Targeting the binding function 3 (BF3) site of the androgen receptor through virtual screening. 2. Development of 2-((2-phenoxyethyl) thio)-1H-benzimidazole derivatives

Article abstract of DOI:10.1021/jm3015712

The human androgen receptor (AR) is a proven therapeutic target in prostate cancer. All current antiandrogens, such as Bicalutamide, Flutamide, Nilutamide, and Enzalutamide, target the buried hydrophobic androgen binding pocket of this protein. However, effective resistance mechanisms against these therapeutics exist such as mutations occurring at the target site. To overcome these limitations, the surface pocket of the AR called binding function 3 (BF3) was characterized as an alternative target for small molecule therapeutics. A number of AR inhibitors directly targeting the BF3 were previously identified by us (J. Med. Chem. 2011. 54, 8563). In the current study, based on the prior results, we have developed structure-activity relationships that allowed designing a series of 2-((2-phenoxyethyl)thio)-1H-benzimidazole and 2-((2-phenoxyethyl)thio)-1H-indole as lead BF3 inhibitors. Some of the developed BF3 ligands demonstrated significant antiandrogen potency against LNCaP and Enzalutamide-resistant prostate cancer cell lines.

Full text of DOI:10.1021/jm3015712

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Article
Targeting the Binding Function 3 (BF3) Site of the Androgen
Receptor Through Virtual Screening. 2. Development of
-((2-phenoxyethyl) thio)-1H-benzimidazole derivatives.
2
Ravi Sashi Nayana Munuganti, Eric Leblanc, Peter Axerio-Cilies, Christophe
Labriere, Kate Frewin, Kriti Singh, Mohamed DH Hassona, Nathan A Lack, Huifang
Li, Fuqiang Ban, Emma Guns, Robert Young, Paul S Rennie, and Artem Cherkasov
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3015712 • Publication Date (Web): 09 Jan 2013
Downloaded from http://pubs.acs.org on January 16, 2013
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Targeting the Binding Function 3 (BF3) Site of the Androgen
Receptor Through Virtual Screening. 2. Development of 2ꢀ((2ꢀ
phenoxyethyl) thio)ꢀ1Hꢀbenzimidazole derivatives.
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a,1
a,1
a
b
M. Ravi Shashi Nayana, Eric Leblanc, Peter AxerioꢀCilies, Christophe Labriere, Kate
a
a
a
c
a
a,*
Frewin, Kriti Singh , Mohamed D.H. Hassona, Nathan A. Lack, Huifang Li, Fuqiang Ban,
a
b
a, 2
a,,2
Emma Tomlinson Guns, Robert Young, Paul S. Rennie, and Artem Cherkasov
[
a] Vancouver Prostate Centre, University of British Columbia, 2660 Oak Street, Vancouver,
British Columbia V6H 3Z6, Canada
[
b] Department of Chemistry, Simon Fraser University, 8888, University Drive, Burnaby, British
Columbia, V5A 1S6, Canada
[c] Koç University School of Medicine, Istanbul, Turkey
1
2
*
Authors contributed equally to this work
Dr. Cherkasov’s and Dr. Rennie’s labs made equal contributions to this work.
Telephone: 604ꢀ875ꢀ4111. Fax: 604ꢀ875ꢀ5654. Eꢀmail: fban@prostatecentre.com
PDB ID: 4HLW
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ABSTRACT
The human androgen receptor (AR) is a proven therapeutic target in prostate cancer. All
current antiꢀandrogens, such as Bicalutamide, Flutamide, Nilutamide and Enzalutamide, target
the buried hydrophobic androgen binding pocket of this protein. However, effective resistance
mechanisms against these therapeutics exist, such as mutations occurring at the target site. To
overcome these limitations, the surface pocket of the AR called Binding Function 3 (BF3) was
characterised as an alternative target for small molecule therapeutics. A number of AR inhibitors
directly targeting the BF3 were previously identified by us (J. Med. Chem. 2011, 54, 8563). In
the current study, based on the prior results, we have developed structureꢀactivityꢀrelationships
that allowed designing a series of 2ꢀ((2ꢀphenoxyethyl) thio)ꢀ1Hꢀbenzimidazole and 2ꢀ((2ꢀ
phenoxyethyl) thio)ꢀ1Hꢀindole as lead BF3 inhibitors. Some of the developed BF3 ligands
demonstrated significant antiꢀandrogen potency against LNCaP and Enzalutamideꢀresistant
prostate cancer cell lines.
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INTRODUCTION
The androgen receptor (AR) plays a pivotal role in the development of the prostate gland
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as well as is involved in occurrence and progression of prostate cancer (PCa). It has also been
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observed that the AR is overexpressed in the majority of castrationꢀresistant prostate cancers
2, 3
(CRPCs).
With recurrent or metastatic PCa, the first line of treatment is some form of
androgen withdrawal therapy that blocks either the production of androgens or their binding to
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the AR.
Unfortunately the effectiveness of this approach is usually temporary due to
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progression of surviving tumor cells to the castration resistant state.
The currently existing antiꢀandrogens, such as Bicalutamide, Flutamide, Nilutamide,
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Enzalutamide (formerly known as MDV3100) and experimental ARN509, have essentially the
same mechanism of action on the AR. Specifically, they all bind to its androgen binding site
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(
ABS) resulting in conformational changes of the receptor that prevent its activation. While
these antagonists suppress the initial cancer growth, they become less effective with longꢀterm
therapy. One of the major causes of resistance is mutations in the ABS. Such mutations not only
weaken ARꢀdrug interactions, but may also turn antagonists into agonists, thereby promoting
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cancer progression. Thus, there is an urgent need for novel therapeutic strategies to modulate
AR, such as direct disruption of its critical interactions with coꢀactivator proteins, mediated by
Activation Function 2 (AF2) and Binding Function 3 (BF3) surface sites.
The AF2 is a hydrophobic groove formed on the surface of AR upon androgen binding.
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The recruitment of coꢀregulators at that site is critical for ARꢀregulated gene expression. Thus,
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the AF2 pocket has been previously characterized as a suitable target site on the AR surface.
In a recent work, Fletterick et al identified an additional surface area adjacent to AF2 groove
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called the Binding Function 3 (BF3). It has been proposed that the BF3 site can allosterically
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affect the AF2 site and is implicated in the receptor’s interaction with a FKPB52 protein – an
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important AR activator. AF2 and BF3 surface pockets are likely to form an allosteric network
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that regulates AR LBD function and represent promising therapeutic targets.
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Previously, we conducted a large scale virtual screen against AF2 and BF3 sites. AR
inhibitors of several chemical scaffolds that target the BF3 site were discovered and
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demonstrated antiꢀAR activity in the subꢀmicromolar range. A comparison of the four reported
coꢀcrystal structures of the selected BF3 inhibitors revealed that residues of BF3 site may
undergo significant conformational changes upon ligand binding. On the basis of the elucidated
structureꢀactivityꢀrelationship of our virtual screening hits and binding poses in the coꢀcrystals,
2ꢀ((2ꢀphenoxyethyl)thio)ꢀ1Hꢀbenzo[d]imidazole was derived and proposed as a proper starting
point for further ligand optimization.
In the present study, we are report on computerꢀaided design, synthesis and experimental
evaluation of 2ꢀ((2ꢀphenoxyethyl) thio)ꢀ1Hꢀbenzo[d]imidazole derivatives and 2ꢀ((2ꢀ
phenoxyethyl) thio)ꢀ1Hꢀindole derivatives as promising new BF3ꢀdirected inhibitors of the AR
representing a potentially novel type of drugs for treating some forms of the CRPCs.
RESULTS
Identification of Analogs of Compound 1 by 2D Similarity Search Method: In the
previous work, our group reported several small molecules that specifically target the ARꢀBF3
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pocket.
Among
them,
(2ꢀ((2ꢀphenoxyethyl)
thio)ꢀ1ꢀ(2ꢀ(pꢀtolyloxy)
ethyl)ꢀ1Hꢀ
benzo[d]imidazole) derivative (compound 1) was selected as a lead candidate. A chemical
template was designed based on the structure of 1 (Figure 1) and a molecular similarity search
was performed to identify compounds with different substitutions at R and 1ꢀ5 positions of the
1
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benzene ring of 1. Instant JChem, a 2D similarity searching tool from ChemAxon, was
2
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employed to search through ZINC database 12.0. All software parameters were set to their
default values. A total of 30 ZINC compounds which generated Tanimoto coefficient above 0.6
with respect to the query structure were selected and tested for their antiꢀAR activity (compounds
2ꢀ31 in Table 1).
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CellꢀBased Testing and In Vitro Characterization: The selected compounds were
screened for their ability to inhibit AR transcriptional activity using a nondestructive, cellꢀbased
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enhanced green fluorescent protein (eGFP) AR transcriptional assay
(see Materials and
Methods). In this assay, the expression of eGFP is under the direct control of an androgen
responsive probasinꢀderived promoter and enables quantification of AR transcriptional activity.
