Antioxidant and Anticancer Activity of Novel Derivatives of 3-[(4-Methoxyphenyl)amino]propanehydrazide

Abstract

Series of novel 3-[(4-methoxyphenyl)amino]propanehydrazide derivatives bearing semicarbazide, thiosemicarbazide, thiadiazole, triazolone, triazolethione, thiophenyltriazole, furan, thiophene, naphthalene, pyrrole, isoindoline-1,3-dione, oxindole, etc. moieties were synthesized and their molecular structures were confirmed by IR, ¹H-, ¹³C-NMR spectroscopy and mass spectrometry data. The antioxidant activity of the synthesized compounds was screened by DPPH radical scavenging method. The antioxidant activity of N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide and 3-((4-methoxyphenyl)amino)-N’-(1-(naphthalen-1-yl)-ethylidene)propanehydrazide has been tested to be ca. 1.4 times higher than that of a well-known antioxidant ascorbic acid. Anticancer activity was tested by MTT assay against human glioblastoma U-87 and triple-negative breast cancer MDA-MB-231 cell lines. In general, the tested compounds were more cytotoxic against U-87 than MDA-MB-231 cell line. 1-(4-Fluorophenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone has been identified as the most active compound against the glioblastoma U-87 cell line.

1. Introduction

Oxidative stress, induced by the generation of reactive oxygen (ROS) and nitrogen (NOS) species, is considered a major causative factor of many contemporary diseases including diabetes, cardiovascular diseases, cancer, viral infection, several neurodegenerative diseases, and various digestive disorders [1]. Antioxidants are involved in the defense mechanism of the organism against pathologies associated with the attack of free radicals by slowing down or inhibiting completely the oxidation processes caused by reactive radicals. In recent years, the interest in synthesis and pharmacological properties of new antioxidant agents has been increasing rapidly. Several classes of organic compounds have attracted attention of the researchers as potential scaffolds for the synthesis of novel biologically active compounds.

1,2,4-Triazole derived compounds usually exhibit a series of pharmacological properties such as anticancer, antimicrobial, antiviral, anticonvulsant, antidepressant, antihypertensive, etc. [2,3,4,5]. 1,2,4-Triazole moiety can influence lipophilicity, polarity, and hydrogen bonding capacity of a molecule, improving pharmacological, pharmacokinetic, toxicological, and physicochemical properties of the compounds [5]. Several drugs, such as ribavirin, estazolam, triazolam, and alprazolam contain the 1,2,4-triazole moiety. Among the biologically active 1,2,4-triazole derivatives, the ones bearing 1,2,4-triazole-3-thione moiety distinguish themselves by a diverse spectrum of activities, including antioxidant, anticancer, antibacterial, and antiviral [6].

Hydrazone derivatives constitute another significant category of compounds in medicinal and pharmaceutical chemistry. It has been established that the biological activity of hydrazone compounds is associated with the presence of the active azomethine -NH-N=CH- pharmacophore [7] and these compounds, in combination with various heterocyclic scaffolds, possess diverse biological activity, including anticancer, antibacterial, antiviral, antiplatelet [8,9], as well as antioxidant one [10,11]. As a model of a hybrid drug, bearing hydrazone and thiophene moieties, 2,5-dimethoxy-terephthalaldehyde bis(thiophene-2-carbonylhydrazone) was synthesized by the condensation reaction between 2,5-dimethoxyterephthalaldehyde and 2-thiophenecarboxylic acid hydrazide to be evaluated by computational pharmacological evaluation for the drug’s pharmacokinetics in the human body and presented a drug score of 45% according to the Lipinski rule of five [12]. Phthalimide ring (isoindoline-1,3-dione) represents another promising pharmacophore subunit for incorporation into hydrazone molecule. The hydrophobic nature of phthalimides increases their potential to cross different biological membranes in vivo [13]. Several isoindoline-1,3-dione and 2-oxindole moieties-containing drugs have been reported as potent anticancer agents in advanced stages of development: RG-108 as DNA methyltransferase inhibitor and promising anticancer agent; orantinib as inhibitor of vascular endothelial growth factor receptors type 2 (VEGFR2), platelet-derived growth factor receptors (PDGFR), and platelet-derived growth factor receptors (FGFR) inhibitor, as well as agent effective against hepatocellular carcinoma; 16PF-00562271 as an inhibitor of focal adhesion kinase (FAK) and protein tyrosine kinase 2 (PYK2) and effective agent against hepatocellular carcinoma. [14]. Introduction of one or several electron donating methoxy groups into benzene ring has been proven to strengthen the anticancer activity of various compounds [15,16]. Incorporation of methoxybenzene and naphthalene moieties into the structure of dihydropyrazole isatin dihydrothiazole hybrid resulted in a very efficient derivative, exhibiting anticancer activity higher than currently used anticancer drug sunitinib [17].

As a continuation of our interest to further explore the structure-activity relationship of the biologically active derivatives of amino acids and nitrogen-containing heterocyclic compounds [18,19,20], we report herein the synthesis of a series of 1-(5-chloro-2-hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives bearing heterocyclic moieties and evaluation of their antioxidant and anticancer activities.

2. Results and Discussion

2.1. Chemistry

2-(3-((4-Methoxyphenyl)amino)propanoyl)-N-phenylhydrazinecarboxamide (2) and its thio analogue were synthesized from 3-((4-methoxyphenyl)amino)propanehydrazide (1) by reaction with phenyl isocyanate and phenyl isothiocyanate, respectively (Scheme 1). Structures of all synthesized compounds have been confirmed by the ¹H- and ¹³C-NMR spectra and HRMS data (Supplementary Material, Figures S1–S120). In the ¹³C-NMR spectrum for hydrazinecarboxamide 2, carbon in C=O group of semicarbazide moiety resonated at 170.34 ppm, whereas analogous carbon (C=S) in thiosemicarbazide moiety gave a signal at 180.83 ppm in the ¹³C-NMR spectrum for 3.

Reactions of 1 with isocyanate and isothiocyanate resulted in formation of diphenylcarbamoyl hydrazide 4 and its thio analogue 5, respectively. In the ¹³C-NMR spectrum of 4, carbon of C=O in the second phenyl carbamoyl moiety resonated at 155.33 ppm, whereas analogous carbon (C=S) in thiocarbamoyl group resonated at 181.67 ppm in the ¹³C-NMR spectrum for 5. In the ¹³C-NMR spectrum for 5, the resonance of carbonyl carbon shifted downfield (170.2 ppm) in comparison with the resonance of this group carbon in the spectrum of 3 (158.59 ppm). Condensation reaction of hydrazinecarbothioamide 5 in concentrated sulfuric acid provided thiadiazole derivative 6 in 77% yield. In the ¹H-NMR spectrum for 6, two singlets of NH group protons are observed at 8.66 ppm and 10.29 ppm in comparison with the ¹H-NMR spectrum of 5, in which four singlets of NH groups are present.

Condensation reactions of phenylhydrazinecarboxamide 2 and its thio analogue 3 in alkaline medium resulted in formation of triazolone 7 and triazolethione 8 derivatives, respectively, in 80% yield. In the ¹³C-NMR spectrum for 7, the resonance of carbonyl group carbon in triazolone ring (154.44 ppm) shifted upfield in comparison with the spectrum of open-chain precursor 2. The same pattern in the resonance of the C=S carbon (172.08 ppm) in triazolethione moiety of 8 has been observed in respect with the resonance of the corresponding carbon in an open-chain derivative 3. Alkylation reactions of triazolone 7 with 2-bromoacetophenone and 2-bromo-4′-fluoroacetophenone afforded derivatives 9 and 10, respectively. In their ¹H-NMR spectra, the singlets at 4.8 ppm have been attributed to the CH₂ group in acetophenone moiety.

Introduction of the acetyl group usually enhances biological activity of the compound. Therefore, triazolethione 8 was heated at reflux with acetyl chloride to afford N-(4-methoxyphenyl)-N-(2-(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)ethyl)acetamide (11). The introduction of acetyl moiety has been confirmed by the carbonyl carbon resonance at 167.71 ppm in its ¹³C-NMR spectrum. Treatment of triazolone 7 and triazolethione 8 with potassium thiocyanate in acetic acid gave corresponding thioureas 12a and 12b. In their ¹³C-NMR spectra, the carbon resonances at 181.93 ppm have been attributed to the carbons in thiourea moiety. Thiazole moiety was introduced into the structure of 12b by its reaction with maleic anhydride in 1,4-dioxane at reflux temperature of the reaction mixture to afford compound 13. Formation of the thiazole ring in 13 has been confirmed by the ¹³C-NMR carbon resonances at 51.21 ppm (CS), 182.95 ppm (NCS), and 187.44 ppm (C=O).

Thione-thiol tautomerism is characteristic of triazolethiones, therefore they easily form S-substituted derivatives, which possess a broad spectrum of biological activities. A series of S-substituted triazolethione derivatives 14–24 were prepared by alkylation reaction of triazolethione 8 with corresponding aliphatic and aromatic halocarbonyl compounds using three methods (A, B, and C) (Scheme 2) [18].

Method A was used to carry out alkylation with ethyl chloroacetate and several acetophenones in DMF in the presence of trimethylamine to afford derivatives 14, 16, 17, and 20. Alkylation of 8 with 2-chloroacetamide in the presence of KOH and K₂CO₃ was employed for the synthesis of 15 (Method B). Compounds 18, 19, and 21–24 were synthesized according to the Method C in acetone in the presence of K₂CO₃. The IR spectra of S-substituted triazole derivatives display absorption bands of carbonyl group in the region of 1649–1753 cm⁻¹, whereas the absorption band of C=S group, which is present at 1237 cm⁻¹ in the IR spectrum for 8, is absent. In the ¹³C-NMR spectra for 14 and 15, C-S- group carbons resonated at approx. 168 ppm, whereas carbons of the same group with aromatic moiety in the attached substituent resonated in the range of 191–193 ppm in the ¹³C-NMR spectra for compounds 16–24. Acetyl group was introduced into the structure of 18 in its reaction with acetyl chloride at reflux temperature to afford acetamide 25. The singlet of CH₃ group protons at 1.61 ppm in the ¹H-NMR spectrum for 25 has been ascribed to the methyl group in acetyl moiety.

Condensation reaction of 1 with hexane-2,5-dione in propan-2-ol in the presence of acetic acid as a catalyst afforded N-(2,5-dimethyl-1H-pyrrol-1-yl)-3-((4-methoxyphenyl)amino)propanamide (26) (Scheme 3). The formation of pyrrole ring has been confirmed by the singlets of two methyl group protons at 1.98 ppm and 2.02 ppm and singlets of the CH group protons at 5.63 ppm and 5.71 ppm in the ¹H-NMR spectrum for 26. Reaction of 1 with isatin or N-benzylisatin in methanol in the presence of glacial acetic acid as a catalyst provided compounds 27a and 27b in 72% and 78% yield, respectively. The singlet at 5.26 ppm has been ascribed to the NH group proton in the 2-oxindole moiety in the ¹H-NMR spectrum for 27a. In the ¹H-NMR spectrum for 27b, the protons of the methylene group in benzyl moiety resonated as a singlet at 5.01 ppm.

