Discovery of novel HCV inhibitors: Design, Synthesis and biological activity of phthalamide derivatives

doi:10.21203/rs.3.rs-1669420/v1

PDF

Research Article

Discovery of novel HCV inhibitors: Design, Synthesis and biological activity of phthalamide derivatives

https://doi.org/10.21203/rs.3.rs-1669420/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases like cirrhosis and hepatocarcinoma, posing a serious worldwide threat when left untreated. Nowadays, direct-acting antivirals (DAAs) constitute core components of anti-HCV treatment. Nonetheless, some DAAs are associated with a growing level of drug resistance as well as adverse reactions. That is why introducing new anti-HCV drugs with higher potency and lower toxicity is so essential. NS5B polymerase is an HCV non-structural protein that acts as a critical target for the development of anti-HCV therapeutics. Based on the necessary pharmacophores for inhibition of HCV NS5B polymerase, a novel series of phthalamide analogs that harbor the potential of NS5B polymerase inhibition to stop HCV proliferation in a cell-based assay were designed, synthesized, and in-vitro evaluated. Interestingly, all compounds displayed low cellular cytotoxicity in Huh 7.5 cells (CC 50 > 100). Compound 28 with EC 50 of 6.0 µM, and an appropriate affinity to NS5B polymerase active site could be considered as a new hit compound, providing useful information for the design of novel HCV inhibitors.

HCV inhibitors

Docking

Synthesis

phthalamide

NS5B polymerase

Hepatitis C caused by the hepatitis C virus (HCV) is considered globally one of the most severe liver diseases that is a leading cause for hepatocellular carcinoma (HCC)^{1, 2}. Based on the reports from the World Health Organization (WHO), nearly a total of 71 million patients is now chronically infected with HCV³. Before 2011, the standard of care (SOC) in antiviral therapy for HCV infection was mainly a combination of subcutaneous pegylated interferon-alpha (pegIFN-α) and oral ribavirin which sometimes may cause patients to experience serious adverse reactions^{4, 5}. Moreover, no vaccine is yet available for the prevention of HCV⁶. Therefore, there is an unmet clinical need to develop novel chemotherapy against HCV with better efficacy and tolerability. With the discovery of additional direct-acting antiviral agents (DAAs), therapy without using pegIFN-α became possible for curing all HCV genotypes while prominently improving unwanted side effects⁷. DAAs specifically target essential proteins in the HCV life cycle, including NS5B RNA dependent RNA polymerase (RdRp) that plays an essential role in the life cycle of the virus^{8, 9}. NS5B is a desirable target for designing new drugs because there is no known NS5B counterpart in the human body¹⁰. In recent years, a diverse range of small NS5B RdRp molecules have been developed which are classified into the nucleoside inhibitors (NIs), targeting enzyme active site, as well as non-nucleoside inhibitors (NNIs), binding with the allosteric sites known as thumb I, thumb II, palm I, palm II and palm β¹¹. At the top of the palm subdomain, the active site of NS5B consists of two divalent ions Mg²⁺ or Mn²⁺ that are coordinated with three conserved aminoacids Asp318, Asp319, and Asp220¹². The metal ions play a pivotal role in the catalysis of the phosphodiester bond formation between sequential nucleotides¹³. Therefore, compounds bearing metal coordinating moieties could be utilized for the NS5B inhibition¹⁴. In general, an aromatic side group is usually connected to the metal-chelating scaffold in the structure of NNIs NS5B inhibitors¹⁰. Following the initial optimization of aminothiazole 5-carboxylic acid scaffold as NS5B inhibitor, a novel series of 4H-pyrazolo[1,5-α]pyrimidin-7-one analogs were developed by Deng et al¹⁵. In this series, the most active compound A showed an IC₅₀of 0.05 µM, against HCV genotypes 1-3 along with single digit micromolar inhibition in the HCV replicon assay. Moreover, in 2013, Venkatraman et al. introduced a novel series of tricyclic indole derivatives as NS5B inhibitors, in which "6H-furo[2,3-e]indole-7-carboxylic acid" B was identified as the most active molecule with promising cellular activity (EC₅₀ = 160 nM) and an IC₅₀ value of 0.031 µM¹⁶. Compounds A and B contain two neighboring carbonyl groups that were found to inhibit HCV NS5B¹⁷ (Fig. 1).

This structural feature was used in our study to introduce phthalamide derivatives as novel anti-HCV agents targeting NS5B activity. Docking studies demonstrated that these structures had high levels of binding affinity for the NS5B active site. Based on that, the phthalamide scaffold is considered as a core structure substituted with a large number of structurally diverse groups. Since most of the NS5B inhibitors possess two hydrophobic moieties, we put two aralkyl fragments on amide NHs (Fig.1) and the effect of various substituted aralkyl moieties on HCV inhibition, using a Japonicum Fulminant Hepatitis virus serotype 1 (JFH1)-derived infectious clone was investigated. The phthalamide scaffold was hypothesized to be responsible for insertion into the active site followed by the metal coordination and an aralkyl fragment probably involved in an interaction with hydrophobic aminoacids of the active site.

Chemistry

Target compounds 3-28 were achieved in moderate to high yields (54-99 %) by using a convenient two-step procedure starting from phthalic anhydride 1. As illustrated in Scheme 1, the procedure began with the condensation of phthalic anhydride, with amine derivatives, in ethyl acetate to generate the key intermediates 2a-2l. The final products 3-28 were obtained upon the reaction with trifluoroacetic anhydride, followed by the reaction with differently-substituted primary amines in THF at room temperature. The structural characterization of the newly-synthesized analogs was confirmed using elemental analysis and IR, ¹H-NMR, ¹³CNMR, and ESI-MS spectroscopies. Based on the ¹H-NMR spectrum, characteristic amide NH signals together with the multiple signals of aromatic ring hydrogens, and the molecular ion signal in the ESI-MS spectrum proved the structure of the synthesized compounds.

