Synthesis, carbonic anhydrase inhibition and cytotoxic activity of novel chromone-based sulfonamide derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.04.033

Four series of sulfonamides incorporating chromone moieties were synthesized and assessed for their cytotoxic activity against MCF-7 and A-549 cell lines, considering the fact that some of these tumors overexpress isoforms of carbonic anhydrase (CA, EC 4.2.1.1) which is inhibited by sulfonamides. Most new sulfonamides showed weak inhibitory activity against the offtarget, cytosolic isoforms hCA I, II but effectively inhibited the tumor-associated hCA IX and XII. The most active compounds featured a primary SO2NH2 group and were active in the low micromolar range against MCF-7 and A-549 cell lines. Compound 4a showed IC50 of 0.72 and 0.50 1/4M against MCF-7 and A-549 cell lines, respectively, and was further evaluated for its proapoptotic activity which proved enhanced in both tumor types.

Full text of DOI:10.1016/j.ejmech.2015.04.033

Accepted Manuscript
Synthesis, carbonic anhydrase inhibition and cytotoxic activity of novel chromone-
based sulfonamide derivatives
Fadi M. Awadallah, Tamer A. El-Waei, Mona M. Hanna, Safinaz E. Abbas,
Mariangela Ceruso, Beyza Ecem Oz, Ozen Ozensoy Guler, Claudiu T. Supuran
PII:
S0223-5234(15)00292-5
10.1016/j.ejmech.2015.04.033
EJMECH 7848
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 January 2015
Revised Date: 31 March 2015
Accepted Date: 14 April 2015
Please cite this article as: F.M. Awadallah, T.A. El-Waei, M.M. Hanna, S.E. Abbas, M. Ceruso, B.E.
Oz, O.O. Guler, C.T. Supuran, Synthesis, carbonic anhydrase inhibition and cytotoxic activity of novel
chromone-based sulfonamide derivatives, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.04.033.
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ACCEPTED MANUSCRIPT
Graphical abstract:
SO2NHR
SO2NHR
O
O
N
N
H
H
O
O
O
General formula A
General formula B
O
O
O
O
O
O
SO2NHR
N
N
N
S
HN
SO2NHR
R1
General formula C
General formula D
Compound 4a (general formula B; R=H) has IC50 of 0.72 and 0.50 µM against MCF-
7 and A-549 cells, respectively. It has Ki of 29.75 and 4.54 nM on hCA IX and XII,
respectively. It raises the % of early apoptotic cells to 94.12 and 74.77% in breast and
lung cells, respectively.

