1 Introduction

Globally, cancer is the second highest cause of death after cardiovascular diseases. Cancer-related deaths have escalated from 8.2 million in 2012 (Ferlay et al., 2015) to 8.7 million in 2015 and 9.5 million in 2018 (Fitzmaurice et al., 2017). In the absence of effective interventions, cancer-related mortality would rise to an estimated 13 million and 16 million deaths among cancer patients by 2030 and 2040, respectively (The International Agency for Research on Cancer, IARC). Cancer is a disorder of abnormal cell growth with the potential of spreading into or invading nearby tissues and organs. Despite many treatment for cancer, chemotherapy is considered the mainstay of cancer therapy. However, chemotherapy has some limitations such as limited efficiency, selectivity, high cast, genotoxicity, and drug resistance. It also produces side effects as a result of damage to normal body cells and organ toxicities which reduced the quality of life in cancer patients (Tang et al., 2017). Despite the advances in chemotherapy, there are no agents that selectively target the cancer cells. This has confirmed the urge to develop new chemotherapeutic agents which are more selective for cancer cells, and thus produce fewer side effects (Al-Anazi et al., 2018; Slaihim et al., 2019; Khairuddean et al., 2020; Salum et al., 2020; Al-Anazi et al., 2021; Mohammad, et al., 2021a; Mohammad, et al., 2021b; Mohammad, et al., 2022; Jumaah et al., 2022). Tyrosine kinase protein-inhibitors are of great interest due to their therapeutic capacity in treating a variety of diseases, especially cancer (Backes et al., 2008a; 2008b; Gschwind et al., 2004).

Among protein tyrosine kinase, the epidermal growth factor receptor (EGFR) has emerged as a key and main target for the development of new anti-cancer agents (Speake et al., 2005; Warnault et al., 2013; Yewale et al., 2013). EGFR kinase inhibition in cancer treatment is performed by blocking this enzyme with small molecules (drugs). To date, over 10 EGFR inhibitors have been approved by the United States Food and Drug Administration (US-FDA) in the past two decades. Some of these inhibitors include Erlotinib (Tarceva®), Gefitinib (Iressa®) and Lapatinib (Tykerb) which is also known as GW-572016, with quinazoline-based derivatives (Dowell et al., 2005; Ganjoo and Wakelee, 2007; Stamos et al., 2002). Although the therapeutic effects of the quinazoline-based agents on different cancers have been well established, many side effects such as skin rashes, diarrhea, vomiting, nausea and hemorrhage were also reported (Sequist and Lynch, 2008). Clearly, as anticancer agents, it is necessary to discover drugs with minimum adverse effects that provide much more hope for mankind. Based on the previously cited studies, the exploration of EGFR with newly synthesized hybrids is a fruitful and fertile research area. It was reported that compounds with chalcone moiety, a class of secondary metabolites of flavonoids exhibit various biological activities, especially as anticancer agents (Karthikeyana et al., 2015; Alman et al., 2020). In addition, nitrogen-containing heterocycles have become important in the development of new effective drugs for the treatment of cancer (Kerru et al., 2020). Nitrogen-based heterocyclic compounds constitute nearly 60% of all drugs approved by the FDA and represent about 73% of the approved anticancer drugs in 2015 (Martins et al., 2015; Hosseinzadeh et al., 2018). Compounds with heterocyclic rings such as pyrazoline (Bhutani et al., 2015; Vincent et al., 2020) and pyrimidine (Kaur et al., 2015; Kumar and Narasimhan, 2018; Mahapatra et al., 2021) have demonstrated a wide variety of promising biological activities especially for anticancer.

Nitrogen-containing compounds, especially pyrimidines, have been screened as potential anti-cancer agents for clinical use because pyrimidine plays an important role in the synthesis of nucleic acid (Parker, 2009). The synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) depends on several pyrimidine derivatives including adenine, guanine, cytosine, thymine, and uracil. In this study, in continuation of the EGFR inhibitors as anticancer agents (Al-Anazi et al., 2018; Al-Anazi et al., 2021), compounds with pyrimidine rings were proposed. The molecular docking and synthesis of a series of pyrazoline and pyrimidine derivatives as anticancer agents are reported. The characterization of these compounds was confirmed using spectroscopic techniques such as FTIR, ¹H NMR, ¹³C NMR and elemental analysis (CHN analysis). Consequently, the biological activity of the synthesized compounds was also evaluated against the EGFR kinase enzyme, and breast cancer cell lines.

