A class of 4-sulfamoylphenyl-ω-aminoalkyl ethers with effective carbonic anhydrase inhibitory action and antiglaucoma effects

Article abstract of DOI:10.1021/jm501497m

We report a series of 4-sulfamoylphenyl-ω-aminoalkyl ethers as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The structure-activity relationship was drawn for the inhibition of four physiologically relevant isoforms: hCA I, II, IX, and XII. Many of these compounds were highly effective, low nanomolar inhibitors of all CA isoforms, whereas several isoform-selective were also identified. X-ray crystal structures of two new sulfonamides bound to the physiologically dominant CA II isoform showed the tails of these derivatives bound within the hydrophobic half of the enzyme active site through van der Waals contacts with Val135, Leu198, Leu204, Trp209, Pro201, and Pro202 amino acids. One of the highly water-soluble compound (as trifluoroacetate salt) showed effective IOP lowering properties in an animal model of glaucoma. Several fluorescent sulfonamides incorporating either the fluorescein-thiourea (7a-c) or tetramethylrhodamine-thiourea (9a,b) moieties were also obtained and showed interesting CA inhibitory properties for the tumor-associated isoforms CA IX and XII.

Full text of DOI:10.1021/jm501497m

Article
pubs.acs.org/jmc
A Class of 4‑Sulfamoylphenyl-ω-aminoalkyl Ethers with Effective
Carbonic Anhydrase Inhibitory Action and Antiglaucoma Effects
Murat Bozdag,† Melissa Pinard,‡ Fabrizio Carta,† Emanuela Masini,§ Andrea Scozzafava,†
Robert McKenna,‡ and Claudiu T. Supuran*,†,∥
†Polo Scientifico, Neurofarba Department and Laboratorio di Chimica Bioinorganica, Rm 188, Universita
̀
degli Studi di Firenze,
Via della Lastruccia 3, Sesto Fiorentino, Florence 50019, Italy
‡Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville,
Florida 32610, United States
§NEUROFARBA Department, Sezione di Farmacologia, Universita
̀
degli Studi di Firenze, Viale Pieraccini 6, Florence, 50139 Italy
∥NEUROFARBA Deptartment, Sezione di Scienze Farmaceutiche, Universita
50019 Sesto Fiorentino Florence, Italy
̀
degli Studi di Firenze, Via Ugo Schiff 6,
ABSTRACT: We report a series of 4-sulfamoylphenyl-ω-aminoalkyl ethers as carbonic
anhydrase (CA, EC 4.2.1.1) inhibitors. The structure−activity relationship was drawn
for the inhibition of four physiologically relevant isoforms: hCA I, II, IX, and XII. Many
of these compounds were highly effective, low nanomolar inhibitors of all CA isoforms,
whereas several isoform-selective were also identified. X-ray crystal structures of two
new sulfonamides bound to the physiologically dominant CA II isoform showed the
tails of these derivatives bound within the hydrophobic half of the enzyme active site
through van der Waals contacts with Val135, Leu198, Leu204, Trp209, Pro201, and
Pro202 amino acids. One of the highly water-soluble compound (as trifluoroacetate
salt) showed effective IOP lowering properties in an animal model of glaucoma. Several
fluorescent sulfonamides incorporating either the fluorescein-thiourea (7a−c) or
tetramethylrhodamine-thiourea (9a,b) moieties were also obtained and showed
interesting CA inhibitory properties for the tumor-associated isoforms CA IX and XII.
The regulation of pH is a highly important process in all life
INTRODUCTION
■
forms, since many biochemical reactions are tightly regulated
by it.1−3 This is probably the reason why so many genetic CA
families are presently known so that in some organisms, a
multitude of different CA families with many isoforms have
been described, each with specialized functions.6−9 The
necessity of a tight/precise pH regulation may thus explain
why most organisms investigated so far contain multiple CA
isoforms, although they differ significantly by their catalytic
activity, susceptibility to various classes of inhibitors, subcellular
localization, and many other such features.1−3,6−9 For example,
in humans, 15 different CA isoforms, all belonging to the
α-class, have been described.1−3
Most mammals (including humans) possess two blood iso-
forms, denominated CA I and CAII, with a total concentration
of these proteins as high as 0.2 mM.10 However, the catalytic
activity of the human (h) isoform hCA I is much lower
compared with that of hCA II, and in addition, hCA I is also
inhibited by the chloride and bicarbonate present in the plasma,
leaving a lot of questions regarding the physiologic function of
this isoform.10,11 On the other hand, the high activity isoform
hCA II (also known as the “rapid” blood enzyme, to distinguish
Carbon dioxide (CO2) is a very stable form of carbon, the
central element of life on this planet and one of the simplest
molecules that was probably highly abundant in the primeval
earth atmosphere. This gas reacts with water, leading to
H2CO3, which is an unstable compound that is spontaneously
transformed into bicarbonate and protons. However, the
reaction between CO2 and water is particularly slow at pH
values of 7.5 or lower, which is usually the physiologic pH value
in many tissues and organisms.1−3 Carbon dioxide hydration
becomes, on the other hand, very effective at higher pH values,
being instantaneous at pH > 12.1−3 Moreover CO2 is an
important molecule in all life processes, being generated in high
amounts in most organisms.3−7 To catalyze its rapid trans-
formation into bicarbonate, catalysts evolved in all life
kingdoms, that is, the enzymes known as carbonic anhydrases
(CAs, EC 4.2.1.1).1−7 Six genetically diverse such enzyme families
are presently knownthe α-, β-, γ-, δ-, ζ-, and η-CAs6−8with
the last class discovered quite recently.9
CAs not only face the conversion of the high amounts of
CO2 formed in the metabolic processes, transforming it in
bicarbonate and protons, but they also manage the acid−base
equilibria connected to this reaction. In fact, the products
formed in the catalyzed reaction are either ions with strong
buffering activity (bicarbonate) or hydrated protons (H+ ions).
Received: September 28, 2014
Published: October 30, 2014
© 2014 American Chemical Society
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it from the “slow” one, hCA I) is involved in the secretion of
electrolytes in a multitude of tissues, such as the bicarbonate-
rich aqueous humor in the anterior chamber of the eyes, and
the cerebrospinal fluid, but also in pH and CO2 homeostasis
all over the body, as mentioned above.11−13 Other functions
include urine formation and bicarbonate reabsorption in the
kidney tubules; biosynthetic reactions, such as gluconeogenesis,
lipogenesis, and ureagenesis; bone resorption and calcification;
and probably many other less well understood physiological/
pathological processes.11−15 Indeed, a dysregulation of the
activity of these isoforms in one or more tissues has important
pathologic consequences, such as glaucoma, when excessive
aqueous humor is secreted within the eye, with the subsequent
increase in the intraocular pressure (IOP) and edema, when not
enough fluids are secreted/eliminated in the urine, leading to
fluid accumulation in the body, processes in which CA II
together with several other isoforms such as CA IV, XII, and
XIV, are involved in the kidneys, epilepsy (the involvement of
CA II and other brain CA isoforms in this disease is poorly
understood and certainly not irrelevant), and some forms of
cancer, in which CA II was observed to be overexpressed,
alone or together with other isoforms such as CA IX and
XII.11−15 CA II is also involved in other pathologies, such as
acute mountain sickness and apparently, atherosclerosis and
osteoporosis.16
Primary sulfonamides constitute the main class of CA
inhibitors (CAIs), with a number of such derivatives in clinical
use for decades, mainly as antiglaucoma agents, diuretics,
antiepileptics, or antiobesity drugs.1c,d,11−16 Recently, some
sulfonamides with CA inhibitory properties entered phase I
clinical trials as antitumor/antimetastatic agents targeting
hypoxic tumors in which two CA isoforms, CA IX and XII,
are overexpressed.1c,d,17 The search for sulfonamide CAIs with
various potentials in therapeutics is a dynamic research field,
with many new classes being reported constantly and
investigated in detail for inhibitory effects against mammalian
and nonmammalian CAs.1,17,18 Here, we report a class of
4-sulfamoylphenyl-ω-aminoalkyl ethers, a poorly investigated
chemotype in the CAIs landscape, with interesting properties as
antiglaucoma agents as well as for the design of fluorescent
enzyme inhibitors with potential use for imaging CAs in various
tissues.
properties against CA isoforms involved in important
physiologic processes, such as CA II and IV.19b These deriv-
atives showed improved water solubility compared with
structurally similar sulfanilamide derivatives, possessed low
nanomolar inhibitory action against CA II, and were shown to
penetrate eye tissues, arriving at the ciliary processes where the
enzyme is present within the eye, and participating in aqueous
humor secretion.19b Unfortunately no in vivo antiglaucoma
studies have been performed with those compounds that
possessed physicochemical properties appropriate for an
antiglaucoma drug candidate.
