Carbonic anhydrase inhibition and cytotoxicity studies of Mannich base derivatives of thymol

Article abstract of DOI:10.3109/14756366.2016.1140755

Mannich bases of thymol were synthesized. The aminomethylation reaction was realised in the ortho position of the phenol for compounds 2 (dipropylamine), 3 (benzylamine), and 4 (dibenzylamine) while it was from para position for 1 (dimethylamine), 5 (pipe

Full text of DOI:10.3109/14756366.2016.1140755

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Carbonic anhydrase inhibition and cytotoxicity
studies of Mannich base derivatives of thymol
Halise Inci Gul, Cem Yamali, Asiye Tugce Yasa, Elif Unluer, Hiroshi Sakagami,
Muhammet Tanc & Claudiu T. Supuran
To cite this article: Halise Inci Gul, Cem Yamali, Asiye Tugce Yasa, Elif Unluer, Hiroshi Sakagami,
Muhammet Tanc & Claudiu T. Supuran (2016): Carbonic anhydrase inhibition and cytotoxicity
studies of Mannich base derivatives of thymol, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2016.1140755
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RESEARCH ARTICLE
Carbonic anhydrase inhibition and cytotoxicity studies of Mannich base
derivatives of thymol
Halise Inci Gul1, Cem Yamali1,2, Asiye Tugce Yasa1, Elif Unluer1, Hiroshi Sakagami3, Muhammet Tanc2, and
Claudiu T. Supuran2
2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey, Polo Scientifico, Laboratorio di Chimica
3
Bioinorganica, Universita degli Studi di Firenze, Sesto Fiorentino, Italy, and Division of Pharmacology, Meikai University School of Dentistry,
Sakado, Saitama, Japan
Abstract
Keywords
Mannich bases of thymol were synthesized. The aminomethylation reaction was realised in
the ortho position of the phenol for compounds 2 (dipropylamine), 3 (benzylamine), and
4 (dibenzylamine) while it was from para position for 1 (dimethylamine), 5 (piperidine),
6 (morpholine) and 7 (N-methylpiperazine). The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory
effects of the compounds were asssessed against hCA I and hCA II. All compounds moderately
inhibited hCA I and hCA II. The cytotoxicity of the compounds against four human oral
squamous cell carcinoma cell lines were compared those against three normal oral cells. Tumor
specificity values were about 2 or slightly more for the compounds 2, 3, 4, 5 and 6. Compound
2 showed cytostatic activity against OSCC cell lines at 16 to 32-fold lower concentrations as
compared with normal cells. This suggests that compound 2 can be considered as cytotoxicity
enhancing drug candidate for further investigations.
Carbonic anhydrase, cytotoxicity, Mannich
bases, phenol, thymol
History
Received 16 December 2015
Accepted 7 January 2016
Published online 3 February 2016
Introduction
anticonvulsant2, anticancer3, analgesic1 and antiinfective2 activ-
ities. But the compounds mentioned above are not selective
inhibitors of CAs and can cause some side effects2.
Carbonic anhydrase (CA, EC 4.2.1.1), a zinc-dependent metal-
1
loenzyme, catalyzes the reversible hydration of CO2 . This
The CA inhibitory effects of some natural products and their
derivatives have been recently investigated5. It was reported that
natural compounds carring phenol or polyphenol moiety had
carbonic anhydrase inhibitory activity at micromolar concentra-
tion6. In addition, the compounds having phenol functional group
are known to have anticancer, antibacterial, antiviral, antifungal
enzyme plays an important role in physiologic and pathologic
processes, such as pH and CO2 homeostasis, electrolyte secretion,
lipogenesis, ureagenesis and calcification2. Sixteen different
isoforms are known in mammals which are cytosolic (CA I, CA
II, CA III, CA VII and CA XIII), membrane-bound (CA IV,
CA IX, CA XII, CA XIV and CA XV), mitochondrial (CA VA and
CA VB), or secreted (CA VI) proteins2,3. There are known
diuretics, antiepileptic, antiglaucoma, anticancer drugs based on
CA inhibitors (CAIs) and they target different human a-CA
isoforms2. Recently, it has been showed that these enzymes are
also present in protozoa, bacteria and fungi1. Therefore, these
enzymes are the target of potential antibacterial, antimalarial and
antifunfal agents4.
and antioxidant activities7–9
.
