Rationale, in silico docking, ADMET profile, design, synthesis and cytotoxicity evaluations of phthalazine derivatives as VEGFR-2 inhibitors and apoptosis inducers (2024)

Hatem Hussein Bayoumi^a, Mohamed-Kamal Ibrahim^a, Mohammed A. Dahab^a, Fathalla Khedr^a and Khaled El-Adl*^ba
aPharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City 11884, Cairo, Egypt. E-mail: eladlkhaled74@yahoo.com; eladlkhaled74@azhar.edu.eg; khaled.eladl@hu.edu.eg
^bPharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University for Sustainable Development, Cairo, Egypt

Received 9th July 2024, Accepted 21st August 2024

First published on 27th August 2024

1. Introduction

Vascular endothelial growth factors (VEGFs) are transmembrane proteins that trigger angiogenesis through VEGF receptor signaling.1 To date, three types of VEGF tyrosine kinase receptors are known, VEGFR-1, VEGFR-2, and VEGFR-3. All of them display a high affinity towards VEGF. However, VEGFR-2 solely remains the only one that transmits the angiogenic signals.2 The VEGFR-2 receptor has been documented to play major roles in both physiological and many pathological angiogenesis, such as cancer.1 Overexpression of this receptor has been ascertained in a number of solid cancers3 and hematological malignancies.4 Consequently, VEGFR-2 has emerged as an ideal target for the development of novel chemotherapeutic agents.3 Tyrosine kinase inhibitors are grouped into three types, type I, II and type III.5 Type II inhibitors interact with the adenosine triphosphate (ATP) binding site as well as the allosteric hydrophobic site and display high selectivity.6,7 The architecture design of VEGFR-2 active site comprises of two pockets, the front and the back. The front pocket has two associated key residues, cysteine919 (Cys919) while the back hydrophobic pocket has another two key residues, glutamate885 (Glu885) and aspartate1046 (Asp1046).8 The pharmacophoric features shared by all of the reported VEGFR-2 inhibitors showed the presence of four main features as shown in Fig. 1;9,10 (i) the core structure occupies the catalytic ATP-binding domain and consists of a flat aromatic ring; (ii) the central hydrophobic spacer occupies the linker region between the ATP-binding domain and the DFG domain; (iii) a hydrophilic linker e.g., amino or urea to act as both H-bond donor and acceptor with the above-mentioned two key amino acid residues, Glu885 and Asp1046; (iv) a terminal hydrophobic moiety to occupy the allosteric hydrophobic pocket.11

Over the last two decades, a number of VEGFR-2 inhibitors have demonstrated success as targeted anticancer therapeutics. Among which, sorafenib (I), the 1,4-disubstituted phthalazine derivatives, vatalanib (II), AAC789 (III), AMG900 (IV), and IM-023911 (V) revealed potent inhibitory activity against VEGFR-2 in nanomolar levels of IC₅₀.12,13 Sorafenib is a food and drug administration (FDA) approved antiangiogenic drug that shares the above pharmacophoric features.14

Phthalazine ring is a vital pharmacophoric scaffold present in the core structures of many anticancer molecules15–19 with potent activity against hepatocellular carcinoma,18 colon cancer,11 breast cancer,19 and as degraders of VEGFR-2.15,20 Many phthalazine derivatives were reported to have VEGFR-2 and EGFR kinase inhibitory activity with IC₅₀ values in the nanomolar range, however, compounds were more selective and better inhibitors of VEGFR-2 compared to epidermal growth factor receptor (EGFR).21,22 Other pharmacophoric heterocycles are implanted in the structures of recently reported anticancer with VEGFR-2 inhibitory effects as pyrazole23,24 and pyrimidine.25,26 These latter were reported to have the spatial configurations that enable both to interact with the VEGFR-2 binding site.26,27 Moreover, the α,β-unsaturated ketonic fragment is also presented in a number of synthetic derivatives with potent VEGFR-2 inhibitory activity.28,29

Over the last few years, our research group members were interested in the construction and evaluation of heterocyclic molecules of expected biological activity particularly as anticancer.30–50

The aim of this study is to develop novel inhibitors of VEGFR-2 protein. Accordingly, the abovementioned facts have encouraged us to design novel phthalazine derivatives linked with fragments of verified VEGFR-2 inhibitory potentials, including α,β-unsaturated ketonic fragment, pyrazole, pyrimidinone and/or pyrimidinthione to investigate the anticancer and VEGFR-2 inhibitory potentials of the designed compounds. All the designed compounds retained the essential pharmacophoric features of the reported and clinically used VEGFR-2 inhibitors (Fig. 1) with two additional bioisosteric modifications: (i) insertion of a new bioisosteric spacer with that reported in the most recent molecular docking and molecular dynamic simulation studies.13 The new spacer has inserted in the form of pharmacophoric fragments of reported anticancer potentials including α,β-unsaturated ketonic fragment, dihydropyrazole, pyrimidinone and/or pyrimidinthione; (ii) attachment of a new substituted tail bioisosteric hydrophobic moieties with that of AMG 900 (IV) which are expected to increase the hydrophobic interaction with VEGFR-2 and consequently the affinity. Substitution pattern of the new tail bioisosteric hydrophobic moieties was selected to ensure different electronic and lipophilic environments that could influence the activity of the target compounds. These modifications were performed in order to carry out further elaboration of the phthalazine scaffolds and to explore a valuable SAR.

The designed target derivatives were synthesized and evaluated as potential VEGFR-2 inhibitors and anti-tumors against Hepatocellular carcinoma G2 (HepG2) and Michigan Cancer Foundation-7 (MCF-7).

2. Results and discussion

2.1. Docking studies

All modeling experiments in the present work were performed using Molsoft software. VEGFR-2 experimental structure was downloaded from the Protein Databank (PDB ID 4ASD).51 All studied ligands have similar position and orientation inside the recognized binding site of VEGFR-2.

