Pyrazolo[3,4-d]pyrimidine derivatives as VEGFR-2 and EGFR^T790M dual inhibitors: design, docking, ADMET, synthesis and anticancer evaluations

Abstract

Novel pyrazolo[3,4-d]pyrimidine derivatives have been designed, synthesized and examined as inhibitors of both EGFR^T790M and VEGFR-2. These compounds exhibit anticancer activities against HCT-116, MCF-7, HepG2 and A549 cancer cell lines. Docking studies were carried out to identify how the proposed molecules interact with both VEGFR-2 and EGFR^T790M. The results of the docking studies showed excellent correlation with the biological screening results. Derivatives 7c, 8b, 7e and 8c exhibit very good anticancer activities against A549, with IC₅₀ values of 5.75, 6.20, 6.55 and 7.10 µM, respectively. Molecules 7c, 8b, 7e, 8c, 7d and 7b showed IC₅₀ values of 5.50, 5.80, 6.15, 7.00, 9.40 and 9.50 µM, respectively, against HCT-116, establishing remarkable anticancer activities, while derivatives 7c, 8b, 7e and 8c showed potent anticancer activities against MCF-7, with IC₅₀ values of 5.90, 6.40, 7.00 and 7.90 µM, respectively. Moreover, molecules 7c, 8b, 7e and 8c, with corresponding IC₅₀ values of 5.00, 5.30, 5.75 and 8.80 µM, demonstrated the highest anticancer activities against HepG2. The particularly active molecules 7b, 7c, 7d, 7e, 8b and 8c were tested against VERO normal cell lines, and their low toxicity was established by IC₅₀ values ranging from 40.00 to 53.99 µM. Furthermore, all the derivatives were studied as inhibitors of both EGFR^T790M and VEGFR-2. Molecules 7c, 8b, 7e and 8c, with IC₅₀ values ranging from 0.90 to 1.25 µM, exhibited very good inhibition against VEGFR-2. In addition, molecules 7c, 8b, 7e, 7b and 8c, with IC₅₀ values of 0.25, 0.32, 0.35, 0.45 and 0.50 µM, respectively, displayed very good EGFR^T790M inhibition. Furthermore, molecules 7c, 7e and 8b exhibited excellent ADMET profile compared to sorafenib and erlotinib.

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1. Introduction

Cancer cells can develop new blood vessels to receive nutrients and oxygen from the neighboring cells. Thus, cancer can spread to other organs, mainly the liver, lung, and bones, through blood vessels and lymphatic systems.^1–4 Inhibition of VEGFR-2 (vascular endothelial growth factor receptor) and EGFR (epidermal growth factor receptor) is crucial for the prevention of tumor metastasis, angiogenesis, and tumor growth.^5,6 The two pathways have comparable signaling routes. VEGF expression and angiogenesis are reduced upon EGFR inhibition. Moreover, elevated VEGFR-2 levels in turn, contribute to the development of resistance to EGFR inhibitors.⁷ EGFR-mutant cancers are influenced by VEGF more than EGFR wild-type tumors.⁸ In nearly 90% of cases, EGFR-mutant NSCLC tumors harbor an exon 21 L858R mutation or an exon 19 deletion, both of which make tumors sensitive to EGFR TKIs in patients.⁹ In the metastatic setting, EGFRTKIs have been recognized as a first-line therapy because of their excellent tolerability and progression-free survival (PFS) benefits.¹⁰ Dual concurrent inhibition of VEGFR-2 and EGFR succeeded by working synergistically to target and treat cancer cells.¹¹ An ATP-binding site is present in both the enzymes, which permits a number of small molecules to bind powerfully and act as dual inhibitors, including afatinib (VI), lapatinib (V), vandetanib (IV), sorafenib (III), gefitinib (II), and erlotinib (I) (Fig. 1).¹² Most of these small molecules consist of quinazoline-based structures, and many challenges have been overcome to modify their structure in order to produce more active anticancer agents. Therefore, the search for new structures that can act as inhibitors of tyrosine kinase continues.^13–16

Fig. 1 Reported dual VEGFR-2 and EGFR inhibitors.

The fused pyrazolo[3,4-d]pyrimidine ring has received much consideration due to its molecular similarity to purines and its broad range of pharmacological potential.^1,17 In the literature, this scaffold has been shown to exhibit anticancer effects through a diverse set of pathways.^1,17–19 Pyrazolo[3,4-d]pyrimidine derivatives have been reported to have anticancer activities as inhibitors of EGFR, VEGFR-2,^20,21 dihydrofolate reductase (DHFR),²² mitogen-activated protein (MAP) kinase,²³ protein kinase target platelet-derived growth factor receptor (PDGFR), and Aurora-A.²¹ These results motivate us to evaluate our novel molecules as dual VEGFR-2 and EGFR inhibitors.

VEGFR-2 inhibitors can be categorized into three main classes. Class I inhibits the ATP region and forms one H-bond with Cys919 in the hinge region. Class II also inhibits the ATP region, but extends beyond the gate region, and inhibits the allosteric hydrophobic pocket. Class III inhibits the allosteric hydrophobic pocket. The specificity and affinity of Class II inhibitors make them superior to Class I and III inhibitors²⁴ (Fig. 2A).

Fig. 2 (A) VEGFR-2 binding site. (B) EGFR-TK binding site.

Moreover, the ATP-binding site of EGFR-TK has five key areas. (a) A phosphate-binding area that is used to enhance the inhibitor's pharmacokinetics. (b) A hydrophobic area I, which has an important role in selectivity and inhibition. (c) A hydrophobic area II, which is employed for inhibition activity. (d) A hydrophilic ribose sugar area, and (e) an adenine binding area, which includes the adenine ring and significant amino acids that form hydrogen bonds²⁵ (Fig. 2B).

In line with our previous work,^26–40 we continue to synthesize and develop new anticancer agents. Therefore, we designed and synthesized new pyrazolo[3,4-d]pyrimidines as dual inhibitors of EGFR-mutant/VEGFR-2. These molecules can be used as antiproliferative agents against HCT-116, MCF-7, HepG2 and A549 cell lines in which VEGFR-2/EGFR-mutants are over-expressed.

1.1. Rationale and structure-based design

Our novel molecules showed the essential structure requirements for both VEGFR-2 and EGFR inhibitors (Fig. 3 and 4). Numerous VEGFR-2 inhibitors, including sorafenib, have four significant parts:^41,42 (i) A hydrophobic tail that inhibited the allosteric hydrophobic pocket.⁴³ (ii) A linker with HBA/HBD (H-bond acceptor/H-bond donor) groups that form H-bonds with Glu885 and Asp1046.⁴⁴ (iii) A central hydrophobic spacer.⁴⁵ (iv) A flat hetero aromatic ring that inhibited ATP-binding area.⁴² Our VEGFR-2 inhibition design depends on bioisosteric replacement techniques based on sorafenib at four points (Fig. 3).

