Synthesis and structure–affinity relationship of chlorinated pyrrolidinone-bearing benzenesulfonamides as human carbonic anhydrase inhibitors

Journal Pre-proofs Synthesis and structure–affinity relationship of chlorinated pyrrolidinonebearing benzenesulfonamides as human carbonic anhydrase inhibitors Benas Balandis, Guostė Ivanauskaitė, Joana Smirnovienė, Kristina Kantminienė, Daumantas Matulis, Vytautas Mickevičius, Asta Zubrienė PII: DOI: Reference:

S0045-2068(19)32104-2 https://doi.org/10.1016/j.bioorg.2020.103658 YBIOO 103658

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

9 December 2019 5 February 2020 10 February 2020

Please cite this article as: B. Balandis, G. Ivanauskaitė, J. Smirnovienė, K. Kantminienė, D. Matulis, V. Mickevičius, A. Zubrienė, Synthesis and structure–affinity relationship of chlorinated pyrrolidinone-bearing benzenesulfonamides as human carbonic anhydrase inhibitors, Bioorganic Chemistry (2020), doi: https://doi.org/ 10.1016/j.bioorg.2020.103658

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© 2020 Published by Elsevier Inc.

Synthesis and structure–affinity relationship of chlorinated pyrrolidinone-bearing benzenesulfonamides as human carbonic anhydrase inhibitors Benas Balandis1, Guostė Ivanauskaitė2, Joana Smirnovienė2, Kristina Kantminienė3, Daumantas Matulis2, Vytautas Mickevičius1, Asta Zubrienė2*

*Corresponding author: Asta Zubrienė E. mail: [email protected], [email protected]

1Department

of Organic Chemistry, Kaunas University of Technology, Radvilėnų pl. 19, Kaunas LT-50254,

Lithuania 2Department

of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, Vilnius LT-10257, Lithuania 3Department

of Physical and Inorganic Chemistry, Kaunas University of Technology, Radvilėnų pl. 19, Kaunas LT-50254, Lithuania

Highlights:  

Pyrrolidinone-bearing benzenesulfonamides with chlorine at position 3 were synthesized. Compounds 12 and 15 exhibited low nanomolar dissociation constants for cancer-related isozyme CA IX.



Disulfonamide 3 was the most selective compound for CA II and exhibited 5 nM binding affinity.



Compounds 14 and 19 were the most potent binders of CA XIV (Kd = 3.3 nM).

Abstract A novel set of pyrrolidinone-based chlorinated benzenesulfonamide derivatives were synthesized and investigated for their binding affinity and selectivity against recombinant human carbonic anhydrases I – XIV using fluorescent thermal shift, p-nitrophenyl acetate hydrolysis and stopped-flow enzymatic inhibition assays. The hydrazones 10–22 prepared from 1-(2-chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3-carboxylic acid exhibited low nanomolar affinity against cancer-related CA IX (Kd in the range of 5.0 – 37 nM). Compounds with triazole or oxadiazole groups attached directly to pyrrolidinone moiety bound all CAs weaker than compounds with more flexible tail groups. Chloro group at the meta position of benzenesulfonamide derivatives increased affinity to all CAs as compared with binding data for nonchlorinated compounds. The compounds have a potential for further development of CA inhibitors with higher selectivity for a particular CA isozyme.

Keywords Carbonic anhydrase; benzenesulfonamide; 5-oxopyrrolidine; fluorescent thermal shift assay; structure-affinity relationship

1. Introduction There are twelve alpha carbonic anhydrase (CA) isozymes in human that catalyze reversible hydration of CO2 to protons and bicarbonate, a vital reaction for the respiration and transport of CO2 between tissues, in pH regulation and homeostasis [1–3]. Abnormal expression levels of several CA isozymes are associated with numerous diseases. Currently CAs are established therapeutic targets of cancer (CA IX and CA XII), glaucoma (CA II, CA IV, CA XII), epilepsy (CA II, CA VII, CA XIV), high altitude sickness (CA II) and obesity (CA VA and CA VB). The clinically used sulfonamide-based drugs against some of these diseases were discovered several decades ago and are still used to date, despite possessing many side effects. There is a need for the development of selective and high affinity CA inhibitors for CA isozymes involved in certain diseases to overcome side effects associated with non-selective inhibition for majority of CAs in human body. Most of the research interest of late years has been focused on design and development of inhibitors against CA IX that show potential for treating solid tumors [4–6]. A wide range of CA inhibitors have been reported [7–9] mainly including sulfonamides and sulfamates, which bind directly to zinc in the active site. Benzenesulfonamides are the most important class of CA inhibitors. A simple unsubstituted benzenesulfonamide binds to CAs with moderate affinity (Kds in the micromolar range). Whitesides et al have shown that about -9 kcal/mol of binding energy is contributed by the Zn-N bond and the hydrogen bond networks between benzenesulfonamide and CA II in the active forms. The contacts between CA II isozyme and unsubstituted benzene ring contributed -2.9 kcal/mol [10]. The functional groups linked to the benzene scaffold were thought to enhance the binding affinity and determine the selectivity towards a particular CA isozyme. We have recently reviewed over 400 benzenesulfonamide binding to 12 catalytically active human CAs and demonstrated the design routes of highly selective compounds towards particular CA isozyme by the variation of substituents size and bulkiness on benzene scaffold and incorporation of electron withdrawing groups [11]. Sulfonamide-based CA inhibitors are often designed by attaching various substituents at para position of benzenesulfonamide ring [12–14]. Thus, benzenesulfonamide derivatives incorporating various heterocyclic tails including pyrazole, imidazole, pyridazinone, isatin etc., have been shown to be CA inhibitors [15–17]. The chemical nature of the tail also allows obtaining the desired physico-chemical properties of inhibitors. Recently, we have described the synthesis and binding properties of 3-substituted 1-[4(aminosulfonyl)phenyl]-5-oxopyrrolidine derivatives [18]. The 5-oxopyrrolidine moiety is often incorporated into the structure of many biologically active compounds [19–21]. The variation of substituents on the 5oxopyrrolidine moiety let us obtain several compounds which bound selectively to CA IX. Later we synthesized the dimethyl substituted benzenesulfonamides with 5-oxopyrrolidine linker at para position exhibiting variable binding affinity and selectivity profiles to human CA isozymes [22]. Some examples of our synthesized most effective compounds are listed in Figure 1.

Figure 1. Structure of clinically used drug acetazolamide and previously synthesized 5-oxopyrrolidine-based benzenesulfonamides [18,22] reported as CA inhibitors. Our current study is focused on the synthesis of analogous pyrrolidinone-based benzenesulfonamides, bearing chlorine at the 3-position of benzenesulfonamide ring. Our motivation to introduce chlorine was the knowledge that halogens as electron-withdrawing substituents decrease the pKa of sulfonamide group and increase the fraction of anion (active form of ligand) leading to the increased binding affinity towards CA [10,23]. The chlorine improved the binding affinity to all CAs as compared with analogous ligands without chlorine at 3-position. We attached various substituents at the 4-position of the pyrrolidinone ring. The structure–affinity relationship is presented for the binding of 31 compounds to all twelve catalytically active human CA isozymes.

2. Results and discussion

2.1. Chemistry

The starting compound, 1-(2-chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3-carboxylic acid (2), was prepared in the reaction of 1-(4-sulfamoylphenyl)-5-oxopyrrolidine-3-carboxylic acid (1) with hydrochloric acid in the presence of subsequently added hydrogen peroxide [22] (Scheme 1). The structures of 2 and all other compounds have been confirmed by the data of IR, 1H and 13C NMR spectroscopy as well as mass spectrometry data.

H2NO2S

H2NO2S

O

Cl

O

N

N 1

7-9 COOH

a H2NO2S

e Cl

H2NO2S

O

Cl

c

N 2

H2NO2S

O

NH N R1

Cl

d

N

O

N

5

6

COOH O

O

O

b H2NO2S

O

NH NH2

f Cl

H2NO2S

O

Cl

O

N

N 3, 4

10-22 O

NH

O

NH N

SO2NH2

R2

3) 4-SO2NH2; 4) 3-SO2NH2 7: R1 = CH3; 8: R1 = C2H5; 9: R1 = C6H5; 10: R2 = H; 11: R2 = 2-OH; 12: R2 = 3-OH; 13: R2 = 4-OH; 14: R2 = 2-Cl; 15: R2 = 3-Cl; 16: R2 = 4-Cl; 17: R2 = 4-F; 18: R2 = 4-OCH3; 19: R2 = 2-NO2; 20: R2 = 3-NO2; 21: R2 = 4-NO2; 22: R2 = 4-COOH; Reaction conditions: (a) 6M HCl, H2O2, r.t., 50 °C  r.t., 1 h; (b) corresponding benzenesulfonamide, 150-160 °C, 4 h; (c) MeOH, conc. H2SO4, reflux, 8 h; (d) N2H4H2O, propan-2-ol, reflux, 5 h; (e) corresponding ketone, propan-2-ol, reflux, 2-4 h; (f) corresponding aldehyde, propan-2-ol, reflux, 1-2 h;

Scheme 1. Synthesis of compounds 2–22. Sulfanilamides 3 and 4 were synthesized by melting 2 with a corresponding benzenesulfonamide at 150–160 °C. The double intensity of the proton signals in the aromatic region of the 1H NMR spectra has confirmed the presence of two benzene rings in 3 and 4. Ester 5 was synthesized by an esterification reaction of the acid 2 with an excess of methanol at reflux temperature in the presence of sulfuric acid as a catalyst. Reaction of ester 5 with hydrazine hydrate in propan-2-ol at reflux temperature gave hydrazide 6, which reactions with corresponding ketones in propan-2-ol at reflux temperature yielded corresponding hydrazones 7–9, whereas the ones with corresponding benzaldehydes provided corresponding hydrazones 10–22. The 1H NMR spectra for hydrazones 7–22 display double sets of resonances for the CO–NH and N=CH group protons due to the restricted rotation around the amide bond. This splitting of the proton resonances indicates that in DMSO-d6 solution hydrazones exist as a mixture of Z/E isomers and, in the majority of cases, the Z isomer predominates [24,25]. Hydrazide 6 was used as a precursor for the synthesis of a series of azole derivatives (Scheme 2).

