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Article

Synthesis, Characterization and In Vitro Evaluation of Novel 5-Ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones as Possible Anticancer Agents

by
Serhii Holota
1,2,*,
Sergiy Komykhov
3,4,
Stepan Sysak
1,
Andrzej Gzella
5,
Andriy Cherkas
6,† and
Roman Lesyk
1,7,*
1
Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010 Lviv, Ukraine
2
Department of Organic Chemistry and Pharmacy, Lesya Ukrainka Volyn National University, Volya Avenue 13, 43025 Lutsk, Ukraine
3
State Scientific Institution “Institute for Single Crystals”, National Academy of Sciences of Ukraine, Nauky Ave 60, 61072 Kharkiv, Ukraine
4
Applied Chemistry Department, Karazin Kharkiv National University, Svobody Sq. 4, 61022 Kharkiv, Ukraine
5
Department of Organic Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
6
Department of Internal Medicine #1, Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010 Lviv, Ukraine
7
Department of Public Health, Dietetics and Lifestyle Disorders, Faculty of Medicine, University of Information Technology and Management in Rzeszow, Sucharskiego 2, 35-225 Rzeszow, Poland
*
Authors to whom correspondence should be addressed.
†
Current address: Team Early Projects Type 1 Diabetes, Therapeutic Area Diabetes and Cardiovascular Medicine, Research & Development, Sanofi-Aventis Deutschland GmbH, Industriepark Höchst-H831, 65926 Frankfurt am Main, Germany.
Molecules 2021, 26(4), 1162; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26041162
Submission received: 10 February 2021 / Revised: 19 February 2021 / Accepted: 20 February 2021 / Published: 22 February 2021
(This article belongs to the Special Issue Design, Synthesis, and Analysis of Potential Drugs)
Browse Figures
Versions Notes

Abstract

:
The present paper is devoted to the search for drug-like molecules with anticancer properties using the thiazolo[3,2-b][1,2,4]triazole-6-one scaffold. A series of 24 novel thiazolo-[3,2-b][1,2,4]triazole-6-ones with 5-aryl(heteryl)idene- and 5-aminomethylidene-moieties has been synthesized employing three-component and three-stage synthetic protocols. A mixture of Z/E-isomers was obtained in solution for the synthesized 5-aminomethylidene-thiazolo[3,2-b]-[1,2,4]triazole-6-ones. The compounds have been studied for their antitumor activity in the NCI 60 lines screen. Some compounds present excellent anticancer properties at 10 μM. Derivatives 2h and 2i were the most active against cancer cell lines without causing toxicity to normal somatic (HEK293) cells. A preliminary SAR study had been performed for the synthesized compounds.

Graphical Abstract

1. Introduction

We are currently witnessing a great progress in research and development of new anti-tumor therapeutic agents. Nevertheless, cancer remains the second most frequent cause of death in the world and the problem is far from being solved [1]. Cancer is a systemic disease and both its internal metabolic and genetic aberrations as well as the efficiency of immunologic protection play a role in tumor development and progression [2]. Chronic inflammation, redox imbalance, metabolic dysfunctions and altered glucose metabolism as well as many other endogenous and exogenous factors play roles in disease biology and define outcomes [3,4,5]. This heterogeneity of cancer requires the application of various approaches to prevention and treatment that may include application of specific small molecules interfering with altered metabolic pathways in cancers, modulation of immune response to transformed cells and precision medicine [6,7,8]. Therefore, the search for novel small molecules capable to modulate selectively metabolic processes including redox regulation remains of significant scientific and practical importance.
The class of 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones bicyclic heteroatom-rich compounds [9] containing a 1,2,4-triazole ring and an enone/chalcone system cross-conjugated through a sulfur atom in the molecule has recently attracted the attention of medicinal chemists due to its diverse biological activities. Some 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones have been studied successfully as potential anti-inflammatory [10,11,12,13,14], analgesic [11,13], antimicrobial [15], antifungal [16], antioxidant [17], anticonvulsant [18,19], antihypertensive [20] and anti-aggregation agents [21] (Figure 1). It is worth mentioning that the structural transformation of the carboxylic group into a 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-one moiety has been proposed as a bioisosteric replacement or structure optimization pathway for the synthesis of novel derivatives to keep the main pharmacological profile and to reduce/improve toxicity parameters and activity profiles [10,22,23,24].
In addition to the abovementioned pharmacological features, some 5-ene-thiazolo-[3,2-b][1,2,4]triazole-6(5H)-ones possess promising antitumor properties. A potential non-camptothecin topoisomerase 1 inhibitor (Top1) with the 5-ene-thiazolo[3,2-b]-[1,2,4]triazole-6(5H)-one scaffold (Figure 2A) had been identified using structure-based virtual screening and in vitro assays [25,26]. The mentioned compound at 10 μM showed superior Top1 inhibitory activity compared with the powerful natural Top1-inhibitor camptothecin. Using a similar screening approach the derivative CCT-196700 (Figure 2B) was identified and reported as a potential phospholipase C-γ2 (PLC-γ2) inhibitor with satisfactory activity parameters at 15 µM in the 3H-phosphatidylinositol 4,5-diphosphate biochemical and calcium release cell-based assays [27]. PLC-γ2 is uniquely expressed in hematopoietic cells and considered a plausible target for the treatment of some cancer types [28]. 5-Ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-one derivatives (Figure 2C,D) with high impact on leukemia lines in the NCI-60 lines screening program were reported [29,30].
In view of our interest in the search for and study of new low-molecular heterocyclic modulators among thiazole-bearing molecules [31,32,33,34], herein, we report the synthesis, structure characterization and in vitro anticancer activity evaluation of some novel heterocyclic derivatives. This manuscript is intended to draw attention towards the chemistry and pharmacology of the 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-one fragment and its further exploration as a prospective source of drug-like molecules.

2. Results and Discussion

2.1. Chemical Synthesis

According to our synthetic goal and using the retrosynthetic approach, a multicomponent one-pot protocol [29] was employed for the synthesis of 5-aryl(heteryl)idene-derivatives 2a–l. The reported data about the impact of substituents in position C-5 on pharmacological properties of thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones and data from our study of anticancer activity with structure-activity relationships for this class of heterocyclic compounds [10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28] were the main reasons for the selection of the substituents R1–R6. Thereby, by the three-component reaction of 1 with chloroacetic acid and aromatic/heteroaromatic aldehydes in a mixture AcONa + AcOH:Ac2O employing convenient heating resulted in compounds 2a–l (Scheme 1) which were purified by recrystallization.
The three-step approach was exploited for the preparation of 5-aminomethylidene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones 5a–h, 6a–d (Scheme 2). Initially the 5-etoxymethylidene derivative 4 was synthesized from 1 based on a published protocol [35]. The aminolysis of 4 by primary or secondary aliphatic/aromatic/heterocyclic amines in an alcoholic medium led to the formation of target derivatives 5a–h, 6a–d. The compounds were obtained in satisfactory yields and were recrystallized for purification (Scheme 2).

