<i>Calotropis gigantea</i> Assisted Synthesis of Zinc Oxide Nanoparticle Catalysis: Synthesis of Novel 3-Amino Thymoquinone Connected 1,4-Dihyropyridine Derivatives and Their Cytotoxic Activity

Gobinath, Perumal; Packialakshmi, Ponnusamy; Hatamleh, Ashraf Atef; Al-Dosary, Munirah Abdullah; Al-Wasel, Yasmeen Abdualrhman; Balasubramani, Ravindran; Surendrakumar, Radhakrishnan; Idhayadhulla, Akbar

doi:https://doi.org/10.1155/2022/9697057

Journal of Nanomaterials

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Phytonanotechnology: Characterization and Medicinal Applications

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Research Article | Open Access

Volume 2022 | Article ID 9697057 | https://doi.org/10.1155/2022/9697057

Calotropis gigantea Assisted Synthesis of Zinc Oxide Nanoparticle Catalysis: Synthesis of Novel 3-Amino Thymoquinone Connected 1,4-Dihyropyridine Derivatives and Their Cytotoxic Activity

Perumal Gobinath,¹Ponnusamy Packialakshmi,¹Ashraf Atef Hatamleh,²Munirah Abdullah Al-Dosary,²Yasmeen Abdualrhman Al-Wasel,²Ravindran Balasubramani,³Radhakrishnan Surendrakumar,¹and Akbar Idhayadhulla¹

Academic Editor: P. Davide Cozzoli

Received21 May 2021

Accepted29 Mar 2022

Published23 Apr 2022

Abstract

The synthesis of biologically active 1,4-dihyripyridine derivatives using a Calotropis gigantea leaf powder and ZnO NPs used as catalyst under solvent-free conditions at room temperature via grinding method has been established in a single-stage, mild, and environmentally friendly green process. The procedure is fast and effective and produces high yields. Three cancer cell lines were used to assess the cytotoxic activity of 1,4-dihyropyridine derivatives. The cytotoxicity of 1,4-dihyropyridine compound 1f (HepG2, LC₅₀-0.50 μM, MCF-7, LC₅₀-0.64 μM, and HeLa, LC₅₀-0.52 μM) was found to be highly active. The synthesized derivatives demonstrated their safety by causing substantially less cytotoxicity in normal cell lines HEK-293, LO2, and MRC5 with g/mL. As a result, compound 1f could serve as a high-impact molecule for the production of anticancer drugs in the future.

1. Introduction

Green synthesis of nanoparticles has received a lot of attention in recent years as a simple, inexpensive, and environmentally friendly alternative to chemical and physical synthesis methods. Cancer is a term used to describe a group of more than 100 diseases that include the irregular and uncontrolled division of cells and affect major body organs [1, 2]. Cancer has surpassed tobacco as the second leading cause of death worldwide, accounting for 1 in every 6 deaths in 2018, with a total of 9.6 million deaths. Different forms of cancers have affected both men and women [3]. Resistance to current multidrug chemotherapy is their slow cure rate, as well as their dreadful and toxic side effects on patients [4]. As a result, exemplary efforts are needed to identify newer, more efficient, and less toxic chemotherapeutic agents for the treatment of cancers [5].

TQ has antioxidant, anti-inflammatory, and cancer-fighting chemical structures which are shown in Figure 1. TQ has been shown to be effective in the treatment of symptoms in a variety of disease models, including cancer, diabetes, asthma, encephalomyelitis, and arthritis [6]. Thymoquinone serves as a potent antioxidant in normal tissues, inhibiting superoxide radical synthesis and lipid peroxidation while also increasing the activities of antioxidant enzymes such as SOD, catalase, GSH, GST, and quinone reductase [7].

TQ is a naturally occurring phytochemical compound that is the main constituent of Nigella sativa (black seed) volatile oil and was first isolated in 1963 by El-Dakhakhny [8]. Both in vitro and in vivo, this monoterpene was found to have potent anticancer properties [9–11]. TQ induces apoptosis in vitro through p53-dependent and p53-independent pathways, while causing no toxicity in normal cells, according to recent research [12, 13]. TQ, as a short-chain ubiquinone derivative, can act as a prooxidant and thus cause oxidative stress by triggering the development of reactive oxygen species (ROS) [14], which has been related to its proapoptotic effect in colon cancer and leukaemia cells [15, 16]. TQ has recently been shown to suppress metastasis in CPT-11-R LoVo colon cancer cells by inhibiting NF-κB and activating JNK and p38, as well as to prevent epithelial–mesenchymal transformation in cancer cells by inhibiting the PI3K/AKT signalling axis [17, 18]. In addition, in vivo tests revealed that TQ has a low overall toxicity [19, 20], making it a viable candidate for clinical applications [21]. TQ has been shown to improve the effectiveness of many chemotherapeutic agents in vitro and in vivo, including in cancers that are immune to them, such as cisplatin in lung cancer and many solid tumours [22, 23]. Many researchers have written on the possible utility of ZnO nanoparticles in the treatment of cancer in recent years in the biological field. It has been investigated as an effective catalyst for several organic transformations [17–21], such as the Mannich reaction and the Knoevenagel condensation reaction, in the synthesis of coumarin, due to various advantages associated with its eco-friendly nature.

