Ligand design, synthesis and biological anti-HCV evaluations for genotypes 1b and 4a of certain 4-(3- & 4-[3-(3,5-dibromo-4-hydroxyphenyl)-propylamino]phenyl) butyric acids and 3-(3,5-dibromo-4-hydroxyphenyl)-propylamino-acetamidobenzoic acid esters

Abstract

4-(4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylamino]phenyl)-4-oxo-butyric acid (V), 4-(3- & 4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylaminophenyl]-2-aryl-4-oxo-butyric acids (Xa–e) and 4-(2-alkyl-2-[N-3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-propylamino]acetamido) benzoate esters (XVa–e) were designed, synthesized and biologically evaluated as anti-HCV for genotypes 1b and 4a. The design was based on their docking scores with HCV NS3/4A protease-binding site of the genotype 1b (1W3C), which is conserved in the genotype 4a structure. The docking scores predicted that most of these molecules have higher affinity to the HCV NS3/4A enzyme more than Indoline lead. These compounds were synthesized and evaluated for their cytopathic inhibitory activity against RAW HCV cell cultures of genotype 4a and also examined against Huh 5–2 HCV cell culture of genotype 1b, utilizing Luciferase and MTS assays. Compounds Xa and Xb have 95 and 80% of the activity of Ribavirin against genotype 4a and compounds XVa, XVb and XVd exerted high percentage inhibitory activity against genotype 1b equal 87.7, 84.3 and 82.8%, respectively, with low EC₅₀ doses.

Keywords::

• Anti-HCV evaluation for genotypes 1b and 4a
• conserved binding site’s structures of HCV proteases of genotypes 1b and 4a
• C-Docker molecular modeling
• new anti-HCV prototypes against genotypes 4a and 1b

Introduction

By the end of the seventies, the clinical picture of the Hepatitis C Viral infection (HCV) disease was well established, but the etiological factor and laboratory tests for identification of the disease were not available [1]. A long awaited discovery using an extensive set of random primers to pick up a specific polynucleotide sequence for the plasma of HCV-infected chimpanzees were made and was found to be completely differ from that of virus A and B [2]. The World Health Organization (WHO) has declared Hepatitis C a global health problem. In 2007 approximately 3% of the world’s population was estimated to be infected with HCV. Unfortunately, some countries in the Middle East (like Egypt) have the highest prevalence in the world [3]. The severity of HCV infection varies from one individual to another and about 80% of those newly infected cases develop a chronic infection, and it is normally 10–30 years before serious life threatening liver diseases emerge. Thus, 10–20% of patients with chronic infection might progress to cirrhosis, and 1–5% of these might develop liver cancer [4]. Liver failure may eventually occur in any of these patients, and liver transplantation is often the only alternative. The HCV life cycle begins with cell entry, uncoating, release of the viral genome into the cytoplasm of the host cell that is used as a template to generate new viral RNA leading to production of new viruses [5]. The viral RNA binds to the ribosomes on the rough endoplasmic reticulum (ER) and translation begins to give a polyprotein that is cleaved into 10 mature viral proteins inside the new viruses [6, 7]. The HCV viral polyprotein is composed of approximately 3000 amino acids and it will be proteolytically cleaved into the structural proteins (C, E1 and E2), P7 and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). Cleavage of such viral polyprotein is mediated by different proteolytic enzymes; the host signal peptide peptidase, the NS2/3 protease and the NS3 protease [8]. The most important proteins that are essential for the life cycle of HCV viruses are the nucleocapsid surrounding the genetic material (core protein), the glycosylated envelope proteins (E1 and E2) [6], the membrane ion channel protein (protein p7) [9], the NS2/3 protease enzyme (that cleave polyprotein between NS2 and NS3) [10], the NS3 serine protease (the key factor in the cleavage at four sites (NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B), the NS3 helicase enzyme (that initiate the RNA replication by unwinding RNA duplexes utilizing adenosine triphosphate) [11], full length NS3 protein (that suggest a mutual influence between the helicase and protease domains) [12], the NS4A enzyme (the essential cofactor to the NS3 protease), the NS4B (that involve in the formation of membrane structures) [11], the phosphorylated metalloprotein (NS5A) (that serve viral RNA transport during RNA replication) [13] and the RNA-dependent RNA polymerase (RdRp) enzyme (NS5B) (that catalyze viral RNA replication) [14].

HCV is an envelope, single-strand positive-sense RNA containing virus that propagates using an RNA-dependent RNA polymerase (RdRp), which lacks a proof reading mechanism, thus causing a great heterogeneity in the HCV genome [5]. Thus, HCV genomes generate eleven major genotypes (designated 1–11), many subtypes (designated a, b, c, etc.), and about 100 different strains (numbered 1, 2, 3, etc.) based on the genomic sequence heterogeneity [15]. Genotypes 1–3 have a worldwide distribution. Types 1a and 1b are the most common, accounting for about 60% of global infections. They predominate in Northern Europe and North America and in Southern and Eastern Europe and Japan, respectively. Type 2 is less frequently represented than type 1. Type 3 is endemic in south-east Asia and is variably distributed in different countries. Genotype 4 is principally found in the Middle East and central Africa and showed extraordinary spread of this type over 90% of sick populations in Egypt [3]. Type 5 is almost exclusively found in South Africa, and genotypes 6–11 are distributed in Asia [16].

The current treatment of Hepatitis C infection involves combination therapy of standard or pegylated α-interferon (a natural immune-agent of 165 amino acid residues) and ribavirin (a synthetic nucleoside analog with broad-spectrum general antiviral properties) [17]. In a continuation of our recently reported work [18] for the use of HCV NS5b polymerase enzyme as a target for designing inhibitors and examine their effects on genotypes 1a, 1b and 4a, the present study involved the use of HCV NS3/4A protease enzyme as a more prominent target to defeat HCV infections of various genotypes. Such enzyme was reported to be the most important selective drug target for potential anti-HCV agents, where various inhibitors for this target were developed and are now in clinical trials [19] (Supplementary materials). The importance of this target are i) It is well-characterized HCV enzymes [20] and have a conserved binding site among all the HCV genotypes [21], ii) it has a successful use in HIV/AIDS therapeutics [22] and iii) It restores the host cell’s antiviral defense [23].

The HCV NS3 protease is a serine protease that utilizes three amino acids during the hydrolysis of a substrate peptide bond: a histidine, an aspartic acid and a serine. These amino acids are recognized as the catalytic triad and are numbered according to the HCV numbering system, His57, Asp81 and Ser139. The reported mechanism of peptide-substrate cleavage by the serine protease enzyme consists of employing such catalytic triad (Ser139, His57 and Asp81) in the stabilization of the oxyanion hole (hemiketal tetrahedral intermediate) during peptide bond cleavage of the protein substrate (Supplementary materials). Most of the reported HCV-protease inhibitors are claimed to exhibit hydrogen bonds (HB) interactions with one or more of the catalytic triad aminoacids (His57, Asp81 and Ser139) and any other adjacent aminoacids at the binding site.

Actually, the 3D crystal structure of HCV protease of genotype 1b is successfully isolated and is available in the www.pdb.org web in many codes including; 1W3C, while 3D crystal structure of the genotype 4a is not recognized up till now and hence it is not available in pdb web. However, this problem was solved by Bahgat et al. finding [21], for the genomic sequence heterogeneity of the HCV proteases enzymes from different isolates (including genotypes 1b and 4a). The same authors demonstrated that the protease enzyme of the genotype 4a has closer homology to that of the genotype 1b. The proteases enzymes of these two genotypes demonstrated multiple alignments of their building blocks where they showed structural conservation in three parts: i) conservation in their catalytic triad (His57, Asp81 and Ser139), ii) conservation in the binding residues for the substrate (Leu135, Phe154, Ala157, Arg161 and Lys165), Zn²⁺ (Cys97, Cys99, Cys145 and His149) and iii) conservation in the 12 N-terminal residues involved in binding to the NS4a cofactor. These conservations in the binding site and specially the catalytic triads, in both genotypes 1b and 4a emphasized that the molecular modeling studies at the catalytic triad of genotype 1b would be comparable to that of genotype 4a and consequently the docking scores at the available 3D crystal structure of HCV protease enzyme of genotype 1b would be considered similar with that of the nonavailable genotype 4a.

The development of new HCV viral NS3 serine protease inhibitors witnessed many progresses including the replacement of the peptide substrate by peptidomimetic molecules. Meanwhile, the availability of detailed information about the 3D structures of HCV proteases together with advances in molecular modeling could allow for deep studies of the active sites and thus would result in more reliable design of inhibitors for HCV [24]. Among the most active new peptidomimitic molecules were the α-ketoacid IRBM 9 (1) [25] and diiodophenol-peptidomimetic-aryl carboxylic acid (2)[26] (Figure 1).

Figure 1.  HCV-protease inhibitors leads.

In the current research, we designed new anti-HCV agents for genotypes 1b and 4a derived from the lead 2 by its manipulation through bioisosteric replacements of the diiodophenol moiety with dibromophenol and introducing of an additional pharmacophoric keto group connected to the phenolic ring, reserving the terminal carboxylic function (or its ester bioprecursor) (to allow the persistence of the stability of the oxyanion hole during cleavage (Supplementary materials), variations in the hydrophobic substituents between the aryl carboxylic acid (or ester) head and the dibromophenol tail by peptidomimetic linkers such as substituted 2-amino-4-oxo-buteric acids chains (as in the series of compounds V and Xa–e) and substituted 2-(aminooxoethylamino)-4-oxo-buteric acids chains (as in the series of compounds XVa–e) and finally reserving the central phenyl moiety to fit the necessary hydrophobic interactions with the complementary amino acids residues in the binding site (Figure 2 and Schemes 1–3).

