A novel and one-pot synthesis of new 1-tosyl pyrrol-2-one derivatives and analysis of carbonic anhydrase inhibitory potencies

Article abstract of DOI:10.1016/j.bmc.2010.04.072

Here we propose a novel one-pot synthesis of new tosyl-pyrrole derivatives. By means of the new developed method, pyrrole derivatives were synthesized at room temperature in a single step, and a useful method is proposed for the synthesis of similar compounds. Moreover, inhibitions of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II by 1-tosyl-pyrrole and 1-tosyl-pyrrol-2-on derivatives were investigated. 1-Tosyl-pyrrole, 1-tosyl-1H-pyrrol-2(5H)-one, 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one and 5-oxo-1-tosyl-2,5-dihydro-1H-pyrrol-2-yl acetate showed inhibitory action with K_i values in the range of 14.6-42.4 μM for hCA I and 0.53-37.5 μM for hCA II, respectively. All pyrrole derivatives were competitive inhibitors with 4-nitrophenylacetate as substrate. Some new synthesized pyrrole derivatives showed very effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors targeting other CA isoforms.

Full text of DOI:10.1016/j.bmc.2010.04.072

Bioorganic & Medicinal Chemistry 18 (2010) 4468–4474
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel and one-pot synthesis of new 1-tosyl pyrrol-2-one derivatives
and analysis of carbonic anhydrase inhibitory potencies
a,d,
a
Cemalettin Alp a,b, Deniz Ekinci c, Mehmet Serdar Gültekin a, , Murat Sßentürk
, Ertan Sßahin ,
*
*
a
_
Ömer Irfan Küfreviog˘lu
a Atatürk University, Science Faculty, Department of Chemistry, Erzurum, Turkey
b Erzincan University, Çayırlı Vocational School, Erzincan, Turkey
c Ondokuz Mayıs University, Faculty of Agriculture, Department of Agricultural Biotechnology, Enzyme and Microbial Biotechnology Division, Samsun, Turkey
d
_
Ag˘rı Ibrahim Çeçen University, Department of Chemistry, Ag˘rı, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we propose a novel one-pot synthesis of new tosyl-pyrrole derivatives. By means of the new devel-
oped method, pyrrole derivatives were synthesized at room temperature in a single step, and a useful
method is proposed for the synthesis of similar compounds. Moreover, inhibitions of two human cyto-
solic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II by 1-tosyl-pyrrole and 1-tosyl-pyrrol-2-on
derivatives were investigated. 1-Tosyl-pyrrole, 1-tosyl-1H-pyrrol-2(5H)-one, 5-hydroxy-1-tosyl-1H-pyr-
rol-2(5H)-one and 5-oxo-1-tosyl-2,5-dihydro-1H-pyrrol-2-yl acetate showed inhibitory action with Ki
Received 4 March 2010
Revised 17 April 2010
Accepted 21 April 2010
Available online 28 May 2010
Keywords:
values in the range of 14.6–42.4 lM for hCA I and 0.53–37.5 lM for hCA II, respectively. All pyrrole deriv-
Carbonic anhydrase
1-Tosyl-1H-pyrrol-2(5H)-one
5-Hydroxy-1-tosyl-1H-pyrrol-2(5H)-one
hCA I
atives were competitive inhibitors with 4-nitrophenylacetate as substrate. Some new synthesized pyrrole
derivatives showed very effective hCA II inhibitory effects, in the same range as the clinically used sulfon-
amide acetazolamide, and might be used as leads for generating enzyme inhibitors targeting other CA
isoforms.
hCA II
Enzyme inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
involved in these processes are important therapeutic targets with
the potential to be inhibited/activated for the treatment of a range
Carbonic anhydrase (EC 4.2.1.1., CA) is a pH regulatory/meta-
bolic enzyme in all life kingdoms, being found in organisms all over
the phylogenetic tree. It catalyzes the hydration of carbon dioxide
to bicarbonate and the corresponding dehydration of bicarbonate
in acidic medium with regeneration of CO2.1 At least 16 CA iso-
zymes have been described up to now in mammals, the most active
ones as catalysts for carbon dioxide hydration being CA II and CA
IX.1–3 The mammalian carbonic anhydrases have generally mono-
meric structures.4 Sixteen isozymes of the zinc-binding enzyme
have been described that differ in their subcellular localization, cat-
alytic activity and susceptibility to different classes of inhibitors.
Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII
and CA XIII), others are membrane bound (CA IV, CA IX, CA XII
and CA XIV), two are mitochondrial (CA VA and CA VB) and one is
secreted in saliva (CA VI). Hilvo et al.5 has reported that CA XV iso-
form is not expressed in humans or in other primates, but it is abun-
dant in rodents and other higher vertebrates.2 These CA isozymes
of disorders such as oedema, glaucoma, obesity, cancer, epilepsy
and osteoporosis.1,3 Thus, carbonic anhydrase inhibitors are valu-
able molecules for drug design and pharmacological applications.