2 Results and discussion

2.1

2.1 Molecular docking

Molecular docking is an important tool in the drug discovery toolbox which predicts the orientation, interaction and binding energies of ligands in their targeted binding sites. Currently, AutoDock 4.2 is reported to be the most popular molecular docking program. Its high accuracy and versatility have expanded its application (Sousa et al., 2006; Meng et al., 2011; Al-Najjar, 2018b). Compounds with chalcone, pyrazoline and pyrimidine moieties were successfully docked against the 3POZ crystal structure, and the results are shown in Table 1. TAK-285 is a novel investigational small molecule (N-[2-[4-[3-chloro-4-[3-(trifluoromethyl)phenoxy] anilino]pyrrolo[3,2-d]pyrimidin-5-yl]ethyl]-3-hydroxy-3-methylbutanamide) which inhibits EGFR and specifically targets HER2, such as lapatinib. It has antiproliferative activity in vitro and in vivo cytotoxic activity against cancer cells (Ishikawa et al., 2011) and was used as a control ligand.

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Table 1 The lowest binding energies from AutoDock 4.2 and interacting amino acids for the pyrazoline and pyrimidine derivatives.

Compound	Binding energy (Kcal/mol)	Interacting amino acids	Compound	Binding energy (Kcal/mol)	Interacting amino acids
TAK-285	−10.15	MET 793, ARG 776, LEU 777, THR790, ARG 841, THR 854,
Chalcone
1	−10.91	MET 793, LYS 745	5	−10.31	MET 793
2	−8.27	MET 793	6	−9.63	MET 793
3	−10.76	MET 793	7	−8.23	LEU 777
4	−10.19	MET 793, LEU 777
Pyrazoline
1Ai	-0.8.49	MET 766, MET 793	4Ai	−7.81	MET 793
1Aii	-0.8.16	MET 793, ASP 855	4Aii	−8.82	LYS 745, MET 793
1Aiii	−9.16	LYS 745, MET 793, ASP 855	4Aiii	−8.80	LYS 745, MET 793, ASP 855
3Ai	−8.26	MET 793, ASP 855	5Ai	−7.66	MET 793
3Aii	−8.80	MET 793, ASN 842, ASP 855	5Aii	−7.32	ASP 855
3Aiii	−8.92	LYS 745, LEU 788, MET 793	5Aiii	−8.92	LYS 745, MET 793, ASN 842
Pyrimidine
1Bi	-0.9.32	MET 793, ASN 842, ASP 855	4Bi	−9.41	MET 793, ASN 842, ASP 855
1Bii	-0.9.22	MET 793, ASN 842, ASP 855	4Bii	−9.43	MET 793, ASN 842, ASP 855
1Biii	−8.96	MET 793, ASN 842, ASP 855	4Biii	−8.40	LYS 745, MET 793, ASP 855
3Bi	−9.23	MET 793, THR 854, ASP 855	5Bi	−7.82	MET 793, ASP 855
3Bii	−7.41	LEU 788, ARG 841	5Bii	−9.71	MET 793, ASN 842, ASP 855
3Biii	−9.42	MET 793, THR 854, ASP 855	5Biii	−9.15	MET 793, ASN 842, ASP 855

Structures of TAK-285 and new chalcone, pyrazoline and pyrimidine derivatives.

The results of the docking score for the chalcone were nearly similar to TAK-285, except for compounds 2 and 7, where the modified extra chains did not affect the affinity of binding inside the pocket. Likewise, compound 6 was not selected for further investigation because of the environmental toxicity, carcinogenicity and mutagenicity of the aromatic nitro compound (Kovacic and Somanathan, 2014). On the other hand, most of the proposed pyrazoline and pyrimidine derivatives have shown good binding energies, ranging from −9.71 to −7.32 kcal/mol. Compound 5Bii revealed the lowest binding energy of −9.71 kcal/mol, while, 5Aii showed the highest binding energy of −7.32 kcal/mol.

