A class of carbonic anhydrase I–selective activators

Article abstract of DOI:10.1080/14756366.2016.1232254

A series of ureido and bis-ureido derivatives were prepared by reacting histamine with alkyl/aryl-isocyanates or di-isocyanates. The obtained derivatives were assayed as activators of the enzyme carbonic anhydrase (CA, EC 4.2.1.1), due to the fact that hi

Full text of DOI:10.1080/14756366.2016.1232254

Journal of Enzyme Inhibition and Medicinal Chemistry
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A class of carbonic anhydrase I – selective
activators
Erol Licsandru, Muhammet Tanc, Istvan Kocsis, Mihail Barboiu & Claudiu T.
Supuran
To cite this article: Erol Licsandru, Muhammet Tanc, Istvan Kocsis, Mihail Barboiu & Claudiu
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Inhibition and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1232254
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Date: 02 November 2016, At: 01:08

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2016
http://dx.doi.org/10.1080/14756366.2016.1232254
RESEARCH ARTICLE
A class of carbonic anhydrase I – selective activators
Erol Licsandrua, Muhammet Tancb, Istvan Kocsisa, Mihail Barboiua and Claudiu T. Supuranb
aAdaptive Supramolecular Nanosystems Group, Institut Europeen des Membranes, University of Montpellier ENSCM-UMR CNRS 5635,
b
Montpellier, France; Department of Neurofarba and Laboratorio di Chimica Bioinorganica, Sezione di Chimica Farmaceutica e Nutraceutica,
ꢀ
Universita degli Studi di Firenze, Sesto Fiorentino (Florence), Italy
ABSTRACT
ARTICLE HISTORY
Received 5 August 2016
Accepted 31 August 2016
Published online 24 October
2016
A series of ureido and bis-ureido derivatives were prepared by reacting histamine with alkyl/aryl-isocya-
nates or di-isocyanates. The obtained derivatives were assayed as activators of the enzyme carbonic anhy-
drase (CA, EC 4.2.1.1), due to the fact that histamine itself has this biological activity. Although inhibition
of CAs has pharmacological applications in the field of antiglaucoma, anticonvulsant, anticancer, and anti-
infective agents, activation of these enzymes is not yet properly exploited pharmacologically for cognitive
enhancement or Alzheimer’s disease treatment, conditions in which a diminished CA activity was reported.
The ureido/bis-ureido histamine derivatives investigated here showed activating effects only against the
cytosolic human (h) isoform hCA I, having no effect on the widespread, physiologically dominant isoform
hCA II. This is the first report in which CA I-selective activators were identified. Such compounds may con-
stitute interesting tools for better understanding the physiological/pharmacological effects connected to
activation of this widespread CA isoform, whose physiological function is not fully understood.
KEYWORDS
Anchoring to zinc-
coordinated water; carbonic
anhydrase; inhibition
mechanism; inhibitors;
occlusion of the active site
entrance; out of the active
site binding
Introduction
reperfusion injury89, and the management of cerebral ischemia90
were also reported, extending thus the applications of these
pharmacological agents. However, the activators of CAs (CAAs),
although known for decades23,24, do not have at this moment
pharmacological applications. This is due to several reasons, the
first of which has to do with the catalyzed reaction (Scheme 1).
As mentioned above, the catalytically effective species of all
CAs has a metal hydroxide species within the active site, which for
the a-CAs is a zinc hydroxide species generated from a water mol-
ecule bound to the Zn2þ ion (Equation (1) in Scheme 1). This is
also the rate-determining step for the catalytic cycle of many CAs
and it is assisted by buffers present in the medium as well as by
an amino acid residue from the middle of the active site cavity,
His64, which has a pKa of about 7 and may shuttle protons
between the active site and the environment23,24. The second step
(Equation (2) in Scheme 1) involves the nucleophilic attack of the
zinc hydroxide to the CO2 molecule bound in a hydrophobic
pocket, with formation of bicarbonate coordinated to zinc, which
is thereafter replaced by an incoming water molecule, with forma-
tion of the acidic species of the enzyme, with water as the fourth
zinc ligand1–3,23,24. Many CAs are highly effective catalysts, with
turnover numbers of >108 s ꢀ 1, close to the limit of diffusion-con-
trolled processes1–3. Thus, many researchers in the period starting
with 50s until the 90s were reluctant to admit that CAs may have
activators. Only in 1997, we reported the first X-ray crystal struc-
ture of an activator bound to the human (h) CA isoform hCA II.
