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. 2023 May 10;8(20):17552–17562. doi: 10.1021/acsomega.2c07569

Design and Synthesis of Thiazole Scaffold-Based Small Molecules as Anticancer Agents Targeting the Human Lactate Dehydrogenase A Enzyme

Dolly Sharma †,‡, Mamta Singh ‡, Jayadev Joshi §, Manoj Garg ‡, Vidhi Chaudhary ∥, Daniel Blankenberg §, Sudhir Chandna ⊥, Vinit Kumar ‡,*, Reshma Rani #,*
PMCID: PMC10210175  PMID: 37251149

Abstract

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A new series of thiazole central scaffold-based small molecules of hLDHA inhibitors were designed using an in silico approach. Molecular docking analysis of designed molecules with hLDHA (PDB ID: 1I10) demonstrates that Ala 29, Val 30, Arg 98, Gln 99, Gly 96, and Thr 94 possessed strong interaction with the compounds. Compounds 8a, 8b, and 8d showed good binding affinity (−8.1 to −8.8 kcal/mol), whereas an additional interaction of NO2 at the ortho position in compounds 8c with Gln 99 through hydrogen bonding enhanced the affinity to −9.8 kcal/mol. Selected high-scored compounds were synthesized and screened for hLDHA inhibitory activities and in vitro anticancer activity in six cancer cell lines. Biochemical enzyme inhibition assays showed the highest hLDHA inhibitory activity observed with compounds 8b, 8c, and 8l. Compounds 8b, 8c, 8j, 8l, and 8m depicted significant anticancer activities, exhibiting IC50 values in the range of 1.65–8.60 μM in HeLa and SiHa cervical cancer cell lines. Compounds 8j and 8m exhibited notable anticancer activity with IC50 values of 7.90 and 5.15 μM, respectively, in liver cancer cells (HepG2). Interestingly, compounds 8j and 8m did not induce noticeable toxicity in the human embryonic kidney cells (HEK293). Insilico absorption, distribution, metabolism, and excretion profiling demonstrates that the compounds possess drug-likeness, and results may pave the way for the development of novel thiazole-based biologically active small molecules for therapeutics.

1. Introduction

Cancer cells present largely different bioenergetics than normal cells and are dependent on an enhanced rate of tumor glycolysis.1 Cancer cell metabolism, specifically tumor glycolysis, has emerged as a unique cancer phenotype due to higher consumption of glucose resulting in higher lactate production in cancer cells than in normal cells even under normoxic conditions. Consequently, tumor glycolysis creates acidosis in the extracellular matrix, which facilitates tumor initiation, progression, invasion, and metastasis.2 Enhanced rate of tumor glycolysis in cancer cells ensures their high energy and metabolite demand, resulting in excess lactate and H+ ion production, which is then transported outside the cell by MCT enzymes and establishes the lactate flux.3−5 Therefore, cancer cells are characterized by an enhanced rate of tumor glycolysis controlled by the overexpression of several enzymes, cofactors, and transporters. A very close association between cancer cell metabolism and cancer stemness was also established.6 Cancer cells represent common characteristic features such as an enhanced rate of aerobic glycolysis, a higher rate of glucose consumption and lactate production, and an increased rate of extracellular acidosis, which can be exploited for drug development.7−10 Therefore, tumor glycolysis is considered a novel target in search of better cancer treatment options. Moreover, in normal cells, the last step of glycolysis produces pyruvate, which is considered an energy hub from where pyruvate goes along with three distinct pathways: (i) formation of lactate; (ii) conversion to acetyl-CoA, and (iii) conversion to alanine. Conversely, in cancer cells, most of the pyruvate is reduced to lactate coupled with the oxidation of NADH to NAD+ catalyzed by the Lactate Dehydrogenase (LDH) enzyme.7−10 The LDH enzyme is a tetrameric protein composed of two different subunits ldha and ldhb, which are encoded by two separate genes ldha and ldhb, respectively. In humans, LDH exists in four isoforms formed by various possible combinations of these two subunits. Among these, the LDHA and LDHB are homoisomers of ldha and ldhb subunits, respectively (Figure 1).7 The human LDHA (hLDHA) enzyme is overexpressed in almost all metabolic cancer and exists at the end of tumor glycolysis7,11 at the bifurcation point of pyruvate, which makes it a viable target from where the selective inhibition of hLDHA can selectively kill the cancer cells via blocking energy and metabolites supply (Figure 1).11

Figure 1.

Figure 1

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LDH inhibition through LDH inhibitors kill cancer cells.

Potential small molecules acting as hLDHA inhibitors can kill cancer cells by serving as starving agents.5 Several small molecules including natural and synthetic compounds with molecular diversity exhibiting significant hLDHA inhibitory activity have been discovered; however, very few have entered clinical trials.7−11 For example, the nonselective natural product gossypol entered into clinical trial but failed due to side effects that may arise due to the presence of aldehyde functional groups (Figure 2). Later, the FX-11 analogue of half gossypol was discovered, which showed potential hLDHA inhibitory activity (Figure 2). A new class of N-hydroxy indole-based hLDHA inhibitor (NHI) was developed, which showed significant selectivity (Figure 2).12−16 Although several molecules have been discovered,17−19 still there is a large chemical gap available to discover new hLDHA inhibitors as potential anticancer agents.

Figure 2.

Figure 2

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Chemical structure of potential hLDH5 inhibitors.

Herein, we have designed a distinguished class of small molecules based on a central scaffold thiazole using an in silico approach20 to disrupt the tumor glycolysis by inhibiting the hLDHA enzyme. Small molecules including thiazole heterocycles displayed a wide range of biological activities and play an important role in medicinal chemistry and drug discovery.21−25 It is an essential part of several natural products such as vitamin B1- Thiamine and several synthetic anticancer drugs; thus, thiazole core-based molecules and hybrid structures would be potential anticancer compounds.26 Lipophilic properties of di-substituted thiazole at two positions make these compounds efficient for transport through a biological membrane. In this paper, we studied the molecular interaction of this different class of small molecules with the hLDHA by molecular docking, synthesized high-scored molecules, and screened them for hLDHA activity evaluation. The most potent molecules were further screened for anticancer activity evaluation in cancer cell lines, and their pharmacokinetic profile was evaluated.

