Synthesis, characterization and docking studies of anti-HCV molecules

Article abstract of DOI:10.14233/ajchem.2020.22618

The present study reports the synthesis and characterization of novel molecules inhibiting the spread of hepatitis C. The molecules were designed as to block the NS3/4A protease enzyme on HCV RNA. The molecules were synthesized using usual peptide synthesis techniques. Compounds with purity more than 95% were characterized and docking studies were also performed. All the compounds were characterized using physico-chemical techniques such as determination of melting point by DSC and NMR, mass, IR spectral studies. The docking studies were also conducted to assess the activity of molecules for inhibition of hepatitis C virus.

Full text of DOI:10.14233/ajchem.2020.22618

Asian Journal of Chemistry; Vol. 32, No. 5 (2020), 1221-1229
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2020.22618
Synthesis, Characterization and Docking Studies of Anti-HCV Molecules
1,*,
2
1
3
SATISH KUMAR
, P.K. SANTRA , J. DWIVEDI and R.C. ARYAN
1Department of Chemistry, Banasthali Vidyapith, Banasthali-304022, India
2Therachem Research Medilab (India) Pvt. Ltd., Sitapura, Jaipur-302022, India
3API Research, Sun Pharmaceutical Industries Ltd., Sector-18, Gurgaon-122015, India
*Corresponding author: E-mail: satte28@gmail.com
Received: 1 January 2020;
Accepted: 13 February 2020;
Published online: 29 April 2020;
AJC-19860
The present study reports the synthesis and characterization of novel molecules inhibiting the spread of hepatitis C. The molecules were
designed as to block the NS3/4A protease enzyme on HCV RNA. The molecules were synthesized using usual peptide synthesis techniques.
Compounds with purity more than 95% were characterized and docking studies were also performed.All the compounds were characterized
using physico-chemical techniques such as determination of melting point by DSC and NMR, mass, IR spectral studies. The docking
studies were also conducted to assess the activity of molecules for inhibition of hepatitis C virus.
Keywords: Hepatitis C virus, Protease enzyme NS3/4A, Telaprevir, Amino acids.
molecules having four different amino acids natural or synthetic
in peptide chain having end capping C-terminal and N-terminal
end with cyclic amines and pyrazine carboxylic or pyridine
acids, respectively. The amino acids chosen are such that they
fit into the cavity of NS3/4A protein thus blocking the site and
interfere the process of HCV RNA replication.
To study the activity of molecules in vitro a lot of time and
resources are needed, hence it was decided to screen molecules
by docking studies. The tool to assess molecules nowadays
various computer aided drug design programs are used. One
such program is given by Schrödinger known as Glide. The
molecules are docked into structure of protein and the energies
are calculated by software on the basis of interaction between
molecules and active site. In docking studies more the negative
energy the more effective is the molecule against the disease.
INTRODUCTION
The hepatitis C virus has (HCV) [1-3] grown multifold in
last few decades, but few options for the treatment of patients
are available. The drugs available in the market are not effective
against all the HCV genotype [4,5]. Researches to find effective
anti HCV drugs is going on fast pace. In recent years drugs
are found in market targeting HCV with different mechanism
of action. Though many drugs failed to achieve desired activity
but one such drug sofosbuvir [6,7] was found successful in
achieving effective anti HCV treatment. These types of drugs
which targets nonstructural protein 5B (NS5B) [8] of HCV
are called NS5B nucleotide polymerase inhibitor. Sofosbuvir
is also an option of treatment in combination [9,10] with other
drugs such as daclatsavir. Other proteins are also present in
HCV RNA which can be blocked to achieve anti HCV acti-
vities. One such protein is nonstructural protein 3/4A (NS3/
4A) [11] serine protease and it is known to be responsible for
cleavage of the HCV protein. If this protein site is blocked it
will inhibit ribonucleic acid (RNA) replication. Many drugs
came into market as HCV NS3/4A protease inhibitors e.g. tela-
previr [12,13] but they all failed to give an effective alternative
option for hepatitis C. This study is also based on anti HCV
drugs targeting NS3/4A protein. We have synthesized various
EXPERIMENTAL
Solvents were of LR grade and further purified by distil-
lation before use. Amino acids and other key raw materials
were of LR grade and also purified before use. The completion
of reaction was monitored by pre-coated TLC plates (TLC
silica gel 60 F254 aluminium sheet by Merck). Synthesized
compounds were purified by column chromatography (using
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1222 Kumar et al.
Asian J. Chem.
silica gel of 60-120 mesh). NMR (BrukerAvance III 400 MHz),
LC/MS (Agilent HP 1100 Series LC/MS) and FT-IR (Perkin
Elmer Spectrum One FT-IR) were used to characterize the
synthesized compounds. Differential scanning calorimetry
(DSC) of Mettler Toledo, Model: DSC 821 e was used to deter-
mine the melting point of molecules. HPLC (Waters E2695,
PDA Detector: 2998) was used to determine the purity of the
molecules. Karl-Fisher Titrator (Analab Scientific Instrument,
µAquaCal50) was used to determine moisture content in raw
materials and in synthesized compounds whenever required.
was washed with 10 times of 5% NaHCO3 solution and 10
times of 1 N HCl and finally concentrated under vacuum to
afford solid residue. The whole reactions chain is presented in
Scheme-I.
Spectral data
N-(Pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-
valyl-L-prolyl-N-cyclohexyl-L-norvalinamide (B-2): White
solid, Yield: 20%, m.f.: C36H51N7O5; m.w.: 661.83; purity by
HPLC: 97.20%; IR (KBr, νmax, cm–1): 3386 (N-H), 3311 (N-
H), 3063 (C-H), 2933 (C-H), 2856 (C-H), 1651 (C=O), 1632
(C=O), 1520 (C=O), 1446 (C-H), 1400 (C-N), 1370 (C-N),
General procedure for the synthesis of anti-HCV molecules
1
Protection of amino acids with N-benzyloxycarbonyl
(CBz): Amino acid (50 g) was added to 100 mL toluene and
the solution was then cooled to 0 ºC and 500 mL lithium hydro-
xide solution (11% in water) was added dropwise at 0-10 ºC.
Benzyl chloroformate solution (240 mL, 50% in toluene) was
then added to the reaction mixture. Reaction mass was stirred at
15-20 ºC for 15 h. Layers were separated and aqueous layer
was washed with 100 mL toluene followed by the addition of
2N HCl, pH was adjusted to1.5. It was then extracted with
400 mL ethyl acetate. Organic extract was treated with 200
mL water and concentrated under vacuum to afford solid
residue. Hexane was added to the solid residue, stirred and
filtered. The solid was dried in oven under vacuum at 40 ºC to
obtain CBz-L-amino acid.
Coupling reaction/amide formation usingTBTU/HBTU:
O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetra-
fluoroborate (TBTU) or O-(benzotriazol-1-yl)-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate (HBTU) 1.2 mol. equiv
was added to a solution of amino acid (1 mol eq) in acetonitrile.
