Synthesis of novel tellurides bearing benzensulfonamide moiety as carbonic anhydrase inhibitors with antitumor activity

Synthesis of novel tellurides bearing benzensulfonamide moiety as carbonic anhydrase inhibitors with antitumor activity

Journal Pre-proof Synthesis of novel tellurides bearing benzensulfonamide moiety as carbonic anhydrase inhibitors with antitumor activity Damiano Tani...

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Journal Pre-proof Synthesis of novel tellurides bearing benzensulfonamide moiety as carbonic anhydrase inhibitors with antitumor activity Damiano Tanini, Lorenzo Ricci, Antonella Capperucci, Lorenzo Di Cesare Mannelli, Carla Ghelardini, Thomas S. Peat, Fabrizio Carta, Andrea Angeli, Claudiu T. Supuran PII:

S0223-5234(19)30720-2

DOI:

https://doi.org/10.1016/j.ejmech.2019.111586

Reference:

EJMECH 111586

To appear in:

European Journal of Medicinal Chemistry

Received Date: 29 June 2019 Revised Date:

23 July 2019

Accepted Date: 4 August 2019

Please cite this article as: D. Tanini, L. Ricci, A. Capperucci, L. Di Cesare Mannelli, C. Ghelardini, T.S. Peat, F. Carta, A. Angeli, C.T. Supuran, Synthesis of novel tellurides bearing benzensulfonamide moiety as carbonic anhydrase inhibitors with antitumor activity, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111586. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

Synthesis of novel tellurides bearing benzensulfonamide moiety as carbonic anhydrase inhibitors with antitumor activity

Damiano Taninia, Lorenzo Riccia, Antonella Capperuccia, Lorenzo Di Cesare Mannellib, Carla Ghelardinib, Thomas S. Peatc, Fabrizio Cartad, Andrea Angelid,e*, Claudiu T. Supurand*

a

University of Florence, Department of Chemistry "Ugo Schiff", Via della Lastruccia 3-13, I-50019

Sesto Fiorentino, Italy. b

NEUROFARBA Department, Section of Pharmacology and Toxicology, Università degli Studi di

Firenze, Viale Pieraccini 6, 50139 Florence, Italy c

CSIRO, 343 Royal Parade, Parkville, Victoria 3052, Australia

d

Department of University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche,

Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy e

Centre of Advanced Research in Bionanoconjugates and Biopolymers Department,“Petru Poni”

Institute of Macromolecular Chemistry, Iasi, Romania

Keywords: carbonic anhydrase; inhibitor, metalloenzymes, tellurium, antitumor, organotelluride compounds.

Corresponding

Authors.

C.T.S.,

E-mail:

[email protected];

A.A.,

E-mail:

[email protected]

Abbreviations Used. CAI(s), carbonic anhydrase inhibitor(s); AAZ, acetazolamide; (h) CA, (human) carbonic anhydrase; KI, inhibition constant.

Abstract. We have synthetized a novel series of β-hydroxy tellurides bearing the benzenesulfonamide group as potent inhibitors of carbonic anhydrase enzymes. In a one pot procedure, we discovered both the ring opening reaction of the three-membered ring and the cleavage of the sulfonamide protecting moiety at the same time. Moreover, the first X-ray co-crystallographic structure of a β-hydroxy telluride derivative with hCA II is reported. The potent effects of these compounds against the tumor-associated hCA IX with low nanomolar constant inhibition values give the possibility to evaluate their activity in vitro using a breast cancer cell line (MDA-MB-231). Compounds 7e and 7g induced significant toxic effects against tumor cells after 48h incubation in normoxic conditions killing over 50% of tumor cells at 3 µM, but their efficacy decreased in hypoxic conditions reaching the 50% of the tumor cell viability only at 30 µM. These unusual features make them interesting lead compounds to act as antitumor agents, not only as Carbonic Anhydrase IX inhibitors, but reasonably in different pathways, where hCA IX is not overexpressed.

