1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic anhydrase II alleviate ocular hypertension in rabbits: Potency is supported by X-ray crystallography of two leads

Article abstract of DOI:10.1016/j.bmc.2017.06.054

Two lead 1,3-oxazole-based carbonic anhydrase inhibitors (CAIs) earlier identified as selective, picomolar inhibitors of hCA II (a cytosolic target for treatment of glaucoma) have been investigated further. Firstly, they were found to be conveniently synt

Full text of DOI:10.1016/j.bmc.2017.06.054

Accepted Manuscript
1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic
anhydrase II alleviate ocular hypertension in rabbits: potency is supported by
X-ray crystallography of two leads
Marta Ferraroni, Laura Luccarini, Emanuela Masini, Mikhail Korsakov, Andrea
Scozzafava, Claudiu T. Supuran, Mikhail Krasavin
PII:
S0968-0896(17)31400-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.054
BMC 13854
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 May 2017
7 June 2017
10 June 2017
Please cite this article as: Ferraroni, M., Luccarini, L., Masini, E., Korsakov, M., Scozzafava, A., Supuran, C.T.,
Krasavin, M., 1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic anhydrase II alleviate
ocular hypertension in rabbits: potency is supported by X-ray crystallography of two leads, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.06.054
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1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic
anhydrase II alleviate ocular hypertension in rabbits: potency is supported by
X-ray crystallography of two leads
b
b
c
Marta Ferraroni,a Laura Luccarini,
Emanuela Masini,
Mikhail Korsakov,
Andrea
Scozzafava,d Claudiu T. Supuran,d,* Mikhail Krasavin e,
*
a Chemistry Dept., Universita degli Studi di Firenze, Florence, 50019, Italy.
b Neurofarba Dept., Sezione di Farmacologia, Universita degli Studi di Firenze, Florence, 50019,
Italy
c The Ushinsky Yaroslavl State Pedagogical University, Yaroslavl, 150000, Russian Federation
d Neurofarba Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di
Firenze, Florence, 50019, Italy
eInstitute of Chemistry, Saint Petersburg State University, Peterhof, 198504, Russian Federation
* Corresponding authors. E-mail addresses: claudiu.supuran@unifi.it (C. T. Supuran),
m.krasavin@spbu.ru (M. Krasavin).
ABSTRACT
Two lead 1,3-oxazole-based carbonic anhydrase inhibitors (CAIs) earlier identified as selective,
picomolar inhibitors of hCA II (a cytosolic target for treatment of glaucoma) have been
investigated further. Firstly, they were found to be conveniently synthesized on multigram scale,
which enables further development. These compounds were found to be comparable in efficacy
to dorzolamide eye drops when applied in the eye drop form as well. Finally, the reasons for
unusually high potency of these compounds became understood from their high-resolution X-ray
crystallography structures. These data significantly expand our understanding of heterocycle-
based primary sulfonamides, many of which have recently emerged from our labs - particularly,
from the corneal permeability standpoint.
Keywords: carbonic anhydrase inhibitors, multigram synthesis, isoform selectivity, picomolar
inhibition, primary sulfonamide, ocular hypertension, X-ray crystallography.