Mixture was stirred for 15 min and then 1.0 mol equiv. amine
was added followed by the addition of triethylamine (1.0 mol
equiv.) drop-wise at 20-25 ºC. Reaction mixture was stirred at
room temperature for 16 h. DCM was added and reaction mass
was then washed successively with 1N HCl, 5% NaHCO3 and
H2O. The layers were separated and the organic extract was
concentrated to yield solid residue. The solid residue was puri-
fied using hexane and ethyl acetate to yield desired compound
which was dried in vacuum oven at 37 2 ºC.
Deprotection of N-benzyloxycarbonyl (CBz):Palladium
on carbon (50% wet with water) was added to the solution of
starting material in methanol (10 vol. equiv.). Reaction mixture
was stirred in high pressure autoclave under hydrogen pressure
of 2 bar at 30 2 ºC for 2-3 h.Reaction mass was then filtered
through the bed of hyflo to remove carbon and the filtrate was
concentrated under vacuum at 36 2 ºC. Solid residue obtained
was further purified using hexane and dried in vacuum oven at
37 2 ºC to afford desired compound.
Coupling reaction/amide formation using EDC·HCl:
Acid derivative (1 mol) was charged to a round bottom flask
and then dichloromethane (10 to 20 vol eq), 1-hydroxybenzo-
triazole (1 mol) and EDC·HCl (1.2 mol) were then added to
the reaction mixture. Reaction mixture was stirred at ambient
temperature for 30 min to get clear solution. Amine (1.0 mol)
was added to the reaction mixture. Reaction mixture was stirred
at ambient temperature for 4 to 24 h. Progress of reaction was
monitored by TLC/HPLC. Reaction was quenched by adding
water(10vol. equiv.). Layerswereseparated and the organic extract
1231 (C-N), 1020 (C-H); H NMR (400 MHz, DMSO-d6, δ,
ppm): 9.14 (s, 1H), 8.88 (d, J = 2.40 Hz, 1H), 8.72 (m, 2H),
8.24 (d, J = 8.92 Hz, 1H), 7.85 (d, J = 8.16 Hz, 1H), 7.70 (d,
J = Hz, 1H), 7.20 (m, 5H), 4.97 (q, J = 7.84 Hz, 1H), 4.53 (d,
J = 8.92 Hz, 1H), 4.44 (m, 1H), 4.17 (q, J = 6.04 Hz, 1H),
3.63-3.48 (m, 3H), 3.31 (d, J = Hz, 2H), 2.02-1.13 (m, 18H),
0.98 (s, 9H), 0.85 (t, J = 7.16 Hz, 3H); MS (ESI-MS) m/z:
662.6 (MH+).
N-(Pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-
prolyl-N-cyclohexyl-L-norvalinamide (B-4):White powder,
Yield: 18%, m.f.: C36H51N7O5; m.w.: 661.83; purity by HPLC:
97.71%; IR (KBr, νmax, cm–1): 3382 (N-H), 3304 (N-H), 3064
(C-H), 2932 (C-H), 2856 (C-H), 1651 (C=O), 1632 (C=O), 1528
(C=O), 1450 (C-H), 1400 (C-N), 1368 (C-N), 1201 (C-N), 1020
(C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 9.12 (s, 1H),
8.88 (d, J = 2.40 Hz, 1H), 8.73 (m, 1H), 8.66 (d, J = 8.52 Hz,
1H), 8.51 (d, J = 8.04 Hz, 1H), 7.77 (d, J = 8.16 Hz, 1H), 7.56
(d, J = 7.88 Hz, 1H), 7.19 (m, 5H), 4.84 (m, 1H), 4.61 (q, J =
4.96 Hz, 1H), 4.37 (m, 1H), 4.16 (q, J = 8.12 Hz, 1H), 3.64 (m,
1H), 3.59 (m, 2H), 3.15 (dd, J = 4.56 Hz, 1H), 3.06 (q, J =
8.40 Hz, 1H), 2.01-1.13 (m, 21H), 0.91 (m, 9H); MS (ESI-
MS) m/z: 662.6 (MH+).
N-(Pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-
prolyl-N-cyclohexyl-L-valinamide (B-8): White powder,
Yield: 21%, m.f.: C36H51N7O5; m.w.: 661.83; purity by HPLC:
94.98%; IR (KBr, νmax, cm–1): 3380 (N-H), 3309 (N-H), 3065
(C-H), 2958 (C-H), 2932 (C-H), 2856 (C-H), 1651 (C=O), 1630
(C=O), 1518 (C=O), 1450 (C-H), 1404 (C-N), 1369 (C-N), 1201
(C-N), 1020 (C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm):
9.12 (s, 1H), 8.88 (d, J = 2.44 Hz, 1H), 8.73 (m, 1H), 8.67 (d,
J = 8.52 Hz, 1H), 8.54 (d, J = 7.96 Hz, 1H), 7.74 (d, J = 7.84
Hz, 1H), 7.68 (d, J = 8.92 Hz, 1H), 7.19 (m, 5H), 4.84 (m, 1H),
4.62 (q, J = 4.82 Hz, 1H), 4.48 (m, 1H), 4.04 (t, J = 8.72 Hz,
1H), 3.63 (m, 1H), 3.51 (m, 2H), 3.11 (dd, J = 4.28 Hz, 1H),
3.07 (q, J = 8.44 Hz, 1H), 2.04-1.03 (m, 18H), 1.00-0.87 (m,
12H); MS (ESI-MS) m/z: 662.5 (MH+).
N-(Pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-
valyl-L-prolyl-N-cyclohexyl-L-alaninamide (B-14): White
powder, Yield: 21%, m.f.: C34H47N7O5; m.w.: 633.78; purity
by HPLC: 97.42%; IR (KBr, νmax, cm–1): 3381 (N-H), 3311
(N-H), 3063 (C-H), 2957 (C-H), 2933 (C-H), 2855 (C-H), 1652
(C=O), 1632 (C=O), 1523 (C=O), 1447 (C-H), 1400 (C-N), 1370
(C-N), 1231 (C-N), 1020 (C-H); 1H NMR (400 MHz, DMSO-
d6, δ, ppm): 9.15 (s, 1H) 8.88 (d, J = 2.44 Hz, 1H), 8.72 (m,
2H), 8.21 (d, J = 8.84 Hz, 1H), 7.91 (d, J = 7.44 Hz, 1H), 7.63
(d, J = 7.84 Hz, 1H), 7.19 (m, 5H), 4.98 (q, J = 7.92 Hz, 1H), 4.53
(d, J = 8.92 Hz, 1H), 4.43 (m, 1H), 4.20 (p, J = 7.20 Hz, 1H),

Vol. 32, No. 5 (2020)
Synthesis, Characterization and Docking Studies of Anti-HCV Molecules 1223
NH2
O
R1
O
R1
OH
LiOH
+
O
NH
O
Cl
OH
Toluene
Water
+
H2N
O
O
HBTU
TEA
ACN
or TBTU
R1
O
R1
NH
NH
Pd/C
Methanol
H2N
O
NH
O
O
+
O
O
EDC.HCl
O
HOBT
O
N
O
N
NH
DIPEA
DCM
OH
NH
O
R1
O
Pd/C
Methanol
O
R3
OH
O
NH
+
O
N
H
O
NH
NH
O
O
R1
DIPEA
DCM
EDC.HCl
HOBT
O
R3
N
O
NH
NH
NH
O
O
R1
Pd/C
Methanol
O
R3
R3
OH
O
N
O
NH
H2N
+
NH
NH
O
O
O
R1
EDC.HCl
DIPEA
DCM
HOBT
N
N
N
R4
O
N
O
or
or
or
or
Ph =
H3C
NH
O
N
O
NH
3
NH
R
O
NH
CH3
O
R1
H3C
CH3
Pd/C
CH3
R1 =
Methanol
R4
O
NH
O
O
R2 =
N
H2N
or
or
+
N
H
3
NH
R
O
Ph
OH
N
H
N
H
NH
O
R1
CH3
CH3
DIPEA
DCM
H3C
H3C
CH3
EDC.HCl
HOBT
R3 =
R4 =
or
or
R4
O
O
NH
O
Ph
O
N
3
NH
R
O
NH
O
R1
Scheme-I: General synthetic reaction of anti-HCV compounds

1224 Kumar et al.
Asian J. Chem.
