Synthesis and carbonic anhydrase I, II, IV and XII inhibitory properties of N-protected amino acid – sulfonamide conjugates

Article abstract of DOI:10.3109/14756366.2016.1147438

N-protected amino acids (Gly, Ala and Phe protected with Boc and Z groups) were reacted with sulfonamide derivatives, leading to the corresponding N-protected amino acid–sulfonamide conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity o

Full text of DOI:10.3109/14756366.2016.1147438

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and carbonic anhydrase I, II, IV and XII
inhibitory properties of N-protected amino acid –
sulfonamide conjugates
F. Zehra Küçükbay, Hasan Küçükbay, Muhammet Tanc & Claudiu T. Supuran
To cite this article: F. Zehra Küçükbay, Hasan Küçükbay, Muhammet Tanc & Claudiu T. Supuran
(2016): Synthesis and carbonic anhydrase I, II, IV and XII inhibitory properties of N-protected
amino acid – sulfonamide conjugates, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.3109/14756366.2016.1147438
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1147438
Published online: 21 Feb 2016.
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Date: 25 February 2016, At: 03:29

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2016 Taylor & Francis. DOI: 10.3109/14756366.2016.1147438
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RESEARCH ARTICLE
Synthesis and carbonic anhydrase I, II, IV and XII inhibitory properties of
N-protected amino acid – sulfonamide conjugates
F. Zehra Ku¨c¸u¨kbay1,2, Hasan Ku¨c¸u¨kbay2,3, Muhammet Tanc2, and Claudiu T. Supuran2
1
2
_
Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Ino¨nu¨ University, Malatya, Turkey, Dipartimento Neurofarba, Sezione Di
`
Scienze Farmaceutiche E Nutraceutiche e Laboratorio Di Chimica Bioinorganica, Universita Degli Studi Di Firenze, Sesto Fiorentino, Florence, Italy,
3
and Department of Chemistry, Faculty of Arts and Science, Ino¨nu¨ University, Malatya, Turkey
_
Abstract
Keywords
N-protected amino acids (Gly, Ala and Phe protected with Boc and Z groups) were reacted with
sulfonamide derivatives, leading to the corresponding N-protected amino acid–sulfonamide
conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds
was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV and hCA XII. Among them,
hCA II, IV and XII are antiglaucoma drug targets, being involved in aqueous humor secretion
within the eye. Low nanomolar inhibition was measured against all four isoforms with the 20
reported sulfonamides, but no selective inhibitory profiles, except for some CA XII-selective
derivatives, were observed. hCA I, II and XII were generally better inhibited by sulfonamides
incorporating longer scaffolds and Gly/Ala, whereas the best hCA IV inhibitors were
homosulfanilamide derivatives, incorporating Phe moieties. The amino acid–sulfonamide
conjugates show good water solubility and effective hCA II, IV and XII inhibition, and may be
considered as interesting candidates for antiglaucoma studies.
Carbonic anhydrase, glaucoma, inhibitor,
N-protected amino acid, sulfonamide
History
Received 5 January 2016
Accepted 25 January 2016
Published online 19 February 2016
Introduction
site coinciding with that where CA activators bind13–20
;
(iv) the compounds which bind out of the active site cavity
(a carboxylic acid derivative was seen to inhibit CA in this
manner)21, and (v) compounds for which the inhibition mechan-
ism is not known, among which the secondary/tertiary sulfona-
mides as well as imatinib/nilotinib are the most investigated
There are six different genetic families encoding for the
metalloprotein carbonic anhydrase (CA, EC 4.2.1.1), a superfam-
ily of metalloenzymes acting as catalysts for the interconversion
between CO2 and bicarbonate. These are the a-, b-, g-, d-, z- and
Z-CA classess1–11. The catalytic/inhibition mechanisms for the
physiologic and other catalyzed by these enzymes are well
understood processes12–21. The discovery of new classes of CA
inhibitors (CAIs), possessing different inhibition mechanisms
compared with the classical inhibitors of the sulfonamide/anion
type1–3,5, has also seen important developments ultimately13. At
least five different CA inhibition mechanisms were reported so
far: (i) the zinc binders are the inhibitors which coordinate to the
catalytically crucial Zn(II) ion from the enzyme active site. The
metal ion may be in a tetrahedral or trigonal bipyramidal
geometries, with the sulfonamides and their isosteres (sulfamides,
sulfamates, etc.), most anions, dithiocarbamates and their
isosteres, carboxylates and hydroxamates binding in this
way1–3,5,13; (ii) the inhibitors that anchor to the zinc-coordinated
water molecule/hydroxide ion, represented by the phenols, some
carboxylates, the polyamines, 2-thioxocoumarins, and sulfocou-
marins1–3,13,14; (iii) the inhibitors which occlude the entrance to
the active site cavity (coumarins and their isosteres), this binding
examples13,22–26
.
The sulfonamides however remain the main class of CAIs with
many clinically used drugs as antiglaucoma agents27–29, diur-
etics30, antiobesity drugs31–33, antiepilpetics34, and more recently
agents for the management of hypoxic tumors35–38, neuropathic
pain39 and ischaemia40,41
.
Continuing our interest in designing biologically active
compounds incorporating N-protected amino acid moieties (the
coumarine19 conjugates as CAIs as well as quinine conjugates
were recently reported42), we focused on the synthesis of amino
acid–sulfonamide conjugates by using the N-(protected a-ami-
noacyl)benzotriazole methodology43 and explore the enzyme
inhibitory activities of such compounds against physiologically
important isoforms, such as the cytosolic human (h) hCAI and II,
as well as the membrane-asssociated/transmembrane hCA IV and
XII1–5
.
Materials and methods
Chemistry
Address for correspondence: Hasan Ku¨c¸u¨kbay and Claudiu T. Supuran,
`
Universita degli Studi di Firenze, Dipartimento Neurofarba, Sezione di
Anhydrous solvents and all reagents were purchased from Sigma-
Aldrich (Milan, Italy), Acros (Milan, Italy) and Merck (Florence,
Italy). All reactions involving air- or moisture-sensitive com-
pounds were performed under a nitrogen atmosphere using dried
Scienze Farmaceutiche e Nutraceutiche e Laboratorio di Chimica
Bioinorganica, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
E-mail: hasan.kucukbay@inonu.edu.tr (H.K.);
claudiu.supuran@unifi.it (C.S.)