This activator was histamine23. The activator was found bound at
The carbonic anhydrases (CAs, EC 4.2.1.1) represent a superfamily
of metalloenzymes, with six distinct genetic families known to
date, the a-, b-, c-, d-, f-, and g-CAs, all of the which efficiently
catalyze the reaction between CO2 and water, with the formation
of bicarbonate and protons1–11. The inhibition and the activation
of CAs are well-understood processes: most types of classical
inhibitors bind to the metal center within the enzyme active
site12–21, whereas the activators bind at the entrance of the active
site cavity and participate in proton shuttling processes between
the metal ion-bound water molecule and the environment22–24
This leads to enhanced formation of the metal hydroxide, catalyt-
ically active species of the enzyme1,21–24
.
.
The substrates/reaction products involved in the CA catalyzed
reaction, i.e. CO2, bicarbonate and protons, are essential mole-
cules/ions in many important physiologic processes in all life king-
doms (Bacteria, Archaea, and Eukarya), throughout the tree of life,
and for this reason, relatively high amounts of these enzymes are
present in different tissues/cell compartments of most investigated
organisms1–11
.
Sulfonamides are the most important class of CA inhibitors
(CAIs)25–43, with at least 20 such compounds in clinical use for
decades, or in clinical development44–79. Sulfonamide/sulfamate
CAIs are used as diuretics, antiglaucoma, anticonvulsant, and anti-
obesity agents80–87, whereas the anticancer and anti-infective use
of such derivatives started to be investigated only recently3,4
.
Furthermore, in the last period, the use of CAIs for the manage-
ment of neuropathic pain88, organ preservation without ischemia the entrance of the CA active site cavity, with the imidazole
CONTACT Mihail Barboiu
mihail-dumitru.barboiu@univ-montp2.fr
Adaptive Supramolecular Nanosystems Group, Institut Europeen des Membranes, University
claudiu.supuran@unifi.it Department
of Montpellier ENSCM-UMR CNRS 5635, Place Eugene Bataillon CC047, Montpellier, F-34095, France; Claudiu T. Supuran
of Neurofarba and Laboratorio di Chimica Bioinorganica, Sezione di Chimica Farmaceutica e Nutraceutica, Universita degli Studi di Firenze, Sesto Fiorentino
(Florence), Italy
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

2
E. LICSANDRU ET AL.
EZn2+-OH-
H+
EZn2+-OH2
+
(1)
-rate determining step-
EZn2+-OH2 + HCO3
+ H2O
EZn2+-OH-
-
EZn2+-HCO3
-
+ CO2
(2)
[EZn2+-OH--AH+]
EZn2+-OH2
[EZn2+-OH2-A]
EZn2+-OH-
AH+ (3)
+
+ A
enzyme-activatorcomplexes
Scheme 1. CA-catalyzed CO2 hydration reactions (steps 1 and 2), and CA activation with an activator molecule A.
moiety participating in shuttling protons between the active site 50 ꢁC and for compounds 2, 7, 12, and 13 was 90 ꢁC. For com-
and the bulk solvent, thus acting as a second proton shuttle of pound 1, the purification method was the same. Ureas 1–22 were
1
the enzyme in addition to His64, whereas the amino group from characterized using H NMR methods and mass spectrometry.
the aminoethyl moiety of histamine did not participate in any
interaction with the enzyme active site23
.
1-(2-(1þH-imidazol-4-yl)ethyl)-3-butylurea 1: (mass spectrometry,
The second reason why CAAs do not have for the moment
pharmacological applications is due to their difficult pharmacol-
ogy. Indeed, it has been reported that some CAAs (such as phenyl-
alanine or imidazole) administered to experimental animals may
produce an important pharmacological enhancement of synaptic
efficacy, spatial learning, and memory, proving that this class of
relatively unexplored enzyme modulators may have important
applications in conditions in which learning and memory are
ꢂ
ES M ¼ 211.1)
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.86 (t, 3H, CH3CH2); 1.30
(m, 4H, CH3CH2CH2CH2); 2.58 (t, 2H, NHCH2CH2); 2.96 (q, 2H,
CH2CH2NH); 3.21 (q, 2H, CH2CH2NH), 5.75 (s mod, 1H, NHCH2);
5.83 (s mod, 1H, NHCH2); 6.78 (s, 1H, C CHNH imidazole); 7.55 (s,
1H, N CHNH imidazole).
impaired, such as for example aging or Alzheimer’s disease91,92
.
