Xanthates and trithiocarbonates strongly inhibit carbonic anhydrases and show antiglaucoma effects in vivo

Article abstract of DOI:10.1021/jm400414j

Dithiocarbamates (DTCs) were recently discovered as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. A series of xanthates and a trithiocarbonate, structurally related to the DTCs, were prepared by reaction of alcohols/thiols with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of four human (h) isoforms, hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar xanthate/trithiocarbonate inhibitors targeting these CAs were detected. A docking study of some xanthates within the CA II active site showed that these compounds bind in a similar manner with the dithiocarbamates, coordinating monodentately to the Zn(II) ion from the enzyme active site. Several xanthates showed potent intraocular pressure lowering activity in two animal models of glaucoma via the topical administration. Xanthates and thioxanthates represent two novel, promising classes of CA inhibitors.

Full text of DOI:10.1021/jm400414j

Article
pubs.acs.org/jmc
Xanthates and Trithiocarbonates Strongly Inhibit Carbonic
Anhydrases and Show Antiglaucoma Effects in Vivo
Fabrizio Carta,† Atilla Akdemir,‡ Andrea Scozzafava,† Emanuela Masini,§ and Claudiu T. Supuran*,†,∥
†Laboratorio di Chimica Bioinorganica, Polo Scientifico, Universita
̀
degli Studi di Firenze, Rm. 188 Via della Lastruccia 3,
50019 Sesto Fiorentino (Florence), Italy
‡Department of Pharmacology, Faculty of Pharmacy, Bezmialem Vakif University, Vatan Caddesi, 34093 Fatih, Istanbul, Turkey
§Preclinical and Clinical Pharmacology Section, NEUROFARBA Department, Universita
50139 Florence, Italy
̀
degli Studi di Firenze, Viale Pierracini 6,
∥Sezione di Scienze Farmaceutiche e Nutraceutiche, NEUROFARBA Department, Universita
50019 Sesto Fiorentino (Florence), Italy
̀
degli Studi di Firenze,
ABSTRACT: Dithiocarbamates (DTCs) were recently dis-
covered as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. A
series of xanthates and a trithiocarbonate, structurally related
to the DTCs, were prepared by reaction of alcohols/thiols with
carbon disulfide in the presence of bases. These compounds
were tested for the inhibition of four human (h) isoforms,
hCA I, II, IX, and XII, involved in pathologies such as
glaucoma (CA II and XII) or cancer (CA IX). Several low
nanomolar xanthate/trithiocarbonate inhibitors targeting these
CAs were detected. A docking study of some xanthates within
the CA II active site showed that these compounds bind in
a similar manner with the dithiocarbamates, coordinating
monodentately to the Zn(II) ion from the enzyme active site.
Several xanthates showed potent intraocular pressure lowering activity in two animal models of glaucoma via the topical
administration. Xanthates and thioxanthates represent two novel, promising classes of CA inhibitors.
means of one of its sulfur atoms. The same sulfur made a
hydrogen bond to the OH of Thr199, whereas a second sulfur
atom participated to another hydrogen bond with the NH group
of the same amino acid residues, Thr199. This binding mode
explained the rather good affinity of this inhibitor to many of
the CA isoforms investigated to date.7a On the basis of this
binding mode of a millimolar inhibitor, trithiocarbonate (CS32−),
we discovered that other compounds incorporating this new
zinc-binding group (ZBG), i.e., the CS2− one, such as the
dithiocarbamates (DTCs), act as even stronger CAIs. These
inhibitors bind to the Zn(II) ion from the enzyme active site
similar to the inorganic anion trithiocarbonate (Figure 1B).9−11
Indeed, we have recently demonstrated that many primary/
secondary dithiocarbamates, possessing the general formula
R1R2N-CS2−M+ act as highly efficient CAIs against many α
and β class CAs (of mammalian, fungal, or bacterial origin).9−11
X-ray crystal structures were also reported9,11 for three DTCs
complexed to hCA II, compounds A−C (Figure 1B). These
DTCs inhibited isoform hCA II with KIs of 25, 41, and
0.95 nM, respectively, and the transmembrane, tumor-
associated hCA IX with KIs of 53, 757, and 6.2 nM, res-
pectively.9,11 As seen from Figure 1B, the binding mode of the
INTRODUCTION
■
The classical carbonic anhydrase (CA, EC 4.2.1.1) inhibitors
(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 which act as prodrugs and bind in
a very different mode compared to sulfonamides and their
isosteres,4 whereas some polyamines (such as spermine),5 as well
as a range of phenols6 were also investigated and showed such
interesting properties and novel mechanisms of inhibition.4−6
Among the CAIs investigated to date, there were also the
inorganic anions which coordinate to the zinc ion from the
enzyme active site.1,7,8 Indeed, trithiocarbonate (CS32−), an
anion similar to carbonate, has recently been investigated and
shown to constitute a “lead” for novel CAIs.8 The X-ray crystal
structure for the adduct of trithiocarbonate (CS32−) bound to
hCA II has been reported, being shown that the inhibitor binds
to the Zn2+ in the hCA II active site in a slightly distorted
tetrahedral geometry of the metal ion,7a occupying a position
similar to that observed in the case of hCA II−bicarbonate
complex (Figure 1A).7b Trithiocarbonate was found mono-
coordinated to the Zn(II) ion from the enzyme active site by
Received: March 21, 2013
© XXXX American Chemical Society
A
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but the organic scaffold present in the DTCs was observed to
make extensive contacts with several amino acid residues from
the active site, which explains the wide range of inhibitory
power of these derivatives (from the subnanomolar to the
micromolar range, for the entire series of around 30 DTCs
reported so far).9−11 For example, the benzyl (hydrophobic)
moiety of A was observed orientated toward the hydrophilic
half of the hCA II active site. For derivative B, the cyano
fragment pointed toward the hydrophilic part whereas the
phenyl moiety was orientated toward the hydrophobic half
of the hCA II cavity (Figure 1B). Interestingly, the small and
compact morpholine dithiocarbamate C was found in the
middle of the active site cavity, and its six-membered ring was
not superposable to the one present in the compound B. Such
a variability of orientation of the scaffold is of great interest
for designing CAIs, as in the case of the sulfonamides this
variability was not observed even considering the huge number
of sulfonamides for which the structure is available in complex
with various CA isoforms.1−3 The highly water-soluble DTC C
was also effective in vivo as an antiglaucoma agent when
administered topically directly to the eye of hypertensive
rabbits,11 a widely used animal model of glaucoma.12
Xanthates and thioxanthates (trithiocarbonates) contain the
same ZBG found in CS32− and in DTCs, and such compounds
have never been investigated as CAIs. Here we report that the
xanthates (RO-CS2 M+) and thioxanthates (organic trithiocar-
−
bonates, RS-CS2 M+) represent two new classes of potent
−
CAIs, with a mechanism of action different from that of the
sulfonamides but similar to that of the DTCs. Furthermore, we
prove that some of these highly water-soluble compounds
possess excellent intraocular pressure (IOP) lowering proper-
ties in an animal model of glaucoma,12 making them interesting
candidates for the development of antiglaucoma drugs based on
inhibitors of CAs.