By employing the most often used method for the synthesis of hydrazone-type compounds, i.e., the reaction of hydrazides with carbonyl compounds, Schiff bases 28–37 were synthesized by condensation reaction of 1 and corresponding disubstituted ketones in methanol at reflux temperature of the reaction mixtures [19,21]. The ¹H-NMR spectra for these compounds display double sets of resonances of the CO–NH group protons with signal intensity ratio 0.6 : 0.4 due to the restricted rotation around the amide bond. This splitting of the proton resonances indicates that in DMSO-d₆ solution hydrazones exist as a mixture of Z/E isomers and, usually, the Z isomer predominates [22,23].

Acetamide 38 was synthesized by treating 30 with acetic anhydride in methanol at reflux temperature of the reaction mixture. The singlet of CH₃ group protons at 1.70 ppm in the ¹H-NMR spectrum for 38 has been ascribed to the methyl group in acetyl moiety. Reaction of 1 with phthalic anhydride in 1,4-dioxane at reflux temperature resulted in formation of propanamide 39 in 81% yield. The proton resonances of the benzene ring of the phthalimide moiety are observed in the range of 7.37–7.70 ppm. In the ¹³C-NMR spectrum for 39, the carbon resonance of double intensity at 173.09 ppm has been attributed to the carbonyl group carbons in phthalimide moiety.

2.2. Evaluation of Antioxidant Activity

Antioxidant properties of compounds 1–39 were evaluated by 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) radical scavenging method [4]. The DPPH radical is a stable free radical that is commonly used as a substrate to evaluate in vitro antioxidant activity [24]. Compound possessing antioxidant property donates a hydrogen atom or electrons to DPPH and converts it to a stable molecule, 1,1-diphenyl-picryl hydrazine. DPPH assay is considered to be an accurate, easy and economic method to evaluate radical scavenging activity of antioxidants, since the radical compound is stable and needs not to be generated [25].

Antioxidant activity of N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide (39) has been tested to be 1.37 times higher than that of a well-known antioxidant ascorbic acid, whereas antioxidant activity of 3-((4-methoxyphenyl)amino)-N’-(1-(naphthalen-1-yl)ethylidene)-propanehydrazide (36) surpassed that of ascorbic acid by 1.35-fold (

Table 1. DPPH scavenging activity of compounds 1–39.

Compound	DPPH Scavenging Activity, %	Compound	DPPH Scavenging Activity, %
1	54.98	21	31.9
2	23.36	22	25.6
3	67.9	23	5.1
4	27.5	24	48.7
5	44.0	25	1.1
6	62.6	26	25.08
7	29.98	27a	66.67
8	45.1	27b	36.09
9	0	28	50.24
10	0	29	73.46
11	28.2	30	55.92
12a	16.36	31	33.18
12b	10.79	32	0
13	13.83	33	0
14	67.3	34	0
15	60.7	35	65.40
16	58.7	36	78.67
17	65.7	37	58.77
18	23.1	38	7.89
19	29.7	39	79.62
20	60.9	Ascorbic acid *	58.2

* Control.

). It is interesting to note that hydrazone bearing 2-naphthalene moiety 37 scavenged DPPH radical weaker than hydrazine 36, but still at the level of vitamin C. Antioxidant activity of hydrazone bearing thiophene moiety 29 was 1.26 times higher than that of the positive control.

Hydrazone 30, which structure differs from the one of 29 just by ethyl group instead of methyl one, exhibited much lower DPPH scavenging activity (55.92%). Introduction of the furan moiety into hydrazone molecule 28 led to an even lower antioxidant activity. The presence of aniline moiety in these molecules led to inactive compounds 33 and 34. Acylation of compound 31 (33.18%) provided almost inactive acetamide 38 (7.89%).

Compounds 3, 14, and 27a have been identified as possessing quite high antioxidant activity as well. The antioxidant activity according to DPPH inhibition decreases in the following order among the investigated compounds: 39 > 36 > 29 > 3 > 14 > 27a > 17 ≈ 35 > 6 > 20 ≈ 15 > 37 ≈ 16 > ascorbic acid > 30 > 1 > 28

1-(4-Bromophenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)-thio)ethanone (17) has been shown to possess the highest antioxidant activity among the series of S-substituted triazolethione derivatives 14–24. Its DPPH radical scavenging activity has been determined to be 1.13 times higher than that of ascorbic acid. Replacement of bromine substituent in 17 with chlorine one in 20 led to a slight decrease in activity, whereas activity of 21 bearing fluorine atom decreased almost twice. Introduction of hydroxyl group led to almost complete inactivity of derivative 23. The same loss of moderate activity of 18 was caused by introduction of acetyl group into the structure of acetamide 25. A similar pattern in the decrease of antioxidant activity has been observed in a pair of triazolethione derivatives 8 (45.1%) and 11 (20.2%), proving the importance of the presence of N-H functional group, which can donate a hydrogen atom. Replacement of this hydrogen atom in triazolone 7 (29.98%) with much bulkier functional groups resulted in completely inactive derivatives 9 and 10.

2.3. Evaluation of Anticancer Activity

The synthesized compounds 1–39 have been tested to possess different activity against human glioblastoma U-87 and triple-negative breast cancer MDA-MB-231 cell lines at 100 µM concentration. In general, compounds showed relatively low activity against the cancer cell lines used in the screening experiments (Figure 1). This could be explained by the presence of drug efflux systems in brain tumours contributing to the drug resistance [26]. Triple-negative breast cancer is typically more resistant to majority of available drugs due to the higher expression of P-glycoprotein that enhances drug efflux from the nucleus [27,28], also epigenetic alterations in histone deacetylase [29], and other mechanisms. Cell line U-87 is quite resistant to temozolomide, which is clinically used drug to treat glioblastoma. It reduces U-87 cell viability up to 60% at 100 µM concentration after 48 h of incubation [30]. It is worthy to note that activity depends a lot on the mechanism of action. For example, topoisomerase inhibitor etoposide at 100 µM concentration reduces MDA-MB-231 cell viability up to approximately 80% after 72 h incubation [31], antimicrotubular drug docetaxel at 100 nM concentration reduces cell viability up to 40% already after 48 h of incubation [32], and novel kinase inhibitor dasatinib at 1 µM concentration reduces the viability up to 30% [33].

A majority of the synthesized compounds were more active against glioblastoma U-87 than the triple-negative breast cancer cell line MDA-MB-231. Cisplatin (a clinically used alkylating agent) also shows similar higher activity towards glioblastoma cells. It almost completely inhibited U-87 cell proliferation after 48 h incubation [30,34], and reduced MDA-MB-231 cell viability only up to 30% at 10 µM concentration after 72 h of incubation [31].

1-(4-Fluorophenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone (21), which reduced cell viability up to 19.6 ± 1.5%, has been identified as the most active compound against glioblastoma cells. However, its’ analogues bearing bromine, methyl, methoxy, chlorine and hydroxyl substituents (17–20 and 23) showed at least twice lower activity. Thiophenyltriazole 16 with no substituent at para-position of benzene ring was even less active in comparison with the para-substituted ones. It can be assumed, that only small substituents (such as fluorine) could be introduced at this position. Possibly, this fluorination prevents metabolism due to which the anticancer activity could be reduced [35].

Thiophenyltriazole 22 and hydrazone 37 were also among the most active compounds against U-87 cell line; they reduced cell viability up to 39.8 ± 3.8% and 40.3 ± 0.8%, respectively. It is worthy to note, that 22, instead of fluorine at the para-position in 21, contains nitro group, which is often associated with anticancer properties as well as mutagenic and genotoxic ones [36]. Hydrazone 37 has been identified as a highly active antioxidant substance, proving that these two properties could be related to each other.

Compounds 36, 19 and 6 have shown the highest activity against MDA-MB-231 cell line. They reduced breast cancer cell viability up to 43.7 ± 7.4%, 44.6 ± 8.0%, and 46.2 ± 5.0%, respectively. Both hydrazones 36 and 37 contain bulky naphthalene moiety in their structure differing only in the position of substitution. Both of them have shown high antioxidant properties. This cross-activity could be non-specific and related to their radical scavenging activity.

Thiophene derivatives 29 and 30, which have been identified as strong antioxidants in DPPH assay, exhibited a very weak effect on cell viability. It could be explained by our previous findings that antioxidant and anticancer activity relationship could not be always explained [37], as many other different mechanisms of action contribute to the anticancer activity of different structures.

In summary, fluorine-substitututed thiophenyltriazole 21, which has shown different activity on different cell lines, possibly due to selectivity on specific targets in glioblastoma cells, has been identified as possessing the promising anticancer activity.

3. Experimental

3.1. General Information

Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and TCI Europe N.V. (Zwijndrecht, Belgium). Reaction course and purity of the synthesized compounds was monitored by TLC using aluminium plates precoated with silica gel 60 F254 (Merck KGaA, Darmstadt, Germany). Melting points were determined on a MEL-TEMP (Electrothermal, A Bibby Scientific Company, Burlington, NJ, USA) melting point apparatus and are uncorrected. FT-IR spectra (ν, cm⁻¹) were recorded on a Spectrum BX FT–IR spectrometer (Perkin–Elmer Inc., Waltham, MA, USA) using KBr pellets. The ¹H- and ¹³C-NMR spectra were recorded in DMSO-d₆ on a Varian Unity Inova (300 MHz, 75 MHz, Palo Alto, CA, USA) and Bruker Avance III (400 MHz, 100 MHz Bruker BioSpin AG, Fällanden, Switzerland ) spectrometers operating in the Fourier transform mode. Chemical shifts (δ) are reported in parts per million (ppm) calibrated from TMS (0 ppm) as an internal standard for ¹H-NMR, and DMSO-d₆ (39.43 ppm) for ¹³C-NMR. Mass spectra were obtained on a maXis UHR-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with ESI ionization.

