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Article

Synthesis, Characterization and Biological Activity Evaluation of Some 1,N-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl) Alkanes

by
Andreea-Anda Alexa
1,
Vasile-Nicolae Bercean
2,
Sabina Elena Boncea
3,
Adriana Ledeţi
4,*,
Valentin Badea
2,
Amalia Ridichie
4,
Marilena Motoc
1 and
Ionuţ Ledeţi
4
1
Discipline of Biochemistry, Department of Biochemistry and Pharmacology, Faculty of Medicine, Victor Babeş University of Medicine and Pharmacy, 2 Eftimie Murgu Square, 300041 Timisoara, Romania
2
Department of Applied Chemistry and Organic and Natural Compounds Engineering, Politehnica, University Timisoara, Vasile Pârvan 6, 300223 Timisoara, Romania
3
Biology and Geology Faculty, Babes-Bolyai University, 5–7 Clinicilor Str., 400015 Cluj-Napoca, Romania
4
Advanced Instrumental Screening Center, Faculty of Pharmacy, Victor Babeş University of Medicine and Pharmacy, 2 Eftimie Murgu Square, 300041 Timisoara, Romania
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(14), 6180; https://doi.org/10.3390/app14146180
Submission received: 18 June 2024 / Revised: 10 July 2024 / Accepted: 12 July 2024 / Published: 16 July 2024
(This article belongs to the Special Issue Advances in Organic Synthetic Chemistry)
Browse Figures
Versions Notes

Abstract

In this paper, we present the synthesis, characterization and evaluation of antiproliferative activity for four compounds carrying the 4-amino-5-mercapto-1,2,4-triazol-3-yl scaffold. The synthesis of 1,n-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl) alkanes was carried out using as starting reagents the dihydrazides of oxalic, malonic, succinic and adipic acids, using mercaptoacetic acid dianion as a leaving group, by a one-pot synthesis method implemented in our research group for the synthesis of 3-substituted-5-mercapto-1,2,4-triazoles. The compounds were obtained with modest yields (12–60%) but with good purity and were characterized by elemental analysis, FTIR, 1H-NMR and 13C-NMR spectroscopy. Also, the stability of the synthesized bis-triazoles was investigated under controlled thermal stress in a dynamic oxidative atmosphere. The last part of the study consisted of biological activity evaluation, by evaluating the antiproliferative activity against the A375 line (human malignant melanoma), as well as on viability of the BJ fibroblast cell line, using MTT and LDH assays.

