Design, synthesis and biological evaluation of novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties as potent carbonic anhydrase IX inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.10.005

A series of novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties were obtained by reacting 4-isocyanato-benzenesulfonamide (2) with 2-amino-4,6-dicholoro-1,3,5-triazine (4). The 4-(3-(4,6-dichloro-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (5) was subsequently derivatized by reaction with various nucleophiles such as, morpholine, ammonia, methyl amine, dimethyl amine, and piperidine. The ureido benzenesulfonamides incorporating triazinyl moieties were investigated as inhibitors of four selected physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, namely, hCA I, II, IX, and XII which are involved in various diseases such as glaucoma, epilepsy, obesity and cancer. The membrane-bound tumor-associated isoform hCA IX was potently inhibited with these compounds with K_is in the range of 0.91–126.2 nM. Specifically, compound 7j showed great potency against hCA IX with sub-nanomolar K_i of 0.91 nM. Since hCA IX is a validated drug target for anticancer agents, these isoform-selective and potent inhibitors may be considered of interest for further medicinal/pharmacologic studies.

Full text of DOI:10.1016/j.bioorg.2018.10.005

Accepted Manuscript
Design, synthesis and biological evaluation of novel ureido benzenesulfona-
mides incorporating 1,3,5-triazine moieties as potent carbonic anhydrase IX
inhibitors
Nabih Lolak, Suleyman Akocak, Silvia Bua, Claudiu T. Supuran
PII:
S0045-2068(18)30903-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.005
YBIOO 2539
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
20 August 2018
26 September 2018
4 October 2018
Please cite this article as: N. Lolak, S. Akocak, S. Bua, C.T. Supuran, Design, synthesis and biological evaluation
of novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties as potent carbonic anhydrase IX
inhibitors, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.005
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Design, synthesis and biological evaluation of novel ureido
benzenesulfonamides incorporating 1,3,5-triazine moieties as potent carbonic
anhydrase IX inhibitors
Nabih Lolak a, Suleyman Akocak a*, Silvia Bua b,and Claudiu T. Supuranb*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Adiyaman University, 02040
Adiyaman, Turkey
b Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche,
Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
Abstract
A series of novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties were
obtained by reacting 4-isocyanato-benzenesulfonamide (2) with 2-amino-4,6-dicholoro-1,3,5-
triazine (4). The 4-(3-(4,6-dichloro-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (5) was
subsequently derivatized by reaction with various nucleophiles such as, morpholine, ammonia,
methyl amine, dimethyl amine, and piperidine. The ureido benzenesulfonamides incorporating
triazinyl moieties were investigated as inhibitors of four selected physiologically relevant human
carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, namely, hCA I, II, IX, and XII which are
involved in various diseases such as glaucoma, epilepsy, obesity and cancer. The membrane-
bound tumor-associated isoform hCA IX was potently inhibited with these compounds with Kis
in the range of 0.91 to 126.2 nM. Specifically, compound 7j showed great potency against hCA
IX with sub-nanomolar Ki of 0.91 nM. Since hCA IX is a validated drug target for anticancer
agents, these isoform-selective and potent inhibitors may be considered of interest for further
medicinal/pharmacologic studies.
Key words: Ureido benzenesulfonamides, 1,3,5-triazine moiety, Carbonic anhydrase, Isoforms,
Isoform-selective inhibitor, cancer
____
*Corresponding authors: Tel: +90 (416) 223 3800-2836, Fax: +90 (416) 223 3842, E-mail:
akocaksuleyman@gmail.com, sakocak@adiyaman.edu.tr, (Suleyman Akocak);
Tel/Fax: +39-055-4573729, E-mail: claudiu.supuran@unifi.it (Claudiu T. Supuran)

