Highly hydrophilic 1,3-oxazol-5-yl benzenesulfonamide inhibitors of carbonic anhydrase II for reduction of glaucoma-related intraocular pressure

Article abstract of DOI:10.1016/j.bmc.2019.115086

Four inhibitors of human carbonic anhydrase II (hCA II) were designed based on the previously reported subnanomolar 1,3-oxazole-based sulfonamide inhibitors of the enzyme to incorporate primary and secondary amine functionality in the carboxamide side cha

Full text of DOI:10.1016/j.bmc.2019.115086

Journal Pre-proofs
Highly Hydrophilic 1,3-Oxazol-5-yl Benzenesulfonamide Inhi bitors of Car-
bonic Anhydrase II for Reduction of Glaucoma-Related Intraocular Pressure
Stanislav Kalinin, Annika Valtari, Marika Ruponen, Elisa Toropainen,
Alexander Kovalenko, Alessio Nocentini, Maxim Gureev, Dmitry Dar'in, Arto
Urtti, Claudiu T. Supuran, Mikhail Krasavin
PII:
S0968-0896(19)31276-3
https://doi.org/10.1016/j.bmc.2019.115086
BMC 115086
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
24 July 2019
29 August 2019
1 September 2019
Please cite this article as: Kalinin, S., Valtari, A., Ruponen, M., Toropainen, E., Kovalenko, A., Nocentini, A.,
Gureev, M., Dar'in, D., Urtti, A., Supuran, C.T., Krasavin, M., Highly Hydrophilic 1,3-Oxazol-5-yl
Benzenesulfonamide Inhi bitors of Carbonic Anhydrase II for Reduction of Glaucoma-Related Intraocular Pressure,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115086
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Highly Hydrophilic 1,3-Oxazol-5-yl Benzenesulfonamide Inhibitors of
Carbonic Anhydrase II for Reduction of Glaucoma-Related Intraocular
Pressure
Stanislav Kalinin,§,a Annika Valtari,§,b Marika Ruponen, Elisa Toropainen, Alexander
b
b
a
c
d
a
,a,b,e
Kovalenko, Alessio Nocentini, Maxim Gureev, Dmitry Dar’in, Arto Urtti,*
Claudiu T.
,
c
,a
Supuran,* and Mikhail Krasavin*
a
Saint Petersburg State University, Saint Petersburg 199034, Russian Federation
School of Pharmacy, University of Eastern Finland, 70211 Kuopio, Finland
Neurofarba Department, Universita degli Studi di Firenze, Florence, Italy
b
c
d
e
I.M. Sechenov First Moscow State Medical University, Moscow, 119991, Russian Federation
Drug Research Programme, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,
University of Helsinki, Helsinki, Finland
§
Equal contribution.
ABSTRACT
Four inhibitors of human carbonic anhydrase II (hCA II) were designed based on the previously
reported subnanomolar 1,3-oxazole-based sulfonamide inhibitors of the enzyme to incorporate
primary and secondary amine functionality in the carboxamide side chain. The new hydrophilic
compounds were found to inhibit the target isoform in sub-nanomolar to low nanomolar range
with a good degree of selectivity to several other hCA isoforms. The hydrophilic character of
these compounds is advantageous for intraocular residence time but not for corneal permeability
which generally requires that a drug be sufficiently lipophilic. Two of the four compounds
investigated, however, were found to exert comparable efficacy as 1% eye drops in PBS to that
®
of the clinically used 2% dorzolamide (Trusopt ) eye drops. This indicated that the absorption of
the compounds may occur via alternative route across conjunctiva and sclera.
Key words: glaucoma; intraocular pressure; hydrophilicity; intraocular delivery; non-corneal
absorption; carbonic anhydrase inhibitors
1
.
Introduction