Introduction. Tellurium is a rare element and, unlike the other group members such as O, S, and Se, which find biological use, it has no apparent role in biology.1 During the last decades, many efforts have been made in the development of innovative and new materials containing inorganic tellurium which may find potential applications in the electronics and medical fields.2-4 At the same time, a range of organotellurium derivatives were developed in medicinal chemistry and possess unique properties such as activity against microorganisms,5 specific cancer cells,6 potent caspase and cathepsin inhibitors7 and antioxidant activity, which are often superior to their selenium analogues.8 In this particular context, our group studied for the first time the employment of different organotelluride scaffolds as modulators of the Carbonic Anhydrases (CAs, EC 4.2.1.1).9 These metalloenzymes, present in all kingdoms life, catalyse the reversible reaction between carbon dioxide (CO2) and water (H2O) to afford bicarbonate anion (-HCO3) and protons (H+).10 This simple reaction works a crucial role in many physiological and pathological processes associated with pH control, ion transport, and fluid secretion.11-13 The human (h) isoform CA IX was found overexpressed in a wide selection of hypoxic tumors acting as a key player in cancer cells survival, proliferation and metastasis,14-16 and for this reason it was validated as a pharmaceutical target.16 In this context large efforts within the Medicinal Chemistry field were carried out with the aim to identify molecular moieties able to potently inhibit the CA isozymes and to discriminate among the various isoform expressed in humans.17,18,31 Among the various inhibiting moieties the primary sulphonamides, and their bioisosteres, still represent the major ones.19 As side note, many synthetic approaches towards the obtainment of sulphonamides or related sulfonyl-containing moieties were also developed with potential application within various fields.20-22 In the current study, we explored the idea to combine moieties able to disrupt the redox balances by means of inhibition of the CA isoforms expressed in hypoxic areas of solid tumors. Our longstanding interest in the study of the reactivity of strained heterocycles with chalcogen-containing

nucleophiles, led us to disclose novel synthetic routes towards functionalised organoselenium and organotellurium derivatives.23-26 Such densely functionalised small molecules are valuable synthetic intermediates which have been employed to develop a wide variety of more complex structures with chain-breaking and catalytic antioxidant properties.27-30 Furthermore, we recently exploited these ring-opening procedures to synthesise novel chalcogen-containing hCA inhibitors9,31-33 and activators.34 In this context, organotellurides are attracting growing interest among synthetic and medicinal chemists and, therefore the development of new tellurium-containing CA inhibitors is highly desirable. Despite their potential interest the synthesis and the evaluation of the CA inhibition activity of organotellurides bearing the benzenesulfonamide moiety haven’t been reported so far. Herein, we report for the first time the synthesis of β-hydroxy tellurides bearing the benzenesulfonamide group 7a-g and 8. We explored their in vitro kinetics on the biologically relevant hCAs (i.e. I, II, IV, VII and IX) and we determined the structural features underpinning the kinetic profile by means of the X-ray crystallographic adduct hCA II-8. Finally the best performing compounds on the tumor associated hCA IX (i.e. compounds 7e, g) were evaluated on breast (MDA-MB-231) cancer cell lines. Results and Discussion. Compounds Design and Synthesis. In order to access β-hydroxy substituted tellurides incorporating the benzenesulfonamide moiety, we sought to investigate the reactivity of a conveniently substituted epoxide with telluriumcontaining

nucleophiles.

Thus,

the

epoxide

5,

was

synthesised

hydroxybenzenesulfonamide 4 and (±) epibromohydrin (Scheme 1).36

from

N-protected

Scheme 1: Synthesis of epoxide 5, bearing the protected benzenesulfonamide moiety. Dialkyl ditellurides 2a,b and diaryl ditellurides 2c-g as precursors of tellurium nucleophiles were prepared from the corresponding bromides according to the literature procedures37-39 via tellurium insertion into carbon-magnesium or carbon-lithium bonds followed by subsequent air oxidation (Scheme 2).

Scheme 2: Synthesis of dialkyl and diaryl ditellurides 2a-g. Conditions A: nBuLi or tBuLi, THF, 78°C, 1 h; Conditions B: Mg(0), Et2O, 3 h. We began our investigations by evaluating the reactivity of the epoxide 5 with aryl tellurolates generated in situ by reduction of the corresponding ditellurides. We were pleased to observe that when dibutyl ditelluride 2a was treated with NaBH4 and then reacted with 5, the tellurium containing N-sulfonylformamidine 6 was formed as the major product. The cleavage of the protecting group under reported conditions40 led to the desired β-hydroxy telluride 7a which was recovered in high yield (Scheme 3).

Scheme 3: Synthesis of β-hydroxy telluride 7a from ditelluride 2a and epoxide 5. In order to further optimize this protocol, we investigated whether β-hydroxy-tellurides could be obtained through a one pot procedure encompassing both the three-membered ring opening reaction and the cleavage of the sulfonamide protecting group. Therefore, the epoxide 5 was reacted with n

BuTe─ generated by treating dibutyl ditelluride 2a with a large excess of NaBH4 (4 eq.) in the

presence of NaOH (2.0 eq.) as reported in Scheme 4.41 We were glad to observe that the hydroxylsubstituted telluride 7a was achieved in good yields. The scope of such a procedure was conveniently extended to include differently substituted diorganyl ditellurides (Scheme 4), allowing the synthesis of unsymmetrical β-hydroxy-substituted dialkyl (7a,b) and alkyl aryl tellurides (7c-g).

R/ArTeTeAr/Ar

i) NaBH4 (4.0 eq.) NaOH (2.0 eq) EtOH, 0 °C to r.t.