Intermolecular interactions of the docked compounds are shown in Supplementary. Fig. 1 shows the binding of TAK-285 within the adenosine triphosphate (ATP) binding pocket of the catalytic tyrosine kinase domain, competing with ATP. Several studies have recently stated that the formation of a hydrogen bond with the ATP binding site of EGFR (MET 793 and/or LYS 745) helps to inhibit its activity (Aertgeerts et al., 2011; Mahajan et al., 2017; Al-Anazi et al., 2021), which can be seen in all of the proposed compounds except in compounds 7, 5Aii and 3Bii. ASN 842 performed conventional hydrogen bonds with TAK-285, matching with compounds 3Aii, 5Aiii, 1B(i-iii), 4B(i-ii) and 4B(ii-iii), by hydrogen bonds interaction. However, amino acid residue LYS 745 has performed a hydrophobic interaction with TAK-285 but formed conventional hydrogen bonds with compounds 1Aiii, 3Aiii, 4Aii, 4Aiii, 5Aiii and 4Biii. Therefore, based on the binding energy study of all the designed compounds, it was decided to synthesize compounds 1, 3, 4 and 5 of chalcone derivatives and some heterocyclic derivatives of pyrazoline (Ai-iii) and pyrimidine (Bi-iii). These compounds have shown good binding energies and strong interactions in the targeted active site.

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Fig. 1

2D intermolecular interactions between docked (TAK-285) and 3POZ protein. Green and pink colored amino acids represent their contribution to hydrogen bonds and hydrophobic interactions.

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3 Chemistry

The series of pyrazoline, (1,3–5)A(i-iii) and pyrimidine, (1,3–5)B(i-iii) derivatives were synthesized from cyclization reactions of chalcone compounds 1, 3–5, as shown in Scheme 1. All the synthesized compounds were confirmed using FT-IR, NMR spectrometers and CHN elemental analysis.

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Scheme 1

Synthesis pathway of chalcone, pyrazoline and pyrimidine derivatives.

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The IR spectra manifested the presence of the diagnostic bands. For example, in compounds 1Aiii and 1Bii, the absorption bands were observed at υ 3070 (C_sp²-H str), 2914–2910 and 2847–2840 cm⁻¹ for (C_sp3-H of the asymmetrical and symmetrical str), 1608–1605 (C = N str), 1553–1521 (C = C str), and 1250–1230 (C-N str) cm⁻¹, respectively. The absorption band at υ 1637–1644 cm⁻¹ was attributed to C = O stretching of the N-acetyl derivative, Aiii and the band at υ 2590–2530 cm⁻¹ was attributed to the C-SH stretching of the 4,6-diphenyl-pyrimidine-2-thiol derivative, Bii. The IR spectra for derivatives Ai-ii, Bi and Biii showed little differences compared to Aiii and Bii compounds. This is due to the disappearance of the stretching absorption bands of the C = O, C-SH and the appearance of the absorption bands of the NH and NH₂ moieties in derivatives Ai-ii and also the appearance of the absorption bands of the NH₂ and OH moieties in derivatives Bi and Biii, respectively, as shown in the Supplementary Figure S2.

The ¹H NMR spectra supported the diagnostic tools for the positional elucidation of the protons. The assignment of the characteristic signals is based on the chemical shifts and intensity patterns. In all the pyrazoline derivatives, two protons of H^a and H^b at C-4 of the pyrazoline ring, which is geminal, were observed and appeared as two doublets of doublets at δ_H 2.86–3.35 and 3.47–3.96. A proton at C-5 of the pyrazoline ring also appeared as a doublet of doublets at δ_H 4.88–6.12 due to the vicinal coupling with two nonequivalent germinal protons of C-4 carbon. However, this proton was observed to be more downfield than those of H^a and H^b in all the products, due to its proximity to a benzene ring. The coupling constants for two nonequivalent geminal protons become smaller (0–3 Hz), as predicted when the HCH angle becomes larger. So, in this study, the small changes in bond angles resulting from stereochemical changes influence the geminal coupling constant (>17 Hz). That means coupling constant become larger when the HCH angle become smaller (a = 120°, ²J_HH = 0–3 Hz & a = 107°, ²J_HH = 17.5 Hz) (Pavia et al., 2014). A singlet centered at δ_H 7.24 and δ_H 7.83 were consistent with the formation of a pyrimidine ring in 2,4,6-trisubstituted pyrimidine derivatives of Bi-iii.

