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1 Supporting Information Further Developments of the Phenyl-Pyrrolyl Pentane Series of Nonsteroidal Vitamin D Receptor Modulators as Anticancer Agents Meixi Hao +, Siyuan Hou +, Lingjing Xue, Haoliang Yuan, Lulu Zhu, Cong Wang, Bin Wang, Chunming Tang, Can Zhang * List: Figure S1. Immunoblot analysis of the VDR protein expression in different cell types. Figure S2. Effects of sw22, 11b, 11g, and 15b on the expression of Cyp24a1 in breast derived MCF7 cells. Figure S3. Superimposition between X-ray crystal structure of the VDR-LBD-LG complex and the in silico docking structure of the VDR-LBD-LG Figure S4. mrna expression change of VDR downstream targets after treatment of 11b, 11d, 15b, and sw22 in MCF7 cells. Figure S5. Immunoblot analysis of the expression of p21 and p27 proteins in MCF7 cells. Table S1. Cell cycle arrest effect of compounds 11b, 11g, and 15b in HepG2 cells. Procedures for Intermediates Preparation 1 H NMR, 13 C NMR and HRMS Spectrums of Phenyl-Pyrrolyl Pentane Derivatives S1

2 Figure S1 Figure S1. Immunoblot analysis of the VDR protein expression in different cell types. VDR protein expression was analyzed in human embryonic kidney cells (HEK293 cells), mouse hepatocarcinoma cells (Heps cells), human hepatocellular carcinoma cells (HepG2 cells), human breast cancer cells (MCF7 cells) and human pancreatic cancer cells (BxPC3 cells). β-actin was used as the internal loading control. S2

3 Figure S2 Relative Expression ** ****** ** *** * ** DMSO Calcitriol sw22 11b 11g 15b 0 Cyp24a1 Figure S2. Effects of sw22, 11b, 11g, and 15b on the expression of Cyp24a1 in breast derived MCF7 cells. Cells were treated with each compound (1 μm) for 24 h, and the expression of Cyp24a1 was determined by quantitative real-time PCR. *, p < 0.05; **, p <0.01; ***, p < versus DMSO control. S3

4 Figure S3 Figure S3. Superimposition between X-ray crystal structure of the VDR-LBD-LG complex and the in silico docking structure of the VDR-LBD-LG (A) Chemical structure of LG which possess a flexible 2-hydroxy-3,3-dimethylbutoxy phenyl side chain; (B) Chemical structure of phenyl-pyrrolyl pentane derivatives which possess a flexible phenyl side chain; (C) X-ray crystal structure of the VDR-LBD-LG (shown in cyan) complex (PDB ID: 2ZFX) superimposed on the in silico docking structure of the VDR-LBD-LG (shown in yellow) complex. S4

5 Figure S4 Relative Expression * * * * * * DMSO Calcitriol sw22 11b 11g 15b 0.0 p21 p27 BAX BAK Figure S4. mrna expression change of VDR downstream targets after treatment of 11b, 11d, 15b, and sw22 in MCF7 cells. The MCF7 cells were treated with compound 11b, 11g, 15b, and sw22 at 1 μm for 24 h. Relative expression of p21, p27, Bax and Bak mrna levels were determined by real-time quantitative PCR in MCF7 cells. Amplification of β-actin cdna was used as a control. *, p < 0.05 versus DMSO control. S5

6 Figure S5 Figure S5. Immunoblot analysis of the expression of p21 and p27 proteins in MCF7 cells. Cells were treated with each compound (1 μm) for 24 h, and p21 and p27 proteins expression was analyzed in MCF7 cells. β-actin was used as the internal loading control. S6

7 Table S1. Cell cycle arrest effect of 11b, 11g and 15b in HepG2 cells. Compound * DMSO 11b 11g 15b Calcitriol G0-G ± ± 0.21*** 74.3 ± 0.56*** 74.2 ± 0.17*** 74.6 ± 0.97*** S 23.7 ± ± ± ± ± 0.47 G2-M 11.3 ± ± ± ± ± 0.67 * The HepG2 cells were treated with 11b, 11g, 15b, and calcitriol at 1 μm for 24 h. Data were presented as Mean ± SD ***, p<0.001 (compared with DMSO control). S7