Intraocular pressure is decreased in glaucoma treatment with eye drops of prostaglandin
1
analogues, beta blocking agents and carbonic anhydrase inhibitors (CAI). The topical CAIs in
the glaucoma treatment include dorzolamide (1) and brinzolamide (2), compounds that are
relatively lipophilic and non-selective as inhibitors of carbonic anhydrase isoforms.2
Acetazolamide (3) and methazolamide (4) are also used as anti-glaucoma agents (Fig. 1), but
they are delivered as per oral tablets; an approach that causes frequent adverse drug reactions.3
Selective and potent inhibition of carbonic anhydrase II isoform (hCA II) is desirable in drug
discovery, because this enzyme is critically important in the reduction of intraocular pressure.4
Potent and selective hCA II inhibitors are expected to be effective drugs in the glaucoma
treatment.
HN
HN
O
O
O
N
N
N
N
O
O
O
S NH2
O
S NH2
O
S NH2
O
N
S NH2
O
S
S
S
N
S
S
N
S
H
O O
O
O O
1
2
3
4
Fig. 1. Examples of clinically used antiglaucoma carbonic anhydrase inhibitors.
Topical ocular drugs are usually lipophilic, because they are delivered to the eye via permeation
5
across the cornea. Lipophilicity tends to decrease water solubility and maximal attainable
soluble drug concentration in the tear fluid, whereas higher concentration gradient between tear
fluid and the eye can be achieved with hydrophilic compounds. Furthermore, instead of ocular
absorption the topically applied drugs are effectively absorbed to the systemic blood stream
across the conjunctiva lining the inner side of the eyelids.6,7 Systemic drug loss reduces ocular
bioavailability, especially in the case lipophilic compounds.7,8,9 In addition, it is known that
ocular drug absorption can take place also via non-corneal route, across the bulbar conjunctiva
10
(
on the ocular surface) and sclera. Hydrophilic compounds can utilize this route, because the
conjunctiva is less tight membrane than the cornea, and the loss of hydrophilic compounds to the
blood stream in the conjunctiva and sclera is slower than in the case of lipophilic compounds.10,11
The site of action of CAIs is in the ciliary body, a tissue that is located next to sclera, making
non-corneal absorption an interesting approach for highly potent, but hydrophilic derivatives.
Compared to the cornea, the conjunctiva has a wider inter-cellular space available for permeation
of hydrophilic compounds.12
Previously, we described a series of 1,3-oxazol-5-yl benzenesulfonamides 5a-c which displayed
a remarkably potent inhibition profile toward human carbonic anhydrase (CA, EC 4.2.1.1) and,
1
3
in particular, its hCA II isoform which is primarily targeted by intraocular pressure-reducing
4
antiglaucoma drugs. The most hCA II-selective compound (5a) has been shown to be

efficacious in vivo in lowering ocular hypertension in rabbits. Furthermore, its high potency and
the pronounced selectivity toward the CA isoform in question has been rationalized by X-ray
crystallographic structure of its complex with the protein.14 In light of these findings, it seemed
worthwhile to select 1a as the scaffold for the introduction of peripheral functional groups which
would increase the resulting compounds’ hydrophilicity and provide also a reactive ‘handle’ for
subsequent chemical conjugation to polymer nanoparticles.
Anti-glaucoma eye drop treatment has poor patient compliance as many patients do not use the
1
5
eyedrops properly, leading to the progression of the disease and loss of vision. Therefore,
increasing interest has been focused on the long acting intraocular drug delivery with polymeric
1
6
systems. The new compounds presented in this work were designed with this goal in mind,
since their structure, on one hand, allows convenient conjugation of the compounds to the
polymeric carriers via amide and other potentially biodegradable linkages. On the other hand, the
hydrophilicity of these compounds was seen as beneficial in this context as hydrophilic
compounds have slower clearance from the intraocular spaces.17-18 Therefore, similar drug cargo
of hydrophilic drug in controlled release system may extend the dosing interval as compared to
the use of lipophilic compound with similar potency. Yet, hydrophilic compounds might be
useful as traditional eye drop medications if they would be delivered across the conjunctiva-
sclera route to the ciliary body.
If both of the latter design requirements are taken into account, synthesis and subsequent
biological investigation of amides 6 prepared from dibasic amines and analogous to compounds
5
b-c became our goal (Figure 2). Foreseeable risks associated with such a structural modification
included potential loss of potency and/or selectivity toward hCA II compared to distinctly
hydrophobic lead compound 5a. Indeed, the active site of carbonic anhydrase is known to have a
very characteristic topology where a hydrophobic half of the protein surface is clearly delineated
from the hydrophilic one.19 Therefore, replacing the small hydrophobic 1,3-oxazole methyl
substituent with a large hydrophilic carboxamides group bearing a basic amine could, in
principle, lead to a loss of the desired affinity to hCA II. Despite these potential risks we set off
to synthesize a set of compounds 6 for investigation of their carbonic anhydrase inhibitory
potency in vitro and subsequent efficacy characterization as glaucoma-associate intraocular
pressure-lowering agents in vivo. Herein, we report the results of these studies.

R'
NH
R
K (hCA II), nM
i
N
O
basic amine
and reactive
handle for conjugation
R
0.050
0.009
N
5
a, R = Me
O
O
N
*
N
N
5b, R =
O
O
6
O
O
*
0.008
S
5c, R =
H N
2
O S O
NH2
O
Fig. 2. Earlier reported potent hCAII inhibitors 5a-c and their modified hydrophilic analogs 6
designed and investigated in this work.
2
.
Results and Discussion
2
.1 Chemistry
Scheme 1. Synthesis of hydrophilic sulfonamides 6a-d investigated in this work.
O
CO Et
O
CO Et
2
2
O
O
O
NH Cl
Cl
CO2Et
NH
POCl3
3
N
o
py, 80 C, 1 h
9%
o
8
0 C, 2 h
7
7
, 48%
HSO Cl/SOCl
3
2
o
60 C
CO Et
2
CO Et
CO Et
2
2
O
O
O
H , Pd/C
2
NaN3
N
N
N
EtOH, r. t.
DMF, r. t.
H NO S
2
2
N O S
ClO S
3
2
2
10, 62%
9
, 80%
8, 59%
R
N
R
N
O
O
O
O
O
O
R'
1
1a-d
TFA
N
H
N
(
2.5 equiv.)
R'
N
MeOH, r. t.
N
1
,4-dioxane
o
6
0 C, 8 h
6a-d, 31-75%
12a-d
O
S
H N
2
O
S
O
O
H N
2
R'
HN
H N
2
H N
2
HN
H
N
O
NH
O
=
N
O
N
O
N
O
O
R
O
NH
O
O
O
11c
11d
11a
11b
The key building block - ethyl 5-phenyloxazole-2-carboxylate (7) was synthesized in two steps
from -aminoacetophenone hydrochloride as described previously.13 This compound was