1. Introduction

Triazoles, particularly mercapto-substituted 1,2,4-triazoles, have become increasingly important in medicine due to their diverse biological activities [1,2], like antibacterial [3,4], antifungal [5,6], antitubercular [7], antioxidant [8], anticancer [9], anti-inflammatory [10], analgesic [11], antidiabetic [12], anticonvulsant [13], antiviral/anti-infective [14] and anxiolytic activity [15]. Numerous compounds from this class of heterocycles are promising candidates in cancer therapy, even if their exact mechanism of action is still under investigation. However, studies suggest that mercapto-substituted 1,2,4-triazoles may interfere with cancer cell growth, division and survival pathways [2].
Beyond the exceptional potential of finding promising candidates from mercapto-substituted 1,2,4-triazole derivatives in oncology and cancer therapy [1,9,16], some triazole derivatives exhibit antiviral activity, making them potential candidates for treating viral, fungal and bacterial infections, with several advantages over existing drugs, like novel mechanism of action, so potentially overcoming resistance issues [17,18,19,20].
Due to the development of efficient synthetic methods for tailoring structural-diverse triazoles, these compounds are more readily available for research in the biomedical field, and some of the derivatives have been incorporated into commercially available drugs including fluconazole, voriconazole (antifungal), trazodone, nefazodone (antidepressant), trapidil (antihypertensive), estazolam (sedative-hypnotic), rufinamide (antiepileptic) [21] and taribavirin (antiviral/anti-infective) [14].
In this study, continuing our work regarding the synthesis of 3-substituted-5-mercapto-1,2,4-triazoles [4,22,23,24,25,26], we describe a facile synthetic pathway for a series of 1,n-bis-[4-amino-5-mercapto-1,2,4-triazol-3-yl] alkanes (1). Our preliminary attempts of obtaining 3-substituted-4-amino-5-mercapto-1,2,4-triazoles followed the synthetic reactions presented in the literature: heating 5-substituted-2-mercapto-1,3,4-oxadiazole with hydrazine hydrate in solvent, under reflux [27,28,29,30,31,32,33], and treating hydrazides with carbon sulfide in ethanolic KOH solution, followed by the addition of hydrazine hydrate, under reflux [34,35,36,37,38]. In both cases, contrary to the results presented in the literature, our synthesis reactions led to a mixture of 3-substituted-4-amino-5-mercapto-1,2,4-triazoles and 5-substituted-2-mercapto-1,3,4-oxadiazole, inseparable by recrystallization; so we adapted the synthesis method used by us previously to obtain several 1,2,4-triazoles [24,39].
Furthermore, there are studies that indicate the fact that a series of 1,n-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl) alkanes were found to be associated with cytotoxic [40] and pharmacological properties, such as antibacterial and anthelmintic [41,42].
Literature data report the synthesis of 1,n-bis-(4-amino-5-mercapto-1, 2, 4-triazol-3-yl) alkanes (1a–d) by the direct reaction of dicarboxylic acids (5), such as malonic [43], succinic [43,44], glutaric [43,44], adipic [44] and other acids with complex structures [41], mainly by melting with thiocarbohydrazide (6) (Scheme 1). For the 1,8-bis-(5-mercapto-4-amino-1,2,4-triazol-3-yl)-octane another synthesis reaction is reported by treating sebacic acid dihydrazide with carbon disulfide and potassium hydroxide, followed by the addition of hydrazine hydrate [45].
Another method used to obtain bis-triazoles of form (1) consists in treating bis-oxadiazoles (8) with hydrazine hydrate in alcoholic medium under reflux [46].
The possible synthetic pathways for the formation of 1,n-bis-(5-mercapto-1,2,4-triazol-3-yl)-alkanes (1), with the above-mentioned properties, are shown in Scheme 1:
Our first attempts using the method from the literature [45] did not lead to obtaining bis-triazoles (1a–d), and following this inconvenience, we aimed towards the synthesis of these compounds by a new method, in which the hydrazides are treated with carbon sulfide, in ethanolic KOH solution, followed by the addition of potassium chloroacetate and treatment of the intermediate 7a–d with hydrazine hydrate, under reflux. This method showed advantages in terms of overall yields, reaction time and number of steps, as well as the purity of the reaction products. According to this approach, the use of mercaptoacetic acid dianion (−S-CH2-COO−) as a leaving group leads to pure bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl)-alkanes and presents the advantage of achieving transformations 2a–d → 3a–d → 4a–d → 7a–d → 1a–d in a single step synthesis, i.e., a “one pot reaction” (Scheme 2).
The hydrazides of oxalic, succinic, malonic and adipic acids (3a–d) were obtained by the hydrazinolysis of the ethyl esters of the corresponding acids (2a–d). All the compounds were characterized by both spectroscopic and thermal techniques. Additionally, all the synthesized compounds (1a–d) were screened for biological activity, namely anticancer activity against the A375 line (human malignant melanoma), as well as on viability of the BJ fibroblast cell line, using MTT and LDH assays.

2. Results and Discussion

2.1. Synthesis of Compounds

2.1.1. Synthesis and Characterization of Dihydrazides (3a–d) by Hydrazinolysis of Esters (2a–d)

In order to obtain the 4H-4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole (1a) and 1,n-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)alkanes (1b–d) derivatives, the synthesis of starting reagents was employed, namely the dihydrazides (3a–d) of the oxalic, malonic, succinic and adipic acids, by the hydrazinolysis of the diesters (2a–d).
Following the protocol presented in Section 3.2.1, the four dihydrazides were obtained with good yields (79–87%) according to the literature [47], and characterized by melting point and FTIR spectroscopy (Table 1).
The measured melting points are in good agreement with literature data for the 3b–d derivatives, while for 3a there is a considerable difference, which can be explained by the fact that this compound is known to exist in at least four polymorphic states [52]. Following this aspect, the existence of several solid forms of organization of this compound determines different physical properties (including melting point), since the formation of hydrogen bonds between donor and acceptor moieties in this molecule is conditioned by the different orientations of the groups with respect to the molecular plane, which are different geometric orientations for hydrogen bonding arrangements in the solid state [52,53].

