Beta and Gamma Amino Acid-Substituted Benzenesulfonamides as Inhibitors of Human Carbonic Anhydrases

Abstract

A series of novel benzenesulfonamide derivatives were synthesized bearing para-N β,γ-amino acid or para-N β-amino acid and thiazole moieties and their binding to the human carbonic anhydrase (CA) isozymes determined. These enzymes are involved in various illnesses, such as glaucoma, altitude sickness, epilepsy, obesity, and even cancer. There are numerous compounds that are inhibitors of CA and used as pharmaceuticals. However, most of them bind to most CA isozymes with little selectivity. The design of high affinity and selectivity towards one CA isozyme remains a significant challenge. The beta and gamma amino acid-substituted compound affinities were determined by the fluorescent thermal shift assay and isothermal titration calorimetry for all 12 catalytically active human carbonic anhydrase isozymes, showing the full affinity and selectivity profile. The structures of several compounds were determined by X-ray crystallography, and the binding mode in the active site of CA enzyme was shown.

1. Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO₂ to bicarbonate and maintain the acid–base balance in tissues and blood and, thus, play a crucial physiological role [1,2,3,4,5]. Human CAs belong to the α-family, which consists of 12 catalytically active (CAI, CAII, CAIII, CAIV, CAVA, CAVB, CAVI, CAVII, CAIX, CAXII, CAXIII, CAXIV) and three non-catalytic isozymes (CAVIII, CAX, and CAXI) that are also called carbonic anhydrase-related proteins (CARPs) [6,7]. Due to their different distribution in the body, the functions performed vary widely [8]. Therefore, disruption of normal function results in illnesses associated with only one or several CAs [9]. Thus, CAs are drug targets for a variety of disorders. There is still a significant challenge to design compounds possessing both high affinity and high selectivity for a specific isozyme, due to the structural similarity of the active sites of CAs [10,11,12]. Sulfonamides were discovered in 1940 as CA inhibitors by Mann and Keilin [13], who also reported some side effects of the newly discovered 4-aminobenzenesulfonamide. This was followed by a series of discoveries of a wide variety of compounds [14,15,16,17,18,19,20,21,22,23,24,25], some of which were approved as drugs. However, the desired properties and selectivity for all targeted CAs have not been achieved, and research in this field is ongoing.

The 5-oxopyrrolidine moiety is a part of a variety of compounds of natural or synthetic origin [26,27]. Recently, we have synthesized and examined the binding properties of 3-substituted 1-(4-sulfamoylphenyl)-5-oxopyrrolidine derivatives [28,29,30]. The different halo-substituents at the 4-sulfamoylphenyl moiety improved the binding properties of the compounds and allowed us to obtain several molecules that selectively bind to CAIX. In addition, we also investigated the binding properties of N-sulfamoylphenyl- and N-sulfamoylphenyl-N-thiazolyl-β-alanines and their derivatives [31], which showed better binding results as compared to compounds with a 5-oxopyrrolidine moiety as a linker. With the aim of further investigating the N-thiazolyl-β-alanine residue, we synthesized some analogous thiazole derivatives with the aliphatic substituents at C-4 and C-5 of the thiazole ring and studied their binding to CA. Moreover, to our knowledge, there were no CA inhibition studies of benzenesulfonamides with a β,γ-amino acid moiety as a linker. Therefore, we decided to explore this idea and incorporated the γ-aminobutyric acid moiety into the N-sulfamoylphenyl-β-alanine structure.

2. Results and Discussion

2.1. Organic Synthesis

Pyrrolidinones 1a,b, which were used in this work as precursors, were synthesized according to the methods described in previous works [29,30]. Compounds 4-((2-(hydrazinecarbonyl)-4-hydrazineyl-4-oxobutyl)amino)benzenesulfonamide (2a) and 3-chloro-4-((2-(hydrazinecarbonyl)-4-hydrazineyl-4-oxobutyl)amino)benzenesulfonamide (2b) were synthesized by nucleophilic ring-opening reactions of pyrrolidones 1a,b by heating them under reflux in an excess of hydrazine monohydrate [32] (Scheme 1). The structures of 2a,b and all of the other compounds have been confirmed by FT-IR, ¹H and ¹³C NMR spectroscopy, and mass spectrometry or elemental analysis data. The triplet signals of NH group at 6.39 ppm (2a) and 6.05 ppm (2b) in the ¹H NMR spectra of compounds 2a,b confirmed the existence of an open pyrrolidone ring structure. Subsequently, β,γ-amino acid derivatives 2a,b were used as the precursors for the synthesis of a series of hydrazone derivatives. Reactions between compounds 2a,b and ketones (acetone and acetophenone) at reflux temperature provided corresponding hydrazones 3a,b and 5a,b, whereas the ones with corresponding benzaldehydes yielded hydrazones 6a,b–18a,b (Scheme 1). ¹H NMR spectra for compounds 3a,b and 5a,b display double sets of resonances for each of the CO-NH group protons, due to the restricted rotation around the amide bond. Similar duplication of CO-NH and N=CH group protons signals was also observed in ¹H NMR spectra of compounds 6–18a,b. Since compounds 6–18a,b possess two isomerism centers, existing in each side chain, 10 isomers of these compounds can form. Therefore, CO-NH or N=CH group proton resonances were observed as the sets of multiplets. For instance, in the ¹H NMR spectrum for 6a, multiplets at 7.94–8.23 ppm and 11.21–11.65 ppm were assigned to N=CH and CO-NH group protons, respectively. This splitting of the proton resonances indicates that in DMSO-d₆ solution hydrazones 3a,b, 5a,b, and 6–18a,b exist as a mixture of Z/E isomers. However, in the attempt to synthesize analogous dihydrazones from compounds 2a,b with methyl ethyl ketone, dioxopyrrolidine derivatives 4a,b were obtained instead of target compounds 4c,d (Scheme 1). The structure of compound 4a was determined by X-ray structural analysis data (Figure 1), thereby confirming the empirical formula—C₁₅H₂₀N₄O₄S. In addition, in the ¹H NMR spectrum of 4a, the multiplet at 2.58–3.14 ppm was assigned to pyrrolidine CH₂ group protons, while the multiplet at 3.25–3.59 ppm to CH and NCH₂ group protons.

The multiplet at 6.45–6.58 ppm has been ascribed to protons of NH group. Furthermore, the multiplet at 0.82–1.24 ppm and a singlet at 1.70 ppm integrated for three protons of methyl groups, respectively. Similar spectral data were observed in compound 4b ¹H and ¹³C NMR spectra.

Hydrazides 2a,b were used for the synthesis of a series of azole derivatives (Scheme 2). Pyrrole or pyrazole derivatives can be synthesized by the condensation reaction of carboxylic acid hydrazides with aliphatic diketones [33,34]. Pyrroles 19a,b were obtained from hydrazides 2a,b by the reactions with hexane-2,5-dione in propan-2-ol at the reflux temperature of the reaction mixture. For instance, in the ¹H NMR spectrum for 19b, singlets at 1.91, 1.94, and 1.98 ppm were assigned to the protons of CH₃ groups, and each of the singlets at 5.61 and 5.63 ppm integrated for two protons of CH groups. These proton resonances have confirmed the presence of 2,5-dimethylpyrrole moiety. The singlets at 10.72 and 10.87 ppm have been ascribed to protons of NH groups. Similar signals are found in the compound 19a ¹H NMR spectrum as well. Furthermore, there was an attempt to synthesize 3,5-dimethylpyrrazoles 20 and 20a using the same method. Unfortunately, only one of the desired products (compound 20) was obtained. In the ¹H NMR spectrum for 20, four singlets integrated for 12 protons of CH₃ groups at 2.18, 2.25, 2.35, and 2.43 ppm, and two singlets assigned to the protons in the CH groups at 6.16 and 6.24 ppm have proven the presence of 3,5-dimethylpyrazole moiety in the molecule. Moreover, the triplet at 6.52 ppm assigned to the proton of NH group proved an open 2-pyrrolidone ring structure of compound 20. To obtain compound 20 non-chlorinated analog, previously synthesized pyrazole 21 [30] was isolated from the reaction mixture. The ¹H and ¹³C NMR spectra data for 21 were very similar, as was described in previous work [30], which confirmed a closed 2-pyrrolidone ring structure in 21. We have proposed a hypothesis that, due to the acidic properties of pentane-2,4-dione and high temperature, the 2-pyrrolidone ring closed during the reaction. These conditions were suitable for compound 20 synthesis. We proposed that some steric hindrance, which occurs due to the chlorine substitute at C-2 of the phenyl ring, prevents the closure of the 2-pyrrolidone ring. In order to synthesize the desired product 20a, we have introduced several changes in reaction conditions. When the reaction was carried out at room temperature with NaOAc (to reduce the acidity of pentane-2,4-dione), we failed to detect new products by TLC after 24 h. Then we raised the reaction temperature to 50 °C, and after that we observed compound 21 by TLC.

Reactions of hydrazides 2a,b with methyl isothiocyanate or its phenyl analog in DMF led to the formation of carbothioamides 22a,b and carbothioamides 23a,b, respectively (Scheme 2). Further condensation reactions of 22a,b and 23a,b in the alkaline medium resulted in the formation of methyl triazolethiones 24a,b and phenyl triazolethiones 25a,b correspondingly. In the ¹H NMR spectra for 24a and 24b, singlets at 3.33, 3.39 ppm (24a) and 3.36, 3.40 ppm (24b) have been assigned to the protons of CH₃ groups. In the ¹³C NMR spectrum for 24a, two signals at 166.49 and 166.74 ppm have been attributed to C=S carbon in triazolethione ring. Similar carbon resonances were observed in compounds 24b and 25a,b ¹³C NMR spectra.

In the next stage of this work, thioureido acid 26 was synthesized by the procedure [31] and used for the synthesis of new functionalized aminothiazoles. Compound 26 was treated with an excess of chloroacetone in water, and sodium acetate was used as a base to obtain thiazole 27 (Scheme 3). In the ¹H NMR spectra for 27, singlets at 2.19 and 6.44 ppm have been assigned to the protons of CH₃ and CH group, respectively. These proton resonances have confirmed the presence of 4-methylthiazol moiety. Later, ester 28 was synthesized by an esterification reaction of thiazole 27 with an excess of methanol at reflux in the presence of sulfuric acid as a catalyst. Hydrazine hydrate reaction with ester 28 in propan-2-ol at reflux temperature yielded hydrazide 29, which was treated with benzaldehyde in propan-2-ol at reflux to afford hydrazone 30. The ¹H NMR spectra 30 display double sets of resonances for the CO-NH and N=CH group protons due to the restricted rotation around the amide bond. The splitting of the proton resonances is also observed for carbon chain protons—two triplets at 2.58 and 3.00 ppm were assigned to the protons of CH₂ group, while the multiplet at 4.06–4.36 ppm region to protons of NCH₂ group. This proton splitting indicates that in DMSO-d₆ solution hydrazone 30 exists as a mixture of Z/E isomers, and, in most cases, the Z isomer predominates [33].

The reaction of carboxylic acid 26 with ethyl 2-chloroacetoacetate in water at reflux gave thiazole 31. Sodium acetate was used as a base (Scheme 3). In the ¹H NMR spectra for 31, a triplet at 2.19 and a singlet at 2.52 ppm have been assigned to the protons of CH₃ groups. In the ¹³C NMR spectrum for 31, two signals at 14.23 and 17.31 ppm have been attributed to CH₃ group carbons and the signal at 60.22 to the carbon of ester CH₂ group. These proton resonances have confirmed the formation of desired product 31. Furthermore, thiazole 31 was treated with an excess of methanol in the presence of sulfuric acid as a catalyst to afford ester 32, which was then used in the reaction with hydrazine hydrate to obtain hydrazide 33. The reaction of sulfonamide 33 with benzaldehyde in propan-2-ol at reflux yielded hydrazone 34. Double sets of resonances are also observed in the ¹H NMR spectra for 34 similar to those in the spectra for 30. Therefore, this indicates that hydrazone 34 also exists as a mixture of Z/E isomers in DMSO-d₆ solution.

Previously synthesized compound 35 [31] was obtained by stirring thioureido acid 26 with 4-chloro-2′-bromoacetophenone in acetone at reflux temperature. Chlorine was introduced into the phenyl ring at the C-3 position of the benzenesulfonamide moiety to determine the halogenation effect on the binding affinity toward carbonic anhydrases. Thiazole 35 was treated with N-chlorosuccinimide in DMF at 0 °C. In the ¹H NMR spectra for 36, the doublets at 7.54 and 7.72 ppm were attributed to the protons of the aromatic ring of 4-sulfamoylphenyl moiety. Moreover, there was no signal observed at the 7.38 ppm region as was described in previous work [31], which was assigned to the proton of CH group in the thiazole ring. This data confirmed that electrophilic substitution only occurred in the thiazole ring, which led to the formation of 5-chlorothiazole 36 but not the desired product 37. To synthesize compound 37, some changes were made to the reaction conditions. Performing the reaction at room temperature in DMF with NClS and monitoring the course of the reaction by TLC, after 1 h the formation of compound 36 was observed. It was then decided to raise the temperature to 80 °C [35], and the reaction was reperformed, but after the 20 h the only compound observed by TLC was thiazole 36. Furthermore, chlorination of compound 35 was attempted in 6 M aqueous HCl solution with hydrogen peroxide overnight [29], although the only product isolated from the reaction mixture was 5-chlorothiazole 36.

2.2. Crystal Structure of Compound 4a

The structure of 4a was examined in more detail. Figure 1 shows a perspective view of molecule 4a with thermal ellipsoids and the atom-numbering scheme followed in the text. The newly obtained 2,5-dioxopyrrolidine ring is planar and forms with the CH₂ (C10) group a dihedral angle of 104.6(7). In the molecular structure, the 2,5-dioxopyrrolidine is almost perpendicular to both the hydrazone fragment (dihedral angle is 115.0(8)) and the benzenesulfonamide system (dihedral angle is equal to 113.3(8)).