13 of the purchased compounds exhibited >50% inhibition of AR transcription at a concentration
of 50ꢁM. These were subjected to concentrationꢀdependent titration to establish corresponding
IC values (Table 1). The observed IC values were estimated to be in the range of 11ꢀ60ꢁM.
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These inhibitors were then tested in SRC2ꢀ3 peptide and androgen displacement assays for their
ability to displace SRC2 peptide from the AF2 site and androgen from the ABS, respectively.
None of the compounds were active in these assays, confirming that they target the BF3 pocket.
From the cell proliferation assay, the compounds were also confirmed to be nontoxic to nonꢀAR
containing cells at a 50ꢁM concentration administered for over 72h (data not shown).
Since the molecules selected from the 2D similarity search did not result in compounds
with improved cellꢀbased activity, the lead optimization was initiated.
Rational Design, Synthesis and Characterization of 2ꢀ((2ꢀphenoxyethyl) thio)ꢀ1Hꢀ
benzimidazole: Based on the crystallographically determined binding pose of a compound 1
(
2YLO) and the corresponding activity profiles of their analogues, we hypothesized that solventꢀ
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exposed substituents at the R position of the ligands are not likely to contribute to target affinity.
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To test this hypothesis, we designed a compound 32 where R = H (Table 1). The structure was
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built using the MOE program and energy minimization was performed by applying the
MMFF94X force field. The compound was then docked into the AR crystal structure (2YLO
structure) using the Glide SP program without applying any constraints. From the docking pose,
we could observe that compound 32 is anchored to the protein site by a hydrogen bond that it
forms with the Glu837 side chain. Compound 32 was synthesized and evaluated by the eGFP
transcriptional assay. As anticipated, it exhibited approximately a 3ꢀfold increase (IC =4.2ꢁM)
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of AR transcriptional activity inhibition compared to the parent substance (IC =13.1ꢁM). The
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doseꢀresponse curve for compound 32 is presented on Figure 2A.
Furthermore, we have tested compound 32 with the AF2 peptide displacement assay and
androgen displacement assay where it did not demonstrate any detectible levels if activity,
confirming that it is a specific BF3 binder. Biolayer interferometry (BLI) studies demonstrated a
direct reversible interaction between this compound and AR LBD (Figure 2B).
Based on these observations, we concluded that the formation of Hꢀbond between NH of
the benzoimidazole moiety of compound 32 and the side chain of the Glu837 residue is a
significant factor for proteinꢀligand affinity. This observation becomes particularly obvious when
compound 32 is compared with the synthetic analogues 33, 34, 35 and 36, which were designed
and tested as negative probes and which also received lower docking scores due to the loss of a
critical Hꢀbond with Glu837. Upon testing for antiꢀAR activity, all these derivatives except
compound 33 turned out to be completely inactive whereas compound 33 showed a 3ꢀfold
decrease in activity (IC =12ꢁM) in the eGFP assay (compounds 33ꢀ36 in Table 1). Similarly,
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effect in activity and binding to the AR LBD. Since compound 32 has a promising experimental
activity profile, it was subjected to structural elucidation using xꢀray crystallography.
Crystallographic Structure of AR in Complex with 2ꢀ((2ꢀphenoxyethyl) thio)ꢀ1Hꢀ
benzimidazole: In an effort to unambiguously confirm the site of the compound’s interaction, xꢀ
ray crystallographic studies were conducted with the AR and compound 32. Following
optimization of the soaking protocol, the structure of the AR in complex with compound 32 was
determined to 2.5Å resolution. The crystallographic data refinement statistics for the
corresponding PDB entry 4HLW are presented in Table 2. In the present crystallographic data
set, there was clear electron density observed at the BF3 site, supporting the presence of the
inhibitor. Interestingly, unlike the cases of previously published BF3 binders, such as compound
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A, compound B (TRIAC), C (T3), and D (FLUF) (Figure 1 in the supplementary data),
compound 32 was found to reside specifically in the BF3 site. Since the crystallographic
information is in agreement with the activity data, compound 32 could be characterized as a
specific BF3 inhibitor. Figure 3A shows a good structural fit of compound 32 inside the target
cavity. Notably, the experimentally determined configuration of the BF3ꢀbound molecule is
similar to its docking predicted pose generated by Glide (r.m.s.d=0.62Å), which gave confidence
to rely on the adopted in silico protocol (Figure 2 in supplementary data).
The BF3 pocket is located on the AR surface close to the AF2 site. Since the AR
androgen binding site is distant from the BF3 site, mutations that usually weaken interactions
between the ABS and the current drugs should not affect the BF3ꢀdirected inhibitors. As it can
be observed from the xꢀray structure, the BF3 pocket is formed by residues from several LBDꢀ
forming helices. The residues contributing to BF3 formation are: Gln670, Pro671, Ile672 and
Phe673 from the NH2ꢀterminal part of a helix 1 (H1), Pro723, Gly724, Arg726 and Asn727 from
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H3 and Phe826, Glu829, Leu830, Asn833, Glu837, and Arg840 from H9. The residues Arg726
and Asn727 act as boundary between the AF2 and BF3 sites and may play an important role in
their cross talk and coordinated action.
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The structure of the ARꢀLBD in complex with compound 32 is generally similar to the
previously reported structures 2YLO. Compound 1 differs slightly in terms of its positioning
inside the BF3. Thus, as predicted compound 32 forms a strong hydrogen bond between the NH
benzimidazole moiety and sideꢀchain carboxyl of the Glu837. Moreover, this compound
maintains strong hydrophobic contacts with the neighboring residues, including Ile672, Phe673
and Leu830. Additional stabilization of the proteinꢀligand complex occurs due to Tꢀshaped
areneꢀarene conjugation between the phenyl ring of compound 32 and the Phe826 side chain.
These interactions possibly explain the increased potency of compound 32 (IC50 =4.2ꢁM)
compared to its parental compound 1 as well as other structural analogues listed in Table 1
(where the corresponding IC50 values range from 11ꢁM to >200ꢁM). Since compound 32
demonstrated improved AR inhibitory activity profile and could be experimentally resolved
inside the BF3 site, it was advanced into further optimization studies.
Structure Activity Relationship (SAR) for 2ꢀ((2ꢀphenoxyethyl) thio)ꢀ1Hꢀ
benzimidazole as AR BF3 ligands: To further explore the relevance of various structural
elements of compound 32, we designed 8 close derivatives of this compound by substituting its
sulfur and oxygen atoms in the linker region (Table 3). Data obtained from BioꢀLayer
Interferometry (BLI) experiments and eGFP cellular assays revealed that subtle changes in the
linker can have profound effects on target binding and inhibitory activity. Accordingly, we
investigated the significance of a sulfur atom in compound 32 by replacing it with nitrogen
(compound 38) and carbon (compound 39). These modifications abolished the cellular activity of
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derivatives 38 and 39 and their binding to the AR. Similarly, the replacement of sulfur with
sulfinyl and sulfonyl fragments caused a significant drop in activity of the corresponding
derivatives 40 and 41. The loss in activity of compounds 40 and 41 was due to the presence of
oxygen which disrupts the critical Van der Waals contacts with Phe673, Tyr834 of the BF3
pocket. Our investigation then focused on oxygen atom of linker region in compound 32. In
particular, the replacement of oxygen with carbon resulted in compound 42, which demonstrated
a 4ꢀfold drop in antiꢀAR potential (12ꢁM). Surprisingly, replacing oxygen with nitrogen
(compound 43) exhibited detrimental effects on its binding and activity. Increasing the length of
the linker fragment from SC H O to SC H O resulted in loss of activity of a derivative 44, likely
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caused by its poor fit inside BF3. Shortening the linker region (i.e. removing oxygen atom and
exchanging the SC H OPh fragment to SC H Ph) did not result in any major alteration of antiꢀ
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AR potential, with the corresponding IC assessed for compound 45 at 7.4ꢁM.
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Another focal point of the study was evaluation of the effect of substitutions at the
benzene ring of compound 32 (Table 4). The starting three analogues, 46, 47 and 48, were
designed and synthesized by introducing a methyl group at R , R and R positions of the core.
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As predicted, compound 47 showed a 2ꢀfold increase in ARꢀsuppressing activity (IC =1.8ꢁM)
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compared to the parental compound 32 whereas compound 46 demonstrated 5ꢀfold decrease
while compound 48 demonstrated only slightly lowered activity (IC =7.0ꢁM). As our docking
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models demonstrated, the presence of a methyl group at the metaꢀ position ensures additional
hydrophobic contacts with the Phe826 and Leu830 residue of BF3 and contributing towards
enhanced ligand binding (Figure 3 in Supplemental data). Similarly, 2, 5ꢀmethyl substitution was
wellꢀtolerated and led to further enhanced activity of the corresponding derivative 49
(
IC =2.7ꢁM). The dose response curve for compounds 47 and 49 is presented in Figure 4A. The
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BLI experiment confirmed a direct reversible interaction between compounds 47, 49 and the AR
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see Figure 4B and 4C). We have also explored the introduction of chlorine atom into R2
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compound 50) and R (compound 51) substitution positions, which appear not to affect the
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overall activity. A larger sulfonamide group was not tolerated at position R (52) and caused a
3
15ꢀfold decrease in the inhibition activity.