H2NO2S

Cl

H2NO2S

O

Cl

N 31

H2NO2S

23 O

H2NO2S

O

N

Cl

N NH

O

N

24

a h

N

H2NO2S

Cl

H2NO2S

O

N

N NH

O

Cl

25

NH NH2

O

HN

d Cl

H2NO2S

NH

COOH

29 N

H2NO2S

Cl

O e

N

O

N 28

26, 27

N NH

N NH

S Cl

f

O

N

c

6

g

NH N

b

O

N

O

N

O

S

30

H2NO2S

N

Cl

O

NH HN

X HN

O

N

N NH

S

26: X = O; 27: X = S; Reaction conditions: (a) acetylacetone, glacial acetic acid, propan-2-ol, reflux, 6 h; (b) hexane-2,5-dione, glacial acetic acid, propan-2-ol, reflux, 6 h; (c) KSCN, HCl, propan-2-ol, reflux, 12 h; (d) corresponding isocyanate, propan-2-ol, reflux, 3 h; (e) 2% KOH, r.t., 8 h, glacial acetic acid, r.t., pH 6; (f) 2% KOH, 60 °C, 6 h, HCl, r.t., pH 5; (g) 10% HCl, reflux, 1 h; (h) CS2, KOH, 50 °C, 24 h, H2O, HCl, pH 6;

Scheme 2. Synthesis of compounds 23–31. Azole derivatives can be obtained by the condensation reaction of acid hydrazides with aliphatic diketones [18,26]. Pyrazole derivative 23 was obtained from hydrazide 6 by the reaction with pentane-2,4-dione in propan-2-ol in the presence of acetic acid at reflux temperature of the reaction mixture, whereas the analogous reaction with hexane-2,5-dione provided pyrrole derivative 24. In the 1H NMR spectrum for 23, a singlet integrated for two protons of CH3 groups at 2.19 ppm and singlet assigned to the proton in the CH group at 6.23 ppm have proven the presence of 3,5-dimethylpyrazole moiety in the molecule. In the 1H NMR spectrum for 24, singlets at 1.98 and 2.01 ppm assigned to the protons of CH3 groups and a singlet integrated for two protons of CH groups at 5.65 ppm have confirmed the presence of 2,5-dimethylpyrrole moiety. The singlet at 10.91 ppm has been ascribed to the proton in the NH group. Reaction of 6 with potassium thiocyanate in propan-2-ol in the presence of hydrochloric acid as a catalyst yielded 3-chloro-4-(2-oxo-4-(5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)pyrrolidin-1-yl)benzenesulfonamide (25). Protons of the NH groups in triazolethione moiety resonated at 6.73 and 12.51 ppm in the 1H NMR spectrum for 25. The carbon attributed to the C=S group resonated at 173.91 ppm in the 13C NMR spectrum. Reaction of hydrazide 6 with phenyl isocyanate or its thio analogue in propan-2-ol led to the formation of carboxamide 26, and carbothioamide 27, respectively. Condensation reactions of 27 in alkaline medium resulted in formation of triazolethione 28. In the 13C NMR spectrum for 28, signal at 168.9 ppm has been attributed to C=S carbon in triazolethione ring. Triazolone 30 was obtained as a result of the reclosure of pyrrolidin-2-one ring in 4-((2-chloro-4-sulfamoylphenyl)amino)-3-(5-oxo-4-phenyl-4,5-dihydro-1H-1,2,4triazol-3-yl)butanoic acid (29) which was synthesized by heating 26 in alkaline medium and subsequently

acidifying the reaction mixture with hydrochloric acid to pH 5. Carbon resonances attributed to C=O group in triazolone ring are observed at approx. 154 ppm in the 13C NMR spectra for 29 and 30. Oxadiazolethione derivative 31 was synthesized by the treatment of 6 with carbon disulfide in alcohol in the presence of KOH followed by cyclization in situ of the formed potassium salt of hydrazinocarbothioate with hydrochloric acid to pH 6 [18,27]. The singlet ascribed to the proton of the NH group in oxadiazolethione moiety is observed at 14.50 ppm in the 1H NMR spectrum for 31. In the 13C NMR spectrum carbon of the C=S group resonated at 178.48 ppm.

2.2.

Binding affinities to CA isozymes

The binding affinities of all compounds for CA isozymes were examined by the fluorescent thermal shift assay (FTSA). The dissociation constants (Kd) are provided in Tables 1 and 2. The binding affinity of 14 synthesized compounds was measured for all twelve human catalytically active CA isozymes. The binding of other compounds was measured against selected five CA isozymes - CA I, CA II, CA IX, CA XII, and CA XIII. The binding affinity values for three compounds 5, 10 and 29 were confirmed by the stopped-flow assay of enzymatic inhibition of CO2 hydration reaction. Moreover, inhibition by the compound 29 of the hydrolysis of p-nitrophenyl acetate was determined. The binding (Fig. 2A, C), inhibition of hydratase activity (Fig. 2B, D), and inhibition of esterase activity (Fig. 2E, F) data were in good agreement, the Kd values measured by three methods for compounds 5, 10 and 29 differed less than several fold what could be explained by slightly different conditions of the assays (Fig. 2G, Table 1, 2).

Table 1. Dissociation constants (Kd, nM) of compounds 2-22 for catalytically active recombinant human CA isozymes, determined by the fluorescent thermal shift assay (pH 7.0, 37 °C) and confirmed by stopped-flow CO2 hydration assay (shown in the parentheses, pH 7.5, 24 °C).

Cpd X 1[18] 2 3 4

58

12

13000

CA I

-OH H N

4200 610 40

CA II 500 105 5.0

SO2 NH2

H N SO2 NH2

CA III

CA IV

20000 20000 20000

2500 1430 1100 670

220

44

2200

330

2600

5

-OCH3

65

14

33000

6

-NHNH2

440

77

67000

1430 (1670) 1700

N

440

110

n.d.

n.d.

500

25

n.d.

N

56

33

N

67

7

N H

8

N H

9 10 11 12 13

N H

N H

16 17 18

OH

N

N H

OH

OH N

N H

N

N H

Cl

N

N H

Cl Cl

N

N H

F N H

N O

N H

19 20

N

N H

14 15

N

N H

N H

C H3

N

N

N

NO2

NO2

Kd, nM CA CA VI VB 4700 8300 3800 3500 83 710

CA VA 10000 3300 420

430

CA VII 680 125 17

CA IX 330 74 19

CA XII 2000 330 500

CA XIII 2500 290 33

CA XIV 720 59 13

13

7.1

440

40

11

110

13

540

59

20

20

770

1700 (770) 1100

100

71

1500 (625) 770

n.d.

n.d.

n.d.

n.d.

83

1000

625

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

72

500

250

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

8.3

420

50

n.d.

10

33000

1400

170

67

290

17

8.3 (5.9)

125

50

7.1

130

20

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

37

330

210

n.d.

83

25

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

5.4

250

100

n.d.

33

10

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

14

590

59

n.d.

31

6.8

13000

710

170

100

120

17

6.7

450

25

3.3

20

10

11000

170

130

50

83

11

5.0

125

33

5.0

32

12

17000

500

130

85

140

20

13

340

37

10

34

14

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

6.7

390

67

n.d.

67

18

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

14

560

91

n.d.

33

8.3

17000

710

330

100

220

17

6.0

600

33

3.3

40

14

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

14

670

25

n.d.

28

17

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

10

660

78

n.d.

NO2

21

N H

N

O

22

33

20

10000

2100

420

540

870

15

18

250

100

17

BSA

7100

1800

>4×105

17000

8300

33000

14000

6700

1500

13000

7500

8700

8a[18]

625

167

>2×105

1250

1670

1250

2000

500

59

2500

830

200

OH N H

N

n.d. not determined. Table 2. Dissociation constants (Kd, nM) of compounds 23-31 for catalytically active recombinant human CA isozymes, determined by the fluorescent thermal shift assay (pH 7.0, 37 °C) and confirmed by stopped-flow CO2 hydration assay (shown in the parentheses, pH 7.5, 24 °C) and p-nitrophenyl acetate hydrolysis assay (shown in the parentheses as second value, pH 7.0, 24 °C).

Cpd CA I

CA II

CA III

CA IV

CA VA

Kd, nM CA CA VI VB

20

7.1

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

63

140

14

n.d.

47

11

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

9.1

240

32

n.d.

330

91

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

125

500

400

n.d.

520

40

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

42

670

200

n.d.

65

15

8300

500

770

330

830

20

25

42

37

20

67

18

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

25

57

34

n.d.

12500 (18000; 59000)

170 (25; 180)

10000

1250 (830)

20000

13000

1400

3000

77 (46)

30 (29)

1900 (500)

33

110

50

3800

1250

1000

3400

2200

30

40

300

310

100

BSA

7100

1800

>4×105

17000

8300

33000

14000

6700

1500

13000

7500

8700

AZM

2400

46

>4×105

87

840

140

220

13

21

130

120

63

X N N

23 24

N N N H

N NH

25 26 27

N H N H

N H

H N

S H N

O H N

H N S

CA VII

CA IX

CA XII

CA XIII

CA XIV

N NH

28

N

S

29 31

N NH O

S

n.d. not determined. We have previously studied a series of benzenesulfonamides bearing pyrrolidinone moiety at para position [18], which showed moderate binding affinity and some selectivity towards cancer related CA IX. The best compounds displayed approximately 50 nM Kd for CA IX. The simplest of the tested compounds, nonhalogenated pyrrolidinone-based benzenesulfonamide 1, displayed 330 nM affinity and higher specificity

for CA IX (Table 1). In this study, we intended to synthesize pyrrolidinone-based benzenesulfonamides with chloro substituent at 3-position. Our motivation for chlorine introduction was the evidence that electronwithdrawing atoms lower the pKa of sulfonamide group and may enhance the binding affinity [23]. Introduction of chlorine lowered the pKa of sulfonamide group by approximately 0.5 pH unit. For example, the pKa value for 1 was 10.1, whereas for 2 it was 9.7. Indeed, the decrease of pKa resulted in the enhancement of binding affinity. The chlorinated compounds 2 and 10 bound to all CA isozymes stronger than previously synthesized [18] analogous nonchlorinated compounds 1 and 8a (Fig. 3A, B). Compound 2 also bound all CAs stronger than the reference compound benzenesulfonamide BSA (Table 1). The contribution of the length and chemical nature of the 5-oxopyrrolidine linker can be seen from the analysis of structure-affinity relationship (SAR) for compounds 3-31. The strongest binding of these compounds towards CA II, CA IX and CA XIV was observed. The Kd ranges for CA II were 5.0 – 170 nM, for CA IX - 5.0 – 125 nM and for CA XIV - 3.3 – 100 nM. The Kd values for CA I spread in a wide ranges from 20 to 12500 nM, similar as for CA VB (44 – 13000 nM). The Kd ranges for CA VII, CA XII and CA XIII were comparable: 11 – 3000 nM for CA VII, 30 – 1500 nM for CA XII and 14 – 1900 for CA XIII. Compounds 3-31 were moderate to weak binders of CA IV (Kd in the range of 170 – 2100 nM), of CA VA (Kd in the range of 130 – 20000 nM) and CA VI (Kd in the range of 83 – 2200 nM). Derivatives 3-31 were weakest inhibitors of CA III (Kds in the range of 3800 – 200000 nM).