Fig. 3 Structural features of VEGFR-2 inhibitors.

Fig. 4 Structural features of EGFR inhibitors.

The main technique is to use the 6-methyl-1-phenyl-pyrazolo[3,4-d]pyrimidine heteroaromatic scaffold to inhibit the ATP region. The second modification is to use phenyl linkers. The third modification is to use formamide spacers to inhibit the gate area. Lastly, the fourth modification is to use an aliphatic and/or aromatic hydrophobic tail to inhibit the allosteric hydrophobic pocket. The elongation of the structure allows our molecules to be considered as Class II VEGFR-2 inhibitors, as they can inhibit the ATP area and extend outside the gate region and inhibit the allosteric hydrophobic pocket (Fig. 3).

On the other hand, EGFR-TKIs, including erlotinib, also have four significant parts. (i) A heteroaromatic structure that inhibits the adenine area.⁴⁶ (ii) A HBA/HBD spacer, e.g. NH group, that forms H-bonds with the amino acids in the linker area. (iii) A hydrophobic domain that inhibits the hydrophobic head I. (iv) A hydrophobic tail that inhibits the hydrophobic pocket II⁴⁷ (Fig. 4).

Our pyrazolopyrimidine molecules with elongated structures contain the basic pharmacophoric characteristics of EGFR-TKIs. Pyrazolopyrimidines replaced the heteroaromatic quinazoline of erlotinib and inhibited the adenine-binding area. Secondly, the 2-methoxyethoxy groups (hydrophobic tail) were replaced with the elongated side chains at position-5 of pyrazolopyrimidine to inhibit the hydrophobic pocket II. Thirdly, the N at position-7 of pyrazolopyrimidine was used as a hydrogen-bond acceptor (HBA). The phenyl ring at position-1 of pyrazolopyrimidine was the fourth modification (hydrophobic head) (Fig. 4).

All of these modifications stimulated our team to investigate the structure–activity relationship (SAR) of the prepared molecules, which served as anticancer agents with EGFR^T790M and VEGFR-2 dual inhibition.

2. Results and discussion

2.1. Chemistry

The targeted molecules 5, 6, 7a–e and 8a–c were gained by following the steps shown in Schemes 1 and 2. Production was started by condensation of ethyl 2-cyanoacetate and triethylorthoformate in the presence of acetic anhydride to give ethyl 2-cyano-3-ethoxyacrylate (1), which underwent an additional cyclization reaction with phenylhydrazine⁴⁸ to give ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate 2. Consequent stirring of 2 with alcoholic 10% potassium hydroxide solution gave 5-amino-1-phenyl-1H-pyrazole-4-carboxylic acid 3.⁴⁹ Acetylation and consequent condensation of 3 in acetic anhydride afforded 6-methyl-1-phenylpyrazolo[3,4-d]^1,3 oxazin-4(1H)-one 4,⁴⁹ which was treated with p-aminobenzoic acid and/or ethyl p-aminobenzoate to yield the respective derivatives 5 and 6 (Scheme 1). Constant stirring of the benzoic acid derivative 5 with ECF (ethyl chloroformate) in the presence of DMF and TEA, followed by the addition of suitable aliphatic and aromatic amines, produced the respective amides 7a–e and 8a–c, by following the mixed anhydride method³⁴ (Scheme 2).

Scheme 1 Synthetic steps for the targeted compounds 5 and 6.

Scheme 2 Synthetic scheme for the synthesis of the targeted compounds 7a–e and 8a–c.

The ¹H NMR spectrum of compound 7d showed the appearance of two new singlet signals corresponding to the (OCH₃) and NH groups at 3.76 and 10.30 ppm, respectively. In addition, compound 7e showed two new singlet signals corresponding to (COCH₃) and NH groups at 2.57 and 10.73 ppm, respectively. Moreover, the mass spectrum for compound 7e showed the molecular ion peak at m/z 463.03, corresponding to its molecular weight. The ¹H NMR spectrum of compound 8c showed new signals at 1.10–3.79 ppm attributed to the cyclohexyl protons. In addition, the ¹³C NMR spectrum showed characteristic signals corresponding to its structure. Moreover, the mass spectrum for compound 8c showed a molecular ion peak at m/z 427.34, corresponding to its molecular weight.

2.2. Docking studies

For molecular docking investigations, MolSoft software was used. The PDB IDs for VEGFR-2 (PDB ID 4ASD)^50–52 and EGFR^T790M (PDB ID 3W2O)⁴³ were used in each experiment.

2.2.1. Docking studies using VEGFR-2 inhibitors. Sorafenib bound in an orientation with a ΔG of −99.50 kcal mol⁻¹ and exhibited 5 H-bonds with Cys919 (2.51 Å and 2.10 Å), Glu885 (1.77 Å and 2.75 Å), and Asp1046 (1.50 Å). The N-methylpicolinamide group occupied and inhibited the hydrophobic pocket generated by Leu840, Val848, Glu917, Lys920, Leu1035, Phe918, and Cys919. Moreover, the central phenoxy linker occupied and inhibited the hydrophobic channel generated by Val848, Lys868, Thr916, Leu1035 and Cys1045. Additionally, the 3-trifluromethyl-4-chlorophenyl tail occupied and inhibited the allosteric hydrophobic channel generated by Glu885, Ile892, Ile888, Hie1026, Asp1046 and Cys1045 (Fig. 5). The urea spacer plays a fundamental role in the high binding affinity of sorafenib with VEGFR-2. These results encouraged us to experiment with formamide linkers in order to gain more effective VEGFR-2 inhibitors.

Fig. 5 Sorafenib binding orientation with 4ASD. Dotted lines represent H-bonds.

The binding orientation of compound 7c is nearly the same as that of sorafenib. It showed a ΔG of −105.02 kcal mol⁻¹ (Table 1) and 6 H-bonds with Cys919 (2.15 Å), Asp1046 (2.23 Å and 2.96 Å), Glu885 (2.98 Å), Ile1025 (1.59 Å) and Hie1026 (2.56 Å). The pocket formed by Leu840, Val848, Glu917, Phe918, Cys919, Lys920 and Leu1035 was occupied and inhibited by the 1-phenylpyrazolopyrimidine scaffold. Additionally, the hydrophobic channel generated by Glu885, Ile888, Ile892, Hie1026 and Ile1025 was occupied and inhibited by the terminal benzenesulfonamide tail (Fig. 6). The strongest anticancer activity of compound 7c may be explained by these interactions.

Fig. 6 Binding orientation of 7c with 4ASD.

