Carbonic anhydrase inhibitors - Part 70. Synthesis and ocular pharmacology of a new class of water-soluble, topically effective intraocular pressure lowering agents derived from nicotinic acid and aromatic/heterocyclic sulfonamides

Article abstract of DOI:10.1016/S0223-5234(99)00212-3

Reaction of twenty aromatic/heterocyclic sulfonamides containing a free amino, imino, hydrazino or hydroxyl group, with nicotinoyl chloride afforded a series of water-soluble (as hydrochloride or triflate salts) compounds. The new derivatives were assayed as inhibitors of three carbonic anhydrase (CA) isozymes, hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound form); h = human, b = bovine isozyme. Efficient inhibition was observed against all three isozymes, but especially against hCA II and bCA IV (in nanomolar range), two isozymes known to play a critical role in aqueous humour secretion within the ciliary processes of the eye. Some of the best inhibitors synthesized were applied as 2% water solutions directly into the eye of normotensive or glaucomatous albino rabbits. Very strong intraocular pressure (IOP) lowering was observed for many of them, and the active drug was detected in eye tissues and fluids. This result prompted us to re-analyse the synthetic work done by other groups for the design of water soluble, topically effective antiglaucoma sulfonamides. According to these researchers, the IOP lowering effect is due to the intrinsic nature of the specific heterocyclic sulfonamide considered, among which the thienothiopyran-2-sulfonamide derivatives represent the best studied case. Indeed, the first agents developed for such applications, such as dorzolamide, are derivatives of this ring system. In order to prove that the tail (in this case the nicotinoyl moiety) conferring water solubility to a sulfonamide CA inhibitor is critically important, similarly to the ring to which the sulfonamido group is grafted, we also prepared a dorzolamide derivative to which the nicotinoyl moiety was attached. This new compound is more water soluble than dorzolamide (as hydrochloride salt), behaves as a strong CA II inhibitor, and acts similarly to the parent derivative in lowering IOP in experimental animals. Thus, it seems that the tail conferring water solubility is more important for topical activity as an antiglaucoma drug than the heterocyclic/aromatic ring to which the sulfonamido moiety is grafted.

Full text of DOI:10.1016/S0223-5234(99)00212-3

Eur. J. Med. Chem. 34 (1999) 799−808
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
799
Original article
Carbonic anhydrase inhibitors – part 70#. Synthesis and ocular pharmacology
of a new class of water-soluble, topically effective intraocular pressure lowering
agents derived from nicotinic acid and aromatic/heterocyclic sulfonamides
Claudiu T. Supurana*, Andrea Scozzafavaa, Luca Menabuonib, Francesco Mincionec,
Fabrizio Brigantia, Giovanna Mincionea
aUniversità degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Firenze, Italy
bOspedale San Giovanni di Dio, S.O. Oculistica, Via Torregalli 3, I-50123, Firenze, Italy
cUniversità degli Studi, Institute of Ophthalmology, Viale Morgagni 85, I-50134, Firenze, Italy
(Received 14 January 1999; revised 30 April 1999; accepted 6 May 1999)
Abstract – Reaction of twenty aromatic/heterocyclic sulfonamides containing a free amino, imino, hydrazino or hydroxyl group, with
nicotinoyl chloride afforded a series of water-soluble (as hydrochloride or triflate salts) compounds. The new derivatives were assayed as
inhibitors of three carbonic anhydrase (CA) isozymes, hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound form); h = human, b
= bovine isozyme. Efficient inhibition was observed against all three isozymes, but especially against hCA II and bCA IV (in nanomolar
range), two isozymes known to play a critical role in aqueous humour secretion within the ciliary processes of the eye. Some of the best
inhibitors synthesized were applied as 2% water solutions directly into the eye of normotensive or glaucomatous albino rabbits. Very strong
intraocular pressure (IOP) lowering was observed for many of them, and the active drug was detected in eye tissues and fluids. This result
prompted us to re-analyse the synthetic work done by other groups for the design of water soluble, topically effective antiglaucoma
sulfonamides. According to these researchers, the IOP lowering effect is due to the intrinsic nature of the specific heterocyclic sulfonamide
considered, among which the thienothiopyran-2-sulfonamide derivatives represent the best studied case. Indeed, the first agents developed for
such applications, such as dorzolamide, are derivatives of this ring system. In order to prove that the tail (in this case the nicotinoyl moiety)
conferring water solubility to a sulfonamide CA inhibitor is critically important, similarly to the ring to which the sulfonamido group is grafted,
we also prepared a dorzolamide derivative to which the nicotinoyl moiety was attached. This new compound is more water soluble than
dorzolamide (as hydrochloride salt), behaves as a strong CA II inhibitor, and acts similarly to the parent derivative in lowering IOP in
experimental animals. Thus, it seems that the tail conferring water solubility is more important for topical activity as an antiglaucoma drug
than the heterocyclic/aromatic ring to which the sulfonamido moiety is grafted. © 1999 Éditions scientifiques et médicales Elsevier SAS
carbonic anhydrase / aromatic, heterocyclic sulfonamides / nicotinoyl chloride / antiglaucoma drugs / hydrochloride salts /
dorzolamide
1. Introduction
sulfonamides [3] continue to play an important role in
chemotherapy, alone or in combination with other
drugs [4], the sulfonamides that inhibit the zinc enzyme
carbonic anhydrase (CA, EC 4.2.1.1) possess many
applications as diuretic, antiglaucoma or antiepileptic
drugs among others [5–7], the hypoglycaemic sulfona-
mides are extensively used in the treatment of some
forms of diabetes [8], whereas the thiazides and high-
ceiling diuretics might be considered as a fortunate
development of the CA inhibitors [9], but these com-
pounds possess a different pharmacological profile, inde-
pendent of CA inhibition [10, 11]. Finally, some antithy-
The sulfonamides represent an important class of
biologically active compounds, with at least five different
classes of pharmacological agents that have been ob-
tained from the sulfanilamide structure as lead, the
derivative initially studied by Domagk [2] as the first
modern chemotherapeutic drug. Indeed, the antibacterial
#
See [1].
