Benzimidazole derivatives as potent and isoform selective tumor-associated carbonic anhydrase IX/XII inhibitors

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Benzimidazole derivatives as potent and isoform selective tumor-associated

carbonic anhydrase IX/XII inhibitors

Azize Gizem Uslu, Tuğ çe Gür Maz, Alessio Nocentini, Erden Banoglu,

Claudiu T. Supuran, Burcu Çalı şkan

PII:

S0045-2068(19)31655-4

DOI:

https://doi.org/10.1016/j.bioorg.2019.103544

YBIOO 103544

Reference:

To appear in:

Bioorganic Chemistry

Received Date:

Revised Date:

Accepted Date:

8 October 2019

6 December 2019

21 December 2019

Please cite this article as: A. Gizem Uslu, T. Gür Maz, A. Nocentini, E. Banoglu, C.T. Supuran, B. Çalı şkan,

Benzimidazole derivatives as potent and isoform selective tumor-associated carbonic anhydrase IX/XII

inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103544

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Benzimidazole derivatives as potent and isoform selective tumor-associated carbonic

anhydrase IX/XII inhibitors

a

b

a

Azize Gizem Uslu , Tuğçe Gür Maz , Alessio Nocentini , Erden Banoglu , Claudiu T.

Supuran ^b, Burcu Çalışkan ^a,*

^aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Taç Sok.

No:3 Yenimahalle 06560 Ankara, Turkey

b

NEUROFARBA Dept., University of Florence, Sezione di Scienze Farmaceutiche, Via Ugo

Schiff 6, 50019 Sesto Fiorentino, Florence, Italy

* Corresponding author: Prof. Dr. Burcu Çalışkan, Department of Pharmaceutical Chemistry,

Faculty of Pharmacy, Gazi University, 06330 Etiler, Ankara, Turkey. Tel: +90 312 2023240;

Fax: +90 312 2235018; E-mail: bcaliska n @gazi.edu.tr.

1

ABSTRACT

We describe the synthesis of a series of 2-arylbenzimidazole derivatives bearing sulfonamide

functionality (4a–d, 7a–c and 10) as well as hydroxamic acid (15a–b), carboxylic acid (16a–

b), carboxamide (17a–b) and boronic acid (22a–b and 26) functionalities, which act as human

carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors. The newly synthesized benzimidazole

derivatives were evaluated against 4 physiologically relevant CA isoforms (hCA I, II, IX, and

XII), and especially the sulfonamide-containing benzimidazoles demonstrated intriguing

inhibitory activity against tumor associated CA IX and XII with K_Ivalues in the range of 5.2–

29.3 nM and 9.9–41.7 nM, respectively. Notably, compound 4c was the most potent and

selective CA IX (K_I= 6.6 nM) and XII (K_I= 9.9 nM) inhibitor with a significant selectivity

ratio over cytosolic CA I and II isoforms in the range of 3.4–25.2. In addition, compounds

having hydroxamic acid (15a-b) or carboxylic acid (16a-b) functionalities resulted in greater

selectivity ratios for CA IX/XII over CAI/II in the range of 4.1-121.5 although with K_Ivalues

in lower micromolar potency (K_Is = 0.36-0.85 M for CA IX/XII).

Keywords: Carbonic anhydrase, benzimidazole, sulfonamide, carboxylic acid, hydroxamic

acid

2

1. Introduction

The carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which catalyze the

reversible hydration of CO₂into protons and bicarbonate [1]. CAs are involved in many

physiological and pathological processes such as respiration and transport of CO₂and

bicarbonate between metabolizing tissues and lungs, homeostasis of pH and CO₂, secretion of

electrolytes in various tissues and organs, biosynthetic reactions (i.e., gluconeogenesis,

lipogenesis and ureagenesis), bone resorption, calcification and tumor growth [1]. Therefore,

many CA isoforms participating these processes are crucial as therapeutic targets, and their

inhibitors have great potential for pharmacological intervention with a wide range of ailments

such as edema, glaucoma, obesity, osteoporosis, epilepsy and cancer [2, 3]. To date, 16 distinct

mammalian α-CA isoforms identified including human isoforms [4]. These CAs can be

classified as cytosolic (CA I, CA II, CA III, CA VII and CA XIII), membrane bound (CA IV,

CA IX, CA XII, CA XIV and CA XV), and mitochondrial (CA VA and CA VB), as well as one

isozyme is secreted in saliva (CA VI). Among them cytosolic hCA I and II are ubiquitous

isoforms, and inhibitors of these isoforms are mainly used as antiglaucoma drugs, diuretics,

antiepileptic drugs, antiedema drugs, and also for the treatment of altitude sickness [1, 4, 5].

However, in recent years, hCA IX and XII isoforms have been the main focus of cancer research

[6, 7]. For instance, hCA IX is found to be overexpressed in hypoxic tumors whereas it is not

abundant in normal tissues [8]. Hypoxic conditions regulate the expression of CA IX through

the hypoxia inducible factor 1 (HIF1) cascade, which controls the external pH to support an

acidic extracellular microenvironment suited for hypoxic tumor cell survival and proliferation,

but detrimental to normal cells [9]. In a similar way, another tumor-associated isozyme hCA

XII decreases the extracellular pH cooperating with hCA IX [10]. Since hCA IX/XII are

reported to play an important role in the progression of tumor growth and metastasis, they have

3

been anticipated as potential targets for the development of novel antineoplastic agents.

However, the main obstacle for developing new cancer therapeutics by targeting hCA IX/XII

occurred as a result of the nonselective inhibition of hCA I/II isoforms, which was envisaged

as the main reason for the unwanted side effects observed with these nonselective inhibitors [1,

5]. The one and only sulfonamide derivative SLC-0111 (Figure 1) with high selectivity against

CA IX isoform has entered to clinical trials alone for solid tumors [11] or as a combination

therapy for pancreatic ductal cancer [12].

Most of the studied inhibitors against one or more hCAs belong to the sulfonamide

containing aromatic/heterocyclic compounds, where the sulfonamide moiety acts as a zinc

binding group (ZBG) whereas the heterocyclic scaffold is considered to selectively promote

interactions with isoform unique residues at the active site cavity [5, 13]. However, the presence

of such a very strong sulfonamide moiety as ZBG in these inhibitors has been reported as one

of the main drawbacks for the encountered non-selectivity issues towards different hCA

isoforms, and distinct ZBGs such as hydroxamic acid [14], benzoxaborole [15], N-hydroxyurea

[16], boronic acid [17], carbohydrazide, carboxylic acid and amide as sulfonamide bioisosteres

have been applied to produce more isoform specific inhibitors [18, 19]. For example, Mori et

al. recently reported a carboxylic acid derivative (GV2-20) with potency in nM range against

CAII, CAVII, CAIX and CAXII [20]. In a follow-up study, the same researchers also

demonstrated that a series of new GV2-20 analogues showed potent CAIX inhibitory activities

with greater selectivity over cytosolic isoforms CAI/II [21]. Moreover, D’Ambroiso et al.

reported in a structural study that 2-benzylsulfinylbenzoic acid was a potent inhibitor of CAII

and CAIX although with a different binding mode [22], indicating that carboxylic acid function

might be promising for developing potent and selective CA inhibitors. Meantime, 5-

sulfonamide congener of the anthelmintic drug thiabendazole was investigated as CA inhibitor

4

by using kinetic and crystallographic approaches (Figure 1) [23]. Thiabendazole-5-sulfonamide

acted as a very effective inhibitor of CA IX (K_I= 6.4 nM), and also inhibited effectively

isozymes CA I, II, IV–VII, and XII with K_Is in the range of 17.8–73.2 nM, showing some levels

of selectivity for inhibition of CA IX isozyme. Based on these observations, we hereby explored

the efficiency of different ZBGs such as sulfonamide, carboxylic acid, hydroxamic acid,

carboxamide and boronic acid in a new series of benzimidazole derivatives on the potency and

selectivity against four physiologically important CA isoforms, the hCA I and II (cytosolic) and

hCA IX/XII (membrane bound).