Table 1 Calculated free energy (ΔG in kcal mol⁻¹) for ligand binding with VEGFR-2

Compound	ΔG [kcal mol^−1]	Compound	ΔG [kcal mol^−1]
5	−65.11	7e	−93.63
6	−76.93	8a	−77.15
7a	−83.51	8b	−101.13
7b	−89.84	8c	−84.87
7c	−105.02	Sorafenib	−99.50
7d	−84.37

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The binding orientation of compound 8b is nearly the same as that of 7c. It showed a binding energy of −101.13 kcal mol⁻¹ and 4 H-bonds with Cys919 (1.92 Å), Asp1046 (2.19 Å and 2.95 Å) and Glu885 (2.97 Å) (Fig. 7). In addition, compound 7e bound in a favorable orientation, with a binding energy of-93.63 kcal mol⁻¹ and forming four H-bonds (Fig. 8).

Fig. 7 Docking orientation of 8b with 4ASD.

Fig. 8 Docking results for 7e with 4ASD.

2.2.2. Docking as EGFR^T790M inhibitors. Erlotinib adopted a binding orientation with a binding energy of −82.77 kcal mol⁻¹ (Table 2) and four H-bonds with Thr854 (2.99 Å), Met793 (1.82 Å), Val726 (2.97 Å) and Cys797 (2.05 Å). The hydrophobic area I, which is generated by Phe723, Ile759, Thr854, Glu762, Leu777, Met790, Glu791, Asp855, and Val726, was occupied and inhibited by the 3-ethynylphenyl head. Additionally, the 2-methoxyethoxy tail occupied and inhibited the hydrophobic area II generated by Leu718, Met793, Pro794, Leu844 and Val845 (Fig. 9).

Table 2 Calculated free energy (ΔG in kcal mol⁻¹) for the binding of the synthesized compounds with EGFR^T790M

Compound	ΔG [kcal mol^−1]	Compound	ΔG [kcal mol^−1]
5	−61.84	7e	−81.85
6	−75.35	8a	−75.57
7a	−76.05	8b	−86.17
7b	−76.66	8c	−79.98
7c	−97.48	Erlotinib	−82.77
7d	−75.88

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Fig. 9 Docking orientation of erlotinib with 3W2O, dotted lines represented H-bonds.

The binding orientation of compound 7c is nearly identical to that of erlotinib. It presented a binding energy of −97.48 kcal mol⁻¹ and 5 H-bonds with Thr854 (2.98 Å), Gln791 (2.96 Å), Pro794 (2.54 Å) and Met793 (2.20 Å and 2.95 Å). The long side-chain occupied and inhibited the hydrophobic area II, generated by Leu718, Met793, Leu844, Pro794, Cys797, Val845, Gly724, Gln791, Met790, and Val726. The 1-phenylpyrazolopyrimidine occupied and inhibited hydrophobic area I, generated by Glu762, Leu777, Ile759, Leu788, Met790, Gly724, Thr854, Asp855, Glu758, and Phe723 (Fig. 10).

Fig. 10 Predicted binding mode for 7c with 3W2O.

Compounds 8b and 7e adopt binding orientations that are almost identical to those of erlotinib and 7c, with binding energies of −86.17 kcal mol⁻¹ (with four H-bonds formed; Fig. 11) and −81.85 kcal mol⁻¹ (with 4 H-bonds formed; Fig. 12), respectively.

Fig. 11 Docking results for compound 8b with 3W2O.

Fig. 12 Docking results for compound 7e with 3W2O.

2.3. Molecular dynamics simulations

Molecular dynamics (MD) simulations were performed for the highly active molecules 7c, 7e, 8b and 8c in EGFR^T790M and VEGFR-2 proteins. The ligand-receptor binding energy was determined by employing Amber's MM/GBSA.py script and the trajectory.⁵³ Erlotinib and sorafenib were used as the respective positive controls. With the help of GAFF2 (ref. 54 and 55) and the AMBERff14SB protein force field, ligand force fields were produced.⁵⁶ The observed root mean square deviation (RMSD) confirmed the stability of the studied molecules as inhibitors within the defined active sites throughout the 50 ns all-atom MD runs (Fig. 13). Molecular divergence of a given ligand from the recognized original/reference structure is assessed by RMSD. The selected MD simulation procedure was valid, and it gave a respectable suggestion of the stability of the ligand-target interactions.

Fig. 13 (A) Analysis of VEGFR-2 protein RMSD throughout 50 ns for the ligand-protein complexes (B) Analysis of EGFR^T790M protein RMSD throughout 50 ns for the ligand-protein complexes.

2.4. In vitro cytotoxic activity

The novel molecules 5, 6, 7a–e and 8a–c were tested against MCF-7, HepG2, HCT-116, A549 and VERO cell lines using the MTT protocol.^57–60 MCF-7, HepG2, HCT-116, A549 and VERO cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Sorafenib and erlotinib were used as reference cytotoxic drugs. The obtained growth inhibitory concentration (IC₅₀) values are shown in Table 3. The results showed that most of the obtained molecules exhibited good to weak IC₅₀ values against the studied cancer cell lines.

Table 3 In vitro cytotoxic effects against the HepG2, MCF-7, MCF-10, HCT-116, A549 and VERO cell lines

Comp.	IC50 (µM)^a
	A549	HCT116	MCF-7	HepG2	MCF-10	VERO
a IC50 values are the mean ± S.D. of three independent experiments.b NT: Not tested compounds.
5	17.20 ± 1.5	16.30 ± 1.5	16.10 ± 1.5	16.95 ± 1.5	^bNT	^bNT
6	13.00 ± 1.5	12.45 ± 1.5	12.75 ± 1.5	14.70 ± 1.5	^bNT	^bNT
7a	10.80 ± 2.5	10.70 ± 1.5	11.15 ± 1.5	12.30 ± 1.5	^bNT	^bNT
7b	10.30 ± 1.5	9.50 ± 1.5	10.25 ± 1.5	11.25 ± 1.5	49.95 ± 2.5	47.11 ± 0.50
7c	5.75 ± 1.5	5.50 ± 1.5	5.90 ± 1.5	5.00 ± 1.5	48.80 ± 2.5	47.16 ± 0.50
7d	10.50 ± 1.5	9.40 ± 1.5	10.25 ± 1.5	10.50 ± 1.5	50.55 ± 2.5	52.77 ± 0.50
7e	6.55 ± 1.5	6.15 ± 1.5	7.00 ± 1.5	5.75 ± 1.5	47.45 ± 2.5	44.60 ± 0.50
8a	11.15 ± 1.5	10.25 ± 1.5	10.70 ± 1.5	12.10 ± 1.5	^bNT	^bNT
8b	6.20 ± 1.5	5.80 ± 1.5	6.40 ± 1.5	5.30 ± 1.5	56.40 ± 2.5	53.99 ± 0.50
8c	7.10 ± 1.5	7.00 ± 1.5	7.90 ± 1.5	8.80 ± 1.5	39.15 ± 2.5	40.00 ± 0.50
Sorafenib	4.04 ± 0.33	5.58 ± 0.55	5.05 ± 0.50	4.00 ± 0.33	^bNT	^bNT
Erlotinib	5.49 ± 0.45	8.20 ± 0.34	13.91 ± 1.3	7.73 ± 0.67	^bNT	^bNT