Our group recently investigated the interaction of hCA I and hCA
II cytosolic isozymes with several types of phenols, such as the sim-
ple phenol and several of its substituted derivatives, for example,
diphenols, salicylates and some of their derivatives.6 In this study,
towards the discovery of new class of inhibitors, we synthesized no-
vel pyrrole derivatives and determined their inhibition effects on
human CA-I and II isozymes, because it is critically important to ex-
plore further classes of potent CAIs in order to detect compounds
with different inhibition profiles.
We investigated a novel series of 1-tosyl pyrrole derivatives,
some of which are widely used as prodrugs. Especially, 5-lactams
such as pyrrol-2-one and its derivatives have shown anti-tumour
activity. Intherecentyears, N-substitutedmaleimidesand5-ylidene
pyrrol-2(5H)-ones have received growing attention,7 due to their
biological activity features and potencies for organic synthesis as
key compounds for synthesis of bio-active molecules, which are
Oteromycin, Talaroconvolutin A, Azaspirene, Fusarin, Clausenamide. . .,
etc.8 Pyrrol-2-one and its derivatives are also active and important
reagents as dienophiles in Diels–Alder reactions, as a 1,3-dipolar re-
agent for Michael addition9 and a material for organic complexes.
_
* Corresponding authors. Address: Ag˘rı Ibrahim Çeçen University, Faculty of Art
and Sciences, Department of Chemistry, 04100 Ag˘rı, Turkey. Tel.: +90 442 2314428;
fax: +90 442 2360948 (M.S.G.); tel.: +90 442 2314414; fax: +90 442 2360948 (M.S.).
E-mail addresses: gultekin@atauni.edu.tr (M.S. Gültekin), senturkm36@gmail.
com (M. Sßentürk).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.072

C. Alp et al. / Bioorg. Med. Chem. 18 (2010) 4468–4474
4469
Many methods have been developed to synthesize these useful
functional heterocycles. There are some studies for the synthesis of
5-hydroxy-2(5H)-pyrrol-2-one skeleton in the literature.10 Most
notably, cyclo addition of phenylisocyanate with dialkyl acetylene
has been extensively studied and pyrrol-2-one has been obtained
with several steps in good yields.10 One of the methods developed
for the synthesis of 1,5-dihydro-pyrrol-2(5H)-one is based on a for-
mal (2+3) cycloaddition reaction of diphenyl-cyclopropenone with
imines or diimines. In general, pyrrol-2-one structure has been
synthesized from acetylene, ketone or aldehyde and 1,3-dipolar
compounds. A number of synthetic routes are available to build
into lactams core structure in pyrrol-2-one skeleton.
Carbonic anhydrase inhibitors are a class of pharmaceuticals
that suppress the activity of carbonic anhydrase. Their clinical
use has been established as antiglaucoma agents, diuretics, anti-
epileptics, in the management of mountain sickness, gastric and
duodenal ulcers, neurological disorders or osteoporosis. Thus, dis-
covery of novel effective carbonic anhydrase inhibitors is a great
afford for pharmacological and medical applications. The aim of
the current study is to develop a novel synthesis method for new
pyrrole-2-one derivatives and to discover novel powerful carbonic
anhydrase inhibitors.
We selected pyrroles as CA inhibitors (CAIs) because the simple
aromatic/heterocyclic atoms have been shown to be inhibitors with
CO2 as substrate for the main isoform of CA, that is, human CA II
(hCA II). In an excellent study, Supuran’s group reported the X-ray
crystal structure for the adduct of hCA II with aromatic/heterocyclic
atoms,12 showing this compound to bind to CA by anchoring its
electronegative moiety to the zinc-bound water/hydroxide ion of
the enzyme active site. All of the inhibitors used in this study were
determined to inhibit the enzyme in competitive manner.
