# Novel Re(I) tricarbonyl coordination compounds based on 2-pyridyl-1,2,3-triazole derivatives bearing a 4-amino-substituted benzenesulfonamide arm: synthesis, crystal structure, computational studies and inhibitory activity against carbonic anhydrase I, II, and IX isoforms†

Yassine Aimene, Romain Eychenne, Sonia Mallet-Ladeira, Nathalie Saffon,

Jean-Yves Winum, Alessio Nocentini, Claudiu T. Supuran, Eric Benoist,

Achour Seridi

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Yassine Aimene, Romain Eychenne, Sonia Mallet-Ladeira, Nathalie Saffon, Jean-Yves Winum, et al. Novel Re(I) tricarbonyl coordination compounds based on 2-pyridyl-1,2,3-triazole derivatives bearing a 4-amino-substituted benzenesulfonamide arm: synthesis, crystal structure, computational studies and inhibitory activity against carbonic anhydrase I, II, and IX isoforms†. Journal of Enzyme Inhibition and Medicinal Chemistry, 2019, 34 (1), pp.773-782. 10.1080/14756366.2019.1585835. hal-02169866

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# Novel Re(I) tricarbonyl coordination compounds based on 2-pyridyl-1,2,3-triazole derivatives bearing a 4-amino-substituted benzenesulfonamide arm: synthesis, crystal structure, computational studies and inhibitory activity against carbonic anhydrase I, II, and IX isoforms†

Yassine Aimene, Romain Eychenne, Sonia Mallet-Ladeira, Nathalie Saffon, Jean-Yves Winum, Alessio Nocentini, Claudiu T. Supuran, Eric Benoist & Achour Seridi

**To cite this article:** Yassine Aimene, Romain Eychenne, Sonia Mallet-Ladeira, Nathalie Saffon, Jean-Yves Winum, Alessio Nocentini, Claudiu T. Supuran, Eric Benoist & Achour Seridi (2019) Novel Re(I) tricarbonyl coordination compounds based on 2-pyridyl-1,2,3-triazole derivatives bearing a 4-amino-substituted benzenesulfonamide arm: synthesis, crystal structure, computational studies and inhibitory activity against carbonic anhydrase I, II, and IX isoforms†, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 773-782, DOI: 10.1080/14756366.2019.1585835

**To link to this article:** https://doi.org/10.1080/14756366.2019.1585835

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# Novel Re(I) tricarbonyl coordination compounds based on 2-pyridyl-1,2,3-triazole derivatives bearing a 4-amino-substituted benzenesulfonamide arm: synthesis, crystal structure, computational studies and inhibitory activity against carbonic anhydrase I, II, and IX isoforms†

Yassine Aimene<sup>a,b,c</sup> ID, Romain Eychenne<sup>b,c</sup>, Sonia Mallet-Ladeira<sup>d</sup>, Nathalie Saffon<sup>d</sup>, Jean-Yves Winume<sup>e</sup> ID, Alessio Nocentini<sup>f</sup>, Claudiu T. Supuran<sup>f</sup> ID, Eric Benoist<sup>b,c</sup> ID and Achour Seridi<sup>a</sup> ID

<sup>a</sup>Laboratoire de Chimie, physique Université du 8 Mai 1945, Guelma, Algérie; <sup>b</sup>CNRS, Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, SPCMIB, Toulouse, France; <sup>c</sup>Université de Toulouse, UPS, Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, SPCMIB, Toulouse, France; <sup>d</sup>Institut de Chimie de Toulouse (FR 2599), Toulouse, France; <sup>e</sup>Institut des Biomolécules Max Mousseron, ENSCM, Université de Montpellier, Montpellier, France; <sup>f</sup>Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, Università degli Studi di Firenze, Florence, Italy

## ABSTRACT

In this work, two bidentate 2-pyridyl-1,2,3-triazole ligands (3a and 3b) containing a 4-substituted benzene-sulfonamide pharmacophore prepared by classical click chemistry procedures, as well as their corresponding rhenium complexes, 4a and 4b of general formula [ReCl(CO₃)L] (L = 3a or 3b) were prepared and fully characterised by spectroscopic methods (IR, NMR, MS, UV-Vis), elemental analysis, X-ray diffraction, and theoretical studies using DFT and TD-DFT methods. In particular, we showed that, in the solid state, the pyridine and the triazole rings of 3b adopted an uncommon *cis* configuration which stems from inter-molecular hydrogen bonds. Preliminary assays demonstrated a promising nanomolar inhibitory activity against carbonic anhydrase isoform IX for both ligands and complexes with a strong affinity Kᵢ of 2.8 nM for ligand 3a. More interestingly, complex 4b exhibited a pronounced selectivity against hCA IX over the off-targets hCA I and hCA II which makes this compound a promising potential anticancer drug candidate.

