Systematic structure modifications of multitarget prostate cancer drug candidate galeterone to produce novel androgen receptor down-regulating agents as an approach to treatment of advanced prostate cancer

Article

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Systematic Structure Modiﬁcations of Multitarget Prostate Cancer

Drug Candidate Galeterone To Produce Novel Androgen Receptor

Down-Regulating Agents as an Approach to Treatment of Advanced

Prostate Cancer

Puranik Purushottamachar,^†,‡,×Abhijit M. Godbole,^{†,‡,×,○}Lalji K. Gediya,^†,‡Marlena S. Martin,^†,‡

Tadas S. Vasaitis,^∥,⊥Andrew K. Kwegyir-Aﬀul,^†,‡Senthilmurugan Ramalingam,^†,‡Zeynep Ates-Alagoz,^#,∞

and Vincent C. O. Njar*^,†,‡,§

‡

§

^†Department of Pharmacology, Center for Biomolecular Therapeutics, and Marlene Stewart Greenebaum Cancer Center,

University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, Maryland 21201-1559, United States

^∥Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States

^⊥Department of Pharmaceutical Sciences, University of Maryland Eastern Shore, Princes Anne, Maryland, United States

^#Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara 06100, Turkey

^∞Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, Pennsylvania

19104, United States

S

* Supporting Information

ABSTRACT: As part of our program to explore the inﬂuence

of small structural modiﬁcations of our drug candidate 3β-

(hydroxy)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (ga-

leterone, 5) on the modulation of the androgen receptor (AR),

we have prepared and evaluated a series of novel C-3, C-16,

and C-17 analogues. Using structure activity analysis, we

established that the benzimidazole moiety at C-17 is essential

and optimal and also that hydrophilic and heteroaromatic

groups at C-3 enhance both antiproliferative (AP) and AR

degrading (ARD) activities. The most potent antiproliferative

compounds were 3β-(1H-imidazole-1-carboxylate)-17-(1H-

benzimidazol-1-yl)androsta-5,16-diene (47), 3-((EZ)-hydroximino)-17-(1H-benzimidazol-1-yl)androsta-4,16-diene (36), and

3β-(pyridine-4-carboxylate)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (43), with GI₅₀values of 0.87, 1.91, and 2.57 μM,

respectively. Compared to 5, compound 47 was 4- and 8-fold more potent with respect to AP and ARD activities, respectively.

Importantly, we also discovered that our compounds, including 5, 36, 43, and 47, could degrade both full-length and truncated

ARs in CWR22rv1 human prostate cancer cells. With these activities, they have potential for development as new drugs for the

treatment of all forms of prostate cancer.

galeterone, 5),^1e,2d,6and two potent AR antagonists,

INTRODUCTION

■

MDV3100 (enzalutamide, 6)⁷and ARN-509 (7).^3aThe

Compelling laboratory and clinical evidence strongly indicates

that incurable castration-resistant prostate cancer (CRPC)

chemical structures of these clinical compounds are presented

in Figure 1.

Despite the substantial clinical eﬃcacy with 3a in patients

remains dependent on functional androgen receptor (AR), AR-

mediated processes,¹and the availability of intraprostatic

with post-docetaxel CRPC,⁸resistance to this therapy has

already been reported.⁹Resistance to 6 treatment has also been

reported.¹⁰Reactivation of AR signaling following 3a or 6

treatments might occur by several mechanisms, prominent of

which is a switching of transcription program under the control

of AR signaling.¹¹Indeed, it may not be possible to inhibit the

new AR-regulated transcription program by currently available

intracellular androgens.²Unlike early stage prostate cancer

(ESPC), CRCP is not responsive to classical AR antagonist

(hydroxyﬂutamide (1) or bicalutamide (2), Figure 1) or

androgen deprivation therapy (luteinizing hormone-releasing

hormone agonists/antagonists). Therefore, recent strategies

have focused on the development of more potent androgen

synthesis inhibitors^2dor AR antagonists.³These research eﬀorts

have led to ongoing clinical evaluations/approvals of three

potent CYP17 inhibitors, abiraterone acetate (Zytiga, 3a),⁴

TAK-700 (orteronel, 4),⁵and VN/124-1 (TOK-001 or

Received: January 10, 2013

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Figure 1. Chemical structures of compounds 1−7.

therapies and some of the promising agents in clinical

development. If so, substantial degradation of AR (full length

and truncated forms) expression would be a promising strategy

for future studies.

understanding of the optimal pharmacophore requirements for

AR degradation/down-regulator (ARD) activity and their

capabilities in regulating the activity of the AR (i.e., AR

inactivation). A preliminary account of part of this work has

recently been reported.¹⁶

Although our clinical agent 5 was originally designed as a

CYP17 inhibitor, we have unequivocally established through

several in vitro and in vivo experiments that it also has other

important desirable antiprostate cancer activities, acting as a

potent anti-androgen and an AR degrading agent.^6,12Because

of our desire to invent more eﬃcacious antiprostate cancer

agents, we were eager to exploit compound 5’s scaﬀold as a

strategy to novel potent/eﬃcacious AR degrading agents

(ARDAs) with improved druglike properties. We also took

into serious consideration published data of crystal structures of

steroid ligand dihydrotestosterone (DHT, 8) and of

metribolone (R1881, 9) (Figure 2) bound to AR LBD in our

RESULTS AND DISCUSSION

■

Design Strategy. Modiﬁcations that allow for additional

interactions between a small molecule and receptor appear to

be key determinants for designing new AR down-regulators

with potential clinical use.¹⁷Synthetic modiﬁcations of 5 were

considered because the resulting fundamental chemical and

physical changes may aﬀect molecular shapes, bond angles, and

partition coeﬃcients. Diﬀerent substituents can have diﬀerent

hydrophobic interactions, size, and electrostatic eﬀects that can

inﬂuence interaction of a ligand with its target receptor.¹⁸

These rational considerations provided the impetus for the

systematic modiﬁcations of moieties tethered to C-17, C-16,

and C-3 as described below.

C-17 Modiﬁcations. To explore the structure−activity

relationship (SAR) of the C-17 benzimidazole moiety of 5,

we synthesized analogues with varied ring nitrogen atoms, with

increased aliphatic/aromatic hydrophobicity and with aromatic

substituents to generate compounds 16−22 as outlined in

Scheme 1.

C-16 Modiﬁcations. On the basis of previous studies by Roy

et al.¹⁹of novel C-16 steroids that exhibit strong AR binding

aﬃnity and antiandrogenic activity, we designed and synthe-

sized several C-16 substituted analogues (compounds 25, 28,

and 31) of 5, tethered with bulky aliphatic and aromatic groups

(Scheme 2).

Figure 2. Chemical structures compounds 8−11.

C-3 Modiﬁcations. On the basis of studies of DHT/

testosterone interactions with AR, it is well established that the

interaction with Arg752 occurs via C-3 ketone.²⁰Arginine is a

polar hydrophilic amino acid that contains a positively charged

guanidine group. On the basis of the hypothesis that any

substitution at C-3 that increases interaction with Arg752 may

increase AR down-regulating activity, we designed and

synthesized various C-3 modiﬁed compounds (33−49, Scheme

3).

medicinal chemistry design strategy.¹³In addition we

considered knowledge of related ERα structure complexed

with known ERα down-regulators such as fulvestrant (10) and

GW5638 (11) (Figure 2).^14,15Speciﬁcally, we conducted a

systematic structure modiﬁcation of compound 5 to see if we

could obtain more potent ARDAs. Herein, we report that lead

optimization of 5 gave rise to several novel compounds that

exhibit the abilities to induce AR (full length and truncated)

ablation at low micromolar concentrations and with improved

antiproliferative (AP) activities. This study expands our current

Chemistry. In this study, 26 novel compounds are described

and are based on the structures of our clinical candidate,

compound 5, as outlined in Scheme 1 (for C-17 modiﬁed

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a

Scheme 1. Synthesis of C-17 Benzimidazole Compounds (5, 16−22)

a

Reagents and conditions: (i) POCl₃-DMF, CHCl₃, Ar, reﬂux; (ii) benzimidazole or indole-3-carbaldehyde or 5,6-dimethylbenzimidazole or 5(6)-

cyanobenzimidazole or 5(6)-methoxybenzimidazole or naphtho⁴⁶imidazole or 2-chlorobenzimidazole, K₂CO₃, DMF, Ar, 80 °C; (iii) 10% Pd on

activated charcoal, PhCN, reﬂux; (iv) 10% methanolic KOH, Ar, rt (2−3 h); (v) 6-chloropurine, TBAF, THF, Ar, 50 °C; (vi)

chlorotri(triphenylphosphine)rhodium[I], PhCH₃, Ar, reﬂux.

a

Scheme 2. Synthesis of C-16 Substituted Compounds (25, 28, and 31)

a

Reagents and conditions: (i) substituted amines, molecular sieves, EtOH, Ar, reﬂux (3−7 h); (ii) MeOH, NaBH₄, ice cold (2 h), rt (3 h); (iii)

MeOH, 10% methanolic−KOH, Ar, rt (2−3 h).

series), Scheme 2 (C-16 modiﬁed series), and Scheme 3 (C-3

modiﬁed series). The preparation of new 17-hetereoaryl

substituted compounds (16−22) from the key intermediate

3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene (13) fol-

lowed the sequence 17-heteroaryl-16-formyl intermediate →

16-deformylated intermediate → 3-deacetylated ﬁnal product

(not shown in Scheme 1), similar to the synthetic route to

compound 5 outlined in Scheme 1. The key intermediate in our

synthesis of all the compounds, 13, was prepared following our

established procedure for Vilsmeier−Haack reaction of the

commercially available 3β-acetoxyandrost-5-en-17-one (12)

with phosphoryl chloride (POCl₃) and dimethylformamide

(DMF) as previously reported.^12a,21For the synthesis of 3β-

acetoxy-16-formyl-17-1H-heteroaryls (14, 17a, 18a, 19a, 20a,

and 22a), the corresponding heteroaryls were each treated with

13 in the presence of K₂CO₃in DMF at approximately 80 °C

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a

Scheme 3. Synthesis of C-3 Modiﬁed Compound (33−49)

a

Reagents and conditions: (i) Al(i-PrO)₃, 1-methyl-4-piperidone, toluene, reﬂux; (ii) Dess−Martin periodinane, MDC, 0 °C, rt (5 h); (iii) mesyl

chloride or tosyl chloride, pyridine, ice cold (5 h), rt (5 h); (iv) substituted hydroxylamine·HCl, sodium acetate, MeOH, EtOH, Ar, reﬂux (2−3 h);

(v) MeLi, THF, Ar, −60 °C (1 h), rt (2 h); (vi) pyridinecarboxylic acid, 2-methyl-6-nitobenzoic anhydride, DMAP, TEA, THF, rt (1 h); (vi*) acid

anhydride, DMAP, pyridine, reﬂux; (vii) 1,1′-carbonylbisimidazole or 1,1′-carbonylbis(2-methylimidazole) or 1,1′-carbonyl-di-(1,2,4-triazole),

CH₃CN, Ar, rt/reﬂux.

to give the desired intermediates (structures of intermediates

not shown except 14) in near-quantitative yields. However,

because of weak basicity of indole, we used indole-3-

carbaldehyde instead for the synthesis of 17-indole-3-

carbaldehyde (16a) intermediate following the same procedure

with excellent yield. Attempts to condense 6-chloropurine with

13 in the presence of K₂CO₃in DMF resulted in inseparable

N⁹/N⁷isomers (∼6/4 ratio as indicated by TLC) in very low

yield. Therefore, we adopted a reported N⁹-purine alkylation

procedure,²²in which 13 was reacted with 6-chloropurine in

the presence of tetrabutylammonium ﬂuoride (TBAF) in THF

at 50 °C to give the desired intermediate (21a) in excellent

yield. TLC analysis indicated that N⁷-purine alkylation was

almost negligible and the N⁹-purine was easily puriﬁed

following recrystallization in ethanol. The positional isomers

of the 16-formyl derivatives (6-methoxy-BzIm 19a1 and 5-

methoxy-BzIm 19a2) were separated at this stage, and their

structures were conﬁrmed on the basis of reported aromatic

proton resonances for related 5- and 6-methoxybenzyl

compounds. Various attempts to separate positional isomers

of 5(6)-nitrilebenzimidazole intermediates of compound 18 at

all stages were unsuccessful. The 5(6)-nitrilebenzimidazole and

2,3-diaminonaphthalene required for synthesis of 18a and 20a

were synthesized by following a reported procedure starting

from 3,4-diaminobenzonitrile and benzo[f ]benzimidazole,

respectively, by reﬂuxing with formic acid.²³The 16-formyl

intermediates (14, 17a−21a; only structure of 14 shown) were

each smoothly deformylated with 10% palladium on activated

charcoal (Pd/C) in reﬂuxing benzonitrile to give the

corresponding deformylated compounds 15, 17b, 18b, 19b,

20b, and 21b, respectively (structures not shown except 15) in

high yields.^12aSimilarly, the two formyl groups of 17-indole-3-

carbaldehyde intermediate (16a) were deformylated with 10%

Pd/C as described above with good yield to give 16b.

Deformylation of 22a was achieved by reﬂuxing with readily

available chlorotris(triphenylphosphine)rhodium(I) in toluene

to give 22b in low yield.^12aUnexpectedly, the 5-methoxy-16-

formyl derivative 19a2 did not undergo deformylation using

both methods. Hydrolysis of 15 and 16b−22b with 10%

methanolic KOH gave target compounds 5, 16, 17, 18, 19, 20,

21, and 22, respectively, in high yields.

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The C-16 substituted compounds were synthesized starting

from 14 as illustrated in Scheme 2. The intermediate imines 23,

26, and 29 were synthesized by reﬂuxing isopentylamine,

aniline, and 3,4-dimethoxyaniline, respectively, with 14 in

ethanol in the presence of molecular sieves. Subsequent

reduction of these imines with sodium borohydride (NaBH₄)

in ice-cold methanol²⁴gave 3-acetoxy-16-alkylamine inter-

mediates 24, 27, and 30, respectively. Following hydrolysis of

the 3β-acetoxy groups in compounds 24, 27, and 30, we

obtained the desired 16-substituted compounds 25, 28, and 31,

respectively, in excellent yields.

approximately 100-fold after 10 nM DHT treatment for 24 h.

The ability of the novel compounds (10 μM) to aﬀect DHT

mediated AR transcription was assessed. Figure 3 shows the

The C-3 modiﬁed compounds were synthesized as depicted

in Scheme 3. Δ⁴-3-Oxo compound (32) was synthesized as we

previously described via modiﬁed Oppenauer oxidation of 5 by

using N-methylpiperidone and aluminum isopropoxide.^12a

Oxidation of 5 with Dess−Martin periodinane in dichloro-

methane (DCM)²⁵aﬀorded the Δ⁵-3-oxo compound 33 in

70% yield. The mesyl (34) and tosyl (35) derivatives of 5 were

readily synthesized by reacting with methanesulfonyl chloride

and toluenesulfonyl chloride, respectively. The C-3 oxime

derivatives (hydroxime, 36; phenyloxime, 37; methyloxime, 38;

benzyloxime, 39) were obtained by reﬂuxing ketone (32) with

the respective substituted hydroxylamine hydrochloride, using

ethanol/methanol solvent mixture in the presence of sodium

acetate.²⁶Of all the oximes, only biologically active oxime 36

was further puriﬁed to separate E and Z geometrical isomers by

combined puriﬁcation methods (column chromatography,

preparative TLC, and recrystallization). Addition of MeLi to

the C-3-keto group of 32 aﬀorded two distereomeric (3α and

3β) alcohols (40) that we did not separate because of modest

biological activity.