8 Procedures for Intermediates Preparation Methyl-4-hydroxy-3-methylbenzoate (1). To a solution of 4-hydroxy-3-methylbenzoic acid (80.0 g, 0.52 mol) in methanol (500 ml) was dropped into conc.h2so4 (80 ml) with stirring. The reaction mixture was refluxed for overnight and then cooled. The solution was adjusted to approximately ph 6 with 2.5 M NaOH, then poured into cold H2O (1 L). With stirring 0.5 hours, having pink solid separated out. The precipitate was filtered off and washed with cold water. The product was collected as a pink solid and dried with vacuum drying oven (84.42 g, 97%). Methyl-4-benzyloxy-3-methylbenzoate (2). Benzyl bromide (95.58 g, 0.56 mol, ml), K2CO3 ( g, 1.02 mol) were added to a solution of compound 1 (84.42 g, 0.51 mol) in acetone (400 ml). The mixture was refluxed for 3 hours and then cooled. The precipitate of K2CO3 was filtered off and the filtrate was evaporated. Then the residue was precipitated in petroleum ether (200 ml) to give compound 2 as pink crystal and dried ( g, 84%). 3-(4-benzyloxy-3-methylphenyl)-pentan-3-ol (3). Mg (8.43 g, 0.35 mol), I2 and anhydrous ether (100 ml) was added into three-necked bottle. With heating and stirring, a solution of bromoethane (42.07 g, 0.38 mol, 28.8 ml) in ether (50 ml) was dropped into the mixture. Until Mg disappeared, the ethyl Grignard reagent (0.35 mol) was produced. Then, to a solution of compound 2 (30.0 g, 0.12 mol) in ether (100 ml) was added ethyl Grignard reagent (0.35 mol) dropwise at 0.The reaction mixture was stirring at 30 for 2 hours. To the mixture was added an aqueous saturated solution of NH4Cl at 0 and the two phases were separated. Aqueous phase was extracted with ether. The combined organic phases were washed with aqueous saturated solution of salt, dried with anhydrous Na2SO4 and evaporated to give compound 3 as white oil (37.09 g, 89%). 2,2,2-trichloro-1-(1H-pyrrol-2-yl) ethanone (4). To solution of pyrrole (33.54 g, 0.49 mol, ml) in anhydrous ether (90 ml). With stirring, the 2,2,2-trichloroacetyl chloride (100.0 g, 0.55 mol) in ether (150 ml) was added the reaction mixture dropwise at 0. The reaction mixture was stirring at r.t for 3 hours. To the mixture was added an aqueous saturated solution of K2CO3 (69.0 g, 0.49 mol) and the two phases were separated. Aqueous phase was extracted with ether. The combined organic phases were washed with brine, separated and S8