*Correspondence and reprints

800
Figure 1. Structures of dorzolamide
1 and brinzolamide 2.
roid drugs have also been developed starting from the
sulfonamide structure as lead molecule [12].
was to explore as many heterocyclic rings as possible on
which the sulfonamido moiety should be grafted, and this
approach was extremely beneficial to the chemistry of
heterocyclic sulfonamides. Still, this approach seemed to
us not a very fortunate one for the design of topically
active IOP lowering agents, and we decided to explore
the opposite one, i.e., to graft moieties that would ensure
water solubility (as salts of a strong acid) on the classical
ring systems of the aromatic/heterocyclic sulfonamides
possessing CA inhibitory properties.
In this paper we report the reaction of twenty aromatic/
heterocyclic sulfonamides containing a free amino,
imino, hydrazino or hydroxyl group, with nicotinoyl
chloride, which afforded a series of water-soluble (as
hydrochloride or triflate salts) sulfonamides with strong
CA inhibitory properties. Moreover, dorzolamide has
been derivatized similarly, at its secondary amino group,
and the obtained compound also possessed a good water
solubility as the hydrochloride salt. The new compounds
reported here were tested for the inhibition of three CA
isozymes, hCA I, hCA II and bCA IV (h = human, b =
bovine isozyme). Affinities in the nanomolar range were
detected for some compounds for isozymes II and IV. The
most active derivatives were assayed in vivo in normo-
tensive and glaucomatous rabbits for their IOP lowering
properties. Very strong intraocular pressure (IOP) lower-
ing was observed for many of them, and the active drug
was detected in eye tissues and fluids. Our conclusion is
that the water-solubilizing tail seems to be more impor-
tant than the ring on which the sulfonamido moiety is
grafted, and that topically active antiglaucoma drugs
might be obtained from many other classes of sulfona-
mides than the thienothiopyran-sulfonamides and their
derivatives.
The second class of the above mentioned pharmaco-
logical agents, ie., the sulfonamides with CA inhibitory
action, have been thoroughly investigated in the last 10
years, mainly in the search for a topically effective
antiglaucoma drug [13–19]. The possibility of adminis-
tering a sulfonamide via the topical route directly into the
eye, although investigated in the 1950s [20, 21], has been
totally unsuccessful, whereas the systemic administra-
tion, quite useful in lowering intraocular pressure (IOP),
was generally accompanied by undesired side effects, due
to CA inhibition in other tissues than the eye [21]. In
1983, Maren’s group [13] postulated that a water-soluble
sulfonamide, also possessing a relatively balanced lipid
solubility, would be an effective IOP lowering drug via
the topical route, but at that moment no inhibitors
possessing such physico-chemical properties existed.
They started to be developed in several laboratories soon
thereafter [13–19], and in 1995 the first such pharmaco-
logical agent, dorzolamide 1 entered into clinical use in
the USA and Europe [22]. A second compound, brinzola-
mide 2, quite similar structurally to dorzolamide has also
recently been approved for the topical treatment of
glaucoma in the USA (figure 1) [23].
Thus, in a series of interesting papers [15, 24–29], the
Merck, Sharp and Dohme group has developed the
synthesis of a large series of generally bicyclic hetero-
cyclic sulfonamides (derivatives of benzothiazole- [24];
benzofuran- [25]; indole- [26]; benzo[b]-thiophene- [27,
28]; thieno-thiopyran [15, 29], etc), which were then
tested as IOP lowering agents, and which led to the above
mentioned drug (dorzolamide). Still, the greatest majority
of the synthesized compounds proved to be potent aller-
gens in vivo since their sulfonamido group was nucleo-
philically displaced by reduced glutathione. More than
that, the only compounds with acceptable water solubility
proved to be hydrochlorides of amino-derivatives of the
thienothiopyran-sulfonamides of the dorzolamide type
[15, 29]. Obviously, the approach followed by this group
2. Experimental protocols
Melting points were determined with a heating plate
microscope and are not corrected; IR spectra were ob-
tained in KBr pellets with a Perkin-Elmer 16PC FTIR

801
1
spectrometer, whereas H-NMR spectra were obtained
(1:1, v/v). Yields were in the range of 70–90%. Hydro-
chlorides of derivatives 23–43 were obtained from the
free bases and a methanolic HCl solution, in methanol as
solvent. The hydrochlorides precipitated by leaving the
above mixtures at 4 °C overnight. The hydrochlorides
were analysed for the presence of Cl– by potentiometric
titrations. The obtained data were ± 0.5% of the theoreti-
cal values calculated for the proposed formulas (data not
shown). Triflate salts were similarly obtained from the
free bases 23–43 and the stoichiometric amount of triflic
acid, in water as solvent.
with a Varian 300CXP apparatus in solvents specified in
each case. Chemical shifts are expressed as δ values
relative to Me4Si as standard. Elemental analyses were
done by combustion for C, H, N with an automated Carlo
Erba analyser, and were ± 0.4% of the theoretical values.