Figure 2.  New designed molecules based on bioisosteric displacements in the diiodophenol-peptidomimetic-aryl carboxylic acid lead (2).

Scheme 1.  Synthetic pathway for compound V. Reagents: i = Br₂/AcOH/H₂O/r.t./2 h, ii = glyoxylic acid/AcOH/reflux 5 days, iii = AlCl₃/DMF/70°C/1 h, iv = HCl/reflux 15 min; v = ethanol/r.t/7 days.

Scheme 2.  Synthetic pathway for final compounds Xa–e. Compounds: VIa =3-acetamido; VIb = 4-acetamido; VII, VIII, IX, X; (NH; R): a = (m-NH-; H), b = (p-NH-; H), c = (p-NH-; 2-Cl), d = (m-NH-; 4-OCH₃), e = (p-NH-; 4-OCH₃). Reagents: i = (CH₃CO)₂O/reflux 2 h, ii = substituted benzaldehyde/NaOH/ethanol/H2O/15ºC/2–3 h, iii = acetone cyanohydrin/TBAH/acetone/reflux 10 h, iv = HCl/reflux 3 h, v = ethanol/r.t./7 days.

Scheme 3.  Synthetic pathways for final compounds XVa-e. Compounds: XI, XII, XIII, XIV, XV: R; a = H; b = CH₃; c = CH(CH₃)₂; d = CH₂CH(CH₃)₂; e = CH₂C₆H₅. Reagents: i = phthalic anhydride/fusion, ii = PCl₅/dry benzene/50ºC/1 h, iii = 4-(NH₂)-C₆H₄CO₂C₂H₅/TEA/CH₂Cl₂/0°C/2 h, iv = hydrazine/ethanol/reflux 1 h, v = 4-(3,5-dibromo-4-hydroxyphenyl)-4-oxo-but-2-enoic acid (II)/ethanol/r.t./7 days.

Secondly; the designed molecules were subjected to molecular modeling docking studies with the 3D structure of HCV NS3 serine protease enzyme in order to pick up the molecules that have high docking energy as active hits. The performed docking studies predicted that most of the proposed designed molecules (V, Xa, Xc, Xd, Xe, XVd and XVe), have higher binding energies than the lead 1, while the remaining proposed molecules (Xb, XVa–c) showed lower but very close docking scores like the lead 1 (Table 1 and Supplementary materials). Accordingly, these proposed molecules were synthesized fulfilling the reaction pathways outlined in Schemes 1–3, to examine their effects on HCV viral cell cultures of genotypes 1b and 4a.

Table 1.  C-Docking energy and HB with leads and test-set molecules (V, Xa–e and XVa–e) with the complementary amino acids of HCV NS3/4A protease-binding site.

Compound	C-Docker energy (-)	Hydrogen bonds with connected amino acids (length in Å)
		With the catalytic triads aminoacids	With the surrounding aminoacids closed to the catalytic triads aminoacids (umbrella effect)
Indoline lead (1)	50.52	Ser139 (3.2), His57 (2.9)	Lys136 (1.78), Gly137 (1.93), Gly137 (2.7), Ala157 (2.36), Arg155 (2.37), Cys159 (2.1)
Peptidomimetic diiodophenol carboxylic acid lead (2)	56.81	–	Leu135 (1.98), Lys136 (1.93), Gly137 (1.97), Ser138 (1.78)
V	54.48	Ser139 (2.86)	Lys136 (1.88), Lys136 (1.86), Gly137 (1.97), Ser138 (2.24), Ser138 (2.23), Arg155 (2.12), Arg155 (2.41)
Xa	55.78	Ser139 (1.87), His57 (2.09)	Lys136 (1.86), Ala156 (1.94)
Xb	48.84	–	Lys136 (1.81), Ala157 (2.10), Cys159 (2.00), Cys159 (2.11), Gln41 (2.23)
Xc	51.64	–	Gly137 (2.43), Ser138 (2.09), Leu135 (2.33),Cys59 (1.89), Cys159 (2.01)
Xd	54.21	Ser139 (2.16)	Lys136 (1.79), Lys136 (2.01), Arg155 (2.44), Arg155 (1.97), Ala157(2.02)
Xe	57.86	–	Gly137 (1.31), Arg155 (1.55)
XVa	48.04	–	Lys136 (2.04), Lys136 (2.35), Gly137 (2.00),
XVb	44.25	–	Leu135 (1.67), Lys136 (1.99), Gly137 (1.95), Ser138 (2.33)
XVc	48.19	Ser139 (2.35), Ser139 (3.20), Ser139 (3.07)	Lys136 (2.02), Gly137 (2.03), Ser138 (2.20), Ala157 (2.43)
XVd	56.75	Ser139 (2.35), Ser139 (3.20), Ser139 (3.05)	Lys136 (1.94), Gly137 (2.25), Ser138 (2.43), Val158 (1.92)
XVe	55.82	Ser139 (2.02), Ser139 (2.99), Ser139 (3.03)	Lys136 (2.04), Gly137 (2.03), Ser138 (2.17)

Results and discussion

Chemistry

Synthesis of the reported intermediates; I [27], II [28], III and IV[29], VIa [30], VIb [31], VIIa [32], VIIb [33], VIIc [34], VIId [32],VIIe [35], VIIIb, IXb [36], XIa, XIb [37], XIc [38], XId and XIe [39], XIIa [40], XIIb [41], XIIc [42], XIId [43], XIIe [44], XIIIa [45], XIIIb [46], XIVa [46], XIVb [47], XIVc [48], XIVd [49] and XIVe [50] were performed according to the corresponding reported procedures outlined in Schemes 1–3. The new intermediates (VIIIa, c, d, e) were prepared by a similar method for preparing VIIIb [37], by hydrocyanation of the appropriate substituted cinamoylacetanilides (VIIa, c–e) by acetone cyanohydrin. FTIR spectra of the obtained intermediates (VIIIa, c, d and e) were characterized by strong nitrile peaks at 2220–2250 cm⁻¹. The ¹H-NMR spectra of the same molecules demonstrated the disappearance of the two doublets of the vinylic functions of their precursors (VIIa, c–e) and appearance of doublet and triplet system of the -CH2-CH- functions of the products (VIIIa, c, d and e). Hydrolysis of the acetamido and the cyano functions of VIIIa, c–e would afford the corresponding 4-(3 or 4-aminophenyl)-2-aryl-4-oxo-butyric acid derivatives (IXa, c–e), in a method similar to that reported for preparing IXb [36]. The FTIR, ¹H-NMR, EIMS spectral data of (IXa, c–e) were matched with their structures. Also, the new p-(phthalimido-acylamino)-benzoate intermediates (XIIIc–e) were prepared by the similar method for preparing XIIIa–b [44, 45], by condensation of the appropriate 2-alkyl-2-(phthalimido-acyl) chloride (XIIc–e) [39, 42, 43], with 4-aminobenzoic acid ethyl ester. Hydrazinolysis of XIIIa–e with hydrazine hydrate followed by acidification by HCl, generated the respective reported aminoacylaminobenzoates as HCl salt (XIVa–e) [46–50].

The new molecule; 4-(1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylamino]-phenyl)-4-oxo-butyric acid (V) was prepared by aza-Micheal addition of 4-(p-aminophenyl)-4-oxo-buteric acid (IV) into 4-(3,5-dibromo-4-hydroxyphenyl)-4-oxo-but-2-enoic acid (II). The FTIR, ¹H-NMR and EIMS spectra of V, were matched with its structure. Furthermore, the new 4-(3-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylamino]phenyl)-2-aryl-4-oxo-butyric acids (Xa, d) and 4-(4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-2-aryl-4-oxo-butyric acids (Xb, c, e), were synthesized by aza-Micheal addition of 4-(3- or 4-aminophenyl)-2-aryl-4-oxo-butanoic acids (IXa–e) into 4-(3,5-dibromo-4-hydroxyphenyl)-4-oxo-but-2-enoic acid (II). The molecular structures of the products (Xa–e) were proved by ¹H-NMR spectroscopy, where they showed the disappearance of the vinylic protons of the precursor compounds (II) and appearance of the doublet and triplet system of the CH2-CH functions of the products (Xa–e). Moreover, the other new series of the designed molecules; ethyl-4-(2-alkyl-2-[N-[3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-proplamino]acetamido)benzoates (XVa–e) were also prepared by aza-Micheal addition reaction of ethyl 4-(2-alkyl-2-aminoacetamido)benzoates (XIVa–e) into the same α-β unsaturated carboxylic acid II. The ¹H-7 NMR spectra of the products (XVa–e) showed disappearance of the two doublets of the vinylic protons of II and instead appearance of the doublet and triplet system of CH2-CH- functions of XVa–e.

