Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo

Article abstract of DOI:10.1021/jm300031j

A series of dithiocarbamates were prepared by reaction of primary/secondary amines with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of four human (h) isoforms of the zinc enzyme carbonic anhydrase, CA (EC 4.2.1.1), hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar inhibitors targeting these CAs were detected. The X-ray crystal structure of the hCA II adduct with morpholine dithiocarbamate evidenced the inhibition mechanism of these compounds, which coordinate to the metal ion through a sulfur atom from the dithiocarbamate zinc-binding function. Some dithiocarbamates showed an effective intraocular pressure lowering activity in an animal model of glucoma.

Full text of DOI:10.1021/jm300031j

Article
pubs.acs.org/jmc
Dithiocarbamates Strongly Inhibit Carbonic Anhydrases and Show
Antiglaucoma Action in Vivo
Fabrizio Carta,† Mayank Aggarwal,‡ Alfonso Maresca,† Andrea Scozzafava,† Robert McKenna,‡
Emanuela Masini,§ and Claudiu T. Supuran*,†
†Laboratorio di Chimica Bioinorganica, Polo Scientifico, Universita
Sesto Fiorentino, Florence, Italy
̀
degli Studi di Firenze, Room 188, Via della Lastruccia 3, 50019
‡Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville, Florida
32610, United States
§Department of Preclinical and Clinical Pharmacology, Universita
̀
degli Studi di Firenze, Viale Pierracini 6, Florence, Italy
S
* Supporting Information
ABSTRACT: A series of dithiocarbamates were prepared by reaction of primary/secondary
amines with carbon disulfide in the presence of bases. These compounds were tested for the
inhibition of four human (h) isoforms of the zinc enzyme carbonic anhydrase, CA (EC
4.2.1.1), hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or
cancer (CA IX). Several low nanomolar inhibitors targeting these CAs were detected. The X-
ray crystal structure of the hCA II adduct with morpholine dithiocarbamate evidenced the
inhibition mechanism of these compounds, which coordinate to the metal ion through a sulfur
atom from the dithiocarbamate zinc-binding function. Some dithiocarbamates showed an
effective intraocular pressure lowering activity in an animal model of glucoma.
INTRODUCTION
■
Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread zinc
metalloenzymes found in higher vertebrates including hu-
mans.1−3 Sixteen isozymes have been characterized to date,
many of which are involved in critical physiological processes.
They catalyze the following reaction: CO2 + H2O ↔ H+ +
1−3
−
HCO3 .
In humans, CAs are present in a large variety of
tissues including the gastrointestinal tract, the reproductive
tract, the nervous system, kidneys, lungs, skin, and eyes.2,3 The
different isozymes are localized in different parts of the cell with
CA I and CA II, important isozymes in normal cells being
localized in the cytosol.1−4
As some solid tumors grow in cancer patients, hypoxic
regions are formed, particularly in the interior of the tumor.13
The gene expression profile of a hypoxic cancer cell is different
from that of other cancer cells in a normally oxygenated
environment, that is, in normoxic conditions.13−15 Under
hypoxia, the distribution of CA isoforms is altered as compared
with normoxic cells.13,14 As a result, CA isozymes IX and XII
are overexpressed in hypoxic tumor cells, in a variety of solid
tumors.13−15 Unlike other CAs, CA IX and CA XII are both
extracellularly localized on hypoxic tumor cells.13−15 These
enzymes play various roles in tumorigenesis, by regulating pH
inside and outside the tumor cell,15 interfering with
Many of the CA isozymes are important therapeutic targets
with the potential to be inhibited to treat a range of
disorders.1−7 CA II plays a role in bicarbonate production in
the eye and is therefore a target for therapy of eye diseases such
as glaucoma.7−9 Indeed, CA inhibitors (CAIs) of the
sulfonamide type such as dorzolamide (DZA) or brinzolamide
(BRZ) are topically used antiglaucoma agents,7−10 whereas the
older drugs, such as acetazolamide (AAZ) or dichlorophena-
mide (DCP) show the same action through systemic
administration, which, however, leads to a wide range of side
effects due to inhibition of the enzyme from other organs than
the target one, that is, the eye.11 CA XII, a transmembrane
isoform with an extracellular active site, was shown to be
overexpressed in glaucomatous patients eyes.12
Received: November 22, 2011
Published: January 26, 2012
© 2012 American Chemical Society
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phosphorylation of various proteins,16 or playing a role in the
cell−cell adhesion.13−15 They therefore provide a target for
cancer therapy because they are relatively specific to the
hypoxic tumor cells and appear to be important in their survival
and proliferation.15 Indeed, several antibodies targeting CA IX
are in phase III clinical development for the treatment of solid
tumors (or for their imaging),17 whereas some small molecule
inhibitors are also in advanced preclinical evaluation.15,18,19
The classical CAIs are the sulfonamides and their isosteres
(sulfamates, sulfamides, etc.).1−4 However, most of these
compounds indiscriminately inhibit many of the 16 CA
isoforms known to date in mammals.1−3 Thus, efforts have
been made to find different CAIs, from the sulfonamide,
sulfamate, and sulfamide ones. Indeed, recently, the coumarins
were discovered as mechanism-based inhibitors that act as
prodrugs and bind in a very different mode as compared to
sulfonamides and their isosteres,20 whereas some polyamines
(such as spermine),21 as well as a range of phenols,22 were also
investigated and showed such interesting properties and novel
mechanisms of inhibition. Among CAIs investigated to date,
there are also the inorganic anions, which coordinate to the zinc
ion from the enzyme active site.1,23,24 Indeed, trithiocarbonate
(CS32−), an anion similar to carbonate, has recently been
investigated and shown to constitute a “lead” for novel CAIs.23
The X-ray crystal structure for the adduct of trithiocarbonate
(CS32−), bound to hCA II, has recently been reported (Figure
1).23 The inhibitor binds to the Zn2+ in the hCA II active site in
demonstrated in a preliminary communication that dithiocar-
bamates (DTCs), compounds possessing the general formula
25
R1R2N-CS2 M+, act as highly efficient CAIs. Here, we report
the first detailed study of the DTCs as a class of potent CAIs,
with a mechanism of action different of that of the
sulfonamides. Furthermore, we prove that some of these highly
water-soluble compounds possess excellent intraocular pressure
(IOP) lowering properties in an animal model of glaucoma,
making them interesting candidates for developing antiglauco-
ma drugs.