Sulfonamides 3–22 used in synthesis were either com-
mercially available compounds (from Sigma, Acros or
Aldrich) or were prepared as described previously:
4-hydrazino-benzenesulfonamide 6 by diazotization of
sulfanilamide followed by reduction of the diazonium salt
with tin(II) chloride [30]; halogenosulfanilamides 9–12
by halogenation of sulfanilamide as reported in the
literature [31]; compound 17 from 5-amino-1,3,4-thia-
diazole-2-sulfonamide (obtained from acetazola-
mide) [32] by acylation with the phthalimido-derivative
of â-alanine, followed by hydrazinolysis [33], whereas
imine 16 by deprotection of methazolamide with concen-
trated hydrochloric acid [34]. The benzothiazole-2-
sulfonamide derivatives 18–20 were prepared as de-
scribed in ref. [35], whereas the alcohols 21 and 22 from
the corresponding amines by diazotization followed by
hydrolysis of the diazonium salts [31]. Dorzolamide 1
was prepared as described in the literature [36]. Nicotin-
oyl chloride hydrochloride, triflic acid and triethylamine
were from Acros. Acetonitrile, acetone (Merck) or other
solvents used in the synthesis were doubly distilled and
kept on molecular sieves in order to maintain them in
anhydrous conditions.
2.1.1.1. 2-(Nicotinoylamido)-benzenesulfonamide 23
White crystals, m.p. 250–252 °C; IR (KBr), cm–1:
1 140 (SO2sym), 1 290 (amide III), 1 370 (SO2as), 1 550
(amide II), 1 680 (amide I), 3 090 (NH); 3 360 (NH2);
1H-NMR (DMSO-d6), δ, ppm: 7.05–7.98 (m, 4H, ArH
from nicotinoyl); 7.15–7.66 (m, 4H, ArH, 1,2-
phenylene); 7.50 (br s, 2H, SO2NH2); 8.14 (br s, 1H,
CONH); Anal., found: C, 51.90; H, 4.10; N, 14.96%;
C12H11N3O3S requires: C, 51.98; H, 4.00; N, 15.15%.
2.1.1.2. 3-(Nicotinoylamido)-benzenesulfonamide 24
White crystals, m.p. 265–266 °C (dec.); IR (KBr),
cm–1: 1 135 (SO2sym), 1 290 (amide III), 1 370 (SO2as),
1 570 (amide II), 1 690 (amide I), 3 080 (NH); 3 360
(NH2); 1H-NMR (DMSO-d6), δ, ppm: 7.05–7.98 (m, 4H,
ArH from nicotinoyl); 7.10–7.50 (m, 4H, ArH, 1,3-
phenylene); 7.56 (br s, 2H, SO2NH2); 8.11 (br s, 1H,
CONH); Anal., found: C, 51.78; H, 3.85; N, 15.07%;
C12H11N3O3S requires: C, 51.98; H, 4.00; N, 15.15%.
2.1. Chemistry
2.1.1.3. 4-(Nicotinoylamido)-benzenesulfonamide 25
White crystals, m.p. 280–281 °C (dec.); IR (KBr),
cm–1: 1 150 (SO2sym), 1 290 (amide III), 1 345 (SO2as),
1 560 (amide II), 1 690 (amide I), 3 060 (NH); 3 360
(NH2); 1H-NMR (DMSO-d6), δ, ppm: 7.05–7.98 (m, 4H,
ArH from nicotinoyl); δA 7.18, δB 7.75 (AA≠BB≠system,
4H, JAB = 7.9 Hz, ArH from 4-sulfamoylphenyl); 7.56 (br
s, 2H, SO2NH2); 8.19 (br s, 1H, CONH); Anal., found: C,
51.67; H, 4.05; N, 14.88%; C12H11N3O3S requires: C,
51.98; H, 4.00; N, 15.15%.
2.1.1. General procedure for the preparation of nico-
tinoyl derivatives of the aromatic/heterocyclic sulfon-
amides 23–43
An amount of 10 mM sulfonamide 3–22 or 1 was
dissolved/suspended in 50 mL of anhydrous acetonitrile
or acetone and then treated with 0.178 g (10 mM) of
nicotinoyl chloride hydrochloride. The stoichiometric
amount (200 µL) of triethylamine was then added and the
reaction mixture was magnetically stirred at 4 °C for
4–10 h. By means of TLC, the conversion of all the
sulfonamides to the corresponding nicotinoyl derivatives
has been monitored. When the reaction was completed,
the solvent was evaporated until a small volume of the
reaction mixture was obtained. Generally the new com-
pounds crystallized spontaneously by leaving the above
mixture at 4 °C overnight. In some cases, the concen-
trated liquor obtained after the evaporation of the solvent
was poured into 50 mL of cold water, then the reaction
products precipitated and filtered. The prepared com-
pounds were recrystallized from ethanol or ethanol-water
2.1.1.4. 4-(Nicotinoylhydrazido)-benzenesulfonamide 26
White crystals, m.p. 265–267 °C; IR (KBr), cm–1: 980
(N–N), 1 150 (SO2sym), 1 290 (amide III), 1 365 (SO2as),
1 555 (amide II), 1 690 (amide I), 3 090 (NH); 3 360
(NH2); 1H-NMR (DMSO-d6), δ, ppm: 7.05–7.98 (m, 4H,
ArH from nicotinoyl); δA 7.18, δB 7.71 (AA≠BB≠system,
4H, JAB = 7.8 Hz, ArH from 4-sulfamoylphenyl); 7.59 (br
s, 2H, SO2NH2); 8.06 (br s, 2H, CONHNH); Anal.,
found: C, 49.40; H, 4.16; N, 19.03%; C12H12N4O3S
requires: C, 49.31; H, 4.14; N, 19.17%.

