Efficient synthesis of new azo-sulfonamide derivatives and investigation of their molecular docking and cytotoxicity results

2.1 Chemistry section

Azo products are usually produced in two stages. The first step of the de-annotation reaction: with the primary type of aromatic amine of sulfonamides, an aqueous mineral acid such as HCl and sodium nitrite (NaNO₂) is mixed as a source of in-situ nitrosonium ion (N⁺ = O) construction in an ice bath to produce diazonium salt. This salt is unstable at r. t., since diazonium salts gradually decompose even at low temperatures, so it is produced at 5° C (Kumar et al., 2010; Sharma et al., 2020). The mate must have active aromatic rings or active nucleophilic groups to participate in the electrophilic substitution reaction and bind to the diazonium salt Fig. 1 (Khan et al., 2021). The present study is to investigate the production and stability of diazonium salts using cellulosic sulfuric acid as catalyst (Gaffer et al., 2017; Lee et al., 1988) and its purpose is to perform a coupling reaction to produce azo dyes under the solvent-free conditions. Cellulose sulfuric acid (CSA) as a solid acid has advantages such as biodegradability, cost-effectiveness, recyclability, and compatibility with green chemical ovens (Ganewatta et al., 2019).

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Fig. 1

Synthesis of azo-sulfonamide derivatives by azotation.

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By grinding sulfonamides and CSA with a few drops of water in a mortar, a homogeneous mixture was obtained. NaNO₂ was then added to the resulting mixture and gently ground for 2–3 min. The reaction mixture was reacted with the coupling agent for 10–20 min and the structure of the products was confirmed by FT-IR, ¹H NMR, and ¹³C NMR spectra, shown in Fig. 2 and Table 1.

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Fig. 2

Synthesis of new azo-sulfonamides in the presence of CSA.

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Table 1 New azo-sulfonamide derivatives synthesized using cellulosic sulfuric acid at room temperature.

In the FT-IR spectrum, the sharp peaks at ∼ 1140 cm⁻¹ and ∼ 1345 cm^-1correspond SO₂. The average peaks in 1435–1480 cm⁻¹ correspond to N⚌N. Existence of a broad band in ∼ 3400 cm⁻¹ corresponds to O—H. Medium twin band at 2700–2850 cm⁻¹ corresponding to H with carbon C⚌O aldehyde (products 5 and 6), N—H bending tape in R₂-NH 1550–1650 cm⁻¹, C—H symmetric tensile tape in CH₃ ∼ 1380 cm⁻¹ (product 4), Peaks in the range of 2860–3000 cm⁻¹ related to C—H aliphatic saturation. Wide and weak peaks in 3000–3100 cm⁻¹ related to aromatic C—H, C⚌O aldehyde stretching band in 1645–1765 cm⁻¹ (products 5 and 6). Medium or strong peaks in 1630–1740 cm⁻¹ are related to C⚌O amide and confirmed the structure of the desired products.

¹H NMR (MHz400) spectra of products were recorded in DMSO‑d₆ solvent. A singlet peak in the range of 2.20–2.60 ppm, confirms the presence of H-methyl of 3-methylisoxazol (products 3 and 6). Aromatic protons appeared in the expected range at 7.00–7.68 ppm, for products 1,2,3 and 4.

The O—H signal is appeared in the range of 15.01–15.90 ppm and confirms the presence of beta-naphthol in the product structure. In products 1 and 3, hydrogen bonds are formed between azo N bonds and O—H hydrogen bonds of beta naphthol and appear in the range 11.17–11.50 ppm. The C—H of thiazole ring appeared in product 4 in the range of 6.80–6.90 ppm and confirms the structure of the products. In the ¹³C NMR spectra (100 MHz), aromatic carbon signals appeared at appropriate 117.00–146.30 ppm.

