Synthesis and biological evaluation of histamine Schiff bases as carbonic anhydrase I, II, IV, VII, and IX activators

Article abstract of DOI:10.1080/14756366.2017.1386660

A series of 20 histamine Schiff base was synthesised by reaction of histamine, a well known carbonic anhydrase (CA, E.C 4.2.2.1.) activator pharmacophore, with substituted aldehydes. The obtained histamine Schiff bases were assayed as activators of five selected human (h) CA isozymes, the cytosolic hCA I, hCA II, and hCA VII, the membrane-anchored hCA IV and transmembrane hCA IX. Some of these compounds showed efficient activity (in the nanomolar range) against the cytosolic isoform hCA VII, which is a key CA enzyme involved in brain metabolism. Moderate activity was observed against hCA I and hCA IV (in the nanomolar to low micromolar range). The structure–activity relationship for activation of these isoforms with the new histamine Schiff bases is discussed in detail based on the nature of the aliphatic, aromatic, or heterocyclic moiety present in the aldehyde fragment of the molecule, which may participate in diverse interactions with amino acid residues at the entrance of the active site, where activators bind, and which is the most variable part among the different CA isoforms.

Full text of DOI:10.1080/14756366.2017.1386660

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of histamine
Schiff bases as carbonic anhydrase I, II, IV, VII, and
IX activators
Suleyman Akocak, Nabih Lolak, Daniela Vullo, Mustafa Durgun & Claudiu T.
Supuran
To cite this article: Suleyman Akocak, Nabih Lolak, Daniela Vullo, Mustafa Durgun & Claudiu
T. Supuran (2017) Synthesis and biological evaluation of histamine Schiff bases as carbonic
anhydrase I, II, IV, VII, and IX activators, Journal of Enzyme Inhibition and Medicinal Chemistry,
32:1, 1305-1312, DOI: 10.1080/14756366.2017.1386660
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1386660
© 2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 26 Oct 2017.
Submit your article to this journal
Article views: 124
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [KSU Kent State University]
Date: 05 November 2017, At: 01:01

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 1305–1312
https://doi.org/10.1080/14756366.2017.1386660
RESEARCH PAPER
Synthesis and biological evaluation of histamine Schiff bases as carbonic
anhydrase I, II, IV, VII, and IX activators
Suleyman Akocaka, Nabih Lolaka, Daniela Vullob, Mustafa Durgunc and Claudiu T. Supuranb
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Adiyaman University, Adiyaman, Turkey; NEUROFARBA Dept., Sezione di
c
ꢀ
Scienze Farmaceutiche, Universita degli Studi di Firenze, Florence, Italy; Department of Chemistry, Faculty of Science and Literature, Harran
University, Sanliurfa, Turkey
ABSTRACT
ARTICLE HISTORY
Received 19 September 2017
Accepted 27 September 2017
A series of 20 histamine Schiff base was synthesised by reaction of histamine, a well known carbonic anhy-
drase (CA, E.C 4.2.2.1.) activator pharmacophore, with substituted aldehydes. The obtained histamine Schiff
bases were assayed as activators of five selected human (h) CA isozymes, the cytosolic hCA I, hCA II, and
hCA VII, the membrane-anchored hCA IV and transmembrane hCA IX. Some of these compounds showed
efficient activity (in the nanomolar range) against the cytosolic isoform hCA VII, which is a key CA enzyme
involved in brain metabolism. Moderate activity was observed against hCA I and hCA IV (in the nanomolar
to low micromolar range). The structure–activity relationship for activation of these isoforms with the new
histamine Schiff bases is discussed in detail based on the nature of the aliphatic, aromatic, or heterocyclic
moiety present in the aldehyde fragment of the molecule, which may participate in diverse interactions
with amino acid residues at the entrance of the active site, where activators bind, and which is the most
variable part among the different CA isoforms.
KEYWORDS
Carbonic anhydrase
activators; histamine; Schiff
bases; isozymes; Alzheimer’s
disease
Introduction
the neurodegenerative disorder of memory and cognitive func-
tion (Alzheimer’s disease) since it has been shown the level of
brain CAs significantly diminished in the brain of Alzheimer’s dis-
ease and older rats as compared to normal and young brain of
Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc containing metal-
loenzymes (present in prokaryotes and eukaryotes) that catalyse
the reversible hydration of carbon dioxide into bicarbonate and
animals20,21
.
proton ions under physiological conditions (CO2þH2O
$
The inhibition and activation processes of CAs are well investi-
gated processes which show different binding modes within the
active site cavity of isozymes (Figure 1)11,22. The classical inhibi-
tors bind deep within the active site cavity by interacting with
the metal centre, which is zinc ion for hCAs (Figure 1(b)). On the
other hand, activators are bound far away from the metal ion, at
the entrance-middle part of the active site cavity and participate
in the proton shuttling process from the active site to the exter-
nal buffer. This is part of the normal catalytic cycle of the
enzyme, with the amino acid residue His 64 in isoforms such as
CA II, IV, VI, VII, IX, XII, found in the middle of the active site cav-
ity, normally participating in this step, which is the rate deter-
mining one for the entire catalytic cycle (Figure 1(a))22,23. The
presence of the activators produces an alternative proton transfer
pathway and enhances the overall catalytic efficiency of the
enzyme. CAAs play thus a role on speeding up the deprotona-
tion of zinc bound water, which decrease the pKa of a coordi-
nated water molecule, in the rate determining step of the
catalytic mechanism (Equation (2) with the generation of the
active form of the enzyme24–36, as described in Equation (3)
–
HCO3 þHþ). Up to now, seven genetically distinct CA families
(a-, b-, c-, d-, f-, g-, and h-CAs), as well as numerous isoforms in
most organisms were discovered1–7. In humans, 16 different CA
and CA related proteins have been described, with different sub-
cellular localisation, catalytic activity, and susceptibility to differ-
ent classes of inhibitors and activators8–12
. Some of these
isoforms are cytosolic (CA I, CA II, CA III, CA VII, and CA XIII),
some of them are transmembrane bound isoforms (CA IV, CA IX,
CA XII, CA XIV, and CA XV), two of them are mitochondrial (CA
VA and CA VB), and one of them is secreted in saliva and milk
(CA VI). On the other hand, catalytically inactive CA related iso-
forms (CARP VIII, CARP X, and CA XI) are also cytosolic
proteins1–4,8,9,11,13–15
.
Carbonic anhydrase has been a therapeutic target for many
years and their inhibitors are clinically used/investigated as diu-
retics, anticonvulsant, antiobesity, antiglaucoma and more
recently antitumour and anti-infective agents16–19. However, the
CA activators (CAAs), although investigated simultaneously with
inhibitors, do not have pharmaceutical applications, yet. Indeed,
it has been proposed that some CAAs might have applications in where the formation of the enzyme–activator complex is shown,
CONTACT Suleyman Akocak
Adiyaman, Turkey; Claudiu T. Supuran
Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
akocaksuleyman@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Adiyaman University, 02040
ꢀ
claudiu.supuran@unifi.it
NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Universita degli Studi di Firenze, Via
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1306
S. AKOCAK ET AL.
Figure 1. Superimposed ribbon diagram (a) and active site detail (b) of the CA II isozyme (PDB codes 1AVN22 and 3HS437) with the activator histamine and the well
known CAI 5-acetamido-1,3,4-thiadiazole-2-sulphonamide (acetazolamide, AZM). Acetazolamide is coordinated to the zinc ion being bound deep within the active site,
whereas histamine does not interact with the metal ion and is bound at the entrance of the cavity. The zinc ion (magenta) is coordinated by three histidine residues
(His 94, His 96, and His 119, in orange) and some key amino acids on active site were shown (in red). The proton shuttle residue His 64 is also shown (in cyan). Figure
made using PyMol (DeLano Scientific).
which leads to an increased rate of the proton transfer reaction
due to the fact that the process became intermolecular and not
intramolecular as in Equation (2).