Chemistry. The rationale of this work was a very simple
one: considering CS3 and DTCs (R1R2N-CS2 M+) as lead
molecules, we replaced the nitrogen atom from the DTCs
by the oxygen or sulfur atoms, leading thus to xanthates
(RO-CS2−M+) and trithiocarbonates (RS-CS2−M+), respectively.
Xanthates 1−18, and the thioxanthate (19) (as well as one
DTC, 20) were prepared as illustrated in Scheme 1, by reaction
2−
−
Figure 1. (A) Trithiocarbonate (CS32−), a recently investigated CAI,8
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 hCA II−bicarbonate complex. The protein
zinc ligands (His94, 96, and 119) and Thr199 are shown (PDB code
3K7K). (B) Structural superposition of dithiocarbamates CAIs bound
to hCA II (as determined by X-ray crystallography).9,11 A is shown in
orange (PDB code 3P58), B in yellow (PDB code 3P5L), and C is
reported in blue (PDB code 3P5A). The hCA II active site is
represented as a surface model (hydrophobic side in red, hydrophilic
side in blue).
Scheme 1. Preparation of Xanthates 1, 2, 4−17,
Trithiocarbonate (Thioxanthate) 19, and DTC 20 from the
Corresponding Alcohol, Thiol or Amine and Carbon
a
Disulfide in the Presence of Base
a
Compounds 3 and 18 were purchased.
of the corresponding nucleophile (alcohol, thiol, or amine) and
CS2 in the presence of a strong base, KOH, by the literature
procedures.9−13
The moieties R present in compounds 1−19 reported here,
include simple aliphatic (C1−C8), cycloaliphatic (cyclopentyl,
cyclohexyl, saturated rings), arylalkyl-, heterocyclyl, as well as
polycyclic rings, possibly substituted with aminoalkyl or Boc-
protected-amino moieties. These scaffolds were incorporated in
these compounds in order to generate chemical diversity, as it
has been observed for the DTCs investigated earlier9−11 that
the nature of the organic scaffold is the main factor influencing
inhibitory activity against various CA isoforms (and also that its
ZBG present in A−C is identical to that of trithiocarbonate
(Figure 1B), with one sulfur atom coordinated to the zinc ion,
B
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binding to the enzyme active site is quite variable, as mentioned
above; see also Figure 1B). A special mention regards the only
DTC investigated here, compound 20, which was obtained
by reaction of cyanamide (H2NCN) with carbon disulfide and
potassium hydroxide (Scheme 1). Compound 20 does not
possess an organic scaffold as compounds 1−19 or the DTCs
investigated earlier,9−11 being thus a special case of quite a simple,
almost “inorganic”, derivative.
CA Inhibition. Compounds 1−20 were assayed14 for the
inhibition of four physiologically relevant CA isoforms, hCA I,
II, IX, and XII. All of them are drug targets: hCA I, II, and XII
are for ophthalmologic diseases, mainly glaucoma,1 whereas CA
IX and XII are for antitumor drugs/tumor imaging agents.1,2
Inhibition data with the sulfonamide, clinically used agent
acetazolamide (AAZ, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide)
are also reported in Table 1 for comparison.
a
Table 1. Human (h) CA I, II, IX and XII Inhibition Data with Xanthates 1−18, Thioxanthate (Trithiocarbonate) 19 and
Dithiocarbamate 2014
a
*From three different CO2 hydrase stopped-flow method assay, mean standard error (from three different determinations).14
C
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The following structure−activity relationship (SAR) can be
observed for the CA inhibition data with 1−20 investigated
here:
Thus, also here SAR is straightforward, with few scaffolds (e.g.,
arylalkyl and hetarylalkyl), leading to highly effective xanthate
hCA IX inhibitors..