HCV replicon assay

Until recent years, it was not possible to detect a whole cycle of HCV replication in cell culture. To study more detailed aspects of HCV reproduction, subgenomic viral RNAs (replicates) able to replicate themselves in human liver cells were developed¹⁸. In the present study, target compounds 3-28 were evaluated in a cell-based viral replication surrogate assay known as the replicon system¹⁹. The FDA-approved nucleotide polymerase inhibitor, sofosbuvir, was used as a control compound. At the same time, the cell viability was identified using an XTT-based colorimetric assay to ensure that the activity of the molecule is entirely independent of cytotoxic effects. The test also included replicon cells receiving vehicle alone (DMSO) and cell culture medium. EC₅₀ and CC₅₀ were determined by at least three experiments (Table 1). As clearly shown in Table 1, all phthalamide derivatives demonstrated low cytotoxicity (CC₅₀ > 100 μM) in Huh-7.5 cells, rendering them viable inhibitors for HCV. Furthermore, most of the target compounds were found active against HCV with the EC₅₀ values in the range from 6 to 230 µM. Interestingly, when the N-phenyl is replaced with 2-hydroxyindanyl, the compound 28 becomes a promising HCV inhibitor with an EC₅₀ of 6 μM and a decent selectivity ratio, serving as a valuable handle for further structure-activity relationship (SAR) explorations. On the other hand, the substitution of the N-aralkyl moieties with halogens improved antiviral activity as well as cytotoxicity profile. This is exemplified by compounds 4, 6, 15, 16, and 27, with a fluorine or chlorine substitution at N-phenyl or N-benzyl rings confers relatively acceptable inhibitory activity (11-19 μM). The anti-HCV assay results revealed a few discernible SAR trends. First of all, the substitution of N-aralkyl was found to enhance anti-HCV activity as compounds 3 and 26 with unsubstituted benzyl and phenyl, were shown to be approximately inactive (EC₅₀ > 100 µM). In contrast, most of the analogs with substituted N-aralkyl groups indicated considerable potency. Also, the properties of substituent appeared crucial for inhibitory activity. For instance, the introduction of a hydrophilic substituent like hydroxyl on the appropriate position of the aralkyl moiety was found to significantly influence anti-HCV activity as compound 28 demonstrated the most anti-HCV activity. Among other molecules, anti-HCV activity increased in the following order: F > Cl > Br > Me > OMe, proposing that substitution with electron-donating groups leads to less active HCV inhibitors. Additionally, it was found that N-substitution with the linear alkyl groups like n-propyl and n-pentyl have a detrimental effect on anti-HCV activity, which may be due to the loss of π interactions between the ligand and the active site. Notably, compound 18 with cyclohexyl was proven to be considerably less potent (EC₅₀ > 200 µM) than its aromatic-counterpart 26 (EC₅₀ = 110 μM). In monochlorinated analogs 15 and 27, anti-HCV activity was not affected by the position of substituent on the phenyl ring while compound 15 with Cl in meta-position was proven to be less cytotoxic (EC₅₀= 18 µM, SI = 18.33) than its para-counterpart 27 (EC₅₀ = 19 µM, SI = 15.53). In compounds 8 and 9 with dimethyl phenyl substitution, changing from 2,3 to 2,6 position increased both the activity (EC₅₀= 63 and 55 µM, respectively) and cytotoxicity (CC₅₀ = 430 and 235 µM, respectively), indicating that the methylation of 6-position is better tolerated by the enzyme active site. As expected, compounds 4 and 6 possessing, 4-fluorobenzyl and 4-fluorophenyl respectively displayed similar activities with an EC₅₀value of 11 µM. Due to this, a fundamental change in binding mode to the enzyme or the physicochemical properties can be reflected in the differences in activity and cytotoxicity between substituents and positions, but further research is required to establish detailed structure-activity relationships. In summary, the biological assay of anti-HCV activity showed the phthalamide scaffold could be considered as a promising core to design potent HCV inhibitors.

Molecular Modeling Studies

The compounds were designed based on the pharmacophore model of NS5B active site inhibitors. Our inhibitors contain a chelating moiety capable of binding to both metal ions at the active site, thus could represent a type of metal-binding NNIs. A five-metal model for NS5B in complex with uridine triphosphate (UTP) (PDB code: 1GX6) as a surrogate for the NS5B/viral genome complex, was used in our docking study to explain the molecular recognition interactions. The binding modes of newly-designed compounds within the HCV RdRp catalytic site at the palm domain were computed. According to the docking results, the amide groups of compounds interacted with two Mn²⁺ that are coordinated by active site residues Asp220, Asp318, and Asp319 (Fig. 2). Compound 28 was found to fit in the active site perfectly, chelating Mn²⁺ions of active site through the carbonyl and hydroxyl groups of phthalamide scaffold with the distances of 2.46 and 2.93 Å (Fig. 2). The aralkyl moiety of compound 28 was inserted into a hydrophobic pocket in the enzyme consisting of Leu159 and Ile160, which are essential residues in interaction with the co-crystallized UTP. Besides, the other amide group of phthalamide scaffold formed interactions with the side chain of Arg48 and Arg158, the residues that were observed to form crucial salt bridge interactions with the terminal phosphate of UTP in the original X-ray crystallographic structure. A special hydrophobic bond was also found between the indanol group of compound 28 and Cys366. These observations correlate positively with the observed inhibitory activity in HCV replicon assay and NS5B could consider as the enzyme target of our inhibitors.

In summary, a novel series of phthalamide analogs were synthesized and evaluated against HCV replication in Huh 7.5 cells. All the newly-synthesized compounds showed reasonable inhibition of the virus multiplication. The results of the investigation demonstrated that compound 28 bearing the indanol moiety possesses the highest potency as anti-HCV with an EC₅₀ of 6 µM and a selectivity ratio of 79.33, which renders this molecule a valuable lead scaffold for the development of more potent anti-HCV agents. More in vitro enzyme-specific studies are needed to elucidate the fundamental molecular details of HCV inhibition by these molecules, especially concerning the inhibition of NS5B activity.

Chemistry

All laboratory-grade chemical reagents and solvents used in this study were obtained commercially from Merck AG or Aldrich Chemical. The reactions were monitored by thin-layer chromatography (TLC) performed on commercially available Merck pre-coated plates (silica gel 60 F254, 0.25 mm), and spots were visualized with UV light. Melting points were determined with an Electrothermal 9100 apparatus and were not corrected. The structures of the newly-synthesized analogs were confirmed by IR, LC-MS, ¹HNMR, and ¹³CNMR spectroscopy methods as well as CHN elemental analysis. Perkin Elmer Model 1420 spectrophotometer, Agilent 6410 triple quadrupole mass spectrometer (LC-MS) with electrospray ionization (ESI) interface, PerkinElmer 843 IR and Costech elemental analyzer (Costech, Italy) were used to obtain Mass spectra, IR, and CHN analysis, respectively. The¹HNMR and ¹³CNMR spectra were generally acquired in d₆-DMSO solutions on a Bruker spectrometer (Bruker Biosciences, USA), operating at 400 MHz for ¹HNMR and 100 MHz for ¹³CNMR. Chemical shifts are given in ppm relative to the TMS solvent signal. Coupling constant (J) values are estimated in hertz (Hz), and spin multiples are given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad).

Procedure for the synthesis of Intermediates 2a-2l

To a mixture of phthalic anhydride in ethyl acetate (1.0 g, 6.8 mmol), various substituted amine derivatives (6.8 mmol) were added. The reaction was stirred at room temperature for 18 hours. After completion of the reaction, an equivalent volume of n-hexane was added to the mixture, and the precipitated material was filtered off. The residue was washed up by the use of n-hexane, diluted aqueous HCl solution, and distilled water to afford the target intermediates 2a-2l.

2-(benzylcarbamoyl) benzoic acid (2a)

Yield: 97%; MP: 108-111 ºC; IR (KBr): 3384 (NH), 2415-3457 (OH), 1677, 1722 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 254.1 (M-H⁺); Anal. Calcd for C₁₅H₁₃NO₃: C, 70.58; H, 5.13; N, 5.49. Found: C, 70.78; H, 5.15; N, 5.47.

2-((4-hydroxyphenyl) carbamoyl) benzoic acid (2b)

Yield: 75%; MP: 135-138 ºC; IR (KBr): 3331 (NH), 2357-3543 (OH), 1677, 1734 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 256.2 (M-H⁺); Anal. Calcd for C₁₄H₁₁NO₄: C, 65.37; H, 4.31; N, 5.45. Found: C, 65.32; H, 4.33; N, 5.48.

2-((4-bromophenyl) carbamoyl) benzoic acid (2c)

Yield: 98%; MP: 145-146 ºC; IR (KBr): 3377 (NH), 2708-3708 (OH), 1671, 1743 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 318.1 (M-H⁺), 320.1 (M+2-H⁺); Anal. Calcd for C₁₄H₁₀BrNO₃: C, 52.52; H, 3.15; N, 4.38. Found: C, 52.49; H, 3.14; N, 4.40.

2-(cyclohexylcarbamoyl) benzoic acid (2d)

Yield: 99%; MP: 154-156 ºC; IR (KBr): 3273 (NH), 2663-3390 (OH), 1738 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 246.2 (M-H⁺); Anal. Calcd for C₁₄H₁₇NO₃: C, 68.00; H, 6.93; N, 5.66. Found: C, 67.95; H, 6.95; N, 5.68.

2-((5,6,7,8-tetrahydronaphthalen-1-yl) carbamoyl) benzoic acid (2e)

Yield: 99%; MP: 165-167 ºC; IR (KBr): 3310 (NH), 2705-3438 (OH), 1664, 1690 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 294.2 (M-H⁺); Anal. Calcd for C₁₈H₁₇NO₃: C, 73.20; H, 5.80; N, 4.74. Found: C, 73.29; H, 5.77; N, 4.73.

