Carbonic anhydrase inhibitors: Synthesis of sulfonamides incorporating dtpa tails and of their zinc complexes with powerful topical antiglaucoma properties

Article abstract of DOI:10.1016/S0960-894X(00)00722-8

Reaction of diethylenetriamino pentaacetic acid (dtpa) dianhydride with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded bis-sulfonamides containing metal-complexing, polyamino-polycarboxylic acid moieties in their molecule. The corresponding mono-sulfonamide derivatives of dtpa were also obtained by an alternative method, from the free acid. Zn(II) complexes of these new sulfonamides were then prepared. Many of these derivatives showed nanomolar affinity towards isozymes I, II and IV of carbonic anhydrase (CA). Some of the best inhibitors were applied as 2% water solutions/suspensions into the eye of normotensive or glaucomatous albino rabbits, when strong and long-lasting intraocular pressure (IOP) lowering was observed.

Full text of DOI:10.1016/S0960-894X(00)00722-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 575±582
Carbonic Anhydrase Inhibitors: Synthesis of Sulfonamides
Incorporating dtpa Tails and of their Zinc Complexes with
Powerful Topical Antiglaucoma Properties
Andrea Scozzafava,a Luca Menabuoni,b Francesco Mincione,c
Giovanna Mincionea and Claudiu T. Supurana,*
a
Á
Universita 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, Florence, Italy
c
Á
Universita degli Studi, Institute of Ophthalmology, Viale Morgagni 85, I-50134, Firenze, Italy
Received 3 October 2000; revised 17 November 2000; accepted 21 December 2000
AbstractÐReaction of diethylenetriamino pentaacetic acid (dtpa) dianhydride with aromatic/heterocyclic sulfonamides possessing a
free amino/imino/hydrazino/hydroxy group a€orded bis-sulfonamides containing metal-complexing, polyamino-polycarboxylic
acid moieties in their molecule. The corresponding mono-sulfonamide derivatives of dtpa were also obtained by an alternative
method, from the free acid. Zn(II) complexes of these new sulfonamides were then prepared. Many of these derivatives showed
nanomolar anity towards isozymes I, II and IV of carbonic anhydrase (CA). Some of the best inhibitors were applied as 2% water
solutions/suspensions into the eye of normotensive or glaucomatous albino rabbits, when strong and long-lasting intraocular
pressure (IOP) lowering was observed. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
clinical medicine.3 They represent a radically new
approach in treating glaucoma with CA inhibitors, as
the undesired side e€ects observed with the systemically
administered compounds are less pronounced.1À3
Inhibitors of the zinc enzyme carbonic anhydrase (CA,
EC 4.2.1.1) are clinically used agents in the treatment of
diverse diseases such as glaucoma; gastroduodenal
ulcers; acid±base disequilibria and diverse neurological/
neuromuscular disorders.1
The clinical success of dorzolamide and brinzolamide
fostered much research in the design and evaluation of
novel agents from this class of enzyme inhibitors.4,5 The
two topically acting antiglaucoma drugs mentioned
above, 6 and 7, are both secondary amines, and the
required water solubility needed for their topical action
is achieved by using their hydrochloride salts. But in
some cases this represents an undesired problem, since
the pH of such solutions becomes rather acidic, and
consequently produces eye irritation after the topical
administration, as already reported for many patients
treated with dorzolamide.6 The most common adverse
e€ects after topical treatment with dorzolamide consist
in local burning or stinging of the eyes, pruritus and
bitter taste, but some more serious problems, such as
nephrolithiasis, anorexia, depression and irreversible
corneal decompensation were also reported.6 Such side-
e€ects might be avoided for compounds that should not
be administered as hydrochloride salts, but this gen-
erally leads to a drastic diminution of water solubility of
Since the discovery that sulfanilamide 1 and related
aromatic/heterocyclic sulfonamides of types 2±5 act as
speci®c CA inhibitors,2 several such compounds were
utilized clinically for more than 45 years in the treat-
ment and prevention of the above-mentioned diseases,
or as physiological tools.1,2 Although systemically
administered sulfonamide inhibitors of types 4 and 5 are
highly e€ective antiglaucoma drugs, their main draw-
back is constituted by severe side-e€ects due to CA
inhibition in tissues other than the eye.1,2 Only recently
some water soluble, topically e€ective sulfonamide CA
inhibitors, such as dorzolamide 6 and brinzolamide 7
have been developed and successfully introduced in
*Corresponding author. Tel.: +39-55-275-7551; fax: +39-055-275-
7555; e-mail: cts@bio.chim.uni®.it
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00722-8

576
A. Scozzafava et al. / Bioorg. Med. Chem. Lett. 11 (2001) 575±582
a sulfonamide drug. Thus, it is clear that novel gen-
eration, more e€ective antiglaucoma sulfonamides act-
ing topically are needed for an ecient treatment of
glaucoma.