Thymol, 2-isopropyl-5-methylphenol, is a bioactive natural
phenolic compound which was isolated from Thymus sulgaris10.
Thymol is known to show antioxidant11–13, anticancer14,15
antidepressant16, antibacterial17,18 and antifungal19 activities.
,
Mannich bases are an important group of compounds in
medicinal chemistry and may be synthsized by Mannich reaction.
There are several type of Mannich bases, such as carbon Mannich
bases and nitrogen Mannich bases20. Phenolic Mannich bases are
Some primary sulfonamides with CA inhibitory action and
their derivatives have been used for a long time in the treatment of
some diseases because of their diuretic and antiglaucom effects1,2
Commercially available CAIs, such as acetazolamide, ethoxzola-
mide and dorzolamide are also known for their potential
a group of carbon Mannich bases20. CAs inhibition9,21,22
,
.
cytotoxic8,23–25, anticonvulsant26–28, diuretic29, antifungal30 and
antioxidant10 activities of several Mannich bases had been
reported.
The aims of this study were as follows:
(i) We aimed to synthesize several phenolic mono Mannich
bases of thymol by using acyclic and cyclic amines having
different chemical structure, properties and pKa values,
which may govern the chemical reactions and bioactivities.
(ii) To investigate the CA inhibitory activity of the compounds
against the human (h) isoforms hCA I and hCA II since it is
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
H. I. Gul et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
a known phenol function, which is a group of carbonic Methods
anhydrase inhibitors. There is no study reporting the
Synthesis of mono Mannich bases, 1–7
carbonic anhydrase inhibitory activities of mono Mannich
bases of natural compound thymol.
The mixture of thymol (6.6 mmol), formaline (6.6 mmol, 37%)
(iii) To investigate the cytotoxic activities of thymol mono and amine (6.6 mmol) [Dimethylamine (1), dipropylamine (2),
Mannich bases against four human oral squamous cell benzylamine (3), dibenzylamine (4), piperidine (5), morpholine
carcinoma cell lines: Ca9-22 (derived from the gingival (6) and N-methylpiperazine (7)] was heated in methanol (15 ml)
tissue), HSC-2, HSC-3 and HSC-4 (derived from the for 15-26 h (Scheme 1). Reactions were monitored by TLC. When
tongue) as compared with three normal oral mesenchymal the reaction was stopped, the solvent was reduced half of volume
cells: HGF (human gingival fibroblasts), HPC (human pulp under vacuum. The content was kept at +4ꢀC for 24 h. The solids
cells) and HPLF (human periodontal ligament fibroblasts). obtained [for the compounds 5, 6 and 7] were crystallized from
The results to be obtained may be useful for new drug suitable solvents [it was dipropylether (5) and acetonitrile (6 and
candidate/s development.
7)]. On the other hand, the compounds 1, 2, 3 and 4 were purified
by column chromatography on silica gel (SiO2) using chloroform
as a mobile phase.
Experimental
5-Methyl-4-[(dimethylamino)methyl]-2-(propan-2-yl)phenol,
compound 1
Materials
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were taken
using a Varian Mercury Plus spectrometer (Varian Inc., Palo Alto,
CA). Chemical shifts (d) were reported in ppm. Melting points
were determined using an Electrothermal 9100 (IA9100, Bibby
Scientific Limited, Staffordshire, UK) instrument and are uncor-
rected. Liquid chromatography ion trap-time of flight tandem
mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an
A viscous liquid, yield 15%. 1H NMR (400 MHz, CDCl3) d: 6.99
(s, 1H), 6.37 (s, 1H), 3.33 (s, 2H), 3.20-3.15 (m, 1H), 2.31–2.20
(m, 9H), 1.23–1.20 (m, 6H).
