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Article

Benzothiazole and Chromone Derivatives as Potential ATR Kinase Inhibitors and Anticancer Agents

by
Mykhaylo Frasinyuk
1,†,
Dimple Chhabria
2,†,
Victor Kartsev
3,‡,
Haritha Dilip
2,‡,
Samvel N. Sirakanyan
4,
Sivapriya Kirubakaran
2,*,
Anthi Petrou
5,
Athina Geronikaki
5,* and
Domenico Spinelli
6,*
1
V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Science of Ukraine, 02094 Kiev, Ukraine
2
Discipline of Chemistry, Indian Institute of Technology Gandhinagar, Gandhinagar 382055, India
3
InterBioScreen, 119019 Chernogolovka, Russia
4
Scientific Technological Center of Organic and Pharmaceutical Chemistry, National Academy of Science of the Republic of Armenia, Institute of Fine Organic Chemistry, Yerevan 0014, Armenia
5
School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
6
Dipartimento di Chimica “G. Ciamician”, Alma Mater Studiorum-Università di Bologna, 40126 Bologna, Italy
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
These authors contributed equally to this work.
Molecules 2022, 27(14), 4637; https://doi.org/10.3390/molecules27144637
Submission received: 17 June 2022 / Revised: 5 July 2022 / Accepted: 8 July 2022 / Published: 20 July 2022
(This article belongs to the Special Issue Design and Synthesis of Organic Molecules as Antineoplastic Agents II)
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Abstract

:
Despite extensive studies and the great variety of existing anticancer agents, cancer treatment remains an aggravating and challenging problem. Therefore, the development of novel anticancer drugs with a better therapeutic profile and fewer side effects to combat this persistent disease is still necessary. In this study, we report a novel series of benzothiazole and chromone derivatives that were synthesized and evaluated for their anticancer activity as an inhibitor of ATR kinase, a master regulator of the DDR pathway. The cell viability of a set of 25 compounds was performed using MTT assay in HCT116 and HeLa cell lines, involving 72 h incubation of the compounds at a final concentration of 10 µM. Cells incubated with compounds 2c, 7h and 7l were found to show viability ≤50%, and were taken forward for dose–response studies. Among the tested compounds, three of them (2c, 7h and 7l) showed higher potency, with compound 7l exhibiting the best IC50 values in both the cell lines. Compounds 2c and 7l were found to be equally cytotoxic towards both the cell lines, namely, HCT116 and HeLa, while compound 7h showed better cytotoxicity towards HeLa cell line. For these three compounds, an immunoblot assay was carried out in order to analyze the inhibition of phosphorylation of Chk1 at Ser 317 in HeLa and HCT116 cells. Compound 7h showed inhibition of pChk1 at Ser 317 in HeLa cells at a concentration of 3.995 µM. Further analysis for Chk1 and pChk1 expression was carried out in Hela cells by treatment against all the three compounds at a range of concentrations of 2, 5 and 10 µM, wherein compound 7h showed Chk1 inhibition at 2 and 5 µM, while pChk1 expression was observed for compound 7l at a concentration of 5 µM. To support the results, the binding interactions of the compounds with the ATR kinase domain was studied through molecular docking, wherein compounds 2c, 7h and 7l showed binding interactions similar to those of Torin2, a known mTOR/ATR inhibitor. Further studies on this set of molecules is in progress for their specificity towards the ATR pathway.

1. Introduction

Cancer remains one of the main leading causes of mortality all over the world. Despite extensive studies and the great variety of existing anticancer agents, cancer treatment remains an aggravating and challenging problem. Therefore, the development of novel anticancer drugs with a better therapeutic profile and fewer side effects to combat this persistent disease is still necessary.
Cells are prone to a multitude of stress factors including endogenous and exogenous factors, such as reactive oxygen species, radiation, etc., which result in genomic instability through either a single strand or a double strand break in their DNA [1]. In order to cope with the stress, and to maintain genomic integrity as well as cell viability, cells develop DNA damage response (DDR) signaling pathways which regulate and repair the damage caused [2,3]. Targeting such crucial signaling pathways in cancer cells can prevent these cells from repairing their damaged single and/or double stranded DNA, hence promoting an efficient therapeutic strategy for cancer. Rad-3 related (ATR) and ataxia telangiectasia mutated (ATM) kinases from the phosphatidylinositol-3-kinase-related kinases (PIKK) family are two major signaling proteins which phosphorylate Ser/Thr residues of their downstream substrates Chk1 and CHk2, respectively [2,3,4]. The significant role of benzothiazoles as potential kinase inhibitors generates interest in exploring the biological activity of these compounds further in the area of kinases and thus, as potential candidates for cancer therapy [5].
Benzothiazole is a heterocyclic organic compound with a wide range of biological activities such as antimicrobial [6,7], anti-inflammatory [8,9], antioxidant [10,11], analgesic [8,12], anticancer [13,14,15,16], antiviral [17,18,19,20], anti-HIV [21,22], antidiabetic [23], anticonvulsant [24,25], antimalarial [26], antitubercular [27,28] and kinase inhibitor [29,30]. It should be mentioned that some FDA-approved drugs involve a benzothiazole carrier scaffold (Figure 1). They include quizartinib, a receptor tyrosine kinase inhibitor, flutemetamol, a diagnostic tool for Alzheimer disease, and riluzole, a drug for treatment of amyotrophic lateral sclerosis.
On the other hand, chromone derivatives possess many different biological activities such as anticancer [31,32,33], antimicrobial [34], anti-inflammatory [35,36], antitubercular [37,38] and others activities [39,40,41], which have generated an interest in developing further this scaffold. The chromone scaffold is also present in some approved drugs such as flavoxate (Figure 2), a muscarinic antagonist and spasmolytic. This drug is used to treat urinary bladder spasms and is indicated for the symptomatic relief of conditions associated with lack of muscle control in the bladder, such as dysuria, urgency, and nocturia.
Taking into account these facts, we designed and synthesized new derivatives incorporating two pharmacophores, benzothiazole and chromone, in the frame of one molecule using the hybridization approach [42]. Hence, in the present study, the synthesized hybrid molecules have been analyzed for their inhibitory potential against cancer cell viability, especially on the colon cancer and cervical cancer cell lines. Further, to identify their potential in inhibiting the DDR signaling pathway, the compounds have also been subjected to assays that specifically inhibit ATR expression in these cancer cells.

2. Results and Discussion

2.1. Chemistry

The general routes for the synthesis of the target 3-hetarylchromones include the synthesis of α-[benzo]thiazolyl acetophenones 1, ring-closure reaction for synthesis of 3-(2[benzo])thiazolyl chromones 2–4, modification of the substituent in position 2 of the chromone ring, and aminomethylation of 7-hydroxychromones. The starting ketones 1a–1f were synthesized by Hoesch reaction of (un)substituted resorcinol with heteroaryl acetonitriles followed by subsequent acidic hydrolysis of intermediate ketone imines [43,44,45].
Subsequent reaction of ketones 1 with ethyl orthoformate, acetic anhydride or ethyl oxalyl chloride in pyridine afforded 3-heteroaryl chromones 2–4.
Thus, refluxing of ketones 1a–1c and 1e with ethyl orthoformate in dry pyridine led to the formation of 2-unsubstituted chromones 2a–2c and 2e. Ring-closure reaction of ketones 1a or 1c with excess of acetic anhydride in dry pyridine with subsequent saponification led to 2-methylchromones 3a and 3c. Reaction of ketones 1a,b,d with ethyl oxalyl chloride in dry pyridine at room temperature afforded 2-ethoxycarbonyl-3-benzotiazolylchromones 4a, 4b, and 4c. Saponification of ester 4c in EtOH gave carboxylic acid 4d.
The reaction of 2-unsubstituted chromones 2a or 2e [46] with hydroxylamine in pyridine afforded 2-aminochromones 5a or 5b as a result of subsequent ring-opening and ring-closure reactions. This unusual behavior of chromones 2a or 2e toward hydroxylamine can be explained by the strong electron-withdrawing effect of the aza-heterocyclic moiety in position 3 of the chromone ring. The bearing of ω-carboxyalkyl group 3-hetarylchromones 6a–6d and related 3-aryl-4-(2-benzothiazolyl)pyrazole 6e were synthesized as we have earlier reported [47] (Scheme 1).
The aminomethylation of chromones with electron-withdrawing substituent in position 3 is possible by applying corresponding aminals [48]. Thus, the reaction of chromones 2–5 with various aminals led to regiospecific formation of 8-aminomethylchromones 7a–7m (Scheme 2) which was confirmed by the disappearance in 1H-NMR spectra of the upfield (for 6-alkyl substituted chromones 2–4) peak or presence of H-6 doublet at 6.84–6.98 ppm with 3J 8.3–8.8 Hz. The best yields were achieved using 1,4-dioxane as solvent.
Thus, in chromone Mannich bases 7 the singlet peak (except for compounds 7c, 7j–7l) of methylene group at 4.02–4.20 ppm was present as well as the secondary amine moiety. In the instance of piperidine and piperazine derivatives, in 1H-NMR spectra signals of cyclic amine protons usually appear as wide non-resolved multiplets. In the case of chromones 7j–7l with 1,3,3-trimethyl-6-azabicyclo [3.2.1]octane moiety, we observed two doublets of diastereotopic 8-CH2 group at 4.00–4.05 ppm and 4.28–4.32 ppm with heminal J 14.3–14.5 Hz. In 1H-NMR peaks of 8-CH2 group, we observed two multiplets at 3.90 and 4.33 ppm due to difficult conversion of the piperidine ring and/or the presence of stereocenter.
Isomeric Mannich bases of 7-hydroxy-3-arylcoumarins were synthesized in similar conditions applying 3-(2-benzothiazolyl)-7-hydroxycoumarin (8) [49] and substituted 3-(2-thiazolyl)coumarins 9a,b [50] (Scheme 3).
The synthesis of 8-aminomethyl derivatives was confirmed by splitting of H-5 and H-6 of coumarin core with 3J 8.6–8.7 Hz. Similar to chromone 7 derivatives, the signal of 8-CH2 group was found as a singlet at 3.99–4.23 ppm for diethanolamine, piperidine or piperazine derivatives. In the case of compound 11f, the signals of 8-CH2 group were found as two doublets at 4.07 and 4.37 ppm with 2J 14.6 Hz.

2.2. Biological Evaluation

2.2.1. Cell Viability Studies—Initial Screening

In order to determine the effect of the compounds on cell viability, the studies were carried out using MTT assay in HCT116 and HeLa cell lines. Initial screening of the compounds was carried out at a final concentration of 10 µM. The cell viability was reduced by ≤50% in the presence of three compounds, namely 2c, 7h and 7l, hence showing cell toxicity in both cell lines at a final concentration of 10 µM. Figure 3 represents the initial screening results for HCT116 cells after 72 h incubation.
Similarly, Figure 4 represents the initial screening results for HeLa cells after 72 h incubation.
Compounds 2c, 7h and 7l showed a significant reduction in cell viability for both the cervical cancer and colon cancer cell lines, suggesting that these compounds could be potent anticancer agents, especially for these two types of cancer. In order to identify the minimum concentration of these compounds required to reduce the cell viability of these cell lines by 50% or more, these compounds were further analyzed for their cytotoxicity effects using dose-response studies. The dose-response studies would be performed under similar conditions as those for the initial screening, for a concentration range of the potent compounds, so as to identify the half maximal inhibitory concentration (IC50) of each compound required to cause cell toxicity.