¨ ¨
F. Z. Kuc¸ukbay et al.
2
J Enzyme Inhib Med Chem, Early Online: 1–8
glassware and syringes techniques to transfer solutions. Nuclear (s, 1H, NH), 8.24 (s, 1H, Ar-H), 7.79 (d,1H, Ar-H, J ¼ 4.0 Hz),
magnetic resonance (1H-NMR, 13C-NMR) spectra were recorded 7.68 (d, 1H, NH, J ¼ 8.0 Hz), 7.57 (s, 2H, NH2), 7.52–7.36 (m,
using a Bruker Advance III 400 MHz spectrometer in DMSO-d6. 7H, Ar-H), 5.08 (s, 2H, OCH2Ph), 4.23 (d, 1H, CHNH, J ¼
Chemical shifts are reported in parts per million (ppm) and the 8.0 Hz), 1.35 (d, 3H, CH3, J ¼ 8.0 Hz). 13C NMR (DMSO-d6,
coupling constants (J) are expressed in Hertz (Hz). Splitting 400 MHz) ꢀ 172.9 (COCH2NH), 156.7 (COOCH2Ph), 145.5,
patterns are designated as follows: s, singlet; d, doublet; t, triplet; 140.2, 137.9, 130.3, 129.2, 128.7, 128.6, 123.0, 121.2, 117.2 (Ar-
m, multiplet; brs, broad singlet; dd, double of doubles. The C), 66.4 (OCH2Ph), 51.8 (CHNH), 18.7 (CH3). HRMS m/z for
assignment of exchangeable protons (OH and NH) was confirmed C17H19N3O5S
[M + H]+ calcd.
378.1,
found
378.0;
by the addition of D2O. Positive-ion electrospray ionization (ESI) [M + NH4]+ calcd. 395.1, found 395.1; [M + Na]+ calcd. 400.1,
mass spectra were recorded on a double-focusing Finnigan MAT found 400.0; [M ꢁ H]ꢁ calcd. 376.1, found 376.0; [M + Cl]ꢁ
95 instrument with BE geometry. Analytical thin-layer chroma- calcd. 412.1, found 412.1.
tography (TLC) was carried out on Merck silica gel F-254 plates.
All microwave-assisted reactions were carried out in a microwave (S)-benzyl (1-oxo-3-phenyl-1-((3-sulfamoylphenyl)amino)
oven system manufactured by GEM (CEM Corporation, propan-2-yl)carbamate, 3
Matthews, NC, USA) under a nitrogen atmosphere. The reaction
White solid (86%); silica gel TLC Rf ¼ 0.29 (MeOH/CH2Cl2 5%
mixtures were transferred into a 10 mL glass pressure microwave
tube equipped with a magnetic stir bar. The tube was closed with a
silicon septum and the reaction mixture was subjected to
microwave irradiation. Melting points (mp) were measured in
1
v/v); mp 139–140 ꢀC; H NMR (DMSO-d6, 400 MHz) 10.46 (s,
1H, NH), 8.21 (s, 1H, Ar-H), 7.80–7.77 (m, 2H, Ar-H + NH), 7.56
(s, 2H, NH2), 7.40–7.25 (m, 12H, Ar-H), 5.01 (s, 2H, OCH2Ph),
4.48–4.43 (m, 1H, CHNH), 3.11–3.07 (m, 1H, CH2), 2.93–2.87
(m, 1H, CH2). 13C NMR (DMSO-d6, 400 MHz) ꢀ 171.9
open capillary tubes and are uncorrected, using a Gallenkamp
MPD350.BM3.5 apparatus. N-protected amino acids19,43–45
;
(COCH2NH), 156.9 (COOCH2Ph), 145.5, 140.1, 138.6, 137.8,
130.4, 130.1, 129.2, 129.0, 128.7, 128.5, 127.3, 123.1, 121.4,
117.3 (Ar-C), 66.3 (OCH2Ph), 57.9 (CHNH), 38.2 (CH2CH).
HRMS m/z for C23H23N3O5S [M + H]+ calcd. 454.1, found 454.1;
[M + NH4]+ calcd. 471.1, found 471.1; [M + Na]+ calcd. 476.1,
found 476.1; [M ꢁ H]ꢁ calcd. 452.1, found 452.2; [M + Cl]ꢁ
calcd. 488.1, found 488.2.
benzyl (2-1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)carbamate
(I), tert-butyl (2-1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)car-
bamate (II), (S)-benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-
oxopropan-2-yl)carbamate (III), (S)-tert-butyl (1-(1H-ben-
zo[d][1,2,3]triazol-1-yl)-1-oxo-3-phenylpropan-2-yl)carbamate
(IV) and sulfonamide derivatives46; 2-(4-sulfamoylphenoxy)etha-
naminium 2,2,2-trifluoroacetate, 3-(4-sulfamoylphenoxy)propan-
1-aminium chloride, 4-(4-sulfamoylphenoxy)butan-1-aminium
2,2,2-trifluoroacetate used as starting materials were prepared
according to literature procedure. Compounds 347, 448, 548 and
General procedure for the synthesis of amino
acid–sulfonamide conjugates, 4–6
1047 were found in the literature but their synthesis methods need A mixture of equivalent amounts of the appropriate N-protected
harsh reaction conditions and cumbersome work-up procedures. aminoacylbenzotriazole and 4-aminobenzenesulfonamide was
Thus, these compounds were synthesized by a new, one-step, easy subjected to microwave irradiation (100 W, 70 ꢀC) in anhydrous
synthetic method, using N-(protected a-aminoacyl)benzotriazoles THF (5 mL) for 30 min. After completion of the reaction, all
in this work.
volatiles were removed by rotavapour and the obtained crude
product was crystallized from methanol.
General procedure for the synthesis of amino acid–
sulfonamide conjugates, 1–3
Benzyl (2-oxo-2-((4-sulfamoylphenyl)amino)ethyl)carbamate, 4
A mixture of equivalent amounts of the appropriate N-protected White solid (84%); silica gel TLC Rf ¼ 0.38 (MeOH/CH2Cl2 5%
1
aminoacylbenzotriazole and 3-aminobenzenesulfonamide was v/v); mp 186–187 ꢀC; H NMR (DMSO-d6, 400 MHz) 10.34 (s,
subjected to microwave irradiation (100 W, 70 ꢀC) in anhydrous 1H, NH), 7.82–7.78 (m, 4H, Ar-H), 7.61 (t, 1H, NH, J ¼ 8.0 Hz),
THF (5 mL) for 30 min. After completion of the reaction, all 7.42–7.37 (m, 5H, Ar-H), 7.27 (s, 2H, NH2), 5.10 (s, 2H,
volatiles were removed by rotavapour and the obtained crude OCH2Ph), 3.88 (d, 2H, CH2NH, J ¼ 8.0 Hz). 13C NMR (DMSO-
product was crystallized from methanol.
d6, 400 MHz) ꢀ 169.4 (COCH2NH), 157.5 (COOCH2Ph), 142.7,
139.3, 137.9, 129.2, 128.7, 128.6, 127.6, 119.5 (Ar-C), 66.4
(OCH2Ph), 45.1 (CH2NH). HRMS m/z for C16H17N3O5S
[M + H]+ calcd. 364.1, found 364.0; [M + Na]+ calcd. 386.1,
found 386.0; [M ꢁ H]ꢁ calcd. 362.1, found 362.1; [M + Cl]ꢁ
calcd. 398.1, found 398.0.
