynyl}-1H-imidazol-2-yl)-pyrrolidine dihydrochlorides 13a-f were obtained according to the synthetic route depicted in Scheme 1. A Sonogashira сross-сoupling reaction between (S)-2[5-(4-iodo-phenyl)-1H-imidazol-2-yl]-pyrrolidine-1-carboxylic acid tert-butyl ester 14a and buta-1,3-diynyl-trimethyl-silane 16, obtained from 1,4-bis-trimethylsilanyl-buta-1,3-diyne 15, yielded the corresponding tert-butyl (S)-2-{5-[4-(4-trimethylsilanyl-buta-1,3-diynyl)-phenyl]1H-imidazol-2-yl}-pyrrolidine-1-carbonate 17a. It should be noted that other synthetic pathways can be applied to intermediate compound 17a; for example, Suzuki cross-coupling reaction via dioxaborolane derivatives24 or via the corresponding boronic acid.25 The trimethylsilyl group in ACS Paragon Plus Environment


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the intermediate compound 17a was removed, and then, Sonogashira сross-сoupling was carried out again between the acid tert-butyl (S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-carboxylate 19a and compound 18a. As a result, the synthetically valuable chiral intermediate 20a was obtained in good yield and used for further versatile transformation. Considering the SAR studies reported previously, the intermediate was subsequently converted into a series of promising amide derivatives. After the protecting group was removed in acidic medium, the acylation of 21a by N-Moc-amino acids 22a-f was conveniently performed, providing the final sub-set of compounds 13a-f in moderate-to-high yields (see exp. section). The desired products were obtained as the optically pure stereomers 13a-c containing four chiral centers, as mixtures of the stereoisomers 13d, e containing two pure (S)-chiral centers, and as the single stereomer 13f bearing two (S)-chiral centers.

Scheme 1 Me3Si

SiMe3 a

15 N

boc N

b I + N H

N

boc N

SiMe3

SiMe3

c

N

boc N

N H

16

N H 18a

17a

14a boc (S)

H N

N H N

N H

d

boc

N

N H

N

N

N

21a

H N

N N H

N N

+

boc

I

b

N H 19a

20a

R-CO2H e +22a-f R

O

H N

N

N

N N

N H

. 2HCl

R O

13a-f NHCO2Me

NHCO2Me

NHCO2Me

NHCO2Me 13, 22: R = i-Pr

i-Pr

O

O a

b

c

d

Me Me O

NHCO2Me

Me

NHCO2Me Me

e

f

(a) Ar, MeLi LiBr, Et2O, 25oC, 15 h; (b) Pd(PPh3)4, CuI, THF, Et3N, 40oC, 15 h; (c) K2CO3, THF, MeOH; (d) HCl, dioxane, DCM; (e) N-Moc-aminoacid, HOAt, EDAC, DIPEA, MeCN, 15 h, 4 oC.

The asymmetrically substituted methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)- 13g and methyl [(R)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((S)-2-methoxycarbonylamino-3-methyl-butyryl)-pyrrolidin-2yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2methyl-propyl]-carbamate dihydrochloride 13 h was prepared starting from the intermediate 18a ACS Paragon Plus Environment

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and methyl {(S)-1-[(S)- 19b methyl {(R)-1-[(S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1carbonyl]-2-methyl-propyl}-carbamate 19c (Scheme 2) under conditions similar to those described above (vide supra, Scheme 1). Scheme 2

H N

18a + I

N N

Boc

i-Pr

* NHCO2Me

O

19b, c

H N

N N

N N

N H

i-Pr

* NHCO2Me

O

20b, c

+ 22a

13g, h

H N

N H N

N N

N H

O

21b, c

i-Pr * NHCO2Me

19b, 20b, 21b: (S,S)-diastereoisomers. 19c, 20c, 21c: (R,S)-diastereoisomers.

Methyl

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

pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1carbonyl)-2-methyl-propyl]-carbamate dihydrochloride 13i was also prepared starting from the intermediate 18a and tert-butyl (R)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-carbonate 19d (Scheme 3) under the same reaction conditions.

Scheme 3 boc N

N

I N H

+ 18a

boc N (S)

(R)

H N

N

N N

N H

H N

N

boc

H N

N H N

N H

21d

20d

19d

+ 22a

MeO2CHN i-Pr

O

H N

N

N

N N

N H

. 2HCl

O

i-Pr NHCO2Me

13i

Finally,

the

symmetrically

substituted

methyl

[1-((R)-2-{5-[4-(4-{2-[(R)-1-(2-

methoxycarbonylamino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-phenyl)-buta-1,3diynyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-carbamate dihydrochlorides 13j,k were prepared following the methodology described above (see Scheme



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1) starting from tert-butyl (R)-2-[5-(4-iodo-phenyl)-1H-imidazol-2-yl]-pyrrolidine-1-carboxylate 14b and using 5-iodo-1H-imidazol 19d as an intermediate compound (Scheme 4).

Scheme 4 boc N

N

I

+16, b

N

boc N

N H

c

SiMe3 N H

boc N

17b

N N H

18b

14b +19d H N

N H N

N H

boc

d

N

N

N H

b H N

N

N N

N H

boc

20e

21e

+22a,b

MeO2CHN

* i-Pr

O N

H N

N N H

N N

O

i-Pr *

. 2HCl

NHCO2Me

13j,k 13j: * = (S)-stereoisomer. 13k: * = (R)-stereoisomer

As shown in Table 2, among the tested compounds, compound 13a was the most active against HCV replicon GT1b and showed an EC50 value of 0.003 nM, which was comparable to that observed for other known NS5A inhibitors with four chiral centers, e.g., 1, 3, 11, and 12 (Table 1, our data for 1 is 0.003 ± 0.001 nM). A clear relationship between the target activity and stereospecificity of compounds 13a-k was revealed. Thus, compound 13h, which contains one (R)-chiral center at the amino acid residue area — CAP1 and three (S)-chiral centers, was more than 120-fold less active than compound 13a under the same biological conditions. Dramatic decreases in activity (23- and 13814-fold reductions) were observed for compounds 13g and 13b, bearing one (R)- and two (R)-chiral points in the Cap1 and Cap2 regions, respectively. An even greater decrease was observed for compounds with (R)-chiral centers incorporated into the pyrrolidine ring. For instance, compound 13i, which contains an (R)-chiral center in the pyrrolidine moiety of Cap2, was found to be 1580-fold less active than compound 13a. Molecules with two and four (R)-chiral centers (13j and 13k) in the pyrrolidine rings (Cap1 and


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Cap2) were almost 260,000 and 290,000-fold less active than the hit compound. The replacement of the N-Moc-L-valine moiety in compound 13a by N-Moc-(S)-3-aminotetrahydrofuran-3carboxylic acid (13c) or N-Moc-2-methylalanine (13f) led to dramatic reductions in the target activity of 7158- and 176176-fold, respectively. Compound 13a showed relatively low cytotoxicity (CC50 = 17.3 µM, Table 2) and an excellent therapeutic index of > 5,000K (SI = CC50/EC50). This compound is also highly active against genotypes 1a (EC50 = 59 pM), 2a (EC50 = 51 pM) and 4a (EC50 = 6.5 pM).26 Compound 13a has a logD = 4.46 (рH = 7.4), 14 heteroatoms, 6 potential hydrogen bond acceptors, 4 potential hydrogen bond donors, and 8 free rotatable bonds. It is a crystalline powder ranging from nearly white to yellow in shade with a melting point of 185-186 °С (above 195 °С, degradation begins). Compound 13a is highly soluble in water (208 µM at pH = 2, 195 µM at pH = 4, 33 µM at pH = 7.4), alcohol (≥ 10 µM), dimethylsulfoxide (≥ 10 µM), and other solvents. To elucidate the possible mechanisms of action and the inhibition potencies of the evaluated compounds, we performed 3D molecular docking studies based on available protein structures and SAR evaluations. The formation of a homodimeric catalytic subunit between the zinc finger domains is crucial for the activity of HCV NS5A.27 The relative dimeric symmetry has recently been revealed for NS5A isoforms28 and several NS5A inhibitors.14 The range of key amino acid residues in the N-terminal region was determined within domain I. Amino acid substitutions in the same positions were associated with resistance to 1 and thiazolidinone derivatives.29-31 For genotype 1a, resistant mutations included M28T (ca), Q30H/R (ca), L31M/V (ca, sa), Y93C/H (ca), and Q54H/R/L (ca); where ca is the cap area, and sa is the spacer area.22,31,32 A similar resistance profile was reported for 12 and included M28T, Q30R, Y93C, Y93H, and Y93N for NS5A GT1a and L28T, L28M+Y93H, L31F+Y93H, and L31V+Y93H for NS5A GT1b.22,32 The major GT1a resistance mutations along the dimer interface as well as in the amphipathic αhelical N-terminus included M28T, L31V/W, Q30D/E/K/H/R, P32L (sa), H58D, Y93C/H/N, and Q54H/R/L as well as L31F/V/W, P32L and Y93C/H/N for GT1b.22,30,32 The common core point



