First example of Azo-Sulfa conjugated chromene moieties: Synthesis, characterization, antimicrobial assessment, docking simulation as potent class I histone deacetylase inhibitors and antitumor agents

Article abstract of DOI:10.1016/j.bioorg.2019.103262

This report presents the development of a novel and primary model of sulfonamide compounds encompassing a chromene azo motif with the intent of becoming applicable for drug candidates in the cases of drug-resistant pathogens. The novel molecules (7a–n) ha

Full text of DOI:10.1016/j.bioorg.2019.103262

Journal Pre-proofs
First example of Azo-Sulfa Conjugated Chromene Moieties: Synthesis, Char-
acterization, Antimicrobial Assessment, Docking Simulation as Potent Class I
Histone Deacetylase Inhibitors and Antitumor Agents
Rawda M. Okasha, Mosa Alsehli, Saleh Ihmaid, Sultan S. Althagfan, Mohamed
S.A. El-Gaby, Hany E. A. Ahmed, Tarek H Afifi
PII:
S0045-2068(19)31061-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.103262
YBIOO 103262
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
3 July 2019
2 September 2019
5 September 2019
Please cite this article as: R.M. Okasha, M. Alsehli, S. Ihmaid, S.S. Althagfan, M.S.A. El-Gaby, H. E. A. Ahmed,
T.H. Afifi, First example of Azo-Sulfa Conjugated Chromene Moieties: Synthesis, Characterization, Antimicrobial
Assessment, Docking Simulation as Potent Class I Histone Deacetylase Inhibitors and Antitumor Agents,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103262
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First example of Azo-Sulfa Conjugated Chromene Moieties: Synthesis,
Characterization, Antimicrobial Assessment, Docking Simulation as Potent
Class I Histone Deacetylase Inhibitors and Antitumor Agents
Rawda M. Okashaa, Mosa Alsehlia, Saleh Ihmaidb, Sultan S. Althagfanc, Mohamed S.A. El-
Gabyd , Hany E. A. Ahmedb,e and, Tarek H Afifia,f,*
aChemistry department, Faculty of Science, Taibah University, 30002, Al-Madinah Al-Munawarah,
Saudi Arabia
bPharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College, Taibah University,
Al-Madinah Al-Munawarah, Saudi Arabia
cClinical and Hospital Pharmacy Department, Taibah University, College of Pharmacy, Taibah
University, Al-Madinah Al-Munawarah 41477, Saudi Arabia
dDepartment of Chemistry, Faculty of Science, Al‐Azhar University at Assiut, Assiut 71524, Egypt
ePharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, 11884
Nasr City, Cairo, Egypt
fChemistry Department, Faculty of Science, Al-Azhar University, 11884 Nasr City, Cairo, Egypt
*Corresponding author.
E-mail address: afifith@yahoo.com
Abstract
This report presents the development of a novel and primary model of sulfonamide compounds
encompassing a chromene azo motif with the intent of becoming applicable for drug candidates in
the cases of drug-resistant pathogens. The novel molecules (7a-n) have been synthesized via a two-
step reaction. First, 4-((2, 4-dihydroxyphenyl)diazenyl)benzenesulfonamide (3a-e) were obtained
through the reaction of their corresponding diazotized 4-aminobenzenesulfonamides (2a-e) with
resorcinol, followed by the heterocyclization of 3a-e with arylidenemalononitriles (6a-d). Upon
structural identification, the newly synthesized compounds were evaluated for their antibacterial and
antifungal activities. Moreover, their cytotoxic screening was performed against three cancer cell
lines: HCT-116, HepG-2, and MCF-7. Further examinations were comprised of the inhibitory effect
analyses of the novel sulfonamide/chromene derivatives against the HDAC classes and the Tubulin
polymerization in order to discern the prime antitumor drug candidates.
Keywords: chromene; sulfonamide; antimicrobial, anticancer activity, HDAC examination.
1

1. Introduction
Drug resistance is a decisive threat to global public health and represents a challenge to a wide
range of disciplines, including; resistance mechanisms, infection control, virulence genes, and drug
design [1,2]. Two of the most common prevalent cases of drug resistance are in the antimicrobial
and antitumor pathogens. The development of antimicrobial resistance has become a major problem
in healthcare, which arises mainly from the overuse and misuse of antibiotics that enhances the
manipulative properties of bacteria. In general, the deceptive mechanism of bacteria could be
achieved through the neutralization of the antibiotic or the alteration of their outer structure that
prevents the antibiotic’s abilities. Therefore, the treatment of infections provides more obstacles as
bacteria continues to evolve and as a lack of progress in developing new antibiotics persists [1,2].
Additionally, in the cancer diagnoses research, drug resistance is one of the greatest
difficulties that causes failure in tumor treatment, a recurrence of the disease, or even the death of a
patient. Resistance to drugs can be present during diagnosis or it can develop after the treatment of
the tumor. However, the development of new medication continues to offer hope to prohibit the
resistance capabilities of the tumor cell lines [3-5].
Recently, the development of new materials with interesting biological activities is a
major target to overcome the resistance toward the present drugs. Chromene molecules are one of
the most demanded compounds that are recognized as one type of ‘privileged medicinal scaffolds’
due to their unique pharmacological and biological activities [6-13]. Derivatives of these molecules
exhibit tremendous medicinal behaviors such as antimicrobial and antifungal [12,14-16],
antiproliferation [9-11,13,17-19], antioxidant [6,20], antispasmolytic, estrogenic [21],
antileishmanial [22], hypotensive [8], vascular-disrupting activity [23], and blood platelet
antiaggregating effects [24]. In addition, these molecules have been used in the enhancement of
cognitive functions for the treatment of neurodegenerative symptom [25], as well as treating
Alzheimer’s disease [26] and Schizophrenia disorder [27]. Moreover, the incorporation of a suitable
heterocyclic moiety such as chromene derivatives with an azo linkage increases their medicinal
performance for particular usage as antibacterial [28,29], antimicrobial [30,31], antioxidant [32],
and other useful chemotherapeutic agents [33].
Additionally, molecules containing sulfonamide moieties, sulfa drugs, have been
acknowledged for their remarkable medicinal and biological applications [34-41]. Moreover,
2

researchers have extensively employed sulfa drugs in infection treatments, particularly for patients
intolerant to antibiotics. The broad spectrum of the sulfonamide’s activities against Gram-positive
and Gram-negative bacteria arises from their capacity to hinder the production process of the folic
acid in the bacterial cells, subsequently, triggering their death. The implementations of sulfonamide
molecules encompass an assortment of assays, including: antibacterial, antifungal, antitumor agents,
anticonvulsants and protease inhibitors, carbonic anhydrase inhibitors, diuretics, hypoglycemic
agents, thyroid inhibitors, and HDAC inhibitors [34-41]. In this report, our foremost passion is to
create a new class of drug-like candidates, established from the merging of the 4H-chromene, azo,
and sulfonamide moieties as proposed in Figure 1. The aforesaid design was propositioned for
synthesis due to the beneficial facets as anticancer agents, resulting from the sulphonamide
functionality as a privileged motif binding to the HDAC target. The antimicrobial and cytotoxicity
effects of these novel materials have been exploited as well as their in vitro HDACs inhibitory
activity.
3

O
O
HN
HO
O
O
NH
NH
N
HO
Vorinostat
Trichostatin A
Natural and synthetic products as inhibitors of histone deacetylase (HDAC Inhibitors)
OH
NH2
N
R
HN
O
N
R
HN
O
S
OH
S
O
O
CN
CN
H
C
Ar
C
ZINC binding group
O
O
R
S
Cyclic Linker
N
H
OH
N
N
O
CAP group
NH2
CN
Figure 1. The design of novel chromene linked to sulfonamide moiety.
4