2.2 Investigation of X-ray diffraction (XRD) spectrum for the synthesized derivatives (4, 5)

The results of X-ray diffraction on the powder samples (4 and 5) were considered. The X-ray diffraction pattern of 4 is considered by the presence of four peaks at 2θ = 13°, 20°, 23° and 45° and for 5 the X-ray diffraction pattern is considered by the presence of seven peaks at 2θ = 6°, 9°, 14°, 20°, 22°, 26° and 45°. Major peaks for both compounds appeared in the range of 2θ = 10°-30°, indicating the relative similarity of the two compounds. XRD is a key technique for pharmaceutical analysis. It plays the main role in all the stages of development, testing and production of drugs. An important part of analytical research and development is quality control of active ingredients, excipients, and final products (Chauhan and Kaith, 2012, 2011). XRD study analyses the peak width along with lattice strain and lattice constants due to dislocation and particles size (Cullity, 1978; Mote et al., 2012). Particles size of the pure azo-sulfonamide are calculated using Scherer’s Equation.

Here, D is the Particles size, βD is full width at half maxima, λ is the wavelength of CuKα radiation (1.54 Å) and 2θ is the Bragg peak position. Bragg peak broadening is the composition of both the effects i.e. instrumental as well as sample dependent. The instrumental peak width was corrected according to each diffraction peak of azo-sulfonamide material using the following relation: $$D=\frac{K\lambda }{\beta Cos\varphi }$$

From the calculations the Particles size, the estimated value of Particles size for samples of azo-sulfonamide (4) and (5), respectively is 12.75 Å and 13.95 Å shown in Fig. 3.

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Fig. 3

X-ray diffraction pattern of samples (4, 5).

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3.1 Human 17-Beta-Hydroxy Steroid Dehydrogenase (PDB ID: 1FDW)

According to the docking results the order of binding energy of the three higher scores molecules appears to be 6 > 1 > 2, respectively. In the case of 6, the nitrogen atom of the isoxazole ring behaves as an H-bond acceptor from the hydroxyl group of Tyr-218 residue by the distance of 2.47 Å, and the binding energy of the amount −7.36 Kcal.mol⁻¹. From the 2D ligand–protein interaction diagram it is realized that the polar isoxazole ring fits very well in the pocket created by residues such as Leu-96, Tyr-218, Leu-219, Gln-221, Ser-222, and Lys-223. Structure 1 is posed in a different orientation relative to 6, thereby getting the advantage of direct interaction with two surrounded residues. As the H-bond acceptor (HBA) between the phenolic group attached to the naphthalene moiety and Gln-221 on one side as well as the H-bond donor (HBD) type between amidic hydrogen and Glu-282. Although the present naphthalene group is unable to fit as well as the isoxazole ring present in 6 in the previously mentioned pocket but the presence of these two effective H-bonding interactions provides considerable stability, which ultimately leads to its high score. There is some extent of similarity when the 2D interaction diagrams of 1 and 2 are compared, but it is immediately apparent that lacking of the second observed interaction for 1 (between amidic hydrogen and Glu-282) in 2 leads to the less binding energy.

3.2 Human Serine/Threonine-Protein Kinase Receptor, (PDB ID: 3FC2)

Interestingly all structures display high Grid Scores and the three highest binding affinities are in the order of 1 > 3 > 6. The 3D ligand–protein interaction indicates appropriate and convenient stacking of the ligands in between the ruptured nucleotides, forming a stable complex with the ability of inhibitory reattachment of the chains. The interchelating section of 1, 2, and 4 structures are mainly due to two rings naphthalene part and that of 3, 5, and 6 are from benzene or from hetero aromatic ring of amide part. Molecule 1 represents an H-bond interaction as HBD to the Arg-487 residue while the naphthalene ring establishes a stabilizing pi-pi stacking with deoxythymidine nucleotide (DT F:9). Molecule 2 shows pi-pi stacking of naphthalene moiety with DG D:13, middle benzene with DT F:9, and pi-cationic interaction between the benzene ring attached to amidic carbonyl group with the Arg-804. Molecule 3 shows pi-pi stacking of hetero aromatic ring with DA D:12 as well as HBD from N—H to DT F:9. Molecule 4 shows pi-pi stacking of naphthalene moiety with DG D:13 and the H-bond interaction as HBD to the Arg-487 residue. Molecule 5 displays pi-pi interactions between the benzene ring attached to the amidic carbonyl group with DA D:12 and DG D:13 nucleotides, respectively. Furthermore, the oxygen atom of the carbaldehyde group accepts an H-bond from DC D:11. Finally, the isoxazole ring of 6 establishes a pi-pi stabilizing interaction with DT F:9 and at the same time, this nucleotide behaves as HBA from the N—H group while at a place farther on the ligand the phenolic hydroxyl group takes part as HBD toward the Asp-541, an interaction not observed for any of the other present ligands.