2-((4-fluorophenyl) carbamoyl) benzoic acid (2f)

Yield: 78%; MP: 129-130 ºC; IR (KBr): 3317 (NH), 2703-3433 (OH), 1651, 1721 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 258.2 (M-H⁺); Anal. Calcd for C₁₄H₁₀FNO₃: C, 64.87; H, 3.89; N, 5.40. Found: C, 64.81; H, 3.88; N, 5.42.

2-((4-fluorobenzyl) carbamoyl) benzoic acid (2g)

Yield: 73%; MP: 133-134 ºC; IR (KBr): 3311 (NH), 2500-3422 (OH), 1647, 1692 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 272.2 (M-H⁺); Anal. Calcd for C₁₅H₁₂FNO₃: C, 65.93; H, 4.43; N, 5.13. Found: C, 65.86; H, 4.42; N, 5.15.

2-((2, 6-dimethylphenyl) carbamoyl) benzoic acid (2h)

Yield: 97%; MP: 136-138 ºC; IR (KBr): 3233 (NH), 2398-3363 (OH), 1642, 1703 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 268.2 (M-H⁺); Anal. Calcd for C₁₆H₁₅NO₃: C, 71.36; H, 5.61; N, 5.20. Found: C, 71.25; H, 5.63; N, 5.23.

2-((2, 3-dimethylphenyl) carbamoyl) benzoic acid (2i)

Yield: 99%; MP: 135-137 ºC; IR (KBr): 3232 (NH), 2709-3413 (OH), 1632, 1696 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 268.2 (M-H⁺); Anal. Calcd for C₁₆H₁₅NO₃: C, 71.36; H, 5.61; N, 5.20; Found: C, 71.30; H, 5.59; N, 5.22.

2-((furan-2-ylmethyl) carbamoyl) benzoic acid (2j)

Yield: 86%; MP: 125-126 ºC; IR (KBr): 3337 (NH), 2468-3462 (OH), 1651, 1693 (C=O), 1450 - 1600 (aromatic C-H); LC-MS (ESI) m/z 244.2 (M-H⁺); Anal. Calcd for C₁₃H₁₁NO₄: C, 63.67; H, 4.52; N, 5.71; Found: C, 63.57; H, 4.50; N, 5.73.

2-((3-chlorophenyl) carbamoyl) benzoic acid (2k)

Yield: 86%; MP: 122-123 ºC; IR (KBr): 3343 (NH), 2553-3435 (OH), 1681, 1736 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 274.1 (M-H⁺); Anal. Calcd for C₁₄H₁₀ClNO₃: C, 60.99; H, 3.66; N, 5.08. Found: C, 60.88; H, 3.66; N, 5.10.

2-((4-(methoxycarbonyl) phenyl) carbamoyl) benzoic acid (2l)

Yield: 86%; MP: 115-116 °C; IR (KBr): 3032 (NH), 2393-3106 (OH), 1695 (C=O), 1450-1600 (aromatic C-H); LC-MS (ESI) m/z 298.2 (M-H⁺); Anal. Calcd for C₁₆H₁₃NO₅: C, 64.21; H, 4.38; N, 4.68; Found: C, 64.29; H, 4.37; N, 4.69.

Procedure for the synthesis of compounds 3-28

The intermediates 2a-2l (1 mmol) were dissolved in super-dried THF (5 ml), and the solution was cooled to 0 °C. After that, equimolar amounts of trifluoroacetic anhydride (TFAA) and triethylamine (TEA) were added to the mixture. After 30 minutes of stirring at 0 °C, amine derivatives were added as well. The resulting mixture was allowed to warm to room temperature and stirred for 20 h until the reaction has been completed. Distilled water (5 ml) and ice were added to the mixture, and the obtained precipitate was filtered off. The solid residue was washed up by distilled water and recrystallized from ethanol 96 to afford the final compounds 3-28.

N¹, N²- dibenzyl phthalamide (3)

Yield: 95%; MP: 174-175 ºC; IR (KBr): 3249, 3309 (NH), 1643 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.48 (s, 4H, CH₂-N), 7.28-7.31 (t, 2H, N-benzyl (p), J = 8 Hz), 7.36-7.44 (m, 8H, N-benzyl (o, m)), 7.54-7.60 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.90 (s, 2H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 42.90, 127.14, 127.35, 127.65, 128.14, 128.69, 129.87, 136.82, 139.97, 168.68; LC-MS (ESI) m/z 343.2 (M-H⁺); Anal. Calcd for C₂₂H₂₀N₂O₂: C, 76.72; H, 5.85; N, 8.13. Found: C, 76.65; H, 5.84; N, 8.17.

N¹-(4-fluorobenzyl)-N²-benzylphthalamide (4)

Yield: 95%; MP: 175-177 ºC; IR (KBr): 3257 (NH), 1633 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.39-4.43 (m, 4H, CH₂-N), 7.11-7.16 (t, 2H, N-benzyl (m), J = 8 Hz), 7.24-7.28 (t, 1H, N-benzyl (p), J = 8 Hz), 7.30-7.43 (m, 6H, 4-fluorobenzyl H₂/H₃/H₅/H₆ & N-benzyl (o)), 7.51-7.53 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.83-8.86 (m, 2H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 42.22, 42.90, 127.63, 128.11, 128.68, 129.57, 129.65, 129.88, 130.27, 130.65, 130.74, 136.11, 136.14, 136.25, 136.76, 136.82, 168.64, 168.73; LC-MS (ESI) m/z 361.2 (M-H⁺); Anal. Calcd for C₂₂H₁₉FN₂O₂: C, 72.91; H, 5.28; N, 7.73. Found: C, 72.85; H, 5.29; N, 7.76.

N¹-benzyl-N²-pentylphthalamide (5)

Yield: 65%; MP: 173-174 ºC; IR (KBr): 3245 (NH), 1629, 1653 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 0.86-0.89 (t, 3H, CH₃, J = 8 Hz), 1.27-1.30 (m, 4H, -CH₂-CH₂-), 1.43-1.48 (q, 2H, CH₂-N, J = 8, 4 Hz), 3.13-3.18 (q, 2H, CH₂-N, J = 8, 4 Hz), 4.42-4.44 (d, 2H, CH₂-N, J = 8 Hz), 7.23-7.26 (t, 1H, N-benzyl (p), J = 8 Hz), 7.31-7.38 (m, 4H, N-benzyl (o, m)), 7.44-7.50 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.23-8.26 (t, 1H, NH, J = 8 Hz), 8.78-8.81 (t, 1H, NH, J = 8 Hz); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 14.39, 22.36, 29.08, 29.22, 119.92, 123.73, 127.96, 128.17, 129.02, 129.89, 129.92, 136.72, 137.37, 140.00, 167.59, 168.07; LC-MS (ESI) m/z 323.2 (M-H⁺); Anal. Calcd for C₂₀H₂₄N₂O₂: C, 74.05; H, 7.46; N, 8.63. Found: C, 74.11; H, 7.44; N, 8.66.

N¹-benzyl-N²-(4-fluorophenyl) phthalamide (6)

Yield: 75%; MP: 164 ºC; IR (KBr): 3230, 3250 (NH), 1638, 1652 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.42-4.43 (d, 2H, CH₂-N, J = 4 Hz), 7.28-7.43 (m, 7H, N-benzyl (o, m, p) & 4-fluorophenyl H₂/H₆), 7.53-7.63 (m, 4H, phenylene H₃/H₄& 4-fluorophenyl H₃/H₅),7.71-7.74 (m, 2H, phenylene H₂/H₅), 8.93-8.96 (t, 1H, NH, J = 8 Hz), 10.41 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 42.79, 127.11, 127.57, 127.65, 128.07, 128.20, 128.63, 128.99, 129.13, 129.31, 129.98, 130.18, 136.30, 136.41, 136.43, 167.55, 168.18; LC-MS (ESI) m/z 347.2 (M-H⁺); Anal. Calcd for C₂₁H₁₇FN₂O₂: C, 72.40; H, 4.92; N, 8.04. Found: C, 72.55; H, 4.90; N, 8.00.