2. Results and Discussion

2.1. Pharmacophore Requirement of the Designed Molecules

The binary and ternary X-ray crystal of hLDH5 or LDHA subunit with the cofactor (NADH) and substrate (pyruvate) shows that the binding pocket is small, and the active site is situated in a deep position; thus, accessibility of the compound to the binding cavity is narrow.12 The binding cavity holds both the substrate and cofactor and is rich in arginine amino acids, which are cationic residues. Thus, the overall binding cavity is polar and cationic; therefore, most of the inhibitors discovered so far have a carboxylate functional group. In some inhibitors, the carboxylate group is in close proximity with hydroxyl and carbonyl groups, which act as the surrogates of the substrate of hLDHA. Considering these pharmacophore requirements, we have designed a series of molecules based on a thiazole central scaffold having a carboxylate group which is part of the aliphatic ring and two aromatic rings that are directly bonded with the thiazole scaffold (Figure 3). This type of series has never been explored before, and such type of unusual class of molecules based on a thiazole scaffold is an important class of molecules with pharmaceutical values.

Figure 3.

Figure 3

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Basic pharmacophore model for target molecules.

2.2. Molecular Docking Study

Molecular docking studies of all designed compounds (Figure 4, Tables 1 and S1) were performed using software ChemDraw (https://chemdrawdirect.perkinelmer), AutoDock (https://autodock.scripps.edu/), Chimera (https://www.cgl.ucsf.edu/chimera), and PyMOL (https://pymol.org/2/). The geometry of the molecules was optimized using Avogadro software (http://avogadro.openmolecules.net.) The structure of all the molecules was made by using ChemDraw and then transformed into a 3D structure suitable for docking using Avogadro (https://two.avogadro.cc/) and OpenBabel software (https://open-babel.soft). To examine the best possible binding modes of a compound in the binding cavity of hLDHA (PDB ID: 1I10), AutoDock was used, and protein-docked compound images were prepared using Chimera or PyMOL software. All the docking poses were ranked to calculate the ΔG bind values to attain acceptable levels of hLDHA inhibition within this chemical class. To obtain the structure–activity relationship (SAR) R1, R2, and R3 were selected as both the electron-withdrawing and electron-donating groups at the ortho, meta, and para positions, respectively (Figure 4, Tables 1 and S1).

Figure 4.

Figure 4

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Structure of target molecules.

Table 1. Defined R1, R2, and R3, Binding Energy (kcal/mol) of Compounds 8a–8m.

S. no. compounds no. R1, R2, R3 binding energy (kcal/mol) S. no. compounds no. R1, R2, R3 binding energy (kcal/mol)
1. 8a R1=R2=R3=H –8.8 8. 8h R1=R2=CH3, R3=H –7.0
2. 8b R1=OCH3, R2=R3=H –8.1 9. 8i R1=H, R2=Cl, R3=H –7.2
3. 8c R1=NO2, R2=R3=H –9.8 10. 8j R1=R2=H, R3=OCH3 –7.1
4. 8d R1=CH3, R2=R3=H –8.5 11. 8k R1=R2=H, R3=NO2 –6.2
5. 8e R1=Cl; R2=R3=H –7.2 12. 8l R1=R2=H, R3=CH3 –7.6
6. 8f R1=H; R2=OCH3, R3=H –6.3 13 8m R1=R2=H; R3=Cl –7.6
7. 8g R1=H; R2=NO2, R3=H –6.4        
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Docking results of the designed molecules with hLDHA were found in the range of −9.8––6.2 kcal/mol and are summarized in Table 1. In compound 8a, R1=R2=R3=H showed strong binding affinity, exhibiting a binding energy of −8.8 kcal/mol. When H of the ortho position was replaced by the OCH3 leading to 8b (R1=OCH3) showed a minor reduction in binding affinity (−8.1 kcal/mol) might be due to −I effect as well as due to the electron pair of O atom of −OCH3, served resonance effect (+R) and can lose an electron. Further, if a stronger electron-withdrawing group, i.e., NO2, was inserted at the ortho position (8c, R1=NO2), the binding energy significantly increases to −9.8 kcal/mol. Besides, the insertion of an electron-donating group CH3 at the ortho position (8d, R1=CH3) slightly decreases the binding energy to −8.5 kcal/mol. In the case of 8e, the electron-withdrawing group “–Cl” (also served resonance effect, +R) at ortho position showed reduced binding affinity, as Cl exhibits a strong −I effect, and +R. Moreover, the substitution of the electron-withdrawing group OCH3, NO2, and Cl and the electron-donating group CH3 at the meta position (R2) showed lower binding affinity (Table 1) than substitution at the ortho position. Further, substitution on the para position (R3) by an electron-withdrawing group as well as an electron-donating group showed better results than substitution on the meta position; however, electron-withdrawing groups at the ortho position were found to be the preferred position for enhanced binding affinity.

Molecular docking analysis of the designed molecules with the hLDHA (PDB ID: 1I10) enzyme revealed that all the molecules showed a common binding mode and presented similar types of binding interactions in the binding cavity of hLDHA (Figure 5). The analysis revealed that amino acids Ala 29, Val 30, Arg 98, Gln 99, Gly 96, and Thr 94 play an important role to possess strong interaction with the compounds (Figure 5). More specifically, the complex structure of hLDHA with 8c indicates that NO2 at the ortho position showed interaction with Gln 99 through hydrogen bonding. The OCH3 of ester exhibited interaction with Ala 29 and Val 30, whereas oxygen of the carbonyl of the ester exhibited interaction with Arg 98 (Figure 5). Based on docking results, 8a–d and 8l were selected for synthesis and biological studies.