−
RESULTS AND DISCUSSION
■
Chemistry. DTCs are well-known metal complexing agents,
and they also possess interesting biomedical and agricultural
applications.26−29 Although this class of compounds (and their
metal complexes) started to be used as fungicides more than 50
years ago,29 few studies investigated their interactions with
metalloenzymes.27 Apart from studies of DTCs as inhibitors of
tyrosinase, a copper enzyme,27 only one such work investigated
the inhibition of N,N-diethyl-DTC with bovine CA (bCA).28
By using Co(II)-substituted CA, Morpurgo et al.28 showed that
the inhibitor does not extrude the metal ion from the enzyme
active site (as it does with the copper ion from the tyrosinase
active site)27 and that it binds to it, probably in a trigonal-
bipyramidal geometry of Co(II). However, no other DTCs
were subsequently investigated for their interaction with CAs
until our group reported that trithiocarbonate and related
compounds containing the new zinc binding group (ZBG)
−
found in it, that is, CS2 , inhibit several CA isoforms in the low
micromolar or submicromolar range.23 Here, we extend those
findings, showing that a wide range of DTCs incorporating
various aliphatic and/or aromatic moieties at the nitrogen atom
acts as low nanomolar and even subnanomolar CAIs.
We prepared a series of 27 DTCs, of types 1a−27a, by the
classical reaction27,30,31 of dithiocarbamoylation between
primary/secondary amines 1−27 and CS2, in the presence of
a base, which most of the time was NaOH, KOH, but in the
case of more basic amines, the amine itself can act as the base
(Scheme 1). As shown in Table 1, a large variety of R1 and R2
Scheme 1. Preparation of DTCs 1a−27a by Reaction of
Amines 1−27 with Carbon Disulfide in the Presence of
Bases
Figure 1. Trithiocarbonate (CS32‑), a recently investigated low
micromolar CAI, binds to the Zn2+ in the hCA II active site in a
slightly distorted tetrahedral geometry of the metal ion, occupying a
position similar to that observed in the case of the hCA II-bicarbonate
complex. The protein zinc ligands (His94, -96, and -119) and Thr199
are shown (PDB code 3K7K).24
a slightly distorted tetrahedral geometry of the metal ion,
occupying a position similar to that observed in the case of the
hCA II−bicarbonate complex.23 Trithiocarbonate was mono-
coordinated to the Zn(II) ion by means of one of the sulfur
atoms. The same sulfur made a hydrogen bond to the OH of
Thr199, whereas a second sulfur atom participated with
another hydrogen bond to the NH group of the same amino
acid residues, Thr199. This binding mode explains the low
micromolar affinity of this inhibitor to many of the CA isoforms
investigated to date.23 On the basis of this binding mode of a
millimolar inhibitor, trithiocarbonate (CS32−), we hypothesized
that compounds incorporating this new zinc-binding function,
moieties are present in DTCs reported here, that is, hydrogen,
alkyl, aryl, aralkyl, hetaryl, and cyclic such moieties, which lead
to the generation of a wide chemical diversity in these
compounds. Presumably, this should be reflected also in varied
physicochemical and biological properties of these DTCs.
Compounds 1a−27a were characterized by physicochemical
1
standard procedures (IR, H an 13C NMR spectroscopy, MS)
and were >99% pure, as determined by HPLC (see the
Experimental Protocols for details)
CA Inhibition. Compounds 1a−27a were assayed32 for the
inhibition of four physiologically relevant CA isoforms, hCA I,
−
CS2 , may act as even stronger CAIs. Indeed, we have recently
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Table 1. CA I, II, IX, and XII Inhibition Data with DTCs 1a−27a by a Stopped-Flow, CO2 Hydrase Assay32
R1R2N-CSS−M+
1a−27a
c
KI (nM)
ab
no.
