Non-small cell lung cancer sensitisation to platinum chemotherapy via new thiazole-triazole hybrids acting as dual T-type CCB/MMP-9 inhibitors

Abstract

Cisplatin remains the unchallenged standard therapy for NSCLC. However, it is not completely curative due to drug resistance and oxidative stress-induced toxicity. Drug resistance is linked to overexpression of matrix metalloproteinases (MMPs) and aberrant calcium signalling. We report synthesis of novel thiazole-triazole hybrids as MMP-9 inhibitors with T-type calcium channel blocking and antioxidant effects to sensitise NSCLC to cisplatin and ameliorate its toxicity. MTT and whole cell patch clamp assays revealed that 6d has a balanced profile of cytotoxicity (IC₅₀ = 21 ± 1 nM, SI = 12.14) and T-type calcium channel blocking activity (⁓60% at 10 μM). It exhibited moderate ROS scavenging activity and nanomolar MMP-9 inhibition (IC₅₀ = 90 ± 7 nM) surpassing NNGH with MMP-9 over −2 and MMP-10 over −13 selectivity. Docking and MDs simulated its receptor binding mode. Combination studies confirmed that 6d synergized with cisplatin (CI = 0.69 ± 0.05) lowering its IC₅₀ by 6.89 folds. Overall, the study introduces potential lead adjuvants for NSCLC platinum-based therapy.

Graphical Abstract

Introduction

Lung cancer is the second most prevalent type of cancer, with approximately two million new cases and over two million deaths worldwide in 2020. It has a high fatality rate, leading to the highest rate of mortality among men with cancer in 93 countries¹. Non-small cell lung cancer (NSCLC), related to tobacco-driven tumorigenesis, accounts for most cases². Surgery is the primary treatment for early-stage lung cancer³. However, the majority of patients suffer advanced incurable NSCLC stages, when they are first diagnosed⁴, or they relapse after surgery, which reflects poor prognosis and aggressiveness⁴. Platinum-based chemotherapy, especially cisplatin, continues to be the standard treatment for most NSCLC patients⁵. However, platinum-based chemotherapy for advanced NSCLC is not completely curative⁶ due to de novo or acquired tumour resistance⁷ and oxidative stress-induced organ toxicity including neurotoxicity, nephrotoxicity, ototoxicity, and cardiovascular damage limiting their long-term clinical outcomes^8–13. It seems that multiple mechanisms are involved. The approaches for circumventing resistance have been the subject of several clinical investigations¹⁴. Notably, extracellular matrix (ECM) proteins are frequently upregulated in platinum-resistant cancer cells according to molecular profiling studies, suggesting that the tumour may remodel the ECM to mediate resistance¹⁵. Matrix metalloproteinases (MMPs) are intracellular membrane-bound endopeptidases that mainly contribute to extracellular matrix remodelling in carcinogenesis¹. There are about 26 known MMPs. One of the most complicated MMPs that has been linked to several malignancies, including lung cancer is MMP-9. In cisplatin-resistant tumours, combined therapy with MMP-9 inhibitors markedly sensitised the tumour to cisplatin^16–18. More in-depth recent studies underlying cisplatin resistance mechanisms directly linked treatment failure and cancer relapse to the emergence of cancer stem cells (CSCs) with self-renewable properties¹⁹. Such features are controlled by various intra- and extracellular pathways, especially calcium signalling that involves different calcium channels including voltage gated calcium channels (VGCCs)^20,21. Special efforts were directed to explore the role of T-type VGCCs in cancer stemness^22,23 inspired by their crucial role in normal embryonic stem cells, where T-type calcium channel knockdown or block led to loss of self-renewal capacities²⁴. Expression of T-type calcium channels has been associated with poorer outcomes in non-small cell lung cancer. Further investigation showed that pharmacological modulation of T-type calcium channels with the calcium channel blocker (CCB), mibefradil inhibited stem cell proliferation²³. Combination studies of various T-type CCBs and cisplatin in A549 cells revealed synergistic anticancer effects through induction of apoptosis and chemosensitization^25,26.

Collectively, it could be proposed that the next frontier is to combine the therapeutic outcome of MMPs inhibition for halting ECM remodelling simultaneously with blocking T-type VGCCs involved in cancer stemness. Such regimen, including MMP inhibitor and T-type CCB, is expected to be efficiently synergistic to cisplatin against NSCLC. Thus, this study aims to tailor multi-targeted hybrids combining features that target MMPs and block T-type calcium channels into a single scaffold. This scaffold will be expected to exhibit uniform pharmacokinetic profile, especially distribution.

Design rationale

Aberrant calcium signals, especially T-type currents, could be responsible for cancer stemness that likely mediates cisplatin chemoresistance, and thus may be considered an efficient target to ameliorate drug resistance²⁷. Combination studies unveiling the role of MMP-9 inhibition in enhancing the anticancer potential of cisplatin¹⁶ were also inspirational. Recent research has linked extracellular calcium to higher levels of MMPs expression^28–31 These findings guided us to target the interplay between T-type calcium signalling and MMP-9 by designing dual inhibitors with the aim of sensitising NSCLC to cisplatin. The study was also extended to evaluate the antioxidant potential of the designed compounds to address the oxidative stress which is a critical component of cisplatin toxicity³². A literature review of selective T-type CCBs revealed versatile lead blockers of diverse chemical identities^33–36. However, the presence of azoles in many lead T-type blockers attracted our interest. On the other hand, MMP inhibitors are designed either as potent mimics of endogenous ligands capped with hydroxamic acid as zinc-binding group (ZBG) or non-hydroxamate inhibitors with more favoured pharmacokinetic profiles³⁷. Indeed, the major challenge facing optimisation of efficient MMPs inhibitor is excluding the problematic hydroxamic acid while maintaining potency. Non-hydroxamate MMPs inhibitors are typically long compounds with attached aromatic rings. Even though these entities are typically hydrophobic, the incorporation of carbonyl and amino groups were recommended to offer opportunities for certain important hydrophilic interactions with the enzymatic active site^38–40. Thus, our target new compounds were designed as thiazole-triazole hybrids connected via amide ligation (Figure 1) conforming with the structural requirements of non-hydroxamate MMP inhibitors and the wide presence of 1,2,3-triazole and thiazole in T-type calcium channel blockers and MMP inhibition^36,41–43. The available space in the structure of the target hybrids was invested by incorporating chalcones and dihydropyrazoles with remarkable antioxidant potential^44,45.

Figure 1.

Rationale design of the target thiazole-triazole hybrids as MMP inhibitors/CCBs/antioxidant agents based on lead MMPs inhibitor⁴⁶, T-type CCB⁴¹, and antioxidant⁴⁷ derivatives.

Results and discussion

Chemistry

2-Amino-4-phenylthiazole (1) was synthesised via one-pot reaction of N-bromosuccinimide, acetophenone, and thiourea in lactic acid⁴⁸. 1 was then allowed to react with chloroacetyl chloride in chloroform to afford the desired chloroacetamide derivative 2a⁴⁹. The bromoacetamide derivative 2b was prepared by a coupling reaction with bromoacetic acid utilising the reagents cocktail DIC and Oxyma⁵⁰. These haloacetamide derivatives 2 were reacted with sodium azide to afford the key intermediate 2-azido-N-(4-phenylthiazol-2-yl)acetamide (3)⁵¹ as confirmed by its IR,¹H- and ¹³C-NMR spectra (Supplementary Figures S1 and S2). The azide derivative 3 was then cyclized to 2-[4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]-N-(4-phenylthiazol-2-yl)acetamide (4) utilising propargyl alcohol, sodium ascorbate, and copper sulphate⁵². IR,¹H- and ¹³C-NMR spectra of 4 displayed signals characteristic to the primary alcoholic group together with other signals attributed to the thematic structural features of the scaffold (Supplementary Figures S3, S4, and S5). The primary alcoholic derivative 4 was selectively oxidised using activated MnO₂ in THF to the corresponding 2–(4-formyl-1H-1,2,3-triazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide (5)⁵³ as confirmed by ¹H-and ¹³C-NMR spectra (Supplementary Figures S6 and S7) showing aldehydic proton and carbonyl carbon signals, respectively. Moreover, its IR spectrum displayed the characteristic aldehydic C = O absorption band (Scheme 1).

Figure 3.

(A) 2D binding mode of 6d, (B) 3D binding mode of 6d (Green sticks), (C) 3D cartoon binding mode of 6d, (D) 2D binding mode of reference reverse hydroxamate inhibitor (NFH), (E) 3D binding mode of reference reverse hydroxamate inhibitor (red sticks), into the active site of MMP-9 catalytic domain (PDB ID: 1GKC⁸¹) The zinc ions are shown as blue spheres.

Figure 4.

(A) RMSD, (B) RMSF, (C) rGyr, of 1GKC with and without 6d during 100 ns MD simulation, and (D) Hydrogen bonding of the simulated 1GKC-6d during 100 ns.

Figure 5.

Average COM between 6d and 1GKC in the complex during 100 ns MD simulation.

Scheme 1.

Synthesis of the key starting compound 2–(4-formyl-1H-1,2,3-triazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide (5).

