Synthesis and paroxonase activities of novel bromophenols

Article abstract of DOI:10.3109/14756366.2012.715287

Three novel bromophenols 10-12 were synthesized. Acylation of veratrole (4) with 2,3-dimethoxy benzoic acid (5) gave a kown diarylmethanone 6. Bromination of 6 with different equivalents of molecular bromine afforded new di and tribrominated compounds 7-9 which were converted to their corresponding bromophenols 10-12 via O-demethylation with BBr3. Paraoxonase-1 (PON1) was purified from human serum with approximately 42% and 3584 U × mg ^-1 specific activity. The synthesized compounds 6-12 showed inhibitory effects on paraoxonase-1 (PON1) which is an organophosphate (OP) hydrolyser and an antioxidant bioscavenger enzyme. IC50 values were determined in the range of 0.123-1.212 mM. Graphical abstract

Full text of DOI:10.3109/14756366.2012.715287

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(5): 1073–1079
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.715287
ReseaRch aRtIcle
Synthesis and paroxonase activities of novel bromophenols
Yusuf Akbaba, Cüneyt Türkeş, Leyla Polat, Hakan Söyüt, Ertan Şahin, Abdullah Menzek,
Süleyman Göksu, and Şükrü Beydemir
Department of Chemistry, Faculty of Science, Atatürk University, Erzurum, Turkey
abstract
Three novel bromophenols 10–12 were synthesized. Acylation of veratrole (4) with 2,3-dimethoxy benzoic acid (5)
gave a kown diarylmethanone 6. Bromination of 6 with different equivalents of molecular bromine afforded new
di and tribrominated compounds 7–9 which were converted to their corresponding bromophenols 10–12 via
O-demethylation with BBr . Paraoxonase-1 (PON1) was purified from human serum with approximately 42% and
3584U × mg−1 specific ac3tivity. The synthesized compounds 6–12 showed inhibitory effects on paraoxonase-1
(PON1) which is an organophosphate (OP) hydrolyser and an antioxidant bioscavenger enzyme. IC50 values were
determined in the range of 0.123–1.212mM.
Graphical abstract
Keywords: Bromination, bromophenols, natural products, demethylation, paroxonase activity
Introduction
activities. Aldose reductase9 and carbonic anhydrase10
inhibitory properties of natural products 2–3 have also
been noted (Figure 1).
e high-density lipoprotein associated serum
esterase, paraoxonase (arylesterase, EC 3.1.8.1, PON1)
is a key enzyme for protection against atherosclerosis
in metabolism. e enzyme includes two Ca2+ ions in its
3D-structure. A Ca2+ is in active region of the enzyme and
another one is structural. Each Ca2+ ions are important
catalytic activity of the enzyme. If one of the Ca2+ ions
is removed from enzyme structure, the enzyme loses
Among natural organohalogen compounds, bromophe-
nols are abundantly found in marine life and frequently
isolated from red algae of the family Rhodomelaceae1.
Recent studies revealed that the marine bromophenols
exhibit a wide spectrum of beneficial biological activi-
ties2. ese compounds have attracted much attention in
the field of functional food and pharmaceutical agents2. It
has been reported that naturally occurring bromophenol
1 shows enzyme inhibition3, cytotoxicity4, feeding deter-
rent5, anti-microbial6, anti-cancer7 and anti-oxidant8
Address for Correspondence: Dr. Süleyman Göksu, Department of Chemistry, Faculty of Science, Atatürk University, 25240 Erzurum, Turkey.