1. Introduction
Glaucoma consists of
a
group of eye diseases showing various clinical
presentations/etiologies, which lead to a loss of visual function due to damage of the optical
nerve.1-5 Although several different types of glaucoma are known, all of them are characterized
by an elevated intraocular pressure (IOP).1-4 The source of this abnormal increase is related to a
malfunctioning of tissues within the trabecular meshwork in the anterior chamber angle of the
eye. Their role is to maintain a balanced pressure in the anterior chamber of the eye by allowing
the outflow of aqueous humor.1-5 Aqueous humor is a transparent liquid, rich in bicarbonate,
which fills the region between the cornea and the lens,5-11 being continuously secreted by the
ciliary body around the lens, and permanently flowing from the ciliary body to the anterior
chamber.5-7 From this eye compartment the aqueous humor passes through the trabecular
meshwork into the Schlemm canal and from it into a multitude of aqueous veins which
eventually merge with the blood carrying veins.5-11 Glaucoma is often characterized by an
excessive retention/secretion of aqueous humor in the anterior chamber, whose effect leads to a
steep increase of the IOP to values as high as 25 – 30 mm Hg.1-5
The relationship between glaucoma and carbonic anhydrase (CA, EC 4.2.1.1), the
enzyme catalyzing the hydration of CO2 to bicarbonate and protons12-16 is rather straightforward
and known in details. The pioneering studies of Friedenwald,6 Kinsey7 and Kinsey and Barany8
on the chemistry and dynamics of aqueous humor showed that the main constituent of this fluid
is sodium bicarbonate. The next step was the identification of CA in the anterior uvea of the eye
by Wistrand9 who demonstrated that it is this enzyme (present in the ciliary processes)
responsible for bicarbonate secretion, as a consequence of the hydration reaction of carbon
dioxide, with formation of bicarbonate (and protons). Becker10 then showed that the sulfonamide
CA inhibitor (CAI) acetazolamide (1) produced a drop of IOP in experimental animals and
humans, whereas Kinsey and Reddy11 proved that this phenomenon is due to a reduced
bicarbonate secretion, as a consequence of CA inhibition by the sulfonamide drug. This was the
beginning of a novel treatment for glaucoma, already in the 50s.17 As this condition is affecting
an increasing number of the population, also being the leading cause of blindness in the Western
countries, it is essential to explore novel medications for its treatment.1-4 In fact, CAIs constitute
one of the main drug classes used for the treatment of glaucoma,12-17 with at least six
sulfonamides in clinical use both as systemic acetazolamide (1), methazolamide (2),
ethoxzolamide (3), dichlorophenamide (4) or topically acting dorzolamide (5) and brinzolamide
(6) drugs (Figure 1).17-20

Figure 1. Classical primary sulfonamide drugs in clinical use for glaucoma.
N
N
N
O
N
N
O
O
O
O
N
EtO
S NH2
S
S
Me
N
H
S
NH2
S
S
NH2
Me
O
O
O
1
2
3
NH2
O
S O
NHEt
NHEt
O
O
O
N
Cl
S NH2
S
MeO(CH2)3
NH2
S
S NH2
Me
S
S
S
O
O
O
O
O
O
Cl
O
4
6
5
Systemic CAIs are useful in reducing elevated IOP characteristic of many glaucoma
forms, as they represent the most efficient physiological treatment of glaucoma. Indeed by
inhibiting the ciliary process enzymes (the sulfonamide susceptible isozymes CA II, CA IV and
CA XII 5, 12, 17-20), a reduced rate of bicarbonate and aqueous humor secretion is achieved, which
leads to a 25 – 30 % decrease of IOP.5, 17 However, these compounds are promiscuous, strong
inhibitors of all CA isoforms (of the 15 ones knows in humans13-15), and the inhibition of various
CA isozymes present in other tissues than the eye leads to an entire range of side effects5,12,17
.
The most prominent ones are: numbness and tingling of extremities, metallic taste, depression,
fatigue, malaise, weight loss, decreased libido, gastrointestinal irritation, metabolic acidosis,
renal calculi and transient myopia.5, 12, 17 Thus, the development of topically acting CAIs is of
great interest, as the two such drugs described so far, dorzolamide (5) and brinzolamide (6), have
a much better safety profile compared to 1-4.21 Among the various strategies for the development
of new CAI chemotypes, the structure-based approach is undoubtedly one of the most versatile
ones, with a large number of effective inhibitors belonging to a variety of classes described in the
last decade.22
We recently described23 a class of sulfonamides incorporating 1,3-oxazolyl benzenes and
thiophenes in their molecules, some of which showed picomolar inhibitory activity against CA
isoforms involved in glaucomagenesis, in particular, hCA II (h = human). For further studies on
which we report herein, we selected two lead compounds 7 and 8 (Figure 2) based on their
showing the best potency (20 and 50 pM, respectively) against this isoform as well as the best
selectivity profile vs. the other isoforms in the panel – hCA I, IX and XII, showing selectivity
about 4 orders of magnitude compared to hCA II (Table 1). Herein, we report these compounds
to have a highly effective IOP lowering profile in an animal model of glaucoma, which is
indicative of this compound class’s being a novel series for the development of topical