SO2NH2 R/ArTe

O

ii) 5 (1.8 eq.) in DMF r.t., 12 h

2a-g

OH 7a-g

SO2NH2 Te

O

Te

OH 7a, 63% Te

Te

SO2NH2 O OH 7b, 58%

SO2NH2

O OH 7c, 64%

Te SO2NH2

O

O

Te

OH 7e, 48% SO2NH2 Te

O OH 7d, 55%

SO2NH2

SO2NH2

O OH 7f, 61%

O OH 7g, 53%

Scheme 4: One pot synthesis of β-hydroxy tellurides 7a-g from ditellurides 2a-g and epoxide 5. With the aim to further investigate the activity of tellurium-containing benzensulfonamides, the symmetric β-hydroxy dialkyl telluride 8 was efficiently synthesised from the epoxide 5 and Li2Te which was generated in situ from elemental tellurium and LiEt3BH, following a slightly modified reported procedure (Scheme 5).24 It is noteworthy, that two ring opening reactions and two protecting group cleavages take place in this one pot route.

Scheme 5: Synthesis of symmetric β-hydroxy telluride 8 from elemental tellurium and epoxide 5. Carbonic anhydrase inhibition.

All compounds herein reported and acetazolamide (AAZ), as standard Carbonic anhydrase inhibitor (CAI), were investigated for their inhibition properties against a panel of hCA isozymes such as I, II, IV, VII and IX isoforms by the stopped-flow carbon dioxide hydration assay41 after 15 min of incubation of the enzyme and inhibitor solutions(Table 1).42–46 Table 1. Inhibition data of human CA isoforms I, II, IV, VII and IX with compounds 7a-g, 8 and AAZ by a stopped flow CO2 hydrase assay.41 KI (nM)* Cmp

hCA I

hCAII

hCA IV

hCA VII

hCA IX

7a

82.1

3.9

915.5

37.2

333.2

7b

188.1

36.2

96.0

63.7

40.7

7c

46.8

0.42

6.8

0.78

14.8

7d

83.9

0.56

1.7

2.7

118.1

7e

36.5

0.64

192.1

0.72

9.5

7f

394.4

4.4

42.1

11.9

7.8

7g

8798.6

8.5

183.2

261.9

6.4

8

67.4

0.62

18.1

0.8

808.9

AAZ

250.0

12.1

74.0

2.5

25.8

* Mean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5-10 % of the reported values). On the basis of the kinetic data reported in Table 1, tellurides bearing benzensulfonamide 7a-g and 8 showed remarkable variation among the different CA isoforms and the following structure−activity-relationships (SARs) can be drawn.

The cytosolic widespread enzyme hCA I was inhibited by compounds 7a-g and 8 with a wide range of potency that spanned KIs 36.5 to 8798.6 nM. The length of alkyl moiety did not influence significantly the potency. On the other hand, the different substituents on the aromatic telluride moiety (7c-g) played a crucial role for the potency. The ortho position with CH3 substituent (7e) showed the best inhibition value (KI 36.5 nM). The activity decreased when a OCH3 moiety was placed in para position (KI 394.4 nM). Finally, the bulky naphtyl scaffold (7g) showed a lower inhibition potency with a KI value in micromolar range (KI 8798.6 nM). The second abundant human cytosolic isoform, hCA II, was strongly inhibited by all compounds here reported. Although low nanomolar CA inhibition was widely known (7a and 7f), subnanomolar inhibition was uncommon (7c-e and 8). It is interesting to note, that the length of the alkyl moiety played an important role for the efficacy against this isoform. Indeed, moving from the ethyl chain (7b) to a butyl one (7a) the potency decreased near 10 fold (KI 3.9 nM and 36.2 nM, respectively). The membrane isoform hCA IV showed an opposite inhibition pattern for compounds 7a and 7b. Indeed, the ethyl moiety, this time, proved to be more efficacious than 7a near ten folds. Moreover, substituents in the aromatic telluride scaffold played a crucial role in efficacy against this isoform. Compound 7d with a 4-methyl moiety showed an inhibition constant value of KI 1.7 nM, when the same group moved to the ortho position, the potency decreased over 100 times. The last cytosolic isoform discussed here, hCA VII, was strongly inhibited by all compounds in the low or sub nanomolar range (KI 0.72 to 63.2 nM) except for the bulky naphtyl moiety (7d) in the high nanomolar range (KI 261.9 nM). Phenyl ring, for this isoform, play an important role for the potency of inhibition, indeed, when substituted with alkyl moiety such as for 7a and 7b the activity decreased. The tumor membrane-associated isoform, hCA IX, was well inhibited by derivatives 7e-g in the low nanomolar range (KI 6.4 to 9.5 nM). Alkyl moieties, such as for compounds 7a, 7b but also for the dimeric derivative 8, did not prove to be efficacious as for the other isoforms mentioned above.

