Carbonic anhydrase inhibitors: Sulfonamides incorporating furan-, thiophene- and pyrrole-carboxamido groups possess strong topical intraocular pressure lowering properties as aqueous suspensions

Article abstract of DOI:10.1016/S0968-0896(00)00143-7

Important physiological and physio-pathological functions are played by several carbonic anhydrase (CA, EC 4.2.1.1) isozymes, which are strongly inhibited by aromatic and heterocyclic sulfonamides. Here we report several new types of such sulfonamides, incorporating furan-, thiophene- and pyrrole-carboxamide moieties in their molecules. Some of these compounds showed very good CA II and CA IV inhibitory properties, with affinities for the enzymes in the low nanomolar range. Due to their relatively low water solubility, some of the most active CA II inhibitors reported here have been formulated as aqueous suspension for topical administration as antiglaucoma agents, in normotensive and glaucomatous rabbits. The derivatives incorporating furan- and pyrrole-carboxamide moieties (but not the corresponding thiophene-substituted derivatives), showed effective and long-lasting intraocular pressure (IOP) lowering both in normotensive as well as glaucomatous animals, with potencies superior to dorzolamide and brinzolamide, the two available topically acting sulfonamide drugs. This is the first example of non-water soluble sulfonamides that significantly lower IOP, being thus similar with the recently introduced drug brinzolamide, which belongs to a completely different chemical family of antiglaucoma sulfonamides. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00143-7

Bioorganic & Medicinal Chemistry 8 (2000) 2145±2155
Carbonic Anhydrase Inhibitors: Sulfonamides Incorporating
Furan-, Thiophene- and Pyrrole-carboxamido Groups Possess
Strong Topical Intraocular Pressure Lowering Properties as
Aqueous Suspensions
Monica Ilies,a Claudiu T. Supuran,b,* Andrea Scozzafava,b Angela Casini,b
Francesco Mincione,c Luca Menabuoni,d Miron T. Caproiu,e Maria Maganue
and Mircea D. Banciuf
aUniversity of Agricultural Sciences and Veterinary Medicine, Faculty of Biotechnologies, Department of Chemistry,
B-dul Marasti 59, 71331-Bucharest, Romania
b
Á
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Florence, Italy
c
Á
Universita degli Studi, Istituto Oculistico, Viale Morgagni 85, I-50134 Florence, Italy
dOspedale San Giovanni di Dio, S. O. Oculistica, Via Torregalli 3, I-50123, Florence, Italy
e`C.D. Nenitzescu' Institute of Organic Chemistry, Splaiul Independentei 202B, Bucharest, Romania
fPolytechnic University, Department of Organic Chemistry, Splaiul Independentei 313, Bucharest, Romania
Received 3 April 2000; accepted 31 May 2000
AbstractÐImportant physiological and physio-pathological functions are played by several carbonic anhydrase (CA, EC 4.2.1.1)
isozymes, which are strongly inhibited by aromatic and heterocyclic sulfonamides. Here we report several new types of such sulfo-
namides, incorporating furan-, thiophene- and pyrrole-carboxamide moieties in their molecules. Some of these compounds showed
very good CA II and CA IV inhibitory properties, with anities for the enzymes in the low nanomolar range. Due to their relatively
low water solubility, some of the most active CA II inhibitors reported here have been formulated as aqueous suspension for topical
administration as antiglaucoma agents, in normotensive and glaucomatous rabbits. The derivatives incorporating furan- and pyr-
role-carboxamide moieties (but not the corresponding thiophene-substituted derivatives), showed e€ective and long-lasting intrao-
cular pressure (IOP) lowering both in normotensive as well as glaucomatous animals, with potencies superior to dorzolamide and
brinzolamide, the two available topically acting sulfonamide drugs. This is the ®rst example of non-water soluble sulfonamides that
signi®cantly lower IOP, being thus similar with the recently introduced drug brinzolamide, which belongs to a completely di€erent
chemical family of antiglaucoma sulfonamides. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
acid production in the proximal tubule of the kidney7), as
well as some biosynthetic reactions such as the lipogenesis
in adypocites8 or gluconeogenesis and ureagenesis in the
liver/kidneys.9 CA inhibition may ensue interesting
medical applications for the prevention or treatment of
disorders caused by an excessive activity of these
enzymes. The inhibitory ability of aromatic/heterocyclic
sulfonamides against a-CAs has been extensively studied
and periodically reviewed3,5a,10 during the last 50 years.
The continuous development of research in this ®eld is
motivated by the practical applications of sulfonamide
CA inhibitors. Such compounds have been used ®rst as
diuretics,11 or afterwards in the therapy of acid-base
disequilibria,5,12 epilepsy,13 mountain sickness,14 gastro-
duodenal ulcers,7,15 glaucoma,16,17 congestive heart
failure,16 osteoporosis.18 Recently, some new approaches
At least 14 di€erent isozymes of the zinc protein car-
bonic anhydrase (CA, EC 4.2.1.1.) have been reported
up to now in higher vertebrates.1 3 By catalyzing the
reversible carbon dioxide hydration to bicarbonate, some
of these enzymes participate in crucial physiological pro-
cesses in these organisms, such as: transport of CO2/
HCO3 between metabolizing tissues and lungs,4 pH
homeostasis,5 cellular signal transduction,5b secretion of
electrolytes in di€erent tissues/organs (e.g. aqueous
humor in the ciliary processes,5a,6 pancreatic7 and gastric
juice7 formation, cerebrospinal ¯uid secretion,5a,7 urinary
*Corresponding author. Tel.: +39-055-275-7552; fax: +39-055-
2757555; e-mail: cts@bio.chim.uni®.it
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00143-7

2146
M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
for the therapy of cancer19 or for designing novel diag-
nostic tools in positron emission tomography (PET) and
magnetic resonance imaging (MRI) have been reported
for some compounds belonging to this class of pharma-
cological agents.20,21
CA isozymes or the organs/tissues in which these are
present.3,34 37Therefore, the synthesis and assay as CA
inhibitors of novel aromatic/heterocyclic sulfonamide
derivatives still represents an interesting challenge in order
to discover isozyme-speci®c or, at least, organ-selective
such pharmacological agents, devoid of severe side e€ects.
Aromatic sulfonamide CA inhibitors such as sulfanilami-
de3,5a,22 1a or dichlorophenamide11a 2 have been used both
as drugs as well as leads for designing other biologically
active compounds (such as the thiazides and high-ceiling
diuretics11a,16a,23 or antithyroid drugs24). Among the
heterocyclic sulfonamides, acetazolamide25 3, methazo-
lamide26 4, and ethoxzolamide27 5 have been widely used
clinically for more than 45 years, mainly as systemic anti-
glaucoma drugs. More recently, dorzolamide28,29 6 and the
structurally related brinzolamide30 7 have been approved
as topical antiglaucoma agents. Benzolamide 8 and chlor-
zolamide 9 Ð sulfonamides containing both aromatic and
heterocyclic rings Ð are also representatives of this class of
CA inhibitors, and possess interesting applications both
for the design of ecient new antiglaucoma agents31 or
for PET-based diagnostic tools.32 All the clinically
available CA inhibitors3,33 possess some relevant side
e€ects, due to their lack of selectivity towards di€erent
In this paper we report several new types of aromatic/
heterocyclic sulfonamide CA inhibitors with potential use
as topical antiglaucoma agents. An ideal antiglaucoma
drug of this type should possess the following features: (i)
it must be a strong CA II and CA IV inhibitor, in order to
achieve a signi®cant reduction of aqueous humor secre-
tion; (ii) it should posses a high enough liposolubility in
order to penetrate through the cornea, but this should
eventually be balanced by an acceptable hydro-
solubility, in order to formulate it in acceptable form for
the topical administration.34 401
Up to now, only dorzolamide 6 seemed to possess such
properties,17 but this drug is not very e€ective, and
many serious side e€ects have been reported after its
administration.41 44 Less eye stinging/reddening than
with dorzolamide has been reported with brinzolamide

M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
2147
7,30 which is applied as an aqueous suspension, due to
its reduced water solubility. Thus, we thought to design
such sulfonamides, possessing a relatively balanced
hydro-/lipo-solubility, that can be formulated either as
solutions, or suspensions for the topical administration.