An effort was also made to replace imidazole with an indole moiety in the developed BF3
inhibitors. Initially, we have made an indoleꢀbased compound 53 which demonstrated a similar
level of AR inhibition compared to compound 32 with a corresponding IC50 of 5.4ꢁM.
Derivative 54 was designed by adding a sulfonamide group to the 7ꢀposition of compound 53.
According to the docking model, sulfonamide forms additional networks of hydrogen bonds with
nearby residues (i.e. side chain of Arg840, Glu837, Asn833 and backbone of Pro671 (Figure
3B)). As a result, the compound demonstrated 4ꢀfold increase its in activity (Figures 4A). The
direct reversible interaction between this compound and the AR LBD was also detected by the
BLI (Figure 4D). Based on the above observations, compounds 32, 47, 49 and 54 were selected
for further evaluation.
Derivatives 32, 47, 49 and 54 Reduce PSA Expression in LNCaP and Enzalutamideꢀ
Resistant Cells: To rule out possible false positive hits in the AR transcriptional eGFP assay, we
validated the activity of compounds 32, 47, 49 and 54 by quantifying their effect on the
2
3
production of the prostate specific antigen (PSA) in prostate cancers cell lines. PSA is a serine
protease whose expression is dependent on AR activity level in the cell. PSA is widely used as a
marker for PCa as its serum concentration is associated with this pathological condition. As
expected, these derivatives induced a doseꢀdependent decrease in PSA levels in LNCaP prostate
2
4
cancer cells with corresponding IC50s value determined as 4.3, 3.3, 1.9 and 1.6ꢁM respectively
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(
Figure 5A). These compounds were also evaluated using in house developed Enzalutamideꢀ
resistant prostate cancer cells (Hidetoshi Kuruma et al. Submitted to cancer research, 2012).
These AR inhibitors are significantly more effective than Casodex and Enzalutamide in these
cells. Figure 5B demonstrates that antiꢀAR drugs are ineffective, with IC s greater than 100ꢁM.
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On the other hand, even though compound 32 (IC =21ꢁM) reduced PSA levels moderately,
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derivatives 47, 49 and 54 (IC =13, 6.8 and 6.4ꢁM) were quite effective in these Enzalutamide
5
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resistant cells. Hence, the inhibition values obtained for inhibition of PSA in LNCaP and
Enzalutamideꢀresistant cells confirms the effectiveness of these inhibitors on the AR signaling
pathway.
Derivatives 32, 47, 49 and 54 Reduces Cell Growth in LNCaP and Enzalutamideꢀ
Resistant Cells: To ascertain the growth inhibitory potential of AR inhibitors 32, 47, 49 and 54,
24
we evaluated their ability to inhibit growth of LNCaP
and Enzalutamideꢀresistant prostate
cancer cells, as well as on ARꢀindependent PC3 cells. The cell viability was assessed after 4 days
of incubation with the test compounds at a concentration of 6ꢂM. Figure 6 shows that compound
32 did not have any significant inhibition effect on these cancer cells whereas its derivatives 47,
49 and 54 suppress cancer cells quite effectively at the concentration measured. Derivatives 49
and 54 exhibit a particularly strong effect on the growth of both LNCaP and Enzalutamideꢀ
resistant cells. Moreover, derivatives 47, 49 and 54 did not show any effect on AR independent
PC3 cell lines, confirming their ARꢀspecific activity.
Derivatives 32, 47, 49 and 54 are Selective AR BF3 inhibitors: We undertook to
profile the selectivity of these derivatives for AR over estrogen receptor α (ERꢀα), the other
member of the steroidal nuclear receptor subfamily. The compounds were tested for their ability
to inhibit 17βꢀEstradiol (E2)ꢀERαꢀmediated gene transcription in MCFꢀ7 human breast cancer
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cells using luciferase reporter whose expression is driven by consensus estrogen response
element. Supplementary Figure 4 shows that the compounds do not inhibit ERꢀα transcriptional
activity compared to Tamoxifen (Tx) measured at 3 different concentrations (10, 5 and 1ꢁM).
This confirms that these inhibitors are ARꢀBF3 specific.
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DISCUSSION
Surface pockets or proteinꢀprotein interaction sites are often considered as attractive
opportunities for therapeutic targeting. However, identifying small molecules that modulate these
sites is often difficult owing to issues such as lack of a wellꢀdefined deep binding pocket.
Although surface sites are challenging drug targets, their adaptive character can provide binding
2
5, 26
grooves for compounds and thus opportunities for drug discovery.
In the case of the AR,
targeting its BF3 pocket offers a promising alternative strategy to create novel therapeutics for
castrationꢀresistant prostate cancer. Since the AR BF3 is surface exposed, identifying compounds
with significant activity profiles and developing structure activity relationship around them is
challenging.
We have previously utilized the power of virtual screening combined with experimental
evaluations to discover a number of small molecules that effectively target the BF3 site of the
AR. On the basis of one of the identified inhibitors (compound 1), we developed a series of
analogues with improved antiꢀAR activity. In particular, a simplified yet more active derivative
32 was synthesized, experimentally evaluated and crystallographically resolved inside the AR
BF3 target cavity. The reported structure 4HLW demonstrated that benzimidazole moiety of the
parent compound 32 makes a strong Hꢀbond with neighboring residue Glu837. The information
obtained by both inhibition experiments and xꢀray crystallography studies indicated that
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compound 32 is a strong BF3ꢀspecific inhibitor. Hence, we synthesized a number of derivatives
of this compound and explored their structureꢀactivity relationship in the context of antiꢀAR
potency.
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We initially modified the linker region of compound 32. Replacement of the oxygen atom
in SC H O was tolerated, but did not result in further improvement of potency. Other
2
4
modifications completely abolished antiꢀAR activity and binding. Hence, we focused on
introducing groups at the benzene ring of the compound 32 template.
By comparison, addition of small hydrophobic substituents such as methyl at various
position of the benzene ring was able to enhance antiꢀAR potency of the corresponding
derivatives of compound 32. In particular, compounds 47 and 49, containing methyl at metaꢀ and
diꢀortho positions, demonstrated IC50 in single digit ꢁꢀmolar range. Replacement of the
benzoimidazole moiety in compound 32 with a synthetically more favorable indole fragment did
not significantly alter the activity of the derivatives. The introduction of a sulfonamide group at
7ꢀposition of the indole core (compound 54) further increased the target affinity by providing
additional hydrogen bonds with Arg840 and Phe673 residues.
These findings culminated in the discovery of rather potent AR inhibitors 32, 47, 49 and
54 with the corresponding IC50s of 4.2, 1.8, 2.7 and 1.5ꢁM respectively, which are 5ꢀ10 times
lower than the IC of 13.1ꢁM for the parental compound 1. The activity of these chemicals was
5
0
further confirmed by their ability to decrease the levels of PSA in LNCaP and Enzalutamideꢀ
resistant PCa cells. Compounds 32, 47, 49 and 54 exhibited IC s of 4.3, 1.9, 3.3 and 1.6ꢁM
5
0
respectively in LNCaP cells. Similar potencies were also observed in Enzalutamideꢀresistant cell
line. Compounds 49 (IC =6.8ꢁM) and 54 (IC =6.4ꢁM) turned out to be especially effective in
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comparison with clinically used Casodex and Enzalutamide (IC >100ꢁM in these resistant
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cells). The PSA inhibition figures were also in agreement with the above numbers giving further
confidence in these BF3 inhibitor prototypes.
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In summary, while we obtained 30 analogues of compound 1 by 2D similarity search,
they were not very active. However, when we rationally developed, synthesized and tested 21
benzimidazole derivatives of compounds 1 and 9 of them showed equivalent or improved
potency against the AR. Similarly, we created and evaluated two indole derivatives which also
exhibited enhanced antiꢀAR potency. These initial results obtained with indoleꢀbased compounds
are encouraging and will be further investigated. Moreover, the structure of the AR in complex
with compound 32 (one of the synthetic derivatives) was elucidated and turned out to be in very
good agreement with our prior predictions, providing additional confidence in our modeling
approach.
27, 28
Drug resistance remains a fundamental cause of therapeutic failure in cancer therapy.
