New sulfonamides containing organometallic-acylhydrazones: synthesis, characterisation and biological evaluation as inhibitors of human carbonic anhydrases

Abstract

A series of organometallic acylhydrazones was prepared, incorporating Re(CO)₃, Mn(CO)₃ and ferrocenyl moieties, which were subsequently reacted with amino-sulfonamides in order to obtain carbonic anhydrase (CA, EC 4.2.1.1) inhibitors possessing organometallic moieties in their molecules. The new derivatives were investigated as inhibitors of four human (h) CA isoforms with pharmaceutical applications, such as the cytosolic hCA I, II and VII and the mitochondrial hCA VA. An interesting inhibitory profile against these isoforms was obtained, with some of these metal complexes acting as subnanomolar or low nanomolar inhibitors. They were also thoroughly characterised from the chemical point of view, making them of interest for further developments in the field of metal complexes of sulfonamides with CA inhibitory action.

1. Introduction

Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) are clinically used for several decades as diuretics¹, antiglaucoma agents², antiobesity drugs³, and more recently, a number of studies showed that CA inhibition has profound antitumor effects by inhibition of hypoxia-inducible isoforms CA IX and XII, overexpressed in many hypoxic tumors⁴. Several proof-of-concept studies demonstrated the involvement of some CA isoforms in neuropathic pain⁵ and arthritis⁶, with inhibitors of the sulfonamide/coumarin⁷ types demonstrating significant effects in vivo, in animal models of these diseases. This is obviously due to the fact that at least 15 different α-class CA isoforms are present in humans, and many of them are drug targets for the treatment or prevention of this large variety of pathologies^1–7. Thus, the field of drug design, synthesis and in vivo investigations of various types of CAIs is a highly dynamic one, with a large number of interesting new chemotypes acting on these widespread enzymes constantly emerging^1–7. Among the clinically used sulfonamide CAIs are acetazolamide (AAZ), methazolamide (MZA), ethoxzolamide (EZA), brinzolamide (BRZ) and dorzolamide (DRZ) – (Figure 1)^1–3. Saccharin (SAC) is a sweetener widely used in beverages and food^1–3.

Figure 1.

Clinically used sulfonamides with CA inhibitory activity^1–3.

Coordination compounds of sulfonamides with CA inhibitory properties in which the sulfonamides act as ligands to various transition or main group metal ions, leading to sulfonamide metal complexes were also investigated for their interactions with these enzymes⁸. Originally investigated for obtaining transition metal ion complexes of acetazolamide AAZ, methazolamide MZA, and ethoxzolamide EZA (the main sulfonamide, clinically used drugs belonging to this class of pharmacological agents)⁸, this approach was subsequently extended to a large set of primary and secondary aromatic/heterocyclic sulfonamides, also including the clinical drugs saccharin (SAC), brinzolamide (BRZ) and dorzolamide (DRZ)^9–14. Other sulfonamides possessing a diverse scaffold but effective CA inhibitory properties were also included in such studies together with metal ions which may add a supplementary pharmacological activity, such as Pt(II), Pd(II) and Ru(II) for the antitumor effects^6,14,15, Zn(II) for the antiglaucoma action¹¹, Al(III) for antacid properties¹⁰, Co(II), Ag(I) and Cu(II) for antifungal activity¹⁰. Imaging tumors overexpressing some CA isoforms (e.g. CA IX and XII) with sulfonamide complexes incorporating isotopes of metal ions which emit positrons (for PET imaging), such as Ga(III), In(III) or Cu(II) were also investigated¹⁴, allowing interesting developments in the field. On the other hand, the organometallic complexes also incorporating sulfonamide CAIs as ligands were less investigated, although some rhenium(I) and ruthenium(II) derivatives were recently reported^14,15.

Here we explored the possibility to prepare organometallic-acylhydrazone incorporating Re(CO)₃, Mn(CO)₃ and ferrocenyl moieties, which were reacted with amino-sulfonamide in order to obtain CAIs possessing organometallic moieties in their molecules.