N¹-benzyl-N²-(4-bromophenyl) phthalamide (7)

Yield: 95%; MP: 221-223 ºC; IR (KBr): 3293 (NH), 1658 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.42-4.44 (d, 2H, CH₂-N, J = 8 Hz), 7.21-7.25 (t, 1H, N- benzyl (p), J = 8 Hz), 7.28-7.36 (m, 4H, N-benzyl (o, m)), 7.51-7.70 (m, 8H, 4-bromophenyl H₂/H₃/H₅/H₆ & phenylene H₂/H₃/H₅/H₆), 8.95-8.98 (t, 1H, NH, J = 8 Hz), 10.50 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 42.93, 122.03, 127.13, 127.57, 128.07, 128.22, 128.65, 130.07, 130.25, 130.43, 131.89, 136.24, 137.44, 139.19, 139.40, 167.82, 168.09; LC-MS (ESI) m/z 407.2 (M-H⁺), 409 (M+2-H⁺); Anal. Calcd for C₂₁H₁₇BrN₂O₂: C, 61.63; H, 4.19; N, 6.84. Found: C, 61.58; H, 4.21; N, 6.86.

N¹-benzyl-N²-(2, 6-dimethylphenyl) phthalamide (8)

Yield: 99%; MP: 194-195 ºC; IR (KBr): 3277 (NH), 1648, 1671 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 2.27 (s, 6H, CH₃), 4.43-4.45 (d, 2H, CH₂-N, J = 8 Hz), 7.07-7.12 (m, 3H, 2,6-dimethylbenzyl H₃/H₄/H₅), 7.21-7.24 (t, 1H, N-benzyl (p), J = 8 Hz),7.28-7.32 (t, 2H, N-benzyl (m), J = 8 Hz), 7.36-7.38 (d, 2H, N-benzyl (o), J = 8 Hz), 7.53-7.60 (m, 3H, phenylene H₃/H₄/H₅), 7.69-7.71 (d, 1H, phenylene H₂, J = 8 Hz), 8.90-8.93 (t, 1H, NH, J = 8 Hz), 9.73 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 18.73, 42.87, 126.99, 127.12, 127.60, 128.10, 128.20, 128.25, 128.66, 128.91, 129.06, 129.41, 129.93, 129.98, 134.56, 135.58, 167.25, 168.56; LC-MS (ESI) m/z 357.3 (M-H⁺); Anal. Calcd for C₂₃H₂₂N₂O₂: C, 77.07; H, 6.19; N, 7.82. Found: C, 77.14; H, 6.21; N, 7.79.

N¹-benzyl-N²-(2, 3-dimethylphenyl) phthalamide (9)

Yield: 98%; MP: 193 ºC; IR (KBr): 3285, 3309 (NH), 1638, 1665 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 2.12 (s, 3H, CH₃), 2.24 (s, 3H, CH₃), 4.42-4.43 (d, 2H, CH₂-N, J = 4 Hz), 7.01-7.64 (m, 12H, 2,3-dimethylphenyl H₄/H₅/H₆ & N-benzyl (o, m, p) & phenylene H₂/H₃/H₄/H₅), 8.88-8.91 (t, 1H, NH, J = 8 Hz), 9.78 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 14.56, 20.65, 42.74, 124.64, 125.53, 127.13, 127.59, 127.63, 128.09, 128.31, 128.65, 129.11, 129.34, 129.87, 130.07, 132.54, 136.52, 136.66, 137.29, 167.71, 168.48; LC-MS (ESI) m/z 357.3 (M-H⁺); Anal. Calcd for C₂₃H₂₂N₂O₂: C, 77.07; H, 6.19; N, 7.82. Found: C, 77.21; H, 6.18; N, 7.80.

N¹-benzyl-N²-((furan-2-yl) methyl) phthalamide (10)

Yield: 97%; MP: 175-176 ºC; IR (KBr): 3278 (NH), 1644 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.37-4.42 (m, 4H, CH₂-N), 6.34-6.39 (m, 2H, furfuryl H₃/H₅), 7.21-7.24 (t, 1H, N-benzyl (p), J = 8 Hz), 7.29-7.37 (m, 4H, N-benzyl (o, m)),7.47-7.56 (m, 5H, furfuryl H₄ & phenylene H₂/H₃/H₄/H₅), 8.76-8.82 (m, 2H, NHs); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 36.64, 42.94, 107.16, 111.02, 127.16, 127.67, 128.14, 128.24, 128.71, 129.35, 129.87, 129.96, 136.53, 136.80, 139.96, 142.40, 168.56, 168.65; LC-MS (ESI) m/z 333.2 (M-H⁺); Anal. Calcd for C₂₀H₁₈N₂O₃: C, 71.84; H, 5.43; N, 8.38. Found: C, 71.78; H, 5.42; N, 8.40.

N¹-benzyl-N²-(2-(hydroxymethyl) phenyl) phthalamide (11)

Yield: 68%; MP: 155-157 ºC; IR (KBr): 3350 (NH), 3050-3400 (OH), 1678 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.45-4.46 (d, 2H, CH₂-N, J = 4 Hz), 4.61-4.62 (d, 2H, CH₂-O, J = 4 Hz), 5.36-5.39 (t, 1H, OH, J = 8 Hz), 7.17-7.25 (m, 2H, N-benzyl (p) & 2-aminobenzylalcohol H₆), 7.28-7.31 (m, 3H, N-benzyl (m) & 2- aminobenzylalcohol H₄), 7.36-7.38 (d, 2H, N-benzyl (o), J = 8 Hz), 7.41-7.43 (d, 1H, 2-aminobenzylalcohol H₅, J = 8 Hz), 7.57-7.63 (m, 3H, phenylene H₃/H₄& 2-aminobenzylalcohol H₃), 7.66-7.69 (m, 2H, phenylene H₂/H₅), 8.96-8.99 (t, 1H, NH, J = 8 Hz), 9.80 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 42.94, 60.96, 124.50, 125.25, 127.14, 127.46, 127.62, 127.79, 128.01, 128.23, 128.66, 129.86, 130.15, 130.25, 135.35, 136.22, 136.54, 137.26, 167.41, 168.36; LC-MS (ESI) m/z 359 (M-H⁺); Anal. Calcd for C₂₂H₂₀N₂O₃: C, 73.32; H, 5.59; N, 7.77; Found: C, 73.44; H, 5.61; N, 7.74.

N¹-benzyl-N²-(1, 2, 3, 4-tetrahydronaphthalen-8-yl) phthalamide (12)

Yield: 99%; MP: 220-222 ºC; IR (KBr): 3263, 3294 (NHs), 1628, 1657 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 1.69 (m, 4H, CH₂-CH₂), 2.69-2.74 (m, 4H, 2 CH₂), 4.44-4.45 (d, 2H, CH₂-N, J = 4 Hz), 6.93-6.95 (d, 1H, tetrahydronaphthylamine H₄, J = 8 Hz), 7.07-7.10 (t, 1H, tetrahydronaphthylamine H₃, J = 8 Hz), 7.21-7.29 (m, 4H, N- benzyl (m, p) & tetrahydronaphthylamine H₂), 7.34-7.36 (d, 2H, N-benzyl (o), J = 8 Hz), 7.54-7.65 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.90-8.93 (t, 1H, NH, J = 8 Hz), 9.60 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 22.86, 22.95, 24.62, 29.71, 42.94, 123.85, 125.40, 126.80, 127.13, 127.60, 128.09, 128.38, 128.65, 129.90, 130.08, 132.30, 136.45, 137.39, 137.78, 139.88, 167.61, 168.49; LC-MS (ESI) m/z 383.2 (M-H⁺); Anal. Calcd for C₂₅H₂₄N₂O₂: C, 78.10; H, 6.29; N, 7.29. Found: C, 78.33; H, 6.28; N, 7.25.