Tel: +90 442 2314389. Fax: +90 442 2360948. E-mail: sgoksu@atauni.edu.tr . Dr. Sukru Beydemir, Department of Chemistry, Faculty of
Science, Atatürk University, 25240 Erzurum, Turkey. Tel.: +90 442 2314388. Fax: +90 442 2360948. E-mail: beydemir@atauni.edu.tr
(Received 28 May 2012; revised 20 July 2012; accepted 21 July 2012)
1073

1074 Y. Akbaba et al.
Figure 1. Some natural bromophenols.
its catalytic activity, intrestingly11–13. PON1 with 354
amino acids has three activities including paraoxonase,
arylesterase and diazoxon activities14. e enzyme is
called as paraoxonase since it was first characterized
as a hydrolyzer for OPs including paraoxon and its
physiological substrate is unknown. PON1 is an esterase
enzyme. It hydrolyses O–P ester linkage in paraoxon15.
Paraoxon is also a strong acetylcholine esterase inhibitor
and is an insecticide. e compound is formed from
mitochondrial oxidation of parathion. is process
is drived by cytochrome p450 enzyme system in the
metabolism16. Moreover, PON1 enzyme has detoxifying,
anti-atherogenic and antioxidant effects17. In addition
to paraoxon substrate, the enzyme can hydrolyze many
OPs, nerve agents like soman and sarin, insecticides such
as chlorpyrifos, oxon and diazoxon and some synthetic
esters like phenyl-acetate17. ese inhibitions on the
enzyme are crucial for especially atherosclerotic lesions.
Because, inhibition of PON1 results increasing oxidative
stress and oxidized low density lipoproteins (LDL),
which is called bad cholesterol. PON1 gain its antioxidant
property from cystein 284 in its structure12. erefore,
decreased activity of PON1 has been acknowledged as
a risk factor for cardiovascular disease and OP toxicity15.
In the present study we focused on the synthesis and
in vitro paroxonase activity of compounds 6–12.
Synthesis of (2,3-dimethoxyphenyl)
(3,4-dimethoxyphenyl)methanone (6)
Polyphosphoric acid (PPA), prepared from concentra-
tion H3PO4 (85%, 9.40 g, 81.60 mmol) and P2O5 (16.9 g,
120.04 mmol) was heated to 80°C in a beaker (100 mL).
Veratrole (4) (2.3 g, 16.47 mmol) and 2,3-dimethoxy-
benzoic acid (5) (3.00 g, 16.47 mmol) were added to
this mixture quickly. e mixture was stirred with a
glass stick at 80°C for 45 min. It was poured onto 35 mL
of ice/water carefully. e organic phase was extracted
with EtOAc (3 × 50 mL). e combined organic layers
were dried over Na2SO4 and the solvent was evaporated.
Crystallization of the residue with EtOAc-hexane gave
618 as white crystals (4.2 g, 85% yield). Mp 124–126°C.
1H-NMR (400 MHz, CDCl3), δ 7.56 (d, J = 1.8 Hz, 1H),
7.25 (dd, J = 1,8 Hz, J = 8.4 Hz, 1H), 7.09 (dd, J = 7.7 Hz,
J = 8.1 Hz, 1H), 7.01 (dd, J = 1.5 Hz, J = 8.1 Hz, 1H), 6.86
(dd, J = 1.5 Hz, J = 7.7 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H),
3.91 (s, methoxide, 3H), 3.90 (s, methoxide, 3H), 3.89
(s, methoxide,3H), 3.73 (s, methoxide, 3H). 13C-NMR
(100 MHz, CDCl3), δ 195.03 (CO), 153.78 (C), 152.94
(C), 149.2 (C), 146.82 (C), 134.74 (C), 130.67 (C), 126.44
(CH), 124.10 (CH), 120.47 (CH), 114.12 (CH), 111.08
(CH), 110.00 (CH), 61.95 (OCH3), 56.27 (OCH3), 56.20
(OCH3), 56.13 (OCH3). Anal. Calcd for C17H18O5: C 67.54,
H 6.00; found: C 67.58, H 6.09.
Synthesis of (2,3-dibromo-5,6-dimethoxyphenyl)
(3,4-dimethoxyphenyl)methanone (7)
Materials and methods
All chemicals and solvents are commercially available.