2. Results and discussions
2.1. Chemistry
2.1.1. Synthesis and Characterization
It is established that the high reactivity of resorcinol is primarily associated with the location
of these two hydroxyl groups in the benzene ring. As far as the reactivity of resorcinol is concerned,
the hydrogen atoms adjacent to the hydroxyl groups, namely at carbon atoms 2, 4, and 6, are
particularly reactive. The hydrogen atom located at the 5-position of the resorcinol molecule is
basically non-reactive and, therefore, does not take part in any chemical reactions under normal
reaction conditions [42]. A series of 4-((2, 4-dihydroxyphenyl)diazenyl)benzenesulfonamide (3a-e)
were synthesized in good yields by the coupling of diazotized 4-aminobenzenesulfonamides (1a-e)
with resorcinol in the presence of 10% sodium hydroxide. The diazotization was carried out in the
presence of nitrosyl chloride at 0-5 oC, Scheme 1.
N2Cl
NH2
O
S
O
S
HCl / NaNO2
0-5oC
RHN
RHN
O
O
1a-e
OH
HO
OH
N
NaOH/ Ethanol
N
O
S
OH
RHN
2
a. R = H
b. R = CH3CO
c. R = NH2-C(=NH)
3a-e
O
d. R = thiazol-2-yl
e. R = 3-methyl-isoxazole-5-yl
Scheme 1. Synthesis of 4-((2, 4-dihydroxyphenyl)diazenyl)benzenesulfonamide 3a-e
The structures of the synthesized compounds 3a-e were established on the basis of their
analytical and spectral data. The infrared spectra of compounds 3a-e displayed absorption band at
5

1597-1627 cm-1, which is a characteristic of the azo group (N=N) in addition to the absorption of
1
the hydroxyl, amino, imino, and sulfone moieties. The representative H NMR spectrum of
compound 3a (DMSO-d6) presented singlet signals at  7.45 and 12.19 ppm, assigned to the NH2
and OH protons, respectively, which is exchangeable with the D2O. A pair of doublets at  6.54 and
7.69 ppm and a singlet at 6.38 ppm was assigned to the aromatic protons of the phenolic ring with
the broad singlet at  8.03 ppm attributable to the aromatic protons of the benzene sulfonamide. The
mass spectrum of compound 3d revealed a molecular ion peak at m/z 376 (2.76%), corresponding
to the molecular formula C15H12N4O4S2. The base peak was discovered to be at m/z 92 (100 %),
which is a characteristic of the C6H5NH moiety.
The
reactivity
of
the
nucleaphilic
molecules,
towards
4-((2,
4-
dihydroxyphenyl)diazenyl)benzenesulfonamide
(3a-e),
the
electrophilic
arylidenemalononitriles (6a-d) was investigated. The reaction of compounds 3a-e with bezaldehyde
derivatives (4a-d) and Malononitrile (5) in ethanol at a reflux temperature in the presence of a
catalytic amount of piperidine furnished the respective 4-((2-amino-3-cyano-4-aryl-7-hydroxy-4-
aryl-4H-chromen-6-yl)diazenyl] benzenesulfonamides (7a-n) in quantitative yields, Scheme 2.
6

Piperidine
H2
C
CN
CN
H
+
Ar CHO
Ethanol / Reflux
CN
NC
Ar
6a-d
4
5
4a, Ar = C6H5
6a, Ar = C6H5
4b, Ar = C6H4CH3-4
4c, Ar = C6H4F-4
4d, Ar = C6H4Cl-2
6b, Ar = C6H4CH3-4
6c, Ar = C6H4F-4
6d, Ar = C6H4Cl-2
HO
OH
Piperidine
N
N
Ethanol / Reflux
O
S
3a-e
RHN
O
O
Ar
RHN S
O
N N
CN
HO
O
NH2
7a-n
7h; R = NH2-C(=NH), Ar = C6H4F-4
7a; R = H, Ar = C6H5
7i; R = thiazol-2-yl, Ar = C6H4CH3-4
7j; R = thiazol-2-yl, Ar = C6H4F-4
7k; R = thiazol-2-yl, Ar = C6H4Cl-2
7l; R = 3-methylisoxazol-5-yl, Ar = C6H4CH3-4
7m; R = 3-methylisoxazol-5-yl, Ar = C6H4F-4
7n; R = 3-methylisoxazol-5-yl, Ar = C6H4Cl-2
7b; R = H, Ar = C6H4F-4
7c; R = COCH3, Ar = C6H4CH3-4
7d; R = COCH3, Ar = C6H4F-4
7e; R = COCH3, Ar = C6H4Cl-2
7f; R = NH2-C(=NH), Ar = C6H5
7g; R = NH2-C(=NH), Ar = C6H4Cl-2
Scheme 2. Synthesis of chromene derivatives 7a-n
The structure of the chromene derivative 7a, as a representative example, was established
based on the foundation of its spectral data. The IR spectra showed the stretching vibration bands at
3439, 2190, and 1589 cm-1 for (OH), (CN), and (N=N), respectively. Furthermore, the (NH2) group
divulged a resonance of a pair of bands at 3357 and 3203 cm-1. The 1H NMR exhibited singlet signals
at  4.77 and 12.01 ppm for the CH-pyran at the 4-position and the (OH) group, respectively [12].
Moreover, a broad signal at  7.05 ppm was attributed to the amino protons while the multiple
signals at  6.80, 7.12-7.20, 7.22-7.33, and 7.76 ppm was attributed to the aromatic protons of the
phenyl group at the 4-position in the pyran ring, while, the two doublets at  8.02 & 8.14 ppm to
7

the aromatic sulfonamide protons. Additionally, the 13C NMR spectra revealed a characteristic
signal at  28.51 ppm, which corresponds to the C-4 of the pyran ring [12].
A plausible mechanism for the formation of the novel chromene derivatives 7a-n was
illustrated in Scheme 3. The proposed strategy produces the suggestion that the deprotonation of the
4-((2, 4-dihydroxyphenyl)diazenyl)benzenesulfonamide (3a-e) with piperidine generated the
aniones 4a-e that can, subsequently, perform the Michael addition with the arylidenemalononitriles
5a-d to give the non-isolable Michael adducts 6a-m, which underwent spontaneous cyclization to
afford the chromene skeletons 7a-n.
CN
H
O
O
Ar
CN
N
H
RHN S
O
N N
HO
5a-d
RHN S
O
N N
4a-e
5a, Ar = C6H5
3a-e
OH
5b, Ar = 4-CH3C6H4
5c, Ar = 4-FC6H4
5d, Ar = 2-ClC6H4
HO
O
O
O
RHN S
O
Ar
H
H
Ar
RHN S
O
N N
N N
HO
CN
CN
H
NH
C
N
HO
OH
O
6a-n
O
Ar
O
RHN S
O
N N
HO
CN
NH2
7a-n
Scheme 3. Proposed mechanism for the formation of Chromene derivaties 7a-n
2.1.2 UV-vis Study
The UV-visible study of the sulfa containing azo chromophores 3a-e and their chromene
counterparts’ 7a-n was performed in a DMF solvent in order to discover their wavelength maximum
(λmax) values. The obtained data, as presented in Table 1, revealed that the formation of the chromene
azo sulfa derivatives, in majority of cases, did not alter the λmax values of their azo precursors due to
the non-conjugated system of the chromene sulfa moieties. All the sulfa azo chromophores and their
chromene analogues portrayed λmax values in the range of 387-445 nm.
8

Table 1. UV-vis study of the new azosulfonamide bearing chromenes 7a-n and their
precursors 3a-e.
O
RHN S
O
O
RHN S
O
Ar
N N
HO
N N
CN
OH
HO
O
NH2
3a-e
7a-n
compound
R
Ar
λmax (nm)
396
3a
7a
7b
3b
H
H
H
395
C6H5
398
C6H4F-4
O
400
H3C C
O
398
390
397
7c
7d
7e
C6H4CH3-4
C6H4F-4
H3C
H3C
C
O
C
O
C6H4Cl-2
H3C C
400
394
387
3c
7f
H2N C
NH
NH
NH
C6H5
H2N
H2N
C
C
7g
C6H4F-4
9

