SUPPORTING INFORMATION

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1 SUPPORTING INFORMATION Rotenoids, Flavonoids and Chalcones from the Root Bark of Millettia usaramensis ssp. usaramensis Tsegaye Deyou, Ivan Gumula, Fangfang Pang, Amra Gruhonjic, Michael Mumo, John Holleran, Sandra Duffy, Paul A. Fitzpatrick, Matthias Heydenreich, Göran Landberg, Solomon Derese, Vicky Avery, Kari Rissanen, Máté Erdélyi and Abiy Yenesew Table of contents S2 Spectroscopic data for 4-O-geranylisoliquiritigenin (1) S6 Spectroscopic data for 12-dihydrousararotenoid-B (2) S10 Spectroscopic data for 12-dihydrousararotenoid-C (3) S14 Spectroscopic data for 4'-O-geranyl-7-hydroxyflavanone (4) S17 Spectroscopic data for 4'-O-geranyl-7-hydroxydihydroflavonol (5) S21 Spectroscopic data for usararotenoid A (6) S25 Spectroscopic data for 12-dihydrousararotenoid A (7) S30 Spectroscopic data for millettosin (8) S35 Spectroscopic data for 12a-epimillettosin (9) S40 Spectroscopic data for usararotenoid C (10) S44 Spectroscopic data for jamaicin (11) S48 Spectroscopic data for 4'-O-geranylisoliquiritigenin (12) S53 Spectroscopic data for 7-O-geranyl-5-hydroxyflavanone (13) S56 Spectroscopic data for tephrosin (14) S61 Spectroscopic data for maximaisoflvone H (15) S64 Spectroscopic data for colenemol (16) S69 Spectroscopic data for 7-hydroxy-8,3',4'-trimethoxyisoflavone (17) S72 X-ray diffraction analyses of 6, 7 and 9. S74 Statistical analyses for the antiplasmodial and cytotoxicity assays S76 References S1

2 Figure S1. The I H NMR of 4-O-geranylisoliquiritigenin (1) observed at MHz for CDCl 3 solution at 25 C. Assignment is given in Table 1. Figure S2. The I3 C NMR of 4-O-geranylisoliquiritigenin (1) observed at MHz for CDCl 3 solution at 25 C. Assignment is given in Table 1. S2

3 Figure S3. The I H, 1 H COSY of 4-O-geranylisoliquiritigenin (1) observed at MHz for CDCl 3 solution at 25 C. Figure S4. The I H, 1 H TOCSY of 4-O-geranylisoliquiritigenin (1) observed at MHz for CDCl 3 solution at 25 C. S3

4 Figure S5. The I H, 1 H NOESY (mixing time 700 ms, d 1 =2.5 s) of 4-O-geranylisoliquiritigenin (1) observed at MHz for CDCl 3 solution at 25 C. Figure S6. The I H, 13 C HSQC of 4-O-geranylisoliquiritigenin (1) observed at MHz/ MHz for CDCl 3 solution at 25 C. S4

5 Figure S7. The I H, 13 C HMBC of 4-O-geranylisoliquiritigenin (1) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S8. The HRMSESI of 4-O-geranylisoliquiritigenin (1). S5

6 Figure S9. The 1 H NMR of 12-dihydrousararotenoid B (2) observed at MHz for CD 2 Cl 2 solution at 25 C. Assignment is given in Table 2. Figure S10. The I3C NMR of 12-dihydrousararotenoid B (2) observed at MHz for CD 2 Cl 2 solution at 25 C. Assignment is given in Table 2. S6

7 Figure S11. The I H, 1 H COSY of 12-dihydrousararotenoid B (2) observed at MHz for CD 2 Cl 2 solution at 25 C. Figure S12. The I H, 1 H NOESY (mixing time 1200 ms, d 1 =2.5 s) of 12-dihydrousararotenoid B (2) observed at MHz for CD 2 Cl 2 solution at 25 C. S7

8 Figure S13. The I H, 13 C HSQC of 12-dihydrousararotenoid B (2) observed at MHz/ MHz for CD 2 Cl 2 solution at 25 C. Figure S14. The 1 H, 13 C HMBC of 12-dihydrousararotenoid B (2) observed at MHz/ MHz for CD 2 Cl 2 solution at 25 C. S8