H

N

H₂NO₂S

N

S

O

N

H

N

F

SO₂NH₂

SLC-0111

Thiabendazole-5-sulfonamide

Figure 1. Structures of SCL-0111 and thiabendazole-5-sulfonamide as selective CA IX

inhibitors

2. Results and discussion

2.1 Chemistry

As a rationale of this study, we used the thiabendazole-5-sulfonamide as a lead

compound and replaced its thiazole group with different aryl moieties along with the change of

the sulfonamide moiety with common bioisosteres. For the first series of sulfonamide

derivatives, we used the synthetic methodology outlined in Scheme 1, following standard

procedures. For N¹-methyl-2-aryl-benzimidazole analogues carrying 5-sulfonamido group (4a-

d), the sulfonamide intermediate 1 was prepared by amination of the 4-chloro-3-

nitrobenzenesulfonyl chloride, which was subsequently used to produce 2 by nucleophilic

substitution with methylamine. After reducing the nitro group in 2, ring closure was realized

by condensation of 3 with the sodium metabisulfite adduct of appropriate benzaldeydes to

produce 4a-c. For the synthesis of benzimidazole derivative 4d carrying bis-sulfonamido

5

groups at both ends, we acquired a two-step procedure whereby 3 was first treated with 4-

sulfamoylbenzoyl chloride to yield the monoacylated derivative, which was subsequently

converted to the target benzimidazole by refluxing in dioxane/HCl. The derivatives 7a-c with

unsubstituted benzimidazole nitrogen were also successfully synthesized under standard

conditions starting from 4-chloro-3-nitrobenzenesulfonyl chloride as outlined in Scheme 1.

Compound 10 carrying sulfonamido group only at the right hand-side of the molecule was

produced starting from 1-fluoro-2-nitrobenzene and following standard conditions in Scheme

1. Compounds having hydroxamic acid 15a-b, carboxylic acid 16a-b and amide 17a-b as

replacements of 5-sulfonamido were synthesized by the series of reactions given in Scheme 2.

Accordingly, starting from 4-fluoro-3-nitrobenzoic acid, esterification to carbomethoxy

derivative followed by nucleophilic displacement of the fluorine and reduction of nitro group

afforded the common intermediate 13, which was condensed with the sodium metabisulfite

adduct of appropriate benzaldehydes to give the advanced intermediates 14a-b. The reaction of

the ester derivatives 14a-b with hydroxyl amine or their hydrolysis provided hydroxamic acids

(15a-b) or carboxylic acids (16a-b), respectively. 16a-b was first reacted with SOCl₂to obtain

acyl chloride and then ammonia to generate amides (17a-b). Finally, to complete our synthetic

work, installation of boronic acid as a sulfonamide replacement at the left (22a-b) and right

(26) hand-side of the BI core were done according to the procedures described in Scheme 3.

Consequently, the nucleophilic displacement of fluorine and reduction of nitro groups in 5-

bromo-2-fluoronitrobenzene were first achieved to generate 4-bromo-1,2-benzenediamine

derivative (19). Condensation of 19 with the sodium metabisulfite adduct of appropriate

benzaldeydes produced the 5-bromo benzimidazoles 20a-b, which underwent a palladium-

catalyzed Suzuki-Miyaura cross-coupling reaction with bis (pinacolato)diboron followed by

hydrolysis to afford 5-boronic acid derivatives 22a-b. Finally, in an attempt to introduce a

boronic acid at the right-hand side, benzimidazole ring closure was realized by reaction of o-

6

phenylenediamine with the sodium metabisulfite adduct of 4-bromobenzaldehyde followed by

N-methylation of the benzimidazole nitrogen to afford 24, which coupled directly with bis

(pinacolato)diboron using Suzuki-Miyaura cross-coupling conditions and hydrolyzed to give

the desired boronic acid derivative 26.

H₂NO₂S

NH₂

H₂NO₂S

NO₂

Cl

H₂NO₂S

NO₂

N

4a R₁=H; X=CH

4b R₁=Cl; X=CH

4c R₁=H; X=N

iv or v

ii

iii

R₁

CH₃

X

N

H

N

H

CH₃

4d R₁=SO₂NH₂; X=CH

1

2

3

i

H₂NO₂S

NH₂

H₂NO₂S

ClO₂S

NO₂

Cl

H₂NO₂S

NO₂

NH₂

N

7a R₁=H; X=CH

7b R₁=Cl; X=CH

7c R₁=H; X=N

iv

vi

iii

R₁

N

H

X

5

6

NH₂

NO₂

F

NO₂

N

v

ii

iii

SO₂NH₂

CH₃

N

H

N

H

CH₃

10

8

9

Scheme 1. Synthesis of compounds 4a-d, 7a-c and 10. i. NH₃(aq), diethylether, rt; ii. CH₃NH₂,

EtOH or DMSO, Δ; iii. SnCl₂.2H₂O, EtOH, Δ; iv (for 4a-c, 7a-c) Benzaldehyde derivatives,

Na₂S₂O₃, DMF, 60^oC; v (for 4d and 10). a) 4-Sulfamoylbenzoyl chloride, DIEA, THF; b) 1,4-

dioxane, HCl, 90^oC; vi. NH₃(aq), 1,4-dioxan, 120^oC, 60 min, MWI.

MeOOC

NO₂

HOOC

NO₂

F

MeOOC

NO₂

F

MeOOC

NH₂

MeOOC

HOOC

N

iii

i

ii

iv

vi

R₁

NHCH₃

CH₃

11

12

13

14a-b

R= H (a); Cl (b)

16a-b

v

vii

O

H₂NOC

N

HO

N

R₁

H

N

CH₃

17a-b

15a-b

Scheme 2. Synthesis of compounds 15-17a-b. i. MeOH, H₂SO₄, Δ; ii. CH₃NH₂, MeOH, 135^oC,

25 min, MWI; iii. SnCl₂.2H₂O, MeOH, Δ; iv. Benzaldehyde derivatives, Na₂S₂O₃, DMF, 60^oC;

7

v. NH₂OH(aq), DMF, rt; vi. LiOH.H₂O, THF:Water (1:1), 60^oC; vii. a) SOCl₂, Δ. b) NH₃,

MeOH, rt.

O

Br

NO₂

F

Br

NO₂

Br

B

Br

NH₂

N

O

iii

i

ii

iv

R

NHCH₃

CH₃

18

19

20a-b

21a-b

R= H (a); Cl (b)

v

OH

B

N

HO

R

N

CH₃

22a-b

NH₂

N

O

N

OH

iii

vi

iv

v

Br

B

N

H

CH₃

24

CH₃

23

26

25

Scheme 3. Synthesis of compounds 22a-b and 26. i. CH₃NH₂, DMSO, 140^oC; ii. SnCl₂.2H₂O,

EtOH, Δ; iii. Na₂S₂O₃, DMF, 60^oC; iv. Pin₂B₂, KOAc, PdCl₂(dppf), 1,4-dioxan, rt to Δ; v.

NaIO₄, THF:Water (4:1), HCl (1N), rt; vi. MeI, KOH, DMSO, rt.

