Novel sulfonamides incorporating 1,3,5-triazine and amino acid structural motifs as inhibitors of the physiological carbonic anhydrase isozymes I, II and IV and tumor-associated isozyme IX

Article abstract of DOI:10.1016/j.bioorg.2018.08.005

A new series of thirty s-triazinyl-substituted aminoalkylbenzenesulfonamides, incorporating a symmetric pair of amino acid moieties, is reported, together with inhibition studies of physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isofo

Full text of DOI:10.1016/j.bioorg.2018.08.005

Accepted Manuscript
Novel sulfonamides incorporating 1,3,5-triazine and amino acid structural mo-
tifs as inhibitors of the physiological carbonic anhydrase isozymes I, II and IV
and tumor-associated isozyme IX
Peter Mikuš, Dominika Krajčiová, Mária Mikulová, Branislav Horváth, Daniel
Pecher, Vladimír Garaj, Silvia Bua, Andrea Angeli, Claudiu T. Supuran
PII:
S0045-2068(18)30641-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.08.005
YBIOO 2462
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 July 2018
27 July 2018
6 August 2018
Please cite this article as: P. Mikuš, D. Krajčiová, M. Mikulová, B. Horváth, D. Pecher, V. Garaj, S. Bua, A. Angeli,
C.T. Supuran, Novel sulfonamides incorporating 1,3,5-triazine and amino acid structural motifs as inhibitors of the
physiological carbonic anhydrase isozymes I, II and IV and tumor-associated isozyme IX, Bioorganic Chemistry
(2018), doi: https://doi.org/10.1016/j.bioorg.2018.08.005
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Novel sulfonamides incorporating 1,3,5-triazine and amino acid structural motifs as inhibitors of
the physiological carbonic anhydrase isozymes I, II and IV and tumor-associated isozyme IX
Glide
docking
model
of
2,2'-((6-((4-
sulfamoylphenethyl)amino)-1,3,5-triazine-2,4-
diyl)bis(imino))disuccinic acid associate with
carbonic anhydrase IX
Peter Mikuša,b,*, Dominika Krajčiováa, Mária Mikulováa, Branislav Horváthc, Daniel Pechera,b, Vladimír
Garajb,d*, Silvia Buae, Andrea Angelie, Claudiu T. Supurane
a
Comenius University in Bratislava, Faculty of Pharmacy, Department of Pharmaceutical Analysis and Nuclear Pharmacy,
Odbojárov
10,
SK-83232
Bratislava,
Slovak
Republic;
mikus@fpharm.uniba.sk,
mikulova43@uniba.sk,
krajciova.dominika@fpharm.uniba.sk
b
Comenius University in Bratislava, Faculty of Pharmacy, Toxicological and Antidoping Centre, Odbojárov 10, SK-83232
Bratislava, Slovak Republic; pecher1@uniba.sk
Comenius University in Bratislava, Faculty of Pharmacy, Department of Nuclear Magnetic Resonance, Odbojárov 10, SK-
c
83232 Bratislava, Slovak Republic; horvath@fpharm.uniba.sk
d
Comenius University in Bratislava, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Odbojárov 10, SK-83232
Bratislava, Slovak Republic; garaj1@uniba.sk
e
Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, University of Florence, Italy;
silvia.bua@unifi.it, andrea.angeli@unifi.it, claudiu.supuran@unifi.it
*Corresponding authors: Peter Mikuš, e-mail address: mikus@fpharm.uniba.sk; Vladimír Garaj, e-mail address: garaj1@uniba.sk
ABSTRACT
A new series of thirty s-triazinyl-substituted aminoalkylbenzenesulfonamides, incorporating a
symmetric pair of amino acid moieties, is reported, together with inhibition studies of physiologically
relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I,
II, transmembrane hCA IV and the tumor-associated, membrane-bound hCA IX.
The compounds were prepared by nucleophilic substitution of chlorine atoms from cyanuric
chloride (2,4,6-trichloro-1,3,5-triazine) using environmentally friendly water-based synthetic conditions.
The products yields ranged in the interval of 43-97%. Purity of the products was verified by the HPLC-
DAD-ESI-Q-TOF MS method. Identity of the products was confirmed by the same method plus NMR and
IR.
The products showed weak inhibition of the cytosolic, off-target isozyme hCA II, but some of them
were low nanomolar (i.e. strong) inhibitors of the tumor-associated hCA IX. The series offered

representatives selective towards isozymes hCA I, IV and IX. 2,2'-((6-((4-sulfamoylphenethyl)amino)-
1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid demonstrated highest selectivity to the tumor-associated
isoform hCA IX over off-target isozymes, with impressive KI ratio (hCA II / hCA IX) 213.9 and inhibition
constant equal to acetazolamide (KI = 25.8 nM). Although the selectivities of some other products, e.g.
those conjugating Leu and Glu, were a bit lower (188.7 and 84.3, respectively) their inhibition constants
were similar to acetazolamide too (24.0 and 27.1, respectively).
The selected most impressive results from the inhibition study were interpreted via molecular
modeling experiment (docking in Glide) revealing different inter-molecular enzyme-substrate interaction
of 2,2'-((6-((4-sulfamoylphenethyl)amino)-1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid within
specific hCA IX and hCA II microregions. Therefore, several selected compounds from this study can be
considered as highly effective and selective inhibitors of hCA IX, worthy to further (preclinical)
investigation.
Keywords:
1,3,5-Triazine
Benzene sulfonamides
Amino acids
Carbonic anhydrase
Enzyme inhibition
Isoform selectivity
1. Introduction
Carbonic anhydrases (CAs) are a family of zinc containing metalloenzymes catalyzing the reversible
hydration of carbon dioxide to bicarbonate ion and hydrogen proton. Sixteen CA isoforms have been well
defined in humans, from which transmembrane hCA IX have shown increased expression in hypoxia-
induced tumor cells [1, 2]. The upregulation of hypoxia inducible factor 1 (HIF-1) directly correlates with
hCA IX overexpression and activation in many tumor cells [3], including head and neck squamous cell
carcinoma [4], non-small lung [5] and breast cancer [6,7], astrocytic [8] and gastrointestinal tumors [9],
colorectal [10], renal [11] and cervical carcinoma [12,13]. Human CA IX plays a major role in tumor cell
proliferation, pH regulation, migration and adhesion, thus the suppression of hCA IX activity results in a
reduction of these processes and metastatic cascade [14]. Since hCA IX is not demonstrated in majority of
normal physiological tissues, but is associated with a wide variety of tumors as mentioned above, hCA IX
and the inhibition of its activity have become a promising and broadly studied target for therapy or
immunotherapy with monoclonal antibodies and for tumor hypoxia imaging.
In comparison with hCA IX, the hCA II isozyme is not regulated by HIF-1. Human CA II is
physiologically dominant and highly active cytosolic isoform [15]. It is present under physiological
conditions in almost every human tissue and organ [16]. Other physiologically relevant human carbonic
anhydrase isoforms include e.g. cytosolic hCA I, that is found at the highest level in erythrocytes and is
also expressed in normal colorectal mucosa [17, 18], or membrane-bound hCA IV, that is present in the
eye [19]. In order to eliminate the hypoxia-induced tumor cells with a minimum impact on the healthy
cells, a big effort is put on the selectivity enhancement of newly developed potential hCA inhibitors.
In recent years, many sulfonamides, phenols, inorganic anions, and coumarin derivatives have been
shaped as main classes of hCA inhibitors, including hCA IX and XII [20-24]. Predominantly benzene
sulfonamides, in which the incorporation of 1,3,5-triazine moiety with 4-aminobenzene sulfonamide has
offered improved specificity and efficacy targeting hCA IX and hCA XII, have become more frequently
explored by working group of Supuran. The conjugates created by an implementation of various moieties
into the 1,3,5-triazinyl benzene sulfonamide intermediates have been investigated in several consecutive
studies [25-30]. In 2005 [26] hydrazino, ethylamino, isopropylamino, N,N-diethylamino- or N-methyl-N-
propylamino- moieties were incorporated into the 1,3,5-triazinyl benzene sulfonamide intermediates
inhibiting hCA I with KIs 31-8500 nM, hCA II with KIs 14-765 nM and hCA IX with KIs 1-640 nM. In

2014 [27] ammonia, anilines, morpholine, tert-butylamine, N-methyl-2-aminoethanol and 1, 3-
diaminopropane were tested as nucleophiles for the substitution of the triazinyl moiety. Therein, hCA II
was moderately inhibited by most of the new compounds (KIs in the range of 12.5–130 nM), and hCA IX
and hCA XII were potently inhibited with KIs in the range of 1.2–34.1 and of 2.1–33.9 nM, respectively.
In 2011 Supuran et al. [26] employed in their synthetic procedures among various nucleophiles
(DOPA, 2-aminoethanol and 4-amino-1-butanol, mono-tert-butyl-dimethylsilyl derivative of
ethyleneglycol) also amino acids such as Gly, β-Ala, L-Ala, Ser as well as methyl esters of Gly and L-Ala.
This series contained a majority of monosubstituted derivatives and some disubstituted derivatives,
showing a moderate-weak inhibition of the cytosolic hCA I and II but a low nanomolar (i.e. strong)
inhibition activity of hCA IX, XII and XIV. The KI values for these isozymes were 4550, 376, 8.4, 6.0, 7.2
nM, respectively, for the 1,3,5-triazinyl aminobenzene sulfonamide derivatives disubstituted with Gly;
673, 368, 8.9, 0.85, 0.92 nM respectively, with β-Ala; and 502, 435, 9.4, 8.7, 7.0 nM, respectively, with
methylester of Gly. The KIs were 1659, 435, 8.5, 9.2, 8.9 nM, respectively, for the 1,3,5-triazinyl
aminomethylbenzene sulfonamide derivatives disubstituted with L-Ala; and 607, 453, 9.2, 9.3, 9.1 nM,
respectively, with methylester of Gly. They were 2040, 409, 8.4, 8.6, 8.8 nM, respectively, for the 1,3,5-
triazinyl aminoethylbenzene sulfonamide derivatives disubstituted with L-Ala; and 3704, 517, 9.0, 7.1, 7.6
nM, respectively, with Ser. In 2013 [29] the fluorine-substituted 1,3,5-triazine moiety was studied in the
reaction with amino acid such as glycine and its methyl ester providing mono-fluoro-substituted products.
Their inhibition studies revealed rather weak inhibition of hCA III, IV, V and XIII with KI in the range of
602-9015 nM, a moderate inhibition of hCA I, VI, and IX with KI in the range of 81.5-250 nM, and strong
inhibition of the physiologically relevant hCA II, VII and XII with KI 4.5-8.3 nM. It is apparent from the
above mentioned results that some conjugates created by an implementation of amino acids into the 1,3,5-
triazinyl aminoalkyl benzene sulfonamide intermediates could be potent inhibitors of the hCA isoforms
related to hypoxia-induced tumor cells. However, no systematic study on the amino acids has been carried
out in this context so far. Therefore, we decided to evaluate such amino acids conjugates in detail and
systematically, covering essential as well as non-essential amino acids and their derivatives. Incorporation
of amino acid moiety enhances water-solubility and was used in design of hundreds of CAIs [31]. General
structures of triazinyl derivatives incorporating amino acid moieties, that have been published so far, are
summarized in Fig. 1.
The aim of this work was to synthesize and characterize novel 1,3,5-triazine (amino-/aminomethyl-
/aminoethyl-)benzene sulfonamide conjugates disubstituted with 10 essential amino acids including (i)
less polar neutral amino acids as well as (ii) acidic amino acids. All amino acids used contained one amino
group as the only nucleophile in the molecule enabling selective chlorine substitution. The optimized
synthetic procedure in a water environment reported in our very recent paper [32] for 1,3,5-triazine
benzene sulfonamide conjugates disubstituted with Gly and β-Ala was adapted/optimized in this work for
the preparation of new amino acid conjugates. The inhibition data of the synthesized products with
approved chemical structures, i.e. the minimum inhibition concentrations towards hCA I, hCA II, hCA IV,
hCA IX and the selectivity expressed as a ratio of hCA IX : hCA II (or other relevant ratios), were
evaluated and discussed.