3.2. Synthesis

2-(3-((4-Methoxyphenyl)amino)propanoyl)-N-phenylhydrazinecarboxamide (2): A mixture of propane-hydrazide (1, 2.09 g, 10 mmol), methanol (30 mL), and phenyl isocyanate (1.35 g, 1.08 mL, 10 mmol) was heated at reflux for 30 min. The reaction mixture was cooled down, precipitate formed was filtered off and recrystallized from DMF/H₂O mixture. Yield 59%; white solid; m.p. 192–193 °C; IR (KBr) ν_max (cm⁻¹): 3375, 3344, 3283, 3206 (NH), 1659, 1638 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.41 (t, J = 7.2 Hz, 2H, CH₂CO), 3.78 (s, 3H, CH₃O), 3.84 (t, J = 7.2 Hz, 2H, CH₂NH), 6.99–7.44 (m, 9H, H_Ar), 7.76, 8.01, 8.71, 9.77 (4s, 4H, 4NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 32.3 (CH₂CO), 46.4 (CH₂NH), 55.3 (CH₃O), 114.9, 119.9, 128.3, 128.7, 129.7, 134.1, 139.6, 140.0, 154.7 (C_Ar), 158.1, 170.3 (C=O); HRMS (ESI): m/z calcd for C₁₇H₂₀N₄O₃ 328.1535 [M + H]⁺, found 328.1537.

2-(3-((4-Methoxyphenyl)amino)propanoyl)-N-phenylhydrazinecarbothioamide (3): A mixture of propanehydrazide 1 (3.15 g, 15 mmol), methanol (60 mL), and phenyl isothiocyanate (0.42 g, 3.71 mL, 20 mmol) was heated at reflux for 4 h. The reaction mixture was cooled down, precipitate formed was filtered off and recrystallized from DMF/H₂O mixture. Yield 78%; white solid; m.p. 164–165 °C; IR (KBr) ν_max (cm⁻¹): 3451–2831 (NH), 1698 (C=O), 1244 (C=S); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.46 (t, J = 7.2 Hz, 2H, CH₂CO), 3.24 (t, J = 7.2 Hz, 2H, CH₂NH); 3.63 (s, 3H, CH₃O), 5.18 (s, 1H, NH), 6.55 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.73 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.21–7.37 (m, 5H, H_Ar‘), 9.54, 9.59 (2s, 2H, NHNHCS), 9.98 (s, 1H, NHAr‘); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 33.5 (CH₂CO), 40.2 (CH₂NH), 55.3 (CH₃O), 113.4, 114.7, 115.29, 126.1, 127.9, 139.1, 142.9, 150.9 (C_Ar), 158.6 (C=O), 180.8 (C=S); HRMS (ESI): m/z calcd for C₁₇H₂₀N₄O₂S 345.1385 [M + H]⁺, found 345.1379.

3.2.1. General Procedure for Synthesis of Compounds 4 and 5

To propanehydrazide 1 (2.09 g, 0.01 mol) dissolved in methanol (70 mL), corresponding cyanate (0.02 mol) was added. The reaction mixture was heated at reflux for 1–2 h. Precipitate formed was filtered off, washed with water, and recrystallized from DMF/H₂O mixture.

2-(3-(1-(4-Methoxyphenyl)-3-phenylureido)propanoyl)-N-phenylhydrazinecarboxamide (4): Prepared from phenyl isocyanate by heating at reflux a reaction mixture for 2 h. Yield 76%; white solid; m.p. 179–180 °C; IR (KBr) ν_max (cm⁻¹): 3386, 3254, 3283, 3206 (NH); 1675, 1661, 1601 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.43 (t, J = 7.2 Hz, 2H, CH₂CO), 3.79 (s, 3H, CH₃O), 3.85 (t, J = 7.2 Hz, 2H, CH₂N), 6.88–7.48 (m, 14H, H_{Ar, Ar’, Ar’’}), 7.72 (s, 1H, NH), 8.00 (s, 1H, NH), 8.69 (s, 1H, NH), 9.76 (s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 32.3 (CH₂CO), 46.2 (CH₂N), 55.3 (CH₃O), 114.9, 118.6, 119.8, 121.9, 122.0, 128.2, 128.6, 129.4, 129.6, 134.1, 139.6, 139.9, 154.6 (C_Ar), 155.3, 158.1, 170.5 (C=O); HRMS (ESI): m/z calcd for C₂₄H₂₅N₅O₄ 447.1985 [M + H]⁺, found 448.1987.

2-(3-(1-(4-Methoxyphenyl)-3-phenylthioureido)propanoyl)-N-phenylhydrazinecarbothioamide (5): Prepared from phenyl isothiocyanate by heating at reflux a reaction mixture for 1 h. Yield 78%; white solid; m.p. 178–179 °C; IR (KBr) ν_max (cm⁻¹): 3333, 3299, 3206, 3154 (NH), 1698 (C=O), 1243, 1216 (C=S); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.64 (t, J = 7.8 Hz, 2H, CH₂CO), 3.77, 3.78 (2s, 3H, CH₃O), 4.34 (t, J = 7.8 Hz, 2H, CH₂N), 6.96–7.34 (m, 14H, H_{Ar, Ar’, Ar’’}), 7.38, 8.65, 9.50, 9.95 (4s, 4H, 4NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 31.0 (CH₂CO), 50.6 (CH₂N), 55.4 (CH₃O), 115.1, 115.2, 124.7, 125.8, 126.0, 127.8, 128.0, 129.1, 134.7, 139.1, 140.5, 158.6 (C_Ar), 170.20 (C=O), 180.8, 181.7 (C=S); HRMS (ESI): m/z calcd for C₂₄H₂₅N₅O₂S₂ 480.1528 [M + H]⁺, found 480.1526.

1-(4-Methoxyphenyl)-3-phenyl-1-(2-(5-(phenylamino)-1,3,4-thiadiazol-2-yl)ethyl)thiourea (6): A mixture of conc. H₂SO₄ (25 mL) and phenylhydrazinecarbothioamide 5 (0.96 g, 2 mmol) was stirred at room temperature until all solid dissolved (approx. 25 min). Afterwards the reaction mixture was added drop by drop to a water-ice mixture. Precipitate formed was filtered off, washed with water, and recrystallized from propan-2-ol. Yield 77%; white solid; m.p. 151–152 °C; IR (KBr) ν_max (cm⁻¹): 3358, 3191 (NH), 1249 (C=S); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 3.29 (t, J = 7.4 Hz, 2H, CH₂C), 3.76 (s, 3H, CH₃O), 4.46 (t, J = 7.4 Hz, 2H, CH₂N), 6.96–7.59 (m, 14H, H_{Ar, Ar’, Ar’’}), 8.66 (s, 1H, NH), 10.29 (s, 1H, NHC=S); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 27.8 (CH₂C), 53.8 (CH₂N), 55.3, 55.5 (CH₃O), 115.0, 115.3, 117.2, 121.7, 124.9, 126.3, 127.8, 129.1, 134.6, 140.5, 140.8, 156.4, 158.6, 164.4 (C_Ar), 181.9 (C=S); HRMS (ESI): m/z calcd for C₂₄H₂₃N₅OS₂ 462.1422 [M + H]⁺, found 462.1419.

3-(2-((4-Methoxyphenyl)amino)ethyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (7): A mixture of phenyl-hydrazinecarboxamide 2 (1.34 g, 3 mmol) and 20% aqueous KOH solution (25 mL) was heated at reflux for 2 h. The reaction mixture was cooled down and acidified with HCl to pH 4. Precipitate formed was filtered off, washed with water, and recrystallized from DMF/H₂O mixture. Yield 81%; white solid; m.p. 156–157 °C; IR (KBr) ν_max (cm⁻¹): 3175, 3017 (NH), 1659 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.62 (t, J =7.2 Hz, 2H, CH₂C), 3.14 (t, J =7.2 Hz, 2H, CH₂NH), 3.62 (s, 3H, CH₃O); 6.39 (d, J = 8.4 Hz, 2H, H_Ar2,6), 6.67 (d, J = 8.4 Hz, 2H, H_Ar3,5), 7.41–7.57 (m, 6H, H_Ar’+NH), 11.76 (s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 25.6 (CH₂C), 40.8 (CH₂NH), 55.3 (CH₃O), 113.2, 114.7, 127.6, 128.8, 129.6, 133.0, 145.2, 145.3, 150.9, 151.5 (C_Ar), 154.4 (C=O); HRMS (ESI): m/z calcd for C₁₇H₁₈N₄O₂ 311.1509 [M + H]⁺, found 311.1503.

3-(2-((4-Methoxyphenyl)amino)ethyl)-4-phenyl-1H-1,2,4-triazole-5(4H)-thione (8): A mixture of phenyl-hydrazinecarbothioamide 3 (1 g, 3 mmol) and 20% aqueous KOH solution (40 mL) was heated at reflux for 4 h. The reaction mixture was cooled down and acidified with HCl to pH 4. Precipitate formed was filtered off, washed with water, and recrystallized from DMF/H₂O mixture. Yield 80%; white solid; m.p. 141–142 °C; IR (KBr) ν_max (cm⁻¹): 3446–2826 (NH), 1237 (C=S); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.63 (t, J = 9.0 Hz, 2H, CH₂C), 3.15 (t, 2H, J = 9.0 Hz, CH₂NH), 3.61 (s, 3H, CH₃O), 5.22 (s, 1H, NHAr’), 6.31 (d, J = 9 Hz, 2H, H_Ar2,6), 6.63 (d, J = 9 Hz, 2H, H_Ar3,5), 7.39–7.58 (m, 5H, H_Ar’), 13.75 (s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 25.4 (CH₂C), 40.4 (CH₂NH), 55.3 (CH₃O), 113.1, 114.7, 122.0, 128.5, 129.5, 133.8, 142.2, 150.7, 150.9, 167.6 (C_Ar), 172.1 (C=S); HRMS (ESI): m/z calcd for C₁₇H₁₈N₄OS 327.1279 [M + H]⁺, found 327.1293.

3.2.2. General Procedure for the Synthesis of 1,2,4-Triazolones 9 and 10

To hydrazinecarboxamide 7 (0.93 g, 3 mmol) dissolved in DMF (15 mL), KOH (0.17 g, 3 mmol), K₂CO₃ (0.37 g, 2.7 mmol), and corresponding bromoacetophenone (3.75 mmol) were added. The reaction mixture was stirred at 40 °C for 24 h. Afterwards water (30 mL) was added to the reaction mixture. Precipitate formed was filtered off, washed with water, and recrystallized from propan-2-ol.

3-(2-((4-Methoxyphenyl)(2-oxo-2-phenylethyl)amino)ethyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (9): Prepared from 2-bromoacetophenone. Yield 63%; white solid; m.p. 142–143 °C; IR (KBr) ν_max (cm⁻¹): 3375 (NH), 1694, 1513 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.70 (t, J = 7.4 Hz, 2H, CH₂C), 3.49 (t, J = 7.4 Hz, 2H, CH₂N), 3.61 (s, 3H, CH₃O), 4.80 (s, 2H, CH₂CO), 6.22 (d, J = 9.2 Hz, 2H, H_Ar2,6), 6.62 (d, J = 9.2 Hz, 2H, H_Ar3,5), 7.41–7.96 (m, 10H, H_{Ar’, Ar’’}), 11.68, 11.71 (2s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.1 (CH₂C), 40.2 (CH₂N), 48.4 (CH₂CO), 55.3 (CH₃O), 112.6, 114.6, 127.5, 127.6, 127.7, 128.7, 128.8, 129.5, 132.9, 133.4, 135.3, 141.6, 145.4, 150.7 (C_Ar), 154.3, 197.2 (C=O); HRMS (ESI): m/z calcd for C₂₅H₂₄N₄O₃ 429.1927 [M + H]⁺, found 429.1930.