Sorafenib exhibited −99.50 kcal mol⁻¹ (Table 1) and 5 H-bonds with Asp1046 (1.50 Å), Glu885 (1.77 Å and 2.75 Å), and Cys919 (2.51 Å and 2.10 Å). The N-methylpicolinamide group placed in the pocket produced by Glu917, Val848, Lys920, Leu1035, Cys919, Phe918 and Leu840. As well, the hydrophobic hollow constructed by Val848, Lys868, Thr916, Leu1035, and Cys1045 filled by the central phenyl spacer. As well, Ile892, Ile888, Hie1026, Glu885, Cys1045 and Asp1046 constructed a hydrophobic canal which filled with the terminal 3-trifluoromethyl-4-chlorophenyl moiety (Fig. 2).

Table 1 The calculated free energy of binding (ΔG in kcal mol⁻¹) for the ligands

As planned, compound 4b showed virtually binding mode as that of sorafenib. It revealed −107.95 kcal mol⁻¹ and formed 10 H-bonds with the key amino acid Glu885 (2.10 Å), Asp1046 (2.54 Å), Cys919 (2.24 Å and 2.31 Å), Glu917 (2.41 Å) and Lys868 (0.96 Å, 1.07 Å, 1.19 Å, 2.12 Å and 2.63 Å). The 4-chlorophenylphthalazine scaffold occupied the hydrophobic ATP binding groove formed by Leu1035, Cys919, Phe918, Glu917, Val848, Lys920 and Leu840. Moreover, the central phenyl group occupied the linker hydrophobic pocket formed by Asp1046, Cys1045, Thr916, Glu917, Lys868 and Val848. Furthermore, the 4-chlorophenyl tail occupied the new hydrophobic groove formed by Asp1046, Cys1045, His1026, Ile1025, Glu885 and Ile888 (Fig. 3). These interactions may explain the highest anticancer activity of compound 4b.

Also compound 3e showed virtually binding mode as that of 4b. It revealed −97.75 kcal mol⁻¹ and formed 7 H-bonds with Glu885 (2.96 Å), Asp1046 (1.01 Å, 1.72 Å and 2.43 Å), Cys919 (1.87 Å and 2.45 Å), and Glu917 (2.61 Å) (Fig. 4).

Moreover, compound 5b revealed −94.27 kcal mol⁻¹ and formed 7 with Glu885 (2.98 Å), Asp1046 (2.97 Å), Cys919 (2.57 Å and 2.95 Å), Glu917 (2.78 Å) and Lys868 (1.93 Å and 2.02 Å) (Fig. 5).

The obtained results showed that our derivatives inhibited the ATP binding domain and forming H-bond with Cys919 and extended over the gate area into the adjacent allosteric hydrophobic which approved that our compound were considered as type II inhibitors of VEGFR-2.

2.2. Validation of the accuracy of docking

As cited in literature52 if the RMSD (root mean square deviation) of the best docked conformation is ≤2.0 Å from the bound ligand in the experimental crystal, the used scoring function is successful. Therefore, the docked results were compared to the crystal structure of the bound ligand–protein complex. The obtained success rates were highly excellent as cited in Table 1. The sorafenib ligand was docked in VEGFR-2 receptor (pdb code: 4ASD). The RMSD of the docked sorafenib was 0.64 Å as it seems exactly superimposed on the co-crystallized native bound one (Fig. 6). These results indicated the high accuracy of the docking simulation in comparison with the biological methods.

2.3. Chemistry

The adopted synthetic strategies for the preparation of target compounds (1–5) are depicted in Schemes 1 and 2. The synthesis was initiated by cyclocondensation of 2-(4-chlorobenzoyl)benzoic acid with hydrazine hydrate to afford the corresponding 4-(4-chlorophenyl)phthalazin-1(2H)-one11,53,54 which underwent chlorination by reaction with phosphorous oxychloride11,55 to afford 1-chloro-4-(4-chlorophenyl)phthalazine. The chloro derivative was heated under reflux with the 4-aminoacetophenone to afford the corresponding acetyl derivative 1. On the other hand, the acetyl derivative 1 was condensed with the appropriate benzaldehyde to yield the corresponding chalcone derivatives 2a–e following the reported procedure53,54,56 (Scheme 1). The produced chalcone derivatives 2a–e underwent binucleophilic cyclocondensation reactions with hydrazine hydrate, urea and/or thiourea57,58 to give the corresponding pyrazoline 3a–e, pyrimidin-2(1H)-one 4a,b and/or pyrimidine-2(1H)-thione 5a,b respectively (Scheme 2).

2.4. In vitro cytotoxic activity

The anti-proliferative activity of the newly synthesized phthalazine derivatives was examined against MCF-7 and HepG2 cell lines using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay.59 Sorafenib was used as a reference cytotoxic drug. The results were expressed as half maximal inhibitory concentration (IC₅₀) values which represent the compound concentrations required to produce a 50% inhibition of cell growth after 72 h of incubation calculated from the concentration-inhibition response curve and summarized in Table 2. From the obtained results, it was explicated that most of the prepared compounds displayed excellent to moderate growth inhibitory activity against the tested cancer cell lines. In particular, compounds 4b and 3e were found to be the most potent derivatives over all the tested compounds against the two MCF-7 and HepG2 cancer cell lines with IC₅₀ = 0.06, 0.06 μM and 0.08, 0.19 μM respectively.

Table 2 In vitro cytotoxic activities of selected synthesized compounds against MCF-7, HepG2 and VERO cell lines and VEGFR-2 kinase assay

With respect to the MCF-7 cell line, compounds 4a and 5b displayed very good anticancer activities with IC₅₀ = 0.16 and 0.11 μM respectively. Compounds 3b, 3c, 3d and 5a, with IC₅₀ ranging from 0.74 to 0.88 μM exhibited good cytotoxicity. Compound 3a with IC₅₀ = 1.16 μM displayed moderate cytotoxicity.

With respect to the HepG2 hepatocellular carcinoma cell line, compounds 3d, 4a and 5b displayed very good anticancer activities with IC₅₀ = 0.15, 0.29 and 0.15 μM respectively. Compounds 3b and 5a with the same IC₅₀ = 0.80 μM for each, exhibited good cytotoxicity. Compounds 3a and 3c, with IC₅₀ = 1.27 and 1.25 μM respectively displayed moderate cytotoxicity.

