Carbonic anhydrase inhibitors: Synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating thioureido-sulfanilyl scaffolds

Article abstract of DOI:10.1016/j.bmcl.2005.02.087

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozyme IX (CA IX) is overexpressed in hypoxic tumors and appears to be involved in acidification of the tumor microenvironment, a process correlated with cancer progression and bad prognosis. The acidification may be reduced by inhibiting the enzyme with potent sulfonamide/sulfamate CA inhibitors. A series of such aromatic sulfonamides incorporating thioureido-sulfanilyl moieties has been prepared and investigated for its interaction with the catalytic domain of the human isozyme hCA IX. The key intermediates in the synthesis were obtained by reacting sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide with 4-acetamido-benzenesulfonyl chloride followed by deacetylation and reaction with thiophosgene. The obtained isothiocyanato sulfonamides were reacted with aliphatic or aromatic primary amines or hydrazines, leading to the corresponding thioureas. Some of these compounds showed excellent inhibitory properties against isozymes I, II, and IX, with several inhibitors also presenting selectivity for the inhibition of CA IX over that of the ubiquitous isozyme CA II. Such sulfonamides may constitute interesting candidates for the development of novel antitumor therapies based on the inhibition of the CA isozymes overexpressed in hypoxic tumors. Due to the highest expression of CA IX in clear renal cell carcinoma and its chemo/radioresistance, our efforts are first of all directed to generate effective therapeutic strategies for the cure of this malignancy.

Full text of DOI:10.1016/j.bmcl.2005.02.087

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2359–2364
Carbonic anhydrase inhibitors: synthesis and inhibition of
cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and
IX with sulfonamides incorporating thioureido-sulfanilyl scaffolds
Luca Puccetti,a Giuseppe Fasolis,a Alessandro Cecchi,b
Jean-Yves Winum,b,c Alessandro Gamberi,b Jean-Louis Montero,c
Andrea Scozzafavab and Claudiu T. Supuranb,*
aOspedale San Lazzaro, Divisione di Urologia, Via Pierino Belli 26, 12051 Alba, Cuneo, Italy
Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3,
50019 Sesto Fiorentino, Florence, Italy
b
`
c
´ ´
Universite Montpellier II, Laboratoire de Chimie Biomoleculaire, UMR 5032, Ecole Nationale Superieure de Chimie de Montpellier,
´
8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France
Received 29 November 2004; revised 23 February 2005; accepted 28 February 2005
Abstract—The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozyme IX (CA IX) is overexpressed in hyp-
oxic tumors and appears to be involved in acidification ofthe tumor microenvironment, a process correlated with cancer progression
and bad prognosis. The acidification may be reduced by inhibiting the enzyme with potent sulfonamide/sulfamate CA inhibitors. A
series of such aromatic sulfonamides incorporating thioureido-sulfanilyl moieties has been prepared and investigated for its inter-
action with the catalytic domain ofthe human isozyme hCA IX. The key intermediates in the synthesis were obtained by reacting
sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide with 4-acetamido-benzenesulfonyl chloride followed by
deacetylation and reaction with thiophosgene. The obtained isothiocyanato sulfonamides were reacted with aliphatic or aromatic
primary amines or hydrazines, leading to the corresponding thioureas. Some ofthese compounds showed excellent inhibitory prop-
erties against isozymes I, II, and IX, with several inhibitors also presenting selectivity for the inhibition of CA IX over that of the
ubiquitous isozyme CA II. Such sulfonamides may constitute interesting candidates for the development of novel antitumor ther-
apies based on the inhibition ofthe CA isozymes overexpressed in hypoxic tumors. Due to the highest expression ofCA IX in clear