Figure 5.

Figure 5

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Compounds 8a, 8b, 8c, and 8d complexed with hLDHA (PDB ID: 1I10).

2.3. Chemistry

Selected high-scored compounds 8a–d, 8j, 8l, and 8m were synthesized from N-alkylation of the final intermediates 7a–d, 7j, 7l, and 7m with the reaction of methyl 2-bromoacetate in the basic medium at 80 °C in dry acetone as a solvent in the presence of potassium carbonate (Figure 6). The thiazole central scaffold-based starting materials 7a–d, 7j, 7l, and 7m for final compounds were synthesized by following a reported synthetic protocol with slight modification to improve the yield using acetophenone.27,28

Figure 6.

Figure 6

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Synthetic scheme for the synthesis of final compounds 8a–d, 8j, 8l, and 8m.

All the starting materials (7a–d, 7j, 7l, and 7m) were characterized by 1HNMR and mass spectrometry for further reaction. Catalytic bromination of acetophenone (1) using bromine in the presence of Lewis catalyst AlCl3 in dry ether resulted in 2-bromo-1-phenylethanone (2), which on treatment with an aqueous solution of ammonium thiocyanate yielded the phenacyl thiocyanate (3, 1-phenyl-2-thiocyanatoethanone). The condensation of phenacyl thiocyanate (3) with various respective amine hydrochlorides (5a–d, 5j, 5l, and 5m) in dry methanol maintaining pH 4–5 under nitrogen resulted in hydrochloride salts of compounds 6a–d, 6j, 6l, and 6m. So obtained hydrochloride salts 6a–d, 6j, 6l, and 6m on treating with aqueous NaOH at pH ∼ 10 led to compounds 7a–d, 7j, 7l, and 7m, which were used as starting materials for the synthesis of target compounds. All the synthesized compounds 8a–d, 8j, 8l, and 8m were purified by column chromatography and the structure of compounds 8a–d, 8j, 8l, and 8m were confirmed by 1H and 13C NMR and mass spectrometry.

2.4. Biological Activities

2.4.1. hLDHA Inhibitory Activities

The pure and well-characterized compounds 8a–h were screened for hLDHA inhibitory activities using the reported protocol.16 For hLDHA inhibitory activities, compounds were dissolved in DMSO, and then dilutions were made using sterile water. In all the biological screenings, the concentration of DMSO was kept below 1%. To determine the inhibitory potencies, compounds were screened on hLDHA in competition with substrate pyruvate by measuring the change in the intensity of NADH via oxidation at pH 7.2. The decrease in NADH fluorescence or absorbance was followed by a spectrofluorometer (BioTek USA) at 340 nm excitation and 460 nm emission wavelengths. The total volume used in each well was 200 μL, which constitutes 152 μL of NADH, sodium pyruvate in PBS buffer, 8 μL compound, and 40 μL of the hLDHA enzyme. The percentage inhibition is calculated assuming 100% inhibition with the reference summarized in Table 2. The hLDHA enzyme inhibitory screening of the selected compounds showed that compounds 8a and 8b showed a moderate inhibitory effect against the hLDHA enzyme in competition with the substrate pyruvate. Compounds 8b, 8c, 8d, 8j, and 8l showed good enzyme inhibitory activities i.e., 48, 53, 43, 49, and 52% in competition with pyruvate. Among all, compounds 8c and 8l endowed approximate same enzyme inhibitory activities, whereas other compounds showed moderate inhibitory effect against the hLDHA enzyme.

Table 2. Percentage Inhibitory hLDHA Activity of 100 μM Repeated in Triplicate and cLogp Values.
compd. no. % inhibitory hLDHA activity (μM) cLogp compd. no. % inhibitory hLDHA activity (μM) cLogp
8a 33 2.489 8j 49 3.555
8b 48 3.555 8l 52 4.135
8c 53 3.379 8m 41 4.349
8d 43 4.135      
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The percentage hLDHA inhibitory activity of compounds 8a–d, 8j, 8l, and 8m was measured at 100 μM concentration using the following equation

2.4.1.

All compounds 8a–d, 8j, 8l, and 8m showed inhibitory hLDHA activities having an acceptable cLogp value and were considered for further biological activities’ evaluation. The cLogp values of all compounds were calculated by software ChemDraw version 12.0 for measurement of hydrophilicity and are reported in Table 2.

2.4.2. In Vitro Cytotoxicity of Target Compounds

In vitro cytotoxicity against various human cancer cell lines was determined using a reported protocol.29 The selected compounds (8a–d, 8j, 8l, and 8m) were screened for anticancer activity in six cancer cell lines including human Pancreatic Ductal Adenocarcinoma (02.03, 04.03 and 03.27), liver cancer cells (HepG2), cervical cancer cell lines (HeLa and SiHa), and human embryonic kidney cells (HEK-293). Cancer cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal serum albumin and 1% antibiotic/antimycotic solution. All the cultures were maintained at 37 °C in an incubator containing 5% CO2. Both normal and cancer cells were seeded in 96-well plates (5.0 × 105cells per well). The cells were exposed to serially diluted concentrations of compounds with a starting concentration of 100 μM. Non-treated control cells were also maintained in the same conditions to compare the growth inhibition. The content in all respective wells including tests and control was decanted after 72 h of treatment and 20 μL of reconstituted MTT (Sigma) was added. After 2 h of dark incubation in a 5% CO2 humidified incubator, the supernatant was removed and 100 μL of MTT solubilization solution was added and kept in a shaking incubator at 37 °C to solubilize formazan crystals. The absorbance was recorded at 570 nm using a microplate reader. The experiments were performed in triplicate, and the cell viability was calculated from the curves of the mean OD values and plotted against the drug concentration. The IC50 value was analyzed using GraphPad Prism 8 reported in Table 3 and Figure 7.

