Discovery of novel thiazolyl-pyrazolines as dual EGFR and VEGFR-2 inhibitors endowed with in vitro antitumor activity towards non-small lung cancer

Abstract

New series of thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) have been synthesised and assessed for their potential EGFR and VEGFR-2 inhibitory activities. Compounds 10b and 10d exerted potent and selective inhibitory activity towards the two receptor tyrosine kinases; EGFR (IC₅₀ = 40.7 ± 1.0 and 32.5 ± 2.2 nM, respectively) and VEGFR-2 (IC₅₀ = 78.4 ± 1.5 and 43.0 ± 2.4 nM, respectively). The best anti-proliferative activity for the examined thiazolyl-pyrazolines was observed against the non-small lung cancer cells (NSCLC). Compounds 10b and 10d displayed pronounced efficacy against A549 (IC₅₀ = 4.2 and 2.9 µM, respectively) and H441 cell lines (IC₅₀ = 4.8 and 3.8 µM, respectively). Moreover, our results indicated that 10b and 10d were much more effective towards EGFR-mutated NSCLC cell lines (NCI-H1650 and NCI-H1975 cells) than gefitinib. Finally, compounds 10b and 10d induce G2/M cell cycle arrest and apoptosis and inhibit migration in A549 cancerous cells.

Graphical Abstract

Keywords:

• Anticancer
• molecular docking
• EGFR inhibitors
• VEGFR-2 inhibitors
• EGFR-mutated NSCLC
• dual kinase inhibitors

1. Introduction

Cancer remains as a major health problem and a life-threatening disease even after decades of fundamental biomedical advances leading to the development of a war chest of anti-tumour therapies. This is largely because of the ability of cancer cells to develop resistance against most antitumor agents¹.

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths in both men and women accounting for about 26% of all cancer deaths². It has been shown that epidermal growth factor receptor (EGFR), the prototypical growth factor receptor tyrosine kinase (RTK)³, is one of the most important key players in the development of many lethal cancers globally^4,5 including: colorectal cancers, ovarian cancers⁶, breast cancers⁷, and non-small-cell lung cancers (NSCLC) in which EGFR overexpression takes place in approximately 43–89% of all cases and relates with diminutive survival and chemoresistance⁸. Accordingly, interruption of the EGFR signalling pathway using small-molecule tyrosine kinase inhibitors (TKIs) is considered as a well-established therapeutic approach in cancer treatment since the approval of gefitinib by the United States Food and Drug Administration (FDA) in 2003^9,10.

Gefitinib and erlotinib (Figure 1) are first-generation EGFR inhibitors that have been approved for the treatment of advanced NSCLC with activating mutations in the EGFR tyrosine kinase domain mainly: L858R (EGFR^L858R)^11–14. However, some patients develop secondary drug resistance mutations which results in a relapse after nearly one year of treatment¹⁵. The “gatekeeper” mutation is the predominant one which is a single point substitution of Thr790 with Met in exon 20 (EGFR^T790M)¹⁶. Hence, afatinib and dacomitinib (Figure 1) have been developed as irreversible second generation EGFR inhibitors^17,18 because they have electrophilic Michael-acceptor systems, such as acrylamide moiety which forms a covalent bond with Cys797¹⁹. This resulted in blocking of T790M resistance mutation by increasing target residence time²⁰.

Figure 1. First-, second- and third generation EGFR inhibitors and dual EGFR/VEGFR-2 inhibitor.

Despite the promising in vitro results in patients with erlotinib-resistant cancers²¹, they showed poor efficacy at clinically achievable concentration²². This is due to the narrow therapeutic window, as the dose required for the inhibition of mutated EGFR^T790M resulted in severe side effects as rash and diarrhoea from inhibiting EGFR^wt23. Recently, this problem was solved by the discovery of third generation EGFR inhibitors as osimertinib (Tagrisso®, AZD9291), rociletinib (CO-1686), naquotinib (ASP8273) and nazartinib (EGF816)^24,25 (Figure 1), however, a resistance has been developed too^26–28. So, exploring more efficient EGFR inhibitors is an urgent and critical demand to surmount the continual evolution of resistance to the current inhibitors.

Besides EGFR, there are other RTKs that have been shown to be important targets in cancer²⁹. For example, the RTK vascular endothelial growth factor receptor-2 (VEGFR-2) stands out as a key target in cancer treatment due to its important role in angiogenesis³⁰. Based on the functional crosstalk of EGFR and VEGFR-2 through shared common downstream signalling pathways³¹, it is inferred that the simultaneous inhibition of both EGFR and VEGFR is an effective approach for overcoming the reported resistance in NSCLC³². In fact, it has been shown that VEGFR-2 inhibition enhances the cytotoxic effect of EGFR inhibitors, whereas, VEGFR-2 activation results in accelerated tumour growth independent of EGFR signalling, thereby facilitating the emergence of resistance to EGFR inhibitors³³. Vandetanib (Caprelsa^®) is an example of FDA approved quinazoline-based drug with dual EGFR and VEGFR-2 inhibitory activity³⁴ (Figure 1).

In the current medical era, thiazole is identified as an important heterocyclic motif that emerged as a promising privileged scaffold in the anticancer drug discovery^35–37, Several FDA approved anticancer drug incorporate the heterocycle thiazole, such as dabrafenib and dasatinib. Pyrazoline moiety, on the other hand, is a highly active heterocyclic nucleus possessing interesting biological activities³⁸ like; anticancer^39–41, anti-inflammatory⁴², antidepressant⁴³, anticonvulsant⁴⁴ and antimicrobial activities⁴⁵.

Recently, some research groups have devoted their efforts to hybridise both the thiazole and pyrazoline into a single frame work, producing novel series based on the thiazolyl-pyrazoline system which thereafter identified as a substantial scaffold in anticancer drug discovery⁴⁶. Interestingly, there is a large number of reported compounds with potent cytotoxic effect based on thiazolyl-pyrazoline scaffold having EGFR (compounds I, II and III, Figure 2)^47–49 and VEGFR-2 (compound IV, Figure 2)⁵⁰ suppressing potentials. It is worth mentioning that none of these studies have developed dual or multiple kinase inhibitors, in addition they have not evaluated the biological activity against EGFR-mutated cancer cell lines.

Figure 2. Structure of some reported thiazolyl-pyrazoline based EGFR and VEGFR-2 inhibitors (I–IV), and the target compounds in this study (7a–7d, 10a–10d and 13a–13f).

Motivated by the above mentioned findings, here we sought to develop new different sets of novel thiazolyl-pyrazoline-based small molecules (7a–7d, 10a–10d and 13a–13f) as potential dual EGFR/VEGFR-2 inhibitors with improved anticancer activities based on the molecular hybridisation between thiazole and pyrazoline motifs (Figure 2).

It is worth to mention that the reported pharmacophoric features required for EGFR inhibition include a central flat hetero-aromatic ring scaffold that should occupy the adenine binding pocket, a terminal hydrophobic head that occupies the hydrophobic sub-pocket, as well as a hydrophobic tail to occupy a second hydrophobic region^51,52. Additionally, certain pharmacophoric features have been identified for the VEGFR-2 inhibitors including a flat hetero-aromatic moiety to be fitted in the ATP binding region, a terminal hydrophobic motif to achieve several hydrophobic interactions in the allosteric hydrophobic pocket, and the presence of H-bond acceptor (HBA) functionalities^53–55. Interestingly, the target thiazolyl-pyrazoline-based small molecules (7a–7d, 10a–10d and 13a–13f) achieved these features required for inhibition of both EGFR and VEGFR-2. Furthermore, the substitution pattern on the pendant phenyl moieties was selected so as to ensure different electronic and lipophilic environments which should manipulate the activity of the target thiazolyl-pyrazolines.

Once created, all derivatives were assessed for their potential inhibitory activity towards EGFR and VEGFR-2 kinases. Also, the inhibitory activities against a panel of 11 kinases (TEK, SYK, EPHB2, ABL1, LCK, CLK1, ROCK1, PKC, AKT1, CDK1, and CDK5) were further investigated to determine the selectivity profile. Then, all compounds were screened for their cytotoxic effect against nine cell lines derived from five tumour subpanels including leukaemia (K562, and KG-1a), breast (MCF-7, BT-549, and HCC70), lung (A549 and H441), colon (HCT116) and liver (HepG2) cancer cell lines. In addition, the anti-proliferative potential of the most potent EGFRWT inhibitors was analysed towards two EGFR mutated cancer cell lines; NCI-H1650 and NCI-H1975. Thereafter, the most efficient cells growth and kinase inhibitors 10b and 10d were selected to explore their possible cellular mechanism of action via cell cycle, apoptosis Annexin-V-FITC and migration assays in NSCLC A549 cells. Finally, in silico studies were carried out to explore the binding interactions of thiazolyl-pyrazolines 10b and 10d within the vicinity of ATP-binding sites on EGFR and VEGFR-2 kinase domains.

2. Results and discussion

2.1. Chemistry

The synthetic pathways adopted for preparation of the intermediates 5a–b and the target thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) were depicted in Schemes 1 and 2. In Scheme 1, chalcones 3a, b were prepared via reaction of 2,4-dichloroacetophenone 1 with the appropriate aromatic aldehydes 2a, b, adopting the base catalysed Claisen–Schmidt condensation reaction as reported⁵⁶ (Scheme 1). As active intermediates, chalcones 3a, b were used to prepare pyrazolines 5a, b by the reaction with thiosemicarbazide 4 in the presence of sodium hydroxide as a catalyst (Scheme 1). Pyrazoline formation occurs via two steps; the first one is the condensation reaction between the chalcones 3a, b and thiosemicarbazide 4, whereas, the second step is the intramolecular cyclisation through Michael addition of the NH group to the double bond⁵⁷ (Scheme 1). Structures of compounds 5a, b were confirmed by microanalyses and spectral data. IR spectra of 5a, b showed NH₂ bands at 3418, 3444 and 3247, 3322 cm⁻¹, respectively, in addition to two bands at 1236, 1254 and 828, 833 cm⁻¹ referring to C=S. ¹H NMR spectra of 5a displayed three doublets of doublets for H_A, H_M and H_X of the pyrazoline ring at δ 3.18, 4.03 and 5.87 ppm, respectively. This AMX pattern confirmed the formation of the pyrazoline ring and the presence of two diastereotopic protons at C-4 (H_A and H_M) and one single proton at C-5 (H_X)⁵⁸. Furthermore, ¹H NMR spectra of 5a, b appeared two D₂O exchangeable singlet signals of NH₂ around δ 7.75, 7.76 and 8.13 ppm, respectively. In addition, ¹³C NMR spectra of 5a, b revealed two signals for C-4 and C-5 carbons of pyrazoline ring at δ 45.7 and 63.0, 63.3 ppm, respectively.