antiglaucoma drugs. Additionally, for these compounds we performed crystallographic study.
The high-resolution crystal structures were obtained to rationalize the high potency of these
compounds against CA II earlier observed in biochemical assays. The results of these studies are
also presented herein.
Figure 2. Two lead compounds investigated in this work.
Table 1. Inhibitory profile of the two lead compounds obtained previously.23
Ki (nM)
Compound
hCA I
96.3
hCA II
0.05
hCA IX
23.1
hCA XII
8.5
7
22.5
36.4
30.3
8
0.02
Acetazolamide
12.1
25.0
5.6
250
2. Results and Discussion
2.1 Chemistry.
Lead candidate selection entails a robust, scalable synthesis is in place in order to support the in
vivo experiments, X-ray crystallographic studies and further preclinical development of the
frontrunner lead(s). The synthesis of compounds 7 annd 8 had been earlier performed on a 10
mmol scale.23 We sought to increase the scale of the synthesis to 100 mmol scale. This was
successfully achieved, without a detrimental effect of compounds’ purity and yields (see
Experimental).
Despite the obvious differences in the structure of 7 and 8 (the former containing sulfonamide
moiety at the phenyl ring while in the latter it is attached to a thiophene moiety; also 8 contains a
potentially hydrolytically prone carboxamide moiety), the syntheses are very similar and
provided highly crystalline, pure multigram batches of both lead compounds for in-depth
characterization.

Scheme 1. Synthesis of lead candidates 7 and 8 investigated in this work.
N-Acylation23 of commercially available α-aminoketones 9 and 12 furnished precursors 10 and
13, respectively, which, without further purification, were cyclodehydrated into key substrates 11
and 14 (using concentrated sulfuric acid or phosphoric oxychloride, respectively) obtained in
high overall yield and chemical purity. The latter compound underwent a clean ester-to-amide
conversion in neat morpholine at elevated temperature to furnish compound 15. Direct
sulfochlorination of 11 and 15 was achieved on the large scale with high regiospecificity and the
respective sulfochlorides were converted to primary sulfonamides 7 and 8 on treatment with
ammonia solution in 1,4-dioxane. (Scheme 1). The product yields and purities in all steps proved
comparable to those published for small-scale syntheses previously.23
2.2 Crystallography.
There are many CA – sulfonamide complexes characterized by means of X-ray
crystallography13-15, 19, 20. However the scaffolds present in compounds 7 and 8 are new and
understanding their binding mode within the enzyme active site may be useful both in the drug
design of potent inhibitors and also for understanding the very high efficacy of these two
compounds towards physiologically relevant isoforms, such as hCA II (involved in glaucoma,
cancer, neuropathic pain, etc.21,22 Inspection of the electronic density for the adducts of
sulfonamides 7 and 8 bound to hCA II; allowed us to evidence the presence of one inhibitor
molecule bound within the enzyme active site cavity (Figures 3 and 4).

Figure 3. Electronic density for the complex of hCA II with 7 bound within the active site. The
Zn(II) ion (gray sphere), its three His ligands (His 94, 96 and 119) as well as amino acid residues
involved in the binding of the inhibitor are also evidenced.
Figure 4. Electronic density for the complex of hCA II with 8 bound within the active site. The
Zn(II) ion (gray sphere), its three His ligands (His 94, 96 and 119) as well as amino acid residues
involved in the binding of the inhibitor are also evidenced.
As observed from Figures 3 and 4, the inhibitors 7 and 8 are bound within the hCA II active site,
coordinated to the Zn(II) ion by means of their deprotonated sulfonamide moiety. The
sulfonamide nitrogens coordinating to the metal ion also make a strong H-bond with the OH of
Thr199. One of the sulfonamide oxygen of the inhibitors makes a hydrogen bond with the amide