9 Relative Abundance m/z Figure S15. The HREIMS of 12-dihydrousararotenoid B (2). Figure S16. The CD curve of 12-dihydrousararotenoid B (2). S9

10 Figure S17. The 1 H NMR of 12-dihydrousararotenoid C (3) observed at MHz for acetone-d 6 solution at 25 C. Assignment is given in Table 2. Figure S18. The I3 C NMR of 12-dihydrousararotenoid C (3) observed at MHz for acetone-d 6 solution at 25 C. Assignment is given in Table 2. S10

11 Figure S19. The I H, 1 H COSY of 12-dihydrousararotenoid C (3) observed at MHz for acetone-d6 solution at 25 C. Figure S20. The 1 H, 1 H NOESY of 12-dihydrousararotenoid C (3) observed at MHz for acetone-d6 solution at 25 C (mixing time 1 sec, d1=4 sec). S11

12 Figure S21. The 1 H, 13 C HSQC of 12-dihydrousararotenoid C (3) observed at MHz / MHz for acetone-d6 solution at 25 C. Figure S22. The 1 H, 13 C HMBC of 12-dihydrousararotenoid C (3) observed at MHz / MHz for acetone-d6 solution at 25 C. S12

13 Relative Abundance m/z Heydenreich_64 # RT: AV: 181 NL: 3.56E4 T: + c Full ms [ ] Relative Abundance Figure S23. The HREIMS of 12-dihydrousararotenoid C (3). m/z Figure S24. The 1 H CD curve for 12-dihydrousararotenoid C (3). S13

14 Figure S25. The 1 H NMR of (S)-4'-O-geranyl-7-hydroxyflavanone (4) observed at MHz for CD 2 Cl 2 solution at 25 C. Assignment is given in Table 3. Figure 26. The 13 C NMR of (S)-4'-O-geranyl-7-hydroxyflavanone (4) observed at MHz for CD2Cl2 solution at 25 C. Assignment is given in Table 3. S14

15 Figure S27. The 1 H, 1 H COSY of (S)-4'-O-geranyl-7-hydroxyflavanone (4) observed at MHz for CD2Cl2 solution at 25 C. Figure S28. The 1 H, 13 C HSQC of of (S)-4'-O-geranyl-7-hydroxyflavanone (4) observed at MHz / MHz for CD2Cl2 solution at 25 C. S15

16 Figure S29. The 1 H, 13 C HMBC of (S)-4'-O-geranyl-7-hydroxyflavanone (4) observed at MHz / MHz for CD2Cl2 solution at 25 C. Figure S30. The HRMS of (S)-4'-O-geranyl-7-hydroxyflavanone (4). S16

17 Figure S31. The 1 H NMR of (2R,3R)-4'-O-geranyl-7-hydroxydihydroflavanol (5) observed at MHz for DMSO-d6 solution at 25 C. Assignment is given in Table 3. Figure S32. The 13 C NMR of (2R,3R)-4'-O-geranyl-7- hydroxydihydroflavanol (5) observed at MHz for DMSO-d6 solution at 25 C. Assignment is given in Table 3. S17

18 Figure S33. The 1 H, 1 H COSY of (2R,3R)-4'-O-geranyl-7- hydroxydihydroflavanol (5) observed at MHz for DMSO-d6 solution at 25 C. Figure S34. The 1 H, 1 H NOESY of (2R,3R)-4'-O-geranyl-7- hydroxydihydroflavanol (5) observed at MHz for DMSO solution at 25 C S18

19 Figure S35. The 1 H, 13 C HSQC of (2R,3R)-4'-O-geranyl-7- hydroxydihydroflavanol (5) observed at MHz / MHz for DMSO-d6 solution at 25 C. Figure S36. The 1 H, 13 C HMBC of (2R,3R)-4'-O-geranyl-7- hydroxydihydroflavanol (5) observed at MHz / MHz for DMSO-d6 solution at 25 C. S19