The toluene moiety of tosyl was found to lay in the hydrophobic
part of the hCA II active site, where CO2, the physiologic substrate
of the CAs, binds in the precatalytic complex explaining thus the
behaviour of tosyl as a unique CO2 competitive inhibitor. Only re-
cently, Supuran’s group investigated the interactions of phenol and
some of its substituted derivatives (as well as bicyclic phenols)
with all mammalian isozymes, CA I–XV,12 evidencing some low
micromolar/submicromolar inhibitors as well as the possibility to
design isozyme selective CAIs. Indeed, the inhibition profile of var-
ious isozymes with this class of agents is very variable, with inhi-
bition constants ranging from the millimolar to the submicromolar
range for many simple phenols.6,11,12 It appeared thus of interest to
extend the previous studies.12
Scheme 2 shows the mechanism proposed for the formation of
2 and 3. In this mechanism, PIFA is added to the olephinic bond of
heterocyclic dien 1 initially and trifluoroacetate (CF3COOÀ) of PIFA
group is removed from iodine, attacks to iodoform ring from oppo-
site side and the addition comes true. Trifluoroacetate is 1,2 added
to the ring, 2-trifloroacetoxy-1-tosyl-pyrrole 6 is formed after
elimination of iodobenzene and trifluoroacetic acid from 5. 1-To-
syl-1H-pyrrol-2(5H)-one 2 is obtained via PIFA oxidation of this
product and 1,3 shift of the proton on the ring. When 2.2 equiv
of PIFA is used, it is added to 6, and 9 is formed via attacking of free
trifloroacetate to iodophor ring. Via elimination of this molecule
and hydrolysis of the product, 3 is formed (Scheme 2). Figure 1
shows the X-ray spectrum for 5-hydroxy-1-tosyl-1H-pyrrol-
2(5H)-one 3.
5-Acetoxy-1-tosyl-1H-pyrrole-2(5H)-one 12 was synthesized in
72% yield via acetylation of hydroxyl group of 3 to pyridine/acetic
anhydride (Scheme 3).
2.2. Crystal structure
An X-ray diffraction analysis of tosylated pyrrole compound (3)
was undertaken. Molecular structure and packing diagram of the
compound are shown in Figure 1a and b, respectively. The com-
pound crystallizes in the monoclinic space group P21/c, with four
molecules in the unit cell. It contains the 1-tosyl pyrrole scaffold
with the substituted hydroxyl and ketone moieties to pyrrole part
and has the configuration shown in Figure 1a, which is the (R)-con-
figuration at the chiral centre (related to the asymmetric C atom at
C11). The bond lengths and angles are within normal ranges. Phenyl
and pyrrole rings are planar and they are oriented at a dihedral an-
gle of 88.10(8)°. C–Oketone bond length is 1.210(3) Å. The structures
of related tosyl pyrroles, that is, 2-bromo-1-(p-toluenesulfonyl)pyr-
role,13 bis(1-tosyl-2-pyrroleyl)ethyne14 and 2-chloromethyl-1-(4-
methylphenylsulfonyl)pyrrole15 have been reported.
The geometry around S atom is significantly deviated from that
of regular tetrahedral. The maximum and minimum angles around
S are 119.9(2) and 103.9(2), respectively. S–O bond lengths differ
by only 0.006 Å which implies that there is no delocalization of
N1 lone pair into the sulfonyl group. S–O distances are similar to
those found in analogous structures.13–15 These distances do not
vary significantly even in the sulfonamide structures, despite the
different interaction patterns observed.4 The S–N bond distance
lies [1.652(2) Å] within the expected range of 1.63–1.69 Å. In all
essential details, the geometry of the molecule regarding bond
lengths and angles of the compound is in good agreement with
the values observed in similar structures.13–16
2. Results and discussion
2.1. Chemistry
The crystal structure consists of 1D polymeric chain along the c-
axis, where the hydroxyl and ketone atoms are involved in intra-
molecular C11–H11Á Á ÁO4 (Àx, À1/2 + y, 1/2 À z) hydrogen bonds,
with HÁ Á ÁO = 2.38 Å and C–HÁ Á ÁN angle of 148°. In addition, the hy-
droxyl group is involved in one intermolecular O4–H4Á Á ÁO1 hydro-
gen bond, with sulfonyl O atom, (HÁ Á ÁN = 2.49 Å and C–HÁ Á ÁN =
122°) (Fig. 1).
We previously investigated the addition reactions of phenylio-
dine bis (trifluoroacetate) (PIFA) to monocyclic and bicyclic alkenes
and synthesized corresponding addition products.10a In this study,
we investigated the reaction between 1-tosyl-pyrrol and PIFA and
synthesized compounds 2 and 3 in high yields. We have developed
a novel method for one-pot synthesis of 1-tosyl-pyrrol-2-one skel-
eton using 1-tosyl-pyrrole as starting material and PIFA (phenylio-
dine bis(trifluoroacetate)) (Scheme 1).
For this purpose, 1 equiv of 1-tosyl-pyrrole molecule was re-
acted with 1.15 and 2.2 equiv of PIFA. Using 1 equiv of 1-tosyl-pyr-
role and 1.15 equiv of PIFA, 1-tosyl-1H-pyrrol-2(5H)-one 2 was
obtained with 81% yield and 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-
one 3 with 19% yield. When 1-tosyl-pyrrole was reacted with
2.2 equiv of PIFA, 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one 3 was
obtained in 97% yield. Both reactions were initiated at 5 °C and stir-
red for 10 h at room temperature.