3-(2-((2-(4-Fluorophenyl)-2-oxoethyl)(4-methoxyphenyl)amino)ethyl)-4-phenyl-1H-1,2,4-triazol-5(4H)-one (10): Prepared from 2-bromo-4′-fluoroacetophenone. Yield 58%; white solid; m.p. 103–104 °C; IR (KBr) ν_max (cm⁻¹): 3494 (NH), 1698, 1514 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.70 (t, J = 7.4 Hz, 2H, CH₂C), 3.48 (t, J = 7.4 Hz, 2H, CH₂N), 3.61 (s, 3H, CH₃O), 4.80 (s, 2H, CH₂CO), 6.23 (d, J = 9.2 Hz, 2H, H_Ar2,6), 6.62 (d, J = 9.2 Hz, 2H, H_Ar3,5), 7.35–7.55 (m, 7H, H_{Ar’, Ar’’}), 8.02–8.05 (m, 2H, H_Ar’’), 11.67, 11.71 (2s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.1 (CH₂C), 40.2 (CH₂N), 48.3 (CH₂CO), 55.3 (CH₃O), 112.7, 114.6, 115.6, 115.9, 127.6, 128.7, 129.5, 130.7, 130.8, 132.0, 132.0, 132.9, 141.6, 145.3, 150.7, 154.3, 163.9 (C_Ar), 166.4, 195.8 (C=O); HRMS (ESI): m/z calcd for C₂₅H₂₃FN₄O₃ 447.1833 [M + H]⁺, found 447.1830.

N-(4-Methoxyphenyl)-N-(2-(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)ethyl)acetamide (11): A mixture of triazolethione 8 (2.61 g, 8 mmol) and acetyl chloride (25 mL) was heated at reflux for 7 h. The reaction mixture was added drop by drop onto ice. Precipitate formed was filtered off, washed with water, and recrystallized from acetone. Yield 66%; white solid; m.p. 182–183 °C; IR (KBr) ν_max (cm⁻¹): 1651, 1510 (C=O), 1255 (C=S); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 1.62, 1.65 (2s, 3H, CH3), 2.60 (t, J = 6.8, 2H, CH₂C), 3.56–3.70 (m, 2H, CH₂N), 3.77 (s, 3H, CH₃O), 6.94–7.56 (m, 9H, H_Ar); 13.70 (s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 22.2, 23.8 (CH₃), 24.7 (CH₂C), 45.4 (CH₂N), 55.4 (CH₃O), 114.7, 128.3, 129.2, 129.3, 129.5, 133.6, 135.0, 149.8, 158.4, 158.5 (C_Ar), 167.7 (C=O), 169.4 (C=S); HRMS (ESI): m/z calcd for C₁₉H₂₀N₄O₂S 369.1386 [M + H]⁺, found 369.1381.

3.2.3. General Procedure for the Synthesis of Compounds 12a, b

A mixture of triazolethione 7 or 8 (2 mmol), potassium thiocyanate (0.39 g, 4 mmol), and acetic acid (10 mL) was heated at reflux for 5 min. The reaction mixture was cooled to room temperature and diluted with water (50 mL). Precipitate formed was filtered off, washed with water, and recrystallized from propan-2-ol.

1-(4-Methoxyphenyl)-1-(2-(5-oxo-4-phenyl-4,5-dihydro-1H-1,2,4-triazol-3-yl)ethyl)thiourea (12a): Prepared from 7 (0.62 g). Yield 76%; white solid; m.p. 203–204 °C; IR (KBr) ν_max (cm⁻¹): 3242, 3176 (NH, NH₂), 1691 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.73 (t, 2H, J = 7.2 Hz, CH₂C), 3.34, 3.61 (2s, 2H, NH₂), 3.77 (s, 3H, CH₃O), 4.07 (t, J = 7.2 Hz, 2H, CH₂N), 6.95 (d, J = 8.8 Hz, 2H, H_Ar2,6), 7.02 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.23–7.46 (m, 5H, H_Ar’), 11.67, 11.71 (2s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.2 (CH₂C), 51.1 (CH₂N), 55.4 (CH₃O), 115.2, 127.2, 128.5, 128.8, 129.3, 132.7, 133.9, 144.4, 154.2 (C_Ar), 158.6 (C=O), 181.9 (C=S); HRMS (ESI): m/z calcd for C₁₈H₁₉N₅O₂S 370.1337 [M + H]⁺, found 370.1337.

1-(4-Methoxyphenyl)-1-(2-(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)ethyl)thiourea (12b): Prepared from 8 (0.65 g). Yield 66%; white solid; m.p. 198–199 °C; IR (KBr) ν_max (cm⁻¹): 3233, 3185 (NH, NH₂), 1520 (C=S); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.73 (t, J = 7.2 Hz, 2H, CH₂C), 3.34 (s, 2H, NH₂), 3.77 (s, 3H, CH₃O), 4.09 (t, J = 7.2 Hz, 2H, CH₂N), 6.95 (d, J = 8.8 Hz, 2H, H_Ar2,6), 7.02 (d, 2H, J = 8.8 Hz, H_Ar3,5), 7.30–7.50 (m, 5H, H_Ar‘), 13.70 (s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 23.7 (CH₂C), 51.2 (CH₂N), 55.4 (CH₃O), 114.7, 115.2, 128.1, 128.8, 129.3, 129.4, 133.5, 133.7, 149.6, 158.6 (C_Ar), 167.6, 181.9 (C=S); HRMS (ESI): m/z calcd for C₁₈H₁₉N₅OS₂ 386.1109 [M + H]⁺, found 386.1104.

2-(2-((4-Methoxyphenyl)(2-(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)ethyl)amino)-4-oxo-4,5-dihydrothiazol-5-yl)acetic acid (13): A mixture of 12b (0.58 g, 1.5 mmol), maleic anhydride (0.29 g, 3 mmol), 1,4-dioxane (15 mL), and DMF (5 mL) was heated at reflux for 7 h. The reaction mixture was cooled down and cold water (10 mL) was added. Precipitate formed was filtered off and recrystallized from DMF/H₂O mixture. Yield 82%; yellow solid; m.p. 159–160 °C; IR (KBr) ν_max (cm⁻¹): 3233, 3185 (NH, NH₂), 1648, 1713 (C=O), 1530 (C=S); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.52–2.60 (m, 1H, CH), 2.73–2.83 (m, 2H, CH₂C), 2.98–3.05 (m, 1H, CH₂), 3.81 (s, 3H, CH₃O), 3.88–4.09 (m, 2H, CH₂N), 4.23–4.35 (m, 1H, CH₂), 6.98–7.57 (m, 9H, H_{Ar, Ar‘}), 12.78 (br s, 1H, OH), 13.75 (s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 23.6 (CH₂C), 37.6 (CH₂), 50.3 (CH₂N), 52.2 (CS), 55.6 (CH₃O), 114.9, 128.3, 129.3, 129.4, 129.5, 132.5, 133.4, 149.1, 159.7 (C_Ar), 167.8 (C=S), 172.4 (COOH), 183.0 (NCS), 187.4 (C=O); HRMS (ESI): m/z calcd for C₂₂H₂₁N₅O₄S₂ 484.1113 [M + H]⁺, found 484.1110.

3.2.4. General Procedure for the Synthesis of S-Substituted 1,2,4-Triazolethiones 14–24

Method A. To triazolethione 8 (0.49 g, 1.5 mmol) dissolved in DMF (5 mL), triethylamine (0.20 g, 0.28 mL, 2 mmol) and corresponding halocarbonyl compound (2 mmol) were added. The reaction mixture was stirred at room temperature for 4 h. Afterwards cold water (30 mL) was added, the precipitate formed was filtered off and recrystallized from propan-2-ol.

Method B. To triazolethione 8 (0.49 g, 1.5 mmol) dissolved in DMF (5 mL), KOH powder (0.11 g, 2 mmol), K₂CO₃ (0.28 g, 2.2 mmol), and corresponding halocarbonyl compound (2 mmol) were added. The reaction mixture was stirred at 35–40 °C for 24 h. Afterwards cold water (30 mL) was added, the precipitate formed was filtered off and recrystallized from propan-2-ol.

Method C. To triazolethione 8 (0.49 g, 1.5 mmol) dissolved in acetone (15 mL), K₂CO₃ (1 g, 7.2 mmol) and corresponding halocarbonyl compound (2 mmol) were added. The reaction mixture was stirred at 40 °C for 3 h. Afterwards water (20 mL) was added, the precipitate formed was isolated by filtration, and recrystallized from propan-2-ol.

((5-(2-((4-Methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)methyl propionate (14): Prepared according to method A from ethyl chloroacetate (0.25 g, 0.21 mL). Yield 80%; white solid; m.p. 71–72 °C; IR (KBr) ν_max (cm⁻¹): 3309 (NH), 1753 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 1.17 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.73 (t, J = 7.2 Hz, 2H, CH₂C), 3.19 (t, 2H, J = 7.2 Hz, 2H, CH₂NH), 3.61, 3.62 (2s, 3H, CH₃O), 4.01 (s, 2H, SCH₂), 4.09 (q, J = 7.2 Hz, 2H, CH₃CH₂), 5.24 (s, 1H, NH), 6.31 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.63 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.44–7.64 (m, 5H, H_Ar’); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 14.0 (CH₃CH₂), 24.7 (CH₂C), 33.9 (SCH₂), 41.3 (CH₂NH); 55.3 (CH₃O), 61.2 (CH₃CH₂), 112.6, 113.1, 114.6, 127.4, 130.0, 130.1, 132.8, 142.2, 149.1, 150.8 (C_Ar), 154.2 (C=O), 168.1 (C-S-); HRMS (ESI): m/z calcd for C₂₁H₂₄N₄O₃S 413.1647 [M + H]⁺, found 413.1660.