2.2.2. Cell Viability Studies—Dose Response

The dose–response studies were conducted using the MTT assay for 2c, 7h and 7l, for a dose range of 0–100 µM similar to the initial screening protocol. Further, the absorbance was measured at 570 nm using the PerkinElmer EnVision Multilabel Reader and the IC50 values calculated for all the three compounds (Table 1).
Figure 5 represents the dose–response curves for the compounds 2c, 7h and 7l in HCT116 cells after 72 h incubation.
Similarly, Figure 6 represents the dose–response curves for the compounds 2c, 7h and 7l in HCT116 cells after 72 h incubation.
Compounds 2c and 7h showed IC50 values of 3.670 µM and 6.553 µM against HCT116 cell lines, while 7l showed an IC50 value of 2.527 µM. However, all three compounds were equally potent against the HeLa cell line with IC50 values of 2.642 µM, 3.995 µM and 2.659 µM, respectively. Further, to analyze the effect of these compounds specifically on the DDR signaling pathway, an essential pathway for cancer cell survival, the compounds were subjected to cell-based assays that would determine the inhibition of these pathways.

2.2.3. Immunoblot Assay

The effect of the synthesized compounds to inhibit the cancer cell’s DNA damage response (DDR) signalling pathway, a common pathway preferred by cancer cells to ensure genomic integrity, as well as cell viability [51], was analyzed by carrying out an immunoblot assay for one of the major kinases involved in the regulation of the pathway. Ataxia telangiectasia mutated and Rad-3 related (ATR) kinase, a member of the the PIKK (phosphatidylinositol-3-kinase-related kinases) family, are involved in the phosphorylation of Ser/Thr residues of its downstream substrate Chk1. The inhibition of Chk1 phosphorylation has been proved to be a clear indication of the inhibition of ATR signalling. Hence, the immunoblot assay helps in the determination of the inhibitory potential of the synthesized compounds against ATR kinase in the DDR pathway.
The analysis of inhibition of phosphorylation of Chk1 at Ser 317 in HeLa and HCT116 cells was carried out by immunoblotting using anti-pChk1 Ser 317 (rabbit) primary antibody and anti-rabbit IgG HRP-linked secondary antibody. Of the three compounds tested, compound 7h showed inhibition of ATR signaling which was observed by checking the status of ChK1 in HeLa cells (Figure 7). However, the results for HCT116 do not show significant inhibition of ATR signaling in the cases of compounds 7h and 2c, while 7l showed inhibition with respect to the control.
Further, to analyze the effect of the concentration of the compounds on the expression of Chk1 and pChk1 at Ser 317 in HeLa cells, immunoblotting assay using ChK1 mouse monoclonal antibody, and anti-pChk1 Ser 317 (rabbit) primary antibody were carried out. Anti-mouse IgG HRP-linked antibody and anti-rabbit IgG HRP-linked secondary antibody were used as secondary antibodies, respectively. The inhibition of Chk1 was observed at 2 µM and 5 µM concentrations of compound 7h. In the cases of compounds 2c and 7l, the ChK1 and pChK1 expression in the control lanes did not provide a clear insight into the inhibition of ATR signaling (Figure 8).

2.3. Molecular Docking

The use of molecular docking as a successful computational tool in drug discovery, to model and visualize the interactions between small molecules and their specific targets, has been prevalent since the 1980s. The conformations and binding interactions corresponding to the behavior of the small molecules at the binding site of the drug target have also been an essential factor in identifying the mechanism of enzyme inhibition, which further enhance the drug development processes [52].
Molecular docking resulted in an in-depth analysis of the major binding interactions between the compounds with the ATR kinase domain. Compound 2c showed the same GScore and DockScore of −5.5 kcal/mol, followed by a GScore and DockScore of −5.2 kcal/mol and −5.1 kcal/mol for Torin2. Compound 7l showed the GScore and DockScore of −5.2 kcal/mol and −4.8 kcal/mol. This was followed by compound 7h with a GScore and DockScore of −4.3 kcal/mol and −3.4 kcal/mol (Table 2).
Two-dimensional ligand interaction diagrams show the major binding interactions between the compounds and the binding site of the ATR kinase domain. Torin2, the standard ligand had hydrogen bonding and π-π stacking interactions as the major interactions at the binding site. Compound 2c had the highest docking score of −5.5 kcal/mol, wherein hydrogen bonding, π-π stacking and π-cation interactions played the major role. Compound 7h had similar interactions in comparison to Torin2, with a lowest docking score of −3.4 kcal/mol. However, unlike all the ligands in the molecular docking study, compound 7l had the highest number of diverse interactions at the binding site, including hydrogen bonding, π-π stacking, π-cation interactions and salt bridge formation. The ligand interaction diagrams for the binding interactions of compounds 2c, 7h and 7l along with Torin2 are shown in Figure 9.
The three-dimensional view of the ligand conformations in the binding site is crucial to visualize the interactions determined through ligand interaction diagrams. The binding poses of the compounds, in comparison to the standard ligand helps in deciphering the nature of binding of these compounds at the binding site. The 3D diagrams indicating the binding poses of the compounds 2c (pink), 7h (yellow), 7l (orange) and Torin2 (green) are depicted in Figure 10.

3. Materials and Methods

3.1. General Information

1H and 13C spectra were recorded on Varian 500 (500/125 MHz) or Varian 400 (400/100 MHz) and/or Varian VXR300 spectrometers in CDCl3 [residual CHCl3 (δH = 7.26 ppm) or CDCl3 (δC = 77.16 ppm) as internal standard] or DMSO-d6 [residual SO(CD3)(CD2H) (δH = 2.50 ppm) or SO(CD3)2 (δC = 39.52 ppm) as internal standard]. Melting points were determined in open capillary tubes using Buchi B-535 apparatus (Buchi Labortechnik AG, Flawil, Switzerland) and were uncorrected. Mass spectra were obtained using an Agilent 1100 spectrometer (Agilent Technologies, Waldbronn, Germany) using APCI (atmospheric-pressure chemical ionization). All spectra are available in Supplementary Materials.
Colon cancer cell line HCT116 and human cervical cancer cell line HeLa were furnished by National Centre for Cell Science (NCCS), Pune, India, DMEM from LONZA, Fetal bovine serum, pen-strap, and trypsin-EDTA from Invitrogen corporation (Carlsbad, CA, USA), MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide] dye from HIMEDIA (Mumbai, India), and transparent 96-well plates from TARSON (Tarsons Products Pvt. Ltd., Mumbai, India).
Sodium dodecyl sulphate (SDS), tetramethylenediamine (TEMED), ammonium persulphate (APS), bromophenol blue and beta-mercaptoethanol were purchased from HIMEDIA (Mumbai, India), Tween-20 and Bovine serum albumin (BSA) from Sigma-Aldrich (Darmstadt, Germany), complete-EDTA free protease inhibitor tablets from Roche (Basel, Switzerland), immunoblot PVDF Western blotting membrane and Clarity ECL Western blotting substrate from Bio-Rad laboratories (Hercules, CA, USA), ChK1- phosphor Ser 317 (Cat. 12302S), ChK1 Mouse (Cat. 2360S) monoclonal antibody, Anti-mouse IgG HRP-linked antibody (Cat. 7076P2) and Anti-rabbit IgG HRP- linked secondary antibody (Cat. 7074) from Cell Signaling Technology (Danvers, MA, USA), and mouse anti-human Beta-actin (Cat. SC47778) from Santa Cruz Biotechnology (Dallas, TX, USA).

3.2. Chemistry

Compounds 1a [43], 1b [45], 1c [44], 1e [46], 2a [43], 2b [45], 2c [44], 2e [46], 3c [44], 6a–6d [47], 8 [48], and 9a,b [49] were synthesized by procedures reported earlier.
2-(1,3-Benzothiazol-2-yl)-1-(2,4-dihydroxy-3-methylphenyl)ethanone (1d) was synthesized according to the literature procedure [44]. Yield 53%, m.p. 255–256 °C. LC-MS: m/z 300.0 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 14.52 (s, 1H), 12.65 (s, 1H), 9.96 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.39–7.30 (m, 2H), 7.21–7.11 (m, 1H), 6.69 (s, 1H), 6.43 (d, J = 8.6 Hz, 1H), 2.01 (s, 3H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 186.0, 162.4, 161.9, 159.9, 138.8, 126.8, 126.7, 125.7, 122.5, 122.3, 112.1, 111.6, 110.4, 106.3, 85.9, 7.9. Elemental analysis for C16H13NO3S; Calc.: C, 64.20; H, 4.38; N, 4.68%. Found: C, 64.22; H, 4.35; N, 4.70%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-2-methyl-4H-chromen-4-one (3a) was synthesized according to the literature procedure [45]. Yield 78%, m.p. 292–293 °C. LC-MS: m/z 310.1 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 10.94 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 8.04–7.93 (m, 2H), 7.53–7.45 (m, 1H), 7.44–7.37 (m, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.85 (s, 1H), 2.95 (s, 3H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 173.7, 168.9, 163.1, 159.9, 156.4, 150.9, 135.0, 127.1, 125.8, 124.8, 122.3, 121.4, 115.6, 114.5, 114.4, 102.0, 21.7. Elemental analysis for C17H13NO3S; Calc.: C, 66.01; H, 3.58; N, 4.53%. Found: C, 66.05; H, 3.60; N, 4.56%.

