Synthesis of coumarin-sulfonamide derivatives and determination of their cytotoxicity, carbonic anhydrase inhibitory and molecular docking studies

Synthesis of Coumarin-Sulfonamide Derivatives and Determination of their

Cytotoxicity, Carbonic Anhydrase Inhibitory and Molecular Docking

Studies

1

,

2

3

4

5

Belma Zengin Kurt *, Fatih Sonmez , Dilek Öztürk , Atilla Akdemir , Andrea Angeli ,

5

Claudiu T. Supuran

1

Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34093,

Istanbul, Turkey

2

Sakarya University of Applied Sciences, Pamukova Vocational School, 54055, Sakarya, Turkey

Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, 34093, Istanbul,

3

Turkey

4

Bezmialem Vakif University, Faculty of Pharmacy, Department of Pharmacology, Computer-aided

drug discovery laboratory, 34093, Istanbul, Turkey

5

Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di ScienzeFarmaceutiche e

Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy

*

Corresponding author: Belma Zengin Kurt. Phone: +902125232288-3249; e-mail:

bzengin@bezmialem.edu.tr

Abstract

Carbonic anhydrases (CA I, II, IX, and XII) are known to be highly expressed in various

human tissues and malignancies. CA IX is a prominent target for especially colorectal

cancers, because it is overexpressed in colorectal cancer and this overexpression leads poor

prognosis. Inhibition of CA IX activity by small molecule CA inhibitors like sulfonamides,

sulfonamide derivative or coumarins leads to inhibition of tumorigenesis. Novel twenty-seven

compounds in three series (sulfonamide-based imines (6a-6i), coumarin-based aldehydes (7a-

7

i), and coumarin-sulfonamide-based target molecules (8a-8i)) were synthesized and

characterized by means of IR, NMR, and mass spectra. All compounds were tested for their

ability to inhibit CA I, CA II, CA IX, and CA XII isoforms. 4-((((2-((1-(3-((2-oxo-2H-

chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1-yl)-

methylene)amino)methyl)benzenesulfonamide (8i) exhibited the highest hCA IX inhibition

with the Ki of 45.5ꢀnM. In addition, 8i was found to be potent in inhibiting cancer cell

proliferation as selective (IC = 17.01±1.35 µM for HT-29, IC = 118.73±1.19 µM for

5

0

50

HEK293T). This novel compound inhibited the CA IX and CA XII protein expression in HT-

9 cells. These findings indicate that 8i can inhibit cellular proliferation in human colon

2

cancer cells by specifically targeting the CA IX and CA XII expression.

Keywords: Coumarin, Sulfonamide, Cytotoxicity, Carbonic anhydrase, Enzyme inhibition,

Molecular Docking.

1

. Introduction

2

+

The carbonic anhydrases (CAs; EC 4.2.1.1) which include a metallic core of Zn ion at their

active centre are a superfamily of metalloenzymes that present in all organisms [1, 2]. The

most important task of carbonic anhydrase for metabolism is to catalyse the reversible

conversion of carbon dioxide into bicarbonate [3-6]. Sixteen different α-CA isoforms were

isolated from mammals, where they act crucial physiological roles. Some of them are

cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA

IX, CA XII, CA XIV and CA XV), CA VA and CA VB are mitochondrial, and CA VI is

secreted in saliva and milk [7, 8]. These CA isoforms have been shown to play an active role

in the mechanism of many diseases. In particular, the expression of the CA IX and CA XII

isoenzymes in hypoxic tumour cells has led to the selection of these enzymes as the target of

the anticancer compound designs by the researchers.

Sulfonamides are one of the most well-known groups of carbonic anhydrase inhibitors. These

compounds have a wide range of applications including diuretic, antiglaucoma, antiobesity,

antiepileptic and antitumor [9-14]. In addition to sulfonamides, coumarin and its derivatives

have been reported to show selective inhibition, particularly on CA IX and XII [15-19].

Coumarin and its bioisosteres (thiocoumarins, 2-thioxocoumarins) act as “prodrugs”, whereas

their hydrolysis products (formed through CA-mediated esterase activity) are the actually CA

inhibitors [20, 21]. Many studies have shown that 7-hydroxycoumarin, 6-nitro-7-

hydroxycoumarin, scopoletin and esculetin can be used in the treatment of cancer [22].

Coumarins have a variety of pharmacological effects and they are mostly used in anticancer

drug design and discovery [23]. Coumarins demonstrate different mechanisms of antitumor

activity at different stages of cancer formation. These mechanisms are; blockage of the cell

cycle, apoptosis of the cell, blockage of the estrogen receptor, or inhibition of DNA-related

enzymes such as topoisomerase [11].

This study aimed to investigate biological activities of novel synthesized compounds bearing

both coumarin and sulfonamide moieties as the effective CA inhibitors. Their effects on CA I,

CA II, CA IX, and CA XII isoforms and cytotoxic properties on the HT-29 cell line were

evaluated. Furthermore, molecular modelling studies were performed to suggest possible

modes of binding of these inhibitors inside the active sites of hCAs.

2

. Result and discussion

.1. Chemistry

The syntheses of the target compounds (8a-8i) are depicted in Scheme 1. Firstly, the

propargyl group was bonded to hydroxyl groups of various aldehydes, and they were attached

to coumarin by the click reactions. Then, the synthesis of target compounds containing both

coumarin and sulfonamide moieties were carried out by forming an imine bond between the

aldehyde fragment of the coumarin derivatives and the aminomethyl moiety of

benzenesulfonamide.

Various hydroxy-aldehydes (4a-i) were reacted with propargyl bromide in DMF at room

temperature and then obtained 5a-i [24] were attached to 4-(aminomethyl)benzene

o

sulfonamide by forming imine group in an alkaline solution of methanol:chloroform at 60 C,

for the synthesis of first series (sulfonamide-based imines (6a-6i)) [25]. On the other hand, the

coumarin derivative (3) bearing propylazide were synthesized from 7-hyroxycoumarin (1)

reacting with 1,3-dibromopropane and sodium azide, respectively [18]. To obtain the second

series (coumarin-based aldehydes (7a-i)) [26], 5a-i and 3 were bonded through triazole-bridge

using CuSO .5H O as a catalyst. The synthesis of the third series (coumarin-sulfonamide-

4

2

based target molecules) (8a-i) was carried out by the reaction of coumarin derivatives (7a-i)

in the second series with 4-(aminomethyl)benzenesulfonamide.

1

13

All of the newly synthesized compounds were characterized by H NMR, C NMR, IR and

MS. According to the IR spectra of the synthesized 6a-i derivatives, it was possible to observe

-1

the absorption at about 3300 cm relating to NH stretch of sulfonamide groups. The CH

2

-1

stretch of the alkyne group present in the 7a-i shows an absorbance about 3150 cm , while

the stretch of the coumarin and aldehyde carbonyl moiety shows the absorbance between

-1

1

710 and 1740 cm . Additionally, relating to NH stretch of sulfonamide groups in the 8a-i

2

-1

have observed the absorption at about 3300 cm and the stretch of the coumarin carbonyl

-1

1

moiety between 1710 and 1700 cm . From the H NMR spectra; for the 6a-i; the signals for

aromatic protons were observed between 7.00 and 7.80 ppm, the signal of CH proton of imine

group was detected at about 8.40-82 ppm and NH proton signals observed about at 7.30-7.36

2

ppm. In addition, the signals of aliphatic protons were determined between 3.52-4.85 ppm.

For the 7a-i; the signals for aromatic protons were observed between 6.30 and 8.38 ppm, the

3

signal of CHO proton of aldehyde group was detected at about 9.90-10.40 ppm and aliphatic

proton signals observed about at 2.20-5.35 ppm. Similarly, for the 8a-i; the signals for

aromatic protons were observed between 6.20 and 8.40 ppm, the signal of CH proton of imine

group was detected at about 8.60-8.80 ppm and aliphatic proton signals observed about at

1

3

2

.20-5.35 ppm. From the C NMR spectra, the signals of the 160.0 and 165.5 ppm can be

seen for carbonyl of lactone groups, C atom of imine group and C atom of near the oxygen

atom. The signals of the aliphatic and aromatic carbons were observed at 13-66 ppm and 101-

1

56 ppm, respectively. In addition, the C atoms of the aldehyde group were observed at about

1

85-192 ppm.

1

13

H NMR, C NMR, and MS spectra of the synthesized compounds are given in

supplementary materials.

Scheme 1. Synthesis of new coumarin-sulfonamide derivatives. Reaction conditions: (i) 1,3-

dibromopropane, K CO , CH CN, reflux, 2h; (ii) NaN , DMF, rt, 16h; (iii) Propargyl bromide, DMF,

2

3

o

K CO , rt, 16h; (iv) 4-(aminomethyl)benzenesulfonamide, NaOH, MeOH:CHCl , 60 C, 18h; (v) 3,

CuSO .5H O, Sodium Ascorbate, THF:H O, 40 C, 2h; (vi) 4-(aminomethyl)benzenesulfonamide,

NaOH, EtOH, 80 C, 18h.