Table 3. In Vitro Cytotoxicity (IC50) of Compounds 8a–d, 8j, 8l, and 8m against Pancreatic, Liver, and Cervical Cancer Cell Linesa.
    cancer cells
    pancreatic IC50 (μM)
 liver IC50 (μM) cCervical IC50 (μM)
S. no tested comp. 02.03 03.27 04.03 HepG2 HeLa SiHa
1. 8a 81.50 72.23 >100 75.50 NT NT
2. 8b 40.12 13.48 52.23 22.66 2.97 4.20
3. 8c >100 25.50 22.10 15.38 6.02 6.78
4. 8d >100 >100 >100 79.20 NT NT
5. 8j 72.20 11.51 75.20 7.90 6.75 2.32
6. 8l 61.23 10.84 50.32 83.68 8.65 6.16
7. 8m 23.22 22.08 52.10 5.15 16.96 1.65
8. doxorubicin       1.130 0.1431 8.332
9. gemcitabine 0.38 0.42 0.19      
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a

Data were obtained as mean SD from 3 independent repeats (n = 3).

Figure 7.

Figure 7

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IC50 values of most active compounds on Panc 03.27, HepG2, HeLa, and SiHa cancer cell lines.

2.4.2.1. In Vitro Cytotoxicity in Human PDAC (02.03, 03.27, and 04.03) Cell Lines

These compounds showed moderate activity against all three (02.03, 03.27, and 04.03) cancer cell lines. Compound 8a, where R1, R2 and R3 are H, showed moderate activity in the high micromolar range against 02.03 and 03.27 cancer cell lines, whereas it was found inactive against 04.03 cell lines (Table 3, Figures 7 and S1). When hydrogen at the ortho position (R1=H) was replaced by electron-withdrawing group OCH3, the resulting compound 8b showed improved inhibitory activity against 02.03, 03.27, and 04.03 cancer cell lines exhibiting IC50 values of 40.12, 13.48, and 52.23 μM, respectively (Table 3, Figures 7, S2 and S3). Insertion of a more electron-withdrawing group −NO2 at the ortho position leads to compound 8c that further enhanced the inhibitory effect on both 03.27 and 04.03 cancer lines; however, for 02.03 cancer cell lines, inhibitory activity decreases (Table 3, Figure 7). When the electron-donating group (−CH3) was inserted at the ortho position in place of the withdrawing group (−OCH3), the resulting compound 8d showed a reduction in inhibitory activity against all the three PDAC cancer cell lines. Upon insertion of the electron-withdrawing group OCH3 at the para position, the resulting compound 8j exhibited a significant inhibitory effect on PDAC 03.27 having an IC50 value of 11.50 μM. However, it showed moderate activity against 02.03 and 04.03 cancer cell lines in the high micromolar range. Compound 8l where the electron-donating group (−CH3) was placed at the para position exhibited an IC50 value of 10.84 μM against the PDAC 03.27 cancer cell lines. In addition, if the -Cl was placed at the para position, the resulting compound 8m showed moderate activity in the low micromolar range (Table 3, Figure 7). These data suggest that the electron-withdrawing groups are a better choice than the electron-donating groups at the ortho position for the development of more potent compounds in this series. Moreover, the substitution at the ortho position is preferable over the para position.

2.4.2.2. In Vitro Cytotoxicity in the Liver (HepG2) Cancer Cell Line

Compound 8a (where R1, R2, and R3 are H) showed moderate activity against HepG2, exhibiting IC50 values of 75.50 μM (Table 3, Figures 7 and S4). Compound 8b, where hydrogen at the ortho position (R1=H) was replaced by the electron-withdrawing group (R1=OCH3), showed a marked increment in inhibitory activity, showing IC50 values of 22.66 μM. Compound 8c showed a significant increment in inhibitory activity, having IC50 value of 15.38 μM in liver cancer cells (HepG2). In the case of compound 8l, R3=CH3 showed a marked reduction in anti-cancer activity, with an IC50 value of 83.68 μM. In compound 8m, Cl is placed at the para position and is endowed an IC50 value of 5.15 μM HepG2 (Table 3, Figure 7). Electron-withdrawing groups are a better choice than the electron-donating groups at the ortho position for the development of more potent compounds.

2.4.2.3. In Vitro Cytotoxicity in the Cervical (HeLa) Cancer Cell Line

Results of in vitro cytotoxicity against the cervical (HeLa) cancer cell line revealed that the substitution on the ortho position by the OCH3 moiety resulted in compound 8b (R1=–OCH3), which showed a marked increment in inhibitory activity, exhibiting IC50 values of 2.97 μM against the HeLa cell line (Table 3, Figures 7 and S5). Further substitution at the same position by the −NO2 group leads to compound 8c showing very good inhibitory activity, with an IC50 value of 6.02 μM in HeLa; however, it showed less activity than 8b. Compound 8j of this series showed almost similar activity to 8c, exhibiting an IC50 value of 6.75 μM (Table 3, Figure 7). Moreover, for compound 8l (R3=CH3), substitution by CH3 at the para position showed a small reduction in anticancer activity, possessing an IC50 value of 8.65 μM, which was found lower than those of 8b, 8c, and 8j. However, it exhibited better activity than compound 8m where Cl is placed at the para position, exhibiting an IC50 value of 16.96 μM in the HeLa cancer cell lines (Table 3, Figure 7). Moreover, substitution on the ortho preferred is preferred position over the para position. These results were found in agreement with the hLDHA inhibitory activities and in silico binding affinities.