Figure 2. The comparison of binding affinity determined by FTSA (panels A, C), inhibition of hydratase activity by SFA (panels B, D) and inhibition of esterase activity by p-NPA hydrolysis assay (panels E, F). (A) Thermal melting curves of CA IX (5 µM) in the presence of compound 10 or 29 at 0, 12.5 and 200 µM concentration determined by FTSA. (B) The decrease of phenol red absorbance due to acidification of medium by CA IX (30 nM) in the presence of inhibitors 10 or 29 at different concentrations (0, 20 and 160 nM). The dotted lines are spontaneous hydration of CO2 in the absence of enzyme. (C) Dependence of melting temperatures (Tm) of CA IX on concentrations of the added compound (red triangles – 10, blue squares – 29). Dosing curves fitted according to [28]. D) CA IX hydratase activity dependence on the added inhibitor concentration (red triangles – 10, blue squares – 29). E) The increase of p-nitrophenol absorbance due to CA I (5.3 µM) or CA II (0.3 µM) catalyzed hydrolysis of p-nitrophenyl acetate in the presence of different concentration of compound 29 (0.0, 0.2 and 0.8 µM for CA II and 0.0, 33.3, 100 µM for CA I). F) CA I (magenta circles) and CA II (violet diamonds) esterase activity dependence on the added inhibitor 29

concentration. Data points in panels D and F were fitted using Morrison equation [29,30]. G) Correlation between Kd’s determined by SFA, p-NPA assay and FTSA.

Figure 3. The influence of chloro group (shown in red) on the binding affinity towards CA isozymes. Addition of chloro group enhanced the binding constant (Kb) for all 12 CA isozymes. Enhancement effect is shown as Kb ratio of chlorinated and nonchlorinated analogous compounds, namely, compounds 2 vs 1 (panel A) and 10 vs 8a (panel B). The binding data of nonchlorinated compounds 1 and 8a were published previously [18].

Next, a more detailed analysis of SAR for some of the tested compounds has revealed that the ester 5 bound most of CA isozymes stronger than the acid 2, for instance, the Kds for CA II and CA XIV were 14 and 13 nM for compound 5, while 105 and 59 nM for compound 2, respectively. The modification of ester 5 to hydrazine 6 decreased binding affinities to all CAs, for instance the Kds for CA II and CA XIV were 77 and 59 nM, respectively. Disulfonamide 3 and 4 bound tightly to CA II, CA VII, CA IX and CA XIV, the Kds were in the range 5.0-19 nM. Compound 3 was the most potent (Kd = 5.0 nM) and selective for CA II from the whole series of tested compounds. It is interesting to compare compounds 7–9, which differ in the substituents R1 of the flexible group. The gradual increase of the size and hydrophobicity of the substituent from methyl (7), ethyl (8), to phenyl (9) groups resulted in the increase of binding strength (decrease in Kd) for overwhelming majority of CAs. For example, the Kds for CA IX decreases in the direction 7→8→9 from 83 nM→72 nM→8.3 nM . Small substituents on the phenyl ring of hydrazones (compounds 10–22) such as OH, Cl, F, OCH3, NO2 and COOH, as well their position in phenyl ring did not affect the affinity towards all CAs significantly (as compared with the compound 10 where R2 is H). In majority of cases, the affinity increased up to 3 times to all of CAs, with exception of CA XII. Introduction of such substituents decreased affinity towards CA XII by 2.0-5.4 times. Also, compound 11 with R2 = 2-OH bound CA IX and CA XIII more than 4 times weaker than compound 10. Compounds 12 and 15 were the most potent binders of CA IX (Kds 5.4 and 5.0 nM, respectively), whereas 14 and 19 were most potent for CA XIV (Kds = 3.3 nM). Figure 4 shows the improvement of binding affinity towards majority of CAs by modification of carboxy group (2) to hydrazine (6) and 3-chlorobenzylidene-hydrazine (15). Compound 15 exhibited the strongest affinity towards CA IX and CA XIV (Kd = 5.0 nM).

Figure 4. Compound chemical structure – binding affinity relationship. Compound structures are shown on the top of the figure, while below the Kd values for compound 2 (panel A), 6 (panel B) and 15 (panel C) binding towards 12 human CA isozymes are presented. Addition of 3-chlorobenzylidene group (15) decreased the Kds for all CAs. Note that lower bars in this figure mean higher affinity. Compounds 25, 28 and 31 with triazole or oxadiazole moieties linked directly to pyrrolidinone ring have more rigid structures. It is interesting to note that compound 31 is the most potent for the weakest enzymatic activity bearing isozyme CA III from the whole series of the tested compounds (Kd = 3.8 µM). Compound 30 exhibited low aqueous solubility; therefore, the binding affinity to CAs could not be determined accurately. However, its derivative, butanoic acid 29, was found to be the most efficient binder of CA XII (Kd = 30 nM). In summary, variation of the substituents on the pyrrolidinone moiety of chlorinated benzenesulfonamides enabled discovery of compounds possessing high binding affinity to isozymes CA II, CA IX, and CA XIV. 3. Conclusion A series of chlorinated pyrrolidinone-bearing benzenesulfonamide derivatives were synthesized and the binding affinities toward all 12 human catalytically active carbonic anhydrase (CA) isozymes were evaluated. The influence of various substituents directly or through the linker bound to the pyrrolidinone moiety was analyzed. Most of the compounds showed nanomolar binding affinities towards CA II, CA IX, and CA XIV. Compound 3 was the most potent binder for CA II, while compound 15 bound the strongest to CAIX, both exhibiting 5 nM binding affinities. Compounds 14 and 19 were the highest affinity binders of CA XIV (Kds = 3.3 nM). The presence of pyrrolidinone moiety was crucial for binding to CA I-VII and CA XIII but did not affect affinity towards transmembrane isoforms CA IX, CA XII, and CA XIV (as shown by 29 affinity profile).

4. Materials and methods

4.1. Chemistry

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 are uncorrected. IR spectra (ν, cm-1) were recorded on a Perkin–Elmer Spectrum BX FT–IR spectrometer using KBr pellets. The 1H and 13CNMR spectra were recorded in DMSO-d6 on BruckerAvance III (400, 101 MHz) spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) calibrated from TMS (0 ppm) as an internal standard for 1H NMR, and DMSO-d6 (39.43 ppm) for 13C NMR. Mass spectra were obtained on 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 precoated with silica gel 60 F254 (MerckKGaA, Darmstadt, Germany). 4.1.1. 1-(2-Chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3-carboxylic acid (2). Carboxylic acid 1 (2.00 g, 7 mmol) was dissolved in 6M HCl solution (50 mL) at 50 °C and 30% hydrogen peroxide (6 mL) was added slowly to the reaction mixture. The solution was cooled to room temperature and stirred for 1 h. The formed precipitate was filtered off, washed with water, diethyl ether, dried, and recrystallized from water to afford white solid, yield 1.73 g (77%); m.p. 225–226 °C; IR (KBr) (v, cm-1):3308, 1728, 1661; 1H NMR (400 MHz, DMSO-d6)  (ppm): 2.62–2.81 (m, 2H, CH2CO), 3.40–3.52 (m, 1H, CH), 3.85–4.01 (m, 2H, NCH2), 7.23– 8.13 (m, 5H, Har, NH2), 12.82 (br. s, 1H, OH); 13C NMR (101 MHz, DMSO-d6) (, ppm): 33.39, 36.53, 51.02, 125.34, 127.25, 130.21, 131.68, 139.00, 144.45, 172.13, 173.94; HRMS (ESI) for C11H11ClN2O5S+H+, calcd. 319.0155, found 319.0150 [M+H+].