2.2. Spectral Characterization

The structures of the derivatives 2a–l, 5a–h, 6a–d were confirmed by the 1H-NMR, 13C-NMR, 2D NMR and LC-MS, (copies of the corresponding spectra are presented in the Supplementary Material).
The 1H-NMR spectra of 5a–h contained signals for protons of the double bond at 8.14–8.17 ppm for 5a–c (R5 = Alk) and 8.26–8.52 for 5d–h (R5 = Ar or Het); for NH proton at 8.78–9.06 ppm for 5a–c (9.29–10.77 for 5d–h); for the 1,2,4-triazole ring proton at 8.14–8.16 ppm for 5a–c (8.30–8.40 ppm for 5d–h) and signals of appropriate substituents. The 13C-NMR spectra contained four respective signals for a bicyclic system and one signal for the ethylene moiety. The assignment in 5b, 5c was made based on HSQC and HMBC experiments.
Compounds 5a–h can exist in four possible stereoisomeric forms A–D (Scheme 3) which significantly vary by stereochemistry of double bond and relative orientation of substituent R5.
The 1H-NMR spectra of pure samples 5b,d–h, beside the signals of the main stereoisomer (I), contained minor signals with relative intensity ~5–15% which were assigned to an additional stereoisomer.
Compound 5c (R5 = cyclopropyl) provided a triple set of signals with relative intensity 62:31:7, presumably for three stereoisomers, I, II and III respectively (Figure 3). The coupling constant 3J between ethylene and NH protons (13.9 Hz) in III isomer indicates the trans-orientation of these protons which corresponds to either A or C isomer (Scheme 3). The assignment between A and C isomers can be performed based on chemical shifts of NH protons. The chemical shift of NH proton in III isomer is shifted downfields by 0.30 ppm as compared to the I isomer which can be explained by intramolecular interactions which are realized in the C isomer (Scheme 3). Therefore, isomers I (62%), II (31%) and III (7%) probably correspond to structures A, B, and C, respectively.
The NOESY spectrum of 5c (Figure 4, Supplementary Figure S1) contained EXSY peaks for ethylene, NH and aliphatic protons of I, II and III isomers which indicates the co-existence of those forms of 5c.
In the 13C-NMR spectrum of 5c signals of A and B form and some signals of C form could be identified (Scheme 4).
The similar pattern of triplicate signals in the 1H-NMR spectrum was observed for 5a (R5 = CH3) and showed the presence of three stereoisomeric/rotameric forms A, B, C with relative intensity 80:10:10, respectively. The coupling constants between NH and ethylene protons for A and C forms were 11.6 and 14.4 Hz, respectively, whereas for form B the 3J was 7.5 Hz which corresponding of the stereochemistry provided in Scheme 3. Only one type of signals for 5a (R5 = CH3) was observed in the 13C-NMR spectrum.
The 1H-NMR for 5b (R5 = CH2COOH) showed the presence of two isomers, A and C, with relative intensity 85:15, respectively. The coupling constants between NH and ethylene protons for A and C isomers were both 14.2 Hz, which corresponds to stereochemistry provided in Scheme 3. The NOESY spectrum has showed proximity between CH2 and ethylene = CH protons as an additional confirmation of the stereochemistry of A and C isomer which allows excluding the B isomer for this compound. 2D NMR data for 5b showed that A and C isomers of 5b exhibited the stereochemistry similar to those for compound 5c (R5 = cyclopropyl).
Taking into account the similarity in chemical shift values for A and C forms, the structure A can be proposed as the main isomer for all derivatives 5a–h, C as a minor isomer and, no B form in most cases (it appears only in 5a and 5c).
Isomer A of 5a–c showed either the presence of AX system with 3J = 11.6…14.2 Hz for NH proton and for the proton of the ethane moiety or two broad singlets for these protons. This can be due to the velocity of A > B transformation which may be susceptible to different factors such as concentration, trace of water, etc.
To evaluate the presence of the rotamer forms we have studied the dependence of the 1H-NMR spectra pattern for compound 5c on the temperature. The tripling of the signals in the spectra of 5c had been gradually lost at 60 °C until the signals coalesce completely at 100 °C, therefore, the complication of 1H-NMR spectra is connected with hindered rotation. Compounds 2a–l, 6a–d showed 1H- and 13C-NMR data similar to those for A isomer of 5a–h, although no traces of additional stereoisomers were found.

2.3. The X-ray Crystal Structure Analysis of (Z)-5-Cyclopropylaminomethylidene-[1,3]thiazolo-[3,2-b][1,2,4]triazol-6(5H)-one (5c)

The molecular structure of 5c and the atom-labelling scheme are illustrated in Figure 5. The molecule consists of the eight-membered thiazolo[3,2-b][1,2,4]triazol-6(5H)-one system and the N-cyclopropylaminomethylidene residue located at the C5 atom. The fused bicyclic system is approximately planar with an r.m.s. deviation of 0.0245 Å. The interatomic C5–C7 distance observed [1.373(2) Å] confirms the presence of a double bond between these atoms. The N-cyclopropylamino moiety is in Z configuration with respect to the bicyclic moiety. The torsion angle S4–C5–C7–N8 is −0.1(3)°.
The molecules of 5c highlight the presence of quite strong resonance effect involving the carbonyl group and the exocyclic N8 atom via the double bond C5–C7. This is indicated by the C6–O12 [1.2293(19) Å], C5–C6 [1.438(2) Å], C5–C7 [1.373(2) Å], and C7-N8 [1.320(2) Å] bonds which are all significantly distorted with respect to the typical lengths of the C=O [1.210(1) Å], Csp2–Csp2 [1.478(4) Å], C=C [1.331(1) Å] and Csp2–N [1.355(3) Å] bonds [36].
In the crystal of 5c, the molecules related by screw axis 21 are connected by the classical N8–H8∙∙∙O12i hydrogen bonds into chains (Supplementary Figure S2a, Table 1). It is worth noting that in the chains, the molecules, or rather their planar thiazolo[3,2-b][1,2,4]triazol-6(5H)-one ring systems, are arranged coplanar to each other to form tapes with the best plane parallel to the plane (−203). The adjacent anti-parallel chains of molecules are further joined by hydrogen bonds C11–H11A∙∙∙N3ii into layers showing a stepped shape, with mean plane parallel to the plane (−102) (Supplementary Figure S2b, Table 1). There are π-electron interactions between the adjacent layers (Supplementary Figure S3). The perpendicular distances between partially overlapping thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (ThiTri) systems are 3.4409 (6) Å and 3.4100 (6) Å for π (ThiTri)∙∙∙ π (ThTr)iii and π(ThiTri)∙∙∙ π (ThTr)iv, respectively [symmetry codes: (iii) x, 3/2 − y, ½ + z, (iv) x, 3/2 − y,−1/2 + z].

2.4. In Vitro Evaluation of the Anticancer Activity and Cytotoxicity. Compare Analysis

Compounds 2a, 2c–f, 2h–l, 3, 5e, 5f were selected for antitumor activity which was performed according to the standard protocols of National Cancer Institute (NCI, Bethesda, MD, USA) Developmental Therapeutic Program (DTP) [37,38,39,40]. At the prescreening stage, the compounds were evaluated for antitumor activity at the concentration of 10 µM against a panel of approximately sixty cancer cell lines representing different types of cancer including leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers. The results of the prescreening assay are summarized in Table 2.
Overall, the 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones exhibited a range of potencies against the tumor cell lines tested. Among them, three compounds (2d, 2h, 2i) presented excellent cytotoxic and four derivatives (2d, 2h, 2i, 2k) cytostatic properties in one or more cell lines when applied at a concentration of 10 µM. It worth noting that compounds 2d, 2h, 2i had been possessing cytotoxic effect against leukemia cell lines. Also, compound 2h has demonstrated a cytotoxic effect on some lines in prostate cancer, NSCLC, breast cancer, renal cancer, melanoma, and CNS cancer subpanels. Since compounds 2h and 2i exhibited the highest cytotoxic and cytostatic activity, they were selected for in-depth studies at five different concentrations (0.01, 0.1, 1.0, 10.0, and 100.0 μM) against the same NCI-60 HTCL panel of cell lines. The outcomes were calculated and presented in the form of three response parameters (GI50, TGI and LC50) for each cell line via growth percentage inhibition curves [41,42]. The GI50 value (growth inhibitory activity) corresponds to the concentration of compound causing 50% decrease in net cell growth, the TGI value (cytostatic activity) represents the concentration of compound resulting in total growth inhibition (100% growth inhibition) and LC50 value (cytotoxic activity) demonstrate the lethal dose of compound causing net 50% death of initial cells. The results calculated are provided in Supplementary Table S2 and Table 3.
Compound 2h inhibited each of the 49 cancer cell lines tested in the micromolar range with GI50 < 10 μM and average GI50, TGI and LC50 values of 3.54 μM, 12.88 μM, and 37.15 μM, respectively (Table 3). The moderate selectivity of 2h was observed for breast cancer cell line MCF7 at GI50 (SI = 4.48) and TGI (SI = 3.19) levels, and for leukemia lines: SR at TGI (SI = 3.16), CCRF-CEM at TGI (SI = 4.08) and LC50 (SI = 4.90) levels. In stark contrast, compound 2i exhibited lower inhibitory activity with GI50 < 10 μM against 13 cell lines with average GI50, TGI, and LC50 values of 10.96 μM, 28.18 μM and 63.09 μM, respectively. A moderate selectivity was observed at GI50 level for leukemia line CCRF-CEM (SI = 3.81) and breast cancer cell line HS 578T (SI = 3.01).
The mean GI50 values (µM) for compounds 2h and 2i have been compared to the same data for standard anticancer agents as the synthetic drug cisplatin and the natural compound curcumin (Figure 6). The data demonstrates that MG_MID (µM) values for 2h are lower than those for cisplatin except in ovarian cancer subpanel and for curcumin except in the colon cancer subpanel when tested in the same experimental setup. Meanwhile, compound 2i showed lower activity and its MG_MID (µM) values were superior only for the colon and breast cancer subpanels in comparison with cisplatin and for the prostate cancer subpanel in comparison with curcumin.
The COMPARE algorithm of the NCI allows the prediction of biochemical mechanisms of action of the novel compounds on the basis of their in vitro activity profiles by comparing these to the ones of standard agents. The COMPARE analysis was performed for 2h and 2i against the NCI “Standard Agents” database at the GI50 level [38,39,40]. The values of the Pearson correlation coefficients (PCC) higher than 0.5 were chosen as noteworthy. However, PCC values obtained did not enable the prediction of the mechanisms of cytotoxicity for the compounds tested with high probability. Both compounds showed the correlation at the GI50 level only with one NCI Standard Agent–“Glycoxalic acid” (NSC:S267213, chemical name: (E)-2-(2-((4-methoxyphenyl)sulfonyl) hydrazono)acetic acid or glyoxylic acid p-methoxybenzenesulfonylhydrazone) and the PCC values were calculated as 0.561 for 2h and 0.647 for 2i. The alkylating mechanism of antineoplastic action has been reported for NSC:S267213 and a set of its structural analogs [43,44,45,46]
Compounds 2h and 2i were also evaluated in vitro for their cytotoxic potential against human embryonic kidney (HEK 293) cell lines using the MTT assay [47]. The IC50 values obtained were 28.99 µM and 24.43 µM for 2h and 2i, respectively, suggest that these derivatives could effectively act against cancer cell lines without causing toxicity to normal somatic cells.