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Synthesis, carbonic anhydrase inhibition and cytotoxic activity of novel chromone-based
sulfonamide derivatives
Fadi M. Awadallah,a* Tamer A. El-Waei,a Mona M. Hanna,a Safinaz E. Abbas,a Mariangela
Ceruso,b Beyza Ecem Oz,b,c Ozen Ozensoy Guler,b,c Claudiu T. Supuranb*
a
Pharmaceutical Chemistry department, Faculty of Pharmacy, Cairo University, Kasr El-Eini street, 11562,
Cairo Egypt
b University of Florence, Neurofarba Department, Via Ugo Schiff 6
(Firenze) Italy
, Polo Scientifico, 50019, Sesto Fiorentino
cYildirim Beyazit University Faculty of Medicine Department of Medical Biology, Cinnah Campus Ankara, Turkey
Abstract.
Four series of sulfonamides incorporating chromone moieties were synthesized and
assessed for their cytotoxic activity against MCF-7 and A-549 cell lines, considering the fact
that some of these tumors overexpress isoforms of carbonic anhydrase (CA, EC 4.2.1.1) which
is inhibited by sulfonamides. Most new sulfonamides showed weak inhibitory activity against
the offtarget, cytosolic isoforms hCA I, II but effectively inhibited the tumor-associated hCA
IX and XII. The most active compounds featured a primary SO2NH2 group and were active in
the low micromolar range against MCF-7 and A-549 cell lines. Compound 4a showed IC50 of
0.72 and 0.50 µM against MCF-7 and A-549 cell lines, respectively, and was further evaluated
for its proapoptotic activity which proved enhanced in both tumor types.
Keywords: Chromone; sulfonamide; breast cancer; lung cancer; carbonic anhydrase.
*Corresponding authors: E-mail address :fadi_mae@hotmail.com (F.M. Awadallah); Telephone/fax: +39-055-
4573729, E-mail:claudiu.supuran@unifi.it (C.T. Supuran)
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1. Introduction.
Chromones are naturally occurring oxygen containing heterocyclic compounds that are
widely distributed in plant kingdom and form the basic nucleus of important compounds such
as anthocyanin and flavonoids. Chromones are also well known for their antimicrobial [1,2],
antitumor [3,4], and antiviral [5,6] activities. Chromone-3-carboxaldehyde are important
synthons in the synthesis of chromone-based bioactive molecules. [7,8]. They are widely used
due to their ease of preparation and modification via Vilsmeier Haack reaction [9,10].
The clinical and medicinal importance of sulfonamides is well documented. The
sulfonamide moiety (–SO2NH-R) is an active pharmacophore, exhibiting a wide variety of
pharmacological activities such as antimicrobial, antimalarial, insulin-releasing antidiabetic,
anti-HIV, high ceiling diuretic, antithyroid, and antitumor activities [11–15]. Among the broad
spectrum of activities elicited by sulfonamides is their role as inhibitors of the zinc containing
metalloenzyme carbonic anhydrase (CA). Many sulfonamide CA inhibitors have been used as
therapeutic agents against various diseases including glaucoma, gastro-duodenal ulcers, acid–
base disequilibria, and various neurological disorders [16–19].
Carbonic anhydrases (CAs, EC 4.2.1.1) are a group of metalloenzymes that are involved
in pH buffering of extra- and intracellular spaces by catalyzing the reversible hydration of
carbon dioxide (cellular waste product) to bicarbonate and a proton [20-25]. This family of
enzymes comprises six genetic families, i.e., α, β, γ, δ, ζ and η-classes. Among the 16 known
human (h) CA isoforms belonging to class α, the human cytosolic isoforms hCA I and hCA II
are ubiquitous in the body and are targets for anticonvulsant, diuretic and anti-glaucoma drugs.
On the other hand, the transmembrane isoforms hCA IX and hCA XII, possessing an
extracellular active site, are associated with some types of cancers being induced by tumor
hypoxia. This being the case, these two isoforms justifiably represent good candidates for
antitumor drug design, with one sulfonamide CA IX/XII inhibitor currently in Phase I clinical
trials for the treatment of primary tumors/metastases overexpressing these enzymes [26-28].
In addition to the aforementioned facts, literature revealed combined chromone-
sulfonamides hybrids as potent antitumor and CA inhibitors as exemplified by compound I and
II which featured a chromone nucleus linked to a sulfonamide moiety either through a methine
(=CH-) group; or through a heterocyclic pyrimidine spacer [29], respectively (Figure 1).
Accordingly, with the goal of producing new antitumor agents, a hybrid pharmacophoric
approach was adopted in the design of the target compounds in which the chromone and an
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appropriately N-(un)substitutedsulfonamide moieties were combined via a methine spacer
(General formula A) in one molecule. Further investigation was performed by including the
reduced methyleneamino (-CH2NH-) linker instead of the methineamino one (=CH-NH-) in
order to impart structure flexibility (General formula B). Another group of target compounds
were designed in such a way to separate the chromone and the sulfonamide pharmacophores
through incorporation of a heterocyclic spacer. Searching for suitable heterocyclic rings of
antitumor importance unveiled the potential activity of the thiazolinone [30-32] and its isosteric
imidazolinone moieties [33,34]. Consequently, and motivated by the positive biological
activities of many derivatives incorporating these heterocyclic rings, compounds of general
formula C and D were synthesized (Figure 2). The new compounds were screened in vitro
against two cell lines: breast MCF-7 and lung A-549. Furthermore, the derivatives that showed
remarkable activity profile were tested for their CA inhibitory activity.
2. Discussion.
2.1. Chemistry.
The key starting compound, chromone-3-carboxaldehyde 2, was prepared from 2-
hydroxyacetophenone 1, dimethylformamide (DMF) and phosphorous oxychloride according
to the Vilsmeier-Haack reaction [35,36]. The first series of target compounds 3a-f was
synthesized via reacting 2 with the appropriate sulfonamide in ethanol in the presence of
catalytic amount of glacial acetic acid. The expected Schiff's base derivatives were not
obtained, but rather the enamine adducts 3a-f were afforded. This could be explained on the
basis of the presence of a very reactive electron deficient center at C2 of the chromone nucleus
which facilitated the nucleophilic attack of an ethanol molecule (solvent) that underwent
addition on the C2-C3 double bond. In addition, it was observed that performing the reaction in
different solvents such as methanol or n-butanol resulted in the 2-methoxy and 2-n-butyloxy
derivatives, respectively, as confirmed by spectral data. Moreover, literature provided similar
explanation for this unexpected reaction [37, 38, 39]. Trials to carry out the reaction using
acetic acid as a solvent or aprotic solvents such as toluene or benzene gave a mixture of two
products that were difficult to be separated. The major product consisted of an enamine adduct
in which a second molecule of the sulfonamide acted as nucleophile that underwent addition to
the C2-C3 olefinic bond of the chromone ring. The minor product was the expected Schiff's
base. Similar unexpected reactions were also reported in literature [1,40] (Scheme 1).
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The second series of target compounds was obtained by reducing the enamine adducts
3a-f with sodium borohydride in ethanol which furnished the methylene-amino derivatives 4a-
f together with the elimination of the ethoxy group at C2 of the chromone ring (Scheme 1).
The chromone-sulfonamide hybrids incorporating a thiazolinone spacer 7a-f were
obtained by reacting the chromone-3-carboxaldehyde 2 with the appropriate thiazolinone-
bearing sulfonamide derivatives 6a-f. This was achieved by reaction of the appropriate
sulfonamide with chloroacetyl chloride in DMF according to the literature procedures to give
the N4-chloroacetamides 5a-f [41-44]. The latter were reacted with ammonium thiocyanate to
undergo intramolecular cyclization with elimination of ammonium chloride to produce the
corresponding thiazolinone intermediates 6a-f. Knoevenagel condensation of the aldehyde 2
with the active methylene protons of the intermediates 6a-f was performed in glacial acetic acid
in the presence of anhydrous sodium acetate to produce the third group of target compounds
7a-f (Scheme 2).
Finally, the last series of compounds comprising the 4-oxo-imidazolyl-
benzenesulfonamides 10a-f and 11a-f were prepared via reaction of the appropriate oxazolone
9a,b with the appropriate sulfonamide as illustrated in Scheme 3. The intermediates 9a,b were
obtained by benzoylation of glycine with the appropriate benzoyl chloride in sodium hydroxide
solution (10%) to afford 8a,b [45,46], followed by Erlenmeyer cyclocondensation reaction with
the aldehyde 2 in acetic anhydride in the presence of anhydrous sodium acetate [47]. The target
imidazolinones 10a-f and 11a-f were obtained by the reaction of the corresponding 9a,b with
the appropriate sulfonamide in glacial acetic acid and anhydrous sodium acetate.
2.2. In vitro cytotoxic activity.
All target compounds were screened in vitro for their cytotoxic activity against breast
(MCF-7) and lung (A-549) cells using the sulforhodamine B (SRB) assay as described by
Skehan et al. [48]. IC50 values (µM) were calculated from the mean value of three separate
experiments. Doxorubicin (DOX) was used as a positive control. Results were listed in Table 1.
Concerning the activity against the breast cell line, most compounds of the first series, 3a-f,
showed higher activity than doxorubicin (IC50= 22.19-30.06 µM) except compounds 3d and 3e.
The reduced derivatives 4a-f demonstrated a decrease in activity compared to compounds 3a-f,
except for compound 4a that exhibited a highly potent activity in a submicromolar value (IC50=
0.72 µM). Substitution of the aminomethine or methylamino spacer by thiazolinone or
imidazolinone moieties as in compounds 7a-f, 10a-f and 11a-f, respectively, was not in favor
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of activity except for the N4-unsubstituted-sulfonamide derivatives 7a, 10a and 11a which
showed increase in activity (IC50= 8.91, 11.50 and 24.31 µM, respectively).
As per the activity of the tested compounds against the lung A-549 cell line, all
compounds were less potent than doxorubicin except for compounds 4a, 7a, 10a and 11a
(IC50= 0.50, 2.23, 1.73 and 13.77 µM, respectively). Compound 4e was almost equipotent to
doxorubicin (IC50= 25.59 µM).
Regarding the effect of the N4-substitution on the sulfonamide moiety, it could be
clearly observed that the unsubstituted derivatives were the most potent within all series.
Compound 4a represented a promising candidate molecule being active against both cell lines
in the submicromolar range.
2.3. Carbonic anhydrase inhibition.
The CA inhibitory ability of some selected sulfonamides that demonstrated good
activity profile in the in vitro antitumor assay was assessed against the cytosolic isoforms hCA
I and II as well as the membrane associated isoforms hCA IX and XII employing a stopped
flow assay method [49] and the results are shown in Table 2. The well known CA inhibitor
acetazolamide (AZA) was used as the reference drug for this assay. Data obtained from the CA
inhibition assay revealed that the tested compounds were selective to the tumor associated
isoforms hCA IX and hCA XII with activity in the nanomolar range. Very weak or no activity
at all was observed against hCA I and II, except for compound 7a (hCA II Ki= 12.95 nM). This
selectivity could be considered a desirable attribute for cytotoxic agents to guard against the
occurrence of untoward side effects. The hCA IX isoform was highly inhibited by compounds
3b, 3e, 3f, 7a, 10a and 11a (Ki= 14.26-23.84 nM) surpassing the activity of AZA. The rest of
the compounds were either equipotent or slightly less potent than AZA. With regards to the
profiling assay against hCA XII, only two compounds, 4a and 10a, displayed slightly higher
activity than AZA (Ki= 4.54 and 5.23 nM, respectively). Compounds of moderate potency
against hCA XII were 4b, 7a, 10b and 11a (Ki= 6.24-10.76 nM). Also, it could be noted that
the most active compounds on hCA IX (7a and 11a) and hCA XII (4a and 10a) were those
derivatives whose sulfonamide pharmacophore possessed a free sulfamoyl group.
2.4. Apoptosis study.
To examine the involvement of apoptosis in the cytotoxic activity of the new
sulfonamide derivatives, MCF-7 and A-549 cells were incubated with the most potent
compound 4a at its IC50 concentration for 24 h. The percentage of apoptotic cells were
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determined by Annexin V-FITC/PI staining and the stained cells were detected by flow
cytometry (Figure 3). The results showed that 4a induced apoptosis in MCF-7 cells where the
percentage of early apoptotic cells reached 94.12%; whereas, the percentage of early apoptotic
cells attained 74.77 % in A-549 cells. The percentage of early apoptotic cells was markedly
elevated as a function of concentration of the tested agent from 0.54 % to 46.85%.
3. Conclusion.
Four series of new chromone-based sulfonamide derivatives were synthesized and
tested for their in vitro cytotoxic activity against breast (MCF-7) and lung (A-549) cell lines.
Compared to doxorubicin, the tested against breast cancer cell lines than lung cancer cell lines.
Compounds in which the chromone and sulfonamide phrmacophores were separated by a small
spacer group such as =CH-N- or –CH2NH-, 3a-f and 4a-f, respectively, were more potent than
those more widely separated by a heterocyclic thiazolinone or imidazolinone rings, 7a-f, 10a-f
and 11a-f, respectively. Also, it was noted that derivatives possessing a free sulfamoyl group,
4a, 7a, 10a and 11a were the most potent. Compounds showing high activity profile in the in
vitro cell line screening were further evaluated for their carbonic anhydrase inhibitory activity
against hCA I, II, IX and XII isoforms. The profiling enzyme assay revealed that the tested
compounds were selective to the tumor associated isoforms hCA IX and XII in the nanomolar
range, and that the compounds that exhibited activity higher than that expressed by
acetazolamide were those having a free sulfamoyl group. Finally, compound 4a, which
possessed the highest activity against the tested cell lines, was further investigated for its
proapoptotic activity where induction of apoptosis was markedly observed in both cancer cell
types. In conclusion, it could be claimed that compound 4a, denoting IC50 of 0.72 and 0.50 µM
against MCF-7 and A-549 cells; and a Ki of 29.75 and 4.54 nM on hCA IX and XII; and which
raised the % of early apoptotic cells to 94.12 and 74.77 % in breast and lung cells, respectively,
could be a promising lead for future investigation.
4. Experimental.
4.1. Chemistry:
Unless otherwise noted, all materials were obtained from commercial suppliers and
used without further purification. TLC was monitored on FLUKA silica gel TLC aluminium
cards (0.2 mm thickness) with fluorescent indicator 254 nm using chloroform: methanol
(9.5:0.5) as eluent. Melting points were performed on Stuart SMP3 version 5 digital melting
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point apparatus and were uncorrected. Elemental microanalyses were performed at the
Microanalytical Center, at the Regional Center for Mycology and Biotechnology, Al-Azhar
1
University. H NMR spectra were measured in dimethylsulphoxide (DMSO-d6) on a Varian
Mercury VX-300 MHz spectrophotometer or Bruker AVANCE III Nano Bay 400 MHz FT-
NMR spectrophotometer. Chemical Shifts were reported in δ as parts per million (ppm)
downfield from tetramethylsilane (TMS) as internal standard. 13C NMR spectra were recorded
on Bruker AVANCE III Nano Bay 400 MHz FT-NMR spectrophotometer at 100 MHz. Mass
spectra were performed on Fennigan MAT, SSQ 7000 mass spectrophotometer at 70eV.
Infrared Spectra were recorded on Schimadzu FT-IR 8400S spectrophotometer (Shimadzu,
Kyoto, Japan), and expressed in wave number (cm-1), using potassium bromide discs.
Compounds 2 [35,36], 3a [28], 3c [39], 5a-f [40-43], 6a [49], 6f [50], 8a,b [44,45], 9a,b [46]
were prepared according to the reported procedures.
4. Experimental:
4.1.
General method for the synthesis of compounds (3a-f)
A solution of chromone-3-carboxaldehyde 2 (1.74 g, 10 mmol), the appropriate
sulfonamide (10 mmol) and glacial acetic acid (0.5 mL) in absolute ethanol (10 mL) was
refluxed for 3.5 h and kept overnight at room temperature. The separated solid was filtered off
and crystallized from a mixture of ethanol:acetone mixture (1:1).
4.1.1. 4-(((2-Ethoxy-4-oxochroman-3-ylidene)methyl)amino)benzenesulfonamide (3a)
(Yield: 85 %, m.p. 158-160 oC as reported) [28].
4.1.2. 4-(((2-Ethoxy-4-oxochroman-3-ylidene)methyl)amino)-N-(5-methylisoxazol-3-
yl)benzenesulfonamide (3b)
Yield: 88%; m.p. 208–211oC. IR (cm-1): 3365 (NH), 3061 (aromatic CH), 2974
(aliphatic CH), 1649 (C=O), 1284(SO2asym), 1165 (SO2sym). 1H NMR (400 MHz) δ: 1.07 (t, 3H,
J= 7.0 Hz, CH2CH3), 2.30 (s, 3H, CH3), 3.81 (q, 2H, J= 7.0 Hz, CH2CH3,), 5.94 (s, 1H, CH-2
chromone), 6.15 (s, 1H, CH, isoxazole), 6.69 (d, 2H, J= 8.7 Hz, CH-3, CH-5 of phenyl), 7.08
(d, 1H, J= 8.2 Hz, CH-8 chromone), 7.13 (t, 1H, J= 8.5Hz, CH-6 chromone), 7.51 (d, 2H, J=
8.7 Hz, CH-2, CH-6 of phenyl), 7.58 (d, 1H, J= 8.8 Hz, CH-5 chromone), 7.86 (t, 1H, J= 8.7
Hz, CH-7 chromone), 8.12 (d, 1H, J= 12.0, CH enamine), 11.35 (s, 1H, SO2NH, exchanged by
D2O), 11.73 (d, 1H, J= 12.0, NH, exchanged with D2O). 13C-NMR δ: 12.4 (CH3 of isoxazole
ring), 15.3 (CH3, -OCH2CH3), 63.6 (CH2,-OCH2CH3), 95.6 (C-4 of isoxazole), 100.2 (C-2 of
7