In PCa, cancer progression to a drugꢀresistant phenotype in the presence of an antagonist
possibly through selection of cells with epigenetics or mutational changes that bypass the
inhibitory action of the drug. Our lead derivatives were tested for their ability to inhibit AR in
LNCaP PCa cell lines including those which have developed resistance to the recently approved
7
potent antiꢀandrogen, Enzalutamide. Results from cell viability assays indicated that the tested
derivatives exhibited effective inhibition of growth in both LNCaP and Enzalutamideꢀresistant
cell lines. There was no significant effect on the growth of PC3 PCa cells which lack the AR.
The effectiveness of these BF3 inhibitors was also confirmed when they were shown to reduce
the endogenous expression levels of PSA in Enzalutamideꢀresistant cell lines. Even though the
specific mechanism for Enzalutamideꢀresistance in these cells is still unclear and may or may not
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be fully related to mutations in the AR, the effectiveness of compounds 47, 49 and 54 in these
Enzalutamideꢀresistant cells substantiates targeting an alternative binding site on the AR such as
the BF3. The results obtained from the ERꢀα luciferase assay confirm that these inhibitors are
specific to AR and do not affect ERꢀα in human breast cancer cells.
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In summary, we have developed a novel class of antiꢀAR drugs chemoꢀtypes with an
alternative mechanism of action which can overcome conventional antiꢀandrogen resistance and
exhibit strong antagonism in PCa cell lines. These BF3 drugs have the potential to provide a
further line of treatment after failure of conventional antiꢀandrogens and progression to castration
resistance.
CONCLUSIONS
In the current study, a series of 2ꢀ((2ꢀphenoxyethyl) thio)ꢀ1Hꢀbenzimidazole and 2ꢀ((2ꢀ
phenoxyethyl)thio)ꢀ1Hꢀindole derivatives were rationally designed, synthesized and evaluated
for their ability to inhibit human AR – a primary drug target in PCa. Importantly, these drugs
bind to a newly characterized target site on the AR called Binding Function 3 (BF3) and
therefore, exhibit a new mechanism for inhibition of the AR. It is anticipated that these novel AR
inhibitors provide an alternative therapeutic strategy that can be applied or complimentary to
current antiꢀandrogen treatments for PCa patients. Furthermore, these BF3 drugs can be used
when resistance arises to conventional antiꢀandrogen therapies. They may also be used in
combination with current antiꢀandrogens to possibly avoid or delay progression to castration
resistance. Since the emergence of castration resistance is the lethal end stage of the disease, we
anticipate that the proposed research will eventually have a substantial impact on patient
survival.
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MATERIALS AND METHODS
ꢀ[(2ꢀphenoxyethyl)thio]ꢀ1Hꢀbenzimidazole (32): To a mixture of 1Hꢀbenzimidazoleꢀ2ꢀ
thiol (0.50 g, 3.33 mmol, 1.0 eq.), K CO (0.93 g, 6.66 mmol, 2.0 eq.) and TBAI (0.37g, 1.00
2
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3
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mmol, 0.3 eq.) in acetonitrile (20mL) was added (2ꢀbromoethoxy)benzene (2.15 g, 9.99 mmol,
.0 eq.). The reaction mixture was stirred at 35°C (oil bath at 40°C) during 16 h and then filtered
3
and evaporated under reduced pressure. The residue was purified by silica gel flashꢀcolumn
chromatography (eluent: heptane/EtOAc, 90/10 to 70/30) to afford compound 32 (530 mg, 59 %)
as a white solid. Note: A less polar compound was also isolated (370mg, 28%) and corresponds
1
to the bisꢀalkylated derivative (characterization not included). H NMR (DMSO, 400 MHz): δ
(
ppm): 3.68 (2H, t, J9’ꢀ8’= 6.4 Hz, 2H9’), 4.32 (2H, t, J8’ꢀ9’= 6.4 Hz, 2H8’), 6.94 (1H, t, J4’ꢀ3’= J4’ꢀ
5’= 7.4 Hz, H4’), 6.99 (2H, dm, J2’ꢀ3’= J6’ꢀ5’= 8.2 Hz, H2’ & H6’), 7.11 to 7.15 (2H, m, H5 & H6),
13
7
.29 (2H, tm, J3’ꢀ2’= J3’ꢀ4’= J5’ꢀ6’= J5’ꢀ4’= 7.4 Hz, H3’ & H5’), 7.45 (2H, bs, H4 & H7) C NMR
(DMSO, 100 MHz): δ (ppm): 30.7 (C9’), 66.8 (C8’), 110.9 (C7), 115.0 (C2’ & C6’), 117.8
C4), 121.3 (C4’), 121.9 (bs, C5 & C6), 130.0 (C3’ & C5’), 150.2 (C2), 158.6 (C1’) MS: ESI:
(
+
+
m/z: 271.1 ([M+H] ), 293.1 ([M+Na] ) HRMS (ESI): calculated for C H N OS: m/z =
15 15
2
2
71.0900, found: 271.0914 calculated for C H N NaOS: m/z = 293.0719, found: 293.0730.
15 14 2
1
ꢀethylꢀ2ꢀ[(phenoxyethyl)thio]ꢀ1Hꢀbenzimidazole (33): To a mixture of compound 32
(
200 mg, 0.74 mmol, 1.0 eq.), K CO (206 mg, 1.48 mmol, 2.0 eq.) and TBAI (111 mg, 0.30
2
3
mmol, 0.4 eq.) in acetonitrile (10 mL) was added ethyl bromide (220 ꢁL, 2.96 mmol, 4.0 eq.).
The reaction mixture was stirred at 35°C (oil bath at 40°C) during 48 h and then filtered over
silica gel pad and evaporated under reduced pressure. The residue was triturated with heptane
1
and diethyl ether to afford compound 33 (160 mg, 72 %) as a beige solid. H NMR (DMSO, 400
MHz): δ (ppm): 1.29 (3H, J= 7.2 Hz, ethyl), 3.74 (2H, t, J9’ꢀ8’= 6.4 Hz, 2H9’), 4.18 (2H, q, J=
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.2 Hz, ethyl), 4.34 (2H, J8’ꢀ9’= 6.4 Hz, 2H8’), 6.94 (1H, t, J4’ꢀ3’= J4’ꢀ5’= 7.8 Hz, H4’), 7.00 (2H,
dm, J2’ꢀ3’= J6’ꢀ5’= 7.8 Hz, H2’ & H6’), 7.16 to 7.20 (2H, m, H5 & H6), 7.29 (2H, tm, J3’ꢀ2’= J3’ꢀ4’
=
1
3
J5’ꢀ6’= J5’ꢀ4’= 7.8 Hz, H3’ & H5’), 7.52 & 7.58 (2H, d & d, H4 & H7) C NMR (DMSO, 100
0
1
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MHz): δ (ppm): 14.4 (ethyl), 30.6 (C9’), 38.4 (ethyl), 66.1 (C8’), 109.4 (C7), 114.5 (C2’ &
C6’), 117.6 (C4), 120.8 (C4’), 121.4 & 121.5 (C5 & C6), 129.5 (C3’ & C5’), 135.7 (C3a), 142.9
+
(C7a), 150.3 (C2), 158.0 (C1’) MS: ESI: m/z: 299.1 ([M+H] ) HRMS (ESI): calculated for
C H N OS: m/z = 299.1213, found: 299.1205
17
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2
