The synthesis of some β-lactams and investigation of their metal-chelating activity, carbonic anhydrase and acetylcholinesterase inhibition profiles

Article abstract of DOI:10.3109/14756366.2016.1170014

β-Lactam antibiotics are a broad class of antibiotics, consisting of all antibiotic agents that contain a β-lactam ring in their molecular structures. Synthesis of β-lactam analogs, which are containing dichloride atoms and N-methyl, N-aromatic rings, was

Full text of DOI:10.3109/14756366.2016.1170014

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
The synthesis of some β-lactams and investigation
of their metal-chelating activity, carbonic
anhydrase and acetylcholinesterase inhibition
profiles
Burhanettin Turan, Kivilcim Şendil, Emin Şengül, Mehmet Serdar Gültekin,
Parham Taslimi, İlhami Gulçin & Claudiu T. Supuran
To cite this article: Burhanettin Turan, Kivilcim Şendil, Emin Şengül, Mehmet Serdar
Gültekin, Parham Taslimi, İlhami Gulçin & Claudiu T. Supuran (2016): The synthesis of
some β-lactams and investigation of their metal-chelating activity, carbonic anhydrase and
acetylcholinesterase inhibition profiles, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.3109/14756366.2016.1170014
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1170014
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Date: 14 April 2016, At: 20:22

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1170014
!
RESEARCH ARTICLE
The synthesis of some b-lactams and investigation of their
metal-chelating activity, carbonic anhydrase and acetylcholinesterase
inhibition profiles
1
1,2
1
1
1
1,3
Burhanettin Turan , Kivilcim S¸ endil , Emin S¸ eng u¨ l , Mehmet Serdar G u¨ ltekin , Parham Taslimi , Ilhami Gul c¸ in ,
_
4
,5
and Claudiu T. Supuran
1
2
Department of Chemistry, Faculty of Science, Ataturk University, Erzurum, Turkey, Department of Chemistry, Faculty of Science and Arts, Kafkas
3
University, Kars, Turkey, Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, Dipartimento Di Chimica Ugo
5
Schiff, Universita Degli Studi Di Firenze, Firenze, Italy, and Department of Neurofarba, Section of Pharmaceutical and Nutriceutical Sciences,
4
Universita Degli Studi Di Firenze, Florence, Italy
Abstract
Keywords
b-Lactam antibiotics are a broad class of antibiotics, consisting of all antibiotic agents that Acetylcholinesterase, b-lactams, carbonic
contain a b-lactam ring in their molecular structures. Synthesis of b-lactam analogs, which are
containing dichloride atoms and N-methyl, N-aromatic rings, was achieved by Schiff bases and
dichloroketene compounds. All the synthesized imines and b-lactam analogs were tested
against two physiologically relevant carbonic anhydrase isozymes (hCA I and II) and
acetylcholinesterase (AChE). They demponstrated effective inhibitory profiles with K values
i
in ranging of 3.22-11.18 nM against hCA I, 3.74-10.41 nM against hCA II, and 0.50-1.57 nM
against AChE. On the other hand, acetazolamide and dorzolamide clinically used as CA
anhydrase, enzyme inhibition, enzyme
purification, Schiff bases
History
Received 12 March 2016
Revised 16 March 2016
Accepted 21 March 2016
Published online 12 April 2016
inhibitors, showed K
against hCA II, respectively. Also, tacrine used as standard AChE inhibitor showed K
.70 nM against AChE.
i
value of 170.34 and 129.26 nM against hCA I, and 115.43 and 135.67 nM
i
value of
5
Introduction
atom containing b-lactam derivatives are interesting compounds
in terms of pharmaceutical and synthetic organic chemistry.
Due to bioacvtivity properties, b-lactam molecules have widely
large area in drug chemistry; new synthetic methods developed for
these molecules are an precious issue in organic chemistry. As
known, the b-lactam structure is part of the moiety of several
antibiotic molecules such as carbapenems (1), pennicillins (2),
and cephalosporins (3), therefore, also called b-lactam anti-
The effect of a substituent such as halogen on the biological
activity of a potential drug structure or bioactive organic
compound still has to be planned for experimentally in biological
studies designed to detect intended activity. In some position on
molecule skeleton, the presence of active atoms such as a chlorine
atom is very important for the specific activity of a compound
obtained by organic reactions. Sometimes chlorine atom demon-
strates to be the optimum substituent of a chemical molecule for
developed activity. As it is known that chlorinated organic
compounds are not generally hazardous and toxic, the chlorine
atom is used as a useful substituent in synthetic organic chemistry
by chemists. Nowadays, it remains an important tool for
investigation between structure and activity relationships in
biochemistry researches and as a substituent in organic molecular
1
biotics . Particularly, all of b-lactam antibiotics inhibit bacteria
cell wall biosynthesis. Up to now, more than thousand different
b-lactamase enzymes have been reported in several species of
bacteria. These enzymes influence widely b-lactam structure and
2
catalytic efficiencies .