3.2.1. General Procedure for the Synthesis of 2-Ethoxycarbonyl Chromones 4a–4c

To a solution of the corresponding starting compounds 1a,b,d (5 mmol) in dry pyridine (10 mL) ethyl oxalyl chloride (15 mmol) was added dropwise under cooling. The reaction mixture was stirred for 24–48 h at room temperature and poured into 200 mL of 1N HCl. The formed precipitate was filtered off, dried, and re-crystallized from EtOH to furnish the desired products 4a–4c.
Ethyl 3-(1,3-benzothiazol-2-yl)-7-hydroxy-4-oxo-4H-chromene-2-carboxylate (4a). Yield 67%, m.p. 223–224 °C, yellow crystals. LC-MS: m/z 368.0 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 11.23 (s, 1H), 8.17 (d, J = 7.9 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.57–7.51 (m, 1H), 7.46 (t, J = 7.4 Hz, 1H), 7.06 (d, J = 8.8 Hz, 1H), 7.01 (s, 1H), 4.45 (q, J = 7.0 Hz, 2H), 1.24 (t, J = 7.0 Hz, 3H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 173.5, 164.1, 161.1, 156.8, 156.6, 154.5, 150.8, 135.6, 127.4, 126.5, 125.4, 122.4, 122.1, 116.7, 115.1, 113.8, 102.5, 63.0, 13.6. Elemental analysis for C19H13NO5S; Calc.: C, 62.12; H, 3.57; N, 3.81%. Found: C, 62.09; H, 3.60; N, 3.79%.
Ethyl 3-(1,3-benzothiazol-2-yl)-6-ethyl-7-hydroxy-4-oxo-4H-chromene-2-carboxylate (4b). Yield 74%, m.p. 192–193 °C, colorless. LC-MS: m/z 396.0 [M + H]+. 1H-NMR (500 MHz, DMSO-d6): δ ppm 11.30 (s, 1H), 8.19 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.92 (s, 1H), 7.59–7.52 (m, 1H), 7.50–7.44 (m, 1H), 7.01 (s, 1H), 4.45 (q, J = 7.1 Hz, 2H), 2.67 (q, J = 7.4 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H), 1.21 (t, J = 7.4 Hz, 3H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 173.3, 162.1, 161.2, 156.9, 154.9, 154.3, 150.8, 135.5, 131.9, 126.5, 125.4, 124.9, 122.4, 122.1, 114.8, 113.7, 101.7, 62.9, 22.4, 13.6, 13.5. Elemental analysis for C21H17NO5S; Calc.: C, 63.79; H, 4.53; N, 3.54%. Found: C, 63.77; H, 4.49; N, 3.57%.
Ethyl 3-(1,3-benzothiazol-2-yl)-7-hydroxy-8-methyl-4-oxo-4H-chromene-2-carboxylate (4c). Yield 69%, m.p. 265–266 °C. LC-MS: m/z 382.0 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 11.07 (s, 1H), 8.16 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.7 Hz, 1H), 7.58–7.50 (m, 1H), 7.49–7.41 (m, 1H), 7.11 (d, J = 8.7 Hz, 1H), 4.47 (q, J = 7.0 Hz, 2H), 2.24 (s, 3H), 1.23 (t, J = 7.1 Hz, 3H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 173.9, 161.6, 161.2, 156.9, 154.5, 154.5, 150.8, 135.6, 126.4, 125.3, 124.0, 122.4, 122.0, 115.2, 115.1, 113.4, 111.5, 62.9, 13.6, 7.9. Elemental analysis for C20H15NO5S; Calc.: C, 62.98; H, 3.96; N, 3.67%. Found: C, 62.94; H, 3.99; N, 3.63%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-8-methyl-4-oxo-4H-chromene-2-carboxylic acid (4d). To a stirred solution of ester 4d (1 mmol) in THF (10 mL), a solution of LiOH⋅2H2O (2 mmol) in water (2 mL) was added at room temperature. After stirring for 24 h, reaction was quenched by 1N HCl (5 mL), formed precipitate was filtered off, dried, and re-crystallized from EtOH to furnish the desired products 4e as yellow crystals. Yield 58%, m.p. 342–343 °C. LC-MS: m/z 354.0 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 11.16 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.62–7.50 (m, 1H), 7.50–7.41 (m, 1H), 7.17 (d, J = 8.7 Hz, 1H), 2.26 (s, 3H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 174.2, 162.4, 161.6, 157.5, 156.0, 154.5, 151.2, 135.7, 126.2, 125.2, 123.9, 122.5, 122.0, 115.2, 115.1, 113.0, 111.5, 7.9. Elemental analysis for C18H11NO5S; Calc.: C, 61.18; H, 3.14; N, 3.96%. Found: C, 61.21; H, 3.12 N, 3.98%.

3.2.2. General Procedure for the Synthesis of 2-Aminochromones 5a,b

A mixture of chromone 2a,e (1 mmol) and hydroxylamine hydrochloride (2 mmol) in pyridine (5 mL) was refluxed for 6–8 h. The reaction mixture was cooled, and the formed precipitate was filtered off, dried, and re-crystallized from EtOH to furnish the desired products 5a,b.
2-Amino-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one (5a). Yield 46%, m.p. 364–365, colorless. °C. LC-MS: m/z 311.0 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 10.86 (s, 1H), 10.68 (s, 1H), 9.46 (s, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.98–7.90 (m, 2H), 7.49–7.40 (m, 1H), 7.36–7.28 (m, 1H), 6.89 (d, J = 8.6 Hz, 1H), 6.77 (s, 1H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 172.2, 163.7, 162.8, 162.1, 153.6, 149.9, 133.0, 127.0, 125.7, 123.7, 121.3, 120.6, 114.0, 113.4, 101.6, 92.0. Elemental analysis for C16H10N2O3S; Calc.: C, 61.93; H, 3.25; N, 9.03%. Found: C, 61.91; H, 3.22; N, 9.00%.
2-Amino-6-ethyl-7-hydroxy-3-(4-methyl-1,3-thiazol-2-yl)-4H-chromen-4-one (5b). Yield 74%, m.p. 290–291 °C. LC-MS: m/z 303.2 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 10.57 (s, 2H), 9.00 (s, 1H), 7.76 (s, 1H), 6.96 (s, 1H), 6.78 (s, 1H), 2.60 (q, J = 7.3 Hz, 2H), 2.39 (s, 3H), 1.17 (t, J = 7.4 Hz, 3H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 171.6, 162.9, 161.1, 159.5, 151.6, 147.9, 128.6, 124.8, 113.2, 111.2, 100.9, 92.2, 22.3, 16.8, 13.8. Elemental analysis for C15H14N2O3S; Calc.: C, 59.59; H, 4.67; N, 9.27%. Found: C, 59.57; H, 4.65; N, 9.30%.