3-Methyl-2-[(dipropylamino)methyl]-6-(propan-2-yl)phenol,
compound 2
1
electrospray ionization (ESI) source, operating in both positive A viscous liquid, yield 6%. H NMR (400 MHz, CDCl3) d: 6.99
and negative ionization mode. Shimadzu’s LCMS Solution (d, J ¼ 7.7 Hz, 1H), 6.60 (d, J ¼ 7.7 Hz, 1H), 3.75 (s, 2H),
software was used for data analysis.
3.33-2.26 (m, 1H), 2.49-2.45 (m, 4H), 2.21 (s, 3H), 1.62-1.53
OH CH
3
A
CH
3
H C
3
OH CH
3
2, 3, 4
O
CH
Methanol
Reflux
3
Amine
+
+
H
H
H C
3
OH CH
3
CH
3
H C
3
A
1, 5, 6, 7
N
CH
3
N
O
N
CH
3
A=
N
N
N
N
CH
N
3
1
2
3
4
5
6
7
Amine used: Corresponding amines of A
Scheme 1. Synthesis of the Mannich bases 1–7.

DOI: 10.3109/14756366.2016.1140755
Mannich base derivatives of thymol
3
(m, 4H), 1.21 (d, J ¼ 7.2 Hz, 6H), 0.90 (t, J ¼ 7.2 Hz, 6H). 13C 15 min at room temperature prior to assay, in order to allow for
NMR (100 MHz, CDCl3) d: 156.1, 133.7, 133.5, 124.5, 120.6, the formation of the E-I complex. The inhibition percentages were
119.9, 55.7, 54.4, 26.6, 22.9, 19.8, 12.1. HRMS (ESI-MS) Calc. obtained by using PRISM 3, as reported earlier33, and represent
for C17H29NO [M + H]+ 264.2322; found 264.2346.
the mean from at least three different determinations. All CA
isoforms were recombinant ones obtained in-house as reported
earlier34. The cell pellets were lysed, and hCA II and hCA I were
purified through affinity chromatography using pAMBS resin.
3-Methyl-2-[(benzylamino)methyl]-6-(propan-2-yl)phenol, com-
pound 3
A viscous liquid, yield 10%. 1H NMR (400 MHz, CDCl3) d: 7.39-
7.28 (m, 5H), 7.01 (d, J ¼ 8.1 Hz, 1H), 6.72 (d, J ¼ 8.1 Hz, 1H),
Cytotoxicity evaluation
3.91(s, 2H), 3.89 (s, 2H), 3.28–3.25 (m, 1H), 2.08 (s, 3H), 1.23 (d, The cytotoxicity of the compounds were assayed towards human
J ¼ 6.6 Hz, 6H). 13C NMR (100 MHz, CDCl3) d: 151.6, 138.6, oral squamous cell carcinoma (Ca9-22, HSC-2, HSC-3, HSC-4)
133.9, 133.7, 129.3, 128.7, 127.6, 123.8, 121.8, 118.4, 81.6, 56.2, and human normal oral cells (HGF, HPLF, HPC) as described35
48.8, 26.5, 22.9, 18.3. HRMS (ESI-MS) Calc. for C18H23NO with some minor modifications. In brief, all cells were cultured in
[M + H]+ 270.1852; found 270.1855.
DMEM supplemented with 10% fetal bovine serum (FBS). The
following concentrations of the compounds in dimethylsulfoxide
(DMSO) were added to the medium and incubated at 37ꢀC for
48 h: compounds 1–7 (0.32, 1, 3.2, 10, 31.6, 100, 316, 1000 mM)
and Melphalan (3.12, 6.25, 12.5, 25, 50, 100, 200, 400 mM). The
media that contained the same concentration of DMSO (0.0078,
0.156, 0.03125, 0.0625, 0.125, 0.25, 0.5 or 1%) were used as
controls, since DMSO above 0.25% is cytotoxic. The viable cell
numbers were determined by the MTT method. The CC50 values
were determined from dose-response curves.
