International Journal of
Molecular Sciences Article
Efficient Lead Finding, Activity Enhancement and Preliminary Selectivity Control of Nuclear Receptor Ligands Bearing a Phenanthridinone Skeleton Yuko Nishiyama 1 , Shinya Fujii 1 , Makoto Makishima 2 and Minoru Ishikawa 1, * ID 1 2
*
ID
, Yuichi Hashimoto 1
Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan;
[email protected] (Y.N.);
[email protected] (S.F.);
[email protected] (Y.H.) Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan;
[email protected] Correspondence:
[email protected]; Tel.: +81-358-417-853
Received: 25 June 2018; Accepted: 17 July 2018; Published: 18 July 2018
Abstract: Background: Nuclear receptors (NRs) are considered as potential drug targets because they control diverse biological functions. However, steroidal ligands for NRs have the potential to cross-react with other nuclear receptors, so development of non-steroidal NR ligands is desirable to obtain safer agents for clinical use. We anticipated that efficient lead finding and enhancement of activity toward nuclear receptors recognizing endogenous steroidal ligands might be achieved by exhaustive evaluation of a steroid surrogate library coupled with examination of structure-activity relationships (SAR). Method: We evaluated our library of RORs (retinoic acid receptor-related orphan receptors) inverse agonists and/or PR (progesterone receptor) antagonists based on the phenanthridinone skeleton for antagonistic activities toward liver X receptors (LXRs), androgen receptor (AR) and glucocorticoid receptor (GR) and examined their SAR. Results: Potent LXRβ, AR, and GR antagonists were identified. SAR studies led to a potent AR antagonist (IC50 : 0.059 µM). Conclusions: Our approach proved effective for efficient lead finding, activity enhancement and preliminary control of selectivity over other receptors. The phenanthridinone skeleton appears to be a promising steroid surrogate. Keywords: phenanthridinone; steroid surrogate; antagonist
1. Introduction Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate DNA transcription by binding small molecular agonists such as hormones. Forty-eight structurally conserved kinds of NRs have been identified, and many of them recognize endogenous steroidal ligands (Figure 1). Because NRs control diverse biological functions including reproduction, differentiation, homeostasis, and the immune system, they were the targets of approximately 5% of FDA-approved drugs in 2011 [1]. However, not only a steroidal ligand, but also its metabolites, brings with it the potential to cross-react with other nuclear receptors, which can result in unwanted side effects, potentially limiting the clinical utility of these agents. Therefore, surrogates for the steroid skeleton are required to develop selective non-steroidal NR ligands for clinical use [2].
Int. J. Mol. Sci. 2018, 19, 2090; doi:10.3390/ijms19072090
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Figure Chemical structures of endogenous steroidal ligands for and NRs and phenanthridinone Figure 1.1. Chemical structures of endogenous steroidal ligands for NRs phenanthridinone ligands ligands for target proteins that recognize endogenous steroidal ligands. for target proteins that recognize endogenous steroidal ligands.
The number of fold structures of human proteins is at least 50 times smaller than the number of The number of fold structures of human proteins is at least 50 times smaller than the number human proteins [2]. This fact indicates that a scaffold that is spatially complementary to one‐fold of human proteins [2]. This fact indicates that a scaffold that is spatially complementary to one-fold structure might serve as a common scaffold for ligands that would interact specifically with more structure might serve as a common scaffold for ligands that would interact specifically with more than than 50 different human proteins, neglecting interactions of the peptide sequences of the proteins. In 50 different human proteins, neglecting interactions of the peptide sequences of the proteins. In other other words, the structures of ligands that bind to one member of fold structures may be useful for the words, the structures of ligands that bind to one member of fold structures may be useful for the development of novel lead compounds for other proteins with the similar fold structure. However, development of novel lead compounds for other proteins with the similar fold structure. However, lead lead compounds obtained based on this approach are likely to possess polypharmacological character. compounds obtained based on this approach are likely to possess polypharmacological character. Therefore, chemical modification of polypharmacological lead compounds, aimed at optimizing Therefore, chemical modification of polypharmacological lead compounds, aimed at optimizing selectivity for a particular target, is required. Since the fold structures of NR ligand‐binding domains selectivity for a particular target, is required. Since the fold structures of NR ligand-binding domains (LBDs) are similar, we have used this strategy to create ligands bearing a diphenylmethane skeleton (LBDs) are similar, we have used this strategy to create ligands bearing a diphenylmethane skeleton for several NRs, including farnesoid X receptor [3], liver X receptor (LXR) [4], vitamin D receptor [5], for several NRs, including farnesoid X receptor [3], liver X receptor (LXR) [4], vitamin D receptor [5], androgen receptor (AR) [6], and estrogen receptor [7]. androgen receptor (AR) [6], and estrogen receptor [7]. We have previously developed LXRs antagonist 1 [8], retinoic acid receptor‐related orphan We have previously developed LXRs antagonist 1 [8], retinoic acid receptor-related orphan receptors (RORs) inverse agonist 2 [9], progesterone receptor (PR) antagonist 3 [10], and receptors (RORs) inverse agonist 2 [9], progesterone receptor (PR) antagonist 3 [10], and pharmacological pharmacological chaperones 4 and 5 for Niemann‐Pick disease type C1 (NPC1) [11] and chaperones 4 and 5 for Niemann-Pick disease type C1 (NPC1) [11] and Niemann-Pick type C1-like 1 Niemann‐Pick type C1‐like 1 (NPC1L1) (Figure 1) [12]. These compounds all contain a (NPC1L1) (Figure 1) [12]. These compounds all contain a phenanthridin-6(5H)-one skeleton as a cyclized phenanthridin‐6(5H)‐one skeleton as a cyclized carba‐analog of the skeleton of LXRs pan agonist carba-analog of the skeleton of LXRs pan agonist T0901317 (6). Because endogenous ligands for the T0901317 (6). Because endogenous ligands for the above proteins (LXRs, RORs, PR, NPLC1 and above proteins (LXRs, RORs, PR, NPLC1 and NPC1L1) all have a steroid skeleton, we hypothesized that NPC1L1) all have a steroid skeleton, we hypothesized that if the phenanthridin‐6(5H)‐one scaffold if the phenanthridin-6(5H)-one scaffold acts as a steroid surrogate, phenanthridinone derivatives would acts as a steroid surrogate, phenanthridinone derivatives would also bind to other NRs that also bind to other NRs that recognize endogenous steroidal ligands. The approach described here is recognize endogenous steroidal ligands. The approach described here is expected to be useful in the expected to be useful in the development of novel steroid surrogates, such as the phenanthridinone development of novel steroid surrogates, such as the phenanthridinone skeleton, enabling efficient skeleton, enabling efficient lead finding of ligands for target proteins that recognize endogenous lead finding of ligands for target proteins that recognize endogenous steroidal ligands, and the steroidal ligands, and the generation of more selective ligands by further molecular modifications of generation of more selective ligands by further molecular modifications of new and convenient new and convenient scaffolds (easy to synthesis, easy to introduce substitutent(s) at any position, etc.), scaffolds (easy to synthesis, easy to introduce substitutent(s) at any position, etc.), compared to the compared to the steroid skeleton. Here, we show that application of this approach to our small library steroid skeleton. Here, we show that application of this approach to our small library of of phenanthridinone-based RORs inverse agonists and PR antagonists led to several selective LXRβ, phenanthridinone‐based RORs inverse agonists and PR antagonists led to several selective LXRβ, AR and glucocorticoid receptor (GR) antagonists. AR and glucocorticoid receptor (GR) antagonists. 2. Results and Discussion 2. Results and Discussion AR and GR, members of NRs, recognize endogenous steroidal ligands as shown in Figure 1. AR and GR, members of NRs, recognize endogenous steroidal ligands as shown in Figure 1. In In addition, because LXRs antagonistic activities of only limited numbers of phenanthridinone addition, because LXRs antagonistic activities of only limited numbers of phenanthridinone analog analog have been reported [8], precise structure-activity relationships (SAR) remain unclear. have been reported [8], precise structure‐activity relationships (SAR) remain unclear. Therefore, we Therefore, we exhaustively evaluated our small library of RORs inverse agonists and/or PR exhaustively evaluated our small library of RORs inverse agonists and/or PR antagonists bearing a antagonists bearing a phenanthridinone skeleton for LXRα, LXRβ, AR and GR antagonistic activities, phenanthridinone skeleton for LXRα, LXRβ, AR and GR antagonistic activities, and examined their and examined their SAR. LXRs regulate ATP-binding cassette proteins (ABCs), ApoE and glucose SAR. LXRs regulate ATP‐binding cassette proteins (ABCs), ApoE and glucose transporter 4 (GLUT4), are involved in lipid metabolism, reverse cholesterol transport, and glucose transport, so