9 dried over anhydrous Na2SO4 and evaporated under reduced pressure to get crude product which was further purified by recrystallization using hexane to give compound 4 as a hoar solid (81.24 g, 76%). Ethyl-1H-pyrrole-2-carboxylate (5). Sodium ethanolate (EtONa) (38.78 g, 0.45 mol) was added to a solution of compound 4 (81.24 g, 0.38 mol) in ethanol (500 ml), the reaction mixture was heated to 70 for 2.5 hours and then cooled. Water (300 ml) was added dropwise to the mixture which was extracted with ethyl acetate. The organic phases was washed by brine and dried with anhydrous Na2SO4. After that evaporated to get crude product which was further purified by column chromatography using ethyl acetate / hexane (1:8, v/v) as eluent to afford corresponding compound 5 as a yellow solid (24.8 g, 46%). Ethyl-5-(3-(4-benzyloxy-3-methylphenyl)-pentan-3-yl)-1H-pyrrole-2-carboxylate (6). BF3 Et2O (15.86 g, 0.13 mol, ml) was added dropwise to a solution of compound 3 (23.55 g, 83 mmol) and compound 5 (9.6 g, 69 mmol) in dichloromethane (CH2Cl2) (150 ml) at 0. The reaction mixture stirred at r.t for 0.5 hours. Water (150 ml) was added to the mixture and the two phases were separated. The aqueous phase was extracted with ethyl acetate, then the combined organic phases was washed with brine and dried over Na2SO4, and evaporated under reduced pressure to give crude product which was further purified with column chromatography using ethyl acetate / hexane (1:10, v/v) as eluent to get a yellow solid (19.34 g, 57%). 1 H NMR (300 MHz, CDCl3) δ: (5H, m), 7.03 (1H, s), 7.01 (1H, d, J=8.5 Hz), 6.79 (1H, d, J=8.5 Hz), 6.70 (1H, d, J=2.0 Hz), 6.50 (1H, d, J=2.0 Hz), 5.04 (2H, s), 4.30 (2H, q, J=7.1 Hz), 2.24 (3H, s), 1.97 (4H, q, J=7.3 Hz), 1.32 (3H, t, J=7.1 Hz), 0.67 (6H, t, J=7.3 Hz). Ethyl-5-(3-(4-benzyloxy-3-methylphenyl)-pentan-3-yl)-1-ethyl-1H-pyrrole-2-carbox ylate (7). To a solution of compound 6 (19.34 g, 47.7 mmol) in N,N- dimethylformamide (DMF) (70 ml). NaH (4.58 g, 95.4 mmol) was added portionwise at 0, after stirring 0.5 hours, iodine ethane (11.15 g, 72 mmol, 5.78 ml) was added. The reaction mixture was stirred at r.t for 0.5 hours. Water (40 ml) was added dropwise to removed excrescent NaH. The organic phase was separated and aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with water followed by brine and then dried over anhydrous Na2SO4 and evaporated to afford compound 7 as yellow oil (19.9 g, 96%). 1 H S9

10 NMR (300 MHz, CDCl3) δ: (5H, m), 7.04 (1H, s), 7.02 (1H, d, J=8.2 Hz), 6.77 (1H, d, J=8.6 Hz), 6.70 (1H, d, J=2.0 Hz), 6.57 (1H, d, J=2.0 Hz), 5.04 (2H, s), 4.27 (4H, m), 2.25 (3H, s), 1.95 (4H, q, J=7.0 Hz), 1.31 (6H, m), 0.67 (6H, t, J=7.0 Hz). Ethyl-5-(3-(4-hydroxy-3-methylphenyl)-pentan-3-yl)-1-ethyl-1H-pyrrole-2-carboxyl ate (8). Ammonium formate (28.94 g, 0.46 mol) and Pd/C (1.99 g) was added to a solution of compound 7 (19.9 g, 46 mmol) in methanol (40 ml) at r.t for overnight. Then the precipitate was filtered off and water and ethyl acetate was added to the solution. The two phases were separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases was washed with brine and dried over Na2SO4 and evaporated under reduced pressure to give compound 8 as white solid (15.15 g, 96%). 1 H NMR (300 MHz, CDCl3) δ: 6.92 (1H, s), 6.88 (1H, d, J=8.6 Hz),6.63 (1H, d, J=2.1 Hz), 6.60 (1H, d, J=8.6 Hz), 6.50 (1H, d, J=2.1 Hz), (4H, m), 2.28 (3H, s), 1.87 (4H, q, J=7.4 Hz), 1.30 (6H, m), 0.60 (6H, t, J=7.4 Hz). 5-(3-(4-hydroxy-3-methylphenyl)-pentan-3-yl)-1-ethyl-1H-pyrrole-2-carboxylicacid (9). A aqueous solution of KOH (8.17 g, 0.14 mol) was added to a solution of compound 8 (10 g, mol) in ethanol (50 ml), the reaction mixture was reflux for 12 hours at 70. Then water (30 ml) was added and the solution which was adjusted to approximately ph 7 with 0.1M HCl and extracted with ethyl acetate was separated into two phases. The combined organic phase was washed with brine and dried over Na2SO4. The solvent was evaporated to give crude product which was further purified by column chromatography using ethyl acetate/hexane (8:1, v/v) to get compound 9 as yellow oil (7.7 g, 83%). 1 H NMR (300 MHz, CDCl3) δ: 7.02 (2H, m), 6.87 (1H, d, J=2.1 Hz), 6.70 (1H, d, J=8.2 Hz), 6.63 (1H, d, J=2.0 Hz), 4.30 (2H, q, J=7.11 Hz), 2.23 (3H, s), 1.95(4H, q, J=7.4 Hz), 1.40 (3H, t, J=7.1 Hz), 0.68 (6H, t, J=7.4 Hz). Synthetic intermediates of 1-bromo-3-ethylpentan-3-ol (Br-OH). To a solution of methyl-3-bromopropanoate (15 g, 89.8 mmol) in anhydrous ether (50 ml), ethyl Grignard reagent (0.19 mol) was added dropwise to the solution at 0. Then the reaction mixture was stirred at 30 for 2 hours. To the mixture was added an aqueous saturated solution of NH4Cl at 0 and the two phases were separated. Aqueous phase was extracted with ether. The combined organic phase was washed with aqueous saturated solution of salt, dried with S10

