A Step Towards Development of Promising Trypanocidal Agents:

Synthesis, Characterization and in Vitro Biological Evaluation of

Ferrocenyl Mannich Base-type Derivatives.

Rocío Paucara, Rubén Martín-Escolanob, Elsa Moreno-Viguria, Nuria Cirauquic, Carlos

Rangel Rodriguesc, Clotilde Marínb, Manuel Sánchez-Morenob, Silvia Pérez-Silanesa,

Mauro Raverad, Elisabetta Gabanod*

a Universidad de Navarra, Department of Technology and Chemistry Pharmaceutics,

Instituto de Salud Tropical, Pamplona 31008, Spain.

b Department of Parasitology, Instituto de Investigación Biosanitaria (ibs.GRANADA),

Hospitales Universitarios De Granada/University of Granada, Granada 18071, Spain

c Department of Pharmaceutical Sciences, Federal University of Rio de Janeiro, Rio de

Janeiro 21949-900, Brazil

d Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte

Orientale, Viale T. Michel 11, 15121, Alessandria, Italy

Table of contents

Intermediates complexes ferrocene characterization. S1

1H and 13C NMR spectra of synthesised compounds. S2

Data of the SOS/umu screening test. S3

1H NMR spectra of the mediums for non-treated and treated parasites. S4

Docking studies. S5

S1. Compounds characterization

Synthesis of acetylferrocene. Yield: 4.34 g, 88 %.1H NMR (CDCl3) = 2.39 (s, 3 H, CH3),

4.20 (s, 5 H, 5 CH, Cp), 4.50 (m, 2 H, 2 CH, substituted Cp), 4.77 (m, 2 H, 2 CH,

substituted Cp) ppm. 13C NMR (CDCl3) = 27.58 (CH3), 69.73 and 72.46 (CH,

substituted Cp), 70.00 (CH, Cp), 79.43 (Cquat, substituted Cp), 202.35 (C=O) ppm. ESI-

MS (positive ion mode): 229 m/z, calc. for [C12H13FeO]+ 229.2 m/z [M+H]+, 228.1 m/z,

calc. for [C12H12FeO]+ 228 m/z [M]+.

Synthesis of 3-chloropropanoylferrocene. Yield: 5.16 g, 75 %. 1H NMR (CDCl3) = 3.17

(t, 2H, 3J = 6.4 Hz, CH2), 3.90 (t, 2H, 3J = 6.4 Hz, Cl-CH2), 4.24 (s, 5 H, 5 CH, Cp), 4.53

(t, 2 H, 3J = 2.0 Hz, 2 CH, substituted Cp), 4.79 (t, 2 H, 3J = 2,0 Hz, 2 CH, substituted Cp)

ppm. 13C NMR (CDCl3) = 38.92 (Cl-CH2), 42.20 (CH2), 69.37 and 72.71 (CH,

substituted Cp), 70.04 (CH, Cp), 78.49 (Cquat, substituted Cp), 200.65 (C=O) ppm. ESI-

MS (positive ion mode): 277.1 m/z, calc. for [C12H14ClFeO]+ 277 m/z [M+H]+; 276.1

m/z, calc. for [C12H13ClFeO]+ 276 m/z [M]+.

Synthesis of Acryloylferrocene. The spectral data were in agreement with Dogan et al.[1]

1-ferrocenylprop-2-en-1-one (Acryloylferrocene). Yield: 62%. Mp: 64-65 ºC. IR (KBr) ν

cm-1: 1658 (s, C=O). 1H NMR (CDCl3, 400 MHz) δ ppm: 6.80 (dd, 1H, CH-a, JCH-a to

olefinic Htrans = 17.0 Hz, JCH-a to olefinic Hcis = 10.4 Hz ); 6.44 (d, 2H, olefinic Htrans, Jolefinic

Htrans to CH-a = 17.0 Hz); 5.71 (d, 1H, olefinic Hcis, Jolefinic Hcis to CH-a = 10.2 Hz); 4.83 (s, 2H,

Hfc); 4.56 (s, 2H, Hfc); 4.19 (s, 5H, Hfc). 13C NMR (APT, CDCl3, 100 MHz) δ ppm:

193.05; 132.96; 126.26; 79.62; 72.82 (2C); 70.05 (5C); 69.71 (2C). Anal. Calc. for

C13H12FeO: C 65.04%, H 5.04%. Found: C 65.43%, H 5.43%.