## ARTICLE HISTORY

Received 22 January 2019
Revised 17 February 2019
Accepted 18 February 2019

## KEYWORDS

Click chemistry; rhenium(I) complexes; crystal structures; DFT calculations; carbonic anhydrase inhibitors

## 1. Introduction

Over recent decades, rhenium(I) tricarbonyl complexes have been intensively studied by the inorganic chemist community, due to their significant photophysical and photochemical properties. These features make them interesting tools for numerous potential practical applications, such as photo sensitisers in solar cells¹, CO₂ reduction catalysts², organic light-emitting devices (OLEDs)³, luminescence sensors⁴, and CO-releasing moieties (CORMs)⁵. Additionally, radioactive fac-[¹⁸⁸Re(CO)₃]⁺ complexes have recently drawn the attention of several research groups for their use as therapeutic radiopharmaceuticals⁶.

In this context, the efficient design and synthesis of chelating ligands represent the pivotal key to the successful application of this type of complexes. Most of the reported ligands which coordinate efficiently to the rhenium(I) tricarbonyl core are based on bidentate and tridentate chelating systems including N-hetero-aromatic nitrogen, oxygen and to a lesser extend sulfur or phosphorus donor atoms⁷. While tridentate ligands lead to the most stable rhenium(I) tricarbonyl complexes, bidentate ligands based on α,α'-diimines are currently very popular. Indeed, their corresponding rhenium complexes exhibit interesting photo-physical

properties which can be tuned by small changes in the ligand scaffold or around the rhenium centre by substituting the chlorine bound to the metal by a ternary ligand (generally, cyanides or N-heterocycles)⁸. Among efficient bidentate α,α'-diimine chelators, 2,2'-bipyridine (bpy) has been originally used⁹. More recently, similar chelating systems based on pyridine-triazole (pyta and tapy systems¹) were developed as alternative ligands to 2,2'-bipyridines¹⁰. Obata et al. first reported that pyta moieties acted like a bipyridine mimic with strongly electron-donating substituents, the corresponding complexes of general formula fac-[Re(pyta)(CO₃)Cl] exhibiting luminescence properties¹¹.

The growing interest on pyta (and tapy) architectures is mainly due to their easier synthetic access and functionalization. Unlikeially, bipy systems, it is possible by using a classical copper-catalysed alkyne-azide cycloaddition (CuAAC reaction) not only to generate a large number of this important class of heterocyclic compounds but also to allow different functionalizations of the triazole ring¹². As an example, we previously reported the preparation of two [M(CO₃)⁺] complexes (M = Re and ⁹⁹mTc) from a pyridyltriazole-based ligand bearing the bioactive (2-methoxyphenyl) piperazine pharmacophore, which is a central nervous system (CNS) receptors-targeted vector. Thus, we demonstrated that (i) both

<figure><img src="image_15.png" /></figure>
<figure><img src="image_16.png" /><figcaption>**Table 1.** Crystal data and structure refinement for **3a**, **3b**, **4a**, and **4b**.</figcaption></figure>

