Thiophene-Linked 1,2,4-Triazoles: Synthesis, Structural Insights and Antimicrobial and Chemotherapeutic Profiles ^†

Abstract

The reaction of thiophene-2-carbohydrazide 1 or 5-bromothiophene-2-carbohydrazide 2 with various haloaryl isothiocyanates and subsequent cyclization by heating in aqueous sodium hydroxide yielded the corresponding 4-haloaryl-5-(thiophen-2-yl or 5-bromothiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 5a-e. The triazole derivatives 5a and 5b were reacted with different secondary amines and formaldehyde solution to yield the corresponding 2-aminomethyl-4-haloaryl-2,4-dihydro-3H-1,2,4-triazole-3-thiones 6a–e, 7a–e, 8, 9, 10a and 10b in good yields. The in vitro antimicrobial activity of compounds 5a–e, 6a–e, 7a–d, 8, 9, 10a and 10b was evaluated against a panel of standard pathogenic bacterial and fungal strains. Compounds 5a, 5b, 5e, 5f, 6a–e, 7a–d, 8, 9, 10a and 10b showed marked activity, particularly against the tested Gram-positive bacteria and the Gram-negative bacteria Escherichia coli, and all the tested compounds were almost inactive against all the tested fungal strains. In addition, compounds 5e, 6a–e, 7a–d and 10a exhibited potent anti-proliferative activity, particularly against HepG-2 and MCF-7 cancer cell lines (IC₅₀ < 25 μM). A detailed structural insight study based on the single crystals of compounds 5a, 5b, 6a, 6d and 10a is also reported. Molecular docking studies of the highly active antibacterial compounds 5e, 6b, 6d, 7a and 7d showed a high affinity for DNA gyrase. Meanwhile, the potent anti-proliferative activity of compounds 6d, 6e and 7d may be attributed to their high affinity for cyclin-dependent kinase 2 (CDK2).

1. Introduction

1,2,4-Triazole heterocycle was early identified as the crucial core of numerous therapeutically interesting drugs with a wide spectrum of chemotherapeutic activities [1,2]. Triazole-based drugs are widely used as a useful medication for the treatment of topical and systemic fungal diseases [3,4]. Fluconazole [5], itraconazole [6], voriconazole [7] and posaconazole [8] are among the currently used antifungal agents. In addition, 1,2,4-triazole-based derivatives were reported to endow potent anticancer activity [9,10]. Bemcentinib [11], letrozole [12], vorozole [13], and anastrazole [14] are currently used as an efficient treatment for different cancers. In addition, the specific tankyrase inhibitor G007-LK was recently approved for clinical trials for the treatment of breast and colorectal cancers [15] (Figure 1).

In recent years, some novel 1,2,4-triazoles have been reported to demonstrate marked antibacterial [16,17,18] and anti-tuberculosis activities [19]. Furthermore, ribavirin [20] and other related 1,2,4-triazole derivatives [21] were recognized as potent antiviral agents (Figure 1).

On the other hand, thiophene heterocycle constitutes a major building block of many drugs [22,23,24]. The thiophene–triazole hybrid analog PF-4989216, a potent and selective PI3K kinase inhibitor, was discovered as an effective anticancer agent [25]. OSI-930 is an orally active anticancer agent acting by inhibitors of c-Kit and VEGFR-2, and it shows broad efficacy in tumor models representative of small cell lung cancer, glioblastoma, colorectal, renal, head and neck, non-small cell lung cancer and gastric cancers [26,27]. MCL0527 was identified as a potent anti-proliferative agent via p53-MDM2 binding inhibition [28]. The thiophene-based antifungal drugs tioconazole [29] and sertaconazole [30] are currently used for the treatment of fungal skin infections. The non-nucleoside polymerase inhibitor lomibuvir (VX-222) is currently used for the treatment of chronic hepatitis C virus (HCV) infections [31] (Figure 2).

In view of the aforementioned findings, we describe herein the synthesis, characterization, preliminary antimicrobial and anti-proliferative activities of a series of thiophene-linked 1,2,4-triazole derivatives. In addition, the single-crystal X-ray structures of five representative compounds were studied to investigate various intermolecular interactions including N–H···S hydrogen bond and C–H···S/N/F/π interactions. Additionally, the σ-hole interactions such as halogen (Br···F) and chalcogen bonds (S···S/π) [32,33,34] were also investigated.

2. Results and Discussion

2.1. Chemical Synthesis

Thiophene-2-carbohydrazide 3a and 5-bromothiophene-2-carbohydrazide 3b were prepared starting with their corresponding carboxylic acids 1a and 1b via esterification to their corresponding esters 2a and 2b, and subsequent hydrazinolysis following the previously reported procedures [35,36]. Treatment of the carbohydrazides 3a and 3b with the corresponding haloaryl isothiocyanate by heating in ethanol yielded the intermediate N-aryl-2-(thiophene-2-carbonyl or 5-bromothiophene-2-carbonyl)hydrazine-1-carbothioamides 4a–e in almost quantitative yields. The target 4-haloaryl-5-(thiophen-2-yl or 5-bromothiophene-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones 5a–e were obtained in good overall yields by cyclization of compounds 4a–e via heating in 10% aqueous sodium hydroxide for two hours (Scheme 1,

Table 1. Crystallization solvents, melting points, yield percentages, molecular formulae and molecular weights of compounds 5a–e, 6a–e, 7a–d, 8, 9, 10a and 10b.

Compound No.	X	Y	R	Cryst. Solv.	M.p. (°C)	Yield (%)	Mol. Formula (Mol. Wt.)
5a	H	4-F	-	EtOH	176–178	72	C12H8FN3S2 (277.34)
5b	H	2-Br,4-F	-	EtOH	256–258	81	C12H7BrFN3S2 (356.23)
5c	Br	3-Cl	-	EtOH	187–189	75	C12H7BrClN3S2 (372.68)
5d	Br	4-Cl	-	EtOH	193–195	82	C12H7BrClN3S2 (372.68)
5e	Br	4-Br	-	EtOH	222–224	89	C12H7Br2N3S2 (417.14)
6a	H	CH2	-	EtOH/H2O	174–176	80	C18H19FN4S2 (374.50)
6b	H	O	-	EtOH/H2O	191–193	72	C17H17FN4OS2 (376.47)
6c	H	S	-	EtOH	185–187	76	C17H17FN4S3 (392.53)
6d	Br	O	-	EtOH/H2O	200–202	78	C17H16BrFN4OS2 (455.36)
6e	Br	S	-	EtOH	180–182	80	C17H16BrFN4S3 (471.43)
7a	H	-	CH3	EtOH/H2O	132–134	64	C18H20FN5S2 (389.51)
7b	H	-	C6H5	EtOH	174–175	82	C23H22FN5S2 (451.58)
7c	Br	-	C6H5	EtOH/H2O	188–190	86	C23H21BrFN5S2 (530.48)
7d	Br	-	2-CH3OC6H4	EtOH/H2O	192–194	90	C24H23BrFN5OS2 (560.50)
8	-	-	-	EtOH	169–171	75	C24H23FN4S2 (450.59)
9	-	-	-	EtOH	191–193	68	C22H19FN4S2 (422.54)
10a	-	-	CH3	EtOH	162–164	82	C20H17FN4S2 (396.50)
10b	-	-	CH2C6H5	EtOH	183–185	66	C26H21FN4S2 (472.60)

).

