Synthesis, characterization and antibacterial assays of new N,N 1 -disubstituted 2,2’- dithiodianiline derivatives

doi:10.21203/rs.3.rs-2397833/v1

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Synthesis, characterization and antibacterial assays of new N,N 1 -disubstituted 2,2’- dithiodianiline derivatives

https://doi.org/10.21203/rs.3.rs-2397833/v1

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A series of novel 1,2-bissubstituted disulfanes bearing beta-amino acid, dihydropyrimidine-2,4-(1H,3H)-dione, hydrazide, hydrazone and azole moieties were synthesized. These disulphides were characterised by spectral and microanalysis data. On the antibacterial evaluation, they were found to have interesting antibacterial properties over a panel of the tested Gram-positive and Gram-negative bacteria: Staphylococcus aureus subs. aureus (ATCC 9144) and zoonotic agent Listeria monocytogenes (ATCC 35152), as well as Gram-negative ones, Escherichia coli (ATCC 13076) and zoonotic agent Salmonella enterica subs. enterica serovar Enteritidis (ATCC 8739). The antibacterial activity was evaluated by determining minimum inhibition (by broth microdilution) and minimum bactericidal (by growth on agar) concentrations. The assessment revealed that MIC values for L. monocytogenes varied between 3.9 and 62.5 µg/mL as well as for S. aureus ranged between 7.8 and 250 µg/mL, with the exception of one compound with much weaker MIC of 500 µg/mL. The MBC values for L. monocytogenes have been found to be of 7.8−250 µg/mL, while S. aureus demonstrated the higher resistance and MBCs varied in the range of 7.8\(\)500 µg/mL. The determined MBC/MIC ratios showed that eleven compounds were classified bactericidal agents for all tested bacteria.

disulfur bridge

β-amino acid

hydrazone

azole

antibacterial activity

The disulfide bond (DSB) plays an important role in the sulfur-containing organic molecules and affects their wide applications in different fields, including medicinal chemistry and pharmaceuticals. The DSB having unique chemical and biophysical properties is one of the essential structural elements of bioactive proteins and peptides, drugs, natural products and other fields [1−9]. Almost all living organisms contain catalytic systems that promote S−S bond formation and are necessary for the biosynthesis of many proteins, such as antibodies, major histocompatibility complex molecules, growth factors, and insulin [10]. The S−S structural functionality strongly influences the stability, folding, and biological function of proteins and peptides as well as maintain the cellular redox balance in the cells of organisms [1] as well as serves for protein modification, furthermore they are widely used in the design of CDDS (controlled drug delivery systems) [11, 12]. Disulfides are components of many proteins, biopolymers including hormones, neurotransmitters, enzyme inhibitors, growth factors, and antimicrobial peptides. The DSB system is a master regulator of bacteria virulence [1, 13−15].

Diaryl disulfides are one of the most widely used structural motifs, performing important functions in bioactive compounds concomitantly in pharmaceuticals [16, 17]. Among numerous drugs containing variously substituted disulfide moiety, for instance, agents for the treatment of cancer [18,] and pulmonary diseases [19], medication for the treatment of chronic alcoholism [20], preparations for the inhibition of the oxytocin and vasopressin [21], analgetic drug [22] can be mentioned. Sulfur − sulfur bonds are prevalent in drugs such as anticancer agent romidepsin, antioxidant lipoic acid, hormone inhibitor atosiban.

Psammaplin A which was isolated from Psammaplysilla sp. attracted considerable attention owning on its potent HDAC and DNA methyltransferase inhibition and antimicrobial properties [23, 24]. Aromatic-Disulfide Based Inhibitors exhibit potent enzymatic activities against SARS-CoV Mpro. [25]. A number of diaryl disulfide Schiff bases demonstrate antibacterial and herbicidal effect [26−28], the bis-chlorophenyl disulfide was reported to be effective against cancer [29, 30], small-molecule PDI (protein disulfide isomerase) appeared to inhibit HIV-1 entry into cells [31].

A disulfide derivative allithiamine (thiamine allyl disulfide) is a derivative of vitamin B₁ and allicin, which was isolated from garlic (Allium sativum) [32], could alleviate the hyperglycaemia-induced endothelial dysfunction [33], improve the sensation of neuropathic pain [34].

Previously, we have synthesized a library of N,N-disubstituted β-aminoacids derivatives containing hydrazone and azole fragments and have evaluated their biological properties, which appeared to possess the convincing anticancer effect against triple-negative breast cancer and glioblastoma cells in vitro [35], demonstrated promising antimicrobial effect [36−39], as well as significant antibacterial [40−43], and in addition, antiviral and antioxidant [42, 43] activities.

As part of our ongoing search for bioactive compounds the main objective of our current research was to design and to synthesize a series of disulfide-bridged derivatives and to evaluate their antibacterial properties.

Synthesis

The family of 2,2’-dithiodianilines are reported in the scientific literature as very important compounds with wide-ranging biological activities, and this encouraged us to synthesize a series of symmetrical N-substituted 2,2‘-dithioaniline derivatives (Scheme 1) and assess their antibacterial properties.

Synthesis of the target 2,2'-((disulfanediylbis(2,1-phenylene))bis(azanediyl))bis(1-phenylethan-1-one) (2a) and 2,2'-((disulfanediylbis(2,1-phenylene))bis(azanediyl))bis(1-(4-chlorophenyl)ethan-1-one) (2b) (Scheme 1) was achieved by applying the initial 2,2‘-dianilinodisulfide and α-haloketones. Reactions with 2-bromoacetophenone and 2-bromo-4‘-chloroacetophenone were performed in propan-2-ol at room temperature for 24 hours and resulted in symmetrical derivatives 2a and 2b, respectively (Supplementary materials, Figures S1−S26).

For the preparation of diacid 3, diamine 1 was reacted with acrylic acid in toluene at 100 °C for 24 hours. The obtained structure was approved by the data of the ¹H NMR spectrum, where a singlet at 12.31 ppm, a signal in the range of 6.48−7.23 ppm, as well as a singlet at 5.42 ppm exactly correspond to OH, aromatic and NH protons (Supplementary materials, Figure S5).

To transform β-alanine 3 into the pyrimidinedione 5, compound 3 was treated with urea in refluxing acetic acid for 18 hours, followed by refluxing with the addition of 18% hydrochloric acid to pH 2 to cyclize the intermediate ureido acid into bis(dihydropyrimidine-2,4(1H,3H)-dione) 5, which then was isolated in the yield of 60%. In the ¹H NMR spectrum of cyclic molecule 5, the proton of the secondary amine adjacent to the phenyl ring (CH₂NH), which resonated as a singlet 5.42 ppm for compound 3 disappeared and a singlet at 10.54 ppm characteristic for C(O)NHC(O) fragment proton arised. The spectrum also shows a down-field shift of the proton signals of the alkyl chain (CH₂CH₂), which is characteristic of a cyclic structure (Supplementary materials, Figures S6 and S7). The mass spectrum also confirmed the formation of a cyclic structure 5 structure (Supplementary materials, Figures S26).

