Design, Microwave-Assisted Synthesis, Antimicrobial and Anticancer Evaluation, and In Silico Studies of Some 2-Naphthamide Derivatives as DHFR and VEGFR-2 Inhibitors.

Naphthamide is a common structural framework with diverse pharmacological activities. Ten novel 2-naphthamide derivatives have been designed, synthesized, and evaluated for their in vitro antibacterial, antifungal, and anticancer activities. The title compounds were synthesized from dimethoxybenzaldehyde derivatives through a four-step microwave-assisted synthesis process. The structures were confirmed by 1H NMR, 13C NMR, and MS spectra. Compound 8b showed good antibacterial activity against Escherichia coli, Streptococcus faecalis, Salmonella enterica, MSSA, and MRSA with MIC values of 16, 16, 16, 8, and 16 μg/mL, respectively, compared to ciprofloxacin (MIC = 8-16 μg/mL). Compounds 5b (IC50 = 3.59-8.38 μM) and 8b (IC50 = 2.97-7.12 μM) exhibited good cytotoxic activity against C26, HepG2, and MCF7 cancer cell lines as compared to paclitaxel (IC50 = 2.85-5.75 μM). Moreover, compounds 5b and 8b exhibited better anticancer activity than PTX against the C26 cell line. In particular, compound 8b showed potent in vitro VEGFR-2 inhibitory activity with the IC50 value of 0.384 μM compared with sorafenib (IC50 = 0.069 μM). Therefore, compound 8b is the most potent compound for anticancer activity as indicated by in vitro cell line inhibition, in silico ADMET, molecular docking, and in vitro VEGFR-2 inhibition studies.

Introduction

Bacterial and fungal resistance can cause life-threatening diseases. In addition, cancer drug resistance is also expanding and posing a great threat to human health and life. This has resulted in research and development in search of new antibiotics and anticancer drugs to maintain an effective drug supply at all times.[1−4] Besides, the commonly used antibacterial drugs such as amoxicillin, norfloxacin, and ciprofloxacin as well as the commonly used antifungal drugs such as clotrimazole, fluconazole, amphotericin, and nystatin are related to severe side effects. Moreover, anticancer drugs including vinca alkaloids (e.g., vinblastine and vincristine) and taxanes (e.g., paclitaxel and docetaxel) always have limited clinical use due to side effects, such as poor solubility, low oral bioavailability, high toxicity, the development of drug resistance, and complex synthesis.[5,6] Therefore, it is important to find out newer, safer, and more effective antibiotics and anticancer drugs with multiple effects, especially showing both good anticancer and antimicrobial activities. This is very beneficial for cancer patients due to their weakened immunity and susceptibility to microbial attack.

Naphthalene is the simplest polycyclic aromatic hydrocarbon consisting of a fused pair of benzene rings. On the other hand, naphthamide derivatives containing a naphthalene nucleus are a common structural framework in various biologically active natural products, pharmaceuticals, and materials.[7,8] In addition, the naphthamide derivatives showed a large spectrum of activities in various pharmacological fields like anticancer,[9−12] antibacterial,[13−15] antifungal,[15−17] anti-inflammatory,[18] and antioxidants.[19] Moreover, many important drugs used therapeutically in the research area contain naphthalene moieties such as nafcillin, bedaquiline, naproxen, tolnaftate, duloxetine, and propanolol (Figure ).

Figure 1

Marketed naphthalene ring containing drug compounds.

Dihydrofolate reductase receptor (DHFR) and vascular endothelial growth factor receptor 2 (VEGFR-2) are important targets in the design and development of novel antitumor agents. In addition, DHFR is also used in the design of antibacterial and antifungal agents. Inhibition of DHFR leads to a decrease in intracellular folate required for one carbon transformation and is therefore important for the biosynthesis of thymidylate, purine nucleotide, methionine, and many other compounds required for RNA and DNA biosynthesis. Furthermore, this receptor has a high binding affinity and selectivity for co-substrates, making it difficult to be displaced by natural substrates.[1,20] On the other hand, VEGFR-2 is strongly associated with poor prognosis and tumor metastasis. VEGFR-2-mediated signaling promotes a number of endothelial responses required for the formation of new blood vessels, such as cell proliferation, metastasis, and survival of cancer cells.[21,22]

Rationale and Structure-Based Design as Antimicrobial and Anticancer Agents

Structure–activity relationship studies of the naphthalene ring system suggested that the C-1 and C-2 positions are very much important for the pharmacological effect.[12] Especially, naphthamide derivatives are potent VEGFR-2 inhibitors, for example, amide groups similar to anticancer drugs (sorafenib, sunitinib, pazopanib, axitinib, vandetanib, and regorafenib). Our designed derivatives and anticancer drug millepachine (potent cytotoxicity against a variety of human cancer cells with an IC50 range of 0.76–4.66 μM), linifanib, and derivatives of Harmange et al., and Lv et al., share three common essential structural features such as a planar naphthalene moiety, the amide group at C-1 and C-2 positions, and the −OR-substituted groups at different positions (Figure ).[12,23]

Figure 2

Rational study design, illustrating the structure of the newly designed N-(4-substituted benzyl) and N-(3-morpholinopropyl) 2-naphthamide derivatives with representative examples for anticancer compounds (IC50, half-maximal inhibitory concentration; VEGFR, vascular endothelial growth factor receptor).

Therefore, the purpose of this study is to synthesize novel N-(4-substituted benzyl) and N-(3-morpholinopropyl) 2-naphthamide derivatives with dimethoxy substituents at positions 5,7 and 6,8 as well as different NH-substituted groups and evaluate their antibacterial, antifungal, and anticancer activities. The active derivatives will be subjected to molecular docking studies to understand potential drug–receptor interactions and the in silico ADMET pharmacokinetic profile as well as to evaluate in vitro DHFR and VEGFR-2 inhibitory activities.

Experimental Section

Materials

All reagents and chemicals were obtained from the commercial supplier Merck (Germany) and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) carried out on silica gel plates (E-Merck Kieselgel 60 F254) using UV light as the visualizing agent. Silica gel (0.040–0.063 mm) from Merck (Germany) was used for column chromatography.

The microwave-assisted synthesis was performed by a microwave synthesizer (CEM Discover) with continuous stirring and infrared temperature sensors. Melting points (mp, °C) were determined in an open capillary using a Gallenkamp melting point apparatus (Sanyo Gallenkamp, U.K.). A Shimadzu FTIR (IRAffinity-1S) spectrometer was used to record the infrared (IR) spectra. Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were recorded on a Bruker Avance 500 (1H, 500 MHz; 13C, 125 MHz) NMR spectrometer at ambient temperature using CDCl3 and DMSO-d6 as solvents. Chemical shifts are reported in parts per million (ppm) relative to the residual solvent peak as following: CDCl3 = 7.26 ppm (1H NMR), DMSO-d6 = 2.50 ppm (1H NMR), CDCl3 = 77.16 ppm (13C NMR), and DMSO-d6 = 40.00 ppm (13C NMR). A liquid chromatography machine (Agilent Technologies 1100 series LC/MSD Trap) was used to record the mass spectra (MS). Optical density (OD) was measured at 570 nm on a Multiskan microplate reader.

Experimental Procedures

General Procedure for the Preparation of (E)-4-(2,4 or 3,5-Dimethoxyphenyl)-3-(ethoxycarbonyl)but-3-enoic Acid (1a–1b)

A mixture of diethyl succinate (10 mmol), 2,4 or 3,5-dimethoxybenzaldehyde (5 mmol), potassium tert-butoxide (t-BuOK, 10 mmol), and tert-butanol (t-BuOH, 10 mL) was dissolved in a sealed reactor vessel. The reaction mixture was microwave-irradiated for 15 min at 60 °C with 200 W energy and a high stirring speed. When the reaction was complete, the mixture was acidified with acetic acid, and then the product was extracted with ethyl acetate. After removing the solvent, a brown crude product was obtained. The pure yellow crystals were obtained by column chromatography on silica gel with hexane and ethyl acetate (5:1 v/v) as the mobile phase. Reaction yields obtained range from 92 to 94%.

General Procedure for the Preparation of Ethyl 4-Acetoxy-6,8 or 5,7-Dimethoxy-2-naphthoate (2a–2b)

A mixture of compound 1 (1 mmol), sodium acetate (NaOAc, 1 mmol), and acetic anhydride (Ac2O, 5 mmol) was dissolved in a sealed reactor vessel. The reaction mixture was microwave irradiated for 10 min at 130 °C with 300 W energy and a high stirring speed. When the reaction was complete, the mixture was cooled, and then 15% NaOH solution was added to neutralize the excess acid. The product was extracted with ethyl acetate, and the solvent was evaporated to yield the crude product. The pure white crystals were obtained by column chromatography on silica gel with hexane and ethyl acetate (6:1 v/v) as the mobile phase. Reaction yields obtained range from 91 to 93%.