3.66-3.47 (m, 3H), 3.11 (d, J = 6.84 Hz, 2H), 2.03-1.03 (m,
17H), 1.09 (s, 9H); MS (ESI-MS) m/z: 634.7 (MH+).
purity by HPLC: 94.3%; IR (KBr, νmax, cm–1): 3305 (N-H),
3063 (C-H), 2958 (C-H), 2932 (C-H), 2871 (C-H), 2855 (C-
H), 1650 (C=O), 1625 (C=O), 1590 (C=O), 1450 (C-H), 1434
(C-H), 1233 (C-N), 997 (C-H); 1H NMR (400 MHz, DMSO-
d6, δ, ppm): 8.63 (m, 2H), 8.54 (d, J = 7.88 Hz, 1H), 7.98 (d,
J = 3.76 Hz, 2H), 7.74 (d, J = 7.76 Hz, 1H), 7.69 (d, 8.84 Hz,
1H), 7.61 (dd, J = 4.20 Hz, 1H), 7.20-7.15 (m, 5H), 4.83 (q, J
= 4.52 Hz, 1H), 4.60 (m, 1H), 4.48 (m, 1H), 4.05 (t, J = 7.44
Hz, 1H), 3.66 (m, 1H), 3.51 (m, 2H), 3.13 (dd, J = 9.64 Hz,
1H), 3.02 (d, J = 8.32 Hz, 1H), 1.98-1.14 (m, 18 H), 0.89 (d, J
= 6.48, 6H), 0.83 (d, J = 6.48, 6H); MS (ESI-MS) m/z: 661.4
(MH+).
N-(Pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-
prolyl-N-cyclohexyl-L-valinamide (C-5): Off white powder,
Yield: 20%, m.f.: C36H51N7O5; m.w.: 661.84; m.p.: 102.3 ºC;
purity by HPLC: 94.6%; IR (KBr, νmax, cm–1): 3379 (N-H),
3307 (N-H), 3064 (C-H), 2958 (C-H), 2932 (C-H), 2871 (C-
H), 1668 (C=O), 1650 (C=O), 1465 (C-H), 1450 (C-H), 1401
(C-H), 1369 (C-H), 1248 (C-N), 1200 (C-N), 1019 (C-H); 1H
NMR (400 MHz, DMSO-d6, δ, ppm): 9.12 (s, 1H), 8.88 (d, J
= 2.08 Hz, 1H), 8.74 (m, 1H), 8.67 (d, J = 8.52 Hz, 1H), 8.54
(d, J = 7.92 Hz, 1H), 7.74 (d, J = 7.72 Hz, 1H), 7.69 (d, J =
8.88 Hz, 1H), 7.19 (m, 5H), 4.84 (m, 1H), 4.60 (m, 1H), 4.48
(m, 1H), 4.05 (t, J = 7.52 Hz, 1H), 3.67 (m, 1H), 3.51 (m, 2H),
3.15 (dd, J = 4.24 Hz, 1H), 3.06 (q, J = 8.52 Hz, 1H), 1.99-
1.11 (m, 18H), 0.91-0.82 (m, 12H); MS (ESI-MS) m/z: 662.4
(MH+).
N-[(5-Methylpyrazin-2-yl)carbonyl]-L-phenylalanyl-
L-leucyl-L-prolyl-N-cyclohexyl-L-valinamide (C-6): Light
brown powder, Yield: 20%, m.f.: C37H53N7O5; m.w.: 675.89;
m.p.: 109.3 ºC; purity by HPLC: 94.4%; IR (KBr, νmax, cm–1):
3306 (N-H), 3030 (C-H), 2958 (C-H), 2932 (C-H), 2870 (C-H),
2855 (C-H), 1649 (C=O), 1620 (C=O), 1475 (C-H), 1449 (C-
H), 1232 (C-N), 1031 (C-H); 1H NMR (400 MHz, DMSO-d6,
δ, ppm): 8.98 (s, 1H), 8.60 (m, 2H), 8.53 (d, J = 7.96 Hz, 1H),
7.74 (d, J = 7.80 Hz, 1H), 7.69 (d, J = 8.84 Hz, 1H), 7.20-7.14
(m, 5H), 4.83 (q, J = 7.96 Hz, 1H), 4.60 (q, J = 8.36 Hz, 1H),
4.48 (m, 1H), 4.05 (t, J = 7.60 Hz, 1H), 3.67 (m, 1H), 3.51
(m, 2H), 3.15 (dd, J = 4.16 Hz, 1H), 3.05 (dd, J = 8.40 Hz,
1H), 2.58 (s, 3H), 2.99-1.11 (m, 18 H), 0.91-0.82 (m, 12H);
MS (ESI-MS) m/z: 676.3 (MH+).
N-(Pyrazin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-
prolyl-N-cyclohexyl-L-alaninamide (B-18): White powder,
Yield: 19%, m.f.: C34H47N7O5; m.w.: 633.78; purity by HPLC:
94.68%; IR (KBr, νmax, cm–1): 3382 (N-H), 3302 (N-H), 3064
(C-H), 2950 (C-H), 2932 (C-H), 2856 (C-H), 1651 (C=O), 1631
(C=O), 1527 (C=O), 1450 (C-H), 1402 (C-N), 1369 (C-N), 1201
(C-N), 1020 (C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm):
9.12 (s, 1H), 8.87 (s, 1H), 8.72 (m, 1H), 8.66 (d, J = 8.40 Hz,
1H), 8.49 (d, J = 7.88 Hz, 1H), 7.87 (d, J = 7.40 Hz, 1H), 7.49
(d, J = 7.68 Hz, 1H), 7.18 (m, 5H), 4.84 (q, J = 4.44 Hz, 1H),
4.60 (q, J = 7.24 Hz, 1H), 4.33 (m, 1H), 4.14 (p, J = 7.16 Hz,
1H), 3.64-3.49 (m, 3H), 3.13 (m, 2H), 2.03-1.08 (m, 20H),
1.09 (d, J = 4.68 Hz, 6H); MS (ESI-MS) m/z: 634.7 (MH+).