2.3. Compound Binding to Carbonic Anhydrases (CA)

The dissociation constants of all synthesized compounds were measured for five CA isozymes, CAI, CAII, CAIX, CAXII, and CAXIII (

Table 1. The observed dissociation constants of CAI, CAII, CAIX, CAXII, and CAXIII with tested compounds at pH 7.0 and 37 °C by the fluorescent thermal shift assay. ND–K_{d_obs} value was not determined. K_{d_obs} data of compounds 21 [30], 26 [31], and 35 [31] were taken from previous publications, as well as compound laboratory names marked with *. The standard deviation of the FTSA measurements is ±1.6-fold in K_{d_obs}.

Cmpd.	Lab. Name	Observed Dissociation Constant (Kd_obs), nM
		CAI	CAII	CAIX	CAXII	CAXIII
2a	2a	78,000	14,000	4000	71,000	130,000
2b	2b	6700	1300	830	14,000	6700
3a	BB2.2-7	170,000	13,000	6700	110,000	100,000
3b	BB2.1-7	10,000	1800	670	14,000	6700
4a	BB2.2-8	18,000	40,000	2000	18,000	20,000
4b	BB2.1-8	2900	330	250	4000	1300
5a	BB2.2-19	8300	1300	590	500	5000
5b	BB2.1-19	4000	250	180	6700	400
6a	3a	14,000	1800	560	25,000	7700
7a	BB2.2-20	6700	2000	500	20,000	10,000
7b	BB2.1-20	1000	170	67	5000	170
8a	BB2.2-21	10,000	2000	670	33,000	5000
8b	BB2.1-21	1000	250	100	20,000	200
9a	BB2.2-22	5900	1300	500	25,000	4300
10a	BB2.2-24	17,000	2500	500	50,000	1700
10b	BB2.1-24	1700	330	130	50,000	130
11a	BB2.2-25	20,000	3300	1000	100,000	5000
11b	BB2.1-25	2500	330	130	33,000	330
12a	4a	36,000	5000	1000	66,000	17,000
12b	4b	6700	1100	670	57,000	1500
13a	5a	22,000	2900	1400	23,000	20,000
13b	5b	2600	390	330	23,000	340
14a	6a	32,000	3400	1400	38,000	23,000
14b	6b	510	59	290	10,000	66
15a	BB2.2-17	13,000	3300	1000	50,000	6700
15b	BB2.1-17	2500	500	140	50,000	400
16a	BB2.2-18	20,000	4000	1400	50,000	14,000
16b	BB2.1-18	3300	500	130	130,000	1000
17a	7a	36,000	5400	3300	79,000	19,000
17b	7b	2600	570	1400	58,000	1400
19a	BB2.2-9	10,000	1700	560	10,000	4000
21	10 *	250	66	220	1400	ND
23a	BB2.2-11	4000	2000	130	2200	10,000
24a	BB2.2-31	43,000	6700	910	3300	19,000
24b	BB2.1-31	2900	250	59	1700	1300
25a	BB2.2-12	100,000	3300	100	4000	25,000
26	19 *	10,000	5000	10,000	33,000	ND
35	31 *	25	100	560	200	530

), by the fluorescent thermal shift assay (FTSA, also referred to as TSA and DSF—differential scanning fluorimetry [36,37,38,39]) (Figure 2).

The binding affinities of 19 compounds for all 12 catalytically active human CA isozymes were measured to test the selectivity (

Table 2. The observed dissociation constants of all 12 catalytically active CAs with tested compounds at pH 7.0 and 37 °C by the fluorescent thermal shift assay. The value ≥ 200,000 is the upper limit of K_{d_obs} detection; ND–K_{d_obs} value was not determined. The standard deviation of the FTSA measurements is ± 1.6-fold in K_{d_obs}. The ITC data are shown in the brackets (pH 7.0, 37 °C).

Cmpd.	Lab. Name	Observed Dissociation Constants (Kd_obs), nM
		CAI	CAII	CAIII	CAIV	CAVA	CAVB	CAVI	CAVII	CAIX	CAXII	CAXIII	CAXIV
6b	3b	1400	240	≥200,000	4200	20,000	130	670	290	100	7900	180	140
9b	BB2.1-22	670	200	≥200,000	1000	2900	830	670	250	100	14,000	250	130
18a	BB2.2-23	20,000	5000	≥200,000	33,000	20,000	14,000	33,000	6700	1400	20,000	25,000	5000
18b	BB2.1-23	5000	1300	≥200,000	13,000	6700	6700	20,000	330	250	13,000	1800	1000
19b	BB2.1-9	290	50	≥200,000	1800	20,000	1400	5000	200	56	1300	130	25
20	BB2.1-10	5000	500	≥200,000	2000	22,000	3300	20,000	10,000	100	13,000	2900	330
22a	BB2.2-30	50,000	2500	≥200,000	6700	33,000	25,000	13,000	10,000	330	11,000	33,000	3300
22b	BB2.1-30	3300	400	≥200,000	2000	6700	≥200,000	3300	670	100	2200	1700	130
23b	BB2.1-11	67	50	15,000	1000	20,000	1100	1000	110	33	50	400	50
25b	BB2.1-12	170	25	5000	1400	38,000	1400	200	170	25	630	400	25
27	BB8.1-7	67	220	63,000	1400	≥200,000	560	18,000	250	48	180	870	170
28	BB8.1-12	37	45 (59)	≥200,000	1100	50,000	46	7100	77	33	250	250	29
29	BB8.1-14	150	380	≥200,000	1500	≥200,000	77	20,000	670	80	830	1700	130
30	BB8.1-15	3300	4000	≥200,000	27,000	≥200,000	11000	67,000	100,000	290	110,000	8300	6700
31	BB8.1-4	2000	910 (860)	≥200,000	6700	≥200,000	20,000	24,000	1700	360	3800	450	2000
32	BB8.1-5	830	53	≥200,000	2000	≥200,000	30 (41)	10,000	130	190	7700	330	770
33	BB8.1-10	3100	500	≥200,000	2800	≥200,000	100	25,000	1100	630	26,000	830	910
34	BB8.1-13	670	150	≥200,000	1000	≥200,000	120	10,000	250	170	14,000	220	1000
36	BB8.3-3	20	40 (130)	17,000	3300	≥200,000	400	6300	33	23	120	40	130

and Figure S1). For clarity we divided our compounds in two groups according to chemical structure: para-N β,γ-amino acid (2a,b, 3a,b, 5a,b–25a,b) and para-N β-amino acid and thiazol (compounds 27–36) bearing benzenesulfonamide derivatives. The binding properties of both groups will be described separately. The affinity of the compounds for five CA isozymes is shown in Figure 3.

For some compounds, the pK_a values for the sulfonamide group were measured and used to calculate the intrinsic, pH-independent dissociation constants or changes in Gibbs energy [40] (Figure 4).

In addition, the binding strength of several compounds was confirmed by isothermal titration calorimetry [41]. The binding data of compounds 28, 31, and 36 to CA II and compound 32 binding to CA VB were in good agreement; the K_d__obs values measured by both methods differed by less than three-fold. Structural characterization of three compounds’ (28, 31, and 36) binding was performed by determining their X-ray crystal structures in complexes with CAII (Figure 5).

2.3.1. para-N β,γ-Amino Acid-Substituted Benzenesulfonamide Derivatives (2–25a,b) Binding to CA

Tested compounds 2–20a,b and 22–25a,b can be grouped into pairs differing by a chlorine atom at meta- position on the benzene ring: compounds b—chlorinated and compounds a—non-chlorinated. Chlorinated compounds exhibited significantly greater observed affinity towards most CA isozymes (Figure 3 or Table 1), with on average a 40-fold increase in affinity for CAI and CAXIII, 15–20-fold for CAII, CAVII, and CAXIV, and marginally increased affinity toward CAIII, CAIV, CAIX, and CAXII.

Compounds 2a and 2b, which are precursors to compounds 3a,b–25a,b, exhibited weak interaction with CAI, CAII, CAIX, CAXII, and CAXIII (K_{d_obs} 0.83–130 μM). Compound 2b had a moderately strong interaction with CAIX (K_{d_obs} = 0.83 μM). The size and hydrophobicity of substituents increases in the direction 3a→6a and 3b→6b, resulting in the increment of affinity toward CAI, CAII, CAIX, CAXII, and CAXIII. However, observed affinity for CAXII of compounds 5a→6a decreased 50 times. Ligand flexibility could also play a role; ligands 5a and 5b having an extra rotatable bond might allow better interaction of the benzene ring with hydrophobic amino acids of CAXII, compared to 6a and 6b. Compound 6b was also tested with all other catalytically active CA isozymes (Table 2); it exhibited weak interaction with CAIII, CAIV, CAVA, and CAXII (K_{d_obs} ≥ 200,000 nM, 4200 nM, 20,000 nM, and 7900 nM, respectively), moderately strong interaction with CAI and CAVI, and strong interaction with CAII, CAVB, CAVII, CAIX, CAXIII, and CAXIV (K_{d_obs} in the range of 100–290 nM). Although compounds 4a and 4b contain closed dioxopyrrolidine ring structure (Figure 3), the affinities for CAI, CAII, CAIX, CAXII, and CAXIII did not differ from the other compounds described in this section. No remarkable selectivity toward any CA isozyme was observed for compounds 2a,b, 3a,b, 5a,b, 6a,b, nor even 4a,b, but usually there was an increase in observed affinity (up to 10–100 times) for CAIX compared to other tested CAs.

Ligands 7–18a and 7–18b are structurally similar to 6a and 6b, only differing by symmetric substituents on the phenyl ring. Small substituents such as -OH, -Cl, -F, -OCH₃, -COOH, and -NO₂ as well as their positions in the phenyl ring did not significantly affect the binding affinity towards CAs. Compounds 7–18a exhibited low affinity for tested CA isozymes, with no significant selectivity for a particular CA. The K_d ranges for CAI were 5.0–36 μM, CAII—1.3–5.0 μM, CAIX—0.5–3.3 μM, CAXII—20–100 μM, and for CAXIII—1.7–23 μM. Chlorinated compounds 7–18b proved to be of more interest, with a generally increased observed affinity toward CAII, CAIX, and CAXIII compared to analogous non-chlorinated compounds 7–18a (Figure 3). Compounds 7–9b, having -OH substituent in, respectively, ortho-, meta-, and para-positions on the benzene ring, interacted similarly with CAI, II, IX, XII, and XIII as compound 6b. Compound 14b, having OCH₃ substitute in para-position on the benzene ring, exhibited the strongest interaction with CAII and CAXIII (K_{d_obs} 59 nM and 66 nM, respectively), although binding was not selective, due to moderately strong interaction with CAI and CAIX (K_{d_obs} 510 nM and 290 nM, respectively).

Thiourea derivatives 22–25a,b (Figure 3) exhibited similar binding patterns to CAI, CAII, CAIX, CAXII, and CAXIII, showing increases in observed binding affinity going from CAI to CAIX. Compounds 22a–25a had a trend of decreasing affinity toward CA isozymes going from CAIX to CAXIII. A similar trend was not observed for their chlorinated analogs 22–25b, which showed a stronger affinity for CAIX but did not have significant differences in binding strength for CAXII and CAXIII (Table 1). Compounds 22a,b, 23b, and 25b were also tested with all catalytically active CA isozymes, and no selectivity was observed. Compound 22a exhibited weak binding to all tested CA isozymes (micromolar K_{d_obs} values). This is consistent with a trend of non-chlorinated compounds having weaker observed binding affinity to CAs than chlorinated analogs. On the other hand, 22a interacted with a stronger affinity with CAIX (K_{d_obs} 330 nM). Compound 22b bound CA isozymes more than three times stronger than 22a and showed the highest affinities for CAIX and CAXIV (K_{d_obs} values of 100 nM and 130 nM, respectively). The K_{d_obs} decreased in the direction 22b→23b→25b for most CAs, especially for CAII (400 nM→50 nM→25 nM), CAIX (100 nM→33 nM→25 nM), and CAXIV (from 130 nM→50 nM→25 nM). Compound 25b also had an unusually strong interaction with CAIII (K_{d_obs} 5000 nM, Table 2), despite having large substituents, which usually contribute to steric hindrance and weak binding to CAIII.

2.3.2. para-N β-Amino Acid and Thiazol Bearing Benzenesulfonamide Derivatives (27–36) Binding to CA

Compounds 27–6 had greater asymmetry than compounds 2–25a,b (Figure 3). Compounds 27–34 and 36 were tested with all catalytically active CA isozymes. In general, compounds from this group exhibited weak interaction with CAIII and CAVA (Table 2), with the K_{d_obs} values ranging from 17 µM to ≥200 µM (200 µM being the weakest detectable K_{d_obs} value). Compounds 27–30 differed by substitutes on β-aminoacid carbonyl group, increasing in size (–OH, –OCH₃, –NHNH₂, –NHN=CPh). Carboxylic acid 27 strongly bound to CAI and CAIX (K_{d_obs} 67 nM and 48 nM, respectively) but also had moderately strong interactions with CAII, CAVII, CAXII, and CAXIV (K_{d_obs} in the ranges 170–250 nM). Esterification of thiazole in 27 to compound 28 increased affinity toward CAII, CAVA, CAVB, and CAXIV (K_{d_obs} values of 45 nM, 50 µM, 46 nM, 29 nM of compound 28, respectively). Of special interest was an increase in affinity toward CAVB in the direction 27→28, with K_{d_obs} value decreasing from 560 nM to 46 nM (Table 2). The hydrazide 29 interacted with all CAs slightly weaker than 28. Hydrazone 30, having by far the largest, hydrophobic, and inflexible –NHN=CPh group, showed a significantly decreased binding strength to all tested CA isozymes (K_ds in the micromolar range). Compounds 31–34 were analogous to compounds 27–30 but had an additional ester substitute on the thiazole ring, which made compounds larger and less flexible than their counterparts (Figure 3). Ligands 31–33 showed similar or significantly weaker interactions with most CA isozymes than their analogous compounds without ethylester group on thiazole ring, 27–29. However, of all tested compounds, the strongest interaction was measured between 32 and CA VB (K_d = 30 nM). Binding affinities of compound 34 towards CAII, CAVB, and CAIX were in the range of 100 nM. Compounds 35 and 36 showed the strongest affinities to CAI (K_{d_obs} 25 nM (Table 1) and 20 nM (Table 2), respectively). Furthermore, compound 36 interacted strongly with CAII, CAVII, CAIX, and CAXIII (K_{d_obs} in the range of 23–40 nM), showing no selectivity toward CAs.