2.4.2.4. In Vitro Cytotoxicity in the Cervical (SiHa) Cancer Cell Line

In vitro cytotoxicity results against the cervical (SiHa) cancer cell line revealed that compound 8b (R1=OCH3, R2=R3=H) showed good inhibitory activity in SiHa, with IC50 values of 4.20 μM (Table 3, Figures 7 and S6). Further substitution at the same position by the NO2 group leads to compound 8c, which showed slightly lower inhibitory activity, having an IC50 value of 6.78 μM. Compound 8j (R3=OCH3), where methoxy group is present at the para position, showed an increment in inhibitory activity, exhibiting an IC50 value of 2.32 μM (Table 3, Figure 7). Moreover, for compound 8l (R3=CH3), substitution by CH3 at the para position showed similar activity to 8c with small reduction with respect to 8j in inhibitory activity possessing an IC50 value of 6.16 μM. Compound 8m, where −Cl is placed at the para position, exhibited an IC50 value of 1.65 μM, which was found to be the most active compound of this series (Table 3, Figure 7).

2.4.2.5. In Vitro Cytotoxicity in Human Embryonic Kidney Cells (HEK293)

To test the selectivity of the most active compounds 8j and 8m, we have carried out the MTT assay in human embryonic kidney cells (HEK293). In vitro cytotoxicity results indicate that these molecules are cytotoxic to cancer cells and do not induce toxicity to healthy cells (Figure S7).

In vitro cytotoxicity data revealed that substitution at the ortho position is preferred over the para position. Moreover, electron-withdrawing groups exhibit better in vitro cytotoxicity than electron-donating moieties. The in vitro activities of the compounds were found in agreement with the hLDHA inhibitory activities and in silico screening. The cLogP values of these compounds were found in the range of 2.489–4.349 and considered acceptable. The hLDHA inhibitory activities and IC50 suggested that clogP in the range of 3.379 to 4.135 favors the inhibitory activities.

2.5. In Silico Predictive ADMET Study

The absorption, distribution, metabolism, excretion, and toxicity (ADMET) are evaluated for 8a, 8b, 8c, 8j, 8l, and 8m using SwissADME (http://www.swissadme.ch/) (Figures S8–S13). Several parameters were considered, viz, number of hydrogen bond donors and acceptors, blood–brain barrier level, absorption level, 2D polar surface area (ADMET 2D PSA), Cytochrome P450 2D6 (CYP2D6), hepatotoxicity probability, aqueous solubility level, and plasma protein binding logarithmic level and calculated by SwissADME. Biorelevant small molecules under consideration are ideal drug-like candidates with good bioavailability and follow the Lipinski rule, which implies the following features, i.e., mw ≤ 500, log P ≤ 5, number of hydrogen bond acceptors ≤ 10 (i.e., N or O atoms), and hydrogen bond donors ≤ 5 (Lipinski et al.(33)). These rules of five were used to investigate the drug-likeness of all 8a–h thiazole-based compounds, and data are summarized in Table 4

Table 4. Molecular Weight, iLOGP, Consensus Log Po/w, Number of Hydrogen Bond Acceptors and Donors.

S. no. compound name molecular weight log Po/w (iLOGP) consensus log Po/w GI absorption drug likeness (Lipinski) log S
1. 8a 324.40 3.37 3.61 high yes –4.49
2. 8b 354.42 3.49 3.57 high yes –4.54
3. 8c 369.39 3.15 2.89 high yes –4.54
6. 8j 354.42 3.63 3.60 high yes –4.54
7. 8l 338.42 3.63 3.95 high yes –4.76
8. 8m 358.84 3.66 4.15 high yes –5.08
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Data summarized in Table 4 suggest that compounds have mw ≤ 500, log P, log P ≤ 5. The number of hydrogen bond acceptors in all the compounds lies in the range of 3 to 5, which follows Lipinski’s rule of five where hydrogen bond acceptors should be ≤ 10 including N or O atoms and hydrogen bond donors ≤ 5. All compounds showed clogP (Table 2) values in the range of 2.62 to 4.15. In silico ADME profiling shows that all the investigated compounds follow Lipinski’s rule of drug-likeness.

2.6. Structure–Activity Relationship

In the thiazole central scaffold-based compounds (8a-d, 8j, 8l, and 8m), the substitution at the ortho, meta, and para positions on the N substituted aromatic ring showed that these compounds showed better activity against cervical SiHa and HeLa cancer cells than the liver (HepG) cancer cell and are least active on pancreatic cancer cells. The substitution by an electron-withdrawing group at ortho position (R1) showed better activity for HepG, SiHa, and HeLa cancer cell lines. Compounds 8b, 8j, and 8l showed good activity on 03.27 pancreatic cancer cell lines and the rest of the molecules showed moderate activity against all three 02.03, 03.27, and 04.03 pancreatic cancer cell lines. All compounds showed better anticancer activity against HepG-2 cell lines than pancreatic cancer cell lines. Compounds 8b, 8c, 8j, 8l, and 8m exhibited good anticancer activity against cervical SiHa and HeLa cancer cell lines.

2.7. Conclusions

A new series of thiazole central scaffold-based small molecules 8a–d, 8j, 8l, and 8m were synthesized and screened for hLDHA inhibitory activities. In silico binding affinity of these compounds was calculated against the hLDHA enzyme. The hLDHA inhibitory activities showed that compounds 8c, 8d, 8e, 8j, and 8m have adequate inhibitory activities, which are consistent with an in silico study. Molecular docking studies depicted that the Ala 29, Val 30, Arg 98, Gln 99, Gly 96, and Thr 94 amino acids of hLDHA strongly interacted with compounds through hydrogen bonding and electrostatic and hydrophobic interaction, which might play important roles in inhibitory activities. In vitro anticancer activity evaluation depicted that compounds 8j and 8m were most active against HepG2 cancer cells possessing IC50 values of 7.9 and 5.15 μM. Compounds 8b, 8j, and 8m showed IC50 values of 4.2, 2.32, and 1.66 μM, respectively, against the SiHa cancer cell line, whereas 8b also showed inhibitory activity against HeLa cancer cell lines having an IC50 value of 2.97 μM. However, in pancreatic cancer cell lines, all the compounds showed a moderate effect. Compounds 8j and 8m did not exhibit toxicity on healthy cells (HEK293). Overall, the compounds of this series showed very good anticancer activities and selectivity in HeLa and SiHa cervical cancer cell lines. Inspired by these results, similar analogues of most active molecules may be explored for cancer therapeutics.

