The first synthesis and antioxidant and anticholinergic activities of 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols including natural products

Article abstract of DOI:10.3906/kim-1709-34

We report herein the first synthesis of biologically active natural bromophenols, namely 1-(2,3,6-tribromo-4,5-dihydroxybenzyl)pyrrolidin-2-one (2), 2RS-1-(2,3-dibromo-4,5-dihydroxybenzyl)-5-oxopyrrolidine-2-carboxylic acid (3), and 2RS-methyl 1-(2,3-dibr

Full text of DOI:10.3906/kim-1709-34

Turk J Chem
Turkish Journal of Chemistry
(2018) 42: 808 – 825
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1709-34
Research Article
The first synthesis and antioxidant and anticholinergic activities of
1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols including
natural products
∗
˙
Mohsen REZAI, C¸ etin BAYRAK, Parham TASLIMI, Ilhami GULC¸ IN, Abdullah MENZEK
¨
˙
Department of Chemistry, Faculty of Science, Atatu¨rk University, Erzurum, Turkey
Received: 20.09.2017
•
Accepted/Published Online: 02.04.2018
•
Final Version: 01.06.2018
Abstract: We report herein the first synthesis of biologically active natural bromophenols, namely 1-(2,3,6-tribromo-
4,5-dihydroxybenzyl)pyrrolidin-2-one (2), 2RS-1-(2,3-dibromo-4,5-dihydroxybenzyl)-5-oxopyrrolidine-2-carboxylic acid
(3), and 2RS-methyl 1-(2,3-dibromo-4,5-dihydroxybenzyl)-5-oxopyrrolidine-2-carboxylate (4), along with their different
derivatives (6 and 8–18). Among the synthesized compounds, 3, 4, and their derivatives (8 and 9) are yielded in
racemic form. Radical scavenging assays of the synthesized molecules were investigated using various bioanalytical
antioxidant methods including 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) and 2,2-diphenyl-1-
picrylhydrazyl (DPPH•). In addition, the reducing power of the novel bromophenols was investigated by Cu2+
–
Cu+ reducing, Fe3+ –Fe2+ reducing, and [Fe3+ –(TPTZ)2 ]3+ –[Fe2+ –(TPTZ)2 ]2+ reducing capacity and ferrous ions
(Fe2+) chelating abilities. The molecules demonstrated powerful antioxidant activities when compared to standard
antioxidant compounds of α-tocopherol, trolox, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT).
Moreover, novel bromophenols were tested against cholinergic enzymes including acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) enzymes in the last part of this study. The presented novel bromophenols showed Ki
values in the range of 2.60 ꢀ 0.75–16.36 ꢀ 2.67 nM against AChE and 13.10 ꢀ 3.33–54.47 ꢀ 13.53 nM against BChE.
Key words: Bromination, bromophenol, pyrrolidin-2-one, natural product, antioxidant activity, acetylcholinesterase,
butyrylcholinesterase
1. Introduction
Natural or synthetic bromophenols exhibit important biological activities such as feeding deterrent1 antioxidant,2−4
antimicrobial,5,6 and inhibition effects on carbonic anhydrase enzyme.7−12 Naturally occurring bromophenols
are abundantly found in marine life and frequently isolated from red algae of the family Rhodomelaceae.13−15
Natural bromophenols 1–4 were isolated from marine red algae2,16−19 and exhibited antioxidant activities
(Figure).2,16−19 It was also reported that compound 2 inhibition had ability on protein tyrosine phosphatase
1B.17,18 The synthesis of compound 1 and its derivatives like racemate as well as their antioxidant activities
were firstly reported by our group.20
Reactive oxygen species (ROS), which contain free radicals such as hydroxyl radicals (OH•), superoxide
anion radicals (O•2−), or nonfree radical species such as singlet oxygen (1 O2) and hydrogen peroxide (H2 O2),
∗Correspondence: amenzek@atauni.edu.tr
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REZAI et al./Turk J Chem
Figure. Some biologically active natural bromophenols.
are diverse types of activated oxygen.21−23 There is an equilibrium between the formation of ROS molecules
and antioxidant defense systems in living organisms.21 The oxidative stress process occurs when the generation
of ROS is higher than the protective ability of the antioxidant defense systems.24 Under pathological situations,
ROS molecules are overproduced and this results in oxidative stress. Additionally, ROS are created when inner
antioxidant defenses are incomplete.25 The imbalance between the antioxidant defense systems and ROS leads
to oxidative correction in the intracellular molecules or cellular membrane.26−28 The oxidative stress has been
defined as a key research concern due to its significant role in human diseases such as atherosclerosis, brain
dysfunction, and cancer.29 Antioxidant compounds are bioactive molecules generally used for the protection of
cellular compounds in living metabolisms. They maintain food quality and prevent oxidative rancidity.30−32
Antioxidant molecules eliminate free radicals and protect the human body from cancers, chronic cardiovascular
diseases, and ageing by removing or neutralizing ROS.33
The most commonly used processes to treat Alzheimer disease (AD) are centralized to impede the
hydrolysis of acetylcholine (ACh) in cholinergic neurons and hinder synaptic depression. This process is called
the cholinergic hypothesis and was recently used in the extension of drugs capable of elevating chemical balance
in the slope of the concentration of ACh in patients with AD.34 AChE performs a main role in ACh hydrolysis in
the neural device.35 The hydrolysis of ACh could be stopped by inhibiting AChE activity. Therefore, different
AChE inhibitors (AChEIs) are used in AD therapy. Among them the compound tacrine was the first drug
recorded by the Food and Drug Administration (FDA), followed by rivastigmine, galantamine, and donepezil.
These compounds as drugs are defined for treating moderate and mild phases of AD, when the patient still enjoys
autonomous cognitive acts.36 AChEIs can be classified as weak or strong inhibitors. Strong inhibitors contain
carbamates and organic phosphates, which are frequently used as pesticides and neural toxins, while weak
inhibitors have been used in the treatment of Parkinson disease, AD, dementia with Lewy bodies, glaucoma,
myasthenia gravis, autism, postural tachycardia syndrome, cognitive disturbances, and insomnia.37 Cholinergic
neurotransmission in the mammalian brain could stop by the hydrolysis of ACh, which is catalyzed by two
cholinesterases (ChEs): AChE and BChE. BChE is also used as drug for the therapy of AD at advanced stage.
Selective BChE inhibition is significantly beneficial, because it does not have classical cholinergic toxicity, which
is a prevalent side effect of AChE inhibitors.38 It has been recorded that some phenolic molecules had a large
potential of biological activity including AChE and BChE inhibition effects and antioxidant properties.39
To the best of our knowledge, the synthesis of natural bromophenols 2–4 was not reported in the literature
previously. The synthesis of them will make a significant contribution to the field because they are natural
products and might exhibit potential biological activity such as antioxidant. Therefore, in this study, the
synthesis of them and their derivatives was realized and their antioxidant and anticholinergic activities were
investigated.
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2. Results and discussion
2.1. Chemistry
Biologically active natural bromophenols 2–4 are N-benzylpyrrolidin-2-one derivatives (Figure). In addition to
OH groups in positions 3 and 4 in their benzene rings, three Br in positions 2, 3, and 6 in 2 and two Br in
positions 2 and 3 in the others (3 and 4) are found. Additionally, COOH and COOMe groups are found in the
pyrrolidone rings of 3 and 4, respectively. For the synthesis of these natural bromophenols 2–4, our method is
based on the preparation of benzylic rings with OMe and Br followed by their connection to the corresponding
pyrrolidinone rings. Hydroxy (OH) groups are converted to OMe moieties because of the better identification and
solubility of the products. It was reported that N-benzyl-2-pyrrolidone could be synthesized from the reaction
of benzyl bromide and 2-pyrrolidinone in the presence of KOH and TBAB (tetrabutylammonium bromide).40
After the synthesis of tetrabromide 5,41,42 the reaction of 5 and 2-pyrrolidinone with KOH and TBAB in THF
(tetrahydrofurane) did not yield compound 6. Compound 6 could be synthesized from the reaction of 5 and
2-pyrrolidone with NaH in THF (Scheme 1).
Scheme 1. a) THF-NaH, 0-25 ◦ C, 24 h; b) BBr3 -CH2 Cl2 , 0–25 ◦ C; c) SOCl2 and then MeOH.
For the synthesis of natural bromophenols 3 and 4, tribromide 7 was obtained from the corresponding
reactions using vanillin as starting material.20,41,42 The reaction of synthesized methyl 5-oxopyrrolidine-2-
carboxylate in the basic condition only gave 8, precursor compounds of natural products 3 and 4. To use
compound 9 in the demethylation reaction of natural products 3 and 4, compound 8 was also converted into
compound 9. Reactions of each compound 6, 8, and 9 with BBr3 in CH2 Cl2 gave the natural bromophenols
2, 3, and 4, respectively.
Derivatives with different numbers of Br, without Br and OMe of biologically active natural bromophenol
2 will be important because they might show biological activities. For the synthesis of derivatives 10–14,
corresponding bromides 5 and 7 were synthesized by known methods.20,41−44 The derivatives 10–14 were
obtained from the reactions of these bromides and 2-pyrrolidinone with NaH in THF (Scheme 2). Similar to
the synthesis of bromophenol 2 from the reaction of 6 with BBr3 , phenol derivatives 15–18 were obtained by
demethylations of derivatives 10–13 (Scheme 2). The corresponding phenol derivative could not be obtained
from the demethylation reaction of 14 with BBr3 .
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Scheme 2. Some derivatives of natural bromphenol 2 and their synthesis.
2.2. Biological activities
2.2.1. Antioxidant results
Antioxidant activity, which reflected the ability of pure compounds, can inhibit the oxidation process in living
systems. In this study, several different antioxidant assays based on different reaction mechanisms were used to
detect the potent antioxidant activity of novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols.
The following results should be noted regarding the inhibition of CA, AChE, and BChE enzymes and antioxidant
activities of novel bromophenols:
i. The first antioxidant assay performed is based on the Fe3+ reduction method. Generally, reducing
properties of a compound depend on its reducing power, which have been shown to exert antioxidant
activity and radical scavenging ability by donating a hydrogen atom.45 The Fe3+ (CN−)6 reduction
procedure detects the antioxidant effect of any molecule as measuring reducing ability in the reaction.
Some 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols including natural products of 2–
4 and some of the other compounds (15–18) had potent reducing activity using Fe3+ (CN−)6 when
compared with the standards (α-tocopherol, trolox, BHT, and BHA). It can be seen in Table 1 that the
Fe3+ reducing capacity of the newly synthesized compounds of 2–4, 15–18, and the standard compounds
was in the following order: BHA (2.322 ꢀ 0.024, r2 : 0.9685) > 4 (1.606 ꢀ 0.006, r2 : 0.9881) > 15
(1.573 ꢀ 0.008, r2 : 0.9638) > 17 (1.520 ꢀ 0.014, r2 : 0.9391) > 2 (1.436 ꢀ 0.049, r2 : 0.9424) >
BHT (1.425 ꢀ 0.007, r2 : 0.9770) > 16 (1.266 ꢀ 0.012, r2 : 0.9037) > 3 (1.177 ꢀ 0.011, r2 : 0.9740) >
trolox (1.123 ꢀ 0.009, r2 : 0.9889) > α-tocopherol (1.005 ꢀ 0.005, r2 : 0.9990) > 18 (0.716 ꢀ 0.004, r2 :
0.9975) at the same concentration (20 µg/mL). The reducing power of bioactive compounds reflects their
electron-donating capacity and is associated with antioxidant activity.
ii. Cupric ion (Cu2+) reducing power of 20 µg/mL concentration of novel 1-(4,5-dihydroxybenzyl)pyrrolidin-
2-one derivative bromophenols and standard reducing agents is shown in Table 1. The results of cupric
ion (Cu2+) reducing power were similar to the results obtained from the Fe3+ (CN−)6 reduction method.
Cu2+ reducing capacity of compounds of 2–4 and some of the other compounds (15–18) and standards
decreased in the following order: BHA (2.323 ꢀ 0.006, r2 : 0.9846) > BHT (2.204 ꢀ 0.027, r2 : 0.9455)
> 3 (2.116 ꢀ 0.013, r2 : 0.9775) > 17 (2.115 ꢀ 0.014, r2 : 0.9563) > 4 (2.064 ꢀ 0.008, r2 : 0.9794)