The drug design has been based on the `tail' strategy
reported previously by our group,37,38 which consists in
attaching certain moieties that would induce the desired
physico-chemical properties to the molecules of aro-
matic/heterocyclic sulfonamides possessing free amino/
hydroxy groups. These moieties should induce among
others high anity to the CA active site, acceptable
water/lipid solubility, and good penetrability through the
biological membranes, to the molecules of the newly
obtained CA inhibitors. The tails chosen to be incorpo-
rated in the compounds reported here are of the hetero-
cyclic-carboxamide type, since such derivatives were
little investigated up to now, and they may be easily
®ne-tuned for obtaining both compounds with an
increased water- as well as lipid solubility.
In vivo IOP lowering after topical administration of
some of the new sulfonamides as well as dorzolamide 6
(as standard), in normotensive rabbits, are shown in Table
3 and Figure 1, whereas data of glaucomatous rabbits are
shown in Table 4.
Discussion
Three related series of compounds, A1±A7, B1±B9 and
C1±C9, incorporating pyrrole-, furan- and thiophene-
carboxamide moieties, respectively, were prepared as
shown in Scheme 1. For the furan and thiophene deri-
vatives, classical acylation procedures involving the acyl
chlorides (method A) led to good results. In the case of
the pyrrole carboxylic acid derivatives (which are highly
sensitive to acidic medium), two di€erent procedures
were employed: a peptide-like coupling with some ami-
nosulfonamides, in the presence of diisopropylcarbodii-
mide (method B),37,38 or the reaction of in situ generated
pyrrole-2-carboxylic acid chloride with the above men-
tioned aminosulfonamides (method C). Mention should
be made that paradoxically, very few sulfonamide CA
inhibitors incorporating heterocyclic-carboxamido moi-
eties have been reported up to now, although several
thousand di€erent sulfonamides have been synthesized
in the search of topically acting antiglaucoma sulfona-
mides in the last 15 years.35 40
Results
The new compounds A1±A7, B1±B9 and C1±C9, incor-
porating pyrrole-, furan- and thiophene-carboxamide
moieties, respectively, reported here, are shown in Scheme
1, and were prepared by non-exceptional procedures,
related to those previously reported by Whiteside's39,40
and our groups.37,38
Inhibition data with the new compounds, the parent sul-
fonamides and standard inhibitors (Table 1) showed the
following: (i) the new compounds A1±A7, B1±B9 and C1±
C9 showed increased anities for the three investigated
CA isozymes, as compared to the corresponding parent
compounds from which they were prepared (the sulfona-
mides 14±22); (ii) the anities of the new inhibitors gen-
erally varied in the following way, based on the parent
sulfonamide from which they were prepared: the deriva-
tive of sulfanilamide<the homosulfanilamides<the p-
Inhibition data against three CA isozymes (hCA I, hCA
II and bCA IV; h=human, b=bovine isozyme) with the
new compounds reported here and standard inhibitors
are shown in Table 1.
Some physico-chemical properties of several highly active
CA inhibitors reported in the paper (as well as data for
some standard compounds) are shown in Table 2.
Scheme 1. Method A: ZˆCl, Net3/MeCN, 0 ꢀC; Method B: ZˆOH, DIPCD/DMAP/dioxane, rt (XˆNH); Method C: ZˆOH, ®rst SOCl2/Py,
1 h 300, rt then aminosulfonamide (XˆNH).

2148
M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
Table 1. CA inhibition data with standard inhibitors, the parent
sulfonamides 14±22 and the new derivatives reported in the present
study, against isozymes I, II and IVc
Table 2. Solubility, chloroform-bu€er partition coecients and in
vitro corneal permeability of some sulfonamide CA inhibitors
1 c
kinÂ103 (h
)
K*I (nM)
Compound
Solubilitya
nM
Log
Pb
Cornea
intact
No
epithelium
Inhibitor
hCA Ia
hCA IIa
bCA IVb
1 (acetazolamide)d
3.2
12
0.001
0.06
0.37
1.90
3.0
4.0
0.6
2.8
0.9
4.1
4.7
4.8
5.0
7.0
13
Acetazolamide 1
Methazolamide 2
Dorzolamide 6
14
15
16
17
18
19
20
21
22
A1
A2
A3
A4
A5
A6
A7
B1
B2
B3
B4
B5
B6
B7
B8
B9
C1
C2
C3
C4
C5
C6
C7
C8
C9
250
50
12
14
9
70
36
45
2 (methazolamide)d
6 (dorzolamide)
60e
5.0
2.0e
1.39
5.2
4.6
1.9
6.0
3.8
7.6
8.5
11.5
10.2
50,000
28,000
8300
9800
6500
6000
25,000
21,000
8600
9300
25,000
500
A2
A6
A7
B8
B9
C2
C8
C9
300
60
110
40
70
170
160
60
19
105
10
21
36
21
8
3000
180
320
66
125
2800
2450
540
355
160
41
56
80
52
40
36
140
37
45
55
48
49
47
32
30
103
23
15
21
17
49
47
15
5.8
9.2
2.4
7.5
0.05
0.04
0.1
0.0008
0.536
0.005
0.633
0.44
0.033
0.750
aSolubility in pH 7.40 bu€er, at 25 ꢀC.
bChloroform-bu€er partition coecient.
cDetermined as described in ref56,57
dData from ref37
550
1000
850
350
eAs hydrochloride, at pH 5.8, from ref17
320
7
15,000
445
510
95
10
17
30
23
31
20
6
6
62
8
7
12
9
Table 3. Fall of IOP of normotensive rabbits (20Æ3 mm Hg), after
treatment with one drop (50 mL) 2% water suspension/solution (for
dorzolamide 6) of CA inhibitor directly into the eye, at 30, 60 and
90 min after administration
520
550
960
800
280
290
ÁIOP (mm Hg)*
Inhibitor
pH
t=0
t=30 min
t=60 min
t=90 min
11,500
365
240
6a
5.5
7.5
7.5
7.0
7.5
7.5
7.5
7.5
7.5
7.5
0
0
0
0
0
0
0
0
0
0
2Æ0.20
4Æ0.30
3Æ0.25
2Æ0.20
2Æ0.15
5Æ0.20
5Æ0.15
7Æ0.30
9Æ0.35
0.5
A2
A6
A7
B5
B8
B9
C2
C8
C9
8Æ0.40
2Æ0.25
140
120
540
270
280
290
0
0
4Æ0.30
8Æ0.40
16
12
3
0
0
3Æ0.10
0
7Æ0.25
9Æ0.45
4
19
0
0
0
0
0
0
0
0
aHuman (cloned) isozyme.
bIsolated from bovine lung microsomes.
cData from ref 30 Standard error for the determination of KI-s was of
10±20% (from 3 di€erent assays).
aAs HCl salt, in solution. *ÁIOP=IOPcontrol
MeanÆSE (n=3).
IOPtreated
;
eye
eye
aminoethyl-benzenesulfonamides the bromo/iodo-sub-
stituted sulfanilamides<the the chloro/¯uoro-substituted
sulfanilamides<the 1,3,4-thiadiazole-2-sulfonamides 4-
methyl-d2-1,3,4-thiadiazoline-2-sulfonamide; (iii) based
on the carboxylic acid from which they were obtained,
the thiophene carboxylic acid derivatives were more
e€ective CA inhibitors as compared to the correspond-
ing furan carboxylic acid derivatives, which in turn were
slightly more active than the corresponding pyrrole
carboxamido derivatives; (iv) all three CA isozymes
investigated here were susceptible to inhibition with this
type of sulfonamide, with hCA II and bCA IV the most
sensitive, whereas hCA I was generally less susceptible
to inhibition as compared to the ®rst two isozymes.
normotensive and glaucomatous rabbits (frequently
used as an animal model of glaucoma) of suspensions of
these CA inhibitors.35 37Mention should be made that
in contrast to dorzolamide 6, the compounds reported
here do not possess a good enough water solubility in
order to formulate them as 2% solutions. This is in fact
also the case of brinzolamide 7, the recently approved
new topical CA inhibitor. Thus, we used 2% water sus-
pensions of the new compounds for the in vivo experiments
discussed shortly.
