Tellurides Bearing Sulfonamides as Novel Inhibitors of Leishmanial Carbonic Anhydrase with Potent Antileishmanial Activity

Article abstract of DOI:10.1021/acs.jmedchem.0c00211

We report for the first time a novel series of tellurides bearing sulfonamide as selective and potent inhibitors of the β-class carbonic anhydrase (CA; EC 4.2.1.1) enzyme expressed in Leishmania donovani protozoa. Such derivatives showed high activity against axenic amastigotes, and among them, compound 5g (4-(((3,4,5-trimethoxyphenyl)tellanyl)methyl)benzenesulfonamide) showed an IC₅₀ of 0.02 μM being highly selective for the parasites over THP-1 cells with a selectivity index of 300. The in vitro and in vivo toxicity experiments showed compound 5g to possess a safe profile and thus paving the way for tellurium-containing compounds as novel drug entities.

Full text of DOI:10.1021/acs.jmedchem.0c00211

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Article
Tellurides Bearing Sulfonamides as Novel Inhibitors of Leishmanial
Carbonic Anhydrase with Potent Antileishmanial Activity
Andrea Angeli, Mikel Etxebeste Mitxeltorena, Carmen Sanmartin, Socorro Espuelas, Amaya
Azqueta, Seppo Parkkila, Fabrizio Carta, Claudiu T. Supuran, and Esther Moreno Amatria
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00211 • Publication Date (Web): 29 Mar 2020
Downloaded from pubs.acs.org on April 4, 2020
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Tellurides Bearing Sulfonamides as Novel Inhibitors of Leishmanial Carbonic
Anhydrase with Potent Antileishmanial Activity
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Andrea Angeli,a,b*† Mikel Etxebeste-Mitxeltorena,c† Carmen Sanmartín,c Socorro Espuelas,c
Esther Moreno,c Amaya Azqueta,c Seppo Parkkila,d Fabrizio Carta,a Claudiu T. Supurana*
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a
University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
b Centre of Advanced Research in Bionanoconjugates and Biopolymers Department,“Petru Poni”
Institute of Macromolecular Chemistry, 700487, Iasi, Romania
c
University of Navarra, School of Pharmacy and Nutrition, Department of Pharmaceutical
Technology and Chemistry, Irunlarrea 1, 31008, Pamplona, Spain.
d Faculty of Medicine and Life Sciences, University of Tampere and Fimlab Ltd, 33100 , Tampere
University Hospital
† These Authors contributed equally.
Abstract. We report for the first time, a novel series of tellurides bearing sulfonamide as selective
and potent inhibitors of the β-class Carbonic Anhydrase (CA; EC 4.2.1.1) enzyme expressed in
Leishmania donovani protozoa. Such derivatives showed high activity against axenic amastigotes,
and among them compound 5g showed an IC50 of 0.02 µM being highly selective for the parasites
over THP-1 cells with a selectivity index of 300. The in vitro and in vivo toxicity experiments
showed compound 5g to possess a safe profile and thus paving the way for tellurium containing
compounds as novel drug entities.
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Keywords: Carbonic Anhydrase; Carbonic Anhydrase Inhibitor (CAI); metalloenzymes;
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tellurium; telluride; leishmania; leishmaniosis.
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INTRODUCTION
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Leishmaniasis comprises a group of infectious diseases having as etiological agents more than 20
obligate intracellular protozoan parasites of the Leishmania genus.1 The transmission of the
parasites to humans occurs through the bites of the infected female phlebotomine sand flies and
the disease may take place after years of incubation.1, 2 Two main clinical forms of the pathology
are known: i) the tegumental leishmaniasis (i.e. cutaneous and mucocutaneous) usually determines
skin sores, deformities and disabilities and ii) the visceral leishmaniasis which affecting internal
organs may be lethal within two years since the clinical appearance of the disease.1, 2 Meta-analysis
data on global scale account for up to 1 million new clinical cases of Leishmania each year with
65.000 deaths connected to the disease itself. Although leishmaniasis is endemic of tropical and
subtropical regions, mass transfer of people and goods make it of global concern.2 According to
the United States Department of Health & Human Services and WHO the most effective way to
prevent any infection still is avoidance of sand fly bites as no pharmacological prophylaxis is
available.1,2 Once leishmania is diagnosed antimonial based drugs such as Meglubine antimoniate
and Sodium stibogluconate are of first choice, followed by Amphotericin B, Paromomycin,
Miltefosine and Pentamidine (Figure 1).3-5
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OH
HN
OH
OH
NH
3
HO
O
O
O
O
OH
O
O
Sb
HO
O
O
3Na
O
O
O
O
Sb Sb
HO
HO
O
OH
H
OH OH H
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OH
OH
OH
Sodium Stibogluconate
Meglubine Antimoniate
HO
HO
HO
OH
OH
O
O
OH
H2N
NH2
OH
O
H
HO
O
OH OH
OH OH
O
OH
NH2
O
H
OH
O
O
O
O
H
HO
NH2
OH
O
HO
OH
Amphotericine
Paromomycin
NH2
H2N
OH
O-
O P O
O
O
O
N+
HN
NH
NH2
NH2
Miltefosine
Pentamidine
Figure 1. Chemical structures of current drugs used in the treatment of leishmaniasis.