Figure 1. Published general structures of triazinyl derivatives incorporating amino acid moieties [31].
2. Results and Discussion
2.1 Chemistry
Final compounds (i.e. amino acids conjugates) were prepared in two steps by nucleophile
substitution of chlorine atoms in cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). For the preparation of
the key monosubstituted s-triazine intermediates (1-3) the previously reported protocol was used [25, 33]
Trisubstituted s-triazines (4-33) were prepared by the reaction of a corresponding intermediate (1-3) with
amino acid (present in its L-enantiomeric form, if applicable) and anhydrous sodium carbonate according
to Scheme 1. This water-based environmentally friendly synthetic procedure, accompanied with a simple
purification, provided very high yields for majority of the products.

Scheme 1. General scheme for the synthesis of target s-triazine derivates.
A series of the synthesized s-triazinyl-amino-/aminomethyl-/aminoethyl-benzenesulfonamide
conjugates substituted with amino acids, including (i) the group of less polar neutral amino acids as well
as (ii) acidic amino acids, was proved for the purity and chemical structure by the HPLC-DAD/MS, NMR,
and IR methods (for the analytical methods see section 4.1). The products were prepared as free acids with
exception of Pro that was present as sodium salt. The reaction yields, ranging in the interval of 42.8-
97.2%, depended on both (i) the length of the linker between sulfonamide and triazine (i.e. amino-,
aminomethyl-, aminoethyl-) and (ii) amino acid structure (for the particular data see section 4.2).
Generally, the prolongation of the linker resulted in the decreasing of the yield (average values for the
amino-, aminomethyl- and aminoethyl-derivatives were 89.2%, 77.3%, and 71.3%, respectively).
Although there was found a correlation between the length of the linker and the yield, the effect of amino
acid type seemed to be random. For example, the highest yields (calculated as an average of the all amino-
/aminoalkyl-benzenesulfonamide derivatives of given amino acid) were found for Gly (90.8%), Phe
(92.2%) and Leu (88.7%) conjugates while the lowest ones for Val (56.9%), Pro (72.3%) and Glu (70.2%)
conjugates. The HPLC-UV measurements did not indicate any significant samples impurities. The purity
of each conjugate was higher than 90% with the average value of the all samples 94.52%. The identity of
the all conjugates was unambiguously confirmed via exact molecular weights and additional structural
information documented by the data in section 4.2 and by Figs. S1-S30 in Supplementary material. Such
approved conjugates were then suitable for testing on their biological activities.
2.2 CA inhibition
Inhibition data against cytosolic hCA isoforms I and II as well as transmembrane hCA IV and IX
with the thirty original benzenesulfonamides with s-triazine moiety disubstituted with amino acids (4 –
33), as well as their intermediates (1 – 3) and the standard sulfonamide CAI acetazolamide (AAZ) are
shown in Table 1. They were obtained by a stopped-flow, CO2 hydrase assay [34]. The following
structure–activity relationship (SAR) data were observed, when considering inhibition data in Table 1 and
comparing them with previously reported inhibition data of intermediates 1–3 monosubstituted with
amino acids [26,27] summarized in Table 2:
(i) Most of the tested compounds 4-33 were ineffective as hCA I inhibitors. Compounds 12, 15 and
22, possessing Asp, Glu and Ile amino acid substituents, are inactive against hCA I. The other derivatives
with these amino acids are weak micromolar inhibitors as well as all Gly derivatives 4-6 and compounds
27 and 33 substituted with Met and Phe respectively and both with 4-aminoethylbenzenesulfonamide (n =
2) on s-triazine core. Substitution with Leu causes weak micromolar inhibition at compounds 19 (n = 0)
and 21 (n = 2), while the compound 20 with intermediate spacer (n = 1) is moderately effective inhibitor
with KI = 96.9 nM. Other effective hCA I inhibitors are compound 32 substituted with Phe and

homosulfanilamide (n = 1) with lowest KI = 67.1 nM, and compound 28 substituted with Pro and
sulfanilamide (n = 0) with KI = 87.0 nM. Compound 28 exhibits also high isozyme selectivity with
selectivity ratio hCA II / hCA I = 35.8. Much more selective hCA I inhibitor already exists [35], although
it happens rarely to find one for this isozyme. For derivatives substituted with Pro, prolongation of linker
between benzenesulfonamide and s-triazine core decreases the activity. Except the three aforementioned
active inhibitors of hCA I 20, 28, 32, disubstitution of Cl atoms with amino acids decreases the inhibition
activity. Monosubstitution of Cl on intermediates 2, 3 with amino acid can increase the activity (Tab. 2;
compounds X2, X3, X5 and X8) [27].
(ii) The investigated sulfonamides showed to be weak inhibitors of hCA II, the physiologically
dominant and highly active cytosolic isoform [15], with KIs in the range of 235.7–7125 nM.
Disubstitution of intermediate 1 with Gly decreases inhibition activity 3.5 times (4) and approximately as
much if any intermediate (1-3) is monosubstituted with amino acid (Tab. 2). A very steep decline of
inhibition activity is observed for the rest of compounds 5-33. Among derivatives substituted with
hydrophobic amino acids Val, Leu, Ile or with Pro, the weakest inhibitors of hCA II are those with
sulfanilamide substituent (n = 0). On the contrary, among derivatives substituted with aromatic amino acid
Phe, the least active inhibitor is compound 33 with long spacer (n = 2). Disubstitution with acidic amino
acids Asp and Glu decreases effectiveness towards hCA II to micromolar KIs except compound 14 with KI
= 0.7 µM.
(iii) The membrane-associated isoform hCA IV was inhibited by the tested sulfonamides 4–33, with
inhibition constants in a wide range. The activity entirely diminished with disubstitution by acidic amino
acid Asp. If disubstituted with Glu, only compound 14 with intermediate linker (n = 1) retained weak
activity (KI = 4.1 µM). For hydrophobic amino acids Val and Leu, increasing the length of linker increases
also the inhibition activity against hCA IV. For derivatives with all other amino acid substituents
including Ile is applicable the same SAR as for Glu derivatives, meaning that compounds with
homosulfanilamide (n = 1) have the best inhibition activity. The best hCA IV inhibitor is compound 26
with Met (KI = 45.6 nM) which is also selective hCA IV inhibitor, as the selectivity ratios hCA I / hCA IV
= 11.7, hCA II / hCA IV = 69.5 and hCA IX / hCA IV = 28.0. Compounds 23 and 32 have inhibition
activity towards hCA IV comparable with AAZ (with KIs 76.3 and 61.9 resp.).
(iv) The tumor-associated hCA IX was the most inhibited isoform among the four investigated
isozymes, with KIs ranging between 8.4 and 2592.4ꢀnM (Tab. 1). The most active hCA IX inhibitor among
the intermediate products 1-3, with chlorine atoms at positions 3 and 5 on s-triazine ring, is compound 1
with sulfanilamide substituent (n = 0) [32]. The same applies to derivatives where both chlorine atoms are
substituted with Gly, β-Ala, Leu or Ile. On the contrary, disubstitution with Val, Pro and acidic amino
acids Asp and Glu demands long linker (n = 2) to retain high activity. In the case of the acidic amino acids
Asp and Glu, prolonging in the linker provides significant effect on the inhibition of hCA IX while there is
almost no effect on the inhibition of hCA II. This makes the compounds 12 and 15 also very selective
towards hCA IX.. Compounds 4 and 7 with Gly and β-Ala substituents and short linker (n = 0) are strong
hCA IX inhibitors with KIs 8.4 and 8.9 nM respectively and selectivity ratio against hCA II >40.
Monosubstituted intermediate 1 with Gly seems to have the same properties towards all tested CAs (Tab.
2; compound X1) [27] as disubstituted compound 4 (Tab. 1). While monosubstitution on intermediates 2
and 3 with Gly strongly increases inhibition activity and selectivity towards hCA IX (Tab. 2; compounds
X1, X2), subsequent substitution of last chlorine atom with Gly cancels this effect (5, 6). Good inhibitors
are also compounds 12, 15, 19 and 30 with KIs in the range 24 – 27 nM, comparable with AAZ. All four
compounds are selective towards isozyme hCA IX and the selectivity of compound 12 is the highest
having selectivity ratio hCA II / hCA IX = 214 (hCA II / hCA IX selectivity was confirmed also by
molecular docking in Glide, see chapter 2.3) and being inactive against isozymes hCA I and IV.
Table 1. Inhibition data for synthesized s-triazine derivatives 4-33 and the standard sulfonamide inhibitor
acetazolamide (AAZ) against hCA I, hCA II, hCA IV and hCA IX; their selectivity ratios for inhibition of
isozyme hCA IX over hCA II. (Methodology based on stopped flow assay was employed for the
evaluation [34]; see section 4.3 Carbonic anhydrase inhibition assay)