2.1.2. Synthesis and Characterization of Bis-Triazoles 1a–d

Synthesis of 4H-4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole (1a) and 1,n-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)alkanes (1b–d) started from dihydrazides (3a–d), as presented in Scheme 2.
Following the protocol presented in Section 3.2.2, the four triazoles were obtained with variable yields (12–60%), and characterized by melting point, elemental analysis, FTIR, 1H-NMR and 13C-NMR spectroscopy (Table 2).
The analysis of the FTIR spectra of 4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole (1a) reveals the appearance of several characteristic bands [40]. For the primary amino group, the N–H stretching vibration is revealed by the band with a maximum at 3330 cm−1. The stretching vibrations of the aromatic C–H bond at 3044 cm−1, as well as the stretching vibrations of the double C=N bonds, can be identified in the spectrum at 1590 cm–1. The existence of the compound in the thione tautomeric form (C=S) is confirmed by the band that appears at 1251 cm−1 [40,54].
The presence of the primary amino groups (–NH2) in the structure of 1,1-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)methane (1b) is indicated by the presence of the bands at 3291 and 3242 cm−1. The stretching vibrations of the aromatic C–H bond are observed at 3076 and 3026 cm−1, as well as the symmetric and asymmetric vibrations corresponding to the CH2 group at 2923 and 2807 cm−1. The stretching vibrations of the C=N bonds are represented by the band observed at 1614, while the C=S vibration is represented by a band at 1235 cm−1.
The IR spectrum of 1,2-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)ethane (1c) shows an absorption band at 3287 cm−1, indicating the presence of the primary amino group (–NH2) in the molecule. The stretching vibrations of the aromatic C–H bonds are observed at 3159 cm−1, while the stretching vibrations of the C=N and C=S bonds appear at 1615 and 1248 cm−1, respectively, as medium-intense bands. The symmetric and asymmetric stretching vibrations for the CH2 groups are represented by the bands seen at 2941 and 2755 cm−1. In the case of 1,4-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-5-yl)butane (1d), the stretching vibrations (N–H) for the amino group are observed at 3284 cm−1, while the bands at 3135 and 3043 cm−1 characterize the stretching vibrations of the aromatic C–H bond. The bands at 2951 and 2771 cm−1 can be assigned to the symmetric and asymmetric vibrations of the CH2 groups, and the stretching vibrations of the C=N and C=S bonds can be seen at 1615 and 1235 cm−1 as medium-intense bands.
The 13C-NMR spectra of compounds 1a–d highlights the presence of only the tautomeric form (C=S) through the signals δ = 165.63–167.07 ppm, corresponding to the C=S exocyclic bonds. The 1H-NMR spectra of compounds 1a–d reveal a singlet in δ = 11.96–13.30 ppm region due to the triazole protons (NH), confirming the closure of the triazole rings. The protons of the NH2 groups appear between δ = 5.51–5.92 ppm, demonstrating the presence of the free amino groups on the triazole ring, while the signals from the alkyl chain of compounds 1b-d are observed at δ = 1.70–4.14 ppm. As further proof, FTIR, NMR 2D COSY 1H-13C, 13C-NMR DEPT-135 and 2D HMBC spectra were recorded, as seen in Supplementary Material, Figures S1–S18.
Also, the other signals observed in both the 13C-NMR and 1H-NMR spectra are presented in Table 2, confirming the identity, structure and purity of the synthesized compounds.

2.2. Thermal Analysis Investigations

The obtained thermoanalytical curves TG/DTG for compounds 1a–d are presented in Figure 1A–D.
The thermal treatment of compound 1a suggests that it is thermally stable up to 194 °C, when the first mass loss occurs up to 284 °C (DTG first process between 194–246 °C, DTGpeak at 204 °C; DTG second process between 246–284 °C, DTGpeak at 267 °C), with a corresponding mass loss Δm = 15.06%. The main mass loss due to thermolysis takes place in the 284–597 °C temperature range (DTG third process between 303–597 °C, DTGpeak at 425 °C), the residual mass being insignificant (less than 1%). Surprisingly, the behavior observed under thermal stress is different when the heating takes place in the furnace of the Setline instrument vs. Optimelt apparatus, a fact that can be explained by the different environments around the solid sample under heating: dynamic air/oxidative atmosphere with considerable flow, vs. static air atmosphere in the capillary glass. However, the browning observed at 201 °C during the melting point investigation can be associated with the first mass loss process from the TG/DTG curve (Figure 1A).
Compound 1b has good thermal stability up to 181 °C when a small mass loss (Δm = 0.19%) takes place up to 232 °C (DTG first process between 181–232 °C, DTGpeak at 197 °C); in the 232–309 °C temperature range, the first considerable mass loss takes place (Δm = 19.38%, DTG process between 232–308 °C, DTGpeaks at 258 and 285 °C, respectively), followed by another considerable mass loss process in the 309–624 °C temperature range (Δm = 80.41%, DTGpeaks at 396 and 501 °C, respectively).
Thermal analysis of compound 1c reveals that is thermally stable up to 238 °C when a multistep mass loss takes place, according to the DTG curve; the first mass loss process occurs in a narrow temperature range (238–266 °C, Δm = 14.44%, DTGpeak at 252 °C—in agreement with the melting indicated by the Optimelt apparatus), followed by several other processes, up to 678 °C (Δm = 83.4%, DTGpeaks at 287, 315 and 647 °C).
Similar thermal behavior to that observed for the 4c derivative is observed for the 1d compound. This triazole is stable up to 232 °C, when a multistep mass loss takes place, according to the DTG curve; the first mass loss process occurs in a narrow temperature range (238–261 °C, Δm = 13.64%, DTGpeak at 252 °C—in agreement with the melting indicated by the Optimelt apparatus), followed by several other processes, up to 750 °C (Δm = 86.34%, DTGpeaks at 264, 276, 330, 448 and 583 °C).

