AR Degrading Agents

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Novel C-4 Heteroaryl 13-Cis Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice Hannah W. Mbatia, Senthilmurugan Ramalingam, Vidya P Ramamurthy, Marlena S Martin, Andrew K. Kwegyir-Afful, and Vincent C. O. Njar J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501792c • Publication Date (Web): 29 Jan 2015 Downloaded from http://pubs.acs.org on February 6, 2015

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Journal of Medicinal Chemistry

Novel C-4 Heteroaryl 13-Cis Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice Hannah W. Mbatia1,2,#, Senthilmurugan Ramalingam1,2,#, Vidya P. Ramamurthy1,2,#, Marlena S. Martin1,2,¥, Andrew K. Kwegyir-Afful1,2 and Vincent C. O. Njar1,2,3,* 1

Department of Pharmacology, 2Center for Biomolecular Therapeutics, Maryland, 3Marlene

Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, MD 21201-1559, USA.

¥

Current Address: Bernard J. Dunn School of Pharmacy, Shenandoah University, 45085

University Drive, Suite 202, Ashburn, VA 20147

1 ACS Paragon Plus Environment


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ABSTRACT The synthesis and in vitro and in vivo anti-breast and anti-prostate cancers activities of novel C-4 heteroaryl 13-cis retinamides that modulate Mnk-eIF4E and AR signaling are discussed. Modifications of the C-4 heteroaryl substituents reveal that the 1H-imidazole is essential for high anti-cancer activity. The most potent compounds against a variety of human breast and prostate cancer (BC/PC) cell lines were compounds 16 (VNHM-1-66), 20 (VNHM-1-81) and 22 (VNHM-1-73). In these cell lines; the compounds induce Mnk1/2 degradation to substantially suppress eIF4E phosphorylation. In PC cells, the compounds induce degradation of both fulllength androgen receptor (fAR) and splice variant AR (AR-V7) to inhibit AR transcriptional activity. More importantly, VNHM-1-81 has strong in vivo anti-breast and anti-prostate cancer activities, while VNHM-1-73 exhibited strong in vivo anti-breast cancer activity, with no apparent host toxicity. Clearly, these lead compounds are strong candidates for development for the treatments of human breast and prostate cancers.


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INTRODUCTION Disruption and/or perturbation of cap-dependent translation is essential for the development of cancers and many fibrotic diseases, the most notable being Alzheimer’s disease.1 Hyper-activation of eukaryotic translation initiation factor 4E (eIF4E), the mRNA 5ꞌ cap-binding protein of cap-dependent translation promotes exquisite transcript-specific translation of key mRNAs that are indispensable in cancer initiation, progression and metastases.2 The oncogenic potential of eIF4E is dependent on serine 209 phosphorylation by MAPK-interacting kinases 1 and 2 (Mnk1/2). Currently, there are several strategies, directed at disruption of the eukaryotic initiation factor 4F (eIF4F) complex to inhibit hyper-activity of oncogenic protein translation as a means to effectively treat a variety of cancers.3 When targeting a critical process such as translation, a major critical question is whether the drug will have significant effects on normal cellular processes, leading to limiting toxicities and too narrow a therapeutic window. This question has been addressed by several experimental and a few clinical studies. Recently, Graff and colleagues elegantly demonstrated that down-regulation of eIF4E by antisense oligonucleotide therapy caused reduction of in vivo tumor growth in MDA-MB-231 breast and PC-3 prostate cancer models without toxicity despite an ~80% eIF4E knockdown in essential organs.4 These data provided in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissue and provided the basis for advancement of eIF4E-specific antisense molecule LY2275796 into Phase 1 clinical studies.5 In other more recent studies, cercosporamide, a potent Mnk1/2 kinase inhibitor was reported to exhibit antitumor efficacies against HCT116 colon carcinoma xenograft tumors6 and MV4-11 acute myeloid leukemia (AML) tumors in animal models,7 without any toxicity.