2.3.3. Intrinsic Thermodynamics of Compounds 27–36 Binding to CAs

We calculated the intrinsic dissociation constants (K_{d_int},

Table 3. The intrinsic dissociation constants (K_{d_int}) for compounds binding to CAs at 37 °C. ND—not determined. *—the pK_a value was not determined and is an average of all measured compounds. Experimental data and statistics of pK_a values are provided in Supplementary Materials.

Cmpd.	Lab. Name	Sulfonamide pKa	Intrinsic Dissociation Constants (Kd_int), nM
			CAI	CAII	CAIII	CAIV	CAVA	CAVB	CAVI	CAVII	CAIX	CAXII	CAXIII	CAXIV
27	BB8.1-7	10.4	0.025	0.039	6.0	0.16	≥27	0.11	0.65	0.038	0.0054	0.028	0.31	0.0076
28	BB8.1-12	10.1	0.027	0.016	≥38	0.25	13	0.018	0.51	0.024	0.0075	0.077	0.18	0.0026
29	BB8.1-14	10.3	0.070	0.084	≥24	0.21	≥33	0.019	0.91	0.13	0.011	0.16	0.77	0.0073
31	BB8.1-4	10.1	1.5	0.32	≥38	1.5	≥53	7.9	1.7	0.52	0.081	1.2	0.32	0.18
32	BB8.1-5	9.7	1.5	0.047	≥96	1.1	≥130	0.030	1.8	0.10	0.11	5.9	0.60	0.17
33	BB8.1-10	10.1 *	2.3	0.18	≥38	0.63	≥53	0.040	1.8	0.34	0.14	8.0	0.60	0.081
36	BB8.3-3	10.2	0.012	0.011	2.6	0.59	≥42	0.13	0.36	0.0081	0.0041	0.029	0.023	0.0092

) and intrinsic Gibbs energy (ΔG_int, Table S1) values of binding between CA isozymes and compounds 27–29, 31–33, and 36 and constructed a compound structure–affinity correlation map (Figure 4) which demonstrates how small changes in functional groups influence compound capability to bind a particular CA isozyme. The intrinsic binding parameters represent the actual binding reaction between the deprotonated sulfonamide and the Zn-bound water form of CA. To calculate the intrinsic binding parameters by Equations (4) and (5), the pK_as of tested sulfonamides were measured spectrophotometrically by measuring absorption at different pH values. The average pK_a values of compounds 27–29, 31, 32, 35, and 36 were determined to be 10.1. Calculation of the intrinsic values has a significant advantage over the observed values, because the intrinsic values eliminate the pH and buffer effects that may be misleading in the structural origins of the altered affinity [5,42,43].

All compounds exhibited relatively weak binding to CAIII (K_{d_int} at limit of detection for compounds 28–34 and K_{d_int} values of 6 and 2.6 nM, respectively, for compounds 27 and 36) and CAVA (K_{d_int} at limit of detection for all tested compounds except compound 28, which binds with K_{d_int} of 13 nM (Table 3)). Figure 4 depicts some compounds in which minor structural changes had a significant effect on affinity for carbonic anhydrases. Esterification of carboxyl group (directions 27→28 and 31→32) (Figure 4) was favorable or had negligible effect on the Gibbs energy for all CAs except for CAXII, which showed a slight decrease in affinity. Addition of methoxy group leads to a large gain in ΔG_int for CAVB (ΔΔG_int (27→28) = -4.6 and ΔΔG_int (31→32) = −14.4 kJ/mol). Further modification of the methoxy with hydrazide group (directions 28→29 and 32→33) decreased the Gibbs energy of binding to all CAs but did not change that to CAVB (ΔΔG_int (28→29) = 0.1 and ΔΔG_int (32→33) = 0.7 kJ/mol). Interestingly, addition of ethoxycarbonyl group on the thiazole ring (directions 27→31, 28→32, and 29→33) resulted in the decrease of ΔG_int or had negligible effect for all CAs, with the largest reduction of affinity for CAXII (ΔΔG_int (28→32) = 11.2 kJ/mol). In summary, the intrinsic binding data showed that esterification increased the binding affinity for the majority of CA isozymes (Table 3). Thus, it would be very interesting to investigate the influence of small and hydrophobic substitutes on the carboxyl group with the perspectives of finding possible new lead compounds for use as inhibitors for CAVB while binding weakly to CAVA isozyme and possibly having increased selectivity for CAVB isozyme.

2.3.4. X-ray Crystallography of Human CAII Complexes with Inhibitors

The crystal structures of compounds 28, 31, and 36 in the complex with CAII were determined by X-ray crystallography (Figure 5). The electron density maps of the ligands bound in the active site of CAII are shown in Figure S2 in Supplementary Materials. The data collection and refinement statistics are presented in Table S2. In all three structures, the benzenesulfonamide ring occupies a similar position (Figure 5) due to the interaction with Leu198; only the positions of the substituents differed. The carboxy group of compounds 31 and 36 faces the hydrophilic side (colored cyan in Figure 5) of the active site of CAII and forms hydrogen bonds with Asn62, Asn67, or water molecules, while the methyl ester in compound 28 adheres to a more hydrophobic surface (colored brown in Figure 5). The 2D schemes of the interactions are shown as LigPlot schemes based on X-ray crystallography in Figure S3. The thiazole-based substituent interacts with Phe131 and was found on either side of the Phe131 side chain depending on the other substituents (Figure 5). Thus, the smallest thiazole substituent of compound 28 occupied the “right side” of the hydrophobic pocket, while the larger thiazole-based substituents of compounds 31 and 36 occupied the more spacious “left side” of the pocket, because they would not fit in the pocket on the “right side” without changing the position of the benzenesulfonamide due to steric hindrance.

3. Materials and Methods

3.1. Organic Synthesis

Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The melting points were determined on a MEL-TEMP (Electrothermal, Bibby Scientific Company, Burlington, NJ, USA) melting point apparatus and were uncorrected. The IR spectra (ν, cm⁻¹) were recorded on a Perkin–Elmer Spectrum BX FT–IR spectrometer using KBr pellets. The ¹H and ¹³C NMR spectra were recorded in DMSO-d₆ medium on Brucker Avance III (400, 101 MHz) spectrometer (see Supplementary Materials, Figures S4–S103). The chemical shifts (δ) were 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 Bruker maXis UHR-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with ESI ionization. Elemental analysis was performed on a CE-440 elemental analyzer (Exeter Analytical Inc., North Chelmsford, MA, USA). The reaction course and purity of the synthesized compounds were monitored by TLC using aluminium plates pre-coated with the silica gel 60 F₂₅₄ (MerckKGaA, Darmstadt, Germany). Single crystals of 4a were investigated on a Rigaku, XtaLAB Synergy, Dualflex, HyPix diffractometer. The crystal was kept at 170.0(1) K during data collection. Using Olex2 [44], the structure was solved with the SIR2014 [45] structure solution program using Direct Methods and refined with the olex2.refine [46] refinement package using Gauss–Newton minimization.

3.1.1. General Procedure for the Synthesis of Hydrazides 2a,b

A mixture of carboxylic acid 1a (3.70 g, 13 mmol) or 1b (4.14 g, 13 mmol) and hydrazine monohydrate (6.3 mL, 130 mmol) was heated at reflux for 4 h. The reaction mixture was cooled down, diluted with propan-2-ol, and heated until reflux. Then, the suspension was left to cool down, and the precipitate was filtered off and recrystallized from propan-2-ol and water mixture.

4-((2-(Hydrazinecarbonyl)-4-hydrazineyl-4-oxobutyl)amino)benzenesulfonamide (2a)

White solid, yield 3.58 g (84%); m.p. 164–165 °C; IR (KBr) (v, cm⁻¹): 3399, 3301, 3213, 1607, 1603; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.11–2.37 (m, 2H, CH₂), 2.79–2.96 (m, 1H, CH), 2.99–3.33 (m, 2H, NCH₂), 4.32 (br. s, 4H, 2NH₂), 6.39 (t, 1H, J = 6.0 Hz, NH), 6.60–7.54 (m, 6H, H_Ar, SO₂NH₂), 9.01, 9.11 (2s, 2H, 2NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 33.66, 44.40, 110.86, 127.29, 130.16, 151.13, 169.84, 172.00; HRMS (ESI) for C₁₁H₁₈N₆O₄S + H⁺, calcd. 330.1110, found 330.1115 [M + H⁺].

3-Chloro-4-((2-(hydrazinecarbonyl)-4-hydrazineyl-4-oxobutyl)amino)benzenesulfonamide (2b)

White solid, yield 3.81 g (80%); m.p. 183–184 °C; IR (KBr) (v, cm⁻¹): 3312, 3213, 1635, 1616; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.16–2.41 (m, 2H, CH₂), 2.90–3.07 (m, 1H, CH), 3.12–3.45 (m, 2H, NCH₂), 4.29 (br. s, 4H, 2NH₂), 6.05 (t, 1H, J = 5.7 Hz, NH), 6.81–7.72 (m, 5H, H_Ar, SO₂NH₂), 9.03, 9.21 (2s, 2H, 2NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 33.46, 44.62, 110.26, 116.83, 126.13, 126.66, 131.25, 146.29, 169.79, 171.89; HRMS (ESI) for C₁₁H₁₁ClN₂O₅S + H⁺, calcd. 319.0155, found 319.0150 [M + H⁺].

3.1.2. General Procedure for the Synthesis of Hydrazones 3a,b

Hydrazide 2a (0.33 g, 1 mmol) or 2b (0.36 g, 1 mmol) was dissolved in acetone (15 mL), and the solution was heated at reflux for 2 h. The reaction mixture was cooled down, and the precipitate was filtered off.

4-((4-Oxo-2-(2-(propan-2-ylidene)hydrazine-1-carbonyl)-4-(2-(propan-2-ylidene)hydrazineyl)butyl)amino)benzenesulfonamide (3a)

White solid, yield 0.28 g (68%); m.p. 79–80 °C; IR (KBr) (v, cm⁻¹): 3391, 3371, 3250, 1687, 1660; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.63–2.10 (m, 12H, 4CH₃), 2.52–3.54 (m, 5H, CH₂CO, CH, NCH₂), 6.38–7.58 (m, 7H, H_ar, NH, NH₂), 9.84–10.19 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 17.02, 17.12, 17.48, 17.60, 24.94, 25.11, 31.94, 33.08, 35.91, 43.51, 44.69, 110.88, 127.26, 130.10, 149.73, 150.31, 150.93, 151.21, 154.96, 156.07, 169.35, 173.19, 174.53; HRMS (ESI) for C₁₇H₂₆N₆O₄S + H⁺, calcd. 411.1814, found 411.1809 [M + H⁺].

3-Chloro-4-((4-oxo-2-(2-(propan-2-ylidene)hydrazine-1-carbonyl)-4-(2-(propan-2-ylidene)hydrazineyl)butyl)amino)benzenesulfonamide (3b)

White solid, yield 0.31 g (70%); m.p. 133–134 °C; IR (KBr) (v, cm⁻¹): 3361, 3227, 1652, 1594; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.67–2.06 (m, 12H, 4CH₃), 2.54–3.53 (m, 5H, CH₂CO, CH, NCH₂), 5.94–6.43 (m, 1H, NH,), 6.84–7.74 (m, 5H, H_ar, NH₂), 9.83–10.25 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 17.04, 17.16, 17.50, 17.61, 24.93, 25.14, 35.26, 36.10, 43.99, 44.80, 110.34, 116.59, 126.10, 126.10, 131.25, 146.21, 149.88, 149.88, 151.10, 163.12, 173.04, 174.54; HRMS (ESI) for C₁₇H₂₅ClN₆O₄S + H⁺, calcd. 445.1425, found 445.1419 [M + H⁺].

3.1.3. General Procedure for the Synthesis of Hydrazones 4a,b

One mmol of hydrazide 2a (0.33 g) or 2b (0.36 g) was dissolved in propan-2-ol (15 mL), and methyl ethyl ketone (0.36 mL, 4 mmol) was added dropwise to the solution. The reaction mixture was heated at reflux for 3 h; then it was cooled down, and the precipitate was filtered off and washed with diethyl ether.

4-(((1-(Butan-2-ylideneamino)-2,5-dioxopyrrolidin-3-yl)methyl)amino)benzenesulfonamide (4a)

White solid, yield 0.25 g (71%); m.p. 117–118 °C; IR (KBr) (v, cm⁻¹): 3332, 3246, 1693, 1597; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 0.82–1.24 (m, 3H, CH₃), 1.70 (s, 3H, CH₂CH₃), 2.01–2.45 (m, 2H, CH₂CH₃), 2.58–3.14 (m, 2H, CH_2pyr), 3.25–3.59 (m, 3H, CH, NCH₂), 6.45–6.58 (m, 1H, NH), 6.68 (dd, 2H, J = 8.6, 5.7 Hz, H_ar), 6.93 (s, 2H, NH₂), 7.52 (dd, 2H, J = 8.9, 2.5 Hz, H_ar); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 10.30, 18.48, 30.81, 31.41, 31.44, 37.79, 42.55, 42.71, 111.04, 111.09, 127.37, 127.40, 130.72, 151.01, 151.06, 171.70, 173.56, 174.33, 176.03, 182.62; HRMS (ESI) for C₁₅H₂₀N₄O₄S + H⁺, calcd. 353.1284, found 353.1289 [M + H⁺].