2. Experimental

2.1. Materials

All manipulations were conducted under an N₂ atmosphere using Schlenk techniques. The compounds (η⁵-C₅H₄CHO)Re(CO)₃¹⁶, (η⁵-C₅H₄CHO)Mn(CO)₃¹⁷, 2 or 4-(hydrazinecarbonyl)benzenesulfonamide¹⁸ and 4-((3-hydrazinyl-3-oxopropyl)amino)benzenesulfonamide¹⁸ were prepared according to published procedures. Ferrocenecarboxaldehyde (98%), sulfanilamide (99%), 4-sulfamoylbenzoic acid (97%), methyl-2-(aminosulfonyl)benzoate (98%) and CF₃COOH (99%) were obtained from Sigma-Aldrich and used without additional purification. Solvents such as CH₂Cl₂, hexane, acetone, EtOH, DMSO, and THF were obtained commercially and purified using standard methods. Infrared spectra were recorded in solid state (KBr pellet) on a Jasco FT-IR 4600 spectrophotometer. ¹H NMR spectra were measured on a Bruker spectrometer model ASCEND TM 400 MHz. All NMR spectra are reported in parts per million (ppm, δ) relative to tetramethylsilane (Me₄Si), with the residual solvent proton resonances used as internal standards. Coupling constants (J) are reported in Hertz (Hz), and integrations are reported as number of protons. The following abbreviations were used to describe the peak patterns: s = singlet, d = doublet, t = triplet, and m = multiplet. Mass spectra were obtained on a Shimadzu model QP5050A GC-MS at the Laboratorio de Servicios Analíticos, Pontificia Universidad Católica de Valparaíso. Elemental analyses were measured on a Perkin Elmer CHN Analyzer 2400.

2.2. Synthesis of organometallic-acylhydrazones. General procedure

The new organometallic-acylhydrazones were prepared following the same procedure as for their organic analogues¹⁹. In each case, the respective acylhydrazide (1 eq.) was charged in a two neck round bottomed flask with dried ethanol (10 mL) and a magnetic stir bar. The solution was stirred under nitrogen atmosphere to obtain a clear solution. To the reaction mixture, formyl organometallic precursor (1 eq.) and four drops of CF₃COOH were added at room temperature and under stirring condition and the reaction was continued for 4 h. After this time, the reaction mixture was filtered and the precipitate was washed with cold hexane (3 × 10 mL) and dried under vacuum for 2 h. The solid obtained was purified using slow diffusion crystallization from THF/hexane (1:5) at −18 °C.

2.2.1. [{(η⁵-C₅H₄)–CH=N–N(H)C(O)–C₆H₄-2–SO₂NH₂}]Re(CO)₃ (1a)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)Re(CO)₃ (121 mg, 0.33 mmol) and 2-(hydrazinecarbonyl)benzenesulfonamide (72 mg, 0.33 mmol). Brown solid, yield 78% (146 mg, 0.26 mmol). IR (KBr, cm⁻¹): 3300–3198 (νNH/NH₂); 2027 (νRe–CO); 1913 (νRe–CO); 1658 (νCO); 1558 (νC = N); 1338 (νS–O). ¹H NMR (DMSO-d₆): δ 5.63 (t, 0.5H, J = 2.2 Hz, C₅H₄); 5.77 (t, 1.5H, J = 2.2 Hz, C₅H₄); 5.87 (t, 0.5H, J = 2.2 Hz, C₅H₄); 6.24 (t, 1.5H, J = 2.2 Hz, C₅H₄); 7.09 (s, 0.5H, NH₂); 7.12 (s, 1.5H, NH₂); 7.69 (m, 3H, Ar–H); 7.95 (m, 1H, Ar–H); 8.02 (s, 0.8H, CH = N); 8.32 (s, 0.2H, CH = N); 11.99 (s, 1H, NH). Mass spectrum (based on ¹⁸⁷Re) (m/z): 560 [M⁺]; 532 [M⁺ - CO]; 504 [M⁺ - 2CO]; 476 [M⁺ - 3CO]. Anal. (%) Calc. for C₁₆H₁₂N₃O₆SRe: C, 34.28; H, 2.16 and N, 7.50; found: C, 34.34; H, 2.17 and N, 7.49.

