S1

SUPPORTING INFORMATION:Small molecule anticancer agents kill cancer cells by

harnessing reactive oxygen species in an iron-dependent manner

Sara R. Fedorkaa, Kevin Soa, Ayad Al-Hamashia, Ibtissam Gadb, Ronit Shahb, Veronika

Kholodovychb, Hanan D. Alqahtanib, William R. Taylorb* and L. M. Viranga Tillekeratnea*

aDepartment of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, 2801, W. Bancroft Street, Toledo, Ohio 43606, United Statesb Department of Biological Sciences, College of Natural Sciences and Mathematics, The University of Toledo, 2801, W. Bancroft Street, Toledo, Ohio 43606, United States*These authors contributing equally to this work and are co-corresponding authors.*These authors contributing equally to this work and are co-corresponding authors.

ltillek@utnet.utoledo.eduwilliam.taylor3@utoledo.edu

List of Contents:Page number

1H and 13C NMR Spectra1) (5R, 6S) and (5S, 6R)-6-hydroxy-3,3,5,7-tetramethyl-oct-1-en-4-one ((+/-) 8) S32) (4R,5S)-5-(tert-butyldimethylsilyloxy)-2,2,4,6-tetramethyl-3-oxoheptanal ((+/-)9) S43) (3S,4R,7S)-3,7-bis-(tert-butyldimethylsilyloxy)-2,4,6,6-tetramethyldec-9-en-5-one

and (3R,4S,7S)- 3,7-bis-(tert-butyldimethylsilyloxy)-2,4,6,6-tetramethyldec-9-en-5-one (10 and 10a) S5

4) (3S,6R,7S)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal (11) S6

5) (3S,6S,7R)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal (11a) S76) (3S,6R,7S)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanoic acid (7) S87) (3S,6S,7R)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanoic acid (7a) S9

8) 2-methyl-4-bromothiazole (13) S109) 2-(methylthio)-4-bromothiazole (14) S11

10) 2-(piperidin-1-yl)-4-bromothiazole (15) S1211) 2-(phenylethynyl)-4-bromothiazole (16) S1312) trimethylsilyl)ethynyl)-4-bromothiazole (17) S1413) 2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enone (20a) S15

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2018

S2

14) 2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enone (20b) S1615) 2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enone (20c) S1716) 2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enone (20d) S1817) 2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone (20e) S1918) 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone (21) S2019) 3-(2-(1H-1,2,3-triazol-4-yl)thiazol-4-yl)-2-methylcyclopent-2-enone (22) S2120) 2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enone (20f) S2221) (S)-2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enol (6a) S2322) (S)-2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enol (6b) S2423) 2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enol (6c) S2524) (S)-2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enol (6d) S2625) (S)-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enol (6e) S2726) (S)-2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enol (6f) S2827) (3S,6R,7S)-((S)-2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-

4,4,6,8-tetramethyl-5-oxononanoate (5a) S2928) (3S,6R,7S)-((S)-2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enyl) 3,7-

dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5b) S3029) (3S,6R,7S)-((R)-2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-

dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate and (3S,6R,7S)-((S)-2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5c) S31

30) (3S,6R,7S)-((S)-2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5d) S32

31) (3S,6R,7S)-((S)-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5e) S33

32) (3S,6R,7S)-((S)-2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5f) S34

33) (R)-3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enol (23) S35

34) 2-Methyl-3-(2-((trimethylsilyl)ethynyl)thiazole-4-yl)cyclopent-2-enol (24) S36

35) 3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enone oxime (25) S37

36) Supplementary Figure S1. Initial assessment toxicity induced by S38

compound 5e and analogues

37) Supplementary Figure S2. Time lapse microscopy of NCI-H522 cells exposed to 6e S39.