The carbonic anhydrases (CAs, EC 4.2.1.1) are widespread
metalloenzymes family and catalyze the reversible hydration of
ꢀ
1
carbondioxide (CO
(
including the a-, b-, g-, d-, z-, and n-CA
2
) and water (H
2
O) to (HCO3 ) and proton
structures in commercialized compounds . Especially, chloro
+ 3–7
H ) . CAs have six genetically and distinct enzyme families,
8
–10
. This regulatory
reaction supports many physiological processes associated with
pH control, fluid secretion, ion transport, and several biosynthetic
1
1–15
Address for correspondence: Prof. Dr. Mehmet Serdar G u¨ ltekin, Faculty processes
of Science, Department of Chemistry, Ataturk University, Erzurum,
Turkey. Tel: +90 442 2314428. Fax: +90 442 2360948. E-mail:
. The inhibition of CAs has been exploited clinic-
ally for many decades, as most CA isoforms of the 16 known in
humans are therapeutic targets with the potential to be inhibited or
gultekin@atauni.edu.tr
_
Prof. Dr. Ilhami Gul c¸ in, Faculty of Science, Department of Chemistry,
16–20
activated
. The two of them (hCA I and II) are cytosolic ones.
The catalytic activity of hCA I, which shows blood enzyme, is
much lower compared to that of hCA II, and in addition hCA I is
Ataturk University, Erzurum, Turkey. Tel: +90 442 2314375. Fax: +90
4
42 2360948. E-mail: igulcin@atauni.edu.tr

2
B. Turan et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
inhibited by the chloride and bicarbonate present in the plasma, washed, and dried. The reaction solution was concentrated to
raising a lot of questions regarding the physiologic function of this about 50 mL and then stirred with 100 mL of n-hexane. The
1
5,21–24
isoform
. The physiologically dominant isoform is the red hexane solution was decanted from the zinc salts and evaporated,
blood cell CA II, which present in many other tissues and its and the residue was purificated on silica-jel coloumn chromatog-
inhibitors have widespread use in a variety of applications as raphy to yield the cycloadduct.
2
5–30
diuretics, antiglaucoma, and antiepileptic agents
. The dis-
covery of new classes of CA inhibitors (CAIs), which possessing
different inhibition mechanisms when compared with the classical
inhibitors . For such reasons, the design of novel classes of Anhydrous solvents and all reagents were purchased from Sigma-
1
potent, possibly isoform-selective inhibitors targeting other Aldrich and Alfa Aesar Nuclear magnetic resonance ( H-NMR
Chemistry
1
5,31–35
1
3
human CA (hCA) isoforms, may lead to clinical applications for and C-NMR); spectra were recorded using a Varian 400 MHz or
treating a multitude of diseases such as edema, epilepsy, obesity, Bruker Advance 400 DPX MHz spectrometer (Erzurum, Turkey)
3
6–40
neuropathic pain, and other neurological disorders
.
Alzheimer’s disease (AD) is a progressive neuro-degenerative and the coupling constants (J) are expressed in Hertz (Hz).
in CDCl ; Chemical shifts are reported in parts per million (ppm),
3
disorder and the fourth leading cause of death in people over Splitting patterns are designated as follows: s, singlet; d, doublet;
6
5-year-old worldwide. AD is characterized by the atrophy sept, septet; t, triplet; q, quartet; m, multiplet; brs, broad singlet;
of cholinergic neurons, behavioral abnormalities, deterioration brm, broad multiplet; dd, double of double; td, triplet of double;
of cognition, memory loss, loss of speech, and, eventually, tt, triplet of triplet; appd, apparent double; appt, apparent triplet.
4
1–44
death
several hallmarks, such as low levels of acetylcholine, oxidative Merck silica gel F-254 plates.
. Although the etiology of this diseae is still elusive, Analytical thin-layer chromatography (TLC) was carried out on
stress, b-amyloid deposits, g-protein aggregation, and the
dyshomeostasis of biometals, are thought to play crucial roles in atoms and N-methyl, N-aromatic rings as substituents, by Schiff
We synthesized the b-lactam analogs containing dichloride
4
1,45–47
the development of AD
. Because of the complex patho- bases and dichloroketene compounds. Synthesis of molecules
genesis of AD, there is no ideal drug for the prevention or comprising Schiff base has long been attracting attentions due to
treatment of AD up to now. Acetylcholinesterase (AChE, EC their biological activities such as anticancer, antibacterial,
3
.1.1.7), a serine protease, is responsible for acetylcholine (ACh) antifungal, antidepressant, analgesic, and cytotoxic effects in
hydrolysis and plays a fundamental role in impulse transmission addition to their regular use in electronic industry, cosmetic, and
by terminating the action of the neurotransmitter ACh at the polymer industry. Schiff bases are frequently used in addition
4
8,49
cholinergic synapses and neuromuscular junction
best primary pharmacological strategies for the treatment of AD and several ligand complexes, thanks to their structural
. One of the reactions, Staudinger Reactions, Hetero–Diels–Alder reactions,
5
1,52
is to improve cholinergic neurotransmission by decreasing the rate characteristics
of decomposition of ACh at synapses in the brain with the use of
.
