Optimization and comparison of synthetic procedures for a group of triazinyl-substituted benzene-sulfonamide conjugates with amino acids

Article abstract of DOI:10.3390/molecules22091533

Sulfonamides incorporating 1,3,5-triazine moieties can selectively and potently inhibit carbonic anhydrase transmembrane isoforms IX, XII, and XIV over cytosolic isoforms I and II. In the present work, a highly effective synthetic procedure was proposed f

Full text of DOI:10.3390/molecules22091533

molecules
Article
Optimization and Comparison of Synthetic
Procedures for a Group of Triazinyl-Substituted
Benzene-Sulfonamide Conjugates with Amino Acids
Dominika Krajcˇiová 1, Daniel Pecher 1,2, Vladimír Garaj 3 and Peter Mikuš 1,2,
*
1
Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy,
Comenius University in Bratislava, Odbojárov 10, SK-832 32 Bratislava, Slovakia;
krajciova.dominika@fpharm.uniba.sk (D.K.); pecher1@uniba.sk (D.P.)
Toxicological and Antidoping Center, Faculty of Pharmacy, Comenius University in Bratislava,
Odbojárov 10, SK-832 32 Bratislava, Slovakia
2
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University in Bratislava,
Odbojárov 10, SK-832 32 Bratislava, Slovakia; garajv@gmail.com
*
Correspondence: mikus@fpharm.uniba.sk; Tel.: +421-250-117-243
Received: 9 August 2017; Accepted: 7 September 2017; Published: 13 September 2017
Abstract: Sulfonamides incorporating 1,3,5-triazine moieties can selectively and potently inhibit
carbonic anhydrase transmembrane isoforms IX, XII, and XIV over cytosolic isoforms I and
II. In the present work, a highly effective synthetic procedure was proposed for this group
of potent cancerostatic drugs and compared with previously used methods. The synthesis
of triazinyl-substituted benzene-sulfonamide conjugates with amino acids can be easily
carried out using sodium carbonate-based water solution as a synthetic medium instead of
N,N-Diisopropylethylamine/Dimethylformamide. The benefits of this synthetic procedure include:
(i) high selectivity of the creation of disubstituted conjugates; (ii) several times higher yield ( 95%)
≥
than that achieved previously; (iii) elimination of organic solvents by the use of an environmental
friendly water medium (green chemistry); (iv) simple and fast isolation of the product. The synthesis
and resulting products were evaluated using TLC, IR, NMR, and MS methods. The present work
demonstrates a significant advantage in providing shortened routes to target structures.
Keywords: 1,3,5-triazine conjugates; sulfonamides; amino acids; carboanhydrase inhibitors;
synthesis optimization
1. Introduction
Carbonic anhydrase (CA) IX and XII, as secondary tumor-related CAs, are found to be
overexpressed in cervical, breast, bladder, and non-small cell lung cancers by instigating hypoxia and
causing acidification in the extracellular region, which leads to metastatic spread of these tumor cells.
Furthermore, the acidification can render classical cancer treatments ineffective, particularly those
utilizing basic antitumor drugs and radiotherapy. It is therefore suggested that the inhibition of CA
IX/CA XII could be an attractive alternative option for anticancer therapy [1–3].
Recently, it was shown that sulfonamides incorporating 1,3,5-triazine moieties can selectively
and potently inhibit carbonic anhydrase transmembrane isoforms IX, XII, and XIV over cytosolic
isoforms I and II [4–8]. The biological activity as well as the water solubility of the triazinyl-substituted
benzene-sulfonamides were changed via the modification of the chemical structure after introducing
various moieties, namely aliphatic amines, methyl esters of amino acids, amino acids and
amino acid derivatives, ammonia, hydrazine, primary and secondary amines, amino-sulfon
amides, alcohols, phenols, anilines, morpholine, tert-butylamine, N-methyl-2-amino ethanol and
Molecules 2017, 22, 1533; doi:10.3390/molecules22091533
www.mdpi.com/journal/molecules

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1,3-diaminopropane, water, methylamine, or aliphatic alcohols (methanol and ethanol). Here, a reaction
of triazinyl-substituted benzene-sulfonamide with amino acids afforded a new series of highly potent
conjugates with enhanced polarity [4,5]. The reaction was carried out in inert atmosphere (N2) at
60 ◦C in dry DMF (Dimethylformamide) and DIPEA (N,N-Diisopropylethylamine). The reaction was
quenched with slush, and the precipitate formed was collected by filtration, washed with H2O, and
dried under high vacuum to give the required compound as a white solid. Declared yields for amino
acid (glycine, β-alanine) conjugates ranged in the interval of 32–55%. These parameters indicate that
there is a need to revise the established synthetic procedure with respect to the reaction yield and
working conditions.