The ¹H NMR spectra of compounds 5a–e showed the NH protons as broad singlets at δ 11.45–11.98 ppm and their ¹³C NMR spectra showed the C=S carbons at δ 168.02–169.79 ppm, confirming the existence of these compounds as the thione tautomers A rather than the thiol tautomers B. Full details of ¹H NMR and ¹³C NMR spectral data of compounds 5a-e, which were in full agreement with their structures, shown in Section 3.2.

5-Substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones were reported to react with primary or secondary amines and formaldehyde to yield the corresponding 2-aminomethyl-2,4-dihydro-3H-1,2,4-triazole-3-thiones (N-Mannich bases) [37,38,39,40,41,42,43]. Thus, compounds 5a and 5b were reacted with piperidine, morpholine, thiomorpholine or 1-substituted piperazines and 37% formaldehyde solution in ethanol to yield the corresponding N-Mannich bases 6a–e and 7a–e in good yields. The N-Mannich bases 8, 9, 10a and 10b were similarly prepared via the reaction of compound 5a with 4-phenylpiperidine, 1,2,3,4-tetrahydroisoquinoline, N-methylaniline or N-benzylaniline and formaldehyde, respectively (Scheme 2, Table 1).

The common ¹H NMR spectral features of the N-Mannich bases 6a–e, 7a–e, 8, 9, 10a and 10b are characterized by the presence of methylene bridge protons (NCH₂N) as sharp peaks at δ 5.02–5.95 ppm. In addition, the ¹³C NMR spectra showed the methylene bridge carbons at δ 66.52–72.70 ppm. Meanwhile, the cyclic thione carbons were shown at δ 169.26–170.64 ppm. Full details of ¹H NMR and ¹³C NMR spectral data of compounds 6a–e, 7a–d, 8, 9, 10a and 10b, which were in full agreement with their structures, are shown in Section 3.3.

2.2. Single-Crystal XRD Study and Structural Insights

The crystallographic data and refinement parameters for compounds 5a and 5b are summarized in Table S1. X-ray analysis revealed that both compounds crystallize in the triclinic crystal system with the space group P-1. The ORTEP representation of compounds 5a and 5b is depicted in Figure 3a and Figure 3b, respectively. In compound 5a, the 1,2,4-triazole ring forms coplanarity with the mean plane of the thiophene ring (2.67°), while such a coplanarity is not observed in compound 5b (12.39°). The dihedral angle between the mean planes of the triazole ring and the substituted phenyl ring is 73.97° and the corresponding angle is found to be 87.71° in compound 5b. The structures of compounds 5a and 5b are overlaid with respect to the triazole ring, indicating a slight rotation around the thiophene and substituted phenyl ring (Figure 3c).

In the crystalline state, both compounds showed similar packing features. Molecules 5a and 5b are arranged in a ladder-like architecture, as shown in Figure 4. The ladder-like pattern is seen along the crystallographic bc plane in 5a, whereas a similar pattern is observed along the crystallographic ac plane in 5b (Figure 4a,b). The intermolecular interactions that stabilize the crystal structures of 5a and 5b are summarized in

Table 2. Intermolecular interaction geometry (Å, °) in compounds 5a and 5b.

D–H···A	D–H	H···A	D···A	∠D–H···A	Symmetry
Compound 5a
N2–H2A···S1	0.83 (3)	2.49 (3)	3.3049 (19)	170 (3)	1 − x, 2 − y, −z
C1–H1···N3	0.93	2.59	3.482 (3)	162	1 − x, 1 − y, −z
C2–H2···N3	0.93	2.63	3.446 (3)	147	−1 + x, y, z
C10–H10···F1	0.93	2.39	3.103 (3)	133	−x, 1 − y, 1 − z
C9–S2···S1			3.4775 (9)	142.66 (9)	−1 + x, 1 + y, z
Compound 5b
N2–H2A···S1	0.83 (4)	2.44 (4)	3.264 (2)	173 (4)	2 − x, 1 − y, −z
C5–H5···N3	0.95	2.59	3.397 (4)	143	1 − x. 1 − y, −z
C10–H10···F1	0.95	2.64	3.060 (4)	107	1 − x, 2 − y, 1 − z
C1–Br1···F1			3.305 (2)	157.63 (11)	x, −1 + y, z
C12–H12···C5	0.95	2.86	3.801 (2)	174	−1 + x, −1 + y, −z
C7–S2···S1			3.339 (1)	166.37 (11)	−1 + x, −1 + y, −z

. The former structure stabilizes with intermolecular N–H···S, C–H···N and C–H···F hydrogen bonds and a C–S···S chalcogen bond. The latter structure also stabilizes with the above interactions in addition to the C12–H12···C5 hydrogen bond and hetero halogen bond (Br···F). This halogen is established due to the presence of an additional organic Br substituent in 5b.

In compound 5a, the amine group of the triazole ring is involved in N–H···S hydrogen bonds with the thione group producing $R_2^2$(8) motif. The adjacent N–H···S hydrogen-bonded dimers are interconnected by C–S···S chalcogen bonds, involving the thiophene S atom acting as a donor (σ-hole) and the thione S atom acting an acceptor. The N–H···S hydrogen bond and C–S···S chalcogen bond generate a supramolecular sheet, as shown in Figure 4c. The same kind of supramolecular sheet is also formed in 5b, utilizing N–H···S hydrogen bond and C–S···S chalcogen bond. This supramolecular sheet is further supported by intermolecular C–H···C(π) interactions (involving thiophene and substituted phenyl rings) as shown in Figure 4d.

In addition to the above interactions, both compounds 5a and 5b also exhibit a dimer, which is formed by intermolecular C–H···N interaction with the graph set motif of $R_2^2$(12) (Figure 5a,b). In compound 5a, the adjacent dimers (mediated by C–H···N interaction) are further interlinked by an intermolecular C–H···N interaction. The triazole N3 atom is involved in three-centered interactions (Figure 5a) and these C–H···N interactions generate alternate $R_2^2$(12)-$R_4^2$(10)-$R_2^2$(12) motifs. In compound 5b, only $R_2^2$(12) motif is formed and it is not extended further (Figure 5b). It is of interest to note that the halogen bond is formed between the Br1 and F1 atoms. This interaction links the 5b molecules into a chain, as shown in Figure 5c.

The crystallographic data and refinement parameters for compounds 6a, 6d and 10a are presented in Table S2. The X-ray analysis revealed that compounds 6a, 6d and 10a crystallize in the monoclinic crystal system with the space group P2₁/c. In the asymmetric unit of 6a, there are two crystallographically independent molecules (molecules A and B), while one molecule is present in the asymmetric units of compounds 6d and 10a. In all three cases, the thiophene ring was disordered over two positions (~180° rotation).

In both the crystallographically independent molecules of compound 6a, the thiophene ring was disordered in two orientations with a refined occupancy ratio of 0.613 (4):0.387 (4) in molecule A. The corresponding occupancy ratio of 0.579 (4):0.421 (4) in molecule B. The ORTEP diagram shows the major and minor disordered components of molecules A and B of 6a (Figure 6).

The major disordered component was used for further analysis. The major disordered components of molecules A and B overlaid very well with the RMSD value of 0.07 Å. In molecule A, the thiophene ring is twisted with respect to the mean plane of the central triazole ring with a dihedral angle of 31.53° (29.52° in molecule B) and the observed twist is relatively large compared to compounds 5a and 5b. The dihedral angle between the mean planes of the triazole and the fluorophenyl rings is 70.24° (70.74° in molecule B). Furthermore, the piperidine ring exhibits a typical chair conformation. The mean plane formed by the piperidine ring and the central triazole ring makes an angle of 59.75° (62.58° in molecule B).