<table><thead><tr><th rowspan="2"></th><th colspan="2">Pyta ligands</th><th colspan="2">Rhenium complexes</th></tr><tr><th>3a</th><th>3b</th><th>4a</th><th>4b</th></tr></thead><tbody><tr><td>Empirical formula</td><td>C<sub>13</sub>H<sub>11</sub>N<sub>5</sub>O<sub>2</sub>S</td><td>C<sub>14</sub>H<sub>13</sub>N<sub>5</sub>O<sub>2</sub>S</td><td>C<sub>16</sub>H<sub>11</sub>ClN<sub>5</sub>O<sub>5</sub>ReS</td><td>C<sub>17</sub>H<sub>13</sub>ClN<sub>5</sub>O<sub>5</sub>ReS, CH<sub>4</sub>O</td></tr><tr><td>Formula weight</td><td>301.33</td><td>315.35</td><td>607.01</td><td>653.08</td></tr><tr><td>T [K]</td><td>193(2)</td><td>193(2)</td><td>193(2)</td><td>193(2)</td></tr><tr><td>Wavelength [Å]</td><td>1.54178</td><td>0.71073</td><td>0.71073</td><td>0.71073</td></tr><tr><td>Crystal system</td><td>Triclinic</td><td>Monoclinic</td><td>Orthorhombic</td><td>Triclinic</td></tr><tr><td>Space group</td><td>P<span style="text-decoration: overline">1</span></td><td>P<sub>21</sub>/c</td><td>Pbcn</td><td>P<span style="text-decoration: overline">1</span></td></tr><tr><td>Unit cell dimensions [Å, °]</td><td>a = 6.3545(2)<br>b = 7.0441(2)<br>c = 29.2801(7)<br>α = 95.856(2)<br>β = 90.497(2)<br>γ = 96.930(2)</td><td>a = 15.8111(9)<br>b = 10.6289(5)<br>c = 8.4974(4)<br>α = 90<br>β = 104.485(2)<br>γ = 90</td><td>a = 19.1728(15)<br>b = 15.1735(11)<br>c = 13.1204(11)<br>α = 90<br>β = 90<br>γ = 90</td><td>a = 7.7195(5)<br>b = 12.1870(7)<br>c = 13.1228(8)<br>α = 108.398(2)<br>β = 98.897(2)<br>γ = 103.123(2)</td></tr><tr><td>V [Å<sup>3</sup>]</td><td>1293.97(6)</td><td>1382.64(12)</td><td>3817.0(5)</td><td>1106.07(12)</td></tr><tr><td>Z</td><td>4</td><td>4</td><td>8</td><td>2</td></tr><tr><td>ρ<sub>calcd</sub> [Mg/m<sup>3</sup>]</td><td>1.547</td><td>1.515</td><td>2.113</td><td>1.961</td></tr><tr><td>μ [mm<sup>-1</sup>]</td><td>2.355</td><td>0.250</td><td>6.657</td><td>5.754</td></tr><tr><td>Max. and min. transm.</td><td>0.7523 and 0.6483</td><td>0.7461 and 0.7194</td><td>0.7461 and 0.5932</td><td>0.7461 and 0.4955</td></tr><tr><td>F(000)</td><td>624</td><td>656</td><td>2320</td><td>632</td></tr><tr><td>Crystal size [mm]</td><td>0.28 × 0.04 × 0.02</td><td>0.22 × 0.12 × 0.08</td><td>0.20 × 0.06 × 0.02</td><td>0.30 × 0.28 × 0.06</td></tr><tr><td>θ range [°]</td><td>3.035–63.142</td><td>3.131–30.590</td><td>3.102–30.581</td><td>3.314–30.535</td></tr><tr><td>Limiting indices</td><td>−7 ≤ h ≤ 7<br>−8 ≤ k ≤ 8<br>−33 ≤ l ≤ 33</td><td>−21 ≤ h ≤ 22<br>−15 ≤ k ≤ 15<br>−12 ≤ l ≤ 12</td><td>−27 ≤ h ≤ 27<br>−20 ≤ k ≤ 21<br>−18 ≤ l ≤ 18</td><td>−11 ≤ h ≤ 11<br>−16 ≤ k ≤ 17<br>−18 ≤ l ≤ 17</td></tr><tr><td>Reflections collected</td><td>12039</td><td>21683</td><td>116299</td><td>36946</td></tr><tr><td>Unique reflections (R<sub>int</sub>)</td><td>4055 [0.0508]</td><td>4237 [0.0442]</td><td>5852 [0.1036]</td><td>6724 [0.0198]</td></tr><tr><td>Completeness to 2θ = 63.142°</td><td>96.3 %</td><td>99.8 %</td><td>99.8 %</td><td>99.3 %</td></tr><tr><td colspan="5" style="text-align: left;">for 3a<br>=25.242° for 3b<br>=25.242° for 4a<br>=25.242° for 4b</td></tr><tr><td>Data / restraints / parameters</td><td>4055 / 6 / 395</td><td>4237 / 0 / 207</td><td>5852 / 0 / 270</td><td>6724 / 0 / 299</td></tr><tr><td>Goodness-of-fit (GOF) on F<sup>2</sup></td><td>1.048</td><td>0.859</td><td>1.005</td><td>1.138</td></tr><tr><td>Final R indices [I > 2σ(I)]</td><td>R<sub>1</sub>=0.0478<br>wR<sub>2</sub>=0.0932</td><td>R<sub>1</sub>=0.0443<br>wR<sub>2</sub>=0.1223</td><td>R<sub>1</sub>=0.0365<br>wR<sub>2</sub>=0.0531</td><td>R<sub>1</sub>=0.0196<br>wR<sub>2</sub>=0.0464</td></tr><tr><td>R indices (all data)</td><td>R<sub>1</sub>=0.0782<br>wR<sub>2</sub>=0.1049</td><td>R<sub>1</sub>=0.0697<br>wR<sub>2</sub>=0.1417</td><td>R<sub>1</sub>=0.0704<br>wR<sub>2</sub>=0.0614</td><td>R<sub>1</sub>=0.0218<br>wR<sub>2</sub>=0.0474</td></tr><tr><td>Largest difference in peak and<br>hole [e Å<sup>-3</sup>]</td><td>0.315 and −0.411</td><td>0.416 and −0.417</td><td>1.279 and −1.402</td><td>2.564 and −1.526</td></tr></tbody></table>

### 2.2.5. [(3a)Re(CO)<sub>3</sub>Cl], (4a)

**3a** (70 mg, 0.23 mmol) and [Re(CO)<sub>5</sub>Cl] (92.5 mg, 0.25 mmol) yielded the desired complex **4a** as a white solid. Suitable crystals of **4a** for X-ray crystal structure determination were grown by slow evaporation of methanol solution.

