Niphatenones, glycerol ethers from the sponge Niphates digitalis block androgen receptor transcriptional activity in prostate cancer cells: Structure elucidation, synthesis, and biological activity

Article abstract of DOI:10.1021/jm2014056

Extracts of the marine sponge Niphates digitalis collected in Dominica showed strong activity in a cell-based assay designed to detect antagonists of the androgen receptor (AR) that could act as lead compounds for the development of a new class of drugs to treat castration recurrent prostate cancer (CRPC). Assay-guided fractionation showed that niphatenones A (3) and B (4), two new glycerol ether lipids, were the active components of the extracts. The structures of 3 and 4 were elucidated by analysis of NMR and MS data and confimed via total synthesis. Biological evaluation of synthetic analogues of the niphatenones has shown that the enantiomers 7 and 8 are more potent than the natural products in the screening assay and defined preliminary SAR for the new AR antagonist pharmacophore, including the finding that the Michael acceptor enone functionality is not required for activity. Niphatenone B (4) and its enantiomer 8 blocked androgen-induced proliferation of LNCaP prostate cancer cells but had no effect on the proliferation of PC3 prostate cancer cells that do not express functional AR, consistent with activity as AR antagonists. Use of the propargyl ether 44 and Click chemistry showed that niphatenone B binds covalently to the activation function-1 (AF1) region of the AR N-terminus domain (NTD).

Full text of DOI:10.1021/jm2014056

Article
pubs.acs.org/jmc
Niphatenones, Glycerol Ethers from the Sponge Niphates digitalis
Block Androgen Receptor Transcriptional Activity in Prostate Cancer
Cells: Structure Elucidation, Synthesis, and Biological Activity
Labros G. Meimetis,† David E. Williams,† Nasrin R. Mawji,‡ Carmen A. Banuelos,‡ Aaron A. Lal,‡
Jacob J. Park,‡ Amy H. Tien,‡ Javier Garcia Fernandez,† Nicole J. de Voogd,§ Marianne D. Sadar,*,‡
and Raymond J. Andersen*,†
†Departments of Chemistry and Earth, Ocean & Atmospheric Sciences, University of British Columbia, 2036 Main Mall, Vancouver,
B.C., Canada V6T 1Z1
‡Genome Sciences Centre, B.C. Cancer Agency, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3
§Netherlands Centre for Biodiversity Naturalis, P.O. Box 9517, 2300 RA Leiden, The Netherlands
S
* Supporting Information
ABSTRACT: Extracts of the marine sponge Niphates digitalis
collected in Dominica showed strong activity in a cell-based
assay designed to detect antagonists of the androgen receptor
(AR) that could act as lead compounds for the development of
a new class of drugs to treat castration recurrent prostate
cancer (CRPC). Assay-guided fractionation showed that
niphatenones A (3) and B (4), two new glycerol ether lipids,
were the active components of the extracts. The structures of 3
and 4 were elucidated by analysis of NMR and MS data and
confimed via total synthesis. Biological evaluation of synthetic analogues of the niphatenones has shown that the enantiomers 7
and 8 are more potent than the natural products in the screening assay and defined preliminary SAR for the new AR antagonist
pharmacophore, including the finding that the Michael acceptor enone functionality is not required for activity. Niphatenone B
(4) and its enantiomer 8 blocked androgen-induced proliferation of LNCaP prostate cancer cells but had no effect on the
proliferation of PC3 prostate cancer cells that do not express functional AR, consistent with activity as AR antagonists. Use of the
propargyl ether 44 and Click chemistry showed that niphatenone B binds covalently to the activation function-1 (AF1) region of
the AR N-terminus domain (NTD).
for 2 to 6 months. Men with CRPC usually succumb to their
disease within 2 to 3 years.
INTRODUCTION
■
One man in six will face the sobering prospect of being
diagnosed with prostate cancer at some point during his life.1
There are approximately 240,000 new diagnoses and 33,000
deaths resulting from prostate cancer each year in the USA
alone. First line treatment for localized low grade prostate
cancer involves radical prostectomy and/or radiation therapy.2
Roughly 30% of these patients will eventually have recurrence
of their cancer accompanied by a sharp rise in tumor burden
and serum concentrations of prostate-specific antigen (PSA).
