Synthesis of 4-[2-(3,4-dimethoxybenzyl)cyclopentyl]-1,2-dimethoxybenzene Derivatives and Evaluations of Their Carbonic Anhydrase Isoenzymes Inhibitory Effects

Article abstract of DOI:10.1111/cbdd.12695

Rearrangement of 1,6-bis(3,4-dimethoxyphenyl)hexane-1,6-dione (8) gave two isomeric products having cyclopentene moiety. Starting from the major product (3,4-dimethoxyphenyl)[2-(3,4-dimethoxyphenyl)cyclopent-1-en-1-yl]methanone (11), eight new compounds (16-23) were obtained by the reactions such as reduction (by catalytic hydrogenation and NaBH₄), nitration, 1,4-addition, bromination, and esterification reactions. Carbonic anhydrases (CA, E.C.4.2.1.1) are ubiquitous metalloenzymes present in almost all living organism that catalyze a simple reaction, the conversion of carbon dioxide (CO₂) and water (H₂O) to bicarbonate ion (HCO₃^-) and a proton (H⁺). CA isoenzymes I and II (hCA I and II) inhibition effects of synthesized eleven new and four known compounds (8-13 and 15-23) were investigated. Inhibition studies of the hCA I and II with 4-[2-(3,4-dimethoxybenzyl)cyclopentyl]-1,2-dimethoxybenzene derivatives revealed that they possess effective inhibitory potency. Cytosolic hCA I and II isoenzymes were potently inhibited by new synthesized 4-[2-(3,4-dimethoxybenzyl)cyclopentyl]-1,2-dimethoxybenzene derivatives with K_is in the range of 313.16-1537.00 nm against hCA I and in the range of 228.31-1927.31 nm against hCA II, respectively.

Full text of DOI:10.1111/cbdd.12695

Chem Biol Drug Des 2016; 87: 594–607
Research Article
Synthesis of 4-[2-(3,4-dimethoxybenzyl)cyclopentyl]-
1,2-dimethoxybenzene Derivatives and Evaluations of
Their Carbonic Anhydrase Isoenzymes Inhibitory
Effects
1
Tekin Artuncß , Yasin Cßetinkaya1,2,*, Hulya
Received 29 July 2015, revised
accepted for publication 12 November 2015
8 October 2015 and
€
Gocßer , Ilhami Gulcßin1,4,*, Abdullah Menzek1,*,
1,3
_
€
€
Ertan Sßahin1 and Claudiu T. Supuran5
1
Aldol condensations using protic or Lewis acids as cata-
lysts and Friedel–Crafts reactions are two widely used
reactions in synthetic organic chemistry. Unsaturated
ketones such as compounds 1 and 2 are obtained as a
result of these main reactions (1–4), and they have anti-
malarial activity (4). It was reported that derivatives of com-
pounds 3 and 4 containing hydroxylated cyclopentenyl
rings showed very potent antitumor activity in a nude
mouse tumor xenograft model implanted with A549 human
lung cancer cells and highly potent antigrowth effects in a
broad range of tumor cell lines. In addition to this, it was
stated that numbers of hydroxyl groups in cyclopentane
units of them were important for the biological activity (5).
€
Department of Chemistry, Faculty of Science, Ataturk
University, Erzurum, Turkey
2Department of Food Technology, Oltu Vocational School,
€
Ataturk University, Oltu-Erzurum, Turkey
Faculty of Sciences and Letters, Agri Ibrahim Cecen
3
ˇ
ˇ
University, Agri, Turkey
4Department of Zoology, College of Science, King Saud
University, Riyadh, Saudi Arabia
5Dipartimento NEUROFARBA, Sezione di Scienze
ꢀ
Farmaceutiche, Universita degli Studi di Firenze, Via Ugo
Schiff 6, Sesto Fiorentino (Firenze), I-50019, Italy
*Corresponding authors: Abdullah Menzek,
amenzek@atauni.edu.tr; Ilhami Gulcßin,
igulcin@atauni.edu.tr; Yasin Cßetinkaya,
yasin.cetinkaya@atauni.edu.tr
_
€
Friedel–Crafts reactions are used in synthesis (6–10) of
bromophenol derivatives such as 5 and 6, and they have
interesting biological activities such as feeding deterrent,
antioxidant, antimicrobial, and cytotoxicity effects (8–17).
Recently, the carbonic anhydrase (CA, EC 4.2.1.1) inhibi-
tory effects of such compounds have been reported (18).
Most of these bromophenols are natural compounds
mostly isolated from red algae (Figure 1).
Rearrangement of 1,6-bis(3,4-dimethoxyphenyl)hexane-
1,6-dione (8) gave two isomeric products having
cyclopentene moiety. Starting from the major product
(3,4-dimethoxyphenyl)[2-(3,4-dimethoxyphenyl)cyclo-
pent-1-en-1-yl]methanone (11), eight new compounds
(16–23) were obtained by the reactions such as reduction
(by catalytic hydrogenation and NaBH4), nitration,
1,4-addition, bromination, and esterification reactions.
Carbonic anhydrases (CA, E.C.4.2.1.1) are ubiquitous
metalloenzymes present in almost all living organism that
catalyze a simple reaction, the conversion of carbon diox-
ide (CO2) and water (H2O) to bicarbonate ion (HCO3ꢀ) and
a proton (H+). CA isoenzymes I and II (hCA I and II) inhibi-
tion effects of synthesized eleven new and four known
compounds (8–13 and 15–23) were investigated. Inhibi-
tion studies of the hCA I and II with 4-[2-(3,4-dimeth-
oxybenzyl)cyclopentyl]-1,2-dimethoxybenzene derivatives
revealed that they possess effective inhibitory potency.
Cytosolic hCA I and II isoenzymes were potently inhibited
by new synthesized 4-[2-(3,4-dimethoxybenzyl)cyclopen-
tyl]-1,2-dimethoxybenzene derivatives with Kis in the
range of 313.16–1537.00 nM against hCA I and in the
range of 228.31–1927.31 nM against hCA II, respectively.
The CAs are zinc metalloenzymes that catalyze a simple
chemical reaction, the reversible interconversion between
carbon dioxide (CO2) and bicarbonate (HCO3ꢀ), which
ꢀ
generates a proton (H+) and HCO3 for the hydration reac-
tion or consumes one equivalent of H+ for the dehydration
reaction (19–21). This makes these isoenzymes crucial for
many physiological processes including electrolyte secre-
tion, pH and CO2 homeostasis, respiration, bone calcifica-
tion,
ureagenesis,
gluconeogeneꢀsis,
tumorigenicity,
lipogenesis, transport of CO2/HCO3 between metaboliz-
ing tissues and the lungs, and some other pathologic or
physiologic processes (22–28). This enzyme class is pre-
sent either in eukaryote or in prokaryote cells. There are
six main genetic families encoding classes of these
enzymes: a-, b-, c-, d-, f-, and g-CAs. It was reported
that a-CAs are normally monomers and rarely dimers;
b-CAs are dimers, tetramers, or octamers; c-CAs are
Key words: Aldol condensation, bromination, carbonic anhy-
drase, cyclopentenyl, enzyme inhibition, Friedel–Crafts reac-
tion
594
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12695

CA Inhibition Profile of Some Dimethoxybenzene Derivatives
R
H
O
H
O
H
O
r
B
NH2
H
O
H
O
O
N
N
O
N
r
B
r
B
X
r
B
N
H
O
r
B
H
O
H
O
r
B
H
O
H
O
H
O
R
H
O
=
1
2
3
4
5
6
R
R
H
X = H
X = F
M
=O e
Figure 1: Structures of some biological active compounds.