N-(Pyridin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-
valyl-L-prolyl-N-cyclohexyl-L-valinamide (C-1): White
powder, Yield: 20%, m.f.: C37H52N6O5; m.w.: 660.84; purity
by HPLC: 93.49%; IR (KBr, νmax, cm–1): 3313 (N-H), 3062
(C-H), 2959 (C-H), 2932 (C-H), 2872 (C-H), 2855 (C-H), 1730
(C=O), 1650 (C=O), 1625 (C=O), 1591 (C=O), 1494 (C-H),
1
1434 (C-H), 1229 (C-N), 997 (C-H); H NMR (400 MHz,
DMSO-d6, δ, ppm): 8.75 (d, J = 4.96 Hz, 1H), 8.63 (d, J =
8.28 Hz, 1H), 8.22 (d, J = 7.96 Hz, 1H), 7.99 (d, J = 6.52 Hz,
2H), 7.77 (dd, J = 4.00 Hz, 2H), 7.60 (d, J = 4.36 Hz, 1H),
7.20-7.10 (m, 5H), 4.95 (q, J = 6.48 Hz, 1H), 4.54 (m, 2H),
4.09 (t, J = 8.2 Hz, 1H), 3.62 (m, 2H), 3.51 (m, 1H), 3.09 (d,
J = 5.88 Hz, 2H), 2.08-1.10 (m, 17 H), 0.97 (s, 9H), 0.86 (d, J
= 6.4, 6H); MS (ESI-MS) m/z: 661.4 (MH+).
N-(Pyrazin-2-ylcarbonyl)-L-phenylalanyl-3-methyl-L-
valyl-L-prolyl-N-cyclohexyl-L-valinamide (C-2): White
powder, Yield: 20%, m.f.: C36H51N7O5; m.w.: 661.83; m.p.:
81.9 ºC; purity by HPLC: 93.74%; IR (KBr, νmax, cm–1): 3382
(N-H), 3313 (N-H), 3063 (C-H), 2959 (C-H), 2933 (C-H), 2872
(C-H), 1668 (C=O), 1653 (C=O), 1626 (C=O), 1466 (C-H), 1444
(C-H), 1400 (C-H), 1370 (C-H), 1247 (C-N), 1199 (C-N), 1019
(C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 9.14 (s, 1H),
8.88 (m, 1H), 8.74 (m, 2H), 8.26 (d, J = 9.00 Hz, 1H), 7.77
(m, 2H), 7.17 (m, 5H), 4.96 (q, J = 7.08 Hz, 1H), 4.54 (m,
2H), 4.08 (t, J = 8.00 Hz, 1H), 3.64-3.59 (m, 3H), 3.10 (d, J =
5.88 Hz, 2H), 2.08-1.10 (m, 15H), 0.97 (s, 9H), 0.86 (d, J =
6.44 Hz, 6H); MS (ESI-MS) m/z: 662.4 (MH+).
N-{(2S)-2-Cyclohexyl-2-[(pyridin-2-ylcarbonyl)amino]-
acetyl}-3-methyl-L-valyl-L-prolyl-N-cyclohexyl-L-valin-
amide (C-7): Off white powder,Yield: 20%, m.f.: C36H56N6O5;
m.w.: 652.86; m.p.: 84.6 ºC; purity by HPLC: 95.6%; IR (KBr,
N-[(5-Methylpyrazin-2-yl)carbonyl]-L-phenylalanyl-
3-methyl-L-valyl-L-prolyl-N-cyclohexyl-L-valinamide (C-3):
Light brown powder, Yield: 20%, m.f.: C37H53N7O5; m.w.:
675.86; m.p.: 79.2 ºC; purity by HPLC: 93.9%; IR (KBr, νmax
,
ν
max, cm–1): 3314 (N-H), 3060 (C-H), 2958 (C-H), 2931 (C-
cm–1): 3314 (N-H), 3063 (C-H), 2959 (C-H).2932 (C-H), 2872
(C-H), 2855 (C-H), 1652 (C=O), 1626 (C=O), 1475 (C-H), 1443
(C-H), 1228 (C-N), 1031 (C-H); 1H NMR (400 MHz, DMSO-
d6, δ, ppm): 8.99 (s, 1H), 8.68 (d, J = 8.32 Hz, 1H), 8.60 (s,
1H), 8.21 (d, J = 8.84 Hz, 1H), 7.77 (m, 2H), 7.20-7.10 (m,
5H), 4.96 (q, J = 6.48 Hz, 1H), 4.53 (m, 2H), 4.48 (m, 1H),
4.08 (t, J = 8.04 Hz, 1H), 3.62 (m, 2H), 3.51 (m, 1H), 3.09 (d,
J = 5.92 Hz, 2H), 2.58 (s, 3H), 2.01-1.10 (m, 15 H), 0.97 (s,
9H), 0.85 (d, J = 6.36, 6H); MS (ESI-MS) m/z: 676.4 (MH+).
N-(Pyridin-2-ylcarbonyl)-L-phenylalanyl-L-leucyl-L-
prolyl-N-cyclohexyl-L-valinamide (C-4): Off white powder,
Yield: 20%, m.f.: C37H52N6O5; m.w.: 660.84; m.p.: 80.5 ºC;
H), 2854 (C-H), 1653 (C=O), 1628 (C=O), 1591 (C=O), 1448
1
(C-H), 1435 (C-H), 1232 (C-N), 997 (C-H); H NMR (400
MHz, DMSO-d6, δ, ppm): 8.70 (m, 2H), 8.17 (d, J = 8.56 Hz,
1H), 8.05-7.99 (m, 2H), 7.76 (d, J = 7.72 Hz, 1H), 7.71 (d, J
= 8.92 Hz, 1H), 7.63 (dd, J = 5.32 Hz, 1H), 4.61 (t, J = 7.56
Hz, 1H), 4.51 (d, J = 8.60 Hz, 1H), 3.40 (m, 1H), 4.06 (t, J =
7.48 Hz, 1H), 3.70 (m, 1H), 3.61 (m, 1H), 3.50 (m, 1H), 1.93-
0.99 (m, 26 H), 0.96 (s, 9H), 0.84 (d, J = 6.64, 6H); MS (ESI-
MS) m/z: 653.4 (MH+).
N-{(2S)-2-Cyclohexyl-2-[(pyrazin-2-ylcarbonyl)amino]-
acetyl}-3-methyl-L-valyl-L-prolyl-N-cyclohexyl-L-valinamide
(C-8): Off white powder,Yield: 22%, m.f.: C35H55N7O5; m.w.:

Vol. 32, No. 5 (2020)
Synthesis, Characterization and Docking Studies of Anti-HCV Molecules 1225
653.85; m.p.: 85.6 ºC; purity by HPLC: 95.1%; IR (KBr, νmax
,
NMR (400 MHz, DMSO-d6, δ, ppm): 9.05 (s, 1H), 8.64 (s,
1H), 8.45 (m, 2H), 7.72 (d, J = 7.72 Hz, 1H), 7.61 (d, J = 8.80
Hz, 1H), 4.54 (m, 1H), 4.47 (t, J = 7.76 Hz, 1H), 4.42 (m,
1H), 4.03 (t, J = 7.44 Hz, 1H), 3.70 (m, 1H), 3.52 (m, 2H),
2.60 (s, 3H), 1.97-0.95 (m, 29 H), 0.95 (m, 12H); MS (ESI-
MS) m/z: 668.5 (MH+).
