Novel sulphamides and sulphonamides incorporating the tetralin scaffold as carbonic anhydrase and acetylcholine esterase inhibitors

Article abstract of DOI:10.1002/ardp.201300273

Reactions of amino, aminomethyl tetralins and benzyl alcohol with chlorosulphonyl isocyanate (CSI) afforded sulphamoyl carbamates. The sulphamoyl carbamates were converted to sulphamides by palladium-catalysed hydrogenolysis. Sulphonamides were synthesize

Full text of DOI:10.1002/ardp.201300273

68
Arch. Pharm. Chem. Life Sci. 2014, 347, 68–76
Full Paper
Novel Sulphamides and Sulphonamides Incorporating the
Tetralin Scaffold as Carbonic Anhydrase and Acetylcholine
Esterase Inhibitors
1,2
2
2
2
_
ꢀ
Akın Akıncıoglu , Meryem Topal , Ilhami Gülçin , and Süleyman Göksu
1
Central Researching Laboratory, Agri Ibrahim Cecen University, Agri, Turkey
Faculty of Science, Department of Chemistry, Atatürk University, Erzurum, Turkey
2
Reactions of amino, aminomethyl tetralins and benzyl alcohol with chlorosulphonyl isocyanate (CSI)
afforded sulphamoyl carbamates. The sulphamoyl carbamates were converted to sulphamides by
palladium-catalysed hydrogenolysis. Sulphonamides were synthesized from the reactions of amines
with MeSO2Cl. Inhibition of human (h) carbonic anhydrase (CA) isoenzymes (hCA I, hCA II) and
acetylcholine esterase (AChE) was investigated with the synthesized compounds. hCA I and hCA II were
inhibited in the low micromolar or sub-micromolar range. The Ki values were in the range of
0.91–9.56 mM against hCA I and of 3.70–27.88 mM against hCA II. Sulphamides 11–13 and
sulphonamides 14–16 had moderate inhibition capacity toward AChE. These findings suggest the
novel sulphamides 11–13 and sulphonamides 14–16 as AChE and CA isoenzyme inhibitory agents.
Keywords: Acetylcholine esterase / Aminotetralins / Carbonic anhydrase / Sulphamides / Sulphamoyl carbamates /
Sulphonamides
Received: July 18, 2013; Revised: September 14, 2013; Accepted: September 17, 2013
DOI 10.1002/ardp.201300273
Introduction
carbon dioxide (CO2) and the dehydration of bicarbonate ion
(HCOꢀ3 ) [8, 9].
Sulphamides [1] and sulphonamides [2] are important
biologically active compounds. In the last decades a lot of
sulphamide and sulphonamide drugs have appeared in
the markets. Quinagolide (1), a dopaminergic drug, is
an antihyperprolactinemia agent [3]. Compound 2 (JNJ-
26990990) has potential use in the treatment of depression,
neuropathic and inflammatory pains [4]. The sulphonamide
drug sultiame (3) is an anticonvulsant used in the treatment
of epilepsy and West syndrome [5]. Acetazolamide (AZA, 4), a
drug also known as carbonic anhydrase (CA) inhibitor, is used
in the treatment of glaucoma [6] and idiopathic intracranial
hypertension [7] (Figure 1).
CA
CO2 þ H2O , HCOꢀ3 þ Hþ
The CA enzyme family is particularly attractive for
biophysical studies of protein-ligand binding and inhibition
studies for many reasons, among which (i) CA is a monomeric
enzyme, single-chain protein of medium molecular weight
(30 kDa). The most widespread isoforms (CA I and CA II) have
no disulphide bonds, pendant sugar or phosphate groups; all
the other isoforms have disulphide bonds, pendant sugar or
phosphate groups. (ii) CA II is an inexpensive enzyme widely
available. (iii) CA II is relatively easy to handle and purify, due
in large part to its excellent stability under standard
laboratory conditions. (iv) The amino acid sequences are
available for most isoenzymes. (v) The structure and active site
geometries have been defined in detail by X-ray diffraction
analysis for most of the 13 catalytically active mammalian
isoforms [8]. Its catalytic mechanism is well understood. (vi) It
not only behaves as a CO2 hydrase/dehydratese activity with a
high turnover number but also possesses esterase activity
with carboxylic acid esters, phosphoric acid esters and even
CA (E.C.: 4.2.1.1) is a protein that is well suited to serve as a
model in many types of biophysical, bioanalytical and
physical-organic chemical studies as well as for inhibitor
drug design studies. This enzyme catalyses the hydration of
Correspondence: Prof. Süleyman Göksu, Faculty of Science, Depart-
ment of Chemistry, Atatürk University, 25240-Erzurum, Turkey.
E-mail: sgoksu@atauni.edu.tr
Fax: þ90 442 2360948
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Arch. Pharm. Chem. Life Sci. 2014, 347, 68–76
New Sulphamides and Sulphonamides as CA and AChE Inhibitors
69
Figure 1. Some selected sulphamide and sulphonamide drugs (1–4).
sulphate esters [10–12]. (vii) Its inhibition mechanism by
inhibitors that bind to the Zn2þ ion is fairly simple and well
characterized [8, 13].
Alzheimer’s disease is the most common form of dementia
and neurodegenerative diseases. It is characterized by memory
dysfunction. Reduction of acetylcholine levels in the brain is
biological purposes. In this context, we report the synthesis of
novel sulphamides 11–13 and sulphonamides 14–16. We also
evaluate CA isoenzymes (hCA I and hCA II) and AChE
inhibitory effects of the synthesized novel sulphamides 11–13
and sulphonamides 14–16.
the most notable biochemical change in Alzheimer’s disease Results and discussion
[14]. Also, Alzheimer’s disease can be treated by use of
pharmaceuticals, which restore the level of acetylcholine
through inhibition of acetylcholine esterase (AChE) [15].