i. . The cytosolic isoform hCA I was modestly inhibited by
compounds 1−9 (xanthates) and DTC 20, with KIs in the
range of 327−795 nM. Thus, the aliphatic, n-alkyl, and
cycloalkyl xanthates were ineffective CAIs, together with the
cyanamide DTC 20. Medium inhibitory activity was seen for
some of the xanthates investigated here, i.e., compounds 10,
13−18, and the trithiocarbonate 19, which showed inhibition
constants in the range of 45.1−81.3 nM, being thus much
more effective CAIs against this isoform compared to the
sulfonamide acetazolamide (AAZ) (KI of 250 nM). These
derivatives incorporate a longer aminoalkyloxy moiety (de-
rivative 10) as well as arylalkyl or polycyclic, bulkier scaffolds
(compounds 13−18). Xanthates 11 and 12, incorporating Boc-
substituted aliphatic alcohols (C4 and C6) were on the other
hand highly effective hCA I inhibitors, with KIs in the range of
5.6−6.3 nM. Thus, the structure−activity relationship (SAR)
for the inhibition of hCA I is rather straightforward: alkyl- and
cycloalkyl xanthates with C1−C8 carbon atoms are weak
inhibitors, whereas activity increases with the incorporation of
heteroatoms in the aliphatic chain (as in 10−12) or the
presence of arylalkyl and polycyclic moieties (as in 13−18)..
ii. . Against the physiologically dominant isoform hCA II, the
C1−C8 aliphatic xanthates 1−8 showed again weak inhibitory
power, with KIs in the range of 293−366 nM (with a rather
irrelevant variatiation in the inhibitory power with the increase
of the chain from C1 to C8, or its ramification/cyclization).
All these compounds incorporate similar alkyl or cycloalkyl
moieties and showed a compact behavior of weak CAIs. A
number of other derivatives, among which the xanthates 12, 13,
16, the thioxanthate 19, and the DTC 20 were medium
potency hCA II inhibitors, with KIs in the range of 21.5−75.7
nM (Table 1). Some of the xanthates investigated here, i.e., 10,
11, 14, 15, 17, and 18, were highly effective, low nanomolar
hCA II inhibitors, with KIs in the range of 5.4−13.1 nM (being
more effective or similar as the clinically used sulfonamide
AAZ). Interestingly, they incorporate various scaffolds, such as
the bulky 1,1-diphenylethyl one in 14, the adamantylethyl one
in 17, the cyclohexyl-norbornyl one in 18, as well as the
N,N-dimethylaminoethoxy-ethyl- or Boc-aminobutyl (in 10 and
11, respectively). To be noted that increasing the length of
the scaffold by an ethylenoxy moiety (from 9 to 10) leads to a
7.45-fold increase of hCA II inhibitory potency (Table 1).
However, for the pair of Boc-aminoalkyl derivatives 11 and 12,
the shorter derivative 11 was 6.2-fold a better hCA II inhibitor
compared to the longer analogue 12..
iv. . A rather similar inhibition profile (with hCA IX) was
seen for the inhibition of hCA XII with the investigated
compounds. Indeed, 1−6, 9−12, 16−18, and 20 were weak
hCA XII inhibitors, with KIs in the range of 561−835 nM.
Three of the investigated xanthates, i.e., 7, 8, and 14, were
medium potency hCA XII inhibitors, with KIs in the range of
63.3−75.6 nM. The best hCA XII inhibitors were again, as for
hCA IX, xanthates, 13 and 15, and the thioxanthate 19, with KIs
in the range of 3.6−6.7 nM..
v. . There is a net distinction in the inhibition profile of the
two cytosolic (hCA I and II) over the two transmembrane CA
isoforms (hCA IX and XII) with these new classes of inhibitors.
Indeed, for the cytosolic enzymes, the best, low nanomolar
inhibitors incorporated Boc-aminoalkyl (hCA I) or dipheny-
lethyl, adamanthylethyl, and other such bulkier polycyclic ring
systems. For the transmembrane isoforms, the best inhibitors
incorporated benzyl and 2-pyridylethyl moieties. For example,
18 was a highly selective hCA II inhibitor, 11 and 12 were
selective hCA I-inhibitors, whereas xanthates 13 and 15 and the
thioxanthate 19 were hCA IX/XII selective (over hCA I and II)
inhibitors.
Inhibition Mechanism of Xanthates and Thioxanthates.
To understand the CA inhibition mechanism with xanthates
and thioxanthates, we performed some theoretical studies.
Compounds 10 and 14 were built and converted to three-
dimensional structures using the MOE software package.15 The
−
zinc-binding function was assigned a negative charge (−CS2 )
because it was expected to interact with the Zn2+ ion of the CA
active site as was also observed for the crystal structure of hCA
II in complex with 4-cyano-4-phenylpiperidine-1-carbodithioate
B (PDB file: 3P5L). Subsequently, partial atomic charges were
calculated and the molecules were energy-minimized according
to a steepest-descent protocol using the MMFF94x force field
in MOE. The ligands were saved as a multi-mol2 file.
The crystal structures of hCA II (PDB 3P5L; 1.50 Å) in
complex with 4-cyano-4-phenylpiperidine-1-carbodithioate (B)
was obtained from the Protein Data Bank. The water
molecules, ligand, and DMSO were deleted. Hydrogen atoms
and charges were added to the protein using the protonate 3D
tool of the MOE software package,15 and a steepest descent
energy minimization was applied (MMFF94x forcefield). The
protein was saved as a mol2-file. Compounds 10 and 14 were
docked into the crystal structure of hCA II (PDB 3P5L; 1.50 Å)
using the GOLD Suite docking package16 and the ChemScore
scoring function (25 docking per ligand) (Figure 2). The binding
pocket was defined as all residues within 12 Å of the N22
nitrogen atom of compound B. No restrictions were applied for
the ligand conformations and binding poses during the docking
procedure.