388
7h
C6H4Cl-2
H2N
C
NH
398
390
392
392
401
445
398
396
3d
7i
thiazol-2-yl
thiazol-2-yl
C6H4CH3-4
C6H4F-4
7j
thiazol-2-yl
7k
3e
7l
C6H4Cl-2
thiazol-2-yl
3-methyl-isoxazol-5-yl
3-methyl-isoxazol-5-yl
3-methyl-isoxazol-5-yl
3-methyl-isoxazol-5-yl
C6H4CH3-4
C6H4F-4
7m
7n
C6H4Cl-2
2.2. Biological screening
2.2.1. Antimicrobial screening
All the newly synthesized compounds (3a-e) and (7a-n) and their precursors (1a-e) were
screened for their antibacterial, antifungal, and antimycobacterium activities via the agar diffusion
well method [35] while the inhibition zones and the minimum inhibitory concentrations (MIC) were
determined by the serial dilution method [36]. It is imperative to cite that the microbial activities of
compounds 1 and 3 have been evaluated in order to derive a comparison between the activity of the
chromene molecules containing azo sulfonamide and their precursor, which consequently clarify the
role of the chromene moieties in this class of activity. The activity of the synthesized compounds
was tested against four Gram-positive bacteria, including Streptococcus pneumoniae (RCMB
010010), Bacillus subtilis(RCMB 010067), Staphylococcus aureus (RCMB 000106), and
Methicillin-Resistant Staphylococcus aureus (MRSA 2658 RCMB); three Gram-negative bacteria,
including Pseudomonas aeruginosa (RCMB 010043), Escherichia coli (RCMB 010052), and
10

Salmonella typhimurium (RCMB 000106); and four fungi, including Aspergillus fumigatus (RCMB
02568), Syncephalastrumracemosum (RCMB 05922), Geotricumcandidum (RCMB 05097), and
Candida albicans (RCMB 05036), with a Mycobacterium tuberculosis strain (RCMB 010094-8).
Ampicillin, Ciprofloxacin, Amphotericin B, and Vancomycin were exploited as control drugs. The
observed antimicrobial data of the target compounds and the reference drugs: the inhibition zone
(IZ) and the minimum inhibitory concentrations (MIC) are given in Tables 2 and 3 and presented in
Figures 2 and 3. The data disclosed that most of the tested compounds exhibited appreciable
bacterial and fungal inhibition in comparison to the reference drugs. In general, the synthesized
compounds were more active against the Gram-negative bacteria, displaying an IZ value of more
than 20. Among the synthesized compounds, 7e-7j were found to be more effective against E. coli,
with the IZ values ranging from 21 to 24 mm and the MIC values from 0.49 to 3.9 µg/ml. Meanwhile,
compounds 7e-7g and 7j were discovered to be more active against S.T with IZ values of 22-26 mm.
In the case of the antibacterial activity against the Gram-positive bacteria, most of the novel
compounds were found to be comparable in activity to the reference drugs, MIC ranging from 3.9-
0.49 µg/mL. In particular, 7g exhibited mild inhibitory activity towards MRSA, IZ = 21.3 mm and
MIC = 3.9 µg/mL. Through in-depth analyses of the novel derivatives, it was detected that the
aromatic substituents possessed an impact on the inhibitory activities of the synthesized chromene
azosulfonamide derivatives. For instance, the most potent antimicrobial compounds 7e-g had a 2-
chloro substituent with terminal amino or acetyl groups, which assures the incidence of broad
activity against a wild bacterial range. Additionally, the combination of the halogen substituent with
the heterocyclic aromatic systems of 3-methylisoxazolyl stemmed an accepted range of their
antimicrobial effects. Overall, the chromene fragments with the different aryl or heteroaryl
substituents revealed significant antimicrobial behavior. Moreover, the desired compounds were
moderately or slightly more active against the examined fungal species. Thus, our investigation of
the antimicrobial activity of the novel derivatives displayed more potency than the reference drugs
against the Gram-negative bacteria and mild activity towards the Gram positive, fungi, and the
mycobacterium strain.
Table 2. Antimicrobial activity of the synthetic compounds (Inhibition Zone, IZ, diameter (mm))
(1 mg/mL in DMSO).
Fungi
Compounds
Gm+ve
Gm-ve
11

S.P B.S S.A MRSA P.A
NA 16.3 18.2 NA
20.4 26.8 19.2 19.2
E.C
S.T
18.9
24.3
20.4
19.8
21.6
22.9
24.2
23.4
21.8
23.4
21.2
20.4
23.2
18.1
15.8
20.9
20.5
20.5
18.3
17.3
18.3
19.1
21.6
23.6
-
A.F S.R
G.C
C.A
NA
19.3
14.2
NA
14.3
NA
NA
20.8
NA
NA
NA
NA
NA
NA
NA
16.6
16.1
21.8
NA
NA
NA
NA
NA
NA
-
1a
NA 17.8
17.6 21.6
NA 18.3
NA 16.4
14.3 19.6
NA 21.8
NA 21.4
NA 20.8
NA 19.3
NA 20.3
NA 19.4
NA 18.6
NA 21.2
NA 16.2
NA 15.2
NA 19.1
NA 18.9
NA 18.9
NA 17.1
NA 14.6
NA 15.6
NA 16.2
NA 19.3
NA 21.1
15.2 17.3 19.3
22.4 NA 24.3
15.7 NA 18.1
13.2 12.3 15.6
18.3 NA 20.2
21.3 23.2 24.4
22.3 21.4 24.6
19.3 NA 21.6
21.3 20.8 21.6
18.3 20.1 22.4
16.3 18.4 19.9
19.3 19.9 21.2
20.6 21.2 22.1
18.6 20.1 20.6
17.3 19.4 20.1
17.2 NA 19.1
16.3 NA 18.6
20.8 NA 22.3
17.6 16.8 19.1
15.2 14.6 15.9
16.2 14.9 16.2
14.6 16.2 16.9
17.6 19.2 21.1
20.3 22.1 23.4
1b
1c
17.1 19.3 16.2
18.6 20.1 17.2
18.3 22.3 16.1
NA 22.3 25.6
NA
NA
NA
NA
1d
1e
3a
3b
21.6 23.4 20.6 21.2
21.5 23.2 20.1 NA
3c
3d
20.4 22.6 19.8 21.3
19.3 21.3 23.3 14.6
3e
7a
NA 20.1 21.3
NA 18.1 20.3
19.8 22.7 19.6
17.6 19.2 17.3
16.3 18.2 16.4
NA
NA
NA
NA
NA
7b
7c
7d
7e
7f
18.9 20.1 21.3 18.3
18.3 19.6 18.4 NA
22.3 23.9 20.8 20.1
7g
7h
7i
19.2 20.9 17.2
17.3 19.2 16.4
17.9 20.1 16.9
16.4 16.9 17.2
NA
NA
NA
NA
7j
7k
7l
7m
18.6 19.3 21.2 15.4
20.4 21.7 23.8 18.4
7n
Ampicillin
ciprofloxacin
Amphotericin B
Vancomycine
23.8 32.4 26.2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
17.3 19.9
22.3
-
-
-
-
-
-
23.7 19.7 28.7
25.4
-
20.3
-
-
-
-
-
Mean zone of inhibition in mm from at least three experiments; (NA) means no activity.
12

Figure 2. Inhibition zone values (IZ) are reported for systematic compound comparisons. The values
are color-coded, according to the following scheme: red 23–33, yellow 18–22, green <15, and grey
(not tested). The color bars mark the matrix positions of the compounds in a particular bacteria type:
gm+ve, gm-ve, fungi, and MRSA.
13