2.3. CA purification, assay and inhibition by 1-tosyl pyrrole (1)
and 1-tosyl pyrrol-2-one derivatives (2, 3, 12)
The purification of the two CA isozymes was performed in a
simple single-step method by means of Sepharose-4B-aniline-sul-
fanilamide affinity column chromatoghrapy.17 hCA I was purified
105.5-fold with a specific activity of 975.92 EU mgÀ1 and overall
yield of 51.7%, and hCA II was purified 646.5-fold with a specific
activity of 5980 EU mgÀ1 and overall yield of 55% (Table 1).18–21
Inhibitory effects of tosylated compounds on enzyme activities
were tested under in vitro conditions; Ki values were calculated
from Lineweaver–Burk graphs and are given in Table 2.22

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C. Alp et al. / Bioorg. Med. Chem. 18 (2010) 4468–4474
PIFA(115 mmol%)
PIFA(220 mmol%)
H
H
H
O
O
N
N
N
HO
Ts
Ts
Ts
2
(81%)
1
3
(97%)
Ts:
Tosyl
PIFA:
Phenyliodine bis(trifluoroacetate)
O
O
S
O
O
CF3
CF3
I
O
O
Scheme 1. Synthesis of compounds 2 and 3 with PIFA (phenyliodine bis(trifluoroacetate)).
1,3 H-shift
O
O
N
N
Ts
Ts
2
7
OCOCF3
Ph
I
OCOCF3
Ph
+
I
-PhI
PIFA
-CF3COOH
OCOCF3
OCOCF3
N
N
N
N
Ts
1
Ts
5
Ts
4
Ts
6
-OCOCF3
PIFA
OCOCF3
OCOCF3
Ph
I
OCOCF3
I+
Ph
I
Ph
F3COCO
OCOCF3
F3COCO
O
N
OCOCF3
N
N
Ts
9
Ts
10
Ts
8
CF3COO-
-PhI,
-CF3COOH
F3COCO
HO
O
O
N
N
Ts
Ts
11
3
Scheme 2. The mechanism for formation of compounds 2 and 3.
We report here the first study on the inhibitory effects of tosyl
pyrroles 1, 2, 3 and 12 on the esterase activity of hCAs I and II. The
acetazolamide (13) was used as reference compound in our exper-
iments for comparison reasons. Data of Table 2 and 3 show the
inhibition of hCAs I and II with tosyl pyrroles 1, 2, 3 and 12, by
an esterase assay,19 with 4-nitrophenylacetate (4-NPA) as
substrate:
(i) Against the slow cytosolic isozyme hCA I, compounds 1–2
behave as moderate inhibitors, with Ki values in the range of
37.3–42.4
showed better inhibitory activity as compared to the previously
mentioned tosyl pyrroles, with Ki values of 14.6–25.7 M, (Table
lM. A second group of derivatives, including 3 and 12,
l
2). Thus, the nature of the groups strongly influences hCA I inhib-
itory activity. Acetazolamide 5 is also a medium-weak CAI with

C. Alp et al. / Bioorg. Med. Chem. 18 (2010) 4468–4474
4471
investigated compounds act as competitive inhibitors with 4-NPA
as substrate, that is, they bind in the same regions of the active site
cavity as compared to the substrate. However, the binding site of
4-NPA itself is unknown, but it is presumed to be in the same re-
gion as that of CO2, the physiological substrate of this enzyme.26
(ii) A better inhibitory activity has been observed with com-
pounds 3–4 investigated here for the inhibition of the rapid cyto-
solic isozyme hCA II (Table 2). Thus, two derivatives, that is, 1
and 2, showed weak hCA II inhibitory activity with Ki-s in the range
of 23.1–37.5
were quite effective hCA II inhibitors, with Ki-s in the range of
0.51–0.73 M, (Table 2). Methoxy 4 is also effective hCA II inhibi-
lM, Table 2), whereas the remaining two derivatives
l
tor. The best hCA II inhibitor in this series of derivatives was the
bulky, 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one 3, which with a Ki
of 0.51 lM is a better inhibitor than acetazolamide, a clinically
used sulfonamide. It must be stressed that Kis measured with the
esterase method is always in the micromolar range because hCAs
I and II are weak esterases.27 It should be noted that esterase meth-
od usually gives Ki-s an order of magnitude higher as compared to
the CO2 hydrase assay.