One must also mention that it was reported that the levels of CA
are significantly diminished in the brain of patients affected by
Alzheimer’s disease93 and these facts strongly support the involve-
ment of different brain CA isozymes in cognitive functions.
However, no clinical trials for the use of CAAs for the management
of these conditions were done at this moment. One should men-
tion that the chemistry and biochemistry of this class of deriva-
1-(2-(1H-imidazol-4-yl)ethyl)-3-hexylurea 2: (mass spectrometry,
ES Mꢂ ¼ 239.1)
þ
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.86 (t, 3H, CH3CH2);
1.24–1.34 (m, 8H, CH3(CH2)4CH2); 2.57 (t, 2H, NHCH2CH2); 2.96 (q,
2H, CH2CH2NH); 3.21 (q, 2H, CH2CH2NH), 5.75 (s mod, 1H, NHCH2);
5.84 (s mod, 1H, NHCH2); 6.75 (s,1H,C CHNH imidazole); 7.54
(s,1H,N CHNH imidazole).
tives was thoroughly investigated, with
a large number of
activators classes (mainly histamine, catecholamine, and amino
acid derivatives94–104) reported and several CA – activator adducts
X-ray crystal structures available23,24,95–97. However, few isoform-
selective CAAs are known to date. Here we report a class of CA I-
selective CAAs, based on the histamine scaffold, which has been
derivatized by using cyanate/dicyanate chemistry.
1-(2-(1H-imidazol-4-yl)ethyl)-3-octylurea 3: (mass spectrometry,
ES Mꢂ ¼ 267.1)
þ
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.86 (t, 3H, CH3CH2);
1.25–1.34 (m, 12H, CH3(CH2)6CH2); 2.58 (t, 2H, NHCH2CH2); 2.95
(q, 2H, CH2CH2NH); 3.21 (q, 2H, CH2CH2NH), 5.74 (s mod, 1H,
NHCH2); 5.82 (s mod, 1H, NHCH2); 6.76 (s,1H,C CHNH imidazole);
7.52 (s,1H,N CHNH imidazole).
Materials and methods
Chemistry
1-(2-(1H-imidazol-4-yl)ethyl)-3-dodecylurea 4: (mass spectrometry,
All the compounds were synthesized following the general pro-
cedure described below. The amine (30 mmol) was mixed with the
corresponding amount of isocyanate, under sonication (1 eq.: 1eq.
for the monourea compounds and 2 eq. for the diurea com-
pounds). The mixture was solubilized in 10 ml of tetrahydrofuran
(THF), 5 ml of ethylacetate, and 10 ml of dimethylacetamide. The
reaction mixture was heated to 120 ꢁC for 15 min. When the pre-
cipitation began, 5 ml of acetonitrile were added and the heating
was maintained for another hour. The resulting product (a white
powder) was filtered and washed with methanol. The exceptions
of the protocol were compounds 1, 6, 10, and 11 for which the
reaction temperature was 60 ꢁC and compounds 2, 7, 12, and 13
for which the reaction temperature was 80 ꢁC. Compound 1 is sol-
uble in the reaction mixture and, therefore, the purification pro-
cedure consisted in evaporation of the solvent under vacuum in a
round bottomed flask and recrystallization from CHCl3.
Alternatively a microwave reactor has been used. The procedure
was the following: the isocyanate was dissolved in 5 ml of aceto-
nitrile and added over the amine in the microwave reactor. The
reaction was performed at 140 ꢁC under energetic stirring, for
ES Mꢂ ¼ 323.2)
þ
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.85 (t, 3H, CH3CH2);
1.25–1.33 (m, 20H, CH3(CH2)10CH2); 2.57 (t, 2H, NHCH2CH2); 2.93
(q, 2H, CH2CH2NH); 3.21 (q, 2H, CH2CH2NH), 5.75 (s mod, 1H,
NHCH2); 5.83 (s mod, 1H, NHCH2); 6.75 (s,1H,C CHNH imidazole);
7.50 (s,1H,N CHNH imidazole).