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Derivatives 7c, 8b, 7e and 8c exhibited very good anticancer effects, with IC₅₀ values of 5.75, 6.20, 6.55 and 7.10 µM, respectively, against A549. Derivatives 6, 7a, 7b, 7d and 8a, with IC₅₀ values of 10.30–13.00 µM, showed good cytotoxicity. Lastly, derivative 5 exhibited a moderate anticancer effect, with IC₅₀ = 17.20 µM.

Derivatives 7c, 8b, 7e, 8c, 7d and 7b exhibited IC₅₀ values of 5.50, 5.80, 6.15, 7.00, 9.40 and 9.50 µM, respectively, showing very good anticancer effects against HCT-116. Derivatives 6, 7a and 8a, with IC₅₀ values of 12.45, 10.70, and 10.25 µM, respectively, showed good cytotoxicity. Lastly, derivative 5 revealed a moderate anticancer effect, with IC₅₀ = 16.30 µM.

Concerning MCF-7, derivatives 7c, 8b, 7e and 8c exhibited very good anticancer effects with IC₅₀ values of 5.90, 6.40, 7.00 and 7.90 µM, respectively. Derivatives 6, 7a, 7b, 7d and 8a, with IC₅₀ values of 10.25–12.75 µM, showed good cytotoxicity. Lastly, derivative 5 revealed a moderate anticancer effect, with IC₅₀ = 16.10 µM.

Regarding HepG2, derivatives 7c, 8b, 7e and 8c, with IC₅₀ values of 5.00, 5.30, 5.75 and 8.80 µM, respectively, exhibited very good anticancer effects. Derivatives 6, 7a, 7b, 7d and 8a showed good cytotoxicity, with IC₅₀ = 10.50–14.70 µM. Lastly, derivative 5 revealed a moderate anticancer effect, with IC₅₀ = 16.95 µM.

Ultimately, six highly potent molecules, 7b, 7c, 7d, 7e, 8b and 8c, were tested against VERO ordinary cell lines to evaluate their cytotoxic potential. The findings revealed that all the assessed agents possessed weak toxicity towards normal VERO cells, with IC₅₀ values of 40.00–53.99 µM. One of the criteria for a good anti-cancer drug is that it should not affect non-cancerous cells. Molecules with low selectivity show SI <2, while those with moderate selectivity present SI values >2. A molecule with high selectivity typically exhibits SI values ≥5.³³ In this research, molecules 7b, 7c, 7d, 7e, 8b and 8c were respectively 4.57, 8.20, 5.03, 6.81, 8.71 and 5.63 times more toxic to A549 than to normal VERO cells and 4.96, 8.57, 5.61, 7.25, 9.31 and 5.71 times more lethal towards HCT-116 than to ordinary VERO cells. In addition, compounds 7b, 7c, 7d, 7e, 8b and 8c are respectively 4.60, 7.99, 5.15, 6.37, 8.44 and 5.06 times more toxic to MCF-7 than to normal VERO cells and 4.19, 9.43, 5.03, 7.76, 10.19 and 4.55 times more toxic to HepG2 than to ordinary VERO cells. All the molecules were highly selective against cancer cell lines, with SI values >5, except for molecule 7b, which demonstrated moderate selectivity against the four cancer cells (SI = 4.19–4.96) and compound 8c against HepG2 (SI = 4.55).

Moreover, molecules 7b, 7c, 7d, 7e, 8b and 8c are respectively 4.85, 8.49, 4.81, 7.24, 9.10 and 5.51 times more toxic towards A549 than to normal MCF-10 cells and 5.26, 8.87, 5.38, 7.72, 9.72 and 5.59 times more lethal to HCT-116 than to ordinary MCF-10 cells. In addition, compounds 7b, 7c, 7d, 7e, 8b and 8c are respectively 4.87, 8.27, 4.93, 6.78, 8.81 and 4.96 times more toxic to MCF-7 than to normal MCF-10 cells and 4.44, 9.76, 4.81, 8.25, 10.64 and 4.45 times more toxic to HepG2 than to ordinary MCF-10 cells. All molecules were highly selective against cancer cell lines, with SI values >5, except molecule 7b, which demonstrated moderate selectivity against A549, MCF-7 and HepG2 cancer cells (SI = 4.85, 4.87 and 4.44, respectively) and compound 7d against the same three cell lines (SI = 4.81, 4.93 and 4.81, respectively). In addition, derivative 8c exhibited moderate selectivity against both MCF-7 and HepG2 cancer cells (SI = 4.96 and 4.45, respectively).

2.5. VEGFR-2 and EGFR^T790M kinases inhibitory assays

The Alpha Screen system (PerkinElmer, USA) using the anti-phosphotyrosine antibody was used to perform the VEGFR-2 inhibition assay with all molecules 5, 6, 7a–e and 8a–c (Table 4).⁶¹ Sorafenib was used as the positive standard. The novel molecules exhibited different degrees of inhibition, with IC₅₀ values of 0.90 to 2.90 µM. Derivatives 7c, 8b, 7e and 8c, with IC₅₀ values of 0.90 to 1.25 µM, were the most active, with very good VEGFR-2 inhibition. Molecules 7a, 7b and 7d exhibited good activity, with IC₅₀ values of 1.50–1.95 µM. In addition, molecules 6 and 8a exhibited moderate VEGFR-2 inhibition, with respective IC₅₀ values of 2.55 and 2.15 µM. Finally, derivative 5, with IC₅₀ = 2.90 µM, exhibited the lowest VEGFR-2 inhibition.