Scheme 1. Synthetic pathway for preparation of intermediates 5a, b and compounds 7a–7d; reagents and reaction conditions: (i) 10% NaOH, EtOH, RT, 4 h; (ii) NaOH, EtOH, reflux, 6 h; (iii) EtOH, reflux, 4 h.

Scheme 2. Synthetic pathway for preparation of compounds 10a–10d and 13a–13f; reagents and reaction conditions: (i) EtOH, reflux, 4 h; (ii) SO₂Cl₂, dry Et₂O, 0–5 °C, 2 h; (iii) (a) NaNO₂-HCl, 0–5 °C, 2 h; (b) NaOAc.3H₂O, EtOH, 0–5 °C, 4 h; (iv) EtOH, reflux, 4 h.

Furthermore, reaction of carbothioamide derivatives 5a, b with 1-aryl-2-bromoethanones 6a, b in refluxing ethanol yielded the corresponding 4-(aryl)-2-(3-(2,4-dichlorophenyl)-5-(4-aryl)-4,5-dihydro-1H-pyrazol-1-yl)thiazoles 7a–7d, respectively, via Hantzsch thiazole synthesis^54,55 (Scheme 1). Initially, nucleophilic substitution of Br in phenacyl bromide by the S-atom of thioamide generates the isothiourea, which subsequently undergoes cyclocondensation and water elimination to furnish the thiazole ring⁵⁷ (Scheme 1). The latter synthesised compounds 7a–7d were confirmed based on their elemental analysis and spectral data. IR spectra showed the disappearance of the characteristic band of NH₂ in the region of 3418, 3444 and 3247, 3322 cm⁻¹. Their ¹H NMR spectra showed an increase in the aromatic integration due to thiazole proton and protons of the extra phenyl ring at δ 6.91–7.82 ppm. ¹³C NMR spectra of 7b, c showed peak around δ 105 ppm corresponding to C-5 of thiazole ring along with an increase in signals assigned for aromatic carbons at δ 111.6–164.8 ppm.

Similarly, thiazole derivatives 10a–10d were obtained from the reaction of the carbothioamides 5a, b with the appropriate α-chloro-1,3-dicarbonyl compounds 9a–9c in absolute ethanol through the Hantzsch thiazole synthesis^49,55 (Scheme 2). Compounds 10a–10d were confirmed based on their elemental analysis and spectral data.

IR spectra of 10a–10d showed a band at 1637–1669 cm⁻¹ due to C=O group, while the spectra of 10c, d revealed a band at 3257 and 3262 cm⁻¹, respectively corresponding to NH group. ¹H NMR spectra of 10a–10d showed a signal at δ 2.36–2.54 ppm attributed to the protons of the methyl group at the 4-position of the thiazole ring. Moreover, ¹H NMR spectrum of 10a showed a singlet signal at δ 2.40 ppm attributed to CH₃ protons of the acetyl group. Furthermore, ¹H NMR spectrum of 10b showed two signals at δ 1.35 and 4.29 ppm, respectively attributed to the CH₃ and CH₂ protons of the ethyl group. Additionally, ¹H NMR spectra of 10c, d displayed a D₂O exchangeable singlet signal of NH proton at δ 9.72, 8.14 ppm, respectively, in addition to an increase in the aromatic integration at δ 6.78–7.81 ppm assignable to the extra phenyl ring. ¹³C NMR of compound 10a showed signals assigned to CH₃ carbon of the thiazole ring, CH₃ and C=O carbons of the acetyl group at δ 19.1, 30.1 and 189.7 ppm, respectively. Additionally, ¹³C NMR spectrum of 10b displayed signals attributed to ethyl carbons at δ 14.7 and 60.7 ppm along with signal due to ester carbonyl carbon at δ 165.3 ppm. Furthermore, ¹³C NMR spectrum of 10d revealed signals at δ 18.2 and 163.8 ppm due to CH₃ carbon of the thiazole ring and C=O, respectively.

Finally, 2-(3-(2,4-dichlorophenyl)-5-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)-4-mthyl-5-(aryldiazenyl)thiazole derivatives 13a–13f were prepared by heating the carbothioamide derivatives 5a, b with 2-oxo-N-arylpropanehydrazonyl chlorides 12a–12c in absolute ethanol^56,57 (Scheme 2). 2-Oxo-N-arylpropanehydrazonyl chlorides 12a–12c were prepared through a coupling reaction between α-chloroacetylacetone and aryl diazonium salts via Japp–Klingeman rearrangement⁵⁹.

IR spectra of compounds 13a–13f showed a band at 1582–1589 cm⁻¹ referring to (N = N) in addition to the absence of the band corresponding to NH₂ group at 3418, 3444 and 3247, 3322 cm⁻¹. ¹H NMR spectra of 13b, e, f revealed singlet signals at δ 2.53–2.61 ppm equivalent to CH₃ protons of the thiazole ring in addition to an increase in the integration of the aromatic protons at δ 6.78–7.90 ppm. Also, ¹H NMR spectra of 13b, e showed additional singlet signals at δ 2.42 and 2.41 ppm attributed to the three protons of 4-CH₃, respectively. ¹³C NMR of 13a–13e showed a signal for CH₃ carbon of the thiazole ring at δ 16.1–16.6 ppm along with an increase in the number of signals of aromatic carbons at δ 109.4–165.3 ppm. Also, ¹³C NMR of 13b, e revealed the presence of a signal at δ 21.4 ppm indicating an additional CH₃ carbon at the phenyldiazenyl ring.

2.2. Biological evaluation

2.2.1. EGFR and VEGFR-2 kinase inhibitory activities of thiazolyl-pyrazoline derivatives

In the current study, the potential inhibitory activity of target thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) towards EGFR and VEGFR-2 were examined, with the aim of exploring the plausible mechanism. The IC₅₀ values of the tested compounds were evaluated compared to reference EGFR inhibitor (gefitinib) and VEGFR-2 inhibitor (vandetanib), Table 1.

Table 1. EGFR and VEGFR-2 kinases inhibitory activity of target thiazolyl-pyrazoline derivatives.

Compound	R	R1	IC50 (nM) ± SEM
			EGFR	VEGFR-2
7a	H	H	145.1 ± 2.0	124.9 ± 1.4
7b	H	Cl	114.2 ± 0.4	195.2 ± 4.6
7c	OCH3	H	511.2 ± 3.2	915.2 ± 4.3
7d	OCH3	Cl	132.9 ± 1.2	185.5 ± 2.3
10a	H	CH3	274.0 ± 2.3	450.3 ± 8.3
10b	H	OCH2CH3	40.7 ± 1.0	78.4 ± 1.5
10c	H	NHC6H5	130.7 ± 1.1	102.2 ± 4.3
10d	OCH3	NHC6H5	32.5 ± 2.2	43.0 ± 2.4
13a	H	H	973.0 ± 2.0	1151.2 ± 4.3
13b	H	CH3	179.4 ± 0.0	214.3 ± 4.0
13c	H	Cl	134.5 ± 1.5	173.3 ± 2.6
13d	OCH3	H	93.6 ± 3.0	335.8 ± 6.3
13e	OCH3	CH3	67.7 ± 2.0	216.4 ± 4.3
13f	OCH3	Cl	107.13 ± 1.2	113.3 ± 1.8
Gefitinib	–	–	55.74 ± 1.0	45.3 ± 1.2
Vandetanib	–	–	43.14 ± 1.3	29.1 ± 1.0

Examination of the obtained results (Table 1) hinted out that the target thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) have better kinase inhibitory activity towards EGFR (IC₅₀ range: 32.5–973.0 nM) than VEGFR-2 (IC₅₀ range: 43.0–1151.2 nM), except for compounds 7a and 10c. As shown in Table 1, compounds 10b and 10d displayed the highest inhibitory activity against EGFR (IC₅₀ = 40.7 and 32.5 nM, respectively) as well as VEGFR-2 (IC₅₀ = 78.4 and 43.0 nM, respectively), due to the ability of the carbonyl group in the ester functionality to form hydrogen bonds with the key amino acids in both EGFR and VEGFR-2 active sites. Moreover, compounds 7a, 7b, 7d, 10a, 10c and 13b–13d exhibited moderate inhibitory activity against EGFR (IC₅₀: 67.7–274.0 nM) and VEGFR-2 (IC₅₀: 102.2–450.3 nM). On the other hand, compounds 7c and 13a displayed the lowest activity against EGFR (IC₅₀ = 511.2 and 973.0 nM, respectively) and VEGFR-2 (IC₅₀ = 915.2 and 1151.2 nM, respectively).

The following structure–activity relationships (SARs) can be concluded from the kinases inhibition data displayed in Table 1. Regarding the EGFR inhibitory activity of the first series 7a–7d, it was found that substitution of the pendant phenyl ring at C-4 of the thiazole moiety (compounds 7b and 7d; IC₅₀ = 114.2 ± 0.4 and 132.9 ± 1.2 nM, respectively) elicited an enhancement of effectiveness towards EGFR in comparison to the unsubstituted analogues (compounds 7a and 7c; IC₅₀ = 145.1 ± 2.0 and 511.2 ± 3.2 nM, respectively). In contrast, the 3,4-dimethoxy substitution for the pyrazoline C-5 phenyl ring (compounds 7c and 7d; IC₅₀ = 511.2 ± 3.2 and 132.9 ± 1.2 nM, respectively) resulted in a worsening of inhibitory activity against EGFR in comparison to the 4-methoxy monosubstituted counterparts (compounds 7a and 7b; IC₅₀ = 145.1 ± 2.0 and 114.2 ± 0.4 nM, respectively).