Methyl 4-(2-(benzylcarbamoyl) benzamido) benzoate (13)

Yield: 70%; MP: 170-171 ºC; IR (KBr): 3033, 3290 (NHs), 1630, 1657 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 3.83 (s, 3H, OCH₃), 4.43-4.44 (d, 2H, CH₂-N, J = 4 Hz), 7.20-7.47 (m, 5H, N-benzyl (o, m, p)), 7.55-7.67 (m, 4H, phenylene H₂/H₃/H₄/H₅), 7.86-7.88 (d, 2H, methyl benzoate H₂/H_6,J = 8 Hz), 7.94-7.96 (d, 2H, methyl benzoate H₃/H₅, J = 8 Hz), 9.02-9.05 (t, 1H, NH, J = 8 Hz), 10.75 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 42.97, 52.35, 128.07, 128.26, 128.64, 128.92, 129.08, 129.32, 129.85, 130.14, 130.30, 130.63, 134.54, 135.05, 136.15, 137.39, 166.37, 168.00, 168.24; LC-MS (ESI) m/z 387.2 (M-H⁺); Anal. Calcd for C₂₃H₂₀N₂O₄: C, 71.12; H, 5.19; N, 7.21. Found: C, 71.39; H, 5.17; N, 7.23.

N¹-benzyl-N²-propylphthalamide (14)

Yield: 91%; MP: 160 ºC; IR (KBr): 3150-3335 (NHs), 1623 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 0.87-0.90 (t, 3H, CH₃, J = 8 Hz), 1.44-1.50 (q, 2H, CH₂, J = 8 Hz), 3.10-3.15 (q, 2H, CH₂-N, J = 8 Hz), 4.41-4.43 (d, 2H, CH₂-N, J = 8 Hz), 7.22-7.26 (t, 1H, N-benzyl (p), J = 8 Hz), 7.31-7.38 (m, 4H, N-benzyl (o, m)), 7.43-7.51 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.24-8.27 (t, 1H, NH, J = 8 Hz), 8.77-8.80 (t, 1H, NH, J = 8 Hz); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 11.95, 22.73, 41.33, 42.89, 127.13, 127.63, 128.11, 128.66, 129.66, 129.82, 136.53, 137.12, 139.95, 168.57, 168.66; LC-MS (ESI) m/z 295.0 (M-H⁺); Anal. Calcd for C₁₈H₂₀N₂O₂: C, 72.95; H, 6.80; N, 9.45. Found: C, 72.67; H, 6.76; N, 9.40.

N¹-benzyl-N²-(3-chlorophenyl) phthalamide (15)

Yield: 89%; MP: 172-173 ºC; IR (KBr): 3287, 3322 (NHs), 1658, 1679 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.42-4.43 (d, 2H, CH₂-N, J = 4 Hz), 7.11-7.37 (m, 7H, N-benzyl (o, m, p) & 3-chlorophenyl H₅/H₆), 7.55-7.65 (m, 5H, 3-chlorophenyl H₄ & phenylene H₂/H₃/H₄/H₅), 7.93 (s, 1H, 3-chlorophenyl H₂), 8.95-8.98 (t, 1H, NH, J = 8 Hz), 10.54 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 42.96, 118.33, 119.39, 123.44, 127.14, 127.58, 128.08, 128.63, 130.13, 130.30, 130.79, 133.46, 136.18, 137.36, 139.88, 141.45, 168.04; LC-MS (ESI) m/z 363.2 (M-H⁺); Anal. Calcd for C₂₁H₁₇ClN₂O₂: C, 69.14; H, 4.70; N, 7.68. Found: C, 69.23; H, 4.71; N, 7.65.

N¹-benzyl-N²-(3, 4-dichlorophenyl) phthalamide (16)

Yield: 97%; MP: 178-179 ºC; IR (KBr): 3257, 3273 (NHs), 1641, 1662 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.42-4.43 (d, 2H, CH₂-N, J = 4 Hz), 7.20-7.35 (m, 5H, N-benzyl (o, m, p)), 7.55-7.66 (m, 6H, 3,4-dichlorophenyl H₅/H₆ & phenylene H₂/H₃/H₄/H₅), 8.10 (s, 1H, 3,4-dichlorophenyl H₂), 8.97-9.00 (t, 1H, NH, J = 8 Hz), 10.64 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm 41.86, 124.04, 126.04, 126.48, 126.56, 127.00, 127.08, 127.30, 127.53, 129.14, 129.26, 129.95, 130.27, 135.02, 136.08, 138.76, 138.99, 166.81, 167.03; LC-MS (ESI) m/z 397.2 (M-H⁺); Anal. Calcd for C₂₁H₁₆Cl₂N₂O₂: C, 63.17; H, 4.04; N, 7.02. Found: C, 63.22; H, 4.03; N, 6.98.

N¹-benzyl-N²-p-tolylphthalamide (17)

Yield: 99%; MP: 175-176 ºC; IR (KBr): 3152-3360 (NHs), 1643 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 2.28 (s, 3H, CH₃), 4.42-4.44 (d, 2H, CH₂-N, J = 8 Hz), 7.13-7.15 (d, 2H, 4-tolyl H₃/H₅, J = 8 Hz), 7.21-7.24 (t, 1H, N-benzyl (p), J = 8 Hz), 7.27- 7.31 (t, 2H, N-benzyl (m), J = 8 Hz), 7.35-7.37 (d, 2H, N-benzyl (o), J = 8 Hz), 7.53-7.61 (m, 6H, 4-tolyl H₂/H₆ & phenylene H₂/H₃/H₄/H₅), 8.91-8.94 (t, 1H, NH, J = 8 Hz), 10.27 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 20.99, 42.91, 119.98, 127.11, 127.58, 128.06, 128.24, 128.64, 129.41, 129.89, 130.10, 132.61, 136.40, 137.54, 137.63, 139.91, 167.38, 168.31; LC-MS (ESI) m/z 343.3 (M-H⁺); Anal. Calcd for C₂₂H₂₀N₂O₂: C, 76.72; H, 5.85; N, 8.13. Found: C, 76.65; H, 5.84; N, 8.11.

N¹-benzyl-N²-cyclohexylphthalamide (18)

Yield: 95%; MP: 175-176 ºC; IR (KBr): 3259, 3320 (NHs), 1643, 1675 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 1.08-1.77 (m, 10H, cyclohexyl H₂/H_2´/H₃/H_3´/H₄/H_4´/H₅/H_5´/H₆/H_6´), 3.59-3.65 (m, 1H, cyclohexyl H₁), 4.41-4.42 (d, 2H, CH₂-N, J = 4 Hz), 7.21-7.24 (t, 1H, N-benzyl (p), J = 8 Hz), 7.29-7.36 (m, 4H, N-benzyl (o, m)), 7.41-7.50 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.09-8.11 (d, 1H, NH, J = 8 Hz), 8.74-8.77 (t, 1H, NH, J = 8 Hz); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 25.15, 25.72, 32.65, 42.91, 48.53, 127.16, 127.65, 128.08, 128.26, 128.66, 129.60, 129.82, 136.33, 137.14, 139.89, 167.77, 168.67; LC-MS (ESI) m/z 335.3 (M-H⁺); Anal. Calcd for C₂₁H₂₄N₂O₂: C, 74.97; H, 7.19; N, 8.33. Found: C, 74.89; H, 7.21; N, 8.37.