2

3

o

4

2

o

4

2

.2. CA inhibition

The CA inhibitory profiles of the synthesized compounds were evaluated by applying a

stopped flow carbon dioxide hydrase assay [27], in comparison to acetazolamide (AAZ) as a

standard CAI against four physiologically significant isoforms CA I, II, IX, and XII.

a) From the Table 1, the K values of the sulfonamide-based compounds 6a-i, the first

i

subseries, were obtained in the range of 15.9-7608 nM, 81.2-2028 nM and 659.3-7345

nM against hCA II, hCA IX and hCA XII, respectively. This subseries weakly

inhibited the cytosolic isoform hCA I (with K >10000 nM). Among them, 6e strongly

i

inhibited the cytosolic isoform hCA II (associated with glaucoma) with K of 15.9 nM,

i

which is close to that of AAZ (K of 12.1 nM). 6h had the best inhibitory activity

i

against hCA IX (tumor associated isoform) with K of 81.2 nM, whereas 6d had the

i

strongest inhibition against isoform hCA XII (associated with tumor) with K of 659.3

i

nM.

b) In terms of structure-activity relationship, 6a (K =826.3 nM), containing the O-

i

propyne group at 2- position of the phenyl ring, has shown almost 8-9-fold higher

inhibition of hCA II as compared to 6b (K =7608 nM) and 6c (K =6493 nM) (O-

i

propyne group at 3- and 4- positions, respectively). Furthermore, 6b (the O-propyne

group at 3- position) exhibited the weakest inhibitory activity against hCA II, hCA IX

and hCA XII with K of 7608 nM, 2028 nM and 2899 nM, respectively (comparing

i

with 6a and 6c). The presence of the methoxy group at 3- position (6d), the

diethylamine group at 4- position (6e) and the bromine at 5- position (6f) of the phenyl

ring of 6a increased dramatically the inhibitory activity against hCA II isoform (with

K of 23.7 nM, 15.9 nM and 35.4 nM for 6d, 6e and 6f, respectively), whereas the nitro

i

group at 5- position (6g) of the phenyl ring of 6a decreased almost 3-fold the hCA II

inhibition (with K of 2154 nM for 6g). Similarly, the presence of the methoxy group

i

at 3- position (6h) of the phenyl ring of 6c enhanced the inhibitory activity against

hCA II and hCA IX isoforms (with K of 438.7 nM and 81.2 nM, against hCA II and

i

hCA IX, respectively, for 6h). On the other hand, the presence of diethylamine group

at 4- position (6e), the bromine at 5- position (6f) and the nitro group at 5- position

(6g) of the phenyl ring of 6a rised strongly the inhibitory activity against hCA IX

isoform (with K of 197.9 nM, 189.8 nM and 211.8 nM for 6e, 6f and 6g,

i

respectively), whereas they reduced the hCA XII inhibition (with K of 921.9 nM,

i

7

345 nM and 5752 nM for 6e, 6f and 6g, respectively). Additionally, the presence of

naphthyl (6i) instead of phenyl ring of 6a didn’t have significant effect on hCA IX and

hCA XII inhibition, while it increased 2.5-fold the hCA II inhibition.

c) From the Table 2, the K values of the coumarin-based compounds, containing triazole

i

as a linker, 7a-i, the second subseries, were obtained in the range of 127.5-1166 nM,

and 60.9->10000 nM against hCA IX and hCA XII, respectively. This subseries

weakly inhibited the cytosolic isoforms hCA I and hCA II (with K >10000 nM).

i

Among them, 7a strongly inhibited the tumor-associated isoforms hCA IX and hCA

XII with K of 127.5 nM and 60.9 nM, respectively.

i

5

d) Regarding with structure-activity relationship, 7a, containing the aldehyde group at 2-

position of the phenyl ring, have shown higher inhibition of hCA IX as compared to

7

b (K =192 nM) and 7c (K =171.1 nM) (aldehyde group at 3- and 4- positions,

i i

respectively). Furthermore, the moving aldehyde group from 2- position to 3- and 4-

positions of the phenyl ring significantly decreased the inhibitory activity against hCA

XII (comparing 7a (K =60.9 nM) with 7b (K =8965 nM) and 7c (K >10000 nM)).

i

The presence of the methoxy group at 6- position (7d) of the phenyl ring of 7a

strongly reduced the inhibitions of both hCA IX and hCA XII (with K of 1166 nM

i

and >10000 nM, respectively, for 7d). Additionally, the presence of diethylamine

group at 5- position (7e), the bromine at 4- position (7f) and the nitro group at 4-

position (7g) of the phenyl ring of 7a decreased dramatically the inhibitory activity

against hCA XII isoform (with K of >10000 nM), whereas they didn’t have significant

i

effect on hCA IX inhibition. Similarly, the presence of naphthyl (7i) instead of phenyl

ring of 7a weakly reduced the inhibitory activity against hCA IX (with K of 189.5

i

nM, for 7i), while it extremely decreased the inhibitory activity against hCA XII (with

K of >10000 nM, for 7i).

i

e) From the Table 3, the K values of the both coumarin and sulfonamide-based

i

compounds, containing triazole and phenyl rings as the linker, 8a-i, the target series,

were obtained in the range of 230-790.6 nM, 45.5-2184 nM and 596.6-6538 nM

against hCA II, hCA IX and hCA XII, respectively. The cytosolic isoform hCA I was

weakly inhibited by this target compounds (with K >10000 nM). Among them, 8e

i

strongly inhibited the cytosolic isoform hCA II with Ki of 267.8 nM, while 8i had the

best inhibitory activity against tumor associated isoforms hCA IX and hCA XII with

K of 45.5 nM and 596.6 nM, respectively.

i

f) With regard to structure-activity relationship, the moving benzylideneamino-sulfonamide

moiety from 3- position to 2- or 4- positions of the phenyl ring decreased the inhibitory

activity against hCA II (comparing 8b (K =383.9 nM) with 8a (K =503.4 nM) and 8c (K

i

=

651.4 nM)) and hCA XII (comparing 8b (K =879.2 nM) with 8a (K =932.7 nM) and 8c (K

i i

=2725 nM)). The hCA IX isoform was the weakest inhibited by 8b (benzylideneamino-

sulfonamide moiety at 3- position of the phenyl ring) with K of 955.3 nM as compared to 8a

i

(

K =187.5 nM) and 8c (K =169.2 nM) (benzylideneamino-sulfonamide moiety at 2- and 4-

i

positions, respectively). The presence of the methoxy group at 6- position (8d) of the phenyl

ring of 8a strongly decreased the inhibitions of both hCA IX and hCA XII (with K of 1479

i

nM and 6538 nM, respectively, for 8d). Additionally, the presence of diethylamine group at

5

- position (8e) and the nitro group at 4- position (8g) of the phenyl ring of 8a decreased

dramatically the inhibitory activity against hCA IX isoform (with K of 2184 nM and 856.7

i

nM, respectively), whereas they didn’t have significant effects on hCA II and hCA XII

inhibitions. Similarly, the presence of bromine group at 4- position (8f) of the phenyl ring

didn’t have major effects on hCA II, hCA IX and hCA XII inhibitions. The presence of

naphthyl (8i) instead of phenyl ring of 8a increased 2- or 3-fold the inhibitory activity against

hCA II, hCA IX and hCA XII inhibitions (with K of 230 nM, 45.5 nM and 569.6 nM,

i

respectively, for 8i).

6

Table 1. In vitro inhibition K values (nM) of 6a-i for the hCA I, II, IX and XII.

i

K (nM)*

i

S.I.*

hCA IX/II)

S.I.

(hCA XII/II)

Compound

hCA I hCA II hCA IX hCA XII

(

>

10000

826.3

7608

1068

2028

900.8

2899

1.29

1.09

0.38

0.266

0.035

>

10000

6493

230.5

888.3

0.136

>

10000

23.7

15.9

35.4

1186

197.9

189.8

659.3

921.9

7345

50.04

12.44

5.36

27.81

59.98

207.48

>

10000

2154

211.8

81.2

5752

2942

0.098

0.185

2.67

6.70

438.7

>

10000

307.7

12.1

1088

25.8

860.7

5.7

3.54

2.13

2.79

0.47

AAZ

250.0

*

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

values). S.I.: Selectivity Index

7

Table 2. In vitro inhibition K values (nM) of 7a-i for the hCA I, II, IX and XII.

i

K (nM)*

i

Compound

hCA I

hCAII

hCA IX

hCA XII

>

10000

>10000

127.5

60.9

>

>10000

192.0

171.1

8965

>10000

>

10000

>10000

1166

>10000

>

10000

>10000

157.9

195.9

>10000

O

H

N

O

N

Br

N

O

7

f

>

10000

>10000

136.6

132.7

>10000

12.1

189.5

25.8

>10000

5.7

AAZ

250.0

*

1

Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-

0 % of the reported values).

8

Table 3. In vitro inhibition K values (nM) of 8a-i for the hCA I, II, IX and XII.

i

K (nM)*

i

S.I.

Compound

hCA I hCA II hCA IX hCA XII

(

hCA IX/II) (hCA XII/II)

>

10000

503.4

383.9

187.5

955.3

932.7

879.2

0.375

2.49

1.85

2.29

>

10000

651.4

388.5

169.2

1479

2725

6538

0.26

3.81

4.18

SO2NH2

N

O

N

16.82

O

8d

SO2NH2

N

O

N

>

10000

267.8

790.6

2184

837.0

918.7

8.16

3.125

1.16

O

N

8e

160.0

0.202

>

10000

466.3

439.8

856.7

760.3

891.9

808.9

1.84

1.73

1.912

1.84

230.0

12.1

45.5

25.8

569.6

5.7

0.197

2.13

2.48

0.47

AAZ

250.0

*

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

values). S.I.: Selectivity Index

9

2

.3 Molecular modelling studies

Compounds 6a – 8i have been tested in enzyme inhibition assays against hCA I, II, IX and

XII (Tables 1 - 3). None of the compounds showed K values in the 0 – 100 nM range for hCA

i

I. For hCA II, hCA IX and hCA XII, the number of compounds with K values in the 0 – 100

i

nM range are 3 (compounds 6d, 6e and 6f), 2 (compounds 6h and 8i) and 1 (compound 7a),

respectively. Interestingly, these compounds inhibited hCA II, IX or XII selectively (Tables 1-

3

).

Docking studies were performed to investigate the possible binding interactions between the

selective inhibitors and the hCA active site. As not all compounds contain a sulfonamide

group, we assumed that besides direct interactions between the ligand and the zinc ion the

ligands could also interact with a zinc-bound water molecule. Therefore, the docking studies

2

+

were performed with either a zinc-bound water molecule or a free Zn -ion [28].