Molecular modeling

The 3D structure of HCV NS3/4A protease enzyme of genotype 1b (that is conserved in the structure of that of genotype 4a) complexes with the inhibitor 1 (code 1W3C) [25], was downloaded from www.pdb.org. Re-docking of 1 with the binding site showed interaction energy = −50.525 kcal/mol with small RMSD deviation in comparison to its crystal structure. The 3D docking diagrams of 1 with its binding site demonstrated 2 hydrogen bonds with catalytic triads aminoacids; Ser139, His57 with distances = 3.2 and 2.9 Å, respectively. Also, it gave 6 HB with the complementary amino acids that are close and surrounding to the catalytic triad with variable distances that may exceed 2 Å, viz; Lys136(1.78 Å), Gly137(1.93 Å), Gly137(2.7 Å), Ala157(2.36 Å), Arg155(2.37 Å), Cys159(2.1 Å) (Figure 3 and Table 1). Such binding pattern could block the cavity surrounding the catalytic triads of the enzyme plus the surrounding aminoacids that are closed to such catalytic triad, by competitive inhibition through umbrella like effect [51], thus preventing the protein substrate from reaching to the catalytic triad and consequently, inhibiting the enzyme. Meanwhile, it was reported [26] that the peptidomimetic diiodophenol carboxylic acid lead (2) have higher anti-HCV protease inhibitor activity than (1), but the 3D x-ray crystal structure of (2) complexed with HCV-protease enzyme is not available at the protein data bank. Thus, docking of (2) with HCV-protease enzyme of genotype 1b, in a parallel process with lead (1) and with the designed compounds V, Xa–e and XVa–e, by applying DS C-Docker protocol, indicated that the lead (2) have higher docking score than (1) and interacted with HCV-protease enzyme binding site through 4 aminoacids closed and surrounded to the catalytic triad of this enzyme with distances not exceed 2 Å, namely, Leu135(1.98), Lys136(1.93), Gly137(1.97) and Ser138(1.78) (Table 1 and Figure 4). These results emphasized that such binding mode of the lead 2 could also, competitively block the cavity containing this catalytic triad, in a similar way like the lead (1) through the umbrella effect and hence inhibit the enzyme. Consequently, this finding proved that the binding with the catalytic triad and/or the surrounding aminoacids that are close to such triad could inhibit the enzyme. Meanwhile, this research witnessed that the docking of the designed molecules (V, Xa–e and XVa–e), showed that some of them, such as V, Xa, Xd, XVc, XVd and XVe were bonded with one or more of the catalytic triad aminoacids plus some of the surrounding amino acids closed to it, in a similar pattern like the lead 1 (Figure 5 and supplementary materials), while the other compounds such as Xb, Xc, Xe, XVa and XVb were bonded with only some of the closed surrounding amionoacids to the catalytic triad in a similar pattern like the leads 2 (Figure 6 and supplementary materials). Also, it was found that compounds V, Xa, Xc, Xd, Xe, XVd and XVe have higher docking scores than the lead 1, while compounds Xb, XVa–c showed slightly lower docking scores than the same lead (Table 1, supplementary materials). Accordingly, it was predicted that such designed compounds were prioritized to have higher or slightly lower inhibitor activity against HCV-protease enzymes of genotypes 1b and 4a, through competitive inhibition of the catalytic triad of the enzyme, respectively.

Figure 3  . Three-dimensional ligand interaction diagram of indoline (1) cocrystallized with HCV NS3/4A protease enzyme (1W3C). It demonstrated docking energy = −50.52 Kcal/mol and HB with Ser139(3.2 Å), His57(2.9 Å) (aminoacids of the catalytic triad) and with Lys136 (1.78 Å), Gly137(1.93 Å), Gly137(2.7 Å), Ala157(2.36 Å), Arg155(2.37 Å), Cys159(2.1 Å) (aminoacids surrounding the catalytic triad).

Figure 4.  Docking pattern of the piptidomimetic diiodophenol lead (2) with HCV NS3-binding site. It showed Dock energy = −56.81 Kcal/mol, and hydrogen bonds with Leu135(1.98 Å), Lys136(1.93 Å), Gly137(1.97 Å), Ser138(2.48 Å) (amino acids surrounding the catalytic triad).

Figure 5.  Docking pattern of the designed compound (Xa) with HCV NS3 binding site. It showed Dock energy = −55.78 Kcal/mol and hydrogen bonds with Ser139(1.87 Å), His57(2.09 Å) (Aminoacid of the catalytic triad) and with Lys136(1.86 Å), Ala156(1.94 Å) (amino acids surrounding the catalytic triad).

Figure 6.  Docking pattern of the designed compound (XVa) with HCV NS3-binding site. It showed Dock energy = −48.04 Kcal/mol and HB with Lys136(2.04 Å), Lys136(2.35 Å), Gly137(2.00 Å) (amino acids surrounding the catalytic triad).

Biological evaluation

Anti-HCV evaluation of the test-set compounds for genotype 4a

Cytotoxicity evaluation of the designed compounds on RAW macrophages

A culture of HCV genotype 4a infected mouse leukaemic monocyte macrophage cell line (RAW macrophages) was used to evaluate the antiviral activities of the synthesized molecules by performing cytotoxic and cytopathic inhibitory activity. The cytotoxicity evaluation was carried out to determine the safety doses of the tested compounds on such macrophages cell line according to Aquino et al. method [52]. The macrophage cells were grown in 96 well plates and the tested compounds (V, Xa–c and XVa–e) and the reference drug; ribavirin were added, separately, at different concentrations (20, 40, 60 and 80 µg/mL) followed by incubation of all the cultures at 37°C for 24 h. Then, the morphology of the macrophages was observed microscopically to determine the percentage of the shrunken (dead) cells. Increase the percentage of viable RAW macrophage on addition of the tested compounds in comparison to the control test, indicates the lower cytotoxicity of the tested compounds and vice versa. The results indicated that seven compounds, namely, V, Xa, Xb, Xd, Xe, XVb and XVe showed no cytotoxic effects at the lowest used dose (20 µg) and hence were considerably safe on macrophages and consequently, they could be tested for their antiviral activity using such RAW macrophage-genotype 4a cell cultures, while compounds Xc, Xd, Xe, XVc and XVd showed high cytotoxicity toward such macrophages at all the tested doses thus, could not be evaluated for their anti-viral activity (Table 2 and Graph 1).

Graph 1.  Results of cytotoxicity of the test-set compounds (V, Xa–c and XVa–e) on RAW macrophage cell line in comparison with ribavirine.

Table 2.  Results of cytotoxicity of the test-set compounds (V, Xa–c and XVa–e) on RAW macrophage cell line in comparison with ribavirine.

Compound	% Viable RAW macrophage cells at different doses of test-set molecules (20, 40, 60 and 80 µg/mL)
	20 µg/mL	40 µg/mL	60 µg/mL	80 µg/mL
Control^a	100	100	100	100
Ribavirin	100	100	100	100
V	100	100	100	90
Xa	100	95	85	80
Xb	95	95	90	80
Xc	95	95	80	70
Xd	100	95	80	65
Xe	100	100	80	100
XVa	0	0	0	0
XVb	100	100	60	50
XVc	70	60	50	50
XVd	100	90	90	80
XVe	100	100	60	50

^aThe control test was performed without adding the test compounds or the reference drug

Cytopathic inhibitory evaluation of the noncytotoxic designed compounds on RAW macrophages-HCV genotype 4a cell culture

Molecules V, Xa, Xb, XVb and XVe were successfully examined for their cytopathic inhibitory effect on HCV genotype 4a viruses, but compounds Xd and Xe could not be examined due to their low solubility in the culture media. The remaining harmful compounds to the RAW macrophages namely; Xc, Xd, Xe, XVc and XVd could not be examined, inspite they have comparative higher docking scores than the lead 1. The cytopathic inhibitory evaluation for V, Xa, Xb, XVb and XVe were measured by incubation of these compounds and the reference drug ribivirin, separately, at different doses lower than the safety dose (i.e. 5, 10, 15 and 20 µg/mL) with the infected HCV RAW macrophage cell cultures of genotype 4a viruses. Then, the cytopathic inhibitory effects were determined microscopically by observation of the percentage of the survival macrophage cells after adding the tested compounds by 48 h. The compounds which were highly inhibiting the virus will leave high percentage of the RAW macrophage cells alive and vice versa. Thus, increase the percentage of the survival macrophages means increases the cytopathic inhibitory effect for the virus and hence could be taken as a reference for their antiviral activity against genotype 4a. The obtained data revealed that compound Xa showed 95% inhibitory effect at 10 µg/mL dose, while compound Xb have 80% inhibition at 15 µg/mL dose in comparison to Ribavirin (100% inhibition at 10 µg/mL). However, compounds V, XVb and XVe showed no activity at all tested doses (5, 10, 15 and 20 µg/mL) (Table 3 and Graph 2).

Graph 2.  Results of anti-HCV genotype 4a activity (cytopathic inhibitory) evaluations.

Table 3.  Results of HCV viral cytopathic inhibitory activity of the test-set compounds against RAW-genotype 4a cell culture.

Compound	% viral cytopathic inhibitory effect at different doses lower than the cytotoxic dose (5, 10, 15, 20 µg/mL)
	5 µg/mL	10 µg/mL	15 µg/mL	20 µg/mL
Control^a	0	0	0	0
Ribavirine	90	100	100	100
V	0	0	0	0
Xa	80	95	95	95
Xb	25	50	80	80
XVb	0	0	0	0
XVe	0	0	0	0

^aThe control test was performed without adding the test compounds or the reference drug.