20 Figure S37. HRESI,MS of (2R,3R)-4'-O-geranyl-7- hydroxydihydroflavanol (5). Figure S38. The CD curve for (2R,3R)-4'-O-geranyl-7- hydroxydihydroflavanol (5). S20

21 Figure S39. The 1 H NMR of usararotenoid A (6) observed at MHz for DMSO-d 6 solution at 25 C. Figure S40. The I3 C NMR of usararotenoid A (6) observed at MHz for DMSO-d 6 solution at 25 C. S21

22 Figure S41. The I H, 1 H COSY of usararotenoid A (6) observed at MHz for DMSO-d 6 solution at 25 C. Figure S42. The I H, 1 H TOCSY of usararotenoid A (6) observed at MHz for DMSO-d 6 solution at 25 C. S22

23 Figure S43. The I H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of usararotenoid A (6) observed at MHz for DMSO-d 6 solution at 25 C. Figure S44. The I H, 13 C HSQC of usararotenoid A (6) observed at MHz/ MHz for DMSO-d 6 solution at 25 C. S23

24 Figure S45. The I H, 13 C HMBC of usararotenoid A (6) observed at MHz/ MHz for DMSO-d 6 solution at 25 C. Figure S46. LC-ESI-MS of usararotenoid A (6). S24

25 Figure S47. The CD curve for usararotenoid A (6). Figure S48. The 1 H NMR of 12-dihydrousararotenoid A (7) observed at MHz for DMSO-d 6 solution at 25 C. S25

26 Figure S49. The I3 C NMR of 12-dihydrousararotenoid A (7) observed at MHz for DMSO-d 6 solution at 25 C. Figure S50. The I H, 1 H COSY of 12-dihydrousararotenoid A (7) observed at MHz for DMSOd6 solution at 25 C. S26

27 Figure S51. The 1 H, 1 H TOCSY of 12-dihydrousararotenoid A (7) observed at MHz for DMSO-d6 solution at 25 C. Figure S52. The 1 H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of 12-dihydrousararotenoid A (7) observed at MHz for DMSO-d6 solution at 25 C. S27

28 Figure S53. The 1 H, 13 C HSQC of 12-dihydrousararotenoid A (7) observed at MHz/ MHz for DMSO-d6 solution at 25 C. Figure S54. The 1 H, 13 C HMBC of dihydrousararotenoid A (7) observed at MHz/ MHz for DMSO-d6 solution at 25 C. S28

29 Figure S55. The 1 H, 13 C HMBC (expanded) of dihydrousararotenoid A (7) observed at MHz/ MHz for DMSO-d6 solution at 25 C. Figure S56. LC-ESI-MS spectrum of 12-dihydrousararotenoid-A (7) S29

30 Figure S57. The CD curve for 12-dihydrousararotenoid-A (7). Figure S58. The I H NMR of millettosin (8) observed at MHz for DMSO-d6 solution at 25 C. S30

31 Figure S59. The 13 C NMR of millettosin (8) observed at MHz for CDCl 3 solution at 25 C. Figure S60. The 1 H, 1 H COSY of millettosin (8) observed at MHz for CDCl 3 solution at 25 C. S31

32 Figure S61. The 1 H, 1 H TOCSY of millettosin (8) observed at MHz for CDCl 3 solution at 25 C. Figure S62. The 1 H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of millettosin (8) observed at MHz for CDCl 3 solution at 25 C S32

33 Figure S63. The 1 H, 13 C HSQC of millettosin (8) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S64. The 1 H, 13 C HMBC of millettosin (8) observed at MHz/ MHz for CDCl 3 solution at 25 C S33

34 Figure S65. The 1 H, 13 C HMBC (expanded-i) of millettosin (8) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S66. The 1 H, 13 C HMBC (expanded-ii) of millettosin (8) observed at MHz/ MHz for CDCl 3 solution at 25 C. S34

35 Figure S67. The 1 H NMR of 12a-epimillettosin (9) observed at MHz for CDCl 3 solution at 25 C. Figure S68. The 13 C NMR of 12a-epimillettosin (9) observed at MHz for CDCl 3 solution at 25 C. S35