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transporter 4 (GLUT4), are involved in lipid metabolism, reverse cholesterol transport, and glucose transport, so LXRs agonists are likely to be of therapeutic value in the treatment of atherosclerosis, hyperlipidemia, and metabolic syndrome. They are also involved in the upregulation of sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS), so LXRs antagonists might have therapeutic value for hepatic steatosis [13]. AR is a receptor of androgens essential for the development and maintenance of the male reproductive system and secondary male sex characteristics. AR antagonists including hydroxyflutamide (OHF) are used for treatment of androgen-dependent tumors, especially prostate tumors. GR is a receptor of cortisol, and selective antagonists of GR could be useful in treating hypercortisolemia associated with Cushing’s syndrome and other conditions in which the endogenous GR is hyperactivated either through higher glucocorticoid levels or increased receptor sensitivity [14]. A steroidal GR antagonist, mifepristone (RU486), shows potent activities toward other steroid receptors. For this work, compounds 1–3, 7–42 and 44 were prepared as described previously [9,10,15]. PR-antagonistic activity was evaluated by assay of PR-regulated alkaline phosphatase activity in human breast cancer cell line T47D [10,16] while RORα, RORβ and RORγ inverse agonistic activities, and LXRα, LXRβ, AR and GR antagonistic activities were evaluated by reporter gene assay in HEK293 cell line [9,10]. Concentration of the agonists were set as around their EC50 values. Reproducibility of our assay systems is shown in Table S1. First, we focused on the SAR at the 2-position of phenanthridinone. The results, including the activities of positive controls T0901317 (6), RU486, and OHF, are shown in Table 1. We have reported that introduction of alkyl groups (8–10) or a hydroxymethyl group (11) at the 2-position of 7 resulted in retention or decrease of the PR-antagonistic activity [10] and RORs inverse agonistic activity [9]. Introduction of a hexafluoropropanol moiety somewhat increased the RORs activity and PR activity. According to the reported X-ray crystal structures of compound 6 and human LXRs LBD, the hydroxyl group of 6 forms a hydrogen bond with a histidine residue (His421 for LXRα and His435 for LXRβ) in helix 11 [17,18]. The SARs for RORs and PR are broadly consistent with those of LXRs, AR, and GR. For AR and PR, introduction of a hydroxymethyl group (11) resulted in retention of the activity, whereas introduction of hexafluoropropanol (12) resulted in 6-fold and 9-fold increases of PR and AR activity, respectively. On the other hand, for LXRs and RORs, 11 showed weaker activity than 7 whereas 12 showed stronger activity than 7, suggesting that the two trifluoromethyl groups might be associated with more potent activity and the hydroxyl group might not form a strong hydrogen bond with histidine in these receptors, in contrast to the other receptors. This idea is consistent with the reported co-crystal structure of T0901317 (6) complexed with RORγ [19]. Overall, we found that compound 12 showed not only PR-antagonistic activity and RORs inverse agonistic activity, but also LXRα, LXRβ, AR and GR antagonistic activity (Table 1). These results support our view that the phenanthridin-6(5H)-one scaffold acts as a steroid surrogate. Next, SAR at the nitrogen atom is shown in Table 2. Similar to the SAR for RORs, introduction of a longer-chain alkyl group on the nitrogen atom (12 and 15) resulted in enhancement of the GR antagonistic activity. SAR for AR was similar to that for PR, that is, hydrogen analog 12 showed potent AR antagonistic activity with an IC50 value of 0.10 µM, and more than 200-fold selectivity for AR over RORs and GR, and about 7.8- and 50-fold selectivity over PR and LXRs, respectively. The longest alkyl analog 17 showed decreased activity toward all NRs, suggesting that the binding pocket hosting the N-alkylated derivatives might not be able to accommodate a group larger than a hexyl group. Next, SAR at the nitrogen atom is shown in Table 2. Similar to the SAR for RORs, introduction of a longer-chain alkyl group on the nitrogen atom (12 and 15) resulted in enhancement of the GR antagonistic activity. SAR for AR was similar to that for PR, that is, hydrogen analog 12 showed potent AR antagonistic activity with an IC50 value of 0.10 µM, and more than 200-fold selectivity for AR over RORs and GR, and about 7.8- and 50-fold selectivity over PR and LXRs, respectively. The longest alkyl analog 17 showed decreased activity towards all NRs, suggesting that the binding pocket hosting the N-alkylated derivatives might not be able to accommodate a group larger than a hexyl group.
bond with histidine in these receptors, in contrast to the other receptors. This idea is consistent with the reported co‐crystal structure of T0901317 (6) complexed with RORγ [19]. Overall, we found that compound 12 showed not only PR‐antagonistic activity and RORs inverse agonistic activity, but also LXRα, LXRβ, AR and GR antagonistic activity (Table 1). These results support our view that the Int. J. Mol. Sci. 2018, 19, 2090 4 of 12 phenanthridin‐6(5H)‐one scaffold acts as a steroid surrogate. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW
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Table 1. SAR at 2‐position. Table 1. SAR at 2-position.
Next, SAR at the nitrogen atom is shown in Table 2. Similar to the SAR for RORs, introduction of a longer‐chain alkyl group on the nitrogen atom (12 and 15) resulted in enhancement of the GR antagonistic activity. SAR for AR was similar to that for PR, that is, hydrogen analog 12 showed potent AR antagonistic activity with an IC50 value of 0.10 μM, and more than 200‐fold selectivity for AR over RORs and GR, and about 7.8‐ and 50‐fold selectivity over PR and LXRs, respectively. The longest alkyl Compound R RORα 1 RORβ 1 RORγ 1 LXRα 2 LXRβ 2 PR 2,3 AR 2 GR 2 analog 17 showed decreased activity toward all NRs, suggesting that the binding pocket hosting the 1 1 1 Compound R RORα RORβ RORγ LXRα 2 4 LXRβ 2 4 PR 2,3 AR 2 GR 2 6 ‐ >20,000 >20,000 6500 290 130 N‐alkylated derivatives might not be able to accommodate a group larger than a hexyl group. 6 - ‐ >20,000 >20,000 6500 290 4 130 4 RU486 0.073 2.0 Next, SAR at the nitrogen atom is shown in Table 2. Similar to the SAR for RORs, introduction RU486 - ‐ 0.073 2.0 OH‐Flu 170 of a OH-Flu longer‐chain on the nitrogen (12 and 15) resulted enhancement of the GR -alkyl 170 7 H group 10,000 13,000 atom 15,000 19,000 15,000 in 7100 17,000 >20,000 7 8 H Me 10,000 17,000 >20,000 18,000 13,000 17,000 15,000 17,000 19,000 17,000 15,000 16,000 7100 7900 11,000 >20,000 antagonistic activity. SAR for AR was similar to that for PR, that is, hydrogen analog 12 showed potent 8 9 Me Et 18,000 17,000 17,000 17,000 16,000 7900 11,000 >20,000 9000 50 value of 0.10 μM, and more than 200‐fold selectivity for AR over 7900 9200 17,000 16,000 7800 19,000 19,000 AR antagonistic activity with an IC 9 10 Et t‐Bu 9000 19,000 19,000 12,000 7900 13,000 9200 10,000 17,000 >20,000 16,000 >20,000 7800 8300 17,000 >20,000 RORs and GR, and about 7.8‐ and 50‐fold selectivity over PR and LXRs, respectively. The longest alkyl 1011 t-Bu 12,000 13,000 10,000 >20,000 >20,000 8300 17,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 8600 9600 >20,000 CH2OH 11 CH2 OH >20,000 >20,000 >20,000 >20,000 >20,000 8600 9600 >20,000 analog 17 showed decreased activity towards all NRs, suggesting that the binding pocket hosting the 12 9800 6500 3900 8400 3700 1200 1800 7400 (CF3)2COH 12 (CF3 )2 COH 9800 6500 3900 8400 3700 1200 1800 7400 N‐alkylated derivatives might not be able to accommodate a group larger than a hexyl group. 1 Inverse agonistic activity (IC50: nM) [9]. 2 Antagonistic activity (IC50: nM). 3 [10]. 4 Agonistic activity 1
Inverse agonistic activity (IC50 : nM) [9]. 2 Antagonistic activity (IC50 : nM). 3 [10]. 4 Agonistic activity (IC50 : nM).