Table 3. The antimicrobial activity of the synthetic compounds (Minimum inhibitory
concentration, MIC, µg/mL).
Gm-ve
S.P B.S S.A MRSA P.A E.C S.T A.F S.R G.C C.A
Compounds
Gm+ve
Fungi
1b
3.9 0.49 3.9
NA 0.98 0.49
0.98 0.98 3.9
1.95 0.98 3.9
3.9 0,98 3.9
3.9
NA
1.95
NA
1.95
15.63 1.95 0.49 0.98 NA 0.49 3.9
NA 0.98 0.98 0.98 0.98 0.49 NA
NA 1.95 0.49 0.98 1.95 0.49 NA
3a
3b
3c
NA 1.95 0.98 3.9
NA 1.95 1.95
3d
NA
NA
NA
3.9 0.98 1.95 1.95 0.98 NA
3.9 0.98 7.81 3.9 0.98 NA
3e
3.9 1.95 0.98 32.5
7b
NA 7.81 3.9
3.9 0.98 3.9
NA
NA
3.9 3.9
3.9
3.9 1.95 NA
7c
7f
NA 1.95 0.98 1.95 1.95 0.98 NA
NA 3.9 1.95 15.63 NA 3.9 15.63
3.9 3.9 1.95 7.81
0.98 0.49 1.95 3.9
3.9 0.98 0.49 7.81
7g
NA 0.98 0.49 1.95 NA 0.98 0.98
NA 1.95 0.49 3.9 0.98 0.98 NA
7n
Ampicillin
ciprofloxacin
Amphotericin B
Vancomycine
0.98 0.24 0.49
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
15.63 3.9 1.95
-
-
-
-
-
-
-
0.98 3.9 0.49 0.49
3.9
-
-
-
-
-
Results are mean values from at least three experiments; (NA) means no activity.
14

Figure 3. Minimum inhibitory concentration values (MIC). The values are color-coded, according
to the following scheme: spectrum colors; violet to yellow to green 6–10, blue to violet 14–17, and
red > 5, with the grey color indicating missing data. The color bars mark the matrix positions of the
compounds in a particular bacteria type: Gram positive, Gram negative, fungi, and MRSA.
2.2.2. Cytotoxic screening
The in vitro cytotoxic activity was performed, utilizing the MTT assay [39] against three
human carcinoma cell lines: human colon carcinoma (HCT-116), human hepatocellular carcinoma
(HepG-2), human breast adenocarcinoma (MCF-7) cell lines. Doxorubicin was used as a positive
control, which has high cytotoxicity. The inhibitory effects of compounds 1a-e, 3a-e, and 7a-n on
the growth of the three cell lines are described in Table 4 and Figure 4. As mentioned previously,
the cytotoxic activities of compounds 1 and 3 have been appraised in order to derive a comparison
between the activity of the chromene molecules containing azo sulfonamide and their precursors,
which consequently elucidate the performance of the chromene moieties in this class of activity.
The sulfa compound class 1a-e exhibited less potency as cell growth inhibitors. As anticipated, the
inhibitory behavior was increased by the 10-fold effect via the incorporation of the azo moieties in
compounds 3a-e. Moreover, the combination of the chromene motifs into these derivatives enhanced
15

the potent cytotoxicity when evaluated against the reference drugs. Within the evaluation of the
azo/chromene-based sulfa molecules, it was discerned that the 4-aryl substituents demonstrate more
activity than their 2-postioned analogues and the non-substituted rings. Hence, the 4-fluro divulged
the highest potent antiproliferative effect linked to the azo derivatives of the sulfaisoxazole moiety,
followed by the 4-methyl derivative of the chromene compounds of the sulfacetamide structure,
Figure 4.
Table 4. Cytotoxicity of the chromene derivatives against the three cancer cell lines.
IC50 (µM)
Compounds
R
Ar
HCT-116
MCF-7
HepG-2
93.5
113.4
97.1
79.9
67.1
39.5
23.5
62.0
58.2
48.1
4.3
143.43
105.9
104.5
81.8
67.1
40.2
24.6
55.2
58.4
42.7
1.8
256.1
90.6
204.9
89.3
84.5
60.7
26.1
89.7
75.4
51.8
9.7
1a
1b
1c
1d
1e
3a
3b
3c
3d
3e
7a
7b
7c
7d
7e
H
COCH3
-
-
-
-
NH2-C=(NH)-
Thiazol-2-yl
3-methyl-isoxazol-5-yl
H
-
COCH3
-
NH2-C=(NH)-
Thiazol-2-yl
3-methyl-isoxazol-5-yl
H
-
-
-
C6H5
H
C6H4F-4
C6H4CH3-4
C6H4F-4
C6H4Cl-2
11.8
8.3
9.6
10.1
9.8
COCH3
5.6
COCH3
5.4
5.7
5.3
COCH3
11.8
17.9
15.1
16

NH2-C=(NH)-
NH2-C=(NH)-
NH2-C=(NH)-
Thiazol-2-yl
C6H5
39.8
10.3
5.3
37.2
13.0
10.3
9.5
45.3
10.4
4.4
7f
C6H4Cl-2
C6H4F-4
7g
7h
C6H4CH3-4
C6H4F-4
10.6
5.5
11.3
5.5
7i
Thiazol-2-yl
4.7
7j
Thiazol-2-yl
C6H4Cl-2
C6H4CH3-4
C6H4F-4
14.8
4.8
18.9
8.6
13.4
4.7
7k
3-methyl-isoxazol-5-yl
3-methyl-isoxazol-5-yl
3-methyl-isoxazol-5-yl
-
7l
7m
3.4
2.2
1.7
C6H4Cl-2
12.6
1.6
13.0
1.9
8.9
7n
2.2
Doxorubicin
The cytotoxic effects of compounds on colon, breast, and liver cell lines, following the exposure to
different concentrations of the target compounds, and the cell viability was assessed, using the MTT
method. Data are presented as IC50 (µg/mL) values.
50
HCT-116
MCF-7
HepG-2
45
40
35
30
25
20
15
10
5
0
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n R. Drug
17

Figure 4. In vitro cytotoxic activity. The IC50 values of the target compounds were introduced in
the bar shape for the comparison of the effect of the aryl substitution on the chromene derivatives
versus the reference drug.
2.2.3. In vitro histone deacetylase (HDACs) inhibitory activity
The selective HDAC inhibitors provide a powerful chemical tool to dissect the individual
functions of the HDAC isoforms and finally provide lead antitumor drug candidates. To assess the
isoform selectivity of the 8-ethoxy-3-nitro-2H-chromene analogues, HDAC1 and HDAC2,
representing class I, and HDAC8, representing class II, were used in the in vitro inhibitory assay.
Only the analogs exhibiting potent cytotoxic effects were used in the assay: 7a, 7c, 7d, 7i, 7j, 7l,
and 7m, in comparison with the referenced HDAC inhibitor Vorinostat. The IC50 values for the
selected compounds that displayed relatively good inhibitory activity toward HADC1, 2, and 8 at
10 μM had been determined in Table 4. As can be witnessed in Table 4, the chromene analogues
7d, 7i, and 7l manifested inhibitory activities against HDAC1 and 2 at, mostly, micromolar and
submicromolar concentrations of 0.92-12.86 µM with a selective tendency towards HDAC1 and 2.
For example, compounds 7d had IC50 values of 1.94 and 2.53 for HDAC 1 and 2, respectively. In
contrast, the chromene analogue 7l displayed effective inhibitory activity against HDAC1 over
HDAC2 and HDAC8. In addition, the enzyme inhibitory activities of the most desired compounds
were well-correlated with their antiproliferative activities. Compounds 7a, 7c, 7d, 7i, 7j, 7l, and 7m
with stronger antiproliferative activities also exerted higher inhibitory activities against the HDACs.
The perceptible rationale behind compound 7d exhibiting non-selectivity against HDAC1 and 2 is
attributable to the electron-withdrawing substituents on the aryl moieties, acetyl and 4-fluro. In
contrast, the derivatives 7i and 7m with a 4-CH3 substituent as an electron-donating group,
accompanied by the heteroaryl, thiazol, and oxazole moieties, presented selectivity tendencies
towards HDAC1 only. Considering the obtained biological data, we could preliminarily arrive at the
conclusion that some target compounds might be potent HDAC enzyme inhibitors. Thus, they
assemble promising lead compounds for the development of novel anti-tumor drugs that can
potentially alter the disease state via inhibiting HDACs.
2.2.4. Tubulin inhibitory effect screening
Microtubule-targeting agents affect microtubule functions and hinder the mitosis process, resulting
in necrosis and apoptosis. Throughout our investigation, we delve into the influence of two chosen
18