R1
R2
hCA I
hCA II
hCA IX
hCA XII
1a
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
Ph
4.8
4.8
4.5
3.6
4.2
29.1
22.1
4.6
4.3
9.2
2a
O[(CH2CH2)]2N
MeN[(CH2CH2)]2N
2-butyl
3a
33.5
21.1
31.8
31.9
4.1
33.0
29.4
36.3
13.5
0.70
16.6
20.3
4.6
17.5
31.7
4.2
4a
5a
O[(CH2CH2)]2N(CH2)2
N[(CH2CH2)N]3
PhCH2
4.5
6a
27.4
19.2
26.0
3.6
9.3
7a
11.5
24.1
20.5
22.0
6.7
8a
4-pyridylCH2
[(CH2)5N]CH2CH2
2-thiazolyl
3.5
9a
4.5
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27a
AAZ
3.9
12.6
57.1
4.3
KOOCCH2
13.1
8.6
325
imidazol-1-yl-(CH2)3
Me
24.7
6910
3100
27.5
0.95
55.5
50.9
51.3
27.8
4.0
6.5
699
790
0.96
0.97
1838
43.1
48.0
157
9.2
714
1413
70.4
4.5
798
1105
46.1
0.99
Et
(CH2)5
iso-Bu
iso-Bu
n-Pr
n-Pr
53.8
50.3
27.4
25.9
4.3
7.0
5.8
n-Bu
n-Bu
n-Hex
Et
n-Hex
n-Bu
16.1
7.5
HOCH2CH2
Me
HOCH2CH2
Ph
4.2
39.6
69.9
0.88
12.6
48.4
2.5
21.5
25.4
0.95
0.92
40.8
17.3
12
28.2
53.0
6.2
7.7
Me
PhCH2
3.0
O[(CH2CH2)]2
3.4
NaS(SC)N[(CH2CH2)]2
37.5
757
4.1
0.78
169
(NC)(Ph)C(CH2CH2)2
(S)-[CH2CH2CH2CH(COONa)]
4.0
5.7
250
25
a
Compounds 1a, 8a, and 10a were thriethylamonium salts; 2a−6a, 9a, 11a, and 12a were potasium salts, and the remaining ones were sodium salts.
b
c
Means from three different assays. Errors were within 5−10% of the reported values (data not shown). Inhibition data against hCA I, II, and IX
for nine DTCs; that is, compounds 13a, 14a, 16−18a, 20a, 22a, 23a, and 26a were reported in ref 25.
II, IX, and XII. All of them are drug targets: hCA I, II, and XII
for ophthalmologic diseases, mainly glaucoma,1,10 whereas CA
IX and XII for antitumor drugs/tumor imaging agents.1,15,17−19
Inhibition data with the sulfonamide, clinically used agent AAZ
are also reported in Table 1, for comparison reasons.
The following structure−activity relationship (SAR) can be
observed for the CA inhibition data with DTCs 1a−27a
investigated here:
increase of potency of 823 times as compared to 14a. It is
also interesting to compare the potencies of 16a and 18a,
which incorporate iso-butyl and n-butyl moieties (and are
isomers) and differ by a factor of 44, unexpectedly, in
favor of the compound with a branched scaffold.
Increasing the length of the aliphatic chains to C6, as
in 19a, leads to a slight loss of potency as compared to
the most active compounds in the aliphatic series, which
are 15a and 16a. Furthermore, the presence of
hydroxyethyl moieties (instead of the ethyl ones) as in
21a leads to a steady increase of potency, 21a being 85.9
times a better hCA I inhibitor as compared to 14a. The
compounds incorporating one alkyl and one aryl moiety
at the nitrogen atom of the DTC function, such as 22a
and 23a, were also effective hCA I inhibitors (KI values
of 39.6−69.9 nM), as were also the heterocyclic
derivatives 24a−27a, some of which showed subnano-
molar activity (the morpholine DTC 24a had a KI of 0.88
nM and was the best DTC hCA I inhibitor and also the
best hCA I inhibitor ever described, as far as we know).
Thus, many of these chemotypes explored here show
excellent hCA I inhibitory activity, which range from the
subnanomolar to the micromolar. Furthemore, many of
the DTCs are much more effective as hCA I inhibitors as
(i) The cytosolic isoform hCA I was strongly inhibited by
DTCs 1a−27a investigated here, with KI values in the
range of 0.88−1838 nM. It may be observed that
irrespective of the nature of R2, the DTCs prepared from
primary amines (R1 = H) 1a−12a were highly effective
hCA I inhibitors, with inhibition constants in the low
nanomolar range (3.5−33.5 nM). On the contrary, the
compounds prepared from secondary amines showed a
more varied biological activity. Thus, the simple
dimethyl- and diethyl-DTCs 13a and 14a were weak
hCA I inhibitors, with KI values in the range of 699−790
nM. The same is true for the di-n-propyl derivative 17a
(KI of 1838 nM). However, the cyclic derivative 15a,
which differs from 14a by the cyclic structure and an
extra carbon atom present in its molecule, is a
subnanomolar hCA I inhibitor, showing a dramatic
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compared to the sulfonamide AAZ, which has a KI of 250
nM against this isoform (Table 1).
Again, the main conclusion is that a large number of
substitution patterns, incorporating varied moieties, lead
to highly effective hCA XII inhibitors.