trimers, whereas the d- and f-CAs are less well under-
stood at this moment (29). a-CAs are found in vertebrates,
bacteria, algae, and cytoplasm of green plants. b-CAs are
present in bacteria, algae, and chloroplasts of monocotyle-
dons and dicotyledons. On the other hand, the d-CAs
exist in diatoms and other marine eukaryotes and f-CAs
are found in diatoms (30). Human CAs (hCAs) all belongs
to the a-family and so far sixteen different CA isoforms dis-
covered in this class (29,31). In humans, CAs are dispersed
in different tissues including the reproductive tract, the gas-
trointestinal tract, kidneys, the nervous system, skin, eyes,
lungs, and among some others (32–34). CA isoenzymes
contain a zinc ion (Zn2+) in their active site, co-ordinated by
three His residues and a H2O molecule/hydroxide ion (-OH)
in the a- and c-CAs or by two Cys and one His residues (in
the b class), with the fourth ligand being a H2O molecule/-
OH ion acting as nucleophile in the catalyzed reactions (35–
41). All human CAs (hCAs) belong to the a-CAs; up to now,
sixteen isozymes have been recognized, among these only
twelve are catalytically active (42). Both human cytosolic CA
isoforms (hCA I and II) are spread throughout the human
body and are drug targets for clinically used diuretics, anti-
convulsants, and antiglaucoma drugs (17,43). Five of them
are cytosolic (CA I, II, III, VII, and XIII), some ones (CA IV, IX,
XII, and XIV) are membrane bound, two isoenzymes (CA VA
and VB) are mitochondrial, and one isoform (CA VI) is
secreted in saliva (43–45). It was recently reported that CA
XV isoform is not expressed in humans or in living primates.
However, it is plentiful in rodents and other higher verte-
brates. Also, three catalytic forms are also known and called
CA-related proteins (CARP) (46–48). All isoenzymes contain
human diseases including glaucoma, cancer, osteoporo-
sis, neurological disorders. Recently, CA isozymes have
become an interesting target for the design of inhibitors or
activators with biomedical applications (21,52). CA inhibi-
tors (CAIs) are well-demonstrated drugs as diuretics, anti-
epileptics, and antiglaucoma, while the novel generation
compounds are undergoing clinical investigation as anti-
obesity, diagnostic tools, or antitumor drugs (22,53–55).
Also, it was reported that to obtain new drugs with less
undesired side-effects mostly produced by the first gener-
ation of inhibitors and for physiological studies in which
specific or selective inhibitors may constitute valuable tools
for understanding the physiology or physiopathology of
hCA isoforms (26,42,56–60). Because of these reasons,
new class of inhibitors containing already known effective
moiety as CAs in their structure have been designed and
synthesized.
Recently, many benzene derivative studies including ureido-
substituted benzene sulfonamide (61), benzene sulfon-
amides incorporating 4- and 3-nitrophthalimide moieties
(62), substituted benzene sulfonamides incorporating 1,3,5-
triazinyl moieties (54), N-substituted N0-(2-arylmethylthio-4-
chloro-5-methylbenzenesulfonyl)guanidines (63), phenyl tail
of benzene sulfonamides (64), aminobenzenesulfonamide–
thiourea conjugates (65), novel benzylamine derivatives (60),
and 4-functionalized 1,3-diarylpyrazoles bearing benzene-
sulfonamide (66) were performed as CAIs. In this study,
we synthesized 4-[2-(3,4-dimethoxybenzyl)cyclopentyl]-1,2-
dimethoxybenzene derivatives and evaluated their inhibition
effects of CA isoenzymes I and II (hCA I and II).
a zinc ion (Zn2+) located at the base of a 15 A deep funnel-
̊
shaped active site cavity that is co-ordinated to the imida-
zole groups of three His residues and to the substrate H2O/
hydroxide (-OH) that reacts with CO2 (26,49–51).
Experimental
Materials
Due to the important roles of CA isoenzymes in a variety
of physiological and pathological processes and the asso-
ciation of abnormal levels or activities of CAs to different
All chemicals, solvents, and silica gel (for column chro-
matography and thick-layer chromatography) were per-
formed as described previously (8,67).
Chem Biol Drug Des 2016; 87: 594–607
595

Artuncß et al.
Experiment
(100 MHz – CDCl3) d 200.58 (CO), 153.56 (C), 149.40 (C),
148.96 (C), 148.55 (C), 141.87 (C), 130.05 (C), 129.03 (C),
128.46 (CH), 123.30 (CH), 118.53 (CH), 111.17 (CH),
111.01 (CH), 110.25 (CH), 109.29 (CH), 56.31 (OCH3),
56.16 (OCH3), 56.03 (OCH3), 55.88 (OCH3), 53.36 (CH),
Values as well as measurements of Mp of all compounds,
IR Spectra, 1H and 13C NMR spectra, chemical shift val-
ues, elemental analyses, and CA inhibitory properties of
samples were performed as described previously (8,67).
PLC is preparative thick-layer chromatography: 1 mm of
silica gel 60 PF (Merck, Darmstadt, Germany) on glass
plates. HRMS were recorded by LC-MS TOF electrospray
ionization technique (1200/6210, Agilent).
32.67 (CH2), 30.56 (CH2); HRMS: m/z (M
+ H) calc. for
C22H24O5 369.16237, found 369.16471.
Synthesis of (3,4-dimethoxyphenyl)[2-(3,4-
dimethoxyphenyl)cyclopent-2-en-1-yl]methanol
(13): Standard procedure for reduction of ketone
with NaBH4
Synthesis
Reaction of diketone 8 in the mixture of HCl/HOAc
To a solution of diketone 8 (0.66 g, 1.71 mmol) in HOAc
(25 mL) was added HCl (37%, 12 mL) at room tempera-
ture and the mixture was stirred for 1 h at room tempera-
ture. To the reaction were slowly added water (15 mL),
NaHCO3 (conc., 20 mL), and CH2Cl2 (50 mL), respec-
tively. The two layers were separated, and the aqueous
layer was extracted with CH2Cl2. (2 9 50 mL). The com-
bined organic layers were washed with water (50 mL),
dried over Na2SO4. After the solvent was evaporated, resi-
due was submitted to column chromatography (silica gel,
40 g) eluting with ethyl acetate (EtOAc)/hexane (1:6) rear-
rangement products 11 (472 mg, 75%) and 12 (101 mg,
16%) that were obtained from the chromatography,
respectively.