renal cell carcinoma and its chemo/radioresistance, our efforts are first ofall directed to generate effective therapeutic strategies for
the cure ofthis malignancy.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
in this and other laboratories for discovering either
small molecule, iRNA-s or immunologic-based agents
targeting these proteins present in a multitude ofhyp-
oxic tumors.7–10
We have recently shown1 that the tumor-associated car-
bonic anhydrase (CA, EC 4.2.1.1) isozyme IX, hCA IX,
is involved in acidification ofhypoxic tumors, and that
this process may be diminished (and probably abol-
ished) by inhibiting the enzyme with potent sulfonamide
inhibitors. This constitutes the proof-of-concept demon-
stration that inhibiting the tumor-associated CAs (two
such isozymes are presently known, CA IX and CA
XII)2–6 may be relevant for the design of novel antitu-
mor therapies. Thus, much work is currently being done
Sulfonamide CA inhibitors (CAIs), such as acetazola-
mide AZA, methazolamide MZA, ethoxzolamide
EZA, or dichlorophenamide DCP among others, ini-
tially played a crucial role in the understanding ofrenal
physiology and pharmacology, leading then to the
development ofwidely used drugs such as the benzothia-
diazine and high ceiling diuretics.1–5 More recently, this
class ofdrugs showed important applications ofr the
2
development ofantiglaucoma agents, being also useful
for the design of other types of pharmacological agents,
as it will be shown here shortly. Thus, a recent and new
*
Corresponding author. Tel.: +39 055 4573005; fax: +39 055
4573385; e-mail: claudiu.supuran@unifi.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.087

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L. Puccetti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2359–2364
N
N
N
O
N
N
O
O
O
O
N
EtO
S
S NH2
S
Me
N
H
S
O
NH2
S
S
O
NH2
Me
O
AZA
MZA
EZA
NH2
SO2NH2
O
S O
H
O
N
S
Cl
N
H
O
O
Cl
S NH2
O
Cl
IND
DCP
N
N
N
N
O
O
S
O
O
S
N
R1
N
H
S
S
S
O
NH2
S
NH2
H
O
O
O
BZA
R1 = NH2 ABA
R1 = ANHCSNH Thioureido-ABA derivatives
field in CAI research has been opened by the report of
the potent antitumor properties ofa rather large number
ofsulfonamide CAIs, as well as by the isolation, purifi-
cation, and characterization ofthe isozymes predomi-
nantly present in tumor cells, such as CA IX and CA
XII.1–6 The mechanisms by which these isozymes parti-
cipate in the tumor cell growth and differentiation, as
well as those by which their inhibitors interfere with
the tumor growth only now begin to be understood,1
but important advances in these directions have recently
been achieved.1 Indeed, several laboratories are involved
in the synthesis, evaluation, and in vitro/in vivo antitu-
mor testing ofnovel types ofCAIs with potential
application as anticancer therapeutic agents.6–12
Furthermore, a compound ofthis type—indisulam
IND—has progressed to Phase II clinical trials for the
amides).1–4 Continuing this work and the same type of
chemistry,13 but using as lead molecule benzolamide
BZA (an orphan drug belonging to the family of CAIs),2
we then reported novel thioureido-containing benzol-
amide-like compounds, which were also shown to act as
very potent inhibitors ofthe transmembrane, tumor-
associated isozyme CA IX (in addition to the strong
inhibition ofthe ubiquitous, cytosolic isozymes I and
II).14 Using these last compounds as lead molecules,14
we report this time novel derivatives incorporating thio-
ureido-sulfanilyl scaffolds. Thus, the thiadiazole scaffold
ofthe above mentioned lead compounds has been chan-
ged to an 1,4-phenylene moiety, and the spacer between
this structural element and the thioureido-sulfanilyl
scaffold has been also varied, with 0, 1, and 2 CH2 moi-
eties present in the new compounds reported in this
paper. Some ofthese compounds showed excellent
inhibitory properties against isozymes CA I, II, and
IX, as well as some specificity for inhibiting the last iso-
zyme over the ubiquitous cytosolic isoform CA II.
6,9,10
treatment ofsolid tumors.