38) Supplementary Figure S3. Effect of compound 6e (10M) on mitochondrial function S39

S3

1H and 13C NMR Spectra

1H NMR of (5R, 6S) and (5S, 6R)-6-hydroxy-3,3,5,7-tetramethyl-oct-1-en-4-one ((+/-) 8)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

6.876.82 3.162.042.02 1.071.00

13C NMR of (5R, 6S) and (5S, 6R)-6-hydroxy-3,3,5,7-tetramethyl-oct-1-en-4-one ((+/-) 8)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

219.

02

141.26

115.

33

77.5

477

.22

76.9

1

76.66

51.7

0

40.76 30.63

23.1822.93

19.02

10.89

(+/-)O

HO

8

S4

1H NMR of (4R,5S)-5-(tert-butyldimethylsilyloxy)-2,2,4,6-tetramethyl-3-oxoheptanal ((+/-)9)

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

15.78 6.766.481.28 1.251.00

13C NMR of (4R,5S)-5-(tert-butyldimethylsilyloxy)-2,2,4,6-tetramethyl-3-oxoheptanal ((+/-)9)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

212.

63

200.72

77.5

477

.23

77.12

76.9

1

61.3

6

46.36

33.34

20.6219.87

18.6

2

16.40 15.70

-3.43

O

TBSO

O

H

(+/-)9

S5

1H NMR of (3S,4R,7S)-3,7-bis-(tert-butyldimethylsilyloxy)-2,4,6,6-tetramethyldec-9-en-5-one and (3R,4S,7S)- 3,7-bis-(tert-butyldimethylsilyloxy)-2,4,6,6-tetramethyldec-9-en-5-one (10 and 10a)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5ppm

24.76 10.862.992.18 1.881.051.00 0.99 0.800.60

13C NMR of (3S,4R,7S)-3,7-bis-(tert-butyldimethylsilyloxy)-2,4,6,6-tetramethyldec-9-en-5-one and (3R,4S,7S)- 3,7-bis-(tert-butyldimethylsilyloxy)-2,4,6,6-tetramethyldec-9-en-5-one (10 and 10a)

\240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

219.

0921

8.48

137.02

116.

50

77.85

77.5

477

.23

76.9

1

76.2275.34

54.4

854

.26

45.9945.43

39.8

139

.32

33.23 32.91

26.4826.31

24.9623.17

21.50

18.8

1

18.50 16.02

-3.13-3.46 -3.75

O

TBSO

OTBS

10, (3S, 4R)10a, (3R, 4S)

**

3

S6

1H NMR of (3S,6R,7S)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal (11)

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

14.60 12.613.412.251.25 1.221.161.00 0.95

13C NMR of (3S,6R,7S)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal (11)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

-4.2

2-4

.00

-3.5

0-3

.23

16

.04

16

.61

18

.31

18

.76

19

.44

21

.31

22

.71

26

.13

26

.43

33

.254

5.4

4

53

.92

70

.41

76

.90

77

.23

77

.54

77

.84

20

1.5

3

21

8.1

6

O

TBSO

OTBS

H

O

11

S7

1H NMR of (3S,6S,7R)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal (11a)

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

21.10 16.096.961.13 1.111.00 0.730.72

13C NMR of (3S,6S,7R)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal (11a)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

-3.4

8-3

.18

15

.85

16

.04

18

.27

18

.72

18

.74

21

.48

24

.06

26

.08

26

.43

32

.854

5.9

8

53

.73

71

.18

76

.90

77

.23

77

.54

77

.80

20

1.1

6

21

8.7

4

O OTBS

H

O

11a

TBSO

S8

1H NMR of (3S,6R,7S)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanoic acid (7)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5ppm

12.04 11.042.981.041.021.011.001.00

13C NMR of (3S,6R,7S)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanoic acid (7)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

217.

92

177.

37

78.00

77.5

4

76.9

0

72.36

54.1

2

45.49

39.8

7

33.24

26.4726.21

22.39 21.3719.27

18.8

018

.40

16.5716.09

-3.20-3.47-4.19

O

TBSO

OTBS

OH

O7

S9

1H NMR of (3S,6S,7R)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanoic acid (7a)

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5ppm

14.60 10.564.763.341.121.05 1.021.00 0.98

13C NMR of (3S,6S,7R)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanoic acid (7a)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

218.