3.2.3. General Procedure for Synthesis of 8-Aminomethyl Derivatives 7

A stirring mixture of the corresponding chromone (1 mmol) and aminal [44] (1.2 mmol) in 1,4-dioxane (5 mL) was refluxed for 6–8 h. The reaction mixture was cooled, diluted with 10 mL of hexane. The formed precipitate was filtered off, dried, and re-crystallized from iPrOH-hexanes mixture to furnish the desired chromone Mannich bases 7.
3-(1,3-Benzothiazol-2-yl)-8-[(dimethylamino)methyl]-7-hydroxy-2-methyl-4H-chromen-4-one (7a). Yield 71%, m.p. 224–225 °C, pale pink. LC-MS: m/z 367.2 [M + H]+. 1H-NMR (500 MHz, CDCl3): δ ppm 8.13 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.52–7.45 (m, 1H), 7.43–7.35 (m, 1H), 6.92 (d, J = 8.8 Hz, 1H), 4.02 (s, 2H), 3.04 (s, 3H), 2.46 (s, 6H). 13C-NMR (125 MHz, CDCl3): δ ppm 175.2, 167.9, 164.7, 160.5, 154.3, 151.8, 136.1, 126.9, 125.8, 124.9, 122.8, 121.4, 116.2, 115.7, 115.2, 107.3, 55.3, 44.7, 22.0. Elemental analysis for C20H18N2O3S; Calc.: C, 65.55; H, 4.95; N, 7.64%. Found: C, 65.57; H, 4.93; N, 7.66%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-2-methyl-8-(pyrrolidin-1-ylmethyl)-4H-chromen-4-one (7b). Yield 67%, m.p. 214–217 °C, colorless. LC-MS: m/z 393.0 [M + H]+. 1H-NMR (300 MHz, CDCl3): δ ppm 11.47 (s, 1H), 8.12 (d, J = 8.8 Hz, 1H), 8.07–8.01 (m, 1H), 8.00–7.94 (m, 1H), 7.51–7.43 (m, 1H), 7.43–7.35 (m, 1H), 6.90 (d, J = 8.8 Hz, 1H), 4.19 (s, 2H), 3.03 (s, 3H), 2.91–2.66 (m, 4H), 2.03–1.83 (m, 4H). 13C-NMR (125 MHz, CDCl3): δ ppm 175.2, 167.9, 164.9, 160.6, 154.0, 151.8, 136.1, 126.7, 125.7, 124.8, 122.8, 121.4, 116.2, 115.7, 115.1, 107.8, 53.9, 51.4, 23.9, 22.0. Elemental analysis for C22H20N2O3S; Calc.: C, 67.33; H, 5.14; N, 7.14%. Found: C, 67.36; H, 5.12; N, 7.17%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-2-methyl-8-[(2-methylpiperidin-1-yl)methyl]-4H-chromen-4-one (7c). Yield 79%, m.p. 204–205 °C, colorless. LC-MS: m/z 421.2 [M + H]+. 1H-NMR (400 MHz, CDCl3): δ ppm 9.83 (s, 1H), 8.08 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.50–7.41 (m, 1H), 7.40–7.32 (m, 1H), 6.86 (d, J = 8.8 Hz, 1H), 4.48–4.23 (m, 1H), 3.90 (d, J = 15.0 Hz, 1H), 3.14–2.85 (m, 4H), 2.75–2.13 (m, 2H), 1.91–1.34 (m, 6H), 1.24 (d, J = 6.1 Hz, 3H); 13C-NMR (125 MHz, CDCl3): δ ppm 175.0, 167.9, 164.5, 160.5, 154.2, 151.7, 136.0, 126.3, 125.6, 124.7, 122.8, 121.3, 116.3, 115.5, 114.9, 107.4, 57.8, 54.5, 50.8, 35.4, 34.2, 25.6, 25.2, 21.9. Elemental analysis for C24H24N2O3S; Calc.: C, 68.55; H, 5.75; N, 6.66%. Found: C, 68.57; H, 5.73; N, 6.67%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-2-methyl-8-[(3-methylpiperidin-1-yl)methyl)]-4H-chromen-4-one (7d). Yield 83%, m.p. 200–201 °C, colorless. LC-MS: m/z 421.0 [M + H]+. 1H-NMR (500 MHz, CDCl3): δ ppm 8.11 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.53–7.43 (m, 2H), 7.41–7.32 (m, 1H), 6.89 (d, J = 8.8 Hz, 1H), 4.03 (s, 2H), 3.02 (s, 3H), 3.00–2.85 (m, 1H), 2.14–1.56 (m, 6H), 1.14–0.95 (m, 1H), 0.92 (d, J = 6.2 Hz, 3H). 13C-NMR (125 MHz, CDCl3): δ ppm 175.2, 167.9, 164.9, 160.5, 154.4, 151.8, 136.1, 126.7, 125.7, 124.8, 122.8, 121.4, 116.2, 115.7, 115.1, 107.0, 61.1, 54.4, 53.7, 32.3, 25.4, 21.9, 19.5. Elemental analysis for C24H24N2O3S; Calc.: C, 68.55; H, 5.75; N, 6.66%. Found: C, 68.57; H, 5.73; N, 6.67%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-2-methyl-8-[(4-methylpiperidin-1-yl)methyl]-4H-chromen-4-one (7e). Yield 85%, m.p. 216–218 °C, colorless. LC-MS: m/z 421.0 [M + H]+. 1H-NMR (400 MHz, CDCl3): δ ppm 8.13 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.54–7.46 (m, 1H), 7.45–7.34 (m, 1H), 6.92 (d, J = 8.8 Hz, 1H), 4.07 (s, 2H), 3.16–3.05 (m, 2H), 3.04 (s, 3H), 2.43–2.20 (m, 2H), 1.85–1.72 (m, 2H), 1.60–1.48 (m, 1H), 1.45–1.30 (m, 2H), 1.00 (d, J = 6.4 Hz, 3H). 13C-NMR (100 MHz, CDCl3): δ ppm 175.0, 168.0, 164.8, 160.4, 154.3, 151.7, 136.0, 126.5, 125.7, 124.8, 122.8, 121.3, 116.1, 115.5, 115.0, 107.0, 54.2, 53.6, 34.0, 30.4, 22.0, 21.6. Elemental analysis for C24H24N2O3S; Calc.: C, 68.55; H, 5.75; N, 6.66%. Found: C, 68.56; H, 5.73; N, 6.65%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-2-methyl-8-(morpholin-4-ylmethyl)-4H-chromen-4-one (7f), pale yellow crystals. Yield 88%, m.p. 243–244 °C. LC-MS: m/z 409.2 [M + H]+. 1H-NMR (400 MHz, CDCl3): δ ppm 8.14 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.54–7.45 (m, 1H), 7.44–7.35 (m, 1H), 6.92 (d, J = 8.8 Hz, 1H), 4.07 (s, 2H), 3.97–3.67 (m, 4H), 3.04 (s, 3H), 2.88–2.53 (m, 4H). 13C-NMR (100 MHz, CDCl3): δ ppm 175.1, 168.1, 163.8, 160.3, 154.5, 151.7, 136.0, 127.1, 125.8, 124.9, 122.9, 121.4, 116.1, 115.8, 115.6, 106.5, 66.7, 54.2, 53.2, 22.0. Elemental analysis for C22H20N2O4S; Calc.: C, 64.69; H, 4.94; N, 6.86%. Found: C, 64.67; H, 4.96; N, 6.85%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-2-methyl-8-[(4-methylpiperazin-1-yl)methyl]-6-propyl-4H-chromen-4-one (7g), colorless. Yield 68%, m.p. 230–231 °C. LC-MS: m/z 464.2 [M + H]+. 1H-NMR (400 MHz, CDCl3): δ ppm 8.04 (d, J = 8.1 Hz, 1H), 8.00–7.94 (m, 2H), 7.51–7.43 (m, 1H), 7.41–7.34 (m, 1H), 4.06 (s, 2H), 3.03 (s, 3H), 2.99–2.40 (m, 10H), 2.35 (s, 3H), 1.80–1.59 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H). 13C-NMR (125 MHz, CDCl3): δ ppm 175.0, 167.8, 162.4, 160.5, 152.8, 151.6, 136.0, 129.5, 125.6, 125.3, 124.6, 122.7, 121.2, 115.3, 114.6, 106.0, 54.7, 53.8, 52.6, 45.9, 31.7, 22.5, 22.0, 14.0. Elemental analysis for C26H29N3O3S; Calc.: C, 67.36; H, 6.31; N, 9.06%. Found: C, 67.32; H, 6.29; N, 9.07%.
2-Amino-3-(1,3-benzothiazol-2-yl)-7-hydroxy-8-[(4-methylpiperazin-1-yl)methyl]-4H-chromen-4-one (7h), yellow. Yield 59%, m.p. 248–250 °C. LC-MS: m/z 423.2 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 10.76 (s, 1H), 9.39 (s, 1H), 8.02 (d, J = 7.4 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.50–7.41 (m, 1H), 7.38–7.29 (m, 1H), 6.84 (d, J = 8.3 Hz, 1H), 4.02 (s, 2H), 2.70–2.57 (m, 4H), 2.46–2.29 (m, 4H), 2.18 (s, 3H). J13C-NMR (125 MHz, CDCl3): δ ppm 172.3, 163.3, 162.7, 162.3, 151.2, 149.9, 132.9, 125.7, 125.3, 123.6, 121.3, 120.5, 113.7, 113.0, 108.1, 91.7, 54.4, 52.1, 51.5, 45.5. Elemental analysis for C22H24N4O3S; Calc.: C, 62.54; H, 5.25; N, 13.26%. Found: C, 62.52; H, 5.27; N, 13.24%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-8-{[4-(2-hydroxyethyl)piperazin-1-yl]methyl}-2-methyl-6-propyl-4H-chromen-4-one (7i), colorless. Yield 76%, m.p. 203–204 °C. LC-MS: m/z 494.0 [M + H]+. 1H-NMR (500 MHz, CDCl3): δ ppm 8.05 (d, J = 8.1 Hz, 1H), 8.01–7.96 (m, 2H), 7.52–7.44 (m, 1H), 7.45–7.34 (m, 1H), 4.08 (s, 2H), 3.69–3.62 (m, 2H), 3.04 (s, 3H), 3.00–2.28 (m, 13H), 1.75–1.60 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H). 13C-NMR (125 MHz, CDCl3): δ ppm 175.2, 167.8, 162.4, 160.7, 153.0, 151.8, 136.1, 129.6, 125.7, 125.7, 124.8, 122.8, 121.4, 115.6, 114.8, 106.0, 59.2, 57.9, 53.8, 52.7, 52.6, 31.8, 22.6, 22.0, 14.1. Elemental analysis for C27H31N3O4S; Calc.: C, 65.70; H, 6.33; N, 8.51%. Found: C, 65.68; H, 6.35; N, 8.53%.
3-(1,3-Benzothiazol-2-yl)-6-ethyl-7-hydroxy-8-[(1,3,3-trimethyl-6-azabicyclo [3.2.1]oct-6-yl)methyl]-4H-chromen-4-one (7j), pale yellow. Yield 84%, m.p. 183–185 °C. LC-MS: m/z 489.2 [M + H]+. 1H-NMR (300 MHz, CDCl3): δ ppm 9.14 (s, 1H), 8.07 (s, 1H), 8.05–7.94 (m, 2H), 7.55–7.44 (m, 1H), 7.42–7.33 (m, 1H), 4.31 (d, J = 14.3 Hz, 1H), 4.04 (d, J = 14.3 Hz, 1H), 3.36–3.28 (m, 1H), 3.27–3.16 (m, 1H), 2.83–2.62 (m, 2H), 2.33–2.24 (m, 1H), 1.93–1.71 (m, 2H), 1.59–1.37 (m, 2H), 1.38–1.23 (m, 7H), 1.13 (s, 4H), 0.96 (s, 3H). 13C-NMR (125 MHz, CDCl3): δ ppm 174.4, 164.4, 159.5, 155.1, 153.5, 151.8, 136.4, 132.1, 126.1, 124.7, 124.5, 122.4, 121.7, 117.6, 115.2, 108.0, 64.8, 62.6, 54.2, 51.5, 44.0, 41.7, 40.7, 36.9, 32.2, 29.6, 25.9, 22.9, 13.8. Elemental analysis for C29H32N2O3S; Calc.: C, 71.26; H, 6.60; N, 5.73%. Found: C, 71.25; H, 6.58; N, 5.76%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-2-methyl-8-[(1,3,3-trimethyl-6-azabicyclo [3.2.1]oct-6-yl)methyl]-4H-chromen-4-one (7k), colorless. Yield 87%, m.p. 158–159 °C. LC-MS: m/z 475.2 [M + H]+. 1H-NMR (400 MHz, CDCl3): δ ppm 10.43 (s, 1H), 8.12 (d, J = 8.8 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.52–7.43 (m, 1H), 7.43–7.33 (m, 1H), 6.91 (d, J = 8.8 Hz, 1H), 4.28 (d, J = 14.5 Hz, 1H), 4.00 (d, J = 14.5 Hz, 1H), 3.34 (d, J = 11.0 Hz, 1H), 3.27–3.16 (m, 1H), 3.02 (s, 3H), 2.32–2.22 (m, 1H), 1.92–1.78 (m, 2H), 1.59–1.48 (m, 1H), 1.47–1.40 (m, 1H), 1.36–1.28 (m, 2H), 1.26 (s, 3H), 1.14 (s, 3H), 0.97 (s, 3H); 13C-NMR (125 MHz, CDCl3): δ ppm 175.0, 167.8, 165.6, 160.5, 154.1, 151.7, 136.1, 126.7, 125.7, 124.8, 122.8, 121.3, 116.3, 115.5, 114.7, 108.1, 65.1, 62.7, 54.4, 51.5, 43.9, 41.7, 40.6, 36.9, 32.1, 29.6, 25.9, 22.0. Elemental analysis for C28H30N2O3S; Calc.: C, 70.86; H, 6.37; N, 5.90%. Found: C, 70.90; H, 6.38; N, 5.88%.
Ethyl 3-(1,3-benzothiazol-2-yl)-6-ethyl-7-hydroxy-4-oxo-8-[(1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-yl)methyl]-4H-chromene-2-carboxylate (7l), colorless. Yield 78%, m.p. 153–154 °C. LC-MS: m/z 561.2 [M + H]+. 1H-NMR (400 MHz, CDCl3): δ ppm 12.15 (s, 1H), 8.04 (s, 1H), 8.01–7.92 (m, 2H), 7.52–7.44 (m, 1H), 7.39 (t, J = 7.5 Hz, 1H), 4.51 (q, J = 7.2 Hz, 2H), 4.32 (d, J = 14.4 Hz, 1H), 4.05 (d, J = 14.4 Hz, 1H), 3.35 (d, J = 11.0 Hz, 1H), 3.28–3.17 (m, 1H), 2.88–2.63 (m, 2H), 2.31 (d, J = 11.0 Hz, 1H), 1.96–1.70 (m, 2H), 1.60–.50 (m, 1H), 1.48–1.39 (m, 1H), 1.35–1.28 (m, 8H), 1.27 (s, 3H), 1.13 (s, 3H), 0.97 (s, 3H); 13C-NMR (125 MHz, CDCl3): δ ppm 174.6, 165.2, 162.2, 157.7, 153.9, 152.9, 151.6, 136.6, 132.7, 126.0, 125.0, 124.4, 122.9, 121.7, 114.9, 114.5, 107.6, 64.7, 63.1, 62.6, 54.1, 51.5, 43.8, 41.7, 40.6, 36.9, 32.2, 29.6, 25.8, 22.9, 13.9, 13.7. Elemental analysis for C32H36N2O5S; Calc.: C, 68.55; H, 6.47; N, 5.00%. Found: C, 68.53; H, 6.51; N, 5.03%.
2-Amino-6-ethyl-7-hydroxy-3-(4-methyl-1,3-thiazol-2-yl)-8-(piperidin-1-ylmethyl)-4H-chromen-4-one (7m), yellow. Yield 49%, m.p. 174–175 °C. LC-MS: m/z 400.2 [M + H]+. 1H-NMR (500 MHz, CDMSO-d6): δ ppm 10.47 (s, 1H), 9.00 (s, 1H), 7.70 (s, 1H), 6.99 (s, 1H), 4.05 (s, 2H), 2.66–2.51 (m, 6H), 2.40 (s, 3H), 1.67–1.55 (m, 4H), 1.53–1.42 (m, 2H), 1.17 (t, J = 7.5 Hz, 3H). 13C-NMR (125 MHz, CDCl3): δ ppm 173.5, 162.5, 161.6, 161.5, 149.6, 148.5, 129.4, 124.1, 113.2, 111.7, 106.0, 93.6, 54.5, 53.9, 25.7, 23.8, 22.7, 17.2, 13.7. Elemental analysis for C21H25N3O3S; Calc.: C, 63.13; H, 6.31; N, 10.52%. Found: C, 63.10; H, 6.15; N, 10.50%.

