Sulfonamides incorporating 1,3,5-triazine moieties selectively and potently inhibit carbonic anhydrase transmembrane isoforms IX, XII and XIV over cytosolic isoforms i and II: Solution and X-ray crystallographic studies

Article abstract of DOI:10.1016/j.bmc.2011.04.005

Reaction of cyanuryl chloride with d,l-amino acids and amino alcohols afforded a new series of triazinyl-substituted benzenesulfonamides incorporating amino acyl/hydroxyalkyl-amino moieties. Inhibition studies of physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA I, II, IX, XII and XIV with these compounds are reported. They showed moderate-weak inhibition of the cytosolic, offtarget isozymes CA I and II, but many of them were low nanomolar inhibitors of the transmembrane, tumor-associated CA IX and XII (and also of CA XIV). The X-ray crystal structure of two of these compounds in adduct with CA II allowed us to understand the features associated with this strong inhibitory properties and possibly also their selectivity. Two of these compounds were also investigated for the inhibition of other human isoforms, that is, hCA IV, VA, VB, VI, VII and XIII, as well as inhibitors of the fungal pathogenic CAs Nce103 (Candida albicans) and Can2 (Cryptococcus neoformans), showing interesting activity. The 1,3,5-triazinyl-substituted benzenesulfonamides constitute thus a class of compounds with great potential for obtaining inhibitors targeting both α-class mammalian, tumor-associated, and β-class from pathogenic organisms CAs.

Full text of DOI:10.1016/j.bmc.2011.04.005

Bioorganic & Medicinal Chemistry 19 (2011) 3105–3119
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Sulfonamides incorporating 1,3,5-triazine moieties selectively and potently
inhibit carbonic anhydrase transmembrane isoforms IX, XII and XIV over
cytosolic isoforms I and II: Solution and X-ray crystallographic studies q
Fabrizio Carta a, , Vladimir Garaj a,b, , Alfonso Maresca a, Jason Wagner c, Balendu Sankara Avvaru c,
a,
Arthur H. Robbins c, Andrea Scozzafava a, Robert McKenna c, , Claudiu T. Supuran
⇑
⇑
a Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University, Odbojarov 10, SK-831 04 Bratislava, Slovak Republic
c Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville, FL 32610, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Reaction of cyanuryl chloride with D,L-amino acids and amino alcohols afforded a new series of triazinyl-
Received 3 March 2011
Revised 31 March 2011
Accepted 1 April 2011
Available online 6 April 2011
substituted benzenesulfonamides incorporating amino acyl/hydroxyalkyl-amino moieties. Inhibition
studies of physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA I, II,
IX, XII and XIV with these compounds are reported. They showed moderate-weak inhibition of the cyto-
solic, offtarget isozymes CA I and II, but many of them were low nanomolar inhibitors of the transmem-
brane, tumor-associated CA IX and XII (and also of CA XIV). The X-ray crystal structure of two of these
compounds in adduct with CA II allowed us to understand the features associated with this strong inhib-
itory properties and possibly also their selectivity. Two of these compounds were also investigated for the
inhibition of other human isoforms, that is, hCA IV, VA, VB, VI, VII and XIII, as well as inhibitors of the
fungal pathogenic CAs Nce103 (Candida albicans) and Can2 (Cryptococcus neoformans), showing interest-
ing activity. The 1,3,5-triazinyl-substituted benzenesulfonamides constitute thus a class of compounds
Keywords:
Carbonic anhydrase
Isoform IX, XII
Sulfonamide
1,3,5-Triazine
X-ray crystallography
Isoform-selective inhibitor
with great potential for obtaining inhibitors targeting both
a
-class mammalian, tumor-associated, and
b-class from pathogenic organisms CAs.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
106–120 nM, whereas CA IX was inhibited in the subnanomolar
range (KI of 0.15 nM), with a selectivity ratio for inhibiting the tu-
mor-associated isoform over the physiologically dominant one CA
II of greater than 700, the highest ever observed for a sulfonamide
inhibitor.1a However its close congeners, 2 and 3, differing by only
one or two extra CH2 moieties, respectively, compared to 1, were
much weaker CA IX inhibitors (KIs of 124–138 nM) but their
efficacy for CA I and II was enhanced compared to 1 (KIs of
13–21 nM against CA II and of 75–136 nM against CA I);1a (ii) the
different reactivity and possibility to sequentially replace one or
two chlorine atoms from the scaffolds of 1–3, by reaction with
various nucleophiles, affords a methodology to prepare large and
chemically diverse classes of CA inhibitors (CAIs), by means of a
facile and straightforward chemistry.1,2
In previous studies from this group1 the synthesis and carbonic
anhydrase (CA, EC 4.2.1.1) inhibition with benzenesulfonamides
incorporating 1,3,5-triazine moieties of types 1–3 have been inves-
tigated.1 Such compounds have been prepared from cyanuric chlo-
ride (2,4,6-trichloro-1,3,5-triazine) and aromatic sulfonamides, by
a procedure already reported in 1959 by D’Alelio and White.2
Although this chemistry is very simple, several interesting facts
emerged regarding compounds 1–3: (i) these derivatives showed
effective inhibition of several CA isoforms, of the 16 presently
known in mammals,3 such as the cytosolic CA I and II, and the
transmembrane, tumor-associated CA IX.3–5 For example,
inhibited CA I and II with inhibition constants in the range of
1
Cl
N
q
Coordinates and structure factors have been deposited in the Protein Data Bank
as entries; compound 7, 3MMF, and compound 11, 3MNA.
(CH2)n
SO 2NH2
N
⇑
Corresponding author. Tel.: +1 352 392 5696; fax: +1 352 392 3422 (R.M.);
N
tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S).
E-mail addresses: rmckenna@ufl.edu (R. McKenna), claudiu.supuran@unifi.it
(C.T. Supuran).
Cl
1-3
n = 0, 1, 2
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.005

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F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
Previous studies have examined the reactivity of sulfonamides
inhibition studies have been expanded to account for two trans-
membrane isoforms, CA XII and XIV, and report the X-ray crystal
structures of two adduct of these new sulfonamides with hCA II,
which afford new insights in the drug design of CAIs targeting var-
ious isoforms.
1–3 towards alcohols,1a phenols,1a primary and secondary aliphatic
amines,1a,b ammonia,1b hydrazine,1b as well as a small number of
amino acids and their derivatives (i.e., the amino acid esters of gly-
cine and b-alanine).1b Similar to the leads 1–3, the compounds ob-
tained by the above-mentioned reactions showed very interesting
properties as CAIs, against the tested isoforms, that is, hCA I and II
(h = human isoform) and hCA IX.1 Indeed, many such compounds
were low nanomolar hCA IX inhibitors whereas their affinity for
the cytosolic offtarget isozymes I and II was low, making them CA
IX-selective inhibitors, with selectivity ratios in the range of be-
tween 10 and 700.1 As CA IX was recently shown to be a promising
antitumor target for the development of therapeutic and imaging
anticancer agents, by us and other groups,3–10 it appeared of inter-
est to prepare more members belonging to this family of com-
pounds and investigate their binding to all the transmembrane CA
isoforms, in addition to CA IX. Indeed, similar to CA IX, CA XII10–13
and XIV10,14 are transmembrane isoforms with an extracellular ac-
tive site, and they share a certain degree of homology with CA IX.10–14
CA XII is also present in many tumors, like CA IX, whereas CA XIV is
not associated with cancers but is widespread in many tissues such
as the kidneys, liver and brain among others.10 The precise function
of these isozymes in all the tissues in which they are present is
poorly understood,10–14 except for tumors,5,6 in which it has been
demonstrated that they participate in the process of pH regulation
during tumorigenesis. In fact all hypoxic tumors overexpress CA
IX5–8 (and many of them also CA XII)5 as a consequence of the hy-
poxia inducible factor-1 (HIF-1) regulatory pathway.7 The overall
consequence of the strong CA IX/XII over-expression is the pH
imbalance of the tumor tissue, with most hypoxic tumors having
acidic pHe values around 6.5, in contrast to the normal tissue which
has characteristic pHe values around 7.4.5–9 Expression of CA IX is
strongly increased in many types of solid tumors, such as glio-
mas/ependymomas, mesotheliomas, papillary/follicular carcino-
mas, as well as carcinomas of the bladder, uterine cervix, kidneys,
esophagus, lungs, head and neck, breast, brain, vulva, and squa-
mous/basal cell carcinomas, among others.5–9,3b CA IX/XII inhibi-
tion with sulfonamides was recently shown to reverse the effect
of tumor acidification,5 leading to inhibition of the primary tumor
and metastases growth, and CA IX/XII have been proposed as novel
therapeutic antitumor targets.3,5,7,8
2. Results and discussion
2.1. Chemistry
The rationale for obtaining novel sulfonamide CAIs based on the
1,3,5-triazine scaffold reported in this paper, was that of incorpo-
rating moieties which may induce an enhanced hydrophilic char-
acter, due to the fact that 1–3 and some of the compounds
reported earlier,1 showed poor water solubility.1 Some
a- and b-
amino acids, aminoalcohols or polyols seemed to be a good option,
as they generally induce favorable such properties, and in the pre-
vious communication1b we showed that three such compounds
(the glycine, glycine methyl ester and b-alanine derivatives of 3)
possess excellent enzyme inhibitory properties against the tu-
mor-associated isoform CA IX. Thus, these findings extend previous
studies, and report here the synthesis of a larger series of such
compounds, obtained by reaction of the dichloro-substituted tria-
zines 1–31 with one or two equivalents of nitrogen or oxygen
nucleophiles, such as a- and b-amino acids and their esters, amino-
alcohols as well as a monosilylated derivative of ethyleneglycol
(Scheme 1). These nucleophiles were chosen in such a way as to
contain moieties which may enhance water solubility and affinity
to the enzyme for the new CAIs, and amino acids as well as amino
alcohols seem to be an interesting choice.
Reaction of the key intermediate sulfonamides 1–3, possessing
two reactive chlorine atoms at the 1,3,5-triazine ring with these
nucleophiles (R1H, R2H), in the molar ratio of 1:1, afforded by
the replacement of one chlorine atom, the formation of a rather
large series of mono-substituted derivatives (5, 7, 9–13 and
16–21) (Scheme 1). When working in molar ratios between the
dichlorotriazines 1–3 and the nucleophile of 1:2, both chlorine
atoms from 1–3 were replaced, leading to the disubstituted com-
pounds 4, 6, 8, 14, 15, 22 and 23 (Scheme 1). The stepwise replace-
ment of one, two or three chlorine atoms from cyanuryl chloride by
means of aromatic nucleophilic substitution reactions was already
documentated in the 50s.2 The nucleophiles R1H, R2H employed in
our syntheses included amino acids such as Gly, b-Ala, Ser, DOPA;
amino acid esters, such as the methyl esters of Gly and Ala; amino-
alcohols such as 2-aminoethanol and 4-amino-1-butanol, as well
as the mono-tert-butyl-dimethylsilyl derivative of ethyleneglycol
(Table 1). All chiral amino acids were racemic mixtures. They were
Reported here are novel sulfonamide CAIs obtained considering
compounds 1–3 as leads, which incorporate the 1,3,5-triazine moi-
ety, connected to potent and selective inhibition of the transmem-
brane, tumor-associated isoforms CA IX over the cytosolic CA I and
II. The synthesis of derivatives which contain amino acyl and ami-
no alcohol moieties in their molecules, due to the enhanced hydro-
philicity of such derivatives, has been explored. Furthermore, the
Cl
N
R1
N
R1H
(CH2)n
SO2NH2
N
(CH2)n
SO2NH2
N
DIPEA
DMF
N
N
Cl
Cl
5, 7, 9-13, 16-21
1-3
DIPEA
DMF
R2H
R1
N
N
(CH2)n
SO2NH2
N
R2
4, 6, 8, 14, 15, 22, 23
Scheme 1. Preparation of sulfonamides 4–23 by reaction of the dichloro-triazines 1–3 with nucleophiles (R1H, R2H) in the presence of diisopropylethylamine (DIPEA), in
DMF.