N¹-benzyl-N²-(1-hydroxybutan-2-yl) phthalamide (19)

Yield: 67%; MP: 136-137 ºC; IR (KBr): 2903-3357 (NHs & OH), 1668, 1681 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 0.88-0.91 (t, 3H, CH₃, J = 8 Hz), 1.33-1.64 (m, 2H, CH₂), 2.49 (s, 1H, OH), 3.31-3.46 (m, 2H, CH₂-O), 3.75-3.78 (m, 1H, CH-N), 4.42-4.44 (d, 2H, CH₂-N, J = 8 Hz), 7.22-7.25 (t, 1H, N-benzyl (p), J = 8 Hz), 7.30-7.38 (m, 4H, N-benzyl (o, m)), 7.48-7.54 (m, 4H, phenylene H₂/H₃/H₄/H₅), 7.96-7.98 (d, 1H, NH, J = 8 Hz), 8.84-8.87 (t, 1H, NH, J = 8 Hz); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 10.24, 24.06, 42.97, 53.38, 63.55, 127.15, 127.64, 127.86, 128.11, 128.24, 128.68, 129.07, 129.59, 129.93, 136.07, 137.43, 139.84, 168.66, 168.79; LC-MS (ESI) m/z 325.0 (M-H⁺); Anal. Calcd for C₁₉H₂₂N₂O₃: C, 69.92; H, 6.79; N, 8.58. Found: C, 69.88; H, 6.81; N, 8.60.

N¹-(4-methoxybenzyl)-N²-benzylphthalamide (20)

Yield: 85%; MP: 174-175 ºC; IR (KBr): 3313, 3351 (NHs), 1665, 1695 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 3.72 (s, 3H, OCH₃), 4.33-4.35 (d, 2H, CH₂-N, J = 8 Hz), 4.41-4.43 (d, 2H, CH₂-N, J = 8 Hz), 6.86-6.88 (d, 2H, 4-methoxybenzyl H₃/H₅, J = 8 Hz), 7.22-7.38 (m, 7H, 4-methoxybenzyl H₂/H₆ & N-benzyl (o, m, p)), 7.48-7.52 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.74-8.77 (t, 1H, NH, J = 8 Hz), 8.80-8.83 (t, 1H, NH, J = 8 Hz); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 42.38, 42.90, 55.50, 127.12, 127.64, 128.12, 128.67, 128.98, 129.81, 131.85, 136.77, 136.83, 139.95, 158.60, 168.52, 168.68; LC-MS (ESI) m/z 373.2 (M-H⁺); Anal. Calcd for C₂₃H₂₂N₂O₃: C, 73.78; H, 5.92; N, 7.48. Found: C, 73.68; H, 5.90; N, 7.49.

N¹-propyl-N²-(5, 6, 7, 8-tetrahydronaphthalen-1-yl) phthalamide (21)

Yield: 98%; MP: 186 ºC; IR (KBr): 3310 (NHs), 1655, 1674 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 0.86-0.90 (t, 3H, CH₃, J = 8 Hz), 1.46-1.52 (q, 2H, CH₂, J = 8 Hz), 1.70-1.72 (m, 4H, tetrahydronaphthyl H₆/H_6'/H₇/H_7'), 2.69-2.74 (m, 4H, tetrahydronaphthyl H₅/H_5'/H₈/H_8'), 3.13-3.17 (q, 2H, CH₂-N, J = 8, 4 Hz), 6.92-6.94 (d, 1H, tetrahydronaphthyl H₄, J = 8 Hz), 7.07-7.10 (t, 1H, tetrahydronaphthyl H₃, J = 8, 4 Hz), 7.26- 7.28 (d, 1H, tetrahydronaphthyl H₂, J = 8 Hz), 7.51-7.62 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.34-8.37 (t, 1H, NH, J = 8 Hz), 9.56 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 11.93, 22.77, 22.87, 22.97, 24.60, 29.70, 41.37, 123.74, 125.40, 126.78, 128.01, 128.35, 129.85, 132.22, 136.47, 136.81, 137.14, 137.76, 167.56, 168.40; LC-MS (ESI) m/z 335.3 (M-H⁺); Anal. Calcd for C₂₁H₂₄N₂O₂: C, 74.97; H, 7.19; N, 8.33. Found: C, 74.87; H, 7.20; N, 8.30.

N¹-(3,4-dimethoxyphenethyl)-N²-(5,6,7,8-tetrahydronaphthalen-1-yl) phthalamide (22)

Yield: 83%; MP: 172-173 ºC; IR (KBr): 3300, 3332 (NHs), 1654, 1683 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 1.71-1.72 (m, 4H, tetrahydronaphthyl H₆/H_6'/H₇/H_7'), 2.72-2.76 (m, 6H, tetrahydronaphthyl H₅/H_5'/H₈/H_8' & CH₂), 3.37-3.42 (q, 2H, CH₂-N, J = 8 Hz), 3.70 (s, 3H, OCH₃), 3.72 (s, 3H, OCH₃), 6.74-6.76 (d, 1H, tetrahydronaphthyl H₄, J = 8 Hz), 6.84-6.86 (m, 2H, dimethoxyphenethyl H₂ & tetrahydronaphthyl H₂), 6.93-6.95 (d, 1H, dimethoxyphenethyl H₆, J = 8 Hz), 7.08-7.11 (t, 1H, tetrahydronaphthyl H₃, J = 8. 4 Hz), 7.27-7.29 (d, 1H, dimethoxyphenethyl H₅, J = 8 Hz), 7.46-7.55 (m, 3H, phenylene H₃/H₄/H₅), 7.62-7.64 (d, 1H, phenylene H₂, J = 8 Hz), 8.42-8.45 (t, 1H, NH, J = 8 Hz), 9.59 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 22.99, 24.64, 29.70, 33.62, 34.97, 41.50, 55.69, 55.78, 120.92, 121.03, 123.49, 123.79, 125.42, 126.81, 127.97, 128.38, 129.83, 129.94, 131.00, 131.96, 132.27, 132.43, 136.49, 136.71, 137.23, 137.79, 168.18, 168.40; LC-MS (ESI) m/z 457.4 (M-H⁺); Anal. Calcd for C₂₈H₃₀N₂O₄: C, 73.34; H, 6.59; N, 6.11. Found: C, 73.24; H, 6.57; N, 6.09.

N¹-benzyl-N²-(4-hydroxyphenyl) phthalamide (23)

Yield: 68%; MP: 150 ºC; IR (KBr): 3100-3400 (NHs, OH), 1660, 1681 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.42-4.44 (d, 2H, CH₂-N, J = 8 Hz), 6.71-6.73 (d, 2H, 4-hydroxyphenyl H₃/H₅, J = 8 Hz), 7.21-7.24 (t, 1H, N-benzyl (p), J = 8 Hz), 7.27-7.31 (t, 2H, N-benzyl (m), J = 8 Hz), 7.35-7.37 (d, 2H, N-benzyl (o), J = 8 Hz), 7.48-7.50 (d, 2H, 4-hydroxyphenyl H₂/H₆, J = 8 Hz), 7.51-7.60 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.87-8.90 (t, 1H, NH, J = 8 Hz), 9.21 (s, 1H, OH), 10.10 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 42.92, 115.40, 121.74, 127.11, 127.59, 128.08, 128.22, 128.64, 129.79, 130.04, 131.72, 136.45, 137.67, 139.92, 153.85, 166.95, 168.44; LC-MS (ESI) m/z 345.2 (M-H⁺); Anal. Calcd for C₂₁H₁₈N₂O₃: C, 72.82; H, 5.24; N, 8.09. Found: C, 72.71; H, 5.22; N, 8.11.