The target chalcones 6a-d were synthesised by condensation of the aldehyde derivative 5 with various ketones utilising NaOH 10% at 0 °C. The ¹H-NMR spectra of the chalcones 6a-d showed the vinylic protons signals (Supplementary Figures S8, S10, S12, and S14), whereas their ¹³C-NMR spectra demonstrated the deshielded signals assigned for chalcone carbonyl carbons as well as other expected chemical shifts (Supplementary Figures S9, S11, S13, and S15). Moreover, the IR spectra of the series showed the diagnostic absorption bands assigned for the chalcone carbonyl groups. The chalcones 6a-d were then cyclized using hydrazine hydrate at room temperature to the corresponding dihydropyrazole derivatives 7a-d. IR, ¹H- and ¹³C-NMR spectra displayed the characteristic signals corresponding to the dihydropyrazole ring (Supplementary Figures S16–S23). On the other hand, the dimeric product 8 was obtained utilising acetone as the ketonic component of the condensation reaction with the aldehyde derivative 5 at room temperature. Its IR spectrum displayed the diagnostic chalcone carbonyl absorption band. ¹H- and ¹³C-NMR spectra (Supplementary Figures S24 and S25) revealed signals attributed to the chalcone group and lacked that of the terminal methyl verifying the structure of the title dimer (Scheme 2). It is worth mentioning that some NMR signals, especially those assigned for the amide NH were duplicated in the spectrum of the chalcone derivative 6c (Supplementary Figure S12). This may be attributed to the amide-imidol tautomeric equilibrium^54–56. Literature review revealed that the relative orientations of groups within an amide molecule have been extensively studied by NMR spectroscopy. Most studies were concerned with the partial double-bond character of the amide bond. This double-bond character arises from the contribution of the tautomeric imidol structure to the amide (Supplementary Fig S12). Accordingly, N-monosubstituted amides may exist in solution as a mixture of cis and trans isomers. The substituents are thus more likely to give well-separated NMR signals whenever the rotation around the carbonyl-to-nitrogen bond is slow⁵⁵.

Scheme 2.

Synthesis of the target chalcones (6a-d and 8) and the dihydropyrazole derivaives (7a-d).

Biological evaluation and molecular modelling

All the newly synthesised derivatives were screened for their potential cytotoxic activities against A549 cells relative to normal human lung fibroblasts by MTT assay^57,58. Simultaneously, all compounds were subjected to the whole-cell patch-clamp technique assay for studying their potential Cav3.2 T-type blocking activities. Based on the screening results, preliminary SAR could be deduced among the evaluated compounds. Accordingly, the most promising derivatives regarding cytotoxic potency, selectivity to cancerous cells over normal fibroblasts and calcium channel blockade capability were then utilised for further in vitro MMPs inhibition assay and binding mode analysis. In this part of the study, the MMP-9 inhibitory potential of the hit compounds was investigated, considering its interplay with abnormal calcium signalling^28–30 and correlation to NSCLC pathogenesis^31,59,60. Moreover, the selectivity profiles of the investigated compounds were studied against other related druggable MMPs isoforms (MMP-2, MMP − 10, and MMP-13). The presumed binding mode of the most potent MMP-9 inhibitor into the enzymatic active site was simulated by docking and molecular dynamics to highlight the most important structural features contributing to activity. Then the ability of the hit derivative to mitigate cisplatin-induced oxidative stress and toxicity was probed via evaluating its free radical scavenging capacity. Finally, the lead compound was subjected to combination studies with cisplatin and evaluated as an adjuvant to therapy offering synergistic anticancer outcome. This workflow is outlined in Chart 1.

Chart 1.

Workflow of biological evaluation and molecular modelling of the synthesised thiazole-triazole hybrids.

Cytotoxicity screening and preliminary SAR

All compounds under test were evaluated for their potential cytotoxic activities against human NSCLC A549 cells compared to normal human lung fibroblasts (Wi-38) using MTT assay^57,58 in reference to cisplatin (Table 1). The overall pattern of cytotoxic activity revealed the designed scaffold’s promising anticancer potential, as all compounds surpassed cisplatin. The MTT assay results revealed that the azide derivative 3 (IC₅₀ = 74.4 nM) was nearly 8-fold more potent than cisplatin (IC₅₀ = 586 nM). Moreover, it exhibited outstanding selectivity to NSCLC A549 cells over normal lung cells Wi-38 (SI = 23.3), therefore offering a promising starting point for structural optimisation. Cyclo-addition reaction of the azide derivative 3 to the primary alcoholic triazole derivative 4 decreased the scaffold’s potency by 3- to 4-fold (IC₅₀ = 268 nM) and was detrimental to the observed selectivity (SI˂1). Selective oxidation of the primary alcohol derivative 4 to the corresponding aldehyde derivative 5 conferred approximately a 2.5-fold increase in cytotoxic activity regarding potency and selectivity (IC₅₀ = 112 and SI = 2.1). Condensation of the aldehyde derivative 5 with various aliphatic and aromatic ketones introduced their respective α-β-unsaturated ketones (chalcones) 6a-d and imparted variable cytotoxic potency and selectivity to the scaffold. The oxobutenyl derivative bearing terminal methyl group 6a exhibited higher potency (IC₅₀ = 82 nM) and excellent selectivity (SI = 23.4) compared to the aldehyde precursor 5. Replacing the terminal methyl by phenyl or tolyl group lowered the detected potency of their respective derivatives 6b (IC₅₀ = 130 nM) and 6c (IC₅₀ = 151 nM). Such transformation also decreased 6b selectivity by approximately 2-fold. However, 6c showed a higher selectivity index than the aldehyde precursor. Interestingly, the potency of the series was optimised in the p-fluorophenyl derivative 6d that was 27-fold more potent than cisplatin recording IC₅₀ = 21 nM and five times more selective than cisplatin in lung cancer cells than normal cells (SI = 12.1). Subsequent cyclisation of the chalcone derivatives 6a, 6c, and 6d into the corresponding dihydropyrazole derivatives 7a, 7c, and 7d led to a significant decrease in cytotoxicity. 7a (IC₅₀ = 213 nM) was 2–3-fold less potent than its precursor 6a, while 7c (IC₅₀ = 200 nM) was around 1.5-fold less potent than the starting chalcone 6c. Furthermore, the fluorinated derivative 7d (IC₅₀ = 425 nM) was far less active than 6d. On the contrary, the dihydropyrazole derivative 7b was the most potent derivative in the current study with 40-fold higher in activity than cisplatin (IC₅₀ = 14 nM) and six times more selective for lung cancer cells (SI = 14.6). Finally, the dimeric chalcone derivative 8 was comparable to the aldehyde precursor 5 in terms of their IC₅₀, but less selective (IC₅₀ = 116 and SI = 0.9). Besides assessing anticancer potency and selectivity of the studied compounds against A549 cells⁵⁷, the screened derivatives were also evaluated for their safe dose (EC₁₀₀) against Wi-38. 6c (EC₁₀₀ = 1177 nM) was at the top of the list followed by 6a (EC₁₀₀ = 460 nM), 6b (EC₁₀₀ = 449 nM), and 3 (EC₁₀₀ = 429 nM) compared to cisplatin (EC₁₀₀ = 595 nM). Other derivatives recorded EC₁₀₀ within a range of 187–38 nM. Collectively, the chalcone 6d and the dihydropyrazole 7b derivatives exhibited the most potent cytotoxic activities against A549 (IC₅₀ = 21 nM and 14 nM, respectively) with excellent selectivity profiles (SI = 12.1 and 14.6 nM, respectively), and were considered as the study lead compounds for further mechanistic studies.

Table 1.

Cytotoxicity screening of the thiazole-triazole hybrides 3–8 against normal human lung fibroblasts (Wi-38), NSCLC cells (A549), and their selectivity index (SI) values.

Compound No.	Structure	Wi-38		A549
		IC50a (nM)*	EC100b (nM)	IC50 (nM)	SIc
3		1728 ± 3	429 ± 32	74 ± 4	23.35
4		249 ± 2	58 ± 4	268 ± 2	0.93
5		243 ± 14	78 ± 4	112	2.17
6a		1924 ± 212	460 ± 110	82 ± 5	23.46
6b		1609 ± 41	449 ± 51	130 ± 6	12.38
6c		3482 ± 297	1177 ± 73	151 ± 3	23.06
6d		255 ± 3	138 ± 9	21 ± 1	12.14
7a		384 ± 18	187 ± 8	213 ± 2	1.80
7b		205 ± 1	122 ± 11	14 ± 3	14.64
7c		230 ± 5	51 ± 7	200 ± 6	1.15
7d		1298 ± 31	108 ± 15	425 ± 17	3.05
8		108 ± 3	38 ± 2	116 ± 1	0.93
Cisplatin	–	1329 ± 82	589 ± 76	586 ± 30	2.26

^*Data are presented as Mean ± SD, n = 3.

^aIC₅₀: the half maximal inhibitory concentration of the tested compound.

^bEC₁₀₀: the effective safe concentration value at 100% cell viability of the tested compound.

^cSI: the ratio of IC₅₀ calculated for healthy and cancer cells.

T-type calcium channel block profiling

All compounds were assayed using the whole cell patch-clamp technique for studying their potential as Cav3.2 T-type CCBs^25,61. Figure 2 displays their overall T-type calcium channel blocking activities, expressed as percentage of inhibition at a concentration of 10 µM. Results showed that primary alcohol 4 exhibited approximately 60% T-type current inhibition. The aldehyde derivative 5 showed only 20% T-type block, whereas the azide derivative 3 was far less active than 4. Within the chalcone series, the methyl, phenyl and tolyl derivatives 6a-c showed relatively moderate blocking activities ranging from 20–40%. On the other hand, the p-fluorophenyl substituted chalcone derivative 6d displayed nearly 55% T-type current inhibition. Dimerisation of 6a into 8 caused a marked decrease in the T-blockade activity. Also, cyclisation of chalcones 6a-d to the corresponding dihydropyrazoles 7a-d resulted in remarkable reduction in the T-current inhibitory effect.

Figure 2.

