The inhibitory effects of phenolic Mannich bases on carbonic anhydrase I and II isoenzymes

Article abstract of DOI:10.3109/14756366.2015.1126715

Phenolic mono Mannich bases [2-[4-hydroxy-3-(aminomethyl)benzylidene]-2,3-dihydro-1H-inden-1-one (8–15)] and bis Mannich bases [2-[4-hydroxy-3,5-bis(aminomethyl)benzylidene]-2, 3-dihydro-1H-inden-1-one (2–7)] were synthesized starting from 2-(4-hydroxyben

Full text of DOI:10.3109/14756366.2015.1126715

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
The inhibitory effects of phenolic Mannich bases
on carbonic anhydrase I and II isoenzymes
Cem Yamali, Mehtap Tugrak, Halise Inci Gul, Muhammet Tanc & Claudiu T.
Supuran
To cite this article: Cem Yamali, Mehtap Tugrak, Halise Inci Gul, Muhammet Tanc &
Claudiu T. Supuran (2016): The inhibitory effects of phenolic Mannich bases on carbonic
anhydrase I and II isoenzymes, Journal of Enzyme Inhibition and Medicinal Chemistry, DOI:
10.3109/14756366.2015.1126715
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1126715
Published online: 10 Jan 2016.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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SHORT COMMUNICATION
The inhibitory effects of phenolic Mannich bases on carbonic anhydrase
I and II isoenzymes
Cem Yamali1,2, Mehtap Tugrak1, Halise Inci Gul1, Muhammet Tanc2, and Claudiu T. Supuran2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey and 2Polo Scientifico, Laboratorio Di Chimica
Bioinorganica, Universita Degli Studi Di Firenze, Sesto Fiorentino, Italy
Abstract
Keywords
Phenolic mono Mannich bases [2-[4-hydroxy-3-(aminomethyl)benzylidene]-2,3-dihydro-1H-
inden-1-one (8–15)] and bis Mannich bases [2-[4-hydroxy-3,5-bis(aminomethyl)benzylidene]-2, 3-
dihydro-1H-inden-1-one (2–7)] were synthesized starting from 2-(4-hydroxybenzylidene)-2,
3-dihydro-inden-1-one (1). This study was designed in order to investigate the carbonic
anhydrase (CA, EC 4.2.1.1) inhibitory properties of a library of compounds incorporating the
phenol functional group. All prepared compounds showed a low inhibition percentages on both
human (h) isoforms hCA I and hCA II compared to the reference sulfonamide acetazolamide.
Mannich bases 2–15 had lower inhibition percentages than the compound 1 on hCA I and hCA II,
except compound 14, which is a Mannich base derivative of dipropylamine, which had a similar
inhibitory power as compound 1 on hCA II. All compounds synthesized 1–15 were 1.3–1.9 times
more effective on hCA II comparing with the effectivenes of the compounds on hCA I.
Carbonic anhydrase, indanone, Mannich
bases, phenol
History
Received 19 November 2015
Revised 27 November 2015
Accepted 28 November 2015
Published online 11 January 2016
Introduction
is to obtain isoform-selective CAIs for the various isoforms involved
specifically in different pathologies5–8
.
Carbonic anhydrases (CA, EC 4.2.1.1) are zinc metalloproteins,
which catalyze a very simple reaction: the carbon dioxide (CO2)
hydration reaction to bicarbonate and proton. CA isoforms are
found in a variety of tissues where they participate in several
important biological processes such as acid–base balance, respir-
ation, CO2 and ion transport, bone resorption, ureagenesis,
gluconeogenesis, lipogenesis, and electrolyte secretion. Many CA
isozymes involved in these processe
Chalcones have a-b-unsaturated ketone moiety in its structure
and they are common structure in natural products such as
flavonoids9. Chalcones and its analogs are excellent scaffolds
for synthetic manipulations and also possess multiple biological
and medicinal properties. Indanone-derived compounds were
also reported with several biological activities such as cytotoxic,
antioxidant, anticholinergic, antifungal, and anticancer activities10.
Mannich bases are synthesized by Mannich reaction. It is an
aminomethylation process of a compound or a drug. They are an
important group of compounds in medicinal chemistry.
Aminomethylation of drugs could be used to improve the delivery
of a drug into the human body. Aminomethylation may increase
the hydrophilic properties of drugs through the introduction of a
polar function into the chemical structure. The aminomethylated
drugs could act as prodrugs releasing the active substance under
controlled hydrolytic conditions via deamination. There are
several type of Mannich bases such as carbon Mannich bases
and nitrogen Mannich bases11. Phenolic Mannich bases are a
s are important therapeutic targets with the potential to be
inhibited/activated for the treatment of a range of disorders such
as edema, glaucoma, obesity, cancer, epilepsy, and osteoporosis1.
Some phenol-bearing compounds are reported with their CA
inhibitory activities2. The simple phenol molecule was reported to
act as an inhibitor of the zinc enzyme CA in a different mechanism
of action3. Indeed, sulfonamides coordinate to the metal ion from
the enzyme active site; phenols and derivatives anchor to the water
molecule/hydroxide ion coordinated to the metal ion4,5
.
The side effects of many compounds belonging to the first-/
second generation of carbonic anhydrase inhibitors (CAIs) of types
1–15 (among which metabolic acidosis, kidney stones, bone loss,
and so on) are due to the potent inhibition of all mammalian CA
isoforms, and not only of the target one. The main scope of the drug
group of carbon Mannich bases. It was reported CAs inhibitory12–14
,
cytotoxic15–18, anticonvulsant19–21, antiinflammatory22, diuretic23,
and antifungal24 activities of Mannich bases.
In this study, it was aimed to investigate the CA inhibitory
activities of new phenolic chalcone analog compounds having
indanone skeleton to make a contribution to CA research library
and give some insight to the researchers who are interested in CA
research. For this aim, it was planned to use some phenolic mono
Mannich bases [(2-[4-hydroxy-3-(aminomethyl)benzylidene]-2,3-
dihydro-1H-inden-1-one (8–15, Scheme 1)] and phenolic bis
Mannich bases [(2-[4-hydroxy-3,5-bis(aminomethyl)benzylidene]
-2,3-dihydro-1H-inden-1-one (2–7, Scheme 1)] synthesized
Address for correspondence: Prof. Halise Inci Gul, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University,
Erzurum, Turkey. E-mail: incigul1967@yahoo.com
Prof. Claudiu T. Supuran, Polo Scientifico, Laboratorio di Chimica
Bioinorganica, Universita degli Studi di Firenze, Sesto Fiorentino
(Firenze), Italy. E-mail: claudiu.supuran@unifi.it