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We previously reported that our proprietary novel C-4 azolyl retinamides (NRs) based on all-trans retinoic acid (ATRA) (1) (Figure 1) scaffold induced apoptosis, potently inhibited the growth, migration and invasion of a variety of human breast and prostate cancer cell lines.8 With respect to the breast cancer cell lines, we demonstrated that the anti-breast cancer activity of our NRs was due mainly to degradation of Mnk1/2 with subsequent suppression of phosphorylated eIF4E (peIF4E).8a However, in the prostate cancer (PC) cell lines, we demonstrated that the antitumor activity of the NRs was due to simultaneous inhibition of the Mnk/eIF4E and androgen receptor (AR) signaling pathways.8b It is important to state here that unlike other reported small molecules which inhibit Mnk1/2 kinase activities,3e, 6, 9 our NRs induced Mnk1/2 degradation via the ubiquitin-proteasome pathway with resultant depletion of peIF4E.8 The structures of promising Mnk 1 and/or 2 kinase inhibitors, including CGP57380 (2),10 cercosporamide (3),6 MNKI-19 (4)11 and MNKI85 (5)11 are presented in Figure 1. In addition, the structures of our lead Mnk degrading agent (MNKDA), VNLG-152 (6)8a and related compounds,12

12

VN/14-1

(7), VN/66-1 (8) and N-(4-hydroxyphenyl) retinamide (4-HPR or fenretinide) (9) are also depicted in Figure 1. In the present study, we have synthesized novel compounds based on 13-cis retinoic acid (13-CRA) (10) (Figure 1) scaffold, and their anti-cancer activities were evaluated in both in vitro and in vivo models of human breast and prostate cancers. These novel compounds target Mnk1/2 degradation in both breast cancer and PC cells and also induce AR degradation in PC cells, which in turn led to induction of apoptosis, cell cycle arrest, inhibition of cell growth, colonization, migration, and invasion. Importantly, compounds 20 and 22 significantly inhibited tumor growth of aggressive MDA-MB-213 breast cancer xenografts, while 20 was shown to substantially suppress castration-resistant prostate cancer CWR2Rv1 xenografts in vivo.


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F F

H

O

NH2 OH

N

HO O

O OH

1

2

N H

NH2

O

HO

O

3

S

N

O

4

F

O

OCHMe2

O N H

H

OH

NHCH2CH2OMe

N

N N

S

O

N N

5

6

N

7

OH

O

OH

O N H N

OH

N

N N

N

H

N

N

F

OCHMe2

N H

9

8

10

O

N

Figure 1: Chemical structures of compounds 1 – 10.

RESULTS AND DISCUSSIONS Design Strategy: Rationale structural modifications of small molecules allow for their interactions with molecular target(s) in ways that could lead to improved drug-like compounds and possibly enhanced in vivo pharmacokinetic profiles.13 Because of our desire to discover more efficacious anti-cancer agents, we were eager to exploit compound 10’s scaffold as a strategy to novel potent/efficacious MNKDAs with improved drug-like properties. The rationale of using this scaffold is based on experimental and clinical reports that unlike ATRA (1), 13-CRA (10) has long elimination half-life in humans14 and most animal species.15 In addition, we also explored rational modification of the terminal amide moiety, C-4 heterocycles and the cyclohexene ring (Figure 2). It is relevant to state here that our previous studies with the ATRA 5 ACS Paragon Plus Environment

OH


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scaffold MNKDAs identified the C-4 azolyl retinamides to be superior to the corresponding carboxylic acid and esters.8 17 1

7

2

6

20

19

16

11

9 8

10

13 14

12

15

5

3 4

18 Cyclohexenyl moiety

O

N

() n X

H Heteroaryl moieties Terminal Aryl amides

Figure 2: Overall Design Strategy for Novel C-4 Heteroaryl 13-Cis Retinamides

C-4 Azoyl/terminal Amide Modifications: Based on the continued successes of the C-imidazole retinamides with the ATRA scaffolds as promising anti-cancer agents,8 we designed and synthesized compounds 16 - 25 as outlined in Scheme 1.

Cyclohexene Rigidification/C-4 Aryl and Heteroaryl Modifications: On the basis of previous studies which showed that structural rigidification that introduces conformational constrains around rotatable bonds contributed to higher specificity and potency, greater metabolic stability, and improved bioavailability,16 we designed and synthesized several cyclohexadiene C-4 aryl substituted retinamides, compounds 33 - 40 (Scheme 2). A potential advantage of this strategy is that the achiral nature of these compounds would not require the tedious characterization of the racemates and pure enantiomers as would be required in advanced preclinical development for the chiral compounds of Scheme 1.17

Chemistry: Herein, we report, for the first time, the synthesis of 18 novel compounds based on the structural scaffold of 13-CRA (10), as outlined in Schemes 1 and 2. 6 ACS Paragon Plus Environment