4-(((1-(Butan-2-ylideneamino)-2,5-dioxopyrrolidin-3-yl)methyl)amino)-3-chlorobenzenesulfonamide (4b)

Brown solid, yield 0.29 g (74%); m.p. 92–93 °C; IR (KBr) (v, cm⁻¹): 3323, 3247, 1694, 1592; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 0.84–1.16 (m, 3H, CH₃), 1.69 (s, 3H, CH₂CH₃), 2.05–2.47 (m, 2H, CH₂CH₃), 2.53–2.93 (m, 2H, CH_2pyr), 3.11–3.79 (m, 3H, CH, NCH₂), 6.32 (br. s, 1H, NH), 6.80–7.0 (m, 1H, H_ar), 7.13 (s, 2H, NH₂), 7.48–7.71 (m, 2H, H_ar); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 10.29, 18.41, 21.90, 24.80, 25.50, 30.77, 31.40, 37.18, 38.14, 43.02, 43.29, 110.24, 110.39, 117.07, 126.14, 126.21, 126.88, 131.63, 146.12, 171.67, 173.62, 174.32, 176.12, 182.71; HRMS (ESI) for C₁₅H₁₉ClN₄O₄S + H⁺, calcd. 387.0894, found 387.0890 [M + H⁺].

3.1.4. General Procedure for the Synthesis of Hydrazones 5a,b

One mmol of hydrazide 2a (0.33 g) or 2b (0.36 g) was dissolved in propan-2-ol (15 mL), and acetophenone (0.35 mL, 3 mmol) was added dropwise to the solution. The reaction mixture was heated at reflux for 2 h; then it was cooled down, and the precipitate was filtered off and washed with diethyl ether and n-hexane.

4-((4-Oxo-2-(2-(1-phenylethylidene)hydrazine-1-carbonyl)-4-(2-(1-phenylethylidene)hydrazineyl)butyl)amino)benzenesulfonamide (5a)

White solid, yield 0.38 g (72%); m.p. 162–163 °C; IR (KBr) (v, cm⁻¹): 3378, 3244, 1652, 1600; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.93–2.38 (m, 6H, 2CH₃), 2.67–3.61 (m, 5H, CH₂CO, CH, NCH₂), 6.42–8.09 (m, 17H, H_ar, NH, NH₂), 10.18–10.90 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 13.57, 13.92, 14.25, 14.76, 26.75, 30.82, 37.81, 42.75, 43.79, 44.72, 110.78, 110.95, 111.07, 118.89, 126.02, 126.13, 126.30, 126.48, 126.50, 127.37, 127.42, 128.17, 128.30, 128.39, 128.46, 128.71, 129.03, 129.79, 130.09, 130.74, 133.22, 138.23, 147.15, 147.82, 151.04, 151.10, 173.81, 174.36, 175.47, 176.06; HRMS (ESI) for C₂₇H₃₀N₆O₄S + H⁺, calcd. 535.2127, found 535.2123 [M + H⁺].

3-Chloro-4-((4-oxo-2-(2-(1-phenylethylidene)hydrazine-1-carbonyl)-4-(2-(1-phenylethylidene)hydrazineyl)butyl)amino)benzenesulfonamide (5b)

White solid, yield 0.43 g (75%); m.p. 158–159 °C; IR (KBr) (v, cm⁻¹): 3345, 3248, 1700, 1592; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.10–2.32 (m, 6H, 2CH₃), 2.54–3.24 (m, 5H, CH₂CO, CH, NCH₂), 6.23–6.42 (m, 1H, NH), 6.85–7.99 (m, 15H, H_ar, NH₂), 10.26–10.83 (m, 2H, 2CONH); HRMS (ESI) for C₂₇H₂₉ClN₆O₄S + H⁺, calcd. 569.1738, found 569.1735 [M + H⁺].

3.1.5. General Procedure for the Synthesis of Hydrazones 6a,b–18a,b

The mixture of hydrazide 2a (0.17 g, 0.5 mmol) or 2b (0.18 g, 0.5 mmol) with corresponding aldehyde (1.5 mmol) and propan-2-ol (15 mL) was heated at reflux for 2 h. Then, the reaction mixture was cooled down, and precipitate was filtered off and recrystallized from 1,4-dioxane.

4-((2-(2-(Benzylidene)hydrazine-1-carbonyl)-4-(2-(benzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (6a)

White solid, yield 0.21 g (84%); m.p. 208–209 °C; IR (KBr) (v, cm⁻¹): 3364, 3239, 1655, 1601; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.79–3.59 (m, 5H, CH₂CO, CH, NCH₂), 6.57–7.75 (m, 17H, H_ar, NH, NH₂), 7.94–8.23 (m, 2H, NCH), 11.21–11.65 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.17, 32.68, 33.64, 36.07, 43.81, 44.70, 110.81, 110.90, 126.68, 126.71, 126.74, 126.92, 126.96, 127.33, 127.40, 128.78, 128.82, 129.69, 129.77, 130.23, 134.19, 134.26, 134.35, 142.68, 142.95, 143.23, 145.80, 146.26, 151.01, 151.12, 167.33, 169.60, 172.67, 172.96, 174.45, 174.62; HRMS (ESI) for C₂₅H₂₆N₆O₄S + H⁺, calcd. 507.1814, found 507.1809 [M + H⁺].

4-((2-(2-(Benzylidene)hydrazine-1-carbonyl)-4-(2-(benzylidene)hydrazineyl)-4-oxobutyl)amino)-3-chlorobenzenesulfonamide (6b)

White solid, yield 0.24 g (89%); m.p. 212–213 °C; IR (KBr) (v, cm⁻¹): 3406, 3243, 1662, 1598; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.63–3.63 (m, 5H, CH₂CO, CH, NCH₂), 6.13–6.40 (m, 1H, NH), 6.92–7.74 (m, 15H, H_ar, NH₂), 7.91–8.27 (m, 2H, NCH), 11.22–11.67 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.23, 33.65, 35.91, 44.27, 109.98, 116.89, 126.06, 126.07, 126.07, 126.67, 126.70, 126.98, 128.78, 129.69, 131.19, 134.14, 134.26, 142.72, 142.97, 143.43, 143.63, 146.22, 153.77, 172.88, 174.68; HRMS (ESI) for C₂₅H₂₅ClN₆O₄S + H⁺, calcd. 541.1425, found 541.1419 [M + H⁺].

4-((2-(2-(2-Hydroxybenzylidene)hydrazine-1-carbonyl)-4-(2-(2-hydroxybenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (7a)

White solid, yield 0.20 g (74%); m.p. 177–178 °C; IR (KBr) (v, cm⁻¹): 3290, 3252, 3163, 1668, 1602; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.56–3.65 (m, 5H, CH₂CO, CH, NCH₂), 6.57–7.78 (m, 15H, H_ar, NH, NH₂), 8.20–8.47 (m, 2H, NCH), 9.81–10.36 (m, 1H, OH), 11.06–11.40 (m, 2H, 2CONH), 11.61–12.01 (m, 1H, OH); HRMS (ESI) for C₂₅H₂₆N₆O₆S + H⁺, calcd. 539.1713, found 539.1709 [M + H⁺].

3-Chloro-4-((2-(2-(2-hydroxybenzylidene)hydrazine-1-carbonyl)-4-(2-(2-hydroxybenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (7b)

White solid, yield 0.22 g (76%); m.p. 204–205 °C; IR (KBr) (v, cm⁻¹): 3294, 3247, 3190, 1667, 1593; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.52–3.75 (m, 5H, CH₂CO, CH, NCH₂), 6.17–6.47 (m, 1H, NH), 6.57–8.47(m, 15H, H_ar, NCH, NH₂), 9.95–10.19 (m, 1H, OH), 11.04–11.43 (m, 2H, 2CONH), 11.66–11.94 (m, 1H, OH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 31.41, 33.82, 35.36, 44.03, 44.60, 116.12, 116.33, 116.54, 116.99, 118.20, 118.57, 118.60, 119.32, 119.62, 120.09, 120.39, 126.10, 126.80, 129.35, 130.85, 131.36, 131.45, 133.25, 141.01, 146.21, 146.64, 147.19, 156.35, 157.28, 157.32, 158.65, 162.81, 166.77, 167.18, 169.03, 169.37, 172.22, 172.49, 174.01, 174.34; HRMS (ESI) for C₂₅H₂₅ClN₆O₆S + H⁺, calcd. 573.1323, found 573.1322 [M + H⁺].

4-((2-(2-(3-Hydroxybenzylidene)hydrazine-1-carbonyl)-4-(2-(3-hydroxybenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (8a)

White solid, yield 0.22 g (81%); m.p. 192–193 °C; IR (KBr) (v, cm⁻¹): 3444, 3373, 3283, 3172, 1657, 1597; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.57–3.60 (m, 5H, CH₂CO, CH, NCH₂), 6.58–7.59 (m, 15H, H_ar, NH, NH₂), 7.85–8.15 (m, 2H, NCH), 9.62 (br. s,, 2H, OH), 11.16–11.62 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 30.98, 31.87, 32.66, 32.67, 34.33, 36.15, 43.71, 44.71, 47.87, 110.93, 112.56, 112.63, 117.00, 117.29, 118.13, 118.37, 118.68, 127.35, 127.47, 129.83, 130.27, 135.54, 135.62, 142.98, 143.20, 146.43, 151.05, 151.14, 157.64, 157.72, 166.92, 169.31, 169.63, 172.56, 172.89, 174.52; HRMS (ESI) for C₂₅H₂₆N₆O₆S + H⁺, calcd. 539.1713, found 539.1707 [M + H⁺].

3-Chloro-4-((2-(2-(3-hydroxybenzylidene)hydrazine-1-carbonyl)-4-(2-(3-hydroxybenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (8b)

White solid, yield 0.21 g (72%); m.p. 119–120 °C; IR (KBr) (v, cm⁻¹): 3412, 3319, 3231, 3067, 1643, 1597; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.61–3.62 (m, 5H, CH₂CO, CH, NCH₂), 6.09–6.32 (m, 1H, NH), 6.68–7.67 (m, 13H, H_ar, NH₂), 7.82–8.15 (m, 2H, NCH), 9.46–9.67 (m, 2H, OH), 11.12–11.63 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.00, 32.58, 35.36, 36.18, 44.32, 44.84, 110.01, 110.46, 112.53, 112.74, 113.57, 116.88, 117.02, 117.55, 118.12, 118.74, 126.12, 126.22, 126.79, 126.87, 129.85, 131.31, 131.37, 135.43, 135.46, 135.56, 143.06, 143.27, 143.93, 146.20, 146.59, 157.64, 157.67, 166.93, 167.22, 169.22, 169.55, 172.58, 172.81, 174.29, 174.63; HRMS (ESI) for C₂₅H₂₅ClN₆O₆S + H⁺, calcd. 573.1323, found 573.1317 [M + H⁺].

4-((2-(2-(4-Hydroxybenzylidene)hydrazine-1-carbonyl)-4-(2-(4-hydroxybenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (9a)

White solid, yield 0.25 g (93%); m.p. 186–187 °C; IR (KBr) (v, cm⁻¹): 3442, 3356, 3288, 3190, 1657, 1602; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.60–3.28 (m, 5H, CH₂CO, CH, NCH₂), 6.51–7.61 (m, 15H, H_ar, NH, NH₂), 7.83–8.12 (m, 2H, NCH), 9.90 (br. s, 2H, OH), 10.99–11.43 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.38, 33.13, 36.47, 37.27, 44.15, 111.31, 116.12, 116.22, 125.66, 125.70, 125.78, 127.79, 127.86, 128.83, 128.88, 129.14, 129.21, 130.61, 130.65, 143.42, 143.72, 144.12, 146.56, 146.87, 147.02, 147.28, 151.48, 151.59, 151.61, 159.50, 159.57, 159.62, 159.71, 159.76, 167.47, 169.75, 172.77, 173.12, 174.44, 174.66; HRMS (ESI) for C₂₅H₂₆N₆O₆S + H⁺, calcd. 539.1713, found 539.1708 [M + H⁺].

3-Chloro-4-((2-(2-(4-hydroxybenzylidene)hydrazine-1-carbonyl)-4-(2-(4-hydroxybenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (9b)

White solid, yield 0.24 g (83%); m.p. 226–227 °C; IR (KBr) (v, cm⁻¹): 3434, 3327, 3236, 3079, 1633, 1593; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.65–3.62 (m, 5H, CH₂CO, CH, NCH₂), 6.11–6.41 (m, 1H, NH), 6.72–7.75 (m, 13H, H_ar, NH₂), 7.80–8.14 (m, 2H, NCH), 9.74–9.99 (m, 2H, OH), 10.99–11.45 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.06, 32.57, 35.31, 36.01, 44.28, 44.86, 109.99, 110.24, 115.67, 115.73, 116.88, 125.17, 125.34, 126.08, 126.77, 126.88, 128.38, 128.47, 128.67, 128.75, 131.18, 143.02, 143.94, 146.25, 159.06, 159.12, 159.21, 159.31, 169.20, 172.33, 172.58, 174.27; HRMS (ESI) for C₂₅H₂₅ClN₆O₆S + H⁺, calcd. 573.1323, found 573.1319 [M + H⁺].