An aim of the present work was to propose a more effective synthetic procedure for the preparation
of triazinyl-substituted benzene-sulfonamide conjugates with amino acids. Current criteria for such
synthesis include: (i) a significant increase in the reaction yield; (ii) the minimization of organic solvents
employed; and (iii) the simplification of the synthetic and isolation procedure. As model amino acids,
glycine and alanine were selected to investigate and compare various synthetic strategies, including
the proposed strategies and those previously employed (based on the DIPEA/DMF procedure).
2. Results and Discussion
We studied the possibilities of effectivization of the synthetic procedure for the preparation
of triazinyl-substituted benzene-sulfonamide conjugates with amino acids. Two previously used
amino acids, i.e., glycine and
β-alanine, were selected in the present work to investigate and
compare various synthetic strategies with the established one based on a 4-(40,60-dichloro-10,30,50-
0
triazin-2 -ylamino)-benzene-sulfonamide precursor
1 (Scheme 1) and a DIPEA/DMF environment [4].
The intermediates and products were synthesized via Schemes 2–6, isolated, purified, and identified
(see corresponding NMR, IR, and HPLC-UV graphical profiles S1-S14 in the Supplementary file).
Firstly, we investigated the synthesis of the amino acid conjugates prepared in the
DIPEA/DMF environment through esters of amino acids and the subsequent hydrolysis of the
substituted products. The representative synthesis of dimethyl 200,2000-[60-(4-sulfamoylphenylamino)-
10,30,50-triazine-20,40-diyl]-bis(azanediyl)diacetate intermediate
hydrolysis of the intermediate is shown in Scheme 3. Although the yield of the intermediate
37%, the yield of the final product from this was only 64%. This means that the real yield of the final
2
is shown in Scheme 2, while the
was
2
2
3
product
3
(pr0e0pared 0according to Schemes 2 and 3) was ca. 24%, which was less than the 32% value
000
0
0
0
0
0
declared for 2 ,2 -[6 -(4-sulfamoylphenylamino)-1 ,3 ,5 -triazine-2 ,4 -diyl]-bis(azanediyl)diacetic acid
conjugate in Reference [4].
Secondly, a water environment (containing NaOH) instead of an organic one was tested
in order to enhance the yield of the product and its isolation. The representative synthesis
of conjugate
3 is shown in Scheme 4. Although the expected product 3 was obtained in a
reasonable amount in solid state (ca. 81% of the theoretical yield), applied NMR as well as
HPLC-UV/MS methods indicated some impurities in the sample. HPLC-UV/MS detected a major
amount (75.1%/53.9%) of
impurities related to containing one substituted glycine and one substituted OH (m/z 341.0663)
(11.0%/9.5%), or two substituted OH groups instead of glycine (m/z 284.0448) (2.7%/36.6%).
According to the quantitative NMR, the amount of pure product from the reaction mixture
was ca. 65%. This is a considerably higher yield compared to that declared in the literature
for 200,2000-[60-(4-sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis(azanediyl)diacetic acid [
].
(prepared according
3 with two substituted glycines (m/z 398.0877), and minor amounts of
3
3
4
However, there is a need for an additional sample preparation to isolate product
to Scheme 4) from its minor structurally related impurities.