Benzyl (2-oxo-2-((3-sulfamoylphenyl)amino)ethyl)carbamate, 1
White solid (83%); silica gel TLC Rf ¼ 0.16 (MeOH/CH2Cl2 5%
v/v); mp 182–183 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 10.32
(s, 1H, NH), 8.21 (s, 1H, Ar-H), 7.79 (d,1H, Ar-H, J ¼ 4.0 Hz),
7.62 (t, 1H, NH, J ¼ 8.0 Hz), 7.56 (s, 2H, NH2), 7.42–7.36 (m,
7H, Ar-H), 5.10 (s, 2H, OCH2Ph), 3.88 (d, 2H, CH2NH, J ¼
8.0 Hz). 13C NMR (DMSO-d6, 400 MHz) ꢀ 169.3 (COCH2NH),
(S)-benzyl (1-oxo-1-((4-sulfamoylphenyl)amino)propan-2-yl)
carbamate, 5
157.6 (COOCH2Ph), 145.6, 140.1, 137.9, 130.4, 129.3, 128.8, White solid (77%); silica gel TLC Rf ¼ 0.25 (MeCOOEt/Hexane
128.7, 122.9, 121.3, 117.1, 117.4 (Ar-C), 66.5 (OCH2Ph), 45.1 50% v/v); mp 240–241 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 10.36
(CH2NH). HRMS m/z for C16H17N3O5S [M + H]+ calcd. 364.1, (s, 1H, NH), 7.81 (brs, 4H, Ar-H), 7.70 (d, 1H, NH, J ¼ 8.0 Hz),
found 364.0; [M + NH4]+ calcd. 381.1, found 381.1; 7.41–7.40 (m, 5H, Ar-H), 7.29 (s, 2H, NH2), 5.08 (s, 2H,
[M + Na]+ calcd. 386.1, found 386.0; [M ꢁ H]ꢁ calcd. 362.1, OCH2Ph), 4.26 (t, 1H, CHNH, J ¼ 8.0 Hz), 1.35 (d, 3H, CH3, J ¼
found 362.1; [M + Cl]ꢁ calcd. 398.1, found 398.0.
8.0 Hz). 13C NMR (DMSO-d6, 400 MHz) ꢀ 173.1 (COCH2NH),
156.8 (COOCH2Ph), 152.8, 142.8, 139.4, 137.9, 129.3, 128.7,
128.6, 127.6, 119.7, 113.4 (Ar-C), 66.4 (OCH2Ph), 51.8 (CHNH),
18.8 (CH3). HRMS m/z for C17H19N3O5S [M + H]+ calcd.
378.1, found 378.0; [M + Na]+ calcd. 400.1, found 400.0;
(S)-benzyl (1-oxo-1-((3-sulfamoylphenyl)amino)propan-2-yl)
carbamate, 2
White solid (76%); silica gel TLC Rf ¼ 0.20 (MeCOOEt/Hexane [M ꢁ H]ꢁ calcd. 376.1, found 376.1; [M + Cl]ꢁ calcd. 412.1,
50% v/v); mp 178–179 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 10.32 found 412.1.

DOI: 10.3109/14756366.2016.1147438
N-protected amino acid–sulfonamide conjugates
3
(S)-benzyl (1-oxo-4-phenyl-1-((3-sulfamoylphenyl)amino)
propan-2-yl)carbamate, 6
143.6, 138.9, 137.9, 130.2, 129.2, 129.0, 128.6, 128.4, 128.3,
127.2, 126.5 (Ar-C), 66.2 (OCH2Ph), 57.3 (CH2Ph), 42.7 (CH),
38.4 (CH3). HRMS m/z for C24H25N3O5S [M + H]+ calcd. 368.2,
found 468.1; [M + Na]+ calcd. 490.1, found 460.1; [M + Cl]ꢁ
calcd. 502.1, found 502.2.1; [M + HCOO]ꢁ calcd. 512.1, found
512.2.
White solid (87%); silica gel TLC Rf ¼ 0.32 (MeOH/CH2Cl2 5%
1
v/v); mp 216–217 ꢀC; H NMR (DMSO-d6, 400 MHz) 10.47 (s,
1H, NH), 7.84–7.78 (m, 6H, Ar-H + NH), 7.38–7.23 (m, 14H, Ar-
H + NH2), 5.02 (s, 2H, OCH2Ph), 4.52–4.47 (m, 1H, CHNH),
3.12–3.07 (m, 1H, CH2), 2.95–2.89 (m, 1H, CH2). 13C NMR
(DMSO-d6, 400 MHz) ꢀ 172.0 (COCH2NH), 156.9 (COOCH2Ph),
142.6, 139.5, 138.5, 137.8, 130.1, 129.2, 129.0, 128.7, 128.5,
General procedure for the synthesis of amino acid–
sulfonamide conjugates, 10–13
127.6, 127.3, 119.8 (Ar-C), 66.3 (OCH2Ph), 57.9 (CHNH), A mixture of equivalent amounts of the appropriate N-protected
38.3 (CH2CH). HRMS m/z for C23H23N3O5S [M + H]+ calcd. aminoacylbenzotriazole and 4-(2-aminoethyl)benzenesulfona-
454.1, found 454.1; [M + Na]+ calcd. 476.1, found 476.1; mide was subjected to microwave irradiation (100 W, 70 ꢀC) in
[M ꢁ H]ꢁ calcd. 452.1, found 452.2; [M + Cl]ꢁ calcd. 488.1, anhydrous THF (5 mL) for 30 min. After completion of the
found 488.2.
reaction, all volatiles were removed by rotavapour and the
obtained crude product was crystallized from methanol.
General procedure for the synthesis of amino
acid–sulfonamide conjugates, 7–9
Benzyl (2-oxo-2-((4-sulfamoylphenethyl)amino)ethyl)carbamate,
10
A mixture of equivalent amounts of the appropriate N-protected
aminoacyl-benzotriazole and (4-sulfamoylphenyl)methanami- Beige solid (95%); silica gel TLC Rf ¼ 0.58 (MeOH/CH2Cl2 5%
nium chloride was subjected to microwave irradiation (100 W, v/v); mp 161–162 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 7.99 (t, 1H,
70 ꢀC) in anhydrous THF (5 mL) for 30 min. After completion of NH, J ¼ 4.0 Hz), 7.78 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.45–7.40 (m,
the reaction, all volatiles were removed by rotavapour and the 8H, Ar-H + NH), 7.33 (s, 2H, NH2), 5.08 (s, 2H, OCH2Ph), 3.61
obtained crude product was crystallized from methanol.