F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
3107
Table 1
Inhibition data of human CA isoforms hCA I, II, IX, XII and XIV with sulfonamides 1–24 and standard sulfonamide inhibitors by a stopped flow CO2 hydrase assay15
R1
N
(CH2)n
SO 2NH2
N
N
R2
1-23
No.
n
R1
R2
KI* (nM)
hCA I
hCA II
hCA IX
hCA XII
hCA XIV
1
2
3
4
5
6
7
8
0
1
2
0
0
0
0
0
0
0
0
0
0
1
1
1
1
2
2
2
2
2
2
—
—
—
—
Cl
Cl
Cl
Cl
Cl
Cl
120a
136a
75a
4550
3032
502
3075
673
1324
109
106a
13a
21a
376
351
435
41
368
561
412
37
119
356
453
435
258
475
32a
33a
29a
371
409
517
12c
12
0.15a
124a
138a
8.4
7.9
9.4
0.35
56.2
43.8
6.0
10.2
8.7
6.7
0.85
5.8
8.5
1.6
43.9
248
9.3
9.2
20.7
80
2.6
2.9
3.5
82
8.6
7.1
1.5
5.7
22
34.3
24.1
15.4
7.2
9.3
7.0
NHCH2COOH
NHCH2COOH
NHCH2COOMe
NHCH2COOMe
NH(CH2)2COOH
NHCHMeCOOH
NHCH(CH2OH)–COOH
NH(CH2)2OH
NH(CH2)4OH
O(CH2)2OSi(Me2)-t-Bu
NHCH2COOMe
NHCHMeCOOH
NH(CH2)2COOH
NHCH[CH2C6H3(OH)2]–COOH
NHCH2COOH
NHCH2COOMe
NH(CH2)2COOH
NHCH[CH2C6H3(OH)2]–COOH
NHCHMeCOOH
NHCH(CH2OH)COOH
—
NHCH2COOH
Cl
NHCH2COOMe
Cl
NH(CH2)2COOH
Cl
Cl
Cl
8.2
8.9
6.5
0.92
10.1
7.4
9
0.96
8.9
0.75
34.8
113
9.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
AAZb
EZAb
DCPb
1098
1245
3472
607
3.8
Cl
Cl
27.7
583
9.1
8.9
16.9
72
10.4
23.5
17.9
77
8.8
7.6
NHCH2COOMe
NHCHMeCOOH
Cl
Cl
Cl
Cl
Cl
Cl
NHCHMeCOOH
NHCH(CH2OH)COOH
—
—
—
—
1659
2360
3021
33a
8.5
34.1
95
1.0a
1.4a
1.7a
91
39a
35a
1549
2040
3704
7c
250
25
8.4
9.0
1.3
25
34
50
4.1
41
2.5
345
—
—
—
8
38
1200
50
a
b
c
From Ref. 1.
From Ref. 3a.
From Ref. 20.
*
Mean from three different assay, by a stopped flow technique (errors were in the range of 5–10% of the reported values).
chosen in such a way as to afford supplementary insights regarding
the structure–activity relationship (SAR) features of this novel fam-
ily of CAIs.
(DCP).15 The isoforms included in the study were the cytosolic
hCA I and II as well as the transmembrane hCA IX, XII and XIV,
all of which are involved in a host of important physiological and
pathological functions in vertebrates.3–10
The following structure–activity relationship (SAR) can be ob-
servedfromdataofTable1, forthe inhibitionofthe CAisoformsinves-
tigated here with the new group of sulfonamides1–23 reported here:
(i) The slow cytosolic isoform hCA I was inhibited by com-
pounds 1–23 with KIs in the range of 33–4550 nM. Most of these
compounds were ineffective as hCA I inhibitors, except for the pre-
viously reported derivatives 18–20 which incorporate the amino-
ethylbenzenesulfonamide moiety (n = 2) and one Gly, GlyOMe or
b-Ala moieties, which had KIs in the range of 33–39 nM, compara-
ble to that of EZA (KI of 25 nM). Medium efficacy as hCA I inhibitors
has been observed for the leads 1–3 and for compound 10, which
showed KIs in the range of 75–136 nM. The remaining compounds
had inhibition constants >500 nM, being thus rather inefficient
hCA I inhibitors (Table 1).
N
N
N
CH3CONH
SO 2NH2
S
EtO
S
SO 2NH2
AAZ
EZA
SO 2NH2
Cl
SO 2NH2
Cl
DCP
(ii) The investigated sulfonamides inhibited hCA II, the physio-
logically dominant and highly active cytosolic isoform,3 with KIs in
the range of 13–517 nM. Several compounds, among which 2, 3, 7,
11 and 18–20 showed effective hCA II inhibition. Similar to the
clinically used derivatives AAZ, EZA and DCP, these compounds
inhibited this isoforms with inhibition constants of 13–41 nM. In
2.2. CA inhibition
The inhibition of five CA isoforms has been investigated with
derivatives 1–23 and clinically used, standard CAIs such as aceta-
zolamide (AAZ), ethoxzolamide (EZA) and dichlorophenamide

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F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
Table 2
addition to the leads 2 and 3, which contain two chlorine atoms on
the 1,3,5-triazine ring, the other effective hCA II inhibitors possess
one chlorine atom and one Gly, GlyOMe or b-Ala moieties in their
molecule. The compounds with a longer spacer (n = 2) were more
effective as hCA II inhibitors compared to the ones incorporating
shorter such linkers (n = 0 or 1). It may be also observed that the
presence of two amino acyl moieties on the triazine ring leads to
less effective inhibitors compared to the compounds with only
one such moiety (compare 6 and 7 for example). The SAR is in fact
not very regular, although generally, for the same/similar substitu-
tion pattern on the triazine ring, the longer spacer compound
(n = 2) was more effective an inhibitor than the compound with
the intermediate spacer (n = 1) which in turn was more effective
than the short spacer derivative (n = 0). Rather bulky amino acid
residues (e.g., DOPA, Ser, etc), or two such groups in the molecule,
also led to less effective hCA II inhibitors compared to compounds
possessing more compact, one such moiety. The short aminoalco-
hol derivative 11 was also more effective than the bulkier com-
pounds 12 (with a longer spacer between the amino and OH
moieties compared to 11) and 13 (incorporating the bulky tert-bu-
tyl-dimethylsilyloxyethyloxy moiety).
(iii) The tumor-associated isoform hCA IX was inhibited by
compounds 1–23 with KIs in the range of 0.15–138 nM (Table 1).
Several subnanomolar and low nanomolar inhibitors of this impor-
tant drug target have been evidenced, in addition to 1, reported
earlier.1 They are: 9, 11 (KIs <1 nM) and 4–8, 10, 14, 15, 18–20,
22 and 23 (KIs in the range of 1.0–9.4 nM). The least effective inhib-
itors were 2, 3, 13, 17 and 21, with KIs in the range of 91–138 nM.
These observations therefore indicate that the sulfonamides incor-
porating the 1,3,5-triazinyl moiety lead to highly effective hCA IX
inhibitors, for a rather large number of substitution patterns. In
the series of reported compounds, the best inhibitors were those
incorporating sulfanilamide (n = 0), one chlorine as substituent of
the triazine moiety and either one Ala or aminoethanol moieties
as the second substituent of the triazine ring (compounds 9 and
11). Highly effective were also the Gly, GlyOMe or b-Ala monode-
rivatives (18–20) with the longer spacer (n = 2) in their molecules,
whereas the loss of activity was correlated with the presence of
bulkier moieties substituting the 1,3,5-triazine ring (as in 13, 17
and 21). It is interesting to note (but rather difficult to explain) that
even if leads 2 and 3 are rather ineffective as hCA IX inhibitors, all
substitutions done in these compounds by replacing one or both
chlorine atoms with amino acyl or amino acyl ester moieties, led
to compounds with enhanced hCA IX inhibitory properties. On
the other hand, not the same can be said on the lead 1. All substi-
tution done on this compound led to less effective hCA IX inhibi-
tors, but the loss of activity was (with few exceptions, 12 and 13)
not significant and an important feature of the new derivatives re-
ported here is their enhanced hydrophilicity compared to 1, which
is poorly soluble in most organic solvents and water.1 It may be
also observed that most of the new triazinyl sulfonamides reported
here show much better hCA IX inhibitory effects compared to the
standard inhibitors AAZ, EZA and DCP.
Inhibition profile of isoforms human (h) CA IV, VA, VB, VI, VII and XIII, as well as the
fungal enzyme Nce103 (Candida albicans) and Can2 (Cryptococcus neoformans) with
two of the triazinyl sulfonamides reported in the paper, compounds 5 and 18. AAZ
(standard inhibitor) inhibition data are also shown
Enzyme
KI* (nM)
5
18
AAZ
hCA IV
hCA VA
hCA VB
hCA VI
hCA VII
hCA XIII
Nce103
Can2
89
48
24
59
9.4
62
9.0
5.5
97
65
23
76
8.3
68
9.7
8.2
74
63
54
11
2.5
5.7
132
10.5
*
Mean from three different assay, by a stopped flow technique (errors were in the
range of 5–10% of the reported values).
14–16, 18–20, 22 and 23), except compounds 17 and 21, both
incorporating the bulky DOPA moieties, which were weaker inhib-
itors (KIs of 80–82 nM) compared to the parent sulfonamides from
which they were obtained. Thus, as for hCA IX discussed above,
many of the new triazinyl sulfonamides reported here showed
highly effective hCA XII inhibitory properties.
(v) The inhibition of the third transmembrane isoform, hCA XIV,
was also investigated here for the first time with triazinyl sulfona-
mides. The leads 1–3 showed significant inhibitory activity, with
KIs in the range of 34.3–15.4 nM. There is a rather regular SAR
for these derivatives, as the inhibition of hCA XIV increases with
the increase of the spacer from n = 0 in the sulfanilamide derivative
1, to n = 2 in the aminoethylbenzenesulfonamide derivative 3,
which was more than two times a better hCA XIV inhibitor com-
pared to 1. Most of the derivatives obtained from 1–3 as leads,
were also highly effective hCA XIV inhibitors except for 12, 13,
17 and 21, all incorporating rarher bulky groups, which were the
least effective inhibitors (KIs in the range of 27.7–583 nM). Thus,
the di-b-Ala derivative 8 was a subnanomolar hCA XIV inhibitor
(and the most potent such inhibitor reported to date).3 Effective
inhibition (KIs <10 nM) was observed for many other such deriva-
tives, among which 4–7, 10, 11, 14, 15, 22 and 23. However, the
SAR for the inhibition of this isoform is rather different compared
to that for the inhibition of hCA IX and XII (which, as outlined
above, were in fact quite similar).
(vi) In order to explore the complete inhibition profile of this
class of sulfonamide CAIs, two of the most active compounds
(against the isoforms discussed above), 5 and 18, have been as-
sayed for the inhibition of the remaining six catalytically active hu-
man isoforms (hCA IV–XIII)3 as well as two b-CAs from the
pathogenic fungi Candida albicans (Nce103) and Cryptococcus neo-
formans (Can2).16 These two enzymes play an important role in
the life cycle of these organisms and their inhibition has recently
been proposed as a new approach to design antifunglas.16 Data
of Table 2 show that hCA IV, hCA VA, hCA VB, hCA VI and hCA XIII
are significantly inhibited by these two sulfonamides, with KIs in
the range of 23–97 nM, but one human isoform (hCA VII) as well
as the two fungal b-CAs are even more sensitive to inhibition, with
KIs in the range of 5.5–9.7 nM. Thus, this family of sulfonamide
CAIs may have potential to also design inhibitors of b-CAs,
enzymes quite widespread in many pathogenic fungi and
bacteria, such as among others Helicobacter pylori, Mycobacterium
tuberculosis, Haemophilus influenzae, Brucella suis, Streptococcus
pneumoniae.3,16,17
(iv) The inhibition of the second isoform associated to tumors,
hCA XII, has not been investigated earlier with this family of deriv-
atives. It may be observed from data of Table 1, that similar to hCA
IX, the lead 1 was a highly effective hCA XII inhibitor (KI of
0.35 nM) whereas its close congeners with longer spacers 2 and
3 (n = 1 and 2) were much less effective, with KIs in the range of
43.8–56.2 nM. The substitution of the chlorine atom(s) from 1, in
compounds such as 4–12 (n = 0) led to a modest loss of hCA XII
inhibitory activity for derivatives 4–11 (KIs of 0.85–10.2 nM) and
to a more important loss for the bulkier derivatives 12 and 13
(KIs of 43.9–248 nM). As for hCA IX, in the case of the leads 2
and 3, all substitution patterns explored here led to more effective
hCA XII inhibitors (KIs in the range of 2.6–9.3 nM for compounds
2.3. X-ray crystallography
In order to rationalize inhibition of CAs with this series of com-
pounds, X-ray crystallographic studies were performed on adducts