3
Thirdly, a water environment replacing NaOH with Na2CO3 was tested in order to prevent
the creation of minor structurally related impurities (mainly hydroxy-derivatives), and further
enhance the yield of the product and improve its isolation. The representative synthesis of conjugate
3
is shown in Scheme 5. Here, the expected product 3 was obtained in very high amounts in

Molecules 2017, 22, 1533
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the solid state (96% of the theoretical yield). Applied NMR as well as HPLC-UV/MS methods
did not indicate any significant impurities in the sample, so no further purification procedure
was necessary. Theoretical and experimental yields were in good agreement, and the HPLC-UV
analysis indicated 95.51% purity of the product
Relative error between the theoretical and HPLC-UV values was 0.51%. According to these
findings, the real yield of pure product (prepared according to Scheme 5) from the reaction
mixture was higher than 95%. It was the highest yield in comparison with those obtained for
200,2000-[60-(4-sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis(azanediyl)diacetic acid
by any
other procedure. The compared procedures included (i) those performed in our work (see Schemes 2–4)
ranging in 24–65% yields as well as (ii) those published in the literature [ ] with the yield value of 32%.
This remarkable improvement in the yield of triazinyl-substituted benzene-sulfonamide
conjugates with amino acids was demonstrated with the glycine conjugate (prepared according to
Scheme 5) in addition to -alanine conjugate (prepared according to Scheme 6). Theoretical and
experimental yields were in good mutual agreement. The HPLC-UV analysis indicated 94.98% purity
of the product (see Figure S14 in the Supplementary Material) while the theoretical value was 98%.
3 (see Figure S13 in the Supplementary material).
3
3
4
3
β
4
4
Relative error between the theoretical and HPLC-UV values was 3.08%. Hence, it was confirmed
that the synthetic strategy based on a water environment with Na2CO3 provided at least 95% yield
of 300,3000-[60-(4-sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]bis(azanediyl)dipropanoic acid
This yield is considerably higher than the value of 55% published in the literature [4].
4.
0
0
0
0
0
0
Scheme 1. Synthesis of 4-(4 ,6 -dichloro-1 ,3 ,5 -triazin-2 -ylamino)-benzene-sulfonamide precursor 1.
◦
Reagents and conditions: 4-aminobenzenesulfanilamide, cyanuric chloride, acetone, at 0 C, was added
to an aqueous solution of NaOH, stirred for 1 h, and the reaction was subsequently quenched by the
addition of slush (100 mL), after which the solid was filtered off. The scheme is reprinted from [4],
with permission of the publisher. Product yield: 95%.
Scheme 2. Synthesis of dimethyl 200,2000-[60-(4-sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-
bis(azanediyl)diacetate intermediate 2. Reagents and conditions: a mixture of methyl 2-aminoacetate
hydrochloride, DMF, and DIPEA was stirred for 2 h at r.t. Then, 4-(40,60-dichloro-10,30,50-triaz◦in-
0
2 -ylamino)-benzene-sulfonamide
1
dissolved DMF was added, and the mixture was stirred at 90 C
under an argon atmosphere overnight. Product yield: 37%.

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00 000
0
0
0
0
0 0
Scheme 3. Hydrolysis of 2 ,2 -[6 -(4-sulfamoylphenylamino)-1 ,3 ,5 -triazine-2 ,4 -diyl]-bis(azanediyl)
diacetate intermediate
2
0 to product 3. Reagents and conditions: a mixture of 200,2000-[60-(4-
0
0
0
0
sulfamoylphenylamino)-1 ,3 ,5 -triazine-2 ,4 -diyl]-bis(azanediyl)diacetate
was stirred at r.t. for 3 h. Product yield: 64%.
2, MeOH, LiOH, and H2O
00 000
0
0
0
0
0
0
0
Scheme 4. Synthesis of 2 ,2 -[6 -(4-sulfamoylphenylamino)-1 ,3 ,5 -triazine-2 ,4 -diyl]-bis(azanediyl)
0
0
0
0
0
diacetic acid 3. Reagents and conditions: 4-(4 ,6 -dichloro-1 ,3 ,5 -triazin-2 -ylamino)-benzene-sulfonamide
1, glycine, NaOH, and H2O were refluxed for 24 h. Product yield: 65%.
00 000
0
0
0
0
0
0
Scheme 5. Synthesis of 2 ,2 -[6 -(4-sulfamoylphenylamino)-1 ,3 ,5 -triazine-2 ,4 -diyl]-bis(azanediyl)
0 0 0 0 0 0
diacetic acid 3. Reagents and conditions: 4-(4 ,6 -dichloro-1 ,3 ,5 -triazin-2 -ylamino)-benzene-sulfonamide
1, glycine, Na2CO3, and H2O were refluxed for 24 h. Product yield: 96%.