The compounds selected for in vivo studies were among
the most active in vitro inhibitors against hCA II and
bCA IV, in the prepared series, such as: A2, A6, A7, B5,
B8, B9, C2, C8 and C9. The following should be noted
regarding data shown in Tables 2±4: (i) the compounds
incorporating thiophene-carboxamide moieties (such as
C2, C8 and C9) are ine€ective as topical IOP lowering
agents, although they were the best CA II and CA IV inhi-
bitors in the new series; (ii) the compounds incorporating
The promising in vitro CA inhibitory activity as well as
other physico-chemical properties (Table 2) for some of
the newly prepared compounds prompted us to investi-
gate their e€ect in vivo, on the intraocular pressure
(IOP), after topical application directly into the eye, in

M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
2149
thiophene-carboxamide derivatives investigated here
were the most e€ective in vitro CA inhibitors in the three
reported subseries, were liposoluble enough so that they
had the best accession rates through the cornea, but due
to their virtually zero water solubility, they did not
show at all any topical action. In fact, these compounds
are quite similar to methazolamide, acetazolamide or
ethoxzolamide, which are all three ine€ective topically,
due to the same problem encountered above, i.e.
absence of appropriate water solubility. The thiadia-
zole-sulfonamide derivatives A6 and B8 act as potent CA
inhibitors, have a low (but acceptable) water solubility,
but being very polar (log P of the same order of magni-
tude as that of acetazolamide or lower), their access
through the cornea is problematic (see kin values in
Table 2), so that they only showed modest IOP lowering
e€ects, which appeared after 90 min or longer periods
from administration (data not shown). In contrast to
these compounds, the closely related thiadiazolines A7
and B9 (possessing an additional methyl group as com-
pared to the previously mentioned thiadiazoles A6 and
B8) possess all the three factors mentioned above in the
right measure: they are strong CA inhibitors (similarly
to the thiadiazoles), possess a low (but acceptable) water
solubility, balanced by a modest lipid solubility (aug-
mented as compared to that of the corresponding thia-
diazoles, due to the presence of the additional methyl
moieties), so that their accession rates through the cornea
are of the same magnitude (or better) than those of
dorzolamide. This seems to be the reason why these
compounds act as more ecient IOP lowering agents
than the standard drug 6. The other compound studied
in some detail (A2) o€ers another examples that may
validate our `theory' exposed above. Thus, A2 is very
ecient in lowering IOP at early periods after adminis-
tration, but then the IOP lowering vanishes rapidly. We
consider this to be due to the relatively high liposolubility
of this compound, which helps a lot its penetration
through the cornea, but at the same time assures its easy
di€usibility into the blood (where high amounts of CA I
and CA II are present), so that the drug may be washed
out from the ciliary processes too rapidly.
Figure 1. E€ect of topically administered sulfonamide inhibitors (2%
water solutions/suspensions) on the IOP of normotensive albino rab-
bits. Curve 1: dorzolamide 1 (hydrochloride salt, pH 5.5; solution);
curve 2: compound A2 (suspension, pH 7.50); curve 3: compound A7
(suspension, pH 7.0), curve 4: compound B9 (suspension, pH 7.50).
Table 4. Fall of IOP of glaucomatous rabbits (36Æ2 mm Hg) after
treatment with one drop (50 mL) 2% suspension of sulfonamide
directly into the eye, at 30, 60 and 90 min after administration
ÁIOP (mm Hg)a*
Inhibitor
pH
t=0
t=30 min
t=60 min
t=90 min
A2
A7
B9
7.5
7.0
7.5
0
0
0
10Æ0.35
7.5Æ0.50
9.5Æ0.35
10.5Æ0.30
11Æ0.25
14Æ0.20
8.5Æ0.40
15Æ0.20
16Æ0.35
a*ÁIOP=IOPcontrol eye IOPtreated eye; MeanÆSE (n=3).
pyrrole-carboxamide- and furan-carboxamide moieties
(such as A2, A6, A7, B5, B8, B9) are show topical
activity in lowering IOP, after administration as aqueous
suspensions. Some of these derivatives, such as A6, B5
and B8 show relatively modest activity, of the same
order of magnitude (or slightly less) as that of dorzolamide
6, used as standard in these measurements (brinzola-
mide 7, although already available clinically in USA,
has not been approved yet in Europe and we could not
include it in our experiments). Other compounds, such
as A7 and B9 showed very e€ective IOP lowering after
topical administration, being 2±4 times more e€ective
than dorzoalmide, both at 30, 60 and 90 min after
administration. Furthermore, their ecacy is much
more prolonged in time, as compared to that of dorzo-
lamide, as observed from the data of Figure 1. Practically
IOP returns at basal values 2 h after administration of
dorzolamide 6, whereas the same e€ect is observed after
6 h with the most active compound in this series, which
is B9. Returning now to the structure±activity relation-
ship, some very interesting facts emerged during this
research. Thus, one may observe that the most active
compounds in lowering IOP in this series were those
possessing the following three features: (1) strong CA II
(and CA IV) inhibitory properties, (2) moderate water
and lipid solubilities; and (3) good accession rates
through the cornea. If only one of these three factors is
absent, the corresponding compound is a failure from
the point of view of topical IOP lowering. Thus, the
The data of Table 4 show that these compounds are highly
active IOP lowering agents also in the glaucomatous rab-
bit, again with B9 acting very eciently, followed by A7
and A2.
Conclusion
We report here some novel aromatic/heterocyclic sulfo-
namides incorporating pyrrole-, furan- and thiophene-
carboxamide moieties in their molecules. Many of the
new compounds showed low nanomolar anities for
isozymes CA II and CA IV, being slightly less e€ective
in inhibiting isozyme CA I. The compounds which
showed strong CA II (and CA IV) inhibitory properties,
correlated with moderate (but not insigni®cant) water
and lipid solubilities; as well as good accession rates
through the cornea, e€ectively lowered IOP in normo-
tensive and glaucomatous rabbits, when administered as
2% water suspensions directly into the eye. Furthermore,

2150
M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
such compounds showed a prolonged duration of action
as compared to the clinically used drug dorzolamide,
administered as 2% solution. As far as we know this
is one of the ®rst studies which ®ne-tunes the in vivo
properties of topically acting sulfonamide CA inhibi-
tors, based on the physico-chemical properties of the
obtained derivatives, allowing thus for a rationale drug
design of more e€ective antiglaucoma therapies.
Method A. An amount of 5 mmoles aminosulfonamide
was suspended/dissolved in 20±30 mL anhydrous MeCN
and 0.78 mL (0.56 g, 5.5 mmoles) triethylamine was
added under stirring. The mixture was cooled to 0±5 ꢀC,
then a solution of 5 mmoles furoyl/thienyl chloride dis-
solved in 3 mL MeCN was added dropwise during
10 min (immediately, a white precipitate appeared). The
reaction was stirred overnight or until a reasonable
conversion was reached (TLC control). The solvent was
evaporated in vacuum and the resulted product was trea-
ted with 15±20 mL water. The crude solid product was
®ltered, washed with 2 mL water and air dried. Yields
were in the range of 35±90%, depending on the reactivity
of the aminosulfonamide. The obtained compounds were
further puri®ed by recrystallisation from the speci®ed
solvent or, in some cases, by column chromatography,
using silicagel 60 as stationary phase, eluted with
MeOH/CHCl3 gradients. The fractions containing the
desired compound were collected, evaporated to dryness
and the obtained product was recrystallized from solvents
speci®ed in each case.
Experimental
Chemistry
Melting points were determined with a Boetius heating
plate microscope and are not corrected; IR spectra were
obtained in KBr pellets with a Carl Zeiss UR 20 spectro-
1
meter. H NMR and 13C NMR spectra were obtained
using a Varian Gemini 300BB apparatus operating at
1
300 MHz for H and 75 MHz for 13C, in d6-DMSO as
solvent. Chemical shifts are expressed as d values (ppm)
relative to Me4Si as internal standard. Attributions were
done by means of chemical shifts, peak integration,
COSY (1H-1H), HETCOR (1H-13C), attached proton test
(APT), model spectra and selective deuteration. Elemental
analyses were done by combustion, for C, H, N, with an
automated Carlo Erba analyzer and the results were
foundÆ0.4% within the theoretical values.
Method B. Five mmoles of aminosulfonamide, dissolved
in 30mL anhydrous dioxane were treated, under stirring,
with 0.78 mL (0.63 g, 5 mmoles) diisopropylcarbodii-
mide (DIPCD) and 0.06 g (0.5 mmoles) dimethylamino-
pyridine (DMAP); the reaction mixture was kept under
argon and 0.56g (5mmoles) of pyrrol-2-carboxylic acid
was added. The obtained homogeneous colorless solution
turned to orange-cream and in around 20min a precipitate
appeared. The stirring was continued overnight, then the
precipitated diisopropylurea (DIPU) was ®ltered, washed
with a small volume of dioxane and discarded. The com-
bined dioxanic solutions were subsequently evaporated to
a small volume and treated with 20 mL of ethyl acetate,
for complete precipitation of DIPU (vide infra). After
®ltering, the precipitate (DIPU) was triturated with
10 mL aqueous NaOH (10%) re®ltered, then discarded.