In this study, first, imine groups known as Schiff bases
5
acetylcholinesterase inhibitors (AChEIs) .
0
53
in literature, 1-(naphthalene-2-yl)-N-phenylmethanimine (10) ,
The aim of this study is to design and synthesize some imines N-(4-ethylphenyl)-1-(naphthalene-2-yl)methanimine (11), N-
(
10–14) and b-lactam analogs (15–19) and to investigate their mesityl-1-(naphthalene-2-yl)methanimine (12), 1-(naphthalene-
5
potential carbonic anhydrase isoenzymes I and II (CA I and II) 2-yl)-N-(p-tolyl) methan-imine (13) , N-methyl-1-(naphthalene-
4
5
5
and acetylcholinesterase (AChE) inhibition properties.
2-yl) methanimine (14) , were synthesized from corresponding
aromatic aldehyde (4) and amine derivatives aniline (5), 4-
ethylaniline (6), 2,4,6-trimethylaniline (7), p-toluidine (8),
methanamine (9). Some Schiff bases (10, 13, 14) as known in
literature and other Schif bases were synthesized in this study.
After characterization of imine compounds by NMR spectrum,
dichloroketene (2,2-dichloroethene-1-one), compounds were
obtained with Zn from trichloride acetyl chloride and active
zinc metal in basic media in high yields (Scheme 1).
These imines were purified by easy methods such as crystal-
lization method or silica-gel colomn chromatography. The yields
and reaction conditions of synthesized imines were shown in
Table 1.
Materials and methods
The synthesis of Schiff bases from 2-naphthalene
aldehyde and amine compounds
Aryl or methyl amines (5–9) (1.0 mmol) were dissolved in 30-
mL ethyl alcohol and added for 10 min in droplets to 0.05%
3
in 20 mL ethyl alcohol. After that, reaction was refluxed at 80–
mL H SO acid and aldehyde (4) (1.0 mmol) solution dissolved
2 4
ꢁ
9
0 C for 24 h. At the end of the reaction followed by TLC,
reaction mixture was taken to a 250 mL beaker glass and added
aqueous Na CO3 solution, so H SO was removed from the
reaction medium. Obtained Schiff base mixture was taken to
organic phase by extracting and using ethyl acetate
2
2
4
In second step, the b-lactam derivatives 3,3-dichloro-4-
naphthalene-2-yl)-1-phenylazetidin-2-one (15), 3,3-dichloro-1-
(
(
4-ethylphenyl)-4-(naphthalene-2-yl)azetidin-2-one (16), 3,3-
(
3 ꢂ 100 mL). The water in organic phase was removed by
dichloro-1-mesityl-4-(naphthalene-2-yl)azetidin-2-one (17), 3,3-
dichloro-4-(naphthalene-2-yl)-1-(p-tolyl) azetidin-2-one (18),
drier Na SO after organic phase was washed off in 100 mL
2
4
saturated NaHCO . Organic solvent was removed in vacuum
3
3
,3-dichloro-1-methyl-4-(naphthalene-2-yl)azetidin-2-one
was synthesized with [2 + 2] ketene addition reaction from
dichloroketene and corresponding imine derivatives
(19)
ꢁ
(20 C, 20 mm Hg). Remnant crude product was purified using
silica gel through column chromatography in ethylacetate:metha-
nol:n-hexane (7:1:2) solvent mixture.
obtained from 10–14 amines and 2-naphtalaldehyde (4) in acidic
reaction condition (Scheme 1). All of the b-lactam structures
5–19 were synthesized first time in literature in this study. The
Typical procedure for dichloroketene cycloadditions
1
A solution of 0.025 mol of freshly distilled trichloroacetyl dichloroketene (2,2-dichloroethene-1-one) was also obtained from
ꢁ
chloride in 250 mL of dry ether was added over 4 h to a stirred, Zn and trichloroacetyl chloride at 40 C in 10 h (Table 2). The
refluxing mixture of 0.025 mol of Schiff base (imine) in 250 mL yields of addition reaction were changed according to the structure
of dry ether and 5 g of activated zinc under a nitrogen atmosphere. of imine compounds. All of the compounds were purified by
The reaction mixture was stirred at reflux for an additional 10 h coloumn chromatography and characterized by NMR
after the addition was complete. The excess zinc was filtered, spectroscopy.

DOI: 10.3109/14756366.2016.1170014
The effects of novel ꢁ-lactams on enzymes
3
Scheme 1. The synthesis route of b-lactam derivatives.
NMR spectral data of first synthesized imines and
b-lactam molecules
1
N-(4-Ethylphenyl)-1-(naphthalene-2-yl)methanimine (11): H-
3,3-Dichloro-4-(naphthalene-2-yl)-1-(p-tolyl)azetidin-2-one (18):
1
H-NMR (400 MHz, CDCl , ppm): ꢀ ¼ 7.0–7.8 (m, 11H), 5.6
3
1
3
(s, 1H), 2.2 (s, 3H).