Moreover, the obtained results for the second series 10a–10d towards EGFR revealed that the incorporation of the ester functionality at C-5 of the thiazole moiety (compound 10b; IC₅₀ = 40.7 ± 1.0 nM) was more beneficial for the activity more than the acetyl (compound 10a; IC₅₀ = 274.0 ± 2.3 nM) and amide (compound 10c; IC₅₀ = 130.7 ± 1.1 nM) functionalities. In addition, the 3,4-dimethoxy substitution for the pyrazoline C-5 phenyl ring in the amide-bearing 10d led to the best EGFR inhibition in this study; IC₅₀ = 32.5 ± 2.2 nM. In a similar fashion, incorporation of a 3,4-dimethoxyphenyl moiety within the third series 13a–13f (compounds 13d–13f; IC₅₀ = 93.6 ± 3.0, 67.7 ± 2.0 and 107.13 ± 1.2 nM, respectively) elicited an enhancement of EGFR inhibitory activity in comparison to the monosubstituted analogues (compounds 13a–13c; IC₅₀ = 973.0 ± 2.0, 179.4 ± 0.0 and 134.5 ± 1.5 nM, respectively).

On the other hand, the SAR outcomes for VEGFR-2 inhibition pointed out that utilisation of the ester group at C-5 of the thiazole moiety within the second series 10a–10d (compound 10 b; IC₅₀ = 78.4 ± 1.5 nM) led to an enhanced activity more than the acetyl (compound 10a; IC₅₀ = 450.3 ± 8.3 nM) and amide (compound 10c; IC₅₀ = 102.2 ± 4.3 nM) groups. Similar to the SAR for EGFR inhibition, grafting a second methoxy group for the amide-bearing counterpart 10c (IC₅₀ = 102.2 ± 4.3 nM) produced compound 10d that identified as the most potent VEGFR-2 inhibitor in this work (IC₅₀ = 43.0 ± 2.4 nM). Further analysis of the VEGFR-2 inhibition data for the third series 13, highlighted that both the para-substitution of the appended phenyl ring at C-5 of the thiazole moiety, and the 3,4-dimethoxy substitution for the pyrazoline C-5 phenyl ring resulted in an improvement of the VEGFR-2 inhibitory activity.

2.2.2. Western blotting of EGFR and VEGFR-2 in A549 cells

To confirm the ability of thiazolyl-pyrazolines derivatives to dual inhibit the activity of EGFR and VEGFR-2 kinases, we examined the effect of the most potent compounds 10b and 10d on the expression levels of p-EGFR, EGFR, p-VEGFR-2 and VEGFR-2 proteins in A549 cells using Western blot analysis. As shown in Figure 3, the protein expression levels of p-EGFR and p-VEGFR-2 were significantly downregulated by compounds 10b and 10d.

Figure 3. Thiazolyl-pyrazoline derivatives downregulated p-EGFR and p-VEGFR-2 protein levels (A) Western blotting analysis of the expression of p-EGFR, EGFR, p-VEGFR-2 and VEGFR-2 in A549 cells treated with 10b or 10d and untreated cells used as a negative control. β-actin served as a loading control. (B) The relative protein expression level of p-EGFR normalised with total EGFR was quantified by the Image J software (C) The relative protein expression level of p-VEGFR-2 normalised with total VEGFR-2. The values are expressed as the mean ± SEM based on three different experiments. ***p < 0.001 indicate a significant difference compared with control.

2.2.3. Kinase selectivity profiling

As compounds 10b and 10d showed high potency against EGFR and VEGFR-2 kinases (Table 1), we further investigated their inhibitory activities against 11 kinases (TEK, SYK, EPHB2, ABL1, LCK, CLK1, ROCK1, PKC, AKT1, CDK1, and CDK5) to determine their selectivity profile (Table 2).

Table 2. Kinase selectivity profile of 10b and 10d.

Kinase	IC50 (nM)
	10b	10d
TEK	>2000	>2000
SYK	1120 ± 22	870 ± 31
EPHB2	>2000	>2000
ABL1	>2000	>2000
LCK	>2000	>2000
CLK1	1069 ± 54	798 ± 19
ROCK1	>2000	>2000
PKC	>2000	>2000
AKT1	816 ± 38	702 ± 24
CDK1	310 ± 12	245 ± 7
CDK5	1136 ± 20	993 ± 15

Interestingly, compounds 10b and 10d displayed moderate inhibition against CDK1 (IC₅₀ = 310 and 245 nM, respectively) and low inhibition against SYK, CLK1, AKT1 and CDK5 (IC₅₀ range: 702–1136 nM). Meanwhile, 10b and 10d did not exhibit measurable inhibition (with IC₅₀ > 2000 nM) against TEK, EPHB2, ABL1, LCK, ROCK1 and PKC kinases, demonstrating significant selectivity of 10b and 10d towards EGFR and VEGFR-2 kinases.

2.2.4. Anti-proliferative activity of thiazolyl-pyrazoline derivatives

To evaluate the anti-proliferative activity of the synthesised thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d, and 13a–13f), a preliminary in vitro one dose (10 µM) anticancer assay was performed. Nine cell lines derived from five tumour subpanels were examined; leukaemia (K562 and KG-1a), breast (MCF-7, BT-549 and HCC70), lung (A549 and H441), colon (HCT116) and liver (HepG2) cancer cell lines. The compounds were added at a single concentration (10 µM) and the cultures were incubated for 24 h, then the per cent growth inhibition (%GI) was calculated.

As shown in Tables 3 and 4, compounds 10b and 10d revealed promising cell growth inhibition activity against almost all the screened cell lines (%GI ranging from 31.0% to 95.3%), especially against the non-small cell lung cancer A549 and H441 cell lines (%GI ranging from 79.1% to 95.3%). Other compounds showed moderate to weak anti-proliferative effect against the tested cell lines; however, the non-small lung cancer cells (A549 and H441) were more sensitive to the target thiazolyl-pyrazoline derivatives.

Table 3. % Growth inhibition ± SEM of thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) towards leukaemia (K562 and KG-1a) and breast cancer (MCF-7, BT-549 and HCC70) cell lines.

Compound	K562	KG-1a	MCF-7	BT-549	HCC70
7a	14.2 ± 2.1	18.4 ± 1.3	0.07 ± 0.0	26.4 ± 3.6	27.3 ± 1.0
7b	21.5 ± 3.0	21.7 ± 4.6	25.9 ± 1.2	5.2 ± 1.0	19.6 ± 0.3
7c	5.2 ± 0.5	4.3 ± 0.7	9.2 ± 0.9	3.1 ± 0.6	7.3 ± 1.2
7d	22.0 ± 1.6	25.1 ± 2.0	2.2 ± 0.6	18.0 ± 2.7	17.3 ± 0.6
10a	12.2 ± 1.9	22.2 ± 1.6	13.6 ± 2.9	9.4 ± 1.0	24.3 ± 2.5
10b	54.2 ± 3.8	31.0 ± 2.5	48.5 ± 2.3	47.2 ± 4.9	41.5 ± 2.1
10c	11.2 ± 1.0	9.1 ± 1.0	2.5 ± 0.8	22.3 ± 0.8	18.3 ± 1.6
10d	60.2 ± 2.3	57.2 ± 3.4	60.9 ± 4.3	40.1 ± 2.0	54.1 ± 2.8
13a	2.2 ± 0.2	4.1 ± 0.9	17.8 ± 1.0	10.3 ± 1.1	15.3 ± 1.4
13b	11.0 ± 0.6	7.1 ± 0.2	3.4 ± 0.5	5.1 ± 0.3	8.3 ± 0.9
13c	5.2 ± 0.9	12.4 ± 1.6	10.4 ± 2.9	8.7 ± 1.9	1.1 ± 0.1
13d	18.2 ± 1.8	24.1 ± 2.7	11.9 ± 1.8	14.2 ± 2.2	22.3 ± 1.3
13e	24.0 ± 2.0	15.2 ± 1.8	1.8 ± 0.6	12.5 ± 1.5	16.1 ± 0.9
13f	31.5 ± 1.0	20.0 ± 2.8	0.8 ± 0.2	24.6 ± 1.3	6.3 ± 1.0

Table 4. % Growth inhibition ± SEM of thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) towards lung (A549 and H441), colon (HCT116) and liver (HepG2) cancer cell lines.

Compound	A549	H441	HepG2	HCT116
7a	34.9 ± 4.3	36.1 ± 2.1	0.5 ± 0.0	0.5 ± 0.1
7b	45.2 ± 2.9	35.2 ± 1.2	34.4 ± 1.4	18.8 ± 1.9
7c	8.9 ± 1.1	16.2 ± 0.3	17.1 ± 2.7	4.9 ± 0.8
7d	31.2 ± 1.4	34.8 ± 2.4	5.1 ± 1.0	0.7 ± 0.0
10a	32.3 ± 2.2	6.3 ± 0.7	28.0 ± 4.3	19.0 ± 2.6
10b	88.3 ± 4.9	79.1 ± 2.6	50.6 ± 2.2	37.7 ± 3.9
10c	28.9 ± 2.0	8.7 ± 0.1	11.9 ± 1.1	7.9 ± 1.1
10d	95.3 ± 5.3	89.2 ± 3.9	65.5 ± 3.9	56.5 ± 2.0
13a	11.0 ± 0.8	22.2 ± 0.9	15.8 ± 2.2	19.5 ± 1.4
13b	16.2 ± 1.3	23.3 ± 2.4	0.08 ± 0.0	0.8 ± 0.2
13c	14.8 ± 1.0	19.9 ± 1.7	14.2 ± 1.0	8.7 ± 0.6
13d	12.6 ± 2.7	38.9 ± 2.4	18.6 ± 2.5	10.1 ± 3.5
13e	30.3 ± 2.4	46.9 ± 3.9	0.3 ± 0.0	9.9 ± 0.8
13f	47.5 ± 3.1	17.3 ± 0.6	0.9 ± 0.3	0.2 ± 0.0

Overexpression of EGFR and VEGFR-2 is well-reported in the literature to be significantly correlated with the induction of the NSCLC cells proliferation³¹. Thereafter, the quantitative IC₅₀ values have been determined for thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) towards the most susceptible cell lines at the preliminary assay (non-small lung cancer A549 and H441 cell lines), at testing concentrations of 1, 5, 10, 25, 50 or 100 µM for 24 h. The antitumor drugs gefitinib and vandetanib were used as reference drugs in this MTT cytotoxicity assay (Table 5).

Table 5. Inhibitory activities of thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d and 13a–13f) against cancerous and normal lung cells. Values represent the mean IC₅₀ values (µM) ± SEM (n = 3) for each drug. Compared IC₅₀s; a compounds versus gefitinib and b compounds versus Vandetanib.

