ZnCl2 catalyzed new coumarinyl-chalcones as cytotoxic agents.

A new series of coumarin-yl-chalcone derivatives (3a-m) had been designed and synthesized through different reactions such as aromatic addition, cyclization and Claisen-Schmidt reactions in good yields (54-78%). 5-acetyl-4-(2-hydroxyphenyl) -6-methyl-3, 4-dihydropyrimidin-2(1H) -one (1) has been synthesized by multi-component one pot reaction of salicylaldehyde, methyl acetoacetate and urea, which was further reacted with malonic acid employing ZnCl2 catalyst to yield 5-acetyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3, 4-dihydropyrimidin-2(1H) -one (2). The title compounds (3a-m) were synthesised by reacting 5-acetyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3, 4-dihydropyrimidin-2(1H)-one (2) with different aromatic aldehydes in the presence of potassium hydroxide. In silico studies, a preliminary screening method for predicting the anti-cancer activity was performed for the synthesized compounds (3a-m) against Src, Alb tyrosine kinase and homology model protein (PDB ID: 4csv). The derivatives 3h and 3m showed moderate binding energies. The in vitro cytotoxic activity was evaluated for the compounds 3h and 3m by using human cancer cell-line morphology and MTT assay against three human cell-lines A549 (Lung), Jurkat (Leukemia) and MCF-7 (Breast). The results indicate that the derivatives 3h and 3m display significant anti-cancer activity, however it was found to be less cytotoxic when compared to the standard used i.e. Imatinib.

Introduction

Many medicinal plants are known to have unique anticarcinogenic, anti-inflammatory, and growth-modulatory effects on the cancer cells (Awan et al., 1908, Shaik et al., 2019, Vaidyanathan et al., 2019). Among them, Coumarin, a benzopyrone class of heterocyclic compound, are pharmacologically important compounds, mostly obtained from natural sources mostly higher plants (Venugopala et al., 2013). Most natural coumarins are oxygenated at C-7; Umbelliferone (7-hydroxycoumarin) being regarded as the structural and biogenetic parent of the more highly oxygenated coumarins (Intekhab and Aslam, 2009). Coumarin and its derivatives were reported to have innumerable pharmacological activities like anti-HIV (Shikishima et al., 2001), anti-tubercular (Karalı et al., 2002), anti-microbial (Kamal et al., 2009), anti-Alzheimer’s (Anand et al., 2012, Piazzi et al., 2008), analgesic anti-inflammatory (Ghate et al., 2005), anti-cancer (Kamal et al., 2010), anti-hyperlipidemic (Madhavan et al., 2003), anti-hypertensive activity (Baek et al., 2000), anti-oxidant (Kharasch et al., 2000), neuroprotective activity (Wang et al., 2012), anti-diabetic (Tegginamath et al., 2016), anti-neurodegenerative (Jameel et al., 2016), anti-psychotics (Chen et al., 2013, Di Carlo et al., 1999), anti-coagulant (Sashidhara et al., 2012) activities. However, it was coupled with another class of naturally occurring pharmacologically important compounds chalcones (1,3-diaryl-2-propane-1-ones), which are secondary metabolites obtained from flavonoids and isoflavonoids mostly isolated from fruits, vegetables, spices, tea and soya-based foodstuff (Rozmer and Perjési, 2016). These chalcones have been reported to possess various beneficial biological properties including anti-bacterial, anti-fungal, anti-malarial, anti-HIV, anti-cancer, antioxidant, anti-tubercular (Kotra et al., 2010, Singh et al., 2014) activities.

Existing scientific literature describes that different types of coumarinyl chalcones are designed, screened for anti-cancer activity against different types of target proteins like tubulin, Falcipian-2 (Pingaew et al., 2014), Cdc25 (Valente et al., 2010) by in-silico screening, screened for anti-cancer activity by in-vitro Glutathione-S-transferase Cdc25 recombinant enzyme assay, screened for anti-cancer activity by in vitro method on different human cancer cell lines like oral, cervical, breast, lung, hepatic, lymph, leukaemia, pancreatic, Nasopharyngeal (Wei et al., 2016).

Another class of compounds, dihydropyrimidinones, has been found to possess a variety of biological activities such as anti-inflammatory, calcium channel antagonism/inhibition other than antitumoral activity, especially after the discovery of the compound monastrol, a dihydropyrimidinone, with Eg5 kinesin inhibitor efficiency (Beena et al., 2016, Vijay et al., 2010). The enzymes tyrosine kinases like Protein Tyrosine kinases, receptor tyrosine kinases (RTKs), Src, a mutant variant protein of tyrosine kinase, Alb tyrosine kinases and non-receptor tyrosine kinases (NRTKs) are the few enzymes responsible for cell proliferation, migration, differentiation and survival. The mutations or over expression of tyrosine kinase leads to uncontrolled cell growth through progressive or uncontrolled activation of intracellular pathway cascades. The relationship between the receptor tyrosine kinase and the cancer development was revealed by many scientific researches (Paul and Mukhopadhyay, 2004).