1-(2-(1H-imþidazol-4-yl)ethyl)-3-octadecylurea 5: (mass spectrom-
etry, ES Mꢂ ¼ 407.3)
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.86 (t, 3H, CH3CH2);
1.24–1.33 (m, 32H, CH3(CH2)16CH2); 2.58 (t, 2H, NHCH2CH2); 2.95
(q, 2H, CH2CH2NH); 3.21 (q, 2H, CH2CH2NH), 5.75 (s mod, 1H,
NHCH2); 5.82 (s mod, 1H, NHCH2); 6,78 (s,1H,C CHNH imidazole);
7,56 (s,1H,N CHNH imidazole).
1,-(Butane-1,4-diyl)bþis(3-(2-(2H-imidazol-4-yl)ethyl)urea) 6: (mass
spectrometry, ES Mꢂ ¼ 363.2)
15 min. The product was filtered and washed with methanol. In 1H-NMR (DMSO-d6, 300 MHz)
the case of compounds 1, 6, 10, and 11, the temperature was CH3(CH2)2CH2); 2.58 (t, 4H, NHCH2CH2); 2.95 (q, 4H, CH2CH2NH);
d
(ppm) ¼ 1.32 (m, 4H,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
3.22 (q, 4H, CH2CH2NH), 5.78 (s mod, 2H, NHCH2); 5.88 (s mod, NHCH2CH2); 3.20 (q, 2H, CH2CH2NH); 3.54 (m, 1H, CH2CH3CHNH);
2H, NHCH2); 6.76 (s,2H,C CHNH imidazole); 7.51 (s,2H,N CHNH 5.63 (s mod, 1H, NHCH2); 5.70 (s mod, 1H, NHCH2); 6.80 (s,1H,C
imidazole).
CHNH imidazole); 7.50 (s,1H,N CHNH imidazole).
(R)-1-(2-(1H-imidazol-4-yl)ethyl)-3-(octan-2-yl)urea 14: (mass spec-
1-(Hexane-1,6-diyl)bþis(3-(2-(2H-imidazol-4-yl)ethyl)urea) 7: (mass
spectrometry, ES Mꢂ ¼ 391.2)
trometry, ES Mꢂ ¼ 267.2)
þ
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.85 (t, 3H, CH3CH2); 0.97
(d, 3H, CH3CHHC2) 1.24 (m, 10H, CH3(CH2)5CH2); 2.57 (t, 2H,
NHCH2CH2); 3.20 (q, 2H, CH2CH2NH); 3.53 (m, 1H, CH2CH3CHNH);
5.67 (s mod, 1H, NHCH2); 5.69 (s mod, 1H, NHCH2); 6.82 (s,1H,C
CHNH imidazole); 7.51 (s,1H,N CHNH imidazole).
1H-NMR (DMSO-d6, 300 MHz)
d
(ppm) ¼ 1.22–1.36 (m, 8H,
CH3(CH2)4CH2); 2.57 (t, 4H, NHCH2CH2); 2.92 (q, 4H, CH2CH2NH);
3.22 (q, 4H, CH2CH2NH) 5.76 (s mod, 2H, NHCH2); 5.85 (s mod, 2H,
NHCH2); 6.76 (s,2H,C CHNH imidazole); 7.55 (s,2H,N CHNH
imidazole).
(S)-1-(2-(1H-imidazol-4-yl)ethyl)-3-(octan-2-yl)urea 15: (mass spec-
trometry, ES Mꢂ ¼ 267.2)
þ
1-(Octane-1,8-diyl)biþs(3–(2-(2H-imidazol-4-yl)ethyl)urea) 8: (mass
spectrometry, ES Mꢂ ¼ 419.3)
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.85 (t, 3H, CH3CH2); 0.97
(d, 3H, CH3CHHC2) 1.23 (m, 10H, CH3(CH2)5CH2); 2.57 (t, 2H,
NHCH2CH2); 3.20 (q, 2H, CH2CH2NH); 3.49 (m, 1H, CH2CH3CHNH);
5.66 (s mod, 1H, NHCH2); 5.69 (s mod, 1H, NHCH2); 6.75 (s,1H,C
CHNH imidazole); 7.51 (s,1H,N CHNH imidazole).