Considering these facts, we decided to explore the synthesis
and properties of ethers incorporating the benzenesulfonamide
“head” and aliphatic ether moieties of the type H2N−(CH2)n−
O−C6H4−SO2NH2. As mentioned above, such ethers were not
investigated as CAIs until now, and considering the aromatic
derivatives reported by the Pfizer group,19b the presence of
aliphatic, amino moieties should also promote the water
solubility of the compounds. In fact, a considerable pharma-
cologic problem of the first generation CAIs, such as
acetazolamide, AAZ; methazolamide, MZA; or dichlorophena-
mide, DCP, was their poor water solubility. Only the second
generation drugs, such as dorzolamide, DRZ; and brinzolamide,
BRZ, have an improved water solubility because these two
topically acting antiglaucoma drugs are administered as
hydrochloride salts (both weak amines).
Thus, we designed the following strategy for obtaining the
4-sulfamoylphenyl ω-aminoalkyl ethers reported in this paper
(Scheme 1). Reaction of ω-amino-alcohols 1a−e (n = 2−6)
with tert-butyloxycarbonyl anhydride afforded the Boc-
protected amines 2a−e,20 which by Mitsunobu reaction with
4-hydroxybenzenesulfonamide 3 led to the Boc-protected
derivatives 4a−e.21 Removal of the protecting group in the
presence of trifluoroacetic acid (TFA) afforded the trifluor-
oacetate salts of the 4-sulfamoylphenyl ω-aminoalkyl ethers
5a−e (Scheme 1). The alkyl chain present in the new
derivatives ranged between 2 and 6 carbon atoms to investigate
the influence of the spacer length for the enzyme inhibitory
properties of the new derivatives.
Another aspect in the design of CAIs is related to the use of
such compounds as diagnostic tools, for example, for the
imaging of tumors in which some CA isoforms are overex-
pressed. We have reported, for example, fluorescein-based
sulfonamides (obtained again from sulfanilamide, homosulfani-
lamide or 4-aminoethylbenzenesulfonamide, which were
reacted with fluorescein isothiocyanate)22 that were essential
for demonstrating the role of CA IX/XII in the acidification of
the extracellular tumor milieu and also in the proof-of-concept
RESULTS AND DISCUSSION
■
Chemistry and Drug Design. Benzenesulfonamides
constitute a highly investigated class of CAIs,19a with most
such compounds reported so far being derivatives of
sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesul-
fonamide. Derivatization of the primary aliphatic/aromatic
amino group from these compounds by its transformation into
carboxamides, secondary sulfonamides, ureas, thioureas, or by
reaction with pyrylium salts has led to a considerable number of
new derivatives that showed excellent inhibitory properties
against CA isoforms of medicinal interest, such as CA II, IV,
VA/VB, IX, or XII.16−18,19a This is generally known as the tail
approach for designing CAIs.18c Surprisingly, very few
benzenesulfonamides incorporating ether or thioether moieties
have been reported so far. In fact, only one paper, by Vernier
et al.,19b considered these chemotypes for the design of
sulfonamide-based CAIs. In a very interesting study, these
authors reported compounds of the type Ar-X-Ar′-SO2NH2,
where X was O or S, and Ar, Ar′ aromatic/heterocyclic six-
membered rings, which showed highly effective inhibitory
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Scheme 1. Preparation of the Sulfonamides 4, 5, 7, and 9 Reported in This Paper
studies regarding the druggability of these novel antitumor
targets.23 However, like most aromatic thioureas, the
fluorescent sulfonamides reported earlier showed a rather low
water solubility, which may be a limiting factor for some of their
applications. Thus, we report here novel derivatives that were
prepared by reaction of the 4-sulfamoylphenyl ω-aminoalkyl
ethers 5a−e with fluorescein isothiocyanate 6 or [9-(2-carboxy-
4-isothiocyanato-phenyl)-6-dimethylaminoxanthen-3-ylidene]-
dimethylammonium chloride 8, leading to the novel fluorescent
compounds of types 7a−c and 9a,b, respectively (Scheme 1).
The last compounds (9a,b) incorporate a fluorophore that was
not investigated earlier for its interaction with CAs.
Carbonic Anhydrase Inhibition. Inhibition data against
four physiologically significant CA isoforms, that is, h (human)
hCA I, II, IX, and XII, are shown in Table 1. The following
structure−activity relationship (SAR) can be drawn from the
data of Table 1:
(i) The slow human isoform hCA I effectively inhibited by
some of the sulfonamides investigated here, such as 4a−4e and
7a, 7b, which showed KI values in the low nanomolar range
(5.3−41.2 nM), whereas other derivatives (e.g., 5d, 5e, and 7c)
were medium potency inhibitors, with KI’s of 52.5−90.9 nM.
Like acetazolamide AAZ, some of the new compounds,
among which 5a−5c and 9a, 9b were less effective hCA I
inhibitors, with KI’s of 151−826 nM. Thus, the best hCA I
inhibitors were the Boc-protected derivatives 4, which
showed a rather compact behavior of very effective inhibitor,
except for the compound with the 4-carbon-atoms linker (4c)
which was less effective compared with its congeners 4a, b, d,
and e. The deprotected amines 5 were less effective as
hCA I inhibitors compared with the corresponding Boc deriv-
atives (Table 1). Among the fluorescent CAIs reported here,
the fluorescein-containing compounds 7a and 7b were
effective hCA I inhibitors, whereas the tetramethylrhodamine
derivatives 9a and 9b were much less effective as hCA I
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and one (9a) was an ineffective inhibitor (KI of 609 nM). The
remaining sulfonamides investigated here showed excellent
hCA XII inhibitory activity, with inhibition constants ranging
between 5.4 and 9.6 nM, again with no obvious SAR to be
discussed (Table 1).
(v) Although most of these sulfonamides were effective CAIs
against all four isoforms investigated here, several interesting
selectivity cases were observed: for example, 9a is a hCA IX-
selective sulfonamide inhibitor, with a KI of 9.6 nM against the
tumor-associated isoform and >215 nM against hCA I, II, and
XII (Table 1). Compound 5b effectively inhibits the two
transmembrane isoforms (KI’s of 9.6−17.9 nM); it is a much
less effective inhibitor of the two cytosolic isoforms hCA I and
II (KI’s of 36.6−452 nM).
X-ray Crystallography. To rationalize some of the
inhibition data presented above, two of the novel sulfonamides
reported here, 4c (incorporating the Boc-aminobutyl moiety)
and 5c (incorporating the 4-aminobutyl fragment) were
cocrystallized with hCA II, and their crystal structures were
resolved at a high resolution (Table 2). Both inhibitors were
Table 1. hCA I, II, IX, and XII Inhibition Data of the Newly
Synthesized Sulfonamides 4a−9b and Acetazolamide AAZ as
Standard, by the Stopped Flow CO2 Hydrase Assay24
a
Ki (nM)
compd
hCA I
hCA II
hCA IX
hCA XII
4a
4b
4c
5.3
6.6
5.0
5.1
8.3
5.8
7.2
6.5
41.2
7.9
5.7
7.7
6.6
4d
4e
5a
5b
5c
5.5
7.1
5.7
6.1
5.2
6.9
6.4
649
452
286
52.5
63.0
18.0
17.1
90.9
826
151
250
66.5
36.6
8.9
8.7
88.5
9.6
17.9
32.6
6.6
7.5
5d
5e
7a
7b
7c
8.8
7.3
3.9
6.5
6.5
5.0
8.5
8.6
4.6
7.2
5.7
3.9
8.8
7.1
9a
9b
AAZ
215
43.3
12.1
9.6
609
36.7
5.7
7.9
25.0
Table 2. Crystallographic Statistics for the hCA II Adducts
a
a
of 4c and 5c
Mean from three different assays; errors are in the range of 10% of
the reported value.
hCA II-4c
4RFC
hCA II-5c
4RFD
PDB ID
inhibitors compared with the fluorescein derivatives men-
tioned above.
space group
P21
P21
unit-cell parameters (Å, deg)
a = 42.4, b = 41.3,
c = 71.7, β = 104.1
a = 42.5, b = 41.3,
c = 72.1, β = 104.3
(ii) The physiologically dominant hCA II very effectively
inhibited by most sulfonamides reported here. Indeed, just
four compounds (5a and 5b) as well as 9a,b showed medium
potency activity, with KI’s of 36.6−215 nM. The remaining
sulfonamides showed very effective hCA II inhibitory proper-
ties, with KI’s ranging between 3.9 and 8.9 nM (Table 1) and
are thus more effective than the clinically used drug
acetazolamide AAZ. The SAR is rather clear-cut: the five
BOC-protected derivatives 4a−4e showed a very compact
behavior with basically no variation of the inhibitory power,
with the length of the linker from 2 to 6 CH2 moieties.