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i 10

O

ii

OH

11

O

OCH3 O

12

O

OCH3

O

OCH3

iii v O N

iv

OH

O

15 N

OCH3 OH

N

14

13

N

vi

O N N

N( H

) n

X

Comp. 16 17 18 19 20

n 0 0 0 0 0

X H p -OH o -OH p -F m -F

Comp. 21 22 23 24 25

n 1 1 1 1 2

X H p -OH p -F m -F p -OH

Scheme 1: Synthesis of C-4 Azoyl/terminal Amide Compounds 17 – 22a a

Reagents and conditions: (i) (CH3)3SiCHN2, MeOH/benzene; (ii) MnO2, CH2Cl2; (iii) NaBH4,

MeOH; (iv) CDI, CH3CN; (v) 2M KOH, MeOH, reflux; (vi) EDC, HOBT, DIEA, appropriate anilines, DMF.

The synthesis of (2Z,4E,6E,8E)-9-(3-(1H-imidazol-1-yl)-2,6,6-trimethylcyclohex-1-en-1yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid (15) was carried out in five steps from the commercially available 13-CRA (10) and the procedure was adopted from our reported procedures of ATRA based retinamides.12b Protection of the carboxylic acid as the methyl ester (11) was done using trimethylsilyldiazomethane in hexanes followed by allylic oxidation using MnO2 to give 4-oxo intermediate (12). Reduction of 12 using NaBH4 gave the (±)-4hydroxymethylterionate (13) which was further treated with carbonyldiimidazole (CDI) at ambient temperature to yield (±)-(1H-imidazol-1-yl)methylterionate (14). Alkaline hydrolysis of 7 ACS Paragon Plus Environment


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14 in refluxing methanol gave the desired free acid 15 in 63% yield. Coupling of the respective amines with 15 was successfully achieved using ethyl(dimethylaminopropyl) carbodiimide (EDC), hydroxybenzotriazole (HOBt), diisopropylethylamine (DIEA) to give compounds 16 - 25 (Scheme 1), respectively, in average yields of ~50%.

i 12

O

O

ii

OCH3 OTf

O

26

O

OCH3

27, R1 =

30, R2 =

O

O

iii O

OH

OCH3

R1

28, R1 =

31, R2 =

N

N

R2

32, R2 =

iv

O

29, R1 =

N

N

N

N( H

) n

N

O X

N( H

) n

N

O X N

N( H

) n

N

O

33, n = 1, X = p -OH 34, n = 2, X = p -OH

35, n = 0, X = H 36, n = 1, X = p -OH 37, n = 2, X = p -OH

38, n = 0, X = H 39, n = 1, X = p -OH 40, n = 2, X = p -OH

Scheme 2: Synthesis of Cyclohexen Rigidification/C-4 Aryl and Heteroaryl Retinamidesa a

Reagents and conditions: (i) 5-Chloro-2-pyridyl triflimide, NaN(SiMe3)2, THF; (ii) 3-

methoxybenzylboronic acid, Pd(PPh3)4, CeCO3, dioxane, reflux; (iii) 2M KOH, MeOH, reflux (iv) EDC, HOBT, DIEA, appropriate benzylamine, DMF.

The synthesis of achiral C-4 aryl retinamides 33-40 (Scheme 2) commenced from the 4oxo intermediate (12). Generation of enolate using sodium bis(trimethylsilyl)amide in THF solution and trapping of the enolate with N-(5-chloro-2-pyridyl)bistrifluoromethanesulfonimide 8 ACS Paragon Plus Environment

X

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furnished the vinyl triflate 26 in 75 % yield according to the reported procedure.18 However, it should be noted that this reaction was carried out at -78°C throughout the reaction time, because, at higher temperatures, decomposition occurred and ≤ 20% of the products were obtained. Regiospecific palladium-catalyzed cross-coupling reaction with boronic acids19 provided compounds 27- 29 in 88, 69 and 60% yields, respectively. Ester hydrolysis using 2M KOH gave the corresponding free acids 30 - 32 in 62, 48 and 70% yields, respectively. Coupling of these respective acids with appropriate amines was successfully achieved using EDC, HOBt and DIEA to give compounds 33 – 40, respectively, in average yields of ~50% (Scheme 2).