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chromanone), 105.8 (C-3+C-5 of phenyl), 113.0 (C-8 of chromanone), 117.0 (C-3 of
chromanone), 120.4, 122.5, 124.4, 125.7, 126.2, 127.2, 129.2, 133.7, 135.5, 136.7 (aromatic
Cs), 153.5 (C-3 of isoxazole), 157.8 (C-8a of chromanone), 170.5 (C-5 of isoxazole), 181.3 (C-
4 of chromanone). EI–MS m/z (%): 455.38 (M+, 22.2%). Anal. Calcd. for C22H21N3O6S
(455.48): C, 58.01; H, 4.65; N, 9.23. Found: C, 58.03; H, 4.67; N, 9.29.
4.1.3. 4-(((2-Ethoxy-4-oxochroman-3-ylidene)methyl)amino)-N-(pyrimidin-2-
yl)benzenesulfonamide (3c) [39]
Yield: 73%; m.p. 244–245oC. IR (cm-1): 3346 (NH), 3034 (aromatic CH), 2935
(aliphatic CH), 1653(C=O), 1280 (SO2asym), 1157 (SO2sym). 1H NMR (300MHz) δ: 1.05 (t, 3H,
J= 7.0 Hz, CH2CH3), 3.67 (q, 2H, J= 7.0 Hz, CH2CH3), 5.94 (s, 1H, CH-2, chromone), 7.03-
7.16 (m, 3H, ArH), 7.52-7.82 (m, 4H, ArH), 7.85-7.97 (m, 5H, ArH), 8.10 (d, 1H, J= 12.3 Hz,
CH enamine), 11.74 (d, 1H, J= 12.6 Hz, NH, exchanged with D2O). Anal. Calcd. for
C22H20N4O5S (452.49): C, 58.40; H, 4.46; N, 12.38. Found: C, 58.52; H, 4.48; N, 12.38.
4.1.4. N-(4,6-Dimethylpyrimidin-2-yl)-4-(((2-ethoxy-4-oxochroman-3-
ylidene)methyl)amino)benzenesulfonamide (3d)
Yield: 75%; m.p. 288-290. IR (cm-1): 3230 (NH), 3082 (aromatic CH), 2974 (aliphatic
CH), 1651(C=O), 1280(SO2asym), 1155 (SO2sym). 1H NMR (400MHz) δ:1.08 (t, 3H, J= 7.2 Hz,
CH2CH3), 2.26 (s, 6H, 2CH3), 3.67 (q, 2H, J= 7.2 Hz, CH2CH3), 5.95 (s, 1H, CH-2,
chromone), 6.77 (s, 1H, CH, pyrimidine), 7.05-7.16 (m, 2H, ArH), 7.44-7.57 (m, 3H, ArH),
7.82-7.98 (m, 4H, ArH), 8.13 (d, 1H, J=12.0 Hz, CH enamine), 11.76 (d, 1H, J= 12.0 Hz, NH,
exchanged with D2O). 13C-NMR δ: 15.4 (CH3), 63.5 (CH2), 100.3 (C-2 of chromanone), 105.5
(C-5 of diazine), 116.0 (C-3 + C-5 of phenyl), 118.5 ( C-8 of chromanone), 122.4, 125.7, 130.4,
130.6, 135.1, 135.2, 138.9 (aromatic Cs), 143.6 (methine), 144.3 (C-4 of phenyl), 156.2 (C-8a
of chromanone), 156.6 (C-4 + C-6 of diazine), 167.8 (C-2 of diazine), 181.1 (C-4 of
chromanone). EI–MS m/z (%): 480.5 (M+, 0.1%). Anal. Calcd. for C24H24N4O5S (480.54): C,
59.99; H, 5.03; N, 11.66. Found: C, 60.08; H, 5.07; N, 11.79.
4.1.5. 4-(((2-Ethoxy-4-oxochroman-3-ylidene)methyl)amino)-N-(thiazol-2-
yl)benzenesulfonamide (3e)
Yield: 80%; m.p. 284–288oC. IR (cm-1): 3118 (NH), 3012 (aromatic CH), 2972
(aliphatic CH), 1658(C=O), 1278(SO2asym), 1147 (SO2sym). 1H NMR (400MHz) δ:1.07 (t, 3H,
J= 7.1 Hz, CH2CH3), 3.68 (q, 2H, J= 7.1 Hz, CH2CH3), 5.95 (s, 1H, CH-2, chromone), 6.45-
6.54 (m, 1H, ArH), 6.82-7.05 (m, 3H, ArH), 7.11-7.50 (m, 3H, ArH), 7.61-7.82 (m, 3H, ArH),
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8.06 (d, 1H, J= 12.0 Hz, CH enamine), 11.77 (d, 1H, J= 12.0 Hz, NH, exchanged by D2O),
12.72 (s, 1H, SO2NH, exchanged with D2O). 13C NMR δ: 15.4 (CH3), 63.5 (CH2), 100.3 (C-2 of
chromanone), 105.3 (C-5 of thiazole), 108.6 (C-3+C-5 of phenyl), 116.9 (C-8 of chromanone),
118.5 (C-3 of chromanone), 122.3, 123.0, 124.9, 126.1, 126.5, 128.2, 135.2 (aromatic Cs).
137.4 (C-4 of thiazole), 143.2 (methine), 144.5 (C-4 of phenyl), 156.1 (C-8a of chromanone),
178.9 (C-2 of thiazole), 181.0 (C-4 of chromanone). EI–MS m/z (%): 459.30 (M++2, 8.7%),
458.35( M++1, 20.16%), 457.35 (M+, 20.14%). Anal. Calcd. for C21H19N3O5S2 (457.52): C,
55.13; H, 4.19; N, 9.18. Found: C, 55.31; H, 4.24; N, 9.34.
4.1.6. 4-(((2-Ethoxy-4-oxochroman-3-ylidene)methyl)amino)-N-(pyridin-2-
yl)benzenesulfonamide (3f)
Yield: 82%; m.p. 185–187oC. IR (cm-1): 3304 (NH), 3030 (aromatic CH), 2927
(aliphatic CH), 1653 (C=O), 1280 (SO2asym), 1159 (SO2sym). 1H NMR (400MHz) δ:1.06 (t, 3H,
J= 7.2 Hz, CH2CH3), 3.65 (q, 2H, J= 7.2 Hz, CH2CH3), 5.94 (s, 1H, CH-2, chromone), 7.09-
8.05 (m, 13H, ArH), 8.11 (d, 1H, J=12.0 Hz, CH enamine), 11.74 (d, 1H, J= 12.0 Hz, NH,
exchanged with D2O). 13C NMR δ: 14.8(CH3), 63.0 (CH2), 99.8 (C-2 of chromanone), 113.6
(C-3 of pyridine), 115.7 (C-3+ C-5 of phenyl), 116.7( C-8 of chromanone ), 119.9, 121.2,
122.3, 125.2, 128.5, 129.1, 134.7, 136.3 (aromatic Cs), 139.0 (C-4 of pyridine ), 142.9
(methine), 145.6 (C-4 of phenyl ), 152.3 (C-6 of pyridine), 155.5 ( C-2 of pyridine), 156.3 (C-
8a of chromanone), 180.9 (C-4 of chromanone). Anal. Calcd. for C23H21N3O5S (451.50): C,
61.19; H, 4.69; N, 9.31. Found: C, 60.09; H, 4.76; N, 9.54.
4.2.
General method for the synthesis of compounds (4a-f)
A mixture of the appropriate enamine derivatives 3a-f (10 mmol) and sodium
borohydride (3.7 g , 10 mmol) in absolute ethanol (20 mL) was stirred overnight at room
temperature. The reaction mixture was diluted with water, neutralized with diluted HCl and the
separated solid was filtered off, washed with water and crystallized from ethanol.
4.2.1. 4-(((4-Oxo-4H-chromen-3-yl)methyl)amino)benzenesulfonamide (4a)
Yield: 60%; m.p. 242–242oC. IR (cm-1): 3304, 3224 (NH, NH2), 3118 (aromatic CH),
2920 (aliphatic CH), 1635 (C=O), 1573 (C=C), 1315(SO2asym), 1147 (SO2sym). 1H NMR
(300MHz) δ: 4.14 (d, 2H, J= 5.7 Hz, CH2), 6.60 (t, 1H, J= 6.0 Hz, NH, exchanged with D2O),
6.89 (s, 2H, SO2NH2, exchanged with D2O), 6.68 (d, 1H, J= 8.7, ArH), 7.48 (m, 4H, ArH), 7.62
(d, 1H, J= 8.7, ArH), 7.78(m, 1H, ArH), 8.08 (d, 1H, ArH) , 8.31 (s. 1H, CH-2, chromone). 13C
NMR δ: 38.4(CH2), 111.6 (C-3+C-5 of phenyl), 118.9 (C-8 of chromone), 121.0, 123.6, 125.4,
9