F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
3109
Table 3
O
O
SO 2NH2
Data and final model statistics for the hCA II adducts with compounds 11 and 7
S
N
H
N
Data-collection statistics
H
O
PDB Accession number
Compound
3MMF
11
3MNA
7
24
Temperature (K)
Wavelength (Å)
Space group
100
1.5418
P21
100
1.5418
P21
Unit-cell parameters (Å,°)
a = 42.34
b = 41.24
c = 72.05
b = 104.31,
42.36
41.23
72.02
b = 104.21,
a
=
c
= 90
a = c = 90
Total number of possible reflections
Total number of unique reflections
Resolution (Å)
38930
38628
38929
38812
(a)
23.8–1.5
(1.55–1.5)*
6.4% (34.1%)
15.8 (3.4)
99.2 (97.1)
3.6 (3.3)
25.3–1.5
(1.55–1.5)
7.1 (31.6)
15.0 (4.5)
99.7 (99.0)
4.0 (3.7)
Q 92
N 67
Rsym
I/r(I)
Completeness
Redundancy
H 119
H 94
F 131
Zn
Final model statistics
aRcryst (%)
15.9
18.2
4–261
2086
14.5
17.0
4–261
2078
bRfree (%)
H 96
N62
Residue Nos.
No. of protein atoms (including alternate
conformations)
L 198
T 200
Bulk solvent
T 199
Zn
No. of drug atoms
22
312
0.010, 1.4
88.9
10.6
0.5
28
340
0.009, 1.3
88.0
11.6
0.5
No. of H2O molecules
R.m.s.d. for bond lengths (Å), angles (°)
Ramachandran statistics (%)
Most favored, additionally allowed and
generously allowed regions
B factors (Å2)
(b)
H 119
Q 92
N 67
H 94
Average, main-, side-chain, compound,
solvent
16.6, 21.2,
21.2, 31.8
13.2, 17.9,
19.9,29.5
F 131
c Rfree is calculated in same manner as bRcryst, except that it uses 5% of the reflection
data omitted from refinement.
a
Rsym = (
Rcryst = (
R
R
|I ꢁ <I>|/
|Fo| ꢁ |Fc|/
Values in parenthesis represent highest resolution bin.
R
<I>) ꢂ 100.
H 96
b
R
|Fobs|) ꢂ 100.
N 62
*
L 198
T 200
Bulk solvent
T 199
of hCA II with two such compounds which showed good affinity for
this isoform, that is, compounds 7 and 11 (KIs of 41 and 37 nM,
respectively, Table 1). The final model statistics for the hCA II ad-
ducts with compounds 7 and 11 are shown in Table 3.
Figure 1. Stick representation of compound (a) 11 (green) and (b) 7 (yellow) bound
in the active site of hCA II. The electron density is represented by a 2 -weighted
r
2Fo ꢁ Fc Fourier map (blue mesh). Amino acids are as labeled. Figure made using
The electron density of all moieties from inhibitors 7 and 11
was clearly seen in the two adducts with the enzyme (Fig. 1). In
the refined models sulfonamides 7 and 11 bind to the zinc ion in
the active site through the N1 atom of the sulfonamide moiety at
distances of ꢀ2.0 Å. As in other published hCA II-sulfonamide com-
plexes,17–20 additional hydrogen bonds from Thr199 N and OG to
the sulfonamide oxygen atoms are present. The inhibitors are both
rigid molecules, and their C10 extensions make no hydrophobic
contacts with the enzyme (Figs. 1 and 2). The chlorine atom of
compounds 7 and 11 contact the side-chain atoms of Ile91 and
Gln92, in addition to making hydrogen bonds with water mole-
cules (Fig. 2). The triazine ring of compounds 7 and 11 stacks upon
the aromatic carbon atoms of the Phe131 phenyl ring. Both com-
pounds are significantly non-planar, possibly due to the steric con-
tact between the benzene and triazine rings across the C4–N7–C8
valence angle. The twist can be described by two torsion angles:
C(5)–C(4)–N(7)–C(8), 161.3° (compound 11) and 156.2° (com-
pound 7), and C(4)–N(7)–C(8)–N(9), ꢁ178.9° and 175.8°, respec-
tively. Neither of the torsion angle C9–C10–N11–C15 deviates by
more than 13° from the triazine ring. Both terminal C15 substitu-
ents are approximately normal to the planes of the triazine ring.
The electron density of the C15 terminal substituent of compounds
7 or 11 are weak and neither interact directly with protein atoms
lining the active site, but rather an array of ordered water mole-
cules. The terminal alcohol oxygen atom in compound 11 is located
PyMOL (DeLano Scientific).
Figure 2. Schematic 2D-representation of hCA II—compound (a) 11 and (b) 7
interactions when bound to the enzyme. Hydrophobic contacts are indicated by red
hash marks. H-bond lengths and the zinc coordination (Å) are indicated by black
dashed lines. Atoms are represented as spheres: zinc, grey; carbon, black; oxygen,
red; nitrogen, blue; sulfur, yellow; chlorine, green). Figure made using Ligplot.

3110
F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
the triazinyl derivative 11 and the tosylureido sulfanilamide 24
(Fig. 4), one may observed that the sulfanilamide fragment of the
two inhibitors are totally superposable when bound to the enzyme
active site. However, the triazinyl-substituted tail from 11 and the
tosylureido one from 24, are orientated towards opposite parts of
the active site. Thus, we hypothesize that these two very different
binding modes of the tails fragments of the two inhibitors are
responsible for the important difference in selectivity for the inhi-
bition of the cytosolic over transmembrane isoforms with these
types of CAIs. The triazinyl portion of the molecule, through the
variable twisting evidenced above for the two adducts described
here, allows for an orientation of the inhibitor molecule in regions
of the active site where no other classes of sulfonamides have been
observed earlier.1,12,13
3. Conclusions
Figure 3. View of compound 7 and 11 superposed in the active site of hCA II. hCA II
is depicted as a surface representation (oxygen, red; nitrogen, blue; carbon, yellow).
Compounds 11 (green) and 7 (yellow) are represented as sticks. Atoms of inhibitor
molecules are colored as in Figure 1. Figure made using PyMOL (DeLano Scientific).
A new series of triazinyl-substituted benzenesulfonamides
incorporating amino acyl moieties are reported, together with inhi-
bition studies of physiologically relevant CA isoforms, such as CA I,
II, IX, XII and XIV. These compounds showed moderate-weak inhi-
bition of the cytosolic, offtarget isozymes CA I and II, but many of
them were low nanomolar inhibitors of the transmembrane, tu-
mor-associated CA IX and XII (and also of CA XIV). The X-ray crystal
structure of two of these compounds in adduct with hCA II allowed
for observing structural features associated with this strong inhib-
itory properties and possibly also their selectivity. Two of these
compounds were also investigated for the inhibition of other hu-
man isoforms, that is, hCA IV, VA, VB, VI, VII and XIII, as well as
inhibitors of the fungal pathogenic CAs Nce103 (Candida albicans)
and Can2 (Cryptococcus neoformans), showing interesting activity.
The 1,3,5-triazinyl-substituted benzenesulfonamides constitute
thus a class of compounds with great potential for obtaining inhib-
itors targeting both mammalian, tumor-associated and pathogenic
organisms b-class CAs.
approximately in the position of the methyl ester of compound 7
(Fig. 3).
In order to rationalize why the triazinyl-substituted benzene-
sulfonamides as an entire class show such strong CA inhibitory
properties against the physiologically dominant isoforms (e.g., CA
II, IX and XII) and in some cases also selectivity for inhibiting trans-
membrane over cytosolic isoforms, we include in our analysis sul-
fonamide 24,20 the tosylureido derivative of sulfanilamide. The
synthesis and hCA I, II and IV inhibition data with this compound
have been reported earlier,20 and alsoits X-ray crystal structure
in adduct with CA II is available (PDB file 1ZFK). Similar to the tri-
azinyl derivatives investigated here, such as among others 7 and
11, compound 24 possesses the 4-amino-benzenesulfonamide
scaffold to which a diverse tail is attached compared to the triazi-
nyl moieties. Furthermore, 24 is a very strong inhibitor of all CA
isoforms investigated here, hCA I, II, IX, XII and XIV, with inhibition
constants in the range of 1.3–12 nM. Thus, the main difference be-
tween the triazinyl substituted compounds 7 and 11 compared to
24, is that the latter demonstrated no significant selectivity for
inhibiting the transmembrane over the cytosolic isoforms. Com-
paring the X-ray crystal structures of the adducts of hCA II with
4. Experimental protocols
4.1. Chemistry
Anhydrous solvents and all reagents were purchased from
Sigma–Aldrich, Alfa Aesar and TCI. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen
atmosphere using dried glassware and syringes techniques to
transfer solutions. NaH 60% in oil dispersion was washed with
n-hexane until a homogeneous white solid was obtained, dried
and stored under a nitrogen atmosphere prior to use. Infrared
(IR) spectra were recorded as KBr plates and are expressed in
m
(cmꢁ1). Nuclear magnetic resonance (1H NMR, 13C NMR, DEPT-
135, DEPT-90, HSQC, HMBC) spectra were recorded using a Bruker
Advance III 400 MHz spectrometer in CDCl3, MeOH-d4 or in
DMSO-d6. Chemical shifts are reported in parts per million
(ppm) and the coupling constants (J) are expressed in hertz
(Hz). Splitting patterns are designated as follows: s, singlet; d,
doublet; sept, septet; t, triplet; q, quadruplet; m, multiplet; br s,
broad singlet; dd, double of doubles, appt, apparent triplet, appq,
apparent quartet. The assignment of exchangeable protons (OH
and NH) was confirmed by the addition of D2O. Analytical thin-
layer chromatography (TLC) was carried out on Merck silica gel
F-254 plates. Flash chromatography purifications were performed
on Merck Silica Gel 60 (230–400 mesh ASTM) as the stationary
phase and ethylacetate/n-hexane or MeOH/DCM were used as elu-
ents. Melting points (mp) were carried out in open capillary tubes
and are uncorrected.
Figure 4. View of compound 11 and 24 (PDB file 1zfk) superposed within the active
site of hCA II. Figure made using PyMOL (DeLano Scientific).