1. Introduction
Cancer is the second most common disease causing death, after cardiovascular diseases
across the world, and its incidence is expected to increase dramatically in the near future. The
high incidence and mortality ratio of cancer is due to the fact that there are more than 200 types
of cancers and it is rather difficult to detect most of them in the early stage. For all these reasons,
the research in the anti-cancer drug discovery focused on cancer treatment with more effective
and less toxic agents [1-3].
Carbonic anhydrase (CA, EC 4.2.1.1) IX and XII (h, human isozymes, hCA IX and XII)
are well-known transmembrane CA isoforms which are highly expressed in different tumor types
and present a rather limited expression in most normal cells [4-7]. These tumor-associated
proteins play important roles in tumor survival, acidification and proliferation under hypoxic
conditions of primary tumors and metastases [4-7]. The extracellular localization of these
isoforms, allows their efficient targeting by antibodies and small molecule inhibitors. Recently,
one of the small molecule CA IX/XII inhibitors, SLC-0111 advanced into Phase I/II clinical
trials for the treatment of hypoxic, metastatic tumors over-expressing these proteins [8-10]. SLC-
0111 belongs to the class of ureido-sulfonamides, which show a very good selectivity for
inhibiting CA IX/XII over CA I and II [8-10], cytosolic isoforms which are off-targets when
considering the antitumor applications of the CA inhibitors.
The 1,3,5-triazine scaffold, also known as s-triazine, is an interesting core for medicinal
chemistry applications due to the broad biological activities and wide variety of applications of
compounds incorporating it, such as antimicrobial, diuretics, antiviral, anti-inflammatory, and
more anti-cancer agents [11-13]. In recent years, sulfonamides incorporating 1,3,5-triazine
moieties were discovered as potent and highly selective hCA IX inhibitors [14-16]. These

compounds showed one of the best selectivity ratio for hCA IX over the widespread, off-target
hCA II, between 166 to 706 fold. The high selectivity ratio of these compounds makes them
good lead compounds for designing other types of selective inhibitors targeting the tumor-
associated isoform hCA IX [14].
In the current work, we combined these two powerful scaffolds (1,3,5-triazine and ureido
substituted benzenesulfonamides) to obtain potent and selective hCA IX and XII inhibitors by
using the tail approach, as described in Figure 1. For this reason, novel ureido
benzensulfonamides incorporating 1,3,5-triazine moieties were synthesized and investigated as
inhibitors of four physiologically and pharmacologically relevant isoforms, which are the
cytosolic isozymes hCA I and II, as well as tumor-associated membrane-bound isoforms hCA IX
and XII.

Figure 1. The design strategy for the ureido benzensulfonamides incorporating 1,3,5-triazine
moieties, starting from SLC-0111 as lead, by using tail approach.
2. Result and Discussion
2.1. Chemistry
In the design of novel and possibly isoform-selective CA inhibitors, the investigation of
hybrid molecules through the combination of different scaffolds and pharmacophores in one
structure may lead to improved potency and selectivity. Considering the versatile chemistry of
cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) and the ureido substituted benzene-sulfonamide
scaffold present in SLC-0111, we have designed and synthesized novel ureido
benzenesulfonamides incorporating the 1,3,5-triazine moiety as CA inhibitors.
The synthesis of this series of ureido benzenesulfonamides was performed according to
the general synthetic route described in Scheme 1. The starting compounds, 4-isocyanato-
benzenesulfonamide (2) and 4,6-dicholoro-1,3,5-triazine-2-amine (4) were synthesized as
previously described [17,18]. The key intermediate of this work, 4-(3-(4,6-dichloro-1,3,5-triazin-
2-yl)ureido) benzenesulfonamide (5) was obtained as shown in Scheme 1 by the reaction of
compounds 2 and 4. Subsequently, the chlorine atoms of the key intermediate 5 were substituted
by using morpholine, piperidine, ammonia, methyl amine, and dimethyl amine as nucleophiles,
in order to generate chemical diversity.

Scheme 1. General synthetic route for the synthesis of benzenesulfonamides incorporating
1,3,5-triazine moieties. Reagents and conditions: (i) nitrobenzene, phosgene, -10 to 90 0C slowly,
0
6h, (ii) Acetone, crushed ice, 25 wt% aqueous ammonia solution, 0 to 5 C, 30 min, yield 95%.
(iii) THF, 48 h, 40 0C, yield 45% (iv) R1H, DMF, 0 to 5 0C, 1h, then R.T. 4h, yields 54-88% (v)
R2H, DMF, room temperature, 1h, then 90 0C, 2h, yields 45-88%.
2.2.Carbonic anhydrase inhibition
The novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties obtained
here were tested as inhibitors of four physiologically and pharmacologically relevant isoforms,
namely, the cytosolic hCA I and II, as well as the transmembrane tumor-associated isoforms
hCA IX and XII, by a stopped-flow CO2 hydrase assay [19]. Acetazolamide (AAZ), a clinically