Table 4 EGFR^T790M and VEGFR-2 kinase assays

Comp	IC50 (µM)^a
	VEGFR-2	EGFR^T790M
a IC50 values are the mean ± S.D. of three independent experiments.b NT: Not tested compounds.
5	2.90 ± 0.50	1.20 ± 0.25
6	2.45 ± 0.50	1.00 ± 0.25
7a	1.70 ± 0.10	0.85 ± 0.25
7b	1.50 ± 0.10	0.45 ± 0.25
7c	0.90 ± 0.50	0.25 ± 0.25
7d	1.95 ± 0.50	0.60 ± 0.25
7e	1.00 ± 0.50	0.35 ± 0.25
8a	2.15 ± 0.50	0.90 ± 0.25
8b	0.95 ± 0.50	0.32 ± 0.25
8c	1.25 ± 0.50	0.50 ± 0.25
Sorafenib	0.84 ± 0.04	^bNT
Erlotinib	^bNT	0.24 ± 0.22

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Furthermore, using the homogeneous time-resolved fluorescence (HTRF) technique,⁶² the EGFR^T790M inhibition assay was used to examine all the derivatives (5, 6, 7a–e and 8a–c; Table 4). Erlotinib (IC₅₀ = 0.24 µM) was employed as the standard. Very good EGFR^T790M inhibition was achieved for compounds 7c, 8b, 7e, 7b and 8c, with IC₅₀ values 0.25, 0.32, 0.35, 0.45 and 0.50 µM, respectively. Furthermore, compound 7d, with an IC₅₀ of 0.60 µM, showed a good inhibition effect. Additionally, compounds 6, 7a and 8a displayed moderate EGFR^T790M inhibition, with IC₅₀ values of 1.00, 0.85 and 0.90 µM, respectively. Finally, derivative 5, with IC₅₀ = 1.20 µM, exhibited the lowest EGFR^T790M inhibition.

2.6. Structure activity relationship (SAR) studies

The pyrazolopyrimidine rings, linker (HBA-HBD), spacer, lipophilicity, and the electronic nature of the substituents all play an essential role in anticancer activities. Compounds 7c, 8b, 7e and 8c exhibited the greatest anticancer activities against the four tested cancer cells.

We can classify the tested derivatives into three groups based on their structure (Table 3). The first group contains compounds 5 and 6. Derivative 6, with a hydrophobic ethyl ester group, showed higher activities against the four cell lines HepG2, MCF-7, A549 and HCT116 than derivative 5, with an electron-withdrawing hydrophilic COOH group (Fig. 14).

Fig. 14 Schematic for the SAR study.

The second group consists of compounds 7a–7e, all of which contain the N-phenyl benzamide moiety. Derivative 7c, with the phenyl substituted with a hydrophilic electron-withdrawing SO₂NH₂ group, demonstrated higher activities against the four cell lines HepG2, MCF-7, A549, and HCT116 than 7e (with an electron-withdrawing hydrophobic acetyl group), 7d (with a hydrophobic electron-donating methoxy group), 7b (with a hydrophilic electron-donating hydroxy group), and the unsubstituted derivative 7a against the four cell lines, except for A549, for which 7b > 7d (Fig. 14).

The third group consists of compounds 8a–8c, which contain the N-alkyl benzamide moiety. Derivative 8b, with the hydrophobic electron-donating n-hexyl group, showed stronger activities against the four cell lines than derivative 8c, with a hydrophobic electron-donating cyclohexyl group, and 8a, with a hydrophobic electron-donating ethyl group (Fig. 14).

2.7. ADMET profile and in silico studies

The in silico ADMET profile analysis of the highly active molecules 7c, 7e and 8b was performed using the pkCSM descriptor⁶³ and related to Lipinski's rule of five.⁶⁴ Good absorption characteristics were expected for compounds with at least three rules: (i) HBD ≤5, (ii) log P ≤5, (iii) molecular weight <500, (iv) and HBA ≤10. The standard anticancer agent sorafenib and compound 7c break one rule, while compounds 7e and 8b, as well as erlotinib, do not break any of the rules (Table 5).

Table 5 ADMET profiles of the most effective compounds 7c, 7e, 8b, sorafenib and erlotinib

Parameter	7c	7e	8b	Sorafenib	Erlotinib
Physicochemical properties
Molecular weight (Daltons)	500.54	463.497	429.524	464.831	393.443
LogP	2.77952	4.33472	3.18992	5.5497	3.4051
Rotatable bonds	5	5	8	5	10
Acceptors	8	7	6	4	7
Donors	2	1	1	3	1
Surface area (Å^2)	205.244	200.035	186.276	185.111	169.532
Absorption
Human intestinal absorption (%)	86.799	100.000	98.092	89.043	94.58
Distribution
BBB permeability (log BB)	−1.158	−0.741	−0.855	−1.684	−0.745
CNS permeability (log PS)	−3.188	−2.043	−2.325	−2.007	−3.216
Metabolism
Inhibition of CYP3A4	No	Yes	Yes	Yes	Yes
Inhibition of CYP2D6	No	No	No	Yes	Yes
Inhibition of CYP1A2	No	Yes	Yes	Yes	Yes
Inhibition of CYP2C19	Yes	Yes	Yes	No	No
Inhibition of CYP2C9	Yes	Yes	Yes	Yes	Yes
Excretion
Clearance (log mL^−1 min kg^−1)	0.531	0.31	0.711	−0.219	0.702
Toxicity
Human max. Tolerated dose (log mg^−1 kg per day)	0.661	0.678	0.601	0.549	0.839
Acute toxic activity (LD50) (mol kg^−1)	3.168	2.97	2.816	2.538	2.393
Hepatotoxic effect	Yes	Yes	Yes	Yes	Yes

---

Our derivatives have excellent GIT absorption in humans (86.8–100.0), which indicates that they can penetrate different cellular membranes easily.⁶⁵ As a result, they may exhibit significantly high bioavailability across the GIT. Our products are capable of penetrating the CNS (CNS permeability = −2.043–3.188), with CNS permeabilities lower than that of sorafenib (CNS permeability = −2.007) but greater than that of erlotinib (CNS permeability = −3.216).

Sorafenib, erlotinib, 7e and 8b may inhibit metabolism through inhibition of CYP3A4. Moreover, sorafenib, 7c and 7e showed poor clearance values. Erlotinib and 8b exhibited greater clearance rates. As a result, erlotinib and 8b have shorter dose intervals, since they can be eliminated from the body more rapidly. Derivatives 7c and 7e, in contrast to erlotinib, exhibited slow clearance rates, which suggests a longer half-life and prolonged dose intervals. Sorafenib, erlotinib, and the new molecules all had unfavorable hepatotoxicity. The three derivatives 7c, 7e and 8b showed maximum tolerated doses higher than that of sorafenib but lower than that of erlotinib. Finally, the novel derivatives showed acute toxic doses higher than those of both sorafenib and erlotinib.

3. Experimental

3.1. Chemistry

3.1.1. General. According to the described processes,^48,49 the starting and intermediate products ethyl 2-cyano-3-ethoxyacrylate (1), ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate (2), 5-amino-1-phenyl-1H-pyrazole-4-carboxylate (3), and 6-methyl-1-phenylpyrazolo[3,4-d][1,3]oxazin-4(1H)-one (4) were synthesized. Each compound was obtained by crystallization from ethanol, and the NMR spectra were recorded at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR in DMSO-d₆.