F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
3111
4.1.1. Synthesis of 4-(40,60-dichloro-10,30,50-triazin-20-ylamino)-
benzenesulfonamide 11,2
4-[(40,60-Dichloro-10,30,50-triazin-20-ylamino)methyl]benzene-
sulfonamide 2: mp 210–221 °C; dH (400 MHz, DMSO-d6) 4.63 (2H,
d, J 6.2, 5-H2), 7.38 (2H, s, SO2NH2, exchange with D2O), 7.51 (2H, d,
2
1
SO2NH2
3
4
SO2NH2
Cl
HN
NaOH 1.7 M aq.
Acetone
2'
N
N
N
N
4'
6'
Cl
N
Cl
Cl
N
Cl
NH2
1
A
1.0 M solution of 4-aminobenzenesulfanilamide (17.2 g,
J 8.4, 2 ꢂ Ar-H), 7.82 (2H, d, J 8.4, 2 ꢂ Ar-H), 9.69 (1H, t, J 6.2, NH,
exchange with D2O); dC (100 MHz, DMSO-d6) 170.6. 169.7, 166.7
(C-20), 144.0 (ipso), 142.5 (ipso), 128.6, 126.8, 44.6 (C-5).
0.1 mol, 1.0 equiv) in acetone (100 ml) was added dropwise to a
vigorously stirred suspension of cyanuric chloride (18.4 g,
1.0 equiv) in the same solvent (100 ml) at 0 °C. The white slurry
was stirred at the same temperature for 30 min. and then a
1.7 M aqueous solution of NaOH (4.0 g, 1.0 equiv) was added over
4.1.3. Synthesis of 4-[6-(40,60-dichloro-10,30,50-triazin-20-yl-
amino)ethyl]benzenesulfonamide 31
SO2NH2
Cl
3
H
5
4'
2'
4
Cl
N
N
NaOH 1.7 M aq.
2
N
N
6
1
Acetone
N
N
SO2NH2
6'
Cl
N
Cl
Cl
3
NH2
a period of 20 min. Stirring was continued for 1 h, the reaction was
quenched by addition of slush (100 ml) and the solid filtered off.
Crystallization from acetone afforded the title compound 1 as a
white solid.
The reaction was carried out according to the previous proce-
dure using 4-(2-aminoethyl)benzenesulfonamide (20.0 g,
1.0 equiv), cyanuric chloride (18.4 g, 1.0 equiv) and NaOH (4.0 g,
1.0 equiv). Crystallization from acetone afforded the title com-
pound 3 as a white solid.
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide
1: 77% yield; mp 190–193 °C; dH (400 MHz, DMSO-d6) 7.34 (2H, s,
SO2NH2, exchange with D2O), 7.80 (2H, d, J 8.8, 2 ꢂ Ar-H), 7.87 (2H,
d, J 8.8, 2 ꢂ Ar-H); dC (100 MHz, DMSO-d6) 170.3, (C-40). 164.9 (C-
20), 140.9 (ipso), 140.8 (ipso), 127.6, 122.1.
4-[6-(40,60-Dichloro-10,30,50-triazin-20-ylamino)ethyl]benzene-
sulfonamide 3: 63% yield; mp 298–300 °C; dH (400 MHz, DMSO-d6)
2.95 (2H, t, J 6.2, 5-H2), 3.59 (2H, q, J 6.2, 6-H2), 7.34 (2H, s, SO2NH2,
exchange with D2O), 7.46 (2H, d, J 8.4, 2 ꢂ 3-H), 7.78 (2H, d, J 8.4,
2 ꢂ 2-H), 9.26 (1H, t, J 6.2, NH, exchange with D2O); dC (100 MHz,
DMSO-d6) 170.0, 166.2 (C-20), 143.8 (ipso), 143.2 (ipso), 130.2 (C-
3), 126.6 (C-2), 42.7 (C-6), 34.6 (C-5).
4.1.2. Synthesis of 4-[(40,60-dichloro-10,30,50-triazin-20-ylamino)-
methyl]benzenesulfonamide 21a
Cl
4'
SO2NH2
Cl
N
3
2
2'
NaOH 4.0 M aq.
Acetone
N
NH
4
1
N
N
N
SO2NH2
6'
5
Cl
Cl
N
Cl
2
NH3Cl
4.1.4. Synthesis of 200,2000-[60-(4-sulfamoylphenylamino)-10,30,50-
triazine-20,40-diyl]-bis(azanediyl)diacetic acid 4
The reaction was carried out according to the previous proce-
dure using 4-aminomethylbenzenesulfonamide hydrochloride
H
3
H
H
4'
6'
4
HO2C
N
N
O
N
2
1
Cl
N
N
DIPEA
DMF
2"
N
2'
OH
N
N
N
SO2NH2
SO2NH2
NH2
NH
CO2H
Cl
2"'
1
4
(26.9 g, 1.0 equiv), cyanuric chloride (22.3 g, 1.0 equiv) and NaOH
(9.7 g, 2.0 equiv).
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide
1 (0.1 g, 1.0 equiv) and glycine (2.4 equiv) were dissolved in dry

3112
F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
DMF (15 ml) and DIPEA (5.0 equiv) was added. The reaction was
stirred at 60 °C under a nitrogen atmosphere until starting material
was consumed (TLC monitoring). Then the reaction was quenched
with slush and the precipitate formed was collected by filtration,
washed whit H2O and dried under high vacuo to give the title com-
pound as a white solid.
6.30 (1H, br s, exchange with D2O), 7.34 (2H, s, exchange with
D2O, SO2NH2), 7.49 (2H, m, , 2 ꢂ 3-H), 7.80 (2H, m, 2 ꢂ 2-H); dC
(100 MHz, DMSO-d6, 80 °C) 173.4 (C@O), 172.3 (C-40), 168.7 (C-
60), 165.8 (C-20), 144.3 (ipso), 143.5 (ipso), 128.3 (C-3), 126.5 (C-
2), 44.4 (C-5), 42.2 (C-200).
200,2000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]-
bis(azanediyl)diacetic acid 4: 32% yield; mp 304 °C with decompo-
4.1.6. Synthesis of dimethyl 20,2000-[60-(4-sulfamoylphenyl-
amino)-10,30,50-triazine-20,40-diyl]bis(azanediyl)diacetate 6
O
H
H
3
H
4
O
N
N
2
N
Cl
N
N
O
DIPEA
DMF
2'
6'
2"
1
O
N
N
N
N
SO2NH2
4'
SO2NH2
NH2HCl
NH
2"'
Cl
O
O
1
GlyOMe.HCl
6
sition; mmax (KBr) cmꢁ1, 3370, 2838, 1730 (C@O, acid), 1598 (aro-
matic); dH (400 MHz, DMSO-d6, 80 °C) 3.95 (2H, d, J 5.0, 200/2000-
H2), 4.10 (2H, d, J 5.0, 200/2000-H2), 7.20 (2H, s, SO2NH2, exchange with
D2O), 7.30 (2H, d, J 8.2, 2 ꢂ 3-H), 7.80 (2H, d, J 8.2, 2 ꢂ 2-H), 7.90
(2H, m, 2 ꢂ CH2NH, exchange with D2O), 9.82 (1H, br s, NH, ex-
change with D2O); dC (100 MHz, DMSO-d6, 80 °C) 171.0 (C@O),
164.8, 157.0, 143.6, 138.2, 127.2, 120.4, 43.3 (C-200).
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfon-
amide 1 (0.1 g, 1.0 equiv) and methyl 2-aminoacetate hydrochlo-
ride (4.2 equiv) were dissolved in dry DMF (15 ml) and DIPEA
(5.0 equiv) was added. The reaction was treated according to the
procedure reported above to afford the title compound as a white
solid.
Dimethyl 20,2000-[60-(4-sulfamoylphenylamino)-10,30,50-triazine-
20,40-diyl]bis(azanediyl)diacetate 6: 36% yield; mp 254 °C with
decomposition; mmax (KBr) cmꢁ1, 3330, 2820, 1760 (C@O, ester),
1566 (aromatic); dH (400 MHz, DMSO-d6) 3.67 (6H, s, 2 ꢂ CH3),
4.1.5. Synthesis of 200-[40-chloro-60-(4-sulfamoylbenzylamino)-
10,30,50-triazin-20-ylamino]acetic acid 5
2
1
SO2NH
3
2
O
H
H
4
Cl
HO2C
N
N
N
DIPEA
DMF
N
N
SO2NH2
2'
OH
6'
2"
5
N
NH
N
N
4'
NH2
Cl
Cl
2
Gly
5
4-[(40,60-Dichloro-10,30,50-triazin-20-ylamino)methyl]benzene-
sulfonamide 2 (0.1 g, 1.0 equiv) and glycine (2.4 equiv) were dis-
solved in dry DMF (15 ml) and DIPEA (5.0 equiv) was added. The
reaction was stirred under a nitrogen atmosphere at rt until start-
ing material was consumed (TLC monitoring). Then the reaction
was quenched with slush and the precipitate formed was collected
by filtration, washed whit H2O and dried under high vacuo to give
the title compound as a white solid.
4.10 (2H, d, J 5.0, 2 ꢂ 200-H2), 7.20 (2H, s, SO2NH2, exchange with
D2O), 7.30 (2H, d, J 8.2, 2 ꢂ 3-H), 7.80 (2H, d, J 8.2, 2 ꢂ 2-H), 8.90
(2H, m, 2 ꢂ CH2–NH, exchange with D2O), 10.40 (1H, br s, –NH–,
exchange with D2O); dC (100 MHz, DMSO-d6) 172.0, 167.3, 149.9,
139.2, 138.0, 127.6, 121.1, 54.0, 44.2.
4.1.7. Synthesis of methyl 200-(40-chloro-60-(4-sulfamoylphenyl-
amino)-10,30,50-triazin-20-ylamino)acetate 7
O
H
H
H
O
3
Cl
N
N
4
2"
6'
N
2'
N
N
2
1
O
DIPEA
DMF
O
N
N
N
N
SO2NH2
NH2HCl
SO2NH2
4'
Cl
Cl
1
GlyOMe.HCl
7
200-[40-Chloro-60-(4-sulfamoylbenzylamino)-10,30,50-triazin-20-
ylamino]acetic acid 5: 28% yield; mp 226–231 °C;
max (KBr) cmꢁ1
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide
1 (0.4 g, 1.0 equiv) and methyl 2-aminoacetate hydrochloride
(0.17 g, 2.2 equiv) were dissolved in dry DMF (15 ml) and DIPEA
(0.36 g, 4.4 equiv) was added. The reddish solution was stirred
m
,
3365, 2838, 1732 (C@O, acid), 1560 (aromatic); dH (400 MHz,
DMSO-d6, 80 °C) 3.69 (2H, d, J 5.0, 200-H2), 4.50 (2H, d, J 5.0, 5-H2),