(d, 2H, CH2Ph, J ¼ 8.0 Hz), 3.33 (t, 2H, CH2CH2NH, J ¼ 8.0 Hz),
2.83 (t, 2H, CH2CH2NH, J ¼ 8.0 Hz). 13C NMR (DMSO-d6,
400 MHz) ꢀ 170.0 (COCH2NH), 157.4 (COOCH2Ph), 144.6,
143.0, 138.0, 130.0, 129.3, 128.7, 128.6, 126.6 (Ar-C), 66.4
(OCH2Ph), 44.5 (CH2NH), 41.1 (CH2CH2Ph), 35.7 (CH2CH2Ph).
HRMS m/z for C18H21N3O5S [M + H]+ calcd. 392.1, found 392.1;
[M + NH4]+ calcd. 409.2, found 409.1; [M + Na]+ calcd. 414.1,
found 414.0; [M + Cl]ꢁ calcd. 426.1, found 426.1; [M + HCOO]ꢁ
calcd. 436.1, found 436.1.
Benzyl (2-oxo-2-((4-sulfamoylbenzyl)amino)ethyl)carbamate, 7
White solid (95%); silica gel TLC Rf ¼ 0.20 (MeOH/CH2Cl2 5%
v/v); mp 182–183 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 8.51 (t, 1H,
NH, J ¼ 4.0 Hz), 7.89 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.54 (t, 1H, NH,
J ¼ 8.0 Hz), 7.47–7.35 (m, 10H, Ar-H + NH2), 5.08 (s, 2H,
OCH2Ph), 4.38 (d, 2H, CH2Ph, J ¼ 4.0 Hz), 3.71 (d,2H, CH2NH,
J
¼
8.0 Hz). 13C NMR (DMSO-d6, 400 MHz)
ꢀ 170.2
(COCH2NH), 157.4 (COOCH2Ph), 144.4, 143.5, 137.9,
129.2, 128.7, 128.6, 128.4, 126.5 (Ar-C), 66.4 (OCH2Ph), 44.5
(CH2Ph), 42.6 (CH2NH). HRMS m/z for C17H19N3O5S
tert-Butyl (2-oxo-2-((4-sulfamoylphenethyl)amino)ethyl)
carbamate, 11
[M + H]+ calcd. 378.1, found 378.0; [M + NH4]+ calcd. 395.1, White solid (89%); silica gel TLC Rf ¼ 0.60 (MeOH/CH2Cl2 30%
found 395.1; [M + Na]+ calcd. 400.1, found 400.0; [M + Cl]ꢁ v/v); mp 201–202 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 7.89 (t, 1H,
calcd. 412.1, found 412.1; [M + HCOO]ꢁ calcd. 422.1, found NH, J ¼ 4.0 Hz), 7.78 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.43 (d, 2H, Ar-
422.1
H, J ¼ 8.0 Hz), 7.32 (s, 2H, NH2), 6.94 (t, 1H, NH, J ¼ 4.0 Hz),
3.52 (d, 2H, CH2NH, J ¼ 4.0 Hz), 3.34 (t, 2H, CH2CH2N, J ¼
8.0 Hz), 2.82 (t, 2H, NCH2CH2, J ¼ 8.0 Hz), 1.42 (s, 9H,
(CH3)3C). 13C NMR (DMSO-d6, 400 MHz) ꢀ 170.2 (COCH2NH),
156.7 (COOCH2Ph), 144.6, 143.0, 130.0, 126.6 (Ar-C), 79.0
((CH3)3CO, 44.2 (CH2NH), 40.9 (NHCH2CH2), 35.7
(NHCH2CH2), 29.1 ((CH3)3C). HRMS m/z for C15H23N3O5S
[M + Na]+ calcd. 380.1, found 380.1; [M + Cl]ꢁ calcd. 392.1,
found 392.1; [M + HCOO]ꢁ calcd. 402.1, found 402.1.
(S)-benzyl (1-oxo-1-((4-sulfamoylbenzyl)amino)propan-2-yl)
carbamate, 8
White solid (93%); silica gel TLC Rf ¼ 0.36 (MeOH/CH2Cl2 5%
v/v); mp 181–182 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 8.55 (t, 1H,
NH, J ¼ 8.0 Hz), 7.80 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.46–7.35 (m,
10H, Ar-H + NH + NH2), 5.07 (s, 2H, CH2Ph), 4.38 (d, 2H,
CH2Ph, J ¼ 8.0 Hz), 4.15–4.10 (m, 1H, CH), 1.28 (d, 3H, CH3, J
¼ 4.0 Hz). 13C NMR (DMSO-d6, 400 MHz) ꢀ 173.7 (COCH2NH),
156.7 (COOCH2Ph), 144.6, 143.5, 137.9, 129.3, 128.8, 128.7, (S)-benzyl (1-oxo-1-((4-sulfamoylphenethyl)amino)propan-2-yl)
128.2, 126.6 (Ar-C), 66.4 (OCH2Ph), 51.2 (CH2Ph), 42.6 (CH), carbamate, 12
19.2 (CH3). HRMS m/z for C18H21N3O5S [M + H]+ calcd. 392.1,
Yellow solid (92%); silica gel TLC Rf ¼ 0.46 (EtOAc/Hexane
found 392.1; [M + NH4]+ calcd. 409.1, found 409.2;
[M + Na]+ calcd. 414.1, found 414.1; [M + Cl]ꢁ calcd. 426.1,
found 426.1; [M + HCOO]ꢁ calcd. 436.1, found 436.1.