nitrogen of Thr199. All these intreactions were observed in all CA – sulfonamide complexes
characterized to date19, 21, 22
The phenyl fragment of sulfonamide 7 makes van der Waals contacts with Leu198 and Val121
and the nitrogen of the oxazolyl ring makes a hydrogen bonds with a water molecule (Figure
3).The thiophene ring of inhibitor 8 is involved in hydrophobic interactions with Leu198. The
oxazolyl oxygen makes a hydrogen bond with Gln92 and the carbonyl oxygen with a water
molecule (Figure 4). A second molecule of 7 was found close to the N-terminus of the protein,
making contact with residue His4, Trp5, Asn11 and Asp19 (data not shown), but probably this is
an artefact due to the high concentration of inhibitor used in the crystallographic experiments
(see Experimental for details).
A superimposition of the two X-ray crystal structures of the adducts of hCA II with 7/8 is shown
in Figure 5.
Fig. 5. Superimposition of the X-ray crystal structures of 7 (brown) and 8 (sky blue) bound
within the hCA II active site. The metal ion, its ligands and the residues involved in inhibitor
binding are also shown.
From Figure 5, it may be observed that only the sulfamoyl as well as the
thiophene/phenylene fragments of the two inhibitors are superimposable, whereas their tails
adopt rather diverse orientation within the enzyme active site, which proves that potent inhibitors
may be obtained even when the binding occurs in different parts of the active site (with the
known hydrophobic/hydrophilic dichotomy23), but may also explain the differences in inhibitory
profile of the two sulfonamides.

2.3 Intraocular pressure lowering studies in rabbits.
Solution of compounds 7 and 8 (1 %) in an ophthalmologically acceptable vehicle (see
Experimental for details) were administered directly into the rabbit eyes and intraocular pressure
(IOP) was measured for 4 hours post-administration, using the standard drug dorzolamide (DRZ)
2 % as reference (Fig. 6). The two sulfonamides 7 and 8 were slightly less effective IOP
lowering agents than DRZ at 60 min post-administration, but their efficacy was better than the
standard drug at later periods of time, and lasted longer than DRZ for which at 4 hours the effect
was closed to 0 (Fig. 6).
Figure 6. The drop in intraocular pressure (∆IOP, mmHg) versus time (min) in hypertonic saline-
induced ocular hypertension in rabbits, treated with 50 µL of 1% solution of compounds 8 (comp
1), 7 (comp 2) using 2% dorzolamide (DRZ) solution as a reference drug and vehicle (saline).
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)
3. Conclusion
The lead hCAII inhibitors (7 and 8) previously identified as selective, picomolar inhibitors of
this glaucoma-related enzyme have been investigated further to show a pronounced efficacy in
the rabbit intraocular hypertension model applied topically as eye drops, even in comparison
with a standard topical drug, 2% dorzolamide. For these leads, high-resolution crystallographic
structures with hCAII were obtained which rationalize the high inhibitory potency toward this

enzyme. Considering the low molecular weight (in particular, for compound 7) and the ease of
synthesis, these compounds are promising development candidates for a novel topical treatment
to relieve glaucoma-related intraocular hypertension. These findings also shed further light on
the potential of heterocycle-containing primary sulfonamides as therapeutically relevant CAIs.
4. Experimental section
4.1 Synthetic chemistry
The synthesis and CA inhibitory data for compounds 7 and 8 were reprted earlier.23 The
synthesis of both compounds for this study was performed on 100 mmol scale as described
below.
4.1.1. 2-Methyl-5-phenyl-1,3-oxazole (11).23
Compound 10 (100 mmol) was added to 94% sulfuric acid (50 mL) the mixture was stirred at 80
ºC for 90 min, cooled down to ambient temperature and poured into ice water (500 mL). The
solution was neutralized with 28% aqueous ammonia and extracted with ethyl acetate (3 x 200
mL). The combined extracts were dried over anhydrous MgSO4, filtered and concentrated under
reduced pressure to provide, after crystallization from ethyl acetate, pure title compound with
analytically data identical to those reported previously.23 Yellow solid, mp = 127-128 ºC. Yield
1
– 12.416 g (78%). H NMR (400 MHz, DMSO-d6) δ ppm 8.21 (s, 1H, Hoxazole), 7.80 (d, J = 7.2
Hz, 2H, 2,6-HAr), 7.43 (t, J = 7.2 Hz, 2H, 3,5-HAr), 7.40 (t, J = 7.2 Hz, 1H, 4-HAr), 2.47 (s, 3H,
CH3). Anal. calcd for C10H9NO: C, 75.45; H, 5.70; N, 8.80; found: C, 75.42; H, 5.66; N, 8.79.
4.1.2. Morpholin-4-yl[5-(thiophen-2-yl)-1,3-oxazol-2-yl]methanone (15).23
Compound 13 (100 mmol) was added promptly to phosphorus (V) oxychloride (1.30 mol) and
stirred at 80 ºC for 90 min and poured over crushed ice (3 L). The resulting precipitate was
collected by filtration, washed with water and air-dired. It was then dissolved in morpholine (50
mL) and the resulting mixture was stirred at 50 ºC for 2.5 h. Upon cooling to r. t. water (250 mL)
was added and the resulting precipitate was collected by filtration, washed by water, air-dried
and further crystallized from isopropyl alcohol to provide the pure title compound with analytical
characteristics identical to those reported previously.23 Pale grey solid, m.p. 66-68 °С, yield
17.972 g (68 %); 1H NMR (400 MHz, DMSO-d6) δ ppm 7.74 (dd, J3-4=4.9 Hz, J3-5=1.0 Hz, 1H,
3-Hthiophene), 7.72 (s, 1H, Hoxazole), 7.59 (dd, J4-5=3.6 Hz, J3-5=1.0 Hz, 1H, 5-Hthiophene), 7.21 (dd,

