Synthesis and carbonic anhydrase inhibitory properties of amino acid – coumarin/quinolinone conjugates incorporating glycine, alanine and phenylalanine moieties

Article abstract of DOI:10.3109/14756366.2015.1113173

N-Protected amino acids (Gly, Ala and Phe) were reacted with amino substituted coumarin and quinolinone derivatives, leading to the corresponding N-protected amino acid–coumarin/quinolinone conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory ac

Full text of DOI:10.3109/14756366.2015.1113173

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 inhibitory
properties of amino acid – coumarin/quinolinone
conjugates incorporating glycine, alanine and
phenylalanine moieties
F. Zehra Küçükbay, Hasan Küçükbay, Muhammet Tanc & Claudiu T. Supuran
To cite this article: F. Zehra Küçükbay, Hasan Küçükbay, Muhammet Tanc & Claudiu T. Supuran
(2015): Synthesis and carbonic anhydrase inhibitory properties of amino acid – coumarin/
quinolinone conjugates incorporating glycine, alanine and phenylalanine moieties, Journal of
Enzyme Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2015.1113173
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1113173
Published online: 19 Nov 2015.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–5
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1113173
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RESEARCH ARTICLE
Synthesis and carbonic anhydrase inhibitory properties of amino
acid – coumarin/quinolinone conjugates incorporating glycine,
alanine and phenylalanine moieties
F. Zehra Ku¨c¸u¨kbay1,2, Hasan Ku¨c¸u¨kbay3,2, Muhammet Tanc2, and Claudiu T. Supuran2
1
2
Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Ino¨nu¨ University, Malatya, Turkey, Laboratorio Di Chimica Bioinorganica E
_
`
Dipartimento Neurofarba, Sezione Di Scienze Farmaceutiche E Nutraceutiche, Universita Degli Studi Di Firenze, Sesto Fiorentino, Florence, Italy, and
3
_
Department of Chemistry, Faculty of Arts and Science, Ino¨nu¨ University, Malatya, Turkey
Abstract
Keywords
N-Protected amino acids (Gly, Ala and Phe) were reacted with amino substituted coumarin and
quinolinone derivatives, leading to the corresponding N-protected amino acid–coumarin/
quinolinone conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new
compounds was assessed against various human (h) isoforms, such as hCA I, hCA II, hCA IV and
hCA XII. The quinolinone conjugates were inactive as enzyme inhibitors, whereas the coumarins
were ineffective hCA I/II inhibitors (KIs450 mM) but were submicromolar hCA IV and XII
inhibitors, with inhibition constants ranging between 92 nM and 1.19 mM for hCA IV, and
between 0.11 and 0.79 mM for hCA XII. These coumarin derivatives, as many others reported
earlier, thus show an interesting selective inhibitory profile for the membrane-bound over the
cytosolic CA isoforms.
Carbonic anhydrase, coumarin, inhibitor,
N-protected amino acid derivatives
History
Received 13 October 2015
Revised 23 October 2015
Accepted 23 October 2015
Published online 19 November 2015
Introduction
Material and methods
Coumarin and quinolinone derivatives are classes of heterocyclic Chemistry
scaffolds exhibiting several biological properties such as carbonic
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
glassware and syringes techniques to transfer solutions. Nuclear
magnetic resonance (1H NMR, 13C NMR) spectra were recorded
using a Bruker Advance III 400 MHz spectrometer in DMSO-d6.
Chemical shifts are reported in parts per million (ppm) and the
coupling constants (J) are expressed in Hertz (Hz). Splitting
patterns are designated as follows: s, singlet; d, doublet; t, triplet;
m, multiplet; brs, broad singlet; dd, double of doubles. The
assignment of exchangeable protons (OH and NH) was confirmed
by the addition of D2O. Positive-ion electrospray ionization mass
spectra were recorded on a double-focusing Finnigan MAT 95
instrument with BE geometry. Analytical thin-layer chromatog-
raphy (TLC) was carried out on Merck silica gel F-254 plates. All
microwave-assisted reactions were carried out in a microwave
oven system manufactured by GEM (CEM Matthews, NC) under
a nitrogen atmosphere. The reaction 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 open capillary tubes
and are uncorrected, using a Gallenkamp MPD350.BM3.5
apparatus. N-protected amino acids; benzyl (2-1H-ben-
zo[d][1,2,3]triazol-1-yl)-2-oxoethyl)carbamate (I), tert-butyl
anhydrase (CA, EC 4.2.1.1) inhibition1, anticoagulant2 and
antioxidant3 properties among others. Discovered initially in a
library of natural products, the first coumarin CA inhibitor (CAI)
was isolated from the Australian plant Leionema elipticum1.