The modern drug development strategy for anti-cancer activity includes in-silico docking of new molecules on different target proteins (Viana et al., 2018). Tyrosine kinases are considered as targets in cancer chemotherapy due to the vital role in altering the growth factor signalling pathway. This paper explains the design and synthesis of some new dihydropyrimidinone substituted coumarinyl chalcones (Scheme 1) by hybridization-based on their docking score obtained by the in-silico studies. The synthesized derivatives were subjected to spectral characterization along with in-vitro anti-cancer studies, using Imatinib, a highly effective non-receptor tyrosine kinase inhibitor, as standard.

Scheme 1

Schematic representation of dihydropyrimidinone derived coumarinyl chalcones.

Experimental

Materials, methods and characterization

The chemicals and materials used in this work. The chemicals used for synthesis were purchased urea was purchased from Loba Chemie Pvt. Ltd., India, 2-hydroxybenzaldehyde was obtained from Fisher Scientific, UK, pentane-2,4-dione was purchased from Merck, Germany and Avra Synthesis Pvt. Ltd. In-silico molecular docking was performed by using Chem office 2016 and VLifeMDS. Melting points of synthesized compounds were determined by Dalal Melting Point Apparatus using open capillary tubes. Infrared spectra were recorded using the KBr Pellet method. 1H NMR and 13C NMR spectra were determined by using Bruker-NMR Spectrometer 400 MHz. Mass spectra were measured with Shimadzu GCMS QP 5000 mass spectrometer. Other chemicals were of commercial grade and used without further purification. The homogeneity of the compounds was checked by thin-layer chromatography (TLC) on TLC silica gel F254aluminum plates (E. Merck, Germany) using chloroform-methanol (3:1) mixture as mobile phase and visualized using iodine vapors.

Completion of synthetic reactions and purity of end products of reactions were confirmed by Thin Layer Chromatography on TLC silica gel F254aluminum plates (E. Merck, Germany), using acetone and ethyl acetate as mobile spots are visualized with UV chamber and iodine vapors.

General procedure for synthesis of 5-acetyl-4-(2-hydroxyphenyl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (1)

A mixture of Salicylaldehyde (1 mmol), Methyl acetoacetate (1.1 mmol), urea (1.3 mmol) and citric acid (0.5 mmol) was taken into a round bottom flask, heated at 80 °C under stirring. Completion of the reaction was monitored by TLC. To the reaction mixture, cold water was added and stirred for 10 min, then filtered, washed with water, dried in vacuum and the obtained product was further recrystallized from ethanol. white crystals; yield 85.41%; m.p. 112–114 °C; IR (KBr, cm−1): v 3479(N—H), 3370(O—H), 3060 (C—H Ar), 2925(C—H), 1734(CO), 1671(CO); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.28(s, 3H, CH), 2.35(s, 3H, CH—CO), 4.91 (s, H, CH), 6.83–7.86 (m, 5H, aromatic), 9.03 (s, H, NH), 9.71 (s, H, OH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 19.4 (CH3), 27.3 (CH3), 41.8 (CH), 106.2, 121.6, 122.3, 128.7 (Ar—C), 145.4 (C—N), 150.7 (CO), 154.2 (C—O), 192.5 (CO). MS (m/z, (relative abundance, %): 247 (M + 1, 14.8), 203 (M+, 33.2), 154 (M+, 100), 94 (M+, 27).

General procedure for synthesis 5-acetyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (2)

A mixture of 5-acetyl-4-(2-hydroxyphenyl) -6-methyl-3,4-dihydropyrimidin-2(1H)-one (0.1 mol) and malonic acid (0.1 mol) was added to a mixture of phosphorus oxychloride (40 mL) and anhydrous zinc chloride (30 g) which was preheated to 60–70 °C and the reaction mixture was heated on a water bath at 70 °C for 20 h. Completion of the reaction was monitored by TLC, then cool and pour the mixture in ice-cold water to afford a solid, washed with cold water and then filtered. It was then treated with 10% sodium carbonate solution and then filtered. The filtrate was slowly acidified with diluted hydrochloric acid. The product was filtered and washed with water and further dried and recrystallized from glacial acetic acid. Brick red crystals; yield 72.45%; m.p. 102–104 °C; IR (KBr, cm−1): v 3456(N—H), 3362(O—H), 3038 (C—H Ar), 2928(C—H), 1732(CO), 1665(CO); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.23(s, 3H, CH), 2.32 (s, 3H, CH), 5.09 (s, H, CH), 6.82–7.69 (m, 5H, aromatic), 9.06 (s, H, NH), 9.88 (s, H, OH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 19.7 (CH3), 27.4 (CH3), 42.3(C—CH), 91.6, 106.2, 117.2, 121.3, 125.2, 126.5, 128.2, (Ar—C), 145. 1 (C—N), 192.3 (CO), 150.2 (CO), 162.1 (CO), 166.8 (C—O). MS (m/z, (relative abundance, %): 315 (M + 1, 18.1), 271 (M+, 36), 154 (M+, 100), 162 (M+, 41).