2.3. Antiproliferative Activity

The compounds showed different activity against human cancer and normal cells at 10 µM and 30 µM concentrations. The analysis of activity on the cancer cell line showed a proportional decrease in viability in rapport with drug concentration, in most cases. Overall, cellular activity was lower than the control group. The untreated control group is considered as 100% mitochondrial viability as they represent cells with no treatment applied. A375 cells treated with 1a, 1b, 1c, or 1d at a 10 µM concentration expressed a viability of 88.94 (±1.64)%, 118.00 (±2.94)% 100.76 (±2.80)%, or 99.98 (±6.71)%, respectively. A375 cells treated with 1a, 1b, 1c, or 1d at a 30 µM concentration expressed a viability of 98.63 (±5.09)%, 92.49 (±10.46)%, 96.61 (±8.25)%, or 94.30 (±6.88)%, respectively. Compound 1a decreased viability by 9.69% for the 10 µM concentration compared to the 30 µM concentration. For the rest of the compounds, viability was decreased by 4.62%, 4.15%, or 5.67% more for the 30 µM concentration than the 10 µM concentration (Figure 2). Fibroblast analysis showed an increase in viability of 17–42% for a 10 µM concentration and 28–81% for a 30 µM concentration compared to the control group. Fibroblast analysis showed an increase in viability for most of the compounds tested. BJ cells treated with 1a, 1b, 1c, or 1d at a 10 µM concentration expressed a viability of 117.96 (±7.81)%, 101.03 (±16.01)% 177.09 (±17.66)%, or 148.61 (±11.45)%, respectively. BJ cells treated with 1a, 1b, 1c, or 1d at a 30 µM concentration expressed a viability of 168.67 (±16.16)%, 96.41 (±6.04)%, 196.90 (±21.07)%, or 122.14 (±4.12)%, respectively. Compounds 1a and 1c increased viability by 50.71% and 19.81%, respectively, for the 30 µM concentration compared to the 10 µM concentration. Compounds 1b and 1d increased viability by 4.61% and 26.47%, respectively for the 10 µM concentration compared to the 30 µM concentration (Figure 3).
LDH assay results showed a decrease in cancer cell growth for the treated group, which resulted in lower LDH release compared with untreated control. Untreated cancer cells released 0.117 (±0.0059) U/mL LDH. A375 cells treated with 1a, 1c, or 1d at a 10 µM concentration released 0.106 (±0.0016) U/mL, 0.108 (±0.0026) U/mL, or 0.106 (±0.0062) U/mL LDH, respectively. A375 cells treated with 1a, 1c, or 1d at a 10 µM concentration released 0.109 (±0.0029) U/mL, 0.107 (±0.0068) U/mL, or 0.108 (±0.0026) U/mL LDH, respectively. Compound 1b was analyzed in another plate and had an untreated control value of 0.0584 U/mL and released 0.0497 U/mL. Compounds 1a, 1b, and 1d decreased LDH release by 0.0026 U/mL, 0.00087U/mL, or 0.0021 U/mL, respectively for the 10 µM concentration compared to the 30 µM concentration, while 1c decreased LDH release by 0.0008 U/mL for the 30 µM concentration compared to the 10 µM concentration. (Figure 4). Data showed a slight cell growth inhibition for treated groups, but higher compound concentration had an insignificant impact compared to a lower dose.
Also, fibroblast showed little to no changes in LDH concentration. BJ cells had an untreated control value of 0.103 (±0.010) U/mL. BJ cells treated with 1a, 1c, or 1d at a 10 µM concentration released 0.104 (±0.003) U/mL, 0.098 (±0.006) U/mL, or 0.114 (±0.007) U/mL, respectively. BJ cells treated with 1a, 1c, or 1d at a 30 µM concentration released 0.094 (±0.007) U/mL, 0.101 (±0.007) U/mL, or 0.122 (±0.008) U/mL, respectively. Compound 1b was analyzed in another plate and had an untreated control value of 0.146 (±0.0068) U/mL and released 0.110 (±0.0028) U/mL at a 10 µM concentration and 0.109 (±0.0055) U/mL at a 30 µM concentration. Compound 1a at 10 µM and 1d at 10 µM and 30 µM increased LDH release by 0.001 U/mL, 0.011 U/mL and 0.0019 U/mL, respectively, compared with the control group. Compounds 1c and 1b at 10 µM and 30 µM decreased LDH release by 0.004 U/mL and 0.002 U/mL and 0.036 U/mL and 0.037 U/mL, respectively compared with the control group (Figure 5).