Many efforts from researchers are currently focus on the development of new and more
effective drugs acting on the infectious protozoa with the aim to drastically reduce the heavy toxic
effects associated to the current pharmacological treatments.6,7 The absence of a vaccine, the high
toxicity of drugs combined with the rapid emergence of drug resistant Leishmania strains make
leishmaniasis an ongoing health threat.8,9 In the last years, several efforts are made to Discover
new therapeutic targets against leishmaniasis. A key note is the recent discovery of a selective cdc-
2-related kinase12 (CRK12) small molecule A in Figure 2, which is under preclinical development
for the management of visceral leishmaniasis.10
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N
NH
N
H
N
CF3
N
O
N
S
O
O
N
H
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A
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Figure 2. Chemical structure of the cdc-2-related kinase12 (CRK12) ligand compound A.10
In this context the validation of new therapeutic targets along with the development of
innovative drugs are of paramount importance and the Carbonic Anhydrases (CAs, EC 4.2.1.1) are
as such.11-13 These metalloenzymes reversibly catalyze the hydration of carbon dioxide to afford
bicarbonate and proton,14 which is an essential reaction to the parasite growth and virulence.15-18
Some of us reported that Leishmania donovani protozoa belonging to the subspecies chagasi and
responsible for the visceral leishmaniasis, encode for an α- and a β-CA isoform.19 The latter which
is not present in humans was expressed, purified and kinetically profiled also by means of various
known CA inhibitors.19
In the current work we made use of the exclusivity of the biological target CA along with
the recent increase of interest of tellurium-containing compounds as potential chemotherapeutic
agents.20-24 The anti-microbial and -parasite activity of tellurium against both flagellate and non-
flagellate forms are well reported 20-24 and represent a significative opportunity in the management
of infectious promoted diseases. Thus we report the synthesis of new organotellurium compounds,
bearing the benzensulfonamide moiety, as Carbonic Anhydrase Inhibitors (CAIs) and we
performed the in vitro kinetic profile against different CA isoforms expressed in bacteria and
protozoa of particular interest. The aim of our study is to build an initial structure−activity
relationship (SARs) platform around the unexplored field of organotellurium compounds for
biomedical purposes.
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RESULTS AND DISCUSSION
Chemistry
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The final compounds here reported 5a-g were obtained in a simple, modular and efficient two-step
synthesis (Schemes 1 and 2). A library of symmetrical aromatic ditellurides 2a-g was obtained by
the appropriate halo aryl via lithiation, tellurium insertion and air oxidation according to, or in
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analogy with, literature procedures (Scheme 1)22-24
.
R
i) t-BuLi
THF, -78°C
Br
Te
Te
ii) Te0 r.t 2h
R
R
1a-g
2a-g
1a
1b
1c
1d
1e
: R= H
2a
2b
2c
2d
2e
: R= H
: R= 4-CH3
: R= 4-OCH3
: 2-Naphtyl
: R= 2-CH3
: R= 2,6-CH3
: R= 4-CH3
: R= 4-OCH3
: 2-Naphtyl
: R= 2-CH3
: R= 2,6-CH3
1f
1g
2f
2g
: R= 3,4,5-OCH3
: R= 3,4,5-OCH3
Scheme 1. General synthesis of symmetrical aromatic ditellurides 2a-g.
In situ reduction of the aromatic ditellurides 2a-g followed by reaction with 4-
(bromomethyl) benzenesulfonamide 4 afforded final compounds 5a-g as outlined in Scheme 2.
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R
SO2NH2
Te
4
NaBH4 (3 equiv)
EtOH
Te
Te
Te
EtOH
R
R
R
9
2a-g
5a-g
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2a
: R= H
5a
: R= H
2b
2c
2d
2e
2f
2g
: R= 4-CH3
: R= 4-OCH3
: 2-Naphtyl
: R= 2-CH3
: R= 2,6-CH3
5b
5c
5d
5e
: R= 4-CH3
: R= 4-OCH3
: 2-Naphtyl
: R= 2-CH3
: R= 2,6-CH3
5f
5g
: R= 3,4,5-OCH3
: R= 3,4,5-OCH3
Scheme 2. General procedure for the synthesis of aryl benzyl tellurides 5a-g
The structures and purity of final compounds as well as all intermediates were confirmed
by spectroscopic data such as 1H, 13C, 125Te nuclear magnetic resonance (NMR), high resolution
mass spectrometry (HRMS) and elemental analysis.
Carbonic Anhydrase Inhibition.
All compounds 5a-g were tested in vitro for their inhibitory activity against the human off-target
CAs I, II and bacterial expressed isoforms such as the Vibrio cholerae (VhCAα, VhCAβ) and
Sulfurihydrogenibium yellowstonense (SspCA), the fungal Cryptococcus neoformans (CAN2) and
Malassezia globosa (MgCA) and finally the protozoal CA enzyme from Leishmania donovani
(LdCA) by means of the stopped-flow carbon dioxide hydration assay.25 All the obtained kinetic
data were compared to those obtained from the standard CAI acetazolamide (AAZ) (Table 1).
Table 1. Inhibition data of human CA isoforms I, II, SspCA, VhCAα, VhCAβ, CAN2, MgCA,
LdCA with compounds 5a-g and AAZ by a stopped flow CO2 hydrase assay.25
KI (nM)*
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Cmp
5a
hCA I
209.8
24.0
hCAII
12.2
4.4
SspCA VhCAα VhCAβ MgCA
CAN2
82.5
34.1
9.4
LdCA
9.8
30.3
4.1
427.9
5.9
6.4
473.8
26.4
6.5
8.9
50.6
406.0
50.0
40.9
9.7
5b
9.6
5c
1.5
2.0
4.2
2.3
31.0
24.7
9.1
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5d
2401
18.0
182.3
0.67
3.3
42.0
14.6
29.6
43.2
4.5
766.9
80.0
42.8
586.1
6.8
10.0
60.4
29.5
77.3
10.5
5e
6.1
5f
256.6
377.1
250
6.4
9.4
5g
13.2
12.1
5.1
31.9
40000
9.6
AAZ
451
91.7
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10 %
of the reported values).