Anti-HCV tests of the designed compounds against Huh2.5-genotype 1b cell line

Luciferase assays

HCV genotype 1b 1389luc-ubi-neo/NS3-3’/5.1 replicon was trans-infected into Huh5.2 cells (a cell line culture with a persistent HCV replicon 1389 luc-ubi-neo/NS3-3’/5.1 replicon with firefly luciferase-lubquitin neomycin phospho-transferase fusion protein EMCV-IRES driven NS3-5B HCV polyprotein) [53]. Such cell culture was used in this research to evaluate the anti-HCV activity for genotype 1b. This is based on the principle that during the growth of the cells, the viral growth will occur with concomitant expression of luciferase as well as fusion protein that is composed of 16 amino acid residues of the HCV core protein, the ubiquitin (ubi), and the selectable marker gene (neo gene), mediated by the EMCV-IRES transition. The antiviral activity is a measure of the activity of the produce luciferase molecules (luciferase assay) [53]. This means that basically, the luciferase reporter assay is used as an indirect measure of HCV replication. The activity of the expressed luciferase is directly proportional to replicated HCV RNA levels. Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence, in which light is produced by oxidation of luciferin pigments by O₂ into oxyluciferin catalysed by luciferase (Figure 7).

Figure 7.  Luciferase assay equation.

In the HCV luciferase assay, an HCV 1b replicon linked to the gene encoding luciferase (luc) is trans-infected into Huh5.2 cells. Cells were grown in the presence of serial dilutions of the inhibitory compounds for a specified period of time, after which the luciferin pigments were injected and the produced luminescences (catalyzed by the produced luciferase) were measured using a luminometer at λ_max 562 nm. Decrease in luciferase activity by antiviral agents (as compared to control), is proportional to inhibition of HCV replication. EC₅₀ values (Effective concentration of antiviral agents that could kill 50% of the virus) could be calculated by plotting the antiviral activity percent versus test-set compounds concentration.

Cytotoxicity tests of the designed compounds on Huh2.5-genotype 1b cell line (MTS assay)

It is worth to mention that any tested compounds for good antiviral activity, they should prevent viral replication while leaving the host cells intact and healthy [54]. But, the vitality of the host cells are always declined on increasing concentrations of tested compounds. The cytotoxicity tests were measured by adding methylated tetrazole sulfonate compound (MTS) to the cell culture, where it is bioreduced by active cells in the presence of phenazine monosulfate (PMS) into a soluble brown dye named formazan. The produced colour can be measured spectrophotometrically, and the quantity of the produced formazan dye, was measured by the amount of absorbance at 490 nm (Figure 8). Such absorbance is directly proportional to the number of living cells in the culture (CC). Thus, cytotoxicity could be quantified by measuring CC₅₀ value (molecular concentration of antiviral agents which can kill 50% of the host cells) [54]. The tests were performed on all the designed compounds except compound Xd due to its rapid re-precipitation during the experiment.

Figure 8.  MTS assay equation.

Other test values for anti-HCV-genotype 1b evaluations

In addition to the measured CC₅₀ and EC₅₀ values, this study involved another test values including; EC₉₀ (concentration of a compound that theoretically inhibits virus replication by 90%), % inh (% of Maximum Inhition of the virus), Max (concentration of a compound at which maximum inhibition of virus replication is observed), Selectivity Index (SI) (= CC₅₀/EC₅₀), Selectivity Surface (SS=integrated surface delineated by the two activities curves and the 50% horizontal line), Therapeutic index (TI = SS × 10log SI) values were performed on the basis of the procedures reported previously [55]. These data would provide complete informations on the overall antiviral potency of the tested compounds. The registered data revealed that compounds XVa, XVb and XVd, showed antiviral activity against the genotype 1b with low EC₅₀ doses (equal: 5.43, 14.80 and 14.70, respectively) and high % maximum viral inhibitions (equal: 87.7, 84.3 and 82.8%, respectively), with maximum doses = 43.4, 49.5 and 50.0 µg/mL, respectively (Table 4).

Table 4.  Results of luciferase and MTS assays and other antiviral values for the test set compounds against Huh-HCV of genotype 1b cell culture.

Compound	Unit	CC50	EC50	EC90	SI	SS	TI	% inh	Max
Ribavirine	µg/mL	>50	22.5	ND	>4.54	>12.6	>5.82	78.5	39.7
XVb	µg/mL	>50	14.8	ND	>3.38	>9.12	>4.82	84.3	49.5
XVa	µg/mL	>50	5.43	ND	>9.20	>17.9	>17.2	87.7	43.4
XVd	µg/mL	>50	14.7	ND	>3.40	>8.73	>4.64	82.8	50.0
XVe	µg/mL	>50	24.5	>50	>2.04	>3.17	>0.98	70.5	50.0
XVc	µg/mL	>50	14.5	>50	>3.44	>3.44	>2.48	67.5	50.0
Xb	µg/mL	>50	>50	>50	ND	ND	ND	10.1	0.40
Xa	µg/mL	>50	>50	>50	ND	ND	ND	32.5	50.0
Xc	µg/mL	>50	>50	>50	ND	ND	ND	5.14	0.40
Xe	µg/mL	>50	>50	>50	ND	ND	ND	36.7	50.0
V	µg/mL	>50	>50	>50	ND	ND	ND	6.24	2.0

Correlation between the in vitro anti-HCV evaluations for genotypes 4a and 1b and the virtual docking scores

The molecular modeling for the designed molecules revealed that compounds V, Xa, Xc, Xd, Xe, XVd and XVe have higher docking scores than 1, while compounds Xb, XVa, XVb and XVc have slightly lower docking scores than the same lead. However, only compounds V, Xa, Xb, Xc, XVb and XVe were tested for their cytopathic activity against genotype 4a-RAW macrophage cell lines, due to their safety toward such macrophages at the tested doses, while the other molecules; Xd, Xe, XVa, XVc and XVd, could not be tested for their cytopathic activity due to their high cytotoxicity against the same macrophages. This evaluation indicated that compounds Xa and Xb that contain substituted 2-amino-4-oxo-buteric acids peptidomoimetic moieties showed in vitro cytopathic activity equal 95 and 80% of the activity of the reference drug; ribavirine, at doses equal 10 and 15 µg/mL, respectively. The molecular modeling score of Xa was just higher than the lead 1, while that of Xb was just lower than that of 1. This means that such in vitro cytopathic inhibitory values for Xa and Xb were nearly coincident with their molecular modeling docking scores. But, the low cytopathic activity of the remaining highly docked and safety molecules (V, XVa and XVe), against genotype 4a-RAW macrophage cell lines, indicated the absence of consistency with their docking scores (Tables 1 and 3). However, the anti-HCV testing against genotype 1b in Huh5.2 host cells lines, for the compounds XVd and XVe (which have higher docking scores than the lead 1) and XVa, XVb and XVd (which have slightly low docking scores than 1), showed high antiviral activity with low EC₅₀ doses (equal: 5.43, 14.80, 14.5, 14.7 and 24.5 µg/mL), high % maximum viral inhibitions (equal: 87.7, 84.3, 67.2, 82.8 and 67.2%) and maximum inhibition doses equal (43.4, 49.5, 50.0, 50.0 and 50.0 µg/mL), respectively. Meanwhile, the other safety designed compounds (V, Xa–c and Xe), (that showed CC₅₀ > 50 µg/mL and gave higher or slightly lower docking values than 1), exhibited low antiviral activity (EC₅₀ ≥ 50 µg/mL) against genotype 1b (Table 4). Thus, the determined CC₅₀, EC₅₀, SI and TI for the designed compounds containing the 2-(amino-oxoethylamino)-4-oxo-buteric acids peptidomimetic linkers, especially compounds XVa, XVb and XVd have the highest activity against genotype 1b in Huh2.5 cell culture, with % inhibitory activity equal 87.7, 84.3 and 82.8% and lowest EC₅₀ doses equal 5.43, 14.80 and 14.7 µg/mL, respectively (Table 4). The deviation in the virtual and real screening in the above mentioned cases, may be explained in terms of what had been reported that “despite the considerable success in molecular modeling scoring functions, yet, accurate and rapid prediction of protein–ligand interactions is still a challenge” [55].

SAR for the anti-HCV activity against genotype 4a

The biological testing for the designed safety compounds of the (V, Xa–e and XVe), against genotyped 4a in RAW-macrophage culture, revealed that compounds containg substituted 2-amino-4-oxo-buteric acids peptidomoimetic moieties such as Xa and Xb have anti-HCV inhibitory activity. The SAR revealed the superior activity of the meta-substituted amino butyric acid derivative (Xa) (95%) than the para-substituted amino butyric acid analogue (Xb) (80%), in comparison to the reference drug; ribavirine toward genotype 4a.

SAR for the anti-HCV activity toward genotype 1b

The biological testing for the second series of the designed that containing the 2-(amino-oxoethylamino)-4-oxo-buteric acids peptidomimetic linkers (XVa–e), have the highest activity against genotype 1b in Huh2.5 cell culture. Replacement of R group in this series (Scheme 3) with H, CH₃ or (CH₃)₂CHCH₂ substituents would give the highest activity, but replacement of R with isopropyl or benzyl substituents would slightly reduce such activity (Table 4).