3. Materials and Methods

3.1. Reagents and Instrumentation

The reagents were commercial products of analytical purity from Chimopar (Bucureşti, Romania), Merck (Darmstadt, Germany), Fluka-Honeywell (Charlotte, NC, USA) and used as received. The synthesis of ethyl esters of oxalic, malonic, succinic and adipic acids (2a–d), was performed according to the indications mentioned in the literature.
Melting points were determined using an Optimelt M100 (Stanford Research Device, Stanford Research Systems, Inc., Sunnyvale, CA, USA) instrument in open capillary tubes, in the heating range 30–350 °C, using a heating rate of 10 °C·min–1 (the values are uncorrected).
Thin-layer chromatography (TLC) was carried out on silica-gel-coated plates 60 F254 Merck using benzene:methanol 7:3 (v/v) as eluant. The chromatographic spots were revealed by exposure to iodine vapors and/or UV light irradiation (λ = 254 nm).
The percentage of C, H and N were obtained by means of elemental analysis using a Vario El Cube apparatus (Elementar Analysensysteme GmbH, Hanau, Germany).
FTIR spectra were recorded in KBr pellets using a Specac Pellet Press (Specac Ltd., Kent, UK) using a Jasco FT/IR-410 spectrophotometer (Jasco Analytical Instruments, Easton, PA, USA).
1H-NMR and 13C-NMR spectra were recorded using a Bruker Avance AC200 spectrometer (Bruker Biospin International, Ag, Aegeristrasse, Switzerland) in DMSO-d6, using TMS as reference; chemical shifts are reported in ppm and the coupling constants in Hz. The multiplicity of the signal and the aspect of the band are abbreviated as follows: br: broad; s: singlet; d: doublet; m: multiplet.
Thermoanalytical curves were obtained using a Setline TGA (SETARAM, Caluire, France) instrument. TG (thermogravimetric/mass curve) and DTG data (derivative thermogravimetric/mass derivative) were recorded on samples placed in an open alumina crucible. The heating process was conducted in dynamic airflow (100 mL·min−1) under non-isothermal conditions at a heating rate β = 10 °C·min−1, from a temperature of 40 °C up to 800 °C, each sample having a mass of approximately 10 mg. Each TG experiment was performed in duplicate and the results were practically identical.

3.2. Synthesis Protocols

3.2.1. Synthesis of Dihydrazides (3a–d) by Hydrazinolysis of Esters (2a–d)

The ethylic ester of each dicarboxylic acid (0.1 mol) was dissolved in absolute ethanol (10 mL), then hydrazine hydrate (0.3 mol) was added and heated under reflux for 8–10 h. After dry distillation of the reaction mixture, at 80–90 °C/5mmHg, the solid residue was crystallized from ethanol.

3.2.2. Synthesis of 4H-4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole (1a) and 1,N-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)alkanes (1b–d)

The dihydrazide of dicarboxylic acid (0.02 mol) was dissolved in an ethanolic potassium hydroxide solution (0.06 mol KOH/40 mL of EtOH), then carbon sulfide (0.06 mole) was added and stirred at room temperature for two hours. The reaction mixture was cooled at 5–10 °C in an ice bath and then, an aqueous potassium chloroacetate solution (0.04 mole ClCH2COOK/30 mL H2O) was added. After 45 min, hydrazine hydrate (0.04 mol) was added at room temperature. After two hours, half of the reaction volume was distilled under vacuum, followed by the addition of hydrazine hydrate (0.04 mol), and left under reflux for 4 h. The solution was cooled to room temperature, brought to pH~1 with concentrated HCl, and the products were crystallized from aqueous ethanol solution.

3.3. Cell Culture

The cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM), a medium containing high glucose, supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% L-glutamate and 1% essential amino acids.
The cellular death and viability experiments were conducted using cells purchased from ATCC, namely, the human melanoma (A375) (ATCC CRL-1619) and fibroblast (BJ) (ATCC CRL-2522) cell lines. The stock culture was preserved at a temperature of −80 °C in a medium containing dimethylsulfoxide (DMSO). The unfrozen cells were maintained in an incubator in a temperature of 37 °C, a 5% concentration of CO2, and a humidified atmosphere.
Assays were conducted roughly after approximately 80% of the bottom flask was populated with cells. The cells were cultivated in a 96-well microplate with a concentration of 104 cells per well for the subsequent analysis. The cells were incubated for 24 h after seeding, allowing them to attach to the well bottom. The treatment was administered at concentrations of 10 µM and 30 µM. Each concentration had 8 replicas for each substance used, besides the control group for each microplate. The positive control consisted of cells without any treatment, while the negative control consisted of cells treated with 10 µL/well of Tween 20. The flask containing the treatment was incubated for a further 24 h, and the results were analyzed using LDH and MTT assays.