All solvents (EtOAc, CH2Cl2, hexane) were distilled and
dried according to standard procedures. Silica gel (SiO2,
60 mesh; Merck, Darmstadt, Germany) was used for col-
umn chromatography (CC). 1 mm of SiO2 60 PF (Merck)
on glass plates were used for preparative thick layer
chromatography. Mp of all compounds was determined
with cap. melting-point apparatus (BUCHI 530; Flawil,
Switzerland) and uncorrected. IR Spectra were recorded
as solns. in 0.1 mm cells with a Mattson 1000 FT-IR
A solution of bromine (0.64 g, 3.97 mmol) in CH2Cl2
(15 mL) was added to a stirred solution of benzophenone
14 (1.00 g, 3.31 mmol) in CH2Cl2 (30 mL) drop wise at
room temperature (RT) over 10min. After the reaction
mixture was stirred at RT for 24 h, the solvent was evapo-
rated. Chromatography of the residue on silica gel (SiO2,
100 g) with ethyl acetate/hexane (1:4) yielded 7 as white
crystals (0.72 g, 47%). Mp 138–140°C. 1H-NMR (400 MHz,
CDCl3), δ 7.59 (d, J = 2.2 Hz, 1H), 7.24 (s, 1H), 7.17 (dd,
A part of AB system, J = 2.2 Hz, J = 8.4 Hz, 1H), 6.92 (d,
B part of AB system, J = 8.4 Hz, 1H), 3.94 (s, methoxide,
3H), 3.91 (s, methoxide, 3H), 3.88 (s, methoxide, 3H), 3.70
(s, methoxide, 3H). 13C-NMR (100 MHz, CDCl3), δ 191.87
(CO), 154.45 (C), 152.71 (C), 149.68 (C), 146.20 (C),
137.54 (C), 129.19 (C), 126.10 (CH), 120.14 (C), 117.81
(CH), 111.79 (C), 110.62 (CH), 110.38 (CH), 61.91 (OCH3),
56.52 (OCH3), 56.30 (OCH3), 56.25 (OCH3). Anal. Calcd
for C17H16Br2O5: C 44.38, H 3.5; found: C 44.47, H 3.67. IR
(CH2Cl2, cm−1): 2938, 1666, 1585, 1513, 1464, 1419, 1300,
1268, 1143, 1003.
spectrophotometer (Cambridge, England). H- and 13C-
1
NMR spectra were recorded on 400 (100)-MHz Varian
spectrometer (Danbury, CT) in deuterated solvents
(CDCl3 and CD3COCD3) with Tetramethylsilane (TMS) as
an internal standard for protons and solvent signals, as
internal standard for carbon spectra. Chemical shift val-
ues were mentioned δ in ppm. Elemental analyses were
recorded on Leco CHNS-932 apparatus (MI). Paroxonase
activities of samples were determined on a spectropho-
tometer (UV-1208, Shimadzu, Japan).
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and paroxonase activities 1075
Bromination of compound 6 with 4 equiv bromine
A solution of bromine (2.08 g, 13.00 mmol) in CH2Cl2
(20 mL) was added to a solution of benzophenone 14
(1.00 g, 3.31 mmol) in CH2Cl2 (30 mL) drop wise at RT over
20 min. e reaction mixture was stirred at RT for 24 h,
and then the solvent was evaporated. Chromatography of
the residue on silica gel (SiO2, 100 g) column with ethyl
acetate/hexane (1:5) gave 7 (0.61 g, 40% yield), 8 (0.46 g,
26% yield) and 9 (0.36 g, 20% yield), respectively.