C-NMR (100 MHz, CDCl ,
3
ppm): ꢀ ¼ 167.5, 135.7, 130.1, 129.1, 128.8, 128.4, 127.9,
27.7, 127.5, 127.3, 127.0, 126.9, 126.8, 126.7, 126.5, 118.2,
74.3, 21.2.
NMR (400 MHz, CDCl , ppm): ꢀ ¼ 8.46 (s, 1H), 8.19 (m, 2H),
3
1
7
2
ꢀ
.92 (m, 3H), 7.56 (m, 2H), 7.28 (m, 4H), 2.71 (q, J ¼ 7.4, 7.6 Hz
1
3
H), 1.3 (t, J ¼ 7.7 Hz, 3H), C-NMR (100 MHz, CDCl , ppm):
3
3,3-Dichloro-1-methyl-4-(naphthalene-2-yl)azetidin-2-one (19):
¼ 159.8, 149.9, 142.6, 135.2, 134.4, 133.4, 131.3, 129.0, 128.9,
1
H-NMR (400 MHz, CDCl , ppm): ꢀ ¼ 7.3–7.9 (m, 7H), 5.2 (s,
3
1
28.2, 127.7, 126.8, 124.2, 121.2, 28.7, 15.9.
N-Mesityl-1-(naphthalene-2-yl)methanimine (12): H-NMR
13
1H), 3.0 (s, 3H). C-NMR (100 MHz, CDCl
34.1, 133.2, 129.4, 129.3, 129.0, 128.7, 128.3, 128.1, 127.4,
27.1, 124.7, 75.9, 28.0.
1
3
, ppm): ꢀ ¼ 162.1,
1
1
(
400 MHz, CDCl , ppm): ꢀ ¼ 8.39 (s, 1H), 8.25 (d, J ¼ 1.5 Hz,
3
8
2
ꢀ
.4 Hz, 1H), 8.18 (s, 1H), 7.95 (m, 3H), 7.57 (m, 2H), 6.9 (s, 2H),
1
.3 (s, 3H), 2.2 (s, 6H). C-NMR (100 MHz, CDCl , ppm):
3
3
Biochemical studies
¼ 162.9, 149.1, 135.3, 134.1, 133.6, 133.3, 131.1, 129.1, 129.0,
1
28.2, 127.7, 127.3, 126.9, 123.9, 21.0, 18.5.
,3-Dichloro-4-(naphthalene-2-yl)-1-phenylazetidin-2-one (15): b-lactam analogs (15–19) on CA isoforms
For the determination of inhibition effects of imines (10–14) and
and II.
3
I
1
H-NMR (400 MHz, CDCl , ppm): d ¼ 7.1–7.9 (m, 12H), 5.6 (s, Both isoenzymes were purified from fresh human erythrocyte
3
1
3
56–60
1
1
1
H). C-NMR (100 MHz, CDCl , ppm): ꢀ ¼ 158.6, 133.7, 136.1, using affinity chromatography technique
. Chromatographic
34.1, 133.2, 129.3, 128.7, 128.4, 128.1, 127.9, 127.6, 127.4, separation technique was widely used for the purification of
27.1, 125.8, 124.5, 118.3, 74.4. biomolecules, including protein and enzymes. To this end,
,3-Dichloro-1-(4-ethylphenyl)-4-(naphthalene-2-yl)azetidin- sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography
3
3
1
61–63
2
7
2
(
1
1
-one (16): H-NMR (400 MHz, CDCl , ppm): ꢀ ¼ 7.3–7.9 (m, was used for the purification of both isoenzymes
as described
3
6
4–66
H), 7.2 (d, J ¼ 8.4 Hz, 2H), 7.1 (d, J ¼ 8.4 Hz, 2H), 5.6 (s, 1H), previously
.
CA activity determination was realized spectrophotometrically
1
3
.5 (q, J ¼ 7.3, 7.5 Hz, 2H), 1.1 ppm (t, J ¼ 7.7 Hz, 3H). C-NMR
6
7
49
100 MHz, CDCl , ppm): ꢀ ¼ 165.1, 142.0, 134.1, 133.8, 133.2, according to Verpoorte et al. as described previously . In this
3
ꢁ
29.9, 129.5, 129.1, 128.9, 128.4, 128.1, 127.8, 127.3, 127.0, method, absorbance change was recorded during 3 min at 25 C.
24.6, 118.2, 74.3, 31.8, 15.7.
p-Nitrophenylacetate (NPA) was used as substrate for both
,3-Dichloro-1-mesityl-4-(naphthalene-2-yl)azetidin-2-one (17): isoenzymes and enzymatically converted to p-nitrophenolate
3
1
68
H-NMR (400 MHz, CDCl , ppm): ꢀ ¼ 7.4–7.8 (m, 7H), 6.8 (bs, ion . These activity determinations are described in detail in
3
1
3
69
2
H), 5.9 (s, 1H), 2.4 (bs, 6H), 2.2 (s, 3H). C-NMR (100 MHz, our previous studies .