2.2 Carbonic anhydrase inhibition

The synthesized compounds 4a-d, 7a-c, 10, 15-17a-b, 22a-b and 26 were investigated for

their inhibitory effects against four physiological relevant isoforms, i.e. hCA I, II, IX and XII,

using acetazolamide (AAZ) as the standard inhibitor by means of a stopped flow CO₂hydrase

assay [24]. The hCA inhibition values and the selectivity ratios for hCA IX and hCA XII

inhibition over the off-target hCA I and hCA II are presented in Table 1. The following structure

activity relationships (SARs) for the aforementioned hCA isoforms can be reported:

i) The ubiquitous cytosolic hCA I was moderately inhibited by all sulfonamide derivatives

except for 4d and 10, which showed a good inhibitory potency with K_Iof 22 and 33.9

nM, respectively. It is noteworthy to point out that the presence of 4-sulfonamidophenyl

8

group at the 2-position of benzimidazole (BI) ring along with (4d) or without (10) 5-

sulfonamido substitution of the BI scaffold caused a considerable increase of the

inhibitory activity against hCA I isoform.

ii) All the synthesized compounds having 5-sulfonamido-BI core (4a-d, 7a-c) strongly

inhibited the cytosolic isoform hCA II with K_Is ranging between 47.7 nM and 10.4 nM,

as compared to reference drug AAZ (K_I= 12.5 nM). Again, derivatives with 4-

sulfonamidophenyl group at the 2-position of BI demonstrated the higher potency

against this isoform with K_Iof 10.4 nM for 4d and 8.8 nM for 10.

iii) The membrane-bound tumor associated isoform CA IX and XII were also strongly

inhibited by 4a-d, 7a-c and 10 with K_Is 5.2-29.3 nM and 9.9-41.7 nM, respectively. The

derivative 4d with bis-sulfonamido substitution at both hand-sides showed even better

inhibitory potency (K_I= 5.2 nM) for CA IX as compared to the reference drug AAZ (K_I

= 25 nM). It is worthy to mention here that N-methyl-BI-5-sulfonamide having

pyridine-2-yl group at 2-position of BI ring (4c) was found to be the best inhibitor of

both CA IX and XII with K_Ivalues of 6.6 and 9.9 nM, respectively, with a pronounced

selectivity over cytosolic isoforms CA I and II (K_I= 166.4 and 33.7 nM, respectively).

In accordance with 4c, compound 7c containing 2-pyridyl moiety at C-2 position of BI

ring without N¹-methyl substitution was also found to be effective inhibitors for all hCA

isoforms (K_I= 14.8-28.3 nM) studied here except for hCA I (K_I= 218.1 nM).

iv) However, bioisosteric replacement of 5-sulfonamido of the BI with hydroxamic acid

(15a-b), carboxylic acid (16a-b), carboxamide (17a-b) and boronic acid (22a-b) had a

detrimental effect on the activity against all hCA isoforms studied here leading to K_I

values at µM range (0.36 - >100 µM). At the same time, several of these bioisosters also

led to better selective inhibition of one isoform over others; e.g., compounds 15a-b with

5-hydroxamic acid and 16a-b with carboxylic acid as 5-sulfonamido replacements were

9

found to be selective inhibitors of isoforms hCA IX and XII respectively, although with

potencies at a lower µM range (K_I= 0.36-0.85 µM). Compound 22a with 5-boronic acid

substitution was also a more selective inhibitor of hCA XII with a K_Iof 0.85 µM.

Analogues with 5-carboxamide substitution (17a-b) were not active against all tested

hCAs implicating that an ionizable group is required at 5-position of the BI ring.

v) With regard to the selectivity ratios, two of the 5-sulfonamido derivatives having pyridyl

moiety at 2 position of BI ring demonstrated good selectivity against cancer associated

hCA IX and/or XII isoforms. For instance, compound 4c was found more selective

towards hCA IX over hCA I (25.2-fold) and hCA II (5.1-fold), while its selectivity index

for hCA XII over hCA I and hCA II was 16.8 and 3.4, respectively. Again, the congener

compound 7c also showed appreciable selectivity for both hCA IX and XII over hCA I

with selectivity ratio of 8.9 and 14.7. On the other hand, compounds with hydroxamate

and carboxylate function at 5 position BI nucleus acted as the highest selective inhibitors

of hCA IX/XII isoforms over hCA I; e.g. 15a with 28.5 and 20.7-fold, 15b with 118.6

and 83.7-fold, 16a with 119.5 and 92.8-fold, 16b with 89.8 and 121.5-fold selectivity

towards hCA IX and XII, respectively, over hCA I. In addition, these compounds were

still quite selective inhibitors of hCA IX/XII isoforms over hCA II with selectivity ratios

in the range of 4.1 to 21.4. Lastly, 5-boronic acid derivative 22a was a very good

selective inhibitor of hCA XII over hCA I (29.3-fold). In a broader sense, it can be said

that these compounds had better selectivity ratios for inhibiting tumor-associated hCA

IX over cytosolic hCA I/II isoforms.

10

Table 1: Inhibition data of CA isoforms I, II, IX and XII with title compounds using AAZ as a standard inhibitor.

K_I(nM)*

CA II

Selectivity ratio

R₁

R₂

R₃

X

CA I

84.8

CA IX

25.4

18.6

6.6

CA XII

41.7

20.4

9.9

CAI/IX

3.3

CAI/XII

2.0

CAII/IX

0.89

1.66

5.1

CAII/XII

0.5

1.5

3.4

1.0

1.2

1.3

1.9

0.3

4.1

8.8

16.6

8.9

-

4a

4b

SO₂NH₂

H

CH₃

H

CH

N

22.7

30.9

Cl

54.3

2.9

2.7

4c

H

166.4

22.0

33.7

25.2

4.2

16.8

2.2

4d

SO₂NH₂

CH

N

10.4

5.2

10.2

30.4

22.6

14.8

33.8

2.0

7a

H

80.2

37.8

21.1

29.3

24.4

22.3

3.8

2.6

1.79

1.6

7b

H

Cl

88.6

47.7

3.0

3.9

7c

H

218.1

33.9

28.3

8.9

14.7

1.0

1.16

0.39

5.64

12.5

21.4

6.6

10

CH₃

SO₂NH₂

CH

8.8

1.5

15a

15b

16a

16b

17a

17b

22a

22b

26

CONHOH

COOH

CONH₂

B(OH)₂

H

Cl

15.7 μM

42.7 μM

78.9 μM

65.6 μM

3.1 μM

4.5 μM

0.55 μM 0.76 μM

0.36 μM 0.51 μM

28.5

118.6

119.5

89.8

-

20.7

83.7

92.8

121.5

-

H

14.1 μM 0.66 μM 0.85 μM

4.8 μM 0.73 μM 0.54 μM

Cl

H

>100 μM >100 μM 50.1 μM >100 μM

-

Cl

>100 μM >100 μM 69.8 μM

65.2 μM

0.81 μM

5.5 μM

10.1 μM

5.7

-

H

23.7 μM

14.3 μM

41.9 μM

250

5.3 μM

31.3 μM

19.5 μM

12.5

4.3 μM

10.1 μM

17.9 μM

25

5.5

29.3

2.6

1.2

6.6

5.7

1.9

2.2

Cl

1.4

3.1

B(OH)₂

2.3

4.15

43.9

1.1

AAZ

10.0

0.5

*Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 % of the reported values)

11

3. Conclusion

In conclusion, we report here a novel series of benzimidazole-5-sulfonamides and their

isosteres incorporating various aryl groups at 2 position of the BI nucleus as hCA inhibitors.

Our work led to several interesting inhibitors against the cytosolic hCA II, as well as the

transmembrane, tumor-associated hCA IX/XII isoforms, and several isoform-selective

sulfonamide inhibitors targeting hCA II, IX and XII were detected (K_Is in the range of 5.2 to

47.7 nM). The SARs obtained for hCA inhibition with this small subset of sulfonamides may

indicate that important changes of activity and selectivity towards hCAs observed even after

minor changes in the 2-aryl tail of the BI nucleus; e.g., the presence of the 2-pyridinyl moiety

in 4c led to a low nanomolar hCA IX/XII inhibition (K_I= 6.6 and 9.9 nM, respectively) with a

pronounced selectivity over cytosolic hCA I/II (Table 1). The isosteric replacement of the 5-

sulfonamido ZBG group with hydroxamate and carboxylate resulted in the highest selective

inhibition of tumor-associated hCA IX/XII isoforms at sub-micromolar range (K_Is in the range

of 0.36 to 0.85 μM), altogether making these BI derivatives of particular interest for further

investigations.