To a solution of ketone 12 (0.1 g, 0.27 mmol) in mixture of
methanol (20 mL) and tetrahydrofuran (THF) (5 mL) at
room temperature was added NaBH4 (538 mg, 14 mmol),
and the mixture was stirred for 3 days. After the reaction
was controlled by TLC and observed to be comple-
mented, the solvents were evaporated. Water (5 mL) and
HCl (10 mL, 0.5%) were added, and the mixture was
extracted with EtOAc (3 9 50 mL). Combined organic lay-
ers were washed with water (2 9 20 mL) and dried over
Na2SO4. After the solvent was evaporated, residue was
submitted to column chromatography (silica gel, 50 g)
eluting with EtOAc/hexane (3:7). Alcohol 13 (82 mg, % 82)
was obtained from the chromatography as brown liquid.
1H NMR (400 MHz – CDCl3) d 7.01 (dd, A part of AB sys-
tem, J = 8.29, 1.72 Hz, aromatic, 1H), 6.96 (d,
J = 1.72 Hz, aromatic, 1H), 6.81 (d, B part of AB system,
(3,4-Dimethoxyphenyl)[2-(3,4-dimethoxyphenyl)cyclo-
pent-1-en-1-yl]methanone (11). M.p 103–105 °C; 1H
NMR (400 MHz – CDCl3) d 7.45–7.41 (m, aromatic, 2H),
6.82 (dd, A part of AB system, J = 8.2, 2.0 Hz, 1H), 6.71
(d, B part of AB system, J = 8.2 Hz, 1H), 6.69–6.66 (m,
aromatic, 2H), 3.87 (s, OCH3, 3H), 3.83 (s, OCH3, 3H),
3.80 (s, OCH3, 3H), 3.61 (s, OCH3, 3H), 3.03–2.98 (m,
CH2, 2H), 2.95–2.88 (m, CH2, 2H), 2.17–2.08 (m,
CH2, 2H); 13C NMR (100 MHz – CDCl3) d 197.89 (CO),
153.51 (C), 149.08 (C), 148.76 (C), 148.34 (C), 143.81 (C),
136.39 (C), 129.69 (C), 129.09 (C), 124.98 (CH), 120.45
(CH), 111.71 (CH), 111.07 (CH), 110.73 (CH), 110.20
(CH), 56.18 (OCH3), 56.13 (OCH3), 55.96 (OCH3), 55.74
(OCH3), 38.35 (CH2), 37.66 (CH2), 22.81 (CH2); HRMS:
m/z (M + H) calc. for C22H24O5 369.16237, found 369.
1654.
J = 8.29 Hz, 1H), 6.75 (dd, A part of AB system,
J = 8.27, 1.44 Hz, aromatic, 1H), 6.70 (d, B part of AB
system, J = 8.27 Hz, aromatic, 1H), 6.59 (d, J = 1.44 Hz,
aromatic, 1H), 5.92–5.88 (m, olefinic, 1H), 4.86 (d, benzylic
CH, J = 4.51 Hz, 1H), 3.85 (s, OCH3, 6H), 3.81 (s, OCH3,
3H), 3.72 (s, OCH3, 3H), 3.66–3.59 (m, methylenic, 1H),
2.18–1.92 (m, methylenic, 4H), 1.73–1.62 (m, methylenic,
1H); 13C NMR (100 MHz – CDCl3) d 141.14 (C), 148.55
(C), 148.20 (C), 148.11 (C), 142.75 (C), 134.65 (C), 129.90
(C), 128.97 (CH), 119.03 (CH), 118.74 (CH), 111.35
(CH),110.44 (CH), 110.20 (CH), 109.63 (CH), 74.96 (CH),
56.10 (CH), 56.05 (OCH3), 56.01 (OCH3), 55.86 (OCH3),
52.49 (OCH3), 31.87 (CH2), 25.65 (CH2); HRMS: m/z
(M + Na) calcd. for C22H26O5: 393.1683; found:
393.1673.
(3,4-Dimethoxyphenyl)[2-(3,4-dimethoxyphenyl)cyclo-
pent-2-en-1-yl]methanone (12). M.p 142–144 °C; 1H
NMR (400 MHz – CDCl3) d 7.74 (dd, A part of AB system,
J = 8.4, 1.9 Hz, aromatic, 1H), 7.59 (d, J = 1.9 Hz, aro-
matic, 1H), 6.94 (d, J = 1.91 Hz, aromatic, 1 H), 6.93 (d,
A part of AB system part of AB system, J = 8.4 Hz, aro-
matic, 1H), (dd, B part of AB system, J = 8.4, 1.91 Hz,
aromatic, 1H), 6.69 (d, B part of AB system, J = 8.4 Hz,
1H), 6.33 (bs, olefin, 1H), 4.94–4.88 (m, aliphatic CH, 1H),
3.95 (s, OCH3, 3H), 3.91 (s, OCH3, 3H), 3.80 (s, OCH3,
3H), 3.76 (s, OCH3, 3H), 2.72–2.56 (m, CH2, 2H), 2.56–
2.43 (m, CH2, 2H), 2.15–2.07 (m, CH2, 2H); 13C NMR
Catalytic hydrogenation of rearrangement product
11
To a solution of compound 11 (1.0 g 2.72 mmol) in EtOAc
(100 mL) at RT was added catalytic amount of Pd/C. The
septum was used as a stopper, and the air inside of the
flask was displaced by hydrogen gas. The reaction was
realized in atmosphere (approximately 1.0 atm) of gas
hydrogen by a balloon filled with hydrogen gas for 20 min.
The reaction was followed by TLC and stopped after
20 min. The reaction mixture was filtered by a filter paper
and the solvent was evaporated. According to 1H NMR of
the mixture, compound 16 was major product and it was
596
Chem Biol Drug Des 2016; 87: 594–607

CA Inhibition Profile of Some Dimethoxybenzene Derivatives
obtained from crystallization of the mixture from hexane/
EtOAc as white crystals (350 mg, 35%).
(OCH3), 48.62 (CH), 46.37 (CH), 30.79 (CH2), 30.62 (CH2),
23.49 (CH2); HRMS: m/z (M
+ Na) calcd. for C22H28O4:
379.1887; found: 379.1880.
The reaction was performed according to above (described
in Synthesis of (3,4-dimethoxyphenyl)[2-(3,4-dimethoxyphe-
nyl)cyclopent-2-en-1-yl]methanol (13): Standard procedure
for reduction of ketone with NaBH4). In the reaction,
Synthesis of (4,5-dimethoxy-2-nitrophenyl)[2-(4,5-
dimethoxy-2-nitrophenyl)cyclopent-1-en-1-yl]
methanone (18): Standard procedure for nitration
To a Ac2O (10 mL), was added HNO3 (1.7 mL, 64%),
dropwise at 0 °C for 5 min. To the mixture was added a
solution of the compound 11 (100 mg, 0.27 mmol) in
CH2Cl2 (5 mL) dropwise at 0 °C for 10 min and then the
mixture was stirred for 3 days without cooling the bath
again. The reaction mixture was quenched by the addition
of a saturated NaHCO3 aqueous solution (20 mL) at 0 °C.