Materials and methods
General
All chemicals and anhydrous solvents were purchased from Sigma-
Aldrich, Merck, Alfa Aesar and TCI and used without further purifi-
cation. Melting points (mp) were determined with SMP30 melting
point apparatus in open capillaries and are uncorrected. Elemental
analysis was carried out on a LEO CHNS model 932 elemental ana-
lyser. FT-IR spectra were recorded by using Perkin Elmer Spectrum
100 FT-IR spectrometer. Nuclear magnetic resonance (1H NMR and
13C NMR) spectra of compounds were recorded using a Bruker
Advance III 300 MHz spectrometer in DMSO-d6 and TMS as an
internal standard operating at 300 MHz for 1H NMR and 75 MHz
for 13C NMR. Thin layer chromatography (TLC) was carried out on
Merck silica gel 60 F254 plates.
þH2O
EZn2þꢀOHꢀþCO2 ꢀ EZn2þꢀHCO3 ꢀ EZn2þꢀOH2þHCO3
(1)
(2)
ꢀ
ꢀ
ENn2ꢀOH2 ꢀ EZn2þꢀOHꢀþHþꢀrate determining stepꢀ
ENn2ꢀOH2 þ A ꢀ ½EZn2þꢀOH2 Aꢁ ꢀ ½EZn2þꢀOHꢀꢀAHþꢁ
ꢀ
(3)
ꢀ ½EZn2þꢀOHꢀꢀAHþꢁ ꢀ EZn2þꢀOHꢀꢀAHþ
In order to possess a good activity, a compound needs both
steric requirements (i.e. to fit within the restricted active site cav-
ity) and electronic factors (to possess an appropriate pKa value of
the proton shuttle moiety) to be present in its structure. The X-ray
crystal structure for the hCA II-histamine adduct22 revealed that
the imidazole moiety is bound in the middle of the cavity, not far
from the natural proton shuttle residue (His 64), whereas the
amino moiety is not involved in any interaction with the enzyme
active site and that is why this amino group might be derivatised
to obtain much more biologically active compounds, as done ear-
lier by some of us.
Schiff base derivatives of sulphonamides were extensively
studied as an efficient and selective inhibitors of several CAs by us
and other researchers38–41. However, a procedure successfully used
to obtain sulphonamide CAIs incorporating Schiff base moieties,
that is, reaction of amino sulphonamide with aldehydes to produce
Schiff base derivatives, has never been approached for preparing
histamine derivatives with potential CA activating properties.
Herein, we present the synthesis and CA activation studies of
histamine Schiff base derivatives on a panel of selected CA iso-
zymes which are cytosolic hCA I, II, VII (involved in many physio-
General procedure for the synthesis of histamine Schiff bases H
(1–20)
The potassium hydroxide (10 mmol) was added to a stirred sus-
pension of histamine dihydrochloride (5 mmol) in dry MeOH
(10–15 ml) at room temperature. After stirring for 2 h, the precipi-
tate salt (KCl) was filtered off and the filtrate was treated with a
solution of aldehydes (5 mmol) in dry MeOH (20–25 ml). The
homogeneous mixture was stirred overnight at room temperature.
The completion of the reaction was monitored by TLC and FT-IR.
The excess solvent was evaporated and the oily residue was crys-
tallised with ethyl acetate and ether to obtain corresponding
Schiff base derivatives. The desired final products H(1–20) were
1
dried under vacuum and fully characterised by FT-IR, H NMR, 13C
logical processes all over the body and also in the brain)1–3 and NMR, elemental analysis, and melting points.
membrane-bound ones hCA IV and IX (the last of which is a vali-
2-(1H-imidazol-4-yl)-N-pentylideneethanamine (H1): Yield:
dated anti-tumour target)16–18
.
55%; colour: white powder, mp: 188–190 ꢂC: FT-IR (cmꢀ1): 1648

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1307
(–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.45 (s, 1H, (s, 1H, H-5 Im), 6.60 (t, 1H, J ¼ 10.8, Ar-H), 3.89 (t, 2H, J ¼ 6.7,
–N¼CH–), 7.75 (d, 1H, J ¼ 1.2, H-2 Im), 7.30 (s, 1H, H-5 Im), 3.62 (t, –CH2CH2-Im), 2.92 (t, 2H, J ¼ 6.7, –CH2CH2-Im), 2.15 (s, 3H, –CH3):
2H, J ¼ 6.5, –CH2CH2-Im), 2.88 (t, 2H, J ¼ 6.5, –CH2CH2-Im), 1.65 (t, 13C NMR (DMSO-d6, 75 MHz, d ppm): 165.38 (–N¼CH–), 162.35,
2H, Val), 1.30 (m, 2H, Val), 1.24 (m, 2H, Val), 0.9 (t, 3H, Val): 13C 133.57, 132.21, 131.78, 117.10, 116.23, 115.32, 112.13, 55.32, 25.15,
NMR (DMSO-d6, 75 MHz, d ppm): 164.38 (–N¼CH–), 142.35, 138.63, 18.34; elemental analysis for C13H15N3O: C, 68.10; H, 6.59; N, 18.33.
118.31, 63.53, 35.13, 29.16, 28.32, 23.25, 14.58; elemental analysis Found: C, 68.08; H, 6.60; N, 18.36.
for C10H17N3: C, 67.00; H, 9.56; N, 23.44. Found: C, 66.98; H, 9.55; N,
23.46.
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-6-methoxyphe-
nol (H8): Yield: 82%; colour: dark yellow powder, mp: 190–192 ꢂC
FT-IR (cmꢀ1): 1632 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm):
N-(furan-2-ylmethylene)-2-(1H-imidazol-4-yl)ethanamine
(H2): Yield: 45%; colour: brown powder, mp: 202–205 ꢂC; FT-IR 14.46 (s, 1H, –OH), 8.51 (s, 1H, –N¼CH–), 7.82 (d, 1H, J ¼ 0.9, H-2
(cmꢀ1): 1645 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.78 Im), 7.63–7.59 (d, 1H, J ¼ 7.8, Ar-H), 7.35–7.32 (d, 1H, J ¼ 7.8, Ar-H),
(s, 1H, –N¼CH–), 7.93 (d, 1H, J ¼ 1.2, H-2 Im), 7.82 (d, 1H, J ¼ 2.2, 6.92 (s, 1H, H-5 Im), 6.58 (t, 1H, J ¼ 10.8, Ar-H), 3.98 (s, 3H, –OCH3),
furan), 7.74 (s, 1H, H-5 Im), 6.98 (d, 1H, J ¼ 2.2, furan), 6.72 (d, 1H, 3.87 (t, 2H, J ¼ 6.6, –CH2CH2-Im), 2.90 (t, 2H, J ¼ 6.6, –CH2CH2-Im):
J ¼ 2.0, furan) 3.48 (t, 2H, J ¼ 6.0, –CH2CH2-Im), 2.87 (t, 2H, J ¼ 6.0, 13C NMR (DMSO-d6, 75 MHz, d ppm): 164.25 (–N¼CH–), 161.65,
–CH2CH2-Im): 13C NMR (DMSO-d6, 75 MHz,
d ppm): 165.71 132.38, 131.24, 130.14, 116.82, 116.12, 115.44, 111.56, 56.32, 54.26,
(–N¼CH–), 151.15, 145.63, 138.42, 134.18, 119.62, 113.34, 56.15, 27.54; elemental analysis for C13H15N3O2: C, 63.66; H, 6.16; N,
32.10; elemental analysis for C10H11N3O: C, 63.48; H, 5.86; N, 22.21. 17.13. Found: C, 63.61; H, 6.18; N, 17.16.