Treatment of this second stage of the disease involves androgen
ablation by chemical or surgical castration and administration of
antiandrogens, resulting in effective tumor regression and a
drop in PSA concentrations. Eventually, after a period of up to
a few years, the cancer will reemerge in all of these patients as
indicated by a second sharp rise in PSA and tumor burden. This
last stage of the disease is referred to as castration recurrent
prostate cancer (CRPC). The only treatments available to
patients with CRPC are administration of cytotoxic agents
(taxanes) or sipuleucel-T (a vaccine), which only prolong life
Growth of prostate cancer cells is driven by the transcrip-
tional activity of the androgen receptor (AR) at all stages of the
disease.2,3 The AR comprises a C-terminal ligand binding
domain (LBD), a DNA binding domain (DBD), a hinge region,
and an N-terminus domain (NTD), containing the activation
function-1 (AF1) region. AR LBD can bind ligands in the
absence of the NTD, but no AR transcriptional activity is
possible without a functioning NTD AF1 region. During the
normal growth of prostate gland tissue and in the early stages of
prostate cancer, transcriptional activity of the AR is initiated by
binding of the endogenous ligand dihydrotestosterone to the
LBD. Androgen ablation by castration and treatment with
potent LBD antagonists (antiandrogens) such as bicalutamide
prevents AR transactivation by endogenous androgens, thereby
providing a highly beneficial although transient therapeutic
effect in the early stages of the disease. All current therapies that
Received: October 18, 2011
Published: December 8, 2011
© 2011 American Chemical Society
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target the AR LBD, either by reducing ligand or directly with
antiandrogens, eventually fail presumably through mechanisms
involving gain-of-function mutations in the LBD, intratumoral
de novo androgen synthesis, expression of constitutively active
AR splice variants that lack LBD, or activation of signaling
pathways that bypass the LBD and target the NTD such as
interleukin-6 (IL6) and protein kinase A (PKA).4 Recognition
of the essential role that the AF1 region plays in the
transactivation of the AR identified the AR NTD as a novel
drug target for treating CRPC. Decoy proteins provided the
first animal model proof-of-principle (POP) demonstration that
blocking activation of the AR NTD was a viable method to
control the growth of CRPC.5
As part of a program designed to find small molecule
antagonists of the AR NTD as lead compounds for the
development of drugs to treat CRPC, we have screened a
library of marine invertebrate extracts with an assay that uses
LNCaP prostate cancer cells containing an engineered PSA
gene with a luciferase reporter.3,6 Stimulation of the cells with
forskolin to stimulate PKA or IL6 activates the AR NTD
leading to AR-dependent production of PSA-luciferase that
generates light upon addition of luciferin. Antagonists of the AR
inhibit light production, a positive hit in the assay. Use of this
assay enabled the discovery of the sintokamides (i.e., 1)6 and
EPI-001 (2),3 the first known small molecule antagonists of the
AR NTD. Extensive characterization of the bioactivity of EPI-
001 has provided the first POP demonstration that small
molecule antagonists of the AR NTD are highly active in vivo
in LNCaP mouse models of CRPC.3
Crude MeOH extracts of the marine sponge Niphates digitalis
collected in Dominica showed strong activity in the screening
assay. Bioactivity-guided fractionation of the extracts led to the
isolation of the glycerol ethers niphatenones A (3) and B (4) as
the active components. The niphatenones represent a new
structural class of AR antagonists that bind covalently to the
AF1 region of the NTD. Details of the isolation, structure
elucidation, synthesis of the natural products 3 and 4 and
various analogues, and the biological activities of the natural
products and synthetic analogues are presented below.
accounted for the two sites of unsaturation required by the
molecular formula.