(ii) The physiologically dominant cytosolic isoform hCA II
also showed an interesting inhibition profile with DTCs
1a−27a. Thus, several primary DTCs (1a, 2a, 7a, and
10a) and several secondary ones (16a, 21a, 24a, and
25a) were excellent hCA II inhibitors, with KI values in
the range of 0.70−4.6 nM, being more effective (even 1
order of magnitude) than the clinically used sulfonamide
AAZ (Table 1). It may be observed that these
compounds incorporate aromatic, arylakyl, hetaryl,
alkyl, and hydroxyalkyl moieties substituting the nitrogen
atom from the DTC moiety. Another rather large group
of derivatives, such as 3a−6a, 8a, 9a, 12a, 15a, 17a−20a,
22a, 23a, 26a, and 27a, were slightly less effective hCA II
inhibitors but still possessed a high efficacy, with KI
values in the range of 13.5−55.5 nM. Again, both
primary and secondary DTCs are present in this
subgroup. They incorporate various types of substituents,
such as alkyl, aryl, aralkyl, and hetaryl ones. It is obvious
that small structural changes in the DTC scaffold
influence dramatically the biological activity. For
example, for the aliphatic secondary DTCs, the isomeric
pair 16a−18a, which differ only by the nature of the
aliphatic chain (iso-Bu moieties in the first compound
and n-Bu in the second derivative), have KI values that
differ by a factor of 53.6. The length of the alkyl chain
also strongly influence activity, with compounds
possessing a medium chain (e.g., 15a−20a) being more
effective than the ones with shorter chains, such as 13a
and 14a, which are rather ineffective as hCA II inhibitors
(KI values of 3.1−6.9 μM). Also, the glycine DTC 11a
was a medium potency hCA II inhibitor, with a KI of 325
nM.
(iii) The tumor-associated isoform hCA IX was highly
inhibited by the DTCs investigated here, with KI values
in the range of 3.6−1413 nM. The simple aliphatic
secondary DTCs 13a/14a and the bulky cyclic derivative
26a were the least effective inhibitors (KI values of
0.714−1.413 μM), and four other compounds (11a, 15a,
17a, and 18a) were effective, medium potency inhibitors,
with KI values in the range of 50.3−70.4 nM. They
incorporate the carboxyalkyl moiety present in glycine
(11a), the five-membered aliphatic ring (from 15a), and
3- or 4-carbon atom n-alkyl chains (17a and 18a). All of
the remaining derivatives showed highly effective hCA IX
inhibitory properties, with KI values < 30 nM. Thus, a
rather high structural diversity (aliphatic, aromatic,
aralkyl, hetaryl moieties) present in primary/secondary
DTCs lead to highly effective hCA IX inhibitors, with
minor structural changes drastically affecting enzyme
inhibition. Many DTCs were more effective hCA IX
inhibitors as compared to AAZ (Table 1).
(v) The DTCs investigated here showed a rather promiscu-
ous inhibitory activity against all four CA isoforms
described here, although each of these CAs had a
different inhibition profile with all of these compounds.
For example, 15a was a subnanomolar inhibitor of hCA I
and inhibited the remaining three isoforms with KI values
in the range of 27.5−70.4 nM, having thus an acceptable
selectivity ratio for the inhibition of hCA I over the
remaining three CAs. Compound 25a was a subnano-
molar inhibitor of hCA II and XII and inhibited hCA I
and IX with higher KI values, of 12.6−37.5 nM.
Compound 23a showed a rather good selectivity for
inhibiting hCA XII over hCA I, II, and IX (Table 1). It
should be also mentioned that being negatively charged,
these compounds show membrane impermeability,33
which may be a favorable pharmacological property in
vivo.
X-ray Crystallography. To explain the potent CA
inhibitory properties of the DTCs, which are a new class of
CAIs, we resolved the X-ray crystal structure of hCA II in
complex with the very potent inhibitor 24a (KI of 0.95 nM).
Compound 24a was well ordered and refined with an
occupancy of 1.0 with B factors that were comparable to the
solvent within the active site (Table 2). The compound is
Table 2. Crystallographic Data Refinement and Model
a
Quality Statistics for the hCA II−24a Complex
PDB accession number
data collection statistics
temperature (K)
3P5A
100
wavelength (Å)
1.5418
P21
space group
unit cell parameters (Å, °)
a = 42.3; b = 41.2 c = 72.1;
β = 104.2
total theoretical reflections
total measured reflections
resolution (Å)
39632
39395
50.0−1.5 (1.54−1.50)
6.4 (18.1); 17.5 (5.8)
99.4 (95.7); 3.7 (3.4)
b
Rsym (%); I/σ(I)
completeness; redundancy
final model statistics
c
d
Rcryst (%); Rfree(%)
0.148; 0.169
residue nos.
4−261
2078
e
no. of protein atoms
no. of compound atoms
no. of H2O molecules
9
300
RMSD bond lengths (Å)
RMSD bond angles (°)
Ramachandran statistics (%)
0.012
1.488
89.4, 10.7, 0.0
13.6, 18.1, 23.9, 29.4
average B factors (Å2) main, side chain,
compound, solvent
a
b
(iv) A rather similar SAR as the one discussed above for hCA
IX was observed for the inhibition of the second
transmembrane isoform, hCA XII, with DTCs 1a−27a.
Thus, 13a/14a and 26a were the least effective inhibitors
(KI values in the range of 169−1105 nM), whereas the
remaining DTCs were highly effective hCA XII
inhibitors, with KI values in the range of 0.78−31.7 nM
(Table 1). Among the best hCA XII inhibitors
(subnanomolar inhibition constants) were the di-
isobutyl-DTC 16a and the piparazine-bis-DTC 25a.