> 15 (1.857 ꢀ 0.004, r2 : 0.9550) > 2 (1.806ꢀ0.005, r2 : 0.9744) ≈ 16 (1.782 ꢀ 0.006, r2 : 0.9684)
> trolox (1.208ꢀ0.005, r2 : 0.9845) > α-tocopherol (1.123 ꢀ 0.006, r2 : 0.9787) > 18 (0.640 ꢀ 0.003,
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Table 1. Determination of reducing power of the same concentration (20 µg/mL) of novel bromophenols and their
methylated precursors by FRAP methods, ferric ion (Fe3+) reducing and cupric ion (Cu2+) reducing capacity by
CUPRAC method.
Fe3+-Fe2+ reducing
λ700
R2
Cu2+-Cu+ reducing
λ450
R2
Fe3+-TPTZ reducing
λ593
R2
Antioxidants
BHA
BHT
2.322 ꢀ 0.024 0.9685 2.323 ꢀ 0.006 0.9846 2.637 ꢀ 0.018 0.9575
1.425 ꢀ 0.007 0.9770 2.204 ꢀ 0.027 0.9455 1.763 ꢀ 0.008 0.9788
α−Tocopherol 1.005 ꢀ 0.005 0.9990 1.123 ꢀ 0.006 0.9787 1.678 ꢀ 0.018 0.9262
Trolox
2
1.123 ꢀ 0.009 0.9889 1.208 ꢀ 0.005 0.9845 2.786 ꢀ 0.018 0.9528
1.436 ꢀ 0.049 0.9424 1.806 ꢀ 0.005 0.9744 2.003 ꢀ 0.004 0.9978
1.177 ꢀ 0.011 0.9740 2.116 ꢀ 0.013 0.9775 1.990 ꢀ 0.009 0.9788
1.606 ꢀ 0.006 0.9881 2.064 ꢀ 0.008 0.9794 2.582 ꢀ 0.010 0.9207
0.314 ꢀ 0.012 0.9615 0.480 ꢀ 0.008 0.9969 0.418 ꢀ 0.006 0.9585
0.207 ꢀ 0.005 0.9775 0.280 ꢀ 0.002 0.9889 0.656 ꢀ 0.010 0.9627
0.297 ꢀ 0.008 0.9832 0.296 ꢀ 0.003 0.9909 0.485 ꢀ 0.007 0.9774
0.287 ꢀ 0.008 0.9979 0.253 ꢀ 0.030 0.9108 0.693 ꢀ 0.019 0.9538
0.225 ꢀ 0.007 0.9919 0.298 ꢀ 0.005 0.9757 0.393 ꢀ 0.006 0.9538
0.242 ꢀ 0.008 0.9440 0.280 ꢀ 0.003 0.9976 0.345 ꢀ 0.011 0.9901
0.223 ꢀ 0.005 0.9885 0.305 ꢀ 0.005 0.9844 0.345 ꢀ 0.007 0.9770
0.203 ꢀ 0.006 0.9920 0.217 ꢀ 0.007 0.9909 0.376 ꢀ 0.011 0.9507
1.573 ꢀ 0.008 0.9638 1.857 ꢀ 0.004 0.9550 1.834 ꢀ 0.006 0.9696
1.266 ꢀ 0.012 0.9037 1.782 ꢀ 0.006 0.9684 1.747 ꢀ 0.009 0.9708
1.520 ꢀ 0.014 0.9391 2.115 ꢀ 0.014 0.9563 2.204 ꢀ 0.006 0.9879
0.716 ꢀ 0.004 0.9975 0.640 ꢀ 0.003 0.9794 0.708 ꢀ 0.023 0.9251
3
4
6
8
9
10
11
12
13
14
15
16
17
18
r2 : 0.9794) at the same concentration (20 µg/mL). The CUPRAC assay is a simple, rapid, cost-effective,
selective, steady, and versatile antioxidant assay useful for a wide variety of polyphenols, as well as for
thiols and synthetic antioxidants.46 The ortho-substitution of HO− groups with electron-donating groups
like methoxy groups (–OCH3) can also increase antioxidant activity. For example the –CH=CH–COOH
groups in cinnamic acid can produce greater H-donating ability and subsequent antioxidant activity than
the –COOH groups in benzoic acids through stabilizing the radical by resonance of –C=C–.
iii. The FRAP method measures the capability of antioxidants to reduce the ferric 2,4,6-tripyridyl-s-triazine
complex [Fe(TPTZ)2 ]3+ to the intensely blue colored ferrous complex [Fe(TPTZ)2 ]2+ in acidic medium.47
According to the results obtained from the FRAP assay (Table 1) the reducing power of novel 1-(4,5-
dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols 2–4 and 15–18 and standards decreased in
the following order: trolox (2.786 ꢀ 0.018, r2 : 0.9528) > BHA (2.637 ꢀ 0.018, r2 : 0.9575) > 4 (2.582
ꢀ 0.010, r2 : 0.9207) > 17 (2.204 ꢀ 0.006, r2 : 0.9879) > 2 (2.003 ꢀ 0.004, r2 : 0.9978) > 3 (1.990 ꢀ
0.009, r2 : 0.9788) > 15 (1.834 ꢀ 0.006, r2 : 0.9696) > BHT (1.763 ꢀ 0.008, r2 : 0.9788) > 16 (1.747
ꢀ 0.009, r2 : 0.9708) > α-tocopherol (1.678 ꢀ 0.018, r2 : 0.9262) > 18 (0.708 ꢀ 0.023, r2 : 0.9251). In
this procedure, higher absorbance indicates higher reducing capability. Furthermore, the highest FRAP
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reduction capability was recorded for the novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bro-
mophenols 2–4, 17. It was reported that the FRAP method offers a well-known index of antioxidant, or
reducing, potential of samples or pure molecules.48−51
iv. The DPPH radical is one of the few stable organic nitrogen radicals and is deep purple. This method is
based on the measurement of the neutralizing capability of antioxidants toward DPPH radicals.52 Table 2
shows half maximal radical scavenging concentrations (IC50) of novel 1-(4,5-dihydroxybenzyl)pyrrolidin-
2-one derivative bromophenols and the reference radical scavenger types including BHA, BHT, trolox, and
α-tocopherol. IC50 amounts for 2–4 and 15–18 were in the following order: 17 (4.71 µg/mL, r2 : 0.9903)
< 3 (5.08 µg/mL, r2 : 0.9204) < 15 (5.68 µg/mL, r2 : 0.9493) < 4 (6.18 µg/mL, r2 : 0.9790) < 2 (6.41
µg/mL, r2 : 0.9993) < 16 (7.01 µg/mL, r2 : 0.9791) < trolox (7.37 µg/mL, r2 : 0.9737) < BHA (8.66
µg/mL, r2 : 0.9594) < BHT (13.07 µg/mL, r2 : 0.9202) < α-tocopherol (16.50 µg/mL, r2 : 0.9490) < 18
(53.30 µg/mL, r2 : 0.9770). It was determined that phenolic moieties in synthesized molecules increased
their radical scavenging activities. These activities can vary depending on the position and number of
hydroxyl groups attached to the aromatic ring.53
Table 2. Determination of half maximal concentrations (IC50 , µg/mL) of novel bromophenols, their methylated
precursors, and standards for DPPH, ABTS radical scavenging, and metal chelating activities.
Compounds
BHA
DPPH· scavenging R2
ABTS•+ scavenging R2
Fe2+ Chelating R2
8.66
0.9594 5.21
0.9934 27.72
0.9728
0.9922
0.9592
0.9102
0.9773
0.9883
0.9393
0.9520
0.9494
0.9190
0.9303
0.9852
0.9022
0.9392
0.9708
0.9912
0.9691
0.9419
0.9889
0.9281
BHT
13.07
0.9202 3.93
0.9490 11.17
0.9737 7.21
0.9737 26.65
0.9550 33.01
0.9749 38.50
α−Tocopherol 16.50
Trolox
7.37
EDTA
2
-
-
-
-
1.88
6.41
0.9993 31.50
0.9204 3.30
0.9790 3.41
0.9833 41.76
0.9939 40.28
0.9838 43.31
0.9039 69.31
0.9945 38.51
0.9844 33.02
0.9033 46.21
0.9334 57.75
0.9493 3.28
0.9791 3.33
0.9903 3.36
0.9770 15.75
0.9588 19.86
0.9905 26.65
0.9748 23.10
0.9506 135.01
0.9518 30.13
0.9338 135.88
0.9277 135.58
0.9424 113.66
0.9748 99.08
0.9946 131.46
0.9686 113.13
0.9980 21.01
0.9248 16.11
0.9910 9.48
3
5.08
4
6.18
6
85.55
138.61
86.62
99.01
84.51
113.60
76.15
83.48
5.68
8
9
10
11
12
13
14
15
16
17
18
7.01
4.71
53.30
0.9736 132.96
v. Another improved technique for the determination of radical scavenging is ABTS·+ scavenging activ-
ity. ABTS radicals were generated in an ABTS/K2 S2 O8 system.54 IC50 values for novel 1-(4,5-
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dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols 2–4, 15–18, and the reference radical scav-
enger compounds such as trolox, α-tocopherol, BHT, and BHA were in the following order: 15 (3.28
µg/mL, r2 : 0.9980) < 3 (3.30 µg/mL, r2 : 0.9905) < 16 (3.33 µg/mL, r2 : 0.9248) < 17 (3.36 µg/mL,
r2 : 0.9910) < 4 (3.41 µg/mL, r2 : 0.9748) < BHT (3.93 µg/mL, r2 : 0.9737) < BHA (5.21 µg/mL,
r2 : 0.9934) < trolox (7.21 µg/mL, r2 : 0.9749) < α-tocopherol (11.17 µg/mL, r2 : 0.9550) < 18 (15.75
µg/mL, r2 : 0.9736) < 2 (31.50 µg/mL, r2 : 0.9588) (Table 2). As in DPPH free radical scavenging
activity, a lower IC50 amount indicates higher ABTS·+ scavenging activity.
Ferrous ion chelating activities of the new 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols,
BHA, BHT, α-tocopherol, and trolox are shown in Table 2. IC50 values of the ferrous ion (Fe2+) chelating
effect of the new 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols and standards decreased as
in the following order: EDTA (1.88 µg/mL, r2 : 0.9773) < 17 (9.48 µg/mL, r2 : 0.9889) < 16 (16.11 µg/mL,
r2 : 0.9419) < 2 (19.86 µg/mL, r2 : 0.9883) < 15 (21.01 µg/mL, r2 : 0.9691) < 4 (23.10 µg/mL, r2 : 0.9520)
< BHT (26.65 µg/mL, r2 : 0.9922) ≈ 3 (26.65 µg/mL, r2 : 0.9393) < BHA (27.72 µg/mL, r2 : 0.9728)
< 8 (30.13 µg/mL, r2 : 0.9190) < α-tocopherol (33.01 µg/mL, r2 : 0.9592) < trolox (38.50 µg/mL, r2 :
0.9102) (Table 2). Chelation of ferrous ions (Fe2+) may protect the cells against molecular oxidative damage
by inhibiting production of ROS.
2.2.2. Results of anticholinesterase activity
I. AD is a life-threatening illness that accounts for 60%–80% of cases of dementia and that is the sixth leading
cause of death in the world.55 Generally, the final assessment comes at a late step of the disease because
clinical signs are difficult to know and AD-relevant biomarkers are not largely accepted. Therapeutic drugs
used to treat AD are ChEIs, which are considered to raise availability of ACh, thereby improving cognitive
processes, i.e. attention and memory.56 Indeed, three separate parts in the active site comprise selectivity
of BChE inhibitors: the first part is acyl binding pocket, second part is determined near the lip of active
site gorge, and the third part is recorded as the choline binding site. Molecules, which can affect these
BChE parts via metal interactions or hydrophobic, hydrogen bonding, and salt bridges can create their
inhibitory action.57 In this work, AChE was also extremely inhibited by novel antioxidant bromophenols
at the low nanomolar concentrations with Ki values in the range of 2.60 ꢀ 0.75–16.36 ꢀ 2.67 nM (Table
3). These results clearly determined that the new synthesized bromophenols had effective AChE inhibition
properties. However, the most powerful AChE inhibition was recorded by novel bromophenol 9 with a
Ki value of 2.60 ꢀ 0.75 nM. Moreover, all the remaining newly synthesized bromophenols reported here
had highly efficient inhibition constants against AChE. Additionally, tacrine (1, 2, 3, 4-tetrahydroacridin-
9-amine), which was the first centrally acting cholinesterase inhibitor recorded for the therapy of AD,
showed a Ki value of 86.37 ꢀ 14.10 nM against AChE.
II. Finally, new antioxidant bromophenols inhibited BChE with Ki values in the range of 13.10 ꢀ 3.33–
54.47 ꢀ 13.53 nM (Table 3). The Ki values of the new antioxidant bromophenols for BChE and AChE
were obtained from Lineweaver–Burk plots. On the other hand, tacrine, which was the first cholinesterase
inhibitor to be discovered for the management of AD symptoms in 1993, had a Ki value of 100.40 ꢀ 15.62
nM. Furthermore, it was considered that donepezil hydrochloride, which is used for the therapy of mild-
to-moderate AD and other diverse memory impairments, was shown to lower AChE inhibition activity
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(IC50 : 55.0 nM).58 As seen in Table 3, IC50 values are in the range of 8.60–29.88 nM and 37.84–83.22
nM towards AChE and BChE, respectively.
Table 3. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes inhibition effects of novel antioxidant
bromophenols.
IC50 (nM)
AChE r2
Ki (nM)
AChE
Inhibitors
BChE r2
BChE
AChE/BChE
0.25
2
13.85
17.10
29.88
13.70
14.87
8.60
0.9722 38.22
0.9832 7.78 ꢀ 1.07
0.9585 9.13 ꢀ 1.31
0.9848 9.80 ꢀ 1.28
0.9433 6.96 ꢀ 1.77
0.9955 4.20 ꢀ 1.24
0.9985 2.60 ꢀ 0.75
0.9794 12.05 ꢀ 2.95
0.9851 11.38 ꢀ 3.17
0.9643 10.83 ꢀ 0.98
0.9681 16.36 ꢀ 2.67
0.9845 9.01 ꢀ 2.94
0.9750 14.78 ꢀ 2.56
0.9723 6.38 ꢀ 2.88
0.9018 7.84 ꢀ 0.63
0.9288 14.25 ꢀ 6.01
29.66 ꢀ 7.67
31.30 ꢀ 6.65
19.53 ꢀ 3.24
26.35 ꢀ 5.78
22.97 ꢀ 3.45
14.97 ꢀ 4.28
39.58 ꢀ 7.34
54.47 ꢀ 13.53
18.75 ꢀ 3.45
13.10 ꢀ 3.33
23.06 ꢀ 2.18
50.18 ꢀ 11.78
36.30 ꢀ 5.65
52.05 ꢀ 6.12
36.26 ꢀ 6.73
3
0.9685 40.57
0.9965 46.51
0.9590 39.22
0.9628 44.58
0.9593 37.84
0.9694 42.43
0.9959 56.20
0.9749 48.38
0.9877 70.07
0.9484 48.26
0.9945 53.72
0.9529 67.15
0.9727 44.73
0.9318 83.22
0.29
4
0.42
6
0.26
8
0.18
9
0.17
10
11
12
13
14
15
16
17
18
TAC*
19.75
25.94
25.87
28.44
22.18
21.98
21.12
21.77
28.88
0.30
0.20
0.57
1.24
0.39
0.28
0.16
0.15
0.38
136.20 0.9796 174.16 0.9711 86.37 ꢀ 14.10 100.40 ꢀ 15.62 0.86
*Tacrine (TAC) was used as positive standard for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
enzymes
2.3. Conclusion
Biologically active natural bromophenols 2–4, tetrabromide 5,41,42 and tribromide 720,41,42 were synthesized
using the procedures described in the literature. Demethylation of compound 6, obtained from the reaction
of tetrabromide 5, yielded natural bromophenol 2. In a similar manner, other natural bromophenols 3 and 4
were also synthesized from 8 and 9. Besides the first synthesis of bromophenols 2–4, the derivatives 10–18 of
2 were synthesized as well. Among these compounds, 3, 4, 8, and 9 are racemic compounds. Additionally, the
synthesized compounds were evaluated for antioxidant and anticholinergic activities.
3. Experimental
3.1. General
Solvents were purified and dried by known methods. For all compounds, values as well as mp, IR spectra,
1 H and 13 C NMR spectra, chemical shift, elemental analyses, and antioxidant activities of samples were
performed as explained previously.12,59 Preparative thick-layer chromatography was used with 1 mm of silica gel
815

