Synthesis and evaluation of new coumarin derivatives as antioxidant, antimicrobial, and anti-inflammatory agents

molecules

Article

Synthesis and Evaluation of New Coumarin

Derivatives as Antioxidant, Antimicrobial,

and Anti-Inﬂammatory Agents

Hanan M. Alshibl ^1,

* ,

, Ebtehal S. Al-Abdullah ¹, Mogedda E. Haiba ^1,2, Hamad M. Alkahtani ¹

Ghada E.A. Awad ³, Ahlam H. Mahmoud ⁴, Bassant M.M. Ibrahim ⁵, Ahmed Bari ¹and

Alexander Villinger ⁶

1

Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,

Riyadh 11451, Saudi Arabia; ealabdullah@ksu.edu.sa (E.S.A.-A.); mogedda.haiba@yahoo.com (M.E.H.);

ahamad@ksu.edu.sa (H.M.A.); abari@ksu.edu.sa (A.B.)

Department of Medicinal Chemistry, National Research Centre, Cairo 12622, Egypt

2

3

Chemistry of Natural and Microbial Product Department, National Research Centre, Cairo 12622, Egypt;

ghadaawadnrc@gmail.com

Department of Therapeutic Chemistry, Pharmaceutical and Drug Industries Research Division,

4

National Research Centre, Dokki, Cairo 12622, Egypt; ahlam@hotmail.co.uk

Pharmacology Department, Medical Research Division, National Research Centre, Cairo 12622, Egypt;

5

bmmih1974@gmail.com

6

Institut für Chemie, Abteilung Anorganische Chemie, Universität Rostock, Albert-Einstein-Str. 3a,

18059 Rostock, Germany; alexander.villinger@uni-rostock.de

*

Correspondence: Halshibl@ksu.edu.sa; Tel.: +966-11805-2756

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Academic Editor: Maria João Matos

Received: 22 June 2020; Accepted: 15 July 2020; Published: 16 July 2020

Abstract: New pyranocoumarin and coumarin-sulfonamide derivatives were prepared and evaluated

for their antioxidant, antimicrobial, and/or anti-inﬂammatory activities. Coumarin-sulfonamide

compounds 8a–d demonstrated signiﬁcant antioxidant activity, while 7c

antimicrobial activity equal to or higher than the standard antimicrobials against at least one tested

microorganism. Regarding the anti-inﬂammatory testing, pyranocoumarins 2b 3a and 5c and

coumarin-sulfonamide compound 9a showed more potent antiproteinase activity than aspirin in vitro

,d, 8c,d, and 9c,d exhibited

,

,b

;

however, ﬁve compounds were as potent as aspirin. The anti-inﬂammatory activity of the promising

compounds was further assessed pharmacologically on formaldehyde-induced rat paw oedema and

showed signiﬁcant inhibition of oedema. For in vitro COX-inhibitory activity of coumarin derivatives,

pyranocoumarin derivative 5a was the most selective (SI = 152) and coumarin-sulfonamide derivative

8d was most active toward COX-2 isozyme. The most active derivatives met the in silico criteria for

orally active drugs; thus, they may serve as promising candidates to develop more potent and highly

eﬃcient antioxidant, antimicrobial, and/or anti-inﬂammatory agents.

Keywords: anti-inﬂammatory; antioxidant; antimicrobial; coumarin; sulfonamide; pyranocoumarin;

synthesis

1. Introduction

Many diseases result from oxidative stress caused by the overproduction of reactive oxygen species

(ROS) [1], which play an essential role in the pathogenesis of cardiovascular and neurodegenerative

diseases as well as in tumour growth, and age-related disorders. Inﬂammatory diseases can aﬀect

the quality of life of many patients; however, the current medicinal drugs are not always eﬀective

and may cause serious adverse eﬀects [2]. Nonetheless, infectious diseases are among the leading

Molecules 2020, 25, 3251; doi:10.3390 /molecules25143251

ww w .mdpi.com /journal /molecules

Molecules 2020, 25, 3251

2 of 28

causes of death globally [3], and antimicrobial resistance has been commonly reported worldwide [3,4].

These obstacles necessitate the search for new therapies with potential antioxidant, anti-inﬂammatory,

and antimicrobial activities.

Coumarin (2H-1-benzopyran-2-one) derivatives are a large class of highly important lactones

containing a fused structure of a benzene ring and

appealed to medicinal chemists because of their potential role in preventing and treating diseases [

Numerous research reports have indicated hydroxycoumarin and its dihydropyran derivatives as

potential candidates for the development of antioxidant (Figures 1 and 2; I_a], II ], III ], and 10]),

anti-inﬂammatory (III , and VI 11]), and antimicrobial (I_b–d12], I_e13], I_f14], and IV 15]) agents.

α

-pyrone. Coumarin derivatives have increasingly

5,6

].

[

7

[

8

[9

V [

,

V

[

Therefore, they are considered as a compelling starting point for a wide range of applications in the

medicinal ﬁeld.

Despite massive eﬀorts by various global academic and industrial research laboratories over

the years, developing antioxidant, anti-inﬂammatory, and antimicrobial agents that are potent, safe,

and selective remains quite challenging. On one hand, coumarin derivatives represent a broad spectrum

of biological activities, which may be useful in developing new compounds with the aforementioned

desired activities [16]. On the other hand, sulfonamides have become an intriguing scaﬀold due to

their broad medicinal applicability. Several sulfonamides are clinically used as therapeutic agents,

such as the anti-inﬂammatory agents celecoxib [17] and sulfasalazine [18] and the antimicrobial agents

sulfamethoxazole [19] and sulfadiazine [20] (Figure 2). Thus, they have been considered as promising

molecules for the design and development of new drugs.

Previous reports have suggested that beginning with modiﬁcation of compounds known to have

pharmacological eﬀects (pyranocoumarins, Figure 1) or conjugating two structures with promising

biological eﬀects into an interesting motif (coumarin-3-sulfonamides, Figure 2) may be useful for

developing new eﬀective therapeutic agents. Therefore, this work aimed to design and synthesise new

coumarin derivatives [21], including pyranocoumarins and coumarin-sulfonamides, in order to evaluate

their antioxidant, anti-inﬂammatory, and antimicrobial activities against medically important bacterial

and fungal strains. In this study, 4-hydroxy-6-methylcoumarin and 6-chloro-4-hydroxycoumarin

moieties were selected as pharmacophores with the aim of obtaining compounds with more powerful

desired eﬀects. In addition, in silico assessment of the drug-likeness of the target compounds

was performed.

Figure 1. Bioactive pyranocoumarin derivatives and design of pyranocoumarins. DPPH, 2,2-diphenyl-

1-picrylhydrazyl.

Molecules 2020, 25, 3251

3 of 28

Figure 2. Bioactive coumarin derivatives and design of coumarin-sulfonamides. * CPE: Carrageenan-

induced paw oedema.

2. Results and Discussion

2.1. Chemistry

In this work, facile and green chemistry methods were used for synthesising new coumarin

derivatives via the routes illustrated in Schemes 1 and 2. Synthetic Scheme 1 shows the procedure

for pyranocoumarin preparation. All of these derivatives have a chiral centre at Position 4, which are

conﬁrmed by one and, in some cases, two dimensional NMR spectroscopy and supported by the

X-ray analysis of 2a. These results suggest the presence of these compounds as racemic mixtures

of ( )-enantiomers. High yields of the new pyranocoumarins 2a,b were prepared with one-pot

±

reaction using water as a solvent by the reaction of diﬀerent aryl aldehydes with malononitrile and

4-hydroxy-6-methylcoumarin, catalysed by potassium hydrogen phthalate (KHP) to generate the

desired carbonitriles according to a previously reported method [22]. The X-ray crystal structure of

compound 2a was studied at 123 K (Figure 3 ). In the crystal structure, NH protons were reﬁned

freely. Moreover, the nitrobenzene moiety in Position 4 is perpendicular to the pyranocoumarin

moiety. This compound has chirality at C-3 and was crystallised in a space group P 21/n with a

chiral monoclinic centrosymmetric arrangement conﬁrming its presence as a racemic mixture (R/S).

The tentative assignments are done on the basis of geometric and atom type data in the cif. The full

crystallographic data are included in the Supplementary Materials (Table S1).

Molecules 2020, 25, 3251

4 of 28

Scheme 1. Synthetic routes of pyranocoumarins 2–5

.

DMF-DMA, N,N-dimethylformamide-

dimethylacetal; M.W., microwave; R.T., room temperature.

Figure 3. X-ray crystal structure of 2a.

The carboxamide derivatives 3a and 3b were obtained in good yields through acidic hydrolysis

of 2a or 2b with sulphuric acid, at room temperature. The reaction of 2a,b with excess

N,N-dimethylformamide-dimethylacetal (DMF-DMA) at 100 ^◦C yielded the corresponding derivatives

4a,b within 15–40 min. Microwave irradiation was used to obtain the target derivatives 5a–f by treating

Molecules 2020, 25, 3251

5 of 28

2a,b with diﬀerent aromatic aldehydes in 1,4-dioxane. Several pilot experiments were carried out to

optimise the irradiation time using the Biotage Initiator+ microwave (400 W). It was revealed that

microwave irradiation for 90–110 min was optimal for this reaction. In this method, the products were

obtained with a short reaction time without using hazardous catalysts; however, all compounds had

low yields (6–37%).

Synthetic Scheme 2 shows the procedure for preparing coumarin-3-sulfonamide compounds.

Coumarin sulfonyl chlorides 6a,b were produced with excellent yields (

≥

97%) by treating 4-hydroxy-

◦

6-substituted coumarin 1a or 1b with chlorosulfonic acid in dichloromethane at 0 C. Despite the

limited available information regarding coumarin-3-sulfonamides, they were generally obtained either

by using a Knoevenagel condensation reaction [23] or through the condensation of coumarin-3-sulfonyl

chlorides with aromatic amines [24]. In the present work, the coumarin-3-sulfonamides 7a–f were

prepared by reﬂuxing an equimolar quantity of 6a or 6b and diﬀerent sulfa-drugs in absolute

ethanol. However, the reaction of 6a or 6b with nucleophilic reagents (1:2), aniline or p-substituted

aniline, in absolute ethanol under reﬂux involves the formation of the corresponding coumarin-3-

sulfonamides 8a–f with yields of 48–92%. Coumarin-sulfonamide chalcone derivatives 9a–f were

synthesised in low yields (15–35%) by Claisen–Schmidt condensation of p-acetyl derivatives of

4-hydroxy-6-(substituted)coumarin-3-sulfonamides 8e or 8f with the p-substituted aromatic aldehyde

through adding sodium hydroxide solution in ethanol at room temperature.

Scheme 2. Synthetic routes of coumarin-sulfonamide derivatives 6–9.

Molecules 2020, 25, 3251

6 of 28

2.2. Antioxidant Testing

The antioxidant eﬀects of the synthesised compounds were assessed using the 2,2-diphenyl-1-

picrylhydrazyl (DPPH) antioxidant assay [25 26]. Table 1 summarises the antioxidant eﬀects of the test

,

samples and ascorbic acid except for compound 5b because it was produced in poor yield. Fourteen

compounds exhibited free radical scavenging activity; however, all compounds showing antioxidant

activity were dose-dependent.

Among the pyranocoumarins, only compounds 3b and 5d showed modest antioxidant activity,

with IC₅₀values of 48.38

coumarin-sulfonamide derivatives 7a–f were comparable to those of compounds 3b and 5d with IC₅₀

±

4.61 and 82.92

±

3.30 µg/mL, respectively. Similarly, the activities of the

values in the range of 61–120

µg/mL. However, antioxidant activity was signiﬁcantly improved in

compounds 8a–f (IC₅₀= ~4–35

µ

g/mL), of which 8c were the most potent. The coumarin-sulfonamide

,d

chalcones 9a–f demonstrated no antioxidant activity, with IC₅₀values >200 µg/mL.

Regarding the newly synthesised coumarin-3-sulfonamides, generally, the chloro-substituted

compounds at Position 6 of the coumarin ring showed higher radical scavenging potential than

the methyl-substituted derivatives. Interestingly, compounds 8c

activity; this improved activity might be explained by the presence of the phenolic hydroxyl group.

In addition, compounds 8a showed good radical scavenging activities when compared to ascorbic

acid. The sulfathiazole-containing compounds 7e and sulphanilamide-containing compounds 7a

,d exhibited the highest antioxidant

,b,e,f

,f

,b

showed higher antioxidant activity than that of sulfadiazine-substituted compounds 7c,d.

Table 1. Antioxidant activity of coumarin compounds and ascorbic acid using DPPH antioxidant assay

(IC₅₀, µg/mL).

Compd

IC₅₀

Compd

IC₅₀

Compd

IC₅₀

2a

2b

3a

3b

4a

4b

5a

5c

—

5f

7a

7b

7c

7d

7e

7f

8a

8b

8c

—

8d

8e

8f

9a

9b

9c

9d

9e

9f

4.30 ± 0.531

93.12 ± 0.727

61.78 ± 2.719

120.12 ± 4.629

112.19 ± 4.391

71.67 ± 3.231

83.52 ± 1.849

14.51 ± 1.827

19.25 ± 4.171

3.87 ± 0.409

32.85 ± 1.322

35.36 ± 3.265

48.38 ± 4.616

—

82.92 ± 3.300

—

5d

5e

Ascorbic acid *

2.83 ± 0.166

Values represent mean

2,2-diphenyl-1-picrylhydrazyl; IC₅₀, half maximal inhibitory concentration; Compd, compound.

±

SD (n = 3). (—): inactive (IC₅₀> 200 µg/mL). * Positive control. DPPH,

2.3. Antimicrobial Testing

The new molecules were individually tested for their antimicrobial activity in vitro against standard

bacteria strains from the American Type Culture Collection (ATCC), namely, Staphylococcus aureus

ATCC 29213, Bacillus subtilis ATCC 6633, Bacillus megaterium ATCC 9885 (Gram-positive), Escherichia coli

ATCC 2592, and Pseudomonas aeruginosa ATCC 27,953 (Gram-negative). The compounds were also

microbiologically tested against locally isolated Saccharomyces cerevisiae (yeast) and the standard

Agricultural Research Service Culture Collection (NRRL) strain of the yeast-like pathogenic fungus

Candida albicans NRRL Y-477. Primary screening was conducted using the agar well diﬀusion

method [27] using nutrient agar (NA) medium and Sabourand dextrose agar (SDA) medium for the

tested pathological bacteria and yeast, respectively. The minimal inhibitory concentration (MIC) for

the most active compounds (having inhibition zones (IZ) >16 mm) against the same microorganisms

used in the primary screening was evaluated using the two-fold serial dilution technique with the

proper nutrient broth [28].

Table 2 represents the preliminary antimicrobial testing results using the agar well diﬀusion method.