Block of Ca_V3.2 (T-type) currents induced by 10 μM application of the test compounds 3–8. Data are presented as Mean ± SD, n = 3.

MMPs inhibition and binding mode analysis

In vitro MMP-2, MMP-9, MMP-10, and MMP-13 inhibition assay

A holistic view of the aforementioned results highlighted the fluorinated chalcone derivative 6d and the dihydropyrazole derivative 7b as the most balanced cytotoxic agents against A549 cells showing T-type calcium channel block. Thus, the two lead compounds were chosen for further detailed mechanistic experiments. Herein, the MMPs inhibitory profiles of 6d and 7b were investigated, with special focus on MMP-9 considering its central role as anticancer target^62,63, its correlation to NSCLC pathogenesis^59,60, interplay with abnormal calcium signals^28–30 and the significance of its modulation in sensitising cisplatin-resistant tumours^16,17. The MMP-9 inhibitory activity of the two lead compounds 6d and 7b was detected at 0.5 µM concentration compared to the reference broad spectrum MMPs inhibitor NNGH utilising the MMP-9 inhibitor assay kit (Abcam, catalogue # ab139448). Results (Table 2) revealed that 6d inhibited 96.2% of the detected MMP-9 activity at the test concentration, surpassing the % inhibition of 7b (82.3%) and the reference NNGH (56.8%). IC₅₀ determination experiments revealed that 6d was nearly 4-fold more active than NNGH recording IC₅₀ = 0.09 μM. Further MMPs inhibition studies were performed to explore the compounds potency and selectivity against the druggable related isoforms MMP −2, −10 and −13^64–67 at 0.5 µM concentration. It is worth mentioning that despite the role of the other isoforms (MMP-10 and −13) in carcinogenesis^68,69, their correlation to calcium channel blockade is seldom described in literature, especially in the context of cancer biology and cisplatin resistance. However, some studies reported different effects of calcium signalling modulation on MMP-2 expression in various cancerous tissues^28–30. As shown in Table 2, 6d exhibited lower MMP-2 inhibition (40.6%) than NNGH (60.9%), whereas 7b showed relatively robust inhibition (95.1%). Both 6d and 7b were nearly equipotent against MMP-10 (% inhibition= 82.4 and 81.8%, respectively) being more active than NNGH (% inhibition = 47.4) at 0.5 µM. On the other hand, 6d inhibited only 35.8% of the measured MMP-13 activity, whereas 7b showed no detectable activity. Herein, it could be deduced that tethering chalcone motif to the main scaffold in 6d favours the selectivity to MMP-9 over MMP-2 (SI = 2.4). Subsequent chalcone cyclisation into the dihydropyrazole derivative 7b led to a significant loss of such selectivity. On the other hand, such transformation didn’t abolish the scaffold’s selectivity to MMP-10 over MMP-13.

Table 2.

The inhibitory profiles of 6d and 7b against MMP-2, MMP-9, MMP-10, and MMP-13.

Compound No.	MMP-9		MMP-2	MMP-10	MMP-13
	% Inhibitiona (0.5 μM)	IC50a (μM)	% Inhibitiona (0.5 μM)	% Inhibitiona (0.5 μM)	% Inhibitiona (0.5 μM)
6d	96.2 ± 0.6	0.09 ± 0.01	40.6 ± 3.5	82.4 ± 1.6	35.8 ± 3.2
7b	82.3 ± 1.7	ND	95.1 ± 1.3	81.8 ± 1.5	Nil
NNGH	56.8 ± 0.8	0.35 ± 0.01	60.9 ± 3.4	47.4 ± 0.8	51.2 ± 1.4

^aData are presented as Mean ± SD, n = 2.

Molecular modelling

Molecular modelling studies were conducted including mainly docking of 6d into MMP-9 active site to simulate its binding modes and possibly elucidate the structural determinants of activity. Molecular dynamic simulations were then performed to further evaluate their binding stability. These studies were preceded by preliminary in silico prediction of the compounds ADMET profiles and drug-likeness scores^70–72. Accordingly, the physicochemical properties formulating Lipinski’s rule of five were computed (Supplementary Table S1) as main parameters for selecting drug-like candidates⁷³. Results showed that all compounds were predicted to fully obey Lipinski’s rule of five, except 8 which displayed 2 violations. The number of rotatable bonds (NROTB) and total polar surface area (TPSA) were also computed for the investigated compounds being good predictors of oral bioavailability⁷⁴. Studies reported that compounds with 10 or fewer rotatable bonds and TPSA equal to or less than 140 Ų will probably exhibit good oral bioavailability and permeability through intestine. Again, all compounds, except 8 fulfilled these criteria⁷⁴. Besides, all compounds were predicted to have acceptable aqueous solubility compared to most of the active pharmaceutical ingredients⁷⁵. Bioactivity prediction results highlighted the drug-likeness of the investigated compounds as indicated by their acceptable drug-likeness scores⁷¹. In silico pharmacokinetics and ADMET profiling calculations (Supplementary Table S2) showed that all compounds were predicted to be well absorbed displaying high human intestinal absorption (HIA > 85%)^76,77. In addition, most compounds recorded low predicted blood–brain barrier (BBB) penetration (C_brain/C_blood ˂ 0.1) reflecting low CNS adverse effects possibility, whereas compounds 5, 6a, 6b, 6d and 7a showed medium BBB penetration possibility (C_brain/C_blood = 0.1–2)⁷⁸. On the other hand, all compounds, except 4, 5 and 7a showed high predicted plasma proteins binding capacity which may affect their pharmacokinetic and pharmacodynamic properties^57,79.

Docking

Molecular Operating Environment (MOE) software version 2019.0102⁸⁰ was employed to conduct docking analysis to pinpoint potential binding modes and key structural characteristics of the active compounds. The coordinates of the MMP-9 catalytic domain (without prodomains and fibronectin) were retrieved from its crystal structure (PDB ID: 1GKC)⁸¹. The optimised domain contained three calcium ions, two zinc ions, and the MMP-9 monomer⁶⁷. An in-silico model of the fluorinated chalcone derivative 6d was created, energy minimised and subjected to geometry optimisation. The active site was located utilising the ‘Site Finder’ function of MOE 2019.0102^80,82. Docking was conducted employing the Triangle Matcher placement method^83–85 and London dG scoring function^85,86. The refined top non-redundant poses were ranked according to their docking scores and analysed in terms of protein-ligand interactions (Supplementary Table S3 and Fig. S26). The best pose was lowest binding energy was then considered for 2D and 3D inspection and further molecular dynamics simulations. Herein the most stable conformer of 6d exhibited four hydrogen bonding interactions involving the amide NH and Ala189, N2 and N3 of the 1,2,3-triazole ring and Leu188 at the backbone of the specificity loop S1’ subsite, as well as the ketone carbonyl group and Tyr423 at the edge of the active site (S1’ subunit). Additional π-π stacking interactions were recognised between the terminal phenyl ring and Phe110. Amidic oxygen also displayed metal interaction with the active site Zn (Figure 3).

Molecular dynamics simulations

The top-ranked 1GKC-6d complex was evaluated for its dynamic stability in comparison to the free protein employing a 100 ns MD simulation at natural room temperature conditions using GROMACS⁸⁷, a free and open-source software used for high-performance MDs and output analysis. Visual molecular dynamics (VMD) software⁸⁸ was utilised for hydrogen bonding and contact frequency analysis. Visualisation of the trajectory’s post-simulation run revealed that 6d remains bound to the ligand binding groove of 1GKC pocket. Calculation of root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (rGyr), hydrogen bonding, average centre of mass (COM) distance between 1GKC and 6d, defined secondary structure analysis (DSSP) and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA)^89,90 were performed to evaluate the stability of the complex compared to the free protein.

Root mean square deviation (RMSD)

The RMSD values of the 1GKC-6d complex, MMP-9 backbone and ligand 6d during 100 ns simulations are shown in Figure 4(A). Both the 1GKC-6d complex and free 1GKC exhibited stable RMSD pattern during most of the simulation time. The average RMSD of 1GKC-6d complex was recorded as 0.3305 ± 0.0756 nm, whereas the values for MMP-9 backbone in the complex and ligand 6d were 0.2661 ± 0.0728 nm and 0.1884 ± 0.0374 nm, respectively.

Root mean square fluctuation (RMSF)

Using GROMACS, the RMSF based on ‘C-alpha’ atoms was determined for the complex and free protein. Except for a few residues that form a loop or turn in the protein, the fluctuation intensity for the studied complex generally stayed below 0.2 nm (Figure 4(B)). The dynamic stability of MMP-9 residues before and after the binding of ligand 6d could be highlighted with minor difference between the average RMSF values for the free protein (0.0916 nm) and the complex (0.1201 nm). The free 1GKC and 1GKC-6d complex exhibited equal drift for most of the amino acid residues in contact with 6d. In other words, the Key amino acids which were predicted to interact with 6d in the studied docking complex such as Leu188 (RMSF = 0.0573 nm) and Ala189 (RMSF = 0.0519 nm) were found at their positions. Moreover, other residues in the pocket recorded RMSF values as exemplified by Tyr179 (RMSF = 0.1514 nm), Gly186 (RMSF = 0.0952 nm) and Leu187 (RMSF = 0.0832 nm).

Radius of gyration (rGyr)

The average rGyr values of the simulated free MMP-9 protein and1GKC-6d complex were recorded as 1.5072 ± 0.0135and 1.5465 ± 0.023 nm, respectively, highlighting the stability of the complex throughout the entire simulation except for minor fluctuations (less or equal to 0.1 nm) detected after approximately 50 ns (Figure 4(C)). The rGyr pattern of the simulated free protein compared to the complex showed minor drifts with around 0.05 nm difference throughout the simulation. These results are compatible with RMSD and RMSF data highlighting that the system was compact.