2.2.2. [{(η⁵-C₅H₄)–CH=N–N(H)C(O)–C₆H₄-4–SO₂NH₂}]Re(CO)₃ (1b)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)Re(CO)₃ (121 mg, 0.33 mmol) and 4-(hydrazinecarbonyl)benzenesulfonamide (72 mg, 0.33 mmol). Yellow solid, yield 83% (155 mg, 0.28 mmol). IR (KBr, cm⁻¹): 3243–3112 (νNH/NH₂); 2027 (νRe–CO); 1950 (νRe–CO); 1664 (νCO); 1556 (νC=N); 1341 (νS–O). ¹H NMR (DMSO-d₆): δ 5.78 (t, 2H, J = 2.2 Hz, C₅H₄); 6.26 (t, 2H, J = 2.2 Hz, C₅H₄); 7.52 (s, 2H, NH₂); 7.94 (d, 2H, J = 8.2 Hz, Ar–H); 8.04 (d, 2H, J = 8.2 Hz, Ar–H); 8.20 (s, 1H, CH=N); 11.96 (s, 1H, NH). Mass spectrum (based on ¹⁸⁷Re) (m/z): 560 [M⁺]; 476 [M⁺ - 3CO]. Anal. (%) Calc. for C₁₆H₁₂N₃O₆SRe: C, 34.28; H, 2.16 and N, 7.50; found: C, 34.33; H, 2.16 and N, 7.52.

2.2.3. [{(η⁵-C₅H₄)–CH=N–N(H)C(O)–CH₂CH₂–NH–C₆H₄-4–SO₂NH₂}]Re(CO)₃ (1c)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)Re(CO)₃ (121 mg, 0.33 mmol) and 4-((3-hydrazinyl-3-oxopropyl)amino)benzenesulfonamide (86 mg, 0.33 mmol). Pale yellow solid, yield 77% (155 mg, 0.26 mmol). IR (KBr, cm⁻¹): 3368-3115 (νNH/NH₂); 2958 (νC_sp3–H); 2025 (νRe–CO); 1912 (νRe–CO); 1641 (νCO); 1605 (νC = N); 1321 (νS–O). ¹H NMR (DMSO-d₆): δ 2.45 (t, 0.9H, J= 6.8 Hz, CH₂CO); 2.80 (t, 1.1H, J= 6.8 Hz, CH₂CO); 3.36 (m, 2H, CH₂NH); 5.73 (m, 2H, C₅H₄); 6.11 (t, 1.1H, J = 2.2 Hz, C₅H₄); 6.16 (t, 0.9H, J = 2.2 Hz, C₅H₄); 6.43 (m, 1H, NH); 6.63 (m, 2H, Ar–H); 6.91 (s, 2H, NH₂); 7.51 (d, 2H, J = 8.2 Hz, Ar–H); 7.68 (s, 0.6H, CH=N); 7.91 (s, 0.4H, CH = N); 11.32 (s, 0.6H, NH); 11.34 (s, 0.4H, NH). Mass spectrum (based on ¹⁸⁷Re) (m/z): 603 [M⁺]; 606 [M⁺ - 3CO]. Anal. (%) Calc. for C₁₈H₁₇N₄O₆SRe: C, 35.82; H, 2.84 and N, 9.28; found: C, 35.85; H, 2.83 and N, 9.30.

2.2.4. [{(η⁵-C₅H₄)–CH=N–N(H)C(O)–C₆H₄-2–SO₂NH₂}]Mn(CO)₃ (2a)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)Mn(CO)₃ (77 mg, 0.33 mmol) and 2-(hydrazinecarbonyl)benzenesulfonamide (72 mg, 0.33 mmol). Yellow solid, yield 76% (108 mg, 0.25 mmol). IR (KBr, cm⁻¹): 3360–3096 (νNH/NH₂); 2020 (νMn–CO); 1940 (νMn–CO); 1683 (νCO); 1537 (νC = N); 1343 (νS–O). ¹H NMR (DMSO-d₆): δ 5.01 (s, 0.5H, C₅H₄); 5.16 (s, 1.5H, C₅H₄); 5.25 (s, 0.5H, C₅H₄); 5.61 (s, 1.5H, C₅H₄); 7.11 (s, 2H, NH₂); 7.62 (m, 4H, Ar–H); 7.96 (s, 0.8H, CH = N); 8.25 (s, 0.2H, CH = N); 12.03 (s, 1H, NH). Mass spectrum (m/z): 429 [M⁺]; 401 [M⁺ - CO]; 373 [M⁺ - 2CO]; 345 [M⁺ - 3CO]. Anal. (%) Calc. for C₁₆H₁₂N₃O₆SMn: C, 44.77; H, 2.82 and N, 9.79; found: C, 44.76; H, 2.81 and N, 9.78.