The compounds showed varying degrees of inhibition against these microorganisms. Generally, potent

Molecules 2020, 25, 3251

7 of 28

antimicrobial eﬀects were observed with compounds 2a,b

,

5a,

c,

f,

7a

,c

,d–f

,

8a

,

c,d

, and 9b–f with IZs

larger than 25 mm against at least one tested microorganism. However, molecules 5e and 8b were

moderately active with IZs between 20 and 24 mm, whereas 3a 4a 5d 8e , and 9a showed

,b,

,

,f

marginal activity (IZs of 15–19 mm). Contrarily, only molecule 7b showed no inhibitory activity against

the tested microorganisms (IZ <15 mm).

The MICs of the active molecules (IZs >16 mm) are summarised in Table 3. Generally, compounds

7c

one or more tested microorganisms when compared to standards, with MICs of 125

compound 7d demonstrated broad-spectrum activity against all microorganisms with a 125

,

d

,

8c

,

d

, and 9c

,

d

which showed the highest IZs, also exhibited potent antimicrobial activity against

g/mL. Interestingly,

g/mL MIC.

µ

Microbiological screening showed that the newly synthesised pyranocoumarins exhibited variable

antimicrobial eﬀects against Gram-positive and Gram-negative bacteria and fungi. These compounds

were generally more sensitive toward Gram-positive bacteria. However, only compounds 5a and 5c

which contained unsubstituted benzylidene and 3,4,5-trimethoxybenzylidene, respectively, displayed

strong antibacterial activity against Gram-negative bacteria (IZ: 24–26 mm) with strong-to-moderate

,

inhibitory activity against Gram-positive bacteria (22–27 mm). Similarly, compounds 2a

showed strong-to-moderate antibacterial eﬀects against Gram-positive bacteria (20–25 mm), with MICs

in the range of 500–1000 g/mL. Amongst the entire series, only compound 5a, which contains an

unsubstituted benzylidene, possessed strong antimicrobial activity against most tested microorganisms

(22–27 mm) with MICs of 250–500 g/mL, as compared to the control drugs. In addition, strong

,b and 5f

µ

antimicrobial activity was observed against S. cerevisiae in compounds 2a (25 mm), 2b (27 mm), and 5c

(27 mm), with a 500 µg/mL MIC.

Most of the newly synthesised coumarin-3-sulfonamides exhibited strong-to-moderate

antimicrobial activity with respect to the reference drugs. Generally, most of these molecules

demonstrated broad-spectrum antimicrobial activity. In addition, compounds 7a,c,d, 8a,c,d, and 9c,d

exhibited antimicrobial activity against at least one microorganism that was equal to or higher than

those of the standard antimicrobials ciproﬂoxacin and ketoconazole.

Samples 7c

higher antibacterial activity (30 mm) than that of standard ciproﬂoxacin (28 mm) against S. aureus,

with an MIC of 125 g/mL. Meanwhile, compounds 7a, and 9c which contain sulfanilamide or

4-chlorophenyl moieties, displayed antibacterial activity equal to that of ciproﬂoxacin (28 mm) against

S. aureus, with a 250 g/mL MIC. The antibacterial activity against B. subtilis by compounds 7c which

are substituted with sulfadiazine, was higher than that of the reference drug (30 mm) by one unit,

whereas the same activity of compounds 7a 8c , and 9c was lower by one unit. Compounds 7c

,d, and 8c,d which contain sulfadiazine or 4-hydroxyphenyl moieties, exhibited slightly

µ

,d

µ

,d

,

,d

,d,

and 9c showed antibacterial activity against B. megaterium equal to that of ciproﬂoxacin (30 mm). On the

other hand, only compounds 8d and 9d, containing a methyl moiety along with 4-hydroxyphenyl

or 4-chlorophenyl, respectively, exhibited higher antibacterial activity than ciproﬂoxacin (30 mm)

against E. coli by one and eight units, respectively. Meanwhile, compounds 7c and 9c, containing a

chloro moiety along with sulfadiazine or 4-chlorophenyl, respectively, possessed antibacterial potential

against E. coli similar to that of the control drug. Only compound 8d, containing a methyl moiety and

4-hydroxyphenyl, exerted antibacterial activity (31 mm) against P. aeruginosa higher than that of the

standard drug, whereas 9c showed activity equal to the standard (30 mm).

The antimicrobial activity against S. cerevisiae of compounds 7c

diameters of 31 or 32 mm, which was slightly higher than that of the control drug, ketoconazole

(30 mm). In addition, compounds 7d 8a , and 9c exhibited antifungal activity against C. albicans

,d, 8c,d, and 9c,d showed IZ

,

,c,d

,d

that was two units higher than that of the reference drug (28 mm).

Lipophilicity is considered one of the most important properties in the optimisation process.

Moreover, lipophilicity aﬀects not only the pharmacodynamic proﬁle of the drug, but also its

pharmacokinetics [29]. However, increasing the lipophilicity of a compound facilitates microbial cell

penetration, hence improving antimicrobial activity [30,31]. In fact, only compound 7b was inactive

against all tested microorganisms and showed the lowest consensus Log P-value (1.05) amongst

Molecules 2020, 25, 3251

8 of 28

all tested compounds, which may be attributed to its low Log P-value. Meanwhile, the remaining

compounds, which showed mild-to-strong activity against one or more microorganism, have consensus

Log P-values in the range of 1.23–5.33. However, lipophilicity is not the only factor that inﬂuences

biological activity; other factors such as electronic eﬀects and steric properties may also contribute to

the biological eﬀects of these compounds.

Table 2. Antimicrobial activity expressed as the diameter (mm) of inhibition zones of chemical

compounds (except 5b), ciproﬂoxacin, and ketoconazole against the pathological strains S. aureus

ATCC 29,213 (SA), B. subtilis ATCC 6633 (BS), B. megaterium ATCC 9885 (BM), E. coli ATCC 2592 (EC),

P. aeruginosa ATCC 27,953 (PA), S. cerevisiae local isolate (SC), and C. albicans NRRL Y-477 (CA) based on

well diﬀusion assay.

Gram +ve Bacteria

Gram −ve Bacteria

Fungi

CA

Compd

Consensus Log P ^a

SA

BS

BM

EC

PA

SC

2a

2b

3a

3b

4a

4b

5a

5c

5d

5e

5f

7a

7b

7c

7d

7e

7f

8a

8b

8c

8d

8e

8f

9a

9b

9c

9d

9e

2.5

24

25

-

22

23

18

-

27

23

-

16

23

29

-

31

25

27

28

19

29

15

18

24

29

21

25

30

-

25

16

-

22

24

-

16

20

25

-

30

25

27

29

24

28

29

16

15

18

25

30

29

20

26

30

-

18

16

-

15

26

24

-

15

20

28

-

30

28

20

23

25

18

27

31

-

20

21

15

16

-

15

24

25

-

15

21

27

-

24

25

22

24

26

17

29

31

-

25

27

-

17

-

22

23

-

18

-

2.39

1.76

1.75

3.03

2.94

4.29

4.21

5.33

5.28

1.23

1.05

1.88

1.71

2.51

2.36

2.52

2.41

2.03

1.90

2.45

2.32

3.5

16

25

23

18

16

25

28

-

30

23

24

26

20

30

-

16

22

28

23

25

28

-

26

27

19

22

24

26

-

31

32

20

26

28

19

32

-

25

24

19

20

22

26

-

27

30

23

24

29

20

30

-

3.15

4.48

4.32

3.88

3.73

26

30

38

21

26

30

-

26

30

29

21

28

30

-

25

31

32

29

26

-

23

30

23

24

-

9f

Ciproﬂoxacin

Ketoconazole

30

28

a

(-): inactive (IZ < 15 mm); Compd, compound. Calculated using SwissADME web tool. Consensus Log P is the

average of ﬁve diﬀerent prediction methods for the calculation of computerised partition coeﬃcient, including ilog

P, Xlog P3, Wlog P, Mlog P, and Log P_SILICOS-IT

.

Molecules 2020, 25, 3251

9 of 28

Table 3. Minimum inhibitory concentration (µg/mL) of the most active compounds (having inhibition

zones >16 mm), ciproﬂoxacin, and ketoconazole against the pathological strains S. aureus ATCC 29,213

(SA), B. subtilis ATCC6633 (BS), B. megaterium ATCC 9885 (BM), E. coli ATCC 2592 (EC), P. aeruginosa

ATCC 27,953 (PA), S. cerevisiae local isolate (SC), and C. albicans NRRL Y-477 (CA) based on two-fold

serial dilution technique.

Gram +ve Bacteria

Gram –ve Bacteria

Fungi

Compd

SA

BS

BM

EC

PA

SC

CA

2a

2b

3a

3b

5a

5c

5d

5e

5f

7a

7c

7d

7e

7f

8a

8b

8c

8d

9a

9b

9c

9d

9e

9f

500

N.D.

250

500

1000

N.D.

500

250

125

500

250

500

1000

125

N.D.

500

250

500

125

500

1000

N.D.

250

500

N.D.

500

1000

N.D.

250

500

1000

N.D.

500

N.D.

1000

500

1000

500

250

125

1000

250

1000

125

N.D.

500

125

250

500

N.D.

1000

500

1000

500

250

125

500

250

1000

125

N.D.

500

125

500

N.D.

125

500

N.D.

500

250

125

500

250

1000

125

1000

500

250

500

125

N.D.

1000

500

125

500

250

1000

250

1000

500

250

500

250

125

N.D.

1000

250

125

500

1000

250

125

N.D.

250

500

250

125

N.D.

1000

250

125

1000

500

1000

250

125

N.D.

250

500

250

125

N.D.

500

N.D.

125

Ciproﬂoxacin

Ketoconazole N.D.

N.D.

N.D., not determined; Compd, compound.

2.4. Anti-Inﬂammatory Activity

The newly synthesised compounds were individually tested for their in vitro anti-inﬂammatory

activity against proteinase enzyme [32]. The most active compounds, 2b, 3a 7f, and 8c, were tested

,

further in vivo for their acute anti-inﬂammatory activity using the formaldehyde-induced paw oedema

method in rats [33]. This test was performed at the Pharmacology Department, Medical Research

Division, National Research Centre, Cairo, Egypt. Furthermore, compounds 2–8 were evaluated for

their COX1/COX2 inhibitory activities.

2.4.1. Proteinase-Inhibitory Activity

Proteinases play a signiﬁcant role in inﬂammatory reactions; in particular, they can contribute to

arthritic reactions. Proteinases mainly exist in the lysosomal granules of neutrophils [34]. However,

proteinase inhibitors were reported to exhibit signiﬁcant levels of protection against tissue damage

and proteolytic activity of neutrophils during inﬂammatory reactions [34–36].

The proteinase-inhibitory activities of the newly synthesised compounds are summarised in

Table 4 . The inhibition of proteinase activity at 250

2b, with 79.72 4.51% and 74.68 3.01% inhibition, respectively. Next, compounds 5c and 9a exhibited

percent inhibition values of 68.5

and 8a showed proteinase-inhibitory activity with 41–49% inhibition. Meanwhile, compounds 4a

µg/mL was found to be highest in compounds 3a and

±

6.23% and 62.14

±

4.87%, respectively. Compounds 3b

,

5d,e

,

7f

,b

,

,c

Molecules 2020, 25, 3251

10 of 28

7d, and 9d showed modest proteinase-inhibitory activity, with percent inhibition values of 31–36%.

The remaining compounds showed weak antiproteinase activity with percent inhibition values below

30%. Compound 9b, however, showed no antiproteinase activity at the tested concentration.

Table 4. Proteinase-inhibitory activity of the coumarin compounds and aspirin.

Compd

% Inhibition

Compd

% Inhibition

Compd

% Inhibition

2a

2b

3a

3b

4a

4b

5a

5c

17.38 ± 1.63

74.68 ± 3.01

79.72 ± 4.51

49.28 ± 10.93

31.99 ± 2.45

33.17 ± 2.79

N.D.

68.5 ± 6.23

43.4 ± 1.52

45.78 ± 1.62

5f

7a

7b

7c

7d

7e

7f

8a

8b

8c

26.83 ± 5.33

18.78 ± 2.05

5.93 ± 2.11

8.9 ± 2.04

8d

8e

8f

9a

9b

9c

9d

9e

9f

20.15 ± 7.31

18.5 ± 3.47

2.0 ± 1.4

62.14 ± 4.87

36.84 ± 4.87

19.8 ± 2.54

43.88 ± 5.6

44.7 ± 8.22

14.3 ± 2.21

41.69 ± 2.83

—

17.6 ± 6.86

31.88 ± 3.82

22.83 ± 4.88

21.7 ± 7.6

45.83 ± 4.21

5d

5e

Aspirin *

Values represent mean ± SD (n = 3). (—): inactive. N.D., not determined; Compd, compound. * Positive control.

The pyranocoumarins 2b

,

3a

,

b

, and 5c showed more potent antiproteinase activity than did

were comparable to the standard drug.

standard aspirin (45.83 4.21%), whereas compounds 5d,e

±

The remaining pyranocoumarins showed moderate-to-poor activity. Compound 2a, containing

4-nitrophenyl, showed weak antiproteinase activity, while the replacement of a cyano group with

carboxamide (3a) signiﬁcantly enhanced this activity. In addition, replacement of the protons of the

amino group with 3,4,5-trimethoxybenzylidene or 2,4-dichlorobenzylidene (5c and 5e, respectively)

signiﬁcantly enhanced the proteinase-inhibitory activity, indicating that the presence of a separate

4-nitrophenyl in pyranocoumarin does not contribute to signiﬁcant antiproteinase activity, but its

presence along with carboxamide, 3,4,5-trimethoxybenzylidene, or 2,4-dichlorobenzylidene does aid

such activity. In contrast, compound 2b, which contains furan-2-yl, showed signiﬁcant antiproteinase

activity; however, hydrolysis of the cyano group resulting in carboxamide (3b) or the replacement of

amino group protons with 3,4,5-trimethoxybenzylidene or 2,4-dichlorobenzylidene (5d,f) reduced the

activity by more than 25%. Compounds 4a,b exhibited modest antiproteinase activity. This is probably

due to the presence of N,N-dimethyl formimidamide at Position 2 of the pyrano ring.

Regarding the newly synthesised coumarin-3-sulfonamides, only compound 9a exhibited higher

antiproteinase activity than aspirin due to the presence of the chloro group at Position 6 of the coumarin

ring along with 4-nitrophenyl in the chalcone moiety. Conversely, replacement of this chloro substituent

with a methyl group diminished the antiproteinase activity. Meanwhile, compounds 7f and 8a,c

possess activity comparable to that of standard aspirin. The remaining compounds of this series

exhibited modest-to-poor antiproteinase activity. The presence of a methyl moiety at Position 6 along

with pyrimidine or thiazole rings in compounds 7d

In contrast, this activity was signiﬁcantly reduced by replacing the methyl moiety with a chloro moiety

or replacing the rings with hydrogen. Compounds 8e showed poor antiproteinase activity due to the

presence of 4-acetylphenyl at Position 3 of the coumarin ring. Meanwhile, compounds 8a showed

,f was essential to their antiproteinase activity.