Alzheimer’s is predicted to affect 1 in 85 people globally by
2050 [16]. For treatment of Alzheimer’s disease, some drugs
such as galanthamine, tacrine and rivastigmine are used as
AChE inhibitors. However, it has been reported that these
drugs have adverse effects like hepatotoxicity and gastrointes-
tinal disturbances [17–20]. Therefore, the safe and active AChE
inhibitors have recently been gaining more attention.
It is well known that aminotetralin derivatives show
beneficial biological activities in CNS such as dopaminer-
gic [21, 22], adrenergic and seratonergic activities [23]. The
synthesis and biological evaluation of some novel sulpha-
mides and sulphonamides derived from these biologically
active tetralins will be useful for further synthetic and
Chemistry
The synthesis of 1-aminotetralin 5 [24], and dopaminergic
compounds 6 [25] and 7 [26] was carried out as described
previously. The reactions of free amines and benzyl alcohol/t-
BuOH with chlorosulphonyl isocyanate (CSI) in the presence
of Et3N to give sulphamoyl carbamates have been reported by
us [9, 27]. By the similar approach, the reactions of amines 5–7
and 1.2 equiv. benzyl alcohol with CSI in the presence of
Et3N afforded novel sulphamide carbamates 8–10 in moderate
yields. New sulphamides 11–13 were synthesized by Pd–C-
catalysed hydrogenolysis of 8–10 with yields of 73, 76 and
80%, respectively (Scheme 1).
On the other hand, the treatment of amines with MeSO2Cl
in the presence of Et3N at 0–25°C yielded novel sulphonamides
14–16 in high yields (Scheme 2). The structures of all novel
Scheme 1. The synthesis of sulphamides 11–13. (i) CSI/NEt3/BnOH, CH2Cl2, 0–25°C, 4 h; (ii) H2/Pd-C, MeOH, 25°C, 4 h.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 68–76
method with Sepharose-4B-L-tyrosine-sulphanilamide affinity
column chromatography [34, 35]. Human erythrocyte CA I
isoenzyme was purified, 154.9-fold with a specific activity of
2088.6 EU mgꢀ1 and overall yield of 22.8%; hCA II isoenzyme
was purified, 245.6-fold with a specific activity of 9335.2 EU
mgꢀ1 and an overall yield of 38.3%.
CA isoenzymes inhibition effects
We report here the first study on the inhibitory effects of novel
sulphamides 11–13 and sulphonamides 14–16 on the esterase
activity of hCA I and hCA II. The inhibitor concentration that
caused 50% inhibition (IC50) was determined from activity
versus (%)-[sulphamides] or [sulphonamides] plots and the
average inhibition constant (Ki) values were calculated from
Lineweaver–Burk plots (Table 1). The data in Table 1 show the
following inhibition of hCA I and hCA II with the novel
sulphamides 11–13 and sulphonamides 14–16 by an esterase
assay, with 4-NPA as substrate.
Scheme 2. The synthesis of novel sulphonamides 14–16. (i)
MeSO2Cl/NEt3, CH2Cl2, 0–25°C, 15 h.
synthesized compounds were determined by 1H and 13C NMR
techniques. The functional groups were characterized by IR.
Inhibition of the CA isoenzymes has pharmacologic
applications in the field of anti-glaucoma, anti-convulsant,
and anti-cancer agents. It is well known that sulphamides and
sulphonamides are the main classes of CA inhibitors, which
bind to the metal ion from the enzyme active site [27]. CA
inhibitors block the function of CA enzyme. The myriad
inhibitors of CA can be divided into four main groups:
i. Sulphamides and sulphonamides
CA isoenzymes purification and activity assay
Methods for isolation and purification of CA are well
established and widely accessible. Commercial sources usually
isolate isoenzymes hCA I and hCA II from fresh human red
blood cells, in which CA is the second most abundant protein
after haemoglobin. Research laboratories typically produce
these isoenzymes by recombinant technology in Escherichia coli
[28, 29]. This method provides the structural flexibility and
specificity of site-directed mutagenesis, which is useful for
biophysical and biochemical studies [18]. The blood protein
and the wild-type recombinant construct are identical except
for the N-terminus, which is a post-translationally acetylated
Ser in the native CA and exists as Met-Ala or Ala in the
recombinant version [9, 30]. The research laboratory procedure
for the purification of hCA from red blood cells involves lysis of
the cells and removal of the cellular remnants by centrifuga-
tion, followed by separation of haemoglobin from hCA on a
sulphonamide-modified agarose affinity chromatography
column [31, 32]. Divalent anions like sulphate do not bind
appreciably in the active site of hCA. Therefore, it was used to
remove nonspecifically bound protein by screening ionic
ii. Sulphonic acid derivatives
iii. Small monoanions such as halides, azides and
thiocyanate
iv. Aromatic rings or phenolic compounds.
The sulphonamides, sulphamides and sulphonic acid
derivatives have Ki values in the picomolar to micromolar
Table 1. Human CA isoenzymes (hCA I and hCA II) and AChE
inhibition data with the novel sulphamides and sulphonamides
11–16.
Ki (mM)
Compounds
hCA-I
hCA-II
AChE
interactions. Then, hCA
I and hCA II can be eluted
11
12
13
14
0.71 ꢁ 0.11
1.24 ꢁ 0.16
0.89 ꢁ 0.18
1.26 ꢁ 0.04
1.03 ꢁ 0.27
1.56 ꢁ 0.05
36.2 ꢁ 0.00b)
–
0.69 ꢁ 0.17
0.72 ꢁ 0.02
1.45 ꢁ 0.35
0.67 ꢁ 0.09
0.87 ꢁ 0.26
0.39 ꢁ 0.13
3.70 ꢁ 0.00b)
–
140.2 ꢁ 26.8
56.7 ꢁ 0.02
63.2 ꢁ 22.5
365.6 ꢁ 61.7
84.5 ꢁ 18.1
537.3 ꢁ 49.1
–
consecutively from the column. hCA I, which has a higher
dissociation constant for sulphonamides than hCA II, is eluted
with Tris-SO4 buffer containing 0.2 M KI; hCA II elutes with
Tris-SO4 buffer containing 0.4 M NaN3. Also, it was shown that
CA isoenzymes from other species could be purified in a
similar way, with only minor modifications. Commercial
suppliers do not use the chromatographic methods, although
their exact procedures are proprietary [31, 33].