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00 000
0
0
0
0
0
0
Scheme 6. Synthesis of 3 ,3 -[6 -(4-sulfamoylphenylamino)-1 ,3 ,5 -triazine-2 ,4 -diyl]-bis(azanediyl)
dipropanoic acid
. Reagents and conditions: 4-(40,60-dichloro-10,30,50-triazin-20-ylamino)-benzene-
4
sulfonamide 1, β-alanine, Na2CO3, and H2O were refluxed for 24 h. Product yield: 95%.
3. Experimental Protocols
3.1. Chemistry
All reagents were purchased from Aldrich (St. Louis, MO, USA) and AppliChem GmbH
(Darmstadt, Germany) and were used without further purification. The reaction was monitored
by thin-layer chromatography carried out on silica gel plates (60 F254, MERCK, Darmstadt, Germany)
as the stationary phase; ethylacetate/n-hexane or MeOH/DCM were used as eluents. Spots were
visualized under UV light (245 nm). Nuclear magnetic resonance (1H-NMR, 13C-NMR) spectra were
recorded using a Varian MERCURY plus 300 MHz spectrometer (Varian, Palo Alto, CA, USA) in
DMSO-d6 at r.t. and at 80 ◦C. Chemical shifts were reported in parts per million (ppm, d) downfield
from TMS (Tetramethylsilane). Splitting patterns are described as singlet (s), doublet (d), triplet
(t), quartet (q), multiple (m), broad (br), and doublet of doublet (dd). Infrared (IR) spectra were
performed on Perkin-Elmer UATR Two (PerkinElmer Ltd., Beaconsfield, UK) and were recorded as
KBr plates. The absorption bands (ν
max) are given in wave numbers (cm−1) and the signal strength
is given as weak (w), strong (s), or medium (m). All of the HPLC-UV/MS analyses were performed
on a chromatographic apparatus consisting of 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). 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. For data acquisition and processing, a computer with Mass Hunter
software(version MassHunter Workstation B 04.00, Agilent Technologies, Santa Clara, CA, USA)
was used. The HPLC-HRMS analysis was carried out using HILIC column—SeQuant® ZIC®-HILIC,
2.1
of acetonitrile and 5 mM solution of ammonium acetate pH 5.8 (adjusted with acetic acid). The flow
rate of the mobile phase was set at 400 L/min and 5.0 L of the sample were injected into the
×
100 mm, 3.5 µm obtained from Merck KGaA, Darmstadt, Germany. The mobile phases consisted
µ
µ
column. The overall time of analysis was 24 min. Gradient elution was used with the column oven
temperature set at 35 ◦C. The following MS parameters were used for all measurements: drying gas
temperature 325 ◦C, drying gas flow 10 L.min-1, nebulizing gas pressure 40 psi, ESI source voltage
3500 V, fragmentor voltage 140 V, collision gas N2.

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3.2. General Procedure for the Synthesis of Compounds 1–4
3.2.1. Synthesis of 4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)-benzene-sulfonamide Precursor 1
(Scheme 1)
A 1.0 M solution of 4-aminobenzenesulfanilamide (17.2 g, 0.1 mol, 1.0 equiv) in acetone (100 mL)
was added dropwise to a v◦igorously stirred suspension of cyanuric chloride (18.4 g, 1.0 equiv) in the
same solvent (100 mL) at 0 C. The white slurry was stirred at the same temperature for 30 min. Then,
a 1.7 M aqueous solution of NaOH (4.0 g, 1.0 equiv) was added over a period of 20 min. Stirring was
continued for 1 h, and the reaction was quenched by the addition of slush (100 mL), after which the
solid was filtered off. Crystallization from acetone afforded the title compound
1
as a white solid [
): The product was obtained as a white
solid in 95% yield. IR (KBr): 3294 (s), 3211 (m), 2343 (w), 1621 (s), 1538 (s), 1494 (s), 1380 (m), 1328 (s),
1226 (s), 1164 (s), 1123 (m), 963 (w), 722 (s), 590 (m), 547 (s), 482 (m); 1H-NMR (DMSO-d6)
: 11.43 (s,
1H, Ar-NH, Notice: The moiety was underlined to indicate the hydrogen to which the signal refers.),
7.86 (d, 2H, J =9.0 Hz, 2 H-2), 7.79 (d, 2H, J = 9.0 Hz, 2
H-3), 7.34 (s, 2H, SO2NH2); 13C-NMR
4].