4.1.2. General procedure for the synthesis of amides 3 and 4 A mixture of carboxylic acid 2 (0.95 g, 3mmol) and corresponding benzenesulfonamide (3 mmol) was heated at 150–160 °C for 4h. The reaction mixture was diluted with 5% aqueous Na2CO3 solution and heated until reflux. Then mixture was left to cool down, the precipitate was filtered off and recrystallized from 1,4dioxane. 4.1.2.1. 1-(2-Chloro-4-sulfamoylphenyl)-5-oxo-N-(4-sulfamoylphenyl)pyrrolidine-3-carboxamide (3). Lightbrown solid, yield 0.48 g (34%); m.p. 274–275 °C; IR (KBr) (v, cm-1): 3326, 3249, 3095, 1694, 1595; 1H NMR (400 MHz, DMSO-d6) : 2.70–2.97 (m, 2H, CH2CO), 3.48–3.67 (m, 1H, CH), 3.85–4.19 (m, 2H, NCH2), 7.19–7.99 (m, 11H, Har, 2NH2), 10.53, 10.55 (2s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.84, 38,36, 51.57,118.80, 118.90, 125.35, 126.48, 126.73, 127.25, 130.32, 131.65, 138.60, 139.01, 141.82, 144.45, 171.11, 172.18; Anal. Calcd. for C17H17ClN4O6S2: C 43.18; H 3.62; N 11.85%. Found: C 43.02; H 3.62; N 11.67%. 4.1.2.2. 1-(2-Chloro-4-sulfamoylphenyl)-5-oxo-N-(3-sulfamoylphenyl)pyrrolidine-3-carboxamide (4). Brown solid, yield 0.76 g (54%); m.p. 252–253 °C; IR (KBr) (v, cm-1):3385, 3338, 3279, 3077, 1683, 1600; 1H NMR (400 MHz, DMSO-d6) : 2.74–2.97 (m, 2H, CH2CO), 3.47–3.65 (m, 1H, CH), 3.88–4.20 (m, 2H, NCH2), 7.24–8.27 (m, 11H, Har, 2NH2), 10.49, 10.52 (2s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.86, 35.84, 36.65, 38.26, 50.50, 51.58, 116.32, 118.80, 120.56, 122.13, 125.36, 126.48, 127.25, 129.54, 130.33, 131.65,

138.98, 139.02, 139.23, 139.26, 141.79, 144.45, 144.66, 170.96, 171.27, 172.20, 172.62; HRMS (ESI) for C17H17ClN4O6S2+H+, calcd. 473.0356, found 473.0349 [M+H+]. 4.1.2.3. Methyl 1-(2-chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3-carboxylate (5). A mixture of acid 2 (5.00 g, 16 mmol), methanol (50 mL), and sulfuric acid (0.5 mL) was heated at reflux for 8 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 (50 mL), the precipitate was filtered off, washed with water, dried, and recrystallized from propan-2-ol to afford white solid, yield 4.17 g (80%); m.p. 133–134 °C; IR (KBr) (v, cm1): 3341, 1734, 1694. 1H NMR (400 MHz, DMSO-d ) : 2.60–2.78 (m, 2H, CH CO), 3.36–3.48 (m, 1H, CH), 6 2 13 3.70 (s, 3H, CH3), 3.85–4.00 (m, 2H, NCH2), 7.37–8.08 (m, 5H, Har, NH2); C NMR (101 MHz, DMSO-d6) : 33.27, 36.32, 50.79, 52.22, 125.31, 127.23, 130.21, 131.64, 138.85, 144.47, 171.83, 172.87; HRMS (ESI) for C12H13ClN2O5S+H+, calcd. 333.0312, found 333.0309 [M+H+]. 4.1.2.4. 3-Chloro-4-(4-(hydrazinecarbonyl)-2-oxopyrrolidin-1-yl)benzenesulfonamide (6). A mixture of ester 5 (12.00 g, 36 mmol), hydrazine hydrate (3.60 g, 72 mmol), and propan-2-ol (70 mL) was heated at reflux for 5 h. The reaction mixture was cooled down, the precipitate was filtered off, washed with propan-2-ol, dried, and recrystallized from propan-2-ol and water mixture to afford white solid, yield 9.68 g (81%); m.p. 178–179 °C; IR (KBr) (v, cm-1): 3459, 3341, 3192, 1734, 1694; 1H NMR (400 MHz, DMSO-d6) : 2.60–2.67 (m, 2H, CH2CO), 3.25–3.31(m, 1H, CH), 3.72–3.92 (m, 2H, NCH2), 4.45 (br. s, 2H, NH2), 7.48–8.04 (m, 5H, Har, NH2), 9.27 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.91, 35.70, 51.66, 125.32, 127.22, 130.30, 131.62, 139.07, 144.39, 170.99, 172.31; HRMS (ESI) for C11H13ClN4O4S+H+, calcd. 333.0424, found 333.0409 [M+H+]. 4.1.3. General procedure for the synthesis of hydrazones 7–9 A mixture of hydrazide 7 (0.32 g, 1 mmol), propan-2-ol (10 mL), and corresponding ketone (3 mmol) was heated at reflux for 2-4 h. The reaction mixture was cooled down, precipitate was filtered off and recrystallized from propan-2-ol. 4.1.3.1. 3-Chloro-4-(2-oxo-4-(2-(propan-2-ylidene)hydrazine-1-carbonyl)pyrrolidin-1-yl)benzenesulfonamide (7). White solid, yield 0.24 g (64%); m.p. 217–218 °C; IR (KBr) (v, cm-1): 3312, 3249, 1697, 1640; 1H NMR (400 MHz, DMSO-d6) : 1.86, 1.87, 1.92, 1.93 (4s, 6H, 2CH3), 2.61–2.81 (m, 2H, CH2CO), 3.46–4.08 (m, 3H, CH, NCH2), 7.50–8.02 (m, 5H, Har, NH2), 10.21 (s, 0.45H, NH), 10.33 (s, 0.55H, NH); 13C NMR (101 MHz, DMSO-d6) :17.14, 17.63, 24.97, 25.25, 32.99, 33.97, 34.57, 36.03, 51.27, 51.87, 125.29, 125.33, 127.22, 130.26, 131.62, 139.11, 144.36, 144.40, 151.34, 156.25, 168.01, 172.34, 172.40, 173.18; HRMS (ESI) for C14H17ClN4O4S+H+, calcd. 373.0737, found 373.0737[M+H+]. 4.1.3.2. 4-(4-(2-(Butan-2-ylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1-yl)-3-chlorobenzenesulfonamide (8). Light-brown solid, yield 0.28 g (72%); m.p. 149–150 °C; IR (KBr) (v, cm-1): 3315, 3231, 1696, 1594; 1H NMR (400 MHz, DMSO-d6) : 0.84–1.13 (m, 3H, CH3), 1.68–2.39 (m, 5H, CH2, CH3), 2.87–2.88 (m, 2H, CH2CO), 3.42–4.08 (m, 3H, CH, NCH2), 7.48–8.00 (m, 5H, Har, NH2), 10.14–10.45 (m, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 10.33, 10.78, 30.77, 31.43, 31.54, 32.87, 34.02, 34.76, 36.07, 37.18, 51.20, 51.86, 110.23, 117.06, 126.21, 126.88, 127.22, 130.34, 131.62, 139.09, 144.39, 146.08, 154.62, 172.48, 173.27, 174.31, 176.11; HRMS (ESI) for C15H19ClN4O4S+H+, calcd. 387.0888, found 387.0885 [M+H+]. 4.1.3.3. 3-Chloro-4-(2-oxo-4-(2-(1-phenylethylidene)hydrazine-1-carbonyl)pyrrolidin-1yl)benzenesulfonamide (9). Light-yellow solid, yield 0.26 g (60%); m.p. 193–194 °C; IR (KBr) (v, cm-1): 3326, 3243, 1691, 1682; 1H NMR (400 MHz, DMSO-d6) : 2.24–2.33 (m, 3H, CH3), 2.66–2.95 (m, 2H,

CH2CO), 3.64–4.30 (m, 3H, CH, NCH2), 7.25–8.08 (m, 10H, Har, NH2), 10.60 (s, 0.3H, NH), 10.80 (s, 0.7H, NH); 13C NMR (101 MHz, DMSO-d6) : 13.71, 13.97, 14.26, 22.07, 30.96, 33.04, 34.20, 34.78, 36.22, 51.24, 51.85, 125.31, 126.05, 126.34, 127.23, 128.32, 128.42, 129.13, 129.32, 130.28, 130.40, 131.61, 138.07, 139.06, 144.40, 148.29, 168.78, 172.30, 172.46, 173.97; HRMS (ESI) for C19H19ClN4O4S+H+, calcd. 435.0894, found 435.0885 [M+H+]. 4.1.4. General procedure for the synthesis of hydrazones 10–22 A mixture of hydrazide 7 (0.32 g, 1 mmol), propan-2-ol (15 mL), and corresponding benzaldehyde (1.5 mmol) was heated at reflux for 1–2h. The reaction mixture was cooled down, the precipitate was filtered off and recrystallized from propan-2-ol. 4.1.4.1. 4-(4-(2-Benzylidenehydrazine-1-carbonyl)-2-oxopyrrolidin-1-yl)-3-chlorobenzenesulfonamide (10). White solid, yield 0.33 g (79%); m.p. 290–291 °C; IR (KBr) (v, cm-1): 3350, 3225, 1695, 1682; 1H NMR (400 MHz, DMSO-d6) : 1.87–2.04(m, 2H, CH2CO), 3.01–3.42 (m, 3H, CHpyr, NCH2), 6.52–7.14 (m, 10H, Har, NH2),7.20, 7.38(2s, 1H, CH), 10.76(s, 0.65H, NH), 10.80 (s, 0.35H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.00, 33.82, 34.39, 36.40, 51.10, 51.53, 125.31, 125.34, 126.86, 127.10, 127.24, 128.83, 128.85, 129.92, 130.32, 130.37, 131.64, 134.07, 134.10, 139.06, 143.70, 144.40, 144.44, 147.07, 168.20, 172.18, 172.34, 173.14; HRMS(ESI) for C18H17ClN4O4S+H+, calcd. 421.0737, found 421.0730 [M+H+]. 4.1.4.2. 3-Chloro-4-(4-(2-(2-hydroxybenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (11). Light-brown solid, yield 0.36 g (82%); m.p. 252–253 °C; IR (KBr) (v, cm-1): 3350, 3226, 3077, 1683, 1621; 1H NMR (400 MHz, DMSO-d6) : 2.66–2.94(m, 2H, CH2CO), 3.41–4.22 (m, 3H, CHpyr, NCH2), 6.78–8.03 (m, 9H, Har, NH2), 8.34, 8.42 (2s, 1H, CH), 10.04 (s, 0.45H, OH), 11.03 (s, 0.55H, OH), 11.52 (s, 0.45H, NH), 11.85 (s, 0.55H, NH); 13C NMR (101 MHz, DMSO-d6) : 32.98, 33.78, 34.40, 51.10, 51.43, 116.13, 116.36, 118.61, 119.37, 119.45, 120.21, 125.31, 125.35, 126.14, 127.25, 129.21, 130.34, 130.36, 131.16, 131.47, 131.65, 139.02, 139.07, 141.07, 144.40, 144.45, 147.27, 156.37, 157.30, 168.05, 172.13, 172.36, 172.81; HRMS (ESI) for C18H17ClN4O5S+H+, calcd. 437.0686, found 437.0675 [M+H+]. 4.1.4.3. 3-Chloro-4-(4-(2-(3-hydroxybenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (12). White solid, yield 0.31 g (71%); m.p. 300–301 °C; IR (KBr) (v, cm-1): 3362, 3219, 3077, 1701, 1682; 1H NMR (400 MHz, DMSO-d6) : 2.69–2.90(m, 2H, CH2CO), 3.76–4.22 (m, 3H, CHpyr, NCH2), 6.78–8.15 (m, 10H, Har, CH, NH2), 9.61, 9.62 (2s, 1H, OH), 11.53 (s, 0.65H, NH), 11.58 (s, 0.35H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.06, 33.83, 34.47, 36.42, 51.06, 51.55, 112.61, 112.63, 117.25, 117.51, 118.41, 118.86, 125.34, 126.49, 127.27, 129.88, 129.92, 130.35, 131.67, 135.55, 135.38, 139.05, 139.07, 143.89, 144.43, 144.45, 147,16, 157.66, 168.16, 172.22, 172.34, 173.02; HRMS (ESI) for C18H17ClN4O5S+H+, calcd. 437.0686, found 437.0683 [M+H+]. 4.1.4.4. 3-Chloro-4-(4-(2-(4-hydroxybenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (13). Light-brown solid, yield 0.34 g (77%); m.p. 283–284 °C; IR (KBr) (v, cm-1): 3288, 3206 3028, 1674, 1614; 1H NMR (400 MHz, DMSO-d6) : 2.69–2.88 (m, 2H, CH2CO), 3.82–4.21 (m, 3H, CHpyr, NCH2), 6.78–6.87 (m, 2H, NH2), 7.48–8.13 (m, 8H, Har, CH), 9.89 (s, 1H, OH), 11.38 (s, 0.6H, NH), 11.43 (s, 0.4H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.02, 33.88, 34.37, 36.39, 51.16, 51.62, 115.71, 125.06, 125.11, 125.32, 125.35, 126.48, 127.26, 128.59, 128.88, 130.33, 130.37, 131.66, 139.07, 139.10, 144.01, 144.40, 144.43, 147.39, 159.26, 159.47, 167.81, 172.26, 172.41, 172.76; HRMS (ESI) for C18H17ClN4O5S+H+, calcd. 437.0686, found 437.0681 [M+H+].