,

f

,

c

good activity that could be due to the presence of the chloro moiety and phenyl or 4-hydroxyphenyl at

Positions 6 and 3, respectively, of the coumarin ring. In contrast, replacement of the chloro moiety

with methyl reduced the activity signiﬁcantly. Compounds 9e,f exhibited weak antiproteinase activity

that might be due to the presence of 4-methoxyphenyl in the chalcone moiety. Compound 9d, which

contains methyl and 4-chlorophenyl, showed modest antiproteinase activity, whereas compound 9c

,

containing chloro and 4-chlorophenyl, exhibited poor activity.

2.4.2. Pharmacological In Vivo Study

Prophylactic anti-inﬂammatory eﬀect on formaldehyde-induced rat paw oedema:

Molecules 2020, 25, 3251

11 of 28

Based on the results of the antiproteinase activity, the most eﬀective compounds, 2b and

3a, and the moderately eﬀective ones, 7f and 8c, were selected for their in vivo evaluation of

anti-inﬂammatory activity.

In the present study, when rat paw volumes were consecutively measured 1, 2, and 3 h after

subplantar injection of 0.2 mL (1%, w/v) formaldehyde in the positive control group, it was found that

formaldehyde signiﬁcantly increased paw volumes by 109.6, 108.7, and 94.89%, respectively, of their

basal volumes (Figure 4).

When used orally as a reference drug for coumarins, indomethacin (25 mg/kg) signiﬁcantly

inhibited inﬂammation, as evidenced by paw volume reduced by 72.09, 74.79, and 71.5% compared to

the positive control group after 1, 2, and 3 h, respectively, following subplantar injection of 0.2 mL

(1% w/v) formaldehyde.

Regarding the anti-inﬂammatory eﬀects of coumarins 2b, 3a, 7f, and 8c, all compounds produced

signiﬁcant protection against inﬂammation throughout the experiment duration (Figure 5). Compound

3a showed signiﬁcantly better inhibition than others (29.2% inhibition) only 1 h after inducing

inﬂammation, followed by compound 7f (17.18% inhibition), 8c (9.45%) and 2b (6.57%). However,

3a was less eﬀective than indomethacin; which is likely due to administering these compounds at

one-tenth of the indomethacin concentration. Moreover, the anti-inﬂammatory eﬀects of the compounds

were signiﬁcantly increased after 2 and 3 h when compared to the positive control group.

Figure 4. Percent of oedema in rats pretreated with indomethacin (25 mg/kg) and compounds 2b

,

3a,

7f, and 8c (2.5 mg/kg). Results are expressed as means

p < 0.0001 (* vs. formaldehyde, # vs. indomethacin).

±

SE, n = 6. Results are statistically signiﬁcant at

Molecules 2020, 25, 3251

12 of 28

Figure 5. Percent inhibition of the standard drug indomethacin (25 mg/kg) and compounds 2b, 3a, 7f,

and 8c (2.5 mg/kg) on rat paw oedema induced by formaldehyde (0.2 mL/kg). Results are expressed as

means ± SE, n = 6. Results are statistically signiﬁcant at p < 0.0001 (# vs. indomethacin).

2.4.3. COX Inhibition

Compounds 2–8 were further tested for their inhibitory activities against COX-1 and COX-2 using

human assay kits. The COX-inhibitory eﬀects of these compounds and the standards indomethacin

and celecoxib are summarised in Table 5. All compounds were screened for their COX-inhibitory

activity except compounds 9a–f because they were obtained in low yield. The selectivity index (ratio of

COX-1/COX-2) of each compound was determined and compared with that of celecoxib, a selective

COX-2 inhibitor.

Compared to indomethacin, all of these molecules were generally less active against COX-1

isozyme and more active toward COX-2. However, potent COX-2-inhibitory activity (IC₅₀

observed in most pyranocoumarin derivatives (2a 3a 4a, and 5a ) and six coumarin-sulfonamide

derivatives (7b , and 8a ). Interestingly, compound 5a showed signiﬁcant selectivity toward

COX-2 isozyme, which was highest amongst the tested compounds (SI = 152) compared to the standard

celecoxib (SI = 243.9). Compounds 2a and 7d followed with SI values of 64, 47.3, and 42.6, respectively.

≤

10µM) was

,

b,

,

b,

,e,f

,d,

f

,d,e

,

b

Regarding the pyranocoumarins, hydrolysis of the cyano group at Position 3 to an amide

or replacement of the amino group at Position 2 with N,N-dimethyl formimidamide was found

to reduce the selectivity toward COX-2 isozyme (2a,b vs. 3a,b and 4a,b). Substitution with

3,4,5-trimethoxybenzylidene or 2,4-dichlorobenzylidene in Position 2 was found to decrease the

selectivity as well (5c–f). Notably, the presence of unsubstituted benzylidene along with the nitrophenyl

at Position 4 improved the selectivity by about 3.6-fold (2a, SI = 42 vs. 5a, SI = 152), whereas a furyl

ring at the same position signiﬁcantly reduced the selectivity (5b, SI = 10.9).

For coumarin-sulfonamides, the presence of a methyl moiety at Position 6 of the coumarin ring

and pyrimidin-2-yl in Position 3 was shown to improve COX-2 selectivity compared to the remaining

derivatives of this series (7d, SI = 64 vs. 7a–c and 7e–f, SI = 1.8–13.7). However, compound 8f

which contains methyl and acetyl moieties, was the only derivative that showed relatively selective

COX-1 activity (SI = 0.3), and replacing the methyl with a chloro moiety changed it into a selective

COX-2 compound (8e, SI = 2.2). Amongst all tested compounds, 8d, containing methyl and hydroxyl

moieties, demonstrated the most potent activity against COX-2 isozyme (IC₅₀= 1.86, SI = 10.5);

,

Molecules 2020, 25, 3251

13 of 28

this potency and selectivity were reduced nearly ten-fold by replacing the methyl with a chloro moiety

(8c, IC₅₀= 11.9, SI = 1.3).

Table 5. In vitro COX-inhibitory activity of compounds 2–8.

IC₅₀(µM) ^a,b

SI ^c

Compd

COX-1

COX-2

2a

2b

3a

3b

116.66

201.16

183.27

7.73

2.74

4.25

9.81

42.58

47.33

18.68

3.16

2.45

4a

4b

5a

5b

5c

5d

5e

5f

7a

7b

7c

7d

7e

7f

8a

8b

12.49

57.46

1126.47

215.39

18.50

27.19

20.80

90.20

239.47

47.60

62.27

214.28

29.62

40.89

25.02

25.99

15.40

19.44

19.90

7.92

7.99

25.23

7.41

19.63

13.30

16.04

8.27

3.10

17.42

8.24

19.94

3.35

16.11

10.07

8.50

1.56

2.28

152.02

10.97

1.39

1.7

2.52

29.1

13.75

5.78

3.12

63.96

1.84

4.06

2.94

1.94

1.29

10.45

2.16

0.31

13.37

11.90

1.86

8c

8d

8e

9.22

8f

25.66

15.14

0.77

Indomethacin

Celecoxib

1.88

187.82

0.12

243.92

a

b

c

The mean of two determinations. The deviation from the mean is <10% of the mean value. Selectivity index

(COX-1/COX-2).

2.5. In Silico Studies

2.5.1. Docking Studies

The major diﬀerence in the COX-2 active site compared to that of COX-1 is the presence of Val 523

instead of Ile 523. As a much smaller amino acid, Val 523 causes a conformational change in Tyr 355,

thus producing a side pocket in COX-2 [37,38]. In addition, it has been reported that His 90, Gln 192,

and Tyr 355 control the access of ligands to this pocket in COX-2 [37,38].

Molecular docking was studied to predict the intermolecular interactions of compounds 5a and

8d with human COX-2 isozyme (PDB ID: 5KIR, Figures 6 and 7 ). The docking simulation shows that

the binding aﬃnity of the coumarin-sulfonamide derivative 8d

(

−

7.3 kcal/mol) was better than that

of the pyranocoumarin derivative 5a

(−

4.4 kcal/mol). These results are consistent with the in vitro

assay ﬁndings.

Compound 8d formed eight hydrogen bonds with the binding site. Oxygen atoms of the coumarin

ring and sulfonamide serve as the acceptors of hydrogen bonds with Typ 355, Arg 513, Phe 518, Ile 517,

and His 90. However, the amino group of the sulfonamide acted as a hydrogen bond donor with Leu

352 and Gln 192. The Val 523 residue formed a Pi-sigma interaction with the pyrone ring of coumarin;

furthermore, other weaker bonds, such as carbon hydrogen, Pi-cation, Pi-sigma, alkyl, and Pi-alkyl

bonds, also accounted for the binding aﬃnity of this compound.

Molecules 2020, 25, 3251

14 of 28

Meanwhile, compound 5a showed only two hydrogen bonds with Ile 517 and Phe 518, and

the oxygen atom of the nitro group served as the hydrogen bond acceptor. Moreover, the Val 523

residue formed a Pi-sigma interaction with the nitrobenzyl ring of this derivative. Other interactions,

including van der Waals, carbon hydrogen, Pi-sigma, and Pi-sulphur bonds, formed between 5a and

other binding site residues. The lower number of hydrogen bonds could contribute to a lower binding

aﬃnity as compared with 8d.

Figure 6. Binding interactions between 8d and human COX-2 enzyme.

Figure 7. Binding interactions between 5a and human COX-2 enzyme.

2.5.2. Drug-Likeness Assessment

In order to study the drug-likeness of the newly synthesised compounds, in silico Lipinski’s Rule of

Five (RO5) and topological polar surface area (TPSA) analyses were conducted using Molinspiration [39].

Molecules 2020, 25, 3251

15 of 28

About 90% of orally active compounds that reach phase II clinical trials or higher satisfy RO5 [40].

This rule states that a molecule likely to be developed as an orally active drug candidate should not

show more than one violation of the following four criteria: Molecular weight

≤

500, octanol-water

10 [40]. Despite

coeﬃcient (LogP) 5, H-bond donors (n-OHNH) 5, and H-bond acceptors (n-ON)

≤

the importance of RO5 for labelling a molecule as “drug-like”, these criteria are restricted to the

topic of oral bioavailability via passive transport alone. Moreover, TPSA is another major factor for

predicting the oral availability of compounds, and its values for intestinal absorption should be less

than 140 Å²[41].

Table 6 shows that all compounds have no more than four hydrogen bond donors, and most

compounds have 10 or fewer hydrogen bond acceptors, except for 5c and 7c,d. The molecular weights

of compounds 5c

ﬁve for most compounds, except 5a

to be oral drug candidates, except compounds 5c

some drugs in clinical use do not obey RO5. The TPSA was less than 140 Å²in all compounds, except

3a 7a–f, and 9a , which indicated easy permeability through the intestinal cell membrane. Based on

,

e,

7c

,e

, and 9a–c

,e

are each greater than 500 Da. The Log P-value is not more than

and 9c . These results indicated that all compounds seem

7c, and 9c which all violated RO5; however,

,e,

f

,d

,

e,

,e

,

,b

these ﬁndings and the results of the biological tests, all tested compounds met the in silico criteria for

orally active drugs and showed promising biological activity; thus, these compounds will be studied

further in vivo for development as orally active agents.

Table 6. The Lipinski parameters and topological polar surface area (TPSA) of target compounds.

TPSA (Å²)

Compd

Mol. Wt

miLogP

n-ON

n-OHNH

RO5 Violations

Rule

2a

2b

3a

3b

4a

4b

5a

5b

5c

5d

5e

5f

7a

7b

7c

7d

7e

7f

8a

8b

8c

8d

8e

8f

9a

9b

9c

≤500

≤5

≤10

≤5

2

4

0

4

2

3

2

3

2

≤1

0

1

0

2

0

2

1

0

2

1

0

1

2

1

2

0

≤140

135.08

102.40

154.39

121.70

124.66

91.98

121.42

88.74

149.13

116.44

121.42

88.74

156.77

168.56

155.67

96.61

375.34

320.30

393.36

338.32

430.42

375.38

463.45

408.41

553.53

498.49

532.34

477.30

430.85

410.43

508.92

488.50

513.96

493.54

351.77

331.35

367.77

347.35

393.80

373.39

526.91

506.49

516.36

495.94

511.94

491.52

3.10

2.40

2.05

1.35

3.58

2.88

5.16

4.45

4.79

4.08

6.44

5.74

1.76

1.53

2.01

1.78

2.89

2.66

3.07

2.84

2.59

2.36

2.97

2.74

4.90

4.67

5.62

5.39

5.00

4.77

8

6

9

7

9

7

8

6

11

9

8

6

9

11

10

6

7

96.61

116.84

113.68

159.50

113.68

122.91

7

10

7

8

9d

9e

9f

8

(n-OHNH), H-bond donors; (n-ON), H-bond acceptors; Compd, compound; Mol. Wt, molecular weight; RO5, rule

of ﬁve.

Molecules 2020, 25, 3251

16 of 28

3. Materials and Methods

3.1. Chemistry

The starting material 4-hydroxy-6-methylcoumarin (1b) was synthesised according to the method

reported by Shah and colleagues [42]. All other reagents and solvents were obtained from commercial

suppliers and used without further puriﬁcation. Melting points (MP; ^◦C, uncorrected) were recorded

using a melting point apparatus (Electrothermal, Staﬀordshire, UK). The infrared (IR) spectra (expressed

in wave number

υ

[cm⁻¹]) were recorded by the SHIMADZU FT/IR S1 Plus Spectrometer (Shimadzu

Europa GmbH, Duisburg, Germany) using potassium bromide (KBr) discs. The Agilent Technologies

600 Ultra Shield NMR Spectrometer (Santa Clara, CA, USA) was utilised to obtain NMR spectra at

600 and 154 MHz for ¹H and ¹³C, respectively. Chemical shifts are expressed in

δ (ppm); coupling

constants are reported in Hz. CDCl₃and DMSO-d₆were used as solvents. The splitting patterns were

designated as: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet),

br (broad), and br s (broad singlet). The Bruker Ascend 700 NMR Spectrometer (Bruker, Fällanden,

Switzerland) was utilised to obtain NMR spectra of compounds 2a, 4a, 5d, and 9c,d,f at 700.17 and

176.08 MHz for ¹H and ¹³C, respectively. The ¹³C-NMR spectrum of compound 5c was obtained at

176.08 MHz. The ¹H and ¹³C-NMR spectra for three compounds in each scheme were presented in the

Supplementary Materials including one intermediate and two target compounds (2a, 4a, 5c; 6b, 8e,

9f; Figures S1–S16). These data include two dimensional NMR spectra of 4a and 8e. The Shimadzu

GC-MS-QP 2010 instrument (Shimadzu Europa GmbH, Duisburg, Germany) was used to record the

electron impact mass spectra (EI-MS) at 70 eV. Elemental analyses (C, H, and N) were carried out at

the Microanalytical Center of the Faculty of Science of Cairo University, Cairo, Egypt. They aligned

with the proposed structures within

±

0.1–0.3% of the theoretical values. The microwave reaction was

conducted using a microwave reactor (Biotage Initiator+, EXP EU, 400 W, 2450 MHz; Biotage, Uppsala,

Sweden). The reactions were monitored by thin-layer chromatography (TLC) using silica gel-precoated

aluminium sheets (60 F254, Merck, Kenilworth, NJ, USA) and visualised with UV at 365 and 254 nm.