General procedure for synthesis of coumarinyl-chalones (3a-m)

Dissolve 0.002 mol of 5-acetyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one and 0.002 mol of substituted aromatic aldehyde in 6 mL of ethanol. While stirring, add 3 mL of 40% KOH dropwise and continue the stirring for about 30 hrs. Completion of the reaction was monitored by TLC. Then add crushed ice to the mixture add to neutralize the mixture with dil. HCl to get precipitate. Recrystallize the product obtained from ethanol.

5-cinnamoyl-4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (3a)

The was found to be Yellow crystals; yield 65.41%; m.p. 153–155 °C; IR (KBr, cm−1): v 3479(N—H), 3370(O—H), 3060 (C—H Ar), 2925(C—H), 1734(CO), 1671(CO); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.50(s, 3H, CH), 4.95(s, H, CH), 6.9–7.65 (m, 13H, aromatic), 9.13(s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 17.7 (CH3), 42.5(C—CH), 117.4, 121.3, 125.2, 126.5, 127.9, 128.4, 128.5, 128.6, 135.2, 148.3, 154.8 (Ar—C), 193.3 (CO), 150.2 (CO), 162.2 (CO), 166.1(C—O), 142.2 (CC). MS (m/z, (relative abundance, %)): 403 (M + 1, 25.6), 271 (47), 243 (M + 2, 100), 162 (M+, 54), 132 (36).

(E) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -5-(3-(4-methoxyphenyl) acryloyl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (3b)

Dark red crystals; yield 62.76%; m.p. 168–170 °C; IR (KBr, cm−1): v3436(N—H), 3362(O—H), 3038 (C—H Ar), 2928(C—H), 1732(CO), 1665(CO), 758 (o-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.54(s, 3H, CH), 3.78 (s, 3H, CH), 4.91(s, H, CH), 6.9–7.84 (m, 12H, aromatic), 9.11(s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 17.9 (CH3), 42.2(C—CH), 54.3(CH), 91.4, 114.4, 117.6, 118.2, 121.1, 123.7, 125.6, 126.1, 127.3, 128.7, 130.4, 154.3, 159.2 (Ar—C), 193.5 (CO), 150.4 (CO), 162.6 (CO), 166.5(C—O), 142.6 (CC). MS (m/z, (relative abundance, %): 433 (M + 1, 26.4), 327 (M + 2, 100), 272 (M+, 36), 162 (M+, 28), 108 (57).

4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-5-((2E,4E) -5-phenylpenta-2,4-dienoyl) -3,4-dihydropyrimidin-2(1H) -one (3c)

Yellow crystals; yield 72.28%; m.p. 151–152 °C;): IR (KBr, cm−1):v3462(N—H), 3269(O—H), 3027 (C—H Ar), 2923(C—H), 1739(CO), 1674(CO), 758(Ar); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.48(s, 3H, CH), 4.74 (s, H, CH), 6.6–7.75 (m, 15H, aromatic), 9.05 (s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 17.6 (CH3), 42.1(C—CH), 91.2, 117.3, 118.5, 121.5, 125.4, 126.3, 127.2, 128.3, 128.6, 135.4, 154.2 (Ar—C), 193.2 (CO), 150.1 (CO), 162.4 (CO), 166.6 (C—O), 141.3 (CC), 151.4 (CC). MS (m/z, (relative abundance, %): 429 (M + 1, 27.5), 327 (M + 2, 100), 272 (M+, 36), 162 (M+, 28), 108 (57).

(E) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-5-(3-(2-nitrophenyl) acryloyl) -3,4-dihydropyrimidin-2(1H) -one (3d)

Green crystals; yield 57.37%; m.p. 117–1119 °C;): IR (KBr, cm−1): v3445(N—H), 3378(O—H), 3042(C—H Ar), 2927(C—H), 1741(CO), 1672(CO), 1531,1384 (Nitro), 756 (p-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.49(s, 3H, CH), 4.93 (s, H, CH), 7.10–8.39 (m, 12H, aromatic), 9.09 (s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 18.3 (CH3), 42.7 (C—CH), 91.2, 117.5, 118.2, 121.1, 123.2, 123.6, 125.1, 126.8, 128.1, 128.4, 134.2, 154.1 (Ar—C), 193.5 (CO), 150.5 (CO), 162.8 (CO), 166.3 (C—O), 147.3 (C—N), 152.6 (CC). MS (m/z, (relative abundance, %): 448 (M + 1, 25.3), 325 (M + 2, 100), 272 (M+, 36), 162 (M+, 28), 108 (57).