REZAI et al./Turk J Chem
60 PF (Merck, Darmstadt, Germany) on glass plates. HRMS data were obtained by LC-MS-TOF electrospray
ionization technique (1200/6210, Agilent).
3.2. Synthesis
3.2.1. 1-(2,3,6-Tribromo-4,5-dimethoxybenzyl)pyrrolidin-2-one (6): standard procedure for the
substitution reaction with 2-pyrrolidinone (8, 10–13)
To NaH (56.43 mg, 2.35 mmol) in a balloon (50 mL), washed with hexane, was added cold THF solution (0
◦ C) of 2-pirrolidone (181.94 mg, 2.14 mmol) in THF (20 mL). The mixture was stirred for 30 min and then
tetrabromide 5 (500 mg, 1.07 mmol) was added to the mixture. After the mixture was stirred at the same
temperature for 30 min more, the cold bath was removed and the mixture was additionally stirred for 24 h.
After the removal of solvent from the reaction mixture under vacuum, cold solution (1%, 0 ◦ C, 15 mL) of NaOH
was slowly added to the resultant mixture. The new mixture was extracted with ethyl acetate (EtOAc) (2 ×
20 mL). After the combined extracts were washed with water (20 mL) and dried over Na2 SO4 , the solvent was
removed under vacuum and compound 6 (0.4 g, 80%, white amorphous solid) was yielded. Mp: 126–127 ◦ C; IR
(CH2 Cl2 , cm−1): 2942, 1691,1395, 1280, 1121, 1010; Rf = 0.65: MeOH/CH2 Cl2 (2:8); 1 H NMR (400 MHz,
CDCl3): 4.94 (s, CH2 , 2H), 3.91 (s, OCH3 , 3H), 3.89 (s, OCH3 , 3H), 3.12 (t, J = 8.1 Hz, CH2 , 2H), 2.40 (t, J
= 8.1 Hz, CH2 , 2H), 1.94 (p, J = 8.1 Hz, CH2 , 2H); 13 C NMR (100 MHz, CDCl3): 174.65 (CO), 151.95 (C),
150.90 (C), 132.23 (C), 123.83 (C), 122.36 (C), 121.91 (C), 61.12 (NCH2), 61.10, NCH2), 49.23 (OCH3), 45.81
(OCH3), 30.92 (CH2), 18.16 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for C13 H14 Br79 Br79 Br81 NO3 :
471.8582; found: 471.8543.
3.2.2. 1-(2,3-Dibromo-4,5-dihydroxybenzyl)pyrrolidin-2-one (2): standard procedure for the demethy-
lation reaction with BBr3 (3, 4, 15–18)
A solution of BBr3 (0.53 g, 2.12 mmol) in CH2 Cl2 (7 mL) was added dropwise into cold (0 ◦ C) solution of
compound 6 (0.50 g, 1.1 mmol) stirred in CH2 Cl2 (15 mL) under N2 (g) over 7 min, and the mixture was
stirred at the same temperature for 20 min. After the cold bath was removed, the mixture was stirred at RT
and under N2 for 16 h. Then methanol (3 mL) was slowly added to the mixture over 15 min at 0 ◦ C. Next,
the solvent was evaporated and then water (25 mL) was added. After the mixture was extracted with EtOAc
(2 × 30 mL), the combined organic phases were dried over Na2 SO4 and the solvent was evaporated. Natural
product 2 (310 mg, 65%) was obtained as white amorphous solid. Mp: 166–168 ◦ C (Lit.2 166–168 ◦ C); Rf
=
0.26: MeOH/CH2 Cl2 (2:8); 1 H NMR (400 MHz, Acetone-d6) : δ 4.88 (bs, CH2 , 2H), 3.14 (t, J = 7.0 Hz,
CH2 , 2H), 2.39 (t, J = 8.0 Hz, CH2 , 2H), 2.06–1.84 (m, CH2 , 2H). 13 C NMR (100 MHz, Acetone-d6) : δ
175.78 (CO), 145.26 (C), 143.84 (C), 125.67 (C), 117.60 (C), 113.40 (C), 113.23 (C), 48.70 (CH2), 45.81 (CH2),
30.67 (CH2), 17.63 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for C11 H10 Br79 Br81 Br81 NO3 : 441.8289;
found: 441.8268.
3.2.3. 2,3-Dibromo-1-(bromomethyl)-4,5-dimethoxybenzene (7)
To a stirred cold (0 ◦ C) mixture of 2,3-dibromo-4,5-dimethoxybenzaldehyde (1.0 g, 3.09 mmol), methanol (10
mL), and diethyl ether (5 mL) NaBH4 (114 mg, 3.09 mmol) was carefully added in portions over 15 min. After
the mixture was stirred at the same temperature for half an hour, the cold bath (ice-water) was removed and
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REZAI et al./Turk J Chem
the reaction progress was monitored by thin-layer chromatography while the mixture was stirred at RT for 4
h. After the solvent was evaporated, water (20 mL) was added and the resulting mixture was extracted with
EtOAc (2 × 15 mL), the combined organic phases were dried over Na2 SO4 , and the solvent was evaporated.
To cold (0 ◦ C) solution of the residue including reduction product in CH2 Cl2 (20 mL) was added Et3 N (0.374
g, 3.70 mmol). After the mixture was stirred at the same temperature for 0.5 h, PBr3 (1.00 g, 3.70 mmol)
was added dropwise and the mixture was additionally stirred for 24 h. As stated above, the remainder of this
substitution reaction was realized according to that of the reduction reaction of the aldehyde. Known20,41,42
tribromide 7 (1.01 g, total 84%) was obtained as a yellow amorphous solid. 1 H NMR (400 MHz, CDCl3) : δ
7.00 (s, 1H), 4.63 (s, CH2 Br, 2H), 3.89 (s, OMe, 3H), 3.86 (s, OMe, 3H); 13 C NMR (100 MHz, CDCl3) : δ
152.68 (C), 147.96 (C), 134.16 (C), 122.55 (C), 117.95 (C), 113.69 (CH), 60.59 (OMe), 56.29 (OMe), 35.11
(CH2 Br).
3.2.4. (2RS)-1-(2,3-dibromo-4,5-dimethoxybenzyl)-5-oxopyrrolidine-2-carboxylic acid (8)
This reaction was realized according to the standard procedure described in the synthesis of 6. Methyl 5-
oxopyrrolidine-2-carboxylate (221 mg, 1.54 mmol), THF (20 mL), tribromide 7 (600 mg, 1.54 mmol), and NaH
(55.54 mg, 2.31 mmol) were used in the reaction. Methyl 5-oxopyrrolidine-2-carboxylate, synthesized from its
acid, was used instead of 2-pirrolidone. The crude compound 8 was purified from CHCl3 /hexane as a white
amorphous solid (280 mg, 40%). Mp: 157–158 ◦ C; IR (CH2 Cl2 , cm−1): 3081, 2992, 1683, 1402, 1318, 1101
cm−1 : Rf = 0.34: MeOH/CH2 Cl2 (5:95) 1 H NMR (400 MHz, CDCl3) : δ 6.95 (s, aromatic, 1H), 5.05 (d,
A part of AB system, J = 14.9 Hz, CH2 , 1H), 4.35 (d, B part of AB system, J = 14.9 Hz, methylenic, 1H),
4.13 (dd, J = 9.2, 2.6 Hz, methylenic, 1H), 3.84 (s, 2 OCH3 6H), 2.64–2.53 (m, methylenic, 1H), 2.50–2.28 (m,
methylenic, 2H), 2.23–2.14 (m, methylenic, 1H); 13 C NMR (100 MHz, CDCl3): δ 176.36 (CO), 175.34 (CO),
152.97 (C), 147.75 (C), 132.68 (C), 122.31 (C), 117.52 (C), 114.19 (CH), 60.76, 59.37, 56.54, 47.68, 29.47 (CH2),
23.38 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for C14 H15 Br79 Br81 NO5 : 437.9375; found: 437.9341.
3.2.5. (2RS)-Methyl 1-(2,3-dibromo-4,5-dimethoxybenzyl)-5-oxopyrrolidine-2-carboxylate (9)
A solution of the compound 8 (100 mg, 0.229 mmol) and SOCl2 (544 mg, 4.58 mmol) in CH2 Cl2 (8 mL) was
stirred at RT for 12 h. After the solvent was removed under vacuum and methanol was added, the formed
solution was stirred for 1 h. Then the solvent was removed under vacuum and compound 9 (90 mg, 87%) was
obtained as brown liquid; IR (CH2 Cl2 , cm−1): 3081, 2934, 1643, 1463, 1421, 1101, 1066, 1002; Rf = 0.65:
MeOH/CH2 Cl2 (5:95); 1 H NMR (400 MHz, CDCl3): 6.92 (s, aromatic, 1H), 5.00 (d, A part of AB system, J
= 15.0 Hz, NCH2 , 1H), 4.32 (d, B part of AB system, J = 15.0 Hz, NCH2 , 1H), 4.08 (dd, A part of AB system,
J = 9.2, 3.0 Hz, NCH, 1H), 3.83 (s, 2 × OCH3), 3.70 (s, OCH3), 2.58–2.49 (m, methylenic, 1H), 2.45–2.33
(m, methylenic, 2H), 2.12–2.03 (m, methylenic, 1H); 13 C NMR (100 MHz, CDCl3): 175.64 (CO), 172.42 (CO),
153.00 (C), 147.65 (C), 133.00 (C), 122.22 (C), 117.42 (C), 113.96 (CH), 60.73 (CH), 59.35 (NCH2), 56.50
(OCH3), 52.71 (OCH3), 47.53 (OCH3), 29.46 (CH2), 23.27 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for
C15 H14 Br79 Br81 NO5 : 449.9531; found: 449.9552.
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3.2.6. (2RS)-1-(2,3-dibromo-4,5-dihydroxybenzyl)-5-oxopyrrolidine-2-carboxylic acid (3)
This reaction was realized according to the standard procedure described in the synthesis of 2. Compound
8 (0.5 g, 1.14 mmol), BBr3 (601.83 mg, 2.40 mmol), CH2 Cl2 (15 mL), and H2 O (5 mL) were used in the
reaction. H2 O was used instead of methanol. The natural product 3 (420 mg, 90%, white amorphous solid)
was obtained. Mp: 160–162 ◦ C, (lit.19 172–174 ◦ C); Rf = 0.25: MeOH/CH2 Cl2 (2:8); 1 H NMR (400 MHz,
Acetone-d6) : δ 9.13 (bs, OH, 1H), 8.45 (bs, OH, 1H), 6.88 (s, aromatic, 1H), 4.93 (d, A part of AB system,
J = 15.2 Hz, methylenic NCH2 , 1H), 4.18 (d, B part of AB system, J = 15.1 Hz, methylenic NCH2 , 1H),
2.51–2.29 (m, methylenic, 3H), 2.21–1.97 (m, methylenic, 2H). 13 C NMR (100 MHz, Acetone-d6) : δ 175.12
(CO), 172.86 (CO), 145.14 (C), 144.31 (C), 128.47 (CH), 115.72 (C), 115.68 (C), 115.15 (C), 58.77 (CHN),
46.53 (CH2 N), 46.47 (CH2), 23.04 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for C12 H11 Br79 Br81 NO5 :
409.9062; found: 409.9038.
3.2.7. (2RS)-Methyl 1-(2,3-dibromo-4,5-dihydroxybenzyl)-5-oxopyrrolidine-2-carboxylate (4)
This reaction was realized according to the standard procedure described in the synthesis of 2. Compound
9 (400 mg, 0.89 mmol), BBr3 (444 mg, 1.79 mmol), CH2 Cl2 (15 mL), and methanol (10 mL) were used in
the reaction. The natural product 4 (330 mg, 88%, pale yellow solid) was obtained. Mp: 190–192 ◦ C (lit.19
183–185 ◦ C); Rf = 0.34: MeOH/CH2 Cl2 (2:8); 1 H NMR (400 MHz, Acetone-d6): 10.07 (bs, OH, 1H), 9.63
(bs, OH, 1H), 6.70 (s, aromatic, 1H), 4.69 (d, A part of AB system, J = 15.1 Hz, methylenic CHN, 1H), 4.02 (m,
methylenic, 2H), 3.62 (s, OMe, 3H), 2.49–2.45 (m, methylenic, 1H), 2.36–2.24 (m, methylenic, 2H), 2.00–1.92
(m, methylenic, 1H). 13 C NMR (100 MHz, Acetone-d6): 175.19 (CO), 172.88 (CO), 146.07 (C), 144.87 (C),
127.50 (C), 115.97 (CH), 114.75 (C), 113.97 (C), 59.07 (CH2 N), 52.92 (CH2 N), 46.76 (OMe), 29.47 (CH2),
23.00 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for C13 H13 Br81 Br81 NO5 : 425.9198; found: 425.9180.
3.2.8. 1-(2,3-Dibromo-4,5-dimethoxybenzyl)pyrrolidin-2-one (10)
This reaction was realized according to the standard procedure described in the synthesis of 6. 2-Pyrrolidinone
(218.85 mg, 2.57 mmol), THF (20 mL), tribromide 7 (0.5 g, 1.29 mmol), and NaH (67.88 mg, 2.83 mmol) were
used in the reaction. Compound 10 (450 mg, 89%) was obtained as a brown liquid; IR (CH2 Cl2 , cm−1):
3316, 2936, 1685, 1467, 1421, 1265, 1160, 1122, 1005; Rf = 0.62: MeOH/CH2 Cl2 (5:95); 1 H NMR (400 MHz,
CDCl3): 6.82 (s, aromatic, 1H), 4.52 (bs, methylenic CH2 N, 2H), 3.77 (s, OMe, 3H), 3.76 (s, OMe, 3H), 3.27
(t, methylenic CH2, J = 6.9 Hz, 2H), 2.36 (t, J = 6.9 Hz, methylenic 2H), 2.00–1.91 (m, methylenic 2H); 13 C
NMR (100 MHz, CDCl3): 175.46 (CO), 153.19 (C), 147.31 (C), 133.65 (C), 122.21 (C), 116.99 (C), 112.74 (CH),
60.68 (CH2 N), 56.44 (CH2 N), 48.10 (OMe), 47.32 (OMe), 30.87 (CH2), 18.15 (CH2); HRMS (APCI-TOF)
(m/z +H) calcd for C13 H15 Br79 Br81 NO3 : 393.9476; found: 393.9446.
3.2.9. 1-(3-Bromo-4,5-dimethoxybenzyl)pyrrolidin-2-one (11)
This reaction was realized according to the standard procedure described in the synthesis of 6. 2-Pyrrolidinone
(274.77 mg, 3.23 mmol), THF (20 mL), 1-bromo-5-(bromomethyl)-2,3-dimethoxybenzene (0.5 g, 1.77 mmol),
and NaH (81.29 mg, 3.39 mmol) were used in the reaction. Compound 11 (450 mg, 89%) was obtained as
colorless liquid; IR (CH2 Cl2 , cm−1): 2934, 1684, 1568, 1413, 1274, 1138, 1043; Rf = 0.68: MeOH/CH2 Cl2
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REZAI et al./Turk J Chem
(5:95); 1 H NMR (400 MHz, CDCl3): 6.80 (s, aromatic, 1H), 6.65 (s, aromatic, 1H), 4.32 (s, methylenic CH2 N,
2H), 3.66 (s, OMe, 3H), 3.64 (s, OMe, 3H), 3.11 (t, methylenic, J = 7.0 Hz, 2H), 2.21 (t, methylenic, J = 8.1
Hz, 2H), 1.85–1.71 (m, methylenic, 2H); 13 C NMR (100 MHz, CDCl3): 175.14 (CO), 149.16 (C), 148.86 (C),
127.89 (C), 115.30 (CH), 114.03 (C), 112.92 (CH), 56.24 (OMe), 56.18 (OMe), 46.87 (CH2 N), 46.03 (CH2 N),
30.87 (CH2), 17.93 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for C13 H16 Br79 NO3 : 314.0392; found:
314.0374.
3.2.10. 1-(2-Bromo-4,5-dimethoxybenzyl)pyrrolidin-2-one (12)
This reaction was realized according to the standard procedure described in the synthesis of 6. 2-Pyrrolidinone
(220 mg, 2.58 mmol), THF (20 mL), 1-bromo-2-(bromomethyl)-4,5-dimethoxybenzene (0.4 g, 1.29 mmol), and
NaH (81.29 mg, 3.39 mmol) were used in the reaction. Compound 12 (360 mg, 89%) was obtained as yellow
liquid; IR (CH2 Cl2 , cm−1): 1674, 1423, 1290, 1120; Rf = 0.53: MeOH/CH2 Cl2 (5:95); 1 H NMR (400 MHz,
CDCl3): 6.91 (s, aromatic, 1H), 6.68 (s, aromatic, 1H), 4.27 (s, methylenic CH2 N, 2H), 3.76 (s, OMe, 3H),
3.74 (s, OMe, 3H), 3.21 (t, J = 7.1 Hz, methylenic, 2 H), 2.35 (t, J = 7.1 Hz, methylenic 2H), 1.93 (p, J = 7.1
Hz, methylenic, 2H); 13 C NMR (100 MHz, CDCl3): 175.27 (CO), 154.06 (C), 145.98 (C), 134.05 (C), 124.24
(CH), 117.75 (C), 111.69 (CH ), 60.70 (OMe), 56.31 (OMe), 46.86 (CH2 N), 46.19 (CH2 N), 31.02 (CH2), 17.89
(CH2); HRMS (APCI-TOF) (m/z +H) calcd for C13 H16 Br79 NO3 : 314.0392; found: 314.0349.
3.2.11. 1-(4-Methoxybenzyl)pyrrolidin-2-one (13)
This reaction was realized according to the standard procedure described in the synthesis of 6. 2-Pyrrolidinone
(423.28 mg, 4.97 mmol), THF (20 mL), 1-(bromomethyl)-4-methoxybenzene (0.5 g, 2.49 mmol), and NaH
(125.32 mg, 5.22 mmol) were used in the reaction. Compound 13 (450 mg, 89%) was obtained as a brown
liquid; IR (CH2 Cl2 , cm−1): 3445, 2086, 1644, 1422, 1120; Rf = 0.5: MeOH/CH2 Cl2 (5:95); 1 H NMR (400
MHz, CDCl3): 7.15 (d, A part of AB system, J = 8.6 Hz, aromatic, 2H), 6.83 (d, B part of AB system, J = 8.6
Hz, aromatic, 2H), 4.36 (s, methylenic CH2 N, 2H), 3.77 (s, OMe, 3H), 3.21 (t, J = 8.1 Hz, methylenic, 2H), 2.40
(t, J = 8.1 Hz, methylenic, 2H), 1.95 (p, J = 8.1 Hz, methylenic, 2H); 13 C NMR (100 MHz, CDCl3); 174.98
(CO), 159.21 (C), 129.64 (2 CH), 128.84 (C), 114.18 (2 CH), 55.44 (OMe), 46.66 (CH2 N), 46.12 (CH2 N), 31.22
(CH2), 17.87(CH2); HRMS (APCI-TOF) (m/z +H) calcd for C11 H12 Br79 NO3 : 206.1181; found: 206.1162.
3.2.12. 1-(3-Bromo-4-methoxybenzyl)pyrrolidin-2-one (14)
To a solution of compound 13 (200 mg, 0.97 mmol) in CH2 Cl2 (15 mL), molecular bromine (622 mg, 3.90
mmol) was added. After the solution was stirred in the dark at RT for 16 h, the solvent was removed under
vacuum and compound 14 (275 mg, 100%) was obtained alone. Mp: 127–129 ◦ C; IR (CH2 Cl2 , cm−1): 2087,
1644, 1422, 1121; Rf = 0.5: MeOH/CH2 Cl2 (5:95); 1 H NMR (400 MHz, CDCl3): 7.47 (d, J = 2.0 Hz,
1H), 7.29 (dd, 1H, J = 8.4, 2.0 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 4.60 (bs, benzylic, 2H), 3.90 (s, OCH3),
3.71–3.50 (m, methylenic, 2H), 3.11 (t, 8.1 Hz, 2H), 2.39–2.19 (m, methylenic, 2H); 1 H NMR (400 MHz,
CDCl3): 178.92 (CO), 156.49 (C), 133.76 (CH), 129.72 (CH), 126.41 (CH), 112.80 (CH), 112.31 (C), 56.74
(OCH3), 50.29 (CH2 N), 48.30 (CH2 N), 31.76 (CH2), 17.90 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for
C12 H14 Br79 NO2 : 284.0286; found: 284.0215.
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REZAI et al./Turk J Chem
3.2.13. 1-(2,3-Dibromo-4,5-dihydroxybenzyl)pyrrolidin-2-one (15)
This reaction was realized according to the standard procedure described in the synthesis of 2. BBr3 (402
mg, 1.60 mmol), CH2 Cl2 (20 mL), and 10 (300 mg, 0.763 mmol) were used in the reaction. Compound 15
(230 mg, 82%) were obtained as pale yellow solid. Mp: 210–212 ◦ C; IR (acetone, cm−1): 3435, 1643, 1406,
1280, 1121; Rf = 0.26: MeOH/CH2 Cl2 (2:8); 1 H NMR (400 MHz, CD3 OD): 6.73 (s, aromatic, 1H), 4.49
(s, methylenic CH2 N, 2H), 3.46–3.21 (m, methylenic 2H), 2.42 (t, J = 8.1 Hz, methylenic 2H), 2.11–1.97 (m,
methylenic 2H); 13 C NMR (100 MHz, CD3 OD): δ 176.46 (CO), 145.51 (C), 144.40 (C), 127.49 (C), 114.60 (C),
114.57 (CH), 113.43 (C), 47.43 (CH2 N), 47.13 (CH2 N), 30.62, 17.58; HRMS (APCI-TOF) (m/z +H) calcd for
C11 H11 Br79 Br81 NO3 : 365.9163; found: 365.9133.
3.2.14. 1-(3-Bromo-4,5-dihydroxybenzyl)pyrrolidin-2-one (16)
This reaction was realized according to the standard procedure described in the synthesis of 2. BBr3 (670 mg,
2.67 mmol), CH2 Cl2 (15 mL), and 11 (0.4 g, 1.27 mmol) were used in the reaction. Compound 16 (300 mg,
80%) was obtained as a brown solid. Mp: 118–120 ◦ C; IR (acetone, cm−1): 3435, 2087, 1643, 1406, 1288, 1121;
Rf = 0.37: MeOH/CH2 Cl2 (2:8); 1 H NMR (400 MHz, Acetone-d6): 6.92 (s, aromatic, 1H), 6.79 (s, aromatic,
1H), 4.28 (s, methylenic CH2 N, 2H), 3.33 (t, J = 8.1 Hz, methylenic, 2H), 2.33 (t, J = 8.1 Hz, methylenic,
2H), 2.05–1.94 (p, J = 8.1 Hz, methylenic, 2H); 13 C NMR (100 MHz, Acetone-d6); 174.78 (CO), 146.39 (C),
142.63 (C), 130.01 (C), 123.15 (CH), 114.21 (CH), 109.04 (C), 46.51 (CH2 N), 45.39 (CH2 N), 30.58 (CH2),
17.73 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for C11 H12 Br79 NO3 : 286.0034; found: 286.0076.
3.2.15. 1-(2-Bromo-4,5-dihydroxybenzyl)pyrrolidin-2-one (17)
This reaction was realized according to the standard procedure described in the synthesis of 2. BBr3 (1.0 g,
4.20 mmol), CH2 Cl2 (15 mL), and 12 (0.6 g 1.91, mmol) were used in the reaction. Compound 17 (460 mg,
84%) was obtained as a yellow solid. Mp: 170–172 ◦ C; IR (acetone, cm−1): 3445, 1644, 1510, 1423, 1282, 1121;
Rf = 0.25: MeOH/CH2 Cl2 (2:8); 1 H NMR (400 MHz, CD3 OD): 9.37 (s, OH, 1H), 9.24 (s, OH, 1H), 6.89 (s,
aromatic, 1H), 6.62 (s, aromatic, 1H), 4.23 (s, methylenic CH2 N, 2H), 3.19 (t, J = 7.6 Hz, methylenic CH2 ,
2H), 2.25 (t, J = 7.6 Hz, methylenic CH2 , 2H), 1.90 (p, J = 7.6 Hz, methylenic CH2 , 2H); 13 C NMR (100
MHz, CD3 OD): 174.60 (CO), 146.52 (C) 145.99 (CH), 126.44 (C), 119.61 (CH), 116.93 (C), 111.12 (C), 46.89
(CH2 N) 45.77 (CH2 N), 30.90 (CH2) 18.10 (CH2); HRMS (APCI-TOF) (m/z +H) calcd for C11 H12 Br79 NO3 :
286.0079; found: 286.0054.
3.2.16. 1-(4-Hydroxybenzyl)pyrrolidin-2-one (18)
The synthesis of compound 18 was realized according to the standard procedure described in the synthesis of 2.
BBr3 (1.07 g, 4.29 mmol), CH2 Cl2 (15 mL), and 13 (0.4 g, 1.95 mmol) were used in the reaction. Compound
18 (310 mg, 83%) was obtained as a brown solid. Mp: 118–120 ◦ C; IR (acetone, cm−1) 3414, 2943, 1641,
1497, 1259, 1110; Rf = 0.31: MeOH/CH2 Cl2 (2:8); 1 H NMR (400 MHz, Acetone-d6): 7.09 (d, A part of
AB system, J = 8.6 Hz, aromatic, 2H), 6.79 (d, B part of AB system, J = 8.6 Hz, aromatic, 2H), 4.31 (s,
methylenic CH2 N, 2H), 3.25 (t, J = 8.1 Hz, methylenic CH2 , 2H), 2.28 (t, J = 8.1 Hz, methylenic CH2 , 2H),
2.05 (p, J = 8.1 Hz, methylenic CH2 , 2H); 13 C NMR (100 MHz, APT, Acetone-d6): 173.97 (CO), 156.84
820