(IC50: nM).
Table 2. SAR at nitrogen atom. Table 2. SAR at nitrogen atom.
Compound Compound 1 113 14 13 15 1416 1517
16 17
R R H HMe Et Me n‐Pr Et n‐Hex n-Pr n‐C9H19
RORα 1 1 RORα >20,000 >20,000 >20,000 >20,000 >20,000 11,000 >20,000 7600 11,000 >20,000
RORβ 1 1 RORβ >20,000 >20,000 >20,000 >20,000 >20,000 8200 >20,000 5400 8200 >20,000
RORγ 1 1 RORγ >20,000 >20,000 >20,000 >20,000 >20,000 4200 >20,000 4700 4200 >20,000
LXRα 2 2 LXRα 5000 4 4 4100 5000 14,000 4100 4 12,000 14,000 16,000 12,000 >20,000
LXRβ 2
PR 2,3
2 LXRβ 5100 4
2,3 PR 780
>20,000 5100 4 4 6600 >20,000 4 6900 6600 10,000 6900 >20,000
340 780 1400 340 3800 1400 2500 3800 10,000
AR 2 AR 2 100 500 100 1700 500 5600 1700 4000 5600 15,000
n-Hex 7600 5400 4700 16,000 10,000 2500 4000 Inverse agonistic activity (IC 50: nM) [9]. 2 Antagonistic activity (IC50: nM). 3 [10].4 [8].
1
n-C9 H19 1
>20,000
>20,000
>20,000 2
>20,000
>20,000
10,000 3
15,000
GR 2 GR 2 >20,000 >20,000 >20,000 >20,000 >20,000 17,000 >20,000 >20,000 17,000 >20,000
>20,000 >20,000
4
Inverse agonistic activity (IC50 : nM) [9]. Antagonistic activity (IC50 : nM). [10]. [8]. Next, the effect of introduction of a methoxy group at every position (methoxy scanning) was investigated (18–24; Table 3) because many types of NR ligands possess a methoxy group(s) [20]. We Next, the effect of introduction of a methoxy group at every position (methoxy scanning) was have reported that 3‐methoxy and 4‐methoxy analogs 19 and 20 showed more than 3‐fold and 2‐fold investigated Table 3) because types of NR analog ligands 12, possess a methoxy group(s) [20]. enhanced PR (18–24; activity compared with many the unsubstituted respectively [10]. In contrast, We have reported that 3-methoxy and 4-methoxy analogs 19 and 20 showed more than 3-fold and 9‐methoxy analog 23 showed 5‐fold weaker PR activity than 12. In the case of RORs, we have 2-fold enhanced PR activity compared with the unsubstituted analog 12, respectively [10]. In contrast, reported that introduction of a methoxy group at the 9‐position (23) enhanced ROR activities [9]. The 9-methoxy analog 23 showed 5-fold weaker PR activity than 12. In the case of RORs, we have reported activities toward LXRs, AR and GR also depended on the position of the methoxy group. 1‐Methoxy that introduction of a methoxy group at the 9-position (23) enhanced ROR activities [9]. The activities analog 18 showed decreased activity for all receptors tested. Enhanced activity was observed when a toward LXRs, AR and GR also depended on the position of the methoxy group. 1-Methoxy analog 18 methoxy group was introduced at the 4‐, 9‐ or 10‐position for LXRα, 9‐ or 10‐position for LXRβ, 3‐ showed decreased activity for all receptors tested. Enhanced activity was observed when a methoxy and 8‐position for AR, and 3‐ or 4‐position for GR. Especially, 3‐methoxy analog 19 showed 5.7‐fold group was GR introduced the 4-, 9- orwith 10-position for LXRα, 9- or 10-position for LXRβ, 3- andresulted 8-position improved activity atcompared 12. Overall, introduction of a methoxy group in for AR, and 3or 4-position for GR. Especially, 3-methoxy analog 19 showed 5.7-fold improved GR increased activity in almost all cases except RORβ, but the most suitable position depended on the activity compared with 12. Overall, introduction of a methoxy group resulted in increased activity in NR (3‐position: PR and GR, 8‐position: AR, 9‐position: RORα, RORγ and LXRβ, 10‐position: LXRα). almost all cases except RORβ, but the most suitable position depended on the NR (3-position: PR and The introduction of a substituent at every position is useful to investigate substituent effects. In this GR, 8-position: AR, 9-position: RORα, RORγ and LXRβ, 10-position: LXRα). The introduction of a context, it is important to note that phenanthridin‐6(5H)‐one analogs bearing substituent(s) at any substituent at every position is useful to investigate substituent effects. In this context, it is important position can be quite easily synthesized, and this represents a considerable advantage compared to to note that phenanthridin-6(5H)-one analogs bearing substituent(s) at any position can be quite easily the steroid skeleton. synthesized, and this represents a considerable advantage compared to the steroid skeleton. The results of methoxy scanning prompted us to investigate the effect of introduction of two The results methoxy scanning prompted us to investigate theGR, effect of introduction of two methoxy groups of (25–31 Table 3). In the cases of RORα, PR, AR and 3,4‐dimethoxy analog 25 methoxy groups (25–31 Table 3). In the cases of RORα, PR, AR and GR, 3,4-dimethoxy analog 25 exhibited similar or weaker activity to the 3‐methoxy and 4‐methoxy analogs 19 and 20. A possible exhibited similar or weaker activity to the 3-methoxy and 4-methoxy analogs 19 and 20. A possible explanation might be the direction of the two methoxy groups, that is, one or both might occupy a explanation might be the direction of the two methoxy groups, that is, one or both might occupy a space space not suitable for receptor interaction due to steric hindrance. This idea is supported by the fact
that 3,4‐dioxolane analog 26 showed stronger activity than 25 toward PR, AR and GR. Compound 26 was the most potent GR antagonist with an IC50 value of 0.76 μM, although this compound also
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showed PR and AR antagonistic activities with IC50 values of 0.23 and 0.54 μM, respectively. When not suitable for receptor interaction due to steric hindrance. This idea is supported by the fact that two methoxy groups were introduced at distal positions, the activity was improved. Thus, Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 12 3,4-dioxolane analog 26 showed stronger activity than 25 toward PR, AR and GR. Compound 26 was 3,8‐dimethoxy analog 27 and 4,8‐dimethoxy analog 28 showed more potent PR‐antagonistic activity the most potent GR antagonist with an IC50 value of 0.76 µM, although this compound also showed PR showed PR and AR antagonistic activities with IC 50 values of 0.23 and 0.54 μM, respectively. When than monomethoxy analogs 19, 20, and 22, and 3,8‐dimethoxy analog 27 showed more potent AR and AR antagonistic activities with IC50 values of 0.23 and 0.54 µM, respectively. When two methoxy two methoxy groups were introduced at distal positions, the activity was improved. Thus, antagonistic activity than monomethoxy analogs 19 and 22. groups were introduced at distal positions, the activity was improved. Thus, 3,8-dimethoxy analog 3,8‐dimethoxy analog 27 and 4,8‐dimethoxy analog 28 showed more potent PR‐antagonistic activity 27 and 4,8-dimethoxy analog 28 showed more potent PR-antagonistic activity than monomethoxy Table 3. SAR of substitution effect of alkoxy groups. than monomethoxy analogs 19, 20, and 22, and 3,8‐dimethoxy analog 27 showed more potent AR analogs 19, 20, and 22, and 3,8-dimethoxy analog 27 showed more potent AR antagonistic activity than antagonistic activity than monomethoxy analogs 19 and 22. monomethoxy analogs 19 and 22. Table 3. SAR of substitution effect of alkoxy groups. Table 3. SAR of substitution effect of alkoxy groups. Compound