2.5. The Preliminary Structure-Anticancer Activity Relationship for Novel 5-Ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones

Based on the biological data obtained so far, structure-activity relationship (SAR) analysis was determined. The SAR data revealed that the anticancer activity of synthesized 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones is dependent upon the nature of substituent connected with a double bond at C-5 of the bicyclic system. Presence of C-5 linked benzylidene moieties resulted in better activity compared to heterocyclic rings (furan (2j), thiophene (2k), N-AcO-indole (2l)). Besides it, the presence and position of a chlorine atom in the benzylidene part of molecules also plays a crucial role in the realization of the anticancer effect. So, the derivatives 2h and 2i with the chlorine atom and with an additional modification by N-(4-R4-phenyl)acetamides fragments linked at the O-2 position have been found to exert excellent antitumor activities. It is worth to note that the introduction of an imine-linker between the 4-chlorophenyl moiety and double bond at C-5 (5f), drastically decreased the inhibitory activity compared with data reported in [29]. Similarly, the moving of the fluorine atom from C-4 to C-2 position in benzylidene moiety (2a) leads to loss of activity [29]. Interestingly, that derivative containing 4-AcO-group in benzylidene core (2d), demonstrated inhibition of leukemia lines CCRF-CEM (−73,92), HL-60 (TB) (−58,44), MOLT-4 (−73,62) against the backdrop of the almost complete lack of impact on other cell lines. In addition, it should be noted that for all active derivatives screened at both stages, the activity against leukemia line(s) was observed which is in agreement with data described in the literature [27,29,30].

3. Materials and Methods

3.1. General Information

Melting points were measured in open capillary tubes on a BÜCHI B-545 melting point apparatus (BÜCHI Labortechnik AG, Flawil, Switzerland) and are uncorrected. The elemental analyses (C, H, N) were performed using the Perkin–Elmer 2400 CHN analyzer (PerkinElmer, Waltham, MA, USA) and were within ±0.4% of the theoretical values. The 500 MHz-1H and 126 MHz-13C spectra were recorded on Bruker AVANCE-500 spectrometer and 2D spectra were recorded on a Bruker AVANCE-600 spectrometer (Bruker, Bremen, Germany). All spectra were recorded at room temperature except where indicated otherwise and were referenced internally to solvent reference frequencies. Chemical shifts (δ) are quoted in ppm and coupling constants (J) are reported in Hz. LC-MS spectra were obtained on a Finnigan MAT INCOS-50 (Thermo Finnigan LLC, San Jose, CA, USA). Solvents and reagents that are commercially available were used without further purification. The synthetic procedure for compound 1 was described in [48], for derivatives 2a–l in [29] and for compound 4 in [35].