F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
3113
O.N. at 90 °C then quenched with a 1.0 M aqueous solution of
hydrochloric acid (40 ml). The precipitate formed was collected
by filtration, washed with H2O and dried under vacuo to afford
the title product as a white solid.
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide
1 (0.1 g, 1.0 equiv) and -alanine (1.2 equiv) were dissolved in dry
D,L
DMF (15 ml) and DIPEA (2.5 equiv) was added. The reaction was
treated according to the procedure reported above to afford the ti-
tle compound as a white solid.
Methyl 200-(40-chloro-60-(4-sulfamoylphenylamino)-10,30,50-tria-
zin-20-ylamino)acetate 7: 35% yield; mp 271–273 °C; mmax (KBr)
D,L
-200-[40-Chloro-60-(4-sulfamoylphenylamino)-10,30,50-triazin-
cmꢁ1
,
3360, 2820, 1762 (C@O, acid), 1559 (aromatic); dH
20-ylamino]propanoic acid 9: 27% yield; mp 322–325 °C; mmax
(KBr) cmꢁ1, 3332, 2850, 1722 (C@O, acid), 1555 (aromatic); dH
(400 MHz, DMSO-d6) 1.40 (3H, d, J 5.4, CH3), 4.40 (1H, br m, 200-
H), 7.20 (2H, d, J 8.2, 2 ꢂ 3-H), 7.80 (2H, d, J 8.2, 2 ꢂ 2-H), 7.90
(2H, s, SO2NH2, exchange with D2O), 9.80 (2H, br s, CH2NH–, ex-
change with D2O); dC (100 MHz, DMSO-d6) 175.2, 170.0, 165.3,
157.8, 143.7, 138.0, 128.2, 120.0, 50.1, 19.8.
(400 MHz, DMSO-d6) 3.70 (3H, s, CH3), 4.12 (2H, d, J 5.0, 200-H2),
7.30 (2H, s, SO2NH2, exchange with D2O), 7.79 (4H, m, Ar-H),
8.70 (1H, br s, CH2–NH–, exchange with D2O), 10.56 (1H, br s, –N
H–, exchange with D2O); dC (100 MHz, DMSO-d6) 171.0, 169.3,
166.7, 164.3, 142.6, 139.0, 127.3, 120.5, 52.8 (CH3), 43.2 (C-200).
4.1.8. Synthesis of 300,3000-[60-(4-sulfamoylphenylamino)-10,30,50-
triazine-20,40-diyl]bis(azanediyl)dipropanoic acid 8
O
2"
H
H
3
H
4
N
N
2
N
Cl
N
N
HO2C
DIPEA
DMF
2'
HO
H2N
6'
3"
1
N
N
N
N
SO2NH2
SO2NH2
4'
NH
3"'
Cl
2"'
HO2C
1
β-Ala
8
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide
1 (0.1 g, 1.0 equiv) and 3-aminopropanoic acid (4.2 equiv) were
dissolved in dry DMF (15 ml) and DIPEA (5.0 equiv) was added.
4.1.10. Synthesis of D,L
-200-[40-chloro-60-(4-sulfamoylphenyl-
amino)-10,30,50-triazin-20-ylamino]-300-hydroxypropanoic acid
10
H
H
N
3
O
H
2'
4
2"
6'
HO2C
HO
N
N
2
Cl
N
N
NH2
DIPEA
DMF
HO
HO
1
N
N
3"
N
N
SO2NH2
SO2NH2
4'
Cl
Cl
1
D,L-Ser
10
The reaction was treated according to the procedure reported
above to afford the title compound as a white solid.
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide
1 (0.1 g, 1.0 equiv) and -serine (1.2 equiv) were dissolved in dry
D,L
300,3000-[60-(4-Sulfamoylphenylamino)-10,30,50-triazine-20,40-diyl]
bis(azanediyl)dipropanoic acid 8: 55% yield; mp 308–310 °C; mmax
(KBr) cmꢁ1, 3348, 2820, 1715 (C@O, acid), 1560 (aromatic); dH
(400 MHz, DMSO-d6) 2.60 (4H, br m, 2 ꢂ 300-H2), 3.63 (4H, br m,
2 ꢂ 200-H2), 7.27 (2H, d, J 8.2, 2 ꢂ 3-H), 7.76 (2H, d, J 8.2, 2 ꢂ 2-H),
7.89 (2H, s, SO2NH2, exchange with D2O), 9.80 (2H, m, 2 ꢂ CH2–
NH, exchange with D2O), 10.40 (1H, br s, NH, exchange with
D2O); dC (100 MHz, DMSO-d6) 174.0, 167.2, 158.2, 143.6, 138.4,
128.5, 120.0, 37.2, 34.7.
DMF (15 ml) and DIPEA (2.5 equiv) was added. The reaction was
treated according to the procedure reported above to afford the ti-
tle compound as a white solid.
D,L
-200-[40-Chloro-60-(4-sulfamoylphenylamino)-10,30,50-triazin-
20-ylamino]-300-hydroxypropanoic acid 10: 32% yield; mp 255–
257 °C; mmax (KBr) cmꢁ1, 3350, 2827, 1712 (C@O, acid), 1560
(aromatic); dH (400 MHz, DMSO-d6) 3.30 (1H, m, 300-HH), 3.72
(1H, br m, 200-H), 4.20 (1H, m, 300-HH), 7.28 (2H, s, SO2NH2, ex-
change with D2O), 7.42 (2H, d, J 8.2, 2 ꢂ 3-H), 7.77 (2H, d, J 8.2,
2 ꢂ 2-H), 8.10 (1H, br s, CHNH-, exchange with D2O), 10.34 (1H,
br s, NH); dC (100 MHz, DMSO-d6) 175.3, 167.0, 166.4, 165.0,
145.2, 141.6, 130.2, 128.5, 68.2, 56.0.
4.1.9. Synthesis of D,L
-200-[40-chloro-60-(4-sulfamoylphenyl-
amino)-10,30,50-triazin-20-ylamino]propanoic acid 9
H
H
H
3
O
Cl
N
N
2"
4
HO2C
N
N
2
N
DIPEA
DMF
2'
6'
OH
N
N
1
N
N
SO2NH2
SO2NH2
NH2
4'
Cl
Cl
1
D,L-Ala
9

3114
F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
4.1.11. Synthesis of 4-[40-chloro-60-(200-hydroxyethylamino)-
10,30,50-triazin-20-ylamino] benzenesulfonamide 11
with D2O), 7.88 (4H, m, Ar-H), 8.39 (1H, br m, CH2NH-, exchange
with D2O), 10.37 (1H, br s, –NH–, exchange with D2O); dC
2"
H
H
3
H
6'
2'
4
N
N
Cl
2
N
N
N
DIPEA
DMF
HO
NH2
3"
HO
1
N
N
N
N
SO2NH2
SO2NH2
4'
Cl
Cl
1
11
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide
1 (1.0 g, 1.0 equiv) was dissolved in dry DMF (20 ml) followed
by addition of ethanolamine (0.19 g, 1.0 equiv) and DIPEA
(0.48 g, 1.2 equiv). The reaction was treated according to the
procedure reported above to afford the title compound as a white
solid.
(100 MHz, DMSO-d6) 173.2, 172.4, 165.1, 145.0, 131.1, 130.5,
118.6, 66.0, 45.1, 32.0, 26.0.
4.1.13. Synthesis of 4-(40-(200-(tert-butyldimethylsilyloxy)-
ethoxy)-60-chloro-10,30,50-triazin-20-ylamino)benzenesulfon-
amide 13
H
2"
H
3
Cl
N
N
4'
2'
4
Si
O
N
2
N
NaH
THF
O
Si
OH
3"
N
N
1
O
N
N
SO2NH2
SO2NH
6'
2
Cl
Cl
1
13
4-[40-Chloro-60-(200-hydroxyethylamino)-10,30,50-triazin-20-yla-
mino] benzenesulfonamide 11: 65% yield; mp 290–293 °C; mmax
(KBr) cmꢁ1, 3260, 2830, 1559 (aromatic); dH (400 MHz, DMSO-d6,
2 atropoisomers were detected in 1/0.5 ratio. Only the major one
is reported herein) 3.42 (2H, m, 300-H2), 3.59 (2H, m, 200-H2), 4.81
(1H, t, J 5.2, O-H, exchange with D2O), 7.30 (2H, s, SO2NH2, ex-
change with D2O), 7.78 (2H, m, Ar-H), 7.92 (2H, m, Ar-H), 8.27
(1H, br m, CH2NH–, exchange with D2O), 10.35 (1H, br s, –NH–, ex-
change with D2O); dC (100 MHz, DMSO-d6) 169.2, 166.5, 164.3,
143.0, 138.7, 127.3, 120.3, 60.1, 44.2.
2-(tert-Butyldimethylsilyloxy)ethanamine (0.22 g, 1.0 equiv)
was added to a suspension of neat NaH (0.036 g, 1.2 equiv) in dry
THF (3.0 ml) and the mixture was stirred under a nitrogen atmo-
sphere at rt for 30 min. The above mixture was slowly added
via cannula to
a
freshly prepared suspension of 4-(40,60-
dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide 1 (0.40 g,
1.0 equiv) in dry THF (17 ml) and the resulting orange mixture
was stirred under a nitrogen atmosphere at rt until starting mate-
rial was consumed (TLC monitoring). The reaction was quenched
with H2O (20 ml), extracted with ethyl acetate (3 ꢂ 15 ml) and
the combined organic layers were washed with brine (2 ꢂ 20 ml),
dried over Na2SO4, filtered off and the solvent was removed in
vacuo to give a residue that was purified by silica gel column
4.1.12. Synthesis of 4-[40-chloro-60-(400-hydroxybutylamino)-
10,30,50-triazin-20-ylamino]benzenesulfonamide 12
H
2''
H
4''
H
3
Cl
N
N
6'
2'
4
1''
N
N
2
N
DIPEA
DMF
NH2
HO
HO
3''
N
N
1
N
N
SO2NH2
SO2N
H2
4'
Cl
Cl
1
12
4-(40,60-Dichloro-10,30,50-triazin-20-ylamino)benzenesulfonamide
1 (0.1 g, 1.0 equiv) and 4-amonobutan-1-ol (1.2 equiv) were dis-
solved in dry DMF (15 ml) and DIPEA (2.5 equiv) was added. The
reaction was treated according to the procedure reported above
to afford the title compound as a white solid.
chromatography eluting with 5% MeOH/DCM to afford the title
compound as a pale yellow solid.
4-(40-(200-(tert-Butyldimethylsilyloxy)ethoxy)-60-chloro-10,30,50-
triazin-20-ylamino) benzenesulfonamide 13: 18% yield; mp 120–
124 °C; silica gel TLC Rf 0.02 (MeOH/DCM 5% v/v); mmax (KBr) cm
ꢁ1, 3280, 2814, 1530 (aromatic); dH (400 MHz, DMSO-d6, 2 atropoi-
somers were detected in 1/0.9 ratio. Only the major one is reported
herein) 0.098 (6H, s, –Si(CH3)2), 0.89 (9H, s, –SiC(CH3)3), 3.92 (2H,
br m, 200-H2), 4.33 (2H, br m, 300-H2), 7.27 (2H, s, SO2NH2, exchange
with D2O), 7.86 (4H, m, Ar-H), 10.37 (1H, br s, –NH–, exchange with
D2O); dC (100 MHz, DMSO-d6) 173.1, 172.6, 165.4, 145.1, 131.0,
130.5, 118.2, 74.2, 65.0, 26.0, 19.1, ꢁ4.2.
4-[40-Chloro-60-(400-hydroxybutylamino)-10,30,50-triazin-20-
ylamino]benzenesulfonamide 12: mp 270–271 °C; silica gel TLC Rf
0.10 (MeOH/DCM 10%); mmax (KBr) cmꢁ1, 3295, 2813, 1560 (aro-
matic); dH (400 MHz, DMSO-d6, 2 atropoisomers were detected in
1/0.4 ratio. Only the major one is reported herein) 1.54 (4H, m,
200-H2, 300-H2), 3.11 (2H, m, 100-H2), 3.47 (2H, m, 400-H2), 4.20 (1H,
t, J 5.4, O-H, exchange with D2O), 7.28 (2H, s, SO2NH2, exchange

