SAR based design of nicotinamides as a novel class of androgen receptor antagonists for prostate cancer

Article abstract of DOI:10.1021/jm3014103

Molecular knowledge of pure antagonism and systematic SAR study offered a direction for structural optimization of DIMN to provide nicotinamides as a novel series of AR antagonists. Nicotinamides with extended linear scaffold bearing sterically bulky alkoxy groups on isoquinoline end were synthesized for H12 displacement. AR binding affinity and molecular basis of antiandrogenic effect establish the optimized derivatives, 7au and 7bb, as promising candidates of second generation AR antagonists for advanced prostate cancer.

Full text of DOI:10.1021/jm3014103

Brief Article
pubs.acs.org/jmc
SAR Based Design of Nicotinamides as a Novel Class of Androgen
Receptor Antagonists for Prostate Cancer
Su Hui Yang,†,§ Chin-Hee Song,‡,§ Hue Thi My Van,† Eunsook Park,‡ Daulat Bikram Khadka,†
Eun-Yeung Gong,‡ Keesook Lee,*,‡ and Won-Jea Cho*,†
†College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwang-ju 500-757, Republic of
Korea
‡Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwang-ju 500-757,
Republic of Korea
S
* Supporting Information
ABSTRACT: Molecular knowledge of pure antagonism and systematic SAR study offered a direction for structural optimization
of DIMN to provide nicotinamides as a novel series of AR antagonists. Nicotinamides with extended linear scaffold bearing
sterically bulky alkoxy groups on isoquinoline end were synthesized for H12 displacement. AR binding affinity and molecular
basis of antiandrogenic effect establish the optimized derivatives, 7au and 7bb, as promising candidates of second generation AR
antagonists for advanced prostate cancer.
short β-turns arranged in three layers to form an antiparallel “α-
helical sandwich”.10,11 Upon agonist binding, the carboxyl-
terminal helix 12 (H12) is repositioned to serve as a lid to
stabilize the ligand, and the second β-turn is then formed to
lock the conformation of H12 to allow the formation of an
activation function 2 (AF2) site which is essential for
recruitment of coactivators, thus controlling transcriptional
activities of the receptor.12 On the other hand, an antagonist
such as bicalutamide causes a partial unfolding of H12, thereby
disrupting the formation of AF2 region in similar pattern as
observed in the crystal structure of estrogen receptor (ER)
LBD in complex with the selective antagonist raloxifene.10,13
In spite of this probable mechanism, chemical modification
of either agonists or first generation antagonists of AR has been
applicable because of the lack of structural information of AR in
the antagonistic mode. This limitation has provided the
majority of drug candidates share similar chemical scaf-
folds.14−16 To address the issue of a possible switch from AR
antagonist to agonist induced by structural similarity, we
performed receptor-based virtual screening (VS) to identify a
totally new chemical scaffold, the nicotinamide DIMN (Figure
1).17 DIMN has been proven to be more potent than the
current drug, BIC, and to significantly reduce cell growth of
both early and late stage prostate cancer. Moreover, to the best
of our knowledge, the nicotinamide DIMN has never been
exposed in the literature with any biological activity, and this is
the first successful attempt to apply VS for a lead identification
by using AR LBD in the agonistic mode.
INTRODUCTION
■
Antiandrogen therapy with first generation androgen receptor
(AR) antagonists, such as bicalutamide (BIC), leads to a
temporary reduction of prostate cancer with a decrease in the
level of serum prostate-specific antigen (PSA), a biomarker of
prostate cancer (Figure 1). Unfortunately, cancer cells grow
Figure 1. Chemical structures of AR antagonists, BIC and DIMN.