The two layers were separated and the aqueous layer was
extracted with CH2Cl2 (3 9 50 mL). The combined organic
layers were washed with water (50 mL), dried over
Na2SO4. After the solvent was evaporated, crystallization
of residue from methanol gave product 18 (0.95 g, %
76.35) as yellow crystals. M.p: 213–215 °C; 1H NMR
(400 MHz – CDCl3) 7.26 (s, aromatic, 1H), 7.19 (s, aro-
matic, 1H), 6.41 (s, aromatic, 1H), 6.38 (s, aromatic, 1H),
3.89 (s, OCH3, 3H), 3.87 (s, OCH3, 3H), 3.83 (s, OCH3,
6H), 3.02 (t, J = 7.5 Hz, methylenic, 4H), 2.14 (m, methy-
lenic, CH2, 2H); 13C NMR (100 MHz – CDCl3) 198.58
(CO), 154.04 (C), 154.00 (C), 153.38 (C), 148.91 (C),
148.10 (C), 138.75 (C), 138.74 (C), 132.36 (C), 128.47 (C),
110.26 (C), 110.21 (CH), 108.56 (CH), 107.12 (CH),
106.21 (CH), 56.73 (OCH3), 56.71 (OCH3), 56.63 (OCH3),
56.57 (OCH3), 42.30 (CH2), 33.91 (CH2), 22.12 (CH2);
HRMS: m/z (M + Na) calcd. for C22H22N2O9: 481.1235;
found: 481.1218.
ꢁ
The product 11 (500 mg, 1.36 mmol) and EtOAc
(30 mL) were used in the reaction. The reaction lasted for
15 min. Crystallization of residue gave ketone 16 (450 mg,
90%).
ꢁ
The product 11 (2.0 g, 5.43 mmol) and EtOAc (45 mL)
were used in the reaction. The reaction lasted for 1.5 h.
According to 1H-NMR of the mixture, alkane 17 was lone
product and likewise its crystallization gave the crystals
(1.82 g, 5.11 mmol, 94%).
ꢁ
The product 11 (1.0 g, 2.72 mmol) and EtOAc (30 mL)
were used in the reaction. The reaction lasted for 45 min.
According to 1H NMR of the mixture, ratio of 16–17 was
5:2.
(3,4-Dimethoxyphenyl)[2-(3,4-dimethoxyphenyl)cyclo-
pentyl]methanone (16). M.p 73–76 °C; 1H NMR
(400 MHz – CDCl3) d 7.37 (dd, A part of AB system,
J = 8.4, 1.92 Hz, aromatic, 1H), 7.15 (d, B part of AB sys-
tem, J = 1.9 Hz, aromatic, 1H), 6.74 (d, J = 8.4 Hz, aro-
matic, 1H), 6.57 (s, aromatic, 2H), 6.46 (s, aromatic, 1H),
4.11–4.05 (m, OCCH, aliphatic, 1H), 3.91 (s, OCH3, 3H),
3.83 (s, OCH3, 3H), 3.76 (s, OCH3, 3H), 3.67 (s, OCH3,
3H), 3.55–3.45 (m, OCCH, aliphatic, 1H), 2.33–2.24 (m,
aliphatic, 1H), 2.20–2.03 (m, aliphatic, 3H), 2.00–1.90 (m,
aliphatic, 1H), 1.83–1.70 (m, aliphatic, 1H); 13C NMR
(100 MHz – CDCl3) d 201.42 (CO), 152.76 (C), 148.79 (C),
148.26 (C),147.39 (C), 134.81 (C), 131.82 (C), 122.74
(CH), 120.38 (CH), 111.04 (CH), 110.76(CH), 110.52 (CH),
109.78 (CH), 56.19 (OCH3), 56.06 (OCH3), 55.94 (OCH3),
55.77 (OCH3), 51.26 (CH), 50.25 (CH), 33.32 (CH2), 28.99
(CH2), 24.85 (CH2); HRMS: m/z (M + Na) calcd. for
C22H26O5: 393.1684; found: 393.1673.
Synthesis of [2-(4,5-dimethoxy-2-nitrophenyl)
cyclopent-1-en-1-yl](3,4-dimethoxyphenyl)
methanone (19)
This reaction was performed according to the standard
procedure described in Synthesis of (4,5-dimethoxy-2-
nitrophenyl)[2-(4,5-dimethoxy-2-nitrophenyl)cyclopent-1-
en-1-yl]methanone (18): Standard procedure for nitration.
In the reaction, Ac2O (8.0 mL), HNO3 (100 mg, 64%), and
compound 11 (135 mg, 3.7 mmol) were used. The reac-
tion lasted for 100 min. The residue was subject to
preparative thick-layer chromatography (PLC) using
EtOAc/hexane (1:4). Compound 19 (30 mg, 20%), the
fastest walking stain, was obtained as brown liquid. 1H
NMR (400 MHz – CDCl3) 7.51 (s, aromatic, 1H), 7.48 (dd,
A part of AB system, J = 8.41, 1.92 Hz, 1H), 7.30 (d,
J = 1.92 Hz, aromatic, 1H), 6.67 (d, B part of AB system,
J = 8.41 Hz, aromatic, 1H), 6.52 (s, aromatic, 1H), 3.85
(s, OCH3, 3H), 3.84 (s, OCH3, 3H), 3.81 (s, OCH3, 6H),
2.96–2.92 (m, methylenic, 2H), 2.73 (bt, J = 7.40, Hz,
methylenic, 2H), 2.17–2.15 (m, methylenic, 2H13); 13C
4-[2-(3,4-Dimethoxybenzyl)cyclopentyl]-1,2-dimethoxy-
benzene (17). M.p 65–67 °C; 1H NMR (400 MHz –
CDCl3) d 6.82 (d, A part of AB system, J = 8.18 Hz, aro-
matic, 1H), 6.76–6.79 (m, aromatic, 3H), 6.58 (dd, A part
of AB system, J = 8.15, 1.53 Hz aromatic, 1H), 3.88 (s,
OCH3, 3H), 3.86 (s, OCH3, 3H), 3.83 (s, OCH3, 3H), 3.82
(s, OCH3, 3H), 3.24–3.18 (m, aliphatic, 1H), 2.34 (m, ali-
phatic, 1H), 2.45–2.33 (m, aliphatic, 2H), 2.13–1.85 (m, ali-
phatic, 5H), 1.75–1.63 (m, aliphatic, 1H); 13C NMR
(100 MHz – CDCl3) d 148.80 (C), 148.64 (C),147.39 (C),
147.14 (C), 136.45 (C), 135.00 (C), 120.76 (CH), 120.50
(CH), 112.47 (CH), 112.36 (CH), 111.16 (CH), 111.04
(CH), 56.11 (OCH3), 56.09 (OCH3), 56.06 (OCH3), 56.00
NMR (100 MHz
– CDCl3) 196.27 (CO), 153.36 (C),
150.153.09 (C), 148.98 (C), 148.24 (C), 145.63 (C),
139.93 (C), 130.70 (C), 128.29 (C), 124.32 (C), 113.38 (C),
Chem Biol Drug Des 2016; 87: 594–607
597

Artuncß et al.
110.66 (C), 110.15 (C), 107.86 (C), 56.71 (OCH3), 56.48
(OCH3), 56.22 (OCH3), 56.07 (OCH3), 38.63 (C11), 36.58
(C), 23.61 (C); Elemental analysis calculated (%) for
C22H23O7: C 63.91, H 5.61, N 3.39 Found C 63.75, H
5.73, N 3.32.