Bradford method was used for the determination of protein
CDCl , ppm): ꢀ ¼ 160.2, 138.6, 133.9, 133.0, 130.4, 130.2, 129.8,
3
7
28.7, 128.4, 128.0, 127.8, 127.3, 126.9, 126.4, 125.2, 124.9, quantity during the purification steps . This spectrophotometrical
0
1
7
7
protein assay was explained as previously . The bovine serum
1
7.9, 21.0, 19.7.

4
B. Turan et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Table 1. The synthesis of imine derivatives (10–14).
substituted
aniline
Reaction
Entry
1
Amine
Schiff bases
Yields (%)
Condition
NH
2
N
95
(
5)
6)
(
10)
NH
2
2
3
N
90
85
(
2
-Naphtaldehyde
(11)
(
4)
NH2
+
N
N
H
2
SO
4
(cat.),
(
7)
8)
EtOH
Reflux, 12 h
(
12)
NH
2
4
5
90
95
(
(
(
13)
14)
NH2
H C
CH
3
3
N
(9)
albumin was used as standard protein which demonstrated by the addition of 50 mL of AChI. The hydrolysis of these
7
1–73
maximum activity at 595 nm
After the purification process of the CA isoenzymes, SDS- the yellow 5-thio-2-nitrobenzoate anion as the result of the
.
substrates was monitored spectrophotometrically by formation of
7
4–76
PAGE has been carried out
%) acrylamide and (0.1%) SDS
process using a Minigel system (Mini-PROTEAN Tetra System, of 412 nm .
China). The method used for visualization of protein has been The effect of imines (10–14) and b-lactam analogs (15–19)
. Stacking gel containing (10 and reaction of DTNB with thiocholine, released by enzymatic
7
7,78
3
was used for running the hydrolysis of AChI, with an absorption maximum at a wavelength
5
0
7
9
explained in detail at previous studies . Briefly, the gel was fixed on both CA isoenzymes was examined using the hydratase
and then stained with Coomassie Brilliant Blue R-250 later on the activity and recorded in triplicate analysis at the each concen-
3
gel destained by using standard methods for detecting protein tration used . For this purpose, different concentrations of
8
8
0,81
bands that are belong to purified CA isoenzymes
.
imines (10–14) and b-lactam analogs (15–19) were determined
The inhibitory effects of imines (10–14) and b-lactam analogs in preliminary assays. CA isoenzyme activities were measured in
15–19) on AChE activitiy were measured according to the the presence of different quantity of them. The control sample
(
8
2
spectrophotometric method of Ellman et al. Acetylthiocholine activity in the absence of a imines (10–14) and b-lactam analogs
iodide (AChI) was used as substrate for the enzymatic reaction. (15–19) was taken as 100%. For each imines (10–14) and
0
5
,5 -Dithio-bis(2-nitro-benzoic) acid (DTNB) was used for the b-lactam analogs (15–19), an activity (%)-[imines or b-lactams]
measurement of the AChE activitiy. Briefly, 100 mL of Tris/HCl was drawn using conventional polynominal regression software.
buffer (1 M, pH 8.0), 10 mL of sample solution dissolved in The half maximal inhibitory concentration (IC ) of each imines
5
0
deionized water at different concentrations, and 50 mL of AChE (10–14) and b-lactam analogs (15–19) was calculated from
ꢁ
50
solution were mixed and incubated for 10 min at 25 C. Then, graphs . IC values are measure of the effectiveness of imines
5
0
5
0 mL of DTNB (0.5 mM) was added. Then reaction was initiated (10–14) and b-lactam analogs (15–19) in inhibiting both CA

DOI: 10.3109/14756366.2016.1170014
The effects of novel ꢁ-lactams on enzymes
5
Table 2. The addition reactions of imines and dichloroketene compounds.
Schiff bases
8
8–91
isoenzymes. For the determination of K values, three different 562 nm
i
. For this purpose, to a mixture of FeCl (0.1 mL,
2
imines (10–14) and b-lactam analogs (15–19) concentrations 0.6 mM), three different concentrations (10–30 mg/mL) of imines
were used. K values reflect the binding affinity of imines (10–14) and b-lactam analogs (15–19) in methanol (0.4 mL) were
i
(10–14) and b-lactam analogs (15–19) to both CA isoenzymes. added. The reactions were started by ferrozine solution addition
In this way, The IC50 value is converted to an absolute inhibition (0.1 mL, 5 mM). After that, the solution was mixed and incubated
constant K value. In this experiment, PNA was used as substrate at room temperature for 10 min. Finally, absorbance value of the
i
at five different concentrations. Finally, Lineweaver–Burk mixture of imines (10–14) and b-lactam analogs (15–19) was
9
2–95
curves were drawn for each imine (10–14) and b-lactam determined spectrophotometrically at 562 nm
analog (15–19) .
.
5
0
Result and discussion
Metal-chelating study
Chemistry
Metal-chelating ability of imines (10–14) and b-lactam analogs
8
4
15–19) was performed according to Dinis et al. with slight Many chlorinated organic compounds are known for their
(
modification
8
5–88
2+
. Fe -binding capacity of imines (10–14) and detrimental effects as a toxicological properties and environmen-
b-lactam analogs (15–19) was spectrophotometrically recorded at tally harmful chemicals. However, chlorinated organic molecules,

6
B. Turan et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Table 3. The enzyme inhibition values of some imines (10–14) and b-lactam analogs (15–19) against human carbonic anhydrase isoenzymes I and II
(
hCA I and II) and acetylcholinesterase (AChE) enzyme.