802
2.1.1.5. 4-(Nicotinoylamidomethyl)-benzenesulfonamide 27
White crystals, m.p. 271–273 °C (dec.); IR (KBr),
cm–1: 1 170 (SO2sym), 1 290 (amide III), 1 372 (SO2as),
1 545 (amide II), 1 690 (amide I), 3 090 (NH); 3 360
(NH2); 1H-NMR (DMSO-d6), δ, ppm: 4.90 (s, 2H, CH2);
7.05–7.98 (m, 4H, ArH from nicotinoyl); δA 7.22, δB
7.79 (AA≠BB≠system, 4H, JAB = 7.9 Hz, ArH from
4-sulfamoylphenyl); 7.67 (br s, 2H, SO2NH2); 8.16 (br s,
1H, CONH); Anal., found: C, 53.81; H, 4.78; N, 14.21%;
C13H13N3O3S requires: C, 53.60; H, 4.50; N, 14.42%.
3H, Ar H the 2-Br-substituted ring); 8.14 (br s, 1H,
CONH); Analysis, found: C, 40.55; H, 3.00; N, 11.50%;
C12H10BrN3O3S requires: C, 40.46; H, 2.83; N, 11.80%.
2.1.1.10.
3-Iodo-4-(nicotinoylamido)-benzenesulfon-
amide 32
White crystals, m.p. 210–212 °C. IR (KBr), cm–1:
1 145 (SO2sym), 1 290 (amide III), 1 360 (SO2as), 1 545
(amide II), 1 690 (amide I), 3 070 (NH); 3 360 (NH2);
1H-NMR (DMSO-d6), δ, ppm: 6.60 (br s, 2H, SO2NH2);
7.05–7.98 (m, 4H, ArH from nicotinoyl); 7.08–7.79 (m,
3H, Ar H the 2-I-substituted ring); 8.14 (br s, 1H,
CONH); Analysis, found: C, 35.87; H, 2.43; N, 10.36%;
C12H10IN3O3S requires: C, 35.75; H, 2.50; N, 10.42%.
2.1.1.6. 4-(Nicotinoylamidoethyl)-benzenesulfonamide 28
White crystals, m.p. 278–280 °C (dec.); IR (KBr),
cm–1: 1 150 (SO2sym), 1 290 (amide III), 1 359 (SO2as),
1 540 (amide II), 1 690 (amide I), 3 080 (NH); 3 360
(NH2); 1H-NMR (DMSO-d6), δ, ppm: 3.10 (t, 2H, αCH2
from the CH2CH2 bridge); 3.70 (t, 2H, âCH2 from the
CH2CH2 bridge); 7.05–7.98 (m, 4H, ArH from nicoti-
noyl); δA 7.15, δB 7.62 (AA≠BB≠system, 4H, JAB = 7.9
Hz, ArH from 4-sulfamoylphenyl); 7.67 (br s, 2H,
SO2NH2); 8.17 (br s, 1H, CONH); Anal., found: C, 55.40;
H, 5.03; N, 13.45%; C14H15N3O3S requires: C, 55.07; H,
4.95; N, 13.76%.
2.1.1.11. 4,5-Dichloro-6-nicotinoylamido-benzene-1,3-
disulfonamide 33
White crystals, m.p. 267–268 °C. IR (KBr), cm–1:
1 140 (SO2sym), 1 290 (amide III), 1 370 (SO2as), 1 550
(amide II), 1 690 (amide I), 3 080 (NH); 3 360 (NH2);
1H-NMR (DMSO-d6), δ, ppm: 7.05–7.98 (m, 4H, ArH
from nicotinoyl); 7.54 (s, 1H, ArH from the pentasubsti-
tuted benzene ring); 7.68 (br s, 4H, 2 SO2NH2); 8.10 (br
s, 1H, CONH); Analysis, found: C, 33.69; H, 2.40; N,
13.08%; C12H10Cl2N4O5S2 requires: C, 33.89; H, 2.37;
N, 13.17%.
2.1.1.7.
3-Fluoro-4-(nicotinoylamido)-benzenesulfon-
amide 29
White crystals, m.p. 234–235 °C. IR (KBr), cm–1:
1 150 (SO2sym), 1 290 (amide III), 1 348 (SO2as), 1 550
(amide II), 1 680 (amide I), 3 060 (NH); 3 360 (NH2);
1H-NMR (DMSO-d6), δ, ppm: 6.60 (br s, 2H, SO2NH2);
7.05–7.98 (m, 4H, ArH from nicotinoyl); 7.05–7.89 (m,
3H, Ar H from the F-substituted ring); 8.15 (br s, 1H,
CONH); Analysis, found: C, 48.54; H, 3.61; N, 14.07%;
C12H10FN3O3S requires: C, 48.81; H, 3.41; N, 14.23%.