3-Methyl-2-[(dibenzylamino)methyl]-6-(propan-2-yl)phenol,
compound 4
A viscous liquid, yield 6%. 1H NMR (400 MHz, CDCl3) d: 11.40
(brs, 1H, –OH), 7.38–7.27 (m, 10H), 7.00 (d, J ¼ 7.9 Hz, 1H),
6.63 (d, J ¼ 7.9 Hz, 1H), 3.76 (s, 2H), 3.62 (s, 2H), 3.58 (s, 1H),
3.49 (d, J ¼ 8.4 Hz, 2H), 3.35–3.33 (m, 1H), 2.24 (s, 2H), 1.24 (d,
J ¼ 6.6 Hz, 6H).
5-Methyl-4-[(piperidin-1-yl)methyl]-2-(propan-2-yl)phenol,
compound 5
Results and discussion
The compounds investigated here were synthesized by the
conventional heating method. The mixture of thymol, formalin
solution and amine was refluxed in methanol; the reagents had the
same mole ratios. Although the same experimental procedure was
applied for the synthesis of phenolic mono Mannich bases of
thymol, the aminomethylation reaction took place at the ortho
position of phenol in the case of the reactions for compounds 2, 3
and 4, in which secondary or primary aliphatic amines were used.
A white solid, yield 12%, m.p. 148 ꢀC, 145 ꢀC31. 1H NMR
(400 MHz, CDCl3) d: 6.99 (s, 1H), 6.26 (s, 1H), 3.32 (s, 2H), 3.16
(q, J ¼ 6.9 Hz 1H), 2.42 (brs, 4H), 2.18 (s, 3H), 1.60–1.55
(m, 4H), 1.43 (brs, 2H), 1.21 (d, J ¼ 6.9 Hz, 6H).
5-Methyl-4-[(morpholino-4-yl)methyl]-2-(propan-2-yl)phenol,
compound 6
1
A white solid, yield 16%, m.p. 152 ꢀC, 152 ꢀC10. H NMR (400 On the other hand, aminomethylation reaction took place at para
MHz, CDCl3) d: 7.00 (s, 1H), 6.48 (s, 1H), 3.69 (brs, 4H), 3.38 position of phenol for the reactions of the compounds 1, 5, 6 and
(s, 2H), 3.16–3.13 (m, 1H), 2.44 (brs, 4H), 2.26 (s, 3H), 1.22 7. Secondary cyclic amines were used for the reactions of 5, 6 and
(d, J ¼ 6.9 Hz, 6H).
7. Compounds obtained with the secondary cyclic amines were
solid apart from the others. Unique pattern of reaction was
observed when the dimethyl amine (for compound 1) was used as
an amine component. Although compound 1 is aliphatic and non-
cyclic amine like dipropylamine (2), benzylamine (3) and
dibenzylamine (4), it gave different reaction from compounds 2
to 4. Aminomethylation reaction took place at para position of
phenol function in a similar way with secondary cyclic amines.
Actually, to occupy the place of aminomethylation, i.e. Mannich
reaction at ortho or para positions of phenol is an expected
scientific fact, since hydroxyl group is a first-class substituent and
direct substituent which will come to ortho or para position by
itself. What are the substituents on nitrogen and how the
substituents are located on it seems to be of great importance to
direct aminomethylation process of thymol to ortho or para
position of phenol function. Aminomethylation took place at para
position of phenol function when the substituents on nitrogen
were two methyls or bigger than this in size and these were
acceptable on the condition that the amine used was in cyclic form
in which free rotation was prevented as in the case of piperidine,
morpholine and N-methylpiperazine. On the other hand, amino-
methylation took place at ortho position of phenol function when
the two substituents on nitrogen had longer chain than methyl
(dipropylamine) and the hydrogens of methyl substituents located
on nitrogen were replaced by phenyl rings (dibenzylamine). Ortho
aminomethylation also occurred when the substituent was
benzylamine. In this case, there is one methyl substituent on
nitrogen and one of its hydrogens was replaced by phenyl
(benzylamine). The common point for the ortho substitution of
5-Methyl-4-[(N-methylpiperazin-1-yl)methyl]-2-(propan-2-
yl)phenol, compound 7
A white solid, yield 29%, m.p. 159 ꢀC. 1H NMR (400 MHz,
CDCl3) d: 7.00 (s, 1H), 6.45 (s, 1H), 3.38 (s, 2H), 3.21–3.14
(m, 1H), 2.47 (brs, 8H), 2.29 (s, 3H), 2.24 (s, 3H), 1.22 (d, J ¼ 6.9
Hz, 6H).