REZAI et al./Turk J Chem
(C), 130.04 (2 CH) 128.42 (C) 115.52 (2 CH) 46.18 (CH2 N) 45.52 (CH2 N) 30.58 (CH2) 17.74 (CH2); HRMS
(APCI-TOF) (m/z +H) calcd for C11 H13 NO2 : 192.1025; found: 192.1004.
3.3. Biological assays
3.3.1. Antioxidant activity assays
3.3.1.1. Fe3+ reducing power assay
For designation of the Fe3+ reducing capability of novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bro-
mophenols, the [Fe(CN)6 ]3− to [Fe(CN)6 ]4− reduction procedure was used.60 Briefly, varied concentrations
of novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols (10–30 µg/mL) in 0.75 mL of deion-
ized water were added to 1.25 mL of phosphate buffer (0.2 M, pH 6.6) and 1.25 mL of potassium ferricyanide
[K3 Fe(CN)6 ] (1%). Then the solution was incubated at 40 ◦ C for 15 min. After the incubation period,
trichloroacetic acid (TCA) was added (1.25 mL, 10%). Lastly, a portion of FeCl3 (0.5 mL, 0.1%) was trans-
ferred to this mixture and the absorbance amount was obtained at 700 nm in a spectrophotometer. According
to the results, when reduction capability increases, absorbance shows greater values.61
3.3.1.2. Cupric ion (Cu2+ ) reducing-CUPRAC assay
Cupric ion (Cu2+) reducing power was used as a second reducing capability procedure for novel 1-(4,5-
dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols. Cu2+ reducing ability was performed conforming
to the procedure explained by Apak et al.62 with slight modification.63 Briefly, aliquots of CuCl2 solution
(0.25 mL, 0.01 M), ethanolic neocuproine solution (0.25 mL, 7.5 × 10−3 M), and NH4 Ac buffer solution (0.25
mL, 1.0 M, pH 6.5) were transferred to a test tube containing novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one
derivative bromophenols at various concentrations (10–30 µg/mL). Total volume was completed with distilled
water to 2 mL followed by vigorous shaking. Absorbance of samples was recorded at 450 nm after 30 min.
3.3.1.3. FRAP Assay
The FRAP method is based upon reduction of Fe3+ -TPTZ complex under acidic medium conditions. Enhanced
absorbance of blue ferrous form (Fe2+ -TPTZ complex) is recorded at 593 nm. TPTZ solution (2.25 mL,
10 mM TPTZ in 40 mM HCl) was freshly prepared and then transferred to 25 mL of acetate buffer (0.3
M, pH 3.6), and FeCl3 solution (2.25 mL, 20 mM) in water. Then various concentrations of novel 1-(4,5-
dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols (10–30 µg/mL) were dissolved in 5 mL of appropriate
buffer solvent, stirred, and incubated at 37 ◦ C for 30 min. Finally the absorbance of the mixture was measured
at 593 nm.64
3.3.1.4. DPPH• scavenging activity
The DPPH• scavenging activity of the novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols
was designated as previously explained.65 Briefly, fresh solution of DPPH radicals (0.1 mM) was prepared in
ethanol. Then 1.5 mL of each novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenol in ethanol
was added to an aliquot of this solution (0.5 mL, 10–30 µg/mL). These mixtures were mixed vigorously and
incubated in the dark for 30 min. Finally the absorbance value was recorded at 517 nm in a spectrophotometer.66
821