Yield: 95 mg (67%). mp >300 °C. ¹H NMR (300 MHz, DMSO-d<sub>6</sub>): δ/ppm = 7.64 (s, 2H, NH<sub>2</sub>), 7.71 (ddd, J = 7.3, 5.5, 1.6, 1H, H<sub>pyr</sub>), 8.14 (d, J = 8.8 Hz, 2H, H<sub>Ar</sub>), 8.25 (d, J = 8.8 Hz, 2H, H<sub>Ar</sub>), 8.31 (ddd, J = 7.9, 1.6, 0.8 Hz, 1H, H<sub>pyr</sub>), 8.38 (td, J = 7.7, 1.5 Hz, 1H, H<sub>pyr</sub>), 9.04 (dd, J = 5.6, 1.2 Hz, 1H, H<sub>pyr</sub>), 10.08 (s, 1H, H<sub>ta</sub>), ¹³C NMR (75 MHz, DMSO-d<sub>6</sub>): δ/ppm = 121.4, 127.8 (CH<sub>Ar</sub>), 122.7, 126.9, 137.4, 149.2 (CH<sub>pyr</sub>), 124.6 (CH<sub>ta</sub>), 141.1, 145.5 (C<sub>Ar</sub>), 148.3 (C<sub>pyr</sub>), 153.3 (C<sub>ta</sub>), 189.4, 196.5, 197.4 (CO). IR: ν<sub>CO</sub>=2027, 1931, 1905 cm<sup>-1</sup>, ESI<sup>+</sup>-MS: m/z = 570.0 [M-Cl]<sup>+</sup>, 627.9 [M+Na]<sup>+</sup>. HRMS calculated for C<sub>16</sub>H<sub>11</sub>N<sub>5</sub>O<sub>5</sub>SClRe [(M+Na)<sup>+</sup>] 627.9597, found 627.9616. Elemental analysis for C<sub>16</sub>H<sub>11</sub>N<sub>5</sub>O<sub>5</sub>SClRe: calculated (found) C, 31.66 (31.37); H, 1.83 (1.44); N, 11.54 (11.10).

### 2.2.6. [(3b)Re(CO)<sub>3</sub>Cl], (4b)

**3b** (75 mg, 0.238 mmol) and [Re(CO)<sub>5</sub>Cl] (95 mg, 0.26 mmol) yielded the desired complex **4b** as a white solid. Suitable crystals of **4b** for X-ray crystal structure determination were grown by slow evaporation of methanol solution.

Yield: 98 mg (66%). mp 268–271 °C. ¹H NMR (300 MHz, DMSO-d<sub>6</sub>): δ/ppm = 6.00 (s, 2H, NCH<sub>2</sub>), 7.41 (s, 2H, NH<sub>2</sub>), 7.60–7.68 (m, 3H, 1H<sub>pyr</sub>, 2H<sub>Ar</sub>), 7.90 (d, J = 8.3 Hz, 2H, H<sub>Ar</sub>), 8.19–8.37 (m, 2H,

2H<sub>pyr</sub>, 8.96 (dd J = 5.4, 1.3 Hz, 1H, H<sub>pyr</sub>), 9.31 (s, 1H, H<sub>ta</sub>), ¹³C NMR (75 MHz, DMSO-d<sub>6</sub>): δ/ppm = 54.1 (CH<sub>2</sub>), 122.8, 126.6, 137.8, 148.6 (CH<sub>pyr</sub>), 126.2 (CH<sub>ta</sub>), 126.4, 129.2 (CH<sub>Ar</sub>), 140.7, 144.5 (C<sub>Ar</sub>), 148.6 (C<sub>pyr</sub>), 153.0 (C<sub>ta</sub>), 189.5, 196.7, 197.5 (CO). IR (KBr): ν<sub>CO</sub>=2029, 1920, 1902 cm<sup>-1</sup>. ESI<sup>+</sup>-MS: m/z = 584.0 [M-Cl]<sup>+</sup>, 642 [M + Na]<sup>+</sup>. HRMS calculated for C<sub>17</sub>H<sub>13</sub>N<sub>5</sub>O<sub>5</sub>NaSClRe [(M + Na)<sup>+</sup>] 641.9753, found 641.9750. Elemental analysis for C<sub>17</sub>H<sub>13</sub>N<sub>5</sub>O<sub>5</sub>SClRe: calculated (found) C, 32.88 (32.79); H, 2.11 (1.84); N, 11.28 (10.97).

### 2.3. Crystal structure determination

X-ray intensity data of ligands **3a**, **3b** and corresponding rhenium complexes **4a**, **4b** were collected on a Bruker D8 VENTURE diffractometer (MA, USA) and using graphite monochromated Mo Kα radiation (λ = 0.71073 Å) for **3b**, **4a** and **4b** and Cu Kα radiation (λ = 1.54178 Å) for **3a** at 193 K. The semi-empirical absorption corrections were employed [SADABS, Programme for data correction, Bruker-AXS]. Crystallographic data and refinement details are given in Table 1. The structures were solved using an intrinsic phasing method¹⁶, and refined by full matrix least squares procedures on F<sup>2</sup>. All non-hydrogen atoms were refined with anisotropic displacement coefficients. The hydrogen atoms bound to carbon atoms were placed in calculated positions and treated as riding on their parent atoms with d(C-H)=0.93 Å, U<sub>iso</sub>(H)=1.2 U<sub>eq</sub>(C) (aromatic); and d(C-H)=0.96 Å, U<sub>iso</sub>(H)=1.5 U<sub>eq</sub>(C) (methyl). The methyl groups were
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Scheme 1.** Synthesis of ligands **3a/3b** and their corresponding benzenesulfonamide rhenium tricarbonyl complexes **4a/4b**. Conditions and reagents: (i) **1a**, NaNO₂, NaN₃, HCl/THF/DMF (v/v/v: 1:1:1), 0–25 °C, 1 night (93%) or **1b**, NaN₃, Tf₂O, CuSO₄·5H₂O, K₂CO₃, CH₂Cl₂, 0–25 °C, 1 night (85%); (ii) Cu(OAc)₂·H₂O, sodium ascorbate, CH₃CN, 45 °C, 1 night, (3a: 69%, 3b: 61%); (iii) [Re(CO)₃Cl], 65 °C, MeOH, 12h, (4a: 67%, 4b: 66%).</figcaption></figure>

allowed to rotate about their local threefold axis. The ShelX software package¹⁶ was used for the calculations.