R
RORα 1
RORβ 1
RORγ 1
LXRα 2
LXRβ 2
PR 2,3
AR 2
GR 2
18
1‐OMe
>20,000
>20,000
>20,000
>20,000
>20,000
4000
>20,000
>20,000
19
3‐OMe
8200
8200
7500
8400
9200
410
1100
20 Compound Compound 21 18 18 22 19 19 23 20 20 24 21 21 22 25 23 22 26 24 23 27 25 24 26 28 25 27 29 28 26 30 29 27 30 31 28 31 29 30
2,3 2 4‐OMe 7500 1 6900 1 5400 1 6900 2 5500 2 560 2400 R RORα RORβ RORγ LXRα LXRβ PR AR R RORα 1 RORβ 1 RORγ 1 LXRα 2 LXRβ 2 PR 2,3 AR 2 7‐OMe 12,000 15,000 13,000 11,000 12,000 12,000 9400 1‐OMe >20,000 >20,000 >20,000 >20,000 >20,000 4000 >20,000 1-OMe >20,000 >20,000 >20,000 >20,000 >20,000 4000 >20,000 8‐OMe 12,000 15,000 13,000 12,000 11,000 1100 1000 3‐OMe 8200 8200 7500 8400 9200 410 1100 3-OMe 8200 8200 7500 8400 9200 410 1100 9‐OMe 6600 7200 2700 7000 2400 5800 >20,000 4-OMe 7500 6900 5400 6900 5500 560 2400 4‐OMe 7500 6900 5400 6900 5500 560 2400 7-OMe 12,000 15,000 13,000 11,000 12,000 12,000 9400 10‐OMe 7700 >20,000 8500 4600 4300 2700 >20,000 7‐OMe 12,000 15,000 13,000 11,000 12,000 12,000 9400 8-OMe 12,000 15,000 13,000 12,000 11,000 1100 1000 3,4‐diOMe >10,000 >10,000 8500 8500 9600 510 6100 8‐OMe 12,000 15,000 13,000 12,000 11,000 1100 1000 9-OMe 6600 7200 2700 7000 2400 5800 >20,000 3,4‐(OCH 2O) >20,000 >20,000 >20,000 >20,000 >20,000 230 540 10-OMe 7700 >20,000 8500 4600 4300 2700 >20,000 9‐OMe 6600 7200 2700 7000 2400 5800 >20,000 3,4-diOMe >10,000 >10,000 8500 8500 9600 510 6100 3,8‐diOMe 9500 13,000 12,000 17,000 18,000 300 580 10‐OMe 7700 >20,000 8500 4600 4300 2700 >20,000 3,4-(OCH2 O) >20,000 >20,000 >20,000 >20,000 >20,000 230 540 4,8‐diOMe 11,000 11,000 9700 >10,000 >10,000 340 1700 3,4‐diOMe >10,000 >10,000 8500 8500 9600 510 6100 3,8-diOMe 9500 13,000 12,000 17,000 18,000 300 580 7,8‐diOMe 7300 7700 8000 3200 3200 4200 4600 3,4‐(OCH 2O) >20,000 >20,000 >20,000 >20,000 >10,000 >20,000 230 540 4,8-diOMe 11,000 11,000 9700 >10,000 340 1700 7,8-diOMe 7300 7700 8000 3200 3200 4200 4600 >20,000 7,9‐diOMe >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 3,8‐diOMe 9500 13,000 12,000 17,000 18,000 300 580 7,9-diOMe >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 8,9‐diOMe 18,000 16,000 19,000 8800 8900 9600 1600 4,8‐diOMe 11,000 11,000 9700 >10,000 >10,000 340 1700 8,9-diOMe 18,000 16,000 19,000 8800 8900 9600 1600 17,8‐diOMe 2 Antagonistic activity (IC 3 [10]. Inverse agonistic activity (IC 50 : nM) [9]. 50 : nM). 7300 7700 8000 3200 3200 4200 4600 1 Inverse agonistic activity (IC : nM) [9]. 2 Antagonistic activity (IC : nM). 3 [10]. 50 50 >20,000 7,9‐diOMe >20,000 >20,000 >20,000 >20,000 >20,000 >20,000
1300 2 3900 GR GR 2 14,000 >20,000 >20,000 8200 1300 1300
10,000 3900 3900 14,000 >20,000 14,000 8200 >20,000 8200 10,000 760 >20,000 10,000 >20,000 1700 >20,000 760 5200 >20,000 1700 7900 760 5200 7900 >20,000 1700 >20,000 17,000 5200 17,000 7900 >20,000
We next examined the SAR of fluoro derivatives 32–35 (Table 4) to investigate the effect of this 31 8,9‐diOMe 18,000 16,000 19,000 8800 8900 9600 1600 17,000
We next examined the SAR of fluoro derivatives 32–35 (Table 4) to investigate the effect of this 1 Inverse agonistic activity (IC 2 Antagonistic activity (IC electron‐withdrawing substituent. For PR, the preferences for fluorine substitution 50: nM) [9]. 50: nM). 3 [10]. position were electron-withdrawing substituent. For PR, the preferences for fluorine substitution position were roughly consistent with the results of the methoxy scanning, that is, 4‐ and 8‐substitued analogs 32 roughly consistent with the results of the methoxy scanning, that is, 4- and 8-substitued analogs 32 and We next examined the SAR of fluoro derivatives 32–35 (Table 4) to investigate the effect of this and 34 showed enhanced PR activity, whereas 9‐substituted analogs 35 showed weaker PR activity 34 showed enhanced PR activity, whereas analogs 35 showed weaker PR activitywere [10]. electron‐withdrawing substituent. For PR, 9-substituted the preferences for fluorine substitution position [10]. For AR, all fluoro analogs 32–35 showed enhanced activity compared with 12, suggesting that For AR, all fluoro analogs 32–35 showed enhanced activity compared with 12, suggesting that an roughly consistent with the results of the methoxy scanning, that is, 4‐ and 8‐substitued analogs 32 an electron‐withdrawing effect at these positions is important for AR antagonistic activity. For electron-withdrawing effect at these positions is important for AR antagonistic activity. For LXRα, and 34 showed enhanced PR activity, whereas 9‐substituted analogs 35 showed weaker PR activity LXRα, 4‐ and 7‐fluoro analogs 32 and 33 showed enhanced activity compared with 12. 4- and 7-fluoro analogs 32 and 33 showed enhanced activity compared with 12. [10]. For AR, all fluoro analogs 32–35 showed enhanced activity compared with 12, suggesting that Table 4. SAR of substitution effect of fluorine atom. an electron‐withdrawing effect at these positions is important for AR antagonistic activity. For Table 4. SAR of substitution effect of fluorine atom. LXRα, 4‐ and 7‐fluoro analogs 32 and 33 showed enhanced activity compared with 12. Table 4. SAR of substitution effect of fluorine atom. Compound
Position
Compound
Position
32
4
4 77 Position 88 99 4
32 33 33 34 34 Compound 35 35 32 33 34
1
1 RORα 1
RORα
15,000
1 RORβ 1
RORβ
14,000
1 RORγ 1
RORγ
18,000
15,000 14,000 18,000 7900 9800 13,000 7900 9800 13,000 1 17,000 1 11,000 11,000 1 >20,000 >20,000 RORα RORβ RORγ 17,000 9800 8300 5000 9800 8300 5000 15,000 14,000 18,000
2 LXRα 2
LXRα
3100
3100 3100 3100 7900 2 LXRα 7900 6400 6400 3100
LXRβ 2
LXRβ 2 4600
4600 3100 3100 6000 2 LXRβ 6000 5400 5400 4600
PR 2,3
PR 2,3 130
130 730 730 2,3 310 PR 310 1000 1000 130
AR 2
AR 2
390
390 460 460 680 2 AR 680 920 920 390
1 Inverse agonistic activity (IC : nM) [9]. 22 Antagonistic activity (IC : nM). 3 [10]. Inverse agonistic activity (IC 50 Antagonistic activity (IC 7 7900 9800 13,000 3100 3100 50 50: nM). 730 3 [10]. 460 50: nM) [9].