4-((2-(2-(2-Chlorobenzylidene)hydrazine-1-carbonyl)-4-(2-(2-chlorobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (10a)

White solid, yield 0.25 g (86%); m.p. 174–175 °C; IR (KBr) (v, cm⁻¹): 3452, 3328, 3081, 1694, 1598; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.57–3.61 (m, 5H, CH₂CO, CH, NCH₂), 6.54–7.76 (m, 15H, H_ar, NH, NH₂), 8.31–8.68 (m, 2H, NCH), 11.43–11.91 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.37, 35.98, 43.92, 110.81, 110.92, 126.58, 126.69, 127.39, 127.59, 127.77, 128.22, 129.89, 130.20, 130.24, 130.32, 130.52, 131.17, 131.42, 131.47, 131.49, 132.86, 133.06, 133.20, 134.69, 138.82, 139.04, 139.25, 142.21, 151.02, 151.10, 158.28, 169.75, 172.79, 173.05, 174.90; HRMS (ESI) for C₂₅H₂₄Cl₂N₆O₄S + H⁺, calcd. 575.1035, found 575.1031 [M + H⁺].

3-Chloro-4-((2-(2-(2-chlorobenzylidene)hydrazine-1-carbonyl)-4-(2-(2-chlorobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (10b)

White solid, yield 0.26 g (85%); m.p. 203–204 °C; IR (KBr) (v, cm⁻¹): 3438, 3337, 3077, 1688, 1602; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.64–3.67 (m, 5H, CH₂CO, CH, NCH₂), 6.17–6.43 (m, 1H, NH), 6.98–8.00 (m, 13H, H_ar, NH₂), 8.29–8.64 (m, 2H, NCH), 11.44–11.92 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.38, 32.53, 35.27, 35.84, 44.28, 110.01, 110.41, 116.85, 126.03, 126.42, 126.55, 126.79, 126.86, 127.41, 127.52, 127.76, 128.21, 129.85, 129.89, 130.19, 131.18, 131.37, 131.47, 132.85, 133.00, 133.07, 138.86, 139.40, 139.64, 142.35, 146.22, 158.26, 167.40, 169.64, 172.80, 172.99, 174.95; HRMS (ESI) for C₂₅H₂₃Cl₃N₆O₄S + H⁺, calcd. 609.0645, found 609.0642 [M + H⁺].

4-((2-(2-(3-Chlorobenzylidene)hydrazine-1-carbonyl)-4-(2-(3-chlorobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (11a)

White solid, yield 0.24 g (83%); m.p. 190–191 °C; IR (KBr) (v, cm⁻¹): 3356, 3240, 3193, 1638, 1600; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.59–3.64 (m, 5H, CH₂CO, CH, NCH₂), 6.56–8.25 (m, 17H, H_ar, NH, NCH, NH₂), 11.33–11.83 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.09, 32.61, 36.16, 36.87, 43.77, 44.64, 110.74, 110.93, 125.22, 125.45, 125.57, 125.93, 126.00, 126.16, 126.93, 127.34, 127.43, 127.87, 129.41, 130.26, 130.30, 130.65, 130.88, 131.17, 133.59, 133.65, 133.72, 135.78, 136.44, 136.61, 141.19, 141.41, 141.71, 144.10, 144.56, 151.02, 151.08, 160.61, 167.18, 167.49, 169.80, 172.81, 173.08, 174.78; HRMS (ESI) C₂₅H₂₄Cl₂N₆O₄S + H⁺, calcd. 575.1035, found 575.1033 [M + H⁺].

3-Chloro-4-((2-(2-(3-chlorobenzylidene)hydrazine-1-carbonyl)-4-(2-(3-chlorobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (11b)

Light-brown solid, yield 0.24 g (79%); m.p. 175–176 °C; IR (KBr) (v, cm⁻¹): 3347, 3244, 3192, 1642, 1593; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.61–3.75 (m, 5H, CH₂CO, CH, NCH₂), 6.15–6.44 (m, 1H, NH), 6.90–8.06 (m, 15H, H_ar, NH₂, NCH), 11.23–11.86 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 31.33, 35.41, 37.52, 43.21, 44.22, 109.85, 110.24, 116.86, 117.14, 125.20, 125.55, 125.85, 126.08, 126.20, 126.28, 126.78, 126.92, 127.40, 127.86, 129.35, 130.65, 130.87, 131.04, 131.16, 131.24, 131.66, 131.79, 133.58, 133.66, 133.71, 133.87, 135.11, 135.78, 136.48, 141.21, 141.90, 146.11, 146.20, 160.22, 160.61, 172.60, 172.82, 173.00, 174.46, 174.90; HRMS (ESI) for C₂₅H₂₃Cl₃N₆O₄S + H⁺, calcd. 609.0645, found 609.0641 [M + H⁺].

4-((2-(2-(4-Chlorobenzylidene)hydrazine-1-carbonyl)-4-(2-(4-chlorobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (12a)

Light-yellow solid, yield 0.25 g (86%); m.p. 179–180 °C; IR (KBr) (v, cm⁻¹): 3401, 3243, 3073, 1663, 1600; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.58–3.57 (m, 5H, CH₂CO, CH, NCH₂), 6.58–7.73 (m, 15H, H_ar, NH, NH₂), 7.86–8.04 (m, 2H, NCH), 11.14–11.76 (m, 2H, 2CONH); HRMS (ESI) C₂₅H₂₄Cl₂N₆O₄S + H⁺, calcd. 575.1035, found 575.1030 [M + H⁺].

3-Chloro-4-((2-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)-4-(2-(4-chlorobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (12b)

White solid, yield 0.25 g (83%); m.p. 211–212 °C; IR (KBr) (v, cm⁻¹): 3404, 3242, 3071, 1661, 1595; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.62–3.66 (m, 5H, CH₂CO, CH, NCH₂), 6.09–6.38 (m, 1H, NH), 6.92–7.76 (m, 13H, H_ar, NH₂), 7.87–8.28 (m, 2H, NCH), 11.20–11.83 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.36, 32.50, 35.19, 35.79, 44.34, 44.72, 110.01, 110.43, 116.85, 126.06, 126.85, 128.24, 128.28, 128.35, 128.54, 128.61, 128.80, 128.86, 131.19, 131.36, 133.05, 133.13, 133.18, 133.29, 134.13, 134.19, 134.26, 134.37, 141.50, 141.73, 142.07, 142.29, 144.55, 145.09, 146.25, 167.04, 167.31, 169.60, 172.72, 172.87, 174.62, 174.87; HRMS (ESI) for C₂₅H₂₃Cl₃N₆O₄S + H⁺, calcd. 609.0645, found 609.0640 [M + H⁺].

4-((2-(2-(4-Fluorobenzylidene)hydrazine-1-carbonyl)-4-(2-(4-fluorobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (13a)

White solid, yield 0.20 g (74%); m.p. 153–154 °C; IR (KBr) (v, cm⁻¹): 3297, 3214, 3071, 1639, 1601; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.08–2.34 (m, 2H, CH₂CO), 2.79–3.30 (m, 3H, CH, NCH₂), 6.39 (t, 1H, J = 6.0 Hz, NH), 6.61–7.76 (m, 14H, H_ar, NH₂), 7.90–8.21 (m, 2H, NCH), 11.10–11.69 (m, 2H, 2CONH); HRMS (ESI) C₂₅H₂₄F₂N₆O₄S + H⁺, calcd. 543.1626, found 543.1621 [M + H⁺].

3-Chloro-4-((2-(2-(4-fluorobenzylidene)hydrazine-1-carbonyl)-4-(2-(4-fluorobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (13b)

White solid, yield 0.23 g (79%); m.p. 207–208 °C; IR (KBr) (v, cm⁻¹): 3410, 3244, 3076, 1662, 1597; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.62–3.67 (m, 5H, CH₂CO, CH, NCH₂), 6.15–6.36 (m, 1H, NH), 6.90–7.80 (m, 13H, H_ar, NH₂), 7.88–8.26 (m, 2H, NCH), 11.24–11.70 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.37, 33.74, 35.19, 44.35, 44.75, 110.02, 110.42, 115.72, 115.94, 116.84, 116.91, 126.06, 126.77, 126.85, 128.73, 128.81, 129.02, 129.10, 129.18, 130.89, 131.16, 141.61, 141.85, 142.16, 142.39, 144.74, 145.30, 146.27, 161.64, 161.80, 164.09, 167.25, 169.52, 172.54, 172.67, 172.83, 174.56, 174.80; HRMS (ESI) for C₂₅H₂₃ClF₂N₆O₄S + H⁺, calcd. 577.1236, found 577.1231 [M + H⁺].

4-((2-(2-(4-Methoxybenzylidene)hydrazine-1-carbonyl)-4-(2-(4-methoxybenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (14a)

White solid, yield 0.21 g (75%); m.p. 168–169 °C; IR (KBr) (v, cm⁻¹): 3344, 3188, 3076, 1651, 1603; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.77–3.52 (m, 5H, CH₂CO, CH, NCH₂), 3.79 (s, 6H, 2CH₃), 6.58–7.67 (m, 15H, H_ar, NH, NH₂), 7.89–8.20 (m, 2H, NCH), 11.02–11.57 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.24, 33.65, 35.98, 43.88, 44.39, 55.24, 55.29, 55.29, 110.86, 114.28, 126.81, 126.90, 127.29, 127.43, 128.25, 128.52, 130.17, 142.54, 143.00, 151.05, 151.12, 160.54, 160.73, 166.70, 169.83, 172.00, 172.36, 172.72, 174.41; HRMS (ESI) C₂₇H₃₀N₆O₆S + H⁺, calcd. 567.2026, found 567.2021 [M + H⁺].

3-Chloro-4-((2-(2-(4-methoxybenzylidene)hydrazine-1-carbonyl)-4-(2-(4-methoxybenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (14b)

Light-yellow solid, yield 0.22 g (73%); m.p. 198–199 °C; IR (KBr) (v, cm⁻¹): 3355, 3243, 3072, 1660, 1595; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.60–3.61 (m, 5H, CH₂CO, CH, NCH₂), 3.79, 3.82 (2s, 6H, 2CH₃), 6.09–6.44 (m, 1H, NH), 6.88–8.20 (m, 15H, H_ar, NCH, NH₂), 11.01–11.55 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.30, 32.57, 35.22, 44.33, 55.21, 55.28, 55.39, 109.97, 114.27, 114.39, 126.11, 126.57, 126.71, 126.87, 128.24, 128.49, 128.57, 129.97, 131.12, 131.31, 142.60, 143.22, 146.28, 160.49, 160.72, 161.66, 172.42, 172.64, 174.23, 174.45; HRMS (ESI) for C₂₇H₂₉ClN₆O₆S + H⁺, calcd. 601.1636, found 601.1631 [M + H⁺].

4-((2-(2-(2-Nitrobenzylidene)hydrazine-1-carbonyl)-4-(2-(2-nitrobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (15a)

Yellow solid, yield 0.24 g (80%); m.p. 197–198 °C; IR (KBr) (v, cm⁻¹): 3361, 3194, 3058, 1641, 1598; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.60–3.57 (m, 5H, CH₂CO, CH, NCH₂), 6.59–8.22 (m, 15H, H_ar, NH, NH₂), 8.33–8.66 (m, 2H, NCH), 11.50–12.07 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.38, 32.63, 33.81, 35.88, 44.03, 44.65, 110.82, 110.94, 124.62, 124.70, 124.79, 127.36, 127.58, 127.67, 127.75, 127.82, 127.91, 128.45, 128.50, 128.60, 128.74, 128.81, 129.43, 130.22, 130.34, 130.52, 132.16, 133.57, 133.69, 133.93, 138.23, 138.45, 138.60, 141.29, 141.57, 141.93, 147.97, 148.00, 148.02, 148.14, 148.88, 151.06, 151.10, 158.67, 167.37, 167.60, 169.93, 172.96, 173.16, 174.96, 175.15; HRMS (ESI) C₂₅H₂₄N₈O₈S + H⁺, calcd. 597.1516, found 597.1513 [M + H⁺].

3-Chloro-4-((2-(2-(2-nitrobenzylidene)hydrazine-1-carbonyl)-4-(2-(2-nitrobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (15b)

Yellow solid, yield 0.29 g (94%); m.p. 178–179 °C; IR (KBr) (v, cm⁻¹): 3358, 3243, 3073, 1646, 1591; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.68–3.69 (m, 5H, CH₂CO, CH, NCH₂), 6.17–6.39 (m, 1H, NH), 6.92–7.76 (m, 13H, H_ar, NH₂), 8.32–8.68 (m, 2H, NCH), 11.59–12.03 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.38, 33.78, 35.26, 35.83, 44.31, 44.70, 110.07, 116.84, 116.95, 124.62, 124.69, 124.78, 126.03, 126.80, 127.43, 127.82, 127.88, 128.45, 128.48, 129.43, 130.35, 131.17, 131.40, 132.16, 133.53, 133.71, 133.92, 138.27, 138.67, 138.98, 146.23, 147.96, 148.00, 148.14, 148.88, 158.67, 167.58, 169.84, 172.95, 173.11, 174.90, 175.16; HRMS (ESI) for C₂₅H₂₃ClN₈O₈S + H⁺, calcd. 631.1126, found 631.1121 [M + H⁺].

4-((2-(2-(3-Nitrobenzylidene)hydrazine-1-carbonyl)-4-(2-(3-nitrobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (16a)

Light-yellow solid, yield 0.26 g (87%); m.p. 218–219 °C; IR (KBr) (v, cm⁻¹): 3352, 3260, 3074, 1667, 1595; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.61–3.58 (m, 5H, CH₂CO, CH, NCH₂), 6.58–8.59 (m, 17H, H_ar, NH, NCH, NH₂), 11.29–11.99 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.40, 32.63, 34.03, 34.30, 36.10, 36.78, 36.82, 43.96, 44.62, 110.73, 110.95, 120.89, 121.06, 121.34, 122.66, 124.00, 125.83, 127.34, 130.17, 130.37, 130.66, 132.10, 132.68, 133.18, 133.25, 134.44, 135.24, 136.03, 136.23, 140.64, 140.85, 143.84, 148.16, 148.21, 151.11, 160.50, 167.36, 167.60, 169.76, 169.98, 172.88, 173.10, 174.85, 175.09; HRMS (ESI) C₂₅H₂₄N₈O₈S + H⁺, calcd. 597.1516, found 597.1514 [M + H⁺].