chlorosulfonylated under somewhat harsh conditions (SOCl /HSO Cl, 60 ºC) to obtain sulfonyl
2
3
chloride 8 in respectable 59% yield. Direct conversion to respective sulfonamide 10 by treatment
with aqueous ammonia failed. Despite our efforts to reduce the reaction temperature and apply
slow addition technique, the carboxylic acid in 7 was non-selectively converted to primary
amide, apparently, being sufficiently activated by the primary sulfonamide group which also
formed at the same time. This obstacle was circumvented via a two-step procedure entailing a
high-yielding synthesis of sulfonyl azide 9 followed by hydrogenation of the latter over Pd/C to
give the target sulfonamide 10 in good yield over two steps. The electron-withdrawing influence
of the sulfonamide group which complicated the preparation of 10 as described above, was a
desirable feature in subsequent synthesis of the target compounds 6a-d. Indeed, on reaction with
2
.5-fold excess of mono-Boc-protected dibasic amines 11a-d at r. t. in MeOH, respective amides
2a-d were obtained in moderate to good yields, deprotected with TFA in 1,4-dioxane at 60 ºC
1
and purified chromatographically to give the target compounds 6a-d (Scheme 1).
2
.2 Carbonic anhydrase inhibition
The inhibitory profile obtained for sulfonamides 6a-d in a stopped-flow kinetics assay against
human CA I, II, IV and XII is shown in Table 1. In addition to hCA II, the other three isoforms
were selected to arbitrarily evaluate the off-target profile of the compounds intended to inhibit
the target isoform.
Table 1. Inhibitory activity of compounds 6a-d against the target (hCA II) as well as selected
off-target (hCA I, IV and XII) isoforms.
a
Compound
Structure
K (nM)
i
hCA I
hCA II hCA IV hCA XII
6
a
O
108.7
5.0
419.8
68.5
O
O
N
H N S
2
NH
N
O
6
b
O
61.9
0.54
256.6
91.5
O
H N S
O
N
2
N
NH2
O

6
c
50.8
0.91
204.3
87.3
NH
O
O
O
N
H N S
H
2
N
O
6
d
O
101.4
250
4.4
12
39.1
75
90.1
5.7
NH2
O
O
N
H N S
H
2
N
O
3b
a
Mean from 3 different assays by stopped flow technique (errors were in the range of  5-10 %
b
of the reported values). Sulfonamide inhibitor acetazolamide (AAZ) used as a reference pan-
CA inhibitor in stopped flow CO hydrase assay.
2
To our delight, all four inhibitors 6a-d preserved the potent inhibition profile against the target
hCA II isoform (although taking some toll on potency compared to the less hydrophilic leads 5a-
c) and a clearly better isoform selectivity profile compared to acetazolamide (4) employed as a
reference inhibitor. Interestingly, sub-nanomolar inhibitory activity was displayed by compounds
6
b and 6c which are isomeric (and nearly isosteric) to each other.
Of course, the ultimate efficacy profile of these inhibitors on the reduction of glaucoma-related
intraocular pressure (IOP) would depend on a multitude of factors among which permeability
characteristics (intrinsically linked to a favorable set of molecular parameters) will have a
superior significance.
2
.3 Docking studies
In order to rationalize the one-order hCA II potency gap between 6a,d and 6b,c docking studies
were performed. Figure 3 shows the docking poses of 6a and 6b in the active site of hCA II. In
the case of both compounds, the phenylsulfonamide moiety acts as a zinc binding group showing
2
0
orientation well documented in a wide range of crystallographic studies. Another common
feature is ionic interaction of the free amino function with Glu69. However the conformation of
6
a enabling the latter interaction is somewhat tense due to the shorter spacer present between the
carboxamide and amino groups in comparison to that of 6b. Additionally, the presence of larger
aliphatic chain in 6b favors enhanced lipophilic interaction of the inhibitor molecule with the
spacy hydrophobic cleft lined up with the residues Leu141, Ile91, Val121, Val143, Trp209 and
Phe131. It is impact of these interactions what we consider to be the crucial factor explaining
higher inhibitory activity of 6b comparing to 6a. Moreover, we strongly believe this conclusion

to be relevant for compounds 6c and 6d (docking poses not shown) exhibiting similar activity
trends.
A
6a
B
Figure 3. Binding poses of 6a (A) and 6b (B) in the hCAII active site. Hydrophobic, positively
and negatively charged regions of the catalytic cleft are surface shown in green, red, and blue
correspondingly. Hydrogen bonds are represented in yellow dashed lines. Salt bridges are
represented as purple dashed lines.
2
.4 In silico ADME properties
ADME descriptors of compounds 6a-d were evaluated and compared to those of commercially
available carbonic anhydrase inhibitors (Table 2). Based on the calculated logD7.4 and logP
values, it can be concluded that 6a-d are indeed much more hydrophilic relative to the topical
carbonic anhydrase inhibitors in the clinical use (1 and 2) whereas orally used drugs (3 and 4) are
similarly hydrophilic.