ACCEPTED MANUSCRIPT
125.9, 127.8, 131.0, 134.7 (aromatic Cs), 151.3 (C-4 of phenyl), 154.9 (C-2 of chromone),
156.4 (C-8a of chromone), 176.85 (C-4 of chromone). EI–MS m/z (%): 331 (M++1, 55.1%),
330 (M+,16.33%). Anal. Calcd. for C16H14N2O4S (330.36): C, 58.17 ; H, 4.27; N, 8.48. Found:
C, 58.24; H, 4.31; N, 8.56.
4.2.2. N-(5-Methylisoxazol-3-yl)-4-(((4-oxo-4H-chromen-3-yl) methyl)
amino)benzenesulfonamide (4b)
Yield: 75%; m.p. 204–206 oC. IR (cm-1): 3348 (NH), 3070 (aromatic CH), 2974 (aliphatic
CH), 1643 (C=O), 1604 (C=C), 1327(SO2asym), 1161(SO2sym). 1H NMR (300MHz) δ: 2.27 (s,
3H, CH3), 4.12 (d, 2H, J= 5.7Hz , CH2), 6.08 (s, 1H, CH, isoxazole), 6.85 (t, 1H, J= 6.0 Hz ,
NH, exchanged with D2O), 6.69-8.07 (m, 8H, ArH), 8.31 (s. 1H, CH-2, chromone), 10.92 (s,
1H, SO2NH, exchanged with D2O). 13C NMR δ: 11.4 (CH3), 37.2 (CH2), 94.6 (C-4 of ioxazole
ring), 110.6 (C-3+C-5 of phenyl), 119.7 ( C-8 of chromone), 120.4, 122.6, 124.1, 124.3, 124.8,
124.9, 128.5, 133.6 (aromatic Cs), 151.4( C-3 of isoxazole), 154.0 ( C-2 of chromone ring),
155.3( C-4 of phenyl), 157.2 (C-8a of chromone), 169.3 (C-5 of isoxazole), 175.7 ( C-4 of
chromone). EI–MS m/z (%): 411 (M+, 53.0%). Anal. Calcd. for C20H17N3O5S (411.43): C,
58.39; H, 4.16; N, 10.21. Found: C, 58.47; H, 4.20; N, 10.36.
4.2.3. 4-(((4-Oxo-4H-chromen-3-yl)methyl)amino)-N-(pyrimidin-2-yl)benzenesulfonamide (4c)
Yield: 40%; m.p. 220–222 oC. IR (cm-1): 3369 (NH), 3086 (aromatic CH), 2929
(aliphatic CH), 1635 (C=O), 1558 (C=C), 1317 (SO2asym), 1149 (SO2sym).1H NMR (400MHz) δ:
4.12 (d, 2H, J= 5.6 Hz, CH2), 6.78 (t, 1H, J= 6.0 Hz, NH, exchanged with D2O), 6.59-8.09 (m,
11H, ArH), 8.33 (s. 1H, CH-2, chromone), 11.04 (s,1H, SO2NH, exchanged with D2O). 13C-
NMR δ: 38.3 (CH2), 111.0 (C-3+ C-5 of phenyl), 114.3 (C-5 of diazine), 117.1 (C-8 of
chromone), 118.9, 120.9, 121.5, 123.6, 125.9, 129.6, 134.7 (aromatic Cs), 150.7 (C-2 of
chromone), 152.1 (C-4 of phenyl), 155.0 (C-8a of chromone), 157.0 (C-4 + C-6 of diazine),
167.8 ( C-2 of diazine), 176.8 (C-4 of chromone). Anal. Calcd. for C20H16N4O4S (408.43): C,
58.82; H, 3.95; N, 13.72. Found; C, 58.97; H, 3.94; N, 13.88.
4.2.4. N-(4,6-Dimethylpyrimidin-2-yl)-4-(((4-oxo-4H-chromen-3-
yl)methyl)amino)benzenesulfonamide (4d)
Yield: 40%; m.p. 165–166 oC. IR (cm-1): 3392 (NH), 3080 (aromatic CH), 2926,
(aliphatic CH), 1635 (C=O), 1558 (C=C), 1319 (SO2asym), 1149 (SO2sym). 1H NMR (400MHz)
δ: 2.23 (s, 6H, 2CH3), 4.12 (d, 2H, J= 5.6 Hz ,CH2), 6.71 (t, 1H, J= 6.0 Hz, NH, exchanged
with D2O), 6.67-8.17 (m, 9 H, ArH), 8.33 (s. 1H, CH-2, chromone), 11.77 (s,1H, SO2NH,
10

ACCEPTED MANUSCRIPT
exchanged with D2O). 13C NMR δ: 14.8 (CH3), 38.6 (CH2), 110.2 (C-5 of pyrimidine), 116.6,
117.9, 120.2, 122.7, 125.4, 126.1, 128.2, 130.3 (aromatic Cs), 150.2 (C-2 of chromone + C-4 of
phenyl), 152.2 (C-8a of chromone), 156.6 (C-4 of chromone). Anal. Calcd. for C22H20N4O4S
(436.49): C, 60.54; H, 4.62; N, 12.84. Found: C, 60.62; H; 4.65; N, 12.97.
4.2.5. 4-(((4-Oxo-4H-chromen-3-yl)methyl)amino)-N-(thiazol-2-yl)benzenesulfonamide (4e)
Yield: 65%; m.p. 165–166 oC. IR (cm-1): 3425 (NH), 3099 (aromatic CH), 2928
(aliphatic CH), 1643 (C=O), 1577 (C=C), 1292 (SO2asym), 1139 (SO2sym). 1H NMR (300MHz)
δ: 4.11 (d, 2H, J= 5.7 Hz, CH2), 6.72 (t, 1H, NH, J= 6.0 Hz, exchanged with D2O), 6.65-8.10
(m, 10H, ArH), 8.30 (s. 1H, CH-2, chromone), 12.40 (s, 1H, SO2NH, exchanged with D2O).
13C NMR δ: 37.9 (CH2), 107.4(C-3+C-5 of phenyl), 110.8 (C-5 of thiazole), 111.1 (C-8 of
chromone), 116.6, 118.4, 120.4, 120.9, 123.1, 124.2, 124.8, 125.4, 134.1 (aromatic Cs),
151.1(C-2 of chromone), 155.9 (C-8a of chromone), 167.9 (C-2 of thiazole), 176.3 ( C-4 of
chromone). EI–MS m/z (%): 413 (M+, 8,7). Anal. Calcd. for C19H15N3O4S2 (413.47): C, 55.19;
H, 3.66; N, 10.16. Found: C, 55.26; H, 3.72; N, 10.30.
4.2.6. 4-(((4-Oxo-4H-chromen-3-yl)methyl)amino)-N-(pyridin-2-yl)benzenesulfonamide (4f)
Yield: 40%; Mp 240–242 oC. IR (cm-1): 3352 (NH), 3070 (aromatic CH), 2925
(aliphatic CH), 1643 (C=O), 1465 (C=C), 1276 (SO2asym), 1141 (SO2sym). 1H NMR (400MHz)
δ: 4.10 (d, 2H, J= 5.6 Hz, CH2), 6.76 (t, 1H, NH, J= 5.6 Hz, exchanged with D2O), 6.66-8.09
(m, 12H, ArH), 8.32 (s. 1H, CH-2, chromone), 11.02 (s, 1H, SO2NH, exchanged with D2O).
EI–MS m/z (%): 407 (M+, 10.0%). Anal. Calcd. for C21H17N3O4S (407.44): C, 61.91; H, 4.21;
N, 10.31. Found: C, 62.03; H, 4.27; N, 10.43.
4.3.
General method for the synthesis of compounds (6a-f)
To a solution of the appropriate N-chloroacetyl sulfonamide derivatives 5a-f (10 mmol)
in ethanol (20 mL), ammonium thiocyanate (0.75 g , 10 mmol) was added and the reaction
mixture was refluxed for 3-12 h. The reaction mixture was filtered while hot and the obtained
solid was crystallized from dioxane to afford the title compounds 6a-f.
4.3.1. 4-((4-Oxo-4,5-dihydrothiazol-2-yl)amino)benzenesulfonamide (6a)
(Yield: 90%; mp. 244-246 oC; reflux time: 3h. as reported) [50].
4.3.2. N-(5-Methylisoxazol-3-yl)-4-((4-oxo-4,5-dihydrothiazol-2-yl)amino)benzenesulfonamide
(6b)
11