In the next stage of the work, dihydrazide 7 synthesis optimization was performed. For this purpose, diacid 3 was converted into the diester 6 by refluxing in methanol for 24 h in the presence of a catalytic amount of concentrated sulfuric acid. The obtained ester was then subjected to hydrazinolysis. Noteworthy, efforts to obtain dihydrazide 7 in high yields by standard methods have not been successful. Therefore, to obtain the best possible yield, several conditions have been examined (Table 1). The stirring of compound 6 in propan-2-ol with the 10-fold excess hydrazine monohydrate (entry A) at room temperature (rt) for 72 hours gave hydrazide in 26% yield, while reaction with the 20-fold excess of hydrazine monohydrate in a solvent-free conditions (entry B) at room temperature for 72 hours resulted in only 19% yield. And finally, the hydrazinolysis was performed in methanol (entry C). Stirring for 48 hours at room temperature gave a yield of 51%.

Table 1

Optimization of the synthesis of dihydrazide 7.
Entry	Solvent	Excess of N₂H₄·H₂O	Temperature	Time, h	Yield, %
A	2-PrOH	10-fold	rt	72	26
B	Solvent free	20-fold	rt	72	19
C	MeOH	10-fold	rt	48	51

The higher reaction temperatures, leading to the formation of various polymers, and complicating the isolation of the pure target product from the reaction mixture, were not favoured. Preparation of dihydrazide 7 directly from diacid 3 was also attempted but the reaction failed. The ¹H NMR spectrum of 7 confirmed the structure, where NHNH₂ singlets were observed at 9.08 and 4.21 ppm, respectively (Supplementary materials, Figure S8). The ¹³C NMR showed an intense characteristic resonance line at 170.09 ppm, which was attributed to the 2C = O of the molecule.

Our attention was next directed to the preparation of the targeted hydrazones 8–21 (Scheme 1). The resulting bishydrazide 7 was subjected to condensation with a series of aromatic and carbaldehydes to generate an appropriate hydrazone-type structure 8–21. The products were obtained in good to excellent yield (71−92%) for aromatic series and in the range of 41−65% for carbaldehyde series. The ¹H NMR spectra of hydrazones 8–21 exhibited NH group singlets in the region of 11.25–11.88 ppm, and in addition CH = N singlets in the range of 7.76–8.41 ppm, which along with the increased intensity of the proton signals in the aromatic field of the spectrum approved the formed new structures (Figures S9−S22 in Supplementary Material). The presence of amide NH−CO bond caused the restricted rotation around it and at the same time determined the splitting of the proton resonances indicating that in DMSO-d₆ solutions hydrazones exist as a mixture of Z/E conformers where usually the Z-form predominates [44−47]. The ¹H NMR spectra of the prepared hydrazones 8–21 displayed double sets of resonances of the CH = N and CO−NH groups protons with signal intensity ratio of 60: 40.

Preparation of the desired bishydrazide allowed us to investigate the synthesis of some azole derivatives. The bis 2,5-dimethylpyrrole derivative 22 was synthesized from the hydrazide 7 and 2,5-hexanedione by refluxing them in propan-2-ol for 1 hour in the presence of an acetic acid catalyst in the reaction mixture. The obtained product was identified (¹H NMR) from the characteristic singlets of the four methyl groups resonating at 1.97 and 2.01 ppm and two singlets at 5.63 and 5.73 ppm, attributed to the CH groups of the pyrrole cycle. Such splitting of the signals may be caused by the restricted rotation of the pyrrole moieties around the amide bonds. The peak at 10.67 ppm was assigned to the protons of 2NH−CO groups (Supplementary materials, Figure S23). For the bis 3,5‐dimethylpyrazole 23, an analogous reaction using pentane-2,4-dione instead of reagent mentioned above was applied. A slightly longer 3-hour interaction gave the target product in 81% yields. The peaks at 2.17 and 2.46 ppm for methyl groups, and singlet at 6.16 ppm for CH_pyr in the ¹H NMR spectrum confirmed formation of two 3,5-dimethylpyrazole cycles (Supplementary materials, Figure S24).

Biological Activity

The increasing prevalence of hospital and community-acquired infections caused by multidrug-resistant (MDR) bacterial pathogens limits the options for effective antibiotic therapy. Gram-negative bacteria are slightly more tolerant than Gram-positive ones, since their outer membrane acts as a barrier preventing cell penetration by hydrophobic chemicals.

The antibacterial properties of synthesized compounds 1–23 were investigated against Gram-positive bacteria: Staphylococcus aureus subs. aureus (ATCC 9144) and zoonotic agent Listeria monocytogenes (ATCC 35152) and Gram-negative Escherichia coli (ATCC 13076) and zoonotic agent Salmonella enterica subs. enterica serovar Enteritidis (ATCC 8739). The minimal inhibitory concentration (MIC) was evaluated by the broth dilution method, while the minimal bactericidal concentration (MBC) was determined by plating. Antibiotic − Cefazolin used as a control (C) for antibacterial activity screening.

The investigations in vitro demonstrated that compounds 1–23 had the antibacterial activity of the broad or narrow spectrum for Gram-positive and Gram-negative bacteria.

All compounds, except 10 had an inhibitory effect on the growth of Gram-positive bacterium Listeria monocytogenes. If compare with another Gram-positive bacterium Staphylococcus aureus, the results showed, that compounds 2b, 12, 13, 14, 15, 16, 23 did not inhibited the growth of this strain (Fig. 1).

MIC values for the L. monocytogenes varied in the interval from 3.9−62.5 µg/mL, and MBC were obtained to be from 7.8 to 250 µg/mL. MIC and MBC values of S. aureus ranged from 7.8 to 500 µg/mL and 31.2 to 500 µg/mL, respectively.

The studies showed that all synthezided compounds inhibited the growth of Gram-negative bacteria E. coli and S. Enteritidis. MIC values for E. coli were found from 62.5 and 125 µg/mL, and for S. Enteritidis only 125 µg/mL was recorded (Fig. 1) meanwhile MBCs for the same strains ranged from 125 to 500 µg/mL (Fig. 2).