3-Chloro-4-((2-(2-(3-nitrobenzylidene)hydrazine-1-carbonyl)-4-(2-(3-nitrobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (16b)

White solid, yield 0.28 g (90%); m.p. 199–200 °C; IR (KBr) (v, cm⁻¹): 3368, 3242, 3071, 1659, 1598; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.66–3.69 (m, 5H, CH₂CO, CH, NCH₂), 6.14–6.33 (m, 1H, NH), 6.83–8.61 (m, 15H, H_ar, NCH, NH₂), 11.48–12.02 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.44, 35.31, 35.92, 44.33, 44.66, 109.93, 116.76, 116.97, 120.83, 120.93, 121.23, 121.37, 123.93, 124.14, 126.07, 126.76, 127.35, 130.11, 130.36, 131.11, 131.40, 131.47, 132.12, 132.72, 133.20, 135.90, 136.06, 136.18, 140.69, 141.00, 143.44, 143.73, 144.01, 146.24, 148.08, 148.17, 167.35, 167.57, 169.92, 172.89, 173.03, 174.93, 175.21; HRMS (ESI) for C₂₅H₂₃ClN₈O₈S + H⁺, calcd. 631.1126, found 631.1122 [M + H⁺].

4-((2-(2-(4-Nitrobenzylidene)hydrazine-1-carbonyl)-4-(2-(4-nitrobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (17a)

Yellow solid, yield 0.28 g (93%); m.p. 256–257 °C; IR (KBr) (v, cm⁻¹): 3413, 3239, 3066, 1665, 1601; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.60–3.60 (m, 5H, CH₂CO, CH, NCH₂), 6.56–8.36 (m, 17H, H_ar, NH, NCH, NH₂), 11.51–12.00 (m, 2H, 2CONH); HRMS (ESI) C₂₅H₂₄N₈O₈S + H⁺, calcd. 597.1516, found 597.1511 [M + H⁺].

3-Chloro-4-((2-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)-4-(2-(4-nitrobenzylidene)hydrazineyl)-4-oxobutyl)amino)benzenesulfonamide (17b)

Yellow solid, yield 0.25 g (81%); m.p. 220–221 °C; IR (KBr) (v, cm⁻¹): 3390, 3247, 3072, 1662, 1596; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.66–3.67 (m, 5H, CH₂CO, CH, NCH₂), 6.17–6.37 (m, 1H, NH), 6.92–8.37 (m, 15H, H_ar, NCH, NH₂), 11.45–12.14 (m, 2H, 2CONH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.49, 35.15, 35.75, 44.38, 44.65, 110.01, 110.45, 116.80, 116.96, 123.87, 124.01, 124.01, 126.06, 126.77, 127.34, 127.57, 127.64, 127.81, 127.90, 131.18, 140.32, 140.52, 140.57, 140.88, 141.11, 143.44, 143.97, 146.24, 147.59, 147.77, 167.64, 173.04, 173.16, 175.37; HRMS (ESI) for C₂₅H₂₃ClN₈O₈S + H⁺, calcd. 631.1126, found 631.1121 [M + H⁺].

4,4′-(((2-(((4-Sulfamoylphenyl)amino)methyl)succinyl)bis(hydrazin-2-yl-1-ylidene))bis(methaneylylidene))dibenzoic acid (18a)

Light-yellow solid, yield 0.27 g (91%); m.p. 308–309 °C; IR (KBr) (v, cm⁻¹): 3319, 3191, 3074, 1667, 1626, 1601; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.70–3.54 (m, 5H, CH₂CO, CH, NCH₂), 6.45–8.51 (m, 17H, H_ar, NH, NCH, NH₂), 11.32–11.94 (m, 2H, 2CONH), 13.03 (br. s, 2H, 2OH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.76, 35.07, 35.75, 49.98, 118.91, 126.49, 126.71, 126.98, 127.13, 127.39, 128.53, 129.55, 129.78, 129.82, 130.32, 131.64, 133.15, 137.48, 138.23, 139.01, 141.87, 142.73, 161.01, 166.86, 166.94, 172.79, 173.65; HRMS (ESI) C₂₇H₂₆N₆O₈S + H⁺, calcd. 595.1611, found 595.1606 [M + H⁺].

4,4′-(((2-(((2-Chloro-4-sulfamoylphenyl)amino)methyl)succinyl)bis(hydrazin-2-yl-1-ylidene))bis(methaneylylidene))dibenzoic acid (18b)

Light-brown solid, yield 0.28 g (90%); m.p. 294–295 °C; IR (KBr) (v, cm⁻¹): 3348, 3245, 3194, 3064, 1671, 1643, 1597; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.65–3.73 (m, 5H, CH₂CO, CH, NCH₂), 6.13–6.40 (m, 1H, NH), 6.84–8.33 (m, 15H, H_ar, NCH, NH₂), 11.36–11.87 (m, 2H, 2CONH), 13.09 (br. s, 2H, 2OH); HRMS (ESI) for C₂₇H₂₅ClN₆O₈S + H⁺, calcd. 629.1221, found 629.1216 [M + H⁺].

3.1.6. General Procedure for the Synthesis of Pyrroles 19a,b

A mixture of hydrazide 2a (0.46 g, 1.4 mmol) or 2b (0.51 g, 1.4 mmol), hexane-2,5-dione (0.8 mL, 7 mmol), and 30 mL of propan-2-ol was heated under reflux for 5h. Then, the reaction mixture was filtered, and the liquid fractions were evaporated under reduced pressure. The residue was diluted with water (20 mL), and the precipitate was filtered off, washed with water, dried, and recrystallized from propan-2-ol.

N¹,N⁴-bis(2,5-Dimethyl-1H-pyrrol-1-yl)-2-(((4-sulfamoylphenyl)amino)methyl)succinamide (19a)

Light-brown solid, yield 0.51 g (75%); m.p. 195–196 °C; IR (KBr) (v, cm⁻¹): 3399, 3362, 3248, 1667, 1598; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.86–2.02 (m, 12H, 4CH₃), 2.55–2.82 (m, 2H, CH₂), 3.15–3.31 (m, 2H, NCH₂), 3.39–3.56 (m, 1H, CH), 5.60, 5.63 (2s, 4H, 4CH_pyr), 6.61–6.77 (m, 3H, H_ar, NH), 6.95 (br. s, 2H, NH₂), 7.48–7.57 (m, 2H, H_ar), 10.70, 10.81 (2s, 2H, 2NNH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 10.88, 10.92, 10.94, 11.04, 32.83, 44.55, 102.79, 102.89, 102.95, 110.88, 126.62, 126.85, 126.99, 130.56, 150.86, 169.93, 172.10; HRMS (ESI) for C₂₃H₃₀N₆O₄S + H⁺, calcd. 487.2127, found 487.2122 [M + H⁺].

2-(((2-Chloro-4-sulfamoylphenyl)amino)methyl)-N¹,N⁴-bis(2,5-dimethyl-1H-pyrrol-1-yl)succinamide (19b)

Light-brown solid, yield 0.59 g (81%); m.p. 232–233 °C; IR (KBr) (v, cm⁻¹): 3401, 3356, 3244, 1660, 1594; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.91, 1.94, 1.98 (3s, 12H, 4CH₃), 2.58–2.84 (m, 2H, CH₂), 3.36–3.43 (m, 2H, NCH₂), 3.58–3.68 (m, 1H, CH), 5.61, 5.63 (2s, 4H, 4CH_pyr), 6.24 (t, 1H, J = 5.5 Hz, NH), 7.00 (d, J = 8.6 Hz, 1H, H_ar), 7.16 (br. s, 2H, NH₂), 7.52–7.71 (m, 2H, H_ar), 10.72, 10.87 (2s, 2H, 2NNH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 10.89, 10.93, 11.00, 11.04, 32.76, 44.69, 102.84, 102.90, 102.96, 110.33, 117.03, 126.15, 126.63, 126.79, 126.84, 126.97, 131.62, 146.12, 169.91, 172.04; HRMS (ESI) for C₂₃H₂₉ClN₆O₄S + H⁺, calcd. 521.1738, found 521.1732 [M + H⁺].

3.1.7. 3-Chloro-4-((4-(3,5-dimethyl-1H-pyrazol-1-yl)-2-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-4-oxobutyl)amino)benzenesulfonamide (20)

A mixture of hydrazide 2b (0.51 g, 1.4 mmol), pentane-2,4-dione (0.6 mL, 6 mmol) and 20 mL of propan-2-ol was heated at reflux for 5 h. Then it was cooled down and the precipitate was filtered off, washed with diethyl ether and recrystallized from propan-2-ol to afford light-brown solid, yield 0.54 g (78%); m.p. 201–202 °C; IR (KBr) (v, cm⁻¹): 3371, 1723, 1595; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.18, 2.25, 2.35, 2.43 (4s, 12H, 4CH₃), 3.34–3.44 (m, 3H, CH, CH₂), 3.61–3.75 (m, 2H, NCH₂), 6.16, 6.24 (2s, 2H, 2CH_pyr), 6.52 (t, 1H, J = 5.9 Hz, NH), 7.13 (br. s, 2H, NH₂), 7.25 (d, 1H, J = 8.7 Hz, H_ar), 7.56–7.65 (m, 2H, H_ar); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 13.56, 13.70, 14.07, 14.20, 35.39, 44.32, 110.76, 111.40, 111.71, 116.99, 126.04, 126.85, 131.50, 143.51, 143.81, 146.19, 151.93, 152.24, 172.07, 173.76; HRMS (ESI) for C₂₁H₂₅ClN₆O₄S + H⁺, calcd. 493.1425, found 493.1419 [M + H⁺].

3.1.8. General Procedure for the Synthesis of Compounds 22a,b and 23a,b

Hydrazide 2a (1 g, 3 mmol) or 2b (1.10 g, 3 mmol) was dissolved in dimethylformamide (5 mL), and methyl isothiocyanate (0.58 g, 8 mmol) or phenyl isothiocyanate (0.9 mL, 7.5 mmol) was added dropwise, respectively. Reaction mixtures were stirred at room temperature for 4–6 h and then diluted with water (20 mL). The precipitate was filtered off, washed with water, dried, and recrystallized from 1,4-dioxane.

2,2′-(2-(((4-Sulfamoylphenyl)amino)methyl)succinyl)bis(N-methylhydrazine-1-carbothioamide) (22a)

White solid, yield 1.13 g (79%); m.p. 192–193 °C; IR (KBr) (v, cm⁻¹): 3381, 3299, 3196, 1657, 1601; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.81–2.89 (m, 6H, 2CH₃), 2.91–3.32 (m, 5H, CH, NCH₂, CH₂), 6.44 (t, 1H, J = 6.0 Hz, ArNH), 6.67 (d, 2H, J = 8.5 Hz, H_ar,), 6.94 (br. s, 2H, NH₂), 7.52 (d, 2H, J = 8.5 Hz, H_ar,), 7.62–7.97 (m, 2H, 2NH), 9.19 (br. s, 2H, 2NH), 9.91, 10.05 (2s, 2H, 2NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 30.87, 33.44, 35.83, 44.32, 111.07, 127.37, 130.64, 150.98, 171.19, 172.71, 182.17; HRMS (ESI) for C₁₅H₂₄N₈O₄S₃ + H⁺, calcd. 477.1161, found 477.1155 [M + H⁺].

2,2′-(2-(((2-Chloro-4-sulfamoylphenyl)amino)methyl)succinyl)bis(N-methylhydrazine-1-carbothioamide (22b)

Light-brown solid, yield 1.19 g (78%); m.p. 212–213 °C; IR (KBr) (v, cm⁻¹): 3386, 3300, 3175, 1660, 1594; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.84, 2.85 (2s, 6H, 2CH₃), 2.93–3.51 (m, 5H, CH, CH₂, NCH₂), 6.17 (t, 1H, J = 5.8 Hz, ArNH), 6.90 (d, 1H, J = 8.8 Hz, H_ar), 7.14 (br. s, 2H, NH₂), 7.57 (d, 1H, J = 8.6 Hz, H_ar), 7.67–7.97 (m, 2H, 2NH), 9.09–9.38 (m, 2H, 2NH), 9.94, 10.10 (2s, 2H, NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 30.88, 33.22, 44.24, 110.38, 117.05, 126.13, 126.77, 131.50, 146.24, 171.29, 172.69, 182.04, 182.17, 182.26; HRMS (ESI) for C₁₅H₂₃ClN₈O₄S₃+H⁺, calcd. 511.0771, found 511.0773 [M + H⁺].

2,2′-(2-(((4-Sulfamoylphenyl)amino)methyl)succinyl)bis(N-phenylhydrazine-1-carbothioamide) (23a)

White solid, yield 1.64 g (91%); m.p. 136–137 °C; IR (KBr) (v, cm⁻¹): 3212, 1660, 1597; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.54–3.27 (m, 5H, NCH₂, CH, CH₂), 6.35–7.85 (m, 17H, NH, NH₂, H_ar), 9.32–10.50 (m, 6H, 6NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 30.80, 33.35, 35.81, 44.28, 111.09, 116.79, 121.03, 123.66, 123.98, 124.45, 125.96, 127.38, 128.14, 128.45, 128.98, 129.93, 130.66, 139.00, 139.03, 141.19, 150.94, 162.32; HRMS (ESI) for C₂₅H₂₈N₈O₄S₃ + H⁺, calcd. 601.1474, found 601.1468 [M + H⁺].