of which also showed excellent water solubility in the
neutral pH range, and promising anti-glaucoma activity
via the topical route in experimental animals. Here we
extend that approach for obtaining water-soluble, high
anity sulfonamide CA inhibitors, which do not owe
their water solubility to formation of hydrochloride
salts. We report here a series of compounds obtained by
reaction of diethylenetriamino pentaacetic acid (dtpa)
dianhydride with aromatic/heterocyclic sulfonamides of
types A±T, possessing free amino/imino/hydrazino
or hydroxy groups, leading to bis-sulfonamides
In recent contributions from this laboratory4,5 it was
shown that by attaching water-solubilizing tails (such as
8-quinoline-sulfonyl-; nicotinoyl-; isonicotinoyl-; 6-car-
boxy-pyridine-2-carboxamido-; amino acyl-, etc.) to the
molecules of aromatic/heterocyclic sulfonamides of
types A±T, ecient CA inhibitors were obtained, some
Scheme 1.

A. Scozzafava et al. / Bioorg. Med. Chem. Lett. 11 (2001) 575±582
577

578
A. Scozzafava et al. / Bioorg. Med. Chem. Lett. 11 (2001) 575±582
incorporating polyamino-polycarboxylic acid moieties
in their molecule. Another series of compounds, more
precisely the corresponding mono-sulfonamide dtpa
derivatives, has been prepared by condensing the free
acid (dtpa) with the sulfonamides A±T in the presence
of carbodiimides. Some Zn(II) complexes of some of
these ligands have also been obtained.
puri®ed by HPLC due to their very similar retention
times (data not shown), and such a mixture has been
used in the enzyme assay as well as for some in vivo
experiments described shortly. The new compounds
reported here were characterized by standard proce-
dures (elemental analysis, spectroscopic data) which
con®rmed the proposed structures.8 Due to the metal
coordinating moieties incorporated in these new com-
pounds, and since metal complexes of sulfonamides
were recently shown to possess interesting topical anti-
glaucoma properties,9 some Zn(II) complexes of several
new bis-sulfonamides of type dtpa(A±T)2 were also pre-
pared, characterized and investigated for their CA inhi-
bition properties in vitro and IOP lowering in
experimental animals (see Tables 1±5).
Reaction of dtpa dianhydride 8 or of the free acid (dtpa)
9 with aromatic/heterocyclic sulfonamides A±T posses-
sing a free amino/imino/hydrazino/hydroxy group in
molar ratios of 1:2, and 1:1, respectively, a€orded the
bis-sulfonamides of type dtpa(A±T)2 and the mono-
sulfonamides dtpa(A±T) (Scheme 1). The procedure for
the synthesis of bis-amides of this type has been repor-
ted previously by Aime's group for aniline derivatives.7
When dianhydride 8 was reacted with the aromatic/het-
erocyclic sulfonamides A±T in molar ratios of 1:1, in the
same conditions as above, derivatives of type dtpa(A±T)
could not be isolated from the reaction mixture (a mix-
ture of bis-derivatized compounds dtpa(A±T)2 and
unreacted dtpa were the reaction products in such
cases), and this prompted us to synthesize the mono-
derivatives by an alternative procedure, as shown in
Scheme 1. Since dtpa contains four equivalent car-
boxylic acid moieties, and the central one, which is non-
equivalent with the other four, the above mentioned
reaction a€orded a mixture of two isomeric sulfona-
mides (in a molar ratio of about 4:1) which could not be
Since a relatively large number of derivatives are repor-
ted here, each compound will be designated by a letter
identifying the sulfonamide from which it was obtained
(A±T) with its stoichiometry (1 or 2, corresponding to
one or two moieties of sulfonamide incorporated), and
`dtpa' preceding it, corresponding to the poly-
aminopolycarboxylic acid moiety contained in its mole-
cule. For instance, dtpa-C is the sulfanilamide derivative
incorporating the dtpa tail, whereas dtpa-C2 corre-
sponds to the bisamide, containing the dtpa moiety
bound to two sulfanilamide groups. Correspondingly,
the `acetazolamide-like' compounds dtpa-M (mono-
amide) and dtpa-M2 are also shown below.