56

178.

48

77.87

77.5

477

.22

76.9

1

73.37

53.8

2

46.08

40.4

2

32.92

29.9

4

26.4826.22

23.75 21.5519.00

18.8

118

.43

16.2215.88

-3.13 -3.45-4.03-4.40

O

TBSO

OTBS

OH

O7a

S10

1H NMR of 2-methyl-4-bromothiazole (13)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

3.000.94

13C NMR of 2-methyl-4-bromothiazole (13)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

19

.41

77

.23

77

.55

77

.87

11

6.2

9

12

3.9

3

16

7.1

9N

S CH3

Br 13

S11

1H NMR of 2-(methylthio)-4-bromothiazole (14)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

3.191.00

13C NMR of 2-(methylthio)-4-bromothiazole (14)

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

168.

09

124.

41

115.6677

.54

77.2

376

.90

16.84N

S S

Br 14

S12

1H NMR of 2-(piperidin-1-yl)-4-bromothiazole (15)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

6.614.000.80

13C NMR of 2-(piperidin-1-yl)-4-bromothiazole (15)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

24

.09

25

.14

49

.26

76

.90

77

.23

77

.54

10

2.9

6

12

1.6

9

17

1.0

1

N

S N

Br 15

S13

1H NMR of 2-(phenylethynyl)-4-bromothiazole (16)

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

3.232.19

13C NMR of 2-(phenylethynyl)-4-bromothiazole (16)

250 200 150 100 50 0ppm

76.9

177

.23

77.5

481

.58

95.6

7

118.82

121.

0412

6.06

128.75130.08132.21

149.

87

N

S

Br16

S14

1H NMR of trimethylsilyl)ethynyl)-4-bromothiazole (17)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5ppm

9.001.11

13C NMR of trimethylsilyl)ethynyl)-4-bromothiazole (17)

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

-0.4

0

76

.91

77

.23

77

.54

95

.52

10

3.0

2

11

8.8

4

12

5.8

6

14

9.5

2N

S

Br

TMS

17

S15

1H NMR of 2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enone (20a)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ppm

2.83 2.752.00 1.970.91

13C NMR of 2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enone (20a)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

209.

94

165.

57 158.

45

152.

02

136.

26

120.28

77.5

477

.23

76.9

1

33.6

6

27.9

9

19.33

10.12

O

N

S

20a

S16

1H NMR of 2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enone (20b)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

2.69 2.651.911.831.00

13C NMR of 2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enone (20b)

250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

10

.41

16

.72

27

.92

33

.93

76

.93

77

.25

77

.57

11

9.6

3

13

7.0

2

15

2.6

3

15

7.8

1

16

6.7

1

21

0.2

5

O

N

SS

20b

S17

1H NMR of 2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enone (20c)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

6.454.00 2.842.00 2.000.91

13C NMR of 2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enone (20c)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

210.

67

170.

83 159.

10

149.

68

136.

06

109.82

77.5

577

.23

76.9

1

49.7

4

33.9

0

27.5

825

.31

24.3

5

10.36

O

N

SN

20c

S18

1H NMR of 2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enone (20d)

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

3.07 2.942.08 2.082.03

13C NMR of 2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enone (20d)

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

10

.45

28

.41

33

.9676

.91

77

.23

77

.54

82

.16

95

.08

12

1.2

2

12

1.8

6

12

8.7

51

30

.03

13

2.2

5

13

7.6

0

14

8.7

5

15

3.0

6

15

7.9

7

21

0.0

7

O

N

S

20d

S19

1H NMR of 2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone (20e)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5ppm

8.292.972.042.000.95

13C NMR of 2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone (20e)

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10ppm

209.

70

157.

65

152.

63

148.

20 137.

36

121.78

102.