4. Experimental Section

4.1 General

Starting materials were purchased from commercial suppliers and used without further

1

13

purification. H and C NMR spectra were recorded in DMSO-d₆on a Varian Mercury 400

MHz High Performance Digital FT-NMR Spectrometer (Agilent Technologies, Santa Clara,

CA, USA) using tetramethylsilane as the internal standard. All chemical shifts were recorded

as δ (ppm). All coupling constants are reported as Hertz. High resolution mass spectra data

(HRMS) were collected using Waters LCT Premier XE Mass Spectrometer (high sensitivity

12

orthogonal acceleration time-of-flight instrument) operating in ESI (+) or ESI (-) method, also

coupled to an AQUITY Ultra Performance Liquid Chromatography system (Waters

Corporation, Milford, MA, USA) using a UV detector monitoring at 254 nm. Purity for all final

compounds were >95%, according to the UPLC/MS method using (A) water + 0.1% Formic

Acid and (B) acetonitrile + 0.1% Formic Acid; flow rate = 0.3 mL/min, Column: Aquity BEH

C18 column (2.1 x 100 mm, 1.7 mm, Waters Corporation, Milford, MA, USA). The reactions

were followed by TLC on precoated Merck silica gel plates purchased from Merck. The

developed plates were visualized using 254 nm or 365 nm UV. Flash column chromatography

on silica gel was performed on RediSep^®prepacked disposable silica gel columns using

Teledyne Isco Combiflash. Preparative liquid chromatography was performed on Buchi Prep

HPLC Column C18 (250 x 21.2 mm, 10 µm, Grace, Columbia, Maryland, USA) using

Reveleris PREP^®purification system. All microwave irradiation experiments were carried out

in a Biotage Initiator + microwave apparatus with Biotage sealed microvawe vials (Biotage,

Sweden AB, Uppsala, Sweden). Melting points of the synthesized compounds were determined

by Stuart SMP50 automatic melting point apparatus. Experimental procedures for the synthesis

of compounds 1-3, 5, 6, 8, 9, 11-13, 18-20a-b, 23 and 24 can be found in Supporting

Information.

4.1.1 1-Methyl-2-phenyl-1H-benzo[d]imidazole-5-sulfonamide (4a)

Benzaldehyde (73 µL, 0.72 mmol, 1 eq) was dissolved in EtOH (2-5 mL), and sodium

metabisulfite (205 mg, 1.08 mmol, 1.5 eq) in H₂O (1-2 mL) was added in dropwise. The

reaction mixture was stirred at rt overnight. Reaction mixture was diluted with EtOH, cooled

and formed precipitate was filtered and dried (128 mg). The mixture of this salt (124.6 mg,

0.596 mmol, 1.5 equiv) and 3-amino-4-(methylamino)benzenesulfonamide (3) (80 mg, 0.397

mmol, 1 equiv) in 5 ml DMF was heated at 60˚ for 6 h. Reaction mixture was diluted with water

13

and formed precipitate was filtered. The crude product was purified by flash chromatography

on silica gel (12 g), eluting with a gradient of 0-11% MeOH in DCM to give 4a (74.5 mg,

64.8% yield) as a white powder. Mp 266-268.6 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 3.92

(3H, s), 7.32 (2H, s), 7.58-7.60 (3H, m), 7.76-7.81 (2H, m), 7.85-7.88 (2H, m), 8.14 (1H, s);

¹³C NMR (100 MHz DMSO-d₆):  32.05, 111.05, 116.93, 119.93, 128.75, 129.41, 129.48,

130.12, 138.25, 138.45, 141.53, 155.41; HRMS m/z calculated for C₁₄H₁₃N₃O₂S [M+H]⁺

288.0807, found: 288.0794.

4.1.2 2-(4-Chlorophenyl)-1-methyl-1H-benzo[d]imidazole-5-sulfonamide (4b)

Prepared from 3-amino-4-(methylamino)benzenesulfonamide (3) (140 mg, 0.695 mmol, 1

equiv) and bisulfite adduct of 4-chlorobenzaldehyde (255 mg, 1.043 mmol, 1.5 equiv) under

the same conditions that were applied to 4a. The crude product was purified by flash

chromatography on silica gel (12 g), eluting with a gradient of 0-5% MeOH in DCM to give 4b

(140 mg, 62% yield) as a white powder. Mp 288.3-290°C. ¹H NMR (400 MHz, DMSO-d₆): δ

3.91 (3H, s), 7.26 (2H, s), 7.64 (2H, d, J = 8.4 Hz), 7.77-7.78 (2H, m), 7.90 (2H, d, J = 8.4 Hz),

8.13 (1H, s); ¹³C NMR (100 MHz, DMSO-d₆):  31.96, 111.00, 116.94, 120.02, 128.31,

128.75, 131.11, 134.97, 138.33, 138.39, 141.41, 154.20; HRMS m/z calculated for

C₁₄H₁₂ClN₃O₂S [M+H]⁺322.0417, found: 322.0415.

4.1.3 1-Methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-sulfonamide (4c)

Prepared from 3-amino-4-(methylamino)benzenesulfonamide (3) (149 mg, 0.742 mmol, 1

equiv) and bisulfite adduct of 2-pyridinecarboxaldehyde (310 mg, 1.468 mmol, 1.98 equiv)

under the same conditions that were applied to 4a. The crude product was purified by flash

chromatography on silica gel (12 g), eluting with a gradient of 0-4.5% MeOH in DCM to give

4c (116 mg, 55% yield) as a white powder. Mp 292.9- 294.5°C. ¹H NMR (400 MHz, DMSO-

14

d₆): δ 4.25 (3H, s), 7.25 (2H, s), 7.53 (1H, bt), 7.80 (2H, s), 8.00 (1H, bt, J = 7.0 Hz), 8.16

13

(1H, s), 8.31 (1H, d, J = 8.0 Hz), 8.75 (1H, s); C NMR (100 MHz, DMSO-d₆):  32.90,

111.18, 117.26, 120.46, 124.53, 124.61, 137.37, 138.54, 138.82, 141.00, 148.79, 149.39,

151.97. HRMS m/z calculated for C₁₃H₁₂N₄O₂S [M+H]⁺289.0759, found: 289.0755.

4.1.4 1-Methyl-2-(4-sulfamoylphenyl)-1H-benzo[d]imidazole-5-sulfonamide (4d)

Step1: DIEA (215 µL, 1.242 mmol, 2 equiv) and 4-sulfamoylbenzoylchloride (176.4 mg, 0.803

mmol, 1.07 equiv) were added to the solution of 3-amino-4-(methylamino)benzenesulfonamide

(3) (150 mg, 0.745 mmol, 1 equiv) in THF. Reaction mixture was stirred at rt under N₂for 24h.

Upon completion, reaction mixture was evaporated, and residue was diluted with water and

extracted with ethyl acetate. Organic layer was dried over Na₂SO₄, filtred and evaporated. The

obtained N-(2-(methylamino)-5-sulfamoylphenyl)-4-sulfamoylbenzamide (225.7 mg, 94%

yield) was used in the next step without purification. Mp 195.2-197.0°C. HRMS m/z calculated

for C₁₄H₁₆N₄O₅S₂[M+H]⁺385.0640, found: 385.0645.

Step 2: N-(2-(methylamino)-5-sulfamoylphenyl)-4-sulfamoylbenzamide (201 mg, 0.523

mmol) in 1,4-dioxane (6 mL) and conc HCl (2 mL) was heated at 90˚C for 4 h. Reaction mixture

was diluted with ice water and neutralized with NaHCO₃solution. The precipitated solid was

filtered and dried. The crude product was purified by preparative LC, eluting with a gradient of

0-42% Acetonitrile in H₂O to give 4d (47 mg, 25% yield) as a white powder. Mp 296- 297.4°C.