2.2.5. [{(η⁵-C₅H₄)–CH = N–N(H)C(O)–C₆H₄-4–SO₂NH₂}]Mn(CO)₃ (2b)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)Mn(CO)₃ (77 mg, 0.33 mmol) and 4-(hydrazinecarbonyl)benzenesulfonamide (72 mg, 0.33 mmol). Yellow solid, yield 83% (118 mg, 0.28 mmol). IR (KBr, cm⁻¹): 3233–3008 (νNH/NH₂); 2025 (νMn–CO); 1933 (νMn–CO); 1663 (νCO); 1557 (νC=N); 1343 (νS–O). ¹H NMR (DMSO-d₆): δ 4.92 (s, 0.5H, C₅H₄); 5.16 (s, 1.5H, C₅H₄); 5.28 (s, 0.5H, C₅H₄); 5.63 (s, 1.5H, C₅H₄); 7.52 (s, 2H, NH₂); 7.94 (d, 2H, J = 8.2 Hz, Ar–H); 8.04 (d, 2H, J = 8.2 Hz, Ar–H); 8.10 (s, 0.25H, CH=N); 8.13 (s, 0.75H, CH=N); 11.90 (s, 0.25H, NH); 11.99 (s, 0.75H, NH). Mass spectrum (m/z): 429 [M⁺]; 345 [M⁺ - 3CO]. Anal. (%) Calc. for C₁₆H₁₂N₃O₆SMn: C, 44.77; H, 2.82 and N, 9.79; found: C, 44.76; H, 2.83 and N, 9.78.

2.2.6. [{(η⁵-C₅H₄)–CH=N–N(H)C(O)–CH₂CH₂–NH–C₆H₄-4–SO₂NH₂}]Mn(CO)₃ (2c)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)Mn(CO)₃ (77 mg, 0.33 mmol) and 4-((3-hydrazinyl-3-oxopropyl)amino)benzenesulfonamide (86 mg, 0.33 mmol). Brown solid, yield 77% (121 mg, 0.26 mmol). IR (KBr, cm⁻¹): 3369–3080 (νNH/NH₂); 2960 (νC_sp3–H); 2025 (νMn–CO); 1929 (νMn–CO); 1646 (νCO); 1602 (νC=N); 1327 (νS–O). ¹H NMR (DMSO-d₆): δ 2.45 (t, 0.9H, J= 6.8 Hz, CH₂CO); 2.81 (t, 1.1H, J= 6.8 Hz, CH₂CO); 3.36 (m, 2H, CH₂NH); 5.11 (s, 2H, C₅H₄); 5.50 (s, 1.1H, C₅H₄); 5.54 (s, 0.9H, C₅H₄); 6.44 (m, 1H, NH); 6.64 (m, 2H, Ar–H); 6.92 (s, 2H, NH₂); 7.51 (d, 2H, J = 7.9 Hz, Ar–H); 7.62 (s, 0.6H, CH=N); 7.83 (s, 0.4H, CH = N); 11.34 (s, 0.6H, NH); 11.36 (s, 0.4H, NH). Mass spectrum (m/z): 472 [M⁺]; 388 [M⁺ - 3CO]. Anal. (%) Calc. for C₁₈H₁₇N₄O₆SMn: C, 45.77; H, 3.63 and N, 11.86; found: C, 45.79; H, 3.62 and N, 11.89.