Evaluation of MBC and MIC ratios showed that compounds 2a, 3, 5, 7, 8, 9, 11, 17, 19, 21, 22 were bactericidal for all tested bacteria (Fig. 3). The effect was regarded as a bactericidal when a ratio of MBC/MIC was ≤ 4 and bacteriostatic − when the ratio of MBC/MIC > 4.

The investigated compounds were defined as bactericidal substances to Gram-negative bacteria E. coli and S. Enteritidis. All compounds were defined as bactericidal substances to S. aureus if exclude seven non applicable (2b, 12−16, 23) ones. Eight compounds (1, 2b, 13−15, 18, 20, 23) were bacteriostatic for Gram-positive motile bacterium L. monocytogenes, while thirteen compounds (2a, 3, 5, 7−9, 11, 12, 16, 17, 19, 21, 22) showed bactericidal properties.

The results showed, that cefazolin was effective at 15.6 µg/mL concentration of MIC and at 31.3 µg/mL of MBC for S. aureus. The antibiotic was effective at 31.3 µg/mL concentration of MIC against L. monocytogenes, E. coli, S. Enteritidis and MBC at the same concentration against the test stains L. monocytogenes and S. Enteritidis. The MBC of E. coli was detected at 62.5 µg/mL.

The results showed that some of the investigated compounds as bactericides were more active than ceftazolin.

This studies showed that investigated derivatives possess broad-spectrum, potent antibacterial activity against selected Gram-positive and Gram-negative bacteria strains.

The comparison of efficacy of compound 1 and its derivates 2a, 2b and 3 showed some difference in the spectrum of antibacterial activity, MIC, and MBC against the tested strains of bacteria. Compound 1 was more potent against the tested bacteria than its derivates in majority of cases using different indices. For example, compound 1 and its derivates 2a and 3 showed the equal spectrum of antibacterial activity, while 2b could not inhibit the growth of S. aureus. Compound 1 inhibited the growth and cause the death of ≥ 99.9% S. aureus at lower concentrations (7.8 µg/mL) than its derivates 2a (62.5 µg/mL) and 3 (250 µg/mL). Despite being ineffective against S. aureus, compound 2b was active at the same MIC (7.8 µg/mL) and MBC (62.5 µg/mL) against L. monocytogenes as initial compound 1. Compounds 2a and 3 could inhibit the growth (31.2 µg/mL and 62.5 µg/mL) and cause the death (125 µg/mL and 250 µg/mL) of ≥ 99.9% L. monocytogenes at higher concentrations than compound 1. Testing with gram-negative bacteria showed, that compounds 2a, 2b and 3 inhibited the growth of E. coli at two-fold higher concentration than compound 1 (125 µg/mL vs 62.5 µg/mL). The death of ≥ 99.9% E. coli was caused by at the same concentrations of compounds 1 and 2a, and two-fold higher ones by compounds 2b and 3. The study of inhibitory potency against S. Enteritidis did not show any differences, while bactericidal effect of compound 2a was found at the lowest concentration (125 µg/mL) in comparison with 1, 2b (both 250 µg/mL) and 3 (500 µg/mL).

The comparison of efficacy of compound 3 and its derivates 5 and 7 did not showed any differences in the spectrum of antibacterial activity. The compared compounds were effective against all four tested strains of bacteria. Compound 3 was less potent against the tested bacteria than its derivates 5 and 7 in more cases using different indices. For example, compound 3 could inhibit the growth of S. aureus at the concentration 250 µg/mL, while 5 and 7–125 µg/mL. Furthermore, compounds 3, 5, and 7 could inhibit the growth of L. monocytogenes at the same concentrations 62.5 µg/mL, as well as E. coli and S. Enteritidis at 125 µg/mL. More obvious differences were found when comparing MBC. All three compounds showed equal low antibacterial activity (500 µg/mL) against E. coli, while differences were found against other bacteria. Compound 5 ant 7 could cause the death of ≥ 99.9% of S. aureus, L. monocytogenes and S. Enteritidis at lower concentrations.

The comparison of efficacy of compound 7 and its derivates 8−21, 22 and 23 showed the highest diversity in the spectrum of antibacterial activity, MIC and MBC against the tested strains of bacteria. Compound 7 was as the same, as more, or as less potent against the tested gram-positive bacteria in comparison with its derivates using different indices. Five derivates of group of compounds 8−21 showed no inhibitory potency against S. aureus and one against L. monocytogenes, while the growth of gram-negative tested bacteria was stopped at the same concentration (125 µg/mL). The killing of ≥ 99.9% of tested bacteria occurred at the diverse concentrations showing that some compounds were more effective than compound 7 against S. aureus, L. monocytogenes and E. coli, but not S. Enteritidis.

In summary, a series of new disulfur-bridged bisderivatives were synthesized and their antibacterial properties were investigated. The antibacterial susceptibility test confirmed all these derivatives to possess an inhibitory effect on the growth of both Gram-negative bacteria species but only 14 of them inhibited both Gram-positive ones. Among the compounds tested, an initial 2,2’-dithiodianiline (1) and hydrazone 19 bearing thien-2-yl fragments exhibited potent inhibitory effect against S. aureus wit MIC values of 7.8 µg/mL. L. monocytogenes appeared to be sensitive to the action of a series of the synthesized compounds, for instance, 1, 2b, 12−14, 18, and 23, which showed MIC of 7.8 µg/mL, while 20 − 3.9 µg/mL; the MIC of derivative 15 was on par with that of control antibiotic (15.6 µg/mL), while the minimum inhibition concentration of 2a, 8, 9, 16, 17 and 19 was slightly higher (31.2 µg/mL). Gram-negative strains demonstrated much stronger resistance against the test compounds. The lowest MIC of 125 µg/mL was showed by almost all compounds on the action of E. coli and S. Enteritidis except 2,2’-dithiodianiline and hydrazone 21 having 5-nitrofuran-2-yl moieties (62.5 µg/mL), however, they were still worse than the control cefazolin (31.2 µg/mL).

As for the minimal bactericidal effect, compounds 1 and 19 were also the most active against S. aureus, showing the significantly higher or comparable efficacy with MBCs of 7.8 and 31.2 µg/mL, respectively. For L. monocytogenes, compounds 12 and 20 demonstrated also the strongest action, which was much stronger or comparable to the control (MBC values of 7.8 and 31.2 µg/mL, respectively, if compare with control MBC of 31.2 µg/mL). Similarly, as for inhibition case, the gram-negative bacteria strains were much resistant to the ability of compounds to kill them. The lowest MBC of 125 µg/mL which destroyed E. coli and S. Enteritidis was demonstrated by hydrazones 13 and 14, bearing 4-Me₂NPh and 2-Cl-5-O₂NPh moieties, and phenylethanone derivative 2a, respectively.

