Evaluation of selenide, diselenide and selenoheterocycle derivatives as carbonic anhydrase I, II, IV, VII and IX inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.03.013

A series of selenides, diselenides and organoselenoheterocycles were evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, IV, VII and IX, involved in a variety of diseases among which glaucoma, retinitis pi

Full text of DOI:10.1016/j.bmc.2017.03.013

Accepted Manuscript
Evaluation of selenide, diselenide and selenoheterocycle derivatives as carbonic
anhydrase I, II, IV, VII and IX inhibitors
Andrea Angeli, Damiano Tanini, Caterina Viglianisi, Lucia Panzella, Antonella
Capperucci, Stefano Menichetti, Claudiu T. Supuran
PII:
S0968-0896(17)30326-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.013
BMC 13609
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 February 2017
27 February 2017
5 March 2017
Please cite this article as: Angeli, A., Tanini, D., Viglianisi, C., Panzella, L., Capperucci, A., Menichetti, S., Supuran,
C.T., Evaluation of selenide, diselenide and selenoheterocycle derivatives as carbonic anhydrase I, II, IV, VII and
IX inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.03.013
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1
Evaluation of selenide, diselenide and selenoheterocycle derivatives as carbonic anhydrase I, II, IV,
VII and IX inhibitors
Andrea Angeli, a Damiano Tanini, b Caterina Viglianisi, b Lucia Panzella,c Antonella Capperucci, b
Stefano Menichetti,b Claudiu T. Supurana*
a Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino
(Florence), Italy
b Università degli Studi di Firenze, Department of Chemistry "Ugo Schiff", Via della Lastruccia 3-13, I-50019 Sesto Fiorentino, Italy.
c University of Naples “Federico II”, Department of Chemical Sciences, Via Cintia 4, I-80126 Naples, Italy
Abstract. A serie of selenides, diselenides and organoselenoheterocycles were evaluated as carbonic
anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, IV, VII and IX,
involved in a variety of diseases among which glaucoma, retinitis pigmentosa, epilepsy, arthritis and
tumors etc. These investigated compounds showed inhibitory action against these isoforms and some of
them were selective for inhibiting the cytosolic over the membrane-bound isoforms, thus making them
interesting leads for the development of isoform-selective inhibitors.
Keywords: carbonic anhydrase; inhibitors; metalloenzymes; selenium; diselenides; selenides;
selenoheterocycles
______
Corresponding author. Tel./fax:+39-055-4573729; e-mail: claudiu.supuran@unifi.it (CTS)

2
1. Introduction
During the last decades, organoselenium compounds were the subject of intense interest, especially from
the point of view of public health. Selenium plays an important role in biological systems as part of the
active site in many proteins.1,2 Organoselenium derivatives show antioxidant,3 antitumor,4 antiviral5-6
and antimicrobial action7. Furthermore, some of them show inhibitory effects on a variety of enzymes
such as nitric oxide synthase (NOS),8-9 inosine monophosphate dehydrogenase (IMDPH)10 and
lipoxygenases (LOX).11 Some of these enzymes are involved in serious diseases, thus leading to possible
applications as therapeutic agents for many selenium-containing derivatives.8-10
The reactive oxygen species ROS are generated by the normal metabolic activity, as well as lifestyle
factors such as smoking, exercise, and diet.12 ROS overproduction is induced by different factors which
lead to a perturbation of the normal cell redox balance, shifting cells into a state of oxidative stress.
Since in conditions of severe stress, survival of the cells depends on their ability to adjust or resist to this
stress,13–15 cells have developed an antioxidant defense system, involving among others gluthathione,
antioxidant vitamins, sulfhydryl groups, and several antioxidant enzymes.16 In this context, a recent
study from our group17 reported that the metalloenzyme carbonic anhydrase (CAs, EC 4.2.1.1),
especially the isoform CA VII, is also involved in the oxidative stress defence processes interfering with
the generation of ROS.18–20
This is the reason why we decided to investigate various selenides, diselenides and selenoheterocycle
compounds with antioxidant properties,21-22 as human (h) CA inhibitors (CAIs). As mentioned above,
CAs are metalloenzymes that catalyse a very simple reaction: the hydration of carbon dioxide to
bicarbonate and proton.23 This reaction plays an important role in many physiological and pathological
processes associated with pH control, ion transport, and fluid secretion.24,25