The ester derivatives (41−46) of 5 were synthesized from 5

by two diﬀerent methods as described below. The pyridine-

carboxylates (41, 42, and 43) and carboxylate of 1,3-

phenyldiacetic acid (44) of 5 were prepared using the mixed

anhydride method via condensations with the respective

anhydrides (pyridinecarboxylic acid/1,3-phenyldiacetic acid

and 2-methyl-6-nitrobenzoic) in the presence of 4-dimethyla-

minopyridine (DMAP) and triethylamine (TEA)²⁷with varying

yields (39−90%). The ester 45 (72% yield) and 46 (28% yield)

were synthesized by reﬂuxing 1,2,3,6-tetrahydrophthalic and

diglycolic anhydrides, respectively, with 5 in the presence of

DMAP in pyridine.²⁸Finally the carbamates (imidazole, 47; 2-

methylimidazole, 48; 1,2,4-triazole, 49) were synthesized in

modest to high yields (67−80%) by reacting 5 with CDI, 1,1-

carbonylbis(2-methylimidazole) and carbonylditriazole (CDT),

respectively, in acetonitrile and DMC solvent mixture.²⁹Except

for the 3-mesyl (34), 5, and 32, all the compounds described in

this study are novel and were rigorously characterized by

physical and spectroscopic (IR, ¹H and ¹³C NMR, and HRMS)

analyses. Most of our novel compounds were then subjected to

in vitro biological activity studies as described in detail in the

following sections.

Figure 3. Eﬀects of compounds at 10 μM on dihydrotestosterone

(DHT) stimulated transcription of AR. LNCaP cells were transfected

with the ARR2 reporter construct + the Renilla luciferase reporting

vector pRL-null and treated with novel compounds for 24 h in the

presence of 10 nM dihydrotestosterone (DHT). Control was baseline

activity without androgen stimulation. Androgen-stimulated luciferase

activity (luminescence) was measured in a Victor 1420 plate reader.

The results are presented as the fold induction (i.e., the relative

luciferase activity of the treated cells divided by that of the control)

normalized to that of the Renilla.

eﬀects of our most potent compounds. These compounds were

able to substantially inhibit DHT mediated transcription, with

inhibition ranging from ∼65% to 100%, and the order of

potency was 32, 5, 47 > 36 > 38 > 28 > 25 > 39 > 34.

Androgen Receptor Binding Assays. In addition to AR

down-regulation, we have previously shown that compound 5

reduces androgen action through inhibition of androgen

binding and subsequently reduces AR mediated transcriptional

activity. We used whole cell competitive binding assays with the

synthetic ligand methyltrienolone (R1881, 9) to assess the AR

binding aﬃnities of our novel compounds in comparison to 5,

the FDA approved anti-androgens 2 and 6, and CYP17

inhibitor 3b as shown in Figure 4A. The compounds with the

greatest ability to displace [³H]9 were 5 and 6, with EC₅₀values

of 670 and 915 nM, respectively. Compound 2 was slightly

weaker with an EC₅₀of 1.4 μM. We did not calculate the EC₅₀

value of 3b because of the shallow steepness of the AR binding

curve, a phenomenon that indicates interaction of 3b with more

than one receptor population.³⁰A recent study also noted

unusual (shallow steepness of the AR binding curve) AR

binding characteristics with 3b.³¹Interestingly, AR-binding

assays using the MDA-MB-453 cell showed that 6 was not as

potent as previously reported for assays using LNCaP cells

transfected with wild type AR^3aand was not signiﬁcantly

diﬀerent from the binding aﬃnity of 2. Speciﬁcally, the binding

aﬃnity data were as follows: 6, EC₅₀= 49 nM; 2, EC₅₀= 31

nM.^3aOur new compounds were not as potent as 5 at

inhibiting androgen binding at the concentrations tested

(Figure 4B). For example, compound 36 showed the strongest

inhibition of [³H]9 binding of all the new compounds tested

(∼40%) at 10 μM. At 30 μM, 36 inhibited [³H]9 binding to by

∼80% while 43 inhibited by ∼53%. Unexpectedly, our most

eﬀective AR antagonist, 47, did not strongly compete for the

AR binding site, exhibiting only 20% displacement at a 30 μM.

Biological Studies: Eﬀects of Compounds on Tran-

scriptional Activation of Androgen Receptor in LNCaP

Cells. After synthesizing the compounds, we used a luciferase

reporter assay to determine whether the novel compounds also

aﬀect AR transcriptional activation (screening assay). Speciﬁ-

cally, we performed a luciferase experiment utilizing LNCaP

cells dual-transfected with the probasin luciferase reporter

construct ARR2-luc and the Renilla luciferase reporting vector

pRL-null as we previously described and reported in the

methods section.^6,12a,dLuciferase expression was increased by

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compound 6 did not aﬀect the expression levels of either full-

length or splice variant forms of AR. It is important to state

here that a number of natural products³⁴and related

analogues³⁵have been shown to degrade both full-length and

truncated AR in several human prostate cancer cell lines.

However, except for the curcumin analogue ASC-J9 that

possesses excellent druglike properties,³⁵most of these

compounds are poor drug candidates because of modest

potencies and/or toxic nature. Irrespective of how our

compounds and others cause degradation of both forms of

AR, such unique AR depleting agents if adequately developed

may be more eﬀective against CRPC than agents that

obligatorily bind to speciﬁc region(s) of AR to elicit

inactivation of AR.³⁶

To determine whether AR degradation or AR transcriptional

deactivation (AR inactivation) was contributing to the

antiproliferative activity, we treated the LNCaP cells with 15

μM selected active compounds (5, 36, 32, 47, and 48) for 24 h

and performed cell viability, AR transcriptional (luciferase)

assay, and AR Western blot analysis. As shown in Figure 6, the

degradation of AR and inhibition of AR mediated transcription

occur before cell growth inhibition, which suggests that

compound-induced AR inactivation contributes to their

antiproliferative activities. These compounds also induced

signiﬁcant PARP cleavage in LNCaP and CWR22rv1 cells,

which suggests their abilities to induce apoptosis (data not

shown).

CYP17 (17α-Hydroxylase Activity) Inhibition Studies.

A few compounds were evaluated for their ability to inhibit

CYP17 enzyme. The assay was kindly performed by Dr. Emily

Scott and colleagues according to their recently reported

procedure in which a truncated version of human CYP17A1

(CYP171dH) was expressed in E. coli and then puriﬁed to

homogeneity.³⁷IC₅₀values of the compounds were determined

from dose−response curves and are listed in Table 1. The IC₅₀

values of abiraterone alcohol (3b, a CYP17 inhibitor recently

approved for prostate cancer therapy), galaterone, and 3β-

hydroxy-17-(1H-imidazole-1-yl)androsta-5,16-diene (VN/85-1,

structure not shown, believed to be the most potent CYP17

inhibitor^12a,21) were also determined in the same assay system

for comparison (used as positive controls). As expected, these

new compounds (16, 36, 43, 47, and 48) with IC₅₀values in

the high micromolar range (93.7−258 μM) were weak

inhibitors of CYP17, reinforcing the previously established

structural requirements for potent steroidal CYP17 inhibitors,

including no tolerance of bulky moieties at C-3 and

appropriately positioned C-17 heterocyclic heteroatom.^21,38

As expected, the well-established CYP17 inhibitors exhibited

exquisite inhibition of the enzyme with IC₅₀values in the

nanomolar range (Table 1).^21,38b

Figure 4. (A) Competitive inhibition of [³H]R1881 binding of

compounds 2, 3b, 5, 6, and 36 to AR in LNCaP cells. Error bars are

SD; n = 3. (B) Competitive inhibition of [³H]R1881 binding of

compounds 5, 16, 36, 43, and 47 to AR in LNCaP cells. Error bars are

SD; n = 3.

It is relevant to state here that other investigators have recently

reported the discovery of small-molecule androgen receptor

down-regulators and anti-androgens that bind weakly to the

AR.³²

Eﬀects of Compounds on AR Degradation, Trans-

activation, and Antiproliferative Activity. To explore the

eﬀects of our compounds on AR degradation, LNCaP cells

were treated with each of the compounds (5, 6, 16−20, 25, 28,

32, 34, 36, 38, 39, 42, 43, 47−49) of interest for 24 h followed

by Western blot analysis. As shown in Figure 5A−C, most of

the new compounds signiﬁcantly caused AR degradation in

LNCaP cells, with compound 47 being the most potent and

greater than 8-fold more active than compound 5 at 15 μM. In

contrast, we note that a few compounds (16, 17, 20, and 49)

caused signiﬁcant up-regulation of AR, a phenomenon that will

be investigated in future studies. The ability of compounds 5

and 47 to suppress protein AR expression was further

demonstrated by immunocytochemical analysis (Figure 5D).

As shown, exposure of LNCaP cells to 5 μM 5 and 47 for 48 h

led to signiﬁcant decrease in AR levels in the nucleus in a

fashion that mimics the Western blot analysis data (vide supra).

These data are similar to those reported for analogues of

ciglitazone, a novel class of AR-ablative agents.^18c

Because of the reported implication of AR splice variants

lacking the ligand-binding domain (truncated AR) in driving

the progression of CRPC,³³we next determined the eﬀects of

our compounds on the down-regulation of AR-3 (also called

AR-V7).^33c,dAs shown in Figure 5E, we observed that

compound 5 and some of our new compounds, 31, 32, 36,

and 47, caused signiﬁcant down-regulation of both full-length

and truncated AR in the CWR22rv1 prostate cancer cell line.

Interestingly, we found that AR-3 was more susceptible to our

compounds than the full-length AR in this cell line. In contrast,

Antiproliferative (Anticancer) and Androgen Recep-

tor Down-Regulating Activities: Structure−Activity

Relationships (SARs). Because of our hypothesis that the

extent of AR degradation induced by compound 5 and possibly

the new analogues would correlate with their ability to inhibit

proliferation of prostate cancer cells (LNCaP), we assessed

these two activities using Western blot analyses and MTT

assays.

C-17 Modiﬁcation. Initially we synthesized and tested indole

16 to assess the eﬀect of decreased polarity at C-17 position

due to absence of N-3 of BzIm ring. Unexpectedly, the

compound caused up-regulation of AR (Figure 5A) and

completely lost anticancer activity (GI₅₀> 100 μM, Table 2)

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Figure 5. (A−E) Diﬀerential eﬀect of compounds on suppressing AR expression in LNCaP and CWR22Rv1 prostate cancer cells. (A−C) Western

blot analysis of AR expression in LNCaP cells treated with various compounds. Cells were exposed to individual compounds (15 μM) for 24 h, and

the protein lysates were subjected to Western blot analysis. (D) Immunocytochemical analysis of the eﬀect of 5 μM each of compounds 5 and 47 on

suppressing AR expression in LNCaP cells after 48 h of exposure. Cells were stained with an-AR x-terminal antibody (green). The nuclear

counterstaining was achieved using 4′,6-diamino-2-phenylindole (DAPI). (E) Eﬀects of compounds on the expression of fAR and tAR-3 in

CWR22Rv1 cells. Cells were exposed to individual compounds (15 μM) for 24 h, and the protein lysates were subjected to Western blot analysis.

Western blots were done in duplicate or triplicate. The numbers below the blots represent respective densitometry intensities.

in comparison to lead compound 5 (GI₅₀= 3.35 μM).

Increasing the number of nitrogen C-17 heterocycle by

substituting with 6-chloropurine (21), caused a 4-fold reduction

in antiproliferative activity (GI₅₀= 13.48 μM). Introducing a

cyano group (18) displayed potent antiproliferative activity

(GI₅₀= 2.81 μM) but with diminished AR down-regulation

(ARD) activity. Introduction of aliphatic hydrophobicity on the

BzIm ring by substituting methyl groups at the 5, 6 positions

ring displayed no modulation of ARDA or anticancer activity

(GI₅₀= 4.26 μM). Increasing aromatic hydrophobicity by

replacing BzIm with naphtho[2,3-d]imidazole ring (20) caused

signiﬁcant loss of ARDA and anticancer activity (GI₅₀= 19.10

μM). Substituting 2-chloro-BzIm (22) caused a 3-fold loss in

antiproliferative activity. None of the C-17 modiﬁed molecules

were superior to our lead compound 5, and this clearly

indicates that the BzIm ring at the C-17 position of lead 5 is

essential and optimal for ARDA and antiproliferative activity.

C-16 Modiﬁcation. Our strategy to increase bulk at the C-16

position by tethering aliphatic hydrophobic groups (isopentyl,

(17) resulted in substantial loss of antiproliferative (GI₅₀

=

42.72 μM) and ARD activities, whereas substituting a

monomethoxy group (19) at the sixth position of the BzIm

G

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Table 3. GI₅₀of C-16 Modiﬁed Compounds

a

compd

GI₅₀(μM)

25

28

31

18.31

22.13

>100

a

The GI₅₀were determined from dose−response curves (by nonlinear

regression analysis using GraphPad Prism) compiled from at least

three independent experiments using LNCaP cells, SEM < 10%, and

represents the compound concentration required to inhibit cell growth

by 50%.

Figure 6. Eﬀects of compounds 5, 32, 36, 43, 47, and 48 on (i) cell

viability (blue), (i) DHT-induced AR transactivation (green), and (iii)

AR protein expression following treatment with 15 μM each

compound for 24 h using LNCaP cells. Error bars are SD; n = 3.

Table 4. GI₅₀of C-3 Modiﬁed Compounds

a

compd

GI₅₀(μM)

compd

GI₅₀(μM)

32

2.64

40

41

42

43

44

45

46

47

48

49

13.34

Table 1. IC₅₀of Select Compounds for Inhibition of CYP17

(17α-Hydroxylase Activity)

b

33

15.96

42.13

47.18

1.91

NT

b

34

NT

a

compd

16

IC₅₀(μM)

35

2.57

7.78

8.22

9.13

0.87

5.34

6.67

36

130

258

132

122

93.7

36E

36Z

37

2.03

36

43

47

48

1.95

b

NT

38

3.38

39

5. 57

For comparison

a

3b

0.206

0.752

0.125

The GI₅₀values were determined from dose−response curves (by

nonlinear regression analysis using GraphPad Prism) compiled from at

least three independent experiments, SEM < 10%, and represents the

compound concentration required to inhibit cell growth by 50%. Not

tested because of insolubility in ethanol.

5

VN/85-1

b

a

IC₅₀value is the concentration of inhibitor that inhibits the CYP17

enzyme activity by 50%, each in duplicate. IC₅₀values were each

determined from dose−response curves.

42.13 and 47.18 μM, respectively. On the contrary,

introduction of oxime moieties at C-3 yielded compounds

(E/Z oxime mixtures) with similar or better activities compared

to compounds 5 and 32. Thus, the simple oxime (36) and the

related methyl (38) and benzyl (39) analogues exhibited GI₅₀

values of 1.91, 3.38, and 5.57 μM, respectively. We could not

assess the biological activities of the phenyloxime (37) because

of its limited solubility in ethanol or DMSO. Considering the

promising and superior activity of an E/Z mixture of oxime 36

and the possibility that the pure E and Z had diﬀerent

antiproliferative activities, we were surprised that 36E and 36Z

isomers exhibited similar potencies, with GI₅₀values of 2.03

and 1.95 μM, respectively.

Table 2. GI₅₀of C-17 Modiﬁed Compounds

a

compd

GI₅₀(μM)

3b

5

1.97

3.35

6

5.12

16

17

18

19

20

21

22

>100

47.72

2.81

4.26

37.10

13.48

10.13

On the basis of known ester based anticancer drugs, such as

docetaxel and cabazitaxel,³⁹and esters in clinical development

such as bevirimat and related analogues,²⁸we ﬁrst synthesized

three pyridinecarboxylate derivatives of compound 5, including

41−43. Of these compounds, the isonicotinoyl derivative 43

exhibited similar antiproliferative activity (GI₅₀= 2.57 μM) as

5. Here again, we could not assess the biological activities of

compounds 41 and 42 because of their limited solubilities in

ethanol or DMSO. The related analogues tethered to the

lipophilic ester side chain with a carboxylic acid terminus (44−

46) exhibited potencies ∼2.5-fold worse than compound 5.

