Microwave-assisted synthesis, characterization, and molecular docking studies of new chlorophthalazine derivatives as anticancer activity

Abstract

A series of phthalazine derivatives have been synthesized using green protocol methods under microwave irradiation. This involved subjecting chlorophthalazine to microwave irradiation and employing various reagents, including hydrazine derivatives, primary and secondary amines, and active methylene, to generate the novel phthalazine derivatives. Subsequently, a comparison was made between the yield percentage and reaction time of the resulting products obtained from conventional methods and those obtained through microwave irradiation. The structures of all new compounds were confirmed by physical properties and spectral analysis. The phthalazine compounds demonstrated significant anticancer activity against three cancer cell lines and showed promising selectivity when compared with doxorubicin. Compound 6 exhibited the best anticancer activity, with IC₅₀ 1.739 µM, 0.384 µM, and 1.52 µM against MCF7, HCT116, and HepG2 cell lines, respectively. The docking results confirmed that the drugs exert their activity by inhibiting PARP-1. The binding scores were in accordance with the experimental IC₅₀, with compound 6 exhibiting the best binding score and retaining the essential binding interactions with the active site. The results of our computational research have revealed that our phthalazine compounds possess a unique structure that effectively inhibits PARP-1.

Keywords:

• Anticancer activity
• chlorophthalazine derivatives
• microwave-assisted
• molecular docking study
• phthalazine derivatives
• one-pot reactions 
  

1. Introduction

Phthalazine derivatives are an important class of heterocyclic compounds that have garnered significant attention due to their diverse range of biological activities and pharmacological properties. Heterocyclic compounds are organic compounds that have structures containing ring systems made up of carbon atoms and at least one or more heteroatoms (Joule & Mills, 2000). They have been proven to possess a diverse array of pharmaceutical and medicinal uses. Moreover, they can be found in natural products from plants and animals such as chlorophyll, folic acid, haemoglobin, amino acids, nucleic acids and vitamins (Eicher, Hauptmann, & Speicher, 2012; Teran, Besada, Vila, Costas-Lago, & Ter, 2019). In addition, phthalazines derivatives have garnered significant interest owing to their intriguing biological features, pharmacological applications, and potential for therapeutic development, particularly in the realm of antibacterial agents (Moustafa, El‐Sayed, Abd El‐Hady, Haikal, & El‐Hashash, 2016; Sangshetti et al., 2019), antifungal (Hashash, 2014), anti-inflammatory activities (Vila, Besada, Costas, Costas-Lago, & Terán, 2015), antiviral (Lu et al., 2018), antileishmanial (Romero, Rodríguez, Oviedo, & Lopez, 2019), antidepressant (Inoue et al., 2015), antitumor (Wasfy, Aly, Behalo, & Mohamed, 2020), anticancer (Behalo, Gad El-Karim, & Rafaat, 2017; Khalifa et al., 2021), and vascular endothelial growth factor receptor 2 (VEGFR-2) activities (El Adl, Ibrahim, Khedr, Abulkhair, & Eissa, 2022; El Rayes, El-Enany, Ali, Ibrahim, & Nafie, 2022). More specifically, the anticancer activity of phthalazine derivatives has been described in various research. Hybrid molecules of steroids and phthalazine were shown to exhibit antiproliferative activity in a dose-dependent manner on various cancer cell lines (Tantawy et al., 2023). New phthalazine derivatives analogues of thalidomide showed far better antiproliferative activity compared to thalidomide against HepG-2, MCF7, and PC3 cell lines where the derivatives were shown to decrease the expression of caspase-8, vascular endothelial growth factor (VEGF), NF-κB P65, and TNF-α in HepG-2 cells (Kotb et al., 2022). Phthalazinone-dithiocarbamate hybrids were described in another work and proved significant antiproliferative activity against A2780, NCI-H460, and MCF7 cell lines (Vila, Besada, Brea, Loza, & Terán, 2022). Additionally, phthalazino[1,2-b]-quinazolinone derivatives were proven to possess broad spectrum histone deacetylase (HDAC) inhibition in the nanomolar range with subsequent activity against solid tumours (Liu, Zhang, Wang, Wang, & Gou, 2022). More importantly, the phthalazine derivative BMN-673 was found to increase cell death through inhibition of DNA repair essentially through trapping of PARP-1 and PARP-1 catalytic inhibition in breast cancer cells (Sethy & Kundu, 2022). Microwave-assisted synthesis is a contemporary organic synthesis technique that offers several advantages for heterocyclic compounds. These advantages include an accelerated reaction rate, reduced reaction time, and increased efficiency in synthesis methods. Additionally, the ability to control reaction parameters, such as temperature, can lead to enhanced yields and higher purity yields. Furthermore, the use of microwave-assisted synthesis allows for milder reaction conditions. Lastly, greener synthesis methods typically require lower energy consumption compared to conventional methods (Daştan, Kulkarni, & Török, 2012; Majumder, Gupta, & Jain, 2013). Previous studies have documented many aspects of organic processes, including the production of rings by cyclization as well as cycloaddition and nucleophilic substitution reactions. Multiple studies have documented the successful synthesis of phthalazine derivatives using microwave irradiation, resulting in high yields (Cao, Qu, Burton, & Rivero, 2008; Rao, Basak, Deb, Sharma, & Reddy, 2013). The field of organic synthesis has garnered considerable interest in recent times due to its application in the production of a wide range of organic compounds, including heterocyclic compounds, including compounds that are commonly found in natural products and possess a wide array of pharmacological and medicinal properties, making them valuable for synthetic purposes (Al Mousawi, El-Apasery, & Elnagdi, 2008; Al Saleh, Hilmy, El-Apasery, & Elnagdi, 2006). The objective of this study is to demonstrate the synthesis and reactions of chlorophthalazine derivatives using various reagents, including hydrazine derivatives, primary and secondary amines, and active methylene compounds. The aim is to generate novel phthalazine derivatives through the utilization of green protocol microwave irradiation methods and, subsequently, compare the yield and reaction time of the resulting products obtained from conventional methods with those obtained through microwave irradiation methods. Next, the anticancer and selective activity of compounds and doxorubicin against three cell lines, and normal fibroblasts are tested. Furthermore, a docking study is also performed to get insight into their mode of action as potential inhibitors of PARP-1 and identify the molecular mechanism of their anticancer activity.

Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in DNA repair. PARP inhibitors have shown promising results in both clinical trials and practice for the treatment of ovarian, breast, prostate, and pancreatic cancers (Rose, Burgess, O'Byrne, Richard, & Bolderson, 2020). In continuation of our work that showed the promising anticancer activity of a series of phthalazinone and phthalazine derivatives and emphasized the importance of the phthalazinone scaffold in binding the PARP-1 target and the potential of exploring the substitution in position 1, a series of 1-substituted-amino and 1-N-heterocycle phthalazinone were designed and tested (Abubshait et al., 2022). Our designed compounds are analogs of the anticancer drug Olaparib and would benefit from the simultaneous 1,4 substitution and the amino functionality in the 4-position, which may result in increased target selectivity and decreased toxicity (Figure 1).

Figure 1. Compound design based on structure similarity with the anticancer drug, olaparib.

2. Materials and methods

2.1. General condition

All the solvents and reagents used were purchased from Sigma-Aldrich Chemicals, USA. The microwave-assisted method is carried out by using a CEM microwave (MARS^™ 6 Synthesis) parallel and Scale-up microwave synthesizer by CEM company in the USA, at 300–400 W and 70–78.37 °C. Melting points determined via the Stuart SMP30 Digital Advanced MP apparatus are uncorrected. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded on an IR Affinity spectrophotometer from Shimadzu. The sample prepared on glass plate contains solid KBr. Nuclear magnetic resonance (NMR) spectra were recorded by a BRUKER spectrometer (400MHZ) by using dimethylsulphoxide (DMSO‐d₆) as solvent, chemical shifts (δ) in part per million (ppm) using tetramethylsilane (TMS) as internal standard. The mass spectra were carried out on LC/MS spectroscopy (LCMS-8040 Shimadzu, model CAT-30A). Elemental microanalysis was done on the Carlo Erba analyser model 110.