2.2.7. [{(η⁵-C₅H₄)–CH=N–N(H)C(O)–C₆H₄-2–SO₂NH₂}]FeCp (3a)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)FeCp (107 mg, 0.50 mmol) and 2-(hydrazinecarbonyl)benzenesulfonamide (107 mg, 0.50 mmol). Red solid, yield 71% (146 mg, 0.36 mmol). IR (KBr, cm⁻¹): 3308–3088 (νNH/NH₂); 1640 (νCO); 1598 (νC = N); 1334 (νS–O). ¹H NMR (DMSO-d₆): δ 4.18 (s, 1H, C₅H₅); 4.26 (s, 4H, C₅H₅); 4.32 (s, 0.4H, C₅H₄); 4.37 (s, 0.4H, C₅H₄); 4.48 (s, 1.6H, C₅H₄); 4.67 (s, 1.6H, C₅H₄); 7.01 (s, 0.5H, NH₂); 7.15 (s, 1.5H, NH₂); 7.72 (m, 3H, Ar–H); 7.96 (m, 1H, Ar–H); 8.15 (s, 0.9H, CH=N); 8.43 (s, 0.1H, CH=N); 11.71 (s, 0.2H, NH); 11.77 (s, 0.8H, NH). Mass spectrum (m/z): 411 [M⁺]. Anal. (%) Calc. for C₁₈H₁₇N₃O₃SFe: C, 52.57; H, 4.17 and N, 10.22; found: C, 52.59; H, 4.18 and N, 10.23.

2.2.8. [{(η⁵-C₅H₄)–CH=N–N(H)C(O)–C₆H₄-4–SO₂NH₂}]FeCp (3b)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)FeCp (107 mg, 0.50 mmol) and 4-(hydrazinecarbonyl)benzenesulfonamide (107 mg, 0.50 mmol). Red solid, yield 78% (160 mg, 0.39 mmol). IR (KBr, cm⁻¹): 3226–3209 (νNH/NH₂); 1635 (νCO); 1567 (νC = N); 1336 (νS–O). ¹H NMR (DMSO-d₆): δ 4.26 (s, 5H, C₅H₅); 4.48 (s, 2H, C₅H₄); 4.69 (s, 2H, C₅H₄); 7.52 (s, 2H, NH₂); 7.95 (d, 2H, J = 8.2 Hz, Ar–H); 8.06 (d, 2H, J = 8.2 Hz, Ar–H); 8.31 (s, 1H, CH=N); 11.68 (s, 1H, NH). Mass spectrum (m/z): 411 [M⁺]. Anal. (%) Calc. for C₁₈H₁₇N₃O₃SFe: C, 52.57; H, 4.17 and N, 10.22; found: C, 52.55; H, 4.19 and N, 10.24.

2.2.6. [{(η⁵-C₅H₄)–CH = N–N(H)C(O)–CH₂CH₂–NH–C₆H₄-4–SO₂NH₂}]FeCp (3c)

This compound was prepared according to the general procedure described above, using in this case: (η⁵-C₅H₄CHO)FeCp (107 mg, 0.50 mmol) and 4-((3-hydrazinyl-3-oxopropyl)amino)benzenesulfonamide (129 mg, 0.50 mmol). Brown solid, yield 74% (168 mg, 0.37 mmol). IR (KBr, cm⁻¹): 3365-3090 (νNH/NH₂); 2949 (νC_sp3–H); 1667 (νCO); 1600 (νC=N); 1316 (νS–O). ¹H NMR (DMSO-d₆): δ 2.42 (t, 0.92H, J= 6.8 Hz, CH₂CO); 2.80 (t, 1.08H, J= 6.8 Hz, CH₂CO); 3.36 (m, 2H, CH₂NH); 5.19 (s, 2.7H, C₅H₅); 5.21 (s, 2.3H, C₅H₅); 4.41 (m, 2H, C₅H₄); 4.59 (m, 2H, C₅H₄); 6.46 (m, 1H, NH); 6.65 (m, 2H, Ar–H); 6.91 (s, 2H, NH₂); 7.52 (m, 2H, Ar–H); 7.83 (s, 0.54H, CH = N); 7.98 (s, 0.46H, CH=N); 11.05 (s, 0.54H, NH); 11.08 (s, 0.46H, NH). Mass spectrum (m/z): 454 [M⁺]. Anal. (%) Calc. for C₂₀H₂₂N₄O₃SFe: C, 52.87; H, 4.88 and N, 12.33; found: C, 52.86; H, 4.89 and N, 12.37.