ꢀ{2ꢀ[(phenoxyethyl)thio]ꢀ1Hꢀbenzimidazoꢀ1ꢀyl}ethanol (34): To a mixture of
compound 32 (2.0 g, 7.4 mmol, 1.0 eq.) and K CO (2.3 g, 16.6 mmol, 2.3 eq.) in NMP (15 mL)
2
3
was added 2ꢀbromoethanol (1.2 mL, 17.7 mmol, 2.4 eq.). The reaction mixture was stirred at
0°C for 15 h, allowed to cool to room temperature and then, diluted with water. The mixture
7
was extracted with ethyl acetate and methyl tertꢀbutyl ether. The organic layer was dried over
Na SO , evaporated under reduced pressure and the residue was purified by silica gel flashꢀ
2
4
column chromatography (eluent: heptane/EtOAc, 90/10 to 60/40) to afford compound 34 (1.1 g,
1
47 %) as a white solid. H NMR (DMSO, 400 MHz): δ (ppm): 3.70 (4H, m, CH ), 4.19 (2H, t,
2
J= 7.0 Hz, CH ), 4.32 (2H, J8’ꢀ9’= 6.4 Hz, 2H8’), 4.97 (1H, t, J= 7.0 Hz, OH), 6.94 (1H, t, J4’ꢀ3’
=
2
J4’ꢀ5’= 7.8 Hz, H4’), 7.00 (2H, dm, J2’ꢀ3’= J6’ꢀ5’= 7.8 Hz, H2’ & H6’), 7.16 to 7.18 (2H, m, H5 &
H6), 7.29 (2H, tm, J3’ꢀ2’= J3’ꢀ4’= J5’ꢀ6’= J5’ꢀ4’= 7.8 Hz, H3’ & H5’), 7.49 & 7.56 (2H, d & d, H4 &
13
H7) C NMR (DMSO, 100 MHz): δ (ppm): 30.6 (C9’), 46.4 (CH ), 59.2 (CH ), 66.2 (C8’),
2
2
109.9 (C7), 114.5 (C2’ & C6’), 117.5 (C4), 120.8 (C4’), 121.3 & 121.4 (C5 & C6), 129.5 (C3’ &
+
C5’), 136.6 (C3a), 142.8 (C7a), 151.2 (C2), 158.0 (C1’) MS: ESI: m/z: 315.1 ([M+H] ) HRMS
(ESI): calculated for C H N O S: m/z = 315.1162, found: 315.1161
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ꢀ{2ꢀ[(2ꢀphenoxyethyl)thio]ꢀ1Hꢀbenzimidazolꢀ1ꢀyl}propanoic acid (35): To a mixture
of 3ꢀ{2ꢀ[(2ꢀphenoxyethyl)thio]methylꢀ1Hꢀbenzimidazolꢀ1ꢀyl}propanoate (450 mg, 1.21 mmol)
in acetic acid (10mL) and water (10mL) was added a concentrated HCl solution (3 mL). The
reaction mixture was stirred at 80°C during 5 h and then coꢀevaporated with heptane and toluene
under reduced pressure. The residue was taken up in ethyl acetate and the organic layer was
dried over Na SO and evaporated to afford, after trituration with methyl tertꢀbutyl ether,
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54
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2
4
1
compound 35 (180 mg, 43 %) as a white solid. H NMR (DMSO, 600 MHz): δ (ppm): 2.75
(
2H, d, J = 7.0 Hz, 2H2), 3.76 (2H, t, J8’’ꢀ9’’= 6.4 Hz, 2H8’’), 4.33 (2H, t, J9’’ꢀ8’’= 6.4 Hz,
2ꢀ3
2
H9’’), 4.42 (2H, d, J = 7.0 Hz, 2H3), 6.92ꢀ6.95 (3H, m, H4’’, H2’’ & H6’’), 7.24ꢀ7.30 (4H, m,
3ꢀ2
1
3
H5’, H6’, H3’’ & H5’’), 7.61 (1H, m, H4), 7.64 (1H, m, H7). C NMR (DMSO, 150 MHz): δ
(
ppm): 32.0 (C2), 33.8 (C9’’), 40.2 (C3), 66.7 (C8’’), 110.9 (C4’), 114.9 (C2’’ & C6”), 117.3
(
C7’), 121.4 (C4’’), 122.9 (C5’ & C6’), 130.0 (C3’’ & C5’’), 135.7 (C7a’), 151.3 (C2’), 158.4
+
(
C1’’), 172.3 (C1). MS: ESI: m/z: 343.1 ([M+H] ) HRMS (ESI): calculated for C H N O S:
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2
3
m/z = 343.1111, found: 343.1081
1ꢀallylꢀ2ꢀ[(2ꢀphenoxyethyl)thio]ꢀ1Hꢀbenzimidazole (36) : To a mixture of compound
2 (300 mg, 1.11 mmol, 1.0 eq.), K CO (309 mg, 2.22 mmol, 2.0 eq.) and TBAI (328 mg, 0.88
3
2
3
mmol, 0.8 eq.) in acetonitrile (10 mL) was added allyl bromide (0.580mL, 6.66 mmol, 6.0 eq.).
The reaction mixture was stirred at 35°C during 20 h and then, diluted with water and extracted
with methyl tertꢀbutyl ether. The organic layer was dried over Na SO , filtered through a silica
2
4
1
gel pad and evaporated to afford compound 36 (330 mg, 96 %) as a white solid. H
NMR (DMSO, 400 MHz): δ (ppm): 3.73 (2H, t, J9’ꢀ8’= 6.4 Hz, 2H9’), 4.33 (2H, J8’ꢀ9’= 6.4 Hz,
2
H8’), 4.80 (2H, m, 2H8), 4.97 (1H, dm, Jtrans= 17.2 Hz, H10), 5.17 (1H, dm, J = 10.2 Hz,
cis
H10), 5.90ꢀ5.99 (1H, m, H9), 6.95 (1H, t, J4’ꢀ3’= J4’ꢀ5’= 7.4 Hz, H4’), 7.00 (2H, dm, J2’ꢀ3’= J6’ꢀ5’
=
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2
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.8 Hz, H2’ & H6’), 7.17 to 7.20 (2H, m, H5 & H6), 7.17 to 7.20 (2H, m, H5 & H6), 7.29 (2H,
1
3
tm, J3’ꢀ2’= J3’ꢀ4’= J5’ꢀ6’= J5’ꢀ4’= 7.4 Hz, H3’ & H5’), 7.46 (1H, dm, H4), 7.60 (1H, dm, H7) C
NMR (DMSO, 100 MHz): δ (ppm): 31.3 (C9’), 46.1 (C8), 66.6 (C8’), 110.2 (C7), 115.0 (C2’
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
&
C6’), 117.7 (C10), 118.2 (C4), 121.3 (C4’), 122.1 & 122.2 (C5 & C6), 130.0 (C3’ & C5’),
+
1
32.7 (C9), 136.6 (C3a), 143.4 (C7a), 151.5 (C2), 158.5 (C1’) MS: ESI: m/z: 311.1 ([M+H] )
HRMS (ESI): calculated for C H N OS: m/z = 311.1213, found: 311.1185
1
8
19
2
2
ꢀ[(2ꢀphenoxyethyl)thio]ꢀ1,3ꢀbenzoxazole (37): To a mixture of 1,3ꢀbenzoxazoleꢀ2ꢀ
thiol (1.0 g, 6.7 mmol, 1.0 eq.), K CO (1.9 g, 13.4 mmol, 2.0 eq.) and TBAI (1.5 g, 4.0 mmol,
2
3
0
.6 eq.) in acetonitrile (40 mL) was added (2ꢀbromoethoxy)benzene (1.6 g, 8.0 mmol, 1.2 eq.).
The reaction mixture was stirred at 35°C (oil bath at 40°C) during 24 h and then, diluted with
water and extracted with ethyl acetate. The organic layer was dried over Na SO and evaporated
2
4
under reduced pressure. The crude was taken up in CH Cl and heptane, filtered through a silica
2
2
gel pad and eluted with methyl tertꢀbutyl ether to afford, after removal of solvent, compound 37
1
(1.1 g, 61 %) as a red solid. H NMR (DMSO, 400 MHz): δ (ppm): 3.73 (2H, t, J9’ꢀ8’= 6.4 Hz,
2H9’), 4.36 (2H, t, J8’ꢀ9’= 6.4 Hz, 2H8’), 6.94 (1H, m, H4’), 6.96 (2H, m, H2’ & H6’), 7.28 (2H,
tm, J3’ꢀ2’= J3’ꢀ4’= J5’ꢀ6’= J5’ꢀ4’= 7.4 Hz, H3’ & H5’), 7.32ꢀ7.35 (2H, m, H5 & H6), 7.6ꢀ7.67 (2H, m,
1
3
H4 & H7). C NMR (DMSO, 100 MHz): δ (ppm): 30.9 (C9’), 65.7 (C8’), 110.2 (C7), 114.5
(C2’ & C6’), 118.2 (C4), 120.9 (C4’), 124.3 & 124.6 (C5 & C6), 129.5 (C3’ & C5’), 141.2
+
(C3a), 151.3 (C2), 157.9 (C1’), 164.0 (C7a). MS: ESI: m/z: 272.1 ([M+H] ) HRMS (ESI):
calculated for C H NO S: m/z = 272.0740, found: 272.0750
1
5
14
2
2
ꢀ[(2ꢀphenoxyethyl) sulfinyl]ꢀ1Hꢀbenzimidazole (40) and 2ꢀ[(2ꢀphenoxyethyl)
sulfonyl]ꢀ1Hꢀbenzimidazole (41): To a mixture of compound 32 (200 mg, 0.74 mmol, 1.0 eq.)