Table 2. Chemical descriptors of carbonic anhydrase inhibitors 6a-d and 1-4 (calculated using
ACDLabs 12.0).a
Compound
MW
324.4
383.5
222.3
236.3
336.4
350.4
350.4
310.3
HBa HBd HBtot LogP LogD7.4 LogD8.0 PSA logPSA
1
2
3
4
6
8
7
7
8
8
8
8
3
3
3
2
3
4
4
5
9
1.67
4.30
-0.26
0.13
0.07
0.11
0.53
-0.21
1.53
4.19
1.62
4.26
151.33 2.180
163.80 2.214
11
10
9
-0.69
-0.21
-0.73
-1.56
-1.78
-1.89
-1.13 151.66 2.181
-0.62 138.87 2.143
-0.30 126.91 2.103
-1.00 140.90 2.149
-1.29 135.70 2.133
-1.79 149.69 2.175
6
a
11
12
12
13
6
b
6
c
6
d
a
MW = molecular weight, HBa = hydrogen bond acceptors, HBd = hydrogen bond donors, HBtot = total amount of
hydrogen bond formers, LogP = logarithmic value of partition coefficient, LogD7.4/LogD8.0 = logarithmic value of
distribution coefficient at pH 7.4/8.0, PSA = polar surface area, LogPSA = logarithmic value of polar surface area.
Computational molecular descriptors presented above were used to further calculate the
conjunctival and corneal permeability values for the same compounds using an earlier reported
approach.21 In general, the calculated corneal and conjunctival permeability values (P ) of the
app
new compounds (6a-d) were similar or lower than those of dorzolamide (1) and brinzolamide (2)
(Table 3).
Table 3. Calculated permeability (P ) values of compounds 1-4 and 6a-d.
app
Compound
Cornea (rabbit)
Cornea (porcine)
Conjunctiva (porcine)
Papp (cm/s)
% of 1
Papp
% of 1 Papp (cm/s) % of 1
(cm/s)
1
2
3
4
7.79E-06
1.83E-05
1.24E-06
2.57E-06
100
235
16
1.75E-07
1.64E-07
1.74E-07
2.54E-07
100
94
1.86E-06
1.77E-06
1.85E-06
2.35E-06
100
95
100
146
100
126
33

6
a
7.94E-07
3.07E-07
2.67E-07
1.63E-07
10
4
2.00E-07
1.35E-07
1.39E-07
9.47E-08
114
77
2.07E-06
1.62E-06
1.65E-06
1.30E-06
111
87
6
b
6
c
3
80
89
6
d
2
54
70
2
.5 Pharmacodynamic effect of intraocular pressure (IOP) in vivo
The newly developed hydrophilic hCAII inhibitors were tested in normotensive rabbits for their
ability to lower the intraocular pressure (IOP). The results are shown as pressure readings (in
mmHg) and percentage changes in Fig. 4 and 5, respectively. Compounds 6a and 6d showed a
clear effect in the treated rabbit eye relative to the untreated control eye. Compounds 6c and 1
(dorzolamide) had some effect in both eyes, whereas the compound 6b did not have any
detectable effects on the IOP.

Figure 4. IOP ± SD (mmHg) after administration of 6a-d and 1 (dorzolamide, DRZ) as well as
phosphate buffered saline (PBS) as negative control, compared to fellow untreated eye in albino
rabbits (n=5).
Figure 5. Percentage change in IOP ± SD after administration of 6a-d, and 1 (dorzolamide,
DRZ) as well as phosphate buffered saline (PBS) as negative control, compared to fellow
untreated eye in albino rabbits (n=5).
Based on the results presented in Fig. 4, the area under curve (AUC) for IOP vs. time curves and
maximum responses (Emax) were determined for each compound (Table 4). For compound 6a,
the percentage decrease of AUC was comparable with the statistical significance of P=0.056.
However, the power of the performed test (0.450) was below the desired power of 0.800, which
resulted in the absence of statistically significant difference in AUC values. On the contrary, the
maximum effects produced by compounds 6a, 6d and 1 (dorzolamide) were significant in
comparison to PBS vehicle. At the same time compounds 6b and 6c did not produce a
statistically significant effect on the IOP in the rabbits. Notably, 6a appeared to have a longer
action in the rabbit eye compared to 1 (dorzolamide) which might be due to the slow clearance
from the eye.
Table 4. Effect of carbonic anhydrase inhibitors 6a-d and 1 on IOP in albino rabbits (n=5).a
Compound
Emax ±SD
AUC0-8h ± SD
159.79 ± 51.95
66.92 ± 43.36
84.04 ± 96.66
113.57 ± 57.57
6
a
35.60** ± 4.88
22.00 ± 5.76
19.40 ± 10.89
29.60* ± 7.79
6
b
6
c
6
d
1
2
7.80* ± 4.96
124.36 ± 35.57
25.90 ± 59.86
(
dorzolamide)
PBS
12.00 ± 6.81