2.1.1.12. 6-Chloro-4-nicotinoylamido-benzene-1,3-disul-
fonamide 34
White crystals, m.p. 290–294 °C (dec.). IR (KBr),
cm–1: 1 150 (SO2sym), 1 290 (amide III), 1 330 (SO2as),
1 540 (amide II), 1 680 (amide I), 3 060 (NH); 3 360
1
(NH2); H-NMR (DMSO-d6), δ, ppm: δA 7.05–7.98 (m,
4H, ArH from nicotinoyl); 7.35 (s, 1H, ArH from disul-
famoylphenyl); 7.59 (s, 1H, ArH from disulfamoylphe-
nyl); 7.75 (br s, 4H, 2 SO2NH2); 8.14 (br s, 1H, CONH);
Analysis, found: C, 36.59; H, 2.90; N, 14.21%;
C12H11ClN4O5S2 requires: C, 36.88; H, 2.84; N, 14.34%.
2.1.1.8.
3-Chloro-4-(nicotinoylamido)-benzenesulfon-
amide 30
White crystals, m.p. 238–239 °C. IR (KBr), cm–1:
1 155 (SO2sym), 1 290 (amide III), 1 339 (SO2as), 1 550
(amide II), 1 690 (amide I), 3 090 (NH); 3 360 (NH2);
1H-NMR (DMSO-d6), δ, ppm: 6.70 (br s, 2H, SO2NH2);
7.05–7.98 (m, 4H, ArH from nicotinoyl); 7.05–7.76 (m,
3H, Ar H the 2-Cl-substituted ring); 8.15 (br s, 1H,
CONH); Analysis, found: C, 46.27; H, 3.37; N, 13.39%;
C12H10ClN3O3S requires: C, 46.23; H, 3.23; N, 13.48%.
2.1.1.13. 5-Nicotinoylamido-1,3,4-thiadiazol-2-sulfon-
amide 35
White crystals, m.p. > 310 °C; IR (KBr), cm–1: 1 180
(SO2sym), 1 295 (amide III), 1 340 (SO2as), 1 545 (amide
II), 1 690 (amide I), 3 060 (NH), 3 375; 1H-NMR
(DMSO-d6), δ, ppm: 6.94 (br s, 2H, SO2NH2); 7.05–7.98
(m, 4H, ArH from nicotinoyl); 8.26 (br s, 1H, CONH);
Anal., found, C, 33.60; H, 2.60; N, 24.45%; C8H7N5O3S2
requires: C, 33.68; H, 2.47; N, 24.55%.
2.1.1.9.
3-Bromo-4-(nicotinoylamido)-benzenesulfon-
amide 31
White crystals, m.p. 230–232 °C. IR (KBr), cm–1:
1 160 (SO2sym), 1 290 (amide III), 1 356 (SO2as), 1 540
(amide II), 1 690 (amide I), 3 060 (NH); 3 360 (NH2);
1H-NMR (DMSO-d6), δ, ppm: 6.65 (br s, 2H, SO2NH2);
7.05–7.98 (m, 4H, ArH from nicotinoyl); 7.05–7.86 (m,
2.1.1.14. 5-Nicotinoylimido-4-methyl-2-sulfonamido-δ2-
1,3,4-thiadiazoline 36
White crystals, m.p. > 310 °C; IR (KBr), cm–1: 1 182
(SO2sym), 1 290 (amide III), 1 366 (SO2as), 1 540 (amide

803
1
II), 1 690 (amide I), 3 080 (NH), 3 380 (NH2); H-NMR
2.1.1.19. 4-(Nicotinoyloxymethyl)-benzenesulfonamide 41
White crystals, m.p. 244–246 °C; IR (KBr), cm–1:
1 040 (CO–O), 1 155 (SO2sym), 1 325 (SO2as), 1 780
(COO), 3 310 (NH2); 1H-NMR (DMSO-d6), δ, ppm: 4.90
(s, 2H, CONHCH2); 7.05–7.98 (m, 4H, ArH from nico-
tinoyl); 7.08–7.41 (m, AA≠BB≠, J = 7.2 Hz; 4H, ArH,
phenylene); 7.49 (s, 2H, SO2NH2); Anal., found, C,
53.19; H, 4.21; N, 9.37%; C13H12N2O4S requires: C,
53.42; H, 4.14; N, 9.58%.
(DMSO-d6), δ, ppm: 3.90 (s, 3H, Me); 6.96 (br s, 2H,
SO2NH2); 7.05–7.98 (m, 4H, ArH from nicotinoyl);
Anal., found, C, 35.99; H, 3.12; N, 23.25%; C9H9N5O3S2
requires: C, 36.11; H, 3.03; N, 23.40%.
2.1.1.15. 5-(Nicotinoylamidoethylcarboxamido)-1,3,4-
thiadiazol-2-sulfonamide 37
White crystals, m.p. 287–289 °C (dec.), IR (KBr),
cm–1: 1 150 (SO2sym), 1 270 and 1 290 (amide III), 1 330
(SO2as), 1 450, 1 570 (amide II), 1 690 and 1 710 (amide
2.1.1.20. 4-(Nicotinoyloxyethyl)-benzenesulfonamide 42
White crystals, m.p. 240–243 °C. IR (KBr), cm–1:
1 040 (CO–O), 1 157 (SO2sym), 1 332 (SO2as), 1 760
(COO), 3 300 (NH2); 1H-NMR (DMSO-d6), δ, ppm: 3.10
(t, 2H, αCH2 from the CH2CH2 bridge); 3.70 (t, 2H,
âCH2 from the CH2CH2 bridge); 6.95 (br s, 2H,
SO2NH2); 7.05–7.98 (m, 4H, ArH from nicotinoyl);
7.05–7.52 (m, AA≠BB≠, J = 7.3 Hz, 4H, ArH, phe-
nylene); Anal., found, C, 54.95; H, 4.67; N, 8.97%;
C14H14N2O4S requires: C, 54.89; H, 4.61; N, 9.14%.