Experimental section

Molecular modeling

All molecular modeling studies were performed using Accelrys, Discovery Studio 2.55 operating system, at Faculty of Pharmacy, Ain Shams University; Cairo, Egypt. Molecules were built within DS and conformational models for each compound were generated automatically. This emphasizes representative coverage over a 20 kcal/mol energy range above the estimated global energy minimum and the best quality generation technique was chosen. Docking study involved the following steps; The 3D structure of HCV NS3/4A protease of genotype 1b cocrystallized with Indoline inhibitor (with code number; 1W3C) was prepared. Docking of the leads 1, 2 and the designed compounds (V, Xa–e and XVe) with the binding site of 1W3C according to DS C-Docker protocol were achieved by selection of the binding site containing 1, then the C-Docker protocol was run and the produced dock energies and numbers of hydrogen bonds of the best docked conformations of each molecule were recorded. Also, the 3D diagrams of the docked designed active hits and the leads molecules were presented (Supplementary materials Figures 4a–b and 5a–k).

Chemistry

All the starting materials were purchased from Sigma-Aldrich. Melting points were determined on Stuart Scientific apparatus and uncorrected. Reactions were monitored using thin layer chromatography (TLC), performed on 0.255-mm silica gel plates, with visualization under UV. light (254 nm). FTIR spectra were recorded on a Perkin-Elmer spectrophotometer. ¹H-NMR spectra were recorded in δ scale on a Perkin-Elmer 300 MHz spectrometer. EIMS spectra were recorded on Finnigan Mat SSQ 7000 (70 ev) mass spectrometer. Elemental analyses were performed at the Microanalytical Center, Cairo University. The reported intermediates were prepared according to the corresponding reported procedures as described in Schemes 1–3. The new intermediates and final molecules were synthesized and their structures were confirmed according to the following:

4-(4-[N-1-Carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylamino]-phenyl)-4-oxo-butyric acid (V)

To a solution of 4-(3, 5-dibromo-4-hydroxyphenyl)-4-oxo-but-2-enoic acid (II) (1.4 g, 4 mmol) in absolute ethanol (15 mL), 4-(4-aminophenyl)-4-oxo-butanoic acid (IV) (0.85 g, 4.4 mmol, 1.1 eq.) was added portionwise, and the reaction mixture was stirred at room temperature, for 7 days. After the reaction completion (as indicated by TLC), HCl (1.0 N, 20 mL) was added and the resulting precipitate was filtered, washed with ether and crystallized from ethanol-water to give white crystal of V; (30% yield), mp 160–162°C; IR 1705 – 1655, (4 C=O), 3335–2490, (3 OH); EIMS (m/z[%]): 543 (M+, 70%), 541 (M-2, 35%), 545, (M+2, 35%); ¹H-NMR (δ): 3.03 (t, 2H, CH₂-CH₂-C=O), 3.06–3.08 (d, 2H, CH-CH₂-C=O), 3.44 (t, 2H, CH₂-CH₂-C=O), 4.55 (t, 1H, CH-CH₂-C=O), 6.64 (d, 2H, CH-CH of Ar-H), 7.72 (d, 2H, CH-CH of Ar-H), 8.05 (s, 2H, Ar-H), 9.13 (s, 1H, NH, D₂O exchangeable), 9.83 (s, 1H, OH, D₂O exchangeable), 12.15 (broad, 2H, 2COOH, D₂O exchangeable). Elemental analysis: Found: C: 44.56, H: 3.15, N: 2.35 for C₂₀H₁₇Br₂NO₇ (543.16); requires, C: 44.23, H: 3.15, N: 2.58.

3-(3-p-Methoxyphenyl-3-cyanopropionyl)acetanilide (VIIId) and 4-(3-Aryl-3-cyanopropion-yl)acetanilides (VIIIc, e)

General procedure; The appropriate 3- or 4-(substituted cinnamoyl) acetanilide (VIIa–e) (36.6 mmol) (viz; 4-(o-chlorocinnamoyl) acetanilide, 3-(p-methoxycinnamoyl)acetanilide and 4-(p-methoxycinnamoyl)acetanilide) was slurred into acetone (40 mL). Acetone cyanohydrin (3.9 g, 45.8 mmol) was then added all at once followed by 40% aqueous solution of tetrabutylammonium hydroxide (1.5 mL, 0.6 gm, 2.32 mmol). The mixture was heated at reflux for 10 h. After cooling to room temperature, water (10 mL) was added to induce crystallization. The slurry was cooled below 10°C and the formed precipitate was filtered, washed with water and crystallized from ethanol to give the compounds (VIIIb–e). Compound VIIIa was separated as oil and was used in the next reaction without further treatment.

Physical and spectral data for compound (VIIIc)

It is separated as white crystals (70% yield), mp 120–122°C; IR 1680–1620 (2 C=O), 2250 (C≡N); EIMS (m/z[%]): M+= 326 (10%); M+2=328 (3%); ¹H-NMR (δ):1.58 (s, 1H, NH, D₂O exchangeable), 2.21 (s, 3H, CH₃-CONH), 3.44–3.68 (d, 2H, CH-CH₂-C=O), 4.51(t, 1H, CH-CH₂-C=O), 6.91–7.12 (m, 4H, Ar-H), 7.52 (d, 2H, CH-CH of Ar-H), 7.80 (d, 2H, CH-CH aromatic). Elemental analysis: Found: C: 65.92, H: 4.33, N: 8.21 for C₁₈H₁₅ClN₂O₂ (326.78); requires, C: 66.16, H: 4.63, N: 8.57.

For compound (VIIId)

It is separated as white crystals (85% yield), mp 148–150°C; IR cm⁻¹; 1700–1650(2 C=O), 2230 (C≡N); EIMS (m/z[%]):M+= 322(15%), ¹H-NMR (δ): 1.56 (s, 1H, NH, D₂O exchangeable), 2.20 (s, 3H, CH₃-CONH), 3.41–3.60 (d, 2H, CH-CH₂-C=O), 3.75 (s, 3H, CH₃-O), 4.48 (t, 1H, CH-CH₂-C=O), 6.94 (d, 2H, CH-CH aromatic), 7.40 (d, 2H, CH-CH aromatic), 7.61 (t, 1H, CH-CH-CH aromatic), 7.90 (d, 1H, CH-CH-CH aromatic), 8.02 (d, 1H, CH-CH-CH aromatic), 8.32 (s, IH, C-CH-C aromatic). Elemental analysis: Found: C: 70.61, H: 5.50, N: 8.92; for C₁₉H₁₈N₂O₃ (326.78), requires; C: 70.79, H: 5.63, N: 8.69.

For compound (VIIIe)

It is separated as white crystals (80% yield), mp 128–130°C; IR cm⁻¹; 1690–1620(2 C=O), 2220 (C≡N); EIMS (m/z[%]):M+= 322(12%), 1H-NMR (δ):1.58 (s, 1H, NH, D₂O exchangeable), 2.21 (s, 3H, CH₃-C=O), 3.44–3.68 (d, 2H, CH-CH₂-C=O), 3.81 (s, 3H, CH₃-O), 4.51 (t, 1H, CH-CH₂-C=O), 6.91 (d, 2H, CH-CH aromatic), 7.34 (d, 2H, CH-CH aromatic), 7.62 (d, 2H, CH-CH aromatic), 7.90 (d, 2H, CH-CH aromatic). Elemental analysis: Found: C: 70.87, H: 5.27, N: 8.77; for C₁₉H₁₈N₂O₃ (326.78); requires, C: 70.79, H: 5.63, N: 8.69.

4-(3-Aminophenyl)-2-p-methoxyphenyl-4-oxo-butyric acid (IXd) and 4-(4-Aminophenyl)-2-aryl-4-oxo-butyric acids (IXc, e)

General procedure; A suspension of the appropriate cyanopropionyl acetanilide (VIIIc–e) (20 mmol) (viz; 4-(3-(o-chlorophenyl)-3-cyanopropionyl)acetanilide, 3-(3-cyano-3-(p-methoxyphenyl)propionyl)acetanilide and 4-(3-cyano-3-(p-methoxyphenyl)propionyl)acetanilide) and HCl (10 N, 200 mL) was refluxed for 3 h. The reaction mixture was cooled to 10ºC and aqueous solution of Na₂CO₃ (10%) was added slowly till precipitation of the product. The solid was filtered, washed with water and crystallized from ethanol-water to give the compounds (IXc–e).

Physical and spectral data for compound (IXc)

White crystals (80% yield), mp 70–172°C; IR cm⁻¹; 1725 – 1680, (2 C=O), 3355–2610 (OH); EIMS (m/z[%]):M+= 303 (42%), M+2=305 (14%) (28%), 1H-NMR (δ): 3.09–3.15 (d, 2H, CH-CH₂-C=O), 4.24 (t, 1H, CH-CH₂-C=O), 5.30 (s, 2H, 2NH₂, D₂O exchangeable), 6.71 (d, 2H, CH-CH aromatic), 6.92–7.31 (m, 4H, aromatic), 7.45 (d, 2H, CH-CH aromatic), 12.31 (s, 1H, COOH, D₂O exchangeable). Elemental analysis: Found: C: 63.51, H: 4.35, N: 4.57; for C₁₆H₁₄ClN₂O₃ (303.07), requires; C: 63.27, H: 4.65, N: 4.57.