Overall, the kinetic data above reported showed remarkable effects on the KI values when the tail moieties were modified. In particular the introduction of linear alkyl moieties, such as in 7a and b, didn’t result in any isoform preferential inhibition, whereas presence of a phenyl ring was particularly efficient in inhibiting the hCA II isoform (i.e. 7c-e). Among such compounds the aryl substituents induced selective inhibition of the remaining isoforms too (i.e. 7c, 7e hCA VII, 7d hCA IV respectively). X-ray Crystallographic investigations Keeping in mind the kinetic data inhibition, the structure of compound 8 in adduct with hCA II was determined by X-ray crystallography at a resolution of 1.35 Å (Statistic summarized in S.I. Table S1), thus allowing us to spot in detail the interactions occurring. A clear electronic density adjacent to the zinc atom in the active site was consistent with the sulfonamide moiety of compound 8 interacting in the canonical way both to the zinc ion and the Thr199 residue (Figure 1).46,47

A

B

Figure 1: Inhibitor 8 bound in the active site of hCA II showing the σA-weighted |Fo−Fc| map (at 2.5 σ) (A) (PDB: 6PGX). Hydrogen bonds, van der Waals interactions and Water Bridges are shown among compound 8 and residues involved (B). Compound 8 made multiple interactions with the protein as it extends out of the catalytic pocket with the second sulfonamide functional group and residues Pro202 and Leu204 making contacts.

These interactions, with the outer rim of hCA II active site residues, give the high affinity for this isoform (Figure 1B). Furthermore, in this complex, one oxygen of 8 is bound to a water molecule. This water molecule is also coordinated by hydrogen bonds to Gln92. Overall the compound showed two additional positive electron densities in the difference maps, showing that monomers, half of the symmetric compound 8, were bound outside the active site in two different (surface) sites of the protein (Figure 2).

Figure 2: X-ray crystal structures of hCA II with compound 8 in the active site and two monomers (halves of the symmetric compound 8) bounded out of the active site. Electron density maps are showed for derivative 8 and its monomers. The first related monomer (Figure S1 in S.I.) showed the sulfonamide group making several hydrogen bond interactions. Indeed, its nitrogen interacted with the side chains of Glu69 and Asn67. Furthermore, the oxygens of the sulfonamide group participated in hydrogen bonds to two

water molecules and Glu69. Other hydrogen bonds were found to the β-hydroxy moiety and Asp71 and Asp72. Van der Waals interactions between 8 and hCA II, are established among the phenyl ring, Glu69 and Ile91. On the contrary, the second monomer (Figure S2 in S.I.) showed fewer interactions with the protein and most are hydrogen bonds between the sulfonamide moiety, the Asp19 sidechain, the His15 backbone carbonyl and a water molecule. Biological assays Regarding the high affinity of compounds 7e-g for hCA IX, in agreement with literature49 we tested two telluride derivatives (7e and 7g) against the human adenocarcinoma cell line MDA-MB-231. This choice was dictated by expression of CA IX than in other cell lines. Moreover, its expression increases with the degree of hypoxia.50 The selected compounds were tested at 3, 10, 30 and 100 µM concentrations, incubated for 48 h both under normoxic and hypoxic conditions (Figure 3).

Figure 3: Effects of telluride derivatives 7e and 7g on viability of the human adenocarcinoma cell line MDA-MB231 following 48h treatment in normoxic (21% O2,) and hypoxic (1% O2,) conditions. ** p<0.001 versus control. In MDA-MB-231 cells, compound 7e was already very effective in normoxia conditions at 3 µM killing over 50% of tumor cells. Increasing the concentration to 10 µM or more, cell viability decreased under the 10% threshold, turning out to be very cytotoxic against this tumor cell line.

Comparably, compound 7g showed the same features. Unexpectedly, these compounds decreased drastically their cytotoxicity in hypoxic condition. Indeed, compound 7e at 3 µM killed 32% of tumor cells and at 30 µM telluride 7e killed over the 50% of cells. The same cytotoxic features were observed for derivative 7g, though at 30 µM showed a stronger effect killing about 90% of MDAMB-231 cells. These unusual results on cell viability in hypoxic conditions could be explained by the high antioxidant propriety of tellurium atom, since analogue compounds without the sulfonamide group are reported in the literature as strong antioxidant agents.29 However, the interesting data against normoxic conditions on MDA-MB231 showed their effects acts on different pathways and not only against hCA IX, giving the possibility to explore these derivatives against other kinds of tumors. Conclusions. In conclusion, we have synthesized the first novel series of telluride bearing sulfonamide as carbonic anhydrase inhibitors and discovered a new methodology of sulfonamide moiety deprotection with mild conditions. To date, no X-ray crystallography are present with these kind of derivatives and CAs, thus we report for the first time a complex among hCA II and compound 8 in order to understand in detail the interactions in the active site. The excellent data inhibition against the tumor associated isoform hCA IX of several derivatives such as 7e and 7g gives an opportunity to explore their antitumor proprieties. The unexpected results show these compounds act as antitumor agents, not only as Carbonic Anhydrase IX inhibitors, but probably in different pathways, thus making them an interesting opportunity to explore different tumors where there is no overexpression of hCA IX. Experimental protocols. Chemistry. 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 or in DMSO-d6 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 125