A. Scozzafava et al. / Bioorg. Med. Chem. Lett. 11 (2001) 575±582
579
The data of Table 1 show that the new inhibitors pre-
pared by attaching dtpa moieties to aromatic/hetero-
cyclic sulfonamides A±T, are more e€ective as
compared to the parent sulfonamides from which they
were prepared, towards the three investigated isozymes,
hCA I, hCA II and bCA IV. The enhanced inhibitory
power of these compounds is presumably due to the
interaction of the long dtpa moiety incorporated in their
molecules with hydrophilic patches at the entrance of
the enzyme active site as observed for inhibitors pre-
viously reported by Whitesides'10 and our groups,4,5,11
and explained by detailed QSAR models.12
The nature of the sulfonamide attached to the dtpa
moiety in the new derivatives reported here greatly
in¯uenced the CA inhibitory power of these sulfona-
mides. Among the synthesized derivatives, the het-
erocyclic sulfonamide derivatives were the most
active, followed by the aromatic sulfonamide deriva-
tives. The eciency of the obtained inhibitor gen-
erally varied in the following way, based on the
parent sulfonamide from which it was prepared: the
derivatives of p-hydrazino-benzenesulfonamide < the
orthanilamides < the metanilamides < the sulfanilami-
des < the
homosulfanilamides<the
p-aminoethyl-
Table 2. Solubility, chloroform±bu€er partition coecients and in
vitro corneal permeability of some sulfonamide CA inhibitors reported
in the paper and dorzolamide as standard
Table 1. Inhibition data for some derivatives reported in the present
paper (data in parentheses represent inhibition by the parent sulfona-
mide A±T)
Compound
Solubilitya Log Pb
(mM)
kinÂ103 (hÀ1 c
)
Inhibitor
KI (nM)
hCA IIa
hCA Ia
bCA IVb
Cornea intact No epithelium
Dorzolamide 6
dtpa-C
dtpa-C2
dtpa-M
dtpa-M2
60d
48e
35e
59e
54e
2.0e
3.0
3.8
3.6
4.3
3.9
5.2
6.9
6.5
8.1
6.2
Acetazolamide 4
Methazolamide 5
Dorzolamide 6
dtpa-A
dtpa-B
dtpa-C
dtpa-D
dtpa-E
dtpa-F
dtpa-G
dtpa-H
dtpa-I
dtpa-J
dtpa-K
dtpa-L
dtpa-M
dtpa-N
dtpa-O
900
780
>50,000
12
14
9
220
240
43
0.983
1.456
1.375
1.961
8600 (45,400)
7200 (25,000)
4100 (28,000)
12,000 (78,500
550 (25,000)
290 (21,000)
180 (8300)
200 (9800)
96 (6500)
79 (6000)
58 (6100)
87 (8400)
55 (8600)
62(9300)
210 (295)
180 (240)
75 (300)
250 (320)
23 (170)
15 (160)
13 (60)
20 (110)
12(40)
16 (70)
10 (28)
12(75)
0.9 (60)
2(19)
550 (1310)
500 (2200)
180 (3000)
630 (3200)
40 (2800)
36 (2500)
29 (180)
32 (320)
27 (66)
29 (125)
36 (175)
52(160)
28 (540)
19 (355)
16 (125)
9 (19)
aSolubility in pH 7.40 bu€er, at 25 ꢀC.
bChloroform±bu€er partition coecient.13
cDetermined as described in ref 13.
dAs hydrochloride, at pH 5.8, from ref 4.
eAs sodium salts.