Synthesis

Reagents, antibiotics, and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. The reaction course and purity of the synthesized compounds were monitored by TLC using aluminum plates precoated with Silica gel with F254 nm (Merck KGaA, Darmstadt, Germany). Melting points were determined with a B-540 melting point analyzer (Büchi Corporation, New Castle, DE, USA) and were uncorrected. NMR spectra were recorded on a Brucker Avance III (400, 101 MHz) spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Chemical shifts were reported in (d) ppm relative to tetramethylsilane (TMS) with the residual solvent as internal reference (DMSO-d₆, δ = 2.50 ppm for ¹H and d = 39.5 ppm for ¹³C). Data were reported as follows: chemical shift, multiplicity, coupling constant (Hz), integration, and assignment. IR spectra (ν, cm^− 1) were recorded on a Perkin–Elmer Spectrum BX FT–IR spectrometer (Perkin– Elmer Inc., Waltham, MA, USA) using KBr pellets. Elemental analyses (C, H, N) were conducted using the Elemental Analyzer CE-440 (Exeter Analytical, Inc., Chelmsford, MA, USA); their results were found to be in good agreement (± 0.3%) with the calculated values. Mass spectra were recorded on Waters SQ Detector 2 Spectrometer using electrospray ionization (ESI) technique.

General procedure for the preparation of bis(1-phenylethanones) 2a, b

To a solution of sodium hydrocarbonate (0.13 g, 1.6 mmol) in water (7 mL) the corresponding bromoacetophenone (3.2 mmol) was added and a solution of diamine 1 (0.4 g, 1.6 mmol) in propan-2-ol (10 mL) was added drop-wise. The mixture was stirred at room temperature for 24 h, then refluxed for 10 min. After cooling, the formed precipitate was filtered off, washed with water and dried.

2,2'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bis(1-phenylethan-1-one) (2a)

White solid, yield 0.55 g (79%), mp 133–134°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ: 4.77, 4.80 (2s, 4H, 2CH₂), 6.36–6.75 (m, 2H, 2NH), 6.70–6.80 (m, 2H, H_Ar), 6.95–7.09 (m, 4H, H_Ar), 7.16–7.24 (m, 2H, H_Ar), 7.59 (t, J = 7.5 Hz, 4H, H_Ar), 7.70 (t, J = 7.2 Hz, 2H, H_Ar), 8.10 (d, J = 7.7 Hz, 4H, H_Ar) ppm.

¹³C NMR (DMSO-d₆, 101 MHz), δ: 49.92 (CH₂), 111.41, 114.86, 115.99, 116.06, 116.21, 116.27, 117.01, 117.65, 127.93, 128.85, 131.30, 131.80, 133.78 134.74, 134.74, 135.82, 135.90, 148.12, 148.29, 149.87 (C_Ar), 195.49, 195.52 (C = O) ppm.

IR (KBr), ν_max: 3071 (2 NH); 1673 (2 C = O) cm⁻¹.

Calcd. for C₂₈H₂₄N₂O₂S₂, %: C 69.39; H 4.99; N 5.78; Found: C 69.30; H 5.03; N 5.71.

2,2'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bis(1-(4-chlorophenyl)ethan-1-one) (2b)

White solid, yield 0.61 g (69%), mp 234–235°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ: 4.33, 4.39 (s, 4H, CH₂), 6.47–6.55 (m, 2H, NH), 6.70–6.88 (m, 5H, H_Ar), 6.90–6.99 (m, 2H, H_Ar), 7.01–7.09 (m, 2H, H_Ar), 7.24–7.28 (m, 2H, H_Ar), 7.36–7.41 (m, 2H, H_Ar), 7.58–7.71 (m, 2H, H_Ar), 8.04 (s, 1H, H_Ar) ppm.

¹³C NMR (DMSO-d₆, 101 MHz), δ: 49.981 (CH₂), 111.85, 114.76, 116.73, 117.20, 117.57, 118.37, 118.98, 119.40, 127.82, 128.30, 129.74, 130.43, 132.80 134.15, 135.47, 136.34, 140.64, 141.07, 142.15, 143.91 (C_Ar), 195.58, 195.74 (C = O) ppm.

IR (KBr), ν_max: 3054 (2 NH); 1597 (2 C = O) cm⁻¹.

Calcd. for C₃₀H₂₆Cl₂N₂O₂S₂, %: C 60.76; H 4.01; N 5.06; Found: C 60.81; H 4.06; N 4.99.

MS, m/z: calcd for C₂₈H₂₂Cl₂N₂O₂S₂: 554.06; Found: 554.89 [M + 2H⁺]⁺.

2,2'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bispropanoic Acid (3)

To a solution of diamine 1 (25.47 g, 0.1 mol) in toluene (400 mL) acrylic acid (21.6 g, 0.3 mol) was added by portions over 20 min. The reaction mixture was stirred at 100°C for 24 h. After cooling, the formed precipitate was filtered of, washed with toluene, diethyl ether and dried.

Green solid, yield 20.02 g (51%), mp 122–123°C (from toluene).

¹H NMR (DMSO-d₆, 400 MHz), δ: 2.45 (t, J = 6.7 Hz, 4H, CH₂CO), 3.32 (dd, J = 10.9, 6.9 Hz, 4H, NHCH₂), 5.42 (s, 2H, NH), 6.48 (t, J = 7.4 Hz, 2H, H_Ar), 6.68 (d, J = 8.3 Hz, 2H, H_Ar), 7.03 (d, J = 7.5 Hz, 2H, H_Ar), 7.23 (t, J = 7.7 Hz, 2H, H_Ar), 12.31 (s, 2H, OH) ppm.

¹³C NMR (DMSO-d₆, 101 MHz), δ: 33.42 (CH₂CO), 38.73 (NCH₂), 110.41, 115.97, 117.49, 131.95, 136.25, 148.74 (C_Ar), 173.19 (OH) ppm.

IR (KBr), ν_max: 3343 (2 NH); 3019 (2 OH); 1708 (2 C = O) cm⁻¹.

Calcd. for C₁₈H₂₀N₂O₄S₂, %: C 55.08; H 5.14; N 7.14; Found: C 55.15; H 5.12; N 7.20.

1,1'-((Disulfanediylbis(2,1-phenylene))bis(Dihydropyrimidine-2,4-(1h,3h)-dione) (5)

A mixture of diacid 3 (1.96 g, 5 mmol), carbamide (1.2 g, 20 mmol) and glacial acetic acid (10 mL) was refluxed for 18 h, then aqueous 18% HCl (10 mL) was added and the mixture was refluxed for 1 h. After completion of the reaction the mixture was diluted with water (20 mL) and left in a refrigerator. The formed precipitate was filtered off, washed with water and dried.