3.1.1. 4-Hydroxy-6-methylcoumarin (1b)

This compound was synthesised following the reported procedure [42]. Yield: 75.67%. MP:

259–261 ^◦C. EI-MS, m/z (Rel. Int.%): 176 (M⁺, 40.7), 147.

3.1.2. 2-Amino-9-methyl-5-oxo-4-aryl-4H,5H-pyrano[3,2-c]chromene-3-carbonitriles (2a–b)

These compounds were synthesised according to the reported procedure [22]. A mixture of

arylaldehyde namely, p-nitrobenzaldehyde or furfuraldehyde, (1mmol), 4-hydroxy-6-methylcoumarin

(0.176 g 1mmol), malononitrile (0.066 g, 1mmol) and KHP (0.2 g) in distilled water (5 mL) was heated

o

at 50 C for 5–6 h. After the completion of the reaction, the reaction mixture was cooled to room

temperature and the solid was ﬁltered oﬀ, washed with cold distilled water, dried, and recrystallised

with CHCL₃/ethanol to give compounds 2a,b.

(±

)-2-Amino-9-methyl-4-(4-nitrophenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (2a): Yield:

98%; yellow solid. MP: 259–261 ^◦C. IR ( _max, cm⁻¹): 3460, 3346 (NH₂), 2204 (CN), 1716 (C=O). ¹H-NMR

(DMSO-d₆, 600 MHz): 2.44 (s, 3H, Ar-CH₃), 4.6 (s, 1H, CH-4), 7.3 (d, J = 8.4, 1H, Ar-H), 7.55–7.59

(m, 5H, Ar-H and NH₂), 7.73 (s, 1H, Ar-H-10), 8.2 (d, J = 9, 2H, Ar-H-3⁰, 5⁰). ¹³C-NMR (DMSO-d₆,

154 MHz): 20.9 (CH₃), 37.2 (C-4), 57.1 (C-3), 103.1 (C-4’a), 113.05, 116.9, 119.3, 122.6, 124.2, 129.6,

υ

δ

134.4, 134.6, 147.0, 150.9, 151.3, 154.3, 158.5, 160.1 (Ar-C, CN and C=O). EI-MS, m/z (%): 377 (M + 2

,

0.13), 73, consistent with the molecular formula C₂₀H₁₃N₃O₅(375.33).

(

±

)-2-Amino-4-(furan-2-yl)-9-methyl-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (2b): Yield:

93.7%; grey solid. MP: 251–253 ^◦C. IR ( _max, cm⁻¹): 3392, 3323 (NH₂), 2198 (CN), 1701 (C=O). ¹H-NMR

(DMSO-d₆, 600 MHz): 2.38 (s, 3H, Ar-CH₃), 4.57 (s, 1H, CH-4), 6.2 (d, J = 2.4, 1H, Ar-H), 6.3 (d, J =

3.2, 1H, Ar-H), 7.3 (d, J = 8.4, 1H, Ar-H), 7.44 (s, 2H, NH₂), 7.49 (m, 2H, Ar-H), 7.6 (s,1H, Ar-H, CH-10).

υ

δ

Molecules 2020, 25, 3251

17 of 28

¹³C-NMR (DMSO-d₆, 154 MHz):

δ

20.95 (CH₃), 37.3 (C-4), 55.6 (C-3), 102.3 (C-4’a), 106.8, 110.6, 113.0,

116.8, 119.1, 122.3, 133.8, 134.4, 142.8, 150.8, 152.9, 153.9, 158.7, 160.3 (Ar-C, CN and C=O). EI-MS, m/z

(%): 321 (M + 1, 0.15), 256, consistent with the molecular formula C₁₈H₁₂N₂O₄(320.3).

3.1.3. 2-Amino-9-methyl-5-oxo-4-aryl-4H,5H-pyrano[3,2-c]chromene-3-carboxamides (3a,b)

A mixture of 2a or 2b (0.01 mol) and concentrated H₂SO₄(15 mL) was kept at room temperature

(25

±

2 ^◦C) with continuous stirring for 10–14 h and then poured on cold water. The solid was ﬁltered,

washed with distilled water, dried, and crystallised with the indicated solvent.

(

±

)-2-Amino-9-methyl-4-(4-nitrophenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carboxamide (3a):

◦

Crystallisation solvent: ethanol (EtOH)/CHCl₃. Yield: 92.9%; yellow solid. MP: 142–144 C. IR

1

(υ

_max, cm⁻¹): 3446, 3408 (NH₂), 1734 (C=O ester), 1676 (C=O amide). H-NMR (DMSO-d₆„ 600 MHz):

δ

2.34 (s, 3H, CH₃), 4.86 (s, 1H, CH-4), 7.0 (s, 2H, NH₂), 7.1 (d, J = 9, 2H, Ar-H, CH-2⁰,6⁰), 7.3 (d, J = 8.4

,

1H, Ar-H), 7.5 (s, 2H, NH₂), 7.6 (d, J = 7.2, 1H, Ar-H), 7.7 (s, 1H, Ar-H, CH-10), 8.1 (d, J = 7.8, 2H,

Ar-H, CH-3⁰,5⁰). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.9 (CH₃), 40.4 (C-4) 54.1 (C-3), 103.4 (C-4’a), 113.0,

116.2, 123.32, 124.6, 129.0, 129.7, 133.2, 146.2, 150.1, 150.7, 151.5, 160.6, 162.4, 169.9 (Ar-C, CN and C=O).

EI-MS, m/z (%): 393 (M⁺, 1.1), 204, consistent with the molecular formula C₂₀H₁₅N₃O₆(393.36).

( )-2-Amino-4-(furan-2-yl)-9-methyl-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carboxamide(3b):Crystallisation

±

solvent: EtOH. Yield: 62.5%; brown solid. MP: 202–204 ^◦C. IR (

υ

_max, cm⁻¹): 3446, 3425 (NH₂), 1724

δ 2.22 (s, 3H, CH₃), 4.83 (s, 1H, H-4),

1

(C=O ester), 1685 (C=O amide). H-NMR (DMSO-d₆„ 600 MHz):

6.49 (d, J = 2.4, 1H, Ar-H), 6.78 (d, J = 3.2, 1H, Ar-H), 7.04–7.51 (m, 7H, Ar-H and NH₂), 7.79 (s, 1H,

Ar-H-10). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.7 (CH3), 38.6 (C-4), 59.4 (C-3), 103. (C-4’a), 109.0, 115.6,

116.5, 121.5, 123.0, 123.4, 133.1, 134.1, 143.6, 151.7, 152.7, 162.7, 163.5, 166.5 (Ar-C, CN and C=O).

EI-MS, m/z (%): 339 (M + 1, 0.1), 338 (M⁺, 0.1), 296 (25.82), 73, consistent with the molecular formula

C₁₈H₁₄N₂O₅(338.31).

3.1.4. N⁰-(3-cyano-9-methyl-5-oxo-4-aryl-4H,5H-pyrano[3,2-c]chromen-2-yl)-N,N-dimethyl

formimidamides (4a,b)

A mixture of 2a or 2b (2.7 mmol) and DMF-DMA (25 mL) was heated at 100 ^◦C for 15–40 min.

The solid was obtained by ﬁltration, washed with EtOH, dried, and then crystallised with CHCl₃/EtOH.

(

±

)-N⁰-(3-cyano-9-methyl-4-(4-nitrophenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromen-2-yl)-N,N-

dimethylformimidamide (4a): Yield: 66.3%; yellow solid. MP: 279–281 ^◦C. IR ( _max, cm⁻¹): 2204 (CN),

1722 (C=O), 1664 (C=N). ¹H-NMR (DMSO-d₆, 600 MHz):

2.4 (s, 3H, Ar-CH₃), 3.06 (s, 3H, N-CH₃),

υ

δ

3.2 (s, 3H, N-CH₃), 4.7 (s,1H, CH-4), 7.3 (d, J = 8.4, 1H, Ar-H), 7.5 (dd, J = 1.4 and J = 8.4, 1H, Ar-H), 7.6

(dd, J = 2.1 and J = 7, 2H, Ar-H), 7.9 (d, J = 1.4, 1H, Ar-H), 8.2 (dd, J = 2.1 and J = 7, 2H, Ar-H), 8.5

(s,1H, N=CH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.8 (Ar-CH₃), 34.9 (N-CH₃), 38.7 (C-4), 41.2 (N-CH₃),

73.0 (C-3), 102.6 (C-4’a), 113.3, 116.7, 119.2, 123.3, 124.2, 129.8, 134.5, 134.8, 147.1, 150.7, 150.9, 154.9,

155.3, 157.9, 160.3 (Ar-C, CN and C=O). EI-MS, m/z (%): 431 (M + 1, 0.1), 430 (M⁺, 0.14), 415 (5.77), 73,

consistent with the molecular formula C₂₃H₁₈N₄O₅(430.41).

(

±

)-N⁰-(3-cyano-4-(furan-2-yl)-9-methyl-5-oxo-4H,5H-pyrano[3,2-c]chromen-2-yl)-N,N-

dimethylformimidamide (4b): Yield: 39%; white solid. MP: 273–275 ^◦C. IR ( _max, cm⁻¹): 2198 (CN),

1724 (C=O), 1654 (C=N). ¹H-NMR (CDCl₃, 600 MHz):

2.4 (s, 3H, Ar-CH₃), 3.1 (s, 3H, N-CH₃), 3.2

υ

δ

(s, 3H, N-CH₃), 4.8 (s, 1H, CH-4), 6.31–6.36 (m, 2H, Ar-H), 7.23–7.26 (m, 2H, Ar-H), 7.37 (d, J = 8.4, 1H,

Ar-H), 7.5 (s, 1H, Ar-H, CH-10), 8.2 (s, 1H, N=CH). ¹³C-NMR (CDCl₃, 154 MHz): 21.0 (Ar-CH₃), 32.6

(C-4), 35.0 (N-CH₃), 41.2 (N-CH₃), 72.9 (C-3), 101.4 (C-4’a), 107.6, 110.8, 113.4, 116.8, 118.7, 121.8, 133.6,

134.1, 142.2, 150.9, 152.7, 153.5, 154.8, 159.1, 160.5 (Ar-C, CN and C=O). EI-MS, m/z (%): 376 (M + 1, 1.9),

99, consistent with the molecular formula C₂₁H₁₇N₃O₄(375.38).

Molecules 2020, 25, 3251

18 of 28

3.1.5. 2-(Arylideneamino)-9-methyl-4-(4-nitrophenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-

carbonitriles (5a–f)

An aromatic aldehyde (benzaldehyde, 2,4-dichlorobenzaldehyde, or 2,3,4-trimethoxybenzaldehyde,

1 mmol) was added to a mixture of 2a or 2b (1mmol) in 1,4-dioxane and then irradiated in a Biotage

Initiator+ microwave for 90–110 min (TLC monitoring). After solvent evaporation, the residue was

washed with toluene to obtain pure product and dried.

( )-2-(benzylideneamino)-9-methyl-4-(4-nitrophenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-

±

carbonitrile (5a): Yield: 23.5%; buﬀ solid. MP: 208–209 ^◦C. IR (

υ

_max, cm⁻¹): 2198 (CN), 1701 (C=O),

1

1670 (C=N). H-NMR (DMSO-d₆, 600 MHz): 2.39 (s, 3H, CH₃), 4.65 (s, 1H, CH-4), 7.2–7.7 (m, 9H,

Ar-H), 8.1–8.3 (m, 4H, Ar-H and N=CH). ¹³C-NMR (DMSO-d_6,154 MHz): 20.9 (CH₃), 37.2 (C-4), 57.1

(C-3), 103.1 (C-4’a), 112.9, 116.8, 119.3, 122.6, 124.2, 128.0, 128.6, 129.0, 129.3, 129.5, 134.2, 134.4, 134.6,

147.0, 150.9, 151.30, 158.4, 160.1 (Ar-C, CN and C=O). EI-MS, m/z (%): 465 (M + 2, 3.7), 57, consistent

with the molecular formula C₂₇H₁₇N₃O₅(463.44).

(

±

)-2-(benzylideneamino)-4-(furan-2-yl)-9-methyl-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile

5b): Yield: 5.8%; brown solid. MP: 250–252 ^◦C. IR ( _max, cm⁻¹): 2194 (CN), 1703 (C=O), 1670 (C=N).

υ

¹H-NMR (DMSO-d₆, 600 MHz): 2.4 (s, 3H, CH₃), 4.4 (s, 1H, CH-4), 7.2–7.5 (m, 11H, Ar-H), 7.7 (s, 1H,

N=CH). ¹³C-NMR (DMSO-d_6,154 MHz): 20.9 (CH₃), 37.3 (C-4), 58.4 (C-3), 104.3 (C-4’a), 107.7, 110.5,

113.0, 116.8, 119.7, 122.5, 127.6, 128.0, 128.6, 129.0, 129.3, 134.2, 134.5, 143.8, 150.7, 153.8, 158.4, 160.1

(Ar-C, CN and C=O). EI-MS, m/z (%): 410 (M + 2, 4), 409 (M + 1, 3), 408 (M⁺, 18), 56, consistent with

the molecular formula C₂₅H₁₆N₂O₄(408.41).