3.2. Preparation and Characterization of Compounds

3.2.1. Characterization of Compounds 2a–l

(Z)-5-(2-Fluorobenzylidene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (2a). Yield 58%, light yellow powder, mp 192–193 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.53 (s, 1H, 2-H), 8.19 (s, 1H, =CH), 7.73 (t, J = 8.5 Hz, 1H, C6H4), 7.64–7.69 (m, 1H, C6H4), 7.46–7.49 (m, 2H, C6H4). 13C-NMR (126 MHz, DMSO-d6, δ): 161.5, 159.4, 155.7, 134.3, 130.1, 129.2, 127.2, 125.5, 119.9, 116.5, 116.2. LCMS (ESI+) m/z 248 [M + H]+. Anal. calc. for C11H6FN3OS: C 53.44%, H 2.45%, N 16.99%. Found: C 53.60%, H 2.70%, N 17.20%.
(Z)-5-(2-Methoxybenzylidene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (2b). Yield 54%, yellow powder, mp 196–198 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.49 (s, 1H, 2-H), 8.34 (s, 1H, =CH), 7.62–7.56 (m, 2H, C6H4), 7.22 (d, J = 8.0 Hz, 1H, C6H4), 7.16 (t, J = 7.5 Hz, 1H, C6H4), 3.95 (s, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 160.0, 159.1, 158.5, 156.5, 134.9, 134.1, 130.2, 124.7, 121.4, 120.7, 112.1, 69.1, 55.9. LCMS (ESI+) m/z 260 [M + H]+. Anal. calc. for C12H9N3O2S: C 55.59%, H 3.50%, N 16.21%. Found: C 55.80%, H 3.70%, N 16.40%.
(Z)-5-(2-(Allyloxy)benzylidene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (2c). Yield 63%, brown-yellow powder, mp 133–136 °C (ethanol). 1H-NMR (500 MHz, DMSO-d6, δ): 8.50 (s, 1H, 2-H), 8.40 (s, 1H, =CH), 7.61 (d, J = 9.3 Hz, 1H, C6H4), 7.59–7.54 (m, 1H, C6H4), 7.23 (d, J = 8.5 Hz, 1H, C6H4), 7.17 (t, J = 7.3 Hz, 1H, C6H4), 6.12 (ddt, J = 15.8, 10.5, 5.2 Hz, 1H, CH2=CH-CH2), 5.45 (d, J = 1.7 Hz, 1H, CH2=CH-CH2), 5.34 (d, J = 1.6 Hz, 1H, CH2=CH-CH2), 4.77–4,79 (m, 2H, CH2=CH-CH2). 13C-NMR (126 MHz, DMSO-d6, δ): 159.8, 159.0, 157.4, 156.3, 134.1, 133.8, 132.7, 129.7, 124.4, 121.5, 120.6, 118.1, 113.4, 68.9. LCMS (ESI+) m/z 286 [M + H]+. Anal. calc. for C14H11N3O2S: C 58.93%, H 3.89%, N 14.73%. Found: C 59.10%, H 4.00%, N 14.90%.
(Z)-4-((6-Oxothiazolo[3,2-b][1,2,4]triazol-5(6H)-ylidene)methyl)phenyl acetate (2d). Yield 53%, white-yellow powder, mp 211–213 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.70 (s, 1H, =CH), 8.47 (s, 1H, 2-H), 8.01 (d, J = 8.5 Hz, 2H, C6H4), 7.58 (d, J = 8.6 Hz, 2H, C6H4), 2.50 (s, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 169.0, 159.5, 159.2, 156.0, 152.7, 138.5, 132.0, 129.9, 124.5, 123.2, 49.7, 20.8. LCMS (ESI+) m/z 288 [M + H]+. Anal. calc. for C13H9N3O3S: C 54.35%, H 3.16%, N 14.63%. Found: C 54.50%, H 3.40%, N 14.80%.
(Z)-5-(4-(Diethylamino)benzylidene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (2e). Yield 67%, light red-purple powder, mp 232–234 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.46 (s, 1H, 2-H), 8.10 (s, 1H, =CH), 7.60 (d, J = 9.1 Hz, 2H, C6H4), 6.86 (d, J = 9.1 Hz, 2H, C6H4), 3.47 (d, J = 7.1 Hz, 4H, 2*CH2), 1.14 (t, J = 7.0 Hz, 6H, 2*CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 158.8, 158.6, 156.5, 150.5, 141.1, 133.9, 118.5, 114.4, 111.9, 44.1, 12.6. LCMS (ESI+) m/z 301 [M + H]+. Anal. calc. for C15H16N4OS: C 59.98%, H 5.37%, N 18.65%. Found: C 60.20%, H 5.50%, N 18.80%.
(Z)-5-(3,4-Dimethoxybenzylidene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (2f). Yield 53%, yellow powder, mp 207–209 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.50 (s, 1H, 2-H), 8.23 (s, 1H, =CH), 7.40 (dd, J = 7.4, 2.2 Hz, 1H, C6H3), 7.36 (d, J = 2.1 Hz, 1H, C6H3), 7.21 (d, J = 8.5 Hz, 1H, C6H3), 3.87 (s, 3H, CH3), 3.85 (s, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 159.3, 159.0, 156.6, 152.4, 152.0, 149.2, 140.4, 125.2, 121.3, 114.0, 112.3, 55.8, 55.5. LCMS (ESI+) m/z 290 [M + H]+. Anal. calc. for C13H11N3O3S: C 53.97%, H 3.83%, N 14.52%. Found: C 54.10%, H 4.00%, N 14.80%.
(Z)-2-(4-Chloro-2-((6-oxothiazolo[3,2-b][1,2,4]triazol-5(6H)-ylidene)methyl)phenoxy)-N-(m-tolyl)acetamide (2g). Yield 68%, white-yellow powder, mp 260–262 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 10.18 (s, 1H, NH), 8.52 (s, 1H, 2-H), 8.40 (s, 1H, =CH), 7.64–7.58 (m, 2H, C6H4 + C6H3), 7.44 (brs, 1H, C6H4 + C6H3), 7.36 (d, J = 8.1 Hz, 1H, C6H4 + C6H3), 7.24–7.08 (m, 2H, C6H4 + C6H3), 6.90 (d, J = 7.4 Hz, 1H, C6H4+C6H3), 4.97 (s, 2H, CH2), 2.27 (s, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 165.5, 159.7, 156.1, 155.9, 138.2, 138.0, 133.5, 132.8, 128.8, 128.6, 126.8, 125.4, 124.4, 122.9, 119.9, 119.9, 116.6, 115.2, 67.5, 21.2. LCMS (ESI+) m/z 428 [M + H]+. Anal. calc. for C20H15ClN4O3S: C 56.27%, H 3.54%, N 13.12%. Found: C 56.50%, H 3.70%, N 13.30%.
(Z)-2-(4-Chloro-2-((6-oxothiazolo[3,2-b][1,2,4]triazol-5(6H)-ylidene)methyl)phenoxy)-N-(4-chlorophenyl)acetamide (2h). Yield 58%, white-yellow powder, mp 224–226 °C (ethanol–DMF 4:1)1H-NMR (500 MHz, DMSO-d6, δ): 10.39 (s, 1H, NH), 8.50 (s, 1H, 2-H), 8.39 (s, 1H, =CH), 7.70–7.54 (m, 4H, C6H4+C6H3), 7.38 (d, J = 8.9 Hz, 2H, C6H4+C6H3), 7.18 (d, J = 8.8 Hz, 1H, C6H4+C6H3), 4.99 (s, 2H, CH2).13C-NMR (126 MHz, DMSO-d6, δ): 166.0, 159.7, 159.4, 156.1, 155.8, 137.4, 133.5, 132.8, 128.8, 128.7, 127.4, 126.9, 125.5, 122.9, 121.1, 115.3, 67.2.LCMS (ESI+) m/z 449 [M + H]+. Anal. calc. for C19H12Cl2N4O3S: C 51.02%, H 2.70%, N 12.53%. Found: C 51.20%, H 2.90%, N 12.70%.
Ethyl(Z)-4-(3-(4-chloro-2-((6-oxothiazolo[3,2-b][1,2,4]triazol-5(6H)-ylidene)methyl)phenyl)propanamido)benzoate (2i). Yield 52%, white-yellow powder, mp 176–178 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 10.60 (s, 1H, NH), 8.51 (s, 1H, 2-H), 8.39 (s, 1H, =CH), 7.93 (d, J = 8.9 Hz, 2H, C6H4+C6H3), 7.74 (d, J = 8.8 Hz, 2H, C6H4+C6H3), 7.57–7.64 (m, 2H, C6H4+C6H3), 7.20 (d, J = 8.7 Hz, 1H, C6H4+C6H3), 5.04 (s, 2H, CH2), 4.28 (q, J = 7.1 Hz, 2H, CH2), 1.30 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 172.0, 166.3, 165.4, 159.7, 159.4, 156.1, 155.8, 142.6, 133.6, 132.5, 130.4, 128.8, 126.8, 125.5, 124.6, 122.9, 118.8, 115.1, 67.5, 60.6, 21.1, 14.4. LCMS (ESI+) m/z 486 [M + H]+. Anal. calc. for C22H17ClN4O5S: C 54.49%, H, 3.53%, N 11.55%. Found: C 54.70%, H 3.80%, N 11.70%.
(Z)-5-(Furan-2-ylmethylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (2j). Yield 61%, brown-yellow powder, mp 230–232 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.47 (s, 1H, 2-H), 8.22 (d, J = 2.3 Hz, 1H, furan), 8.13 (s, 1H, =CH), 7.35 (d, J = 4.0 Hz, 1H, furan), 6.86 (dd, J = 3.6, 1.8 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6, δ): 160.1, 159.2, 156.3, 149.3, 148.8, 125.7, 122.0, 120.8, 114.1. LCMS (ESI+) m/z 220 [M + H]+. Anal. calc. for C9H5N3O2S: C 49.31%, H 2.30%, N 19.17%. Found: C 49.50%, H 2.50%, N 19.30%.
(Z)-5-(Thiophen-2-y-lmethylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (2k). Yield 64%, brown powder, mp > 250 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.57 (s, 1H, =CH), 8.50 (s, 1H, 2-H), 8.19 (d, J = 7.0 Hz, 1H, thiophen), 7.90 (d, J = 3.1 Hz, 1H, thiophen), 7.39 (dd, J = 3.7 Hz, 1H, thiophen). 13C-NMR (126 MHz, DMSO-d6, δ): 159.3, 156.2, 137.1, 136.7, 136.2, 135.3, 135.2, 132.7, 129.6. LCMS (ESI+) m/z 236 [M + H]+. Anal. calc. for C9H5N3OS2: C 45.94%, H 2.14%, N 17.86%. Found: C 46.10%, H 2.30%, N 18.00%.
(Z)-5-((1-Acetyl-1H-indol-3-yl)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (2l). Yield 66%, dark-yellow powder, mp > 250 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.37–8.43 (m, 3H, 2-H, =CH, indole), 8.15 (brs, 1H, indole), 7.91–8.11 (m, 1H, indole), 7.38–7.45 (m, 2H, indole), 2.82 (s, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 170.4, 166.4, 160.5, 159.2, 156.6, 135.5, 130.6, 130.0, 126.9, 125.1, 124.4, 119.5, 116.7, 114.6, 23.4. LCMS (ESI+) m/z 311 [M + H]+. Anal. calc. for C15H10N4O2S: C 58.06; H 3.25; N 18.05%. Found: C 58.30%, H 3.50%, N 18.20%.

3.2.2. Synthesis of 2-((1H-1,2,4-Triazol-3-yl)thio)acetic Acid (3)

A mixture of compound 1 (10 mmol) with the chloroacetic acid (10 mmol) and anhydrous sodium acetate was refluxed for 2 h in glacial acetic acid (10 mL). The white solid obtained was filtered, washed with ethanol and recrystallized from glacial acetic acid. Yield 76%, white powder, mp 132–134 °C (acetic acid). 1H-NMR (500 MHz, DMSO-d6, δ): 13.99 (brs, NH), 12.80 (brs, COOH), 8.47 (brs, 2-H), 3.91 (2H, s, CH2). 13C-NMR (126 MHz, DMSO-d6, δ): 170.0, 158.1, 144.8, 33.7. LCMS (ESI+) m/z 160 [M + H]+. Anal. calc. for C4H5N3O2S: C 30.19; H 3.17; N 26.40. Found: C 30.30%, H 3.40%, N 26.60%.