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Page 12 of 48

mutations for both genotypes included Y93C/H/N, L31V/W and P32L, suggesting the major role of these amino acid residues in drug binding. The distance between the two cap binding regions within the dimeric interface is approximately 15−20 Å, as reported by Makonen Belema and colleagues,14 or 15−18 Å, as reported by Cordek and co-workers.34 This distance is closely related to the linker length in the structures of many dimeric inhibitors, providing a unique binding mode for these compounds. The common topological pharmacophore includes a spacer (linker) surrounded by crucial amino acid residues L31 and P32 and two caps that are in tight interaction with the loop regions on either side of the dimer interface in proximity to Y93, M28/L28, L31 and Q54.14,34 In highly potent inhibitors, these caps generally include Pro, Val (or Phe) residues or their direct analogues.

We also performed a related in vitro mutagenesis study to investigate the resistance profile of compound 13a (vide infra). During the study, we identified the major mutation points L31F, T56A and Y93H located in the HCV NS5A gene. These mutations significantly enhanced the resistance of HCV replicons toward 13a inhibition activity. The obtained results confirmed that compound 13a is a direct-acting antiviral agent that binds to the NS5A protein. The disclosed resistance profile is similar to that described above for other NS5A inhibitors, suggesting a similar binding mode.

The molecular docking study for compound 13a was performed using MOE software23 (Figure 2a) following the approach reported by Cordek and colleagues.34 The binding site within the fulllength homology dimeric model of GT1b NS5A domain I and the amphipathic helix anchor domain was reconstructed based on available crystallographic data [PDB codes: 3FQM and 1R7C]. Compound 13a was then docked into the defined binding site using the static mode. The compound locates symmetrically across the dimer interface with the hydrophobic diynyl-phenyl core, lying along T95 and captured by Q54, T56 and Q62. Y93 stacks face-to-face with the


Page 13 of 48

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imidazole ring, while Q62 and/or T56 form hydrogen bonds with the imidazole N atoms. This binding mode (Figure 2a, colored in orange) is very similar to that reported previously by Belema and colleagues,14 particularly for compounds 12,22,32 1,14,34 3,35 and 11.21,36,37 We also revealed an alternative binding mode (Figure 2a, colored in white) in which one methyl group of the valine moiety is above the imidazole ring, thereby providing an additional hydrophobic interaction exclusively in the case of S,S-isomers with Y93, while the imidazole itself is located appropriately for slanted t-stacking with Y93 supported by a weak direct (or water-mediated) hydrogen bond between the OH group of Y93 and the imidazole NH or the O of the N-acetyl Pro fragment (Figure 2b). The second methyl group of valine provides hydrophobic interactions with T56 and P58. A similar alternative binding mode was also observed for the other NS5A inhibitors mentioned above (Figure 3). These results correlate well with the obtained biological data and explain the differences in activity between the diastereomers. Notably, stereospecificity was found to be much more crucial within the Cap of ethynyl-imidazole (Сap2) than within the phenyl-imidazole side (Сap1, Table 2), suggesting that the binding of Сap1 is more flexible and tolerant toward both drugs and the target. Note also that the structures and stereochemistries of Сap1 and Сap2 have a more drastic effect on the activity than does the linker length (Table 2). This resulted in a relatively high tolerance for linker length (up to 21 Å) within an extensive group of NS5A inhibitors, including compounds 13a, 1, 3, 12, and 11. Thus, the (R,S,S,S)configuration of 13g (EC50 = 0.079 nM) was highly active, while the (S,S,S,R)-analogue 13h, with the same spacer, was less active (EC50 = 0.4089 nM).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tyr93

Thr59 ~3A

T95

~4.9A

P32

L31 N

Pro97

L31`

Pro97

H

O

N

N

O 2.7A

Q54

Tyr93

P32`

N

N

O

Q62

L28

Pro58 O

Page 14 of 48

N

N

2.5A

~3A

O

F37`

F37

P58`

L28

P58 Q62`

Tyr93

T95`

O

N

O

Thr59 Pro58

Tyr93

Q54`

I52

o

18 ± 1A

(a)

(b)

Figure 2. Binding hypothesis for compound 13a; the α-helix domain is not shown: (a) the first (orange, Y93 stacks face-to-face with the imidazole) and alternative (white, t-stacking of Y93 and imidazole) binding modes of 13a, the compound is positioned along the extended cavity formed within the dimeric interface of NS5A; (b) 2D-supramolecular interface proposed for alternative binding mode of 13a; (`) denotes the other subunit in the dimer interface. o

19 ± 1A

o

18.5 ± 2.5A O

Tyr93

O

P29

L31

N

O

O

N

*

(S)

Q62

L28

R30` L31

F37 P58`

H N

N

L28

O N

Compound 11

O

F37` P58

Q62

P29`

O

N L28`

O

O

Tyr93`

Tyr93

F37 P58`

O

N

H N

N

N

N

L31`

P32

O

L28` N

F37` P58

O R30

N Q62`

N

N

N

O

P32 O

Q62` Tyr93`

Compound 12

O

o

17.5 ± 0.5A

o

20.5 ± 1.5A L28 L31 O O

P29

N N O

P32

P32` L31`

N

P29` O

N

N

Tyr93

Tyr93`

N

F37`

F37

P58

P58`

N N

O

L31`

P32

O

P29

Tyr93

Tyr93`

S N

N N

S

N

F37` L28

N

O

O

O

O

L31

N

N

L28`

Compound 1

O

P58

O

P29`

N

P32` P58` F37

L28`

Compound 3

Figure 3. 2D supramolecular interface predicted for compounds 11, 12, 1, and 3.

It was shown that the incorporation of an N-arylated pyrrolidine core (12 and related compounds) in the center of the linker area led to dramatic gains in activity against both the GT1a and GT1b genotypes compared to the unsubstituted pyrrolidine analogues.31 Among the major resistant mutations in GT1b, L28T/M, L31F/V/M, Y93H and R30Q were identified as the


Page 15 of 48

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most crucial for good binding, while for GT1a the mutations were Y93H/C/N, M28V/T, Q30R, L31V and H58D. Interestingly, inverted resistant mutations (R30Q and Q30R) were observed for GT1b and GT1a, respectively. Although arginine and glutamine have comparable lengths, arginine can provide π-cationic interactions, particularly with phenyl or heteroaryl fragments, while glutamine provides H-bonding acceptor functionality. Both residues share H-bonding donor potential. These amino acids were suggested to be in close proximity with the cap fragment, providing crucial contacts, as highlighted in Figure 3.