(2,3-Dibromo-5,6-dihydroxyphenyl)
(3,4-dihydroxyphenyl)methanone (10)
Pale yellow solid. Mp 228–230°C. 1H-NMR (400 MHz,
CD3COCD3), δ 9.36 (bs, OH, 1H), 8.80 (bs, OH, 1H), 8.45
(bs, OH, 1H), 8.26 (bs, OH, 1H), 7.35 (d, J = 1.8 Hz, 1H),
7.29 (s,1H), 7.23 (dd, A part of AB system, J = 1.8 Hz, J =
8.4 Hz, 1H), 6.92 (d, B part of AB system, J = 8.4 Hz, 1H).
13C-NMR (100 MHz, CD3COCD3), δ 191.18 (CO), 151.20
(C), 145.79 (C), 145.32 (C), 143.41 (C), 131.53 (C), 129.05
(C), 123.77 (CH), 119.52 (CH), 116.08 (CH), 115.42 (CH),
113.69 (C), 110.34 (C). Anal. Calcd for C13H8Br2O5: C 38.65,
H 2.00; found: C 38.55, H 2.09. IR (Acetone, cm−1): 3198,
2947, 1644, 1589, 1522, 1443, 1402, 1300, 1122, 953, 846.
(2-Bromo-4,5-dimethoxyphenyl)(2,3-dibromo-5,6-
dimethoxyphenyl)methanone (8)
White crystals, Mp 142–144°C. 1H-NMR (400 MHz,
CDCl3), δ 7.31 (s, 1H), 7.23 (s, 1H), 7.08 (s, 1H), 3.92 (s,
methoxide, 3H), 3.88 (s, methoxide, 3H), 3.84 (s, methox-
ide, 3H), 3.71 (s, methoxide, 3H). 13C-NMR (100 MHz,
CDCl3), δ 190.99 (CO), 153.24 (C), 152.76 (C), 148.47(C),
146.30 (C), 138.22 (C), 128.86 (C), 120.25 (C), 118.05
(CH), 117.50 (CH), 115.23 (C), 114.95 (CH), 112.13 (C)
61.72 (OCH3), 56.61 (OCH3), 56.52 (OCH3), 56.47 (OCH3).
Anal. Calcd for C17H15Br3O5: C 37.88, H 2.80; found: C
37.88, H 2.74. IR (CH2Cl2, cm-1): 2938, 2841, 1681, 1588,
1508, 1465, 1419, 1367, 1337, 1298, 1260, 1219, 1199, 1164,
1041, 1005, 925, 841.
(2-Bromo-4,5-dihydroxyphenyl)(2,3-dibromo-5,6-
dihydroxyphenyl)methanone (11)
Brownish solid. Mp 227–229°C. 1H-NMR (400 MHz,
CD3COCD3), δ 9.40 (bs, OH, 1H), 9.15 (bs, OH, 1H), 8.63
(bs, OH, 1H), 8.41 (bs, OH, 1H), 7.29 (s, 1H), 7.20 (s, 1H),
7.16 (s, 1H). 13C-NMR (100 MHz, CD3COCD3), δ 190.13
(CO), 150.44 (C), 145.92 (C), 144.43 (C), 143.67 (C),
131.72 (C), 127.62 (C), 121.97 (CH), 120.17 (CH), 119.81
(CH), 113.89 (C), 112.46 (C), 110.54 (C). Anal. Calcd for
C13H7Br3O5: C 32.33, H 1.46 found: C 32.28, H 1.50. IR
(Acetone, cm−1): 3228, 2955, 1659, 1585, 1505, 1403, 1364,
1284, 1217, 1053, 972, 853, 810, 707, 672.
(3-Bromo-4,5-dimethoxyphenyl)(2,3-dibromo-5,6-
dimethoxyphenyl)methanone (9)
White crystals, Mp 149–151°C. 1H-NMR (400 MHz,
CDCl3), δ 7.53 (d, J = 1.8 Hz, 1H), 7.35 (d, J = 1.8 Hz, 1H),
7.27 (s, 1H), 3.93 (s, methoxide, 3H), 3.92 (s, methoxide,
3H), 3.90 (s, methoxide, 3H), 3.71 (s, methoxide, 3H). 13C-
NMR (100 MHz, CDCl3), δ 191.23 (CO), 154.08 (C), 152.69
(C), 151.71(C), 146.19 (C), 136.53 (C), 132.52 (C), 128.07
(CH), 120.31 (C), 118.19 (CH), 111.75 (C), 111.62 (CH),
111.42 (C) 61.92 (OCH3), 60.98 (OCH3), 56.54 (OCH3),
56.49 (OCH3). Anal. Calcd for C17H15Br3O5: C 37.88, H
2.80; found: C 37.87, H 2.94. IR (CH2Cl2, cm−1): 2939, 1677,
1562, 1465, 1419, 1403, 1280, 1551, 1082, 1044, 1004.