The aqueous solution was brought to pH=1.5 with 20%
aqueous HCl. The precipitated crude product was ®l-
tered, washed with 2 mL water and dried. An additional
amount of crude product was obtained by extracting
twice the previous ethyl acetate mother liquor with
10 mL 10% aqueous NaOH, separating the aqueous
layer, treating it with 20% aqueous HCl and ®ltering,
washing and drying the resulting precipitate. Total yields
were in the range of 45±55%. The pure products were
obtained after column chromatography, using a similar
procedure as in method A.
All reactions were monitored by thin-layer chromato-
graphy (TLC), using 0.25 mm-thick precoated silica gel
plates (E. Merck) eluted with MeOH:CHCl3 1:4 v/v. 2-
Pyrrole-carboxylic acid, homosulfanilamide (hydro-
chloride) and 4-(2-aminoethyl)benzenesulfonamide were
from Sigma-Aldrich (Milan, Italy). 2-Thiophene- and 2-
furancarboxylic acid chlorides, sulfanilamide, as well as
pyridine, acetonitrile and other solvents used in syntheses
were from E. Merck (Darmstadt, Germany). 5-Amino-
1,3,4-thiadiazole-2-sulfonamide was obtained from acet-
azolamide (Sigma) by deprotection with concentrated
hydrochloric acid followed by neutralization, using a
previously reported procedure.41,43 5-Imino-4-methyl-
Á2-1,3,4-thiadiazoline-2-sulfonamide hydrochloride was
similarly prepared from methazolamide (Sigma). Halo-
genosulfonamides were prepared by literature proce-
dures,45 optimized in our laboratory. Preparative column
chromatography was performed on silica gel 60 (0.063±
0.200 mm)-from Merck-eluted with MeOH:CHCl3 1:9 v/v.
General procedure for the preparation of compounds
Method C. An amount of 0.56 g (5 mmoles) of pyrrol-2-
carboxylic acid was dissolved under stirring in 10 mL of
anhydrous pyridine, then 0.4 mL (0.654 g, 5.5 mmoles)
freshly distilled thionyl cloride was added dropwise.
After the ¯ask was septum stoppered and ¯ushed with
argon, three drops of dimethylformamide were added
with a syringe needle, left afterwards into the septum.
The generation of pyrroyl chloride was completed in
one hour (TLC control); meanwhile a precipitate of
pyridinium chloride was formed. The reaction mixture
was treated with the stoechiometric amount (5 mmoles)
of the corresponding aminosulfonamide/aminosulfona-
mide hydrochloride and stirred under inert atmosphere
The compounds were obtained from the corresponding
hetharylcarboxylic acid chlorides, commercially available
for thiophene and furan, using classical acylation proce-
dures26 in acetonitrile as solvent (method A). In the case of
pyrrol carboxylic acid Ð very sensitive to acidic medium,
two procedures were used: peptide-like coupling using
carbodiimides (DIPCD/DMAP), which works well with
aminosulfonamides with an aliphatic amino group
(method B) and coupling of the corresponding pyrroyl
chloride, generated in situ (by treating the pyrrol-2-car-
boxylic acid with SOCl2 in anhydrous pyridine) with
amino(halogeno)sulfonamides (method C).

M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
2151
until a reasonable conversion was obtained (TLC control).
The solvent was then evaporated in vacuum and the
resulted glue was treated with 15±20 mL of water, when
the crude product precipitated. This was ®ltered,
washed with 2 mL water and air-dried. Yields were in
the range of 15±55%, depending on the reactivity of the
aminosulfonamide. The pure products were obtained as
described in method A.
NMR (DMSO-d6), d, ppm: 41.58 (CH2), 108.64 (C4-
pyrrole), 110.14 (C3-pyrrole), 125.68 (C2/C3-Ph), 121.56
(C5-pyrrole), 125.94 (C2-pyrrole), 127.41 (C3/C2-Ph),
142.53(C1/C4-Ph), 144.19 (C4/C1-Ph), 160.75 (CONH).
Anal. (C12H13N3SO3) C, H, N, S.
4-(Pyrrole-2-carboxamidoethyl)benzenesulfonamide, A5,
(method B). White crystals, mp 263±6 ꢀC (MeOH). IR
(KBr), cm 1: 1160 (SO2sym), 1330 (SOa2s), 1560 (amide
1
4-(Pyrrole-2-carboxamido)benzenesulfonamide, A1, (method
C). White crystals, mp 297±9 ꢀC (MeOH). IR (KBr),
cm 1: 1160 (SO2sym), 1330 (SOa2s), 1585 (amide II), 1640
(amide I); 1H NMR (d6-DMSO), d, ppm; J, Hz: 6.19 (m,
1H, H4-pyrrole, 3.7, 2.3, 1.4 etc.), 7.01 (m, 1H, H3-pyr-
role, 2.7, 1.5 etc.), 7.12 (m, 1H, H5-pyrrole, 2.4, 1.4 etc.),
7.23 (s, 2H, SO2NH2), 7.78 (d, 2H, AA0BB0, 8.9), 7.91 (d,
2H, AA0BB0, 8.9), 10.03 (s, 1H, NHCO), 11.72 (s, 1H,
H1-pyrrole); 13C NMR (d6-DMSO), d, ppm: 109.07 (C4-
pyrrole), 112.00 (C3-pyrrole), 119.20 (C2/C3-Ph), 123.18
(C5-pyrrole), 125.60 (C2-pyrrole), 126.5 (C3/C2-Ph),
137.92 (C1/C4-Ph), 142.39 (C4/C1-Ph), 159.27 (CONH).
Anal. (C11H11N3SO3) C, H, N, S.
II), 1610 (amide I); H NMR (d6-DMSO), d, ppm; J,
Hz: 2.90 (t, 2H, 7.2), 3.47 (q, 2H, 6.5), 6.07 (m, 1H, H4-
pyrrole, 3.6, 2.5, 2.2 etc.), 6.73 (m, 1H, H3-pyrrole, 3.7, 1.5
etc.), 6.84 (m, 1H, H5-pyrrole, 2.6, 1.4 etc.), 7.29 (s, 2H,
SO2NH2), 7.42 (d, 2H, AA0BB0, 8.2), 7.75 (d, 2H, AA0BB0,
8.2), 8.08 (t, 1H, NHCO, 5.7), 11.39 (s, 1H, H1-pyrrole);
13C NMR (DMSO-d6), d, ppm: 35.14 (CH2-Ph), 39.70,
(N-CH2), 108.50 (C4-pyrrole), 109.77 (C3-pyrrole), 125.68
(C2/C3-Ph), 121.23 (C5-pyrrole), 126.25 (C2-pyrrole),
129.14 (C3/C2-Ph), 141.99 (C1/C4-Ph), 143.89 (C4/C1-
Ph), 160.66 (CONH). Anal. (C13H15N3SO3) C, H, N, S.
5-(Pyrrole-2-carboxamido)-1,3,4-thiadiazole-2-sulfona-
mide, A6, (method C). Dark yellow crystals, mp 291±
4 ꢀC (MeOH). IR (KBr), cm 1: 1170 (SO2sym), 1320 (SOa2s),
1580 (amide II), 1650 (amide I); 1H NMR (d6-DMSO), d,
ppm; J, Hz: 6.26 (dt, 1H, H4-pyrrole, 3.9, 2.3 etc.), 7.16
(m, 1H, H3-pyrrole, 3.9, 1.5 etc.), 7.43 (m, 1H, H5-pyr-
role, 2.5, 1.4 etc.), 8.31 (s, 2H, SO2NH2), 12.12 (s, 1H,
H1-pyrrole), 13.07 (s, 1H, NHCO); 13C NMR (DMSO-
d6), d, ppm: 110.11 (C4-pyrrole), 115.11 (C3-pyrrole),
125.69 (C5-pyrrole), 122.95 (C2-pyrrole), 158.07 (CONH),
161.93 (C-thiadiazole), 164.22 (C-thiadiazole). Anal. (C7
H7N5S2O3) C, H, N, S.
4-(Pyrrole-2-carboxamido)-3-¯uorobenzenesulfonamide,
A2, (method C). White crystals, mp 284±6 ꢀC (MeOH).
