Novel potent metallocenes against liver stage malaria
Joana Matos,1 Filipa P. da Cruz,2 Élia Cabrita,3 Jiri Gut,4 Fátima Nogueira,3 Virgílio E.
do Rosário,3 Rui Moreira,5 Philip J. Rosenthal,4 Miguel Prudêncio2 and Paula Gomes1,1
1
CIQUP – Centro de Investigação em Química da Universidade do Porto, Departamento de Química,
Faculdade de Ciências, Universidade do Porto, P-4169-007 Porto, Portugal;
2
Unidade de Malária,
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, P-1649-028 Lisboa,
Portugal;
3
Centro de Malária e Outras Doenças Tropicais, IHMT - Universidade Nova de Lisboa, P-
1349-008 Lisboa, Portugal;
4
Department of Medicine, San Francisco General Hospital, University of
California, CA 94143-0811, USA;
5
iMed.UL, CECF, Faculdade de Farmácia, Universidade de Lisboa,
P-1600-083 Lisboa, Portugal.
SUPPLEMENTAL INFORMATION:
1. Detailed procedures and spectroscopic data for noval compounds 4f,g and 8
S2
2. Figure S1
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1
Corresponding author: Paula Gomes, Centro de Investigação em Química da Universidade do Porto,
Rua do Campo Alegre 687, P-4169-007 Porto, Portugal; Phone: +351 220402563; Fax: +351 220402659;
E-mail: [email protected].
1. Detailed synthetic procedures and spectroscopic data for novel compounds 4f,g and 8
Synthesis of compounds 4f-g were carried out as previously described (15). Compound
8 was synthesized as follows: Primaquine (0.2000 g; 0.7717 mmol) was dissolved in
TEA (3.0 mL). In a separate flask, 6-bromohexylferrocene (0.1500 g; 0.4297 mmol)
was dissolved in TEA (6.0 mL) and 3.00 mL of this solution were then added to the
primaquine solution. The reaction mixture was taken to 120 °C and, after 1 h, the other
3.0 mL-portion of 6-bromohexylferrocene in TEA was added. The reaction was allowed
proceeding at 120 °C for more 24 h. The reaction mixture was poured into 20.0 mL of
ethyl acetate (AcOEt), to which 50.0 mL of aqueous NaOH 2 M were added. The
aqueous layer was washed with AcOEt (4×30.0 mL) and the resulting organic layers
were pooled and dried over anhydrous Na2SO4. After removal of the drying agent by
filtration, and evaporation of the filtrate to dryness, the crude product was submitted to
column chromatography on silica using DCM/acetone. Product 8 was isolated as yellow
oil (14%).
Spectroscopic data for compounds 4f-g and 8:
4f: 1H NMR (DMSO-d6, 400 MHz) 8.53 (dd, J=4.22 Hz, J=1.66 Hz; 1H, Q2); 7.92 (dd,
J=8.26 Hz, J=1.61 Hz; 1H, Q4); 7.40 and 7.38 (t+t, J=4.99 Hz and J=4.95 Hz; 1H, CH2-NH-CO-); 7.30 (dd, J=8.24 Hz, J=4.22 Hz; 1H, Q3); 6.33 (d, J=2.49 Hz; 1H, Q5);
6.27 (d, J=2.32 Hz; 1H, Q7); 6.04 to 5.96 (bs+bs; 2H, -NH-CH(CH3)- and –NH-COCpa-); 4.69 to 4.65 (m; 2H, Cpa2 and Cpa5); 4.31 (t, J=1.99 Hz; 2H, Cpa3 and Cpa4);
4.18 (s; 5H, Cpb); 3.88 (s; 3H, CH3-O-); 3.65 to 3.59 (m; 1H, -CH((CH2)4NH2)-); 3.38
to 3.33 (m; 2H, -CH2-NH2); 3.31 to 3.25 (m; 2H, -CH2-NH-CO-); 1.86 to 1.54 (m; 12H,
-CH(CH3)-(CH2)2- and –(CH2)3-CH2-NH2); 1.29 (d, J=6.35 Hz; 3H, -CH(CH3)-).
13
C
NMR (DMSO, 100 MHz) 174.9 (-CH2-NH-CO-); 170.3 (-NH-CO-Cpa); 159.4 (QC6);
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144.9 (QC2); 144.3 (QC8); 135.3 (QC4); 134.8 (QC10); 129.9 (QC9); 121.8 (QC3);
96.7 (QC7); 91.6 (QC5); 70.3 (CpaC1+C2); 69.6 (Cpb); 68.1 (CpaC3-C5); 55.2 (CH((CH2)4NH2)-); 54.8 (CH3-O-); 47.8 and 47.7 (-CH(CH3)- and –CH2-NH2); 38.9 (CH2-NH-); 34.3 (-CH(CH3)-CH2-); 33.9 (-CH2-(CH2)3-NH2); 29.4 (-CH2-CH2-NH2);
26.3 (-CH2-CH2-CH2-NH-); 22.7 (-CH2-CH2-CH2-NH-); 20.5 (-CH(CH3)-). ESI-MS:
[M+H]+ found: 600.3333 (C32H41FeN5O3 requires 599.54). HPLC: tr = 5.05 min (% area
= 98.3 %).