Values in parentheses represent the highest resolution bin. Rsym
=
c
d
∑|I − ⟨I⟩|/∑⟨I⟩. Rcryst = (∑|Fo| − |Fc|/∑|Fobs|) × 100 . Rfree is
calculated in same manner as Rcryst, except that it uses 5% of the
e
reflection data omitted from refinement. Includes alternate
conformations.
buried deep into the active site, displacing the catalytic zinc-
bound solvent, such that one of the sulfur atoms coordinates
directly to the zinc ion of the enzyme. The overall zinc
coordination (3N from the coordinating histidine residues
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His94, -96, and -119 and 1S from the inhibitor ligand) can be
described as a distorted tetrahedron (Figure 2). The details of
this tetrahedral geometry are provided in Table 3. The zinc-
bound sulfur also interacts with the O atom of Thr199 in a
similar manner to that observed in the more classical clinically
used sulfonamides and sulfamates CAIs.34−36 Compound 24a
possesses a puckered ring and binds with a slightly higher B
factor of 23.9 Å2, as compared to the two other DTCs for which
the X-ray structure in complex with hCA II was reported
earlier,25 that is, 23a and 26a. For a structural comparison, the
three compounds were superposed onto each other (Figure 3).
For unbound hCA II, the side chain conformation of His64 has
been shown to be dependent upon the buffer pH, which affects
the protonation state of the imidazole ring. It is widely believed
that this side chain flips from an “in” to “out” conformation as
part of the proton transfer mechanism in hCA II; hence, two
conformations of the residue are often observed in crystal
structures.34−36 His64 in the hCA II structure in complex with
compounds 26a (PDB ID: 3P5L) and 24a (PDB ID: 3P5A)
has a dual conformation. In the case of 26a, the terminal six-
membered hydrophobic ring sits close to F131, V135, and P202
at the rim of the active site in a hydrophobic pocket of hCA II.
Whereas for 24a, the six-membered ring does not extend far
enough out of the active site to either reach this hydrophobic
pocket or close enough to the in-conformation of His64.
Hence, compounds 24a and 26a are 5.6 and 5.0 Å, respectively,
from His64 and therefore do not affect its conformation.
Whereas in the hCA II−23a structure (PDB ID: 3P58), the six-
membered planar ring forms a T-shaped π-stacking with the
imidzole ring of His64 and stabilizes this amino acid in the “in”
conformation.25 In addition, 24a is positioned 3.2 Å from
Thr200 but does not form hydrogen bonds with either the
protein main or the side chain. However, the endocyclic oxygen
atom in the tail ring of 24a was within hydrogen bond distance
of 3.4 and 3.2 Å from water393 and water540, respectively
(Figure 2), which probably also contributes to its high affinity
to hCA II.
Figure 2. Stick representation of the hCA II active site with compound
24a (green) complexed within it. The active-site zinc is depicted as a
blue sphere. Water molecules are depicted as red spheres. The electron
density is represented by a σ-weighted 2Fo − Fc Fourier map (gray
mesh). Amino acids involved in the binding of inhibitor are also
shown. The figure was made using PyMOL (DeLano Scientific).
Table 3. Geometry of the Zn(II) Ion and Bound S Atom (of
the DTCs) for Compounds 23a, 24a, and 26a Complexed
within the hCA II Active Site (Angle Defined as His−Zn−S)
23a
2.3
24a
2.3
26a
2.3
Intraocular Pressure Lowering in Hypertensive
Rabbits. Two of the new CAIs investigated here, compounds
24a and 25a, which show excellent hCA II and XII inhibitory
properties (Table 1), were investigated in vivo, for their ability
to lower IOP in carbomer-induced glaucoma in rabbits.10c
Normal IOP in rabbits, like in humans, is around 15−20
distance (Å)
angle (deg) with His94
angle (deg) with His96
angle (deg) with His119
111.3
114.2
119.4
107.3
112.2
128.3
108.8
112.9
126.1
Figure 3. View of compounds 23a (cyan), 24a (green), and 26a (magenta) superposed in the active site of hCA II. The left and right panel depict
overall and zoomed active-site views, respectively. hCA II is depicted as a gray surface representation. The active-site zinc is depicted as a blue sphere.
The figure was made using PyMOL (DeLano Scientific).
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mmHg. In this model, the IOP is quite elevated, thus
mimicking the pathologic situation observed in the human
disease.10 Clinically used drugs such as DZA induce a maximal
IOP lowering of 4−5 mmHg, as reported recently by us.10 The
two DTCs investigated here in detail were chosen both due to
their excellent enzyme inhibitory activity in vitro and also
because they show very good water solubility, being formulated
at 2% eye drop solution at neutral pH (due to their salt
character, whereas DZA is formulated at pH 5.5, as a
hydrochloride salt, and induces eye irritation).10 In fact, water
solubility of eye drugs is a significant problem,8−10 with many
classes of drugs achieving an acceptable solubility only as salts
with strong acids, such as HCl, which leads to acidic pH values
causing eye irritation (DZA is a well-known case).10
micromolar. SARs for the inhibition of isoforms hCA I, II,
IX, and XII were straightforward and slightly different, with
small modifications in the backbone of the compound leading
to dramatic changes of biological activity. The inhibition
mechanism of the DTCs was also explained, by resolving the X-
ray crystal structure for hCA II complexed with a heterocyclic
−
DTC. The CS2 moiety present in DTCs represents a new
zinc-binding function. It is directly coordinated to the Zn(II)
ion from the enzyme active site and also participates in an
interaction (hydrogen bond) with the OH moiety of Thr199,
an amino acid essential for the binding of many classes of CAIs
(and of the substrates). The organic scaffold of the DTC is
deeply buried within the enzyme active site and also
participates in favorable interactions with it, which leads to a
high stabilization of the enzyme−inhibitor adduct. Some of the
most potent CAIs detected here showed favorable IOP
lowering effects in an animal model of glaucoma. Being
water-soluble, with the pH of the solution in the neutral range
and with duration of action lasting up to 4−8 h, this new class
of CAIs may constitute interesting candidates for developing
novel antiglaucoma therapies, a field in which no new drug has
emerged in the last 15 years.