CA IX gene regulation is tightly dependent on the HIF-
1a gene expression, which is evoked by environmental
hypoxic stimuli. Initially, the strategy to counteract
hypoxic tumor growth was by targeting ofthe HIF-1 a
protein, but its expression is usually transient and its
half-life is very short.5–7 On the contrary, CA IX is a
very stable protein. The activity ofCA IX, whose hal-f
life is of at least 96 h, is long lasting, even in the case
ofhypoxia abrogation. Finally, CA IX is anchored to
the outermost layer ofthe cell and not placed in the
inner nuclear compartment (as HIF-1a is), making this
enzyme a druggable interesting target.1,5–7
2. Chemistry
The reaction ofamino-substituted sulfonamides such as
sulfanilamide 1, homosulfanilamide 2, and 4-aminoeth-
ylbenzenesulfonamide 3 with alkyl/arylsulfonyl halides
has been investigated in detail by our group.15 During
the previous studies, we also reported the sulfanylated
derivatives 5–7,15a which were obtained by treating
the parent amino sulfonamides 1–3 with 4-acet-
amidobenzenesulfonyl chloride 4, followed by deacetyl-
ation with concentrated hydrochloric acid for removal
ofthe protecting group. Derivatives 5–7 obtained as
reported earlier15a were treated with thiophosgene in
acidic medium, in order to prepare the key intermediates
needed in the present study, the isothiocyanato deriva-
tives 8–10 (Scheme 1), by the procedure already reported
for preparing the isothiocyanato derivatives of the sim-
pler sulfonamides 1 and 2.13,16
In previous work from this laboratory,13 we have devel-
oped thioureido-containing sulfonamide CAIs derived
from simple aromatic sulfonamides, such as sulfanil-
amide 1 and homosulfanilamide 2, which showed excel-
lent CA I, II, and IV inhibitory properties and were
effective topically acting antiglaucoma agents in an ani-
mal model ofthis disease (some ofthe above-mentioned
isozymes are involved in aqueous humor secretion with-
in the eye, being the targets ofantiglaucoma sulofn-

L. Puccetti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2359–2364
2361
O
O
SO2Cl
1)
AcNH
( )n
H2N
S
( )n
N
4
H
H2N
2) HCl 35%
SO2NH2
SO2NH2
5: n = 0
6: n = 1
7: n = 2
1: n = 0
2: n = 1
3: n = 2
CSCl2
O
O
O
O
R2NH2
Et3N
S
( )n
N
S
S
R2
( )n
N
H
H
N
H
N
SCN
H
SO2NH2
SO2NH2
11: n = 0
12: n = 1
13: n = 2
8: n = 0
9: n = 1
10: n = 2
Scheme 1.
The isothiocyanates 8–10 prepared as outlined above
were then converted to thioureas 11–13 by reaction with
primary amines/hydrazines in the presence oftriethyl-
amine as catalyst, by the procedure reported for the
preparation ofother thioureido-substituted suolfn-
amides.13,14 The amines/hydrazines used for obtaining
the novel derivatives reported here, oftypes 11–13, were
chosen in such a way as to incorporate moieties, which:
(i) were previously shown to lead to potent CA IX
inhibitors,14 such as dimethylaminoethyl, morpholinyl,
N-methylpiperazinyl, morpholinyl-N-ethyl, sulfamoyl-
phenylethyl,17,18 etc.; and (ii) may assure an acceptable
water solubility to such compounds, eventually as salts
with pharmaceutically acceptable acids, such as hydro-
chlorides, trifluoroacetates, triflates, etc.19 Indeed, many
ofthe moieties mentioned above (dimethylaminoethyl,
morpholinyl, N-methylpiperazinyl, and morpholinyl-N-
ethyl) are able to be protonated, leading to salts with en-
hanced water solubility as compared to the sulfonamides
from which they are derived. A strong and possibly
water-soluble CA IX inhibitor would constitute an
excellent tool for investigating in more detail the role(s)
ofthe tumor-associated CA isozymes in tumor growth
and differentiation, as well as potential therapeutic
options exploiting their specific inhibition.