1H-NMR (DMSO-d6, 300 MHz)
d
(ppm) ¼ 1.24–1.36 (m, 12H,
CH3(CH2)6CH2); 2.58 (t, 4H, NHCH2CH2); 2.95 (q, 4H, CH2CH2NH);
3.21 (q, 4H, CH2CH2NH), 5.76 (s mod, 2H, NHCH2); 5.84 (s mod,
2H, NHCH2); 6.77 (s, 2H,C CHNH imidazole); 7.54 (s, 2H, N CHNH
imidazole).
1,1'-((1r,4r)-cyclohexane-1,4-diyl)bis(3-(2-(1H-imidazol-4-yl)ethy-
1-(Dodecane-1,12-diyl)bis(3-(2-(2H-imidazol-4-yl)ethyl)urea)
9:
l)urea) 16: (mass spectrometry, ES Mꢂ ¼ 389.3)
þ
(mass spectrometry, ES Mꢂ ¼ 475.2)
þ
1H-NMR (DMSO-d6, 300 MHz)
d
(ppm) ¼ 1.09 (t mod, 5H,
1H-NMR (DMSO-d6, 300 MHz)
d
(ppm) ¼ 1.24–1.33 (m, 16H,
CHCH2CH2); 1.09 (dd mod, 5H, CHCH2CH2); 2.58 (t, 4H,
NHCH2CH2); 3.32 (q, 4H, CH2CH2NH); 5.69 (s mod, 2H, NHCH2);
5.74 (s mod, 2H, NHCH2); 6.68 (s,2H,C CHNH imidazole); 7.56
(s,2H,N CHNH imidazole).
CH3(CH2)8CH2); 2.59 (t, 4H, NHCH2CH2); 2.95 (q, 4H, CH2CH2NH);
3.22 (q, 4H, CH2CH2NH), 5.77 (s mod, 2H, NHCH2); 5.83 (s mod,
2H, NHCH2); 6.82 (s,2H,C CHNH imidazole); 7.62 (s,2H,N CHNH
imidazole).
1-(2-(1H-imidazol-4-yl)ethyl)-3-phenylurea 17: (mass spectrometry,
(R)-1-(2-(1H-imidazol-4-yl)eþthyl)-3–(3-methylbutan-2-yl)urea
(mass spectrometry, ES Mꢂ ¼ 225.2)
10:
ES Mꢂ ¼ 231.1)
þ
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 2.66 (t, 2H, NHCH2CH2);
2.58 (t, 2H, NHCH2CH2) 3.31 (q, 2H, CH2CH2NH); 6.18 (s mod,1H,C
CHNH imidazole); 6.87 (t,1H, p-CH Ph); 7.20 (t,2H, m-CH Ph); 7.375
(d,2H,o-CH Ph); 7.55 (s,1H,N CHNH imidazole); 8.48 (s,1H, CHNH
imidazole).
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.81 (q, 6H, CH3CHCH3);
0.92 (d, 3H, CH3CHHC2); 1.57 (h, 1H, CH3CHCH3); 2.60 (t, 2H,
NHCH2CH2); 3.21 (q, 2H, CH2CH2NH); 3.45 (m, 1H, CH2CH3CHNH);
5.68 (s mod, 1H, NHCH2); 5.71 (s mod, 1H, NHCH2); 6.82 (s,1H,C
CHNH imidazole); 7.51 (s,1H,N CHNH imidazole).
1,1'-(2,2'-(1,3-phenylene)bis(propane-2,2-diyl))bis(3-(2-(1H-imida-
(S)-1-(2-(1H-imidazol-4-yl)eþthyl)-3-(3-methylbutan-2-yl)urea
(mass spectrometry, ES Mꢂ ¼ 225.2)
11:
zol-4-yl)ethyl)urea) 18: (mass spectrometry, ES Mꢂ ¼ 467.2)
þ
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 1.61 (s,12H, CH3); 2.08
(s,2H, CH); 2,56 (t, 4H, NHCH2CH2) 3.17 (q, 4H, CH2CH2NH); 5.80 (s
mod, 2H, NHCH2); 6.24 (s,1H,C CC phenyl); 6.75 (s, 2H, C CHNH imid-
azole); 7.13–7.18 (m, 3H, CCHCH phenyl and s mod, 2H, NHCH2);
7.33 (s mod, 2H, CHCHCH phenyl); 7.52 (s,1H,N CHNH imidazole).