derivatives on the Tubulin polymerization, using the HCT-116 cancer cell line. The data exhibited
a moderate effect on the inhibition of the Tubulin polymerization in comparison to Colchicine,
reported in Table 5. As witnessed from the table, the derivative 7c with an electron-donating
substituent (4-CH3) demonstrated more activity in the selective comparison with 7d with an electron-
withdrawing group (4-F) for the inhibition of the Tubulin polymerization. The intention of selecting
7c and 7d for this evaluation was as they are prime instances of selectivity and non-selectivity against
the HDAC behavior, which is perceptible from the HDAC activity panel.
Table 5. The inhibitory effect analyses of chromene derivatives against the HDAC classes and the
Tubulin polymerization.
Inhibitory activity
HDAC (IC50, µM)
Class I Class II
Tubulin polymerization
Compounds
HDAC1
3.82
7.18
1.94
1.36
3.82
0.92
7.18
0.82
-
HDAC2
4.74
4.4
HDAC8
7.2
HCT-116 (µM) Inhibition %
-
-
7a
>10
9.5
7.18
92.44
7c
7d
2.53
3.64
1.2
8.84
87.12
>10
>10
>100
6.5
-
-
7i
-
-
7j
7l
12.86
5.01
0.93
-
-
-
-
-
-
-
7m
1.98
-
Vorinostat
Colchicine
0.16
86.3
Data are presented as average IC50 (µM) values for at least three experiments.
2.3. Docking studies of 7d, 7i, and 7m into the HDAC2 enzyme
To further investigate the interactions between these compounds and the HDACs, we
performed the docking simulations, using the MOE Suite. Following the ligand preparation, the
compounds were docked into the binding pockets in the HDAC2 isoform as an example of class I
HDACs. As shown in Figure 5A (PDB ID: 4LY132) [46], the docking simulations suggested that
19

the chromene compounds 7d, 7i, and 7m interact with the active site in a fashion similar to the
benzamide ligand observed in the reported crystal structure of the HDAC 2 (PDB ID: 4LXZ), Figure
5. Firstly, the docking pose of the benzamide ligand in the complex with HDAC2, Figure 5A,
consists of a long, narrow tunnel, leading to a cavity that contains the catalytic Zn2+ ion, which is
pentacoordinated with Asp181, His183, and Asp269 as well as with the carbonyl oxygen and the
NH2 of the benzamide. In addition to the simultaneous bidentate chelate formation with Zn2+, there
is also a hydrogen bonding interaction with His145, Asp181, and Tyr308 through its benzamide NH
and NH2 fragments. According to the detailed interaction analyses reported in Table 6, the active
derivatives 7d, 7i and 7m chelate mainly to the Zn2+ ion through its sulfonamide group by dual metal
interactions. In addition, these molecules experienced interesting hydrogen bonding interactions
with three important amino acid residues Tyr308, Gly154, and His146 through the NH and SO2
fragments by a 1.5-3.1 Å distance range. Moreover, the aryl chromene moiety forms a π-π
interaction with certain aromatic amino acids: Pro211, Phe155, Phe210, Leu276, and Tyr209, with
the p-position substituents (F and Methyl) that might play an important role in increasing their
binding affinity for the HDAC2 enzyme. This interaction analyses represented the role of the aryl
substitution in HDAC selectivity and might be responsible for their antiproliferative effects.
A)
20

B)
C)
D)
Figure 5. Docking modes of active compounds in the binding pocket of HDAC2. Interactions
between the protein (PDB ID: 4LY1), the ligands of compound 7d, 7i, 7m, and the benzamide inhibitor
are shown as dotted lines; the zinc ion as a black ion; the residues as colored codes as presented in the
software; and the ligands as black line models. (A) Predicted binding mode of benzamide inhibitor with
HDAC2. (B) Predicted binding mode of 7d with HDAC2. (C) Predicted binding mode of 7i with
HDAC2. (D) Predicted binding mode of 7m with HDAC2.
Table 6. Description of the docking data of selected target compounds.
Comp.
Fragment
Target residues
(Distance, Å)
Interaction
Binding energy
(dG)
21

Zn+2 (Asp269,
Asp181,
Coordination
bonding
Acetyl C=O
His183), 1.92,
2.03, 2.05
Tyr308, 2.74
Tyr308, 2.74
His146, 2.65
Hydrogen bonding
Hydrogen bonding
Sulphonamide-
SO2
7d
-16.5
Sulphonamide-
NH
Gly154, 1.85
Hydrogen bonding
Sulphonamide- Phe210, Phe155,
Hydrophobic
interaction
Benzene
Chromene
Thiazole
, Tyr308
Asp104, Pro211,
Glu208
Hydrophobic
Hydrophobic
Phe155, Met35
Zn+2 (Asp269,
Asp181,
Coordination
bonding
His183), 1.92,
2.03, 2.05
Sulphonamide-
SO2
His146 , 3.11
Tyr308, 2.57
Hydrogen bonding
Hydrogen bonding
7i
-16.4
Sulphonamide-
NH
Asp269, 2.05
Sulphonamide-
Benzene
His183, Leu276 Aromatic stacking
Pro211, Phe210,
Hydrophobic
Tyr209
Chromene
Isoxazole
Leu144,
Hydrophobic
Cys156, Gly306
Zn+2 (Asp269,
Coordination
bonding
Asp181,
His183), 1.92,
2.03, 2.05
-15.6
7l
Sulphonamide-
SO2
His146 , 2.41
Tyr308, 2.28
Hydrogen bonding
22

Sulphonamide-
Benzene
His183, Phe155,
Phe210
Aromatic stacking
Hydrophobic
Asp104,
Chromene
Gly154, Leu276
His183, Tyr308,
Phe155
Benzamide ring
Aromatic stacking
Zn+2 (Asp269,
Asp181), 3.19,
2.69
Coordination
bonding
Benzamide C=O
-14.5
Hydrogen bonding
Hydrogen bonding
Hydrogen bonding
Hydrogen bonding
Hydrophobic
Tyr308, 2.69
His145, 2.83
Asp181, 2.85
His185, 3.7
His146, Phe155,
Leu144
Aniline NH2
Thiophene
interaction
The data reported in the table are extracted from MOE program showing the corresponding amino
acids residues in enzyme pocket, corresponding fragments of ligands, interaction distances, types of
interaction, and their binding energy to selected drugs.
3. Conclusion
This research depicts a breakthrough of a novel series of azosulfonamides bearing 4H-chromenes, which
were designed, synthesized, and evaluated for their inhibitory activity against the HCT-116, MCF-7, and
HepG-2 cell lines. The incorporation of the chromene moieties into the azosulfonamide molecules
instigated a substantial triumph in their cytotoxicity evaluation against their corresponding precursors.
Furthermore, some of these 4H-chromene/sulfonamide derivatives displayed potent antiproliferative
activities and were more potent towards the examined tumor cell lines in comparison to the Doxorubicin
reference drug. Additionally, certain mechanisms were explored such as the HDAC inhibitory analyses,
which established the existence of the submicromolar inhibitory concentrations against the HDAC 1 and
2 subtypes. In order to gain insight into the inhibition procedure of the HDAC enzymes and to verify the
ligand-binding interactions, the blind docking protocol was adopted. The application of the sulfa drugs
as both a zinc binding group (ZBG) and internal cavity motifs, attached to the chromene substructure,
demonstrated high potential as potent anticancer molecules, mediated through the selective HDAC
23