KI* (nM)
hCA II hCA IV hCA IX hCA II / hCA IX
selectivity
Compound
n
-R
hCA I
250.0
AAZ
1
12.0
74.0
NT
NT
NT
25.8
0.15 a
124.0 a
138.0 a
8.4
145.8
330.4
8.9
1818.5
134.2
211.9
2364.1
25.8
202.4
193.2
27.1
2111.1
1371.8
130.9
24.0
167.3
123.8
92.6
164.3
189.3
2222.2
1274.9
2592.4
295.0
265.5
25.7
0.5
706.7
0.1
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
0
1
2
Cl
120.0a
136.0 a
75.0 a
106.0 a
13.0 a
21.0 a
2
Cl
3
Cl
0.2
4
Gly
Gly
Gly
β-Ala
β-Ala
β-Ala
Asp
Asp
Asp
Glu
Glu
Glu
Val
Val
Val
Leu
Leu
Leu
Ile
4550.0
9362.4
4023.8
673.0
655.4
960.7
8259.8
3700.0
>10000
9260.8
6214.1
>10000
398.7
377.0
932.2
4191.9
96.9
4854.5
>10000
664.5
7337.2
346.7
531.3
376.0 >10000
44.8
3.3
1.3
41.3
0.4
6.6
29.3
2.5
213.9
35.2
3.6
84.3
2.5
5
478.4
428.1
368.0
661.6
867.3
3415.2
9596.0
4476.0
6
7
8
9
892.1 >10000
6219.1 >10000
5953.7 >10000
5519.6 >10000
7125.0 >10000
4125.8
2284.3 >10000
5335.4 >10000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
695.2
839.3
804.1
4528.8
396.0
912.5
2948.9
628.5
1556.2
803.7
3170.6
5017.9
3112.8
773.1
1070.8
866.1
656.8
476.3
2380.9
516.8
367.1
4021.2
76.3
1421.3
3294.0
45.6
2336.1
9315.0
350.4
2206.9
9387.0
61.9
0.6
6.1
188.7
2.4
7.4
31.8
3.8
Ile
Ile
8.2
Met
Met
Met
Pro
Pro
Pro
Phe
Phe
Phe
0.4
2.5
4527.8
87.0
1.9
10.6
2.9
256.4
958.4
41.7
4.5
305.4
191.5
119.6
223.1
67.1
235.7
6161.8
2.0
27.6
4893.8
374.5
NT = not tested
a From ref. 25
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10% of the
reported values).
Table 2. Inhibition data for s-triazine derivatives monosubstituted with amino acids against hCA I, hCA
II, and hCA IX reported in previous works [26,27]; their selectivity ratios for inhibition of isozyme hCA
IX over hCA II.
KI* (nM)
hCA II
351 a
29 b
selectivity
Compound
n
-R1 -R2
hCA II / hCA IX
hCA I
3032 a
35 b
hCA IX
7.9 a
X1
0
1
2
0
2
0
1
2
Gly Cl
Gly Cl
Gly Cl
44
17
32
X2
1.7 b
X3
33 b
32 b
1.0 b
0.96 a
1.0 a
X4
Ala
Ala
Ser
Cl
Cl
Cl
1324 a
33 a
561 a
32 a
584
32
X5
X6
109 a
2360 a
31 b
412 a
258 a
28 b
8.9 a
12
X7
β-Ala Cl
β-Ala Cl
34.1 a
1.2 b
7.6
23
X8
a From ref. 27

b From ref. 26
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10% of the
reported values) [26,27].
2.3 Molecular modeling
The docking of compound 12 into the human isozymes II and IX showed interaction with residues in
the same region as similar compounds docked by Saluja et al. [28]. When compared with crystal structure
of methyl N-{4-chloro-6-[(4-sulfamoylphenyl)amino]-1,3,5-triazin-2-yl}glycinate complexed to hCA II
(Fig. 2) [27], it is obvious that substitution of chlorine atom on s-triazine with bulkier amino acid prevents
the compound 12 from π-π interaction of s-triazine with Phe-131 and moves it closer to Trp-5 and His-64
(marked as HIE 64 in the Fig. 2). The weak H-bond between Cl and Gln-92 residue is substituted with
stronger H-bond provided by carboxylic group of compound 12.
Figure 2. Superposition of molecule 12 (light blue carbons) docked in the active site of hCA II and crystal
structure of N-{4-chloro-6-[(4-sulfamoylphenyl)amino]-1,3,5-triazin-2-yl}glycinate (white carbons)
complexed with hCA II (PDB ID: 3MNA) [27].
The pose of molecule 12 in hCA IX is slightly different from the pose in hCA II (Fig. 3, 4). While
Arg-60 residue in hCA IX creates H-bond with carboxylic group of 12 (1.86 Ǻ), Leu-60 residue in hCA II
is repulsive due to steric hindrance and lipophilic character. This repulsion prevents the other carboxylic
groups to create H-bonds with Arg-58 and Asn-62 residues. This explains the higher affinity of the
compound towards isozyme IX.

The computed scores were in agreement with the experimentally found selectivity. The docking score
of the molecule 12 in hCA IX was -8.33 while in hCA II only -4.35.
Figure 3. Superposition of molecule 12 docked in the active site of hCA IX (light blue carbons of all
associating groups) creating H-bond with Arg-60 and the same molecule docked in the active site of hCA
II (green carbons of all associating groups) repulsed by Leu-60.

Figure 4. 2D interaction diagram of compound 12 dockes into the active site of hCA IX (upper panel) and
hCA II (lower panel).
3. Conclusion
A new set of thirty triazinyl-substituted benzenesulfonamides, incorporating a symmetric pair of
amino acid moieties was prepared by easy, environment friendly procedure with high yields. The
inhibition studies of physiologically relevant hCA isoforms, such as hCA I, II, IV and tumor-associated
hCA IX with the set of sulfonamide inhibitors are reported. The results demonstrate that the tested
compounds showed only modest activity as inhibitors of hCA I (KIs ≥ 67.1 nM) and hCA IV (KIs ≥ 45.6
nM) and their activity towards hCA II was weak (KIs in the range 235.7 – 7125 nM). However, this set of
compounds showed high diversity in isozyme selectivity, offering selective hCA I inhibitor 28 with two
Pro residues, selective hCA IV inhibitor 26 with two Met residues and selective hCA IX inhibitors 15, 19
and 12 with two Glu, Leu and Asp residues, respectively (selectivity ratios hCA II / hCA IX were 84, 189
and 214, respectively). Best inhibitors of hCA IX, considering KI values, are compounds 4 and 7 (KIs with
values 8.4 and 8.9 nM, respectively, and selectivity against physiologically most relevant isozyme hCA II
over 40). Undoubtedly, the presented findings will stimulate a further more detail investigation of some of
these new highly potent hCA inhibitors. In a continuation of the structure – activity study, following
valuable modifications are proposed for this group of conjugates: (i) the substitution of triazinyl-
aminoalkyl-benzenesulfonamides with other amino acids (and their derivatives) and (ii) peptides, and,
especially, (iii) complexing such conjugates (ligands) with metal cations [36]. With the selectivity and
capability to chelate radioactive metal isotopes by free carboxylate groups, also some derivatives from this
study could constitute excellent leads for the development of new, unconventional group of anticancer
drugs or diagnostics targeting hypoxia-induced CA isoforms such as CA IX. For example, compound 12
has good inhibition activity (KI = 25.8 nM) and high selectivity towards hCA IX (selectivity ratio hCA II /
hCA IX = 214) and possesses four carboxylic groups that can serve as chelating groups for radioactive
metal isotopes. Such metal complexes could constitute new class for treatment/diagnostics of hypoxic
tumors and will be the subject of our further investigations.
4. Experimental section
4.1 Experimental materials and methods
All reagents and solvents were purchased from AppliChem GmbH (Darmstadt, Germany) and
Sigma Aldrich (St.Louis, MO, USA) and used without further purification. All the reactions were
monitored by the thin-layer chromatography (TLC) with the UV visualization (245 nm) using Merck
Silica gel plates 60 F254 as a stationary phase and hexane/ethyl acetate or dichloromethane/methanol as
13
mobile phases. The nuclear magnetic resonance (1H-NMR, C-NMR) spectra were recorded on a Varian
MERCURY plus 300 MHz spectrometer at 90 °C using DMSO-d6 as a solvent and tetramethylsilane
(TMS) as an internal standard. Chemical shifts are reported in parts per million (ppm). Splitting patterns
are designated as follows: s- singlet, d- doublet, t- triplet, q- quartet, m- multiplet, br-broad and dd-doublet
of doublet. Infrared (IR) spectra (in KBr plates) were recorded on Perkin-Elmer UATR Two (PerkinElmer
Ltd., Beaconsfield, UK). The measured data are expressed in wavenumbers (cm-1) and the signal strength
is given as weak (w), medium (m) or strong (s). All of the HPLC-UV/MS measurements were performed
on an LC Agilent Infinity System (Agilent Technologies, Santa Clara, CA, USA) equipped with a gradient
pump (1290 Bin Pump VL), an automatic injector (1260 HiPals), and a column thermostat (1290 TCC).
Zorbax Extend-C18 column, 2.1x50 mm, 1.7 µm (Agilent Technologies, Santa Clara, CA, USA) and
SeQuant® ZIC®-HILIC column, 2.1x100 mm, 3.5 µm (Merck KGaA, Darmstadt, Germany), were
employed for the HPLC analyses. The LC system was coupled with a photodiode array detector (Infinity
1290 DAD) and a quadrupole time-of-flight mass spectrometer (6520 Accurate Mass Q-TOF LC/MS). Q-

TOF was equipped with an electrospray ionization source operated in positive ionization mode. All the
samples were dissolved in 20% acetonitrile and filtered through 0.22 µm nylon syringe filter before the
HPLC-HRMS analyses. The HPLC-HRMS methods were optimized with respect to particular groups of
the analyzed compounds, see Table 3. The flow rate was set at 0.5 mL/min and 1.0 µL of the sample was
injected into the column. Gradient elution was used with the column oven temperature 35 °C. All
measurements were performed with following MS parameters: drying gas temperature 350 °C, drying gas
flow 10 L.min-1, nebulizing gas pressure 60 psi, ESI source voltage 3500 V, fragmentor voltage 140 V,
collision gas N2.
Table 3. The HPLC-HRMS methods used for the analyses of the amino acid conjugates 4-33.
Mobile
phase B
Analyzed compound
Majority of compounds
Compounds 9 and 25
Column type
Mobile phase A
0.1% formic acid in demineralized
water
10 mM ammonium acetate in
demineralized water with pH 6.8
10 mM ammonium acetate in
demineralized water with pH 6.8
acetonitrile
Zorbax Extend-C18
Zorbax Extend-C18
SeQuant® ZIC®-HILIC
acetonitrile
acetonitrile
Dicarboxylic compounds
10-15
4.2 Synthetic chemistry and products characterization
4.2.1 General procedure for the synthesis
of
4-(4´,6´-dichloro-1´,3´,5´-triazine-2´-
ylamino/aminomethyl/aminoethyl)benzenesulfonamide intermediates 1-3 [27]
The 1.0 M solution of 4-amino-/aminomethyl-/aminoethyl-benzenesulfonamide (1 equiv) in acetone (100
ml) was added dropwise into a vigorously stirred suspension of cyanuric chloride (1 equiv) in acetone
(100 ml) at 0°C. The white reaction mixture was stirred at the same temperature for 30 min. After then the
1.7 M aqueous solution of NaOH (1 equiv) was added during a period of 20 min. Stirring was continued
for 1 h, and the reaction was quenched by the addition of slush (100 mL). After reaction was completed,
the resulted solid was filtered off, washed and dried under high vacuum. The product was crystalized from
acetone affording the compounds 1-3 as a white solid.
4.2.2 General procedure for the synthesis of 2,2´-((6-(4-Sulfamoylphenyl-/benzyl-/phenethyl-amino)-1,3,5-
triazine-2,4-diyl)bis(imino)) conjugates with monocarboxylic acids 4-9, 16-33
Reaction
mixture
containing
4-(4´,6´-dichloro-1´,3´,5´-triazine-2´-
ylamino/aminomethyl/aminoethyl)benzenesulfonamide (0.2 g, 1 equiv) and amino acid (3 equiv) was
stirred in H2O (2 mL) at room temperature for 10 min. The aqueous solution of Na2CO3 (4 equiv) was
added dropwise into the reaction mixture. Then the mixture was refluxed for 24 h and evaluated by TLC
during the reaction. The synthesis was terminated by 1M HCl addition (up to pH=2.0-4.5, depending on
amino acid) until a maximum amount of precipitate was produced. The product was isolated by filtration
and dried under high vacuum. After then the product purity and structural characterization were
established by a combination of IR spectra, HPLC-UV/MS and NMR (for the recorded spectra see Figures
S1-S24 in the Supplementary material).
4.2.2.1 2,2´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))diacetic acid 4 [32]: White
solid; yield 96.4%. IR (KBr, cm-1): 2968 (w), 1683 (m), 1598 (m), 1507 (s), 1407 (s), 1159 (s). 1H NMR
(300 MHz, DMSO-d6, 90 °C) δ: 12.10 (br s, 2H, COOH), 9.17 (s, 1H, Ar-NH), 7.89 (d, 2H, J = 8.9 Hz,
Ar-H(a)), 7.67 (d, 2H, J = 8.9 Hz, Ar-H(b)), 6.94 (br s, 4H, SO2NH2, 2 x CH2-NH), 3.95 (d, 4H, J = 5.2
Hz, 2 x CH2-NH); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 172.02, 166.23, 164.39, 144.03, 136.91,
126.65, 119.32, 42.69. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C13H15N7SO6H]+ 398.0877; Found:
398.0878.