36 Figure S69. The 1 H, 1 H COSY of 12a-epimillettosin (9) observed at MHz for CDCl 3 solution at 25 C. Figure S70. The 1 H, 1 H TOCSY of 12a-epimillettosin (9) observed at MHz for CDCl 3 solution at 25 C. S36

37 Figure S71. The 1 H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of 12a-epimillettosin (9) observed at MHz for CDCl 3 solution at 25 C. Figure S72. The 1 H, 13 C HSQC of 12a-epimillettosin (9) observed at MHz/ MHz for CDCl 3 solution at 25 C. S37

38 Figure S73. The 1 H, 13 C HSQC of 12a-epimillettosin (9) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S74. The 1 H, 13 C HMBC 12a-epimillettosin (9) observed at MHz/ MHz for CDCl 3 solution at 25 C. S38

39 Figure S75. The 1 H, 13 C HMBC (expanded) of 12a-epimillettosin (9) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S76. LC-ESI-MS spectrum of 12a-epimillettosin (9) S39

40 Figure S77. The CD curve for 12a- epimillettosin (9) Figure S78. The 1 H NMR of usararotenoid C (10) observed at 500 MHz for CDCl 3 solution at 25 C. S40

41 Figure S79. The 13 C NMR of usararotenoid C (10) observed at 125 MHz for CDCl 3 solution at 25 C. Figure S80. The 1 H, 1 H COSY of usararotenoid C (10) observed at 500 MHz for CDCl 3 solution at 25 C. S41

42 Figure S81. The 1 H, 1 H NOESY of usararotenoid C (10) observed at 500 MHz for CDCl 3 solution at 25 C. Figure S82. The 1 H, 13 C HSQC of usararotenoid C (10) observed at 500 MHz/125 MHz for CDCl 3 solution at 25 C S42

43 Figure S83. The 1 H, 13 C HMBC usararotenoid C (10) observed at 500 MHz/125 MHz for CDCl 3 solution at 25 C. Figure S84. LC-ESI-MS spectrum usararotenoid C (10) S43

44 Figure S85. The 1 H NMR of jamaicin (11) observed at MHz for CDCl 3 solution at 25 C. Figure S86. The 13 C NMR of jamaicin (11) observed at MHz for CDCl 3 solution at 25 C. S44

45 Figure S87. The 1 H, 1 H COSY of jamaicin (11) observed at MHz for CDCl 3 solution at 25 C. Figure S88. The 1 H, 1 H TOCSY of jamaicin (11) observed at MHz for CDCl 3 solution at 25 C. S45

46 Figure S89. The 1 H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of jamaicin (11) observed at MHz for CDCl 3 solution at 25 C. Figure S90. The 1 H, 13 C HSQC of jamaicin (11) observed at MHz/ MHz for CDCl 3 solution at 25 C. S46

47 Figure S91. The 1 H, 13 C HMBC of jamaicin (11) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S92. LC-ESI-MS spectrum of jamaicin (11) S47

48 Figure S93. The I H NMR of 4'-O-geranylisoliquiritigenin (12) observed at 400 MHz for CDCl 3 solution at 25 C. Figure S94. The 13 C NMR of 4'-O-geranylisoliquiritigenin (12) observed at MHz for CDCl 3 solution at 25 C. S48

49 Figure S95. The 1 H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of 4'-O-geranylisoliquiritigenin (12) observed at MHz for CDCl 3 solution at 25 C. Figure S96. The HSQC of 4'-O-geranylisoliquiritigenin (12) observed at MHz/ MHz for CDCl 3 solution at 25 C S49

50 Figure S97. The HSQC (expanded-i) of 4'-O-geranylisoliquiritigenin (12) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S98. The HSQC (expanded-ii) of 4'-O-geranylisoliquiritigenin (12) observed at MHz/ MHz for CDCl 3 solution at 25 C. S50

51 Figure S99. The HMBC of 4'-O-geranylisoliquiritigenin (12) observed at MHz/ for CDCl 3 solution at 25 C. Figure S100. The HMBC (expanded-i) of 4'-O-geranylisoliquiritigenin (12) observed at MHz/ MHz for CDCl 3 solution at 25 C. S51