Binding interactions of compounds 6d, 6e and 6f with hCA II

Compound 6e shows the lowest measured K value for hCA II. The sulfonamide group

i

2

+

interacts with the Zn -ion and Thr199 and Thr200 (Figure 1). The phenyl group adjacent to

the sulfonamide moiety forms hydrophobic interactions with the side chains of His94 and

Leu198. The nitrogen atom between the two phenyl groups of the ligand forms a hydrogen

bond with the side chain of Gln92. Hydrophobic interactions are formed with the side chains

of Ile91 and Phe131. Compounds 6d and 6f form similar binding interactions with the hCA II

active site. No poses have been identified for these compounds in which the ligand forms an

interaction with a zinc-bound water molecule. Many compounds of series 6a – 6i show a

similar docked pose as described for compound 6e (Figure 1). As such, the difference in the

measured K values for these compounds cannot be explained by docking studies alone.

i

1

0

2

+

Figure 1. The docked pose of compound 6e (turquoise) in the active site of hCA II. The Zn -ion is

2

+

indicated with a turquoise sphere. Hydrogen bonds and interaction with the Zn -ion are indicated in

dashed red lines. The active site surface is indicated with a gray mesh.

Binding interactions of compounds 6h and 8i with hCA IX

For compound 6h, docked poses have been obtained with either a direct interaction with the

2

+

Zn -ion and with the zinc-bound water molecule. For the first poses, the sulfonamide tail of

2

+

compound 6h interacts with the Zn -ion and Thr199, while its phenyl group forms

hydrophobic interactions with the side chain of Leu198 (Figure 2A). The rest of the molecule

is flexible and adopts different conformations and forms hydrophobic interactions with

Leu98, Val131, Leu135 or Pro202. No hydrogen bonds are formed between ligand and active

site residues.

Another docked pose has been obtained for compound 6h in which the sulfonamide group

forms hydrogen bonds with the zinc-bound water molecule, Gln92 and Thr200 (Figure 2B).

In addition, a hydrogen bond is formed with Trp5. Hydrophobic interactions are formed with

Ser3 and Pro202. Similar docked poses have been obtained for the other members of

compound series 6.

2

+

For compound 8i only one docked pose with a direct interaction with the Zn -ion has been

obtained for the coumarin-closed conformation. The sulfonamide tail is able to form

2

+

interactions with the Zn -ion and Thr199 (Figure 3). Only one weak hydrogen bond may be

formed between the backbone Nitrogen atom of Val131 and the ether oxygen of the ligand.

Hydrophobic interactions are formed with Leu91, Val131 and Pro202.

1

2

+

Figure 2. The docked poses of compound 6h forming direct binding interactions to the Zn -ion (panel

A, purple and green) or to the zinc-bound water molecule (panel B; purple) in the active site of hCA

2+

IX. The Zn -ion is indicated with a turquoise sphere and the water molecule oxygen atom is indicated

2

+

with a red sphere. Hydrogen bonds and interaction with the Zn -ion are indicated in dashed red lines.

The active site surface is indicated with a gray mesh.

Figure 3. The docked pose of compound 8i (turquoise) in the closed coumarin form in the active site

2

+

of hCA IX. The Zn -ion is indicated with a turquoise sphere. Hydrogen bonds and interaction with the

2

+

Zn -ion are indicated in dashed red lines. The active site surface is indicated with a gray mesh.

Binding interactions of compound 7a with hCA XII

For compound 7a, only one docked pose has been obtained for the open coumarin form in

2

+

which the ligand’s carboxylic acid moiety forms a direct interaction with the Zn -ion and

1

2

Thr199 of the hCA XII active site (Figure 4). The terminal carbonyl group of the ligand

forms hydrogen bonds with Ser132, while the triazole moiety forms a hydrogen bond with

Gln92. The phenyl group of the ligand forms hydrophobic interactions with Leu198.

Figure 4. The docked pose of compound 7a (turquoise) in the open coumarin form in the hCA XII

2

+

active site. The Zn -ion is indicated with a turquoise sphere. Hydrogen bonds and interaction with the

2

+

Zn -ion are indicated in dashed red lines. The active site surface is indicated with a gray mesh.

Understanding the low K values of the tested ligands.

I

Many of the analogs were able to bind in a similar way as described in Figures 1 - 4. As such,

the difference in the measured K values for these compounds cannot be explained by docking

i

studies alone. The inaccuracy in the calculation of the ligand-protein binding energies with

molecular mechanics based force fields as well as induced-fit effects that are not included in

these docking protocols are probably amongst the reason for this. In addition, the

-

deprotonation efficiency of the sulfonamide group to yield the R-SO NH group as well as the

2

efficiency in the opening of the coumarin rings are important as they influence the shape and

-

2+

binding characteristics of the molecule (e.g., R-SO NH is required for binding to the Zn -

2

ion). Finally, our docking protocols focussed on inhibitors binding to the active site either

2

+

directly to the Zn -ion or to the zinc-bound water molecule. However, inhibitors may also

bind to allosteric sites of the enzymes, which were not included in this study. Nevertheless,

1

3

possible binding interactions between these inhibitors and the hCA active sites have been

suggested.

2

.4 Biological activities

Cell viability against normal and cancer cell lines

Cell viability assay was carried out using human colorectal adenocarcinoma cell line HT-29,

HT-29 is CA IX and CA XII positive cell line which they are overexpressed under hypoxic

conditions [18]. HT-29 cells were treated with the synthesized compounds for 24 hours and 3-

(

4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to

determine the viability of cells. We have used two reference compounds, namely doxorubicin

dox) and acetazolamide (aaz). GraphPad Prism software (GraphPad Software, San Diego,

CA, USA) was used to calculate the median inhibition concentration (IC ) for all compounds.

(

5

0

As shown in Table 4, the nine most active compounds 6c, 7a, 7d, 7e, 7f, 8a, 8e, 8f and 8i

show potent inhibition of cancer cells growth with IC₅₀ranging from 9.03ꢀ±ꢀ1.33 to

2

3.34ꢀ±ꢀ1.51ꢀµM compared to doxorubicin (IC₅₀value of 5.38ꢀ±ꢀ1.40ꢀµM). Compound 7e

possessed highest anticancer activity among all new hybrids against cancer cell growth with

IC 9.03ꢀ±ꢀ1.33ꢀµM for the HT-29 colorectal adenocarcinoma cell line. It is non-selective as it

5

0

also shows potent cytotoxicity on HEK293T cells. According to these results, especially

coumarin-aldehyde (7a-7i) and coumarin-sulfonamide (8a-8i), based derivatives showed

better cytotoxicity. All other compounds showed low activity against the growth of cancer

cell line. 7a and 8i which showed high activity in enzyme inhibition showed similarly high

cytotoxicity. It has also been found that compounds exhibiting enzyme inhibition close to

these compounds also exhibit good cytotoxicity. The 11 compounds selected were also tested

on healthy cell lines considering both enzyme inhibition values and cell viability values of the

compounds. These compounds are 6c, 6d, 6h, 7a, 7d, 7e, 7f, 8a, 8e, 8f and 8i. HEK293T

(embryonic kidney) cell line was used for this purpose. HEK293T cells were treated with

selected compounds for 24 hours and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay was used to determine the viability of cells.

1

4

Table 4. IC values calculated from viability at HT-29 cell line results of synthesized molecules

50

IC (µM)

50

5

0

Comp.

HT-29

Comp.

7g

HT-29

6

a

31.04 ± 1.18

103.82 ± 1.54

14.11 ± 1.43

81.28 ± 1.57

62.78 ± 1.45

45.68 ± 1.25

34.83 ± 1.35

136.1 ± 2.10

46.08 ± 1.33

18.89 ± 1.33

129.3 ± 1.83

100.3 ± 1.56

13.3 ± 1.24

9.03 ± 1.33

7.47 ± 1.43

53.63 ± 1.53

65.36 ± 1.90

149.48 ± 2.57

14.29 ± 1.31

106.9 ± 2.82

125.4 ± 1.31

52.46 ± 1.46

23.34 ± 1.51

17.7 ± 1.33

117 ± 4.60

6

b

7h

7i

6

c

6

d

8a

6

e

8b

8c

6

f

6

g

8d

8e

6

h

6

i

8f

7

a

8g

7

b

8h

8i

31.55 ± 1.68

17.01 ± 1.35

7

c

7

d

AAZ

Dox

53.78 ± 1.75

5.38 ± 1.40

7

e

7

f

The cell viability was represented as a percentage (%) relative to untreated

cells as a control.

The results obtained are given in Table 5. All of the compounds selected according to these

results showed lower cytotoxicity than doxorubicin. In particular, it is seen that 8i does not

nearly decrease viability (IC value of 118.73ꢀ±ꢀ1.19ꢀµM) of healthy cells. Also, sulfonamide

5

0

based derivatives (6c, 6d, 6h) allowed healthy cells to maintain viability (IC value ranging

50

from 135.07ꢀ±ꢀ1.29 to 537.04 ± 1.31 µM). 6h and 8i show selective cytotoxicity on HT-29,

while 7a displays cytotoxicity both on HT-29 and HEK293T as non-selective. Compounds

6

a and especially 7d indicate cytotoxicity on HT-29, while they do not seem to potently

inhibit hCAs. It is thought that the cytotoxic effect is carried out through different

mechanisms. 7a and 8i were selected for western blotting studies according to the results of

enzyme inhibition, cell viability analysis on healthy and cancer cells and modelling studies.

1

5

Table 5. IC values calculated from viability at HEK293T cell line results of selected molecules

50

IC (µM)

50

5

0

HEK293T

Comp.