cm–1): 3380 (N-H), 3316 (N-H), 3061 (C-H), 2956 (C-H), 2931
(C-H), 2854 (C-H), 1668 (C=O), 1650 (C=O), 1627 (C=O), 1466
(C-H), 1448 (C-H), 1399 (C-H), 1369 (C-H), 1234 (C-N), 1202
(C-N), 1019 (C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm):
9.19 (s, 1H), 8.91 (m, 1H), 8.76 (m, 1H), 8.66 (d, J = 9.28 Hz,
1H), 8.17 (d, J = 8.60 Hz, 1H), 7.76 (d, J = 7.84 Hz, 1H), 7.72
(d, J = 8.88 Hz, 1H), 4.62 (t, J = 7.92 Hz, 1H), 4.51 (d, J =
8.64 Hz, 1H), 4.40 (m, 1H), 4.06 (t, J = 7.72 Hz, 1H), 3.70
(m, 1H), 3.61 (m, 1H), 3.50 (m, 1H), 1.93-1.10 (m, 26H),
0.96 (s, 9H), 0.84 (d, J = 6.64 Hz, 6H); MS (ESI-MS) m/z:
654.4 (MH+).
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-3-methyl-1-
oxobutan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-
[(pyridin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]-
octahydrocyclopenta[c]pyrrole-1-carboxamide (D-1):
White powder, Yield: 21%, m.p. by DSC: 79.3 ºC; m.f.:
C40H56N6O5; m.w.: 700.9; purity by HPLC: 92.53%; IR (KBr,
N-[(2S)-2-Cyclohexyl-2-{[(5-methylpyrazin-2-yl)carb-
onyl]amino}acetyl]-3-methyl-L-valyl-L-prolyl-N-cyclo-
hexyl-L-valinamide (C-9): Light brown powder;Yield: 23%,
m.f.: C36H57N7O5; m.w.: 667.88; m.p.: 84.6 ºC; purity by HPLC:
95.3%; IR (KBr, νmax, cm–1): 3316 (N-H), 3075 (C-H), 2959
(C-H), 2931 (C-H), 2854 (C-H), 1649 (C=O), 1627 (C=O), 1475
(C-H), 1448 (C-H), 1247 (C-N), 1202 (C-N), 1031 (C-H); 1H
NMR (400 MHz, DMSO-d6, δ, ppm): 9.05 (s, 1H), 8.64 (s,
1H), 8.60 (d, J = 9.40 Hz, 1H), 8.15 (d, J = 8.52 Hz, 1H), 7.76
(d, J = 7.72 Hz, 1H), 7.71 (d, J = 8.80 Hz, 1H), 4.62 (t, J =
7.72 Hz, 1H), 4.50 (d, J = 8.72 Hz, 2H), 4.39 (m, 1H), 4.06 (t,
J = 7.64 Hz, 1H), 3.60 (m, 1H), 3.50 (m, 1H), 2.60 (s, 3H),
1.92-0.99 (m, 26 H), 0.96 (s, 9H), 0.84 (d, J = 6.68 Hz, 6H);
MS (ESI-MS) m/z: 668.4 (MH+).
N-{(2S)-2-Cyclohexyl-2-[(pyridin-2-ylcarbonyl)amino]-
acetyl}-L-leucyl-L-prolyl-N-cyclohexyl-L-valinamide (C-10):
Off white powder;Yield: 20%, m.f.: C36H56N6O5; m.w.: 652.86;
m.p.: 81.9 ºC; purity by HPLC: 95.6%; IR (KBr, νmax, cm–1):
3305 (N-H), 3066 (C-H), 2958 (C-H), 2930 (C-H), 2854 (C-
H), 1650 (C=O), 1625 (C=O), 1591 (C=O), 1449 (C-H), 1435
(C-H), 1235 (C-N), 997 (C-H); 1H NMR (400 MHz, DMSO-
d6, δ, ppm): 8.68 (d, J = 4.56 Hz, 1H), 8.59 (d, J = 9.32 Hz, 1H),
8.45 (d, J = 7.60 Hz, 1H), 8.06-8.00 (m, 2H), 7.72 (d, J = 7.84
Hz, 1H), 7.63 (m, 2H), 4.55-4.40 (m, 3H), 4.03 (t, 7.40 J = Hz,
1H), 3.71 (m, 1H), 3.52 (m, 2H), 1.86-1.34 (m, 29H), 0.89-
0.81 (m, 12H); MS (ESI-MS) m/z: 653.4 (MH+).
ν
max, cm–1): 3303 (N-H), 3063 (C-H), 2957 (C-H), 2933 (C-H),
2868 (C-H), 2855 (C-H), 1649 (C=O), 1629 (C=O), 1590 (C=O),
1451 (C-H), 1433 (C-H), 1225 (C-N), 997 (C-H); 1H NMR (400
MHz, DMSO-d6, δ, ppm): 8.65 (m, 2H), 8.57 (d, J = 7.88 Hz,
1H), 7.98 (d, J = 3.68 Hz, 2H), 7.74 (dd, J = 3.64 Hz, 2H),
7.60 (dd, J = 4.68 Hz, 1H), 7.19 (m, 5H), 4.83 (m, 1H), 4.59
(q, J = 7.32 Hz, 1H), 4.36 (s, 1H), 4.07 (t, J = 7.60 Hz, 1H),
3.72 (dd, J = 9.00 Hz, 1H), 3.54 (m, 2H), 3.10 (dd, 9.36 J = Hz,
1H), 3.02 (dd, J = 5.68 Hz, 1H), 2.68 (m, 1H), 1.92-1.10 (m,
21H), 0.86 (m, 12H); MS (ESI-MS) m/z: 701.4 (MH+).
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-3-methyl-1-
oxobutan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-
[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)-
pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
(D-2): Off white powder, Yield: 21%, m.p. by DSC: 79.9 ºC;
m.f.: C39H55N7O5; m.w.: 701.8; purity by HPLC: 91.02%; IR
(KBr, νmax, cm–1): 3381 (N-H), 3302 (N-H), 3064 (C-H), 2957
(C-H), 2933 (C-H), 2869 (C-H), 1662 (C=O), 1650 (C=O), 1629
(C=O), 1466 (C-H), 1451 (C-H), 1400 (C-H), 1368 (C-H), 1249
(C-N), 1152 (C-N), 1019 (C-H); 1H NMR (400 MHz, DMSO-
d6, δ, ppm): 9.19 (s, 1H), 8.87 (d, J = 1.92 Hz, 1H), 8.72 (m,
1H), 8.67 (d, J = 8.48 Hz, 1H), 8.57 (d, J = 7.92 Hz, 1H), 7.74
(m, 2H), 7.16 (m, 5H), 4.84 (m, 1H), 4.58 (q, J = 7.28 Hz,
1H), 4.35 (m, 1H), 4.05 (t, J = 7.64 Hz, 1H), 3.72 (t, J = 9.28
Hz, 1H), 3.54 (m, 2H), 3.14-3.02 (m, 2H), 2.68 (m, 1H), 1.92-
1.10 (m, 21H), 0.90-0.82 (m, 12H); MS (ESI-MS) m/z: 702.4
(MH+).