White solid, yield 1.32 g (60%), mp 121–122°C (from acetone/hexane mixture, 1:2).

¹H NMR (DMSO-d₆, 400 MHz), δ: 2.64–2.81 (m, 4H, CH₂CO), 3.38–3.58 (m, 2H, CH₂N), 3.77–3.94 (m, 2H, CH₂N), 7.34–7.44 (m, 6H, H_Ar), 7.54–7.68 (m, 2H, H_Ar), 10.54 (s, 2H, 2NH) ppm.

¹³C NMR (DMSO-d₆, 101 MHz), δ: 31.14, 44.66, 44.70 (CH₂CO, NCH₂), 127.92, 127.96, 128.07, 128.51, 128.69, 134.71, 134.91, 139.81, 139.90, 152.21 (C_Ar), 170.49, 170.51 (4CO) ppm.

IR (KBr), ν_max: 3212 (2NH); 1703 (4C = O) cm⁻¹.

MS, m/z: calcd for C₂₀H₁₈N₄O₄S₂Na: 465.07; Found: 465.12 [M + Na]⁺.

Calcd. for C₂₀H₁₈N₄O₄S₂, %: C 54.29; H 4.10; N 12.66; Found: C 54.24; H 4.14; N 12.62.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bispropanehydrazide) (7)

To a solution of diacid 3 (4 g, 10 mmol) in methanol (100 mL) sulfuric acid (1 mL) was added and the mixture was refluxed for 24 h. Then methanol was evaporated under reduced pressure, and the residue was poured with aqueous 5% sodium carbonate (40 mL). The obtained mixture was extracted with diethyl ether (3 x 50 mL). The combined organic layer was left in a refrigerator for 24 h, then filtered of and the filtrate was evaporated under reduced pressure. The obtained ester 6 (2.93 g, 7 mmol) was dissolved in methanol (30 mL) and very slowly hydrazine monohydrate (3.5 g, 70 mmol) was added. The mixture was stirred at room temperature for 48 h. The formed bishydrazide 7 was filtered of, washed with methanol, hexane and dried.

White solid, yield 1.53 g (51%), mp 131–132°C (from MeOH).

¹H NMR (DMSO-d₆, 400 MHz), δ: 2.30 (t, J = 6.7 Hz, 4H, CH₂CO), 3.30–3.35 (m, 4H, NHCH₂), 4.21 (s, 4H, NH₂), 5.56 (t, J = 5.7 Hz, 2H, 2PhNH), 6.46 (t, J = 7.4 Hz, 2H, H_Ar), 6.68 (d, J = 8.3 Hz, 2H, H_Ar), 6.99 (d, J = 7.6 Hz, 2H, H_Ar), 7.22 (t, J = 7.8 Hz, 2H, H_Ar), 9.08 (s, 2H, (NHCO) ppm.

¹³C NMR (DMSO-d₆, 101 MHz), δ: 32.83 (CH₂CO), 39.47 (NCH₂), 110.34, 115.78, 117.33, 131.84, 136.09, 148.75 (C_Ar), 170.09 (CO) ppm.

IR (KBr), ν_max: 3204 (4 NH); 1655 (2 C = O) cm⁻¹.

Calcd. for C₁₈H₂₄N₆O₂S₂, %: C 51.41; H 5.75; N 19.98; Found: C 51.50; H 5.79; N 20.01.

General Procedure For The Preparation Of Hydrazones 8−21

To a solution of bishydrazide 7 (0.42 g, 1 mmol) in propan-2-ol (10 mL) the corresponding aromatic or carbaldehyde (2.5 mmol) was added and the mixture was refluxed for 2–3 h. After cooling, the formed precipitate was filtered off, washed with propan-2-ol and dried.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(benzylidene)propanehydrazide) (8)

White solid, yield 0.44 g (74%), mp 169–170°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.46–2.49 (m, 1.4H, CH₂CO), 2.83−2.92 (m, 2.6H, CH₂CO), 3.38–3.50 (m, 4H, 2NHCH₂), 5.49–5.62 (m, 2H, 2PhNH), 6.39–6.52 (m, 2H, H_Ar), 6.70–6.80 (m, 2H, H_Ar), 6.95–7.04 (m, 2H, H_Ar), 7.19–7.26 (m, 2H, H_Ar), 7.39–7.46 (m, 6H, H_Ar), 7.62–7.69 (m, 4H, H_Ar), 8.00, 8.01, 8.17 (3s, 2H, CH = N), 11.39, 11.44, 11.48 (3s, 2H, NHCO) ppm.

IR (KBr), ν_max: 3432 (4 NH); 1648 (2 C = O); 1501 (2 C = N) cm⁻¹.

Calcd. for C₃₂H₃₂N₆O₂S₂, %: C 64.41; H 5.41; N 14.08; Found, %: C 64.35; H 5.43; N 13.99.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(4-fluorobenzylidene)propanehydrazide) (9)

White solid, yield 0.52 g (82%), mp 176–177°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.45–2.49 (m, 1.4H, CH₂CO), 2.80–2.91 (m, 2.6H, CH₂CO), 3.36–3.49 (m, 4H, 2NHCH₂), 5.47–5.60 (m, 2H, 2PhNH), 6.38–6.52 (m, 2H, H_Ar), 6.68–6.78 (m, 2H, H_Ar), 6.92–7.05 (m, 2H, H_Ar), 7.16–7.29 (m, 6H, H_Ar), 7.65–7.76 (m, 4H, H_Ar), 7.98, 7.99, 8.15 (3s, 2H, 2CH = N), 11.38, 11.39, 11.43, 11.47 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3432 (4 NH); 1648 (2 C = O); 1509 (2 C = N) cm⁻¹.

Calcd. for C₃₂H₃₀F₂N₆O₂S₂, %: C 60.74; H 4.78; N 13.28; Found, %: C 60.81; H 4.79; N 13.34.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(4-chorobenzylidene)propanehydrazide) (10)

White solid, yield 0.52 g (80%), mp 162–163°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.45−2.49 (m, 1.4H, CH₂CO), 2.79–2.95 (m, 2.6H, CH₂CO), 3.34–3.54 (m, 4H, 2NHCH₂), 5.43−5.62 (m, 2H, 2PhNH), 6.34–6.57 (m, 2H, H_Ar), 6.66–6.81 (m, 2H, H_Ar), 6.89–7.08 (m, 2H, H_Ar), 7.16–7.29 (m, 2H, H_Ar), 7.40−7.73 (m, 8H, H_Ar), 7.98, 7.99, 8.15 (3s, 2H, 2CH = N), 11.44, 11.49, 11.52 (3s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3030 (4 NH); 1647 (2C = O); 1591 (2C = N) cm⁻¹.