CCDC 1871452 (**3a**), CCDC 1871453 (**3b**), CCDC 1871454 (**4a**) and CCDC 1871455 (**4b**) contain the supplementary crystallographic data. These data can be obtained free of charge from http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; tel: +44 (0)1223 336408; fax: +44 (0)1223 336033; or e-mail: deposit@ccdc.cam.ac.uk.

## 2.4. Computational details

The reported calculations for the studied tricarbonyl rhenium(I) complexes **4a** and **4b** were carried out using the GAUSSIAN 09 programme package¹⁷ with the aid of the GaussView visualisation program¹⁸. Full geometries optimizations were performed in gas-phase and methanol solvent without any symmetry restrictions with the DFT method using the hybrid B3LYP (Becke, three-parameter, Lee-Yang-Parr) functional¹⁹. The pseudo-potential (ECP) LANL2DZ²⁰ was employed to describe the electrons of Re atom while the other atoms (H, C, O, N and S) were assigned by the standard 6–31G (d) basis set. The optimised geometries were evaluated using vibrational frequencies calculations to ensure that the true local minimum was attained and all eigenvalues are positive. Natural bond orbital analysis was carried out using NBO code included in GAUSSIAN 09²¹. At the optimised structures, time-dependent density functional theory (TD-DFT) method²² using B3LYP functional was applied to calculate the vertical excitation energies and corresponding oscillator strengths. In addition, the solvent effect (methanol) was modelled using the polarisable continuum model with the integral equation formalism (IEF-PCM)²³. The initial geometries were taken from X-ray structures, and all calculations were based on the optimised geometries.

## 2.5. Carbonic anhydrase inhibition assays

An Applied Photophysics stopped-flow instrument was used for assaying the CA catalysed CO₂ hydration activity. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na₂SO₄ (for maintaining the constant ionic strength), following the initial rates of the CA-catalysed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7–17 mM for the determination of the kinetic parameters and inhibition constants. In particular, CO₂ was

bubbled in distilled deionised water for 30 min so that the water was saturated (the concentration at a specific temperature is known from literature). In addition, a CO₂ assay kit (from Sigma) was used to measure the concentration in variously diluted solutions obtained from the saturated one (which was kept at the same temperature and a constant bubbling during the experiments). For each inhibitor at least six traces of the initial 5–10% of the reaction was used for determining the initial velocity²⁴. The uncatalysed 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-deionised water and dilutions up to 0.01 nM were done thereafter with distilled-deionised water. Inhibitor and enzyme solutions were pre-incubated together for 15 min–2 h (or longer, i.e. 4–6 h) at room temperature (at 4 °C for the incubation periods longer than 15 min) 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 equation²⁵,²⁶, as reported earlier, and represent the mean from at least three different determinations. hCA I was purchased by Sigma-Aldrich and used without further purification, whereas all the other hCA isoforms were recombinant ones obtained in-house as reported earlier²⁷,²⁸.

# 3. Results and discussion

## 3.1. Synthesis and structural characterisation

The synthesis of rhenium tricarbonyl complexes **4a** and **4b** were readily synthesised following a three-step procedure as depicted in Scheme 1. The ligands **3a** and **3b** were prepared by classical copper-catalysed Huisgen cycloaddition (CuAAC reaction) using the corresponding azido intermediates and commercial 2-ethynylpyridine derivatives. Both azido compounds **2a** and **2b** were obtained in good yields (>80%) by following a procedure reported earlier¹⁵. By applying classical CuAAC conditions we previously reported for pyta ligands preparation¹⁰b (i.e. catalytic system: Cu(OAc)₂·H₂O/sodium ascorbate in tert-butanol/water mixture or in acetonitrile at room temperature), **3a** and **3b** were obtained in low yield or at trace level. In turn, the yield of the cycloaddition was noticeably improved by performing the reaction at 45 °C in acetonitrile (average yield of 65%). Pyta-based rhenium complexes **4a** and **4b** were finally obtained in modest yields (c.a. 66%) by the reaction of commercial rhenium pentacarbonyl chloride with chelating ligands in refluxing methanol during 12 h. All our compounds are stable in the solid state as well as in organic solvents
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Figure 1.** Unprecedented *cis*-configuration of the 2-pyridyl-1,2,3-triazole unit of ligand **3b**; a partial view of the crystal packing of **3b** with N-H···N and C-H···O hydrogen bonds and π-π contacts shown as dashed lines. The purple spheres represent the centroids of the rings involved in π-π interactions.</figcaption></figure>