S2. 1H and 13C NMR spectra of synthesized ferrocene Mannich base-typederivatives

Figure S2.1. 1H NMR of acetylferrocene.

Figure S2.2. 13C NMR of acetylferrocene.

Figure S2.3. 1H NMR of compound i1.

Figure S2.4. 13C NMR of compound i1.

Figure S2.5. 1H NMR of compound i2.

Figure S2.6. 13C NMR of compound i2.

Figure S2.7. 1H NMR of compound 1.

Figure S2.8. 13C NMR of compound 1.

Figure S2.9. 1H NMR of compound 2.

Figure S2.10. 13C NMR of compound 2.

Figure S2.11. 1H NMR of compound 3.

Figure S2.12. 13C NMR of compound 3.

Figure S2.13. 1H NMR of compound 5.

Figure S2.14. 13C NMR of compound 5.

Figure S2.15. 1H NMR of compound 7.

Figure S2.16. 13C NMR of compound 7.

Figure S2.17. 1H NMR of compound 8.

Figure S2.18. 13C NMR of compound 8.

Figure S2.19. 1H NMR of compound 9.

Figure S2.20. 13C NMR of compound 9.

S3. Data of the SOS/umu screening test.

Table S3.1. % survival

Testingconc.(mg.mL-1)

1 0.5 0.25 0.125 0.063 0.031 0.016 0.008 0.004 0.002 0.001

Without metabolic activation2 242.8 127.0 118.8 208.2 71.0 81.2 54.3 52.5 141.9 84.2 64.5With metabolic activation (S9 fraction)2 120.93 118.13 113.87 110.80 126.53 91.20 94.40 87.07 114.53 118.67 107.20

Table S3.2: Induction factor

Testingconc.(mg.mL-1)

1 0.5 0.25 0.125 0.063 0.031 0.016 0.008 0.004 0.002 0.001

Without metabolic activation2 0.35P 0.82 P 0.76 P 0.41 P 1.13 P 0.97 P 1.36 P 1.53 0.53 0.86 1.47With metabolic activation (S9 fraction)2 0.73

P 0.73 P 0.82 P 0.90 P 0.81 P 1.04 P 1.06 P 1.15 0.71 0.79 1.03

P: the compound precipitate

S4. 1H NMR spectra of the mediums for non-treated and treated parasites.

Figure S4. 1H NMR spectra of the mediums for non-treated (control) parasites and parasites

treated with BZN and compound 2. The table shows the chemical shifts of the peaks (expressed

in parts per million, ppm) qualitatively and quantitatively identified for each metabolite.

S5. Docking studies.

Figure S5.1. Proposed binding mode of the nine new derivatives on the T.cruzi Fe-SOD enzyme

(PDB entry 4DVH), as obtained in our docking study: compounds 1(a), 2(b), 3(c), 4(d), 5(e), 6(f),

7(g), 8(h), 9(i). For the protein, one protomer is shown in yellow and the other in cyan. The

colouring scheme used is: oxygen (red), nitrogen (blue), compounds fluoride (pale blue),

compounds chloride (green), iron (brown). The enzyme iron ions are depicted as brown spheres.

Hydrogen bonds between protein and ligand are shown as yellow dashed lines. The figure was

created with The PyMOL Molecular Graphics System, Version 1.7 Schrödinger, LLC.

Table S5.1. MolDock score predicted by the Molegro Virtual Docker (MVD) program, version

6.0[2] for the best-scored pose of each compound, shown in Supplementary Figure S5.1.

Compound MolDock score1 -145,9692 -162,0303 -148,5754 -139,9715 -142,7446 -138,5057 -144,2168 -152,2619 -137,648

BIBLIOGRAPHY

[1] Ö. Doğan, S. Zeytinci, A. Bulut, Synthesis of New Ferrocenyl‐Substituted Aziridines via the Gabriel–Cromwell Reaction, Synth. Commun. 35 (2005), 1067-1076.[2] R. Thomsen, M.H. Christensen, MolDock:  A New Technique for High-Accuracy Molecular Docking,J. Med. Chem. 49 (2006), 3315-3321.