A large number of synthetic and natural product coumarins were
subsequently investigated as inhibitors of these enzymes4–10, due
to the fact that they showed a high degree of selectivity for
inhibiting a number of isoforms of the 15 presently described in
humans, hCA I–hCA XIV (there are two V-type isoforms, hCA
VA and hCA VB)11–19. Furthermore, these enzymes are wide-
spread in organisms all over the phylogenetic tree, and their
inhibition has pharmacologic applications11–20
.
With the hope to obtain new biologically active coumarin and
quinolinone derivatives, the current work focused on the synthesis
of amino acid–coumarin/quinolinone conjugates by using the
N-(protected a-aminoacyl)benzotriazole methodology21 and the
exploration of 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-associated/
transmembrane hCA IV and XII.
Address for correspondence: Claudiu T. Supuran. Laboratorio Di Chimica
Bioinorganica
E Dipartimento Neurofarba, Sezione Di Scienze
`
Farmaceutiche E Nutraceutiche, Universita Degli Studi Di Firenze,
50019 Sesto Fiorentino, Florence, Italy. E-mail: claudiu.supuran@unifi.it
(2-1H-benzo[d][1,2,3]triazol-1-yl)-2-oxoethyl)carbamate
(II),

¨ ¨
F. Z. Kuc¸ukbay
2
J Enzyme Inhib Med Chem, Early Online: 1–5
(S)-benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-yl) (S)-benzyl (1-oxo-1-((2-oxo-2H-chromen-6-yl)amino)-3-phenyl-
carbamate (III), (S)-tert-butyl (1-(1H-benzo[d][1,2,3]triazol-1- propan-2-yl)carbamate, 4
yl)-1-oxo-3-phenylpropan-2-yl)carbamate (IV) used as starting
Brown solid (94%); silica gel TLC Rf ¼ 0.33 (MeOH/CH2Cl2 5%
v/v); mp 137–138 ꢀC; 1H NMR (DMSO-d6, 400 MHz) ꢀ 10.38 (s,
1H, NH), 8.11 (m, 2H, CH¼CH–CO + Ar–H), 7.96 (t,1H, NH,
J ¼ 4.0 Hz), 7.95–7.94 (m, 2H, Ar–H), 7.80–7.71 (m, 10H, Ar–H),
6.52 (d, 1H, ¼CH–CO, J ¼ 12.0 Hz), 5.02 (s, 2H, CH2Ph), 4.47
materials were prepared according to literature procedure22–24
.
Compounds 525 and 626 were found in the literature but their
synthesis methods need harsh reaction conditions and cumber-
some work-up procedures. Thus, these compounds were synthe-
sized by
a new, one-step, easy synthetic method using
(m, 1H, CHNH), 3.11 (m, 1H, CH2Ph), 2.93 (m, 1H, CH2Ph). 13
C
N-(protected a-aminoacyl)benzotriazoles in this study.
NMR (DMSO-d6, 400 MHz)
ꢀ 171.6 (COCH2NH), 160.9
(O–C¼O), 156.9 (COOCH2Ph), 150.4, 145.2, 138.6, 137.8,
136.1, 130.1, 129.2, 129.0, 128.6, 128.5, 127.3, 124.5, 119.6,
119.0, 117.6, 117.4 (Ar–C + C ¼ C), 66.3 (OCH2Ph), 57.9
(CHNH), 38.3 (CH2Ph). HRMS m/z for C26H22N2O5 [M + H]+
calcd. 443.2, found 443.1.0; [M + Na]+ calcd. 465.2, found 465.1.
General procedure for the synthesis of amino acid–coumarin
conjugates, 1–6
A mixture of equivalent amounts of the appropriate N-protected
aminoacylbenzotriazole and 6-amino-2H-chromen-2-one or
7-amino-4-methyl-2H-chromen-2-one was subjected to micro-
Benzyl (2-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-2-oxoethyl)-
carbamate, 5
ꢀ
wave irradiation (100 W, 70 C) in anhydrous THF (5 mL) for
30 min. After completion of the reaction, all volatiles were
removed by rotavapor and the obtained crude product was
crystallized from methanol.