As reported in Table 1 the substituents introduced into the tail were essential for both the
kinetic activity and isoform selectivity.
The unsubstituted compound 5a exhibited good inhibition potency against the β-CA
isoforms of Vibrio cholerae, Malassezia globosa and Leishmania donovani with KIs of 6.4, 8.9
and 9.8 nM and thus far more potent of the standard CAI AAZ (KIs of 451, 40000 and 91.7
respectively). The Cryptococcus neoformans CAN2 was the only exception as AAZ resulted 7.9
fold more potent (Table 1). The introduction of the methyl moiety in para-position as in 5b
enhanced the inhibition potency on the α-CAs whereas it was detrimental for the β-ones with the
CAN2 and LdCA isoforms as the exceptions. A quite interestingly kinetic profile was obtained
when the methyl group in 5b was replaced with the methoxy moiety instead (i.e. 5c). In this case
the human expressed α-CAs I and II (KIs 1.5 and 2.0 nM respectively), the Vibrio cholerae VhCAs
(KI 2.3 and 26.4 nM) and the Cryptococcus neoformans β-CAN2 (KI 9.4 nM) showed the strongest
KI value lowering effect among all the enzymes tested. The compound 5c was almost effective as
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the 5b on the α-SspCA (KIs of 4.2 and 4.1 nM respectively) whereas resulted less effective against
the MgCA and the LdCA (KIs of 406 and 31.0 nM respectively).
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The ortho substituted regioisomer of 5b, (i.e. 5e) changed considerably the enzymatic
inhibition profiles. As reported in Table 1 both the hCAs and VhCA were potently inhibited with
KIs up to subnanomolar values as for the hCA II. 5b was 1.2 fold more potent that 5a as MgCA
inhibitor (40.9 and 50.6 nM respectively) and remarkably less potent on the α-isoforms SspCA,
VhCA and the β-CAN2 (Table 1). Again the LdCA KI values was almost constant (KIs of 9.6 and
9.1 nM respectively).
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The addition of a second methyl methyl moiety in para to 5e to afford 5f allowed to better
discriminate the between the hCAs I and II. As reported in Table 1 the hCAs II was 77.8 fold more
inhibited when compared to the isoform I (KIs of 3.3 nM and 256.6 nM respectively). In analogy
to the hCAs, compound 5f maintained a good selectivity also for the VhCAs although the KI value
against the α-isoform was almost halved and the β-one remained almost constant (KIs of 42.8nM
and 6.4 nM respectively). Important increases of inhibition potency were observed also for CAN2
and MgCA with KIs of 29.5 and 9.7 nM respectively, whereas the SspCA and LdCA were 2 and
1.03 fold less inhibited (Table 1).
A drastic change in the substitution pattern as in compound 5g didn’t disrupt the inhibition
profiles against the hCAs and the VhCAs. Data in Table 1 showed the hCA II and the β-VhCA
being preferentially inhibited over their counterparts with selectivity indexes (SI) of 28.6 (i.e. KI
CA II/KI CA I) and 114.9 (i.e. KI β-VhCA/KI α-VhCA respectively). As for the SspCA, MgCA
and CAN2 medium/high nanomolar inhibition values were obtained (KIs of 43.2, 31.9 and 77.3
nM respectively). Also for compound 5g the inhibition value against the LdCA was constant and
perfectly matched with the methyl-substituted compound 5b (KI 9.6 nM).
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The bulkiest organotellurium derivative among the series (i.e. 5d) showed inhibition
potencies against the CA isoforms tested spanning between the medium/high nanomolar range
with the exceptions of β-VhCA and CAN2 which were nanomolar. In particular 5d inhibited the
β-VhCA with a KI of 6.5 nM (i.e. close-fitted with 5a, and 5e-g) and CAN2 with a KI of 10.0 nM
(i.e. matched with the standard CAI AAZ 10.5 nM).
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Notably, all telluride compounds 5a-g in our study revealed to be potent and selective
protozoan LdCA inhibitors when compared to AAZ. The kinetic profile of such compounds
represents a significant finding for the provision of novel leads with promising in vitro anti-
protozoan activities useful to validate LdCA as antileishmania target.
Antileishmanial Activity
The synthesized tellurides 5a-d and 5f were initially tested against L. infantum axenic amastigotes
according to the procedure previously described by our group by three independent experiments.26,
27
In order to assess the selectivity against L. infantum axenic amastigotes, the compounds were
tested against leukemia cells derived from monocytes (THP-1). Miltefosine and Edelfosine were
used as reference drugs and the IC50 obtained are summarized in Table 2.