N¹-benzyl-N²-(1-phenylethyl) phthalamide (24)

Yield: 77%; MP: 193 ºC; IR (KBr): 3256, 3313 (NHs), 1646, 1663 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 1.39-1.41 (d, 3H, CH₃, J = 8 Hz), 4.40-4.42 (d, 2H, CH₂-N, J = 8 Hz), 5.07-5.10 (m, 1H, CH), 7.21-7.29 (m, 2H, N-benzyl (p) & phenethyl (p)), 7.30-7.37 (m, 6H, N-benzyl (o, m) & phenetyhl (m)), 7.41-7.42 (d, 2H, phenethyl (o), J = 4 Hz), 7.47-7.54 (m, 4H, phenylene H₂/H₃/H₄/H₅), 8.74-8.76 (d, 1H, NH, J = 8 Hz), 8.78-8.81 (t, 1H, NH, J = 8 Hz); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 22.93, 42.93, 48.73, 126.57, 127.03, 127.17, 127.65, 128.13, 128.33, 128.70, 129.78, 129.88, 136.55, 136.92, 139.91, 145.12, 167.84, 168.65; LC-MS (ESI) m/z 357.3 (M-H⁺); Anal. Calcd for C₂₃H₂₂N₂O₂: C, 77.07; H, 6.19; N, 7.82. Found: C, 77.23; H, 6.20; N, 7.84.

N¹-benzyl-N²-(3, 4-dimethoxybenzyl) phthalamide (25)

Yield: 85%; MP: 160-161 ºC; IR (KBr): 3210, 3310 (NHs), 1643, 1675 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 3.37-3.39 (d, 2H, CH₂-N, J = 5.6 Hz), 3.71 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 4.42-4.44 (d, 2H, CH₂-N, J = 6 Hz), 6.75-6.77 (dd, 1H, dimethoxybenzyl H₅, J = 6.4, 1.6 Hz), 6.86 - 6.88 (m, 2H, dimethoxybenzyl H₂/H₆), 727.22-7.26 (t, 1H, N-benzyl (p), J = 7.2 Hz), 7.31-7.35 (t, 2H, N-benzyl (m), J = 7.6 Hz), 7.37-7.41 (m, 3H, phenylene H₄ & N-benzyl (o)), 7.47-7.51 (m, 3H, phenylene H₂/H₃/H₅), 8.34-8.37 (t, 1H, NH, J = 5.6 Hz), 8.81-8.84 (t, 1H, NH, J = 6 Hz); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 41.46, 42.89, 55.80, 55.94, 112.30, 113.01, 120.93, 127.13, 127.62, 128.06, 128.14, 128.66, 129.74, 129.80, 132.47, 136.62, 137.00, 139.98, 147.62, 149.02, 168.58, 168.65; LC-MS (ESI) m/z 403.2 (M-H⁺); Anal. Calcd for C₂₄H₂₄N₂O₄: C, 71.27; H, 5.98; N, 6.93. Found: C, 71.15; H, 6.00; N, 7.00.

N¹-benzyl-N²-phenylphthalamide (26)

Yield: 67%; MP: 174-175 ºC; IR (KBr): 3299, 3305 (NHs), 1651, 1677 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.42-4.44 (d, 2H, CH₂-N, J = 6 Hz), 7.20-7.40 (m, 8H, N-benzyl (o, m, p) & phenyl (m, p)), 7.53-7.64 (m, 4H, phenyl (o) & phenylene H₃/H₄), 7.74-7.76 (m, 2H, phenylene H₂/H₅), 8.96-8.99 (t, 1H, NH, J = 6 Hz), 10.51 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 42.95, 121.49, 127.13, 127.30, 127.58, 127.66, 128.08, 128.23, 128.65 ,128.97, 130.05, 130.23, 136.26, 137.44, 138.99, 167.79, 168.11; LC-MS (ESI) m/z 329.1 (M-H⁺); Anal. Calcd for C₂₁H₁₈N₂O₂: C, 76.34; H, 5.49; N, 8.48. Found: C, 76.55; H, 5.46; N, 8.49.

N¹-benzyl-N²-(4-chlorophenyl) phthalamide (27)

Yield: 72%; MP: 178-181 ºC; IR (KBr): 3301, 3322 (NHs), 1645, 1663 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 4.42-4.44 (d, 2H, CH₂-N, J = 5.6 Hz), 7.09-7.11 (t, 1H, N-benzyl (p), J = 7.2 Hz), 7.19-7.41 (m, 6H, N-benzyl (o, m) & 4-chlorophenyl H₃/H₅), 7.48-7.63 (m, 4H, 4-chlorophenyl H₂/H₆ & phenylene H₃/H₄), 7.74-7.76 (m, 2H, phenylene H₂/H₅), 8.96-8.99 (t, 1H, NH, J = 5.6 Hz), 10.39 (s, 1H, NH); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 45.79, 123.75, 127.11, 127.59, 128.08, 128.26, 128.64, 128.90, 129.05, 129.41, 129.93, 130.15, 134.56, 136.33, 137.63, 167.67, 168.26; LC-MS (ESI) m/z 363.1 (M-H⁺); Anal. Calcd for C₂₁H₁₇ClN₂O₂: C, 69.14; H, 4.70; N, 7.68. Found: C, 69.25; H, 4.68; N, 7.71.

N¹-benzyl-N²-((1R, 2R)-2-hydroxy-2, 3-dihydro-1H-inden-1-yl) phthalamide (28)

Yield: 54%; MP: 174-175 ºC; IR (KBr): 3299, 3305 (NHs, OH), 1651, 1677 (C=O), 1450-1600 (aromatic C-H); ¹H-NMR (d₆-DMSO, 400 MHz) δ ppm, 2.70-2.76 (q, 1H, aminoindanol H₃, J = 8 Hz), 3.10-3.16 (q, 1H, aminoindanol H_3', J = 8 Hz), 4.30-4.48 (m, 3H, CH₂-N & CH-O), 5.19-5.23 (t, 1H, CH-N, J = 8 Hz), 5.35-5.36 (d, 1H, OH, J = 4 Hz), 7.15-7.57 (m, 13H, phenylene H₂/H₃/H₄/H₅& aminoindanol H₄/H₅/H₆/H₇ & N-benzyl (o ,m, p)), 8.67-8.70 (d, 1H, NH, J = 12 Hz), 8.89-8.92 (t, 1H, NH, J = 8 Hz); ¹³C-NMR (d₆-DMSO, 100 MHz) δ ppm; 42.78, 46.04, 61.70, 78.08, 127.03, 127.13, 127.65, 127.93, 128.08, 128.34, 128.69, 128.89, 129.08, 129.28, 129.76, 129.82, 134.73, 136.72, 137.16, 139.94, 168.77, 169.22; LC-MS (ESI) m/z 385.1 (M-H⁺); Anal. Calcd for C₂₄H₂₂N₂O₃: C, 74.59; H, 5.74; N, 7.25. Found: C, 74.45; H, 5.73; N, 7.28.

Docking studies

The protein structure was prepared for docking by the use of AutoDockTools®²⁰. The docking analysis was performed using Autodock VINA® operating system²¹. Molecules were built within chemdraw® and subsequently optimized using the Hyperchem 8.0 software. The 3D protein structure of the HCV polymerase enzyme with its cognate ligand, UTP (PDB code: 1GX6), was downloaded from the Protein Data Bank of the Research Collaboration for Structural Bioinformatics (RCSB) website²². Co-crystallized ligand and water molecules were removed from the protein structure, while Mn²⁺ ions remained intact. The receptors and ligands were treated as rigid and flexible structures, respectively. Non-polar hydrogens were merged. Polar hydrogens and Kollman united partial atom charges were added to the individual protein atoms. Grid map dimensions (20×20×20) were set surrounding the active site. Docking results were clustered with a root mean square deviation (RMSD) of 0.5 Å and evaluated by Pymol software version 1.5.0.1.