However, the situation is changed for the amines 5a−5e, which
on one hand were weaker hCA II inhibitors compared with the
corresponding Boc-protected derivatives and on the other hand
showed an increase in the inhibitory power with an increase in
the linker chain from 2 to 6 CH2 moieties. Indeed, between
compounds 5a and 5e, there is a 17-fold difference in the
inhibitory activity against this isoform. As for hCA I inhibition,
again, the fluorescein-tailed sulfonamides 7a−7c were much
more inhibitory compared with the tetramethylrhodamine
derivatives 9a and 9b.
resolution (Å)
total no. reflections
individual reflections
redundancy
1.80 (1.86−1.80)
71 626
1.63 (1.69−1.63)
105 407
22 386
30 415
3.2
3.5
completeness
98.8 (99.5)
0.163
99.7 (97.9)
0.086
b
Rsym
c
Rcryst/Rfree
0.224/0.260
0.178/0.206
0.010/1.29
rmsd for bond lengths/angles 0.006/1.10
(Å, deg)
av B-factors (Å2)
main/side/ligand
12.3/16.7/
5.3/9.1/
no. protein atoms
2086
54
2114
no. water molecules
127
Ramachandran statistics most 89.4/10.5/0.5
favored and additional/
generously allowed
87.6/11.5/0.9
a
b
Values in parentheses represent highest resolution bin. Rsym
=
c
(∑|I - ⟨I⟩|/∑ ⟨I⟩). Rcryst = (∑|F0 − Fc|/∑ |F0|). Rfree is calculated in
the same way as Rcryst except it is for data omitted from refinement
(5% of reflections for all data sets).
(iii) The tumor-associated, transmembrane isoform hCA IX
was very well inhibited by all derivatives reported here, with KI’s
of 5.8−32.6 nM. The SAR is almost impossible to delineate
because all these compounds show excellent inhibitory
activity. For example, the Boc-protected derivatives 4a−e
have a minimal variation of the inhibition constants, ranging
between 5.8 and 8.3 nM. This variation is slightly higher for the
amines 5 (between 6.5 and 32.6 nM) and is again almost absent
for the fluorescent sulfonamides 7 and 9. It is interesting
to note that for this isoform, both the fluorescein and the tetra-
methylrhodamine derivatives were equally effective as CAIs.
(iv) The other transmembrane isoform investigated here,
hCA XII, was also effectively inhibited by most of the new
sulfonamides reported in this paper. Two compounds, 5a and
9b, were medium potency inhibitors (KI’s of 36.7−88.5 nM),
observed bound within the enzyme active site, coordinating
to the Zn(II) ion by means of the deprotonated nitrogen of
the sulfonamide moiety (Figures 1 and 2), like all other
sulfonamide or sulfamates investigated so far by means of this
technique.1,25,26 The phenyl ring and the rather long,
hydrophobic alkyl tails of both inhibitors were observed to
interact only with residues of the hydrophobic half of the hCA
II active site (as shown in Figures 1−3), such as Val121,
Phe131, Leu198, Pro201, and Pro202. The tail of compound 5c
extends farther out into the enzyme’s hydrophobic cleft,
allowing it to form more stabilizing interactions with amino
acids, such as Pro201 and Leu204; however, the shorter tail of
compound 4c is unable to perform such interactions (Figure 3).
It should be mentioned that for the Boc-protected derivative
4c, the electron density of the tail region was not completely
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Figure 3. Overlay of sulfonamides 4c (orange) and 5c (teal) bound
within the active site of hCA II. Hydrophobic region, red; hydrophilic
region, blue; zinc ion, gray sphere at the bottom of the activity.
Figure 1. Electron density of compound 4c bound within the hCA II
active site. The Zn(II) ion (gray sphere) and its three His ligands
(His94, 96, and 119) as well as other residues involved in the catalytic
cycle or binding with inhibitors are shown. The 2F0 − Fc electron
density is represented by a 0.6σ-weighted gray mesh. Because of the
less-ordered electron density for the tail region of 4c, its map was
contoured at a lower sigma level compared with compound 5c.
properties. In fact, we observed earlier that compounds that
have their tails lying only in the hydrophobic half generally do
not possess isoform-selective inhibitory properties, and this is
also confirmed by the present findings (although neither 4c
nor 5c is highly effective as hCA I inhibitors, Table 1). In fact,
these two compounds inhibit hCA II, IX, and XII in rather
similar ranges and are effective inhibitors against these three
isoforms. Because our interest was to obtain compound with
antiglaucoma activity, for which both hCA II and XII27 effective
inhibitory properties are desired, we consider the present
observations of real interest.
Antiglaucoma Activity. Both the Boc-protected and the
amino derivative sulfonamides reported here showed excellent
water solubility and could be formulated as 2% eye drops at the
neutral pH value (dorzolamide, DRZ, the clinically used drug
is a hydrochloride salt with a pH of the eye drops of 5.5 which
produces eye irritation and stinging as side effects).11 We have
investigated the intraocular pressure (IOP) lowering proper-
ties of some of these compounds, more precisely, 4c and 5c
(for which the X-ray in adduct with hCA II was reported; see
the Discussion section, above), in an animal model of
glaucoma.28 Indeed, both compounds were low nanomolar
inhibitors of isoforms hCA II (responsible for aqueous humor
secretion) and hCA XII (isoform that is overexpressed in the
eyes of glaucomatous patients).27 As seen from the data of
Figure 4, the Boc-protected derivative 4c showed a small
decrease of IOP (of 1−2.5 mmHg) when given topically to
the eye of the animals, whereas the free amine 5c (as a
trifluoroacetate salt) was more effective, with an IOP decrease
of 4.4 mmHg at 2 h postadministration, more effective
than DRZ, the standard drug, which caused an IOP drop of
4 mmHg at 60 min postadministration. Another notable
difference between 5c and DRZ was the fact that the new
compound investigated here had a prolonged efficacy
compared to DRZ, for which after 4 h no IOP decrease was
seen. In contrast, 5c showed efficacy even after 4 h
postadministration, with an IOP drop of 3 mmHg at that
time point. It should be mentioned that the animal model
employed here is of normotensive rabbits,28 and this is why
the absolute IOP drops are not very high, but the advantage of
Figure 2. Electron density of compound 5c bound within the hCA II
active site. The Zn(II) ion (gray sphere) and its three His ligands
(His94, 96, and 119) as well as other residues involved in the catalytic
cycle or binding with inhibitors are shown. The 2F0 − Fc electron
density is represented by a 1.0σ-weighted gray mesh.
defined (Figure 1), probably because of its high flexibility and
disorder when complexed to the enzyme. In contrast, for the
amine 5c, all atoms from the tail region had the electron density
well-defined, proving that this region is less disordered
compared with the Boc-protected compound 4c (Figure 2).
To account for this disorder and to ensure the ligand was built
into the density correctly, the map for compound 4c was
contoured at a lower sigma level (0.6) than that of 5c (1.0).