2.3. CA inhibition studies

An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic activity of various CA isozymes for CO₂ hydration reaction^10,11,20. Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na₂SO₄ (for maintaining constant ionic strength), following the CA-catalyzed CO₂ hydration reaction for a period of 10 s at 25 ◦C. 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 uncatalysed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) were prepared in distilled-deionised water and dilutions up to 1 nM were done thereafter with the assay buffer. Enzyme and inhibitor solutions were pre-incubated together for 15 min (standard assay at room temperature) prior to assay, in order to allow for the formation of the enzyme-inhibitor complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation, as reported earlier^10–12. All CAs were recombinant proteins produced as reported earlier by our group^10–12,20.

3. Results and discussion

3.1. Synthesis and characterisation of organometallic-acylhydrazones containing sulfonamide fragments

The preparation of these new family of organometallic-acylhydrazones described in the experimental section involved the synthesis of the appropriated organic acylhydrazide precursor 2-(hydrazinecarbonyl)benzenesulfonamide, 4 (hydrazinecarbonyl)benzenesulfonamide and 4-((3-hydrazinyl-3-oxopropyl)amino)benzenesulfonamide, which were prepared according to published procedures^18,19. The organometallic-acylhydrazones containing sulfonamide moieties were obtained as described in Scheme 1, following the same procedure reported for some organic analogues¹⁹, that is, by the condensation reaction of the appropriate acylhydrazide and the corresponding formyl organometallic complex in anhydrous EtOH. All compounds were isolated in good yields (71–83%) as solids, after crystallisation from THF/hexane mixture. These products are air-stable and slightly soluble in most polar organic solvents (e.g. CH₂Cl₂, acetone, CH₃CN).

Scheme 1.

Synthesis of organometallic-acylhydrazones containing sulfonamide fragments.

In all cases, the infrared spectral analysis of these compounds showed the characteristic absorption corresponding stretching vibration of the ν(C = N) bond in the range of 1605–1537 cm⁻¹ in KBr disk. Similar ν(C = N) frequency values have been previously reported for other organometallic-acylhydrazones derived from ferrocenyl²¹ and cymantrenyl groups²². The absence of the band assigned to the aldehyde carbonyl group of organometallic complexes confirmed the formation of the organometallic Schiff bases. Moreover, all compounds showed the expected absorption bands for the νN–H, νCO, and νSO₂ stretches in the ranges of 3369–3080 cm⁻¹, 1683–1635 cm⁻¹ and 1343–1321 cm⁻¹, respectively. In addition, the spectra for 1c, 2c, and 3c exhibited an absorption band for νC_sp3–H at ∼2950 cm⁻¹. Furthermore, the IR spectra of cyrhetrenyl (1a–c) and cymantrenyl (2a–c) acylhydrazones revealed the presence of terminal metal carbonyl groups in the region of 2027–1912 cm^−123–25. A strong molecular ion was shown in the mass spectrum of each organometallic-acylhydrazones, in addition to the detection of notable successive losses of CO ligands for the cyrhetrenyl (1a–c) and cymantrenyl (2a–c) derivatives. The elemental analysis data determined for all compounds are in agreement with their proposed formulas.

For all complexes, the ¹H NMR spectra showed the presence of a sharp singlet in the range of 8.43–7.62 ppm, and it was assigned to the iminic proton. These results are in agreement with the values reported for organometallic Schiff bases^26–28. Moreover, ¹H NMR spectra for 1a–c and 2a–c showed sets of resonances in the region of 6.26–4.92 ppm, which are ascribed to the protons of the cyrhetrenyl and cymantrenyl moieties²⁹. On this regard, the ferrocenyl derivatives 3a–c exhibited resonances around δ 4.69–4.32 due to the non-equivalent alpha and beta protons containing in the substituted Cp ring and a singlet in the region of 4.26–4.18 ppm, which was assigned to the proton resonances of the unsubstituted cyclopentadienyl group. For all compounds, the multiplets observed between 8.06 and 6.63 ppm were assigned to the hydrogen atoms of the C₆H₄ ring. As per literature reports, the broad singlet observed at 7.52–6.91 ppm was assigned to the hydrogen nuclei of the SO₂NH₂ group^18,30,31.