1
30% v/v); mp 130–131 ꢀC; H NMR (DMSO-d6, 400 MHz) 7.99
(t, 1H, NH, J ¼ 8.0 Hz), 7.93 (d, 1H, NH, J ¼ 4.0 Hz), 7.81–7.78
(m, 2H, Ar-H), 7.46–7.35 (m, 9H, Ar-H + NH2), 5.08 (s, 2H,
OCH2Ph), 4.02 (t, 2H, CH2CH2Ph, J ¼ 8.0 Hz), 3.39–3.31 (m,
1H, CHCH3), 2.82 (t, 2H, PhCH2CH2, J ¼ 8.0 Hz), 1.20 (d, 3H,
CH3, J ¼ 8.0 Hz). 13C NMR (DMSO-d6, 400 MHz) ꢀ 173.4
(S)-benzyl (1-oxo-3-phenyl-1-((4-sulfamoylbenzyl)amino)
propan-2-yl)carbamate, 9
White solid (94%); silica gel TLC Rf ¼ 0.29 (MeOH/CH2Cl2 5% (COCH2NH), 155.7 (COOCH2Ph), 144.6, 143.0, 138.0, 130.1,
v/v); mp 230–231 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 8.68 (t, 1H, 130.0, 129.3, 128.7, 126.6, 126.0, 115.9 (Ar-C), 66.3 (OCH2Ph),
NH, J ¼ 8.0 Hz), 7.77 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.64 (d,1H, NH, 51.1 (CHNH), 42.9 (NCH2CH2), 35.7 (CH2CH2Ph), 19.2
J ¼ 8.0 Hz), 7.36–7.30 (m, 14H, Ar-H + NH2), 5.01 (s, 2H, (CH3). HRMS m/z for C19H23N3O5S [M + H]+ calcd. 406.1,
OCH2Ph), 4.39–4.30 (m, 2H, CH2Ph + CH), 3.07–3.03 (m, 1H, found 406.1; [M + Na]+ calcd. 428.1, found 428.1; [M + Cl]ꢁ
CH2CH), 2.88–2.82 (m, 1H, CH2CH). 13C NMR (DMSO-d6, calcd. 440.1, found 440.1; [M + HCOO]ꢁ calcd. 450.1, found
400 MHz) ꢀ 172.5 (COCH2NH), 156.8 (COOCH2Ph), 144.3, 450.2.

¨ ¨
F. Z. Kuc¸ukbay et al.
4
J Enzyme Inhib Med Chem, Early Online: 1–8
(S)-benzyl (1-oxo-3-phenyl-1-((4-sulfamoylphenethyl)amino)
propan-2-yl)carbamate, 13
[M + Cl]ꢁ calcd. 456.1, found 456.1; [M + HCOO]ꢁ calcd.
466.1, found 466.1.
Beige solid (97%); silica gel TLC Rf ¼ 0.66 (MeOH/CH2Cl2 30%
v/v); mp 195–196 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 8.15 (t, 1H,
NH, J ¼ 8.0 Hz), 7.77 (d, 2H, ArH, J ¼ 4.0 Hz), 7.75 (d, 1H, NH,
(S)-benzyl (1-oxo-3-phenyl-1-((3-(4-sulfamoylphenoxy)propyl)
amino)propan-2-yl)carbamate, 16
J ¼ 4.0 Hz), 7.37–7.24 (m, 14H, Ar-H + NH2), 4.99 (s, 2H, White solid (89%); silica gel TLC Rf ¼ 0.45 (MeOH/CH2Cl2 5%
OCH2Ph), 4.23–4.22 (m, 1H, NHCHCH2Ph), 3.36 (t, 2H, v/v); mp 183–184 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 8.11 (t, 1H,
PhCH2CH2, J ¼ 8.0 Hz), 3.54 (t, 2H, PhCH2CH2, J ¼ 8.0 Hz), NH, J ¼ 4.0 Hz), 7.77 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.55 (d, 1H, NH,
2.85–2.76 (m, 3H, CH2), 2.97–2.93 (m, 1H, CHCH2), 2.81–2.78 J ¼ 8.0 Hz), 7.39–7.28 (m, 10H, Ar-H), 7.23 (s, 2H, NH2), 7.08 (d,
(m, 3H, CHCH2 + PhCH2CH2) . 13C NMR (DMSO-d6, 400 MHz) 2H, Ar-H, J ¼ 8.0 Hz), 4.99 (s, 2H, OCH2Ph), 4.22 (m, 1H,
ꢀ 172.3 (COCH2NH), 156.7 (COOCH2Ph), 144.6, 143.0, 138.0, CHCH2), 4.01 (t, 2H, CH2CH2O, J ¼ 8.0 Hz), 3.24 (m, 2H,
139.0, 138.0, 130.1, 129.2, 129.0, 128.6, 128.4, 127.2, 126.6 (Ar- CH2CH2NH), 3.01–2.96 (m, 1H, CHCH2), 2.81–2.78 (m, 1H,
C), 66.2 (OCH2Ph), 57.2 (CHNH), 40.7 (CH2CH2Ph), 38.5 CHCH2), 1.86 (m, 2H, CH2CH2CH2). 13C NMR (DMSO-d6,
(NCHCH2), 35.7 (CH2CH2Ph). HRMS m/z for C25H27N3O5S 400 MHz)
ꢀ
172.2 (COCH2NH), 161.8 (OC6H4), 156.7
[M + H]+ calcd. 482.2, found 482.2; [M + Na]+ calcd. 504.2, (COOCH2Ph), 138.9, 137.9, 137.0, 130.0, 129.2, 128.9, 128.6,
found 504.1; [M + Cl]ꢁ calcd. 516.1, found 516.2; 128.5, 128.0, 127.1, 115.3 (Ar-C), 66.6 (OCH2Ph), 66.1
[M + HCOO]ꢁ calcd. 526.2, found 526.3.
(CHCH2), 57.2 (CH2CH2O), 38.6 (NHCH2CH2), 36.3 (CHCH2),
29.5
(CH2CH2CH2).
HRMS
m/z
for
C26H29N3O6S
Benzyl (2-oxo-2-((2-(4-sulfamoylphenoxy)ethyl)amino)ethyl)
carbamate, 14
[M + H]+ calcd. 512.2, found 512.1; [M + Na]+ calcd. 534.2,
found 534.1.
A
mixture of benzyl (2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-
General procedure for the synthesis of amino
acid–sulfonamide conjugates, 17–20
oxoethyl)carbamate (0.12 g; 0.39 mmol), 2-(4-sulfamoylphenox-
y)ethanaminium 2,2,2-trifluoroacetate (0.13 g; 0.39 mmol) and
Et3N (0.06 mL; 0.43 mmol) was subjected to microwave irradi- A mixture of equivalent amounts of the appropriate N-protected
ation (100 W, 70 ꢀC) in anhydrous THF (5 mL) for 30 min. After aminoacylbenzotriazole,
4-(4-sulfamoylphenoxy)butan-1-ami-
completion of the reaction, all volatiles were removed by nium 2,2,2-trifluoroacetate and Et3N was subjected to microwave
rotavapour and the obtained crude product was crystallized irradiation (100 W, 70 ꢀC) in anhydrous THF (5 mL) for 30 min.
from methanol.