N-{(2S)-2-Cyclohexyl-2-[(pyrazin-2-ylcarbonyl)-
amino]acetyl}-L-leucyl-L-prolyl-N-cyclohexyl-L-valinamide
(C-11): Off white powder;Yield: 20%, m.f.: C35H55N7O5; m.w.:
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-3-methyl-1-
oxobutan-2-yl]-2-[(2S)-4-methyl-2-{[(2S)-2-{[(5-methyl-
pyrazin-2-yl)carbonyl]amino}-3-phenylpro-panoyl]amino}-
pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
(D-3): Light brown powder,Yield: 21%, m.p. by DSC: 91.8 ºC;
m.f.: C40H57N7O5; m.w.: 715.9; purity by HPLC: 91.8%; IR
(KBr, νmax, cm–1): 3304 (N-H), 3064 (C-H), 2957 (C-H), 2933
(C-H), 2869 (C-H), 1649 (C=O), 1630 (C=O), 1474 (C-H), 1451
653.85; m.p.: 82.9 ºC; purity by HPLC: 95.8%; IR (KBr, νmax
,
cm–1): 3389 (N-H), 3309 (N-H), 3072 (C-H), 2956 (C-H), 2931
(C-H), 2854 (C-H), 1648 (C=O), 1465 (C-H), 1449 (C-H), 1401
(C-H), 1368 (C-H), 1248 (C-N), 1157 (C-N), 1019 (C-H); 1H
NMR (400 MHz, DMSO-d6, δ, ppm): 9.19 (s, 1H), 8.91 (d, J
= 1.92 Hz, 1H), 8.76 (m, 1H), 8.52 (d, J = 9.24 Hz, 1H), 8.44
(d, J = 7.48 Hz, 1H), 7.72 (d, J = 7.76 Hz, 1H), 7.61 (d, J =
8.80 Hz, 1H), 4.55-4.40 (m, 3H), 4.03 (t, J = 7.24 Hz, 1H),
3.72 (m, 1H), 3.52 (m, 2H), 1.98-0.96 (m, 29H), 0.93-0.80
(m, 12H); MS (ESI-MS) m/z: 654.4 (MH+).
1
(C-H), 1249 (C-N), 1226 (C-N), 1031 (C-H); H NMR (400
MHz, DMSO-d6, δ, ppm): 8.97 (s, 1H), 8.60-8.53 (m, 3H), 7.75
(d, J = 9.24 Hz, 2H), 7.17 (m, 5H), 4.82 (q, J = 4.24 Hz, 1H),
4.58 (q, J = 7.20 Hz, 1H), 4.35 (m, 1H), 4.05 (t, J = 7.56 Hz,
1H), 3.72 (t, J = 8.96 Hz, 1H), 3.53 (m, 2H), 3.13-3.01 (m,
2H), 2.68 (m, 1H), 2.58 (s, 3H), 1.92-1.10 (m, 21 H), 0.90-0.82
(m, 12H); MS (ESI-MS) m/z: 716.4 (MH+).
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-3-methyl-1-
oxobutan-2-yl]-2-[(2S)-2-({(2S)-2-cyclohexyl-2-[(pyridin-2-
ylcarbonyl)amino]acetyl}amino)-3,3-dimethylbutanoyl]-
octahydrocyclopenta[c]pyrrole-1-carboxamide (D-7): Light
N-[(2S)-2-Cyclohexyl-2-{[(5-methylpyrazin-2-yl)-
carbonyl]amino}acetyl]-L-leucyl-L-prolyl-N-cyclohexyl-L-
valinamide (C-12): Light brown powder; Yield: 20%, m.f.:
C36H57N7O5; m.w.: 667.88; m.p.: 82.9 ºC; purity by HPLC:
95.63%; IR (KBr, νmax, cm–1): 3308 (N-H), 3076 (C-H), 2959
(C-H), 2931 (C-H), 2854 (C-H), 1650 (C=O), 1627 (C=O), 1474
(C-H), 1449 (C-H), 1248 (C-N), 1204 (C-N), 1031 (C-H); 1H

1226 Kumar et al.
Asian J. Chem.
brown powder, Yield: 21%, m.p. by DSC: 121.9 ºC; m.f.:
C39H60N6O5; m.w.: 792.93; purity by HPLC: 88.95%; IR (KBr,
ν
4.58 (q, J = 7.44 Hz, 1H), 4.24 (d, J = 3.16 Hz, 1H), 4.10 (m,
1H), 3.72 (t, J = 9.68 Hz, 1H), 3.66-3.48 (m, 2H), 3.13 (dd, J
= 4.32 Hz, 1H), 3.07 (q, J = 8.36 Hz, 1H), 2.68 (m, 1H), 1.92-
1.01 (m, 24 H), 0.95-0.78 (m, 9 H); MS (ESI-MS) m/z: 702.5
(MH+).
max, cm–1): 3317 (N-H), 3062 (C-H), 2959 (C-H), 2932 (C-H),
2855 (C-H), 1655 (C=O), 1623 (C=O), 1591 (C=O), 1449 (C-
H), 1434 (C-H), 1228 (C-N), 1201 (C-N), 997 (C-H);1H NMR
(400 MHz, DMSO-d6, δ, ppm): 8.67 (d, J = 4.56Hz, 1H), 8.59
(d, J = 9.24 Hz, 1H), 8.25 (d, J = 9.20 Hz, 1H), 8.03 (m, 2H),
7.78-7.72 (m, 2H), 7.63 (d, J = 5.24 Hz, 1H), 4.67 (t, J = 2.72
Hz, 1H), 4.57 (d, J = 9.16 Hz, 1H), 4.32 (d, J = 3.00 Hz, 1H),
4.06 (q, J = 8.36 Hz, 1H), 3.76 (m, 1H), 3.65 (m, 1H), 3.32
(m, 1H), 2.74 (m, 1H), 1.90-1.00 (m, 29H), 0.93 (s, 9H), 0.84
(d, J = 6.72 Hz, 6H); MS (ESI-MS) m/z: 693.7 (MH+).
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-1-oxo-
propan-2-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-
[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butanoyl]-
octahydrocyclopenta[c]pyrrole-1-carboxamide (V-14): Off
white powder; Yield: 21%, m.f.: C37H51N7O5; m.w.: 673.84;
m.p.: 67.8 ºC; purity by HPLC: 98.5%; IR (KBr, νmax, cm–1):
3392 (N-H), 3319 (N-H), 3030 (C-H), 2961 (C-H), 2934 (C-H),
2856 (C-H), 1654 (C=O), 1624 (C=O), 1522 (C=O), 1449 (C-
H), 1399 (C-H), 1370 (C-H), 1228 (C-N), 1154 (C-N), 1019
(C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 9.14 (s, 1H),
8.87 (d, J = 1.24 Hz, 1H), 8.72 (d, J = 1.32 Hz, 1H), 8.67 (d,
J = 8.40 Hz, 1H), 8.24 (d, J = 8.72 Hz, 1H), 7.93 (d, J = 7.32
Hz, 1H), 7.60 (d, J = 7.76 Hz, 1H), 7.19 (m, 5H), 4.99 (q, J =
7.60 Hz, 1H), 4.49 (d, J = 8.68 Hz, 1H), 4.27 (d, J = 3.28 Hz,
1H), 4.17 (p, J = 7.12 Hz, 1H), 3.76 (t, J = 9.64 Hz, 1H), 3.58
(m, 1H), 3.49 (m, 1H), 3.08 (m, 2H), 2.63 (m, 1H), 2.54 (m,
1H), 1.52-1.23 (m, 18 H), 1.19 (d, J = 6.96 Hz, 1H), 0.98 (s, 9
H); MS (ESI-MS) m/z: 674.6 (MH+).