employed sulfonamide CAI and SLC-0111 (Phase I/II clinical trials for the treatment of
advanced, metastatic breast cancer) were also included in the assays as standard drugs. The
following structure-activity relationship (SAR) may be drawn regarding the inhibition data of
Table 1 for this series of ureido benzenesulfonamides incorporating the 1,3,5-triazine scaffold, 5,
6 (a-e), and 7 (a-k):
i.
The widely abundant slow cytosolic isoform hCA I was moderately inhibited by all the
novel inhibitors that are presented in this work, with the inhibition constants in the range
of 91.7-8374.8 nM. Only one compound (6e) showed efficient inhibition, being more
effective than the clinically used drug acetazolamide (AAZ), with a Ki of 91.7 nM. The
least potent inhibitors from the series were compound 7a (R1= morpholine, R2= NH2),
compound 7e (R1, R2= morpholine) and 7i (R1= N(Me)2, R2 = NH2), with Kis of 8343.1,
4186.7 and 8374.8 nM, respectively.
ii.
All compounds reported here were more efficient as hCA II inhibitors compared to SLC-
0111, which is a weak inhibitor of this isoform with a Ki of 960 nM. In general, all the
new compounds reported here showed low nanomolar to subnanomolar inhibition of hCA
II, with Kis ranging between 0.69 nM and 420.9 nM. The best hCA II inhibitors were
derivatives 6d (R1= morpholine, R2= Cl), 6e (R1= piperidine, R2= Cl) and 7j (R1=
N(Me)2, R2= NHMe), with Kis of 1.5, 0.69, and 3.1 nM.
iii.
The transmembrane tumor-associated isoform hCA IX was efficiently inhibited by most
of the compounds reported in this work. Only three compounds from the series were less
potent than SLC-0111, which is an effective hCA IX inhibitor, namely compound 6a, 7a,
and 7e with Kis of 48.5, 126.2, 46.5 nM, respectively. One of the most important findings
of the current work is that compound 7j showed subnanomolar activity (Ki, 0.91 nM)

against hCA IX, with good selectivity over hCA I and hCA XII, and reasonable
selectivity over hCA II. Other hCA IX potent inhibitors were derivatives 5 (R1, R2= Cl),
6c (R1= N(Me)2, R2= Cl), 6e (R1= piperidine, R2= Cl), and 7f (R1= piperidine, R2=
NHMe) with Kis of 4.4, 4.5, 2.3, and 2.7 nM, respectively.
iv.
The other tumor-associated membrane bound isoform hCA XII was moderately inhibited
by most of the ureido benzenesulfonamides incorporating 1,3,5-triazine moieties reported
here, with Kis ranging from 80.5 to 901.3 nM, except derivatives 7a and 7e, which did
not inhibit the enzyme up to 10,000 nM. Among this series, two compounds showed
potent inhibition against hCA XII, i.e., compound 5 (R1=R2= Cl) and compound 7f (R1=
piperidin, R2= NHMe) with Kis of 84.2 and 80.5 nM, respectively.
Table 1: Inhibition data of human CA isoforms hCA I, II, IX and XII with derivatives 5, 6 (a-e),
and 7 (a-k) reported here and the standard sulfonamide inhibitors acetazolamide (AAZ) and
SLC-0111 (phase I/II clinical trials for the treatment of advanced metastatic breast cancer) by a
stopped flow CO2 hydrase assay [19].
KI* (nM)
Comp. R1
R2
hCAI
hCA II
hCA IX
hCA XII
5
Cl
Cl
Cl
Cl
Cl
873.0
816.8
676.5
660.2
93.9
178.6
9.0
4.4
84.2
6a
6b
6c
-NH2
48.5
26.8
4.5
901.3
579.0
346.5
-NHMe
-N(Me)2
12.4