(±

)-9-methyl-4-(4-nitrophenyl)-5-oxo-2-((3,4,5-trimethoxybenzylidene)amino)-4H,5H-pyrano[3,2-c]

chromene-3-carbonitrile (5c): Yield: 30.5%; buﬀ solid. MP: 221–223 ^◦C. IR ( _max, cm⁻¹): 2200 (CN),

υ

1702 (C=O), 1672 (C=N). ¹H-NMR (DMSO-d₆, 600 MHz): 2.3 (s, 3H, CH₃), 3.6 (s, 3H, OCH₃), 3.9 (s,

6H, 2OCH₃), 4.6 (s, 1H, CH-4), 6.4 (s, 1H, Ar-H), 6.5 (s, 1H, Ar-H), 7.2–7.7 (m, 5H, Ar-H), 8.0–8.1

(m, 3H, Ar-H and N=CH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.8 (CH₃), 37.2 (C-4), 56.2 (2OCH₃, 3⁰,

5⁰-trimethoxyphenyl), 57.2 (C-3), 60.7 (OCH₃), 102.9 (C-4’a), 107.1, 112.8, 116.8, 119.4, 122.7, 123.8, 124.2,

129.5, 131.1, 134.6, 136.8, 139.5, 147.0, 151.2, 153.2, 158.5, 160.4, 160.5 (Ar-C, CN and C=O). EI-MS, m/z

(%): 555 (M + 2, 0.1), 554 (M + 1, 0.23), 204, consistent with the molecular formula C₃₀H₂₃N₃O₈(553.52).

(

±

)-4-(furan-2-yl)-9-methyl-5-oxo-2-((3,4,5-trimethoxybenzylidene)amino)-4H,5H-pyrano[3,2-c]

chromene-3-carbonitrile (5d): Yield: 17.5%; brown solid. MP: 248–250 ^◦C. IR ( _max, cm⁻¹): 2196 (CN),

1701 (C=O), 1664 (C=N). ¹H-NMR (DMSO-d₆, 600 MHz):

2.4 (s, 3H, CH₃), 3.3 (s, 3H, OCH₃), 3.6–3.7

υ

δ

(m, 6H, 2OCH₃), 4.4 (s, 1H, CH-4), 6.42–6.49 (m, 4H, Ar-H), 7.2–7.7 (m, 4H, Ar-H), 9.4 (s, 1H, N=CH).

¹³C-NMR (DMSO-d₆, 154 MHz): 20.9 (CH₃), 37.6 (C-4), 56.3 (2OCH₃, 3⁰, 5⁰-trimethoxyphenyl), 58.3

(C-3), 60.3 (OCH₃), 104.0 (C-4’a), 105.2, 113.1, 116.8, 119.7, 122.6, 134.1, 134.4, 137.0, 139.5, 150.7, 153.2,

153.9, 158.4, 160.2, 160.3 (Ar-C, CN and C=O). EI-MS, m/z (%): 496 (M

the molecular formula C₂₈H₂₂N₂O₇(498.48).

−

2, 1.18), 135, consistent with

(

±

)-2-((2,4-dichlorobenzylidene)amino)-9-methyl-4-(4-nitrophenyl)-5-oxo-4H,5H-pyrano[3,2-c]

chromene-3-carbonitrile (5e): Yield: 37.2%; buﬀ solid. MP: 236–238 ^◦C. IR ( _max, cm⁻¹): 2198 (CN),

1718 (C=O), 1676 (C=N). ¹H-NMR (DMSO-d₆, 600 MHz):

2.4 (s, 3H, CH₃), 4.6 (s, 1H, CH-4), 7.03–8.4

υ

δ

(m, 10H, Ar-H), 10.2 (s, 1H, N=CH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.96 (CH₃), 37.2 (C-4), 57.1 (C-3),

103.1 (C-4’a), 113.0, 116.9, 119.3, 122.6, 123.7, 124.2, 128.6, 129.1, 129.6, 129.7, 130.7, 131.1, 131.9, 134.4,

134.6, 147.0, 150.9, 151.3, 158.5, 160.1(Ar-C, CN and C=O). EI-MS, m/z (%): 533 (M + 1, 0.2), 532 (M⁺, 3.7),

531 (M

)-2-((2,4-dichlorobenzylidene)amino)-4-(furan-2-yl)-9-methyl-5-oxo-4H,5H-pyrano[3,2-c]chromene-

3-carbonitrile (5f): Yield: 17.8%; brown solid. MP: >300 ^◦C. IR ( _max, cm⁻¹): 2220 (CN), 1718 (C=O),

1647 (C=N). ¹H-NMR (DMSO-d₆, 600 MHz):

2.38 (s, 3H, CH₃), 4.4 (s, 1H, CH-4), 7.2–8.1 (m, 9H,

Ar-H), 10.1 (s, 1H, N=CH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.8 (CH₃), 37.4 (C-4), 58.5 (C-3), 100.1

−

1, 8.7), 530 (M

−

2, 27.8), 291, consistent with the molecular formula C₂₇H₁₅Cl₂N₃O₅(532.33).

(±

υ

δ

Molecules 2020, 25, 3251

19 of 28

(C-4’a), 107.2, 113.5, 116.68, 117.1, 119.7, 122.2, 122.8, 125.6, 126.2, 128.6, 129.3, 130.9, 131.7, 133.7, 134.3,

148.2, 149.5, 152.2, 158.9, 160.3 (Ar-C, CN and C=O). EI-MS, m/z (%): 478 (M + 1, 1.12), 476 (M 1, 2.72),

−

475 (M − 2, 6), 474 (M − 3, 8), 91, 73, consistent with the molecular formula C₂₅H₁₄Cl₂N₂O₄(477.3).

3.1.6. 6-substituted-4-hydroxyl-2-oxo-2H-chromene-3-sulfonyl chloride (6a,b)

Chlorosulfonic acid (13.5 mmol) was added dropwise to a solution of 1a or 1b (11.3 mmol) in

dichloromethane (22.7 mL) cooled to 0 ^◦C. The reaction mixture was continuously stirred at ambient

temperature overnight. The precipitates were ﬁltered, washed with dichloromethane and petroleum

ether, and then dried.

6-Chloro-4-hydroxy-2-oxo-2H-chromene-3-sulfonyl chloride (6a): Yield: 97%; white solid. MP:

◦

118–120 C. IR (

υ

_max, cm⁻¹):3317 (br., OH), 1685 (C=O), 1375 (asymmetric, S-O), 1176 (symmetric,

1

S-O). H-NMR (DMSO-d₆, 600 MHz):

δ 7.3–7.6 (m, 2H, Ar-H), 7.7 (s, 1H, Ar-H-5), 14.0 (br. s, 1H,

OH). ¹³C-NMR (DMSO-d₆, 154 MHz): 108.54 (C-3), 116.7, 118.9, 123.8, 128.6, 133.7, 151.6, 156.9, 161.6

(Ar-C, CN and C=O). EI-MS, m/z (%): 299 (M + 4, 0.2), 298 (M + 3, 0.4), 297 (M + 2, 2.1), 296 (M + 1,

3.5), 295 (M⁺, 13.21), 294 (M

C₉H₄Cl₂O₅S (295.1).

−

1, 0.5), 293 (M

−

2, 0.1), 207, consistent with the molecular formula

4-Hydroxy-6-methyl-2-oxo-2H-chromene-3-sulfonyl chloride (6b): Yield: 98%, white solid. MP:

116–117 ^◦C. IR ( _max, cm⁻¹): 3381 (br., OH), 1695 (C=O), 1328 (asymmetric, S-O), 1131 (symmetric,

S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

υ

δ

2.34 (s, 3H, CH₃), 7.2 (d, J = 7.8, 1H, Ar-H-7), 7.4 (d, J = 8.4,

1H, Ar-H-8), 7.6 (s, 1H, Ar-H-5), 13.9 (br. s, 1H, OH). ¹³C-NMR (DMSO-d₆, 154 MHZ): 20.7 (CH₃),

107.9 (C-3), 114.9, 116.4, 124.2, 133.9, 134.8, 151.1, 157.5, 162.7(Ar-C, CN and C=O). EI-MS, m/z (%): 278

(

M + 4, 0.07), 277 (M + 3, 0.52), 276 (M + 2, 2.72), 275 (M + 1, 0.23), 274 (M⁺, 0.05), 44, consistent with

the molecular formula C₁₀H₇ClO₅S (274.68).

3.1.7. 4-Hydroxy-6-(substituted) coumarin-3-sulfonamides (7a–f)

A mixture of 6a or 6b (0.002 mol), sulfa compounds (sulfanilamide, sulfadiazine, or sulfathiazole,

0.002 mol), and absolute EtoH (15 mL) was heated under reﬂux for 3–6 h (TLC monitoring). After cooling,

the solid was ﬁltered out, washed with absolute EtOH and petroleum ether, and then dried to obtain

the pure products.

6-Chloro-4-hydroxy-2_◦-oxo-N-(4-sulfamoylphenyl)-2H-chromene-3-sulfonamide (7a): Yield: 27%, white

solid. MP: 245–247 C. IR (

(asymmetric S-O), 1165 (symmetric S-O). H-NMR (DMSO-d₆, 600 MHz):

υ

_max, cm⁻¹):3346–3257 (NH₂, NH), 3101 (br, OH), 1726 (C=O), 1338

1

δ 5.62 (br. s, 2H, NH₂),

6.9–7.0 (m, 3H, Ar-H-2’, 6’ and NH), 7.4 (s, 1H, Ar-H-5), 7.6–7.8 (m, 4H, Ar-H). ¹³C-NMR (DMSO-d₆,

154 MHz): 108.5 (C-3), 116.8, 118.9, 119.0, 122.8, 123.8, 127.8, 128.6, 132.8, 133.7, 151.7, 157.0, 161.6 (Ar-C,

CN and C=O) EI-MS, m/z (%): 433 (M + 3, 0.1), 432(M + 2, 0.22), 431 (M +1, 0.85), 430 (M⁺, 1.9), 73,

consistent with the molecular formula C₁₅H₁₁ClN₂O₇S₂(430.84).

4-Hydroxy-6-methyl-2-oxo-N-(4-sulfamoylphenyl)-2H-chromene-3-sulfonamide (7b): Yield: 56%;

white solid. MP: 241–243 ^◦C. IR ( _max, cm⁻¹): 3352, 3250 (NH₂, NH), 3142 (br., OH), 1680 (C=O), 1355

υ

(asymmetric S-O), 1157 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

δ 2.36 (s, 3H, CH₃), 2.49 (s,

1H, NH), 4.55 (br. s, 2H, NH₂), 6.9 (d, J = 8.4, 2H, Ar-H-2’, 6’), 7.2 (d, J = 8.4, 1H, Ar-H-7), 7.4 (d, J = 7.8

,

1H, Ar-H-8), 7.6 (m, 3H, Ar-H- 3’, 5’ and 5), 13.9 (br. s, 1H, OH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.78

(CH₃), 107.9 (C-3), 114.9, 116.5, 117.8, 124.2, 127.8, 133.9, 134.9, 151.1, 157.6, 162.7 (Ar-C, CN and C=O).

EI-MS, m/z (%): 411 (M + 1, 0.51), 410 (M⁺, 2.62), 408 (M

−

2, 12.76), 133, consistent with the molecular

formula C₁₆H₁₄N₂O₇S₂(410.42).

6-Chloro-4-hydroxy-2-oxo-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)-2H-chromene-3-sulfonamide

7c): Yield: 78.2%; yellow solid. MP: 230–232 ^◦C. IR ( _max, cm⁻¹): 3373 (2NH), 3084 (br., OH), 1691

(C=O), 1346 (asymmetric S-O), 1151 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

4.7 (br. s, 1H,

NH), 6.6 (d, J = 9.6, 2H, Ar-H-2’, 6’), 7.0 (m, 1H, diazine-H-4), 7.4–7.7 (m, 5H, Ar-H and NH), 7.8 (s, 1H,

(

υ

δ

Molecules 2020, 25, 3251

20 of 28

Ar-H-5), 8.4 (m, 2H, diazine-H-3, 5). ¹³C-NMR (DMSO-d₆, 154 MHz): 108.6 (C-3), 113.7, 116.0, 116.8,

118.9, 123.8, 126.8, 128.6, 130.2, 133.6, 151.7, 156.9, 157.6, 158.7, 161.6 (Ar-C, CN and C=O). EI-MS, m/z

(%): 511 (M + 3, 0.6), 315, consistent with the molecular formula C₁₉H₁₃ClN₄O₇S₂(508.9).

4-Hydroxy-6-methyl-2-oxo-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)-2H-chromene-3-sulfonamide

(

7d): Yield: 86.3%.; yellow solid. MP: 207–209 ^◦C. IR ( _max, cm⁻¹): 3620.39 (OH), 3423, 3356 (2NH),

υ

1691 (C=O), 1334 (asymmetric S-O), 1166 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

δ 2.39 (s,

3H, CH₃), 6.6 (d, J = 9.6, 2H, Ar-H-2’, 6’), 7.0 (m, 1H, diazine-H-4), 7.2 (d, J = 10.8, 1H, Ar-H-8), 7.4 (d,

J = 9.6, 1H, Ar-H-7), 7.64–7.67 (m, 3H, Ar-H-5, 3’, 5’), 8.4 (m, 2H, diazine-H-3,5). ¹³C-NMR (DMSO-d₆,

154 MHz): 20.78 (CH₃), 108.0 (C-3), 113.3, 114.9, 116.0, 116.5, 124.2, 126.2, 128.2, 128.4, 130.2, 133.9,

134.8, 151.1, 157.6, 158.7, 162.7 (Ar-C, CN and C=O) EI-MS, m/z (%): 490 (M + 2, 2.54), 489 (M + 1, 5.08),

355, consistent with the molecular formula C₂₀H₁₆N₄O₇S₂(488.4).

6-Chloro-4-hydroxy-2-oxo-N-(4-(N-(thiazol-2-yl)sulfamoyl)phenyl)-2H-chromene-3-sulfonamide (7e):

Yield: 74.13%; white solid. MP: 215–217 ^◦C. IR ( _max, cm⁻¹): 3448 (2NH), 3140 (br., OH), 1676 (C=O),

υ

1338 (asymmetric S-O), 1147 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

δ 5.3 (br. s, 1H, NH),

5.4 (br. s, 1H, NH), 6.7 (d, J = 3.6, 1H, thiazole-H), 6.8 (d, J = 8.4, 2H, Ar-H-2’, 6’), 7.2 (d, J = 3.6, 1H,

thiazole-H), 7.42 (d, J = 8.4, 1H, Ar-H-8), 7.58 (d, J = 9.6, 2H, Ar-H-3’ and 5’), 7.72 (d, J = 7.8, 1H, Ar-H-7),

7.8 (s, 1H, Ar, CH-5). ¹³C-NMR (DMSO-d₆, 154 MHz): 108.3 (C-3), 108.6, 116.6, 116.8, 118.9, 123.8, 124.8,

128.1, 128.6, 132.8, 133.7, 147.1, 151.7, 157.0, 161.6, 168.7, (Ar-C, CN and C=O). EI-MS, m/z (%): 514

(M + 1, 0.2), 513 (M⁺, 1.2), 73, consistent with the molecular formula C₁₈H₁₂ClN₃O₇S₃(513.95).