An isolated linear H spin system in 3 comprising four
1
contiguous methylenes (H2-1 to H2-4) was identified from the
COSY data. A multiplet at δ 3.12 (H2-1) was correlated to a
pentet at δ 1.40 (H2-2) that was in turn correlated to a pentet at
δ 1.64 (H2-3), which showed a correlation to a triplet at δ 2.23
(H2-4). The triplet at δ 2.23 (H2-4) and the pentet at δ 1.64
(H2-3) both showed strong HMBC correlations to the ketone
carbonyl resonance at δ 198.5 (C-5), demonstrating that C-4 of
the linear methylene chain was attached to the ketone carbon.
A second isolated spin system identified in the COSY
spectrum of 3 started with a methylene resonance at δ 3.19
(H2-1′; HSQC correlation to δ 72.6) that showed a correlation
to a methine at δ 3.63 (H-2′; HSQC correlation to δ 70.7),
which was in turn weakly correlated to a methylene resonance
at δ 3.42 (H-3′; HSQC correlation to δ 64.2). The resonance at
δ 3.42 (H-3′) also showed an HSQC correlation to the carbon
resonance at δ 64.2 (C-3′) and a strong COSY correlation to
the resonance at δ 3.51 (H-3′), assigned to its geminal partner.
This isolated spin system was assigned to a glycerol moiety.
HMBC correlations observed between the glycerol methylene
resonance at δ 3.19 (H2-1′) and the C-1 carbon resonance at δ
71.2, and between the H2-1 methylene resonance at δ 3.12 and
the C-1′ glycerol resonance at δ 72.6 showed that the glycerol
moiety was connected to C-1 via an ether linkage.
ISOLATION AND STRUCTURE ELUCIDATION
■
Specimens of Niphates digitalis (Lamarck, 1814) were collected
by hand using SCUBA on shallow reefs near Pennville in the
Commonwealth of Dominica and frozen on site for transport to
UBC. Freshly thawed sponge (160 g) was extracted repeatedly
with MeOH, and the combined extracts were evaporated in
vacuo to give a residue that was partitioned between H2O and
EtOAc. The bioactive EtOAc soluble material was fractionated
by assay-guided sequential application of reversed-phase flash
chromatography, Sephadex LH20 chromatography, and C18
reversed-phase HPLC to give small amounts of pure
niphatenones A (3) (0.1 mg) and B (4) (0.1 mg).
Niphatenone A (3) was isolated as a clear oil that gave a
[M + Na]+ ion in the HRESIMS at m/z 379.2808 consistent
with a molecular formula of C21H40O4 (calcd for C21H40O4Na,
379.2824), requiring 2 sites of unsaturation. The limited
quantity of niphatenone A (3) that was available precluded a
measurement of its specific rotation or a 1D 13C NMR
spectrum. A pair of olefinic methine resonances at δ 6.01 (H-6)
The remaining fragment of 3 had to be saturated, acyclic, and
account for C11H23. A COSY correlation was observed between
the resonance at δ 6.69 (H-7), assigned to the β proton of the
αβ-unsaturated ketone, and a methylene resonance at δ 1.87
(H2-8). The H2-8 (δ 1.87) resonance showed a COSY
correlation into an unresolved envelope of resonances at ≈ δ
1.1 to 1.3 in the COSY spectrum. A lone triplet methyl
resonance at δ 0.92 (H3-18) was also correlated in the COSY
1
and 6.69 (H-7) in the H NMR spectrum of 3 were correlated
to each other in the COSY spectrum and to a carbon resonance
at δ 198.5 in the HMBC spectrum, indicating the presence of
an αβ-unsaturated ketone substructure in 3. The enone
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Table 1. NMR Data (600 MHz, C6D6) for Natural Niphatenones A (3) and B (4)
3
4
position