a
All values presented are mean ±SD; decrease in area under curve (AUC) was calculated from the curve of decrease
in IOP (%) vs. time as compared to the baseline; * = statistically significant P<0.05; ** = statistically significant
P<0.001.
Possible eye irritation caused by the administration of carbonic anhydrase inhibitors was
followed visually throughout the experiment. Compounds 6a, 6c, 6d and 1 (dorzolamide) did not
induce blinking or head shaking after drug administration to the rabbits’ eyes, whereas
compound 6b caused mild irritation (eye lid closure for 5 to 10 seconds). All the eye drops of
new compounds were close to neutrality (pH 6.80 – 7.36) and isotonicity (osmolality 254-337
mOsm/kg).
hCA II inhibitors 6a and 6d (administered as 1% eye drops) displayed the IOP lowering efficacy
that comparable to that of the clinically used dorzolamide 2% eye drops (1). Due to the low
quantity of the compounds 6b and 6c they were formulated as 0.5% eye drops and the lack of
efficacy observed with these hCA II inhibitors can potentially be overcome at higher doses. The
compounds which displayed efficacy (at half the dose compared to 1) are hydrophilic and were
predicted to have 10-50 times lower corneal permeability in the rabbits than 1 (dorzolamide)
(Table 3). The comparable pharmacological effect displayed by hydrophilic compounds 6a and
6
d and more lipophilic 1 may indeed be attributed to the conjunctival permeation (not feasible
for lipophilic compounds) and reduced systemic loss which is characteristic for hydrophilic
compounds.
3
. Conclusion
We have described next-generation 1,3-oxazole-based carbonic anhydrase inhibitors endowed
with a primary or secondary amine periphery. The compounds were designed with a dual goal of
increasing compounds’ hydrophilicity and provide a reactive ‘handle’ for potential conjugation
to sustained-release nanoparticles. Increased hydrophilicity, while desirable for increased drug
residence in the intraocular space was generally viewed as an obstacle for corneal drug
absorption. However, hydrophilic compounds may be efficiently absorbed via conjunctiva and
thus have greater efficacy which may be expected if corneal absorption alone is considered.
Compounds described herein displayed a potent and selective inhibition of hCA II isoform, a
glaucoma target and two of these compounds (6a and 6d) showed comparable efficacy as 1%
eye drops in reducing the intraocular pressure in normotensive rabbit to that of clinically used
2
% dorzolamide eye drops. This is despite the fact that the corneal permeability of these
hydrophilic compounds was predicted to be significantly lower than that of dorzolamide. The
data support the concept of hydrophilic compounds permeating across the conjunctiva and sclera
into the ciliary body.