04

96.1

4

77.5

477

.22

76.9

1

33.7

4 28.1

8

10.22

-0.45

O

N

S

20e

TMS

S20

1H NMR of 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone (21)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

3.002.22 2.150.95 0.85

13C NMR of 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone (21)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

209.

96

157.

5415

3.06

147.

49 137.

82

121.89

83.2

477

.54

77.2

376

.91

76.3

1

33.9

2

28.3

1

10.38

O

N

S

21

S21

1H NMR of 3-(2-(1H-1,2,3-triazol-4-yl)thiazol-4-yl)-2-methylcyclopent-2-enone (22)

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

2.062.00 1.960.78 0.66

13C NMR of 3-(2-(1H-1,2,3-triazol-4-yl)thiazol-4-yl)-2-methylcyclopent-2-enone (22)

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

208.

85

158.

4615

7.43

152.

30

141.

24

135.

46

122.24

40.1

339

.92

39.7

139

.50

39.2

939

.08

38.8

8

33.2

5

27.4

6

9.99

O

N

SN

NNH

22

S22

1H NMR of 2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enone (20f)

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

3.28 3.142.22 2.221.091.00

13C NMR of 2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enone (20f)

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

210.

20

158.

02

153.

39

143.

06

137.

35

132.35

120.4777

.54

77.2

376

.91

42.30

33.9

3

28.1

8

10.41

O

N

SN

NN

20f

S23

1H NMR of (S)-2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enol (6a)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

2.712.69 1.091.091.000.89

13C NMR of (S)-2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enol (6a)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

164.

96 153.

09

138.

66

131.

64

115.25

82.37

77.5

477

.22

76.9

1

32.9

1 32.7

2

19.55 13.37N

S

HO 6a

S24

1H NMR of (S)-2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enol (6b)

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

2.702.63 1.48 1.421.321.00

13C NMR of (S)-2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enol (6b)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

165.

23 153.

34

139.

22

130.

68

114.71

82.14

77.5

477

.23

76.9

1

32.4

5

16.6813.41

N

SS

HO 6b

S25

1H NMR of 2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enol (6c)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5ppm

7.704.23 2.951.171.151.081.00

13C NMR of 2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enol (6c)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

170.

89 150.

07 137.

95

131.

74

104.63

82.61

77.5

477

.23

76.9

1

49.7

0

32.7

132

.19

25.3

624

.44

13.39N

SN

HO 6c

S26

1H NMR of (S)-2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enol (6d)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

2.75 2.641.81 1.021.00 1.001.000.930.90

13C NMR of (S)-2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enol (6d)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

154.

15

147.

77

139.

97

132.18

131.

13

129.69128.67

121.

64

117.36

93.9

5

82.7

7

82.30

77.5

477

.22

76.9

1

32.9

932

.69

13.51N

S

HO 6d

S27

1H NMR of (S)-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enol (6e)

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0ppm

2.60 1.231.181.101.091.071.00 1.00

N

S

OH

6e

13C NMR of (S)-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enol (6e)

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10ppm

154.

17

146.

55

140.

25

130.

85

117.49

82.30

82.1

477

.44 77

.22

77.0

176

.91

32.9

032

.66

13.47

N

S

OH6e

S28

1H NMR of (S)-2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enol (6f)

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0ppm

2.422.34 1.16 1.121.12 1.081.000.800.74

13C NMR of (S)-2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enol (6f)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

157.

2515

4.26

143.

56

139.

65

132.20

131.

08

115.84

82.36

77.5

476

.91

42.20

32.7

1

13.47N

S

HO 6fN N

N

S29

1H NMR of (3S,6R,7S)-((S)-2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5a)

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

5.734.10 3.66 3.20 3.042.92 1.211.061.01 1.011.00

13C NMR of (3S,6R,7S)-((S)-2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5a)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

222.

36

173.

27

165.

13

152.

46

134.