δC
71.2
δH(J in Hz)
δC
70.5
δH(J in Hz)
3.01, m
1
3.12, m
a
2
29.4
1.40, p (7.1)
1.64, p (7.1)
2.23, t (7.1)
28.3
1.33, nd
1.88, m
3
20.9
29.2
4
39.8
145.1
6.66, dt (15.8, 7.0)
5.99, d (15.8)
5
198.5
130.7
6
130.6
6.01, d (15.8)
6.69, dt (15.8, 7.0)
1.87, m
198.6
7
146.3
40.6
2.28, t (7.4)
1.68, m
8
32.5
24.4
9
28.5
1.23, nd
29.6
1.27, nd
10
11
12
13
14
15
16
17
18
1′
2′
3′
29.5−30.1
29.5−30.1
29.5−30.1
29.5−30.1
29.5−30.1
29.5−30.1
32.3
1.1−1.3
1.1−1.3
1.1−1.3
1.1−1.3
1.1−1.3
1.1−1.3
1.30, nd
29.5−30.0
29.5−30.0
29.5−30.0
29.5−30.0
29.5−30.0
29.5−30.0
32.2
1.18−1.35
1.18−1.35
1.18−1.35
1.18−1.35
1.18−1.35
1.18−1.35
1.31, nd
23.1
1.30, nd
23.1
1.31, nd
14.4
0.92, t (6.8)
3.19, m
14.4
0.91, t (6.7)
3.15, m
72.6
72.6
70.7
3.63, m
70.6
3.61, m
64.2
3.42, dd (10.7, 5.2) 3.51, m
64.1
3.39, m 3.48, m
a
Multiplicity was not determined due to the overlapping signals/chemical shifts determined from 2D data.
spectrum of 3 into the unresolved envelope of resonances
at ≈ δ 1.1 to 1.3. This data was consistent with the C11H23 frag-
ment of niphatenone A being a linear saturated hydrocarbon chain
attached to C-7 of the αβ-unsaturated ketone as shown in 3. The
observed H-6/H-7 scalar coupling constant of 15.8 Hz indicated
that the Δ6,7-alkene had the E configuration.
linked to the glycerol fragment via a C-1 to C-1′ ether bond as
shown in 4.
The remaining fragment of niphatenone B had to be
saturated, acyclic, and account for C12H25. A triplet methylene
resonance at δ 2.28 (H2-7) and a methylene multiplet at δ 1.68
(H2-8), which were coupled to each other in the COSY
spectrum, both showed HMBC correlations to the C-6 ketone
carbonyl resonance (δ 198.6). The H2-8 methylene resonance
(δ 1.68) and a methyl triplet at δ 0.91 (H3-18) both showed
COSY correlations into an unresolved envelope between δ 1.18
and 1.35. The above data was consistent with a linear 12 carbon
alkyl group attached to the enone carbonyl to give the structure
4 for niphatenone B.
Niphatenones A (3) and B (4) are new glycerol ether sponge
natural products. They are homologues of the ceratodictyols A
(5) and B (6) isolated by Matsunaga and co-workers from the
red alga/sponge assemblage Ceratodictyon spongiosum/Haliclona
cymaeformis.7 The ceratodictyols were reported to have modest
in vitro cytotoxicity against a human cervical cancer cell line
(IC50 ≈ 67 μM). Niphatenones A (3) and B (4) showed strong
activity in a cell-based screening assay designed to find
anatagonists of AR NTD.3,5 They represent a new pharmaco-
phore with this promising biological activity, and they are,
therefore, potential lead compounds for the development of a
new class of drugs to treat CRPC.
Niphatenone B (4) was isolated as a clear oil that gave a
[M + Na]+ ion in the HRESIMS at m/z 379.2811 consistent
with a molecular formula of C21H40O4 (calcd for C21H40O4Na,
379.2824), identical to the molecular formula of niphatenone
1
A (3). The H NMR spectrum recorded for 4 showed a very
strong resemblance to the corresponding spectrum obtained for
3 (Table 1), suggesting that 3 and 4 were closely related
isomers. A pair of scalar coupled (J = 15.8 Hz) olefinic methine
resonances at δ 5.99 (H-5) and 6.66 (H-4) were correlated in
the HMBC spectrum to a carbonyl resonance at δ 198.6 (C-6),
revealing that niphatenone B also contained an αβ-unsaturated
ketone substructure with the E configuration.