(3-Bromo-4,5-dihydroxyphenyl)(2,3-dibromo-5,6-
dihydroxyphenyl)methanone (12)
Brownish solid. Mp 237–239°C. 1H-NMR (400 MHz,
CD3COCD3), δ 9.43 (bs, OH, 1H), 9.25 (bs, OH, 1H),
9.05 (bs, OH, 1H), 8.38 (bs, OH, 1H), 7.49 (d, J = 1.8 Hz,
1H), 7.33 (d, J = 1.8 Hz, 1H), 7.32 (s,1H). 13C-NMR (100
MHz, CD3COCD3), δ 190.37 (CO), 148.76 (C), 145.88
(C), 145.85 (C), 143.53 (C), 130.63 (C), 129.25 (C), 126.38
(CH), 119.86 (CH), 115.03 (CH), 113.84 (C), 110.13 (C),
109.37(C). Anal. Calcd for C13H7Br3O5: C 32.33, H 1.46;
found: C 32.32, H 1.46. IR (Acetone, cm−1): 3230, 2963,
1645, 1582, 1489, 1426, 1403, 1302, 966, 852, 727.
General procedure for the synthesis of
bromophenols 10–12
X-ray structure determination
A solution of BBr3 (0.60 mL) in CH2Cl2 (10.0 mL) was
added to a stirred solution of dibromide 7 (0.40 g, 0.87
mmol) in CH2Cl2 (15 mL) drop wise at 0°C under N2(g)
over 5–10 min. e reaction mixture was stirred at the
same temperature for 2 h and then at RT for 1 day under
N2. e reaction was monitored by TLC and after the
reaction was completed, methanol (35 mL) was slowly
added over 15 min to the mixture at 0°C. e solvent
was evaporated, water (45 mL) and EtOAc (40 mL)
were added to the residue. e organic layer was sepa-
rated and the water phase was extracted with EtOAc
(2 × 40 mL). e combined organic layers were dried
over Na2SO4 and the evaporation of the solvent gave
bromophenol 10 (0.34 g, 96%). Compounds 11 (from 8,
98% yield) and 12 (from 9, 98% yield) were also synthe-
sized by the same procedure.
For the crystal structure determination, the single-
crystal of the compounds 7, 8 and 9 was used for data
collection on a four-circle Rigaku R-AXIS RAPID-S dif-
fractometer (equipped with a 2D-area IP detector). e
graphite-monochromatized Mo Kα radiation (λ = 0.71073
Å) and oscillation scans technique with Δω = 5° for one
image were used for data collection. e lattice parameters
were determined by the least-squares methods on the
basis of all reflections with F2 > 2σ(F2). Integration of the
intensities, correction for Lorentz and polarization effects
and cell refinement was performed using CrystalClear
(Rigaku/MSC Inc., 2005) software. e structures were
solved by direct methods using SHELXS-97 and refined
by a full-matrix least-squares procedure using the pro-
gram SHELXL-97. H atoms were positioned geometrically
and refined using a riding model. Final difference Fourier
© 2013 Informa UK, Ltd.