IC50 (nM)
K
I
(nM)
2
2
2
r
Compounds
hCA I
r
hCA II
r
AChE
hCA I
hCA II
AChE
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
7.79
6.30
6.93
8.25
10.83
4.59
3.59
4.15
5.09
7.37
101.19
88.45
–
0.9816
0.9829
0.9646
0.9783
0.9727
0.9688
0.9911
0.9856
0.9831
0.9727
0.9509
0.9760
–
10.21
7.79
8.66
9.12
14.74
7.37
5.29
5.02
6.13
7.88
113.75
93.01
–
0.9516
0.9611
0.9655
0.9644
0.9339
0.9561
0.9709
0.9606
0.9616
0.9382
0.9791
0.9910
–
3.21
2.76
3.01
2.44
3.03
1.35
1.18
1.39
1.52
1.97
–
0.9695
0.9933
0.9714
0.9776
0.9434
0.9632
0.9760
0.9631
0.9496
0.9593
–
5.31 ± 0.39
4.67 ± 0.54
9.09 ± 4.06
7.45 ± 1.42
11.18 ± 1.98
4.81 ± 1.78
3.22 ± 0.22
3.40 ± 0.82
3.74 ± 0.71
4.99 ± 0.69
170.34 ± 2.48
129.26 ± 0.21
–
7.27 ± 0.91
9.93 ± 4.19
8.98 ± 1.80
10.29 ± 3.55
10.41 ± 2.69
6.59 ± 2.51
3.74 ± 0.39
4.37 ± 1.74
5.88 ± 2.32
7.08 ± 1.46
115.43 ± 1.63
135.67 ± 0.32
–
1.57 ± 0.55
1.48 ± 0.06
1.01 ± 0.46
0.82 ± 0.34
1.36 ± 0.54
0.82 ± 0.39
0.50 ± 0.20
0.89 ± 0.37
0.61 ± 0.15
0.94 ± 0.35
–
AZA*
DZA*
TAC**
–
9.76
–
0.9463
–
5.70 ± 1.09
*
*
Acetazolamide (AZA) and dorzolamide (DZA) were used as a standard inhibitor for all hCA I and II.
*Tacrine (TAC) was used as a standard inhibitor for AChE enzyme.
molecular weight range from 200 to 600, are used as pharma- (1) Cytosolic hCA I isoenzyme was potently inhibited by imines
ceutical or biologically active substances in drug chemistry and
plant protection agents, which are important and essential
chemicals in green chemistry. As a result of the scientific
investigation, it has been experimentally found that the introduc-
tion of a chlorine atom into several specific positions of a
biologically active molecule may be essential to improve the basic
biological activity.
(10–14) and b-lactam analogs (15–19). K values were found
i
in ranging between 3.22 ± 0.22 and 11.18 ± 1.98 nM
(Table 3). The best inhibition for this isoform was observed
by novel b-lactam analog 16 (3,3-dichloro-1-(4-ethylphenyl)-
4-(naphthalene-2-yl)azetidin-2-one), which possess bio-
logical active groups of –C¼O, –CH , and Cl with K
3
i
value of 3.22 ± 0.22 nM. It is well-known that CA isoen-
zymes easily inhibited by the compounds, which had these
biologically active groups. On the other hand, as can seen in
Figure 1 acetazolamide (AZA, 5-acetamido-1,3,4-thiadia-
zole-2-sulfonamide) and dorzolamide (DZA, N-(5-sulfamoyl-
1,3,4-thiadiazol-2-yl)acetamide) used as CAI for the medical
treatment of some diseases including idiopathic intracranial
hypertension, epileptic seizure, glaucoma, altitude sickness,
periodic paralysis, cystinuria, central sleep apnea, and dural
ectasia, showed Ki values of 170.34 ± 2.48 and
129.26 ± 0.21 nM, respectively. The hCA I is highly abundant
in red blood cells and is found in many tissues but its precise
physiological function is unknown. CA I is associated with
cerebral and retinal edema, and the inhibition of CA I may be
Biochemistry
The discovery of new classes of CAIs, possessing different
inhibition mechanisms compared with the classical inhibitors of
the sulfonamide and anion type, has also seen important
9
6
developments ultimately . So far, five different CA inhibition
2
mechanisms were reported: (i) the zinc ion (Zn ) binders are the
+
2
inhibitors which coordinate to the catalytically crucial Zn from
+
2
+
the enzyme active site. In this inhibition type, Zn may be in a
tetrahedral or trigonal bipyramidal geometries, with the sulfona-
mides and sulfamides, sulfamates, most anions, dithiocarbamates,
9
7
carboxylates, and hydroxamates binding in this way ; (ii) the
2
inhibitors that anchor to the Zn -coordinated water molecule
+
6
a valuable tool for fighting these conditions .