Finally, we considered evaluation of C-3 carbamates because of

(1) the precedence of drugs with carbamate moieties such as

the widely used anthelmintics albendazole, fenbendazole, and

mebendazole;⁴⁰(2) the added feature of lowering the

lipohilicity of compound 5, which should also increase

solubilities and perhaps physiological relevance.⁴¹Of the

three heteroaryl carbamates tested, the imidazolyl carbamate

47 with a GI₅₀value of 0.87 μM was shown to be the most

active, being ∼4-fold superior to compound 5. Introduction of

a

The GI₅₀values were determined from dose−response curves (by

nonlinear regression analysis using GraphPad Prism) compiled from at

least three independent experiments, SEM < 10%, and represents the

compound concentration required to inhibit cell growth by 50%.

25) and aromatic groups (benzyl, 28; dimethoxybenzyl, 31)

resulted in signiﬁcant loss of ARD and anticancer activities

(GI_50sof 18.31, 22.13, and >100 μM, respectively; Table 3).

C-3 Modiﬁcation. In an attempt to better understand the

role played by OH and O in the ARD/antiproliferative activities

of compounds 5 and 32 and to possibly achieve enhanced

interaction with Arg in the AR ligand biding domain, we

designed, synthesized, and tested a number of C-3 modiﬁed

analogues. First, oxidation of 5 or reductive alkylation of 32 to

give 3-oxo-Δ⁵compound 33 or 3-hydroxy-3-methyl compound

40, respectively, led to signiﬁcant loss (∼5-fold) of anti-

proliferative activity (Table 4). Conversion of compound 5 to

the mesyl (34) and tosyl (35) derivatives also gave compounds

with mediocre antiproliferative activities, with GI₅₀values of

H

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Figure 7. In vitro SAR of 3,16/17-substituted steroid derivatives as androgen receptor degrading agents..

2¹-methyl as in carbamate 48 caused a 6-fold decrease in

activity relative to 47, similar to ∼8-fold decrease in activity.

spectrometer. High-resolution mass spectra were obtained on a Bruker

12 T APEX-Qe FTICR-MS instrument by positive ion ESI mode by

Susan A. Hatcher, Facility Director, College of Sciences Major

Instrumentation Cluster, Old Dominion University, Norfolk, VA.

Epiandrosterone acetate and all other chemicals and reagents were

purchased from Sigma-Aldrich. Dihydrotestosterone (DHT) used in

the biological experiments was synthesized following our recently

reported procedure.⁴³Tritiated [³H]R1881 was purchased from

Perkin-Elmer LAS, while MDV3100 was purchased from Sequoiq

Resrach Products Ltd., Pangbourne, U.K. Compounds 3a and 3b were

synthesized in our lab. All compounds were stored in the cold (0−8

°C). Silica gel plates (Merck F254) were used for thin-layer

chromatography, while ﬂash column chromatography (FCC) was

performed on silica gel (230−400 mesh, 60 Å). Preparative TLC was

performed using silica gel GF (Analtec 500 μm) plates. Petroleum

ether refers to light petroleum, bp 40−60 °C. The purity of all ﬁnal

compounds was determined to be at least 95% pure by a combination

of HPLC, NMR, and HRMS.

CONCLUDING REMARKS

■

Our study has shown that potent antiproliferative (AP) and AR

down-regulating (ARD) activities can be retained in compound

5 by modiﬁcation of the 3β-OH to appropriate carbamate (47).

We also establish that the C-17 benzimidazole group is essential

and optimal for both AP and ARD activities and substituents

tethered to C-16 are not tolerated. A summary of the in vitro

structure−activity relationship (SAR) of these steroidal

compounds as androgen receptor degrading agents (ARDAs)

is presented in Figure 7. Importantly, we show that binding

aﬃnity to the LBD of AR is not essential for potent AP/ARD

activities and that some of our compounds also exhibit exquisite

depletion of both full-length and splice variant ARs. The

signiﬁcance of these novel ﬁndings has important implications

because our novel compounds, including galeterone currently

in phase 2 clinical trials in prostate cancer patients, can prevent

androgen receptor activation by any known means. Although

we have yet to conduct in vivo anti-prostate tumor eﬃcacy

studies with our carbamate compound 47, on the basis of the

strong promising data presented in this study and also that the

carbamate moiety plays an important role in medicinal

chemistry, being found in many drugs and prodrugs,^28,39,42

we strongly believe that 47 is a strong candidate for further

development as a potential drug for the treatment of all forms

of prostate cancer in humans. Consequently, in vivo antitumor

eﬃcacy evaluations of compound 47 and other promising

compounds in castration resistant models of prostate cancer are

in progress.

3β-Acetoxy-17-chloro-16-formylandrosta-5,16-diene (13).

This compound, prepared from 3β-acetoxyandrost-5-en-17-one

(epinadrosterone acetate, 12) as previously described, provided

spectral and analytical data as reported.²¹

General Method A: Synthesis of 3β-Acetoxy-17-(1H-hetero-

aryl-1-yl)-16-formylandrosta-5,16-dienes (14, 16a−18a, 19a1,

19a2, 20a, and 22a).^12aA 25 mL round-bottom ﬂask equipped with

a magnetic stir bar and condenser was charged with 3β-acetoxy-17-

chloro-16-formylandrosta-5,16-diene (13, 0.38 g, 1 mmol), the

corresponding heteroaryl (3 mmol), and K₂CO₃(0.41 g, 3 mmol)

in dry DMF (∼7.5 mL), and the mixture was stirred at 80 °C under Ar

and monitored by TLC. After cooling to room temperature, the

reaction mixture was poured onto ice-cold water (50 mL) and the

resulting precipitate was ﬁltered, washed with water, and dried to give

crude product. Puriﬁcation by FCC [petroleum ether/EtOAc/TEA

(6:4:0.3)] gave the desired pure compounds. The above listed

intermediate compounds were synthesized (using reactants, reagent,

and solvent ratio), isolated, and puriﬁed by using this method unless

otherwise stated.

3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-formylandrosta-

5,16-diene (14). Compound 14 was prepared by following general

method A, reacting 13 (2.5 g, 6.65 mmol) with benzimidazole (2.35 g,

19.9 mmol) in the presence of K₂CO₃(2.76 g, 19.9 mmol) in dry

DMF at 80 °C for 1.5 h. Followed by FCC puriﬁcation provided pure

14 with identical spectral and analytical data as we previously

reported.^12a

EXPERIMENTAL SECTION

■

Chemistry. Melting points (mp) were determined with a Fischer-

Johns melting point apparatus and are uncorrected. Proton magnetic

resonance spectra (¹H NMR) spectra were recorded in CDCl₃or

DMSO-d₆at 500 or 400 MHz with Me₄Si as an internal standard using

a Varian Inova 500 or Bruker 400 MHz spectrometer. ¹³C NMR

spectra were recorded in CDCl₃using a Bruker 400 or 500 MHz

I

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3β-Acetoxy-17-(3-formyl-1H-indol-1-yl)-16-formylandrosta-

5,16-diene (16a). Compound 16a was prepared by following general

method A, reacting 13 (1 g, 2.66 mmol) with indole-3-carbaldehyde

(0.5 g, 3.44 mmol) in the presence of K₂CO₃(0.5 g, 3.62 mmol) in dry

DMF (15 mL) at 80 °C for 8 h. Puriﬁcation by FCC [petroleum

ether/EtOAc (7:3)] gave 1.1 g (85%) of pure 16a: mp 206−208 °C;

IR (neat) 2935, 2852, 1729, 1665, 1635, 1453, 1374, 1239, 1032, 783

cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.01 (s, 3 H, 18-CH₃), 1.09 (s, 3

H, 19-CH₃), 2.06 (s, 3 H, 3β-OCOCH₃), 4.65 (dt, J = 12.2, 6.5 Hz, 1

H, 3 α-H), 5.46 (br, 1 H, 6-H), 7.29 (s, 1 H, 2′-H), 7.39 (m, 2 H,

aromatic-Hs), 7.80 (d, J = 14.9 Hz, 1 H, aromatic-H), 8.36 (m, 1 H,

aromatic-H), 9.58 (br, 1 H, 16-CHO), and 10.15 (s, 1 H, indole-

CHO).

3β-Acetoxy-17-(5, 6-dimethyl-1H-benzimidazol-1-yl)-16-for-

mylandrosta-5,16-diene (17a). Compound 17a was prepared by

following general method A, reacting 13 (0.5 g, 1.33 mmol) with 5,6-

dimethylbenzimidazole (0.54 g, 4.0 mmol) in the presence of K₂CO₃

(0.55 g, 4.0 mmol) in dry DMF (10 mL) at 80 °C for 5 h. Puriﬁcation

by FCC gave 0.46 g (70.7%) of pure 17a: mp 174−175 °C; IR (neat)

2941, 2852, 1727, 1672, 1622, 1463, 1487, 1365, 1236, 1029, 897, 843,

717, 657 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 1.06 (s, 3 H, 18-CH₃),

1.16 (br s, 3 H, 19-CH₃), 2.03 (s, 3 H, 3β-OCOCH₃), 2.35 (s, 3 H,

aromatic-CH₃) 2.38 (s, 3 H, aromatic-CH₃), 4.64 (m, 1 H, 3 α-H),

5.44 (br, 1 H, 6-H), 7.02 (br s, 1 H, aromatic-Hs), 7.59 (s, 1 H,

aromatic-H), 7.87 (s, 1 H, 2′-H), and 9.60 (s, 1 H, 16-CHO).

3β-Acetoxy-17-(5(6)-nitrile-1H-benzimidazol-1-yl)-16-formy-

landrosta-5,16-diene (18a). Compound 18a was prepared by

following general method A, reacting 13 (0.5 g, 1.33 mmol) with 5(6)-

nitrilebenzimidazole²³(0.38 g, 2.65 mmol) in the presence of K₂CO₃

(0.55 g, 4.0 mmol) in dry DMF (10 mL) at 80 °C for 5 h. Puriﬁcation

by short column [petroleum ether/EtOAc/TEA (6:4:0.1)] gave 0.28 g

(43.5%) of pure 18a: mp 146−147 °C; IR (neat) 2935, 2226, 1726,

1673, 1470, 1238 1032, 906, 728 cm⁻¹; ¹H NMR (400 MHz, CDCl₃)

δ 1.07 (s, 3 H, 18-CH₃), 1.19 (br s, 3 H, 19-CH₃), 2.04 (s, 3 H, 3β-

OCOCH₃), 4.62 (dt, J = 10.1, 5.3 Hz, 1 H, 3 α-H), 5.44 (br, 1 H, 6-

H), 7.61−7.96 (m, 3 H, aromatic-H), 8.21 (s, 1 H, 2′-H), and 9.52 (s,

1 H, 16-CHO).

2951, 2359, 1725, 1670, 1604, 1491, 1452, 1375, 1253, 1032, 897, 852,

818, 700, 657, 618, 576, 565, 550, 529, 511, 476 cm⁻¹; ¹H NMR (500

MHz, CDCl₃) δ 1.05 (s, 6H, 18 and 19-CH₃), 2.04 (s, 3 H, 3α-

OCH₃), 4.62 (m, 1 H, 3β-H), 5.44 (br, s, 6-H), 7.46 (br s, 2 H,

aromatic-H), 7.94 (s, 2 H, aromatic-H), 8.04 (m, 1 H, aromatic-H),

8.15 (s, 1 H, aromatic-H) 8.33 (s, 1 H, 2′-H), and 9.71 (s, 1 H, 16-

CHO).

3β-Acetoxy-17-(6-Chloro-9H-purin-9-yl)-16-formylandrosta-

5,16-diene (21a).²²A mixture of 13 (2.43 g, 6.46 mmol), 6-

chloropurine (0.5 g, 3.23 mmol), and TBAF (1.69 g, 6.46) in dry THF

(40 mL) was stirred at 50 °C under Ar for 48 h. After cooling to room

temperature, the reaction mixture was concentrated and poured onto

ice-cold water (250 mL) and the resulting precipitate was ﬁltered,

washed with water, and dried to give a crude product. Puriﬁcation by

FCC [DCM/methanol (9.7:0.3)] and then recrystallization with hot

ethanol gave 0.82 g (51.3%) of pure 21a: mp 140−142 °C; IR (neat)

2943, 2853, 1729, 1672, 1584, 1556, 1435, 1236, 1032, 939, cm⁻¹; ¹H

NMR (500 MHz, CDCl₃) δ 1.07 (s, 3 H, 18-CH₃), 1.09 (s, 3 H, 19-

CH₃), 2.04 (s, 3 H, 3β-OCOCH₃), 4.61 (m, 1 H, 3 α-H), 5.43 (br, 1

H, 6-H), 8.20 (s, 1 H, 2′-H), 8.79 (s, 1 H, aromatic-H), and 9.53 (s, 1

H, 16-CHO).

3β-Acetoxy-17-(2-chloro-1H-benzimidazol-1-yl)-16-formy-

landrosta-5,16-diene (22a). Compound 22a was prepared by

following general method A, reacting 13 (0.5 g, 1.33 mmol) with 2-

chlorobenzimidazole (0.6 g, 4.0 mmol) in the presence of K₂CO₃

(0.55 g, 4.0 mmol) in dry DMF (10 mL) at 80 °C for 50 h. After

cooling to room temperature, the reaction mixture was poured onto

ice-cold water (250 mL) and the resulting emulsion was extracted with

DCM. The organic layer was dried and evaporated. Puriﬁcation by

FCC [petroleum ether/EtOAc (8:2)] gave 0.27 g (41.1%) of pure

22a: mp 203 °C; IR (neat) 2936, 1731, 1679, 1448, 1244, 1033, 734

cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.06 (s, 3 H, 18-CH₃), 1.16 (s, 3

H, 19-CH₃), 2.04 (s, 3 H, 3β-OCOCH₃), 4.62 (m, 1 H, 3 α-H), 5.43

(br, 1 H, 6-H), 7.17 (d, 1 H, J = 7.9 Hz, aromatic-H), 7.34 (m, 2 H,

aromatic-Hs), 7.74 (d, 1 H, J = 7.4 Hz, aromatic-H), and 9.37 (s, 1 H,

16-CHO).

General Method B: Synthesis of 3β-Acetoxy-17-(1H-hetero-

aryl-1-yl)androsta-5,16-dienes (15, 16b−21b). A solution of 3β-

acetoxy-17-(1H-heteroaryl-1-yl)-16-formylandrosta-5,16-diene (14,

17a−21a) in dry benzonitrile (10 mL) was reﬂuxed in the presence

of 10% Pd/C (50% weight of reactant) under Ar and monitored by

TLC. After the mixture was cooled to room temperature, the catalyst

was removed by ﬁltration through a Celite pad. The ﬁltrate was

evaporated, and the residue was puriﬁed by FCC on silica gel, using

petroleum ether/EtOAc/TEA (7.5:3:0.5) solvent system. The above

listed intermediate compounds were synthesized (using reactants,

reagent, and solvent ratio), isolated, and puriﬁed by using this method

unless otherwise stated.

3β-Acetoxy-17-(6-methoxy-1H-benzimidazol-1-yl)-16-for-

mylandrosta-5,16-diene (19a1) and 3β-Acetoxy-17-(5-me-

thoxy-1H-benzimidazol-1-yl)-16-formylandrosta-5,16-diene

(19a2). Compounds 19a1 and 19a2 were prepared by following

general method A, reacting 13 (0.5 g, 1.33 mmol) with 5(6)-

methoxybenzimidazole (0.59 g, 4.0 mmol) in the presence of K₂CO₃

(0.55 g, 4.0 mmol) in dry DMF (10 mL) at 80 °C for 3 h. Puriﬁcation

by FCC [petroleum ether/EtOAc/TEA (7.5:2:0.5)] gave ﬁrst the less

polar 6-methoxy derivative (19a1) and subsequently the more polar 5-

methoxy derivative (19a2).