4.2.2.2 2,2´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))diacetic acid 5:
White solid; yield 97.1%. IR (KBr, cm-1): 3288 (w, NH), 1709 (w, C=O), 1621 (s), 1557 (s), 1399 (m),
1331 (m, SO2NH2), 1152 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.77 (d, 2H, J = 8.3 Hz,
2 x Ar-H(a)), 7.68 (br s, 1H, Ar-NH), 7.47 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.13 (br s, 2H, NH-CH), 7.02
(s, 2H, SO2NH2), 4.50 (d, 2H, J = 3.6 Hz, NH-CH2), 3.92 (s, 4H, 2 x H-C(2)); 13C NMR (75 MHz,
DMSO-d6, 90 °C) δ: 171.51, 164.62, 163.74, 144.22, 143.24, 128.22, 126.15, 43.79, 42.70. HRMS
(ESI/QTOF, m/z): [M + H]+ Calcd. for [C14H17N7SO6H]+ 412.1034; Found: 412.1036.
4.2.2.3 2,2´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))diacetic acid 6:
White solid; yield 78.9%. IR (KBr, cm-1): 3288 (m, NH), 1716 (w, C=O), 1620 (s), 1557 (s), 1514 (s),
1407 (m), 1308 (m, SO2NH2), 1150 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.74 (d, 2H, J
= 8.2 Hz, 2 x Ar-H(a)), 7.39 (d, 2H, J = 8.2 Hz, 2 x Ar-H(b)), 7.01 (s, 2H, SO2NH2), 6.61 (br s, 3H, 3 x
13
Ar-NH), 3.90 (d, 4H, J = 3.1 Hz, 2 x H-C(2)), 3.46 (m, 2H, NH-CH2), 2.89 (m, 2H, CH2-Ar); C NMR
(75 MHz, DMSO-d6, 90 °C) δ: 172.05, 165.52, 150.49, 144.42, 142.56, 129.40, 126.18, 42.92, 42.70,
35.57. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C15H19N7SO6H]+ 426.1190; Found: 426.1191.
4.2.2.4 3,3´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))dipropanoic acid 7 [32]:
White solid; yield 94%. IR (KBr, cm-1): 3290 (w, NH), 2967 (m), 1717 (w, C=O), 1630 (m), 1592 (s),
1509 (s), 1399 (m), 1163 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 9.00 (br s, 1H, Ar-NH),
7.92 (d, 2H, J = 8.8 Hz, 2 x Ar-H(a)), 7.66 (d, 2H, J = 8.8 Hz, 2 x Ar-H(b)), 6.88 (s, 2H, SO2NH2), 6.57
(br s, 2H, 2 x NH-CH), 3.51 (dt, 4H, J = 7.1 Hz, J = 5.8 Hz, 2 x H-C(3)), 2.52 (t, 4H, J = 7.1 Hz, 2 x H-
13
C(2)); C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.24, 166.30, 164.62, 144.33, 136.72, 126.71, 119.10,
36.95, 34.72. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C15H19N7SO6H]+ 426.1190; Found:
426.1190.
4.2.2.5 3,3´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))dipropanoic acid 8:
White solid; yield 73.7%. IR (KBr, cm-1): 3430 (m, NH), 3266 (w, NH), 1667 (m), 1634 (s), 1551 (s),
1
1521 (m), 1408 (m), 1329 (m, SO2NH2), 1149 (s, SO2NH2). H NMR (300 MHz, DMSO-d6, 90 °C) δ:
7.74 (d, 2H, J = 8.1 Hz, 2 x Ar-H(a)), 7.45 (d, 2H, J = 8.1 Hz, 2 x Ar-H(b)), 6.99 (s, 2H, SO2NH2), 6.92
(br s, 1H, Ar-NH), 6.21 (br s, 2H, 2 x NH-CH), 4.47 (d, 2H, J = 6.2 Hz, NH-CH2), 3.42 (dt, 4H, J = 7.1
Hz, J = 5.5 Hz, 2 x H-C(3)), 2.45 (t, 4H, J = 7.1 Hz, 2 x H-C(2)); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ:
173.32, 166.34, 166.18, 145.37, 142.94, 127.98, 126.00, 43.70, 36.80, 34.81. HRMS (ESI/QTOF, m/z):
[M + H]+ Calcd. for [C16H21N7SO6H]+ 440.1347; Found: 440.1349.
4.2.2.6 3,3´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))dipropanoic acid 9:
White solid; yield 50.6%. IR (KBr, cm-1): 3301 (m, NH), 2939 (m), 1723 (w, C=O), 1622 (m), 1548 (s),
1
1408 (m), 1331 (m), 1307 (m, SO2NH2), 1153 (s, SO2NH2). H NMR (300 MHz, DMSO-d6, 90 °C) δ:
7.75 (d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.39 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.00 (s, 2H, SO2NH2), 6.42
(br s, 1H, Ar-NH), 6.26 (br s, 2H, 2 x NH-CH), 3.46 (m, 6H, NH-CH2, 2 x H-C(3)), 2.90 (m, 2H, CH2-
Ar), 2.48 (t, 4H, J = 7.1 Hz, 2 x H-C(2)); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.32, 165.88,
165.81, 144.52, 142.55, 129.39, 126.19, 41.78, 36.81, 35.72, 34.81. HRMS (ESI/QTOF, m/z): [M + H]+
Calcd. for [C17H23N7SO6H]+ 454.1503; Found: 454.1506.
4.2.2.7 2,2´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-methylbutanoic acid)
16:
White solid; yield 80.6%. IR (KBr, cm-1): 3262 (w, NH), 2967 (w), 1717 (w, C=O), 1622 (m), 1590 (m),
1
1558 (s), 1497 (s), 1404 (m), 1327 (m, SO2NH2), 1154 (s, SO2NH2). H NMR (300 MHz, DMSO-d6, 90
°C) δ: 9.22 (br s, 1H, Ar-NH), 7.90 (d, 2H, J = 8.9 Hz, 2 x Ar-H(a)), 7.70 (d, 2H, J = 8.9 Hz, 2 x Ar-
H(b)), 6.93 (br s, 2H, SO2NH2), 6.68 (br s, 2H, 2 x NH-CH), 4.42 (dd, 2H, J = 8.1 Hz, J = 6.0 Hz, 2 x H-

C(2)), 2.17 (m, 2H, 2 x H-C(3)), 0.98 (d, 6H, J = 6.8 Hz, 2 x CH3), 0.97 (d, 6H, J = 6.8 Hz, 2 x CH3); 13C
NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.44, 165.42, 163.81, 143.57, 137.37, 126.77, 119.65, 59.32,
30.44, 19.47, 18.85. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C19H27N7SO6H]+ 482.1820; Found:
482.1821.
4.2.2.8 2,2´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-methylbutanoic acid)
17:
White solid; yield 42.8%. IR (KBr, cm-1): 3283 (w, NH), 2967 (w), 1717 (w, C=O), 1622 (s), 1558 (s),
1471 (w), 1394 (w), 1328 (m, SO2NH2), 1156 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.77
(d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.61 (br s, 1H, Ar-NH), 7.48 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.02 (s,
2H, SO2NH2), 6.78 (br s, 2H, 2 x NH-CH), 4.52 (br s, 2H, NH-CH2), 4.35 (m, 2H, 2 x H-C(2)), 2.12 (m,
13
2H, J = 6.6 Hz, 2 x H-C(3)), 0.93 (d, 6H, J = 6.8 Hz, 2 x CH3), 0.92 (d, 6H, J = 6.8 Hz, 2 x CH3); C
NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.25, 164.20, 163.29, 144.29, 143.26, 128.15, 126.09, 59.25,
43.81, 30.36, 19.39, 18.71. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C20H29N7SO6H]+ 496.1973;
Found: 496.1975.
4.2.2.9 2,2´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-methylbutanoic
acid) 18:
White solid; yield 47.4%. IR (KBr, cm-1): 3284 (w, NH), 2966 (w), 1717 (w, C=O), 1622 (s), 1558 (s),
1471 (w), 1395 (w), 1331 (m, SO2NH2), 1157 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.75
(d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.41 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.02 (s, 3H, SO2NH2, Ar-NH),
6.73 (br s, 2H, 2 x NH-CH), 4.37 (dd, 2H, J = 7.7 Hz, J = 5.9 Hz, 2 x H-C(2)), 3.51 (dt, 2H, J = 7.4 Hz, J
= 7.2 Hz, NH-CH2), 2.91 (t, 2H, J = 7.4 Hz, CH2-Ar), 2.13 (m, 2H, 2 x H-C(3)), 0.95 (d, 12H, J = 6.9 Hz,
4 x CH3); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.32, 163.79, 163.17, 144.14, 142.69, 129.43,
126.18, 59.29, 46.25, 35.37, 30.42, 19.40, 18.77. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for
[C21H31N7SO6H]+ 510.2129; Found: 510.2131.
4.2.2.10 2,2´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(4-methylpentanoic acid)
19:
White solid; yield 92.3%. IR (KBr, cm-1): 3263 (br, w, NH), 2959 (w), 1717 (w, C=O), 1623 (m), 1590
(m), 1558 (s), 1496 (s), 1404 (m), 1318 (m, SO2NH2), 1154 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6,
90 °C) δ: 9.07 (br s, 1H, Ar-NH), 7.91 (d, 2H, J = 8.8 Hz, 2 x Ar-H(a)), 7.68 (d, 2H, J = 8.8 Hz, 2 x Ar-
H(b)), 6.91 (br s, 2H, SO2NH2), 6.76 (d, 2H, J = 8.2 Hz, 2 x NH-CH), 4.53 (dt, 2H, J = 8.2 Hz, J = 6.6 Hz,
2 x H-C(2)), 1.81-1.56 (m, 6H, 2 x H-C(4), 2 x H-C(3)), 0.93 (d, 6H, J = 6.4 Hz, 2 x CH3), 0.90 (d, 6H, J
= 6.4 Hz, 2 x CH3); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 174.77, 165.78, 164.19, 143.91, 137.09,
126.72, 119.41, 52.28, 25.06, 23.20, 22.09. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for
[C21H31N7SO6H]+ 510.2129; Found: 510.2137.
4.2.2.11 2,2´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(4-methylpentanoic acid)
20:
White solid; yield 87.9%. IR (KBr, cm-1): 3263 (br, w, NH), 2957 (w), 1723 (w, C=O), 1622 (s), 1539 (s),
1456 (w), 1403 (w), 1333 (m, SO2NH2), 1157 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.75
(d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.46 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.32 (br s, 1H, Ar-NH), 7.01 (br s,
2H, SO2NH2), 6.71 (br s, 2H, 2 x NH-CH), 4.56-4.42 (m, 4H, 2 x H-C(2), NH-CH2), 1.76-1.51 (m, 6H, 2
x H-C(3), 2 x H-C(4)), 0.89 (d, 6H, J = 6.4 Hz, 2 x CH3), 0.86 (d, 6H, J = 6.4 Hz, 2 x CH3); 13C NMR (75
MHz, DMSO-d6, 90 °C) δ: 174.66, 164.74, 164.52, 144.66, 143.15, 128.11, 126.08, 52.24, 43.70, 24.98,
23.15, 22.10. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C22H33N7SO6H]+ 524.2286; Found:
524.2294.