H O)/hydroxide ion (–OH), represented by the phenols, some (2) The ubiquitous and physiologically predominant cytosolic
(
carboxylates, the polyamines, 2-thioxocoumarins, and sulfocou-
2
isoform hCA II is associated with several diseases. For hCA
II, b-lactam analogs (15–19) had K values ranging in
9
6,98
marins
which occlude the entrance to the active site cavity, this binding
; (iii) the inhibitors-like coumarins and their isosteres
i
3.74 ± 0.39–10.29 ± 3.55 nm. On the other hand, acetazola-
mide (AZA, 5-aceta-mido-1,3,4-thiadiazole-2-sulfonamide),
a clinically used compound, was a medium potency CA II
inhibition for this isoform, with an inhibition constant of
115.43 ± 1.63 nM. Also, dorzolamide (DZA), a carbonic
9
9
site coinciding with that where CA activators bind ; (iv) the
compounds which bind out of the active site cavity, and (v)
compounds for which the inhibition mechanism is not known,
among which the secondary or tertiary sulfonamides are the most
1
00
investigated examples
.
The hCA I and II isoenzyme inhibitory activity of the imines
anhydrase
inhibitor,
demonstrated
Ki
value
135.67 ± 0.32 nM. DZA was used as antiglaucoma agent
of
1
02
(10–14) and b-lactam analogs (15–19) was shown in Table 3. CA
and acts by decreasing the production of aqueous humour
.
II isoform was chosen because of its antiglaucoma drug targets,
however CA I, due to its diffuse distribution in the blood and
gastrointestinal tract is one of the main off-targets for such
pharmacologic agents . On the other hand, AChE was chosen
for its significant contribution in drug discovery and development
for AD. All the synthesized imines (10–14) and b-lactam analogs
All of the derivatives showed highly effective hCA II
inhibitory activity, comparable to that of AZA and DZA.
As can be seen in CA I, the most inhibition effect of CA II
was observed by b-lactam analog 16 (3,3-dichloro-1-(4-
ethylphenyl)-4-(naphthalene-2-yl)azetidin-2-one) with Ki
values of 3.74 ± 0.39 nM.
1
01
(
15–19) were tested for the evaluation of their inhibitory activity (3) Effective AChE inhibitors can be used for AD treatment.
Most of the currently available drugs on the market including
tacrine, rivastigmine, donepezil, and galantamine intended to
toward the slower cytosolic hCA I isoform, the more rapid
cytosolic hCA II isoenzyme and AChE enzyme. Also, these novel
derivatives showed effective ferrous ions (Fe )-chelating effects.
The following results can be observed from the inhibition data of
Table 3:
2
+
103
treat AD are AChE inhibitors . The inhibition effects of
some imines (10–14) and b-lactam analogs (15–19) against
AChE
activities
were
measured
according
to

DOI: 10.3109/14756366.2016.1170014
The effects of novel ꢁ-lactams on enzymes
7
Figure 1. Standard compounds used for carbonic anhydrase I and II isoenzymes (acetazolamide and dorzolamide) and acetylcholinesterase (tacrine)
inhibitors.
8
2
spectrophotometric Ellman method . AChI was used as
substrates of the reaction. This method is based on the
amount of thiocholine released when the enzyme AChE
hydrolyzes the AChI to thiocholine and acetate. The product
thiocholine reacts with DTNB to produce a yellow compound
Table 4. Determination of half maximal concentrations (IC50, mg/mL) of
Fe chelating of some imines (10–14) and b-lactam analogs (15–19) and
standard compounds.
2+
2
Fe chelating
+
2
Compounds
r
(5-thio-2-nitrobenzoate) anion, which can be detected at a
wavelength of 412 nm. AChE was very effectively inhibited
by imines (10–14) and b-lactam analogs (15–19). It was
BHA
BHT
a-Tocopherol
Trolox
36.47
57.75
18.73
28.88
12.83
76.15
113.60
85.56
49.50
57.75
138.60
230.01
98.89
77.01
115.50
0.9837
0.9922
0.9861
0.9828
0.9924
0.9855
0.9944
0.9813
0.9737
0.9983
0.9889
0.9783
0.9828
0.9911
0.9728
found that K values were ranging between 0.50 ± 0.20 and
1
i
.57 ± 0.55 nM (Table 3). All imines (10–14) and b-lactam EDTA
analogs (15–19) demonstrated similar inhibiton profile 10
1
1
1
1
1
1
2
3
4
5
against AChE. On the other hand, tacrine, a first centrally
acting cholinesterase inhibitor approved for the treatment of
AD, was used as a standard AChE with K values of
i
5
.70 ± 1.09 nM. Imines 10 (1-(naphthalene-2-yl)-N-phenyl-
methanimine), which shown the weakest AChE inhibiton, 16
had 3.63-times AChE inhibiton effects than that of Tacrine. 17
1
1
8
9
The K values of imines (10–14) and b-lactam analogs (15–
i
1
plots .