Compound	IC50 (µM) ± SEM
	A549	H441	WI-38
7a	22.5 ± 1.2^a ns, ^b***	38.4 ± 2.3^a***, ^b***	88.2 ± 4.2 ^a***, ^b***
7b	12.0 ± 0.4^a***, ^b***	26.5 ± 1.1^a***, ^b***	60.6 ± 3.1^a***, ^b***
7c	>100	>100	>100
7d	20.2 ± 2.0^a ns, ^b***	29.1 ± 3.5^a***, ^b***	40.1 ± 2.7^a*, ^b***
10a	49.1 ± 3.2^a***, ^b***	>100	>100
10b	4.2 ± 0.8^a***, ^bns	4.8 ± 0.1^a*, ^bns	72.4 ± 7.2^a***, ^b***
10c	23.5 ± 1.3^a ns, ^b***	45.2 ± 2.3^a***, ^b***	>100
10d	2.9 ± 0.2^a***, ^bns	3.8 ± 0.5^a*, ^bns	51.4 ± 3.5^a***, ^b***
13a	>100	94.4 ± 5.5^a***, ^b***	>100
13b	59.1 ± 3.2^a***, ^b***	24.2 ± 1.8^a***, ^b***	>100
13c	46.3 ± 2.5^a***, ^b***	33.5 ± 3.9^a***, ^b***	91.4 ± 8.9^a***, ^b***
13d	57.1 ± 4.1^a***, ^b***	21.7 ± 1.3^a***, ^b***	64.2 ± 2.8^a***, ^b***
13e	19.1 ± 1.3^a*, ^b***	15.2 ± 3.6^a***, ^b***	87.0 ± 4.2^a***, ^b***
13f	13.7 ± 2.2^a**, ^b***	24.8 ± 1.4^a***, ^b***	>100
Gefitinib	22.3 ± 1.4	7.1 ± 1.3	35.1 ± 2.0
Vandetanib	3.4 ± 1.0	3.9 ± 0.8	8.6 ± 0.4

*p < 0.05, **p < 0.01, ***p < 0.001 and ns = non-significant.

The results illustrated in Table 5 showed that compounds 10b and 10d have the best anti-proliferative activity, herein reported, against A549 (IC₅₀ = 4.2 and 2.9 µM, respectively) and H441 (IC₅₀ = 4.8 and 3.8 µM, respectively) cell lines, whereas compounds 7c and 13a have non-significant anti-proliferative activity (IC₅₀ more than 100 μM). Other compounds showed moderate to low anti-proliferative effect against A549 (IC₅₀ range: 12.0–59.1 µM) and H441 (IC₅₀ range: 15.2–94.4 µM). Interestingly, the anti-proliferative effect of compounds 10b and 10d was higher than the reference drug gefitinib and was comparable to vandetanib. In addition, compounds 7a, 7b, 7d, 10c, 13e and 13f exerted moderate anticancer activity against A549 cell lines with IC₅₀s values spanning between 12.0 and 23.5 µM, whereas compounds 7b, 7d, 13b and 13d–13f showed moderate growth inhibitory activity towards H441 cells with IC₅₀s spanning in the range 15.2–29.1 µM, Table 5.

It is noteworthy that the synthesised thiazolyl-pyrazoline derivatives (7a–7d, 10a–10d, and 13a–13f) showed weak or non-significant cytotoxicity to human normal WI-38 lung fibroblast cells (IC₅₀ range: 40.1 ± 2.7 – >100 µM) (Table 5). The mean tumour selectivity value calculated for compounds 10b and 10d was found to be 16.1 and 15.3, respectively, which reveals good selectivity towards tumour cells with efficient safety index (Table 6).

Table 6. Cytotoxic effect of 10b and 10d towards human non-tumorigenic lung WI-38 cells and mean tumour selectivity index (S. I.) (WI-38/A549 and H441).

Compound	IC50 (µM) ± SEM			Mean tumour selectivity
	WI-38	A549	H441
10b	72.4 ± 7.2	4.2 ± 0.8	4.7 ± 0.1	16.1
10d	51.4 ± 3.5	2.9 ± 0.2	3.8 ± 0.5	15.3

2.2.5. Effect of thiazolyl-pyrazoline derivatives on EGFR-mutated NSCLC cell lines

The prolonged use of the TKIs targeting EGFR or VEGFR-2 kinases led to drug resistance due to mutations development and toxicity. In this study, we analysed the anti-proliferative potential of the most potent EGFRWT inhibitors 10b and 10d towards two EGFR mutated cancer cell lines; NCI-H1650 possesses exon19 deletion (delE746-A750) mutation of EGFR and NCI-H1975 carries L858R/T790M double mutation of EGFR. Interestingly, compounds 10b and 10d exhibited significant growth inhibitory activity against NCI-H1650 (IC₅₀ = 5.7 and 3.5 µM, respectively) and NCI-H1975 (IC₅₀ = 6.2 and 4.4 µM, respectively) Table 7 and Figure 4.

Figure 4. Effect of thiazolyl-pyrazoline derivatives on EGFR mutated NSCLC cell lines; dose–response curves for 10b and 10d in comparison with gefitinib control. The percentage of viable cells is shown relative to that of untreated control cells. Data were presented as means ± standard error mean (SEM) of three independent experiments.

Table 7. Inhibitory activity of thiazolyl-pyrazoline derivatives 10 b and 10d against EGFR-mutated NSCLC cell lines.

Compound	IC50 (µM)
	NCI-H1650	NCI-H1975
10b	5.7 ± 0.5	6.2 ± 0.9
10d	3.5 ± 0.2	4.4 ± 0.6
Gefitinib	>20	>20

Moreover, the obtained results indicated that both 10b and 10d were much more sensitive towards NCI-H1650 and NCI-H1975 cells than gefitinib (IC₅₀ > 20 µM towards the two examined cell lines). Accordingly, 10b and 10d may serve as a lead compounds for the treatment of gefitinib-resistant EGFR mutant NSCLC.

2.2.6. Thiazolyl-pyrazoline derivatives inhibited cancer cell migration

As the thiazolyl-pyrazoline derivatives 10b and 10d showed potent inhibitory effect against VEGFR-2 Kinase that plays an important role in cancer cell migration, we investigated the effect of 10b and 10d on the A549 cancer cell migration using wound healing assay. As shown in Figure 5, compounds 10b and 10d were effective in reducing the cancer cell migration compared to the control, highlighting that the VEGFR-2 kinase inhibitory effect of 10b and 10d is the engine for the inhibition of the cancer cell migration.

Figure 5. Effect of compounds 10b and 10d on A549 cells migration. Scratch wound healing assay was used to analyse the inhibition of cell migration in A549 cells treated for 36 h with either 10b or 10d and the untreated cells served as a negative control. Representative images of scratched areas in confluent A549 cell layers.

2.2.7. Thiazolyl-pyrazoline derivatives induced G₂/M cell cycle arrest

In general, the anticancer agents inhibit the proliferation of cancerous cells by arresting cell division at various checkpoints. These checkpoints present at G₁/S phase, S-phase and G₂/M phases. Therefore, we tested the effect of the most potent compounds (10b and 10d) on cell cycle progression in A549 cell line. Results in Figure 6 obviously indicate that compounds 10b and 10d arrested the cell cycle at a G₂/M phase (29.2% and 33.92%, respectively) when compared to the untreated controls (6.06% and 5.93%, respectively). Parallel to these findings, the cell population in G₁ and S phases decreased after treatment with 10b and 10d (Figure 6(A,B)).

Figure 6. Compounds 10b and 10d arrested the cell cycle at a G2/M phase while gefitinib arrested the cell cycle at a G1 phase in A549 cells. (A) Representative cell cycle distribution data are shown by flow cytometric analysis upon incubation with 0 (control), 2.5 or 5 μM of 10b or 10d or with 0 (control), 5 or 20 μM of gefitinib for 24 h. (B) Quantification of cell cycle distribution upon 24 h incubation with 10b or 10d. The values are means ± SEM of three different experiments. *p < 0.05 and **p < 0.01 indicate significant differences compared with control. ns = non-significant.

On the other hand, gefitinib (20 µM) significantly arrested the cell cycle at G1 phase. This difference between the synthesised compounds and gefitinib in the arrested cell cycle phase could be attributed to the moderate inhibitory effect of 10b and 10d on the CDK1 kinase (IC₅₀ = 310 and 245 nM, respectively) that regulates the G2/M phase transition. This result strongly suggests that the synthesised thiazolyl-pyrazoline derivatives inhibit the proliferation of A549 cells by arresting cell cycle at G₂/M phase.

2.2.8. Thiazolyl-pyrazoline derivatives induced apoptosis

Furthermore, the pro-apoptotic effect of compounds 10b and 10d was also investigated on A549 cells using the Annexin V-FITC/PI double staining. The proportion of annexin V-positive apoptotic cells increased gradually from 5.4% at control to 16.5% at 5 μM 10b (Figure 7(A,B)) and from 6.3% at control to 28.5% at 5 μM 10d. These data indicate that the synthesised thiazolyl-pyrazoline derivatives treatment provokes the induction of apoptosis in A549 cells.

Figure 7. Apoptotic effect of compounds 10b and 10d on A549 cells. (A) Representative cytograms of apoptotic A549 cells stimulated with 0 (control), 2.5 or 5 μM 10b or 10d, for 24 h. The lower-right (annexin V + PI- cells) and the upper-right (annexin V + PI + cells) quadrants show early and late apoptotic cells, and the lower-left (annexin V-PI- cells) and the upper-left (annexin V-PI- cells) quadrants represent viable and necrotic cells, respectively. (B) Quantification of annexin V-positive apoptotic cells upon stimulation with 10b or 10d. The values are the means ± SEM of three different experiments. *p < 0.05 and *p < 0.01 indicate significant differences compared with control.