3.2.3. Synthesis and Characterization of Compounds 5a–h, 6a–d

A mixture of compound 4 (10 mmol) with the appropriate amine (10 mmol) was refluxed for 2 h in ethanol. The solid products obtained were filtered, washed with ethanol and recrystallized from ethanol or the mixture of ethanol–dimethylformamide (4:1).
(E/Z)-5-((Methylamino)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (5a). Yield 53%, bright red-purple powder, mp 239–241 °C (ethanol–DMF 4:1). A form (~80%), 1H-NMR (500 MHz, DMSO-d6, δ): 8.76 (br s, 1H, NH), 8.29 (s, 1H, 2-H), 8.16 (d, 3J = 11.6 Hz, 1H, =CH), 3.09 (3H, s, CH3). B form (~10%), 1H-NMR (500 MHz, DMSO-d6, δ): 8.69 (brs, 1H, NH), 8.30 (s, 1H, 2-H), 8.05 (d, 3J = 7.5 Hz, 1H, =CH), 3.10 (s, 3H, CH3). C form (~10%), 1H-NMR (500 MHz, DMSO-d6, δ): 9.12–9.24 (m, 1H, NH), 8.33 (s, 1H, 2-H), 7.95 (d, 3J = 14.4 Hz, 1H, =CH), 3.11 (s, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 157.7, 156.2, 155.9, 151.6, 88.5, 35.2. LCMS (ESI+) m/z 183 [M + H]+. Anal. calc. for C6H6N4OS: C 39.55%, H 3.32%, N 30.75%. Found: C 39.70%, H 3.50%, N 30.90%.
(E/Z)-((6-Oxothiazolo[3,2-b][1,2,4]triazol-5(6H)-ylidene)methyl)glycine (5b). Yield 62%, yellow powder, mp 229 (with decomp.) °C (ethanol–DMF 4:1). A form (~85%), 1H-NMR (500 MHz, DMSO-d6, δ): 13.07 (brs, 1H, COOH), 8.95–9.08 (m, 1H, NH), 8.32 (s, 1H, 2-H), 8.17 (d, 3J = 14.2 Hz, 1H, =CH), 4.19 (d, 3J = 5.6 Hz, 2H, CH2). 13C-NMR (126 MHz, DMSO-d6, δ): 170.9 (COOH), 157.9 (C-2), 156.5 (C-6), 156.4 (C-3a), 151.6 (=CH), 89.95 (C-5), 49.0 (CH2). NOESY: 8.17–9.03 (=CH…HN), 4.19–8.17 (CH2…=CH), 4.19–9.03 (CH2…NH). C form, (~15%), 1H-NMR (500 MHz, DMSO-d6, δ): 13.07 (brs, 1H, OH), 9.22–9.29 (m, 1H, NH), 8.33 (s, 1H, 2-H), 7.91 (d, 3J = 14.2 Hz, 1H, =CH), 4.16 (d, 3J = 6.3 Hz, 2H, CH2). 13C-NMR (126 MHz, DMSO-d6, δ): 170.7 (COOH), 157.7 (C-2), 157.4 (C-3a), 156.1 (C-6), 154.9 (=CH), 88.9 (C-5), 49.7 (CH2). NOESY: 9.26–7.91 (weak, NH…HC=), 9.26–4.16 (NH…H2C), 4.16–7.91 (CH2…HC=). EXSY: 9.02–9.25. LCMS (ESI+) m/z 227 [M + H]+. Anal. calc. for C7H6N4O3S: C 37.17%, H 2.67%, N 24.77%. Found: C 37.40%, H 2.90%, N 24.90%.
(E/Z)-5-((Cyclopropylamino)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (5c). Yield 54%, beige powder, mp 199–201 °C (ethanol–DMF 4:1). A form (~62%), 1H-NMR (500 MHz, DMSO-d6, δ): 9.04 (brs, 1H, NH), 8.31 (s, 1H, 2-H), 8.12 (brs, 1H, =CH), 3.04–3.10 (m, 1H, cyclopropyl), 0.87–0.98 (m, 1H, cyclopropyl), 0.66–0.80 (m, 3H, cyclopropyl).13C-NMR (126 MHz, DMSO-d6, δ): 157.9 (C-6), 157.8 (C-2), 156.2 (C-3a), 150.1 (=CH), 89.51 (C-5), 30.3 (cyclopropyl), 6.0 (cyclopropyl). NOESY: 8.14–9.05 (=CH…HN), 3.07–9.05 (NH…HC<), 3.07–8.14 (=CH…HC<), 0.72–9.05, 0.72–8.14, 0.72–3.07, 0.72–0.95. B form (~31%), 1H-NMR (500 MHz, DMSO-d6, δ): 8.88–8.98 (m, 1H, NH), 8.35 (s, 1H, 2-H), 7.97 (d, 3J = 7.3 Hz, 1H, =CH), 2.84–2.93 (m, 1H, cyclopropyl), 0.66–0.80 (m, 4H, cyclopropyl). 13C-NMR (126 MHz, DMSO-d6, δ): 157.9 (C-3a), 157.8 (C-2), 157.1 (C-6), 148.8 (=CH), 89.48 (C-5), 25.2 (cyclopropyl), 8.84 (cyclopropyl). C form (~7%), 1H-NMR (500 MHz, DMSO-d6, δ): 9.33–9.39 (m, 1H, NH), 8.06 (d, 3J = 13.9 Hz, 1H, =CH), 3.01–3.04 (m, 1H, cyclopropyl), 0.80–0.85 (m, 2H, cyclopropyl), 0.66–0.80 (m, 2H, cyclopropyl). 13C-NMR (126 MHz, DMSO-d6, δ): 157.6 (C-3a), 157.4 (C-2), 153.7 (C-6), 31.0 (cyclopropyl), 6.2 (cyclopropyl). EXSY: 9.36–8.93 (C-B), 9.36–9.06 (C-A), 8.93–9.06 (B-A), 8.06–7.97 (C-B), 8.14–8.06 (A-C), 7.97–8.14 (B-A), 2.87–3.07 (B-A). LCMS (ESI+) m/z 209 [M + H]+. Anal. calc. for C8H8N4OS: C 46.14%, H 3.87%, N 26.90%. Found: C 46.30%, H 4.00%, N 27.10%.
(E/Z)-5-((Phenylamino)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (5d). Yield 56%, brown-yellow powder, mp 262–263 °C (ethanol–DMF 4:1). A form (~90%), 1H-NMR (500 MHz, DMSO-d6, δ): 10.76 (brs, 1H, NH), 8.52 (brs, 1H, =CH), 8.38 (s, 1H, 2-H), 7.37–7.42 (m, 4H, C6H5), 7.13–7.17 (m, 1H, C6H5). 13C-NMR (126 MHz, DMSO-d6, δ): 158.3, 156.9, 156.8, 155.5, 141.8, 129.7, 124.5, 117.1, 94.7. C form (~10%), 1H-NMR (500 MHz, DMSO-d6, δ): 10.72 (brs, 1H, NH), 8.68 (d, 3J = 13.6 Hz, 1H, =CH), 8.39 (s, 1H, 2-H), 7.37–7.42 (m, 4H, C6H5), 7.13–7.17 (m, 1H, C6H5). LCMS (ESI+) m/z 245 [M + H]+. Anal. calc. for C11H8N4OS: C 54.09%, H 3.30%, N 22.94%. Found: C 54.20%, H 3.50%, N 23.10%.
(E/Z)-5-(((3-Hydroxyphenyl)amino)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (5e). Yield 55%, brown-yellow powder, mp 269–271 °C (ethanol–DMF 4:1). A form (~90%), 1H-NMR (500 MHz, DMSO-d6, δ): 10.68 (brs, 1H, NH), 9.69 (s, 1H, OH), 8.40 (brs, 1H, =CH), 8.38 (s, 1H, 2-H), 7.13–7.23 (m, 1H, C6H4), 6.78–6.85 (m, 1H, C6H4), 6.69–6.78 (m, 1H, C6H4), 6.52–6.62 (m, 1H, C6H4). 13C-NMR (126 MHz, DMSO-d6, δ): 158.5, 158.3, 157.0, 156.8, 141.5, 140.9, 130.6, 111.8, 107.6, 104.2, 94.5. C form (~10%), 1H-NMR (500 MHz, DMSO-d6, δ): 10.61 (brs, 1H, NH), 9.73 (s, 1H, OH), 8.63 (d, 3J = 12.6 Hz, 1H, =CH), 8.38 (s, 1H, 2-H), 6.84–6.88 (m, 2H, C6H4), 6.57–6.61 (m, 2H, C6H4). LCMS (ESI+) m/z 261 [M + H]+. Anal. calc. for C11H8N4O2S: C 50.76%, H 3.10%, N 21.53%. Found: C 50.90%, H 3.40%, N 21.70%.
(E/Z)-5-(((4-Chlorophenyl)amino)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (5f). Yield 64%, yellow powder, mp > 280 °C (ethanol–DMF 4:1). A form (~90%), 1H-NMR (500 MHz, DMSO-d6, δ): 10.77 (1H, s, NH), 8.51 (brs, 1H, =CH), 8.40 (s, 1H, 2-H), 7.39–7.45 (m, 4H, C6H4). 13C-NMR (126 MHz, DMSO-d6, δ): 158.3, 157.0, 156.7, 155.6, 141.6, 129.5, 128.2, 118.9, 95.6. C form (~10%), 1H-NMR (500 MHz, DMSO-d6, δ): 10.74 (brs, 1H, NH), 8.62 (d, 3J = 13.2 Hz, 1H, =CH), 8.40 (s, 1H, 2-H), 7.49–7.52 (m, 2H, C6H4), 7.45–7.48 (m, 2H, C6H4). LCMS (ESI+) m/z 280 [M + H]+. Anal. calc. for C11H7ClN4OS: C 47.40%, H 2.53%, N 20.10%. Found: C 47.60%, H 2.70%, N 20.30%.
(E/Z)-5-(((Furan-2-ylmethyl)amino)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (5g). Yield 71%, light pink powder, mp 176–178 °C (ethanol). A form (~90%), 1H-NMR (500 MHz, DMSO-d6, δ): 9.29 (s, 1H, NH), 8.30 (s, 1H, 2-H), 8.26 (brs, 1H, =CH), 7.66–7.69 (m, 1H, furan), 6.44–6.46 (m, 1H, furan), 6.41–6.44 (m, 1H, furan), 4.62 (s, 2H, CH2). 13C-NMR (126 MHz, DMSO-d6, δ): 157.9. 156.5, 156.3, 150.6, 150.3, 143.3, 110.7, 108.6, 89.8, 44.7. C form (~10%), 1H-NMR (500 MHz, DMSO-d6, δ): 9.43–9.50 (m, 1H, NH), 8.31 (s, 1H, 2-H), 8.06 (d, 3J = 13.9, 1H, =CH), 7.65–7.66 (m, 1H, furan), 6.39–6.41 (m, 1H, furan), 4.58 (d, 3J = 4.73, 2H, CH2). LCMS (ESI+) m/z 249 [M + H]+. Anal. calc. for C10H8N4O2S: C 48.38%, H 3.25%, N 22.57%. Found: C 48.50%, H 3.40%, N 22.80%.
(E/Z)-5-(((5-Methylisoxazol-3-yl)amino)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (5h). Yield 54%, yellow powder, mp 243–245 °C (ethanol–DMF 4:1). A form (~95%) 1H-NMR (500 MHz, DMSO-d6, δ): 11.32 (brs, 1H, NH), 8.40 (s, 1H, 2-H), 8.32 (brs, 1H, =CH), 6.41 (s, 1H, isoxazol), 2.39 (s, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 171.2, 159.9, 158.7, 157.7, 156.8, 140.5, 98.5, 94.2, 12.3. C form (~5%) 1H-NMR (500 MHz, DMSO-d6, δ): 10.79 (d, 3J = 13.2 Hz, 1H, NH), 8.46 (d, 3J = 13.2 Hz, 1H, =CH). LCMS (ESI+) m/z 250 [M + H]+. Anal. calc. for C9H7N5O2S: C 43.37%, H 2.83%, N 28.10%. Found: C 43.50%, H 3.00%, N 28.30%.
(Z)-5-(Indolin-1-ylmethylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (6a). Yield 57%, yellow-brown powder, mp > 250 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.62 (s, 1H, =CH), 8.43 (s, 1H, 2-H), 7.58 (d, J = 8.1 Hz, 1H, C6H4), 7.36 (d, J = 7.3 Hz, 1H, C6H4), 7.29 (t, J = 7.7 Hz, 1H, C6H4), 7.14 (t, J = 7.3 Hz, 1H, C6H4), 4.42 (t, J = 8.0 Hz, 2H, CH2), 3.37 (t, J = 8.0 Hz, 2H, CH2). 13C-NMR (126 MHz, DMSO-d6, δ): 158.3, 157.8, 156.7, 142.3, 138.9, 131.5, 128.1, 126.1, 125.0, 111.0, 93.1, 49.2, 27.7. LCMS (ESI+) m/z 271 [M + H]+. Anal. calc. for C13H10N4OS: C 57.76%, H 3.73%, N 20.73%. Found: C 57.90%, H 3.90%, N 20.90%.
(Z)-5-(Piperidin-1-ylmethylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (6b). Yield 61%, beige powder, mp 190–192 °C (ethanol). 1H-NMR (500 MHz, DMSO-d6, δ): 8.35 (s, 1H, 2-H), 8.12 (s, 1H, =CH), 3.64 (s, 4H, piperidine), 1.65 (s, 6H, piperidine). 13C-NMR (126 MHz, DMSO-d6, δ): 157.8, 157.1, 156.7, 149.1, 148.7, 96.8, 86.2, 22.9. LCMS (ESI+) m/z 237 [M + H]+. Anal. calc. for C10H12N4OS: C 50.83%, H 5.12%, N 23.71%. Found: C 51.10%, H 5.40%, N 23.90%.
(Z)-5-((4-(2-Hydroxyethyl)piperazin-1-yl)methylene)thiazolo[3,2-b][1,2,4]triazol-6(5H)-one (6c). Yield 67%, light pink powder, mp 189–192 °C (ethanol). 1H-NMR (500 MHz, DMSO-d6, δ): 8.35 (s, 1H, 2-H), 8.13 (s, 1H, =CH), 4.47 (t, J = 5.3 Hz, 1H, OH), 3.65 (d, J = 5.6 Hz, 4H, piperazine), 3.52 (q, J = 6.0 Hz, 2H, CH2), 2.58 (dd, J = 6.1, 4.2 Hz, 4H, piperazine), 2.45 (t, J = 6.1 Hz, 2H, CH2). 13C-NMR (126 MHz, DMSO-d6, δ): 157.9, 157.0, 156.0, 155.8, 148.9, 86.8, 59.7, 59.2, 58.4. LCMS (ESI+) m/z 282 [M + H]+. Anal. calc. for C11H15N5O2S: C 46.96%, H 5.37%, N 24.89%. Found: C 47.10%, H 5.60%, N 25.00%.
Ethyl(Z)-4-((6-oxothiazolo[3,2-b][1,2,4]triazol-5(6H)-ylidene)methyl)piperazine-1-carboxylate (6d). Yield 62%, beige powder, mp 204–206 °C (ethanol–DMF 4:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.37 (s, 1H, 2-H), 8.19 (s, 1H, =CH), 4.07 (q, J = 7.1 Hz, 2H, CH2), 3.69 (brs, 4H, piperazine), 3.55 (brs, 4H, piperazine), 1.20 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (126 MHz, DMSO-d6, δ): 158.1, 157.3, 156.2, 154.5, 149.2, 111.6, 87.5, 61.2, 14.5. LCMS (ESI+) m/z 310 [M + H]+. Anal. calc. for C12H15N5O3S: C 46.59%, H 4.89%, N 22.64%. Found: C 46.90%, H 5.00%, N 22.80%.