1076 Y. Akbaba et al.
maps showed no peaks of chemical significance. Crystal
data for dibromide 7: C17H16Br2O5, crystal system, space
group: monoclinic, P21/n; (no:14); unit cell dimensions: a =
8.4968(2), b = 9.4886(2), c = 22.2020(6) Å, β = 92.02(2)˚; vol-
ume: 1788.88(8)Å3; Z = 4; calculated density: 1.708g/cm3;
absorption coefficient: 4.554/mm−1; F(000): 912; θrange for
data collection 2.3–30.6°; refinement method: full-matrix
least-square on F2; data/parameters: 4195/218; goodness-
of-fit on F2: 1.271; final R indices [I > 2σ(I)]: R1 = 0.083, wR2
= 0.189; largest diff. peak and hole: 0.458 and −0.463 e Å−3;
CCDC-877660. Crystal data for tribromide 8: C17H15Br3O5,
crystal system, space group: monoclinic, P21/c; (no:14);
unit cell dimensions: a = 9.4465(7), b = 21.0003(18), c =
9.6253(9) Å, 훼 = 90 β = 106.08(4), γ = 90°; volume: 1834.8(3)
Å3; Z = 4; calculated density: 1.951g/cm3; absorption coeffi-
cient: 6.620/mm−1; F(000): 1048; θ range for data collection
2.4–26.4°; refinement method: full-matrix least-square on
F2; data/parameters: 2604/228; goodness-of-fit on F2: 1.252;
final R indices [I > 2σ(I)]: R1 = 0.0865, wR2 = 0.197; largest
diff. peak and hole: 0.773 and −0.111 e Å−3; CCDC-878580.
Crystal data for tribromide 9: C17H15Br3O5, crystal system,
space group: monoclinic, P21/c; (no:14); unit cell dimen-
sions: a = 13.5992(5), b = 9.4949(2), c = 14.7357(7) Å, 훼 = 90
β = 104.37(2), γ = 90°; volume: 1843.23(12)Å3; Z = 4; calcu-
lated density: 1.942g/cm3; absorption coefficient: 6.590/
mm−1; F(000): 1048; θ range for data collection 2.6–30.9°;
refinement method: full-matrix least-square on F2; data/
parameters: 2668/226; goodness-of-fit on F2: 1.078; final R
indices [I > 2σ(I)]: R1 = 0.0957, wR2 = 0.108; largest differ-
ence peak and hole: 0.438 and −0.766 e Å−3; CCDC-879013.
spectrophotometrically using the same procedure in our
previous studies19. One enzyme unit was defined as the
amount of enzyme that catalyzes the hydrolysis of 1 μmol
of substrate at 25°C.
Purification of PON1
Twenty five milliliters of human serum was precipitated
with ammonium sulphate. e precipitation intervals
for paraoxonase were 60–80%20. e precipitate was col-
lected by centrifugation at 15,000 rpm for 20 min and
redissolved in 100 mM Na-phosphate buffer (pH 7.0). e
enzyme solution, which had been dialyzed in the pres-
ence of 1 mM Na-phosphate buffer (pH 7.0) at 4°C, was
loaded onto the DEAE-Sephadex A50 anion exchange
column (3 cm2 × 30 cm), which had been equilibrated
with 100 mM Na-phosphate buffer (pH 7.0). e column
procedure was applied like previous study21. Enzyme
solution from the DEAE-Sephadex A50 column were
mixed with glycerol and loaded onto the Sephadex G-200
chromatography (60 cm × 2 cm2). For each fraction quali-
tative protein assay was performed at 280 nm and activity
determination at 412 nm spectrofotometrically. Active
fractions were combined for other kinetic studies12.
Quantitative protein assay
Quantitative presence of protein in the solution was
determined at 595 nm spectrophotometrically22.
SDS polyacrylamide gel electrophoresis
e purifiy of PON1 was controlled by SDS polyacryl-
amide gel electrophoresis as in previous study23.