General Procedure for the Preparation of 4-Hydroxy-6,8 or 5,7-Dimethoxynaphthalene-2-carboxylic Acid (3a–3b)

A mixture of compound 2 (5 mmol), 3M KOH solution (5 mL), and 95% ethanol (EtOH, 5 mL) was dissolved in a sealed reactor vessel. The reaction mixture was microwave irradiated for 15 min at 80 °C with 300 W energy and a high stirring speed. When the reaction was complete, the mixture was neutralized with a 10% HCl solution. The product was extracted with ethyl acetate, and the solvent was evaporated to yield the crude product. The pure yellow crystals were obtained by column chromatography on silica gel with hexane and ethyl acetate (3:1 v/v) as the mobile phase. Reaction yields obtained range from 95 to 96%.

General Procedure for the Preparation of N-(4-Substituted benzyl) and N-(3-Morpholinopropyl) 2-Naphthamide Derivatives (4a–4b and 5–8(a–b))

Reflux Method

To a mixture of compound 3 (1 mmol) and acetonitrile (MeCN, 20 mL) was added drop by drop of thionyl chloride (SOCl2, 5 mL). The reaction mixture was stirred in a nitrogen atmosphere at room temperature for 3.5 h. After removing the solvent, the solid was redissolved in acetonitrile (10 mL) and then gradually added 3-morpholinopropylamine or 4-substituted benzylamine (3 mmol) to the reaction flask and continued stirring the mixture in nitrogen gas at room temperature for 5 min. The product was extracted with ethyl acetate, and the solvent was evaporated to yield the crude product. The pure white crystals were obtained by column chromatography on silica gel with ethyl acetate and methanol (6:1 v/v) as the mobile phase. Reaction yields obtained range from 80 to 86%.

Microwave-Assisted Method

A mixture of compound 3 (1 mmol) and acetonitrile (MeCN, 20 mL) was dissolved in a sealed reactor vessel. To the mixture was added drop by drop of thionyl chloride (SOCl2, 5 mL) and irradiated for 30 min at 80 °C with a power of 300 W. After removing the solvent, the solid was redissolved in acetonitrile (10 mL) and then added 3-morpholinopropylamine or 4-substituted benzylamine (3 mmol) and continued to irradiate for 2 min at 130 °C with 300 W energy and a high stirring speed. The product was extracted with ethyl acetate, and the solvent was evaporated to yield the crude product. The pure white crystals were obtained by column chromatography on silica gel with ethyl acetate and methanol (6:1 v/v) as the mobile phase. Reaction yields obtained range from 93 to 97%.

(E)-4-(2,4-dimethoxyphenyl)-3-(ethoxycarbonyl)but-3-enoic Acid (1a)

Yellow solid, mp 116–117 °C. 1H NMR (500 MHz, CDCl3, δ ppm): 7.98 (1H, s, -CH=C-), 7.27 (1H, s, HAr), 6.54 (1H, d, J = 2.0 Hz, HAr), 6.47 (1H, d, J = 2.0 Hz, HAr), 4.29 (2H, q, J = 7.0 Hz, -CH2-OCO-), 3.83 (3H, s, -OCH3), 3.75 (3H, s, -OCH3), 3.52 (2H, s, -CH2-COOH), 1.35 (3H, t, J = 7.0 Hz, -CH3). 13C NMR (125 MHz, CDCl3, δ ppm): 168.0, 162.1, 159.0, 138.4, 130.8, 123.7, 116.6, 110.0, 104.6, 98.5, 61.6, 56.5, 55.5, 34.2, 14.2. LC-MS (m/z) [M – H]− calcd for C15H17O6 293.1031, found 293.1039.

(E)-4-(3,5-dimethoxyphenyl)-3-(ethoxycarbonyl)but-3-enoic Acid (1b)

Yellow solid, mp 118–119 °C. 1H NMR (500 MHz, CDCl3, δ ppm): 7.96 (1H, s, -CH=C-), 7.28 (1H, s, HAr), 6.55 (1H, d, J = 1.5 Hz, HAr), 6.44 (1H, d, J = 2.0 Hz, HAr), 4.31 (2H, q, J = 7.5 Hz, -CH2-OCO-), 3.82 (3H, s, -OCH3), 3.77 (3H, s, -OCH3), 3.54 (2H, s, -CH2-COOH), 1.36 (3H, t, J = 7.0 Hz, -CH3). 13C NMR (125 MHz, CDCl3, δ ppm): 168.3, 161.9, 159.2, 138.5, 130.6, 123.8, 116.9, 110.3, 104.5, 98.6, 61.4, 56.6, 55.4, 34.3, 14.5. LC-MS (m/z) [M – H]− calcd for C15H17O6 293.1031, found 293.1028.

Ethyl 4-Acetoxy-6,8-dimethoxy-2-naphthoate (2a)

White solid. 1H NMR (500 MHz, CDCl3, δ ppm): 8.80 (1H, d, J = 0.5 Hz, HAr), 7.81 (1H, d, J = 1.5 Hz, HAr), 6.67 (1H, d, J = 2.0 Hz, HAr), 6.54 (1H, d, J = 2.0 Hz, HAr), 4.41 (2H, q, J = 7.0 Hz, -CH2-), 4.02 (3H, s, -OCH3), 4.01 (3H, s, -OCH3), 1.43 (3H, t, J = 7.0 Hz, -CH3). 13C NMR (125 MHz, CDCl3, δ ppm): 169.2, 166.3, 161.0, 158.0, 145.4, 131.2, 124.5, 123.3, 122.3, 119.2, 98.6, 91.7, 61.1, 55.8, 55.4, 21.0, 14.4. LC-MS (m/z) [M – H]− calcd for C17H17O6 317.1031, found 317.1045; [M + H]+ calcd for C17H19O6 319.1176, found 319.1169.

Ethyl 4-Acetoxy-5,7-dimethoxy-2-naphthoate (2b)

White solid. 1H NMR (500 MHz, CDCl3, δ ppm): 8.79 (1H, d, J = 1.0 Hz, HAr), 7.86 (1H, d, J = 2.0 Hz, HAr), 6.68 (1H, d, J = 2.0 Hz, HAr), 6.53 (1H, d, J = 2.0 Hz, HAr), 4.42 (2H, q, J = 7.0 Hz, -CH2-), 4.04 (3H, s, -OCH3), 4.02 (3H, s, -OCH3), 1.45 (3H, t, J = 7.0 Hz, -CH3). 13C NMR (125 MHz, CDCl3, δ ppm): 169.4, 166.5, 161.1, 157.9, 145.2, 131.3, 124.7, 123.2, 122.4, 119.5, 98.5, 91.9, 61.2, 55.9, 55.5, 21.2, 14.6. LC-MS (m/z) [M – H]− calcd for C17H17O6 317.1031, found 317.1048; [M + H]+ calcd for C17H19O6 319.1176, found 319.1181.

4-Hydroxy-6,8-dimethoxy-2-naphthoic Acid (3a)

Yellow solid. 1H NMR (500 MHz, CDCl3, δ ppm): 8.47 (1H, s, HAr), 7.36 (1H, d, J = 1.5 Hz, HAr), 7.14 (1H, d, J = 2.0 Hz, 1H, HAr), 6.53 (1H, d, J = 2.0 Hz, HAr), 3.98 (3H, s, -OCH3), 3.95 (3H, s, -OCH3). 13C NMR (125 MHz, CDCl3, δ ppm): 169.9, 159.8, 157.5, 151.8, 129.3, 124.1, 121.9, 117.4, 108.3, 98.4, 92.7, 55.5, 55.4. LC-MS (m/z) [M – H]− calcd for C13H11O5 247.0612, found 247.0653.

4-Hydroxy-5,7-dimethoxy-2-naphthoic Acid (3b)

Yellow solid. 1H NMR (500 MHz, CDCl3, δ ppm): 8.43 (1H, s, HAr), 7.34 (1H, d, J = 2.0 Hz, HAr), 7.13 (1H, d, J = 2.0 Hz, 1H, HAr), 6.56 (1H, d, J = 2.0 Hz, HAr), 3.99 (3H, s, -OCH3), 3.96 (3H, s, -OCH3). 13C NMR (125 MHz, CDCl3, δ ppm): 169.6, 159.7, 157.3, 151.9, 129.4, 124.2, 121.8, 117.5, 108.4, 98.6, 92.8, 55.7, 55.5. LC-MS (m/z) [M – H]− calcd for C13H11O5 247.0612, found 247.0625.