8
17,000
11,000
>20,000
7900
6000
310
680
GR 2
GR 2
6900
6900 9800 9800 >20,000 GR 2 >20,000 7000 7000 6900 9800 >20,000
We then examined examined various substituents at the 9-position 9‐position (Table 5). 1000 We reported that 35 9 9800 substituents 8300 6400 5400 5). 7000 We then various at5000 the (Table We have have920 reported that 1 Inverse agonistic activity (IC 2 Antagonistic activity (IC 3 [10]. introduction of a chlorine atom at the 9‐position (2) enhanced inverse agonistic activity toward 50 : nM) [9]. 50 : nM). introduction of a chlorine atom at the 9-position (2) enhanced inverse agonistic activity toward RORγ [9]. The SARs of RORγ were roughly consistent with the results for LXRβ, and 2 also exhibited We then examined various substituents at the 9‐position (Table 5). We have reported that introduction of a chlorine atom at the 9‐position (2) enhanced inverse agonistic activity toward RORγ [9]. The SARs of RORγ were roughly consistent with the results for LXRβ, and 2 also exhibited
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potent LXRβ antagonistic activity with the IC50 value of 0.88 μM. Replacement of fluorine at the RORγ [9]. The of RORγ (2) were roughly consistent with the results for LXRβ, and 2but alsodecreased exhibited 9‐position (35) SARs with chlorine caused enhanced activity toward RORs and LXRβ, potent LXRβ antagonistic activity with the IC50 value of 0.88 μM. Replacement of fluorine at the potent LXRβ antagonistic activity with the IC value of 0.88 µM. Replacement of fluorine at the activity toward PR, AR, and GR, suggesting that molecular modification at the 9‐position would be 50 9‐position (35) with chlorine (2) caused enhanced activity toward RORs and LXRβ, but decreased 9-position (35) with chlorine (2) caused enhanced activity toward RORs and LXRβ, but decreased important to improve selectivity for metabolic NRs over steroid receptors. activity toward PR, AR, and GR, suggesting that molecular modification at the 9‐position would be activity toward PR, AR, and GR, suggesting that molecular modification at the 9-position would be important to improve selectivity for metabolic NRs over steroid receptors. Table 5. SAR of substitution effect at 9‐posiiton. important to improve selectivity for metabolic NRs over steroid receptors. Table 5. SAR of substitution effect at 9‐posiiton. Table 5. SAR of substitution effect at 9-posiiton.
Compound 36 Compound Compound 2 36 37 36 2 38 2 37 37 38 38
R Me R
RCl
Me
RORα 1
RORβ 1
RORγ 1
LXRα 2
LXRβ 2
7600 RORα 1
7100 RORβ 1
1000 RORγ 1
12,000 LXRα 2
3000 LXRβ 2
7600
7100
1000
12,000
3000
1 1 1 2 2 RORα 5600 RORβ4900 RORγ690 LXRα9200 LXRβ880
PR 2,3 3700 PR 2,3
2,3 PR 4300
3700
AR 2 6400 AR 2
2 AR5200
6400
Me 7600 5500 71005600 10002600 12,000 6400 7100 3000 4500 3700 4200 >20,000 CF3 Cl 4900 690 9200 880 4300 5200 Cl 5600 5600 5200 OH 6400 49005500 6904100 92007000 8805500 4300 5100 >3000 4500 4200 >20,000 >20,000 CF CF 5500 5500 56005600 26002600 71007100 4500 4200 3 3 1 Inverse agonistic activity (IC 50: nM) [9]. 2 Antagonistic activity (IC50: nM). 3 [10]. OH 6400 6400 55005500 41004100 70007000 5500 5100 OH 5500 5100 >3000 >3000 1
GR 2 16,000 GR 2
2 GR 10,000
16,000
16,000 >20,000 10,000 10,000 >20,000 >20,000 >20,000 >20,000 >20,000
1 Inverse agonistic activity (IC : nM) [9]. 22 Antagonistic activity (IC : nM). 3 [10]. Inverse agonistic activity (IC 50 50: nM) [9]. Antagonistic activity (IC 50 50: nM). 3 [10].
Next, we focused on the 4‐position (Table 6), because 4‐methoxy analog 20 showed enhanced RORα, LXRα and PR activity, and 4‐fluoro analog 32 showed enhanced LXRα, PR and AR activity. Next, we focused on the 4‐position (Table 6), because 4‐methoxy analog 20 showed enhanced Next, we focused on the 4-position (Table 6), because 4-methoxy analog 20 showed enhanced Compounds 39–44 and 2 showed weak activity toward RORs and GR, indicating that the alkyl RORα, LXRα and PR activity, and 4‐fluoro analog 32 showed enhanced LXRα, PR and AR activity. RORα, LXRα and PR activity, and 4-fluoro analog 32 showed enhanced LXRα, PR and AR activity. group on the nitrogen atom is important for activity toward these receptors. We have reported that Compounds 39–44 and and 22 showed showed weak activity toward RORs indicating that the group alkyl Compounds 39–44 weak activity toward RORs andand GR, GR, indicating that the alkyl 4‐alkyl analogs 40–42 showed greater PR activity than the unsubstituted analog 1, and 4‐methyl group on the nitrogen atom is important for activity toward these receptors. We have reported that on the nitrogen atom is important for activity toward these receptors. We have reported that 4-alkyl analog 40 had an IC50 value of 0.15 μM [10]. As for LXRs and AR, 4‐alkyl analogs 40–42 showed 4‐alkyl 40–42 showed greater PR than activity the unsubstituted 1, and analog 4‐methyl analogsanalogs 40–42 showed greater PR activity the than unsubstituted analog 1, analog and 4-methyl 40 decreased activity compared with the unsubstituted analog 1, whereas 4‐fluoro analog 32 showed analog 40 had an IC 50 value of 0.15 μM [10]. As for LXRs and AR, 4‐alkyl analogs 40–42 showed had an IC50 value of 0.15 µM [10]. As for LXRs and AR, 4-alkyl analogs 40–42 showed decreased greater activity than 1. Based on the reported SARs of non‐steroidal AR antagonists, an decreased activity compared with the unsubstituted analog 1, activity compared with the unsubstituted analog 1, whereas whereas 4‐fluoro analog 32 showed 4-fluoro analog 32 showed greater electron‐withdrawing substituent neighboring the amide bond is expected to be important for greater activity than 1. Based on the reported SARs of non‐steroidal AR antagonists, an activity than 1. Based on the reported SARs of non-steroidal AR antagonists, an electron-withdrawing potent AR antagonism [21]. Therefore, 4‐chloro analog 43 was designed and synthesized as shown in electron‐withdrawing substituent the amide bond is expected important [21]. for substituent neighboring the amideneighboring bond is expected to be important for potentto ARbe antagonism Scheme 1. Interestingly, 43 showed greater AR activity and weaker RORs, LXRs, PR and GR activity potent AR antagonism [21]. Therefore, 4‐chloro analog 43 was designed and synthesized as shown in Therefore, 4-chloro analog 43 was designed and synthesized as shown in Scheme 1. Interestingly, than 4‐fluoro analog 32. Compound 43 showed the IC50 value of 0.059 μM, being 2.9‐fold more Scheme 1. Interestingly, 43 showed greater AR activity and weaker RORs, LXRs, PR and GR activity 43 showed greater AR activity and weaker RORs, LXRs, PR and GR activity than 4-fluoro analog 32. potent than clinically used OHF under our assay conditions. Compound 43 showed more than than 4‐fluoro 32. Compound the IC50 2.9-fold value of more 0.059 potent μM, being 2.9‐fold more Compound 43analog showed the IC50 value43 of showed 0.059 µM, being than clinically used 340‐fold selectivity over RORs and LXRβ, and 90‐fold and 6‐fold selectivity over LXRα and PR, potent than clinically used OHF under our assay conditions. Compound 43 showed more than OHF under our assay conditions. Compound 43 showed more than 340-fold selectivity over RORs respectively. This result indicates that molecular modification at the 4‐position can increase 340‐fold selectivity over RORs LXRβ, and and 6‐fold selectivity LXRα and PR, and LXRβ, and 90-fold and 6-foldand selectivity over 90‐fold LXRα and PR, respectively. Thisover result indicates that selectivity for AR over the other NRs. respectively. This result indicates that can molecular modification at the can increase molecular modification at the 4-position increase selectivity for AR over4‐position the other NRs. selectivity for AR over the other NRs. Table 6. SAR at 4‐posiiton and other substitution effects. Table 6. SAR at 4-posiiton and other substitution effects. Table 6. SAR at 4‐posiiton and other substitution effects.