6d
6e
7a
7b
7c
7d
7e
7f
Cl
548.1
91.7
1.5
31.6
2.3
301.4
277.5
>10000
872.9
747.7
831.8
>10000
80.5
Cl
0.69
420.9
33.6
3.9
-NH2
-NHMe
-N(Me)2
8343.1
803.2
602.9
625.9
4186.7
427.7
551.3
126.2
32.0
7.4
33.2
6.9
28.3
46.5
2.7
-NHMe
5.2
7g
-N(Me)2
8.5
4.9
743.7
7h
474.2
8374.8
394.9
923.8
250
78.9
299.4
3.1
12.7
23.8
0.91
11.4
25
494.7
692.9
554.7
626.3
5.7
7i
-N(Me)2
-N(Me)2
-N(Me)2
-
-NH2
7j
-NHMe
7k
-N(Me)2
7.5
AAZ
-
-
12
SLC-0111 -
5080
960
45.1
4.5
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10
% of the reported values).
3. Conclusions
In conclusion, we report here a novel series of ureido benzenesulfonamides incorporating
1,3,5-triazine moieties, which also contain with morpholine, piperidine, ammonia, methyl amine,

and dimethyl amine moieties in their molecules. The novel compounds were investigated as
inhibitors of four physiologically and pharmacologically relevant isoforms, the cytosolic
isoforms hCA I and II, as well as tumor-associated membrane-bound isoforms hCA IX and XII.
All compounds showed potent inhibition against the tumor-associated isozyme hCA IX with low
nanomolar to subnanomolar potency, with Kis in the range of 0.91 to 126.2 nM. For other
isoforms, distinct inhibition profiles and interesting structure-activity relationship were observed,
depending on the nature of the amine that was appended on the 1,3,5-triazine scaffold. As hCA
IX is a validated drug target for metastatic hypoxic tumors and SLC-0111 advanced to Phase I/II
clinical trials for the treatment of breast cancer, these hCA IX potent ureido
benzenesulfonamides incorporating 1,3,5-triazine moieties might be of interest for further
medicinal/pharmacologic studies.
4. Experimental
4.1. Chemistry
All chemicals and anhydrous solvents were purchased from Sigma-Aldrich, Merck, Alfa
Aesar and TCI and used without further purification. FT-IR spectra were obtained by using
Perkin Elmer Spectrum 100 FT-IR spectrometer. Nuclear Magnetic Resonance (1H-NMR and
13C-NMR) spectra of compounds were recorded using a Bruker Advance III 300 MHz
1
spectrometer in DMSO-d6 and TMS as an internal standard operating at 300 MHz for H-NMR
13
and 75 MHz for C-NMR. Thin layer chromatography (TLC) was carried out on Merck silica
gel 60 F254 plates.

4.1.1. Syntesis
of
4-(3-(4,6-dichloro-1,3,5-triazin-2-yl)ureido)
benzenesulfonamide (5)
The starting compounds, 4-isocyanato-benzenesulfonamide (2) and 4,6-dicholoro-1,3,5-
triazine-2-amine (4) were synthesized as previously described [15,16]. A solution of 2 (10 mmol)
in 5 mL of THF was slowly added over the solution of 4 (10 mmol) under stirring at room
temperature. The solution was left under stirring for 48 h at 40 0C (TLC monitoring). After that,
the solvent was evaporated and crude residue was purified by column chromatography (ethyl
acetate/ petroleum ether) to give title compound 5. The obtained product was dried under
vacuum and fully characterized by FT-IR, 1H-NMR, 13C-NMR, and melting point.
0
Yield: 25%; Color: white solid; mp: 244-246 C; FT-IR (cm-1): 3213, 1653, 1535 (asymmetric),
1
1342, 1164 (symmetric) (S=O), 1090; H-NMR (DMSO-d6, 300 MHz, δ ppm): 10.65 (s, 1H, -
NH-), 9.10 (s, 1H, -NH-), 8.00 (d, 2H, J = 8.4, Ar-H), 7.93 (d, 2H, J = 6.9, Ar-H), 7.58 (s, 2H, -
13
SO2NH2): C-NMR (DMSO-d6, 75 MHz, δ ppm): 165.3, 156.1, 150.4, 147.1, 135.5, 129.2,
126.0;
4.1.2. General procedure for the synthesis of compounds 6(a-e).
At 0 0C, a 10 mmol solution of R1-H (morpholine, piperidine, 25 wt% ammonia, 40 wt%
methyl amine, dimethyl amine) was added to 5 mmol of 5 in DMF under stirring. After complete
addition, the mixture was allowed to warm to room temperature for 4h. Then, the product was
0
filtered off washed with water and dried under vacuum at 40 C. The obtained final pure
products were fully characterized by FT-IR, 1H-NMR, 13C-NMR, and melting points.
4-(3-(4-amino-6-chloro-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (6a).
0
Yield: 54%; Color: white solid; mp: 288-290 C; FT-IR (cm-1): 3304, 3148, 1643, 1547
1
(asymmetric), 1413, 1155 (symmetric) (S=O), 1093; H-NMR (DMSO-d6, 300 MHz, δ