4.2.2.12 2,2´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(4-methylpentanoic
acid) 21:
White solid; yield 85.8%. IR (KBr, cm-1): 3286 (w, NH), 2958 (m), 1723 (w, C=O), 1622 (s), 1557 (s),
1470 (w), 1441 (w), 1334 (m, SO2NH2), 1157 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.75
(d, 2H, J = 8.2 Hz, 2 x Ar-H(a)), 7.41 (d, 2H, J = 8.2 Hz, 2 x Ar-H(b)), 7.02 (br s, 5H, SO2NH2, 2 x Ar-
NH, NH-CH2), 4.48 (m, 2H, NH-CH2), 3.50 (br s, 2H, 2 x H-C(2)), 2.91 (t, 2H, J = 7.4 Hz, CH2-Ar),
1.78-1.54 (m, 6H, 2 x H-C(3), 2 x H-C(4)), 0.92 (d, 6H, J = 6.4 Hz, 2 x CH3), 0.89 (d, 6H, J = 6.4 Hz, 2 x
13
CH3); C NMR (75 MHz, DMSO-d6, 90 °C) δ: 174.44, 164.08, 163.36, 144.12, 142.70, 129.42, 126.21,
52.35, 35.36, 25.00, 23.15, 22.09. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C23H35N7SO6H]+
538.2442; Found: 538.2450.
4.2.2.13 2,2´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-methylpentanoic acid)
22:
White solid; yield 79.9%. IR (KBr, cm-1): 3266 (w, NH), 2966 (w), 1717 (w, C=O), 1623 (m), 1590 (m),
1
1558 (s), 1497 (s), 1406 (m), 1328 (m, SO2NH2), 1154 (s, SO2NH2). H NMR (300 MHz, DMSO-d6, 90
°C) δ: 9.21 (br s, 1H, Ar-NH), 7.91 (d, 2H, J = 8.8 Hz, 2 x Ar-H(a)), 7.70 (d, 2H, J = 8.9 Hz, 2 x Ar-
H(b)), 6.93 (br s, 2H, SO2NH2), 6.68 (br s, 2H, 2 x NH-CH), 4.48 (m, 2H, 2 x H-C(2)), 1.92 (m, 2H, 2 x
H-C(3)), 1.52 (m, 2H, H-C(4)), 1.28 (m, 2H, H-C(4)), 0.95 (d, 6H, J = 6.8 Hz, 2 x C(3)-CH3), 0.90 (t, 6H,
J = 7.4 Hz, 2 x H-C(5)); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.44, 165.33, 163.83, 143.57, 137.41,
126.74, 119.68, 58.26, 37.04, 25.59, 16.01, 11.64. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for
[C21H31N7SO6H]+ 510.2129; Found: 510.2133.
4.2.2.14 2,2´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-methylpentanoic acid)
23:
White solid; yield 89.3%. IR (KBr, cm-1): 3284 (w, NH), 2966 (w), 1716 (w, C=O), 1622 (s), 1557 (s),
1463 (w), 1408 (w), 1334 (m, SO2NH2), 1157 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.76
(d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.66 (br s, 1H, NH-CH2), 7.48 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.02 (s,
2H, SO2NH2), 6.84 (br s, 2H, 2 x NH-CH), 4.52 (br s, 2H, NH-CH2), 4.43 (m, 2H, 2 x H-C(2)), 1.87 (m,
2H, 2 x H-C(3)), 1.49 (m, 2H, H-C(4)), 1.47 (m, 2H, H-C(4)), 0.90 (d, 6H, J = 7.0 Hz, 2 x C(3)-CH3),
13
0.86 (t, 6H, J = 7.5 Hz, 2 x H-C(5)); C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.22, 163.68, 162.47,
144.22, 143.26, 128.16, 126.12, 58.19, 43.77, 37.03, 25.50, 15.96, 11.61. HRMS (ESI/QTOF, m/z): [M +
H]+ Calcd. for [C22H33N7SO6H]+ 524.2286; Found: 524.2290.
4.2.2.15 2,2´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-methylpentanoic
acid) 24:
White solid; yield 84.6%. IR (KBr, cm-1): 3265 (w, NH), 2965 (w), 1719 (w, C=O), 1621 (s), 1551 (s),
1459 (w), 1406 (w), 1332 (m, SO2NH2), 1157 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.75
(d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.41 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.02 (br s, 3H, SO2NH2, Ar-NH),
6.72 (br s, 2H, 2 x NH-CH), 4.43 (m, 2H, 2 x H-C(2)), 3.51 (m, 2H, NH-CH2), 2.91 (m, 2H, CH2-Ar),
1.89 (m, 2H, 2 x H-C(3)), 1.49 (m, 2H, H-C(4)), 1.25 (m, 2H, H-C(4)), 0.92 (d, 6H, J = 6.9 Hz, 2 x C(3)-
CH3)), 0.88 (t, 6H, J = 7.5 Hz, 2 x H-C(5)); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.31, 166.35,
163.87, 144.13, 142.68, 129.43, 126.17, 58.19, 47.18, 37.06, 35.36, 25.53, 15.99, 11.61. HRMS
(ESI/QTOF, m/z): [M + H]+ Calcd. for [C23H35N7SO6H]+ 538.2442; Found: 538.2449.
4.2.2.16
2,2´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(4-methylthiobutanoic
acid) 25:
White solid; yield 80.4%. IR (KBr, cm-1): 3243 (w, NH), 1717 (w, C=O), 1623 (m), 1589 (m), 1558 (s),
1496 (s), 1404 (m), 1320 (m, SO2NH2), 1154 (s, SO2NH2). 1H NMR (300MHz, DMSO-d6, 90 °C) δ: 9.26
(br s, 1H, Ar-NH), 7.89 (d, 2H, J = 8.8 Hz, 2 x Ar-H(a)), 7.69 (d, 2H, J = 8.8 Hz, 2 x Ar-H(b)), 7.11 (br s,
4H, SO2NH2, 2 x NH-CH), 4.60 (br s, 2H, 2 x H-C(2)), 2.57 (m, 4H, 2 x H-C(4)), 2.15-1.97 (m, 4H, 2 x

H-C(3)), 2.05 (s, 6H, 2 x CH3); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.82, 165.09, 163.68, 143.56,
137.41, 126.75, 119.67, 53.09, 31.60, 30.61, 15.15. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for
[C19H27N7S3O6H]+ 546.1258; Found: 546.1259.
4.2.2.17
2,2´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(4-methylthiobutanoic
acid) 26:
White solid; yield 88.4%. IR (KBr, cm-1): 3282 (w, NH), 2969 (w), 1717 (w, C=O), 1623 (m), 1558 (s),
1
1507 (m), 1398 (m), 1330 (m, SO2NH2), 1156 (s, SO2NH2). H NMR (300 MHz, DMSO-d6, 90 °C) δ:
7.76 (d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.47 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.25 (br s, 1H, Ar-NH), 7.01
(s, 2H, SO2NH2), 6.75 (br s, 2H, 2 x NH-CH), 4.51 (m, 4H, 2 x H-C(2), NH-CH2), 2.52 (m, 4H, 2 x H-
13
C(4)), 2.09-1.90 (m, 4H, 2 x H-C(3)), 2.03 (s, 6H, 2 x CH3); C NMR (75 MHz, DMSO-d6, 90 °C) δ:
173.92, 165.19, 165.00, 144.74, 143.12, 128.14, 126.08, 53.02, 43.70, 31.72, 30.55, 15.14. HRMS
(ESI/QTOF, m/z): [M + H]+ Calcd. for [C20H29N7S3O6H]+ 560.1419; Found: 560.1423.
4.2.2.18 2,2´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(4-methylthiobutanoic
acid) 27:
White solid; yield 83.2%. IR (KBr, cm-1): 3281 (w, NH), 2919 (w), 1717 (w, C=O), 1617 (s), 1558 (m),
1540 (m), 1507 (m), 1398 (w), 1328 (m, SO2NH2), 1154 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90
°C) δ: 7.75 (d, 2H, J = 8.4 Hz, 2 x Ar-H(a)), 7.41 (d, 2H, J = 8.4 Hz, 2 x Ar-H(b)), 7.12 (br s, 3H, 2 x NH-
CH, Ar-NH), 7.02 (s, 2H, SO2NH2), 4.56 (m, 2H, 2 x H-C(2)), 3.50 (m, 2H, NH-CH2), 2.91 (m, 2H,
13
CH2-Ar), 2.55 (m, 4H, 2 x H-C(4)), 2.08-1.91 (m, 4H, 2 x H-C(3), 2.05 (s, 6H, 2 x CH3); C NMR (75
MHz, DMSO-d6, 90 °C) δ: 173.64, 163.01, 162.79 , 144.11, 142.68, 129.44, 126.16, 53.05, 41.81, 35.36,
31.60, 30.51, 15.14. HRMS (ESI/QTOF) m/z: [M + H]+ Calcd. for [C21H31N7S3O6H]+ 574.1571; Found:
574.1577.
4.2.2.19
N,N´-(6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(pyrrolidine-2-carboxylic
acid) 28:
White solid; yield 97.2%. IR (KBr, cm-1): 2978 (w), 2882 (w), 1728 (w, C=O), 1624 (m), 1582 (m), 1539
1
(s), 1475 (s), 1343 (s, SO2NH2), 1156 (s, SO2NH2). H NMR (300 MHz, DMSO-d6, 90 °C) δ: 9.05 (br s,
1H, Ar-NH), 7.90 (d, 2H, J = 8.7 Hz, 2 x Ar-H(a)), 7.67 (d, 2H, J = 8.7 Hz, 2 x Ar-H(b)), 6.88 (s, 2H,
SO2NH2), 4.46 (br s, 2H, 2 x H-C(2)), 3.60 (br s, 4H, 2 x H-C(4)), 2.25 (m, 2H, H-C(6)), 2.04-1.90 (m,
6H, H-C(6), 2 x H-C(5)); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 174.30, 164.10, 163.60, 144.14,
136.77, 126.73, 119.16, 59.04, 46.71, 30.25, 23.67. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for
[C19H23N7SO6H]+ 478.1508; Found: 478.1511.
4.2.2.20 N,N´-(6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(pyrrolidine-2-carboxylic acid) 29:
Beige solid; yield 62.3%. IR (KBr, cm-1): 2969 (w), 1733 (w), 1717 (w, C=O), 1653 (m), 1601 (s), 1539
(s), 1457 (m), 1396 (w), 1331 (m, SO2NH2), 1156 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ:
8.28 (br s, 1H, NH-CH2), 7.79 (d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.50 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)),
7.03 (br s, 2H, SO2NH2), 4.55 (br s, 2H, 2 x H-C(2)), 4.43 (br s, 2H, NH-CH2 ), 3.59 (br s, 4H, 2 x H-
C(4)), 2.23 (dq, 2H, J = 8.2 Hz, H-C(6)), 2.02-1.92 (m, 6H, H-C(6), 2 x H-C(5)); 13C NMR (75 MHz,
DMSO-d6, 90 °C) δ: 173.25, 167.19, 164.54, 143.45, 134.15, 128.38, 126.25, 59.72, 47.22, 43.78, 29.88,
23.71. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C20H25N7SO6H]+ 492.1660; Found: 492.1668.
4.2.2.21 N,N´-(6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(pyrrolidine-2-carboxylic acid)
30:
Beige solid; yield 57.5%. IR (KBr, cm-1): 2968 (w), 2880 (w), 1733 (w), 1717 (w, C=O), 1653 (m), 1598
(s), 1539 (s), 1457 (m), 1396 (w), 1332 (m, SO2NH2), 1156 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6,
90 °C) δ: 7.74 (d, 2H, J = 8.3 Hz, 2 x Ar-H(a)), 7.41 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.02 (br s, 2H,
SO2NH2), 4.44 (br s, 2H, 2 x H-C(2)), 3.57 (m, 6H, 2 x H-C(4), NH-CH2), 2.92 (t, 2H, J = 7.1 Hz, CH2-