2,2′-(2-(((2-Chloro-4-sulfamoylphenyl)amino)methyl)succinyl)bis(N-phenylhydrazine-1-carbothioamide) (23b)

White solid, yield 1.71 g (90%); m.p. 158–159 °C; IR (KBr) (v, cm⁻¹): 3244, 1683, 1593; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.40–4.00 (m, 5H, CH₂, CH, NCH₂), 6.17 (br. s, 1H, NH), 6.83–7.87 (m, 15H, NH₂, H_ar), 9.38–10.55 (m, 6H, 6NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 30.79, 30.97, 33.17, 35.80, 44.17, 110.34, 116.80, 117.05, 123.82, 124.40, 126.13, 126.24, 126.77, 128.11, 128.51, 131.09, 131.53, 139.02, 146.19, 158.23, 162.31; HRMS (ESI) for C₂₅H₂₇ClN₈O₄S₃ + H⁺, calcd. 635.1084, found 635.1079 [M + H⁺].

3.1.9. General Procedure for the Synthesis of Compounds 24a,b and 25a,b

A mixture of thiosemicarbazide 22a,b (0.5 mmol) or 23a,b (0.5 mmol) and 2% aqueous KOH solution (20 mL) was stirred at room temperature for 8 h. Afterwards, it was acidified with glacial acetic acid to pH 6. The formed precipitate was filtered off, washed with water, and recrystallized from propan-2-ol.

4-((2,3-bis(4-Methyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)propyl)amino)benzenesulfonamide (24a)

Brown solid, yield 0.16 g (73%); m.p. 170–171 °C; IR (KBr) (v, cm⁻¹): 3332, 3215, 3087; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.13–3.23 (m, 2H, CH₂), 3.33, 3.39 (2s, 6H, 2CH₃), 3.49–3.61 (m, 2H, NCH₂), 3.61–3.71 (m, 1H, CH), 6.57–6.73 (m, 3H, ArNH, H_ar), 6.95 (br. s, 2H, NH₂), 7.52 (d, J = 8.4 Hz, 2H, H_ar), 13.52, 13.64 (2s, 2H, NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 26.50, 29.73, 29.99, 32.58, 45.84, 110.92, 127.52, 130.77, 150.71, 150.84, 153.36, 166.49, 166.74; HRMS (ESI) for C₁₅H₂₀N₈O₂S₃ + H⁺, calcd. 441.0950, found 441.0944 [M + H⁺].

4-((2,3-bis(4-Methyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)propyl)amino)-3-chlorobenzenesulfonamide (24b)

Light-brown solid, yield 0.17 g (71%); m.p. 151–152 °C; IR (KBr) (v, cm⁻¹): 3308, 3241, 3078; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.15–3.22 (m, 2H, CH₂), 3.36, 3.40 (2s, 6H, 2CH₃), 3.57–3.67 (m, 2H, NCH₂), 3.71–3.83 (m, 1H, CH), 6.39 (t, 1H, J = 6.1 Hz, ArNH), 6.95 (d, J = 8.7 Hz, 1H, H_ar), 7.13 (br. s, 2H, NH₂), 7.54 (d, J = 8.7 Hz, 1H, H_ar), 7.65 (s, 1H, H_ar), 13.51, 13.63 (2s, 2H, NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 26.32, 29.73, 30.00, 32.36, 45.69, 110.20, 116.94, 126.17, 126.98, 131.61, 146.13, 150.68, 153.15, 166.52, 166.74; HRMS (ESI) for C₁₅H₁₉ClN₈O₂S₃+H⁺, calcd. 475.0560, found 475.0557 [M + H⁺].

4-((2,3-bis(4-Phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)propyl)amino)benzenesulfonamide (25a)

White solid, yield 0,19 g (68%); m.p. 258–259 °C; IR (KBr) (v, cm⁻¹): 3381, 3201, 3074; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.79–2.87 (m, 2H, CH₂), 3.03–3.21 (m, 2H, NCH₂), 3.25–3.38 (m, 1H, CH), 6.09 (d, J = 8.4 Hz, 2H, H_ar), 6.59 (t, 1H, J = 6.2 Hz, ArNH), 6.86–7.68 (m, 14H, NH₂, H_ar), 13.44 (br. s, 2H, 2NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 26.94, 32.43, 45.31, 110.49, 127.20, 127.94, 129.42, 129.56, 130.63, 133.14, 133.17, 149.96, 149.98, 152.45, 167.62, 167.76; HRMS (ESI) for C₂₅H₂₄N₈O₂S₃+H⁺, calcd. 565.1263, found 565.1257 [M + H⁺].

4-((2,3-bis(4-Phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)propyl)amino)-3-chlorobenzenesulfonamide (25b)

White solid, yield 0,22 g (73%); m.p. 175–176 °C; IR (KBr) (v, cm⁻¹): 3351, 3192, 3065; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.76–3.79 (m, 5H, CH_2, CH, NCH_2,), 5.90 (d, J = 8.8 Hz, H, H_ar), 6.34 (t, 1H, J = 6.2 Hz, ArNH), 6.92–7.75 (m, 15H, NH₂, H_ar), 12.82 (br. s, 2H, 2NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 26.98, 31.92, 45.22, 109.03, 116.84, 117.01, 125.60, 126.86, 127.77, 128.07, 129.06, 129.23, 129.44, 129.49, 131.39, 133.04, 133.27, 145.15, 150.01, 152.32, 167.52, 167.78; HRMS (ESI) for C₂₅H₂₃ClN₈O₂S₃+H⁺, calcd. 599.0873, found 599.0867 [M + H⁺].

3.1.10. 3-((4-Methylthiazol-2-yl)(4-sulfamoylphenyl)amino)propanoic acid (27)

Carboxylic acid 26 (0.45 g, 1.5 mmol) and NaOAc (0.20 g, 2.5 mmol) were dissolved into 10 mL of hot water. Then, chloroacetone (0.20 mL, 2.5 mmol) was added, and reaction mixture was heated at reflux for 4h. Then, it was cooled down, and the precipitate was filtered off and washed with diethyl ether and n-hexane to afford light-pinkish solid, yield 0.50 g (98%); m.p. 162–163 °C; IR (KBr) (v, cm⁻¹): 3300, 1680, 1497; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.19 (s, 3H, CH₃), 2.63 (t, 2H, J = 7.2 Hz, CH₂), 4.14 (t, 2H, J = 7.3 Hz, CH₂), 6.44 (s, 1H, CH), 7.39 (s, 2H, NH₂), 7.62 (d, 2H, J = 8.3 Hz, H_ar), 7.85 (d, 2H, J = 8.3 Hz, H_ar), 12.36 (br. s, 1H, OH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 17.39, 32.25, 48.53, 103.51, 125.15, 127.33, 140.99, 147.39, 148.45, 167.23, 172.48; Anal. Calcd. for C₁₃H₁₅N₃O₄S₂: C 45.74; H 4.43; N 12.31%. Found: C 45.82; H 4.48; N 12.30%.

3.1.11. Methyl 3-((4-Methylthiazol-2-yl)(4-sulfamoylphenyl)amino)propanoate (28)

A mixture of thiazole 27 (0.41 g, 1.2 mmol), H₂SO₄ (0.1 mL), and 15 mL of methanol were heated under reflux for 6 h. Then, the reaction mixture was filtered, and the liquid fractions were evaporated under reduced pressure. The residue was diluted with 5% aqueous Na₂CO₃ solution (20 mL), and the precipitate was filtered off, washed with water, dried, and recrystallized from propan-2-ol to afford white solid, yield 0,32 g (74%); m.p. 72–73 °C; IR (KBr) (v, cm⁻¹): 1679, 1507; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.19 (s, 3H, CH₃), 2.72 (t, 2H, J = 7.0 Hz, CH₂), 3.51 (s, 3H, CH₃), 4.18 (t, 2H, J = 7.0 Hz, CH₂), 6.43 (s, 1H, CH), 7.40 (s, 2H, NH₂), 7.61 (d, 2H, J = 8.3 Hz, H_ar), 7.85 (d, 2H, J = 8.3 Hz, H_ar); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 17.37, 32.12, 48.48, 51.43, 103.52, 125.35, 127.36, 141.17, 147.31, 148.44, 167.22, 171.39; Anal. Calcd. for C₁₄H₁₇N₃O₄S₂: C 47.31; H 4.82; N 11.82%. Found: C 47.56; H 4.83; N 11.81%.

3.1.12. 4-((3-Hydrazineyl-3-oxopropyl)(4-methylthiazol-2-yl)amino)benzenesulfonamide (29)

Ester 28 (0.53 g, 1.5 mmol) and hydrazine monohydrate (0.15 g, 3 mmol) were dissolved into 5 mL of propan-2-ol, and the reaction mixture was heated under reflux for 5 h. Then, it was cooled down, and the precipitate was filtered off, washed with diethyl ether, and n-hexane to afford white solid, yield 0.47 g (89%); m.p. 167–168 °C; IR (KBr) (v, cm⁻¹): 3382, 3260, 1644, 1498; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.19 (s, 3H, CH₃), 2.44 (t, 2H, J = 7.3 Hz, CH₂), 3.92–4.72 (m, 4H, NH₂, CH₂), 6.45 (s, 1H, CH), 7.44–7.96 (m, 6H, NH₂, H_ar), 9.06 (s, 1H, NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 17.40, 31.77, 49.21, 103.58, 124.93, 127.22, 127.26, 140.77, 147.41, 148.43, 167.18, 169.14; Anal. Calcd. for C₁₃H₁₇N₅O₃S₂: C 43.93; H 4.82; N 19.70%. Found: C 44.04; H 4.75; N 19.50%.

3.1.13. 4-((3-(2-Benzylidenehydrazineyl)-3-oxopropyl)(4-methylthiazol-2-yl)amino)benzenesulfonamide (30)

Hydrazide 29 (0.53 g, 1.5 mmol), benzaldehyde (0.19 g, 1.8 mmol), and 10 mL of propan-2-ol were mixed under reflux for 1 h. Then, the reaction mixture was cooled down, and the precipitate was filtered off, washed with diethyl ether, and recrystallized from propan-2-ol to afford white solid, yield 0.58 g (87%); m.p. 170–171 °C; IR (KBr) (v, cm⁻¹): 3247, 1671, 1595, 1499; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.08 (s, 3H, CH₃), 2.58 (t, 0.70H, J = 7.2 Hz, CH₂), 3.00 (t, 1.30H, J = 7.4 Hz, CH₂) 4.06–4.36 (m, 2H, NCH₂), 7.13–8.14 (m, 13H, NH₂, CH, N=CH, H_Ar), 11.35, 11.38, 11.41, 11.44 (4s, 1H, NH); Anal. Calcd. for C₂₀H₂₁N₅O₃S₂: C 54.16; H 4.77; N 15.79%. Found: C 54.36; H 4.65; N 15.90%.

3.1.14. 3-((5-(Ethoxycarbonyl)-4-methylthiazol-2-yl)(4-sulfamoylphenyl)amino)propanoic acid (31)

Carboxylic acid 26 (1.00 g, 3 mmol) and NaOAc (0.30 g, 3.6 mmol) were dissolved into 10 mL of hot water. Then, ethyl 2-chloroacetoacetate (0.70 g, 4.25 mmol) was added, and the reaction mixture was heated at reflux for 12h. Then, it was cooled down, and the precipitate was filtered off and washed with water and n-hexane to afford white solid, yield 1.05 g (85%); m.p. 221–222 °C; IR (KBr) (v, cm⁻¹): 3546, 1708, 1677, 1509; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.20 (t, 3H, J = 7.1 Hz, CH₃), 2.52 (s, 3H, CH₃), 2.65 (t, 2H, J = 7.1 Hz, CH₂), 4.08–4.28 (m, 4H, 2CH₂), 7.48 (s, 2H, NH₂), 7.70 (d, 2H, J = 8.4 Hz, H_ar), 7.95 (d, 2H, J = 8.4 Hz, H_ar), 12.38 (s, 1H, OH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 14.23, 17.31, 32.14, 48.34, 60.22, 109.47, 127.19, 127.69, 143.05, 146.02, 159.13, 161.56, 169.25, 172.23; Anal. Calcd. for C₁₆H₁₉N₃O₆S₂: C 46.48; H 4.63; N 10.16%. Found: C 46.29; H 4.56; N 10.10%.

3.1.15. Ethyl 2-((3-Methoxy-3-oxopropyl)(4-sulfamoylphenyl)amino)-4-methylthiazole-5-carboxylate (32)

A mixture of thiazole 31 (0.42 g, 1 mmol), H₂SO₄ (0.1 mL) and 15 mL of methanol were heated under reflux for 5 h. Then, the reaction mixture was filtered, and the liquid fractions were evaporated under reduced pressure. The residue was diluted with 5% aqueous Na₂CO₃ solution (20 mL); the precipitate was filtered off, washed with water, dried, and recrystallized from propan-2-ol to afford white solid, yield 0.32 g (74%); m.p. 140–141 °C; IR (KBr) (v, cm⁻¹): 3371, 1732, 1678, 1520; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.19 (t, 3H, J = 7.1 Hz, CH₃), 2.48 (s, 3H, CH₃), 2.72 (t, 2H, J = 6.9 Hz, CH₂), 3.51 (s, 3H, CH₃), 4.13 (q, 2H, J = 7.1 Hz, CH₂), 4.21 (t, 2H, J = 6.9 Hz, CH₂), 7.47 (s, 2H, NH₂), 7.68 (d, 2H, J = 8.3 Hz, H_ar), 7.94 (d, 2H, J = 8.2 Hz, H_ar); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 14.21, 17.28, 32.01, 48.28, 51.48, 60.22, 109.54, 127.24, 127.70, 143.14, 145.94, 159.08, 161.53, 169.21, 171.15; Anal. Calcd. for C₁₇H₂₁N₃O₆S₂: C 47.76; H 4.95; N 9.83%. Found: C 47.77; H 4.94; N 9.99%.