for the parent sulfonamides from which the new deriva-
tives were obtained (such as 1–3 and 5–7) are also pro-
vided for comparison, as they were recently reported
by our group.8
The following SAR should be noted from, data of Table
1: (i) the cytosolic slow isozyme hCA I is inhibited by all
the new compounds 11–13 investigated here, with inhi-
bition constants in the range of3.1–355 nM. Thus,
whereas the simple sulfonamides 1–3 act as weak CA I
inhibitors (KIs in the range of21–28 lM), the corre-
sponding sulfanylated derivatives 5–7 are much better
inhibitors, with KIs in the range of95–109 nM ( Table
1).8 It may be seen that for all these compounds the
inhibitory properties increase with the increase ofthe
number ofcarbon atoms (from 0 to 2) between the benz-
ene ring bearing the primary sulfamoyl group and the
amino moiety. For the thioureas 11–13, SAR is more
complicated. Thus, a group ofderivatives, such as 11a,
11c, 11d, 12a, 12c, 12d, 12f, and 13a, behave as weaker
hCA I inhibitors as compared to the corresponding par-
ent sulfonamides from which they were obtained (5–7),
showing KIs in the range of105–355 nM. It is also note-
worthy to observe that the most active derivative in such
a subseries possessing the same substitution pattern
(e.g., 11a, 12a, and 13a) is the sulfanilamide compound
(n = 0), which is more active than the homosulfanil-
amide derivative (n = 1), which in turn is more active
than the compound with n = 2 (just the reverse situation
as compared to the parent sulfonamides 5–7 discussed
above). Another group ofderivatives, including 11b,
12b, 12e, and 12g act as very potent hCA I inhibitors,
with KIs in the range of3.1–9.5 nM. These compounds
are stronger inhibitors than the parent sulfonamides
from which they were derived (5–7) or than the clini-
cally used sulfonamides, which showed inhibition cons-
tants in the range of25 nM (ethoxzolamide)–1.2 lM
3. Carbonic anhydrase inhibition
Inhibition data against three physiologically relevant
isozymes, that is, the cytosolic isoforms hCA I and II
and the membrane-bound, tumor-associated isozyme
hCA IX (all ofthem ofhuman origin) with the new com-
pounds 11–13 as well as the standard, clinically used CA
inhibitors acetazolamide AZA, methazolamide MZA,
ethoxzolamide EZA, dichlorophenamide DCP, and
indisulam IND, are shown in Table 1. Inhibition data

2362
L. Puccetti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2359–2364
Table 1. Inhibition data for sulfonamides 1–13 investigated in the present paper and standard sulfonamide CA inhibitors, against isozymes I, II, and
IX
Inhibitor
R2
KIa (nM)
Selectivity ratio KI (hCA II)/KI (hCA IX)
hCA Ib
hCA IIb
hCA IXc
AZA
MZA
EZA
DCP
IND
1d
—
—
900
780
12
14
25
27
0.48
0.52
—
—
25
8
38
34
50
0.23
0.76
1200
31
—
—
15
24
0.62
1.02
28,000
25,000
21,000
164
300
170
160
46
294
103
33
2d
—
—
1.65
6.49
3d
5d
—
—
34
31
1.35
1.06
6d
109
95
33
30
7d
—
24
1.25
1.20
11a
11b
11c
11d
12a
12b
12c
12d
12e
12f
Me2NCH2CH2
[O(CH2CH2)2N]CH2CH2
Me–[N(CH2CH2)2N]
[O(CH2CH2)2N]
Me2NCH2CH2
[O(CH2CH2)2N]CH2CH2
Me–[N(CH2CH2)2N]
[O(CH2CH2)2N]
PhCH2CH2
105
8.1
180
7.2
290
12
6.7
5.6
4.8
6.7
5.4
4.8
5.0
6.4
5.0
5.2
4.6
6.1
9.5
32.14
1.50
175
233
210
43.28
2.22
3.1
220
10.3
278
640
290
46
45.83
2.06
246
268
43.43
128.00
55.76
10.00
1.65
8.6
355
3.7
308
2-Pyridyl-CH2
4-H2NO2SC6H4CH2CH2
Me2NCH2CH2
12g
13a
10.1
a Errors in the range of5–10% ofthe reported value (from three different assays).
b Human (cloned) isozymes, by the CO2 hydration method.
c Catalytic domain ofhuman, cloned isozyme, by the CO hydration method.
2
d From Ref. 8.