ppm): 10.60 (s, 1H, -NH-), 9.15 (s, 1H, -NH-), 8.11 (d, 2H, J = 7.2, Ar-H), 7.95 (d, 2H, J
= 7.5, Ar-H), 7.56 (s, 2H, -SO2NH2), 6.62 (s, 2H, -NH2): 13C-NMR (DMSO-d6, 75 MHz,
δ ppm): 165.7, 164.1, 156.4, 150.2, 147.3, 136.1, 129.1, 126.3;
4-(3-(4-chloro-6-(methylamino)-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (6b).
0
Yield: 78%; Color: white solid; mp: 282-285 C; FT-IR (cm-1): 3255, 3117, 1647, 1551
1
(asymmetric), 1319, 1155 (symmetric) (S=O), 1104; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 10.62 (s, 1H, -NH-), 9.18 (s, 1H, -NH-), 8.08-7.94 (m, 4H, Ar-H), 7.55 (s, 2H, -
SO2NH2), 6.52 (s, 2H, -NHCH3), 2.81-2.75 (m, 3H, -NHCH3) : 13C-NMR (DMSO-d6, 75
MHz, δ ppm): 165.7, 164.4, 156.5, 150.7, 146.7, 136.1, 128.9, 126.2, 27.6;
4-(3-(4-chloro-6-(dimethylamino)-1,3,5-triazin-2-yl)ureido)
benzenesulfonamide
0
(6c). Yield: 83%; Color: white solid; mp: 249-251 C; FT-IR (cm-1): 3361, 3257, 1669,
1
1577 (asymmetric), 1345, 1166 (symmetric) (S=O), 1113; H-NMR (DMSO-d6, 300
MHz, δ ppm): 10.65 (s, 1H, -NH-), 9.15 (s, 1H, -NH-), 8.08 (d, 2H, J = 7.2, Ar-H), 7.92
(d, 2H, J = 6.9, Ar-H), 7.54 (s, 2H, -SO2NH2), 3.12 (s, 6H, -CH3): 13C-NMR (DMSO-d6,
75 MHz, δ ppm): 165.9, 164.7, 156.2, 150.5, 146.8, 136.3, 128.8, 126.1, 35.5;
4-(3-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (6d).
0
Yield: 88%; Color: white solid; mp: 265-267 C; FT-IR (cm-1): 3337, 3201, 1669, 1577
1
(asymmetric), 1345, 1166 (symmetric) (S=O), 1113; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 10.68 (s, 1H, -NH-), 9.10 (s, 1H, -NH-), 8.12 (d, 2H, J = 6.9, Ar-H), 7.90 (d, 2H, J
= 6.3, Ar-H), 7.51 (s, 2H, -SO2NH2), 3.80-3.45 (m, 8H, morpholine): 13C-NMR (DMSO-
d6, 75 MHz, δ ppm): 165.6, 164.3, 156.5, 150.2, 146.7, 136.2, 128.7, 126.3, 66.3, 43.4;
4-(3-(4-chloro-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (6e).
0
Yield: 66%; Color: white solid; mp: 229-231 C; FT-IR (cm-1): 3341, 3240, 1673, 1558