¹H NMR (400 MHz, DMSO-d₆): δ 3.95 (3H, s), 7.42 (4H, bs), 7.78 (1H, dd, J = 8.8, 1.6 Hz),

7.84 (1H, d, J = 8.8 Hz), 8.00 (2H, d, J = 8.4 Hz), 8.08 (2H, d, J = 8.4 Hz), 8.14 (1H, d, J =1.6

Hz); ¹³C NMR (100 MHz, DMSO-d₆):  32.17, 111.32, 117.12, 120.31, 125.98, 130.02,

132.52, 138.51, 141.45, 145.28, 154.02; HRMS m/z calculated for C₁₄H₁₄N₄O₄S₂[M+H]⁺

367.0535, found: 367.0520.

15

4.1.5 2-Phenyl-1H-benzo[d]imidazole-5-sulfonamide (7a)

Prepared from 3,4-diaminobenzenesulfonamide (6) (101 mg, 0.540 mmol, 1 equiv) and

bisulfite adduct of benzaldehyde (170 mg, 0.810 mmol, 1.5 equiv) under the same conditions

that were applied to 4a. The crude product was purified by preparative LC, eluting with a

gradient of 0-57% Acetonitrile in H₂O to give 7a (28 mg, 20% yield) as a white powder. Mp

282-283°C. ¹H NMR (400 MHz, DMSO-d₆): δ 7.28 (2H, s), 7.50-7.58 (3H, m), 7.67-7.72 (2H,

m), 8.04 (1H, s), 8.18 (2H, d, J = 6.8 Hz), 13.28 (1H, s); ¹³C NMR (100 MHz, DMSO-d₆): 

119.89, 126.73, 129.06, 129.46, 130.49, 138.07, 153.88; HRMS m/z calculated for

C₁₃H₁₁N₃O₂S [M+H]⁺274.0650 , found 274.0647.

4.1.6 2-(4-Chlorophenyl)-1H-benzo[d]imidazole-5-sulfonamide (7b)

Prepared from 3,4-diaminobenzenesulfonamide (6) (130 mg, 0.695 mmol, 1 equiv) and

bisulfite adduct of 4-chlorobenzaldehyde (254 mg, 1.043 mmol, 1.5 equiv) under the same

conditions that were applied to 4a. The crude product was purified by preparative LC, eluting

with a gradient of 0-50% Acetonitrile in H₂O to give 7b (107 mg, 50% yield) as a white powder.

Mp 313.4-315.3°C. ¹H NMR (400 MHz, DMSO-d₆): δ 7.25 (2H, s), 7.64 (2H, d, J = 8.8 Hz),

7.69 (1H, dd, J = 8.4, 1.6 Hz), 7.73 (1H, d, J = 8.4 Hz), 8.04 (1H, bs), 8.19 (2H, d, J = 8.8 Hz);

¹³C NMR (100 MHz, DMSO-d₆):  119.98, 128.31, 128.39, 129.09, 135.11, 138.23, 152.76;

HRMS m/z calculated for C₁₃H₁₀ClN₃O₂S [M+H]⁺308.0261, found: 308.0250.

4.1.7 2-(Pyridin-2-yl)-1H-benzo[d]imidazole-5-sulfonamide (7c)

Prepared from 3,4-diaminobenzenesulfonamide (6) (63 mg, 0.337 mmol, 1 equiv) and bisulfite

adduct of 2-pyridinecarboxaldehyde (106.7 mg, 0.505 mmol, 1.5 equiv) under the same

conditions that were applied to 4a. The crude product was purified by preparative LC, eluting

with a gradient of 0-50% Acetonitrile in H₂O to give 7c (28 mg, 30% yield ) as a white powder.

16

Mp 273.5-275°C. ¹H NMR (400 MHz, DMSO-d₆): δ 7.29 (2H, s), 7.53-7.56(1H, m), 7.69-7.74

(2H, m), 7.99-7.03 (1H, m), 8.07 (1H, s), 8.34 (1H, d, J = 7.6 Hz), 8.75 (1H, d, J = 4.4 Hz),

13

13.51 (1H, bs); C NMR (100 MHz, DMSO-d₆): 120.24, 121.84, 125.30, 137.74, 138.47,

147.83, 149.52, 153.30; HRMS m/z calculated for C₁₄H₁₄N₄O₄S₂[M+H]⁺275.0603, found:

275.0592.

4.1.8 4-(1-Methyl-1H-benzo[d]imidazol-2-yl)benzenesulfonamide (10)

Step1: DIEA (627.3 µL, 3.624 mmol, 2 equiv) and N¹-methylbenzene-1,2-diamine (437.85 mg,

1.993 mmol, 1.1 equiv) were added to the solution of N¹-methylbenzene-1,2-diamine (9) (222.3

mg, 1.812 mmol, 1 equiv) in THF. It was stirred at rt under N₂for 24h and upon completion of

the reaction it was evaporated. Reaction mixture was diluted with water and extracted with

ethyl acetate. Organic layer was dried over Na₂SO₄, filtered and evaporated. The crude product

was purified by flash chromatography on silica gel (24 g), eluting with a gradient of 0-4.8%

DCM in MeOH to give N-(2-(methylamino)phenyl)-4-sulfamoylbenzamide) (385 mg, 70%

yield). Mp 258.4-259.9 °C. HRMS m/z calculated for C₁₄H₁₅N₃O₃S [M+H]⁺306.0912, found:

306.0912.

Step 2: Prepared from N-(2-(methylamino)phenyl)-4-sulfamoylbenzamide (306.6 mg, 1.004

mmol) under the same conditions that were applied to 4d. The crude product was purified by

preparative LC, eluting with a gradient of 0-56% Acetonitrile in H₂O to give 10 (134 mg,

1

46.5% yield) as a white powder. Mp 281-283.6 ˚C. H NMR (400 MHz, DMSO-d₆): δ 3.91

(3H, s), 7.27 (1H, t, J = 7.2 Hz), 7.32 (1H, t, J = 7.2 Hz), 7.54 (2H, s), 7.64 (1H, d, J = 7.8 Hz),

13

7.71 (1H, d, J = 7.8 Hz), 8.01 (2H, d, J = 8.2 Hz), 8.07 (2H, d, J = 8.2 Hz); C NMR (100

MHz, DMSO-d6):31.68, 110.67, 119.12, 122.12, 122.71, 125.84, 129.74, 133.13, 136.61,

142.32, 144.71, 151.50; HRMS [M+H]⁺m/z calculated for C₁₄H₁₃N₃O₂S 288.0807, found:

288.0809.

17

4.1.9 N-Hydroxy-1-methyl-2-phenyl-1H-benzo[d]imidazole-5-carboxamide (15a)

Step 1: Prepared from methyl 3-amino-4-(methylamino)benzoate (13) (403 mg, 12.236 mmol,

1 equiv) and bisulfite adduct of benzaldehyde (701.112 mg, 3.355 mmol, 1.5 equiv) under the

same conditions that were applied to 4a. The obtained methyl 1-methyl-2-phenyl-1H-

benzo[d]imidazole-5-carboxylate (14a) (CAS: 1497331-60-8) (466 mg, 78% yield) was used

1

in the next step without purification. Mp 152.4-154.1°C. H NMR (400 MHz, DMSO-d₆): δ

3.90 (s, 3H), 3.93 (s, 3H), 7.60–7.62 (m, 3H), 7.76 (d, J = 8.4 Hz, 1H), 7.88-7.90 (m , 2H), 7.95

(dd, J = 8.4, 1.6 Hz, 1H), 8.29 (d, J = 1.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 32.47,

52.52, 111.29, 121.12, 123.87, 123.96, 129.21, 129.89, 130.01, 130.55, 140.36, 142.46, 155.63,