15
16
AZAa)
GALc)
3.70 ꢁ 0.00c)
a)
AZA was used as positive standard inhibitor for both CA
isoenzymes (CA I and CA II).
In the present study, hCA I and hCA II isoenzymes were
purified from fresh human erythrocytes. The purification of
both CA isoenzymes was performed using a simple one-step
b)
From ref. [37].
c)
Galantamine was used as a reference compound for AChE
inhibitory properties.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 68–76
New Sulphamides and Sulphonamides as CA and AChE Inhibitors
71
range, whereas the inorganic monoanions bind CA with Ki
values in the micromolar to millimolar range [33]. Recently, it
was observed by us that sulphamides show greater inhibitory
effects than that of sulphonamides [27]. Similarly, it has been
reported that phenolic inhibitors with aromatic rings have Ki
values in the micromolar to millimolar range [10, 36–40].
The most obvious bioisosteres of sulphonamides are
sulphamides, in which an additional electron-withdrawing
atom/group is directly attached to the sulphamoyl function,
generating compounds with general formula R–NH–SO2NH2.
These groups were considered as interesting candidates for
obtaining CAIs. In first, Supuran’s group showed that the
inhibition of the rapid cytosolic isoenzymes hCA I (Ki:
0.71 ꢁ 0.11 mM). On the other hand, the strongest inhibitory
effect has been observed with novel sulphonamide 16, for
hCA II (Ki: 0.39 ꢁ 0.13 mM).
It had been reported that sulphamide and sulphamic acid
act as moderate hCA II inhibitors, with inhibition constants of
1130 mM for sulphamide and 390 mM for sulphamic acid at
the physiological pH (7.4), respectively [51]. In contrast to
these literatures, in the current study, we observed strong
inhibitory effect of new synthesized sulphamides 11–13 and
sulphonamides 14–16 on hCA I and hCA II. Ki values of these
sulphamide and sulphonamide compounds (11–16) are in the
range of 0.89–1.56 mM for hCA I, and 0.39–1.45 mM for hCA II
(Table 1). The arithmetic average Ki values of novel synthesized
sulphamides and sulphonamides (11–16) are 1.12 mM for hCA
I and 1.29 mM for hCA II. The half maximal inhibitory
concentration (IC50) is a measure of the effectiveness of novel
sulphamides or sulphonamides 11–16 in inhibiting CA
isoenzyme function. These values are commonly used as a
measure of inhibition potency in biological and pharmaco-
logical research. Ki values were calculated by making use of
IC50 values. A lower IC50 and Ki value reflects strong CA
isoenzyme inhibition effect. The results obtained from Table 1
clearly showed that both the cytosolic isoenzymes hCA I and
hCA II were effectively inhibited by sulphamide and
sulphonamide compounds, with Kis within the range of
0.39–1.56 mM with a comparable potency as the reference
compound AZA with Ki value of 36.2 mM. All novel synthesized
sulphamides (11–13) and sulphonamides (14–16) acted as
strong hCA I and hCA II inhibitors. The structure–activity
relationship and inhibition properties of hCA II isoenzyme are
particularly comparable to that what is outlined above for
hCA I. Due to the two isoenzymes having a high sequence
homology of amino acid present within the active site, all new
synthesized sulphamide and sulphonamide compounds (11–
16) had much stronger CA I and CA II inhibition effects than
the clinically used AZA included in the assays as a standard
inhibitor (Table 1).
simple sulphamide binds to the metal ion in the Co2þ
-
substituted CA II [41], whereas the X-ray structures of the
compound in adduct with CA II have been reported several
years later [42]. Nowadays, there are a large number of aromatic,
heterocyclic, aliphatic and sugar-based sulphamides, which
were shown to possess highly effective inhibitory properties
against all known mammalian isoforms [13]. Therefore,
sulphamides constitute a highly important class of CAIs, with
some derivatives clinically used for the treatment of epilepsy
and obesity.
There have been several attempts to illuminate the
inhibition mechanism of CA and to synthesize new com-
pounds with higher inhibition potential. Novel CA inhibitors
have been designed for pharmacological and medicinal
approaches, and many inhibitors have been synthesized
recently [43]. There are many studies in the literature on the
interactions of different compounds and CA isoenzymes.
Recently, the inhibitory effects of dantrolene [44], melato-
nin [45], vitamin E [46], morphine [47], ethanol [48], antioxi-
dant phenols [49], phenolic acids [36], distinguished natural
polyphenols products, prominent phenolic compounds [38]
and bromophenols [50] with hCA I and hCA II have been
investigated by our groups. Indeed, the inhibition profiles of
hCA I and hCA II with novel sulphamides 11–13 and
sulphonamides 14–16 are very variable, with inhibition
constants ranging from the nanomolar (390 nM) to the sub-
micromolar (1.56 mM) level. For this purpose, hCA I and hCA II
inhibitory effects of the novel sulphamides 11–13 and
sulphonamides 14–16 were tested under in vitro conditions
and theirs Ki values are calculated and presented in Table 1.