2.3. Molecular docking

In view of the promising kinase inhibitory activity of the thiazolyl-pyrazoline derivatives, herein reported, a molecular docking study was carried out for the most potent derivatives 10b and 10d to provide insights for their potential binding interactions within the examined kinases binding sites. The molecular operating environment (MOE) 2019.02 was implemented in the conduction, analysis and visualisation of the entire docking studies. The 3D structural co-ordinates of EGFR and VEGFR-2 were downloaded from the protein databank PDB IDs 1M17 and 4ASD, respectively. Both the selected PDB IDs have excellent resolution of 2.6 Å and 2.03 for 1M17 and 4ASD, respectively. In addition, 1M17 contains EGFR in complex with potent inhibitor erlotinib, while 4ASD contains VEGFR-2 in complex with the potent inhibitor sorafenib, making them optimum choice for the docking studies. Pose retrieval step of the co-crystallised ligands resulted in values of 0.85 and 0.63 Å between the docked and the co-crystallised poses for erlotinib and sorafenib, respectively, indicating a valid docking protocol (Figure 8).

Figure 8. Superimposition of the co-crystallised (blue) and the docking pose (pink) of erlotinib in the EGFR (A) and sorafenib in the VEGFR-2 (B) binding sites.

Moreover, the previous step resulted in an energy scores (S) of −11.7 and −14.9 kcal/mol, for erlotinib and sorafenib, respectively. The retrieved docking scores of the co-crystalized ligands were used as comparative means to benchmark the docking score values of compounds 10b and 10d.

2.3.1. Docking of compounds 10b and 10d into EGFR active site

Visual inspection of erlotinib binding with EGFR active site, revealed the formation of two hydrogen bonds with the key residues Leu768 and Met769, in addition, two carbon hydrogen bonds were noticed with Glu738 and Gln767. Through a water molecule, erlotinib was able also to interact with Cys751 and Thr766 (Figure 9).

Figure 9. 2D diagram for the binding of erlotinib in the EGFR active site (PDB: 1M17).

Docking simulations for thiazolyl-pyrazoline derivatives 10b and 10d showed that they fit well into the EGFR active site with good docking scores (−12.9 and −14.1 kcal/mol, respectively) comparable to that of erlotinib (−11.7 kcal/mol). The general binding patterns of compounds 10b and 10d are consistent with crystallographic binding mode of erlotinib within the EGFR-TK active site (PDB: 1M17), Figure 10.

Figure 10. 2D diagram for the binding interactions of compound 10b (A) and compound 10d (B) in the EGFR active site (PDB: 1M17).

For instance, the carbonyl group in the amide functionality of compound 10d was engaged in two hydrogen bonds with the key residues Leu768 and Met769. Similarly, the carbonyl group in the ester functionality of compound 10b was engaged in hydrogen bond interaction with Met769, in addition to three hydrogen bonds with Gln767, Cys751 and Thr766 through a water molecule (Figures 10 and 11).

Figure 11. 3D diagram of compounds 10b (A) and 10d (B) showing their binding interaction with the EGFR active site (PDB: 1M17).

Compounds 10b and 10d formed a carbon hydrogen bond with Cys773 and Val693, respectively. The ortho-chloride atom of compound 10b formed a halogen interaction with Gly695, while the ortho-chloride atom of compound 10d formed a halogen interaction with Lys692. On the other hand, only compound 10d was engaged in arene bond interactions with Leu694, Lys704 and Leu802, which explains the superior activity and docking score of compound 10d over compound 10b. Also, it highlights the important role of the additional phenyl ring attached to the amide linker in compound 10d in EGFR inhibition (Figures 10 and 11).

2.3.2. Docking of compounds 10b and 10d into VEGFR-2 active site

The efficient VEGFR-2 inhibitory activity of compounds 10b and 10d were illustrated by their good docking scores (−13.7 and −14.4 kcal/mol) comparable to that of sorafenib (−14.9 kcal/mol). As depicted in Figure 12, sorafenib exerts its VEGFR-2 inhibition activity through formation of hydrogen bonds with the essential residues; Glu885, Cys1045, Asp1046 and Cys919, in addition to other multiple interactions.

Figure 12. 2D diagram for the binding interactions of sorafenib in the VEGFR-2 active site (PDB: 4ASD).

Interestingly, both compounds 10b and 10d were proven to maintain multiple essential interactions upon docking into VEGFR-2 active site (Figures 13 and 14). For instance, compound 10b was engaged in hydrogen bond interactions with Leu889 and Asp1046 via the nitrogen and sulphur atoms of the thiazole ring, respectively. The nitrogen of pyrazoline ring formed two hydrogen bonds with Cys1045, in addition, the carbonyl group in the ester functional contributed to a hydrogen bond with Phe1047. The ortho-chloride and the para-methoxy substituents contributed to bonding interactions with Ile888 and Glu885, respectively. In a similar manner, all the previous interactions were achieved by compound 10d in addition to an extra hydrogen bond interaction with Lys868, in addition to an arene interaction with Leu1035 (Figures 13 and 14).

Figure 13. 2D diagram of compounds 10b (A) and 10d (B) showing their binding interaction with the VEGFR-2 active site (PDB: 4ASD).

Figure 14. 3D diagram of compounds 10b (A) and 10d (B) showing their binding interaction with the VEGFR-2 active site (PDB: 4ASD).

3. Conclusion

In conclusion, this study reports the facile synthesis of potent anticancer series of thiazolyl-pyrazoline derivatives acting as dual EGFR/VEGFR-2 inhibitors with significant proapoptotic properties. All the synthesised thiazolyl-pyrazolines have been screened for their inhibitory activities against EGFR and VEGFR-2 kinases and for their cytotoxic effect against nine cell lines derived from five tumour subpanels. Compounds 10b and 10d displayed potent and selective inhibitory activity towards EGFR-TK (IC₅₀ = 40.7 ± 1.0 and 32.5 ± 2.2 nM, respectively) and VEGFR-2 (IC₅₀ = 78.4 ± 1.5 and 43.0 ± 2.4 nM, respectively). Furthermore, compounds 10b and 10d showed a promising anticancer activity against non-small lung cell lines as they were the most potent derivatives with IC₅₀ values equal 4.2 and 2.9 µM against A549 cells, and equal 4.8 and 3.8 µM against H441 cells, respectively. Moreover, our results indicated that 10b and 10d were much effective towards EGFR-mutated NSCLC cell lines (NCI-H1650 and NCI-H1975 cells) than gefitinib. Finally, compounds 10b and 10d induce G₂/M cell cycle arrest and apoptosis and inhibit migration in A549 cancerous cells. Furthermore, the molecular docking study explored the binding mode and possible different interactions between the target compounds and the active sites of EGFR and VEGFR-2 enzymes. Accordingly, thiazolyl-pyrazoline scaffold can be considered as promising scaffold for further development of more potent dual EGFR and VEGFR-2 inhibitors.

4. Experimental

4.1. Chemistry

4.1.1. General

Melting points were measured with a Stuart melting point apparatus (Stuart Scientific, Redhill, UK) and were uncorrected. Infra-red (IR) spectra were recorded on FT-IR spectrometer using KBr discs (Perkin Elmer, Waltham, MA). Mass spectra (MS) were performed on a Varian electron impact EI mass spectrometer (EI-MS) at 70 eV at Regional Centre for Mycology and Biotechnology, Al-Azhar University utilising Thermo Scientific ISQ LT mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). NMR Spectra were recorded on a Bruker NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) at Center for Drug Discovery Research and Development, Faculty of Pharmacy, Ain Shams University. ¹H spectra were run at 400 MHz and ¹³C spectra were run at 100 MHz in deuterated dimethyl sulfoxide (DMSO-d₆) or chloroform CDCl₃. Chemical shifts are expressed in δ values (ppm) using the solvent peak as internal standard. All coupling constant (J) values are given in hertz. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were carried out at the Regional Centre for Mycology and Biotechnology, Al-Azhar University utilising Thermo Scientific Flash 2000 elemental analyser (Thermo Fisher Scientific Inc., Waltham, MA). Analytical thin-layer chromatography (TLC) was employed routinely to follow the course of reactions and to check the purity of products using aluminium sheets pre coated with silica gel (Kieselgel, F254, pore size 60 Å, Merck, Darmstadt, Germany) and observed under a UV lamp (short-wavelength, 254 nm). All reagents and solvents were purified and dried by standard techniques.

4.1.2. General procedure for preparation of 1-(2,4-dichlorophenyl)-3-(aryl) prop-2-en-1-one 3a, b

To a solution of 2,4-dichloroacetophenone 1 (1.76 g, 9.3 mmol) and an appropriate aldehyde derivative 2a, b (9.3 mmol) in absolute ethanol (40 ml), 10% sodium hydroxide solution (15 ml) was added portion wise. The mixture was stirred for 4 h at room temperature. The separated precipitate was filtered, washed with water, dried and crystallised from ethanol to afford compounds 3a, b, respectively.

4.1.2.1. 1-(2,4-Dichlorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (3a)

Yellow powder in 87% yield, m.p. 102–103 °C (reported m.p. 106–107 °C⁶⁰) IR (KBr, ν cm⁻¹): 1653 (C=O); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.81 (s, 3H, OCH₃), 6.99 (d, J = 7.6 Hz, 2H, Ar-H), 7.11 (d, J = 16.8 Hz, 1H, olefinic H), 7.38 (d, J = 15.2 Hz, 1H, olefinic H), 7.58 (s, 2H, Ar-H), 7.72–7.76 (m, 3H, Ar-H); MS (EI) m/z (%): 307.28 (M⁺, 9.98), 131.17 (100); Anal. Calcd. for C₁₆H₁₂Cl₂O₂ (307.17): C, 62.56; H, 3.94; Found; C, 62.50; H, 3.97%.

4.1.2.2. 1-(2,4-Dichlorophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3b)

Yellow powder in 96% yield, m.p. 138–139 °C (reported m.p. 134–136 °C⁶¹) IR (KBr, ν cm⁻¹): 1657 (C=O); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.81 (s, 6H, 2 (OCH₃)), 7.01 (d, J = 8.4 Hz, 1H, Ar-H), 7.16 (d, J = 16 Hz, 1H, olefinic H), 7.31–7.41 (m, 3H, Ar-H + olefinic H), 7.58 (s, 2H, Ar-H), 7.78 (s, 1H, Ar-H); MS (EI) m/z (%): 337.14 (M⁺, 7.37), 83.13 (100); Anal. Calcd. for C₁₇H₁₄Cl₂O₃ (337.2): C, 60.55; H, 4.18; Found; C, 60.62; H, 4.13%.