After completion of the reaction, all volatiles were removed by
White solid (0.14 g, 93%); silica gel TLC Rf ¼ 0.75 (MeOH/ rotavapour and the obtained crude product was crystallized from
CH2Cl2 5% v/v); mp 183–184 ꢀC; 1H NMR (DMSO-d6, 400 MHz) methanol.
7.99 (t, 1H, NH, J ¼ 4.0 Hz), 7.78 (d, 2H, Ar-H, J ¼ 8.0 Hz),
7.45–7.40 (m, 8H, Ar-H + NH), 7.33 (s, 2H, NH2), 5.08 (s, 2H, Benzyl (2-oxo-2-((4-(4-sulfamoylphenoxy)butyl)amino)ethyl)
OCH2Ph), 3.61 (d, 2H, CH2Ph, J ¼ 8.0 Hz), 3.33 (t, 2H, carbamate, 17
CH2CH2NH, J ¼ 8.0 Hz), 2.83 (t, 2H, CH2CH2NH, J ¼ 8.0 Hz).
White solid (94%); silica gel TLC Rf ¼ 0.55 (MeOH/CH2Cl2 5%
v/v); mp 135–136 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 7.97 (t, 1H,
NH, J ¼ 4.0 Hz), 7.78 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.45 (t, 1H, NH,
J ¼ 4.0 Hz), 7.40–7.36 (m, 5H, Ar-H), 7.23 (s, 2H, NH2), 7.11 (d,
2H, Ar-H, J ¼ 8.0 Hz), 5.07 (s, 2H, OCH2Ph), 4.08 (t, 2H,
CH2CH2OPh, J ¼ 8.0 Hz), 3.63 (d, 2H, CH2NH, J ¼ 4.0 Hz), 3.16
(q, 2H, CH2CH2NH, J ¼ 8.0 Hz), 1.78–1.74 (m, 2H, CH2CH2O),
1.60–1.57 (m, 2H, NCH2CH2). 13C NMR (DMSO-d6, 400 MHz) ꢀ
169.9 (COCH2NH), 162.0 (OC6H4), 157.4 (COOCH2Ph), 138.0,
13C NMR (DMSO-d6, 400 MHz) ꢀ 170.0 (COCH2NH), 157.4
(COOCH2Ph), 144.6, 143.0, 138.0, 130.0, 129.3, 128.7, 128.6,
126.6 (Ar-C), 66.4 (OCH2Ph), 44.5 (CH2NH), 41.1 (CH2CH2Ph),
35.7 (CH2CH2Ph). HRMS m/z for C18H21N3O6S [M + Na]+ calcd.
430.1, found 430.0.
General procedure for the synthesis of amino
acid–sulfonamide conjugates, 15 and 16
A mixture of equivalent amounts of the appropriate N-protected 137.0, 129.3, 128.7, 128.6, 128.5, 115.4 (Ar-C), 68.6 (OCH2Ph),
aminoacylbenzotriazole, 3-(4-sulfamoylphenoxy)propan-1-ami- 66.4 (CH2CH2O), 44.5 (CH2NH), 39.1 (CH2CH2NH), 26.9
nium chloride and Et3N was subjected to microwave irradiation (CH2CH2O), 26.6 (NCH2CH2). HRMS m/z for C20H25N3O6S
(100 W, 70 ꢀC) in anhydrous THF (5 mL) for 30 min. After [M + Na]+ calcd. 458.1, found 458.1; [M + Cl]ꢁ calcd. 470.1,
completion of the reaction, all volatiles were removed by found 470.1; [M + HCOO]ꢁ calcd. 480.1, found 480.1.
rotavapour and the obtained crude product was crystallized
from methanol.
tert-butyl (2-oxo-2-((4-(4-sulfamoylphenoxy)butyl)amino)ethyl)-
carbamate, 18
Benzyl (2-oxo-2-((3-(4-sulfamoylphenoxy)propyl)amino)ethyl)-
carbamate, 15
White solid (88%); silica gel TLC Rf ¼ 0.29 (EtOAc/Hexane 30%
1
v/v); mp 75–76 ꢀC; H NMR (DMSO-d6, 400 MHz) 7.80 (t, 1H,
White solid (91%); silica gel TLC Rf ¼ 0.5 (MeOH/CH2Cl2 5% v/ NH, J ¼ 4.0 Hz), 7.77 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.22 (s, 2H,
1
v); mp 125–126 ꢀC; H NMR (DMSO-d6, 400 MHz) 7.97 (t, 1H, NH2), 7.10 (d, 2H, Ar-H, J ¼ 8.0 Hz), 6.93 (t, 1H, NH, J ¼
NH, J ¼ 4.0 Hz), 7.77 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.46–7.35 (m, 8.0 Hz), 4.08 (t, 2H, CH2CH2OPh, J ¼ 8.0 Hz), 3.53 (d, 2H,
6H, Ar-H + NH), 7.22 (s, 2H, NH2), 7.10 (d, 2H, Ar-H, J ¼ CH2NH, J ¼ 8.0 Hz), 3.16 (q, 2H, CH2CH2NH, J ¼ 8.0 Hz),
8.0 Hz), 5.06 (s, 2H, OCH2Ph), 4.09 (t, 2H, CH2CH2OPh, J ¼ 1.78–1.74 (m, 2H, CH2CH2O), 1.60–1.56 (m, 2H, NCH2CH2),
8.0 Hz), 3.80 (d, 2H, CH2NH, J ¼ 4.0 Hz), 3.27 (t, 2H, 1.42 (s, 9H, (CH3)3). 13C NMR (DMSO-d6, 400 MHz) ꢀ 170.0
CH2CH2NH, J ¼ 8.0 Hz), 1.91 (t, 2H, CH2CH2CH2, J ¼ (COCH2NH), 161.9 (OC6H4), 156.8 (COOCH2Ph), 137.0, 128.6,
8.0 Hz). 13C NMR (DMSO-d6, 400 MHz) ꢀ 170.0 (COCH2NH), 115.3 (Ar-C), 78.9 (OC(CH3)3), 68.7 (CH2CH2O), 52.2 (CH2NH),
161.9 (OC6H4), 157.4 (COOCH2Ph), 137.9, 137.0, 129.3, 128.7, 44.2 (CH2CH2NH), 38.9 (CH2CH2O), 29.1 (NCH2CH2), 26.8
128.6, 128.5, 115.4 (Ar-C), 66.6 (OCH2Ph), 66.4 (CH2CH2O), (CH3). HRMS m/z for C17H27N3O6S [M+] calcd. 401.2, found
44.5 (CH2NH), 36.9 (NHCH2CH2), 29.6 (CH2CH2CH2). HRMS 401.2; [M + Na]+ calcd. 424.2, found 424.1; [M + HCOO]ꢁ calcd.
m/z for C19H23N3O6S [M + Na]+ calcd. 444.1, found 444.1; 446.2, found 446.1.