1
(asymmetric), 1332, 1159 (symmetric) (S=O), 1084; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 10.66 (s, 1H, -NH-), 9.02 (s, 1H, -NH-), 8.08 (d, 2H, J = 7.2, Ar-H), 7.88 (d, 2H, J
= 6.9, Ar-H), 7.53 (s, 2H, -SO2NH2), 3.45-3.21 (m, 4H, piperidine), 1.74-1.48 (m, 6H,
13
piperidine): C-NMR (DMSO-d6, 75 MHz, δ ppm): 165.9, 164.7, 156.4, 150.5, 146.8,
136.5, 128.4, 126.2, 43.6, 25.8, 24.3;
4.1.3. General procedure for the synthesis of compounds 7 (a-k).
Under stirring, a 2 mmol solution of R2-H (morpholine, piperidine, 25 wt% ammonia, 40
wt% methyl amine, dimethyl amine) was added to 1 mmol of 6 (a-e) in DMF at room
0
temperature. Then, the reaction temperature was raised to 90 C for 2h. After cooling to room
temperature, the mixture was filtered and the precipitate was washed with water and dried at 50
1
13
0C. The obtained final pure products 7 (a-k) were fully characterized by FT-IR, H-NMR, C-
NMR, and melting points.
4-(3-(4-amino-6-morpholino-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (7a).
0
Yield: 45%; Color: white solid; mp: 248-251 C; FT-IR (cm-1): 3273, 3205, 1635, 1529
1
(asymmetric), 1338, 1179 (symmetric) (S=O), 1091; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 10.55 (s, 1H, -NH-), 8.88 (s, 1H, -NH-), 8.15 (d, 2H, J = 7.2, Ar-H), 7.92 (d, 2H, J
= 6.9, Ar-H), 7.53 (s, 2H, -SO2NH2), 6.48 (s, 2H, -NH2), 3.79-3.65 (m, 4H, morpholine),
13
3.42-3.35 (m, 4H, morpholine): C-NMR (DMSO-d6, 75 MHz, δ ppm): 165.2, 164.1,
156.2, 150.5, 146.8, 136.1, 128.3, 126.1, 66.5, 43.2;
4-(3-(4-(methylamino)-6-morpholino-1,3,5-triazin-2-yl)ureido) benzenesulfonamide
0
(7b). Yield: 85%; Color: white solid; mp: 248-251 C; FT-IR (cm-1): 3423, 3319, 3213,
1
1663, 1514 (asymmetric), 1330, 1157 (symmetric) (S=O), 1108; H-NMR (DMSO-d6,

300 MHz, δ ppm): 10.58 (s, 1H, -NH-), 8.85 (s, 1H, -NH-), 8.07 (d, 2H, J = 7.5, Ar-H),
7.88 (d, 2H, J = 7.2, Ar-H), 7.52 (s, 2H, -SO2NH2), 6.58 (s, 2H, -NHCH3), 3.75-3.62 (m,
13
4H, morpholine), 3.45-3.37 (m, 4H, morpholine), 2.99-2.85 (m, 3H, -NHCH3) : C-
NMR (DMSO-d6, 75 MHz, δ ppm): 166.0, 164.7, 156.8, 150.7, 146.5, 136.4, 128.2,
126.6, 66.2, 42.8, 28.3;
4-(3-(4-(dimethylamino)-6-morpholino-1,3,5-triazin-2-yl)ureido)
benzenesulfonamide (7c). Yield: 88%; Color: white solid; mp: 262-2650C; FT-IR (cm-
1): 3330, 3203, 1674, 1513 (asymmetric), 1306, 1167 (symmetric) (S=O), 1111; 1H-NMR
(DMSO-d6, 300 MHz, δ ppm): 10.41 (s, 1H, -NH-), 8.76 (s, 1H, -NH-), 8.02 (d, 2H, J =
7.5, Ar-H), 7.93 (d, 2H, J = 7.2, Ar-H), 7.53 (s, 2H, -SO2NH2), 3.84-3.43 (m, 8H,
13
morpholine), 3.05 (s, 6H, -CH3) : C-NMR (DMSO-d6, 75 MHz, δ ppm): 166.8, 165.2,
156.9, 150.8, 146.3, 136.7, 128.5, 126.9, 66.6, 43.4, 35.8;
4-(3-(4-morpholino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)ureido) benzenesulfonamide
(7d). Yield: 82%; Color: white solid; mp: 222-2250C; FT-IR (cm-1): 3216, 3064, 1656,
1
1505 (asymmetric), 1331, 1162 (symmetric) (S=O), 1102; H-NMR (DMSO-d6, 300
MHz, δ ppm): 10.41 (s, 1H, -NH-), 8.75 (s, 1H, -NH-), 8.00 (d, 2H, J = 8.1, Ar-H), 7.93
(d, 2H, J = 8.4, Ar-H), 7.53 (s, 2H, -SO2NH2), 3.80-3.43 (m, 12H, morpholine and
piperidine), 1.71-1.44 (m, 6H, piperidine) : 13C-NMR (DMSO-d6, 75 MHz, δ ppm):
166.7, 165.1, 156.7, 150.4, 146.6, 136.3, 128.4, 126.6, 66.5, 44.0, 43.7, 25.9, 24.8;
4-(3-(4,6-dimorpholino-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (7e). Yield:
87%; Color: white solid; mp: 298-3000C; FT-IR (cm-1): 3244, 3170, 1668, 1501
1
(asymmetric), 1339, 1165 (symmetric) (S=O), 1067; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 10.45 (s, 1H, -NH-), 8.72 (s, 1H, -NH-), 8.00 (d, 2H, J = 7.5, Ar-H), 7.91 (d, 2H, J