Paroxonase activity of compounds 6, 7, 8, 9, 10, 11, 12
Enzyme activity assay
In vitro studies of synthesized compounds
e enzyme assay was based on the estimation of p-nitro-
phenol at 412 nm. Paraoxonase activity was determined
We examined the inhibitory effects of novel synthesized
compounds 6, 7, 8, 9, 10, 11, 12. PON activities were
Scheme.1. Synthesis of novel bromophenols (i) PPA, 80°C, 85%; (ii) 1.2 eq Br2/CH2Cl2, 25°C, 47%; (iii) 4 eq Br2/CH2Cl2, 25°C; (iv) 1. BBr3/
CH2Cl2, 0–25°C, 2. MeOH or H2O, 0–25°C.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and paroxonase activities 1077
benzophenon 6 with 1.2 eq Br2 in CH2Cl2 at RT followed
by chromatographic separation of the residue gave 7 in
47% yield. e bromination of 6 with excess amount of Br2
(4 eq) in CH2Cl2 at RT was also carried out. Dibrominated
product 7, three brominated pruducts 8 and 9 were syn-
thesized in yields of 40, 26 and 20%, respectively by the
bromination of 6 with 4 eq Br2 (Scheme 1). NMR analysis
of compounds 7, 8 and 9 did not allow us to determine
their exact structures. erefore, the structures of these
compounds were determined by X-ray diffraction analy-
sis (Figure 2).
Interestingly, further analysis of the X-ray crystal
structure revealed the presence of oxygen bromine
halogen bonding. is intermolecular attraction was
observed between the bromine atom and the oxygen
atom of the carbonyl (for 9) and OMe groups (for 7 and
8). e geometrical parameters of the contacts included
an interatomic distance (Br∙∙∙O) of 3.36, 3.29 and 3.05
Å and a bond angle (C–Br∙∙∙O) of 146.7, 152 and 172.1°
for the structure 7, 8 and 9, respectively. e Br∙∙∙O dis-
tances were smaller than the sum of the van der Waals
radius (3.37 Å) for Br∙∙∙O, especially for the structure 9
suggesting the presence of halogen bonding. e halo-
gen bonding is typically observed between a polarized
halogen atom (a Lewis acid) and negatively-charged
oxygen, nitrogen or sulfur atom (a Lewis base). Oxygen-
derived Lewis bases include the carbonyl, hydroxyl,
charged carboxylate and phosphate groups. Halogen
bonding has been reported to exist in many biological
molecules, such as protein and nucleic acid structures,
and has also been demonstrated to play significant
roles in intermolecular recognition and self-assembly
processes27.
Cleavage of ethers is an integral functional group trans-
formation, as it is important for the synthesis of natural
products, pharmaceuticals and fine chemicals. Cleavage
of alkyl aryl ethers with HBr28 or BBr38,10,24 are well-known
in the literature. In the present study, O-demethylation
of aryl methyl ethers was carried out according to the
procedure described by us previously8,10,24. Reaction of
7–9 with BBr3 in CH2Cl2 at 0–25°C under N2 followed by
addition of H2O or MeOH provided novel synthetic bro-
mopehols 10, 11 and 12 with yields of 96, 98 and 98%,
respectively (Scheme 1).
PON1 were purified with three steps from human
serum by ammonium sulfate precipitation, DEAE-
Sephadex A50 ion exhange chromatography and
Sephadex G-200 gel filtration chromatography (Table 1).
e purification results for PON1 depicted a 42.1% yield,
3584 U × 1/mg−1 proteins specific activity and ~230 puri-
fication fold. However, we did not encounter any study
about paraoxonase and novel synthesized compounds
interactions. In the present study, our compounds indi-
cate inhibitory effects on PON1. All compounds inhib-
ited the enzyme activity and IC50 values for 6, 7, 8, 9, 10,
11, 12 were calculated as 1.212, 0.174, 0.123, 0.125, 0.832,
1.110, 0.701 mM, respectively (Table 2). Inhibition rank
can expressed as 8>9>7>12>10>11>6.
Figure 2. e molecular structures of the compounds 7, 8 and 9,
showing the atom numbering scheme.
measured in the presence of different compound con-
centrations. Control activity was assumed to be 100% in
the absence of inhibitor. Experiments of all compounds
were tested in triplicate at each concentration used
and Activity%-[Chemical] graphs were drawn for each
compounds12.