(CH), 117.57 (CH), 111.04 (CH), 110.62 (CH), 109.03
(CH), 108.95 (CH), 78.93 (C-N), 61.35 (C), 55.97 (2
OCH3),55.92 (OCH3), 55.87 (OCH3), 55.80 (CH), 37.12
(CH2), 33.64 (CH2), 25.94 (CH2), 23.19 (CH3); HRMS: m/z
(M + H) calcd. for C24H28N2O5: 425.2080; found:
425.2071.
Reaction of compound 17 with Br2
To
a
solution of compound 17 (100 mg) in CH2Cl2
Reduction of ketone 16 with NaBH4
(20 mL), excess Br2 (2 mL) was added dropwise at RT for
5 min. After stirring for 30 min, the solvent was evaporated
and crystallization of residue from hexane/EtOAc gave
1,2,4-tribromo-3-[(2-(2,3-dibromo-3,4-dimethoxyphenyl)
cyclopentyl)methyl]-5,6-dimethoxybenzene (20) as the sole
product (60 mg, 29%). M.p 154–156 °C; 1H NMR
(400 MHz – CDCl3) d 6.90 (s, aromatic, 1H), 3.90 (s,
OCH3, 3H), 3.86 (s, OCH3, 3H)), 3.85 (s, OCH3, 3H), 3.85
(s, OCH3, 3H), 3.74 (dd, J = 9.9, 5.7 Hz, 1H), 3.03–2.93
(m, aliphatic, 1H), 2.79 (dd, J = 12.4, 8.8 Hz, aliphatic,
1H), 2.53 (dd, B part of AB system, J = 13.3, 3.8 Hz, ali-
phatic, 1H), 2.10–1.95 (m, aliphatic, 3H), 1.76–1.58 (m, ali-
phatic, 3H); 13C NMR (100 MHz – CDCl3) d 152.18 (C),
150.33 (C), 146.30 (C), 139.63 (C), 138.67 (C), 122.88 (C),
122.21 (C),121.77 (C), 119.43 (C), 112.43 (C), 112.40 (C),
112.30 (CH), 61.04 (OCH3), 60.95 (OCH3), 60.82 (OCH3),
56.57 (OCH3), 50.35 (CH), 40.88 (CH), 38.81 (CH2), 30.31
(CH2), 29.10 (CH2), 23.48 (CH2). Elemental analysis calcu-
lated (%) for C22H23Br5O4: C 35.19, H 3.09. Found:
35.10, H 3.11.
This reaction was performed according to the standard
procedure described in Synthesis of (3,4-dimethoxyphenyl)
[2-(3,4-dimethoxyphenyl)cyclopent-2-en-1-yl]methanol (13):
Standard procedure for reduction of ketone with NaBH4.
In the reaction, ketone 16 (150 mg, 0.42 mmol) and
NaBH4 (155 mg, 4.1 mmol) was used. The reaction lasted
for 5 days. Crystallization of residue from EtOAc/hexane
gave (3,4-dimethoxyphenyl)[2-(3,4-dimethoxyphenyl)cyclo-
pentyl]methanol (22) (90 mg, 60%) as white crystals. M.p
102–104 °C; 1H NMR (400 MHz – CDCl3) d 6.78 (d, A
part of AB system, J = 8.2 Hz, aromatic, 1H), 6.73 (bs,
aromatic, 1H), 6.64 (dd, B part of AB system, J = 8.2,
2.0 Hz, aromatic, 1H), 6.61–6.59 (m, aromatic, 1H), 6.44
(bs, aromatic, 1H), 3.94 (d, J = 3.8 Hz, 1H), 3.89 (s,
OCH3, 3H), 3.85 (s, OCH3, 3H), 3.80 (s, OCH3, 3H), 3.75
(s, OCH3, 3H), 3.72 (dd, J = 8.3, 2.8 Hz, 1H), 2.78 (ddd,
J = 4.0, 8.1, 12.2 Hz, 1H), 2.05–1.81 (m, 2H), 1.78–1.67
(m, 1H), 1.67–1.47 (m, 3H); 13C NMR (100 MHz – CDCl3)
d 148.97 (C),148.82 (C), 148.39 (C), 147.31 (C), 140.12
(C), 139.64 (CH), 137.61 (C), 119.43 (CH), 111.09 (CH),
110.74 (CH), 107.60 (CH), 106.82 (CH), 58.76 (OCH3),
55.93 (OCH3), 55.91 (OCH3), 55.88 (OCH3), 55.83 (CH),
54.88 (CH), 49.28 (CH2), 34.30 (CH2), 33.88 (CH2), 25.84
(CH2). Elemental analysis calculated (%) for C22H23O5: C
70.94, H 7.58. Found: 69.27, H 7.66.
Reaction of compound 11 with hydrazine hydrate
To a solution of compound 11 (0.5 g, 1.36 mmol) in HOAc
(15 mL) was hydrazine hydrate (204 mg, 3.92 mmol) was
added. The mixture was refluxed for 5 days, and then, it
was poured into mixture of ice-water (250 g). A solution
(concerted) of NH3 was used for neutralization of the mix-
ture and it was controlled with pH paper. One day later,
precipitation appeared in the mixture, and then it was col-
lected with a filter paper. It was allowed to dry completely.
The mixture (535 mg) was submitted to column chro-
matography (silica gel, 50 g) eluting hexane/EtOAc (9:1).
Products 11 (275 mg) and 21 (260 mg, 45%) were
obtained from column chromatography, respectively.
Synthesis of (3,4-dimethoxyphenyl)[2-(3,4-
dimethoxyphenyl)cyclopentyl]methyl 4-
nitrobenzoate (23)
A solution of alcohol 22 (0.4 g, 1.1 mmol) in CH2Cl2
(10 mL) was cooled at 30 °C by ice-salt bath. To the solu-
tion was added pyridine (0.8 g, 0.01 mol) and p-nitro ben-
zoyl chloride (0.44 g, 2.4 mmol) in CH2Cl2 (15 mL)
dropwise at same temperature for 10 min, respectively.
The mixture was stirred for 1 day for without cooling the
bath again, cooled to 0 °C and poured into a cold mixture
(1.0%, 250 mL) of HCl. The two layers were separated
and the aqueous layer was extracted with CH2Cl2.