Table 2. IC50 values for the compounds 5a-f on amastigotes and cytotoxic activity in THP-1 cell
lines
IC50 (µM)
Compound
Amastigotes
THP-1
SIa
5a
5b
0.56 ± 0.05
3.44 ± 0.02
6.37 ± 1.09
>25
11.37
>7.26
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5c
0.73 ± 0.19
1.98 ± 0.68
2.71
>17
2.98
5d
1.47 ± 0.55
2.23 ± 0.55
>25
5f
6.64 ± 0.42
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5g
0.02 ± 0.01
0.82 ± 0.13
2.84 ± 0.10
6.01 ± 0.06
4.96 ± 0.16
18.50 ± 0.60
300.50
6.05
Edelfosine
Miltefosine
6.51
a Selectivity index (SI) is the ratio of IC50 values of compounds against THP-1 cells relative to
those against L. infantum amastigotes.
Compounds 5a, 5c and in particular 5g showed high potency and displayed IC50 values
lower than the reference drug Edelfosine. The remaining derivatives (i.e. 5b, 5d and 5f) reported
higher IC50 values when compared to Edelfosine and close related to the second reference drug
Miltefosine. In light of these results, we considered also the selectivity against L. infantum with
respect THP-1. The substitution in para position as for compounds 5b and 5c or the presence of
two methyl groups in 2 and 6 position (i.e. 5f) did not induce any relevant selectivity against the
protozoa species. On the other hand, compound 5a results near two fold more selective against L.
infantum when compared to both the reference drugs. The selectivity was deeply increased when
to the telluride scaffold was added in position 3, 4, and 5 of the aromatic tail ring the methoxyl
moieties (i.e. 5g). As reported in Table 2 the SI index of 5g for the L. infantum axenic
amastigotes over the THP1 cells was over 300 fold higher.
On considering the high potency and selectivity values over the reference drugs, compounds 5a,
5b, 5d and 5g were selected and further tested at 3 µM concentration for their leishmanicidal
activity on infected macrophages. Again, edelfosine was used as a comparative reference
(Figure 2).
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e
5a
5b
5d
5g
Control
Edelfosin
Figure 2. Leishmanicidal activity on infected macrophages for compounds 5a-d and 5g
As reported above, compound 5g showed the best leishmanical activity against infected
macrophages with selectivity values up to 50 times higher than reference drug edelfosine and far
superior to the other selected compounds 5a, 5b and 5d.
In Vitro Toxicity Assessment
Compound 5g activity and toxicity was evaluated against Vero and Caco-2 cells lines. The
results are below reported in Table 3.
Table 3: Cytotoxic activity for compound 5g on THP-1, Vero and Caco-2 cell lines
IC50 mean ± s.d. (µM)
L. infantum
amastigotes
Compound
THP-1
Vero
Caco-2
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5g
0.02 ± 0.01
6.01± 0.06 21.89 ± 0.93
>100
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9
Compound 5g did not affect the intestinal epithelium cells Caco-2 (IC50 >100 µM), however the
Vero cells cytotoxicity value of 21.89 µM suggested that a possible nephrotoxicity should be
considered.
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Compound 5g genotoxicity was assessed by the SOS/UMU test. Salmonella typhimurium
bacteria were exposed to different concentrations of the compound, with and without S9
metabolic activation. Cell survival percentage (%) was measured spectrophotometrically. More
than 80 % of the bacteria survived at every tested concentration of the compound without S9.
However, concentrations > 1mg/mL of metabolized compound killed the bacteria. As shown in
Figure 3, the compound 5g did not show genotoxicity values (IF < 2) even with the S9
metabolic activation and therefore suggesting its safe administration.
100
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%
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IF
% S9
IF S9
2.5
2.0
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0.0
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Concentration (mg/mL)
Figure 3. Cell survival % with and without S9 metabolic activation. Genotoxicity values (IF) with
the S9 metabolic activation of compound 5g
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Since the oral bioavailability of any drug is dependent of its intestinal absorption,
compounds 5g ability to cross the intestinal mucosa was studied by the Ussing Chambers method
and determined by Tellurium inductively coupled plasma (ICP). The results in Table 4 showed
that less than 2% of the molecule was able to cross the intestinal mucosa. Therefore, other
administration route such as intraperitoneal (i.p.) had to be selected in order to explore the toxicity
and efficacy of the compound also in consideration of its low solubility in water (data not shown).
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Table 4. Predicted oral bioavailability of compound 5g
Te concentration (ppm) ± s.d.
Compound
5g
Donor
Receptor
Absorption
36.36 ± 0.74
0.43 ± 0.24
<2%
In vivo Toxicity Assessment
The acute toxicity of compound 5g was assessed with a starting dose of 15mg/kg via i.p.
administration. Since the animals did not show any behavioral, physical or neurological anomalies
up to 48 h the dose was triplicated (i.e. 45mg/Kg) and resulted well tolerated. Further scale up to
90mg/Kg determined death of all mice treated. The repeat dose toxicity study was assessed by
administration of 5g at 20 mg/Kg via i.p. for five consecutive days. All treated mice survived to
treatment and did not show any anomality when closely inspected. The anatomopathological
inspections on liver specimens of the animal exposed to repeat dose toxicity studies revealed
normal morphology and no significant alterations associated with a cytotoxic effect induced from
the administration of 5g (Figure 4).
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Figure 4. Histological samples of liver treated with 20 mg/Kg of compound 5g intraperitoneally
for 5 days. Livers were fixed in 4% paraformaldehyde, embedded in paraffin and stained with
hematoxylin-eosin.
Closer inspection revealed accumulation of glycogen in the cytoplasm of hepatocytes,
which is not related to any degenerative process but to nocturnal feeding as normally occurs in
rodents. Kidney specimens reported a tubule nephrosis characterized by loss of nuclei of the cells
that form the contiguous tubules, as well as the eosinophilia acquired by the cellular cytoplasm
(Figure 5).