3.3 Human Topoisomerase II alpha (5GWK)

Interestingly all structures show high Grid Scores and the three highest binding affinities are 1 > 3 > 6, respectively. The 3D ligand–protein interaction indicates appropriate and convenient stacking of ligands between the ruptured nucleotides forming a stable complex with the ability of inhibitory reattachment of the chains. The interchelating section of 1, 2, and 4 structures are mainly due to two rings naphthalene part and that of 3, 5, and 6 is benzene or hetero aromatic ring of amide part. Molecule 1 represents an H-bond interaction as HBD to the Arg-487 residue while the naphthalene ring establishes a stabilizing pi-pi stacking with deoxythymidine nucleotide (DT F:9). Molecule 2 shows pi-pi stacking of naphthalene moiety with DG D:13, middle benzene with DT F:9, and pi-cationic interaction between the benzene ring attached to amidic carbonyl group with the Arg-804. Molecule 3 shows pi-pi stacking of hetero aromatic ring with DA D:12 as well as HBD from N—H to DT F:9. Molecule 4 shows pi-pi stacking of naphthalene moiety with DG D:13 and an H-bond interaction as HBD to the Arg-487 residue. Molecule 5 displays pi-pi interactions between the benzene ring attached to the amidic carbonyl group with DA D:12 and DG D:13 nucleotides, respectively. Furthermore, the oxygen atom of the carbaldehyde group accepts an H-bond from DC D:11. Finally, the isoxazole ring of 6 establishes a pi-pi stabilizing interaction with DT F:9 and at the same time, this nucleotide behaves as HBA from the N—H group while at a place farther on the ligand the phenolic hydroxyl group takes part as HBD toward the Asp-541, an interaction not observed for any of the other present ligands.

4.1 Computational methods

System Preparation. The crystal structure of Human Serine/Threonine-Protein Kinase Receptor (PDB ID: 3FC2) was used as the initial structure and hydrogen atoms of the protein residues were added using LeaP (Song et al., 2019). The ligands were placed in the position determined by the previous docking process. The system was then soaked in a cubic box of 15,166 TIP3P water molecules extending to at least 15 Å from the protein atoms. The ff99SB force field (Ganjali et al., 2008) was used to describe the protein, together with General Amber Force Field (GAFF) (Wang et al., 2004) parameters for ligand.

MD Simulation. The simulation was carried out using the Ambertools 19 Molecular Dynamics package (Song et al., 2019). The system, prepared as described above, was first subjected to 1000 steps of steepest descent and 1000 steps of conjugate gradient minimization. The system was then heated to the target temperature of 300 K for a period of 20 ps under constant volume periodic boundary conditions (NVT). Subsequently, approximately 30 ps of constant pressure and temperature simulation (NPT) was carried out to equilibrate the system, which was followed by 30 ps of production simulation performed under the same conditions, using periodic boundary conditions with constant pressure, and the Berendsen barostat for constant pressure simulation. The temperature was controlled via Langevin dynamics (Boys and Bernardi, 1970) with collision frequency of 1 ps⁻¹. A cutoff of 16 Å was used for nonbonded interactions and long-range electrostatic interactions were treated by means of the Particle Mesh Ewald (PME) method (Rassolov et al., 1998). All bonds involving hydrogen were constrained by the SHAKE method (Ryckaert et al., 1977) and the time step for numerical integration was 2 fs. The simulation results were analyzed using the cpptraj program in the Ambertools19 package (Roe and Cheatham, 2013). The diagram of density, energies, temperature and RMSD obtained by MD study of the 3FC2 protein along with molecules 1, 2 and 4 are displayed in Figs. 8-10.

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Fig. 8

Properties of the system 3FC2 protein and ligand 1 during the MD production.

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Fig. 9

Properties of the system 3FC2 protein and ligand 2 during the MD production.

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Fig. 10

Properties of the system 3FC2 protein and ligand 4 during the MD production.