Table 3. IOP lowering in normotensive rabbits (23.5 Æ 2.6 mmHg)
after treatment with one drop (50 mL) 2% solution/suspension of CA
inhibitor (the pH of the solution shown), at 30, 60 and 90 min after
administration directly into the eye
110 (455)
36 (70)
32(55)
1 (3)
Inhibitor
ÁIOP (mmHg)a
t=30 min t=60 min
dtpa-P
dtpa-Q
dtpa-R
dtpa-S
0.8 (9)
0.8 (8)
0.6 (7)
80 (125)
61 (110)
175 (295)
155 (240)
64 (300)
215 (320)
19 (170)
8 (160)
7 (60)
15 (110)
10 (40)
9 (70)
9 (28)
11 (75)
1 (60)
1.5 (19)
0.6 (3)
0.5 (9)
0.6 (8)
0.5 (7)
43 (125)
34 (110)
16
6 (17)
5 (15)
pH
t=0
t=90 min
30 (50)
510 (24,000)
360 (18,000)
5400 (45,400)
6400 (25,000)
3750 (28,000)
9800 (78,500)
380 (25,000)
170 (21,000)
135 (8300)
155 (9800)
79 (6500)
70 (6000)
43 (6100)
63 (8400)
50 (8600)
54 (9300)
102(455)
25 (70)
21 (55)
16 (50)
325 (24,000)
290 (18,000)
350
39
36
40
43
265 (560)
190 (450)
330 (1310)
425 (2200)
156 (3000)
430 (3200)
37 (2800)
25 (2500)
18 (180)
27 (320)
21 (66)
24 (125)
25 (175)
41 (160)
7 (540)
9 (355)
8 (125)
6 (19)
5 (17)
4 (15)
170 (560)
115 (450)
100
21
16
4
Dorzolamide 6
dtpa-C
dtpa-C2
5.5
7.0
7.0
7.4
7.5
7.5
7.0
0
0
0
0
0
0
0
1.9 Æ0.24.0
3.1 Æ 0.24.5
4.4 Æ 0.27.5
3.1 Æ 0.5
Æ 0.3
Æ 0.27.2
2.1 Æ 0.2
Æ 0.3
dtpA±T
dtpa-A2
dtpa-B2
dtpa-C2
dtpa-D2
dtpa-E2
dtpa-F2
dtpa-G2
dtpa-H2
dtpa-I2
Æ 0.29.5
Æ 0.3
dtpa-M
6.8 Æ 0.24.5
13.0 Æ 0.28.1
9.2 Æ 0.3
Æ 0.3
dtpa-M2
dtpa-N
Zn- dtpa-M2
6.5 Æ 0.1
Æ 0.6
6.7 Æ 0.1
13.0 Æ 0.4
Æ 0.3
b
7.4 Æ 0.214.5
Æ 0.29.5
aÁIOP=IOPcontrol eyeÐIOPtreated eye; Mean Æ SEM (n=3).
bSuspension.
dtpa-J2
dtpa-K2
dtpa-L2
dtpa-M2
dtpa-N2
dtpa-O2
dtpa-P2
dtpa-Q2
dtpa-R2
dtpa-S2
dtpA-T2
Zn-dtpa-C2
Zn-dtpa-E2
Zn-dtpa-F2
Zn-dtpa-M2
Zn- dtpa-N2
Table 4. Fall of IOP of glaucomatous rabbits (33.5 Æ 3.0 mmHg),
after treatment with one drop (50 mL) solution/suspension of 2% of
CA inhibitor (as hydrochloride or sodium salt, with the pH value shown
below) directly into the eye, at 30, 60 and 90 min after administration
Inhibitor
pH
ÁIOP (mmHg)a
t=0
t=30 min
t=60 min
t=90 min
Dorzolamide
dtpa-C2
5.5
7.0
7.5
7.5
7.0
0
0
0
0
0
3.6 Æ 0.26.7
6.0 Æ 0.4
Æ 0.3
12.5 Æ 0.15
13.0 Æ 0.5
16.0 Æ 0.4
19.2 Æ 0.8
4.2 Æ 0.15
11.0 Æ 0.2
14.9 Æ 0.5
17.5 Æ 0.3
18.9 Æ 0.5
9
7
0.5
dtpa-M2
dtpa-N
Zn-dtpa-M2
7.4 Æ 0.3
8.8 Æ 0.6
b
19.8 Æ 0.7
0.4
5
aÁIOP=IOPcontrol eyeÀIOPtreated eye; Mean SEM (n=3).
aHuman (cloned) isozymes.
bSuspension.