IR (KBr), cm 1: 1160 (SO2sym), 1320 (SOa2s), 1600 (amide
1
II), 1650 (amide I); H NMR (d6-DMSO), d, ppm; J,
Hz: 6.20 (m, 1H, H4-pyrrole, 3.7, 2.4, 1.4 etc.), 7.01 (m,
1H, H3-pyrrole, 3.7, 2.1 etc.), 7.10 (m, 1H, H5-pyrrole,
2.2, 1.5 etc.), 7.46 (s, 2H, SO2NH2), 7.65 (ddd, 1H, H2-
Ph, 6.1, 2.0), 7.68 (dtr, 1H, H6-Ph, 8.4, 2.0), 7.92 (ddtr,
1H, H5-Ph, 8.3, 4.1, 1.6), 9.84 (s, 1H, NHCO), 11.79 (s,
1H, H1-pyrrole); 13C NMR (DMSO-d6), d, ppm; JC,F
,
Hz: 109.24 (C4-pyrrole), 112.59 (C3-pyrrole), 113.40 (d,
C2-Ph, 22.9), 121.13 (C5-Ph), 123.28 (C6-Ph), 125.12
(C2-pyrrole), 125.98 (C5-pyrrole), 129.39 (d, C4-Ph,
11.8), 140.81 (d, C1-Ph, 6.2), 153.89 (d, C3-Ph, 249.9),
159.01 (CONH). Anal. (C11H10N3SO3F) C, H, N, S.
2
5-(Pyrrole-2-carboximido)-4-methyl-D -1,3,4-thiadiazo-
line-2-sulfonamide, A7, (method C). White crystals, mp
294±7 ꢀC (MeOH). IR (KBr), cm 1: 1170 (SOs2ym), 1325
as
1
(SO2 ), 1520 (amide II), 1600 (amide I); H NMR (d6-
DMSO), d, ppm; J, Hz: 6.22 (dt, 1H, H4-pyrrole, 3.7, 2.3
etc.), 6.99 (m, 1H, H3-pyrrole, 3.8, 1.6, 0.5 etc.), 7.07 (m,
1H, H5-pyrrole, 2.6, 1.6 etc.), 8.44 (s, 2H, SO2NH2), 11.84
(s, 1H, H1-pyrrole); 13C NMR (DMSO-d6), d, ppm: 37.72
(CH3), 109.60 (C4-pyrrole), 114.99 (C3-pyrrole), 123.89
(C5-pyrrole), 128.55 (C2-pyrrole), 157.09 (CONH), 163.45
(C-thiadiazole), 166.52 (C-thiadiazole). Anal. (C8H9N5S2
O3) C, H, N, S.
4-(Pyrrole-2-carboxamido)-3-chlorobenzenesulfonamide,
A3, (method C). White crystals, mp 254±7 ꢀC (MeOH).
IR (KBr), cm 1: 1160 (SO2sym), 1320 (SOa2s), 1580 (amide
1
II), 1660 (amide I); H NMR (d6-DMSO), d, ppm; J,
Hz: 6.20 (dtr, 1H, H4-pyrrole, 3.7, 2.3), 7.01 (dtr, 1H,
H5-pyrrole, 2.6, 1.4), 7.09 (ddd, 1H, H3-pyrrole, 3.8, 2.4,
1.4), 7.49 (s, 2H, SO2NH2), 7.78 (dd, 1H, H6-Ph, 8.4, 2.1),
7.89 (d, 1H, H5-Ph, 8.5), 7.92 (d, 1H, H2-Ph, 2.1), 9.64 (s,
1H, NHCO), 11.80 (s, 1H, H1-pyrrole); 13C NMR
(DMSO-d6), d, ppm: 109.25 (C3-pyrrole), 112.33 (C2-
pyrrole), 123.33 (C6-Ph), 124.82 (C4-pyrrole), 125.08 (C4-
Ph), 126.82 (C5-Ph), 127.09 (C2-Ph), 127.79 (C1-pyrrole),
138.14 (C1-Ph), 141.39 (C3-Ph), 158.95 (CONH). Anal.
(C11H10N3SO3Cl) C, H, N, S.
4-(Furan-2-carboxamido)benzenesulfonamide, B1, (method
A). White crystals, mp 283±4 ꢀC (MeOH). IR (KBr),
cm 1: 1180 (SO2sym), 1320 (SOa2s), 1555 (amide II), 1660
1
(amide I); H NMR (d6-DMSO), d, ppm; J, Hz: 6.72
(dd, 1H, H4-furan, 3.5, 1.7), 7.40 (d, 1H, H3- furan,
3.6), 7.96 (d, 1H, H5-furan, 1.7), 7.29 (s, 2H, SO2NH2),
7.80 (d, 2H, AA0BB0, 8.8), 7.94 (d, 2H, AA0BB0, 8.8),
10.51 (s, 1H, NHCO); 13C NMR (DMSO-d6), d, ppm:
112.40 (C4-furan), 115.60 (C3-furan), 119.96 (C2/C3-
Ph), 146.26 (C5-furan), 147.12 (C2-furan), 126.64 (C3/
C2-Ph), 138.83 (C1/C4-Ph), 141.64 (C4/C1-Ph), 156.22
(CONH). Anal. (C11H10N2SO4) C, H, N, S.
4-(Pyrrole-2-carboxamidomethyl)benzenesulfonamide, A4,
(method B). White crystals, mp 256±8,5oC (MeOH). IR
(KBr), cm 1: 1160 (SO2sym), 1330 (SOa2s), 1560 (amide
1
II), 1630 (amide I); H NMR (d6-DMSO), d, ppm; J,
Hz: 4.49 (d, 2H, 6.0), 6.10 (m, 1H, H4-pyrrole, 3.7, 2.3
etc.), 6.83 (m, 1H, H3-pyrrole, 3.7, 1.5 etc.), 6.87 (m,
1H, H5-pyrrole, 2.7, 1.4 etc.), 7.31 (s, 2H, SO2NH2),
7.46 (d, 2H, AA0BB0, 8.3), 7.78 (d, 2H, AA0BB0, 8.3),
8.62 (t, 1H, NHCO, 6.1), 11.47 (s, 1H, H1-pyrrole); 13C
4-(Furan-2-carboxamido)-3-¯uorobenzenesulfonamide, B2,
(method A). White crystals, mp 239±41.5 ꢀC (MeOH).
IR (KBr), cm 1: 1160 (SO2sym), 1340 (SOa2s), 1600 (amide

2152
M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
II), 1670 (amide I); 1H NMR (d6-DMSO), d, ppm; J, Hz:
6.73 (dd, 1H, H4-furan, 3.5, 1.7), 7.40 (ddd, 1H, H3-furan,
3.5, 1.8, 0.4), 7.49 (s, 2H, SO2NH2), 7.67 (dd, 1H, H2-Ph,
5.8, 2.0), 7.70 (dd, 1H, H6-Ph, 8.3, 1.9), 7.88 (ddd, 1H,
H5-Ph, 8.3, 7.4, 4.7), 7.98 (dd, 1H, H5-furan, 0.7, 1.7),
10.23 (s, 1H, NHCO); 13C NMR (DMSO-d6), d, ppm;
JC,F, Hz: 112.34 (C4-furan), 113.48 (d, C2-Ph, 23.16),
115.74 (C3-furan), 121.95 (d, C5-Ph, 3.1), 126.5 (C6-Ph),
128.44 (d, C4-Ph, 11.8), 141.81 (d, C1-Ph, 6.0), 146.32
(C5-furan), 146.71 (C2-furan), 154.30 (d, C3-Ph, 251.3),
156.2 (CONH). Anal. (C11H9N2SO4F) C, H, N, S.
furan), 113.79 (C3-furan), 125.78 (C2/C3-Ph), 127.58 (C3/
C2-Ph), 142.66 (C1/C4 Ph), 143.62 (C4/C1-Ph), 145.29
(C5-furan), 147.69 (C2-furan), 157.97 (CONH). Anal.
(C12H12N2SO4) C, H, N, S.
4-(Furan-2-carboxamidoethyl)benzenesulfonamide, B7,
(method A). White crystals, mp 231±2.5 ꢀC (MeOH). IR
(KBr), cm 1: 1165 (SO2sym), 1300 (SOa2s), 1535 (amide II),
1640 (amide I); 1H NMR (d6-DMSO), d, ppm; J, Hz: 2.91
(t, 2H, 7.1), 3.49 (q, 2H, 6.7), 6.61 (dd, 1H, H4-furan, 3.4,
1.7 etc.), 7.08 (d, 1H, H3-furan, 3.5), 7.82 (d, 1H, H5-
furan, 1.6), 7.32 (s, 2H, SO2NH2), 7.43 (d, 2H, AA0BB0,
8.2), 7.76 (d, 2H, AA0BB0, 8.2), 8.49 (t, 1H, NHCO, 5.6);
13C NMR (DMSO-d6), d, ppm: 34.85 (CH2-Ph), 39.70 (N-
CH2), 111.91 (C4-furan), 113.38 (C3-furan), 125.79 (C2/
C3-Ph), 129.22 (C3/C2-Ph), 142.09 (C1/C4-Ph), 143.71
(C4/C1-Ph), 145.03 (C5-furan), 147.93 (C2-furan), 157.85
(CONH). Anal. (C13H14N2SO4) C, H, N, S.