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The noteworthy points with the energy plot are that all of the energy terms increased during the first 20 ps, corresponding to heating from 0 K to 300 K. Then all energies are leveled off and remained constant for the remainder of the simulation indicating that the relaxation was completed and that an equilibrium has been reached. The system behaves in an expected manner, the temperature started at 0 K and then increased to 300 K over a period of about 20 ps. The temperature then remained constant for the remainder of the simulation which means that the use of Langevin dynamics for temperature regulation is successful. The system has equilibrated at a density of approximately 1.02 g cm⁻³, as the density of pure liquid water at 300 K is approximately 1.00 g cm⁻³, this seems to be reasonable so adding the 3FC2 protein has increased the density by around 2 %. The RMSD increases very fast in the first 20 ps. There is not any significant conformational change in dihedral angles in the protein. RMSD values less than 1.5-0.2.0 Å are indicating a stable structure. The structures obtained at the end of MD production are represented in Fig. 11.

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Fig. 11

Structure of the system after MD production for 3FC2 protein along with ligands 1, 2 and 4.

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5.1 Cytotoxic activity

One way to treat cancer is to use strong chemical drugs or chemotherapy. Today, several chemical drugs have been used and researched to treat cancer. It is important to find new drugs that can fight cancer cells.

Cell lines: We obtained breast cancer cells (MCF-7) from the Pasteur Institute of Iran (Tehran, Iran). The cells were stored at 37 °C in a humid atmosphere (90 %) containing 5 % CO₂ and then in DMEM (Dulbecco modified Eagle medium) with 10 % (v /v) FBS (bovine fetal serum), 100 units per ml different concentrations of samples 1–6 and vinblastine (Vin) were incubated with seed cells overnight.

Cell viability assay: The MTT reduction method has been used to estimate cell viability after exposure to synthetic compounds (Abd Alhameed et al., 2019; Yazdani Nyaki et al., 2021). Cells (MCF-7) were implanted in 96-well plates per 10,000 cells in each well containing a 200 μl medium. After 24 hr. of incubation at 37 °C, different concentrations of test compounds were added to the wells. Samples were tested 1–6 with Vin at the following concentrations: 20, 40, 60, 80, and 100 μg / mL. The samples were dissolved in dimethyl sulfoxide (DMSO) and then diluted with a cell culture medium, and after cell destruction, formazan dye was formed, indicating the mitochondria of living cells. In all treatments, including blank, the final concentration of DMSO was 1 % of the total volume of the medium. Cell viability was then calculated by MTT as a percentage of control (untreated cells) Fig. 12.

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Fig. 12

MTT assay synthesized derivatives diagram.

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The anti-cancer activity of azo-sulfonamides designed in vitro against MCF-7 cells was tested by MTT assay and Vin was used as the reference drug. The tested compounds were used in different concentrations and cell survival was determined after incubation for 24 h. Inhibition of cell proliferation in each treatment is reported as a percentage of the number of cells treated with untreated control cells Fig. 12. Cytotoxic activity in new derivatives is shown as IC₅₀ (μM) values ​​Fig. 13.

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Fig. 13

Graph IC₅₀ values of synthesized derivatives.

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In the designed azo-sulfonamide series, various aliphatic and aromatic alternatives showed promising cytotoxicity, with some compounds showing significant IC₅₀ values of the reference compound, Vin.

Fig. 13 shows IC₅₀ values ​​1-6. As a positive control, Vin with an IC₅₀ of approximately 11.46 μM was the same under all conditions. As can be seen in Fig. 13, the results of the MTT Assay test show that sample 5 has a stronger toxin property than other compounds. However, all IC₅₀ values ​​showed almost reasonable activity for the compounds. Among the tested compounds, derivatives 5, 4, and 6 showed proliferative activity with IC₅₀ of 32.28, 32.66, and 35.35 μM, respectively.

Based on the obtained results, it seems that compound 5 contains a benzene ring and salicylic aldehyde coupling group, as well as also in samples 4 and 6 the presence of thiazole, isoxazole, 2-naphthol, and salicylic aldehyde groups, shows more toxicity and activity in inhibiting MCF-7 cell human proliferation. Thus, these compounds are less effective against breast cells than positively controlled Vin. The MTT toxicity of compounds 1–6 compared to Vin is thus 5 > 4 > 6 > 2 > 3 > 1 (Fig. 13).

In addition, the findings of the MTT experiment showed that the technique adopted between the sulfonamide and the mating group provided highly active compounds, making them very promising candidates for further research. The present study focused on investigating the potential anticancer mechanism of a new sulfasalazine analog compound in MCF-7 cells.