3.4. Cellular Viability Assay

The proliferation was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution, at a concentration of 0.5 mg/mL, which was added in a concentration of 100 µL/well, after the media was removed, and incubated for 2 h. Tetrazolium salt enters cell membranes and undergoes reduction by mitochondrial and cytosolic enzymes, resulting in the formation of purple formazan crystals. After incubation, MTT solution was removed by pipetting, and formazan crystals were dissolved by adding 100 µL isopropanol to each well. The microplate was analyzed using a spectrophotometer at a wavelength of 550 nm, with a reference wavelength of 630 nm to eliminate cellular debris errors.
The viability percentage was calculated by multiplying the optical density at 570 nm of the experimental sample by 100 and dividing it by the optical density at 570 nm of the control sample.
Viability assay by LDH release measures the level of plasma membrane damage. LDH oxidizes L-lactate to pyruvate and the reaction is mediated by the hydrogen acceptor, Nicotinamide Adenine Dinucleotide (NAD+). The process produces NADH, which then will reduce a yellow tetrazolium salt called iodonitrotetrazolium (INT). A total of 50 µL of culture medium from the treated microplate was transferred to another microplate, onto which reagents were then added. Reagent concentrations were 22.2 mg/mL TRIS, 19.6 mg/mL Li-Lactate and 3.44 mg/mL NAD. The reagent solution in each well consisted of 50 µL of TRIS, 50 µL Li-Lactate and 50 µL NAD. The microplate was placed in a microplate reader after 5 min, and the absorbance was measured at 490 nm using a reference wavelength of 630 nm to eliminate errors caused by cellular debris.
The unit of measurement [U/mL] was calculated using the formula (A *Vt)/(ε*d*Vp), where: Absorbance is multiplied by the total volume of reagents in the well, which is up to 0.2 mL. Millimolar extinction coefficient (ε) of INT to formazan at a wavelength of 490 nm is 18.4 mM−1cm−1. The reading light path (d) of the reaction is determined by the diameter of the 96-well plate, which is 6.4 mm, resulting in an area of 0.32 cm2. The volume of the reaction liquid is 0.2 cm3, so the light path is calculated as 0.2 cm3 divided by 0.32 cm2, resulting in a light path of 0.625 cm. Vp represents the volume of the sample that is being examined for the reaction, which is equal to 50 microliters or 0.05 milliliters.

4. Conclusions

Four compounds carrying the 4-amino-5-mercapto-1,2,4-triazol-3-yl moiety were synthesized, characterized and evaluated for their biological activity. The synthesis of 1,n-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl) alkanes was carried out by a new method, previously employed in our research group for the synthesis of 3-substituted-4-amino-5-mercapto-1,2,4-triazoles. In this study, simple and highly accessible starting reagents were used, namely, the dihydrazides of oxalic, malonic, succinic and adipic acids, using mercaptoacetic acid dianion as a leaving group. Initially, the compounds were characterized by elemental analysis, which confirmed the theoretical composition, and by FTIR spectroscopy, which revealed the existence of characteristic bands of the moieties from bis-triazole structures. Although the compounds were obtained with modest yields (12–60%), their purity was high.
Later, 1H-NMR and 13C-NMR spectroscopy were employed, the spectral results confirming the identity and purity of the designed compounds. The last part of our study aimed toward determining the stability profile of these compounds under controlled thermal stress in oxidative atmosphere. The biological activity evaluation consisted of studying the anticancer activity against the A375 line (human malignant melanoma), as well as on viability of the BJ fibroblast cell line, using MTT and LDH assays.
As future directions of research, we aim toward optimization of the reaction yields and extend the homolog series of the bis-triazoles by using long-chained dicarboxylic acids. Also, a study for the reaction mechanism of this synthetic protocol will be investigated.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app14146180/s1. Figure S1. FT-IR spectra of 1a, 4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole; Figure S2. 1H-NMR spectra of 1a, 4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole; Figure S3. 13C-NMR spectra of 1a, 4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole; Figure S4. 2D COSY 1H-13C spectra of 1a, 4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole; Figure S5. 13C-RMN DEPT-135 spectra of 1a, 4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole; Figure S6. 2D HMBC NMR spectra of 1a, 4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole; Figure S7. FT-IR spectra of 1b, 1,1-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)methane; Figure S8. 1H-NMR spectra of 1b, 1,1-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)methane; Figure S9. 13C-NMR spectra of 1b, 1,1-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)methane; Figure S10. FT-IR spectra of 1c, 1,2-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)ethane; Figure S11. 1H-NMR spectra of 1c, 1,2-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)ethane; Figure S12. 13C-NMR spectra of 1c, 1,2-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)ethane; Figure S13. FT-IR spectra of 1d, 1,4-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-5yl)butane; Figure S14. 1H-NMR spectra of 1d, 1,4-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-5yl)butane; Figure S15. 13C-NMR spectra of 1d, 1,4-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-5yl)butane; Figure S16. 2D COSY 1H-13C spectra of 1d, 1,4-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-5yl)butane; Figure S17. 13C-RMN DEPT-135 spectra of 1d, 1,4-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-5yl)butane; Figure S18. 2D HMBC NMR spectra of 1d, 1,4-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-5yl)butane.