3
2. Results and discussion
2.1. Chemistry
Our drug design strategy consisted in synthesizing or testing as CA inhibitors compounds incorporating
various selenium-containing functionalities, such as selenides, diselenides and selenium heterocycles
reported in Chart 1. It should be mentioned that many of the analogous sulfur-containing compounds
were shown to strongly inhibit many CA isoforms26.
Chart 1: Selenium-containing chemotypes investigated s CA inhibitors in the paper.
Benzo[b]selenophenes 1, 2 and 3 were synthesised from resveratrol, elemental selenium and sulfuryl
chloride following our reported procedure (Scheme 1).21

4
β-Hydroxy- and β-amino selenides 4 and 14 as well as β-hydroxy- and β -amino-diselenides 5, 9, 16 and
17 were obtained from the corresponding epoxides or N-Tosyl protected aziridines and
bis(trimethylsilyl)selenide (Me3Si-Se-SiMe3) according to the literature (Scheme 2).27
β -Phenylseleno-alcohols 6, 7 and -amine 15 were synthesised from epoxides and N-Tosyl protected
aziridines, respectively, upon treatment with phenylselenotrimethylsilane as reported in the left part of
the Scheme 2.28 All spectroscopic data matched those previously reported for these types of compounds.
2-Aminoaryl diselenides 10-12, 2-nitroaryl diselenide 13 and selenazines 18-22 were prepared according
to literature procedures (Scheme 3).22

5
1,2-Bis(2-hydroxyphenyl)diselenide
8
was prepared according to literature procedures.29
Benzeneseleninic acid 23 was commercially available and provided by Sigma Aldrich.
2.2. Carbonic anhydrase inhibition
All compounds 1-23 were tested in vitro for their inhibitory activity against the physiologically relevant
hCA isoforms I, II, IV, VII and IX by means of the stopped-flow carbon dioxide hydration assay30 and
their activities were compared to the standard CAI acetazolamide (AAZ) (Table 1).
Table 1. Inhibition data of human CA isoforms I, II, IV, VII and IX with compounds 1-23 and AAZ by
a stopped flow CO2 hydrase assay.30
KI (µM)*
hCA IV
Cmp
hCA I
hCA II
hCA VII
hCA IX
1
2
3
4
5
49.5
29.1
15.9
17.1
52.1
19.1
17.7
0.91
>100
>100
56.8
9.1
52.4
>100
>100
6.5
1.8
0.66
>100
>100
37.0
22.9
9.1
>100
>100

6
6
7
8
>100
>100
29.6
>100
81.3
63.6
75.9
>100
89.8
67.3
23.2
>100
>100
>100
>100
80.1
79.1
>100
0.25
>100
>100
23.7
>100
30.9
61.6
>100
>100
5.6
>100
>100
26.6
>100
>100
5.9
>100
>100
>100
>100
1.6
9
>100
78.9
6.2
>100
>100
>100
>100
23.5
>100
>100
>100
>100
>100
>100
>100
0.074
>100
5.8
4.6
>100
>100
>100
8.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
8.4
>100
>100
>100
>100
29.5
>100
>100
>100
>100
>100
>100
>100
0.025
3.2
13.1
96.1
>100
>100
>100
59.6
>100
>100
0.012
>100
7.8
>100
>100
>100
>100
>100
>100
0.006
AAZ
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5-10 % of the
reported values).
We have investigated a range of selenides, diselenides and seleno-heterocycle derivatives for their
interaction with the five hCA here considered, after a period of 15 min of incubation of the enzyme and
inhibitor solutions.30–33 The following structure activity relationship (SAR) may be noted regarding the
inhibition data of Table 1:
(i) The cytosolic hCA I was inhibited by benzoselenophene compounds 1-3 with Ki ranging between
15.9 and 49.5 µM. The interesting case for these molecules was the presence of chlorine on the
benzoselenophene ring. The inhibition potency increased when one or two halogen atoms were present
in the molecule, 1.7 and 3 times, respectively, compared to the unsubstituted derivative. β-Hydroxy
Selenide 4 was 3 times more potent than the analog diselenide 5 (Ki 17.1 µM for 4 and Ki 52.1 µM for 5,
respectively). On the other hand, the β-phenylseleno-alcohols 6 and 7 did not inhibit hCA I. Phenol
diselenide 8 showed an inhibition constant of 29.6 µM, but a further addition of an ortho-methoxyl
group (9), led to a loss of activity. The introduction of a methyl group on the aromatic diselenide