Figure 7a shows the columnar packing mode of compound 6a along the crystallographic bc plane. The intermolecular interactions (C–H···S/F/π interactions and a short S_(lp)···C_(π) contact) that stabilize the crystal structure of 6a are summarized in

Table 3. Intermolecular interaction geometry (Å, °) in compounds 6a, 6d and 10a. Cg1: centroid of the thiophene ring (major disordered component; molecule A of 6a. Cg2: centroid of the thiophene ring (major disordered component; molecule B of 6a, Cg3: centroid of the F-substituted phenyl ring.

D–H···A	D–H	H···A	D···A	∠D–H···A	Symmetry
Compound 6a
C12–H12···S2	0.95	2.83	3.748 (4)	163	1 + x, y, z
S1A···C2			3.374 (5)		1 + x, y, z
C8–H8···Cg1	0.95	2.70	3.565 (4)	1.52	−1 + x, y, z
C29–H29···S4	0.95	2.87	3.738 (4)	152	1 − x, 2 − y, −z
C34–H34A···F2	0.99	2.59	3.480 (3)	149	1 − x, 2 − y, −z
C27–H27···F1	0.95	2.45	3.347 (4)	158	1 + x, 1 + y, z
C26–H26···Cg2	0.95	2.60	3.479 (4)	155	−1 + x, 1 + y, z
Compound 6d
O1···C1			3.207 (6)		1 − x, 1 − y, 1 – z
C12–Br1···F1			3.209 (2)	146.19 (15)	−x, ½ + y, 3/2 – z
C9–H9···S1	0.93	2.96	3.736 (2)	142	x, ½ − y, −½ + z
C3A–H3A···F1	0.93	2.65	3.401 (2)	138	−1 + x, −1 + y, −z
C12–H12···C5	0.95	2.86	3.801 (2)	174	−1 + x, −1 + y, −z
C7–S2···S1			3.339 (1)	166.37 (11)	−1 + x, −1 + y, −z
Compound 10a
C2–S1···Cg3			3.8074 (9)	159.49 (6)	1 − x, 1 − y, −z
C3A–H3A···F1	0.95	2.52	3.247 (12)	133	−x, −y, −z
C17–H17···N2	0.95	2.71	3.637 (2)	165	1 − x, ½ + y, ½ − z
C5–S2A···C17			3.3782 (19)	166.92 (8)	1 − x, ½ + y, ½ − z
C11–H11···S1	0.95	2.97	3.853 (2)	156	1 − x, −y, −z
C14–H14A···F1	0.98	2.56	3.398 (2)	143	1 − x, −y, −z

Cg1: centroid of the thiophene ring (major disordered component; molecule A of 6a. Cg2: centroid of the thiophene ring (major disordered component; molecule B of 6a, Cg3: centroid of the F-substituted phenyl ring).

. In the solid state, molecule A and its counterparts related to symmetry generate a supramolecular chain by intermolecular C–H···S, C–H···π (involving thiophene ring π-center as an acceptor) interactions and a short S_(lp)···C_(π) contact (Figure 7b). A similar type of supramolecular chain and interactions formed between molecule B and its partners related to symmetry (Figure 7c). The intermolecular S_(lp)···C_(π) contact was also observed earlier in 1,2,4-triazole derivatives [44]. Furthermore, molecule B interacts with its symmetry equivalent molecule through intermolecular C–H···S and C–H···F interactions to produce a molecular dimer (Figure 7d). Molecular A and molecule B interact via intermolecular C–H···F interactions, as shown in Figure 7e.

In compound 6d, the thiophene ring was disordered in two orientations with a refined occupancy ratio of 0.667 (5):0.333 (5). The ORTEP diagram shows the major and minor disordered components of 6d (Figure 8). The major disordered component was used for further analysis. The morpholine ring adopts a typical chair conformation. The thiophene and the central triazole ring are oriented at an angle of 17.92°. The disubstituted phenyl ring is inclined at an angle of 83.58° with respect to the mean plane of the triazole ring. The corresponding angle is 72.31° between the mean planes of the triazole and morpholine rings. As shown in Figure 8c, 6d molecules were columnarly packed along the crystallographic ac plane with an interesting packing feature. In each column, the morpholine rings come closer to each other. The disubstituted phenyl rings are closer together between two adjacent columns. The intermolecular interactions that stabilize the crystal structure of 6d are summarized in Table 3. In the solid state, centrosymmetrically related molecules form a molecular dimer stabilized by O_(lp)···C_(π) contacts (involving morpholine ring and triazole rings) (Figure 8d). This structure also exhibits a halogen bond involving Br and F atoms that link the molecules into a supramolecular chain (Figure 8e). In addition to these interactions, the intermolecular C–H···S/F interactions generate a molecular dimer of 6d, as shown in Figure 8f.

In compound 10a also, the thiophene ring was disordered over two orientations with a refined occupancy ratio of 0.634 (2):0.366 (2). The ORTEP diagram shows the major and minor disordered components of 10d (Figure 9). The major disordered component was used for further analysis. The dihedral angle between the mean planes of the thiophene and the central triazole rings is 27.17°. The triazole ring makes an angle of 79.50° and 64.86° with respect to the mean plane of the fluorophenyl and phenyl rings, respectively. Figure 9a shows the columnar packing mode of compound 10a along the crystallographic ac plane. The intermolecular interactions that stabilize the crystal structure of 10a are summarized in Table 3.

In the crystalline state of compound 10a, molecules related by center of inversion (−x, −y, −z) form a dimer through intermolecular C–H···F interactions (involving the thiophene and the F-substituted rings). In addition, a molecular dimer is formed by intermolecular C–H···N interaction and a chalcogen bond between the S atom of the thiophene ring and one of the C atoms of the phenyl ring. A similar type (C–S···π) of chalcogen bond was observed in the 1,2,4-triazolo [3,4-b][1,3,4]thiadiazole derivative [45]. As shown in Figure 10a, this dimer and the former center of the inversion-related dimer are alternately linked, generating a supramolecular chain. Further, there are other two dimers also observed in 10a, which help to stabilize the crystal structure. One of the dimers stabilizes with chalcogen bonds involving the thione S atom and centroid of the F-substituted phenyl ring (Figure 10b) and other dimer stabilizes with C–H···S/F interactions in which methyl and F-substituted phenyl ring act as a donor for these interactions (Figure 10c).

2.3. In Vitro Antimicrobial Activity

The in vitro antibacterial and antifungal activities of the newly synthesized compounds 5a–e, 6a–e, 7a–d, 8, 9, 10a and 10b were evaluated against a panel of standard pathogenic bacterial and fungal strains of the Institute of Fermentation of Osaka (IFO), namely Staphylococcus aureus IFO 3060, Bacillus subtilis IFO 3007 and Micrococcus luteus IFO 3232 (Gram-positive bacteria); Escherichia coli IFO 3301 and Pseudomonas aeruginosa IFO 3448 (Gram-negative bacteria); and the pathogenic fungi Candida albicans IFO 0583, Aspergillus oryzae IFO 4177 and Aspergillus niger IFO 4414. The initial screening was carried out using the semi-quantitative agar disk-diffusion method using the Mueller–Hinton agar medium [46]. The results of the initial antimicrobial screening of compounds 5a–e, 6a–e, 7a–e, 8, 9, 10a and 10b (200 μg/disc); the antibacterial antibiotics Ampicillin trihydrate and Ciprofloxacin; and the antifungal drug Fluconazole (100 μg/disc) are shown in

Table 4. In vitro activity of compounds 5a–e, 6a–e, 7a–d, 8, 9, 10a and 10b (200 μg/8 mm disc); the broad-spectrum antibacterial drugs Ampicillin trihydrate and Ciprofloxacin and the antifungal drug Fluconazole (100 μg/8 mm disc) against Staphylococcus aureus IFO 3060 (SA), Bacillus subtilis IFO 3007 (BS) and Micrococcus luteus IFO 3232 (ML); the Gram-negative bacterial strains Escherichia coli IFO 3301 (EC) and Pseudomonas aeuroginosa IFO 3448 (PA) and the standard fungi Candida albicans IFO 0583 (CA), Aspergillus oryzae IFO 4177 (AO) and Aspergillus niger IFO 4414 (AN).