19a1. Yield, 0.15 g (24%); mp 242−245 °C; IR (neat) 2935, 1721,

1

1673, 1502, 1440, 1249, 1220, 1032, 805, 759 cm⁻¹; H NMR (400

3β-Acetoxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene

(15). Compound 15 was prepared by reﬂuxing 14 (2.04 g, 4.45 mmol)

with 10% Pd/C (1.0 g) in dry benzonitrile (10 mL) for 5 h followed

by FCC puriﬁcation, which provided pure 15 with identical spectral

and analytical data as we previously reported.^12a

MHz, CDCl₃) δ 1.07 (s, 3 H, 18-CH₃), 1.18 (br s, 3 H, 19-CH₃), 2.03

(s, 3 H, 3β-OCOCH₃), 3.82 (s, 3 H, -OCH₃), 4.62 (dt, J = 11.2, 6.6

Hz, 1 H, 3 α-H), 5.44 (t, 1 H, J = 1.84 Hz, 6-H), 6.70 (m, 1 H,

aromatic-H), 6.95 (m, 1 H, aromatic-H), 7.70 (m, 1 H, aromatic-H),

7.87 (s, 1 H, 2′-H), and 9.61 (s, 1 H, 16-CHO).

3β-Acetoxy-17-(1H-indol-1-yl)androsta-5,16-diene (16b).

Compound 16b was prepared by following general method B,

reﬂuxing 16a (0.17 g, 0.36 mmol) with 10% Pd/C (0.085 g) in dry

benzonitrile (3 mL) for 24 h. Then about 0.030 g of Pd/C and solvent

(1 mL) were added, and the mixture was further reﬂuxed for 12 h.

Puriﬁcation by FCC gave 0.12 g (77.5%) of pure 16b: mp 182−185

°C; IR (neat) 2936, 2854, 1727, 1631, 1455, 1368, 1249, 1030, 721,

cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 0.95 (s, 3 H, 18-CH₃), 1.03

(s, 3 H, 19-CH₃), 1.99 (s, 3 H, 3β-OCOCH₃), 4.47 (m, 1 H, 3α-H),

5.42 (br, 1 H, 6-H), 5.88 (s, 1 H, 16-H), 6.57 (m, 1 H, 3′-H), 7.05 (m,

1 H, 2′-H), 7.15 (m, 1 H, aromatic-H), 7.37 (d, J = 3.2 Hz, 1 H,

aromatic-H), 7.50 (d, J = 8.0 Hz, 1 H, aromatic-H), and 7.57 (d, J =

7.7 Hz, 1 H, aromatic-H).

19a2. Yield, 0.13 g (20%); mp 228−231 °C; IR (neat) 2936, 2852,

1

1722, 1673, 1481, 1341, 1245, 1031, 897, 800, 739 cm⁻¹; H NMR

(400 MHz, CDCl₃) δ 1.06 (s, 3 H, 18-CH₃), 1.16 (br s, 3 H, 19-CH₃),

2.04 (s, 3 H, 3β-OCOCH₃), 3.88 (s, 3 H, -OCH₃), 4.63 (m, 1 H, 3 α-

H), 5.44 (d, J = 5.6 Hz, 1 H, 6-H), 6.98 (m, 1 H, aromatic-H), 7.29

(m, 1 H, aromatic-H), 7.30 (m, 1 H, aromatic-H), 7.92 (s, 1 H, 2′-H),

and 9.61 (s, 1 H, 16-CHO).

About 0.11 g of mixture of 19a1 and 19a2 was also collected

(overall yield is 61%).

3β-Acetoxy-17-(1H-benzo[f ]benzimidazol-1-yl)-16-formy-

landrosta-5,16-diene (20a). Compound 20a was prepared by

following general method A, reacting 13 (0.38 g, 1 mmol) with 1H-

benzo[f ]benzimidazole (0.2 g, 1.2 mmol) in the presence of K₂CO₃

(0.207g, 1.5 mmol) in dry DMF (3 mL) at 80 °C for 2 h. Puriﬁcation

by FCC [petroleum ether/EtOAc/TEA (6:4:0.3)] gave 0.37 g (72%)

of pure compound 20a: mp 158−160 °C; IR (CHCl₃) 3691, 3024,

3β-Acetoxy-17-(5,6-dimethyl-1H-benzimidazol-1-yl)-

androsta-5,16-diene (17b). Compound 17b was prepared by

following general method B, reﬂuxing 17a (0.15 g, 0.308 mmol)

with 10% Pd/C (0.075 g) in dry benzonitrile (2 mL) for 7 h.

J

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Puriﬁcation by FCC gave 0.12 g (84.8%) of pure 17b: mp 159−162

°C; IR (neat) 2926, 2852, 1729, 1626, 1491, 1462, 1369, 1236, 1030,

846, cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ1.03 (s, 3 H, 18-CH₃), 1.09

(s, 3 H, 19-CH₃), 2.06 (s, 3 H, 3β-OCOCH₃), 2.40 (s, 6H, 2 ×

aromatic-CH₃), 4.64 (m, 1 H, 3α-H), 5.45 (br, 1 H, 6-H), 5.96 (s, 1 H,

16-H), 7.26 (s, 1 H, aromatic-H), 7.58 (s, 1 H, aromatic-H), and 7.87

(s, 1 H, 2′-H).

petroleum ether/EtOAc (6:4) to obtain pure target compounds. The

above listed ﬁnal compounds were synthesized (using reactants,

reagent, and solvent ratio), isolated, and puriﬁed by using this method

unless otherwise stated.

3β-Hydroxy-17-(1H-benzimidazol-1-yl)-androsta-5,16-diene

(5).^12aCompound 5 was prepared by following general method C,

treating acetate solution of 15 (1 g 3.02 mmol) in methanol (15 mL)

with 10% methanolic KOH (5 mL) for 1.5 h. Puriﬁcation by FCC over

a short column provided pure 5 with identical spectral and analytical

data as we previously reported. Previously unreported ¹³C NMR data

5: ¹³C NMR (400 MHz, CDCl₃) δ147.1, 143.1, 141.6, 141.3, 134.5,

124.1, 123.4, 122.5, 120.9, 120.1, 111.1, 71.4, 55.8, 50.5, 47.2, 42.2,

37.1, 36.7, 34.8, 31.6, 31.1, 30.3, 30.3, 20.6, 19.3, 16.0.

3β-Acetoxy-17-(5(6)-nitrile-1H-benzimidazol-1-yl)androsta-

5,16-diene (18b). Compound 18b was prepared by following general

method B, reﬂuxing 18a (0.15 g, 0.31 mmol) with 10% Pd/C (0.075

g) in dry benzonitrile (2 mL) for 24 h. Puriﬁcation by FCC gave 0.09 g

(63.5%) of pure 18b: mp 204−206 °C; IR (neat) 2939, 2222, 1731,

1487, 1247, 1030, 822, cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ1.07 (s, 3

H, 18-CH₃), 1.18 (s, 3 H, 19-CH₃), 2.04 (s, 3 H, 3β-OCOCH₃), 4.62

(m, 1 H, 3α-H), 5.44 (m, 1 H, 6-H), 6.03 (m, 1 H, 16-H), 7.54 −8.15

(m, 4 H, aromatic-H).

3β-Hydroxy-17-(1H-indol-1-yl)-androsta-5,16-diene (16).

Compound 16 was prepared by slightly modifying general method

C. The acetate solution of 16b (0.09 g 0.2 mmol) in methanol (1.5

mL) was reﬂuxed with 10% methanolic KOH (1 mL) for 3 h.

Puriﬁcation by FCC over short column gave pure 16 (0.076 g, 98.7%):

mp 142−145 °C; IR (neat) 3305, 2931, 2836, 1625, 1455, 1327, 1225,

3β-Acetoxy-17-(6-methoxy-1H-benzimidazol-1-yl)androsta-

5,16-diene (19b). Compound 19b was prepared by following general

method B, reﬂuxing 19a1 (0.15 g, 0.307 mmol) with 10% Pd/C (0.075

g) in dry benzonitrile (2 mL) for 72 h. Then about 0.030 g of Pd/C

was added, and the mixture was further reﬂuxed for 12 h. Puriﬁcation

by FCC gave 0.05 g (35%) of pure sticky compound 19b: IR (neat)

1

10598, 1042, 740 cm⁻¹; H NMR (500 MHz, CDCl₃) δ 1.00 (s, 3 H,

18-CH₃), 1.06 (s, 3 H, 19-CH₃), 3.54 (m, 1 H, 3α-H), 5.41 (br, 1 H, 6-

H), 5.85 (s, 1 H, 16-H), 6.55 (m, 1 H, 3′-H), 7.11 (m, 1 H, 2′-H), 7.19

(dd, J = 8.4, 5.7 Hz, 2 H, aromatic-Hs), 7.51 (d, 1 H, J = 8.3 Hz,

aromatic-H), and 7.60 (d, 1 H, J = 7.8 Hz, aromatic-H); ¹³C NMR

(500 MHz, CDCl₃) δ 149.6, 141.2, 137.2, 128.4, 126.9, 122.0, 121.7,

120.6, 119.6, 111.3, 102.4, 71.7, 55.9, 50.6, 47.3, 42.0, 37.2, 36.8, 35.1,

31.6, 30.2, 20.8, 19.4, 16.0; HRMS calcd 410.2454 (C₂₇H₃₃ON·Na⁺),

found 410.2460.

3β-Hydroxy-17-(5, 6-dimethyl-1H-benzimidazol-1-yl)-

androsta-5,16-diene (17). Compound 17 was prepared by following

general method C by treating acetate solution of 17b (0.1 g 0.22

mmol) in methanol (2 mL) with 10% methanolic KOH (1 mL) for 3

h. Puriﬁcation by FCC over a short column provided pure 17 (0.05 g,

55%): mp 194−196 °C; IR (neat) 3262, 2925, 2896, 2848, 1628, 1493,

1481, 1371, 1058, 834, cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.02 (s,

3 H, 18-CH₃), 1.06 (s, 3 H, 19-CH₃), 2.38 (s, 6H, 2 × aromatic-CH₃),

3.55 (m, 1 H, 3α-H), 5.41 (m, 1 H, 6-H), 5.95 (t, J = 2.6 Hz, 16-H),

7.25 (s, 1 H, aromatic-H), 7.57 (s, 1 H, aromatic-H), and 7.87 (s, 1 H,

2′-H); ¹³C NMR (500 MHz, CDCl₃) δ 147.3, 141.3, 132.7, 131.6,

123.4, 121.1, 119.9, 111.3, 71.6, 55.9, 50.5, 47.2, 42.3, 37.2, 34.9, 31.6,

30.37, 20.6, 19.3, 16.0; HRMS calcd 439.2719 (C₂₈H₃₆ON₂·Na⁺),

found 439.2726.

1

2940, 1713, 1496, 1363, 1237, 1216, 1030, 816, cm⁻¹; H NMR (400

MHz, CDCl₃) δ1.01 (s, 3 H, 18-CH₃), 1.07 (s, 3 H, 19-CH₃), 2.04 (s,

3 H, 3β-OCOCH₃), 3.88 (s, 3 H, -OCH₃), 4.63 (m, 1 H, 3α-H), 5.44

(s, 1 H, 6-H), 5.96 (br, 1 H, 16-H), 6.92 (m, 2 H, aromatic-Hs), 7.69

(d, 1 H, J = 8.7 Hz, aromatic-H), and 7.85 (s, 1 H, 2′-H).

3β-Acetoxy-17-(1H-benzo[f ]benzimidazol-1-yl)androsta-

5,16-diene (20b). Compound 20b was prepared by following general

method B, reﬂuxing 20a (0.2 g, 4.45 mmol) with 10% Pd/C (0.1 g) in

dry benzonitrile (4 mL) for 5 h. Puriﬁcation by FCC gave 0.14 g

(73.8%) of pure 20b: mp 144−146 °C; IR (CHCl₃) 3687, 2947, 2854,

2358, 2340, 1725, 1633, 1609, 1557, 1489, 1454, 1373, 1291,1253,

1195, 1136, 1031, 985, 910, 839, 735, 665, 590, 544, 533,513, 502, 488

cm⁻¹; 1H NMR (500 MHz, CDCl₃) δ 1.08 (s, 3 H,18-CH₃), 1.10 (s, 3

H, 19-CH₃), 2.01 (s, 3 H, 3β-OCH₃), 4.62 (m,1H, 3α-H), 5.45 (br,s,6-

H), 6.11 (s, 1 H, 16-H), 7.42 (m, 2 H, aromatic-Hs), 7.92 (m, 2 H,

aromatic-H), 8.04 (m, 1 H, aromatic-H), 8.15 (s, 1 H, aromatic-H),

and 8.29 (s, 1 H, 2′-H).

3β-Acetoxy-17-(6-Chloro-9H-purin-9-yl)androsta-5,16-diene

(21b). Compound 21b was prepared by following general method B,

reﬂuxing 21a (0.4 g, 0.81 mmol) with 10% Pd/C (0.4 g, i.e., equal

weight of 21a) in dry benzonitrile (7.5 mL) for 4 h. After the mixture

was cooled to room temperature, the catalyst was removed by ﬁltration

through a Celite pad. The ﬁltrate was evaporated and carried to the

next step without puriﬁcation.

3β-Hydroxy-17-(5(6)-nitrile-1H-benzimidazol-1-yl)androsta-

5,16-diene (18). Compound 18 was prepared according to general

method C by treating acetate solution of 18b (0.075 g 0.165 mmol) in

methanol (1.5 mL) with 10% methanolic KOH (1 mL) for 2 h.

Puriﬁcation by FCC over a short column provided pure 18 (0.055 g,

80.8%): mp 192−193 °C; IR (neat) 3409, 3285, 2928, 2226, 1654,

3β-Acetoxy-17-(2-chloro-1H-benzimidazol-1-yl)androsta-

5,16-diene (22b). A solution of 3β-acetoxy-17-(2-chlorobenzimida-

zol-1-yl)-16-formylandrosta-5,16-diene (22a) (0.15 g, 0.304 mmol) in

dry toluene (3 mL) was reﬂuxed in the presence of chlorotris-

(triphenylphosphine)rhodium(I) (0.29 g, 0.311 mmol) for 60 h. After

the mixture was cooled to room temperature, the catalyst was removed

by ﬁltration through a Celite pad. The ﬁltrate was evaporated, and the

residue was puriﬁed by FCC [petroleum ether/EtOAc (8:2)] to give

0.04 g (28%) of pure 22b: mp 161−165 °C; IR (neat) 2926, 2853,

1

1614, 1469, 1229, 1059, 801, cm⁻¹; H NMR (500 MHz, CDCl₃) δ

1.01 (d, 3 H, 18-CH₃), 1.06 (d, 3 H, 19-CH₃), 3.55 (tdq, J = 9.0, 4.7,

2.6 Hz, 1 H, 3α-H), 5.40 (dp, J = 4.8, 1.7 Hz, 6-H), 6.02 (m, 1 H, 16-

H), 7.52−8.15 (m, 4 H, aromatic-H); ¹³C NMR (500 MHz, CDCl₃) δ

146.7, 144.8, 141.5, 127.0, 126.4, 125.5, 121.5, 119.8, 116.4, 112.4,

106.8, 106.1, 71.7, 56.1, 50.6, 47.5, 42.4, 37.3, 36.9, 34.9, 31.7, 30.6,

20.8, 19.5, 16.2, 15.0; HRMS calcd 414.2539 (C₂₇H₃₁ON_3.H⁺), found

414.2532.

1

1629, 1403, 1462, 1369, 1233, 1035, 847 cm⁻¹; H NMR (500 MHz,

CDCl₃) δ 1.05 (d, 6H, 18- and 19-CH₃), 2.04 (s, 3 H, 3β-OCOCH₃),

4.62 (m, 1 H, 3α-H), 5.44 (m, 1 H, 6-H), 6.06 (s, 1 H, 16-H), 7.33 (m,

1 H, aromatic-H), 7.52 (m, 1 H, aromatic-H), and 7.68 (m, 2 H,

aromatic-H).