4-(Furan-2-carboxamido)-3-chlorobenzenesulfonamide, B3,
(method A). White crystals, mp 224±5 ꢀC (MeOH). IR
(KBr), cm 1: 1160 (SO2sym), 1310 (SOa2s), 1575 (amide II),
1660 (amide I); 1H NMR (d6-DMSO), d, ppm; J, Hz: 6.74
(dd, 1H, H4-furan, 3.5, 1.7), 7.39 (d, 1H, H3-furan, 3.5),
7.52 (s, 2H, SO2NH2), 7.81 (dd, 1H, H6-Ph, 8.5, 2.1), 7.93
(dd, 1H, H5-Ph, 8.5, 0.9), 7.95 (dd, 1H, H2-Ph, 2.1, 0.9),
7.99 (dd, 1H, H5- furan, 1.7, 0.7), 10.02 (s, 1H, NHCO);
13C NMR (DMSO-d6), d, ppm: 112.49 (C4-furan), 115.84
(C3-furan), 125.01 (C6-Ph), 126.87 (C5-Ph), 127.06 (C2-
Ph), 128.14 (C4-Ph), 137.35 (C1-Ph), 142.08 (C3-Ph),
146.35 (C5-furan), 146.73 (C2-furan), 156.14 (CONH).
Anal. (C11H9N2SO4Cl) C, H, N, S.
5-(Furan-2-carboxamido)-1,3,4-thiadiazole-2-sulfonamide,
B8, (method A). White crystals, mp 299±300 ꢀC (MeOH).
IR (KBr), cm 1: 1170 (SO2sym), 1320 (SOa2s), 1580 (amide
1
II), 1680 (amide I); H NMR (d6-DMSO), d, ppm; J,
Hz: 6.78 (dd, 1H, H4-furan, 3.6, 1.7 etc.), 7.77 (d, 1H, H3-
furan, 3.6), 8.07 (d, 1H, H5-furan, 1.7), 8.38 (s, 2H,
SO2NH2), 13.53 (br s, 1H, NHCO); 13C NMR (DMSO-
d6), d, ppm: 112.77 (C4-furan), 118.38 (C3-furan), 144.83
(C2-furan), 148.32 (C5-furan), 156.00 (CONH), 161.57
(C-thiadiazole), 164.75 (C-thiadiazole). Anal. (C7H6N4
S2O4) C, H, N, S.
4-(Furan-2-carboxamido)-3-bromobenzenesulfonamide, B4,
(method A). White crystals, mp 216±8oC (MeOH). IR
(KBr), cm 1: 1165 (SO2sym), 1310 (SOa2s), 1585 (amide II),
1650 (amide I); 1H NMR (d6-DMSO), d, ppm; J, Hz: 6.74
(dd, 1H, H4-furan, 3.5, 1.7), 7.39 (dd, 1H, H3-furan,
3.5, 0.7), 7.56 (vbrs, 2H, SO2NH2), 7.84 (dd, 1H, H6-Ph,
8.5, 2.0), 7.91 (d, 1H, H5-Ph, 8.4), 7.99 (dd, 1H, H5-
furan, 1.7, 0.7), 8.10 (d, 1H, H2-Ph, 2.0), 9.80 (vbrs, 1H,
NHCO); 13C NMR (DMSO-d6), d, ppm: 112.49 (C4-
furan), 115.76 (C3-furan), 118.66 (C4-Ph), 125.55 (C6-
Ph), 127.11 (C5-Ph), 129.91 (C2-Ph), 138.71 (C1-Ph),
142.26 (C3-Ph), 146.30 (C5-furan), 146.76 (C2-furan),
156.02 (CONH). Anal. (C11H9N2SO4Br) C, H, N, S.
5-(Furan-2-carboxamido)-4-methyl-D2 -1,3,4-thiadiazo-
line-2-sulfonamide, B9, (method A). White crystals, mp
28±7ꢀC (MeOH). IR (KBr), cm 1: 1175 (SOs2ym), 1325
as
1
(SO2 ), 1510 (amide II), 1600 (amide I); H NMR (d6-
DMSO), d, ppm; J, Hz: 6.71 (m, 1H, H4-furan, 3.4, 1.7
etc.), 7.42 (d, 1H, H3-furan, 3.4), 7.97 (br s, 1H, H5-
furan), 8.51 (s, 2H, SO2NH2); 13C NMR (DMSO-d6), d,
ppm: 112.37 (C4-furan), 117.79 (C3-furan), 147.34 (C5-
furan), 150.01 (C2-furan), 157.94 (CONH), 164.91 (C-
thiadiazole), 164.94 (C-thiadiazole). Anal. (C8H8N4S2O4)
C, H, N, S.
4-(Furan-2-carboxamido)-3-iodobenzenesulfonamide, B5,
(method A). White crystals, mp 205±6oC (MeOH). IR
(KBr), cm 1: 1170 (SO2sym), 1330 (SOa2s), 1570 (amide II),
1650 (amide I); 1H NMR (d6-DMSO), d, ppm; J, Hz: 6.74
(dd, 1H, H4-furan, 3.5, 1.8), 7.37 (d, 1H, H3-furan, 3.5),
7.48 (s, 2H, SO2NH2), 7.76 (d, 1H, H5-Ph, 8.4), 7.85 (dd,
1H, H6-Ph, 8.4, 2.1), 7.99 (dd, 1H, H5-furan, 1.8, 0.9),
8.29 (d, 1H, H2-Ph, 2.1), 9.92 (s, 1H, NHCO); 13C NMR
(DMSO-d6), d, ppm: 96.98 (C4-Ph), 112.49 (C4-furan),
115.58 (C3- furan), 126.15 (C6-Ph), 126.84 (C5-Ph), 136.03
(C2-Ph), 141.97 (C1-Ph), 142.26 (C3-Ph), 146.23 (C5-
furan), 146.99 (C2-furan), 156.09 (CONH). Anal. (C11
H9N2SO4I) C, H, N, S.
4-(Thiophene-2-carboxamido)benzenesulfonamide, C1,
(method A). White crystals, mp 284±6 ꢀC (MeOH). IR
(KBr), cm 1: 1160 (SO2sym), 1320 (SOa2s), 1585 (amide II),
1650 (amide I); 1H NMR (d6-DMSO), d, ppm; J, Hz: 7.25
(dd, 1H, H4-thiophene, 5.0, 3.8), 7.89 (dd, 1H, H3-thio-
phene, 1.1 5.0), 8.08 (dd, 1H, H5-thiophene, 3.8, 1.1), 7.30
(s, 2H, SO2NH2), 7.82 (d, 2H, AA0BB0, 8.9), 7.92 (d, 2H,
AA0BB0, 8.9), 10.54 (s, 1H, NHCO); 13C NMR (DMSO-
d6), d, ppm: 119.90 (C4-thiophene), 126.67 (C2/C3-Ph),
128.26 (C3/C2-Ph), 129.77 (C3-thiophene), 132.61 (C5-
thiophene), 138.79 (C2-thiophene), 139.49 (C1/C4-Ph),
141.80 (C4/C1-Ph), 160.28 (CONH). Anal. (C11H10
N2S2O3) C, H, N, S.
4-(Furan-2-carboxamidomethyl)benzenesulfonamide, B6,
(method A). White crystals, mp 214±7 ꢀC (MeOH). IR
(KBr), cm 1: 1185 (SO2sym), 1320 (SOa2s), 1530 (amide
1
II), 1635 (amide I); H NMR (d6-DMSO), d, ppm; J,
4-(Thiophene-2-carboxamido)-3-¯uorobenzenesulfonamide,
C2, (method A). White crystals, mp 237±9 ꢀC (MeOH).