again in the absence of androgens and progress to castration-
resistant prostate cancer (CRPC).1 CRPC is attributed to
elevated AR gene expression which can be driven by AR gene
amplification,2,3 AR gene mutation,4,5 or ligand-independent
AR activation through other factors such as increased
expression of transcriptional coactivators.6,7 In addition, the
first generation AR antagonists acquire agonistic property in
cells engineered to express higher AR amounts. This switch
from antagonism to partial agonism is demonstrated by the
antiandrogen withdrawal syndrome; the serum concentration of
PSA decreases in patients after discontinuation of antiandro-
gens.8 Besides, the current combination therapy for CRPC
involving docetaxel and prednisone increases survival by 2.4
months on average, but it is not curative and causes significant
adverse effects.9
AR, by itself, is not transcriptionally active; however,
androgen signaling is activated by binding of a natural or
synthetic ligand to the active site of AR LBD. It in turn
indicates that the pharmacological activity of AR ligands can be
determined by the impact of ligand binding on AR. Like other
nuclear receptors (e.g., estrogen receptor), AR shares a similar
three-dimensional structure containing 11 α-helices and two
In this study, we present a SAR study of DIMN analogues on
AR, the structural optimization of nicotinamides, and their
biological evaluation, to suggest that nicotinamides may
become a new series of AR antagonists effective for the
treatment of advanced prostate cancer.
Received: September 28, 2012
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
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a
Scheme 1. Synthesis of DIMN Analogues (7−9) and Synthesis of Substituted Isoquinolines (6c−m)
a
Reagents and conditions: (a) SOCl2, reflux; (a′) SOCl2, cat. DMF, reflux; (b) amine (R2-NH2, 3), Et3N, CH2Cl2, rt; (c) cyclic amine (R1-H, 6),
iPrOH, reflux; (d) cyclic amine (R1-H, 6), DMSO, 100 °C; (e) MeI, NaH, THF, 60 °C; (f) alkyl halide, K2CO3, DMSO or THF; (g) CH3NO2,
AcONH4, AcOH, reflux; (h) LiAlH4, dry THF, reflux; (i) HCHO, 1N HCl, 30% NaOH, rt; (j) MTBE, conc HCl, iPrOH, rt; (k) NaHCO3 or NaOH
sol, rt; (l) ClCOOMe, Et3N, dry THF, rt; (m) PPA, 145 °C; (n) SO2Cl2, acetic acid, rt; (o) HBr (48% in H2O), reflux; (p) Boc anhydride, Et3N,
THF, rt; (q) alcohol, PPh3, DIAD, dry THF, rt; (r) alkyl halide, NaH, DMF, rt; (s) 3 M HCl, NaOH, EtOH, rt.
thoxyisoquinolinyl-nicotinamides (7ao−7as), 6′-methyl pyr-
idine substitution (C) mostly displayed the strongest
antagonistic activity (>89.9% inhibition), probably due to
their involvement in hydrogen bonding (Figure 2; see Figure
S1 of Supporting Information (SI)).
The active analogues are expected to have similar binding
mode to DIMN.17 They also have a stretched scaffold linked
with the isoquinoline end which can dislocate H12.
Considering the development of AR antagonists with a long
chain facing H12,19 we envisioned that the introduction of
sterically bulky groups on the hydrophobic isoquinoline of
nicotinamides would improve AR antagonistic potency.
Optimization was further carried out to add large functional
groups on the isoquinoline by varying the size, position, or
numbers of the substituents. Benzaldehydes as starting
materials were prepared; some aldehydes (10c−d, 10j) were
commercially available and others (10e−g) were simply
synthesized (Scheme 1). Henry reaction on benzaldehydes 10
and a subsequent reduction resulted in phenethylamines 12.