(E) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -5-(3-(4-hydroxyphenyl) acryloyl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (3e)

Brown crystals; yield 66.18%; m.p. 127–129 °C; IR (KBr, cm−1): v3469(N—H), 3274(O—H), 3056(C—H Ar), 2936(C—H), 1729(CO), 1670(CO), 758 (o-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.27(s, 3H, CH), 4.88(s, H, CH), 6.78–8.17 (m, 12H, aromatic), 9.02 (s, H, NH), 9.56 (s, H, OH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 17.95 (CH3), 43.3(C—CH), 115.2, 117.6, 118.7, 121.7, 123.5, 125.5, 126.3, 127.4, 128.7, 130.4, 154.3 (Ar—C), 193.5 (CO), 150.1 (CO), 162.2 (CO), 166.7 (C—O), 142.6 (CC), 157.2 (CO). MS (m/z, (relative abundance, %): 419 (M + 1, 27.3), 326 (M+, 37), 272 (M+, 100), 147 (M+, 41), 106 (23).

(E) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-5-(3-(4-nitrophenyl) acryloyl) -3,4-dihydropyrimidin-2(1H) -one (3f)

Brown crystals; yield 54.29%; m.p. 173–174 °C; IR (KBr, cm−1): v3479(N—H), 3113(O—H), 3081(C—H Ar), 2927(C—H), 1763(CO), 1692(CO), 1522,1345 (Nitro), 758 (o-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.42(s, 3H, CH), 4.75(s, H, CH), 7.05–8.30 (m, 12H, aromatic), 9.05 (s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 20.13 (CH3), 45.6(C—CH), 95.2, 117.9, 118.1, 121.9, 123.5, 124.3, 125.7, 126.8, 128.6, 129.2, 130.1, 141.5, 148.5, 154.2 (Ar—C), 193.6 (CO), 150.5 (CO), 162.4 (CO), 166.4 (C—O), 142.3 (CC). MS (m/z, (relative abundance, %): 448 (M + 1, 27.3), 326 (M+, 28), 272 (M+, 100), 177 (M+, 19), 123 (31).

(E) -5-(3-(4-chlorophenyl) acryloyl) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (3g)

Golden yellow crystals; yield 78.72%; m.p. 182–184 °C; IR (KBr, cm−1): v3438(N—H), 3238(O—H), 3062(C—H Ar), 2928(C—H), 1732(CO), 1672(CO), 758 (o-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.49 (s, 3H, CH), 4.92 (s, H, CH), 7.03–7.75 (m, 12H, aromatic), 9.09 (s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 20.4 (CH3), 43.2 (C—CH), 92.1, 117.2, 118.7, 121.5, 123.1, 125.5, 126.2, 128.3, 129.2, 129.9, 133.5, 133.9, 148.5, 154.4 (Ar—C), 193.3 (CO), 150.1 (CO), 162.8 (CO), 166.7 (C—O), 143.5 (CC). MS (m/z, (relative abundance, %): 438 (M + 2, 32.1), 326 (M+, 37), 272 (M+, 100), 162 (M+, 26), 111 (41).

(E) -5-(3-(4-(dimethylamino) phenyl) acryloyl) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (3h)

Dark brown crystals; yield 58.38%; m.p. 144–146 °C; IR (KBr, cm−1): v3402(N—H), 3225(O—H), 3032(C—H Ar), 2925(C—H), 1713(CO), 1668(CO), 765(o-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.47 (s, 3H, CH), 3.10 (s, 6H, CH), 5.05 (s, H, CH), 6.50–7.95 (m, 12H, aromatic), 9.05 (s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 20.7 (CH3), 40. 2 (N—CH), 45.1 (C—CH), 92.5, 117.6, 118.2, 121.7, 123.4, 124.4, 125.1, 126.5, 128.7, 129.1, 148.2 (Ar—C), 150.6, 154.4 (Ar—C—N), 193.6 (CO), 150.4 (CO), 162.2 (CO), 166.3 (C—O), 142.1 (CC). MS (m/z, (relative abundance, %): 446 (M + 1, 27.5), 326 (M+, 28), 272 (M+, 100), 174 (M+, 15), 120 (32).