167.24. HRMS m/z calculated for C₁₆H₁₄N₂O₂[M+H]⁺267.1134, found: 267.1135.

Step 2: A mixture 14a (150 mg, 0.563 mmol) and hydroxylamine solution (50 wt. % in H₂O,

3mL) in DMF (3 mL) was stirred at rt for 24h. Reaction mixture was diluted with ice water and

pH was adjusted to 5 with 6 N HCl. The formed precipitate was filtered and dried. The crude

product was purified by preparative LC, eluting with a gradient of 0-46% Acetonitrile in H₂O

to give 15a (58 mg, 39% yield) as a white powder. Mp 281.5-283°C. ¹H NMR (400 MHz,

DMSO-d₆): δ 3.87 (3H, s), 7.56-7.57 (3H, m), 7.65 (1H, d, J = 8.8 Hz), 7.75 (1H, d, J = 8.8

Hz), 7.83-7.85 (2H, m), 8.10 (1H, s), 8.91 (1H, bs), 11.17 (1H, bs); ¹³C NMR (100 MHz,

DMSO-d₆):  31.75, 110.28, 117.78, 121.49, 126.66, 128.59, 129.24, 129.72, 129.78, 138.39,

141.85, 154.42, 164.81; HRMS m/z calculated for C₁₅H₁₃N₃O₂[M+H]⁺268.1086, found:

268.1094.

4.1.10 2-(4-Chlorophenyl)-N-hydroxy-1-methyl-1H-benzo[d]imidazole-5-carboxamide

(15b)

Step 1: Prepared from methyl 3-amino-4-(methylamino)benzoate (13) (297.6 mg, 1.65 mmol,

1 equiv) and bisulfite adduct of 4-chlorobenzaldehyde (606 mg, 2.477 mmol, 1.5 equiv) under

18

the same conditions that were applied to 4a. The obtained methyl 2-(4-chlorophenyl)-1-methyl-

1H-benzo[d]imidazole-5-carboxylate (14b) (490 mg, 98% yield) was used in the next step

without purification. Mp 154.6-156.1°C. ¹H NMR (400 MHz, DMSO-d₆): δ 3.90 (s, 3H), 3.93

(s, 3H), 7.68 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 1H), 7.92-7.97 (m, 3H), 8.29 (d, J = 1.6

13

Hz, 1H). C NMR (100 MHz, DMSO-d₆): δ 32.49, 52.54, 111.39, 121.19, 124.02, 124.08,

128.88 129.32, 131.69, 135.48, 140.38, 142.39, 154.53, 167.20. HRMS m/z calculated for

C₁₆H₁₃N₂O₂Cl [M+H]⁺301.0744, found: 301.0743.

Step 2: Prepared from 14b (77 mg, 0.256 mmol) and hydroxylamine solution (50 wt. % in H₂O,

3mL) under the same conditions applied to 15a. The crude product was purified by preparative

LC, eluting with a gradient of 0-46% Acetonitrile in H₂O to give 15b (30 mg, 39% yield) as a

white powder. Mp 199-201°C. ¹H NMR (400 MHz, DMSO-d₆): δ 3.88 (3H, s), 7.62 (2H, d, J

= 8.4 Hz), 7.66 (1H, d, J = 8.4 Hz), 7.75 (1H, d, J = 8.4 Hz), 7.88 (2H, d, J = 8.4 Hz), 8.10 (1H,

s), 8.91 (1H, bs), 11.17 (1H, bs); ¹³C NMR (100 MHz, DMSO-d₆):  31.77, 110.36, 117.84,

121.65, 126.77, 128.56, 128.69, 131.01, 134.74, 138.41, 141.77, 153.27, 164.73; HRMS m/z

calculated for C₁₅H₁₂ClN₃O₂[M+H]⁺302.0696, found: 302.0708.

4.1.11 1-Methyl-2-phenyl-1H-benzo[d]imidazole-5-carboxylic acid (16a)

The mixture of methyl 1-methyl-2-phenyl-1H-benzo[d]imidazole-5-carboxylate (14a) (120

mg, 0.451 mmol, 1 equiv) and LiOH.H₂O (56.73 mg, 1.352 mmol, 3 equiv) in THF: H₂O (3

mL : 3 mL) was stirred at 60˚C overnight. THF was evaporated and reaction mixture was diluted

with water and acidified with 6 N HCl, the precipitated solid was filtered off under vacuum and

dried. These solid was crystallized in methanol and ethyl acetate to give 16a (80 mg, 70%

yield) as white crystals. Mp 295-296 °C. ¹H NMR (400 MHz, DMSO-d₆): δ 3.90 (3H, s), 7.56-

7.60 (3H, m), 7.70 (1H, d, J = 8.4 Hz), 7.85-7.88 (2H, m), 7.92 (1H, dd, J = 8.4, 1.6 Hz), 8.27

(1H, d, J = 1.6 Hz); ¹³C NMR (100 MHz, DMSO- d₆):  31.95, 110.52, 120.79, 123.67, 124.69,

19

128.72, 129.38, 129.63, 130.01, 139.66, 141.98, 154.94, 167,88; HRMS m/z calculated for

C₁₅H₁₂N₂O₂[M+H]⁺253.0977, found: 253.0970.

4.1.12 2-(4-Chlorophenyl)-1-methyl-1H-benzo[d]imidazole-5-carboxylic acid (16b)

Prepared from methyl 2-(4-chlorophenyl)-1-methyl-1H-benzo[d]imidazole-5-carboxylate

(14b) (120 mg, 0.399 mmol, 1 equiv) under the same conditions that applied to 16a. The crude

product was washed with hot methanol:THF mixture to give 16b (75 mg, 65% yield) as a white

powder. Mp: 301.1-302.3°C. 1H NMR (400 MHz, DMSO- d₆): δ 3.88 (3H, s), 7.62 (2H, d, J =

8.4 Hz), 7.68 (1H, d, J = 8.0 Hz), 7.87-7.92 (3H, m), 8.25 (1H, s); ¹³C NMR (100 MHz, DMSO-

d₆):  31.86, 110.46, 120.78, 123.71, 124.77, 128.44, 128.72, 131.06, 134.87, 139.60, 141.86,

153.73, 167.71; HRMS m/z calculated for C₁₅H₁₁ClN₂O₂[M+H]+ 287.0587, found: 287.0582.

4.1.13 1-Methyl-2-phenyl-1H-benzo[d]imidazole-5-carboxamide (17a)

The mixture of 1-methyl-2-phenyl-1H-benzo[d]imidazole-5-carboxylic acid (16a) (80.97mg,

0.321 mmol) in SOCl₂(2 mL) was refluxed for 4 h. The reaction mixture was then concentrated

in vacuo, and to the resulting residue was added ammonia solution (7N in MeOH, 2 mL) and

stirred at rt under N₂for 3h. The reaction mixture was evaporated, diluted with ice water and

precipitated solid was filtered and dried. The crude product was purified by preparative LC,

eluting with a gradient of 0-48% Acetonitrile in H₂O to give 17a (40 mg, 50% yield) as a white

powder. Mp 254-255.6°C. ¹H NMR (400 MHz, DMSO-d₆): δ 3.89 (3H, s), 7.28 (1H, bs), 7.57-

7.61 (3H, m), 7.66 (1H, d, J = 8.8 Hz), 7.85-7.89 (3H, m), 8.00 (1H, bs), 8.27 (1H, s); ¹³C NMR

(100 MHz, DMSO-d₆):  31.85, 110.05, 118.64, 122.24, 128.28, 128.67, 129.32, 129.81,

129.84, 138.55, 141.94, 154.42, 168.30; HRMS m/z calculated for C₁₅H₁₃N₃O [M+H]⁺

252.1137, found: 252.1125.