in CH Cl (20 mL) was added, at 0°C, mꢀCPBA (383 mg, 2.22 mmol, 3.0 eq.). The reaction
2
2
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mixture was stirred at 0°C during 10 min and then, quenched with a saturated aqueous sodium
sulfite solution. The mixture was stirred at room temperature for several minutes, the organic
layer dried over Na SO and the solvent removed under reduced pressure. The residue was
2
4
10
11
12
13
14
15
16
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18
19
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36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
purified by silica gel flashꢀcolumn chromatography (eluent: heptane/EtOAc, 80/20 to 40/60) to
1
afford compounds 40 (110 mg, 52 %) and 41 (80 mg, 36 %) as white solids. Compound 40: H
NMR (DMSO, 400 MHz): δ (ppm): 3.68 (1H, m, 1H9’), 3.78 (1H, m, 1H9’), 4.39ꢀ4.47 (2H, m,
2
H8’), 6.81 (2H, d, J2’’ꢀ3’’= J6’’ꢀ5’’= 8.2 Hz, H2’ & H6’), 6.94 (1H, t, J4’ꢀ3’= J4’ꢀ5’= 8.2 Hz, H4’),
.26 (2H, t, J3’ꢀ2’= J3’ꢀ4’= J5’ꢀ4’= J5’ꢀ6’= 8.2 Hz, H3’ & H5’), 7.31ꢀ7.33 (2H, m, H5 & H6), 7.66
7
1
3
(
2H, bs, H4 & H7), 13.57 (1H, s, benzimidazolic H) C NMR (DMSO, 100 MHz): δ (ppm):
53.7 (C9’), 63.2 (C8’), 114.9 (C2’ & C6), 121.5 (C4’), 123.6 (C5 & C6), 130.0 (C3’’ & C5’’),
+
+
1
54.6 (C2), 158.1 (C1’) MS: ESI: m/z: 287.1 ([M+H] ), 309.1 ([M+Na] ) HRMS (ESI):
calculated for C H N O S: m/z = 287.0849, found: 287.0833 calculated for C H N NaO S:
1
5
15
2
2
15 14
2
3
ꢀ
m/z = 309.0668, found: 309.0653 ESI: m/z: 285.1 ([MꢀH] ) HRMS (ESI): calculated for
1
C H N O S: m/z = 285.0703, found: 285.0676 Compound 41: H NMR (DMSO, 400 MHz):
15 13
2
2
δ (ppm): 4.11 (2H, t, J8’ꢀ9’= 5.4 Hz, 2H8’), 4.38 (2H, t, J9’ꢀ8’= 5.4 Hz, 2H9’), 6.48 (2H, d, J2’’ꢀ3’’
=
J6’’ꢀ5’’= 8.2 Hz, H2’ & H6’), 6.88 (1H, t, J4’ꢀ3’= J4’ꢀ5’= 8.2 Hz, H4’), 7.15 (2H, t, J3’ꢀ2’= J3’ꢀ4’= J5’ꢀ
1
3
4’= J5’ꢀ6’= 8.2 Hz, H3’ & H5’), 7.40ꢀ7.42 (2H, m, H5 & H6), 7.71 (2H, bs, H4 & H7) C NMR
(DMSO, 100 MHz): δ (ppm): 54.6 (C9’), 61.9 (C8’), 114.6 (C2’ & C6), 121.5 (C4’), 129.8
+
(C3’’ & C5’’), 157.7 (C1’) MS: ESI: m/z: 303.1 ([M+H] ) HRMS (ESI): calculated for
ꢀ
C H N O S: m/z = 303.0798, found: 303.0780 ESI: m/z: 301.1 ([MꢀH] ) HRMS (ESI):
15
15
2
3
calculated for C H N O S: m/z = 301.0624, found: 301.0652
1
5
13
2
3
2
ꢀ[(3ꢀphenylpropyl)thio]ꢀ1Hꢀbenzimidazole (42): To a mixture of 1Hꢀbenzimidazoleꢀ2ꢀ
thiol (200 mg, 1.33 mmol, 1.0 eq.), K CO (370 mg, 2.66 mmol, 2.0 eq.) and TBAI (295 mg,
2
3
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0
.80 mmol, 0.6 eq.) in acetonitrile (6 mL) was added (3ꢀbromopropyl)benzene (606 ꢁL, 3.99
mmol, 3.0 eq.). The reaction mixture was stirred at 35°C (oil bath at 40°C) during 48 h and then,
diluted with water and extracted with ethyl acetate. The organic layer was dried over Na SO ,
2
4
0
1
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0
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6
7
8
9
0
evaporated under reduced pressure and the residue was purified by silica gel flashꢀcolumn
chromatography (eluent: heptane/EtOAc, 90/10 to 70/30) to afford compound 42 (260 mg, 73 %)
as a white solid. Note: A less polar compound was also isolated (120mg, 25%) and corresponds
1
to the bisꢀalkylated derivative (characterization not included). H NMR (DMSO, 400 MHz): δ
(ppm): 2.03 (2H, m, 2H8’), 2.74 (2H, t, J7’ꢀ8’= 7.6 Hz, 2H7’), 3.28 (2H, t, J9’ꢀ8’= 7.6 Hz, 2H9’),
7
.09 to 7.13 (2H, m, H5 & H6), 7.18 (1H, m, H4’), 7.22 (2H, m, H2’ & H6’), 7.29 (2H, m, H3’
1
3
& H5’), 7.44 (2H, bs, H4 & H7) C NMR (DMSO, 100 MHz): δ (ppm): 36.0 (C9’), 36.2
(C8’), 39.2 (C7’), 126.5 (bs, C5 & C6), 131.1 (C4’), 133.6 (C2’, C3’, C5’ & C6’), 146.3 (C1’),
+
1
55.2 (C2) MS: ESI: m/z: 269.1 ([M+H] ) HRMS (ESI): calculated for C H N S: m/z =
16 17
2
269.1107, found: 269.1120
2
ꢀ[(3ꢀphenoxypropyl)thio]ꢀ1Hꢀbenzimidazole (44): To a mixture of 1Hꢀbenzimidazoleꢀ
2ꢀthiol (200 mg, 1.33 mmol, 1.0 eq.), K CO (370 mg, 2.66 mmol, 2.0 eq.) and TBAI (147 mg,
2
3
0.40 mmol, 0.3 eq.) in acetonitrile (6 mL) was added 3ꢀphenoxypropyl bromide (630 ꢁL, 3.99
mmol, 3.0 eq.). The reaction mixture was stirred at 35°C (oil bath at 40°C) during 48 h and then,
diluted with water and extracted with methyl tertꢀbutyl ether. The organic layer was dried over
Na SO , the solvent removed under reduced pressure and the residue purified by silica gel flashꢀ
2
4
column chromatography (eluent: heptane/EtOAc, 90/10 to 70/30) to afford compound 44 (260
1
mg, 69 %) as a white solid. H NMR (DMSO, 400 MHz): δ (ppm): 2.19 (2H, q, J9’ꢀ8’= J9’ꢀ10’
=
6.4 Hz, 2H9’), 3.42 (2H, t, J10’ꢀ9’= 6.4 Hz, 2H10’), 4.10 (2H, t, J8’ꢀ9’= 6.4 Hz, 2H8’), 6.91ꢀ6.96
(3H, m, H4’, H2’ & H6’), 7.12 (2H, m, H5 & H6), 7.28 (2H, m, H3’ & H5’), 7.37 (1H, bs, H7),
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.51 (1H, bs, H4), 12.6 (1H, s, benzimidazolic H) C NMR (DMSO, 100 MHz): δ (ppm): 28.5
(C10’), 29.4 (C9’), 66.2 (C8’), 110.7 (C7), 114.9 (C2’ & C6’), 117.8 (C4), 121.0 (C4’), 121.6 &
1
22.0 (C5 & C6), 130.0 (C4’), 129.9 (C3’ & C5’), 150.3 (C2), 158.9 (C1’) MS: ESI: m/z: 285.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
(
[M+H] ) HRMS (ESI): calculated for C H N OS: m/z = 285.1069, found: 285.1054
16 17
2
2
ꢀ[(2ꢀphenylethyl)thio]ꢀ1Hꢀbenzimidazole (45): To a mixture of 1Hꢀbenzimidazoleꢀ2ꢀ
thiol (200 mg, 1.33 mmol, 1.0 eq.), K CO (370 mg, 2.66 mmol, 2.0 eq.) and TBAI (295 mg,
2
3
0.80 mmol, 0.6 eq.) in acetonitrile (6 mL) was added (3ꢀbromopropyl)benzene (606 ꢁL, 3.99
mmol, 3.0 eq.). The reaction mixture was stirred at 35°C (oil bath at 40°C) during 48 h and then,
diluted with water and extracted with ethyl acetate. The organic layer was dried over Na SO ,
2
4
evaporated under reduced pressure and the residue was purified by silica gel flashꢀcolumn
chromatography (eluent: heptane/EtOAc, 90/10 to 70/30) to afford compound 45 (260 mg, 77 %)
as a white solid. NB: A less polar compound was also isolated (80 mg, 17 %, white solid) and
1
corresponds to the N,Sꢀbisꢀalkylated derivative (characterization not included).