Calcd. for C₃₂H₃₀Cl₂N₆O₂S₂, %: C 57.74; H 4.54; N 12.63; Found, %: C 57.80; H 4.59; N 12.69.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(4-bromobenzylidene)propanehydrazide) (11)

White solid, yield 0.67 g (89%), mp 117–118°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.46−2.49 (m, 1.4H, CH₂CO), 2.79–2.93 (m, 2.6H, CH₂CO), 3.35–3.50 (m, 4H, 2NHCH₂), 5.40−5.68 (m, 2H, 2PhNH), 6.32–6.59 (m, 2H, H_Ar), 6.64–6.79 (m, 2H, H_Ar), 6.89–7.06 (m, 2H, H_Ar), 7.14–7.26 (m, 2H, H_Ar), 7.48−7.71 (m, 8H, H_Ar), 7.96, 8.13 (2s, 2H, 2CH = N), 11.44, 11.49, 11.53 (3s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3400 (4 NH); 1670 (2C = O); 1589 (2C = N) cm⁻¹.

Calcd. for C₃₂H₃₀Br₂N₆O₂S₂, %: C 50.94; H 4.01; N 11.14; Found, %: C 50.88; H 3.96; N 11.19.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(4-nitrobenzylidene)propanehydrazide) (12)

White solid, yield 0.63 g (92%), mp 188–189°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.52−2.57 (m, 1.4H, CH₂CO), 2.82–2.98 (m, 2.6H, CH₂CO), 3.35–3.55 (m, 4H, 2NHCH₂), 5.39−5.67 (m, 2H, 2PhNH), 6.30–6.62 (m, 2H, H_Ar), 6.64–6.84 (m, 2H, H_Ar), 6.87–7.10 (m, 2H, H_Ar), 7.15–7.31 (m, 2H, H_Ar), 7.80−8.38 (m, 10H, H_Ar, 2CH = N), 11.67, 11.69, 11.72, 11.75 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 2955 (4 NH); 1672 (2C = O); 1519 (2C = N) cm⁻¹.

Calcd. for C₃₂H₃₀N₈O₆S₂, %: C 55.97; H 4.40; N 16.32; Found, %: C 56.02; H 4.36; N 16.28.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(4-(dimethylamino)benzylidene)propanehydrazide) (13)

White solid, yield 0.49 g (75%), mp 118–119°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.43−2.47 (m, 1.4H, CH₂CO), 2.79–2.89 (m, 2.6H, CH₂CO), 2.94 (s, 12H, 4CH₃), 3.34–3.48 (m, 4H, 2NHCH₂), 5.46−5.62 (m, 2H, 2PhNH), 6.39–6.53 (m, 2H, H_Ar), 6.64–6.78 (m, 6H, H_Ar), 6.94–7.06 (m, 2H, H_Ar), 7.17–7.26 (m, 2H, H_Ar), 7.41−7.51 (m, 4H, H_Ar), 7.87, 8.00, 8.01 (3s, 2H, 2CH = N), 11.09, 11.12, 11.18 (3s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3411 (4 NH); 1609 (2C = O); 1524 (2C = N) cm⁻¹.

Calcd. for C₃₂H₃₀N₈O₆S₂, %: C 63.32; H 6.20; N 16.41; Found, %: C 63.28; H 6.24; N 16.40.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(2-chloro-5-nitrobenzylidene)propanehydrazide) (14)

White solid, yield 0.61 g (81%), mp 139–140°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.51−2.53 (m, 1.4H, CH₂CO), 2.78–3.01 (m, 2.6H, CH₂CO), 3.36–3.51 (m, 4H, 2NHCH₂), 5.41−5.60 (m, 2H, 2PhNH), 6.34–6.52 (m, 2H, H_Ar), 6.73 (d, J = 8.2 Hz, 2H, H_Ar), 6.90–7.25 (m, 4H, H_Ar), 7.72–7.85 (m, 2H, H_Ar), 8.12–8.24 (m, 2H, H_Ar), 8.32−8.61 (m, 4H, H_Ar, 2CH = N), 11.71, 11.75, 11.83, 11.88 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3444 (4 NH); 1637 (2C = O); 1505 (2C = N) cm⁻¹.

Calcd. for C₃₂H₂₈Cl₂N₈O₆S₂, %: C 50.86; H 3.74; N 14.83; Found, %: C 50.91; H 3.68; N 14.77.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(4-methoxybenzylidene)propanehydrazide) (15)

White solid, yield 0.49 g (75%), mp 97–98°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.45−2.47 (m, 1.4H, CH₂CO), 2.77–2.93 (m, 2.6H, CH₂CO), 3.39–3.45 (m, 4H, 2NHCH₂), 3.78 (s, 6H, 2OCH₃), 5.46−5.62 (m, 2H, 2PhNH), 6.34–6.58 (m, 2H, H_Ar), 6.68–6.79 (m, 6H, H_Ar), 6.93–7.07 (m, 6H, H_Ar), 7.18–7.26 (m, 2H, H_Ar), 7.54−7.65 (m, 4H, H_Ar), 7.94, 7.95, 8.10, 8.11 (4s, 2H, 2CH = N), 11.27, 11.39, 11.48 (3s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3440 (4 NH); 1665 (2C = O); 1511 (2C = N) cm⁻¹.

Calcd. for C₃₄H₃₆N₆O₄S₂, %: C 62.17; H 5.52; N 12.80; Found, %: C 62.24; H 5.49; N 12.75.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(2,3,4-trimethoxybenzylidene)propanehydrazide) (16)

White solid, yield 0.57 g (73%), mp 95–96°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.46 (t, J = 6.5 Hz, 1.4H, CH₂CO), 2.78–2.89 (m, 2.6H, CH₂CO), 3.39–3.44 (m, 4H, 2NHCH₂), 3.75, 3.79, 3.82 (3s, 18H, 6OCH₃), 5.39−5.71 (m, 2H, 2PhNH), 6.41–6.50 (m, 2H, H_Ar), 6.68–7.26 (m, 9H, H_Ar), 7.47–7.55 (m, 2H, H_Ar), 8.18, 8.32 (2s, 2H, 2CH = N), 11.25, 11.26, 11.37, 11.43 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3440 (4 NH); 1665 (2C = O); 1511 (2C = N) cm⁻¹.