Yellow solid (87%); silica gel TLC Rf ¼ 0.61 (MeOH/CH2Cl2 5%
v/v); mp 234–235 ꢀC; 1H NMR (DMSO-d6, 400 MHz) ꢀ 10.46 (s,
1H, NH), 7.79–7.76 (m, 2H, Ar–H), 7.65 (t,1H, NH, J ¼ 4.0 Hz),
7.52 (m, 1H, Ar–H), 7.44–7.36 (m, 5H, Ar–H), 6.30 (s, 1H, ¼CH–
CO), 5.10 (s, 2H, CH2Ph), 3.90 (d, 2H, CH2NH, J ¼ 4,0 Hz), 2.44
(s, 3H, CH3). 13C NMR (DMSO-d6, 400 MHz) ꢀ 169.7
(COCH2NH), 160.9 (O-C¼O), 157.6 (COOCH2Ph), 154.6,
154.0, 143.1, 137.9, 129.3, 128.8, 128.7, 126.9, 116.1, 116.0,
113.2, 106.5 (Ar-C + C ¼ C), 66.5 (CH2Ph), 45.2 (CH2NH), 18.9
(CH3). HRMS m/z for C20H18N2O5 [M + H]+ calcd. 367.1, found
367.1; [M + Na]+ calcd. 389.1, found 389.1.
Benzyl (2-oxo-2-((2-oxo-2H-chromen-6-yl)amino)ethyl)carba-
mate, 1
Beige solid (95%); silica gel TLC Rf ¼ 0.20 (MeOH/CH2Cl2 5%
1
ꢀ
v/v); mp 160–161 C; H NMR (DMSO-d6, 400 MHz) ꢀ 10.23
(s, 1H, NH), 8.10 (d, 1H, CH ¼ CH–CO), J ¼ 12.0 Hz), 8.06
(s, 1H, Ar–H), 7.71 (m, 1H, Ar-H), 7.62 (t,1H, NH, J ¼ 8.0 Hz),
7.40-7.35 (m, 6H, Ar–H), 6.52 (d, 1H, ¼CH–CO, J ¼ 12.0 Hz),
5.10 (s, 2H, CH2Ph), 3.88 (d, 2H, CH2NH, J ¼ 8,0 Hz). 13C NMR
(DMSO-d6, 400 MHz) ꢀ 169.1 (COCH2NH), 160.9 (O-C¼O),
157.5 (COOCH2Ph), 150.3, 145.2, 137.9, 136.2, 129.3, 128.7,
128.6, 124.2, 119.6, 118.7, 117.6, 117.4 (Ar-C + C ¼ C), 66.4
(CH2Ph), 45.0 (CH2NH). HRMS m/z for C19H16N2O5 [M + H]+
calcd. 353.1, found 353.1; [M + Na]+ calcd. 375.1, found 375.0.
(S)-benzyl (1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-oxo-
propan-2-yl)carbamate, 6
Yellow solid (84%); silica gel TLC Rf ¼ 0.65 (MeOH/CH2Cl2 5%
v/v); mp 175–177 ꢀC; 1H NMR (DMSO-d6, 400 MHz) ꢀ 10.48 (s,
1H, NH), 7.81–7.73 (m, 3H, Ar–H + NH), 7.55 (d,1H, Ar–H,
J ¼ 8.0 Hz), 7.41–7.35 (m, 5H, Ar–H), 6.31 (s, 1H, ¼CH–CO),
5.08 (s, 2H, CH2Ph), 4.26 (t, 1H, CHNH, J ¼ 8,0 Hz), 2.44 (s, 3H,
CH3), 1.35 (d, 3H, CHCH3, J ¼ 8.0 Hz). 13C NMR (DMSO-d6,
tert-butyl (2-oxo-2-((2-oxo-2H-chromen-6-yl)amino)ethyl)carba-
mate, 2
400 MHz)
ꢀ
173.2 (COCH2NH), 160.9 (O–C¼O), 157.0
Yellow solid (96%); silica gel TLC Rf ¼ 0.77 (MeOH/CH2Cl2 5%
v/v); mp 201–202 ꢀC; 1H NMR (DMSO-d6, 400 MHz) ꢀ 10.17 (s,
1H, NH), 8.12 (d, 1H, CH¼CH–CO), J ¼ 12.0 Hz), 8.07 (s, 1H,
Ar–H), 7.71 (m, 1H, Ar–H), 7.11 (t,1H, NH, J ¼ 4.0 Hz), 6.52 (d,
1H, ¼CH–CO, J ¼ 12.0 Hz), 3.78 (d, 2H, CH2NH, J ¼ 4,0 Hz).