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Figure 5. Histological samples of kidney treated with 20 mg/Kg of compound 5g intraperitoneally
for 5 days. Kidneys were fixed in 4% paraformaldehyde, embedded in paraffin and stained with
hematoxylin-eosin.
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Such a histological finding is compatible with a nephrotoxic action of compound 5g and
in agreement with the Vero cells cytotoxicity assay previously reported which determined an
IC50 value of 21.89 µM.
Conclusions
In summary, we investigated a novel series of tellurides containing sulfonamides as inhibitors of
the CA isoforms expressed in various etiological infectious diseases such as the Vibrio cholerae
(VhCAα, VhCAβ) and Sulfurihydrogenibium yellowstonense (SspCA), the fungal Cryptococcus
neoformans (CAN2) and Malassezia globosa (MgCA) the protozoal CA enzyme from
Leishmania donovani (LdCA) in comparison with the ubiquitarian hCAs I and II. Preliminary in
vitro SAR studies of compounds 5a-g showed potent inhibition activity against LdCA which was
retained despite various substitutions patterns on the tail aromatic ring were adopted. The activity
of such compounds against infected macrophages showed 5g as comparable or better performing
when to the reference drugs Edelfosine and Miltefosine and thus providing the evidence of
significant antileishmanial activity. Furthermore the in vitro and in vivo toxicity experiments
assessed 5g as safe to be administered and thus paving the way for advanced studies to be
performed.
EXPERIMENTAL SECTION
Materials and Methods
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All reactions were carried out in an oven-dried glassware under inert atmosphere (N2). 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, Mercury 400, and Bruker 400 Ultrashield spectrometers operating
at 200 and 400 MHz (for 1H), 50 and 100 MHz (for 13C), and 126 MHz (for 125Te). NMR signals
were referenced to nondeuterated residual solvent signals (7.26 ppm for 1H, 77.0 ppm for 13C).
(PhTe)2 was used as an external reference for 125Te NMR (δ= 420 ppm). 1H NMR data are reported
as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, ap d = apparent
doublet, m = multiplet, dd = doublet of doublet, ecc.), coupling constant (J) or line separation (ls),
and assignment. The solvents used in MS measures were acetone, acetonitrile (Chromasolv grade),
and mQ water 18 MU. The high resolution mass spectrometry (HRMS) analysis was performed
with a Thermo Finnigan LTQ Orbitrap mass spectrometer coupled with an electrospray ionization
source (ESI). Analysis was carried out in positive ion or negative mode ([M+H]+, [M+NH4]+,
[M+Na]+, [M+Li]+, [M-H]-), was used a proper dwell time acquisition to achieve 60,000 units of
resolution at full width at half maximum (fwhm). Elemental composition of compounds was
calculated on the basis of their measured accurate masses, accepting only results with an attribution
error less than 5 ppm and a not integer RDB (double bond/ring equivalents) value.28 Stock
solutions of analytes were prepared using acetone (1.0 mg mL−1) and stored at 4 °C. Then working
solutions of each analyte were prepared by dilution of the stock solutions using mQ H2O/ACN 1/1
(v/v) up to a concentration of 1.0 μg mL−1. The HRMS analysis was performed by introducing the
analyte working solution via syringe pump at 10 μL min−1. NMR and Elemental composition
analysis showed that the purity of all the final products was more than 95%.
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General procedure for the synthesis of aryl ditellurides (2a-g).
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To a stirred solution of appropriate bromo derivatives 1a-g (8.5 mmol) in dry THF (40 mL) under
N2 at -78°C was added ter-butyllithium (7.5 mL, 1.7 M; 12.75 mmol). After 1 h, the cooling bath
was removed and freshly crushed finely ground elemental tellurium (8.5 mmol) was added while
a brisk stream of nitrogen was passed through the open system. After 1 hour, when only trace
amounts of tellurium remained, the dark solution was quenched with a saturated solution of NH4Cl.
After extraction with diethyl ether, separation, drying of the organic phase with Na2SO4 and
evaporation, the pure product was obtained by flash chromatography with appropriate mixture of
hexane/ethyl acetate.
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2-bis (3,4,5-Trimethoxyphenyl) ditellane (2g)
Following the general procedure, the product gave after flash chromatography (hexane/ethyl
acetate 9:1) as a red solid 2g (1,7 g, 68%). 1H NMR (400 MHz, CDCl3) δ(ppm): 6.99 (2H, s), 3.86
(3H, s), 3.79 (6H, s) 13C NMR (100 MHz, CDCl3) δ(ppm): 153.4, 145.4, 116.0, 102.2, 61.3, 56.5
125Te NMR (126 MHz, CDCl3) δ(ppm) : 489.5 HRMS (ESI negative) m/z: 593 [M-H]-
4-(Bromomethyl)benzenesulfonamide (4).
Compound 4 was obtained according to the reported procedures.29
General Procedure for the synthesis of 5a-g
A solution of ditelluride 2a-g (1.0 mmol) in ethanol (5 mL) under a nitrogen atmosphere at r.t. was
treated with NaBH4 (4.0 mmol) and the mixture was stirred until ditelluride was reduced. Then, a
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THF solution (3 mL) of 4-(bromomethyl)benzenesulfonamide (4) (2 mmol) was added and the
mixture was stirred for 2 h. Afterwards, the reaction was treated with saturated NH4Cl aq. (3 mL),
extracted with EtOAc (10 mL) and washed with water (2 x 10 mL) and brine (2 x 10 mL). The
organic phase was dried over Na2SO4, the solvent was evaporated under vacuum and the crude
product was purified by flash column chromatography.