2.4.1. Selectivity index (SI). The cytotoxicity against normal VERO cells of the nine compounds 3a–e, 4a,b and 5a,b were evaluated. Our compounds exhibited low toxicity concerning normal VERO cells with IC₅₀ = 3.00–4.75 μM. The good anticancer drug would not affect the normal cells. The highly selective anticancer agent should displayed SI value ≥ 5. The moderate selective one displayed SI value > 2 while low selective one displayed SI < 2.50 In this research, compounds 3a, 3b, 3c, 3d, 3e, 4a, 4b, 5a and 5b are correspondingly 4.09, 5.12, 4.67, 5.88, 40.25, 25.00, 50.00, 4.79 and 28.64 folds more toxic regarding MCF-7 than VERO cells. Regularly, structures 3a, 3b, 3c, 3d, 3e, 4a, 4b, 5a and 5b are consequently 3.74, 5.31, 3.29, 29.00, 16.95, 13.79, 50.00, 5.15 and 21.00 folds toxic in HepG2 than in VERO cells. All compounds displayed high selectivity except compounds 3a and 3c against both tested cancer cell lines and compound 5a against MCF-7 which exhibited moderate selectivity.

2.5. In vitro VEGFR-2 kinase assay

The nine derivatives 3a–e, 4a, 4b, 5a and 5b were evaluated for their inhibitory activities against VEGFR-2 by using an anti-phosphotyrosine antibody with the Alpha Screen system (PerkinElmer, USA).60 The results were reported as a 50% inhibition concentration value (IC₅₀) calculated from the concentration-inhibition response curve. Results of VEGFR-2 enzyme assay are summarized in Table 2. Sorafenib was used as positive control in this assay. The tested compounds displayed high and low inhibitory activities with IC₅₀ values ranging from 0.09 ± 0.02 to 1.25 ± 0.05 μM. Among them, compound 4b was found to be the most potent derivative that inhibited VEGFR-2 at IC₅₀ value of 0.09 ± 0.02 μM. Compounds 3e, 4a and 5b exhibited good activity with IC₅₀ values = 0.12 ± 0.02, 0.15 ± 0.03 and 0.13 ± 0.03 μM respectively. Finally, the other compounds 3a, 3b, 3c, 3d and 5a exhibited low activities with IC₅₀ values ranging from 0.70 ± 0.05 to 1.25 ± 0.05 μM.

2.6. Structure activity relationship (SAR)

The preliminary SAR study has focused on the effect of position, hydrophobic and/or electronic nature of the substituents used in this study. Also, it focused on the effect of the type, length and number of spacers used. Generally, the 4-(4-chlorophenyl)phthalazine scaffold, bearing different 4-substituted anilines joined to the hydrophobic tails moieties through the new spacers; pyrazoline, pyrimidin-2(1H)-one and/or pyrimidin-2(1H)-thione.

In molecular docking studies, generally compound 4b with the new pyrimidin-2(1H)-one spacer impart higher VEGFR-2 binding affinity and consequently higher anticancer activity (Fig. 7) than compounds 3e with pyrazoline and compounds 5b with pyrimidin-2(1H)-thione respectively.

The data obtained from biological testing highly matched with that obtained from molecular modeling studies. Compound 4b with pyrimidin-2(1H)-one spacer joined to 4-chlorophenyl tail moiety exhibited higher anticancer activities against both HepG2 and MCF-7 cell lines than derivative 3e with pyrazoline spacer and 4-methoxyphenyl tail moiety and derivative 5b with pyrimidin-2(1H)-thione spacer and 4-chlorophenyl tail moiety.

From the structure of the synthesized derivatives and the data shown in Table 2 we can divide these tested compounds into three groups. The first group contains the new pyrazoline spacers as in compounds 3a–e. Generally, in this group the substituents at position-4 of the tail moieties exhibited higher activities than that at position-2 against the two cancer cell lines. Compound 3e with the hydrophobic electron donating +mesomeric (+M) and +inductive (+I) 4-methoxy group showed higher anticancer activities than compound 3b with hydrophobic electron withdrawing (+M) and (−I) 4-chloro one and compound 3c with hydrophilic electron donating 2-OH group against the two cancer cell lines. Also, compound 3e (+M and +I) showed higher anticancer activities than compound 3d with hydrophilic electron donating (+M and −I) 4-OH group against MCF-7 while against HepG2 3d showed higher activities than 3e. Compound 3d with 4-OH substitution exhibited higher anticancer activities than compound 3c with 2-OH substituent which enables us to conclude that position-4 play an important role in anticancer activities. The unsubstituted compound 3a displayed the lowest anticancer activities among this group against both tested cell lines.

The second group 4a,b contains the new pyrimidin-2(1H)-one spacers. Compound 4b with hydrophobic electron withdrawing (+M and −I) 4-chloro group at tail moiety showed higher anticancer activities than the unsubstituted one 4a against the two cancer cell lines.

In the same manner, in the third group compound 5b with pyrimidin-2(1H)-thione spacer and hydrophobic electron withdrawing (+M and −I) 4-chloro group at tail moiety showed higher anticancer activities than the unsubstituted one 5a against the two cancer cell lines.

2.7. Effect on cell cycle progression

To get a better insight regarding the effect of 4b on growth inhibition of cancer cells, its effect on the cell cycle distribution and apoptosis induction were evaluated in MCF-7 cells according to the procedure described by Wang et al.61 MCF-7 cells were treated with 2.3 μM of compound 4b for 24 h. Then, the cells were harvested, stained with propidium iodide, and analyzed for cell distribution during the various phases of the cell cycle. The results (Table 3 and Fig. 8A) showed that compound 4b arrested cell growth in growth 2-mitosis (G2-M) phase; accumulation of cells at that phase became 6.92% after being 13.2 in control cells.