Found: C, 63.45; H, 5.85; N, 22.24.
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-6-bromophenol
2-(1H-imidazol-4-yl)-N-(4-methylbenzylidene)ethanamine
(H9): Yield: 85%; colour: yellow powder, mp: 145–147 ꢂC; FT-IR
(H3): Yield: 72%; colour: white powder, mp: 190–193 ꢂC; FT-IR (cmꢀ1): 1635 (–C¼N–); H NMR (DMSO-d6, 300 MHz, d ppm): 14.60
(cmꢀ1): 1640 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.62 (s, 1H, –OH), 8.53 (s, 1H, –N¼CH–), 7.85 (d, 1H, J ¼ 0.9, H-2 Im),
(s, 1H, –N¼CH–), 7.88 (d, 1H, J ¼ 1.0, H-2 Im), 7.78 (d, 2H, J ¼ 8.2, 7.64–7.60 (d, 1H, J ¼ 7.8, Ar-H), 7.36–7.33 (d, 1H, J ¼ 7.8, Ar-H), 6.96
Ar-H), 7.35 (s, 1H, H-5 Im), 7.28 (d, 2H, J ¼ 8.2, Ar-H), 3.65 (t, 2H, (s, 1H, H-5 Im), 6.61 (t, 1H, J ¼ 10.8, Ar-H), 3.92 (t, 2H, J ¼ 6.6,
J ¼ 6.2, –CH2CH2-Im), 2.90 (t, 2H, J ¼ 6.2, –CH2CH2-Im), 1.85 (s, 3H, –CH2CH2-Im), 2.92 (t, 2H, J ¼ 6.6, –CH2CH2-Im): 13C NMR (DMSO-d6,
–CH3): 13C NMR (DMSO-d6, 75 MHz, d ppm): 166.41 (–N¼CH–), 75 MHz, d ppm): 166.13 (–N¼CH–), 162.72, 136.46, 134.63, 133.61,
163.82, 133.51, 132.93, 131.65, 116.31, 115.64, 114.73, 111.42, 132.14, 117.58, 116.62, 116.41, 112.76, 54.28, 27.54; elemental ana-
55.24, 26.72, 18.38; elemental analysis for C13H15N3: C, 73.21; H, lysis for C12H12BrN3O: C, 49.00; H, 4.11; N, 14.29. Found: C, 49.03;
1
7.09; N, 19.70. Found: C, 73.19; H, 7.10; N, 19.73.
H, 4.08; N, 14.31.
2-(1H-imidazol-4-yl)-N-(4-methoxybenzylidene)ethanamine
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-4-bromophenol
(H4): Yield: 78%; colour: light brown powder, mp: 178–181 ꢂC; FT- (H10): Yield: 67%; colour: yellow powder, mp: 168–170 ꢂC; FT-IR
IR (cmꢀ1): 1636 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm): (cmꢀ1): 1640 (–C¼N–); H NMR (DMSO-d6, 300 MHz, d ppm): 14.45
1
8.66 (s, 1H, –N¼CH–), 7.92 (d, 1H, J ¼ 1.2, H-2 Im), 7.82 (d, 2H, (s, 1H, –OH), 8.78 (s, 1H, –N¼CH–), 7.88 (d, 1H, J ¼ 1.0, H-2 Im),
J ¼ 7.8, Ar-H), 7.42 (s, 1H, H-5 Im), 7.33 (d, 2H, J ¼ 7.8, Ar-H), 3.85 (s, 7.72–7.68 (d, 1H, J ¼ 8.2, Ar-H), 7.55–7.46 (m, 2H, Ar-H), 7.12 (s, 1H,
3H, –OCH3), 3.62 (t, 2H, J ¼ 6.5, –CH2CH2-Im), 2.92 (t, 2H, J ¼ 6.5, H-5 Im), 3.95 (t, 2H, J ¼ 6.8, –CH2CH2-Im), 2.95 (t, 2H, J ¼ 6.8,
–CH2CH2-Im): 13C NMR (DMSO-d6, 75 MHz,
d d ppm): 167.48
ppm): 167.33 –CH2CH2-Im): 13C NMR (DMSO-d6, 75 MHz,
(–N¼CH–), 162.62, 134.48, 133.35, 131.83, 116.68, 115.38, 112.32, (–N¼CH–), 162.89, 136.45, 134.12, 133.54, 132.68, 117.45, 116.14,
56.65, 54.98, 26.35; elemental analysis for C13H15N3O: C, 68.10; H, 115.58, 112.23, 55.57, 27.43; elemental analysis for C12H12BrN3O: C,
6.59; N, 18.33. Found: C, 68.08; H, 6.60; N, 18.35.
49.00; H, 4.11; N, 14.29. Found: C, 49.01; H, 4.06; N, 14.32.
4-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-N,N-dimethyla-
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-4-chlorophenol
niline (H5): Yield: 73%; colour: light red powder, mp: 194–196 ꢂC; (H11): Yield: 70%; colour: light yellow powder, mp: 184–186 ꢂC;
FT-IR (cmꢀ1): 1638 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm): FT-IR (cmꢀ1): 1639 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm):
8.76 (s, 1H, –N¼CH–), 7.95 (d, 1H, J ¼ 0.9, H-2 Im), 7.86 (d, 2H, 14.48 (s, 1H, –OH), 8.75 (s, 1H, –N¼CH–), 7.84 (d, 1H, J ¼ 1.0, H-2
J ¼ 8.2, Ar-H), 7.45 (s, 1H, H-5 Im), 7.28 (d, 2H, J ¼ 8.2, Ar-H), 3.70 (t, Im), 7.70–7.67 (d, 1H, J ¼ 8.2, Ar-H), 7.52–7.47 (m, 2H, Ar-H), 7.10 (s,
2H, J ¼ 6.6, –CH2CH2-Im), 3.35 (s, 6H, –N(CH3)2): 2.95 (t, 2H, J ¼ 6.6, 1H, H-5 Im), 3.92 (t, 2H, J ¼ 6.8, –CH2CH2-Im), 2.91 (t, 2H, J ¼ 6.8,
–CH2CH2-Im): 13C NMR (DMSO-d6, 75 MHz,
d d ppm): 166.95
ppm): 166.35 –CH2CH2-Im): 13C NMR (DMSO-d6, 75 MHz,
(–N¼CH–), 162.48, 134.83, 133.35, 131.83, 116.68, 115.38, 112.32, (–N¼CH–), 162.47, 136.32, 134.08, 133.38, 132.92, 117.12, 116.59,
56.65, 54.98, 26.35; elemental analysis for C14H18N4: C, 69.39; H, 115.46, 112.81, 56.12, 27.18; elemental analysis for C12H12ClN3O: C,
7.49; N, 23.12. Found: C, 69.40; H, 7.45; N, 23.15.
57.72; H, 4.84; N, 14.20. Found: C, 57.70; H, 4.80; N, 14.25.