Te). NMR signals were referenced to nondeuterated residual solvent signals (CDCl3: 7.26 ppm

for 1H, 77.0 ppm for

13

C; DMSO: 2.50 ppm for 1H, 39.5 ppm for

external reference for

125

13

C). (PhTe)2 was used as an

Te 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. Dialkyl ditellurides 2a,b and diaryl ditellurides 2c-g, were prepared according to literature reported procedures from the corresponding bromides following literature reported procedures via tellurium insertion into carbon-magnesium or carbon-lithium bonds and subsequent air oxidation.37-39 Spectroscopic data of all synthesised compounds matched those previously reported in the literature.50-52 Synthesis of β-hydroxy tellurides bearing the benzenesulfonamide moiety Synthesis

of

N'-((4-(3-(butyltellanyl)-2-hydroxypropoxy)phenyl)sulfonyl)-N,N-

dimethylformimidamide (6) NaBH4 (15 mg, 0.40 mmol, 4.0 eq.) was portionwise added to a suspension of ditelluride 2a (28 mg, 0.10 mmol, 1.0 eq.) in EtOH (2.0 mL) under inert atmosphere (N2). After 10 min, a DMF solution (0.5 mL) of the epoxide 5 (51 mg, 0.18 mmol, 1.8 eq.) was slowly added and the reaction mixture was stirred at room temperature for 12 h. The reaction was quenched by addition of saturated aq. NH4Cl (4 mL) and diluted with EtOAc (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 x 5 mL). The organic phases were then collected, washed with brine (2 x 10 mL), dried over Na2SO4, filtered and concentrated under vacuum. The crude material was purified by flash chromatography (petroleum ether/EtOAc 3:2) to yield the

substituted β-hydroxy telluride 6 bearing the protected benzenesulfonamide moiety (66 mg, 78%). 1

H NMR (400 MHz, CDCl3) δ (ppm) 0.88 (3H, t, J = 7.4 Hz), 1.32-1.40 (2H, m), 1.67-1.75 (2H,

m), 2.65 (2H, t, J = 7.6 Hz, CH2CH2Te), 2.76 (1H, bs, OH), 2.86 (1H, dd, J = 5.6, 12.6 Hz, CHaHbTe) 2.93 (1H, dd, J = 4.4, 12.6 Hz, CHaHbTe), 2.98 (3H, s), 3.10 (3H, s), 4.02-4.11 (3H, m, CH2O overlapped with CHOH), 6.93 (2H, ap d, J = 8.8 Hz), 7.79 (2H, ap d, J = 8.8 Hz), 8.09 (1H, s).

13

C NMR (100 MHz, CDCl3) δ (ppm): 4.0, 7.9, 13.3, 24.9, 34.1, 35.4, 41.4, 69.9, 71.8, 114.4,

128.5, 134.8, 158.9, 161.1. MS (ESI positive) m/z (%): 473.4 [M+H]+ (100). General procedure for the synthesis of substituted β-hydroxy tellurides 7a-g NaBH4 (30 mg, 0.80 mmol, 4.0 eq.) was portionwise added to a suspension of ditelluride 2a-g (0.20 mmol, 1.0 eq.) and NaOH (0.40 mmol, 2.0 eq.) in EtOH (5 mL) at 0°C under inert atmosphere (N2). After 10 min, the epoxide 5 (102 mg, 0.36 mmol, 1.8 eq.) was slowly added and the reaction mixture was stirred at room temperature for 12 h. The reaction was quenched by addition of saturated aq. NH4Cl (5 mL) and diluted with EtOAc (10 mL), The layers were separated and the aqueous layer was extracted with EtOAc (2 x 5 mL), dried over Na2SO4, filtered and concentrated under vacuum. The crude material was purified by flash chromatography to yield substituted βhydroxy tellurides (7a-g). 4-(3-(butyltellanyl)-2-hydroxypropoxy)benzenesulfonamide (7a) Following the general procedure, 2a (22 mg, 0.06 mmol) and 5 (31 mg, 0.11 mmol) gave, after flash column chromatography (petroleum ether/EtOAc 1:1), telluride 7a (29 mg, 63%). 1H NMR (400 MHz, CDCl3) δ (ppm) 0.91 (3H, t, J = 7.4 Hz), 1.33-1.42 (2H, m), 1.68-1.77 (2H, m), 2.59 (1H, bs, OH), 2.68 (2H, t, J = 7.6 Hz, CH2CH2Te), 2.86-2.90 (1H, m, CHaHbTe), 2.94-2.98 (1H, m, CHaHbTe), 4.09-4.15 (3H, m, CH2O overlapped with CHOH), 4.74 (2H, bs, SO2NH2), 7.00 (2H, ap d, J = 8.8 Hz), 7.87 (2H, ap d, J = 8.8 Hz).