2-((5-(2-((4-Methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)acetamide (15): Prepared according to method B from 2-chloroacetamide (0.19 g). Yield 75%; white solid; m.p. 77–78 °C; IR (KBr) ν_max (cm⁻¹): 3346–2826 (NH, NH₂), 1696 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.74 (t, J = 7.2 Hz, 2H, CH₂C), 3.20 (q, 2H, J = 7.2 Hz, CH₂NH), 3.62 (s, 3H, CH₃O), 3.88 (s, 2H, SCH₂), 5.23 (t, J = 6.2 Hz, 1H, NH), 6.32 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.64 (d, J = 8,8 Hz, 2H, H_Ar3,5), 7.23 (s, 1H, NH₂), 7.46–7.61 (m, 5H, H_Ar’), 7.67 (s, 1H, NH₂); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.7 (CH₂C), 35.9 (SCH₂), 41.3 (CH₂NH), 55.3 (CH₃O), 113.1, 114.6, 127.4, 129.9, 130.1, 132.9, 142.2, 149.9, 150.8 (C_Ar), 154.0 (C=O), 168.7 (C-S-); HRMS (ESI): m/z calcd for C₁₉H₂₁N₅O₂S 384.1494 [M + H]⁺, found 384.1500.

2-((5-(2-((4-Methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-1-phenylethanone (16): Prepared according to method A from 2-bromoacetophenone (0.4 g). Yield 69%; white solid; m.p. 159–160 °C; IR (KBr) ν_max (cm⁻¹): 3292 (NH), 1687 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.74 (t, J = 7.2 Hz, 2H, CH₂C), 3.15–3.25 (m, 2H, CH₂NH), 3.61 (s, 3H, CH₃O), 4.86 (s, 2H, SCH₂), 5.23 (s, 1H, NH), 6.32 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.64 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.46–8.02 (m, 10H, H_{Ar’, Ar”}); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.6 (CH₂C), 40.0 (SCH₂), 41.2 (CH₂NH), 55.2 (CH₃O), 112.9, 114.5, 127.3, 128.3, 128.7, 129.9, 132.8, 133.6, 135.2, 142.1, 149.3, 150.7 (C_Ar), 154.0 (C=O), 193.1 (C-S-); HRMS (ESI): m/z calcd for C₂₅H₂₄N₄O₂S 445.1698 [M + H]⁺, found 445.1708.

1-(4-Bromophenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone (17): Prepared according to method A from 4-bromophenacyl bromide (0.56 g). Yield 63%; white solid; m.p. 168–169 °C; IR (KBr) ν_max (cm⁻¹): 3291 (NH), 1694 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.73 (t, J = 7.2 Hz, 2H, CH₂C), 3.19 (t, 2H, J = 7.2 Hz, CH₂NH), 3.61 (s, 3H, CH₃O), 4.82 (s, 2H, SCH₂), 5.22 (s, 1H, NH), 6.31 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.63 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.45–7.94 (m, 9H, H_{Ar’, Ar”}); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.8 (CH₂C), 40.2 (SCH₂), 41.3 (CH₂NH), 55.3 (CH₃O), 113.1, 114.6, 127.4, 127.8, 130.0, 130.1, 130.4, 131.9, 132.9, 134.3, 142.2, 149.2, 150.8 (C_Ar), 154.2 (C=O), 192.5 (C-S-); HRMS (ESI): m/z calcd for C₂₅H₂₃BrN₄O₂S 523.0803 [M + H]⁺, found 523.0799.

2-((5-(2-((4-Methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-1-(p-tolyl)ethanone (18): Prepared according to method C from 2-bromo-4′-methylacetophenone (0.43 g). Yield 71%; white solid; m.p. 136–137 °C; IR (KBr) ν_max (cm⁻¹): 3282 (NH), 1689 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.39 (s, 3H, CH₃), 2.74 (t, J = 7.4 Hz, 2H, CH₂C), 3.19 (q, J = 6.8, 14.0 Hz, 2H, CH₂NH), 3.61 (s, 3H, CH₃O), 4.82 (s, 2H, SCH₂), 5.22 (t, J = 6.0 Hz, 1H, NH), 6.31 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.63 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.35 (d, J = 8.0 Hz, 2H, H_Ar’’), 7.45–7.50 (m, 2H, H_Ar’), 7.58–7.63 (m, 3H, H_Ar’), 7.90 (d, J = 8.0 Hz, 2H, H_Ar’’); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 21.2 (CH₃), 24.7 (CH₂C), 40.1 (SCH₂), 41.3 (CH₂NH), 55.3 (CH₃O), 113.1, 114.6, 127.4, 128.5, 129.4, 130.0, 130.1, 132.8, 132.9, 142.2, 144.3, 149.4, 150.8 (C_Ar), 154.1 (C=O), 192.7 (C-S-); HRMS (ESI): m/z calcd for C₂₆H₂₆N₄O₂S 459.1854 [M + H]⁺, found 459.1853.

1-(4-Methoxyphenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone (19): Prepared according to method C from 2-bromo-4’-methoxyacetophenone (0.30 g). Yield 76%; white solid; m.p. 121–122 °C; IR (KBr) ν_max (cm⁻¹): 3287 (NH), 1683 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.76 (t, J = 7.2 Hz, 2H, CH₂C), 3.25 (t, J = 7.2 Hz, 2H, CH₂NH), 3.61, 3.64 (2s, 3H, CH₃O), 3.85, 3.86 (2s, 3H, CH₃O), 4.79, 4.80 (2s, 2H, SCH₂), 5.22 (s, 1H, NH), 6.45–7.99 (m, 14H, H_{Ar, Ar‘, Ar‘}‘+NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.3 (CH₂C), 40.0 (SCH₂), 42.2 (CH₂NH), 55.3, 55.3 (CH₃O), 55.6, 55.6 (CH₃O), 112.5, 114.0, 114.7, 127.4, 128.1, 130.0, 130.1, 130.8, 132.9, 141.6, 149.6, 150.6, 153.8 (C_Ar), 163.6 (C=O), 191.5 (C-S-); HRMS (ESI): m/z calcd for C₂₆H₂₆N₄O₃S 475.1804 [M + H]⁺, found 475.1802.

1-(4-Chlorophenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone (20): Prepared according to method A from 2-bromo-4’-chloroacetophenone (0.47 g). Yield 61%; white solid; m.p. 152–153 °C; IR (KBr) ν_max (cm⁻¹): 3291 (NH), 1694 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.73 (t, J = 7.2 Hz, 2H, CH₂C), 3.19 (t, 2H, J = 7.2 Hz, CH₂NH), 3.61 (s, 3H, CH₃O), 4.83 (s, 2H, SCH₂), 5.25 (s, 1H, NH), 6.31 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.63 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.44–8.06 (m, 9H, H_{Ar′, Ar″}); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.72 (CH₂C), 40.22 (SCH₂), 41.29 (CH₂NH), 55.28 (CH₃O), 113.10, 114.62, 127.37, 128.92, 129.80, 130.00, 130.07, 130.32, 132.90, 134.01, 138.62, 142.15, 149.24, 150.82 (C_Ar), 154.15 (C=O), 192.29 (C-S-); HRMS (ESI): m/z calcd for C₂₅H₂₃ClN₄O₂S 479.1308 [M + H]⁺, found 479.1306.

1-(4-Fluorophenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone (21): Prepared according to method C from 2-bromo-4′-fluoroacetophenone (0.43 g). Yield 73%; white solid; m.p. 145–146 °C; IR (KBr) ν_max (cm⁻¹): 3290 (NH); 1693 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.74 (t, J = 7.4 Hz, 2H, CH₂C), 3.19 (t, J = 7.4 Hz, 2H, CH₂NH), 3.61 (s, 3H, CH₃O), 4.84 (s, 2H, SCH₂), 5.22 (s, 1H, NH), 6.31 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.63 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.36–7.61 (m, 7H, H_{Ar’, Ar’’}), 8.07–8.11 (m, 2H, H_Ar’’); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.7 (CH₂C), 40.2 (SCH₂), 41.3 (CH₂NH), 55.3 (CH₃O), 113.1, 114.6, 115.8, 115.9, 127.4, 130.0, 130.1, 131.4, 132.1, 132.9, 142.2, 149.3, 150.8 (C_Ar), 154.1 (C=O), 191.8 (C-S-); HRMS (ESI): m/z calcd for C₂₅H₂₃FN₄O₂S 463.1604 [M + H]⁺, found 463.1601.

2-((5-(2-((4-Methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-1-(4-nitrophenyl)ethanone (22): Prepared according to method C from 2-bromo-4′-nitroacetophenone (0.49 g), recrystallized from propan-2-ol. Yield 88 %; brown solid; m.p. 169–170 °C IR (KBr) ν_max (cm⁻¹): 3306 (NH), 1698 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.74 (t, J = 7.4 Hz, 2H, CH₂C), 3.19 (t, J = 7.0 Hz, 2H, CH₂NH), 3.61 (s, 3H, CH₃O), 4.89 (s, 2H, SCH₂), 5.22 (s, 1H, NH), 6.31 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.63 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.46–7.49 (m, 2H, H_Ar’), 7.60–7.62 (m, 3H, H_Ar’), 8.23 (d, J = 8,7 Hz, 2H, H_Ar’’), 8.36 (d, J = 8.7 Hz, 2H, H_Ar’’); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.7 (CH₂C), 40.2 (CH₂NH), 41.3 (SCH₂), 55.3 (CH₃O), 113.1, 114.6, 123.9, 127.4, 129.8, 130.0, 130.1, 132.9, 140.1, 142.2, 149.1, 150.1, 150.8 (C_Ar), 154.2 (C=O), 192.7 (C-S-); HRMS (ESI): m/z calcd for C₂₅H₂₃N₅O₄S 490.1549 [M + H]⁺, found 490.1544.

1-(4-Hydroxyphenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone (23): Prepared according to method C from 2-bromo-4’-hydroxyacetophenone (0.43 g). Yield 84%; white solid; m.p. 198–199 °C; IR (KBr) ν_max (cm⁻¹): 3380, 3300 (OH, NH), 1649 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.74 (t, J = 7.4 Hz, 2H, CH₂C), 3.20 (q, J = 6.8, 13.0 Hz, 2H, CH₂NH), 3.61 (s, 3H, CH₃O), 4.76 (s, 2H, SCH₂), 5.23 (t, J = 5.6 Hz, 1H, NH), 6.32 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.64 (d, J = 8.8 Hz, 2H, H_Ar3,5), 6.88 (d, J = 8.7 Hz, 2H, H_Ar’’), 7.46–7.61 (m, 5H, H_Ar’), 7.88 (d, J = 8.7 Hz, 2H, H_Ar’’), 10.58 (s, 1H, OH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.7 (CH₂C), 40.2 (CH₂NH), 41.3 (SCH₂), 55.3 (CH₃O), 113.1, 114.6, 115.4, 126.7, 127.4, 129.9, 130.0, 131.1, 133.0, 142.2, 149.6, 150.8, 154.1 (C_Ar), 162.7 (C=O), 191.1 (C-S-); HRMS (ESI): m/z calcd for C₂₅H₂₄N₄O₃S 461.1647 [M + H]⁺, found 461.1653.

