Synthesis and carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine

Article abstract of DOI:10.1016/j.bmc.2013.03.077

A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate (CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six α-carbonic anhydrases (CAs, EC 4.2.1.1), that is, CA I, CA II, CA VA, CA IX, CA XII and CA XIV, and two β-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis (Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low micromolar to nanomolar range by the dopamine sulfamide analogues. K_is were in the range of 0.061-1.822 μM for CA I, 1.47-2.94 nM for CA II, 2.25-3.34 μM for CA VA, 0.041-0.37 μM for CA IX, 0.021-1.52 μM for CA XII, 0.007-0.219 μM for CA XIV, 0.35-5.31 μM for CgCA and 0.465-4.29 μM for Rv3588. The synthesized sulfamides may lead to inhibitors targeting medicinally relevant CA isoforms with potential applications as antiepileptic, antiobesity antitumor agents or anti-infective.

Full text of DOI:10.1016/j.bmc.2013.03.077

Accepted Manuscript
Synthesis and carbonic anhydrase inhibitory properties of sulfamides structur‐
ally related to dopamine
Kadir Aksu, Meryem Nar, Muhammet Tanc, Daniela Vullo, İlhami Gülçin,
Süleyman Göksu, Ferhan Tümer, Claudiu T. Supuran
PII:
S0968-0896(13)00299-X
http://dx.doi.org/10.1016/j.bmc.2013.03.077
BMC 10734
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Please cite this article as: Aksu, K., Nar, M., Tanc, M., Vullo, D., Gülçin, İ., Göksu, S., Tümer, F., Supuran, C.T.,
Synthesis and carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine, Bioorganic
& Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.03.077
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and carbonic anhydrase inhibitory properties of sulfamides
structurally related to dopamine
Kadir Aksu1, Meryem Nar1, Muhammet Tanc2, Daniela Vullo2 İlhami Gülçin1,
Süleyman Göksu1*, Ferhan Tümer3 and Claudiu T. Supuran*2
1Faculty of Science, Department of Chemistry, Atatürk University, 25240-Erzurum, Turkey
2Università degli Studi di Firenze, NEUROFARBA Department, Sezione di Scienze
Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019-Sesto Fiorentino (Florence), Italy
3Faculty of Science and Arts, Department of Chemistry, Sutcu Imam University,
46100-Kahramanmaras, Turkey
__________
*Correspondence authors: Phone :+90 4422314389, Fax :+90 4422360948, E-mail:
sgoksu@atauni.edu.tr (SG); Phone: +39-055-4573005; Fax: +39-055-4573385; e-mail:
claudiu.supuran@unifi.it. (CTS)
1

ABSTRACT
A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction
of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate
(CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by
treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six
α-carbonic anhydrases (CAs, EC 4.2.1.1), i.e., CA I, CA II, CA VA, CA IX, CA XII and CA
XIV, and two ß-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis
(Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low
micromolar to nanomolar range by the dopamine sulfamide analogues. KIs were in the range
of 0.061-1.822 µM for CA I, 1.47-2.94 nM for CA II, 2.25-3.34 µM for CA VA, 0.041-0.37
µM for CA IX, 0.021-1.52 µM for CA XII, 0.007-0.219 µM for CA XIV, 0.35-5.31 µM for
CgCA and 0.465-4.29 µM for Rv3588. The synthesized sulfamides may lead to inhibitors
targeting medicinally relevant CA isoforms with potential applications as antiepileptic,
antiobesity antitumor agents or anti-infective.
Keyword: Dopamine; sulfamoyl carbamates; sulfamide; carbonic anhydrase; enzyme
inhibition
2

1. INTRODUCTION
Sulfamides have interesting biological activities,1 this functional group being present in
several drugs. The dopaminergic drug quinagolide (1), also known as norprolac, is used in the
treatment of hyperprolactinemia.2 Doripenem (2), (tradename doribax) also has a sulfamide
moiety in its scaffold, being a broad-spectrum injectable antibiotic.3 The recently developed
drug candidate 3 (JNJ-26990990) is a powerful anticonvulsant.4 Compound 3 has the potential
to be used in the treatment of depression, neuropathic pain, and inflammatory pain.5
Sulfamide 4 and its derivatives possess protein tyrosine kinase inhibitory activity,6 therapeutic
actions against diabetes, HCV replication inhibition7, and as endothelin receptor antagonists8.
The autacoid dopamine (5) is a neurotransmitter and plays an important role in the
central nervous system-related disorders such as schizophrenia and Parkinson’s disease.9 The
carbonic anhydrase (CA, EC 4.2.1.1) activatory properties of dopamine (5) related
compounds have also been reported10,11 (Figure 1).
Figure 1 here
CA is a well-suited protein 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 catalyzes the hydration of carbon dioxide (CO2) and the dehydration of
-
bicarbonate ion (HCO3 ).12
The CA enzyme family is particularly attractive for biophysical studies of protein-ligand
binding and inhibition studies for the following reasons: (i) CA, a monomeric enzyme, is a
single-chain protein of medium-sized molecular weight (around 30 kDa). Although, the most
widespread CA II have no disulfide bonds, pendant sugar or phosphate groups, all the other
isoforms have disulfide bonds, pendant sugar or phosphate groups, (ii) CA II is an widely
available inexpensive enzyme, (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 isozymes. (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 isoforms12, and their catalytic mechanisms are well understood, (vi) It has not
only a CO2 hydrase/dehydratese activity with a high turnover number, but also possesses an
esterase activity toward carboxylic acid esters, phosphoric acid esters, and even sulfate
3

esters,13-15 (vii) its inhibition mechanism with ligands that can bind to the Zn2+ ion is fairly
simple and well characterized.12,16
-
CA catalyzes the reversible hydration of CO2 to bicarbonate (HCO3 ) (Scheme 1) in a
two-step “ping-pong” mechanism.17 The first step involves the direct nucleophilic attack of a
Zn2+-bound hydroxyl group on CO2 to form a metal-bound HCO3 , followed by displacement
-
of HCO3 by a water molecule. In the second step, the Zn2+-bound water molecule transfers a
-
proton to molecules of buffer in the solvent and regenerates the zinc-hydroxide form of CA.18
Scheme 1 here
So far, 16 different α-CA isoforms have been identified in mammals.13 These
isoenzymes are inhibited by three main inhibition mechanisms: (i) coordination of the
inhibitor to the Zn2+ from the enzyme active site by replacing the zinc-bound water/hydroxide
ion and leading to a tetrahedral geometry of Zn2+, or by addition of the inhibitor to the metal
coordination sphere, when the Zn2+ ion is in a trigonal bipyramidal geometry;16,18-20 (ii) by
anchoring the inhibitor to the Zn2+- bound solvent molecule, that is, a water or a hydroxide
13,21-23
ion, phenols
and polyamines;24 or (iii) by occlusion of the entrance to the active site
cavity,25 when the inhibitors bind in the activator binding region from the CA active site
(Scheme 2).26-31
Scheme 2 here
As sulfamides and dopamine related compounds show important biological activities,
the synthesis and CA inhibitory properties of sulfamides derived from dopamine derivatives
will be useful for further synthetic and biological purposes. In this context, here we report on
the synthesis of sulfamides 19-23. We also evaluate eight CA isoenzymes (CA I, CA II, CA
VA, CA IX, CA XII, CA XIV, CgCA and Rv3588) inhibitory effects of the synthesized
compounds 19-23.
2. RESULTS AND DISCUSSION
2.1. Chemistry
Curtius reaction of carboxylic acids with diphenylphosphoryl azide has been described clearly
in the literature.32,33 By applying this literature procedure, the reaction of acid 6 with diphenyl
o
phosphoryl azide (DPPA) in the presence Et3N at 80 C for 6 hours followed by addition of
o
PhCH2OH and heating at 80 C for 30 hours afforded a new carbamate 7 in excellent yield.
Hydrogenolysis of benzyloxycarbamates in the presence of CHCl3 is a convenient method for
the synthesis of amine hydrochloride salts.34 Pd-C catalyzed hydrogenolysis of 7 in MeOH-
4