HEK293T

397.8 ± 1.53

135.07 ± 1.29

537.04 ± 1.31

13.73 ± 1.09

30.4 ± 1.18

Comp.

7f

6

c

6.08 ± 1.17

13.91 ± 1.13

29.54 ± 1.20

8.25 ± 1.23

6d

6h

7a

7d

8a

8e

8f

8i

Dox

118.73 ± 1.19

1.051 ± 0.57

7

e

19.21 ± 1.11

The cell viability was represented as a percentage (%) relative to

untreated cells as a control.

The effects of 7a and 8i on protein levels of CA IX and CA XII

In order to examine the effects of test substances on protein levels of CA IX and CA XII,

human HT-29 cells were treated with compounds 7a and 8i and western blotting assay was

performed [29, 30]. Cells were seeded in DMEM/F12 (normoxic cells) or DMEM/F12

containing 200µM CoCl (hypoxic and treated cells) to create a hypoxic environment. As

2

shown in the Figure 5, both 7a and 8i inhibited protein expression of CA IX and CA XII. Low

dose of 7a (1.56 µM) was not enough to statistically inhibited CA IX expression, despite a

reduction in the graph. However higher doses of 7a (6.25 and 25 µM) reduced CA IX protein

expression and it was found to be statistically significant (p<0.05) (Figure 5A). When cells

treaded with 8i, it is seen from the graph that all three doses (1.56, 6.25 and 25 µM) inhibited

CA IX expression gradually (p<0.05, p<0.01 and p<0.001, respectively) (Figure 5A). When

looking at the Figure 5B, similarly low dose of 7a (1.56 µM) was not enough to statistically

inhibited CA XII expression, as we saw with CA IX. However again, higher doses of 7a (6.25

and 25 µM) reduced CA XII protein expression. It was found to be statistically significant at

dose 6.25 µM (p<0.01), however because of high standard error it is not significant at dose 25

µM (Figure 5B). Reduction in protein expression of CA XII when treated with 8i was seen at

all doses with statistically significance at lower doses (1.56 and 6.25 µM) (p<0.05), however

not at dose 25 µM because of high standard error (Figure 5B).

1

6

Figure 5. The effects of 7a and 8i on protein levels of CA IX and CA XII in HT-29 cells. (A) CA IX

and (B) CA XII protein levels of normoxic (N), hypoxic (H) and treated cells at three doses (1.56, 6.25

and 25 µM). One-way ANOVA with Tukey’s post-hoc test, *, p<0.05; **, p<0.01; ***, p<0.001.

Mean ± SEM. C. Representative images of the bands (n=3).

3

. Conclusion

In tumor cells anaerobic glycolysis is increased to meet the increased metabolic needs. This

caused an acidic environment and hypoxic conditions. Carbonic anhydrases (CAs) are a group

of zinc-binding enzymes; catalyse the reversible hydration of CO to bicarbonate. Tumor cells

2

need CA enzymes to maintain neutral intracellular pH. To be able to tolerate hypoxic

conditions; CA IX and CA XII are overexpressed in many cancers, including colorectal

cancer. Clinical evidence shows that CA IX expression is associated with the poor survival of

patients with colorectal cancer. Because of these concerns, the synthesis of new potential

anticancer agents has been realized by targeting CA IX and CA XII enzyme inhibition.

This study demonstrates that this new derivative of coumarin-sulfonamide inhibits cellular

proliferation of colorectal cancer cells which is overexpressing CA IX and CA XII in hypoxic

conditions as well as the new compounds also shows CA IX and CA XII enzyme inhibition.

Three groups of new compound derivatives were synthesized as sulfonamide-based (6a-6i),

1

7

coumarin-aldehyde-based (7a-7i) and coumarin-sulfonamide-based derivatives (8a-8i);

investigated for the inhibition of four physiologically relevant CA isoforms, CA I (cytosolic

isoform) and CA II (associated with glaucoma), and CA IX and XII (tumor-associated

isoforms). The effect of these compounds on four different carbonic anhydrase enzymes

varied according to the isoform of carbonic anhydrases. Compounds that inhibit CA I isoform

at more micromolar levels inhibited CA II, CA IX and CA XII isoforms at nanomolar levels.

Compounds were evaluated in vitro on HT-29 human colorectal adenocarcinoma and healthy

HEK293T embryonic kidney cell lines. Among all the molecules, 8i demonstrated higher

anti-proliferation effect on colorectal cancer cell line HT-29. Additionally, 8i showed lower

cytotoxicity on the healthy HEK293T cell line, which is a desirable feature for a drug

candidate. Its inhibitory effects on CA IX and CA XII were displayed both enzyme inhibition

assays and its possible binding interactions with hCAs were suggested with molecular

modelling studies. 8i caused a dose-dependent decrease in the level of CA IX protein while

for CAXII it decreases the protein levels in a non-dose dependent manner. In addition,

western blotting studies indicate that the mechanism of action of compound 8i is not only

inhibition of hCA IX, but also by downregulation of the tumour-associated hCA IX and XII.

The data indicated that 8i may be promising drug candidate.

4

. Experimental

.1. Material and Method

The device STUART SMP40 model was used for the determination of melting points. To

measure infrared spectra, Alfa Bruker brand device is used. A Varian Infinity Plus

1

13

spectrometer was used at 300 and 75 Hz, respectively, to record the H and C NMR spectra,

while the Thermo Fisher Scientific LC-HRMS was used to determine the mass spectra. Other

spectrophotometric measurements were performed with BioTek Power Wave XS (BioTek,

USA). Cell lines were purchased from American Type Culture Collection (ATCC),

Dulbecco's Modified Eagle's Medium-F12, RPMI Medium, fetal calf serum and PBS GIBCO

BRL Invitrogen (Carlsbad, CA). Chemicals and solvents were purchased from Merck, Sigma-

Aldrich, Across, and Alfa Aesar. The solvents used were used without purification.

4

.2. General procedures and spectral data

Compounds 2 and 3 were synthesized according to the procedure shown in our previous study

18].

[

1

8

Synthesis of compounds 5a-5i

Propargyl bromide (7.40 mmol) and anhydrous K CO (1.00 g) were added to a solution of

2

3

aldehyde derivatives (6.17 mmol) in DMF (15 mL). The mixture was stirred at room

temperature for 16h, and then it was poured on crushed ice. The precipitated product was

filtered and dissolved in EtOAc (100 mL). The solution was washed with water (50 mL),

brine (50 mL), and water (50 mL), respectively. The organic layer was dried over anhydrous

Na SO and concentrated under vacuum to give propargyl aldehyde derivatives. The crude

2

4

products were crystallized with petroleum ether [24].

-(prop-2-yn-1-yloxy)benzaldehyde (5a): White powder, 90% yield, H NMR (CDCl , 300

1

2

3

MHz) δ/ppm: 2.57 (1H, s), 4.83 (2H, s), 7.06-7.13 (2H, m), 7.57 (1H, td, J=7.3, 1.7 Hz), 7.86

1

3

(

1H, d, J=7.6 Hz), 10.48 (1H, s ); C NMR (CDCl , 75 MHz) δ/ppm: 56.5, 113.3, 121.9,

3

1

25.6, 135.9, 159.9, 189.8.

-(prop-2-yn-1-yloxy)benzaldehyde (5b): White powder, 90% yield, H NMR (CDCl , 300

1

3

MHz) δ/ppm: 2.54 (1H, s), 4.69 (2H, s), 7.17-7.20 (1H, s), 7.37-7.47 (3H, m), 9.91 (1H, s );

1

3

C NMR (CDCl , 75 MHz) δ/ppm: 56.1, 113.8, 121.2, 130.3, 137.9, 158.2, 192.1.

3

1

4

-(prop-2-yn-1-yloxy)benzaldehyde (5c): Yellow powder, 85% yield, H NMR (CDCl , 300

3

MHz) δ/ppm: 2.57-2.59 (1H, m), 4.77 (2H, d, J= 2.3 Hz), 7.09 (2H, d, J=8.7 Hz), 7.85 (2H, d,

1

3

J=8.4 Hz), 9.90 (1H, s ); C NMR (CDCl , 75 MHz) δ/ppm: 56.1, 115.3, 130.7, 132.1, 162.5,

3

1

91.0.

1

3

-methoxy-2-(prop-2-yn-1-yloxy)benzaldehyde (5d): White powder, 80% yield, H NMR

(CDCl , 300 MHz) δ/ppm: 2.46-2.48 (1H, m), 3.89 (3H, s), 4.87 (2H, d, J= 2.3 Hz), 7.13-7.25

3

13

(

2H, m), 7.42-7.46 (1H, m), 10.48 (1H, s ); C NMR (CDCl , 75 MHz) δ/ppm: 56.2, 61.0,

3

1

17.9, 119.1, 125.1, 131.3, 149.7, 153.0, 190.7.

-(diethylamino)-2-(prop-2-yn-1-yloxy)benzaldehyde (5e): Pink powder, 80% yield, H NMR

1

4

(

CDCl , 300 MHz) δ/ppm: 1.14 (6H, t, J= 7.3 Hz), 2.50-2.52 (1H, m), 3.31-3.38 (4H, m), 4.70

3

2H, d, J= 2.3 Hz), 6.13 (1H, s), 6.23 (1H, dd, J= 9.0, 2.3 Hz), 7.63 (1H, d, J= 8.7 Hz), 10.04

1

3

1H, s); C NMR (CDCl , 75 MHz) δ/ppm: 12.8, 45.1, 56.3, 94.5, 105.2, 114.7, 130.8, 153.8,

3

1

62.2, 187.0.