Rabbits were treated with 2% solutions of DTCs 24a and
25a, and their IOP was monitored for 48 h (Figure 4a). The
EXPERIMENTAL PROTOCOLS
■
1
Chemistry. H, 13C, DEPT, COSY, HMQC, and HMBC spectra
were recorded using a Bruker Advance III 400 MHz spectrometer. The
chemical shifts are reported in parts per million (ppm), and the
coupling constants (J) are expressed in Hertz (Hz). For all new
compounds, DEPT, COSY, HMQC, and HMBC were routinely used
1
to definitely assign the signals of H and 13C. Infrared spectra were
recorded on a Perkin-Elmer Spectrum R XI spectrometer as solids on
KBr plates. Melting points (mp) were measured in open capillary
tubes, unless otherwise stated, using a Buchi Melting Point B-540
̈
melting point apparatus and are uncorrected. Thin-layer chromatog-
raphy (TLC) was carried out on Merck silica gel 60 F254 aluminum-
backed plates. Elution of the plates was carried out using ethyl acetate/
n-hexane or MeOH/DCM systems. Visualization was achieved with
UV light at 254 nm, by dipping into a 0.5% aqueous potassium
permanganate solution, by Hanessian's stain solution, and heating with
a hot air gun or by exposure to iodine.
All other solvents and chemicals were used as supplied from Aldrich
Chemical Co., Acros, Fisher, Alfa Aesar, or Lancaster Synthesis.
Aniline 1, morpholin-4-amine 2, 4-methylpiperazin-1-amine 3, ( ) sec-
butylamine 4, 2-morpholinoethanamine 5, N1,N1-bis(2-aminoethyl)-
ethane-1,2-diamine 6 (Tris), benzylamine 7 (CAS 100-46-9), pyridin-
4-ylmethanamine 8 (CAS 3731-53-1), 2′-(piperidin-1-yl)ethanamine 9,
2-aminothiazole 10, glycine 11, 3-(1H-imidazol-1-yl)propan-1-amine
12, sodium dimethyldithiocarbamate 13a, sodium diethyldithiocarba-
mate 14a, pyrrolidine 15, diisobutylamine 16, dipropylamine 17,
dibutylamine 18, dihexylamine 19, ethylbutyamine 20, diethanolamine
21, N-methylbenzenamine 22, N,N-benzylmethylamine 23, morpho-
line 24, piperazine 25, 4-cyano-4-phenylpiperidine hydrochloride 26,
and L-proline 27 were purchased from Sigma-Aldrich (Milan, Italy)
and were of the highest available purity. The purity of the prepared
DTCS has been determined by HPLC and was >99%.
General Procedure for the Synthesis of Compounds 1a−
27a.30 Secondary/primary amines 1−27 (1.0 g, 1.0 equiv) were
treated with a NaOH, KOH, or Et3N (1.0−2.2 equiv), 4.0 mL of
MeOH as a cosolvent was used, and the solutions were stirred at 0 °C
for 20 min (Scheme 1). Then, carbon disulfide (1.2−2.4 equiv) was
added dropwise, and the mixture was stirred at r.t. until starting
material was consumed (TLC monitoring). The solvents were
removed under vacuo at r.t., and the residues obtained were dissolved
in MeOH and filtered off trough Celite, and the filtrate was
concentrated in vacuo not exceeding 20 °C.
Figure 4. (A) IOP lowering vs time of glaucomatous rabbits treated
with one drop (50 μL) of a 2% water solution of DTC 24a and 25a.
(B) IOP in the eyes treated with vehicle (IOP are the means from
three different animals). Error bars are shown in both figures,
accounting for an average of 0.8−1.5 mmHg.
contralateral eye was treated with vehicle and was used as
control (Figure 4B). As observed from Figure 4, both
compounds were effective in reducing elevated IOP time
dependently for a rather long period. The maximal effect (of
−6−10 mmHg) has been observed after 2 h postadministra-
tion, and it lasted for up to 4−8 h, being almost double that
reported for DZA (of 4−5 mmHg and lasting only for about
3−4 h).10 DTC 24a was slightly more effective than 25a as an
IOP lowering agent. The IOP in vehicle-treated eyes was rather
constant during the entire duration of the experiments, varying
between 37 and 39 mmHg for animals treated with 25a and
between 37 and 40 mmHg for animals treated with 24a (the
data of the figures are the mean for three different animals, and
the error range is shown in Figure 4).
CONCLUSIONS
■
We report here that DTCs represent a novel class of highly
effective CAIs. DTCs are easy to prepare from simple starting
materials, they can incorporate a very high chemical diversity,
and they act as inhibitors of several physiologically relevant CA
isoforms, with potencies from the subnanomolar to the
1726
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Journal of Medicinal Chemistry
Article
Synthesis of Triethylammonium Phenylcarbamodithioate 1a.