20

4.1.14 2-(4-Chlorophenyl)-1-methyl-1H-benzo[d]imidazole-5-carboxamide (17b)

Prepared from 2-(4-chlorophenyl)-1-methyl-1H-benzo[d]imidazole-5-carboxylic acid (16b)

(113.8 mg, 0.397 mmol) under the same conditions that were applied to 17a. The crude product

was purified by preparative LC, eluting with a gradient of 0-48% Acetonitrile in H₂O to give

17b (58 mg, 51% yield) as a white powder. Mp 296-297.4°C. ¹H NMR (400 MHz, DMSO-d₆):

δ 3.90 (3H, s), 7.29 (1H, bs), 7.64-7.68 (3H, m), 7.87-7.91 (3H, m), 8.00 (1H, bs), 8.27 (1H, s);

¹³C NMR (100 MHz, DMSO-d₆):  31.87, 110.14, 118.71, 122.40, 128.40, 128.66, 128.77,

131.10, 134.78, 138.57, 141.86, 153.29, 168.24; HRMS m/z calculated for C₁₅H₁₂ClN₃O

[M+H]⁺286.0747, found: 286.0750.

4.1.15 (1-Methyl-2-phenyl-1H-benzo[d]imidazol-5-yl)boronic acid (22a)

Step 1: 5-bromo-1-methyl-2-phenyl-1H-benzo[d]imidazole (20a) (556.2 mg, 1.937 mmol, 1

equiv), KOAc (950.5 mg, 9.685 mmol, 5 equiv) and Pin₂B₂(737.8 mg, 2.905 mmol, 1.5 equiv)

were dissolved in dioxane and degassed via N₂for 15 minutes. Then PdCl₂(dppf) (354.3 mg,

0.484 mmol, 0.25 equiv) was added onto that mixture and degassed for another 10 minutes and

it was refluxed for 5 h. The reaction mixture was partioned between EA and water, filtered from

Celite pad. The phases was seperated and aqueus phase extracted with EA. The combined

organic phase was dried, filtered and concentrated to give 1-methyl-2-phenyl-5-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (21a) was used in the next step

without purification.

Step 2: 21a (1 equiv) dissolved in THF: H₂O (4:1), NaIO₄(3 equiv) was added and stirred at rt

for 30 minutes. 1N HCl (200 µL) was added and reaction mixture stirred at rt for 6 h. Upon

completion THF was evaporated, the residue was extracted with ethyl acetate. Organic layer

was dried, filtered and evaporated. The crude product was purified by preparative LC, eluting

with a gradient of 0-50% Acetonitrile in H₂O to give 22a (33.6 mg, 7.5% yield) as a white

21

powder. Mp 225.1-227.3°C. ¹H NMR (400 MHz, DMSO-d₆): δ 3.85 (3H, s), 7.53-7.58 (4H,

m), 7.73 (1H, d, J = 8.4 Hz), 7.82-7.84 (2H, m), 7.96 (2H, s), 8.16 (1H, s); ¹³C NMR (100 MHz,

DMSO-d₆):  31.62, 109.44, 125.45, 128.33, 128.62, 129.28, 129.59, 130.16, 138.11, 142.20,

153.01; HRMS m/z calculated for C₁₄H₁₃BN₂O₂[M+H]⁺252.1185 , found 252.1184.

4.1.16 (2-(4-Chlorophenyl)-1-methyl-1H-benzo[d]imidazol-5-yl)boronic acid (22b)

Step 1: Prepared from 5-bromo-2-(4-chlorophenyl)-1-methyl-1H-benzo[d]imidazole (20b)

(300 mg, 0.9331 mmol) under the same conditions that were applied to 21a. The crude 2-(4-

chlorophenyl)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo

[d]imidazole (21b) was used in the next step without purification.

Step 2: Prepared from 21b under the same conditions applied to step 2 of 22a. The crude

product was purified by preparative LC, eluting with a gradient of 0-50% Acetonitrile in H₂O

to give 22b (11.8 mg, 4.4% yield) as a white powder. Mp 225-226.3°C. ¹H NMR (400 MHz,

DMSO-d₆): δ 3.86 (3H, s), 7.55 (1H, d, J = 8.0 Hz), 7.63 (2H, d, J = 8.2 Hz), 7.74 (1H, d, J =

8.0 Hz), 7.87 (2H, d, J = 8.2 Hz), 7.97 (2H, s), 8.15 (1H, s); ¹³C NMR (100 MHz, DMSO-d₆):

 31.68, 109.60, 125.35, 128.58, 128.74, 131.094, 134.60, 138.03, 141.79, 151.78; HRMS m/z

calculated for C₁₄H₁₂BClN₂O₂[M+H]⁺287.0759, found 287.0749.

4.1.17 (4-(1-Methyl-1H-benzo[d]imidazol-2-yl)phenyl)boronic acid (26)

Step 1: Prepared from 2-(4-bromophenyl)-1-methyl-1H-benzo[d]imidazole (24) (530 mg,

1.846 mmol) under the same conditions that were applied to 21a. The obtained 1-methyl-2-(4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-benzo[d]imidazole (25) was used in

the next step without purification.

Step 2: Prepared from 25 under the same conditions applied to step 2 of 22a. The crude product

was purified by preparative LC, eluting with a gradient of 0-50% Acetonitrile in H₂O to give

22

1

26 (35.4 mg, 7.6% yield) as a white powder. Mp 211-213.2°C. H NMR (400 MHz, DMSO-

d₆): δ 3.87 (3H, s), 7.23 (1H, t, J = 7.6 Hz), 7.28 (1H, t, J = 7.6 Hz), 7.60 (1H, d, J = 7.6 Hz),

7.67 (1H, d, J = 7.6 Hz), 7.80 (2H, d, J = 7.6 Hz), 7.96 (2H, d, J = 7.6 Hz), 8.22 (2H, s); ¹³C

NMR (100 MHz, DMSO-d₆):  31.68, 110.54, 118.97, 121.93, 122.37, 128.20, 131.45, 134.21,

136.60, 142.46, 153.02; HRMS m/z calculated for C₁₄H₁₃BN₂O₂[M+H]⁺252.1185, found

252.1176.

4.2 Carbonic anhydrase I, II, IX, and XII inhibition studies

An Applied Photophysics stopped-flow instrument was utilized for assaying the CO₂

hydration activity catalyzed by CA. Phenol red (0.2 mM) was used as an indicator, working at

the absorbance maximum of 557 nm, with 20 mM Hepes (pH = 7.5) as buffer, and 20 mM

Na₂SO₄(for maintaining constant the ionic strength), following the initial rates of the CA-

catalyzed CO₂hydration reaction for a period of 10–100 s. The concentration range of CO₂

from 1.7 to 17 mM to determine the kinetic parameters as well as inhibition constants. For each

inhibitor at least six traces of the initial 5–10% of the reaction were used for determination of

the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted

from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-

deionized water and serial dilutions up to 0.01 nM were completed with the assay buffer.

Inhibitor and enzyme solutions were preincubated for 15 min at room temperature for allowing

for the E–I complex formation. The inhibition percentages were determined by the PRISM 3,

as reported earlier, and represent the mean from at least three different determinations. All

recombinant CA isoforms were obtained in-house as reported previously [25-31].

Acknowledgement

We acknowledge the Turkish Academy of Sciences (TÜBA) for financial support.

23

Supporting Information

Experimental procedures for the synthesis of intermediate compounds 1-3, 5, 6, 8, 9, 11-13, 18-

20a-b, 23 and 24, NMR spectra and Graphical presentations of Stopped Flow Kinetic Assays

for the compounds tested can be found in Supporting Information.

Declaration of interest

The authors declare that they have no competing interests.

References

[1]

[2]

C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and

activators, Nat Rev Drug Discov 7(2) (2008) 168-81.