3.1.2. General procedure for the syntheses of compounds 5 and 6. 6-Methyl-1-phenylpyrazolo[3,4-d][1,3]oxazin-4(1H)-one (4) (2.27 g, 0.01 mol) was refluxed for 6 hours with an equimolar amount of either 4-aminobenzoic acid or ethyl 4-aminobenzoate (0.01 mol). The mixture was allowed to reach room temperature, then poured on an ice-water mixture to separate the solids, which were then filtered and crystallized from ethanol to obtain the desired compounds 5 and 6, respectively.
3.1.2.1. 4-(6-Methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzoic acid (5). Yield 85%; m.p. 237–9 °C; IR_νmax (cm⁻¹): 3421 (OH), 3050 (CH aromatic), 2964 (CH aliphatic), 1713, 1631 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 2.18 (s, 3H, CH₃), 7.42–7.45 (m, H, H-4 of C₆H₅), 7.52–7.64 (m, 4H, H-3, H-5 of C₆H₄ and H-3, H-5 of C₆H₅), 8.09–8.15 (m, 4H, H-2, H-6 of C₆H₄ and H-2, H-6 of C₆H₅), 8.37 (s, 1H, CH of pyrazole), 12.62 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆): 26.68, 110.95, 121.76 (2), 122.30 (2), 126.61, 129.15 (2), 129.40, 130.51 (2), 137.86, 140.92, 142.68, 143.86, 150.19, 165.79, 167.19; Anal. Calcd. For C₁₉H₁₄N₄O₃ (m.w. 346.35): C, 65.89; H, 4.07; N, 16.18. Found: C, 65.80; H, 4.00; N, 16.05.
3.1.2.2. Ethyl 4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzoate (6). Yield 80%; m.p. 180–2 °C; IR_νmax (cm⁻¹): 3085 (CH aromatic), 2979 (CH aliphatic), 1725, 1658 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 1.34–1.37 (t, 3H, CH₂CH₃), 2.18 (s, 3H, CH₃), 4.35–4.40 (q, 2H, CH₂CH₃), 7.41–7.45 (m, H, H-4 of C₆H₅), 7.58–7.64 (m, 4H, H-3, H-5 of C₆H₄ and H-3, H-5 of C₆H₅), 8.09–8.16 (m, 4H, H-2, H-6 of C₆H₄ and H-2, H-6 of C₆H₅), 8.37 (s, 1H, CH of pyrazole); ¹³C NMR (100 MHz, DMSO-d₆): 14.57, 25.02, 61.64, 105.75, 120.71 (2), 122.29, 126.99, 127.73 (2), 129.62 (2), 130.95 (2), 136.77, 138.59, 142.19, 151.18, 157.94, 158.97, 165.62; Anal. Calcd. For C₂₁H₁₈N₄O₃ (m.w. 374.14): C, 67.37; H, 4.85; N, 14.96. Found: C, 67.50; H, 4.95; N, 14.85.