2-((5-(2-((4-Methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)-1-(naphthalen-2-yl)ethanone (24): Prepared according to method C from 2-bromo-2′-acetonaphthone (0.50 g). Yield 91%; yellow solid; m.p. 154–155 °C; IR (KBr) ν_max (cm⁻¹): 3283 (NH); 1688 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.74 (t, J = 7.4 Hz, 2H, CH₂C), 3.19 (q, J = 6.8, 14.0 Hz, 2H, CH₂NH), 3.61 (s, 3H, CH₃O), 5.01 (s, 2H, SCH₂), 5.23 (t, J = 5.7 Hz, 1H, NH), 6.31 (d, J = 8.8 Hz, 2H, H_Ar2,6), 6.63 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.47–8.76 (m, 12H, H_{Ar’, Ar’’}); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 24.7 (CH₂C), 40.3 (CH₂NH), 41.3 (SCH₂), 55.3 (CH₃O), 113.1, 114.6, 123.7, 127.1, 127.4, 127.7, 128.4, 128.9, 129.7; 130.0, 130.1, 130.6, 132.1, 132.6, 133.0, 142.2, 149.4, 150.8 (C_Ar), 154.1 (C=O), 193.1 (C-S-); (HRMS (ESI): m/z calcd for C₂₉H₂₆N₄O₂S 495.1854 [M + H]⁺, found 495.1849.

N-(4-Methoxyphenyl)-N-(2-(5-((2-oxo-2-(p-tolyl)ethyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)ethyl)acetamide (25): A mixture of triazolethione 18 (0.2 g, 0.44 mmol) and acetyl chloride (10 mL) was heated at reflux for 1 h. The reaction mixture was poured into a water-ice mixture. Precipitate formed was filtered off, washed with water, and recrystallized from propan-2-ol. Yield 96%; white solid; m.p. 163–164 °C; IR (KBr) ν_max (cm⁻¹): 1651, 1510 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 1.61 (s, 3H, CH₃); 2.38 (s, 3H, CH₃), 2.82 (t, J = 6.8 Hz, 2H, CH₂C), 3.66 (t, J = 6.8 Hz, 2H, CH₂N); 3.77 (s, 3H, CH₃O), 4.92 (s, 2H, SCH₂), 6.94 (d, J = 8.8 Hz, 2H, H_Ar2,6), 7.08 (d, J = 8.8 Hz, 2H, H_Ar3,5), 7.35–7.43 (m, 4H, H_{Ar’, Ar’’}), 7.56–7.63 (m, 3H, H_Ar’), 7.91 (d, J = 8.0 Hz, 2H, H_Ar’’); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 21.3 (CH₃), 22.3 (CH₃), 23.2 (CH₂C), 40.5 (SCH₂), 46.2 (CH₂N), 55.4 (CH₃O), 114.8, 127.2, 128.6, 129.4, 130.2, 130.6, 132.1, 132.7, 135.2, 144.5, 150.8, 153.5 (C_Ar), 158.5, 169.6 (C=O), 192.4 (C-S-); HRMS (ESI): m/z calcd for C₂₈H₂₈N₄O₃S 501.1960 [M + H]⁺, found 501.1963.

N-(2,5-Dimethyl-1H-pyrrol-1-yl)-3-((4-methoxyphenyl)amino)propanamide (26): A mixture of propanehydrazide 1 (1.05 g, 5 mmol), propan-2-ol (75 mL), hexane-2,5-dione (0.69 g, 6 mmol), and acetic acid (1.5 mL) was heated at reflux for 20 h. Afterwards cold water (25 mL) was added. Precipitate formed was filtered off and recrystallized from ethanol. Yield 93%; white solid; m.p. 119–120 °C; IR (KBr) ν_max (cm⁻¹): 3346, 2843 (NH), 1663 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 1.98, 2.02 (2s, 6H, 2CH₃), 2.54 (t, J = 6.9 Hz, 2H, CH₂CO), 3.32 (t, J = 6.9 Hz, 2H, CH₂NH), 3.65 (s, 3H, CH₃O), 5.23 (s, 1H, NH), 5.63, 5.71 (2s, 2H, 2CH), 6.54–6.78 (m, 4H, H_Ar), 10.60 (s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 10.9 (СH₃), 33.3 (CH₂CO), 40.1 (CH₂NH), 55.2 (CH₃O), 102.8, 113.3, 114.6, 126.7, 142.5, 150.9 (C_Ar), 170.4 (C=O); HRMS (ESI): m/z calcd for C₁₆H₂₁N₃O₂ 289.1790 [M + H]⁺, found 289.1790.

3.2.5. General Procedure for the Synthesis of Compounds 27a,b

To propanehydrazide 1 (2.09 g, 10 mmol) dissolved in methanol (30 mL), the corresponding isatin (11 mmol) dissolved in methanol (15 mL) and glacial acetic acid (2–3 drops) were added. The reaction mixture was heated at reflux for 10–15 min until precipitate formed. The precipitate was filtered off while hot and recrystallized from DMF/H₂O mixture.

3-((4-Methoxyphenyl)amino)-N’-(2-oxoindolin-3-ylidene)propanehydrazide (27a): Prepared from isatin (1.62 g). Yield 72%; yellow solid; m.p. 203–204 °C; IR (KBr) ν_max (cm⁻¹): 1694, 1728 (C=O), 3389, 3342, 3262 (NH); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 3.01–3.34 (m, 4H, CH₂CO+ CH₂NH), 3.63 (s, 3H, CH₃O), 5.26 (s, 1H, NHAr), 6.55–7.48 (m, 8H, H_{Ar, Ar‘}), 11.23 (s, 1H, NHC), 12.54 (s, 0.7H, NHCO), 12.98 (s, 0.3H, NHCO); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 31.2 (CH₂CO), 40.2 (CH₂NH), 55.3 (CH₃O), 111.1, 113.3, 114.6, 118.8, 119.8, 122.5, 131.4, 133.9, 142.3, 142.7, 150.9 (C_Ar), 162.5, 173.9 (C=O); HRMS (ESI): m/z calcd for C₁₈H₁₈N₄O₃ 339.1457 [M + H]⁺, found 339.1467.

N’-(1-Benzyl-2-oxoindolin-3-ylidene)-3-((4-methoxyphenyl)amino)propanehydrazide (27b): Prepared from N-benzylisatin (2.61 g). Yield 78%; yellow solid; m.p. 123–124 °C; IR (KBr) ν_max (cm⁻¹): 1678, 1614 (C=O), 3384, 3219 (NH); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.68–3.43 (m, 4H, CH₂CO + CH₂NH), 3.64 (s, 3H, CH₃O), 4.90–4.98 (m, 2H, CH₂), 5.82 (s, 1H, NHAr), 6.60–7.61 (m, 13H, H_{Ar, Ar′, Ar″}), 11.29, 12.48 (2s, 0.7H, NHCO), 12.91 (s, 0.3H, NHCO); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 31.1 (CH₂CO), 39.9 (CH₂), 42.5 (CH₂NH), 55.3 (CH₃O), 110.4, 113.8, 114.6, 114.8, 119.3, 120.2, 123.2, 127.4, 127.6, 128.7, 131.3, 135.7, 142.0, 142.5, 151.3 (C_Ar), 160.6, 173.8 (C=O); HRMS (ESI): m/z calcd for C₂₅H₂₄N₄O₃ 429.1926 [M + H]⁺, found 429.1929.

3.2.6. General Procedure for the Synthesis of Compounds 28–37

To propanehydrazide 1 (0.63 g, 3 mmol) dissolved in methanol (20 mL), corresponding acetophenone (3 mmol) and acetic acid (4 drops; in the case of compounds 29, 30, 32, 33, and 36) were added. The reaction mixture was heated at 95 °C for 4–24 h. Precipitate formed was filtered off, washed with water, and recrystallized from methanol.

N’-(1-(Furan-2-yl)ethylidene)-3-((4-methoxyphenyl)amino)propanehydrazide (28): Prepared from 2-acetylfuran (0.33 g, 0.30 mL) by heating the reaction mixture for 22 h. Yield 76%; white solid; m.p. 142–143 °C; IR (KBr) ν_max (cm⁻¹): 3361, 3389 (NH), 1679 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.16 (s, 3H, CH₃), 2.56 (t, J = 7.2 Hz, 0.8H, CH₂CO), 2.85 (t, J = 7.2 Hz, 1.2H, CH₂CO), 3.26 (t, J = 7.2 Hz, 2H, CH₂NH), 3.63 (s, 3H, CH₃O), 5.15, 5.20 (2t, J = 4.8 Hz, 1H, NHAr), 6.53–6.89 (m, 7H, H_{Ar, furan}), 10.30 (s, 0.4H, NH), 10.40 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 13.2, 13.5 (CH₃), 32.6, 34.1 (CH₂CO), 48.6 (CH₂NH), 55.3 (CH₃O), 110.6, 111.8, 113.1, 114.6, 139.6, 142.8, 144.2, 150.7 (C_Ar), 151.9 (C=N), 173.7 (C=O); HRMS (ESI): m/z calcd for C₁₆H₁₉N₃O₃ 302.1504 [M + H]⁺, found 302.1501.

3-((4-Methoxyphenyl)amino)-N’-(1-(thiophen-2-yl)ethylidene)propanehydrazide (29). (Z/E isomeric mixture, 60 : 40%): Prepared from 2-acetylthiophene (0.38 g, 0.32 mL) by heating the reaction mixture for 12 h. Yield 69%; white solid; m.p. 160–162 °C; IR (KBr) ν_max (cm⁻¹): 3363, 3389 (NH), 1679 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.26 (s, 3H, CH₃), 2.56 (t, J = 8.0 Hz, 0.8H, CH₂CO), 2.86 (t, J = 8.0 Hz, 1.2H, CH₂CO), 3.28 (t, J = 8.0 Hz, 2H, CH₂NH), 3.63 (s, 3H, CH₃O), 5.17 (s, 1H, NH), 6.55–6.74 (m, 4H, H_Ar), 7.05–7.07 (m, 1H, CH), 7.40–7.56 (m, 2H, CH), 10.36 (s, 0.4H, NH), 10.51 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 13.9, 14.4 (CH₃), 32.6, 34.1 (CH₂CO), 40.2 (CH₂NH), 55.3 (CH₃O), 113.2, 113.4, 114.6, 127.1, 127.4, 127.6, 127.7, 128.0, 128.6 (C_thiophene), 142.8, 142.9, 143.5, 143.6, 143.9, 147.9, 150.7, 150.9 (C_Ar), 167.6 (C=N), 173.6 (C=O); HRMS (ESI): m/z calcd for C₁₆H₁₉N₃O₂S 318.1276 [M + H]⁺, found 318.1273.