IR (KBr), cm 1: 1160 (SO2sym), 1340 (SOa2s), 1600 (amide
Hz: 4.48 (d, 1H, 6.0), 6.63 (dd, 1H, H4-furan, 3.4, 1.7
etc.), 7.14 (d, 1H, H3-furan, 3.4), 7.85 (d, 1H, H5-furan,
1.8), 7.31 (s, 2H, SO2NH2), 7.46 (d, 2H, AA0BB0, 8.3),
1
II), 1650 (amide I); H NMR (d6-DMSO), d, ppm; J,
Hz: 7.25 (dd, 1H, H4-thiophene, 5.0, 3.8), 7.50 (s, 2H,
SO2NH2), 7.68 (dd, 1H, H2-Ph, 6.7, 2.0), 7.71 (dd, 1H,
7.77 (d, 2H, AA0BB0, 8.3), 9.04 (t, 1H, NHCO, 6.1); 13
C
NMR (DMSO-d6), d, ppm: 41.66 (CH2), 111.98 (C4-

M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
2153
H6-Ph, 8.3, 2.0), 7.85 (ddd, 1H, H5-Ph, 8.3, 7.1, 3.3),
7.91 (dd, 1H, H3-thiophene, 5.0, 1.1), 8.06 (dd, 1H, H5-
thiophene, 3.8, 1.1), 10.40 (s, 1H, NHCO); 13C NMR
(DMSO-d6), d, ppm; JC,F, Hz: 113.55 (d, C2-Ph, 23.1),
121.97 (C5-Ph), 126.85 (C6-Ph), 128.30 (C4-thiophene),
128.70 (d, C4-Ph, 12.2), 130.21 (C3-thiophene), 132.65
(C5-thiophene), 138.50 (C2-thiophene), 141.87 (d, C1-
Ph, 5.3), 154.42 (d, C3-Ph, 250.9), 160.02 (CONH).
Anal. (C11H9N2S2O3F) C, H, N, S.
NHCO, 5.8); 13C NMR (DMSO-d6), d, ppm: 42.23 (CH2),
125.82 (C2/C3-Ph), 127.59 (C3/C2-Ph), 128.05 (C4-thio-
phene), 128.39 (C3-thiophene), 131.10 (C5-thiophene),
139.60 (C1/C4-Ph), 142.70 (C2-thiophene), 143.61 (C4/C1-
Ph), 161.32 (CONH). Anal. (C12H12N2S2O3) C, H, N, S.
4-(Thiophene-2-carboxamidoethyl)benzenesulfonamide, C7,
(method A). White crystals, mp 239±40.5 ꢀC (MeOH).
IR (KBr), cm 1: 1160 (SO2sym), 1330 (SOa2s), 1550 (amide
1
II), 1610 (amide I); H NMR (d6-DMSO), d, ppm; J,
4-(Thiophene-2-carboxamido)-3-chlorobenzenesulfonamide,
C3, (method A). White crystals, mp 222±3 ꢀC (MeOH).
IR (KBr), cm 1: 1170 (SO2sym), 1320 (SOa2s), 1580 (amide
Hz: 2.91 (t, 2H, 7.2), 3.49 (q, 2H, 6.4), 7.13 (dd, 1H, H4-
thiophene, 5.0, 3.7), 7.73 (dd, 1H, H3-thiophene, 5.0,
1.2), 7.71 (dd, 1H, H5-thiophene, 3.8, 1.2), 7.31 (s, 2H,
SO2NH2), 7.43 (d, 2H, AA0BB0, 8.3), 7.75 (d, 2H, AA0BB0,
8.1), 8.62 (t, 1H, NHCO, 5.7); 13C NMR (DMSO-d6), d,
ppm: 34.85 (CH2-Ph), 40.22 (N-CH2), 125.77 (C4-thio-
phene), 127.92 (C2/C3-Ph), 129.20 (C3/C2-Ph), 130.68
(C3-thiophene), 130.72 (C5-thiophene), 139.99 (C1/C4-
Ph), 142.06 (C2-thiophene), 143.7 1 (C4/C1-Ph), 161.16
(CONH). Anal. (C13H14N2S2O3) C, H, N, S.
1
II), 1660 (amide I); H NMR (d6-DMSO), d, ppm; J,
Hz: 7.25 (dd, 1H, H4-thiophene, 5.0, 3.8), 7.54 (brs, 2H,
SO2NH2), 7.81 (m, 2H (H2+H6)-Ph, 1.5, 0.9 etc.), 7.91
(dd, 1H, H3-thiophene, 5.0, 1.1), 7.95 (dd, 1H, H5-Ph,
1.5, 0.9), 8.05 (dd, 1H, H5-thiophene, 3.8, 1.1), 10.26 (s,
1H, NHCO); 13C NMR (DMSO-d6), d, ppm: 124.89 (C6-
Ph), 126.91 (C5-Ph), 128.30 (C2-Ph), 128.35 (C4-thio-
phene), 129.13 (C4-Ph), 130.03 (C3-thiophene), 132.58
(C5-thiophene), 137.72 (C1-Ph), 138.56 (C2-thiophene),
142.39 (C3-Ph), 160.01 (CONH). Anal. (C11H9N2S2
O3Cl) C, H, N, S.
5-(Thiophene-2-carboxamido)-1,3,4-thiadiazole-2-sulfon-
amide, C8, (method A). White crystals, mp 272±5 ꢀC
(EtOH). IR (KBr), cm 1: 1170 (SO2sym), 1310 (SOa2s),
1
1540 (amide II), 1650 (amide I); H NMR (d6-DMSO),
4-(Thiophene-2-carboxamido)-3-bromobenzenesulfonamide,
C4, (method A). White slightly pink crystals, mp 206±
7 ꢀC (MeOH). IR (KBr), cm 1: 1160 (SOs2ym), 1310
d, ppm; J, Hz: 7.30 (dd, 1H, H4-thiophene, 5.0, 3.9),
8.06 (dd, 1H, H3-thiophene, 5.0, 1.1), 8.33 (dd, 1H, H5-
thiophene, 3.8, 1.1), 8.38 (s, 2H, SO2NH2), 13.62 (br s, 1H,
NHCO); 13C NMR (DMSO-d6), d, ppm: 128.92 (C4-
thiophene), 132.34 (C3-thiophene), 135.13 (C5-thio-
phene), 135.80 (C2-thiophene), 160.13 (CONH), 162.01
(C-thiadiazole), 164.71 (C-thiadiazole). Anal. (C7H6N4S3
O3) C, H, N, S.
as
1
(SO2 ), 1580 (amide II), 1650 (amide I); H NMR (d6-
DMSO), d, ppm; J, Hz: 7.26 (dd, 1H, H4-thiophene,
5.0, 3.7), 7.54 (brs, 2H, SO2NH2), 7.77 (d, 1H, H5-Ph,
8.4), 7.86 (dd, 1H, H6-Ph, 8.4, 2.0), 7.91 (dd, 1H, H3-
thiophene, 5.0, 1.2), 8.04 (dd, 1H, H5-thiophene, 3.8, 1.2),
8.11 (d, 1H, H2-Ph, 2.0), 10.26 (s, 1H, NHCO); 13C NMR
(DMSO-d6), d, ppm: 119.95 (C4-Ph), 125.46 (C6-Ph),
128.30 (C5-Ph), 128.75 (C4- hiophene), 129.93 (C2-Ph),
129.98 (C3-thiophene), 132.52 (C5-thiophene), 138.61
(C2-thiophene), 139.17 (C1-Ph), 142.77 (C3-Ph), 159.96
(CONH). Anal. (C11H9N2S2O3Br) C, H, N, S.
5-(Thiophene-2-carboxamido)-4-methyl-D2-1,3,4-thiadiaz-
oline-2-sulfonamide, C9, (method A). White crystals, mp
279±81 ꢀC (EtOH). IR (KBr), cm 1: 1170 (SOs2ym), 1325
as
1
(SO2 ), 1520 (amide II), 1580 (amide I); H NMR (d6-
DMSO), d, ppm; J, Hz: 7.23 (dd, 1H, H4-thiophene,
5.0, 3.7), 7.91 (dd, 1H, H3-thiophene, 5.0, 1.2), 7.93 (dd,
1H, H5-thiophene, 3.7, 1.2), 8.54 (s, 2H, SO2NH2); 13C
NMR (DMSO-d6), d, ppm: 128.54 (C4-thiophene), 132.63
(C3-thiophene), 133.59 (C5-thiophene), 140.73 (C2-thio-
phene), 158.10 (CONH), 164.94 (C-thiadiazole), 168.57
(C-thiadiazole). Anal. (C8H8N4S3O3) C, H, N, S.