52 Figure S101. The HMBC (expanded-ii) of 4'-O-geranylisoliquiritigenin (12) observed at MHz/ for CDCl 3 solution at 25 C. Figure S102. LC-ESI-MS spectrum of 4'-O-geranylisoliquiritigenin (12). S52

53 Figure S103. The 1 H NMR of 7-O-geranyl-5-hydroxyflavanone (13), observed at 400 MHz for CDCl 3 solution at 25 C. Figure S104. The 1 H NMR (expanded) of 7-O-geranyl-5-hydroxyflavanone (13), observed at 400 MHz for CDCl 3 solution at 25 C. S53

54 Figure S105. The 13 C NMR of 7-O-geranyl-5-hydroxyflavanone (13) observed at 125 MHz for DMSO-d 6 solution at 25 C. Figure S106. The 1 H, 1 H COSY of 7-O-geranyl-5-hydroxyflavanone (13) observed at 400 MHz MHz for CDCl 3 solution at 25 C. S54

55 Figure S107. The 1 H, 1 H NOESY of 7-O-geranyl-5-hydroxyflavanone (13) observed at 400 MHz for CDCl 3 solution at 25 C. Figure S108. The 1 H, 13 C HSQC of 7-O-geranyl-5-hydroxyflavanone (13) observed at 400/125 MHz at 25 C. S55

56 Figure S109. The 1 H, 13 C HMBC of 7-O-geranyl-5-hydroxyflavanone (13), observed at 400/125 MHz at 25 C. Figure S110. The 1 H NMR of tephrosin (14), observed at MHz for CDCl 3 solution at 25 C. S56

57 Figure S111. The 13 C NMR of tephrosin (14) observed at MHz for CDCl 3 solution at 25 C. Figure S112. The 1 H, 1 H COSY of tephrosin (14) observed at MHz for CDCl 3 solution at 25 C. S57

58 Figure S113. The 1 H, 1 H TOCSY of tephrosin (14) observed at MHz for CDCl 3 solution at 25 C. Figure S114. The 1 H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of tephrosin (14) observed at MHz for CDCl 3 solution at 25 C. S58

59 Figure S115. The 1 H, 13 C HSQC of tephrosin (14) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S116. The 1 H, 13 C HMBC of tephrosin (14) observed at MHz/ MHz for CDCl 3 solution at 25 C. S59

60 Figure S117. The 1 H, 13 C HMBC (expanded) of tephrosin (14) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S118. LC-ESI-MS spectrum of tephrosin (14). S60

61 Figure S119. The 1 H NMR of maximaisoflavone H (15), observed at MHz for CDCl 3 solution at 25 C. Figure S120. The 1 H NMR of maximaisoflavone H (15), observed at MHz for CDCl 3 solution at 25 C. S61

62 Figure S121. The 1 H, 1 H COSY of maximaisoflavone H (15) observed at MHz for CDCl 3 solution at 25 C. Figure S122. The 1 H, 1 H TOCSY of maximaisoflavone H (15) observed at MHz for CDCl 3 solution at 25 C. S62

63 Figure S123. The 1 H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of maximaisoflavone H (15) observed at MHz for CDCl 3 solution at 25 C. Figure S124. The 1 H, 13 C HSQC of maximaisoflavone H (15) observed at MHz/ MHz for CDCl 3 solution at 25 C. S63

64 Figure S125. The 1 H, 13 C HMBC of maximaisoflavone H (15) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S126. The 1 H NMR of colenemol (16) observed at MHz for CDCl 3 solution at 25 C. S64

65 Figure S127. The 13 C NMR of colenemol (16) observed at MHz for CDCl 3 solution at 25 C. Figure S128. The 1 H, 1 H COSY of colenemol (16) observed at MHz for CDCl 3 solution at 25 C S65

66 Figure S129. The 1 H, 1 H TOCSY of colenemol (16) observed at MHz for CDCl 3 solution at 25 C. Figure S130. The 1 H, 13 C HSQC of colenemol (16) observed at MHz/ MHz for CDCl 3 solution at 25 C. S66