3.1.3. General procedure for synthesis of compounds 7a–e. Et₃N (0.1 g, 0.2 mL, 0.001 mol) was added to a suspension of 4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzoic acid (5) (0.35 g, 0.001 mol) in DCM (10 mL). The reaction mixture was stirred in an ice-salt bath for 10 min. To the clear solution, ethyl chloroformate (0.11 g, 0.1 mL, 0.001 mol) was added in a dropwise manner over a period of 20 min, and the reaction mixture was stirred for 1 h in the ice-salt bath. Then, a solution of the appropriate aromatic amine (0.001 mol), namely, aniline, 4-aminophenol, 4-aminobenzenesulfonamide, 4-methoxyaniline and/or 4-aminoacetophenone and TEA (0.1 g, 0.2 mL, 0.001 mol) in DCM (10 mL) was added to the previous mixture. The whole mixture was stirred overnight at r.t. and the obtained precipitate was filtered, washed with water followed by hot ethanol, and dried to provide the corresponding amide derivatives 7a–e, respectively.
3.1.3.1. 4-(6-Methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)-N-phenylbenzamide (7a). Yield 87%; m.p. 202–4 °C; IR_νmax (cm⁻¹): 3318 (NH), 3068 (CH aromatic), 2979 (CH aliphatic), 1695, 1641 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 2.24 (s, 3H, CH₃), 7.12–7.16 (m, H, H-4 of NHC₆H₅), 7.35–7.47 (m, 2H, H-3, H-5 of NHC₆H₅), 7.55–7.57 (m, 2H, H-2, H-6 of C₆H₄), 7.61–7.66 (m, H, H-4 C₆H₅), 7.79–7.83 (m, 2H, H-3, H-5 of C₆H₅), 7.88–7.91 (m, 2H, H-2, H-6 of NHC₆H₅), 7.99–8.01 (m, 2H, H-3, H-5 of C₆H₄), 8.12–8.14 (m, 2H, H-2, H-6 of C₆H₄), 8.42 (s, 1H, CH of pyrazole), 10.15 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆): 21.55, 108.50, 110.06, 115.83 (2), 118.39 (2), 119.06 (2), 121.26 (2), 122.39 (2), 128.97, 129.31, 130.62, 131.75, 138.78 (2), 147.97, 148.08, 149.08, 152.75, 153.11, 153.73; Anal. Calcd. For C₂₅H₁₉N₅O₂ (m.w. 421.15): C, 71.25; H, 4.54; N, 16.62; O, 7.59. Found: C, 71.20; H, 4.54; N, 16.75; O, 7.70.
3.1.3.2. N-(4-Hydroxyphenyl)-4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzamide (7b). Yield 78%; m.p. 205–7 °C; IR_νmax (cm⁻¹): 3439 (OH), 3315 (NH), 3075 (CH aromatic), 2950 (CH aliphatic), 1711, 1695 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 2.18 (s, 3H, CH₃), 7.38–7.48 (m, 2H, H-3, H-5 of NHC₆H₅), 7.55–7.61 (m, 3H, H-2, H-6 of NHC₆H₄ and H-4 C₆H₅), 7.66–7.68 (m, 2H, H-2, H-6 of C₆H₄), 7.72–7.77 (m, 2H, H-3, H-5 of C₆H₅), 8.01–8.02 (m, 2H, H-3, H-5 of C₆H₄), 8.08–8.10 (m, 2H, H-2, H-6 of C₆H₅), 8.35 (s, 1H, CH of pyrazole), 10.33 (s, 1H, NH, D₂O exchangeable), 12.09 (s, 1H, OH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆): 26.74, 106.07 (2), 106.53, 117.11 (2), 118.95 (2), 120.14 (2), 121.95, 128.57 (2), 129.34 (2), 130.97, 139.96, 142.87, 143.90, 145.01 (2), 150.18, 154.38, 165.12, 167.20; Anal. Calcd. For C₂₅H₁₉N₅O₃ (m.w. 437.15): C, 68.64; H, 4.38; N, 16.01. Found: C, 68.55; H, 4.45; N, 15.95.
3.1.3.3. 4-(6-Methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)-N-(4-sulfamoylphenyl)-benzamide (7c). Yield 83%; m.p. 232–4 °C; IR_νmax (cm⁻¹): 3310, 3240 (NH₂ and NH), 3066 (CH aromatic), 2986 (CH aliphatic), 1715, 1699 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 2.23 (s, 3H, CH₃), 6.88 (s, 2H, NH₂, D₂O exchangeable), 7.27–7.45 (m, 2H, H-2, H-6 of C₆H₄), 7.58–7.67 (m, 5H, H-3, H-4, H-5 of C₆H₅ and H-3, H-5 of C₆H₄SO₂NH₂), 7.75–7.98 (m, 2H, H-3, H-5 of C₆H₄), 8.09–8.15 (m, 4H, H-2, H-6 of C₆H₅ and H-2, H-6 of C₆H₄SO₂NH₂), 8.38 (s, 1H, CH of pyrazole), 10.42 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆): 21.53, 108.47, 109.27, 113.34, 115.77, 116.83 (2), 117.30 (2), 119.96 (2), 121.66 (2), 122.80 (2), 127.58, 129.09 (2), 130.70, 138.79, 139.02, 147.93, 149.19, 152.70, 153.69; Anal. Calcd. For C₂₅H₂₀N₆O₄S (m.w. 500.13): C, 59.99; H, 4.03; N, 16.79. Found: C, 60.12; H, 4.10; N, 16.88.
3.1.3.4. N-(4-Methoxyphenyl)-4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzamide (7d). Yield 86%; m.p. 200–2 °C; IR_νmax (cm⁻¹): 3402 (NH), 3068 (CH aromatic), 2930 (CH aliphatic), 1711, 1654 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 2.18 (s, 3H, CH₃), 3.76 (s, 3H, OCH₃), 6.84–6.97 (m, 2H, H-3, H-5 of C₆H₅OCH₃), 7.33–7.46 (m, 2H, H-2, H-6 of C₆H₄), 7.59–7.72 (m, 5H, H-3, H-4, H-5 of C₆H₅ and H-2, H-6 of C₆H₅OCH₃), 8.09–8.16 (m, 4H, H-3, H-5 of C₆H₄ and H-2, H-6 of C₆H₅), 8.38 (s, 1H, CH of pyrazole), 10.30 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆): 25.09, 61.57, 105.85, 122.19 (4), 127.65, 129.69 (4), 129.80 (2), 130.92 (2), 131.08 (2), 136.84 (2), 138.70, 142.29, 151.21, 157.84, 158.95, 165.55; Anal. Calcd. For C₂₆H₂₁N₅O₃ (m.w. 451.16): C, 69.17; H, 4.69; N, 15.51. Found: C, 69.25; H, 4.75; N, 15.50.
3.1.3.5. N-(4-Acetylphenyl)-4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzamide (7e). Yield 84%; m.p. 220–2 °C; IR_νmax (cm⁻¹): 3316 (NH), 3064 (CH aromatic), 2985 (CH aliphatic), 1716, 1698, 1668 (3C=O); ¹H NMR (400 MHz, DMSO-d₆): 2.22 (s, 3H, CH₃), 2.57 (s, 3H, COCH₃), 7.42–7.46 (m, H, H-4 of C₆H₅), 7.59–7.67 (m, 4H, H-2, H-6 of C₆H₄ and H-3, H-5 of C₆H₅), 7.96–8.02 (m, 4H, H-3, H-5 of C₆H₄ and H-2, H-6 of C₆H₅COCH₃), 8.09–8.16 (m, 4H, H-2, H-6 of C₆H₅ & H-3, H-5 of C₆H₅COCH₃), 8.39 (s, 1H, CH of pyrazole), 10.73 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆): 21.58, 23.78, 106.05, 108.53, 114.60 (2), 115.68, 118.34 (2), 121.30 (2), 127.73 (2), 129.35 (2), 130.82, 138.43 (2), 144.19, 147.65, 152.91, 153.77, 154.03, 155.32, 163.64, 172.34, 174.55; MS (m/z): 463.03 (M⁺, 27.20%), 445.57 (70.52%), 363.49 (71.93%), 318.85 (70.56%), 183.37 (100%, base beak); Anal. Calcd. For C₂₇H₂₁N₅O₃ (m.w. 463.16): C, 69.97; H, 4.57; N, 15.11. Found: C, 70.10; H, 4.66; N, 15.00.