Table 3 Effect of compound 4b on cell cycle progression in MCF-7 cells after 24 h treatment

2.8. Induction of apoptosis and necrosis

To explore the mode of induced cell death, induced apoptosis in MCF-7 cells by compound 4b was evaluated using Annexin V and propidium iodide (PI) double staining assay.62 MCF-7 cells were treated with the IC₅₀ of compound 4b for 24 h, harvested, stained with Annexin-V/PI and analyzed for apoptosis using Flowing Software. The obtained results are represented in Table 4 and Fig. 8B. The obtained results showed that compound 4b induced early apoptosis (15.06%) by more than 25 folds over the control (0.59%). On the other hand, the obtained results showed that compound 4b induced necrotic effect (4.66%) twice the effect of the control (1.75%).

Table 4 Effect of compound 4b on stages of the cell death process in MCF-7 cells

2.9. Effects on mitochondrial apoptosis pathway (Bcl-2 family) proteins

Mitochondrial apoptotic pathway is chiefly regulated by the members of the B-cell lymphoma-2 (BCL-2) family.63 Among these, Bcl2 and Bcl2 associated X, apoptosis regulator (BAX) finely tune this programmed process. The Bcl2 protein inhibits apoptosis (anti-apoptotic) while Bax stimulates it (pro-apoptotic). Thus, the balance between these two different opposing proteins regulates the cell fate.64,65 Increments in the Bax/Bcl2 ratio trigger the release of mitochondrial cytochrome C into the cytosol which in turn potentiates a cascade of caspases that ultimately leads to activation of caspase 3; the apoptosis executioner.66,67 Accordingly, in the current study, MCF-7 cells were treated with the IC₅₀ of compound 4b and their effect on the expression levels of Bcl-2 and Bax were determined as illustrated in Table 5 and Fig. 9. As shown by the results, compound 4b boosted the level of the pro-apoptotic protein; Bax by approximately 3 folds. Moreover, compound 4b markedly reduced the levels of the anti-apoptotic proteins Bcl-2 by approximately 3 folds compared to the control. A rather more precise value for apoptosis induction is the Bax/Bcl2 ratio as it gives a more accurate estimation of the overall proapoptotic activity of the molecule. Analyzing the results reveals that compound 4b interestingly boosted the Bax/Bcl2 ratio by approximately 8 folds, as compared to the control. Moreover, compound 4b markedly reduced the levels of the anti-apoptotic proteins Bcl-2 by approximately 4 folds compared to the control. A relatively more indicative and precise value for apoptosis induction is the Bax/Bcl2 ratio as it gives a more accurate estimation of the overall proapoptotic activity of the molecule. Collectively, these findings that compound 4b markedly increased Bax level and downregulated Bcl2 level, concomitantly with tremendously augmenting the Bax/Bcl2 ratio proved undoubtedly their pro-apoptotic effect.

Table 5 Effect of compound 4b on levels of BAX and Bcl-2 proteins expression in MCF-7 cells treated for 24 h

It should be noted that all experiments were performed in compliance with relevant laws or guidelines; all experiments followed institutional guidelines; Research Ethics Committee of Al-Azhar University approved the experiments as there is no human subjects were used.

2.10. ADMET profiling study

In silico report of the highly active derivatives 3e, 4b and 5b was conducted for their physicochemical character evaluation and the proposed absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile. It was predicted using pkCSM descriptor algorithm procedures68 and matched to the rule of five described by Lipinski.69 Good absorption properties were expected for the molecules that accomplish at least three rules: (i) hydrogen bond donors ≤5, (ii) hydrogen bond acceptors ≤10, (iii) molecular weight <500, (iv) logP ≤ 5. In the current work, the standard anticancer agent sorafenib violated logP rule and our compounds violated molecular weight and logP rules.

As a result of obtaining data from Table 6, we can assume that compounds 3e, 4b and 5b have excellent gastrointestinal tract (GIT) absorption in human (90.574–87.184) which indicates easier to cross different biological membranes.70 So, they may show a significantly high bioavailability through GIT. Concerning central nervous system (CNS) penetrability, our prepared compounds can reach CNS (CNS permeability values −1.384 to −0.863).

Table 6 ADMET profile of the four most active derivatives and Sorafenib

Parameter	3e	4b	5b	Soraf.
Physicochemical properties
Molecular weight	506.009	536.422	552.49	464.831
LogP	7.1411	7.7645	9.13379	5.5497
Rotatable bonds	6	5	5	5
Acceptors	6	5	5	4
Donors	2	2	2	3
Surface area	219.528	227.506	233.704	185.111
Absorption
Water solubility	−3.586	−2.97	−2.969	−4.822
Human intest. Absorption	90.574	89.92	87.184	89.043
Distribution
Permeability throughout BBB	0.015	−1.61	1.027	−1.684
Permeability to CNS	−1.384	−1.211	−0.863	−2.007
Metabolism
CYP2D6 substrate	No	No	No	No
CYP3A4 substrate	Yes	Yes	Yes	Yes
Inhibition of CYP1A2	Yes	No	No	Yes
Inhibition of CYP2C19	Yes	Yes	Yes	Yes
Inhibition of CYP2C9	Yes	Yes	Yes	Yes
Inhibition of CYP2D6	No	No	No	No
Inhibition of CYP3A4	No	No	No	Yes
Excretion
Clearance	0.107	−0.026	−0.067	−0.219
Toxicity
Human max. Tolerated dose	0.523	0.431	0.431	0.549
Acute toxic activity (LD50)	3.443	2.751	2.747	2.538
Chronic toxic activity (LOAEL)	1.643	1.62	1.418	1.198
Hepatotoxic effect	Yes	Yes	No	Yes

It is well known that cytochrome P450 3A4 (CYP3A4), the major drug-metabolizing enzyme, could be inhibited by sorafenib, but our derivatives couldn't. Elimination was expected depending on the total clearance which is a considerable factor in deciding dose intervals. Unlike, sorafenib our compounds exhibited slowly clearance rate, which signifies a long duration of action and extended dosing intervals. Toxicity is the final ADMET profile studied factor. Sorafenib and the novel compounds 3e and 4b shared the drawback of unwanted hepatotoxic actions while our derivative 5b showed no hepatotoxicity. Sorafenib and our compounds demonstrated high maximum tolerated dose. These involve the advantage of the broad therapeutic index of, sorafenib, and our derivatives. The oral acute and chronic toxic doses of the novel compounds 3e, 4b and 5b are higher than that of sorafenib.