CHCl3 (CHCl3 is used for producing HCl)35 afforded dopamine analogue 8 as the
hydrochloride salt which was neutralized to its free amine 9 with 10% NaOH (Scheme 3).
Scheme 3 here
Dopamine related compounds 10-13 are commercially available. The synthesis of sulfamides
from the reaction of amines with chlorosulfonyl isocyanate (CSI) has been reported.36,37
Following these synthetic methodologies, the reactions of 1 eq. of amines 9-11 and 1.5 eq. of
t-BuOH and the reactions of 1 eq. of amines 12, 13 and 1.5 eq. of PhCH2OH with CSI at 0-25
oC for 4 hours afforded novel sulfamoyl carbamates 14-18 in high yields. The cleavage of N-
tert-butylamides with trifluoroacetic acid has been reported by Catt and Matier.38 By a similar
approach, the reaction of sulfamide carbamates 14-16 with trifluoroacetic acid at room
temperature afforded novel sulfamides 19, 20, and a known sulfamide 2139,40 in excellent
yields. On the other hand, palladium catalyzed hydrogenolysis of carbamates 17 and 18 gave
sulfamides 227 and 237 in good yields (Scheme 4). The structures of all synthesized
compounds were characterized by 1H- and 13C-NMR spectroscopic techniques. The functional
groups were characterized by IR.
Scheme 4 here
2.2. CA isoenzymes inhibition studies
2.2.2 CA isoenzymes inhibition
Methods for isolation and purification of CA are well established and widely accessible.
Commercial sources usually isolate isozymes hCA I and hCA II from fresh human red blood
cells, in which CA is the second most abundant protein after hemoglobin. Research
laboratories typically produce these isozymes by recombinant technology in E. coli.41,42 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.43
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 centrifugation, followed by
separation of hemoglobin from hCA on a sulfonamide-modified agarose affinity column.44,45
Divalent anions like sulfate do not bind appreciably in the active site of CA. Therefore, they
are used to remove nonspecifically bound protein by screening ionic interactions. Also, it was
5

shown that CA isozymes 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.18,44
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 sulfonamides
constitute the main class of CA inhibitors, which can bind to the metal ion from the enzyme
active site.46 CA inhibitors block the function of the CA enzyme. The myriad inhibitors of CA
can be divided into four main groups: (i) sulfonamides / sulfamides, (ii) other sulfonic acid
derivatives, (iii) small monoanions such as halides, azides, and thiocyanate and, (iv) inhibitors
with aromatic rings / phenolic compounds. The sulfonamides, sulfamides and sulfonic acid
derivatives have values of Ki in the picomolar to micromolar range, whereas the inorganic
monoanions bind CA with values of Ki in the micromolar to millimolar range.18 Similarly,
phenolic inhibitors with aromatic rings had Ki values in the micromolar to millimolar
range.13,23,47-51
Inhibition data against all the catalytically active CA isozymes, that is, CA I, CA II,
CA VA, CA IX, CA XII, CA XIV, CgCA and Rv3588 with sulfamides 19-23 are presented in
Table 1 and they were obtained at pH 7.4 in 10 mM Hepes buffer, at 25 °C, by a stopped-flow
assay monitoring the CO2 hydration reaction by Khalifah52 as described previously.13,23 The
reference compound acetazolamide (AAZ) has been tested for enzyme inhibition against all
eight CA isoforms (Table 1). All tested sulfamides 19-23 showed Ki values in the lower µM
to the nanomolar range (Ki <5 µM, see Table 1).
Insert Table 1 here
The following should be noted regarding the inhibition of these CA isozymes with sulfamides
19-23:
(i) The hCA I enzyme was effectively inhibited well by all tested sulfamides 19-23. Against
isozyme hCA I, sulfamides 19-23 exhibited excellent inhibitory activity, with inhibition
constants in the range of 61 nM –1.82 µM. The best inhibitor was sulfamide 22 (with a Ki
of 61 nM), whereas the worst inhibition was observed for sulfamides 19 having three
methoxy groups (-OCH3) (with a Ki of 1.82 µM). Compound 22, which has a methoxy
group on the para- position (Scheme 4.), showed a better Ki value compared to the other
compounds. Thus, para- position of sulfamide and methoxy group (-OMe) lead to
6

effective hCA I inhibitors. Despite all this, the differences in Ki value between sulfamides
19-23 were rather small.
(ii) The physiologically dominant16,20 cytosolic human isoenzyme hCA II was very effectively
inhibited by sulfamides 19-23, with Kis in the range of 1.47-2.95 nM. The Ki values of
sulfamides 19-23 were similar to each other. The differences between highest and lowest
Ki values of sulfamides 19-23 were only two-fold. The structure–activity relationship is
rather similar to what outlined above for hCA I, because the two enzymes (hCA I and
hCA II) have a high sequence homology of amino acid present within the active site), but
these sulfamides were much more effective as hCA II inhibitors than as hCA I
inhibitors.16,20 The Ki values of all sulfamides 19-23 were lower than that of
acetazolamide (Ki of 4.9 nM).
(iii) Isoform CA VA was poorly inhibited by sulfamides 19 and 20 (KIs > 50 µM) The other
sulfamides 21-23 showed Kis in the range of 2.28 – 3.34 µM. Sulfamides 21 was the best
hCA VA inhibitor.
(iv) The two transmembrane, tumor-associated isozymes hCA IX and XII were also
effectively inhibited by sulfamides 19-23 with Kis in the range of 41 nM –0.376 µM for
hCA IX, and of 21 nM –1.52 µM for hCA XII, respectively. Sulfamides 20 was a,
inefficient CA IX inhibitor. As these isozymes are overexpressed in hypoxic tumors
where there are also reactive oxygen species damaging the tissue,13,23 the inhibition of
these isoforms with sulfamides also possessing antioxidant activity might be of interest to
be studied in more details. CA XIV, another transmembrane isoform, was also effectively
inhibited by sulfamides 19-23, with inhibition constants in the range of 7 nM-0.22 µM.
(v) Another studied CA isoform was the ß-CA (CgCA) purified from the pathogenic yeast
Candida glabrata. CgCA exhibited a particular inhibition profile with sulfamides 19-23.
All investigated sulfamides 19-23 were effective inhibitors, with Kis in the range of 0.35-
5.31 µM. The most potent CgCA inhibitor was sulfamide 22 with an inhibitor constant of
0.35 µM.
7