Compound	Diameter of Inhibition Zone (mm) a
	X	Y	R	SA	BS	ML	EC	PA	CA	AO	AN
5a	H	4-F	-	24	32	-	18	11	-	-	-
5b	H	2-Br,4-F	-	15	28	10	20	15	-	-	-
5c	Br	3-Cl	-	12	11	-	-	-	-	-	-
5d	Br	4-Cl	-	14	14	-	-	-	-	-	-
5e	Br	4-Br	-	28	34	15	18	-	-	-	-
6a	H	CH2	-	18	20	-	18	-	12	-	-
6b	H	O	-	28	25	-	20	12	-	-	-
6c	H	S	-	21	18	-	14	-	-	-	-
6d	Br	O	-	28	34	15	18	-	-	-	-
6e	Br	S	-	26	32	17	22	-	-	-	-
7a	H	-	CH3	35	27	-	22	16	12	-	-
7b	H	-	C6H5	20	22	-	17	-	-	-	-
7c	Br	-	C6H5	20	22	15	-	-	-	-	-
7d	Br	-	2-CH3OC6H4	30	36	20	18	17	-	-	-
8	-	-	-	18	22	-	18	-	-	-	-
9	-	-	-	22	28	-	16	-	-	-	-
10a	-	-	CH3	20	24	-	-	-	-	-	-
10b	-	-	CH2C6H5	16	20	-	15	-	-	-	-
Ampicillin trihydrate				28	30	25	24	22	NT	NT	NT
Ciprofloxacin				34	38	32	38	36	NT	NT	NT
Fluconazole				NT	NT	NT	NT	NT	21	22	24

^a (-): Inactive (inhibition zone < 10 mm), (NT): Not tested.

.

The results showed variable grades of inhibition against the tested microorganisms. In general, marked antibacterial activity was shown by compounds 5a, 5b, 5e, 5f, 6a–e, 7a–d, 8, 9, 10a and 10b, which showed growth inhibition zones ≥ 18 mm, particularly against the tested Gram-positive bacteria S. aureus and B. subtilis. Meanwhile, marked inhibitory activity was displayed by compounds 5a, 5b, 5e, 6a, 6d, 6e, 6d, 6e, 7a, 7d and 8 against the Gram-negative bacteria E. coli with lower activity against P. aeuroginosa. All the tested compounds were found to be almost inactive against all the tested fungal strains.

In the 4-aryl-5-(thiophen-2-yl or 5-bromothiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones series 5a–e, it was noticed that replacement of the 4-fluorophenyl, 2-bromo-4-fluorophenyl and 4-bromophenyl substituents at position 4 of the core triazole ring with 3- or 4-chlorophenyl moieties (compounds 5c and 5d) greatly deteriorated the antibacterial activity. In addition, an additional bromine atom on the thiophene ring (compounds 5c–e) did not influence the potency and spectrum of the antibacterial activity.

The antibacterial activity of the 2-aminomethy derivatives 6a–e, 7a–d, 8, 9, 10a and 10b (N-Mannich bases) was generally superior to their precursors 5a and 5b. In the piperidinomethyl, morpholinomethyl and thiomorpholinomethyl derivatives 6a–e, it was observed that replacement of the 4-fluorophenyl substituent at position 4 of the core triazole ring with 2-bromo-4-fluorophenyl enhanced activity against M. luteus. Potent and broad-spectrum antibacterial activity was attained in the piperzinomethyl derivatives 7a–d. As noticed in compounds 6a–e, replacement of the 4-fluorophenyl substituent at position 4 of the core triazole ring with the 2-bromo-4-fluorophenyl moiety in compounds 7a–d enhanced activity against M. luteus. The optimum antibacterial activity was shown by the 4-methylpiperazino and the 4-(2-methoxyphenyl)piperazino analogs 7a and 7d, which exhibited potent antibacterial activity against S. aureus, B. subtilis and E. coli and retained moderate activity against Pseudomonas aeuroginosa (growth inhibition zones 14–17 mm). The antibacterial activity of the 4-phenylpiperazino derivative 8 and its annulated derivative 9 was almost similar, with potent activity against S. aureus, B. subtilis and E. coli. The N-methylanilino derivative 10a displayed potent activity against the Gram-positive bacteria S. aureus and B. subtilis. Meanwhile, the activity of the N-benzylanilino analog 10b was slightly altered against the Gram-positive bacteria with moderate potency (growth inhibition zones 10–13 mm) against Gram-negative bacteria E. coli.

The minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) for the active compound against the same microorganism used in the primary screening were carried out using the microdilution susceptibility method in Mueller–Hinton Broth and Sabouraud Liquid Medium [47,48]. The MIC and MBC values of compounds 5a, 5b, 5e, 6b–e, 7a–d, 8, 9 and 10a (which showed inhibition zones > 20 mm) and the antibacterial antibiotics Ampicillin trihydrate and Ciprofloxacin (

Table 5. The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of compounds 5a, 5b, 5e, 6b–e, 7a–d, 8, 9 and 10a in comparison with the broad-spectrum antibacterial drugs Ampicillin trihydrate and Ciprofloxacin against the Gram-positive bacteria Staphylococcus aureus IFO 3060 (SA) and Bacillus subtilis IFO 3007 (BS) and the Gram-negative bacterial strain Escherichia coli IFO 3301 (EC).

Comp. No.	MIC/MBC (μg/mL)
	SA	BS	EC
5a	4.0/8.5	1.0/1.8	ND
5b	ND	3.0/4.8	ND
5e	3.2/5.4	3.5/5.2	ND
6b	3.8/7.2	1.2/2.5	ND
6c	5.0/8.6	ND	ND
6d	2.0/3.8	3.4/6.2	ND
6e	4.8/8.2	3.4/5.2	24.0/68.2
7a	3.2/6.0	4.2/7.2	22.4/78.0
7b	ND	16.0/48.0	ND
7c	ND	18.5/64.0	ND
7d	2.1/3.8	1.0/2.1	ND
8	ND	18.2/32.0	ND
9	18.0/36.2	8.2/24.0	ND
10a	ND	12.4/22.6	ND
Ampicillin trihydrate	2.0/3.5	1.0/3.0	ND
Ciprofloxacin	0.75/1.5	0.5/1.0	0.25/1.0

ND: Not determined.

) were consistent with the results of the initial screening.

Antimicrobial standards state that an agent is typically categorized as fungicidal or bactericidal if the MIC/MBC ratio is less than 4 [49]. The MIC/MBC ratio for all the active compounds 5a, 5b, 5e, 6b–e, 7a–d, 8, 9 and 10a were found to be less than 4. Accordingly, these compounds are considered potential antibacterial candidates for further studies.