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.81 (q, 6H, CH3CHCH3);
0.92 (d, 3H, CH3CHHC2); 1.56 (h, 1H, CH3CHCH3); 2.57 (t, 2H,
NHCH2CH2); 3.20 (q, 2H, CH2CH2NH); 3.45 (m, 1H, CH2CH3CHNH);
5.68 (s mod, 1H, NHCH2); 5.70 (s mod, 1H, NHCH2); 6.76 (s,1H,C
CHNH imidazole); 7.51 (s,1H,N CHNH imidazole).
(R)-1-(2-(1H-imidazoþl-4-yl)ethyl)-3–(1-phenylethyl)urea 19: (mass
spectrometry, ES Mꢂ ¼ 259.1)
(R)-1-(2-(1H-imidazol-4-yl)ethyl)-3-(hexan-2-yl)urea 12: (mass spec-
trometry, ES Mꢂ ¼ 239.3)
þ
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 1.29 (d,3H, CH3); 2.57 (t,
2H, NHCH2CH2); 3.21 (q, 2H, CH2CH2NH); 4.70 (qv, 1H, CH3CHNH);
5.79 (s mod, 1H, NHCH2); 6.37 (d mod,1H,NH-CH-Ph); 6.77 (s,1H,C
CHNH imidazole); 7.17–7.33 (m,5H, phenyl); 7.56 (s,1H,N CHNH
imidazole).
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.86 (t, 3H, CH3CH2); 0.97
(d, 3H, CH3CHHC2) 1.24 (m, 6H, CH3(CH2)3CH2); 2.57 (t, 2H,
NHCH2CH2); 3.21 (q, 2H, CH2CH2NH); 3.54 (m, 1H, CH2CH3CHNH);
5.64 (s mod, 1H, NHCH2); 5.69 (s mod, 1H, NHCH2); 6.81 (s,1H,C
CHNH imidazole); 7.51 (s,1H,N CHNH imidazole).
(S)-1-(2-(1H-imidazol-4-yl)ethyl)-3-(1-phenylethyl)urea 20: (mass
(S)-1-(2-(1H-imidazol-4-yl)ethyl)-3-(hexan-2-yl)urea 13: (mass spec-
trometry, ES Mꢂ ¼ 239.2)
spectrometry, ES Mꢂ ¼ 259.1)
þ
þ
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 0.84 (t, 3H, CH3CH2); 0.97 1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 1.29 (d,3H, CH3); 2.60 (t,
(d, 3H, CH3CHHC2) 1.23 (m, 6H, CH3(CH2)3CH2); 2.56 (t, 2H, 2H, NHCH2CH2); 3.19 (q, 2H, CH2CH2NH); 4.72 (qv, 1H, CH3CHNH);

4
E. LICSANDRU ET AL.
5.79 (s mod, 1H, NHCH2); 6.37 (d mod,1H,NH-CH-Ph); 6.80 (s,1H,C experiments were purified recombinant proteins obtained as
reported earlier by our group23,24
.
CHNH imidazole); 7.19–7.33 (m,5H, phenyl); 7.61 (s,1H,N CHNH
imidazole).
Results and discussion
(R)-1-(2-(1H-imidazol-4-yl)eþthyl)-3-(1-(4-fluorophenyl)ethyl)urea 21:
(mass spectrometry, ES Mꢂ ¼ 277.1)
Chemistry
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 1.28 (d,3H, CH3); 2.58 (t,
2H, NHCH2CH2); 3.20 (q, 2H, CH2CH2NH); 4.78 (qv, 1H, CH3CHNH);
5.80 (s mod, 1H, NHCH2); 6.40 (d mod, 1H, NH-CH-Ph); 6.80 (s, 1H,
C CHNH imidazole); 7.13–7.18 (m, 2H, CHCCH phenyl); 7.27–7.32
(m, 2H, CHCFCH phenyl); 7.52 (s,1H,N CHNH imidazole).
The rationale for designing new CAAs reported in this paper is
based on the reported X-ray crystal structure for the hCA II – hista-
mine adduct23. As mentioned above, the aminoethyl moiety of the
activator does not make relevant contacts with the enzyme and is
free to be derivatized as it points out towards the exit of the
active site. In this way, the imidazole moiety of the activator can
participate to the proton shuttling processes crucial for enhancing
the catalytic efficiency of the enzyme, whereas the derivatized
amino group may lead to a further stabilization of the enzyme-
activator adduct. In an earlier work106, we showed that sulfona-
mido, carboxamido, and ureido/thioureido derivatives of histamine
(at the aliphatic portion of the molecule) act as efficient activators
of several CA isoforms, such as hCA I, hCA II, and bCA IV
(b ¼ bovine isoform). As only a few (more exactly 5) ureido deriva-
tives of histamine were reported, all of them incorporating aro-
matic R moieties, here we decided to investigate a larger such
series of ureas and diureas, obtained by reacting histamine with
alkyl/aryl isocyanates and di-isocyanates, as describes in Scheme 2.