3.1.16. Ethyl 2-((3-(Hydrazineyloxy)-3-oxopropyl)(4-sulfamoylphenyl)amino)-4-methylthiazole-5-carboxylate (33)

A mixture of ester 32 (0.43 g, 1 mmol), hydrazine monohydrate (0.15 g, 3 mmol), and 15 mL of propan-2-ol were heated under reflux for 12 h. Then, the reaction mixture was cooled down, and the precipitate was filtered off and recrystallized from propan-2-ol to afford white solid, yield 0.38 g (88%); m.p. 206–207 °C; IR (KBr) (v, cm⁻¹): 3325, 3223, 1712, 1679, 1507; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.19 (t, 3H, J = 7.1 Hz, CH₃), 2.43 (t, 2H, J = 7.1 Hz, CH₂), 2.48 (s, 3H, CH₃), 4.06–4.24 (m, 6H, NH₂, 2CH₂), 7.47 (s, 2H, SO₂NH₂), 7.67 (d, 2H, J = 8.1 Hz, H_ar), 7.93 (d, 2H, J = 8.2 Hz, H_ar), 9.07 (s, 1H, NH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 14.25, 17.33, 31.71, 49.20, 60.21, 109.45, 127.14, 127.63, 142.92, 146.11, 159.17, 161.58, 168.86, 169.21; Anal. Calcd. for C₁₆H₂₁N₅O₅S₂: C 44.95; H 4.95; N 16.38%. Found: C 45.20; H 4.90; N 16.23%.

3.1.17. Ethyl 2-((3-(2-Benzylidenehydrazineyl)-3-oxopropyl)(4-sulfamoylphenyl)amino)-4-methylthiazole-5-carboxylate (34)

A mixture of hydrazide 33 (0.43 g, 1 mmol), benzaldehyde (0.13 g, 1.2 mmol), and 15 mL of propan-2-ol were heated under reflux for 3 h. Then, the reaction mixture was cooled down and diluted with diethyl ether. The precipitate was filtered off to afford white solid, yield 0.44 g (85%); m.p. 195–196 °C; IR (KBr) (v, cm⁻¹): 3269, 1678, 1655, 1553 1513; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.11–1.27 (m, 3H, CH₃), 2.51 (s, 3H, CH₃), 2.64 (t, 0.75H, J = 7.1 Hz, CH₂), 3.07 (t, 1.25H, J = 7.3 Hz, CH₂), 4.01–4.50 (m, 4H, 2CH₂), 7.15–8.15 (m, 12H, H_ar, CH_, NH₂), 11.39 (s, 0.7H, NH), 11.45 (s, 0.3H, NH); Anal. Calcd. for C₂₃H₂₅N₅O₅S₂: C 53.58; H 4.89; N 13.58%. Found: C 53.23; H 4.67; N 13.34%.

3.1.18. 3-((5-Chloro-4-(4-chlorophenyl)thiazol-2-yl)(4-sulfamoylphenyl)amino)propanoic acid (36)

Thiazole 35 (0.70 g, 1.6 mmol) and N-chlorosuccinimide (0.60 g, 4.5 mmol) were dissolved into 3 mL of DMF, and the reaction mixture was stirred at 0 °C for 6 h. Then, it was diluted with cold water, and the precipitate was filtered off and dissolved into 20 mL of 5% aqueous NaOH solution. The mixture was acidified with glacial acetic acid to pH 6 to afford light-yellowish solid, yield 0.53 g (71%); m.p. 109–110 °C; IR (KBr) (v, cm⁻¹): 3353, 1727, 1510; ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.67 (t, 2H, J = 7.1 Hz, CH₂), 4.20 (t, 2H, J = 7.1 Hz, CH₂), 7.46 (s, 2H, NH₂), 7.54 (d, 2H, J = 8.4 Hz, H_ar), 7.72 (d, 2H, J = 8.4 Hz, H_ar), 7.91 (d, 4H, J = 8.4 Hz, H_ar), 12.32 (br. s, 1H, OH); ¹³C NMR (101 MHz, DMSO-d₆) (δ, ppm): 32.30, 48.13, 107.25, 126.10, 127.65, 128.60, 129.44, 131.42, 132.97, 142.31, 144.05, 146.30, 163.41, 172.46; Anal. Calcd. for C₁₉H₁₆Cl₂N₂O₄S₂: C 48.41; H 3.42; N 5.94%. Found: C 48.29; H 3.35; N 5.99%.

3.2. Determination of Compounds Binding to Human CA Isozymes

3.2.1. Protein Preparation

Human CA isozymes were expressed in E. coli (CAI, II, III, IV, VA, VB, VII, XII, XIII, XIV) or mammalian cells (CAVI and CAIX), chromatographically purified following the published protocols [47], and stored at −80 °C. The protein concentration was determined by UV absorption at 280 nm using Beer–Lambert law. The CAII used for crystallization was additionally purified by affinity chromatography.

3.2.2. Fluorescent Thermal Shift Assay (FTSA)

The fluorescent thermal shift assay was performed to obtain the observed binding constants (inversely proportional to the observed dissociation constants (K_{d_obs}s)) between CA isozymes and synthesized compounds. The QIAGEN Rotor-Gene Q was used to carry out the experiments, with a blue channel used for excitation (365 nm) and detection (460 nm). FTSA in this case relies on an extrinsic probe, 8-anilino-1-naphthalenesulfonate (ANS), to detect the fluorescence increase during protein denaturation, from which protein melting temperature (T_m) can be determined. Since the binding compounds usually thermally stabilize a protein, an increase in T_m is observed when increasing the compound concentration. The observed affinity of ligands for CAs was determined by plotting the T_m as a function of ligand concentration and then fitting a curve as described previously [48]. Samples consisted of constant concentration of CAs (5 µM for all proteins except 7.5 or 10 µM of CAIV) and varied concentrations of ligand ((0–200) µM) in 50 mM sodium phosphate buffer at pH 7.0 containing 100 mM NaCl, 50 µM ANS, and 2% (v/v) DMSO. Samples were heated from 25 °C to 99 °C at the speed of 1 °C/min. An example of an experiment is given in Figure 2.

3.2.3. Isothermal Titration Calorimetry (ITC)

ITC determined the observed standard enthalpy changes upon binding as well as the K_{d_obs} values [41] that were compared to ones obtained by FTSA. The ITC experiments were performed by adding the proteins to the calorimeter cell and the compound solutions to the syringe at 37 ºC. The CAII binding with compounds 28, 31, and 36 and CAVB with compound 32 (Figure S104) were determined using a MicroCal PEAQ-ITC calorimeter (Northampton, MA, USA). Experiments consisted of 19 injections, the first injection of 0.4 μL over 0.8 s, with the remaining injections of 2 μL over 4 s with spacing of 150 s between injections, at temperature 37 °C, reference power 10 μcal/s, and stirring speed of 750 rpm. The protein concentrations for CAII and CAVB were 10 μM and 8 μM, respectively; the compound concentration was 10 times higher than the protein (100 μM for compounds tested with CAII, 80 μM for compound tested with CAVB). Proteins and compounds were diluted in sodium phosphate 50 mM buffer at pH 7.0, containing 100 mM NaCl and 2% (v/v) DMSO. Data were analyzed using NITPIC (version 1.2.7) [49,50] and Sedphat (v. 14.0) [51] software; the heat of dilution was subtracted from the titration curves.

3.2.4. Determination of Compound Sulfonamide Group pK_a

To calculate the intrinsic thermodynamic parameters of compound binding to CA isozymes, the pK_a values of the sulfonamide group and the water molecule bound to the Zn(II) ion at the active site of CA proteins had to be determined. The pK_a values for CA isozyme-bound water molecules were taken from [40], CAI—8.1; CAII—6.9; CAIII—6.5; CAIV—6.6; CAVA—6.7; CAVB—7.0; CAVI—6.0; CAVII—6.8; CAIX—6.6; CAXII—6.8; CAXIII—8.0; CAXIV—6.1. Compound pK_a values were measured by obtaining UV-Vis spectra at 37 °C at different pH values (from 7.0 to 12.0 with 0.5 pH increment) using BMG Labtech CLARIOstar^Plus plate reading spectrophotometer. Compounds were diluted to a constant concentration of 50–200 µM (concentration was chosen depending on compound solubility and absorbance peak) in a universal buffer, consisting of 50 mM sodium phosphate, 50 mM sodium acetate, 25 mM sodium borate, and 50 mM sodium chloride, and also contained 2% (v/v) DMSO. The pK_a values were calculated as described previously in [28,52] by normalizing the absorbance, plotting it as a function of pH, then fitting to the Henderson–Hasselbalch equation using the least square method (Figure S105).

The pK_as of compounds 27, 28, 29, 31, 32, 35, and 36 were measured, with an average pK_a value of 10.1 for all compounds. Since the pK_a values were only able to be measured for compound group 26–36, due to universal buffer pH limitations, intrinsic thermodynamic parameters were not calculated for compounds 2(a,b)–25(a,b), only for compounds 26–36 using experimentally obtained pK_a values when possible and using the average pK_a value mentioned above for compounds not measured.

3.2.5. Calculation of the Intrinsic Thermodynamic Parameters

The intrinsic thermodynamic parameters were calculated as described in [40]. The intrinsic reaction of inhibitor binding to CA is shown in Equation (1). The observed reaction includes the associated protonation–deprotonation reactions of protein, ligand, and buffer and thus has to be accounted for in order to make correct conclusions about compound binding affinities. It is known that CA proteins bind sulfonamides only when water molecules are coordinated to the Zn(II) ion, and sulfonamides only bind to CA proteins when their amino group is deprotonated (confirmed by neutron crystallography [53,54,55]):

$${\mathrm{CA} \mathrm{Zn}}^{\mathrm{II}}-{\mathrm{H}}_2\mathrm{O}+{\mathrm{RSO}}_2{\mathrm{NH}}^{-}\rightleftarrows { \mathrm{CA} \mathrm{Zn}}^{\mathrm{II}}-{\mathrm{NH} \mathrm{SO}}_2\mathrm{R}+{\mathrm{H}}_2\mathrm{O}$$

(1)

When calculating K_{d_int} values, these protein and ligand fractions from Equation (1) have to be accounted for (Equations (2) and (3)). The intrinsic dissociation constant is calculated by multiplying K_{d_obs} value by the corresponding fractions of protein and ligand (Equation (4)).

$$f_{RS{O}_{2}N{H}^{-}}=\frac{{10}^{pH-p{K}_{a\_RS{O}_{2}N{H}_2}}}{1+{10}^{pH-p{K}_{a\_RS{O}_{2}N{H}_2}}}$$

(2)

$$f_{CAZn{H}_{2}O}=1-\frac{{10}^{pH-p{K}_{a\_CAZn{H}_{2}O}}}{1+{10}^{pH-p{K}_{a\_CAZn{H}_{2}O}}}$$

(3)

$$K_{d\_int}=K_{d\_obs}\times f_{RS{O}_{2}N{H}^{-}}\times f_{CAZn{H}_{2}O}$$

(4)

$$\Delta G_{int}^0=RTln\left(K_{d\_int}\right)$$

(5)

The intrinsic standard Gibbs energy changes upon binding were calculated using Equation (5). Here, R—universal ideal gas constant (8.3144 J/mol·K), T—absolute temperature (310.15 K).

3.2.6. X-ray Crystallography: Crystallization, Data Collection, and Structure Determination

CAII was concentrated by ultrafiltration to 31 mg/mL and mixed with the crystallization solution (0.1 M sodium bicine (pH 9.0), 0.2 M ammonium sulfate, and 2 M sodium malonate (pH 7.0)) at a ratio 1:1 and crystallized by the sitting drop method. One μL of 50 mM compound solution in DMSO was combined with 50 μL of reservoir solution for crystals soaking.

X-ray diffraction data were collected at the EMBL beamline P13 at PETRA III ring of DESY synchrotron in Hamburg. Crystals were flash cooled to 100 K without additional cryo-protection. The data were processed and scaled using XDS [56], MOSFLM [57], and SCALA [58]. MOLREP [59] was used for molecular replacement, with an initial model of 4HT0. The model was refined with REFMAC [60] and fitted in the electron density map using COOT [61]. The 3D models of compounds were constructed by the AVOGADRO [62] program, while ligand parameter files were created using LIBCHECK [63,64]. The PDB IDs, data collection, and refinement statistics are listed in Table S2 in Supplementary Materials. The 2D schemes based on X-ray crystallography are shown as LigPlot+ [65] schemes in Figure S3.

4. Conclusions

A series of benzenesulfonamides bearing para-N β,γ-amino acid or para-N β-amino acid and thiazole moieties were synthesized, and the binding affinities for the entire family of 12 catalytically active human CA isozymes were determined by the fluorescent thermal shift assay. Selected compound affinities were also determined by isothermal titration calorimetry, while X-ray crystallography helped to determine the structural arrangement of the inhibitors in the active site of CAII. The intrinsic affinities that are independent of protonation reactions were determined according to the described model. The analysis showed that the chlorine atom in the meta-position of para-N β-amino acid bearing benzenesulfonamide derivatives increases the observed affinity for CA over non-chlorinated compounds, while the substituents located further away from this group of benzenesulfonamides have lower influence on the affinity. The most potent compound, 36, bearing para-N β-amino acid and thiazole moieties bound CAI and CAIX with 20 nM K_{d_obs}. Correlation of the chemical structures of the compounds with the intrinsic affinities showed that small changes in the chemical structure of the compound can have significant effects on the affinity.