Author Contributions

Conceptualization, A.-A.A., V.-N.B., A.L. and I.L.; Data curation, A.L., V.B., A.R. and M.M.; Formal analysis, A.R. and M.M.; Investigation, A.-A.A., V.-N.B., S.E.B., A.L., V.B., A.R. and I.L.; Methodology, A.-A.A., V.-N.B., S.E.B., A.L. and V.B.; Resources, S.E.B.; Software, A.L. and V.B.; Supervision, V.-N.B. and I.L.; Validation, V.-N.B. and A.L.; Visualization, M.M.; Writing—original draft, A.-A.A., V.-N.B., S.E.B., A.L., A.R. and I.L.; Writing—review and editing, A.-A.A., V.-N.B., A.L., M.M. and I.L. Synthesis was performed by A.-A.A. and V.-N.B.; Structural Characterization and Physiochemical Evaluations were performed by A.L., V.B., A.R. and I.L.; Biological Assays were performed by S.E.B. and M.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding. We would like to acknowledge “Victor Babes” University of Medicine and Pharmacy Timisoara for their support in covering the costs of publication for this research paper.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to acknowledge “Victor Babes” University of Medicine and Pharmacy Timisoara for their support in covering the costs of publication for this research paper.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 06180 sch001
Scheme 1. Synthetic pathways for 1,n-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl)-alkanes (1).
Scheme 1. Synthetic pathways for 1,n-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl)-alkanes (1).
Applsci 14 06180 sch001
Applsci 14 06180 sch002
Scheme 2. Synthesis of 1,n-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl)-alkanes (1).
Scheme 2. Synthesis of 1,n-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl)-alkanes (1).
Applsci 14 06180 sch002
Applsci 14 06180 g001
Figure 1. TG (red line)/DTG (black line) data recorded for synthesized triazoles, namely 1a (A), 1b (B), 1c (C) and 1d (D) in a dynamic air atmosphere at β = 10 °C·min−1.
Figure 1. TG (red line)/DTG (black line) data recorded for synthesized triazoles, namely 1a (A), 1b (B), 1c (C) and 1d (D) in a dynamic air atmosphere at β = 10 °C·min−1.
Applsci 14 06180 g001
Applsci 14 06180 g002
Figure 2. The results of the MTT assay for A375 cell line for compounds 1a, 1c and 1d (A) and 1b, respectively (B).
Figure 2. The results of the MTT assay for A375 cell line for compounds 1a, 1c and 1d (A) and 1b, respectively (B).
Applsci 14 06180 g002
Applsci 14 06180 g003
Figure 3. The results of the MTT assay for BJ cell line for compounds 1a, 1c and 1d (A) and 1b, respectively (B).
Figure 3. The results of the MTT assay for BJ cell line for compounds 1a, 1c and 1d (A) and 1b, respectively (B).
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Applsci 14 06180 g004
Figure 4. The results of the LDH assay for A375 cell line for compounds 1a, 1c and 1d (A) and 1b, respectively (B).
Figure 4. The results of the LDH assay for A375 cell line for compounds 1a, 1c and 1d (A) and 1b, respectively (B).
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Figure 5. The results of the LDH assay for BJ cell line for compounds 1a, 1c and 1d (A) and 1b, respectively (B).
Figure 5. The results of the LDH assay for BJ cell line for compounds 1a, 1c and 1d (A) and 1b, respectively (B).
Applsci 14 06180 g005
Table 1. Structure and characterization of the obtained dihydrazides of oxalic, malonic, succinic and adipic acids.
Table 1. Structure and characterization of the obtained dihydrazides of oxalic, malonic, succinic and adipic acids.
Structure of DihydrazideCharacterization of Compound
Molecular Formula,
Molar Mass, Aspect and
Synthesis Yield		Melting Point (°C) Measured/LiteratureFTIR Bands
(KBr, cm–1):
Applsci 14 06180 i001
Oxalyl dihydrazide
(Oxalic acid dihydrazide) (3a)		C2H6N4O2
M = 118 g·mol–1
White powder
yield η = 82%		190–194/
242–244 [48] 		3286, 3195, 1650, 1612, 1504, 748
Applsci 14 06180 i002
Malonic dihydrazide