The COSY, HMBC, and HSQC data confirmed the presence
of a glycerol moiety in niphatenone B (4) [δ 3.15 (2H), 72.6
(C-1′); 3.61 (1H), 70.6 (C-2′); 3.39 (1H), 3.48 (1H), 64.1 (C-3′)].
COSY correlations were observed between the H-4 alkene
methine resonance at δ 6.66 and a methylene quartet at δ 1.88
(H2-3). The methylene quartet was further correlated in the
COSY spectrum to a poorly resolved methylene resonance at δ
1.33 (H2-2) that was in turn correlated to a deshielded
methylene resonance at δ 3.01 (H2-1). Both of the methylene
proton resonances at δ 1.88 (H2-3) and 1.33 (H2-2) showed
HMBC correlations to the olefinic methine carbon resonance at
δ 145.1 (C-4) and the methylene carbon resonance at δ 70.5
(C-1), in agreement with the presence of three contiguous
methylenes attached to the β carbon of the enone substructure.
HMBC correlations observed between the H2-1 resonance at δ
3.01 and the C-1′ resonance at δ 72.6, and between the H2-1′
resonance at δ 3.15 and the C-1 resonance at δ 70.5 showed
that the distal terminus of the C-1 to C-3 methylene chain was
SYNTHESIS OF NIPHATENONES A (3) AND B (4)
■
The extremely small amounts of niphatenones A (3) and B (4)
available from the N. digitalis extract made it impossible to
determine the absolute configurations of the natural products
or carry out further evaluation of their biological activities. In
order to provide reference materials for the absolute
configuration determination and biological evaluation, we
have completed a total synthesis of S-(3) and (R)-niphatenone
A (7) and S-(4) and (R)-niphatenone B (8).
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Scheme 1
The synthesis of (R)-niphatenone A (7) started with
commercially available (S)-4-hydroxymethyl-2,2-dimethyl-1,3-
dioxolane (9) as shown in Scheme 1. Deprotonation of 9 with
sodium hydride in the presence of crown ether followed by
treatment with the primary bromide 10 gave the protected
glycerol ether 11 in acceptable yield.8 Hydrogenolysis gave the
primary alcohol 12 that was converted to the primary bromide
13 by treatment with carbon tetrabromide and triphenylphos-
phine. Deprotonation of the phosphonium salt 14 with n-butyl
lithium followed by alkylation with the bromide 13 gave the
Wittig reagent 15.9 Reaction of 15 with dodecanal 16 gave the
enone 17. Deprotection of 17 with HCl in H2O/THF gave
(R)-niphatenone A (7). Repeating the synthesis using (R)-4-
hydroxymethyl-2,2-dimethyl-1,3-dioxolane as the starting ma-
terial gave (S)-niphatenone A (3).
via NMR analysis. Chiral HPLC was able to resolve the
synthetic enantiomers of both niphatenones A and B. Using the
Chiral HPLC method, we showed that naturally occurring
niphatenones A (3) and B (4) both had the S configuration.
The synthetic niphatenone enantiomers 3, 4, 7, and 8 were
tested in an assay that measures AR transcriptional activity
(Figure 1). Compounds 4, 7, and 8 showed strong activity in
the assay. It is interesting to note that the configuration of the
glycerol C-2′ chiral center is an important determinant of
potency in the niphatenones, with the unnatural R enantiomers
7 and 8 being more active than the natural S enantiomers 3 and
4, respectively.
SYNTHESIS OF ANALOGUES OF NIPHATENONES A
■
AND B
The synthesis of (R)-niphatenone B (8) also started with
(S)-4-hydroxymethyl-2,2-dimethyl-1,3-dioxolane (9) as shown
in Scheme 2. Deprotonation of 9 with sodium hydride followed
by alkylation with the primary bromide 18 gave ether 19.
Removal of the benzyl protecting group in 19 via hydro-
genolysis gave the primary alcohol 20 that was oxidized with
DMP to give the aldehyde 21.10 The starting material for the
preparation of the Horner−Wadsworth−Emmons (HWE)
Wittig reagent 22 was epoxide 23. Treatment of 23 with
cerous chloride and sodium bromide gave bromohydrin 24,11
which was oxidized with DMP to give bromo ketone 25.