4.1.3. General procedure for preparation of carbothioamide derivatives 5a, b

To a mixture of 3a, b (1 mmol) and thiosemicarbazide 4 (1 mmol) in absolute ethanol (50 ml), sodium hydroxide (0.29 g, 7 mmol) was added. The reaction mixture was heated under reflux with stirring for 6 h. After cooling, the formed product was filtered, washed with ethanol, dried and crystallised from ethanol to give the corresponding pyrazoline derivatives 5a, b, respectively.

4.1.3.1. 3-(2,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (5a)

Yellow powder in 70% yield, m.p. 168–169 °C (reported m.p. 175–178 °C⁶²) IR (KBr, ν cm⁻¹): 3418 and 3247 (NH₂), 1599 (C = N), 1236 and 828 (C = S); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.18 (dd, J = 2.8, 16 Hz, 1H, H_A), 3.73 (s, 3H, OCH₃), 4.03 (dd, J = 12.6, 17.2 Hz, 1H, H_M), 5.87 (dd, J = 3.2, 12 Hz, 1H, H_X), 6.88 (d, J = 10.4 Hz, 2H, Ar-H), 7.08 (d, J = 8.4 Hz, 2H, Ar-H), 7.52 (dd, J = 2.4, 8.4 Hz, 1H, Ar-H), 7.71 (d, J = 1.6 Hz, 1H, Ar-H), 7.75 (s, 1H, NH, D₂O exchangeable), 8.00 (d, J = 8 Hz, 1H, Ar-H), 8.13 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 45.7 (CH₂ pyrazoline), 55.5 (OCH₃), 63.0 (CH pyrazoline), 114.3 (2 C), 127.1 (2 C), 128.1, 129.3, 130.7, 132.7, 133.4, 135.2, 135.7, 153.3, 158.7, 177.0 (C=S); MS (EI) m/z (%): 380.16 (M⁺, 28.54), 182.72 (100), Anal. Calcd. for C₁₇H₁₅Cl₂N₃OS (380.29): C, 53.69; H, 3.98; N, 11.05; Found; C, 53.74; H, 3.95; N, 11.13%.

4.1.3.2. 3-(2,4-Dichlorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (5b)

Light yellow powder in 98% yield, m.p. 198–200 °C; IR (KBr, ν cm⁻¹): 3444 and 3322 (NH₂), 1581 (C=N), 1254 and 833 (C=S); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.19–3.23 (m, 1H, H_A), 3.72 (s, 6H, 2OCH₃), 3.99–4.07 (dd, J = 17.6, 11.6 Hz, 1H, H_M), 5.86–5.89 (m, 1H, H_X), 6.65 (d, J = 8.8 Hz, 1H, Ar-H), 6.80 (s, 1H, Ar-H), 6.88 (d, J = 7.6 Hz, 1H, Ar-H), 7.52 (d, J = 9.6 Hz, 1H, Ar-H), 7.71 (s, 1H, Ar-H), 7.76 (s, 1H, NH, D₂O exchangeable), 7.96 (d, J = 9.6 Hz, 1H, Ar-H), 8.13 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 45.7 (CH₂ pyrazoline), 55.9 (OCH₃), 56.0 (OCH₃), 63.3 (CH pyrazoline), 110.0, 112.3, 117.6, 128.1, 129.4, 130.7, 132.7, 133.4, 135.5, 135.7, 148.3, 149.2, 153.5 (C=N), 177.1 (C=S); MS (EI) m/z (%): 410 (M⁺, 24.38), 216.30 (100); Anal. Calculated for C₁₈H₁₇Cl₂N₃O₂S (410): C, 52.69; H, 4.18; N, 10.24; Found; C, 52.73; H, 4.11; N, 10.29%.

4.1.4. General procedure for preparation of compounds 7a–7d

A mixture of carbothioamides 5a, b (1 mmol) and the appropriate 1-aryl-2-bromoethanone 6a, b (1.1 mmol) in absolute ethanol (20 ml) was heated under reflux for 4 h. After cooling, the formed precipitate was filtered and crystallised from ethanol to afford the corresponding compounds 7a–7d.

4.1.4.1. 2-(3-(2,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (7a)

Yellow powder in 55% yield, m.p. 72–75 °C; IR (KBr, ν cm⁻¹): 1583 (C=N); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.38–3.44 (m, 1H, H_A), 3.70 (s, 3H, OCH₃), 4.05–4.12 (m, 1H, H_M), 5.64 (s, 1H, H_X), 6.91 (d, J = 8.8, 2H, Ar-H), 7.26–7.35 (m, 6H, 5 Ar-H and thiazole H) , 7.51 (d, J = 12 Hz, 1H, Ar-H), 7.69–7.79 (m, 4H, Ar-H); MS (EI) m/z (%): 480 (M⁺, 16.80), 76.10 (100); Anal. Calcd. for C₂₅H₁₉Cl₂N₃OS (480): C, 62.50; H, 3.99; N, 8.75; Found; C, 62.41; H, 4.17; N, 8.82%.

4.1.4.2. 4-(4-Chlorophenyl)-2-(3-(2,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thiazole (7b)

Yellow powder in 60% yield, m.p. 140–141 °C; IR (KBr, ν cm⁻¹): 1583 (C=N); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.42–3.46 (m, 1H, H_A), 3.72 (s, 3H, OCH₃), 4.10 (dd, J = 17.6, 12 Hz, 1H, H_M), 5.63 (dd, J = 14, 5.2 Hz, 1H, H_X), 6.91 (d, J = 8.8 Hz, 2H, Ar-H), 7.34–7.42 (m, 5H, Ar-H and thiazole H), 7.53 (d, J = 10.4 Hz, 1H, Ar-H), 7.72–7.80 (m, 4H, Ar-H); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 45.8 (CH₂ pyrazoline), 55.5 (OCH₃), 64.3 (CH pyrazoline), 106.0, 114.4 (2 C), 127.7 (2 C), 128.1, 128.5 (2 C), 129.0 (2 C), 129.2, 130.9, 132.2, 132.5, 132.9, 133.7, 133.7, 135.0, 149.8, 150.8, 159.2, 164.8; MS (EI) m/z (%): 514 (M⁺, 10.28), 89.34 (100); Anal. Calcd. for C₂₅H₁₈Cl₃N₃OS (514): C, 58.32; H, 3.52; N, 8.16; Found; C, 58.44; H, 3.76; N, 8.41%.

4.1.4.3. 2-(3-(2,4-Dichlorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-phenylthiazole (7c)

Yellow powder in 69% yield, m.p. 120–122 °C; IR (KBr, ν cm⁻¹): 1584 (C=N); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.50 (dd, J = 17.6, 8 Hz, 1H, H_A), 3.72 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 4.12 (dd, J = 17.6, 12.4 Hz, 1H, H_M), 5.64 (dd, J = 12.4, 7.2 Hz, 1H, H_X), 6.92–7.09 (m, 3H, Ar-H), 7.26–7.38 (m, 4H, 3 Ar-H, thiazole H), 7.56 (dd, J = 9.2, 2.8 Hz, 1H, Ar-H), 7.76–7.78 (m, 3H, Ar-H), 7.82 (d, J = 8.8 Hz, 1H, Ar-H); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 45.8 (CH₂ pyrazoline), 55.9 (2OCH₃), 64.7 (CH pyrazoline), 105.2, 111.6, 112.3, 119.2, 126.0 (2 C), 128.1, 128.2, 129.0, 129.4 (2 C), 130.8, 132.3, 132.9, 134.2, 134.9, 135.0, 148.8, 149.0, 150.8, 151.0, 164.77; MS (EI) m/z (%): 510 (M⁺, 15.80), 96.39 (100); Anal. Calcd. for C₂₆H₂₁Cl₂N₃O₂S (510): C, 61.18; H, 4.15; N, 8.23; Found; C, 61.02; H, 4.39; N, 8.47%.

4.1.4.4. 4-(4-Chlorophenyl)-2-(3-(2,4-dichlorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)thiazole (7d)

Yellow powder in 79% yield, m.p. 158–159 °C; IR (KBr, ν cm⁻¹): 1584 (C=N); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.48 (dd, J = 15.2, 8 Hz, 1H, H_A), 3.72 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 4.11 (dd, J = 14.8, 9.6 Hz, 1H, H_M), 5.62 (dd, J = 15.2, 9.2 Hz, 1H, H_X), 6.93 (s, 2H, Ar-H), 7.06 (s, 1H, Ar-H), 7.40–7.43 (m, 3H, 2Ar-H and thiazole H), 7.54 (d, J = 7.6 Hz, 1H, Ar-H), 7.75–7.81 (m, 4H, Ar-H); MS (EI) m/z (%): 544 (M⁺, 21.11), 218.49 (100); Anal. Calcd. for C₂₆H₂₀Cl₃N₃O₂S (544): C, 57.31; H, 3.70; N, 7.71; Found; C, 57.53; H, 3.86; N, 7.89%.

4.1.5. General procedure for preparation of compounds 10a–10d

A mixture of carbothioamides 5a, b (1 mmol) and 3-chloropentane-2,4-diones 9a–9c (1.1 mmol) in absolute ethanol (30 ml) was heated under reflux for 4 h. After cooling, the formed precipitate was filtered and crystallised from ethanol to afford the corresponding compounds 10a–10d.

4.1.5.1. 1-(2-(3-(2,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methylthiazol-5-yl)ethan-1-one (10a)

Yellow powder in 72% yield, m.p. 205–207 °C; IR (KBr, ν cm⁻¹): 1647 (C=O); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 2.38 (s, 3H, CH₃), 2.40 (s, 3H, CH₃), 3.73 (s, 3H, OCH₃), 4.15 (dd, J = 18.8, 11.6 Hz, 1H, H_M) 5.70 (dd, J = 11.6, 5.6 Hz, 1H, H_X), 6.90 (d, J = 10.4 Hz, 2H, Ar-H), 7.21 (d, J = 8.8 Hz, 2H, Ar-H), 7.55 (d, J = 8.8 Hz, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.81 (d, J = 9.6 Hz, 1H, Ar-H); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 19.1 (CH₃), 30.1 (CH₃), 46.1 (CH₂ pyrazoline), 55.5 (OCH₃), 63.1 (CH pyrazoline), 114.6 (2 C), 124.6, 127.8, 128.2, 128.8, 131.0, 132.5, 133.2, 133.3, 135.5, 153.4, 157.5, 159.2, 165.2 (aromatic carbons), 189.7 (C=O); MS (EI) m/z (%): 460 (M⁺, 15.52), 153.75 (100); Anal. Calcd. for C₂₂H₁₉Cl₂N₃O₂S (460): C, 57.40; H, 4.16; N, 9.13; Found; C, 57.31; H, 4.30; N, 9.25%.