J3-4=4.9 Hz, J4-5=3.6 Hz, 1H, 4-Hthiophene), 4.06 (m, 2H, Hmorpholine), 3.66 (m, 6H, Hmorpholine. Anal.
calcd for C12H12N2O3S: C, 54.53; H, 4.58; N, 10.60; found: C, 54.51; H, 4.55; N, 10.63.
4.1.3 General procedure (GP) for the synthesis of compounds 7 and 8.
The starting 1,3-oxazole 11 or 15 (the entire quantity obtained in the previous step) was added
portion-wise to a cooled mixture of chlorosulfonic acid (600 mmol) and thionyl chloride (60.0
mmol). The resulting mixture was heated at 60 °С for 2 hours, cooled to room temperature and
poured over crushed ice (3 L). The precipitate formed was filtered off, washed with water and
dissolved in chloroform (1 L). The solution was washed with 5% aqueous potassium carbonate,
dried over anhydrous calcium chloride, filtered and concentrated. The residue was flash-
chromatographed on a short plug of silica gel using an appropriate gradient of ethyl acetate in
hexanes as eluent. The intermediate sulfonyl chloride was dissolved in 1,4-dioxane (100 mL) and
treated with 25% aqueous ammonia (7.5 mL, 100 mmol). The resulting mixture was heated at 75
°C for 2 hours. The volatiles were removed in vacuo and the residue was treated with water (100
mL). The resulting precipitate was filtered off and crystallized from isopropyl alcohol to give
sulfonamides 7 or 8. The yield of sulfonamides is reported for two consecutive steps
(sulfochlorination and sulfonamide synthesis).
4.1.3.1. 4-(2-Methyl-1,3-oxazol-5-yl)benzenesulfonamide (7)23
Obtained according to GP as a white solid, m.p. 223-225 °С (i-PrOH), yield 13.923 g (75 %)
with analytical characteristics identical to those reported previously:23 1H NMR (400 MHz,
DMSO-d6) δ ppm 7.88 (d, J=8.8 Hz, 2H, HAr), 7.84 (d, J=8.8 Hz, 2H, HAr), 7.70 (s, 1H, Hoxazole),
7.40 (s, 2H, NH2), 2.49 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6) δ ppm 162.6, 149.9,
144.0, 131.4, 127.3, 125.5, 124.7, 14.6. MS m/z 238 (relative intensity) 238 (M+, 100), 174 (21),
158 (19), 130 (10), 120 (5), 103 (7), 77 (7), 76 (7), 63 (7), 54 (9), 50 (5), 43 (5), 27 (5). Anal.
calcd for C10H10N2O3S: C, 50.41; H, 4.23; N, 11.76; found: C, 50.40; H, 4.21; N, 11.81.
4.1.3.2. 5-[2-(Morpholin-4-ylcarbonyl)-1,3-oxazol-5-yl]thiophene-2-sulfonamide (8)23
Obtained according to GP as a white solid, m.p. 190-192 °С (i-PrOH), yield 16.093 g (69 %). 1H
NMR (400 MHz, DMSO-d6) δ ppm 7.91 (s, 1 H, Hoxazole), 7.86 (s, 2 H, NH2), 7.60 (d, J=3.8 Hz,
1H, Hthiophene), 7.58 (d, J=3.8 Hz, 1H, Hthiophene), 4.03 (m, 2H, Hmorpholine), 3.66 (m, 6H,
Hmorpholine). 13C NMR (126 MHz, DMSO-d6) δ ppm 155.3, 154.2, 147.1, 146.7, 132.6, 131.8,
126.8, 125.1, 67.1, 66.8, 47.9, 43.5. MS m/z (relative intensity) 343 (M+, 12), 258 (6), 257 (17),
256 (30), 201 (7), 187 (11), 137 (6), 114 (22), 86 (100), 72 (6), 70 (40), 64 (8), 56 (21), 45 (6),