3.3. Crystal Structure Determination of (E/Z)-5-((Cyclopropylamino)methylenethiazolo-[3,2-b]-[1,2,4]-triazol-6(5H)-one (5c)

3.3.1. Crystal Data

C8H8N4OS, Mr = 208.24, monoclinic, space group P21/c, a = 9.8291(4), b = 12.1556(4), c = 7.4814(3) Å, β = 95.025(4)°, V = 890.44(6) Å3, Z = 4, Dcalc = 1.553 g/cm3, µ = 3.005 mm−1, T = 130.0(1) K.

3.3.2. Data Collection

A yellow lath crystal (MeOH) of 0.24 × 0.13 × 0.03 mm was used to record 9342 (Cu Ka-radiation, θmax = 76.52°) intensities on a SuperNova Dual Atlas diffractometer (Rigaku, Oxford, UK) [49] using mirror monochromatized Cu Kα radiation from a high-flux microfocus source (λ = 1.54178 Å). Accurate unit cell parameters were determined by least-squares techniques from the θ values of 3457 reflections, θ range 4.48–76.30°. The data were corrected for Lorentz polarization and for absorption effects [49]. The 1850 total reflections (Rint = 0.034) were used for structure determination.

3.3.3. Structure Solution and Refinement

The structure was solved was solved by dual-space algorithm (SHELXT) [50], and refined against F2 for all data (SHELXL-97) [51]. The positions of the H atom bonded to N atom were obtained from the difference Fourier map and were refined freely. The remaining H atoms were placed geometrically in calculated positions and were refined with a riding model, with C–H = 0.99 Å (CH2), 1.00 Å (Csp3H), 0.95 Å (Csp2H) and Uiso(H) = 1.2Ueq(C). Final refinement converged with R = 0.0305 (for 1639 data with F2 > 4σ(F2)), wR = 0.0836 (on F2 for all data), and S = 1.105 (on F2 for all data). The largest difference peak and hole was 0.322 and −0.250 eÅ3. The molecular illustrations were drawn using ORTEP-3 for Windows [52]. Software used to prepare material for publication was WINGX [52], OLEX [53] and PLATON [54].
The supplementary crystallographic data of 5c have been deposited at the Cambridge Crystallography Data Centre (CCDC) as supplementary publication CCDC2046355. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-(0)1223–336033 or e-mail: [email protected]).