4-Hydroxy-6,8-dimethoxy-N-(3-morpholinopropyl)-2-naphthamide (4a)

White solid, mp 172–174 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 8.54 (1H, s, -NH-), 8.00 (1H, s, HAr), 7.32 (1H, s, HAr), 7.04 (1H, d, J = 2.0 Hz, HAr), 6.63 (1H, d, J = 2.5 Hz, HAr), 3.96 (3H, s, -OCH3), 3.89 (3H, s, -OCH3), 3.58 (4H, t, J = 4.5 Hz, -CH2-), 3.31 (2H, q, J = 6.5 Hz, -CH2-), 2.34 (6H, t, J = 7.0 Hz, -CH2-), 1.72–1.68 (2H, m, -CH2-). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.9, 158.5, 156.5, 152.3, 129.8, 127.5, 121.3, 111.6, 107.9, 98.6, 92.9, 66.3, 56.4, 55.8, 55.3, 53.1, 38.4, 25.8. LC-MS (m/z) [M – H]− calcd for C20H25N2O5 373.1769, found 373.1754; [M + H]+ calcd for C20H27N2O5 375.1914, found 375.1898.

4-Hydroxy-5,7-dimethoxy-N-(3-morpholinopropyl)-2-naphthamide (4b)

White solid, mp 174–175 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 8.52 (1H, s, -NH-), 7.98 (1H, s, HAr), 7.30 (1H, s, HAr), 7.03 (1H, d, J = 2.0 Hz, HAr), 6.63 (1H, d, J = 2.5 Hz, HAr), 3.94 (3H, s, -OCH3), 3.86 (3H, s, -OCH3), 3.59 (4H, t, J = 4.5 Hz, -CH2-), 3.30 (2H, q, J = 6.5 Hz, -CH2-), 2.35 (6H, t, J = 7.0 Hz, -CH2-), 1.71–1.67 (2H, m, -CH2-). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.8, 158.4, 156.7, 152.2, 129.7, 127.5, 121.2, 111.4, 108.0, 98.5, 92.7, 66.2, 56.5, 55.7, 55.2, 53.4, 38.2, 25.7. LC-MS (m/z) [M – H]− calcd for C20H25N2O5 373.1769, found 373.1746; [M + H]+ calcd for C20H27N2O5 375.1914, found 375.1901.

4-Hydroxy-6,8-dimethoxy-N-(4-methylbenzyl)-2-naphthamide (5a)

White solid, mp 135–136 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 10.16 (1H, s, -OH), 9.01 (1H, s, -NH-), 8.05 (1H, d, J = 1.0 Hz, HAr), 7.33 (1H, d, J = 2.0 Hz, HAr), 7.22 (2H, d, J = 8.0 Hz, HAr), 7.12 (2H, d, J = 8.0 Hz, HAr), 7.02 (1H, d, J = 2.0 Hz, HAr), 6.65 (1H, d, J = 2.0 Hz, HAr), 4.43 (2H, d, J = 6.0 Hz, -CH2-), 3.94 (3H, s, -OCH3), 3.88 (3H, s, -OCH3), 2.29 (3H, s, -CH3). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.6, 158.5, 156.9, 152.2, 136.8, 135.5, 129.5, 128.6, 127.7, 127.3, 121.2, 111.9, 107.7, 98.6, 92.8, 55.6, 55.3, 42.2, 20.5. LC-MS (m/z) [M – H]− calcd for C21H20NO4 350.1398, found 350.1376; [M + H]+ calcd for C21H22NO4 352.1543, found 352.1512.

4-Hydroxy-5,7-dimethoxy-N-(4-methylbenzyl)-2-naphthamide (5b)

White solid, mp 136–138 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 10.14 (1H, s, -OH), 8.99 (1H, s, -NH-), 8.06 (1H, d, J = 1.0 Hz, HAr), 7.32 (1H, d, J = 2.0 Hz, HAr), 7.22 (2H, d, J = 8.0 Hz, HAr), 7.13 (2H, d, J = 8.0 Hz, HAr), 7.04 (1H, d, J = 2.0 Hz, HAr), 6.64 (1H, d, J = 2.0 Hz, HAr), 4.42 (2H, d, J = 6.0 Hz, -CH2-), 3.95 (3H, s, -OCH3), 3.87 (3H, s, -OCH3), 2.27 (3H, s, -CH3). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.7, 158.4, 156.7, 152.0, 137.0, 135.6, 129.4, 128.7, 127.5, 127.2, 121.2, 111.8, 107.9, 98.4, 92.6, 55.7, 55.2, 42.4, 20.6. LC-MS (m/z) [M – H]− calcd for C21H20NO4 350.1398, found 350.1382; [M + H]+ calcd for C21H22NO4 352.1543, found 352.1529.

4-Hydroxy-6,8-dimethoxy-N-(4-methoxybenzyl)-2-naphthamide (6a)

White solid, mp 151–152 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 10.17 (1H, s, -OH), 9.04 (1H, s, -NH-), 8.08 (1H, d, J = 1.5 Hz, HAr), 7.34 (1H, d, J = 2.0 Hz, HAr), 7.13 (2H, d, J = 7.5 Hz, HAr), 7.03 (1H, d, J = 2.0 Hz, HAr), 6.93 (2H, d, J = 8.0 Hz, HAr), 6.65 (1H, d, J = 2.0 Hz, HAr), 4.34 (2H, d, J = 6.0 Hz, -CH2-), 3.98 (3H, s, -OCH3), 3.93 (3H, s, -OCH3), 3.90 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6, δ ppm): 167.0, 158.7, 156.9, 154.4, 152.3, 136.9, 135.4, 129.8, 128.6, 127.7, 127.3, 112.3, 107.7, 98.7, 92.8, 55.6, 55.3, 43.2. LC-MS (m/z) [M – H]− calcd for C21H20NO4 366.1347, found 366.1368; [M + H]+ calcd for C21H22NO4 368.1492, found 368.1487.

4-Hydroxy-5,7-dimethoxy-N-(4-methoxybenzyl)-2-naphthamide (6b)

White solid, mp 154–155 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 10.14 (1H, s, -OH), 9.01 (1H, s, -NH-), 8.09 (1H, d, J = 2.0 Hz, HAr), 7.33 (1H, d, J = 2.0 Hz, HAr), 7.13 (2H, d, J = 8.0 Hz, HAr), 7.04 (1H, d, J = 2.0 Hz, HAr), 6.93 (2H, d, J = 8.0 Hz, HAr), 6.66 (1H, d, J = 2.0 Hz, HAr), 4.33 (2H, d, J = 6.0 Hz, -CH2-), 3.99 (3H, s, -OCH3), 3.92 (3H, s, -OCH3), 3.88 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.8, 159.0, 156.8, 155.0, 153.0, 137.3, 135.9, 129.5, 128.6, 127.5, 127.4, 112.7, 107.9, 98.6, 92.7, 56.0, 55.5, 42.1. LC-MS (m/z) [M – H]− calcd for C21H20NO5 366.1347, found 366.1351; [M + H]+ calcd for C21H22NO5 368.1492, found 368.1502.

N-(4-bromobenzyl)-4-hydroxy-6,8-dimethoxy-2-naphthamide (7a)

White solid, mp 142–143 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 10.17 (1H, s, -OH), 9.01 (1H, s, -NH-), 8.08 (1H, d, J = 2.0 Hz, HAr), 7.86 (2H, d, J = 8.0 Hz, HAr), 7.33 (1H, d, J = 2.0 Hz, HAr), 7.17 (2H, d, J = 8.5 Hz, HAr), 7.03 (1H, d, J = 2.0 Hz, HAr), 6.65 (1H, d, J = 2.0 Hz, HAr), 4.44 (2H, d, J = 5.5 Hz, -CH2-), 3.94 (3H, s, -OCH3), 3.89 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.9, 158.6, 156.7, 152.6, 136.6, 135.2, 133.3, 129.8, 128.7, 127.8, 127.2, 112.3, 107.7, 98.8, 92.6, 55.6, 55.4, 43.2. LC-MS (m/z) [M – H]− calcd for C20H17BrNO4 414.0346, found 414.0340; [M + H]+ calcd for C20H19BrNO4 416.0492, found 416.0485.

N-(4-bromobenzyl)-4-hydroxy-5,7-dimethoxy-2-naphthamide (7b)

White solid, mp 145–146 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 10.16 (1H, s, -OH), 9.02 (1H, s, -NH-), 8.08 (1H, d, J = 2.0 Hz, HAr), 7.87 (2H, d, J = 7.0 Hz, HAr), 7.32 (1H, d, J = 2.0 Hz, HAr), 7.16 (2H, d, J = 7.0 Hz, HAr), 7.03 (1H, d, J = 2.5 Hz, HAr), 6.65 (1H, d, J = 2.0 Hz, HAr), 4.43 (2H, d, J = 5.5 Hz, -CH2-), 3.95 (3H, s, -OCH3), 3.90 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.8, 158.7, 156.7, 152.1, 137.2, 135.9, 129.5, 128.8, 127.5, 127.4, 121.3, 111.6, 107.8, 98.6, 92.8, 55.8, 55.3, 42.5. LC-MS (m/z) [M – H]− calcd for C20H17BrNO4 414.0346, found 414.0351; [M + H]+ calcd for C20H19BrNO4 416.0492, found 416.0497.