1.44 (s, (CH3)3C, 9H). 13C NMR (DMSO-d6, 400 MHz) ꢀ 169.3
(COCH2NH), 160.9 (O–C¼O), 156.8 (COOCH2Ph), 145.3, 137.9,
136.3, 124.2, 119.6, 118.6, 117.6, 117.4 (Ar–C + C ¼ C), 79.0
(C(CH3)3), 44.7 (CH2NH), 29.1 ((CH3)3C. HRMS m/z for
C19H18N2O5 [M + H]+ calcd. 319.1, found 319.1; [M + Na]+
calcd. 341.1, found 341.1.
(COOCH2Ph), 154.5, 154.0, 143.2, 129.2, 128.7, 128.6, 126.8,
116.1, 116.0, 113.2, 106.4 (Ar–C + C ¼ C), 66.4 (CH2Ph), 51.9
(CH2NH), 18.9 (CH3), 18.8 (CHCH3). HRMS m/z for
C21H20N2O5 [M + H]+ calcd. 381.1, found 381.1; [M + Na]+
calcd. 403.1, found 403.1.
General method for the synthesis of amino acid-quinolin-2-one
conjugates, 7–10
A mixture of equivalent amounts of the appropriate N-protected
aminoacylbenzotriazole and 7-amino-1,2,3,4-tetrahydro-2-quino-
ꢀ
linone was subjected to microwave irradiation (100 W, 70 C) in
anhydrous THF (5 mL) for 30 min. After completion of the
reaction, all volatiles were removed by rotary evaporator and the
obtained crude product was crystallized from methanol.
(S)-benzyl (1-oxo-1-((2-oxo-2H-chromen-6-yl)amino)propan-2-
yl)carbamate, 3
Beige solid (92%); silica gel TLC Rf ¼ 0.36 (MeOH/CH2Cl2 5%
v/v); mp 87–88 ꢀC; 1H NMR (DMSO-d6, 400 MHz) ꢀ 10.24
(s, 1H, NH), 8.11 (d, 1H, CH¼CH–CO), J ¼ 8.0 Hz), 8.07 (s, 1H,
Ar–H), 7.73 (m, 1H, Ar–H), 7.67 (t,1H, NH, J ¼ 8.0 Hz), 7.42–
7.40 (m, 6H, Ar–H), 6.52 (d, 1H, ¼CH–CO, J ¼ 8.0 Hz), 5.75
(s, 2H, CH2Ph), 4.24 (t, 2H, CH2NH, J ¼ 8,0 Hz), 1.35 (d, 3H,
CH3, J ¼ 8.0 Hz. 13C NMR (DMSO-d6, 400 MHz) ꢀ 172.6
(COCH2NH), 160.9 (O–C¼O), 157.2 (COOCH2Ph), 150.3,
145.3, 137.9, 136.3, 129.3, 128.7, 128.6, 124.4, 119.6, 118.9,
117.6, 117.4 (Ar–C + C ¼ C), 66.4 (CH2Ph), 51.7 (CH2NH), 18.8
(CH3). HRMS m/z for C20H18N2O5 [M + H]+ calcd. 367.1, found
367.0; [M + Na]+ calcd. 389.1, found 389.1.
