SUPPORTING INFORMATION

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SUPPORTING INFORMATION

1. Reagents and general experimental procedure S2

2. 1_{H NMR spectrum (DMSO) of 17} _S3

3. 13_{C NMR spectrum (DMSO) of 17} _S4

4. COSYspectrum of (DMSO) of 17 S5

5. HSQC spectrum of (DMSO) of 17 S6

6. HMBC spectrum of (DMSO) of 17 S7

7. ESI mass spectrum of 17 S8

8. 1_{H NMR spectrum (DMSO) of 20} _S9

9. 13_{C NMR spectrum (DMSO) of 20} _S10

10. COSYspectrum of (DMSO) of 20 S11

11. HSQC spectrum of (DMSO) of 20 S12

12. HMBC spectrum of (DMSO) of 20 S13

13. ESI mass spectrum of of 20 S14

14.1_{H NMR spectrum (DMSO) of 18} _S15

15.13_{C NMR spectrum (DMSO) of 18} _S16

19.1_{H ESI mass spectrum of of 21} _S20

26. Purity criteria for tested compounds S27

27. Table 1SI S28 28. Table 2SI S29 29. Table 3SI S30 30. Figure 1SI S31 31. Figure 2SI S32 32. Figure 3SI S33

Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2015

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S2

Reagents and general experimental procedure

Commercial reagents: Sigma–Aldrich. Solvents: Carlo Erba. TLC: Silica Gel 60 F254 (plates 5 x 20, 0.25 mm) Merck. Preparative TLC: Silica Gel 60 F254 plates (20 x 20, 2 mm). Spots revealed by UV lamp then by spraying with 2 N sulfuric acid and heating at 120 °C. ‘Acidic’ silica gel was prepared by treating Silica Gel 60 Merck with 1 N HCl for 24 h, washing with water until the chlorine test was negative, activating for 48 h at 120 °C, then equilibrating with 10% of water. Anhydrous solvents: Sigma–Aldrich or prepared by distillation according to standard procedures. ESI mass spectra were performed on a hybrid quadrupole-TOF mass spectrometer, dissolving the sample in MeOH. The spectra were recorded by infusion into the ESI source using MeOH as the solvent. EI mass spectra were obtained on GC-MS HP 5890, HP 5971A Mass selective detector. 1_H

(700 MHz) and 13_{C (175 MHz) NMR spectra were recorded on a Varian INOVA spectrometer}

respectively; chemical shifts were referenced to the residual solvent signal (CDCl3: δH=7.26,

δC=77.0). For an accurate measurement of the coupling constants, the one-dimensional 1H NMR

spectra were transformed at 64 K points (digital resolution: 0.09 Hz). Homonuclear (1_H-1_{H) and}

heteronuclear (1_H-13_{C) connectivities were determined by COSY and HSQC experiments,}

respectively. Two and three bond 1_H-13_{C connectivities were determined by gradient 2D HMBC}

experiments optimized for a 2,3_{J of 8 Hz. Routine}1_{H and}13_{C NMR spectra were recorded with a}

Varian Gemini 200 MHz or a Varian Mercury 300 MHz or a Bruker Avance 400 MHz. High performance liquid chromatography (HPLC) separations were achieved on a Knauer 501 apparatus equipped with an RI detector.

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1

_{H NMR in DMSO of 17}

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S4

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S6

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H R E S I N 1 + _ 1 4 1 1 1 0 1 3 1 2 5 2 # 9 8 R T : 0 ,7 5 A V : 1 N L : 6 ,7 4 E 7 T : F T M S + c E S I F ull m s [ 3 0 0 ,0 0 -6 0 0 ,0 0 ] 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0 5 6 0 5 8 0 6 0 0 m /z 0 5 10 15 20 52 30 35 40 45 05 55 60 65 70 75 80 85 90 95 1 0 0 Relative Abundance 3 6 3 ,0 9 8 2 3 7 9 ,0 7 2 1 3 5 8 ,1 4 2 8 3 4 1 ,1 1 6 3 5 2 6 ,3 3 0 3 5 5 8 ,3 2 0 1 3 0 5 ,1 5 6 9 4 8 7 ,2 4 4 6 4 6 5 ,1 4 7 2 5 8 0 ,3 0 2 0 3 9 3 ,2 9 7 2 5 1 4 ,2 9 3 7 4 1 3 ,2 6 5 9 4 2 9 ,3 1 8 0 3 2 8 ,2 4 5 4

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S8

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S10

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S12

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S13

ESI mass spectrum of 20

H R E S I N 1 + _ 1 4 1 1 1 0 1 3 1 2 5 2 # 8 8 R T : 0 ,6 7 A V : 1 N L : 6 ,7 1 E 7 T : F T M S + c E S I F ull m s [ 3 0 0 ,0 0 -6 0 0 ,0 0 ] 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 m /z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 08 85 90 95 100 Relative Abundance 3 6 3 ,0 9 8 4 3 7 9 ,0 7 2 3 3 5 8 ,1 4 3 0 3 4 1 ,1 1 6 5 4 8 7 ,2 4 5 1 3 6 8 ,0 7 6 0 4 9 8 ,2 9 9 1 4 6 5 ,1 4 7 5 3 9 3 ,2 9 7 7 5 1 4 ,2 9 4 1 3 ,2 6 6 0 3 1 8 ,3 0 0 2 4 2 3 ,1 3 6 8 3 8 5 ,2 9 2 1 4 4 9 ,3 6 0 0 3 2 8 ,2 4 5 4