Biological Studies: Effects of 13-Cis retinamides on the Growth of Breast and Prostate Cancers Cells in Vitro. We determined the effects of the 13-cis NRs on human cancer cell proliferation using a variety of breast and prostate cancer cells lines using our previously described MTT assay procedures.8, 12b, 20

The results (GI50 values) are summarized in Table 1. Growth inhibitory concentrations

(GI50 values) are the concentrations of compounds that cause 50% growth inhibition obtained from dose-response curves. The clinically relevant retinoids, ATRA and 4-HPR and the Mnk inhibitor, cercosporamide were used as comparators in the assays.

Anti-proliferative activities in breast cancer cell lines: As stated earlier, the structural modification focused on modifications of the terminal amide, cyclohexe ring and C-4 substituents. As presented in Table 1, several important generalizations emerge from the antiproliferative data. First, most of our compounds, including compounds 16 – 25, possess higher



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anti-proliferative activities towards all four breast cancer cell lines than the positive controls, ATRA, 4-HPR and the Mnk inhibitors, as evidenced by the lower GI50 values (Table 1).

Table 1: Inhibitory concentrations (GI50, µM) of NRs and reference compounds on the Growth of Human Breast Cancer Cells in Vitro Cpds.

GI50s (µM)

GI50s (µM)

GI50s (µM)

GI50s (µM)

MCF-7

MDA-MB-231

MDA-MB-468

SKBR-3

16

1.90 ± 0.00

1.74 ± 0.22

2.54 ± 0.12

1.73 ± 0.09

17

1.99 ± 0.26

13.05 ± 1.06

6.83 ± 0.33

3.89 ± 0.12

18

1.09 ± 0.00

4.67 ± 0.14

3.03 ± 0,39

3.89 ± 0.06

19

0.40 ± 0.08

4.95 ± 0.24

4.57 ± 0.00

25.11 ± 1.03

20

5.49 ± 0.11

8.51 ± 0.16

6.76 ± 0.23

16.98 ± 0.79

21

9.77 ± 0.64

5.88 ± 0.27

7.24 ± 0.14

10.71 ± 0.98

22

0.16 ± 0.00

2.23 ± 0.07

2.70 ± 0.35

2.45 ± 0.09

23

1.93 ± 0.16

1.41 ± 0.06

2.45 ± 0.06

5.50 ± 0.00

24

1.81± 0.15

1.65 ± 0.04

3.09 ± 0.18

4.24 ± 0.21

25

1.90 ± 0.08

2.88 ± 0.08

13.48 ± 0.97

1.41 ± 0.03

33

*

*

*

*

34

*

*

*

*

35

8.32 ±

25.11 ± 0.76

39.81 ± 1.33

47.40 ±

36

*

*

*

*

37

8.75 ±

26.91 ± 0.98

50.11 ± 2.40

42.24 ±

38

7.01 ±

26.91 ± 1.23

22.90 ± 1.03

30.98 ±


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39

4.96 ±

11.48 ± 1.07

19.05 ± 1.22

11.48 ±

40

2.96 ±

14.45 ± 0.92

23.44 ± 0.96

23.99 ±

14.12 ± 0.86

14.12 ± 0.55

22.90 ± 0.15

11.05 ±

6.02 ±

3.98 ±

43.65 ± 0.17

47.86 ± 0.23

26.02 ± 0.87

ATRAb 4-HPR Cerco.b

a

4.65 ±

The GI50 were determined from dose-response curves (by nonlinear regression analysis using

GraphPad Prism) compiled from at least three independent experiments using indicated breast cancer cells, SEM < 10%, and represents the compound concentration required to inhibit cell growth by 50%. b

Previously reported in Ramalingam S, et al.8a * represents ≥ 60% inhibition of breast cancer

cell growth at 10 µM.

Second, for each compound, the anti-proliferative activities towards the breast cancer cell lines varied significantly. In general, the MCF-7 cell line was the most sensitive, while the SKBR-3 were the least sensitive to these compounds. Third, for a given breast cancer cell line, the antiproliferative activity depends largely on the structure of the NR, which in turn depends largely on the nature of the C-4 heterocycle and cyclohexen/cyclohexadiene ring. [1] Compounds with the imidazole tethered to C-4 of the cyclohexene ring (16 – 25) exhibit more potent antiproliferative activities than compounds with phenyl (33 and 34), pyridine (35 – 37) or pyrimidine (38 – 40) tethered to C-4 of the cyclohexadiene ring. [2] Compared to compound 16 which possess an unsubstituted amide phenyl ring, compounds with substituted hydroxyl (17 and 18) or fluoro (19 and 20) groups displayed mixed anti-proliferative activities, but in general were either 11 ACS Paragon Plus Environment