In a recent study, thioxolone, a simple compound lacking the
sulfonamide, sulfamate or related functional groups that are typi-
cally found in all known CA inhibitors, was reported to act as a
CAI inhibitor and could represent the starting point for a new class
of inhibitors that may have advantages for patients with sulfon-
amide allergies (thioxolone acts as a prodrug, being hydrolyzed
in situ with the formation of a mercapto phenol derivative which
is the real enzyme inhibitor).28 However, Innocenti et al.29 showed
that compared to sulfonamides, thioxolone was inefficient for gen-
erating isozyme-selective inhibitors, since except for hCA I which
was inhibited in the nanomolar range (Ki of 91 nM), the remaining
12 mammalian CA isoforms (CA II–CA XV) were inhibited with a
very flat profile by this compound (Ki-s in the range of only
4.93–9.04 lM). Thus, it is critically important to explore further
classes of potent CAIs in order to detect compounds with a differ-
ent inhibition profile as compared to the sulfonamides and their
bioisosteres and to find novel applications for the inhibitors of
these widespread enzymes.
3. Conclusions
We have developed a novel and one-pot synthesis for 1-tosyl-
1H-pyrrol-2(5H)-one, 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one. By
means of the new developed method, 2 and 3 were synthesized
at room temperature using only PIFA reactive in a single step,
and also an available method is proposed for the synthesis of mol-
ecules which have similar structures. Tosylated pyrrole com-
pounds 1, 2, 3 and 12 affect the activity of CA isozymes due to
the presence of the different functional groups. Our findings here
indicate thus another class of possible CAIs of interest, in addition
to the well-known sulfonamides/sulfamates/sulfamides, the phe-
nols/biphenyl diphenols. Indeed, some tosylpyrroles investigated
here showed effective hCAs I and II inhibitory activity, in the low
micromolar range, by the esterase method which usually gives
Ki-s an order of magnitude higher as compared to the CO2 hydrase
assay.30 These findings point out that these compounds may lead
to generate potent CAIs against all CA isoforms and might be used
for the treatment of CA-related disorders.
Figure 1. (a) The molecular structure of 3, with the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level. (b) The crystal
packing of (3), viewed down the b-axis. H-bonds are shown as dashed lines.
Ac2O
O
AcO
HO
O
N
N
Pyridine
Ts
3
Ts
12 (72%)
Scheme 3. Acetylation of compound 3 with pyridine/acetic anhydrate.
4. Experimental
4.1. General
this assay and substrate against hCA I (Ki of 36.2 l
M).8b Kinetic
investigations (Lineweaver–Burk plots, data not shown) indicate
All chemicals used were of reagent grade. Solvents were dried
over appropriate drying agents and freshly distilled before use.
that similar to sulfonamides and inorganic anions,4–6,23–25 all the

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C. Alp et al. / Bioorg. Med. Chem. 18 (2010) 4468–4474
Table 1
Summary of purification procedure for human carbonic anhydrase isozymes (hCA I and hCA II) by a Sepharose-4B-aniline-sulfanilamide affinity column chromatography
Purification steps
Activity
(EU/mL)
Total
volume
(mL)
Protein
(mg/mL)
Total
protein (mg)
Total
activity (EU)
Specific activity
(EU/mg)
Yield
(%)
Purification
factor
Hemolysate
163
43
18.88
811.84
7009
8.63
100
1
Sepharose-4B-aniline-sulfanilamide affinity
column chromatography
hCA I
hCA II
486
794
8
5
0.52
0.13
4.16
0.65
3888
3970
934.62
6107.70
55.47
56.64
108.30
707.73
The progress of synthetic reactions was monitored by thin layer
chromatography (TLC) in a suitable solvent system. Column chro-
matography was performed on Fluka Silica Gel 60 (70–230 mesh,
254–366 mesh). Melting points were determined by a Gallen Kamp
melting point apparatus. 1H and 13C NMR spectra were recorded on
a Varian 400 spectrometer in deuterated chloroform (CDCl3),
depending on the solubility of the product. Chemical shifts are re-
ported in d ppm downfield with respect to tetramethylsilane as an
internal standard in 1H and 13C NMR spectra. Elementary analyzed
spectra were recorded using a Leco CHNS-932 spectrometer. Infra-
red spectra were recorded using Perkin–Elmer 337 Greating
Spectrophotometer.
droxy-1-tosyl-1H-pyrrol-2(5H)-one (3) was recrystallized from
ether–hexane solution (mp: 140–142 °C).
1H NMR (200 MHz, CDCl3, ppm): d 2.42 (s, 3H), 6.06 (dd, 1H, J
= 6.1, 0.9 Hz), 7.05 (dd, 1H, J = 6.1, 1.83 Hz) 6.24 (m, 1H) 7.33 (d,
2H, J = 8.23 Hz), 7.95 (d, 2H, J = 8.23 Hz). 13C NMR (50 MHz, CDCl3,
ppm): 23.6, 78.4 129.4, 130.1, 131.7, 137.5, 147.3, 149.9, 168.5. IR
(KBr, cmÀ1): 3456, 3103, 2918, 1731, 1597, 1490, 13-401, 1354,
1168, 1112, 1055. Anal. Calcd for C11H11NO4S (253.27): C, 52.16;
H, 4.38; N, 5.53; S, 12.66. Found: C, 51.88; H, 4.33; N, 5.47; S, 12.47.