4.1.5.2. Ethyl-2-(3-(2,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methylthiazole-5-carboxylate (10b)

Yellow powder in 63% yield, m.p. 192–195 °C; IR (KBr, ν cm⁻¹): 1669 (C=O); ¹H NMR (CDCl₃, 400 MHz) δ ppm: 1.35 (t, J = 7.6 Hz, 3H, CH₃), 2.54 (s, 3H, CH₃), 3.53 (dd, J = 18.4, 8 Hz, 1H, H_A), 3.81 (s, 3H, OCH₃), 4.11 (dd, J = 20.4, 9.2 Hz, 1H, H_M), 4.29 (q, J = 7.6 Hz, 2H, CH₂), 5.90 (s, 1H, H_X), 6.88 (d, J = 8.4 Hz, 2H, Ar-H), 7.27–7.29 (m, 2H, Ar-H), 7.34 (dd, J = 9.2, 3.6 Hz, 1H, Ar-H), 7.48 (d, J = 2.4 Hz, 1H, Ar-H), 7.85 (d, J = 7.6 Hz, 1H, Ar-H); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 14.7 (CH₃), 17.7 (CH₃), 46.3 (CH₂ pyrazoline), 55.5 (OCH₃), 60.7 (CH₂), 63.2 (CH pyrazoline), 111.4, 114.6, 127.8 (2 C), 128.2, 128.8, 131.0, 132.5, 133.1, 133.3, 135.5, 153.2, 159.2, 159.35, 162.2 (aromatic carbons), 165.3 (C=O); MS (EI) m/z (%): 490 (M⁺, 10.41), 55.10 (100); Anal. Calcd. for C₂₃H₂₁Cl₂N₃O₃S (490): C, 56.33; H, 4.32; N, 8.57; Found; C, 56.60; H, 4.47; N, 8.78%.

4.1.5.3. 2-(3-(2,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-N-phenylthiazole-5-carboxamide (10c)

White powder in 74% yield, m.p. 195–197 °C; IR (KBr, ν cm⁻¹): 3257 (NH), 1637 (C=O); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 2.36 (s, 3H, CH₃), 3.35–3.40 (m, 1H, H_A), 3.71 (s, 3H, OCH₃), 4.15 (dd, J = 17.6, 12 Hz, 1H, H_M) 5.68–5.71 (m, 1H, H_X), 6.92 (d, J = 8.8 Hz, 2H, Ar-H), 7.06 (t, J = 8.8 Hz, 1H, Ar-H), 7.23 (d, J = 8.8 Hz, 2H, Ar-H), 7.30 (t, J = 8.8 Hz, 2H, Ar-H), 7.55 (d, J = 10.4 Hz, 1H, Ar-H), 7.62 (d, J = 8.8 Hz, 2H, Ar-H), 7.76 (s, 1H, Ar-H), 7.80 (d, J = 9.6 Hz, 1H, Ar-H), 9.72 (s, 1H, NH, D₂O exchangeable); MS (EI) m/z (%): 537 (M⁺, 36.86), 245.37 (100); Anal. Calcd. for C₂₇H₂₂Cl₂N₄O₂S (537): C, 60.34; H, 4.13; N, 10.42; Found; C, 60.49; H, 4.22; N, 10.68%.

4.1.5.4. 2-(3-(2,4-Dichlorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-N-phenylthiazole-5-carboxamide (10d)

Buff powder in 50% yield, m.p. 166–167 °C; IR (KBr, ν cm⁻¹): 3262 (NH), 1637 (C=O); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 2.38 (s, 3H, CH₃), 3.72 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 4.14 (dd, J = 18.4, 12 Hz, 1H, H_M), 5.68 (dd, J = 12, 5.6 Hz, 1H, H_X), 6.78 (d, J = 7.6 Hz, 1H, Ar-H), 6.87–6.95 (m, 2H, Ar-H), 7.06 (t, J = 6.4 Hz, 1H, Ar-H), 7.30 (t, J = 7.2 Hz, 2H, Ar-H), 7.55 (d, J = 11.6 Hz, 1H, Ar-H), 7.63 (d, J = 7.2 Hz, 2H, Ar-H), 7.75–7.81 (m, 2H, Ar-H), 8.14 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 18.2 (CH₃), 46.1 (CH₂ pyrazoline), 56.0 (2OCH₃), 63.7 (CH pyrazoline), 110.7, 112.3, 116.5, 118.3, 120.8, 124.0 (2 C), 128.2, 129.0 (2 C), 130.9, 132.4, 133.0, 133.7, 135.3, 139.5, 148.7, 149.2, 152.3, 154.6, 160.9 (aromatic carbons), 163.8 (C=O); MS (EI) m/z (%): 567 (M⁺, 34.07), 299.67 (100); Anal. Calcd. for C₂₈H₂₄Cl₂N₄O₃S (567): C, 59.26; H, 4.26; N, 9.87; Found; C, 59.40; H, 4.38; N, 9.82%.

4.1.6. General procedure for preparation of compounds 13a–13f

A mixture of carbothioamides 5a, b (1 mmol) and 2-oxo-N-arylpropanehydrazonoyl chlorides 12a–12c (1.1 mmol) in absolute ethanol (30 ml) was heated under reflux for 4 h. The produced product was filtered and washed with hot ethanol to give the corresponding compounds 13a–13f.

4.1.6.1. 2-(3-(2,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-(phenyldiazenyl)thiazole (13a)

Red powder in 50% yield, m.p. 168–170 °C; IR (KBr, ν cm⁻¹): 1582 (N = N); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.46–3.47 (m, 1H, H_A), 3.74 (s, 3H, CH₃), 4.19 (dd, J = 18, 12 Hz, 1H, H_M), 5.81 (dd, J = 12.4, 5.6 Hz, 1H, H_X), 6.93 (d, J = 6.8 Hz, 2H, Ar-H), 7.25 (d, J = 12 Hz, 2H, Ar-H), 7.38 (t, J = 6 Hz, 1H, Ar-H), 7.48 (t, J = 7.6 Hz, 2H, Ar-H), 7.56 (d, J = 7.6 Hz, 1H, Ar-H), 7.69 (d, J = 7.6, 2H, Ar-H), 7.78 (s, 1H, Ar-H), 7.85 (d, J = 9.6 Hz, 1H, Ar-H); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 16.5 (CH₃ of thiazole), 46.3 (CH₂ pyrazoline), 55.6 (OCH₃), 63.0 (CH pyrazoline), 114.7 (2 C), 122.1 (2 C), 127.8 (2 C), 128.2 (2 C), 128.6, 129.8, 131.1 (2 C), 132.7, 133.2, 133.3, 135.7, 141.4, 152.6, 154.3, 158.3, 159.3, 165.0; MS (EI) m/z (%): 524 (M⁺ + 2, 1.17), 522 (M⁺, 4.86), 40.16 (100); Anal. Calcd. for C₂₆H₂₁Cl₂N₅OS (522): C, 59.77; H, 4.05; N, 13.40; Found; C, 59.86; H, 4.13; N, 13.62%.

4.1.6.2. 2-(3-(2,4-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-(p-tolyldiazenyl)thiazole (13b)

Red powder in 80% yield, m.p. 170–171 °C; IR (KBr, ν cm⁻¹): 1582 (N=N); ¹H NMR (CDCl₃, 400 MHz) δ ppm: 2.42 (s, 3H, CH₃), 2.61 (s, 3H, CH₃), 3.53 (dd, J = 18, 7.2 Hz, 1H, H_A), 3.81 (s, 3H, OCH₃), 4.12 (dd, J = 19.2, 9.6 Hz, 1H, H_M), 5.77–5.80 (m, 1H, H_X), 6.89 (d, J = 9.2 Hz, 2H, Ar-H), 7.23–7.29 (m, 4H, Ar-H), 7.32 (dd, J = 9.2, 2 Hz, 1H, Ar-H), 7.47 (d, J = 2.8 Hz, 1H, Ar-H), 7.67 (d, J = 6.4 Hz, 2H, Ar-H), 7.90 (d, J = 10 Hz, 1H, Ar-H); ¹³C NMR (CDCl₃, 100 MHz) δ ppm: 16.1 (CH₃ of thiazole), 21.4 (CH₃), 46.2 (CH₂ pyrazoline), 55.3 (OCH₃), 63.5 (CH pyrazoline), 114.3 (2 C), 122.0 (2 C), 127.3 (2 C), 127.5 (2 C), 128.7, 129.6, 130.8 (2 C), 131.4, 132.6, 133.5, 136.3, 139.4, 142.2, 150.9, 152.3, 159.4, 164.8; MS (EI) m/z (%): 536 (M⁺, 32.38), 110.11 (100); Anal. Calcd. for C₂₇H₂₃Cl₂N₅OS (536): C, 60.45; H, 4.32; N, 13.05; Found; C, 60.31; H, 4.49; N, 13.28%.

4.1.6.3. 5-((4-Chlorophenyl)diazenyl)-2-(3-(2,4-dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methylthiazole (13c)

Red powder in 80% yield, m.p. 170–171 °C; IR (KBr, ν cm⁻¹): 1582 (N=N); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.44 (dd, J = 16.8, 6.4 Hz, 1H, H_A), 3.73 (s, 3H, CH₃), 4.18 (dd, J = 16, 8 Hz, 1H, H_M), 5.80 (dd, J = 9.6, 6 Hz, 1H, H_X), 6.93 (d, J = 7.2 Hz, 2H, Ar-H), 7.24 (d, J = 9.6 Hz, 2H, Ar-H), 7.51 (d, J = 8.8 Hz, 2H, Ar-H), 7.55 (d, J = 8 Hz, 1H, Ar-H), 7.68 (d, J = 8 Hz, 2H, Ar-H), 7.77 (s, 1H, Ar-H), 7.84 (d, J = 7.6 Hz, 1H, Ar-H); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 16.6 (CH₃), 46.3 (CH₂ pyrazoline), 55.6 (OCH₃), 63.0 (CH pyrazoline), 114.7 (2 C), 120.2, 123.6 (2 C), 127.8, 128.3, 128.5, 129.8, 131.1, 132.7 (2 C), 133.1, 133.3, 133.8, 135.8, 141.3, 151.3, 154.7, 159.3, 159.4, 165.3; MS (EI) m/z (%): 556 (M⁺, 35.77), 311.62 (100); Anal. Calcd. for C₂₆H₂₀Cl₃N₅OS (556): C, 56.07; H, 3.62; N, 12.58; Found; C, 56.20; H, 3.88; N, 12.61%.