Compound Compound 39 Compound 40 39 39 41 40 40 42 41 41 43 42 42 43 3 44 3 44 43 3 3 3 3 3 44 3 3
R R 4‐OMe R 4-OMe 4‐Me 4‐OMe 4-Me 4‐Et 4-Et 4‐Me 4‐n‐Pr 4-n-Pr 4‐Et 4‐Cl 4-Cl 4‐n‐Pr 4‐Me, 8‐F 4-Me, 8-F 4‐Cl ‐ 4‐Me, 8‐F
RORα 1 RORα 1 >20,000 RORα >20,0001 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000
RORβ 1 RORγ 1 LXRα 2 LXRβ 2 PR 2,3 AR 2 RORβ 1 RORγ 1 LXRα 2 LXRβ 2 PR 2,3 AR 2 >20,000 9600 >20,000 >20,000 270 >20,000 1 2 2 2,3 RORβ RORγ LXRα LXRβ PR AR 2 >20,000 9600 1 >20,000 270 >20,000 >20,000 >20,000 >20,000 >20,000 150 >20,000 >20,000 >20,000 9600 >20,000 >20,000 270 >20,000 >20,000 >20,000 >20,000 >20,000 150 >20,000 >20,000 >20,000 >20,000 >20,000 210 14,000 >20,000 >20,000 >20,000 210 14,000 >20,000 >20,000 >20,000 >20,000 150 >20,000 11,000 9900 >20,000 >20,000 20,000 360 5400 11,000 9900 >20,000 20,000 5400 >20,000 >20,000 >20,000 >20,000 210 14,000 >20,000 >20,000 5300 >20,000 360 350 59 >20,000 >20,000 5300 >20,000 350 59 11,000 9900 >20,000 20,000 360 5400 >20,000 >20,000 >20,000 >20,000 46 1700 >20,000 >20,000 >20,000 46 1700 >20,000 >20,000 5300 >20,000 >20,000 350 59 >20,000 >20,000 >20,000 >20,000 27 300 >20,000 >20,000 >20,000 >20,000 27 >20,000 >20,000 >20,000 >20,000 46 3 300 1700 1 Inverse agonistic activity (IC 2 Antagonistic activity (IC50: nM). [10]. 1 Inverse agonistic activity (IC50: nM) [9] [9] 2 Antagonistic (IC50 : nM). ‐ >20,000 >20,000 >20,000 >20,000 activity >20,000 27 3 [10]. 300 50 : nM)
Inverse agonistic activity (IC50: nM) [9] 2 Antagonistic activity (IC50: nM). 3 [10].
1
GR 2 GR 2 >20,000 GR 2 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 16,000 16,000 >20,000 >20,000 >20,000 16,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000 >20,000
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Scheme Reagents and conditions: (a) CF 33COCF 33 ∙1.5H 2O, p‐TsOH∙H 2O, reflux, 27%; (b) ·1.5H Scheme 1. Reagents and conditions: (a)CF COCF p-TsOH2·O, H O,toluene, toluene, reflux, 27%; 2 O, 2toluene, Scheme 1. 1. Reagents and conditions: (a) 3COCF 3∙1.5H 2O, p‐TsOH∙H reflux, 27%; (b) ◦ C; ◦°C (b) 2-iodobenzoic acid, EDC, DMAP, DMF, 100 (c) SEMCl, NaH, DMF, 0 C to RT; (d) Pd(OAc) 2‐iodobenzoic acid, EDC, DMAP, DMF, 100 °C ; (c) SEMCl, NaH, DMF, 0 to RT; (d) Pd(OAc) 2 2‐iodobenzoic acid, EDC, DMAP, DMF, 100 °C; (c) SEMCl, NaH, DMF, 0 °C to RT; (d) Pd(OAc)2, ,2, ◦ C, 24% in 2 steps; (e) TBAF, THF, reflux, 18%. PCy ·3HBF ,4, Cs Cs22CO DMA, 130°C PCy ∙HBF 2CO CO °C , 24% in 2 steps; (e) TBAF, THF, reflux, 18%. 33∙HBF 33,, DMA, 130 PCy 44, Cs 3, DMA, 130 , 24% in 2 steps; (e) TBAF, THF, reflux, 18%.
Chronic administration administration of AR antagonists antagonists often often leads leads to the development development of resistance. Chronic administration Chronic ofof AR AR antagonists often leads toto the the development ofof resistance. resistance. Mutations in the LBD of AR, such as T877A, have been identified in patients treated with flutamide Mutations in the LBD of AR, such as T877A, have been identified in patients treated with flutamide Mutations in the LBD of AR, such as T877A, have been identified in patients treated with flutamide (Figure 2), and the activated metabolite, hydroxyflutamide, is an agonist of AR bearing T877A [22]. (Figure 2), and the activated metabolite, hydroxyflutamide, is an agonist of AR bearing T877A [22]. (Figure 2), and the activated metabolite, hydroxyflutamide, is an agonist of AR bearing T877A [22]. In contrast, another AR antagonist, enzalutamide, was reported to antagonize antagonize AR mutant mutant T877A In contrast, another AR antagonist, enzalutamide, was reported to T877A In contrast, another AR antagonist, enzalutamide, was reported to antagonize AR AR mutant T877A [23]. [23]. Thus, development of a novel chemical class of AR antagonists, different from flutamide [23]. Thus, development of a chemical novel chemical of AR antagonists, from flutamide Thus, development of a novel class ofclass AR antagonists, differentdifferent from flutamide analogs analogs seems an attractive approach for the treatment of AR antagonist‐resistant cancer, providing analogs seems an attractive approach for the treatment of AR antagonist‐resistant cancer, providing seems an attractive approach for the treatment of AR antagonist-resistant cancer, providing further further motivation motivation to evaluate evaluate steroid surrogates. surrogates. Therefore, we investigated investigated the antiandrogenic antiandrogenic further to steroid we the motivation to evaluate steroid surrogates. Therefore,Therefore, we investigated the antiandrogenic activity of activity of several potent AR antagonists by means of PSA (prostate‐specific antigen, an activity potent of several potent AR by (prostate-specific means of PSA antigen, (prostate‐specific antigen, an several AR antagonists by antagonists means of PSA an AR-regulated gene) AR‐regulated gene) ELISA (enzyme‐linked immunosorbent assay) in human prostate cancer cell line AR‐regulated gene) ELISA (enzyme‐linked immunosorbent assay) in human prostate cancer cell line ELISA (enzyme-linked immunosorbent assay) in human prostate cancer cell line LNCaP, which has LNCaP, which has has T877A mutation in the AR LBD. LBD. Potent AR antagonist 43 did did activity not show show LNCaP, which T877A mutation the AR antagonist 43 not T877A mutation in the AR LBD. Potentin AR antagonist 43Potent did notAR show antiandrogenic in antiandrogenic activity in LNCaP, whereas several other AR antagonists including 33 decreased the antiandrogenic activity in LNCaP, whereas several other AR antagonists including 33 decreased the LNCaP, whereas several other AR antagonists including 33 decreased the PSA level with an IC50 of 50 of 190 nM (Table 7). This result suggests that the new non‐flutamide type AR PSA level with an IC of 190 nM (Table 7). This result suggests that the new non‐flutamide type AR PSA level with an IC 190 nM (Table 7). This50result suggests that the new non-flutamide type AR antagonist 33 is a promising antagonist 33 is a promising candidate for antiandrogen therapy of prostate cancer. antagonist 33 is a promising candidate for antiandrogen therapy of prostate cancer. candidate for antiandrogen therapy of prostate cancer.