(3 9 40 mL). The combined organic layers were washed
with water (50 mL), dried over Na2SO4. After the solvent
was evaporated, crystallization of residue from EtOAc/hex-
ane gave ester 23 (0.48 g, 91%) as yellow crystals. M.p:
149–152 °C; 1H NMR (400 MHz – CDCl3) 8.23 (d, A part
of AB system, J = 8.9 Hz, aromatic, 2H), 8.05 (d, B
part of AB system, J = 8.9 Hz, aromatic, 2H), 6.92 (d, A
part of AB system, J = 8.3 Hz, aromatic, 2H), 6.84
1-[3,6a-Bis(3,4-dimethoxyphenyl)-4,5,6,6a-tetrahydro-
cyclopenta[c]pyrazol-1(3aH)-yl]ethanone (21). M.p
(pale yellow solid) 150–152 °C; 1H NMR (400 MHz –
CDCl3) d 7.44 (d, J = 1.9 Hz, aromatic, 1H), 7.15 (dd,
J = 8.3, 2.0 Hz, aromatic, 1H), 6.88–6.78 (m, aromatic,
4H), 3.95 (s, OCH3, 3H), 3.92 (s, OCH3, 3H), 3.84 (s,
OCH3, 3H), 3.84 (s, OCH3, 3H), 3.79 (dd, J = 9.5, 3.2 Hz,
aliphatic, 1H), 3.00–2.92 (m, aliphatic, 1H), 2.53 (ddd,
J = 13.6, 10.8, 5.9 Hz, aliphatic, 1H), 2.41 (s, CH3, 3H),
2.23–2.13 (m, aliphatic, 1H), 1.98–1.86 (m, aliphatic, 1H),
1.1.73–1.63 (m, aliphatic, 1H); 13C NMR (100 MHz –
CDCl3) d 168.26 (CO), 157.77 (C=N), 150.81 (C), 149.18
(C), 148.83 (C), 148.11 (C), 135.75 (C), 123.82 (C), 120.61
(bs, aromatic, 1H), 6.78 (d,
B part of AB system,
J = 8.3 Hz, aromatic, 2H), 6.67 (d, A part of AB system,
598
Chem Biol Drug Des 2016; 87: 594–607

CA Inhibition Profile of Some Dimethoxybenzene Derivatives
J = 8.2 Hz, aromatic, 2H), 6.62 (d, B part of AB system,
J = 8.2 Hz, aromatic, 2H), 5.29 (bs, aromatic, 1H), 3.85
(s, OCH3, 3H), 3.82 (s, OCH3, 3H), 3.71 (s, OCH3, 3H),
3.56 (s, OCH3, 3H), 3.48–3.41 (m, methylenic, 1H), 2.91–
2.81 (m, methylenic, 1H), 2.26–2.16 (m, methylenic, 2H),
2.04–1.86 (m, methylenic, 2H), 1.77–1.64 (m, methylenic,
2H); 13C NMR (100 MHz – CDCl3) 163.28 (CO), 150.53
(C), 149.09 (C), 149.03 (C), 148.6 (C), 147.54 (C), 136.13
(C), 135.77 (C), 132.85 (C), 130.64 (CH), 123.67 (CH),
120.45 (CH), 119.9 (CH), 112.21 (CH), 111.23 (CH),
111.11 (CH), 110.52 (CH), 79.23 (OCH3), 56.18 (OCH3),
56.05 (OCH3), 55.88 (OCH3), 55.62, 50.01, 47.31, 32.92,
28.88, 24.05; HRMS: m/z (M + Na) calcd. for C29H31NO8:
544.1956; found: 544.1942.
(equipped with a two-dimensional area IP detector). Gra-
ꢀ
phite-monochromated Mo-Ka radiation (k = 0.71073 A)
and oscillation scans technique with Dw = 5ꢀ for one
image were used for data collection. The lattice parame-
ters were determined by the least-squares methods on the
basis of all reflections with F2 > 2r(F2). Integration of the
intensities, correction for Lorentz and polarization effects,
and cell refinement was performed using CRYSTALCLEAR soft-
ware (Rigaku/MSC Inc.a, 2005). The structures were
solved by direct methods using SHELXS-97b and refined by
a full-matrix least-squares procedure using the program
SHELXL-97b. All non-hydrogen atoms were refined
anisotropically, and H atoms were positioned geometrically
and refined using a riding model. The final difference Four-
ier maps showed no peaks of chemical significance. Crys-
tal data for 16: C22H26O5, crystal system, space group:
monoclinic, P21/c; (no:14); unit cell dimensions:
Biological activity
ꢀ
ꢀ3
The purification of cytosolic CA isoenzymes (CA I and II)
was previously described (48–50) with a simple one-step
method by a Sepharose-4B-L tyrosine-sulfanilamide affinity
chromatography (52). The quantity of protein in the column
effluents was determined spectrophotometrically at 280 nM
previously reported (68–71). Sodium dodecyl sulfate–poly-
acrylamide gel electrophoresis (SDS-PAGE) describes a
technique widely used in biochemistry and pharmacology
for separation of biological macromolecule such as pro-
teins and nucleic acids, according to their electrophoretic
mobility (68,72). SDS-PAGE was applied with a Bio-Rad
Mini Gel system after purification of both CA isoenzymes.
This protein separation method was described previously
(73). Briefly, it was performed in acrylamide for the running
(10%) and the stacking gel (3%) contained SDS (0.1%),
respectively (25,74,75).
a = 5.5112 (4), b = 19.0654(13), c = 18.2400(12) A,
a = 90, b = 91.430(3), c = 90ꢀ; volume: 1915.9 (2) A ;
Z = 4; calculated density: 1.284 g/cm3; absorption coeffi-
cient: 0.090 mmꢀ1; F(000): 792; h-range for data collec-
tion 1.5–24.9ꢀ; refinement method: full-matrix least-square
on F2; data/parameters: 2471/244; goodness of fit on F2:
1.047; final R-indices [I > 2r(I)]: R1 = 0.047, wR2 = 0.106;
ꢀꢀ3
largest diff. peak and hole: 0.295 and ꢀ0.240 e A
.
CCDC-1056789. Crystal data for 20: C22H23N5O4Br5,
crystal system, space group: triclinic, P-1; (no:2); unit cell
dimensions: a = 9.3536(3), b = 9.7641(3), c = 14.5395(4)
ꢀ
A, a = 77.672(2), b = 76.553(2), c = 72.583(2)ꢀ; volume:
3
ꢀ3
1217.36(10) A ; Z = 2; calculated density: 2.05 g/cm ;
absorption coefficient: 8.280 mmꢀ1; F(000): 724; h-range
for data collection 1.5–26.4ꢀ; refinement method: full-
matrix least-square on F2; data/parameters: 3310/282;
goodness of fit on F2: 1.037; final R-indices [I > 2r(I)]:
R1 = 0.074, wR2 = 0.197; largest diff. peak and hole:
CA isoenzymes activities were determined in according to
the method of Verpoorte et al. (76) described previously
(77). The increasing in absorbance of reaction medium
was spectrophotometrically recorded at 348 nM. Also, the
quantity of protein was determined at 595 nM according to
Bradford method (78). Bovine serum albumin was used as
standard protein given previously in detail (79). For the
determination of inhibition effect of each 4-(2-(3,4-
dimethoxybenzyl)cyclopentyl)-1,2-dimethoxybenzene
derivatives, an activity (%)-[4-(2-(3,4-dimethoxybenzyl)
cyclopentyl)-1,2-dimethoxybenzene derivatives] graph was
drawn. For calculation of Ki values, three different 4-[2-
(3,4-dimethoxybenzyl)cyclopentyl]-1,2-dimethoxybenzene
derivatives concentrations were used. In these experi-
ments, as a substrate NPA was used at five different con-
centrations. Finally, the Lineweaver–Burk curves were
drawn (80). The drawings calculations were described in
detailed previously (81).
ꢀꢀ3
1.481 and ꢀ1.296 e A .CCDC-1056866.