ACCEPTED MANUSCRIPT
Yield: 85%; m.p. 259–261 oC; reflux time: 8h. IR (cm-1): 3334 (NH), 3089 (aromatic
CH), 2933 (aliphatic CH), 1689 (C=O), 1244 (SO2asym), 1170 (SO2sym). 1H NMR (300MHz) δ:
2.29 (s, 3H, CH3), 4.02 (s, 2H, CH2), 6.11 (s, 1H, CH, isoxazole), 7.74-7.85 (m, 4H, ArH),
11.35 (s, 1H, NH, exchanged with D2O), 11.89 (s, 1H, SO2NH, exchanged with D2O). EI–MS
m/z (%): 352 (M++1.1 %). Anal. Calcd. for C13H12N4O4S2 (352.38): C, 44.31; H, 3.43; N,
15.90. Found: C, 44.39; H, 3.47; N; 16.04.
4.3.3. 4-((4-Oxo-4,5-dihydrothiazol-2-yl)amino)-N-(pyrimidin-2-yl)benzenesulfonamide (6c)
Yield: 73%; m.p. 297(dec.); reflux time: 10h. IR (cm-1): 3277 (NH), 3039 (aromatic
CH), 2941 (aliphatic CH), 1687 (C=O), 1330 (SO2asym), 1161 (SO2sym). 1H NMR (400MHz) δ:
4.03 (s, 2H, CH2), 7.03-7.1 (m, 2H, ArH), 7.73-7.97 (m, 3H, ArH), 8.49-8.54 (m, 2H, ArH),
11.79 (br s, 2H, NH and SO2NH, exchanged with D2O). EI–MS m/z (%): 349 (M+, 0.5 %).
Anal. Calcd. for C13H11N5O3S2 (349.38): C, 44.69; H, 3.17; N, 20.05. Found: C, 44.81; H, 3.20;
N, 20.21.
4.3.4. N-(4,6-Dimethylpyrimidin-2-yl)-4-((4-oxo-4,5-dihydrothiazol-2-
yl)amino)benzenesulfonamide (6d)
Yield: 68%; m.p. 275-277 oC; reflux time: 12h. IR (cm-1): 3263 (NH), 3041 (aromatic
CH), 2978 (aliphatic CH), 1670 (C=O), 1244(SO2asym), 1143, (SO2sym). 1H NMR (400MHz) δ:
2.25 (s, 6H, 2 CH3), 4.02 (s, 2H, CH2), 7.72-7.96 (m, 5H, ArH), 11.76 (br, s, 2H, NH and
SO2NH, exchanged with D2O). EI–MS m/z (%): 378 (M++1, 0.6 %), 377 (M+, 0.6 %). Anal.
Calcd. for C15H15N5O3S2 (377.44): C, 47.73; H, 4.01; N, 18.56. Found: C, 47.88; H, 4.07; N,
18.69.
4.3.5. 4-((4-Oxo-4,5-dihydrothiazol-2-yl)amino)-N-(thiazol-2-ylbenzenesulfonamide (6e)
Yield: 88%; m.p. > 300 oC; reflux time: 10h. IR (cm-1): 3269 (NH), 3045 (aromatic
CH), 2989 (aliphatic CH), 1687 (C=O), 1240 (SO2asym), 1136 (SO2sym). 1H NMR (400MHz) δ:
4.02 (s, 2H, CH2), 6.81(d, 1H, J= 4.0 Hz, CH-5, thiazole), 7.25(d, 1H, J= 4.0 Hz, CH-4,
thiazole), 7.71-7.90 (m, 4H, ArH), 11.48 (s, 1H, NH exchanged with D2O), 12.64 (s, 1H,
SO2NH exchanged with D2O). EI–MS m/z (%): 354 (M+, 37.7 %). Anal. Calcd. for
C12H10N4O3S3 (354.42): C, 40.67; H, 2.84; N, 15.81. Found: C, 40.75; H, 2.82; N, 15.97.
4.3.6. 4-((4-Oxo-4,5-dihydrothiazol-2-yl)amino)-N-(pyridin-2-yl)benzenesulfonamide (6f)
(Yield: 85%; m.p. 240-242 oC; reflux time: 5h as reported) [51].
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4.4.
General method for the synthesis of compounds (7a-f)
A mixture of chromone-3-carboxaldehyde 2 (1.74 g , 10 mmol), the appropriate
thiazolinone derivative 6a-f (10 mmol) and anhydrous sodium acetate (0.82 g, 10 mmol) in
glacial acetic acid (50 mL) was heated in a boiling water bath for 3-12 h. The separated solid
was filtered off while hot, washed with hot water and crystallized from DMF/H2O.
4.4.1. 4-((4-Oxo-5-((4-oxo-4H-chromen-3-yl)methylene)-4,5-dihydrothiazol-2-
yl)amino)benzenesulfonamide (7a)
Yield: 80 %; m.p. > 300 oC; reaction time: 3h. IR (cm-1): 3325 (NH), 3290, 3242
(NH2), 3049 (aromatic CH), 2950 (aliphatic CH), 1676 (C=O amide), 1643 (C=O, chromone),
1290(SO2asym), 1159 (SO2sym). 1H NMR (400MHz) δ: 7.34 (s, 2H, SO2NH2, exchanged with
D2O), 7.44-8.16 (m, 9H, ArH+ methine H), 8.81 (s, 1H, CH-2, chromone), 12.12 (s, 1H, NH
exchanged with D2O). 13C NMR δ: 118.9 (C-3 of chromone), 119.0 (C-8 of chromone), 121.3,
122.0, 123.4, 125.2, 125.9, 126.7, 127.0, 127.6, 129.7, 135.5, 138.0 (aromatic Cs), 144.3 (C-9,
methine), 154.4 (C-2 of chromone), 155.8 (C-8a of chromone), 160.4 (C-2 of thiazolinone),
166.04 (C-4, C=O of chromone), 175.27 (C-4, C=O of thiazolinone). EI–MS m/z (%): 428
(M++1, 2.3%), 427 (M+, 0.5 %). Anal. Calcd. for C19H13N3O5S2 (427.45): C, 53.39; H; 3.07; N,
9.83. Found: C, 53.48; H, 3.12; N, 9.98.
4.4.2. N-(5-Methylisoxazol-3-yl)-4-((4-oxo-5-((4-oxo-4H-chromen-3-yl) methylene)-4,5-
dihydrothiazol-2-yl)amino)benzenesulfonamide (7b)
Yield: 85 %; m.p. > 300 oC; reaction time: 8h. IR (cm-1): 3263 (NH), 3051(aromatic
CH), 2964 (aliphatic CH), 1680 (C=O, amide), 1651 (C=O, chromone), 1238 (SO2asym), 1161
(SO2sym). 1H NMR (300MHz) δ: 2.31 (s, 3H, CH3), 6.16 (s, 1H,CH, isoxazole), 7.20 (m, 1H,
ArH), 7.50-7.70(m, 3H, ArH), 7.82-8.10 (m, 6H, ArH+ methine H+ NH, exchanged with D2O),
8.79 (s, 1H, CH-2, chromone), 11.44 (s, 1H, SO2NH, exchanged with D2O). 13C NMR δ: 11.4
(CH3), 94.8 (C-4 of isoxazole), 117.4 (C-8 of chromone), 119.9, 121.4, 122.3, 124.9, 125.7,
127.7, 134.3 (aromatic Cs), 154.7 (C-2 of chromone), 156.4 (C-8a of chromone), 160.0 (C-2 of
thiazolinone), 161.7 (C-4 of chromone), 169.7 (C-4 of thiazolinone). EI–MS m/z (%): 509
(M++1, 48%), 508 (M+, 48%). Anal. Calcd. for C23H16N4O6S2 (508.52): C, 54.32; H, 3.17; N,
11.02. Found: C, 54.39; H, 3.21; N, 11.13.
4.4.3. 4-((4-Oxo-5-((4-oxo-4H-chromen-3-yl)methylene)-4,5-dihydrothiazol-2-yl)amino)-N-
(pyrimidin-2-yl)benzenesulfonamide (7c)
13

ACCEPTED MANUSCRIPT
Yield: 78 %; m.p. > 300 oC; reaction time: 12h. IR (cm-1): 3250 (NH), 3041 (aromatic
CH), 2955 (aliphatic CH), 1680 (C=O, amide), 1649 (C=O, chromone), 1247 (SO2asym), 115
7(SO2sym). 1H NMR (300MHz) δ: 7.04-7.18 (m, 2H, ArH), 7.53-7.71(m, 3H, ArH), 7.83-
8.11(m, 5H, ArH), 8.51-8.53(m, 2H, ArH, +methine H), 8.79 ( s, 1H, CH-2, chromone), 11.85
(s, 2H, NH and SO2NH, exchanged with D2O ). 13C NMR δ: 116.2 (C-8 of chromone), 118.5,
119.0, 120.5, 121.9, 123.0, 123.3, 126.0, 126.7, 129.6, 135.4 (aromatic Cs), 155.8 (C-8a of
chromone), 157.3 (C-2 of thiazolinone + C-4 and C-6 of diazine), 158.8 (C-4 of thiazolinone +
C-2 of diazine), 160.9 (C-4 of chromone). EI–MS m/z (%): 505 (M+, 5%). Anal. Calcd. for
C23H15N5O5S2 (505.52): C, 54.65; H, 2.99; N, 13.85. Found: C, 54.72; H, 3.02; N, 14.03.
4.4.4. N-(4,6-Dimethylpyrimidin-2-yl)-4-((4-oxo-5-((4-oxo-4H-chromen-3-yl)methylene)-4,5-
dihydrothiazol-2-yl)amino)benzene sulfonamide (7d)
Yield: 69%; m.p. > 300 oC; reaction time: 12h. IR (cm-1): 3313 (NH), 3043 (aromatic
CH), 2957 (aliphatic CH), 1680 (C=O, amide), 1647 (C=O, chromone), 1246 (SO2asym), 1163
(SO2sym). 1H NMR (400MHz) δ: 2.27 (s, 6H, 2CH3), 6.76 (s, 1H, CH-5, pyrimidine), 7.45-7.56
(m, 3H, ArH+ methine H), 7.69-7.85 (m, 3H, ArH), 7.94-8.12 (m, 3H, ArH), 8.69 (s, 1H, CH-
2, chromone), 11.89 (s, 2H, NH and SO2NH, exchanged with D2O). 13C NMR δ: 22.7 (CH3),
113.2 (C-5 of pyrimidine), 118.4 (C-3+C-5 of phenyl), 121.0 (C-8 of chromone), 122.3, 122.9,
125.1, 126.0, 126.4, 127.1, 128.2, 128.9, 130.4, 131.2, 134.8 (aromatic Cs), 155.2 (C-2 of
chromone), 156.0 ( C-8a of chromone), 160.3 (C-2 of thiazolinone), 169.6 (C-4+C-6 of
pyrimidine), 185.7 (C-4 of thiazolinone), 193.2 (C-4 of chromone). EI–MS m/z (%): 533 (M+,
12.0%). Anal. Calcd. for C25H19N5O5S2 (533.58): C, 56.28; H, 3.59; N, 13.13. Found: C, 56.39;
H, 3.64; N, 13.28.
4.4.5. 4-((4-Oxo-5-((4-oxo-4H-chromen-3-yl)methylene)-4,5-dihydrothiazol-2-yl)amino)-N-
(thiazol-2-yl)benzenesulfonamide (7e)
Yield: 65%; m.p. > 300 oC; reaction time: 8h. IR (cm-1): 3246 (NH), 3043 (aromatic
CH), 2950 (aliphatic CH), 1674 (C=O, amide), 1651 (C=O chromone), 1321 (SO2asym),1184
(SO2sym). 1H NMR (300MHz) δ: 6.84 (d, 1H, J= 4.0 Hz, CH-5, thiazole), 7.14-7.15(m, 1H,
ArH), 7.27 (d, 1H, J= 4.0 Hz, CH-4, thiazole), 7.50-7.92(m, 7H, ArH), 8.08(s, 1H, methine H),
8.79 (s, 1H, CH-2, chromone), 12.40, 12.75 (s, 2H, NH and SO2NH, exchanged with D2O). 13C
NMR δ: 108.7 (C-5 of thiazole + C-3 and C-5 of phenyl), 118.5 (C-8 of chromone), 119.0,
120.7, 122.0, 122.8, 123.4, 124.9, 126.0, 126.7, 127.7, 135.4, 138.7 (aromatic Cs), 155.8 (C-8a
of chromone), 160.9 (C-4 of thiazolinone), 169.2 (C-4 of chromone). EI–MS m/z (%): 510 (M+,
14