Calcium channel blockers, cardiovascular, antihypertensive, antitumor, anti-inflammatory, analgesic, neuroprotectant, platelet antiaggregator, anti-ischemic, anti-Alzheimer, antimicrobial, and insecticidal are some of the biological functions of 1,4-DHP structures which are shown in Figure 1 [24–30].

Aside from their biological significance, 1,4-DHPs are useful sources of hydride for reductive amination and synthetic intermediates for the production of biologically significant molecules [31]. Since Hantzsch’s first classical study on the synthesis of 1,4-DHPs by multicomponent reactions (MCR) in 1882 [32], there has been a lot of interest in improving and developing new methods to generate therapeutically relevant molecules [33]. MCR has been focusing on the development of highly diverse and functionalized molecules in recent years [34–43]. In certain cases, these synthetic hybrids with partial natural compound structures are more active than their parent compounds [44, 45]. To our knowledge, no thymoquinone-linked 1,4-dihydropyridine derivatives have been identified using the grindstone method in the presence of ZnO NP catalyst. As a result, the natural product TQ can be considered a promising anticancer active compound that is ideal for using the Hantzsch method to create new potent anticancer agents.

2. Materials and Methods

2.1. General Methods

All the melting points were determined in open capillary tubes and are uncorrected. The FT-IR spectra were recorded the KBr disk technique on a Shimadzu 8201pc (4000–1000 cm⁻¹). The ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker DRX-300 MHz. For SEM research, a Scanning Electron Microscope (SEM) model VP-1450 (LEO, Co., Germany) was used. An LEO 912 AB transmission electron microscopy (TEM) instrument was used for the study. Mass spectra were recorded by Perkin Elmer GCMS model Clarus SQ8 (EI). The elemental analysis (C, H, and N) was performed using an elemental analyser model (Varian EL III). TLC was used to verify the purity of each compound using silica-gel, 60F254 aluminium sheets as adsorbent, and iodine used for visualization.

2.1.1. Preparation of the Calotropis gigantea Leaf Extract

The extract for the reduction of zinc ions (Zn²⁺) to zinc nanoparticles (ZnO) was made by combining 60 g of washed dried fine cut leaves with 200 mL of sterile distilled water in a 250 mL glass beaker. After that, the mixture was boiled for 60 minutes or until the aqueous solution’s colour changed from watery to light yellow. The extract was allowed to cool to room temperature before being filtered onto filter paper. To be used in future research, the extract was kept in the refrigerator.

2.1.2. Preparation of Zinc Nanoparticles

A stirrer-heater was used to boil 60 mL of C. gigantea leaf extract to 70-80 degrees Celsius for the synthesis nanoparticle. As the temperature reached 70 degrees Celsius, 5 grammes of zinc nitrate was applied to the solution. This mixture is then reduced to a deep yellow paste by boiling it. This paste was then deposited in a ceramic crucible and heated for 3 hours at 350 degrees Celsius in an air-heated furnace. For characterization purposes, a light-yellow powder was obtained and carefully collected and packed. To obtain a finer nature for characterization, the substance was mashed in a mortar-pestle. Scheme 1 depicts the synthesis of ZnO-NPs.

2.1.3. General Procedure for the Synthesis of Diethyl 1-(2-Isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (1a)

A reaction mixture consisting of ethyl acetoacetate (0.01 mol, 1.3 mL), benzaldehyde (0.01 mol, 1.06 mL), and 3-aminothymoquinone (0.01 mol, 1.80 g) with green catalyst ZnO NPs was mixed in a mortar and ground for up to 15-20 min at room temperature. Subsequently, the product was washed with excess ice-cold water, and then, the product was filtered and washed with water and dried to afford the crude product. The crude precipitate dissolved in DMF and centrifuged to separate the catalyst and later the catalyst was washed several times with EtOH, then dried, and reutilized three times for the same reaction. Then, the product was confirmed via TLC. The precipitate was recrystallized with ethanol. The product was obtained in excellent yield. The same method was followed for the synthesis of compounds 1b–j.

(1) Synthesis of Diethyl 1-(2-Isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (1a). The results are as follows: dark red solid, yield 85%; mp 46°C; IR (kBr) (cm^-1); 3028 (Ar-H), 2948, 2820 (C-H str of CH₃), 1745 (C=O in ester), 1650, 1620 (C=O); ¹H NMR (300 MHz, DMSO-d₆): 7.26-7.23 (m, 5H, Ph), 6.29 (s, 1H, -CH in TQ), 4.93 (s, 1H, CH-Ph), 4.16 (m, 4H, -(CH₂)₂), 2.52 (m, 1H, CH in TQ), 2.26 (s, 6H, 2,6-CH₃), 1.73 (s, 3H, -CH₃), 1.22 (t, 6H, (-CH₃)₂), 1.06 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.21, 178.53, 167.25, 153.57, 144.69, 144.41, 143.82, 142.73, 133.84, 128.65, 127.76, 125.77, 102.38, 61.79, 43.53, 21.02, 15.44, 14.26, 14.18; EI-MS: 491 (M+, 20%); elemental analysis (C₂₉H₃₃NO₆): calculated: C, 70.86; H, 6.77; N, 2.85%; found: C, 70.84; H, 6.75; N, 2.83%.