9) for AChE were calculated from Lineweaver–Burk
3
8
(
4) Metal-chelating measurement is an antioxidant method,
which based on the absorbance measurement of ferrous ion
2
Fe )-ferrozine complex after prior treatment of
+
(
Fe solution with test material
a
. Ferrozine forms a
were found in the range of 49.50–230.01 mg/mL (Table 4).
2
+
104–106
2
+
Whereas, IC50 values belonging to Fe ion-chelating
capacity of positive controls like a-tocopherol, trolox,
BHT, BHA, and EDTA were found to be in range of
2
complex with free Fe , but not with Fe bound to other
+
2+
2
+
chelators; thus, a decrease in the amount of Fe -ferrozine
complex formed after treatment indicates the presence of
1
Fe ions-binding activity. These results clearly show that
2.83–57.75 mg/mL. A lower IC value reflects a higher
5
0
1
07,108
2+
. The ferrozine–Fe complex
2+
antioxidant chelators
produced a red chromophore with absorbance that can be
2
+
Fe ion-chelating effect of imines (10–14) and b-lactam
analogs (15–19) had effective Fe ions chelating; however,
these values were lower than that of standard metal chelators.
1
09,110
2
+
measured at 562 nm
. A significant drawback of this
complexation reaction in measuring the presence of antioxi-
dant chelator is that the reaction is affected by both the
2
+
Fe ions are the most efficient pro-oxidants in pharmacol-
2
+
2+
1
15,116
antioxidant-Fe and ferrozine–Fe complex formation con-
stants, and the competition between the two chelators for
binding to iron. Thus, a weak antioxidant iron chelator would
be seriously underestimated in quantitative determin-
ogy systems and food
. Ferrozine can create complexes
2
with Fe . In the presence of Fe -chelating compounds,
ferrozine-Fe complex formation is a broken down, resulting
in a decrease in the red color formation of Ferrozine-
+
2+
2
+
1
11,112
2
Fe complex
+
117,118
ation
. The metal-chelating capacity was significant
.
since it reduced the concentration of the catalyzing transition
metal in lipid peroxidation. It was reported that chelating
agents are effective as secondary antioxidants because they
reduce the redox potential, and, thereby, stabilize the
Conclusion
1
13,114
oxidized form of the metal ion
. EDTA is a strong In the present study, practical and efficient method has been
metal chelator; hence, it was used as standard metal chelator developed for N-aryl b-lactams the first time in literature from
agent in this study. The data obtained from Table 4 reveal Schiff bases and simple dichloroketene compound. Schiff base
that imines (10–14) and b-lactam analogs (15–19) possess reactions are widely used in chemistry due to their mild reaction
marked iron-binding capacity, suggesting that their main conditions and high reaction rates. The synthesis of b-lactams
action as a peroxidation inhibitor may be related to their iron- derived 15–19 from these Schif bases and the dichloroketene 4 by
binding capacity. The distinction between different concen- using [2 + 2] addition reaction. It is evident from the results of the
trations (10–30 mg/mL) of imines (10–14) and b-lactam investigation that a effective method has been obtained, by EtOH
analogs (15–19) and the control value was fixed to be solvent under acidic conditions, for the synthesis of Schiff bases
statistically important (p50.01). Furthermore, it is found that from corresponding amines 10–14 and 2-naphtalaldehyde (4) in
IC50 values for imines (10–14) and b-lactam analogs (15–19) high yields in our study. The imines (10–14) and b-lactam analogs

8
B. Turan et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
20. Go
¨
c¸ er H, Akıncıo g˘ lu A, G o¨ ksu S, et al. Carbonic anhydrase and
(15–19) used in the present study showed effective inhibition
profiles against AChE enzyme and both hCA isoenzymes. Also,
acetylcholinesterase inhibitory effects of carbamates and sulfa-
moylcarbamates. J Enzyme Inhib Med Chem 2015;30:316–20.
1. Supuran CT. Carbonic anhydrases: novel therapeutic applications
for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81.
2. Akbaba Y, Bastem E, Topal F, et al. Synthesis and carbonic
anhydrase inhibitory effects of novel sulfamides derived from
1-aminoindanes and anilines. Arch Pharm 2014;347:950–7.
2
these compounds demonstrated effective ferrous ion (Fe )-
+
2
2
chelating activity. In this study, nanomolar level of K values was
i
observed for each novel imines (10–14) and b-lactam analogs
(15–19), and these compounds can be selective inhibitor of both
cytosolic CA isoenzymes and AChE enzyme. Also, they can be
used as metal chelators in related applications.
2
3. G o¨ ksu S, Naderi A, Akbaba Y, et al. Carbonic anhydrase inhibitory
properties of novel benzylsulfamides using molecular modeling
and experimental studies. Bioorg Chem 2014;56:75–82.
Declaration of interest
24. G u¨ ney M, Co s¸ kun A, Topal F, et al. Oxidation of cyanobenzocy-
cloheptatrienes: synthesis, photooxygenation reaction and carbonic
anhydrase isoenzymes inhibition properties of some new benzo-
tropone derivatives. Bioorg Med Chem 2014;22:3537–43.
The authors declare no conflict of interest.
2
5. 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.
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