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S14

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S16

ESI mass spectrum of 18

H R E S I N 4 + _ 1 4 1 1 1 0 1 3 1 2 5 2 # 2 0 R T : 0 ,1 5 A V : 1 N L : 2 ,0 5 E 7 T : F T M S + c E S I F ull m s [ 1 5 0 ,0 0 -6 0 0 ,0 0 ] 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0 5 6 0 5 8 0 6 0 0 m /z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 08 85 90 95 100 Relative Abundance 4 0 5 ,1 4 5 1 3 8 3 ,1 6 3 2 5 6 7 ,2 7 0 6 4 1 0 ,1 2 2 7 4 0 0 ,1 8 9 9 3 2 2 ,0 7 1 7 4 2 1 ,1 1 9 1 5 4 0 ,3 4 6 1 5 1 2 ,3 1 4 9 4 4 2 ,2 3 6 7 5 9 3 ,7 1 5 9 4 6 3 ,1 8 6 9 3 0 5 ,1 5 6 9 5 5 9 ,6 6 3 1 4 8 4 ,1 9 7 1 3 6 8 ,5 8 5 7 3 4 9 ,1 8 2 9 5 7 3 ,3 4 1 0

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S18

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S19

ESI mass spectrum of 21

H R E S I N 4 + _ 1 4 1 1 1 0 1 3 1 2 5 2 # 1 2 8 R T : 0 ,9 9 A V : 1 N L : 2 ,3 2 E 7 T : F T M S + c E S I F ull m s [ 1 5 0 ,0 0 -6 0 0 ,0 0 ] 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0 m /z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 08 85 90 95 100 Relative Abundance 4 0 5 ,1 4 4 6 3 8 3 ,1 6 2 8 4 1 0 ,1 2 2 2 4 0 0 ,1 8 9 3 3 2 2 ,0 7 1 3 4 2 1 ,1 1 8 3 5 4 0 ,3 4 5 1 2 ,3 1 4 1 4 6 3 ,1 8 6 2 4 4 2 ,2 3 5 9 3 0 5 ,1 5 6 6 3 6 8 ,5 8 5 6 4 8 4 ,1 9 6 6 3 4 8 ,3 4 6 4 3 9 5 ,1 4 4 5

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S20

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S22

ESI mass spectrum of 19

N ic o le tti II H P L C 1 1 _ 1 2 _ 0 9 F r5 N 6 H R + _ 1 5 0 1 1 2 1 5 5 6 4 7 # 8 3 R T : 0 ,6 5 A V : 1 N L : 1 ,4 0 E 7 T : F T M S + c E S I F ull m s [ 1 0 0 ,0 0 -1 4 0 0 ,0 0 ] 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 0 0 1 0 5 0 m /z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 08 85 90 95 100 Relative Abundance 4 8 9 ,2 3 9 7 9 5 5 ,4 9 1 5 4 1 3 ,2 6 6 7 5 0 5 ,2 1 3 7 8 7 9 ,5 1 8 1 5 2 6 ,3 3 1 5 3 9 9 ,3 5 8 6 8 0 3 ,5 4 4 5 9 7 1 ,4 6 1 6 4 6 7 ,2 5 8 0 4 2 7 ,3 9 0 1 7 1 9 ,3 5 6 6 6 0 0 ,6 5 4 2 5 6 1 ,3 9 7 6 6 4 9 ,4 5 0 0 8 9 3 ,6 4 2 3 9 3 1 ,4 8 8 2 7 6 3 ,4 9 3 7 1 0 8 7 ,5 3 1 1

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S24

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S25 N ic o le tti H P L C 9 _ 1 2 _ 0 9 F r3 N 5 H R + _ 1 5 0 1 1 2 1 5 5 6 4 7 # 1 5 R T : 0 ,1 1 A V : 1 N L : 4 ,9 4 E 7 T : F T M S + c E S I F ull m s [ 1 0 0 ,0 0 -1 0 0 0 ,0 0 ] 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 8 5 0 9 0 0 9 5 0 1 0 m /z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 08 85 90 95 100 Relative Abundance 4 8 9 ,2 3 9 6 9 5 5 ,4 9 1 3 4 1 3 ,2 6 6 7 5 0 5 ,2 1 3 6 8 7 9 ,5 1 7 9 9 7 1 ,4 6 5 6 8 0 3 ,5 4 4 6 8 6 8 ,5 1 3 5 4 2 9 ,2 4 0 2 5 2 6 ,3 3 1 6 7 0 0 ,4 5 7 9 4 7 3 ,3 4 5 7 5 6 1 ,3 9 7 6 6 0 1 ,1 9 6 0 6 5 2 ,4 7 2 0 9 0 7 ,5 5 0 2 7 6 3 ,4 9 2 1 9 2 7 ,5 8 9 9 9 7 9 ,6 6