Hydrogen bonding analysis

Figure 4(D) displays the total number of hydrogen bonds formed between MMP-9 active site and the bound ligand 6d throughout the simulation. 6d retained an average of four hydrogen bonds, with no notable gaps of hydrogen bonding-free periods. Further analysis showed that Tyr423, Ala189 and Leu188 displayed hydrogen bonding interactions with high occupancies (85.91, 61.34 and 69.43%, respectively).

The average centre of mass distance (COM)

Analysis of the average COM distance between 1GKCand 6d within the 100 ns simulation (Figure 5) revealed that the 1GKC-6d complex system recorded an average COM distance equals to 12.115 ± 0.414 Å with minor COM distance fluctuations between 2.5–3 Å or less demonstrating that the ligand remained occupying the active site.

The contact frequency (CF) analysis

Using VMD software, CF was analysed to further assess the binding mode between 1GKC and 6d (Figure 6). An amino acid is considered in contact when being within a distance cut-off of 4 Å as determined by a VMD script (contact Freq. tcl module). The ligand 6d displayed CF% approaching 100% with the active site S1 pocket residues Asp185, Leu188 and Pro421-Tyr 423. The common active site key residues Tyr179, Ala189, His401, His 411 were also recorded at high CF.

Figure 6.

Contact frequency analysis of the complex 1GKC-6d during 100 ns MD simulation.

Defined secondary structure protein (DSSP) analysis

The DSSP algorithm was utilised for analysing the secondary structure changes that occurred due to the ligand binding in comparison to the free 1GKC throughout the simulations (Figure 7). As illustrated, negligible secondary structural variations occurred in the S1 pocket (Leu188, Pro421 and Tyr423) or the S1 wall cavity (Leu397, Val398, His401, Leu418, Met422 and Tyr423).

Figure 7.

Defined secondary structure protein (DSSP) analysis of (A) Free 1GKC and (B) 1GKC-6d complex structures during 100 ns MD simulation.

Molecular mechanics-Poisson Boltzman surface area (MM-PBSA) calculations

The Molecular Mechanics-Poisson Boltzman Surface Area (MM-PBSA) method was selected for rescoring complex using G-mmpbsa software. Table 3 shows the ΔG binding energy, Van der Waal, electrostatic, polar solvation and solvent accessible surface area (SASA) energies of the studied complex. Van der Waal and electrostatic energies represented the highest contributors to the computed binding energy. The overall outcome demonstrated that 6d remained bound in stable form within the active site during simulation and possibly justified its promising MMP-9 inhibitory activity.

Table 3.

Calculated binding free energies of 1GKC-6d complex [kJ/mol] during 100 ns MD simulation.

Complex	ΔG	Van der Waal energy	Electrostatic energy	Polar solvation energy	SASA energy
1GKC-6d	−50.567 ± 31.494	−163.354 ± 11.300	−109.747 ± 24.550	240.782 ± 51.131	−18.248 ± 1.149

DPPH radical scavenging assay

The antioxidant ability of the hit thiazole derivative 6d, in the context of its free radical scavenging capacity, was evaluated by the α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method⁹¹. In this assay, the antioxidant activity of 6d was measured at five concentrations (500, 250, 125, 100, 62.5 and 31.25 µg/mL) (Figure 8). Results showed that 6d exhibited a concentration-dependent antioxidant potential as detected by the recorded DPPH radical scavenging % at 250 (70.9 ± 6.6) and 500 µg/mL (85.4 ± 7.9%). The calculated IC₅₀ of 6d was 146.9 ± 22.54 µg/mL (Supplementary Figure S27) highlighting its ability to ameliorate cisplatin-induced oxidative stress and toxicity.

Figure 8.

DPPH scavenging % of serial concentrations of 6d. Data is presented as Mean ± SD, n = 3.

Combination studies with cisplatin

In the light of the aforementioned MTT assay (Table 1), whole patch clamp technique (Figure 2) and MMPs inhibition results (Table 2), the lead compound 6d was selected for combination studies with cisplatin and evaluated as an adjuvant against A549 utilising MTT assay^25,58,92. Herein, 6d was combined with cisplatin at their IC₅₀ doses in order to examine the possible synergistic cytotoxic potential of the combination on A549 cells. The adjuvant potency of 6d was evaluated as the decrease in combination IC₅₀ compared to cisplatin IC₅₀ when used alone, followed by estimation of combination and dose reduction indices. Results revealed that the combination IC₅₀ against A549 cells was 162 nM, that is, lowered by > 72.3% compared to that of cisplatin as single agent (IC₅₀ = 586 nM) (Table 4). Accordingly, the combination index (CI) was computed to quantitatively describe the drug combination effect as synergism, additive, or antagonism^93,94. The estimated CI value was computed as 0.69 ± 0.05 indicating a synergistic interaction between 6d and cisplatin. It is worth mentioning that when the CI value is under 1, the doses of both agents (cisplatin and 6d) necessary to produce a combined cytotoxic effect are lower than those predicted from additivity, therefore such CI is indicative of synergistic interactions between the two agents^93–96. We also computed the dose reduction index (DRI) that describes how many folds the dose of each drug was reduced in the combination compared to each agent alone⁹⁷. Interestingly, 6d was able to reduce the IC₅₀ dosage of cisplatin by 6.89 folds in combination.

Table 4.

IC₅₀ (nM) of 6d as single anticancer agent and as an adjuvant of cisplatin against A549 cells compared to that of cisplatin.

Compound No.	IC50 (nM)
6d	21 ± 1
Cisplatin	586 ± 30
Cisplatin + 6d	162 ± 15

Data are presented as Mean ± SD, n = 3.

The CI usually has a different value at a different effect level. Thus, a plot of CI values at different fraction affected (Fa; A549 growth inhibition % affected by cisplatin and 6d) was automatically generated⁹⁸ (Figure 9). The simulated Fa-CI plot showed that CI tends to increase as the Fa increases, indicating a possible higher level of synergism at lower effect level for combination. However, in accordance with previous reports^99–101, some data points may be observed relatively scattered or even out-of-scale. This may be attributed to the complicated nature of drug¹⁰¹ and possible lack of ideally constant correlation between growth inhibition effect (Fa) and CI as generally seen in case of growth inhibition curves and other cell-based assays.

Figure 9.

Fraction affected (Fa)-combination index (CI) plot of interaction between 6d and cisplatin in terms of % growth inhibition (Fa) of the treated A549 cells (Fa value of 0.5 indicates 50% A549 growth inhibition). The mean values of three independent experiments were used.

This promising synergistic activity is illustrated in Figure 10, where the A549 cells treated with the combination of 6d and cisplatin shrank more than those treated with other individual agents and displayed relatively higher degree of morphological collapse.

Figure 10.

Morphological alterations of A549 after 72 h treatment with IC₅₀ doses of 6d and cisplatin as single agents and in combination compared to untreated control.

Collectively, 6d reduced the calcium currents by nearly 60% via blocking T-type calcium channel associated with emergence of lung cancer stem cells, tumour self-renewal and treatment failure. It concomitantly inhibited MMP-9 (IC₅₀ = 0.09 ± 0.01 μM) that correlates with cisplatin resistance in lung cancer as previously discussed. Thus, as a multitarget probe, 6d synergized with cisplatin (CI = 0.69), enhanced its potency and sensitised A549 cells to it as indicated by DRI of 6.89 in combination of their IC₅₀ doses. Besides, the adjuvant application of 6d could be extended to mitigate cisplatin toxicity by utilising its ROS scavenging activities (IC₅₀ = 146.9 ± 22.54 µg/mL) and high selectivity (SI = 12.14) to A549 cells over normal human lung fibroblast (Wi-38).

Conclusion

In this study, a series of rationally designed substituted thiazole-1,2,3-triazole hybrids were synthesised and evaluated as potential adjuvants for cisplatin-based lung cancer therapy, possibly, via a dual MMP-9 inhibition and/or T-type calcium channel block. MTT assay against A549 cells and whole patch clamp experiments elected 6d as the most balanced derivative with promising anticancer potency (IC₅₀ = 21 ± 1 nM, SI = 12.14) and moderate T-type calcium channel blocking activities (60% blockade at 10 μM). The study lead fluorinated chalcone derivative 6d surpassed the hydroxamate reference inhibitor NNGH against MMP-9 recording nanomolar IC₅₀ (IC₅₀ = 90 ± 7 nM). Docking studies simulated the possible binding mode with the MMP-9 catalytic domain and predicted multiple key interactions. Molecular dynamics revealed the stability of the ligand-receptor complex. 6d showed high % inhibition against MMP-10 (82.4%), and relatively lower activities against MMP-2 (40.6%) and −13 (35.8%) at 0.5 μM, highlighting its selectivity to MMP-9 over MMP-2 and MMP-10 over MMP-13. Furthermore, 6d demonstrated free radical scavenging activity with IC₅₀ = 146.9 ± 22.54 µg/mL. Combination studies of 6d and cisplatin demonstrated its promising adjuvant activities. 6d lowered the IC₅₀ of the combination by >72.32% against A549 cells recording CI = 0.69 ± 0.05 and DRI for cisplatin = 6.89 confirming its synergistic activity. This study could possibly inspire preclinical studies combining dual MMPs inhibitors/T-type CCBs. Indeed, the major challenge facing clinical investigations of MMPs inhibitor is the problematic hydroxamic acid. Herein, we introduced not only new potent non-hydroxamate inhibitors, but also multi-target adjuvants blocking T-type calcium currents and scavenging ROS.