Ar), 2.22 (m, 2H, H-C(6)), 2.03-1.89 (m, 6H, H-C(6), 2 x H-C(5)); 13C NMR (75 MHz, DMSO-d6, 90 °C)
δ: 173.60, 164.60, 160.96, 143.92, 142.73, 129.49, 126.21, 59.56, 47.07, 41.80, 35.26, 29.98, 23.69.
HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C21H27N7SO6H]+ 506.1816; Found: 506.1825.
4.2.2.22
2,2´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-
phenylpropanoic acid) 31:
White solid; yield 93.8%. IR (KBr, cm-1): 3281 (br, w, NH), 1724 (w, C=O), 1623 (m), 1588 (m), 1557
1
(s), 1495 (s), 1404 (m), 1327 (m, SO2NH2), 1154 (s, SO2NH2). H NMR (300 MHz, DMSO-d6, 90 °C) δ:
9.09 (br s, 1H, Ar-NH), 7.83 (d, 2H, J = 8.9 Hz, 2 x Ar-H(a)), 7.67 (d, 2H, J = 8.9 Hz, 2 x Ar-H(b)), 7.25
– 7.14 (m, 10H, Ar-H), 6.91 (br s, 2H, SO2NH2), 6.70 (br s, 2H, 2 x NH-CH) 4.72 (ddd, 2H, J = 8.6 Hz, J
= 7.8 Hz, J = 5.2 Hz, 2 x H-C(2)), 3.17 (dd, 2H, J = 14.0 Hz, J = 5.2 Hz, 2 x H-C(3)), 3.05 (dd, 2H, J =
14.0 Hz, J = 8.6 Hz, 2 x H-C(3)); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.59, 165.57, 164.11,
143.76, 138.31, 137.16, 129.54, 128.61, 126.66, 119.56, 119.28, 55.15, 37.45. HRMS (ESI/QTOF, m/z):
[M + H]+ Calcd. for [C27H27N7SO6H]+ 578.1821; Found: 578.1824.
4.2.2.23 2,2´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-phenylpropanoic acid)
32:
White solid; yield 92.5%. IR (KBr, cm-1): 3287 (w, NH), 1717 (w, C=O), 1625 (s), 1551 (s), 1397 (m),
1333 (m, SO2NH2), 1158 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.74 (d, 2H, J = 8.6 Hz,
2 x Ar-H(a)), 7.41 (d, 2H, J = 8.6 Hz, 2 x Ar-H(b)), 7.28-7.13 (m, 10H, Ar-H), 7.00 (br s, 3H, SO2NH2,
Ar-NH), 6.27 (br s, 2H, 2 x NH-CH) 4.64 (ddd, 2H, J = 8.4 Hz, J = 7.8 Hz, J = 5.3 Hz, 2 x H-C(2)), 4.44
(d, 2H, J = 6.21 Hz, NH-CH2), 3.09 (dd, 2H, J = 14.0 Hz, J = 5.4 Hz, 2 x H-C(3)), 2.99 (dd, 2H, J = 14.0
13
Hz, J = 8.4 Hz, 2 x H-C(3)); C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.77, 165.95, 165.67, 145.00,
143.02, 138.34, 129.48, 128.51, 126.68, 126.17, 125.94, 55.00, 43.65, 37.52. HRMS (ESI/QTOF, m/z):
[M + H]+ Calcd. for [C28H29N7SO6H]+ 592.1973; Found: 592.1979.
4.2.2.24 2,2´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))di(3-phenylpropanoic
acid) 33:
White solid; yield 90.4%. IR (KBr, cm-1): 3285 (w, NH), 1717 (w, C=O), 1623 (s), 1551 (s), 1455 (w),
1398 (w), 1332 (m, SO2NH2), 1157 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.74 (d, 2H, J
= 8.4 Hz, 2 x Ar-H(a)), 7.38 (d, 2H, J = 8.4 Hz, 2 x Ar-H(b)), 7.28-7.14 (m, 10H, Ar-H), 7.01 (br s, 2H,
SO2NH2), 6.74 (br s, 1H, Ar-NH), 6.55 (br s, 2H, 2 x NH-CH), 4.68 (ddd, 2H, J = 8.4 Hz, J = 7.9 Hz, J =
5.3 Hz, 2 x H-C(2)), 3.44 (dt, 2H, J = 7.4 Hz, J = 4.8 Hz, NH-CH2), 3.13 (dd, 2H, J = 14.0 Hz, J = 5.3 Hz,
13
2 x H-C(3)), 3.02 (dd, 2H, J = 14.0 Hz, J = 8.4 Hz, 2 x H-C(3)), 2.86 (t, 2H, J = 7.4 Hz, CH2-Ar); C
NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.59, 164.82, 164.61, 144.28, 142.62, 138.26, 129.50, 128.60,
126.78, 126.32, 126.02, 55.16, 41.75, 37.50, 35.45. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for
[C29H31N7SO6H]+ 606.2129; Found: 606.2135.
4.2.3 General procedure for the synthesis of 2,2´-((6-(4-Sulfamoylphenyl-/benzyl-/phenethyl-amino)-1,3,5-
triazine-2,4-diyl)bis(imino)) conjugates with dicarboxylic acids 10-15
Reaction
mixture
containing
4-(4´,6´-dichloro-1´,3´,5´-triazine-2´-
ylamino/aminomethyl/aminoethyl)benzenesulfonamide (0.2 g, 1 equiv) and glutamic or aspartic acid (3
equiv) was stirred in H2O (3 mL) at room temperature for 10 min. The aqueous solution of Na2CO3 (5
equiv) was added dropwise into the reaction mixture. Then the mixture was refluxed for 24 h and
evaluated by TLC during the reaction. The synthesis was terminated by 1M HCl addition (up to pH=3.0-
4.0) until a maximum amount of precipitate was produced. The product was isolated by filtration and
dried under high vacuum. Preliminary analysis indicated some impurities in the samples so that further
purification (recrystallization from water) was needed to achieve the purity higher than 90%. After then
the product purity and characterization were established by a combination of IR spectra, HPLC-UV/MS
and NMR (for the recorded spectra see Figures S25-S30 in the Supplementary material).