N-(4-chlorobenzyl)-4-hydroxy-6,8-dimethoxy-2-naphthamide (8a)

White solid, mp 147–148 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 10.17 (1H, s, -OH), 9.02 (1H, s, -NH-), 8.08 (1H, d, J = 1.0 Hz, HAr), 7.68 (2H, d, J = 8.0 Hz, HAr), 7.33 (1H, d, J = 2.0 Hz, HAr), 7.13 (2H, d, J = 7.5 Hz, HAr), 7.03 (1H, d, J = 2.0 Hz, HAr), 6.65 (1H, d, J = 2.0 Hz, HAr), 4.44 (2H, d, J = 6.0 Hz, -CH2-), 3.92 (3H, s, -OCH3), 3.90 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.7, 158.6, 156.9, 152.4, 136.9, 135.4, 133.2, 129.7, 128.6, 127.7, 127.3, 112.2, 107.6, 98.7, 92.6, 55.6, 55.4, 43.3. LC-MS (m/z) [M – H]− calcd for C20H17ClNO4 370.0852, found 370.0873; [M + H]+ calcd for C20H19ClNO4 372.0997, found 372.0976.

N-(4-chlorobenzyl)-4-hydroxy-5,7-dimethoxy-2-naphthamide (8b)

White solid, mp 149–150 °C. 1H NMR (500 MHz, DMSO-d6, δ ppm): 10.19 (1H, s, -OH), 9.01 (1H, s, -NH-), 8.07 (1H, d, J = 2.0 Hz, HAr), 7.61 (2H, d, J = 7.5 Hz, HAr), 7.33 (1H, d, J = 2.5 Hz, HAr), 7.14 (2H, d, J = 8.0 Hz, HAr), 7.03 (1H, d, J = 2.0 Hz, HAr), 6.65 (1H, d, J = 2.0 Hz, HAr), 4.49 (2H, d, J = 5.5 Hz, -CH2-), 3.98 (3H, s, -OCH3), 3.84 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6, δ ppm): 166.8, 158.6, 156.8, 152.0, 137.2, 135.8, 129.5, 128.6, 127.6, 127.3, 121.3, 111.7, 107.8, 98.5, 92.7, 55.8, 55.4, 42.3. LC-MS (m/z) [M – H]− calcd for C20H17ClNO4 370.0852, found 370.0859; [M + H]+ calcd for C20H19ClNO4 372.0997, found 372.0990.

In Vitro Antimicrobial Activity

Antimicrobial assays were performed using a microtitre broth dilution method with ciprofloxacin (an antibacterial drug) and fluconazole (an antifungal drug) as positive controls.[1] Briefly, the test trays were incubated for 24–48 h at 37 °C for bacterial strains on nutrient agar medium and for 48 h at 25 °C for fungal strains on potato dextrose agar. The different concentration gradients of tested compounds and positive controls in the media were set to 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 μg/mL. The inoculum was prepared by dilution in broth media of each bacterium and fungus to give a final concentration of 5 × 105 CFU/mL. The trays were then incubated at 35 °C for 18–20 h with bacteria and at 25 °C for 72 h with fungi. The MIC was determined to be the lowest concentration that completely inhibits the growth of the organism. All MIC determinations were repeated in triplicate in independent experiments.

In Vitro Anticancer Activity

The cytotoxic activity was performed using the methyl thiazolyl tetrazolium (MTT) method with paclitaxel (anticancer drug) as the positive control. The MTT assay detects the reduction of yellow tetrazolium by metabolically active cells to be purple formazan measured using spectrophotometry.[24] The tumor cell lines were grown on 96-well plates with media consisting of 10% fetal calf serum (FCS), Eagle’s minimum essential medium, 2 mM l-glutamine, 100 μg/mL streptomycin, and 100 IU/mL penicillin with a density of 5 × 103 cells per well. The 96-well plates were incubated at 37 °C for 24 h in 5% CO2. The eight concentrations of tested compounds and paclitaxel (0.5, 1, 5, 10, 25, 50, 80, and 100 μM) in DMSO were then added to each well of 96-well plates (six replicates) using the control DMSO at the same concentration and incubated for 48 h. After that, 10 μL of a fresh solution of MTT reagent was added to each well and incubated at 37 °C for 4 h in 5% CO2. The cells were dissolved in ethanol after the purple precipitate was obtained. The optical density (OD) was measured at 570 nm using a microplate reader. The percent of proliferation inhibition was calculated using the following formulawhere ODt is the optical density of the test compound, ODb is the optical density of blank, and ODc is the optical density of control.

The percent of proliferation inhibition versus the corresponding concentrations of the compound was plotted. The 50% inhibitory concentration (IC50) of each compound was obtained using Graphpad Prism version 8.30.

In Silico ADMET Prediction

The physicochemical properties of all compounds were calculated using the SwissADME web tool. In silico prediction of the absorption, distribution, metabolism, and excretion (ADME) properties and the toxicity (T) risks was performed using an ADMETlab 2.0 descriptor algorithm protocol.[1,25]

Molecular Docking Studies

The structure and energy of ligands were drawn and minimized using a ChemBioDraw Ultra 19.0. The ligand molecules with minimized energy were carried out in the docking simulation using AutoDock Vina.[26,27] Protein molecules of dihydrofolate reductase (PDB ID 3GHW) and vascular endothelial growth factor receptor 2 (PDB ID 2HO4) were retrieved from the protein data bank. The receptors were added to only polar hydrogen and Kollman charges after all of the water molecules have been removed. The grid box for docking simulations was set by AutoDock tools. The ligands were docked with the target to determine the docking parameters using AutoDock Vina with the help of grid-based ligand docking. The pictorial representation of the interaction between the ligands and the target protein was performed by BIOVIA Discovery Studio 2021 software.

In Vitro Dihydrofolate Reductase Inhibition Assay

The dihydrofolate reductase (DHFR) inhibition assay was performed by the CS0340 DHFR assay kit (Sigma). Stock solutions (10 mM) of dihydrofolic acid and NADPH (reduced nicotinamide adenine dinucleotide phosphate) were prepared in assay buffer, pH 7.5. Stock solutions of the test compounds were prepared in DMSO and added to the mixture in different concentrations such as 10–8, 10–7, 10–6, 10–5, and 10–4 M in the respective wells of 96-well plates containing assay buffer. The final concentration of DMSO in each experiment was 0.4%. The changes in absorbance at 340 nm were monitored as a function of time using the test compounds and methotrexate (as a positive control). Percentage inhibition of enzymatic activity was calculated after nullifying the effects of NADPH, folate, and solvent. The 50% inhibitory concentration (IC50) of each compound was calculated by plotting a graph between percentage inhibition and the corresponding concentration of the compound using Graphpad Prism version 8.30.

In Vitro VEGFR-2 Inhibition Assay

The VEGFR-2 tyrosine kinase activity was tested using the VEGFR-2 kinase assay kit (BPS-Bioscience). In brief, the different dilutions (10–8, 10–7, 10–6, 10–5, and 10–4 M) of the VEGFR-2 enzyme and tested compounds were added to the wells of a 96-well plate and incubated at 30 °C for 30 min. Next, 25 mL of ADP-GloTM reagent was added and incubated at room temperature for 45 min. Finally, the kinase detection reagent (Kinase-Glo MAX, Promega) was added to each well. A luminescence signal was detected and quantified by a microplate reader for the determination of the IC50 value of tested compounds after subtracting the blank control value.

Statistical Analysis

All values are expressed in mean ± SEM (standard error of the mean). The differences in IC50 value between tested compounds and paclitaxel were analyzed by one-way ANOVA with Tukey’s HSD post-hoc test using Minitab 19.0 software. The p-value < 0.05 indicates statistically significant.