We report here the initial study on the inhibitory effects of
synthesized novel sulphamides 11–13 and sulphonamides 14–
16 on the hydratase activity of hCA I and hCA II. The data in
Table 1 show the following regarding the inhibition of hCA I
and hCA II activity by novel sulphamides 11–13 and
sulphonamides 14–16. The novel sulphamides 11–13 and
sulphonamides 14–16 investigated here showed effective
inhibitory properties against the cytosolic isoforms hCA I and
hCA II; however, the strongest inhibitory effect has been
observed with novel sulphamide 11, investigated here for the
All of the synthesized novel sulphamides and sulphonamides
(11–16) have one aromatic ring with two methoxy groups. The
aromatic ring in a chemical compound is known to be in
favour of CA inhibition. These new synthesized sulphamides
compounds have been investigated as CA isoenzyme inhibitors
in this study. The rationale of investigating these compounds
as CA inhibitors exists in the fact that the compounds
with aromatic rings have been shown to be the only
competitive inhibitor with CO2 as the substrate for the
main isoform of CA, i.e., human cytosolic isoenzymes (CA I
and CA II).
CA inhibitors are a class of pharmaceuticals that suppress
the activity of CA. Their clinical use has been established as
anti-glaucoma agents, diuretics, antiepileptics, and in the
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management of mountain sickness, gastric and duodenal
ulcers, neurological disorders, or osteoporosis [52]. Sulpha-
mides are indicated for the reduction of elevated intraocular
pressure in patients with open-angle glaucoma or ocular
moderate Alzheimer’s disease and various other memory
impairments, has been shown to lower AChE inhibition
activity (EC50: 3.70 mM) [66].
hypertension and those who are insufficiently responsive to Conclusions
beta-blockers. The metal-complexing anions and the sulpha-
mides with a terminal –SO2NH2 group coordinate to the Zn2þ
ion. Simple anions such as HS–, CN–, NCO–, N3–, HSOꢀ3 , Iꢀ and
HCOOꢀ may bind with tetrahedral geometry or form trigonal-
bipyramidal coordination [53] whereas the sulphamides
replace the water coordinated to zinc and the ‘deep-water’
hydrogen-bonded to Thr199-NH. Both waters are present in
the uncomplexed state [54]. Although sulphonamides were
considered the moiety par excellence to coordinate the
catalytic zinc and for designing potent CAIs, in recent years
related functional groups such as sulphamide, sulphonamide
and others have proven to be successful in the design of
selective CAIs [55].
Inhibition effects of novel sulphamides 11–13 and sulpho-
namides 14–16 on AChE were determined by commercially
available purified AChE (E.C. 3.1.1.7, Product no: C0663-50 UN,
Sigma–Aldrich) from human erythrocytes based on the
spectrophotometric method of Ellman et al. [56]. It was
reported that the inhibition of AChE has been used for the
determination of neurotoxic properties of chemicals capable
of interfering with normal neurotransmission of the para-
sympathetic and sympathetic nervous system. It was well
known that some chemical compounds like organophosphate
and carbamate pesticides [57–60], enantiomeric inhibi-
tors [61], metals and non-pesticide contaminants have also
been reported to inhibit AChE enzymatic activity [62–65].
Also, it was demonstrated that the main AChE inhibitory
effects were primarily associated with aromatic compounds
and, to lesser degree, with aliphatic compounds [62].
All of these studies clearly showed that an investigation into
the mechanism of action of AChE might lead to the design of
the mechanism-based inhibitors, which could be of future
therapeutic use. Different types of AChE inhibitors have been
studied for the treatment of Alzheimer’s disease. Some of the
AChE inhibitors differ in their mechanism of action,
metabolism and brain selectivity.
AChE was very effectively inhibited by novel sulphamides
11–13 and sulphonamides 14–16, with Kis in the range of
56.04 ꢁ 0.02 to 537.3 ꢁ 49.1 mM. The Ki values of novel
sulphamides 11–13 and sulphonamides 14–16 were similar
to each other. The differences between the highest and the
lowest Ki values of novel sulphamides 11–13 and sulphona-
mides 14–16 were only tenfold. The most active sulphamide
was novel sulphamides 12, and sulphonamides 15 with same
functional groups demonstrated Ki values with 56.7 ꢁ 0.02
and 84.5 ꢁ 18.1 mM, respectively. On the other hand, galant-
amine (GAL), which is used for the treatment of mild-to-
In summary, three sulphamides 11–13 and three sulphona-
mides 14–16 were synthesized starting from known biologi-
cally active amines 5–7 for the first time. The synthesized
sulphamoyl carbamates 8–10, sulphamides 11–13 and
sulphonamides 14–16 may be important synthons for further
biological and synthetic purposes. In addition, several
sulphamides 11–13 and sulphonamides 14–16 have been
assayed for the inhibition of the physiologically relevant
human CA isoenzymes (hCA I and hCA II). The compounds
investigated here showed CA isoenzyme inhibitory effects, in
the nanomolar to low micromolar range, by the esterase
method. Toward hCA I sulphamide and sulphonamide
compounds (11–16) the inhibition constant was in the range
of 0.71–1.56 mM, and against hCA II it was in the range of 0.39–
1.45 mM. In general, the compounds had comparable
inhibitory activity with the clinically used AZA. Also, these
findings clearly showed that novel synthesized sulphamides
and sulphonamides (11–16) might be used as leads for
generating potent hCA I and hCA II inhibitors, eventually
targeting other isoforms that have not been assayed as yet for
their interactions with such agents. Additionally, from this
study it can be concluded that tested novel sulphamides 11–
13 and sulphonamides 14–16 exhibited moderate inhibitory
activity on AChE.
Experimental
All chemicals and solvents are commercially available and were
used after distillation or treatment with drying agents. Melting
points were determined on a capillary melting apparatus (BUCHI
530) and are uncorrected. IR spectra were obtained from solutions
in 0.1 mm cells with a Perkin-Elmer spectrophotometer. The
1H and 13C NMR spectra were recorded on a 400 (100)-MHz Varian
and 400 (100)-MHz Bruker spectrometer (d in ppm, Me4Si as the
internal standard). Elemental analyses were performed on a Leco
CHNS-932 apparatus. Column chromatography was performed
on silica gel (60-mesh, Merck). PLC is preparative thick-layer
chromatography: 1 mm of silica gel 60 PF (Merck) on glass plates.