0
0
0
0
0
0
4-(4 ,6 -Dichloro-1 ,3 ,5 -triazin-2 -ylamino)-benzene-sulfonamide (
1
δ
×
×
(DMSO-d6) δ
: 170.25, 169.47, 164.40, 140.44, 140.32, 127.11, 121.55. HRMS (ESI/QTOF) m/z: [M + H]+
Calcd. for [C9H7N5SO2Cl2H]+ 319.9770; Found: 319.9772.
3.2.2. Synthesis of Dimethyl 200,2000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis
(azanediyl) Diacetate Intermediate 2 (Scheme 2)
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)-benzene-sulfonamide 1. (0.2 g, 1.0 equiv) was dissolved in
2 mL dry DMF. The mixture of methyl 2-aminoacetate hydrochloride (4.5 equiv) in 2 mL DMF and
DIPEA (9.0 equiv) was stirred for 2 h at r.t. After 2 h, a solution of 4-(40,60-dichloro-10,30,50-triazin-20-
ylamino)-benzene-sulfonamide
1
was added dropwise and the reaction was stirred at 90 ◦C
under an argon atmosphere. The reaction ran overnight and was monitored by TLC (Thin Layer
Chromatography). The next day, the reaction was quenched with slush and the precipitate formed was
collected by filtration, washed with H2O, and dried under high vacuum to give the title compound as
a white solid.
00 000
0
0
0
0
0
0
2 ,2 -[6 -(4-Sulfamoylphenylamino)-1 ,3 ,5 -triazine-2 ,4 -diyl]-bis(azanediyl)diacetate (2): The product was
obtained as a white solid in 37% yield. IR (KBr): 3427 (m), 3367 (m), 3263 (m), 3005 (w), 2361 (w), 1734
(s), 1569 (s), 1◦500 (s), 1411 (s), 1317 (s), 1153 (s), 987 (m), 903 (w), 807 (s), 590 (m), 541 (m); 1H-NMR
(DMSO-d6, 80 C) δ: 9.16 (s, 1H, Ar-NH), 7.86 (d, 2H, J = 8.8 Hz, H-2), 7.66 (d, 2H, J = 8.8 Hz, H-3), 7.07
(s, 2H, CH2-NH-), 6.95 (s, 2H, SO2NH2), 4.01 (d, 4H, J = 5.3 Hz, H-200, H-2000), 3.64 (s, 6H, 2
×
CH3);
: 171.27, 166.33, 164.50, 143.99, 137.00, 126.54, 119.35, 51.80, 42.65. HRMS
(ESI/QTOF) m/z: [M + H]+ Calcd. for [C15H19N7SO6H]+ 426.1190; Found: 426.1190.
◦
13C-NMR (DMSO-d6, 80 C)
δ
00 000 0 0 0 0
2 (Scheme 2) to Product 2 ,2 -[6 -(4-Sulfamoylphenylamino)-1 ,3 ,5 -
3.2.3. Hydrolysis of Intermediate
triazine-20,40-diyl]-bis(azanediyl)diacetic Acid 3 (Scheme 3)
200,2000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis(azanediyl) diacetate 2. (0.10048 g) was
dissolved in 4 mL MeOH. LiOH (4.0 eguiv) was dissolved in 4 mL H2O and added to the methanol
solution of compound 2. The mixture was stirred at r.t. and monitored by TLC. After 3 h, the hydrolysis
was terminated (before the addition of 1M HCl, methanol was evaporated) by the addition of a few
drops of 1M HCl until a white precipitate was no longer produced. The precipitated mixture was
filtered and the precipitate was dried under high vacuum.