The fact that the tails of these compounds lie only in the
hydrophobic cleft of the hCA II active site is a noteworthy
finding, because we showed in earlier papers25,26 that the active
site region (hydrophobic versus hydrophilic halves) in which a
sulfonamide binds is indicative of its isoform-selectivity
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Figure 4. Drop of intraocular pressure (ΔIOP, mmHg) versus time (min) in hypertonic saline-induced ocular hypertension in rabbits, treated with
50 μL of 2% solution of compounds 4c, 5c, and DRZ as the standard drug and vehicle. Errors were within 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 the Student’s t test).
dissolved in dichloromethane (DCM) and treated with a 1 M NaOH
aqueous solution or diisopropyl ethylamine (DIPEA) (1.0 equiv), then
di-tert-butyl dicarbonate (1.0 equiv) was added, and the mixture was
vigorously stirred O.N. until consumption of starting materials (TLC
monitoring). The reaction was quenched with a 1 M hydrochloric acid
aqueous solution, neutralized with NaHCO3 aqueous solution, and
extracted with ethyl acetate (3 × 15 mL). The combined organic layers
were washed with H2O (3 × 20 mL), dried over Na2SO4, filtered, and
concentrated under vacuo to give the titled product.
b. General Procedure for Mitsunobu Coupling. Boc-aminoalchol
2a−e (1.0 equiv) was dissolved in dry THF and transferred to a two
neck-flask via cannula, followed by addition of Ph3P (1.0 equiv) and
4-hydroxybenzenesulfonamide (1.0 equiv). Then the solution was
cooled to 0 °C, and diisopropyl azodicarboxylate (DIAD) (1.1 equiv)
was added dropwise. The reaction was warmed to r.t. and stirred at the
same temperature until the starting materials were consumed (TLC
monitoring), quenched with slush, and extracted with ethyl acetate
(3 × 15 mL). The combined organic layers were washed with H2O
(3 × 20 mL), dried over Na2SO4, filtered, and concentrated under
vacuo to give a residue that was purified by silica gel column
chromatography followed by crystallization when necessary.
this model is that the measurements can be done rapidly and
are highly reproducible.14a
CONCLUSIONS
■
We report a series of new 4-sulfamoylphenyl-ω-aminoalkyl
ethers that have been prepared by Mitsunobu reaction.
Interesting SAR has been observed for the inhibition of four
physiologically relevant CA isoforms: hCA I, II, IX, and XII.
Many of the new compounds were highly effective inhibitors of
all these isoforms, in the low nanomolar range, with few
isoform-selective compounds also identified. These findings
have been rationalized by resolving the X-ray crystal structures
of two of the new sulfonamides. The tails of these derivatives
were observed bound only in the hydrophobic half of the
enzyme active site, making van der Waals contacts with amino
acids such as Val135, Leu198, Leu204, Trp209, Pro201. and
Pro202. One of the compounds incorporating a free amine
moiety, which was highly water-soluble as a trifluoroacetate salt,
also showed effective IOP lowering properties in an animal
model of glaucoma. Several fluorescent sulfonamides have also
been reported that incorporate either fluorescein−thiourea or
tetramethylrhodamine−thiourea moieties, which also effectively
inhibited some CA isoforms investigated here.
c. General Procedure for Boc Deprotection. Compounds 4a−e
(1.0 equiv) was dissolved in DCM or 1,4-dioxane and treated with
TFA. The reaction was stirred at r.t. until the starting material was
consumed (TLC monitoring). The solvent was removed under vacuo,
and the obtained residue was crystallized from IPA or triturated from
diethyl ether to obtain the titled compound as a white solid.
Synthesis of tert-Butyl 2-Hydroxyethylcarbamate 2a. Ethanol-
amine 1a (1.0 g, 1.0 equiv) was dissolved in DCM (16.5 mL) and
EXPERIMENTAL PROTOCOLS
■
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 syringe 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 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
measured in open capillary tubes with a Gallenkamp MPD350.BM3.5
apparatus and are uncorrected. All compounds reported here were
>95% HPLC pure.
treated with a 1 M aqueous solution of NaOH (1.0 equiv), and then di-
tert-butyl dicarbonate (1.0 equiv) was added. The reaction mixture was
treated according to the general procedure a, previously reported, to give
the titled compound 2a as a colorless liquid, which was used as it is.
tert-Butyl 2-Hydroxyethylcarbamate 2a: 66% yield; silica gel TLC
Rf 0.18 (ethyl acetate/n-hexane 50% v/v); δH (400 MHz, DMSO-d6)
1.41 (9H, s), 3.0 (2H, q, J 6.0), 3.38 (2H, t, J, 6.0), 4.6 (1H, t, J 6.0,
exchange with D2O, OH), 6.71 (1H, brt, exchange with D2O, NH); δC
(100 MHz, DMSO-d6) 29.2, 43.6, 61.0, 78.4, 156.6. Experimental data
are in agreement with reported data.29
Synthesis of tert-Butyl 3-Hydroxypropylcarbamate 2b. 3-Amino-
1-propanol 1b (0.98 g, 1.0 equiv) was dissolved in DCM (13 mL) and
General Procedure for the Synthesis of O-Alkylbenzene-
sulfonamides 5a−e via Mitsunobu coupling. a. General
Procedure for Boc Protection. Aminoalcohol 1a−e (1.0 equiv) was
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treated with a 1 M aqueous solution of NaOH (1.0 equiv), and then
di-tert-butyl dicarbonate (1.0 equiv) was added. The reaction mixture
was treated according to the general procedure a, previously reported,
to give the titled compound 2b as a colorless liquid, which was used
as it is.
tert-Butyl 3-Hydroxypropylcarbamate 2b: 80% yield; silica gel TLC
Rf 0.18 (ethyl acetate/n-hexane 50% v/v); δH (400 MHz, DMSO-d6) 1.41
(9H, s), 1.55 (2H, pent, J 6.4), 3.0 (2H, q, J 6.4), 3.42 (2H, q, J, 6.0), 4.4
(1H, t, J 6.0, exchange with D2O, OH), 6.76 (1H, brt, exchange with
D2O, NH); δC (100 MHz, DMSO-d6) 29.2, 33.7, 38.1, 60.7, 78.3, 156.5.
Experimental data are in agreement with reported data.30
Synthesis of tert-Butyl 4-Hydroxybutylcarbamate 2c. 4-Amino-1-
butanol 1c (1.45 g, 1.0 equiv) was dissolved in DCM (16 mL) and
according to the general procedure b, previously reported. The
reaction was stirred at r.t. until starting materials were consumed
(TLC monitoring). The reaction was quenched with slush and
extracted with ethyl acetate (3 × 15 mL). The combined organic layers
were washed with H2O (3 × 20 mL), dried over Na2SO4, filtered, and
concentrated under vacuo, and the obtained residue was purified by
silica gel column chromatography eluting with ethyl acetate/n-hexane
60% v/v, followed by crystallization in EtOH/H2O mixture to afford
the titled compound 4a as a white solid.
treated with DIPEA (1.0 equiv) and then di-tert-butyl dicarbonate
(1.0 equiv). The reaction mixture was treated according to the general
procedure a, previously reported, to give the titled compound 2c as a
yellow liquid, which was used as it is.
tert-Butyl 4-Hydroxybutylcarbamate 2c: 70% yield; silica gel TLC
Rf 0.16 (ethyl acetate/n-hexane 50% v/v); δH (400 MHz, DMSO-d6)
1.41 (13H, s), 2.93 (2H, m), 3.40 (2H, m), 4.39 (1H, t, J, 5.0,
exchange with D2O, OH), 6.80 (1H, t, J 6.0, exchange with D2O, NH);
δC (100 MHz, DMSO-d6) 27.2, 29.2, 30.8, 40.7, 61.4, 78.2, 156.5.
Experimental data are in agreement with reported data.31
tert-Butyl 2-(4-Sulfamoylphenoxy)-ethylcarbamate 4a: 40%
yield, silica gel TLC Rf 0.4 (ethyl acetate/n-hexane 60% v/v); mp
148−149 °C; δH (400 MHz, DMSO-d6) 1.42 (9H, s), 3.33 (2H, t,
J 5.6), 4.07 (2H, t, J, 5.6), 7.07 (1H, brt, exchange with D2O, NH), 7.1
(2H, d, J 8.8), 7.24 (2H, s, exchange with D2O, SO2NH2), 7.77 (2H, d,
J 8.8); δC (100 MHz, DMSO-d6) 29.4, 40.3, 68.0, 79.2, 115.7, 128.9,
137.2, 156.9, 162.0. Elemental analysis: calcd C 49.35, H 6.37, N 8.85,
S 10.14; found C 49.43, H 6.03, N 8.68, S 9.97; m/z (ESI negative)
315.6 [M − H]−.