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equipotent or less active than compound 16. The only exception was p-fluoro compound (19) with a GI50 = 0.4 µM against MCF-7 cells was ~4.8 fold more potent than 16 (GI50 = 1.90 µM). [3] Insertion of a methylene moiety between the amide and phenyl group clearly led to decreased anti-proliferative activities (compare GI50s of 16: 1.74 – 2.54 µM to GI50s of 21: 5.88 – 10.71 µM). [4] However, ortho/para hydroxyl or fluoro substitutions to benzyl group of 21 to give compounds 22 – 24 led to enhanced anti-proliferative activities across the four breast cancer cell lines. Amongst these three compounds, 22 was the most potent with GI50 values of 0.16, 2.23, 2.70 and 2.45 µM versus MCF-7, MDA-MB-231, MDA-MB-468 and SKBR-3 cell lines, respectively. Related compound 25 with an ethylene moiety between the amide and p-hydroxyl group led to varied anti-proliferative activities. It is notable that 25 amongst these compounds, exhibited the best activity (GI50 = 1.41 µM) against SKBR-3 cell line.

Anti-proliferative activities in prostate cancer cell lines: The GI50 values of compounds 16 – 22 against three human prostate cancer cell lines, including, LNCaP, CWR22Rv1 and PC-3 were determined and compared to the GI50 values of clinically relevant drugs, ATRA, 4-HPR, casodex and MDV3100 (enzalutamide) (Table 2). In general, the compounds were more potent than ATRA, but were equipotent to 4-HPR, casodex and MDV3100. The PC-3 cell line was the least sensitive to all the compounds tested. Compounds 20 and 22 exhibited the most potent antiproliferative activities across the three cell lines.


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Table 2:

Inhibitory concentrations (GI50, µM) of NRs and reference compounds on the Growth of Human Prostate Cancer Cells in Vitro GI50s (µM)a

GI50s (µM)a

GI50s (µM)a

LNCaP

CWR22Rv1

PC-3

16

2.69 ± 0.09

1.28 ± 0.03

8.91 ± 0.51

17

3.54 ± 0.18

3.89 ± 0.08

11.74 ± 0.76

18

3.23 ± 0.19

2.23 ± 0.11

15.48 ± 0.87

19

3.89 ± 0.23

8.12 ± 0.52

30.19 ± 0.94

20

2.69 ± 0.14

2.04 ± 0.01

5.62 ± 0.03

21

*

*

*

22

1.69 ± 0.07

1.86 ± 0.06

3.54 ± 0.02

ATRAb

47.86 ± 0.21

25.11 ± 0.16

36.3 ± 0.27

4-HPRb

2.69 ± 0.14

3.23 ± 0.08

3.54 ± 0.09

Casodexb

2.61 ± 0.09

3.81 ± 0.18

9.15 ± 0.14

MDV3100b

2.88 ± 0.13

3.34 ± 0.25

9.15 ± 0.26

Cpds.

a

The GI50 were determined from dose-response curves (by nonlinear regression analysis using

GraphPad Prism) compiled from at least three independent experiments using LNCaP cells, SEM < 10%, and represents the compound concentration required to inhibit cell growth by 50%. b

Previously reported in Ramamurthy V et al.8b * Indicates compound requirement > 100 µM to

induce 50 % inhibition of cell growth in PC cells



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In vitro characterization of compounds 16, 20 and 22 in breast cancer cell lines: We previously reported that the anti-proliferative effects of the 1st generation MNKDAs in breast cancer cells was due to degradation of Mnk1/2 with subsequent depletion of peIF4E and induction of apoptosis.8a To determine if these compounds modulate Mnk1/2 and related oncogenic proteins, we performed western blot of lysates from MCF-7, MDA-MB-231 and MDA-MB-468 human breast cancer cells treated with compounds 16, 20, and 22 or with vehicle (DMSO, negative control) or compound 6 (positive control). Figure 3A-C shows that the three compounds significantly and dose-dependently, reduced the expressions of Mnk1, Mnk2 and peIF4E, with no noticeable effects on the expression of total eIF4E. The compounds also caused dose-dependent formation of c-PARP which indicates induction of cell death (apoptosis).