(dichlorophenamide). Thus, it may be observed that the
morpholinyl-N-ethyl as well as phenethyl moieties
substituting the thiourea group ofcompounds 11–13
lead to the best hCA I inhibitors, irrespective ofthe
spacer between the benzenesulfonamide and the substi-
tuted-thioureido moieties, whereas other substitution
patterns investigated here lead to such less effective
inhibitors; (ii) against the rapid cytosolic isozyme hCA
II, the compounds reported here showed inhibition con-
stants in the range of8.1–640 nM. Again, as for isozyme
I, the simple aromatic sulfonamides 1–3 show moderate
inhibitory properties (KIs in the range of160–300 nM),
which increase with the length ofthe (CH 2)nNH2 moiety
for n from 0 to 2 (Table 1). The same is true for the sul-
fanylated derivatives 5–7, which act as stronger hCA II
inhibitors (KIs in the range of30–46 nM) than the sul-
fonamides 1–3 from which they are derived. For the thio-
ureas 11–13, several substitution patterns, such as
those present in compounds 11b, 11d, 12b, and 12d–f,
lead to a decrease ofthe inhibitory properties as com-
pared to the parent sulfonamides 5–7, as these thioureas
showed KIs in the range of180–640 nM. On the con-
trary, derivatives 11a, 11c, 12a, 12c, 12g, and 13a
showed excellent hCA II inhibitory properties (similar
to those ofthe clinically used sulofnamides shown in
Table 1), with KIs in the range of8.1–46 nM. It may
be seen that the best substitution patterns for obtaining
good hCA II inhibitors include the dimethylaminoethyl-
and N-methyl-piperazine moieties as substituents ofthe
thioureido functionality. It is also important to note that
these are just the moieties that led to ineffective hCA I
inhibitors. Thus, we are for the first time in the presence
ofa series ofcompounds in which we obtained good
hCA I inhibition and ineffective hCA II inhibition for
some representatives (such as 11b, 12b), whereas for
other compounds we obtained just the inverse profile,
with good hCA II inhibition and ineffective hCA I inhi-
bition (e.g., 11a, 11c, 12a, 12c, and 13a). It is difficult to
explain these important differences ofactivity at this
point, and work is in progress in this laboratory for
resolving the X-ray crystal structures ofsome ofthese
derivatives with the two isozymes discussed above; (iii)
the compounds 11–13 reported here showed excellent
hCA IX inhibitory properties, with KIs in the range of
4.6–6.7 nM, being thus much more effective inhibitors
as compared to the parent sulfonamides 5–7 (KIs in
the range of24–34 nM), the simple sulofnamides 1–3
or the clinically used compounds shown in Table 1. It
may be seen that the entire class ofthioureas showed
this compact behavior, similarly to the ABA-thioureido
compounds reported earlier.14 It may be thus stated that
probably the thioureido scaffolds attached to sulfanyl-
ated-aromatic/hetetrocyclic sulfonamide scaffolds lead
to effective and rather selective hCA IX inhibitors, with
many optimal substitution patterns detected; (iv) some
ofthe compounds reported here (such as 11a, 11c,
12a, 12c, and 13a) showed selectivity ratios for inhibit-
ing hCA II over hCA IX in the range of1.20–2.06, being
thus only slightly more effective hCA IX than hCA II
inhibitors. Most ofthe new compounds, including 11b,
11d, 12b, and 12d–g, showed on the other hand much
better selectivity ratios, in the range of10.00–128.00,
being thus much more effective inhibitors ofthe
tumor-associated isozyme over the ubiquitous cytosolic

L. Puccetti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2359–2364
2363
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Scozzafava, A. Curr. Med. Chem.—Cardiovasc. Hematol.
Agents 2004, 2, 49–68.
isozyme hCA II. Still, most ofthese compounds behave
as potent hCA I inhibitors, but the real physiological
role ofthis last isozyme is rather uncertain at this point.
4. Conclusions
5. Pastorekova, S.; Pastorek, J. Cancer-related carbonic
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T. J. Enzyme Inhib. Med. Chem. 2003, 18, 333–338; (b)
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Chem. 2003, 46, 2197–2204.
A series ofaromatic sulofnamides incorporating thio-
ureido-sulfanilyl moieties have been prepared and inves-
tigated for its interaction with the catalytic domain of
the human isozyme hCA IX as well as with the cytosolic
isozymes I and II. The key intermediates in the synthe-
sis were obtained by reacting sulfanilamide, homosulf-
anilamide, or 4-aminoethylbenzenesulfonamide with 4-
acetamido-benzenesulfonyl chloride followed by deacet-
ylation and reaction with thiophosgene. The obtained
isothiocyanato sulfonamides were then reacted with
aliphatic or aromatic primary amines or hydrazines,
leading to the corresponding thioureas. Some ofthese
compounds showed excellent inhibitory properties
against isozymes I, II, and IX, with several inhibitors
also presenting selectivity for the inhibition of CA IX
over that ofthe ubiquitous isozyme CA II. Such sulfon-
amides may constitute interesting candidates for the
development ofnovel antitumor therapies based on
the inhibition ofthe CA isozymes overexpressed in hyp-
oxic tumors. Clear renal cell carcinoma is the only hu-
man malignancy sharing both the highest CA IX
expression and chemo/radiotherapy resistance.20 To
date, partial treatment response is achieved only with
immunotherapy. Furthermore, CA IX is a tumor-associ-
ated antigen only in renal cell carcinoma. As a conse-
quence, the present and previous results from our
group attempt a new approach to be added to the main-
stay ofrenal cell carcinoma therapy, based on inhibition
ofCA IX.