As seen from Figure 2, xanthates 10 and 14 coordinate mono-
dentately to the Zn(II) from the enzyme active site, almost in
a superposable manner with the DTC B for which the X-ray
crystal structure was reported earlier.9 The zinc−sulfur
distances, evidenced in Figure 2B, were of 2.46 Å for xanthate
10, of 2.12 Å for xanthate 14, and of 2.31 Å for DTC B,
respectively. The sulfur atoms of these xanthates/DTC also
form hydrogen bonds with the side chain hydroxyl of Thr199
(for compound 10, of 3.0 Å, for compound 14, of 3.5 Å, for
DTC B, of 3.1 Å; Figure 2). The organic scaffolds of xanthates
10 and 14 adopt extended conformation within the enzyme
iii. . The tumor-associated hCA IX was also modestly
inhibited by xanthates 1−5, 9−12, 16−18, and 20, with KIs in
the range of 322−831 nM. SAR was here more complicated
compared to hCA I and II discussed above, as the ineffective
inhibitors incorporate many types of scaffolds (alkyl, amino-
alkyl, Boc-amino-alkyl, adamantyl, and other polycyclic rings).
The simple DTC 20 was also an ineffective hCA IX inhibitor
(KI of 678 nM). Four of the investigated xanthates, i.e., 6−8
and 14, were medium potency hCA IX inhibitors, with KIs in
the range of 47.2−78.8 nM. They incorporate the long n-alkyl
chain with 8 carbon atoms (compound 8), the cycloalkyl
C5 and C6 rings as well as the bulky diphenylethyl moieties.
Two xanthates, 13 and 15, and the thioxanthate 19, were
highly effective hCA IX inhibitors, with KIs in the range of 4.3−
8.1 nM. They incorporate the benzyl or 2-pyridylethyl moieties.
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IOP Lowering with Xanthate CAIs. Some of the best in
vitro hCA II inhibitors (hCA II is the predominant isoform
from the ciliary bodies, being responsible of aqueous humor
secretion),18 such as derivatives 10, 11, 14, 15, and 17, were
initially screened in a normotensive rabbit model19 for their
possible effects as IOP lowering agents (Figure 3). Data of
Figure 3 show promising activities for most of these
compounds, with a decrease of the IOP of 1.0−3.5 mmHg
after administration of 50 μL of solution of 2% xanthate directly
into the eye. Mention should be made that the standard,
clinically used drug dorzolamide (DZA), a sulfonamide CAI, is
ineffective as an IOP lowering agent in this model.
Furthermore, to establish whether this new class of CAI
may be an alternative to the sulfonamides with antiglaucoma
action,18 we have tested their efficacy in a hypertensive rabbit
Figure 2. (a). The docked poses of compounds 10 (green) and 14
(turquoise) inside the active site of hCA II (PDB 3P5L).9 Both
xanthates have their Zn2+-binding CS2− moieties at a similar location as
the one from the DTC 4-cyano-4-phenylpiperidine-1-carbodithioic
acid (ligand B of 3P5L, shown brown). The phenyl groups of ligands
14 and B interact hydrophobically with Phe131 (π-stacking). (b)
Alternative view for the binding of compounds 10, 14, and B to the
hCA II active site (the Zn−sulfur interaction shown as black dotted
lines).
model described earlier by us.12 In the carbomer model,12
a
high IOP of 35−40 mmHg can be achieved, which simulates
better the human diseases characterized by IOP levels in this
range. As seen from data of Figure 4, administration of
xanthates 10, 11, 14, 15, and 17 (50 μL solution of 2% xanthate
directly into the eye) as well as DZA (as standard drug) led to a
strong IOP lowering of 4.0−12.0 mmHg postadministration.
The maximal effect of DZA was of 4 mmHg lowering, at
around 1 h postadministration, whereas many of the xanthate
CAIs were 2−3 times more effective compared to DZA.
The peak of IOP lowering with the xanthates was at around
50−100 min postadministration (depending on the xanthate),
and the effect was also longer lasting compared to DZA
(Figure 4). In fact, an appreciable IOP lowering was still seen
up to 200−250 min postadministration (in the case of DZA,
the effect vanished or was very minor after 150 min). The
structure (and CA inhibitory power of the xanthate) clearly
influenced the IOP lowering effects. The best compound was
14 (KI of 5.4 nM against hCA II), which led to an IOP lowering
of 12 mmHg (3 times as high as the equivalent concentration of
DZA), t = 100 min postadministration. This effect was rather
prolonged, for more than 250 min (Figure 4). The next best
active site (Figure 2A), similar to the binding of the DTC B,
determined by X-ray crystallography.9 Indeed, one phenyl group
of 14 and the phenyl moiety present in the DTC B interacted
with Phe131 through an interesting π-stacking observed earlier
for some sulfonamide derivatives incorporating aromatic
moieties.17 Other amino acid residues involved in the binding
of xanthates 10 and 14, as determined through these docking
studies, were His64, Leu198, and, as mentioned above, Thr199
(Figure 2A,B). All these favorable interactions, in which the two
compounds participate with the metal ion from the enzyme
active site and amino acid residues, may explain the excellent in
vitro inhibition measured against this isoform (of 8.1 nM for 10,
and of 5.4 nM for 14, respectively, see Table 1).
Figure 3. ΔIOP (mm Hg) versus time (min), in normotensive rabbits, treated with 50 μL of 2% solution of xanthates 10, 11, 14, 15, and 17. Errors
were in the range of 10−15% of the reported IOP values (from three different measurements for each of the four animals in the study group) and
were statistically significant (p = 0.045, by Student’s t test). Dorzolamide DZA was ineffective as IOP lowering agent in this model, and its IOP
lowering curve was superimposable to that of the vehicle (within the limits of the experimental error, data not shown).
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Figure 4. ΔIOP (mm Hg) versus time (min), in hypertensive rabbit eyes treated with 50 μL of 2% solution of xanthates 10, 11, 14, 15, and 17, and
dorzolamide DZA as standard drug. Errors were in the range of 10−15% of the reported IOP values (from three different measurements for each of
the four animals in the study group) and were statistically significant (p = 0.045, by Student’s t test).
follows: s, singlet; d, doublet; sept, septet; t, triplet; q, quadruplet; m,
multiplet; brs, 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 ethyl
acetate/n-hexane were used as eluents. Melting points (mp) were carried
out in open capillary tubes and are uncorrected. Purity was determined
by HPLC and was >95% for all the compounds reported in the paper.