Biological activity
Carbonic anhydrase enzyme assay
An Applied Photophysics stopped-flow instrument has been used
for assaying the CA catalyzed CO2 hydration activity32. Phenol
red (at a concentration of 0.2 mM) has been used as an indicator,
working at the absorbance maximum of 557 nm, with 20 mM
Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining
constant the ionic strength), following the initial rates of the CA-
catalyzed CO2 hydration reaction for a period of 10–100 s. The
CO2 concentrations ranged from 1.7 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

4
H. I. Gul et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
phenol function was that substituents present on nitrogen have the calculated. First, TS was calculated by dividing the mean CC50
ability of free rotation.
value of each compound against three human oral normal cells
1H NMR spectra of the ortho substituted thymols gave two (Column D) by the mean CC50 value against four human
doublets for the hydrogens located at 4 and 5 position of phenol OSCC cell lines (Column B). Second, TS was also calculated
1
function, while H NMR spectra of the para substituted thymol by dividing the value CC50 of each compound against HGF
derivatives gave two singlets for the hydrogens located at 3 and 6 cells (Column C) by the CC50 value against Ca9-22 cell lines
position of phenol function as reported in the ‘‘Experimental’’ (Column A), both cells being originated from the same tissue
section. The compounds 2 and 3 were reported for the first time. (gingiva). Tumor-specificity values are presented at Table 2.
Chemical structure of the compounds were confirmed by 1H All compounds showed lower TS values than reference drug
NMR and/or melting points for the literature registered com- melphalan by these two types of criteria. According to TS
pounds (1, 4, 5, 6 and 7). 1H NMR, 13C NMR and HRMS for the values obtained by first calculation method, the order of the
compounds 2 and 3 reported here for the first time.
potency of TS values of the compounds was as follow: The
The CA inhibition (% inhibition at 0.1 mM concentration of compound number (TS value): 5 (2.2)46 (2.0)43 (1.9)42
inhibitor) of the Mannich bases prepared here is shown in Table 1. (1.8). The calculation of TS value for 4 was difficult due to
This percentage was in the range of 18–26% for hCA I and of much lower cytotoxicity of this compound to both malignant
28–33% for hCA II. Contrary to other phenols investigated and non-malignant cells. When the second calculation was
earlier5,6,9, the Mannich bases reported here show poor inhibitory considered, compound 4 showed TS value greater than 2.1 due
activity against these two CA isoforms. This is probably due to the to the scale over of CC50 value, while the compound 5
fact that in the ortho position to the OH moiety there is one or two (TS¼2.6) had higher TS value than the compounds 2 and 3,
rather bulky functionalities, which may interfere with the binding which showed comparable tumor-specificity (TS ¼ 2.2). On the
of the compound to the enzyme active site.
other hand, compound 2 showed some selectivity against
The cytotoxic activity of the compounds were compared human oral squamous cell lines, especially at lower concen-
between four human oral squamous cell carcinoma (OSCC) tration. The growth of all four OSCC cell lines declined at
cell lines (Ca9-22, HSC-2, HSC-3, HSC-4) and three human 7.8–15.6 mg/ml, whereas the growth of all three normal cells
normal oral cells (HGF, HPLF, HPC). The results are declined at 16–32-fold higher concentration (250 mg/ml)
presented in Table 2. Tumor-specificity (TS) value reflects (Figure 1 and Table 3). The TS values may be significantly
the selectivity of the compounds against cancer tissues rather affected by the type of growth inhibition, either cytotoxic or
than normal ones. In this study, two types of TS values were cytostatic.