Benzyl (2-oxo-2-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)amino)
ethyl)carbamate, 7
White solid (97%); silica gel TLC Rf ¼ 0.81 (MeOH/CH2Cl2 5%
1
ꢀ
v/v); mp 224–225 C; H NMR (DMSO-d6, 400 MHz) ꢀ 10.15
(s, 1H, NHlactam), 9.94 (s, 1H, NH), 7.55 (t,1H, NH, J ¼ 8.0 Hz),
7.42–7.25 (m, 4H, Ar–H), 7.24 (s, 1H, Ar–H), 7.12–7.11 (m, 2H,
Ar–H), 5.09 (s, 2H, CH2Ph), 3.82 (d, 2H, CH2NH, J ¼ 8,0 Hz),
2.82 (t, 2H, COCH2, J ¼ 8.0 Hz), 2.44 (t, 2H, COCH2CH2,
J ¼ 8.0 Hz). 13C NMR (DMSO-d6, 400 MHz)
ꢀ
171.2
(COCH2NH), 168.6 (NH–C¼O), 157.5 (COOCH2Ph), 139.4,

DOI: 10.3109/14756366.2015.1113173
Synthesis and CA inhibitory properties of amino acid
3
138.8, 138.0, 129.3, 128.7, 128.6, 119.3, 113.7, 107.2 (Ar–C), 6 h prior to assay, as for all other couamrin/sulfocoumarin
66.4 (CH2Ph), 44.9 (CH2NH), 31.5 (COCH2), 25.2 (COCH2CH2). derivatives investigated earlier1,5–10. Enzyme concentrations in
HRMS m/z for C19H19N3O4 [M + H]+ calcd. 354.1, found 354.1; the assay system were in the range of 8–16 nM for all the enzymes
[M + Na]+ calcd. 376.1, found 376.1.
considered in this study. All CAs were recombinant proteins,
28–31
obtained as reported earlier by our group
.
tert-butyl (2-oxo-2-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)ami-
no)ethyl)carbamate, 8
Results and discussion
Beige solid (94%); silica gel TLC Rf ¼ 0.26 (Ethyl acetate/Hexane
30% v/v); mp 135–136 ꢀC; 1H NMR (DMSO-d6, 400 MHz) ꢀ
10.13 (s, 1H, NHlactam), 9.87 (s, 1H, NH), 7.24 (s, 1H, Ar–H), 7.10
(d, 2H, Ar–H, J ¼ 4.0 Hz), 7.02 (t,1H, NH, J ¼ 4.0 Hz), 3.72 (d,
2H, CH2NH, J ¼ 4,0 Hz), 2.83 (t, 2H, COCH2, J ¼ 4.0 Hz), 2.45
(t, 2H, COCH2CH2, J ¼ 4.0 Hz), 1.43 (s, 9H, (CH3)3C). 13C NMR
(DMSO-d6, 400 MHz) ꢀ 171.2 (COCH2NH), 168.9 (NH–C¼O),
156.8 (COOCH2Ph), 139.4, 138.8, 119.3, 113.7, 107.1,(Ar–C),
79.0 (CO(CH3)3), 44.6 (CH2NH), 31.6 (COCH2), 29.1
(COCH2CH2), 25.2 (C(CH3)3). HRMS m/z for C16H21N3O4
[M]+ calcd. 319.1, found 319.1; [M + Na]+ calcd. 342.1, found
342.1.
The new N-protected amino acid–coumarin conjugates (1–6) were
synthesized by the treatment of the appropriate coumarin
derivatives with N-protected aminoacylbenzotriazole under
ꢀ
microwave heating at 70 C for 30 min. They were obtained in
good yields, of 84–96% (Scheme 1). New N-protected amino
acid–quinolinone conjugates (7–10) were also synthesized by a
similar method mentioned above for the coumarin conjugates,
using appropriately N-protected aminoacylbenzotriazoles and 7-
amino-1,2,3,4-tetrahydro-2-quinolinone32 under microwave heat-
ing at 70 ꢀC for 30 min with good yields of 94–98%. The synthesis
pathways of the N-protected amino acid–coumarin or –quinoli-
none derivatives (1–10) are summarized in Scheme 1.