For compound (IXd)

White crystals (85% yield), mp 196–198°C; IR cm⁻¹; 1725–1680 (2 C=O), 3355–2610 (OH); EIMS (m/z[%]):M+= 299 (28%), 1H-NMR (δ): 3.16–3.21 (d, 2H, CH-CH₂-C=O), 3.67 (s, 3H, CH₃-O), 4.12 (t, 1H, CH-CH₂-C=O), 5.45 (s, 2H, NH₂, D₂O exchangeable), 6.83 (d, 2H, CH-CH aromatic), 7.10–7.25 (m, 3H, Ar-H), 7.38 (d, 1H, CH-CH-CH aromatic), 7.47 (d, 2H, CH-CH Ar-H), 12.41 (s, 1H, COOH, D₂O exchangeable). Elemental analysis: Found: C: 67.91, H: 5.32, N: 4.34; for C₁₇H₁₇NO₄ (299.12)), requires; C: 68.21, H: 5.72, N: 4.68.

For compound (IXe)

White crystals (75% yield), mp 80–82°C; IR cm⁻¹; 1715 – 1670 (2 C=O), 3350–2570 (OH); EIMS (m/z[%]):M+= 299 (26%), 1H-NMR (δ): 3.12–3.19 (d, 2H, CH-CH₂-C=O), 3.71 (s, 3H, CH₃-O), 4.07 (t, 1H, CH-CH₂-C=O), 5.41 (s, 2H, NH₂, D₂O exchangeable), 6.62 (d, 2H, CH-CH aromatic), 6.83 (d, 2H, CH-CH aromatic), 7.45 (d, 2H, CH-CH aromatic), 7.68 (d, 2H, CH-CH aromatic), 12.29 (s, 1H, COOH, D₂O exchangeable). Elemental analysis: Found: C: 68.01, H: 5.97, N: 4.44; for C₁₇H₁₇NO₄ (299.12)), requires; C: 68.21, H: 5.72, N: 4.68.

4-(3-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylamino]phenyl)-2-aryl-4-oxo-butyric acids (Xa,d) and 4-(4-[N-1-carboxy-3-(3,5 dibromo-4-hydroxyphenyl)-3-oxo-propylamino]phenyl)-2-aryl-4-oxo-butyric acids (Xb,c,e)

General procedure: To a solution of 4-(3, 5-dibromo-4-hydroxyphenyl)-4-oxo-but-2-enoic acid II (1.4 g, 4 mmol) in absolute ethanol (15 mL) was added the appropriate carboxypropionylanilines (IXa–e) (4.4 mmol, 1.1 eq.) (viz; 4-(3-aminophenyl)-4-oxo-2-phenylbutanoic acid, 4-(4-aminophenyl)-4-oxo-2-phenyl butanoic acid, 4-(4-aminophenyl)-2-(2-chlorophenyl)-4-oxo-butanoic acid, 4-(3-aminophenyl)-2-(4-methoxyphenyl)-4-oxo-butanoic acid and 4-(4-aminophenyl)-2-(4-methoxyphenyl)-4-oxo-butanoic acid), and the mixture was stirred at room temperature for 7 days. After completion of the reaction (as indicated by TLC), HCl (1.0 N, 20 mL) was added and the resulting precipitate was filtered, washed with ether and crystallized from ethanol-water to give the titled compounds (Xa–e).

Physical and spectral data for compound (Xa)

White crystals (20% yield), mp 108–110°C; IR cm⁻¹; 1740–1680(4 C=O), 3230–2470(3 OH); EIMS (m/z[%]): M+=619 (50%), M-2=617 (25%), M+2=621(25%), 1H-NMR (δ): 3.61–3.63 (d, 2H, HOOC-CH-CH₂-C=O), 3.82–3.84 (d, 2H, HOOC-CH-CH₂-C=O), 4.10 (t, 1H, N-CH-CH₂-C=O), 4.45 (t, 1H, N-CH-CH₂-C=O), 7.01 (d, 1H, CH-CH-CH aromatic), 7.18 (s, 1H, C-CH-C aromatic), 7.34–7.43 (m, 7H, Ar-H), 8.09 (s, 2H, Ar-H), 8.96 (s, 1H, NH, D₂O exchangeable), 9.75 (s, 1H, OH, D₂O exchangeable), 12.11 (broad, 2H, 2COOH, D₂O exchangeable). Elemental analysis: Found: C: 50.16, H: 3.09, N: 2.19; for C₂₆H₂₁Br₂NO₇ (619.26), requires; C: 50.43, H: 3.42, N: 2.26.

For compound (Xb)

White crystals (28% yield), mp 110–112°C; IR cm−1; 1730–1600 (4 C=O), 3390–2500 (3 OH); EIMS (m/z[%]): (M+= 619 (60%), M-2=617 (30%), M+2=621(, 30%). 1H-NMR (δ):3.06–3.08 (d, 2H, HOOC-CH-CH₂-C=O), 3.46–3.48 (d, 2H, N-CH-CH₂-C=O), 4.04 (t, 1H, HOOC-CH-CH₂-C=O), 4.54 (t, 1H, N-CH-CH₂-C=O), 6.62 (d, 2H, CH-CH aromatic), 7.25–7.36 (m, 5H, Ar-H), 7.75 (d, 2H, CH-CH aromatic), 8.09 (s, 2H, Ar-H), 9.11 (s, 1H, NH, D₂O exchangeable), 9.79 (s, 1H, OH, D₂O exchangeable), 12.17 (broad, 2H, 2COOH, D₂O exchangeable). Elemental analysis: Found: C: 50.37, H: 3.45, N: 2.16; for C₂₆H₂₁Br₂NO₇ (619.26), requires; C: 50.43, H: 3.42, N: 2.26.

For compound (Xc)

White crystals (30% yield), mp 186–188°C; IR cm⁻¹; 1740–1610 (4 C=O), 3580–2650 (3 OH); EIMS (m/z[%]): (M+= 653 (60%), M-2=651 (40%), M+2= 655(20%). 1H-NMR (δ): 3.06–3.08 (d, 2H, HOOC-CH-CH₂-C=O), 3.60–3.63 (d, 2H, N-CH-CH₂-C=O), 3.46 (t, 1H, HOOCCH-CH₂-C=O), 4.55 (t, 1H, N-CH-CH₂-C=O), 6.62 (d, 2H, CH-CH aromatic), 7.27–7.30 (m, 3H, Ar-H), 7.44 (d, 2H, CH-CH aromatic), 7.74 (d, 2H, CH-CH aromatic), 8.07 (s, 2H, Ar-H), 9.11 (s, 1H, NH, D₂O exchangeable), 9.79 (s, 1H, OH, D₂O exchangeable), 12.35 (broad, 2H, 2COOH, D₂O exchangeable). Elemental analysis: Found: C: 47.82, H: 3.00, N: 2.16; for C₂₆H₂₀Br₂ClNO₇ (653.7), requires; C: 47.77, H: 3.08, N: 2.14.

For compound (Xd)

White crystals (20% yield), mp 160–162°C; IR cm⁻¹; 1720 – 1670 (4 C=O), 3480–2540 (3 OH); EIMS (m/z[%]): M+ = 649(48%), M-2= 647(24%), M+2= 651(24%). 1H-NMR (δ): 3.46–3.48 (d, 2H, HOOC-CH-CH₂-C=O), 3.64–3.68 (d, 2H, N-CH-CH₂-C=O), 3.90 (s, 3H, CH₃-O), 3.99 (t, 1H, NCH-CH₂-C=O), 4.27 (t, 1H, CH-CH₂-C=O), 6.83 (d, 1H, CH-CH-CH aromatic), 6.88 (d, 2H, CH-CH aromatic), 7.10–7.22 (m, 3H, Ar-H), 7.29 (d, 2H, CH-CH aromatic), 8.09 (s, 2H, Ar-H), 9.26 (s, 1H, NH, D₂O exchangeable), 9.65 (s, 1H, OH, D₂O exchangeable), 12.31 (broad, 2H, 2COOH, D₂O exchangeable). Elemental analysis: Found: C: 49.63, H: 3.40, N: 2.24; for C₂₇H₂₃Br₂NO₈ (649.28), requires; C: 49.95, H: 3.57, N: 2.16.

For compound (Xe)

White crystals (25% yield), mp 65–67°C; IR cm⁻¹; 1730 – 1690 (4 C=O), 3580–2660 (3 OH); EIMS (m/z[%]): M+= 649 (50%), M-2 = 647(25%), M+2 = 651(25%). 1H-NMR (δ):3.42–3.44 (d, 2H, HOOC-CH-CH₂-C=O), 3.62–3.64 (d, 2H, N-CH-CH₂-C=O), 3.85 (s, 3H, CH₃-O), 4.03 (t, 1H, HOOCCH-CH₂-C=O), 4.65 (t, 1H, NCH-CH₂-C=O), 6.65 (d, 2H, CH-CH aromatic), 6.98 (d, 2H, CH-CH aromatic), 7.32 (d, 2H, CH-CH aromatic), 7.80 (d, 2H, CH-CH aromatic), 8.11 (s, 2H, Ar-H), 9.13 (s, 1H, NH, D₂O exchangeable), 9.69 (s, 1H, OH, D₂O exchangeable), 12.33 (broad, 2H, 2COOH, D₂O exchangeable). Elemental analysis: Found: C: 50.19, H: 3.77, N: 2.50; for C₂₇H₂₃Br₂NO₈ (649.28), requires; C: 49.95, H: 3.57, N: 2.16.