Cocrystallization and X-ray Data Collection of CA II
Complex. Cocrystals for the hCA II−24a complex were obtained
using the hanging drop vapor diffusion method.37 Drops of 10 μL (0.3
mM hCA II, 0.7 mM DTC 24a, 0.1% dimethyl sulfoxide, 0.8 M
sodium citrate, and 50 mM Tris-HCl; pH 8.0) were equilibrated
against the precipitant solution (1.6 M sodium citrate and 50 mM
Tris-HCl; pH 8.0) at room temperature (∼20 °C). Crystals were
observed after 5 days. On the basis of visual selection, a crystal of the
complex was cryoprotected by quick immersion into 20% glycerol
precipitant solution and flash-cooled by exposing to a gaseous stream
of nitrogen at 100 K. The X-ray diffraction data were collected using
an R-AXIS IV+2 image plate system on a Rigaku RU-H3R Cu rotating
anode operating at 50 kV and 22 mA, using Osmic Varimax HR optics.
The detector−crystal distance was set to 80 mm. The oscillation steps
were 1° with a 5 min exposure per image. Indexing, integration, and
scaling were performed using HKL2000.38
Aniline 1 (0.5 g, 1.0 equiv) was treated with triethylamine (1.0
equiv) in benzene (0.5 mL) followed by the addition of carbon
disulfide (1.0 equiv) at 0 °C. The mixture was warmed to r.t.
and stirred O.N. at r.t. The solid formed was washed with
diethyl ether and dried under vacuo to afford the titled
compound as a light yellow solid in 51% yield.
Triethylammonium Phenylcarbamodithioate 1a. νmax (KBr)
cm−1: 2960, 2886, 1648, 1599, 1520, 1451. δH (400 MHz, DMSO-
d6): 1.13 (9H, t, J = 6.8, 3 × CH2CH3), 2.98 (6H, brs, 3 × CH2CH3),
6.97 (1H, t, J = 8.0, 4-H), 7.22 (2H, dd, J = 8.3, 8.0, 2 × 3-H), 7.93
(2H, d, J = 8.3, 2 × 2-H), 9.00 (1H, brs, exchange with D2O,
(CH3CH2)3N+-H), 10.10 (1H, brs, exchange with D2O, N-H). δC (100
MHz, DMSO-d6): 10.0, 46.6, 114.8, 122.9, 128.4, 143.2, 215.5 (CS).
m/z (ESI), 168 [M − Na]−.
Structure Determination of CA II Drug Complex. Starting
phases were calculated from Protein Data Bank (PDB) entry 3KS339
with waters removed. Refinement using the Phenix package,40 with 5%
of the unique reflections selected randomly and excluded from the
refinement data set for the purpose of Rfree calculations,41 was
alternated with manual refitting of the model in Coot.42 The validity of
the final model was assessed by PROCHECK.43 Complete refinement
statistics and model quality are included in Table 2.
Diisobutylcarbodithioic Acid Sodium Salt 16a. mp 220 °C with
dec. νmax (KBr) cm−1: 2961, 2933, 2867, 1640, 1601, 1520, 1480, 1090.
δH (400 MHz, DMSO-d6) 0.84 (12H, d, J = 6.8, 4 × CH3), 2.43 (4H,
m, 2 × CH), 3.86 (4H, d, J = 7.2, 2 × CH2). δC (100 MHz, DMSO-
d6): 21.2, 27.4, 61.5, 215.4 (CS). m/z (ESI), 204 [M − Na]−. Data
are in agreement with reported data.30
Animals and Glaucoma Induction. Adult male New Zealand
Albino rabbits weighing 2−2.5 kg were employed in this study. The
animals were utilized in groups of eight for each of the chosen specific
treatments. The experimental procedures conformed to those of the
Declaration of Helsinki and with the Guideline for the Care and Use of
Laboratory Animals as adopted and promulgated by the U.S. National
Institute of Health and were conducted upon authorization of the
Italian Regulations on Protection of Animals used for experimental
and other scientific purpose (DM 116/1992) as well as with the
European Union Regulations (OJ of ECL 358/1, 12/12/1986), and
the experimental protocol was approved by the local animal care
committee of the University of Florence (Florence, Italy). The rabbits
were kept in individual cages; food and water were provided ad
libitum. The animals were identified with a tattoo on the ear,
numbered consecutively, and maintained on a 12−12 h light/dark
cycle in a temperature-controlled room (22−23 °C). All selected
animals were examined before the beginning of the study and were
determined to be normal on ophthalmic and general examinations.
Glaucoma was induced by injection of 0.1 mL of 0.25% carbomer
(Siccafluid, FarMilaTHEA Pharmaceuticals) into anterior eye
chamber bilaterally in New Zealand albino rabbits] anesthetized with
tiletamine and zolazepam (Zoletil 100, 0.05 mg/kg b.w.) plus xilazine
(Xilor 2%, 0.05 mL/kg b.w.) i.m., by the procedure previously
reported.10c IOP was measured before carbomer injection and after 1,
2, and 4 h the first day and three times a day until stabilization and
then every 24 h. All rabbits treated with carbomer presented a net
increase in IOP. One drop of 0.2% oxybuprocaine hydrochloride
(Novesine, Sandoz) diluted 1:1 with sterile saline was instilled in each
eye immediately before each set of pressure measurements. IOP was
measured using a Tono-Pen XL tonometer (Medtronic Solan, United
States) as reported by earlier.10 The pressure readings were matched
with two-point standard pressure measurements at 1, 2, 4, and 8 h after
the instillation of the drug and once a day for the following days using
a Digilab calibration verifier. All IOP measurements were done by the
same investigators using the same tonometer. As soon as a stable IOP
increase was obtained, the animals were treated with the drugs in
study. The efficacy of the different drugs in lowering IOP was
evaluated after drug administration over 4 hours, with the following
schedule: before and after 30, 60, 90, 120, and 240 min after drug
administration. The treatment was performed in three animals per
drug in one eye and compared to the contralateral eye treated with
vehicle. A group of four nonglaucomatous albino rabbits was treated
with the drugs of this study and used as control. At the end of the
experiments, the animals were killed with a lethal dose of Pentothal
(Abbott S.p.A., Campoverde di Aprilia, LT).