4-Hydroxy-6-methyl-2-oxo-N-(4-(N-(thiazol-2-yl)sulfamoyl)phenyl)-2H-chromene-3-sulfonamide (7f):

Yield: 84.7%; white solid. MP: 167–169 ^◦C. IR ( _max, cm⁻¹): 3421(OH) overlapped with 3338 (2NH),

υ

1683 (C=O), 1350 (asymmetric S-O), 1153 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

δ 2.37 (s,

3H, CH₃), 6.7 (m, 3H, Ar, CH-2’, 6’ and thiazole-H), 7.1 (d, J = 3.6, 1H, thiazole-H), 7.2 (d, J = 8.4, 1H,

Ar-H-8), 7.4 (d, J = 7.8, 1H, Ar-H-7), 7.5 (d, J = 8.4, 2H, Ar-H-3’, 5’), 7.6 (s, 1H, Ar-H-5), 13.9 (br. s, 1H,

OH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.78 (CH₃), 107.9 (C-3), 108.1, 114.9, 115.3, 116.5, 124.2, 124.8,

128.1, 131.2, 133.9, 134.9, 145.9, 151.1, 157.5, 168.6, 162.7 (Ar-C, CN and C=O). EI-MS, m/z (%): 493 (M⁺,

0.1), 491(M

−

2, 3.2), 490 (M

−

3, 3.2), 73, consistent with the molecular formula C₁₉H₁₅N₃O₇S₃(493.53).

3.1.8. 4-Hydroxy-6-(substituted)coumarin-3-sulfonamides (8a–f)

A mixture of 6a or 6b (0.002 mol), 4-substituted-aniline (aniline, 4-hydroxyaniline, or 4-acetylaniline,

0.004 mol) and absolute EtOH (15 mL) was reﬂuxed for 6–8 h. While warm, the solid was collected by

ﬁltration and then washed with hot absolute EtOH and petroleum ether, respectively. After drying, the

solid was recrystallised if necessary.

6-Chloro-4-hydroxy-2-oxo-N-phenyl-2H-chromene-3-sulfonamide (8a): Yield: 92.65%. MP: 337–339

^◦C. IR ( _max, cm⁻¹): 3367 (NH), 3169 (br., OH), 1683 (C=O), 1348 (asymmetric S-O), 1168 (symmetric

υ

S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

δ 7.2–7.4 (m, 6H, Ar-H), 7.72 (d, J = 8.4, 1H, Ar-H), 7.8 (s, 1H,

Ar-H-5). ¹³C-NMR (DMSO-d₆, 154 MHz): 108.6 (C-3), 116.8, 117.6, 118.9, 122.7, 123.8, 127.5, 128.6,

130.2, 133.7, 151.7, 157.0, 161.6 (Ar-C, CN and C=O). EI-MS, m/z (%): 350 (M

with the molecular formula C₁₅H₁₀ClNO₅S (351.76).

−

1, 1.18), 57, consistent

4-Hydroxy-6-methyl-2-oxo-N-phenyl-2H-chromene-3-sulfonamide (8b): Yield: 84.5%; white solid.

MP: 324–325 ^◦C. IR ( _max/cm⁻¹): 3356 (NH), 3111 (br., OH), 1685 (C=O), 1354 (asymmetric S-O), 1159

(symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

2.37 (s, 3H, CH₃), 7.3–7.6 (m, 8H, Ar-H), 9.6 (br.

υ

δ

s, 1H, NH), 13.9 (br. s, 1H, OH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.79 (CH₃), 107.9 (C-3), 114.9,

116.5, 123.0, 124.2, 127.9, 130.2, 133.1, 133.9, 134.9, 151.1, 157.6, 162.7 (Ar-C, CN and C=O). EI-MS, m/z

(%): 434 (M + 3, 1.65), 333 (M⁺², 3.92), 332 (M⁺¹, 26.25), 207, consistent with the molecular formula

C₁₆H₁₃NO₅S (331.34).

Molecules 2020, 25, 3251

21 of 28

6-Chloro-4-hydroxy-N-(4-hydroxyphenyl)-2-oxo-2H-chromene-3-sulfonamide (8c): Crystallisation

solvent: CHCl₃. Yield: 48.3%; white solid. MP: 250–251 ^◦C. IR ( _max, cm⁻¹): 3278 (br., OH overlapped

with NH), 1681 (C=O), 1346 (asymmetric S-O), 1168 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

υ

δ

6.8 (m, 2H, Ar-H-2’, 6’), 7.1 (m, 2H, Ar-H-3’, 5’), 7.4 (d, J = 8.4, 1H, Ar,CH-8), 7.7 (d, J = 7.8, 1H,

Ar,CH-7), 7.8 (s, 1H, Ar,CH-5), 9.7 (s, 1H, NH), 10.0 (br. s, 1H, OH). ¹³C-NMR (DMSO-d₆, 154 MHz):

108.5 (C-3), 116.5, 116.8, 118.9, 123.8, 124.2, 128.6, 133.7, 151.7, 157.0, 160.1, 161.7 (Ar-C, CN and C=O).

EI-MS, m/z (%): 366 (M

formula C₁₅H₁₀ClNO₆S (367.76).

−

1, 0.1), 363 (M

−

4, 28.19), 332 (5.86), 196, consistent with the molecular

4-Hydroxy-N-(4-hydroxyphenyl)-6-methyl-2-oxo-2H-chromene-3-sulfonamide (8d): Crystallisation

solvent: CHCl₃. Yield: 63.13%; white solid. MP: 253–254 ^◦C. IR ( _max, cm⁻¹): 3298 (br., OH overlapped

with NH), 1662 (C=O), 1354 (asymmetric S-O), 1166 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

υ

δ

2.38 (s, 3H, CH₃), 6.8 (m, 2H, Ar, CH-2’, 6’), 7.1–7.2 (m, 3H, Ar, CH-3’, 5’ and 8), 7.4–7.6 (m, 2H, Ar,

CH-7 and 5), 9.7 (br. s, 1H, NH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.79 (CH₃), 107.9 (C-3), 114.9, 116.5,

123.4, 124.2, 124.3, 133.9, 134.9, 151.1, 157.2, 157.6, 162.7, 173.7 (Ar-C, CN and C=O). EI-MS, m/z (%):

345 (M

−

2, 0.04), 344 (M

−

3, 0.48), 343 (M

−

4, 3.31), 342 (M

−

5, 9.7), 341 (M

−

6, 52.35), 209, consistent

with the molecular formula C₁₆H₁₃NO₆S (347.34).

N-(4-acetylphenyl)-6-chloro-4-hydroxy-2-oxo-2H-chromene-3-sulfonamide (8e): Crystallisation

solvent: CHCl₃/isopropanol. Yield: 81.25%; white solid. MP: 238–240 ^◦C. IR ( _max, cm⁻¹): 3344 (NH),

3088 (br., OH), 1718 (C=O ketone), 1678 (C=O ester), 1357 (asymmetric S-O), 1166 (symmetric S-O).

¹H-NMR (DMSO-d₆, 600 MHz):

2.38 (s, 3H, CH₃C=O), 6.6 (d, J = 8.4 2H, Ar, CH-2’, 6’), 7.3 (d, J = 7.8

υ

δ

,

1H, Ar,CH-8), 7.6 (m, 3H, Ar,CH-3’, 5’ and 7), 7.7 (s, 1H, Ar,CH-5), 13.9 (br. s, 1H, OH). ¹³C-NMR

(DMSO-d₆, 154 MHz): 26.4 (CH₃), 108.5 (C-3), 115.0, 116.7, 118.8, 123.8, 127.4, 128.5, 130.8, 133.6, 150.6,

151.6, 156.9, 161.6 (Ar-C, CN and C=O). 195.8 (C=O ketone). EI-MS, m/z (%): 397 (M + 4, 0.13), 391

(M − 2, 0.13), 390 (M − 3, 0.83), 54, consistent with the molecular formula C₁₇H₁₂ClNO₆S (393.8).

N-(4-acetylphenyl)-4-hydroxy-6-methyl-2-oxo-2H-chromene-3-sulfonami_◦de (8f): Crystallisation

solvent: CHCl₃/isopropanol. Yield: 74.5%; white solid. MP: 231–232 C. IR (υ

_max, cm⁻¹): 3342

(NH), 3068 (br., OH), 1728 (C=O ketone), 1707 (C=O ester), 1359 (asymmetric S-O), 1163 (symmetric

S-O). ¹H-NMR (DMSO-d₆, 600 MHz):

2.37 (s, 3H, CH₃), 2.38 (s, 3H, CH₃C=O), 6.6 (d, J = 7.8, 2H,

δ

Ar,CH-2’, 6’), 7.2 (d, 1H, J = 9, Ar,CH-8), 7.4 (d, J = 8.4, 1H, Ar,CH-7), 7.66–7.69 (m, 3H, Ar,CH-5, 3’, 5’),

13.9 (br. s, 1H, OH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.7 (CH₃), 26.6 (CH₃-C=O), 107.9 (C-3) 114.9,

116.4, 116.9, 124.2, 129.4, 130.7, 133.9, 134.9, 147.5, 151.1, 157.6, 162.77 (Ar-C, CN and C=O). 196.18

(C=O ketone). EI-MS, m/z (%): 377 (M + 4, 0.1), 476 (M + 3, 0.5), 375 (M + 2, 2.4), 374 (M + 1, 4), 373

(M⁺, 15.3), 73, consistent with the molecular formula C₁₈H₁₅NO₆S (373.38).

3.1.9. Coumarin-Sulfonamide Chalcones (9a–f)

Equimolar quantities of 8e or 8f (0.01 mol) and an aromatic aldehyde (4-nitrobenzaldehyde,

4-chlorobenzaldehyde, or 4-methoxybenzaldehyde, 0.01 mol) were dissolved in a minimal amount of

EtOH. Sodium hydroxide solution (2 mL, 0.02 M) was added slowly and stirred at ambient temperature

for 24 h. The mixture was poured slowly into 400 mL of ice water with constant stirring and then

refrigerated for 24 h. The formed precipitates were ﬁltered, washed with distilled water, dried,

and recrystallised with CHCl₃if necessary.

(E)-6-Chloro-4-hydroxy-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)-2-oxo-2H-chromene-3-sulfonamide

◦

(

9a): Yield: 35.34%; orange solid. MP: 205–207 C. IR (

υ

_max, cm⁻¹): 3487 (OH), 3388 (NH), 1703

(C=O ester), 1637 (C=O ketone), 1342 (asymmetric S-O), 1180 (symmetric S-O). ¹H-NMR (DMSO-d₆,

600 MHz):

δ 6.3–6.6 (m, 4H, Ar-H), 7.7 (d, J = 9, 1H, Ar-H), 7.9–8.3 (m, 9H, CH_α=CH_β, Ar-H and

NH). ¹³C-NMR (DMSO-d₆, 154 MHz): 101.1 (C-3), 113.2, 115.9, 122.0, 124.4, 124.6, 125.4, 127.2, 130.0,

130.4, 130.5, 130.8, 131.9, 139.1, 142.34, 148.2, 151.20, 157.7, 161.9 (Ar-C, CN and C=O), 185.92 (C=O

Molecules 2020, 25, 3251

22 of 28

ketone). EI-MS, m/z (%): 527 (M + 1, 8), 526 (M⁺, 11), 373, consistent with the molecular formula

C₂₄H₁₅ClN₂O₈S (526.9).

(E)-4-Hydroxy-6-methyl-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)-2-oxo-2H-chromene-3-sulfonamide

(

9b): Yield: 30%; orange solid. MP: 220–220 ^◦C. IR ( _max, cm⁻¹): 3487 (OH), 3388 (NH), 1707 (C=O

υ

ester), 1637 (C=O ketone), 1340 (asymmetric S-O), 1182 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600

MHz):

δ 2.38 (s, 3H, CH₃), 6.67–6.7 (m, 4H, Ar-H), 7.25 (s, 1H, Ar, CH-5), 7.4–8.3 (m, 9 H, CH_α=CH_β,

Ar-H, and NH), 13.9 (br. s, 1H, OH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.82 (CH₃), 107.9 (C-3) 114.9,

116.4, 116.8, 121.67, 123.5, 124.3, 128.4, 129.3, 130.7, 133.9, 134.9, 138.2, 140.7, 141.94, 145.9, 151.1, 157.6,

162.32 (Ar-C, CN and C=O), 186.10 (C=O ketone). EI-MS, m/z (%): 506 (M⁺, 0.24), 503 (M

consistent with the molecular formula C₂₅H₁₈N₂O₈S (506.48).

−

3, 1.61), 73,

(E)-6-Chloro-N-(4-(3-(4-chlorophenyl)acryloyl)phenyl)-4-hydroxy-2-oxo-2H-chromene-3-sulfonamide

9c): Yield: 22.32%; yellow solid. MP: 157–159 ^◦C. IR ( _max, cm⁻¹): 3460 (OH), 3340 (NH), 1703 (C=O

(

υ

ester), 1629 (C=O ketone), 1346 (asymmetric S-O), 1176 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600

MHz): 5.6 (s, 1H, NH), 6.6 (m, 2H, Ar-H), 7.4 (d, J = 8.4, 1H, Ar-H), 7.51 (d, J = 8.4, 1H, Ar-H), 7.61 (d,

J = 16.1, 1H, C=CH_α), 7.7 (dd.,J = 2.5, J = 6.3, 2H, Ar-H), 7.8 (d, J = 8.4, 1H, Ar-H), 7.89–7.92 (m, 3H,

Ar-H and C=CH_β), 7.95 (d, J = 8.4, 2H, Ar-H), 14.1 (br. s, 1H, OH). ¹³C-NMR (DMSO-d₆, 154 MHz):

108.6, 113.4, 116.8, 118.9, 122.8, 123.6, 123.8, 128.6, 129.3, 130.6, 131.6, 132.8, 133.7, 134.6, 134.8, 140.4,

151.7, 156.9, 161.6 (Ar-C, CN and C=O). 186.1 (C=O ketone). EI-MS, m/z (%): 519 (M + 3, 0.3), 518

(M + 2, 0.6), 517 (M + 1, 1.6), 221, consistent with the molecular formula C₂₄H₁₅Cl₂NO₆S (516.35).