Ethyl 4-[2-alkyl-2-(N-phthalimidyl)acetamido]benzoates (XIIIc–e)

General procedure; To an ice-cold solution of ethyl 4-aminobenzoate (2.9 g, 17.6 mmol) in methylene chloride (50 mL) and triethylamine (5 mL, 36 mmol), N-substitutedphthalimidylacetyl chlorides (XIIc–e) (5.38, 17.05 mmol) (viz; 2-isopropyl-2-(N-phthalimidyl) acetyl chloride, 2-isobutyl-2-(N-phthalimidyl)acetyl chloride and 2-benzyl-2-(N-phthalimidyl)acetyl chloride) dissolved in methylene chloride (50 mL) were added over 2 h. The reaction mixture was stirred for another hour, the solvent was evaporated under reduced pressure and the residual solid was crystallized from ethanol to give the titled compounds (XIIIc–e).

Physical and spectral data for compound (XIIIc)

White crystals (70% yield), mp 170–172°C; IR cm⁻¹; 1770–1690(4 C=O), 3300–3120 (NH); EIMS (m/z[%]): M+= 394 (70%). 1H-NMR (δ):1.01 (d, 6H, (CH₃)₂-CH), 1.46 (t, 3H, CH₂-CH₃), 1.54 (m, 1H, (CH₃)₂-CH), 4.44 (q, 2H, CH₂-CH₃), 5.16 (d, 1H, CH-CH-(CH₃)₂), 7.63 (d, 2H, CH-CH aromatic), 7.77 (d, 2H, CH-CH aromatic), 7.89 (d, 2H, CH-CH aromatic), 7.97 (d, 2H, CH-CH aromatic), 8.85 (s, 1H, NH, D₂O exchangeable). Elemental analysis: Found: C: 66.90, H: 5.32, N: 7.20; for C₂₂H₂₂N₂O₅ (394.42), requires; C: 66.99, H: 5.62, N: 7.10.

For compound (XIIId)

White crystals (63% yield), mp 180–182°C; IR cm⁻¹; 1750–1680 (4 C=O), 3330–3130 (NH); EIMS (m/z[%]): M+ =408 (75%). 1H-NMR (δ):0.97 (d, 6H, (CH₃)₂-CH), 1.36 (t, 3H, CH₂-CH₃), 1.52 (m, 1H, (CH₃)₂-CH), 2.00 – 2.20 (m, 2H, CH-CH₂-CH), 4.34 (q, 2H, CH₂-CH₃), 5.06 (t, 1H, CH-CH₂-CH), 7.60 (d, 2H, CH-CH aromatic), 7.75 (d, 2H, CH-CH aromatic), 7.87 (d, 2H, CH-CH aromatic), 7.97 (d, 2H, CH-CH aromatic), 8.75 (s, 1H, NH, D₂O exchangeable). Elemental analysis: Found: C: 66.90, H: 5.32, N: 7.20; for C₂₃H₂₄N₂O₅ (408.45), requires; C: 66.99, H: 5.62, N: 7.10.

For compound (XIIIe)

White crystals (75% yield), mp 166–168°C; IR cm⁻¹; 1730–1650 (4 C=O), 3350–3170 (NH); EIMS (m/z[%]): M+ =442 (72%). 1H-NMR (δ): 1.36 (t, 3H, CH₂-CH₃), 3.69–3.75 (d, 2H, N-CH₂-CH-C=O), 4.35 (q, 2H, CH₂-CH₃), 4.54 (t, 1H, N-CH₂-CH-C=O), 7.21–7.32 (m, 5H, Ar-H), 7.65 (d, 2H, CH-CH aromatic), 7.79 (d, 2H, CH-CH aromatic), 7.91 (d, 2H, CH-CH aromatic), 7.99 (d, 2H, CH-CH aromatic), 8.76 (s, 1H, NH, D₂O exchangeable). Elemental analysis: Found: C: 70.33, H: 5.11, N: 6.12; for C₂₆H₂₂N₂O₅ (442.46), requires; C: 70.58, H: 5.62, N: 6.33.

Ethyl 4-(2-alkyl-2-[N-3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-propylamino]acetamido)benzoates (XVa–e)

General procedure: To a solution of 4-(3, 5-dibromo-4-hydroxyphenyl)-4-oxo-but-2-enoic acid II (1.4 g, 4 mmol) in absolute ethanol (15 mL) was added the appropriate arylaminooxoalkylamines (XIVa–e) (4.4 mmol, 1.1 eq.) (viz; ethyl 4-(2-aminoacetamido)benzoate, ethyl 4-(2-amino-2-methylacetamido) benzoate, ethyl 4-(2-amino-2-isopropylacetamido)benzoate, ethyl 4-(2-amino-2-isobutylacetamido)benzoate and ethyl 4-(2-amino-2-benzylacetamido)benzoate). The reaction mixture was stirred at room temperature for 7 days. After completion of the reaction (as indicated by TLC), HCl (1.0 N, 20 mL) was added and the resulting precipitate was filtered, washed with ether and crystallized from ethanol-water to give the titled compounds (XVa–e).

Physical and spectral data for compound (XVa)

White crystals (65% yield), mp 152–154°C; IR cm⁻¹; 1700–1610(4 C=O), 3390–2470 (2 OH); EIMS (m/z[%]): M+= 572 (42%), M-2= 570 (21%), M=574 (21%). 1H-NMR (δ): 1.22 (t, 3H, CH₂-CH₃), 3.22–3.24 (d, 2H, N-CH-CH₂-C=O), 3.59 (s, 2H, NH-CH₂-C=O), 3.70 (q, 2H, CH₂-CH₃), 4.24 (t, 1H, N-CH-CH₂-C=O), 7.68 (d, 2H, CH-CH Ar-H), 7.90 (d, 2H, CH-CH Ar-H), 7.93 (s, 2H, Ar-H), 9.11 (s, 2H, 2NH, D₂O exchangeable), 9.80 (s, 1H, OH, D₂O exchangeable), 12.27 (s, 1H, COOH, D₂O exchangeable). Elemental analysis: Found: C: 44.50, H: 3.65, N: 5.21; for C₂₁H₂₀Br₂N₂O₇ (572.20), requires; C: 44.08, H: 3.52, N: 4.90.

For compound (XVb)

White crystals (60% yield), mp 126–128°C; IR cm⁻¹; 1730–1615 (4 C=O), 3400–2510 (2 OH); EIMS (m/z[%]): M+=586 (62%), M-2= 584(31%), M+2= 588(31%).

1H-NMR (δ): 1.18 (d, 3H, CH-CH₃), 1.35 (t, 3H, CH₂-CH₃), 3.42–3.44 (d, 2H, N-CH-CH₂-C=O), 3.90 (q, 1H, CH-CH₃), 4.00 (t, 1H, NCH-CH₂-C=O), 4.24 (q, 2H, CH₂-CH₃), 7.70 (d, 2H, CH-CH Ar-H), 7.90 (d, 2H, CH-CH Ar-H), 8.06 (s, 2H, Ar-H), 9.13 (s, 2H, 2NH, D₂O exchangeable), 9.50 (s, 1H, OH, D₂O exchangeable), 12.53 (s, 1H, COOH, D₂O exchangeable). Elemental analysis: Found: C: 44.71, H: 3.57, N: 4.54; for C₂₂H₂₂Br₂N₂O₇ (586.23), requires; C: 45.07, H: 3.78, N: 4.78.

For compound (XVc)

White crystals (60% yield), mp 124–126°C; IR cm⁻¹; 1710 – 1600, (4 C=O), 3350–2550, (2 OH) (4 C=O), 3400–2510 (2 OH); EIMS (m/z[%]): M+=614(32%), M-2= 612(16%), M+2= 616 (16%)). 1H-NMR (δ):.95 (d, 6H, CH-(CH₃)₂), 1.26 (t, 3H, CH₂-CH₃), 2.17 (m, 1H, CH-(CH₃)₂), 3.33–3.36 (d, 2H, N-CH-CH₂-C=O), 3.72 (d, 1H, CH-CH-(CH₃)₂), 3.83 (t, 1H, N-CH-CH₂-C=O), 4.25 (q, 2H, CH₂-CH₃), 7.70 (d, 2H, CH-CH Ar-H), 7.86 (d, 2H, CH-CH Ar-H), 8.07 (s, 2H, Ar-H), 9.15 (s, 2H, 2NH, D₂O exchangeable), 9.40 (s, 1H, OH, D₂O exchangeable), 12.33 (s, 1H, COOH, D₂O exchangeable). Elemental analysis: Found: C: 46.47, H: 4.31, N: 4.66; for C₂₄H₂₆Br₂N₂O₇, (614.28), requires; C: 46.93, H: 4.27, N: 4.56.

For compound (XVd)

White crystals (40% yield), mp 128–130°C; IR cm⁻¹; 1720 – 1650, (4 C=O), 3420–2540 (2 OH); EIMS (m/z[%]): M+=628(50%), M-2=626 (25%), M+2= 630 (25%). 1H-NMR (δ): 0.86 (d, 6H, CH₂-CH-(CH₃)₂), 1.04 (t, 3H, CH₂-CH₃), 1.28 (m, 1H, CH₂-CH-(CH₃)₂), 1.67 (t, 2H, CH₂-CH-(CH₃)₂), 3.35 (q, 2H, CH₂-CH₃), 3.58–3.60 (dd, 2H, CH-CH₂-C=O), 3.96 (t, 1H, CH₂-CH-C=O), 4.26 (t, 1H, CH-CH₂-C=O), 7.73 (d, 2H, CH-CH Ar-H), 7.94 (d, 2H, CH-CH Ar-H), 8.14 (s, 2H, Ar-H), 9.10 (s, 2H, 2NH, D₂O exchangeable), 9.45 (s, 1H, OH, D₂O exchangeable), 12.76 (s, 1H, COOH, D₂O exchangeable). Elemental analysis: Found: C: 47.60, H: 4.91, N: 4.40; for C₂₅H₂₈Br₂N₂O₇ (614.28), requires; C: 47.79,, H: 4.49, N: 4.46.