4.1.6.4. 2-(3-(2,4-Dichlorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-(phenyldiazenyl)thiazole (13d)

Reddish orange powder in 50% yield, m.p. 187–188 °C; IR (KBr, ν cm⁻¹): 1587 (N=N); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 3.44–3.50 (m, 1H, H_A), 3.72 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 4.18 (dd, J = 17.2, 11.6 Hz, 1H, H_M), 5.80 (dd, J = 11.2, 5.6 Hz, 1H, H_X), 6.79 (d, J = 6.4 Hz, 1H, Ar-H), 6.91–6.96 (m, 2H, Ar-H), 7.37 (t, J = 7.6 Hz, 1H, Ar-H), 7.48 (t, J = 8.4 Hz, 2H, Ar-H), 7.55 (d, J = 8.8 Hz, 1H, Ar-H), 7.68 (d, J = 8.4 Hz, 2H, Ar-H), 7.76 (s, 1H, Ar-H), 7.83 (d, J = 7.6 Hz, 1H, Ar-H); ¹³C NMR (DMSO-d₆, 100 MHz) δ ppm: 16.4 (CH₃ of thiazole), 46.2 (CH₂ pyrazoline), 55.9 (2OCH₃), 63.3 (CH pyrazoline), 110.5 (2 C), 112.3, 118.1, 122.1 (2 C), 128.3 (2 C), 129.8, 131.0 (2 C), 132.6, 133.2 (2 C), 133.4, 135.8 (2 C), 148.8, 149.2 (2 C), 152.6, 165.1; MS (EI) m/z (%): 552 (M⁺, 15.69), 329.74 (100); Anal. Calcd. for C₂₇H₂₃Cl₂N₅O₂S (552): C, 58.70; H, 4.20; N, 12.68; Found; C, 58.92; H, 3.37; N, 12.68%.

4.1.6.5. 2-(3-(2,4-Dichlorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methyl-5-(p-tolyldiazenyl)thiazole (13e)

Reddish orange powder in 73% yield, m.p. 176–177 °C; IR (KBr, ν cm⁻¹): 1589 (N=N); ¹H NMR (CDCl₃, 400 MHz) δ ppm: 2.41 (s, 3H, CH₃), 2.59 (s, 3H, CH₃), 3.55 (dd, J = 18, 4 Hz, 1H, H_A), 3.87 (s, 6H, 2OCH₃), 4.10 (dd, J = 17.2, 13.2 Hz, 1H, H_M), 5.73 (dd, J = 13.2, 4 Hz, 1H, H_X), 6.83–6.86 (m, 3H, Ar-H), 7.22–7.34 (m, 3H, Ar-H), 7.46 (s, 1H, Ar-H), 7.66 (d, J = 9.6 Hz, 2H, Ar-H), 7.87 (d, J = 8.8 Hz, 1H, Ar-H); ¹³C NMR (CDCl₃, 100 MHz) δ ppm: 16.1 (CH₃), 21.4 (CH₃), 46.1 (CH₂ pyrazoline), 55.9 (OCH₃), 56.0 (OCH₃), 63.7 (CH pyrazoline), 109.4, 111.4, 118.1, 122.0 (2 C), 127.5, 128.7, 129.6 (2 C), 130.8, 131.3, 133.0, 133.5, 136.3, 139.4, 148.8, 149.2, 150.9, 152.3, 156.5, 164.8; MS (EI) m/z (%): 566 (M⁺, 16.00), 46.07 (100); Anal. Calcd. for C₂₈H₂₅Cl₂N₅O₂S (566): C, 59.36; H, 4.45; N, 12.36; Found; C, 59.46; H, 4.38; N, 12.59%.

4.1.6.6. 5-((4-Chlorophenyl)diazenyl)-2-(3-(2,4-dichlorophenyl)-5-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-methylthiazole (13f)

Orange powder in 70% yield, m.p. 190–191 °C; IR (KBr, ν cm⁻¹): 1586 (N=N); ¹H NMR (DMSO-d₆, 400 MHz) δ ppm: 2.53 (s, 3H, CH₃), 3.48 (dd, J = 17.6, 5.6 Hz, 1H, H_A), 3.73 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 4.19 (dd, J = 19.6, 12.8 Hz, 1H, H_M), 5.81 (dd, J = 12, 5.6 Hz, 1H, H_X), 6.78 (d, J = 7.6 Hz, 1H, Ar-H), 6.92–6.96 (m, 2H, Ar-H), 7.53 (d, J = 9.2 Hz, 2H, Ar-H), 7.57 (d, J = 7.6 Hz, 1H, Ar-H), 7.71 (d, J = 9.2 Hz, 2H, Ar-H), 7.79 (d, J = 3.2 Hz, 1H, Ar-H), 7.85 (d, J = 10 Hz, 1H, Ar-H); MS (EI) m/z (%): 586 (M⁺, 55.20), 458.78 (100); Anal. Calcd. for C₂₇H₂₂Cl₃N₅O₂S (586): C, 55.25; H, 3.78; N, 11.93; Found; C, 55.43; H, 3.89; N, 12.14%.

4.2. Biological evaluation

4.2.1. Cell culture and reagents

Human leukaemia (K562 and KG-1a), breast (MCF-7, BT-549 and HCC70), lung (A549, H441, NCI-H1650 and NCI-H1975), colon (HCT116), liver (HepG2) cancer cell lines and WI-38 human lung fibroblast cell line were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and were cultured in their suitable media containing 10% foetal bovine serum (FBS; Sigma-Aldrich) in a humidified atmosphere with 5% CO₂ at 37 °C. Gefitinib and vandetanib were purchased from Sigma-Aldrich. All chemicals used in this study were analytical or cell-culture grade.

4.2.2. Kinase inhibition assay

The EGFR kinase assay was carried out in 96-well plates coated with PGT (poly l-glutamic acid l-tyrosine, 4:1, Sigma Aldrich, St. Louis, MO) as previously described⁶³. IC₅₀ values were calculated using GraphPad Prism 5. Each experiment was carried out at least three times. The VEGFR-2 kinase assay was carried out in 96-well streptavidin coated plate using recombinant human VEGFR-2/KDR ELISA kit according to manufacturer’s instructions. Moreover, the IC₅₀ values of 10b and 10d on 11 different kinases (TEK, SYK, EPHB2, ABL1, LCK, CLK1, ROCK1, PKC, AKT1, CDK1 and CDK5) were determined using Z'-LYTE® technology which is based on FRET (Invitrogen/Life Technologies).

4.2.3. Western blot analysis

The protein extraction and quantification were done as previously described⁶⁴.

4.2.4. Anti-proliferative assay

Cytotoxicity was measured using the MTT assay. Human leukaemia (K562 and KG-1a), breast (MCF-7, BT-549 and HCC70), lung (A549, H441, NCI-H1650 and NCI-H1975), colon (HCT116), liver (HepG2) cancer cell lines and WI-38 human lung fibroblast cell line were plated at a density of 1 × 10⁴ cells per well in 96-well plates overnight and then treated with the compounds. After 24 h treatment, 20 μL of MTT solution (2 mg/mL in phosphate-buffered saline [PBS]) was added to each well and the cells were cultured for another 4 h at 37 °C. The medium was aspirated and 150 μL DMSO was added to solubilise MTT formazan crystals. The plates were then shaken, and the optical density was determined at 570 nm using an ELISA plate reader (Model 550, Bio-Rad, Hercules, CA). At least three independent experiments were performed. The IC₅₀ values were calculated using GraphPad Prism 5 (Version 5.01, GraphPad Software, San Diego, CA).

4.2.5. Cell cycle and apoptosis analyses

DNA content was measured using 7-aminoactinmycin D (7AAD) (Biotium, Inc., Hayward, CA) staining and is commonly used for cell cycle phase analysis as previously described⁶⁵. Apoptosis was evaluated using the annexin V/propidium iodide (PI) staining kit (BioLegend, San Diego, CA) according to the manufacturer’s instructions and as previously described.

4.3. Molecular docking

In this work, all the docking studies were conducted using Molecular Operating Environment (MOE 2019.02) software^67,68. The crystal structures of EGFR in complex with erlotinib and VEGFR-2 in complex with sorafenib were downloaded from the protein databank PDB IDs 1M17 and 4ASD, respectively. All the receptors and ligands were prepared using the default parameters of MOE software. The binding sites in the two targets were determined by selecting the pocket surrounding the binding domain of the co-crystallised ligands. Prior commencing the docking of compounds 10b and 10d, a pose-retrieval docking experiments for the X-ray coordinates of the co-crystallised ligands in their corresponding binding sites was carried out. The RMSD values between the co-crystallised pose and the docking pose from the previous step were 0.85 and 0.63 Å for erlotinib and sorafenib, respectively. After the positive indication on the docking validity, compounds 10b and 10d were docked into the two receptors using the same conditions. Finally, the results of the docking were visualised using the 2 D and 3 D interaction diagrams generated by MOE.

4.4. Scratch wound healing assay

A549 cells were seeded and incubated in 6-well cell culture plates till reaching confluence. Then, the confluent monolayers were scratched to form a “wound” using a sterile needle. The cells were treated with either vehicle as a control, 10b or 10d for 36 h. The images were recorded at 0 and 36 h to monitor the migration of cells into the wounded area using a light photomicroscope.

4.5. Statistical analysis

Data were presented as the means ± standard error of mean (SEM). Student’s t-test was performed to determine the statistical significance compared to the vehicle treated control, gefitinib or vandetanib. Statistical significance was defined as *p < 0.05, **p < 0.01 or ***p < 0.001. Data are representative of three independent experiments.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Amer Ali Abd El-Hafeez was supported by an NIH-funded Cancer Therapeutics Training Program [CT2, T32 CA121938] and Pradipta Ghosh by the NIH [CA238042, CA100768 and CA160911].