(E) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -5-(3-(2-hydroxyphenyl) acryloyl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (3i)

Orange crystals; yield 68.83%; m.p. 155–157 °C; IR (KBr, cm−1): v3452(N—H), 3234(O—H), 3062(C—H Ar), 2928(C—H), 1742(CO), 1667(CO), 757(p-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.38 (s, 3H, CH), 4.74 (s, H, CH), 6.72–8.21 (m, 12H, aromatic), 9.07 (s, H, NH), 11.21 (s, H, OH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 20.2 (CH3), 43.4 (C—CH), 91.3, 117.4, 118.6, 121.2, 122.2, 123.1, 125.6, 126.3, 128.2, 129.1, 129.5, 148.4 (Ar—C), 154.6 (Ar—C—N), 193.1 (CO), 150.7 (CO), 162.8 (CO), 157.4, 166.7 (C—O), 152.4 (CC). MS (m/z, (relative abundance, %): 419 (M + 1, 25.3), 326 (M+, 17), 272 (M+, 100), 162 (M+, 35), 94 (26).

(E) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -5-(3-(3-hydroxyphenyl) acryloyl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (3j)

Yellow crystals; yield 53.11%; m.p. 136–138 °C; IR (KBr, cm−1): v3458(N—H), 3237(O—H), 3046(C—H Ar), 2930(C—H), 1730(CO), 1672(CO), 757(m-sub);1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.44 (s, 3H, CH), 5.03 (s, H, CH), 6.58–8.05 (m, 12H, aromatic), 9.07 (s, H, NH), 9.58 (s, H, OH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 20.4 (CH3), 45.2 (C—CH), 91.2, 115.3, 117.6, 117.8, 118.4, 121.2, 123.5, 125.1, 126.9, 128.4, 130.4, 148.2 (Ar—C), 154.3 (Ar—C—N), 193.4 (CO), 150.2 (CO), 162.3 (CO), 158.2, 166.3 (C—O), 142.5 (CC). MS (m/z, (relative abundance, %): 419 (M + 1, 25.3), 326 (M+, 17), 272 (M+, 100), 162 (M+, 35), 94 (26).

(E) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-5-(3-(3-nitrophenyl) acryloyl) -3,4-dihydropyrimidin-2(1H) -one (3k)

Greenish white crystals; yield 59.73%; m.p. 103–105 °C; IR (KBr, cm−1): v3385(N—H), 3238(O—H), 3049(C—H Ar), 2926(C—H), 1737(CO), 1673(CO), 1489,1282 (nitro), 753(m-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.38 (s, 3H, CH), 4.89 (s, H, CH), 6.49–8.52 (m, 12H, aromatic), 9.07 (s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 17.8 (CH3), 41.9 (C—CH), 92.4, 117.9, 118.1, 121.5, 123.8, 125.6, 126.9, 128.1, 130.1, 136.2, 137.9, 148.5 (Ar—C), 147.5, 154.3 (Ar—C—N), 193.2 (CO), 150.6 (CO), 162.1 (CO), 166.7 (C—O), 142.7 (CC). MS (m/z, (relative abundance, %): 448 (M + 1, 25.3), 326 (M+, 25), 272 (M+, 100), 177 (M+, 42), 123 (15).

(E) -5-(3-(2-chlorophenyl) acryloyl) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-3,4-dihydropyrimidin-2(1H) -one (3l)

Browncrystals; yield 57.25%; m.p. 106–107 °C; IR (KBr, cm−1): v3379(N—H), 3167(O—H), 3058(C—H Ar), 2926(C—H), 1739(CO), 1673(CO), 757(p-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.41 (s, 3H, CH), 4.95 (s, H, CH), 6.81–8.20 (m, 12H, aromatic), 9.03 (s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 20.2 (CH3), 42.3 (C—CH), 91.7, 117.7, 118.8, 121.3, 123.2, 125.2, 126.4, 127.3, 128.9, 129.1, 130.2, 133.2, 134.4, 148.1 (Ar—C), 154.3 (Ar—C—N), 193.2 (CO), 150.2 (CO), 162.6 (CO), 166.3 (C—O), 122.2 (CC), 134.5 (C—Cl). MS (m/z, (relative abundance, %): 438 (M + 2, 32.5), 326 (M+, 31), 272 (M+, 100), 166 (M+, 38), 112 (44).