Lambert and colleagues have recently discussed the crystal structure of GT1a-D1 and suggested a novel mechanism of action for dimeric HCV inhibitors.38 The obtained crystal structure is highly exposed to water molecules, having a Matthews coefficient of 4.73Å3/Dalton and a solvent content of 73.97%, similar to the recently disclosed GT1b-D1 dimer crystal structure.27 Therefore, ligand binding could be dependent largely on the re-solvation energy and thermodynamic terms. Two novel head-to-head dimeric forms of NS5A-D1 were observed, and the cavity surface was described in terms of the key amino acid content responsible for ligand binding and symmetry. Molecular dynamic simulations were performed based on the obtained X-Ray and available NMR data to construct a full-length 3D model. The amino acid residues Val75, Gly76, and Pro77, located along the spacer area and capturing the biphenyl core, as well as His54, Arg56, Glu62, His66, Arg73, and Thr79, surrounding the cap regions, were suggested to be the most crucial for 1 binding affinity. The hydrogen bond periodically formed between Glu62 and the valine carbonyls (or the imidazole NH) was identified in the docking study. Dynamic modeling (40 ns) was then carried out and revealed some oscillation along the dimer axis. It was also revealed through the modeling that the key Tyr93 did not directly interact with 1 but rather via a tight amino acid frame. Thus, the salt bridge with Arg56 maintained by Glu62 was observed and thereby adjusted Tyr93 to complete the network. This allosteric mode of resistance/binding is partially supported by the relatively high dynamics observed within the



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Page 16 of 48

ligand-capture amino acid interface upon binding, providing tentative evidence against rigid interactions in a lock and key mechanism.39 Although in our work the static docking simulation was actually performed only with a flexible ligand (not the target), the calculated scores correlated well with the target activity of the tested compounds. A detailed in silico study with a relatively wide range of novel NS5A inhibitors supported by the dynamic docking procedure will be reported elsewhere (manuscript in preparation).

The flexible 3D alignment of 11 and 12, as revealed using ICM Pro software,40 out of the target clearly showed that the cap areas and phenyl moieties in the center of the spacers, as well as the two-sided aromatic fragments, are well superposed (Figure 4). In this case, the imidazole ring can be regarded as a good bioisosteric replacement for the amide fragment, providing similar contacts maintained by the H-bond donor (NH in imidazole and carboxamide) and the H-bond acceptor points (N in imidazole, O – in carboxamide). A detailed binding picture can be developed on the basis of the 2D- (Figure 3) and 3D-representations (Figure 4) of these compounds. Thus, the introduction of an aromatic functionality in the linker area of compound 13a can be reasonably regarded as a beneficial modification that will, presumably, enhance target activity or/and selectivity.

Figure 4. 3D alignment of 11 (orange) and 12 (yellow); HBA – H-bond donor, HBD – H-bond acceptor, Aro – aromatic moiety, Hyd – hydrophobic fragment.

The point mutation L31F/V in GT1b NS5A conferred resistance to compound 12. Based on the performed docking study, it was suggested that the optimal distance for hydrophobic interaction ACS Paragon Plus Environment

Page 17 of 48

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between res 31 and the iso-butyl phenyl moiety of compound 12 corresponds to leucine. The isoform with a bulky Phe can contribute to steric clashes upon binding, while the small Val residue provides insufficient hydrophobic contact. The substitution of P32 by L32 is also associated with resistance types, thereby suggesting that stacking interactions, presumably with aromatic moieties in the NS5A inhibitors, are more preferable than a Leu probe. Similar speculations can be made for the core and side chain interactions (with phenyl groups) of compound 11, but in this case binding is achieved by the same amino acid sharing by the second subunit.

The addition of 40% NHS had little effect on the activities of compounds 13 against the HCV GT1b replicon, suggesting their weak binding to human plasma proteins.41 The effect of NHS on compound 13a potency was moderate. Its activity decreased only 3-4-fold in the presence of 40% NHS. As mentioned above, 13a has a relatively high TI against HCV replicons in vitro and was inactive against other flaviviruses, including West Nile virus, yellow fever virus and dengue virus, at the highest concentration tested (30 µM). These data suggest that compound 13a is a specific HCV inhibitor (Tables 2 and 3).

Table 3. The spectrum of antiviral activity of compound 13a in Vero-76 cells, infected in vitro as well as cytotoxicity West Nile virus (strain Yellow fever virus

Dengue virus

NY99) Compound EC50 CC50[a]

EC50

CC50[a]

SI50

EC50

CC50[a]

SI50

µM

SI50

µM

µM

> 26.3

26.3

n/a

> 23.5

23.5

n/a

> 28.4

28.4

n/a

IFN-α (ng/ml) 0.011

> 10

> 910

0.11

> 10

> 93

0.014

> 10

> 714

13a

a

CC50 was determined in Vero-76 cell line



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Page 18 of 48

The 13a-resistant HCV replicons were selected by continuous passaging of the Huh7 cells harboring the HCV replicon (GT1b, clone Con1)41 in the presence of the drug at concentrations exceeding the EC50 values by 5- to 40-fold for 3-6 weeks. Some drug-resistant cell populations were cloned. The primary structure of the NS5A gene was determined by direct sequencing of the HCV RNA pools. As a result, several NS5A mutations were identified (Table 4). In general, the NS5A mutations conferring resistance to compound 13a were similar to those reported previously for other NS5A inhibitors.30 In the reference cell culture (not treated with 13a), none of the mutations described above were found. Then, cultures carrying 100% of one of the mutations (L51F, T56A or Y93H) were cloned. All cloned cell cultures harboring mutant HCV replicons were significantly more resistant to compound 13a than were the cultures harboring the wild type HCV replicon (not shown).

Table 4. HCV NS5A mutations, identified after selection with compound 13a in vitro. Numbers of independent cell cultures with a given mutation are shown Mutation weight, % Mutation 20-39

40-59

≥ 60

Total

CGA89CCA (Q30P)

-

-

1

1

TTG93TTT (L31F)

3

-

1

TTG93ATG (L31M)

1

-

-

AAG131AGG (K44R)

-

-

1

ACC166TCC (T56S)

-

1

-

ACC166GCC (T56A)

-

3

1

AGG233AAG (R78K)

-

1

-

TAC277CAC (Y93H)

1

1

2

5

1

5

1

6 TAC277AAC (Y93N)

2

-

-


Page 19 of 48

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CGG1067CAG (R356Q)

-

-

1

1

CTG1088CCG (L363P)

-

-

1

1

ACC1099CCC (T367P)

-

1

-

1

GTG1103GCG (V368L)

-

1

1

2

Pharmacokinetics studies performed in mice, rats and dogs (Table 5)42 have revealed that compound 13a achieves a relatively high level in plasma (Cmax) and possesses a long half-live (t1/2) as well as high oral bioavailability (F%), which in rats reached 65%, compared to 11% observed for 1.34 Lower bioavailability was also reported for other NS5A inhibitors, including 12 (F = 6.2%),22 10 (F = 32.5%),16 and 11 (38%).21 The t1/2 values for compound 13a upon oral administration were 3 h and 4.5 h in rats and dogs, respectively. However, in dogs (2.5·10-3 g/kg, p.o.) and monkeys (2.5·10-3 g/kg, p.o.), the bioavailability of 12 was 57% and 35%, respectively.22 Another compound developed by Abbott containing a pyrido[2,3-d]pyrimidin-4amino core showed F = 18.8% in dogs (2.5·10-3 g/kg p.o. s.d.).43 1 and 11, as PEG formulations, demonstrated F = 108%44 and F = 35% (in dogs, 2·10-3 g/kg p.o. s.d.),21 respectively.

Table 5. Key pharmacokinetic features of 13a and other NS5A inhibitors[a] after a single p.o. and i.v. administration Administration

Dose,

Cmax, mg/L

Compound route

mg/kg

[b]

t1/2, h

F, %[c]

(µM)

rats 13a

11.2

2097 (2950)

3.0

65

11

30

318 (360)

-

9

5

-

-

50

1 (solution)

3

0.538 (0.728)

1.5

-

10

1

-

4.7

58.5

1 (PEG)

p.o.



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Page 20 of 48

12

3

3 (3.36)

15.9

6.2

MK-4882

2

241 (310)

-

38

13a

2.8

2566 (3610)

3.4

-

1

-

2

-

5

-

4.2

-

MK-4882

i.v.