2
C. Yamali et al.
J Enzyme Inhib Med Chem, Early Online: 1–4
OH
H
O
i
+
O
O
OH
1
ii
ii
A
A
OH
OH
A
O
O
8-15
2-7
N
N
8
N
O
4
N
5
N
N
A=
N
7
N
6
N
2
3
N
N
N
N
N
N
N
O
9
10
11
12
13
14
15
Scheme 1. General structures of the compounds synthesized. i: Ethanol, aq. NaOH 10% ii: Paraformaldehyde, amine (A), acetonitrile, 200W, 13.8 barr,
120ꢀC Amine used: Corresponding amines of A.
according to our previous studies17,25 starting from 2–(4-hydro-
xybenzylidene)-2,3-dihydro-inden-1-one (1, Scheme 1).
Table 1. Inhibition percentages of compound 1–15 on hCA I and hCA II
isoenzymes at 1 mM inhibitor concentration in the assay system.
% Inhibition % Inhibition
Material and methods
Compound No.
hCA I
A
hCA II
B
hCA II/hCA I
CA enzyme assay
1
2
3
4
5
6
7
8
35
25
26
31
26
26
28
23
23
31
24
26
29
25
23
79
–
45
38
39
42
41
36
37
41
39
42
41
43
43
45
44
95
–
1.3
1.5
1.5
1.4
1.6
1.4
1.3
1.8
1.7
1.4
1.7
1.7
1.5
1.8
1.9
–
An Applied Photophysics stopped-flow instrument has been used
for assaying the CA-catalyzed CO2 hydration activity26. Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining
constant ionic strength), following the initial rates of the CA-
catalyzed CO2 hydration reaction for a period of 10–100s. The
CO2 concentrations ranged from 1.7 to 17 mM for the determin-
ation of the kinetic parameters and inhibition constants. For each
inhibitor, at least six traces of the initial 5–10% of the reaction
have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of
inhibitor (0.1 mM) were prepared in distilled–deionized water and
dilutions up to 0.01 nM were done thereafter with the assay buffer.
Inhibitor and enzyme solutions were preincubated together for
1.4
1.3
1.1
1.3
1.3
1.3
1.5
1.5
1.1
1.5
1.3
1.2
1.4
1.5
–
1.2
1.2
1.1
1.1
1.3
1.2
1.1
1.2
1.1
1.1
1.0
1.0
1.0
1.0
–
9
10
11
12
13
14
15
AAZ
AAZ: Acetazolamide used as a positive control.
Inhibition concentration was 10ꢁ6 M for all compounds.
15 min at room temperature prior to assay, in order to allow for ‘‘A’’ values were calculated by dividing the inhibition percentage value of
the compound 1 against hCA I to inhibition percentage value of the
Mannich bases against hCA I.
‘‘B’’ values were calculated by dividing the inhibition percentage value
of the compound 1 against hCA II to inhibition percentage value of the
Mannich bases against hCA II.
the formation of the E–I complex. The inhibition percantage was
obtained by using PRISM 3, as reported earlier27, and represent
the mean from at least three different determinations. All CA
isoforms were recombinant ones obtained in-house as reported
earlier28. The cell pellets were lysed, and hCA II and hCA I were
purified through affinity chromatography using pAMBS resin.
Table 1, Mannich bases 2–15 had lower inhibition percentage on
hCA I than the compound 1 from which Mannich bases were
derived from. Only the compound 14 had equal inhibition
percentage with the compound 1 on hCA II. All compounds
had lower inhibition percentages than reference drug
Results and discussion
The hCA I and hCA II inhibition percentages of the compounds
1–15 are shown in Table 1 and also in Figure 1. According to