Intramolecular cyclization followed by the use of concentrated
HCl produced isoquinoline salts 13 which were basified to
provide the desired free isoquinolines 6c−g (Scheme 1).20
As another route for intramolecular cyclization, amine 12j
was converted into carbamic acid methyl ester 14j in the
presence of methyl chloroformate. Further cyclization under
acidic condition afforded isoquinolone 15j, which was reduced
to give the desired isoquinoline 6j.21 Treatment of isoquino-
lines 6f−g with sulfuryl chloride produced chlorinated
isoquinolines 6h−i. In addition, some isoquinolines with an
alkoxy group on C6 were derived from the synthetic
isoquinoline salt 13c. Demethylation of 13c followed by N-
protection with a Boc group conferred the phenolic OH, which
is susceptible to selective O-alkylation for incorporation of
different alkoxy groups. Application of either Mitsunobu
RESULTS AND DISCUSSION
■
To enable a rational design of AR antagonists, a SAR study of
DIMN analogues was performed to identify chemical sites
which could be derivatized without a significant loss in activity.
Variations of the lead DIMN structure were possible in three
parts (Figure 1); the isoquinoline was replaced by heterocyclic
amine or substituted isoquinoline, the nicotinamide by
terephthalamide, and the pyridylamine by a variety of amines.
N-Methylation on the amide bond of nicotinamides was also
planned. Such DIMN analogues as nicotinamides or tereph-
thalamides (7, 8) were prepared by a similar method which has
been recently reported to be favorable for easy isolation of final
products with moderate to good chemical yield,17 and N-
methylation of nicotinamides (9) was carried out by applying a
typical procedure (Scheme 1).18
According to the AR antagonistic effect of 31 synthetic
DIMN analogues (4b−e, 7ab−as, 7bl−bn, 8a−b, 9ac, 9aj−al)
(see Table 1), compounds which were well tolerated or
conferred stronger potency compared to DIMN have isoquino-
line or 6,7-dimethoxy isoquinoline (7ab, 7ae, 7ao, 7aq, 7as).
Replacement of the isoquinoline (A) by saturated monocyclic
amine (7bl−bn) led to lower inhibition (max 65.7%
inhibition). The activity was further diminished on removal
of the amine (4b−e) (max 43.1% inhibition), suggesting a
requirement of a bulky hydrophobic moiety (A). Substitution
of the nicotinamide core (B) by terephthalamide (8a−b) or by
N-methyl nicotinamide (9ac, 9aj−9al) led to a considerable
loss in activity (max 48.8% inhibition). It indicates that the
nicotinamide scaffold is necessary to retain the potency as it
possibly confer the best positioning of amide CO, NH (B) to
associate with the receptor by hydrogen bonding. Next, the
inhibitory effect was not relied on the substitution pattern of
the amine (C). However, in the analysis of the effect of
isoquinolinyl-nicotinamides (DIMN, 7ab−an) and 6,7-dime-
B
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Table 1. AR Agonist/Antagonist Effect of DIMN Analogues
Figure 2. SAR of DIMN analogues for AR antagonistic effects.
substitution at positions 5 or 6 of the isoquinoline ring (R1)
(7au, 7bb−bc, 7bf−bg, 7bk) led to the improved potency with
more than 95% inhibition. However, some derivatives which
contain a benzyloxy group at position 5 or 6 of the isoquinoline
(7bd, 7bj, 9ax) and chlorinated isoquinoline (7ay, 7az)
afforded the reduced activity (30.5−86.4% inhibition). These
observations suggest that the benzyloxy group or chlorine of
the isoquinoline moiety would be too bulky to fit into the
ligand binding pocket of AR. On the other hand, the flexible
alkoxy chain is presumed to ensure proper binding into the
active site and ultimately affect H12 positioning by a strong
steric clash with Met895 in closest proximity (Figure S1,
SI).13,22
Prior to verifying the probability of nicotinamides as potent
AR antagonists, cytotoxicity on growth of mouse embryonic
fibroblast (MEF) cells as normal cells was investigated. In
particular, among those which inhibited the ligand-induced AR
transactivation by more than 95%, two compounds (7au and
7bb) exhibited little or no cytotoxic effect (Figure 3) and were
Figure 3. Cytotoxicity of nicotinamides on MEF cell proliferation.