200,2000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis(azanediyl)diacetic acid (3): The product
was obtained as a white solid in 64% yield. IR (KBr): 3264 (w), 2361 (w), 1734 (w), 1591 (s), 1504 (s),
1406 (s), 1312 (m), 1229 (m), 1155 (s), 905 (w), 779 (m), 587 (m), 544 (m); 1H-NMR (DMSO-d6, 80 ◦C)
δ:
9.29 (s, 1H, Ar-NH), 7.88 (d, 2H, J = 8.9 Hz, H-2), 7.68 (d, 2H, J = 8.9 Hz, H-3), 7.01 (br s, 4H, SO2NH2,

Molecules 2017, 22, 1533
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◦
2 × CH2-NH), 3.96 (br s, 4H, 2 × CH2-NH); 13C-NMR (DMSO-d6, 80 C)
δ
: 171.87, 165.72, 163.98,
143.83, 137.12, 126.65, 119.33, 42.70. HRMS (ESI/QTOF) m/z: [M + H]+ Calcd. for [C13H15N7SO6H]+
398.0877; Found: 398.0877.
3.2.4. Synthesis of 200,2000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis(azanediyl)
diacetic Acid 3 (Scheme 4)
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)-benzene-sulfonamide 1. (0.1 g, 1.0 equiv) was stirred in H2O
(2 ml) at r.t. for 10 min. Then alkaline solution of glycine (2.4 equiv of Gly and 5.0 equiv of NaOH)
was added dropwise into precursor 1. The mixture was refluxed for 24 h and monitored by TLC.
The synthesis was terminated by the addition of a few drops of 1M HCl until a white precipitate was
no longer produced. The product was filtered off and dried under high vacuum.
200,2000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis(azanediyl)diacetic acid (
was obtained as a white solid in 81% yield. IR (KBr): 2968 (s), 2361 (s), 2343 (m), 1560 (m), 1498 (s),
1405 (s), 1308 (m), 1157 (s), 837 (w), 779 (m), 587 (m), 544 (s); 1H-NMR (DMSO-d6, 80 ◦C)
: 9.14 (s,
1H, Ar-NH), 7.89 (d, 2H, J = 8.9 Hz, H-2), 7.67 (d, 2H, J = 8.9 Hz, H-3), 6.94 (s, 2H, SO2NH2), 6.88
3). The product
δ
(br s, 2H, 2 × CH2-NH), 3.94 (d, 4H, J = 6.2 Hz 2
×
CH2-NH); 13C-NMR (DMSO-d6, 80 ◦C)
δ: 172.06,
166.40, 164.50, 144.09, 136.83, 126.60, 119.28, 42.67. HRMS (ESI/QTOF) m/z: [M + H]+ Calcd. for
[C13H15N7SO6H]+ 398.0877; Found: 398.0877 [C13H15N7SO6H]+; 341.0663 [C11H12N6SO5H]+; 284.0448
[C9H9N5SO4H]+.
3.2.5. Synthesis of 200,2000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis(azanediyl)
diacetic Acid 3 (Scheme 5)
0
0
0
0
0
0
4-(4 ,6 -Dichloro-1 ,3 ,5 -triazin-2 -ylamino)-benzene-sulfonamide 1. (0.2 g, 1.0 equiv) and glycine (3.0 equiv)
were stirred in H2O (2 mL) at r.t. for 10 min. Then aqueous solution of Na2CO3 (4.0 equiv) was added
dropwise into the mixture of precursor 1 and glycine. The mixture was refluxed for 24 h and monitored
by TLC. The synthesis was terminated by the addition of a few drops of 1M HCl (up to pH = 2.0) until a
white precipitate was no longer produced. The product was filtered off and dried under high vacuum.