11

dogs 13a

10

5810 (8174)

4.5

58

11 (PEG)

2

256 (290)

-

35

MK-4882

1

148 (190)

-

26

1 (PEG)

2.3

-

-

108

10

1

-

7.4

58.5

12

2.5

0.64 (0.72)

7.3

57

13a

5

6900 (9707)

4.9

-

1

148 (190)

4.7

-

1

-

7.7

-

5

0.14 (0.18)

-

12

2.5

0.29 (0.32)

5

35

10.3

58.5

p.o.

MK-4882

i.v.

11

monkeys MK-4882 12 p.o. 10

1

1

3

0.37 (0.50)

3

38

1

-

3

-

1

-

16

-

MK-4882 i.v. 11 a

Data for all compounds except 13a was obtained from Integrity Database.42 bConcentration was calculated for non saline composition; cF = 100% ⋅ [AUC0→∞(p.o.)⋅Dose(i.v.)] / [AUC0→∞(i.v.)⋅Dose(p.o.)].


Page 21 of 48

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As shown in Table 6, at a dose of 10 µM, compound 13a did not inhibit any of the studied human liver CYP450 enzymes. These results clearly suggest the absence of clinically significant interactions between 13a and other medicinal products metabolized by the abovementioned human cytochromes. Compound 13a was stable when it was incubated for 60 min in the presence of 1 µM concentrations of rat, dog or human liver microsomal fractions (Table 7).

Table 6. The affinity of 13a and 1 towards CYP450 panel

a

Compound

13a

1[a]

CYP450

IC50, µM

IC50, µM

1A2

> 10

> 100

2C9

> 10

59.5 ± 13.6

2C19

> 10

-

2D6

> 10

> 100

3A4

> 10

7.20 ± 1.70

Data for compound 1 was obtained from Integrity Database.42

Table 7. Metabolic stability of 13a, evaluated after incubation for 60 min in the presence of 1 µM rat, dog or human liver microsomal fractions Residual substance, % Substance

13a

Rat

Dog

Human

70.8 + 5.2

84.7 + 4.1

81.5 + 4.6

The inhibition potency of 13a toward cardiac hERG potassium channels was investigated at 30 µM and 3 µM concentrations using Invitrogen’s Predictor hERG test system. The binding did not exceed 10% and was not dose-dependent, suggesting a relatively low risk of cardiotoxicity. We also investigated the acute toxicity of 13a in mice and rats. It was found that the LD50 value



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Page 22 of 48

in mice (intraperitoneal administration) exceeded the maximum studied dose (1600 mg/kg) and was equal to 3435.4 ± 502.3 mg/kg in males and 3005.5 ± 678.7 mg/kg in females upon oral administration (Table 8). The LD50 values in outbred Wistar rats upon the oral administration of compound 13a were as follows: 2902.0 ± 488.1 mg/kg for males, 2829.2 ± 311.4 mg/kg for females and 2860.8 ± 227.0 mg/kg for both males and females.

Table 8. The LD values determined for compound 13a in SHK mice (p.o.) LD

Mice

Values, mg/kg

males

2320.3

females

1160.1

males

2565.4

females

1565.7

males

3435.4 ± 502.3

females

3005.5 ± 678.7

males

4305.3

females

4445.3

LD10

LD16

LD50

LD84

The LD10 values for the intraperitoneal administration of 13a in rats were similar in both males and females, while the LD50 and LD84 were higher in females (Table 9). These differences were not statistically significant; however, they indicated the tendency for females to be more tolerant to 13a upon intraperitoneal administration. These results suggest that compound 13a could be classified45 as a low toxicity substance (group I).

Table 9. The LD values for intreperitoneal administration (i.p.) of compound 13a in rats Lethal dose

Rats

Values, mg/kg

LD10

males

228.6 ± 87.7


Page 23 of 48

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females

228.4 ± 63.9

males and females

234.2 ± 38.5

males

349.4 ± 69.3

females

412.1 ± 68.4

males and females

395.2 ± 40.8

males

485.6 ± 123.7

females

651.3 ± 87.2

males and females

593.2 ± 51.9

LD50

LD84

In a 6-week toxicity study in dogs and a 12-week study in rats, no significant signs of toxicity were observed for compound 13a. In split studies, the compound was found to be non-mutagenic in vitro and not immunogenic or allergenic in vivo. No adverse effects on the reproductive systems of male or female rats were observed. These results will be published elsewhere. Based on the preclinical data obtained during an extensive preclinical evaluation, compound 13a was selected for subsequent clinical development.

CONCLUSIONS SAR studies with a novel series of hepatitis C virus NS5A inhibitors containing 5-[4-(4imidazol-4-yl-phenyl)-buta-1,3-diynyl]-1H-imidazole linkers led to dramatic improvements in antiviral activity in the HCV replicon model. Compound 13a, containing four (S) chiral centers, showed excellent activity and selectivity (EC50 = 3 pM for GT1b, CC50 = 17.3 µM). Human serum did not significantly reduce the antiviral activity (< 4-fold). No potency was observed against other flaviviruses (yellow fever, West Nile, dengue) in the in vitro infection experiments. In vitro studies clearly showed that the 13a resistance profile overlaps with but is not identical to those of other known HCV NS5A inhibitors. A 3D molecular docking study revealed two potential binding modes for the most active compounds within this series as well as for several



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Page 24 of 48

other NS5A inhibitors. The optimal spacer length for ensuring tight binding was predicted to be 17-20 Å. Animal studies revealed a favorable pharmacokinetic profile and good oral bioavailability for compound 13a. It was well-tolerated in rodents (in male mice, LD50 = 3435.4 ± 502.3 mg/kg or higher). In safety pharmacology and sub-chronic toxicity studies in rats and dogs, 13a was not associated with any significant pathological or clinical findings. Further clinical development of compound 13a is strongly warranted.

EXPERIMENTAL SECTION Chemistry General Procedures. All chemicals and solvents were used as received from the suppliers without further purification. The intermediate compounds were obtained following the synthetic procedures described in the references.10–13,46–48 The crude reaction mixtures were concentrated under reduced pressure by removing the organic solvents in a rotary evaporator. Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker DPX-400 spectrometer at room temperature (rt) with tetramethylsilane as an internal standard. The chemical shifts (δ) are reported 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 greater than 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; wavelength, UV 214 and 254 nm. The preparative HPLC system included two sets of Shimadzu LC-8A pumps, a Shimadzu Controller SCL 10Avp, and a Shimadzu Detector SPD 10Avp. A Reprosil-Pur C-18-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 was conducted on a PE Sciex API 165 system using electrospray in positive ion mode, [M + H]+, and a Shimadzu HPLC System equipped with a Waters XBridge C18 3.5 µm column (4.6 mm × 150 mm). High resolution mass spectra (HRMS) were acquired


Page 25 of 48

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on an Orbitrap Elite mass spectrometer (Thermo, Bremen, Germany) equipped with an HESI ion source. The N-Moc-amino acids 22a-f were obtained by reaction between the corresponding amino acid and methyl chloroformate under basic conditions.49 The 3-amino-tetrahydrofuran-3carboxylic acid used for the synthesis of intermediates 22c,d was prepared in accordance with the synthetic procedure described in ref 50 and was separated to provide enantiomers by the method published in ref 51. The 2-amino-3-methoxy-2-methylpropanoic acid used for the synthesis of intermediate compound 22e was prepared from 1-methoxypropan-2-one by the procedure reported for 3-amino-tetrahydrofuran-3-carboxylic acid.50

General procedure 1. Substituted (S)-2-(5-{4-[4-((S)-2-pyrrolidin-2-yl-3H-imidazol-4-yl)phenyl]-buta-1,3-diynyl}-1H-imidazol-2-yl)-pyrrolidine

dihydrochlorides

(13a-f).