REZAI et al./Turk J Chem
3.3.1.5. ABTS•+ scavenging activity
The ABTS radical scavenging capacity of the novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bro-
mophenols was characterized using the spectroscopic procedure explained by Re et al.67 The ABTS radical
cation (ABTS•+) was acquired by reacting 7 mM solution of ABTS with 2.45 mM K2 S2 O8 . Prior to the
assay, the ABTS• radical cation solution was diluted with ethyl alcohol to an absorbance of 0.750 ꢀ 0.05 at
734 nm. Then 1 mL of ABTS•+ solution was added to 3 mL of each novel 1-(4,5-dihydroxybenzyl)pyrrolidin-
2-one derivative bromophenol (20–60 µg/mL) and control solution. The extent of decolorization was obtained
as percentage reduction of absorbance. Percentage of radicals scavenging effects (RSEs) of each novel 1-(4,5-
dihydroxybenzyl)pyrrolidin-2-one derivative bromophenol was computed using the following equation: RSEs
(%) = [1– (As/Ac)] × 100, where Ac is the absorbance value of control and As is absorbance value of sample.
3.3.1.6. Metal chelating assay
Ferrous ion (Fe2+) chelating activities of the novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bro-
mophenols were evaluated conforming to the procedure explained previously.68 Diverse concentrations (10–30
µg/mL) of the novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols in 0.25 mL of ethanol,
0.25 mL of FeSO4 solution (2 mM), 1 mL of ferrozine solution (0.2% in 0.2 M HCl), 1 mL of Tris-HCl buffer
solution (pH 7.4), and 2.5 mL of ethanol solution were placed into a test tube, in that order. The absorbance
was measured at 562 nm.
3.3.2. AChE/BChE activity determination and inhibition studies
The inhibitory efficacy of the novel 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols on BChE/AChE
activities was obtained conforming to the spectrophotometric procedure of Ellman et al.69 as described
previously.70 Butrylcholine iodide (BChI) and also acetylthiocholine iodide (AChI) compounds were used as
substrates of both reactions. In this work, 5,5′-dithio-bis(2-nitro-benzoic)acid (DTNB) was used for the esti-
mation of the BChE/AChE activities. Briefly, 100 µL of buffer solution (pH 8.0, Tris-HCl, 1.0 M) and diverse
concentration of sample solutions (1.1–48 nM for AChE and 11–115 nM for BChE) dissolved in deionized water
were added to 50 µL of BChE/AChE solutions (5.32 × 10−3 EU). Then the mixture was incubated for 10 min
at 20 ◦ C. Finally, 50 µL of DTNB (0.5 mM and 25 mL) of BChI/AChI was added to the incubated mixture.
Furthermore, the reaction was initiated by the addition of 50 µL of BChI/AChI. Activities of these enzymes
were evaluated spectrophotometrically at a wavelength of 412 nm.
Acknowledgments
¨
˙
This study was financed by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project
No: 113Z702) and realized at Atatu¨rk University, Faculty of Science, Department of Chemistry. We are grateful
to both institutes for their support.
References
1. Kurata, K.; Taniguchii, K.; Takashima, K.; Hayashi, I.; Suzuki, M. Phytochemistry. 1997, 45, 485-487.
2. Duan, X. J.; Li, X. M.; Wang, B. G. J. Nat. Prod. 2007, 70, 1210-1213.
˙
3. Balaydın, H. T.; Gu¨lc¸in, I.; Menzek, A.; G¨oksu, S.; Sahin, E. J. Enzyme Inhib. Med. Chem. 2010, 25, 685-695.
822