Calcd. for C₃₈H₄₄N₆O₈S₂: C 58.75; H 5.71; N 10.82; Found, %: C 58.69; H 5.76; N 10.79.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(3,4,5-trimethoxybenzylidene)propanehydrazide) (17)

White solid, yield 0.55 g (71%), mp 196–197°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.46−2.49 (m, 1.4H, CH₂CO), 2.83–3.00 (m, 2.6H, CH₂CO), 3.36–3.50 (m, 4H, 2NHCH₂), 3.67, 3.68, 3.78, 3.81 (4s, 18H, 6OCH₃), 5.49−5.63 (m, 2H, 2PhNH), 6.35–6.51 (m, 2H, H_Ar), 6.67–6.78 (m, 2H, H_Ar), 6.91–7.06 (m, 6H, H_Ar), 7.16−7.25 (m, 2H, H_Ar), 7.91, 8.08 (2s, 2H, 2CH = N), 11.39, 11.41, 11.42, 11.52 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3402 (4 NH); 1652 (2C = O); 1503 (2C = N) cm⁻¹.

Calcd. for C₃₈H₄₄N₆O₈S₂: C 58.75; H 5.71; N 10.82; Found, %: C 58.69; H 5.68; N 10.87.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(naphtalen-1-ylmethylene)propanehydrazide) (18)

White solid, yield 0.56 g (81%), mp 160–161°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.52−2.58 (m, 1.4H, CH₂CO), 2.87–3.02 (m, 2.6H, CH₂CO), 3.40–3.55 (m, 4H, 2NHCH₂), 5.50−5.67 (m, 2H, 2PhNH), 6.35–6.52 (m, 2H, H_Ar), 6.70–6.81 (m, 2H, H_Ar), 6.94–7.27 (m, 4H, H_Ar), 7.51−7.65 (m, 6H, H_Ar), 7.82−8.02 (m, 6H, H_Ar), 8.49−8.82 (m, 4H, H_Ar, 2CH = N), 11.44, 11.45, 11.53, 11.59 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3437 (4 NH); 1669 (2C = O); 1507 (2C = N) cm⁻¹.

Calcd. for C₄₀H₃₆N₆O₂S₂: C 68.94; H 5.21; N 12.06; Found, %: C 68.87; H 5.17; N 12.07.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-(thien-2-ylmethylene)propanehydrazide) (19)

White solid, yield 0.25 g (41%), mp 183–184°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 55/45, 2.47, 2.79 (2t, J = 6.5 Hz, 2H, 2CH₂CO), 3.35–3.50 (m, 4H, 2NHCH₂), 5.48−5.62 (m, 2H, 2PhNH), 6.42−6.78 (m, 4H, H_Ar), 6.97–7.28 (m, 6H, H_Ar, H_Th), 7.36−7.68 (m, 4H, H_Th), 8.18, 8.38 (2s, 2H, 2CH = N), 11.35, 11.36, 11.38, 11.41 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3365 (4 NH); 1675 (2C = O); 1500 (2C = N) cm⁻¹.

Calcd. for C₂₈H₂₈N₆O₂S₄: C 55.24; H 4.64; N 13.80; Found, %: C 55.30; H 4.65; N 13.89.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-((5-nitrothien-2-yl)methylene)propanehydrazide) (20)

White solid, yield 0.39 g (56%), mp 162–163°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.51−2.53 (m, 1.4H, CH₂CO), 2.71–2.87 (m, 2.6H, CH₂CO), 3.37–3.47 (m, 4H, 2NHCH₂), 5.34−5.74 (m, 2H, 2PhNH), 6.34–6.85 (m, 4H, H_Ar), 6.96–7.31 (m, 4H, H_Ar), 7.41–7.51 (m, 2H, H_Ar), 8.02–8.09 (m, 2H, H_Ar), 8.10, 8.15, 8.41 (3s, 2H, 2CH = N), 11.76, 11.78, 11.79, 11.81 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3367 (4 NH); 1674 (2C = O); 1505 (2C = N) cm⁻¹.

Calcd. for C₂₈H₂₆N₈O₆S₄: C 48.13; H 3.75; N 16.04; Found, %: C 48.08; H 3.72; N 15.99.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(azanediyl))bis( N '-((5-nitrofuran-2-yl)methylene)propanehydrazide) (21)

White solid, yield 0.43 g (65%), mp 167–168°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ, Z/E 60/40, 2.51−2.56 (m, 1.4H, CH₂CO), 2.76–2.91 (m, 2.6H, CH₂CO), 3.36–3.46 (m, 4H, 2NHCH₂), 5.44−5.59 (m, 2H, 2PhNH), 6.46 (t, J = 7.4 Hz, 2H, H_Ar), 6.75 (d, J = 8.0 Hz, 2H, H_Ar), 6.95–7.04 (m, 2H, H_Ar), 7.14−7.26 (m, 4H, H_Ar, H_Fur), 7.76 (s, 2H, H_Fur), 7.91, 7.93, 8.12 (3s, 2H, 2CH = N), 11.75, 11.78, 11.80, 11.83 (4s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3208 (4 NH); 1676 (2C = O); 1511 (2C = N) cm⁻¹.

Calcd. for C₂₈H₂₆N₈O₈S₂: C 50.44; H 3.93; N 16.81; Found, %: C 50.52; H 3.99; N 16.78.

3,3'-((disulfanediylbis(2,1-phenylene))bis(azanediyl))bis(N-(2,5-dimethyl-1 H -pyrrol-1-yl)propanamide) (22)

To a solution of bishydrazide 7 (0.84 g, 2 mmol) in propan-2-ol (20 mL) hexane-2,5-dione (0.57 g, 5 mmol) and glacial acetic acid (2 mL) were added and the mixture was heated at reflux for 1 h. After completion of the reaction, the mixture was cooled down, the formed precipitate was filtered of, washed with propan-2-ol and dried.

White solid, yield 0.82 g (71%), mp 146–147°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ: 1.97, 2.01 (2s, 12H, CH₃), 2.57 (t, J = 6.6 Hz, 4H, CH₂CO), 3.42–3.54(m, 4H, NHCH₂), 5.56 (t, J = 5.9 Hz, 2H, PhNH), 5.63, 5.73 (2s, 4H, 4CH), 6.49 (t, J = 7.4 Hz, 2H, H_Ar), 6.77 (d, J = 8.2 Hz, 2H, H_Ar), 6.98 (d, J = 7.6 Hz, 2H, H_Ar), 7.26 (t, J = 7.7 Hz, 2H, H_Ar), 10.67 (s, 2H, 2NHCO) ppm.

IR (KBr), ν_max: 3439 (4 NH); 1663 (2 C = O) cm⁻¹.

Calcd. for C₃₀H₃₆N₆O₂S₂, %: C 62.47; H 6.29; N 14.57; Found, %: C 62.40; H 6.25; N 14.61.