6.1 Chemicals and devices

The chemicals and solvents used in this study were prepared by Merck & Aldridge companies as follows: cellulose, chlorine and sulfonic acid, MeOH, acetone, EtOAc, and NaNO₂, as well as sulfonamide derivatives from the drug company (Caspiantamin) prepared, this material was used without further purification. To follow the reaction process, thin layer chromatography (TLC) with aluminum plates and silica gel 60 F254 and ultraviolet lamps were used. The melting points of the synthesized compounds were measured with the melting point measuring device of Model 9100 Electrothermal and reported as uncorrected. IR spectra were recorded by JASCO FT/IR-4700 infrared spectrometer. ¹H NMR and ¹³ C NMR spectra were obtained with Bruker Advance 500 MHz using TMS as internal standard and in DMSO solvent at r. t.

6.2 Catalyst preparation

The cellulose-sulfuric acid was prepared according to the literature (Shaabani and Maleki, 2007). After completion of the reaction and separation of the organic compounds, cellulosic sulfuric acid (CSA) was recovered. For this purpose, the CSA was first washed with EtOH and MeOH, then placed at 70 °C for 24 h to dry completely. The recovered catalyst was used three more times for the above reaction, which could be performed without significant change in CSA activity.

6.3 Method for the synthesis of new azo-sulfonamide derivatives

At first, a homogeneous mixture was obtained by grinding sulfonamides (type 1 amine) (2 mmol) and cellulosic sulfuric acid (1.5 g) with a few drops of water (0.2 ml) in a mortar. NaNO₂ (4 mmol, 0.138 g) was then added to the resulting mixture and gently ground for 2–3 min. The presence of diazonium salt was confirmed by the beta naphthol. The reaction mixture was reacted with the coupling agent for 10–20 min. Reaction progress was tracked using TLC. After the reaction was complete, the mixture contained in acetone (10 ml) was dissolved and separated from sulfuric acid by filtration, then washed 3 more times with acetone (10 ml). The solution was evaporated under filtration and the remaining solids were crystallized with EtOH and H₂O. The newly obtained azo-sulfonamide derivatives with good yield (60–93 %) and a short reaction time 20–35 min. In Table 1, the general results of azo-sulfonamide derivatives with various substitutions are reported. Fig. 2 and Table 1.

Physical properties and spectral data for synthesized products.

(E)-N-((4-((2-hydroxynaphthalen-1-yl)diazenyl)phenyl)sulfonyl)acetamide (1).

Orange powder, efficiency: 81 %, m.p: 287 °C; FT-IR (KBr, υ/cm⁻¹): 3428 (O—H stretch), 2859 (aliphatic C—H stretch), 1723 (C⚌O stretch), 1592 (aromatic C⚌C stretch), 1438 (N⚌N stretch), 1254 (C—O stretch), 1337, 1147 (symmetric SO₂ streach), 856 ، 660 (aromatic C—H out-of-plane bend). ¹H NMR (400 MHz, DMSO‑d₆) δ: 1.98 (t, J = 4.2, 2.2 Hz, 3H), 6.80 (d, J = 9.6 Hz, 1H), 7.52–7.55 (t, J = 6.2, 2.0 Hz, 1H), 7.64–7.67 (t, J = 4.0, 1.2 Hz, 1H), 7.84 (d, J = 7.56 Hz, 1H), 8.49 (d, J = 7.28 Hz, 1H), 7.98–8.04 (m, 5H), 12.17 (s, 1H), 15.80 (s, 1H). ¹³C NMR (100 MHz, DMSO‑d₆) δ: 178, 169.43, 147.24, 143.39, 36.15, 133.13, 131.02, 130.15, 129.97, 129.79, 128.71, 127.69, 126.31, 122.46, 17.77, and 23.82. HRMS-ESI (mlz) [M⁺] Calcd. for C₁₈H₁₅N₃O₄S 369.0825, found 369.0911. Elemental Analysis Calcd. for C₁₈H₁₅N₃O₄S (%): C, 58.53; H, 4.09; N, 11.38; O, 17.32; S, 8.68 Found: C, 59.13; H, 4.33; N, 10.68; O, 18.65; S, 7.21.