3-((4-Methoxyphenyl)amino)-N’-(1-(thiophen-2-yl)propylidene)propanehydrazide (30). (Z/E isomeric mixture, 60 : 40%): Prepared from 2-propionylthiophene (0.42 g, 0.37 mL) by heating the reaction mixture for 19 h. Yield 86%); white solid; m.p. 143–145 °C; IR (KBr) ν_max (cm⁻¹): 3345, 3390 (NH), 1675 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 1.05 (t, 3H, J = 8.0 Hz, CH₃CH₂), 2.58 (t, 0.8H, J = 8.0 Hz, CH₂CO), 2.77 (qui, 2H, J = 8.0 Hz, CH₃CH₂), 2.87 (t, 1.2H, Hz, CH₂CO), 3.28 (qui, 2H, J = 8.0 Hz, CH₂NH), 3.63, 3.64 (2s, 3H, CH₃O), 5.17, 5.18 (2s, 1H, NH), 6.55–6.75 (m, 4H, H_Ar), 7.04 (t, 1H, J = 4.0 Hz, CH), 7.39–7.56 (m, 2H, CH), 10.42 (s, 0.4H, NH), 10.65 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 11.1 (CH₃CH₂), 20.1, 20.4 (CH₃CH₂), 32.7, 34.1 (CH₂CO), 40.3 (CH₂NH), 55.3 (CH₃O), 113.2, 113.4, 114.6, 126.6, 127.2, 127.5, 127.7, 127.9, 128.5 (C_thiophene), 142.7, 142.8, 142.8, 142.9, 148.3, 150.7, 150.9, 151.7 (C_Ar), 167.7 (C=N), 173.7 (C=O); HRMS (ESI): m/z calcd for C₁₇H₂₁N₃O₂S 332.1432 [M + H]⁺, found 332.1431.

3-((4-Methoxyphenyl)amino)-N’-(1-phenylethylidene)propanehydrazide (31): Prepared from acetophenone (0.36 g, 0.35 mL) by heating the reaction mixture for 4h. Yield 75%; white solid; m.p. 155–156 °C; IR (KBr) ν_max (cm⁻¹): 3385, 3358 (NH), 1680 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.25 (s, 3H, CH₃), 2.59 (t, J = 6.8 Hz, 0.8H, CH₂CO), 2.93 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.24–3.32 (m, 2H, CH₂NH), 3.63, 3.64 (2s, 3H, CH₃O), 5.20 (s, 1H, NH), 6.52–6.77 (m, 4H, H_Ar), 7.33–7.82 (m, 5H, H_acetophenone), 10.39 (s, 0.4H, NH), 10.51 (s, 0.6H, NH), ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 13.6, 14.1 (CH₃), 32.7, 34.1 (CH₂CO), 40.3 (CH₂NH), 55.3 (CH₃O), 113.2, 113.4, 114.6, 126.0, 126.3, 128.3, 128.3, 128.9, 129.1 (C_acetophenone), 138.3, 142.8, 147.3, 150.7, 150.9 (C_Ar), 167.9 (C=N), 173.9 (C=O); HRMS (ESI): m/z calcd for C₁₈H₂₁N₃O₂ 312.1712 [M + H]⁺, found 312.1710.

3-((4-Methoxyphenyl)amino)-N’-(1-phenylpropylidene)propanehydrazide (32) (Z/E isomeric mixture, 60 : 40%): Prepared from propiophenone (0.4 g, 0.39 mL) by heating the reaction mixture for 12 h. Yield 75%; white solid; m.p. 136–138 °C; IR (KBr) ν_max (cm⁻¹): 3345, 3395 (NH), 1676 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 1.01 (t, J = 8.0 Hz, 3H, CH₃CH₂), 2.61 (t, J = 8.0 Hz, 0.8H, CH₂CO), 2.75–2.83 (m, 2H, CH₃CH₂), 2.95 (t, J = 8.0 Hz, 1.2H, CH₂CO), 3.25–3.33 (m, 2H, CH₂NH), 3.63, 3.64 (CH₃O), 5.20 (s, 1H, NH), 6.51–6.78 (m, 4H, H_Ar), 7.34–7.81 (m, 5H, H_acetophenone), 10.49 (s, 0.4H, NH), 10.65 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 10.8 (CH₃CH₂), 19.2, 19.5 (CH₃CH₂), 32.7, 34.2 (CH₂CO), 40.3 (CH₂NH), 55.3 (CH₃O), 113.2, 113.4, 114.6, 126.1, 126.4, 128.4, 128.5, 128.9, 129.0 (C_acetophenone), 137.1, 142.8, 142.9, 150.7, 150.9, 151.5, 154.8 (C_Ar), 168.1 (C=N), 174.1 (C=O); HRMS (ESI): m/z calcd for C₁₉H₂₃N₃O₂ 326.1868 [M + H]⁺, found 326.1864.

N’-(1-(4-Aminophenyl)ethylidene)-3-((4-methoxyphenyl)amino)propanehydrazide (33) (Z/E isomeric mixture, 60 : 40%): Prepared from 4′-aminoacetophenone (0.41 g) by heating the reaction mixture for 24 h. Yield 68%; white solid; m.p. 137–138 °C; IR (KBr) ν_max (cm⁻¹): 3464, 3363, 3332 (NH, NH₂), 1660 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.13 (s, 3H, CH₃), 2.54 (t, J = 6.8 Hz, 0.8H, CH₂CO), 2.89 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.26 (t, J = 6.8 Hz, 2H, CH₂NH), 3.64 (s, 3H, CH₃O), 5.11–5.22 (m, 1H, NH), 5.42 (s, 2H, NH₂), 6.52–6.59 (m, 4H, H_Ar), 6.68–6.75 (m, 2H, H_acetophenone), 7.45–7.53 (m, 2H, H_acetophenone), 10.14 (s, 0.4H, NH), 10.21 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 13.2, 13.7 (CH₃), 32.7, 34.2 (CH₂CO), 39.7, 40.2 (CH₂NH), 55.3 (CH₃O), 113.1, 113.2, 113.3, 114.6, 125.3, 125.5, 127.1, 127.5 (C_acetophenone), 142.9, 148.2, 149.9, 150.1, 150.7, 150.8 (C_acetophenone), 152.3 (C_Ar), 167.3 (C=N), 173.4 (C=O); HRMS (ESI): m/z calcd for C₁₈H₂₂N₄O₂ 327.1821 [M + H]⁺, found 327.1842.

N’-(1-(3-Aminophenyl)ethylidene)-3-((4-methoxyphenyl)amino)propanehydrazide (34): Prepared from 3′-aminoacetophenone (0.41 g) by heating the reaction mixture for 24 h. Yield 71%; white solid; m.p. 109–110 °C; IR (KBr) ν_max (cm⁻¹): 3464, 3379, 3358 (NH, NH₂), 1679 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.17 (s, 3H, CH₃), 2.57 (t, J = 6.8 Hz, 0.8H, CH₂CO), 2.91 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.28 (t, J = 6.8 Hz, 2H, CH₂NH), 3.63, 3.64 (2s, 3H, CH₃O), 5.13 (s, 3H, NH+NH₂), 6.51–7.08 (m, 8H, H_{Ar, acetophenone}), 10.27 (s, 0.4H, NH), 10.39 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 13.8, 14.2 (CH₃), 32.8, 34.2 (CH₂CO), 40.2, 40.3 (CH₂NH), 55.3, 55.3 (CH₃O), 111.5 (C-2_acetophenone), 113.2, 113.4, 113.9 (C-4_acetophenone), 114.3, 114.7, 115.0 (C-6_acetophenone), 128.7, 128.7, 138.9, 139.0 (C-5_acetophenone), 142.9 (C-1_acetophenone), 148.2, 148.5, 148.6 (C-3_acetophenone), 150.7, 150.9, 151.8 (C_Ar), 167.8 (C=N), 173.8 (C=O); HRMS (ESI): m/z calcd for C₁₈H₂₂N₄O₂ 327.1821 [M + H]⁺, found 327.1835.

N’-(1-(3,4-Dimethoxyphenyl)ethylidene)-3-((4-methoxyphenyl)amino)propanehydrazide (35): Prepared from 3′,4′-dimethoxyacetophenone (0.54 g) by heating the reaction mixture for 48 h. Yield 27%; white solid; m.p. 73–74 °C; IR (KBr) ν_max (cm⁻¹): 3465, 3366 (NH), 1675 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.21, 2.22 (2s, 3H, CH₃), 2.57 (t, J = 6.8 Hz, 0.8H, CH₂CO), 2.92 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.24–3.32 (m, 2H, CH₂NH), 3.62, 3.64 (2s, 3H, CH₃O), 3.72, 3.78, 3.81, 3.84 (4s, 6H, 2CH₃O), 5.15–5.23 (m, 1H, NH), 6.50–6.75 (m, 4H, H_Ar), 6.92–7.65 (m, 3H, H_acetophenone), 10.41 (s, 1H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 13.5, 14.0 (CH₃), 32.8, 34.2 (CH₂CO), 39.6, 40.4 (CH₂NH), 55.2, 55.3, 55.3, 55.4, 55.5, 55.7 (CH₃O), 109.1, 109.2, 110.2, 110.8, 111.0, 111.2 (C-2,5_acetophenone), 113.2, 113.3, 114.6, 114.6, 119.2, 119.6 (C-6_acetophenone), 129.9, 130.9, 131.0 (C-1_acetophenone), 142.9, 148.4, 148.5, 148.5 (C-3_acetophenone), 149.8, 150.0, 150.7, 150.8, 151.3, 153.0 (C-4_acetophenone) (C_Ar), 167.7 (C=N), 173.8 (C=O); HRMS (ESI): m/z calcd for C₂₀H₂₅N₃O₄ 372.1923 [M + H]⁺, found 372.1921.