(2) Synthesis of Diethyl 4-(4-Chlorophenyl)-1-(2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1b). The results are as follows: white solid, yield 76%; mp 168°C; IR (kBr) (cm^-1); 3041 (Ar-H), 2963, 2871 (C-H str of CH₃), 1755 (C=O in ester), 1630, 1640 (C=O), 840 (C-Cl); ¹H NMR (300 MHz, DMSO-d₆):7.37-7.17 (m, 4H, Ph-Cl), 6.32 (s, 1H, -CH in TQ), 4.95 (s, 1H, CH-Ph), 4.16 (m, 4H, -(CH₂)₂), 2.53 (m, 1H, CH in TQ), 2.28 (s, 6H, 2,6-CH₃), 1.74 (s, 3H, -CH₃), 1.28 (t, 6H, (-CH₃)₂), 1.08 (d, J Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆):187.22, 178.54, 167.26, 153.58, 144.70, 143.83, 142.74, 142.50, 133.85, 131.30, 130.40, 128.70, 102.39, 61.80, 43.51, 21.03, 15.45, 14.27, 14.19; EI-MS: 526 (M+, 20%); elemental analysis (C₂₉H₃₂ClNO₆): calculated: C, 66.22; H, 6.13; N, 2.66%; found: C, 66.20; H, 6.11; N, 2.64%.

(3) Synthesis of Diethyl 4-(2-Chlorophenyl)-1-(2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1c). The results are as follows: white solid, yield 89%; mp 184°C; IR (kBr) (cm^-1); 3034 (Ar-H), 2960, 2871 (C-H str of CH₃), 1741 (C=O in ester), 1665, 1623 (C=O), 840 (C-Cl); ¹H NMR (300 MHz, DMSO-d₆):7.65-7.18 (m, 4H, Ph-Cl), 6.34 (s, 1H, -CH in TQ), 4.96 (s, 1H, CH-Ph), 4.19 (m, 4H, -(CH₂)₂), 2.54 (m, 1H, CH in TQ), 2.29 (s, 6H, 2,6-CH₃), 1.75 (s, 3H, -CH₃), 1.29 (t, 6H, (-CH₃)₂), 1.09 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.23, 178.55, 167.27, 153.59, 144.71, 143.84, 143.70, 142.75, 133.86, 131.40, 128.71, 127.10, 126.70, 126.41, 102.40, 61.81, 38.40, 21.04, 15.46, 14.28, 14.20; EI-MS: 526 (M+, 20%); elemental analysis (C₂₉H₃₂ClNO₆): calculated: C, 66.22; H, 6.13; N, 2.66%; found: C, 66.20; H, 6.11; N, 2.64%.

(4) Synthesis of Diethyl 4-(4-Hydroxyphenyl)-1-(2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1d). The results are as follows: white solid, yield 80%; mp 204°C; IR (kBr) (cm^-1); 3098 (OH),3032 (Ar-H), 2964, 2865 (C-H str of CH₃), 1764 (C=O in ester), 1652, 1638 (C=O); ¹H NMR (300 MHz, DMSO-d₆):7.06-6.63 (m, 4H, Ph-OH), 6.35 (s, 1H, -CH in TQ), 5.37 (s, 1H, OH), 4.97 (s, 1H, CH-Ph), 4.20 (m, 4H, -(CH₂)₂), 2.55 (m, 1H, CH in TQ), 2.30 (s, 6H, 2,6-CH₃), 1.76 (s, 3H, -CH₃), 1.30 (t, 6H, (-CH₃)₂), 1.10 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.24, 178.56, 167.28, 155.50, 153.60, 144.72, 143.85, 142.76, 137.71, 133.87, 130.40, 115.81, 102.41, 61.82, 43.52, 21.05, 15.47, 14.29, 14.21; EI-MS: 508 (M+, 20%); elemental analysis (C₂₉H₃₃NO₇): calculated: C, 68.62; H, 6.55; N, 2.76%; found: C, 68.60; H, 6.53; N, 2.74%.

(5) Synthesis of Diethyl 4-(2-Hydroxyphenyl)-1-(2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1e). The results are as follows: yellow solid, yield 90%; mp 112°C; IR (kBr) (cm^-1); 3089 (OH), 3069 (Ar-H), 2958, 2825 (C-H str of CH₃), 1762 (C=O in ester), 1662, 1658 (C=O); ¹H NMR (300 MHz, DMSO-d₆):7.09-6.83 (m, 4H, Ph-OH), 6.29 (s, 1H, -CH in TQ), 5.30 (s, 1H, OH), 4.98 (s, 1H, CH-Ph), 4.21 (m, 4H, -(CH₂)₂), 2.56 (m, 1H, CH in TQ), 2.31 (s, 6H, 2,6-CH₃), 1.77 (s, 3H, -CH₃), 1.31 (t, 6H, (-CH₃)₂), 1.11 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.25, 178.57, 167.29, 156.10, 153.61, 144.73, 143.86, 142.77, 133.88, 130.41, 127.12, 122.61, 121.14, 102.42, 61.83, 37.52, 21.06, 15.48, 14.30, 14.22; EI-MS: 508 (M+, 20%); elemental analysis (C₂₉H₃₃NO₇): calculated: C, 68.62; H, 6.55; N, 2.76%; found: C, 68.60; H, 6.53; N, 2.74%.