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-1-oxo-
propan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-[(pyra-
zin-2-ylcarbonyl)amino]propanoyl}amino)pentanoyl]-
octahydrocyclopenta[c]pyrrole-1-carboxamide (V-18): Off
white powder; Yield: 18%, m.f.: C37H51N7O5; m.w.: 673.84;
m.p.: 99.31 ºC; purity by HPLC: 95.3%; IR (KBr, νmax, cm–1):
3392 (N-H), 3319 (N-H), 3030 (C-H), 2961 (C-H), 2934 (C-H),
2856 (C-H), 1654 (C=O), 1624 (C=O), 1522 (C=O), 1449 (C-
H), 1399 (C-H), 1370 (C-H), 1228 (C-N), 1154 (C-N), 1019
(C-H);1H NMR (400 MHz, DMSO-d6, δ, ppm): 9.14 (s, 1H),
8.87 (d, J = 1.24 Hz, 1H), 8.72 (d, J = 1.32 Hz, 1H), 8.67 (d,
J = 8.40 Hz, 1H), 8.24 (d, J = 8.72 Hz, 1H), 7.93 (d, J = 7.32
Hz, 1H), 7.60 (d, J = 7.76 Hz, 1H), 7.19 (m, 5H), 4.99 (q, J =
7.60 Hz, 1H), 4.49 (d, J = 8.68 Hz, 1H), 4.27 (d, J = 3.28 Hz,
1H), 4.17 (p, J = 7.12 Hz, 1H), 3.76 (t, J = 9.64 Hz, 1H), 3.58
(m, 1H), 3.49 (m, 1H), 3.08 (m, 2H), 2.63 (m, 1H), 2.54 (m,
1H), 1.52-1.23 (m, 18 H), 1.19 (d, J = 6.96 Hz, 1H), 0.98 (s, 9
H); MS (ESI-MS) m/z: 674.6 (MH+).
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-3-methyl-1-
oxobutan-2-yl]-2-[(2S)-2-({(2S)-2-cyclohexyl-2-[(pyridin-2-
ylcarbonyl)amino]acetyl}amino)-4-methylpentanoyl]-
octahydrocyclopenta[c]pyrrole-1-carboxamide (D-10):
Light brown powder,Yield: 21%, m.p. by DSC: 124.5 ºC; m.f.:
C39H60N6O5; m.w.: 692.93; purity by HPLC: 80.82%; IR (KBr,
ν
max, cm–1): 3384 (N-H), 3311 (N-H), 3061 (C-H), 2961 (C-H),
2874 (C-H), 1727 (C=O), 1681 (C=O), 1660 (C=O), 1620
(C=O), 1460 (C-H), 1434 (C-H), 1240 (C-N), 1201 (C-N), 997
(C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.68 (d, J =
4.64 Hz, 1H), 8.57 (d, J = 9.44 Hz, 1H), 8.50 (d, J = 7.92 Hz,
1H), 8.03 (m, 2H), 7.65 (m, 3H), 4.67 (t, J = 2.70 Hz, 1H),
4.57 (dd, J = 9.13 Hz, 1H), 4.29 (d, J = 2.84 Hz, 1H), 4.02
(m, 1H), 3.72 (m, 1H), 3.63 (m, 1H), 3.54 (m, 1H), 2.72 (m,
1H), 1.90-1.00 (m, 32H), 0.85 (m, 12H); MS (ESI-MS) m/z:
693.5 (MH+).
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-1-oxo-
pentan-2-yl]-2-[(2S)-3,3-dimethyl-2-({(2S)-3-phenyl-2-
[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)-
butanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
(V-2): White powder; Yield: 20%, m.f.: C39H55N7O5; m.w.:
701.89; m.p.: 133.6 ºC; purity by HPLC: 93.33%; IR (KBr,
ν
max, cm–1): 3380 (N-H), 3309 (N-H), 3063 (C-H), 2963 (C-
H), 2934 (C-H), 2870 (C-H), 1654 (C=O), 1620 (C=O), 1527
(C=O), 1449 (C-H), 1399 (C-H), 1370 (C-H), 1228 (C-N), 1153
(C-N), 1020 (C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm):
9.13 (s, 1H), 8.88 (d, J = 2.24 Hz, 1H), 8.71 (m, 1H), 8.66 (d,
J = 8.32 Hz, 1H), 8.27 (d, J = 8.88 Hz, 1H), 7.90 (d, J = 8.04
Hz, 1H), 7.66 (d, J = 8.00 Hz, 1H), 7.20 (m, 5H), 4.99 (m,
1H), 4.48 (d, J = 8.84 Hz, 1H), 4.29 (d, J = 3.56 Hz, 1H), 4.18
(m, 1H), 3.79 (q, J = 9.80 Hz, 1H), 3.64-3.48 (m, 2H), 3.08
(m, 2H), 2.66 (m, 1H), 1.84-1.10 (m, 21 H), 0.97 (s, 9 H),
0.83 (t, J = 6.92 Hz, 3H); MS (ESI-MS) m/z: 702.5 (MH+).
(1S,3aR,6aS)-N-[(2S)-1-(Cyclohexylamino)-1-oxo-
pentan-2-yl]-2-[(2S)-4-methyl-2-({(2S)-3-phenyl-2-
[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)-
pentanoyl]octahydrocyclopenta[c]pyrrole-1-carboxamide
(V-4): White powder; Yield: 20%, m.f.: C39H55N7O5; m.w.:
RESULTS AND DISCUSSION
Using the above mentioned procedure, compounds were
synthesized in 8-10 steps using natural and synthetic amino
acids. The end capping for C-terminal of peptide chain was
used is cyclohexyl amine. Pyrazine-2-carboxlic acid, pyridine-
2-carboxylic acid and 4-methyl-2-pyrazine carboxylic acids
were used to end cap N-terminal of peptide chain. The peptide
bond formation was carried out in solution phase synthesis
using EDC·HCl/HOBT orTBTU as coupling reagent and triethyl-
amine or diisopropylamine as base. Palladium on carbon was
used for deprotection of carbobenzyloxy group to get free
amine group. The yield of peptide bond formation step ranged
from 60-90% for different intermediate steps and the yield for
deprotection of carbobenzyloxy group was between 80-95%.
The overall yield of all the steps for different molecule ranged
from 15-25%.