and their stability is not affected by the presence of air or moisture. They were fully characterised by NMR, IR, MS and elemental analysis and exhibited classical spectroscopic features for pyta-based compounds (see experimental section and ESI part, Tables S1-2 for NMR data). Briefly, upon complexation with the tricarbonyl rhenium core, a significant down-field shift (~0.6 ppm) of the singlet due to the triazole proton and very minor shifts of the hydrogens of the phenyl ring compared to those of the free ligand were observed in proton NMR for each complex, confirming the coordination of the rhenium by the triazolyl unit and the pentident nature of the 4-substituted arylsulfonamide moiety. The pattern of the CO stretching frequencies in the IR spectra (three intense bands in the 2030-1900 cm⁻¹ region) confirms the facial arrangement of the CO ligands in the complexes. More interestingly, as reported recently by Sarkar et al., the average value of the CO stretching frequencies gave information on the overall donor strength of the chelating pyta pincer²⁹. In our case, both complexes exhibited an average value of 1950 cm⁻¹, this value being consistent with those reported for other pyta ligands²⁹ and indicating an overall donor strength similar to bpy ligands. Fortunately, we obtained crystals of ligands **3a/3b** and complexes **4a/4b** suitable for single crystal X-ray diffraction from the slow evaporation of a concentrated sample in methanol solution. Selected crystallographic parameters are listed in Table 1 and selected bond lengths and angles values are gathered in the ESI part (Tables S3-S5).

Both ligands (**3a** and **3b**) were crystallized in triclinic and monoclinic system and their crystals were solved in space groups *P*\`1 and *P*2\_1/c, respectively (see ORTEP diagrams in ESI part, Figure S1). As expected, the structural characteristics of **3a/3b** are similar to those reported for related pyta ligands¹⁰a,b,11,12,30. The azo character of the triazolyl ring is confirmed by the N(2)-N(3) distance of the 1,2,3-triazole unit which is shorter than the N(3)-N(4) and N(2)-C(6) bonds (1.314(3) Å vs. 1.357(3) Å and 1.366(4) Å for **3a** with similar bond length values for both molecules of the asymmetric unit, and 1.313(2) Å vs. 1.343(2) Å and 1.363(2) Å for **3b**.

While **3b** is essentially planar with an angle between the least-square planes of the 2-pyridyl and triazole rings of 8.1(1)°, **3a** displays an exceptional deviation between the pyridyl part and the triazolyl entity. The values of the dihedral angle (33.3(1)° and 27.9(1)° for both molecules of the asymmetric unit) are the largest observed for the pyta scaffold compared to the previously

reported values (range from 1.56°-15°)¹⁰a,11,30. In turn, one of the two molecules of **3a** exhibits a singular planar geometry with respect to the triazolyl moiety and the phenyl group of the benzenesulfonamide unit (dihedral angle value of 3.2(1)° vs. 30.3(2)° for the second molecule of the asymmetric unit). Generally, in pyta scaffold bearing a phenyl group directly connected to the triazole ring, the dihedral angle between the pyridyl and triazole rings is shorter than that between the triazole and phenyl rings (dihedral angle value: c.a. 20-30°)¹¹,30a. A network of inter-molecular hydrogen interactions (see ESI part, Table S6) and π-π interactions could explain these unexpected structural features for **3a** (see ESI part, Table S7).

More interestingly, while the nitrogen atoms N(1)/N(2) and N(6)/N(7) of **3a** exhibits a classical anti configuration¹¹,12,30 with minimal electronic repulsions between both nitrogens, an unprecedented *cis* arrangement is clearly adopted by the nitrogen atoms of the pyridine and triazole rings of ligand **3b**, as illustrated in Figure 1. Stabilisation through a network of non-covalent interactions has been invoked to explain thus unexpected arrangement. The single-crystal X-ray structure of **3b** shows two types of hydrogen-bonding interactions (see ESI part, Table S6): stronger intermolecular hydrogen-bonding interactions of the type N-H···N [2.939(2) Å] between the N(5)-H(5b) proton of the sulfonamide moiety and the N(1) atom of the pyridine ring, as well as C-H···O [3.260(2) Å] between the C(7)-H(7) proton of the 1,2,3-triazolyl group and O(1) atom of the sulfonamide moiety. π-π interactions have also been detected in the crystallographic structure for **3b** (see ESI part, Table S7).