(E)-N-((4-((2-hydroxynaphthalen-1-yl)diazenyl)phenyl)sulfonyl)benzamide (2).

Orange powder, efficiency: 83 %, m.p: 291 °C; FT-IR (KBr, υ/cm⁻¹): 2982 (aliphatic C—H stretch), 1447 (N⚌N stretch), 1232 (C—O stretch), 1146 (symmetric SO₂ streach). ¹H NMR (400 MHz, DMSO‑d₆) δ: 6.8 (d, J = 9.4 Hz, 1H), 7.28–7.35 (m, 2H), 7.36–7.38 (m, 1H), 7.45–7.49 (m, 1H), 7.60–7.64 (m, 1H), 7.7 (d, J = 7.6 Hz, 1H), 7.8 (d, J = 8.1 Hz, 2H), 7.89–7.92 (m, 2H)), 8.53 (d, J = 8.1 Hz, 1H)), 7.94–7.97 (m, 3H). ¹³C NMR (100 MHz, DMSO‑d₆) δ: 172.35, 170.20, 145.52, 145.40, 141.42, 139.70, 133.22, 130.32, 130.10, 129.79, 129.49, 129.11, 128.78, 128.38, 127.84, 126.72, 125.12, 122.00, and 117.90. HRMS-ESI (mlz) [M⁺] Calcd. for C₂₃H₁₇N₃O₄S 431.0945, found 431.1138. Elemental Analysis Calcd. for C₂₃H₁₇N₃O₄S (%): C, 64.03; H, 3.97; N, 9.74; O, 14.83; S, 7.43 Found: C, 63.88H, 4.11; N, 9.56; O, 15.03; S, 7.42.

(E)-N-((4-((2-hydroxynaphthalen-1-yl)diazenyl)phenyl)sulfonyl)-5-methylisoxazole-3-carboxamide (3).

Orange powder, efficiency: 64 %, m.p: 207 °C; FT-IR (KBr, υ/cm⁻¹): 3071 (aromatic C—H stretch), 2859 (aliphatic C—H stretch), 1614 (N—H stretch in R2-NH(, 1593 (aromatic C⚌C stretch), 1466 (N⚌N stretch), 1149 (asymmetric SO₂ streach), 1255 (C—O stretch) 1149 (symmetric SO₂ streach). ¹H NMR (400 MHz, DMSO‑d₆) δ: 2.38 (s, 3H), 6.79 (d, J = 9.06 Hz, 1H), 6.08 (s, 1H), 7.47–7.51 (t, J = 6.2, 3.1 Hz, 2H), 7.59–7.63 (t, J = 4.5, 2.1 Hz, 1H), 8.47 (d, J = 8.0 Hz, 1H), 7.88–7.97 (m, 5H), 11.49 (s, 1H), 15.76 (s, 1H). ¹³C NMR (100 MHz, DMSO‑d₆) δ: 177.56, 170.60, 158.47, 147, 143.25, 136.72, 136.35, 133.11, 130.94, 130.12, 129.76, 129.08, 128.68, 127.62, 126.22, 122.45, 118.14, 96.01, and 12.55. HRMS-ESI (mlz) [M⁺] Calcd. for C₂₁H₁₆N₄O₅S 436.4474, found 436.6521. Elemental Analysis Calcd. for C₂₁H₁₆N₄O₅S (%): C, 57.79; H, 3.70; N, 12.84; O, 18.33; S, 7.35 found: C, 56.88; H, 4.15; N, 13.25; O, 17.04; S, 8.68.

(E)-N-((4-((2-hydroxynaphthalen-1-yl)diazenyl)phenyl)sulfonyl)thiazole-2-carboxamide (4).