2.4. In Vitro Anti-Proliferative Activity

Compounds 5a–e, 6a–e, 7a–e, 8, 9, 10a and 10b were tested for in vitro anti-proliferative activity against four human cancer cell lines, namely hepatocellular carcinoma (HePG-2), breast cancer (MCF-7), prostate cancer (PC-3) and colorectal cancer (HCT-116), by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [50,51].

Table 6. In vitro anti-proliferative activity of the tested compounds 5a–e, 6a–e, 7a–e, 8, 9, 10a and 10b and Doxorubicin expressed as IC₅₀ values against HePG-2, MCF-7, PC-3 and HCT-116 cancer cell lines.

Comp. No.	IC50 (µM) 1
	HePG-2	MCF-7	PC-3	HCT-116
5a	42.23 ± 4.0	80.22 ± 10.0	68.82 ± 4.8	84.11 ± 729
5b	49.20 ± 3.4	>100	88.82 ± 4.0	>100
5c	69.0 ± 7.2	>100	>100	>100
5d	92.02 ± 10.0	>100	>100	>100
5e	19.24 ± 1.4	30.30 ± 3.6	52.66 ± 4.1	46.10 ± 2.2
6a	22.02 ± 1.4	50.24 ± 4.4	46.50 ± 4.8	86.11 ± 5.8
6b	10.12 ± 1.1	28.16 ± 2.2	33.42 ± 2.5	68.06 ± 3.6
6c	12.66 ± 1.3	24.20 ± 2.6	28.18 ± 1.5	54.20 ± 3.9
6d	6.80 ± 0.1	9.58 ± 6.0	28.56 ± 2.1	38.18 ± 2.9
6e	8.62 ± 0.6	29.56 ± 3.6	44.20 ± 3.9	69.28 ± 5.4
7a	12.20 ± 0.16	19.20 ± 1.6	40.60 ± 3.3	56.44 ± 3.6
7b	14.42 ± 0.18	16.68 ± 1.5	52.28 ± 4.3	44.0 ± 2.8
7c	8.86 ± 0.1	28.88 ± 3.2	28.88 ± 1.6	39.14 ± 2.6
7d	6.60 ± 0.4	6.40 ± 1.8	27.06 ± 1.9	28.82 ± 1.4
8	54.06 ± 4.4	89.16 ± 7.4	>100	>100
9	79.16 ± 3.6	>100	>100	>100
10a	20.02 ± 1.6	32.18 ± 4.0	56.4 ± 3.5	>100
10b	66.04 ± 5.2	>100	>100	>100
Doxorubicin	4.50 ± 0.2	4.17 ± 0.2	8.87 ± 0.6	5.23 ± 0.3

¹ IC₅₀ values presented as the mean ± SD of three separate determinations.

presents the results of the anti-proliferative activity of compounds 5a–e, 6a–e, 7a–e, 8, 9, 10a and 10b as well as the anticancer drug Doxorubicin [52].

As indicated by the anti-proliferative activity results, the tested compounds displayed variable degrees of activity against the tested cancer cell lines. Generally speaking, the compounds exhibited potent anti-proliferative activity against HePG-2 and MCF-7. In addition, the N-Mannich bases 6a–e, 7a–e, 8, 9, 10a and 10b had higher activity than their precursors 5a and 5b. Compounds 5e, 6a–e, 7a–d and 10a exhibited potent activity with IC₅₀ < 25 μM.

Within the 5-substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones 5a–e, the optimal activity was attained by compound 5e, which showed potent activity against HePG-2 and retained moderate activity (IC₅₀ 26–50 μM) or weak activity (IC₅₀ 51–100 μM) against MCF-7, PC-3 and HCT-116 cell lines.

In the 2-aminomethyl derivatives 6a–e, 7a–d, 8, 9, 10a and 10b, the anti-proliferative activity was mainly dependent on the nature of the haloaryl substituents at position 4 of the core triazole nucleus and the peripheral amino moieties. The anti-proliferative activity of the 4-(2-bromo-4-fluorophenyl) analogs (6d, 6e, 7c and 7d) was higher than the 4-(4-fluorophenyl) analogs (6a–c, 7a and 7b). Regarding the peripheral amino moieties, the anti-proliferative activity of the piperidine, morpholine and piperazine derivatives (6a–e and 7a–d) was generally higher than their 4-phenylpiperidine 8, tetrahydroisoquinoline 9, N-methylaniline 10a and N-benzylaniline 10b analogs. However, N-methylaniline 10a retained good activity against HePG-2, moderate activity against MCF-7, weak activity against PC-3 and lacked activity against HCT-116 cell lines.

2.5. Molecular Docking Analysis

To corroborate the in vitro antibacterial activity of the most active compounds (5e, 6b, 6d and 7d), we performed a molecular docking simulation to predict the favorable pose of these compounds at the active site of the DNA gyrase subunit B of Staphylococcus aureus. The CB-Dock2 program combines cavity detection and molecular docking with Autodock vina for the given protein target [53,54,55]. In this study, the 3D structure of DNA gyrase subunit B (PDB ID: 3G75) from Staphylococcus aureus was retrieved from the protein data bank. The co-crystallized ligand (ligand ID: B38) was used as a control to assess the binding affinity of select title compounds. Figure 11 shows the predicted pose of these compounds and overlaps with the position of the control inhibitor B38 (4-methyl-5-[3-(methylsulfanyl)-1H-pyrazol-5-yl]-2-thiophen-2-yl-1,3-thiazole). The docking score of these compounds and the co-crystallized inhibitor B38 is summarized in

Table 7. Docking score of compounds 5e, 6b, 6d, 7a, 7d and co-crystallized ligand B38.

Compound	Docking Score (kcal mol−1)
5e	−6.5
6b	−5.7
6d	−6.0
7a	−6.7
7d	−7.8
B38	−6.4

. The result suggests that compound 7d showed a relatively better affinity for the target DNA gyrase subunit B compared to our other compounds and the control inhibitor. The intermolecular interactions between protein–ligand complexes were analyzed using the predicted poses with the PLIP web server [56].

Compound 5e establishes four hydrophobic interactions and one hydrogen bond. Residues Asn 54, Ile 86 and Ile 175 are involved in hydrophobic interactions. The backbone O atom and the triazole N atom are engaged in a hydrogen bond. It is noted that in the remaining compounds (6b, 6d, 7a and 7d), the active site residues Asn 54 are involved in a hydrogen bond with the ligands, mostly with the bulky substitution region introduced in the triazole nucleus. The residues Glu 50 and Val 131 are involved in hydrophobic contacts with 6b. Similarly, residues Glu 58 and Pro 87 are hydrophobic, whereas the side chain O atom of Asp 57 acts as an acceptor for a halogen bond with the Br atom in 6d. In 7a, the side chain of Asp 81 (O atom) establishes a short contact with F of 7a. Furthermore, Ile 86 and Asn 54 also participate in hydrophobic interactions. As seen in Figure 11f, there is a relatively greater number of interactions between 7d and the active site residues; therefore, the affinity is slightly stronger compared to other compounds. In this complex, the methoxy group is involved in a hydrogen bond with the side chain of Asn 54. The other residues are involved in hydrophobic contacts.