3.4. Anticancer Activity Screening (NCI-60 Human Tumor Cell Lines Screen)

The primary anticancer assay was performed on a panel of approximately sixty human tumor cell lines derived from nine neoplastic diseases (leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers), in accordance with the protocol of the National Cancer Institute Drug Evaluation Branch (Bethesda, MD, USA) [37,38,39,40]. Tested compounds were added to the culture at a single concentration (10−5 M) and the cultures were incubated for 48 h. End-point determinations were made with a protein-binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percentage of the growth of the treated cells when compared to the untreated control cells. The percentage of the growth was evaluated spectrophotometrically versus controls not treated with test agents. The cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of human tumor cell lines at concentrations ranging from 10−4 to 10−8 M. 48-h continuous drug exposure protocol was followed and an SRB protein assay was used to estimate cell viability or growth. Using absorbance measurements [time zero (Tz), control growth in the absence of drug (C), and test growth in the presence of the drug (Ti)], the percentage growth was calculated for each drug concentration. Percentage growth inhibition was calculated as:
[(Ti − Tz)/(C − z)] × 100
for concentrations for which Ti ≥ Tz, [(Ti − Tz)/Tz] × 100 for concentrations for which Ti < Tz. Dose-response parameters (GI50, TGI) were calculated for each compound. Growth inhibition of 50% (GI50) was calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. Values were calculated for each of these parameters if the level of activity was reached; however, if the effect was not reached or was excessive, the value for that parameter was expressed as more or less than the maximum or minimum concentration tested. The lowest values were obtained with the most sensitive cell lines. Compounds having GI50 values ≤ 100 μM were declared to be active. The selectivity index (SI) calculated by dividing the full panel MG_MID (full-panel mean-graph midpoint) (μM) of the compounds 2h and 2i by their individual subpanel MG_MID of a cell line (μM) was considered as a measure of compounds’ selectivity. Ratios between 3 and 6 mean moderate selectivity, ratios greater than 6 indicate high selectivity toward the corresponding cell line, while compounds not meeting either of these criteria are rated nonselective [38,55].

3.5. Cytotoxicity Study

The cytotoxicity of compounds 2h and 2i was analyzed in vitro by the MTT colorimetric assay on human embryonic kidney cell lines (HEK 293) [47]. HEK cell lines were cultured in DMEM supplemented with 10% fetal calf serum and 1% antibiotic-antimycotic mixture culture plates at 37 °C and 5% CO2-humidified incubator. Cells were seeded at a density of 1 × 105 cells/well in 96-well plate for 24 h; then, cells were washed and incubated in fresh medium. Compounds 2h and 2i were added at a concentration of 1, 5, 10, 25, and 50 μg/mL to triplicate wells and kept for 24 h, after which cells were washed three times with PBS. After washing, 20 μL of MTT solution (5 mg/mL stock solution) were added to each well, and cells were then incubated for an additional 4 h. The unreacted MTT dye and medium were aspirated off, and 100 μL of DMSO was added to each well to ensure solubilization of formazan crystals. The contents of the plates were mixed for 15 min to achieve complete solubilization of the formazan crystals, and the measurement of optical density was carried out at 570 nm with a micro plate spectrophotometer (MRX Microplate Reader, Dynatech Laboratories Inc., Chantilly, VA, USA) at 570 nm.

4. Conclusions

The synthesis of a series of novel 5-aryl(heteryl)idene- and 5-aminomethylidene derivatives of thiazolo[3,2-b][1,2,4]triazole-6(5H)-one has been achieved and the synthetic pathway for 5-aminomethylidene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones confirmed using X-ray analysis. The stereochemistry and E/Z-isomerization of 5-aminomethylidene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones have been studied using 1H-, 13C- and 2D NMR spectral analysis. The derivatives synthesized have been evaluated for their anticancer activity and it has been possible to identify several hits with anticancer properties and low cytotoxicity to the mammalian cells which should be study further as part of more detailed investigations into this special and especially promising class of heterocyclic compounds. The obtained data contributes to the SAR profile of 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones for further exploration to design improved and optimized molecules with antitumor activity.

Supplementary Materials

The following are available online. Table S1: NCI numbers of compouds selected for screening. Table S2: Influence of compounds 2h and 2i on the growth of tumor panels, Figures S1–S49: Copies of 1H, 13C-NMR spectra of compounds 2a, 2c, 2d, 2h, 2i, 2l, 3, 5a–h, 6b, 6c; 2D NMR and LC-MS spectra of compounds 5b,c.

Author Contributions

Conceptualization, S.H. and R.L.; methodology, S.H. and R.L.; software, A.G.; validation, S.H., S.K. and A.C.; formal analysis, S.H., A.G. and A.C.; investigation, S.H., S.K., S.S., A.C. and A.G.; writing—original draft preparation, S.H., S.K., A.G., A.C. and R.L.; writing—review and editing—S.H., A.G., A.C. and R.L.; supervision, R.L.; project administration, R.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Ministry of Healthcare of Ukraine (0121U100690).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in this article.

Acknowledgments

Authors are grateful to G. Morris (Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD, USA), for in vitro evaluation of anticancer activity; A. Luzhetskyy, A. Palusczak, M. Stierhof (Department of Pharmaceutical Biotechnology, Saarland University, Germany), V. Chebanov, V. Musatov (STC “Institute for Single Crystals” NAS of Ukraine), N. Trotsko (Department of Organic Chemistry, Medical University of Lublin) for LC-MS, NMR spectra and cytotoxicity studies. This paper is dedicated to the memory of our two colleagues that recently passed away. Sergiy Komykhov, who is a co-author of this paper passed away when the paper was in the final stage of preparation for submission. His contribution to this work as well as help and valuable suggestions in the past are highly appreciated. Sergiy Komykhov will be remembered as an outstanding scientist, bright mind, and good friend that loved what he was doing and shared his passion with everyone who was privileged to work with him. Peter Eckl our friend, professor from the University of Salzburg, who was our collaborator, research partner and scientific mentor for many years and passed away last year.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds 2a–l, 4, 5a–h, 6a–d are available from the authors.