3-((4-Methoxyphenyl)amino)-N’-(1-(naphthalen-1-yl)ethylidene)propanehydrazide (36). (Z/E isomeric mixture, 60 : 40%): Prepared from 1-acetonaphthone (0.51 g, 0.46 mL) by heating the reaction mixture for 5 h. Yield 65%; white solid; m.p. 152–154 °C; IR KBr) ν_max (cm⁻¹): 3337, 3290 (NH), 1667 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.37, 2.38 (2s, 3H, CH₃), 2.64 (t, J = 6.8 Hz, 0.8H, CH₂CO), 2.79 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.19–3.32 (m, 2H, CH₂NH), 3.57, 3.65 (2s, 3H, CH₃O), 5.15 (s, 0.6H, NH), 5.22 (s, 0.4H, NH), 6.43–6.76 (m, 4H, H_Ar), 7.48–7.58 (m, 4H, H_{acetonaphthone}), 7.90–8.21 (m, 3H, H_{acetonaphthone}), 10.51 (s, 0.4H, NH), 10.61 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 18.8, 19.1 (CH₃), 32.9, 34.2 (CH₂CO), 40.2 (CH₂NH), 55.2, 55.3 (CH₃O), 113.1, 113.4, 114.4, 114.7, 125.2, 125.3, 125.3, 125.6, 125.9, 126.0, 126.5, 126.6, 128.4, 128.6, 128.7 (C_{acetonaphthone}), 130.2, 133.4, 137.7, 137.9, 142.6, 142.9, 149.4, 150.6, 150.9 (C_Ar), 153.5 (C=N), 167.9, 173.9 (C=O); HRMS (ESI): m/z calcd for C₂₂H₂₃N₃O₂ 362.1868 [M + H]⁺, found 362.1868.

3-((4-Methoxyphenyl)amino)-N’-(1-(naphthalen-1-yl)ethylidene)propanehydrazide (36). (Z/E isomeric mixture, 60 : 40%): Prepared from 1-acetonaphthone (0.51 g, 0.46 mL) by heating the reaction mixture for 5 h. Yield 65%; white solid; m.p. 152–154 °C; IR KBr) ν_max (cm⁻¹): 3337, 3290 (NH), 1667 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.37, 2.38 (2s, 3H, CH₃), 2.64 (t, J = 6.8 Hz, 0.8H, CH₂CO), 2.79 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.19–3.32 (m, 2H, CH₂NH), 3.57, 3.65 (2s, 3H, CH₃O), 5.15 (s, 0.6H, NH), 5.22 (s, 0.4H, NH), 6.43–6.76 (m, 4H, H_Ar), 7.48–7.58 (m, 4H, H_{acetonaphthone}), 7.90–8.21 (m, 3H, H_{acetonaphthone}), 10.51 (s, 0.4H, NH), 10.61 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 18.8, 19.1 (CH₃), 32.9, 34.2 (CH₂CO), 40.2 (CH₂NH), 55.2, 55.3 (CH₃O), 113.1, 113.4, 114.4, 114.7, 125.2, 125.3, 125.3, 125.6, 125.9, 126.0, 126.5, 126.6, 128.4, 128.6, 128.7 (C_{acetonaphthone}), 130.2, 133.4, 137.7, 137.9, 142.6, 142.9, 149.4, 150.6, 150.9 (C_Ar), 153.5 (C=N), 167.9, 173.9 (C=O); HRMS (ESI): m/z calcd for C₂₂H₂₃N₃O₂ 362.1868 [M + H]⁺, found 362.1868.

3-((4-Methoxyphenyl)amino)-N’-(1-(naphthalen-2-yl)ethylidene)propanehydrazide (37): Prepared from 2-acetonaphthone (0.51 g) by heating the reaction mixture for 16 h. Yield 69%; white solid; m.p. 141–142 °C; IR (KBr) ν_max (cm⁻¹): 3367, 3310 (NH) 1662 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.37 (s, 3H, CH₃), 2.64 (t, J = 6.8 Hz, 0.8H, CH₂CO), 3.01 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.27–3.39 (m, 2H, CH₂NH), 3.61, 3.64 (2s, 3H, CH₃O), 5.23 (s, 1H, NH), 6.53–6.78 (m, 4H, H_Ar), 7.48–8.27 (m, 7H, H_{acetonaphthone}), 10.50 (s, 0.4H, NH), 10.62 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 13.4, 13.8 (CH₃), 32.8, 34.3 (CH₂CO), 39.6, 40.3 (CH₂NH); 55.2, 55.3 (CH₃O), 113.2, 113.4, 114.6, 123.4, 123.7, 125.9, 126.2, 126.4, 126.7, 126.7, 127.5, 127.6, 127.7, 128.4, 132.8, 132.8, 133.1, 133.2, 135.6, 135.7 (C_{acetonaphthone}); 142.9, 142.9, 150.6, 150.7, 150.9 (C_Ar), 153.5 (C=N), 168.1, 174.0 (C=O); HRMS (ESI): m/z calcd for C₂₂H₂₃N₃O₂ 362.1868 [M + H]⁺, found 362.1868.

N-(4-Methoxyphenyl)-N-(3-oxo-3-(2-(1-phenylethylidene)hydrazinyl)propyl)acetamide (38): To propanehydrazide 31 (0.19 g, 0.6 mmol) dissolved in methanol (5 mL), acetic anhydride (5 mL) was added and the reaction mixture was heated at reflux for 2 h. Afterwards water (10 mL) was added. Precipitate formed was filtered off, washed with methanol, and recrystallized from methanol. Yield 59 %; white solid; m.p. 145–146 °C; IR (KBr) ν_max (cm⁻¹): 3176 (NH) 1658; 1607 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 1.70 (s, 3H, CH₃), 2.19, 2.23 (2s, 3H, CH₃), 2.54 (t, J = 6.8 Hz, 0.8H, CH₂CO), 2.84 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.77 (s, 3H, CH₃O), 3.87 (t, 2H, CH₂N), 6.93–7.78 (m, 9H, H_Ar), 10.37 (s, 0.4H, NH), 10.47 (s, 0.6H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 13.4, 14.1 (CH₃), 22.5 (CH₃), 31.5, 32.8 (CH₂CO), 44.9, 45.1 (CH₂N), 55.3 (CH₃O), 114.7, 125.9, 126.3, 128.3, 128.9, 129.4 (C_acetophenone), 135.4, 138.0, 138.3, 147.2, 158.4 (C_Ar), 167.1, 169.2, 169.2, 173.1 (C=O); HRMS (ESI): m/z calcd for C₂₀H₂₃N₃O₃ 354.1817 [M + H]⁺, found 354.1844.

N-(1,3-Dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide (39): To propanehidrazyde 1 (0.63 g, 3 mmol) dissolved in 1,4-dioxane (20 mL), phthalic anhydride (0.89 g, 6 mmol) was added and the reaction mixture was heated at reflux for 4 h. Afterwards Na₂CO₃ was added until pH 7. Precipitate formed was filtered off, washed with water, and recrystallized from methanol. Yield 81%; white solid; m.p. 147–148 °C; IR (KBr) ν_max (cm⁻¹): 3331, 3290 (NH), 1664 (C=O); ¹H-NMR (DMSO-d₆, 400 MHz) δ: 2.46 (t, J = 6.8 Hz, 0.8H, CH₂CO), 2.89 (t, J = 6.8 Hz, 1.2H, CH₂CO), 3.25–3.30 (m, 2H, CH₂NH), 3.64 (s, 3H, CH₃O), 5.14–5.24 (m, 1H, NH), 6.54–6.75 (m, 4H, H_Ar), 7.37–7.70 (m, 4H, H_phthal), 11.32 (s, 0.6H, NH), 11.38 (s, 0.4H, NH); ¹³C-NMR (DMSO-d₆, 101 MHz) δ: 32.2 (CH₂CO), 34.3 (CH₂NH); 55.3, 55.3 (CH₃O), 113.2, 113.2, 114.6, 126.6, 126.9, 128.8, 129.7, 129.9, 134.3, 134.3, 142.7, 150.7 (C_Ar), 167.3, 173.1 (C=O); HRMS (ESI): m/z calcd for C₁₈H₁₇N₃O₄ 340.1297 [M + H]⁺, found 340.1299.

3.3. Evaluation of Antioxidant Activity

The free radical scavenging activity of compounds was screened by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay [4]. Briefly, 1 mM solution of DPPH in ethanol was prepared, and this solution (1 mL) was added to the solutions of the tested compounds (1 mg/mL of DMSO). The mixture was shaken vigorously and allowed to stand at room temperature for 20 min. Afterwards, the absorbance was measured at 517 nm with a UV-200-RS spectrophotometer (MRC Ltd., Israel). The lower absorbance of the reaction mixture indicated a higher free radical scavenging activity. The capability to scavenge the DPPH radical was calculated according to the following equation:

$$DPPHscavengingeffect(\%)=\frac{A_0-A_1}{A_0}\times 100$$

(1)

where A₀—the absorbance of the control reaction and A₁—the absorbance in the presence of the compounds.

3.4. Evaluation of Anticancer Activity

The anticancer activity of the synthesized compounds was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Co.) assay as described elsewhere [37]. Briefly, human glioblastoma U-87 and human triple-negative breast cancer cell line MDA-MB-231 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were grown in Dulbecco’s Modified Eagle’s Medium GlutaMAX (DMEM GlutaMAX) (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS and 1% antibiotics (10,000 U/mL penicillin and 10 mg/mL streptomycin; Gibco) at 37 °C in a humidified atmosphere containing 5% CO₂. Cell cultures were grown to 70% confluence and trypsinized with 0.125% TrypLE™ Express solution (Gibco) before passage. They were used until passage 20.

One hundred μL of cancer cells were seeded in 96-well plates in triplicate (5 × 10³ cells/well) and incubated at 37 °C for 24 h. Next day the tested compounds were added into cells at a concentration of 100 µM in triplicate. Final dimethylsulfoxide (DMSO, solvent of tested compounds) concentration in cells was 0.5%. Free medium without cells was used as a positive control. Cells treated with medium containing 0.5% DMSO served as a blank, or negative control. After 72 h incubation, the medium was aspirated from the plate/Fresh cell culture medium containing 0.5 mg/mL of MTT was added into each well and incubated for 4 h at 37 °C in a humidified atmosphere containing 5% CO₂. Then the liquid was aspirated from the wells. Formazan crystals were dissolved in 100 μL of DMSO. Absorbance was measured at 570 nm and a reference wavelength of 630 nm using a multi-detection microplate reader Multiskan go (Thermo Fisher Scientific Oy, Ratastie, Finland).

Compound effect on cell viability was calculated using the formula:

$$Relativecellviability(\%)=\frac{A-A_0}{A_{NC}-A_0}\times 100$$

(2)

where A—mean of absorbance of the tested compound, A₀—mean of absorbance of blank (no cells, positive control) and A_NC—mean of absorbance of a negative control (only cells, no treatment).

4. Conclusions

A series of novel 3-[(4-methoxyphenyl)amino]propanehydrazide derivatives were synthesized. Antioxidant activity of the synthesized compounds was screened by DPPH radical scavenging method. The antioxidant activity of N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)-amino)propanamide (39) and 3-((4-methoxyphenyl)amino)-N’-(1-(naphthalen-1-yl)ethylidene)-propanehydrazide (36) surpassed that of ascorbic acid ca. 1.4 times. 1-(4-Fluorophenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone (21), which reduced cell viability at 100 µM concentration up to 19.6 ± 1.5%, has been identified as the most active compound against glioblastoma U-87 cell line.