1
I), 3 090 (NH); 3 360 (NH2); H-NMR (DMSO-d6), δ,
ppm: 2.25–2.60 (m, 4H, CH2CH2); 6.88 (br s, 3H, CONH
+ SO2NH2); 7.05–7.98 (m, 4H, ArH from nicotinoyl);
8.24 (br s, 1H, CONH from nicotinoylamido moiety);
Analysis, found: C, 37.15; H, 3.19; N, 23.46%;
C11H12N6O4S2 requires: C, 37.07; H, 3.39; N, 23.58%.
2.1.1.16. 6-Nicotinoylamido-benzothiazol-2-sulfonamide 38
White crystals, m.p. 290–294 °C (dec.), IR (KBr),
cm–1: 1 165 (SO2sym), 1 290 (amide III), 1 344 (SO2as),
1 540 (amide II), 1 680 (amide I), 3 060 (NH); 3 360
(NH2); 1H-NMR (DMSO-d6), δ, ppm: 7.05–7.98 (m, 4H,
ArH from nicotinoyl); 6.94 (dd, 1H, J = 9 Hz; J = 3 Hz,
H-5 of benzothiazole); 7.10 (d, 1H, J = 3 Hz, H-7 of
benzothiazole); 7.78 (d, 1H, J = 9 Hz, H-4 of benzothia-
zole); 8.10 (br s, 2H, SO2NH2); 8.18 (br s, 1H, CONH);
Analysis, found: C, 46.85; H, 2.94; N, 16.47%;
C13H10N4O3S2 requires: C, 46.70; H, 3.01; N, 16.76%.
2.1.1.21. 5,6-Dihydro-4-[N-nicotinoyl-(ethylamido)]-6-
methyl-4H-thieno-[2,3-b]thiopyran-2-sulfonamide 7,7-
dioxide 43
White crystals, m.p. 270–272 °C; IR (KBr), cm–1:
1 135 (SO2sym), 1 290 (amide III), 1 345 (SO2as), 1 545
(amide II), 1 680 (amide I), 3 366 (NH2); 1H-NMR
(DMSO-d6), δ, ppm: 1.29 (d, 3H, Me); 1.39 (t, 3H, Me
from ethyl); 2.55 (m, 1H, CH); 2.80 (m, 1H, CH);
3.05–3.20 (m, 2H, CH2 from ethyl); 4.37 (m, 2H, CH2);
7.05–7.98 (m, 4H, ArH from nicotinoyl); 8.03 (s, 1H, CH,
ArH from thienyl); 8.25 (br s, 2H, SO2NH2); Anal.,
found, C, 44.57; H, 4.39; N, 9.66%; C16H19N3O5S3
requires: C, 44.74; H, 4.46; N, 9.78%.
2.1.1.17. 6-Nicotinoyloxy-benzothiazol-2-sulfonamide 39
White crystals, m.p. 281–283 °C (dec.), IR (KBr),
cm–1: 1 030 (CO–O), 1 160 (SO2sym), 1 350 (SO2as),
1 450, 1 775 (COO), 3 360 (NH2); 1H-NMR (DMSO-d6),
δ, ppm: 7.05–7.98 (m, 4H, ArH from nicotinoyl); 6.90
(dd, 1H, J = 9 Hz; J = 3 Hz, H-5 of benzothiazole); 7.11
(d, 1H, J = 3 Hz, H-7 of benzothiazole); 7.79 (d, 1H, J =
9 Hz, H-4 of benzothiazole); 8.10 (br s, 2H, SO2NH2);
Analysis, found: C, 46.40; H, 2.90; N, 12.38%;
C13H9N3O4S2 requires: C, 46.56; H, 2.71; N, 12.53%.
2.2. Pharmacology
2.2.1. Enzyme assay
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 Forsman et
al. [37] (the two plasmids were a gift from Prof. Sven
Lindskog, Umea University, Sweden). Cell growth con-
ditions were those described by Lindskog’s group [38]
and enzymes were purified by affinity chromatography
according to the method of Khalifah et al. [39]. Enzyme
concentrations were determined spectrophotometrically
2.1.1.18. 6-Nicotinoyloxyethyloxy-benzothiazol-2-sulfon-
amide 40
White crystals, m.p. 245–246 °C, IR (KBr), cm–1:
1 030 (CO–O), 1 175 (SO2sym), 1 341 (SO2as), 1 450,
1
1 770 (COO), 3 360 (NH2); H-NMR (DMSO-d6), δ,
ppm: 2.89 (t, 3H, CH2); 3.14 (t, 3H, CH2); 6.95 (dd, 1H,
J = 9 Hz; J = 3 Hz, H-5 of benzothiazole); 7.05–7.98 (m,
4H, ArH from nicotinoyl); 7.10 (d, 1H, J = 3 Hz, H-7 of
benzothiazole); 7.79 (d, 1H, J = 9 Hz, H-4 of benzothia-
zole); 8.15 (br s, 2H, SO2NH2); Analysis, found: C,
47.58; H, 3.66; N, 10.89%; C15H13N3O5S2 requires: C,
47.49; H, 3.45; N, 11.07%.
at 280 nm, utilizing
a
molar absorptivity of
49 mM–1.cm–1 for CA I and 54 mM–1.cm–1 for CA II,
respectively, based on Mr = 28.85 kDa for CA I, and
29.30 kDa for CA II, respectively [40, 41]. CA IV was
isolated from bovine lung microsomes as described by

804
Maren et al., and its concentration has been determined
by titration with ethoxzolamide [42].
each set of pressure measurements. IOP was measured
three times at each time interval, and the means reported.