ACCEPTED MANUSCRIPT
2.8%). Anal. Calcd. for C22H14N4O5S3 (510.56): C, 51.76; H, 2.76; N, 10.97. Found: C, 51.79;
H, 2.78; N, 11.13.
4.4.6. 4-((4-Oxo-5-((4-oxo-4H-chromen-3-yl)methylene)-4,5-dihydrothiazol-2-yl)amino)-N-
(pyridin-2-yl)benzenesulfonamide (7f)
Yield: 70%; m.p. > 300 oC; reaction time: 4h. IR (cm-1): 3244 (NH), 3022 (aromatic
CH), 2945 (aliphatic CH), 1681 (C=O, amide), 1649 (C=O chromone), 1280 (SO2asym), 1178
(SO2sym). 1H NMR (300MHz) δ: 6.80-6.90 (m, 1H, ArH), 7.17-7.20 (m, 2H, ArH), 7.47-7.52
(m, 2H, ArH), 7.66-7.87 (m, 6H, ArH), 8.02-8.04 (m, 2H, ArH+methine H), 8.71 (s, 1H, CH-2,
chromone), 12.00 (s, 2H, NH and SO2NH, exchanged with D2O). 13C NMR δ: 102.1(C-3 of
pyridine ), 113.7 (C-3+C-5 of phenyl), 115.5 (C-8 of chromone), 118.4, 118.7, 120.5, 121.3,
122.8, 125.4, 126.1, 126.8, 127.5, 128.3, 129.4, 130.5, 134.7 (aromatic Cs), 140.2 (C-6 of
pyridine), 147.4 (C-2 of chromone), 153.0 (C-8a of chromone), 155.3 (C-4 of thiazolinone),
170.0 (C-4 of chromone). Anal. Calcd. for C24H16N4O5S2 (504.54): C, 57.13; H, 3.20; N, 11.10.
Found: C, 57.27; H, 3.18; N, 11.27.
4.5.
General method for the synthesis of compounds (10a-f) and (11a-f).
An equimolar mixture of the oxazolone 9a or 9b and the appropriate sulfonamide
derivative (10 mmol) and anhydrous sodium acetate (0.03 g, 0.36 mmol) in glacial acetic acid
(20 mL) was heated in a boiling water bath while stirring for 8-24 h. The separated solid was
filtered off while hot, washed with water, and crystallized from DMF/H2O.
4.5.1. 4-(5-Oxo-4-((4-oxo-4H-chromen-3-yl)methylene)-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)benzenesulfonamide (10a)
Yield: 82%; m.p. 292-294 oC; reaction time: 8h. IR (cm-1): 3311, 3265 (NH2), 3050
(aromatic CH), 2950 (aliphatic CH), 1683 (C=O, imidazolinone), 1653 (C=O, chromone), 1593
(C=C), 1315 (SO2asym), 1159 (SO2sym). 1H NMR (300MHz) δ: 6.79-7.10 (m, 2H, ArH), 7.23 (s,
2H, SO2NH2, exchanged with D2O), 7.52-7.99 (m, 12H, ArH), 8.72 (s, 1H, CH-2 of chromone).
13C NMR δ: 119.9 (C-8 of chromone), 123.6, 126.0, 126.5, 126.9, 128.2, 128.9, 132.4, 133.6,
139.0 (aromatic Cs), 142.6 (C-2 of chromone), 155.8 (C-8a of chromone), 158.5 (C-2 of
imidazolinone), 164.6 (C-4 of imidazolinone), 175.9 (C-4 of chromone). EI–MS m/z (%): 472
(M++1, 4.6%). Anal. Calcd. for C25H17N3O5S (471.49): C, 63.69; H, 3.63; N, 8.91. Found: C,
63.81; H, 3.65; N, 8.99.
4.5.2. N-(5-Methylisoxazol-3-yl)-4-(5-oxo-4-((4-oxo-4H-chromen-3-yl) methylene)-2-phenyl-
4,5-dihydro-1H-imidazol-1-yl)benzenesulfonamide (10b)
15

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Yield: 75%; m.p. 295-296 oC; reaction time: 8h. IR (cm-1): 3251 (NH), 3010 (aromatic
CH), 2926 (aliphatic CH), 1687 (C=O, imidazolinone), 1656 (C=O, chromone), 1591 (C=C),
1317 (SO2asym), 1166 (SO2sym). 1H NMR (300MHz) δ: 2.30 (s, 3H, CH3), 6.12 (s, 1H, CH,
isoxazole), 6.76 (s, 1H, methine H), 7.51- 8.19 (m, 13H, ArH), 8.73 (s, 1H, CH-2 of
chromone), 10.60 (s, 1H, SO2NH, exchanged with D2O). 13C NMR δ: 12.0 (CH3), 95.3 (C-4 of
isoxazole), 115.4 (C-8 of chromone), 119.6, 123.1, 125.4, 126.0, 127.7, 127.8, 128.4, 131.9,
133.1, 133.1, 133.2, 134.6 (aromatic Cs), 149.5 (C-1 of phenyl sulfonamide), 155.3 (C-2 of
chromone), 157.5 (C-3 of isoxazole), 157.5 (C-8a of chromone), 158.1 (C-2 of imidazolinone),
164.3 (C-5 of isoxazole), 170.2 (C-4 of imidazolinone), 175.4 (C-4 of chromone). EI–MS m/z
(%): 552 (M+, 40.7 %). Anal. Calcd. for C29H20N4O6S (552.56): C, 63.04; H, 3.65; N, 10.14.
Found: C, 63.08; H, 3.64; N, 10.22.
4.5.3. 4-(5-Oxo-4-((4-oxo-4H-chromen-3-yl)methylene)-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)-N-(pyrimidin-2-yl)benzenesulfonamide (10c)
Yield: 60%; m.p. 280-282 oC; reaction time: 10h. IR (cm-1): 3421 (NH), 3034 (aromatic
CH), 2932 (aliphatic CH), 1680 (C=O, imidazolinone), 1653 (C=O, chromone), 1593 (C=C),
1319(SO2asym), 1165 (SO2sym). 1H NMR (300MHz) δ: 6.75 (s, 1H, methine H), 7.03 (m, 1H,
ArH), 7.48-7.53 (m, 12H, ArH), 8.16-8.17(m, 1H, ArH), 8.49-8.51 (m, 2H, ArH), 8.72 (s, 1H,
CH-2 of chromone), 10.55 (s, 1H, SO2NH, exchanged with D2O). 13C NMR δ: 115.4 (C-5 of
pyrimidine), 118.4 (C-8 of chromone), 118.8, 119.1, 120.2, 122.2, 122.9, 123.8, 125.7, 127.1,
128.6, 129.5, 130.4, 132.7, 133.1, 134.6 (aromatic Cs), 143.1 (C-2 of chromone), 155.2 (C-8a
of chromone), 158.1 (C-4 + C-6 of pyrimidine + C-2 of imidazolinone), 164.2 (C-2 of
pyrimidine), 164.9 (C-4 of imidazolinone), 175.4 (C-4 of chromone). Anal. Calcd. for
C29H19N5O5S (559.56): C, 63.38; H, 3.48; N, 12.74. Found: C, 63.52; H, 3.46; N, 12.91.
4.5.4. N-(4,6-Dimethylpyrimidin-2-yl)-4-(5-oxo-4-((4-oxo-4H-chromen-3-yl)methylene)-2-
phenyl-4,5-dihydro-1H-imidazol-1-yl)benzenesulfonamide (10d)
Yield: 45%; m.p. 212-213 oC; reaction time: 12h. IR (cm-1): 3242 (NH), 3062 (aromatic
CH), 2926 (aliphatic CH), 1718 (C=O, imidazolinone), 1651 (C=O, chromone), 1595 (C=C),
1313(SO2asym), 1159(SO2sym). 1H NMR (300MHz) δ: 2.24 (s, 6H, 2CH3), 6.75 (s, 1H, methine
H), 7.41- 8.01 (m, 14 H, ArH), 8.70 (s, 1H, CH-2 of chromone), 10.60 (s, 1H, SO2NH,
exchanged with D2O). EI–MS m/z (%): 577 (M+, 8.0 %). Anal. Calcd. for C31H23N5O5S
(577.62): C, 64.46; H, 4.01; N, 12.12; S, 5.55. Found: C, 64.59; H, 4.09; N, 12.21; S, 5.62.
16