We also explored the anti-proliferative potential of compounds 6d, 6e and 7d against one of the cancer targets, namely cyclin-dependent kinase 2 (CDK2), a class of cell cycle regulators implicated in multiple cancers [57]. The 3D structure of human CDK2 was retrieved from the protein data bank with accession ID: 8OY2. This protein was complexed with an inhibitor molecule (ligand id: W5W; (1S,2S,11aS)-1-methoxy-1,4,7,10-tetramethyl-2,9-bis(oxidanyl)-2,11a-dihydrobenzo[b][1,4]benzodioxepine-3,6-dione. The molecular docking simulation correctly identified the active site and placed the ligand at the active site. The experimental conformation of W5W and the predicted pose of this molecule overlap very well, indicating the effectiveness of the program. The docking score revealed that compounds 6d, 6e and 7d showed a relatively better binding affinity than the control inhibitor W5W (

Table 8. Docking score of compounds 6d, 6e and 7d and co-crystallized ligand W5W.

Compound	Docking Score (kcal mol−1)
6d	−7.6
6e	−7.3
7d	−7.6
W5W	−5.6

). The predicted pose of these molecules was used to analyze the intermolecular interactions formed between active site residues and the ligand molecules (Figure 12).

Compound 6d interacts with the active site residues via hydrophobic (Asn 132 and Val 18) interactions and a salt bridge between the N atom of the morpholine and carboxylate of the Asp 86. The same set of residues is also involved in interactions with compound 6e. Relatively, compound 7d establishes a greater number of contacts with the active site residues. Residues Leu 134, Ala 31, Ile 10, Val 18, Ala 149 and Asp 127 are involved in hydrophobic interactions, while Asp 145 participates in salt bridge interaction with one of the N atoms of the piperazine.

Taken as a whole, compound 7d exhibits both antibacterial and anti-proliferative potentials, as revealed by in vitro and in silico studies.

3. Materials and Methods

3.1. General Information

Melting points (°C, uncorrected) were determined in open-glass capillaries using a Stuart SMP30 electro–thermal melting point apparatus (Nottingham, UK). Nuclear magnetic resonance (NMR) spectra were determined in CDCl₃ on Bruker RMN AV600 and Bruker Avance III HD FT-high resolution NMR instruments (Billerica, MA, USA) at δ 600.15 MHz for ¹H and 150.36 MHz for ¹³C, δ 400.20 MHz for ¹H and 100.64 MHz for ¹³C, respectively. Elemental analyses (C, H, N and S) were in agreement with the proposed structures within ±0.3% of the theoretical values (Table S3). Monitoring of the reactions and checking of the purity of the final products was carried out with thin layer chromatography (TLC) using silica gel-precoated aluminum sheets 60 F₂₅₄ (Merck, Darmstadt, Germany) and visualization with ultraviolet light (UV) at 365 and 254 nm. All chemicals and solvents were purchased from commercial suppliers and used without additional purification. The reference drugs Ampicillin trihydrate (CAS # 7177-48-2) and Ciprofloxacin (CAS # 85721-33-1), Fluconazole (CAS # 86386-73-4) and Doxorubicin (CAS 23214-92-8) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). The synthesis of compound 5a was previously reported via the reaction of ethyl thiophene-2-carboxylate with thiosemicarbazide in anhydrous methanol in the presence of sodium methylate followed by heating for 5 min [58].

3.2. General Procedure for the Synthesis of 4-Haloaryl-5-(thiophen-2-yl or 5-Bromothiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones 5a–e

The appropriate haloaryl isothiocyanate (0.01 mole) was added to a solution of thiophene-2-carbohydrazide 3a or 5-bromothiophene-2-carbohydrazide 3b (0.01 mole) in ethanol (15 mL) and the mixture was heated under reflux with stirring for 1 h. The solvent was distilled off in vacuo to yield the intermediates N-aryl-2-(thiophene-2-carbonyl or 5-bromothiophene-2-carbonyl)hydrazine-1-carbothioamides 4a–e in almost quantitative yields. Aqueous sodium hydroxide solution (10%, 15 mL) was added to compounds 4a–e and the mixture was heated under reflux for 2 h and then filtered hot. On cooling, the mixture was acidified with hydrochloric acid and the precipitated crude product was filtered, washed with water, dried and crystallized from ethanol to yield the target products 5a–e.

4-(4-Fluorophenyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 5a. ¹H NMR (600.15 MHz): δ 6.86 (d, 1H, Thiophene-H, J = 3.9 Hz), 6.94 (t, 1H, Thiophene-H, J = 3.9 Hz), 7.26–7.40 (m, 5H, Ar-H and Thiophene-H), 11.45 (br. s, 1H, NH). ¹³C NMR (150.91 MHz): δ 117.22, 126.38, 127.66, 129.12, 129.84, 130.77, 144.41, 147.05, 162.71 (Ar-C, Thiophene-C and Triazole-C5), 169.78 (C=S).

4-(2-Bromo-4-fluorophenyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 5b. ¹H NMR (400.20 MHz): δ 6.90 (d, 1H, Thiophene-H, J = 4.0 Hz), 6.96 (t, 1H, Thiophene-H, J = 4.0 Hz), 7.28–7.44 (m, 4H, Ar-H and Thiophene-H), 11.65 (br. s, 1H, NH). ¹³C NMR (100.64 MHz): δ 116.94, 122.03, 124.68, 128.05, 129.78, 132.58, 135.38, 143.95, 149.16, 162.37, 164.36 (Ar-C, Thiophene-C and Triazole-C5), 169.79 (C=S).

5-(5-Bromothiophen-2-yl)-4-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 5c. ¹H NMR (400.20 MHz): δ 7.08 (d, 1H, Thiophene-H, J = 4.0 Hz), 7.10–7.22 (m, 4H, Ar-H and Thiophene-H), 7.38 (s, 1H, Ar-H), 11.68 (br. s, 1H, NH). ¹³C NMR (100.64 MHz): δ 116.20, 126.0, 127.22, 128.06, 129.36, 130.12, 130.84, 131.88, 133.0, 135.22, 138.42 (Ar-C, Thiophene-C and Triazole-C5), 168.62 (C=S).

5-(5-Bromothiophen-2-yl)-4-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 5d. ¹H NMR (400.20 MHz): δ 7.01 (d, 1H, Thiophene-H, J = 3.9 Hz), 7.08–7.36 (m, 5H, Ar-H and Thiophene-H), 11.80 (br. s, 1H, NH). ¹³C NMR (100.64 MHz): δ 115.98, 126.68, 127.0, 128.02, 129.24, 133.10, 136.66, 139.58 (Ar-C, Thiophene-C and Triazole-C5), 168.44 (C=S).

4-(4-Bromophenyl)-5-(5-bromothiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 5e. ¹H NMR (400.20 MHz): δ 7.33–7.42 (m, 2H, Ar-H and Thiophene-H), 7.04 (d, 1H, Thiophene-H, J = 4.0 Hz), 7.32 (d, 2H, Ar-H), 7.35–7.37 (m, 3H, Ar-H and Thiophene-H), 11.98 (br. s, 1H, NH). ¹³C NMR (100.63 MHz): δ 114.98, 123.68, 127.0, 128.02, 129.24, 133.10, 135.12, 136.66, 139.58 (Ar-C, Thiophene-C and Triazole-C5), 168.02 (C=S).