Results and Discussion

Chemistry

2,4-Dimethoxybenzaldehyde and 3,5-dimethoxybenzaldehyde are the starting materials for the preparation of N-(4-substituted benzyl) and N-(3-morpholinopropyl) 2-naphthamide derivatives. The synthesis of the compounds consists of four steps (Scheme ). The Stobbe condensation was carried out with diethyl succinate using potassium tert-butoxide and tert-butanol by a microwave-assisted method to yield two (dimethoxyphenyl)-3-(ethoxycarbonyl)but-3-enoic acid derivatives (1a–1b). In the second step, cyclization was carried out with sodium acetate and acetic anhydride agents to form 2-naphthoate derivatives (2a–2b) by the microwave-assisted method with excellent yields (91–93%). Next, the ester hydrolysis was carried out in an alkaline environment to yield naphthalene-2-carboxylic acid derivatives (3a–3b). Finally, two carboxylic acid derivatives were converted to acyl halide derivatives with thionyl chloride, followed by an amidation reaction to yield N-(4-substituted benzyl) and N-(3-morpholinopropyl) 2-naphthamide derivatives (4a–4b, 5a–8a, and 5b–8b). In the final stage, the reaction time has been dramatically reduced, as using conventional heating, the reaction is carried out in 3.5 h compared with 30 min heating in the microwave. In addition, the reaction yield has increased ranging between 12 and 13% with microwave assistance (Table ). All compounds have physical–chemical properties of fragments (M. Wt < 500) that follow Lipinski’s rules, which can lead to potent compounds for further development,[23] especially 10 derivatives (4–8) are new compounds.

Scheme 1

Synthesis of N-(4-substituted benzyl) and N-(3-morpholinopropyl) 2-Naphthamide Derivatives

MW, microwave irradiation; t-BuOK, potassium tert-butoxide; t-BuOH, tert-butanol; NaOAc, sodium acetate; Ac2O, acetic anhydride; EtOH, ethanol; MeCN, acetonitrile.

Table 1

Yields and Physicochemical Parameters of N-(4-substituted Benzyl) and N-(3-morpholinopropyl) 2-Naphthamide Derivatives (4a–4b and 5a–5b)a

						yield
entry	group	R	code	physicochemical parameters		Re	MW
1	6,8-(OCH3)2		4a	m. wt: 374.43	NRB: 8	81	93
				NHA: 6	Log P: 2.19
				NHD: 2	PSA: 80.26
2	5,7-(OCH3)2		4b	m. wt: 374.43	NRB: 8	82	94
				NHA: 6	Log P: 2.11
				NHD: 2	PSA: 80.26
3	6,8-(OCH3)2	-CH3	5a	m. wt: 351.40	NRB: 6	82	96
				NHA: 4	Log P: 3.58
				NHD: 2	PSA: 67.79
4	5,7-(OCH3)2	-CH3	5b	m. wt: 351.40	NRB: 6	83	95
				NHA: 4	Log P: 3.60
				NHD: 2	PSA: 67.79
5	6,8-(OCH3)2	-OCH3	6a	m. wt: 367.40	NRB: 7	86	97
				NHA: 5	Log P: 3.20
				NHD: 2	PSA: 77.02
6	5,7-(OCH3)2	-OCH3	6b	m. wt: 367.40	NRB: 7	85	97
				NHA: 5	Log P: 3.28
				NHD: 2	PSA: 77.02
7	6,8-(OCH3)2	-Br	7a	m. wt: 416.27	NRB: 6	80	94
				NHA: 4	Log P: 3.86
				NHD: 2	PSA: 67.79
8	5,7-(OCH3)2	-Br	7b	m. wt: 416.27	NRB: 6	81	95
				NHA: 4	Log P: 3.90
				NHD: 2	PSA: 67.79
9	6,8-(OCH3)2	-Cl	8a	m. wt: 371.81	NRB: 6	83	96
				NHA: 4	Log P: 3.77
				NHD: 2	PSA: 67.79
10	5,7-(OCH3)2	-Cl	8b	m. wt: 371.81	NRB: 6	84	95
				NHA: 4	Log P: 3.82
				NHD: 2	PSA: 67.79

Re and MW, yields of conventional heating (or reflux) and microwave-assisted method (%); Re, reflux; MW, microwave; M. Wt, molecular weight; NHA, number of hydrogen bond acceptors; NHD, number of hydrogen bond donors; NRB, number of rotatable bonds; PSA, polar surface area (Angstroms squared).

1H NMR, 13C NMR, and mass spectra of the synthesized compounds are in accordance with the assigned structures. The 1H NMR spectra of compounds 4–8 indicated the characteristic NH protons of the amide group as a singlet in the δ 9.04–8.52 ppm region as well as the distinctive aromatic protons in the δ 8.09–6.63 ppm region. On the other hand, 1H NMR spectra of compounds 4–8 revealed the appearance of a singlet in the 3.99–3.84 ppm region of the methoxy (-OCH3) moiety. In addition, 1H NMR spectra revealed the appearance of the 3-morpholinopropyl group of compound 4 in the 3.59–1.67 ppm region as well as the methylene (-CH2-Ar) group in the 4.49–4.33 ppm region and the methyl (-CH3) group in the 2.29–2.27 ppm region of compound 5. Furthermore, the C=O moiety of the amide group (δ 167.0–166.6 ppm), CAr (δ 159.0–92.6 ppm), methoxy group (δ 56.0–55.2 ppm), the methylene moiety of the benzyl group (δ 43.3–42.1 ppm), Calkyl of the 3-morpholinopropyl group (δ 66.3–38.2 ppm), and the methyl group (δ 25.8–25.7 ppm) were identified in the 13C NMR spectrum of compounds 4–8. The molecular ion peak M (m/z) of compounds 4–8 was observed in the mass spectrum, confirming the hypothesized structure.

In Vitro Antimicrobial Activities

Antimicrobial activities (exhibited by MIC values) including antifungal activities (CA, Candida albicans and AN, Aspergillus niger) and antibacterial activities against three Gram-negative strains (EC, Escherichia coli; PA, Pseudomonas aeruginosa; SE—Salmonella enterica) and three of Gram-positive strains (SF—Streptococcus faecalis, MSSA, MRSA) of all synthesized compounds are summarized in Table .

Table 2

Antimicrobial Activity (MIC, μg/mL) of Synthesized Compounds 4a–4b, 5a–8a, and 5b–8ba

		antibacterial						antifungal
entry	code	EC	PA	SF	SE	MSSA	MRSA	CA	AN
1	4a	128	128	128	64	64	256	128	256
2	4b	128	128	128	64	64	256	64	256
3	5a	64	64	64	64	32	128	128	256
4	5b	64	64	32	32	16	32	64	256
5	6a	64	64	64	64	32	128	256	256
6	6b	64	128	32	32	32	32	64	256
7	7a	64	64	64	64	32	128	128	256
8	7b	64	64	32	32	32	64	64	256
9	8a	64	64	64	64	32	128	64	256
10	8b	16	64	16	16	8	16	64	256
11	Cipro	16	16	8	8	8	16	ND	ND
12	Flu	ND	ND	ND	ND	ND	ND	4	128

ND, not determined; Cipro, ciprofloxacin; Flu, fluconazole; MIC (μg/mL), ±0.5 μg/mL; EC, Escherichia coli ATCC 25922; PA, Pseudomonas aeruginosa ATCC 27853; SF, Streptococcus faecalis ATCC 29212; SE, Salmonella enterica ATCC 10428; MSSA, methicillin-susceptible strains of Staphylococcus aureus ATCC 29213; MRSA, methicillin-resistant strains of Staphylococcus aureus ATCC 43300; CA, Candida albicans ATCC 10321; AN, Aspergillus niger ATCC 16404. The values in bold highlight the best compounds with the best MIC values compared to positive controls.

Compounds 4a, 4b, 6a–8a, and 6b–7b showed weak to moderate activities against six strains of bacteria (EC, PA, SF, SE, MSSA, and MRSA) and two strains of fungi (CA and AN) with MIC ≥ 32 μg/mL. Compound 5b (5,7-dimethoxy, N-(4-methylbenzyl)) showed good antibacterial activities against one Gram-negative strain SE and three Gram-positive strains SF, MSSA, and MRSA with MIC ranging between 16 and 32 μg/mL as compared to ciprofloxacin (Cipro, MIC = 8–16 μg/mL) but showed moderate activities against the bacterial strains EC and PA with MIC of 64 μg/mL. Moreover, compound 8b (5,7-dimethoxy, N-(4-chlorobenzyl)) showed potent antibacterial activities against all tested Gram-negative (EC and SE) and Gram-positive (SF, MSSA, and MRSA) strains, except for the PA strain with MIC ranging between 8 and 16 μg/mL as compared to ciprofloxacin. In addition, compounds 5b and 8b showed moderate activities against two fungi strains CA and AN with MIC of 64 and 256 μg/mL, respectively, as compared to Flu (MIC of 4 μg/mL at CA and 128 μg/mL at AN). From the structure–activity relationship (SAR), the presence of the 5,7-dimethoxy groups in the naphthalene ring and the N-(4-methylbenzyl)/ N-(4-chlorobenzyl) group is more desirable for enhanced antibacterial activities against SE, SF, MSSA, and MRSA strains in 5b and 8b.