4g: 1H NMR (CDCl3, 400 MHz) 8.52 (dd, J=4.21 Hz, J= 1.61 Hz; 1H, Q2); 7.91 (dd,
J=8.27 Hz, J=1.61 Hz; 1H, Q4); 7.79 to 7.71 (bs; 1H, -NH-CO-Fc); 7.30 (dd, J= 8.24
Hz and J= 4.22 Hz; 1H, Q3); 7.10 (t, J=5.03 Hz; 1H, -CH2-NH-CO-); 6.33 (d, J=2.49
Hz; 1H, Q5); 6.27 (d, J=2.47 Hz; 1H, Q7); 6.01 (d, J= 8.31 Hz; 1H, -NH-CH(CH3)-);
4.75 to 4.67 (m; 2H, Cpa2 and Cpa5); 4.41 to 4.36 (m; 1H, -CH(CH2-NH2)-); 4.35 to
4.33 (m; 2H, Cpa3 and Cpa4); 4.18 and 4.17 (s+s; 5H, Cpb); 3.88 (s; 3H, CH3-O-); 3.68
to 3.58 (m; 1H, -NH-CH(CH3)-); 3.42 to 3.22 (m; 4H, -CH2-NH-CO- and –CH2-NH2);
2.00 to 1.92 (bs; 2H, -CH2-NH2); 1.80 to 1.61 (m; 4H, -(CH2)2-CH2-); 1.30 (d, J=6.37
Hz; 3H, -CH(CH3)-). 13C NMR (CDCl3, 100 MHz) 171.2(-NH-CO-CH<); 170.99 (-NHCO-Fc); 159.4 (QC6); 144.9 (QC2); 144.3 (QC8); 135.3 (QC4); 134.8 (QC10); 129.9
(QC9); 121.8 (QC3); 96.7 (QC7); 91.6 (QC5); 75.3 (CpaC1); 70.6 (CpaC2-C5); 69.7
(Cpb); 68.3 (CpaC3-C4); 55.2 (CH3-O); 53.2 (-CH(CH2-NH2)-); 47.7 (-CH(CH3)-); 43.3(CH2-NH2); 39.3 (-CH2-NH-CO-); 33.8 (-CH2-CH2-CH2-); 26.2 (-CH2-CH2-CH2-); 20.5
(-CH(CH3)-). ESI-MS: [M+H]+ found: 558.2060 (C29H35FeN5O3 requires 557.46).
HPLC: tr = 4.45 min (% area = 99.3 %).
8: 1H NMR (CDCl3, 400 MHz) 8.52 (dd, J=4.22 Hz, J= 1.64 Hz; 1H, Q2); 7.91 (dd,
J=8.27 Hz, J=1.61 Hz; 1H, Q4); 7.29 (dd, J= 8.24 Hz and J= 4.22 Hz; 1H, Q3); 6.32 (d,
J=2.49 Hz; 1H, Q5); 6.28 (d, J=2.47 Hz; 1H, Q7); 6.04 (d, J= 8.19 Hz; 1H, -NH-
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CH(CH3)-); 4.08 (s; 5H, Cpb); 4.03 and 4.02 (d+d, J= 1.62 Hz and J= 1.64 Hz; 4H,
Cpa2 and Cpa5 + Cpa3 and Cpa4); 3.89 (s; 3H, CH3-O-); 3.67 to 3.58 (m; 1H, -NHCH(CH3)-); 2.64 (t, J= 7.04 Hz; 2H, -CH2-NH-); 2.58 (t, J= 7.20 Hz; 2H, -NH-CH2(CH2)5-); 2.32 to 2.28 (m; 2H, -CH2-CH2-(CH2)4-); 1.78 to 1.60 (m; 4H, -CH(CH3)(CH2)2-CH2-); 1.52 to 1.44 (m; 5H, -CH2-(CH2)2-CH2-Fc and –CH2-NH-CH2-); 1.35 to
1.27 (m; 7H, -CH(CH3)- and –(CH2)2-CH2-Fc).

C NMR (CDCl3, 100 MHz) 159.4
(QC6); 145.0 (QC2); 144.2 (QC8); 135.4 (QC4); 134.7 (QC10); 129.9 (QC9); 121.8
(QC3); 96.6 (QC7); 91.5 (QC5); 89.4 (CpaC1); 68.4 (Cpb); 68.0 (CpaC2-
C4-C5);
67.0
(CpaC3); 55.2 (CH3-O); 50.0 (-CH2-NH-CH2-); 48.0 (-CH(CH3)- and -CH2-Fc); 34.5 (CH2-CH2-CH2-NH-); 31.1 (-CH2-CH2-Fc); 30.1 (-NH-CH2-CH2-); 29.5 (-(CH2)3-CH2(CH2)2-Fc); 27.2 (-NH-CH2-CH2-CH2-); 26.7 (-CH2-CH2-CH2-NH-); 20.5 (-CH(CH3)-).
ESI-MS: [M+H]+ found 528.2667 (C31H41FeN3O requires 527.52). HPLC: tr = 10.95
min (% area = 98.7 %).
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150.00
Luminescence
140.00
AlamarBlue
120.00
100.00
80.00
100.00
60.00
50.00
40.00
20.00
0.00
Cell confluence (% of control)
Infection (% of control)
200.00
0.00
Figure S1. In vitro evaluation of primacene 8 concerning its (a) cytotoxicity towards Huh-7 cells
(AlamarBlue assay, cell confluence scale), and (b) activity against liver-stage P. berghei
parasites (luminescence measurements, % of infection scale).
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