(vi) Finally another β-class CA (Rv3588) encoded in the genome of Mycobacterium
tuberculosis was investigated here. Rv3588 was effectively inhibited by all investigated
sulfamides (19-23), with inhibition constants in the range of 0.46-4.23 µM. As well as
CA IX, Rv3588 is not regularly inhibited by sulfonamide 20. Hence, the inhibition
profile of sulfonamide 20 was not determined on Rv3588.
Sulfamides are among the possible isoesteres of sulfonamides, in which an additional
electron-withdrawing atom such as oxygen or grup such as NH is directly attached to the
sulfamoyl function, generating compounds with general formula of R-NH-SO2NH2. These
groups were considered as interesting candidates for CA inhibitors. First, Supuran’s group
showed that a simple sulfamide binds to the metal ion in the Co2+-substituted CA II,53
whereas the X-ray structures of the compound in adduct with CA II was reported several
years later.54 Nowadays, there are a large number of aromatic, heterocyclic, aliphatic, and
sugar-based sulfamides, which were shown to possess highly effective inhibitory properties
against all known mammalian isoforms.16 Therefore, sulfamides constitute a highly important
class of CA inhibitors, with some derivatives clinically used for the treatment of epilepsy and
obesity.
Novel CA inhibitors have been designed for pharmacological and medicinal
approaches, and many inhibitors have been synthesized recently.55 There are many studies in
the literature on the interactions of different compounds and CA isoenzymes. Recently, the
inhibitory effects of dantrolene,56 melatonin,57 vitamin E,58 morphine,59 ethanol,60 antioxidant
phenols,50 phenolic acids,23,48 distinguished natural polyphenols products, prominent phenolic
compounds,49 bromophenols61 with hCA I and hCA II has been investigated by our group.
Indeed, the inhibition profiles of hCA I, and hCA II with compounds 19-23 are very variable,
with inhibition constants ranging from the subnanomolar to the submicromolar levels. For this
purpose, hCA I and hCA II inhibitory effects of the sulfamide compounds 19-23 were tested
under in vitro conditions and their Ki values were calculated and presented in Table 1.
We report here the initial study on the inhibitory effects of synthesized sulfamides 19-
23 and on CA I, CA II, CA VA, CA IX, CA XII, CA XIV, CgCA and Rv3588. Our data show
that sulfamides 19-23 possess effective inhibitory properties against the various CA
isoenzymes, the strongest inhibitory effect being observed with against the rapid cytosolic
isoenzymes CA II (Kis: 1.47-2.94 nM).
8

It has been reported that sulfamide and sulfamic acid act as moderate hCA II
inhibitors, with inhibition constants of 1130 μM for sulfamide and 390 μM for sulfamic acid
at the physiological pH (7.4), respectively.46 In the current study, we observed strong
inhibitory effects of the newly synthesized sulfamides 19-23 on eight CA enzymes.
3. CONCLUSIONS
In summary, dopamine analogue 8, mescaline hydrochloride, was synthesized starting from 3-
(3,4,5-trimethoxyphenyl)propanoic acid (6) via Curtius reaction by an alternative method. By
this study, a series of sulfamides 19-23 were synthesized starting from the corresponding
dopamine analogues. The synthesized sulfamoyl carbamates 14-18 and novel sulfamides 19,
20 may be important for further synthetic and biological purposes. In addition, several
sulfamide compounds 19-23 have been assayed for the inhibition of the physiologically
relevant human carbonic anhydrase isoforms like CA I, CA II, CA VA, tumour-associated
isozymes CA IX and CA XII, CA XIV, two ß-carbonic anhydrase isoforms like CgCA from
Candida glabrata and Rv3588 from Mycobacterium tuberculosis. The sulfamides
investigated here showed effective CA inhibitory effects, in the low-micromolar to nanomolar
range. In general, the compounds 19-23 had comparable inhibitory activity with the clinically
used sulfonamide AAZ. These findings clearly showed that the synthesized sulfamides 19-23
may pave the way for generating potent CA inhibitors eventually targeting isoforms that have
not been investigated in detil for thei inhibition profile by sulfamides.
4. 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
1
mm cells with a Perkin-Elmer spectrophotometer. The H and 13C NMR spectra were
recorded on a 400 (100)-MHz Varian and 400 (100)-MHz Bruker spectrometer; δ in ppm,
Me4Si as the internal standard. Elemental analyses were performed on a Leco CHNS-932
apparatus. All 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.
9

4.1. Synthesis
4.1.1. Synthesis of benzyl 3,4,5-trimethoxyphenethylcarbamate (7)
A solution of DPPA (2.75 g, 9.99 mmol) and NEt3 (1.01 g, 9.99 mmol) in benzene (10 ml)
were added to a solution of 3-(3,4,5-trimethoxyphenyl)propanoic acid (6) (2.00 g, 8.32 mmol)
o
in benzene (30 mL). After the reaction mixture was stirred at 80 C for 6 h, PhCH2OH (2.70
o
g, 24.97 mmol) was added to the reaction mixture and heated at 80 C for 30 h. The solvent
was evaporated. The column chromatography of the residue on silica gel (100 g) with 15%
o
EtOAc-Hexane yielded carbamate 7 (2.05 g, 71%). White solid. Mp 78-80 C.; IR (CH2Cl2,
1
cm-1) 3368, 2929, 1720, 1591, 1547, 1462, 1345, 1264, 1127. H NMR (400 MHz, CDCl3) δ
7.36-7.38 (m, 5H, Ph-H), 6.41 (s, 2H, Ar-H), 5.13 (s, 2H, CH2), 4.82 (bs, 1H, NH), 3.87 (s,
3H, OCH3), 3.85 (s, 6H, OCH3), 3.50 (m, 2H, CH2), 2.79 (m, 2H, CH2). 13C NMR (100 MHz,
CDCl3 ) δ 156.3 (CO), 153.3 (2C), 136.5 (2C), 134.4 (C), 128.6 (2CH), 128.19 (CH), 128.15
(2CH), 105.6 (2CH), 66.7 (OCH2), 60.9 (OCH3), 56.1 (2OCH3), 42.2 (CH2NH), 36.5 (CH2).
Anal. Calcd for (C19H23NO5): C 66.07, H 6.71, N 4.06; Found C 66.03, H 6.74, N 4.07.
4.1.2. Synthesis of 2-(3,4,5-trimethoxyphenyl)ethanamine hydrochloride (8)
Pd-C (100 mg) and benzyl 3,4,5 trimethoxyphenethylcarbamate (7) (1.75 g 5.07 mmol) in
MeOH-CHCl3 (4:1, 100 mL) were placed into a 250-mL flask. A balloon filled with H2 gas
(3 L) was fitted to the flask. The mixture was deoxygenated by flushing with H2. The
reaction mixture was hydrogenated at rt for 24 h. The catalyst was removed by filtration.
Recrystallization of the residue from MeOH-Et2O afforded hydrochloride salt 8 (1.05 g, 84%
yield). White solid. Mp 184-186 oC. (lit67 Mp: 183-184) 1H NMR (400 MHz, CD3OD) δ 6.59
(s, 2H, Ar-H), 4.91 (bs, H of NH3 and MeOH), 3.84 (s, 6H, OCH3), 3.73 (s, 3H, OCH3), 3.18
(t, 2H, J=7.6 Hz, CH2), 2.90 (t, 2H, J=7.6 Hz, CH2). 13C NMR (100 MHz, CD3OD) δ 153.7
(2C), 140.3 (C), 132.8 (C), 105.8 (2CH), 59.9 (OCH3), 55.4 (2OCH3), 40.8 (CH2N), 33.7
(CH2).
4.1.3 The synthesis of 3,4,5-trimethoxyphenylethanamine (9)
Amine hydrochloride salt 8 (0.95 g, 3.83 mmol,) was dissolved in MeOH (30 ml) and cooled
o
to 0 C. To this solution was added a solution of 10% NaOH (15 ml). The reaction mixture
was stirred at room temp. for 3 h. After most of the MeOH was evaporated, CH2Cl2 (50 ml)
and H2O (20 mL) were added to the residue. Organic layer was separated and H2O layer was
10