Both the pyrazoline and pyrimidine derivatives exhibited aromatic protons in the region of δ_H 8.82–9.27 and δ_H 9.11–9.25. In all the compounds, a singlet was assigned to H^6′ and H^7′ of the terminal pyrimidine ring. Signals for NH (Ai), NH₂ (Aii, Bi), SH (Bii), acetyl-CH₃ (Aiii) and OH (Biii) protons resonated as a singlet at δ_H 7.61–10.23, 7.23–9.82, 6.72–6.83, 9.25–9.29, 2.34–2.46 and 7.36–5.42, respectively. The aromatic protons were observed within the expected region along with the integral values. The ¹H NMR spectrum of compound 1Aiii (Supplementary Figure S2.13(b)) showed two signals for acetyl-CH₃ and methoxy-H^5′' protons which resonated as a singlet at δ_H 2.36 and δ_H 3.78, respectively. The presence of the pyrazoline ring was proven by the diastereotopic protons of H^a and H^b on C-4 which appeared as a pair of doublets at δ_H 3.09 and δ_H 3.71 while the proton at C-5 was observed as a doublet of doublets at δ_H 5.61.

The ¹H NMR spectrum of 1Bii pyrimidine derivative (Supplementary Figure S2.21(b)) has shown a signal for methoxy-H^5′' proton which resonated as a singlet at δ_H 3.86. The presence of 2,4,6-trisubstituted pyrimidine ring was proven by proton H⁵ which appeared as a singlet at δ_H 7.42. In both derivatives, 1Aiii and 1Bii, the signals for protons H^6′ and H^7′ of the terminal pyrimidine ring appeared as a singlet at δ_H 8.82–9.16 and δ_H 9.11–9.18. Lastly, the remaining signals were observed as the aromatic protons which resonated as doublets at δ_H 7.32–828 (H^2′), δ_H 7.45–7.87 (H^3′), δ_H 7.62–8.11 (H^2′') and δ_H 6.87–7.02 (H^3′').

The ¹³C NMR spectra of all derivatives (Ai-iii and Bi-iii) (Supplementary Figure S2.(13,21)c) have confirmed the absence signals of trans-alkene carbons and the appearing signals of the pyrazoline ring which were observed at δ_C 37.5–43.0 and δ_C 54.9–63.8, attributed to C-4 and C-5, respectively. On the other hand, the appearing signal at δ_C 94.8–106.4 was attributed to C⁵ on the pyrimidine ring, Other characteristic aromatic carbons have appeared in the expected region.

Furthermore, the carbon skeleton of compound 1Aiii was confirmed by ¹³C NMR which showed the aliphatic carbons at δ_C 21.9, 55.4, 42.3, 59.5 for the acetyl-CH₃, methoxy-C^5′', C-4 and C-5 of pyrazoline, respectively. The carbonyl carbon was observed at δ_C 168.8 and the aromatic carbons appeared in the range of δ_C 161.4–114.2. Likewise, the carbon skeleton of compound 1Bii was confirmed by ¹³C NMR that showed the aliphatic carbon at δ_C 55.6 for the methoxy-C^5′', C-5 for the 2,4,6-trisubstituted pyrimidine ring at δ_C 101.9 and the aromatic carbon at δ_C 114.1–189.5. The DEPT-90 and DEPT-135 NMR spectra (Supplementary Figure S2.(13,21)d,e) have confirmed all the methine (CH), methylene (CH₂), methyl (CH₃) and quaternary carbons (C) in both of 1Aiii and 1Bii. The extreme downfield signals at δ_C 168.8 and δ_C 189.5 were assigned to the carbonyl carbon and C² (C-SH) carbon of 1Aiii and 1Bii, respectively. The extreme upfield signals at δ_C 59.5, 55.4, 42.3 and 21.9 were assigned to the methine (C⁵), methoxy carbon (C^5′'), methylenic (C⁴) which appeared as a negative signal in the DEPT-135) and methyl carbon of the acetyl moiety in 1Aiii, respectively. Similarly, for derivative 1Bii, the extreme upfield signal at δ_C 55.6 and δ_C 101.9 were assigned to the methoxy (C^5′') and the methine (C⁵) carbons of the pyrimidine ring, respectively. The quaternary carbons (C^4′', C^1′', C³, C^1′, C^4′, C^5′) and (C^4′', C^1′', C⁴, C², C⁶, C^1′, C^4′, C^5′) in 1Aiii and 1Bii, respectively, were identified through their absence in the DEPT-90 and DEPT-135 spectra.