2.2. Synthetic chemistry procedure

2.2.1. Synthesis of 1-chloro-4-(4-isopropyl-phenyl)-phthalazine (2)

Compound 1 [4-(4-isopropyl-phenyl)-2H-phthalazin-1-one] was confirmed before, as in the previous paper (Abubshait et al., 2022). A mixture of 1 (2.64 gm, 10 mmol) dissolved in (30 ml) of dry benzene, phosphorus oxychloride (3.00 ml, 30 mmol), and phosphorus pentachloride (2.08 gm,10 mmol), under the air of nitrogen gas, was refluxed (30 min). Then the mixture poured in crushed ice with NaHCO₃ to give compound 2 after filtered and crystallized from dry benzene to give beige powder; mp.: 240–242 °C; FT‐IR (KBr, ν, cm⁻¹): 2922, 2865 (-CH, -CH₃), 3068-3010 (=CH, Ar), 1635, 1448 (C = C); ¹H NMR, δ (ppm) in DMSO-d₆: 1.28 (d, 6H, 2CH₃), 2.98 (m, 1H, -CH(CH₃)₂), 7.24-8.54 (m, 8H, -CH-, ArH); ¹³C NMR, δ (ppm) in DMSO-d₆: 23.91 (2 C, -CH₃), 30.9 (1 C, -CH(CH₃)₂), 126.7, 127.5, 128.5, 132.0, 148.0 (10 C, =CH-, Ar), 130.6, 134.0, 151.6 (3 C,=CH-, pyridazine), 171 (1 C, -C-Cl); MS (m/z): 282.09 (M⁺,100.0), 283.10 (M⁺ + 1,19.1); Anal. calcd. for C₁₇H₁₅ClN₂/(282.77): C, 72.21; H, 5.35; N, 9.91; Cl, 12.54%; found: C, 72.22; H, 5.36; N, 9.92; Cl, 12.53%.

2.2.2. Synthesis of hydrazine derivatives (3–10)

A mixture of 2 (2.84 gm, 10 mmol) and hydrazine hydrate (0.65 ml, 20 mmol), and/or (10 mmol) phenylhydrazine (0.98 ml), 3,4-dimethylphenylhydrazine hydrochloride (1.72 gm), 3-nitrophenylhydrazine hydrochloride (1.89 gm), 2,4-dinirtophenyl hydrazine (1.98 gm), and 2-thiophene carboxylic hydrazide, benzoyl hydrazine (1.36 gm), and semicarbazide hydrochloride (1.12 gm) in (20 ml) dry benzene with Et₃N (1 ml) was refluxed at 60 °C for 5–6 h (MW, 9–25 min). After that the precipitated products were collected, washed with cold benzene, dried, and then recrystallized from benzene.

2.2.2.1. [4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-hydrazine (3)

White powder; mp.: 260–263 °C; FT‐IR (KBr, ν, cm⁻¹): 2956, 2868 (-CH, -CH₃), 3064-3010 (=CH, Ar), 1606, 1506 (C = C), 3304-3273 (-NH-NH₂); ¹H NMR, δ (ppm) in DMSO-d₆: 1.24 (d, 6H, 2CH₃), 3.47 (m, 1H, -CH(CH₃)₂), 7.34–8.75 (m, 8H, -CH-, Ar-H), 12.03, 13.01 (br, 3H, -NH-NH₂); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.27 (2 C, -CH₃), 33.7 (1 C, -CH(CH₃)₂), 125.6, 127.5, 128.7, 132.0, 143.0 (10 C, =CH-, Ar), 130.8, 135.0, 155.6 (3 C,=C-, pyridazine), 168.0 (1 C,=C-NH-NH₂); MS (m/z): 278.15 (M⁺,100.0), 279.16 (M⁺ + 1, 56.0); Anal. calcd. for C₁₇H₁₈N₄/(278.35); C, 73.35; H, 6.52; N, 20.13%; found: C, 73.33; H, 6.53; N, 20.14%.

2.2.2.2. N-[4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-N'-phenyl-hydrazine (4)

Brown powder; mp.:147–149 °C; FT‐IR (KBr, ν, cm⁻¹): 2957, 2862 (-CH, -CH₃), 3050-3019 (=CH, Ar), 1606, 1491 (C = C), 3320-3210 (-NH-NH); ¹H NMR, δ (ppm) in DMSO-d₆: 2.48 (d, 6H, 2CH₃), 3.42 (m, 1H, -CH(CH₃)₂), 6.71–8.55 (m, 13H, -CH-, ArH), 11.86 (br, 2H, -NH-NH); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.4 (2 C, -CH₃), 33.7 (1 C, -CH(CH₃)₂), 126.4, 128.4, 128.7, 132.0 (15 C, =CH-, Ar), 130.0, 134.0, 155.6 (3 C,=C-, pyridazine), 147.3, 166.0 (2 C, =C-NH-NH-C=); MS (m/z): 354.18 (M⁺,100.0), 355.19 (M⁺ + 1, 25.1); Anal. calcd. for C₂₃H₂₂N₄/(354.45): C, 77.94; H, 6.26; N, 15.81%; found: C, 78.00; H, 6.27; N, 15.83%.

2.2.2.3. N-[4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-N'-(3,4-dimethyl phenyl-hydrazine (5)

Light brown powder; mp.:115-116 °C; FT‐IR(KBr, ν, cm⁻¹): 2956, 2868 (-CH, -CH₃), 3060-3022 (=CH, Ar), 1606, 1475 (C = C), 3320-3208 (-NH-NH); ¹H NMR, δ (ppm) in DMSO-d₆: 2.09 (d, 6H, 2CH₃), 2.50 (s, 6H, 2CH₃), 3.43 (m, 1H, -CH(CH₃)₂), 7.2–8.00 (m, 11H, -CH-, ArH), 8.40 (br, 2H, -NH-NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 8.9 (2 C, -CH₃), 20.0 (2 C, -CH₃), 39.5 (1 C, -CH(CH₃)₂), 123.9, 127.8, 128.7, 130.4 (15 C, =CH-, Ar), 130.0, 135.0, 155.6 (3 C,=CH-, pyridazine), 167.1, 145.2 (2 C, =C-NH-NH-C=); MS (m/z): 382.22 (M⁺,100.0), 381.22 (M⁺ + 1,47.7); Anal. calcd. for C₂₅H₂₆N₄/(382.50); C, 78.50; H, 6.85; N, 14.65%; found: C, 78.48; H, 6.85; N, 14.67%.

2.2.2.4. N-[4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-N'-(3-nitro-phenyl)-hydrazine (6)

Light orange powder; mp.: 196–198 °C; FT‐IR (KBr, ν, cm⁻¹): 2933, 2850 (-CH, -CH₃), 3088-3010 (=CH, Ar), 1599, 1457 (C = C), 1550,1372 (-NO₂), 3321-3319 (-NH-NH); ¹H NMR, δ (ppm) in DMSO-d₆: 1.26 (d, 6H, 2CH₃), 3.42 (m, 1H, -CH(CH₃)₂), 7.35-8.91 (m, 12H, -CH-, ArH), 10.33,12.80 (br, 2H, -NH-NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.3 (2 C, -CH₃), 32.7 (1 C, -CH(CH₃)₂), 108.4, 116, 121, 128.8, 132.0 (14 C, =CH-, Ar), 130.0, 133.0, 155.6 (3 C,=CH-, pyridazine), 148.9 (1 C,=CH-NO₂), 147.5, 169.0 (2 C, =C-NH-NH-C=); MS (m/z): 399.17 (M⁺,62.4), 401.17 (M⁺ + 2,16.1), 401.18 (M⁺²,3.2); Anal. calcd. for C₂₃H₂₁N₅O₂/(399.45): C, 69.16; H, 5.30; N, 17.53%; found: C, 69.16; H, 5.28; N, 17.52%.