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)phenol
(H6):
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-4,6-dibromo-
Yield: 88%; colour: light yellow powder, mp: 180–182 ꢂC; FT-IR phenol (H12): Yield: 62%; colour: yellow powder, mp: 165–167 ꢂC;
1
(cmꢀ1): 1640 (–C¼N–); H NMR (DMSO-d6, 300 MHz, d ppm): 14.42 FT-IR (cmꢀ1): 1643 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm):
(s, 1H, –OH), 8.49 (s, 1H, –N¼CH–), 7.78 (d, 1H, J ¼ 1.0, H-2 Im), 14.68 (s, 1H, –OH), 8.82 (s, 1H, –N¼CH–), 7.92 (d, 1H, J ¼ 1.0, H-2
7.60–7.57 (d, 1H, J ¼ 8.2, Ar-H), 7.31–7.28 (d, 1H, J ¼ 783, Ar-H), Im), 7.80 (s, 1H, Ar-H), 7.65 (s, 1H, Ar-H), 7.32 (s, 1H, H-5 Im), 3.98
6.88 (s, 1H, H-5 Im), 6.81–6.74 (m, 2H, Ar-H), 3.78 (t, 2H, J ¼ 6.8, (t, 2H, J ¼ 6.6, –CH2CH2-Im), 2.96 (t, 2H, J ¼ 6.6, –CH2CH2-Im): 13C
–CH2CH2-Im), 2.88 (t, 2H, J ¼ 6.8, –CH2CH2-Im): 13C NMR (DMSO-d6, NMR (DMSO-d6, 75 MHz, d ppm): 168.12 (–N¼CH–), 163.37, 137.28,
75 MHz, d ppm): 163.18 (–N¼CH–), 160.42, 132.11, 131.22, 130.12, 135.06, 134.42, 132.98, 117.65, 116.34, 115.80, 112.39, 56.63, 27.87;
115.48, 115.10, 114.78, 111.15, 54.83, 26.25; elemental analysis for elemental analysis for C12H11Br2N3O: C, 38.64; H, 2.97; N, 11.26.
C12H13N3O: C, 66.96; H, 6.09; N, 19.52. Found: C, 66.98; H, 6.05; N, Found: C, 38.60; H, 3.00; N, 11.31.
19.55.
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-4,6-dichlorophe-
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-6-methylphenol
nol (H13): Yield: 60%; colour: yellow powder, mp: 134–136 ꢂC; FT-
(H7): Yield: 78%; colour: white powder, mp: 168–171 ꢂC; FT-IR IR (cmꢀ1): 1642 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm):
(cmꢀ1): 1636 (–C¼N–); H NMR (DMSO-d6, 300 MHz, d ppm): 14.45 14.70 (s, 1H, –OH), 8.80 (s, 1H, –N¼CH–), 7.91 (d, 1H, J ¼ 1.0, H-2
1
(s, 1H, –OH), 8.53 (s, 1H, –N¼CH–), 7.81 (d, 1H, J ¼ 0.9, H-2 Im), Im), 7.82 (s, 1H, Ar-H), 7.63 (s, 1H, Ar-H), 7.30 (s, 1H, H-5 Im), 3.96
7.64–7.60 (d, 1H, J ¼ 7.5, Ar-H), 7.38–7.35 (d, 1H, J ¼ 7.5, Ar-H), 6.93 (t, 2H, J ¼ 6.6, –CH2CH2-Im), 2.94 (t, 2H, J ¼ 6.6, –CH2CH2-Im): 13C

1308
S. AKOCAK ET AL.
NMR (DMSO-d6, 75 MHz, d ppm): 168.05 (–N¼CH–), 163.13, 137.69, 135.47, 133.28, 118.35, 116.56, 115.77, 112.42, 58.24, 27.85; elem-
135.47, 134.43, 132.32, 117.38, 116.04, 115.86, 112.21, 56.38, 27.59; ental analysis for C13H12N4: C, 69.62; H, 5.39; N, 24.98. Found: C,
elemental analysis for C12H11Cl2N3O: C, 50.72; H, 3.90; N, 14.79.
Found: C, 50.70; H, 3.92; N, 14.81.
69.60; H, 5.35; N, 25.01.
N-(4-bromo-2-methylbenzylidene)-2-(1H-imidazol-4-yl)
ethanamine (H20): Yield: 78%; colour: light yellow powder, mp:
208–210 ꢂC; FT-IR (cmꢀ1): 1640 (–C¼N–); 1H NMR (DMSO-d6,
300 MHz, d ppm): 8.88 (s, 1H, –N¼CH–), 7.86 (d, 1H, J ¼ 1.0, H-2
Im), 7.74–7.70 (d, 1H, J ¼ 7.8, Ar-H), 7.58–7.52 (m, 2H, Ar-H), 7.33 (s,
1H, H-5 Im), 3.90 (t, 2H, J ¼ 6.0, –CH2CH2-Im), 2.89 (t, 2H, J ¼ 6.0,
–CH2CH2-Im), 2.43 (s, 3H, –CH3): 13C NMR (DMSO-d6, 75 MHz, d
ppm): 165.67 (–N¼CH–), 160.43, 136.85, 135.01, 133.69, 132.46,
117.58, 116.31, 115.79, 112.32, 57.11, 28.01, 19.44; elemental ana-
lysis for C13H14BrN3: C, 53.44; H, 4.83; N, 14.38. Found: C, 53.40; H,
4.85; N, 14.41.
2-(1H-imidazol-4-yl)-N-((perfluorophenyl)methylene)ethan-
amine (H14): Yield: 75%; colour: white powder, mp: 170–172 ꢂC;
FT-IR (cmꢀ1): 1644 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm):
8.75 (s, 1H, –N¼CH–), 7.92 (d, 1H, J ¼ 1.0, H-2 Im), 7.32 (s, 1H, H-5
Im), 3.95 (t, 2H, J ¼ 6.8, –CH2CH2-Im), 2.92 (t, 2H, J ¼ 6.8, –CH2CH2-
Im): 13C NMR (DMSO-d6, 75 MHz, d ppm): 167.95 (–N¼CH–), 163.37,
134.49, 133.12, 131.18, 130.67, 116.82, 115.89, 114.29, 111.95,
56.69, 27.92; elemental analysis for C12H8F5N3: C, 49.84; H, 2.79; N,
14.53. Found: C, 49.80; H, 2.80; N, 14.56.
N-(2-bromobenzylidene)-2-(1H-imidazol-4-yl)ethanamine
(H15): Yield: 68%; colour: white powder, mp: 199–201 ꢂC; FT-IR
(cmꢀ1): 1639 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.52
(s, 1H, –N¼CH–), 7.75 (d, 1H, J ¼ 1.2, H-2 Im), 7.63–7.59 (d, 1H,
J ¼ 7.8, Ar-H), 7.35–7.30 (d, 1H, J ¼ 7.8, Ar-H), 6.92 (s, 1H, H-5 Im),
6.85–6.78 (m, 2H, Ar-H), 3.82 (t, 2H, J ¼ 6.5, –CH2CH2-Im), 2.90 (t,
2H, J ¼ 6.5, –CH2CH2-Im): 13C NMR (DMSO-d6, 75 MHz, d ppm):
165.68 (–N¼CH–), 161.45, 133.12, 131.88, 130.32, 130.58, 115.21,
115.01, 114.34, 111.55, 55.43, 27.42; elemental analysis for
C12H12BrN3: C, 51.82; H, 4.35; N, 15.11. Found: C, 51.85; H, 4.30; N,
15.15.
N-(5-bromo-2-methoxybenzylidene)-2-(1H-imidazol-4-yl)e-
thanamine (H16): Yield: 72%; colour: white powder, mp:
204–206 ꢂC; FT-IR (cmꢀ1): 1641 (–C¼N–); 1H NMR (DMSO-d6,
300 MHz, d ppm): 8.75 (s, 1H, –N¼CH–), 7.83 (d, 1H, J ¼ 1.0, H-2
Im), 7.70–7.66 (d, 1H, J ¼ 7.8, Ar-H), 7.52–7.45 (m, 2H, Ar-H), 7.21 (s,
1H, H-5 Im), 3.93 (t, 2H, J ¼ 6.6, –CH2CH2-Im), 3.88 (s, 3H, –OCH3),
2.91 (t, 2H, J ¼ 6.6, –CH2CH2-Im): 13C NMR (DMSO-d6, 75 MHz, d
ppm): 166.41 (–N¼CH–), 161.79, 136.02, 134.45, 133.28, 132.78,
117.11, 116.41, 115.29, 112.72, 56.38, 55.93, 27.12; elemental ana-
lysis for C13H14BrN3O: C, 50.67; H, 4.58; N, 13.64. Found: C, 50.70;
H, 4.55; N, 13.68.