IOP was measured first, immediately before drug admin-
istration, then at 30 min after the instillation of the
pharmacological agent, and then every 30 min for a
period of several hours. For all IOP experiments, drug
was administered to only one eye, leaving the contralat-
eral eye as an untreated control. The ocular hypotensive
activity is expressed as the average difference in IOP
between the treated and control eye, in this way mini-
mizing the diurnal, seasonal and interindividual varia-
tions commonly observed in the rabbit [45, 46]. All data
are expressed as mean ± SE, using a one-tailed t test.
Ocular hypertension was elicited in the right eye of albino
rabbits by the injection of α-chymotrypsin (from Sigma)
as described by Melena et al. [48]. The IOP of operated
animals was checked after approximately four weeks, and
animals with an elevated pressure of 30–36 mm Hg were
used at least one month after the injection of
α-chymotrypsin.
Initial rates of 4-nitrophenyl acetate hydrolysis cataly-
sed by different CA isozymes were monitored spectro-
photometrically, at 400 nm, with a Cary 3 instrument
interfaced with an IBM compatible PC [43]. Solutions of
substrate were prepared in anhydrous acetonitrile; the
substrate concentrations varied between 2 × 10–2 and
1 × 10–6 M, working at 25 °C. A molar absorption coef-
ficient e of 18 400 M–1.cm–1 was used for the
4-nitrophenolate formed by hydrolysis, in the conditions
of the experiments (pH 7.40), as reported in the litera-
ture [43]. Non-enzymatic hydrolysis rates were always
subtracted from the observed rates. Duplicate experi-
ments 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 pre-
pared 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
distilled-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 [43]. Enzyme
concentrations were 3.5 nM for hCA II, 12 nM for hCA
I and 36 nM for bCA IV (this isozyme has a decreased
esterase activity [44] and higher concentrations had to be
used for the measurements).
2.2.3. Drug distribution in ocular fluids and tissues
The general procedure of Maren’s group has been
followed [45, 46]. The animals were killed with an
intracardiac air injection. Aqueous humour (both poste-
rior and anterior chamber fluids) were withdrawn. Then,
the cornea and anterior uvea (iris plus attached ciliary
body) were dissected, rinsed well with water, blotted,
weighed and put into 1–2 mL of water. For isolation of
the ciliary processes, intact anterior uvea rings were
placed on a parafilm covered piece of polystyrene foam in
a Petri dish. The tissue was wetted with normal saline and
dissected under a microscope, then ciliary processes were
liberated from their attachment to the iris, cut, weighed
and put in 0.5 mL of distilled water. The tissue from 4
eyes (average weight of 8 mg/eye) was pooled for drug
analysis. Samples were boiled for 5 min (in order to
denature CA, and free drug from the E-I complex),
diluted and then incubated with a known amount of
enzyme. The activity of the free enzyme and in the
presence of the inhibitor were determined as described
above. A calibration curve has been used in order to
determine the fractional inhibition in the different tissues,
as described in [45, 46].
2.2.2. Measurement of tonometric IOP
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 tempera-
ture controlled room, at 22–26 °C. Solutions of inhibitors
(2%, as hydrochlorides, by weight) were obtained in
distilled deionized water. The pH of these solutions was
around 5.50–6.40.
IOP was measured using a Digilab 30R pneumatonom-
eter (BioRad, Cambridge, MA, USA) as described by
Maren’s group [45, 46]. The pressure readings were
matched with two-point standard pressure measurements
at least twice each day using a Digilab calibration verifier.
All IOP measurements were done by the same investiga-
tor with the same tonometer. One drop of 0.2% oxybup-
rocaine hydrochloride (novesine, Sandoz) diluted 1:1
with saline was instilled in each eye immediately before
3. Results
Compounds prepared by reaction of nicotinoyl chlo-
ride with aromatic/heterocyclic sulfonamides, of type
23–43, are shown below (figure 2). Inhibition data against
three CA isozymes, hCA I, hCA II and bCA IV with
compounds 1–43 are presented in table I. In vivo IOP

805
Table I. CA inhibition data with standard inhibitors 1–2, the
parent sulfonamides 3–22 and the new derivatives 23–43 reported
in the present study, against isozymes I, II and IV.
Inhibitor
KI* (nM)
hCA Ia
50 000
–
hCA IIa
9
bCA IVb
45
Dorzolamide 1
Brinzolamidec 2
3.2
295
240
300
320
170
160
60
110
40
70
28
75
60
19
3
45.3
1 310
2 200
3 000
3 215
2 800
2 450
180
320
66
125
175
160
540
355
125
19
3
4
5
6
7
8
9
45 400
25 000
28 000
78 500
25 000
21 000
8 300
9 800
6 500
6 000
6 100
8 400
8 600
9 300
455
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
70
55
50
9
8
7
17
15
24 000
18 000
21 800
20 500
16 000
23 200
1 200
1 100
547
630
650
650
540
628
36
28
21
13
9
9
125
110
310
285
142
324
79
66
38
49
45
42
36
39
2
560
450
570
310
165
400
115
102
70
78
80
69
77
65
9
11
12
19
8
7
3
5
5
3
2
2 160
2 100
2 000
79
64
5
140
116
11
Figure 2. Structure of derivatives 3–43.