Error bars indicate the mean SEM n.s., not significant; **, p < 0.01;
***, p < 0.001.
further tested for AR inhibitory activity and binding affinity
toward AR. 7au and 7bb showed IC50 values of 0.99 and 0.46
μM, respectively, which are much lower than that of DIMN
(IC50 = 4.45 μM) (Figure 4A); this result is in agreement with
their potent inhibitory effect on AR transactivation (Table 1).
In a competitive ligand binding assay performed with [3H] 5α-
DHT, 7au and 7bb were bound to AR with reduced affinity
(Figure 4B). On the basis of the apparent equilibrium
dissociation constant (Kd) for DHT−AR complex,23 the
equilibrium binding constants (Ki), calculated by Cheng−
Prusoff equation, are 25.6 and 1.5 μM for 7au and DIMN,
respectively. Next, the inhibitory effect of the nicotinamides on
the proliferation of human prostate cancer cells was evaluated.
In androgen-dependent LNCaP cells, 7au showed a stronger
inhibitory effect by suppressing up to 90% while 7bb inhibited
the proliferation by 80% which is similar to that of DIMN
(Figure 4C). The IC50 values of 7au and 7bb are 0.68 and 1.47
μM, respectively (Figure S2, SI). More interestingly, in
androgen-independent C4-2 cells which have similar character-
istics with CRPC cells,24,25 growth suppression by 7au with
IC50 value of 2.50 μM was more apparent whereas 7bb with
IC50 of 11.8 μM has comparable effect with respect to DIMN
(Figure 4D and Figure S3, SI). Furthermore, the nicotinamides
a
Data from triplicate experiments. AR transcriptional activity was
determined as percentage after treatment with 10 μM compounds in
the absence (AR agonistic effect) or presence of 10 nM DHT (AR
antagonistic effect). N.E., no antagonistic effect.
reaction or an SN2 reaction on the hydroxy isoquinoline 17c
and further deprotection of the Boc protecting group produced
the desired O-alkylated isoquinoline 6k−m.
A total of 19 nicotinamide derivatives (7at−az, 7ba−bk, 9ax)
were prepared and examined for their AR agonistic/
antagonistic effects (Table 1). As expected, most nicotinamides
bearing alkoxy isoquinoline resulted in comparable or stronger
antagonistic activity compared to DIMN whereas they did not
show agonistic effect. Particularly, the introduction of an alkoxy
C
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2 was accessed, and, encouragingly, they thoroughly blocked
the AR transactivation (Figure 5B). Taken together, these
results suggest that the optimized nicotinamides effectively
inhibit the AR activation steps, ultimately exhibiting potent
antiandrogenic activity.
To further elucidate our presumption of antagonism of the
nicotinamides toward AR by dispositioning an amino acid
residue of H12, a molecular modeling study was performed.
According to the docking modes of DIMN and 7au, 7au has
the same hydrogen bonding pattern of DIMN but with an
additional hydrogen bond with Gln711 (Figure 6). While the
Figure 4. Effects of the optimized nicotinamides in human prostate
cancer cells in vitro. (A) Dose-dependent AR antagonistic activity. (B)
Dose−response competitive ligand binding curves of [3H] 5α-DHT
equilibrium binding to AR. (C) Inhibitory effects on androgen-
dependent LNCaP cell growth, (D) androgen-independent, but AR-
positive, C4-2 cell growth, and (E) androgen-independent and AR-
negative PPC-1 cell growth. IC50 is the concentration of compounds,
which inhibits 50% of AR transactivation (A) and radioligand DHT
binding to AR (B). Error bars indicate the mean
SEM n.s., not
significant; ***, p < 0.001.
Figure 6. Molecular docking modes of DIMN (A) and 7au (B).
hardly inhibited the growth of androgen-independent but AR-
negative PPC-1 cells, implying their selective inhibitory activity
on prostate cancer cells which are AR-positive (Figure 4E).