3,3'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bis(1-(3,5-dimethyl-1h-pyrazol-1-yl)propan-1-one) (23)

To a solution of bishydrazide 7 (0.84 g, 2 mmol) in propan-2-ol (20 mL) pentane-2,4-dione (0.8 g, 8 mmol) and glacial acetic acid (2 mL) were added and the mixture was heated at reflux for 3 h. After completion of the reaction, the mixture was cooled down, the formed precipitate was filtered of, washed with propan-2-ol and dried.

White solid, yield 0.92 g (84%), mp 59–60°C (from 2-PrOH).

¹H NMR (DMSO-d₆, 400 MHz), δ: 2.17 (s, 6H, 2CH₃), 2.46 (s, 6H, 2CH₃), 3.26 (t, J = 6.6 Hz, 4H, CH₂CO), 3.45–3.52 (m, 4H, NHCH₂), 5.54 (t, J = 6.0 Hz, 2H, PhNH), 6.16 (s, 2H, 2CH), 6.44 (t, J = 7.4 Hz, 2H, H_Ar), 6.74 (d, J = 8.3 Hz, 2H, H_Ar), 6.96 (d, J = 7.6 Hz, 2H, H_Ar), 7.19 (t, J = 7.7 Hz, 2H, H_Ar) ppm.

IR (KBr), ν_max: 3440 (4 NH); 1724 (2 C = O) cm⁻¹.

Calcd for C₂₈H₃₂N₆O₂S₂, %: C 61.29; H 5.88; N 15.32; Found, %: C 61.22; H 5.92; N 15.28.

Biology

Strains and Culturing Conditions

Gram-positive bacteria Staphylococcus aureus subs. aureus (ATCC 9144) and Listeria monocytogenes (ATCC 35152) as well as Gram-negative ones, Escherichia coli Crooks (ATCC 8739) and Salmonella enterica subs. enterica serovar Enteritidis (ATCC 13076) were used.

Bacteria were cultured on Tryptone soya agar (OXOID, England) for 24-hours before the test. A turbidity concentration of 0.5 Mac Farland (10⁸ CFU/ml) was prepared from the grown bacterial cultures.

Determination of minimum inhibitory concentration (MIC).

The broth microdilution method [48, 49] was used to study the antibacterial activity of symmetrical N-substituted 2,2‘-dithioaniline derivatives. Bacterial growth was assessed in 96-well microplates (OXOID, England) in Muller Hinton (MH) broth. Serial two-fold dilutions of the compounds were used to determine the MIC, ranging from 500 µg/mL to 0.244 µg/mL concentrations. The concentration of bacteria used for the study was 5 x 10⁵ CFU/mL. Each well contained 100 µL of the mixture (different concentrations of compounds and bacteria 5 x 10⁴ CFU). The sterility of the medium, the growth of the tested bacteria and the sensitivity to the antibiotic cefazolin were also controlled. Microtitration plates with the test mixture were incubated at + 37 °C for 24 hours. MIC was determined as the lowest concentration of the compound at which no bacterial growth (turbidity) can be seen in the plate wells.

Cefazolin used as a control (C) for antibacterial activity screening.

Determination of minimum bactericidal concentration (MBC)

MBC was considered as the lowest concentration of the compound causing ≥ 3 log₁₀ reduction (≥ 99.9% kill) in number (5 x 10⁴ CFU/100 µL) of bacteria [50]. Ten microliters of mixture were taken from the well in which the MIC value was determined and from up to three wells in which the concentration of the compound was 2, 4 and 8 times higher. The tested mixtures were spread on Mueller Hinton agar. The growth of bacteria and number of colonies were evaluated after 24 hours of incubation at + 37 °C. MBC was considered as the lowest concentration of a compound when bacteria did not grow or formed up to 5 colonies.

Interpretation of MBC/MIC ratios

If the ratio MBC/MIC ≤ 4, the effect was considered as bactericidal but if the ratio MBC/MIC > 4, the effect was defined as bacteriostatic.

The antibiotic cefazolin was effective for S. aureus at concentrations − 15.6 µg/mL (MIC) and 31.3 µg/mL (MBC). The MIC and MBC of cefazolin for L. monocytogenes and S. Enteritidis were 31.3 µg/mL. The MIC and MBC of cefazolin for E. coli were respectively 31.3 µg/mL and 62.5 µg/mL.

Conflict of interest None.

Ethical Approval I consciously assure that for the manuscript in titled up the following items are fulfilled: The paper is not currently being considered for publication elsewhere. All authors have been personally and actively involved in substantial work leading to the paper and will take public responsibility for its content.

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author contributions Conceptualization, V.M. and B.G.; methodology, R.V., J. Š. and R.L.; software, R.V.; validation, K.S., B.G., R.V., J. Š. and R. L.; formal analysis, B.G. and V.M.; investigation, K.S., B.G., R.V., J. Š. and R. L.; resources, K.S. and B.G.; data curation, V.M., J. Š. and R. L.; writing—original draft preparation, R.V. and J. Š.; writing—review and editing, B.G. and V.M.; visualization, R.V. and J.Š.; supervision and project administration, V.M. All authors have read and agreed to the published version of the manuscript.

Funding No funding was received to assist with the preparation of this manuscript.

Data availability No data were used for the research described in the manuscript.

Supplementary Information The online version contains supplementary material available at

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Scheme 1 is available in Supplementary Files section.

No competing interests reported.

Scheme1.png
Scheme 1. Synthesis of 2,2’-dithioaniline derivatives 2-23.
Supplementarymaterial.docx

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Synthesis, characterization and antibacterial assays of new N,N 1 -disubstituted 2,2’- dithiodianiline derivatives

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Abstract

Figures

Introduction

Results And Discussion

Synthesis

Biological Activity

Conclusion

Experimental

Synthesis

2,2'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bis(1-phenylethan-1-one) (2a)

2,2'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bis(1-(4-chlorophenyl)ethan-1-one) (2b)

2,2'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bispropanoic Acid (3)

1,1'-((Disulfanediylbis(2,1-phenylene))bis(Dihydropyrimidine-2,4-(1h,3h)-dione) (5)

3,3'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bispropanehydrazide) (7)

General Procedure For The Preparation Of Hydrazones 8−21

3,3'-((Disulfanediylbis(2,1-phenylene))bis(Azanediyl))bis(1-(3,5-dimethyl-1h-pyrazol-1-yl)propan-1-one) (23)

Biology

Strains and Culturing Conditions

Determination of minimum bactericidal concentration (MBC)

Interpretation of MBC/MIC ratios

Declarations

References

Scheme

Additional Declarations

Supplementary Files

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