F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
3115
4.1.14. Synthesis of dimethyl 200,2000-[60-(4-sulfamoylbenzyl-
amino)-10,30,50-triazine-20,40-diyl]bis(azanediyl)diacetate 14
ing to the procedure reported above to afford the title product as
a white solid.
2
1
3
SO2NH2
O
H
H
Cl
4
O
N
N
N
O
DIPEA
DMF
N
N
SO2NH2
2'
6'
2"
5
N
NH
O
N
N
4'
NH2HCl
NH
Cl
2"'
O
O
14
2
4-[(40,60-Dichloro-10,30,50-triazin-20-ylamino)methyl]benzenesul-
fonamide 2 (0.1 g, 1.0 equiv) and methyl 2-aminoacetate hydro-
chloride (4.2 equiv) were dissolved in dry DMF (15 ml) and
DIPEA (5.0 equiv) was added. The reaction was stirred under a
nitrogen atmosphere at rt until starting material was consumed
(TLC monitoring) and treated according to the procedure reported
above to afford the title product as a white solid.
D,L
-20,2000-[60-(4-Sulfamoylbenzylamino)-10,30,50-triazine-20,40-diyl]
bis(azanediyl)dipropanoic acid 15: 34% yield; mp 260–261 °C; mmax
(KBr) cmꢁ1, 3350, 2827, 1718 (C@O, acid), 1551 (aromatic); dH
(400 MHz, DMSO-d6) 1.32 (6H, d, J 5.6, 2 ꢂ CH3), 4.50 (4H, m, 5-
H2, 2 ꢂ 200-H), 7.28 (2H, s, SO2NH2, exchange with D2O), 7.50 (2H,
d, J 8.2, 2 ꢂ 3-H), 7.80 (2H, d, J 8.2, 2 ꢂ 2-H); dC (100 MHz, DMSO-
d6) 174.3, 169.6, 165.1, 158.0, 144.1, 138.0, 128.6, 50.1, 44.2, 19.3.
Dimethyl 200,2000-[60-(4-sulfamoylbenzylamino)-10,30,50-triazine-
20,40-diyl]bis(azanediyl)diacetate 14: 34% yield; mp 245–246 °C;
mmax (KBr) cmꢁ1, 3332, 2835, 1762 (C@O, ester), 1560 (aromatic);
4.1.16. Synthesis of 300-[40-chloro-60-(4-sulfamoylbenzylamino)-
10,30,50-triazin-20-ylamino]propanoic acid 16
2
1
SO2NH2
3
O
2"
H
H
Cl
4
N
N
N
DIPEA
DMF
HO
HO2C
N
N
SO2NH2
2'
6'
5
3"
N
NH
N
N
H2N
4'
Cl
Cl
2
16
dH (400 MHz, DMSO-d6, 3 atropoisomers were detected in 1/0.3/
0.29 ratio. Only the major one is reported herein) 3.52 (6H, s,
2 ꢂ CH3), 3.65 and 3.92 (4H, d, J 5.6, 2 ꢂ 20-H2), 4.50 (2H, d, J 5.6,
5-H2), 7.30 (2H, s, SO2NH2, exchange with D2O), 7.42 (2H, d, J 8.2,
2 ꢂ 3-H), 7.78 (2H, d, J 8.2, 2 ꢂ 2-H), 8.20 and 8.60 (1H, t, J 5.6,
2 ꢂ CH2–NH, exchange with D2O), 10.52 (1H, br s, –NH–, exchange
with D2O); dC (100 MHz, DMSO-d6) 171.9, 169.0, 165.2, 144.5.
129.0, 126.8, 53.0, 44.3, 43.0, 42.4.
4-[(40,60-Dichloro-10,30,50-triazin-20-ylamino)methyl]benzenesul-
fonamide (0.1 g, 1.0 equiv) and 3-aminopropanoic acid
(2.4 equiv) were dissolved in dry DMF (15 ml) and DIPEA
(5.0 equiv) was added. The reaction was stirred under a nitrogen
atmosphere at rt until starting material was consumed (TLC mon-
itoring) and treated according to the procedure reported above to
afford the title product as a light brown solid.
2
300-[40-Chloro-60-(4-sulfamoylbenzylamino)-10,30,50-triazin-20-
ylamino]propanoic acid 16: 35% yield; mp 165–167 °C; mmax (KBr)
cmꢁ1, 3322, 2833, 1716 (C@O, acid), 1555 (aromatic); dH (400 MHz,
DMSO-d6) 2.58 (2H, br m, 300-H2), 3.65 (2H, br m, 200-H2), 4.53
4.1.15. Synthesis of D,L
-20,2000-[60-(4-sulfamoylbenzylamino)-
10,30,50-triazine-20,40-diyl]-bis(azanediyl)dipropanoic acid 15
2
1
SO2NH2
3
O
H
H
4
Cl
2"
DIPEA
DMF
HO2C
N
N
N
N
N
SO2NH2
2'
6'
OH
5
N
NH
N
N
NH2
4'
Cl
NH
2"'
2
15
CO2H
4-[(40,60-Dichloro-10,30,50-triazin-20-ylamino)methyl]benzenesul-
fonamide 2 (0.1 g, 1.0 equiv) and -alanine (4.2 equiv) were dis-
solved in dry DMF (15 ml) and DIPEA (5.0 equiv) was added. The
reaction was stirred under a nitrogen atmosphere at rt until start-
ing material was consumed (TLC monitoring) and treated accord-
(2H, br s, 5-H2), 7.28 (2H, s, SO2NH2, exchange with D2O),
7.40 (2H, d, J 8.2, 2 ꢂ 3-H), 7.80 (2H, d, J 8.2, 2 ꢂ 2-H), 9.78
(1H, br s, -NH-, exchange with D2O); dC (100 MHz, DMSO-d6)
175.0, 170.1, 167.2, 162.3, 145.2, 139.6, 128.2, 119.7, 45.3,
37.2, 34.7.
D,L

3116
F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
4.1.17. Synthesis of
D
,
L
-200-[40-chloro-60-(4-sulfamoylbenzyl-
200-[40-Chloro-60-(4-sulfamoylphenethylamino)-10,30,50-triazin-
amino)-10,3’,50-triazin-20-ylamino]-300-(600,700-dihydroxy-
phenyl)propanoic acid 17
20-ylamino]acetic acid 18: 30% yield; mp 199–201 °C; mmax (KBr)
cmꢁ1
,
3350, 2827, 1730 (C@O, acid), 1565 (aromatic); dH
2
1
SO2NH2
3
4
5
O
H
NH2
Cl
2"
2'
DIPEA
DMF
HO2C
N
N
NH
HO
6'
N
N
SO2NH2
5"
6"
4"
HO
HO
N
N
NH
N
3"
4'
Cl
HO
Cl
9"
7"
8"
OH
17
2
4-[(40,60-Dichloro-10,3’,50-triazin-20-ylamino)methyl]benzene-
sulfonamide 2 (0.1 g, 1.0 equiv) and -3,4-dihydroxyphenylala-
(400 MHz, DMSO-d6, 80 °C) 2.90 (2H, m, 5-H2), 3.48 (2H, m, CH2
glycine), 3.92 (2H, m, 6-H2), 6.80 (1H, br s, exchange with D2O),
7.32 (2H, s, SO2NH2, exchange with D2O), 7.44 (2H, d, J 8.2,
2 ꢂ 3-H), 7.78 (2H, d, J 8.2, 2 ꢂ 2-H); dC (100 MHz, DMSO-d6,
80 °C) 172.2, 169.0, 166.9 (C-6), 166.0 (C-20), 144.4 (ipso), 143
(ipso), 130.1 (C-3), 126.7 (C-2), 43.4, 42.2, 35.1 (C-6).
D,L
nine (2.4 equiv) were dissolved in dry DMF (15 ml) and DIPEA
(5.0 equiv) was added. The reaction was stirred under a nitrogen
atmosphere at rt until starting material was consumed (TLC mon-
itoring) and treated according to the procedure reported above to
afford the title product as a light brown solid.
D
,
L
-200-[40-Chloro-60-(4-sulfamoylbenzylamino)-10,30,50-triazin-
4.1.19. Synthesis of methyl 200-[40-chloro-60-(4-sulfamoyl-
phenethylamino)-10,30,50-triazin-20-ylamino]acetate 191b
20-ylamino]-300-(600,700-dihydroxyphenyl)propanoic acid 17: 37%
O
H
H
3
H
5
O
Cl
N
N
N
N
4
N
2
1
DIPEA
DMF
O
2'
6'
6
2"
O
N
N
N
N
SO2NH2
SO2NH2
NH2HCl
4'
Cl
Cl
3
19
yield; mp 299–301 °C; mmax (KBr) cmꢁ1, 3360, 2812, 1700 (C@O,
acid), 1567 (aromatic); dH (400 MHz, DMSO-d6) 3.57 (2H, m, 200-
H, 300-HH), 4.55 (3H, m, 300-HH, 5-H2), 6.53 (3H, m, 500-H, 800-H, 900-
H), 7.29 (2H, s, SO2NH2, exchange with D2O), 7.40 (2H, d, J 8.2,
2 ꢂ 3-H), 7.78 (2H, d, J 8.2, 2 ꢂ 2-H), 8.30 (2H, br s, CHNH–, ex-
change with D2O); dC (100 MHz, DMSO-d6) 174.3, 166.8, 167.1,
165.2, 148.1, 145.0, 144.8, 143.0, 136.0, 129.8, 129.0, 121.6,
119.5, 114.0, 67.8, 45.6, 38.0.
4-[6-(40,60-Dichloro-10,30,50-triazin-20-ylamino)ethyl]benzene-
sulfonamide 3 (0.1 g, 1.0 equiv) and methyl 2-aminoacetate hydro-
chloride (2.2 equiv) were dissolved in dry DMF (15 ml) and DIPEA
(5.0 equiv) was added. The reaction was stirred under a nitrogen
atmosphere at rt until starting material was consumed (TLC mon-
itoring) and treated according to the procedure reported above to
afford the title product as a white solid.
Methyl 200-[40-chloro-60-(4-sulfamoylphenethylamino)-10,30,50-
triazin-20-ylamino]acetate 19: 42% yield; mp 234–236 °C; mmax
(KBr) cmꢁ1, 3340, 2829, 1766 (C@O, ester), 1561 (aromatic); dH
(400 MHz, DMSO-d6, 80 °C) 2.90 (2H, m, 5-H2), 3.44 (2H, m, CH2
4.1.18. Synthesis of 200-[40-chloro-60-(4-sulfamoylphenethyl-
amino)-10,30,50-triazin-2’-ylamino]acetic acid 181b
H
H
H
3
5
Cl
N
N
4
2'
HO2C
N
6'
N
2
N
O
DIPEA
DMF
6
2"
1
N
N
N
N
OH
SO2NH2
SO2NH2
4'
NH2
Cl
Cl
3
18
4-[6-(40,60-Dichloro-10,30,50-triazin-20-ylamino)ethyl]benzene-
sulfonamide 3 (0.1 g, 1.0 equiv) and glycine (2.2 equiv) were
dissolved in dry DMF (15 ml) and DIPEA (5.0 equiv) was added.
The reaction was stirred under a nitrogen atmosphere at rt until
starting material was consumed (TLC monitoring) and treated
according to the procedure reported above to afford the title prod-
uct as a white solid.
glycine), 3.62 (3H, s, CH3), 4.04 (2H, m, 6-H2), 7.32 (2H, s, SO2NH2,
exchange with D2O), 7.43 (2H, d, J 8.2, 2 ꢂ 3-H), 7.79 (2H, d, J 8.2,
2 ꢂ 2-H), 8.11 (2H, m, exchange with D2O); dC (100 MHz, DMSO-
d6, 80 °C) 171.3 (C@O), 168.7 (C-40), 166.6 (C-60), 166.0 (C-20),
144.3 (ipso), 143.0 (ipso), 130.0, 126,6, 52.7, 43.3, 42.4, 35.0 (CH3).
4.1.20. Synthesis of 300-[40-chloro-60-(4-sulfamoylphenethyl-
amino)-10,30,50-triazin-20-ylamino]propanoic acid 201b
H
2"
O
H
H
3
5
Cl
N
N
N
4
N
N
2
1
HO2C
2'
6'
DIPEA
DMF
6
HO
H2N
3"
N
N
N
N
SO2NH2
SO2NH
4'
2
Cl
Cl
3
20