3. Experimental Section

3.1. General

All the chemicals and reagents were purchased from Sigma-Aldrich, SRL, and Sd fine and were used without further purification. The hLDHA enzyme was purchased from Sigma-Aldrich. Pancreatic cancer cells (02.03, 04.03, 03.27) and liver cancer cells (HepG2) were purchased from ATCC and NCCS, respectively. Gemcitabine was procured from Selleckchem. Stock solution of 1 mM was prepared in DMSO and used for in vitro cytotoxicity analysis in pancreatic cancer cells. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 Merck KGaA Germany, and spots were visualized by iodine vapor or by irradiation with ultraviolet light (254 nm). Silica gel of 100–200 mesh was obtained for column chromatography. Melting points (mp) of all 7a–d, 7j, 7l, and 7m and 8a–d, 8j, 8l, and 8m were calculated on a JSGW apparatus and are uncorrected. Solvent DMSO was used for NMR purchased from Sigma. NMR spectra were recorded on a Bruker WH-400 spectrometer or JEOL 400 MHz instrument at a ca. 5–15% (w/v) solution in DMSO-d6. Mass spectra were recorded on a Q EXACTIVE PLUS, Thermo Scientific spectrometer. Elemental analysis was carried out on a Vario ELIII elementor. HPLC analysis was performed on Shimadzu LC-2030C 3D plus using column XTIMATE C18 and flow rate 1.0 mL/min.

3.1.1. General Procedure for the Synthesis of Phenacyl Thiocyanate (3)

Phenacyl thiocyanate was synthesized in 96% yield by the reaction of phenacyl bromide with ammonium thiocyanate in methanol by using a literature protocol with slight modifications.27,28 Herein, saturated ammonium thiocyanate solution was used to generate thiocyanate nucleophiles. Nucleophilic substitution reaction of phenacyl bromide with thiocyanate nucleophile in a methanol/water solution resulted in the desired phenacyl thiocyanate. However, in the literature protocol, KSCN/SiO2–RNH3OAc/Al2O3, NaSCN, and KSCN have been used to generate nucleophiles.

3.1.2. General Procedure for the Synthesis of Various 3,4-Diphenylthiazol-2(3H)-imine (7a–h)

These starting materials were prepared by following a reported synthetic procedure with a slight modification.28 Phenacyl thiocyanate 3 (1 mmol) was placed in a round bottom flask in dry methanol under nitrogen. Once the clear solution formed, aniline hydrochlorides (5a–d, 5j, 5l, and 5m) (1 mmol) were added to the reaction vessel, and then the reaction mixture was allowed to heat at 70 °C with continuous stirring for 7 h. Reaction progress was monitored by TLC. Once the reaction was completed, the solvent was removed under reduced pressure. The solid residue so obtained was dissolved in methanol/water at a (1:1) ratio and the reaction content was basified using an aqueous solution of NaOH up to pH 10–11 to precipitate out the desired compound. The precipitate so obtained was filtered and recrystallized in an appropriate solvent to give pure products 7a–d, 7j, 7l, and 7m. Melting points and NMR spectral data of reported compounds 7a–d, 7j, 7l, and 7m were inconsistent with the synthesized compounds.

3.1.2.1. 3,4-Diphenylthiazol-2(3H)-imine (7a)

The solvent of crystallization was EtOH; yield 90%; solid; mp: 85.3 °C; 1H NMR (400 MHz; DMSO-d6) δ 6.48 (s, 1H, >C=CH), 7.14–7.16 (m, 2H, Ar), 7.33–7.34 (m, 3H, Ar), 7.42–7.44 (m, 2H, Ar), 8.18–8.19 (m, 2H, Ar), 8.45 (s, 1H, NH exch).

3.1.2.2. 3-(2-Methoxyphenyl)-4-phenylthiazol-2(3H)-imine (7b)

The solvent of crystallization was EtOH; yield 83%; solid; mp: 91.4 °C; 1H NMR (400 MHz; DMSO-d6) δ 3.52 (s, 3H, OCH3), 6.13 (s, 1H, >C=CH), 6.89–6.95 (m, 2H, Ar), 7.04–7.06 (m, 2H, Ar), 7.14–7.18 (m, 3H, Ar), 7.23–7.28 (m, 2H, Ar), 7.65 (s, 1H, NH exch).

3.1.2.3. 3-(2-Nitrophenyl)-4-phenylthiazol-2(3H)-imine (7c)

The solvent of crystallization was EtOH; yield 86%; solid; mp: 125.3 °C; 1H NMR (400 MHz; DMSO-d6) δ 6.24 (s, 1H, >C=CH), 7.10–7.12 (m, 3H, Ar), 7.20–7.22 (m, 3H, Ar), 7.46–7.56 (m, 2H, Ar), 7.97–7.99 (m, 1H, Ar), 8.21 (s, 1H, NH exch).

3.1.2.4. 4-Phenyl-3-(o-tolyl)thiazol-2(3H)-imine (7d)

The solvent of crystallization was EtOH; yield 89%; solid; mp: 108.1 °C; 1H NMR (400 MHz; DMSO-d6) δ 2.23 (s, 3H, CH3), 6.18 (s, 1H, >C=CH), 6.97–7.99 (m, 2H, Ar), 7.09–7.12 (m, 4H, Ar), 7.15–7.18 (m, 3H, Ar), 7.65 (s, 1H, NH exch).