Synthesis of Morpholinecarbamodithioate Sodium Salt 24a.
Morpholine 24 (1.0 g, 1.0 equiv) was treated according to the
general procedure with 1.0 M aqueous solution of NaOH (1.0
equiv) followed by the addition of carbon disulfide (1.2 equiv).
The title compound was obtained as a white solid in
quantitative yield.
Morpholinecarbamodithioate Sodium Salt 24a. mp 320 °C with
dec. νmax (KBr) cm−1: 2966, 2901, 2854, 1625, 1520, 1416, 1215. δH
(400 MHz, DMSO-d6): 3.52 (4H, t, J = 8.0 CH2), 4.33 (2H, t, J = 8.0,
CH2). δC (100 MHz, DMSO-d6): 50.6, 67.1, 215.4 (CS). m/z (ESI),
162 [M − Na]−.
CA Inhibition. An Applied Photophysics stopped-flow instrument
has been used for assaying the CA-catalyzed CO2 hydration activity.
Phenol red (at a concentration of 0.2 mM) has been used as an
indicator, working at the absorbance maximum of 557 nm, with 20
mM Hepes (pH 7.5) as the buffer, and 20 mM Na2SO4 (for
maintaining constant the ionic strength), following the initial rates of
the CA-catalyzed CO2 hydration reaction for a period of 10−100 s.32
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 determined in the same manner and subtracted
from the total observed rates. Stock solutions of inhibitor (0.1 mM)
were prepared in distilled−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
temperature prior to assay, to allow for the formation of the E−I
complex. The inhibition constants were obtained by nonlinear least-
squares methods using PRISM 3, as reported earlier,33 and represent
the mean from at least three different determinations. All CA isofoms
were recombinant ones obtained in house as reported earlier.9,10
1727
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Journal of Medicinal Chemistry
Article
Pastorek, J. Hypoxia activates the capacity of tumor-associated
carbonic anhydrase IX to acidify extracellular pH. FEBS Lett. 2004,
577, 439−444. (b) 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,
ASSOCIATED CONTENT
■
S
* Supporting Information
Complete characterization of compunds 1a−27a. This material
is available free of charge via the Internet at http://pubs.acs.org.
P.; Kanopka, A.; Gorlach, A.; Gasmann, M.; Harris, A. L.; Maxwell, P.;
̈
Accession Codes
The atomic coordinates of the hCA II-dithiocarbmate adduct
(code 3P5A) have been deposited in the Protein Data Base.
Scozzafava, A. Taking advantage of tumor cell adaptations to hypoxia
for developing new tumor markers and treatment strategies. J. Enzyme
Inhib. Med. Chem. 2009, 24 (S1), 1−39.
(6) (a) Dubois, L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran,
C. T.; Scozzafava, A.; Wouters, B. G.; Lambin, P. Imaging of CA IX
with fluorescent labelled sulfonamides distinguishes hypoxic and (re)-
oxygenated cells in a xenograft tumour model. Radiother. Oncol. 2009,
AUTHOR INFORMATION
Corresponding Author
*Tel: +39-055-457 3005. Fax: +39-055-4573385. E-mail:
claudiu.supuran@unifi.it.
■
92, 423−428. (b) Ahlskog, J. K. J.; Dumelin, C. E.; Trussel, S.;
̈
Marlind, J.; Neri, D. In vivo targeting of tumor-associated carbonic
anhydrases using acetazolamide derivatives. Bioorg. Med. Chem. Lett.
2009, 19, 4851−4856. (c) Buller, F.; Steiner, M.; Frey, K.; Mircsof, D.;
Notes
The authors declare no competing financial interest.
Scheuermann, J.; Kalisch, M.; Buhlmann, P.; Supuran, C. T.; Neri, D.
̈
ACKNOWLEDGMENTS
■
Selection of Carbonic Anhydrase IX Inhibitors from One Million
DNA-Encoded Compounds. ACS Chem. Biol. 2011, 6, 336−344.
This research was financed by an FP7 EU project (Metoxia)
and a grant from the NIH GM 25154. We thank Dr. G.
Formicola and Dr. T. Somma for their collaboration in
intraocular pressure measurements.
̀
(d) Chiche, J.; Ilc, K.; Laferriere, J.; Trottier, E.; Dayan, F.; Mazure, N.
́
M.; Brahimi-Horn, M. C.; Pouyssegur, J. Hypoxia-inducible carbonic
anhydrase IX and XII promote tumor cell growth by counteracting
acidosis through the regulation of the intracellular pH. Cancer Res.
2009, 69, 358−368. (e) Swietach, P.; Wigfield, S.; Cobden, P.;
Supuran, C. T.; Harris, A. L.; Vaughan-Jones, R. D. Tumor-associated
carbonic anhydrase 9 spatially coordinates intracellular pH in three-
dimensional multicellular growth. J. Biol. Chem. 2008, 283, 20473−
20483.
ABBREVIATIONS USED
CA, carbonic anhydrase; CAI, carbonic anhydrase inhibitor;
DTC, dithiocarbamate
■
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