Rhenium complexes **4a** and **4b** shown in Figure 2 (molecular views), crystallise as neutral molecular complexes in the orthorhombic space groups *P*bcn and triclinic space groups *P*\`1, respectively. As suggested by the spectroscopic data, each rhenium centre adopts a distorted octahedral coordination geometry. The rhenium atoms are coordinated to two nitrogens of the pyta pincer, three carbonyl donors in a *fac*-orientation and one chlorine atom. The bond lengths and angles for both complexes **4a** and **4b** are unexceptional and similar to those previously reported for other rhenium tricarbonyl complexes based on bidentate ligands¹⁰⁻¹²,³⁰. As an example, the average Re-carbonyl bond length is 1.92 Å, and the OC-Re-CO angles range from 87.4(2)° to 90.3(2)° and 88.8(1)° to 90.3(1)° for **4a** and **4b**, respectively. The *trans* bond angles exhibit a moderate distortion from an idealised octahedral geometry (range of 170.3-179.0°), as expected. The Re-Cl bond lengths [2.490(1) Å for **4a**, 2.485(1) Å for **4b**] lie in the upper side of the range (2.454-2.505 Å) found for other rhenium tricarbonyl complexes based on pyta derivatives¹⁰g,11,12a. For both complexes **4a** and **4b**, the chelate part is essentially planar with a very slight deviation of 2.5(1)° and 8.2(2)° between the mean planes through the triazolyl and pyridyl rings, respectively. As observed previously, the co-planarity was extended to the benzenesulfonamide pendent arm in **4a**. The 4-substituted aromatic arm shows a dihedral angle of 2.1(1)° with the mean plane of the triazolyl ring, as a result of conjugation¹⁰b,³¹. In both complexes, a network of π-π interactions and inter-molecular hydrogen bonds between two adjacent complex molecules for **4a** and between two adjacent complex molecules plus the lattice methanol molecule for **4b** are involved in the crystal cohesion (see ESI part, Table S6, and Figure S2).

## 3.2. Computational study

The density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations were performed in order to
<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>**Figure 2.** The molecular structures of rhenium complexes **4a** and **4b**. Displacement ellipsoids are drawn at 50% probability, solvent molecule (**4b**) and hydrogen atoms (except H atoms on nitrogen) have been omitted for clarity.</figcaption></figure>

<figure><img src="image_23.png" /></figure>

assess the impact of the methylene linker (—CH₂—) between the benzenesulfonamide moiety and the chelate unit of **3b** on the electronic structures of its rhenium complexes (See Tables S8–S10 and Figure S3 in ESI part for more details). While HOMOs energy levels exhibit small changes, the introduction of the methylene spacer in the complex **4b** has an effect on LUMOs energy, as illustrated in Figure 3. Compared to **4a**, the introduction of this methylene linker increased the energy level of LUMO (−2.29 eV) causing wider energy gap (3.22 eV for **4b** vs. 3.09 eV for **4a**) and a slight blue-shift of the lowest energy absorption band (331 nm for **4b** vs. 333 nm for **4a**). The lowest virtual orbitals of complexes are mainly concentrated on the π-anti-bonding orbitals of the chelate and CO ligands. The lowest virtual molecular orbital (LUMO) is centred on π-anti-bonding orbitals of the 2-pyridyl-1,2,3-triazole part and CO ligands. The LUMO + 1 and LUMO + 4 are mainly localised on the full pyta ligand for both complexes. The LUMO + 2 is located on the 2-pyridyl-1,2,3-triazole unit in **4a**, and with a slight contribution from the phenyl ring in the case of **4b**. The LUMO + 3 is wholly centred on phenyl ring for the complex **4a**, whereas for **4b**, it is localised on the full connected ligand.

For **4a** and **4b**, the low-energy absorption bands at 333 and 331 nm have mixed metal-to-ligand [MLCT] and ligand-to-ligand [LLCT] charge transfer character. Accordingly, these electronic transitions originate mainly from the mixed orbitals of the rhenium centre and chlorine to the π-antibonding orbitals of the chelating ligand and carbonyl group, which can be described as {d/π(Cl) → π*(L)/π*(CO) or π*(L)}. The band assignments were presented like those previously reported by other researchers, for related Re(CO)₃-complexes with similar bidentate ligands³².

### 3.3. Carbonic anhydrase inhibitory activity

As mentioned in the introduction, the group of Alberto demonstrated that compact tricarbonyl rhenium complexes acted as excellent carbonic anhydrase IX and XII inhibitors, with nanomolar affinities¹⁴c. More recently, Tim Storr et al., reported that tricarbonyl rhenium complexes based on a dipyridylamine (DPA) or an iminodiacetate (IDA) ligands with a pendant benzenesulfonamide pharmacophore exhibited interesting CA-IX inhibition ($K_i$ of 37 nM for the best candidate)¹⁴d.

In our case, both ligands **3a** and **3b** and their corresponding complexes **4a** and **4b** were tested for their efficacy to inhibit physiologically relevant hCA I, II and the tumour-associated hCA

<table><caption>**Table 2.** Inhibition data of human CA I, II and IX isoforms with compounds **3a**, **3b**, **4a**, and **4b** in comparison with the standard sulfonamide inhibitor acetazolamide (AAZ) by a stopped-flow CO₂ hydrase assay.</caption><thead><tr><th rowspan="2">Compound</th><th colspan="3">K<sub>i</sub> (nM)<sup>a</sup></th><th colspan="2">Selectivity ratio</th></tr><tr><th>hCA I</th><th>hCA II</th><th>hCA IX</th><th>I/IX</th><th>II/IX</th></tr></thead><tbody><tr><td>3a</td><td>181.3</td><td>4.1</td><td>2.8</td><td>64.75</td><td>1.46</td></tr><tr><td>3b</td><td>305.2</td><td>56.1</td><td>22.5</td><td>13.56</td><td>2.49</td></tr><tr><td>4a</td><td>3220.5</td><td>194.9</td><td>18.7</td><td>172.2</td><td>10.4</td></tr><tr><td>4b</td><td>7146.0</td><td>836.5</td><td>27.3</td><td>261.75</td><td>30.64</td></tr><tr><td>AAZ</td><td>250</td><td>12</td><td>25</td><td>10</td><td>0.48</td></tr></tbody></table>