(E)-N-(4-(3-(4-chlorophenyl)acryloyl)phenyl)-4-hydroxy-6-methyl-2-oxo-2H-chromene-3-sulfonamide

(

9d): Yield: 22.41%; yellow solid. MP: 141–143 ^◦C. IR ( _max, cm⁻¹): 3462 (OH), 3342 (NH), 1701 (C=O

υ

ester), 1647 (C=O ketone), 1346 (asymmetric S-O), 1178 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600

MHz): 2.39 (s, 3H, CH₃), 6.7 (d, J = 8.4, 2H, Ar-H), 7.2 (d, J = 8.4, 1H, Ar-H), 7.4–7.5 (m, 3H, Ar-H), 7.6

δ

(d, J = 15.4, 1H, C=CH_α), 7.67 (s, 1H, Ar-H), 7.8–7.9 (m, 3H, Ar-H and C=CH_β), 7.99 (d, J = 9.1, 2H,

Ar-H), 14.0 (s, 1H, OH). ¹³C-NMR (DMSO-d₆, 154 MHz): 20.80 (CH₃), 108.03 (C-3), 114.5, 114.9, 116.5,

123.5, 124.3, 129.3, 129.8, 130.7, 131.5, 131.6, 133.9, 134.5, 134.8, 134.9, 140.7, 151.2, 157.5, 162.7 (Ar-C,

CN and C=O). 186.44 (C=O ketone). EI-MS, m/z (%): 497 (M + 2, 1.1), 496 (M + 1, 10.4), 495 (M⁺,

60.46), 55, consistent with the molecular formula C₂₅H₁₈ClNO₆S (495.93).

(E)-6-Chloro-4-hydroxy-N-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)-2-oxo-2H-chromene-3-sulfonamide

(

9e): Yield: 30.23%; yellow solid. MP: >300 ^◦C. IR ( _max, cm⁻¹): 3468 (OH), 3329 (NH), 1695 (C=O

υ

ester), 1626 (C=O ketone), 1350 (asymmetric S-O), 1163 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600

MHz): 3.8 (s, 3H, OCH₃), 6.6 (d, J = 7.7, 2H, Ar-H), 6.9 (m, 2H, Ar-H), 7.09 (m, 2H, Ar-H), 7.35 (d,

δ

J = 8.4, 1H, Ar-H), 7.57 (d, J = 8.4, 1H, Ar-H), 7.7–7.9 (m, 3H, Ar-H, CH_α=C and C=CH_β), 8.2 (d,

J = 8.4, 2H, Ar-H), 8.5 (s, 1H, NH). ¹³C-NMR (DMSO-d₆, 154 MHz): 55.7 (OCH₃), 108.3 (C-3), 113.1,

114.7, 119.9, 120.3, 121.6, 125.9, 128.2, 129.1, 130.4, 130.7, 131.2, 131.4, 135.2, 144.1, 141.8, 161.3, 157.1,

162.0 (Ar-C, CN and C=O). 186.3 (C=O ketone). EI-MS, m/z (%): 513 (M + 2, 0.12), 73, consistent with

the molecular formula C₂₅H₁₈ClNO₇S (511.93).

(E)-4-Hydroxy-N-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)-6-methyl-2-oxo-2H-chromene-3-sulfonamide

(

9f): Yield: 15.2%; yellow solid. MP: >300 ^◦C. IR ( _max, cm⁻¹):3468 (OH), 3331 (NH), 1701 (C=O ester),

υ

1626 (C=O ketone), 1342 (asymmetric S-O), 1163 (symmetric S-O). ¹H-NMR (DMSO-d₆, 600 MHz): 2.39

(s, 3H, CH₃), 3.81 (s, 3H, OCH₃), 6.8 (d, J = 7.7, 2H, Ar-H), 7.01 (d, J = 7.7, 2H, Ar-H), 7.2 (d, J = 8.4, 1H,

Ar-H), 7.5 (d, J = 8.4, 1H, Ar-H), 7.6 (d, J = 15.4, 1H, C=CH_α), 7.67 (s, 1H, Ar-H-5), 7.7 (d, J = 15.4,

1H, C=CH_β), 7.8 (d, J = 7.7, 2H, Ar-H), 8.0 (d, J = 8.2, 2H, Ar-H), 14.04 (br. s, 1H, OH). ¹³C-NMR

(DMSO-d₆, 154 MHz): 20.79 (CH₃), 55.8 (OCH₃), 108.0 (C-3), 114.8, 114.9, 115.5, 116.5, 120.1, 124.3,

128.5, 130.8, 130.9, 131.2, 132.3, 133.9, 134.9, 142.5, 151.2, 157.6, 161.4, 162.7 (Ar-C, CN and C=O). 186.8

(C=O ketone). EI-MS, m/z (%): 491 (M⁺, 0.8), 490 (M

formula C₂₆H₂₁NO₇S (491.51).

−

1, 5.29), 78, consistent with the molecular

Molecules 2020, 25, 3251

23 of 28

3.1.10. X-Ray Crystallographic Study of Compound 2a

The Bruker-Nonius Apex X8 CCD Diﬀractometer was used to analyse the X-ray single-crystal

structure. Yellow needles of compound 2a were obtained by crystallisation from EtOH by allowing

the solvent to slowly evaporate. Crystallographic data for the structure of compound 2a have been

deposited at the Cambridge Crystallographic Data Center (deposit CCDC 1985063).

3.2. Antioxidant Testing

The radical scavenging activity of the synthesised compounds was measured using the DPPH

microplate-based method [25

(Spectramax M5, Molecular Devices, San Jose, CA, USA). Reference standard ascorbic acid was utilised

as the positive control for this assay. Test samples were prepared at a starting concentration of 200 g/mL

after mixing with 100 L of 0.2 mM DPPH methanolic solution. A series of concentrations (1.563 to

,26]. The absorbance was measured using a microplate/cuvette reader

µ

200

µ

g/mL) were used to determine the 50% inhibitory concentration (IC₅₀_◦) of samples. Samples were

incubated with DPPH in the dark for 30 min at room temperature (25 C). The methanolic DPPH

solution was prepared daily and stored in a ﬂask covered with aluminium foil. DPPH radicals have an

absorption maximum (λ_max) at 515 nm, at which the absorbance values were measured and converted

into a percentage of antioxidant activity [43].

The methanolic DPPH solution without antioxidants was used as a control, and samples in

methanol alone served as a background control. IC₅₀was determined using GraphPad Prism 7

(GraphPad Software Inc., San Diego, CA, USA). The percent inhibition was calculated using the

following Equation (1):

"

#

(Sample Absorbance − Sample background Absorbance)

% Inhibition = 100 −

× 100

(1)

DPPH only Absorbance

The assay was carried out in triplicate and performed as much as possible in an area protected

against light. The data points are presented as the mean ± standard deviation (SD, n = 3).

3.3. Antimicrobial Activity

3.3.1. Agar Well Diﬀusion Method

Antimicrobial tests were conducted via the agar well diﬀusion method [27] using 100

suspension containing 1

10⁸CFU/mL pathological tested bacteria and 1 10⁶CFU/mL yeast spread

on NA and SDA, respectively. After the media cooled and solidiﬁed, wells (10 mm in diameter) were

made in the solidiﬁed agar and loaded with 100 L of the tested compound solution prepared by

µL of

×

µ

dissolving 10 mg of the chemical compound in 1 mL dimethyl sulfoxide (DMSO). The inoculated plates

were then incubated for 24 h at 37 ^◦C for bacteria and yeast growth. DMSO served as negative control.

Ciproﬂoxacin (10 mg/mL) and ketoconazole (10 mg/mL) were used as standards for antibacterial and

antifungal activity, respectively. After incubation, antimicrobial activity was evaluated by measuring

the zone of inhibition against the test organisms and compared with that of the standard. Antimicrobial

activities were expressed as the diameter of the inhibition zone (IZ) in mm. The experiment was carried

out in triplicate and the average zone of inhibition was calculated.

3.3.2. MIC Measurement

The bacteriostatic activity of the active compounds (having IZ >16 mm) was then evaluated

using the two-fold serial dilution technique [28]. Two-fold serial dilutions of the tested compounds

solutions were prepared using the proper nutrient broth to reach ﬁnal concentrations of 1000, 500, 250,

and 125 µg/mL. The tubes were then inoculated with the test microorganisms grown in their suitable

broth at 37 ^◦C for 24 h (1

×

10⁸CFU/mL for bacteria and 1

×

10⁶CFU/mL of yeast), 0.1 mL of the above

Molecules 2020, 25, 3251

24 of 28

inoculum was administered per 5 mL, and the tubes were incubated at 37 ^◦C for 24 h. The lowest

concentration showing no growth was taken as the MIC.

3.4. Anti-Inﬂammatory Activity

3.4.1. Proteinase-Inhibitory Activity

The test was performed according to El-Serwy et al. [32] with minor modiﬁcations. The reaction

mixture (2 mL) contained 0.06 mg trypsin, 20 mM Tris HCl buﬀer (pH 7.4), and 1 mL test sample

(250 µg/mL for each compound). After incubating for 5 min, 1 mL of 0.8% (w/v) casein was added.

The mixture was incubated for an additional 20 min. The reaction was arrested by adding 1 mL of

10% perchloric acid. The cloudy suspension was centrifuged, and the absorbance of the supernatant

was read at 280 nm. Reference standard aspirin was used as the positive control. The experiment

was performed in triplicate. The percent inhibition of protein denaturation was calculated using the

following Equation (2):

"

#

(A control − A test)

% Inhibition = 100 −

× 100

(2)

A control

where Acontrol and Atest represent the absorbance of the control reaction without and with the

sample, respectively.

3.4.2. Pharmacological In Vivo Study

Prophylactic anti-inﬂammatory eﬀect on formaldehyde-induced rat paw oedema:

Animals: Female Wister albino rats (150–175 g) were obtained from the animal house colony of the

National Research Centre, Egypt. The animals were kept in standard plastic cages in an air-conditioned

room at 22

±

3 ^◦C and 55

±

5% humidity and supplied with a standard laboratory diet and water ad

libitum. All animal experimental procedures were carried out according to the Ethics Committee of

the National Research Centre, Cairo, Egypt and followed the guidelines of the National Institutes of

Health Guide for Care and Use of Laboratory Animals.

Tested derivatives and the reference drug were orally administered to rats one hour before

induction of paw oedema and all hind footpad thickness were measured at zero time (basal reading)

immediately before, then every hour for three consecutive hours, after induction of left hind paw

oedema. The paw thickness was measured by using Vernier calliper [33].

The diﬀerence between initial and subsequent readings gave the change in oedema thickness for

the corresponding time. Oedema volume of the paw of the positive control group (Vc) and volume of

treated group (Vt) were used to calculate percentage (%) inhibition and (%) oedema volume using the

following Equations (3) and (4):

% Oedema volume = (Oedema volume after drug treatment/base time volume) × 100 − 100 (3)

% Inhibition = (Vt/Vc) × 100 − 100

(4)

Thirty-six female rats were classiﬁed into the following groups (6 rats each):

•

Positive control group: Paw oedema was induced by 0.2 mL (1%, w/v) formaldehyde injected in

the subplantar area of the left hind paw of the rat [33].

Prophylactic groups:

All drug regimens were given orally one hour before induction of paw oedema, as follows:

(a) Indomethacin (Reference) group: Rats were given indomethacin orally at 25 mg/kg [44].

(b) Test groups: rats were given coumarin derivative 2b 3a 7f, and 8c orally in a dose of 2.5 mg/kg each.

,

Molecules 2020, 25, 3251

25 of 28

Statistical analysis: values were expressed as means

±

standard error (SE). Comparisons between

means were carried out using two-way analysis of variance followed by the Tukey Kramer multiple

comparisons test for all acute toxicity tests. p < 0.0001 was considered signiﬁcant. All statistical tests

were performed in GraphPad Prism (version 6).

3.4.3. COX Inhibition

COX assay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA) and used

following the manufacturer’s instructions (COX-1 Catalogue no. 701070, COX-2 Catalogue no. 701080).

The activity was expressed in IC₅₀, which is the mean of two determinations, and the deviation from

the mean is <10% of the mean value.

3.5. In Silico Studies

3.5.1. Molecular Docking Study

The protein crystal structure was obtained from the RCSB Protein Databank. The 3D-crystal

structure used for docking was 5KIR, cyclooxygenase-2 (prostaglandin synthase-2) complexed with

the selective inhibitor rofecoxib, with a resolution of 2.697 Å. Discovery Studio, AutoDock Tools,

Vina, and PyRx (The Scripps Research Institute, La Jolla, CA, USA) software programs were used for

molecular docking. Using Discovery Studio, the protein crystal structure was processed by removing

water, ligands, and sulphate molecules. The protein was then saved as a PDB ﬁle format. Second,

polar hydrogens were added using AutoDock Tools, and the ﬁle was saved in PDBQT format. Third,

the original ligand was isolated and saved as a PDB using Discovery Studio. Compounds 5a and 8d

were also saved in PDB ﬁle format. Finally, docking was performed by PyRx, and the docking scores

were obtained.

3.5.2. Drug-Likeness Assessment

In silico Lipinski Rule of Five (RO5) and topological polar surface area (TPSA) analyses were

conducted using Molinspiration (accessed 24 February 2018) [39], an online cheminformatics software

that provides web-based interactive calculation of molecular properties, including molecular weight,

hydrogen bond donors and acceptors, and calculated partition coeﬃcients of the molecules.

4. Conclusions

In the present investigation, the newly synthesised compounds were screened for antioxidant,

antimicrobial, and anti-inﬂammatory activities. Compounds 8a–d demonstrated the highest antioxidant

activity, while compounds 7c,d, 8c,d, and 9c,d exhibited antimicrobial activity equal to or higher than the

standard antimicrobials of ciproﬂoxacin and ketoconazole against one or more tested microorganisms.

Notably, compound 7d exhibited broad-spectrum antimicrobial activity. Regarding anti-inﬂammatory

activity, compounds 2b

,

3a

,

b

,

5c, and 9a showed more potent in vitro antiproteinase activity than

were as potent as aspirin. Moreover, compound 3a showed

aspirin; however, 5d

,e

,

7f, and 8a,c

signiﬁcant in vivo anti-inﬂammatory activity. Compounds 5a and 8d were the most selective and

potent coumarin derivatives, respectively, toward COX-2 isozyme. These potent compounds, except

5c, 7c, and 9c, met the in silico criteria for orally active drugs. Potent compounds such as 3a, 8c,d,

and 7d are promising starting points for the development of more potent antioxidant, antimicrobial,

and/or anti-inﬂammatory agents as orally active compounds. However, further studies are needed to

determine the toxicity and pharmacokinetic properties of these compounds.

Supplementary Materials: Figures S1–S16: ¹H and ¹³C-NMR spectra of compounds 2a

Table S1: X-ray Crystallographic data of compound 2a.

, 4a, 5c, 6b, 8e, and 9f.

Author Contributions: H.M.A. (Hanan M. Alshibl), E.S.A.-A., and M.E.H. designed the study. E.S.A.-A.

supervised this work and reviewed the manuscript. M.E.H. contributed to the synthesis and reviewed the

chemistry section of the manuscript. G.E.A.A. performed the antimicrobial testing. A.H.M. and B.M.M.I.

Molecules 2020, 25, 3251

26 of 28

carried out the anti-inﬂammatory testing (proteinase-inhibitory activity and in vivo pharmacological study).