(6) Synthesis of Diethyl 4-(4-Hydroxy-3-methoxyphenyl)-1-(2-isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1f). The results are as follows: white solid, yield 74%; mp 118°C; IR (kBr) (cm^-1); 3089 (OH),3069 (Ar-H), 2961 (O-CH₃), 2918, 2865 (C-H str of CH₃),1742 (C=O in ester), 1663, 1713 (C=O); ¹H NMR (300 MHz, DMSO-d₆):7.09-6.83 (m, 4H, Van), 6.36 (s, 1H, -CH in TQ), 5.35 (s, 1H, OH), 4.99 (s, 1H, CH-Ph), 4.22 (m, 4H, -(CH₂)₂), 3.83 (s, 3H, -OCH₃), 2.57 (m, 1H, CH in TQ), 2.32 (s, 6H, 2,6-CH₃), 1.78 (s, 3H, -CH₃), 1.32 (t, 6H, (-CH₃)₂), 1.12 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.26, 178.58, 167.30, 153.62, 147.42, 145.70, 144.74, 143.87, 142.78, 135.80, 133.89, 122.71, 115.50, 114.51, 102.43, 61.84, 56.10, 21.07, 15.49, 14.31, 14.23; EI-MS: 538 (M+, 20%); elemental analysis (C₃₀H₃₅NO₈): calculated: C, 67.02; H, 6.56; N, 2.61%; found: C, 67.00; H, 6.54; N, 2.59%.

(7) Synthesis of Diethyl 1-(2-Isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (1g). The results are as follows: white solid, yield 95%; mp 256°C; IR (kBr) (cm^-1); 3041 (Ar-H), 2960, 2875 (C-H str of CH₃), 1707 (C=O in ester), 1630, 1652 (C=O), 1540 (-NO₂); ¹H NMR (300 MHz, DMSO-d₆):8.12-7.59 (m, 4H, PH-NO₂), 6.37 (s, 1H, -CH in TQ), 5.00 (s, 1H, CH-PH), 4.23 (m, 4H, -(CH₂)₂), 2.58 (m, 1H, CH in TQ), 2.34 (s, 6H, 2,6-CH₃), 1.79 (s, 3H, -CH₃), 1.33 (t, 6H, (-CH₃)₂), 1.13 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.27, 178.59, 167.31, 153.63, 147.90, 145.21, 144.75, 143.88, 142.79, 134.20, 133.90, 121.80, 121.12, 120.91, 102.44, 61.85, 42.52, 21.08, 15.50, 14.32, 14.24; EI-MS: 537 (M+, 20%); elemental analysis (C₂₉H₃₂N₂O₈): calculated: C, 64.64; H, 7.16; N, 5.22%; found: C, 64.62; H, 7.14; N, 5.00%.

(8) Synthesis of Diethyl 4-(4-(Dimethylamino)phenyl)-1-(2-isopropyl-5-methyl-3,6-dioxo cyclohexa-1,4-dien-1-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1h). The results are as follows: yellow yield 79%; mp 184°C; IR (kBr) (cm^-1); 3072 (Ar-H), 2968, 2875, 2953, 2966 (C-H str of CH₃),1761 (C=O in ester), 1665, 1623 (C=O); ¹H NMR (300 MHz, DMSO-d₆):6.95-6.64 (m, 4H, Ph), 6.38 (s, 1H, -CH in TQ), 5.01 (s, 1H, CH-Ph), 4.24 (m, 4H, -(CH₂)₂), 3.06 (s, 6H, -(CH₃)₂), 2.59 (m, 1H, CH in TQ), 2.35 (s, 6H, 2,6-CH₃), 1.80 (s, 3H, -CH₃), 1.34 (t, 6H, (-CH₃)₂), 1.14 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.29, 178.61, 167.33, 153.65, 148.10, 144.77, 143.90, 142.81, 133.99, 133.92, 128.10, 112.01, 102.46, 61.87, 41.73, 21.10, 15.52, 14.34, 14.26; EI-MS: 535 (M+, 20%); elemental analysis (C₃₁H₃₈N₂O₆): calculated: C, 69.64; H, 7.16; N, 5.24%; found: C, 69.62; H, 7.14; N, 5.22%.