13

C NMR (100 MHz, CDCl3) δ (ppm): 4.1, 8.0, 13.4,

25.0, 34.2, 69.9, 72.0, 114.8, 128.7, 134.3, 161.8. MS (ESI positive) m/z (%): 440.2 [M+Na]+ (100).

4-(3-(ethyltellanyl)-2-hydroxypropoxy)benzenesulfonamide (7b) Following the general procedure, 2b (31 mg, 0.1 mmol) and 5 (51 mg, 0.18 mmol) gave, after flash column chromatography (petroleum ether/EtOAc 1:1), telluride 7b (40 mg, 58%). 1H NMR (400 MHz, CDCl3) δ (ppm) 1.01 (3H, t, J = 7.2 Hz), 2.66 (2H, q, J = 7.2 Hz), 2.85-2.93 (1H, m, CHaHbTe), 2.96-3.04 (1H, m, CHaHbTe), 4.05-4.21 (3H, m, CH2O overlapped with CHOH), 4.76 (2H, bs, SO2NH2), 7.00 (2H, ap d, J = 8.8 Hz), 7.87 (2H, ap d, J = 8.8 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): -2.4, 7.7, 17.5, 69.8, 72.0, 114.8, 128.7, 134.4, 161.6. MS (ESI positive) m/z (%): 412.4 2 [M+Na]+ (100). 4-(2-Hydroxy-3-(phenyltellanyl)propoxy)benzenesulfonamide (7c) Following the general procedure, 2c (66 mg, 0.16 mmol) and 5 (82 mg, 0.29 mmol) gave, after flash column chromatography (petroleum ether/EtOAc 1:1), telluride 7c (81 mg, 64%). 1H NMR (400 MHz, CDCl3) δ (ppm) 2.61 (1H, bd, J = 4.6 Hz, OH), 3.15 (1H, dd, J = 6.8, 12.6 Hz, CHaHbTe), 3.20 (1H, dd, J = 6.1, 12.6 Hz, CHaHbTe), 4.04 (1H, dd, J = 6.0, 9.4 Hz, CHaHbO), 4.08 (1H, dd, J = 4.1, 9.4 Hz, CHaHbO), 4.15-4.23 (1H, m, CHOH), 4.78 (2H, bs, NH2), 6.90 (2H, ap d, J = 8.9 Hz), 7.19 (2H, ap t, J = 7.4 Hz), 7.26-7.30 (1H, m), 7.45-7.78 (2H, m), 7.83 (2H, ap d, J = 8.9 Hz). 13

C NMR (100 MHz, CDCl3) δ (ppm): 13.7, 69.8, 71.9, 111.0, 114.7, 128.1, 128.6, 129.4, 134.2,

138.6, 161.7. 125Te NMR (126 MHz, CDCl3) δ (ppm): 392.1. MS (ESI negative) m/z (%): 436 [MH]- (100). 4-(2-hydroxy-3-(p-tolyltellanyl)propoxy)benzenesulfonamide (7d) Following the general procedure, 2d (52 mg, 0.12 mmol) and 5 (61 mg, 0.22 mmol) gave, after flash column chromatography (petroleum ether/EtOAc 3:1), telluride 7d (53 mg, 55%). 1H NMR (400 MHz, CDCl3) δ (ppm) 2.31 (3H, s), 2.60 (1H, bs, OH), 3.11 (1H, dd, J = 6.6, 12.5 Hz, CHaHbTe), 3.16 (1H, dd, J = 5.9, 12.5 Hz, CHaHbTe), 4.02 (1H, dd, J = 6.0, 9.4 Hz, CHaHbO), 4.06 (1H, dd, J = 4.1, 9.4 Hz, CHaHbO), 4.11-4.20 (1H, m, CHOH), 4.76 (2H, bs, NH2), 6.89 (2H, ap d,

J = 9.0 Hz), 6.99 (2H, ap d, J = 7.6 Hz), 7.65 (2H, ap d, J = 7.6 Hz), 7.82 (2H, ap d, J = 9.0 Hz). 13

C NMR (100 MHz, CDCl3) δ (ppm): 13.8, 21.2, 69.8, 71.7, 106.8, 114.7, 128.6, 130.4, 134.1,

138.4, 139.0, 161.7.