7
incorporating the secondary sulfonamide 10 decreased the potency of inhibition compared to the analog
without it, i.e., compound 11 (Ki 81.3 and 63.6 µM respectively). The amino aromatic diselenide 12
inhibited in the high micromolar range (Ki 75.9 µM) hCA I, whereas the compound with nitro group 13
did not inhibit this isoform. The β-amino selenides and diselenides 14-17, bearing a secondary
sulfonamide moiety, inhibited hCA I in the medium - high micromolar range (23.2- 89.9 µM).
Furthermore, the diselenide 16 was three and respectively four times more potent than 14 and 15. The
seleno heterocycles inhibited this cytosolic isoform in the high micromolar range (compounds 21-22: Ki
79.1 µM and Ki 80.1 µM).
(ii) The dominant cytosolic human isoform, hCA II, was inhibited in low or low-medium micromolar
range by the benzoselenophene derivatives 1-3 (Ki 0.91-19.1 µM). The presence of one and two chlorine
atoms in the benzoselenophene ring, also for this isoform, increased the potency. Compound 3 was
nearly twenty times more potent than the derivative without chlorine atoms 1. β-Hydroxy selenides and
diselenides 4-7 did not inhibit this cytosolic isoform. hCA II was inhibited by the aromatic diselenide
incorporating a phenol moiety (8) with a Ki of 23.7 µM, and the secondary sulfonamide group (10-11) in
medium - high micromolar range. In the case of the last two compounds, the introduction of a methyl
group on the aromatic ring halved the inhibition potency (Ki 61.6 to 30.9 µM). The selenides 4-15
inhibited in the low micromolar range (Ki 3.2 µM -5.6 µM) hCA II, whereas symmetrical diselenides 16
and 17 inhibited this cytosolic isoform in the medium (Ki 13.1 µM) or high micromolar ranges (Ki 96.1
µM). The only selenoheterocycle here studied, 21, inhibited this isoform in the medium-high
micromolar range.
(iii) The membrane-bound hCA IV was inhibited by benzoselenophene compounds 1-3 in the low -
medium micromolar range (Ki 9.1 µM -56.8 µM). The presence of chlorine atoms in these compounds
increases the potency, but this time, compound 2 with one chlorine atom showed the best activity. β-
Hydroxy selenide and diselenide 4-7 and β-amino selenide and diselenide 14-17 did not inhibited this