CA enzyme activation assay
An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow
instrument has been used to assay the catalytic activity of various
CA isozymes for CO2 hydration reaction42. Phenol red (at a con-
centration of 0.2 mM) was used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM HEPES (pH 7.5) as
buffer, 0.1 M Na2SO4 (for maintaining constant ionic strength), fol-
lowing the CA-catalysed CO2 hydration reaction for a period of
10 s at 25 ꢂC. The CO2 concentrations ranged from 1.7 to 17 mM
for the determination of the kinetic parameters and activation
constants. For each activator at least six traces of the initial 5–10%
of the reaction have been used for determining the initial velocity.
The uncatalysed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of activa-
tors (10 mM) were prepared in distilled-deionised water and dilu-
tions up to 0.001 mM were done thereafter with distilled-deionised
water. Activator and enzyme solutions were pre-incubated
together for 15 min (standard assay at room temperature, or for
prolonged periods of 24–72 h, at 4 ꢂC) prior to assay, in order to
allow for the formation of the E–A complex. The activation con-
stant (KA), defined similarly with the inhibition constant KI, can be
obtained by considering the classical Michaelis–Menten equation
(Equation (4), which has been fitted by non-linear least squares by
using PRISM 3):
2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)benzoic
acid)
(H17): Yield: 68%; colour: cream powder, mp: 168–170 ꢂC; FT-IR
1
(cmꢀ1): 1638 (–C¼N–); H NMR (DMSO-d6, 300 MHz, d ppm): 13.40
(br.s, 1H, –OH), 8.82 (s, 1H, –N¼CH–), 8.12 (d, 1H, J ¼ 0.9, H-2 Im),
7.90 (d, 1H, J ¼ 6.5, Ar-H), 7.83–7.80 (m, 2H, Ar-H), 7.72–7.69 (d, 1H,
J ¼ 7.2, Ar-H), 7.12 (s, 1H, H-5 Im), 3.88 (t, 2H, J ¼ 6.4, –CH2CH2-Im),
2.94 (t, 2H, J ¼ 6.4, –CH2CH2-Im): 13C NMR (DMSO-d6, 75 MHz, d
ppm): 169.34 (–C¼O), 163.72 (–N¼CH–), 146.21, 139.38, 136.92,
133.72, 132.11, 130.48, 129.01, 127.43, 117.22, 59.32, 30.28; elem-
ental analysis for C13H13N3O2: C, 64.19; H, 5.39; N, 17.27. Found: C,
64.15; H, 5.40; N, 17.30.
v ¼ vmax=f1 þ KM=½Sꢁð1 þ ½Aꢁf=KAÞg
where [A]f is the free concentration of activator.
Working at substrate concentrations considerably lower than
KM ([S] ꢃ KM), and considering that [A]f can be represented in the
form of the total concentration of the enzyme ([E]t) and activator
([A]t), the obtained competitive steady-state equation for deter-
mining the activation constant is given by Equation (5):
(4)
2-(1H-imidazol-4-yl)-N-(4-isopropylbenzylidene)ethanamine
(H18): Yield: 74%; colour: white powder, mp: 196–198 ꢂC; FT-IR
(cmꢀ1): 1634 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.72
(s, 1H, –N¼CH–), 7.98 (d, 1H, J ¼ 1.2, H-2 Im), 7.87 (d, 2H, J ¼ 7.4,
Ar-H), 7.48 (s, 1H, H-5 Im), 7.39 (d, 2H, J ¼ 7.8, Ar-H), 3.78 (t, 2H,
J ¼ 6.2, –CH2CH2-Im), 2.95 (t, 2H, J ¼ 6.2, –CH2CH2-Im), 2.82 (m, 1H,
–CH(CH3)2), 1.28 (d, 6H, –CH(CH3)2): 13C NMR (DMSO-d6, 75 MHz, d
ppm): 165.31 (–N¼CH–), 161.23, 135.78, 133.93, 132.36, 117.12,
115.65, 112.45, 57.84, 31.22, 28.65, 24.32; elemental analysis for
C15H19N3: C, 74.65; H, 7.94; N, 17.41. Found: C, 74.61; H, 7.92; N,
17.45.
½ ꢁ
½ ꢁ
½ ꢁ
½ ꢁ ½ ꢁ
½ ꢁ
v ¼ v0:KA=fKA þ ð A t ꢀ 0:5fð A t þ E t þ KAÞ
(5)
2
1=2
½ ꢁ
ꢀð A t þ E t þ KAÞ ꢀ 4 A t: E tÞ gg
where v0 represents the initial velocity of the enzyme-catalysed
reaction in the absence of activator19,25,40,43–46
.
Results and discussion
4-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)benzonitrile
(H19): Yield: 85%; colour: white powder, mp: 188–191 ꢂC; FT-IR
(cmꢀ1): 1638 (–C¼N–); 1H NMR (DMSO-d6, 300 MHz, d ppm): 8.78
(s, 1H, –N¼CH–), 8.08 (d, 1H, J ¼ 1.8, H-2 Im), 7.94 (d, 2H, J ¼ 7.0,
Ar-H), 7.54 (s, 1H, H-5 Im), 7.46 (d, 2H, J ¼ 7.4, Ar-H), 3.88 (t, 2H,
Chemistry
The rationale for designing new CAAs presented in this work is
based on previous data which showed efficient CA activating
effects for derivatised histamine based compounds. Furthermore,
J ¼ 6.4, –CH2CH2-Im), 2.98 (t, 2H, J ¼ 6.4, –CH2CH2-Im): 13C NMR as mentioned above, the X-ray crystal structure for the hCA II-his-
(DMSO-d6, 75 MHz, d ppm): 165.31 (–N¼CH–), 162.78, 139.38, tamine adduct has been used to infer the fact that derivatisation

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1309
of the amino moiety of histamine with not interfere with the bind- but they were not investigated so far as CAAs. This is the reason
ing of the compound in the CAA binding site22–24,28. According to
why the histamine Schiff base derivatives H(1–20) were obtained
and assessed as activators of selected CA isoforms, involved in cru-
cial physiologic and pathologic processes. We expect that the
nature of aliphatic/aromatic/heterocyclic moiety of the aldehyde
reagent will affect the biologic activity of these compounds, and
for this reason we used variously substituted aldehydes, in order
to explore a wider chemical space within the reported series.
Activation data of five physiologically relevant, cytosolic hCA I,
II, and VII, as well as the membrane-anchored (hCA IV) and the
transmembrane hCA IX, with compounds, H(1–20) and histamine
(HST) as standard activator, are shown in Table 1. To the best of
our knowledge, this is the first study which evaluates the activa-
tion profile of histamine Schiff bases on isoforms hCA I, II, IV, VII,
and IX. The following SAR can be observed regarding the activa-
tion data of Table 1:
X-ray results, the amino ethyl moiety of histamine does not have
any interactions with enzyme and it might be derivatised in such
a way as to make new contacts within the active site of the
enzyme, which may lead to more efficient activators, as already
demonstrated for sulphonylated, carboxamide and ureido deriva-
tives of histamine22–25,27,28,47,48
.