REZAI et al./Turk J Chem
˙
4. C¸etinkaya, Y.; G¨oc¸er, H.; Menzek, A.; Gu¨lc¸in, I. Arch. Pharm. 2012, 345, 323-334.
5. Choudhary, M.; Patel, R. N.; Rawat, S.P. J. Mol. Struc. 2014, 1070, 94-105.
6. Bharti, S.; Choudhary, M.; Mohan, B.; Rawat, S. P.; Sharma, S. R.; Ahmad K. J. Mol. Struc. 2017, 1049, 846-861.
7. Balaydın, H. T.; S¸entu¨rk, M.; G¨oksu, S.; Menzek, A. Eur. J. Med. Chem. 2012, 54, 423-428.
˙
8. C¸etinkaya, Y.; G¨oc¸er, H.; Gu¨l¸cin, I.; Menzek, A. Arch. Pharm. 2014, 347, 354-359.
˙
9. Bozta¸s; M.; C¸etinkaya, Y.; Topal, M.; Gu¨lc¸in, I.; Menzek, A.; S¸ahin, E.; Tan¸c, M.; Supuran, C. T. J. Med. Chem.
2015, 58, 640-650.
˙
10. Nar, M.; C¸etinkaya, Y.; Gu¨lc¸in, I.; Menzek, A. J. Enzyme Inhib. Med. Chem. 2013, 28, 402-406.
˙
11. Artun¸c, T.; C¸etinkaya, Y.; G¨oc¸er, H.; Gu¨l¸cin, I.; Menzek, A.; S¸ahin, E.; Supuran, C. T. Chem. Biol. Drug. Des.
2016, 87, 594-607.
˙
12. Bayrak, C¸.; Taslimi, P.; Gu¨l¸cin, I.; Menzek, A. Bioorg. Chem. 2017, 72, 359-366.
13. Gribble, G. W. Chem. Soc. Rev. 1999, 28, 335-346.
14. Akbaba, Y.; Tu¨rke¸s, C.; Polat, L.; S¨o˘gu¨t, H.; S¸ahin, E.; G¨oksu, S.; Menzek, A.; Beydemir, S¸. J. Enzyme Inhib.
Med. Chem. 2013, 28, 1073-1079.
15. Balaydın, H. T.; Akbaba, Y.; Menzek, A.; S¸ahin, E.; G¨oksu, S. Arkivoc. 2009, XIV, 75-87.
16. Choi, J. S.; Park, H. J.; Jung, H. A.; Chung, H. Y.; Jung, J. H.; Choi, W. C. J. Nat. Prod. 2000, 63, 1705-1706.
17. Liu, X.; Li, X.; Gao, L.; Cui, C.; Li, C.; Li, J.; Wang, B. Chin. J. Oceanol. Limnol. 2011, 29, 686-690.
18. Zhao, J.; Ma, M.; Wang, S.; Li, S.; Cao, P.; Yang, Y.; Lu, Y.; Shi, J.; Xu, N.; Fan, X.; et al. J. Nat. Prod. 2005,
68, 691-694.
19. Li, K.; Li, X. M.; Gloer, J. B.; Wang, B. G. Food Chem. 2012, 135, 868-872.
20. Balaydın, H. T.; S¸entu¨rk, M.; Menzek, A. Bioorg. Med. Chem. Lett. 2012, 22, 1352-1357.
˙
21. Gu¨lc¸in, I. Arch. Toxicol. 2012, 86, 345-391.
˙
22. Gu¨lc¸in, I.; Beydemir, S. Mini Rev. Med. Chem. 2013, 13, 408-430.
¨
23. G¨oc¸er, H.; Akıncıo˘glu, A.; Ozta¸skın, N.; G¨oksu, S.; Gu¨lc¸in, I. Arch. Pharm. 2013, 346, 783-792.
˙
˙
24. G¨oc¸er, H.; Gu¨lc¸in, I. Int. J. Food Sci. Nutr. 2011, 62, 821-825.
˙
25. Gu¨lc¸in, I.; Topal, F.; C¸akmak¸cı, R.; G¨oren, A. C.; Bilsel, M.; Erdo˘gan, U. J. Food Sci. 2011, 76, C585-C593.
˙
26. Bursal, E.; Gu¨lc¸in, I. Food. Res. Int. 2011, 44, 482-1489.
˙
27. Gu¨lc¸in, I.; Topal, F.; Oztu¨rk Sarikaya, S. B.; Bursal, E.; G¨oren, A. C.; Bilsel, M. Rec. Nat. Prod. 2011, 5, 158-175.
˙
28. K¨oksal, E.; Bursal, E.; Dikici, E.; Tozog˘lu, F.; Gu¨lc¸in, I. J. Med. Plants Res. 2011, 5, 217-222.
˙
29. Topal, F.; Topal, M.; G¨oc¸er, H.; Kalın, P.; Ko¸cyi˘git, U. M.; Gu¨lc¸in, I.; Alwasel, S. H. J. Enzyme Inhib. Med. Chem.
2016, 31, 674-683.
˙
30. Aksu, K.; Topal. F.; Gu¨l¸cin, I.; Tu¨mer, F.; G¨oksu, S. Arch. Pharm. 2015, 348, 446-455.
¨
˙
31. G¨oc¸er, H.; Akıncıo˘glu, A.; Ozta¸skın, N.; G¨oksu, S.; Gu¨lc¸in, I. Arch. Pharm. 2013, 346, 783-792.
˙ ¨ ˙
32. Gu¨lc¸in, I.; Oktay, M.; Kire¸cci, E.; Ku¨freviog˘lu, O. I. Food Chem. 2003, 83, 371-382.
˙ ¨ ˙
33. Gu¨lc¸in, I.; Ku¨freviog˘lu, O. I.; Oktay, M.; Bu¨yu¨kokurog˘lu, M. E. J. Ethnopharmacol. 2004, 90, 205-215.
˙
34. Gu¨lc¸in, I.; Scozzafava, A.; Supuran, C. T.; Akıncıo˘glu, H.; Koksal, Z.; Turkan, F.; Alwasel, S. H. J. Enzyme Inhib.
Med. Chem. 2016, 31, 1060-1066.
˙
35. G¨oc¸er, H.; Topal, F.; Topal, M.; Ku¨c¸u¨k, M.; Teke, D.; Gu¨l¸cin, I.; Alwasel, S. H.; Supuran, C. T. J. Enzyme Inhib.
Med. Chem. 2016, 31, 441-447.
823