The resultant strong antiandrogenic effect of the nicotina-
mides can be explained not only by ligand−receptor binding
affinity but also by molecular basis for their antagonism. To
explore molecular basis of the nicotinamides, the dynamic of
AR subcellular distribution was analyzed by green fluorescent
protein (GFP) expression. When a natural agonist DHT binds
to AR, the DHT−AR complex translocates from the cytoplasm
into the nucleus, causing the localization of the liganded AR
proteins in the nucleus (Figure 5A). However, treatment of 7au
linear structure of DIMN (left panel) helps the isoquinoline
end to face H12, the sterically bulky methoxy substituents in
the isoquinoline of 7au (right panel) are far more likely to
displace Met895 of H12. Illustrated by the predicted docking
results, it is obvious that the nicotinamides containing alkoxy
groups in the isoquinoline end locate themselves to be close
enough to have a stronger steric clash with Met895 to exhibit
potent antagonism.
CONCLUSION
■
A novel chemical entity, nicotinamides, has been designed and
developed as potent candidates of second generation AR
antagonists. On the basis of the lead DIMN structure, an initial
SAR exploration could be rationalized as well as the importance
of having a nicotinamide core. As expected, the optimized
nicotinamides bearing alkoxy substituents at positions 5 or 6 of
the isoquinoline led to a significant inhibition in growth of
androgen-dependent or androgen-independent prostate cancer
cells. This strong antiandrogenic effect of the nicotinamides
would be well explained by their AR binding affinity as well as
the potent inhibition effect on AR activation steps.
Furthermore, encouragingly, the assumption of the nicotina-
mides retaining strong AR antagonism seems to be clarified as
the alkoxy substituents of the isoquinoline moiety are well
closely packed on the pocket and located in just a short
distance, causing a dislocation of Met895. It in turn leads to the
impairment in the reposition of mobile carboxyl-terminal H12
and the consequent disruption of AF2 formation. All these
findings highlight the nicotinamide nucleus containing alkoxy
groups on isoquinoline as an excellent scaffold for a novel class
of AR antagonists. The provided SAR data and docking models
can offer a useful guidance for designing of pure AR antagonists
as effective therapeutic agents for advanced prostate cancer.
Figure 5. Molecular basis of antiandrogenic effect of the nicotinamides
(7au and 7bb). (A) Inhibitory effect on the nuclear translocation of
GRP-AR, and (B) SRC-1 and SRC-2 mediated enhancement of AR
transactivation.
or 7bb in presence of DHT resulted in a distinct distribution
pattern in which the liganded AR proteins were instead
dispersed throughout the cytoplasm, indicating that they
effectively interfere with nuclear translocation of the AR
proteins (Figure 5A). Once entering the nucleus, the liganded
AR proteins bind to their target gene promoter as a homodimer
which can be formed by recruiting AR coactivators. Such
coactivators as SRC-1 and SRC-2 have been reported to be
elevated in the development of more aggressive prostate
cancer.26 Next, the inhibition effect of the nicotinamides on AR
transactivation enhanced by overexpression of SRC-1 and SRC-
ASSOCIATED CONTENT
* Supporting Information
Preparation and characterization of all synthesized compounds,
physical and spectral data, biological assays details and
■
S
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AUTHOR INFORMATION
Corresponding Author
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E-mail, wjcho@jnu.ac.kr. For K.L.: phone, +82-62-530-0509;
fax, +82-62-530-0500; E-mail, klee@jnu.ac.kr.
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Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
T e c h n o l o g y ( N R F - 2 0 1 1 - 0 0 1 5 5 5 1 a n d N R F -
2012R1A2A2A01008388).
ABBREVIATIONS USED
■
AR, androgen receptor; BIC, bicalutamide; PSA, prostate-
specific antigen; CRPC, castration-resistant prostate cancer;
AF2, activation function 2; DHT, dihydrotestosterone; MEF,
mouse embryonic fibroblast; SRC, steroid receptor coactivator
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