4.1.4.5. 3-Chloro-4-(4-(2-(2-chlorobenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (14). White solid, yield 0.31 g (68%); m.p. 262–263 °C; IR (KBr) (v, cm-1): 3288, 3189, 1682, 1602; 1H NMR (400 MHz, DMSO-d6) : 2.69–2.91 (m, 2H, CH2CO), 3.82–4.30 (m, 3H, CHpyr, NCH2), 7.35–8.05 (m, 9H, Har, NH2), 8.43, 8.60 (2s, 1H, CH), 11.77 (s, 0.65H, NH), 11.87 (s, 0.35H, NH); 13C NMR (101 MHz, DMSO-d ) : 33.00, 33.74, 34.33, 36.46, 51.03, 51.42, 125.30, 125.34, 126.86, 127.23, 6 127.65, 129.92, 130.32, 130.36, 131.28, 131.30, 131.33, 131.65, 132.96, 133.15, 139.04, 139.79, 142.93, 144.41, 144.44, 168.36, 172.13, 172.28, 173.33; HRMS (ESI) for C18H16Cl2N4O4S+H+, calcd. 455.0348, found 455.0341 [M+H+]. 4.1.4.6. 3-Chloro-4-(4-(2-(3-chlorobenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (15). White solid, yield 0.37 g (82%); m.p. 246–247 °C; IR (KBr) (v, cm-1): 3288, 3196, 1683, 1665; 1H NMR (400 MHz, DMSO-d6) : 2.71–2.86 (m, 2H, CH2CO), 3.84–4.28 (m, 3H, CHpyr, NCH2), 7.43–7.97 (m, 9H, Har, NH2), 8.01, 8.19 (2s, 1H, CH), 11.70 (s, 0.65H, NH), 11.77 (s, 0.35H, NH); 13C NMR (101 MHz, DMSO-d ) : 33.02, 33.78, 34.32, 16.38, 51.07, 51.47, 125.31, 125.34, 125.68, 125.72, 6 126.03, 126.45, 127.24, 129.57, 129.75, 130.31, 130.42, 130.72, 131.62, 131.64, 133.62, 133.69, 136.32, 136.38, 139.02, 139.04, 142.12, 144.41, 144.44, 145.37, 168.42, 172.15, 172.36, 173.36; HRMS (ESI) for C18H16Cl2N4O4S+H+, calcd. 455.0348, found 455.0339 [M+H+]. 4.1.4.7. 3-Chloro-4-(4-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (16). White solid, yield 0.35 g (77%); m.p. 241–242 °C; IR (KBr) (v, cm-1): 3208, 3064, 1693, 1668; 1H NMR (400 MHz, DMSO-d6) : 2.67–2.88 (m, 2H, CH2CO), 3.45–4.41 (m, 3H, CHpyr, NCH2), 7.44–7.98 (m, 9H, Har, NH2), 8.02, 8.21 (2s, 1H, CH), 11.65 (s, 0.7H, NH), 11.70 (s, 0.3H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.02, 33.79, 34.32, 36.39, 51.05, 51.50, 125.31, 125.34, 127.25, 128.52, 128.73, 128.71, 130.31, 130.35, 131.65, 133.03, 133.07, 134.34, 134.58, 139.02, 139.05, 142.46, 144.41, 144.44, 145.75, 168.30, 172.16, 172.30, 173.23; HRMS (ESI) for C18H16Cl2N4O4S+H+, calcd. 455.0348, found 455.0340 [M+H+]. 4.1.4.8. 3-Chloro-4-(4-(2-(4-fluorobenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (17). White solid, yield 0.36 g (82%); m.p. 256–257 °C; IR (KBr) (v, cm-1): 3213, 3061, 1668, 1604; 1H NMR (400 MHz, DMSO-d6) : 2.69–2.86 (m, 2H, CH2CO), 3.83–4.26 (m, 3H, CHpyr, NCH2), 7.23–7.98 (m, 9H, Har, NH2), 8.03, 8.21 (2s, 1H, CH), 11.60 (s, 0.65H, NH), 11.64 (s, 0.35H, NH); 13C NMR (101 MHz, DMSO-d ) : 33.00, 33.80, 34.33, 36.38, 51.07, 51.51, 115.78, 116.00, 125.30, 125.34, 6 127.23, 129.01, 129.09, 129.23, 129.32, 130.34, 130.69, 130.72, 131.64, 139.02, 139.06, 142.58, 144.40, 144.43, 145.94, 161.75, 164.21, 168.21, 172.17, 172.31, 173.15; HRMS (ESI) for C18H16ClFN4O4S+H+, calcd. 439.0643, found 439.0634 [M+H+]. 4.1.4.9. 3-Chloro-4-(4-(2-(4-methoxybenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (18). Yellow solid, yield 0.34 g (76%); m.p. 198–199 °C; IR (KBr) (v, cm-1): 3446, 3253, 1680, 1606; 1H NMR (400 MHz, DMSO-d6) : 2.69–2.87 (m, 2H, CH2CO), 3.72–4.24 (m, 6H, CHpyr, NCH2, CH3), 6.91–8.20 (m, 11H, Har, CH, NH2), 11.46 (s, 0.6H, NH), 11.52 (s, 0.4H, NH); 13C NMR (101 MHz, DMSO-d6) : 32.99, 33.86, 34.39, 36.38, 51.14, 51.59, 55.30, 55.39, 114.33, 114.40, 125.31, 125.34, 126.67, 127.24, 128.44, 128.70, 129.98, 130.32, 130.36, 131.64, 139.05, 139.08, 143.59, 144.40, 144.43, 146.95, 160.50, 160.68, 160.87, 167.93, 172.22, 172.38, 172.86; HRMS (ESI) for C19H19ClN4O5S+H+, calcd. 451.0843, found 451.0840 [M+H+].