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4-((Phenyltellanyl)methyl)benzenesulfonamide (5a)
Following the general procedure, 2a (205 mg, 0.5 mmol) and 4 (250 mg, 1 mmol) gave after flash
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chromatography (Hex/EtOAc 1:1) 5a (247 mg, 66%). H NMR (400 MHz, DMSO-d6) δ(ppm):
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7.69-7.64 (4H, m), 7.34-7-25 (7H, m), 4.37 (2H, s) C NMR (100 MHz, DMSO-d6) δ(ppm):
147.0, 142.5, 138.8, 130.2, 129.3, 128.7, 126.5, 114.1, 11.6. 125Te NMR (126 MHz, DMSO-d6)
δ(ppm) : 682.3 HRMS (ESI positive) m/z: 395 [M+NH4]+ Elemental analysis: calculated C, 41.65;
H, 3.50; N, 3.74; S, 8.55 Found C, 41.68; H, 3.52; N, 3.71; S, 8.57
4-((p-Tolyltellanyl)methyl)benzenesulfonamide (5b)
Following the general procedure, 2b (218 mg, 0.5 mmol) and 4 (250 mg, 1 mmol) gave after flash
chromatography (Hex/EtOAc 1:1.5) 5b (245 mg, 63%). 1H NMR (400 MHz, DMSO-d6) δ(ppm):
7.65 (2H, d, J=8.3 Hz), 7.57 (2H, d, J=7.9 Hz), 7.32-7.29 (5H, m), 7.10 (2H, d, J=7.7 Hz), 4.32
(2H, s), 2.33 (3H, s) 13C NMR (100 MHz, DMSO-d6) δ(ppm): 147.1, 142.5, 139.2, 138.3, 131.1,
129.3, 126.5, 109.8, 21.7, 11.7. 125Te NMR (126 MHz, DMSO-d6) δ(ppm) : 672.9 HRMS (ESI
positive) m/z: 407 [M+NH4]+ Elemental analysis: calculated C, 43.23; H, 3.89; N, 3.60; S, 8.24
Found C, 43.21; H, 3.90; N, 3.58; S, 8.27
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4-(((4-Methoxyphenyl)tellanyl)methyl)benzenesulfonamide (5c)
5
6
Following the general procedure, 2c (235 mg, 0.5 mmol) and 4 (250 mg, 1 mmol) gave after flash
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chromatography (Hex/EtOAc 1:1) 5c (271 mg, 67%). H NMR (400 MHz, DMSO-d6) δ(ppm):
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7.64 (2H, d, J=8.3 Hz), 7.58 (2H, d, J=8.7 Hz), 7.29 (2H, brs, NH, exchange with D2O), 7.25 (2H,
d, J=8.3 Hz), 6.85 (2H, d, J=8.7 Hz), 4.27 (2H, s), 3.79 (3H, s) 13C NMR (100 MHz, DMSO-d6)
δ(ppm): 160.4, 147.1, 141.6, 130.2, 129.2, 126.4, 116.2, 102.7, 55.9, 12.0. 125Te NMR (126 MHz,
DMSO-d6) δ(ppm) : 672.9 HRMS (ESI positive) m/z: 425 [M+NH4]+ Elemental analysis:
calculated C, 41.53; H, 3.73; N, 3.46; S, 7.92 Found C, 41.52; H, 3.74; N, 3.45; S, 7.90
4-((Naphthalen-2-yltellanyl)methyl)benzenesulfonamide (5d)
Following the general procedure, 2d (255 mg, 0.5 mmol) and 4 (250 mg, 1 mmol) gave after flash
chromatography (Hex/EtOAc 1:1) 5d (259 mg, 61%). 1H NMR (400 MHz, DMSO-d6) δ(ppm):
8.26 (1H, s), 7.93 (1H, dd, J=6.1, 2.9 Hz), 7.87 (1H, dd, J=6.2, 2.9 Hz) 7.82-7.76 (2H, m), 7.64
(2H, d, J=8.3 Hz), 7.57-7.54 (2H, m), 7.38 (2H, d, J=8.3 Hz), 7.24 (2H, brs, NH, exchange with
D2O), 4.47 (2H, s) 13C NMR (100 MHz, DMSO-d6) δ(ppm): 147.1, 142.5, 137.9, 135.4, 134.7,
133.0, 129.4, 129.1, 128.5, 128.0, 127.4, 127.3, 126.5, 11.8. 125Te NMR (126 MHz, DMSO-d6)
δ(ppm) : 684.5 HRMS (ESI negative) m/z: 426 [M-H]- Elemental analysis: calculated C, 48.05; H,
3.56; N, 3.30; S, 7.54 Found C, 48.20; H, 3.53; N, 3.34; S, 7.52
4-((o-Tolyltellanyl)methyl)benzenesulfonamide (5e)
Following the general procedure, 2e (218 mg, 0.5 mmol) and 4 (250 mg, 1 mmol) gave after flash
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chromatography (Hex/EtOAc 1:1) 5e (256 mg, 66%). H NMR (400 MHz, DMSO-d6) δ(ppm):
7.75-7.72 (2H, m), 7.66 (2H, d, J=8.3 Hz), 7.38 (2H, d, J=8.3 Hz) 7.27-7.23 (3H, m), 7.08 (1H,
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td, J=7.3, 1.1 Hz), 4.36 (2H, s), 2.31 (3H, s) C NMR (100 MHz, DMSO-d6) δ(ppm): 146.5,
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138.6, 130.2, 129.5, 129.1, 127.6, 127.3, 126.5, 118.3, 26.8, 11.2. 125Te NMR (126 MHz, DMSO-
d6) δ(ppm) : 564.0 HRMS (ESI positive) m/z: 396 [M+Li]+ Elemental analysis: calculated C,
43.23; H, 3.89; N, 3.60; S, 8.24 Found C, 43.25; H, 3.91; N, 3.59; S, 8.23
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4-(((2,6-Dimethylphenyl)tellanyl)methyl)benzenesulfonamide (5f)
Following the general procedure, 2f (232 mg, 0.5 mmol) and 4 (250 mg, 1 mmol) gave after flash
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chromatography (Hex/EtOAc 1:1) 5f (241 mg, 60%). H NMR (400 MHz, DMSO-d6) δ(ppm):