In published studies, N-(-4-aryloxyphenyl) 1-naphthamide derivatives exhibited potent antibacterial and antifungal activities against Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli and two pathogenic fungal strains Aspergillus niger and Candida albicans with the zone of inhibition in (mm) equivalent to that of the reference standard drugs ciprofloxacin and voriconazole.[15] In addition, N-pyridin-4-ylmethyl alkyl-2-naphthamide and phenylpropan-2-yl alkyl-2-naphthamide derivatives showed excellent broad-spectrum antifungal properties (including Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis) with MIC50 ranging between 0.125 and 0.250 μg/mL comparable to fluconazole and naftifine (MIC50 = 0.25–2.00 μg/mL) and also exhibited the obvious antifungal effects against drug-resistant fungi with MIC50 ranging between 2 and 8 μg/mL comparable to fluconazole (MIC50 > 16 μg/mL) and naftifine (MIC50 = 8 to >16 μg/mL).[17] Moreover, N-(4-phenylpiperazin-1-y)/N-cyclohexyl/N-(2-trifluoromethylphenyl) 4-alkoxy-2-naphthamide derivatives were able to synergize with the antibiotics tested and inhibit Nile Red efflux by AcrB in the resistant phenotype as well as displayed a significant increase in efficacy as efflux pump inhibitors against the Escherichia coli strain BW25113.[14] Similar to reported potent compounds in the literature, our active 2-naphthamide derivatives (5b and 8b) contain the 4-hydroxy group on the naphthalene ring and the N-arylmethyl group of the 2-naphthamide nucleus. Compound 8b also showed good antibacterial activity against Escherichia coli with the MIC value of 16 μg/mL similar to N-(-4-aryloxyphenyl) 1-naphthamide and 4-alkoxy-2-naphthamide derivatives.[14,15] Besides, compound 8b (N-(4-chlorobenzyl)) exhibited more than 2-fold potential antibacterial activity against bacterial strains SF, SE, MSSA, and MRSA than compound 5b (N-(4-methylbenzyl)). This may be due to the different four-substituted moieties of the N-benzyl group (Figure ).

Figure 3

Structure of potentially anticancer active compounds 5b and 8b.

Anticancer Activity

The potent anticancer activity of synthesized compounds was tested against four cancer cell lines (C26, colon carcinoma cell line; HepG2, hepatocellular carcinoma cell line; MCF7, human breast cancer cell line; and H69PR, human small cell lung cancer cell line) using paclitaxel (PTX) as a nonselective positive control. The results are summarized in Table .

Table 3

Anticancer Activity (IC50, μM) of Synthesized Compounds 4a–4b, 5a–8a, and 5b–8ba

		anticancer
entry	code	C26	HepG2	MCF7	H69PR
1	4a	23.84 ± 2.93	14.47 ± 2.02	16.09 ± 2.42	52.24 ± 4.81
2	4b	25.05 ± 3.23	17.82 ± 2.46	18.56 ± 1.84	53.68 ± 3.55
3	5a	9.21 ± 2.30	13.61 ± 1.37	10.34 ± 2.11	34.18 ± 5.64
4	5b	3.59 ± 1.04	8.38 ± 1.14	6.75 ± 1.95	28.45 ± 3.70
5	6a	14.38 ± 1.62	17.40 ± 1.51	15.91 ± 1.28	29.24 ± 2.65
6	6b	12.33 ± 2.15	13.96 ± 1.40	13.34 ± 2.69	28.49 ± 3.29
7	7a	18.50 ± 1.73	16.72 ± 1.68	19.82 ± 1.59	22.98 ± 2.87
8	7b	9.83 ± 1.54	12.05 ± 1.09	11.45 ± 1.61	21.29 ± 3.04
9	8a	8.61 ± 1.49	10.08 ± 1.17	12.36 ± 1.34	25.45 ± 3.02
10	8b	2.97 ± 0.98	7.12 ± 1.25	3.58 ± 1.06	12.79 ± 2.83
11	PTX	3.96 ± 0.54	5.75 ± 0.60	2.85 ± 0.43	7.29 ± 0.91

IC50 ± SEM (μM, SEM—standard error of the mean); PTX, paclitaxel; C26, colon carcinoma cell line; HepG2, human hepatocyte carcinoma cell line; MCF7, human breast cancer cell line; H69PR, human small cell lung cancer cell line. The values in bold highlight the best compounds with the best IC50 values compared to positive controls.

Compounds 4a–8a and 4b–7b exhibited weak activity (IC50 = 21.29–53.68 μM) against the H69PR cell line, but only compound 8b (5,7-dimethoxy, N-(4-chlorobenzyl)) showed good anticancer activity against the H69PR cell line with an IC50 value of 12.79 μM as compared to PTX (IC50 = 7.29 μM). In addition, compounds 4a, 4b, 6a, and 7a exhibited moderate activity (IC50 = 14.47–25.05 μM) against C26, HepG2, and MCF7 cell lines. Compounds 5a (6,8-dimethoxy, N-(4-methylbenzyl)), 6b (5,7-dimethoxy, N-(4-methoxybenzyl)), 7b (5,7-dimethoxy, N-(4-bromobenzyl)), and 8a (6,8-dimethoxy, N-(4-chlorobenzyl)) showed moderate activity (IC50 = 10.34–13.34 μM) against the MCF7 cell line with IC50 values less potential than PTX (IC50 = 2.85 μM) about 3.83 to 4.68 times but exhibited good anticancer activity against C26 (IC50 = 8.61–12.33 μM) and HepG2 (IC50 = 10.08–13.96 μM) cell lines with IC50 values larger than PTX (IC50 = 3.96–5.75 μM) about 1.75–2.43 times. Moreover, compound 5b (5,7-dimethoxy, N-(4-methylbenzyl)) showed potent anticancer activity against C26, HepG2, and MCF7 with IC50 of 3.59, 8.38, and 6.75 μM, respectively, as compared to PTX. However, compound 5b exhibited weak anticancer activity against H69PR with the IC50 of 28.45 μM. In particular, compound 8b showed the strongest anticancer activity among all compounds against C26, HepG2, and MCF7 with IC50 of 2.97, 7.12, and 3.58 μM, respectively, as compared to PTX. As seen in Figure , compounds 5b and 8b exhibited better anticancer activity than PTX on the C26 cell line but exhibited weaker anticancer activity than PTX on HepG2, MCF7, and H69PR cell lines. Target engagement with electron-donating substituent -OCH3 at positions 5 and 7 on the naphthalene ring and the N-(4-methylbenzyl)/N-(4-chlorobenzyl) group may be responsible for its anticancer activity as compared to other compounds.

Figure 4

Comparison of anticancer activity (IC50 values) between active compounds and PTX (C26, colon carcinoma cell line; HepG2, human hepatocyte carcinoma cell line; MCF7, human breast cancer cell line; H69PR, human small cell lung cancer cell line; PTX, paclitaxel; (*), significantly different compared with IC50 of 8b and PTX with p < 0.05).

In published studies with similar structures, one chalcone containing a naphthalene moiety with a diethylamino group at the para position of the phenyl ring showed potent anticancer activity against HCT116 and HepG2 cell lines with IC50 values of 1.20 and 1.02 μM, respectively.[11] Moreover, 6-aryloxy N-methyl 1-naphthamide exhibited selective and equally high potency against FGFR1/2 and VEGFR-2 with IC50 values less than 5.0 nM.[10] In particular, the anilinopyrimidine-based 1-naphthamide derivative with the N-phenyl group showed high VEGFR-2 inhibitory potency in both enzymatic and VEGF-induced HUVEC (human umbilical vein endothelial cell) proliferation assays with IC50 values of 0.5 and 9.8 nM, respectively.[12] Similar to reported potent compounds in the literature, our active 2-naphthamide derivatives (5b and 8b) contain the N-alkyl or N-arylmethyl group (Figure ). Besides, compounds 5b–8b (5,7-dimethoxy) exhibited better anticancer activity than compounds 5a–8a (6,8-dimethoxy) against C26, HepG2, and MCF7 cancer cell lines. This result has shown that the positions of two methoxy groups on the naphthalene nucleus have an important role in enhancing the antitumor activity.

In Silico ADMET Profile

In the present study, a computational study of the two most active compounds was conducted to determine the surface area and other physicochemical properties, according to the directions of Lipinski’s rule.[24] Lipinski suggested that the absorption capacity of a compound is much better if the molecule achieves at least three out of four of the following rules: (i) HB donor groups ≤ 5, (ii) HB acceptor groups ≤ 10, (iii) M. Wt less than 500, and (iv) log P less than 5. In this study, compounds 5a and 5b follow all Lipinski’s rules. All of the highest active derivatives have a number of hydrogen bonding acceptor groups ranging between 4 and 6 and hydrogen bonding donor groups of 2. Also, molecular weights range from 351.50 to 374.43 and log P values range between 2.12 and 2.89, and all of these values agree with Lipinski’s rules.