Orange powder, efficiency: 74 %, m.p: 205 °C; FT-IR (KBr, υ/cm⁻¹): 3209 (O—H stretch), 1722 (C⚌O stretch), 1590 (aromatic C⚌C stretch), 1434 (N⚌N stretch), 1363 (asymmetric SO₂ streach), 1140 (symmetric SO₂ streach). ¹H NMR (400 MHz, DMSO‑d₆) δ: 15.81 (s, 1H), 12.86 (s, 1H), 8.47 (d, J = 8.0, 1H), 7.96 (d, J = 9.6 Hz, 1H), 7.88–7.93 (m, 4H), 7.76 (d, J = 7.4 Hz, 1H), 7.59–7.63 (m, 1H), 7.46–7.50 (m, 1H), 7.25 (d, J = 4.52 Hz, 1H), 6.80–6.83 (t, J = 8.2, 4.2 Hz, 2H). ¹³C NMR (100 MHz, DMSO‑d₆) δ: 175.7, 169.3, 146.3, 142.6, 140.3, 133.1, 130.5, 130.0, 129.6, 128.5, 128.08, 127.02, 108.7, 118.2, 122.02, 125.7,and 126.2. HRMS-ESI (mlz) [M⁺] Calcd. for C₂₀H₁₄N₄O₄S₂ 438.0572, found 438.1065. Elemental Analysis Calcd. for C₂₀H₁₄N₄O₄S₂ (%): C, 54.79; H, 3.22; N, 12.78; O, 14.60; S, 14.62 found: C, 55.02; H, 4.26; N, 12.55; O, 13.28; S, 14.89.

(E)-N-((4-((3-formyl-4-hydroxyphenyl)diazenyl)phenyl)sulfonyl)benzamide (5).

Orange powder, efficiency: 87 %, m.p: 225 °C; FT-IR (KBr, υ/cm⁻¹): 3605 (O—H stretch), 3059 (aromatic C—H stretch), 1692 (aldehyde C⚌O stretch), 1582 (aromatic C⚌C stretch), 1477 (N⚌N stretch), 1344 (asymmetric SO₂ streach), 1265 (C—O stretch). ¹H NMR (400 MHz, DMSO‑d₆)δ: 10.32 (s, 1H), 8.13 (d, J = 2.7 Hz, 1H), 8.02 (dd, J = 9.0, 2.7 Hz, 1H), 7.93 (d, J = 8.6 Hz, 2H), 7.84 (d, J = 8.6 Hz, 2H), 6.97 (d, J = 9.0 Hz, 1H), 1.76 (s, 3H). ¹³C NMR (100 MHz, DMSO‑d₆)δ: 190.82, 166.76, 164.51, 154.56, 145.28, 142.41, 133.28, 130.41, 129.54, 128.91, 124.90, 123.21, 123.04, and 119.06. HRMS-ESI (mlz) [M⁺] Calcd. For C₂₀H₁₅N₃O₅S 409.0741, found 409.1158. Elemental Analysis Calcd. For C₂₀H₁₅N₃O₅S (%): C, 58.67; H, 3.69; N, 10.26; O, 19.54; S, 7.83 Found: C, 57.99; H, 3.75; N, 11.32; O, 19.20; S, 7.74.

(E)-N-((4-((3-formyl-4-hydroxyphenyl)diazenyl)phenyl)sulfonyl)-5-methylisoxazole-3-carboxamide (6).

Orange powder, efficiency: 79 %, m.p: 213 °C; FT-IR (KBr, υ/cm⁻¹): 3794 (O—H stretch), 1727 و 1659 (C⚌O stretch), 1581 (aromatic C⚌C stretch), 1479 (N⚌N stretch), 1349, 1137 (symmetric SO₂ streach), 1283 (C—O stretch). ¹H NMR (400 MHz, DMSO‑d₆) δ: 10.37 (s, 1H), 8.2 (d, J = 5.1 Hz, 1H), 8.11–8.14 (dd, J = 7.5, 3.4 Hz, 1H), 8.01–8.06 (d, J = 8.1 Hz, 4H), 7.2 (dd, J = 8.88, 3.8 Hz, 1H), 2.51–2.52 (t, J = 7.4, 4.1 Hz, 3H), 6.18 (s, 1H). ¹³C NMR (100 MHz, DMSO‑d₆) δ:190.80, 170.97, 164.67, 154.69, 145.20, 141.16, 130.40, 128.72, 124.97, 123.49, 123.21, 119.08, 95.96, and 12.56. HRMS-ESI (mlz) [M⁺] Calcd. for C₁₈H₁₄N₄O₆S 414.0623, found 414.0847. Elemental Analysis Calcd. for C₁₈H₁₄N₄O₆S (%): C, 52.17; H, 3.41; N, 13.52; O, 23.17; S, 7.74 Found: C, 52.25; H, 4.68; N, 13.11; O, 22.84; S, 7.12.