3
. Experimental section
4
.1 Synthetic chemistry
1
NMR spectroscopic data were recorded with 400 spectrometer (400.13 MHz for H and 100.61
1
3
MHz for C) in DMSO-d or in CDCl and were referenced to residual solvent proton signals
6
3
(
δ = 2.50 and 7.26 ppm, respectively) and solvent carbon signals (δ = 39.52 and 77.00 ppm,
H
C
respectively). Mass spectra were recorded with a Bruker Maxis HRMS-ESI-qTOF spectrometer
electrospray ionization). Melting points were determined with a Stuart SMP50 instrument in
(
open capillary tubes and are uncorrected. Column chromatography was carried out with silica gel
grade 60 (0.0400.063 mm) 230400 mesh. Preparative HPLC was carried out on Shimadzu LC-
2
0AP chromatograph, equipped with spectrophotometric detector. Column: Agilent Zorbax
prepHT XDB-C18, 5 lm, 21.2 150 mm. Eluent: A) 0.1% TFA in water, B) 0.1% TFA in
acetonitrile. All reagents and solvents were used as received from commercial sources unless
otherwise noted.
4
.1.1. Ethyl 5-(4-(chlorosulfonyl)phenyl)oxazole-2-carboxylate (8)
A mixture of chlorosulfonic acid (6.13 mL, 92.1 mmol) and thionyl chloride (0.67 mL, 9.21
1
3
mmol) was cooled in an ice bath and substrate 7 (2.00 g, 9.21 mmol) was added portionwise.
The mixture was stirred for 30 minutes, then warmed to 60°C and stirred for 12 hours. The
course of the reaction was monitored by TLC. The reaction mass was then poured into
chloroform over crashed ice. Organic layer was separated, washed with 10% NaHCO aqueous
3
solution (15.0 ml), H O (3*15.0 ml) and brine (15.0 ml), dried over Na SO , filtered and
2
2
4
concentrated. The mixture was purified via column chromatography to give 1.92 g (66%) of
1
product as yellow powder. M.p. 85-88°C. H NMR (400 MHz, DMSO-d ) δ 8.00 (s, 1H,
6
CHOxazole), 7.80 (d, J = 8.4 Hz, 2H, H ), 7.74 (d, J = 8.4 Hz, 2H, H ), 4.40 (q, J = 7.1 Hz, 2H,
Ar
Ar
1
3
CH ), 1.35 (t, J = 7.1 Hz, 3H, CH ) ppm. C NMR (100 MHz, CDCl ) δ 155.3, 153.0, 151.5,
2
3
3
1
44.5, 132.9, 128.0 (2C), 127.0, 125.9 (2C), 63.1, 14.2 ppm. HRMS (ESI), m/z calcd for
+
+
C H ClNO S 316.0041 [M+H] found 316.0039.
1
2
11
5
4
.1.2. Ethyl 5-(4-sulfamoylphenyl)oxazole-2-carboxylate (10)
To a solution of 8 (1.50 g, 4.75 mmol) in dimethylformamide (5.0 mL) NaN (0.32 g, 5.0 mmol)
3
was added. The reaction mixture was stirred for 2 hours. The course of the reaction was
monitored by TLC. The reaction mixture was poured onto H O, the precipitate formed was
2
filtered and dissolved in hot ethanol (20.0 ml) followed by addition of 10% Pd/C (0.15 g). The
suspension was stirred under H atmosphere at 80°C and the course of the reaction was
2
monitored by TLC. After the reaction completion, the catalyst was filtered off and the solvent

was removed by rotary evaporation. The residue was purified by flash chromatography to
provide sulfonamide 10 (49% over 2 steps, 0.69 g; 2.3 mmol) as white powder. M.p. 180-183°C.
1
H NMR (400 MHz, Acetone-d ) δ 8.05 (s, 4H, H ), 7.98 (s, 1H, CHOxazole), 6.72 (br.s, 2H,
6
Ar
1
3
SO NH ), 4.48 (q, J = 7.1 Hz, 2H, CH ), 1.43 (t, J = 7.1 Hz, 3H, CH ) ppm. C NMR (100
2
2
2
3
MHz, DMSO) δ 155.5, 152.4, 152.3, 145.2, 129.7, 127.1 (2C), 127.0, 125.7 (2C), 62.7, 14.5
+
+
ppm. HRMS (ESI), m/z calcd for C H N O S 297.0540 [M+H] found 297.0536.
1
2
13
2
5
4
.1.3. Preparation of compounds 6a–d; General Procedure (GP)
To a solution of 10 (0.10 g, 0.34 mmol) in methanol corresponding mono-Boc-protected diamine
1 (0.68 mmol) was added and the mixture was refluxed for 12 hours. The solution was then
removed by rotary evaporation, the residue was dissolved in chloroform (5.0 mL), washed by
0% solution of HCl (2x2.5 mL) and H O (3x2.5 mL), dried over Na SO and concentrated. The
1
1
2
2
4
residue was dissolved in 1,4-dioxane (3.0 mL) and trifluoroacetic acid (3.0 mL) was added
slowly. The mixture was stirred for 4 hours. The formation of amine salt was monitored by TLC.
The white precipitate formed was filtered off, washed with 1,4-dioxane and additionally purified
by reverse-phase HPLC using acetonitrile:H O system containing trifluoroacetic acid (0.1%).
2
4
.1.3.1. 4-(2-(4-Sulfamoylphenyl)oxazole-5-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate (6a)
1
Prepared according to GP1. Yield: 0.049 g (32%). M.p. 165-168°C. H NMR (400 MHz,
+
DMSO) δ 8.99 (s, 2H, NH ), 8.10 (s, 1H, H
), 8.00 (d, J = 8.5 Hz, 2H, H ), 7.96 (d, J = 8.5
Oxazole Ar
2
Hz, 2H, H ), 7.48 (br.s, 2H, SO NH ), 4.33 (t, J = 5.0 Hz, 2H, CH ), 3.87 (t, J = 5.0 Hz, 2H,
Ar
2
2
2
1
3
CH ), 3.28 – 3.21 (m, 4H, CH ) ppm. C NMR (126 MHz, DMSO, the signal of TFA not
2
2
included) δ 155.1, 154.1, 151.1, 145.0, 129.8, 127.2 (2C), 125.5 (2C), 125.5, 43.9 (2C), 43.4,
+
+
4
3.0 ppm. HRMS (ESI), m/z calcd for C H N O S 337.0971 [M+H] found 337.0980.
14 17 4 4
4
.1.3.2. 1-(5-(4-Sulfamoylphenyl)oxazole-2-carbonyl)piperidin-4-aminium 2,2,2-trifluoroacetate
(6b)
1
Prepared according to GP1. Yield: 0.117 g (75%). M.p. 158-161°C. H NMR (400 MHz, D O) δ
2
7
.86 – 7.79 (m, 2H, H ), 7.76 – 7.68 (m, 2H, H ), 7.60 (s, 1H, HOxazole), 4.89 – 4.79 (m, 1H,
Ar Ar
+
CH ), 4.70 (s, SO NH NH in exchange with water, 5H), 4.62 – 4.52 (m, 1H, CH ), 3.56 (tt, J
2
2
2,
3
2
=
11.0, 4.0 Hz, 1H, CH), 3.42 – 3.27 (m, 1H, CH ), 3.08 – 2.97 (m, 1H, CH ), 2.26 – 2.10 (m,
2 2
1
3
2
H, CH ), 1.83 – 1.61 (m, 2H, CH ) ppm. C NMR (100 MHz, D O, the signal of TFA was not
2 2 2
included) δ 156.2, 153.6, 151.2, 141.6, 130.2, 126.6 (2C), 125.4 (2C), 124.4, 47.8, 45.3, 41.6,
+
+
2
9.9, 29.0 ppm. HRMS (ESI), m/z calcd for C H N O S 351.1127 [M+H] found 351.1124.
15 19 4 4
4
.1.3.3. 4-(5-(4-Sulfamoylphenyl)oxazole-2-carboxamido)piperidin-1-ium 2,2,2-trifluoroacetate
(6c)
1
Prepared according to GP1. Yield: 0.097 g (62%). M.p. 156-159°C. H NMR (400 MHz, D O) δ
2
+
7
.83 – 7.77 (m, 2H, H ), 7.73 – 7.68 (m, 2H, H ), 7.55 (s, 1H, H
), 4.70 (s, SO NH NH ,
Ar
Ar
Oxazole
2
2,
2