1

5

-bromo-2-(prop-2-yn-1-yloxy)benzaldehyde (5f): Yellow powder, 85% yield, H NMR

(

DMSO-d , 300 MHz) δ/ppm: 3.66-3.68 (1H, m), 4.99 (2H, d, J= 3.8 Hz), 7.26 (1H, d, J= 8.7

6

1

9

1

3

Hz), 7.4 (1H, s), 7.82 (1H, d, J= 8.7 Hz), 10.22 (1H, s ); C NMR (DMSO-d , 75 MHz)

6

δ/ppm: 57.4, 78.9, 80.1, 114.0, 117.6, 126.9, 130.7, 138.9, 159.1, 188.5.

1

5

-nitro-2-(prop-2-yn-1-yloxy)benzaldehyde (5g): Yellow powder, 85% yield, H NMR

(

DMSO-d , 300 MHz) δ/ppm: 3.76-3.77 (1H, m), 5.16 (2H, d, J= 2.0 Hz), 7.50 (1H, d, J= 9.0

6

1

3

Hz ), 8.43 (1H, d, J= 2.3 Hz), 8.53 (1H, dd, J= 9.9, 2.9 Hz), 10.29 (1H, s); C NMR (DMSO-

d₆, 75 MHz) δ/ppm: 58.0, 78.3, 80.6, 115.9, 124.4, 125.0, 131.4, 142.0, 163.9, 188.4.

1

3

-methoxy-4-(prop-2-yn-1-yloxy)benzaldehyde (5h): Yellow powder, 85% yield, H NMR

(

CDCl , 300 MHz) δ/ppm: 2.46-2.5 (1H, m), 3.98 (3H, s), 4.98 (2H, d, J= 2.3 Hz), 7.18 (1H,

3

1

3

d, J= 8.7 Hz), 7.40-7.54 (2H, m), 9.9 (1H, s); C NMR (CDCl , 75 MHz) δ/ppm: 56.2, 56.7,

3

1

09.5, 112.6, 126.5, 131.0, 150.1, 152.3, 191.1.

1

2

-(prop-2-yn-1-yloxy)-1-naphthaldehyde (5i): Brown powder, 85% yield, H NMR (CDCl ,

3

00 MHz) δ/ppm: 2.56-2.58 (1H, m), 4.90 (2H, d, J= 2.6 Hz), 7.33 (1H, d, J= 9.0 Hz), 7.42

(1H, t, J= 7.0 Hz), 7.61 (1H, t, J= 7.9 Hz), 7.75 (1H, d, J= 8.2 Hz), 8.02 (1H, d, J= 9.3 Hz),

13

9

.26 (1H, d, J= 8.4 Hz), 10.87 (1H, s); C NMR (CDCl , 75 MHz) δ/ppm: 57.5, 114.1, 118.0,

3

1

25.2, 125.4, 128.5, 129.2, 130.1, 131.6, 137.5, 162.1, 192.2.

Synthesis of compounds 6a-i

mmol of 4-(aminomethyl)benzenesulfonamide and 1.1 mmol of aldehyde derivatives were

1

dissolved in 50 mL of CHCl :MeOH (5:1, v/v). The solution was refluxed for 18 h. It was

3

cooled and evaporated under vacuum. The pure imine compounds were obtained after washed

by ether [25].

4

-(((2-(prop-2-yn-1-yloxy)benzylidene)amino)methyl)benzenesulfonamide

(6a):White

powder, 82% yield; mp. 140-141°C; IR: 3300, 3282, 3040, 2959, 2130, 1625, 1600, 1458,

-1 1

1

338, 1152, 1028, 922, 753 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.63 (1H, t, J=1.1

6

Hz), 4.85 (2H, s), 4.93 (2H, d, J=2.3 Hz), 7.04 (1H, t, J=7.6 Hz), 7.20 (1H, d, J=8.4 Hz), 7.33

(

2H, s, NH ), 7.45-7.53 (3H, m), 7.80 (2H, d, J=8.2 Hz), 7.91 (1H, t, J=7.6 Hz), 8.82 (1H, s);

2

1

3

C NMR (DMSO-d , 75 MHz) δ/ppm: 56.8, 64.2, 79.4, 79.6, 114.2, 122.1, 125.0, 126.4,

6

+

1

27.5, 128.8, 132.8, 143.2, 144.5, 157.2, 158.0; HRMS (ESI): C H N O S [M+Na] ,

1

7

16

2

3

calculated for 351.078, found 351.0760.

4

-(((3-(prop-2-yn-1-yloxy)benzylidene)amino)methyl)benzenesulfonamide

(6b):White

powder, 86% yield; mp. 136-138°C; IR: 3337, 3291, 3038, 2956, 2128, 1641, 1583, 1492,

2

0

-1 1

1

313, 1270, 1151, 1043, 672 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.60 (1H, t, J=1.1

6

Hz), 4.84 (4H, s), 7.09-7.11 (1H, m), 7.34 (2H, s, NH ), 7.39-7.42 (3H, m), 7.52 (2H, d, J=7.0

2

1

3

Hz), 7.80 (2H, d, J=8.2Hz), 8.52 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 56.1, 63.7,

6

7

9.1, 79.7, 113.9, 118.5, 122.2, 126.4, 128.8, 130.5, 138.0, 143.3, 144.3, 158.1, 163.0;

+

HRMS (ESI): C H N O S [M+Na] , calculated for 351.078, found 351.0760.

1

7

16

2

3

4

-(((4-(prop-2-yn-1-yloxy)benzylidene)amino)methyl)benzenesulfonamide

(6c):White

powder, 76% yield; mp. 188-189°C; IR: 3322, 3282, 3038, 2900, 2124, 1642, 1604, 1513,

-1 1

1

344, 1151, 1026, 829 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.36 (1H, t, J=0.5 Hz),

6

4

.80 (2H, s), 4.86 (2H, d, J=0.8 Hz), 7.06 (2H, d, J=8.2 Hz), 7.32 (2H, s, NH ), 7.50 (2H, d,

2

1

3

J=8.2 Hz), 7.74-7.81 (4H, m), 8.46 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 56.2, 63.7,

6

7

9.2, 79.6, 115.6, 126.4, 128.7, 130.0, 130.2, 143.2, 144.6, 159.9, 162.3; HRMS (ESI):

+

C H N O S [M+Na] , calculated for 351.078, found 351.0759.

17

16

2

3

4

-(((3-methoxy-2-(prop-2-yn-1-yloxy)benzylidene)amino)methyl)benzenesulfonamide

(6d):Yellow powder, 80% yield; mp. 143-144°C; IR: 3292, 3255, 3040, 2956, 2120, 1636,

-1 1

1

583, 1480, 1330, 1154, 1077, 785 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.53 (1H, t,

6

J=2.3 Hz), 3.82 (3H, s), 4.77 (2H, d, J=2.3 Hz), 4.82 (2H, s), 7.10-7.18 (2H, m), 7.33 (2H, s,

13

NH ), 7.46-7.50 (3H, m), 7.78 (2H, d, J=8.4 Hz), 8.80 (1H, s); C NMR (DMSO-d , 75

2

6

MHz) δ/ppm: 56.5, 60.7, 64.3, 79.7, 79.9, 115.6, 118.5, 125.4, 126.4, 128.8, 130.8, 143.3,

+

1

44.3, 146.7, 153.2, 159.0; HRMS (ESI): C H N O S [M+Na] , calculated for 311.088,

1

8

18

2

4

found 381.0865.

4

-(((4-(diethylamino)-2-(prop-2-yn-1-yloxy)benzylidene)amino)methyl)benzenesulfonamide

(6e):Browne powder, 76% yield; mp. 120-121°C; IR: 3307, 3287, 3035, 2971, 2121, 1600,

-1 1

1

518, 1402, 1272, 1092, 821 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 1.12 (6H, t, J=6.7

6

Hz ), 3.39-3.43 (4H, m), 3.63 (1H, s), 4.75 (2H, s), 4.91 (2H, s), 6.31 (1H, s), 6.35 (1H, d,

J=9.0 Hz), 7.33 (2H, s, NH ), 7.48 (2H, d, J=7.0 Hz), 7.72 (1H, d, J=8.7 Hz), 7.79 (2H, d,

2

1

3

J=8.2 Hz), 8.60 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 13.2, 44.6, 56.4, 61.2, 79.2,

6

8

0.0, 95.9, 105.6, 118.6, 125.5, 126.3, 128.7, 141.4, 144.5, 146.7, 153.5, 159.0; HRMS(ESI):

+

C H N O S [M+H] , calculated for 400.162, found 400.1675.

21

25

3

4

-(((5-bromo-2-(prop-2-yn-1-yloxy)benzylidene)amino)methyl)benzenesulfonamide

(6f):White powder, 82% yield; mp. 174-175°C; IR: 3309, 3284, 3040, 2884, 2125, 1636,

-1 1

1

588, 1475, 1329, 1151, 1016, 822 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.68 (1H, t,

6

2

1

J=8.7 Hz), 4.87 (2H, s), 4.95 (2H, d, J=2.3 Hz), 7.36 (2H, s, NH ), 7.52(2H, d, J=7.9 Hz),

2

7

.66 (1H, dd, J=9.3, 3.2 Hz), 7.81 (2H, d, J=7.9 Hz), 7.98 (1H, d, J=2.6 Hz ), 8.75 (1H, s);

1

3

C NMR (DMSO-d , 75 MHz) δ/ppm: 57.2, 64.0, 79.2, 79.7, 114.0, 116.9, 126.4, 127.1,

6

1

28.8, 129.5, 135.1, 143.3, 144.2, 156.3, 156.8; LC-HRMS (m/z): 430,9841 for:. HRMS(ESI):

+

C H BrN O S [M+Na] , calculated for 428.988, found 430.9841.