H
NMR (DMSO, 400 MHz): δ (ppm): 3.73 (2H, t, J9’ꢀ8’= 6.4 Hz, 2H9’), 4.36 (2H, t, J8’ꢀ9’= 6.4 Hz,
2H8’), 6.94 (1H, m, H4’), 6.96 (2H, m, H2’ & H6’), 7.28 (2H, tm, J3’ꢀ2’= J3’ꢀ4’= J5’ꢀ6’= J5’ꢀ4’
=
13
7
.4 Hz, H3’ & H5’), 7.32ꢀ7.35 (2H, m, H5 & H6), 7.6ꢀ7.67 (2H, m, H4 & H7). C NMR
(
DMSO, 100 MHz): δ (ppm): 30.9 (C9’), 65.7 (C8’), 110.2 (C7), 114.5 (C2’ & C6’), 118.2
(
C4), 120.9 (C4’), 124.3 & 124.6 (C5 & C6), 129.5 (C3’ & C5’), 141.2 (C3a), 151.3 (C2), 157.9
+
(
C1’), 164.0 (C7a). MS: ESI: m/z: 272.1 ([M+H] ) HRMS (ESI): calculated for C H NO S:
1
5
14
2
m/z = 272.0740, found: 272.0750.
2
{[2ꢀ(oꢀtolyloxy)ethyl]thio}ꢀ1Hꢀbenzimidazole (46): To
a
mixture of 1Hꢀ
benzimidazoleꢀ2ꢀthiol (0.50 g, 3.33 mmol, 1.0 eq.), K CO (0.93 g, 6.66 mmol, 2.0 eq.) and
2
3
TBAI (0.37 g, 1.00 mmol, 0.3 eq.) in acetonitrile (20 mL) was added 1ꢀ(2ꢀbromoethoxy)ꢀ2ꢀ
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methylbenzene (2.15 g, 9.99 mmol, 3.0 eq.). The reaction mixture was stirred at 35°C (oil bath at
40°C) during 16 hours and then diluted with water and extracted with ethyl acetate. The organic
layer was dried over Na SO and evaporated under reduced pressure. The residue was purified
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
by silica gel flashꢀcolumn chromatography (eluent: heptane/EtOAc, 90/10 to 70/30) to afford
compound 46 (580 mg, 61 %) as a white solid. NB: A less polar compound was also isolated
(300 mg, 22 %, pale oil) and corresponds to the N,Sꢀbisꢀalkylated derivative (characterization not
1
included). H NMR (DMSO, 400 MHz): δ (ppm): 2.11 (3H, s, methyl), 3.70 (2H, t, J9’ꢀ8’=
6.4 Hz, 2H9’), 4.32 (2H, t, J8’ꢀ9’= 6.4 Hz, 2H8’), 6.84 (1H, t, J4’ꢀ3’= J4’ꢀ5’= 7.8 Hz, H4’), 7.01 (1H,
d, J3’ꢀ4’= J5’ꢀ4’= 7.8 Hz, H3’), 7.10 to 7.16 (4H, m, H5, H6, H5’ & H6’), 7.38 (1H, bs, H4), 7.51
1
3
(1H, bs, H7). C NMR (DMSO, 100 MHz): δ (ppm): 16.3 (methyl), 31.0 (C9’), 67.1 (C8’),
110.6 (C7), 112.0 (C3’), 117.7 (C4), 121.0 (C4’), 121.8 (bs, C5 & C6), 126.3 (C2’), 127.5 (C3’),
+
1
30.9 (C5’), 150.3 (C2), 156.6 (C1’). MS: ESI: m/z: 285.1 ([M+H] ) HRMS (ESI): calculated
for C H N OS: m/z = 285.1056, found: 285.1034.
1
6
17
2
2
ꢀ{[2ꢀ(pꢀtolyloxy)ethyl]thio}ꢀ1Hꢀbenzimidazole (48): To
a
mixture of 1Hꢀ
benzimidazoleꢀ2ꢀthiol (200 mg, 1.33 mmol, 1.0 eq.), K CO (370 mg, 2.66 mmol, 2.0 eq.) and
2
3
TBAI (147 mg, 0.40 mmol, 0.3 eq.) in acetonitrile (12 mL) was added (2ꢀbromoethoxy)ꢀ4ꢀ
methylbenzene (860 mg, 3.99 mmol, 3.0 eq.). The reaction mixture was stirred at 35°C (oil bath
at 40°C) during 20 h and then filtered and evaporated under reduced pressure. The residue was
purified by silica gel flashꢀcolumn chromatography (eluent: heptane/EtOAc, 90/10 to 70/30) to
afford compound 48 (130 mg, 34 %) as white solid. Note : Another compound was also isolated
(300 mg, 54 %) %) and corresponds to the bisꢀalkylated derivative (characterization not
1
included). H NMR (DMSO, 400 MHz): δ (ppm): 2.22 (3H, s, methyl), 3.65 (2H, t, J9’ꢀ8’
=
6.4 Hz, 2H9’), 4.27 (2H, t, J8’ꢀ9’= 6.4 Hz, 2H8’), 6.88 (2H, dm, J2’ꢀ3’= J6’ꢀ5’= 8.2 Hz, H2’ & H6’),
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.08 (2H, d, J3’ꢀ2’= J5’ꢀ6’= 8.6 Hz, H3’ & H5’), 7.12 (2H, m, H5 & H6), 7.45 (2H, bs, H4 & H7)
1
3
C NMR (DMSO, 100 MHz): δ (ppm): 20.5 (methyl), 30.7 (C9’), 66.9 (C8’), 114.9 (C2’ &
C6’), 121.8 (C5 & C6), 130.0 (C4’), 130.3 (C3’ & C5’), 150.2 (C2), 156.5 (C1’) MS: ESI: m/z:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
2
85.1 ([M+H] ) HRMS (ESI): calculated for C H N OS: m/z = 285.1056, found: 285.1054
16 17
2
2
{[2ꢀ(2,6ꢀdimethylphenoxy)ethyl]thio}ꢀ1Hꢀbenzimidazole (49): To a mixture of 1Hꢀ
benzimidazoleꢀ2ꢀthiol (0.50 g, 3.33 mmol, 1.0 eq.), K CO (0.93 g, 6.66 mmol, 2.0 eq.) and
2
3
TBAI (0.37 g, 1.00 mmol, 0.3 eq.) in acetonitrile (20 mL) was added 2ꢀ(2ꢀbromoethoxy)ꢀ1,3ꢀ
dimethylbenzene (2.29 g, 9.99 mmol, 3.0 eq.). The reaction mixture was stirred at 35°C (oil bath
at 40°C) during 16 h and then diluted with water and extracted with ethyl acetate. The organic
layer was dried over Na SO and evaporated under reduced pressure. The residue was purified
2
4
by silica gel flashꢀcolumn chromatography (eluent: heptane/EtOAc, 90/10 to 70/30) to afford
compound 49 (450 mg, 45 %) as a white solid. Note: A less polar compound was also isolated
(
350 mg, 24 %, orange oil) and corresponds to the N,Sꢀbisꢀalkylated derivative (characterization
1
not included). H NMR (DMSO, 400 MHz): δ (ppm): 2.24 (6H, s, methyl), 3.70 (2H, t, J9’ꢀ8’
=
6.4 Hz, 2H9’), 4.08 (2H, t, J8’ꢀ9’= 6.4 Hz, 2H8’), 6.91 (1H, t, J4’ꢀ3’= J4’ꢀ5’= 7.8 Hz, H4’), 7.01 (2H,
d, J3’ꢀ4’= J5’ꢀ4’= 7.8 Hz, H3’ & H5’), 7.10 to 7.14 (2H, m, H5 & H6), 7.37 (1H, bs, H4), 7.50 (1H,
13
bs, H7) C NMR (DMSO, 100 MHz): δ (ppm): 16.5 (2 methyl), 31.8 (C9’), 70.7 (C8’), 110.8
(
C7), 117.7 (C4), 121.6 & 122.1 (C5 & C6), 124.3 (C4’), 129.2 (C3’ & C5’), 130.8 (C2’ & C6’),
+
1
36.0 (C7a), 144.1 (C3a), 150.4 (C2), 155.5 (C1’) MS: ESI: m/z: 299.1 ([M+H] ) HRMS
(
ESI): calculated for C H N OS: m/z = 299.1213, found: 299.1190
17 19 2
2
ꢀ[(2ꢀphenoxyethyl)thio]ꢀ1Hꢀindole (53): To a mixture of indolineꢀ2ꢀthione (0.60 g, 4.0
mmol, 1.0 eq.), K CO (1.1 g, 8.0 mmol, 2.0 eq.) and TBAI (0.9 g, 2.4 mmol, 0.6 eq.) in
2
3
acetonitrile (30 mL) was added (2ꢀbromoethoxy)benzene (1.6 g, 8.0 mmol, 2.0 eq.). The reaction
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mixture was stirred at room temperature during 12 h then, diluted with water and extracted with
ethyl acetate. The organic layer was dried over Na SO , evaporated under reduced pressure, and
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the residue was purified by silica gel flashꢀcolumn chromatography (eluent: heptane/EtOAc, 95/5
0
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to 85/15) to afford compound 53 (0.7 g, 65 %) as a white solid. H NMR (DMSO, 400 MHz): δ
(ppm): 3.29 (2H, t, J9’ꢀ8’= 6.4 Hz, 2H9’), 4.15 (2H, t, J8’ꢀ9’= 6.4 Hz, 2H8’), 6.57 (1H, bs, H3),
6.90 (2H, dm, J2’ꢀ3’= J6’ꢀ5’= 7.4 Hz, H2’ & H6’), 6.93 (1H, tm, J4’ꢀ3’= J4’ꢀ5’= 7.4 Hz, H4’), 6.99
(
1H, tm, J = J = 7.8 Hz, H6), 7.09 (1H, tm, J = J = 7.8 Hz, H5), 7.26 (2H, tm, J3’ꢀ2’= J3’ꢀ4’=
6ꢀ5
6ꢀ7
5ꢀ4
5ꢀ6
J5’ꢀ6’= J5’ꢀ4’= 7.4 Hz, H3’ & H5’), 7.32 (1H, d, J = 7.8 Hz, H4), 7.46 (1H, d, J = 7.8 Hz, H7),
4ꢀ5
7ꢀ6
1
3
1
1.44 (1H, s, indolic H) C NMR (DMSO, 100 MHz): δ (ppm): 34.4 (C9’), 66.7 (C8’), 106.8
(C3), 111.3 (C4), 114.9 (C2’ & C6’), 119.7 (C6), 119.9 (C7), 121.3 (C4’), 122.1 (C5), 128.5
+
(
C3a), 128.8 (C2), 130.0 (C3’ & C5’), 137.9 (C7a), 158.6 (C1’) MS: ESI: m/z: 270.1 ([M+H] )
HRMS (ESI): calculated for C H NOS: m/z = 270.0947, found: 270.0956
1
6
16
Synthetic procedure for Nꢀ(2ꢀphenoxyethyl)ꢀ1Hꢀbenzo[d]imidazolꢀ2ꢀamine (38), 2ꢀ(3ꢀ
phenoxypropyl)ꢀ1Hꢀbenzo[d]imidazole (39), Nꢀ(2ꢀ((1Hꢀbenzo[d]imidazolꢀ2ꢀyl)thio)ethyl)aniline
(43),
2ꢀ((2ꢀ(mꢀtolyloxy)ethyl)thio)ꢀ1Hꢀbenzo[d]imidazole
(47),
2ꢀ((2ꢀ(3ꢀ
chlorophenoxy)ethyl)thio)ꢀ1Hꢀbenzo[d]imidazole (50), 2ꢀ((2ꢀ(4ꢀchlorophenoxy)ethyl)thio)ꢀ1Hꢀ
benzo[d]imidazole (51) is mentioned in supporting information (Supporting Figure 19ꢀ20).