*Standard error for the determination of KI-s was of 5–10% (from
a
two different assays). Human (cloned) isozyme; bIsolated from
lowering data with some of the most active CA inhibitors
reported here, in normotensive and glaucomatous rabbits,
after topical administration of the drug, are shown in
tables II and III, respectively. Ex vivo distribution data of
compound 35 in ocular tissues and fluids after the topical
administration in normotensive rabbits, are shown in
table IV.
c
bovine lung microsomes; From [47].
4. Discussion
Reaction of sulfonamides 3–22 or 1 with nicotinoyl
chloride afforded a series of new compounds of type

806
Table II. Fall of IOP of normotensive rabbits (20.1 ± 2.0 mm Hg), after treatment with one drop (50 µL) of a solution 2 % of CA inhibitor
(as hydrochloride salt, with the pH value shown below) directly into the eye, at 30, 60 and 90 min after administration.
Inhibitor
pH
∆IOP (mm Hg)*
t = 0
t = 30 min
2.2 ± 0.10
5.4 ± 0.12
6.2 ± 0.10
5.1 ± 0.12
2.1 ± 0.05
2.4 ± 0.05
2.5 ± 0.06
t = 60 min
4.1 ± 0.15
10.9 ± 0.11
12.4 ± 0.14
8.1 ± 0.11
4.0 ± 0.10
4.3 ± 0.11
4.5 ± 019
t = 90 min
2.7 ± 0.08
12.5 ± 0.17
14.1 ± 0.12
8.6 ± 0.12
4.5 ± 0.10
3.5 ± 0.08
7.0 ± 0.14
1 (dorzolamide)
5.5
5.5
5.8
5.5
5.7
5.5
5.5
0
0
0
0
0
0
0
35
36
37
39
40
43
* ∆IOP = IOPcontrol eye – IOPtreated eye; Mean ± average spread (n = 3).
23–43. The reaction was generally performed in acetone
or acetonitrile as solvent, in the presence of triethylamine
as base. In the case of compounds 15 and 16, the above
procedure led to very low yields of nicotinoylamido
derivatives, and Schotten-Baumann conditions were ap-
plied for obtaining 35 and 36 in good yields. Hydrochlo-
rides of the new derivatives were then prepared by
reacting the free bases 23–43 with a methanolic HCl
solution. Similarly were obtained the triflate salts, by
reaction of bases 23–43 with triflic acid in water as
solvent. These salts possess a very good water solubility,
generally in the range of 3–5% (data not shown). The pH
of such solutions were generally around 5.5–6.0, making
them appropriate for topical application directly into the
eye.
Compounds 3–43 were characterized by standard
chemical and physical methods that confirmed their
structure (see Experimental protocols for details) and
were assayed for the inhibition of isozymes hCA I, hCA
II and bCA IV (table I).
Inhibition data against the three CA isozymes, hCA I,
hCA II and bCA IV with the new derivatives (table I)
prove that the nicotinoylamido-sulfonamides 23–43 re-
ported here generally behave as strong inhibitors, with
greatly increased efficiencies as compared to the parent
compounds from which they were prepared (the sulfon-
amides 3–22). The efficiency of the obtained inhibitor
generally varied in the following way, based on the parent
sulfonamide from which it was prepared: the derivative
of p-hydrazino-benzenesulfonamide 26 < the orthanil-
amide 23 the metanilamide 24 < the sulfanilamide 25 <
the homosulfanilamides 27 < the p-aminoethyl-benzene-
sulfonamides 28 < the 1,3-benzene-disulfonamides 33
and 34 the halogeno-substituted sulfanilamides 29–32 <
the 1,3,4-thiadiazole-2-sulfonamides 35 and 37
4-methyl-δ2-1,3,4-thiadiazoline-2-sulfonamide 36 the
dorzolamide derivative 43 < the benzothiazole-2-
sulfonamides 38–40. All three CA isozymes investigated
here were susceptible to inhibition with this type of
sulfonamide, with hCA II and bCA IV the most inhib-
itable, followed by hCA I, generally less susceptible to
inhibition as compared to the first two isozymes.
The promising in vitro CA inhibitory activity of some
of the newly prepared compounds prompted us to inves-
tigate their effect in vivo, on the intraocular pressure
(IOP), after topical application directly into the eye, in
normotensive and glaucomatous rabbits, frequently used
as an animal model of glaucoma [13–15, 22, 23, 45].
Some of these results are shown in tables II and III.
The inhibitors selected for in vivo studies were among
the most active against hCA II and IV, in the prepared
series, such as compounds 35–40, and 43. The following
Table III. Fall of IOP of glaucomatous rabbits (34.1 ± 2.0 mm Hg), after treatment with one drop (50 µL) of a solution 2 % of CA inhibitor
(as hydrochloride salt, with the pH value shown below) directly into the eye, at 30, 60 and 90 min after administration.
Inhibitor
pH
∆IOP (mm Hg)*
t = 0
t = 30 min
4.3 ± 0.25
10.3 ± 0.20
8.5 ± 0.10
4.8 ± 0.10
t = 60 min
7.1 ± 0.30
15.2 ± 0.20
13.2 ± 0.20
6.6 ± 0.10
t = 90 min
5.0 ± 0.25
19.1 ± 0.15
20.4 ± 0.20
11.6 ± 0.15
1
5.5
5.5
5.8
5.5
0
0
0
0
35
36
43
* ∆IOP = IOPcontrol eye – IOPtreated eye; Mean ± average spread (n = 3).

807
Table IV. Ocular tissue concentrations (µM) after 1 and 2 h,
following corneal application of one drop (50 µL) of a 2 % solution
of the compound 35 in normotensive albino rabbits.
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