125

Te NMR (126 MHz, CDCl3) δ (ppm): 376.5. MS (ESI negative) m/z (%):

450 [M-H]- (100). 4-(2-Hydroxy-3-(o-tolyltellanyl)propoxy)benzenesulfonamide (7e) Following the general procedure, 2e (52 mg, 0.12 mmol) and 5 (61 mg, 0.22 mmol) gave, after flash column chromatography (petroleum ether/EtOAc 1:1), telluride 7e (48 mg, 48%). 1H NMR (400 MHz, CDCl3) δ (ppm) 2.46 (3H, s), 2.87 (1H, bs, OH), 3.10-3.18 (2H, m, CH2Te), 3.99-4.07 (2H, m, CH2O), 4.14-4.24 (1H, m, CHOH), 5.11 (2H, bs, NH2), 6.86 (2H, ap d, J = 8.4 Hz), 6.97 (1H, t, J = 7.2 Hz), 7.15-7.21 (2H, m), 7.69 (1H, d, J = 7.5 Hz ), 7.78 (2H, d, J = 8.4 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 12.5, 26.6, 69.9, 71.9, 114.7, 126.7, 128.3, 128.5, 129.3, 134.2, 134.2, 137.8, 142.6, 161.6.

125

Te NMR (126 MHz, CDCl3) δ (ppm): 283.6. MS (ESI negative) m/z (%):

450 [M-H]- (100). 4-(2-Hydroxy-3-((4-methoxyphenyl)tellanyl)propoxy)benzenesulfonamide (7f) Following the general procedure, 2f (47 mg, 0.10 mmol) and 5 (51 mg, 0.18 mmol) gave, after flash column chromatography (petroleum ether/EtOAc 1:1), telluride 7f (51 mg, 61%). 1H NMR (400 MHz, CDCl3) δ (ppm) 2.64 (1H, bd, J = 5.1 Hz, OH), 3.05 (1H, dd, J = 6.5, 12.5 Hz, CHaHbTe), 3.13 (1H, dd, J = 6.0, 12.5 Hz, CHaHbTe), 3.77 (3H, s, CH3O), 4.01 (1H, dd, J = 4.6, 8.2 Hz, CHaHbO), 4.04 (1H, dd, J = 3.1, 8.2 Hz, CHaHbO), 4.10-4.17 (1H, m, CHOH), 4.92 (2H, bs, NH2), 6.68 (2H, ap d, J = 8.7 Hz), 6.85 (2H, ap d, J = 9.0 Hz), 7.67 (2H, ap d, J = 8.7 Hz), 7.79 (2H, ap d, J = 9.0 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 14.1, 55.2, 69.9, 71.5, 99.9, 114.7, 115.3, 128.5, 134.1, 141.0, 159.9, 161.6. 125Te NMR (126 MHz, CDCl3) δ (ppm): 369.4. MS (ESI negative) m/z (%): 466 [M-H]- (100). 4-(2-Hydroxy-3-(naphthalen-2-yltellanyl)propoxy)benzenesulfonamide (7g)

Following the general procedure, 2g (51 mg, 0.10 mmol) and 5 (51 mg, 0.18 mmol) gave, after flash column chromatography (petroleum ether/EtOAc 1:1), telluride 7g (47 mg, 53%). 1H NMR (400 MHz, CDCl3) δ (ppm) 2.65 (1H, bd, J = 5.3 Hz, OH), 3.22 (1H, dd, J = 6.5, 12.6 Hz, CHaHbTe), 3.28 (1H, dd, J = 6.0, 12.5 Hz, CHaHbTe), 4.02 (1H, dd, J = 4.4, 8.2 Hz, CHaHbO), 4.06 (1H, dd, J = 3.0, 8.2 Hz, CHaHbO), 4.17-4.25 (1H, m, CHOH), 4.78 (2H, bs, NH2), 6.79 (2H, ap d, J = 8.8 Hz), 7.44-7.51 (2H, m), 7.62 (1H, d, J = 8.4 Hz), 7.68-7.63 (1H, m), 7.71 (2H, ap d, J = 8.8 Hz), 7.76-7.79 (2H, m), 8.26 (1H, s). 13C NMR (100 MHz, CDCl3) δ (ppm): 13.9, 69.9, 71.6, 108.3, 114.6, 126.5, 126.6, 127.2, 127.8, 128.5, 128.6, 132.6, 134.1, 135.0, 138.4, 161.5. 125Te NMR (126 MHz, CDCl3) δ (ppm): 393.6. MS (ESI negative) m/z (%): 486 [M-H]- (100). 4,4'-((Tellurobis(2-hydroxypropane-3,1-diyl))bis(oxy))dibenzenesulfonamide (8) A solution of the epoxide 5 (141 mg, 0.5 mmol, 2.0 eq.) in dry DMF was slowly added to a suspension of Li2Te in THF, generated in situ from elemental tellurium (32 mg, 0.25 mmol., 1.0 eq.) and LiEt3BH (1.0 mL of a 1M THF solution; 1.0 mmol, 4.0 eq.), according to a literature reported procedure.18 The reaction was stirred for 12 h at ambient temperature and then treated with a NaOH 1M aqueous solution. The reaction mixture was stirred at ambient temperature for 4 h and then diluted with EtOAc (10 mL), filtered through a short pad of celite, washed with NH4Cl (5 mL) and with H2O (2 x 5 mL).The organic phase was dried over Na2SO4, filtered and evaporated under reduced pressure. The crude material was purified by flash column chromatography (petroleum ether/EtOAc 1:1) to afford β-hydroxy telluride 8 as a pale yellow solid (79 mg, 74%). 1H NMR (400 MHz, DMSO-d6) δ (ppm) 2.84 (4H, dd, J = 5.5, 12.0 Hz, CHaHbTe), 2.90 (4H, dd, J = 3.9, 12.0 Hz, CHaHbTe), 3.97-4.08 (12H, m, CH2O and CHOH), 5.32 (4H, bd, J = 3.8 Hz, OH), 7.08 (2H, ap d, J = 8.9 Hz), 7.18 (2H, bs, NH2), 7.75 (2H, ap d, J = 8.9 Hz).