A large number of structurally diverse histamine Schiff base
derivatives were synthesised according to general synthetic route
illustrated in Scheme 1. In order to generate chemical diversity,
different substituted aldehydes were chosen, possessing aliphatic,
aromatic and heterocyclic moieties, and they were reacted with
histamine leading to the new histamine Schiff base derivatives
H(1–20) (Scheme 1). All the newly synthesised Schiff bases
H(1–20) were fully characterised by using several analytical and
spectral data (see experimental part for details).
i. The widely abundant cytosolic slow red cell isozyme hCA I
was moderately activated by most of the histamine Schiff
bases H(1–20), with activation constants in the range of
0.24–24.3 mM. Potent hCA I activation has been obtained
with the following compounds: H3, H4, H7, and H8, which
showed KAs of 0.85, 0.24, 0.94, and 0.80 mM, respectively,
CA activation studies
Schiff base derivatives are known for their important biological
applications, especially as efficient and selective CAIs targeting
several isozymes among which CA I, IV, and IX isozymes38–41,49
,
Scheme 1. General synthetic route for the synthesis of histamine Schiff bases H(1–20).

1310
S. AKOCAK ET AL.
of the complex51. Compounds H3–10 showed better activity
(KAs in the range of 0.93–2.15 mM) than histamine (HST) (KA
4.03 mM). The remaining derivatives were medium potency
(KAs in the range of 18.9–47.1 mM, such as H1, and H11–20).
iv. The third cytosolic isoform investigated here, hCA VII, was effi-
ciently activated by all compounds reported here with KAs in
the range of 6 nM to 24.6 mM which all showed better activa-
tion potency then starting reference compound histamine
(HST) (KA 37.6 mM). Specifically, derivatives H4–10 showed nM
potency with KAs ranging from 6 nM to 61 nM against this iso-
form, which is a key isozyme involved in brain metabolism.
They incorporate variously substituted phenyl moieties at the
aldehyde fragment of the Schiff base, among which 4-MeO–,
4-Me2N–, 2-OH, 2-OH-3-Me-phenyl, etc. (Table 1 and Scheme 1).
v. The transmembrane isoform CA IX showed a different activa-
tion profile (low micromolar range) compared to the other
isoforms discussed here (Table 1). The best hCA IX activator
was compound H2 with KA 7.81 mM which also showed the
best activation profile against another membrane-bound iso-
form hCA IV with KA 1.8 nM. However H2 is a medium
potency hCA IX activator and highly efficient hCA IV activa-
tor. The remaining derivatives showed a micromolar activity
with KAs in the range of 15.8–65.4 mM.
Table 1. In vitro hCA I, hCA II, hCA IV, hCA VII, and hCA IX activation data with
histamine Schiff bases H(1–20) by a stopped-flow CO2 hydrase assay.
KA (mM)a
Comp.H
hCA I
hCA II
hCA IV
hCA VII
hCA IX
1
2
3
4
5
6
7
8
18.4
1.60
0.85
0.24
1.17
1.02
0.94
0.80
2.83
4.14
10.7
8.52
11.2
12.7
20.4
24.3
14.1
9.64
17.8
10.4
2.10
54.2
21.7
36.8
37.9
34.2
25.8
14.5
8.62
9.27
10.4
21.9
11.2
24.3
21.2
45.2
30.7
58.9
63.4
44.5
41.8
125
47.1
0.0018
1.24
1.03
2.15
1.70
1.86
0.93
0.96
1.51
36.8
18.9
25.7
33.5
29.1
20.0
24.1
28.3
29.0
30.5
4.03
20.6
21.9
0.81
0.052
0.061
0.012
0.038
0.006
0.012
0.007
12.6
24.6
7.13
11.4
8.23
7.16
6.26
9.51
8.44
0.78
37.6
31.4
7.81
15.8
39.9
26.4
41.7
20.5
31.8
30.0
62.6
65.4
47.9
48.1
51.2
49.0
47.9
31.6
47.9
58.2
57.6
35.2
9
10
11
12
13
14
15
16
17
18
19
20
HST
Histamine (HST) has been used as standard activator42
.
aMean from three different determinations (errors in the range of 5–10% of the
vi. It is important to mention that the compounds H4–10 were
three orders of magnitude more selective for hCA VII as com-
pared with other isozymes hCA I, II, IV, and IX investigated
here. Since hCA VII is a key isoform involved in brain metab-
olism, cognition52, and neuropathic pain53, some of these
newly synthesised compounds might be investigated as a
leads for such neurologic conditions in the search of efficient
pharmacologic agents54.
reported values, data not shown).
which incorporate aromatic moieties with methyl- and/or
methoxy moieties at the phenyl ring. The presence of OH,
carboxy, or halogeno moieties on the phenyl, as well as the
aliphatic or heterocyclic Schiff bases were less effective as
hCA I activators (Table 1).
ii. All compounds reported here H(1–20), were more efficient as
hCA II activators compared to histamine (HST), which is a
weak activator of this isoform with a KA of 125 mM. However,
in general, all compounds showed a moderate activation
against this isozyme with KAs ranging between 8.62 mM and
63.4 mM. The best hCA II activators were H8–10, and H12
with KAs in the range of 8.62–11.2 mM. These derivatives
incorporate phenyl moieties substituted with OH, methoxy,
and bromine, being thus very diverse of the best hCA II acti-
vators discussed above. The compounds H1, H15, and
H17–20 (incorporating aliphatic, 2-bromophenyl-, as well as
2-COOH, 4-i-Pr, 4-CN, and 2-Me-5-Br-phenyl moieties) were
the least effective activators in the series, with KAs in the
range of 41.8–63.4 mM. This structure–activity data show that
small differences in the structure of the activator lead to
impressive differences of the activating properties, due to the
fact that the presence of just one small substituent may lead
to clashes or favourable interactions with amino acid residues
in the activator binding site, which is situated at the entrance
of the cavity, as demonstrated by extensive X-ray structural
Conclusions
We report here a series of 20 histamine Schiff bases which were
synthesised by the reaction of histamine with a large number of
substituted aldehydes incorporating aromatic, heterocyclic, or ali-
phatic moieties. The obtained histamine Schiff bases were investi-
gated as activators of five physiologically relevant CA isozymes,
the cytosolic CA I, CA II, and CA VII, as well as the membrane-
anchored CA IV and transmembrane CA IX isoforms. All com-
pounds showed a better potency than histamine against isozymes
CA I and CA VII with a distinct activation profile and an interesting
structure–activity relationship, dependent on the nature of the
aldehyde fragment present in the molecule. Many of the com-
pounds showed nanomolar efficacy against isozyme CA VII (KAs in
the range of 6 nM to 24.6 mM) which is a key CA isoform involved
in the brain metabolism, cognition, and neuroptic pain. As CAAs
may be used in the memory therapy and cognitive neurodegener-
ative disorders52, these histamine Schiff bases reported here may
be considered of interest for in vivo investigations for possible
therapeutic applications of both activators and inhibitors of these
enzymes.17,52–63
data for CA-activator complexes22,23,30–36,50
.
iii. The membrane-bound isoform hCA IV was activated in a dif-
ferent manner by these compounds. The best activator of
this isoform was H2 with an activation constant of KA 1.8 nM.
This is the only derivative possessing a furan moiety in the
aldehyde fragment of the Schiff base, which confers this
excellent, nanomolar affinity for this isoform. It should be
mentioned that no X-ray crystal structures of hCA IV in com-
plex with activators are available so far, and the modelling
studies of CA-activator complexes were not successful so far,
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
due to the fact that activators do not interact with the metal We would like to thank TUBITAK (The Scientific and Technological
ion, which leads to important distortion due to the param-
eterisation of zinc, which dominates over the remaining parts
Research Council of Turkey) under grant no. 215Z484 for financial
support.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1311
19. Akocak S, Alam MR, Shabana AM, et al. PEGylated bis-sul-
fonamide carbonic anhydrase inhibitors can efficiently con-
trol the growth of several carbonic anhydrase IX-expressing
carcinomas. J Med Chem 2016;59:5077–88.