A rather large number of such derivatives were obtained
(Table 1), which incorporate various alkyl moieties of variable
length, cycloalkyl, and aryl moieties. All compounds were thor-
oughly characterized by physico-chemical procedures which con-
formed their structure (see Materials and methods for details).
(S)-1-(2-(1H-imidazol-4-yl)eþthyl)-3-(1-(4-fluorophenyl)ethyl)urea 22:
(mass spectrometry, ES Mꢂ ¼ 277.1)
1H-NMR (DMSO-d6, 300 MHz) d (ppm) ¼ 1.28 (d,3H, CH3); 2.58 (t,
2H, NHCH2CH2); 3.22 (q, 2H, CH2CH2NH); 4.72 (qv, 1H, CH3CHNH);
5.79 (s mod, 1H, NHCH2); 6.39 (d mod,1H,NH-CH-Ph); 6.79 (s,1H,C
CHNH imidazole); 7.09–7.15 (m,2H, CHCCH phenyl); 7.27–7.31
(m,2H,CHCFCH phenyl); 7.58 (s,1H,N CHNH imidazole)
Carbonic anhydrase assay
A stopped-flow method105 has been used for assaying the CA cat-
alyzed CO2 hydration activity with Phenol red as indicator, working
at the absorbance maximum of 557 nm, following the initial rates
of the CA-catalyzed CO2 hydration reaction for 10–100 s. For each
activator, at least six traces of the initial 5–10% of the reaction
have been used for determining the initial velocity. The uncata-
lyzed rates were determined in the same manner and subtracted
from the total observed rates. Stock solutions of activator (0.1 mM)
were prepared in distilled–deionized water with 5% DMSO and
dilutions up to 0.1 nM were done thereafter with distilled-deion-
ized water. The activation constant (KA), defined similarly with the
inhibition constant KI, was obtained by considering the classical
Michaelis–Menten equation (Equation (4)), which has been fitted
by non-linear least squares by using PRISM 3:
CA activation
Ureas 1–22 and histamine were assayed for the activation of the
physiologically most important cytosolic isoforms hCA I and II
(Table 1). It should be mentioned that these are widespread iso-
forms in many tissues (e.g. red blood cells contain approximately
150 lM of hCA I and 20 lM of hCA II)1, including not only the
blood but also the gastro-intestinal tract, kidneys, lungs, and the
brain1,7
.
À
Á
v ¼ vmax=f1 þ KM=½Sꢃ 1 þ ½Aꢃf=KA
where [A]f is the free concentration of activator.
g
(4)
Data of Table 1 show some very interesting structure-activity
relationship (SAR) data for the activation of these two isoforms
with histamine and its ureido/bisureido derivatives 1–22. The most
salient feature is that unlike histamine, which is a poor hCA II acti-
vator (KA of 125 lM) but a rather efficient hCA I activator (KA of
2.0 lM), the ureas 1–22 do not activate at all hCA II, but are all
of them effective hCA I activators, with KAs in the range of
0.73–3.4 lM (Table 1). The second rather interesting feature of this
class of CAAs is the fact that the activation constants against hCA
I show a rather modest range, with a minimal variation of potency,
irrespective of the rather diverse substitution pattern at the ureido
moiety, or whether they are mono- or bis-urea derivatives (and as
a consequence they contain one or two imidazole moieties able
Working at substrate concentrations considerably lower than
KM ([S] ꢄ 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 the following equa-
tion23,24,95–104
:
À
Á
v ¼ v0:KA=fKA þ ð½Aꢃtꢀ0:5f ½Aꢃtþ½EꢃtþKA
(5)
À
Á
2
1=2
ꢀ ½Aꢃtþ½EꢃtþKA ꢀ4½Aꢃt:½EꢃtÞ gg
where v0 represents the initial velocity of the enzyme-catalyzed
reaction in the absence of activator. All CA isozymes used in the to participate in proton shuttling processes). Thus, the most
Scheme 2. Synthesis of ureas 1–22 from histamine and isocyanates/diisocyanates.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
Table 1. CA activation against isoforms hCA I and II with ureas 1–22 and histamine as standard, by a stopped-flow CO2
hydrase assayk105. KA ¼activation constant.