11 anhydrous Na2SO4 and evaporated solvent to afford crude product as white oil which was purified to give intermediates Br-OH as a yellow liquid (6 g, 43%). 1 H NMR (300 MHz, CDCl3) δ: 3.62 (2H, t, J=6.9 Hz), 1.90 (2H, t, J=6.9 Hz), 1.35 (4H, m), 0.90 (6H, t, J=2.1 Hz). 5-(3-(4-((3-ethyl-3-hydroxypentyl)oxy)-3-methylphenyl)-pentan-3-yl)-1-ethyl-1H-py rrole-2-carboxylic acid (10). NaH (1.92 g, 0.08 mol) was added to a solution of compound 9 (7.7 g, mmol) in DMF (30 ml) at 0 for 0.5 hours, then the intermediates Br-OH (6 g, 0.03 mol) was dropped into the mixture. The reaction mixture was stirring at 25 for 3 hours. Water was added with stirring and the two phases were separated which was adjusted to approximately ph 7 with 0.1M HCl. The aqueous phase was extracted with ethyl acetate. Then the combined organic phase was washed with water followed by brine, dried over Na2SO4 and evaporated under reduced pressure to give crude product which was further purified by column chromatography using dichloromethane/methanol (50:1, v/v) to afford compound 10 as a yellow oil (7.88 g, 75%). 1 H NMR (300 MHz, CDCl3) δ: 7.04 (2H, m), 6.84 (1H, d, J=2.0 Hz), 6.72 (1H, d, J=8.4 Hz), 6.60 (1H, d, J=2.0 Hz), 4.34 (2H, q, J=7.2 Hz), 4.13 (2H, t,j=6.0 Hz), 2.17 (3H, s), 1.96 (4H, m), 1.58 (4H, q, J=7.3 Hz), 1.35 (3H, t, J=7.2 Hz), 0.91 (6H, t, J=7.5 Hz), 0.66 (6H, t, J=7.2 Hz). S11

12 1 H NMR, 13 C NMR and HRMS spectrums of phenyl-pyrrolyl pentane derivatives Intermediate 6 Ethyl-5-(3-(4-benzyloxy-3-methylphenyl)-pentan-3-yl)-1H-pyrrole-2-carboxylate S12

13 Intermediate 7 Ethyl-5-(3-(4-benzyloxy-3-methylphenyl)-pentan-3-yl)-1-ethyl-1H-pyrrole-2-carboxylate Intermediate 8 Ethyl-5-(3-(4-hydroxy-3-methylphenyl)-pentan-3-yl)-1-ethyl-1H-pyrrole-2-carboxylate S13