701.89; m.p.: 67.9 ºC; purity by HPLC: 92.7%; IR (KBr, νmax
,
cm–1): 3378 (N-H), 3299 (N-H), 3064 (C-H), 2962 (C-H), 2933
(C-H), 2869 (C-H), 1651 (C=O), 1621 (C=O), 1530 (C=O), 1453
(C-H),1385(C-H), 1362 (C-H), 1228 (C-N), 1153 (C-N), 1020
(C-H); 1H NMR (400 MHz, DMSO-d6, δ, ppm): 9.11 (s, 1H),
8.88 (d, J = 2.36 Hz, 1H), 8.73 (d, J = 1.36 Hz, 1H), 8.65 (d,
J = 8.60 Hz, 1H), 8.54 (d, J = 7.88 Hz, 1H), 7.87 (d, J = 8.04
Hz, 1H), 7.58 (d, J = 7.80 Hz, 1H), 7.20 (m, 5H), 4.86 (m, 1H),

Vol. 32, No. 5 (2020)
Synthesis, Characterization and Docking Studies of Anti-HCV Molecules 1227
Sharp melting point and HPLC purity of synthesized mole-
cules ensured the high purity of synthesized compounds.
Synthesized molecules with purity more than 90% were
reported in the present communication. Molecular mass
observed in the mass spectrum of molecules supported the
molecular formula and molecular formula of the synthesized
compounds given in Table-1. Similarly, a characteristic group
frequencies observed in the IR spectra and the position of
chemical shifts for different protons, their intensities and their
fine structure in the 1H NMR spectra of synthesized compounds
were substantially supported the suggested structure of synthe-
sized compounds.
In the IR spectra of synthesized compounds, the peaks for
the stretching vibration of NH of amide group were observed
in the ranged 3393-3306 cm-1 and the peaks for the stretching
vibration for CO present in amide group were observed in the
ranged 1700-1650 cm-1, which substantially supported the
presence of peptide bond in the synthesized compounds. Stret-
ching frequencies for ketonic C=O groups present in the mole-
cules were observed at around 1625 cm-1. Characteristic peaks
for other functional group present in the molecules were also
observed as these were reported in the elsewhere.
In 1H NMR spectra of the synthesized compounds, a typical
chemical shifts for -CH protons of pyrazine ring were observed
TABLE-1
STRUCTURE OF MOLECULES WITH DOCKING SCORE
Code
Structure
Code
Structure
O
O
O
O
O
NH
N
N
NH
N
N
N
NH
NH
CH
B-2
C-9
NH
3
CH3
O
NH
O
O
CH
3
NH
O
H C
H3C
N
H3C
CH3
3
O
NH
H3C
H C
3
CH3
O
O
O
O
O
NH
N
NH
N
N
NH
NH
N
B-4
C-10
NH
CH
O
3
NH
N
O
O
NH
O
H3C
O
CH
NH
3
H3C
CH3
H C
3
CH3
H3C
CH3
N
N
N
O
O
O
NH
NH
O
N
N
NH
NH
NH
N
B-8
B-14
B-18
C-1
C-11
C-12
D-1
O
O
O
NH
O
O
NH
H3C
H3C
O
NH
H3C
CH3
CH3
CH3
CH3
CH3
N
N
H3C
O
O
O
NH
N
NH
O
NH
N
N
NH
NH
N
O
O
O
O
NH
NH
O
H3C
H3C
H3C
O
NH
H3C
CH3
CH3
H3C
CH3
N
N
N
O
O
O
NH
NH
O
NH
NH
NH
N
O
O
O
NH
O
N
O
NH
H3C
O
H3C
NH
H3C
CH3
CH3
O
O
O
O
NH
N
N
NH
NH
N
NH
N
D-2
CH3
O
O
NH
NH
N
O
O
H3C
CH3
O
O
H3C
NH
NH
H3C
H3C
CH3
CH3
CH3

1228 Kumar et al.
Asian J. Chem.
O
O
O
O
N
N
NH
N
NH
NH
N
NH
N
C-2
D-3
D-7
CH3
O
O
NH
NH
O
O
N
H3C
CH3
H3C
O
O
H3C
NH
NH
H3C
H3C
CH3
CH3
CH3
O
O
O
O
NH
N
NH
NH
N
NH
N
C-3
CH3
O
CH3
O
NH
NH
N
O
O
H3C
H3C
CH3
H3C
N
CH3
O
O
NH
NH
H3C
H3C
CH3
CH3
O
O
O
O
NH
NH
NH
N
NH
N
C-4
C-5
D-10
V-2
O
O
NH
NH
N
O
N
O
O
H3C
O
H3C
NH
NH
H3C
H3C
CH3
CH3
CH3
CH3
CH3
CH3
H3C
N
N
O
O
O
NH
N
NH
O
NH
N
N
NH
NH
N
O
O
O
O
NH
NH
O
H3C
O
NH
H3C
H3C
CH3
CH3
H3C
CH3
N
N
N
O
O
O
NH
N
N
NH
NH
O
NH
NH
N
C-6
C-7
C-8
V-4
V-14
V-18
O
O
O
NH
O
O
NH
H3C
H3C
O
NH
H3C
H3C
CH3
CH3
CH3
CH3
N
N
H3C
O
O
O
NH
N
NH
O
NH
NH
NH
N
CH3
O
O
O
O
NH
N
O
NH
H3C
H3C
CH3
O
NH
H3C
CH3
H3C
CH3
N
N
N
O
O
O
NH
N
N
NH
NH
O
NH
NH
N
CH3
O
O
O
NH
O
O
NH
H3C
CH3
H3C
O
NH
H3C
CH3
in the range δ 9.5 to 8.5 ppm, peaks for phenyl protons were
observed in the range of δ 6.0 to 8.5 ppm. Peaks of CH proton
of amino acid beside amide bond were observed in the range
δ 5.0 to 3.5 ppm. Peaks in the range δ 1.8 to 1.1 ppm confirmed
the presence of cyclohexyl group.All these supported the struc-
ture of synthesized compounds given in Table-1.
The molecular docking studies of all the molecules were
conducted to determine the affinity between molecules and
active site of NS3/4A protein of hepatitis C virus. The docking
studies were carried out by using Glide program of Schrödinger.
The molecules were docked into the 3D structure of NS3/4A
domain of HCV protein. The docking score of all the molecules
are also given in Table-1. The docking of telaprevir drug was
also conducted along with new molecules and the docking score
of telaprevir as predicted by software was found to be -5.110482.
More the negative the docking score, more is the interaction
between molecule and binding site. There are many molecules
which have comparable energies to that of telaprevir such as
B-8, B-18, C-3, C-4, C-7 and V-2 and other molecules such as
C-10 andV-8 have slightly more negative energies than telaprevir.
This implies that these compounds may be potent as anti-HCV
molecules.

Vol. 32, No. 5 (2020)
Synthesis, Characterization and Docking Studies of Anti-HCV Molecules 1229
4. P. Simmonds, J. Bukh, C. Combet, G. Deléage, N. Enomoto, S. Feinstone,
P. Halfon, G. Inchauspé, C. Kuiken, G. Maertens, M. Mizokami, D.G.
Conclusion
All the molecules were characterized using physico-
chemical techniques and supported the suggested structures
of the synthesized compounds. Docking studies indicated that
these molecules might have significant anti HCV properties
on comparison with NS3/4A inhibitor HCV drug telaprevir.
Few molecules were found to have either equal or more docking
score, which means that they may be used as potent HCV
inhibitor in future. There is need for further investigation of
these molecules.
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CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
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