DOI: 10.3109/14756366.2015.1126715
CA inhibitory effects of phenolic Mannich bases
3
Figure 1. CA inhibition percentages of the compounds on graphical model.
acetazolamide (AAZ) on both hCA I and hCA II. Accroding to the inhibition on hCA I. The Mannich bases reported here show more
results in Table 1, bis Mannich bases compound 2 (with N-methyl poor inhibitory activity against these two CA isoforms than
piperazine), compound 3 (with pyrrolidine), compound 4 (with starting compound which is chalcone analog. This is probably due
morpholine), and compound 7 (with dipropylamine) did more to the fact that in the ortho position to the OH moiety there are
inhibition on hCA I than their corresponding mono Mannich one or two rather bulky functionalities which may interfere with
bases.
the binding of the compound to the enzyme active site. By this
The inhibition potency of the bis Mannich bases at issue in study, CA inhibitory acitivities of new phenolic compounds were
terms of times for the compound 2, 3, and 7 were 1.1 times while reported to give some insight to the scientists focusing on CA
it was 1.3 times for the compound 4 comparing to their research.
corresponding mono Mannich bases. On the other hand, mono
Mannich base 10 (with piperidine) had shown 1.2 times more
potent hCA I inhibition than its corresponding bis Mannich base
analog, the compound 5.
Acknowledgements
The authors H.I.G and C.T.S thank to ERASMUS Office of Ataturk
University, Turkey, for their financial support to Yamali C.
When inhibitory activities of the compounds on hCA II were
considered, mono Mannich bases were more effective than bis
Mannich bases on the contrary to hCA I’s. It can be easily noticed
from Table 1 that mono Mannich bases 8 (with N-methyl
piperazine), 11 (with pyrrolidine), 12 (with dimethylamine), and
14 (with dipropylamine) were more effective than their corres-
ponding bis Mannich bases on hCA II. The potency ratios were
1.1 times stronger for the compounds 8 and 11, and 1.2 times
stronger for the compounds 12 and 14 comparing to their
corresponding bis Mannich bases.
The compound 1 was more effective on hCA I (1.1–1.5
times) and hCA II (1.1–1.3), compared with the Mannich bases
2–15. The Mannich bases reported here show poor inhibitory
activity against these two CA isoforms. This is probably due to
the fact that in the ortho position/s to the OH moiety there are
one or two rather bulky functionalities which may interfere with
the binding of the compound to the enzyme active site. In
addition, the compounds 1–15 were 1.3–1.9 times more effective
on hCA II than on hCA I without exception according to results
in Table 1 and Figure 1.
Declaration of interest
The authors report no conflict of interest and are responsible for
the contents and writing of the paper.
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