Acknowledgments
12. Supuran, C. T. Expert Opin. Invest. Drugs 2003, 12, 283–
287.
This research was financed in part by a grant ofthe 6th
Framework Programme ofthe European Union (EUR-
OXY project) and by a grant from Miroglio Vestebene
S.p.A Via Santa Barbara 11 12051 (Alba, Cuneo, Italy).
J.Y.W. is grateful to CSGI, University of Florence and
University ofMontpellier II ofr a travel grant to
Florence.
13. (a) Casini, A.; Scozzafava, A.; Mincione, F.; Menabuoni,
L.; Ilies, M. A.; Supuran, C. T. J. Med. Chem. 2000, 43,
4884–4892; (b) Casini, A.; Scozzafava, A.; Mincione, F.;
Menabuoni, L.; Supuran, C. T. J. Enzyme Inhib. Med.
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14. Cecchi, A.; Winum, J.-Y.; Innocenti, A.; Vullo, D.;
Montero, J.-L.; Scozzafava, A.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2004, 14, 5775–5780.
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A. Eur. J. Med. Chem. 1998, 33, 739–752.
16. Reaction ofthe isothiocyanates 8–10 with amines or
hydrazines R2NH2 has been done in diethylether, water or
N,N-dimethylacetamide as solvents, as previously
described.14
References and notes
ˇ
1. Svastova, E.; Hulikova, A.; Rafajova, M.; ZatÕovicova,
M.; Gibadulinova, A.; Casini, A.; Cecchi, A.; Scozzafava,
A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S. FEBS
Lett. 2004, 577, 439–445.
2. Carbonic Anhydrase—Its Inhibitors and Activators; Supu-
ran, C. T., Scozzafava, A., Conway, J., Eds.; CRC (Taylor
and Francis Group): Boca Raton, FL, 2004; pp 1–363,
and references cited therein.
3. (a) Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Pat.
2000, 10, 575–600; (b) Supuran, C. T.; Scozzafava, A.
Expert Opin. Ther. Pat. 2002, 12, 217–242; (c) Supuran, C.
T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23,
1-[4-N-(2-(4-Sulfamoylphenyl)-phenylethyl)-sulfamoyl-
phenyl]-3-(2-dimethylaminoethyl)-thiourea 13a: mp 108–
110 (EtOH–MeOH 1.1); H NMR (DMSO-d6, 400 MHz)
d 2.30 (s, 6H, Me2N), 2.57 (t, J = 5.8 Hz, 2H, CH2NMe2);
2.77 (t, J = 6.6 Hz, 2H, C6H4CH2); 2.98 (t, J = 6.3 Hz, 2H,
CH2NHSO2); 3.60 (m, 2H, CSNHCH2); 7.33 (s, 2H,
SO2NH2); 7.42–7.95 (m, 8H, ArH from 2C6H4 moieties);
8.21 (s, 1H, SO2NH). Elem. anal., found: C, 47.12; H,
1

2364
L. Puccetti et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2359–2364
5.33; N, 14.27; C19H27N5O4S3 requires: C, 46.99; H, 5.60;
N, 14.42.
17. Casini, A.; Abbate, F.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2003, 13, 2759–2763.
18. Abbate, F.; Casini, A.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2004, 14, 2357–2361.
19. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti,
F.; Mincione, G.; Supuran, C. T. J. Med. Chem. 1999, 42,
2641–2650.
20. Puccetti, L.; Manetti, R.; Parrochi, P.; Piccinni, M. P.;
Mavilia, C.; Carini, M.; Romagnani, S.; Maggi, E. Int. J.
Cancer 2002, 98(5), 674–681.