Figure 2. Chemical structures of AR antagonists. Figure 2. Chemical structures of AR antagonists. Figure 2. Chemical structures of AR antagonists. Table 7. Antiandrogenic activity toward AR antagonist‐resistant cell line LNCaP. Table 7. Antiandrogenic activity toward AR antagonist‐resistant cell line LNCaP. Table 7. Antiandrogenic activity toward AR antagonist-resistant cell line LNCaP. Compound AR Reporter Gene IC AR Reporter Gene IC 50 (nM) LNCaP IC 50 (nM) ± SEM Compound 50 (nM) LNCaP IC 50 (nM) ± SEM 1 100 >10,000 100 IC50 (nM) >10,000 Compound1 AR Reporter Gene LNCaP IC50 (nM) ± SEM 13 500 >10,000 13 500 >10,000 1 100 540 >10,000 26 560 ± 260 (N = 2) 26 540 560 ± 260 (N = 2) 13 500 >10,000 32 390 ± 30 (N = 2) 490 ± 260 (N = 2) 32 390 ± 30 (N = 2) 490 ± 260 (N = 2) 26 540 560 ± 260 (N = 2) 33 460 190 ± 95 (N = 2) 33 460 190 ± 95 (N = 2) 32 390 ± 30680 (N = 2) 490 ± 260 (N = 2) 34 680 >10,000 34 >10,000 33 460 190 ± 95 (N = 2) 35 920 550 ± 110 (N = 2) 35 920 550 ± 110 (N = 2) 34 680 >10,000 43 59 ± 9.5 (N = 2) >10,000 43 59 ± 9.5 (N = 2) >10,000
35 920 550 ± 110 (N = 2) Mean IC 50 values with standard error of mean (SEM) from 1 or N times independent experiments. Mean IC 50 values with standard error of mean (SEM) from 1 or N times independent experiments. 43 59 ± 9.5 (N = 2) >10,000 Mean IC50 values with standard error of mean (SEM) from 1 or N times independent experiments. 3. Materials and Methods 3. Materials and Methods
3.3.1. Chemistry Materials and Methods 3.1. Chemistry 3.1. Chemistry 3.1.1. General 3.1.1. General 1H NMR spectra were recorded on a JNM‐ECA500 (500 MHz) spectrometer (JEOL Ltd., Tokyo, 3.1.1.1H NMR spectra were recorded on a JNM‐ECA500 (500 MHz) spectrometer (JEOL Ltd., Tokyo, General Japan). Chemical shifts (δ) are reported reported in parts parts per per million. million. Flash column chromatography chromatography was 1 H Chemical Japan). shifts (δ) are Flash column was NMR spectra were recorded on a in JNM-ECA500 (500 MHz) spectrometer (JEOL Ltd., Tokyo, performed on silica gel 60N (40–50 mm) (Kanto Chemical Co., Inc., Tokyo, Japan). HPLC analyses to performed on silica gel 60N (40–50 mm) (Kanto Chemical Co., Inc., Tokyo, Japan). HPLC analyses to Japan). Chemical shifts (δ) are reported in parts per million. Flash column chromatography was check purity were performed on an analytical column (GL Science Inc., Tokyo, Japan) Inertsil ODS‐4 check purity were performed on an analytical column (GL Science Inc., Tokyo, Japan) Inertsil ODS‐4
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performed on silica gel 60N (40–50 mm) (Kanto Chemical Co., Inc., Tokyo, Japan). HPLC analyses to check purity were performed on an analytical column (GL Science Inc., Tokyo, Japan) Inertsil ODS-4 reversed-phase column, 5 µm, 4.6 mm × 150 mm) eluted with a mobile phase consisting of CH3 CN/water at a flow rate of 1.0 mL/min, with UV (ultraviolet) monitoring at 254 nm, at 37 ◦ C. 3.1.2. 2-(4-Amino-3-chlorophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (46) To a solution of 2-chloroaniline (45) (1.58 mL, 15.0 mmol) in toluene (11 mL) were added hexafluoroacetone trihydrate (2.06 mL, 18.0 mmol) and p-TsOH (258 mg, 1.50 mmol). The mixture was stirred for 8.5 h at 110 ◦ C, and then cooled to room temperature. Water was added, and the resulting mixture was extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2 SO4 , and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/AcOEt = 10:1-3:1) to give 46 as a pink solid (9%). 1 H NMR (500 MHz, CDCl3 ) δ: 7.58 (s, 1H), 7.35 (d, J = 8.6 Hz, 1H), (d, J = 9.2 Hz, 1H). 3.1.3. N-(2-Chloro-4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)-2-iodobenzamide (47) To a solution of EDC (742 mg, 3.87 mmol) and DMAP (473 mg, 3.87 mmol) in DMF (4.0 mL) was added 2-iodobenzoic acid (640 mg, 2.58 mmol) under an Ar atmosphere. The mixture was stirred for 30 min at room temperature, then 46 (379 mg, 1.29 mmol) was added to it, and stirring was continued at 100 ◦ C for 4 h. The resulting mixture was cooled, diluted with water, and extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2 SO4 , and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane/AcOEt = 4:1) to give 47 (181 mg, 0.346 mmol, 27%) as a brown solid. 1 H NMR (500 MHz, DMSO-d6 ) δ: 7.94 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 1.7 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 4.6 Hz, 2H), 7.24 (td, J = 3.9, 1.3 Hz, 1H). 3.1.4. N-(2-Chloro-4-(1,1,1,3,3,3-hexafluoro-2-((2-(trimethylsilyl)ethoxy)methoxy)propan-2-yl)phenyl)2-iodo-N-((2-(trimethylsilyl)ethoxy)methyl)benzamide (48) Sodium hydride (41.6 mg, 1.04 mmol) was added to a solution of 47 (181 mg, 0.346 mmol) in DMF (1.5 mL) at 0 ◦ C. The mixture was stirred for 1 h at 0 ◦ C, then SEMCl (184 µL, 1.04 mmol) was added at 0 ◦ C, and stirring was continued for 6 h at room temperature. The resulting mixture was diluted with AcOEt, quenched with water, and extracted with AcOEt. The combined organic layer was washed with water, dried over Na2 SO4 , and concentrated. The residue was purified by column chromatography (n-hexane/AcOEt = 20:1 to 10:1) to give 48 (219 mg, 0.279 mmol) as a colorless oil. The compound was used for the next reaction without further purification. 3.1.5. 4-Chloro-2-(1,1,1,3,3,3-hexafluoro-2-((2-(trimethylsilyl)ethoxy)methoxy)propan-2-yl)-5((2-trimethylsilyl ethoxy)methyl)phenanthridin-6(5H)-one (49) To a solution of 48 (219 mg, 0.279 mmol) in DMA (1.0 mL) were added PCy3 ·HBF4 (63.7 mg, 0.173 mmol), Cs2 CO3 (620 mg, 1.90 mmol) and Pd(OAc)2 (12.0 mg, 0.0532 mmol) under an Ar atmosphere. The mixture was stirred for 3 h at 130 ◦ C, then cooled to room temperature, and water was added. The resulting mixture was extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2 SO4 , and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane/AcOEt = 20:1) to give 49 as a brown oil (45.2 mg, 0.0824 mmol, 24% in 2 steps). 1 H NMR (500 MHz, CDCl3 ) δ: 8.56 (s, 1H), 8.53 (dd, J = 8.0, 1.1 Hz, 1H), 8.26 (d, J = 7.7, 1.5 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.47 (s, 1H), 5.85 (s, 2H), 4.95 (s, 2H), 3.88 (t, J = 8.6 Hz, 2H), 3.74 (t, J = 8.3 Hz, 2H), 1.02 (t, J = 8.3 Hz, 2H), 0.98 (m, 2H), 0.02 (s, 9H), −0.05 (s, 9H). 3.1.6. 4-Chloro-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenanthridin-6(5H)-one (43) TBAF (1 M in THF, 0.779 mL, 0.779 mmol) was added to a solution of 49 (42.7 mg, 0.0779 mmol) in THF (0.8 mL) at room temperature. The reaction mixture was stirred under reflux for 9 h, then diluted