Chemistry
1-Aminotetralin 5 [24], 2-aminotetralin 6 [22], and 1-aminomethyl
tetralin 7 [26] were synthesized as described in the literature.
Standard procedure for the synthesis of
sulphamoylcarbamates: Benzyl N-(6,7-dimethoxy-1,2,3,4-
tetrahydronaphthalen-1-yl)sulphamoylcarbamate (8)
Benzylalcohol (0.50 g, 4.63 mmol) was added to a solution of CSI
(0.66 g, 4.60 mmol) in CH2Cl2 (10 mL) at 0°C. A solution of amine 5
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Arch. Pharm. Chem. Life Sci. 2014, 347, 68–76
New Sulphamides and Sulphonamides as CA and AChE Inhibitors
73
(0.80 g, 3.86 mmol) in CH2Cl2 (30 mL) and NEt3 (0.55 g, 5.40 mmol)
was added to the solution of CSI dropwise and stirred at 0°C for
1 h, then at room temperature for 3 h. The reaction mixture was
cooled to 0°C and to this mixture a solution of 0.1 N HCl (50 mL)
was added. Organic phase was separated and H2O phase was
extracted with CH2Cl2 (2 ꢂ 30 mL). Combined organic layers were
dried over Na2SO4 and the solvent was evaporated. Column
chromatography of the residue on silica gel (30 g) with 30%
EtOAc–hexane yielded carbamate 8 (0.98 g, 60%) as white solid.
The same experimental procedure was also used for the synthesis
of compounds 9 (%54) and 10 (50%).
1256, 1219, 1159, 1119, 1089, 1068, 1028. Anal. calcd. for
(C21H26N2O6S): C, 58.05; H, 6.03; N, 6.45; S, 7.38. Found: C, 58.10;
H, 5.99; N, 6.48; S, 7.32.
Standard procedure for the synthesis of sulphamides
(hydrogenolysis of sulphamoylcarbamates): N-(6,7-
Dimethoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-
sulphamide (11)
Into a 100-mL flask Pd-C (50 mg) and sulphamoylcarbamate 8
(0.7 g, 1.66 mmol) in MeOH (50 mL) were added. A balloon filled
with H2 gas (3 L) was fitted to the flask. The mixture was
deoxygenated by flushing with H2 and then hydrogenated at
room temperature for 4 h. The catalyst was removed by filtration.
Recrystallization of the residue from EtOAc–hexane gave 11
(0.35 g, 73% yield).
Benzyl N-(6,7-dimethoxy-1,2,3,4-tetrahydronaphthalen-
1-yl)sulphamoylcarbamate (8)
White solid. Mp 88–90°C. 1H NMR (400 MHz, CDCl3):
d (ppm) ¼ 7.36–7.31 (m, 6H, 5Ph-H and NH), 6.85 (s, 1H, Ar–H),
6.52 (s, 1H, Ar–H), 5.37 (d, 1H, NH, J ¼ 7.6 Hz), 5.20 (d, A part of AB;
1H, Ha of OCH2, J ¼ 12.1 Hz), 5.14 (d, B part of AB; 1H, Hb of OCH2,
J ¼ 12.1 Hz), 4.53–4.51 (m, 1H, CH–N), 3.83 (s, 3H, OCH3), 3.78
(s, 3H, OCH3), 2.68–2.60 (m, 2H, CH2), 1.91–1.87 (m, 2H, CH2),
1.78–1.72 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3): d (ppm) ¼
151.6 (CO), 149.1 (C), 147.9 (C), 134.8 (C of Ph), 130.1 (C), 129.1 (CH
of Ph), 129.0 (2CH of Ph), 128.6 (2CH of Ph), 126.5 (C), 111.7 (2CH),
68.7 (OCH2), 56.12 (OCH3), 56.06 (OCH3), 53.2 (CH–N), 30.0 (CH2),
28.7 (CH2), 19.4 (CH2). IR (CH2Cl2, cmꢀ1): 3417, 3332, 3272, 3193,
3056, 2928, 2855, 1688,1608, 1445, 1402, 1343, 1265, 1163, 1120,
1084, 1071. Anal. calcd. for (C20H24N2O6S): C, 57.13; H, 5.75; N,
6.66; S, 7.63. Found: C, 57.08; H, 5.82; N, 6.58; S, 7.65.
Sulphamides 12–13 were also synthesized by this procedure
with yields of 76 and 80%, respectively.
N-(6,7-Dimethoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-
sulphamide (11)
White solid. Mp: 152–155°C. 1H NMR (400 MHz, CDCl3): d
(ppm) ¼ 6.95 (s, 1H, Ar–H), 6.52 (s, 1H, Ar–H), 4.78 (bs, 2H,
NH2), 4.62–4.54 (m, 2H, NH and CH–N), 3.84 (s, 3H, OCH3), 3.83
(s, 3H, OCH3), 2.69–2.63 (m, 2H, CH2), 2.03–1.98 (m, 2H, CH2), 1.82–
1.78 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3): d (ppm) ¼ 148.8 (C),
147.8 (C), 130.1 (C), 127.5 (C), 111.8 (CH), 111.5 (CH), 56.3 (OCH3),
56.1 (OCH3), 52.3 (CH–N), 30.7 (CH2), 28.8 (CH2), 19.4 (CH2). IR
(CH2Cl2, cmꢀ1): 3495, 3417, 3325, 2969, 2952, 2926, 2845, 1706,
1647, 1609, 1512, 1453, 1420, 1353, 1317, 1286, 1255, 1216, 1155,
1116, 1088, 1074, 1051, 1037, 1017. Anal. calcd. for (C12H18N2O4S):
C, 50.33; H, 6.34; N, 9.78; S, 11.20. Found: C, 50.38; H, 6.40; N, 9.72;
S, 11.18.