Experimental

Chemistry

Material and equipment

The materials and equipment are reported in the supplementary data.

Synthesis

2-Amino-4-phenylthiazole (1)

A mixture of acetophenone (1.45 ml, 12.5 mmol) and lactic acid (10 ml) was stirred at 90 °C. When mixture became homogeneous, N-bromosuccinimide (NBS) (2.67 g, 15 mmol) was added in 4 portions. On the addition of each portion of NBS (3.75 mmol), the colour of the reaction mixture changed from colourless to orange red. Within a few minutes, the orange-red colour disappeared. After completion of α-bromination as per chromatographic analysis (TLC), thiourea (1.14 g, 15 mmol) was added and the mixture was stirred at the same temperature for 10 min. Then, the reaction mixture was slowly cooled down to room temperature, poured into ice-cold water, then NaHCO₃ was added till neutralisation of lactic acid. The separated product was filtered, washed with water and dried. The produced 2-amino-4-phenylthiazole (1) was sufficiently pure and directly used in the next step. Yield; 2.3 g (90%), mp: 150 °C (reported mp: 149–151 °C)⁴⁸.

2-Chloro-N-(4-phenylthiazol-2-yl)acetamide (2a)

A mixture of the 2-Amino-4-phenylthiazole (1) (0.4 g, 2.3 mmol) and chloroacetyl chloride (0.85 g, 0.6 ml, 7.5 mmol) in chloroform (7 ml) was stirred in the presence of K₂CO₃ (1.036 g, 7.5 mmol) for 24 h at room temperature. The reaction mixture was poured into ice-cold water (50 ml). The formed ppt was filtered and dried. The produced 2-chloro-N-(4-phenylthiazol-2-yl)acetamide (2a) was sufficiently pure. Yield; 0.28 g (49%), mp: 151 °C (reported mp: 152 °C)¹⁰².

2-Bromo-N-(4-phenylthiazol-2-yl)acetamide (2b)

A solution of bromoacetic acid (0.077 g, 0.56 mmol) in dry CH₂Cl_{2 (}2.5 ml) was stirred at 0 °C for 5 min. An equimolar amount of a mixture of ethyl cyanohydroxyiminoacetate (Oxyma) (0.079 g, 0.56 mmol) and N,N′-diisopropylcarbodiimide (DIC) (0.07 g, 0.08 ml, 0.56 mmol) was added, and the mixture was stirred at 0 °C for 1 h. The aminothiazole derivative 1 (0.098 g, 0.56 mmol) was added portion-wise to the reaction solution and stirred at room temperature for 18 h. The reaction mixture was poured into ice-cold water and the precipitate was filtered, washed with water (3 × 30 ml) and dried. The produced 2-bromo-N-(4-phenylthiazol-2-yl)acetamide (2b) was sufficiently pure for use in next experiments in quantitative yield mp: 180 °C (reported mp: 181–182 °C)¹⁰³.

2-Azido-N-(4-phenylthiazol-2-yl)acetamide (3)

A solution of sodium azide (0.1 g, 1.53 mmol) in the least amount of water was added to a solution of the haloacetamide derivative (2a or 2b) (0.158 mmol) in acetone (5 ml). The mixture was stirred for 12 h at room temperature till reaction completion then poured into ice-cold water. The separated product was filtered, washed with water (3 × 30 ml), dried and crystallised from ethanol. Yield; 0.035 g (86%), mp: 131 °C¹⁰⁴. ¹H-NMR (400 mHz, DMSO-D₆) δ 4.23 (s, 2H, CH₂); 7.34 (t, J = 8 Hz, 1H, phenyl-C₄-H); 7.45 (t, J = 8 Hz, 2H, phenyl-C₃,₅-H_s); 7.70 (s, 1H, Thiazole-CH); 7.92 (d, J = 4 Hz, 2H, phenyl-C₂,₆-H_s); 12.55 (br s, 1H, NH).¹³C-NMR (100 mHz, DMSO-D₆) δ 51.15 (CH₂); 108.94 (Thiazole-C₅); 126.22 (Phenyl-C_3,5); 128.39 (Phenyl-C₄); 129.28 (Phenyl-C_2,6); 134.70 (Phenyl-C₁); 149.53 (Thiazole-C₄); 157.87 (Thiazole-C₂); 167.46 (NH-C = O). Anal. Calc for C₁₁H₉N₅OS (259.29): C, 50.95; H, 3.50; N, 27.01; S, 12.37. Found. C, 50.79; H, 3.64; N, 26.86; S; 12.29. IR (KBr, cm⁻¹):3437 (NH); 2111 (N₃); 1673 (C = O); 1552 (Thiazole-C = N).

2-[4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl]-N-(4-phenylthiazol-2-yl)acetamide (4)

A mixture of azide derivative 3 (0.9 g, 3.47 mmol), CuSO₄.5H₂O (0.111 g, 0.7 mmol), sodium ascorbate (0.275 g, 1.38 mmol), propargyl alcohol (0.2 ml, 3.47 mmol) in THF (24 ml) and H₂O (24 ml) was stirred for 3 h at room temperature. The reaction mixture was poured in ice-cold water (50 ml). The formed product was filtered, dried and crystallised from ethanol. Yield; 0.8 g (73%), mp: 211 °C.¹H-NMR (400 mHz, DMSO-D₆) δ 4.56 (s, 2H, CH₂), 5.24 (s, 1H, OH), 5.48 (s, 2H, CH₂-C = O), 7.35 (s, 1H, phenyl-C₄-H), 7.45 (s, 2H, phenyl-C_3,5-Hs), 7.69 (s, 1H, Triazole-CH), 7.91 (s, 2H, phenyl-C₂,₆-Hs), 8.06 (s, 1H, Thiazole-CH), 12.81 (s, 1H, NH).¹³C-NMR (125 mHz, DMSO-D₆) δ 52.00 (CH₂), 55.59 (CH₂-OH), 109.11 (Thiazole-C₅), 125.36 (Triazole-C₅), 126.24 (Phenyl-C_3,5), 128.46 (Phenyl-C₄), 129.32 (Phenyl-C_2,6), 134.67 (Phenyl-C₁), 145.96 (Triazole-C₄), 149.57 (Thiazole-C₄), 157.89 (Thiazole-C₂), 165.50 (NH-C = O). Anal. Calc. for C₁₄H₁₃N₅O₂S: C, 53.32; H, 4.16; N, 22.21; S, 10.17. Found. C, 53.54; H, 4.29; N, 22.49; S, 10.28. IR (KBr, cm⁻¹): 3448 (NH), 3222 (OH), 3100 (Triazole-CH), 1684 (C = O), 1572 (Triazole N = N and Thiazole-C = N).

2–(4-Formyl-1H-1,2,3-triazol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide (5)

A solution of the alcohol derivative 4 (0.6 g, 1.9 mmol) in ethyl acetate (15 ml) was treated with activated MnO₂ (1.15 g, 13.3 mmol) and stirred at room temperature for 24 h. After reaction completion, the reaction mixture was filtered and washed with ethyl acetate. The filtrate was evaporated in vacuo and the residue was washed with H₂O, dried and crystallised from ethanol. Yield; 0.3 g (50%), mp: 193 °C. ¹H-NMR (400 mHz, DMSO-D₆) δ 5.64 (s, 2H, CH₂-C = O), 7.36 (t, J = 8 Hz, 1H, phenyl-C₄-H), 7.46 (t, J = 8 Hz, 2H, phenyl-C₃,₅-H_s), 7.72 (s, 1H, Triazole-CH), 7.93 (d, J = 8 Hz, 2H, phenyl-C₂,₆-Hs), 8.94 (s, 1H, Thiazole-CH), 10.09 (s, 1H, CHO), 12.92 (s, 1H, NH).¹³C-NMR (125 mHz, DMSO-D₆) δ 51.83 (CH₂), 108.71 (Thiazole-C₅), 125.72 (Triazole-C₅), 128.00 (Phenyl-C_3,5), 128.85 (Phenyl-C₄), 130.12 (Phenyl-C_2,6), 134.09 (Phenyl-C₁), 146.87 (Triazole-C₄), 149.07 (Thiazole-C₄); 157.32 (Thiazole-C₂), 164.52 (NH-C = O); 185.03 (CHO). Anal. Calc. for C₁₄H₁₁N₅O₂S: C, 53.66; H, 3.54; N, 22.35; S, 10.28. Found: C, 53.89; H, 3.70; N, 22.59; S, 10.47. IR (KBr, cm⁻¹): 3420 (NH), 3136 (Triazole-CH), 2846, 2778 (aldehyde-CH), 1693 (Aldehyde-C = O, Amide-C = O), 1569 (Triazole N = N and Thiazole-C = N).

General procedure for the preparation of chalcone derivatives 6a-d

A mixture of aldehyde derivative 5 (0.2 g, 0.64 mmol) and an equimolar amount of the appropriate ketone derivative in absolute ethanol (15 ml) was stirred in ice bath for 5 min, then NaOH 10% (0.64 ml) was added gradually. Stirring continued for 24 h. Then the mixture was neutralised by glacial acetic acid. The separated precipitate was filtered, washed, dried and crystallised from ethanol.