67 Figure S131. The 1 H, 13 C HMBC of colenemol (16) observed at MHz/ MHz for CDCl 3 solution at 25 C. Figure S132. The 1 H, 13 C HMBC spectrum (expansion-i) of colenemol (16) observed at MHz/ MHz for CDCl 3 solution at 25 C. S67

68 Figure S133. The 1 H, 13 C HMBC spectrum (expansion-ii) of colenemol (16) observed at MHz/ MHz for CDCl 3 solution at 25 C. [ ] Relative Abundance m/z Figure S134. Mass spectrum of colenemol (16). S68

69 Figure S135. The 1 H NMR of 7-hydroxy-8,3',4'-trimethoxyisoflavone (17) observed at MHz for DMSO-d 6 solution at 25 C. Figure S136. The 13 C NMR of 7-hydroxy-8,3',4'-trimethoxyisoflavone (17) observed at MHz for DMSO-d 6 solution at 25 C. S69

70 Figure S137. The 1 H, 1 H COSY of 7-hydroxy-8,3',4'-trimethoxyisoflavone (17) observed at MHz for DMSO-d 6 solution at 25 C. Figure S138. The 1 H, 1 H NOESY (mixing time 700ms, d 1 =2.5 s) of 7-hydroxy-8,3',4'- trimethoxyisoflavone (17) observed at MHz for DMSO-d 6 solution at 25 C. S70

71 Figure S139. The 1 H, 13 C HSQC of 7-hydroxy-8,3',4'-trimethoxyisoflavone (17) observed at MHz/ MHz for DMSO-d 6 solution at 25 C. Figure S140. The 1 H, 13 C HMBC of 7-hydroxy-8,3',4'-trimethoxyisoflavone (17) observed at MHz/ MHz for DMSO-d 6 solution at 25 C. S71

72 X-ray diffraction analysis Single crystals were obtained by slow diffusion of tetrahydropyrane into a 3:1 mixture of dichloromethane/acetonitrile solution of the complex. X-ray data for usararotenoid A (6) and 12a- Epimillittosin (9) were collected on an Agilent SuperNova Dual diffractometer with Atlas detector at T = 123.0(1) K using mirror-monochromatized Cu-Kα radiation (λ = Å). CrysAlisPro 1 software was used for data collection, integration and reduction as well as applying the Analytical numeric absorption correction using a multifaceted crystal model 2. For 12-dihydrousararotenoid A (7), the X-ray data collection was performed on an Bruker-Nonius KappaCCD diffractometer with an APEX-II detector at 173.0(1) K using graphite monochromatorized Mo-Kα radiation (λ = Å), where, the data collection and reduction were done using the program COLLECT 3 and HKL DENZO AND SCALEPACK 4, respectively. The intensities were corrected for absorption using SADABS 5.The structures were solved by Direct method with SHELXS 6 and refined by full-matrix least-squares using SHELXL within OLEX2 7 package. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms in usararotenoid A (6) and 12a-Epimillittosin (9) were located from difference Fourier maps and their positions and thermal parameters were refined freely. All the hydrogen atoms in 12- dihydrousararotenoid A (7) were refined using riding models with U eq (H) of 1.5U eq (parent) for terminal groups and 1.2 U eq (parent) for non-terminal groups. Usararotenoid A (6): mm, C 18 H 12 O 8, M = , orthorhombic, space group P , a = (7) Å, b = (11) Å, c = (12) Ǻ, α = 90, β = 90, γ = 90, V = (2) Ǻ 3, Z = 4, ρ = g cm 3, μ = mm 1, F(000) = 736, reflections (θ max = ) measured (3023 unique, R int = , completeness = 99.7%), Final R indices (I > 2σ(I)): R 1 = , wr 2 = , R indices (all data): R 1 = , wr 2 = GOF = for 283 parameters and 0 restraints, largest diff. peak and hole 0.218/ eǻ 3. CCDC contains the supplementary data for this structure. An ORTEP image of the molecule of usararotenoid A is shown below. 12-dihydrousararotenoid A (7): mm, C 18 H 14 O 8, M = , monoclinic, space group P2 1, a = (19) Å, b = (15) Å, c = (2) Ǻ, α = 90, β = (3), γ = 90, V = 736.9(3) Ǻ 3, Z = 2, ρ = g cm 3, μ = mm 1, F(000) = 372, 4298 reflections (θ max = ) measured (3291 unique, R int = , completeness = 91.0%), Final R indices (I > 2σ(I)): R 1 = , wr 2 = , R indices (all data): R 1 = , wr 2 = GOF = for 237 parameters and 1 restraints, largest diff. peak and hole 0.241/ eǻ 3. CCDC contains the supplementary S72