Methyllithium lithium bromide complex solution (1.5 М) in ether was added to a solution of 5.83 g (30 mmol) of 1,4-bis(trimethylsilyl)buta-1,3-diyne 15 dissolved in 40 mL of ether in an argon atmosphere (aa) under vigorous stirring. The reaction mixture was continuously stirred at rt for 15 h, then cooled in an ice bath and quenched with 40 mL of a saturated NH4Cl solution. The organic layer was washed with brine and dried over Na2SO4, and the solvent was evaporated in a rotovap under vacuum. Tert-butyl (S)-2-[5-(4-iodophenyl)-1H-imidazol-2-yl]-pyrrolidine-1carboxylate 14a46 (8.8 g, 20 mmol), triethylamine (20 mL), Pd(PPh)4 (1.16 g, 1 mmol) and CuI (0.19 g, 1 mmol) were added to the solution of the liquid residual 16 dissolved in THF (70 mL). The resulting mixture was stirred under aa at 40 °C for 15 h. Then, the mixture was filtered through Celite, rotovapped and subjected to column chromatography on SiO2 (eluent = chloroform: acetone 15:1) to afford 7.76 g (85%) of 17a. The LC-MS molecular ion peak was at 434 (M+H)+. Potassium carbonate (K2CO3) (7.04 g, 51 mmol) was added to the solution of compound 17a (7.36 g, 17 mmol) in THF (120 mL) and methanol (120 mL), and the reaction mixture was stirred under aa for 2 h, then filtered and rotovapped. A mixture of tert-butyl (S)-2(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-carboxylate (19a)10 (6.17 g, 17 mmol) in triethylamine



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(20 mL) with Pd(PPh)4 (0.93 g, 0.8 mmol) and CuI (0.15 g, 0.8 mmol) was added to the solution of the obtained compound 18a dissolved in THF (60 mL). The resulting mixture was then stirred under aa at 40 °C for 15 h. After the reaction was completed, the mixture was filtered, the precipitate was washed with a chloroform:methanol 3:1 solution, the filtrate was rotovapped, and the residue was boiled in 100 mL of methanol. The mixture was then cooled to rt and stirred in a fridge for 4 h. The formed precipitate (compound 20a) was then filtered off, washed with cold methanol and ether and dried in air. The desired product, tert-butyl [2-((S)-2-{5-[4-(4-{2-[(S)-1(tert-buthoxycarbonyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1Himidazol-2-yl}-pyrrolidin-1-yl)-carboxylate (20a), was obtained in 57% yield (5.78 g) and purity. The LC-MS molecular ion peak was at 597 (M + H)+. 1H NMR (DMSO-d6, 400 MHz) δ 12.67, 12.73 (2s, 0.2H), 12.21, 12.28 (2s, 0.9H), 11.94, 12.01 (2s, 0.9H), 7.77 (m, 2H), 7.55 (m, 3.7H), 7.35 (m, 0.3H), 4.77 (m, 2Н), 3.51 (m, 2Н), 3.34 (m, 2Н), 2.18 (m, 2Н), 1.95 (m, 3Н), 1.84 (m, 3Н), 1.39 (s, 7.5Н), 1.14, 1.17 (2s, 10.5Н). Hydrochloric acid (HCl) (4 M, 25 mL) was added dropwise to the solution of compound 20a (5.78 g, 9.7 mmol) in dioxane (25 mL). The resulting mixture was then stirred for 15 h. The formed precipitate was filtered off, washed with ether and dried under vacuum to obtain 5.04 g (96%)

of

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

pyrrolidin-2-yl]-1H-imidazole tetrahydrochloride (21a). The LC-MS molecular ion peak was at 397 (M + H)+. 1H NMR (DMSO-d6, 400 MHz) δ 10.38 (br s, 1H), 10.27 (br s, 1H), 9.86 (br s, 1H), 9.22 (br s, 1H), 8.18 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.80 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 5.02 (m, 1Н), 4.74 (m, 1Н), 3.45 (m, 1Н), 3.37 (m, 1Н), 3.29 (m, 2Н), 2.47 (m, 2Н), 2.35 (m, 1Н), 2.17 (m, 2Н), 2.09 (m, 1Н), 2.00 (m, 2Н). The mixture of N-Мос-amino acid 22a-f (0.283 mmol), 1-hydroxy-7-azabenzotriazole (40 mg, 0.295 mmol) and EDAC (53 mg, 0.277 mmol) in acetonitrile (1 mL) was stirred in a fridge for 1 h, then compound 21a (64 mg, 0.118 mmol) and DIPEA (61 mg, 0.472 mmol, 82 mL) were added, and the resulting mixture was continuously stirred in a fridge for 15 h. The mixture was rotovapped, dissolved in dichloromethane, washed


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twice with a 5% Na2CO3 solution, dried over Na2SO4, rotovapped again, and subjected to HPLC. Compounds 13a-f were then readily converted into the corresponding salt compositions following the procedure described above and were isolated as the dihydrochloride salts (13af⋅⋅2HCl) through the addition of acetone.

Methyl

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

pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine1-carbonyl)-2-methylpropyl]-carbamate dihydrochloride (13a). Yield 62 mg (67%). 1H NMR (DMSO-d6, 400 MHz) δ 15.49 (br s, 0.3H), 14.95 (br s, 0.8H), 8.23 (s, 0.1H), 8.21 (s, 0.9H), 8.01 (m, 3H), 7.77 (m, 2H), 7.26 (m, 1.8H), 6.84 (m, 0.1H), 5.78 (m, 0.06H), 5.50 (m, 0.05H), 5.19 (t, J = 7.0 Hz, 0.9Н), 5.07 (m, 0.9H), 4.06 (m, 2.7H), 3.81 (m, 3H), 3.53 (s, 5.4H), 3.43 (s, 0.4H), 3.30 (s, 0.2H), 2.15 (m, 10H), 0.82 (m, 12H). ESIHRMS m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found 711.3590. Methyl

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

pyrrolidin-2-yl]-3H-imidazol-4-yl}-phenyl)-buta-1,3-diynyl]-1H-imidazol-2-yl}-pyrrolidine1-carbonyl)-2-methyl-propyl]-carbamate dihydrochloride (13b). Yield 63 mg (68%).1H NMR (DMSO-d6, 400 MHz) δ 14.77 (m, 1.1H), 8.22 (s, 0.75H), 8.20 (s, 0.25H), 7.96 (m, 2.8H), 7.78 (m, 2.2H), 7.63 (d, J = 8.0 Hz, 0.05Н), 7.48 (d, J = 8.0 Hz, 0.2Н), 7.24 (d, J = 8.0 Hz, 1.5Н), 6.73 (m, 0.02H), 5.98 (m, 0.03H), 5.63 (m, 0.2H), 5.22 (m, 1Н), 5.07 (m, 0.8H), 4.17 (m, 2H), 3.90 (m, 1H), 3.83 (m, 1H), 3.63 (m, 2H), 3.55, 3.56 (2s, 6H), 2.40 (m, 1H), 2.27 (m, 1H), 2.03 (m, 8H), 0.87 (m, 10.1H), 0.73 (m, 1.1H), 0.43 (m, 0.2H), 0.31 (m, 0.6H). ESIHRMS m/z calcd for C38H47N8O6 [M+H]+ 711.3613; found 711.3638. Methyl

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

carbonyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl)-1H-imidazol-2-yl]pyrrolidine-1-carbonyl}-tetrahydro-furan-3-yl}-carbamate dihydrochloride (13c). Yield 39 mg (41%). 1H NMR (DMSO-d6, 400 MHz) δ 14.67 (m, 1.6H), 8.18 (m, 2.8Н), 7.97 (m,3H), 7.79



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(m, 2.2H), 5.67 (m, 0.05H), 5.44 (m, 0.07H), 5.16 (m, 0.93H), 5.02 (m, 0.91H), 4.14 (m, 2Н), 3.87 (m, 1H), 3.72 (m, 9H), 3.60 (s, 3H), 3.56 (s, 3H), 2.34 (m, 3H), 2.14 (m, 6H), 1.95 (m, 3H). ESIHRMS m/z calcd for C38H43N8O8 [M + H]+ 739.3198; found 739.3190. Methyl