In-vitro anti-HCV and cytotoxicity assays

Cell Culture and virus production

Huh7.5, a Huh7 variant cell line, originated from HCC that is highly permissive to efficient HCV RNA multiplication (kindly provided by Rice C) was cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco®) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin and 100 μg/mL of streptomycin²³. The full-length HCV JFH1 genome (kindly provided by Wakita T) was used for in-vitro transcription to produce approximately high-titer stocks of infectious particles in cell culture (HCVcc) as previously described^{24, 25}.

RT-qPCR assay

The RT-qPCR analysis was used to explore the inhibitory effects of our newly synthesized compounds. Briefly, 24 h before infection, 3 × 10⁵ Huh7.5 cells were seeded in 48-well plates with DMEM containing 10% FBS. Then the huh7.5 cells were treated with HCV viral stock (10⁵ IU/ml) for 3 h, at which point the infection was stopped. The supernatant was discarded, and each well was washed three times with phosphate buffer saline (PBS) to removed non penetrated virions. Then the fresh medium was added to each well. Subsequently, the infected cells were treated with different levels of newly-synthesized compounds as well as the sofosbuvir (Sigma-Aldrich, USA) as the standard. 72 h post-inoculation, the supernatant medium had been collected and viral RNA extracted, under the manufacturer's protocol, using the viral QIAamp viral mini kit of RNA (Qiagen, Düsseldorf, Germany). Then, the number of HCV RNA copies was measured using a quantitative RT-PCR assay by HCV Viral load kit (Gene Proof, Czech Republic, with CE, IVD) as directed by the manufacturer. The 50% inhibitory concentration (IC₅₀), which was defined as the concentration of analog at which the secreted HCV virions were decreased by 50% relative to the control reactions (infected wells in the absence of drug), was determined by nonlinear regression analysis using GraphPad Prism5.0 software (GraphPad Software, San Diego, CA).

Cytotoxicity Assay

In parallel with the in vitro evaluation of anti-HCV effects, the XTT (sodium 3-[1(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid) assay was carried out to evaluate the cytotoxicity of compounds to Huh-7.5 cells, according to XTT proliferation kit (Sigma-Aldrich) instructions²⁶. In this regard, cells were treated with various concentrations (500, 250, 100, 25, and 12 μm/ml) of newly-synthesized analogs and incubated for 72 hours. Then, the amount of orange formazan dye generated via the cleavage of the yellow tetrazolium salt XTT by the viable cells was measured using the UV-visible spectrophotometry method²⁷.

Conflicts of interest

The authors declare that they have no involvement in any organization or entity with an interest in the subject matter or materials covered in this manuscript.

Acknowledgments

This work was supported by a grant from the Research Council of Shaheed Beheshti University of Medical Sciences [30030].

P. Axley, Z. Ahmed, S. Ravi and A. K. Singal, Journal of clinical and translational hepatology, 2018, 6, 79.
F. Zoulim, T. J. Liang, A. L. Gerbes, A. Aghemo, S. Deuffic-Burban, G. Dusheiko, M. W. Fried, S. Pol, J. K. Rockstroh and N. A. Terrault, Gut, 2015, 64, 1824-1833.
M. Jefferies, B. Rauff, H. Rashid, T. Lam and S. Rafiq, World J Clin Cases, 2018, 6, 589-599.
E. Palumbo, Ther Adv Chronic Dis, 2011, 2, 39-45.
M. P. Manns, H. Wedemeyer and M. Cornberg, Gut, 2006, 55, 1350-1359.
K. S. Abdelwahab and Z. N. Ahmed Said, World J Gastroenterol, 2016, 22, 862-873.
V. Gupta, A. Kumar, P. Sharma and A. Arora, Indian J Med Res, 2017, 146, 23-33.
N. Alazard-Dany, S. Denolly, B. Boson and F.-L. Cosset, Viruses, 2019, 11, 30.
K. Kumthip and N. Maneekarn, Virol J, 2015, 12, 217-217.
S. M. I. M. Rabbani, R. Vahabpour, Z. Hajimahdi and A. Zarghi, Iran J. Pharm. Res., 2019, 18, 1790.
M. H. Powdrill, J. A. Bernatchez and M. Götte, Viruses, 2010, 2, 2169-2195.
T. C. Appleby, J. K. Perry, E. Murakami, O. Barauskas, J. Feng, A. Cho, D. Fox, D. R. Wetmore, M. E. McGrath and A. S. Ray, Science, 2015, 347, 771-775.
K. K. S. Ng, J. J. Arnold and C. E. Cameron, Curr Top Microbiol Immunol, 2008, 320, 137-156.
A. Bhatt, K. R. Gurukumar, A. Basu, M. R. Patel, N. Kaushik-Basu and T. T. Talele, European journal of medicinal chemistry, 2011, 46, 5138-5145.
Y. Deng, G. W. Shipps Jr, T. Wang, J. Popovici-Muller, K. E. Rosner, M. A. Siddiqui, J. Duca, A. B. Cooper and M. Cable, Bioorganic & medicinal chemistry letters, 2009, 19, 5363-5367.
S. Venkatraman, F. Velazquez, S. Gavalas, W. Wu, K. X. Chen, A. G. Nair, F. Bennett, Y. Huang, P. Pinto and Y. Jiang, Bioorganic & medicinal chemistry, 2013, 21, 2007-2017.
C. Zhao, Y. Wang and S. Ma, European journal of medicinal chemistry, 2015, 102, 188-214.
V. Lohmann, F. Körner, J.-O. Koch, U. Herian, L. Theilmann and R. Bartenschlager, Science, 1999, 285, 110-113.
K. J. Blight, A. A. Kolykhalov and C. M. Rice, Science, 2000, 290, 1972-1974.
G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew and A. J. Olson, Journal of computational chemistry, 1998, 19, 1639-1662.
O. Trott and A. J. Olson, Journal of computational chemistry, 2010, 31, 455-461.
S. Bressanelli, L. Tomei, F. A. Rey and R. De Francesco, Journal of virology, 2002, 76, 3482-3492.
Z. Cai, T. J. Liang and G. Luo, Journal of virology, 2004, 78, 3633-3643.
T. Wakita, T. Pietschmann, T. Kato, T. Date, M. Miyamoto, Z. Zhao, K. Murthy, A. Habermann, H.-G. Kräusslich and M. Mizokami, Nature medicine, 2005, 11, 791-796.
G. Mateu, R. O. Donis, T. Wakita, J. Bukh and A. Grakoui, Virology, 2008, 376, 397-407.
D. A. Scudiero, R. H. Shoemaker, K. D. Paull, A. Monks, S. Tierney, T. H. Nofziger, M. J. Currens, D. Seniff and M. R. Boyd, Cancer research, 1988, 48, 4827-4833.
J. C. Stockert, R. W. Horobin, L. L. Colombo and A. Blázquez-Castro, Acta histochemica, 2018, 120, 159-167.

Scheme 1 is available in the Supplementary Files section.

scheme1.png
Scheme 1. Reagents and conditions: (a) Ethyl acetate, r.t, 18h; (b) Trifluoroacetic anhydride (TFAA), Triethylamine (TEA), THF, 0 ºC, 30 min; (c) R₂-NH₂, r.t, 24 h.
Supplementary.pdf

PDF

Editorial decision: Major Revision
23 Jun, 2022
Reviewers agreed at journal
25 May, 2022
Reviewers invited by journal
25 May, 2022
Editor assigned by journal
19 May, 2022
First submitted to journal
18 May, 2022

You are reading this latest preprint version

Discovery of novel HCV inhibitors: Design, Synthesis and biological activity of phthalamide derivatives

Status:

Version 1

Abstract

Figures

Introduction

Results And Discussion

Chemistry

HCV replicon assay

Molecular Modeling Studies

Conclusions

Materials And Methods

Declarations

References

Scheme

Supplementary Files

Status:

Version 1