3. Conclusion

In this work, we describe the design and synthesis of fifteen new phthalazine derivatives. In the designed compounds, new spacers in the form of fragments with verified VEGFR-2 inhibitory potentials, including α,β-unsaturated ketonic fragment, pyrazole, pyrimidinone and/or pyrimidinthione were introduced at position-4 of the phenyl tail. Also, new tail hydrophobic moieties were attached to these spacers that are expected to increase the hydrophobic interaction with VEGFR-2 enzyme. These structural optimizations have led to the identification of the novel derivatives 4b and 3e as the most promising hit molecules. The designed compounds were evaluated for their anticancer activities against two human tumor cell lines: HepG2 and MCF-7. All the tested compounds showed low to high anticancer activities against the selected cancer cells. Also, molecular docking study was performed to investigate the proposed binding mode of the new compounds with VEGFR-2 active site. The data obtained from biological testing were highly correlated with that obtained from docking study. In particular, compounds 4b and 3e were found to be the most potent derivatives over all the tested compounds against the two MCF-7 and HepG2 cancer cell lines with IC₅₀ = 0.06, 0.06 μM and 0.08, 0.19 μM respectively. Our final compounds 3a–e, 4a,b and 5a,b were evaluated for their cytotoxicity against normal VERO cells. Our compounds exhibited low toxicity concerning normal VERO cells with IC₅₀ = 3.00–4.75 μM. All compounds displayed high selectivity except compounds 3a and 3c against both tested cancer cell lines and compound 5a against MCF-7 which exhibited moderate selectivity. The nine derivatives 3a–e, 4a, 4b, 5a and 5b were evaluated for their inhibitory activities against VEGFR-2. Compound 4b was found to be the most potent derivative that inhibited VEGFR-2 at IC₅₀ value of 0.09 ± 0.02 μM. Compounds 3e, 4a and 5b exhibited good activity with IC₅₀ values = 0.12 ± 0.02, 0.15 ± 0.03 and 0.13 ± 0.03 μM respectively. To get a better insight regarding the effect of 4b on growth inhibition of cancer cells, its effect on the cell cycle distribution and apoptosis induction were evaluated in MCF-7 cells. Compound 4b arrested cell growth in G2-M phase; accumulation of cells at that phase became 6.92% after being 13.2 in control cells. In addition, our derivatives 3e, 4b and 5b showed good in silico calculated ADMET profile in comparing to sorafenib.

4. Experimental

All melting points were carried out by open capillary method on a Gallen kamp Melting point apparatus at faculty of pharmacy Al-Azhar University and were uncorrected. The infrared spectra were recorded on pye Unicam SP 1000 IR spectrophotometer at Pharmaceutical analytical Unit, Faculty of Pharmacy, Al-Azhar University using potassium bromide disc technique. Proton magnetic resonance ¹H NMR spectra were recorded on a Bruker 400 Megahertz-nuclear magnetic resonance (400 MHZ-NMR) spectrophotometer at Faculty of pharmacy, Mansoura University. Carbon-13 (C13) nuclear magnetic resonance (¹³C NMR) spectra were recorded on a Bruker 100 Megahertz-nuclear magnetic resonance (100 MHZ-NMR) spectrophotometer at Faculty of pharmacy, Mansoura University. The mass spectra were carried out on Direct Probe Controller Inlet part to Single Quadropole mass analyzer in Thermo Scientific Gas chromatography-mass spectrometry (GCMS) model ISQ LT using Thermo X-Calibur software at the Mycology and Biotechnology Regional Center, Al-Azhar University. Elemental analyses (C, H, N) were performed on a carbon hydrogen and nitrogen (CHN) analyzer at Mycology and Biotechnology Regional Center, Al-Azhar University.

4.1. Chemistry

4-(4-Chlorophenyl)phthalazin-1(2H)-one, and 1-chloro-4-(4-chlorophenyl)phthalazine were obtained according to the reported procedures.11