3.2.4. General Procedure for Synthesis of Coumarin Aminomethyl Derivatives 10,11

A stirring mixture of the corresponding 3-hetarylcoumarin 9 or 10a,b chromone (1 mmol) and aminal (1.2 mmol) in 1,4-dioxane (5 mL) was refluxed for 6–8 h. The reaction mixture was cooled, diluted with 10 mL of hexane. The formed precipitate was filtered off, dried, and re-crystallized from toluene-hexane mixture to furnish the desired chromone Mannich bases 7.
3-(1,3-Benzothiazol-2-yl)-8-{[bis(2-hydroxyethyl)amino]methyl}-7-hydroxy-2H-chromen-2-one (10a). Yield 56%, m.p. 186–187 °C, yellow. LC-MS: m/z 413.2 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 9.01 (s, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.54–7.48 (m, 1H), 7.45–7.32 (m, 1H), 6.69 (d, J = 8.6 Hz, 1H), 4.23 (s, 2H), 3.65 (t, J = 5.5 Hz, 4H), 2.87 (t, J = 5.5 Hz, 4H). 13C-NMR (125 MHz, CDCl3): δ ppm 168.4, 160.9, 159.8, 153.9, 152.1, 142.9, 135.4, 130.8, 126.3, 124.6, 122.0, 121.9, 116.0, 111.4, 109.6, 107.4, 57.3, 55.1, 49.8. Elemental analysis for C21H20N2O5S; Calc.: C, 61.15; H, 4.89; N, 6.79%. Found: C, 61.12; H, 4.91; N, 6.77%.
3-(1,3-Benzothiazol-2-yl)-8-[(3,3-dimethylpiperidin-1-yl)methyl]-7-hydroxy-2H-chromen-2-one (10b). Yield 85%, m.p. 188–189 °C, green (fluorescent). LC-MS: m/z 421.0 [M + H]+. 1H-NMR (500 MHz, CDCl3): δ ppm 8.97 (s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.54–7.47 (m, 2H), 7.41–7.35 (m, 1H), 6.83 (d, J = 8.6 Hz, 1H), 4.17–3.95 (m, 2H), 3.19–2.45 (m, 2H), 2.32–1.91 (m, 1H), 1.91–1.16 (m, 5H), 1.02 (s, 6H). 13C-NMR (125 MHz, CDCl3): δ ppm 166.0, 161.0, 160.4, 153.5, 152.6, 142.8, 136.6, 130.1, 126.4, 125.0, 122.6, 121.8, 115.5, 114.8, 111.3, 107.5, 65.8, 54.7, 53.8, 36.7, 31.2, 21.9. Elemental analysis for C24H24N2O3S; Calc.: C, 68.55; H, 5.75; N, 6.66%. Found: C, 68.52; H, 5.77; N, 6.67%.
3-(1,3-Benzothiazol-2-yl)-7-hydroxy-8-{[4-(2-hydroxyethyl)piperazin-1-yl]methyl}-2H-chromen-2-one (10c). Yield 64%, m.p. 207–208 °C, yellow. LC-MS: m/z 438.0 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 9.06 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.56–7.48 (m, 1H), 7.45–7.37 (m, 1H), 6.79 (d, J = 8.6 Hz, 1H), 3.99 (s, 2H), 3.50 (t, J = 6.0 Hz, 2H), 2.80–2.53 (m, 8H), 2.45–2.36 (m, 2H). 13C-NMR (125 MHz, DMSO-d6): δ ppm 165.9, 160.6, 159.7, 153.7, 152.0, 142.9, 135.5, 130.8, 126.4, 124.8, 122.0, 118.8, 115.2, 112.7, 110.4, 107.4, 59.8, 58.4, 52.6, 51.9, 51.7. Elemental analysis for C23H23N3O4S; Calc.: C, 63.14; H, 5.30; N, 9.60%. Found: C, 63.12; H, 5.28; N, 9.63%.
7-Hydroxy-8-{[4-(2-hydroxyethyl)piperazin-1-yl]methyl}-3-(4-methyl-1,3-thiazol-2-yl)-2H-chromen-2-one (11a). Yield 58%, m.p. 157–159 °C, yellow. LC-MS: m/z 402.2 [M + H]+. 1H-NMR (500 MHz, CDCl3): δ ppm 8.74 (s, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.04–6.98 (m, 1H), 6.81 (d, J = 8.6 Hz, 1H), 4.11 (s, 2H), 3.68–3.60 (m, 2H), 3.20–2.53 (m, 10H), 2.52 (s, 3H). 13C-NMR (125 MHz, CDCl3): δ ppm 163.8, 160.2, 159.2, 152.9, 152.8, 139.9, 129.6, 116.4, 115.8, 115.0, 111.8, 107.4, 59.3, 58.0, 53.7, 52.8, 52.6, 17.3. Elemental analysis for C20H23N3O4S; Calc.: C, 59.83; H, 5.77; N, 10.47%. Found: C, 59.85; H, 5.79; N, 10.44%.
3-[4-(4-Bromophenyl)-1,3-thiazol-2-yl]-7-hydroxy-8-{[4-(2-hydroxyethyl)piperazin-1-yl]methyl}-2H-chromen-2-one (11b). Yield 71%, m.p. 224–225 °C, colorless. LC-MS: m/z 542.0 [M + H]+. 1H-NMR (300 MHz, CDCl3): δ ppm 8.88 (s, 1H), 7.88 (d, J = 8.5 Hz, 2H), 7.62 (s, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H), 4.13 (s, 2H), 3.68–3.59 (m, 2H), 3.03–2.29 (m, 10H). 13C-NMR (100 MHz, CDCl3): δ ppm 164.1, 160.2, 159.8, 154.1, 152.9, 140.5, 133.5, 131.9, 129.7, 128.0, 122.2, 115.8, 115.5, 115.1, 111.7, 107.4, 59.2, 57.9, 53.7, 52.8, 52.6. Elemental analysis for C25H24BrN3O4S; Calc.: C, 55.35; H, 4.46; N, 7.75%. Found: C, 55.32; H, 4.49; N, 7.73%.
3-[4-(4-Bromophenyl)-1,3-thiazol-2-yl]-7-hydroxy-8-[(2-methylpiperidin-1-yl)methyl]-2H-chromen-2-one (11c). Yield 68%, m.p. 184–186 °C, yellow. LC-MS: m/z 511.0 [M + H]+. 1H-NMR (300 MHz), δ 11.31 (s, 1H), 8.85 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.61–7.54 (m, 3H), 7.47 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 4.55–4.23 (m, 1H), 4.08–3.91 (m, 1H), 3.20–2.15 (m, 3H), 1.91–1.34 (m, 6H), 1.25 (d, J = 5.5 Hz, 3H). 13C-NMR (125 MHz, CDCl3–TFA 1:1): δ ppm 165.1, 160.6, 155.7, 148.4, 148.1, 135.8, 133.8, 129.5, 128.7, 127.7, 125.1, 118.1, 116.8, 112.7, 109.0, 105.0, 64.4, 54.0, 47.4, 32.1, 23.3, 21.8, 18.1. Elemental analysis for C25H23BrN2O3S; Calc.: C, 58.71; H, 4.53; N, 5.48%. Found: C, 58.68; H, 4.50; N, 5.50%.
3-[4-(4-Bromophenyl)-1,3-thiazol-2-yl]-7-hydroxy-8-[(3-methylpiperidin-1-yl)methyl]-2H-chromen-2-one (11d). Yield 83%, m.p. 216–217 °C, yellow. LC-MS: m/z 511.0 [M + H]+. 1H-NMR (300 MHz, CDCl3): δ ppm 11.45 (s, 1H), 8.84 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.61–7.53 (m, 3H), 7.48 (d, J = 8.6 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 4.06 (s, 2H), 3.10–2.85 (m, 2H), 2.39–1.52 (m, 6H), 1.15–0.73 (m, 4H); 13C-NMR (125 MHz, DMSO-d6): δ ppm 162.4, 159.4, 159.2, 154.5, 153.2, 140.5, 133.3, 132.8, 131.9, 128.4, 121.6, 117.4, 115.2, 114.1, 111.7, 103.6, 58.5, 52.4, 48.7, 29.7, 29.0, 22.7, 18.8. Elemental analysis for C25H23BrN2O3S; Calc.: C, 58.71; H, 4.53; N, 5.48%. Found: C, 58.72; H, 4.51; N, 5.50%.
3-[4-(4-Bromophenyl)-1,3-thiazol-2-yl]-7-hydroxy-8-[(4-methylpiperidin-1-yl)methyl]-2H-chromen-2-one (11e). Yield 85%, m.p. 229–230 °C, yellow. LC-MS: m/z 511.0 [M + H]+. 1H-NMR (400 MHz, DMSO-d6): δ ppm 8.94 (s, 1H), 8.18 (s, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 8.2 Hz, 2H), 6.75 (d, J = 8.7 Hz, 1H), 4.07 (s, 2H), 3.08–2.97 (m, 2H), 2.48–2.36 (m, 2H), 1.79–1.65 (m, 2H), 1.58–1.42 (m, 1H), 1.33–1.16 (m, 2H), 0.94 (d, J = 6.1 Hz, 3H). 13C-NMR (125 MHz, CDCl3–TFA 1:1): δ ppm 165.3, 164.9, 160.6, 155.6, 148.4, 148.0, 135.8, 133.8, 128.7, 127.7, 125.0, 118.0, 116.8, 112.5, 108.8, 104.5, 55.3, 50.3, 31.6, 29.0, 20.3. Elemental analysis for C25H23BrN2O3S; Calc.: C, 58.71; H, 4.53; N, 5.48%. Found: C, 58.70; H, 4.54; N, 5.51%.
7-Hydroxy-3-(4-methyl-1,3-thiazol-2-yl)-8-[(1,3,3-trimethyl-6-azabicyclo [3.2.1]oct-6-yl)methyl]-2H-chromen-2-one (11f). Yield 70%, m p. 159–160 °C, yellow (fluorescent). LC-MS: m/z 425.0 [M + H]+. 1H-NMR (500 MHz, CDCl3): δ ppm 8.73 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H), 6.99 (s, 1H), 6.80 (d, J = 8.6 Hz, 1H), 4.37 (d, J = 14.6 Hz, 1H), 4.07 (d, J = 14.6 Hz, 1H), 3.36 (d, J = 11.2 Hz, 1H), 3.30–3.19 (m, 1H), 2.52 (s, 3H), 2.30 (d, J = 11.2 Hz, 1H), 1.96–1.76 (m, 2H), 1.53 (d, J = 14.0 Hz, 1H), 1.42 (d, J = 13.7 Hz, 1H), 1.32 (dd, J = 14.3, 2.2 Hz, 1H), 1.28 (d, J = 11.5 Hz, 1H), 1.24 (s, 3H), 1.12 (s, 3H), 0.95 (s, 3H). 13C-NMR (125 MHz, CDCl3): δ ppm 166.6, 160.5, 159.6, 152.8, 152.8, 140.4, 129.8, 116.0, 115.7, 114.6, 110.7, 108.1, 64.9, 63.1, 54.8, 51.5, 43.8, 41.7, 40.6, 36.9, 32.1, 29.6, 25.8, 17.4. Elemental analysis for C24H28rN2O3S; Calc.: C, 67.90; H, 6.65; N, 6.60%. Found: C, 67.88; H, 6.67; N, 6.63%.