Synthesis of tert-Butyl 5-Hydroxypentylcarbamate 2d. 5-Amino-
1-pentanol 1d (1.5 g, 1.0 equiv) was dissolved in DCM (14.5 mL) and
Synthesis of tert-Butyl 3-(4-Sulfamoylphenoxy)-propylcarbamate
4b. tert-Butyl 3-hydroxypropylcarbamate 2b (0.88 g, 1.0 equiv) was
treated with a 1 M aqueous solution of NaOH (1.0 equiv) and then di-
tert-butyl dicarbonate (1.0 equiv). The reaction mixture was treated
according to the general procedure a, previously reported, to give the
titled compound 2d as a yellow liquid, which was used as it is.
tert-Butyl 5-Hydroxypentylcarbamate 2d: 77% yield; silica gel
TLC Rf 0.3 (ethyl acetate/n-hexane 60% v/v); δH (400 MHz, DMSO-d6)
1.22−1.32 (2H, m), 1.34−1.48 (13H, m) 2.92 (2H, q, J 6.4), 3.39
(2H, m), 4.36 (1H, t, J 5.2, exchange with D2O, OH), 6.79 (1H, brt,
exchange with D2O, NH); δC (100 MHz, DMSO-d6) 23.8, 29.2, 30.3,
33.1, 40.8, 61.6, 78.2, 156.5. Experimental data are in agreement with
reported data.30b
tert-Butyl 6-Hydroxyhexylcarbamate 2e. 6-Amino-1-hexanol 1e
(0.1 g, 1 equiv) was dissolved in DCM (8.5 mL) and treated with a
dissolved in dry THF (4.7 mL) and was treated with Ph3P (1.0 equiv),
4-hydroxybenzenesulfonamide 3 (1.0 equiv), and DIAD (1.1 equiv)
according to the general procedure b, previously reported. The reac-
tion was stirred at r.t. until starting materials were consumed (TLC
monitoring). The reaction was quenched with slush and extracted
with ethyl acetate (3 × 15 mL). The combined organic layers were
washed with H2O (3 × 20 mL), dried over Na2SO4, filtered, and
concentrated under vacuo, and the obtained residue was purified by
silica gel column chromatography eluting with ethyl acetate/n-hexane
55% v/v, followed by crystallization in IPA to afford the titled
compound 4b as a white solid.
DIPEA (1.0 equiv) and di-tert-butyl dicarbonate (1.0 equiv). The
reaction mixture was treated according to the general procedure a,
previously reported, to give the titled compound 2e as a colorless oil,
which was used as it is.
tert-Butyl 6-Hydroxyhexylcarbamate 2e: 81% yield; silica gel TLC
Rf 0.2 (ethyl acetate/n-hexane 50% v/v); δH (400 MHz, DMSO-d6)
1.23−1.34 (4H, m), 1.34−1.48 (13H, m), 2.89 (2H, q, J 6.7), 3.36
(2H, q, J 5.2), 4.3 (1H, t, J 5.2, exchange with D2O, OH), 6.76 (1H,
brt, exchange with D2O, NH); δC (100 MHz, DMSO-d6) 26.2, 27.1,
29.2, 30.5, 33.4, 40.7, 61.6, 78.2, 156.5. Experimental data are in
agreement with reported data.32
Synthesis of tert-Butyl 2-(4-Sulfamoylphenoxy)-ethylcarbamate
4a. tert-Butyl 2-hydroxyethylcarbamate 2a (1.0 g, 1.0 equiv) was
dissolved in dry THF (9.5 mL) and was treated with Ph3P (1.0 equiv),
4-hydroxybenzenesulfonamide 3 (1.0 equiv), and DIAD (1.1 equiv)
tert-Butyl 3-(4-sulfamoylphenoxy)-propylcarbamate 4b: 21%
yield, silica gel TLC Rf 0.35 (ethyl acetate/n-hexane 55% v/v); mp
134−135 °C; δH (400 MHz, DMSO-d6) 1.41 (9H, s), 1.88 (2H, pent,
J 6.4), 3.10 (2h, q, J 6.4), 4.08 (2H, t, J, 6.4), 6.95 (1H, t, J 6.4,
exchange with D2O, NH), 7.09 (2H, d, J 8.8), 7.23 (2H, s, exchange
with D2O, SO2NH2), 7.77 (2H, d, J 8.8); δC (100 MHz, DMSO-d6)
29.2, 30.0, 37.7, 66.6, 78.5, 115.3, 128.6, 137.0, 156.6, 161.9. Elemental
analysis: calcd C 50.89, H 6.71, N 8.48, S 9.70; found C 51.09, H 7.01,
N 8.64, S 9.54; m/z (ESI negative) 329.40 [M − H]−.
Synthesis of tert-Butyl 4-(4-Sulfamoylphenoxy)-butylcarbamate
4c. tert-Butyl 4-hydroxybutylcarbamate 2c (1.11 g, 1.0 equiv) was
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Synthesis of tert-Butyl 6-(4-Sulfamoylphenoxy)-hexylcarbamate
4e. tert-Butyl 6-hydroxyhexylcarbamate 2e (1.37 g, 1.0 equiv) was
dissolved in dry THF (10.0 mL) and was treated with Ph3P
(1.0 equiv), 4-hydroxybenzenesulfonamide 3 (1.0 equiv), and DIAD
(1.1 equiv) according to the general procedure b, previously reported.
The reaction was stirred at r.t. until starting materials were consumed
(TLC monitoring). The reaction was quenched with slush and
extracted with ethyl acetate (3 × 15 mL). The combined organic layers
were washed with H2O (3 × 20 mL), dried over Na2SO4, filtered, and
concentrated under vacuo, and the obtained residue was purified by
silica gel column chromatography eluting with 60% v/v ethyl acetate/
n-hexane to afford the titled compound 4c as a white solid.
dissolved in dry THF (9.0 mL) and was treated with Ph3P (1.0 equiv),
4-hydroxybenzenesulfonamide 3 (1.0 equiv), and DIAD (1.1 equiv)
according to the general procedure b, previously reported. The re-
action was stirred at r.t. until starting materials were consumed (TLC
monitoring). The reaction was quenched with slush and extracted with
ethyl acetate (3 × 15 mL). The combined organic layers were washed
with H2O (3 × 20 mL), dried over Na2SO4, filtered, and concentrated
under vacuo, and the obtained residue was purified by silica gel column
chromatography, eluting with 50% v/v ethyl acetate/n-hexane to afford
the titled compound 4e as a white solid.
tert-Butyl 4-(4-Sulfamoylphenoxy)-butylcarbamate 4c: 22% yield,
silica gel TLC Rf 0.40 (ethyl acetate/n-hexane 60% v/v); mp 93−94 °C;
δH (400 MHz, DMSO-d6) 1.41 (9H, s), 1.56 (2H, m), 1.74 (2H, m),
3.10 (2h, q, J 6.5), 4.08 (2H, t, J 6.5), 6.95 (1H, t, J 5.2, exchange with
D2O, NH), 7.09 (2H, d, J 8.8), 7.23 (2H, s, exchange with D2O,
SO2NH2), 7.77 (2H, d, J 8.8); δC (100 MHz, DMSO-d6) 26.9, 27.0,
29.2, 30.2, 68.6, 78.4, 115.4, 128.6, 137.0, 156.6, 162.0; Elemental
analysis: calcd C 52.31, H 7.02, N 8.13, S 9.31; found C 52.65, H 6.76,
N 8.01, S 8.91; m/z (ESI negative) 343.17 [M − H]−.
tert-Butyl 6-(4-Sulfamoylphenoxy)-hexylcarbamate 4e: 42%
yield; silica gel TLC Rf 0.35 (ethyl acetate/n-hexane 50% v/v);
101−102 °C; δH (400 MHz, DMSO-d6) 1.41 (17H, m), 1.75 (2H, q,
J 8.0), 2.94 (2H, q, J 8.0), 4.08 (1H, t, J 5.2, exchange with D2O, OH),
6.80 (1H, brt, exchange with D2O, NH), 7.11 (2H, d, J 8), 7.23 (2H, s,
exchange with D2O, SO2NH2), 7.77 (2H, d, J 8); δC (100 MHz,
DMSO-d6) 23.6, 24.8, 26.4, 29.2, 30.1, 39.5, 68.3, 78.2, 115.4, 128.7,
136.9, 156.3, 162.1. Elemental analysis: calcd C 54.82, H 7.58, N 7.52,
S 8.61; found C 54.66, H 7.58, N 7.36, S 8.56; m/z (ESI negative)
371.25 [M − H]−.