42 (25), 29 (6). Anal. calcd for C12H13N3O5S2: C, 41.97; H, 3.82; N, 12.24; found: C, 41.94; H,
3.86; N, 12.19.
4.2 Co-crystallization and X-ray data collection
Crystals of hCA II complexed with compounds 7 and 8 were obtained using the sitting drop
vapor diffusion method. An equal volume of 0.8 mM solution of hCA II in Tris pH=8.0 and 1.6
mM of the inhibitors in Hepes 20mM pH=7.4 was mixed and incubated for 15 minutes. 2 µl of
the complex solution were mixed with 2 µl of a solution of 1.6 M sodium citrate, 50 mM Tris pH
8.0 and were equilibrated against the same solution at 296 K. Crystals of the complex grew in a
few days. The crystals were flash-frozen at 100K using a solution obtained by adding 25% (v/v)
glycerol to the mother liquor solution as cryoprotectant. A data set on a crystal of the complex
with the inhibitor 7 was collected using synchrotron radiation at the BM30A beamline at ESRF
(Grenoble, France) with a wavelength of 0.980 Å and a ADSC Q315r CCD detector. A data set
on a crystal of the complex with the inhibitor 8 was collected at the Centro di Cristallografia
Strutturale (CRIST) in Florence using an Oxford Diffraction instrument equipped with a sealed
tube Enhance Ultra (Cu) and a Onyx CCD detector.
Data were integrated and scaled using the program XDS.24 Data processing statistics are showed
in Table 2.
a
Table 2. Summary of Data Collection and Atomic Model Refinement Statistics.
hCA II + 7
hCA II + 8
PDB ID
5NEA
5NEE
Wavelength (Å)
Space Group
0.980
P21
0.980
P21
Unit cell (a,b,c,β) (Å, °)
42.38, 41.26, 72.07, 104.45 42.32, 41.24, 71.83, 104.28
Limiting resolution (Å)
Unique reflections
40.0 - 1.30 (1.30- 1.38)
58311 (9234)
69.6 - 1.70 (1.80-1.70)
25205 (3580)
Rsym (%)
6.4 (57.0)
2.88 (2.69)
98.2 (96.7)
11.60 (1.80)
6.8 (71.2)
2.45 (1.46)
94.6 (84.3)
12.22 (1.07)
Redundancy
Completeness overall (%)
<I/(I)>