3β-Hydroxy-17-(6-methoxy-1H-benzimidazol-1-yl)androsta-

5,16-diene (19). Compound 19 was prepared according to general

method C by treating acetate solution of 19b (0.05 g 0.11 mmol) in

methanol (1 mL) with 10% methanolic KOH (1 mL) for 3 h.

Puriﬁcation by FCC over a short column provided pure 19 (0.03 g,

55%): mp 169−179 °C; IR (neat) 3339, 2933, 1614, 1501, 1450, 1283,

General Method C: Synthesis of 3β-Hydroxy-17-(1H-hetero-

aryl-1-yl)androsta-5,16-dienes (5, 16−22) and 3β-Hydoxy-17-

(1H-benzimidazol-1-yl)-16-((alkyl/arylamino)methyl)androsta-

5,16-dienes (25, 28, and 31). The acetate (15, 16b−22b, 24, 27,

30) (1 g) was dissolved in methanol (15 mL) under an inert Ar

atmosphere, and the resulting solution was treated with 10%

methanolic KOH (5 mL). The mixture was stirred at room

temperature and monitored by TLC. Reaction mixture was

concentrated under vacuum. Ice−water (100 mL) was added, and

the resulting white precipitate was ﬁltered, washed with water, and

dried. FCC was done on a short silica gel column, eluting with

1

1068, 906, 813, 728 cm⁻¹; H NMR (400 MHz, CDCl₃) δ 1.01 (s, 3

H, 18-CH₃), 1.06 (s, 3 H, 19-CH₃), 3.58 (m, 1 H, 3α-H), 3.86 (s, 3 H,

-OCH₃), 5.41 (t, 1 H, J = 2.42 Hz, 6-H), 5.95 (t, 1 H, J = 1.48 Hz,16-

H), 6.92 (m, 2 H, aromatic-H), 7.67 (m, 1 H, aromatic-H), and 7.58

(s, 1 H, 2′-H); ¹³C NMR (500 MHz, CDCl₃) δ 157.32, 147.6, 141.5,

137.9, 135.4, 124.0, 121.2, 120.7, 111.6, 95.2, 71.7, 56.2, 50.7, 47.5,

42.5, 37.4, 35.1, 31.8, 30.6, 20.9, 19.5, 16.2; HRMS calcd 441.2512

(C₂₇H₃₄O₂N₂·Na⁺), found 441.2507.

K

dx.doi.org/10.1021/jm400048v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3β-Hydroxy-17-(1H-benzo[f ]benzimidazol-1-yl)androsta-

5,16-diene (20). Compound 20 was prepared according to general

method C by treating acetate solution of 20b (0.1 g, 0.32 mmol) in

methanol (5 mL) with 10% methanolic KOH (1 mL) for 1.5 h.

Puriﬁcation by crystallization from EtOAc/methanol gave 20 (0.075 g,

74%): mp 150−152 °C; IR (CHCl₃) 2934, 2339, 1609, 1490, 1453,

3 β - A c e t o x y - 1 7 - ( 1 H - b e n z i m i d a z o l - 1 - y l ) - 1 6 -

((isopentylamino)methyl)androsta-5,16-diene (24). Compound

24 was prepared by following general method E, reacting 23 (0.1 g, 0.2

mmol) in methanol (1.5 mL) with NaBH₄(0.0035 g, 0.09 mmol) at

°C for 1.5 h. The crude product 24 (0.09 g, 89%) was carried to the

next step without puriﬁcation.

3β-Hydoxy-17-(1H-benzimidazol-1-yl)-16-((isopentylamino)-

methyl)androsta-5,16-diene (25). Compound 25 was prepared by

following general method C, treating methanolic solution (1 mL) of

crude acetate 24 (0.08 g 0.15 mmol) with 10% methanolic KOH (0.75

mL) for 3 h. Puriﬁcation followed by passage through a short silica bed

[DCM/ethanol (9.5:0.5)] gave 25 (0.065 g, 88%): mp 111−113 °C;

IR (neat) 3281, 2927, 2850, 1487, 1454, 1374, 1224, 1061, 1007, 765

cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 0.81 (d, 6H, alphatic-CH₃), 1.04

(s, 6H, 18, 19-CH₃), 3.55 (m, 1 H, 3α-H), 5.41 (br, 1 H, 6-H), 7.19−

7.43 (m, 3 H, aromatic-Hs), 7.75−7.82 (m, 1 H, aromatic-H), and 8.1

(s, 1 H, 2′-H); ¹³C NMR (500 MHz, CDCl₃) δ 142.8, 140.0, 134.8,

123.4, 122.4, 120.2, 110.8, 71.5, 55.9, 50.7, 48.9, 42.3, 38.9, 36.8, 34.6,,

32.4, 31.6, 30.3, 26.0, 22.6, 20.5, 19.3, 16.0, 15.8; HRMS calcd

510.3454 (C₃₂H₄₅ON₃·Na⁺), found 510.345 09.

3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-((EZ)-

(phenylimino)methyl)androsta-5,16-diene (26). Compound 26

was prepared by following synthetic method D, reﬂuxing 14 (0.15 g,

0.33 mmol), aniline (0.06 g, 0.65 mmol), and molecular sieves (0.04 g)

in ethanol (2 mL) for 3 h. Puriﬁcation by passing through a silica bed

gave 0.15 g (85.9%) of 26: sinters at 85−90 °C, melts at 125 °C; IR

(neat) 2973, 2932, 2822, 1727, 1635, 1589, 1486, 1453, 1239, 1219,

1029, 764 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 1.10 (s, 3 H, 18-CH₃)

1.23 (s, 3 H, 19-CH₃), 2.06 (s, 3 H, 3β-OCOCH₃), 4.65 (m, 1 H, 3α-

H), 5.49 (br, 1 H, 6-H), 6.96 (m, 2 H, aromatic-Hs) 7.17 (m, 1 H,

aromatic-H) 7.26 (s, 1 H, imine-CH), 7.35 (m, 4 H, aromatic-Hs),

7.87 (m, 1 H, aromatic-H), 7.94 (m, 1 H, aromatic-H), and 7.99 (s, 1

H, 2′-H).

3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-((phenylamino)-

methyl)androsta-5,16-diene (27). Compound 27 was prepared by

following general synthetic method E, reacting 26 (0.1 g, 0.19 mmol)

in methanol (1.5 mL) with NaBH₄(0.0035 g, 0.09 mmol) at °C for

1.5 h. The crude 27 was carried to the next step without puriﬁcation.

3β-Hydoxy-17-(1H-benzimidazol-1-yl)-16-((phenylamino)-

methyl)androsta-5,16-diene (28). Compound 28 was prepared by

following general method C, treating methanolic solution (1 mL) of

crude acetate 27 with 10% methanolic KOH (0.75 mL) for 3 h.

Puriﬁcation followed by passage through a short silica bed [DCM/

ethanol (9.5:0.5)] gave 28 (0.08 g, 86%): mp 130−132 °C; IR (neat)

3329, 2928, 2852, 1602, 1418, 1375, 1217, 1058, 1007, 833, cm⁻¹; ¹H

NMR (500 MHz, CDCl₃) δ 1.03 (s, 3 H, 18-CH₃), 1.04 (s, 3 H, 19-

CH₃), 3.54 (m, 1 H, 3α-H), 3.65 (br s, 2 H, -CH₂), 5.38 (t, 1 H, J =

2.62 Hz, 6-H), 6.40 (t, 2 H, J = 8.8 Hz, aromatic-Hs), 6.69 (d, 1 H, J =

7.3 Hz, aromatic-H), 7.08 (m, 2 H, aromatic-Hs), 7.20−7.33 (m, 3 H,

aromatic-Hs), 7.74−7.84 (m, 1 H, aromatic-H), and 7.79 (s, 1 H, 2′-

H); ¹³C NMR (500 MHz, CDCl₃) δ 147.6, 141.3, 138.7, 123.7, 122.5,

129.9, 120.4, 118.0, 113.0, 110.8, 71.6, 54.7, 50.6, 48.0, 42.2, 36.8, 34.4,

32.4, 31.1, 30.3, 20.5, 19.3, 15.8. HRMS calcd 516.2985

(C₃₃H₃₉ON₃·Na⁺), found 516.2981

1

1291, 1040, 837, 808, 705, 663, 608, 578, 550, 517 cm⁻¹; H NMR

(500 MHz, CDCl₃) δ 1.09 (s, 6H, 18 and 19-CH₃), 3.57 (m, 1 H, 3α-

H), 5.44 (br, s, 6-H), 6.13 (s,1 H, 16-H), 7.44 (m, 2 H, aromatic-Hs),

7.94 (m, 2 H, aromatic-H), 8.03 (m, 1 H, aromatic-H), 8.18 (s, 1 H,

aromatic-H), and 8.31 (s, 1 H, 2′-H). HRMS calcd 461.2563

(C₃₀H₃₄N₂O·Na+), found 461.2570.

3β-Hydroxy-17-(6-chloro-9H-purin-9-yl)androsta-5,16-diene

(21). Compound 21 was prepared according to general method C by

treating acetate solution of 21b (0.04 g 0.085 mmol) in methanol (1

mL) with 10% methanolic KOH (1 mL) for 3 h. Puriﬁcation by FCC

over a short column [DCM/methanol/TEA (9.7:0.3:0.05)] gave pure

21 (0.03 g, 82.6%): mp 272−274 °C; IR (neat) 3385, 2928, 2604,

2498, 1664, 1516, 1433, 1346, 1040, 805, cm⁻¹; ¹H NMR (500 MHz,

CDCl₃) δ 1.12 (s, 3 H, 18-CH₃), 1.23 (s, 3 H, 19-CH₃), 3.50 (m, 1 H,

3α-H), 5.41 (br, 1 H, 6-H), 5.59 (s, 1 H, 16-H), 8.11 (s, 1 H, 2′-H),

8.40 (s, 1 H, aromatic-H); ¹³C NMR (500 MHz, CDCl₃) δ 164.1,

153.4, 141.6, 139.4, 121.9, 120.8, 71.2, 56.3, 53.1, 50.1, 47.0, 46.0, 36.9,

31.2, 19.5, 15.0, 11.7, 8.9, 8.8; HRMS calcd 871.3952

(C₂₄H₂₉ClON₄)₂·Na⁺, found 871.3972

3β-Hydroxy-17-(2-chloro-1H-benzimidazol-1-yl)androsta-

5,16-diene (22). Compound 22 was prepared according to general

method C by treating acetate solution of 22b (0.03 g 0.064 mmol) in

methanol (0.75 mL) with 10% methanolic KOH (1 mL) for 3 h.

Puriﬁcation by FCC over short column [petroleum ether/EtOAc

(7:3)] gave pure 22 (0.025 g, 91.6%): mp 83−86 °C; IR (neat) 3346,

1

2929, 1449, 1267, 1121, 1071, 1040, 742 cm⁻¹; H NMR (500 MHz,

CDCl₃) δ 1.05 (br, 6H, 18 and 19-CH₃), 3.54 (m, 1 H, 3α-H), 5.41

(br, 1 H, 6-H), 6.04 (m, 1 H, 16-H), 7.25 (m, 1 H, aromatic-H), 7.31

(m, 1 H, aromatic-H), and 7.68 (m, 2 H, aromatic-H); ¹³C NMR (500

MHz, CDCl₃) δ 141.5, 133.2, 129.9, 123.3, 121.2, 111.5, 71.9, 55.9,

50.8, 42.5, 38.9, 37.3, 37.0, 34.0, 31.8, 30.6, 24.0, 23.2, 20.73, 19.5,

17.3, 16.4; HRMS calcd 445.2017 (C₂₈H₃₆ON₂·Na⁺), found 445.2020.

General Method D: Synthesis of 3β-Acetoxy-17-(1H-benzi-

midazol-1-yl)-16-((alkyl/arylimino)methyl)androsta-5,16-di-

enes (23, 26, and 29). The title compounds were prepared by

reﬂuxing a solution of 3β-acetoxy-17-(1H-benzimidazol-1-yl)-16-

formylandrosta-5,16-diene (14) (1 equivalent), corresponding primary

amine (2 equivalent), molecular sieves (∼25% weight of 14), and

ethanol under Ar for 3−12 h. Reaction mixture was ﬁltered and

concentrated under vacuum. The residue was stirred with water and

the resulting crude product ﬁltered. Puriﬁcation by the FCC on silica

gel column [petroleum ether/EtOAc (1:1)] gave the desired pure

compounds. The above listed compounds were synthesized (using

reactants, reagent, and solvent ratio), isolated, and puriﬁed by using

this method unless otherwise stated.

3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-((EZ)-

(isopentylimino)methyl)androsta-5,16-diene (23). Compound

23 was prepared by following general method D, reﬂuxing 14 (0.4

g, 0.87 mmol), isopentylamine (0.15 g, 1.7 mmol), molecular sieves

(0.2 g) in ethanol (5 mL) for 3 h. Puriﬁcation followed by FCC gave

0.41 g (89%) of 23: sinters at 135 °C, melts at 145 °C; IR (neat) 2934,

3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-((EZ)-((3,4-

dimethoxyphenyl)imino)methyl)androsta-5,16-diene (29).

Compound 29 was prepared by following general method D, reﬂuxing

14 (0.3 g, 0.65 mmol), 3,4-dimethoxyaniline (0.2 g, 1.3 mmol), and

molecular sieves (0.075 g) in ethanol (2 mL) overnight. Puriﬁcation by

FCC [petroleum ether/EtOAc (1:1)] gave 0.29 g (74.5%) of 29:

sinters at 115 °C, melts at 130 °C; IR (neat) 2937, 2904, 2852, 1729,

1

2851, 1726, 1676, 1640, 1490, 1453, 1247, 1219, 1032, 744 cm⁻¹; H

NMR (400 MHz, CDCl₃) δ 0.87 (d, 6H, aliphatic-CH₃), 1.07 (s, 3 H,

18-CH₃), 1.16 (s, 3 H,19-CH₃), 2.06 (s, 3 H, 3β-OCOCH₃), 4.64 (m,

1 H, 3 α-H), 5.46 (br s, 1 H, 6-H), 7.30 (s, 1 H, imine-CH), 7.34 (m, 2

H, aromatic-Hs), 7.72 (s, 1 H, aromatic-H), 7.87 (s, 1 H, aromatic-H),

and 7.94 (s, 1 H, 2′-H).

1

1586, 1509, 1451, 1372, 1233, 1125, 1026, 765 cm⁻¹; H NMR (400

MHz, CDCl₃) δ 1.10 (s, 3 H, 18-CH₃) 1.23 (s, 3 H, 19-CH₃), 2.06 (s,

3 H, 3β-OCOCH₃), 3.84 (m, 6H, 2 × OCH₃), 4.64 (m, 1 H, 3α-H),

5.48 (br s, 1 H, 6-H), 6.56 (m, 2 H, aromatic-Hs), 6.73 (m, 1 H,

aromatic-H), 7.36 (m, 3 H, aromatic-2Hs and imine-CH), 7.88 (m, 1

H, aromatic-H), 7.95 (m, 1 H, aromatic-H), and 8.00 (s, 1 H, 2¹-H).

3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-(((3,4-

dimethoxyphenyl)amino)methyl)androsta-5,16-diene (30).

Compound 30 was prepared by following general synthetic method

General Method E: Synthesis of 3β-Acetoxy-17-(1H-benzi-

midazol-1-yl)-16-((alkyl/arylamino)methyl)-androsta-5,16-di-

enes (24, 27, and 30). To an ice cold solution of 16-enamines (23/

26/30) (1 mol equiv) in methanol was added NaBH₄(0.5 mol equiv)

in three portions over 30 min. Reaction continued for 1.5−5 h and

then neutralized with acetic acid. The mixture was evaporated and the

residue treated with water and ﬁltered. Crude product was carried to

the next step without puriﬁcation.

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E, reacting 29 (0.15 g, 0.25 mmol) in methanol (2.5 mL) with NaBH₄

(0.05 g, 0.126 mmol) at °C for 5 h. The crude 30 was carried to the

next step without puriﬁcation.