DOI: 10.3109/14756366.2016.1147438
N-protected amino acid–sulfonamide conjugates
5
(S)-benzyl (1-oxo-1-((4-(4-sulfamoylphenoxy)butyl)amino)
propan-2-yl)carbamate, 19
at 70 ꢀC for 30 min, with good yields of 83%–97%. The synthetic
pathway of sulfonamides 1–20 is summarized in Scheme 1.
We have included in the study amino-sulfonamides having the
NH2 group directly linked to the benzene ring on which the
sulfonamide zinc-binding group is found (for example, metanila-
mide and sulfanilamide) as well as derivatives having various
linkers between the benzenesulfonamide fragment and the
primary amine group. These were of the alkylene type (methy-
lene, ethylene) or longer ones, of the -(CH2)nO- type, with n
varying between 2 and 4 (Scheme 1). The amino acids chosen for
derivatization were Gly, Ala and Phe, in order to investigate
whether this part of the inhibitor scaffold influences the CA
inhibitory properties. Furthermore, the amino group from the
amino acid reagent was protected with the benzyloxycarbonyl (Z)
or tert-butyloxycarbonyl (Boc) protecting groups, as shown in
Scheme 1.
Beige solid (93%); silica gel TLC Rf ¼ 0.45 (EtOAc/Hexane 30%
1
v/v); mp 76–77 ꢀC; H NMR (DMSO-d6, 400 MHz) 8.29 (t, 1H,
NH, J ¼ 8.0 Hz), 7.78 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.44–7.35 (m,
6H, Ar-H + NH), 7.22 (s, 2H, NH2), 7.10 (d, 2H, Ar-H, J ¼
8.0 Hz), 5.05 (s, 2H, OCH2Ph), 4.09–3.99 (m, 3H,
CH2CH2OPh + CH), 3.16–3.12 (m, 2H, CH2CH2NH), 1.77–1.74
(m, 2H, CH2CH2O), 1.60–1.56 (m, 2H, NCH2CH2), 1.23 (d, 3H,
CH3, J ¼ 8.0 Hz). 13C NMR (DMSO-d6, 400 MHz) ꢀ 173.2
(COCH2NH), 161.9 (OC6H4), 156.5 (COOCH2Ph), 138.0, 137.0,
129.2, 128.7, 128.6, 128.5, 115.3 (Ar-C), 68.6 (OCH2Ph), 66.2
(CH2CH2O), 51.1 (CHNH), 39.0 (CH2CH2NH), 26.8
(CH2CH2O), 26.5 (NCH2CH2), 19.3 (CH3). HRMS m/z for
C21H27N3O6S
[M + Na]+ calcd. 472.2, found 472.1.
[M + H]+ calcd.
450.2
Found
450.1;
The structures of N-protected amino acid–sulfonamide conju-
1
gates (1–20) were elucidated by H NMR, 13C NMR and mass
spectrometric analyses. All spectral data were in agreement with
the proposed structures. The characteristic NH resonances of the
(S)-benzyl (1-oxo-3-phenyl-1-((4-(4-sulfamoylphenoxy)butyl)
amino)propan-2-yl)carbamate, 20
White solid (97%); silica gel TLC Rf ¼ 0.77 (MeOH/CH2Cl2 30% sulfonamide part of the conjugates 1–6 and 2–20 were observed
v/v); mp 197–198 ꢀC; 1H NMR (DMSO-d6, 400 MHz) 8.04 (t, 1H, between 10.32–10.47 and 7.80–8.68 ppm, respectively. Other
NH, J ¼ 4.0 Hz), 7.78 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.52(d, 1H, NH, amino group resonances for the sulfonamide conjugates 1–20
J ¼ 8.0 Hz), 7.37–7.28 (m, 10H, Ar-H), 7.23 (s, 2H, NH2), 7.11 were observed between 6.93–8.24 ppm. Carbonyl resonances of
(d, 2H, Ar-H, J ¼ 8.0 Hz), 4.99 (s, 2H, OCH2Ph), 4.27–4.21 (m, the amide carbonyl and carbamate carbonyl were observed around
1H, CHCH2), 4.06 (t, 2H, CH2CH2O), 3.19–3.12 (m, 2H, 170 and 156 ppm, respectively. The characteristic NH2 resonances
CH2CH2N), 3.01–2.97 (m, 1H, CHCH2Ph), 2.84–2.78 (m, 1H, for sulfonamide functional group were observed between 7.22–
CHCH2Ph), 1.71 (t, 2H, CH2CH2O, J ¼ 8.0 Hz), 1.55 (t, 2H, 7.57 ppm in the 1H NMR spectrum. All other aliphatic and
NCH2CH2, J ¼ 8.0 Hz). 13C NMR (DMSO-d6, 400 MHz) ꢀ 172.1 aromatic protons and carbons were observed in the expected
(COCH2NH), 162.0 (OC6H4), 156.7 (COOCH2Ph), 139.0, 138.7, regions (see Materials and Methods).
138.0, 137.0, 130.1, 129.0, 128.6, 128.4, 127.2, 115.4 (Ar-C),
The hCA I, II, IV and XII enzyme inhibitory activity of the N-
68.6 (OCH2Ph), 66.1 (CHNH), 57.2 (CH2CH2O), 39.1 protected amino acid–sulfonamide conjugates 1–20 are shown in
(CH2CH2NH), 26.8 (CH2CH2O), 26.5 (NCH2CH2). HRMS m/z Table 1. We have chosen these isforms as three of them (CA II, IV
for C27H31N3O6S [M + H]+ calcd. 526.2 Found 526.1; and XII) are antiglaucoma drug targets1,27–29 whereas CA I, due
[M + Na]+ calcd. 548.2, found 548.1.
to its diffuse distribution in the blood and gastrointestinal tract is
one of the main off-targets for such pharmacologic agents57.
The following structure–activity relationship (SAR) can be
observed from the inhibition data of Table 1.