(9) Synthesis of Diethyl 1-(2-Isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-2,6-dimethyl-4-(4-oxo-4H-chromen-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate (1i). The results are as follows: white solid, yield 70%; mp 128°C; IR (kBr) (cm^-1); 3065 (Ar-H), 2948, 2871 (C-H str of CH₃), 1750 (C=O in ester), 1713, 1625 (C=O); ¹H NMR (300 MHz, DMSO-d₆): 8.08-6.90 (m, 5H, Chromene-3-carbaldehyde), 6.39 (s, 1H, -CH in TQ), 5.02 (s, 1H, CH-Ph), 4.25 (m, 4H, -(CH₂)₂), 3.00 (m, 1H, CH in TQ), 2.36 (s, 6H, 2,6-CH₃), 1.81 (s, 3H, -CH₃), 1.35 (t, 6H, (-CH₃)₂), 1.15 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.28, 183.01, 178.60, 167.32, 157.20, 153.64, 151.60, 144.76, 143.89, 142.80, 135.20, 133.91, 125.80, 123.91, 123.42, 116.10, 115.31, 102.45, 61.86, 43.71, 21.09, 15.51, 14.33, 14.25; EI-MS: 560 (M+, 20%); elemental analysis (C₃₂H₃₃NO₈): calculated: C, 68.68; H, 5.94; N, 2.50%; found: C, 68.66; H, 5.92; N, 2.48%.

(10) Synthesis of Diethyl 1-(2-Isopropyl-5-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1j). The results are as follows: white solid, yield 89%; mp 152°C; IR (kBr) (cm^-1); 3070 (Ar-H), 2961 (O-CH₃), 2960, 2851 (C-H str of CH₃), 1747 (C=O in ester), 1743, 1630 (C=O); ¹H NMR (300 MHz, DMSO-d₆):7.12-6.87 (m, 4H, Ph-OCH₃), 6.40 (s, 1H, -CH in TQ), 5.03 (s, 1H, CH-Ph), 4.25 (m, 4H, -(CH₂)₂), 3.83 (s, 3H, OCH₃), 3.01 (m, 1H, CH in TQ), 2.37 (s, 6H, 2,6-CH₃), 1.82 (s, 3H, -CH₃), 1.36 (t, 6H, (-CH₃)₂), 1.18 (d, Hz, CH₃); ¹³C NMR (300 MHz, DMSO-d₆): 187.30, 178.62, 167.34, 157.60, 153.66, 144.78, 143.91, 142.82, 136.70, 133.93, 130.00, 114.20, 102.46, 61.87, 55.80, 43.59, 21.11, 15.53, 14.35, 14.27; EI-MS: 522 (M+, 20%); elemental analysis (C₃₀H₃₅NO₇): calculated: C, 69.08; H, 6.76; N, 2.69%; found: C, 69.06; H, 6.74; N, 2.67%.

2.1.4. Cytotoxic Activity

The cytotoxicity test was carried out according to a protocol established by the National Cancer Institute in the United States [46].

3. Results and Discussion

3.1. Characterization of Silver Nanoparticles

3.1.1. Scanning Electron Microscopy (SEM)

SEM image has showed individual zinc particles as well as a number of aggregates. The SEM image revealed a spherical-shaped nanoparticle with a diameter of 12-27 nm. Figure 2 shows the formation of aggregated molecules in the 12 m range.

3.1.2. Transmission Electron Microscopy (TEM)

To learn more about the size and morphology of the ZnO NPs, a TEM study was performed. TEM images of ZnO NPs at different magnifications are shown in Figures 3(a) and 3(b). The average particle size of ZnO NPs can be seen in the TEM images in the range of 16–27 nm.

(a)

(b)

3.1.3. Catalyst Recovery Studies

Figure 4 demonstrates the recovery of the catalyst with a small loss of catalytic activity after at least 12-15 run times. By optimising the reaction conditions, the application of the catalyst was examined. Table 1 shows the yield of a number of aldehydes used in the condensation reaction with the ZnO-NP (1 mole percent) catalyst at room temperature in a solvent-free setting.

3.2. Chemistry

All the newly synthesized thymoquinone derivatives were characterized by FT-IR, which showed various functional groups. The ¹H-NMR spectra of compounds (1a-1j) indicate frequency observed at 7.16-7.07 and 6.79-5.54, corresponding to the NH-CH and CH-Ph protons. The ¹³C-NMR spectra exhibit the peak at 144.42-118.76 and 40.60-40.53, corresponding to the NH-CH and CH-Ph carbon, respectively. Optimization of reaction condition (1a-1j) is shown in Figure 5. The synthetic pathway of thymoquinone derivatives is shown in Scheme 2. The possible mechanism for the coupling of aldehydes, dimedone, and amines in the presence of ZnO NPs as an effective catalyst is shown in Scheme 3. To the best of our knowledge, ZnO NPs catalyze the reaction by electrophilic activation of the carbonyl groups of aldehydes and ethyl acetoacetate; this makes them susceptible to nucleophilic attack.

3.2.1. Cytotoxic Activity

The cytotoxic activity of the newly prepared compounds 1a-1j is tested using the US NCI protocol, which was previously recorded [46]. The values for growth inhibition at 50% (GI₅₀), tumour growth inhibition (TGI), and lethal concentration (LC₅₀) were calculated. The compounds 1f have a significant activity against HepG2, LC₅₀-0.50 μM, MCF-7, LC₅₀-0.64 μM, HeLa, and LC₅₀-0.52 μM. Doxorubicin was used as a standard drug. Using the MTT assay, the compounds were tested for cytotoxicity in human embryonic kidney cells (HEK-293), lung cells (MRC-5), and liver cells (LO2). Since most of the compound’s IC50 values are greater than 100, the assay results indicated that these compounds had no significant effect on normal kidney cell growth. As a result, compound 1f can be used as a lead compound for the production of more potent agents for cancer cell lines HepG2 (liver), MCF-7 (breast), and HeLa (cervical). Table 2 shows the effects of cytotoxic screening of compounds (1a-1j), and Table 3 shows the cytotoxicity screening of normal cell lines.