(E) -4-(4-hydroxy-2-oxo-2H-chromen-8-yl) -6-methyl-5-(3-(3,4,5-trimethoxyphenyl) acryloyl) -3,4-dihydropyrimidin-2(1H) -one (3m)

Brick red crystals; yield 65.41%; m.p. 153–155 °C; IR (KBr, cm−1): v3414(N—H), 3234(O—H), 3059(C—H Ar), 2939(C—H), 1737(CO), 1675(CO), 758, 645(3,4,5-sub); 1H NMR (400 MHz, DMSO‑d6, δ/ppm): 2.47 (s, 3H, CH), 3.91 (s, 9H, CH), 4.78 (s, H, CH), 6.75–8.10 (m, 10H, aromatic), 9.08 (s, H, NH). 13C NMR (100 MHz, DMSO‑d6) δ/ppm: 20.5 (CH3), 40.4 (C—CH), 56.5, 60.7 (O—CH3), 92.4, 103.1, 117.3, 118.5, 121.6, 123.5, 125.7, 126.3, 128.7, 148.5 (Ar—C), 154.5 (Ar—C—N), 193.6 (CO), 150.7 (CO), 162.4 (CO), 138.5, 153.1, 166.3 (C—O), 142.5 (CC). MS (m/z, (relative abundance, %): 493 (M + 1, 28.7), 326 (M+, 45), 272 (M+, 100), 223 (M+, 27), 168 (32).

Docking studies

Docking study was performed for the evaluation of the anticancer activity of synthesized derivatives 3a-m against tyrosine kinase protein (4csv) using VLife MDS software. The ligands were prepared by the VLife engine tool. The X-ray crystal structure of protein tyrosine kinase (4csv) was downloaded from rcsb.com/pdb site. The protein crystal structure optimized by removal of water molecules, addition hydrogen, Ramachandran plot analysis using Biopredicta module tolls of VLifeMDS. To identify the co-crystal ligand bound to the amino acid sequence of the protein, save the protein structure and the extracted co-crystal ligand in mol2 format, which can be used as reference ligand. The grid docking was performed using GRIP docking under batch docking option in the Biopredicta module. The interpretation of the result was done based on the score obtained by conformers of the ligands. The conformer which has the least score should be the one which has the high binding energy to the active site in the protein. We can examine the interaction between the ligand and protein by selecting the different scoring options like hydrogen bond, hydrophobic interaction π-stacking, and van der Waals force, etc.

In vitro anticancer activity

The anticancer activity was evaluated for compounds 3h and 3m by in vitro methods using morphology and MTT assay against Human malignant cell lines like A549 (Lung), Jurkat (Leukemia) and MCF-7 (Breast). A549, Jurkat and MCF-7 cells were sub-cultured in-house, in PGP Life Sciences, Hyderabad. The source of the cell line is ATCC. Cells were grown in 75 cm2 bottle canted necked vented flasks (Corning) with RPMI and the cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Cells (passages 25–35) were grown in Dulbecco’s Modified Eagles medium (Gibco Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% penicillin (1000U/mL), 1% streptomycin (1000 μg/mL) and 1% amphotericin (250 U/mL). The cells were passaged enzymatically with 0.25% trypsin- 1 mM EDTA and sub-cultured on 75 cm2 plastic flasks at a density of 2.2 × 104 cells/cm2. The culture medium was replaced every 2 days. Subculturing was done by briefly rinsed the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor after Removing and discarding culture medium. 2.0–3.0 mL of Trypsin-EDTA solution was added to the flask and observed, cells under an inverted microscope until cell layer is dispersed (usually within 5–15 min). 6.0–8.0 mL of complete growth medium was added and aspirated the cells by gently pipetting. Appropriate aliquots of the cell suspension were added to new culture vessels. Cultures can be established between 2 × 103 and 1 × 104 viable cells/cm2. Do not exceed 7 × 104 cells/cm2. Cultures were incubated at 37 °C.

The morphology was done by the subcultured human cell lines, Jurkat, A549 and MCF-7 cells were treated 12 h post-seeding with 150 & 300 mg/mL concentration of 3h and 3m for morphological study. Cells were observed for 24, 48 and 72 h, after treatment of test samples. Cell confluence (80%) was confirmed by microscopic observance. Images were taken by the Axiovert 200 M phase-contrast microscope at the magnification of 10x. Axiovision Rel.4.2 software was used to acquire the images.

The MTT assay was performed by inoculating cultured Jurkat, A549 and MCF-7 cells (100 µL per well) in a clear bottom 96-well tissue (abdos) culture plates (The number of cells was 105 cells per well). All three cell lines Jurkat, A549, and MCF-7 were used for 3h; A549 and Jurkat cell lines were used for 3m to determine IC50 of their cytotoxicity. The compounds 3h and 3m were added in different concentrations ranging from 10 to 150 μg/mL (10, 25, 50, 75, 100, 125 and 150 μg/mL) in triplicate after 24 h of seeding and incubated the cells for 24 h, 48 h and 72 h period of time. A volume of 20 µL culture medium was used for all test samples. Remove the medium and wash the cell twice with PBS. Add 15 µL MTT reagent per well which was made up in PBS medium to a final concentration of 0.5 mg/mL. The volume of the reagent should be adjusted depending on the volume of the cell culture. Cells were incubated for 3 h at 37 °C until intracellular purple formazan crystals are visible under the microscope. MTT reagent removed and 100 µL of the DMSO was added to each well and mixed gently on an orbital shaker for one hour at room temperature. The volume of DMSO should be adjusted depending on the volume of cell culture. The absorbance was measured at 570 nm for each well on an absorbance plate reader and IC50 values were calculated. A positive control Imatinib was used as a known cytotoxic activity agent. Dimethyl sulfoxide was the vehicle used for dissolution of the testing compound, and its final concentration on the cells was less than 0.2%.