Benzyl N-(6,7-dimethoxy-1,2,3,4-tetrahydronaphthalen-
2-yl)sulphamoylcarbamate (9)
White solid. Mp: 140–142°C. 1H NMR (400 MHz, CDCl3):
d (ppm) ¼ 7.49 (bs, 1H, NH), 7.41–7.34 (m, 5H, Ph-H), 6.54 (s,
1H, Ar–H), 6.45 (s, 1H, Ar–H), 5.23 (d, 1H, NH, J ¼ 6.9 Hz), 5.20 (s,
2H, OCH2), 3.87 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.77–3.69 (m, 1H,
CH–N), 2.97 (dd, A part of AB; 1H, Ha of CH2, J ¼ 5.0 and 16.0 Hz),
2.78–2.74 (m, 2H, CH2), 2.63 (dd, B part of AB; 1H, Hb of CH2, J ¼ 8.0
and 16.0 Hz), 2.04–1.96 (m, 1H, H of CH2), 1.81–1.72 (m, 1H, H of
CH2). 13C NMR (100 MHz, CDCl3): d (ppm) ¼ 151.2 (CO), 147.8 (C),
147.5 (C), 134.7 (C of Ph), 128.9 (CH of Ph), 128.8 (2CH of Ph), 128.5
(2CH of Ph), 126.8 (C), 124.7 (C), 112.0 (CH), 111.5 (CH), 68.6 (OCH2),
56.0 (OCH3), 55.9 (OCH3), 50.7 (CH-N), 35.5 (CH2), 29.1 (CH2), 26.4
(CH2). IR (CH2Cl2, cmꢀ1): 3257, 3064, 3030, 3002, 2935, 2840, 1727,
1610, 1517, 1452, 1352, 1295, 1241, 1226, 1159, 1114, 1071, 1017.
Anal. calcd. for (C20H24N2O6S): C, 57.13; H, 5.75; N, 6.66; S, 7.63.
Found: C, 57.11; H, 5.78; N, 6.64; S, 7.60.
N-(6,7-Dimethoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-
sulphamide (12)
White solid. Mp: 158–160°C. 1H NMR (400 MHz, CDCl3):
d (ppm) ¼ 6.56 (s, 1H, Ar–H), 6.54 (s, 1H, Ar–H), 4.76 (bs, 3H, NH
and NH2), 3.81–3.76 (m, 7H, 2OCH3 and CH–N), 3.09 (dd, A part of
AB; 1H, Ha of CH2, J ¼ 4.6 and 15.7 Hz), 2.82 (t, 2H, CH2, J ¼ 6.3 Hz),
2.71 (dd, B part of AB; 1H, Hb of CH2, J ¼ 8.0 and 15.7 Hz), 2.11–
2.04 (m, 1H, H of CH2), 1.87–1.78 (m, 1H, H of CH2). 13C NMR
(100 MHz, CDCl3): d (ppm) ¼ 147.6 (C), 147.4 (C), 126.9 (C), 125.2
(C), 111.9 (CH), 111.4 (CH), 55.9 (OCH3), 55.88 (OCH3), 50.1 (CH–N),
36.0 (CH2), 29.4 (CH2), 26.5 (CH2). IR (CH2Cl2, cmꢀ1): 3501, 3338,
3252, 3098, 2927, 2851, 1735, 1610, 1547, 1517, 1460, 1449, 1345,
1328, 1265, 1247, 1222, 1208, 1158, 1112, 1071, 1015. Anal. calcd.
for (C12H18N2O4S): C, 50.33; H, 6.34; N, 9.78; S, 11.20. Found: C,
50.27; H, 6.29; N, 9.83; S, 11.26.
Benzyl-N-((6,7-dimethoxy-1,2,3,4-tetrahydronaphthalen-
1-yl)methyl)sulphamoylcarbamate (10)
White solid. Mp: 62–64°C. 1H NMR (400 MHz, CDCl3):
d (ppm) ¼ 7.51 (bs, 1H, NH), 7.36–7.31 (m, 5H, Ph–H), 6.62
(s, 1H, Ar–H), 6.55 (s, 1H, Ar–H), 5.25 (t, 1H, NH, J ¼ 6.3 Hz), 5.16 (s,
2H, OCH2), 3.83 (s, 6H, 2OCH3), 3.28–3.23 (m, 2H, CH and 1H of
CH2NH), 2.96–2.93 (m, 1H, CH2NH), 2.67–2.64 (m, 2H, CH2), 1.83–
1.73 (m, 4H, 2CH2). 13C NMR (100 MHz, CDCl3): d (ppm) ¼ 151.5
(CO), 148.0 (C), 147.5 (C), 134.7 (C of Ph), 130.2 (C), 129.1 (CH of Ph),
129.0 (2CH of Ph), 128.6 (2CH of Ph), 127.8 (C), 112.3 (CH), 111.5
(CH), 68.8 (OCH2), 56.3 (OCH3), 56.0 (OCH3), 48.9 (CH2–N), 37.2
(CH), 29.3 (CH2), 25.9 (CH2), 19.7 (CH2). IR (CH2Cl2, cmꢀ1): 3270,
3064, 3034, 2932, 2856, 2094, 1735, 1610, 1514, 1464, 1454, 1353,
N-((6,7-Dimethoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-
methyl)sulphamide (13)
White solid. Mp: 105–107°C. 1H NMR (400 MHz, CDCl3):
d (ppm) ¼ 6.71 (s, 1H, Ar–H), 6.55 (s, 1H, Ar–H), 4.89 (bs, 2H,
NH2), 4.80 (t, 1H, NH, J ¼ 6.4 Hz), 3.84 (s, 3H, OCH3), 3.82 (s, 3H,
OCH3), 3.34–3.25 (m, 2H, CH and 1H of CH2–N), 2.96–2.94 (m, 1H,
1H of CH2–N), 2.67–2.65 (m, 2H, CH2), 1.83–1.73 (m, 4H, 2CH2).