extracted with CH2Cl2 (2x30 mL). Combined organic layers were dried over Na2SO4 and
CH2Cl2 was evaporated to give oily mescaline 9 (0.79 g, 98%).
1H- and 13C-NMR data is in agreement with data given in the literature.68
4.1.4. Standard procedure for the synthesis of tert-butyl sulfamoylcarbamates: tert-butyl N-
(3,4,5-trimethoxyphenethyl)sulfamoylcarbamate (14)
t-BuOH (0.49 g, 6.63 mmol) was added to a solution of CSI (0.67 g, 4.73 mmol) in CH2Cl2
o
(10 mL) at 0 C. A solution of amine (9) (1.00 g, 4.73 mmol) in CH2Cl2 (30 ml) and NEt3
(0.53g, 5.21 mmol) were added to the solution of CSI-t-BuOH drop wise and it was stirred at
o
o
0 C for 1 h. then at room temp for 3 h. The reaction mixture was cooled to 0 C and to this
mixture was added a solution of 0.1 N HCl (50 mL). Organic phase was separated and H2O
phase was extracted with CH2Cl2 (2x30 mL). Combined organic layers were dried over
Na2SO4 and the solvent was evaporated. Column chromatography of the residue on silica gel
(50 g) with 25% EtOAc-Hexane gave carbamate 14 (1.52 g, 82%). Yellow oil. IR (CH2Cl2,
1
cm-1) 3361, 3274, 2977, 2939, 1702, 1593, 1460, 1367, 1349, 1244, 1146, 1127. H NMR
(400 MHz, CDCl3) δ 7.31 (bs, 1H, NH), 6.36 (s, 2H, Ar-H), 5.14 (t, 1H, NH, J=6.1 Hz), 3.79
(s, 6H, OCH3), 3.76 (s, 3H, OCH3), 3.26 (q, 2H, CH2NH, J=6.7 Hz), 2.76 (t, 2H, CH2; J=7.0
Hz), 1.38 (s, 9H, CH3). 13C NMR (100 MHz, CDCl3) δ 153.5 (2C), 150.16 (CO), 136.9 (C),
133.3 (C), 105.6 (2CH), 83.9 (OC), 60.9 (OCH3), 56.1 (2OCH3), 44.9 (CH2NH), 35.8 (CH2),
27.9 (3CH3). Anal. Calcd for (C16H26N2O7S): C 49.22, H 6.71, N 7.17, S 8.21; Found C
49.20, H 6.74, N 7.12, S 8.23.
t-Butyl sulfamoylcarbamates 15 and 16 were also synthesized by this procedure with yields
of 76% and 78% respectively.
4.1.5. tert-butyl N-(2,3-dimethoxyphenethyl)sulfamoylcarbamate (15)
o
White solid. Mp 113-115 C. IR (CH2Cl2, cm-1) 3274, 2974, 1720, 1655, 1586, 1480, 1351,
1268, 1144. 1H NMR (400 MHz, CDCl3) δ 7.53 (bs, 1H, NH), 6.98-7.02 (m, 1H, Ar-H), 6.83
(dd, 1H, Ar-H, J=8.2, J=1.4 Hz), 6.77 (dd, 1H, Ar-H, J=7.6, J=1.3 Hz), 5.40 (t,1H, NH, J=5.8
Hz), 3.85 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.30 (m, 2H, CH2), 2.90 (t, 2H, CH2, J=7.0 Hz),
1.43 (s, 9H, CH3). 13C NMR (100 MHz, CDCl3) δ 152.8 (CO), 150.2 (C), 147.3 (C), 131.5
(C), 124.3 (CH), 122.3 (CH), 111.5 (CH), 83.6 (OC), 60.7 (OCH3), 55.7 (OCH3), 44.4
(CH2NH), 30.1 (CH2), 27.9 (3CH3). Anal. Calcd for (C15H24N2O6S): C 49.99, H 6.71, N 7.77,
S 8.90; Found C 50.01, H 6.73, N 7.78, S 8.88.
4.1.6. Tert-butyl N-(3,4-dimethoxyphenethyl)sulfamoylcarbamate (16)
11

o
White solid. Mp 163-164 C. IR (CH2Cl2, cm-1) 3442, 2926, 1726, 1656, 1518, 1459, 1346,
1262, 1143. 1H NMR (400 MHz, CDCl3) δ 7.20 (bs, 1H, NH), 6.82 (d, 1H, Ar-H, J=8.1 Hz),
6.76-6.72 (m, 2H, Ar-H), 5.12-5.10 (m, 1H, NH), 3.88 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
3.33-3.30 (m, 2H, CH2), 2.84 (t, 2H, CH2, J=7.0 Hz), 1.45 (s, 9H, CH3). 13C NMR (100 MHz,
CDCl3) δ 150.0 (CO), 149.3 (C), 148.1 (C), 130.0 (C), 120.7 (CH), 111,9 (CH), 111.6 (CH),
83.91 (OC), 56.0 (OCH3), 55.9 (OCH3), 45.1 (CH2NH), 35.0 (CH2), 27.9 (3CH3). Anal. Calcd
for (C15H24N2O6S): C 49.99, H 6.71, N 7.77, S 8.90; Found C 49.94, H 6.69, N 7.80, S 8.93.
4.1.7. Standard procedure for the synthesis of benzyl sulfamoylcarbamates: benzyl N-(4-
methoxyphenethyl)sulfamoylcarbamate (17)
PhCH2OH (0.27 g, 2.48 mmol) was added to a solution of CSI (0.24 g, 1.65 mmol) in CH2Cl2
o
(10 ml) at 0 C. A solution of amine (12) (0.25 g, 1.65 mmol) in CH2Cl2 (15 ml) and NEt3
(0.19 g, 1.82 mmol) were added to the solution of CSI-PhCH2OH drop wise and it was stirred
at 0 oC for 1 h. then at room temp for 3 h. The reaction mixture was cooled to 0 oC. A solution
of 0.1 N HCl (50 mL) was added to the mixture. Organic phase was separated and H2O phase
was extracted with CH2Cl2 (2x30 mL). Combined organic layers were dried over Na2SO4. The
solvent was evaporated. Carbamate 17 was purified by column chromatography of the residue
on silica gel (30 g) with 20% EtOAc-Hexane (0.48 g, 80%). White solid. Mp 137-139 oC; IR
(CH2Cl2, cm-1) 3422, 3288, 2926, 1731, 1449, 1341, 1265, 1149; 1H NMR (400 MHz, CDCl3)
δ 7.76 (bs, 1H, NH), 7.38-7.32 (m, 5H, Ar-H), 7.08 (A part of AB, d, 2H, J=8.6 Hz, Ar-H),
6.85 (B part of AB, d, 2H, J=8.6 Hz, Ar-H), 5.32 (t, 1H, J=6.0 Hz, NH), 5.14 (s, 2H, OCH2),
3.80 (s, 3H, OCH3), 3.32-3.27 (m, 2H, CH2NH), 2.79 (t, 2H, CH2, J=7.2 Hz). 13C NMR (100
MHz, CDCl3) δ 158.5 (CO), 151.2 (C), 134.5 (C), 129.7 (2CH), 129.3 (C), 128.9 (CH), 128.8
(2CH), 128.5 (2CH), 114.2 (2CH), 68.6 (OCH2), 55.3 (OCH3), 45.1 (CH2NH), 34.5 (CH2);
Anal. Calcd for (C17H20N2O5S): C 56.03, H 5.53, N 7.69, S 8.80; Found C 56.07, H 5.56, N
7.66, S 8.79.
Benzyl-sulfamoylcarbamate 18 was also synthesized by this procedure with yields of 85%.
4.1.8. Benzyl N-(2-methoxyphenethyl)sulfamoylcarbamate (18)
o
White solid. Mp: 105-108 C; IR (CH2Cl2, cm-1) 3494, 3279, 2937, 1722, 1495, 1455, 1351,
1244, 1158. 1H NMR (400 MHz, CDCl3) δ 7.64 (bs, 1H, NH), 7.39-7.32 (m, 5H, Ar-H), 7.26
(dt, 1H, Ar-H, J=8.0, J= 1.7 Hz), 7.12 (dd, 1H, Ar-H, J=7.4, J=1.5 Hz), 6.93-6.87 (m, 2H, Ar-
H), 5.46 (t, 1H, NH, J=6.1 Hz), 5.13 (s, 2H, OCH2), 3.83 (s, 3H, OCH3), 3.36-3.31 (m, 2H,
CH2NH), 2.87 (t, 2H, CH2, J=6.9 Hz). 13C NMR (100 MHz, CDCl3) δ 157.4 (CO), 151.2 (C),
12