7.59 (2H, d, J=8.3 Hz), 7.28 (2H, brs, NH, exchange with D2O), 7.21-7.17 (3H, m), 7.13 (2H, d,
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J=8.3 Hz), 4.13 (2H, s), 2.47 (6H, s) C NMR (100 MHz, DMSO-d6) δ(ppm): 147.0, 146.0,
142.4, 130.2, 129.0, 127.0, 126.3, 122.1, 30.2, 11.0. 125Te NMR (126 MHz, DMSO-d6) δ(ppm) :
460.4 HRMS (ESI positive) m/z: 412 [M+Li]+ Elemental analysis: calculated C, 44.71; H, 4.25;
N, 3.48; S, 7.96 Found C, 44.69; H, 4.22; N, 3.46; S, 7.93
4-(((3,4,5-Trimethoxyphenyl)tellanyl)methyl)benzenesulfonamide (5g)
Following the general procedure, 2g (295 mg, 0.5 mmol) and 4 (250 mg, 1 mmol) gave after flash
1
chromatography (Hex/EtOAc 1:1) 5g (335 mg, 72%). H NMR (400 MHz, DMSO-d6) δ(ppm):
7.68 (2H, d, J=8.3 Hz), 7.36 (2H, d, J=8.3 Hz), 7.28 (2H, brs, NH, exchange with D2O), 6.85 (2H,
s), 4.39 (2H, s), 3.75 (6H, s), 3.68 (3H, s) 13C NMR (100 MHz, DMSO-d6) δ(ppm): 154.0, 146.9,
142.5, 138.7, 129.6, 126.5, 116.6, 106.9, 61.0, 56.9, 12.2. 125Te NMR (126 MHz, DMSO-d6)
δ(ppm) : 719.5 HRMS (ESI positive) m/z: 485 [M+Na]+ Elemental analysis: calculated C, 41.33;
H, 4.12; N, 3.01; S, 6.89 Found C, 41.30; H, 4.15; N, 3.02; S, 6.87
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Carbonic Anhydrase Inhibition.
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An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
CO2 hydration activity.25 Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4 for the α-CAs) or
TRIS (pH 8.3 for the β-CAs) as buffers, 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,30-34 and represent the mean from at
least three different determinations. All CA isofoms were recombinant ones obtained in-house as
reported earlier.30-34
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Biological assay.
Cells and culture conditions.
L. infantum axenic amastigotes were grown in M199 (Invitrogen, Leiden, The Netherlands)
medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 1 g/liter β-alanine, 100
mg/liter L-asparagine, 200 mg/liter sacarose, 50 mg/liter sodium pyruvate, 320 mg/liter malic
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acid, 40 mg/liter fumaric acid, 70 mg/liter succinic acid, 200 mg/liter α-ketoglutaric acid, 300
mg/liter citric acid, 1.1 g/liter sodium bicarbonate, 5 g/liter morpholine ethanesulfonic acid, 0.4
mg/liter hemin, and 10 mg/liter gentamicin, pH 5.4, at 37°C. THP-1 cells were kindly provided
by T. Michel (Université Nice Sophia Antipolis, Nice, France) and were grown in RPMI 1640
medium (Gibco, Leiden, The Netherlands) supplemented with 10% heat-inactivated FCS,
antibiotics, 1mM HEPES, 2mM glutamine, and 1mM sodium pyruvate, pH 7.2, at 37°C and 5%
CO2.
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Leishmanicidal activity and cytotoxicity assays.
Drug treatment of amastigotes was performed during the logarithmic growth phase at a
concentration of 2 x 106 parasites/ml at 26°C or 1 x 106 parasites/ml at 37°C for 24 h. Drug
treatment of Jurkat and THP-1 cells was performed during the logarithmic growth phase at a
concentration of 4X105 cells/ml at 37°C and 5% CO2 for 24 h. The percentage of living cells was
evaluated by flow cytometry by the propidium iodide (PI) exclusion method.35
Leishmania infection assay.
THP-1 cells were seeded at 120,000 cells/ml in 24-multidish plates (Nunc, Roskilde, Denmark)
and differentiated to macrophages for 24 h in 1 ml of RPMI 1640 medium containing 10 ng/ml
phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA). Medium culture
was removed, and 1.2 x 106 Leishmania amastigotes in 1ml of THP-1 medium were added to
each well. Four h later all medium with noninfecting amastigotes was removed, washed 3 times
with 1 x phosphate-buffered saline (1 x PBS), and replaced with new THP-1 medium and
corresponding treatment. After 48 h of treatment, medium was removed; THP-1 cells were
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washed 3 times with 1 x PBS and detached with TrypLE Express (Invitrogen, Leiden, The
Netherlands) according to the manufacturer’s instructions. Infection was evaluated by flow
cytometry.