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4.5.5. 4-(5-Oxo-4-((4-oxo-4H-chromen-3-yl)methylene)-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)-N-(thiazol-2-yl)benzenesulfonamide (10e)
Yield: 80%; m.p. 241-246 oC; reaction time: 8h. IR (cm-1): 3379 (NH), 3032(aromatic
CH), 2925 (aliphatic CH), 1672 (C=O, imidazolinone), 1649 (C=O, chromone), 1587 (C=C),
1317(SO2asym), 1143 (SO2sym). 1H NMR (400MHz) δ: 6.77 (s, 1H, methine H), 6.81 (d,1H, CH-
5, J= 4.0 Hz, thiazole), 7.24 (d,1H, CH-4, J= 4.0 Hz, thiazole), 7.52- 7.61 (m, 4H, ArH), 7.71-
7.78 (m, 3H, ArH), 7.85-7.89 (m, 3H, ArH), 7.97-7.99 (m, 2H, ArH), 8.17-8.19 (m, 1H, ArH),
8.73 (s, 1H, CH-2 of chromone), 10.53 (s, 1H, SO2NH, exchanged with D2O). 13C NMR δ:
108.3 (C-5 of thiazole), 115.8 (C-8 of chromone), 118.7, 119.1, 119.7, 123.4, 125.8, 126.3,
127.0, 128.0, 128.7, 132.2, 133.5, 135.0, 136.8 (aromatic Cs), 142.7 (C-4 of thiazole + C-4 of
phenyl), 148.4 (C-2 of chromone), 155.6 (C-8a of chromone), 158.3 (C-2 of imidazolinone),
164.4 (C-4 of imidazolinone), 171.3 (C-2 of thiazole), 175.8 (C-4 of chromone). EI–MS m/z
(%): 554 (M+, 1.1%). Anal. Calcd. for C28H18N4O5S2 (554.60): C, 60.64; H, 3.27; N, 10.10.
Found: C, 60.68; H, 3.29; N, 11.13.
4.5.6. 4-(5-Oxo-4-((4-oxo-4H-chromen-3-yl)methylene)-2-phenyl-4,5-dihydro-1H-imidazol-1-
yl)-N-(pyridin-2-yl)benzenesulfonamide (10f)
Yield: 70%; m.p. 246-248 oC; reaction time: 8h. IR (cm-1): 3261 (NH), 3051 (aromatic
CH), 2931 (aliphatic CH), 1685 (C=O, imidazolinone), 1655 (C=O, chromone), 1591 (C=C),
1319(SO2asym), 1163 (SO2sym). 1H NMR (300MHz) δ: 6.75 (s, 1H, methine H), 7.15-7.98 (m, 17
H, ArH), 8.71 (s, 1H, CH-2 of chromone), 10.50 (s, 1H, SO2NH, exchanged with D2O).13C
NMR δ: 115.9 (C-3 of pyridine), 119.9 (C-8 of chromone), 123.6, 126.0, 126.5, 128.1, 128.3,
128.9, 132.4, 133.6, 135.2 (aromatic Cs), 143.0 (C-6 of pyridine), 155.8 (C-2 of chromone),
158.5 (C-8a of chromone), 164.6 (C-2 of imidazolinone), 165.5 (C-4 of imidazolinone), 175.9
(C-4 of chromone). EI–MS m/z (%): 548 (M+, 65.3%). Anal. Calcd. for C30H20N4O5S (548.57):
C, 65.68; H, 3.67; N, 10.21. Found: C, 65.76; H, 3.64; N, 10.40.
4.5.7. 4-(2-(4-Chlorophenyl)-5-oxo-4-((4-oxo-4H-chromen-3-yl) methylene)-4,5-dihydro-1H-
imidazol-1-yl)benzenesulfonamide (11a)
Yield: 62%; m.p. > 300 oC; reaction time: 18h. IR (cm-1): 3338, 3288 (NH2), 3059
(aromatic CH), 2930 (aliphatic CH), 1681 (C=O, imidazolinone), 1656 (C=O, chromone), 1595
(C=C), 1300 (SO2asym), 1161 (SO2sym). 1H NMR (300MHz) δ: 6.83 (s, 1H, methine H), 7.24 (s,
2H, SO2NH2, exchanged with D2O), 7.55-7.97 (m, 11H, ArH), 8.15-8.19 (m, 1H, ArH), 8.72 (s,
1H, CH-2, chromone). 13C NMR δ: 115.8 (C-8 of chromone), 119.6, 123.1, 125.4, 126.0, 127.8,
17

ACCEPTED MANUSCRIPT
128.5, 129.6, 132.0, 132.8, 133.3, 134.6, 136.6 (aromatic Cs), 143.4 (C-2 of chromone), 157.5
(C-8a of chromone), 158.1 (C-2 of imidazolinone), 164.1 (C-4 of imidazolinone), 175.8 (C-4 of
chromone). EI–MS m/z (%): 507.02 (M++2, 0.7 ), 505.03 (M+, 2.0%). Anal. Calcd. for
C25H16ClN3O5S (505.93): C, 59.35; H, 3.19; N, 8.31. Found: C, 59.42; H, 3.21; N, 8.44.
4.5.8. 4-(2-(4-Chlorophenyl)-5-oxo-4-((4-oxo-4H-chromen-3-yl) methylene)-4,5-dihydro-1H-
imidazol-1-yl)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (11b)
Yield: 75%; m.p. 295-297 oC; reaction time: 12h. IR (cm-1): 3250 (NH), 3161 (aromatic
CH), 2924 (aliphatic CH), 1687 (C=O, imidazolinone), 1654 (C=O, chromone), 1591 (C=C),
1317(SO2asym), 1165 (SO2sym). 1H NMR (300MHz) δ: 2.29 (s, 3H, CH3), 6.10 (s, 1H, CH,
isoxazole), 6.79 (s, 1H, methine H), 7.55-7.99 (m, 11H, ArH), 8.17-8.18 (m, 1H, ArH), 8.73 (s,
1H, CH-2 chromone), 10.57 (s, 1H, SO2NH, exchanged with D2O). 13C NMR δ: 12.0 (CH3),
95.3 (C-4 of isoxazole), 115.9 (C-8 of chromone), 119.6, 123.1, 125.4, 126.0, 127.8, 128.5,
129.6, 132.0, 132.8, 133.3, 134.6, 136.8 (aromatic Cs), 155.3 (C-2 of chromone), 157.5 ( C-3
of isoxazole), 158.1 (C-8a of chromone + C-2 of imidazolinone), 164.1 (C-5 of isoxazole),
170.2 (C-4 of imidazolinone), 175.3 (C-4 of chromone). EI–MS m/z (%): 588.01 (M++2,
2.2%), 586 (M+, 6.9%). Anal. Calcd. for C29H19ClN4O6S (586.07): C, 59.34; H, 3.26; N, 9.54.
Found: C, 59.42; H, 3.29; N, 9.68.
4.5.9. 4-(2-(4-Chlorophenyl)-5-oxo-4-((4-oxo-4H-chromen-3-yl) methylene)-4,5-dihydro-1H-
imidazol-1-yl)-N-(pyrimidin-2-yl)benzenesulfonamide (11c)
Yield: 40%; m.p. 284-288 oC; reaction time: 14h. IR (cm-1): 3284 (NH), 3041 (aromatic
CH), 2950 (aliphatic CH), 1683 (C=O, imidazolinone), 1656 (C=O, chromone), 1591 (C=C),
1315(SO2asym), 1163 (SO2sym). 1H NMR (300MHz) δ: 6.79 (s, 1H, methine H), 7.01-7.04 (m,
2H, ArH), 7.52-7.99 (m, 8H, ArH), 8.15-8.18 (m, 2H, ArH), 8.49-8.51 (m, 3H, ArH), 8.73 (s,
1H, CH-2, chromone), 10.54 (s, 1H, SO2NH, exchanged with D2O). 13C NMR δ: 115.9 (C-5 of
pyrimidine), 118.4 (C-8 of chromone), 118.7 (C-3 of chromone), 119.2, 123.1, 125.4, 126.0,
128.5, 128.6, 129.6, 132.0, 132.8 , 134.3, 134.6, 136.7 (aromatic Cs), 143.0 (C-1 of phenyl),
155.3 (C-2 of chromone), 156.9 (C-8a of chromone), 158.0 (C-4 + C-6 of pyrimidine + C-2 of
imidazolone), 158.2 (C-2 of pyrimidine), 164.0 (C-4 of imidazolinone), 175.3 (C-4 of
chromone). Anal. Calcd. for C29H18ClN5O5S (583.07): C, 59.64; H, 3.11; N, 11.99. Found: C,
59.73; H, 3.14; N, 12.13.
4.5.10. 4-(2-(4-Chlorophenyl)-5-oxo-4-((4-oxo-4H-chromen-3-yl) methylene)-4,5-dihydro-1H-
imidazol-1-yl)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (11d)
18