2.2.2.5. N-[4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-N'-(2,4-dinitro-phenyl)-hydrazine (7)

Brown powder; mp.: 186–188 °C; FT‐IR (KBr, ν, cm⁻¹): 2930, 2853 (-CH, -CH₃), 3078-3010 (=CH, Ar), 1597, 1458 (C = C), 1550,1370 (-NO₂), 3421-3319 (-NH-NH); ¹H NMR, δ (ppm) in DMSO-d₆: 1.29 (d, 6H, 2CH₃), 3.12 (m, 1H, -CH(CH₃)₂), 7.19–8.90 (m, 11H, -CH-, ArH), 10.33,12.81 (br, 2H, -NH-NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.4 (2 C, -CH₃), 31.7 (1 C, -CH(CH₃)₂), 113.4, 117, 126.7, 127, 133.7,148.7 (15 C, =CH-, Ar), 143.6 (1 C,=CH-, pyridazine), 139.9 (2 C,=CH-NO₂), 143.5, 166.0 (2 C, =C-NH-NH-C=); MS (m/z): 444.15 (M⁺,62.4), 446.17 (M⁺ + 2,16.1), Anal. calcd. for C₂₃H₂₀N₆O₄/(444.44): C, 62.16; H, 4.54; N, 18.91%; found: C, 62.19; H, 5.58; N, 18.82%.

2.2.2.6. N-[4-(4-isopropyl-phenyl)-phthalazin-1-yl]-hydrazide-N'-thiophene-2-carboxylic acid (8)

Light brown powder; mp.: 190–192 °C; FT‐IR (KBr, ν, cm⁻¹): 2930, 2852 (-CH, -CH₃), 3079-3010 (=CH, Ar), 1599, 1457 (C = C), 3421-3319 (-NH-NH); 1680 (C = O); ¹H NMR, δ (ppm) in DMSO-d₆: 1.26 (d, 6H, 2CH₃), 3.42 (m, 1H, -CH(CH₃)₂), 7.06–7.93 (m, 11H, -CH-, ArH), 10.33,12.80 (br, 2H, -NH-NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.4 (2 C, -CH₃), 31.7 (1 C, -CH(CH₃)₂), 119, 126, 128.5, 133.0, 136.7 (13 C, =CH-, Ar), 117.0, 127.0,143.3 (3 C,=CH-, pyridazine),145.5 (1C, -C-CO) 167.3 (1 C,-C = O), 166.0 (1 C, NH-NH-C=); MS (m/z): 388.49 (M⁺,100), 389.14.17 (M⁺ + 1,15.1), Anal. calcd. For C₂₂H₂₀N₄OS/(388.49): C, 68.02; H, 5.19; N, 14.42; S, 8.25%; found: C, 68.05; H, 5.20; N, 14.52, S, 8.22%.

2.2.2.7. 6-(4-Isopropyl-phenyl)-3-phenyl-[1,2,4]triazolo[3,4-a]-phthalazine (9)

Light orange powder; mp.: 186–188 °C; FT‐IR (KBr, ν, cm⁻¹): 2928, 2850 (-CH, -CH₃), 3086-3011 (=CH, Ar), 1597, 1459 (C = C); ¹H NMR, δ (ppm) in DMSO-d₆: 1.29 (d, 6H, 2CH₃), 3.12 (m, 1H, -CH(CH₃)₂), 7.19–7.93 (m, 13H, -CH-, ArH); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.2 (2 C, -CH₃), 31.7 (1 C, -CH(CH₃)₂), 126.1, 128.8, 129.0, 133.0, 136.5 (18 C,=CH-,Ar, pyridazine), 127.2 (2 C,=CH-, pyridazine), 148.1 (1 C,=CH-, triazole), 152.1 (2 C, =C-N-N-C=); MS (m/z): 364.44 (M⁺,100), 365.40 (M⁺ + 1,18.1), Anal. calcd. for C₂₄H₂₀N₄/(364.44): C, 79.10; H, 5.53; N, 15.37%; found: C, 79.14; H, 5.55; N, 15.32%.

2.2.2.8. 6-(4-Isopropyl-phenyl)-[1,2,4]triazolo[3,4-a]-phthalazin-3-yl-amine (10)

Light yellow; mp.: 176–178 °C; FT‐IR (KBr, ν, cm⁻¹): 2932, 2853 (-CH, -CH₃), 3090-3030 (=CH, Ar), 1595, 1457 (C = C), 3386-3330 (-NH₂); ¹H NMR, δ (ppm) in DMSO-d₆: 1.29 (d, 6H, 2CH₃), 3.12 (m, 1H, -CH(CH₃)₂), 7.19-7.93 (m, 8H, -CH-, ArH), 10.3 (br, 2H, -NH₂); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.3 (2 C, -CH₃), 31.7 (1 C, -CH(CH₃)₂), 126.7,133.0,148.7 (10 C, =CH-, Ar), 127.0, 125.1 (4 C,=CH-, pyridazine), 148.9 (1 C,=C-NH₂); MS (m/z): 303.36 (M⁺,65.4), 305.16 (M⁺ + 2,16.1), Anal. calcd. for C₁₈H₁₇N₅/(303.36): C, 71.27; H, 5.65; N, 23.09%; found: C, 71.29; H, 5.69; N, 23.10%.

2.2.3. Synthesis of compound (11–17)

A mixture of (2.84 gm, 10 mmol) of 2 and (10 mmol) of methyl-amine (0.31 ml), aniline (0.93 ml), p-toluidine (1.07 gm), p-anisidine (1.23 gm), β-naphthylamine (1.43 gm), anthranilic acid (1.37 gm) and diphenylamine (1.69 gm) in dry benzene (30 ml) placed in an oil bath and refluxed 4–6 h at 80 °C and (MW, 9–20 min). The residues were filtered to crystalline from, the mixture was extracted with ether, and ether layer was dried with anhydrous MgSO₄. Finally, the residue was distilled off to be crystallized from a suitable solvent to give 11–17.

2.2.3.1. [4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-methyl-amine (11)

Beige powder; mp.:160–162 °C; FT‐IR (KBr, ν, cm⁻¹): 2970, 2868 (-CH, -CH₃), 3070-3010 (=CH, Ar), 1607, 1456 (C = C), 3450 (-NH), 1188 (-C-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.27 (d, 6H, 2CH₃), 2.31 (d, 3H, -CH₃), 3.16 (m, 1H, -CH(CH₃)₂), 7.19–8.03 (m, 8H, -CH-, ArH), 10.57 (br, 1H, -NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.1 (2 C, -CH₃), 35.3 (1 C, -CH₃), 31.5 (1 C, -CH(CH₃)₂), 126.7, 127.4, 132.1,133.4 (10 C, =CH-, Ar),117, 127.1, 143.1 (3 C,=CH-, pyridazine), 166.0 (1 C, =C-NH_-); MS (m/z): 277.36 (M⁺,98.0); Anal. calcd. for C₁₈H₁₉N₃/(277.36): C, 77.95; H, 6.90; N, 15.15%; found: C, 77.93; H, 6.87; N, 15.17%.