CA inhibition
An Applied Photophysics stopped-flow instrument has been used
for assaying the CA catalyzed CO2 hydration activity by using
method of Khalifah49. 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 HEPES (pH 7.5) as buffer, and 20 mM
Na2SO4 (for maintaining constant the ionic strength), following
the initial rates of the CA-catalyzed CO2 hydration reaction for a
period of 10–100 s. The CO2 concentrations ranged from 1.7–
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 0.01 nM were done thereafter
with the assay buffer. Inhibitor and enzyme solutions were pre-
incubated 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-square
methods using PRISM (www.graphpad.com), and non-linear least
squares methods, values representing the mean of at least three
hCA I was potently inhibited by sulfonamides 11, 14, 15, 17–
19 (inhibition constants ranging between 5.6 and 24.5 nM),
moderately inhibited by 6, 7, 9, 10, 12, 13 and 16 (KIs in the range
of 45.9–102.4) and poorly inhibited by the remaining derivatives,
which showed KIs in the range of 351.1–1946 nM (Table 1). Thus,
best inhibition for this isoform was associated with compounds
incorporating a long linker between the amino and benzenesulfo-
namide moieties (14, 15, 17–19), whereas the nature of the
protecting group (Pg) seem to be less important for activity
(compare the activity of 17–19). The Gly and Ala derivatives
were generally much better hCA I inhibitors compared with the
corresponding Phe derivatives. Acetazolamide (AZA, 5-aceta-
mido-1,3,4-thiadiazole-2-sulfonamide), a clinically used com-
pound, was a medium potency CAI for this isoform, with an
inhibition constant of 250 nM.
Only three derivatives showed highly effective hCA II
inhibitory activity, comparable to that of AZA, that is, 17–19
(KIs in the range of 8.6–11.9 nM). Again they incorporate the
longest scaffold, being Gly or Ala derivatives with Z- or Boc-
protecting groups. A number of other compounds (1, 4–6, and
8–15) were medium potency hCA II inhibitors, with KIs in the
range of 24.5–97.2 nM (Table 1). They incorporate all the
sulfonamide scaffolds employed in this study and this reveals
that in this case the nature of the linker and that of the amino acid
different determinations, as described earlier by us50–56
.
Results and discussion
New N-protected amino acid–sulfanamide conjugates (1–20) were moiety/protecting groups are the most important factors influen-
synthesized by the treatment of the appropriate sulfonamide cing the CA II inhibitory power. The remaining derivatives (2, 3,
derivatives, incorporating primary amine moieties, with 7 and 20) were the least effective inhibitors, with KIs in the range
N-protected aminoacylbenzotriazoles under microwave heating of 139–694 nM.

¨ ¨
F. Z. Kuc¸ukbay et al.
6
J Enzyme Inhib Med Chem, Early Online: 1–8
H2N
SO2NH2
O
H
N
Pg
N
H
SO2NH2
R
1
2
3
Pg= Z, R= H
Pg= Z, R= CH3
Pg= Z, R= CH2Ph
H2N
SO2NH2
SO2NH2
O
H
N
Pg
N
H
R
4
5
6
Pg= Z, R= H
Pg= Z, R= CH3
Pg= Z, R= CH2Ph
HCl
.
H2N
SO2NH2
O
H
N
Pg
N
H
R
SO2NH2
7
8
9
Pg= Z, R= H
Pg= Z, R= CH3
Pg= Z, R= CH2Ph
H2N
O
SO2NH2
H
N
SO2NH2
O
N
Pg
N
H
N
N
R
Pg
N
H
R
10 Pg= Z, R= H
Pg= Benzyloxycarbonyl (Z)
11 Pg= Boc, R= H
t-Butoxycarbonyl (Boc)
TFA
H2N
.
12 Pg= Z, R= CH3
13 Pg= Z, R= CH2Ph
I
Pg= Z, R= H
II Pg= Boc, R= H
SO2NH2
SO2NH2
SO2NH2
O
O
O
III Pg= Z, R= CH3
IV Pg= Z, R= CH2Ph
O
H
N
O
Pg
Pg
Pg
N
H
R
SO2NH2
14 Pg= Z, R= H
HCl
.
H2N
SO2NH2
O
H
N
N
H
O
R
TFA
.
15 Pg= Z, R= CH3
16 Pg= Z, R= CH2Ph
H2N
O
H
N
O
N
H
R
SO2NH2
17 Pg= Z, R= H
18 Pg= Boc, R= H
19 Pg= Z, R= CH3
20 Pg= Z, R= CH2Ph
Scheme 1. Synthesis pathways of the new sulfonamide conjugates of N-protected amino acids.
hCA IV, a membrane-anchored isoform1, was the least elongated molecule, all the best inhibitors of this isoform had a
sensitive CA among the investigated ones to inhibition by these more compact scaffold, being derivatives of metanilamide or
compounds. A group of derivatives, 1, 4–6, 9, 13 and 17 showed homosufanilamide. The remaining derivatives were either inactive
inhibitory activity in the same range or better than AZA, with KIs (2 and 20 had KIs410 mM) or were poorly active CAIs, with KIs
in the range of 27.9–93.5 nM (KI of AZA is of 74 nM). in the range of 174.9–1453 nM (Table 1).
Interestingly, the most potent CA IV inhibitor, 6, was a Phe
hCA XII, a transmembrane isoforms with an out of the cell
derivative (also incorporating Z as protecting group and the active site1 was more sensible to inhibition by the reported
homosufanilamide scaffold). Thus, apart 17 which has an sulfonamides, which with few exceptions (8, 9, 11 and 18) had

DOI: 10.3109/14756366.2016.1147438
N-protected amino acid–sulfonamide conjugates
7
Table 1. hCA I, II, IV and XII inhibition data with amino acid–
sulfonamide conjugates 1–20, by a stopped-flow CO2 hydrase assay49
As the amino acid–sulfonamide conjugates show good water
solubility and effective hCA II, IV and XII inhibition, they may be
considered as interesting candidates for antiglaucoma studies.
.
KI (nM)*
Acknowledgements
Compound
hCA I
hCA II
hCA IV
hCA XII
_
`
¨ ¨
We thank Inonu University, Turkey (BAPB), Universita degli Studi di
Firenze, Italy, and the European Union of the 7th Framework Program
Dynano for financial support.
1
2
3
4
5
6
7
8
851.7
1946
1915
391.6
359.2
60.9
51.4
351.1
95.3
93.9
5.6
95.4
102.4
15.0
24.5
45.9
17.1
9.3
52.3
224.1
451.7
31.9
42.9
26.6
139.0
97.2
62.2
70.6
35.9
69.1
28.4
29.1
50.3
24.5
8.6
81.2
410 000
174.9
89.6
72.3
27.9
545.0
365.2
93.5
362.0
383.0
1453
76.7
692.1
1358
401.5
43.5
838.9
825.1
410 000
74
89.8
73.1
9.5
61.2
95.7
72.1
90.8
109.0
101.8
9.7
951.2
34.7
10.3
99.1
83.9
76.0
91.2
803.1
96.3
85.7
5.7
Declaration of interest
The authors declare no conflict of interest.
9
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10
11
12
13
14
15
16
17
18
19
20
AZA
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*Mean from 3 different assays, errors in the range of 5%–10% of the
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