3.1.2.5. 3-(4-Methoxyphenyl)-4-phenylthiazol-2(3H)-imine (7j)

The solvent of crystallization was EtOH; yield 88%; solid; mp: 98.4 °C; 1H NMR (400 MHz; DMSO-d6) δ 3.67 (s, 3H, OCH3), 6.19 (s, 1H, >C=CH), 6.82–6.84 (m, 2H, Ar), 7.00–7.07 (m, 2H, Ar), 7.08–7.09 (m, 2H, Ar), 7.16–7.19 (m, 3H, Ar), 7.68 (s, 1H, NH exch).

3.1.2.6. 4-Phenyl-3-(p-tolyl)thiazol-2(3H)-imine (7l)

The solvent of crystallization was EtOH; yield 89%; solid; mp: 108.4 °C; 1H NMR (400 MHz; DMSO-d6) δ 2.21 (s, 3H, CH3), 6.19 (s, 1H, >C=CH), 6.96–7.98 (m, 2H, Ar), 7.06–7.08 (m, 4H, Ar), 7.16–7.18 (m, 3H, Ar), 7.62 (s, 1H, NH exch).

3.1.2.7. 3-(4-Chlorophenyl)-4-phenylthiazol-2(3H)-imine (7m)

The solvent of crystallization was EtOH; yield 78%; solid; mp: 105.1 °C; 1H NMR (400 MHz; DMSO-d6) δ 6.24 (s, 1H, >C=CH), 7.11–7.13 (m, 2H, Ar), 7.19–7.20 (m, 2H, Ar), 7.20–7.32 (m, 3H, Ar), 7.32–7.35 (m, 2H, Ar), 8.02 (s, 1H, NH exch).

3.1.3. General Procedure for the Synthesis of Methyl 2-((3,4-Diphenylthiazol-2(3H)-ylidene)amino)acetate (8a–d, 8j, 8l, and 8m)

Compound 7a (750 mg, 2 mmol) was placed in a round bottom flask in dry acetone (10 mL) under nitrogen. Once the clear solution was formed, 2.5 mmol (345 mg) of potassium carbonate was placed in a reaction vessel on continuous stirring at room temperature. After 30 min of incubation of the reaction mixture, 2.2 mmol (306 mg) of methyl 2-bromoacetate was added to the reaction mixture and stirring continued at 80 °C. Reaction progress was monitored by TLC. Once the reaction was completed, the solvent was removed under reduced pressure. The solid residue so obtained was quenched with ice cold aqueous solution of sodium bicarbonate. The so-obtained solid was purified by column chromatography to give the pure product.

3.1.3.1. Methyl 2-((3,4-Diphenylthiazol-2(3H)-ylidene)amino)acetate (8a)

The yield was 85%; solid; mp: 132.3 °C; 1H NMR (400 MHz; DMSO-d6) δ 2.74 (s, 3H, CH3), 3.57 (s, 2H, CH2), 6.50 (s, 1H, >C=CH), 7.25–7.27 (m, 2H, Ar), 7.36–7.38 (m, 5H, Ar), 7.45–7.46 (m, 3H, Ar). 13C NMR (400 MHz; DMSO-d6) δ 170.44, 160.92, 146.10, 137.71, 133.56, 132.17, 130.50, 130.90, 129.84, 129.32, 129.15, 129.03, 128.58, 128.56, 125.80, 99.05, 55.30, 52.01. MS (m/z) 325.09. Elemental Analysis: (calculated), found, (C, 66.64%; H, 4.97%; N, 8.64%; S, 9.88%) C, 66.63%; H, 4.95%; N, 8.61%; S, 9.87%.

Other compounds were prepared by following a similar procedure.

3.1.3.2. Methyl 2-((3-(2-Methoxyphenyl)-4-phenylthiazol-2(3H)-ylidene)amino)acetate (8b)

The elution solvent was hexane/ethyl acetate (8:2); yield 82%; solid; mp: 105.3 °C; 1H NMR (400 MHz; DMSO-d6) δ 3.53 (s, 3H, OCH3), 3.61 (s, 3H, OCH3), 3.77–3.89 (q, 2H, CH2), 6.39 (s, 1H, >C=CH), 6.92–6.96 (m, 2H, Ar), 7.11–7.13 (m, 2H, Ar), 7.19–7.28 (m, 5H, Ar). 13C NMR (400 MHz; DMSO-d6) δ 171.06, 162.60, 140.32, 131.75, 131.63, 130.21, 128.85, 128.43, 128.20, 127.10, 121.01, 113.21, 96.63, 56.02, 55.46, 52.01. MS (m/z) 345.61 (M + H)+. Elemental Analysis: (calculated), found, (C, 66.64%; H, 4.97%; N, 8.64%; S, 9.88%), C, 66.61%; H, 4.95%; N, 8.63%; 9.85%.

3.1.3.3. Methyl 2-((3-(2-Nitrophenyl)-4-phenylthiazol-2(3H)-ylidene)amino)acetae (8c)

The elution solvent was hexane/ethyl acetate (8:2); yield 78%; solid; mp: 97.4 °C; 1H NMR (400 MHz; DMSO-d6) δ 3.55 (s, 3H, OCH3), 3.76 (s, 2H, CH2), 6.54 (s, 1H, >C=CH), 7.15–7.17 (m, 2H, Ar), 7.20–7.23 (m, 4H, Ar), 7.49–7.52 (td, 1H, Ar), 7.57–7.60 (m, 1H, Ar), 7.98–8.00 (m, 1H, Ar).13C NMR (400 MHz; DMSO-d6) δ 170.95, 155.49, 140.85, 138.71, 134.56, 132.17, 131.50, 130.90, 129.86, 129.34, 129.05, 129.03, 128.59, 128.57, 125.70, 99.07, 55.31, 52.02. MS (m/z) 371.61(M + H)+. Elemental Analysis: (calculated), found, (C, 58.53%; H, 4.09%; N, 11.38%; S, 8.68%), C, 58.51%; H, 4.07%; N, 11.37%; S, 8.68%.