= 8.1, Ar-H), 7.52 (s, 2H, -SO2NH2), 3.79-3.65 (m, 8H, morpholine), 3.54-3.41 (m, 8H,
13
morpholine): C-NMR (DMSO-d6, 75 MHz, δ ppm): 166.4, 156.8, 150.2, 146.3, 136.6,
128.7, 126.2, 66.8, 43.5;
4-(3-(4-(methylamino)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)ureido)
0
benzenesulfonamide (7f). Yield: 78%; Color: white solid; mp: 239-241 C; FT-IR (cm-
1): 3404, 3334, 1667, 1515 (asymmetric), 1329, 1160 (symmetric) (S=O), 1088; 1H-NMR
(DMSO-d6, 300 MHz, δ ppm): 10.65 (s, 1H, -NH-), 9.08 (s, 1H, -NH-), 8.05 (d, 2H, J =
7.5, Ar-H), 7.85 (d, 2H, J = 7.2, Ar-H), 7.54 (s, 2H, -SO2NH2), 6.55 (s, 2H, -NHCH3),
3.42-3.29 (m, 4H, piperidine), 2.79-2.72 (m, 3H, -NHCH3), 1.72-1.52 (m, 6H,
13
piperidine): C-NMR (DMSO-d6, 75 MHz, δ ppm): 166.1, 164.5, 156.7, 150.8, 146.9,
136.2, 128.7, 126.5, 43.9, 28.1, 25.5, 24.2;
4-(3-(4-(dimethylamino)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)ureido)
benzenesulfonamide (7g). Yield: 86 %; Color: white solid; mp: 236-239 0C; FT-IR (cm-
1): 3322, 3170, 1695, 1503 (asymmetric), 1335, 1159 (symmetric) (S=O), 1094; 1H-NMR
(DMSO-d6, 300 MHz, δ ppm): 10.69 (s, 1H, -NH-), 9.05 (s, 1H, -NH-), 8.10 (d, 2H, J =
8.1, Ar-H), 7.83 (d, 2H, J = 7.2, Ar-H), 7.53 (s, 2H, -SO2NH2), 3.40-3.27 (m, 4H,
13
piperidine), 3.10 (s, 6H, -CH3), 1.75-1.52 (m, 6H, piperidine): C-NMR (DMSO-d6, 75
MHz, δ ppm): 166.4, 164.7, 156.5, 150.4, 146.6, 136.4, 128.2, 126.3, 43.7, 35.4, 25.7,
24.3;
4-(3-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-yl)ureido)
benzenesulfonamide
(7h).
0
Yield: 82 %; Color: white solid; mp: 233-236 C; FT-IR (cm-1): 3339, 3225, 1664, 1501
1
(asymmetric), 1327, 1169 (symmetric) (S=O), 1103; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 10.60 (s, 1H, -NH-), 9.01 (s, 1H, -NH-), 8.01 (d, 2H, J = 7.5, Ar-H), 7.79 (d, 2H, J