For compound (XVe)

White crystals (20% yield), mp 108–110°C; IR cm⁻¹; 1740–1640, (4 C=O), 3370–2620 (2 OH); EIMS (m/z[%]): M+ = 663(44%) M-2=661(22%) M+2= 665(22%). 1H-NMR 1.25 (t, 3H, CH₂-CH₃), 3.73–3.75 (dd, 2H, CH-CH₂-C=O), 3.88–3.92 (dd, 2H, CH₂-CH-C=O), 3.12 (q, 2H, CH₂-CH₃), 4.18 (t, 1H, CH₂-CH-C=O), 4.26 (t, 1H, CH-CH₂-C=O), 7.21–7.32 (m, 5H, Ar-H), 7.61 (d, 2H, CH-CH Ar-H), 7.87 (d, 2H, CH-CH Ar-H), 8.04(s, 2H, Ar-H), 9.19 (s, 2H, 2NH, D₂O exchangeable), 9.23 (s, 1H, OH, D₂O exchangeable), 12.11 (s, 1H, COOH, D₂O exchangeable). Elemental analysis: Found: C: 50.53, H: 4.03, N: 3.88; for C₂₈H₂₆Br₂N₂O₇ (662.32), requires; C: 50.78, H: 3.96, N: 4.23.

Biological evaluation

Anti-HCV evaluation for genotype 4a

The experimental procedures to perform anti-HCV activity of the tested compounds against genotype 4a virus were done according to Aquino et procedures [52] which comprise the following: i) Sera were collected from HCV genotype 4a infected patients. The diagnosis of these patients was based on physical examination, testing their liver function biochemically and serological testing for the presence of anti-HCV-antibodies. The RT-PCR counting of the viruses and characterization of the genotype 4a virus were also performed. The only sera with high viremia were included in the present study; ii) The RAW macrophage cells (mouse origin) were propagated in a growth media (RPMI-1640), supplemented with 15% Fetal Bovine Serum (FBS), 1% antibiotic-antimycotic mixture was added. The pH was adjusted at 7.2–7.4 by 7.5% by adding suitable amounts of NaHCO₃ solution. The mixture was sterilized by filtration through 0.2 µm pore size nitrocellulose membrane; iii) Antibiotic-Antimycotic Mixture (GIBCO-BRL), 10,000 U Penicillin G sodium 10,000 µg, Streptomycin sulfate and 250 µg amphotericin B, were added to the growth media to protect them from further infection and iv) The growth media was prepared by dissolving RPMI-1640 media (developed by SIGMA-ALDRICH lab by Rose well Park Memorial Institute). The formulation was based on the RPMI-1640 series of media utilizing a bicarbonate buffering system and alterations in the amounts of amino acids and vitamins. (10.41 g) in 1-L deionized water then the medium was supplemented with 1% antibiotic-antimycotic mixture, 15% fetal bovine serum (FBS). The pH was adjusted at 7.4 by 7.5% NaHCO₃ and then the mixture was filtered by a sterile membrane of 0.2 µm pore size nitrocellulose membrane and finally sterility test was carried out in nutrient agar. v) The viral cytopathic inhibitory effect evaluation was performed by adding 100 µL serum-free medium in all wells, then the test-set compounds and the reference drug; ribavirin were added in their safety doses in quadruplicates, followed by 50 µL of the virus dilution to all wells, the cultured plates were incubated for 2 h at 37°C under 5% CO₂ incubator. The well plates were lined with RAW macrophage cell line as previously described, and then discard the medium covering the adhered living RAW macrophage. Wash the plate with serum-free medium and transfer 150 µL of each virus dilution to corresponding wells in the macrophage cell plate and then add 100 µL of culture medium to the last column as a cell control. Allow the virus to grow inside the RAW macrophages for 1–2 h at 37°C under 5% CO₂ incubation. Add 200 µL of growth medium to each well and incubate at 37°C under 5% CO₂ incubation. Observe the morphology of the RAW macrophage cells microscopically to determine their survival.

Anti-HCV evaluations for genotype 1b (luciferase and MTS assays)

One day before addition of compounds, Huh 5.2 cells, containing the hepatitis C virus genotype 1b (I389luc-ubi-neo/NS3-3’/5.1 replicon) was incubated. A replicon based bioassay for the measurement of interferons in patients with chronic hepatitis C and sub-cultured in cell growth medium (DMEM, [Cat. N°41965039] supplemented with 10% FCS, 1% nonessential amino acids [11140035], 1% penicillin/streptomycin [15140148] and 2% Geneticin [10131027]; Invitrogen) at a ratio of 1:3–1:4 and grown for 3–4 days in 75 cm² tissue culture flasks (Techno Plastic Products), were harvested and seeded in assay medium (DMEM, 10% FCS, 1% nonessential amino acids, 1% penicillin/streptomycin) at a density of 6500 cells/well (100 µL/well) in the well tissue culture microtiter plates (Falcon, Beckton Dickinson for evaluation of anti-metabolic effect and Cultur-Plate, Perkin-Elmer for evaluation of antiviral effect). The microtiter plates were incubated overnight (37°C, 5% CO₂, 95–99% relative humidity), yielding a nonconfluent cell monolayer.

The evaluation of the cytotoxicity as well as the antiviral effect of each compound was performed in parallel. Four-steps, 1–5 compound dilution series were prepared. Following assay setup, the microtiter plates are incubated for 72 h (37°C, 5% CO₂, 95–99% relative humidity).

For the evaluation of cytotoxicity effects, the assay medium is aspirated, replaced with 75 µL of a 5% MTS (Promega) solution in phenol red-free medium and incubated for 1.5 h (37°C, 5% CO₂, 95–99% relative humidity). Absorbance is measured at a wavelength of 498 nm (Safire, Tecan) and optical densities (OD values) are converted to percentage of untreated controls.

For the evaluation of antiviral effects, assay medium is aspirated and the cell monolayers are washed with PBS. The wash buffer is aspirated, 25 µL of Glo Lysis Buffer (Cat. N°. E2661, Promega) is added after which lysis is allowed to proceed for 5 min at room temperature. Subsequently, 50 µL of Luciferase Assay System (Cat. N° E1501, Promega) is added and the

Luciferase luminescence signal is quantified immediately (1000 ms integration time/well, Safir, Tecan). Relative luminescence units are converted to percentage of untreated controls.

Final conclusion for anti-HCV for genotypes 1b and 4a

In this investigation, the in vitro anti-HCV activity testing for genotype 4a-RAW macrophage cell lines, for the tested compounds revealed that compounds 4-(3-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylaminophenyl]-2-aryl-4-oxo-butyric acid (Xa) and 4-(4-[N-1-carboxy-3-(3,5-dibromo-4-hydroxyphenyl)-3-oxo-propylaminophenyl]-2-aryl-4-oxo-butyric acid (Xb) that have the 2-amino-4-oxo-buteric acid peptidomimetic linkers), exerted 95 and 80% viral inhibition in comparison to the Indoline reference drug. Thus, these compounds (Xa and Xb) would be reported here, as the first time, as prototypes for defeating genotype 4a of HCV infections. Such results were consistent with their docking scores at the 3D structure of binding site of the HCV NS3 protease enzyme that is conserved in both genotypes 1b and 4a [21]. In the other hand, compound 4-(2-[N-3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-propylamino]acetamido)benzoate ethyl ester (XVa), 4-(2-methyl-2-[N-3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-propylamino]acetamido)benzoate ethyl ester (XVb) and 4-(2-isobutyl-2-[N-3-(3,5-dibromo-4-hydroxyphenyl)-1-carboxy-3-oxo-propylamino]acetamido)-benzoate ethyl ester (XVd), that have the 2-amino-oxoethylamino-4-oxo-buteric acid peptidomimetic linkers, gave high antiviral activity against genotype 1b, with CC₅₀ values more than 50 µg/mL, low EC₅₀ doses equal 5.43, 14.80 and 14.70 µg/mL, and with high % viral inhibitions equal 87.7, 84.3 and 82.8%, respectively. Thus, these molecules would be considered as new prototypes for treating HCV of genotype 1b.

It is recommended; in the future that further optimizations for compounds Xa and Xb should be performed to develop more effective and selective anti-HCV agents for genotype 4a. Also, manipulations of compounds XVa, XVb and XVd should be followed up to develop new selective anti-HCV inhibitors for genotype 1a.

Acknowledgement

The authors are deeply acknowledging Bahgat M., M., professor of microbiology department of NCR of Egypt, for performing anti-HCV evaluation of genotype 4a. Also, it’s an honor to appreciate Dr. Jeffrey S. Glenn, Associate Professor of Medicine, Division of Gastroenterology and Hepatology, Director, Center for Hepatitis and Liver Tissue Engineering, Stanford University, School of Medicine, Stanford, USA, for her kind collaboration in performing anti-HCV testing of genotype 1b.

Declaration of interest

All the authors have no interest and no personal relationships or financial with any peoples or organizations that could inappropriately influence (bias) their work directly or indirectly, during the subject matter or materials discussed in this manuscript.