2-[4–(3-Oxobut-1-en-1-yl)-1H-1,2,3-triazol-1-yl]-N-(4-phenylthiazol-2-yl)acetamide (6a)

Yield: 0.2 g (88.5%), mp: 207 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 2.35 (s, 3H, CH₃),5.57 (s, 2H, CH₂-C = O), 6.85 (d, J = 16 Hz, 1H, CH=CH-CO), 7.34 (t, J = 6 Hz, 1H, phenyl-C₄-H), 7.45 (t, J = 8 Hz, 2H, phenyl-C₃,₅-H_s), 7.65 (d, J = 16 Hz, 1H, CH=CH-CO), 7.69 (s, 1H, Triazole-CH), 7.92 (d, J = 8 Hz, 2H, phenyl-C₂,₆-H_s), 8.58 (s, 1H, Thiazole-CH), 12.84 (s, 1H, NH).¹³C-NMR (125 mHz, DMSO-D₆) δ 27.65 (CH₃), 52.10 (CH₂), 109.1 (Thiazole-C₅), 126.16 (Phenyl-C_3,5), 127.93 (Triazole-C₅), 128.19 (CH=CH-CO), 128.40 (Phenyl-C₄), 129.26 (Phenyl-C_2,6), 132.39 (CH=CH-CO), 134.55 (Phenyl-C₁), 143.23 (Triazole-C₄), 149.5 (Thiazole-C₄), 157.78 (Thiazole-C₂), 165.21 (NH-C = O), 198.42 (C = O). Anal. Calc. for C₁₇H₁₅N₅O₂S: C, 57.78; H, 4.28; N, 19.82; S, 9.07. Found: C, 58.02; H, 4.53; N, 19.70; S, 9.21. IR (KBr, cm⁻¹): 3454 (NH), 3078 (Triazole-CH), 2991 (CH₃), 1701, 1667 (Chalcone-C = O), 1632 (Amide-C = O), 1562 (Triazole N = N and Thiazole-C = N).

2-[4–(3-Oxo-3-phenylprop-1-en-1-yl)-1H-1,2,3-triazol-1-yl]-N-(4-phenylthiazol-2-yl) acetamide (6b)

Yield; 0.23 g (86.5%), mp: 168 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 5.61 (s, 2H, CH₂-C = O), 7.34 (t, J = 8 Hz, 1H, phenyl-C₄-H), 7.45 (dist. t, 3H, phenyl-C₃,₅-H_s, CO-phenyl-C₄-H), 7.53 (t, J = 8 Hz, 1H, CO-phenyl-C₃-H), 7.59 (t, J = 8 Hz, 1H, CO-phenyl-C₅-H), 7.66 (dist. d, 1H, CH=CH-CO), 7.71 (s, 1H, Triazole-CH), 7.75,7.79 (2s, 1H, CO-phenyl-C₂-H), 7.86–7.93 (m, 2H, phenyl-C₂,₆-H_s), 7.99 (dist. d, 1H, CO-phenyl-C₆-H), 8.11 (d, J = 8 Hz, 1H, CH=CH-CO), 8.74 (s, 1H, Thiazole-CH), 12.88 (s, 1H, NH). ¹³C-NMR (125 mHz, DMSO-D₆) δ 52.05 (CH₂), 109.18 (Thiazole-C₅), 123.19 (Triazole-C₅), 126.26 (Phenyl-C_3,5), 128.23 (CH=CH-CO), 128.49 (Phenyl-C₄), 128.98 (CO-Phenyl-C_2,6), 129.38 (Phenyl-C_2,6), 129.51 (CO-Phenyl-C_3,5), 133.21 (CO-Phenyl-C₄), 133.85 (CH=CH-CO), 134.52 (Phenyl-C₁), 137.78 (CO-Phenyl-C₁), 143.95 (Triazole-C₄), 149.50 (Thiazole-C₄), 157.60 (Thiazole-C₂), 164.95 (NH-C = O), 189.81 (C = O). Anal. Calc. for C₂₂H₁₇N₅O₂S: C, 63.60; H, 4.12; N, 16.86; S, 7.72. Found: C, 63.87; H, 4.05; N, 17.09; S, 7.80. IR (KBr, cm⁻¹): 3450 (NH), 3063 (Triazole-CH), 1684 (Chalcone-C = O), 1607 (Amide-C = O), 1558 (Triazole N = N and Thiazole-C = N).

2-{4-[3-Oxo-3-(p-tolyl)prop-1-en-1-yl]-1H-1,2,3-triazol-1-yl}-N-(4-phenylthiazol-2-yl) acetamide (6c)

Yield; 0.25 g (91%), mp: 240 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 2.39, 2.40 (2s, 3H, CH₃), 5.41, 5.62 (2s, 2H, CH₂-C = O), 7.28–7.38 (m, 4H, CO-phenyl-C_2,3,5,6-Hs), 7.40–7.46 (m, 3H, phenyl-C₃,_4,5-H_s), 7.71–7.78 (m, 2H, Triazole-CH, CH=CH-CO), 7.89–7.96 (m, 3H, phenyl-C₂,₆-H_s, CH=CH-CO), 8.03, 8.05 (2s, 1H, Thiazole-CH), 12.79, 12.85 (2s, 1H, NH). ¹³C-NMR (125 mHz, DMSO-D₆) δ 21.66 (CH₃), 51.94 (CH₂), 109.17 (Thiazole-C₅), 122.23 (Triazole-C₅), 126.23 (Phenyl-C_3,5), 128.25 (CO-Phenyl-C_3,5), 128.46 (Phenyl-C₄), 128.58 (CH=CH-CO), 129.09 (CO-Phenyl-C₄), 129.31 (Phenyl-C_2,6), 129.75 (CO-Phenyl-C_2,6), 129.98 (CH=CH-CO), 132.50 (CO-Phenyl-C₁), 134.66 (Phenyl-C₁), 144.18 (Triazole-C₄), 149.78 (Thiazole-C₄), 157.84 (Thiazole-C₂), 165.36 (NH-C = O), 198.49 (C = O). Anal. Calc. for C₂₃H₁₉N₅O₂S: C, 64.32; H, 4.46; N, 16.31; S, 7.47. Found: C, 64.58; H, 4.61; N, 16.49; S, 7.39. IR (KBr, cm⁻¹): 3449 (NH), 3063 (Triazole-CH), 2925 (CH₃), 1677 (Chalcone-C = O), 1606 (Amide-C = O), 1559 (Triazole N = N and Thiazole-C = N).

2-{4-[3–(4-Fluorophenyl)-3-oxoprop-1-en-1-yl]-1H-1,2,3-triazol-1-yl}-N-(4-phenylthiazol-2-yl)acetamide (6d)

Yield; 0.25 g (90%), mp: decomposes at 163 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 5.63 (s, 2H, CH₂-C = O), 7.07 (dist. d, 1H, CH=CH-CO), 7.36 (s, 2H, CO-phenyl-C_3,5-Hs), 7.45 (s, 3H, phenyl-C₃,_4,5-H_s), 7.72 (s, 1H, Triazole-CH), 7.81 (s, 1H, CO-phenyl-C₂-H), 7.93 (s, 3H, phenyl-C_2,6-Hs, CO-phenyl-C₄-H), 8.22 (s, 1H, CH=CH-CO), 8.76 (s, 1H, Thiazole-CH), 12.93 (s, 1H, NH). ¹³C-NMR (125 mHz, DMSO-D₆) δ 52.15 (CH₂), 109.13 (Thiazole-C₅), 116.28 (CO-phenyl-C₃), 116.50 (CO-Phenyl-C₅), 122.81 (CH=CH-CO), 125.98 (Triazole-C₅), 126.15 (Phenyl-C_3,5), 127.62 (CH=CH-CO), 128.40 (Phenyl-C₄), 128.90 (Triazole- C₄), 129.25 (Phenyl-C_2,6), 131.87 (CO-phenyl-C₂), 131.96 (CO-phenyl-C₆), 133.27 (CO-Phenyl-C₁), 134.53 (Phenyl-C₁), 149.51 (Thiazole-C₄), 157.77 (Thiazole-C₂), 165.21 (NH-C = O), 166.81 (CO-phenyl-C₄), 188.05 (C = O). Anal. Calc. for C₂₂H₁₆FN₅O₂S: C, 60.96; H, 3.72; N, 16.16; S, 7.40. Found: C, 61.23; H, 3.85; N, 16.42; S, 7.56. IR (KBr, cm⁻¹): 3446 (NH); 3072 (Triazole-CH); 1692, 1682 (Chalcone-C = O); 1600 (Amide-C = O); 1561 (Triazole N = N and Thiazole-C = N); 1234 (C-F).

General procedure for the preparation of dihydropyrazole derivatives 7a-d

A mixture of chalcone derivative 6a-d (0.96 mmol) and hydrazine hydrate 99% (0.048 g, 0.05 ml, 0.96 mmol) in absolute ethanol (7 ml) was stirred for 18 h. The formed precipitate was filtered, washed with water, dried and crystallised from ethanol.