Crystallographic data that were deposited in CSD under
CCDC registration numbers contain the supplementary
crystallographic data for this Letter. These data can be
obtained free of charge from the Cambridge Crystallo-
graphic
Data
Centre
(CCDC)
via
www.ccdc.
cam.ac.uk/data_request/cif and are available free of
charge upon request to CCDC, 12 Union Road,
Cambridge, UK (fax: +441223 336033, e-mail:
deposit@ccdc.cam.ac.uk).
Result and Discussion
Chemistry
According to the literature method (2), the reaction of 7
with adipoyl chloride gave 8. Thereafter, reduction (with
NaBH4) and nitration (with Ac2O)/HNO3) reactions of dike-
tone 8 were performed. The diol 9c and dinitro derivative
10 [2] were obtained from the reactions (Scheme 1). Treat-
ment of compound 8 with HCl/HOAc gave two rearranged
products, 11 and 12. One of them is the major product 11
Crystallography
For the crystal structure determination, single crystal of the
compounds 16 and 20 was used for data collection on a
four-circle Rigaku R-AXIS RAPID-S diffractometer
Chem Biol Drug Des 2016; 87: 594–607
599

Artuncß et al.
O
O
4
NO2
OH OH
MeO
MeO
OMe
OMe
HO
HO
MeO
MeO
OMe
OMe
4
NO2
14
15
9
10
Ac2O
HNO3
AcCl
NaBH
4
AcO
AcO
O
O
4
MeO
MeO
MeO
OMe
OMe
Adipoyl chloride
AlCl3/CH2Cl2
MeO
7
8
HCl/HOAc
OMe
OMe
OMe
OMe
OMe
O
OH
O
NaBH4
MeO
MeO
MeO
MeO
MeO
MeO
+
OMe
11
12
13
Scheme 1: Synthesis of compound 8–13 and 15.
Br
Br
O2N
O2N
Br
OMe
OMe
OMe
OMe
OMe
OMe
O
O
MeO
MeO
MeO
MeO
MeO
MeO
Br
NO2
Br
18
19
20
c
b
d
OMe
OMe
OMe
OMe
OMe
OMe
O
O
MeO
MeO
MeO
MeO
MeO
MeO
+
a
11
16
17
e
NO2
f
Ac
N
OMe
OMe
OMe
OMe
O
OH
O
N
g
MeO
MeO
MeO
MeO
MeO
OMe
OMe
MeO
21
22
23
Scheme 2: Reagents and conditions: (a) H2, Pd-C, HCO2Et, percent yields of products depend on the conditions. See Experimental
section. (b) HNO3, Ac2O, 0 °C – RT, 3 days, 77% .(c) HNO3, Ac2O, 0 °C -RT, 100 min, 20%. (d) Excess Br2, CH2Cl2, RT, 29%. (e)
NH2NH2 .H2O, HOAc, reflux, 5 days, 45%. (f) NaBH4, MeOH, RT, 60%. (g) p-Nitrobenzoyl chloride, ꢀ30 °C – RT, 1 days, 91%.
having the similar to compound 2. The alcohol 13 was
obtained by the reduction of the minor rearranged product
12. Compound 8 and its derivatives incorporate two vera-
trole-like units. One another remarkable point of this type
reaction is the synthesis of acetylated derivative of the
starting material and examining its biological activities. For
600
Chem Biol Drug Des 2016; 87: 594–607

CA Inhibition Profile of Some Dimethoxybenzene Derivatives
this purpose, the diacetate 15 (82,83) was synthesized the
reaction of 14 and acetyl chloride (Scheme 1).
the corresponding compounds and positions of bromine
atoms in 20, X-ray analysis of ketone 16 and bromide 20
was performed. The R,S configuration of the chiral car-
bons C10(R), C11(S) and C9(R), C10(R) in Figures 2 and 3
was established, respectively.
Compounds 16 and 17 were synthesized by reduction of
compound 11 via catalytic hydrogenation reaction (H2, Pd/
C). The ratios of 16 and 17 depend on the reaction time.
As the reaction time increases, the ration of 17–16 also
increases (Scheme 2). According to the reaction condition,
nitrated products 18 and 19 were obtained from the reac-
tion of 11 with a mixture of Ac2O and HNO3. Bromination
of 17 with excess Br2 gave a mixture of products. Crystal-
lization of the mixture from EtOAc/hexane gave bromide
20. To synthesize the dihydropyrazole derivative 21, com-
pound 11 was treated with hydrazine monohydride
(NH2NH2.H2O) and Ac2O. By reduction of compound 16
with NaBH4 in MeOH, the benzyl alcohol 22 was synthe-
sized. To be sure for the formation of the hydroxyl group
and determination of the structure, it was converted to the
corresponding ester 23 with p-nitrobenzoyl chloride
(Scheme 2).
Biological activity
Two physiologically relevant CA isoforms, hCA I and hCA
II, were included in our study. New 4-[2-(3,4-dimethoxy-
benzyl)cyclopentyl]-1,2-dimethoxybenzene derivatives were
tested for their inhibitory properties against hCA I, and
hCA II, showing generally an efficient inhibition (Table 1). It
was well known that developing isoenzyme-specific CAIs
should be highly beneficial in obtaining novel classes of
drugs devoid of various undesired side-effects (30). We
report here the first study on the inhibitory effects of new
4-(2-(3,4-dimethoxybenzyl)cyclopentyl)-1,2-dimethoxyben-
zene derivatives on the esterase activity of hCA I and II.
The low-activity cytosolic isoenzyme hCA I is ubiquitously
expressed in the body and can be found in high concen-
trations in the blood and gastrointestinal tract (84). Specif-
NMR data of synthesized compounds are consistent with
the proposed structures of them. To ensure from configu-
rations of substituents in saturated cyclopentane rings of
ically, hCA
I
is found in many tissues, and it was
Figure 2: Up: Mercury drawing of the
molecule ketone-16. Thermal ellipsoids are
drawn at the 50% probability level. Down:
Unit cell and the intramolecular short-contact
geometry along the a-axis.
Chem Biol Drug Des 2016; 87: 594–607
601

Artuncß et al.
Figure 3: Up: Mercury drawing of the molecule bromide-20. Thermal ellipsoids are drawn at the 40% probability level. Down: Unit cell
and the intramolecular short-contact geometry along the a-axis.