8
isoform, except for compound 16 which showed a Ki of 23.5 µM. Aromatic diselenide 8 inhibited hCA
IV in the medium micromolar range (Ki 26.6 µM), 10 and 11 between Ki 6.2 - 78.9 µM. The presence of
methyl groups on the aromatic ring led to a significant decrease of the inhibitory potency. The other
aromatic diselenides, seleno heterocycles and benzenseleninic acid 23 did not inhibit this isoform.
(iv) The other cytosolic human isoform studied, hCA VII, was inhibited by benzoselenophene
compounds 1 and 2 in the low micromolar range (Ki 6.5 µM -1.8 µM), whereas compound 3 in the high
nanomolar (Ki 0.66 µM). Secondary sulfonamide (SO2NHR) compounds here considered, 10, 11, 15 and
17 inhibited this isoform in the low micromolar range (Ki 4.6 µM -8.9 µM). The substitution of the
methyl group on aromatic diselenide 10 and 11, this time did not influence the potency of inhibition.
(v) Transmembrane hCA IX was inhibited by compounds 1-3 in the medium and low micromolar range
(Ki 37.0 µM -9.1 µM). The chlorine substitution on the benzoselenophene scaffold increased the potency
as for the cytosolic isoforms discussed previously. The aromatic diselenides 10 and 11 inhibited hCA IX
in low micromolar range (Ki 1.6 µM -8.4 µM). Methyl substitution on aromatic scaffold led to a
decrease the potency of inhibition nearly four times.
(vi) An interesting inhibition profile was observed for compounds 4, 5, 12 and 22, which showed
selectivity for isoform hCA I. On the other hand, 14 and 21, showed selectivity for the two abundant
cytosolic isoforms hCA I and hCA II. All isoforms here considered, except for the transmembrane hCA
IX, were inhibited by the aromatic diselenide 8. Another interesting point, was the inhibitory activity of
the secondary sulfonamides 15 and 17, as well as their selectivity for the cytosolic isoforms here
investigated.
3. Conclusions
We have investigated a series of selenides, diselenides and selenoheterocycles as inhibitors on five α-
carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, i.e., hCA I, II, IV, VII and IX.

9
These isoforms are drug targets for antiglaucoma (hCA I, II and IV), antiepileptic (hCA VII) or
antitumor (hCA IX) agents. Recently, a study from our group reported another potential role for isoform
hCA VII as oxygen radical scavenger.20 In this contest, the investigated organoselenium compounds
showed inhibitory action and in same case selectivity against the cytosolic over membrane-bound
isoforms, among which aslo hCA VII.
4. Experimental Part
4.1. Chemistry
All reactions were carried out in an oven-dried glassware under inert atmosphere (N2). Tetrahydrofuran
(THF), dichloromethane (DCM) and N,N-dimethylformamide (DMF) were dried using a solvent
purification system (Pure-Solv™). All commercial materials were used as received without further
purification. Flash column chromatography purifications were performed on Silica gel 60 (230-400
mesh). Thin layer chromatography was performed on TLC plates Silica gel 60 F254. NMR spectra were
recorded in CDCl3 with Varian Gemini 200 and Mercury 400 spectrometers operating at 200 and 400
1
MHz (for H), 50 and 100 MHz (for 13C). NMR signals were referenced to nondeuterated residual
solvent signals (7.26 ppm for 1H, 77.0 ppm for 13C).
4.1.1 General procedure for preparation of the benzoselenophene derivatives 1-3.
Fresh distilled SO2Cl2 (0.8 mmol, 1 mmol or 2 mmol to obtain 1, 2 or 3 as major products, in that order)
was added dropwise to selenium powder (79 mg, 1 mmol) and stirred at rt for 10 min, and then 2.5mL of
distilled THF was added. After 1 h, resveratrol (94 mg, 0.4 mmol) dissolved in 0.8 mL of dry DMF was
added. The mixture was stirred for 24 h at rt. The brownish red product was filtered over Celite before
extraction with ethyl acetate (3 × 15 mL), and the organic layer was washed with water and brine and