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

furan-3-carbonyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl)-1Himidazol-2-yl]-pyrrolidine-1-carbonyl}-tetrahydro-furan-3-yl}-carbamate dihydrochloride (13d). 1H NMR (DMSO-d6, 400 MHz) δ 14.56 (br s, 1.8H), 8.18 (m, 2.7Н), 7.99 (d, J = 8.0 Hz, 2H), 7.88 (br s, 1.3H), 7.77 (d, J = 8.0 Hz, 2H), 5.71 (m, 0.03H), 5.44 (m, 0.06H), 5.18 (m, 0.96H), 5.04 (m, 0.94H), 4.16 (m, 2Н), 3.77 (m, 8H), 3.59 (m, 8H), 2.34 (m, 3H), 2.14 (m, 6H), 1.95 (m, 3H). ESIHRMS m/z calcd for C38H43N8O8 [M + H]+ 739.3198; found 739.3224. Methyl

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

propionyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-phenyl)-buta-1,3-diynyl]-1H-imidazol-2-yl}pyrrolidin-1-yl)-1-methoxymethyl-1-methyl-2-oxo-ethyl]-carbamate dihydrochloride (13e). 1

H NMR (DMSO-d6, 400 MHz) δ 14.44 (m, 1.8H), 8.19 (d, J = 9.6 Hz, 1H), 7.98 (m, 2Н), 7.89

(m, 1.5H), 7.78 (m, 3H), 7.69 (m, 0.5H), 5.22 (m, 1H), 5.06 (m, 1H), 3.85 (m, 1Н), 3.77 (m, 2H), 3.68 (m, 4H), 3.58 (m, 5H), 3.43 (m, 2H), 3.24, 3.26 (2s, 6H), 2.31 (m, 1H), 2.14 (m, 3H), 1.94 (m, 4H), 1.35 (m, 6H). ESIHRMS m/z calcd for C38H47N8O8 [M + H]+ 743.3514; found 743.3546. Methyl

[2-((S)-2-{5-[4-(4-{2-[(S)-1-(2-methoxycarbonylamino-2-methyl-propionyl)-

pyrrolidin-2-yl]-3H-imidazol-4-yl}-phenyl)-buta-1,3-diynyl]-1H-imidazol-2-yl}-pyrrolidin1-yl)-1,1-dimethyl-2-oxo-ethyl]-carbamate dihydrochloride (13f). 1H NMR (DMSO-d6, 400 MHz) δ 14.44 (br s, 1.7H), 8.19 (s, 1H), 7.98 (d, J = 8.4 Hz, 2Н), 7.93 (m, 1H), 7.90 (m, 1H), 7.83 (m, 1H), 7.78 (d, J = 8.4 Hz, 2Н), 5.23 (m, 1H), 5.06 (m, 1H), 3.81 (m, 1Н), 3.70 (m, 3H), 3.63 (s, 3H), 3.57 (s, 3H), 2.31 (m, 1H), 2.14 (m, 3H), 2.02 (m, 1H), 1.92 (m, 3H), 1.33 (m, 12H). ESIHRMS m/z calcd for C36H43N8O6 [M + H]+ 683.3300; found 683.3334.


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Methyl

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

pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine1-carbonyl)-2-methyl-propyl]-carbamate dihydrochloride (13g). Compound 13g was prepared according to the procedure described above for compounds 13a-f starting from compound 18а and the corresponding methyl {2-[(S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidin-1yl]-2-oxo-ethyl}-carbate 19b,g-i, followed by the conversion of the formed product 20b,g-i to the intermediate compound 21b,g-i and the acylation of amino acid 22a as well as the following transformation of the acylation products to the corresponding dihydrochlorides 13g-j. Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)-13g and methyl [(R)-1-((S)-2-{5-[4-(4-{2-[(S)-1((S)-2-methoxycarbonylamino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]carbamate dihydrochloride (13h). Compounds 13g,h were prepared starting from the intermediate compound 18a and methyl {(S)-1-[(S)-19b methyl {(R)-1-[(S)-2-(5-iodo-1Himidazol-2-yl)-pyrrolidine-1-carbonyl]-2-methyl-propyl}-carbamate 19c (see Scheme 2) using the conditions applied for compounds 13a-f (vide supra). Methyl

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

pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine1-carbonyl)-2-methyl-propyl]-carbamate dihydrochloride (13g). 1H NMR (DMSO-d6, 400 MHz) δ 14.79 (m, 0.9H), 8.20 (m, 1.1H), 7.95 (m, 2.3H), 7.85 (br s, 1H), 7.77 (m, 2.1H), 7.60 (m, 0.06H), 7.26 (m, 1.4Н), 7.10 (m, 0.04Н), 6.86 (m, 0.01H), 6.71 (m, 0.01Н), 5.95 (m, 0.06H), 5.37 (m, 0.1H), 5.22 (m, 0.94Н), 5.04 (m, 0.9H), 4.19 (m, 1H), 4.06 (m, 1H), 3.90 (m, 1H), 3.80 (m, 2H), 3.67 (m, 1H), 3.54, 3.55 (2s, 6H), 2.39 (m, 1H), 2.23 (m, 1H), 2.11 (m, 3H), 1.99 (m, 5H), 0.85 (m, 11.3H), 0.74 (m, 0.45H), 0.42 (m, 0.25H). ESIHRMS m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found 711.3601. [(R)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((S)-2-methoxycarbonylamino-3-methyl-butyryl)-pyrrolidin2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-



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carbonyl)-2-methyl-propyl]-carbamate dihydrochloride (13h). 1H NMR (DMSO-d6, 400 MHz) δ 15.29 (br s, 0.5H), 14.79 (br s, 0.8H), 8.18, 8.20 (2s, 1H), 7.97 (m, 2.8H), 7.76 (m, 2.3H), 7.47 (m, 0.2H), 7.25 (m, 1.6Н), 6.78 (m, 0.1H), 5.72 (m, 0.05H), 5.63 (m, 0.2H), 5.17 (m, 0.95Н), 5.07 (m, 0.8H), 4.13 (m, 2H), 3.96 (m, 1H), 3.83 (m, 2H), 3.62 (m, 1H), 3.54, 3.56 (2s, 6H), 2.32 (m, 2H), 2.17 (m, 2H), 2.01 (m, 6H), 0.86 (m, 8H), 0.74 (m, 3.4H), 0.31 (m, 0.6H). ESIHRMS m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found 711.3602. Methyl

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

pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine1-carbonyl)-2-methyl-propyl]-carbamate

dihydrochloride

(13i).

Compound

13i

was

synthesized in accordance with the procedure provided above for compounds 13a-f starting from compound 18a and 5-iodimidazola 19d. Subsequent removal of the Boc protecting group in 20d resulted in 21d. The acylation of the formed intermediate compound 21d by N-Moc-L-valine 22a yielded the desired product 13i, which was easily transformed into the corresponding dihydrochloride salt following the procedure described above. 1H NMR (DMSO-d6, 400 MHz) δ 15.35 (br s, 0.8H), 14.81 (m, 0.8H), 8.20 (m, 1H), 7.98 (m, 2.8H), 7.76 (m, 2.25H), 7.47 (d, J = 8.0 Hz, 0.2H), 7.26 (m, 1.65Н), 6.84 (m, 0.05H), 6.74 (m, 0.05H), 5.76 (m, 0.07H), 5.63 (m, 0.2H), 5.18 (m, 0.94Н), 5.08 (m, 0.8H), 4.14 (m, 2H), 4.00 (m, 1H), 3.81 (m, 2H), 3.62 (m, 1H), 3.54, 3.56 (2s, 5.8H), 3.31 (s, 0.2H), 2.37 (m, 1H), 2.26 (m, 1H), 2.17 (m, 2H), 2.02 (m, 5.5H), 1.86 (m, 0.3H), 1.67 (m, 0.2H), 0.86 (m, 8H), 0.74 (m, 3.5H), 0.31 (d, J = 6.8 Hz, 0.5H). ESIHRMS m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found 711.3638.

General procedure 2. Methyl [1-((R)-2-{5-[4-(4-{2-[(R)-1-(2-methoxycarbonylamino-3methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-phenyl)-buta-1,3-diynyl]-1H-imidazol2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-carbamate

dihydrochlorides

13j,k.