The presence of the –NH–CO– group registered as a broad singlet in the range of 12.03–11.05 ppm. Similar δ have been reported for other organometallic hydrazones^21,22,32. On this regard, it is an important remark that the chemical shifts of the –NH– resonance showed a dependence on the presence of the organometallic moiety bound to the iminic entity. In fact, the downfield shift observed for the cyrhetrenyl (1a–c) and cymantrenyl (2a–c) derivatives (Δδ∼0.5) compared with ferrocenyl analogues (3a–c) can be related to the electron-withdrawing properties of the (η⁵-C₅H₄)M(CO)₃ moieties^33,34, which produce a deshielding of the NH resonance, thus suggesting that the nature of the organometallic framework modifies the degree of electronic delocalization on the –C(H)=N–NH– unit. We have found similar results for ferrocenyl and cyrhetrenyl hydrazones^19,35 and 1,3,4-thiadiazoles³⁶. In the case of the acylhydrazones 1c, 2c and 3c, additional signals were observed at 6.44 ppm, 3.36 ppm and 2.80–2.42 ppm, respectively. These resonances were attributed to the presence of the –CH₂CH₂NH– group³⁷.

It is important to mention that acylhydrazones can form four isomers owing to the presence of amide (–CONH–) and azomethine groups (–CH = N–) in their structure^38,39. The geometrical isomers (E/Z) originate from the azomethine group and rotamer (cis/trans) formation is due to the restricted rotation of the amide group (see Figure 2). However, the survey of the literature reveals that the N-acyl hydrazones synthesised from aromatic carbaldehyde are essentially planar and exist completely in the form of geometric (E)-configuration about the C = N bond due to steric hindrance on the imine bond^38–41. Therefore, we discarded the formation of Z, cis and Z, trans isomers.

Figure 2.

General structure of possible Z/E geometrical isomers and cis/trans amide rotamers of organometallic-acylhydrazones.

Based on ¹H NMR data, the organometallic-acylhydrazones (1a–c), (2a–c) and (3a–c) reported in this work exist as a mixture of cis/trans isomers in DMSO-d₆ solutions. On this regard, ¹H NMR spectra for all compounds show resonances for the CO–NH and CH = N group protons are present in double sets and the signal intensity ratio is ∼0.6 cis: 0.4 trans. This splitting signals pattern was also observed for cyclopentadienyl (C₅H₅, C₅H₄) and ethyl (–CH₂CH₂–) protons. The cis isomer predominates because of a hindered rotation around the CO–NH bond¹⁹. Similar results have been reported for organic-acylhydrazones derived from other benzenesulfonamide derivatives³⁷.

In order to confirm the existence of cis-/trans-amide rotamers, we carried out ¹H NMR spectra of 1a measurements at several temperatures (Figure 3). Variable temperature (VT) ¹H NMR spectra showed that increasing the temperature within the range of 296-346 K led to coalescence of the –CONH– resonance of cis- and trans-amide rotamers. Similar results have been published previously by Barhoumi-Slimi and co-workers in organic-acylhydrazones derived from cinnamaldehyde and β-chloro-α,β-unsaturated aldehydes⁴².

Figure 3.

NH region of ¹H NMR spectra of complex 1a in DMSO-d₆ registered at variable temperatures.

Unfortunately, the low solubility of all the compounds in deuterated solvents precluded us to measure their 13C NMR and bidimensional NMR spectra to complement their characterisation.

3.2. CA inhibition studies

The sulfonamide containing organometallic-acylhydrazones have been evaluated in vitro as CAIs. Three cytosolic human (h) isoforms (hCA I, II, and VII) and one mitochondrial (hCA VA) have been included for the screening, and the results revealed interesting selectivity profiles for some of the evaluated compounds. Inhibition data obtained with the standard stopped-flow CO₂ hydrase assay are compared to those of the standard sulfonamide inhibitor acetazolamide (AAZ)^43–47 (Table 1). Structure-activity relationships have been delineated dividing the compounds into three classes, depending on the organic portion responsible for the CA inhibition.