6413

4.09

116.06 85.50

77.5

477

.22

76.9

1

76.6072.74

52.3

5

40.99

37.0

533

.50

30.74

29.4

9

21.6519.7119.18

13.6510.60

O

HO

OH

O

O

N

S

5a

S30

1H NMR of (3S,6R,7S)-((S)-2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5b)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

5.524.01 3.103.05 2.89 2.881.321.00 1.000.97

13C NMR of (3S,6R,7S)-((S)-2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5b)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

222.

35 173.

23

165.

58 152.

75

134.

6613

3.88

115.65

85.46

77.5

4

76.9

1

76.6172.75

52.3

6

41.02

37.0

8 33.1

4

30.74

29.4

7

21.64 19.6919.24

16.7313.72

10.60

O

HO

OH

O

O

N

S

S

5b

S31

1H NMR of (3S,6R,7S)-((R)-2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate and (3S,6R,7S)-((S)-2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5c)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

7.58 5.894.61 3.24 3.142.971.04 1.03 1.021.000.99

13C NMR of (3S,6R,7S)-((R)-2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate and (3S,6R,7S)-((S)-2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5c)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

222.2

8

173.3

017

0.89 14

9.46

134.7

613

3.27

105.45

85.81

77.54

77.22 76

.91

76.5972.72

72.65

52.37

49.70

40.97

37.06

37.02

32.82

30.74

29.45

25.35

24.42

21.64 19.7319.18

13.6710.60

O

HO

OH

O

O

N

S

N

5c

S32

1H NMR of (3S,6R,7S)-((S)-2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5d)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

5.894.31 3.24 3.203.07 2.852.03 1.581.151.06 1.001.001.000.98

13C NMR of (3S,6R,7S)-((S)-2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5d)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

222.

34

173.

19

153.

49

147.

96 135.

4813

4.06

132.19129.75

128.69

121.

58

118.0594

.12

85.30

82.6

777

.55

77.2

376

.91

76.6272.75

52.3

7

41.03

37.0

833

.60

30.76

29.4

9

21.67 19.7119.21

13.79 10.62

O

HO

OH

O

O

N

S

5d

S33

1H NMR of (3S,6R,7S)-((S)-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5e)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

5.373.98 2.99 2.862.81 1.261.02 1.001.00 0.980.94 0.88

13C NMR of (3S,6R,7S)-((S)-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5e)

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

222.

35 173.

16

153.

47

146.

69

135.

7713

3.70

118.19

85.23

82.3

077

.54 77

.23

76.9

0

76.6172.75

52.3

6

41.02

37.0

633

.51

30.76

29.4

6

21.6719.71 19.21

13.7610.62

O

HO

OH

O

O

N

S

5e

S34

1H NMR of (3S,6R,7S)-((S)-2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5f)

13C NMR of (3S,6R,7S)-((S)-2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5f)

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0ppm

6.384.714.21 3.63 3.38 3.281.481.211.201.181.171.141.00

240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0ppm

10.6213.74

19.2019.69

21.66

29.5

0

30.74

33.4

337

.08

41.0142.23

52.3

6

72.7476.60

76.9

177

.23

77.5

5

85.39

116.58

132.18

134.

0313

5.07

143.

45

153.

5815

7.39

173.

20

222.

33

O

HO

OH

O

O

N

S

5f

N NN

S35

1H NMR of (R)-3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enol (23)

Neptune\data1\kso\vxrs400\KS-1-81i

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0ppm

1.211.08 1.071.071.000.95

N

S

OH

30

13C NMR of (R)-3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enol (23)

Avance600\nmrdata\tillekes\KS-1-81i_C13_APT\KS-1-81i_C13_APT_001000fid

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10ppm

154.

23

146.

57

140.

31

130.