inhibition. To broaden our comprehensive study, we administrated assessments for the novel derivatives’
antibacterial, antifungal, and antimycobacterium activities, which illustrated palpable bacterial and
fungal inhibition when employing an evaluation against the reference drugs.
4. Experimental
4.1. Chemistry
The starting reagents and solvents were purchased from the Aldrich chemical company and were
used as received. The melting points were determined with a Mel-Temp apparatus and are
1
uncorrected. All the new compounds were characterized by the H-NMR and 13C-NMR, using
Varian Gemini NMR spectrometer (Varian, Inc., Palo Alto, CA) (300 MHz, internal standard:
tetramethylsilane), and 1H-NMR (400 MHz), 13C-NMR (100 MHz) and 13C DEPT at 135o and 19F
NMR spectra on a Gemini 200 NMR spectrometer, and were measured in DMSO-d6 at room
temperature. The chemical shifts (δ) were reported in ppm to a scale calibrated for tetramethylsilane
(TMS), which is used as an internal standard. The IR spectra (KBr pellets) were recorded using a
FTIR Bruker Vector 22 spectrophotometer (Bruker Corp., Billerica, MA) ( max in cm−1) at the
Microanalytical Center, Faculty of Science, Cairo University. The mass spectra were determined on
a Shimadzu GC/MS-QP5050A spectrometer. Elemental analysis was carried out at the Regional
Center for Mycology and Biotechnology (RCMP), Al-Azhar University, Cairo, Egypt and the results
were within ± 0.25%. The follow up of the reactions and the check of the purity of the compounds
were made by the TLC on silica gel-protected aluminum sheets (Type 60 GF254, Merck), and the
spots were detected by exposure to a UV-lamp at λ 254 nm for a few seconds.
4.1.1.
General
procedure
for
the
synthesis
of
4-((2,4-
dihydroxyphenyl)diazenyl)benzenesulfonamide (3a-e)
4-Aminobenzenesulfonamide(1a-e) (10 mmol) was suspended in water (50 mL). HCl (10 mL, 36.5
%) was added dropwise to this solution with stirring. The mixture was gradually heated up to 70 oC
till a clear solution was obtained. The solution was cooled to 0-5 oC in an ice bath. A sodium nitrite
solution (0.5 g in 5 mL H2O) was then added over a period of 5 minutes with stirring. Resorcinol
(1.1 g, 10 mmol) was dissolved in 10% of the NaOH solution and kept at 0-5 oC. Subsequently, the
diazonium salt solution was added with the occasional slow stirring to the resorcinol solution for
15-30 minutes. After acidification, the formed precipitate was filtered, dried, and recrystallized from
absolute ethanol to give the pure products 3a-e.
24

4-((2, 4-dihydroxyphenyl)diazenyl)benzenesulfonamide (3a)
Yellow crystals (87 %), m.p. 130 °C; IR (KBr) cm-1: max 3518 (OH), 3401, 3180 (NH2), 1627
(N=N), 1328, 1156 (S=O); 1H NMR (DMSO-d6, ppm) δ: 1.95 (2H, s, NH2, D2O exchangeable),6.38
(1H, s, Ar-H), 6.65 (2H, d, J = 7Hz, Ar-H), 7.45 (1H, s, OH, D2O, exchangeable) 7.68 (2H, d, J =
7Hz, Ar-H), 7.97 (3H, m, Ar-H), 12.18 (1H, s, NH, D2O exchangeable); 13C NMR (DMSO-d6, ppm)
δ: 104.2, 107.5, 122.1, 124.4, 126.3, 131.2, 140.5, 153.1, 154.6, 160.1 (Ar-C). MS (m/z), 293; Anal.
Calcd; for C12H11N3O4S: %C: 49.14, %H: 3.78; %N: 14.33. Found: %C: 49.10, %H: 3.64; %N:
14.41.
N-((-4-[(2,4-dihydroxyphenyl) diazenyl)phenyl) sulfonyl)acetamide (3b)
Red crystals (84 %), m.p. 150 °C; IR (KBr) cm-1: max 3480 (OH), 3250 (NH), 1625(C=O), 1597
(N=N), 1308, 1142 (S=O); 1H NMR (DMSO-d6, ppm) δ: 2.25 (3H, s, CH3), 6.20 (1H, s, OH, D2O
exchangeable), 6.35 (1H, d, J = 7.1Hz, Ar-H), 6.50 (1H, d, Ar-H), 6.72 (1H, s,Ar-H), 7.66 (1H, d, J
= 7.1Hz, Ar-H), 7.97 (2H, d, J = 8.4 Hz, Ar-H), 8.12 (2H, d, J = 8.4 Hz, Ar-H), 10.6 (1H, s, OH,
13
D2O exchangeable), 12.30 (1H, s, NH, D2O exchangeable); C NMR (DMSO-d6, ppm) δ: 16.2
(CH3), 104.1, 107.3, 122.6, 124.5, 126.2, 130.2, 140.3, 156.5, 152.1, 160.5 (Ar-C), 170.10 (C=O).
MS (m/z), 335; Anal. Calcd; for C14H13N3O5S: %C: 50.14, %H: 3.91; %N: 12.53. Found: %C: 65.23,
%H: 3.82; %N: 12.63.
N-carbamimidoyl-4-((2,4-dihydroxyphenyl) diazenyl) benzenesulfonamide (3c)
Orange crystals (92 %), m.p. 136 °C; IR (KBr) cm-1: max 3594, 3436, 3338 (OH, NH, NH2), 1626
(N=N), 1340, 1132 (S=O); 1H NMR (DMSO-d6, ppm) δ: 6.41 (1H, bs, Ar-H), 6.55 (1H, d, J = 7.1Hz,
Ar-H), 6.89 (2H, bs, NH2), 7.69 (1H, d, J = 7.1Hz, Ar-H), 7.93 (4H, bs, Ar-H), 9.80 (1H, s, OH,
D2O exchangeable), 10.6 (1H, s, NH, D2O exchangeable), 12.80 (1H, s, NH, D2O exchangeable);
13C NMR (DMSO-d6, ppm) δ: 103.0, 109.6, 121.7, 126.9, 129.6, 132.1, 144.1, 152.5, 157.2, 157.9
(Ar-C), 163.9 (C=N). 13C NMR-DEPT (135o) δ: 102.8, 109.4, 121.5, 126.5, 126.7, 129.4 for
ArCH’s. MS (m/z), 335; Anal. Calcd; for C13H13N5O4S: %C: 46.56, %H: 3.91; %N: 20.88. Found:
%C: 46.63, %H: 3.73; %N: 20.90.
4-((2,4-Dihydroxyphenyl) diazenyl)-N-(thiazol-2-yl) benzenesulfonamide (3d)
25