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with water, and extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2 SO4 , and concentrated under reduced pressure. The residue was purified by column chromatography (CHCl3 /MeOH = 40:1) to give 43 as a pale yellow solid (18%). 1 H NMR (500 MHz, DMSO-d ) δ: 8.35 (d, J = 8.6 Hz, 1H), 8.27 (dd, J = 8.0, 1.1 Hz, 1H), 7.86–7.83 6 (m, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.41 (d, J = 8.6 Hz, 1H). HLPC purity 97.0% (area %). 3.2. Biology 3.2.1. T-47D Alkaline Phosphatase Assay T-47D alkaline phosphatase assays were performed as described in the literature [10]. Briefly, human ductal breast epithelial tumor (T-47D) cells were treated with fresh medium containing test compound plus progesterone (final concentration 1 nM), and incubated for 24 h. The fixed cells were washed with PBS and assay buffer was added. After the reaction was terminated, the absorbance at 405 nm was measured. All data points were measured in triplicate. 3.2.2. Reporter Gene Assay Human embryonic kidney (HEK) 293 cells (RIKEN BRC, Ibaraki, Japan) were maintained in DMEM (Dulbecco’s Modified Eagle’s medium) containing 5% (v/v) fetal bovine serum, and penicillin/streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. Cells were seeded in clear-bottomed white 96-well plates at a density of 1 × 104 cells/well and incubated for 6 h prior to transfection. Cells were co-transfected with 30 ng of a NR expression plasmid, 50 ng of a luciferase reporter and 10 ng of CMX-β-galactosidase expression vector per well, using the calcium phosphate co-precipitation method. After 24 h, transfected cells were treated with dimethyl sulfoxide (DMSO) or DMSO solution of test compounds for 24 h. T0901317 (0.3 µM), T0901317 (0.1 µM), dihydrotestosterone (0.3 nM), and dexamethasone (1 nM) were used as agonists for LXRα, LXRβ, AR and GR, respectively. After addition of a luciferase substrate, luminescence was detected with a luminometer. The luciferase activity of each sample was normalized by β-galactosidase activity: 2-nitrophenyl-β-D-galactopyranoside was added, and the absorbance was measured at 405 nm. Each sample was carried out in triplicate. A six-point sigmoidal dose-response curve was generated for each compound. The IC50 value for each compound was calculated by linear approximation of two points adjacent to 50% inhibition with logarithmic scale. In Table S1, biological reproducibility are reported as the mean IC50 ± SEM. Nuclear receptor plasmids: GR (CMX-hGR), AR (CMX-hAR), ROR (pcDNA3.1(-)-hRORα1, pcDNA3.1(-)-hRORβ1, pcDNA3.1(-)-hRORγ1), LXR (CMX-GAL4N-hLXRα-LBD, CMX-GAL4NhLXRβ-LBD). Luciferase reporter plasmids: GR (MTV-Luc), AR (ARE-Luc), ROR (RORE-TK-Luc), LXR (TK-MH100x4-Luc). Plasmids for AR, GR and LXRs were provided by Prof. Dr. Makishima, and plasmids for RORs were provided by Itsuu Institute (Kanagawa, Japan). 3.2.3. PSA Level in LNCaP Cell Line The human prostate cancer cell line LNCaP (ECACC, London, UK) was routinely maintained in RPMI1640 containing 10% (v/v) fetal bovine serum, and penicillin/streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. Cells were cultured in RPMI1640 without phenol red containing 10% (v/v) charcoal-stripped fetal bovine serum, and penicillin/streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2 in air for 3 days. Then cells were plated in 96-well plates at 1 × 104 cells/well and incubated overnight. On the next day, cells were treated with fresh media containing test compound plus dihydrotestosterone (final concentration 10 nM), and incubated for 48 h. Then, PSA levels were measured in the culture supernatants, after 100-fold dilution with the
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medium, using ELISA (Immunospec Corp., CA, USA Free prostate-specific antigen cat. #E29-212) according to the manufacturer’s instructions. All data points were carried out twice in triplicate. 4. Conclusions Development of non-steroidal NR ligands is desirable to obtain safer, more selective agents for clinical use. Here, non-steroidal antagonists for multiple target proteins including LXRβ (IC50 : 0.88 µM), AR (IC50 : 0.10 µM), GR (IC50 : 0.76 µM) were efficiently identified by exhaustive evaluation of a small steroid surrogate library. In addition, a potent AR antagonist 43 (IC50 : 0.059 µM) belonging to a new chemical class was developed. Further, AR antagonist 33 showed antiandrogenic activity toward AR antagonist-resistant cell line LNCaP with the IC50 value of 190 nM. Structure-activity relationship studies of phenanthridinone analogs revealed that (1) hydrogen at the nitrogen is important for AR and PR activity, and a longer alkyl group is favorable for RORs and GR activity, (2) molecular modification at the 9-position can increase selectivity for metabolic NRs over steroid receptors, and (3) an electron-withdrawing substituent on the phenyl group is important for AR antagonistic activity, while modification at the 4-position can increase selectivity for AR over other NRs. The phenanthridinone skeleton is more readily modifiable than the steroid skeleton. Thus, our strategy of efficient lead finding, activity enhancement and preliminary control of selectivity over other receptors should expand the utility of the multi-template approach, even though the compounds obtained here still require further optimization. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/7/ 2090/s1. Author Contributions: Conceptualization, Y.H.; methodology, Y.N. and M.I.; validation, Y.N., S.F., Y.H. and M.I.; organic synthesis, Y.N.; plasmid preparation, M.M.; data curation, Y.N. and M.I.; writing—original draft preparation, M.I.; supervision, S.F., M.M., Y.H. and M.I.; funding acquisition, S.F., Y.H. and M.I. Funding: The work described in this Letter was partially supported by Grants-in Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Japan Society for the Promotion of Science (KAKENHI Grant No. 17H03996 (Y.H.), No. 17H03997(S.F.) and No. 25293027 (M.I.)). Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations NR SAR PR LXR AR GR LBD ROR NPC1 NPC1L1 OHF Ts EDC DMAP DMF SEM Cy DMA TBAF THF
Nuclear receptors Structure-activity relationship Progesterone receptor Liver X receptor Androgen receptor Glucocorticoid receptor Ligand-binding domains retinoic acid receptor-related orphan receptors Niemann-Pick disease type C1 Niemann-Pick disease type C1-like 1 Hydroxyflutamide p-Toluenesulfonyl N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide 4-(N,N-Dimethylamino)pyridine Dimethylformamide 2-(Trimethylsilyl)ethoxymethyl Cyclohexyl Dimethylacetamide Tetrabutylammonium fluoride Tetrahydrofuran
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