17

15

2

3

4

-(((5-nitro-2-(prop-2-yn-1-yloxy)benzylidene)amino)methyl)benzenesulfonamide

(6g):Yellow powder, 68% yield; mp. 149-150°C; IR: 3341, 3278, 3038, 2891, 2126, 1635,

-1 1

1

608, 1518, 1331, 1150, 999 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.77 (1H, s), 4.90

6

(2H, s), 5.12 (2H, s), 7.31-7.43 (3H, m), 7.51 (2H, d, J=8.2 Hz), 7.80 (2H, d, J=7.9 Hz), 8.35

1

3

(

1H, dd, J=9.3, 2.9 Hz), 8.62 (1H, d, J=2.9 Hz ), 8.79 (1H, s); C NMR (DMSO-d , 75 MHz)

6

δ/ppm: 57.7, 64.0, 78.6, 80.4, 114.9, 122.8, 125.3, 126.5, 128.0, 128.9, 142.1, 143.4, 143.9,

+

1

56.4, 161.5; HRMS (ESI): C H N O S [M+Na] , calculated for 396.063, found 396.0610.

1

7

15

3

5

4

-(((3-methoxy-4-(prop-2-yn-1-yloxy)benzylidene)amino)methyl)benzenesulfonamide

(6h):White powder, 85% yield; mp. 175-176°C; IR: 3337, 3305, 3036, 2995, 2120, 1639,

-1 1

1

597, 1510, 1270, 1022, 804 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.61 (1H, t, J=1.4

6

Hz), 3.80 (3H, s), 4.81 (2H, s), 4.85 (2H, d, J=1.1 Hz), 7.10 (1H, d, J=8.2 Hz), 7.30 (1H, d,

J=8.4 Hz), 7.31 (2H, s), 7.44 (1H, s), 7.51 (2H, d, J=7.6 Hz), 7.80 (2H, d, J=8.2 Hz), 8.44

1

3

(

1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 56.1, 56.6, 70.2, 79.2, 79.6, 113.9, 122.7,

6

1

26.4, 127.4, 128.0, 128.9, 130.4, 144.5, 149.9, 156.4, 162.6; HRMS (ESI): C H N O S

18 18 2 4

+

[

M+Na] , calculated for 381.088, found 381.0864.

4

-((((2-(prop-2-yn-1-yloxy)naphthalen-1-yl)methylene)amino)methyl)benzenesulfonamide

(6i): Yellow powder, 81% yield; mp. 129°C; IR: 3317, 3300, 2978, 2121, 1637, 1592, 1464,

-

1 1

1

5

334, 1165, 995, 803 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 3.64 (1H, s), 4.94 (2H, s),

6

.06 (2H, s), 7.35-7.42 (3H, m), 7.47-7.58 (4H, m), 7.81 (2H, d, J=8.2 Hz), 7.88 (1H, d, J=7.9

1

3

Hz), 8.06 (1H, d, J=9.0 Hz), 9.14 (1H, s), 9.23 (1H, d, J=8.4 Hz); C NMR (DMSO-d , 75

6

MHz) δ/ppm: 57.8, 65.9, 79.6, 79.8, 115.7, 117.8, 125.0, 126.5, 128.7, 128.8, 129.0, 129.7,

+

1

3

31.9, 133.5, 143.3, 144.6, 157.1, 160.5; HRMS (ESI): C H N O S [M+H] , calculated for

2

1

18

2

3

79.104, found 379.1097.

Synthesis of 7a-i derivatives

The dissolved 0.1 mmol CuSO .5H O and 0.2 mmol sodium ascorbate in water (5 mL) was

4

2

added to the solution of 7-(3-azidopropoxy)-2H-chromen-2-one (3) (1 mmol) and aldehyde

2

o

derivatives (5a-i) (1 mmol) in 20 mL of THF:H O (4:1, v/v). The mixture was stirred at 40 C

2

for 2h. It was concentrated under vacuum; the residue was dissolved in EtOAc (100 mL). The

solution was washed with water (50 mL), EDTA (50 mL), and water (50 mL), respectively.

The organic layer was concentrated under vacuum. The obtained 7a-i derivatives were

crystallized from ethanol [26].

2

-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde

(7a): White powder, 82% yield; mp. 137°C; IR: 3153, 3069, 2947, 1714, 1686, 1612, 1597,

-

1 1

1

4

394, 1248, 1121, 1050, 834 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 2.29-2.36 (2H, m),

6

.09 (2H, t, J=5.8 Hz), 4.58 (2H, t, J=7.0 Hz), 5.35 (2H, s), 6.30 (1H, d, J=9.3 Hz ), 6.88-7.03

(2H, m), 7.10 (1H, t, J=7.3 Hz), 7.43 (1H, d, J=8.4 Hz), 7.59-7.70 (3H, m), 7.98 (1H, d, J=9.3

1

3

Hz), 8.38 (1H, s), 10.33 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 29.8, 47.3, 62.9, 66.0,

6

1

4

01.8, 113.1, 113.2, 113.3, 114.8, 121.8, 125.1, 125.4, 128.2, 130.1, 137.0, 143.0, 144.9,

+

56.0, 160.9, 161.0, 162.1, 189.8; HRMS (ESI): C H N O [M+Na] , calculated for

2

19

3

5

28.122, found 428.1205.

3

-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde

(7b): Yellow powder, 77% yield; mp. 56°C; IR: 3140, 3083, 2946, 1720, 1694, 1609, 1394,

-1

1

256, 1121, 1016, 832 cm ; H NMR (CDCl , 300 MHz) δ/ppm: 2.45-2.49 (2H, m), 4.04

3

(2H, t, J=5.8 Hz), 4.63 (2H, t, J=7.0 Hz), 5.25 (2H, s), 6.25 (1H, d, J=9.6 Hz ), 6.75 (1H, s),

6

7

1

4

.80 (1H, dd, J=8.4, 2.3 Hz), 7.23-7.28 (2H, m), 7.36-7.48 (3H, m), 7.63 (1H, d, J=9.3 Hz),

1

3

.69 (1H, s), 9.95 (1H, s); C NMR (CDCl , 75 MHz) δ/ppm: 29.8, 47.3, 62.2, 64.9, 101.7,

3

12.7, 113.1, 113.6, 122.0, 123.5, 124.0, 129.1, 130.4, 137.9, 143.6, 143.7, 155.9, 158.9,

+

61.2, 161.6, 192.2; HRMS (ESI): C H N O [M+Na] , calculated for 428.122, found

2

19

3

5

28.1202.

4

-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde

(7c): White powder, 80% yield; mp. 118°C; IR: 3150, 3076, 2944, 1708, 1687, 1607, 1392,

-1 1

1

254, 1155, 1122, 1046, 994 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 2.31-2.36 (2H, m),

6

4

.10 (2H, t, J=5.8 Hz), 4.59 (2H, t, J=6.7 Hz), 5.29 (2H, s), 6.30 (1H, d, J=9.6 Hz ), 6.90-6.97

(2H, m), 7.25 (2H, d, J=8.7 Hz), 7.62 (1H, d, J=8.4 Hz), 7.87 (2H, d, J=8.7 Hz), 8.0 (1H, d,

13

J=9.3 Hz), 8.35 (1H, s), 9.88 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 29.8, 47.3, 62.1,

6

6.0, 101.8, 113.1, 113.2, 113.3, 115.8, 125.7, 130.1, 130.4, 132.4, 142.7, 145.0, 156.0, 160.9,

2

3

+

1

4

62.1, 163.6, 192.0; HRMS (ESI): C H N O [M+Na] , calculated for 428.122, found

22 19 3 5

28.1203.

3

-methoxy-2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzaldehyde (7d): White powder, 86% yield; mp. 144°C; IR: 3142, 3002, 2954,

-1 1

1

729, 1684, 1627, 1483, 1252, 1140, 953 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 2.24-

6

2

.29 (2H, m), 3.92 (3H, s), 4.01 (2H, t, J=5.8 Hz), 4.53 (2H, t, J=7.0 Hz), 5.26 (2H, s), 6.30

(1H, d, J=9.6 Hz ), 6.90-6.95 (2H, m), 7.19-7.20(2H, m), 7.39-7.42 (1H, m), 7.63 (1H, d,

1

3

J=8.4 Hz), 8.0 (1H, d, J=9.3 Hz), 8.24 (1H, s), 10.0 (1H, s); C NMR (DMSO-d , 75 MHz)

6

δ/ppm: 29.9, 47.0, 56.8, 65.7, 66.6, 101.8, 113.1, 113.3, 118.5, 119.3, 125.3, 125.8, 130.1,

1

30.4, 142.9, 145.0, 150.4, 153.6, 156.0, 160.9, 162.1, 190.6; HRMS (ESI): C H N O

23 21 3 6

+

[

M+Na] , calculated for 488.133, found 458.1306.

4

-(diethylamino)-2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzaldehyde (7e): White powder, 72% yield; mp. 125-126°C; IR: 3149, 3080,

-1 1

2

968, 1726, 1680, 1614, 1587, 1271, 1117, 1030, 846 cm ; H NMR (DMSO-d , 300 MHz)

6

δ/ppm:1.09 (6H, t, J=6.7 Hz), 2.28-2.32 (2H, m), 3.37-3.44 (4H, m), 4.06 (2H, t, J=5.5 Hz),

.55 (2H, t, J=6.7 Hz), 5.29 (2H, s), 6.24-6.37 (3H, m), 6.85-6.93 (2H, m), 7.47(1H, d, J=8.7

4

1

3

Hz), 7.56 (1H, d, J=8.4 Hz), 7.95 (1H, d, J=9.3 Hz), 8.31 (1H, s), 9.94 (1H, s); C NMR

DMSO-d , 75 MHz) δ/ppm: 13.1, 29.8, 44.7, 47.2, 62.5, 66.0, 94.9, 101.8, 105.2, 113.1,

(

6

1

13.2, 113.3, 113.9, 125.2, 130.1, 144.9, 154.2, 156.0, 160.9, 162.1, 163.2, 185.9; HRMS

+

(

ESI): C H N O [M+Na] , calculated for 499.196, found 499.1936.