The 2D-NMR correlation using ¹H–¹H COSY (Supplementary Figure S2.29) and ¹H–¹³C HSQC (Supplementary Figure S2.30) spectra were used for unambiguous assignment. The ¹H–¹H COSY spectrum of pyrazoline derivative 1Aiii showed the correlations of H^4a with H^4b, H^4a with H⁵, H^4b with H^4a, H^4b with H⁵, H⁵ with H^4b, H⁵ with H^4a, H^3′' with H^2′', H^2′ with H^3′, H^3′ with H^2′ and H^2′' with H^3′'. The ¹H–¹H COSY spectrum of pyrimidine derivative 1Bii has shown the correlations of H^3′' with H^2′', H^3′ with H^2′, H^2′' with H^3′' and H^2′ with H^3′. On the other hand, the ¹H–¹³C HSQC spectrum has revealed the correlations between the proton and carbon and confirmed the assigned structure of pyrazoline 1Aiii and pyrimidine 1Bii. In pyrazoline 1Aiii, a cross peak was observed between both protons H^4a and H^4b at δ_H 3.09 and δ_H 3.71 with the methylene carbon C⁴ (CH₂) at δ_C 42.3. In addition to this, two protons at δ_H 3.78 and δ_H 2.36 have shown two cross-peaks with the aliphatic carbons of C^5′' and CH₃-CO at δ_C 55.4 and δ_C 21.9, respectively. Also, a cross-peak of the pyrimidine derivative was observed between proton H⁵ at δ_H 7.59 with methine carbon C⁵ (CH) at δ_C 101.9. In addition to this, a proton at δ_H 3.83 has shown a cross-peak with the aliphatic carbon (C^5′') at δ_C 55.6. Furthermore, two protons of H^6′ and H^7′ of the terminal pyrimidine ring in derivatives 1Aiii and 1Bii have appeared at δ_H 8.82–9.25 and δ_H 9.11–9.21 which showed two cross-peaks for the methine carbons at δ_C 155.2–154.8 and δ_C 157.4–157.8, respectively. The remaining four aromatic protons H^3′', H^2′, H^3′, H^2′' appeared at δ_H 6.87–7.03, δ_H 7.32–8.33, δ_H 7.45–7.95 and δ_H 7.62–8.16, showing a cross-peak with methine carbon at δ_C 114.1–114.2, δ_C 126.8–128.0, δ_C 127.3–127.6 and δ_C 128.2–128.7, respectively. Finally, All the carbons were determined and figuratively represented in Supplementary Figure S2.

4 In-vitro assays

4.1

4.1 Recombinant EGFR kinase assay

To confirm the molecular docking results of the ability of compounds 4Aiii and 5Bii to bind and inhibit EGFR kinase, the luminescence of ATP conversion was measured using ADP-Glo^TM kinase assay at two different concentrations of 50 and 0.19 μM. Results indicate a significant inhibition of the recombinant kinase when incubated with the inhibitors. Compound 4Aiii has managed to inhibit the enzyme with 83% and 82% of the inhibition rate, respectively for both concentrations. On the other hand, compound 5Bii showed an inhibition rate of 89% at the concentration of 50 μM. However, when the concentration drops to 0.19 μM its inhibition rate falls to 72% which is still considered as a significant inhibitory activity (Fig. 2). This goes consistently with the molecular docking results where both compounds gave a very comparable binding affinity and the same number of interactions with the surrounding amino acids seated in the EGFR binding pocket. Conceptually, this indicates the high potential of these pyrazoline and pyrimidine derivatives to be further investigated for their true cytotoxic activity.

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Fig. 2

Recombinant kinase activity measured using ADP-Glo^TM for compounds 4Aiii and 5Bii at three different concentrations (0, 0.19 and 50 μM).