2-[4–(3-Methyl-4,5-dihydro-1H-pyrazol-5-yl)-1H-1,2,3-triazol-1-yl]-N-(4-phenylthiazol-2-yl) acetamide (7a)

The title compound was prepared from 2-[4–(3-oxobut-1-en-1-yl)-1H-1,2,3-triazol-1-yl]-N-(4-phenylthiazol-2-yl)acetamide (6a). Yield; 0.25 g (71%), mp: 239 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 1.91 (s, 3H, CH₃), 2.69–2.75 (m, 1H, dihydropyrazole-CH₂), 2.94–3.01 (m, 1H, dihydropyrazole-CH₂), 4.75 (s, 1H, dihydropyrazole-CH), 5.48 (s, 2H, CH₂-C = O), 7.36 (t, J = 6 Hz, 2H, phenyl-C₄-H, dihydropyrazole-NH), 7.47 (t, J = 6 Hz, 2H, phenyl-C_3,5-Hs), 7.71 (s, 1H, Triazole-CH), 7.93 (d, J = 8 Hz, 2H, phenyl-C_2,6-Hs), 8.06 (s, 1H, Thiazole-CH), 12.81 (s, 1H, NH). ¹³C-NMR (125 mHz, DMSO-d₆) δ 16.12 (CH₃), 43.23 (Dihydropyrazole-C₄), 51.98 (CH₂), 55.87 (Dihydropyrazole-C₅), 109.21 (Thiazole-C₅), 124.01 (Triazole-C₅), 126.16 (Phenyl-C_3,5), 128.50 (Phenyl-C₄), 129.28 (Phenyl-C_2,6), 134.22 (Triazole-C₄), 134.63 (Phenyl-C₁), 148.25 (Dihydropyrazole-C₃), 149.51 (Thiazole-C₄), 157.86 (Thiazole-C₂), 165.57 (NH-C = O). Anal. Calc. for C₁₇H₁₇N₇OS: C, 55.57; H, 4.66; N, 26.68; S, 8.73. Found: C, 55.73; H, 4.82; N, 26.51; S, 8.80. IR (KBr, cm⁻¹): 3421 (Amide-NH), 3221 (Dihydropyrazole-NH), 3080 (Triazole-CH), 2955 (CH₃), 1688 (C = O), 1626 (Dihydropyrazole-C = N), 1564 (Triazole N = N and Thiazole-C = N).

2-[4–(3-Phenyl-4,5-dihydro-1H-pyrazol-5-yl)-1H-1,2,3-triazol-1-yl]-N-(4-phenylthiazol-2-yl) acetamide (7b)

The title compound was prepared from 2-[4–(3-oxo-3-phenylprop-1-en-1-yl)-1H-1,2,3-triazol-1-yl]-N-(4-phenylthiazol-2-yl) acetamide (6b). Yield; 0.28 g (68%), mp: 219 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 3.13–3.19 (m, 2H, dihydropyrazole-CH₂), 5.00 (t, J = 10 Hz, 1H, dihydropyrazole-CH), 5.49 (s, 2H, CH₂-C = O), 7.36 (s, 2H, phenyl-C₄-H, dihydropyrazole-phenyl-C₄-H), 7.39–7.48 (m, 4H, dihydropyrazole-phenyl-C_3,5-Hs, phenyl-C_3,5-Hs), 7.60 (s, 1H, dihydropyrazole-NH), 7.67 (d, J = 8 Hz, 2H, dihydropyrazole-phenyl-C_2,6-Hs), 7.71 (s, 1H, Triazole-CH), 7.93 (d, J = 4 Hz, 2H, phenyl-C_2,6-Hs), 8.13 (s, 1H, Thiazole-CH), 12.83 (s, 1H, NH). ¹³C-NMR (125 mHz, DMSO-D₆) δ 28.94 (Dihydropyrazole-C₄), 51.81 (CH₂), 55.60 (Dihydropyrazole-C₅), 108.55 (Thiazole-C₅), 123.68 (Triazole-C₅), 125.48 (dihydropyrazole-phenyl-C_2,6), 126.64 (Phenyl-C_3,5), 127.89 (Phenyl-C₄), 128.18 (Phenyl-C_2,6), 128.47 (dihydropyrazole-phenyl-C_3,5), 128.75 (Triazole-C₄), 134.59 (Phenyl-C₁), 138.21 (dihydropyrazole-phenyl-C₄), 148.84 (dihydropyrazole-phenyl-C₁), 148.97 (Thiazole-C₄), 149.85 (Dihydropyrazole-C₃), 157.78 (Thiazole-C₂), 165.45 (NH-C = O). Anal. Calc. for C₂₂H₁₉N₇OS: C, 61.52; H, 4.46; N, 22.83; S, 7.47. Found: C, 61.75; H, 4.62; N, 22.71; S, 7.73. IR (KBr, cm⁻¹): 3449 (Amide-NH), 3210 (Dihydropyrazole-NH), 3061 (Triazole-CH), 1684 (C = O, Dihydropyrazole-C = N), 1568 (Triazole N = N and Thiazole-C = N).

2-{4-[3-(p-tolyl)-4,5-dihydro-1H-pyrazol-5-yl]-1H-1,2,3-triazol-1-yl}-N-(4-phenylthiazol-2-yl)acetamide (7c)

The title compound was prepared from 2-{4-[3-oxo-3-(p-tolyl)prop-1-en-1-yl]-1H-1,2,3-triazol-1-yl}-N-(4-phenylthiazol-2-yl) acetamide (6c). Yield; 0.3 g (70.6%), mp: 263 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 2.33 (s, 3H, CH₃), 3.09–3.16 (m, 2H, dihydropyrazole-CH₂), 4.97 (t, J = 10 Hz, 1H, dihydropyrazole-CH), 5.49 (s, 2H, CH₂-C = O), 7.22 (d, J = 8 Hz, 2H, dihydropyrazole-phenyl-C_3,5-Hs), 7.35 (t, J = 6 Hz, 1H, phenyl-C₄-H), 7.44–7.49 (m, 3H, dihydropyrazole-NH, phenyl-C_3,5-Hs), 7.56 (d, J = 8 Hz, 2H, dihydropyrazole-phenyl-C_2,6-Hs), 7.71 (s, 1H, Triazole-CH), 7.92 (d, J = 8 Hz, 2H, phenyl-C_2,6-Hs), 8.12 (s, 1H, Thiazole-CH), 12.84 (br s, 1H, NH). ¹³C-NMR (125 mHz, DMSO-D₆) δ 21.37 (CH₃), 38.88 (Dihydropyrazole-C₄), 51.91 (CH₂), 56.05 (Dihydropyrazole-C₅), 109.05 (Thiazole-C₅), 124.16 (Triazole-C₅), 125.98 (tolyl-C_2,6), 126.15 (Phenyl-C_3,5), 128.39 (Phenyl-C₄), 129.25 (Phenyl-C_2,6), 129.55 (tolyl-C_3,5), 130.85 (Triazole-C₄), 134.57 (Phenyl-C₁), 138.14 (tolyl-C₄), 149.21 (tolyl-C₁), 149.48 (Thiazole-C₄), 149.92 (Dihydropyrazole-C₃), 157.80 (Thiazole-C₂), 165.41 (NH-C = O). Anal. Calc. for C₂₃H₂₁N₇OS: C, 62.28; H, 4.77; N, 22.11; S, 7.23. Found: C, 62.47; H, 4.83; N, 21.98; S, 7.44. IR (KBr, cm⁻¹): 3449 (Amide-NH); 3213 (Dihydropyrazole-NH); 3061 (Triazole-CH); 2954 (CH₃); 1686 (C = O, Dihydropyrazole-C = N); 1568 (Triazole N = N and Thiazole-C = N).

2-{4-[3–(4-Fluorophenyl)-4,5-dihydro-1H-pyrazol-5-yl]-1H-1,2,3-triazol-1-yl}-N-(4-phenylthiazol-2-yl)acetamide (7d)

The title compound was prepared from 2-{4-[3–(4-fluorophenyl)-3-oxoprop-1-en-1-yl]-1H-1,2,3-triazol-1-yl}-N-(4-phenylthiazol-2-yl)acetamide (6d). Yield 0.25 g (58.2%), mp: 238 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 3.12–3.18 (m, 2H, dihydropyrazole-CH₂), 5.00 (dist. t, 1H, dihydropyrazole-CH), 5.49 (s, 2H, CH₂-C = O), 7.25 (t, J = 8 Hz, 2H, dihydropyrazole-phenyl-C_3,5-Hs), 7.35 (t, J = 6 Hz, 1H, phenyl-C₄-H), 7.46 (t, J = 8 Hz, 2H, phenyl-C_3,5-Hs), 7.59 (s, 1H, dihydropyrazole-NH), 7.71 (br s, 3H, Triazole-CH, dihydropyrazole-phenyl-C_2,6-Hs), 7.92 (d, J = 8 Hz, 2H, phenyl-C_2,6-Hs), 8.13 (s, 1H, Thiazole-CH), 12.84 (s, 1H, NH). ¹³C-NMR (125 mHz, DMSO-D₆) δ 38.85 (Dihydropyrazole-C₄), 51.91 (CH₂), 56.21 (Dihydropyrazole-C₅), 109.05 (Thiazole-C₅), 115.82 (F-phenyl-C₃), 116.04 (F-phenyl-C₅), 124.21 (Triazole-C₅), 126.16 (Phenyl-C_3,5), 127.99 (F-phenyl-C_2,6), 128.07 (F-phenyl-C₁), 128.40 (Phenyl-C₄), 129.26 (Phenyl-C_2,6), 130.18 (Triazole-C₄), 134.57 (Phenyl-C₁), 143.19 (Dihydropyrazole-C₃), 149.48 (Thiazole-C₄), 157.80 (Thiazole-C₂), 161.29 (F-phenyl-C₄), 165.41 (NH-C = O). Anal. Calc. for C₂₂H₁₈FN₇OS: C, 59.05; H, 4.05; N, 21.91; S, 7.17. Found: C, 59.32; H, 4.23; N, 22.15; S, 7.29. IR (KBr, cm⁻¹): 3449 (Amide-NH); 3213 (Dihydropyrazole-NH); 3068 (Triazole-CH); 1686 (C = O, Dihydropyrazole-C = N); 1567 (Triazole N = N and Thiazole-C = N); 1232 (C-F).