REZAI et al./Turk J Chem
¨
36. Ozta¸skın, N.; Taslimi, P.; Mara¸s, A.; G¨oksu, S.; Gu¨lc¸in, I. Bioorg. Chem. 2017, 74, 104-114.
˙
˙
37. Akıncıo˘glu, A.; Kocaman, E.; Akıncıo˘glu, H.; Salmas, R. E.; Durda˘gı, S.; Gu¨lc¸in, I.; Supuran, C. T.; G¨oksu, S.
Bioorg. Chem. 2017, 74, 238-250.
¨
38. Aksu, K.; Ozgeris, B.; Taslimi, P.; Naderi, A.; Gu¨lc¸in, I.; G¨oksu, S. Arch. Pharm. 2016, 349, 944-954.
˙
39. Taslimi, P.; Sujayev, A.; Garibov, E.; Nazarov, N.; Huyut, Z.; Alwasel, S. H.; Gu¨l¸cin, I. J. Biochem. Mol. Toxicol.
2017, 31, e21897.
40. Mun, J.; Smith, M. B. Synthetic Commun. 2007, 37, 813-819.
41. Mori, T.; Bando, H.; Kanaiwa, Y.; Amiya, T.; Kurata, K. Chem. Pharm. Bull. 1983, 3, 1754-1756.
42. Akbaba, Y.; Balaydin, H. T.; G¨oksu, S.; Sahin, E.; Menzek A. Helv. Chim. Acta. 2010, 93, 1127-1135.
43. S¸ahin, E.; Balaydın, H. T.; G¨oksu S.; Menzek, A. Acta Crystallogr. 2010, E66, o3029.
44. C¸etinkaya, Y.; Menzek, A.; S¸ahin, E.; Balaydın, H. T. Tetrahedron 2011, 67, 3483-3489.
˙
45. Gu¨lc¸in, I.; Tel, A. Z.; Kirecci, E. Int. J. Food Propert. 2008, 11, 450-471.
˙
46. K¨ose, L. P.; Gu¨lc¸in. I.; G¨oren, A. C.; Namiesnik, J.; Martinez-Ayala, A. L.; Gorinstein, S. Ind. Crops Prod. 2015,
74, 712-721.
˙
47. Gu¨lc¸in, I.; Bursal, E.; S¸ehito˘glu, H. M.; Bilsel, M.; G¨oren, A. C. Food Chem Toxicol. 2010, 48, 2227-2238.
¨
48. Ozta¸skın, N.; C¸etinkaya, Y.; Taslimi, P.; G¨oksu, S.; Gu¨l¸cin, I·. Bioorg. Chem. 2015, 60, 49-57.
˙
49. Gu¨lc¸in; I.; Elmasta¸s, M.; Aboul-Enein, H. Y. Arab. J. Chem. 2012, 5, 489-499.
˙
50. C¸etinkaya, Y.; G¨oc¸er, H.; Menzek, A.; Gu¨lc¸in, I. Arch. Pharm. 2012, 345, 323-334.
˙
51. Gu¨lc¸in, I.; Beydemir, S.; Topal, F.; Gagua, N.; Bakuridze, A.; Bayram, R.; Gepdiremen, A. J. Enzyme Inhib. Med.
Chem. 2012, 27, 587-594.
52. Blois, M. S. Nature 1958, 181, 1199-1200.
˙
53. Gu¨lc¸in, I.; Huyut, Z.; Elmasta¸s, M.; Aboul-Enein, H. Y. Arab. J. Chem. 2010, 3, 43-53.
54. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. 1999, Free Radical Biol. Med. 26,
1231-1237.
55. De Ferrari, G.V.; Canales, M. A.; Shin, I.; Weiner, L. M.; Silman, I.; Inestrosa, N. C.; Biochemistry 2001, 40,
10447-10457.
56. Cacabelos, R. Neuropsychiatr. Dis. Treat. 2007, 3, 303-333.
57. Kamal, A.; Shaik, A. B.; Reddy, G. N.; Kumar, G. N.; Joseph, J.; Kumar, G. B.; Purushotham, U.; Sastry, G. N.
Med. Chem. Res. 2014, 23, 2080-2092.
58. Sugimoto, H.; Yamanishi, Y.; Iimura, Y.; Kawakami, Y. Curr. Med. Chem. 2000, 7, 303-333.
¨
59. Kaya, O.; S¸engu¨l, M. E.; Menzek, A.; S¸ahin, E.; Gu¨r, B. Tetrahedron 2016, 72, 2828-2837.
60. Veliog˘lu, Y. S.; Mazza, G.; Gao, L.; Oomah, B. D. J. Agric. Food Chem. 1998, 46, 4113-4117.
61. Yen, G. C.; Hung, C. Y. Food Res. Int. 2000, 33, 487-492.
¨
62. Apak, R.; Gu¸clu, K.; Ozyurek, M.; Karademir, S. E.; Er¸ca, E. Int. J. Food Sci. Nutr. 2006, 57, 292-304.
¨
63. Apak, R.; Gu¸clu, K.; Ozyurek, M.; Karademir, S.E. J. Agric. Food Chem. 2004, 52, 7970-7981.
64. Benzie, I. F. F.; Strain, J. J. Anal. Biochem. 1996, 239, 70-76.
65. Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Lebensm. Wiss. Technol. 1995, 28, 25-30.
66. Bondet, V.; Brand-Williams, W.; Berset, C. Food Sci. Technol. 1997, 30, 609-615.
67. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Free Radical Biol. Med. 1999, 26,
1231-1237.
824