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5 Cytotoxicity assay

The newly synthesized chalcone, pyrazoline and pyrimidine derivatives were evaluated for their in-vitro cytotoxic activity (0–100 μg/mL) against two human breast cancer cell lines (MCF7 and MDA-MB-231) and one non-cancerous breast (MCF-10A), with the aid of tamoxifen as reference drugs. The concentration required for 50% inhibition of cell viability (IC₅₀) alongside the selectivity index (SI) was calculated and is presented in Table 2.

In general, results showed that pyrazoline derivatives have better anti-cancer potentials compared to the pyrimidine-derived entities. For the chalcone attached to the pyrimidine ring (compounds 1, 3, 4 and 5, Supplementary Figure S3.1), they displayed different cytotoxic activity according to the attached substituent, in the order of C₆H₅- > thiophene ring > C₆H₄-OMe > C₆H₄F against MCF-7. Likewise, for the MDA-MB-231 cell line, all chalcone compounds (1, 3–5) exhibited moderate cytotoxic (IC₅₀: 100 µM) activity. Compounds 5 also displayed a high cytotoxic selectivity of 3.66 compared to tamoxifen. On the other hand, compounds 1, 3, and 4 exhibited a lower degree of cytotoxic selectivity against MCF-7, which shows increased cytotoxicity toward the healthy cell.

Among the pyrazoline derivatives (Supplementary Figure S3.2–3), 5Ai showed the lowest IC₅₀ value (26.30 ± 0.61 μM) which was consistent with tamoxifen IC₅₀ (26.95 ± 3.01 μM), indicating an excellent cytotoxic activity. Also, 5Ai showed a high cytotoxic selectivity index toward MCF-7 avoiding massive toxicity of normal cells (MCF-10A). This enhanced the activity of 5Ai which might be due to the presence of 1H and the 3-phenyl within the pyrazole ring. Another pyrazoline, 4Ai (IC₅₀: 40.27 ± 1.08, SI: 2.48) has 1H and 3-(p-fluorophenyl) while pyrazoline 4Aii (IC₅₀: 38.01 ± 2.96, SI: 2.63) has 1-thioamide and 3-(p-fluorophenyl) within the ring. Remarkably, pyrazoline 1Aiii was the only compound that reasonably appears to be effective against the MDA-MB-231 cell line (IC₅₀: 51.29 ± 6.75 μM, SI: 1.95) with 1-ethanone and 3-(p-methoxyphenyl).

On the other hand, compound 3Bi (Supplementary Figure S3.2–3) was the only pyrimidine derivative that expressed the best cytotoxic activity. This compound has conveyed a superior activity against the MCF-7 cell line (IC₅₀: 5.5 ± 0.07 μM, SI: 18.18) compared to the other tested pyrimidine derivatives including the tamoxifen standard which 3Bi topped it with magnitudes of selectivity and specificity. The superior activity of 3Bi was due to the presence of thiophen-2-yl over position no. 4 of the pyrimidine ring.

6 Conclusion

Several chalcone, pyrazoline and pyrimidine derivatives which may function as EGFR inhibitors were studied. Some selected compounds of pyrazoline (1,3–5)A(i-iii) and pyrimidine (1,3–5)B(i-iii) derivatives with promising EGFR inhibitory activity were synthesized and characterized using spectroscopic techniques such as FTIR, ¹H NMR, ¹³C NMR and elemental analysis (CHN analysis). These synthesized compounds were investigated in vitro for their activity against the EGFR kinase enzyme, then for their anti-proliferative and cytotoxic activities using multiple cell lines of the breast. Interestingly, the molecular docking study revealed that the chalcone, pyrazoline and pyrimidine derivatives have good binding affinities towards EGFR kinase. These results are consistent with their effect on the enzyme where dropping the concentration of the pyrazoline compound 4Aiii did not affect its inhibitory activity. Moreover, the cytotoxic activity of pyrazoline derivatives against breast cancer cell lines revealed that 5Ai is the best-proposed candidate against hormonal breast cancer, while 1Aiii is the most potent against non-hormonal breast cancer. On the other hand, only one compound of the pyrimidine derivatives exhibited excellent activity against hormonal breast cancer while other pyrimidine derivatives appeared to be active against non-hormonal breast cancer. Further investigations and studies are encouraged to test these compounds against other cancer cell lines and other possible modifications can also be done to improve their activity.

7 Materials and methodology

8 Biological assay