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Figure 1. The current state of 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-one bearing compounds in medicinal chemistry.
Figure 1. The current state of 5-ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-one bearing compounds in medicinal chemistry.
Molecules 26 01162 g001
Molecules 26 01162 g002 550
Figure 2. 5-Ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones with anticancer properties.
Figure 2. 5-Ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones with anticancer properties.
Molecules 26 01162 g002
Molecules 26 01162 sch001 550
Scheme 1. Three-component synthesis of 5-aryl(heteryl)idene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones (2a–l). Reagents and conditions: i) 1 (10 mmol), ClCH2COOH (10 mmol), R1-CHO (12 mmol), AcONa (20 mmol), AcOH:Ac2O (5 + 5 mL), reflux, 3 h.
Scheme 1. Three-component synthesis of 5-aryl(heteryl)idene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones (2a–l). Reagents and conditions: i) 1 (10 mmol), ClCH2COOH (10 mmol), R1-CHO (12 mmol), AcONa (20 mmol), AcOH:Ac2O (5 + 5 mL), reflux, 3 h.
Molecules 26 01162 sch001
Molecules 26 01162 sch002 550
Scheme 2. Synthesis of 5-aminomethylidene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones 5a–h, 6a–d. Reagents and conditions: (i) 1 (10 mmol), ClCH2COOH (10 mmol), AcONa (10 mmol), AcOH 5 mL, reflux, 2 h, 76%; (ii) 3 (10 mmol), HC(OEt)3, (12 mmol), Ac2O (10 mL), reflux, 2 h, 78%; (iii) 4 (10 mmol), appropriate amino derivatives (10 mmol), EtOH, reflux, 2 h.
Scheme 2. Synthesis of 5-aminomethylidene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones 5a–h, 6a–d. Reagents and conditions: (i) 1 (10 mmol), ClCH2COOH (10 mmol), AcONa (10 mmol), AcOH 5 mL, reflux, 2 h, 76%; (ii) 3 (10 mmol), HC(OEt)3, (12 mmol), Ac2O (10 mL), reflux, 2 h, 78%; (iii) 4 (10 mmol), appropriate amino derivatives (10 mmol), EtOH, reflux, 2 h.
Molecules 26 01162 sch002
Molecules 26 01162 sch003 550
Scheme 3. Possible stereoisomeric/rotameric forms of 5a–h.
Scheme 3. Possible stereoisomeric/rotameric forms of 5a–h.
Molecules 26 01162 sch003
Molecules 26 01162 g003 550
Figure 3. 1H-NMR spectrum of 5c. Signals of A, B, C forms of 5c are shown.
Figure 3. 1H-NMR spectrum of 5c. Signals of A, B, C forms of 5c are shown.
Molecules 26 01162 g003
Molecules 26 01162 g004 550
Figure 4. Interpretation of exchange cross-peaks between A, B and C isomers of 5c.
Figure 4. Interpretation of exchange cross-peaks between A, B and C isomers of 5c.
Molecules 26 01162 g004
Molecules 26 01162 sch004 550
Scheme 4. 1H and 13C-NMR chemical shifts of (A–C) isomers of 5c with key interactions in the relevant HMBC spectra.
Scheme 4. 1H and 13C-NMR chemical shifts of (A–C) isomers of 5c with key interactions in the relevant HMBC spectra.
Molecules 26 01162 sch004
Molecules 26 01162 g005 550
Figure 5. A view of 5c, showing the atomic labelling scheme. Non-H atoms are drawn as 30% probability displacement ellipsoids and H atoms are drawn as spheres of an arbitrary size.
Figure 5. A view of 5c, showing the atomic labelling scheme. Non-H atoms are drawn as 30% probability displacement ellipsoids and H atoms are drawn as spheres of an arbitrary size.
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Molecules 26 01162 g006 550
Figure 6. Mean growth inhibitory concentration (MG_MID GI50, µM) of compounds 2h and 2i in comparison with cisplatin and curcumin. *NSCLC—Non-small Cell Lung Cancer, #CNS—Central nervous system.
Figure 6. Mean growth inhibitory concentration (MG_MID GI50, µM) of compounds 2h and 2i in comparison with cisplatin and curcumin. *NSCLC—Non-small Cell Lung Cancer, #CNS—Central nervous system.
Molecules 26 01162 g006
Table
Table 1. Hydrogen-bond geometry (A) for 5c.
Table 1. Hydrogen-bond geometry (A) for 5c.
D—H∙∙∙AD—HH∙∙∙AD∙∙∙AD—H∙∙∙A
N8—H8∙∙∙O12 i0.85 (2)1.95 (2)2.7844 (17)168 (2)
C11—H11B∙∙∙N3 ii0.992.613.603 (2)179
Symmetry codes: i 1 − x, −1/2 + y,1/2 − z; ii 2 − x, 1 − y, 1 − z.
Table
Table 2. Anticancer screening data in concentration 10 μM.
Table 2. Anticancer screening data in concentration 10 μM.
Compound60 Cell Lines Assay in One Dose, 10 μM
Mean Growth, %Range of Growth, %Most Sensitive Cell Line(s)
(Growth Inhibition Percent)/Panel		Positive Cytostatic Effect aPositive Cytotoxic Effect b
2a89.9852.79 to 122.91NCI-H522 (52.79)/NSCLC#0/590/59
2c102.5777.10 to 146.05NCI-H522 (77.10)/NSCLC#0/590/59
2d99.67−73.92 to 147.73CCRF-CEM (-73.92), HL-60 (TB) (−58.44), MOLT-4 (−73.62) all Leukemia2/583/58
2e101.8261.87 to 133.86SR (61.87)/Leukemia0/580/58
2f108.1686.65 to 261.80OVCAR-5 (86.65)/Ovarian Cancer0/570/57
2h28.98−48.01 to 121.70HL-60 (TB) (−32.79), MOLT-4 (−11.15), RPMI-8226 (−16,52), SR (−48.01)/all Leukemia;
HOP-92 (−21.66)/NSCLC#;
BT-549 (−16.67), HS 578T (−15.94)/all Breast Cancer;
RXF 393 (−15.19)/Renal Cancer;
LOX IMVI (−9.62)/Melanoma;
DU-145 (−21.71)/Prostate Cancer;
SF-539 (−33.43), SNB-75 (−11.46)/CNS Cancer		30/5712/57
2i61.05−22.54 to 122.94CCRF-CEM (−3.49); SR (−25.54) all Leukemia17/582/58
2j106.3282.41 to 135.61K-562 (82.41)/Leukemia0/560/56
2k110.5125.23 to 180.21SR (25.23)/Leukemia1/560/56
2l105.4166.88 to 141.12PC-3 (66.88)/Prostate Cancer0/590/59
397.2174.85 to 122.06SR (74.85)/Leukemia0/590/59
5e100.5980.34 to 137.75T-47D (80.34)/Breast Cancer0/590/59
5f93.6154.41 to 111.61T-47D (54.41)/Breast Cancer0/590/59
NSCLC#: Non-Small Cell Lung Cancer. a Ratio between number of cell lines with percent growth from 0 to 50 and total number of cell lines. b Ratio between number of cell lines with percent growth of <0 and total number of cell lines.
Table
Table 3. Influence of compounds 2h and 2i on the growth of tumor panels (GI50, TGI, LC50) and selectivity index (SI) values.
Table 3. Influence of compounds 2h and 2i on the growth of tumor panels (GI50, TGI, LC50) and selectivity index (SI) values.
Panel, MG_MIDGI50, μMSI (GI50)TGI, μMSI (TGI)LC50, μMSI (LC50)
2h2i2h2i2h2i2h2i2h2i2h2i
Leukemia1.9211.321.840.976.8551.551.880.5526.8199.251.390.64
Range*1.35–2.692.88–18.19--3.16–
14.45		9.99–
>100		--7.58–
48.97		95.49–
>100		--
NSCLC6.9814.130.510.7818.2230.030.710.9444.0164.550.840.97
Range*2.29–15.4810.23–19.49--9.99–
30.90		25.70–
39.81		--31.62–
60.25		50.11–
83.17		--
Colon cancer6.1312.420.580.8817.3926.510.741.0641.3956.320.901.12
Range*2.45–9.339.55–16.59--10.96–
21.37		21.37–
33.11		--33.11–
45.70		46.77–
57.60		--
CNS Cancer2.879.851.231.1110.7627.421.191.0338.5866.250.960.95
Range*1.77–4.076.02–13.18--4.46–
15.48		19.05–
44.66		--12.59–
87.09		43.65–
>100		--
Melanoma4.7911.770.740.9316.4925.610.781.1041.2055.370.901.14
Range*2.63–8.315.62–14.79--11.22–
20.89		18.19–
33.11		--33.11–
51.28		42.65–
77.62		--
Ovarian Cancer8.0513.690.440.8020.3431.150.630.9052.0968.650.710.92
Range*2.11–
15.48		6.60–12.88--5.62–
30.90		20.89–
38.90		--19.49–
97.72		51.28–
>100		--
Renal Cancer5.3713.020.660.8416.4027.130.781.0340.4655.020.921.15
Range*1.62–
12.58		5.75–17.37--8.12–
25.11		21.37–
31.62		--28.84–
50.11		51.28–
58.88		--
Prostate Cancer2.677.61.331.4410.3427.111.251.0430.7768.151.210.93
Range*1.54–3.803.98–11.22--4.46–
16.21		16.21–
38.01		--15.84–
45.70		36.30–
>100		--
Breast Cancer4.489.720.791.1315.6130.290.820.9346.0573.180.800.86
Range*<0.01–
15.48		3.54–21.87--4.07–
41.68		15.84–
67.06		--19.49–
>100		41.68–
>100		--
Total MG_MID3.5410.96--12.8828.18--37.1563.09--
Range*—Values range inside panel, μM; GI50—molar concentration of the compound that inhibits 50% net cell growth; TGI—molar concentration of the compound leading to the total inhibition; LC50 molar concentration of the compound leading to 50% net cell death.
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Holota, S.; Komykhov, S.; Sysak, S.; Gzella, A.; Cherkas, A.; Lesyk, R. Synthesis, Characterization and In Vitro Evaluation of Novel 5-Ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones as Possible Anticancer Agents. Molecules 2021, 26, 1162. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26041162

AMA Style

Holota S, Komykhov S, Sysak S, Gzella A, Cherkas A, Lesyk R. Synthesis, Characterization and In Vitro Evaluation of Novel 5-Ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones as Possible Anticancer Agents. Molecules. 2021; 26(4):1162. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26041162

Chicago/Turabian Style

Holota, Serhii, Sergiy Komykhov, Stepan Sysak, Andrzej Gzella, Andriy Cherkas, and Roman Lesyk. 2021. "Synthesis, Characterization and In Vitro Evaluation of Novel 5-Ene-thiazolo[3,2-b][1,2,4]triazole-6(5H)-ones as Possible Anticancer Agents" Molecules 26, no. 4: 1162. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26041162

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