4. Biological Evaluation

4.1. Cell Viability Studies

A set of 25 compounds was tested for cell viability studies using MTT assay, in HCT116 and HeLa cell lines. All experiments were carried out in triplicate as per previously reported methods [53,54]. For the viability studies, HCT116 and HeLa cells were seeded at the count of 7 × 103 cells per well in a transparent 96-well plates, and incubated for 24 h at 37 °C, in a humidified incubator with 5% CO2. Following this, the cells were treated with the compounds at a final concentration of 10 µM and incubated further for 72 h at 37 °C. After incubation, MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide] was added at a final concentration of 0.5 mg/mL. The cells were further incubated for 4 h at 37 °C in an incubator. The formazan crystals formed were dissolved using 50 µL of DMSO. Thereafter, the absorbance was measured at 570 nm using the PerkinElmer EnVision Multilabel Reader (Perkin Elmer, Inc., Waltham, MA, USA). The cells treated with DMSO were used as a control for all the experiments. Both the cell lines showed viability ≤ 50% for three compounds in the initial screening, namely 2c, 7h and 7l. Hence, these three compounds were selected from the initial screening and were sensitized against HCT116 and HeLa, across a dose range of 0–100 µM by performing another cell viability assay. Finally, the results obtained were plotted using Graphpad Prism 8.0.2 to obtain the respective IC50 values.

4.2. Immunoblot Assay

The immunoblot assay was carried out as per previous reports [2]. HCT116 and HeLa cells were seeded at the count of 0.5 × 105 cells per ml in a 6-well plate and incubated overnight at 37 °C, in a humidified CO2 incubator. For this assay, the cells were treated with the hit compounds with their concentration range approximately equal to their IC50 values (Table 3), and incubated for 1 h at 37 °C. After the incubation, the cells were given UV radiation energy via UVP cross linker at 50 mJ/cm2 and further incubated for 1 h at 37 °C. The media was stored before UV radiation and added back after the treatment. The cells were then trypsinized, washed with ice cold 1X PBS, and lysed with RIPA buffer. The cell lysates were isolated by centrifugation at 4 °C, 13,500 rpm for 10 min. The protein concentration was normalized by Bradford assay. The samples were loaded on SDS-PAGE gel and subjected to immunoblotting. β-actin was taken as a loading control in the experiment.
Further, to identify the pattern of inhibition of Chk1 phosphorylation, the immunoblot assay was repeated for a range of concentrations of the compounds 2c, 7h, and 7l in HeLa cell line against Chk1 and pChk1 antibodies. The experiments were conducted in a similar way for a concentration range of 2 µM, 5 µM and 10 µM. β-actin was taken as a loading control in the experiment.

4.3. Molecular Docking

In the present study, the compounds 2c, 7h and 7l were subjected to molecular docking studies at the Torin2 binding site of homology modelled ATR kinase domain [55], using the Glide module (XP) of Maestro 12.7.156 version of Schrödinger software [56]. The homology modelled ATR kinase domain was further prepared for molecular docking, using the Protein Preparation Wizard by including Epik state penalties (pH 7.0 ± 2.0), and by performing the minimization using 0.3 Å RMSD and OPLS4 force field [57,58,59]. Further, the compounds 2c, 7h and 7l were included in the project using the 2D sketcher option in the software, following which the 3D structures were incorporated in the workspace. The standard molecule for the study was selected to be Torin2, the standard inhibitor in the modelled structure, which is a well-known mTOR and ATR/ATM inhibitor known for its potency against the p-Chk1 Ser 317 and p70 S6K Thr 389 substrates in the DDR pathway through cell studies. The ligands were then prepared using the LigPrep module [57].
The ligand preparation was followed by receptor grid generation, wherein the grid was generated using the Glide module, by selecting the Torin2 ligand from the minimized protein structure. Ligand docking for the standard molecule and the synthesized compounds was conducted in the Glide XP (Extra Precision) module [56].
The docking results were analyzed in the XP Visualizer tool, wherein the binding energies were analyzed using the docking score instead of GlideScore, since the Epik state penalties were included during the protein and ligand preparations. The best pose of each of the compounds 2c, 7h and 7l with higher negative value of DockScore were considered for the study.

5. Conclusions

A series of benzothiazole and chromone derivatives were synthesized and evaluated for their anticancer activity as inhibitors of ATR kinase. Cell viability of a set of 25 compounds was performed using MTT assay in HCT116 and HeLa cell lines, involving 72 h incubation of the compounds at a final concentration of 10 µM. Cells incubated with compounds 2c, 7h and 7l were found to show a viability ≤50, and were taken forward for dose–response studies. The three compounds showed IC50 values in micromolar range. Among all the compounds tested, 7l had the best IC50 values in both the cell lines. Compounds 2c and 7l were found to be equally cytotoxic towards both the cell lines, namely, HCT116 and Hela, while compound 7h showed better cytotoxicity towards HeLa cell line.
To further identify the inhibitory potential of the synthesized compounds in the DNA damage response (DDR) signalling pathway, an immunoblot assay was carried out for these three compounds in order to analyze the inhibition of phosphorylation of Chk1 at Ser 317 in HeLa and HCT116 cells. Compound 7h showed inhibition of pChk1 at Ser 317 in HeLa cells at a concentration of 3.995 µM. Further analysis for Chk1 and pChk1 expression was analyzed in Hela cells by treatment against all the three compounds at a range of concentrations such as 2, 5 and 10 µM, wherein compound 7h showed Chk1 inhibition at 2 and 5 µM, while pChk1 expression was observed for compound 7l at a concentration of 5 µM. The binding interactions of the compounds with the ATR kinase domain were studied through molecular docking, wherein compound 2c, 7h and 7l showed binding interactions similar and/or lesser than the standard ligand Torin2. Thus, these compounds can serve as starting point for further modifications in order to improve the activity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27144637/s1. The copies of 1H- and 13C-NMR spectra for all new synthesized compounds have been submitted along with the manuscript.

Author Contributions

Conceptualization, A.G. and V.K.; software, H.D. and A.P.; validation, D.C. and H.D.; investigation, M.F. and D.C.; formal analysis, S.N.S.; data curation, A.G. and S.K.; writing—original draft preparation, A.G., M.F. and S.K.; writing—review and editing, A.G., S.K. and D.S.; supervision, S.K. and A.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Acknowledgments

Sivapriya Kirubakaran sincerely thanks Kankuben Bakshirambhai Gelot Chair position for the great support. Authors thank Enamine Ltd. for data collection for LC MS spectra.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