O-Isopropylxanthic acid potassium salt 3 and O-tricyclo[5.2.1.02,6]-
dec-9-yl dithiocarbonate potassium salt 18 were purchased from
Aldrich as used
compounds as IOP lowering agents were 11 and 10 (KIs of
9.5 and 8.2 nM, respectively). It is interesting to note that
these derivatives possess a balanced hydro-/lipophilicity, as they
contain both a highly hydrophilic moiety (the xanthate group)
as well as hydrophobic organic moieties.
CONCLUSIONS
■
The xanthates and thioxanthates represent a new class of CAIs.
Compounds with all type of activity were reported from
micromolar to low nanomolar inhibitors against four isoforms:
the cytosolic hCA I and II and the transmembrane hCA IX and
XII. Furthermore, isoform-selective compounds were identified
against all these CAs: 18 was a selective hCA II-inhibitor, 11
and 12 were selective hCA I-inhibitors, whereas xanthates 13
and 15 and the thioxanthate 19 were hCA IX/XII selective
(over hCA I and II) inhibitors. The binding mode of these
compounds to hCA II closely resembles the binding of the
DTCs, which has been investigated by means of X-ray crystallo-
graphy. One sulfur atom of the CS2− ZBG was found coordinated
to the metal ion at a distance of 2.1−2.5 Å; furthermore, the same
sulfur formed a hydrogen bond with the side chain hydroxyl of
Thr199 (of 3.0−3.5 Å), whereas the rest of the scaffold interacted
hydrophobically with amino acid residues involved in the binding
of other classes of CAIs (such as the sulfonamides and the
DTCs), among which are His64, Phe131, and Leu198. In vivo, in
animal models of glaucoma in normotensive/hypertensive rabbits,
some of the effective hCA II inhibitor xanthates showed effective
IOP lowering after direct administration within the eye (as 2%
solutions).
General Procedure for the Synthesis of Compounds 1−20.
Primary alcohols (1.0 equiv) were treated with KOH (1.0 equiv for
1−17, 2.0 equiv for 20) and carbon disulfide (1.2 equiv) in diethyl
ether. The precipitates formed were collected by filtration, washed
with diethyl ether, and dried in vacuo to afford the title products.
Synthesis of Potassium O-Methyl Carbonodithioate Salt 1.
Methanol (3.0 mL) was treated with crushed potassium hydroxide
(1.0 equiv) followed by addition of carbon disulfide (1.2 equiv). The
solution was stirred at rt and then concentrated in vacuo to give a
residue that was triturated from diethyl ether to afford the title product
as a pale-yellow solid. Potassium O-methyl carbonodithioate salt (1):
89% yield; δH (400 MHz, DMSO-d6) 3.40 (s); δC (100 MHz, DMSO-d6)
58.5, 231.4 (CS); m/z (ESI Negative), 106.96 [M − K]−.
EXPERIMENTAL SECTION
■
Synthesis of Potassium (Carbodithioethoxy)ethane 2. Ethanol
(3.0 mL) was treated with crushed potassium hydroxide (1.0 equiv),
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. 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 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
and the solution was treated with carbon disulfide (1.2 equiv). The
solution was stirred at rt and then concentrated in vacuo to give a
residue that was triturated from diethyl ether to afford the title product
F
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as a pale-yellow solid. Potassium (carbodithioatooxy)ethane (2): 90%
yield; δH (400 MHz, DMSO-d6) 2.21 (3H, t, J 6.2, CH3), 4.26 (2H, q,
J 6.2, CH2); δC (100 MHz, DMSO-d6) 15.4, 67.0, 230.8 (CS); m/z
(ESI Negative), 120.98 [M − K]−.
Synthesis of Potassium O-(3-Methyl-butyl) Carbonodithioate
Salt 4. 3-Methyl-butan-1-ol (0.2g, 1.0 equiv) was treated with freshly
Synthesis of Potassium O-Cyclohexyl Carbonodithioate Salt 8.
Cyclohexan-1-ol (0.2g, 1.0 equiv) was treated with freshly crushed
potassium hydroxide (1.0 equiv) and carbon disulfide (1.1 equiv) in
diethyl ether (15 mL). The suspension was stirred at rt for 3 h, and the
white precipitate formed was collected by filtration, washed with
diethyl ether (2 × 15 mL), and dried in vacuo to afford the desired
product as a pale-yellow solid. Potassium O-cyclohexyl carbon-
odithioate salt 8: 67% yield; δH (400 MHz, DMSO-d6) 1.20−2.00
(10H, m), 5.22 (1H, m, 1-H); δC (100 MHz, DMSO-d6) 24.9, 26.2,
32.5, 78.4, 230.2 (CS); m/z (ESI Negative), 175.03 [M − K]−.
Synthesis of Potassium O-(2-Dimethylamino-ethyl) Carbon-
odithioate Salt 9. 2-Dimethylaminoethan-1-ol (0.2g, 1.0 equiv) was
crushed potassium hydroxide (1.0 equiv) and carbon disulfide (1.1
equiv) in diethyl ether (15 mL). The suspension was stirred at rt for
3 h, and the white precipitate formed was collected by filtration,
washed with diethyl ether (2 × 15 mL), and dried in vacuo to afford
the desired product as a light-brown solid. Potassium O-(3-methyl-
butyl) carbonodithioate salt 4: 78% yield; δH (400 MHz, DMSO-d6)
0.91 (6H, d, J 6.8, 2 × CH3), 1.50 (2H, q, J 6.8, 2-H2), 1.69 (1H, m,
3-H), 4.26 (2H, t, J 6.8, 1-H2); δC (100 MHz, DMSO-d6) 23.5, 25.7,
38.4, 69.9, 230.9 (CS); m/z (ESI Negative), 163.03 [M − K]−.