4.1.2. General procedure for synthesis of chalcone derivatives (2a–e). To a mixture of the acetyl derivative (1) (0.40 g, 0.001 mol) and the appropriate aromatic aldehyde namely, benzaldehyde, 4-chlorobenzaldehyde, 2-hydroxybenzaldehyde, 4-hydroxybenzaldehyde and/or 4-methoxybenzaldehyde (0.001 mol) in ethyl alcohol (30 ml), 40% aqueous NaOH (10 ml) was added dropwise within 20 min. The reaction mixture was stirred at rt for 24 h. After completion of the reaction detected by TLC, the formed solid was collected by filtration, washed with water and air dried, crystallized from absolute ethanol/DMF mixture to give the corresponding chalcones 2a–e respectively.
4.1.2.1. 1-(4-[{4-(4-Chlorophenyl)phthalazin-1-yl}amino]phenyl)-3-phenyl-prop-2-en-1-one (2a). Light yellow powder; yield, 75%; m.p. 245–247 °C; IR ν_max (cm⁻¹): 3440 (NH), 2917 (CH aromatic), 2851 (CH aliphatic), 1654 (CO), 1600 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 9.73 (s, 1H), 8.72 (t, J = 7.3 Hz, 1H), 8.22 (s, 2H), 8.11 (d, J = 8.8 Hz, 2H), 7.99 (dq, J = 9.0, 3.3 Hz, 4H), 7.90 (dd, J = 6.8, 3.8 Hz, 2H), 7.78–7.69 (m, 3H), 7.70–7.56 (m, 4H), 7.47 (dd, J = 5.1, 1.9 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.84, 187.72, 152.49, 143.48, 135.84, 134.16, 133.15, 132.52, 132.05, 130.29, 129.81, 129.41, 129.27, 129.05, 128.78, 126.24, 122.59, 119.80, 119.67; anal. calcd for C₂₉H₂₀ClN₃O (461.95): C, 75.40; H, 4.36; N, 9.10. Found: C, 75.62; H, 4.51; N, 9.27%.
4.1.2.2. 3-(4-Chlorophenyl)-1-(4-[{4-(4-chlorophenyl)phthalazin-1-yl}amino]phenyl)prop-2-en-1-one (2b). Light yellow powder, yield, 82%; m.p. 244–246 °C; IR ν_max (cm⁻¹): 3443 (NH), 2918 (CH aromatic), 2851 (CH aliphatic), 1604 (CO), 1447 (CN); ¹H NMR (400 MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.23 (s, 2H), 8.09 (t, J = 7.4 Hz, 2H), 8.03 (d, J = 10.6 Hz, 2H), 8.00–7.96 (m, 2H), 7.93 (d, J = 10.1 Hz, 2H), 7.77–7.74 (m, 2H), 7.70 (d, J = 10.9 Hz, 1H), 7.68–7.63 (m, 2H), 7.55 (d, J = 8.4 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.84, 187.50, 141.99, 135.83, 135.33, 134.36, 134.15, 133.14, 132.52, 132.50, 132.06, 132.04, 130.98, 130.84, 130.32, 129.79, 129.43, 129.04, 126.21, 123.33, 119.67; anal. calcd for C₂₉H₁₉Cl₂N₃O (496.39): C, 70.17; H, 3.86; N, 8.47. Found: C, 70.39; H, 4.03; N, 8.70%.
4.1.2.3. 1-(4-[{4-(4-Chlorophenyl)phthalazin-1-yl}amino]phenyl)-3-(2-hydroxyphenyl)prop-2-en-1-one (2c). Reddish yellow powder; yield, 77%; m.p. 246–248 °C; IR ν_max (cm⁻¹): 3419 (OH), 3261 (NH), 3057 (CH aromatic), 2959, 2918 (CH aliphatic), 1674 (CO), 1601 (CN); ¹H NMR (400 MHz, DMSO-d₆) δ 9.75 (s, 1H), 9.39 (s, 1H), 8.73 (d, 1H), 8.16–7.65 (m, 17H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.84, 157.58, 153.78, 152.19, 148.83, 145.93, 135.84, 134.15, 133.13, 132.50, 132.04, 130.85, 129.81, 129.04, 126.23, 126.17, 123.40, 119.67, 119.32, 116.07; anal. calcd for C₂₉H₂₀ClN₃O₂ (477.95): C, 72.88; H, 4.22; N, 8.79. Found: C, 73.04; H, 4.29; N, 9.02%.
4.1.2.4. 1-(4-[{4-(4-Chlorophenyl)phthalazin-1-yl}amino]phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (2d). Yellow powder; yield, 78%; m.p. 247–249 °C; IR ν_max (cm⁻¹): 3417 (OH), 3263 (NH), 2918 (CH aromatic), 2851 (CH aliphatic), 1674 (CO), 1601 (CN); ¹H NMR (400 MHz, DMSO-d₆) δ 9.78 (s, 1H), 9.24 (s, 1H), 8.73 (d, 1H), 8.16–7.64 (m, 17H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.83, 186.67, 153.78, 152.14, 148.89, 146.09, 135.83, 134.14, 133.10, 132.48, 132.03, 130.83, 129.79, 129.03, 126.20, 126.16, 123.40, 119.67, 119.36, 115.87. Anal. calcd for C₂₉H₂₀ClN₃O₂ (477.95): C, 72.88; H, 4.22; N, 8.79. Found: C, 73.07; H, 4.31; N, 9.05%.
4.1.2.5. 1-(4-[{4-(4-Chlorophenyl)phthalazin-1-yl}amino]phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (2e). Orange powder; yield, 75%; m.p. 250–252 °C; IR ν_max (cm⁻¹): 3421 (NH), 2919 (CH aromatic), 2851 (CH aliphatic), 1602 (CO), 1447 (CN); ¹H NMR (400 MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.75 (d, 1H), 8.22–8.07 (m, 4H), 8.01–7.86 (m, 4H), 7.76–7.65 (m, 7H), 7.03 (d, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.83, 187.64, 161.67, 145.88, 143.47, 135.84, 134.16, 133.15, 132.52, 132.05, 131.14, 130.85, 130.12, 129.81, 129.05, 128.00, 126.24, 126.18, 123.39, 120.05, 119.78, 119.65, 114.88, 55.86; anal. calcd for C₃₀H₂₂ClN₃O₂ (491.98): C, 73.24; H, 4.51; N, 8.54. Found: C, 73.45; H, 4.63; N, 8.81%.