Results

Chemistry

Dihydropyrimidinone derived coumarinyl chalcones were synthesised according to the reported protocols (Scheme 2).

Scheme 2

Synthesis of compounds 3(a-m).

Briefly, the dihydropyrimidinone (1) was obtained by one pot synthesis from 2 hydroxy-benzaldehyde, pentane 2, 4 dione, urea using citric acid as catalyst. The obtained 2-hydroxy dihydropyrimidinone derivatives were then treated with malonic acid in presence of anhydrous ZnCl2. Finally, the obtained Dihydropyrimidino coumarins were treated with various aromatic aldehydes to yield the titled compounds (3a-m).

The results had shown that the compounds were fitted into the active pocket by forming hydrogen bonding, hydrophobic and π-stacking interactions with amino acids of tyrosine protein were shown in Fig. 1, Fig. 2. The docked compounds binding energies are exhibited in Table 1. The microscopic images displaying the morphological changes in cancer cell upon treatment with the compounds 3h and 3m are depicted in Fig. 3. Based on the morphological studies, we conducted the MTT assay for 3h against Jurkat, A549, and MCF-7 and 3m against Jurkat and A549 human cell-lines. The anti-cancer activity of compounds 3h and 3m are shown in Table 2. These obtained results are comprehensively explained below in the discussion section.

Fig. 1

Docking interactions 2D view: a) π-stacking interaction and hydrophobic interaction of derivative 3h; b) Hydrogen bonding and Hydrophobic of derivative 3m.

Fig. 2

Docking interactions 3D view: a) π-stacking interaction (Blue) and Hydrophobic interaction (Grey) of derivative 3h; b) Hydrogen bonding (Blue) and Hydrophobic (Grey) of derivative 3m.

Table 1

Binding energies of docked compounds.

S. No.	Compound	Binding energy
1	3h	−60.162
2	3m	−50.521
3	3f	−46.002
4	3k	−44.220
5	3a	−12.586
6	3i	0.475
7	3c	31.794
8	3l	66.242
9	3d	67.660
10	3j	69.478
11	3g	–
12	3b	–
13	3e	–
14	Imatinib	−118.260

Fig. 3

Digital microscopic images of morphological changes in cancer cell upon treatment with the compounds 3h and 3m.

Table 2

Anti-cancer activity of compounds 3h and 3m.

S. No.	Sample	Cytotoxicity at time (h)	IC50* (μg/mL) ± SD in different cell lines
			Jurkat (Leukaemia) Cell line	A549 (Lung) Cell line	MCF-7 (Breast) Cell line
1	3h	24 h	77.74 ± 1.21	90.82 ± 1.25	89.90 ± 1.27
		48 h	75.01 ± 1.36	85.31 ± 1.42	84.18 ± 0.85
		72 h	70.90 ± 1.14	79.34 ± 0.87	79.13 ± 1.41
2	3m	24 h	75.72 ± 1.36	86.91 ± 1.16	–
		48 h	70.17 ± 0.85	80.29 ± 0.73	–
		72 h	65.56 ± 0.75	74.28 ± 1.28	–
3	Imatinib	24 h	3.46 ± 1.57	2.84 ± 0.37	6.12 ± 1.05
		48 h	3.13 ± 1.47	2.62 ± 1.29	5.80 ± 0.26
		72 h	2.91 ± 0.82	2.43 ± 0.78	5.57 ± 0.92

Each value indicate three replicates.