These compounds were synthesized at Enamine (http://www.enamine.net/). 4ꢀ(2ꢀ((1Hꢀ
benzo[d]imidazolꢀ2ꢀyl)thio)ethoxy) benzenesulfonamide (52), 2ꢀ((2ꢀphenoxyethyl)thio)ꢀ1Hꢀ
indoleꢀ7ꢀsulfonamide (54) were obtained from Enamine’s stock.
Preparation of the Protein Structure for Docking: AR crystal structure complexed
1
9
with compound 1, 2YLO (2.50Å resolution) and 4HLW (2.50Å resolution) were used for
molecular docking studies. For protein structure preparation, all solvent molecules have been
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deleted and the bond order for the ligand and protein has been adjusted. The missing hydrogen
atoms have been added, and side chains have then been energyꢀminimized using the OPLSꢀ2005
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force field, as implemented by Maestro. The ligand binding region has been defined by a 12Å
box centered on the crystallographic ligands of the crystal structures. No Van der Waals scaling
factors were applied; the default settings were used for all other adjustable parameters.
Ligand Preparation: All the compounds were built using MOE version
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009. Hydrogen atoms were added after these structures were “washed” (a procedure including
salt disconnection, removal of minor components, deprotonation of strong acids, and protonation
of strong bases). The following energy minimization was performed with the MMFF94x force
field, as implemented by the MOE, and the optimized structures were exported into the Maestro
suite in SD file format.
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Molecular docking: Docking experiments were performed using Glide included in
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Schrodinger Package, Maestro interface version 9.0. For docking, standardꢀprecision (SP)
docking method was adopted to generate the minimized pose, and the Glide scoring function
(Glide Score) was used to select the final poses for each ligand.
Heterologous Expression of the AR: The AR ligand binding domain (LBD) was
1
6
expressed and purified as previously described.
eGFP Cellular AR Transcription Assay: AR transcriptional activity was assayed as
2
2
previously described. Briefly, stably transfected eGFPꢀexpressing LNCaP human prostate
cancer cells (LNꢀARR2PBeGFP) containing an androgenꢀresponsive probasinꢀderived promoter
(ARR2PB) were grown in phenolꢀredꢀfree RPMI 1640 supplemented with 5% CSS for 5 days.
The cells were then seeded into a 96ꢀwell plate (35,000cells/well) and treated the next day with
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.1nM R1881 and increasing concentrations (0ꢀ100ꢂM) of compound. After 3 days of treatment
the fluorescence was measured (excitation, 485 nm; emission, 535 nm).
Prostate Surface Antigen assay: The evaluation of PSA excreted into the media was
performed in parallel to the eGFP assay using the same plates (see above description). After the
cells were incubated for 3 days 150ꢁl of the media was taken from each well, and added to 150ꢁl
of PBS. PSA levels were then evaluated using Cobas e 411 analyzer instrument (Roche
Diagnostics) according to the manufacturer’s instructions.
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MTS Assay: Cell proliferation was determined using the MTS cell proliferation assay
following incubation with the compound (0ꢀ100ꢂM) over 72h (CellTiter 961 Aqueous One
Solution Reagent, Promega). In brief, 30ꢂL of the reagent was added to cells in each well of the
96ꢀwell plate containing 200ꢂL of media and incubated for 90 minutes at 37°C in 5% CO . The
2
production of formazan was measured at 490 nm.
Luciferase ERꢀα transcriptional assay: ERꢀα positive MCFꢀ7 human breast cancer
cells were grown in phenolꢀredꢀfree RPMI 1640 supplemented with 10% CSS for 5 days. The
cells were seeded on a 96ꢀwell plate (30,000cells/well). After 24 hours, the cells were transfected
with 50 ng luciferase plasmid using Lipofectamine 2000 reagent (Invitrogen) and treated the next
day with either the test compounds or Tamoxifen in the presence of 1 nM E2. The final
concentration of the compounds was 10 ꢁM and Tamoxifen was added at 3 different
concentrations, 10 ꢁM, 5 ꢁM and 1 ꢁM. The medium contained 0.1% (v/v) EtOH and 0.1 %
(v/v) DMSO. 24 hours after treatment the medium was aspirated off and the cells were lysed by
adding 65 ꢁL of 1X passive lysis buffer (Promega). The plates were placed on a shaker at room
temperature for 15 mins and then subjected to two freeze thaw cycles to help lyse the cells. 20
ꢁ
L of the lysate from each treatment was transferred onto a 96 well white flat bottom plate
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(
Corning) and the luminescence signal was measured after adding 50 ꢁL of the luciferase assay
reagent (Promega).
BioꢀLayer Interferometry (BLI) assay: The direct reversible interaction between small
molecules and the AR was quantified by BLI using OctetRED (ForteBio). The LDB of the
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biotinylated androgen receptor (bAR) was produced in situ with AviTag technology. The
AviTag sequence (GLNDIFEAQKIEWHE) followed by a six residue glycine serine linker
(
GSGSGS) was incorporated at the Nꢀterminus of the AR LBD (669ꢀ919). Escherichia coli BL21
containing both biotin ligase and AR LBD vectors were induced with 0.5mMisopropylꢀβꢀDꢀ
thiogalactopyranoside (IPTG) in the presence of dihydrotestosterone (DHT) and biotin at 16°C
overnight. The bacteria were then lysed by sonication, and the resulting lysate was purified by
immobilized metal ion affinity chromatography (IMAC) with nickel_nitrilotriacetic acid
(Ni_NTA) resin and cationꢀexchange chromatography (HiTrap SP).
Purified bAR LBD (50ꢂg/mL) was bound to the superꢀstreptavidin sensors overnight at
room temperature. The sensor was kept in assay buffer [150 mM Lithium Sulfate, 50 mM
HEPES, 1 mM DTT and 10 ꢂM DHT]. In all experiments, a known AF2ꢀinteracting peptide was
used as a control to confirm functionality of the bAR LBD.
Androgen Displacement Assay: Androgen displacement was assessed with the Polar
Screen Androgen Receptor Competitor Green Assay Kit as per the instructions of the
manufacturer.
Peptide Displacement Assay: Peptide displacement was assessed as described in our
1
2, 19
previous work.
Determination of Compound Purity: Compound identity and purity were confirmed by
LC_MS/MS. Briefly, an Acquity ultraperformance liquid chromatograph (UPLC) with a 2.1*
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