13

C NMR (100 MHz,

DMSO-d6) δ (ppm): 9.8, 70.5, 73.7, 115.6, 128.7, 137.3, 162.0. 125Te NMR (126 MHz, DMSO-d6) δ (ppm): 117.5, 118.5. MS (ESI negative) m/z (%): 589 [M-H]- (100). Carbonic Anhydrase Inhibition.

An Applied Photophysics stopped-flow instrument has been used for assaying the CA-catalyzed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as a pH indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as the buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CAcatalyzed CO2 hydration reaction for a period of 10−100 s.42 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 distilled−deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, to allow for the formation of the E−I complex. The inhibition constants were obtained by nonlinear least squares methods using PRISM 3, as reported earlier,43-46 and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in house as reported earlier.43-46 Biological Assays. Cell viability of the human adenocarcinoma cell line MDA-MB-231 assay. Human adenocarcinoma cell line MDA-MB-231 were obtained from American Type Culture Collection (Rockville, MD). PC3 and MDA-MB-231 were cultured in DMEM high glucose with 20% FBS in 5% CO2 atmosphere at 37° C. Media contained 2 mM L-glutamine, 1% essential aminoacid mix, 100 IU ml-1 penicillin and 100 µg ml-1 streptomycin (Sigma, Milan, Italy). Cells were plated in 960 wells cell culture (1.104/well) and, 24 h after, treated with the tested compounds for 48 h. Low oxygen conditions were acquired in a hypoxic workstation (Concept 400 anaerobic incubator, Ruskinn Technology Ltd., Bridgend, UK). The atmosphere in the chamber consisted of 1% O2 (hypoxia), 5% CO2, and residual N2. In parallel, normoxic (21% O2) dishes were incubated

in air with 5% CO2. Cell vitality was assessed via MTT assay. Viability is expressed as % in comparison to the control cells (arbitrarily set 100 % of viable cells). Data are presented as mean ± SEM. One-way ANOVA with a Bonferroni post-hoc test was used to compare the treated samples to the control. A p-value less than 0.05 was considered to indicate a significant difference. Protein X-ray Crystallography hCA II protein was purified as previously described.53 The protein was concentrated to about 7 mg/mL and set up in SD2 crystallization plates (Molecular Dimensions) with the following ratio of protein plus reservoir: 200 nL + 200 nL. The plate was incubated at 8 ºC and the reservoir conditions consisted of 2.6 to 3.0 M ammonium sulfate with 0.1 M Tris buffer at pH 8.0 to pH 8.5. Compound in DMSO was added to the crystallization drops after crystals had formed and several days before data were collected. 360 frames of one degree oscillation were obtained from the MX1 beamline of the Australian Synchrotron. The data were indexed using XDS54 and scaled using Aimless.55 Molecular replacement was done using Phaser56 using 4CQ0 as the initial starting model. The model was manually rebuilt using Coot57 and refined using Refmac.58 The compound was placed in density using the program Afitt (OpenEye Scientific Software) and further refined using Refmac. Acknowledgements. We thank the Australian Synchrotron and the beamline scientists for their help with data collection and thank OpenEye Scientific Software for a license to the program Afitt. Funding Sources This work was supported by a grant of the Romanian Ministry of Research and Innovation, CNCS– UEFISCDI, project number PN-III-P4-ID-PCCF-2016–0050, within PNCDI II

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• • • • •

Development of an easy procedure for the synthesis of novel β-hydroxy tellurides bearing the benzenesulfonamide moiety. Evaluation of their inhibitory activity against hCAs I, II, IV, VII, IX. Potent inhibitory activity was found against the tumor-associated hCA IX. X-rays co-crystallographic studies. Interesting activities were found in vitro against breast cancer cell line (MDA-MB-231).