3.3. General Procedure for the Synthesis of 2-Aminomethyl-4-aryl-5-(thiophen-2-yl or 5-Bromothiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thiones 6a–e, 7a–d, 8, 9, 10a and 10b

The appropriate secondary amine piperidine, morpholine, thiomorpholine, 1-methylpiperazine, 1-phenylpiperazine, 1-(2-methoxyphenyl)lpiperazine, 4-phenylpiperidine, 1,2,3,4-tetrahydroisoquinoline, N-methylaniline or N-benzylaniline (0.01 mole) and 37% formaldehyde solution (1.0 mL) were added to a hot solution of 4-(4-fluorophenyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 5a or 4-(2-bromo-4-fluorophenyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 5b (0.01 mole) in ethanol (15 mL) and the mixture was heated under reflux for 10 min then stirred at room temperature for 5 h and allowed to stand overnight. The precipitated crude products were filtered, washed with water, dried and crystallized from ethanol or aqueous ethanol.

4-(4-Fluorophenyl)-2-(piperidin-1-ylmethyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 6a. ¹H NMR (600.15 MHz): δ 1.41–1.44 (m, 2H, Piperidine-CH₂), 1.59–1.63 (m, 4H, Piperidine-CH₂), 2.85 (t, 4H, Piperidine-CH₂, J = 5.4 Hz), 5.88 (s, 2H, NCH₂N), 6.88 (d, 1H, Thiophene-H, J = 2.0 Hz), 6.93 (t, 1H, Thiophene-H, J = 2.0 Hz), 7.25–7.27 (m, 2H, Ar-H), 7.36–7.38 (m, 3H, Ar-H and Thiophene-H). ¹³C NMR (150.36 MHz): δ 23.80, 25.99, 51.87 (Piperidine-C), 71.04 (NCH₂N), 117.09, 126.71, 127.58, 129.0, 130.78, 130.89, 130.94, 144.82, 162.59, 164.26 (Ar-C, Thiophene-C and Triazole-C5), 170.38 (C=S).

4-(4-Fluorophenyl)-2-(morpholinomethyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 6b. ¹H NMR (400.20 MHz): δ 2.68 (t, 4H, Morpholine-CH₂, J = 5.0 Hz), 3.18 (t, 4H, Morpholine-CH₂, J = 5.0 Hz), 5.20 (s, 2H, NCH₂N), 6.90–6.98 (m, 2H, Ar-H and Thiophene-H), 7.16–7.34 (m, 2H, Ar-H and Thiophene-H), 7.39–7.39 (m, 3H, Ar-H and Thiophene-H). ¹³C NMR (100.46 MHz): δ 50.70, 71.19 (Morpholine-C), 71.0 (NCH₂N), 116.42, 121.52, 127.73, 128.60, 129.22, 132.20, 132.84, 144.58, 162.14, 164.20 (Ar-C, Thiophene-C and Triazole-C5), 169.64 (C=S).

4-(4-Fluorophenyl)-2-(thiomorpholinomethyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 6c. ¹H NMR (400.20 MHz): δ 2.70 (t, 4H, Thiomorpholine-CH₂, J = 4.0 Hz), 3.18 (t, 4H, Morpholine-CH₂, J = 4.0 Hz), 5.21 (s, 2H, NCH₂N), 6.84 (d, 1H, Thiophene-H, J = 4.0 Hz), 6.92 (t, 1H, Thiophene-H, J = 4.0 Hz), 7.24–7.38 (m, 5H, Ar-H and Thiophene-H). ¹³C NMR (100.64 MHz): δ 28.05, 52.84 (Thiomorpholine-C), 71.39 (NCH₂N), 117.22, 126.48, 127.73, 129.20, 130.87, 130.96, 145.10, 162.24. 164.74 (Ar-C, Thiophene-C and Triazole-C5), 170.32 (C=S).

4-(2-Bromo-4-fluorophenyl)-2-(morpholinomethyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 6d. ¹H NMR (600.15 MHz): δ 2.87–97 (m, 4H, Morpholine-CH₂), 3.72 (t, 4H, Morpholine-CH₂, J = 4.0 Hz), 5.34 (d, 2H, NCH₂N, J = 6.0 Hz), 6.94–6.98 (m, 2H, Ar-H and Thiophene-H), 7.27–7.30 (m, 2H, Ar-H and Thiophene-H), 7.39–7.55 (m, 2H, Ar-H and Thiophene-H). ¹³C NMR (150.36 MHz): δ 50.67, 66.93 (Morpholine-C), 69.80 (NCH₂N), 116.47, 121.57, 124.74, 126.19, 127.77, 128.66, 129.26, 130.29, 144.63, 162.37, 164.07 (Ar-C, Thiophene-C and Triazole-C5), 169.98 (C=S).

4-(2-Bromo-4-fluorophenyl)-2-(thiomorpholinomethyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 6e. ¹H NMR (400.20 Hz): δ 2.64 (t, 4H, Thiomorpholine-CH₂, J = 4.7 Hz), 3.11–3.21 (m, 4H, Thiomorpholine-CH₂), 5.26 (d, 2H, NCH₂N, J = 6.2 Hz), 6.90–6.94 (m, 2H, Ar-H and Thiophene-H), 7.22–7.26 (m, 1H, Ar-H), 7.35 (d, 1H, Thiophene-H, J = 4.5 Hz), 7.36–51 (m, 2H, Ar-H). ¹³C NMR (100.64 Hz): δ 28.75, 52.86 (Thiophene-C), 71.35 (NCH₂N), 116.86, 121.68, 124.85, 127.90, 128.76, 129.39, 132.37, 132.45, 144.74, 162.31, 164.35 (Ar-C, Thiophene-C and Triazole-C5), 169.80 (C=S).

4-(4-Fluorophenyl)-2-[(4-methylpiperazin-1-yl)methyl]-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 7a. ¹H NMR (400.20 MHz): δ 1.76–1.91 (m, 7H, CH₃ and Piperazine-CH₂), 2.44–2.67 (m, 4H, Piperazine-CH₂), 5.30 (s, 2H, NCH₂N), 6.89 (d, 1H, Thiophene-H, J = 4.0 Hz), 6.95 (t, 1H, Thiophene-H, J = 4.0), 7.20–7.43 (m, 5H, Ar-H and Thiophene-H). ¹³C NMR (100.64 MHz): δ 33.53, 51.75 (Piperazine-C), 42.09 (CH₃), 70.66 (NCH₂N), 117.25, 126.30, 127.77, 128.56, 129.20, 130.97, 146.32, 162.30, 164.81 (Ar-C, Thiophene-C and Triazole-C5), 170.51 (C=S).

4-(4-Fluorophenyl)-2-[(4-phenylpiperazin-1-yl)methyl]-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 7b. ¹H NMR (400.20 Hz): δ 3.12 (s, 4H, Piperazine-CH₂), 3.28 (t, 4H, Piperazine-CH₂, J = 5.2 Hz), 5.30 (s, 2H, NCH₂N), 6.84 (d, 1H, Thiophene-H, J = 3.6 Hz), 6.87–6.93 (m, 2H, Ar-H), 6.96 (d, 1H, Thiophene-H, J = 4.0 Hz), 7.23–7.26 (m, 5H, Ar-H and Thiophene-H), 7.33–7.37 (m, 3H, Ar-H). ¹³C NMR (100.63 Hz): δ 49.68, 50.55 (Piperazine-C), 69.82 (NCH₂N), 116.67, 117.30, 126.52, 127.78, 129.30, 129.34, 130.77, 130.80, 130.94, 131.03, 145.18, 162.32, 164.83 (Ar-C, Thiophene-C and Triazole-C5), 170.64 (C=S).