After assessing ADMET profiles of active compounds (Table ), we can suggest that these derivatives have the advantage of better intestinal absorption in humans than Cipro, Flu, and PTX, as all compounds showed Caco-2 permeability higher than the control drugs and higher than −5.15 log unit. Besides, compounds 5b and 8b showed high passive MDCK permeability (>20 × 10–6 cm/s) as compared to the reference drugs. In addition, all compounds showed good plasma protein binding capacity with PPB > 98.0% as compared to Cipro (PPB = 37%), Flu (PPB = 62%), and PTX (95%). As for prediction of the BBB (blood–brain barrier) permeability, compounds 5b and 8b demonstrated the inability to penetrate BBB similar to Cipro and PTX. Therefore, toxicity and side effects on the brain as well as the central nervous system may not appear.

Table 4

ADMET Profile of the Most Active Compounds Ciprofloxacin, Fluconazole, and Paclitaxela,ab

parameter	5b	8b	Cipro	Flu	PTX
Absorption
Caco-2 permeability	–4.946	–4.955	–5.269	–4.950	–5.461
MDCK permeability	1.2 × 10–5	1.5 × 10–5	3.0 × 10–6	2.8 × 10–5	5.4 × 10–5
Pgp-inhibitor	++	–	–––	–––	+++
Pgp-substrate	–––	–––	+++	–––	+++
HIA	+++	+++	+++	+++	+++
F20%	+++	+++	+++	+++	–––
F30%	––	++	+++	+++	–––
Distribution
PPB (%)	98.627	99.350	37.456	61.763	94.571
VD (L/kg)	0.665	0.678	2.324	0.835	0.907
BBB penetration	––	–––	–––	+++	––
Fu (%)	1.631	1.067	78.856	51.002	6.779
Log Kp (cm/s)	–5.67	–5.61	–9.090	–7.920
Metabolism
CYP1A2 inhibitor	++	+++	––	-	–––
CYP1A2 substrate	+++	+++	––	-	–––
CYP2C19 inhibitor	++	+++	–––	+	–––
CYP2C19 substrate	-	––	––	–––	–––
CYP2C9 inhibitor	++	++	–––	––	++
CYP2C9 substrate	++	++	–––	+	–––
CYP2D6 inhibitor	+	++	–––	–	–––
CYP2D6 substrate	++	++	––	––	–––
CYP3A4 inhibitor	++	++	–––	–	++
CYP3A4 substrate	–	––	––	––	+
Excretion
CL (mL/min/kg)	7.876	7.201	3.214	5.960	3.416
T1/2	0.504	0.358	0.056	0.228	0.028
Toxicity
hERG blockers	+	++	––	–––	––
H-HT	––	––	+++	+++	+++
DILI	–	+	+++	+++	+++
AMES toxicity	–	–	––	++	–––
rat oral acute toxicity	–––	–––	––	+++	–
FDAMDD	+	++	++	++	++
skin sensitization	+	+	+	+++	–––
carcinogenicity	–––	–––	–	+++	–––
eye corrosion	–––	–––	–––	–––	–––
eye irritation	–––	–––	–––	––	–––
respiratory toxicity	–––	–––	++	++	+++
Tox21 Pathway
NR-AR	–––	–––	++	–––	––
NR-AR-LBD	–––	–––	–––	–––	+++
NR-AhR	+++	+++	––	+	–––
NR-Aromatase	–––	––	–––	+++	++
NR-ER	––	––	–	–––	+
NR-ER-LBD	–––	–––	–––	–––	+
NR-PPAR-γ	++	++	–––	–––	+++
SR-ARE	+	++	–	––	++
SR-ATAD5	+	+	–––	–––	+++
SR-HSE	––	––	–––	–––	–
SR-MMP	+	++	–––	––	+++
SR-p53	+	++	–––	–––	+++
Toxicophore Rules
acute toxicity rule	0 alert	0 alert	1 alert	0 alert	0 alert
genotoxic carcinogenicity rule	1 alert	1 alert	1 alert	0 alert	1 alert
nongenotoxic carcinogenicity rule	0 alert	1 alert	1 alert	0 alert	0 alert
skin sensitization rule	2 alerts	2 alerts	0 alert	0 alert	6 alerts
aquatic toxicity rule	0 alert	1 alert	1 alert	1 alert	2 alerts
nonbiodegradable rule	0 alert	1 alert	2 alerts	1 alert	2 alerts
SureChEMBL rule	0 alert	0 alert	0 alert	0 alert	0 alert
FAF-Drugs4 rule	1 alert	1 alert	1 alert	1 alert	1 alert

Cipro—ciprofloxacin, Flu—fluconazole, PTX—paclitaxel, Caco-2 permeability (optimal: higher than −5.15 Log unit), MDCK permeability (low permeability: <2 × 10–6 cm/s, medium permeability: 2–20 × 10–6 cm/s, high passive permeability: >20 × 10–6 cm/s), Pgp—P-glycoprotein, HIA—human intestinal absorption (−: <30%, +: ≥30%), F—bioavailability (−: 20%), Log Kp (skin permeation), CL—clearance (low: <5 mL/min/kg, moderate: 5–15 mL/min/kg, high:>15 mL/min/kg), T (category 1: long half-life (>3 h), category 0: short half-life (<3 h)), H-HT—human hepatotoxicity, DILI—drug-induced liver injury, FDAMDD—maximum recommended daily dose, AR—androgen receptor, AR-LBD—androgen receptor ligand-binding domain, AhR—aryl hydrocarbon receptor, ER—estrogen receptor, ER-LBD—estrogen receptor ligand-binding domain, PPAR-γ—peroxisome proliferator-activated receptor γ, ARE—antioxidant response element, ATAD5—ATPase family AAA domain-containing protein 5, HSE—heat shock factor response element, MMP—mitochondrial membrane potential.

The output value is the probability of being inhibitor/substrate/active/positive/high toxicity/sensitizer/carcinogens/corrosives/irritants (category 1) or noninhibitor/nonsubstrate/inactive/negative/low toxicity/nonsensitizer/noncarcinogens/noncorrosives/nonirritants (category 0). For the classification endpoints, the prediction probability values are transformed into six symbols: 0–0.1(−–−), 0.1–0.3(−−), 0.3–0.5(−), 0.5–0.7(+), 0.7–0.9(++), and 0.9–1.0(+++).

Two active compounds 5b (log Kp of −5.67 cm/s) and 8b (log Kp of −5.61 cm/s) showed better skin permeation than Cipro (log Kp of −9.09 cm/s) and Flu (log Kp of −7.92 cm/s). The cytochrome enzymes could be moderate to strongly inhibited under the effect of active compounds especially CYP1A2 and CYP2D6, while Cipro and Flu could not. These compounds also showed the effect of CYP2C9 and CYP3A4 inhibition as compared with PTX. Moreover, compounds 5b (7.876 mL/min/kg) and 8b (7.201 mL/min/kg) were classified as a moderate clearance level ranging between 5 and 15 mL/min/kg as compared with Flu (CL = 5.69 mL/min/kg). On the other hand, two new ligands did not show rat oral acute toxicity, carcinogenicity, eye corrosion, eye irritation, and respiratory toxicity. Therefore, these potent compounds showed lower overall toxicity than the reference drugs Cipro, Flu, and PTX. Finally, these compounds also exhibited good “Tox21 pathway” and “toxicophore rules” profiles as compared to the reference drug PTX.

In Silico Molecular Docking Studies

After ADMET profiling, docking studies were carried out to predict the most suitable binding pose and inhibition mechanism of the most active compounds. Based on the principle that similar compounds tend to bind to the same proteins as well as in vitro enzyme inhibition of the reported homologous naphthamide structures, two target proteins were selected, including dihydrofolate reductase (DHFR) for antibacterial and anticancer activities and vascular endothelial growth factor receptor 2 (VEGFR-2) for anticancer activity.[12,23] The protein–ligand complex is formed through hydrogen bonds (both from side chains and backbones), electrostatic interactions (π–cation and π–anion), and hydrophobic interactions (π–σ, π–π, alkyl, π–alkyl) of the binding interface (Table ).[24]