= 6.9, Ar-H), 7.52 (s, 2H, -SO2NH2), 3.41-3.29 (m, 8H, piperidine), 1.73-1.54 (m, 12H,
13
piperidine): C-NMR (DMSO-d6, 75 MHz, δ ppm): 166.3, 156.4, 150.2, 146.3, 136.5,
128.7, 126.4, 43.9, 25.3, 24.1;
4-(3-(4-amino-6-(dimethylamino)-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (7i).
0
Yield: 51 %; Color: white solid; mp: 222-225 C; FT-IR (cm-1): 3273, 3103, 1639, 1528
1
(asymmetric), 1334, 1161 (symmetric) (S=O), 1091; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 10.45 (s, 1H, -NH-), 8.82 (s, 1H, -NH-), 8.01 (d, 2H, J = 7.2, Ar-H), 7.93 (d, 2H, J
13
= 7.5, Ar-H), 7.53 (s, 2H, -SO2NH2), 6.60 (s, 2H, -NH2), 3.08 (s, 6H, -CH3): C-NMR
(DMSO-d6, 75 MHz, δ ppm): 166.1, 164.7, 156.6, 150.2, 147.5, 136.2, 129.3, 126.8,
35.6;
4-(3-(4-(dimethylamino)-6-(methylamino)-1,3,5-triazin-2-yl)ureido)
0
benzenesulfonamide (7j). Yield: 82 %; Color: white solid; mp: 245-248 C; FT-IR (cm-
1): 3425, 3312, 3213, 1661, 1535 (asymmetric), 1331, 1158 (symmetric) (S=O), 1093;
1H-NMR (DMSO-d6, 300 MHz, δ ppm): 10.48 (s, 1H, -NH-), 8.80 (s, 1H, -NH-), 8.03 (d,
2H, J = 6.9, Ar-H), 7.94 (d, 2H, J = 7.2, Ar-H), 7.54 (s, 2H, -SO2NH2), 6.50 (s, 2H, -
NHCH3), 3.12 (s, 6H, -CH3), 2.75-2.69 (m, 3H, -NHCH3): 13C-NMR (DMSO-d6, 75
MHz, δ ppm): 166.4, 164.5, 156.5, 150.2, 147.4, 136.8, 129.5, 126.3, 35.6, 28.7;
4-(3-(4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)ureido) benzenesulfonamide (7k).
0
Yield: 86 %; Color: white solid; mp: 262-264 C; FT-IR (cm-1): 3317, 3270, 1678, 1517
1
(asymmetric), 1339, 1162 (symmetric) (S=O), 1089; H-NMR (DMSO-d6, 300 MHz, δ
ppm): 10.47 (s, 1H, -NH-), 8.79 (s, 1H, -NH-), 8.05 (d, 2H, J = 6.9, Ar-H), 7.95 (d, 2H, J
= 7.5, Ar-H), 7.53 (s, 2H, -SO2NH2), 3.09 (s, 12H, -CH3): 13C-NMR (DMSO-d6, 75
MHz, δ ppm): 166.5, 156.7, 150.1, 147.8, 136.4, 129.3, 126.2, 35.8;

4.1.4. CA inhibition
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been
used to assay the catalytic/inhibition of various CA isozymes [19]. Phenol Red (at a
concentration of 0.2 mM) has been used as an indicator, working at the absorbance maximum of
557 nm, with 10 mM Hepes (pH 7.4) as a buffer, 0.1 M Na2SO4 or NaClO4 (for maintaining
constant the ionic strength; these anions are not inhibitory in the used concentration), following
the CA-catalyzed CO2 hydration reaction for a period of 5-10 s. Saturated CO2 solutions in water
at 25 °C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of
10 mM (in DMSO-water 1:1, v/v) and dilutions up to 0.01 nM done with the assay buffer
mentioned above. At least 7 different inhibitor concentrations have been used for measuring the
inhibition constant. Inhibitor and enzyme solutions were pre-incubated together for 10 min at
room temperature prior to assay, in order to allow for the formation of the E-I complex.
Triplicate experiments were done for each inhibitor concentration, and the values reported
throughout the paper is the mean of such results. The inhibition constants were obtained by
nonlinear least-squares methods using the Cheng-Prusoff equation, as reported earlier, and
represent the mean from at least three different determinations [20-22]. All CA isozymes used
here were recombinant proteins obtained as reported earlier by our group.
Acknowledgment
The authors would like to thank to TUBITAK (The Scientific and Technological
Research Council of Turkey) for financial support (grant no: 315S103).

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Graphical Abstract

Highlights




The synthesis of a series of novel ureido benzenesulfonamides incorporating 1,3,5-triazine
moieties was reported.
The ureido benzenesulfonamides incorporating 1,3,5-triazine derivatives were investigated as
hCA I, II, IX and XII inhibitors.
The derivatives showed to be subnanomolar to nanomolar inhibitors of hCA IX isozyme with Kis
in the range of 0.91 to 126.2 nM.
These derivatives showed some selectivity for hCA IX over hCA I, II and XII isoforms