580
A. Scozzafava et al. / Bioorg. Med. Chem. Lett. 11 (2001) 575±582
benzenesulfonamides  the halogeno-substituted sulfa-
nilamides  the 1,3-benzene-disulfonamides < the 1,3,4-
thiadiazole-2-sulfonamides 4-methyl-d2-1,3,4-thiadiazo-
line-2-sulfonamide  the benzothiazole-2-sulfonamides.
The monoamides/esters were generally less active than
the corresponding bis-amides/esters. The Zn(II) com-
plexes of some bis-sulfonamides that were also pre-
pared, were much more active than the corresponding
ligands, as already reported in the literature.9 All three
CA isozymes investigated here were susceptible to inhi-
bition with this type of sulfonamide, with hCA II and
bCA IV the most sensitive, whereas hCA I was gen-
erally less susceptible to inhibition as compared to the
®rst two isozymes. Mention should be made that the
two susceptible isozymes (CA II and CA IV) are just
those involved in aqueous humor secretion,1 whereas
CA I inhibition may explain the presence or the lack of
side e€ects due to inhibitor washed out from the eye.14
Thus, our compounds, in contrast to dorzolamide also
inhibit the slow isozyme, CA I, which may be a positive
property, since once in the systemic circulation (after
being washed out from the eye, after a prolonged use of
eye drops containing such CA inhibitors) they should
bind to the predominant isozyme present in blood, i.e.,
hCA I, and will thus inhibit to a slighter degree the
rapid isozyme hCA II, which is involved in bicarbonate
transport, electrolyte secretion, and other vital physio-
logical processes.1,14 Indeed, the blood contains up to
150 mM of hCA I, whereas the hCA II concentration is
much lower, of about 20 mM.15 The fact that dorzola-
mide shows so many systemic side e€ects6 after topical
administration may be due in part to its low anity just
for hCA I.
Some physico-chemical properties of several strong in
vitro inhibitors were investigated in detail (Table 2),
showing that many of these new derivatives possess
excellent water solubility, at pH values in the range of
7.0±7.5 pH units. This is correlated with a balanced
hydro- and lipophilicity, and in consequence optimal
accession rates across the cornea, which is typical for
the e€ective topically acting sulfonamides.1À5 The pre-
pared Zn(II) complexes of such derivatives were on the
other hand less water soluble (data not shown), but
brinzolamide 6 is administered as a suspension (due to
its poor water solubility), and thus we also used water
suspensions of these compounds for the in vivo experi-
ments.
Table 5. Ocular tissue concentrations (mM) after
1 and 2h,
following corneal application of one drop (50 mL) of 2% solution of
compounds dtpa-M2 and dtpa-N in normotensive albino rabbits
Compound
Time (h)
Cornea
Drug concentration (mM)a
Aqueous humor Ciliary process
dtpa-M2
dtpa-N
1 h
2
1 h
2
150 Æ 10
241 Æ 13
50 Æ 3
19 Æ 1
46 Æ 5
2 Æ 2
h
Æ 5 45 39 Æ 3
In vivo, in normotensive rabbits, some of the new
mono- and bis-sulfonamides reported here showed very
e€ective IOP lowering after topical administration, with
pressure reductions of 3.1±6.7 mmHg at half an hour
164 Æ 9
2
Æ 1500
h
Æ 5 56 49 Æ 4
3
aMean Æ SEM (n=3).