2.2.3.2. [4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-phenyl-amine (12)

Beige powder; mp.:150–151 °C; FT‐IR (KBr, ν, cm⁻¹): 2955, 2870 (-CH, -CH₃), 3061-3010 (=CH, Ar), 1607, 1456 (C = C), 3450 (-NH), 1188 (-C-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.27 (d, 6H, 2CH₃), 3.46 (m, 1H, -CH(CH₃)₂), 6.58–8.33 (m, 13H, -CH-, ArH), 11.57 (br, 1H, -NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.3 (2 C, -CH₃), 31.7 (1 C, -CH(CH₃)₂), 115.4, 117.0, 126.9, 128.4, 129.4, 133.1, 147.1 (15 C, =CH-, Ar), 131.0, 133.3, 151.1 (3 C,=CH-, pyridazine), 166.2, 166.0 (2 C, =C-NH-C=); MS (m/z): 339.17 (M⁺,61.0); Anal. calcd. for C₂₃H₂₁N₃/(339.43): C, 81.38; H, 6.24; N, 12.38%; found: C, 81.40; H, 6.22; N, 12.40%.

2.2.3.3. [4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-p-tolyl-amine (13)

Beige powder; mp.:140–141 °C; FT‐IR (KBr, ν, cm⁻¹): 2965, 2868 (-CH, -CH₃), 3050-3020 (=CH, Ar), 1604, 1479 (C = C), 3450 (-NH), 1170 (-C-N); ¹H NMR, δ (ppm) in DMSO-d₆: 2.18 (d, 6H, 2CH₃), 2.51 (s,3H, 2CH₃), 3.54 (m, 1H, -CH(CH₃)₂), 6.71–8.07 (m, 12H, -CH-, ArH), 9.0 (br, 1H, -NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 20.7 (1 C, -CH₃), 24.3 (2 C, -CH₃), 34.7 (1 C, -CH(CH₃)₂), 117.0, 128.4, 129.4, 133.1, 141.1 (15 C, =CH-, Ar), 130.0, 133.3, 151.1 (3 C,=CH-, pyridazine), 168.2, 169.0 (2 C,=C-NH-C=); MS (m/z): 353.19 (M⁺,100.0), 350.19 (M⁺-3,7.8); Anal. calcd. for C₂₄H₂₃N₃/(353.46): C, 81.55; H, 6.56; N, 11.89%; found: C, 81.54; H, 6.56; N, 11.90%.

2.2.3.4. [4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-(4-methoxy-phenyl)-amine (14)

Light purple powder; mp.: 126–128 °C; FT‐IR (KBr, ν, cm⁻¹): 2960, 2868 (-CH, -CH₃), 3060-3020 (=CH, Ar), 1600, 1475 (C = C), 1270 (-C-O-C), 3450 (-NH), 1170 (-C-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.27 (d, 6H, 2CH₃), 3.46 (m, 1H, -CH(CH₃)₂), 1.27 (s,3H, -OCH₃), 6.77-8.07 (m, 12H, -CH-, ArH), 11.34 (br, 1H, -NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.3 (2 C, -CH₃), 31.7 (1 C, -CH(CH₃)₂), 55.8 (1 C, -OCH₃), 115.4, 118, 125.6, 128.4, 129.4, 133.1, 147.1 (14 C, =CH-, Ar), 131.0, 133.3, 151.1 (3 C,=CH-, pyridazine), 154.0 (1 C, =C-OCH₃), 139.0, 169.0 (2 C, =C-NH-C=); MS (m/z): 369.18 (M⁺,100.0), 370.19 (M⁺ + 1,27.1); Anal. calcd. for C₂₄H₂₃N₃O/(369.46): C, 78.02; H, 6.27; N, 11.37%; found: C, 78.03; H, 6.27; N, 11.40%.

2.2.3.5. [4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-naphthalen-2-yl-amine (15)

Beige powder; mp.:180–183 °C; FT‐IR (KBr, ν, cm⁻¹): 2958, 2878 (-CH, -CH₃), 3071-3030 (=CH, Ar), 1609, 1459 (C = C), 3450 (-NH), 1188 (-C-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.28 (d, 6H, 2CH₃), 3.45 (m, 1H, -CH(CH₃)₂), 6.76–8.01 (m, 15H, -CH-, ArH), 10.57 (br, 1H, -NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.3 (2 C, -CH₃), 31.7 (1 C, -CH(CH₃)₂), 115.4, 119.0, 126.7, 127.4, 133.1, 148.1 (19 C, =CH-, Ar), 117.1, 127.3, 127.1 (3 C,=CH-, pyridazine), 142.6, 166.6 (2 C, =C-NH-C=); MS (m/z): 389.19 (M⁺,100.0), 390.19 (M⁺ + 1,27.1); Anal. calcd. for C₂₇H₂₃N₃/(389.49): C, 83.26; H, 5.95; N, 10.79%; found: C, 83.21; H, 5.98; N, 10.80%.

2.2.3.6. 5-(4-Isopropyl-phenyl)-6,6a,12-triaza-benzo[a]anthracen-7-one (16)

White powder; mp.: 190–191 °C; FT‐IR (KBr, ν, cm⁻¹): 2957, 2862 (-CH, -CH₃), 3060-3010 (=CH, Ar), 1600, 1475 (C = C), 1670 (C = O); ¹H NMR, δ (ppm) in DMSO-d₆: 1.20 (d, 6H, 2CH₃), 3.42 (m, 1H, -CH(CH₃)₂), 7.2–7.5 (m, 12H, -CH-, ArH); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.4 (2 C, -CH₃), 31.7 (1 C, -CH(CH₃)₂), 110, 116, 126.4, 128.4, 131, 133 (16 C, =CH-, Ar), 134.3, 155.6 (2 C,=CH, pyridazine), 151, 169 (2 C, -C = N); 170 (1 C, -C = O); MS (m/z): 365.15 (M⁺,100), 366.16 (M⁺ + 1,27.0) Anal. calcd. for C₂₄H₁₉N₃O(365.43): C, 78.88; H, 5.24; N, 11.50%; found: C, 78.90; H, 5.26; N, 11.52%.

2.2.3.7. [4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-diphenyl-amine (17)

White powder; mp.:203–204 °C; FT‐IR(KBr, ν, cm⁻¹): 2962, 2852 (-CH, -CH₃), 3070-3010 (=CH, Ar), 1600, 1475 (C = C), 1360 (-C-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.25 (d, 6H, 2CH₃), 3.43 (m, 1H, -CH(CH₃)₂), 7.35–8.07 (m, 18H, -CH-, ArH),¹³C NMR, δ (ppm) in DMSO-d₆: 24.3 (2 C, -CH₃), 33.7 (1 C, -CH(CH₃)₂), 117, 120, 125.6, 127, 128.8, 129.6, 133.1, 147.1 (20 C, =CH-, Ar), 131, 133.3, 151.1 (3 C,=CH-, pyridazine), 143, 169 (3 C, -N-C=); MS (m/z): 415.20 (M⁺,100.0), 416.21 (M⁺ + 1,15.1); Anal. calcd. for C₂₉H₂₅N₃/(415.53): C, 83.82; H, 6.06; N, 10.11%; found: C, 83.84; H, 6.04; N, 10.09%.

2.2.4. Synthesis compounds pyrazol phthalazin derivatives (18–21)

A mixture (2.84 gm,10 mmol) of 2 (10 mmol) of acetyl acetone (1.00 ml), and/or ethyl acetoacetate (1.27 ml), ethyl cyanoacetate (1.13 ml), and malononitrile (0.55 gm) was dissolved in 30 ml ethanol with C₂H₅ONa, then refluxed in an oil bath for 5 h at 80 °C (MW, 10 min). After an excess solvent is distilled, off, poured into cold water, then extracted with diethyl ether. The solid crystallized from benzene, then dissolved in 30 ml of ethanol, and (0.9 ml, 20 mmol) of hydrazine hydrate, and refluxed to give 18–21, The separated solid crystallized from a suitable solvent.