COONHCH in exchange with water, 5H), 4.11 (tt, J = 11.0, 4.1 Hz, 1H, CH), 3.51 (dt, J = 13.5,
3
1
1
.8 Hz, 2H, CH ), 3.15 (td, J = 12.8, 3.0 Hz, 2H, CH ), 2.21 (dd, J = 14.8, 3.8 Hz, 2H, CH ),
2 2 2
1
3
.94 – 1.80 (m, 2H, CH ) ppm. C NMR (100 MHz, D O, the signal of TFA not included) δ
2
2
55.7, 154.0, 151.8, 141.6, 130.2, 126.6 (2C), 125.3 (2C), 125.2, 45.0, 42.9 (2C), 27.5 (2C) ppm.
+
+
HRMS (ESI), m/z calcd for C H N O S 351.1127 [M+H] found 351.1139.
1
5
19
4
4
4
.1.3.4.
2-(5-(4-Sulfamoylphenyl)oxazole-2-carboxamido)ethan-1-aminium
2,2,2-
trifluoroacetate (6d)
1
Prepared according to GP1. Yield: 0.045 g (31%). M.p. 160-163°C. H NMR (400 MHz, D O) δ
2
+
7
.90 – 7.83 (m, 2H, H ), 7.83 – 7.76 (m, 2H, H ), 7.66 (s, 1H, H
), 4.71 (s, SO NH NH ,
Ar
Ar
Oxazole
2
2,
3
COONHCH in exchange with water, 6H), 3.77 – 3.67 (m, 2H, CH ), 3.31 – 3.23 (m, 2H, CH )
2
2
1
3
ppm. C NMR (100 MHz, D O, the signal of TFA not included) δ 156.9, 153.8, 151.8, 141.5,
2
+
1
3
30.0, 126.5 (2C), 125.3, 125.2 (2C), 39.0, 37.1 ppm. HRMS (ESI), m/z calcd for C H N O S
12 15 4 4
+
11.0814 [M+H] found 311.0809.
4
.2 Carbonic anhydrase inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
2
2
CO hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,
2
working at the absorbance maximum of 557 nm, with 20 mM Tris (pH 8.3) as buffer, and 20
mM Na SO (for maintaining constant the ionic strength), following the initial rates of the CA-
2
4
catalyzed CO hydration reaction for a period of 10-100 s. The CO concentrations ranged from
2
2
1
.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the
initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from
the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-
deionized water and dilutions up to 0.005 nM were done thereafter with the assay buffer.
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 non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as
reported earlier, and represent the mean from at least three different determinations. All CA
isoforms were recombinant ones obtained in-house.23-26
4
.3 Docking studies
2
7
The crystal structure of Hca II (PDB code 2AW1 ) was downloaded from the Protein Data
Bank.28 Protein-ligand complex was preprocessed using Schrodinger Protein PrepWizard.29