13C NMR (100 MHz, CDCl3): d (ppm) ¼ 147.5 (C), 147.1 (C), 130.1 (C),
128.3 (C), 111.9 (CH), 111.6 (CH), 56.2 (OCH3), 55.8 (OCH3), 48.4
(CH2–N), 37.1 (CH), 29.1 (CH2), 25.7 (CH2), 19.6 (CH2). IR (CH2Cl2,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Akıncıoglu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 68–76
cmꢀ1): 3495, 3338, 3271, 2935, 2861, 2829, 1644, 1611, 1560,
1513, 1465, 1406, 1330, 1255, 1218, 1188, 1157, 1118, 1090, 1064,
1026. Anal. calcd. for (C13H20N2O4S): C, 51.98; H, 6.71; N, 9.33; S,
10.68. Found: C, 51.93; H, 6.75; N, 9.38; S, 10.63.
1735, 1609, 1514, 1464, 1448, 1404, 1317, 1255, 1217, 1195, 1150,
1118, 1088, 1062, 1026. Anal. calcd. for (C14H21NO4S): C, 56.16; H,
7.07; N, 4.68; S, 10.71. Found: C, 56.10; H, 7.08; N, 4.72; S, 10.73.
Haemolysate preparation
Standard procedure for the synthesis of sulphonamides:
N-(6,7-Dimethoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-
methanesulphonamide (14)
Amine 5 (0.5 g, 2.41 mmol) was dissolved in CH2Cl2 (30 mL) and
this solution was cooled to 0°C. To this solution Et3N (0.29 g,
2.89 mmol) and MeSO2Cl (0.28 g, 2.41 mmol) were added. The
reaction mixture was stirred at room temperature. for 15 h. After
the solvent was evaporated, the residue was chromatographed on
silica gel (30 g) column with 20% EtOAc–hexane. Sulphonamide
14 was synthesized as colourless crystal (0.57 g, 82% yield).
Erythrocytes were isolated from fresh human blood, which was
obtained from Ataturk University Hospital Blood Centre follow-
ing low-speed centrifugation at 3000ꢂg for 15 min (HERMLE Z
383 K) by removal of plasma and buffy coat. The red cells were
washed twice with NaCl (0.9%) and haemolysed with 1.5 volumes
of ice-cold water. Ghost and intact cells were then removed by
high-speed centrifugation at 15,000ꢂg for 30 min at 4°C and the
pH of the haemolysate was adjusted to 8.7 with solid Tris [67].
Purification of hCA I and hCA II by affinity
chromatography
Sepharose-4B-L-tyrosine affinity chromatography column was
prepared according to our previous studies [35]. The pH-adjusted
haemolysate was then subjected to affinity chromatography
using a column packed with Sepharose 4B-L-tyrosine sulphanil-
amide resin [47]. For this purpose, pH value of affinity column
was adjusted to 8.7 with solid Tris. Sepharose-4B-L-tyrosine-
sulphanylamide affinity column was equilibrated with Tris-HCl
(25 mM)/Na2SO4 (0.1 M, pH 8.7). The affinity gel was washed with
Tris-HCl (25 mM)/Na2SO4 (22 mM, pH 8.7). hCA I and hCA II
isoenzymes were eluted with NaCl (1.0 M)/sodium phosphate
(0.25 M; pH 6.3) and sodium acetate (0.1 M)/NaClO4 (0.5 M, pH 5.6),
respectively. Column flow rate was 20 mL/h and 4-mL fractions
were collected. All procedures were performed at 4°C.
N-(6,7-Dimethoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-
methanesulphonamide (14)
Mp: 147–149°C. 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 6.86 (s, 1H,
Ar–H), 6.48 (s, 1H, Ar–H), 4,86 (d, 1H, NH, J ¼ 8.4 Hz), 4.52–4.47
(m, 1H, CH–N), 3.78 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 2.96 (s, 3H,
CH3), 2.69–2.59 (m, 2H, CH2), 1.99–1.94 (m, 1H, H of CH2), 1.87–
1.73 (m, 3H, CH2 and H of CH2). 13C NMR (100 MHz, CDCl3):
d (ppm) ¼ 148.7 (C), 147.7 (C), 129.9 (C), 127.7 (C), 111.7 (CH), 111.6
(CH), 56.2 (OCH3), 56.0 (OCH3), 52.0 (CH–N), 42.3 (CH3), 31.4 (CH2),
28.7 (CH2), 19.7 (CH2). IR (CH2Cl2, cmꢀ1): 3512, 3287, 3053, 3002,
2935, 2857, 2834, 1728, 1610, 1514, 1462, 1450, 1321, 1256, 1219,
1188, 1148, 1118, 1090, 1079, 1054, 1022. Anal. calcd. for
(C13H19NO4S): C, 54.72; H, 6.71; N, 4.91; S, 11.24. Found: C, 54.68;
H, 6.78; N, 4.96; S, 11.30.
Hydratase activity assay
Compounds 15 and 16 were also synthesized by the same
procedure with the yields of 85 and 88%, respectively.
CA hydratase activity was assayed by following the hydration of
CO2 according to the method described by Wilbur and
Anderson [68]. The activity of CO2-hydratase in enzyme units
(EU) was calculated by using the equation (to ꢀ tc/tc) where to and tc
are the times for pH change of the non enzymatic and the
enzymatic reactions, respectively [69].