Discussions

Molecular docking

The title compounds (3a-m) were docked into the active pocket of Src, Albtyrosine kinase homology model protein (PDB ID: 4csv) co-crystallized with Imatinib standard drug for elucidating the anti-cancer activity. The docking was performed by utilizing VLife MDS software to observe the binding interaction as docking score. Among all the derivatives synthesized, 3h and 3m had shown moderate interaction with docking scores as −60.162 and −50.521 respectively when compared to Imatinib docking score −118.26. The results had shown in Fig. 1& 2. indicating that the compounds were fitted into the active pocket by forming hydrogen bonding, hydrophobic and π-stacking interactions with amino acids of tyrosine protein. The derivative 3h has the π-stacking interaction between B ring of chalcone moiety and PHE148, Hydrophobic bond with ASP147, VAL65, MET65, LEU59, AL35, LYS37, PHE148, and VAL22. The derivative 3m has the Hydrogen bond interaction between the oxygen of paramethoxy and LYS37 amino acid, hydrophobic interaction with LEU64, LEU59, MET56, ASP147, VAL65, ALA146, LEU136, THR81, LYS37, GLU52 and VAL22 amino acids of the protein. Hence, the results proved that the compound 3h with dimethylamino substitution at the para position on the B ring of chalcone, compound 3m with methoxy substitution at the meta and para positions on the B ring of chalcone were found to have the moderate binding efficiency in the active pockets of the target protein. It indicates that the derivatives with methyl group substitution on the B ring of chalcone has the capability to moderately fit into active pocket of protein when compared to standard drug Imatinib and there may be a chance of compounds with more number of hydrophobic substitutions on the B ring of chalcone may increase the binding interaction with protein (Table 1).

In vitro anticancer activity

Based on the results of in-silico studies, it has been observed that the compounds (3a-m) might possess moderate to potent cytotoxic activity. Hence, the aim of our study is to validate the in-silico cytotoxic studies with the in vitro human anti-cancer activity against the human cancer cell lines A549 (Lung), Jurkat (Leukemia) and MCF-7 (Breast). Compounds 3a-m were screened for anti-cancer activity by in-vitro method using Morphology and MTT assay (Balakrishna and Kumar, 2015). The morphological evaluation was performed for the compounds 3a-m against three human malignant cell lines (Jurkat, A549 and MCF-7). Compound 3h had shown moderate cellular changes like rounding, vacuole formation against A549 and shrinking, abnormal rounding against MCF-7 and shrinking, blebbing against Jurkat cell line at both the concentrations tested (75 & 150 μg/mL) (Balakrishna and Kumar, 2015). Compound 3m shown moderate cellular changes like rounding, vacuole formation against A549, and shrinking, abnormal rounding against MCF-7, but doesn’t show any significant cellular changes against Jurkat cell line at both the concentrations tested (75 & 150 μg/mL). Microscopic images are depicted in Fig. 3. Based on the Morphological studies, we conducted the MTT assay for 3h against Jurkat, A549, and MCF-7 and 3m against Jurkat and A549 human cell-lines. The IC50 values were determined by Non-linear regression method and presented in Table 2.

Compound 3h which has a dimethylamino group at the para position in aromatic portion showed cytotoxic activity against three human cell lines Jurkat, A549 and MCF-7 with IC50values of 70.90, 79.34, and 79.13 μg/mL respectively. Compound 3m which has 3, 4, 5-trimethoxy substitution in aromatic portion showed cytotoxic activity against two human cell lines Jurkat and A549 with IC50 values of 65.56 and 74.28 μg/mL. Results indicating that the cytotoxic activity of compound 3h and 3m was less when compared to standard drug Imatinib which has potent cytotoxic activity against three human cell lines Jurkat, A549 and MCF-7 with IC50 values of 2.91, 2.43, and 5.57 μg/mL respectively. The in vitro MTT assay results were found to be in accordance with in-silico docking studies, which indicates that compound 3h and 3m possess less cytotoxic activity when compared with standard drug Imatinib.

Moreover, when the cell lines subjected to the compounds 3h and 3m were observed under the microscope, it was evident that the compounds caused skunking and blebbing of cells, which leads to the cell death. Vacuoles were formed in cells A549 which lead to cell death, cell deformities induced in the cells tested by the compounds 3h and 3m is indicated by arrows.

Conclusion

Some new coumarinylchalcones derivatives (3a-m) were synthesized and evaluated for anticancer activity. The in silico docking results described that the derivatives with dimethylamino substitution at para position (3h) and 3, 4, 5 trimethoxy substitution (3m) on B ring of chalcone moiety shows good binding energies. Further in vitro anticancer activity was performed using MTT assay methods for compound showing good results in in silico studies. Morphology evaluation indicates that the compound 3h is potent against all three human malignant cell lines A549 (Lung), Jurkat (Leukemia) and MCF-7 (Breast), while compound 3m is moderate against only two human malignant cell lines A549 (Lung) and Jurkat (Leukemia). MTT assay indicates that the compound 3h is potent against all three human malignant cell lines A549 (Lung), Jurkat (Leukemia) and MCF-7 (Breast) with IC50 values as 70.90, 79.34 79.13 μg/mL respectively. The compound 3m showed moderate activity against two human malignant cell lines A549 (Lung) and Jurkat (Leukemia) with IC50 values as 65.56 and 74.28 μg/mL. Future research is required to explain the mechanism of action for anti-cancer activity.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.