The structures of the N-protected amino acid–coumarin/
1
(S)-benzyl (1-oxo-1-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)ami-
no)propan-2-yl)carbamate, 9
quinolinone conjugates (1–10) were elucidated by H NMR, 13C
NMR and mass spectrometry (MS) analyses. All spectral data
White solid (96%); silica gel TLC Rf ¼ 0.63 (MeOH/CH2Cl2 5% were in accordance with the assumed structures (see ‘‘Materials
v/v); mp 220–221 ꢀC; 1H NMR (DMSO-d6, 400 MHz) ꢀ 10.14 (s, and Methods’’ section). The characteristic NH resonances for the
1H, NHlactam), 9.94 (s, 1H, NH), 7.58 (d,1H, NH, J ¼ 8.0 Hz), coumarin part of the conjugates were observed between 10.17 and
7.40–7.36 (m, 5H, Ar–H), 7.26 (s, 1H, Ar–H), 7.13–7.09 (m, 2H, 10.48 ppm. Other amino group resonances for the cuomarin
Ar–H), 5.07 (s, 2H, CH2Ph), 4.22 (t, 1H, CH2NH, J ¼ 8,0 Hz), conjugates were observed between 7.11 and 7.98 ppm. Carbonyl
2.84 (t, 2H, COCH2, J ¼ 8.0 Hz), 2.45 (t, 2H, COCH2CH2, resonances of the amide carbonyl, lactone carbonyl and carba-
J ¼ 8.0 Hz), 1.31 (d, 3H, CH3, J ¼ 8.0 Hz). 13C NMR (DMSO-d6, mate carbonyl were observed around 169.1–173.2, 169.0 and
400 MHz) ꢀ 171.3 (COCH2NH), 171.2 (NH–C¼O), 156.7 156.8–157.6 ppm, respectively. The characteristic NH resonances
(COOCH2Ph), 139.4, 138.9, 138.0, 129.3, 128.7, 128.6, 119.4, for carbamate and quinolinone part of the conjugates were
113.8, 107.3, (Ar–C), 66.3 (CH2Ph), 51.6 (CH2NH), 31.6 observed between 10.13–10.17 and 9.87–10.10 ppm, respectively.
(COCH2), 25.2 (COCH2CH2), 19.0 (CH3). HRMS m/z for Other amino group resonances for the quinolinone conjugates
C20H21N3O4 [M + H]+ calcd. 368.1, found 368.1; [M + Na]+ were observed between 7.02 and 7.95 ppm. Carbonyl resonances
calcd. 390.1, found 390.1.
of the amide carbonyl, lactame carbonyl and carbamate
carbonyl were observed around 171.2–173.3, 168.9–171.2 and
156.7–157.5 ppm, respectively. All other aliphatic and aromatic
protons and carbons were observed at expected regions.
The hCA I, II, IV and XII enzyme inhibitory activity results
of the coumarin/quinolinone amino acid conjugates are shown
in Table 1.
Data of Table 1 show that the quinolinone conjugates 7–10
were totally inactive as CAIs, although the parent compound,
7-amino-1,2,3,4-tetrahydro-2-quinolinone32 showed interesting
such properties. On the other hand, the coumarin derivatives
showed interesting enzyme inhibitory properties. As many other
coumarins1,5–10, they did not inhibit the cytosolic, off-target
isoforms hCA I and II (KIs450 mM) but showed submicromolar
affinity for the membrane-associated (hCA IV) and transmem-
barne (hCA XII) isoforms. Indeed, against hCA IV apart 2 and 6
which was inactive, the other derivatives showed inhibition
constants in the range of 92 nM–1.19 mM, with the best activity
being shown by the benzyloxycarbonyl-protected (Cbz) Gly
(S)-benzyl (1-oxo-1-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)
amino)-3-phenylpropan-2-yl)carbamate, 10
White solid (98%); silica gel TLC Rf ¼ 0.58 (MeOH/CH2Cl2 5%
v/v); mp 150–151 ꢀC; 1H NMR (DMSO-d6, 400 MHz) ꢀ 10.17 (s,
1H, NHlactam), 10.10 (s, 1H, NH), 7.95 (d,1H, NH, J ¼ 4.0 Hz),
7.38–7.12 (m, 13H, Ar–H), 5.00 (s, 2H, CH2Ph), 4.22 (m, 1H,
CHNH), 3.05 and 2.91 (m, 2H, CHCH2Ph), 2.86 (t, 2H, COCH2,
J ¼ 8.0 Hz), 2.46 (t, 2H, COCH2CH2, J ¼ 8.0 Hz). 13C NMR
(DMSO-d6, 400 MHz) ꢀ 171.3 (COCH2NH), 171.2 (NH–C¼O),
156.9 (COOCH2Ph), 139.4, 138.8, 138.7, 138.0, 130.2, 129.2,
129.0, 128.7, 128.6, 128.5, 127.3, 119.5, 114.0, 107.4 (Ar–C),
66.2 (CH2Ph), 57.8 (CHNH), 38.5 (CHCH2Ph, 31.6 (COCH2),
25.2 (COCH2CH2). HRMS m/z for C26H25N3O4 [M + H]+ calcd.
444.1, found 444.1; [M + Na]+ calcd. 466.1, found 466.1.