3.3. Structure-Activity Relationship

The aim of a structure-activity relationship study (SAR) was to discover a correlation between a dynamic molecule’s chemical structure and its cytotoxic activity. The chemical group/atom that plays a critical role in modulating the cytotoxic activity of compounds within a particular system can be identified using SAR analysis. Using the 1,4-dihydropyridine derivative’s cytotoxic behaviour findings, the data of the selected 1,4-dihydropyridine derivatives (1a-1j) showed that compound 1f is the most effective (HepG2, LC₅₀-0.50 μM, MCF-7, LC₅₀-0.64 μM, HeLa, and LC₅₀-0.52 μM) control doxorubicin. The fundamental SAR could be assessed and is shown in Figure 6.

It was discovered that the compound 1f has a high cytotoxic activity against cancer cell lines due to the presence of a 1,4-dihydropyridine ring fused to a vanillin. The presence of a OH moiety on a phenyl ring attached to a 1,4-dihydropyridine skeleton caused this. The remaining compounds have only moderate cytotoxic activity against all of the cancer cell lines.

4. Conclusion

We have developed an environmentally friendly new method for the high yield synthesis of 3-amino thymoquinone connected 1,4-dihyropyridine derivatives (1a-1j) using ZnO as nanoparticle catalyst. Three cancer cell lines and normal cell lines were used to assess the cytotoxic activity of the 1,4-dihyropyridine derivatives. MTT assay was performed using doxorubicin as the standard drug on human embryonic kidney cell (HEK293), liver cell (LO2), and lung cell (MRC5). Compound 1f (HepG2, LC₅₀-0.50 μM, MCF-7, LC₅₀-0.64 μM, HeLa, and LC₅₀-0.52 μM) was found to be highly active when compared with other compounds. Therefore, compound 1f could provide as a high momentous molecule for further development of an anticancer drug.

Data Availability

Supplementary information is associated with this submission.

Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgments

The authors extend their appreciation to the Researchers supporting project number RSP-2021/316, King Saud University, Riyadh, Saudi Arabia.

Supplementary Materials

The supplementary material used to support the findings of this study is included within the supplementary information files. (The supplementary file contains ¹H-NMR and ¹³C-NMR spectra). (Supplementary Materials)