86

117.46

82.31

82.1

2

77.4

677

.25

77.0

476

.96

32.9

432

.69

13.48

N

S

OH

30

23

23

23

S36

1H NMR of 2-Methyl-3-(2-((trimethylsilyl)ethynyl)thiazole-4-yl)cyclopent-2-enol (24)

Neptune\data1\kso\inova600\KS-1-95d

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0ppm

3.02 2.261.05 1.051.051.000.95 0.90

N

S

OH31

13C NMR of 2-Methyl-3-(2-((trimethylsilyl)ethynyl)thiazole-4-yl)cyclopent-2-enol (24)

Avance600\nmrdata\tillekes\KS-1-95d_C13_APT\KS-1-95d_C13_APT_001000fid

150 140 130 120 110 100 90 80 70 60 50 40 30 20ppm

154.

23

146.

57

140.

31 130.

85

117.

46

82.1

3

77.4

777

.26

77.0

476

.96

32.9

432

.69

N

S

OH 3124

23

23

24

23

S37

1H NMR of 3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enone oxime (25)

Neptune\data1\kso\inova600\KS-1-85h

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5ppm

2.76 2.171.500.72 0.590.38

N

S

NOH 32

13C NMR of 3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enone oxime (25)

Avance600\nmrdata\tillekes\KS-1-85h_C13_APT\KS-1-85h_C13_APT_001000fid

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10ppm

206.

25

168.

12

154.

67

147.

50

141.

45

135.

52

120.2484

.41

77.4

0

31.7

730

.07

29.8

229

.69

24.4

9

11.63

N

S

NOH

32

25

23

25

23

S38

5e (M)

0

20

40

60

80

100

120

0 5 10 15 20 25

HOP62

NCI-H522

* p<0.05

* * *

Norm

aliz

ed ce

ll vi

abili

ty

0

20

40

60

80

100

120

140

Norm

aliz

ed ce

ll vi

abili

ty HOP62NCI-H522

5e (All at 20 M)5d 5a

-0.10

0.10.20.30.40.50.60.7

DMSO 5e 6e 23 24 25

21

6e

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10Concentration (M)

Abso

rban

ceAb

sorb

ance

A

B

C

D

Supplemental Figure S1. Initial assessment toxicity induced by compound 5e and analogues. (A and B) NCI-H522 or HOP-62 cells were plated and exposed to the indicated compounds at the doses specified. Cells treated for 5 days were analyzed by methylene blue staining to determine viable cells. Average values are shown with bars to represent standard deviation. *p<0.05. (C and D) NCI-H522 were exposed to the compounds indicated. Three days later, viability was determined either by measuring absorbance after methylene blue staining (in C) or using the MTS assay (in D) as described in materials and methods. In “C”, significantly lower absorbance was observed after either 2.5M or 5M of 6e compared to compound 21 (p<0.02). In “D” all compounds resulted in significantly lower absorbance compared to DMSO (p<0.005, students t-test).

S39

H:MIN

0:24

2:12

0:36 0:48 1:00 1:12

1:24 1:36 1:48 2:00

Supplemental Figure S2. Time lapse microscopy of NCI-H522 cells exposed to 6e. Images were captured every 12 min. In the example shown, mitosis begins 48 min after drug addition. Two other cells in the same field completed mitosis, and by the end of the movie (19 h post drug addition) all cells were dead.

TMRE Phase Contrast

0h

2h

4h

8h

0

1

2

3

4

0

5

10

15

20

25

30

0

5

10

15

20

25

0 2 4 8 0 2

TMR

E in

tens

ity

TMR

E in

tens

ity

hr post treatment

Average whole cellIndividual mitochondria

hr post treatment

num

ber o

f cel

ls

increasing TMRE

8hDMSO

A B

Supplemental Figure S3. Effect of compound 6e (10M) on mitochondrial function. NCI H522 cells treated for times indicated with compound 6e and stained with TMRE to indicate mitochondrial potential. (A) Representative microscopic fields of stained cells. Arrowheads: two cells that have lost mitochondrial membrane potential; phase contrast indicates that these are dead cells. (B) Quantitation of TMRE staining from microscopic images in whole cells or individual mitochondria.