Figure 1. E€ect of topically administered IOP lowering drugs (50 mL solutions/suspensions) on the IOP of glaucomatous albino rabbits. Curve 1:
Dorzolamide hydrochloride solution 2% (standard); Curve 2: dtpa-M2 2% solution (pH 7.0) Curve 3: Zn-dtpa-M2 suspension 2% (pH 7.0).

A. Scozzafava et al. / Bioorg. Med. Chem. Lett. 11 (2001) 575±582
581
(compared to 1.9 mmHg for dorzolamide), 4.5±13.0
mmHg at 1 h (4.0 for the standard drug), and 4.5±13
mmHg at 90 min after administration (compared to 2.1
for dorzolamide) (Table 3). An important feature of the
new class of CA inhibitors reported here is that IOP
remained low for longer periods (3±6 h) after their
topical administration, as compared to the standard
drug (data not shown). IOP generally returned at the
baseline values after 5±6 h after administration of the
drug. The above ®ndings also apply for the Zn(II)
complexes as well as to the glaucomatous rabbit experi-
ments (Table 4 and Fig. 1) but the IOP reductions were
much more important as compared to those seen in
normotensive rabbits. Thus, IOP reductions of 6.0±19.8
mmHg were generally observed after 30 min, whereas at
1 h, these amounted to 12.0±19.2 mmHg, and in the case
of the Zn(II) complex Zn-dtpa-M2 remained at these
low values for prolonged periods (Fig. 1). Thus, all
these derivatives are longer lasting and much more
e€ective IOP lowering agents as compared to the clini-
cally available drug dorzolamide.
Mincione, F.; Briganti, F.; Mincione, G. Eur. J. Pharm. Sci.
1999, 8, 317.
5. (a) Borras, J.; Scozzafava, A.; Menabuoni, L.; Mincione,
F.; Briganti, F.; Mincione, G.; Supuran, C. T. Bioorg. Med.
Chem. 1999, 7, 2397. (b) Renzi, G.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2000, 10, 673. (c) Scozzafava,
A.; Briganti, F.; Ilies, M. A.; Supuran, C. T. J. Med. Chem.
2000, 43, 292.
6. (a) Konowal, A.; Morrison, J. C.; Brown, S. V.; Cooke,
D. L.; Maguire, L. J.; Verdier, D. V.; Fraunfelder, F. T.;
Dennis, R. F.; Epstein, R. J. Am. J. Ophthalmol. 1999, 127,
403. (b) Aalto-Korte, K. Contact Dermatitis 1998, 39, 206. (c)
Carlsen, J.; Durcan, J.; Swartz, M.; Crandall, A. Arch. Oph-
thalmol. 1999, 117, 1087. (d) Thoe Schwartzenberg, G. W.;
Trope, G. E. Can. J. Ophthalmol. 1999, 34, 93.
7. Aime, S.; Fasano, M.; Paoletti, S.; Terreno, E. Gazz. Chim.
Ital. 1995, 125, 125.
8. For example: 14-[4-(Aminosulfonyl)phenyl]-3-[2-[2-[4-(amino-
sulfonyl)phenyl]ethyl]amino]-2-oxoethyl]-6,9-bis(carboxymethyl)-
11-oxo-3,6,9,12-tetraazatetradecanoic acid, dtpa-F2: An
amount of 5.35 g (15 mmol) dtpa dianhydride (N,N-bis-[2-
(2,6-dioxo-4-morpholinyl)ethyl]glycine was added to a solu-
tion of 6.0 g (30 mmol) of 4-(2-aminoethyl)-benzenesulfona-
mide F dissolved in 100 mL of anhydrous DMF. The mixture
was magnetically stirred at room temperature for 4 h, then the
reaction mixture was poured in 300 mL of methylene chloride
and the obtained solid was ®ltered and thoroughly washed
with methylene chloride and then with acetone. HPLC pur-
i®cation was by elution with potassium phosphate/MeCN 2:1,
v/v (1 mL/min); mp 121±122 ꢀC (dec.); IR (KBr), cmÀ1: 1173
(SOs2ym), 1340 (SOa2s), 1560 (amide II), 1600 (amide I), 1760
(COOH), 3335 (NH, NH2); 1H NMR (300 MHz, D2O±KOD),
d, ppm: 2.90 (t, 2H, CH2 of aminoethylbenzenesulfonamide,