10
dried over anhydrous Na2SO4. The solvent was evaporated under vacuum to afford the crude product,
which was purified by flash column chromatography (chloroform/methanol 9 : 1, 1% acetic acid).
4.1.2 General Procedure for preparation of β-hydroxy diselenides 5, 9.
A solution of epoxide (1 mmol) and bis(trimethylsilyl)selenide (HMDSS) (1.6 mmol) in anhydrous THF
(3 mL), was treated under an inert atmosphere with TBAF (1 M in THF, 0.64 mL, 0.64 mmol). After
stirring for 4 h, the solution was diluted with diethyl ether, washed with water, and dried with Na2SO4.
The solvent was evaporated under vacuum to afford a crude product, which was purified by flash
column chromatography eluting with an appropriate mixture of petroleum ether/ethyl acetate.
4.1.3 General Procedure for the preparation of N-Ts protected β-amino diselenides 16, 17.
A solution of aziridine (1 mmol) and bis(trimethylsilyl)selenide (HMDSS) (1.6 mmol) in anhydrous
THF (3 mL) was cooled under an inert atmosphere at 0 °C, and treated with TBAF (1 M in THF, 0.48
mL, 0.48 mmol). After warming to room temperature and stirring for ca. 1 h, the solution was diluted
with diethyl ether, washed with water, and dried with Na2SO4. The solvent was evaporated under
vacuum to afford a crude product, which was purified by flash column chromatography eluting with an
appropriate mixture of petroleum ether/ethyl acetate.
4.1.4 General Procedure for the preparation of β-hydroxy selenide 4.
A solution of epoxide (1 mmol) and bis(trimethylsilyl)selenide (HMDSS) (0.6 mmol) in anhydrous THF
(3 mL), was treated under an inert atmosphere with TBAF (1 M in THF, 0.24 mL, 0.24 mmol). After
stirring for 4 h, the solution was diluted with diethyl ether, washed with water, and dried with Na2SO4.
The solvent was evaporated under vacuum to afford a crude product, which was purified on silica gel
eluting with petroleum ether/ethyl acetate 2:1.
4.1.5 General Procedure for the preparation of N-Ts protected β-amino selenide 14.
A solution of aziridine (1 mmol) and bis(trimethylsilyl)selenide (HMDSS) (0.6 mmol) in anhydrous
THF (3 mL) was cooled under inert atmosphere at 0 °C, and treated with TBAF (1 M in THF, 0.3 mL,

11
0.3 mmol). After warming to room temperature and stirring for ca. 1 h, the solution was diluted with
diethyl ether, washed with water, and dried with Na2SO4. The solvent was evaporated under vacuum to
afford a crude product, which was purified by flash column chromatography (petroleum ether/ethyl
acetate 3:1).
4.1.6 General Procedure for the preparation of 2-Nitroaryl diselenide 13.
In a round-bottomed flask, a mixture of elemental selenium (158 mg, 2.0 mmol) and KOH (224 mg, 4.0
mmol) was melted with a thermal blower. The resulting mixture was cooled to room temperature, and
diluted with distilled water (4 mL). Then the corresponding o-halonitrobenzene (1.0 mmol) and THF (1
mL) were added, and the mixture was stirred for 0.5 to 2 h, depending on the substrate. When the
reaction was complete, the product was extracted with EtOAc (20 mL), and the organic phase was
washed with H2O (3 × 20 mL). The organic phase was dried with MgSO4, and filtered, and the solvent
was removed under reduced pressure. The product was purified by flash column chromatography
(petroleum ether/ethyl acetate 2:1).
4.1.7 General Procedure for the preparation of 2-Aminoaryl diselenide 12
The appropriate 2-nitroaryl diselenide (1.0 mmol), methanol (20 mL), FeSO4·7H2O (5.0 mmol, 1.390 g),
and distilled water (20 mL) were added to a two-necked round-bottomed flask equipped with a reflux
condenser. The reaction mixture was stirred at reflux for 1.5 h. After this time, NH4OH (10 mL) was
added, and the mixture was stirred under reflux for 10 min. A black mixture was formed after the
addition of the NH4OH. Then the mixture was cooled to room temperature, diluted with ethyl acetate (50
mL), filtered, and washed with H2O (3 × 20 mL). The organic phase was dried with MgSO4, and filtered,
and the solvent was removed under reduced pressure. The product was purified by flash column
chromatography (petroleum ether/ethyl acetate 2:1).
4.1.8 General Procedure for preparation of 2-N-Sulfonylaminoaryl diselenides 10, 11.