ACCEPTED MANUSCRIPT
Yield: 43%; m.p. 261-268 oC; reaction time: 24h. IR (cm-1): 3363 (NH), 3089 (aromatic
CH), 2927 (aliphatic CH), 1697 (C=O, imidazolinone), 1653 (C=O, chromone), 1591 (C=C),
1315(SO2asym), 1149 (SO2sym). 1H NMR (400MHz) δ: 2.26 (s, 6H, 2CH3), 6.76 (s, 1H, ArH,
pyrimidine), 6.80 (s, 1H, methine H), 7.54-7.58 (m, 3H, ArH), 7.61-7.63 (m, 1H, ArH),7.71-
7.73 (m, 3H, ArH), 7.95-7.99 (m, 4H, ArH),8.15-8.17 (m, 1H, ArH), 8.73 (s, 1H, CH-2
chromone), 10.54 (s, 1H, SO2NH, exchanged with D2O). 13C NMR δ: 23.3 (CH3), 114.0 (C-5
of pyrimidine), 118.9 (C-8 of chromone), 119.2, 119.3, 123.6, 125.9, 126.5, 129.0, 129.5,
130.2, 132.5, 133.3, 135.1, 137.3 (aromatic Cs), 143.3 (C-2 of chromone), 155.8 (C-8a of
chromone), 156.7 (C-2 of imidazolinone), 158.5 (C-4 + C-6 of pyrimidine), 164.5 (C-2 of
pyrimidine), 164.6 (C-4 of imidazolinone), 175.8 (C-4 of chromone). Anal. Calcd. for
C31H22ClN5O5S (611.06): C, 60.83; H, 3.62; N 11.44; S, 5.24. Found: C, 60.90; H, 3.69; N,
11.58; S, 5.33.
4.5.11. 4-(2-(4-Chlorophenyl)-5-oxo-4-((4-oxo-4H-chromen-3-yl) methylene)-4,5-dihydro-1H-
imidazol-1-yl)-N-(thiazol-2-yl)benzenesulfonamide (11e)
Yield: 72%; m.p. 268-271oC; reaction time: 24h. IR (cm-1): 3300 (NH), 3084 (aromatic
CH), 2935 (aliphatic CH), 1672 (C=O, imidazolinone), 1651 (C=O, chromone), 1587 (C=C),
1313(SO2asym), 1143 (SO2sym). 1H NMR (400MHz) δ: 6.80 (s, 1H, methine H), 6.81 (d, 1H, CH-
5, J = 4.0 Hz , thiazole), 7.43 (d, 1H, CH-4, J= 4.0 Hz, thiazole), 7.46-7.99 (m, 11H, ArH),
8.15-8.17 (m, 1H, ArH) , 8.73 (s, 1H, CH-2, chromone),10.52 (s, 1H, SO2NH, exchanged with
D2O). 13C NMR δ: 108.5 (C-5 of thiazole), 116.5 (C-8 of chromone), 119.9, 123.6, 124.8,
125.9, 126.5, 127.2, 127.4, 128.6, 129.0, 130.1, 131.4, 132.5, 133.3, 135.1, 137.0, 137.2
(aromatic Cs), 138.4 (C-4 of thiazole), 142.8 (C-2 ofchromone), 155.8 (C-8a of chromone),
158.5 ( C-2 of imidazolinone), 164.5 (C-4 of imidazolinone), 168.7 (C-2 of thiazole), 175.8
(C-4 of chromone). EI–MS m/z (%): 590 (M++2, 7.5%), 588.01 (M+, 22.8%). Anal. Calcd. for
C28H17ClN4O5S2 (589.04): C, 57.09; H , 2.91; N, 9.51. Found: C, 57.20; H, 2.97; N, 9.62
4.5.12. 4-(2-(4-Chlorophenyl)-5-oxo-4-((4-oxo-4H-chromen-3-yl) methylene)-4,5-dihydro-1H-
imidazol-1-yl)-N-(pyridin-2-yl)benzene sulfonamide (11f)
Yield: 45%; m.p. 285-287oC; reaction time: 15h. IR (cm-1): 3269 (NH), 3059 (aromatic
CH), 2926 (aliphatic CH), 1683 (C=O, imidazolinone), 1653 (C=O, chromone), 1593 (C=C),
1313(SO2asym), 1166 (SO2sym). 1H NMR (300MHz) δ: 6.79 (s, 1H, methine H), 7.03-7.04 (m,
1H, ArH), 7.55-7.99 (m, 14H, ArH), 8.15-8.17 (m, 1H, ArH), 8.73 (s, 1H, CH-2, chromone),
10.54 (s, 1H, SO2NH, exchanged with D2O). EI–MS m/z (%): 584 (M++2, 3.5%), 582 (M+,
19

ACCEPTED MANUSCRIPT
10.7%) . Anal. Calcd. for C30H19ClN4O5S (583.01): C, 61.80; H, 3.28, N; 9.61; S, 5.50 . Found:
C, 61.92; H, 3.24; N,9.73; S, 5.61.
4.2. In vitro cytotoxic activity:
4.2.1. Cell culture:
MCF-7 human breast cancer cells, grown in RPMI-1640 medium; and A-549 human
lung cancer cells, grown in DMEM, were supplemented with 10% heat inactivated FBS, 50
units/mL of penicillin and 50 mg/mL of streptomycin and were maintained at 37° C in a
humidified atmosphere containing 5% CO2. The cells were maintained as “monolayer culture”
by serial subculturing.
4.2.2. Sulforhodamine B assay (SRB):
Exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded in
96-well plates at 1000-2000 cells/well in RPMI supplemented medium or in DMEM
supplemented medium. After 24 h, the cells were incubated for 72 h with various
concentrations of the tested compounds. Following 72 h treatment, the cells were fixed with
10% trichloroacetic acid for 1 h at 4 ºC. Wells were stained for 10 min at room temperature
with 0.4% SRB dissolved in 1% acetic acid. The plates were air dried for 24 h and the dye was
solubilized with Tris-HCl for 5 min on a shaker at 1600 rpm. The optical density (OD) of each
well was measured spectrophotometrically at 564 nm with an ELISA microplate reader
(ChroMate-4300, FL, USA). The IC50 values were calculated according to the equation for
Boltzman sigmoidal concentration–response curve using the nonlinear regression fitting models
(Graph Pad, Prism Version 5).
4.3. Carbonic anhydrase inhibition:
An Applied Photophysics stopped-flow instrument was used for assaying the CA
catalysed CO2 hydration activity [49]. Phenol red (at a concentration of 0.2 mM) was used as
indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as
buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial
rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s. The CO2
concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction
was used for determining the initial velocity. The uncatalyzed rates were determined in the
same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1
20

ACCEPTED MANUSCRIPT
mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done
thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for
15 min at room temperature prior to assay, in order to allow for the formation of the E-I
complex. The inhibition constants were obtained by non-linear least-squares methods using the
Cheng-Prusoff equation, as reported earlier [52-54], and represent the mean from at least three
different determinations. All four CA isoforms were recombinant ones obtained in-house as
reported earlier [51-53].
4.4. Apoptosis study:
MCF-7 or A-549 cells (1 x 105 cells/ml) were plated in a 24-well culture plate
(Corning) for 24 h at 37 oC with 5% CO2 and 95% humidity. The tested compound 4a was
added. Cells were incubated for another 24 h. Cells were trypsinized with 0.25% trypsin in
Ca2+/Mg2+-free 2% BSA-PBS (Sigma, St. Louis, MO,USA) and washed twice with Ca2+/Mg2+-
free PBS. Cells were stained and adjusted to 1 x 106 cells/ml with annexin V-FITC/PI. The
reaction was allowed to proceed in the dark for 30 min at room temperature. Finally, 400 ml of
1 x binding buffer was added and flow cytometry carried out within 1 h.
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Figure captions:
Figure 1. Examples of chromone-sulfonamide hybrids as antitumor and CA inhibitors.
Figure 2. General formulae of target compounds.
Figure 3. Flow cytometry histograms of MCF-7 and A-549 cells after 24h treatment with 4a.
Scheme captions:
Scheme 1. Synthesis of compounds 3a-f and 4a-f.
Scheme 2. Synthesis of compounds 7a-f.
Scheme 3. Synthesis of compounds 10a-f and 11a-f.
25

ACCEPTED MANUSCRIPT
Table captions:
Table 1. IC50 values ± SEM of the new compounds against MCF-7 and A-549 cell lines
Table2. Ki values of tested compounds on hCA I, II, IX and XII.
26

Table 1.
ACCEPTED MANUSCRIPT
IC50 (µM) ± SEM
MCF-7
IC50 (µM) ± SEM
A-549
Compound
22.19±1.01
25.80±0.58
30.06±1.13
45.62±1.56
48.61±4.10
28.81±1.57
0.72±0.03
34.50±1.64
> 100
33.39±6.74
57.23±0.63
> 100
36.35±1.70
> 100
39.05±1.13
0.50±0.02
59.66±0.29
85.25±4.24
55.83±2.73
25.59±2.70
> 100
3a
3b
3c
3d
3e
3f
4a
4b
4c
93.57±5.71
58.61±4.33
> 100
4d
4e
4f
8.91±1.08
>100
2.23±55.91
>100
7a
7b
82.60±7.20
>100
>100
>100
7c
7d
>100
>100
7e
47.10±3.17
11.50±1.19
30.01±1.40
>100
99.71±5.90
88.48±8.25
>100
24.31±2.83
>100
>100
93.92±5.49
>100
>100
1.73±40.36
>100
40.47±1.84
63.21±2.78
88.40±3.02
>100
13.77±2.08
>100
59.03±7.14
52.61±2.47
>100
>100
26.81±2.50
7f
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
DOX
>100
33.13±2.90

ACCEPTED MANUSCRIPT
Table 2.
Ki (nM)*
Compound
hCA I
>10000
>10000
>10000
>10000
965.03
>10000
398.78
>10000
>10000
8003.15
>10000
>10000
250.00
hCA II
>10000
>10000
>10000
>10000
370.02
>10000
12.95
hCA IX
23.84
37.62
19.91
20.93
29.75
32.09
14.26
24.74
25.58
23.04
25.24
18.39
25.00
hCA XII
424.04
79.35
22.88
62.02
4.54
3b
3d
3e
3f
4a
10.76
9.91
4b
7a
>10000
1064.56
75.22
85.83
546.55
5.23
7c
7f
10a
10b
11a
AZA
1112.0
722.05
12.00
9.71
6.24
5.70
AZA (Acetazolamide), a well-known CAI, was used as a standard for comparison.
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
± 5-10 % of the reported values).