Synthesis of Potassium O-Pentyl Carbonodithioate Salt 5.
Pentan-1-ol (0.2g, 1.0 equiv) was treated with freshly crushed
treated with freshly crushed potassium hydroxide (1.0 equiv) and
carbon disulfide (1.1 equiv) in diethyl ether (15 mL). The suspension
was stirred at rt for 3 h, and the white precipitate formed was collected
by filtration, washed with diethyl ether (2 × 15 mL), and dried in
vacuo to afford the desired product as a pale-yellow solid. Potassium
O-(2-dimethylamino-ethyl) carbonodithioate salt 9: 85% yield; δH
(400 MHz, DMSO-d6) 2.19 (6H, s, 2 × CH3), 2.51 (2H, t, J 6.6,
2-H2), 4.31 (2H, t, J 6.6, 1-H2); δC (100 MHz, DMSO-d6) 46.5, 58.6,
69.4, 230.6 (CS); m/z (ESI Negative), 164.02 [M − K]−.
potassium hydroxide (1.0 equiv) and carbon disulfide (1.1 equiv) in
diethyl ether (15 mL). The suspension was stirred at rt for 3 h, and the
white precipitate formed was collected by filtration, washed with
diethyl ether (2 × 15 mL), and dried in vacuo to afford the desired
product as a pale-yellow solid. Potassium O-pentyl carbonodithioate
salt 5: 95% yield; δH (400 MHz, DMSO-d6) 0.92 (3H, t, J 6.4, CH3),
1.34 (4H, m), 1.62 (2H, m), 4.21 (2H, t, J 6.8, 1-H2); δC (100 MHz,
DMSO-d6) 14.9, 22.9, 28.9, 29.2, 71.4, 231.0 (CS); m/z (ESI
Negative), 163.03 [M − K]−.
Synthesis of Potassium O-[2-(4-Dimethylamino-ethoxy)-ethyl]
Carbonodithioate Salt 10. 2-(4-Dimethylamino-ethoxy)-ethanol
Synthesis of Potassium O-Octyl Carbonodithioate Salt 6. Octan-1-ol
(0.2g, 1.0 equiv) was treated with freshly crushed potassium hydroxide
(0.2g, 1.0 equiv) was treated with freshly crushed potassium hydroxide
(1.0 equiv) and carbon disulfide (1.1 equiv) in diethyl ether (15 mL).
The suspension was stirred at rt for 3 h, and the white precipitate
formed was collected by filtration, washed with diethyl ether (2 ×
15 mL), and dried in vacuo to afford the desired product as a pale-
yellow solid. Potassium O-[2-(4-dimethylamino-ethoxy)-ethyl] car-
bonodithioate salt 10: 80% yield; δH (400 MHz, DMSO-d6) 2.18
(6H, s, 2 × CH3), 2.42 (2H, t, J 6.4, 3-H2), 3.62 (2H, t, J 6.4, 2-H2),
4.33 (2H, t, J 6.4, 1-H2); δC (100 MHz, DMSO-d6) 46.5, 59.3, 69.5,
69.6, 70.7, 230.5 (CS); m/z (ESI Negative), 208.05 [M − K]−.
Synthesis of Potassium O-4-(tert-Butoxycarbonylamino)butyl
Carbonodithioate 11. tert-Butyl 4-hydroxybutylcarbamate (0.1g,
(1.0 equiv) and carbon disulfide (1.1 equiv) in diethyl ether (15 mL).
The suspension was stirred at rt for 3 h, and the white precipitate
formed was collected by filtration, washed with diethyl ether (2 ×
15 mL), and dried in vacuo to afford the desired product as a pale-
yellow solid. Potassium O-octyl carbonodithioate salt 6: 97% yield;
δH (400 MHz, DMSO-d6) 0.90 (3H, t, J 6.4, CH3), 1.31 (10H, m),
1.61 (2H, m), 4.20 (2H, t, J 6.8, 1-H2); δC (100 MHz, DMSO-d6)
14.3, 23.1, 28.7, 28.9, 29.0, 29.1, 29.2, 72.0, 230.9 (CS); m/z (ESI
Negative), 205.07 [M − K]−.
Synthesis of Potassium O-Cyclopentyl Carbonodithioate Salt 7.
Cyclopentan-1-ol (0.2g, 1.0 equiv) was treated with freshly crushed
1.0 equiv) was treated with freshly crushed potassium hydroxide
(1.0 equiv) and carbon disulfide (1.1 equiv) in diethyl ether (15 mL).
The suspension was stirred at rt for 3 h, and the white precipitate
formed was collected by filtration, washed with diethyl ether (2 ×
15 mL), and dried in vacuo to afford the desired product as a pale-
yellow solid. Potassium O-4-(tert-butoxycarbonylamino)butyl carbon-
odithioate 11: 68% yield; δH (400 MHz, DMSO-d6) 1.41 (12H, 2-H2/
3-H2, 3 × CH3), 1.59 (2H, m, 2-H2/3-H2), 2.96 (2H, q, J 6.2, 4-H2),
4.19 (2H, t, J 6.2, 1-H2), 6.84 (1H, brt, J 6.2, exchange with D2O,
NH); δC (100 MHz, DMSO-d6) 25.4, 26.2, 28.2, 28.4, 42.0, 70.0,
154.6, 230.2 (CS); m/z (ESI Negative), 264.07 [M − K]−.
potassium hydroxide (1.0 equiv) and carbon disulfide (1.1 equiv) in
diethyl ether (15 mL). The suspension was stirred at rt for 3 h, and the
white precipitate formed was collected by filtration, washed with
diethyl ether (2 × 15 mL), and dried in vacuo to afford the desired
product as a pale-yellow solid. Potassium O-cyclopentyl carbon-
odithioate salt 7: 67% yield; δH (400 MHz, DMSO-d6) 1.50−1.86
(8H, m, 2 × 2-H2, 2 × 3-H2), 5.61 (1H, m, 1-H); δC (100 MHz,
DMSO-d6) 24.6, 33.3, 82.6, 230.4 (CS); m/z (ESI Negative),
161.01 [M − K]−.