134.6 (C), 130.7 (CH), 128.8 (2CH), 128.7 (2CH), 128.6 (C), 128.4 (CH), 128.3 (CH), 126.0
(C), 120.8 (CH), 110.5 (CH), 68.4 (OCH2), 55.3 (OCH3), 44.0 (CH2NH), 30.3 (CH2). Anal.
Calcd for (C17H20N2O5S): C 56.03, H 5.53, N 7.69, S 8.80; Found C 56.02, H 5.57, N 7.71, S
8.79.
4.1.9. Standard procedure for the synthesis of sulfamides (Hydrolysis of tert-butyl
sulfamoylcarbamates): N-(3,4,5-trimethoxyphenethyl) sulfamide (19)
t-Butyl sulfamoylcarbamate (14) (0.73 g, 1.87 mmol) was added to a solution of TFA-CH2Cl2
(1:1 10 mL). The mixture was stirred at room temperature for 12 h. Then, the solvent was
evaporated. Recrystallization of the residue from CH2Cl2-hexane gave 19 (0.45 g, 83% yield).
White solid. 109-111 oC. IR (CH2Cl2, cm-1) 3269, 2936, 1737, 1593, 1509, 1460, 1423, 1332,
1
1153, 1125. H NMR (400 MHz, CDCl3) δ 6.42 (s, 2H, Ar-H), 4.80 (bs, 3H, NH and NH2),
3.82 (s, 6H, OCH3), 3.79 (s, 3H, OCH3), 3.32 (t, 2H, CH2, J=7.1 Hz), 2.79 (t, 2H, CH2, J=7.1
Hz). 13C NMR (100 MHz, CDCl3) δ 153.5 (2C), 136.7 (C), 134.2 (C), 106.0 (2CH), 61.1
(OCH3), 56.3 (2OCH3), 44.8 (CH2NH), 36.2 (CH2). Anal. Calcd for (C11H18N2O5S): C 45.51,
H 6.25, N 9.65, S 11.04; Found C 45.49, H 6.28, N 9.63, S 11.05.
Sulfamides 20 and 21 were also synthesized by this procedure with yields of 78% and 75%
respectively.
4.1.10. N-(2,3-dimethoxyphenethyl)sulfamide (20)
o
White solid. Mp 107-108 C. IR (CH2Cl2, cm-1) 3350, 3274, 2929, 1736, 1630, 1548, 1481,
1
1266, 1157. H NMR (400 MHz, CDCl3) δ 7.02-6.98 (m, 1H, Ar-H), 6.77-6.83 (m, 2H, Ar-
H), 4.81 (t, 1H, NH, J=5.8 Hz), 4.74 (bs, 2H, NH2), 3.85 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
3.35 (q, 2H, CH2, J=6.7 Hz), 2.89 (t, 2H, J=6.8 Hz, CH2). 13C NMR (100 MHz, CDCl3) δ
152.8 (C), 147.2 (C), 132.1 (C), 124.4 (CH), 122.3 (CH), 111.2 (CH), 60.7 (OCH3), 55.7
(OCH3), 44.2 (CH2NH), 30.1 (CH2). Anal. Calcd for (C10H16N2O4S): C 46.14, H 6.20, N
10.76, S 12.32; Found C 46.10, H 6.23, N 10.74, S 12.34.
4.1.11. N-(3,4-dimethoxyphenethyl)sulfamide (21)
o
White solid. Mp 96-98 C. IR (CH2Cl2, cm-1) 3391, 2925, 2845, 1636, 1517, 1465, 1330,
1
1262, 1156. H NMR (400 MHz, CDCl3) δ 6.81 (d, 1H, Ar-H, J=8.3 Hz), 6.76-6.74 (m, 2H,
Ar-H), 4.74 (bs, 3H, NH and NH2), 3.87 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.35 (t, 2H,
CH2NH, J=7.0 Hz), 2.82 (t, 2H, CH2, J=7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 149.3 (C),
148.1 (C), 130.0 (C), 120.7 (CH), 111.9 (CH), 111.6 (CH), 56.0 (OCH3), 55.9 (OCH3), 45.1
13