REZAI et al./Turk J Chem
68. Dinis, T. C.; Maderia, V. M.; Almeida, L. M. Arch. Biochem. Biophys. 1994, 315, 161-169.
69. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherston, R. M. Biochem. Pharmacol. 1961, 7, 88-95.
¨
70. Ozbey, F.; Taslimi, P.; Gu¨lcin, I.; Mara¸s, A.; G¨oksu, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2016, 31,
˙
79-85.
825

The first synthesis and antioxidant and anticholinergic activities of
1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols including
natural products
∗
˙
Mohsen REZAI, C¸ etin BAYRAK, Parham TASLIMI, Ilhami GULC¸ IN, Abdullah MENZEK
¨
˙
Department of Chemistry, Faculty of Science, Atatu¨rk University, Erzurum, Turkey
Abstract: We report herein the first synthesis of biologically active natural bromophenols, namely 1-(2,3,6-
tribromo-4,5-dihydroxybenzyl)pyrrolidin-2-one (2), 2RS-1-(2,3-dibromo-4,5-dihydroxybenzyl)-5-oxopyrrolidine-
2-carboxylic acid (3), and 2RS-methyl 1-(2,3-dibromo-4,5-dihydroxybenzyl)-5-oxopyrrolidine-2-carboxylate (4),
along with their different derivatives (6 and 8–18). Among the synthesized compounds, 3, 4, and their
derivatives (8 and 9) are yielded in racemic form. Radical scavenging assays of the synthesized molecules were
investigated using various bioanalytical antioxidant methods including 2,2’-azino-bis (3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS•+) and 2,2-diphenyl-1-picrylhydrazyl (DPPH•). In addition, the reducing power of the
novel bromophenols was investigated by Cu2+ –Cu+ reducing, Fe3+ –Fe2+ reducing, and [Fe3+ –(TPTZ)2 ]3+
–
[Fe2+ –(TPTZ)2 ]2+ reducing capacity and ferrous ions (Fe2+) chelating abilities. The molecules demonstrated
powerful antioxidant activities when compared to standard antioxidant compounds of α-tocopherol, trolox,
butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). Moreover, novel bromophenols were
tested against cholinergic enzymes including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
enzymes in the last part of this study. The presented novel bromophenols showed Ki values in the range of 2.60
0.75–16.36
2.67 nM against AChE and 13.10
3.33–54.47
13.53 nM against BChE.
Key words: Bromination, bromophenol, pyrrolidin-2-one, natural product, antioxidant activity, acetylcholineste-
rase, butyrylcholinesterase
∗Correspondence: amenzek@atauni.edu.tr
1

Supplementary material contains NMR spectra of synthesized compounds.
1H NMR spectrum of compound 2 (400 MHz, Acetone-d6)
13C NMR spectrum of compound 2 (100 MHz, Acetone-d6)
2

1H NMR spectrum of compound 3 (400 MHz, Acetone-d6)
13C NMR spectrum of compound 3 (100 MHz, Acetone-d6)
3

1H NMR spectrum of compound 4 (400 MHz, Acetone-d6)
13C NMR spectrum of compound 4 (100 MHz, Acetone-d6)
4

1H NMR spectrum of compound 6 (400 MHz, CDCl3)
13C NMR spectrum of compound 6 (100 MHz, CDCl3)
5

1H NMR spectrum of compound 8 (400 MHz, CDCl3)
13C NMR spectrum of compound 8 (100 MHz, CDCl3)
6

COOMe
Br
Br
N
O
MeO
OMe
5.00
4.90
4.80
4.70
4.60
4.50
4.40
4.30
4.20
4.10
ppm (f1)
2.60
2.50
2.40
2.30
2.20
2.10
2.00
ppm (f1)
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm (f1)
1H-NMR Spectrum of the compound 9 (400 MHz, CDCl3)
COOMe
N
Br
Br
MeO
O
OMe
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
ppm (f1)
13C NMR spectrum of compound 9 (100 MHz, CDCl3)
7

13C NMR spectrum of compound 10 (100 MHz, CDCl3)
13C-NMR spectrum of compound 10 (100 MHz, CDCl3)
8

13C NMR spectrum of compound 11 (100 MHz, CDCl3)
13C NMR spectrum of compound 11 (100 MHz, CDCl3)
9

13C NMR spectrum of compound 12 (100 MHz, CDCl3)
13C NMR spectrum of compound 12 (100 MHz, CDCl3)
10

1H NMR spectrum of compound 13 (100 MHz, CDCl3)
13C NMR spectrum of compound 13 (100 MHz, CDCl3)
11

O
Br
N
MeO
7.40
7.30
7.20
7.10
7.00
ppm (f1)
3.50
ppm (f1)
3.00
2.50
8.50
8.00
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
ppm (f1)
1H NMR spectrum of compound 14 (100 MHz, CDCl3)
O
Br
N
MeO
112.90
112.80
112.70
112.60
112.50
112.40
112.30
ppm (f1)
150
100
50
ppm (f1)
13C NMR spectrum of compound 14 (100 MHz, CDCl3)
12