20. Sun MK, Alkon DL. Pharmacological enhancement of synap-
tic efficacy, spatial learning, and memory through carbonic
References
1. Supuran CT. Advances in structure-based drug discovery of
carbonic anhydrase inhibitors. Expert Opin Drug Discov
2017;12:61–88.
2. Supuran CT. How many carbonic anhydrase inhibition mech-
anisms exist? J Enzym Inhib Med Chem 2016;31:345–60.
3. Supuran CT. Structure and function of carbonic anhydrases.
Biochem J 2016;473:2023–32.
4. Supuran CT. Carbonic anhydrases: novel therapeutic applica-
tions for inhibitors and activators. Nat Rev Drug Discov
2008;7:168–81.
5. Gul HI, Yamali C, Yesilyurt F, et al. Microwave-assisted syn-
thesis and bioevaluation of new sulfonamides. J Enzyme
Inhib Med Chem 2017;32:369–74.
anhydrase activation in rats.
J Pharmacol Exp Ther
2001;297:961–7.
21. Sun MK, Alkon DL. Carbonic anhydrase gating of attention:
memory therapy and enhancement. Trends Pharmacol Sci
2002;23:83–9.
22. Briganti F, Mangani S, Orioli P, et al. Carbonic anhydrase
activators: X-ray crystallographic and spectroscopic investiga-
tions for the interaction of isozymes I and II with histamine.
Biochemistry 1997;36:10384–92.
23. Clare BW, Supuran CT. Carbonic anhydrase activators. 3:
structure–activity correlations for a series of isozyme II acti-
vators. J Pharm Sci 1994;83:768–73.
24. Dave K, Ilies MA, Scozzafava A, et al. An inhibitor-like bind-
ing mode of a carbonic anhydrase activator within the active
site of isoform II. Bioorg Med Chem Lett 2011;21:2764–8.
25. Draghici B, Vullo D, Akocak S, et al. Ethylene bis-imidazoles
are highly potent and selective activators for isozymes VA
and VII of carbonic anhydrase, with a potential nootropic
effect. Chem Commun (Camb) 2014;50:5980–3.
26. Ilies M, Scozzafava A, Supuran CT. Carbonic anhydrase acti-
vators. In: Supuran CT, Scozzafava A, Conway J, eds.
Carbonic anhydrase – its inhibitors and activators. Boca
Raton: CRC Press; 2004:317–52.
27. Licsandru E, Tanc M, Kocsis I, et al. A class of carbonic anhy-
drase I – selective activators. J Enzyme Inhib Med Chem
2017;32:37–46.
€
6. Kocyigit UM, Budak Y, Gurdere MB, et al. Synthesis of chal-
cone-imide derivatives and investigation of their anticancer
and antimicrobial activities, carbonic anhydrase and acetyl-
cholinesterase enzymes inhibition profiles. Arch Physiol
Biochem 2017 [Aug 9]. [Epub ahead of print]. doi: 10.1080/
13813455.2017.1360914
_
7. Gulc¸in I, Abbasova M, Taslimi P, et al. Synthesis and biological
evaluation of aminomethyl and alkoxymethyl derivatives as
carbonic anhydrase, acetylcholinesterase and butyrylcholines-
terase inhibitors. J Enzym Inhib Med Chem 2017;32:1174–82.
8. Neri D, Supuran CT. Interfering with pH regulation in
tumours as a therapeutic strategy. Nat Rev Drug Discov
2011;10:767–77.
9. Akocak S, Ilies MA, Next-generation primary sulfonamide car-
bonic anhydrase inhibitors. In: Supuran CT, Cappasso C, eds.
Targeting carbonic anhydrases. London: Future Science;
2014:35–51.
10. Capasso C, Supuran CT. An overview of the alpha-, beta-
and gamma-carbonic anhydrases from bacteria: can bacterial
carbonic anhydrases shed new light on evolution of bac-
teria? J Enzym Inhib Med Chem 2015;30:325–32.
11. Alterio V, Di Fiore A, D’Ambrosio K, et al. Multiple binding
modes of inhibitors to carbonic anhydrases: how to design
specific drugs targeting 15 different isoforms? Chem Rev
2012;112:4421–68.
12. Guzel-Akdemir O, Akdemir A, Karali N, et al. Discovery of
novel isatin-based sulfonamides with potent and selective
inhibition of the tumor-associated carbonic anhydrase iso-
forms IX and XII. Org Biomol Chem 2015;13:6493–9.
13. Supuran C, Scozzafava A, Conway J, et al. Carbonic anhydrase:
its inhibitors and activators. Boca Raton, FL: CRC Press; 2004.
14. Supuran CT. Carbonic anhydrases as drug targets—an over-
view. Curr Top Med Chem 2007;7:825–33.
15. Supuran CT, Scozzafava A. Carbonic anhydrases as targets
for medicinal chemistry. Bioorg Med Chem 2007;15:4336–50.
16. Lou Y, McDonald PC, Oloumi A, et al. Targeting tumor hyp-
oxia: suppression of breast tumor growth and metastasis by
novel carbonic anhydrase IX inhibitors. Cancer Res
2011;71:3364–76.
28. Dave K, Scozzafava A, Vullo D, et al. Pyridinium derivatives of
histamine are potent activators of cytosolic carbonic anhy-
drase isoforms I, II and VII. Org Biomol Chem 2011;9:2790–800.
29. Supuran CT. Carbonic anhydrases: from biomedical applica-
tions of the inhibitors and activators to biotechnological use
for CO(2) capture. J Enzyme Inhib Med Chem 2013;28:229–30.
30. Temperini C, Innocenti A, Scozzafava A, et al. Carbonic anhy-
drase activators: l-adrenaline plugs the active site entrance
of isozyme II, activating better isoforms I, IV, VA, VII, and XIV.
Bioorg Med Chem Lett 2007;17:628–35.
31. Temperini C, Scozzafava A, Puccetti L, et al. Carbonic anhy-
drase activators: X-ray crystal structure of the adduct of
human isozyme II with l-histidine as a platform for the
design of stronger activators. Bioorg Med Chem Lett
2005;15:5136–41.
32. Temperini C, Scozzafava A, Supuran CT. Carbonic anhydrase
activators: the first X-ray crystallographic study of an adduct
of isoform I. Bioorg Med Chem Lett 2006;16:5152–6.
33. Temperini C, Scozzafava A, Supuran CT. Carbonic anhydrase
activation and the drug design. Curr Pharm Des
2008;14:708–15.
34. Temperini C, Scozzafava A, Supuran CT. Drug design studies
of carbonic anhydrase activators. In: Supuran CT, Winum JY,
eds. Drug design of zinc-enzyme inhibitors – functional, struc-
tural, and disease applications. Hoboken: Wiley; 2009:473–86.
17. Pacchiano F, Aggarwal M, Avvaru BS, et al. Selective hydro-
phobic pocket binding observed within the carbonic anhy-
drase II active site accommodate different 4-substituted-
ureido-benzenesulfonamides and correlate to inhibitor 35. Temperini C, Scozzafava A, Vullo D, et al. Carbonic anhydrase
potency. Chem Commun (Camb) 2010;46:8371–3.