IX isoforms, as a preliminary study. The synthesised compounds were screened for their inhibition potential by means of stopped-flow carbon dioxide hydration assay and compared with the clinically used reference drug acetazolamide (AAZ) (Table 2).

Both complexes **4a** and **4b** and the reference drug acetazolamide (AAZ) exhibited similar nanomolar affinities (ca. 25 nM) against hCA IX isozyme. Compared to reported affinity values with different tricarbonyl rhenium systems bearing an arylsulfonamide moiety, our values are better than those recently reported by the group of Storr (37–220 nM), in the lower range than those obtained by Babich et al. (3–116 nM) but higher than those obtained by Alberto et al. (3–7 nM)¹⁴b-d. The introduction of a methylene group between the metallic part and the sulfonamide unit has a slight negative effect on the inhibition values (18.7 nM for **4a** vs. 27.3 nM for **4b**), as previously reported¹⁴c. Additionally, a better inhibition is observed with ligands **3a** and **3b** compared to their corresponding metallic analogs. In particular, a very strong inhibition is found for ligand **3a**, which displayed 7-fold higher affinity than its rhenium complex **4a** ($K_i$(**3a**)=2.8 nM vs. $K_i$(**4a**)=18.7 nM). This unexpected result should be confirmed on other hCA isoforms, the non-metaled free ligands exhibiting generally weaker affinity than that of the corresponding rhenium complexes¹⁴b. In turn, this first study showed that both rhenium complexes were highly inactive against hCA I and exhibited a low affinity against hCA II but a nanomolar affinity against hCA IX. In terms of selectivity, complex **4b** was our best candidate with promising hCA I/hCA IX and hCA II/hCA IX ratios of 261.75 and 30.64, respectively.

<footnote>a mean from 3 different assays, by a stopped-flow technique (errors in the range of ±5–10% of the reported value).</footnote>

<footnote>Italic values are the references values of AAZ (acetalozamide). These are used to compare with our results.</footnote>
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>Figure 3. Molecular orbital diagrams of 4a (Left) and 4b (Right).</figcaption></figure>

4. Conclusion

In short, two new bidentate 2-pyridyl-1,2,3-triazole ligands with a pendent 4-substituted benzenesulfonamide arm and their corresponding Re(CO)₃-complexes were successfully synthesised. All the prepared compounds were fully characterised by classical spectroscopic methods and X-ray, as well as theoretical studies for both complexes. While in the solid state, rhenium complexes showed classical distorted octahedral geometry, ligand 3b adopted an uncommon cis-configuration arising from a network of intermolecular hydrogen-bonding interactions. DFT calculations revealed that the presence of methylene (—CH₂—) linker between the benzenesulfonamide moiety and the chelate

unit has a slight effect on the absorption bands position of 4b as well as the HOMO-LUMO gap which is slightly blue-shifted compared to 4a.

Preliminary investigation on the inhibitory activity of these compounds against the cytosolic human carbonic anhydrase I, II and the membrane-associated isoforms IX (hCAs I, II and IX) revealed an activity in the nanomolar range for hCA IX isozyme. Surprisingly, better inhibition was found with ligands compared to rhenium complexes. In particular, this preliminary study showed that ligand 3a exhibited a strong affinity Kᵢ of 2.8 nM for hCA IX. Additionally, complex 4b exhibited a pronounced selectivity against hCA IX over the off-targets isoforms hCA I and hCA II
<figure><img src="image_26.png" /></figure>

which makes this compound a promising potential anticancer drug candidate. Further *in vitro* studies against other hCA isoforms with both ligands and rhenium complexes and other derivatives based on the same scaffold are under progress in order to rationalise these first biological results.

### Note

1. Pyta and tapy mean 4-(2-pyridyl)-1,2,3-triazole and 1-(2-pyridyl)-1,2,3-triazole, respectively.

### Disclosure statement

No potential conflict of interest was reported by the authors.

### Funding

This research was supported in part by the Algerian Minister of High Education and Scientific Research through the National Programme Exceptional (PNE, Ph.D. fellowship A.Y.) and CNEPRUE project B00L01UN2401201400148.

### ORCID

Yassine Aimene ID http://orcid.org/0000-0001-7067-2644

Jean-Yves Winum ID http://orcid.org/0000-0003-3197-3414

Claudiu T. Supuran ID http://orcid.org/0000-0003-4262-0323

Eric Benoist ID http://orcid.org/0000-0003-0764-9024

Achour Seridi ID http://orcid.org/0000-0002-2288-544X

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