3.1.3.4. Methyl 2-((4-Phenyl-3-(o-tolyl)thiazol-2(3H)-ylidene)amino)acetate (8d)

The elution solvent was hexane/ethyl acetate (8:2); yield 78%; solid; mp: 98.2 °C; 1H NMR (400 MHz; DMSO-d6) δ 2.26 (s, 3H, CH3), 3.62 (s, 3H, OCH3), 3.85 (s, 2H, CH2), 6.46 (s, 1H, >C=CH), 7.01–7.03 (m, 3H, Ar), 7.10–7.16 (m, 4H, Ar), 7.23–7.25 (m, 3H, Ar). 13C NMR (400 MHz; DMSO-d6) δ 171.04, 161.61, 141.31, 136.34, 134.97, 130.93, 129.82, 129.32, 128.94, 128.86, 128.73, 97.93, 55.67, 52.19, 21.05. MS (m/z) 339.59(M + H)+. Elemental Analysis: (calculated), found, (C, 67.43%; H, 5.36%; N, 8.28%; S, 9.47%), C, 67.41%; H, 5.34%; N, 8.27%; S, 9.45%.

3.1.3.5. Methyl 2-((3-(4-Methoxyphenyl)-4-phenylthiazol-2(3H)-ylidene)amino)acetate (8j)

The elution solvent was hexane/ethyl acetate (8:2); yield 80%; solid; mp: 96.3 °C; 1H NMR (400 MHz; DMSO-d6) δ 3.68 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.92 (s, 2H, CH2), 6.50 (s, 1H, >C=CH), 6.91–6.93 (m, 2H, Ar), 7.12–7.14 (m, 2H, Ar), 7.21–7.22 (m, 2H, Ar), 7.30–7.31 (m, 3H, Ar). 13C NMR (400 MHz; DMSO-d6) δ 171.96, 162.66, 158.59, 140.34, 131.80, 131.09, 130.64, 128.83, 128.74, 128.66, 114.42, 97.44, 55.74, 55.56, 52.04. MS (m/z) 355.62 (M + H)+. Elemental Analysis: (calculated), found, (C, 64.39%; H, 5.12%; N, 7.90%; S, 9.05%), C, 64.37%; H, 5.11%; N, 7.90%; S, 9.03%.

3.1.3.6. Methyl 2-((4-Phenyl-3-(p-tolyl)thiazol-2(3H)-ylidene)amino)acetate (8l)

The elution solvent was hexane/ethyl acetate (8:2); yield 75%; solid; mp: 99.2 °C; 1H NMR (400 MHz; DMSO-d6) δ 2.31 (s, 3H, CH3), 3.67 (s, 3H, OCH3), 3.91 (s, 2H, CH2), 6.51 (s, 1H, >C=CH), 7.07–7.09 (m, 2H, Ar), 7.16–7.21 (m, 4H, Ar), 7.28–7.29 (m, 3H, Ar). 13C NMR (400 MHz; DMSO-d6) δ 171.06, 162.60, 140.32, 137.35, 135.98, 131.93, 129.85, 129.36, 128.96, 128.87, 128.72, 97.92, 55.68, 52.18 21.04. MS (m/z) 339.55 (M + H)+. Elemental Analysis: (calculated), found, (C, 67.43%; H, 5.36%; N, 8.28%; S, 9.47%), C, 67.41%; H, 5.33%; N, 8.27%; S, 9.46%.

3.1.3.7. Methyl 2-((3-(4-Chlorophenyl)-4-phenylthiazol-2(3H)-ylidene)amino)acetate (8m)

The elution solvent was hexane/ethyl acetate (8:2); yield 75%; solid; mp: 98.3 °C; 1H NMR (400 MHz; DMSO-d6) δ 3.58 (s, 3H, CH3), 3.83 (s, 2H, CH2), 6.47 (s, 1H, >C=CH), 7.10–7.12 (m, 2H, Ar), 7.13–7.15 (m, 2H, Ar), 7.22–7.23 (m, 3H, Ar), 7.32–7.35 (m, 2H, Ar). 13C NMR (400 MHz; DMSO-d6) δ 170.45, 160.93, 145.10, 138.71, 134.56, 132.17, 131.50, 130.90, 129.86, 129.34, 129.05, 129.03, 128.59, 128.57, 125.70, 99.07, 55.31, 52.02. MS (m/z) 359.57(M + H)+. Elemental Analysis: (calculated), found, (C, 60.25%; H, 4.21%; N, 7.81%; S, 8.94%), C, 60.23%; H, 4.19%; N, 7.79%; S, 8.92%.

Acknowledgments

D.S., M.S., V.K., and R.R. thank Amity University UP India for providing necessary support. R.R. also acknowledges the support of SERB to carry out this work.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c07569.

  • Molecular docking; in vitro cytotoxicity of the compounds; in silico ADMET profiling of the compounds; NMR spectra of compounds, mass spectrograms, and HPLC analysis of the compounds (PDF)

Author Contributions

R.R. and V.K. performed the conceptualization, supervision, investigation, data analysis, and writing. D.S. conducted the synthesis, data curation, data analysis, and writing. M.S. completed the in vitro studies. V.C. was in charge of the NMR. J.S. and D.B. took over the molecular docking. M.G. took charge of the anticancer activities in pancreatic cancer cells. S.C. executed the in vitro studies. All authors approved the submitted version.

This work was funded by a SERB grant (ECR/2017/000548) to R.R.

The authors declare no competing financial interest.

Supplementary Material

ao2c07569_si_001.pdf (1.6MB, pdf)

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ao2c07569_si_001.pdf (1.6MB, pdf)

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