7.2); 3.47 (q, 2H, CH2 of aminoethylbenzenesulfonamide 6.5);
3.20 (4H, t, ethylenic CH2 near lateral nitrogens); 3.35 (4H, s,
CH2 of the lateral acetates); 3.48 (4H, t, ethylenic CH2 near
central nitrogen); 3.56 (4H, s, CH2 of the acetamido groups);
3.91 (2H, s, CH2 of the central acetate); 7.42(d, 2H, AA 0BB0,
8.2); 7.75 (d, 2H, AA0BB0, 8.2); 13C NMR (D2O-KOD), d,
ppm: (see structural formula dtpa-F2 for the numbering of the
carbon atoms) 36.15 (C-10); 42.07 (C-9); 53.12 (C-4); 53.68 (C-
3); 60.03 (C-2); 60.15 (C-7); 61.05 (C-5); 128.12 (C-13); 132.47
(C-12); 146.79 (C-14); 147.20 (C-11), 177.04 (C-8); 180.53 (C-
1); 181.20 (C-6); Elemental analysis, found: C, 47.13; H, 5.89;
N, 12.61; S, 8.30%; C30H43N7O12S2 requires: C 47.55; H, 5.72;
N, 12.94; S, 8.46%. The Zn(II) complex of the above ligand
was prepared as follows: A suspension of 14-[4-(aminosulfo-
nyl)phenyl]-3-[2-[2-[4-(aminosulfonyl)phenyl]ethyl]amino]-2-
oxoethyl]-6,9-bis(carboxymethyl)-11-oxo-3,6,9,12-tetra-azate-
tradecanoic acid (1.51 g, 2mmol) in 50 mL water was treated
with the stoichiometric amount of 1 N NaOH solution in
order to obtain the disodium salt. The obtained solution was
treated with a solution of ZnCl2 (1 mmol) in 5 mL of water,
maintaining the pH at 6.5. The reaction was monitored by
HPLC, on a stationary phase of Lichrospher 100 RP-18.5 mm,
with a 250Â4 mm column packed by E. Merck, at 40 ꢀC. Iso-
cratic elution with premixed mobile phase (1 g octylamine was
added to 100 mL of acetonitrile mixed with 900 mL of water)
has been performed. The eluent was bu€ered with phosphoric
acid, maintaining the pH at 6, the ¯ow rate was 1.5 mL/min.
After 4 h the solution was loaded onto an Amberlite XAD
1600 resin column (250 mL) and eluted with MeCN/water
(1:10, v/v). The fractions containing the complex were evapo-
rated to give a white solid of the complex with an overall yield
of 95%; mp >300 ꢀC; IR (KBr), cmÀ1: 1170 (SOs2ym); 1336
(SOa2s); 1560 (amide II); 1610 (amide I); 1745 (COOÀ); 3335
(NH2); Elemental analysis, found: Zn, 8.14; C, 43.84; H, 4.75;
N, 11.67; S, 7.54%; C30H41N7O12S2Zn requires: Zn, 7.94; C,
43.73; H, 4.98; N, 11.90; S, 7.77%.
Table 5 shows ex vivo data obtained in normotensive
rabbits after the topical administration of two of the
most potent topical inhibitors in the prepared series. It
can be observed that at 1 and 2h after topical adminis-
tration of drug, high levels of inhibitors were found in
the cornea, aqueous humor and ciliary processes. Based
on the inhibition constant of these compounds, the
fractional inhibition estimated in these tissues/¯uids is
of 99.5±99.9%, indicating the fact that the powerful
IOP decrease observed is indeed due to CA inhibition.
In conclusion, we report here a novel class of very
powerful, water soluble, topically acting sulfonamide
CA inhibitors, incorporating metal-complexing dtpa
moieties in their molecules, as well as some of their
zinc(II) complexes. Some of these inhibitors were very
ecient IOP lowering agents in normotensive and glau-
comatous rabbits after topical administration as water
solutions/suspensions.
Acknowledgements
This research was ®nanced by the EU grant ERB
CIPDCT 940051 and by a grant from the Italian CNR-
Target Project Biotechnology.
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