Orange crystals (89 %), m.p. 133 °C; IR (KBr) cm-1: max 3447 (OH), 3150 (NH), 1623 (N=N),
1326, 1142 (S=O); 1H NMR (DMSO-d6, ppm) δ: 6.38 (1H, s, ArH), 6.52 (1H, d, J = 7.1Hz, Ar-H),
6.84(1H, d, Ar-H), 7.25 (1H, d, J = 8Hz, thiazol-H), 7.68 (1H, d, J = 8Hz, thiazole-H), 7.94 (4H, bs,
13
Ar-H), 9.46 (1H, s, OH, D2O exchangeable), 12.27 (1H, bs, NH, D2O exchangeable); C NMR
(DMSO-d6, ppm) δ: 102.9, 108.2, 109.5, 121.7, 124.3, 126.9, 129.2, 132.6, 142.4, 152.4, 157.3,
163.9, 168.8 (Ar-C); 13C NMR-DEPT (135o) δ: 102.6, 108.0, 109.3, 121.5, 124.1, 126.7, 128.9 for
ArCH’s. MS (m/z), 376; Anal. Calcd; for C15H12N4O4S2: %C: 47.86, %H: 3.21; %N: 14.88. Found:
%C: 47.70, %H: 3.34; %N: 14.78.
4-((2,4-Dihydroxyphenyl)diazenyl)-N-(3-methylisoxazol-5-yl)benzenesulfonamide (3e)
Red crystals (82 %), m.p. 153 °C; IR (KBr) cm-1: max 3422 (OH), 3150 (NH), 1616 (N=N), 1346,
1
1154 (S=O); H NMR (DMSO-d6, ppm) δ: 2.30 (3H, s, CH3), 6.17 (1H, s, ArH), 6.30 (1H, d, J =
7.1Hz, Ar-H), 6.50(1H, d, Ar-H), 6.39 (1H, s, isoxazole-H), 7.67 (1H, d, J = 7.1Hz, Ar-H), 8.01 (4H,
bs, Ar-H), 10.70 (1H, s, OH, D2O exchangeable), 11.50 (1H, s, NH, D2O exchangeable); 12.1 (1H,
bs, NH, D2O exchangeable); 13C NMR (DMSO-d6, ppm) δ: 11.98(CH3), 95.4, 103.0, 109.8, 122.1,
128.2, 128.9, 132.9, 139.3, 153.5, 157.3, 157.9, 164.4 (Ar-C). 13C NMR-DEPT (135o) δ: 95.2, 102.7,
109.6, 121.9, 127.8, 128.6 for ArCH’s. MS (m/z), 374; Anal. Calcd; for C16H14N4O5S: %C: 51.33,
%H: 3.77; %N: 14.97. Found: %C: 51.30, %H: 3.82; %N: 14.86.
4.1.2. General procedure for the synthesis of 4-((2-amino-3-cyano-4-aryl-7-hydroxy-4-aryl-
4H-chromen-6-yl)diazenyl] benzenesulfonamides (7a-n)
To a mixture of 4-((2, 4-dihydroxyphenyl)diazenyl)benzenesulfonamide (3a-e) (10 mmol) and
arylidenemalononitriles (5a-d) (10 mmol) in 20 mL ethanol, a few drops of piperidine was added.
The reaction mixture was refluxed for 1 hour. After cooling, the precipitate was filtered, washed
with water, and recrystallized from the appropriate solvent to yield the pure products 7a-n.
4-((2-Amino-3-cyano-7-hydroxy-4-phenyl-4H-chromen-6-yl) diazenyl) benzenesulfonamide
(7a)
Brown crystals (86 %), m.p. 165 °C (EtOH); IR (KBr) cm-1: max 3439 (OH), 3357, 3203 (NH2),
1
2190 (CN), 1589 (N=N), 1341, 1152 (S=O); H NMR (DMSO-d6, ppm) δ: 4.77 (1H, s, pyran-H),
6.80 (1H, d, Ar-H), 7.05 (2H, bs, NH2, D2O exchangeable), 7.18-7.20 (2H, m, Ar-H), 7.22-7.33 (2H,
m, Ar-H),7.76 (1H, d, Ar-H), 8.02 (2H, d, Ar-H), 8.14 (2H, d, Ar-H), 11.97 (1H, s, NH, D2O
26

13
exchangeable); C NMR (DMSO-d6, ppm) δ: 28.5 (pyran-CH), 60.1, 104.1, 116.1 (CN), 123.1,
124.5, 125.2, 126.0, 128.1, 129.0, 129.3, 132.3, 140.1, 142.1, 148.2, 154.2, 160.1, 170.5 (Ar-C +
pyran-C2 & pyran-C3). MS (m/z), 447; Anal. Calcd; for C22H17N5O4S: %C: 59.05, %H: 3.83; %N:
15.65. Found: %C: 59.15, %H: 3.63; %N: 14.53.
4-((2-Amino-3-cyano-7-hydroxy-4-(4-fluorophenyl)-4H-chromen-6- yl) diazenyl) benzene
sulfonamide (7b)
Brown crystals (83 %), m.p. 210 °C (EtOH); IR (KBr) cm-1: max 3495 (OH), 3355, 3180 (NH2),
1
2193 (CN), 1604 (N=N), 1340, 1153 (S=O); H NMR (DMSO-d6, ppm) δ: 4.80 (1H, s, pyran-H),
6.81 (1H, d, Ar-H), 7.07-7.26 (4H, m, Ar-H and NH2; D2O exchangeable ), 7.94 (2H, d, J = 7Hz,
Ar-H), 8.02 (2H, d, J = 7Hz, Ar-H), 8.02 (2H, d, J = 7Hz, Ar-H), 11.97 (1H, s, NH, D2O
exchangeable); 13C NMR (DMSO-d6, ppm) δ: 29.0 (pyran-CH), 59.1, 105.1, 115.1, 116.0, 117.1
(CN), 123.2, 124.1, 124.6, 130.2, 133.9, 137.1, 140.5, 148.1, 154.1, 159.2, 160.3, 177.1 (Ar-C +
pyran-C2 & pyran-C3). MS (m/z), 465; Anal. Calcd; for C22H16FN5O4S: %C: 56.77, %H: 3.46; %N:
15.05. Found: %C: 56.67, %H: 3.32; %N: 15.10.
N-4-((2-amino-3-cyano-7-hydroxy-4-(p-tolyl)-4H-chromen-6-yl)diazenyl)phenyl)sulfonyl)
acetamide (7c)
Orange crystals (83 %), m.p. 155 °C (EtOH); IR (KBr) cm-1: max 3490 (OH), 3350, 3150 (NH2),
2217 (CN), 1723 (C=O), 1598 (N=N), 1367, 1143 (S=O); 1H NMR (DMSO-d6, ppm) δ: 2.18 (3H,
s, COCH3), 2.38 (3H, s, Ar-CH3), 5.06 (1H, s, pyran-H), 6.84 (1H, s, Ar-H), 6.99 (1H, d, Ar-H),
7.05 (2H, d, Ar-H ), 7.19(1H, S, NH), 7.39 (2H, d, Ar-H ), 7.93(2H, d, Ar-H ), 8.33(1H, s, Ar-H ),
8.05( 1H, s, NH, D2O exchangeable ), 12.13 (1H, s, NH (Hydrazo), D2O exchangeable); 13C NMR
(DMSO-d6, ppm) δ: 16.1 (CH3), 21.1 (CH3), 30.1 (pyran-CH), 59.5, 105.1, 116.0 (CN), 121.9, 124.2,
124.5, 125.3, 128.1, 129.6, 134.1, 134.3, 138.5, 140.3, 150.1, 154.1, 160.1, 172.5 (Ar-C + pyran-C2
& pyran-C3), 176.4 (C=O). MS (m/z), 503; Anal. Calcd; for C25H21N5O5S: %C: 59.63, %H: 4.20;
%N: 13.91. Found: %C: 59.52, %H: 4.37; %N: 13.97.
N-4-((2-amino-3-cyano-4-(4-fluorophenyl)-7-hydroxy-4H-chromen-6-yl)diazenyl)
phenylsulfonyl) acetamide (7d)
27