4.1.4.10. 3-Chloro-4-(4-(2-(2-nitrobenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (19). Light-yellow solid, yield 0.33 g (71%); m.p. 277–278 °C; IR (KBr) (v, cm-1): 3288, 3197, 1682, 1598; 1H NMR (400 MHz, DMSO-d6) : 2.71–2.84 (m, 2H, CH2CO), 3.80–4.22 (m, 3H, CHpyr, NCH2,), 7.50–8.18 (m, 9H, Har, NH2), 8.40, 8.62 (2s, 1H, CH), 11.87 (s, 0.67H, NH), 11.97 (s, 0.33H, NH); 13C NMR (101 MHz, DMSO-d6) : 32.99, 33.73, 34.31, 36.43, 50.99, 51.41, 124.52, 124.73, 125.31, 127.24, 128.06, 128.38, 128.63, 130.33, 130.53, 130.74, 131.67, 133.52, 133.81, 139.03, 139.27, 142.62, 144.42, 148.03, 148.15, 168.56, 172.12, 172.24, 173.46; HRMS (ESI) for C18H16ClN5O6S+H+, calcd. 466.0588, found 466.0583 [M+H+]. 4.1.4.11. 3-Chloro-4-(4-(2-(3-nitrobenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (20). Light-yellow solid, yield 0.38 g (82%); m.p. 224–225 °C; IR (KBr) (v, cm-1): 3345, 3218, 1674, 1614; 1H NMR (400 MHz, DMSO-d6) : 2.65–2.93 (m, 2H, CH2CO), 3.85–4.30 (m, 3H, CHpyr, NCH2), 7.50–8.36 (m, 9H, Har, NH2), 8.48, 8.54 (2s, 1H, CH), 11.83 (s, 0.6H, NH), 11.89 (s, 0.4H, NH); 13C NMR (101 MHz, DMSO-d6) : 32.99, 33.77, 34.38, 36.38, 51.03, 51.46, 121.05, 121.13, 124.13, 124.32, 125.31, 127.24, 130.32, 130.42, 131.65, 132.93, 133.24, 135.92, 136.03, 139.01, 141.51, 144.44, 144.66, 148.21, 148.26, 168.62, 172.13, 172.32, 173.43; HRMS (ESI) for C18H16ClN5O6S+H+, calcd. 466.0588, found 466.0578 [M+H+]. 4.1.4.12. 3-Chloro-4-(4-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1yl)benzenesulfonamide (21). Light-yellow solid, yield 0.41 g (87%); m.p. 265–266 °C; IR (KBr) (v, cm-1): 3302, 3187, 1701, 1682; 1H NMR (400 MHz, DMSO-d6) : 2.66–2.88 (m, 2H, CH2CO), 3.83–4.33(m, 3H, CHpyr, NCH2), 7.42–7.97 (m, 9H, Har, NH2), 8.01, 8.20 (2s, 1H, CH), 11.70 (s, 0.65H, NH), 11.77 (s, 0.35H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.02, 33.78, 34.32, 36.38, 51.06, 51.47, 125.31, 125.34, 125.68, 125.72, 126.03, 126.45, 127.24, 129.56, 129.75, 130.31, 130.42, 130.72, 131.62, 131.64, 133.62, 133.69, 136.32, 136.38, 139.02, 139.04, 142.12, 144.41, 144.44, 145.37, 168.42, 172.14, 172.36, 173.36; HRMS (ESI) for C18H16ClN5O6S+H+, calcd. 466.0588, found 466.0582 [M+H+]. 4.1.4.13. 4-((2-(1-(2-Chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3carbonyl)hydrazineylidene)methyl)benzoic acid (22). White solid, yield 0.32 g (69%); m.p. 263–264 °C; IR (KBr) (v, cm-1): 3182, 3076, 1682, 1601; 1H NMR (400 MHz, DMSO-d6) : 2.69–2.90 (m, 2H, CH2CO), 3.85–4.29 (m, 3H, CHpyr, NCH2), 7.51–8.03 (m, 9H, Har, NH2), 8.09, 8.26 (2s, 1H, CH), 11.74 (s, 0.65H, NH), 11.79 (s, 0.65H, NH), 13.08 (s, 1H, OH); 13C NMR (101 MHz, DMSO-d6) : 33.02, 33.79, 34.34, 36.44, 51.05, 51.49, 125.32, 125.36, 126.91, 127.13, 127.26, 129.77, 130.33, 130.38, 131.56, 134.67, 131.76, 138.11, 138.18, 139.03, 139.05, 142.65, 144.42, 144.45, 145.87, 166.89, 168.45, 172.16, 172.31, 173.41; HRMS (ESI) for C19H17ClN4O6S+H+, calcd. 464.0557, found 465.0627[M+H+]. 4.1.5. 3-Chloro-4-(4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-2-oxopyrrolidin-1-yl)benzenesulfonamide (23). A mixture of hydrazide 6 (1.00 g, 3 mmol), pentane-2,4-dione (0.92 g, 9 mmol), acetic acid (0.2 mL), and propan-2-ol (25 mL) was heated at reflux for 6h. Afterwards, the volatile fraction was removed under reduced pressure and the residue was diluted with water. The precipitate was filtered off and recrystallized from propan-2-ol to afford white solid, yield 0.76 g (64%); m.p. 120–121 °C; IR (KBr) (v, cm-1): 3303, 1694, 1682; 1H NMR (400 MHz, DMSO-d ) : 2.19 (s, 6H, 2CH ), 2.79–2.88 (m, 2H, CH CO), 3.89–4.17 (m, 2H, NCH ) 6 3 2 2 4.49–4.63 (m, 1H, CHpyrrol), 6.23 (s, 1H, CHpyrazol), 7.56 (s, 2H, NH2), 7.68 (d, 1H, J = 8.3 Hz, Har), 7.83 (d, 1H, J = 8.3 Hz, Har), 7.94 (s, 1H, Har); 13C NMR (101 MHz, DMSO-d6) : 13.56, 14.07, 33.28, 36.95, 51.19, 111.61, 125.33, 127.23, 130.33, 131.68, 138.85, 143.87, 144.50, 152.20, 171.82, 172.21; Anal. Calcd. for C16H17ClN4O4S: C 48.43; H 4.32; N 14.12 %. Found: C 48.19; H 4.65; N 13.93%.

4.1.6. 1-(2-Chloro-4-sulfamoylphenyl)-N-(2,5-dimethyl-1H-pyrrol-1-yl)-5-oxopyrrolidine-3-carboxamide (24). A mixture of hydrazide 6 (1.00 g, 3 mmol), hexane-2,5-dione (0.7 g, 6 mmol), acetic acid (0.2 mL), and propan-2-ol (20 mL) was heated at reflux for 6h. Afterwards, the reaction mixture was cooled down, the precipitate was filtered off and recrystallized from propan-2-ol to afford orange solid, yield 0.76 g (62%); m.p. 214–215 °C; IR (KBr) (v, cm-1): 3543, 3226, 1727, 1668; 1H NMR (400 MHz, DMSO-d6) : 1.98, 2.01 (2s, 6H, 2CH3), 2.70–2.88 (m, 2H, CH2CO), 3.52–3.65 (m, 1H, CHpyrrolidin) 3.85–4.12 (m, 2H, NCH2), 5.65 (s, 2H, 2CHpyrrol), 7.57 (s, 2H, NH2), 7.67 (d, 1H, J = 8.3 Hz, Har), 7.84 (d, 1H, J = 8.3 Hz, Har), 7.96 (s, 1H, Har);10.91 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 10.95, 33.58, 35.52, 51.64, 103.10, 125.36, 126.72, 126.75, 127.23, 130.29, 131.67, 138.95, 144.49, 171.41, 172.00; HRMS (ESI) for C17H19ClN4O4S+H+, calcd. 411.0894, found 411.0886 [M+H+]. 4.1.7. 3-Chloro-4-(2-oxo-4-(5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)pyrrolidin-1-yl)benzenesulfonamide (25). A mixture of hydrazide 6 (0.32 g, 1 mmol), KSCN (0.5g, 5 mmol), conc. HCl (1 mL), and propan-2-ol (15 mL) was heated at reflux for 12 h. Then the mixture was cooled down, the precipitate was filtered off and recrystallized from propan-2-ol to afford light-yellow solid, yield 0.18 g (49%); m.p. 208–209 °C; IR (KBr) (v, cm-1): 3401, 3297, 1622, 1609; 1H NMR (400 MHz, DMSO-d6) : 2.61–2.82 (m, 2H, CH2CO), 3.42–3.52 (m, 1H, CH), 3.85–4.01 (m, 2H, NCH2), 6.73 (s, 1H, NH), 7.52–8.04 (m, 5H, Har, NH2), 12.51 (br. s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.39, 36.52, 51.02, 125.34, 127.25, 130.19, 131.65, 138.97, 144.44, 172.12, 173.91; Anal. Calcd. for C12H12ClN5O3S2: C 38.56; H 3.24; N 18.73 %. Found: C 38.74; H 3.02; N 18.46%. 4.1.8. General procedure for the synthesis of compounds 26 and 27 To hydrazide 6 (1.00 g, 3 mmol) dissolved in propan-2-ol (30 mL), phenyl isocyanate (0.54 g, 4.5 mmol) or phenyl isothiocyanate (0.6 g, 4.5 mmol) was added dropwise. The reaction mixture was heated at reflux for 3 h and left to cool down. The formed precipitate was filtered off, washed with diethyl ether, and recrystallized from propan-2-ol. 4.1.8.1. 2-(1-(2-Chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3-carbonyl)-N-phenylhydrazine-1-carboxamide (26). White solid, yield 1.07 g (79%); m.p. 192–193 °C; IR (KBr) (v, cm-1): 3332, 3253, 3086, 1701, 1683, 1668; 1H NMR (400 MHz, DMSO-d6) : 2.59–2.96 (m, 2H, CH2CO), 3.40–4.17 (m, 3H, CHpyr, NCH2), 6.90– 7.99 (m, 10H, Har, NH2), 8.12 (s, 1H, NH), 8.79 (s, 1H, NH), 9.95 (d, J = 8.3 Hz, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 33.72, 35.38, 35.67, 50.40, 51.46, 125.34, 126.51, 127.25, 128.62, 128.68, 130.30, 131.65, 138.98, 139.05, 139.54, 141.79, 144.43, 155.24, 172.03, 172.19, 172.38, 172.64; HRMS (ESI) for C18H18ClN5O5S+H+, calcd. 452.0795,found 452.0787 [M+H+]. 4.1.8.2. 2-(1-(2-Chloro-4-sulfamoylphenyl)-5-oxopyrrolidine-3-carbonyl)-N-phenylhydrazine-1carbothioamide (27). White solid, yield 1.18 g (84%); m.p. 156–157 °C; IR (KBr) (v, cm-1): 3308, 3262, 3090, 1693, 1682; 1H NMR (400 MHz, DMSO-d6) : 2.64–2.82 (m, 2H, CH2CO), 3.38–3.50 (m, 1H, CH), 3.82–4.03 (m, 2H, NCH2), 7.08–8.05 (m, 10H, Har, NH2), 9.51–10.32 (m, 3H, 3NH); 13C NMR (101 MHz, DMSO-d6) : 33.68, 35.60, 51.36, 125.33, 127.25, 128.16, 128.20, 130.28, 131.61, 139.03, 139.06, 144.42, 172.20; HRMS (ESI) for C18H18ClN5O4S2+H+, calcd. 468.0567, found 468.0564 [M+H+]. 4.1.9. 3-Chloro-4-(2-oxo-4-(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)pyrrolidin-1yl)benzenesulfonamide (28). A mixture of thiosemicarbazide 27 (0.47 g, 1 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 to