H.M.A. (Hamad M. Alkahtani) contributed to the computational studies and reviewed the manuscript. A.B.

and A.V. conducted the X-ray crystal structure determination and contributed to the chemical analysis. H.M.A.

(

Hanan M. Alshibl) performed the chemistry and remaining experiments and wrote the manuscript. All authors

have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Acknowledgments: This research project was supported by a grant from the “Research Center of the Female

Scientiﬁc and Medical Colleges”, Deanship of Scientiﬁc Research, King Saud University. The authors thank the

Deanship of Scientiﬁc Research and RSSU at King Saud University for their technical support.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1.

Liguori, I.; Russo, G.; Curcio, F.; Bulli, G.; Aran, L.; Della-Morte, D.; Gargiulo, G.; Testa, G.; Cacciatore, F.;

Bonaduce, D.; et al. Oxidative Stress, Aging, and Diseases. Clin. Interv. Aging 2018, 13, 757–772. [CrossRef]

[PubMed]

2.

3.

4.

Chen, L.; Deng, H.; Cui, H.; Fang, J.; Zuo, Z.; Deng, J.; Li, Y.; Wang, X.; Zhao, L. Inﬂammatory Responses and

Inﬂammation-Associated Diseases in Organs. Oncotarget 2017, 9, 7204–7218. [CrossRef] [PubMed]

Pal, C.; Bengtsson-Palme, J.; Kristiansson, E.; Larsson, D.G.J. The Structure and Diversity of Human, Animal

and Environmental Resistomes. Microbiome 2016, 4, 54. [CrossRef] [PubMed]

Laxminarayan, R.; Duse, A.; Wattal, C.; Zaidi, A.K.M.; Wertheim, H.F.L.; Sumpradit, N.; Vlieghe, E.; Hara, G.L.;

Gould, I.M.; Goossens, H.; et al. Antibiotic Resistance—the Need for Global Solutions. Lancet Infect. Dis.

2013, 13, 1057–1098. [CrossRef]

5.

6.

7.

Pereira, T.M.; Franco, D.P.; Vitorio, F.; Kummerle, A.E. Coumarin Compounds in Medicinal Chemistry: Some

Important Examples from the Last Years. Curr. Top. Med. Chem. 2018, 18, 124–148. [CrossRef]

Kontogiorgis, C.; Detsi, A.; Hadjipavlou-Litina, D. Coumarin-Based Drugs: A Patent Review (2008–Present).

Expert Opin. Ther. Pat. 2012, 22, 437–454. [CrossRef]

Dehkordi, M.F.; Dehghan, G.; Mahdavi, M.; Hosseinpour Feizi, M.A. Multispectral Studies of DNA Binding,

Antioxidant and Cytotoxic Activities of a New Pyranochromene Derivative. Spectrochim. Acta. A. Mol.

Biomol. Spectrosc. 2015, 145, 353–359. [CrossRef]

8.

9.

Vukovic, N.; Sukdolak, S.; Solujic, S.; Niciforovic, N. Substituted Imino and Amino Derivatives of

4-Hydroxycoumarins as Novel Antioxidant, Antibacterial and Antifungal Agents: Synthesis and in Vitro

Assessments. Food Chem. 2010, 120, 1011–1018. [CrossRef]

Arora, R.K.; Kaur, N.; Bansal, Y.; Bansal, G. Novel Coumarin–Benzimidazole Derivatives as Antioxidants

and Safer Anti-Inﬂammatory Agents. Acta Pharm. Sin. B 2014, 4, 368–375. [CrossRef]

10. Bylov, I.E.; Vasylyev, M.V.; Bilokin, Y.V. Synthesis and Anti-Inﬂammatory Activity of N-Substituted

2-Oxo-2H-1-Benzopyran-3-Carboxamides and Their 2-Iminoanalogues. Eur. J. Med. Chem. 1999, 34,

997–1001. [CrossRef]

11. Sashidhara, K.V.; Kumar, M.; Modukuri, R.K.; Sonkar, R.; Bhatia, G.; Khanna, A.K.; Rai, S.; Shukla, R.

Synthesis and Anti-Inﬂammatory Activity of Novel Biscoumarin–Chalcone Hybrids. Bioorg. Med. Chem.

Lett. 2011, 21, 4480–4484. [CrossRef] [PubMed]

12. Bedair, A.H.; El-Hady, N.A.; El-Latif, M.S.A.; Fakery, A.H.; El-Agrody, A.M. 4-Hydroxycoumarin in

Heterocyclic Synthesis: Part III. Synthesis of Some New Pyrano[2,3-d]Pyrimidine, 2-Substituted[1,2,4]

Triazolo[1,5-c]Pyrimidine and Pyrimido[1,6-b][1,2,4]Triazine Derivatives. Il Farm. 2000, 55, 708–714. [CrossRef ]

13. Shi, X.; Lv, C.; Li, J.; Hou, Z.; Yang, X.; Zhang, Z.; Luo, X.; Yuan, Z.; Li, M. Synthesis, Photoluminescent,

Antibacterial Activities and Theoretical Studies of Three Novel Coumarin and Dihydropyran Derivatives

Containing a Triphenylamine Group. Res. Chem. Intermed. 2015, 41, 8965–8974. [CrossRef]

14. Al-Masoudi, N.A.; Mohammed, H.H.; Hamdy, A.M.; Akrawi, O.A.; Eleya, N.; Spannenberg, A.;

Pannecouque, C.; Langer, P. Synthesis and Anti-HIV Activity of New Fused Chromene Derivatives Derived

from 2-Amino-4-(1-Naphthyl)-5-Oxo-4H,5H-Pyrano[3,2- c]Chromene-3-Carbonitrile. Z. Für Naturforschung B

2014, 68, 229–238. [CrossRef]

15. Sahoo, J.; Kumar Mekap, S.; Sudhir Kumar, P. Synthesis, Spectral Characterization of Some New 3-Heteroaryl

Azo 4-Hydroxy Coumarin Derivatives and Their Antimicrobial Evaluation. J. Taibah Univ. Sci. 2015, 9,

187–195. [CrossRef]

Molecules 2020, 25, 3251

27 of 28

16. Alshibl, H.M.; Al-Abdullah, E.S.; Alkahtani, H.M. Coumarin: A Promising Scaﬀold for Design and

Development of Bioactive Agents. Curr. Bioact. Compd. 2019, 15, 1–15. [CrossRef]

17. Silverstein, F.E.; Faich, G.; Goldstein, J.L.; Simon, L.S.; Pincus, T.; Whelton, A.; Makuch, R.; Eisen, G.;

Agrawal, N.M.; Stenson, W.F.; et al. Gastrointestinal Toxicity With Celecoxib vs. Nonsteroidal Anti-

Inﬂammatory Drugs for Osteoarthritis and Rheumatoid Arthritis The CLASS Study: A Randomized

Controlled Trial. JAMA 2000, 284, 1247–1255. [CrossRef]

18. Peppercorn, M.A. Sulfasalazine. Pharmacology, Clinical Use, Toxicity, and Related New Drug Development.

Ann. Intern. Med. 1984, 101, 377–386. [CrossRef]

19. Fischi, M.A.; Dickinson, G.M.; Vole, L.L. Safety and Eﬃcacy of Sulfamethoxazole and Trimethoprim

Chemoprophylaxis for Pneumocystis Carinii Pneumonia in AIDS. JAMA 1988, 259, 1185–1189. [CrossRef]

20. Fox, C.L. Pharmacology and Clinical Use of Silver Sulfadiazine and Related Topical Antimicrobial Agents.

Pahlavi Med. J. 1977, 8, 45–64.

21. Alshibl, H.M.; Al-Abdullah, E.S.; Haiba, M.E. Coumarin Derivatives. U.S. Patent Application Pending no.

16/399,950, 30 April 2019.

22. Kiyani, H.; Ghorbani, F. Eﬃcient Tandem Synthesis of a Variety of Pyran-Annulated Heterocycles,

3,4-Disubstituted Isoxazol-5(4H)-Ones, and α,β-Unsaturated Nitriles Catalyzed by Potassium Hydrogen

Phthalate in Water. Res. Chem. Intermed. 2015, 41, 7847–7882. [CrossRef]

23. Reddy, N.S.; Mallireddigari, M.R.; Cosenza, S.; Gumireddy, K.; Bell, S.C.; Reddy, E.P.; Reddy, M.V.R. Synthesis

of New Coumarin 3-(N-Aryl) Sulfonamides and Their Anticancer Activity. Bioorg. Med. Chem. Lett. 2004, 14,

4093–4097. [CrossRef] [PubMed]

24. Behrami, A. Antibacterial Activity of Coumarine Derivatives Synthesized from 4-Chloro-Chromen-2-One.

The Comparison with Standard Drug. Orient. J. Chem. 2014, 30, 1747–1752. [CrossRef]

25. Yu, L.; Haley, S.; Perret, J.; Harris, M.; Wilson, J.; Qian, M. Free Radical Scavenging Properties of Wheat

Extracts. J. Agric. Food Chem. 2002, 50, 1619–1624. [CrossRef] [PubMed]

26. Qazi, S.S.; Li, D.; Briens, C.; Berruti, F.; Abou-Zaid, M.M. Antioxidant Activity of the Lignins Derived from

Fluidized-Bed Fast Pyrolysis. Mol. Basel Switz. 2017, 22. [CrossRef] [PubMed]

27. Perez-Eid, C.; Pauli, W.M.; Bazerque, P. An Antibiotic Assay by Agar-Well Diﬀusion Method. Acta Biologiae

et Med. Experimentalis. 1990, 15, 113–115.

28. Scott, A.C. Laboratory Control of Antimicrobial Therapy. In Practical Medical Microbiology; Collee, J., Duguid, J.,

Fraser, A., Marmion, B., Eds.; Churchill Livingstone: Edinburgh, UK, 1989; Volume 2, pp. 161–181.

29. Rutkowska, E.; Pajak, K.; Józ´wiak, K. Lipophilicity-Methods of Determination and Its Role in Medicinal

Chemistry. Acta Pol. Pharm. 2013, 70, 3–18.

30. Kalinowska, M.; Bajko, E.; Matejczyk, M.; Kaczyn´ski, P.; Łozowicka, B.; Lewandowski, W. The Study

of Anti-/Pro-Oxidant, Lipophilic, Microbial and Spectroscopic Properties of New Alkali Metal Salts of

5-O-Caﬀeoylquinic Acid. Int. J. Mol. Sci. 2018, 19, 463. [CrossRef]

31. Kocabalkanli, A.; Ates, Ö.; Ötük, G. Synthesis of Mannich Bases of Some 2,5-Disubstituted 4-Thiazolidinones

and Evaluation of Their Antimicrobial Activities. Arch. Pharm. (Weinheim) 2001, 334, 35–39. [CrossRef]

32. El-Serwy, W.S.; Mohamed, N.A.; El-Serwy, W.S.; Mahmoud, A.H. Synthesis, biological evaluation and

molecular modeling studies of some 5-methylisoxazole derivatives as anti-Inﬂammatory agents. J. Chem.

Pharmaceut. Res. 2017, 9, 117–127.

33. Ibrahim, B.M.M.; Yassin, N.A.Z.; Hetta, M.H.; Ta, K.F.; Mohammed, W.I.; Hassan, M.E.S. Phytochemical

and pharmacological studies on newly-suggested herbal formulations for potential protection against

inﬂammatory conditions. Int. J. Pharmacogn. Phytochem. Res. 2016, 8, 2044–2055.

34. Owen, C.A.; Campbell, E.J. Neutrophil Proteinases and Matrix Degradation. The Cell Biology of Pericellular

Proteolysis. Semin. Cell Biol. 1995, 6, 367–376. [CrossRef]

35. Leelaprakash, G.; Dass, S.M. In vitro anti-inﬂammatory activity of methanol extract of Enicostemma axillare.

Int. J. Drug Dev. Res. 2011, 3, 189–196.

36. Ruiz-Ruiz, J.C.; Matus-Basto, A.J.; Acereto-Escoﬃé, P.; Segura-Campos, M.R. Antioxidant and

Anti-Inﬂammatory Activities of Phenolic Compounds Isolated from Melipona Beecheii Honey.

Food Agric. Immunol. 2017, 28, 1424–1437. [CrossRef]

37. Garavito, R.M.; DeWitt, D.L. The Cyclooxygenase Isoforms: Structural Insights into the Conversion of

Arachidonic Acid to Prostaglandins. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1999, 1441, 278–287.

[CrossRef]

Molecules 2020, 25, 3251

28 of 28

38. Llorens, O.; Perez, J.J.; Palomer, A.; Mauleon, D. Structural Basis of the Dynamic Mechanism of Ligand

Binding to Cyclooxygenase. Bioorg. Med. Chem. Lett. 1999, 9, 2779–2784. [CrossRef]

39. Molinspiration Cheminformatics. Available online: http://www.molinspiration.com (accessed on

24 February 2018).

40. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and Computational Approaches to

Estimate Solubility and Permeability in Drug Discovery and Development Settings. Adv. Drug Deliv. Rev.

2001, 46, 3–26. [CrossRef]

41. Clark, D.E. Rapid Calculation of Polar Molecular Surface Area and Its Application to the Prediction of

Transport Phenomena. 2. Prediction of Blood–Brain Barrier Penetration. J. Pharm. Sci. 1999, 88, 815–821.

[CrossRef]

42. Bhatt, N.S.; Shah, A.K.; Raval, R.V.; Thakor, V. Novel Synthesis of Benzofurans. Curr. Sci. 1984, 53, 1289–1290.

43. Kapewangolo, P.; Omolo, J.J.; Bruwer, R.; Fonteh, P.; Meyer, D. Antioxidant and Anti-Inﬂammatory Activity

of Ocimum Labiatum Extract and Isolated Labdane Diterpenoid. J. Inﬂamm. Lond. Engl. 2015, 12, 1–13.

[CrossRef]

44. Moharram, F.A.-E.; Al-Gendy, A.A.; El-Shenawy, S.M.; Ibrahim, B.M.; Zarka, M.A. Phenolic proﬁle,

anti-inﬂammatory, antinociceptive, anti-ulcerogenic and hepatoprotective activities of Pimenta racemosa

leaves. BMC Complement Altern Med. 2018, 18, 208. [CrossRef] [PubMed]

Sample Availability: Samples of the compounds are available from the authors.

©

2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access

article distributed under the terms and conditions of the Creative Commons Attribution

(CC BY) license (http://creativecommons.org /licenses /by /4.0 /).

Article Doi

Synthesis and evaluation of new coumarin derivatives as antioxidant, antimicrobial, and anti-inflammatory agents

DOI: 10.3390/molecules25143251

Source and publish data:

Authors:

Article abstract of DOI:10.3390/molecules25143251

Full text of DOI:10.3390/molecules25143251

Products guided by the article

R&D Labs maybe for 1338566-33-8

Relevant to this article

Hot Product