Compounds 13j,k were prepared according to the procedures provided above for compounds 13a-f starting from compound 14b and through the subsequent interaction of intermediate 18b


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with the corresponding 5-iodimidazole 19d. The dihydrochlorides 13j,k were obtained as describe above. Methyl

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

pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine1-carbonyl)-2-methyl-propyl]-carbamate dihydrochloride (13j). 1H NMR (DMSO-d6, 400 MHz) δ 15.14 (br s, 0.4H), 14.50 (br s, 0.6H), 8.20, 8.22 (2s, 1H), 7.96 (m, 2.9H), 7.79 (m, 2.2H), 7.63 (m, 0.04H), 7.48 (d, J = 8.0 Hz, 0.13Н), 7.24 (d, J = 8.0 Hz, 1.1Н), 6.73 (m, 0.03H), 5.98 (m, 0.04H), 5.63 (m, 0.19H), 5.22 (m, 0.95Н), 5.08 (m, 0.8H), 4.17 (m, 2H), 3.91 (m, 1H), 3.83 (m, 1H), 3.64 (m, 2H), 3.55 (s, 3H), 3.56 (s, 3H), 2.39 (m, 1H), 2.27 (m, 1H), 2.03 (m, 7.9H), 1.66 (m, 0.1H), 0.87 (m, 10.3H), 0.73 (m, 0.9H), 0.43 (m, 0.2H), 0.31 (m, 0.6H). ESIHRMS m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found 711.3613. Methyl

[(R)-1-((R)-2-{5-[4-(4-{2-[(R)-1-((R)-2-methoxycarbonylamino-3-methyl-butyryl)-

pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine1-carbonyl)-2-methyl-propyl]-carbamate dihydrochloride (13k). 1H NMR (DMSO-d6, 400 MHz) δ 15.28 (br s, 0.4H), 14.78 (br s, 0.6H), 8.20 (s, 1H), 7.95 (m, 3H), 7.77 (d, J = 8.4 Hz, 2H), 7.29 (t, J = 8.0 Hz, 1.3H), 6.87 (m, 0.05H), 5.71 (m, 0.03H), 5.41 (m, 0.05H), 5.16 (t, J = 6.8 Hz, 0.9Н), 5.04 (m, 0.9H), 4.09 (m, 2H), 3.95 (m, 1H), 3.82 (m, 3H), 3.54 (s, 5.4H), 3.43 (s, 0.4H), 3.31 (s, 0.2H), 2.37 (m, 1H), 2.25 (m, 1H), 2.16 (m, 3H), 2.01 (m, 5H), 0.81 (m, 12H). ESIHRMS m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found 711.3635.

Properties calculation. The selected molecular descriptors were calculated for 13a and other compounds using SmartMining Software52 and ChemoSoft Software.53 The LogD was calculated in Marvin (ChemAxon).54

Solubility of compound 13a. The aqueous solubility was determined using a filtration-based kinetic HTS assay with spectroscopic readout.55 The compound stock was prepared in DMSO



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and diluted in pION universal buffer at the desired pH to a final concentration of 200 µM (DMSO concentration — 2%). The mixture was then incubated at rt for 1 h under constant shaking, then filtered through a 96w Millipore MultiScreen Solubility Filter Plate. The optical density spectrum from 250-400 nm of the tested compound in the filtrate was recorded using a SpectraMax UV/V microplate reader (Molecular Devices – Sunnyvale, CA) after the 1.67-fold dilution of the filtrate with acetonitrile. To quantify the aqueous solubility, a standard calibration curve from 0–200 µM was constructed in 40% acetonitrile in buffer. The solubility was calculated by:56 Solubility (µM) = 1.67 ⋅ (ODλ(f) - ODλ) / k, where ODλ(f) is the optical density of the filtrate at the selected wavelength, ODλ is the optical density of the solvent, k is the slope ratio of the calibration curve (µM-1), and 1.67 is the dilution coefficient of the filtrate by acetonitrile.

Molecular modeling. The 3D molecular docking study was performed in MOE software23 following the approach reported by Cordek and colleagues.34

Replicon antiviral assays. Compounds 13a-k were tested in the HCV GT1b subgenomic replicon assays with or without the addition of 40% normal human serum (Table 2).41,57,58 The human hepatoma cell line Huh7 harboring the HCV replicon (genotype 1b) was used as the test line to perform the assay. Cells were seeded at a density of 7.5⋅103 cells per well in 96-well plates containing 50 µL of assay media. The test compound (2X stock solution) was prepared fresh in the assay medium (DMEM 1X, Cellgro, cat. № 10-013-CV). A total of 11 serial 3-fold dilutions of the test compounds were prepared from the 2X stock in assay media ranging from 20 nM to 0.2 pM final concentrations. No earlier than 4 hours after the cells were seeded, compound treatment was initiated by adding the test solution (50 µL) to the plates. The final concentration of the compound therefore ranged from 10 nM to 0.1 pM when diluted 1:1 in the existing culture


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media. The final DMSO concentration was 0.5%. Test examples were then incubated for 3 days at 37 °C in a 5% CO2 atmosphere. The medium was removed from the plates via gentle aspiration. Cells were fixed with 100 µL of 1:1 acetone:methanol for 1 minute, washed three times with PBS buffer, and then blocked with 150 µL/well 10% FBS in PBS for 1 hour at rt. The cells were washed three times with PBS buffer and incubated with 100 µL/well anti-hepatitis C core mAb (Affinity BioReagents; cat. № MA1-080, 1 mg/mL stock diluted 1:4,000 in 10% FBSPBS) for 2 hours at 37 °C. The cells were then washed three times with PBS and incubated with 100 µL/well HRP-Goat Anti-Mouse antibody (diluted 1:3,500 in 10% FBS-PBS) for 1 hour at 37 °C. Finally, the cells were washed three times with PBS and developed with 100 µL/well OPD solution (1 OPD tablet + 12 mL citrate/phosphate buffer + 5 µL 30% H2O2 per plate) for 30 minutes in the dark at rt. The reaction was stopped with 100 µL/well of 2N H2SO4, and the absorbance was measured at A490 X on a Victor3 V 1420 multilabel counter (Perkin Elmer). The EC50 values for the test compounds were calculated from the resulting best-fit equations determined by Xlfit software.

Cytotoxicity. The cytotoxicities of the tested compounds were evaluated in parallel in the same human hepatoma cell line Huh7 as described previously.45

Antiviral spectrum studies. The antiviral activity of compound 13a against West Nile virus, yellow fever virus and dengue virus (Table 3) was studied in African green monkey kidney cells (VERO) obtained from the American Type Culture Collection (Manassas, VA, USA) and routinely passaged in minimal essential medium (MEM with 0.15% NaCHO3) supplemented with 5% fetal bovine serum (FBS, Hyclone). The serum was reduced to a final concentration of 1%, and then gentamicin, at a final concentration of 50 µg/mL, was added to the test medium. All viruses were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The tested compounds were diluted in MEM using one-half log dilutions, and the final



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concentrations were evaluated in an eight-dilution test, starting with 30 µM. Interferon alphacon1 (Infergen) was used as a standard inhibitor. Cells were seeded onto 96-well flat-bottomed tissue culture plates (Corning Glass Works, Corning, NY), 0.1 mL/well, at the proper cell concentration, and incubated overnight at 37 °C. Various dilutions of the test compound were added to each well (3 wells/dilution, 0.1 mL/well). The resulting medium was added to cell and virus control wells (0.1 mL/well). Virus samples diluted in test medium were added to the compound test wells (3 wells/dilution of compound) and to virus control wells at 0.1 mL/well. The test medium without the virus was added to all the toxicity control wells (2 wells/dilution of each test compound) and cell control wells at 0.1 mL/well. The plates were then incubated at 37 °C in a humidified incubator under a 5% CO2, 95% air atmosphere until the virus control wells had developed an adequate cytopathic effect (typically 3 days). The cell viabilities of the compound-treated and untreated cell cultures were determined as reported previously,45 and the EC50 and CC50 values were calculated as described above.