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In vitro genotoxicity assay
Salmonella typhimurium TA1535/pSK1002 test strain (DSMZ, Germany) was employed to
determine the genotoxic potential of 5g as some of its possible metabolites.36 Bacteria were
exposed to different concentrations of compounds either with or without metabolic activation (S9
mix; MutazymeTM) to determine the bacterial survival percentage, calculated by measuring the
absorbance at 600 nm. Then, β-galactosidase activity was measured using a colorimetric method,
with ONPG as a substrate. Positive and negative controls were included in the experiments.
Genotoxicity activity was measured in terms of β-galactosidase activity by calculating an induction
factor (IF) relative to the negative control (i.e., bacteria exposed to the solvent). A compound was
considered genotoxic if the value of IF ≥ 2 at non-cytotoxic concentration (i.e., bacterial survival
≥ 80 %). The experiment was considered valid for positive controls if the obtained IF ≥ 2.
Intestinal permeation assay
All animal manipulations were carried out according to the European Community guidelines for
animal care [DL 116/92, application of the European Communities Council Directive of 24
November 1986 (86/609/EEC)]. The ethical policy of the University of Navarra (Pamplona, Spain)
complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes
of Health (NIH Publication No. 85-23, revised 1996).
Intestinal permeation experiments were carried out by the Ussing Chamber method.37 Wistar
female rats were fasted for 16 h with free access to water before the experiment. Rats were
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anesthetized with isoflurane and sacrificed by cervical dislocation. Jejunum was aseptically
extracted, excised in 2 cm segments avoiding Peyer’s patches and placed in cold 0.9 % NaCl
solution. Jejunum segments were anchored into the Ussing Chambers. All experiments were
realized from the apical side or donor compartment (DC) to the basolateral or receptor (RC)
direction. 5 mL of deionized water at pH 7.4 containing 5% PEG 400 (w/v) were added to both
compartments to ensure sink conditions. Chambers were continuously oxygenated and maintained
at 37 ºC. After 10 min of preincubation, the solution in the DC was replaced by 5 mL of compound
solution with a final concentration of 5 mM. Samples of 500 µL were collected from the RC every
20 min up to 120 min and replaced by equal volume of the RC solution. Samples were freezed at
-20 ºC until analysis. The membrane integrity was evaluated by the addition of yellow lucifer to
the DC and measuring the fluorescence at the end of the experiment.38 The concentration of
compound that permeated the jejunum was determined by Tellurium inductively coupled plasma
(ICP). Experiments were carried out for triplicate for each compound. Appropriate dilution (1:10)
of compound 5g with 1% nitric acid solution in acid-washed polyethylene tubes was prepared
before analyzing. Solution samples were then assayed for tellurium content by a Horiba scientific
model of ULTIMA-2 Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES).
Tellurium was determined with 214.281 nm wavelength, plasma gas flow rate of 12.1 L.min-1,
nebulizer gas flow rate of 0.83 L.min-1, auxiliary gas flow rate of 0.52 L.min-1, and 1.1 kW of
power. A Bandelin model of SONOREX DIGITEC 35 kHz ultrasonic bath with heater (20– 80
ºC) was used.39
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In vivo toxicity assay
Acute toxicity was carried out following the Up-and-Down procedure proposed by the OECD
425.40 Three female mice were treated with a dose of 15 mg/Kg with compound 5g. As all mice
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survived, triplicate dose was administered to other new three mice. If one or two of them died, the
same dose was administered to one or two mice more. Compound was solubilized in PEG
400:H2O (60:40). For the repeat dose toxicity assay, 20 mg/Kg of compound 5g was administered
intraperitoneally, for 5 consecutive days. If survived, mice were then sacrificed and liver and
kidney were fixed in 4% (w/v) paraformaldehyde for 24 h, embedded in paraffin and stained with
hematoxylin-eosin (Merk) for histological observation analysis.
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ASSOCIATED CONTENT
Supporting information is available free of charge on the ACS Publications website: SMILES
1
13
representation for compounds (CSV), Supporting Information file (PDF) with H, C and 125Te
NMR spectra of compounds 5a-g.
AUTHOR INFORMATION
Corresponding Authors
Andrea Angeli (AA): Phone: +39-055-4573666; E-mail: andrea.angeli@unifi.it;
https://orcid.org/0000-0002-1470-7192
Claudiu T. Supuran (CTS): Phone: +39-055-4573729; E-mail: claudiu.supuran@unifi.it;
https://orcid.org/0000-0003-4262-0323
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
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This work was partially supported by a grant of the Romanian Ministry of Research and
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Innovation, CNCS–UEFISCDI, project number PN-III-P4-ID-PCCF-2016–0050, within PNCDI
II.
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Abbreviations used
CA, Carbonic Anhydrase; CAI, Carbonic Anhydrase Inhibitor; TLC, thin layer chromatography;
NMR, Nuclear Magnetic Resonance; PBS, Phosphate Buffered Saline; PMA, phorbol 12-
myristate 13-acetate.
Supporting Information Availability: 1H, 13C and 125Te NMR Spectra of compounds 2g and 5a-
g; List of molecular formula strings
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