Results and discussion
Alkylation or acylation of aromatic compounds with
PPA (plyphosphoric acid) prepared from phosphoric
acid and P2O5 have been given in the literature clearly24.
By applying the same procedure, acylation of veratrole
(4) with 2,3-dimethoxybenzoic acid (5) in PPA at 80°C
furnished 618 with a yield of 85%. As it is known, bro-
mination of aromatic compounds can be achieved by
either N-bromosuccinimide (NBS)25, and LiBr/Ceric
ammonium nitrate8, or molecular bromine (Br2)26. Here,
Br2 was chosen as brominating agent. Bromination of
© 2013 Informa UK, Ltd.

1078 Y. Akbaba et al.
Table 1 Purification steps of PON1 enzyme from human serum by Ammonium sulfate precipitation, anion exchange chromatography and
gel filtration chromatography.
Activity Total volume Protein
Total protein Total activity Specific activity
Purification
fold
Purification steps
Serum
(U/mL)
(mL)
(mg/mL)
(mg)
163.25
75.15
(U)
(EU/mg)
Yield (%)
100,0
102
25
6.53
2550
15.62
1,0
Ammonium sulfate
precipitation (60–80%)
113.5
15
5.01
1702.5
22.65
66.7
1,45
DEAE-Sephadex A50 anion
exchange chromatography
137.7
179.2
9
6
0.18
0.05
1.62
0.3
1239.3
1075.2
765
48.6
42.1
49
Sephadex G-200 gel filtration
chromatography
3584
~229
bromophenols, isocitrate lyase inhibitors, from the red alga
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Feeding-deterrent bromophenols from Odonthalia corymbifera.
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Med Chem Lett 2008;18:104–108.
Table 2. IC50 values of chemicals on PON1.
Compound
IC50 mM
1.212
0.174
0.123
0.125
0.832
1.110
0.701
6
7
8
9
10
11
12
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conclusions
In summary, starting from veratrole (4) and
2,3-diemthoxybenzoic acid (5) three bromophenols
10–12 were synthesized for the first time, the procur-
sores of these compounds can be important synthons for
further biological and synthetic purposes. In addition,
PON1 enzyme was purified by simple chromatographic
methods, rapidly. Enzyme-chemical compound interac-
tion experiments were performed and IC50 values were
calculated from Activity%-[bromophenol] curves. All
synthesized compounds 6–12 used in this study affect
the activity of PON1 because of the presence of the differ-
ent functional groups (OMe, OH and Br) present in their
aromatic scaffold. Our findings here indicate that these
compounds and their derivatives may be used as PON1
inhibitors.
12. Alici HA, Ekinci D, Beydemir S. Intravenous anesthetics inhibit
human paraoxonase-1 (PON1) activity in vitro and in vivo. Clin
Biochem 2008;41:1384–1390.
13. Ates O, Azizi S, Alp HH, Kiziltunc A, Beydemir S, Cinici E et al.
Decreased serum paraoxonase 1 activity and increased serum
homocysteine and malondialdehyde levels in age-related macular
degeneration. Tohoku J Exp Med 2009;217:17–22.
14. Isgör MM, Beydemir S. Some cardiovascular therapeutics inhibit
paraoxonase 1 (PON1) from human serum. Eur J Pharmacol
2010;645:135–142.
acknowledgment
Authors are indebted to the Department of Chemistry
(Atatürk University) for research conditions.
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SL, La Du BN. Paraoxonase inhibits high-density lipoprotein
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16. La Du BN. (1992). Pharmo genetics of drug metabolism. New York:
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Declaration of interest
is research was financed by grants from TÜBİTAK (e
Scientific and Technological Research Council of Turkey)
(Project no: TBAG-107T348).
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to influence lipid peroxidation. Atherosclerosis 1995;115:243–253.
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on PON 1; a major bioscavenger against cardiovascular diseases.
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