demonstrated that this isoenzyme is involved in retinal
and cerebral edema, and its inhibition may be a valuable
tool for fighting these conditions (68,84). Our results indi-
cate that new compounds with 4-[2-(3,4-dimethoxybenzyl)
cyclopentyl]-1,2-dimethoxybenzene scaffold (8–13 and 15–
23) had effective inhibition profile against this cytosolic
isoform hCA I, as well as the cytosolic dominant rapid iso-
zymes hCA II. hCA I was effectively inhibited by the all of the
newly synthesized 4-[2-(3,4-dimethoxybenzyl)cyclopentyl]-
1,2-dimethoxybenzene derivatives (8–13 and 15–23). These
new compounds bind to hCA I in the nanomolar range. Ki
values are ranging in 313.2 ꢂ 74.7–1537.0 ꢂ 99.3 nM for
hCA I isoenzyme. On the other hand, acetazolamide (AZA),
considered being a broad-specificity CA inhibitor owing to
its widespread inhibition of CAs, showed Ki value of
52.98 ꢂ 10.72 nM against hCA I. 4-[2-(3,4-dimethoxyben-
zyl)cyclopentyl]-1,2-dimethoxybenzene of 20, possessing
with tetramethoxy groups and five Br substituents at the
602
Chem Biol Drug Des 2016; 87: 594–607

CA Inhibition Profile of Some Dimethoxybenzene Derivatives
Table 1: Human carbonic anhydrase isoenzymes (hCA I and II) inhibition values with new 4-(2-(3,4-dimethoxybenzyl)cyclopentyl)-1,
2-dimethoxybenzene derivatives given by an esterase assay
IC50 (nM)
hCA I
KI (nM)
Compounds
r2
hCA II
r2
hCA I
hCA II
8
434.48
625.45
394.87
739.51
624.32
601.56
814.62
472.07
559.77
526.66
853.97
274.89
563.87
513.95
394.64
141.11
1583.63
0.966
0.963
0.883
0.961
0.873
0.889
0.957
0.919
0.962
0.882
0.889
0.968
0.960
0.970
0.981
0.966
0.958
345.63
696.62
543.11
599.48
421.78
517.55
736.21
655.62
504.00
647.59
577.50
474.98
440.83
505.83
389.76
110.28
26.61
0.964
0.866
0.832
0.929
0.970
0.854
0.880
0.864
0.969
0.831
0.862
0.840
0.848
0.972
0.895
0.955
0.952
841.26 ꢂ 23.15
778.81 ꢂ 49.55
1343.39 ꢂ 48.32
365.60 ꢂ 21.97
735.41 ꢂ 88.43
655.77 ꢂ 96.30
1537.00 ꢂ 99.30
609.81 ꢂ 22.91
420.98 ꢂ 84.86
848.64 ꢂ 58.13
573.18 ꢂ 69.49
313.16 ꢂ 74.70
492.02 ꢂ 88.89
454.91 ꢂ 98.23
616.33 ꢂ 44.86
52.98 ꢂ 10.72
1686.87 ꢂ 46.94
902.54 ꢂ 74.96
1927.31 ꢂ 75.46
1261.45 ꢂ 73.22
228.31 ꢂ 46.92
561.02 ꢂ 98.14
1541.46 ꢂ 57.19
1809.47 ꢂ 86.58
1789.19 ꢂ 45.11
1027.20 ꢂ 76.19
690.22 ꢂ 81.87
1575.79 ꢂ 89.24
509.76 ꢂ 44.02
606.49 ꢂ 89.56
1552.00 ꢂ 43.69
587.64 ꢂ 29.00
40.81 ꢂ 17.06
9
10
11
12
13
15
16
17
18
19
20
21
22
23
AZAa
DRZa
25.02 ꢂ 10.76
aAcetazolamide (AZA) and Dorzolamide (DRZ) were used as a standard inhibitor for both CA isoenzymes.
aromatic part, was the best hCA II inhibitor (Ki:
313.16 ꢂ 74.70 nM). The inhibition effects of 4-[2-(3,4-
dimethoxybenzyl)cyclopentyl]-1,2-dimethoxybenzene
derivatives(8–13 and 15–23) (8–21) are lower than that of
acetazolamide (AZA; 52.98 ꢂ 10.72 nM), but higher than
that of Dorzolamide (DRZ, 1686.87 ꢂ 46.94 nM), which
used to lower increased intraocular pressure in open-angle
glaucoma and ocular hypertension. It was reported that the
presence of halogen atoms in CA inhibitors was a good
strategy toward hCA-selective inhibitors discovery. In this
study, the fluorinated analogues showed a better inhibition
with interesting inhibition constants at the nanomolar level
against the tumor-associated isoforms (85). Recently, an
example of the success of such a strategy has been
reported and a fluorine substitution effect on cytosolic hCA
isoforms inhibition has been shown (86). This effect was not
only attributed to the modification of both hydrophilic/lipo-
philic balance between the inhibitor and the enzyme pocket,
but also to a modification of the molecule direction in the
active site, leading to specific interactions with the enzyme
(46).
sized compounds for hCA II were found as 1141.58 nM.
These results clearly showed that newly synthesized 4-[2-
(3,4-dimethoxybenzyl)cyclopentyl]-1,2-dimethoxybenzene
derivatives (8–13 and 15–23) had higher inhibition affinity
toward hCA I than that of hCA. AZA, which may interact
with the distinct hydrophobic and hydrophilic halves of the
CA II active site, and DRZ, which used for decreasing fluid
production and pressure inside the eye, showed Kis of
40.81 ꢂ 17.06 and 25.02 ꢂ 10.76 nM, respectively.
Conclusion
The diketone 8 was obtained from the reaction of 1,2-
dimethoxybenzene (7), adipoyl dichloride, and AlCl3. Com-
pounds 9c and 10 (2) were synthesized from the reactions
of 8 with the corresponding reagents. The reaction of dike-
tone 8 with the mixture of HCl and HOAc gave the
isomeric rearranged products 11 and 12 on the cyclopen-
tene ring. From the reduction (catalytic hydrogenation and
NaBH4), nitration, 1,4-addition, bromination, and esterifica-
tion reactions of the compound 11, which is the major
product in the rearrangement reaction, eight new com-
pounds 16–23 were obtained. As a result, eleven new and
four known compounds for the literature were synthesized
in this work. The synthesized 4-[2-(3,4-dimethoxybenzyl)
cyclopentyl]-1,2-dimethoxybenzene derivatives (8–13 and
15–23) were then evaluated for their hCA I and II isoen-
zymes inhibition properties. These compounds were found
to be active enzyme inhibitors. The most active inhibitors
in the both series were found to be as compound 20
which had Ki value of 313.16 ꢂ 74.70 nM for hCA I
and compound 11 with Ki value of 228.31 ꢂ 46.92 nM for
hCA II.
CA II is involved in several diseases including glaucoma,
epilepsy, edema, and altitude sickness. Against the physi-
ologically dominant isoform hCA II, 4-[2-(3,4-dimethoxy-
benzyl)cyclopentyl]-1,2-dimethoxybenzene derivatives (8–
13 and 15–23) showed Kis of 228.3–1927.3 nM (Table 1).
The compound 11, possessing with tetramethoxy groups
at the aromatic part and one carbonyl group, was the best
hCA II inhibitor (Ki: 228.31 ꢂ 46.92 nM). However, the
average Ki values of newly synthesized 4-[2-(3,4-dimethox-
ybenzyl)cyclopentyl]-1,2-dimethoxybenzene derivatives (8–
13 and 15–23) were found to be 712.13 nM for hCA I.
Conversely, the average Ki values of these new synthe-
Chem Biol Drug Des 2016; 87: 594–607
603

Artuncß et al.
including natural products: Vidalol B. Eur
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J Med
Acknowledgments
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€
We thank to Ataturk University for the financial supports of
this work (BAP, Project No: 2012/154). I.G extends his sin-
cere appreciation to the Research Chairs Program at King
Saud University for funding this research.
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The authors declare that there is no conflict of interest.
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Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Synthesis of known compounds 8–10 and
14.
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