afford white solid, yield 0.37 g (84%); m.p. 164–165 °C; IR (KBr) (v, cm-1): 3363, 3243, 1694; 1H NMR (400 MHz, DMSO-d6) : 2.53–2.86 (m, 2H, CH2CO), 3.61–3.96 (m, 3H, CH, NCH2), 7.38–7.95 (m, 10H, Har, NH2),13.81 (br. s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 29.72, 34.71, 51.88, 125.73, 127.67, 129.01, 130.08, 130.21, 130.72, 131.94, 134.00, 139.13, 144.93, 152.87, 168.90, 171.94; HRMS (ESI) for C18H16ClN5O3S2+H+, calcd. 450.0461, found 450.0457 [M+H+]. 4.1.10. 4-((2-Chloro-4-sulfamoylphenyl)amino)-3-(5-oxo-4-phenyl-4,5-dihydro-1H-1,2,4-triazol-3-yl)butanoic acid (29). A mixture of semicarbazide 26 (0.45 g, 1 mmol) and 2% aqueous KOH solution (20 mL) was stirred at 60 °C for 8 h. Afterwards, it was acidified with HCl to pH 5. The formed precipitate was filtered off, washed with water, and recrystallized from propan-2-ol to afford brown solid, yield 0.16 g (35%); m.p. 225– 226 °C; IR (KBr) (v, cm-1): 3401, 3302, 3217, 1713, 1667; 1H NMR (400 MHz, DMSO-d6) : 2.61–2.85 (m, 2H, CH2CO), 3.11–3.31 (m, 3H, CH, NCH2), 5.94 (d, 1H, J = 8.7 Hz, Har), 6.31 (t, 1H, J = 6.0 Hz, NH) 7.07– 7.58 (m, 9H, Har, NH2), 11.78 (s, 1H, NH), 12.37 (br. s, 1H, OH); 13C NMR (101 MHz, DMSO-d6) : 31.08, 34.07, 45.44, 116.91, 125.68, 126.84, 127.81, 128.67, 129.40, 131.17, 132.57, 145.63, 148.01, 154.34, 172.91; HRMS (ESI) for C18H18ClN5O5S+H+, calcd. 452.0795, found 452.0790 [M+H+]. 4.1.11. 3-Chloro-4-(2-oxo-4-(5-oxo-4-phenyl-4,5-dihydro-1H-1,2,4-triazol-3-yl)pyrrolidin-1yl)benzenesulfonamide (30). A mixture of acid 29 (0.45 g, 1 mmol) and 10% aqueous HCl solution (20 mL) was heated at reflux for 1 h and was allowed to cool down. The formed precipitate was filtered off, washed with water, and recrystallized from propan-2-ol to afford light-brown solid, yield 0.35 g (81%); m.p. 161–162 °C; IR (KBr) (v, cm-1): 3382, 3077, 1695; 1H NMR (400 MHz, DMSO-d6) : 2.55–2.83 (m, 2H, CH2CO), 3.64–4.02 (m, 3H, CH, NCH2), 7.37–8.00 (m, 10H, Har, NH2), 11.88 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 29.34, 33.74, 51.19, 125.28, 127.21, 127.78, 129.01, 129.64, 131.47, 132.65, 144.45, 146.93, 154.64, 171.67; HRMS (ESI) for C18H16ClN5O4S+H+, calcd. 434.0690, found 434.0683 [M+H+]. 4.1.12. 3-Chloro-4-(2-oxo-4-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)pyrrolidin-1-yl)benzenesulfonamide (31). A mixture of KOH (0.17 g, 3 mmol), methanol (20 mL), and CS2 (0.2 mL, 3mmol) was stirred at room temperature for 20 min. Hydrazide 6 (0.50 g, 1.5mmol) was added into the mixture and the obtained reaction mixture was stirred at 50 °C for 24 h. Afterwards, the liquid fractions were evaporated under reduced pressure and the residue was dissolved in water (10 mL). The obtained solution was acidified with HCl to pH 6, the precipitate was filtered off, washed with ice-cold water, and recrystallized from water to afford light-yellow solid, yield 0.39 g (70%); m.p. 121–122 °C; IR (KBr) (v, cm-1): 3227, 3081, 1682; 1H NMR (400 MHz, DMSO-d6) : 2.76–3.07 (m, 2H, CH2CO), 3.92–4.19 (m, 3H, CH, NCH2), 7.50–8.09 (m, 5H, Har, NH2), 14.50 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6) : 29.80, 33.92, 51.36, 125.79, 127.71, 130.75, 132.11, 139.06, 145.04, 164.21, 171.78, 178.48; HRMS (ESI) for C12H11ClN4O4S2+H+, calcd. 374.9988, found 374.9981 [M+H+].

4.2.

Protein preparation

Production and purification of recombinant human CAs has been previously described in [31,32], their molecular weight has been confirmed by HR mass-spectrometry, purity by SDS-PAGE and their concentrations have been determined spectrophotometrically (at 280 nm).

4.3.

Determination of ligand binding affinity to CAs

4.3.1. Fluorescent thermal shift assay (FTSA) FTSA (which is also termed ThermoFluor® or Differential scanning fluorimetry (DSF)) experiments were carried out in a Corbett Rotor-Gene 6000 (QIAGEN Rotor-Gene Q) instrument using the blue channel (365±20 nm excitation and 460±15 nm detection). The protein solution in the absence and presence of various compound concentrations was heated from 25 to 99 °C (heating rate 1ºC/min) and the melting temperature Tm shift was determined by following the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS). The samples consisted of a constant concentration of protein (5 μM for all CAs, except 10 μM for CA IV), different concentrations of compound (usually 0-200 μM), 50 µM ANS and 50 mM sodium phosphate buffer (at pH 7.0) containing 100 mM sodium chloride and 2% (v/v) of DMSO. Compound binding constant was obtained from protein Tm as a function of the added ligand concentration. Data analysis was performed as previously described in[33] at 37 °C. 4.3.2. Stopped-flow CO2 hydration assay (SFA). Carbonic anhydrase I, II, IV, VI, IX, XII, XIII hydratase activity inhibition experiments were performed using Applied Photophysics SX.18 MV-R instrument at 24 ⁰C. Saturated substrate solution was prepared by bubbling carbon dioxide gas into Milly-Q water for 1 hour at room temperature. Phenol red was used as a pH indicator to follow the absorbance (λ-557 nm) while CA acidified the medium. The samples consisted of 400 nM CA I, 45 nM CA II, 50-100 nM CA IV, 100 nM CA VI, 30 nM CA IX, 50-100 nM CA XII or 400 nM CA XIII and 0-40 µM compound 5, 0-0.625 µM compound 10 or 0-33.3 µM compound 29 (≤0.33% DMSO), 30 µM phenol red, 25 mM Hepes buffer (at pH 7.5) containing 0.2 M sodium sulfate. Raw curves were fitted using a single exponential model and the dissociation constants were determined using Morrison equation[29,30]: ([𝐶𝐴] + [𝐼] + 𝐾𝑑 ― ([𝐶𝐴] + [𝐼] + 𝐾𝑑)2 ― 4[𝐶𝐴][𝐼] 𝐶𝐴 𝑎𝑐𝑡.(%) = (1 ― ) ⋅ 100% 2[𝐶𝐴] Where [CA] is total added concentration of active CA, [I] – total added inhibitor 5, 10 or 29 concentration and Kd is inhibitor binding affinity.

4.3.3 p-nitrophenyl acetate (p-NPA) hydrolysis assay. Carbonic anhydrase I and II esterase activity inhibition experiments were performed using Agilent 8453 UVvisible Spectroscopy System at 24 ⁰C. The product of p-nitrophenyl acetate hydrolysis was monitored at 405 nm. The samples consisted of 5.3 µM CA I or 0.3 µM CA II and 0-100 µM compound 29 (≤1 % DMSO) in 25 mM sodium phosphate buffer (pH 7.0) containing 50 mM sodium chloride, and 100 µM p-nitrophenyl acetate . The slopes of raw curves were estimated and dissociation constants were determined using Morrison equation as described in a previous paragraph. 4. 4. Determination of pKa of sulfonamide group of compound 2 The pKa of sulfonamide group of compound 2 was determined spectrophotometrically as previously described[22]. Briefly, 50 μM compound solutions in universal buffer (50 mM sodium acetate, 25 mM sodium borate, 50 mM sodium phosphate and 50 mM sodium chloride) containing 2 % (v/v) of DMSO at different pH (from pH 6.9 to 11.3) were made and UV/Vis spectra were recorded. The normalized ratio of absorbancies approximately 10 nm above and 10 nm below the isosbestic point was plotted as a function of pH. The pKa was determined as a midpoint of the fitted Henderson–Hasselbach curve using least-square method.

Acknowledgements This research was supported by the grant S-MIP-17-87 from the Research Council of Lithuania.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

[12]

[13] [14]

[15]