Table 1. hCA I and hCA II inhibition percentage of the Mannich bases
1–7.
% Inhibition
Compound
Inhibitor concentration, M
hCA I
hCA II
1
2
3
4
5
6
10ꢁ7
10ꢁ7
10ꢁ7
10ꢁ7
10ꢁ7
10ꢁ7
10ꢁ7
10ꢁ7
26
19
19
18
23
21
23
80
32
32
29
28
33
32
30
80
7
Acetazolamide
Figure 1. Viable cell number percentange of human OSCC and normal
cells treated with increasing concentrations with compound 2.
Table 2. Cytotoxic activities of the Mannich bases 1–7.
CC50 (mM)
Human OSCC cell lines
Human oral normal cells
Ca9-22
HSC-2
HSC-3
HSC-4
mean
HGF
HPLF
HPC
mean
TS
TS
Compound
1
2
3
4
(A)
734
125
98
467
321
728
575
27.8
(B)
(C)
726
271
(D)
767 36
321 83
(D/B)
1.1
1.8
1.9
51.2
2.2
2
1.6
11.1
(C/A)
1
698
250
150
632
54
102
719
281
99
696 45
178 106
112 25
4867 267
367 47
409 273
485 291
15.3 8.4
785
417
207
791
276
203
2.2
2.2
42.1
2.6
1
220
210
9
41000
419
417
41000
335
61
41000
394
428
41000
836
753
41000
851
970
41000
722
766
41000
803 71
830 122
765 31
170 27
5
6
7
Melphalan
632
11.2
53
10.9
679
11.1
747
144
800
168
747
198
1.3
5.2
CC50 values refer to the concentrations of the compounds in micromoles which reduce the viable cell number by 50%. Human oral squamous cell
carcinoma (OSCC) cell lines used are Ca9-22 (derived from gingiva), HSC-2, HSC-3, HSC-4 (derived from tongue). Normal oral cells used are
human gingival fibroblasts (HGF), human periodontal ligament fibroblasts (HPLF), and human pulp cells (HPC). Tumor-specificity (TS) value is
calculated by dividing the mean CC50 value of each compound against normal cells by the mean CC50 value against OSCC. CC50 value was
determined from the growth curves plotted at different concentrations of each compounds in triplicate wells.

DOI: 10.3109/14756366.2016.1140755
Mannich base derivatives of thymol
5
Table 3. Cytotoxic activities of the compound 2 against human OSCC and normal oral cells.
Viable cell number (%)*
Human OSCC cell lines
Human oral normal cells
HPLF
Concentration (mM)
Ca9-22
HSC-2
HSC-3
HSC-4
HGF
HPC
0
7.8
15.6
31.3
62.5
125
100
72
68
63
54
50
40
4
100
107
80
61
58
54
50
1
100
58
55
58
43
48
45
1
100
73
72
67
60
62
57
0
100
94
94
103
99
94
54
6
100
100
98
100
98
109
127
103
107
55
99
102
105
125
13
250
500
7
1000
3
3
1
0
2
5
7
*Each value represents the cell number determined by triplicate assays.
11. Aeschbach R, Loliger J, Scott BC, et al. Antioxidant actions of
thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food
Chem Toxicol 1994;32:31–6.
12. Aboelwafa HR, Yousef HN. The ameliorative effect of thymol
against hydrocortisone-induced hepatic oxidative stress injury in
adult male rats. Biochem Cell Biol 2015;93:282–9.
13. Huang MH, Liao LF, Kuo SH, et al. Effects of 4-piperidinomethyl-
2-isopropyl-5-methylphenol on oxidative stress and calcium current.
J Pharm Pharmacol 2005;57:1191–7.