CA inhibition
An Applied Photophysics stopped-flow instrument was used for derivative 1. Interestingly, the corresponding Boc-protected
assaying the CA catalyzed CO2 hydration activity27. Phenol red derivative 2 was ineffective as an hCA IV inhibitor, whereas the
(at 0.2 mM) was used as indicator, working at the absorbance inhibitory activity diminished for the remaining Cbz-protected
maximum of 557 nm with 10 mM HEPES (pH 7.5) as buffer and Ala and Phe derivatives 3 and 4. Thus, bulkier amino acyl
0.1 M NaClO4 (for maintaining constant ionic strength), at 20 ꢀC, moieties than the Gly one were detrimental in this position of the
following the CA-catalyzed CO2 hydration reaction for a period of coumarin ring for the hCA IV inhibitory activity.
10–100 s (the uncatalyzed reaction needs around 60–100 s in the
Against hCA XII the new coumarin conjugates showed an
assay conditions, whereas the catalyzed ones are of around 6–10 s). effective inhibitory power, with KIs ranging between 0.11 and
The CO2 concentrations ranged from 1.7 to 17 mM for the 0.79 mM (Table 1). The best inhibitor was 6 (KI of 0.11 mM) which
determination of the kinetic parameters. The uncatalyzed rates incorporates the amino acyl moiety in position 7 of the coumarin
were determined in the same manner and subtracted from the total ring, and contains a Cbz–Ala moiety. The corresponding Gly
observed rates. Enzyme and inhibitor were incubated together for derivative was around 5 times less effective as a hCA XII

¨ ¨
F. Z. Kuc¸ukbay
4
J Enzyme Inhib Med Chem, Early Online: 1–5
H N
H
2
Scheme 1. Synthesis pathways of the new
coumarin and quinolinone conjugates of
N-protected amino acids.
N
R
O
O
O
O
O
NH
Pg
1 Pg= Z, R= H
2 Pg= Boc, R= H
3 Pg= Z, R= CH
3
4 Pg= Z, R= CH Ph
2
CH
O
3
CH
O
3
O
N
N
R
N
H
O
N
O
H N
2
NH
Pg
R
O
NH
5
6
Pg= Z, R= H
Pg= Z, R= CH
Pg
3
Pg= Benzyloxycarbonyl (Z)
t-Butoxycarbonyl (Boc)
I
II
III
Pg= Z, R= H
Pg= Boc, R= H
CH
3
CH
3
Pg= Z,
R=CH
3
IV Pg=Z, R= CH Ph
2
HN
O
R
N
H
O
N
H
O
H N
2
NH
Pg
7 Pg= Z, R= H
Pg= Boc, R= H
8
9
Pg= Z, R= CH
3
Pg= Z, R= CH Ph
2
10
Table 1. hCA I, II, IV and XII inhibition with amino acid conjugates
reported in the paper, by a stopped-flow, CO2 hydrase assay, after 6 h
N-protected amino acid–coumarin/quinolinone conjugates. The
CA inhibitory activity of the new compounds was assessed against
various isoforms, such as hCA I, hCA II, hCA IV and hCA XII.
The quinolinone conjugates were inactive as enzyme inhibitors,
whereas the coumarins were ineffective hCA I/II inhibitors
hCA XII (KIs450 mM) but were submicromolar hCA IV and XII inhibitors,
ioncubation period between enzyme and inhibitor27
.
KI (mM)*
Compound
hCA I
hCA II
hCA IV
with inhibition constants ranging between 92 nM and 1.19 mM for
hCA IV, and between 0.11 and 0.79 mM for hCA XII. These
coumarin derivatives, as many others reported earlier, thus show
an interesting selective inhibitory profile for the membrane-bound
over the cytosolic CA isoforms.
1
2
3
4
5
6
7
8
450
450
450
450
450
450
450
450
450
450
0.25
450
450
450
450
450
450
450
450
450
450
0.012
0.092
450
0.14
1.19
0.39
450
450
450
450
450
0.074
0.49
0.79
0.60
0.23
0.58
0.11
450
450
450
450
0.006
Acknowledgements
9
10
AAZ*
_
We thank Inonu University, Turkey (BAPB), and Universita degli Studi di
`
¨ ¨
Firenze, Italy, for financial support.
Acetazolamide (AAZ) was used as a standard inhibitor for all CAs
investigated here.
Declaration of interest
*Mean from three different assays. Errors were in the range of 10% of
the reported values (data not shown).
The authors declare no conflict of interest.
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