References

C. B. Blackadar, “Historical review of the causes of cancer,” World Journal of Clinical Oncology, vol. 7, no. 1, pp. 54–86, 2016.
View at: Publisher Site | Google Scholar
P. S. Roy and B. J. Saikia, “Cancer and cure: a critical analysis,” Indian Journal of Cancer, vol. 53, no. 3, pp. 441-442, 2016.
View at: Publisher Site | Google Scholar
Report, W, WHO Cancer Report, WHO, 2019.
S. K. Carter, M. Bakowski, and K. Hellman, Chemotherapy of Cancer, John Wiley and Sons Inc, USA, 3rd edition, 1987.
S. Srivastava, E. J. Koay, A. D. Borowsky et al., “Cancer overdiagnosis: a biological challenge and clinical dilemma,” Nature Reviews. Cancer, vol. 19, no. 6, pp. 349–358, 2019.
View at: Publisher Site | Google Scholar
C. C. Woo, A. P. Kumar, G. Sethi, and K. H. B. Tan, “Thymoquinone: potential cure for inflammatory disorders and cancer,” Biochemical Pharmacology, vol. 83, no. 4, pp. 443–451, 2012.
View at: Publisher Site | Google Scholar
H. U. Gali-Muhtasib, W. G. Abou Kheir, L. A. Kheir, N. Darwiche, and P. A. Crooks, “Molecular pathway for thymoquinone-induced cell-cycle arrest and apoptosis in neoplastic keratinocytes,” Anti-Cancer Drugs, vol. 15, no. 4, pp. 389–399, 2004.
View at: Publisher Site | Google Scholar
M. El–Dakhakhny, “Studies on the chemical constitution of Egyptiannigella satival. seeds. The essential oil,” Planta Medica, vol. 11, no. 4, pp. 465–470, 1963.
View at: Publisher Site | Google Scholar
O. A. Badary, O. A. Al-Shabanah, M. N. Nagi, A. C. Al-Rikabi, and M. M. Elmazar, “Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone,” European Journal of Cancer Prevention, vol. 8, no. 5, pp. 435–440, 1999.
View at: Publisher Site | Google Scholar
S. Attoub, O. Sperandio, H. Raza et al., “Thymoquinone as an anticancer agent: evidence from inhibition of cancer cells viability and invasion in vitro and tumor growthin vivo,” Fundamental & Clinical Pharmacology, vol. 27, no. 5, pp. 557–569, 2013.
View at: Publisher Site | Google Scholar
R. Schneider-Stock, I. H. Fakhoury, A. M. Zaki, C. O. El-Baba, and H. U. Gali Muhtasib, “Thymoquinone: fifty years of success in the battle against cancer models,” Drug Discovery Today, vol. 19, no. 1, pp. 18–30, 2014.
View at: Publisher Site | Google Scholar
H. Gali-Muhtasib, M. Diab-Assaf, C. Boltze et al., “Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism,” International Journal of Oncology, vol. 25, no. 4, pp. 857–866, 2004.
View at: Google Scholar
M. Roepke, A. Diestel, K. Bajbouj et al., “Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells,” Cancer Biology & Therapy, vol. 6, no. 2, pp. 160–169, 2007.
View at: Publisher Site | Google Scholar
P. S. Koka, D. Mondal, M. Schultz, A. B. Abdel-Mageed, and K. C. Agrawal, “Studies on molecular mechanisms of growth inhibitory effects of thymoquinone against prostate cancer cells: role of reactive oxygen species,” Experimental Biology and Medicine, vol. 235, no. 6, pp. 751–760, 2010.
View at: Publisher Site | Google Scholar
N. El-Najjar, M. Chatila, H. Moukadem et al., “Reactive oxygen species mediate thymoquinone-induced apoptosis and activate ERK and JNK signaling,” Apoptosis, vol. 15, no. 2, pp. 183–195, 2010.
View at: Publisher Site | Google Scholar
E. M. Dergarabetian, K. I. Ghattass, S. B. El-Sitt et al., “Thymoquinone induces apoptosis in malignant T-cells via generation of ROS,” Frontiers in Bioscience, vol. 5, no. 2, pp. 706–719, 2013.
View at: Google Scholar
M. C. Chen, N. H. Lee, H. H. Hsu et al., “Inhibition of NF-κB and metastasis in irinotecan (CPT-11)-resistant LoVo colon cancer cells by thymoquinone via JNK and p38,” Environmental Toxicology, vol. 32, no. 2, pp. 669–678, 2017.
View at: Publisher Site | Google Scholar
B. Iskender, K. Izgi, and H. Canatan, “Novel anti-cancer agent myrtucommulone-A and thymoquinone abrogate epithelial–mesenchymal transition in cancer cells mainly through the inhibition of PI3K/AKT signalling axis,” Molecular and Cellular Biochemistry, vol. 416, no. 1-2, pp. 71–84, 2016.
View at: Publisher Site | Google Scholar
O. A. Badary, O. A. Al-Shabanah, M. N. Nagi, A. M. Al-Bekairi, and M. M. A. Elmazar, “Acute and subchronic toxicity of thymoquinone in mice,” Drug Development Research, vol. 44, no. 2-3, pp. 56–61, 1998.
View at: Publisher Site | Google Scholar
A. Al-Ali, A. A. Alkhawajah, M. A. Randhawa, and N. A. Shaikh, “Oral and intraperitoneal LD50 of thymoquinone, an active principle of Nigella sativa, in mice and rats,” Journal of Ayub Medical College, Abbottabad, vol. 20, pp. 25–27, 2008.
View at: Google Scholar
M. AbuKhader, “Thymoquinone in the clinical treatment of cancer: fact or fiction?” Pharmacognosy Reviews, vol. 7, no. 14, pp. 117–120, 2013.
View at: Publisher Site | Google Scholar
M. Tan, A. Norwood, M. May, M. Tucci, and H. Benghuzzi, “Effects of (-)epigallocatechin gallate and thymoquinone on proliferation of a PANC-1 cell line in culture,” Biomedical Sciences Instrumentation, vol. 42, pp. 363–371, 2006.
View at: Google Scholar
S. H. Jafri, J. Glass, R. Shi, S. Zhang, M. Prince, and H. Kleiner-Hancock, “Thymoquinone and cisplatin as a therapeutic combination in lung cancer: In vitro and in vivo,” Journal of Experimental & Clinical Cancer Research, vol. 29, no. 1, pp. 87–97, 2010.
View at: Publisher Site | Google Scholar
J. P. Wan and Y. Pan, “Recent advance in the pharmacology of dihydropyrimidinone,” Mini-Reviews in Medicinal Chemistry, vol. 12, no. 4, pp. 337–349, 2012.