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S26

Purity criteria for tested compounds.

a _{The degree of purity of compounds 11-22 was measured by silica gel high performance liquid chromatography} (HPLC). The chromatography data were obtained on a SiO2 column (Luna 3 µm, 150 x 4.60 mm) eluting with

EtOAc/hexane 6:4 (v/v) the compounds 11-16 (flow rate of 1 mL/min) and eluting with EtOAc/hexane 7:3 (v/v) the compounds 17-22 (flow rate of 1 mL/min).

b _{Retention times (t}

R) are expressed in mintes

Compounds Puritya _t Rb Compounds Puritya tRb 11 95.8% 13.9 17 97.2% 9.1 12 96.8% 13.1 18 97.6% 7.4 13 97.1% 12.1 19 96.8% 5.7 14 96.5% 23.7 20 97.3% 22.3 15 95.8% 24.3 21 97.7% 18.3 16 95.5% 21.1 22 96.5% 14.5

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Table 1. NMR data of compounds 18/21, 19/22.

18 21 19 22

Pos. _

C _{(J in Hz)}H, mult. C _{(J in Hz)}H, mult. C _{(J in Hz)}H, mult. C _{(J in Hz)}H, mult.

1 - - - -2 48.8 3.34 m 48.6 3.33 m 48.8 3.34 m 48.7 3.33 m 3 40.0 3.80 m 39.9 3.76 m 40.0 3.80 m 39.5 3.76 m 4 - 9.05, brs - 9.06, brs - 9.05, brs - 9.06, brs 4a 146.1 - 145.7 - 145.0 - 144.5 -5 180.6 - 176.7 - 179.8 - 175.7 -6 137.9 - 137.1 - 136.0 - 136.1 -7 140.7 - 148.1 - 139.7 - 145.5 -8 173.1 - 176.6 - 172.1 - 175.6 -8a 108.4 - 109.6 - 107.2 - 108.5 -1’ 26.0 2.24, t (7.5) 25.5 2.31, t (7.5) 26.0 2.24, t (7.5) 25.5 2.31, t (7.5) 2’ 26.8 1.30 28.0 1.28 27.1 1.30 27.9 1.30 3’ 31.7 1.20 31.8 1.20 29.0 1.20 29.0 1.20 4’ 22.2 1.19 22.1 1.19 29.2 1.19 29.2 1.19 5’ 14.2 0.80, t (7.5) 14.2 0.80, t (7.5) 29.3 1.19 29.3 1.19 6' - - - - 29.4 1.19 29.4 1.19 7’ - - - - 31.7 1.19 31.8 1.19 9’ - - - - 22.5 1.20 22.3 1.20 10’ - - - - 14.4 0.81, t (7.5) 14.3 0.81, t (7.5) 1’’ 177.2 - 177.9 - 177.1 - 177.9 -2’’ 39.1 - 39.0 - 39.1 - 39.0 -Me 27.4 1.22 27.3 1.22 27.4 1.21 27.3 1.21 NH - 8.90, brs - 9.00, brs - 8.88, brs - 9.00, brs

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S28

Table 2SI. Prevalent ionic forms of new thiazinoquinones. Compounds Prevalent ionic form (%)a

pH 7.2 pH 5.5

12 N(100) N(100)

15 N(100) N(100)

19 N(100) N(100)

22 N(100) N(100)

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Table 3SI. Inhibition of BH formation by chloroquine and test compounds Compounds % inhibition BH formation*

Hemin/ compound ratios 1:0.5 1:1 1:2 1:4 1:8 1:16 17 0 1.01 1.78 3.57 14.6 19.15 20 0 0 1.04 8.91 13.68 25.31 22 0 0 1.12 7.47 12.15 CQ 0 44.11 89.46 80.02 90.83

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S30

Figure 1SI. QH

i (top) and QHii (bottom) species of 12 GM (green; A) and 15 GM (magenta; B).

The ligands are colored by atom type (O = red, N = blue, S = yellow and H = white). Hydrogen bonds are highlighted by green dashed lines. Hydrogens are omitted for the sake of clarity with the exception of those involved in hydrogen bonds.

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Figure 2SI. QH

i (top) and QHii (bottom) species of 19 GM (magenta; A) and 22 GM (green; B).

The ligands are colored by atom type (O = red, N = blue, S = yellow and H = white). Hydrogen bonds are highlighted by green dashed lines. Hydrogens are omitted for the sake of clarity with the exception of those involved in hydrogen bonds.

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S32

Figure 3SI. PM7 GM conformer of 12 (green; A); PM7 GM conformer of 15 (magenta; B). The

ligands are colored by atom type (O = red, N = blue, S = yellow and H = white). Hydrogens are omitted for the sake of clarity with the exception of those of amine function.