2.2.4.1. 1-(3,5-Dimethyl-4H-pyrazol-4-(4-isopropyl-phenyl)-phthalazine (18)

White crystals; mp.:270–271 °C; FT‐IR(KBr, ν, cm⁻¹): 2962,2852 (-CH, -CH₃), 3060, 3010 (=CH, Ar), 1598,1440 (C = C), 1610 (C-N-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.29 (d, 6H, 2-CH₃), 2.00 (s, 6H, 2-CH₃, pyrazole), 2.50 (d,1H, -CH-, pyrazole), 3.12 (m, 1H, -CH(CH₃)₂), 7.4–8.3 (m, 8H, -CH-, ArH); ¹³C NMR, δ (ppm) in DMSO-d₆: 18.2 (2 C, -CH₃, pyrazole), 24.3 (2 C, -CH₃), 33.7 (1 C, -CH(CH₃)₂), 39.9 (1 C, -CH-, pyrazole), 126.5, 127.1, 128.7, 129 (10 C, =CH-, Ar), 132, 134.5, 150.6 (4 C,=CH-, pyridazine), 160 (2 C, -C-CH₃, pyrazole); MS (m/z): 342.18 (M⁺,100.0), 341.19 (M⁺-1,24.8); Anal. calcd. for C₂₂H₂₂N₄/(342.44): C, 77.16; H, 6.48; N, 16.36%; found: C, 77.18; H, 6.45; N, 16.38%.

2.2.4.2. 4-[4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-5-methyl-2,4-dihydro-pyrazol-3-one (19)

White crystal; mp.:250–252 °C; FT‐IR(KBr, ν, cm⁻¹): 2963,2853 (-CH, -CH₃), 3067-3020 (=CH, Ar), 1598,1440 (C = C), 1680 (C = O), 1610 (C-N-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.29 (d, 6H, 2CH₃), 2.00 (s, 3H, -CH₃), 3.43 (m, 1H, -CH(CH₃)₂), 5.21 (s,1H, -CH-, pyrazole), 7.243–8.3 (m, 8H, -CH-, ArH), 12.85 (br, 1H, -NH); ¹³C NMR, δ (ppm) in DMSO-d₆: 15.3 (1 C, -CH₃), 24.3 (2 C, -CH₃), 33.8 (1 C, -CH(CH₃)₂), 40.3 (1 C, -CH-, pyrazole), 126.6, 127, 128.3, 129.7, 134 (10 C, =CH-, Ar), 132, 147, 150 (4 C,=CH-, pyridazine), 155.6 (1 C, -CH-, pyrazole), 159.7 (1 C, -C = O); MS (m/z): 344.1 6 (M⁺, 100.0); 345.17 (M⁺ + 1, 20); Anal. calcd. for C₂₁H₂₀N₄O/(344.41): C, 73.23; H, 5.85; N, 16.27%; found: C, 73.20; H, 5.86; N, 16.27%.

2.2.4.3. 5-Amino-4-[4-(4-isopropyl-phenyl)-phthalazin-1-yl]-2,4-dihydro-pyrazol-3-one (20)

Light brown crystals; mp.:169–170 °C; FT‐IR(KBr, ν, cm⁻¹): 2962,2850 (-CH, -CH₃), 3065-3025 (=CH, Ar), 1625,1475 (C = C), 1682 (C = O), 3409, 3350 (-NH_2), 1625 (C-N-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.28 (d, 6H, 2CH₃), 3.01 (m, 1H, -CH(CH₃)₂), 4.7 (s,1H, -CH-, pyrazole), 7.43–8.34 (m, 8H, -CH-, ArH), 12.85, 11.9 (br, 3H, -NH_{2, -}NH-); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.3 (2 C, -CH₃), 33.8 (1 C, -CH(CH₃)₂), 51.0 (1 C, -CH-, pyrazole), 126.5, 127, 128.3, 128.7, 129 (10 C, =CH-, Ar); 132.6, 134.5, 155.6 (4 C,=CH-, pyridazine),149.7 (1C, =C-NH_2, pyrazole), 159.7 (1 C, -C = O); MS (m/z): 345.16 (M⁺,51.39), 347.17 (M⁺ + 2,31.0); Anal. calcd. for C₂₀H₁₉N₅O/(345.40):C, 69.55; H, 5.54; N, 20.28%; found: C, 69.57; H, 5.55; N, 20.29%.

2.2.4.4. 4-[4-(4-Isopropyl-phenyl)-phthalazin-1-yl]-4H-pyrazole-3,5-diamine (21)

Light brown powder; mp.:172–173 °C; FT‐IR(KBr, ν, cm⁻¹): 2960,2856 (-CH, -CH₃), 3070-3030 (=CH, Ar), 1600,1465 (C = C), 3450,3300 (-NH_2), 1625 (C-N-N); ¹H NMR, δ (ppm) in DMSO-d₆: 1.28 (d, 6H, 2CH₃), 3.42 (m, 1H, -CH(CH₃)₂), 2.65 (d,1H, -CH-, pyrazole), 7.36–8.27 (m, 8H, -CH-, ArH), 12.85, 11.87 (br, 4H, -NH₂); ¹³C NMR, δ (ppm) in DMSO-d₆: 24.4 (2 C, -CH₃), 33.8 (1 C, -CH(CH₃)₂), 40.3 (1 C, -CH-, pyrazole), 126.5, 127.1, 128.4, 129, 146 (10 C, =CH-, Ar), 132.1, 134.1, 150 (4 C,=CH-, pyridazine), 160 (2 C,=CH-NH₂); MS (m/z): 344.17 (M⁺,100.0), 345.18 (M⁺ + 1, 22.5); Anal. calcd. for C₂₀H₂₀N₆ (344.41): C, 69.75; H, 5.85; N, 24.40%; found: C, 69.77; H, 5.88; N, 24.38%.

2.3. Biological evaluation

2.3.1. Cell culture

In the present study, we have utilized three cancer cell lines with the utmost range of 15 passages. These cell lines represent three organs starting with the breast (MCF7), colon (HCT116), and liver (HepG2). To examine the selectivity of compounds, a normal foetal lung fibroblast was used with almost 10 passages (MRC5). All cells were sources from the American Type Culture Collection (ATCC), USA. The three cancer cells were maintained in RPMI-1640 media (10% Fetal bovine serum (FBS)), while MRC5 was maintained in Eagle’s minimum essential medium (10% FBS), with the standard conditions (37 °C, 5% CO₂, and 100% relative humidity (Abdalla, Abdallah, et al., 2020).

2.3.2. Anticancer and selectivity studies

In order to determine the anticancer activity of an investigational compound, the 19 compounds, in addition to doxorubicin were tested using the MTT assay according to a previous study (Abdalla, Qattan, et al., 2020). Each cell line was subcultured to 80%–90% confluency before adding each compound or doxorubicin at final concentrations of 0–50 µM for 72h. Next, MTT was added to each well including negative controls and incubated for 3h at 37 °C. MTT was then removed, and the remaining precipitation was dissolved with DMSO. The absorbance (A₅₅₀) for each well/concentration was read on multi-plate reader (Bio-Rad, PR 4100, Hercules, CA, USA), and the inhibition concentration (IC₅₀) was calculated using GraphPad Prism software (version 5). The selectivity index (SI) for each of the 19 compounds and doxorubicin was calculated by dividing the compound IC₅₀ against MRC-5 cells/IC₅₀ against either MCF7, HCT116 or HepG2 cells (Abdalla et al., 2021).