View at: Publisher Site | Google Scholar
J. T. David, “The 1,4-dihydropyridine nucleus: a pharmacophoric template part 1. Actions at ion channels,” Mini Reviews in Medicinal Chemistry, vol. 3, no. 3, pp. 215–223, 2003.
View at: Publisher Site | Google Scholar
L. Yet, “1,4-Dihydropyridines,” 1,4-Dihydropyridines. In Privileged Structures in Drug Discovery: Medicinal Chemistry and Synthesis, Wiley, pp. 59–82, 2018.
View at: Google Scholar
A. K. Samrat and B. A. Pratibha, “1, 4-Dihydropyridines: a class of pharmacologically important molecules,” Mini-Reviews in Medicinal Chemistry, vol. 14, no. 3, pp. 282–290, 2014.
View at: Publisher Site | Google Scholar
E. Carosati, P. Ioan, M. Micucci et al., “1,4-Dihydropyridine scaffold in medicinal chemistry, the story so far and perspectives (part 2): action in other targets and antitargets,” Current Medicinal Chemistry, vol. 19, no. 25, pp. 4306–4323, 2012.
View at: Publisher Site | Google Scholar
M. Abhinav Prasoon, B. Ankit, and R. Awani Kumar, “1,4-Dihydropyridine: a dependable heterocyclic ring with the promising and the most anticipable therapeutic effects,” Mini Reviews in Medicinal Chemistry, vol. 19, pp. 1219–1254, 2019.
View at: Publisher Site | Google Scholar
G. Li and J. C. Antilla, “Highly enantioselective hydrogenation of enamides catalyzed by chiral phosphoric acids,” Organic Letters, vol. 11, no. 5, pp. 1075–1078, 2009.
View at: Publisher Site | Google Scholar
A. Hantzsch, “Ueber die synthese pyridinartiger verbindungen aus acetessigäther und aldehydammoniak,” Justus Liebigs Annalen der Chemie, vol. 215, no. 1, pp. 1–82, 1882.
View at: Publisher Site | Google Scholar
U. Eisner and J. Kuthan, “Chemistry of dihydropyridines,” Chemical Reviews, vol. 72, no. 1, pp. 1–42, 1972.
View at: Publisher Site | Google Scholar
A. Dömling and I. Ugi, “Multicomponent reactions with isocyanides,” Angewandte Chemie International Edition, vol. 39, no. 18, pp. 3168–3210, 2000.
View at: Publisher Site | Google Scholar
A. Dömling, “Recent developments in isocyanide based multicomponent reactions in applied chemistry,” Chemical Reviews, vol. 106, no. 1, pp. 17–89, 2006.
View at: Publisher Site | Google Scholar
S. G. Cedric, V. R. Felipe, R. R. Kamilla, and E. K. Arthur, “Multicomponent reactions for the synthesis of bioactive compounds: a review,” Current Organic Synthesis, vol. 16, no. 6, pp. 855–899, 2019.
View at: Publisher Site | Google Scholar
E. M. de Marigorta, J. M. D. L. Santos, A. M. Ochoa de Retana, J. Vicario, and F. Palacios, “Multicomponent reactions (MCRs): a useful access to the synthesis of benzo-fused γ-lactams,” Beilstein Journal of Organic Chemistry, vol. 15, pp. 1065–1085, 2019.
View at: Publisher Site | Google Scholar
O. Ghashghaei, F. Seghetti, and R. Lavilla, “Selectivity in multiple multicomponent reactions: types and synthetic applications,” Beilstein Journal of Organic Chemistry, vol. 15, pp. 521–534, 2019.
View at: Publisher Site | Google Scholar
C. Lambruschini, A. Basso, and L. Banfi, “Integrating biocatalysis and multicomponent reactions,” Drug Discovery Today: Technologies, vol. 29, pp. 3–9, 2018.
View at: Publisher Site | Google Scholar
M. Driowya, A. Saber, H. Marzag, L. Demange, K. Bougrin, and R. Benhida, “Microwave-assisted syntheses of bioactive seven-membered, Macro-Sized Heterocycles and Their Fused Derivatives,” Molecules, vol. 21, no. 8, p. 1032, 2016.
View at: Publisher Site | Google Scholar
E. Ruijter and R. V. Orru, “Multicomponent reactions - opportunities for the pharmaceutical industry,” Drug Discovery Today: Technologies, vol. 10, no. 1, pp. e15–e20, 2013.
View at: Publisher Site | Google Scholar
B. B. Touré and D. G. Hall, “Natural product synthesis using multicomponent reaction strategies,” Chemical Reviews, vol. 109, no. 9, pp. 4439–4486, 2009.
View at: Publisher Site | Google Scholar
M. A. Zolfigol, E. Kolvari, A. Abdoli, and M. Shiri, “Synthesis of new tripodal Hantzsch 1,4-dihydropyridines under solvent-free condition and their conversion to the corresponding tripodal pyridines,” Molecular Diversity, vol. 14, no. 4, pp. 809–813, 2010.
View at: Publisher Site | Google Scholar
G. Mehta and V. Singh, “Hybrid systems through natural product leads: an approach towards new molecular entities,” Chemical Society Reviews, vol. 31, no. 6, pp. 324–334, 2002.
View at: Publisher Site | Google Scholar
C. Reiter, A. Hermann, A. Çapcı, T. Efferth, and S. B. Tsogoeva, “New artesunic acid homodimers: Potent reversal agents of multidrug resistance in leukemia cells,” Bioorganic & Medicinal Chemistry, vol. 20, no. 18, pp. 5637–5641, 2012.
View at: Publisher Site | Google Scholar
T. Fröhlich, A. Çapcı Karagöz, C. Reiter, and S. B. Tsogoeva, “Artemisinin-Derived Dimers: Potent Antimalarial and Anticancer Agents,” Journal of Medicinal Chemistry, vol. 59, no. 16, pp. 7360–7388, 2016.
View at: Publisher Site | Google Scholar
P. Skehan, R. Storegng, D. Scudiero et al., “New colorimetric cytotoxicity assay for anticancer-drug screening,” Journal of the National Cancer Institute, vol. 82, no. 13, pp. 1107–1112, 1990.
View at: Publisher Site | Google Scholar

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Copyright © 2022 Perumal Gobinath et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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