References

  1. Srinivas, U.S.; Tan, B.W.Q.; Vellayappan, B.A.; Jeyasekharan, A.D. ROS and the DNA damage response in cancer. Redox Biol. 2019, 25, 101084. [Google Scholar] [CrossRef]
  2. Bhakuni, R.; Shaik, A.; Priya, B.; Kirubakaran, S. Characterization of SPK 98, a Torin2 analog, as ATR and mTOR dual kinase inhibitor. Bioorg. Med. Chem. Lett. 2020, 30, 127517. [Google Scholar] [CrossRef] [PubMed]
  3. Dietlein, F.; Thelen, L.; Reinhardt, H.C. Cancer-specific defects in DNA repair pathways as targets for personalized therapeutic approaches. Trends Genet. 2014, 30, 326–339. [Google Scholar] [CrossRef] [PubMed]
  4. Fokas, E.; Prevo, R.; Hammond, E.M.; Brunner, T.B.; McKenna, W.G.; Muschel, R.J. Targeting ATR in DNA damage response and cancer therapeutics. Cancer Treat. Rev. 2014, 40, 109–117. [Google Scholar] [CrossRef] [PubMed]
  5. Cilibrasi, V.; Spanò, V.; Bortolozzi, R.; Barreca, M.; Raimondi, M.V.; Rocca, R.; Maruca, A.; Montalbano, A.; Alcaro, S.; Ronca, R.; et al. Synthesis of 2H-Imidazo[2′,1′:2,3] [1,3]thiazolo[4,5-e]isoindol-8-yl-phenylureas with promising therapeutic features for the treatment of acute myeloid leukemia (AML) with FLT3/ITD mutations. Eur. J. Med. Chem. 2022, 235, 114292. [Google Scholar] [CrossRef] [PubMed]
  6. Morsy, M.A.; Ali, E.M.; Kandeel, M.; Venugopala, K.N.; Nair, A.B.; Greish, H.; El-Daly, M. Screening and Molecular Docking of Novel Benzothiazole Derivatives as Potential Antimicrobial Agents. Antibiotics 2020, 9, 221. [Google Scholar] [CrossRef]
  7. Haroun, M.; Tratrat, C.; Petrou, A.; Geronikaki, A.; Ivanov, M.; Ciric, A.; Soković, M.; Nagaraja, S.; Venugopala, K.N.; Nair, A.B.; et al. Exploration of the Antimicrobial Effects of Benzothiazolylthiazolidin-4-One and In Silico Mechanistic Investigation. Molecules 2021, 26, 4061. [Google Scholar] [CrossRef]
  8. Kumar, G.; Singh, N.P. Synthesis, anti-inflammatory and analgesic evaluation of thiazole/oxazole substituted benzothiazole derivatives. Bioorg. Chem. 2021, 107, 104608. [Google Scholar] [CrossRef]
  9. Ugwu, D.I.; Okoro, U.C.; Ukoha, P.O.; Gupta, A.; Okafor, S.N. Novel anti-inflammatory and analgesic agents: Synthesis, molecular docking and in vivo studies. J. Enzym. Inhib. Med. Chem. 2018, 33, 405–415. [Google Scholar] [CrossRef] [Green Version]
  10. Djuidje, E.N.; Barbari, R.; Baldisserotto, A.; Durini, E.; Sciabica, S.; Balzarini, J.; Liekens, S.; Vertuan, S.; Manfredini, S. Benzothiazole Derivatives as Multifunctional Antioxidant Agents for Skin Damage: Structure–Activity Relationship of a Scaffold Bearing a Five-Membered Ring System. Antioxidants 2022, 11, 407. [Google Scholar] [CrossRef]
  11. Cabrera-Pérez, L.C.; Padilla-Martínez, I.I.; Cruz, A.; Mendieta-Wejebe, J.E.; Tamay-Cach, F.; Rosales-Hernández, M.C. Evaluation of a new benzothiazole derivative with antioxidant activity in the initial phase of acetaminophen toxicity. Ar. J. Chem. 2019, 12, 3871–3882. [Google Scholar] [CrossRef]
  12. Kumar, K.R.; Karthik, K.N.S.; Begum, P.R.; Prasada, R.C.M.M. Synthesis, characterization and biological evaluation of benzothiazole derivatives as potential antimicrobial and analgesic agents. Asian J. Res. Pharm. Sci. 2017, 7, 115–119. [Google Scholar] [CrossRef]
  13. Uremis, N.; Uremis, M.M.; Tolun, F.I.; Ceylan, M.; Doganer, A.; Kurt, A.H. Synthesis of 2-Substituted Benzothiazole Derivatives and Their In Vitro Anticancer Effects and Antioxidant Activities against Pancreatic Cancer Cells. Anticancer Res. 2017, 37, 6381–6389. [Google Scholar] [PubMed]
  14. Irfan, A.; Batool, F.; Naqvi, S.Z.; Islam, A.; Osman, S.M.; Nocentini, A.; Alissa, S.A.; Supuran, C.T. Benzothiazole derivatives as anticancer agents. J Enzym. Inhib. Med. Chem. 2020, 35, 265–279. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Islam, M.K.; Baek, A.-R.; Sung, B.; Yang, B.-W.; Choi, G.; Park, H.-J.; Kim, Y.-H.; Kim, M.; Ha, S.; Lee, G.-H.; et al. Synthesis, Characterization, and Anticancer Activity of Benzothiazole Aniline Derivatives and Their Platinum (II) Complexes as New Chemotherapy Agents. Pharmaceuticals 2021, 14, 832. [Google Scholar] [CrossRef]
  16. Osmaniye, D.; Levent, S.; Karaduman, A.B.K.; Ilgın, S.; Özkay, Y.; Kaplancıklı, Z.A. Synthesis of New Benzothiazole Acylhydrazones as Anticancer Agents. Molecules 2018, 23, 1054. [Google Scholar] [CrossRef] [Green Version]
  17. Asiri, Y.I.; Alsayari, A.; Muhsinah, A.B.; Mabkhot, Y.N.; Hassan, M.Z. Benzothiazoles as potential antiviral agents. J. Pharm. Pharmacol. 2020, 72, 1459–1480. [Google Scholar] [CrossRef]
  18. Elamin, M.B.; Abd Elaziz, A.A.E.S.; Abdallah, E.M. Benzothiazole moieties and their derivatives as antimicrobial and antiviral agents: A mini-review. Int. J. Res. Pharm. Sci. 2020, 3, 3309–3315. [Google Scholar] [CrossRef]
  19. Kumar, M.; Chung, S.-M.; Enkhtaivan, G.; Patel, R.V.; Shin, H.-S.; Mistry, B.M. Molecular Docking Studies and Biological Evaluation of Berberine–Benzothiazole Derivatives as an Anti-Influenza Agent via Blocking of Neuraminidase. Int. J. Mol. Sci. 2021, 22, 2368. [Google Scholar] [CrossRef]
  20. Khyati Bhagdev, K.; Sarkar, S. Benzothiazole Moiety and Its Derivatives as Antiviral Agents. Med. Sci. Forum 2021, 7, 2–7. [Google Scholar]
  21. Al-Masoudia, N.A.; Jafar, N.N.A.; Abbas, L.J.; Baqir, S.J.; Pannecouque, C. Synthesis and anti-HIV Activity of New Benzimidazole, Benzothiazole and Carbohyrazide Derivatives of the anti-Inflammatory Drug Indomethacin. Z. Naturforsch. 2011, 66b, 953–960. [Google Scholar] [CrossRef]
  22. Petrou, A.; Eleftheriou, P.; Geronikaki, A.; Akrivou, M.G.; Vizirianakis, I. Novel thiazolidin-4-ones as potential non-nucleoside inhibitors of HIV-1 reverse transcriptase. Molecules 2019, 24, 3821. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Ullah, S.; Mirza, S.; Salar, U.; Hussain, S.; Javaid, K.; Khan, K.M.; Khalil, R.; Atia-Tul-Wahab; Ul-Haq, Z.; Perveen, S.; et al. 2-Mercapto Benzothiazole Derivatives: As Potential Leads for the Diabetic Management. Med. Chem. 2020, 16, 826–840. [Google Scholar] [CrossRef] [PubMed]
  24. Nath, R.; Shahar, Y.M.; Pathania, S.; Grover, G.; Debnath, B.; Akhtar, M.J. Synthesis and anticonvulsant evaluation of indoline derivatives of functionalized aryloxadiazole amine and benzothiazole acetamide. J. Mol. Struct. 2021, 1228, 129742. [Google Scholar] [CrossRef]
  25. Khokra, S.L.; Arora, K.; Khan, S.A.; Kaushik, P.; Saini, R.; Husain, A. Synthesis, Computational Studies and Anticonvulsant Activity of Novel Benzothiazole Coupled Sulfonamide Derivatives. Iran. J. Pharm. Res. 2019, 18, 826–840. [Google Scholar]
  26. Hadanu, R.; Idris, S.; Sutapa, I.W. QSAR analysis of benzothiazole derivatives of antimalarial compounds based on am1 semi-empirical method. Indones. J. Chem. 2015, 15, 86–92. [Google Scholar] [CrossRef]
  27. Suresh, A.J.; Bharathi, K.; Surya, P.R. Design, Synthesis, Characterization and Biological Evaluation of Some Novel Benzothiazole Derivatives as Anti Tubercular Agents Targeting Glutamine Synthetase-I. And Cyclopropanemycolic acid synthase-2. J. Pharm. Chem. Biol. Sci. 2018, 5, 312–319. [Google Scholar]
  28. Mehra, R.; Rajput, V.S.; Gupta, M.; Chib, R.; Kumar, A.; Wazir, P.; Ali Khan, I.; Nargotra, A. Benzothiazole Derivative as a Novel Mycobacterium tuberculosis Shikimate Kinase Inhibitor: Identification and Elucidation of Its Allosteric Mode of Inhibition. J. Chem. Inf. Model. 2016, 56, 930–940. [Google Scholar] [CrossRef]
  29. Shahare, H.V.; Talele, G.S. Designing of benzothiazole derivatives as promising EGFR tyrosine kinase inhibitors: A pharmacoinformatics study. J. Biomol. Struct. Dyn. 2020, 38, 1365–1374. [Google Scholar] [CrossRef]
  30. Cao, S.; Cao, R.; Liu, X.; Luo, X.; Zhong, W. Design, Synthesis and Biological Evaluation of Novel Benzothiazole Derivatives as Selective PI3KβInhibitors. Molecules 2016, 21, 876. [Google Scholar] [CrossRef] [Green Version]
  31. Sugita, Y.; Takao, K.; Uesawa, Y.; Nagai, J.; Iijima, Y.; Sano, M.; Sakagami, H. Development of Newly Synthesized Chromone Derivatives with High Tumor Specificity against Human Oral Squamous Cell Carcinoma. Medicines 2020, 7, 50. [Google Scholar] [CrossRef] [PubMed]
  32. Reis, J.; Gaspar, A.; Milhazes, N.; Borges, F. Chromone as a Privileged Scaffold in Drug Discovery: Recent Advances. J. Med. Chem. 2017, 60, 7941–7957. [Google Scholar] [CrossRef] [PubMed]
  33. Bouhenna, M.M.; Mameri, N.; Pérez, M.V.; Talhi, O.; Bachari, K.; Silva, A.M.S.; Luyten, W. Anticancer Activity Study of Chromone and Coumarin Hybrids using Electrical Impedance Spectroscopy. Anticancer Agents. Med. Chem. 2018, 18, 854–864. [Google Scholar]
  34. Zhan, Q.; Xu, Y.; Zhan, L.; Wang, B.; Guo, Y.; Wu, X.; Ai, W.; Song, Z.; Yu, F. Chromone Derivatives CM3a Potently Eradicate Staphylococcus aureus Biofilms by Inhibiting Cell Adherence. Infect. Drug Resist. 2021, 14, 979–986. [Google Scholar] [CrossRef]
  35. Chu, Y.-C.; Chang, C.-H.; Liao, H.-R.; Fu, S.-L.; Chen, J.J. Anti-Cancer and Anti-Inflammatory Activities of Three New Chromone Derivatives from the Marine-Derived Penicillium citrinum. Mar. Drugs 2021, 19, 408–426. [Google Scholar] [CrossRef]
  36. Matta, A.; Sharma, A.K.; Tomar, S.; Cao, P.; Kumar, S.; Balwani, S.; Ghosh, B.; Prasad, A.K.; Van der Eycken, E.V.; DePass, A.L.; et al. Synthesis and anti-inflammatory activity evaluation of novel chroman derivatives. New J. Chem. 2020, 44, 13716–13727. [Google Scholar]
  37. Mohsin, N.u.A.; Irfan, M.; Hassan, S.u.; Saleem, U. Current Strategies in Development of New Chromone Derivatives with Diversified Pharmacological Activities: A Review. Pharm. Chem. J. 2020, 54, 241–257. [Google Scholar] [CrossRef]
  38. Nalla, V.; Shaikh, A.; Bapat, S.; Vyas, R.; Karthikeyan, M.; Yogeeswari, P.; Sriram, D.; Muthukrishnan, M. Identification of potent chromone embedded [1,2,3]-triazoles as novel anti-tubercular agents. R. Soc. Open Sci. 2018, 5, 171750. [Google Scholar] [CrossRef] [Green Version]
  39. Wang, G.; Chen, M.; Qiu, J.; Xie, Z.; Cao, A. Synthesis, in vitro α-glucosidase inhibitory activity and docking studies of novel chromone-isatin derivatives. Bioorganic Med. Chem. Lett. 2018, 8, 113–116. [Google Scholar] [CrossRef]
  40. Sugiyama, T.; Narukawa, Y.; Shibata, S.; Masui, R.; Kiuchi, F. New 2-(2-Phenylethyl)chromone Derivatives and Inhibitors of Phosphodiesterase (PDE) 3A from Agarwood. Nat. Prod. Commun. 2016, 11, 795–797. [Google Scholar] [CrossRef] [Green Version]