Orange crystals (85 %), m.p. 162 °C (EtOH); IR (KBr) cm-1: max 3412 (OH), 3316, 3188 (NH2),
2192 (CN), 1700 (C=O), 1604 (N=N), 1314, 1138 (S=O); 1H NMR (DMSO-d6, ppm) δ: 2.16 (3H,
s, CH3CO), 4.77 (1H, s, pyran-H), 6.39 (1H, s, Ar-H), 6.72 (1H, s, Ar-H), 6.73 (2H, d, Ar-H), 7.08-
7.22(4H, m, Ar-H and NH2), 7.74(2H, d, Ar-H), 7.96 (2H, d, Ar-H), 8.00 (2H, d, Ar-H ), 13C NMR
(DMSO-d6, ppm) δ: 21.1 (CH3), 30.0 (pyran-CH), 58.8, 105.1, 115.1, 116.0 (CN), 123.1, 124.1,
124.6, 125.2, 128.1, 130.2, 134.2, 138.4, 140.1, 150.3, 155.3, 160.2, 173.0 (Ar-C + pyran-C2 &
pyran-C3), 177.2 (C=O). MS (m/z), 507; Anal. Calcd; for C24H18FN5O5S: %C: 56.80, %H: 3.58;
%N: 13.80. Found: %C: 56.91, %H: 3.45; %N: 13.70.
N-(4-((2-amino-4-(2-chlorophenyl)-3-cyano-7-hydroxy--4H-chromen-6-yl)diazenyl)
phenylsulphonyl)acetamide (7e)
Orange crystals (80 %), m.p. 177 °C (EtOH); IR (KBr) cm-1: max 3447 (OH), 3350, 3100 (NH2),
2193 (CN), 1690 (C=O), 1598 (N=N), 1398, 1150 (S=O); 1H NMR (DMSO-d6, ppm) δ: 2.23 (3H,
s, CH3CO), 5.29 (1H, s, pyran-H), 6.72 (1H, s, Ar-H), 6.79(1H, d, Ar-H), 7.05 (1H, s, NH, D2O
exchangeable), 7.09-7.41(4H, m, Ar-H and NH2), 7.75 (1H, d, Ar-H), 8.08 (4H, br s, Ar-H), 11.86
(1H, s, NH, D2O exchangeable); 13C NMR (DMSO-d6, ppm) δ: 20.4 (CH3), 30.0 (pyran-CH), 58.8,
105.0, 117.0 (CN), 123.1, 124.1, 124.6, 125.6, 127.1, 127.3, 129.2, 129.7, 133.7, 141.2, 142.0,
148.0, 154.6, 173.0 (Ar-C + pyran-C2 & pyran-C3), 176.0 (C=O). Anal. Calcd; for C24H18ClN5O5S:
%C: 55.02, %H: 3.46; %N: 13.37. Found: %C: 55.23, %H: 3.56; %N: 13.26.
4-((2-Amino-3-cyano-7-hydroxy-4-phenyl-4H-chromen-6-yl)diazenyl)-N-carbamimidoyl
benzenesulfonamide (7f)
Orange crystals (88 %), m.p. 156 °C (EtOH); IR (KBr) cm-1: max 3428 (OH), 3343, 3208 (NH2),
1
2190 (CN), 1589 (N=N), 1398, 1137 (S=O); H NMR (DMSO-d6, ppm) δ: 4.78 (1H, s, pyran-H),
6.77 (2H, br, NH2), 6.80 (1H, s, NH), 6.82 (1H, s, NH), 7.03 (2H, s, NH2), 7.22 (3H, t, Ar-H), 7.31
13
(3H, t, Ar-H), 7.77 (1H, d, Ar-H), 7.9 (2H, d, Ar-H), 8.05 ( 2H, d, Ar-H), 11.90 (1H, s, NH); C
NMR (DMSO-d6, ppm) δ: 36.3 (pyran-CH), 57.3, 108.4, 112.4 (CN), 119.9, 122.5, 124.2, 126.5,
126.6, 127.0, 128.3, 134.0, 144.0, 145.8, 151.9, 152.7, 153.4, 158.0, 159.4 (Ar-C + pyran-C2 &
pyran-C3). 13C NMR-DEPT (135o) δ: 36.0, 108.1, 122.2, 123.9, 126.2, 126.3, 126.7, 128.0 for
ArCH’s. MS (m/z), 489; Anal. Calcd; for C23H19N7O4S: %C: 56.43, %H: 3.91; %N: 20.03. Found:
%C: 56.55, %H: 3.84; %N: 20.12.
28

4-((2-amino-4-(2-chlorophenyl)-3-cyano-7-hydroxy-4H-chromen-6-yl)diazenyl)-N-
carbamimidoylbenzenesulfonamide (7g)
Orange crystals (86 %), m.p. 182 °C (EtOH/Dioxane); IR (KBr) cm-1: max 3422 (OH), 3343, 3216
(NH2), 2194 (CN), 1586 (N=N), 1398, 1139 (S=O); 1H NMR (DMSO-d6, ppm) δ: 5.30 (1H, s, pyran-
H), 6.78 (2H, br, NH2), 6.80 (2H, s, NH2), 7.03 (2H, s, NH2), 7.13 (1H, s, NH), 7.20-7.28(2H, m,
Ar-H), 7.41 (1H, d, Ar-H), 7.77 (1H, d, Ar-H), 7.88( 2H, d, Ar-H), 8.05( 2H, d, Ar-H), 11.82(1H, s,
NH Hydrazo); 13C NMR (DMSO-d6, ppm) δ: 33.4 (pyran-CH), 55.9, 108.0, 111.3 (CN), 119.4,
122.5, 123.6, 126.5, 127.5, 128.1, 129.2, 130.0, 131.9, 134.9, 141.2, 145.7, 152.1, 153.0, 153.09,
158.0, 159.4 (Ar-C + pyran-C2 & pyran-C3). 13C NMR-DEPT (135o) δ: 33.4, 107.9, 122.3, 123.3,
126.3, 127.2, 127.8, 129.0, 129.8 for ArCH’s. Anal. Calcd; for C23H18ClN7O4S: %C: 52.72, %H:
3.46; %N: 18.71. Found: %C: 52.74, %H: 3.50; %N: 18.63.
4-((2-amino-3-cyano-4-(4-fluorophenyl)-7-hydroxy-4H-chromen-6-yl)diazenyl)-N-
carbamimidoylbenzenesulfonamide (7h)
Orange crystals (83 %), m.p. 230 °C (EtOH); IR (KBr) cm-1: max 3427 (OH), 3342, 3209 (NH2),
1
2191 (CN), 1588 (N=N), 1399, 1139 (S=O); H NMR (DMSO-d6, ppm) δ: 4.81 (1H, s, pyran-H),
6.76 (2H, b s, NH2), 6.79 (1H, s, Ar-H), 6.82 (1H, s, NH), 7.05 (2H, s, NH2), 7.13 (2H, t, Ar-H),
7.25 (2H, m, Ar-H), 7.77(1H, d, Ar-H), 7.89 (2H, d, Ar-H), 8.05(2H, d, Ar-H), 11.98 (1H, NH,
Hydrazo); 13C NMR (DMSO-d6, ppm) δ: 35.7 (pyran-CH), 57.0, 108.4, 112.2, 114.9 (CN), 115.1,
119.8, 122.5, 124.0, 126.6, 128.8, 128.9, 134.9, 141.0, 145.8, 151.9, 152.5, 153.5, 158.0, 159.3,
159.5, 162.0 (Ar-C + pyran-C2 & pyran-C3). 13C NMR-DEPT (135o) δ: 35.4, 108.1, 114.6, 114.8,
122.2, 123.8, 126.3, 128.6, 128.7 for ArCH’s. MS (m/z), 507; Anal. Calcd; for C23H18FN7O4S: %C:
54.43, %H: 3.57; %N: 19.32. Found: %C: 54.47, %H: 3.65; %N: 19.41.
4-((2-amino-3-cyano-7-hydroxy-4-p-tolyl-4H-chromen-6-yl)diazenyl)-N-(thiazol-2-yl)
benzenesulfonamide (7i)
Orange crystals (82 %), m.p. 175 °C (EtOH); IR (KBr) cm-1: max 3460 (OH), 3347, 3100 (NH2),
1
2192 (CN), 1586 (N=N), 1328, 1142 (S=O); H NMR (DMSO-d6, ppm) δ: 2.23 (3H, s, Ar-CH3),
4.72 (1H, s, pyran-H), 6.80 (1H, d, J = 8 Hz, thiazole-H), 6.84 (1H, d, J = 8 Hz, thiazole-H), 7.0
(2H, s, NH2), 7.08 (4H, bs, Ar-H), 7.25(1H, dd, Ar-H), 7.55 (1H, dd, NH, Ar-H), 7.85 (1H, dd, NH,
29