G
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Synthesis of Potassium O-6-(tert-Butoxycarbonylamino)hexyl
Carbonodithioate 12. tert-Butyl 6-hydroxyhexylcarbamate (0.1g,
137.3, 149.9, 159.8, 230.6 (CS); m/z (ESI Negative), 198.01
[M − K]−.
Synthesis of Potassium O-Methyl-1-adamantyl Carbonodithioate
16. Adamantane-methyl-1-ol (0.1g, 1.0 equiv) was treated with freshly
1.0 equiv) was treated with freshly crushed potassium hydroxide
(1.0 equiv) and carbon disulfide (1.1 equiv) in diethyl ether (15 mL).
The suspension was stirred at rt for 3 h, and the white precipitate
formed was collected by filtration, washed with diethyl ether (2 ×
15 mL), and dried in vacuo to afford the desired product as a pale-
yellow solid. Potassium O-6-(tert-butoxycarbonylamino)hexyl carbon-
odithioate 12: 70% yield; δH (400 MHz, DMSO-d6) 1.30 (4H, m, 2 ×
CH2), 1.41 (12H, s, CH2, 3 × CH3), 1.60 (2H, m, CH2), 2.93 (2H, q,
J 6.2, 6-H2), 4.20 (2H, t, J 6.2, 1-H2), 6.80 (1H, brt, J 6.2, exchange
with D2O, NH); δC (100 MHz, DMSO-d6) 26.4, 27.1, 29.2, 29.5, 30.4,
40.7, 71.4, 78.2, 156.5, 230.9 (CS); m/z (ESI Negative), 292.10
[M − K]−.
crushed potassium hydroxide (1.0 equiv) and carbon disulfide (1.1
equiv) in diethyl ether (15 mL). The suspension was stirred at rt for
3 h, and the white precipitate formed was collected by filtration,
washed with diethyl ether (2 × 15 mL), and dried in vacuo to afford
the desired product as a white solid. Potassium O-methyl-1-adamantyl
carbonodithioate 16: 60% yield; δH (400 MHz, DMSO-d6) 1.56
(6H, s, 3 × 2-H2), 1.67 (6H, m, 3 × 4-H2), 1.98 (3H, brs, 3 × 3-H),
3.84 (2H, s, 1′-H2); δC (100 MHz, DMSO-d6) 26.8, 28.4, 37.8, 40.0,
72.0, 230.0 (CS); m/z (ESI Negative), 241.07 [M − K]−.
Synthesis of Potassium O-Benzyl Carbonodithioate 13. Benzyl
alcohol (0.2g, 1.0 equiv) was treated with freshly crushed potassium
Synthesis of Potassium O-Ethyl-1-adamantyl Carbonodithioate
17. Adamantane-ethyl-1-ol (0.1g, 1.0 equiv) was treated with freshly
hydroxide (1.0 equiv) and carbon disulfide (1.1 equiv) in diethyl ether
(15 mL). The suspension was stirred at rt for 3 h, and the white
precipitate formed was collected by filtration, washed with diethyl
ether (2 × 15 mL), and dried in vacuo to afford the desired product as
a pale-yellow solid. Potassium O-benzyl carbonodithioate 13: 87%
yield; δH (400 MHz, DMSO-d6) 5.38 (2H, s, CH2), 7.32 (5H, m,
Ar−H); δC (100 MHz, DMSO-d6) 72.9, 128.1, 128.8, 129.0, 139.2,
230.2 (CS); m/z (ESI Negative), 182.99 [M − K]−.
crushed potassium hydroxide (1.0 equiv) and carbon disulfide (1.1
equiv) in diethyl ether (15 mL). The suspension was stirred at rt for
3 h, and the white precipitate formed was collected by filtration,
washed with diethyl ether (2 × 15 mL), and dried in vacuo to afford
the desired product as a white solid. Potassium O-2-(pyridin-2-yl)ethyl
carbonodithioate 17: 65% yield; δH (400 MHz, DMSO-d6) 1.43
(2H, t, J 6.8, 2′-H2), 1.54 (6H, s, 3 × 2-H2), 1.69 (6H, m, 3 × 4-H2),
1.95 (3H, brs, 3 × 3-H), 4.28 (2H, t, J 6.8, 1′-H2); δC (100 MHz,
DMSO-d6) 26.4, 28.2, 37.9, 40.0, 42.2, 68.0, 230.2 (CS); m/z (ESI
Negative), 255.09 [M − K]−.
Synthesis of Potassium O-(2,2-Diphenyl-ethyl) Carbonodithioate
Salt 14. 2,2-Diphenyl-ethanol 1 (0.1g, 1.0 equiv) was treated with
Synthesis of Potassium Benzyl Carbonotrithioate 19. Benzylmer-
captan (0.2g, 1.0 equiv) was treated with freshly crushed potassium
freshly crushed potassium hydroxide (1.0 equiv) and carbon disulfide
(1.1 equiv) in diethyl ether (15 mL). The suspension was stirred at rt
for 3 h, and the white precipitate formed was collected by filtration,
washed with diethyl ether (2 × 15 mL), and dried in vacuo to afford
the desired product as a light-brown solid. Potassium O-(2,2-diphenyl-
ethyl) carbonodithioate salt 14: 71% yield; δH (400 MHz, DMSO-d6)
4.48 (1H, t, J 7.6, 2-H), 4.79 (2H, d, J 7.6, 1-H2), 7.22 (2H, m, Ar−H),
7.32 (8H, m, Ar−H); δC (100 MHz, DMSO-d6) 50.5, 73.8, 127.2, 123.0,
129.3, 143.5, 230.4 (CS); m/z (ESI Negative), 295.37 [M − K]−.