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Figure 3: Effect of compounds 16, 20 and 22 on Mnk1 and 2, peIF4E, eIF4E and cPARP proteins in MCF-7 cells. Equal protein concentrations from MCF-7 (A), MDA-MB-231 (B) and MDA-MB-468 (C) cells treated for 24 h with 16, 20 and 22 at the various concentrations as indicated and 6 (20 µmol/L) were separated by SDS-PAGE and western blots probed with antibodies to Mnk1 and 2, peIF4E, eIF4E and cPARP. Vehicle treated cells were included as control and all blots were reprobed for GAPDH for loading control. Based on previous studies with closely related compound,8 we evaluated the inhibitory effects of compounds 16, 20 and 22 on cell migration, using the human metastatic breast cancer cell line, MDA-MB-231 by wound-healing assay. Figure 4 shows that the compounds 20 and 22 significantly inhibited migration of MDA-MB-231 cells compared to the control. At 24 h after cell monolayers were wounded, control cells had completely filled in the scratched area.

Figure 4: Effects of compounds 16, 20 and 22 on MDA-MB-231 cell migration: MDA-MB231 cells (5 x 105 cells/well) were seeded on Boyden chamber, grown to confluence and scratches made at experimental time zero. The cells were treated with indicated compounds (5 µM each) for 24h. Representative photomicrographs of initial and final wounds are shown at 100x magnification.



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In vitro characterization of compounds 16, 20 and 22 in prostate cancer cell lines: Previously, we demonstrated that the anti-tumor activity of the early NRs in prostate cancer cells was due to simultaneous inhibition of the Mnk/eIF4E and androgen receptor (AR) signaling pathways.8b Similar to studies described above, we performed western blot of lysates from LNCaP human prostate cancer cells treated with compounds 16, 20, and 22 or with vehicle (DMSO, negative control). Figure 5A shows that the three compounds significantly and in a dose-dependent fashion, reduced the expressions of fAR, Mnk1 and Mnk2, with no noticeable effects on the expression of total eIF4E. In addition, the compounds caused significant depletion of oncogenic cyclin D1 and they each induced cleaved PARP (c-PARP), a reliable marker of apoptotic cell death (Figure 5B).

Figure 5: Effects of compounds 16, 20 and 22 on the expression of fAR, Mnk, peIF4E, cyclin D1 and cleaved PARP in LNCaP cells. Equal protein concentrations from LNCaP cells treated with 16, 20 and 22 at different concentrations (0.6-20 µM) for 24 h were separated by SDS-PAGE and western blots probed with antibodies to fAR, Mnk1/2, peIF4E, cyclin D1 and


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cleaved PARP. Vehicle treated cells were included as a control and all blots were reprobed for βactin for loading control.

Based on previous studies with closely related compound,8 we evaluated the inhibitory effects of compounds 16, 20 and 22 on cell migration, using the human metastatic PC cell line, PC-3 by wound-healing assay. Figure 6 shows that the three compounds significantly inhibited migration of PC-3 cells compared to the control. At 24 h after cell monolayers were wounded, control cells had completely filled in the scratched area.

Figure 6: Effects of compounds 16, 20 and 22 on PC-3 cell migration: PC-3 cells (5 x 105 cells/well) were seeded on Boyden chamber, grown to confluence and scratches made at experimental time zero. The cells were treated with indicated compounds (5 µM each) for 24h. Representative photomicrographs of initial and final wounds are shown at 100x magnification.

Since AR is a major driver of proliferation in PCa,21 we next examined the effect of these three compounds on AR transcriptional activity in LNCaP cells. As shown in Figure 7, 24 h exposure of LNCaP cells to lead NRs, 16, 20, and 22 (10 µM) resulted in a 2 – 4-fold dramatic



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inhibition of DHT induced AR transcriptional activity that was however less potent than that observed upon clinically available MDV3100 treatment. Of these leads, compound 20 was the most potent.

Figure 7: Effects of compounds 16, 20, and 22 on DHT-induced AR transactivation in LNCaP cells. LNCaP cells dual transfected with ARR2-Luc and the Renilla luciferase reporting vector pRL-null and treated with 10 µM of specified compounds for 18 h in the presence of 10 nmol/L dihydrotestosterone (DHT). Control represents baseline activity without androgen stimulation. Androgen stimulated luciferase activity (luminescence) was measured in a victor 1420 plate reader. The results are presented as the fold induction (i.e., the relative luciferase ♣

activity of the treated cells divided by that of control) normalized to that of Renilla. *, P< 0.05; ,

P

AR Degrading Agents

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