2

6

28

4

5

-bromo-2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzaldehyde (7f): White powder, 80% yield; mp. 157-158°C; IR: 3158, 3073,

-

1 1

2

3

970, 1738, 1682, 1663, 1589, 1400, 1273, 1232, 1123, 1047, 846 cm ; H NMR (DMSO-d ,

6

00 MHz) δ/ppm: 2.31-2.35 (2H, m), 4.08 (2H, t, J=5.8 Hz), 4.58 (2H, t, J=7.0 Hz), 5.36 (2H,

s), 6.30 (1H, d, J=9.6 Hz), 6.88-6.95 (2H, m), 7.45(1H, d, J=8.4 Hz), 7.60 (1H, J=8.7 Hz),

7

1

.72 (1H, d, J=2.6 Hz), 7.82 (1H, dd, J=7.9, 2.6 Hz ), 7.98 (1H, d, J=9.6 Hz ), 8.38 (1H, s),

1

3

0.22 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 29.8, 47.3, 63.3, 66.0, 101.8, 113.1,

6

13.3, 113.6, 117.7, 125.5, 126.6, 130.1, 130.4, 139.0, 142.7, 144.9, 156.0, 160.0, 160.9,

+

62.1, 188.7; HRMS (ESI): C H BrN O [M+Na] , calculated for 506.033, found 506.0305.

2

18

3

5

-nitro-2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzaldehyde (7g): Yellow powder, 66% yield; mp. 181°C; IR: 3160, 3080,

2

4

-1 1

2

946, 1723, 1683, 1611, 1588, 1341, 272, 1121, 1034, 838 cm ; H NMR (DMSO-d , 300

6

MHz) δ/ppm: 2.32-2.36 (2H, m), 4.09 (2H, t, J=5.8 Hz), 4.59 (2H, t, J=7.0 Hz), 5.54 (2H, s),

.30 (1H, d, J=9.6 Hz ), 6.88-7.03 (2H, m), 7.60(1H, d, J=8.4 Hz), 7.70 (1H, J=9.3 Hz), 7.98

1H, d, J=9.3 Hz), 8.40 (1H, d, J=2.9 Hz ), 8.43 (1H, s), 8.51 (1H, dd, J=9.3, 2.9 Hz ), 10.27

6

(

1

3

1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 29.8, 47.3, 63.9, 66.0, 101.8, 113.1, 113.2,

6

1

15.9, 124.1, 124.7, 125.8, 130.1, 131.4, 141.6, 144.9, 155.9, 160.9, 162.0, 164.9, 188.5;

+

HRMS (ESI): C H N O [M+Na] , calculated for 473.107, found 473.1210.

2

18

4

7

3

-methoxy-4-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzaldehyde (7h): White powder, 82% yield; mp. 117-118°C; IR: 3153, 3083,

-1

1

2

947, 1723, 1674, 1613, 1507, 1279, 1227, 1132, 1048, 835 cm ; H NMR (CDCl , 300

3

MHz) δ/ppm: 2.44-2.48 (2H, m), 3.90 (3H, s), 4.04 (2H, t, J=5.8 Hz), 4.62 (2H, t, J=7.0 Hz),

.38 (2H, s), 6.26 (1H, d, J=9.6 Hz), 6.75-6.81 (2H, m), 7.21 (1H, d, J=8.2 Hz), 7.35-7.44

5

1

3

(

3H, m), 7.64 (1H, d, J=9.6 Hz), 7.72 (1H, s), 9.84 (1H, s); C NMR (DMSO-d , 75 MHz)

6

δ/ppm: 29.8, 47.4, 56.2, 62.9, 64.8, 101.7, 109.2, 112.5, 112.7, 113.1, 113.6, 127.0, 129.1,

+

1

30.7, 143.6, 149.9, 153.1, 155.9, 161.3, 161.6, 191.2; HRMS (ESI): C H N O [M+Na] ,

2

3

21

3

6

calculated for 458.133, found 458.1304.

2

-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy)-1-

naphthaldehyde (7i): Orange powder, 77% yield; mp. 173-174°C; IR: 3152, 3072, 2958,

-1

1

725, 1682, 1610, 508, 1229, 1117, 1043, 829 cm ; H NMR (CDCl , 300 MHz) δ/ppm:

3

2

.44-2.48 (2H, m), 4.01 (2H, t, J=5.5 Hz), 4.63 (2H, t, J=6.7 Hz), 5.47 (2H, s), 6.23 (1H, d,

J=9.3 Hz ), 6.74-6.77 (2H, m), 7.31 (1H, d, J=8.2 Hz), 7.40-7.58 (2H, m), 7.62-7.64 (2H, m),

.70 (1H, s), 7.76 (1H, d, J=7.6 Hz), 8.04 (1H, d, J=9.0 Hz ), 9.20 (1H, d, J=8.7 Hz ), 10.82

7

1

3

(

1H, s); C NMR (CDCl , 75 MHz) δ/ppm: 29.8, 47.4, 63.6, 64.8, 101.7, 112.6, 113.1, 113.6,

3

1

4

14.0, 117.4, 125.1, 125.3, 128.5, 129.0, 129.2, 130.2, 131.6, 137.8, 143.5, 155.9, 161.3,

+

61.6, 162.7, 192.0; HRMS (ESI): C H N O [M+Na] , calculated for 478.138, found

2

6

21

3

5

78.1356.

Synthesis of compound 8a-i

mmol of 4-(aminomethyl)benzenesulfonamide, 1 mmol of 7a-i derivatives and 1 mmol NaOH

1

were dissolved in 50 mL of EtOH. The solution was refluxed for 18 h. It was cooled and

evaporated under vacuum. The final compounds were obtained after washing by ether.

2

5

4

-(((2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzylidene)amino)methyl)benzenesulfonamide (8a):White powder, 76% yield;

-1

mp. 107-108°C; IR: 3338, 3063, 2973, 1722, 1614, 1599, 1336, 1283, 1154, 1125, 996 cm ;

1

H NMR (DMSO-d , 300 MHz) δ/ppm: 2.31-2.35 (2H, m), 4.09 (2H, t, J=6.1 Hz), 4.58 (2H,

6

t, J=6.7 Hz), 4.80 (2H, s), 5.28 (2H, s), 6.28 (1H, d, J=9.3 Hz ), 6.88-7.04 (4H, m), 7.33 (2H,

d, J=7.9 Hz), 7.43-7.54 (3H, m), 7.60 (1H, d, J=8.7 Hz), 7.80 (2H, d, J=8.2 Hz), 7.89 (1H, d,

1

3

J=7.6 Hz), 7.94 (1H, d, J=9.3 Hz ), 8.37 (1H, s ), 8.78 (1H, s); C NMR (DMSO-d , 75 MHz)

6

δ/ppm: 29.8, 47.2, 62.6, 64.2, 65.6, 66.0, 101.8, 113.1, 113.2, 113.3, 114.1, 121.7, 124.7,

1

25.4, 126.4, 127.4, 128.7, 130.1, 133.0, 143.2, 144.5, 145.0, 155.9, 158.0, 158.1, 160.9,

+

62.1; HRMS (ESI): C H N O S [M+Na] , calculated for 596.188, found 596.1548.

2

9

27

5

6

4

-(((3-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzylidene)amino)methyl)benzenesulfonamide (8b): White powder, 72% yield;

mp. 134-135°C; IR: 3333, 3070, 2957, 1730, 1611, 1475, 1271, 1228, 1158, 1120, 1036, 832

-1 1

cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 2.30-2.34 (2H, m), 4.09 (2H, t, J=5.8 Hz), 4.57

6

(2H, t, J=7.0 Hz), 4.84 (2H, s), 5.19 (2H, s), 6.28 (1H, d, J=9.6 Hz ), 6.89-6.96 (2H, m), 7.13-

7

.17 (1H, m), 7.35-7.39 (3H, m), 7.46 (1H, s), 7.51-7.63 (4H, m), 7.80 (2H, d, J=8.2 Hz), 7.98

1

3

(

1H, d, J=9.6 Hz), 8.30 (1H, s ), 8.51 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 29.8,

6

4

1

7.2, 61.8, 63.7, 66.0, 101.8, 113.1, 113.2, 113.3, 114.1, 118.2, 121.7, 125.4, 126.2, 126.4,

28.8, 130.1, 130.5, 138.0, 143.2, 143.3, 144.3, 145.0, 155.9, 158.9, 160.9, 162.1, 163.0;

+

HRMS (ESI): C H N O S [M+Na] , calculated for 596.188, found 596.1550.

2

9

27

5

6

4

-(((4-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzylidene)amino)methyl)benzenesulfonamide (8c):Yellow powder, 81% yield;

-1 1

mp. 58-59°C; IR: 3272, 3067, 2951, 1718, 1603, 1508, 1328, 1229, 1156, 1094, 830 cm ; H

NMR (DMSO-d , 300 MHz) δ/ppm: 2.28-2.34 (2H, m), 4.09 (2H, t, J=6.1 Hz), 4.57 (2H, t,

6

J=6.7 Hz), 4.80 (2H, s), 5.20 (2H, s), 6.30 (1H, d, J=9.6 Hz ), 6.89-6.96 (2H, m), 7.12 (2H, d,

J=8.7 Hz), 7.25-7.30 (1H, m), 7.50 (2H, d, J=8.2 Hz), 7.62 (1H, d, J=8.4 Hz), 7.70-7.88 (5H,

13

m), 7.98 (1H, d, J=9.6 Hz), 8.31 (1H, s ), 8.45 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm:

6

2

1

9.8, 47.2, 56.6, 61.8, 63.7, 66.0, 101.8, 113.1, 113.2, 113.3, 115.5, 125.5, 126.4, 128.7,

29.7, 130.2, 130.3, 143.0, 143.2, 144.6, 145.0, 156.0, 160.8, 160.9, 162.1, 162.4; HRMS

+

(

ESI): C H N O S [M+Na] , calculated for 596.188, found 596.1550.