Synthesis of Potassium O-2-(Pyridin-2-yl)ethyl Carbonodithioate
15. 2-(Pyridin-2-yl)ethan-1-ol (0.2g, 1.0 equiv) was treated with
hydroxide (1.0 equiv) and carbon disulfide (1.1 equiv) in diethyl ether
(15 mL). The suspension was stirred at rt for 3 h, and the white
precipitate formed was collected by filtration, washed with diethyl
ether (2 × 15 mL), and dried in vacuo to afford the desired product
as a bright-yellow solid. Potassium benzyl carbonotrithioate 19: 66%
yield; δH (400 MHz, DMSO-d6) 4.40 (2H, s, CH2), 7.28 (5H, m, Ar−
H); δC (100 MHz, DMSO-d6) 46.1, 127.1, 129.0, 129.1, 129.6, 140.2,
239.6 (CS); m/z (ESI Negative), 198.97 [M − K]−.
Synthesis of Dipotassium Cyanodithioimidocarbonate 20.12
Cyanamide (0.2g, 1.0 equiv) was dissolved in absolute ethanol (2.0 mL)
freshly crushed potassium hydroxide (1.0 equiv) and carbon disulfide
(1.1 equiv) in diethyl ether (15 mL). The suspension was stirred at rt
for 3 h, and the white precipitate formed was collected by filtration,
washed with diethyl ether (2 × 15 mL), and dried in vacuo to afford
the desired product as a pale-yellow solid. Potassium O-2-(pyridin-2-
yl)ethyl carbonodithioate 15: 90% yield; δH (400 MHz, DMSO-d6)
3.10 (2H, t, J 6.8, 2-H2), 4.56 (2H, d, J 6.8, 1-H2), 7.25 (1H, m, Ar−
H), 7.34 (1H, d, J 8.0, Ar−H), 7.47 (1H, t, J 8.0, Ar−H), 8.52 (1H, d,
J 8.0, Ar−H); δC (100 MHz, DMSO-d6) 38.1, 70.5, 122.4, 124.2,
and treated with carbon disulfide (1.1 equiv), followed by addition of a
solution of potassium hydroxide (2.0 equiv) in the same solvent (5.0 mL).
The solution was stirred at rt for 4 h, and the precipitate formed was
collected by filtration, washed with diethyl ether (3 × 15 mL), and dried
under vacuo to afford the title compounds as a white solid. Dipotassium
cyanodithioimidocarbonate 20: 76% yield; δC (100 MHz, DMSO-d6)
122.4, 228.2 (CS); m/z (ESI Negative), 115.95 [M − 2K]−.
H
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CA Inhibition. An Applied Photophysics stopped-flow instrument
was used for assaying the CA catalyzed CO2 hydration activity. Phenol
red (at a concentration of 0.2 mM) was used as indicator, working
at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5)
as 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.13 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 were 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 in order 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,11
and represent the mean from at least three different determinations.
All CA isoforms were recombinant ones obtained in-house as reported
earlier.9−11
Normotensive Rabbit IOP Lowering Studies. Male white New
Zealand rabbits weighing 1500−2000 g were used in these studies.
Animals were anaesthetized using Zoletil (Tiletamine chloride plus
Zolazepam chloride, 3 mg/kg body wt, im) and injected with 0.1 mL
hypertonic saline solution (5% in distilled water) into the vitreous of
both eyes. IOP was determined using a tonometer (Tono-pen Avia
Tonometer, Reichhert Inc. Depew, NY 14043, USA) prior to hyper-
tonic saline injection (basal) at 0.5, 1.0, 1.5, 3, and 6 h thereafter.
Vehicle (phosphate buffer 7.00 plus DMSO 2%) or drugs were
instilled immediately after the injection of hypertonic saline. Eyes
were randomly assigned to different groups. Vehicle or drug (0.50 mL)
were directly instilled into the conjunctival pocket at the desired doses
(usually 2%).19
Hypertensive Rabbit IOP Lowering Studies. 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 Guidelines for the
Care and Use of Laboratory Animals as adopted and promulgated by
the U.S. National Institutes 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 h−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 the anterior eye chamber
bilaterally in New Zealand albino rabbits anesthetized with tiletamine
and zolazepam (Zoletil 100, 0.05 mg/kg body wt) plus xilazine (Xilor
2%, 0.05 mL/kg body wt) im, by the procedure previously reported.12
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, USA) as reported by earlier.12 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 h, with
the following schedule: before and after 30, 60, 90, 120, 240, and
300 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 SpA, Campoverde di Aprilia, IT).
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +39-055-457-3005. Fax: +0-39-055-457-3385. E-mail:
claudiu.supuran@unifi.it.
Notes
The authors declare the following competing financial
interest(s): The compounds described in the manuscript and
their antiglaucoma action have been patented.
ACKNOWLEDGMENTS
This work was financed in part by two EU projects, Metoxia
and Dynano.
■
ABBREVIATION USED
■
AAZ, acetazolamide; CA, carbonic anhydrase; CAI, CA
inhibitor; hCA, human CA; DZA, dorzolamide; DTC,
dithiocarbamate; IOP, intraocular pressure
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