4-(Thiophene-2-carboxamido)-3-iodobenzenesulfonamide,
C5, (method A). White slightly pink crystals, mp 232.5±
4 ꢀC (MeOH). IR (KBr), cm 1: 1160 (SOs2ym), 1335
as
1
(SO2 ), 1580 (amide II), 1650 (amide I); H NMR (d6-
DMSO), d, ppm; J, Hz: 7.26 (dd, 1H, H4-thiophene,
5.0, 3.7), 7.51 (s, 2H, SO2NH2), 7.65 (d, 1H, H5-Ph,
8.4), 7.87 (dd, 1H, H6-Ph, 8.4, 2.1), 7.90 (dd, 1H, H3-
thiophene, 5.0, 1.1), 8.03 (dd, 1H, H5-thiophene, 3.7, 1.1),
8.32 (d, 1H, H2-Ph, 2.0), 10.21 (s, 1H, NHCO); 13C NMR
(DMSO-d6), d, ppm: 98.12 (C4-Ph), 126.12 (C6-Ph), 128.32
(C4-thiophene), 129.79 (C3-thiophene), 132.38 (C5-thio-
phene), 136.02 (C5-Ph), 136.06 (C2-Ph), 138.87 (C2-thio-
phene), 142.53 (C1-Ph), 142.96 (C3-Ph), 159.96 (CONH).
Anal. (C11H9N2S2O3I) C, H, N, S.
Pharmacology
Human CA I and CA II cDNAs were expressed in
Escherichia coli strain BL21 (DE3) from the plasmids
pACA/hCA I and pACA/hCA II described by Lidskog et
al.46 (the two plasmids were a gift from Prof. Sven Linds-
kog, Umea University, Sweden). Cell growth conditions
were those described by this group,47 and enzymes were
puri®ed by anity chromatography according to the
method of Khalifah et al.48 Enzyme concentrations were
4-(Thiophene-2-carboxamidomethyl)benzenesulfonamide,
C6, (method A). Cream crystals, mp 223±5 ꢀC (EtOH).
IR (KBr), cm 1: 1160 (SO2sym), 1310 (SOa2s), 1550 (amide
determined spectrophotometrically at 280 nM, utilizing a
1
molar absorptivity of 49mM 1 cm
for CA I and
1
1
II), 1620 (amide I); H NMR (d6-DMSO), d, ppm; J,
54mM 1 cm for CA II, respectively, based on Mr=
28.85 kDa for CA I, and 29.30 kDa for CA II, respec-
tively.49,50 CA IV was isolated from bovine lung micro-
somes as described by Maren et al, and its concentration
has been determined by titration with ethoxzolamide.51
Hz: 4.51 (d, 1H, 6.0), 7.17 (dd, 1H, H4-thiophene, 4.9,
3.7), 7.78 (dd, 1H, H3-thiophene, 4.9, 1.0), 7.82 (dd, 1H,
H5-thiophene, 3.7, 1.0), 7.33 (s, 2H, SO2NH2), 7.49 (d,
2H, AA0BB0, 8.3), 7.80 (d, 2H, AA0BB0, 8.3), 9.15 (t, 1H,

2154
M. Ilies et al. / Bioorg. Med. Chem. 8 (2000) 2145±2155
Initial rates of 4-nitrophenyl acetate hydrolysis cata-
lysed by di€erent CA isozymes were monitored spectro-
photometrically, at 400 nM, with a Cary 3 instrument
interfaced with an IBM compatible PC.52 Solutions of
Sigma) as described in ref55 The IOP of operated animals
was checked after approximately four weeks, and animals
with an elevated pressure of 30±35 mm Hg were used at
least one month after the injection of a-chymotrypsin.
substrate were prepared in anhydrous acetonitrile; the
2
substrate concentrations varied between 2.10
and
Determination of water (bu€er) solubility: A standard
solution was prepared by dissolving a precisely weighted
amount (generally 1 mg) of inhibitor in 10 mL of metha-
nol. The UV absorption maximum of each compound
has been determined (with a Cary 3 spectrophotometer)
eventually diluting the solution (with MeOH) as necessary.
A saturated solution of each compound was then pre-
pared by stirring magnetically a small volume of 0.039 M
phosphate bu€er (pH 7.4) in the presence of excess inhi-
bitor for 3 h. The obtained saturated solution was ®ltered
in order to remove solid compound through a Millipore
0.45 mM ®lter and scanned by UV at the wavelength of
the absorption maximum previously determined. Total
solubility was determined by the relationship: C0=A0C/A,
where C=concentration of standard solution (mg/mL);
A=absorbance of standard solution; A0=absorbance of
the saturated solution; C0=concentration of the satu-
rated solution (mg/mL).32
1.10 6 M, working at 25ꢀC. A molar absorption coecient
1
E of 18,400M 1 cm was used for the 4-nitrophenolate
formed by hydrolysis, in the conditions of the experi-
ments (pH 7.40), as reported in the literature.52 Non-
enzymatic hydrolysis rates were always subtracted from
the observed rates. Duplicate experiments were done for
each inhibitor concentration, and the values reported
throughout the paper are the mean of such results.
Stock solutions of inhibitor (1 mM) were prepared in
distilled-deionized water with 10±20% (v/v) DMSO
(which is not inhibitory at these concentrations) and
dilutions up to 0.01 nM were done thereafter with dis-
tilled-deionized water. Inhibitor and enzyme solutions
were preincubated together for 10 min at room tempera-
ture prior to assay, in order to allow for the formation of
the E±I complex. The inhibition constant KI was deter-
mined as described by Pocker and Stone.52 Enzyme
concentrations were 3.1 nM for hCA II, 13 nM for hCA
I and 33 nM for bCA IV.
Partition coecient determinations: Chloroform bu€er
partition coecients were obtained by equilibrating the
test compound between chloroform and 0.1-ionic strength
pH 7.4 phosphate bu€er. The concentration in each phase
was determined by UV spectrophotometry or HPLC.37
Adult male New Zealand albino rabbits weighing 3±3.5 kg
were used in the experiments (three animals were used for
each inhibitor studied). The experimental procedures
conform to the Association for Research in Vision and
Ophthalmology Resolution on the use of animals. The
rabbits were kept in individual cages with food and water
provided ad libitum. The animals were maintained on a
12 h: 12 h light/dark cycle in a temperature controlled
room, at 22±26 ꢀC. Suspensions of inhibitors (2%, by
weight) were obtained in distilled deionized water. The
pH of these solutions was in the range of 7.0±7.5.
Transcorneal penetration of drugs: The method of
Maren et al.56 with the modi®cations of Pierce's group57
(for the HPLC assay of sulfonamides) have been used.
Excised rabbit corneas with either intact or denuded
epithelium were used in these experiments. The pH was
7.4 and exposed area was of 1.2 cm2. Concentrations of
drug of 40±2000 mM were placed in the epithelial chamber
and samples of ¯uid were collected from the endothelial
chamber at di€erent intervals, up to 4 h. Both chambers
contained 6 mL. Drugs present in these ¯uids were
assayed both by the HPLC method of Pierce et al.,57 or
enzymatically.37 The results of the drug analyses were
used to calculate the rate constant of transfer across the
cornea (kin). As described by Pierce,57 this value was
determined by using the formula:
IOP was measured using a Digilab 30R pneumatonometer
(BioRad, Cambridge, MA, USA) as described by Maren's
group.53,54 The pressure readings were matched with
two-point standard pressure measurements at least twice
each day using a Digilab Calibration veri®er. All IOP
measurements were done by the same investigator with
the same tonometer. One drop of 0.2% oxybuprocaine
hydrochloride (novesine, Sandoz) diluted 1:1 with saline
was instilled in each eye immediately before each set of
pressure measurements. IOP was measured three times
at each time interval, and the means reported. IOP was
measured ®rst immediately before drug administration,
then at 30min after the instillation of the pharmacological
agent, and then each 30 min for a period of 4±6 h. For
all IOP experiments drug was administered to only one
eye, leaving the contralateral eye as an untreated control.
The ocular hypotensive activity is expressed as the average
di€erence in IOP between the treated and control eye, in
this way minimizing the diurnal, seasonal and inter-
individual variations commonly observed in the rabbit.53,54
All data are expressed as meanÆSE, using a one-tailed t
test.
1
kinꢀÂ103 h † ˆ ‰drugŠendo=‰drugŠepiÂ60=t  1000
where ‰drugŠendo=concentration of drug on endothelial
side; ‰drugŠepi=concentration of drug on epithelial side;
t=time (in min).
Acknowledgements
This research was ®nanced by the EU grant ERB
CIPDCT 940051. Thanks are addressed to Dr. M. A.
Ilies for expert technical assistance.
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Ocular hypertension was elicited in the right eye of
albino rabbits by the injection of a-chymotrypsin (from

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