4.2.3.1 2,2´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid 10:
White solid; yield 90.2%. IR (KBr, cm-1): 3286 (w, NH), 2967 (w), 1715 (m, C=O), 1589 (s), 1554 (s),
1
1496 (s), 1380 (m), 1154 (s, SO2NH2). H NMR (300 MHz, DMSO-d6, 90 °C) δ: 9.12 (s, 1H, Ar-NH),
7.89 (d, 2H, J = 8.8 Hz, 2 x Ar-H(a)), 7.68 (d, 2H, J = 8.8 Hz, 2 x Ar-H(b)), 6.89 (s, 2H, SO2NH2), 6.60
13
(br s, 2H, 2 x NH-CH), 4.68 (t, 2H, J = 6.4 Hz, 2 x H-C(2)), 2.72 (d, 4H, J = 6.4 Hz, 2 x H-C(3)); C
NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.44, 172.41, 165.77, 164.55, 144.00, 136.95, 126.80, 119.29,
50.59, 38.24. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C17H19N7SO10H]+ 514.0992; Found:
514.0996.
4.2.3.2 2,2´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid 11:
White solid; yield 78.9%. IR (KBr, cm-1): 3290 (m, NH), 1717 (m, C=O), 1623 (s), 1559 (s), 1541 (s),
1396 (w), 1328 (m, SO2NH2), 1151 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.75 (d, 2H, J
= 8.3 Hz, 2 x Ar-H(a)), 7.47 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.09 (br s, 1H, Ar-NH), 6.99 (s, 2H,
SO2NH2), 6.38 (br s, 2H, 2 x NH-CH), 4.73 (dd, 2H, J = 6.4 Hz, J = 5.9 Hz, 2 x H-C(2)), 4.48 (d, 2H, J =
6.3 Hz, CH2-Ar), 2.71 (dd, 2H, J = 16.2 Hz, J = 5.9 Hz, 2 x Ha-C(3)), (dd, 2H, J = 16.2 Hz, J = 6.4 Hz, 2
x Hb-C(3)); 13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.25, 172.20, 166.22, 165.80, 145.09, 143.00,
128.15, 126.11, 50.45, 43.66, 37.06. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C18H21N7SO10H]+
528.1143; Found: 528.1149.
4.2.3.3 2,2´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))disuccinic acid 12:
Beige solid; yield 68.2%. IR (KBr, cm-1): 3281 (w, NH), 1717 (m, C=O), 1617 (s), 1559 (s), 1541 (s),
1396 (m), 1328 (m, SO2NH2), 1152 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.75 (d, 2H, J
= 8.3 Hz, 2 x Ar-H(a)), 7.41 (d, 2H, J = 8.3 Hz, 2 x Ar-H(b)), 7.00 (br s, 2H, SO2NH2), 6.60 (br s, 1H, Ar-
NH), 6.41 (br s, 2H, 2 x NH-CH), 4.76 (dd, 2H, J = 6.4 Hz, J = 5.9 Hz, 2 x H-C(2)), 3.46 (m, 2H, NH-
CH2), 2.89 (m, 2H, CH2-Ar ) 2.75 (dd, 2H, J = 16.2 Hz, J = 5.9 Hz, 2 x Ha-C(3)), 2.72 (dd, 2H, J = 16.2
13
Hz, J = 6.4 Hz, 2 x Hb-C(3)); C NMR (75 MHz, DMSO-d6, 90 °C) δ: 173.24, 172.18, 165.86, 165.57,
144.44, 142.55, 129.43, 126.19, 50.49, 41.79, 37.19, 37.01, 35.63. HRMS (ESI/QTOF, m/z): [M + H]+
Calcd. for [C19H23N7SO10H]+ 542.1300; Found: 542.1309.
4.2.3.4 2,2´-((6-(4-Sulfamoylphenylamino)-1,3,5-triazine-2,4-diyl)bis(imino))diglutaric acid 13:
White solid; yield 87.6%. IR (KBr, cm-1): 3242 (m, NH), 2967 (br, w), 1713 (m, C=O), 1622 (m), 1589
1
(s), 1557 (s), 1495 (s), 1404 (m), 1318 (m, SO2NH2), 1151 (s, SO2NH2). H NMR (300 MHz, DMSO-d6,
90 °C) δ: 9.02 (br s, 1H, Ar-NH), 7.89 (d, 2H, J = 8.9 Hz, 2 x Ar-H(a)), 7.68 (d, 2H, J = 8.9 Hz, 2 x Ar-
H(b)), 6.89 (br s, 2H, SO2NH2), 6.66 (br s, 2H, 2 x NH-CH), 4.45 (t, 2H, J = 6.8 Hz, 2 x H-C(2)), 2.34 (t,
13
4H, J = 7.6 Hz, 2 x H-C(4)), 2.12-1.91 (m, 4H, 2 x H-C(3)); C NMR (75 MHz, DMSO-d6, 90 °C) δ:
174.21, 174.06, 166.11, 164.49, 144.04, 136.88, 126.75, 119.23, 53.51, 31.28, 27.48. HRMS (ESI/QTOF,
m/z): [M + H]+ Calcd. for [C19H23N7SO10H]+ 542.1305; Found: 542.1308.
4.2.3.5 2,2´-((6-(4-Sulfamoylbenzylamino)-1,3,5-triazine-2,4-diyl)bis(imino))diglutaric acid 14:
Beige solid; yield 59.8%. IR (KBr, cm-1): 3242 (m, NH), 1716 (m, C=O), 1622 (s), 1558 (s), 1404 (m),
1324 (m, SO2NH2), 1152 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.75 (d, 2H, J = 8.4 Hz,
2 x Ar-H(a)), 7.46 (d, 2H, J = 8.4 Hz, 2 x Ar-H(b)), 6.99 (br s, 3H, SO2NH2, Ar-NH), 6.36 (br s, 2H, 2 x
NH-CH), 4.46 (m, 4H, 2 x H-C(2), CH2-Ar), 2.30 (m, 4H, 2 x H-C(4)), 2.16-1.83 (m, 4H, 2 x H-C(3));
13C NMR (75 MHz, DMSO-d6, 90 °C) δ: 174.17, 174.11, 166.23, 166.14, 145.17, 143.00, 128.16, 126.07,
53.22, 43.64, 30.97, 27.28. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C20H25N7SO10H]+ 556.1456;
Found: 556.1462.
4.2.3.6 2,2´-((6-(4-Sulfamoylphenethylamino)-1,3,5-triazine-2,4-diyl)bis(imino))diglutaric acid 15:

Beige solid; yield 66.3%. IR (KBr, cm-1): 3242 (m, NH), 2968 (w), 1717 (m, C=O), 1622 (s), 1558 (s),
1540 (s), 1405 (m), 1312 (m, SO2NH2), 1152 (s, SO2NH2). 1H NMR (300 MHz, DMSO-d6, 90 °C) δ: 7.75
(d, 2H, J = 8.4 Hz, 2 x Ar-H(a)), 7.40 (d, 2H, J = 8.4 Hz, 2 x Ar-H(b)), 7.00 (s, 2H, SO2NH2), 6.57 (br s,
1H, Ar-NH), 6.51 (br s, 2H, 2 x NH-CH), 4.43 (br s, 2H, 2 x H-C(2)), 3.46 (m, 2H, NH-CH2), 2.89 (t, 2H,
J = 7.4 Hz, CH2-Ar), 2.33 (m, 4H, 2 x H-C(4)), 2.12-1.86 (m, 4H, 2 x H-C(3)); 13C NMR (75 MHz,
DMSO-d6, 90 °C) δ: 174.05, 165.46, 153.82, 144.42, 142.56, 129.44, 126.15, 53.25, 41.77, 35.52, 30.91,
27.21. HRMS (ESI/QTOF, m/z): [M + H]+ Calcd. for [C21H27N7SO10H]+ 570.1613; Found: 570.1621.
4.3 Carbonic anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity [34]. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at
the absorbance maximum of 557 nm, with 20 mM Tris (pH 8.3) 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 have been 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 mM) were prepared in distilled-deionized water and dilutions up to 0.005 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 PRISM 3 and the ChengPrusoff
equation, as reported earlier, and represent the mean from at least three different determinations. All CA
isoforms were recombinant ones obtained in-house. [37-40]
4.4.
Protein preparation
The hCA IX isoenzyme structure was prepared for modeling using the 2.2ꢀÅ resolution hCA IX-
acetazolamide crystal complex (PDB ID: 3IAI) [41]. The hCA II isoenzyme structure was prepared for
modeling using the 1.5ꢀÅ resolution hCA II- 4-({4-chloro-6-[(2-hydroxyethyl)amino]-1,3,5-triazin-2-
yl}amino)benzenesulfonamide crystal complex (PDB ID: 3MMA) [27]. Schrödinger's ‘Protein
Preparation Wizard’ [42] was used for the preparation with water molecules within 5ꢀÅ of the ligand
initially retained, but deleted for subsequent docking. Bond orders were assigned and hydrogen atoms
added, with protonation states for basic and acidic residues based on residue pKa’s at normal pH (7.0).
However, subsequent optimization of hydroxyl groups, histidine C/N atom “flips” and protonation states,
and side chain O/N atom “flips” of Asn and Gln residues was based on optimizing hydrogen bonding
patterns, so that the final assignments were checked on visual inspection of the protein.
4.5
Molecular docking
LigPrep [43] was used to prepare ligand 12 (geometry optimization by OPLS 3, generation of all optical
isomers, low-energy ring conformations) and Epik [44–46] for the generation of the probable ionized and
tautomerized structures within a pH range of 7 ±2. Sulfonamide group was N-deprotonated as it is well
known that in this form it serves as zinc binding site [47].
Docking to hCA IX and hCA II was performed in program Glide with XP precision [48,49]. Penalization
for low probable ionization state and constraint for bond between zinc and zinc binding group were used.
Acknowledgements
This work was supported by the projects VEGA 1/0463/18, APVV-15-0585, and UK/330/2018.

References
[1] A. Thiry, J.M. Dogné, B. Masereel, C.T. Supuran, Targeting tumor-associated carbonic anhydrase IX
in cancer therapy, Trends Pharmacol. Sci. 27 (2006) 566-73. doi:10.1016/j.tips.2006.09.002.
[2] C.T. Supuran, Inhibition of carbonic anhydrase IX as a novel anticancer mechanism, World J. Clin.
Oncol. 3 (2012) 98-103. doi:10.5306/wjco.v3.i7.98.
[3] C.C. Wykoff, N.J. Beasley, P.H. Watson, K.J. Turner, J. Pastorek, et al., Hypoxia-inducible expression
of tumor-associated carbonic anhydrases, Cancer Res. 60 (2000) 7075-83.
[4] I.J. Hoogsteen, H.A.M. Marres, K. Wijffels, P. Rijken, J. Peters, et al., Colocalization of carbonic
anhydrase 9 expression and cell proliferation in human head and neck squamous cell carcinoma, Clin.
Cancer Res. 11 (2005) 97-106.
[5] A. Giatromanolaki, M.I. Koukourakis, E. Sivridis, J. Pastorek, C.C. Wykoff, K.C. Gatter, A.L.Harris,
Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently
to poor outcome in non-small cell lung cancer, Cancer Res. 61 (2001) 7992-98.
[6] S.K. Chia, C.C. Wykoff, P.H. Watson, Ch. Han, R.D. Leek, J. Pastorek, et al., Prognostic significance
of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma, J. Clin. Oncol.
19 (2001) 3660-68. doi: 10.1200/JCO.2001.19.16.3660.
[7] C. Trastour, E. Benizri, F. Ettore, A. Ramaioli, E. Chamorey, J. Pouyssegur, E. Berra, HIF-1α and CA
IX staining in invasive breast carcinomas: Prognosis and treatment outcome, Int. J. Cancer. 120 (2007)
1451-58. doi:10.1002/ijc.22436.
[8] J.A. Haapasalo, K.M. Nordfors, M. Hilvo, I.J. Rantala, Y. Soini, A.K. Parkkila, et al., Expression of
carbonic anhydrase IX in astrocytic tumors predicts poor prognosis, Clin. Cancer Res. 12 (2006) 473-477.
doi: 10.1158/1078-0432.
[9] A.J. Kivelä, J. Kivelä, J. Saarnio, S. Parkkila, Carbonic anhydrases in normal gastrointestinal tract and
gastrointestinal tumours, World J. Gastroenterol. 11 (2005) 155-63. doi: 10.3748/wjg.v11.i2.155.
[10] P. Viikilä, A.J. Kivelä, H. Mustonen, S. Koskensalo, A. Waheed, W.S. Sly, et al., Carbonic anhydrase
enzymes II, VII, IX and XII in colorectal carcinomas, World J. Gastroenterol. 22 (2016) 8168-77.
doi:10.3748/wjg.v22.i36.8168.
[11] T. Dorai, I.S. Sawczuk, J. Pastorek, P.H. Wiernik, J.P. Dutcher, The role of carbonic anhydrase IX
overexpression in kidney cancer, Eur. J. Cancer. 41 (2005) 2935-47. doi:10.1016/j.ejca.2005.09.011.
[12] J.A. Loncaster, A.L. Harris, S.E. Davidson, J.P. Logue, R.D. Hunter, C.C. Wycoff, et al., Carbonic
anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: Correlations with tumor
oxygen measurements and prognosis in locally advanced carcinoma of the cervix, Cancer Res. 61 (2001)
6394-99.
[13] D. Hedley, M. Pintilie, J. Woo, A. Morrison, D. Birle, A. Fyles, M. Milosevic, R. Hill, Carbonic
Anhydrase IX Expression, Hypoxia, and Prognosis in Patients with Uterine Cervical Carcinomas, Clin.
Cancer Res. 9 (2003) 5666-74.
[14] J. Pastorek, S. Pastoreková, Hypoxia-induced carbonic anhydrase IX as a target for cancer therapy:
From biology to clinical use, Semin. Cancer Biol. 31 (2015) 52-64. doi:10.1016/j.semcancer.2014.08.002.
[15] C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and activators, Nat.
Rev. Drug Disc. 7 (2008) 168-181. doi:10.1038/nrd2467.
[16] K. Nordfors, J. Haapasalo, M. Korja, et al., The tumour-associated carbonic anhydrases CA II, CA IX
and CA XII in a group of medulloblastomas and supratentorial primitive neuroectodermal tumours: an
association of CA IX with poor prognosis, BMC Cancer. 10 (2010) 148-57. doi:10.1186/1471-2407-10-
148.
[17] National Center for Biotechnology Information, NCBI report for CA1 carbonic anhydrase 1.
https://www.ncbi.nlm.nih.gov/gene/759, 2018 (updated 7 Jan 2018).
[18] M. Mori, R.J. Staniunas, G.F. Barnard, J.M. Jessup, G.D. Steele, L.B. Chen, The significance of
carbonic anhydrase expression in human colorectal cancer, Gastroenterol. 105 (1993) 820-26.