3.1.4. General procedure for synthesis of compounds 8a–c. TEA (0.1 g, 0.2 mL, 0.001 mol) was added to a suspension of 4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzoic acid (5) (0.35 g, 0.001 mol) in DCM (10 mL). The reaction mixture was stirred in an ice-salt bath for 10 min. To the clear solution, ethyl chloroformate (0.11 g, 0.1 mL, 0.001 mol) was added in a dropwise manner over a period of 20 min, and the reaction mixture was stirred for 1 h in the ice-salt bath. Then, a solution of the appropriate aliphatic amine (0.001 mol), namely, ethylamine, n-hexylamine and/or cyclohexylamine and TEA (0.1 g, 0.2 mL, 0.001 mol) in DCM (10 mL) was added to the previous mixture. The whole mixture was stirred overnight at r.t., and the obtained precipitate was filtered, washed with water followed by hot ethanol, and dried to provide the corresponding amide derivatives 8a–c, respectively.
3.1.4.1. N-Ethyl-4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzamide (8a). Yield 83%; m.p. 210–2 °C; IR_νmax (cm⁻¹): 3400 (NH), 3066 (CH aromatic), 2981 (CH aliphatic), 1714 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 1.18 (t, 3H, CH₂CH₃), 2.18 (s, 3H, CH₃), 3.30–3.35 (q, 2H, CH₂CH₃), 7.41–7.44 (m, H, H-4 of C₆H₅), 7.52–7.64 (m, 4H, H-2, H-6 of C₆H₄CO and H-3, H-5 of C₆H₅), 8.00–8.03 (m, 2H, H-3, H-5 of C₆H₄CO), 8.09–8.15 (m, 2H, H-2, H-6 of C₆H₅), 8.36 (s, 1H, CH of pyrazole), 8.64 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆): 14.62, 25.12, 34.63, 105.88, 122.15 (2), 127.60 (2), 128.93 (2), 129.78 (2), 135.87, 136.82, 138.73, 140.30, 142.35, 144.53, 157.89, 158.93, 165.70; MS (m/z): 373.21 (M⁺, 15.17%), 343.47 (88.40%), 264.30 (53.38%), 180.37 (100%, base beak); Anal. Calcd. For C₂₁H₁₉N₅O₂ (m.w. 373.15): C, 67.55; H, 5.13; N, 18.76. Found: C, 67.48; H, 4.98; N, 18.69.
3.1.4.2. N-Hexyl-4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzamide (8b). Yield 81%; m.p. 233–5 °C; IR_νmax (cm⁻¹): 3308 (NH), 3066 (CH aromatic), 2986 (CH aliphatic), 1714, 1649 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 1.14–1.22 (t, 3H, CH₂CH₃), 1.31–1.38 (m, 4H, CH₂CH₂), 1.75–1.85 (m, 4H, CH₂CH₂), 2.18 (s, 3H, CH₃), 3.05–3.09 (t, 2H, NHCH₂CH₂), 7.43–7.45 (m, H, H-4 of C₆H₅), 7.52–7.64 (m, 4H, H-2, H-6 of C₆H₄ and H-3, H-5 of C₆H₅), 8.00–8.03 (m, 2H, H-3, H-5 of C₆H₄), 8.09–8.11 (m, 2H, H-2, H-6 of C₆H₅), 8.37 (s, 1H, CH of pyrazole), 9.15 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆): 14.56, 19.06, 22.04, 24.42, 28.06, 31.61, 36.73, 110.95, 121.77 (2), 122.23 (2), 126.42, 126.83, 128.43, 129.08 (2), 129.66 (2), 136.33, 140.24, 142.16, 150.20, 165.46, 167.06; Anal. Calcd. For C₂₅H₂₇N₅O₂ (m.w. 429.22): C, 69.91; H, 6.34; N, 16.31. Found: C, 70.00; H, 6.30; N, 16.25.
3.1.4.3. N-Cyclohexyl-4-(6-methyl-4-oxo-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)benzamide (8c). Yield 77%; m.p. 217–9 °C; IR_νmax (cm⁻¹): 3227 (NH), 3077 (CH aromatic), 2930 (CH aliphatic), 1715, 1633 (2C=O); ¹H NMR (400 MHz, DMSO-d₆): 1.10–1.20 (m, 2H, CH₂), 1.30–1.40 (m, 4H, CH₂CH₂), 1.62–1.85 (m, 4H, CH₂CH₂), 2.19 (s, 3H, CH₃), 3.79 (m, H, NHCH), 7.42–7.45 (m, 2H, H-2, H-6 of C₆H₄), 7.53–7.55 (m, H, H-4 of C₆H₅), 7.58–7.64 (m, 3H, H-3, H-5 of C₆H₅ and NH, D₂O exchangeable), 7.99–8.16 (m, 4H, H-3, H-5 of C₆H₄ and H-2, H-6 of C₆H₅), 8.37 (s, 1H, CH of pyrazole); ¹³C NMR (100 MHz, DMSO-d₆): 14.61, 25.09 (2), 25.37, 32.84 (2), 61.57, 105.85, 122.17 (2), 127.62, 128.91 (2), 129.10 (2), 129.79, 130.92, 135.94, 136.83 (2), 138.70, 151.20, 157.93, 158.95, 165.15; MS (m/z): 427.34 (M⁺, 19.19%), 395.49 (100%, base beak), 371.64 (44.64%), 281.49 (74.54%), 95.60 (86.91%), 57.12 (74.21%), 43.01 (66.11%); Anal. Calcd. For C₂₅H₂₅N₅O₂ (m.w. 427.20): C, 70.24; H, 5.89; N, 16.38. Found: C, 70.30; H, 5.95; N, 16.25.

4. Conclusion

In conclusion, the newly designed pyrazolo[3,4-d]pyrimidine derivatives were synthesized and tested against HepG2, A549, MCF-7 and HCT-116 as dual VEGFR-2 and EGFR^T790M inhibitors. The proposed mode of their interactions with VEGFR-2 and EGFR^T790M was investigated using molecular design. The data from the docking studies and the results of the biological screening were excellently correlated. In general, derivatives 7c, 8b, 7e and 8c exhibited very good anticancer effects, with IC₅₀ values of 5.75, 6.20, 6.55 and 7.10 µM, respectively, against A549. Derivatives 6, 7a, 7b, 7d and 8a, with IC₅₀ values of 10.30–13.00 µM, showed good cytotoxicity. Against HCT-116, derivatives 7c, 8b, 7e, 8c, 7d and 7b, with IC₅₀ values of 5.50, 5.80, 6.15, 7.00, 9.40 and 9.50 µM, respectively, exhibited very good anticancer effects. Derivatives 6, 7a and 8a, with IC₅₀ values of 12.45, 10.70, and 10.25 µM, respectively, showed good cytotoxicity. Concerning MCF-7, derivatives 7c, 8b, 7e and 8c exhibited very good anticancer effects with IC₅₀ values of 5.90, 6.40, 7.00 and 7.90 µM, respectively. Derivatives 6, 7a, 7b, 7d and 8a, with IC₅₀ values of 10.25–12.75 µM, showed good cytotoxicity. Regarding HepG2, derivatives 7c, 8b, 7e and 8c, with IC₅₀ values of 5.00, 5.30, 5.75 and 8.80 µM, respectively, exhibited very good anticancer effects. Derivatives 6, 7a, 7b, 7d and 8a showed good cytotoxicity, with IC₅₀ values of 10.50–14.70 µM. Moreover, the six extremely viable structures 7b, 7c, 7d, 7e, 8b and 8c were tested against VERO normal cell lines to estimate their cytotoxic capabilities. All the results confirm that each of the compounds exhibit poor toxicity toward normal VERO cells, with IC₅₀ values ranging from 40.00 to 53.99 µM. Furthermore, all the derivatives 5–8a–c were tested as VEGFR-2 and EGFR^T790M inhibitors. The most active derivatives, which showed very good inhibition against VEGFR-2, were found to be derivatives 7c, 8b, 7e and 8c, with IC₅₀ values of 0.90 to 1.25 µM. The IC₅₀ for compounds 7a, 7b and 7d were 1.50–1.95 µM and showed good activity. In addition, derivatives 7c, 8b, 7e, 7b and 8c, with IC₅₀ values of 0.25, 0.32, 0.35, 0.45 and 0.50 µM, respectively, showed very good EGFR^T790M inhibition. Furthermore, compound 7d, with IC₅₀ = 0.60 µM, showed good inhibition activity. The ADMET profiles for the three most active compounds 7c, 7e and 8b, were computed in silico, using sorafenib and erlotinib as reference medicines, and they presented excellent ADMET profiles.

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

The data supporting this article are available upon request.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra00037a.

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