Synthesis of tert-Butyl 5-(4-Sulfamoylphenoxy)-pentylcarbamate
4d. tert-Butyl 5-hydroxypentylcarbamate 2d (1.2 g, 1.0 equiv) was
Synthesis of 2-(4-Sulfamoylphenoxy)-ethylammonium Trifluo-
roacetate Salt 5a. TFA (7.0 equiv) was added to a stirring mixture of
dissolved in dry THF (9.0 mL) and was treated with Ph3P (1.0 equiv),
4-hydroxybenzenesulfonamide 3 (1.0 equiv), and DIAD (1.1 equiv)
according to the general procedure b, previously reported. The re-
action was stirred at r.t. until starting materials were consumed
(TLC monitoring). The reaction was quenched with slush and
extracted with ethyl acetate (3 × 15 mL). The combined organic
layers were washed with H2O (3 × 20 mL), dried over Na2SO4,
filtered, and concentrated under vacuo, and the obtained residue
was purified by silica gel column chromatography, eluting with 60%
v/v ethyl acetate/n-hexane to afford the titled compound 4d as
a white solid.
tert-Butyl 5-(4-Sulfamoylphenoxy)-pentylcarbamate 4d: 27%
yield, silica gel TLC Rf 0.42(ethyl acetate/n-hexane 60% v/v); mp
95−96 °C; δH (400 MHz, DMSO-d6) 1.37−1.51 (13H, m), 1.73 (2H,
pent, J 6.8), 2.94 (2H, q, J 6.4), 4.04 (2H, t, J 6.4), 6.80 (1H, t, J 5.7,
exchange with D2O, NH), 7.08 (2H, d, J 8.8), 7.20 (2H, s, exchange
with D2O, SO2NH2), 7.75 (2H, d, J 8.8); δC (100 MHz, DMSO-d6)
23.6, 29.1, 29.2, 30.1, 40.6, 68.7, 78.2, 115.3, 128.6, 136.9, 156.5, 162.0;
Elemental analysis: calcd C 53.61, H 7.31, N 7.82, S 8.95; found C
53.50, H 7.01, N 7.87, S 8.69; m/z (ESI negative) 357.60 [M − H]−.
tert-butyl 2-(4-sulfamoylphenoxy)-ethylcarbamate 4a (0.5 g, 1.0 equiv)
in 10 mL of DCM. The reaction was stirred at r.t. according to the
general procedure c, previously reported until starting material was
consumed (TLC monitoring). The solvents were evaporated under
vacuo, and the obtained residue was triturated with diethyl ether and
dried under vacuo to afford the titled compound 5a as a white solid.
2-(4-Sulfamoylphenoxy)-ethylammonium Trifluoroacetate Salt
5a: 88% yield, mp 142−143 °C; δH (400 MHz, DMSO-d6) 3.30 (2H, t,
J 5.2), 4.27 (2H, t, J 5.2), 7.17 (2H, d, J, 8.8), 7.29 (2H, s, exchange with
D2O, SO2NH2), 7.81 (2H, d, J 8.8), 8.02 (3H, brt, exchange with D2O,
NH3); δC (100 MHz, DMSO-d6) 39.2, 65.7, 115.7, 118.2 (d, J1C−F 299),
128.7, 137.8, 159.4 (q, J2
31), 161.2; δF (376 MHz, DMSO-d6)
C−F
−73.5 (3F, s). Elemental analysis: calcd C 36.37, H 3.97, N 8.48, S 9.71;
found C 36.76, H 3.72, N 8.08, S 10.02; m/z (ESI positive) 217.08
[M − CF3COO−]+.
Synthesis of 3-(4-Sulfamoylphenoxy)-propylammonium Trifluo-
roacetate Salt 5b. tert-Butyl 3-(4-sulfamoylphenoxy)-propylcarbamate
4b (0.8 g, 1.0 equiv) was dissolved in 1,4-dioxane (10.0 mL), followed
by addition of TFA (50.0 equiv). The reaction was treated according
to the general procedure c, previously reported (TLC monitoring).
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triturated from diethyl ether to afford the titled compound 5d as a
white solid.
5-(4-Sulfamoylphenoxy)-pentylammonium Trifluoroacetate Salt
5d: 90% yield; mp 121−122 °C; δH (400 MHz, DMSO-d6) 1.50
(2H, m), 1.63 (2H, m), 1.79 (2H, m), 2.85 (2H, m), 4.09 (2H, t,
J 6.0), 7.10 (2H, d, J 9.0), 7.24 (2H, s, exchange with D2O, SO2NH2),
+
7.71 (3H, brt, exchange with D2O, NH3 ), 7.77 (2H, d, J 9.0); δC (100
MHz, DMSO-d6) 23.4, 27.6; 28.9, 39.6, 68.6, 115.3, 118.1 (d, J1
C−F
298), 128.6, 137.0, 159.1 (q, J2C−F 31), 161.9; δF (376 MHz, DMSO-d6)
−73.5 (3F, s). Elemental analysis: calcd C 41.93, H 5.14, N 7.52, S
8.61; found C 41.73, H 5.23, N 7.25, S 8.34; m/z (ESI positive) 259.17
[M-CF3COO−]+.
The solvents were evaporated under vacuo, and the obtained residue
was triturated with diethyl ether and dried under vacuo to afford the
titled compound 5b as a white solid.
Synthesis of 6-(4-Sulfamoylphenoxy)-hexylammonium Trifluo-
roacetate Salt 5e. tert-Butyl 6-(4-sulfamoylphenoxy)-hexylcarbamate
3-(4-Sulfamoylphenoxy)-propylammonium Trifluoroacetate Salt
5b: 91% yield, mp 129−130 °C; δH (400 MHz, DMSO-d6) 2.05 (2H,
pent, J 6.4), 3.00 (2H, m,), 4.17 (2H, t, J 6.0), 7.12 (2H, d, J, 8.8), 7.26
(2H, s, exchange with D2O, SO2NH2), 7.78 (3H, brt, exchange with
D2O, −NH3), 7.79 (2H, d, J 8.8); δC (100 MHz, DMSO-d6) 27.6,
37.1, 66.0, 115.43, 118.2 (d, J1
297), 128.6, 137.3, 159.4 (q,
C−F
J2
31) 161.6; δF (376 MHz, DMSO-d6) −73.64 (3F, s), Elemental
C−F
analysis: calcd C 38.37, H 4.39, N 8.14, S 9.31; found C 38.42, H 4.60,
N 7.95, S 9.66; m/z (ESI positive) 231.30 [M − CF3COO−]+.
Synthesis of 4-(4-Sulfamoylphenoxy)-butylammonium Trifluo-
roacetate Salt 5c. tert-Butyl 4-(4-sulfamoylphenoxy)-butylcarbamate
4e (0.10 g, 1.0 equiv) was dissolved in DCM (1.8 mL), followed by
addition of TFA (5.0 equiv). The reaction was treated according to the
general procedure c, previously reported (TLC monitoring). The
solvents were evaporated under vacuo, and the obtained residue
was crystallized from IPA to afford the titled compound 5e as a
white solid.
6-(4-Sulfamoylphenoxy)-hexylammonium Trifluoroacetate Salt
5e: 30% yield; mp 122−123 °C; δH (400 MHz, DMSO-d6) 1.45 (4H,
m), 1.59 (2H, pent, J 7.5), 1.76 (2H, pent, J 7.5), 2.82 (2H, t, J 7.5),
4.08 (2H, t, J 6.5), 7.11 (2H, d, J 9.0), 7.25 (2H, brs, exchange with
+
D2O, SO2NH2), 7.63 (3H, brt, exchange with D2O, NH3 ), 7.77 (2H,
4c (0.38 g, 1.0 equiv) was dissolved in DCM (5.0 mL), followed by
addition of TFA (12.0 equiv). The reaction was treated according
to the general procedure c previously reported (TLC monitoring).
The solvents were evaporated under vacuo, and the obtained res-
idue was crystallized from IPA to afford the titled compound 5c as a
white solid.
d, J 9.0); δC (100 MHz, DMSO-d6) 25.9, 26.4, 27.9, 30.7, 39.7, 68.7,
115.4, 118.1 (d, J1C−F 298), 128.6, 137.0, 159.3 (q, J2C−F 31), 162.0; δF
(376 MHz, DMSO-d6) −73.5 (1F, s). Elemental analysis: calcd C
43.52, H 5.48, N 7.25, S 8.30; found C 43.19, H 5.24, N 6.95, S 8.16;
m/z (ESI positive) 273.40 [M − CF3COO−]+.