Table 5

In Silico Molecular Docking Results of Active Compounds and Standard Drugsa

receptor	compound	affinity (Kcal/mol)	hydrogen bond number	interaction types
DHFR	5b	–9.9	2	Hydrogen (ASP21 - 2.86 Å); C-Hydrogen (GLY17 - 3.49 Å); π–π (PHE34); alkyl (ILE60, LEU67, LYS55); π-alkyl (ILE16, ILE60, PHE34, VAL115)
	8b	–9.8	1	C-Hydrogen (GLU30 - 3.46 Å); π–π (PHE31, PHE34); alkyl (ALA9, PRO61, VAL8, ILE7); π-alkyl (PHE31, PHE34, ALA9, PRO61)
	PTX	–9.9	4	Hydrogen (GLY117 - 2.65 Å; THR56 - 2.09; 2.39 Å); C-Hydrogen (SER119 - 2.97 Å); π-cation (LYS55); π-alkyl (ILE16, LYS55)
VEGFR-2	5b	–9.5	3	Hydrogen (LEU838 - 2.03 Å; CYS917 - 1.92 Å); C-Hydrogen (GLU915 - 3.79 Å); π-σ (LEU838); π-Sulfur (CYS1043); π–π (PHE1045); alkyl (LYS866, VAL846); π-alkyl (ALA864, LEU838, VAL846, VAL914)
	8b	–9.8	1	C-Hydrogen (LYS918 - 3.73 Å); π-σ (LEU838); π–π (PHE1045); alkyl (LEU887, VAL897, CYS1043); π-alkyl (PHE916, VAL897, VAL914, CYS1043)
	PTX	–8.2	8	Hydrogen (ARG1049 - 2.68; 2.98 Å; ASN921 - 2.78 Å; LEU838 - 2.30 Å; LYS1053 - 2.39; 2.60 Å); C-Hydrogen (ASP1062 - 3.21 Å; GLY920 - 3.46 Å); π-σ (LEU838); π–π (PHE916); π-alkyl (ALA864, VAL846, VAL914)

DHFR, dihydrofolate reductase; VEGFR-2, vascular endothelial growth factor receptor 2; PTX, paclitaxel.

On the DHFR receptor, compounds 5b (−9.9 Kcal/mol) and 8b (−9.8 Kcal/mol) showed similar high affinity for PTX (−9.9 Kcal/mol) at the active site. Compound 5b established a conventional hydrogen bond (2.86 Å) with ASP21 amino acid and one carbon–hydrogen bond with GLY17 amino acid at the 4-hydroxy (4-OH) group with a bond length of 3.49 Å. Compound 5b also showed hydrophobic interactions (π–π stacking, alkyl, π–alkyl) with PHE34, ILE60, LEU67, LYS55, ILE16, and VAL115 amino acids at the aromatic rings and the methyl (-CH3) group with bond lengths of 3.94, 3.94, 4.63, 4.71, 4.63, and 5.26 Å, respectively. Hydrophobic interaction with LYS55 and ILE16 amino acids of compound 5b is similar to that of PTX. Besides, compound 8b established one carbon–hydrogen bond (3.46 Å) with GLU30 amino acid and the 7-methoxy (7-OCH3) group. Similarly, compound 8b also showed hydrophobic interactions (π–π stacking, alkyl, π–alkyl) with PHE31, PHE34, ALA9, PRO61, VAL8, and ILE7 amino acids at the aromatic rings and 4-chloro (4-Cl) group with bond lengths of 3.74, 3.97, 4.11, 4.91, 5.42, and 4.36 Å, respectively (Figure ). These results suggested that DHFR is the most likely target for the anticancer activity of compounds 5b and 8b.

Figure 5

2D and 3D representation of the interaction of active molecules 5b and 8b and paclitaxel (PTX) with dihydrofolate reductase (DHFR).

On the VEGFR-2 receptor, compounds 5b and 8b showed strong interactions with the affinities of −9.5 and −9.8 Kcal/mol, respectively, compared with the reference drug PTX (−8.2 Kcal/mol). Compound 5b established two conventional hydrogen bonds (1.92–2.03 Å) with LEU838 and CYS917 amino acids at the 4-hydroxy (4-OH) and amide (-CONH-) groups and one carbon–hydrogen bond with GLU915 amino acid at the methylene (-CH2-) moiety of the N-(4-methylbenzyl) group with a bond length of 3.79 Å. In addition, compound 5b showed hydrophobic interactions (π–σ, π–sulfur, π–π, alkyl, π–alkyl) with LEU838, CYS1043, PHE1045, LYS866, VAL846, ALA864, and VAL914 amino acids with bond lengths of 3.93, 5.32, 4.77, 4.35, 4.77, 5.02, and 4.88 Å, respectively (Figure ). On the other hand, compound 8b established one carbon-–hydrogen bond (3.46 Å) with LYS918 amino acid and the 5-methoxy (5-OCH3) group. Compound 8b also showed hydrophobic interactions (π–σ, π–π T-shaped, alkyl, π–alkyl) with LEU838, PHE1045, LEU887, VAL897, CYS1043, PHE916, and VAL914 amino acids at the benzene and naphthalene rings and 4-chloro (4-Cl) group with bond lengths of 3.97, 5.37, 5.13, 3.53, 4.60, 4.98, and 4.83 Å, respectively. In particular, compounds 5b and 8b showed interactions with LEU838 and VAL914 with the crucial residue of the VEGFR-2 protein from Escherichia coli that resembles the cocrystallization ligand and PTX.

Figure 6

2D and 3D representation of the interaction of active molecules 5b and 8b and paclitaxel (PTX) with vascular endothelial growth factor receptor 2 (VEGFR-2).

In summary, from the in silico molecular docking study results, it can be concluded that compounds 5b and 8b were considered the best dock ligand in antitumor targets such as DHFR and VEGFR-2. Moreover, compound 8b showed a higher affinity than compound 5b on the VEGFR-2 receptor. This may explain the result that compound 8b showed better in vitro antitumor activity than compound 5b.

In Vitro DHFR and VEGFR-2 Inhibitory Activities

The results of in silico molecular docking studies have predicted that DHFR and VEGFR-2 are potential receptors to explain the mechanism of antibacterial and anticancer activities for the active derivatives. Therefore, compounds 5b and 8b were subjected to further assay for their ability to inhibit DHFR and VEGFR-2 using methotrexate and sorafenib as positive controls. The results presented in Table show that compounds 5b and 8b managed to inhibit DHFR kinase with IC50 < 10 μM and VEGFR-2 kinase with IC50 < 1 μM. Compounds 5b and 8b showed good DHFR inhibitory activity with IC50 values of 9.085 and 7.881 μM, respectively, compared with positive control methotrexate (IC50 = 0.022 μM). Especially, it has been observed that compounds 5b and 8b showed excellent VEGFR-2 inhibitory activity with IC50 values of 0.623 and 0.384 μM, respectively, which is comparable to sorafenib with the IC50 value of 0.069 μM. In addition, these compounds exhibited 15–20 times better inhibitory activity on the VEGFR-2 receptor than the DHFR receptor. Furthermore, many naphthamide derivatives have also shown highly selective and strong VEGFR-2 inhibitory activity in some published studies.[12,23,28] Therefore, the study results may suggest that the potent anticancer activity of our active compounds is mediated by interaction with the VEGFR-2 protein.

Table 6

50% Inhibitory Concentration (IC50, μM) of Active Compounds for DHFR and VEGFR-2 Inhibitory Activitiesa

compound	DHFR	VEGFR-2
5b	9.085 ± 0.511	0.623 ± 0.056
8b	7.881 ± 0.442	0.384 ± 0.047
methotrexate	0.022 ± 0.008	ND
sorafenib	ND	0.069 ± 0.021

ND, not determined; IC50 ± SEM (μM, SEM—standard error of the mean).

Conclusions

In summary, 10 novel 2-naphthamide derivatives have been designed, synthesized, and evaluated for their antimicrobial and anticancer activities. The reaction yield was greatly increased as well as the reaction time was significantly reduced using a microwave-assisted method. Compound 8b showed potent antibacterial activity against five strains EC, SF, SE, MSSA, and MRSA with MIC ranging between 8 and 16 μg/mL as compared to ciprofloxacin. Moreover, compounds 5b and 8b also exhibited potent anticancer activity with IC50 < 8.5 μM against C26, HepG2, and MCF7 cell lines compared with the reference drug PTX. From the structure–activity relationship, the presence of the N-(4-methyl/4-chlorobenzyl), 4-hydroxy, and 5,7-dimethoxy groups of the 2-naphthamide scaffold is more desirable for enhanced antibacterial and antitumor activities in 5b and 8b. Molecular docking predicted that DHFR and VEGFR-2 proteins from Escherichia coli are the most suitable targets for antibacterial and anticancer activities. Compound 8b being the most potent anticancer displayed good interactions against DHFR and VEGFR-2 with the affinity of −9.8 Kcal/mol as compared with PTX (−9.9 Kcal/mol at DHFR and −8.2 Kcal/mol at VEGFR-2). Moreover, compound 8b showed excellent in vitro VEGFR-2 inhibitory activity with the IC50 value of 0.384 μM compared with the positive control sorafenib (IC50 = 0.069 μM). The obtained ADMET results suggest that these derivatives showed a good ADMET profile. This work paved the way for the synthesis of more potent compounds based on 2-naphthamide scaffolds and explored their various potential biological activities as well as their mechanism of action.