4-(2-Bromo-4-fluorophenyl)-2-[(4-phenylpiperazin-1-yl)methyl]-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 7c. ¹H NMR (400.20 Hz): δ 2.36–2.42 (m, 4H, Piperazine-CH₂), 3.28–3.36 (m, 4H, Piperazine-CH₂), 5.20 (d, 2H, NCH₂N, J = 6.2 Hz), 6.90 (d, 1H, Thiophene-H, J = 3.8 Hz), 6.98–7.44 (m, 6H, Ar-H andThiophene-H), 7.47–7.67 (m, 4H, Ar-H and Thiophene-H). ¹³C NMR (100.63 Hz): δ 50.08, 52.40 (Piperazine-C), 72.70 (NCH₂N), 114.02, 116.46, 120.68, 122.0, 124.48, 126.98, 127.50, 128.60, 130.98, 137.46, 145.22, 148.0, 162.90, 164.62 (Ar-C, Thiophene-C and Triazole-C5), 169.88 (C=S).

4-(2-Bromo-4-fluorophenyl)-2-{[(4-(2-methoxyphenyl)piperazin-1-yl]methyl}-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 7d. ¹H NMR (400.20 MHz): δ 2.86 (t, 4H, Piperazine-CH₂, J = 4.8 Hz), 3.20 (t, 4H, Piperazine-CH₂, J = 4.8 Hz), 3.96 (s, 3H, OCH₃), 5.02 (d, 2H, NCH₂N, J = 6.0 Hz), 6.89–7.13 (m, 5H, Ar-H and Thiophene-H), 7.23–7.39 (m, 3H, Ar-H and Thiophene-H), 7.40–7.55 (m, 2H, Ar-H and Thiophene-H). ¹³C NMR (100.63 MHz): δ 51.86, 52.44 (Piperazine-C), 54.20 (OCH₃), 70.28 (NCH₂N), 114.04, 115.84, 121.10, 121.86, 123.0, 124.02, 124.98, 126.42, 128.46, 129.86, 130.46, 136.82, 139.90, 140.96, 148.20, 161.46, 163.02 (Ar-C, Thiophene-C and Triazole-C5), 169.40 (C=S).

4-(4-Fluorophenyl)-2-[(4-phenylpiperidin-1-yl)methyl]-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 8. ¹H NMR (400.20 MHz): δ 1.76–1.91 (m, 5H, Piperidine-H), 2.67–2.74 (m, 2H, Piperidine-H), 3.36–3.41 (m, 2H, Piperidine-H), 5.30 (s, 2H, NCH₂N), 6.89 (d, 1H, Thiophene-H, J = 4.0 Hz), 6.95 (t, 1H, Thiophene-H, J = 4.0 Hz), 7.20–7.40 (m, 10H, Ar-H and Thiophene-H). ¹³C NMR (100.64 MHz): δ 33.53, 42.10, 51.76 (Piperidine-C), 70.66 (NCH₂N), 117.25, 126.30, 126.94, 127.77, 128.56, 129.20, 129.24, 130.97, 131.06, 145.07, 146.32, 162.30, 164.81 (Ar-C, Thiophene-C and Triazole-C5), 170.51 (C=S).

2-[(3,4-Dihydroisoquinolin-2(1H)-yl)methyl]-4-(4-fluorophenyl)-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 9. ¹H NMR (400.20 Hz): δ 2.96 (t, 2H, Isoquinoline-CH₂, J = 6.0 Hz), 3.27 (t, 2H, Isoquinoline-CH₂, J = 6.0 Hz), 4.16 (s, 2H, Isoquinoline-CH₂), 5.45 (s, 2H, NCH₂N), 6.89 (d, 1H, Thiophene-H, J = 3.2 Hz), 6.95 (t, 1H, Thiophene-H, J = 3.6 Hz), 7.13–7.15 (m, 5H, Ar-H), 7.26–7.40 (m, 5H, Ar-H and Thiophene-H). ¹³C NMR (100.63 Hz): δ 29.35, 48.72, 52.56 (Isoquinoline-CH₂), 69.89 (NCH₂N), 117.15, 125.72, 126.16, 126.54, 126.71, 127.68, 128.85, 129.22, 130.74, 130.90, 133.91, 134.48, 145.11, 162.22, 164.72 (Ar-C, Thiophene-C, Triazole-C5 and Triazole-C5), 170.49 (C=S).

4-(4-Fluorophenyl)-2-[(N-methylanilino)methyl]-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 10a. ¹H NMR (600.15 MHz): δ 3.40 (s, 3H, CH₃), 5.85 (s, 2H, NCH₂N), 6.83–6.86 (m, 3H, Ar-H and Thiophene-H), 6.91 (t, 1H, Thiophene-H, J = 4.3), 7.18 (d, 2H, Ar-H, J = 8.4 Hz), 7.24–7.36 (m, 6H, Ar-H and Thiophene-H). ¹³C NMR (150.36 MHz): δ 39.56 (CH₃), 66.52 (NCH₂N), 113.60, 117.12, 117.31, 118.58, 126.58, 127.53, 129.11, 129.24, 130.88, 130.94, 145.34, 147.35, 162.63, 164.40 (Ar-C, Thiophene-C and Triazole-C5), 169.26 (C=S).

4-(4-Fluorophenyl)-2-[(N-benzylanilino)methyl]-5-(thiophen-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 10b. ¹H NMR (400.20 MHz): δ 5.10 (s, 2H, Benzylic CH₂), 5.95 (s, 2H, NCH₂N), 6.81–6.93 (m, 3H, Ar-H and Thiophene-H), 7.17–7.36 (m, 15H, Ar-H and Thiophene-H). ¹³C NMR (100.64 MHz): δ 55.13 (CH₂), 64.97 (NCH₂N), 114.05, 117.16, 118.87, 126.61, 126.99, 127.67, 129.26, 129.29, 129.52, 130.53, 130.94, 131.03, 138.54, 145.46, 147.02, 162.28, 164.78 (Ar-C, Thiophene-C and Triazole-C5), 169.39 (C=S).

3.4. Single-Crystal XRD Studies

Suitable single crystals for X-ray diffraction were obtained by slow evaporation of a solution of compounds 5a, 5b, 6a, 6d and 10a in ethanol:chloroform (1:1, v/v) at room temperature. The crystal data and structure refinement parameters are shown in Table S1 (compounds 5a and 5b) and Table S2 (compounds 6a, 6d and 10a). The X-ray intensity data were collected on a Rigaku OD SuperNova/Atlas area-detector diffractometer using Cu Kα radiation (λ = 1.54184 Å) from a micro-focus X-ray source (for compound 5b) and on Excalibur, Ruby and Gemini diffractometers for the remaining crystals. Using Olex2 [59], the structure was solved with the SHELXT small molecule structure solution program [60] and refined with the SHELXL2018/3 program package [61] by full-matrix least-squares minimization on F². In 5a and 5b, the amino H atom was located from a difference Fourier map and refined freely with its isotropic displacement parameters. In the remaining compounds, the H atoms were placed in calculated positions and were constrained to ride on their parent atoms, with U_iso(H) = 1.2U_eq(C). The methyl H atoms were constrained to an ideal geometry with U_iso(H) = 1.5U_eq(C) but were allowed to rotate freely about the C–C bonds. In compounds 6a, 6d and 10d, the thiophene ring was disordered over two orientations. These structures were refined with suitable disorder models using the appropriate restraints and satisfactory models were obtained for these compounds. The crystal structure of 5b was refined as a two-component twin. The twin matrix is (−1.000 0 0 0 −1 0 0.4610.838 1) and the twin scales are 0.759(3) and 0.241(3).