(Malonic acid dihydrazide) (3b)		C3H8N4O2
M = 132 g·mol–1
White powder
yield η = 79%		152–157/
152–154 [49]		3330, 3035, 1626; 1076; 1122; 1504
Applsci 14 06180 i003
Succinic dihydrazide
(Succinic acid dihydrazide) (3c)		C4H10N4O2
M = 146 g·mol–1
White powder
yield η = 84%		166–171/
170–171 [50]		3289; 3195; 1540; 1509
Applsci 14 06180 i004
Adipic dihydrazide
(Adipic acid dihydrazide) (3d)		C6H14N4O2
M = 174 g·mol–1
White powder
yield η = 87%		174–181/
180–182 [51]		3200, 3311, 3181, 3047, 2925; 1636, 1536
Table 2. Structure and characterization of the obtained triazoles.
Table 2. Structure and characterization of the obtained triazoles.
Structure of TriazoleCharacterization of Compound
Molecular Formula, Molar Mass, Aspect and Synthesis YieldMelting Point (°C)FTIR Bands (KBr, cm–1):Elemental Analysis % (Calcd./Found)
1H-NMR δH (DMSO-d6, 200 MHz)
13C-NMR δC (DMSO-d6, 50 MHz)
Applsci 14 06180 i005
4-amino-5-mercapto-3-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)-1,2,4-triazole (1a)		C4H6N8S2
M = 230.72 g·mol–1
White powder
yield η = 33%		>350 *
(201 °C)		3330; 3044; 1590; 1508; 1449; 1251; 991C-20.86; H-2.63; N-48.66/
C-20.49; H-2.48; N-48.14
11,96 (s, 2H, NH); 5,92 (s, 4H, NH2)
167,07 (3-C); 139,30 (5-C);
Applsci 14 06180 i006
1,1-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)methane (1b)		C5H8N8S2,
M = 244.30 g·mol–1
White powder
yield η = 12%		281–282 *
(181 °C)		3291; 3242; 3076; 3026; 2923; 2807; 1614; 1572; 1235; 980C-24.58; H-3.30; N-45.87/
C-24.11; H-3.18; N-45.26
13,39 (s, 1H, NH); 5,51 (s, 2H, -NH2) 4,14 (s, 2H, -CH2-);
166,19 (3-C); 147,15 (5-C);21,50 (-CH2-)
Applsci 14 06180 i007
1,2-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-3-yl)ethane (1c)		C6H10N8S2,
M = 258.32 g·mol–1
White powder
yield η = 12%		252–254 *3287; 3159; 2941; 2755; 1615; 1561; 1477;
969		C-27.90; H-3.90; N-43.38/
C-27.77; H-3.84; N-43.02
13,33 (s, 1H, NH); 5,54 (s, 2H, -NH2); 3,07 (s, 2H, -CH2-)
165,86 (3-C); 150,68 (5-C);
20,79 (-CH2-)
Applsci 14 06180 i008
1,4-bis-(4H-4-amino-5-mercapto-1,2,4-triazol-5-yl)butane (1d)		C8H14N8S2,
M = 286.38 g·mol–1
White powder
yield η = 60%		252–253 *3284; 3135; 3043; 2951; 2771; 1615; 1562; 1488; 1296; 743C-33.55; H-4.93; N-39.13/
C-33.18; H-4.74; N-38.94
13,25 (s, 2H, NH); 5,51 (s, 4H, NH2); 2,60–2,67 (m, 2H, Tz-CH2-CH2-);
1,54–1,70 (m, 2H, Tz-CH2-CH2-)
165,63 (3-C); 151,89 (5-C); 24,97 (Tz-CH2-CH2-); 23,68 (Tz-CH2-CH2-);
* melting with decomposition (browning temperature).
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Alexa, A.-A.; Bercean, V.-N.; Boncea, S.E.; Ledeţi, A.; Badea, V.; Ridichie, A.; Motoc, M.; Ledeţi, I. Synthesis, Characterization and Biological Activity Evaluation of Some 1,N-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl) Alkanes. Appl. Sci. 2024, 14, 6180. https://doi.org/10.3390/app14146180

AMA Style

Alexa A-A, Bercean V-N, Boncea SE, Ledeţi A, Badea V, Ridichie A, Motoc M, Ledeţi I. Synthesis, Characterization and Biological Activity Evaluation of Some 1,N-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl) Alkanes. Applied Sciences. 2024; 14(14):6180. https://doi.org/10.3390/app14146180

Chicago/Turabian Style

Alexa, Andreea-Anda, Vasile-Nicolae Bercean, Sabina Elena Boncea, Adriana Ledeţi, Valentin Badea, Amalia Ridichie, Marilena Motoc, and Ionuţ Ledeţi. 2024. "Synthesis, Characterization and Biological Activity Evaluation of Some 1,N-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl) Alkanes" Applied Sciences 14, no. 14: 6180. https://doi.org/10.3390/app14146180

APA Style

Alexa, A.-A., Bercean, V.-N., Boncea, S. E., Ledeţi, A., Badea, V., Ridichie, A., Motoc, M., & Ledeţi, I. (2024). Synthesis, Characterization and Biological Activity Evaluation of Some 1,N-bis-(4-amino-5-mercapto-1,2,4-triazol-3-yl) Alkanes. Applied Sciences, 14(14), 6180. https://doi.org/10.3390/app14146180

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