73 data for this structure. An ORTEP image of the molecule of 12-dihydrousararotenoid A is shown below. 12a-Epimillittosin (9): mm, C 22 H 18 O 7, M = , monoclinic, space group P2 1, a = (12) Å, b = (15) Å, c = (19) Ǻ, α = 90, β = (16), γ = 90, V = (2) Ǻ 3, Z = 2, ρ = g cm 3, μ = mm 1, F(000) = 412, reflections (θ max = ) measured (3580 unique, R int = 0.022, completeness = 99.5%), Final R indices (I > 2σ(I)): R 1 = , wr 2 = , R indices (all data): R 1 = , wr 2 = GOF = for 334 parameters and 1 restraints, largest diff. peak and hole 0.190/ eǻ 3. CCDC contains the supplementary data for this structure. An ORTEP image of 12a-epimillittosin is shown below. These data can be obtained free of charge via or by ing or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: Note: The X-ray structures of Compound 6, in CCDC as ITEGET and Compound 7, in CCDC as ITEGIX have been published earlier. [8] S73

74 Statistical analyses for the antiplasmodial and cytotoxicity assays Standard deviations for the antiplasmodial (chloroquine sensitive 3D7 and chloroquine resistant Dd2 Plasmodium falciparum strains), and cytotoxicity (HEK-293) assays. The IC 50 values are given in nm. 3D7 3D7 3D7 3D7 Dd2 Dd2 Dd2 Dd2 HEK293 HEK293 n=1 n=2 average stdev n=1 n=2 average stdev n=1 n=2 Pyrimethamine % at 40uM 63% at 40uM 62% at 40uM % at 40uM Chloroquine % at 40uM 42% at 40uM Pyronaridine Puromycin Artesunate % at 20uM 71% at 20uM DHA % at 20uM 58% at 20uM Compound % at 40uM 100% at 40uM Compound 6 98% at 100uM 99% at 100uM 99% at 100uM 89% at 100uM 90% at 100uM 90% at 100uM Compound 9 36% at 100uM 44% at 100uM 28% at 100uM 44% at 100uM 14% at 100uM 29% at 100uM Compound 11 93% at 100uM 94% at 100uM 94% at 100uM 81% at 100uM 81% at 100uM 81% at 100uM Compound S74

75 Confidence intervals for the cytotoxicity data (MDB-MB-231 cells). Sample IC 50 MDB-MB % CE um ug/ml ug/ml Usararotenoid A (6) '-O-Geranylisoliquiritigenin (12) E E 5 Millettosin (8) a-Epimillettosin (9) Usararotenoid C (10) E E 5 12-Dihydrousararotenoid A (7) >279.3 >100 - Colenemol (16) S75

76 References [1] CrysAlisPro, ed., Agilent Technologies, Ltd., Yarton, UK, [2] R. C. Clark, J. S. Reid, Acta Crystallogr., Sect. A: Found. Crystallogr., 1995, 51, [3] Bruker AXS BV, Madison, WI, USA; [4] Z. Otwinowski, W. Minor, Methods Enzymol. 1997, 276, [5] R.H. Blessing, J. Appl. Cryst. 1997, 30, [6] G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2008, 64, 112. [7] O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard and H. Puschmann J. Appl. Cryst. 2009, 42, [8] Yenesew, A., Derese, S.; Midiwo, J. O.; Oketch-Rabah, H.A.; Lisgarten, J.; Palmer, R.; Meydenreich, M.; Peter, M.G.; Akala, H.; Wangui, J.; Liyiala, P.; Waters, N. C. Phytochemistry 2003, 64, S76