F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
3117
4-[6-(40,60-Dichloro-10,30,50-triazin-20-ylamino)ethyl]benzene-
sulfonamide (0.1 g, 1.0 equiv) and 3-aminopropanoic acid
(2.2 equiv) were dissolved in dry DMF (15 ml) and DIPEA
(5.0 equiv) was added. The reaction was stirred under a nitrogen
atmosphere at rt until starting material was consumed (TLC mon-
itoring) and treated according to the procedure reported above to
afford the title product as a white solid.
(2H, d, J 8.2, 2 ꢂ 2-H), 8.27 (2H, br s, CHNH-, exchange with
D2O); dC (100 MHz, DMSO-d6) 175.2, 167.0, 166.8, 165.3, 148.0,
145.2, 145.1, 142.2, 135.4, 130.1, 128.7, 122.0, 118.0, 115.3, 68.6,
44.1, 38.4, 36.2.
3
4.1.22. Synthesis of D,L
-200,2000-[60-(4-sulfamoylphenethylamino)-
10,30,50-triazine-20,40-diyl] bis(azanediyl)dipropanoic acid 22
O
H
H
H
5
3
2"
DIPEA
DMF
HO2C
N
4
Cl
N
N
N
N
2
1
2'
6'
6
OH
N
N
N
N
NH2
SO2NH2
SO2NH
4'
2
Cl
NH
CO2H
2"'
3
22
300-[40-Chloro-60-(4-sulfamoylphenethylamino)-10,30,50-triazin-20-
ylamino]propanoic acid 20: 28% yield; mp 265–269 °C; mmax (KBr)
cmꢁ1, 3350, 2827, 1712 (C@O, acid), 1560 (aromatic); dH (400 MHz,
DMSO-d6) 2.60 (2H, br m, 300-H2), 2.90 (2H, m, 5-H2), 3.65 (2H, br m,
200-H2), 3.99 (2H, m, 6-H2), 7.35 (2H, s, SO2NH2, exchange with
D2O), 7.38 (2H, d, J 8.2, 2 ꢂ 3-H), 7.72 (2H, d, J 8.2, 2 ꢂ 2-H), 9.65
(1H, br s, CH2NH-, exchange with D2O); dC (100 MHz, DMSO-d6)
175.3, 169.0, 167.1, 162.2, 145.0, 140.0, 128.1, 120.2, 44.6, 38.1,
37.1, 36.5.
4-[6-(40,60-Dichloro-10,30,50-triazin-20-ylamino)ethyl]benzene-
sulfonamide 3 (0.1 g, 1.0 equiv) and -alanine (4.2 equiv) were
D,L
dissolved in dry DMF (15 ml) and DIPEA (5.0 equiv) was added.
The reaction was stirred under a nitrogen atmosphere at rt until
starting material was consumed (TLC monitoring) and treated
according to the procedure reported above to afford the title prod-
uct as a white solid.
D,L
-200,2000-[60-(4-Sulfamoylphenethylamino)-10,30,50-triazine-20,40-
diyl]bis(azanediyl)dipropanoic acid 22: 48% yield; mp 274–276 °C;
max (KBr) cmꢁ1, 3362, 2818, 1717 (C@O, acid), 1558 (aromatic); dH
4.1.21. Synthesis of D,L
-200-[40-chloro-60-(4-sulfamoylphenethyl-
amino)-10,30,50-triazin-20-ylamino]-300-(600,700-dihydroxyphenyl)-
propanoic acid 21
m
O
H
NH2
H
H
3
5
Cl
N
N
DIPEA
DMF
2"
HO
HO2C
N
2'
N
6' N
4
2
1
6
5"
N
N
6"
4"
HO
HO
3"
N
N
SO2NH2
SO2N
H2
4'
Cl
Cl
HO
9"
7"
21
OH
3
8"
4-[6-(40,60-Dichloro-10,30,50-triazin-20-ylamino)ethyl]benzene-
sulfonamide 3 (0.1 g, 1.0 equiv) and DL-3,4-dihydroxy-phenylala-
nine (2.2 equiv) were dissolved in dry DMF (15 ml) and DIPEA
(5.0 equiv) was added. The reaction was stirred under a nitrogen
atmosphere at rt until starting material was consumed (TLC mon-
itoring) and treated according to the procedure reported above to
afford the title product as a white solid.
(400 MHz, DMSO-d6) 1.35 (6H, d, J 5.6, 2 ꢂ CH3), 2.88 (2H, m, 5-H2),
4.00 (2H, m, 6-H2), 4.35 (2H, m, 2 ꢂ 200-H), 7.32 (2H, s, SO2NH2, ex-
change with D2O), 7.41 (2H, d, J 8.2, 2 ꢂ 3-H), 7.80 (2H, d, J 8.2,
2 ꢂ 2-H); dC (100 MHz, DMSO-d6) 174.1, 168.0, 165.0, 160.2,
144.0, 135.7, 129.0, 55.2, 44.2, 36.4, 19.3.
4.1.23. Synthesis of D,L
-200,2000-[60-(4-sulfamoylphenethylamino)-
D
,L
-200-[40-Chloro-60-(4-sulfamoylphenethylamino)-10,30,50-tria-
10,30,50-triazine-20,40-diyl]bis(azanediyl)bis(300-hydroxyprop-
anoic acid) 23
zin-20-ylamino]-300-(600,700-dihydroxyphenyl)propanoic acid 21:
28% yield; mp 319–321 °C with decomposition; mmax (KBr) cmꢁ1
,
O
H
H
H
3
5
2"
HO2C
HO
N
N
4
N
Cl
N
N
2
1
NH2
2'
6'
6
DIPEA
DMF
HO
HO
N
N
N
N
3"
SO2NH2
SO2NH2
4'
HN
2"'
CO2H
23
Cl
3
OH
3366, 2818, 1703 (C@O, acid), 1563 (aromatic); dH (400 MHz,
DMSO-d6) 2.90 (2H, br m, 5-H2), 3.62 (4H, m, 6-H2, 200-H, 300-HH),
4.50 (1H, m, 300-HH), 6.50 (3H, m, 500-H, 800-H, 900-H), 7.33 (2H, s,
SO2NH2, exchange with D2O), 7.42 (2H, d, J 8.2, 2 ꢂ 3-H), 7.81
4-[6-(40,60-Dichloro-10,30,50-triazin-20-ylamino)ethyl]benzene-
sulfonamide 3 (0.1 g, 1.0 equiv) and -serine (4.2 equiv) were dis-
solved in dry DMF (15 ml) and DIPEA (5.0 equiv) was added. The
reaction was stirred under a nitrogen atmosphere at rt until start-
D,L

3118
F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
ing material was consumed (TLC monitoring) and treated accord-
ing to the procedure reported above to afford the title product as
a white solid.
drops (0.2 mM hCA II; 0.4 mM compound either 1 or 11; 0.8 M so-
dium citrate; 50 mM Tris-Cl; pH 8.0) were equilibrated against pre-
cipitant solution (1.6 M sodium citrate; 50 mM Tris-Cl; pH 8.0) at
room temperature (ꢀ20 °C).26 Useful crystals were observed 4 days
after the crystallization setup. A crystal was cryoprotected by quick
immersion into 25% glycerol precipitant solution and flash-cooled
by exposure to a gaseous stream of nitrogen at 100 K. X-ray diffrac-
tion data were obtained using an R-AXIS IV++ image plate system
with Osmic Varimax HR optics and a Rigaku RU-H3R Cu rotating
anode operating at 50 kV and 22 mA. The detector-crystal distance
was set to 80 mm. The oscillation steps were 1° with a 6 min expo-
sure per image. Indexing, integration, and scaling were performed
using HKL2000.27
D,L
-200,2000-[60-(4-Sulfamoylphenethylamino)-10,30,50-triazine-20,40-
diyl]bis(azanediyl)bis(300-hydroxypropanoic acid) 23: 42% yield; mp
278–280 °C with decomposition;
m
max (KBr) cmꢁ1, 3342, 2830, 1710
(C@O, acid), 1562(aromatic);dH (400 MHz, DMSO-d6)2.88(2H, brm,
5-H2), 3.34 (4H, m, 6-H2, 2 ꢂ 300-HH), 3.77 (2H, br m, 2 ꢂ 200-H), 4.40
(2H, m, 2 ꢂ 300-HH), 7.32 (2H, s, SO2NH2, exchange with D2O), 7.45
(2H, d, J 8.2, 2 ꢂ 3-H), 7.76 (2H, d, J 8.2, 2 ꢂ 2-H), 8.00 (2H, br s,
CHNH-, exchange with D2O); dC (100 MHz, DMSO-d6) 174.9, 165.3,
164.0, 145.0, 141.4, 129.0, 127.5, 69.4, 58.0, 44.2, 36.0.
The preparation of some of the intermediates used in the syn-
theses are also shown below.
4.4. Structure determination
4.1.24. Synthesis of methyl 2-aminoacetate hydrochloride21
Thionyl chloride (1.58 g, 1.0 equiv) was added drop-wise to a
solution of glycine (1.0 g, 1.0 equiv) in MeOH (50 ml) at 0 °C. The
solution was stirred under reflux until starting material was con-
sumed (TLC monitoring). Then solvent was evaporated in vacuo
to afford the title product as a white solid and does not need fur-
ther purification.
The crystal structure of hCA II (PDB accession code: 2ILI)26 was
used to obtain initial phases of the structures using PHENIX.28 The
solvent molecules were removed to avoid model bias and 5% of the
unique reflections were selected randomly and excluded from the
refinement data set for the purpose of Rfree calculations. For both
compounds, the initial |Fo ꢁ Fc| difference electron density maps
unambiguously showed the triazine, 4-aminobnzene, and sulfon-
amide positions. Atomic models for compounds 7 and 11 were cal-
culated and energy minimized using the PRODRG2 server.29
Refinements proceeded using normal protocols of positional, iso-
tropic atomic displacement parameters, TLS refinement, and auto-
mated solvent additions, alternating with manual refitting of the
models using COOT.30 Final rounds of refinement for both models
included riding hydrogen positions, which decreased Rwork and
Rfree values by about 1% each. The quality of the final models were
assessed with PROCHECK.31 Crystal and refinement data are sum-
marized in Table 2.
4.1.25. Synthesis of 2-(tert-butyldimethylsilyloxy)ethanol22
2
TBDMS
NaH
THF
OH
Si
OH
HO
O
1
Dry ethane-1,2-diol (1.0 g, 1.0 equiv) was added to a suspen-
sion of neat NaH (0.65 g, 1.0 equiv) in dry THF (20 ml). The sus-
pension was stirred at rt for 30 min followed by addition over
20 min of a solution of tert-butyldimethylsilylchloride (2.43 g,
1.0 equiv) in the same solvent (20 ml) and the resulting mixture
was stirred under a nitrogen atmosphere at rt for 50 min. Then
the reaction was treated with diethyl ether (70 ml), washed with
a 10% aqueous solution of K2CO3 (3 ꢂ 30 ml), brine (3 ꢂ 20 ml)
and the organic layers were dried over Na2SO4, filtered off and
the solvent evaporated under vacuo to give a pale yellow oil that
was purified by silica gel column chromatography eluting with
10% ethyl acetate in n-hexane to afford the title compound as a
thin colourless oil. 2-(tert-Butyldimethylsilyloxy)ethanol: 89%
yield; silica gel TLC Rf 0.22 (ethyl acetate/petroleum ether 10%
v/v); dH (400 MHz, DMSO-d6) -0.98 (6H, s, –Si(CH3)2), 0.92 (9H,
s, –SiC(CH3)3), 3.46 (2H, q, J 5.6, 1-H2), 3.63 (2H, t, J 5.6, 2-H2),
4.5. CA inhibition
An Applied Photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.15 Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
Hepes (pH 7.4) and 20 mM NaBF4 (for maintaining constant the io-
nic strength), following the initial rates of the CA-catalyzed CO2
hydration reaction for a period of 10–100 s. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants. For each inhibitor, at least
six traces of the initial 5–10% of the reaction have been used for
determining the initial velocity. The uncatalyzed rates were deter-
mined in the same manner and subtracted from the total observed
rates. Stock solutions of inhibitor (10 mM) were prepared in dis-
tilled–deionized water and dilutions up to 0.01 nM were done
thereafter with distilled–deionized water. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temper-
ature prior to assay, in order to allow for the formation of the E–I
complex. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3, whereas the kinetic param-
eters for the uninhibited enzymes from Lineweaver–Burk plots, as
reported earlier,1,3 and represent the mean from at least three dif-
ferent determinations. All CAs were recombinant proteins obtained
as reported earlier by these groups.1,12,13,16
4.59 (1H, t,
J 5.6, O-H, exchange with D2O); dC (100 MHz,
DMSO-d6) 65.5 (C-1), 63.4 (C-2), 26.8 (–SiC(CH3)3), 19.0 (–
SiC(CH3)3), ꢁ4.3 (–Si(CH3)2).
4.2. Expression and Purification of hCA II
The plasmid encoding hCA II was transformed into E. coli BL21
cells through standard procedures and the transformed cells were
expressed at 37 °C in LB medium containing 100 l
g/ml ampicillin.23
hCA II production was induced by the addition of isopropyl thioga-
lactoside to a final concentration of 1 mM at an O.D600 of 0.6 AU.
The cells were harvested after 4hrs of post induction. The cell pellets
were lysed and hCA II was purified through affinity chromatography
using pAMBS resin as has been described elsewhere.24
Acknowledgments
4.3. Co-crystallization and X-ray data collection
This research was financed in part by a 7th FP EU grant
(METOXIA) to A.S. and C.T.S., and in part by an NIH (GM 25154)
and Maren Foundation grant to R.M. R.M. would also like to thank
Co-crystals of hCA II complexed with compounds 7 and 11 were
obtained using the hanging drop vapor diffusion method.25 10
ll