  41. Rao, Y.J.; Abhijit, K. Synthesis of Novel Functionalized Pyrano Annulated/Oxazolone Pendent Chromone Derivatives as Potent Anti-Diabetic Agents. Russ. J. Gen. Chem. 2020, 90, 1074–1082. [Google Scholar] [CrossRef]
  42. Ivasiv, V.; Albertini, C.; Gonçalves, A.E.; Rossi, M.; Bolognesi, M.L. Molecular hybridization as a tool for designing multitarget drug candidates for complex diseases. Curr. Top. Med. Chem. 2019, 19, 1694–1711. [Google Scholar] [CrossRef] [PubMed]
  43. Khilya, V.P.; Grishko, L.G.; Sokolova, T.N. Chemistry of heteroanalogs of isoflavones. III. Synthesis of benzimidazole and benzothiazole analogs of isoflavones. Chem. Heterocycl. Compd. 1975, 11, 1353–1355. [Google Scholar] [CrossRef]
  44. Gorbulenko, N.V.; Frasinyuk, M.S.; Khilya, V.P. Chemistry of heteroanalogs of isoflavones. 16. Benzthiazole analogs of isoflavones. Chem. Heterocycl. Compd. 1994, 30, 405–412. [Google Scholar] [CrossRef]
  45. Frasinyuk, M.S.; Turov, A.V.; Khilya, V.P. Chemistry of the hetero analogs of isoflavones. 22. Mannich reaction in the benzimidazole and benzothiazole analogs of isoflavones. Chem. Heterocycl. Compd. 1998, 34, 923–928. [Google Scholar] [CrossRef]
  46. Andrews, S.P.; Mason, J.S.; Hurrell, E.; Congreve, M. Structure-based drug design of chromone antagonists of the adenosine A2A receptor. MedChemComm 2014, 5, 571–575. [Google Scholar] [CrossRef]
  47. Frasinyuk, M.S.; Bondarenko, S.P.; Gorbulenko, N.V.; Turov, A.V.; Khilya, V.P. Cyclic Carboxylic Anhydrides as New Reagents for Formation of Chromone Ring. J. Heterocycl. Chem. 2014, 51, 768–774. [Google Scholar] [CrossRef]
  48. Mrug, G.P.; Bondarenko, S.P.; Khilya, V.P.; Frasinyuk, M.S. Synthesis and aminomethylation of 7-hydroxy-5-methoxyisoflavones. Chem. Nat. Compd. 2013, 49, 235–241. [Google Scholar] [CrossRef]
  49. Khilya, O.V.; Frasinyuk, M.S.; Turov, A.V.; Khilya, V.P. Chemistry of 3-Hetarylcoumarins. 1. 3-(2-Benzazolyl)coumarins. Chem. Heterocycl. Compd. 2001, 37, 1029–1037. [Google Scholar] [CrossRef]
  50. Khilya, O.V.; Shablykina, O.V.; Frasinyuk, M.S.; Turov, A.V.; Ishchenko, V.V.; Khilya, V.P. Chemistry of 3-hetarylcoumarins. 2. 3-(2-thiazolyl)coumarins. Chem. Heterocycl. Compd. 2004, 40, 1408–1420. [Google Scholar] [CrossRef]
  51. Katoueezadeh, M.; Pilehvari, N.; Fatemi, A.; Hassanshahi, G.; Torabizadeh, S.A. Inhibition of DNA damage response pathway using combination of DDR pathway inhibitors and radiation in treatment of acute lymphoblastic leukemia cells. Future Oncol. 2021, 17, 2803–2816. [Google Scholar] [CrossRef] [PubMed]
  52. Meng, X.Y.; Zhang, H.X.; Mezei, M.; Cui, M. Molecular docking: A powerful approach for structure-based drug discovery. Curr. Comput.-Aided Drug Des. 2011, 7, 146–157. [Google Scholar] [CrossRef] [PubMed]
  53. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  54. van Meerloo, J.; Kaspers, G.J.; Cloos, J. Cell sensitivity assays: The MTT assay. Methods Mol. Biol. 2011, 731, 237–245. [Google Scholar] [PubMed]
  55. Shaik, A.; Bhakuni, R.; Kirubakaran, S. Design, Synthesis, and Docking Studies of New Torin2 Analogs as Potential ATR/mTOR Kinase Inhibitors. Molecules 2018, 23, 992. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; Halgren, T.A.; Sanschagrin, P.C.; Mainz, D.T. Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes. J. Med. Chem. 2006, 49, 6177–6196. [Google Scholar] [CrossRef] [Green Version]
  57. Sastry, G.M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. J. Comput.-Aided Mol. Des. 2013, 27, 221–234. [Google Scholar] [CrossRef]
  58. Greenwood, J.R.; Calkins, D.; Sullivan, A.P.; Shelley, J.C. Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution. J. Comput.-Aided Mol. Des. 2010, 24, 591–604. [Google Scholar] [CrossRef]
  59. Shelley, J.C.; Cholleti, A.; Frye, L.; Greenwood, J.R.; Timlin, M.R.; Uchimaya, M. Epik: A software program for pKa prediction and protonation state generation for drug-like molecules. J. Comput.-Aided Mol. Des. 2007, 21, 681–691. [Google Scholar] [CrossRef]
Molecules 27 04637 g001 550
Figure 1. FDA-approved drugs.
Figure 1. FDA-approved drugs.
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Molecules 27 04637 g002 550
Figure 2. Flavoxate, FDA-approved drug.
Figure 2. Flavoxate, FDA-approved drug.
Molecules 27 04637 g002
Molecules 27 04637 sch001 550
Scheme 1. Synthesis of the key intermediates 3-hetaryl chromones 2–5 and target compounds (2c, 4a,b, and 5a). Reagents and conditions: (a) HC(OEt)3, pyridine, 120 °C, 4–6 h; (b) i (CH3CO)2O, pyridine, r.t. 24–48 h; ii NaOH, EtOH, reflux, 1 min; (c) EtOOCCOCl, pyridine, r.t., 24–48 h; (d) LiOH, THF, r. t. 72 h; (e) NH2OH⋅HCl, pyridine, reflux, 6–8 h; (f) succinic, glutaric or diglycolic anhydride, pyridine, r.t. 72 h; (g) hydrazine, EtOH, reflux, 4 h.
Scheme 1. Synthesis of the key intermediates 3-hetaryl chromones 2–5 and target compounds (2c, 4a,b, and 5a). Reagents and conditions: (a) HC(OEt)3, pyridine, 120 °C, 4–6 h; (b) i (CH3CO)2O, pyridine, r.t. 24–48 h; ii NaOH, EtOH, reflux, 1 min; (c) EtOOCCOCl, pyridine, r.t., 24–48 h; (d) LiOH, THF, r. t. 72 h; (e) NH2OH⋅HCl, pyridine, reflux, 6–8 h; (f) succinic, glutaric or diglycolic anhydride, pyridine, r.t. 72 h; (g) hydrazine, EtOH, reflux, 4 h.
Molecules 27 04637 sch001
Molecules 27 04637 sch002 550
Scheme 2. Synthesis of compounds 7a–7m.
Scheme 2. Synthesis of compounds 7a–7m.
Molecules 27 04637 sch002
Molecules 27 04637 sch003 550
Scheme 3. Synthesis of coumarin Mannich bases 10–11.
Scheme 3. Synthesis of coumarin Mannich bases 10–11.
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Figure 3. Initial screening results for HCT116 cells after 72 h incubation.
Figure 3. Initial screening results for HCT116 cells after 72 h incubation.
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Figure 4. Initial screening results for HeLa cells after 72 h incubation.
Figure 4. Initial screening results for HeLa cells after 72 h incubation.
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Figure 5. Dose response curves for the compounds in HCT116 cells after 72 h incubation.
Figure 5. Dose response curves for the compounds in HCT116 cells after 72 h incubation.
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Figure 6. Dose–response curves for the compounds in HeLa cells after 72 h incubation.
Figure 6. Dose–response curves for the compounds in HeLa cells after 72 h incubation.
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Figure 7. Inhibition of Chk1 at Ser 317 in HeLa and HCT116 cells analyzed by immunoblot assay.
Figure 7. Inhibition of Chk1 at Ser 317 in HeLa and HCT116 cells analyzed by immunoblot assay.
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Figure 8. Expression of Chk1 and pChk1 at Ser 317 in cells analyzed by immunoblot assay with respect to treatment against compounds 7l, 7h and 2c.
Figure 8. Expression of Chk1 and pChk1 at Ser 317 in cells analyzed by immunoblot assay with respect to treatment against compounds 7l, 7h and 2c.
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Figure 9. Ligand interaction diagrams for the compounds (a) 2c, (b) 7h, (c) 7l and (d) Torin2.
Figure 9. Ligand interaction diagrams for the compounds (a) 2c, (b) 7h, (c) 7l and (d) Torin2.
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Figure 10. Binding pose for the compounds (a) 2c, (b) 7h, (c) 7l and (d) Torin2 at the binding site.
Figure 10. Binding pose for the compounds (a) 2c, (b) 7h, (c) 7l and (d) Torin2 at the binding site.
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Table
Table 1. IC50 values of compounds in HCT116 and HeLa.
Table 1. IC50 values of compounds in HCT116 and HeLa.
Sl. No.CompoundIC50 Value (µM)
HCT116HeLa
12c3.6702.642
27h6.5533.995
37l2.5272.659
Table
Table 2. Molecular docking results for Torin2 and compounds 2c, 7h and 7l.
Table 2. Molecular docking results for Torin2 and compounds 2c, 7h and 7l.
Sl. No.InteractionPoint
Interaction		Donor
Atom		Acceptor
Atom		Type of
Interaction		Bond
Distance
(Å)		Binding
Energy
(Dock Score) (kcal/mol)
1.2c2c OH–Val 882c: OVal 88Hydrogen bond1.72−5.5
2c aromatic ring–Trp 872c: aromatic ringTrp 87π-π stacking3.37, 3.81, 3.87
2c benzothiazole–Lys 162c: benzothiazoleLys 16π-Cation
interaction		5.15, 5.37
2.Torin2Lys 100–Torin2 OLys 100Torin2: OHydrogen bond1.80−5.1
Torin2 NH2–Val 88Torin2: NVal 88Hydrogen bond2.31
Lys 16–Torin2 NLys 16Torin2: NHydrogen bond2.01
Torin2 pyridine–Trp 87Torin2: pyridineTrp 87π-π stacking3.47, 3.77
3.7h7h OH–Asn 897h: OAsn 89Hydrogen bond1.90−3.4
7h NH–Thr 917h: NThr 91Hydrogen bond1.93
7h benzothiazole–Trp 872c: benzothiazoleTrp 87π-π stacking4.00
4.7lLys 16–7l O Lys 167l: OHydrogen bond1.94−4.8
7l benzothiazole–Trp 877l: benzothiazoleTrp 87π-π stacking5.02
Lys 16–7l benzothiazoleLys 167l: benzothiazoleπ-Cation
interaction		5.01
Lys 100–7l OLys 1007l: OSalt Bridge2.80
Table
Table 3. Concentration of compounds 2c, 7h and 7l for immunoblot assay in HCT116 and HeLa cell lines.
Table 3. Concentration of compounds 2c, 7h and 7l for immunoblot assay in HCT116 and HeLa cell lines.
Sl. No.CompoundConcentration for Blot (µM)
HCT116HeLa
12c3.62.6
27h6.54.0
37l2.52.6
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Frasinyuk, M.; Chhabria, D.; Kartsev, V.; Dilip, H.; Sirakanyan, S.N.; Kirubakaran, S.; Petrou, A.; Geronikaki, A.; Spinelli, D. Benzothiazole and Chromone Derivatives as Potential ATR Kinase Inhibitors and Anticancer Agents. Molecules 2022, 27, 4637. https://doi.org/10.3390/molecules27144637

AMA Style

Frasinyuk M, Chhabria D, Kartsev V, Dilip H, Sirakanyan SN, Kirubakaran S, Petrou A, Geronikaki A, Spinelli D. Benzothiazole and Chromone Derivatives as Potential ATR Kinase Inhibitors and Anticancer Agents. Molecules. 2022; 27(14):4637. https://doi.org/10.3390/molecules27144637

Chicago/Turabian Style

Frasinyuk, Mykhaylo, Dimple Chhabria, Victor Kartsev, Haritha Dilip, Samvel N. Sirakanyan, Sivapriya Kirubakaran, Anthi Petrou, Athina Geronikaki, and Domenico Spinelli. 2022. "Benzothiazole and Chromone Derivatives as Potential ATR Kinase Inhibitors and Anticancer Agents" Molecules 27, no. 14: 4637. https://doi.org/10.3390/molecules27144637

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