18. Pacchiano F, Carta F, McDonald PC, et al. Ureido-substituted
benzenesulfonamides potently inhibit carbonic anhydrase IX
and show antimetastatic activity in a model of breast cancer
metastasis. J Med Chem 2011;54:1896–902.
activators. Activation of isozymes I, II, IV, VA, VII, and XIV
with l- and d-histidine and crystallographic analysis of their
adducts with isoform II: engineering proton-transfer proc-
esses within the active site of an enzyme. Chemistry
2006;12:7057–66.

1312
S. AKOCAK ET AL.
36. Temperini C, Scozzafava A, Vullo D, et al. Carbonic anhydrase
activators. Activation of isoforms I, II, IV, VA, VII, and XIV
anhydrases with Schiff bases incorporating sulfonamide moi-
eties. Bioorg Med Chem 2015;23:4181–7.
with l- and d-phenylalanine and crystallographic analysis of 50. Temperini C, Winum JY, Montero JL, et al. Carbonic anhy-
their adducts with isozyme II: stereospecific recognition
within the active site of an enzyme and its consequences for
the drug design. J Med Chem 2006;49:3019–27.
drase inhibitors: the X-ray crystal structure of the adduct of
N-hydroxysulfamide with isozyme II explains why this new
zinc binding function is effective in the design of potent
inhibitors. Bioorg Med Chem Lett 2007;17:2795–801.
37. Sippel KH, Robbins AH, Domsic J, et al. High-resolution struc-
ture of human carbonic anhydrase II complexed with aceta- 51. Bernadat G, Supuran CT, Iorga BI. Carbonic anhydrase bind-
zolamide reveals insights into inhibitor drug design. Acta
ing site parameterization in OPLS-AA force field. Bioorg Med
Crystallogr F 2009;65:992–5.
Chem 2013;21:1427–30.
38. Durgun M, Turkmen H, Ceruso M, et al. Synthesis of 4-sulfa- 52. de Souza LC, Provensi G, Vullo D, et al. Carbonic anhydrase
moylphenyl-benzylamine derivatives with inhibitory activity
against human carbonic anhydrase isoforms I, II, IX and XII.
Bioorg Med Chem 2016;24:982–8.
activation enhances object recognition memory in mice
through phosphorylation of the extracellular signal-regulated
kinase
in
the
cortex
and
the
hippocampus.
39. Durgun M, Turkmen H, Ceruso M, et al. Synthesis of Schiff
Neuropharmacology 2017;118:148–56.
base derivatives of 4-(2-aminoethyl)-benzenesulfonamide 53. Carta F, Mannelli LD, Pinard M, et al. A class of sulfonamide
with inhibitory activity against carbonic anhydrase isoforms
carbonic anhydrase inhibitors with neuropathic pain modu-
I, II, IX and XII. Bioorg Med Chem Lett 2015;25:2377–81.
lating effects. Bioorg Med Chem 2015;23:1828–40.
40. Akocak S, Lolak N, Nocentini A, et al. Synthesis and bio- 54. Supuran CT. Carbonic anhydrase inhibition and the manage-
logical evaluation of novel aromatic and heterocyclic bis-sul-
fonamide Schiff bases as carbonic anhydrase I, II, VII and IX
inhibitors. Bioorg Med Chem 2017;25:3093–7.
ment of neuropathic pain. Expert Rev Neurother
2016;16:961–8.
55. Briganti F, Pierattelli R, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors. Part 37. Novel classes of isozyme I and
II inhibitors and their mechanism of action. Kinetic and spec-
troscopic investigations on native and cobalt-substituted
enzymes. Eur J Med Chem 1996;31:1001–10.
56. Supuran CT, Clare BW. Carbonic anhydrase inhibitors – Part
57: quantum chemical QSAR of a group of 1, 3, 4-thiadia-
zole-and 1, 3, 4-thiadiazoline disulfonamides with carbonic
41. Sarikaya B, Ceruso M, Carta F, et al. Inhibition of carbonic
anhydrase isoforms I, II, IX and XII with novel Schiff bases:
identification of selective inhibitors for the tumor-associated
isoforms over the cytosolic ones. Bioorg Med Chem
2014;22:5883–90.
42. Khalifah RG. The carbon dioxide hydration activity of car-
bonic anhydrase. I. Stop-flow kinetic studies on the native
human isoenzymes B and C. J Biol Chem 1971;246:2561–73.
43. Abbate F, Casini A, Owa T, et al. Carbonic anhydrase inhibi-
anhydrase inhibitory properties. Eur
J
Med Chem
1999;34:41–50.
tors: E7070, a sulfonamide anticancer agent, potently inhibits 57. Masini E, Carta F, Scozzafava A, Supuran CT. Antiglaucoma
cytosolic isozymes I and II, and transmembrane, tumor-asso-
carbonic anhydrase inhibitors: a patent review. Expert Opin
ciated isozyme IX. Bioorg Med Chem Lett 2004;14:217–23.
Ther Pat 2013;23:705–16.
44. Scozzafava A, Supuran CT. Carbonic anhydrase activators – 58. Monti SM, Supuran CT, De Simone G. Anticancer carbonic
part 21. Novel activators of isozymes I, II and IV incorporat-
ing carboxamido and ureido histamine moieties. Eur J Med
Chem 2000;35:31–9.
anhydrase inhibitors: a patent review (2008–2013). Expert
Opin Ther Pat 2013;23:737–49.
59. Scozzafava A, Menabuoni L, Mincione F, Supuran CT.
Carbonic anhydrase inhibitors. A general approach for the
preparation of water-soluble sulfonamides incorporating pol-
yamino-polycarboxylate tails and of their metal complexes
possessing long-lasting, topical intraocular pressure-lowering
properties. J Med Chem 2002;45:1466–76.
45. Scozzafava A, Supuran CT. Carbonic anhydrase activators.
Part 24. High affinity isozymes I, II and IV activators, deriva-
tives of 4-(4-chlorophenylsulfonylureido-amino acyl)ethyl-1H-
imidazole. Eur J Pharm Sci 2000;10:29–41.
46. Scozzafava A, Supuran CT. Carbonic anhydrase activators:
high affinity isozymes I, II, and IV activators, incorporating a 60. Carta F, Supuran CT. Diuretics with carbonic anhydrase
beta-alanyl-histidine scaffold. J Med Chem 2002;45:284–91.
47. Ilies M, Banciu MD, Ilies MA, et al. Carbonic anhydrase activa-
inhibitory action: a patent and literature review (2005–2013).
Expert Opin Ther Pat 2013;23:681–91.
tors: design of high affinity isozymes I, II, and IV activators, 61. Scozzafava A, Supuran CT, Carta F. Antiobesity carbonic
incorporating tri-/tetrasubstituted-pyridinium-azole moieties.
anhydrase inhibitors: a literature and patent review. Expert
J Med Chem 2002;45:504–10.
Opin Ther Pat 2013;23:725–35.
48. Ilies MA, Banciu MD, Ilies M, et al. Carbonic anhydrase activa- 62. Dubois L, Peeters S, Lieuwes NG, et al. Specific inhibition of
tors. Part 17. Synthesis and activation study of a series of 1-
(1,2,4-triazole-(1H)-3-yl)-2,4,6-trisubstituted-pyridinium salts
against isozymes I, II and IV. Eur J Med Chem 1997;32:911–8.
carbonic anhydrase IX activity enhances the in vivo thera-
peutic effect of tumor irradiation. Radiother Oncol
2011;99:424–31.
49. Ceruso M, Carta F, Osman SM, et al. Inhibition studies of 63. Carta F, Scozzafava A, Supuran CT. Sulfonamides: a patent
bacterial, fungal and protozoan beta-class carbonic review (2008–2012). Expert Opin Ther Pat 2012;22:747–58.