[19] S. Angapelly, P.V. Sri Ramya, A. Angeli, S.M. Monti, M. Buonanno, M. Alvala, C.T. Supuran,
M.Arifuddin, Discovery of 4-sulfamoyl-phenyl-β-lactams as a new class of potent carbonic anhydrase
isoforms I, II, IV and VII inhibitors: The first example of subnanomolar CA IV inhibitors, Bioorg. Med.
Chem. 25 (2017) 539-44. doi:10.1016/j.bmc.2016.11.027.
[20] S. Pastorekova, A. Casini, A. Scozzafava, D. Vullo, J. Pastorek, C.T. Supuran, Carbonic anhydrase
inhibitors: the first selective, membrane-impermeant inhibitors targeting the tumor-associated isozyme IX,
Bioorg. Med. Chem. Lett. 14 (2004) 869-73. doi:10.1016/j.bmcl.2003.12.029.
[21] A. Cecchi, A. Hulikova, J. Pastorek, S. Pastoreková, A. Scozzafava, J.Y. Winum, J.L. Montero, C.T.
Supuran, Carbonic anhydrase inhibitors. Design of fluorescent sulfonamides as probes of tumor-associated
carbonic anhydrase IX that inhibit isozyme IX-mediated acidification of hypoxic tumors, J. Med. Chem.
48 (2005) 4834-41. doi:10.1021/jm0501073.
[22] B.L. Wilkinson, L.F. Bornaghi, T.A. Houston, A. Innocenti, C.T. Supuran, S.A. Poulsen, A novel
class of carbonic anhydrase inhibitors: glycoconjugate benzene sulfonamides prepared by “click-tailing”,
J. Med. Chem. 49 (2006) 6539-48. doi:10.1021/jm060967z.
[23] F. Pacchiano, F. Carta, P.C. McDonald, Y. Lou, D. Vullo, A. Scozzafava, S. Dedhar, C.T. Supuran,
Ureido-substituted benzenesulfonamides potently inhibit carbonic anhydrase IX and show antimetastatic
activity in a model of breast cancer metastasis, J. Med. Chem. 54 (2011) 1896-1902. doi:
10.1021/jm101541x.
[24] A. Maresca, C. Temperini, H. Vu, N.B. Pham, S.A. Poulsen, A. Scozzafava, R.J. Quinn, C.T.
Supuran, Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide
inhibitors, J. Am. Chem. Soc. 131 (2009) 3057-62. doi: 10.1021/ja809683v.
[25] V. Garaj, L. Puccetti, G. Fasolis, J.Y. Winum, J.L. Montero, A. Scozzafava, et al., Carbonic
anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes
I, II, and IX with sulfonamides incorporating 1,2,4-triazine moieties, Bioorg. Med. Chem. Lett. 14 (2004)
5427-33. doi.org/10.1016/j.bmcl.2004.07.087.
[26] V. Garaj, L. Puccetti, G. Fasolis, J.Y. Winum, J.L. Montero, A. Scozzafava, et al., Carbonic
anhydrase inhibitors: Novel sulfonamides incorporating 1,3,5-triazine moieties as inhibitors of the
cytosolic and tumour-associated carbonic anhydrase isozymes I, II and IX, Bioorg. Med. Chem. Lett. 15
(2005) 3102-08. doi.org/10.1016/j.bmcl.2005.04.056.
[27] F. Carta, V. Garaj, A. Maresca, J. Wagner, B.S. Avvaru, A.H. Robbins, et al., Sulfonamides
incorporating 1,3,5-triazine moieties selectively and potently inhibit carbonic anhydrase transmembrane
isoforms IX, XII and XIV over cytosolic isoforms I and II: Solution and X-ray crystallographic studies,
Bioorg. Med. Chem. 19 (2011) 3105-19. doi:10.1016/j.bmc.2011.04.005.
[28] A.K. Saluja, M. Tiwari, D. Vullo, C.T. Supuran, Substituted benzene sulfonamides incorporating
1,3,5-triazinyl moieties potently inhibit human carbonic anhydrases II, IX and XII, Bioorg. Med. Chem.
Lett. 24 (2014) 1310-14. doi:10.1016/j.bmcl.2014.01.048.
[29] M. Ceruso, D. Vullo, A. Scozzafava, C.T. Supuran, Inhibition of human carbonic anhydrase isoforms
I–XIV with sulfonamides incorporating fluorine and 1,3,5-triazine moieties, Bioorg. Med. Chem. 21
(2013) 6929-36. doi:10.1016/j.bmc.2013.09.031.
[30] E. Havránková, J. Csöllei, D. Vullo, V. Garaj, P. Pazdera, C.T. Supuran, Novel sulfonamide
incorporating piperazine, aminoalcohol and 1,3,5-triazine structural motifs with carbonic anhydrase I, II
and IX inhibitory action, Bioorg. Chem. 77 (2018) 25-37. doi:10.1016/j.bioorg.2017.12.034.
[31] N. Chiaramonte, M.N. Romanelli, E. Teodori, C.T. Supuran, Amino acids as building blocks for
carbonic anhydrase inhibitors, Metabolites, 8 (2018) 36-58. doi:10.3390/metabo8020036.
[32] D. Krajčiová, D. Pecher, V. Garaj, P. Mikuš, Optimization and Comparison of Synthetic Procedures
for a Group of Triazinyl-Substituted Benzene-Sulfonamide Conjugates with Amino Acids, Molecules. 22
(2017) 1533-41. doi:10.3390/molecules22091533.
[33] G.F. D´Alelio, H.J. White, Preparation of Some Sulfanilamide Derivatives of s-Triazine, J. Org.
Chem. 24 (1959) 643-44. doi: 10.1021/jo01087a018.

[34] R.G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic
studies on the native human isoenzymes B and C, J. Biol. Chem. 246 (1971) 2561-73.
[35] M. Sechi, A. Innocenti, N. Pala, D. Rogolino, M. Carcelli, A. Scozzafava, C.T. Supuran, Inhibition of
α-class cytosolic human carbonic anhydrases I, II, IX and XII, and β-class fungal enzymes by carboxylic
acids and their derivatives: new isoform-I selective nanomolar inhibitors, Bioorg. Med. Chem. Lett. 22
(2012) 5801-5806. doi: 10.1016/j.bmcl.2012.07.094.
[36] P. Mikuš, et al., Metal complexes of triazinyl-substituted aminoalkylbenzenesulfonamides conjugated
with chelating moieties as a new class of potential (radio)pharmaceuticals, In preparation.
[37] A. Maresca, F. Carta, D. Vullo, C.T. Supuran, Dithiocarbamates strongly inhibit the β- class carbonic
anhydrases from Mycobacterium tuberculosis, J. Enzyme Inhib. Med. Chem. 28 (2013) 407–411. doi:
10.3109/14756366.2011.641015.
[38] D. Ekinci, N.I. Kurbanoglu, E. Salamci, M. Senturk, C.T. Supuran, Carbonic anhydrase inhibitors:
inhibition of human and bovine isoenzymes by benzenesulphonamides, cyclitols and phenolic compounds,
J. Enzyme Inhib. Med. Chem. 27 (2012) 845–848. doi: 10.3109/14756366.2011.621122.
[39] D. Ekinci, L. Karagoz, D. Ekinci, M. Senturk, C.T. Supuran, Carbonic anhydrase inhibitors: in vitro
inhibition of α isoforms (hCA I, hCA II, bCA III, hCA IV) by flavonoids, J. Enzyme Inhib. Med. Chem.
28 (2013) 283–288. doi: 10.3109/14756366.2011.643303.
[40] C. Alp, A. Maresca, N.A. Alp, M.S. Gültekin, D. Ekinci, A. Scozzafava C.T. Supuran,
Secondary/tertiary benzenesulfonamides with inhibitory action against the cytosolic human carbonic
anhydrase isoforms I and II, J. Enzyme Inhib. Med. Chem. 28 (2013) 294–298. doi:
10.3109/14756366.2012.658788.
[41] V. Alterio, M. Hilvo, A. Di Fiore, C.T. Supuran, P. Pan, S. Parkkila, A. Scaloni, J. Pastorek, S.
Pastorekova, C. Pedone, A. Scozzafava, S.M. Monti, G. De Simone, Crystal structure of the catalytic
domain of the tumor-associated human carbonic anhydrase IX, PNAS, 106 (2009), 16233-16238, doi:
10.1073/pnas.0908301106.
[42] G.M. Sastry, M. Adzhigirey, T. Day, R. Annabhimoju, W. Sherman, Protein and ligand preparation:
Parameters, protocols, and influence on virtual screening enrichments, J. Comput. Aid. Mol. Des., 27
(2013), 221-234, doi: 10.1007/s10822-013-9644-8.
[43] LigPrep, version 3.5, Schrödinger, LLC, New York, NY, 2015.
[44] Epik, version 3.3, Schrödinger, LLC, New York, NY, 2015.
[45] J.C. Shelley, A. Cholleti, L.L. Frye, J.R. Greenwood, M.R. Timlin, M. Uchiyama, Epik: a software
program for pKa prediction and protonation state generation for druglike molecules, J. Comput. Aided
Mol. Des., 21 (2007), pp. 681-691, 10.1007/s10822-007-9133-z.
[46] J.R. Greenwood, D. Calkins, A.P. Sullivan, J.C. Shelley, Towards the comprehensive, rapid, and
accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution, J.
Comput. Aided Mol. Des., 24 (2010), pp. 591-604, 10.1007/s10822-010-9349-1.
[47] R.W. King, A.S.V. Burgen, Sulphonamide complexes of human carbonic anhydrases ultraviolet
difference spectroscopy, Biochim. Biophys. Acta, 207 (1970), pp. 3583-3587, 10.1021/jm011131t.
[48] Glide, version 6.8, Schrödinger, LLC, New York, NY, 2015.
[49] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood, T.A. Halgren, P.C.
Sanschagrin, D.T. Mainz, Extra precision glide: docking and scoring incorporating a model of
hydrophobic enclosure for protein-ligand complexes, J. Med. Chem., 49 (2006), pp. 6177-6196,
10.1021/jm051256o

Graphical abstract

Highlights
 Novel sulfonamides incorporating 1,3,5-triazine and amino acid structural motifs
 Green synthesis of disubstituted amino acid products with high selectivity and yield
 Study on inhibition activity of products toward carbonic anhydrases I, II, IV, IX
 Found highly effective inhibitors toward tumor-associated hCA IX
 Docking study on the most promising enzyme-substrate complex