2.4. Molecular modeling

Molecular docking simulation and ligand binding energy calculation were performed using the molecular operating environment (MOE) 2014 (CCG^®; Montreal, Canada). PyMol 2.0.6 Graphical Software (Schrödinger^®, New York, NY, USA) was used for binding interaction visualization and figures generation. Ligand preparation, energy minimization, and potential energy calculation were performed using MOE interface. The crystal structure of human PARP-1 (PDB code: 7aad), co-crystallized with Olaparib was selected for the docking studies. The selected target and ligands were prepared using protein preparation and LigX tools, respectively. The protein underwent three-dimensional protonation, auto-correction of atom types and connections, and partial charge assignment at a physiological neutral pH of 7.4. The ligand PDB file was modified by adding hydrogens, the partial charges and ionization state were calculated, and was energy minimized using MMFF94X forcefield. The active site for docking was defined by residues within 5 Angstrom from the co-crystallized ligand. The docking procedure was executed using the MOE dock tool within the MOE software, utilizing the default parameters. This process utilized rigid docking, in which the protein remained inflexible while the ligands were kept flexible and the ligand’s conformation was generated via ligand placement and bond rotation. The docking output was set to 10 docking poses per ligand. The results were assessed by identifying potential ligand interactions with the protein active site using the MOE–ligand interactions wizard and bond distance calculation in PyMol. The binding score (S score) in MOE and the comparative docking with the crystal ligand were also used to identify the best binders. The selection was made using a comprehensive approach that included the best docking score, low root mean square deviation (RMSDs) compared to the co-crystalline ligand, and important interactions with the active site.

3. Results and discussion

3.1. Chemistry

Phthalazine and phthalazinone derivatives have garnered significant attention in medicinal chemistry and drug discovery and had been reported to have a wide range of biological actions and medical applications such as antimicrobial (Ibrahim, Eldehna, Abdel-Aziz, Elaasser, & Abdel-Aziz, 2014; Mourad, Makhlouf, Soliman, & Mohamed, 2020), anticancer (El Sayed et al., 2024; Emam, Rayes, Ali, Soliman, & Nafie, 2023; Vila et al., 2022), antitumor (Eldehna, Ibrahim, Abdel-Aziz, Farrag, & Youssef, 2015; Marzouk, Shaker, Hafiz, & El-Baghdady, 2016), anti-Alzheimer’s inhibitors (Ezzat, Abdelhamid, Fouad, Abdel‐Aziz, & Allam, 2023), anti of acquired immunodeficiency syndrome (anti-AIDS) (Sroczyński, Malinowski, Szcześniak, & Pakulska, 2016), anti-inflammatory, antiproliferative agents (Hameed et al., 2016), anticonvulsant (Ayyad, Sakr, & El-Gamal, 2016), and antidiabetic (Agrawal et al., 2013). In addition, phthalazinone 1 was synthesized in a prior study conducted by Abubshait et al. (2022). Chlorophthalazine 2 (Scheme 1) was produced by refluxing compound 1 with a combination of phosphorus pentachloride and phosphorus oxychloride (Abubshait, 2007; Zhang et al., 2010).

Scheme 1. General methods for preparation chlorophthalazine 2.

Additionally, chlorophthalazine structure for FT-IR spectra showed conforming stretching bands for aliphatic and aromatic groups, major bands at 2922, 2865 cm⁻¹ (-CH, -CH₃), 3068-3010 cm⁻¹ (=CH, Ar), 1635, 1448 cm⁻¹ (C = C), and disappearance of the carbonyl group of phthalazinone 1. Moreover, the (¹H,¹³C) NMR spectra of 2 showed the presence of obvious bands for -CH₃, -CH(CH₃)₂, related to pyridazine, aromatic groups also new protons and carbon bands are presented, at 1.28 (d, 6H, 2CH₃), 2.98 (m, 1H, -CH(CH₃)₂), 7.24–8.54 (m, 8H, -CH-, ArH); ¹³C NMR, δ (ppm) 23.91 (2 C, -CH₃), 30.9 (1 C, -CH(CH₃)₂), 126.7, 127.5, 128.5, 132.0, 148.0 (10 C, =CH-, Ar), 130.6, 134.0, 151.6 (3 C,=CH-, pyridazine), 171 (1 C, -C-Cl). The one-pot reaction of chlorophthalazine is an important synthetic application to produce a heterocyclic nucleus possessing broad pharmaceutical and biological activity (Ahmad, Varshney, Rauf, Husain, & Ahmad, 2014; Khidre, Mostafa, Mukhrish, Salem, & Behalo, 2022; Romero et al., 2020). This study investigates the nucleophilic displacement reactions of chlorophthalazine with different hydrazine derivatives, primary and secondary amines, and active methylene compounds in order to produce novel phthalazine derivatives. The reactions were conducted using both traditional methods and microwave irradiation to synthesize new phthalazine derivatives from 2 with high yields and a rapid reaction rate. Microwave irradiation was employed as a green and efficient technology to assist in the synthesis process (Almahli et al., 2018; Boraei et al., 2019; Bunce, Harrison, & Nammalwar, 2012). The use of microwave irradiation in synthetic of heterocycle compounds has several advantages over traditional synthetic methods is a promising method for accelerating reaction rates and improving yields and improve the selectivity of various organic transformations (Damera, Pagadala, Rana, & Jonnalagadda, 2023). The traditional synthetic methods of heterocycles have relied on multi-step processes involving distinct reaction steps and purification procedures, these conventional methods often encounter drawbacks such as decreased productivity, extended reaction times, and the production of by-products. However, using microwave irradiation as green strategy for the improved synthesis of biologically and pharmaceutically interesting annulated heterocycles has further highlighted the potential of this approach in accessing novel heterocyclic systems (Javahershenas, Makarem, & Klika, 2024). Scheme 2 shows the reaction of chlorophthalazine 2 with hydrazine hydrate, phenylhydrazine, 3,4-dimethylphenylhydrazine, 3-nitrophenylhydrazine, 2,4-dinitrophenylhydrazine, 2-thiophenecarboxylic hydrazine, benzoylhydrazine and semicarbazide experimented under reflux for 5–6 h and under microwave for 9–25 min in dry benzene to give 3–10, respectively. The FT-IR spectra of hydrazine derivatives showed new (-NHNH_2, -NHNH-) stretching bands at regions 3421-3304 and 3319-3210 cm⁻¹.

Scheme 2. Reactions of chlorophthalazine 2 with various hydrazine derivatives.

The displacement of chloride from chlorophthalazine can be achieved by nucleophile attacks on electron-rich groups, specifically primary and secondary amines such as methyl-amine, aniline, p-toluidine, p-anisidine, α-naphthylamine, anthranilic acid, and diphenylamine. This can be achieved through reflux for a duration of 4–6 h or microwave (MW) for 9–10 min to give the resulting products 11–17, respectively. Scheme 3 (Abou-Seri, Eldehna, Ali, & Abou El Ella, 2016; Abubshait, Kassab, Al-Shehri, & Abubshait, 2011; Behalo et al., 2017).

Scheme 3. Reactions of chlorophthalazine 2 with primary and secondary amines.

Compounds 11–17 exhibited bands at 3450 and 1188–1360 cm⁻¹, which can be attributed to the presence of secondary and tertiary amine groups (-NH, -C-N). The reaction between compound 2 and carbon nucleophiles, in which active methylene was involved, resulted in the formation of pyrazolo derivatives. Phthalazine derivatives were obtained through the reactions of compound 2 with acetyl acetone, ethyl acetoacetate, ethyl cyanoacetate, and malononitrile in the presence of sodium ethoxide. These derivatives were further subjected to a cyclization process with hydrazine hydrate in ethanol, resulting in the formation of pyrazolo derivatives 18–21, respectively. Scheme 4 (Abubshait, 2007; El Shamy et al., 2023).

Scheme 4. General methods for preparation of compounds 18–21.