(CH2NH), 35.0 (CH2). Anal. Calcd for (C10H16N2O4S): C 46.14, H 6.20, N 10.76, S 12.32;
Found C 46.17, H 6.16, N 10.73, S 12.33.
4.1.12. Standard procedure for the synthesis of sulfamides (Hydrogenolysis of benzyl
sulfamoylcarbamates): N-(4-methoxyphenethyl) sulfamide (22)
Into a 100 mL flask were placed Pd-C (50 mg) and benzyl sulfamoylcarbamate 17 (0.45 g
1.23 mmol) in MeOH (50 mL). 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 rt for 4 h. The
catalyst was removed by filtration. Recrystallization of the residue from EtOAc-hexane gave
22 (0.21 g, 74%). White solid. Mp 95-98oC. IR (CH2Cl2, cm-1) 3283, 2927, 2851, 1735, 1612,
1
1547, 1514, 1247, 1157. H NMR (400 MHz, CDCl3) δ 7.13 (d, 2H, Ar-H, J=8.5 Hz), 6.85
(d, 2H, J=8.5 Hz, Ar-H), 4.63 (bs, 2H, NH2), 4.49 (bs, 1H, NH), 3.78 (s, 3H, OCH3), 3.34 (t,
2H, CH2NH, J=6.9 Hz), 2.82 (t, 2H, CH2, J=6.9 Hz). 13C NMR (100 MHz, CDCl3) δ 158.5
(C), 129.94 (C), 129.85 (2CH), 114.2 (CH), 55.3 (OCH3), 44.8 (CH2NH), 34.7 (CH2). Anal.
Calcd for (C9H14N2O3S): C 46.94, H 6.13, N 12.16, S 13.92; Found C 46.90, H 6.12, N 12.14,
S 13.94.
Sulfamide 23 was also synthesized by this procedure with yields of 70%.
4.1.13. N-(2-methoxyphenethyl)sulfamide (23)
Brown Oil. IR (CH2Cl2, cm-1) 3282, 3002, 2955, 1601, 1587, 1495, 1464, 1439, 1327, 1245,
1158. 1H NMR (400 MHz, CDCl3) δ 7.21-7.13 (m, 2H, Ar-H), 6.89-6.82 (m, 2H, Ar-H), 4.75
(bs, 3H, NH and NH2), 3.79 (s, 3H, OCH3), 3.31 (t, 2H, CH2NH, J=7.0 Hz), 2.86 (t, 2H, CH2,
J=7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 157.6 (C), 130.8 (CH), 128.1 (CH), 126.7 (C),
120.7 (CH), 110.6 (CH), 55.4 (OCH3), 43.5 (CH2NH), 30.5 (CH2). Anal. Calcd for
(C9H14N2O3S): C 46.94, H 6.13, N 12.16, S 13.92; Found C 46.91, H 6.11, N 12.13, S 13.94.
4.2. Inhibition studies
An Applied Photophysics stopped-flow instrument has been used for assaying the CA
catalysed CO2 hydration activity.52 Phenol red (at a concentration of 0.2 mM) has been used
as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) as
buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial
rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s.52 The CO2
concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and
14

inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction
have been used for determining the initial velocity. The uncatalyzed rates were determined in
the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (10
mM) were prepared in distilled-deionized water and dilutions up to 0.001 nM were done
thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together
for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I
complex. The inhibition constants were obtained by non-linear least-squares methods using
PRISM 3, as reported earlier,11.13 and represent the mean from at least three different
determinations. All CA isoforms were recombinant ones obtained in house as reported earlier.
10-15
The concentration of the enzymes in the assays was in the range of 5.3 – 12.5 nM. All
enzymes were recombinant ones pèrepared as described earlier by one of our groups.23-25
Acknowledgments
We are greatly indebted to Ataturk University (BAP2012/471) for financial support of this
study for (KA, SG). This research was financed by two 7th FP EU projects (Metoxia and
Dynano) for (CTS).
15

REFERENCES
1.
Winum, J.Y.; Scozzafava, A.; Montero, J.L.; Supuran C.T. Mini Rev. Med. Chem. 2006,
6, 921.
2.
Winum, J.Y.; Scozzafava, A.; Montero, J.L.; Supuran, C.T. Med. Res. Rev. 2006, 26,
767.
3.
4.
Brown, S.D.; Traczewski, M.M. J. Antimicrob. Chemother. 2005, 55, 944.
Parker, M.H.; Smith-Swintosky, V.L.; McComsey, D.F.; Huang, Y.; Brenneman, D.;
Klein, B.; Malatynska, E.; White, H.S.; Milewski, M.E.; Herb, M.; Finley, M.F.; Liu, Y.;
Lubin, M. L.; Qin, N.; Iannucci, R.; Leclercq, L.; Cuyckens, F.; Reitz, A.B.; Maryanoff,
B.E. J. Med. Chem. 2009, 52, 7528.
5.
6.
7.
Smith-Swintosky, V.L. US Patent 20070191449A1, 2007, Chem. Abstr. 2007, 147,
277426.
Schou, S.C.; Greve, D.R.; Nielsen, S.F.; Jensen, J.B.; Dack, K.N. PCT Int. Appl. WO
2012093169A1, 2012, Chem. Abstr. 2012, 157, 198297.
Seiwert, S.D.; Blatt, L.M.; Andrews, S.W.; Martin, P.; Schumacher, A.; Barnett, B.R.;
Eary, T.C.; Kaus, R.; Kercher, T.; Liu, W.; Lyon, M.; Nichols, P.; Wang, B.; Sammakia,
T.; Kennedy, A.; Jiang, Y. PCT Int. Appl., WO 2007015824A2, 2007, Chem. Abstr.
2007, 146, 229612.
8.
9.
Bolli, M.; Boss, C.; Fischli, W.; Clozel, M.; Weller, T. PCT Int. Appl., WO 2002053557
A1, 2002, Chem. Abstr. 2002, 137, 93766.
Haadsma-Svensson, S.R.; Svensson, K.A. CNS Drug Rev. 1998, 4, 42.
10. Senturk, M.; Ekinci, D.; Göksu, S.; Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2012,
27, 365.
11. Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C.T. J. Med. Chem.
2010, 53, 5511.
12. Alterio, A.; Fiore, A.D.; D’Ambrosio, K.; Supuran, C.T.; Simone, G.D. Chem. Rev.
2012, 112, 4421.
13. Innocenti, A.; Öztürk Sarıkaya, S.B.; Gülçin, İ.; Supuran C.T. Bioorg. Med. Chem.,
2010, 18, 2159.
14. Ekinci, D.; Kurbanoglu, N.I.; Salamci, E.; Şenturk M.; Supuran, C.T. J. Enzyme Inhib.
Med. Chem. 2012, 27, 845.
15. Ekinci, D.; al-Rashida, M.; Abbas, G.; Şentürk, M.; Supuran, C.T. J. Enzyme Inhib.
Med. Chem. 2012, 27, 744.
16. Supuran, C.T. Nat. Rev. Drug Disc. 2008, 7, 168.
16