4.1.3. General procedure for synthesis of compounds (3a–e). A mixture of the appropriate chalcone 2a–e (0.1 mol) in 30 ml absolute ethanol and hydrazine 99% (0.5 mol) in 100 ml round bottom flask with magnetic stirrer was heated under reflux while stirring for 8 h, after cooling the separated solid was filtered and washed with water then air dried and crystallized from absolute ethanol to give the corresponding pyrazole derivatives 3a–e respectively.
4.1.3.1. 4-(4-Chlorophenyl)-N-(4-(5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)phthalazin-1-amine (3a). Yellow powder; yield, 70%; m.p. 271–273 °C; IR ν_max (cm⁻¹): 3424 (NH), 2919 (CH aromatic), 2851 (CH aliphatic), 1600 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 9.48 (s, 1H), 8.75 (s, 1H), 8.07–7.85 (m, 7H), 7.74–7.58 (m, 7H), 7.49–7.13 (m, 3H), 6.21 (m, 2H), 4.84 (m, 1H); MS (m/z): 475.29 (M⁺, 12.81%), 417.60 (43.00%), 395.22 (57.82%), 368.38 (100%, base peak); anal. calcd for C₂₉H₂₂ClN₅ (475.16): C, 73.18; H, 4.66; N, 14.71. Found: C, 73.40; H, 4.83; N, 14.85%.
4.1.3.2. 4-(4-Chlorophenyl)-N-(4-(5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)phthalazin-1-amine (3b). Yellow powder; yield, 70%; m.p. 270–272 °C; IR ν_max (cm⁻¹): 3424 (NH), 2919 (CH aromatic), 2852 (CH aliphatic), 1597, 1548 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 9.48 (s, 1H), 8.74 (s, 1H), 8.05–7.90 (m, 8H), 7.73–7.71 (m, 2H), 7.68–7.65 (m, 4H), 7.44–7.43 (m, 2H), 6.26 (m, 2H), 4.67 (m, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 158.67, 152.36, 145.86, 142.92, 136.10, 134.40, 132.53, 132.24, 132.01, 129.83, 129.05, 129.03, 127.89, 125.36, 123.57, 123.26, 120.87, 118.93, 113.91, 109.82, 99.73, 43.54, 36.38; MS (m/z): 509.47 (M⁺, 4.00%), 132.12 (46.22%), 77.09 (100%, base peak), 43.09 (97.05%); anal. calcd for C₂₉H₂₁Cl₂N₅ (509.12): C, 68.24; H, 4.15; N, 13.72. Found: C, 68.43; H, 4.37; N, 13.99%.
4.1.3.3. 2-(3-[4-{(4-[4-Chlorophenyl]phthalazin-1-yl)amino}phenyl]-4,5-dihydro-1H-pyrazol-5-yl)phenol (3c). Reddish yellow powder; yield, 69%; m.p. 266–268 °C; IR ν_max (cm⁻¹): 3753 (OH), 3422 (NH), 2918 (CH aromatic), 2852 (CH aliphatic), 1643, 1620 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 9.76 (s, 1H), 9.38 (s, 1H), 8.72 (s, 1H), 8.18–7.64 (m, 16H), 6.14 (m, 2H), 4.75 (m, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.85, 153.84, 152.77, 152.35, 142.96, 140.13, 136.09, 134.39, 134.15, 133.14, 132.80, 132.23, 131.98, 130.86, 129.81, 129.04, 128.99, 125.36, 120.87, 119.65, 118.92, 43.18, 31.19; MS (m/z): 491.94 (M⁺, 12.81%), 165.80 (49.57%), 129.96 (53.75%), 103.84 (100%, base peak), 76.17 (69.04%); anal. calcd for C₂₉H₂₂ClN₅O (491.15): C, 70.80; H, 4.51; N, 14.24. Found: C, 71.02; H, 4.67; N, 14.51%.
4.1.3.4. 4-(3-[4-{(4-[4-Chlorophenyl]phthalazin-1-yl)amino}phenyl]-4,5-dihydro-1H-pyrazol-5-yl)phenol (3d). Orange powder; yield, 61%; m.p. 265–267 °C; IR ν_max (cm⁻¹): 3439 (OH), 3303 (NH), 3081 (CH aromatic), 2962 (CH aliphatic), 1599, 1574 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 9.76 (s, 1H), 9.38 (s, 1H), 8.72 (s, 1H), 8.18–7.65 (m, 16H), 6.26 (m, 2H), 4.74 (m, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.85, 134.40, 134.16, 133.94, 133.16, 132.53, 132.25, 132.05, 131.99, 130.87, 129.82, 129.05, 129.00, 126.25, 126.18, 126.07, 125.36, 123.38, 120.87, 119.64, 45.00, 31.19; MS (m/z): 491.96 (M⁺, 0.58%), 127.06 (60.97%), 115.07 (98.22%), 91.10 (97.78%), 43.08 (100%, base peak); anal. calcd for C₂₉H₂₂ClN₅O (491.15): C, 70.80; H, 4.51; N, 14.24. Found: C, 68.69; H, 4.68; N, 14.45%.
4.1.3.5. 4-(4-Chlorophenyl)-N-(4-[5-{4-methoxyphenyl}-4,5-dihydro-1H-pyrazol-3-yl]phenyl)phthalazin-1-amine (3e). Yellow powder; yield, 67%; m.p. 353–355 °C; IR ν_max (cm⁻¹): 3436 (NH), 2918 (CH aromatic), 2851 (CH aliphatic), 1601, 1547 (CN); ¹³C NMR (101 MHz, DMSO-d₆) δ 197.03, 153.41, 134.92, 134.22, 134.21, 133.99, 133.65, 132.27, 132.19, 131.89, 131.71, 129.92, 129.30, 129.24, 129.13, 128.33, 127.15, 126.90, 126.67, 123.96, 120.75, 43.89, 31.19, 26.99; MS (m/z): 505.71 (M⁺, 13.43%), 476.19 (65.66%), 413.94 (100%, base peak), 312.93 (73.97%), 295.66 (67.05%); anal. calcd for C₃₀H₂₄ClN₅O (505.17): C, 71.21; H, 4.78; N, 13.84. Found: C, 71.38; H, 5.01; N, 14.02%.