ꢂ
ꢂ
No.
Structure
KA hCA I (lM)
KA hCA II (lM)
1
n ¼ 1
3.1
>200
2
3
4
5
6
7
8
9
n ¼ 2
n ¼ 3
n ¼ 5
n ¼ 8
n ¼ 1
n ¼ 2
n ¼ 3
n ¼ 5
2.7
3.0
3.4
2.8
2.9
3.1
2.2
1.7
>200
>200
>200
>200
>200
>200
>200
>200
10
11
1.6
1.5
>200
>200
12
13
3.0
2.2
>200
>200
14
15
16
3.1
3.0
1.6
>200
>200
>200
17
18
1.1
>200
>200
0.73
(continued)

6
E. LICSANDRU ET AL.
Table 1. Continued
ꢂ
ꢂ
No.
19
Structure
KA hCA I (lM)
KA hCA II (lM)
>200
1.2
20
21
1.4
>200
>200
0.98
22
0.97
2.0
>200
–
Histamine
125
ꢂ
Errors in the range of 10% of the reported values, from three different determinations (data not shown).
effective activator is indeed a bis-urea (compound 18), which con- steps of the life cycle of many organisms, including eukaryotes,
tains two imidazoles in its molecule and showed a KA of 0.73 lM,
being thus 2.74 times a more effective hCA I activator compared
to histamine. However, the other submicromolar CAAs detected
here, compounds 21 and 22 (KAs of 0.97–0.98 lM) were monour-
eas, containing only one imidazole moiety. Several of the ureas
investigated here (e.g. 1–8 and 12–15) were slightly less effective
hCA I activators compared with histamine, with KAs in the range
of 2.2–3.4 lM. It is difficult to explain this loss of activity, also con-
sidering the fact that some aryl-ureido histamines reported ear-
lier106, possessing a very diverse substitution pattern compared
with these compounds, possessed a much more efficient activat-
ing profile against hCA I (and also activated hCA II and bCA IV).
Probably the rather long aliphatic moieties present in the ureas
investigated here were detrimental for the binding of the activator
at the entrance of the active site cavity, a region of the enzyme
rich in hydrophilic amino acid residues1,18. However, although
slightly less effective than histamine, these compounds did show
activating properties against this isoform, but not at all against
hCA II, which is probably even more difficult to explain. However,
as explained earlier by us23, the entrance of the active site cavity
of the two isoforms are very diverse, with hCA II possessing a clus-
ter of at least 6 histidine residues (His3, 4, 10, 15, 17, and 64)
which is absent in hCA I. The much more hydrophilic environment
at the entrance of hCA II active site probably explains why the
hydrophobic ureas reported here 1–22 do not efficiently bind to
this enzyme, and do not show any CA activating effect. This is, as
far as we know, the only example of isoform-selective CAA, and
may be of relevance for better understanding the physiology/
pharmacology of hCA I.
Bacteria and Archaea. A large number of CA inhibitors have
pharmacological applications in the field of antiglaucoma, anticon-
vulsant, anticancer, and anti-infective agents, whereas activation of
these enzymes is not yet properly exploited pharmacologically for
cognitive enhancement or Alzheimer’s disease, conditions in which
a diminished CA activity was reported. We report here a series of
ureido/bis-ureido histamine derivatives which were investigated
for their activating effects against the cytosolic human (h) isoform
hCA I and II. We observed that all these compounds, unlike hista-
mine or other activator classes, show no activating effects on the
widespread, physiologically dominant isoform hCA II, but were
rather effective hCA I activators. This is the first report in which CA
I-selective activators were identified. Such compounds may consti-
tute interesting tools for better understanding the physiological/
pharmacological effects connected to activation of this widespread
CA isoform.
Disclosure statement
One author (C. T. S.) declares conflict of interest, being author of
several patents in the field of CA inhibitors/activators. This
research was financed by several EU projects (Euroxy, Metoxia,
DeZnIt, and Dynano). The other authors do not declare conflict of
interest.
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