17. Lindskog, S.; Silverman, D.N. In The Carbonic Anhydrases: New Horizons;
Chegwidden, W.R., Carter, N.D., Edwards, Y.H., Eds.; Birkhauser Verlag: Basel,
Switzerland, Vol. 90, 2000.
18. Krishnamurthy, V.M.; Kaufman, G.K.; Urbach, A.R.; Gitlin, I.; Gudiksen, K.L.; Weibel,
D.B.; Whitesides G.M. Chem. Rev. 2008, 108, 946.
19. Supuran, C.T. Curr. Pharm. Des. 2008, 14, 603.
20. Supuran, C.T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336.
21. Nair, S.K.; Ludwig, P.A.; Christianson, D.W. J. Am. Chem. Soc. 1994, 116, 3659.
22. Simonsson, I.; Jonsson, B.H.; Lindskog, S. Biochem. Biophys. Res. Commun. 1982, 108,
1406.
23. Innocenti, A.; Gülçin, İ.; Scozzafava, A.; Supuran C.T. Bioorg. Med. Chem. Lett. 2010,
20, 5050.
24. Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran C.T. J. Med.
Chem. 2010, 53, 5511.
25. Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C.T. J.
Med. Chem. 2010, 53, 335.
26. Çoban T.A.; Beydemir, S.; Gülçin, İ.; Ekinci, D.; Innocenti, A.; Vullo, D.; Supuran, C.
T.; Bioorg. Med. Chem. 2009, 17, 5791.
27. Temperini, C.; Scozzafava, A.; Puccetti, L.; Supuran, C.T. Biorg. Med. Chem. Lett.
2005, 15, 5136.
28. Temperini, C.; Scozzafava, A.; Vullo, D.; Supuran, C.T. Chemistry, 2006, 12, 7057.
29. Temperini, C.; Vullo, D.; Scozzafava, A.; Supuran, C.T. J. Med. Chem. 2006, 49, 3019.
30. Temperini, C.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C.T. Bioorg.
Med. Chem. Lett. 2007, 17, 628.
31. Temperini, C.; Innocenti, A.; Scozzafava, A.; Supuran, C.T. Bioorg. Med. Chem. 2008,
16, 8373.
32. Göksu, S.; Kazaz, C.; Sütbeyaz, Y.; Seçen, H. Helv. Chim. Acta, 2003, 86, 3310.
33. Göksu, S.; Seçen, H.; Sütbeyaz, Y. Helv. Chim. Acta 2006, 89, 270.
34. Öztaşkın, N.; Göksu, S.; Seçen, H. Synth. Commun. 2011, 41, 2017.
35. Göksu, S.; Seçen, H. Tetrahedron 2005, 61, 6801.
36. Park, J.D.; Kim, D.H.; Kim, S.J.; Woo, J.R.; Ryu, S.E. J. Med. Chem., 2002, 45, 5295.
37. Tite, T.; Tomas, L.; Docsa, T.; Gergely, P.; Kovensky, J.; Gueyrard, D.; Wadouachi, A.
Tetrahedron Lett. 2012, 53, 959.
38. Catt, J.D.; Matier, W.L. J. Org. Chem. 1974, 39, 566.
17

39. Lee, C.H.; Kim, S.H.; Chung, K.W.; Kim, T.W. J. Kor. Chem. Soc. 1998, 42, 245.
40. Lee, J.D.; Lee, C.H.; Nakamura, H.; Ko, J.; Kang, S. O. Tetrahedron Lett. 2002, 43,
5483.
41. Murakami, H.; Marelich, G.P.; Grubb, J.H.; Kyle, J.W.; Sly, W.S. Genomics, 1987, 1,
159.
42. Alam, M.T.; Yamada, T.; Carlsson, U.; Ikai, A. FEBS Lett. 2002, 519, 35.
43. Nair, S.K.; Calderone, T.L.; Christianson, D.W.; Fierke, C.A. J. Biol. Chem. 1991, 266,
17320.
44. Osborne, W.R.A.; Tashian, R.E. Anal. Biochem. 1975, 64, 297.
45. Khalifah, R.G.; Strader, D.J.; Bryant, S.H.; Gibson, S.M. Biochemistry 1977, 16, 2241.
46. Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759.
47. Akıncıoğlu, A.; Akbaba, Y.; Göçer, H.; Göksu, S.; Gülçin, İ.; Supuran, C.T. Bioorg.
Med. Chem. 2013, 21, 1379.
48. Öztürk Sarikaya, S.B.; Gülçin, İ.; Supuran C.T. Chem. Biol. Drug Des. 2010, 75, 515.
49. Öztürk Sarikaya, S.B.; Topal, F.; Şentürk, M.; Gülçin, İ.; Supuran, C.T. Bioorg. Med.
Chem. Lett. 2011, 21, 4259.
50. Şentürk, M.; Gülçin, İ.; Daştan A.; Küfrevioğlu, Ö.İ.; Supuran C.T. Bioorg. Med. Chem.
2009, 17, 3207.
51. Şentürk, M.; Gülçin, İ.; Beydemir, Ş.; Küfrevioğlu Ö.İ.; Supuran C.T. Chem. Biol. Drug
Design 2011, 77, 494.
52. Khalifah, R.G. J. Biol. Chem. 1971, 246, 2561.
53. Briganti, F.; Pierattelli, R.; Scozzafava, A.; Supuran, C.T. Eur. J. Med. Chem. 1996, 31,
1001.
54. Abbate, F.; Supuran, C.T.; Scozzafava, A.; Orioli, P.; Stubbs, M.T.; Klebe, G. J. Med.
Chem. 2002, 45, 3583.
55. Balseven, H.; İşgör, M.M.; Mert, S.; Alım, Z.; Beydemir, Ş.; Ok, S.; Kasımoğulları, R.
Bioorg. Med. Chem. 2013, 21, 21.
56. Gülçin, İ.; Beydemir, Ş.; Büyükokuroğlu, M.E. Biol. Pharm. Bull. 2004, 27, 613.
57. Beydemir, Ş.; Gülçin, İ. J. Enzyme Inhib. Med. Chem. 2004, 19, 193.
58. ArasHisar, Ş.; Hisar, O.; Beydemir, Ş.; Gülçin, İ.; Yanık, T. Acta Vet. Hung. 2004, 52,
413.
59. Çoban, T.A.; Beydemir, Ş.; Gülçin, İ.; Ekinci, D. Biol. Pharm. Bull. 2007, 30, 2257.
60. Çoban, T.A.; Beydemir, Ş.; Gülçin, İ.; Ekinci, D. J. Enzyme Inhib. Med. Chem. 2008,
23, 266.
18

61. Balaydın, H.T.; Şentürk, M.; Göksu, S.; Menzek, A. Eur. J. Med. Chem. 2012, 54, 423.
62. Brzozowski, Z.; Slawinski, J.; Saczewski, F.; Innocenti, A.; Supuran C.T. Eur. J. Med.
Chem. 2010, 45, 2396.
63. Ho, Y.T.; Purohit, A.; Vicker, N.; Newman, S.P.; Robinson, J.J.; Leese, M.P.;
Ganeshapillai, D.; Woo, L. W.L.; Potter, B.V.L.; Reed, M.J. Biochem. Biophysic. Res.
Co. 2003, 305, 909.
64. Rodríguez, O.M.; Maresca, A.; Témpera, C.A.; Bravo, R.D.; Colinas, P.A.; Supuran,
C.T. Bioorg. Med. Chem. Lett. 2011, 21, 4447.
65. Colinas, P.A.; Bravo, R.D.; Vullo, D.; Scozzafava, A.; Supuran, C.T. Bioorg. Med.
Chem. Lett. 2007, 17, 5086.
66. Gülçin, İ.; Beydemir, S. Mini Rev. Med. Chem. 2013, 13, 408.
67. Supuran C.T.; Scozzafava A. Curr. Med. Chem. Immunol. Endocr. Metab. Agents, 2001,
1, 61.
68. Pradhan, P.K.; Dey, S.; Jaisankar, P.; Giri, V.S. Synth. Commun. 2005, 35, 913.
69. Beydemir, Ş.; Gülçin, İ. J. Enzyme Inhib. Med. Chem. 2004, 19, 193.
70. Verpoorte, J.A.; Mehta, S.; Edsall, J.T. J. Biol. Chem. 1967, 242, 4221.
71. Beydemir, Ş.; Gülçin, İ.; Küfrevioğlu, Ö.İ.; Çiftçi, M. Pol. J. Pharmacol. 2003, 55, 787.
72. Şentürk, M.; Gülçin, İ.; Çiftci, M.; Küfrevioğlu, Ö.İ. Biol. Pharm. Bull. 2008, 31 2036.
73. Nar, M.; Çetinkaya, Y.; Gülçin, I.; Menzek, A. J. Enzyme Inhib. Med. Chem. 2013, 28
402.
74. Köksal, E.; Gülçin, İ. Protein Peptide Lett. 2008, 15, 320.
75. Gülçin, İ.; Beydemir, Ş.; Çoban, T.A.; Ekinci, D. Fresen. Environ. Bull. 2008, 17, 1283.
76. Gülçin, İ.; Küfrevioğlu, Ö.İ.; Oktay, M. J. Enzyme Inhib. Med. Chem. 2005, 20, 297.
77. Hisar, O.; Beydemir, Ş.; Gülçin, İ.; Küfrevioğlu, Ö.İ.; Supuran, C.T. J. Enzyme Inhib.
Med. Chem. 2005, 20, 35.
78. Çetinkaya, Y.; Göçer, H.; Göksu, S.; Gülçin, I. J. Enzyme Inhib. Med. Chem. 2013, doi:
10.3109/14756366.2012.763163.
79. Akbaba, Y.; Akıncıoğlu, A.; Göçer, H.; Göksu, S.; Gülçin, I.; Supuran C.T. J. Enzyme
Inhib. Med. Chem. 2013, doi: 10.3109/14756366.2012.750311.
19