14. Deb DD, Parimala G, Saravana Devi S, Chakraborty T. Effect of
thymol on peripheral blood mononuclear cell PBMC and acute
promyelotic cancer cell line HL-60. Chem Biol Interact 2011;193:
97–106.
15. Karkabounas S, Kostoula OK, Daskalou T, et al. Anticarcinogenic
and antiplatelet effects of carvacrol. Exp Oncol 2006;28:121–5.
16. Deng XY, Li HY, Chen JJ, et al. Thymol produces an antidepressant-
like effect in a chronic unpredictable mild stress model of depression
in mice. Behav Brain Res 2015;291:12–19.
17. Wei Z, Zhou E, Guo C, et al. Thymol inhibits Staphylococcus aureus
internalization into bovine mammary epithelial cells by inhibiting
NF-kappaB activation. Microb Pathog 2014;71:15–19.
Conclusion
The chemical structure and tumor-specific cytotoxicity of com-
pounds 2 and 3 are reported in this study for the first time. At the
same experimental condition ortho and para aminomethylation
occurred depending on the nature of amine used. The compounds
were slightly more selective against hCA II rather than hCA I.
Based on the cytostatic property of compound 2, it is important to
consider the possibility that this compound may enhance the
cytotoxicity of popular chemotherapeutic agents.
Acknowledgements
Prof. Gul and Prof. Supuran thank ERASMUS office of Ataturk
University, Turkey for their financial support to Yamali C.
Declaration of interest
The authors report no conflict of interest and are responsible for
the contents and writing of the paper.
18. Didry N, Dubreuil L, Pinkas M. Activity of thymol, carvacrol,
cinnamaldehyde and eugenol on oral bacteria. Pharm Acta Helv
1994;69:25–8.
References
19. Mahmoud AL. Antifungal action and antiaflatoxigenic properties of
some essential oil constituents. Lett Appl Microbiol 1994;19:
110–13.
20. Roman G. Mannich bases in medicinal chemistry and drug design.
Eur J Med Chem 2015;89:743–816.
21. Sieger GM, Barringer WC, Krueger JE. Mannich derivatives of
medicinals. 2. Derivatives of some carbonic anhydrase inhibitors.
J Med Chem 1971;14:458–60.
22. Buyukkidan N, Buyukkidan B, Bulbul M, et al. Synthesis and
characterization of phenolic Mannich bases and effects of these
compounds on human carbonic anhydrase isozymes I and II.
J Enzyme Inhib Med Chem 2013;28:337–42.
23. Gul HI, Yerdelen KO, Das U, et al. Synthesis and cytotoxicity of
novel 3-aryl-1-(30-dibenzylaminomethyl-40-hydroxyphenyl)-prope-
nones and related compounds. Chem Pharm Bull 2008;56:1675–81.
24. Tugrak M, Yamali C, Sakagami H, Gul HI. Synthesis of mono
Mannich bases of 2-(4-hydroxybenzylidene)-2,3-dihydroinden-1-
one and evaluation of their cytotoxicities. J Enzyme Inhib Med
Chem 2015. [Epub ahead of print]. doi:10.3109/14756366.
2015.1070263.
25. Yerdelen KO, Gul HI, Sakagami H, Umemura N. Synthesis and
biological evaluation of 1,5-bis(4-hydroxy-3-methoxyphenyl)penta-
1,4-dien-3-one and its aminomethyl derivatives. J Enzyme Inhib
Med Chem 2015;30:383–8.
26. Gul HI, Calis U, Vepsalainen J. Synthesis and evaluation of
anticonvulsant activities of some bis Mannich bases and corres-
ponding piperidinols. Arzneimittelforschung 2002;52:863–9.
27. Gul HI, Calis U, Vepsalainen J. Synthesis of some mono-Mannich
bases and corresponding azine derivatives and evaluation of their
anticonvulsant activity. Arzneimittelforschung 2004;54:359–64.
1. Supuran CT, Scozzafava A. Carbonic anhydrases as targets for
medicinal chemistry. Bioorg Med Chem 2007;15:4336–50.