200,2000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-bis(azanediyl)diacetic acid (
3). The product
was obtained as a white solid in 96% yield. IR (KBr): 2968 (w), 2360 (w), 2343 (w), 1683 (m), 1598
(m), 1507 (s), 1407 (s), 1159 (s), 1096 (w), 847 (w), 779 (s), 588 (m), 544 (s); 1H-NMR (DMSO-d6, 80 ◦C)
δ
: 9.17 (s, 1H, Ar-NH), 7.89 (d, 2H, J = 8.9 Hz, H-2), 7.67 (d, 2H, J = 8.9 Hz, H-3), 6.94 (br s, 4H,
SO2NH2, 2 × CH2-NH), 3.95 (d, 4H, J = 5.2 Hz, 2 × CH2-NH); 13C-NMR (DMSO-d6, 80 ◦C)
δ: 172.03,
166.23, 164.40, 144.04, 136.92, 126.66, 119.32, 42.70. HRMS (ESI/QTOF) m/z: [M + H]+ Calcd. for
[C13H15N7SO6H]+ 398.0877; Found: 398.0878.
3.2.6. Synthesis of 300,3000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]bis(azanediyl)
Dipropanoic acid 4 (Scheme 6)
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)-benzene-sulfonamide
(3.0 equiv) were stirred in H2O (2 mL) at r.t. for 10 min. Then aqueous solution of Na2CO3 (4.0 equiv)
was added dropwise into the mixture of precursor and -alanine. The mixture was refluxed for 24 h
1. (0.2 g, 1.0 equiv) and β-alanine
1
β
and monitored by TLC. The synthesis was terminated by the addition of a few drops of 1 M HCl (up
to pH = 3.0) until a white precipitate was no longer produced. The product was filtered off and dried
under high vacuum.
300,3000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]bis(azanediyl) dipropanoic acid (
was obtained as a white solid in 98% yield. IR (KBr): 2967 (m), 2361 (s), 2343 (m), 1630 (m), 1592 (s), 1509
(s), 1399 (m), 1163 (s), 889 (w), 678 (w), 581 (m), 552 (m); 1H-NMR (DMSO-d6, 80 ◦C)
: 9.32 (br s, 1H,
Ar-NH), 7.92 (d, 2H, J = 8.8 Hz, H-2), 7.69 (d, 2H, J = 8.8 Hz, H-3), 6.96 (br s, 4H, SO2NH2, 2 CH2-NH),
3.53 (t, J = 7.1 Hz, 4H, H-200, H-2000), 2.54 (t, 4H, J = 7.1 Hz, H-300, H-3000); 13C-NMR (DMSO-d6, 80 ◦C)
4). The product
δ
×
δ:

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173.19, 164.70, 163.70, 143.83, 137.17, 126.68, 119.42, 36.98, 34.54. HRMS (ESI/QTOF) m/z: [M + H]+ Calcd.
for [C15H19N7SO6H]+ 426.1190; Found 426.1190.
4. Conclusions
In this work, three synthetic procedures for a group of triazinyl-substituted benzene-sulfonamide
conjugates with amino acids were designed and tested as alternatives to the already established
procedure used for the preparation of these highly potent CA-inhibitors. It was clearly demonstrated
that the synthetic procedure based on an amino acid (here glycine and
β-alanine) attachment
(i.e., nucleophilic substitution into two equivalent positions) to 4-(40,60-dichloro-10,30,50-triazin-
20-ylamino)-benzene-sulfonamide in a water medium containing sodium carbonate provided
significant improvement in terms of (i) the maximization of the reaction yield ( 95%, i.e., ca.
≥
2–3-fold higher as that declared in the literature), (ii) the minimization of organic solvents
employed (green synthesis), and (iii) the simplification of the synthetic and isolation procedure.
The proposed procedure is also attractive for the effective preparation of required triazinyl-substituted
benzene-sulfonamide conjugates with other amino acids containing an amino group as the only
significant nucleophile in the structure. In this way, new analogues in the studied group of
CA-inhibitors can be easily prepared.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This work was supported by the projects VEGA 1/0873/15, KEGA 022UK-4/2015, and
APVV-15-0585. The authors would like to give their great thanks to Dr. Branislav Horváth for his valuable
assistance and advice during the measurements.
Author Contributions: P.M. proposed and coordinated all the work. D.K. performed the experiments. D.P. and
D.K. analyzed data. P.M. and D.K. wrote the manuscript. V.G. provided consultations concerning the established
synthetic method for triazinyl-substituted benzene-sulfonamide conjugates. All authors read and approved the
final version of the manuscript.
Conflicts of Interest: The authors do not have any conflict of interest concerning the present work.
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Sample Availability: Samples of the compounds are not available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).