14 Intermediate 9 5-(3-(4-hydroxy-3-methylphenyl)-pentan-3-yl)-1-ethyl-1H-pyrrole-2-carboxylicacid 9 S14

15 Intermediate Br-OH 1-bromo-3-ethylpentan-3-ol Br-OH Intermediate S15

16 Compound 11a N-(3-(dimethylamino)propyl)-1-ethyl-5-(3-(4-((3-ethyl-3-hydroxypentyl)oxy)-3- methylphenyl)pentan-3-yl)-1h-pyrrole-2-carboxamide 11a S16

17 S17

18 Compound 11b N-(3-(diethylamino)propyl)-1-ethyl-5-(3-(4-((3-ethyl-3-hydroxypentyl)oxy)-3- methylphenyl)pentan-3-yl)-1h-pyrrole-2-carboxamide 11b S18

19 S19

20 Compound 11c N-(2-(dimethylamino)ethyl)-1-ethyl-5-(3-(4-((3-ethyl-3-hydroxypentyl)oxy)3- methylphenyl)pentan-3-yl)-1h-pyrrole-2-carboxamide 11c S20

21 S21

22 Compound 11d N-(3-(diethylamino)ethyl)-1-ethyl-5-(3-(4-((3-ethyl-3-hydroxypentyl)oxy)-3- methylphenyl)pentan-3-yl)-1h-pyrrole-2-carboxamide 11d S22

23 S23

24 Compound 11e 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid cyanomethyl-amide 11e S24

25 S25

26 Compound 11f 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid (2-amino-ethyl)-amide 11f S26

27 S27

28 Compound 11g 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid (3-morpholin-4-yl-propyl)-amide 11g S28

29 S29

30 Compound 11h 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid bis-(2-cyano-ethyl)-amide 11h S30

31 S31

32 Compound 11i 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid allyl-methyl-amide 11i S32

33 S33

34 Compound 12a 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid 1-methyl-prop-2-ynyl ester 12a S34

35 S35

36 Compound 12b 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid but-3-ynyl ester 12b S36

37 S37

38 Compound 13a 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-4h-pyrrole-2-carboxylic acid 4-ethoxycarbonylmethyl-phenyl ester 13a S38

39 S39

40 Compound 13b 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-4h-pyrrole-2-carboxylic acid (4-methoxy-phenyl)-amide 13b S40

41 S41

42 Compound 13c 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid (4-trifluoromethyl-phenyl)-amide 13c S42

43 S43

44 Compound 13d 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid (4-ethoxy-phenyl)-amide 13d S44

45 S45

46 Compound 14a 2-[(1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carbonyl)-amino]-3-methyl-pentanoic acid methyl ester 14a S46

47 S47

48 Compound 14b [(1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carbonyl)-methyl-amino]-acetic acid ethyl ester 14b S48

49 S49

50 Compound 14c 2-[(1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carbonyl)-amino]-succinic acid dimethyl ester 14c S50

51 S51

52 Compound 15a 2-[(1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carbonyl)-amino]-3-methyl-pentanoic acid 15a S52

53 S53

54 Compound 15b [(1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carbonyl)-methyl-amino]-acetic acid 15b S54

55 S55

56 Compound 15c 2-[(1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carbonyl)-amino]-succinic acid 1-methyl ester 15c S56

57 S57

58 Compound 15d 2-[(1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carbonyl)-amino]-propionic acid 15d S58

59 S59

60 Compound 16a 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid (1-hydroxymethyl-2-methyl-butyl)-amide 16a S60

61 S61

62 Compound 16b 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid (2-hydroxy-ethyl)-methyl-amide 16b S62

63 S63

64 Compound 16c 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid (3-hydroxy-1-hydroxymethyl-propyl)-amide 16c S64

65 S65

66 Compound 16d 1-Ethyl-5-{1-ethyl-1-[4-(3-ethyl-3-hydroxy-pentyloxy)-3-methyl-phenyl]- propyl}-1h-pyrrole-2-carboxylic acid (2-hydroxy-1-methyl-ethyl)-amide 16d S66

67 S67