CC(1/2)
99.8 (62.5)
99.5 (68.7)
Refinement statistics
Resolution range (Å)
30.0 -1.3
30.0 - 1.7
Unique reflections,
working/free
54724/2796
23640/1185
Rfactor (%)
16.58
18.63
2142
380
18.41
22.23
2081
289
Rfree(%)
No. of protein atoms
No. of water molecules
No. of heterogen atoms
r.m.s.d. bonds(Å)
r.m.s.d. angles (°)
Average B factor (Å2)
All atoms
42
28
0.007
1.296
0.010
1.431
13.28
11.43
26.42
13.55
11.55
23.94
inhibitor
solvent
1
Values in parentheses are for the highest resolution shell.
4.3 Structure determination
The crystal structure of hCA II (PDB accession code: 4FIK) without solvent molecules and other
heteroatoms was used to obtain initial phases of the structures using Refmac5.25 5% of the
unique reflections were selected randomly and excluded from the refinement data set for the
purpose of Rfree calculations. The initial |Fo - Fc| difference electron density maps
unambiguously showed the inhibitor molecules.
An electron density, which could be interpreted as a second molecule of inhibitor 7, was present
near the N-terminus of the protein. Thus a second 7 molecule was introduced in the model with
0.75 occupancy. Electron density was not present for the morpholine ring of inhibitor 8 probably
due to disorder and occupancy equal to 0 was attributed to its atoms.
Atomic models for inhibitors were calculated and energy minimized using the program JLigand
1.0.39. Refinements proceeded using normal protocols of positional, isotropic atomic
displacement parameters alternating with manual building of the models using COOT.26 Solvent
molecules were introduced automatically using the program ARP.27 The quality of the final

models were assessed with COOT and Rampage.28 Crystal parameters and refinement data are
summarized in Table 2. Atomic coordinates were deposited in the Protein Data Bank (PDB
accession code: 5NEA, 5NEE). Graphical representations were generated with Chimera.29
4.3 Intraocular pressure studies in rabbits
Adult male New Zealand Albino rabbits weighing 2-2.5 Kg were employed in this study. The
animals were utilized in groups of eight for each of the chosen specific treatments. The
experimental procedures were conform to those of the Declaration of Helsinki and with the
Guideline for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S.
National Institute of Health, and were conducted upon authorization of the Italian Regulations on
Protection of Animals used for experimental and other scientific purpose (DM 116/1992) as well
as with the European Union Regulations (OJ of ECL 358/1, 12/12/1986), and the experimental
protocol was approved by the local animal care committee of the University of Florence
(Florence, Italy). The rabbits were kept in individual cages; food and water were provided ad
libitum. The animals were identified with a tattoo on the ear, numbered consecutively and
maintained on a 12-12h light/dark cycle in a temperature controlled room (22°-23°C). All
selected animals were examined before the beginning of the study and were determined to be
normal on ophthalmic and general examinations. The increase in IOP was induced by the
injection of 50 µl of sterile hypertonic saline (5%) into the vitreous bilaterally in pre-anesthetized
rabbits with tiletamine/zolazepam (Zoletil 50/50 mg/mL, Virbac) i.m. 0.05 ml/kg, plus xylazine
(Xylor 2%,) i.m. 0.02 ml/kg.30 Drugs were dissolved in pirogen-free appropriate vehicle
(buffered aqueous solution pH 7, polyoxyl 40, dimethylsulphoxyde (DMSO) 2%, hydrogenated
castor oil 5 mg/ml, tromethamine 10 mg/ml, edetate disodium 0.5mg/ml and benzalkonium
chloride 0.15 mg/mL) and administered as eye drops at different concentrations immediately
after hypertonic saline injection. IOP was measured using a Tono-Pen AVIA® (Reichert Inc.
Depew, NY) prior (basal) and post hypertonic saline injection 60, 120, 240 min after drug or
vehicle treatment. One drop of 0.2% oxybuprocaine hydrochloride, 4 mg/ml, was instilled in
each eye immediately before each set of pressure measurements. The ocular hypotensive
activity of studied compounds is expressed in mmHg as average difference in IOP between drug-
and vehicle-treated eyes.30

Acknowledgement
This research was supported by the Russian Science Foundation (project grant 14-50-00069).
The authors are grateful to Mr. Stanislav Kalinin of Saint Petersburg State University for
facilitating the research communication between this University and University of Florence.
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1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic
anhydrase II alleviate ocular hypertension in rabbits: potency is supported by
X-ray crystallography of two leads
Marta Ferraroni, Laura Luccarini, Emanuela Masini, Mikhail Korsakov, Andrea Scozzafava,
Claudiu T. Supuran,* Mikhail Krasavin *