84.5%): mp 139−141 °C; IR (neat) 2948, 2850, 1490, 1451, 1329,

1171, 917, 740 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 0.99 (s, 3 H, 18-

CH₃), 1.01 (s, 3 H, 19-CH₃), 2.44 (s, 3 H, 4″-CH₃), 4.35 (m, 1 H, 3α-

H), 5.37 (m, 1 H, 6-H), 5.97 (m, 1 H, 16-H), 7.25−7.34 (m, 3 H,

aromatic-Hs), 7.35−7.37 (m, 2 H, 2″, 6″-Hs), 7.48 (m, 1 H, aromatic-

H), 7.79 (m, 3 H, aromatic-H and 3″-, 5″-H), and 7.95 (s, 1 H, 2′-H);

¹³C NMR (500 MHz, CDCl₃) δ 147.0, 144.5, 141.6, 139.3, 134.6,

129.8, 127.6, 123.5, 122.5, 120.6, 111.1, 82.1, 55.7, 50.3, 47.2, 38.9,

36.8, 34.8, 30.3, 28.5, 21.7, 20.57, 19.1. HRMS calcd 565.2495

(C₃₃H₃₈O3N₂S·Na⁺), found 565.2506.

General Method F: Synthesis of 3-(Substituted-oximino)-17-

(1H-benzimidazol-1-yl)androsta-4,16-dienes (36−39). To a

reﬂuxing solution of ketone 32 (1 mol equivalent) in ethanol−

methanol (2:1) solvent mixture was added a solution of sodium

acetate (9.4 mol equiv) and the corresponding substituted oxamine

hydrochloride (10.5 mol equiv) in distilled water (10 mol equiv).

Reﬂux continued for 2−3 h. Then the mixture was concentrated. The

residue was treated with water and the crude product ﬁltered.

Puriﬁcation FCC over silica using 5% ethanolic DCM gave pure

oximes.

3β-Hydoxy-17-(1H-benzimidazol-1-yl)-16-(((3,4-

dimethoxyphenyl)amino)methyl)androsta-5,16-diene (31).

Compound 31 was prepared by following method C, treating

methanolic solution of (2 mL) of crude acetate 30 with 10%

methanolic KOH (0.75 mL). Subsequent puriﬁcation by FCC [DCM/

ethanol (9.7: 0.3)] gave 31 (0.11 g, 79.6%): sinters at 120 °C melts

135 °C; IR (neat) 3351, 2929, 2852, 1612, 1514, 1454, 1229, 1136,

1

1025, 765, cm⁻¹; H NMR (500 MHz, CDCl₃) δ 1.03 (s, 3 H, 18-

CH₃), 1.09 (s, 3 H, 19-CH₃), 3.53 (m, 1 H, 3α-H), 3.61 (br, 2 H, N-

CH₂), 3.74−3.77 (s, 6H, 2 × OCH₃), 5.37 (br, 1 H, 6-H), 5.95 (br, 1

H, aromatic-1″-H), 6.04 (d, J = 2.6 Hz, 1 H, aromatic-5″-H), 6.64 (br,

1 H, aromatic-6″-H), 7.21−7.31 (m, 3 H, aromatic-Hs), 7.74−7.83 (m,

1 H, aromatic-H), and 7.79 (s, 1 H, 2′-H); ¹³C NMR (500 MHz,

CDCl₃) δ 149.9, 142.2, 138.8, 123.7, 122.5, 120.9, 112.9, 110.3, 103.8,

99.4, 71.5, 56.6, 55.7, 50.6. 48.3, 42.8, 4.1, 34.7, 32.2, 31.1, 30.0, 20.5,

19.3, 15.8. HRMS calcd 576.3196 (C₃₅H₄₃O₃N₃·Na⁺), found

576.3188.

17-(1H-Benzimidazol-1-yl)androsta-4,16-dien-3-one (32).

This compound was prepared from 5 as previously described,

providing spectral and analytical data as reported.^12a¹³C NMR (500

MHz, CDCl₃) δ 199.4, 170.5, 147.2, 143.5, 141.1, 134.7, 124.3, 124.3,

123.5, 122.6, 122.5, 111.3, 54.3, 54.2, 47.4, 38.9, 35.9, 35.8, 34.1, 33.8,

32.8, 31.4, 30.4, 17.5, 17.3, 16.3.

3-((EZ)-Hydroximino)-17-(1H-benzimidazol-1-yl)androsta-

4,16-diene (36). Compound 36 was prepared by following general

method F. To a reﬂuxing solution of 32 (0.08 g, 0.194 mmol) in

ethanol−methanol (2 mL) was added a solution of sodium acetate

(0.15 g, 1.83 mmol) and hydroxylamine·HCl (0.07 g, 2.04 mmol) in

0.75 mL of distilled water. The reﬂux continued for 2 h, and

subsequent puriﬁcation by FCC gave compound 36 (mixture of EZ

isomers) (0.06 g, 77%): sinters at 145 °C, melts 155−160 °C; IR

(neat) 3181, 2929, 2853, 1609, 1453, 1226, 847 cm⁻¹; ¹H NMR (500

MHz, CDCl₃) δ 1.02 (s, 3 H, 18-CH₃), 1.11−1.15 (s, 3 H, 19-CH₃),

5.81 and 6.52 (∼57% and 33% for E and Z isomers, respectively) (s, 1

H, 4-H), 5.95 (br, 1 H, 16-H), 7.30 (m, 2 H, aromatic-Hs), 7.47 (m, 1

H, aromatic-H), 7.81 (m, 1 H, aromatic-H), and 7.95 (s, 1 H, 2′-H);

¹³C NMR (500 MHz, CDCl₃) δ 158.64, 156.6, 154.5, 147.0, 142.9,

17-(1H-Benzimidazol-1-yl)androsta-5,16-dien-3-one (33). To

an ice cold solution of 5 (0.05 g, 0.13 mmol) in dry DCM (3 mL) was

added Dess−Martin periodinane (0.11 g, 0.26 mmol), and the mixture

was stirred at ice cold temperature for 5 h. Then it was diluted with

ether and was quenched with a mixture of saturated aqueous

NaHCO₃/Na₂S₂O₃(1:3). The organic layer was washed with brine

and dried over Na₂SO₄. Then solvent was evaporated under vacuum

and the crude product was puriﬁed by FCC [DCM/ethanol/TEA

(30:1:0.05)] to give the title compound 33 (0.035 g, 70%): mp 170−

172 °C; IR (neat) 2941, 1711, 1491, 1451, 1226, 751 cm⁻¹; ¹H NMR

(500 MHz, CDCl₃) δ 1.05 (s, 3 H, 18-CH₃), 1.24 (s, 3 H, 19-CH₃),

5.41 (t, 1 H, J = 2.5 Hz, 6-H), 5.99 (br, 1 H, 16-H), 7.30 (m, 2 H,

aromatic-Hs), 7.49 (d, J = 6.9 Hz, 1 H, aromatic-H), 7.81 (m, 1 H,

aromatic-H), and 7.96 (s, 1 H, 2′-H); ¹³C NMR (500 MHz, CDCl₃) δ

209.9, 147.3, 143.5, 139.2, 134.8, 124.3, 123.5, 122.8, 122.6, 122.0,

120.5, 111.3, 56.0, 49.9, 49.7, 47.5, 37.74, 37.4, 37.0, 31.3, 31.1, 30.4,

19.3, 19.2, 16.8, 16.2. HRMS calcd 409.2250 (C₂₆H₃₀ON₂·Na⁺), found

409.2258.

3β-Mesyloxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene

(34). To ice cold solution of 5 (0.4 g, 1.03 mmol) in pyridine (5 mL)

was added methanesulfonyl chloride (0.68 g, 6 mmol). Reaction

mixture stirred at 0 °C for 5 h and then at room temperature for 8 h

and quenched with 75 mL of ice−water. The resulting yellow solid was

ﬁltered, washed, dried and the crude product was puriﬁed by FCC

[DCM/ethanol (1.5%)] to give the title compound 34 (0.4 g, 83%):

mp 177−179 °C (lit.⁶149−150 °C); IR (neat) 2944, 1486, 1452,

1326, 1170, 938, 765 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.03 (s, 3

H, 18-CH₃), 1.09 (s, 3 H, 19-CH₃), 3.03 (s, 3 H, mesyl-Hs), 4.56 (m,

1 H, 3α-H), 5.49 (br, 1 H, 6-H), 6.0 (m, 1 H, 16-H), 7.30 (m, 2 H,

aromatic-Hs), 7.49 (m, 1 H, aromatic-H), 7.82 (m, 1 H, aromatic-H),

and 7.97 (s, 1 H, 2′-H); ¹³C NMR (500 MHz, CDCl₃) δ 147.1, 143.3,

141.6, 139.1, 134.6, 123.4, 120.2, 81.6, 55.7, 50.3, 47.2, 39.2, 36.8, 34.8,

31.1, 28.9, 20.6, 19.1, 16.0. HRMS calcd 955.4472 (C₂₆H₃₀ON₂)₂Na⁺,

found 955.4468.

134.5, 124.3, 122.6, 117.8, 111.2, 55.3, 54.2, 47.3, 38.1, 34.6, 32.8, 30.3,

24.6, 20.9, 18.7, 17.9, 16.1. HRMS calcd 424.2359 (C₂₆H₃₁ON₃·Na⁺),

found 424.2363.

Separation of E and Z Isomers of 36. Initially the EZ mixture

was puriﬁed by FCC using petroleum ether and EtOAc (1:1) mixture.

This provided better purity of individual isomers with slight

contamination of each in one another. The major product 36E was

further puriﬁed by crystallization with hot EtOAc which resulted in

pure single isomer 36E: mp 218−221 °C; ¹H NMR (400 MHz,

CDCl₃) δ ppm 1.02 (s, 3 H, 18-CH₃), 1.11 (s, 3 H, 19-CH₃), 5.85 (s, 1

H, 4-H), 5.98 (s, 1 H, 16-H), 7.28−7.36 (m, 2 H, aromatic-Hs), 7.44−

7.55 (m, 1 H, aromatic-H), 7.79−7.88 (m, 1 H, aromatic-H), 7.97 (s, 1

H, 2′-H), 9.04 (br s, 1 H, -OH); ¹³C NMR (101 MHz, CDCl₃) δ ppm

156.7, 154.4, 147.1, 143.1, 141.6, 134.5, 124.1, 123.5, 122.5, 120.2,

117.9, 111.1, 55.3, 54.0, 47.3, 38.1, 34.8, 34.6, 34.2, 32.2, 31.5, 30.2,

21.1, 18.7, 17.6, 16.1. 36Z was further puriﬁed by preparative TLC

using petroleum ether/EtOAc (1:1) as solvent system: mp 158−162

1

°C; H NMR (400 MHz, CDCl₃) δ ppm 1.02 (s, 3 H, 18-CH₃), 1.15

(s, 3 H, 19-CH₃), 5.97 (br s, 1 H, 16-H), 6.53 (s, 1 H, 4-H), 7.27−7.34

(m, 2 H, aromatic-Hs), 7.44−7.52 (m, 1 H, aromatic-H), 7.76−7.87

(m, 1 H, aromatic-H), 7.7 (s, 1 H, 2′-H), 8.87 (br s, 1 H, -OH); ¹³C

NMR (101 MHz, CDCl₃) δ ppm 158.5, 147.0, 143.1, 141.6, 134.5,

124.2, 123.5, 122.6, 120.2, 117.7, 111.1, 55.2, 54.2, 47.3, 39.0, 38.1,

36.1, 34.8, 34.2, 32.8, 31.8, 30.2, 24.7, 20.9, 17.9, 16.1.

3-((EZ)-O-Phenyloxime)-17-(1H-benzimidazol-1-yl)androsta-

4,16-diene (37). Compound 37 was prepared by following general

method F. To a reﬂuxing solution of 32 (0.05g, 0.13 mmol) in

ethanol−methanol (2 mL) was added a solution of sodium acetate

(0.1 g, 1.22 mmol) and phenoxamine·HCl (0.2 g, 1.35 mmol) in 0.5

mL of distilled water. The reﬂux continued for 2 h, and subsequent

puriﬁcation by FCC gave compound 37 (mixture of EZ isomers) (0.04

g, 64%): mp 96−98 °C; IR (neat) 2935, 2854, 1627, 1590, 1487, 1216,

3β-Tosyloxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene

(35). To a cold (0 °C) solution of 5 (0.1 g, 0.26 mmol) in pyridine (3

mL) was added tosyl chloride (0.06 g, 0.31 mmol). Reaction mixture

was stirred at 0 °C for 5 h and then at room temperature for 3 h and

quenched with 30 mL of ice−water. The resulting yellow solid was

ﬁltered, washed, and dried, and the crude product was puriﬁed by FCC

[DCM/ethanol (1.0%)]. Resulting sticky solid was dissolved in 1.5 mL

of EtOAc and about 10 mL of petroleum ether added slowly with

stirring. The resulting turbid solution was stirred at room temperature

for 30 min to give free-ﬂowing solid of title compound 35 (0.115 g,

1

897 cm⁻¹; H NMR (500 MHz, CDCl₃) δ 1.05 (s, 3 H, 18-CH₃),

1.16−1.20 (s, 3 H, 19-CH₃), 6.00 (s, 1 H, 4-H and 16-H), 6.00 and

6.67 (∼55% and 45% for E and Z isomers, respectively) (s, 1 H, 4-H),

7.01 (m, 1 H, aromatic-H), 7.22 (m, 2 H, aromatic-Hs), 7.32 (m, 4 H,

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aromatic-Hs), 7.52 (m, 1 H, aromatic-H), 7.83 (m, 1 H, aromatic-Hs),

and 7.97 (s, 1 H, 2′-H); ¹³C NMR (500 MHz, CDCl₃) δ 160.6, 159.5,

158.0, 156.0, 147.1, 129.2, 124.2, 123.5, 121.7, 120.2, 117.4, 114.7,

111.2, 55.3, 55.0, 47.3, 38.2, 36.0, 34.1, 32.4, 30.2, 24.6, 21.0, 20.0,

17.6, 16.1. HRMS calcd 500.2672 (C₃₂H₃₅ON₃·Na⁺), found 500.2677.

3-((EZ)-O-Methyloxime)-17-(1H-benzimidazol-1-yl)androsta-

4,16-diene (38). Compound 38 was prepared by following general

method F. To a reﬂuxing solution of 32 (0.075g, 0.194 mmol) in

ethanol−methanol (2 mL) was added a solution of sodium acetate

(0.15 g, 1.83 mmol) and methoxyamine·HCl (0.17 g, 2.04 mmol) in

0.75 mL of distilled water. The reﬂux continued for 3 h, and

subsequent puriﬁcation by FCC gave compound 38 (mixture of EZ

isomers) (0.072 g, 89%): mp 94−96 °C; IR (neat) 2935, 2854, 1628,

esters derivatives were synthesized in a manner similar to the above.

TLC and H NMR and HRMS analyses revealed that the presence of

1

other esters derived from 2-methyl-6-nitrobenzoic anhydride is absent.