2,2’-[(3-Oxopenta-1,4-diene-1,5-diyl)bis(1H-1,2,3-triazole-4,1-diyl)]bis(N-(4-phenylthiazol-2-yl)acetamide) (8)

A half equimolar amount of acetone (0.02 g, 0.02 ml, 0.32 mmol) was added gradually to a solution of aldehyde derivative 5 (0.2 g, 0.64 mmol) in absolute ethanol (15 ml). The mixture was stirred at room temperature for 5 min then NaOH 10% (0.64 ml) was added gradually. The mixture was stirred for 24 h then neutralised with glacial acetic acid. The formed precipitate was filtered, washed, dried and crystallised from ethanol. Yield 0.15 g (82.9%), mp: 225 °C, ¹H-NMR (400 mHz, DMSO-D₆) δ 5.13 (br s, 4H, 2x CH₂-C = O), 7.02 (s, 2H, 2x CH=CH-CO), 7.08 (s, 2H, 2x CH=CH-CO), 7.26 (t, J = 6 Hz, 2H, 2x phenyl-C₄-H), 7.37 (t, J = 8 Hz, 4H, 2x phenyl-C₃,₅-H_s), 7.44 (s, 2H, 2x Triazole-CH), 7.80 (d, J = 8 Hz, 4H, 2x phenyl-C₂,₆-H_s), 7.92 (s, 2H, 2x Thiazole-CH), 12.87 (s, 2H, 2x NH). ¹³C-NMR (125 mHz, DMSO-D₆) δ 51.75 (2x CH₂), 102.10, 109.10 (2x Thiazole-C₅), 125.97 (2x Triazole-C₅), 126.53 (2x Phenyl-C_3,5), 127.82 (2x Phenyl-C₄), 128.24 (2x Phenyl-C_2,6), 129.15 (2x CH=CH-CO), 129.53 (2x Triazole-C₄), 134.69 (2x Phenyl-C₁), 135.20 (2x CH=CH-CO), 150.12 (2x Thiazole-C₄), 157.95 (2x Thiazole-C₂), 165.86 (2x NH-C = O), 169.24 (C = O). Anal. Calc. for C₃₁H₂₄N₁₀O₃S₂: C, 57.40; H, 3.73; N, 21.59; S, 9.89. Found: C, 57.61; H, 3.85; N, 21.67; S, 9.95. IR (KBr, cm⁻¹): 3400 (NH); 3080 (Triazole-CH); 1701 (C = O); 1628 (Amide-C = O, Triazole N = N and Thiazole-C = N).

Biology

MTT assay

Wi-38 cells are human foetal lung fibroblast cells derived from the lung tissue of a 3-month-old, female, embryo^105–108. A549 cells are cancerous epithelial cells isolated from the lung tissue of a white, 58-year-old male with lung cancer^109–113. Both Wi-38 (CCL-75) and A549 (CCL-185) cell lines were purchased from the American Type Culture Collection (ATCC, USA). Firstly, Wi-38 cell line was utilised to assess the compounds’ cytotoxicity. The Wi-38 cell line was cultivated in DMEM media with 10% FBS, seeded in 96-well cell culture plates at a density of 5 x10³ cells per well, and incubated at 37 °C in a 5% CO₂ incubator. Serial doses of the synthesised compounds and Cisplatin were incubated with Wi-38 cells for 72 h after 24 h had passed for cell attachment. The MTT technique was used to measure cell viability⁵⁸. Each well received 20 µL of a 5 mg/ml MTT solution from Sigma, USA, and the plate underwent a 3-h incubation period at 37 °C. Following the removal of the MTT solution, 100 µL of DMSO was added, and the absorbance of each well was assessed at 570 nm using a microplate reader (BMG LabTech, Germany). The Graphpad Instat software was used to estimate the dose (EC₅₀ and EC₁₀₀) values (at 50% and 100% cell viability, respectively) of the investigated substances. DMEM (Lonza, USA) supplemented with 10% FBS was used to culture the human lung cancer cell line (A549) in order to test the anticancer effects of the aforementioned substances. The MTT technique was used as previously mentioned. Using the Graphpad Instat program, IC₅₀ values were determined. Additionally, cellular morphological alterations were examined using a phase contrast inverted microscope and a digital camera (Olympus, Japan) before and after treatment with the studied anticancer agents.

T-type calcium channel blocking activity

All compounds were evaluated at concentration of 10 µM for their abilities to block T-type (Ca_V3.2) calcium channels through employing the whole cell patch-clamp assay⁶¹ using Human embryonic kidney cells (HEK) tsA-201 cells purchased from ATCC, USA (293 T cells: CRL-3216). Chemicals, and protocols for cell culture and transient transfection as well as electrophysiological recordings are detailed as previously reported^114–116 in the supplementary data.

In vitro MMP-2, MMP-9, MMP-10 and MMP-13 inhibition assays

The assay was performed utilising MMP-9 Inhibitor Assay Kit (Abcam, catalog # ab139448), MMP-2 inhibitor screening colorimetric assay kit (Abcam; catalogue # ab139446), MMP-10 Assay Kit (Abcam, catalogue # ab139457), and MMP-13 Inhibitor Screening Assay Kit (Abcam, catalog # ab139450), respectively, following the manufacturer’s instructions^92,117,118.

DPPH radical scavenging assay

The antioxidant activity of the synthesised compounds was evaluated in vitro by 1,1-diphenyl-2-picrylhydrazyl DPPH radical scavenging assay. 1 mg of the powdered compounds was weighed and dissolved in 1:1 Water: DMSO (v/v) to prepare various concentrations. Next, 0.6 mM of DPPH solution was prepared by dissolving 24 mg of DPPH in 100 ml of absolute ethanol. Then, 1 ml of the ethanol solution of DPPH was mixed with an equal solution of the synthesised compound to obtain the following final concentrations of 500, 250, 125, 100, 62.5 and 31.25 µg/mL. The selected compound 6d was left with the dye for 12 h at room temperature with complete dark conditions to allow the compound to react with stable free radicals. The absorbance of the solution was read at a wavelength of 517 nm using UV-Visible spectrophotometer, and the scavenging efficiency was measured. The inhibition ratio (I%) of the synthesised compounds was estimated according to the following equation:

$$\text{I\hspace{0.17em}}\%\hspace{0.17em}=\hspace{0.17em}\left(A_0\hspace{0.17em}-\hspace{0.17em}{A}_1\right)/A_0\hspace{0.17em}\times \text{\hspace{0.17em}1}00,$$

where A₀ stands for the absorbance of the control, while A₁ stands for the absorbance of the compounds. Then, IC₅₀ was calculated (Supplementary Figure S27).

Combination studies and data analysis

MTT method was done as described above⁵⁸. Combination studies and data analysis were performed as previously reported⁹² and detailed in the supplementary data.

Molecular modelling

Docking

Docking study was performed employing Molecular Operating Environment (MOE) 2019.0102 software⁸⁰. The 3D structure of the investigated compound 6d was built in silico using MOE, optimised by adding hydrogens then subjected to energy minimisation using Amber10:EHT force field with reaction-field electrostatics (an interior dielectric constant of 1 and an exterior dielectric of 80) using an 8–10 Å cut-off distance. The coordinates of the MMP-9 catalytic domain were retrieved from the RCSB PDB (PDB ID: 1GKC⁸¹) Unwanted residues were removed, then the "Structure Preparation" module was used employing the default settings to correct structural problems, followed by adding hydrogens and assigning the protonation states employing the Protonate3D application. Three calcium ions, two zinc ions, and one MMP-9 monomer made up the optimised domain. The ‘Site Finder’ function of MOE 2019.0102 was then used to locate the active site while taking the reported key amino acids and binding sites into account⁸². The "Triangle Matcher" placement method^83–85 generated 100 docked modes with the top 5 London dG scores^85,86 being refined according to default rigid refinement settings and re-scored employing the GBVI/WSA dG scoring function¹¹⁹. The poses were ranked by the scores from the GBVI/WSA binding free energy calculation. This docking output was then inspected, and the complexes were analysed in terms of protein-ligand interactions employing the standard protein–ligand interaction fingerprints (PLIF) tool implemented in MOE (Supplementary Table S3 and Fig. S26). The pose of the least energy conformer with the lowest docking score and protein-ligand interactions was selected among the cluster for further molecular dynamics simulations. Repeated docking experiments nearly reproduced the best selected binding mode at comparable scores.

Molecular dynamics simulations

MDs, utilised software, trajectory analysis and MM-PBSA Calculations are detailed in the supplementary data.

Statistical analysis

The data were presented as mean ± standard error of the mean (SEM). The significant values were considered at p < 0.05. ANOVA by Tukey’s test was used for evaluating the difference between the mean values. Analysis was done for three measurements employing SPSS software version 16 unless otherwise indicated.

Funding Statement

The authors would like to thank the Science, Technology & Innovation Funding Authority (STIFA) for funding this work through the Young Researcher Grant (Proposal ID 43024). This research project also was funded by the Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Sciences, South Dakota State University including support from the Rollins Juhnke Fund and Francis Miller Fund. Gerald W. Zamponi is supported by Grants from the Canadian Institutes of Health Research and by a grant from Alberta Innovates, and holds a Canada Research Chair.

Authors’ contributions

Conception and design: K. A. Ismail, A-Mohsen M. E. Omar, M. Teleb, G. W. Zamponi, H. Fahmy; methodology, analysis and interpretation of data: H. Gamal, K. A. Ismail, A-Mohsen M E Omar, M. Teleb, M. M. Abu-Serie, S. Huang, A. S. Abdelsattar, G. W. Zamponi, H. Fahmy; writing –drafting of the paper: H. Gamal, M. Teleb, M. M. Abu-Serie; revision for intellectual content and final approval of the version: K. A. Ismail, M. Teleb, G. W. Zamponi, H. Fahmy. All authors agreed to be accountable for all aspects of the work.

Disclosure statement

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

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.