General Procedure for Synthesis of Fluorescent Tagged
Sulfonamides 7a−c. The O-alkylbenzenesulfonamide salt 5 (1.0
equiv) and 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-isothiocyanatoben-
zoic acid 6 (1.0 equiv) were poured into a two-neck flask, and dry
DMA (1.0 mL) was added, followed by addition of TEA (1.5 equiv).
The reaction was stirred at r.t. until starting materials were con-
sumed (TLC monitoring). It was then quenched with slush and a
6 M aqueous hydrochloric acid solution and then extracted with
ethyl acetate (3 × 15 mL). The combined organic layers were
washed with H2O (3 × 20 mL), dried over Na2SO4, filtered, and
concentrated in vacuo to afford the titled compound 7 as an orange
powder.
4-(4-Sulfamoyl-phenoxy)-butylammonium Trifluoroacetate Salt
5c: 50% yield; mp 110−111 °C; δH (400 MHz, DMSO-d6) 1.74
(2H, m), 1.83 (2H, m), 2.90 (2H, t, J 7.4), 4.11 (2H, t, J 6.0), 7.10
(2H, d, J 8.0), 7.25 (2H, s, exchange with D2O, SO2NH2), 7.79 (5H,
+
m, Ar−H, NH3 ); δC (100 MHz, DMSO-d6) 24.8, 26.4, 39.5, 68.3,
115.4, 118.2 (d, J1C−F 298) 128.6, 137.1, 159.15 (q, J2C−F 31), 161.8; δF
(376 MHz, DMSO-d6) −73.4 (3F, s). Elemental analysis: calcd C
40.22, H 4.78, N 7.82, S 8.95; found C 40.30, H 4.63, N 7.72, S 9.56;
m/z (ESI positive) 245.17 [M − CF3COO−]+.
Synthesis of 5-(4-Sulfamoylphenoxy)-pentylammonium Trifluo-
roacetate Salt 5d. tert-Butyl 5-(4-sulfamoylphenoxy)-pentylcarbamate
Synthesis of 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-[2-(4-sul-
famoylphenoxy)-ethyl]-thioureido}-benzoic Acid 7a. 2-(4-Sulfa-
moylphenoxy)-ethylammonium trifluoroacetate salt 5a (50 mg,
1.0 equiv) and 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-isothiocyanato-
benzoic acid 6 (1.0 equiv) were treated according to the general
procedure previously reported afford the titled compound 7a as an
orange powder.
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-[2-(4-sulfamoylphe-
noxy)-ethyl]-thioureido}-benzoic Acid 7a. 76% yield; silica gel TLC
Rf 0.10 (MeOH/DCM 10% v/v); mp 172−173 °C (dec.); δH (400
MHz, DMSO-d6) 3.98 (2H, brt), 4.32 (2H, t, J 5.2), 6.61−6.66 (4H,
m), 6.71 (2H, d, J 2.0), 7.23 (5H, m, 3H Ar−H, 2H exchange with
D2O, SO2NH2), 7.76 (1H, exchange with D2O, NH), 7.80 (2H Ar−H,
d, J 8.8), 8.28 (1H, s), 8.35 (1H, exchange with D2O, NH), 10.11 (1H,
exchange with D2O, NH), 10.15 (2H, exchange with D2O, OH); δC
(100 MHz, DMSO-d6) 44.0, 67.1, 84.1, 103.2, 110.7, 113.6, 115.5,
4d (0.46 g, 1.0 equiv) was dissolved in DCM (5.0 mL), followed by
addition of TFA (7.0 equiv). The reaction was treated according to the
general procedure c, previously reported (TLC monitoring). The
solvents were evaporated under vacuo, and the obtained residue was
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6.62 (4H, m), 6.71 (2H, s), 7.15 (2H, d, J 8.8), 7.23 (2H, s, exchange
with D2O, SO2NH2), 7.79 (2H, d, J 8.8), 8.26 (1H, exchange with
D2O, NH), 10.00 (1H, exchange with D2O, NH), 10.15 (2H, exchange
with D2O, OH); δC (100 MHz, DMSO-d6) 29.0, 41.9, 66.9, 85.2,
103.3, 110.8, 113.7, 115.5, 117.7, 125.1, 127.6, 128.7, 130.1, 130.6,
137.2, 142.4, 148.0, 153.0, 160.6, 162.0, 169.5, 181.6 (CS); m/z
(ESI positive) 621.0 [M + 2H]2+.
Synthesis of 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-[4-(4-sul-
famoylphenoxy)-butyl]-thioureido}-benzoic Acid 7c. 4-(4-Sulfamoyl-
117.6, 125.1, 127.6, 128.7, 130.0, 130.6, 137.4, 142.1, 148.3, 152.9,
160.5, 161.7, 169.5, 181.8 (CS). Elemental analysis: calcd C 57.51,
H 3.83, N 6.94, S 10.59; found C 57.56, H 4.03, N 6.68, S 10.84; m/z
(ESI positive) 606.50 [M + H]+.
Synthesis of 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-[3-(4-sul-
famoylphenoxy)-propyl]-thioureido}-benzoic Acid 7b. 3-(4-Sulfa-
phenoxy)-butylammonium trifluoroacetate salt 5c (50 mg, 1.0
equiv)and 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-isothiocyanatobenzoic
acid 6 (1.0 equiv) were treated according to the general procedure e,
previously reported, to afford the titled compound 7c as an orange powder.
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-[4-(4-sulfamoylphe-
noxy)-butyl]-thioureido}-benzoic Acid 7c. 79% yield; silica gel TLC
Rf 0.70 (MeOH/DCM 20% v/v); mp 200−201 °C (dec); δH (400
MHz, DMSO-d6) 1.77−1.87 (4H, m), 3.64 (2H, brs), 4.14 (2H, t,
J 6.1), 6.60−6.66 (4H, m), 6.71 (2H, d, J 2.2), 7.13 (2H, d, J 8.8), 7.22
(3H, d, J 8.3, Ar−H, 2H exchange with D2O, SO2NH2), 7.77 (2H, d,
J 8.8), 8.30 (2H, s, 1H exchange with D2O, NH), 10.09 (3H, brs,
exchange with D2O, 2xOH, NH); δC (100 MHz, DMSO-d6) 26.4,
26.9, 45.4, 68.5, 85.3, 103.2, 110.0, 113.7, 115.4, 117.1, 126.0, 128.1,
128.7, 129.8, 130.0, 137.1, 141.9, 148.9, 153.1, 160.8, 161.9, 168.6,
182.6 (CS); m/z (ESI positive) 634.5 [M + H]+.
General Procedure for Synthesis of Florescent Tagged O-
Alkyl Benzenesulfonamides 9a,b. Compound 5 (1.0 equiv) and
[9-(2-carboxy-4-isothiocyanatophenyl)-6-dimethylaminoxanthen-3-
ylidene]dimethylammonium chloride 8 (1.0 equiv) were poured into a
two-neck flask, and dry DMA (1.0 mL) was added, followed by
addition of TEA (1.5 equiv). The reaction was stirred at r.t. until
starting materials were consumed (TLC monitoring), and then it was
quenched with slush and a 6 M aqueous hydrochloric acid solution.
The precipitate formed was centrifuged, collected, dried under vacuo,
washed with diethyl ether (3 × 10 mL), and dried under vacuo to
afford the titled compound 9 as a red powder.
moylphenoxy)-propylammonium trifluoroacetate salt 5b (50 mg,
1.0 equiv) and 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-isothiocyana-
tobenzoic acid 6 (1.0 equiv) were treated according to the general
procedure e, previously reported, to afford the titled compound 7b as
an orange powder.
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-{3-[3-(4-sulfamoylphe-
noxy)-propyl]-thioureido}-benzoic Acid 7b. 83% yield; silica gel TLC
Rf 0.40 (MeOH/DCM 20% v/v); mp 185−186 °C (dec); δH (400
MHz, DMSO-d6) 2.11 (2H, t, J 6.4), 3.72 (2H, brt), 4.18 (2H, t; J 5.8),
Synthesis of 9-(2-Carboxy-4-{3-[2-(4-sulfamoylphenoxy)ethyl]-
thioureido}phenyl)-6-dimethylaminoxanthen-3-ylidene]-dimethyl-
ammonium Chloride 9a. 2-(4-Sulfamoyl-phenoxy)ethylammonium
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of this reservoir solution was added to 5 μL of CA II at a final
concentration of 10 mg/mL so that the final drug concentration was at
0.24 mM. Hanging drops were set up, and crystals were seen within
5 days. This was repeated for 5c.