Coupling of 2 with various active methylene compounds afforded the corresponding coupling products by applying conventional methods in ethanol under reflux for 6h (Henary et al., 2020; Richard et al., 1986). In comparison, the reaction was successfully performed within a time frame under microwave irradiation. In addition to a short-time reaction, the collected products were obtained in high yields compared with the conventional method. Scheme 4 represents the pathway for preparing the target compounds through the nucleophilic attack of 2 with active methylene compounds by using MW irradiation. The synthesized compounds were found to undergo a cyclization process as a result of the nucleophilic attack within the nucleophile hydrazinehydrate which led to the formation of the corresponding pyrazolyl derivatives. A comprehensive analysis of the unique products is conducted using their spectral data. The IR spectra exhibited absorption bands within the range of 1680-1680 cm⁻¹, which can be attributed to the (C = O) functional group associated with compounds 19 and 20. Compounds 20 and 21 exhibited new stretching bands at positions 3409-3450-3350-3000, which can be attributed to the (NH₂) group. The ¹H NMR spectra exhibited prominent bands corresponding to isopropyl, aliphatic, and aromatic protons. Additionally, novel bands were seen for methyl and pyrazole protons at a wavenumber of 2.00 and 2.50 ppm, respectively. The spectra of compounds 18–21 exhibit new signals for the (NH₂, -NH-) group. Table 1 displays the yield and reaction time (in minutes) of compounds 2–21, obtained using MW irradiation and the conventional approach.

Table 1. Yield (%) and reaction time (min) of compounds 2–21 by using MW irradiation and conventional method.

Compound No.	Reaction time (min)		Yield (%)
	Conventional methods	MW irradiation	Conventional methods	MW irradiation
2	30	–	(1.67 mg) 60.0%	–
3	360	11	(2.39 mg) 86.0%	(2.73 mg) 98.0%
4	360	10	(2.73 mg) 77.0%	(3.47 mg) 98.0%
5	360	10	(2.62 mg) 68.4%	(3.72 mg) 97.0%
6	360	10	(3.03 mg) 76.0%	(3.94 mg) 98.8%
7	360	9	(3.33 mg) 75.0%	(3.93 mg) 88.6%
8	360	9	(3.34 mg) 76.1%	(4.21 mg) 95.0%
9	360	9	(3.25 mg) 70.0%	(4.17 mg) 89.8%
10	360	25	(2.42 mg) 80.1%	(2.79 mg) 92.2%
11	300	9	(2.11 mg) 76.3%	(2.35 mg) 85.0%
12	300	9	(2.71 mg) 80.0%	(3.29 mg) 97.0%
13	240	10	(2.71 mg) 76.9%	(3.42 mg) 97.0%
14	360	10	(3.23 mg) 87.5%	(3.41 mg) 92.3%
15	300	9	(3.00 mg) 77.2%	(3.27 mg) 84.0%
16	300	20	(3.07 mg) 84.0%	(3.58 mg) 98.0%
17	300	15	(1.94 mg) 46.7%	(4.03 mg) 97.0%
18	300	10	(2.10 mg) 61.3%	(2.99 mg) 87.3%
19	300	10	(1.89 mg) 55.0%	(2.58 mg) 75.8%
20	300	10	(1.73 mg) 50.0%	(2.74 mg) 79.5%
21	300	10	(1.98 mg) 57.5%	(2.94 mg) 85.5%

3.2. Biological evaluation

3.2.1. Anticancer and selectivity studies

To determine the anticancer effect of compounds 3–21 against MCF7, HCT116, and HepG2 cancer cells, an MTT assay was performed. The most active compound against MCF7 cells was 13 with IC₅₀ 0.286 µM, which was less effective but comparable with the IC₅₀ of doxorubicin against MCF7 (0.041 µM), while the least active compound was No. 4 with IC₅₀ 5.370 µM. The range of anticancer activity against HCT116 was wider, as compound 6 showed IC₅₀ 0.384 µM, which was 10 times better than the anticancer effect of doxorubicin against HCT116 (IC₅₀ 3.964 µM), while compound 3 was less active against HCT116 with IC₅₀ 25.228 µM. Regarding HepG2, the most active compound was 16 with IC₅₀ 0.484 µM which was comparable with the IC₅₀ of doxorubicin against HepG2 cells (0.381 µM), while the least active compound was 4 with IC₅₀ 5.808 µM. Thus, the most active compounds were 13, 6, and 16, respectively (Table 2). Table 2 also includes the anticancer effect of each compound against MRC5 normal fibroblast cells. The IC₅₀ ranges between 0.839 µM and 25.800 µM. This data was used to calculate the SI of each compound towards MRC5 normal fibroblast cells (Table 3) compared with each of the three cell lines, and the best SI was for compound 6 (Figure 2), followed by compound 13, all of which was comparable with the selectivity of doxorubicin.

Figure 2. Dose response curve of compound 6 against MCF7, HCT116, HePG2 and MRC5 cells. X-axis: dose, Y-axis: absorbance.

Table 2. Anticancer activity of compounds 3–21 and doxorubicin against three cell lines, and MRC5 normal fibroblast cells (MTT 72 h, IC₅₀ “µM” ± SD, n = 3).

Compound No.	MCF7	HCT116	HepG2	Average cytotoxicity* (IC50)	MRC5
3	1.965 ± 0.393	25.228 ± 2.713	4.715 ± 1.098	10.636	12.145 ± 0.205
4	5.370 ± 0.282	1.954 ± 1.310	5.808 ± 0.997	4.377	19.270 ± 1.456
5	2.881 ± 0.844	27.126 ± 1.447	2.978 ± 0.169	10.995	7.894 ± 0.474
6	1.739 ± 0.295	0.384 ± 0.045	1.52 ± 0.025	1.214	25.800 ± 1.004
7	2.578 ± 0.074	5.087 ± 0.369	3.668 ± 0.804	3.777	20.935 ± 1.152
8	1.255 ± 0.188	16.592 ± 3.157	4.277 ± 1.324	7.375	16.885 ± 0.629
9	1.485 ± 0.363	17.389 ± 0.310	2.031 ± 0.033	6.968	8.808 ± 0.596
10	5.097 ± 0.014	2.584 ± 0.443	4.162 ± 0.415	3.947	3.168 ± 0.237
11	1.313 ± 0.231	4.160 ± 0.691	4.336 ± 0.796	3.270	11.030 ± 1.173
12	1.390 ± 0.468	5.358 ± 0.364	5.065 ± 1.044	3.937	6.077 ± 0.343
13	0.286 ± 0.038	4.846 ± 0.101	1.006 ± 0.143	2.046	11.620 ± 1.527
14	0.988 ± 0.067	14.031 ± 1.143	2.110 ± 0.580	5.710	11.555 ± 0.756
15	0.843 ± 0.752	5.022 ± 0.472	3.367 ± 0.364	3.077	6.288 ± 0.581
16	1.702 ± 0.649	10.992 ± 0.682	0.484 ± 0.377	4.393	7.929 ± 0.382
17	1.460 ± 0.235	13.884 ± 2.268	1.550 ± 0.264	5.631	13.050 ± 0.862
18	1.456 ± 0.295	12.255 ± 1.887	2.179 ± 0.774	5.296	5.205 ± 0.840
19	1.056 ± 0.027	2.619 ± 1.429	1.735 ± 0.333	1.803	9.023 ± 0.603
20	1.625 ± 0.259	1.780 ± 0.710	1.624 ± 0.327	1.676	4.896 ± 0.571
21	1.512 ± 0.497	5.728 ± 1.571	3.136 ± 0.189	3.459	0.839 ± 0.084
Doxorubicin	0.041 ± 0.002	3.964 ± 0.360	0.381 ± 0.133	1.462	2.476 ± 0.033

* Average cytotoxicity (IC₅₀) of each compound against the three cancer cells. When SI is more than 1= selectivity.