4.1.4. General procedure for synthesis of pyrimidin-2(1H)-one (4a,b). A mixture of the appropriate chalcone 3a–e (0.008 mol) and urea (0.50 g, 0.008 mol) in absolute ethanol (30 ml) was heated under reflux while stirring for 10 h in the presence of conc. HCl (5 ml). The reaction mixture was concentrated to the half of its volume, cooled and neutralized with NH₄OH solution. The precipitated solid was filtered, washed with water, air dried and recrystallized from ethanol to give the corresponding pyrimidin-2-one derivatives 4a,b respectively.
4.1.4.1. 4-(4-[{4-(4-Chlorophenyl)phthalazin-1-yl}amino]phenyl)-6-phenylpyrimidin-2(1H)-one (4a). Yellow powder; yield, 65%; m.p. 265–267 °C; IR ν_max (cm⁻¹): 3437 (NH), 3070 (CH aromatic), 2917 (CH aliphatic), 1652 (CO), 1602 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 9.75 (s, 1H), 8.72 (s, 1H), 8.26–7.48 (m, 18H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.85, 187.74, 143.52, 135.67, 135.37, 134.23, 133.24, 132.63, 132.07, 131.71, 130.97, 130.30, 129.82, 129.55, 129.41, 129.28, 129.13, 129.07, 126.33, 126.22, 123.41, 122.57, 119.75; MS (m/z): 501.84 (M⁺, 48.68%), 493.76 (52.76%), 187.40 (100%, base peak), 149.91 (74.31%), 130.84 (97.80%); anal. calcd for C₃₀H₂₀ClN₅O (501.97): C, 71.78; H, 4.02; N, 13.95. Found: C, 72.04; H, 4.13; N, 14.17%.
4.1.4.2. 6-(4-Chlorophenyl)-4-(4-[{4-(4-chlorophenyl)phthalazin-1-yl}amino]phenyl)pyrimidin-2(1H)-one (4b). Yellow powder; yield, 56%; m.p. 270–272 °C; IR ν_max (cm⁻¹): 3429 (NH), 3067 (CH aromatic), 2917 (CH aliphatic), 1657 (CO), 1598 (CN); ¹H NMR (300 MHz, DMSO-d₆) δ 10.42 (s, 1H), 8.95 (s, 1H), 8.30–7.55 (m, 17H); ¹³C NMR (101 MHz, DMSO-d₆) δ 187.81, 153.29, 152.62, 142.39, 135.45, 135.20, 134.29, 134.22, 134.06, 132.27, 131.71, 131.05, 130.71, 130.44, 129.94, 129.45, 129.30, 129.14, 127.45, 126.88, 124.26, 123.23, 121.22, 113.29; MS (m/z): 536.28 (M⁺, 26.02%), 533.62 (55.08%), 367.24 (100%, base peak), 296.64 (79.61%), 232.05 (93.84%); anal. calcd for C₃₀H₁₉Cl₂N₅O (536.42): C, 67.17; H, 3.57; N, 13.06. Found: C, 67.41; H, 3.62; N, 13.29%.

4.1.5. General procedure for synthesis of pyrimidin-2(1H)-thione (5a,b). A mixture of the appropriate chalcone 3a–e (0.007 mol) and thiourea (0.5 g, 0.007 mol) in absolute ethanol (30 ml) was heated under reflux while stirring for 12 h in the presence of 0.5 g. of NaOH. The reaction mixture was concentrated to the half of its volume, cooled and neutralized with dilute HCl. The precipitated solid was filtered, washed with water, air dried and recrystallized from ethanol to give the corresponding pyrimidin-2-thione derivatives 5a,b respectively.
4.1.5.1. 4-(4-[{4-(4-Chlorophenyl)phthalazin-1-yl}amino]phenyl)-6-phenylpyrimidin-2(1H)-thione (5a). Yellow powder; yield, 60%; m.p. 251–253 °C; IR ν_max (cm⁻¹): 3423 (NH), 2917 (CH aromatic), 2850 (CH aliphatic), 1597, 1543 (CN), 1402 (CS); ¹H NMR (300 MHz, DMSO-d₆) δ 10.08 (s, 1H), 8.83 (s, 1H), 8.19–7.69 (m, 18H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.98, 135.06, 134.82, 134.20, 133.88, 133.51, 132.95, 132.17, 131.80, 131.76, 130.19, 129.91, 129.31, 129.21, 127.04, 125.65, 126.62, 123.89, 123.87, 120.62, 120.05; MS (m/z): 518.06 (M⁺, 22.25%), 471.49 (88.13%), 451.77 (73.13%), 448.63 (100%, base peak), 152.60 (98.44%); anal. calcd for C₃₀H₂₀ClN₅S (518.04): C, 69.56; H, 3.89; N, 13.52. Found: C, 69.78; H, 4.06; N, 13.74%.
4.1.5.2. 6-(4-Chlorophenyl)-4-(4-[{4-(4-chlorophenyl)phthalazin-1-yl}amino]phenyl)pyrimidin-2(1H)-thione (5b). Light yellow powder; yield, 71%; m.p. 260–262 °C; IR ν_max (cm⁻¹): 3424 (NH), 3065 (CH aromatic), 2918 (CH aliphatic), 1596, 1547 (CN), 1402 (CS); ¹H NMR (300 MHz, DMSO-d₆) δ 10.03 (s, 1H), 8.82 (s, 1H), 8.36–8.11 (m, 4H), 8.06–8.02 (m, 4H), 7.96–7.90 (m, 2H), 7.76–7.68 (m, 6H), 7.63 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 196.94, 159.72, 152.39, 145.84, 145.15, 134.69, 134.41, 134.29, 134.20, 133.73, 133.30, 132.15, 131.70, 131.58, 129.87, 129.28, 129.17, 129.13, 128.33, 126.87, 126.62, 126.52, 123.82, 120.43; MS (m/z): 552.91 (M⁺, 6.08%), 531.17 (58.53%), 348.27 (100%, base peak), 121.10 (99.95%); anal. calcd for C₃₀H₁₉Cl₂N₅S (552.48): C, 65.22; H, 3.47; N, 12.68. Found: C, 65.43; H, 3.65; N, 12.90%.

4.2. Docking studies

VEGFR-2 (PDB ID 4ASD)51 was used by Molsoft program to carry out docking studies.

4.3. Biological testing

4.3.1. In vitro cytotoxic activity. Our derivatives were tested against two cell lines, HepG2 and MCF-7 using MTT colorimetric assay.59

4.3.2. In vitro VEGFR-2 assay. The highly active compounds were assessed for their inhibitory activities against VEGFR-2.60

4.3.3. Cell cycle progression. The effect of 4b on growth inhibition of cancer cells, its effect on the cell cycle distribution and apoptosis induction was evaluated in MCF-7 cells according to the reported procedures.61,62

4.4. ADMET profile

In silico ADMET profile of the highly active derivatives 3e, 4b and 5b was predicted using pkCSM descriptor algorithm procedures.68

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There is no interest to declare.

Acknowledgements

The authors express their gratitude and admiration Dr Mohamed Morsy Faculty of Science, Al-Azhar University for supporting the pharmacological part.

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