Diffraction Data and Collection. Diffraction data for CA II-4c and
CA II-5c complexes were collected on an in-house R-Axis IV+2 image
plate detector using a RU-H3R rotating Cu anode (Kα = 1.5418 Å)
operating at 50 kV and 22 mA. Images were collected every 1° with an
exposure time of 5 min at a detector distance of 100 mm. The crystal
data were integrated, merged and scaled using HKL2000.33
trifluoroacetate salt 5a (6.9 mg, 1.0 equiv) and [9-(2-carboxy-4-
isothiocyanatophenyl)-6-dimethylaminoxanthen-3-ylidene]-dimethyl-
ammonium chloride 8 (1.0 equiv) were treated according to the
previously reported procedure to afford the titled compound 9a as a
red powder.
9-(2-Carboxy-4-{3-[2-(4-sulfamoylphenoxy)-ethyl]-thioureido}-
phenyl)-6-dimethylaminoxanthen-3-ylidene]-dimethylammonium
Chloride 9a. 70% yield; silica gel TLC Rf 0.40 (MeOH/DCM 20%
v/v); mp 177−178 °C (dec); δH (400 MHz, DMSO-d6) 3.30 (12H, s),
3.99 (2H, brt), 4.33 (2H, t, J 5.4), 7.00 (4H, m), 7.14 (3H, m), 7.21
(2H, d, J 8.8), 7.26 (2H, s, exchange with D2O, SO2NH2), 7.41 (1H,
m), 7.72 (1H, d, J 8.8), 7.81 (2H, d, J 8.8), 8.46 (2H, brs, 1H,
exchange with D2O, NH), 10.38 (1H, exchange with D2O, NH); δC
(100 MHz, DMSO-d6) 35.1, 41.5, 43.9, 67.1, 97.2, 114.1, 115.3, 122.6,
124.5, 125.9, 128.7, 131.7, 137.5, 142.6, 157.6, 160.2, 161.4, 161.6,
166.8, 167.1 (CO), 181.5 (CS); m/z (ESI positive) 660.50
[M − Cl]+.
Synthesis of 9-(2-Carboxy-4-{3-[3-(4-sulfamoyl-phenoxy)-propyl]-
thioureido}-phenyl)-6-dimethylaminoxanthen-3-ylidene]-dimethyl-
ammonium Chloride 9b. 3-(4-Sulfamoylphenoxy)-propylammonium
trifluoroacetate salt 5b (7.2 mg, 1.0 equiv) and [9-(2-carboxy-4-
isothiocyanatophenyl)-6-dimethylamino-xanthen-3-ylidene]-dimethyl-
ammonium chloride 8 (1.0 equiv) were treated according to the
previously reported procedure to afford the titled compound 9b as a
red powder.
Structure Determination. Phasing was carried out in the
PHENIX34 suite of programs using the Auto Molecular Replacement
procedure to obtain the initial phases using a previously solved HCA II
structure with water molecules removed (PDB code 3KS3).35 The
graphics program Coot36 was used to view the electron density map,
and the structure was adjusted on the basis of the calculated electron
density. Topology files of the inhibitors were generated using the
PRODRG37 server, and these files were used to model the drug into
the density generated. Refinement was continued using PHENIX.REFINE
until the Rcrys and Rfree were minimized. The geometric restraints of the
final model were analyzed using PROCHECK.38 The data diffraction and
final model refinement statistics are summarized in Table 2.
Normotensive Rabbit IOP Lowering Studies. Male New Zeland
albino rabbits weighing 1500−2000 g were used in these studies.
Animals were anaesthetized using Zoletil (tiletamine chloride +
zolazepam chloride, 3 mg/kg body weight, i.m. injection) and injected
with 0.05 mL hypertonic saline solution (5% in distilled water) into
the vitreous of both eyes. IOP was measured by using a digital
tonometer (Tomo-Pen Avia Tonometer, Reichert Inc. Depew, NY
14043, USA) prior to hypertonic saline injection (basal) at 1, 2, and 4
h after administration of the drug.14a Vehicle (phosphate buffer, pH
7.0 plus DMSO 2%) or drugs were instilled immediately after
the injection of hypertonic saline into the conjunctive pocket. Eyes
were randomly assigned to different groups. Four different animals
were used for the tested compounds. Experiments with animals were
conducted in agreement with current ethical guidelines and norms
approved by the ethical committee of our university.
9-(2-Carboxy-4-{3-[3-(4-sulfamoyl-phenoxy)-propyl]-thioureido}-
phenyl)-6-dimethylamino-xanthen-3-ylidene]-dimethylammonium
Chloride 9b. 74% yield; silica gel TLC Rf 0.30 (MeOH/DCM 20%
v/v); mp 192−193 °C (dec); δH (400 MHz, DMSO-d6) 3.30 (12H, s),
4.13 (2H, t, J 6.2), 4.21 (2H, t, J 6.2), 6.98 (3H, m), 7.08−7.25 (8H,
m), 7.40 (1H, d, J 8.8), 7.75 (1H, d, J 8.8), 7.79 (2H, d, J 8.8), 7.87
(1H, brs), 7.94 (1H, d, J 8.8), 8.58 (1H, brt, exchange with D2O, NH),
10.51 (1H, brs, exchange with D2O, NH), 10.65 (1H, s); δC (100
MHz, DMSO-d6) 28.8, 35.0, 41.4, 41.5, 66.8, 97.2, 114.2, 115.5, 122.6,
124.2, 127.0, 128.6, 131.7, 137.1, 142.6, 157.7, 160.8, 161.9, 161.6,
166.8, 167.1 (CO), 181.5 (CS); m/z (ESI positive) 674.42
[M − Cl]+.
ASSOCIATED CONTENT
Accession Codes
4RFC and 4RFD.
■
X-ray Crystallography. Co-Crystallization. Two microliters of a
600 mM concentration of 4c was added to a 500 μL 1.6 M sodium
citrate, 50 mM Tris−HCL pH 7.8 reservoir solution. One microliter
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(4) (a) Xu, Y.; Feng, L.; Jeffrey, P. D.; Shi, Y.; Morel, F. M. Structure
and metal exchange in the cadmium carbonic anhydrase of marine
diatoms. Nature 2008, 452, 56−61. (b) Supuran, C. T. Bacterial
carbonic anhydrases as drug targets: towards novel antibiotics? Front.
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carbonic anhydrase inhibitors: A patent and literature review. Expert
Opin. Ther. Pat. 2013, 23, 693−704. (d) Hirohashi, N.; Alvarez, L.;
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H.; Supuran, C. T.; Kotzur, N.; Kakiuchi, Y.; Kaupp, U. B.; Baba, S. A.
Sperm from sneaker male squids exhibit chemotactic swarming to
CO2. Curr. Biol. 2013, 23, 775−781.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39-055-4573005. Fax: +39-055-4573385. E-mail:
claudiu.supuran@unifi.it.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was financed by two EU grants of the seventh
framework program (Metoxia and Dynano projects to AS and
CTS) and the National Institutes of Health Grant CA165284
(to R.M.).
(5) (a) Cottier, F.; Leewattanapasuk, W.; Kemp, L. R.; Murphy, M.;
Supuran, C. T.; Kurzai, O.; Muhlschlegel, F. A. Carbonic anhydrase
̈
regulation and CO2 sensing in the fungal pathogen Candida glabrata
involves a novel Rca1p ortholog. Bioorg. Med. Chem. 2013, 21, 1549−
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NONSTANDARD ABBREVIATIONS
■
M.; Supuran, C. T.; Muhlschlegel, F. A.; Steegborn, C. Structure and
̈
CA, carbonic anhydrase; CAI, CA inhibitor; DCM, dichloro-
methane; DIAD, diisopropyl azodicarboxylate; DIPEA, diiso-
propylethylamine; DMA, dimethylacetamide; IOP, intraocular
pressure; IPA, propan-2-ol; KI, inhibition constant; SAR,
structure−activity relationship; TFA, trifluoroacetic acid
inhibition of the CO2-sensing carbonic anhydrase Can2 from the
pathogenic fungus Cryptococcus neoformans. J. Mol. Biol. 2009, 385,
1207−1220. (c) Moya, A.; Tambutte, S.; Bertucci, A.; Tambutte, E.;
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