Compound 13a-resistant HCV replicons. The replicons were selected by continuous passaging of the Huh7 cells harboring the HCV replicon (GT1b, clone Con1)41 in the presence of the tested compound at concentrations exceeding the EC50 values by 5- to 40-fold for 3-6 weeks. Some drug-resistant cell populations were cloned. The primary structure of the NS5A gene was determined by direct sequencing of the HCV RNA pools. As a result, several NS5A mutations were identified (Table 4).

Animals. Mice, rats, and dogs were used in the pharmacokinetics studies; in the acute, subchronic, and chronic toxicity studies; and in the mutagenicity studies. The main principles of animal housing and care were in full compliance with the approved guide59 following the federal and Institutional Animal Care and Use Committee (IACUC) guidelines.


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Pharmacokinetics. Pharmacokinetic studies were performed in male naive BALB/C mice, male naive Sprague−Dawley rats and 2 male and 2 female naive English-breed beagle dogs. Intravenous administration was dosed via infusion over 30 min in a vehicle containing 20% 2HP-β-CD in water (mice and rats) and sterile saline solution (0.9% NaCl in water) (dogs). Oral dosing was administered by gavage in a vehicle containing 0.5% Tween 80 in water (mice and dogs) and 0.5% DMSO in PBS, pH = 7.4. Blood samples were collected over a 24 h period postdose into Eppendorf tubes containing 5% EDTA in water. The plasma was isolated, and the concentrations of the test compounds in plasma were determined by LC/MS/MS (QTRAP 5500, Applied Biosystems and chromatograph Agilent 1290, Agilent Technologies) after protein precipitation with acetonitrile. Noncompartmental PK was performed on the plasma concentration data to calculate the PK parameters using WinNonlin Professional 6.1 software (Pharsight Corporation).

Interaction with CYP450 enzymes. The interaction of compound 13a with human cytochrome CYP450 was studied on a cytochrome panel consisting of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A4 (Invitrogen Vivid CYP450 Screening Kits) in 1% DMSO according to the manufacturer’s protocols. The final compound concentrations were 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0.003 µM, and each concentration was tested in duplicate. The EC50 values were determined with Graph Pad Prism 5 sigmoidal dose-response fitting.

Metabolic stability of the 13a. The metabolic stability of the hit compound was evaluated in the presence of 0.5 mg/mL pooled microsomal protein from rat, dog and human livers (BD Gentest, USA) and NADPH over 1 h using 1 µM of the test compound (Table 7). The residual compound was determined by LC-MS/MS analysis. The t1/2 was calculated as described previously.60



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Cardiotoxicity in vitro. The inhibition potency of compound 13a toward the K+ channel myocardium of hERG was studied using the Invitrogen Predictor hERG Fluorescence Polarization Assay Kit. Tests were carried out at 13a concentrations of 3 µM and 30 µM in accordance with the manufacturer's instructions.61 The total reaction volume was 20 µL, and the DMSO content was 0.5%. The Z-factor value was 0.5, the S/N was 8.3, and the assay window ∆mP was 140. The antiarrhythmic drug Е-4031, which blocks hERG-type potassium channels, was used as a control sample. The obtained IC50 value calculated for Е-4031 was 17 nМ, which correlated well with the data reported previously.62 For both concentrations, the binding did not exceed 10%; no dose-dependence was observed. Acute toxicity. Acute toxicity studies were performed in mice and rats according to standard protocols.63 All groups included six males and six females. The individual body weight variation within each sex did not exceed ± 10%. Working solutions were prepared on the day of administration. For i.p. administration, physiological saline (0.9% NaCl in water) was used as a vehicle; hydroxypropyl methylcellulose in water (0.2%) was used for oral administration. The final solutions of compound 13a were administered orally using gavage at 10 mL/kg. Animals from the control group received equivalent volumes of the vehicle. The clinical signs of toxicity were monitored twice a day, at approximately 8-10 a.m. and again at the end of the workday, at 3-4 p.m. The doses leading to the death of 50% and 10% of the animals (LD50 and LD10) and the grade of acute toxicity were derived based on the mortality reported within 14 days after the administration of 13a. Statistical processing of the data, obtained by the registration of animal mortality rate for the purpose of (LD50 ± standard error), LD10, LD16, and LD84 determination, was carried out by probit analysis.64 Calculations were performed by Finney’s method using BioStat 2006 software.

Mutagenicity (in vitro). The mutagenic effect of compound 13a was estimated using histidinedependent strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) Ames


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MPF (Xenometrix, Switzerland). The assay was performed in full compliance with the manufacturer's manual (Xenometrix, Switzerland). Approximately 107 his–bacteria of each strain were treated with 13a at two concentrations (20 and 200 µM) and with positive (specific mutagen) and negative (vehicle – 2% DMSO) controls. The assay was repeated in triplicate to provide sufficient data for statistical analysis. The statistically significant increase in the number of colonies in comparison with the vehicle confirmed the mutagenic action of 13a in Ames test. The following positive controls recommended by the manufacturer were used: 2-nitrofluorene, 4-nitroquinolile-N-oxide, N4-aminocytidine, and 9-aminoacridine for the TA98, TA100, TA1535, and TA1537 strains, respectively.

Mutagenicity (in vivo). Male and female C57BL/6 mice weighing 18-20 g at the age of 8-12 weeks were used to estimate the mutagenicity of compound 13a. In the chromosome aberration test, 13a was administered orally in the form of a suspension in Tween-80. Single doses of 20 mg/kg and 200 mg/kg were given to male mice, and the cell material was preserved 24 hours after drug administration; 20 mg/kg doses were given to male and female mice daily during 5 days with cell material preservation 6 hours after the last drug administration. Mice from the control group (vehicle control) received equivalent volumes of oral Tween-80 solution. Cisplatin was used as a positive control. The drug was administered intraperitoneally in the dose of 5 mg/kg. The animals were then autopsied 24 hours after exposure. Cytogenetic preparations of femoral marrow were obtained by the standard air-dry technique.65 Cytogenetic analysis was conducted using a Standart-20 (Carl Zeiss) microscope in accordance with the principles of cytogenetic analysis, as previously described.66

ASSOCIATED CONTENT Supporting Information.



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1

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H spectra of compounds 13a-k; pharmacokinetic graphs for 13a. This material is available free

of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *e-mail: [email protected] Phone: +89057320053

Notes The authors of this study declare no conflicts of interest.

ABBREVIATIONS AUC0→∞, area under the concentration–time curve from time zero to infinity; F, bioavailability (%);

Boc;

tert-butoxycarbonyl;

Cmax,

maximum

plasma

concentration;

DIPEA,

diisopropylethylamine; EDAC, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; GT1a, hepatitis C virus genotype 1a, a subtype of hepatitis C virus genotype 1; GT1b, hepatitis C virus genotype 1b, a subtype of hepatitis C virus genotype 1; hERG, the human Ether-à-go-go-Related Gene; 2-HP-βCD, 2-hydroxypropyl-beta-cyclodextrin; IFN-α, Infergen; LD, lethal dose; µM, micromolar; MOE, molecular operating environment; 40% NHS, normal human serum; N-Moc; Nmetoxycarbonyl; NS5A, hepatitis C virus nonstructural protein 5A; nM, nanomolar; OPD, ophenylenediamine; pM, picomolar; PSA, molecular polar surface area (Å); RNA, ribonucleic acid; t1/2, half-life time after administration of a drug; THF, tetrahydrofuran.

ACKNOWLEDGMENTS


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We sincerely thank Nikita Polyakov from the Vernadsky Institute of Geochemistry and Analytical Chemistry of the Russian Academу of Sciences for obtaining the HRMS data.

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GRAPHICAL ABSTRACT . 2HCl 1 2

. 2HCl

p.o. EC50 = 3.4 ± 0.2 pM

rats

Cmax (mg/L) 2097 3 T1/2 (h) F (%) 65 Metab. stab. (%) 70.8 LD50 (mg/kg) in mice:

dogs 5810 4.5 58 84.7 2326.8-3937.7


Discovery of Novel Highly Potent Hepatitis C Virus NS5A Inhibitor

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