M. Aggarwal, C.D. Boone, B. Kondeti, R. McKenna, Structural annotation of human carbonic anhydrases, Journal of Enzyme Inhibition and Medicinal Chemistry. 28 (2013) 267–277. https://doi.org/10.3109/14756366.2012.737323. S.C. Frost, R. McKenna, eds., Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications, Springer Netherlands, Dordrecht, 2014. https://doi.org/10.1007/978-94-007-7359-2. W.R. Chegwidden, N.D. Carter, Y.H. Edwards, eds., The Carbonic Anhydrases, Birkhäuser Basel, Basel, 2000. https://doi.org/10.1007/978-3-0348-8446-4. M.Y. Mboge, B.P. Mahon, R. McKenna, S.C. Frost, Carbonic Anhydrases: Role in pH Control and Cancer, Metabolites. 8 (2018). https://doi.org/10.3390/metabo8010019. N.K. Tafreshi, M.C. Lloyd, M.M. Bui, R.J. Gillies, D.L. Morse, Carbonic Anhydrase IX as an Imaging and Therapeutic Target for Tumors and Metastases, Subcell Biochem. 75 (2014) 221–254. https://doi.org/10.1007/978-94-0077359-2_12. M. Y. Mboge, R. McKenna, S. C. Frost, Advances in Anti-Cancer Drug Development Targeting Carbonic Anhydrase IX and XII, in: Atta-ur-Rahman, K. Zaman (Eds.), Topics in Anti-Cancer Research, BENTHAM SCIENCE PUBLISHERS, 2016: pp. 3–42. https://doi.org/10.2174/9781681083339116050004. C.T. Supuran, How many carbonic anhydrase inhibition mechanisms exist?, Journal of Enzyme Inhibition and Medicinal Chemistry. 31 (2016) 345–360. https://doi.org/10.3109/14756366.2015.1122001. A. Nocentini, C.T. Supuran, Advances in the structural annotation of human carbonic anhydrases and impact on future drug discovery, Expert Opinion on Drug Discovery. 14 (2019) 1175–1197. https://doi.org/10.1080/17460441.2019.1651289. C.T. Supuran, Advances in structure-based drug discovery of carbonic anhydrase inhibitors, Expert Opin Drug Discov. 12 (2017) 61–88. https://doi.org/10.1080/17460441.2017.1253677. V.M. Krishnamurthy, B.R. Bohall, C.-Y. Kim, D.T. Moustakas, D.W. Christianson, G.M. Whitesides, Thermodynamic parameters for the association of fluorinated benzenesulfonamides with bovine carbonic anhydrase II, Chem Asian J. 2 (2007) 94–105. https://doi.org/10.1002/asia.200600360. V. Linkuvienė, A. Zubrienė, E. Manakova, V. Petrauskas, L. Baranauskienė, A. Zakšauskas, A. Smirnov, S. Gražulis, J.E. Ladbury, D. Matulis, Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design, Quarterly Reviews of Biophysics. 51 (2018). https://doi.org/10.1017/S0033583518000082. M. Bozdag, M. Ferraroni, E. Nuti, D. Vullo, A. Rossello, F. Carta, A. Scozzafava, C.T. Supuran, Combining the tail and the ring approaches for obtaining potent and isoform-selective carbonic anhydrase inhibitors: Solution and X-ray crystallographic studies, Bioorganic & Medicinal Chemistry. 22 (2014) 334–340. https://doi.org/10.1016/j.bmc.2013.11.016. C.T. Supuran, A. Scozzafava, A. Casini, Carbonic anhydrase inhibitors, Med. Res. Rev. 23 (2003) 146–189. https://doi.org/10.1002/med.10025. Z. Hou, B. Lin, Y. Bao, H. Yan, M. Zhang, X. Chang, X. Zhang, Z. Wang, G. Wei, M. Cheng, Y. Liu, C. Guo, Dual-tail approach to discovery of novel carbonic anhydrase IX inhibitors by simultaneously matching the hydrophobic and hydrophilic halves of the active site, European Journal of Medicinal Chemistry. 132 (2017) 1–10. https://doi.org/10.1016/j.ejmech.2017.03.023. R. Kumar, S. Bua, S. Ram, S. Del Prete, C. Capasso, C.T. Supuran, P.K. Sharma, Benzenesulfonamide bearing imidazothiadiazole and thiazolotriazole scaffolds as potent tumor associated human carbonic anhydrase IX and XII inhibitors, Bioorganic & Medicinal Chemistry. 25 (2017) 1286–1293. https://doi.org/10.1016/j.bmc.2016.12.047.

[16] R. Yaseen, D. Ekinci, M. Senturk, A.D. Hameed, S. Ovais, P. Rathore, M. Samim, K. Javed, C.T. Supuran, Pyridazinone substituted benzenesulfonamides as potent carbonic anhydrase inhibitors., Bioorg Med Chem Lett. 26 (2016) 1337–1341. https://doi.org/10.1016/j.bmcl.2015.12.016. [17] H.S. Ibrahim, S.M. Abou-Seri, M. Tanc, M.M. Elaasser, H.A. Abdel-Aziz, C.T. Supuran, Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic anhydrase isoforms IX and XII., Eur J Med Chem. 103 (2015) 583–593. https://doi.org/10.1016/j.ejmech.2015.09.021. [18] K. Rutkauskas, A. Zubrienė, I. Tumosienė, K. Kantminienė, V. Mickevičius, D. Matulis, Benzenesulfonamides bearing pyrrolidinone moiety as inhibitors of carbonic anhydrase IX: synthesis and binding studies, Medicinal Chemistry Research. 26 (2017) 235–246. https://doi.org/10.1007/s00044-016-1741-5. [19] P. Moutevelis-Minakakis, E. Papavassilopoulou, G. Michas, K. Georgikopoulou, M.-E. Ragoussi, N. Neophytou, P. Zoumpoulakis, T. Mavromoustakos, D. Hadjipavlou-Litina, Synthesis, in silico docking experiments of new 2pyrrolidinone derivatives and study of their anti-inflammatory activity, Bioorganic & Medicinal Chemistry. 19 (2011) 2888–2902. https://doi.org/10.1016/j.bmc.2011.03.044. [20] N. Siddiqui, W. Ahsan, M.S. Alam, R. Ali, K. Srivastava, Design, Synthesis and Evaluation of Anticonvulsant Activity of Pyridinyl-Pyrrolidones: A Pharmacophore Hybrid Approach, Arch. Pharm. Pharm. Med. Chem. 345 (2012) 185– 194. https://doi.org/10.1002/ardp.201100140. [21] K. Winnicka, M. Tomasiak, A. Bielawska, PIRACETAM - AN OLD DRUG WITH NOVEL PROPERTIES?, (n.d.) 5. [22] I. Vaškevičienė, V. Paketurytė, N. Pajanok, Š. Žukauskas, B. Sapijanskaitė, K. Kantminienė, V. Mickevičius, A. Zubrienė, D. Matulis, Pyrrolidinone-bearing methylated and halogenated benzenesulfonamides as inhibitors of carbonic anhydrases, Bioorganic & Medicinal Chemistry. 27 (2019) 322–337. https://doi.org/10.1016/j.bmc.2018.12.011. [23] V.M. Krishnamurthy, G.K. Kaufman, A.R. Urbach, I. Gitlin, K.L. Gudiksen, D.B. Weibel, G.M. Whitesides, Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding, Chem. Rev. 108 (2008) 946–1051. https://doi.org/10.1021/cr050262p. [24] I. Parašotas, E. Urbonavičiūtė, K. Anusevičius, I. Tumosienė, I. Jonuškienė, K. Kantminienė, R. Vaickelionienė, V. Mickevičius, Synthesis and Biological Evaluation of Novel Di- and Trisubstituted Thiazole Derivatives, HETEROCYCLES. 94 (2017) 1074. https://doi.org/10.3987/COM-17-13714. [25] I. Tumosienė, A. Peleckis, I. Jonuškienė, R. Vaickelionienė, K. Kantminienė, J. Šiugždaitė, Z.J. Beresnevičius, V. Mickevičius, Synthesis of novel 1,2- and 2-substituted benzimidazoles with high antibacterial and antioxidant activity, Monatsh Chem. 149 (2018) 577–594. https://doi.org/10.1007/s00706-017-2066-x. [26] I. Tumosienė, K. Kantminienė, I. Jonuškienė, A. Peleckis, S. Belyakov, V. Mickevičius, Synthesis of 1-(5-Chloro-2hydroxyphenyl)-5-oxopyrrolidine-3-carboxylic Acid Derivatives and Their Antioxidant Activity, Molecules. 24 (2019) 971. https://doi.org/10.3390/molecules24050971. [27] I. Tumosienė, Z.J. Beresnevičius, Synthesis of azoles from 3,3′-[(4-alkoxyphenyl)imino]bis(propanoic acid hydrazides), Monatsh Chem. 140 (2009) 1523–1528. https://doi.org/10.1007/s00706-009-0210-y. [28] P. Cimmperman, L. Baranauskienė, S. Jachimovičiūtė, J. Jachno, J. Torresan, V. Michailovienė, J. Matulienė, J. Sereikaitė, V. Bumelis, D. Matulis, A Quantitative Model of Thermal Stabilization and Destabilization of Proteins by Ligands, Biophys. J. 95 (2008) 3222–3231. https://doi.org/10.1529/biophysj.108.134973. [29] J.F. Morrison, Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et Biophysica Acta (BBA) - Enzymology. 185 (1969) 269–286. https://doi.org/10.1016/00052744(69)90420-3. [30] J.W. Williams, J.F. Morrison, [17] The kinetics of reversible tight-binding inhibition, in: Methods in Enzymology, Academic Press, 1979: pp. 437–467. https://doi.org/10.1016/0076-6879(79)63019-7. [31] V. Dudutienė, J. Matulienė, A. Smirnov, D.D. Timm, A. Zubrienė, L. Baranauskienė, V. Morkūnaite, J. Smirnovienė, V. Michailovienė, V. Juozapaitienė, A. Mickevičiūtė, J. Kazokaitė, S. Bakšytė, A. Kasiliauskaitė, J. Jachno, J. Revuckienė, M. Kišonaitė, V. Pilipuitytė, E. Ivanauskaitė, G. Milinavičiūtė, V. Smirnovas, V. Petrikaitė, V. Kairys, V. Petrauskas, P. Norvaišas, D. Lingė, P. Gibieža, E. Capkauskaitė, A. Zakšauskas, E. Kazlauskas, E. Manakova, S. Gražulis, J.E. Ladbury, D. Matulis, Discovery and characterization of novel selective inhibitors of carbonic anhydrase IX, J. Med. Chem. 57 (2014) 9435–9446. https://doi.org/10.1021/jm501003k. [32] A. Mickevičiūtė, V. Juozapaitienė, V. Michailovienė, J. Jachno, J. Matulienė, D. Matulis, Recombinant Production of 12 Catalytically Active Human CA Isoforms, in: D. Matulis (Ed.), Carbonic Anhydrase as Drug Target:

Thermodynamics and Structure of Inhibitor Binding, Springer International Publishing, Cham, 2019: pp. 15–37. https://doi.org/10.1007/978-3-030-12780-0_2. [33] V. Petrauskas, A. Zubrienė, M.J. Todd, D. Matulis, Inhibitor Binding to Carbonic Anhydrases by Fluorescent Thermal Shift Assay, in: D. Matulis (Ed.), Carbonic Anhydrase as Drug Target: Thermodynamics and Structure of Inhibitor Binding, Springer International Publishing, Cham, 2019: pp. 63–78. https://doi.org/10.1007/978-3-03012780-0_5.

Graphical abstract

Conflicts of interest: The authors declare that they have no conflicts of interest, including no financial, personal or other relationships with other people or organisations.