3β-(Pyridine-2-carboxylate)-17-(1H-benzimidazol-1-yl)-

androsta-5,16-diene (41). Compound 41 was prepared by following

general method G, using 2-methyl-6-nitrobenzoic anhydride (0.13 g,

0.39 mmol), picolinic acid (0.05 g, 0.39 mmol), 4-DMAP (0.04 g, 0.29

mmol), THF (1 mL), 5 (0.075 g, 0.19 mmol), THF (1 mL), and TEA

(0.1 mL). FCC gave pure 41 (0.09 g, 90%): mp 243−44 °C; IR (neat)

1

2942, 2852, 1729, 1496, 1286, 1227, 1139, 754 cm⁻¹; H NMR (400

MHz, CDCl₃) δ 1.03 (s, 3 H, 18-CH₃), 1.12 (s, 3 H, 19-CH₃), 4.99

(m, 1 H, 3α-H), 5.49 (t, 1 H, J = 1.98 Hz, 6-H), 5.99 (t, 1 H, J = 1.42

Hz, 16-H), 7.32 (m, 2 H, aromatic-Hs), 7.46−7.50 (m, 2 H, picolinoyl-

5-H and aromatic-H), 7.80−7.84 (m, 1 H, aromatic-H; 1H, picolinoyl-

4-H), 7.96 (s, 1 H, 2′-H), 8.15 (br, 1 H, picolinoyl-3-H), 8.79 (m, 1 H,

picolinoyl-6-H); ¹³C NMR (500 MHz, CDCl₃) δ 164.9, 150.1, 148.7,

143.4, 141.8, 140.2, 137.2, 134.7, 127.0, 125.4, 124.4, 123.6, 122.7,

120.3, 111.4, 75.6, 56.0, 50.6, 47.4, 38.2, 37.2, 35.0, 31.3, 30.5, 27.8,

20.82, 19.5, 17.0. HRMS calcd 516.2621 (C₃₂H₃₅O₂N₃·Na⁺), found

516.2614.

1

1489, 1452, 1226, 1050, 743 cm⁻¹; H NMR (500 MHz, CDCl₃) δ

1.04 (s, 3 H, 18-CH₃), 1.11 (s, 3 H, 19-CH₃), 3.89 (s, 3 H, OCH₃),

5.83 and 6.44 (∼69% and 31% for E and Z isomers, respectively) (s, 1

H, 4-H), 6.03 (m, 1 H, 16-H), 7.35 (m, 2 H, aromatic-Hs), 7.53 (m, 1

H, aromatic-H), 7.87 (m, 1 H, aromatic-H), and 8.06 (s, 1 H, 2′-H);

¹³C NMR (500 MHz, CDCl₃) δ 158.7, 156.0, 154.5, 153.1, 146.7,

125.2, 124.0, 123.3, 119.6, 117.7, 111.2, 61.6, 55.3, 54.2, 47.3, 38.0,

34.2, 32.2, 31.5, 30.3, 24.7, 21.0, 19.2, 17.6, 16.1. HRMS calcd

438.2515 (C₂₇H₃₃ON₃·Na⁺), found 438.2520.

3β-(Pyridine-3-carboxylate)-17-(1H-benzimidazol-1-yl)-

androsta-5,16-diene (42). Compound 42 was prepared by following

general method G, using 2-methyl-6-nitrobenzoic anhydride (0.13 g,

0.39 mmol), nicotinic acid (0.05 g, 0.39 mmol), 4-DMAP (0.035 g,

0.29 mmol), THF (1 mL), 5 (0.075 g, 0.19 mmol), THF (1 mL), and

TEA (0.1 mL). FCC gave pure 42 (0.85 g, 89%): mp 206−207 °C; IR

3-((EZ)-(O-Phenylmethyl)oxime)-17-(1H-benzimidazol-1-yl)-

androsta-4,16-diene (39). Compound 39 was prepared by following

general method F. To a reﬂuxing solution of 32 (0.075g, 0.194 mmol)

in ethanol−methanol (2 mL) was added a solution of sodium acetate

(0.15 g, 1.83 mmol) and benzyloxyamine·HCl (0.33 g, 2.04 mmol) in

0.75 mL of distilled water. The reﬂux continued for 3 h, and

subsequent puriﬁcation by FCC gave compound 39 (mixture of EZ

isomers) (0.092 g, 96%) which solidiﬁes on storage: sinters 66−68 °C,

melts 77−79 °C; IR (neat) 2935, 2854, 1627, 1609, 1489, 1452, 1225,

1

(neat) 3435, 2942, 2851, 1710, 1496, 1285, 1120, cm⁻¹; H NMR

(400 MHz, CDCl₃) δ 1.03 (s, 3 H, 18-CH₃), 1.13 (s, 3 H, 19-CH₃),

4.93 (m, 1 H, 3α-H), 5.49 (br, 1 H, 6-H), 5.99 (t, 1 H, J = 1.46 Hz, 16-

H), 7.32 (m, 2 H, aromatic-Hs), 7.41 (m,1H, nicotinoyl-5-H), 7.50

(m, 1 H, aromatic-H), 7.83 (m, 1 H, aromatic- H), 7.98 (s, 1 H, 2′-H),

8.33 (m, 1 H, nicotinoyl-4-H), 8.79 (m, 1 H, nicotinoyl-6-H), 9.23 (br

s, 1 H, nicotinoyl-2-H); ¹³C NMR (500 MHz, CDCl₃) δ 164.9, 153.5,

151.1, 147.3, 141.8, 140.0, 137.3, 126.8, 124.4, 123.6, 122.7, 120.4,

111.4, 75.2, 55.0, 50.6, 47.4, 38.3, 37.1, 35.0, 31.3, 30.5, 20.8, 19.5,

16.2. HRMS calcd 516.2621 (C₃₂H₃₅O₂N₃·Na⁺), found 516.2617.

3β-(Pyridine-4-carboxylate)-17-(1H-benzimidazol-1-yl)-

androsta-5,16-diene (43). Compound 43 was prepared by following

general method G, using 2-methyl-6-nitrobenzoic anhydride (0.13 g,

0.39 mmol), isonicotinic acid (0.05 g, 0.39 mmol), 4-DMAP (0.035 g,

0.29 mmol), THF (1 mL), 5 (0.075 g, 0.19 mmol), THF (1 mL), and

TEA (0.1 mL). FCC gave pure 43 (0.064 g, 67%): mp 184−85 °C; IR

(neat) 2944, 2953, 1719, 1489, 1282, 1124, 745 cm⁻¹; ¹H NMR (400

MHz, CDCl₃) δ 1.03 (s, 3 H, 18-CH₃), 1.13 (s, 3 H, 19-CH₃), 4.90

(m, 1 H, 3α-H), 5.49 (br, 1 H, 6-H), 5.99 (s, 1 H, 16-H), 7.30 (m, 2

H, aromatic-Hs), 7.49 (m, 1 H, aromatic-H), 7.81 (m, 1 H, aromatic-

H), 7.85 (m, 2 H, isonicotinoyl-3, 5-Hs), 7.96 (s, 1 H, 2′-H), and 8.78

(m, 2 H, isonicotinoyl-2, 6-Hs); ¹³C NMR (500 MHz, CDCl₃) δ

164.7, 150.8, 147.4, 143.5, 141.8, 139.9, 138.1, 134.8, 124.3, 123.6,

122.7, 120.4, 111.3, 75.6, 56.0, 50.6, 47.4, 38.2, 37.0, 35.0, 31.3, 30.5,

27.9, 19.5, 16.2. HRMS calcd 516.2621 (C₃₂H₃₅O₂N₃·Na⁺), found

516.2615.

3β-(3-(Oxycarbonyl)phenylacetic acid)-17-(1H-benzimida-

zol-1-yl)androsta-5,16-diene (44). Compound 41 was prepared

by following general method G. 2-Methyl-6-nitrobenzoic anhydride

(0.18 g, 0.51 mmol) was added to a solution of 1,3-phenyldiacetic acid

(0.1 g, 0.51 mmol) and DMAP (0.05 g, 0.39 mmol) in THF (2 mL), 5

(0.1 g, 0.26 mmol), THF (1 mL), and TEA (0.15 mL). FCC gave pure

44 (0.055 g, 39.81%): mp 222−23 °C; IR (neat) 2944, 1734, 1610,

1454, 1337, 1204, 1165, 1003, 749 cm⁻¹; ¹H NMR (500 MHz,

CDCl₃) δ 0.99 (s, 3 H, 18-CH₃), 1.05 (s, 3 H, 19-CH₃), 3.59 (s, 2 H,

CH₂-Hs), 3.64 (s, 2 H, CH₂-Hs), 4.63 (m, 1 H, 3α-H), 5.40 (br, 1 H,

6-H), 5.98 (m, 1 H, 16-H), 7.18−7.23 (m, 3 H, aromatic-Hs), 7.27−

7.31 (m, 3 H, aromatic-H), 7.47 (m, 1 H, aromatic-H), 7.81 (m, 1 H,

aromatic-H), 8.01 (s, 1 H, 2′-H); ¹³C NMR (400 MHz, CDCl₃) δ

171.2, 147.1, 141.8, 140.3, 135.0, 134.6, 130.5, 128.9, 128.0, 125.0,

123.9, 122.16, 120.0, 111.5, 74.4, 56.0, 50.5, 47.4, 45.6, 41.8, 38.2, 37.0,

37.0, 31.3, 30.5, 27.82, 20.8, 19.4, 16.1, 8.7. HRMS calcd 587.2880

(C₃₆H₄₀O₄N₂·Na⁺), found 587.2876

1

1015, 864 cm⁻¹; H NMR (500 MHz, CDCl₃) δ 1.03 (s, 3 H, 18-

CH₃), 1.10 (s, 3 H, 19-CH₃), 5.10 (s, 2 H, OCH₂), 5.83 and 6.52

(∼69% and 31% for E and Z isomers, respectively) (s, 1 H, 4-H), 5.97

(s, 1 H, 16-H), 7.25 (br, 3 H, aromatic-Hs), 7.37 (m, 4 H, aromatic-

Hs), 7.48 (m, 1 H, aromatic-H), 7.82 (m, 1 H, aromatic-H) and 7.95

(s, 1 H, 2′-H); ¹³C NMR (500 MHz, CDCl₃) δ 156.4, 154.6, 153.5,

147.0, 138.1, 127.9, 122.8, 120.0, 117.8, 111.3, 55.4, 54.0, 47.3, 38.0,

34.6, 32.2, 30.3, 24.7, 21.0, 19.6, 17.9, 16.1. HRMS calcd 514.2828

(C₃₃H₃₇ON₃·Na⁺), found 514.2834.

3-Methyl-3-hydroxy-17-(1H-benzimidazol-1-yl)androsta-

4,16-diene (40). To a solution of ketone (32) (0.1 g, 0.26 mmol) in

dry THF (3 mL) was added MeLi (1.6 M solution in ether, 0.41 mL,

0.60 mmol) at −60 °C, and the resulting mixture was stirred at 0 °C

for 1 h and then at room temperature for 3 h. The reaction was

quenched with saturated aqueous NH₄Cl and was extracted with

EtOAc. The organic layer was washed with brine and dried over

Na₂SO₄, and the solvent was removed under vacuum. The residue was

puriﬁed by short FCC [petroleum ether/EtOAc/TEA (60:40:0.5)] to

aﬀord product 40 (0.05 g, 48%); mp 95−97 °C; IR (neat) 3329, 2827,

1

2853, 1489, 1453, 1376, 1292, 1226, 1133, 918, 741 cm⁻¹; H NMR

(500 MHz, CDCl₃) δ 1.00 (s, 3 H, 18-CH₃), 1.07 (s, 3 H, 19-CH₃),

1.27 (s, 3 H, C3-CH₃), 5.25 (t, J = 1.6 Hz, 1 H, 6-H), 5.96 (t, 1 H, J =

1.52 Hz, 16-H), 7.29 (m, 2 H, aromatic-Hs), 7.49 (m, 1 H, aromatic-

H), 7.82 (dd, J = 7.0, 2.6 Hz, 1 H, aromatic- H), and 7.95 (s, 1 H, 2′-

H); ¹³C NMR (500 MHz, CDCl₃) δ 145.3, 127.6, 124.4, 123.6, 122.7,

120.4, 111.4, 70.1, 55.7, 54.8, 37.8, 35.6, 35.3, 34.7, 32.5, 30.4, 28.5,

21.1, 18.8, 16.3. HRMS calcd 425.2563 (C₂₇H₃₄ON₂·Na⁺), found

425.2570.

General Method G. Mixed Anhydride Method for the

Synthesis of Aromatic/Heteroaromatic Esters (41−44). 2-

Methyl-6-nitrobenzoic anhydride (0.39 mmol) was added to a solution

of pyridinecarboxylic acid (0.386 mmol) and DMAP (0.29 mmol) in

THF (1 mL), and the resulting mixture was allowed to stand at room

temperature for 5 min. A solution of 5 (0.193 mmol) in THF (1 mL)

was mixed with the above reagent mixture and then with TEA (0.1

mL). This reaction mixture was allowed to stand at room temperature

for 2 h. Reaction mixture was absorbed on silica and puriﬁed by FCC

using 2% ethanol in DCM in the presence of traces of TEA (0.06%).

The picolinoyl, nicotinoyl, isonoctinoyl, and 1,3-phenyldiacetic acid

N

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3β-(6-(Cyclohex-3-enecaboxylic acid)carboxylate)-17-(1H-

benzimidazol-1-yl)androsta-5,16-diene (45). A mixture of 5

(0.1 g, 0.26 mmol), DMAP (0.035 g, 0.28 mmol), 1,2,3,6-

tetrahydrophthalic anhydride (0.13 g, 0.85 mmol), and pyridine (3

mL) was reﬂuxed for 3 h. The mixture was cooled to room

temperature, and the reaction was quenched with water. Precipitate

was extracted with EtOAc, dried with Na₂SO₄, evaporated, and the

residue was puriﬁed by FCC [petroleum ether/EtOAc/TEA

(9.5:0.3:0.2)] to give 0.1 g (71.9%) of pure compound 45: mp

178−179 °C; IR (neat) 2931, 1724, 1453, 1225, 1195, and 743 cm⁻¹;

¹H NMR (400 MHz, CDCl₃) δ 0.99 −1.04 (m, 6H, 18-CH₃and19-

CH₃), 4.64 (m, 1 H, 3α-H), 5.40 (br, 1 H, 6-H), 5.69 (m, 2 H, c-hexyl-

4, c-hexyl-5, Hs), 5.96 (s, 1 H, 16-H), 7.30 (m, 2 H, aromatic-Hs), 7.50

(d, 1 H, aromatic-H), 7.84 (1 H, m, aromatic-H), 8.05 (s, 1 H, 2′-H);

¹³C NMR (500 MHz, CDCl₃) δ 177.3, 173.5, 1401.0, 126.0, 125.3,

124.8, 123.8, 123.0, 121.9, 120.0, 111.4, 73.8, 55.9, 50.5, 47.4, 45.4,

40.7, 38.2, 37.1, 34.9, 31.3, 30.5, 27.7, 26.4, 19.4, 16.2, 8.8. HRMS

calcd 563.2880 (C₃₄H₄₀N₂O₄·Na⁺), found 563.2879.

30.3, 27.7, 20.6, 19.3, 16.9, 16.0. HRMS calcd 519.2730

(C₃₁H₃₆O₂N₄·Na⁺), found 519.2730.

3β-(1H-1,2,4-Triazole-1-carboxylate)-17-(1H-benzimidazol-

1-yl)androsta-5,16-diene (49). A solution of 5 (0.15 g, 0.386

mmol), CDT (0.19 g, 1.16 mmol) in anhydrous acetonitrile (3 mL),

and DCM (1.5 mL) was reﬂuxed for 3 h. The solvent was evaporated

and the residue treated with water and extracted with DCM. The

crude white product obtained on evaporation of solvent was puriﬁed

by FCC using 4% ethanol in DCM in the presence of traces of TEA

(0.06%). The product was triturated with petroleum ether to give 49

(0.15 g, 80%): mp 205−206 °C; IR (neat) 2950, 2855, 1776, 1489,

1375, 1289, 978, 750 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.03 (s, 3

H, 18-CH₃), 1.12 (s, 3 H, 19-CH₃), 4.96 (m, 1 H, 3α-H), 5.52 (m, 1

H, 6-H), 5.99 (s, 1 H, 16-H), 7.30 (m, 2 H, aromatic-Hs), 7.50 (t, 1 H,

J = 3.8 Hz, aromatic-H), 7.81 (m, H, aromatic-H), 7.96 (s, 1 H, 2′-H),

8.07 (s, 1 H, 5″-H), and 8.83 (s, 1 H, 3″-H); ¹³C NMR (500 MHz,

CDCl₃) δ 153.8, 147.3, 145.8, 143,5, 141.8, 139.2, 134.7, 124.3, 123.6,

122.7, 120.4, 111.3, 80.0, 55.9, 50.5, 47.4, 37.9, 37.0, 35.0, 31.3, 30.5,

27.6, 20.8, 19.4, 16.2. HRMS calcd 506.2526 (C₂₉H₃₃O₂N₅·Na⁺),

found 506.2525.