2. Supuran CT. Carbonic anhydrases: novel therapeutic applications for
inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81.
3. Supuran CT, Scozzafava A, Casini A. Carbonic anhydrase inhibitors.
Med Res Rev 2003;23:146–89.
4. Innocenti A, Hall RA, Schlicker C, et al. Carbonic anhydrase
inhibitors. Inhibition of the beta-class enzymes from the fungal
pathogens Candida albicans and Cryptococcus neoformans with
aliphatic and aromatic carboxylates. Bioorg Med Chem 2009;17:
2654–7.
5. Bayram E, Senturk M, Kufrevioglu OI, Supuran CT. In vitro
inhibition of salicylic acid derivatives on human cytosolic carbonic
anhydrase isozymes I and II. Bioorg Med Chem 2008;16:9101–5.
6. Innocenti A, Beyza Ozturk Sarikaya S, Gulcin I, Supuran CT.
Carbonic anhydrase inhibitors. Inhibition of mammalian isoforms
I-XIV with a series of natural product polyphenols and phenolic
acids. Bioorg Med Chem 2010;18:2159–64.
7. Gorlach A, Kietzmann T. Superoxide and derived reactive oxygen
species in the regulation of hypoxia-inducible factors. Methods
Enzymol 2007;435:421–46.
8. Gul HI, Cizmecioglu M, Zencir S, et al. Cytotoxic activity of 40-
hydroxychalcone derivatives against Jurkat cells and their effects on
mammalian DNA topoisomerase I. J Enzyme Inhib Med Chem
2009;24:804–7.
9. Bilginer S, Unluer E, Gul HI, et al. Carbonic anhydrase inhibitors.
Phenols incorporating 2- or 3-pyridyl-ethenylcarbonyl and tertiary
amine moieties strongly inhibit Saccharomyces cerevisiae beta-
carbonic anhydrase. J Enzyme Inhib Med Chem 2014;29:495–9.
10. Ai-Yu S, Mei-Han H, Li-Fang L, Try-Shy W. Thymol analogues
with antioxidant and L-type calcium current inhibitory activity. 28. Gul HI, Calis U, Ozturk Z, et al. Evaluation of anticonvulsant
Drug Dev Res 2005;64:195–202. activities of bis(3-aryl-3-oxo-propyl) ethylamine hydrochlorides and

6
H. I. Gul et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
4-aryl-3-arylcarbonyl-1-ethyl-4-piperidinol hydrochlorides. Arznei 32. Khalifah RG. The carbon dioxide hydration activity of carbonic
mittelforschung 2007;57:133–6. anhydrase. I. Stop-flow kinetic studies on the native human
29. Koechel DA, Rankin GO. Diuretic activity of Mannich base isoenzymes B and C. J Biol Chem 1971;246:2561–73.
derivatives of ethacrynic acid and certain ethacrynic acid analogues. 33. Maresca A, Temperini C, Pochet L, et al. Deciphering the
J Med Chem 1978;21:764–9.
mechanism of carbonic anhydrase inhibition with coumarins and
thiocoumarins. J Med Chem 2010;53:335–44.
activity of 1-aryl-3-phenethylamino-1-propanone hydrochlorides 34. D’Ambrosio K, Smaine FZ, Carta F, et al Development of potent
30. Mete E, Ozelgul C, Kazaz C, et al. Synthesis and antifungal
and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols. Arch Pharm 2010; carbonic anhydrase inhibitors incorporating both sulfonamide and
343:291–300. sulfamide groups. J Med Chem 2012;55:6776–83.
31. Moehrle H, Miller C. Mannich bases. Part 14. Aminomethylation of 35. Motohashi N, Wakabayashi H, Kurihara T, et al. Biological activity
different substituted phenols by secondary amines. Pharmazie 1978;
33:500–5.
of barbados cherry (acerola fruits, fruit of Malpighia emarginata
DC) extracts and fractions. Phytother Res 2004;18:212–23.