4.3.3. Synthesis of 5-oxo-1-tosyl-2,5-dihydro-1H-pyrrole-2-yl
acetate (12)
To a mixture of 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one (3)
(1 g, 3.95 mmol) and pyridine (5 ml, 60 mmol) in dichloromethane
(20 ml) was added slowly acetic anhydride (0.81 g, 7.90 mmol,
2 equiv). The reaction mixture was stirred at room temperature
for 12 h. The reaction mixture was washed with 5% HCl. Then the
organic layer was washed with NaHCO3 solution and dried over so-
dium sulfate, filtered and evaporated to afford a residue. Purifica-
tion of the residue was by silica gel column chromatography
using ethyl acetate–hexane to yield 72% (0.84 g, 2.84 mmol) of 5-
oxo-1-tosyl-2,5-dihydro-1H-pyrrole-2-yl acetate (12). Compound
12 was recrystallized from ether–hexane solution (mp: 136–
138 °C).
4.2. Chemicals
Tosylchloride, Sepharose 4B, protein assay reagents and 4-
nitrophenylacetate were obtained from Sigma–Aldrich Co. All
other chemicals were of analytical grade and obtained from Merck.
4.3. Synthesis of 1-tosyl-pyrrol-2-one derivatives
4.3.1. Synthesis of 1-tosyl-1H-pyrrol-2(5H)-one (2)
1 g (4.51 mmol) of 1-tosyl pyrrole (1) was dissolved in 30 ml of
dichloromethane and stirred magnetically at À15 °C. 2.23 g
(5.20 mmol, 1.15 equiv) of PIFA were added slowly to the reaction
solution at À15 °C. Then the reaction mixture was warmed to room
temperature and stirred at room temperature for 16 h. The reaction
mixture was washed with NaHCO3 solution and dried over sodium
sulfate, filtered and evaporated to afford a residue. The crude res-
idue was purified on column chromatography (silica gel) using
ethyl acetate-hexane to yield 81% (0.87 g, 3.66 mmol) of 1-tosyl-
1H-pyrrol-2(5H)-one (2) and 19% (0.22 g, 0.86 mmol) of 5-hydro-
xy-1-tosyl-1H-pyrrol-2(5H)-one (3). 1-Tosyl-1H-pyrrol-2(5H)-one
(2) was recrystallized from ether–hexane solution (mp: 127–
129 °C).
1H NMR (200 MHz, CDCl3, ppm): d 2.16 (s, 3H), 2.43(s, 3H), 6.12
(dd, 1H, J = 6.8, 1.4 Hz), 7.10 (m, 2H), 7.34 (d, 2H, J = 8.24 Hz) 7.94
(d, 2H, J = 8.24 Hz). 13C NMR (50 MHz, CDCl3, ppm): 22.5, 23.5,
83.0, 130.0, 130.1, 131.5, 137.8, 147.1, 147. 168.2, 171.0. IR (KBr,
cmÀ1): 3105, 2920, 1749, 1596, 1367, 1229, 1170, 1097. Anal. Calcd
for C13H13NO5S (295.31): C, 52.87; H, 4.44; N, 4.74; S, 10.86. Found:
C, 52.88; H, 4.50; N, 5.65; S, 10.77.
4.4. Purification of carbonic anhydrase isozymes from human
erythrocytes by affinity chromatography
1H NMR (200 MHz, CDCl3, ppm): d = 2.41 (s, 6H), 4.47 (t, 4H,
J = 2.0 Hz), 6.04 (dt, 2H, J = 6.1, 2.0 Hz), 7.25 (dt, 2H, J = 6.1,
2.0 Hz), 7.32 (d, 4H, J = 8.42 Hz), 7.93 (d, 4H, J = 8.42 Hz). 13C
NMR (50 MHz, CDCl3, ppm): 23.5, 54.3, 129.0, 129.9, 131.7,
137.7, 147.0, 148.4, 170.1. IR (KBr, cmÀ1): 3101, 2925, 1726,
1596, 1444, 1400, 1359, 1169, 1092. Anal. Calcd for C11H11NO3S
(237.27): C, 55.68; H, 4.67; N, 5.90; S, 13.51. Found: C, 55.92; H,
4.52; N, 5.81; S, 13.26.