I. Nishimori, T. Minakuchi, S. Onishi, D. Vullo, A. Cecchi, A. Scozzafava, C.T.

Supuran, Carbonic anhydrase inhibitors: cloning, characterization, and inhibition

studies of the cytosolic isozyme III with sulfonamides, Bioorg Med Chem 15(23) (2007)

7229-36.

[3]

[4]

[5]

C.T. Supuran, A. Scozzafava, A. Casini, Carbonic anhydrase inhibitors, Med Res Rev

23(2) (2003) 146-89.

C.T. Supuran, Structure and function of carbonic anhydrases, Biochem J 473(14) (2016)

2023-32.

V. Alterio, A. Di Fiore, K. D'Ambrosio, C.T. Supuran, G. De Simone, Multiple binding

modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15

different isoforms?, Chem Rev 112(8) (2012) 4421-68.

[6]

[7]

C.T. Supuran, V. Alterio, A. Di Fiore, D.A. K, F. Carta, S.M. Monti, G. De Simone,

Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and cancer stem

cells: Three for the price of one, Med Res Rev 38(6) (2018) 1799-1836.

A. Waheed, W.S. Sly, Carbonic anhydrase XII functions in health and disease, Gene

623 (2017) 33-40.

24

[8]

[9]

D. Neri, C.T. Supuran, Interfering with pH regulation in tumours as a therapeutic

strategy, Nat Rev Drug Discov 10(10) (2011) 767-77.

J. Pastorek, S. Pastorekova, Hypoxia-induced carbonic anhydrase IX as a target for

cancer therapy: from biology to clinical use, Semin Cancer Biol 31 (2015) 52-64.

[10] O.O. Guler, G. De Simone, C.T. Supuran, Drug design studies of the novel antitumor

targets carbonic anhydrase IX and XII, Curr Med Chem 17(15) (2010) 1516-26.

[11] https://clinicaltrials.gov/ct2/show/NCT02215850.

[12] https://clinicaltrials.gov/ct2/show/NCT03450018.

[13] 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,

Bioorg Med Chem 22(1) (2014) 334-40.

[14] A. Di Fiore, A. Maresca, C.T. Supuran, G. De Simone, Hydroxamate represents a

versatile zinc binding group for the development of new carbonic anhydrase inhibitors,

Chem Commun (Camb) 48(70) (2012) 8838-40.

[15] V. Alterio, R. Cadoni, D. Esposito, D. Vullo, A.D. Fiore, S.M. Monti, A. Caporale, M.

Ruvo, M. Sechi, P. Dumy, C.T. Supuran, G. De Simone, J.Y. Winum, Benzoxaborole

as a new chemotype for carbonic anhydrase inhibition, Chem Commun (Camb) 52(80)

(2016) 11983-11986.

[16] C. Temperini, A. Innocenti, A. Scozzafava, C.T. Supuran, N-hydroxyurea--a versatile

zinc binding function in the design of metalloenzyme inhibitors, Bioorg Med Chem Lett

16(16) (2006) 4316-20.

[17] C.T. Supuran, Bortezomib inhibits mammalian carbonic anhydrases, Bioorg Med Chem

25(19) (2017) 5064-5067.

[18] C.T. Supuran, Advances in structure-based drug discovery of carbonic anhydrase

inhibitors, Expert Opin Drug Discov 12(1) (2017) 61-88.

[19] C.T. Supuran, Carbon- versus sulphur-based zinc binding groups for carbonic

anhydrase inhibitors?, J Enzyme Inhib Med Chem 33(1) (2018) 485-495.

[20] M. Mori, Y. Cau, G. Vignaroli, I. Laurenzana, A. Caivano, D. Vullo, C.T. Supuran, M.

Botta, Hit Recycling: Discovery of a Potent Carbonic Anhydrase Inhibitor by in Silico

Target Fishing, ACS Chem Biol 10(9) (2015) 1964-9.

[21] Y. Cau, D. Vullo, M. Mori, E. Dreassi, C.T. Supuran, M. Botta, Potent and Selective

Carboxylic Acid Inhibitors of Tumor-Associated Carbonic Anhydrases IX and XII,

Molecules 23(1) (2017).

25

[22] K. D'Ambrosio, S. Carradori, S.M. Monti, M. Buonanno, D. Secci, D. Vullo, C.T.

Supuran, G. De Simone, Out of the active site binding pocket for carbonic anhydrase

inhibitors, Chem Commun (Camb) 51(2) (2015) 302-5.

[23] L. Crocetti, A. Maresca, C. Temperini, R.A. Hall, A. Scozzafava, F.A. Muhlschlegel,

C.T. Supuran, A thiabendazole sulfonamide shows potent inhibitory activity against

mammalian and nematode alpha-carbonic anhydrases, Bioorg Med Chem Lett 19(5)

(2009) 1371-5.

[24] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-

flow kinetic studies on the native human isoenzymes B and C, J Biol Chem 246(8)

(1971) 2561-73.

[25] S. Bilginer, E. Unluer, H.I. Gul, E. Mete, S. Isik, D. Vullo, O. Ozensoy-Guler, S.

Beyaztas, C. Capasso, C.T. Supuran, Carbonic anhydrase inhibitors. Phenols

incorporating 2- or 3-pyridyl-ethenylcarbonyl and tertiary amine moieties strongly

inhibit Saccharomyces cerevisiae beta-carbonic anhydrase, J Enzyme Inhib Med Chem

29(4) (2014) 495-9.

[26] H.I. Gul, E. Mete, S.E. Eren, H. Sakagami, C. Yamali, C.T. Supuran, Designing,

synthesis and bioactivities of 4-[3-(4-hydroxyphenyl)-5-aryl-4,5-dihydro-pyrazol-1-

yl]benzenesulfonamides, J Enzyme Inhib Med Chem 32(1) (2017) 169-175.

[27] H.I. Gul, M. Tugrak, H. Sakagami, P. Taslimi, I. Gulcin, C.T. Supuran, Synthesis and

bioactivity studies on new 4-(3-(4-Substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-

c]pyrazol-2-yl) benzenesulfonamides, J Enzyme Inhib Med Chem 31(6) (2016) 1619-

24.

[28] H.I. Gul, C. Yamali, H. Sakagami, A. Angeli, J. Leitans, A. Kazaks, K. Tars, D.O.

Ozgun, C.T. Supuran, New anticancer drug candidates sulfonamides as selective hCA

IX or hCA XII inhibitors, Bioorg Chem 77 (2018) 411-419.

[29] E. Unluer, H.I. Gul, A. Demirtas, H. Sakagami, N. Umemura, M. Tanc, C. Kazaz, C.T.

Supuran, Synthesis and bioactivity studies of 1-aryl-3-(2-hydroxyethylthio)-1-

propanones, J Enzyme Inhib Med Chem 31(sup3) (2016) 105-109.

[30] C. Yamali, H.I. Gul, H. Sakagami, C.T. Supuran, Synthesis and bioactivities of halogen

bearing phenolic chalcones and their corresponding bis Mannich bases, J Enzyme Inhib

Med Chem 31(sup4) (2016) 125-131.

[31] C. Yamali, M. Tugrak, H.I. Gul, M. Tanc, C.T. Supuran, The inhibitory effects of

phenolic Mannich bases on carbonic anhydrase I and II isoenzymes, J Enzyme Inhib

Med Chem 31(6) (2016) 1678-81.

26

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be

considered as potential competing interests:

27

Highlights

. Synthesis of novel benzimidazole derivatives as carbonic anhydrase (CA) are reported.

. Compounds are evaluated against four CA isoforms (hCA I, II, IX, XII).

. 4c is the most potent and selective CA IX (K_I=6.6 nM) and XII (K_I=9.9 nM) inhibitor.

28

29

Article Doi

Benzimidazole derivatives as potent and isoform selective tumor-associated carbonic anhydrase IX/XII inhibitors

DOI: 10.1016/j.bioorg.2019.103544

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bioorg.2019.103544

Full text of DOI:10.1016/j.bioorg.2019.103544

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