Table 3. Selectivity index (SI) of compounds 3–21* and doxorubicin against MRC5 normal fibroblast cells.

Compound No.	MCF7	HCT116	HepG2
3	0.57	0.48	2.57
4	3.62	9.88	3.31
5	2.73	0.29	2.65
6	15.17	67.89	16.97
7	8.37	4.12	5.71
8	14.06	1.01	3.95
9	6.28	0.5	4.33
10	0.62	1.22	0.75
11	8.41	2.65	2.54
12	4.36	1.13	1.19
13	41.5	2.39	11.62
14	11.78	0.82	5.47
15	4.51	1.25	1.86
16	4.65	0.72	16.5
17	8.93	0.93	8.41
18	3.58	0.42	2.39
19	8.59	3.45	5.21
20	3.01	2.74	3.01
21	0.54	0.14	0.26
Doxorubicin	60.39	0.62	6.48

* A compound is considered selective when SI ≥1.

The best selective compounds were compound 6 selective against three cells compared to MRC5 cells, followed by compounds 13, 16, 8, and 14, best cytotoxic and selective compounds 6 and 13 (Table 3).

3.3. Molecular modeling

Molecular docking simulations performed on the synthesized compounds with poly ADP- ribose polymerase (PAPR-1) revealed a favourable binding affinity. The results confirmed the experimental IC₅₀, which predominantly fell within the one-digit micromolar range, and successfully identified the compounds with the highest experimental activity (6, 13, 19, 20) as the best binders. Those compounds were especially perfectly aligned with the standard co-crystallized inhibitor, Olaparib in the active site (Figure 3).

Figure 3. Comparative binding positions of compound 6 (green), compound 13 (blue), compound 19 (magenta), compound 20 (orange) and olaparib (red) bound to PARP-1 (7aad) active site.

The docking studies showed that the most active compounds were able to bind the gatekeeper residues, Tyr 896, and Tyr 907, which were identified in our previous study as being crucial for enzyme binding. The initial binding with these two residues is the starting point for ligands to access the active site, and the π–π interactions formed with these residues give affinity and selectivity to the interacting ligand. In the most active compounds, one of the aromatics interacts with Tyr 907 while the phthalazinone ring performs the interaction with Tyr 896. The less lipophilic phthalazinone ring is the part of the structure that is solvent exposed while the more lipophilic isopropyl-phenyl at position-4 is directed deeper in the binding pocket which stabilizes the compounds in the active site. This is either in the tiny binding pocket of the enzyme formed by Gly 863, Ser 907 and Glu 988 in the case of compounds 19 and 20 (Figure 4(c,d), respectively) or in the more spacious hydrophobic pocket formed by Tyr 710, Asp 766, Leu 769, Arg 878, Ala 880, Pro 881, and Tyr 889 in case of compounds 6 and 13 (Figure 4(a,b), respectively). The substituted nitrogen moiety at position-1 on the other hand occupies the opposite side of the binding pocket. This interaction was noted for compounds 6 and 13 possibly because of the higher flexibility of the anilino or phenylhyarazino moiety compared to the more rigid pyrazole ring in compounds 19 and 20, which allows them to fit better in the tiny pocket.

Figure 4. Surface representation of compound 6 (a), compound 13 (b), compound 19 (c) and compound 20 (d) bound to PARP-1 (7aad) active site.

Compound 6 (IC₅₀, 1.214) established two additional electrostatic interactions with its phthalazinone and side chain hydrazino nitrogens and His 862 sidechain and Gly 863 backbone, respectively (Figure 5(a)). Compound 13 (IC₅₀, 2.046) did not show additional electrostatic bonds, however, a potential and characteristic π–H bonding was noted with its anilino nitrogen and Tyr 896 which may explain the comparable activity with the other compounds despite the lack of direct H-bonding (Figure 5(b)). Compound 19 (IC₅₀, 1.803) was able to gain high affinity through H-bonding with its pyrazole ring and Arg 878 and Gly 894 backbones, where it acted as a strong H-bond donor (Figure 5(c)). Compound 20 (IC₅₀, 1.676) was identified as one of the best binder due to formation of several H-bonds with residues in the binding pocket more specifically with its pyrazole and Ala 880 backbone and its amine and Asp 766 backbone and its phthalzinone nitrogen and His 862 sidechain (Figure 5(d)).

Figure 5. Binding interactions of compound 6 (a), compound 13 (b), compound 19 (c) and compound 20 (d) with PARP-1 (7aad) active site. Potential electrostatic interactions are represented as yellow dotted lines and are measured in angstrom.

The results of the biological testing and the docking studies formulated a primary structure–activity relationship for the phthalazine derivatives. The phthalazine core is important and is involved in anchoring the compounds at the active site. Substitution at position 1 with a phenyl ring gives more active compounds as the aromatic ring digs deeper in the active site and establishes hydrophobic interactions and π–π stacking. Substitution at position 4 with amino functionality is important for the activity, and it can accommodate aminophenyl, hydrazinophenyl, and aminohetreocycles as examples. However, increasing the steric hindrance in this part such as disubstituted amine (compound 17) or ring expansion (compound 16), reduced the activity.

The fact that PARP is overexpressed in the tested cancer cells (Siraj et al., 2018) together with the high affinity of the synthesized compounds, imparted a high selectivity on cancer cells compared to normal cells with compounds 6, 8, 13, 14, and 16 being the most selective. Compounds 6 and 13 especially showed selectivity comparable to the clinically used drug doxorubicin in MCF7 cells (15.17 and 41.5, respectively, compared to 60.39) and superior selectivity on HCT116 (67.89 and 2.39, respectively, compared to 0.62) and HepG2 cell lines (16.97 and 11.62, respectively, compared to 6.48). The docking scores also correlated well with the experimental activity and selectivity data. A comparison of the binding scores, average IC₅₀, and average selectivity of the most active compounds is presented in Table 4.

Table 4. Binding scores, average IC₅₀, and average selectivity of the most active compounds.

Compound No.	Binding score	Average IC50	Average selectivity
6	−8.467	1.214	33.34
13	−7.952	2.046	18.5
19	−7.062	1.803	4.02
20	−6.889	1.676	2.92
Doxorubicin	–	1.462	22.5

In conclusion, the optimization of our phthalzinone compounds through exploring the substitution on position 1 with various alkyl and aryl substituted nitrogen and nitrogen heterocycles enhanced both the affinity and selectivity to the PARP target. The docking scores were aligned with the experimental data, which validated the compounds’ target interactions and selectivity. Future research directions will focus on testing our phthalazines experimentally for PARP-1 activity together with synthesizing more derivatives that explore substitution at the other positions of the phthalazine core, which together will guide the drug design and optimization for in vivo preclinical testing.

4. Conclusions

A new series of phthalazine derivatives have been produced by utilizing environmentally friendly processes that use microwave irradiation in high yield and short reaction times. The utilization of the dynamic microwave power system resulted in highly effective heating of the materials, leading to decreased durations of chemical reactions and enhanced yields of reactions in the preparation of the target phthalazine derivatives. The synthesized phthalazine derivatives showed broad anticancer activity against three different cell lines namely, MCF7, HCT116, and HepG2. Compound 6 was identified as the most active and selective compound that showed comparable results with doxorubicin. The docking results concurred with the experimental data and highlighted the influence of substituting position 1 of the phthalazine derivatives on their binding affinity to the PARP-1 active site. Our computational analysis findings have identified our phthalazine compounds as a novel scaffold for inhibiting PARP-1.

Acknowledgements

We would like to thank the College of Science and the Basic and Applied Scientific Research Center at Imam Abdulrahman Bin Faisal University for the co-workers and making the spectral analyses.

Disclosure statement

The authors declare that there is no conflict of interest regarding this publication.