Erythrocytes were purified from fresh human blood obtained
from the Blood Centre of the Research Hospital at Atatürk Univer-
sity. The blood samples were centrifuged at 1500 rpm for 15 min
and the plasma and buffy coat were removed. The red cells were
isolated and washed twice with 0.9% of NaCl and hemolyzed with
1.5 volumes of ice-cold water. The ghost and intact cells were re-
moved by centrifugation at 20,000 rpm for 30 min at 4 °C. The
pH of the hemolysate was adjusted to 8.7 with solid Tris. Firstly,
Sepharose-4B was oxidized by KMnO4 and subsequently activated
by SOCl2. After that aniline was attached to the activated gel as a
spacer arm and finally diazotized sulfanilamide was clamped to
the para position of aniline molecule as ligand. The hemolysate
was applied to the prepared Sepharose 4B-aniline-sulfanylamide
affinity column which had been equilibrated with 25 mM Tris–
HCl/0.1 M Na2SO4 (pH 8.7). The affinity gel was washed with
25 mM Tris–HCl/22 mM Na2SO4 (pH 8.7). The human carbonic
anhydrase (hCA I and hCA II) isozymes were eluted with 1 M
NaCl/25 mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4
(pH 5.6), respectively. All procedures were performed at 4 °C.13
4.3.2. Synthesis of 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one (3)
1 g (4.51 mmol) of 1-tosyl pyrrole (1) was dissolved in 50 ml of
dichloromethane and stirred magnetically at room temperature.
4.27 g (9.92 mmol, 2.2 equiv) of PIFA were added slowly to the
reaction solution at room temperature and stirred at room temper-
ature for 18 h. The reaction mixture was washed with NaHCO3
solution and dried over sodium sulfate, filtered and evaporated
to afford a residue. The crude residue was purified on column chro-
matography on silica gel ethyl acetate–hexane to yield 97% (1.11 g,
4.38 mmol) of 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one (3). 5-Hy-

C. Alp et al. / Bioorg. Med. Chem. 18 (2010) 4468–4474
4473
Table 2
hCAs I and II inhibition data with pyrrole derivatives 1–3, 12 and acetazolamide6b 13, by an esterase assay with 4-nitrophenylacetate as substrate
Structure
Compounds
R
Kia
(lM)
hCA I
hCA II
O
N S
O
1
—
42.4 3.1
37.5 3.3
2
3
12
H
OH
OAc
37.3 2.7
14.6 1.6
25.7 3.7
23.1 2.9
0.51 0.04
0.73 0.02
O
O
N S
O
R
N
N
O
SO2NH2
13
—
36.2
0.37
N
H
S
a
Mean from at least three determinations. Errors in the range of 5–10% of the reported value (data not shown).
Table 3
I50 values from in vitro study of hCA-I and hCA-II in the presence of different tosylpyrrole derivatives
Inhibitor
I50 values for hCA-I (
l
M)
I50 values for hCA-II (lM)
N-p-tosyl-pyrrole (1)
1-Tosyl-1H-pyrrole-2(5H)-one (2)
5-Hydroxy-1-tosyl-1H-pyrrole-2(5H)-one (3)
5-Oxo-1-tosyl-2,5-dihydro-1H-pyrrole-2-yl acetate (12)
23.14
18.18
6.03
14.29
11.24
0.21
7.97
0.37
4.5. Hydratase activity assay
graph was drawn. To determine Ki values, three different inhibitor
concentrations were tested. In these experiments, 4-nitrophenylac-
etate was used as substrate at five different concentrations (0.15–
0.75 mM). The Lineweaver–Burk curves were drawn.22
Carbonic anhydrase activity was assayed by following the
hydration of CO2 according to the method described by Wilbur
and Anderson.18 CO2-hydratase activity as an enzyme unit (EU)
was calculated by using the equation (to À tc/tc) where t0 and tc
are the times for pH change of the non-enzymatic and the enzy-
matic reactions, respectively.