12
A solution of sulfonyl chloride (1.75 mmol) in dry CH2Cl2 (5 mL) was added dropwise under a nitrogen
flow to a solution of bis(2-aminophenyl) diselenide (0.73· mmol) in dry CH2Cl2 (5 mL) and dry pyridine
(0.118 mL, 1.46 mmol). The reaction mixture was heated at reflux for 44 h, then it was cooled to room
temp. and diluted with CH2Cl2 (40 mL). The organic phase was washed with H2O (3 × 40 mL), and
saturated aq. NH4Cl (2 × 40 mL), and dried with anhydrous Na2SO4. The solvents were evaporated
under vacuum and the crude material was purified by flash column chromatography eluting with an
appropriate mixture of petroleum ether/ethyl acetate.
4.1.9 General Procedure for the preparation of β-phenylseleno- alcohols 6, 7 and amine 15.
A solution of epoxide or aziridine (1 mmol) in dry THF (4 mL) was treated under inert atmosphere with
phenylseleno-trimethylsilane (1.1 mmol) and TBAF or PhONnBu4 (0.2 mmol).28 The mixture was stirred
at rt. and progression of the reaction was monitored by TLC. After quenching with water, the product
was extracted with diethyl ether. The resulting organic phase was washed with brine, dried over Na2SO4
and the solvent evaporated under vacuum. TLC purification with an appropriate mixture of petroleum
ether/ethyl acetate afforded products 6, 7, 15.
4.1.10 General Procedure for the preparation of selenazines 18-22.
Cu(OTf)2 (0.016 mmol, 20 mol%), p-methoxystyrene (21 mg, 0.16 mmol, 1 equiv.) and Et3N (8 mg,
0.08 mmol, 0.5 equiv.) were added in sequence to a solution of 2-N-sulfonyl diselenide (0.08 mmol, 1
equiv.) in dry solvent (0.03 M) in a reaction vial. The mixture was heated at the right temperature until
TLC showed complete disappearance of the diselenide. Then, the reaction mixture was diluted with
dichloromethane (40 mL), washed with saturated NH4Cl (40 mL), and dried with anhydrous Na2SO4,
and the solvents were evaporated to dryness. Purification by flash column chromatography eluting with
an appropriate mixture of petroleum ether/ethyl acetate gave derivatives 18-22.
4.1.11 Procedure for preparation of 1,2-Bis(2-hydroxyphenyl)diselane 8.

13
To a stirred solution of Se0 (2.0 mmol) and halides (1.0 mmol) in dry DMSO (2.0 mL) was added CuO
nanoparticles (10.0 mol%) followed by KOH (2.0 equiv) under nitrogen atmosphere at 90 °C. The
progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture
was allowed to cool, which was subjected to column chromatographic (petroleum ether/ethyl acetate
2:1) separation to give pure diselenide 8.
4.2. Carbonic anhydrase inhibition
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2
hydration activity.30 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at
the absorbance maximum of 557 nm, with 20 mMHepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for
maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration
reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of
the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed
rates were determined in the same manner and subtracted from the total observed rates. Stock solutions
of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done
thereafter with 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,31–33 and represent the mean from at least three different determinations. All
CA isofoms were recombinant ones obtained in-house as reported earlier.31–33
Acknowledgments. This work was financed in part by two EU projects of the 7th Framework
programme, Metoxia and Dynano (to CTS).

14
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19
Evaluation of selenide, diselenide and selenoheterocycle derivatives as carbonic anhydrase I, II, IV,
VII and IX inhibitors
Andrea Angeli, a Damiano Tanini, b Caterina Viglianisi, b Lucia Panzella,c Antonella Capperucci, b
Stefano Menichetti,b Claudiu T. Supurana*