2

9

27

5

6

2

6

4

-(((2-methoxy-6-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzylidene)amino)methyl)benzenesulfonamide (8d):Yellow powder, 85% yield;

-1 1

mp. 64-65°C; IR: 3320, 3079, 2941, 1728, 1610, 1476, 1267, 1156, 1124, 1072, 833 cm ; H

NMR (DMSO-d , 300 MHz) δ/ppm: 2.19-2.23 (2H, m), 3.85 (3H, m), 3.96 (2H, t, J=5.8 Hz),

6

4

.47 (2H, t, J=6.7 Hz), 4.69 (2H, s), 5.12 (2H, s), 6.25 (1H, d, J=9.3 Hz ), 6.84-6.90 (2H, m),

.05 (1H, t, J=7.9 Hz), 7.15 (1H, dd, J=8.2, 1.4 Hz), 7.36-7.44 (5H, m), 7.57 (1H, d, J=8.4

7

1

3

Hz), 7.77 (2H, d, J=8.2 Hz), 7.95 (1H, d, J=9.6 Hz), 8.18 (1H, s ), 8.53 (1H, s); C NMR

DMSO-d , 75 MHz) δ/ppm: 29.9, 47.0, 56.5, 56.6, 65.8, 66.3, 101.8, 113.1, 113.2, 113.3,

(

6

1

6

15.6, 118.5, 125.0, 125.6, 126.4, 128.7, 130.1, 130.2, 143.2, 144.3, 147.4, 153.4, 155.9,

+

58.9, 160.9, 162.0; HRMS (ESI): C H N O S [M+H] , calculated for 604.179, found

3

0

29

5

7

04.1839.

4

-(((4-(diethylamino)-2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzylidene)amino)methyl)benzenesulfonamide (8e): Brown powder, 65% yield;

mp. 76-77°C; IR: 3264, 3060, 2969, 1722, 1597, 1509, 1332, 1271, 1155, 1120, 1016, 833

-1 1

cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 1.00-1.10 (6H, m), 2.27-2.31 (2H, m), 3.37-

6

3.42 (4H, m), 4.06 (2H, t, J=5.8 Hz), 4.55 (2H, t, J=6.7 Hz), 4.66 (2H, s), 5.23 (2H, s), 6.24-

6.30 (2H, m ), 6.39 (1H, s), 6.86 (1H, dd, J=8.7, 2.3 Hz), 6.91 (1H, d, J=2.6 Hz), 7.32 (2H, s),

7.41 (2H, d, J=8.2 Hz), 7.56 (1H, d, J=8.7 Hz), 7.68 (1H, d, J=9.0 Hz), 7.75 (2H, d, J=8.2

1

3

Hz), 7.94 (1H, d, J=9.3 Hz), 8.30 (1H, s ), 8.55 (1H, s); C NMR (DMSO-d , 75 MHz)

6

δ/ppm:13.1, 19.2, 29.8, 44.5, 47.2, 56.6, 62.4, 66.0, 101.8, 113.1, 113.2, 113.3, 118.5, 125.2,

1

25.6, 126.3, 128.6, 130.1, 130.2, 143.0, 143.6, 145.0, 156.0, 159.9, 160.9, 162.1; HRMS

+

(

ESI): C H N O S [M+H] , calculated for 645.242, found 645.2464.

3

36

6

4

-(((5-bromo-2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzylidene)amino)methyl)benzenesulfonamide (8f):Yellow powder, 79% yield;

-1 1

mp. 70-71°C; IR: 3249, 3080, 2942, 1723, 1609, 1555, 1328, 1229, 1156, 1124, 833 cm ; H

NMR (DMSO-d , 300 MHz) δ/ppm: 2.30-2.32 (2H, m), 4.08 (2H, t, J=5.8 Hz), 4.58 (2H, t,

6

J=6.7 Hz), 4.82 (2H, s), 5.30 (2H, s), 6.28 (1H, d, J=9.3 Hz), 6.88-7.02 (2H, m), 7.32-7.35

(3H, m), 7.48 (2H, d, J=8.2 Hz), 7.59-7.65 (2H, m), 7.80 (2H, d, J=7.6 Hz), 7.94-8.01 (2H,

1

3

m), 8.36 (1H, s ), 8.70 (1H, s); C NMR (DMSO-d , 75 MHz) δ/ppm: 29.8, 47.3, 56.6, 64.0,

6

1

6.0, 101.8, 113.1, 113.3, 113.6, 116.8, 125.6, 126.4, 126.7, 128.7, 129.5, 130.1, 135.2, 142.8,

43.2, 144.2, 145.0, 155.9, 156.9, 157.1, 160.9, 162.1; HRMS (ESI): C H BrN O S

2

9

26

5

6

+

[

M+Na] , calculated for 674.068, found 676.0631.

2

7

4

-(((5-nitro-2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzylidene)amino)methyl)benzenesulfonamide (8g):Orange powder, 56% yield;

-

mp. 68-69°C; IR: 3310, 3080, 2950, 1720, 1609, 1510, 1335, 1265, 1156, 1125, 990, 833 cm

1

;

H NMR (DMSO-d , 300 MHz) δ/ppm: 2.32-2.36 (2H, m), 4.10 (2H, t, J=5.8 Hz), 4.60

6

(2H, t, J=6.7 Hz), 4.87 (2H, s), 5.48 (2H, s), 6.26-6.31 (1H, m), 6.85-6.97 (3H, m), 7.40-7.66

(4H, m), 7.78-7.83 (2H, m), 7.90-8.04 (3H, m), 8.32-8.44 (1H, m), 8.64 (1H, s ), 8.77 (1H, s);

1

3

C NMR (DMSO-d , 75 MHz) δ/ppm: 29.8, 47.3, 56.6, 64.0, 66.0, 101.8, 113.1, 113.3,

6

1

6

13.6, 116.8, 125.6, 126.4, 126.7, 128.7, 129.5, 130.1, 135.2, 142.8, 143.2, 144.2, 145.0,

+

55.9, 156.9, 157.1, 160.9, 162.1; HRMS (ESI): C H N O S [M+Na] , calculated for

2

9

26

6

8

41.143, found 641.1398.

4

-(((3-methoxy-4-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)benzylidene)amino)methyl)benzenesulfonamide (8h):Yellow powder, 74% yield;

-1 1

mp. 86-87°C; IR: 3304, 3040, 2948, 1724, 1613, 1508, 1268, 1155, 1093, 814 cm ; H NMR

(

DMSO-d , 300 MHz) δ/ppm: 2.25-2.32 (2H, m), 3.73 (3H, s), 4.07 (2H, t, J=5.8 Hz), 4.55

6

(2H, t, J=7.0 Hz), 4.77 (2H, s), 5.16 (2H, s), 6.26 (1H, d, J=9.3 Hz), 6.87-6.93 (2H, m), 7.19-

7

.27 (2H, m), 7.35-7.39 (3H, m), 7.48 (2H, d, J=7.9 Hz), 7.59 (1H, d, J=8.4 Hz ), 7.78 (2H, d,

1

3

J=7.9 Hz ), 7.96 (1H, d, J=9.3 Hz ), 8.30 (1H, s ), 8.40 (1H, s); C NMR (DMSO-d , 75

6

MHz) δ/ppm: 29.9, 47.2, 55.9, 56.6, 63.8, 66.0, 101.8, 109.8, 113.1, 113.2, 113.3, 123.3,

1

25.7, 126.4, 128.9, 129.9, 130.2, 143.0, 143.2, 144.5, 145.0, 149.7, 150.5, 156.0, 161.0,

+

62.1, 162.6; HRMS (ESI): C H N O S [M+Na] , calculated for 626.169, found 626.1654.

3

0

29

5

7

4

-((((2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-

yl)methoxy)naphthalen-1-yl)methylene)amino)methyl)benzenesulfonamide (8i): Orange

powder, 75% yield; mp. 93-94°C; IR: 3236, 3070, 2944, 1728, 1609, 1508, 1328, 1230, 1155,

-1 1

1

124, 932 cm ; H NMR (DMSO-d , 300 MHz) δ/ppm: 2.29-2.33 (2H, m), 4.06 (2H, t, J=5.8

6

Hz), 4.57 (2H, t, J=6.7 Hz), 4.90 (2H, s), 5.45 (2H, s), 6.27 (1H, d, J=9.3 Hz), 6.86-6.94 (3H,

m), 7.20-7.59 (7H, m), 7.70 (1H, d, J=9.3 Hz), 7.81-7.98 (3H, m), 8.06 (1H, d, J=9.0 Hz),

1

3

8

2

1

.36 (1H, s ), 9.13 (1H, s), 9.26 (1H, d, J=8.4 Hz); C NMR (DMSO-d , 75 MHz) δ/ppm:

6

9.8, 47.2, 55.9, 62.8, 66.0, 101.8, 113.1, 113.2, 113.3, 116.8, 117.5, 123.3, 123.5, 124.3,

25.7, 126.5, 128.6, 129.6, 130.1, 132.6, 133.6, 143.2, 144.6, 145.0, 155.9, 158.0, 160.9,

+

62.1; HRMS (ESI): C H N O S [M+Na] , calculated for 646.174, found 646.1702.

3

29

5

6

4

.3. CA inhibition assays

2

8

Article Doi

Synthesis of coumarin-sulfonamide derivatives and determination of their cytotoxicity, carbonic anhydrase inhibitory and molecular docking studies

DOI: 10.1016/j.ejmech.2019.111702

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Article abstract of DOI:10.1016/j.ejmech.2019.111702

Full text of DOI:10.1016/j.ejmech.2019.111702

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