4.7. Protein determination
Protein quantity was determined spectrophotometrically at
595 nm according to the Bradford method during the purification
steps, using bovine serum albumin as the standard.20
4.6. Esterase activity assay
Carbonic anhydrase activity was assayed by following the change
in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-
nitrophenylate ion over a period of 3 min at 25 °C using a spectro-
photometer (CHEBIOS UV–VIS) according to the method described
by Verpoorte et al.19 The enzymatic reaction, in a total volume of
3.0 mL, contained 1.4 mL of 0.05 M Tris–SO4 buffer (pH 7.4), 1 mL
of 3 mM 4-nitrophenylacetate, 0.5 mL of H2O and 0.1 mL of enzyme
solution. A reference measurement was obtained by preparing the
same cuvette without enzyme solution. The inhibitory effects of 1-
tosyl-pyrrole (1), 1-tosyl-1H-pyrrol-2(5H)-one (2), 5-hydroxy-1-to-
syl-1H-pyrrol-2(5H)-one (3) and 5-oxo-1-tosyl-2,5-dihydro-1H-
pyrrol-2-yl acetate (12) were examined. All compounds were tested
in triplicate at each concentration used. Different inhibitor concen-
trations were used. HCA-I enzyme activities were measured for N-p-
4.8. SDS polyacrylamide gel electrophoresis
SDS polyacrylamide gel electrophoresis was performed after
purification of the enzymes. It was carried out in 10% and 3% acryl-
amide for the running and the stacking gel, respectively, containing
0.1% SDS according to Laemmli procedure. A 20-mg sample was
applied to the electrophoresis medium. Gels were stained for
1.5 h in 0.1% Coommassie Brilliant Blue R-250 in 50% methanol
and 10% acetic acid, then destained with several changes of the
same solvent without the dye.21
4.9. Crystal structure determination
For the crystal structure determination, the single-crystal of
5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one 3 was used for data col-
lection on a four-circle Rigaku R-AXIS RAPID-S diffractometer
(equipped with a two-dimensional area IP detector). The graph-
tosyl-pyrrole (1) (10.50–39.5
(2) (12.75–25.6 M), 5-hydroxy-1-tosyl-1H-pyrrol-2(5H)-one (3)
(2.5–10.2 M) and 5-oxo-1-tosyl-2,5-dihydro-1H-pyrrol-2-yl ace-
tate (12) (5.0–10.7) at cuvette concentrations and HCA-II enzyme
activities were measured for 1-p-tosyl-pyrrol (1) (10.15–21.5 M),
1-tosyl-1H-pyrrol-2(5H)-one (2) (7.65–15.8 M), 5-hydroxy-1-to-
syl-1H-pyrrol-2(5H)-one (3) (0.05–0.55 M) and 5-oxo-1-tosyl-
lM), 1-tosyl-1H-pyrrole-2(5H)-one
l
l
ite-monochromatized Mo K radiation (k = 0.71073 Å) and oscilla-
a
l
tion scans technique with Dx = 5° for one image were used for
l
data collection. The lattice parameters were determined by the
least-squares methods on the basis of all reflections with
l
2,5-dihydro-1H-pyrrol-2-yl acetate (12) (0.10–0.75) at cuvette
concentrations. Control cuvette activity in the absence of inhibitor
was taken as 100%. For each inhibitor, an Activity (%)–[Inhibitor]
F2 > 2 (F2). Integration of the intensities, correction for Lorentz
r
and polarization effects and cell refinement was performed using
CrystalClear (Rigaku/MSC Inc., 2005) software.31 The structures

4474
C. Alp et al. / Bioorg. Med. Chem. 18 (2010) 4468–4474
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Crystal data and structure refinement
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C11H11NO4S
253.3
293(2)
0.71073
Monoclinic
P21/c
12.2054(2)
7.3819(2)
12.8944(4)
90
93.93(3)
90
1159.0(1)
4
1.45
0.281
528
Plate; colourless
0.20 Â 0.02 Â 0.02
3.2–30.6
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
Volume (Å3)
Z
Density (calcd) (Mg/m3)
Absorption coefficient (mmÀ1
F(0 0 0)
)
Crystal
Crystal size (mm3)
h range for data collection (°)
Index ranges
À17 6 h 6 17, À9 6 k 6 10, À18 6 l 6 18
Reflections collected
Independent reflections
Completeness to h = 30.2°
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
33639
3558 [Rint = 0.079]
99.90%
Full-matrix least-squares on F2
2561/0/156
1.054
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Final R indices [F2 > 2
R indices (all data)
r
(F2)]
R1 = 0.058, wR2 = 0.159
R1 = 0.084, wR2 = 0.179
0.216 and À0.414 e ÅÀ3
Largest diff. peak and hole
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were solved by direct methods using SHELXS-9732 and refined by a
full-matrix least-squares procedure using the program SHELXL
-
97.32 Hydrogen atom involved in hydrogen bonding was located
in the difference Fourier map and allowed to refine freely. All other
hydrogen atoms were added at calculated positions and refined
using a riding model. Their isotropic temperature factors were
fixed to 1.2 times (1.5 times for methyl groups) the equivalent
isotropic displacement parameters of the parent carbon atom.
Anisotropic thermal displacement parameters were used for all
non-hydrogen atoms. The final difference Fourier maps showed
no peaks of chemical significance. Crystal data and structure
refinement parameters of compound (3) are given in Table 4.
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Supplementary data
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Crystallographic data (excluding structure factors) for the struc-
tures reported in this article have been deposited with the Cam-
bridge Crystallographic Data Centre as supplementary
publication number CCDC 765673. Copies of the data can be ob-
tained free of charge on application to CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK (fax: +44-1223-336-033; e-mail:
deposit@ccdc.cam.ac.uk).
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Acknowledgement
This research was financed by grants from the Scientific and
Technological Research Council of Turkey (Project no: TBAG-
107397) to CA and MSG.
References and notes
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