Biology Experiments. Cell Culture. LNCaP cells were purchased

from American Type Culture Collection (ATCC; Rockville, MD,

U.S.). Cells were maintained in ATCC recommended culture medium

with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville,

GA, U.S.) and 1% penicillin/streptomycin (Invitrogen). Cells were

grown as a monolayer in T75 or T150 tissue culture ﬂasks in a

humidiﬁed incubator (5% CO₂, 95% air) at 37 °C. CWR22rv1 cells are

a gift from Dr. Marja Nevalainen of Thomas Jeﬀerson University,

Philadelphia, PA.

Cell Growth Inhibition (MTT Colorimetric Assay). The cells

were seeded in 96-well plates (Corning Costar) at a density of 5 × 10³

cells per well. Cells were allowed to adhere to the plate for 24 h and

then treated with various concentrations of compounds dissolved in

95% EtOH. Cells were treated for 7 days with renewal of test

compound and medium on day 4. On the seventh day, medium was

renewed and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-

tetrazolium bromide) (Sigma, St. Louis, MO, U.S.) solution (0.5 mg

of MTT per mL of medium) was added to the medium such that the

ratio of MTT/medium was 1:10. The cells were incubated with MTT

for 2 h. The medium was then aspirated, and DMSO was added to

solubilize the violet MTT−formazan product. The absorbance at 562

nm was measured by spectrophotometry (Biotek Inc.).

Transcriptional Activation Luciferase Assay. LNCaP cells were

transferred to steroid-free medium 3 days before the start of the

experiment and plated at 1 × 10⁵per well in steroid-free medium. The

cells were dual transfected with ARR2-Luc and the Renilla luciferase

reporter vector pRL-null. After a 24 h incubation period at 37 °C, the

cells were incubated in fresh phenol red free RPMI 1640 containing

5% charcoal-stripped fetal bovine serum and treated with 10 nmol/L

dihydrotestosterone, ethanol vehicle, and/or the selected compounds

in triplicate. After an 18-h treatment period, the cells were washed

twice with ice-cold Dulbecco’s PBS and assayed using the dual

luciferase kit (Promega) according to the manufacturer’s proto-

col.Cells were lysed with 100 μL of luciferase lysing buﬀer, collected in

a microcentrifuge tube, and pelleted by centrifugation. Supernatants

(20 μL aliquots) were transferred to corresponding wells of opaque

96-well multiwall plates. Luciferase assay reagent was added to each

well, and the light produced during the luciferase reaction was

measured in a Victor 1420 scanning multiwell spectrophotometer

(Wallac, Inc.). After measurement, Stop and Glo reagent (Promega)

was added to quench the ﬁreﬂy luciferase signal and initiate the Renilla

luciferase luminescence. Renilla luciferase luminescence was also

measured in the Victor 1420.The results are presented as the fold

induction (i.e., the relative luciferase activity of the treated cells divided

by that of the control) normalized to that of the Renilla.

3β-(Oxycarbonyl(methoxy)acetic acid)-17-(1H-benzimida-

zol-1-yl)androsta-5,16-diene (46). A mixture of 5 (0.1 g, 0.26

mmol), DMAP (0.035 g, 0.28 mmol), diglycolic anhydride (0.1 g, 0.85

mmol), and pyridine (3 mL) was reﬂuxed for 3 h. The mixture was

cooled to room temperature, and the reaction was quenched with

water. Precipitate was extracted with EtOAc, dried with Na₂SO₄,

evaporated and the residue was puriﬁed by FCC [petroleum ether/

EtOAc/TEA (9.5:0.3:0.2)] to give 0.05 g (28.6%) of pure compound

46: mp 214−215 °C; IR (neat) 2934, 1722, 1456, 1225, 1147, and 745

cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.01 (s, 3 H, 18-CH₃), 1.07 (s, 3

H, 19-CH₃), 4.25 (s, 2 H, CH₂), 4.26 (s, 2 H, CH₂), 4.74 (m, 1 H, 3α-

H), 5.45 (br, 1 H, 6-H), 6.00 (m, 1 H, 16-H), 7.32 (m, 2 H, aromatic-

Hs), 7.49 (m,1H, aromatic-H), 7.82 (m, 1 H, aromatic-H), 8.06 (s, 1

H, 2′ aromatic- H); ¹³C NMR (500 MHz, CDCl₃); δ 172.9, 169.9,

147.0, 141.7, 140.0, 134.4, 125.4, 124.2, 123.4, 119.7, 111.6, 75.0, 69.1,

68.8, 56.0, 50.5, 47.4, 38.2, 37.0, 34.9, 31.3, 31.1, 30.5, 27.8, 20.8, 19.4,

16.2. HRMS calcd 527.2516 (C₃₀H₃₆N₂O₅·Na⁺), found 527.2516.

3β-(1H-Imidazole-1-carboxylate)-17-(1H-benzimidazol-1-yl)-

androsta-5,16-diene (47). A solution of 5 (0.15 g, 0.38 mmol), CDI

(0.125 g, 0.77 mmol) in anhydrous acetonitrile (2 mL), and DCM (1

mL) was stirred at room temperature for 2 h. Then the solvent was

evaporated and the residue treated with water and extracted with

DCM. The crude white product obtained on evaporation of solvent

was puriﬁed by FCC using 1.7% methanol in DCM in the presence of

traces of TEA (0.06%) to give 47 (0.135 g, 72%): mp 194−96 °C; IR

(neat) 2965, 2923, 2839, 1754, 1488, 1452, 1392, 1292, 834, 773

cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.03 (s, 3 H, 18-CH₃), 1.12 (s, 3

H, 19-CH₃), 4.85 (m, 1 H, 3α-H), 5.51 (br, 1 H, 6-H), 5.99 (s, 1 H,

16-H), 7.07 (s, 1 H, 4″-H), 7.30 (m, 2 H, aromatic-Hs), 7.43 (s, 1 H,

aromatic-H), 7.49 (m, 1 H, 5″-H), 7.81 (m, 1 H, aromatic- H), 7.96 (s,

1 H, 2′-H), and 8.13 (s, 1 H, 2″-H); ¹³C NMR (500 MHz, CDCl₃) δ

148.1, 147.1, 143.3, 141.3, 139.1, 137.1, 134.6, 130.6, 124.1, 123.1,

120.2, 117.1, 111.1, 78.4, 55.7, 50.6, 47.2, 37.9, 36.8, 34.8, 31.1, 30.3,

27.6, 20.6, 19.3, 16.0. HRMS calcd 505.2573 (C₃₀H₃₄O₂N₄·Na⁺),

found 505.2577.

3β-(2-Methyl-1H-imidazole-1-carboxylate)-17-(1H-benzimi-

dazol-1-yl)androsta-5,16-diene (48). A solution of 5 (0.075 g,

0.193 mmol) and 1,1-carbonylbis(2-methylimidazole) (0.05 g, 0.214

mmol) in anhydrous acetonitrile (1.5 mL) and DCM (0.75 mL) was

reﬂuxed overnight. The solvent was evaporated. The residue was

treated with water and extracted with DCM. The crude white product

obtained on evaporation of solvent was puriﬁed by FCC using 4%

ethanol in DCM in the presence of traces of TEA (0.06%). The

product was triturated with petroleum ether to give 48 (0.065 g, 67%):

mp 186−187 °C; IR (neat) 2935, 2855, 1749, 1452, 1394, 1291, 1146,

983 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.03 (s, 3 H, 18-CH₃), 1.12

(s, 3 H, 19-CH₃), 2.64 (s, 3 H, 2″-CH₃), 4.80 (m, 1 H, 3α-H), 5.51

(m, 1 H, 6-H), 5.99 (m, 1 H, 16-H), 6.84 (s, 1 H, 5″-H), 7.29 (m, 2 H,

aromatic-Hs), 7.35 (s, 1 H, aromatic-H), 7.48 (m, H, aromatic-H),

7.81 (m, 1 H, 4″-H), and 7.96 (s, 1 H, 2′- H); ¹³C NMR (500 MHz,

CDCl₃) δ 149.0, 147.9, 147.1, 143. 3, 141.6, 139.2, 134.6, 127.8, 123.4,

122.5, 120.2, 118.1, 111.1, 78.0, 55.7, 50.3, 47.2, 38.0, 36.8, 34.8, 31.1,

Western Blot Analysis. For immunoblot detection of various

proteins, LNCaP or CWR22v1 prostate cancer cells were cultured.

Cells were treated with indicated compounds, and whole cell lysates

were prepared as described in ref 44 using RIPA lysis buﬀer (Sigma

Aldrich) and protease and phosphatase inhibitors (Sigma Aldrich). All

O

dx.doi.org/10.1021/jm400048v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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of the antibodies were ordered from Cell Signaling Technology.

Protein content was determined using the Bradford assay (Bio-Rad,

Hercules, CA, U.S.). Protein was subjected to SDS−PAGE and

transferred onto nitrocellulose membrane. Membranes were then

incubated with secondary antibody (Cell Signaling Technology) at

room temperature for 1 h. Bands were visualized by chemilumines-

cence (Millipore). Protein expression was normalized to β-actin, and

densitometry was carried out using Image J or ImageQuant 5.0

(Molecular Dynamics, Sunnyvale, CA, U.S.). CWR22Rv1 cells were

used for endogenous levels of splice variant AR-3. Protein levels were

analyzed with respective antibodies. Full length AR and β-actin

antibodies were purchased from Cell Signaling. Antibody speciﬁc for

splice variant AR-3 was obtained from Dr. Yun Qiu, University of

Maryland, School of Medicine, Baltimore, MD.^33c

AUTHOR INFORMATION

Corresponding Author

*Phone: 410-706 6364. Fax: 410-706 0032. E-mail: vnjar@som.

umaryland.edu.

■

Present Address

^○A.M.G.: Department of Internal, Medicine St. Barnabas

Hospital, Mills Building, 3rd Floor, 4422 Third Avenue, Bronx,

NY 10457.

Author Contributions

^×P.P and A.M.G. contributed equally to the work performed in

this manuscript.

Notes

Androgen Receptor Competitive Binding Assay. Competitive

binding assays were performed with the synthetic androgen

methyltrienolone [³H]R1881 essentially as described by Wong et al.

and Yarbrough et al.⁴⁵Wells in 24-well multiwell dishes were coated

with poly ^L-lysine (0.05 mg/mL) for 30 min, rinsed with sterilized,

distilled water, and dried for 2 h. To determine the kinetics of

[³H]R1881 binding to the LNCaP, AR cells were plated ((2−3) × 10⁵

cells/well) in 24-well multiwell dishes in steroid-free medium and

allowed to attach. The following day the medium was replaced with

serum-free, steroid-free RPMI supplemented with 0.1% BSA and

containing [³H]R1881 (0.01−10 nM) in the presence or absence of a

200-fold excess of cold DHT, to determine nonspeciﬁc binding, and 1

μM triamcinolone acetonide to saturate progesterone and glucocorti-

coid receptors. Following a 2 h incubation period at 37 °C, cells were

washed twice with ice-cold DPBS and solubilized in DPBS containing

0.5% SDS and 20% glycerol. Extracts were removed and cell associated

radioactivity counted in a scintillation counter. The data were

analyzed, and K_dand B_maxdetermined, by nonlinear regression using

Graphpad Prism software (GraphPad Software, Inc., San Diego, CA).

When the concentration of [³H]R1881 required to almost saturate AR

in both cell lines was established (5.0 nM), the ability of the test

compounds (1 nM to 10 μM) to displace [³H]R1881 (5.0 nM) from

the receptors was determined as described above. The IC₅₀of each

compound was determined by nonlinear regression with Graphpad

Prism software (GraphPad Software, Inc., San Diego, CA).

Immunocytochemical Analysis. LNCaP cells were plated in

eight-chamber vessel tissue culture treated glass slide (0.025 × 10⁶

cells/well) for 12 h and then treated with 5 μM VN/124-1 or

VNPT55 for 48 h. Cells were washed twice with PBS and ﬁxed in 3.7%

formaldehyde for 10 min and permeabilized with 0.25% Triton in PBS

for another 5 min after several washes. Cells were blocked with 5%

BSA with 0.5% NP40 in PBS and incubated with anti-AR (1:600

dilution, Cell Signaling) in 2.5% BSA in PBS overnight. Cells were

incubated for 1 h with secondary antibody Alexa Fluor 488 conjugate

anti-rabbit IgG(H+L) at 1:1000 (Cell Signaling) and nuclear

counterstain for 5 min (DAPI at 1:5000). All images were taken

using the Nikon TE2000 microscope.

The authors declare the following competing ﬁnancial

interest(s): Vincent C. O. Njar is the lead inventor of

compound 5 patents and technologies thereof owned by the

University of Maryland, Baltimore, and licensed to Tokai

Pharmaceuticals, Inc. The other authors declare no potential

conﬂict of interest. A patent application to protect the novel

compounds has been ﬁled.

ACKNOWLEDGMENTS

■

This work was supported in part by grants from NIH and NCI

(Grants R21 CA11791-01, 1RO1CA129379-01 A2, and

5RO1CA129379-02) and from start-up funds from Jeﬀerson

School of Pharmacy, Thomas Jeﬀerson University, Philadelphia,

PA, U.S., and University of Maryland School of Medicine and

the Center for Biomolecular Therapeutics (CBT), Baltimore,

MD, U.S., to V.C.O.N.. We also thank Dr. Adejare Adeboye,

Professor of Pharmaceutical Sciences, University of the

Sciences, Philadelphia, PA, U.S., for help with some NMR

acquisitions. A.K.K.-A. was supported in part by University of

Maryland School of Medicine Toxicology Program.

ABBREVIATIONS USED

■

AR, androgen receptor; ARD, androgen receptor down-

regulation; AP, antiproliferative; BzIm, benzimidazole; BSA,

bovine serum albumin; CRPC, castration resistant prostate

cancer; CYP, cytochrome P450; CYP17A, 17α-hydroxylase/

17,20-lyase; DAPI, 6-diamino-2-phenylindole; DHT, dihydro-

testosterone; DBPS, Dulbecco’s phosphate buﬀered saline;

ESPC, early stage prostate cancer; FBS, fetal bovine serum;

FCC, ﬂash column chromatography; GI₅₀, compound concen-

tration required to inhibit cell growth by 50%; HRMS, high-

resolution mass spectrometry; hAR, human androgen receptor;

IC₅₀, compound concentration required to inhibit cell growth

by 50%; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-

tetrazolium bromide; PDB, Protein Data Bank; SDS, sodium

dodecyl sulfate

CYP17 Inhibition Assay. The assay was kindly performed by Dr.

Emily Scott and colleagues according to their recently reported

procedure in which a truncated version of human CYP17A1

(CYP171dH) was expressed in E. coli and then puriﬁed to

homogeneity.³⁷IC₅₀values of the compounds were determined

from dose−response curves. The IC₅₀values of abiraterone alcohol

(3b, a CYP17 inhibitor recently approved for prostate cancer therapy),

galaterone, and 3β-hydroxy-17-(1H-imidazole-1-yl)androsta-5,16-

diene (VN/85-1, structure not shown, believed to be the most potent

CYP17 inhibitor^12a,21) were also determined in the same assay system

for comparison (used as positive controls).

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ASSOCIATED CONTENT

■

S

* Supporting Information

HPLC chromatograms and high resolution mass spectral data

for compounds 16−22, 25, 28, 31, and 33−49. This material is

available free of charge via the Internet at http://pubs.acs.org.

P

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Systematic structure modifications of multitarget prostate cancer drug candidate galeterone to produce novel androgen receptor down-regulating agents as an approach to treatment of advanced prostate cancer

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