N-(6,7-Dimethoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-
methanesulphonamide (15)
White solid. Mp: 137–139°C. 1H NMR (400 MHz, CDCl3):
d (ppm) ¼ 6.57 (s, 1H, Ar–H), 6.53 (s, 1H, Ar–H), 4.65 (d, 1H, NH,
J ¼ 7.6 Hz), 3.82–3.78 (m, 7H, 2OCH3 and CH–N), 3.08 (dd, A part of
AB; 1H, Ha of CH2, J ¼ 5.2 and 15.8 Hz), 3.01 (s, 3H, CH3), 2.84 (t, 2H,
CH2, J ¼ 6.3 Hz), 2.70 (dd, B part of AB; 1H, Hb of CH2, J ¼ 8.1 and
15.8 Hz), 2.13–2.07 (m, 1H, H of CH2), 1.87–1.82 (m, 1H, H of CH2).
13C NMR (100 MHz, CDCl3): d (ppm) ¼ 147.7 (C), 147.5 (C), 126.8 (C),
125.0 (C), 112.0 (CH), 111.5 (CH), 55.93 (OCH3), 55.92 (OCH3), 49.8
(CH–N), 41.9 (CH3), 36.6 (CH2), 30.0 (CH2), 26.6 (CH2). IR (CH2Cl2,
cmꢀ1): 3512, 3275, 3064, 3002, 2932, 2852, 1736, 1610, 1547,
1518, 1462, 1448, 1411, 1316, 1265, 1250, 1226, 1150, 1114, 1073,
1017. Anal. calcd. for (C13H19NO4S): C, 54.72; H, 6.71; N, 4.91; S,
11.24. Found: C, 54.71; H, 6.71; N, 4.90 S, 11.26.
Esterase activity assay
Esterase activity of hCA I and hCA II was assayed by following
the change in absorbance at 348 nm of 4-nitrophenyl acetate to
4-nitrophenolate ion over a period of 3 min at 25°C using a
spectrophotometer according to the method described by
Verpoorte et al. [70]. A reference measurement was obtained by
preparing the mixture without the enzyme solution. All
measurements were recorded in triplicate.
Quantitative protein determination
Quantitative protein determination was done by measuring the
absorbance at 595 nm according to Bradford [71], explained
previously [72] using bovine serum albumin as a standard.
N-((6,7-Dimethoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-
methyl)methanesulphonamide (16)
Mp: 142–144°C. 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 6.68 (s, 1H,
Ar–H), 6.58 (s, 1H, Ar–H), 4.31 (t, 1H, NH, J ¼ 6.2 Hz), 3.86 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 3.35 (t, 2H, CH2–N, J ¼ 6.2 Hz), 2.97–2.94
(m, 1H, CH), 2.92 (s, 3H, CH3), 2.70–2.68 (m, 2H, CH2), 1.85–1.74
(m, 4H, 2CH2). 13C NMR (100 MHz, CDCl3): d (ppm) ¼ 148.0 (C),
147.5 (C), 130.3 (C), 128.0 (C), 112.3 (CH), 111.6 (CH), 56.3 (OCH3),
56.1 (OCH3), 48.5 (CH2–N), 40.7 (CH3), 37.8 (CH), 29.3 (CH2), 26.0
(CH2), 19.9 (CH2). IR (CH2Cl2, cmꢀ1): 3434, 3274, 3008, 2928, 2851,
CA inhibition
The inhibitory effects of the novel sulphamides 11–13 and
sulphonamides 14–16 on CA enzyme activity purified from
human erythrocytes were tested in triplicate at each concentra-
tion by using esterase activity assay. CA activities were measured
in the presence of different substrate concentrations as previously
described. The Ki values were determined from a series of
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Arch. Pharm. Chem. Life Sci. 2014, 347, 68–76
New Sulphamides and Sulphonamides as CA and AChE Inhibitors
75
experiments using the novel sulphamides 11–13 and sulphona-
mides 14–16 and 4-nitrophenylacetate as the substrate at five
different concentrations to construct Lineweaver–Burk [73]
curves described previously [74].
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Acetyl cholinesterase inhibition
The inhibitory effects of the novel sulphamides 11–13 and
sulphonamides 14–16 on AChE activities were measured by
slightly modifying the spectrophotometric method of Ellman
et al. [56]. Acetylthiocholine iodide (AChI) was used as substrate of
the reaction. 5,50-Dithio-bis(2-nitrobenzoic)acid (DTNB, Product
no: D8130-1G, Sigma–Aldrich) was used for the measurement of
the AChE activity. Briefly, 100 mL of Tris/HCl buffer (1 M, pH 8.0),
10 mL of sample solution dissolved in deionized water at different
concentrations, and 50 mL of AChE (5.32 ꢂ 10ꢀ3 U) solution were
mixed and incubated for 10 min at 25°C. Then 50 mL of DTNB
(0.5 mM) was added. The reaction was then initiated by the
addition of 50 mL of AChI (10 mM, Product no: 01480-1G, Sigma–
Aldrich). The hydrolysis of these substrates was monitored
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nitrobenzoate anion as the result of the reaction of DTNB with
thiocholine, released by the enzymatic hydrolysis of AChI, at a
wavelength of 412 nm [17]. Absorbance was measured at 405 nm
every 45 s, three times consecutively.
In order to determine the effect of the novel sulphamides 11–13
and sulphonamides 14–16 on AChE, different concentrations of
these novel compounds were added into the reaction medium.
The enzyme activity was measured, and an experiment in the
absence of drug was used as control. The IC50 values were
obtained from activity (%) versus the novel sulphamides 11–13
and sulphonamides 14–16 concentration plots.
To determine the Ki constants in the media with the novel
sulphamides 11–13 and sulphonamides 14–16 as inhibitor,
different ACh concentrations were used. Inhibitor solution was
added into the reaction medium, resulting in three different fixed
concentrations of inhibitors. Lineweaver–Burk graphs [72] were
drawn using 1/V versus and 1/[S] values. Ki constants were
calculated from these graphs. Galantamine was generally used as
a reference compound.
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_
Council of Turkey (TÜBITAK, Grant No. 109T241) and Ataturk
University for their financial support of this work. We also thank Prof.
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