FIGURES, SCHEMES AND TABLES
OH
O
N
O
H
N
S
O
N
O
S
H
N
S
NH2
H
H
N
HO
H
O
N
H
H
O
OH
2
1
O
S
H
O
N
NH2
H
NH2
N
S
NH2
HO
HO
O
O
S
3
4
5
Fig. 1. Chemical structures of selected sulfamides (1-4) and of dopamine (5)
20

SCHEME
O
O
O
O
O
O
O
O
O
H
H
O
N
O
N
H
H2O
O
N
H
H
H
H
H
H
H
H
H
HN
HN
N
N
H
H
O
O
H
O
H
HN
O
N
O
O
H
O
O
O
C
O
O
Zn2+
Zn2+
O
His
Zn2+
His
His
His
His
His
His
His
His
O
O
O
O
O
O
O
O
H
O
O
H
N
H
Proton
BH+
B
O
N
H
O
N
O
shuttling
H
H
H
H
O
HN
H
N
H
O
H
H
HN
O
N
H
H
H
O
H
HN
N
O
H
H
O
H
O
H
Zn2+
O
H
His
His
Zn2+
Zn2+
H
His
His
His
CA-Zn2+-OH
His
His
His
His
CA-Zn2+-OH2
+
Scheme 1. Mechanism of catalysis of the hydration of CO2 by hCA II.17 The formal charge
was shown only on the Zn2+-bound water (and not the histidine residues) to emphasize
that this water ligand is acidic.
21

His 119
Zn2+
His 119
His 119
2
HO
His 96
2+
Inh-
His 96
HO
Zn2+ His 96
+InhH
O
C
O
His 94
His 94
His 94
B
A
E
B
Inh_
OH2
His 119
His 119
His 119
O
-
InhH
-H+
HCO3
2
C
H
O
2
+
Z
n
His 96
His 94
His 96
His 94
OH2
Zn2+ His 96
O
His 94
C
D
F
Scheme 2. General catalytic and inhibition mechanisms of carbonic anhydrases
22

O
H
N
O
Ph
MeO
MeO
MeO
MeO
OH
i
O
71%
OMe
OMe
6
7
ii
84%
NH2
NH3Cl
MeO
MeO
MeO
MeO
iii
98%
OMe
OMe
9
8
Scheme 3. Synthesis of mescaline 9. (i) DPPA/Et3N, benzene, 80 oC, 6 h, then PhCH2OH, 80
oC, 30 h; (ii) H2/Pd-C, MeOH-CHCl3, rt, 24 h; (iii) 10% solution of NaOH
23

R1
O
R1
O
H
H
N
R2
R3
N S
O
R2
R3
NH2
i
OR
R4
1
R4
R1=H, R2=R3=R4=OMe
2
3
4
14
9
R=t-Bu, R =H, R =R =R =OMe
82%
76%
15 R=t-Bu, R3=R4=H, R1=R2=OMe
10 R3=R4=H, R1=R2=OMe
11 R1=R2=H, R3=R4=OMe
12 R1=R2=R4=H, R3=OMe
16 R=t-Bu, R1=R2=H, R3=R4=OMe 78%
17 R=PhCH2, R1=R2=R4=H, R3=OMe 80%
2
3
4
1
2
3
4
1
18
13
R=PhCH2, R =R =R =H, R =OMe 85%
R =R =R =H, R =OMe
ii
R1
O
1
2
3
4
19
H
R =H, R =R =R =OMe 83%
R2
R3
N S NH2
O
20 R3=R4=H, R1=R2=OMe 78%
21 R1=R2=H, R3=R4=OMe 75%
22 R1=R2=R4=H, R3=OMe 74%
R4
2
3
4
1
23
R =R =R =H, R =OMe 70%
Scheme 4. Synthesis of sulfamides. (i) For the synthesis of 14-16: CSI/ NEt3/t-BuOH,
CH2Cl2, 0-25 oC, 4 h; For the synthesis of 17 and 18: CSI/ NEt3/PhCH2OH, CH2Cl2, 0-25 oC,
o
4 h; (ii) For the synthesis of 19-21: CF3CO2H, 25 C, 12 h; for the synthesis of 22 and 23;
H2/Pd-C, MeOH, rt, 4 h.
24

Table 1. CA I, CA II, CA VA, CA IX, CA XII, CA XIV and two ß-CAs from Candida
glabrata (CgCA) and Rv3588 from Mycobacterium tuberculosis inhibition data with some
sulfamides 19-23 by an hydratase method.
KI (µM)
Sulfamides
CA-I
(µM)
1.82
CA II CA VA CA IX CA XII CA XIV CgCA
Rv3588
(µM)
2.20
(nM)
2.03
2.95
2.40
1.47
2.04
12.0
(µM)
>50
(µM)
0.221
>50
(µM)
1.52
(µM)
0.22
(µM)
3.34
4.59
5.31
0.35
2.71
1.97
19
20
0.25
>50
0.047
0.044
0.021
0.416
0.006
0.18
>50
21
0.28
2.28
3.25
3.34
0.063
0.092
0.376
0.041
0.025
0.031
0.007
0.16
3.61
22
0.061
0.25
4.23
23
AAZ*
0.46
0.250
0.041
0.010
*: Acetazolamide (AAZ) was used as a standard inhibitor for all CAs investigated here
25

GRAPHICAL ABSTRACT
Carbonic anhydrase (CA) inhibitory properties of sulfamide derivatives of dopamine related
compounds were investigated. These compounds showed promising CA inhibitory properties.
R2
R3
R4
R1
R1
R1
O
S
R2
R3
NH2
R2
R3
NH
NH2
OMe OMe
OMe
H
H
O
OMe
H
H
H
OMe
OMe
H
R4
R4
H
H
OMe
OMe
H
H
H
OMe
26