F. Carta et al. / Bioorg. Med. Chem. 19 (2011) 3105–3119
3119
P.; Blackburn, G. M.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 999; (c)
Casey, J. R.; Morgan, P. E.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.; Supuran,
C. T. J. Med. Chem. 2004, 47, 2337.
the Centre of Structure Biology, University of Florida, for their con-
tinued support to help maintain the in-house X-ray facilities.
13. Feldshtein, M.; Elkrinawi, S.; Yerushalmi, B.; Marcus, B.; Vullo, D.; Romi, H.;
Ofir, R.; Landau, D.; Sivan, S.; Supuran, C. T.; Birk, O. S. Am. J. Hum. Genet. 2010,
87, 713.
Supplementary data
14. (a) Vullo, D.; Innocenti, A.; Nishimori, I.; Pastorek, J.; Scozzafava, A.;
Pastorekova, S.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 963; (b)
Nishimori, I.; Vullo, D.; Innocenti, A.; Scozzafava, A.; Mastrolorenzo, A.;
Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 3828; (c) Vullo, D.; Voipio, J.;
Innocenti, A.; Rivera, C.; Ranki, H.; Scozzafava, A.; Kaila, K.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2005, 15, 971.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.04.005.
References and notes
15. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561.
16. (a) Hall, R. A.; Mühlschlegel, F. A. Fungal and Nematode Carbonic
Anhydrases: Their Inhibition in Drug Design. In Drug Design of Zinc–Enzyme
Inhibitors: Functional, Structural, and Disease Applications; Supuran, C. T.,
Winum, J. Y., Eds.; John Wiley & Sons: Hoboken, 2009; pp 301–322; (b)
Ohndorf, U. M.; Schlicker, C.; Steegborn, C. Crystallographic Studies on
Carbonic Anhydrases from Fungal Pathogens for Structure-assisted Drug
Development. In Drug Design of Zinc–Enzyme Inhibitors: Functional, Structural,
and Disease Applications; Supuran, C. T., Winum, J. Y., Eds.; John Wiley & Sons:
Hoboken, 2009; pp 323–334.
1. (a) Garaj, V.; Puccetti, L.; Fasolis, G.; Winum, J. Y.; Montero, J. L.; Scozzafava, A.;
Vullo, D.; Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2004, 14, 5427;
(b) Garaj, V.; Puccetti, L.; Fasolis, G.; Winum, J. Y.; Montero, J. L.; Scozzafava, A.;
Vullo, D.; Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 3102.
2. D’Alelio, G. F.; White, H. J. J. Org. Chem. 1959, 24, 643.
3. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Özensoy, O.; De Simone,
G.; Supuran, C. T. Curr. Med. Chem. 2010, 17, 1516; (c) De Simone, G.; Supuran,
C. T. Biochim. Biophys. Acta 2010, 1804, 404; (d) Supuran, C. T. Bioorg. Med. Chem.
Lett. 2010, 20, 3467.
17. (a) Burghout, P.; Vullo, D.; Scozzafava, A.; Hermans, P. W. M.; Supuran, C. T.
Bioorg. Med. Chem. 2011, 19, 243; (b) Supuran, C. T. Curr. Pharm. Des. 2010, 16,
3233; (c) Nishimori, I.; Minakuchi, T.; Maresca, A.; Carta, F.; Scozzafava, A.;
Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3300; (d) Winum, J. Y.; Kohler, S.;
Supuran, C. T. Curr. Pharm. Des. 2010, 16, 3310.
18. (a)Alterio, V., Di Fiore, A., D’Ambrosio, K., Supuran, C. T., De Simone, G., Eds.X-
ray Crystallography of CA Inhibitors and its Importance in Drug Design of Zinc–
Enzyme Inhibitors: Functional, Structural, and Disease Applications; Supuran,
C. T., Winum, J. Y., Eds.; Wiley: Hoboken, 2009; pp 73–138; (b) Avvaru, B. S.;
Wagner, J. M.; Maresca, A.; Scozzafava, A.; Robbins, A. H.; Supuran, C. T.;
McKenna, R. Bioorg. Med. Chem. Lett. 2010, 20, 4376; (c) Winum, J. Y.;
Temperini, C.; El Cheikh, K.; Innocenti, A.; Vullo, D.; Ciattini, S.; Montero, J. L.;
Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2006, 49, 7024.
19. (a) Wagner, J. M.; Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; Supuran, C. T.;
McKenna, R. Bioorg. Med. Chem. 2010, 18, 4873; (b) Pacchiano, F.; Aggarwal, M.;
Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem.
Commun. (Camb.) 2010, 46, 8371; (c) Köhler, K.; Hillebrecht, A.; Wischeler, J. S.;
Innocenti, A.; Heine, A.; Supuran, C. T.; Klebe, G. Angew. Chem., Int. Ed. 2007, 46,
7697; (d) Eriksson, E.; Jones, T. A.; Liljas, A. Proteins: Struct. Funct., Genet. 1988,
4, 274.
20. Supuran, C. T.; Scozzafava, A. J. Enzyme Inhib. 1999, 14, 343.
21. McKerrow, J. D.; Al-Rawi, J. M. A.; Brooks, P. Synth. Commun. 2010, 40, 1161.
22. Procopiou, P. A.; Barrett, V. J.; Bevan, N. J.; Biggadike, K.; Box, P. C.; Butchers, P.
R.; Coe, D. M.; Conroy, R.; Emmons, A.; Ford, A. J.; Holmes, D. S.; Horsley, H.;
Kerr, F.; Li-Kwai-Cheung, A.-M.; Looker, B. E.; Mann, I. S.; McLay, I. M.;
Morrison, V. S.; Mutch, P. J.; Smith, C. E.; Tomlin, P. J. Med. Chem. 2010, 53, 4522.
23. Forsman, C. A.; Behravan, G.; Osterman, A.; Jonsson, B. H. Acta Chem. Scand.
1988, 42, 314.
4. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.;
Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De Simone,
G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233.
5. (a) Švastová, E.; Hulıková, A.; Rafajová, M.; Zat’ovicová, M.; Gibadulinová, A.;
´
ˇ
Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C.; Pastorek, J. FEBS Lett. 2004,
577, 439; (b) Dubois, L.; Douma, K.; Supuran, C. T.; Chiu, R. K.; van Zandvoort,
M. A. M. J.; Pastoreková, S.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Radiother.
Oncol. 2007, 83, 367; (c) Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.;
Supuran, C. T.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Radiother. Oncol. 2009,
92, 423.
6. (a) Swietach, P.; Wigfield, S.; Cobden, P.; Supuran, C. T.; Harris, A. L.; Vaughan-
Jones, R. D. J. Biol. Chem. 2008, 283, 20473; (b) Swietach, P.; Wigfield, S.;
Supuran, C. T.; Harris, A. L.; Vaughan-Jones, R. D. BJU Int. 2008, 101, 22; (c)
Swietach, P.; Hulikova, A.; Vaughan-Jones, R. D.; Harris, A. L. Oncogene 2010, 29,
6509.
7. Ahlskog, J. K. J.; Dumelin, C. E.; Trüssel, S.; Marlind, J.; Neri, D. Bioorg. Med.
Chem. Lett. 2009, 19, 4851.
8. (a) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.;
Dedhar, S.; Supuran, C. T. J. Med. Chem. 2011, 54, 1896; (b) Hilvo, M.;
Baranauskiene, L.; Salzano, A. M.; Scaloni, A.; Matulis, D.; Innocenti, A.;
Scozzafava, A.; Monti, S. M.; Di Fiore, A.; De Simone, G.; Lindfors, M.; Janis, J.;
Valjakka, J.; Pastorekova, S.; Pastorek, J.; Kulomaa, M. S.; Nordlund, H. R.;
Supuran, C. T.; Parkkila, S. J. Biol. Chem. 2008, 283, 27799; Lou, Y.; McDonald, P.
C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.;
Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland, B. W.; Waterhouse, D.; Bally,
M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton, A.; Pacchiano, F.; Carta, F.;
Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Cancer Res.
2011, in press (Epub Mar 17, 2011).
9. (a) Bartosova, M.; Parkkila, S.; Pohlodek, K.; Karttunen, T. J.; Galbavy, S.; Mucha,
V.; Harris, A. L.; Pastorek, J.; Pastorekova, S. J. Pathol. 2002, 197, 1; (b)Cancer-
related Carbonic Anhydrase Isozymes and their Inhibition; Pastoreková, S.,
Pastorek, J., Eds.Carbonic Anhydrase: Its Inhibitors and Activators; Supuran,
C. T., Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton, FL, 2004; pp 255–
281; (c) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.; Williams, K.;
Kienninger, J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.; Lambin, P.; Wouters, B.
G.; Supuran, C. T.; Poellinger, L.; Ratcliffe, P.; Kanopka, A.; Görlach, A.;
Gasmann, M.; Harris, A. L.; Maxwell, P.; Scozzafava, A. J. Enzyme Inhib. Med.
Chem. 2009, 24, 1.
24. Khalifah, R. G.; Strader, D. J.; Bryant, S. H.; Gibson, S. M. Biochemistry 1977, 16,
2241.
25. McPherson, A. Preparation and Analysis of Protein Crystals, 1st Ed., 1982, Wiley,
New York.
26. Fisher, S. Z.; Maupin, C. M.; Budayova-Spano, M.; Govindasamy, L.; Tu, C. K.;
Agbandje-McKenna, M.; Silverman, D. N.; Voth, G. A.; McKenna, R. Biochemistry
2007, 42, 2930–2937.
27. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
28. Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.;
Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.;
Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.;
Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr., Sect. D 2010, 66, 213.
29. Schuettelkopf, A. W.; van Aalten, D. M. F. Acta Crystallogr., Sect. D 2004, D60,
1355.
10. Kyllönen, M. S.; Parkkila, S.; Rajaniemi, H.; Waheed, A.; Grubb, J. H.; Shah, G. N.;
Sly, W. S. J. Histochem. Cytochem. 2003, 51, 1217.
11. Whittington, D. A.; Grubb, J. H.; Waheed, A.; Shah, G. N.; Sly, W. S.;
Christianson, D. W. J. Biol. Chem. 2004, 279, 7223.
30. Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D 2004, 60, 2126.
31. Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. J. Appl.
Crystallogr. 1993, 26, 283.
12. (a) D’Ambrosio, K.; Vitale, R. M.; Dogne´, J. M.; Masereel, B.; Innocenti, A.;
Scozzafava, A.; De Simone, G.; Supuran, C. T. J. Med. Chem. 2008, 51, 3230; (b)
Temperini, C.; Cecchi, A.; Boyle, N. A.; Scozzafava, A.; Cabeza, J. E.; Wentworth,