Complexed ligand is removed from active site and the protein structure was minimized using
Schrodinger Prime module and OPLS3e force field at 310 K. Glide Grid program was used to
prepare docking grid with included positional constraints. As a constraining factor interaction of
sulfamide group with zinc ion in active site and hydrogen bond with Thr 200 residue was used.
Grid spacing was set up as 0.375Ȧ. Grid box size was selected as 8 x 8 x 8 Ȧ. Docking
3
0
procedure was carried out with Schrodinger Glide program using OPLS3e force field. Before
docking receptor constraints was included, where needed ligand features is marked to use.
Energy window of 2 kcal/mol for conformer generation was chosen. Docked compounds
subjected to 15 docking runs, with generation of 5000 poses per structure. Reference ligand
geometry was used for binding pose control and best docked conformations were taken into
account.
4
.4 QSPR modeling
The permeability properties of the molecules were calculated using the formulae presented in
Table 5.
Table 5. Formulae for estimating permeability properties of carbonic anhydrase inhibitors.21,31
Permeability (Papp, cm/s)
Cornea (rabbit)
Formulaa
Reference
LogPapp = -3.885-0.183(HBtot)+0.277(logD7.4)
LogPapp = -4.6823−0.7670(logPSA)−0.1346
25
9
Cornea (porcine)
(HBd)+3.0024(Halogen ratio)
Conjunctiva (porcine)
LogPapp = -4.1594-0.6121(logPSA)-
.0792(HBd)+3.2914(Halogen ratio)
26
0
a
LogPapp = logarithmic value of apparent permeability, HBtot = total amount of hydrogen bond formers, LogD7.4
logarithmic value of distribution coefficient at pH 7.4, LogPSA = logarithmic value of polar surface area, HBd =
=
hydrogen bond donors, Halogen ratio = sum of all halogens divided by the sum of all heavy atoms excluding
hydrogen (zero for all compounds in this work).
4
.5. Intraocular pressure studies
New Zealand White young adult female rabbits from Envigo Laboratories (UK) were used in the
experiments. The animals were housed under standard laboratory conditions of 12-hour dark-
light cycles and were provided with normal pellet diet with water ad libitum. Animals were
handled in accordance with the statement of the Animals in Research Committee of the ARVO

(
Association for Research in Vision and Ophthalmology, Rockville, Maryland, USA) and all
animal experiments were approved by the Finnish National Animal Experiment Board
Eläinkoelautakunta, ELLA).
(
®
Commercial dorzolamide was used as reference drug (Trusopt , dorzolamide 20 mg/mL, Santen
Pharmaceutical Co., Ltd). The compounds investigated (6a-d) were dissolved in PBS at 10
mg/mL (6a and 6d) or 5 mg/mL (6b and 6c) concentrations. The pH and osmolality (Auto-
Osmometer Osmostat OM-6020, Kagaku Ca. Ltd.) of the eye drop solutions were measured to
ensure that the eye drops were close to neutrality and isotonicity.
Before the start of the experiment, validation of animal model and some pre-experiments were
done to habituate the animals for measuring and to get background information of the variability
in individual animals, fellow eyes, days and the circadian rhythm.
Each drug was applied as single eye drop at volumes of 25 µl to the left eye of the rabbit. The
right eye was left untreated. The rabbits were held immobile for one minute after administration.
Tonometer (TonoLab®, Icare Finland Ltd.) was used to measure intraocular pressure (IOP). In
all experiments, the same eye was measured six times, the smallest and largest values were
excluded and the average of four centremost values were used to calculate the value for the time
point. The IOP of five normotensive rabbits were measured from both eyes before the
administration of the molecule and at the timepoints 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 hours after
treatment. All experiments were conducted between 8:00am and 5:00pm. The wash-out period
maintained between each test was at least four days. The change from baseline value(%) and the
corresponding area under curve (AUC) of each molecule were compared with the vehicle and the
statistical differences in the maximum effect (Emax) and AUC were tested with One-Way
repeated measurement ANOVA with Dunnett’s method.
Acknowledgements
This research was supported by the Russian Federation Government Megagrant
1
4.W03.031.0025. We thank the Research Center for Magnetic Resonance, the Centers for
Chemical Analysis and Materials Research of Saint Petersburg State University Research Park
for the analytical data. Dr. Kati-Sisko Vellonen is acknowledged for her advice on QSPR
modeling. We also thank Dr. Veli-Pekka Ranta for advice on statistical analysis.
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Conflict of Interest
None of the authors declares any conflict of interest.
3
2.
Highly Hydrophilic 1,3-Oxazol-5-yl Benzenesulfonamide Inhibitors of
Carbonic Anhydrase II for Reduction of Glaucoma-Related Intraocular
Pressure
Stanislav Kalinin, Annika Valtari, Marika Ruponen, Alexander Kovalenko, Alessio Nocentini,
Dmitry Dar’in, Arto Urtti, Claudiu T. Supuran, and Mikhail Krasavin
basic amine for hydrophilicity
and subsequent conjugation to
slow-release carries
NH
NH2
K (hCA II), nM
HN
O
N
i
O
O
R
0.050
0.009
N
N
R = Me
O
O
N
*
N
N
R =
O
O
O
*
R =
0.008
S
H N
2
O S O
NH2
O S O
NH2
O
O
efficacious as eye drops in vivo
3
3.