Table 1.

Inhibition of human (h) CA isoforms hCA I, II, VA, and VII with acetazolamide (AAZ) and the organometallic derivatives reported here, by a stopped-flow, CO₂ hydrase assay⁴⁷.

	KI (nM)*
Cmp	hCA I	hCA II	hCA VA	hCA VII
1a	>10000	2595.6	58.7	69.7
1b	595.4	0.48	69.5	74.2
1c	7736.8	6.59	35.7	9.6
2a	>10,000	>10,000	73.8	76.1
2b	373.6	0.47	30.3	77.7
2c	136.6	1.54	32.9	9.2
3a	>10000	6624.2	66.3	77.8
3b	2817.3	4.02	34.2	27.2
3c	8090.9	18.2	21.8	9.5
AAZ	250	12.1	63	2.5

*Mean from 3 different assays. Errors were in the range of ±5–10% of the reported values (data not shown).

1. The 2-(hydrazinecarbonyl)benzenesulfonamide derivatives (series a) revealed to be ineffective in inhibiting hCA I (Ki > 10,000 nM) and showed poor active against hCA II, with Ki values in the micromolar range (2595.6 < Ki < 10,000 nM), regardless of the different metal substituted Cp ring contained in them. On the other hand, the insertion of the sulfonamide group in ortho position of the aromatic ring turned out to be favourable for the inhibition of hCA VA and VII, which were strongly inhibited by all the compounds investigated here, with Ki values in the nanomolar range for the cyrhetrenyl 1a, cymantrenyl 2a and ferrocenyl 3a derivatives. Therefore, compounds 1a, 2a, and 3a were potent and selective hCA VA and VII inhibitors (over the cytosolic enzymes hCA I and II).

2. The insertion of 4 (hydrazinecarbonyl)benzenesulfonamide fragment on the molecular scaffolds (series b) led to a dramatic enhancement of inhibition potency against hCA II, particularly for compounds 1b and 2b, which were 8-fold more potent than the ferrocenyl derivative 3b. A slight enhancement of potency against hCA I was also observed for the cyrhetrenyl 1b and cymantrenyl 2b derivatives, which were effective in the high nanomolar range (Ki values of 595.4 nM and 373.6 nM respectively). The same isosteric substitution on the organic scaffold did not affect the inhibition potency against hCA VA and VII, with the best Ki values showed by the cymantrenyl 2b (30.3 nM) and ferrocenyl 3b (34.2 nM) derivatives. For hCA VII, the ferrocenyl derivative 3b turned out to be 3-fold more potent (Ki of 27.2 nM) than the other organometallic analogues investigated here.

3. The expansion of the organic scaffold due to the insertion of 4-((3-hydrazinyl-3-oxopropyl)amino)benzenesulfonamide moiety in the derivatives of series c revealed to be detrimental for the inhibition potency against hCA I for the cyrhetrenyl 1c and ferrocenyl 3c derivatives, that possessed Ki-s in the micromolar range. On the other hand, the cymantrenyl 2c inhibition potency was not affected by this structural modification. The potency against hCA II was retained with a slight worsening of the Ki values, mostly for the ferrocenyl derivative 3c (4.5 fold potency decrease when compared to 3b). hCA VA was strongly inhibited by all derivatives investigated here. Noteworthy, the same modification in the organometallic-acylhydrazones scaffold led to a clear enhancement of the potency against hCA VII, with low nanomolar Ki values showed by all derivatives, (ranging between 9.2 nM and 9.6 nM).

In conclusion, we report a new class of organometallic CAIs possessing an interesting inhibitory profile against isoforms with important pharmacologic applications, such as CA II, VA, and VII. Some of these metal complexes were subnanomolar or low nanomolar inhibitors of many such enzymes. They were also thoroughly characterised from the chemical point of view, making them of interest for further developments in the field of metal complexes of sulfonamides with CA inhibitory action.

Funding Statement

R.A and N.N acknowledge FONDECYT-Chile [Project 11130443, 1110669]. R.A and Y.H. acknowledge VRID-UdeC [grant 216.021.034-1.0] for the funding of this study.

Disclosure statement

No potential conflict of interest was reported by the authors.