Reaction of 25 with triethylphosphite gave the HWE Wittig
reagent precursor 22.12 Deprotonation of 22 with barium
hydroxide followed by the addition of aldehyde 21 gave enone
26.13 Removal of the acetonide protecting group in 26 with
HCl in H2O/THF gave authentic (R)-niphatenone B (8).14
Repeating the synthesis with (R)-4-hydroxymethyl-2,2-dimethyl-
1,3-dioxolane as the starting material gave authentic (S)-
niphatenone B (4).
A number of analogues of niphatenones A and B were prepared
in order to explore the SAR for this new AR antagonist
pharmacophore. The niphatenones contain enone function-
alities that are potential Michael acceptors. In order to probe if
covalent bonding of the niphatenones to the AR played a role
in their biological activity, (R)-dihydroniphatenone B (28) was
prepared from acetonide 26 via catalytic hydrogenation to give
the saturated ketone 27,15 followed by acid catalyzed hydrolysis
of the acetonide as shown in Scheme 3.14
To examine the effect of changing the length of the linear
saturated alkyl substituent on the ketone functionality in
niphatenone B (4), the analogue (R)-32 with a C-18 ketone
substituent and the analogue (R)-35 with a one carbon ketone
substituent were prepared using the same approach as that
followed in the synthesis of (R)-niphatenone B (8) as shown in
Scheme 4.
The niphatenones are very nonpolar compounds that are
poorly soluble in water. In an attempt to make a more water-
soluble version, analogue (S)-41 containing a PEG tail attached
to the glycerol moiety was prepared as shown in Scheme 5. (S)-
Glycidol (36) was deprotonated by treatment with NaH and
crown ether in THF, and the resulting alkoxide was alkylated
with the primary bromide 18 to give the ether (R)-37.
Hydrogenolysis removed the benzyl protecting group in 37 to
Comparison of the NMR and MS data obtained for the
synthetic enantiomers of niphatenones A (3 and 7) and B (4
and 8) with the data collected for the natural products 3 and 4
showed that they were identical (Supporting Information),
confirming the constitutions assigned to the natural products
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Scheme 2
give the primary alcohol (R)-38. Oxidation of the primary
alcohol 38 with DMP to give to the corresponding aldehyde
followed by a HWE Wittig reaction with deprotonated 22 gave
the epoxide (R)-39. Opening of the epoxide in 39 with bismuth
triflate in the PEG diol 40 as a solvent gave the desired PEG
ether (S)-41. The glycerol fragment of the niphatenones is also
found in EPI-001 (2), a highly effective antagonist of the AR
NTD.3 The chlorohydrin functionality in 2 is required for its
AR-NTD blocking properties. Therefore, we decided to make
the EPI-001/niphatenone B hybrid analogue (S)-42 from the
epoxide (R)-39 by opening with cerium(III) chloride
heptahydrate as shown in Scheme 5. In order to explore the pos-
sibility that the niphatenones bind covalently to the AR via
the Michael acceptor enone substructures, we prepared
analogue (S)-44 containing an alkyne functionality by opening
epoxide (R)-39 with bismuth triflate in propargyl alcohol as a
solvent (Scheme 5). It was anticipated that Click chemistry
could be used to add biotin or fluorescent tags to this probe
after exposure to target proteins in order to provide direct
evidence for binding of the niphatenones to the AR NTD.
Finally, we made the all carbon chain analogue (S)-45 as
shown in Scheme 6. Primary alcohol (S)-46 was converted to
the primary bromide (S)-47 by treatment with triphenylphos-
phine in carbon tetrabromide. Conversion of bromide 18 into
the corresponding Grignard reagent followed by Cu catalyzed
coupling with 47 gave the benzyl protected dioxolane 48.
Removal of the benzyl protecting group in 48 via hydro-
genolysis gave the primary alcohol 49 that was oxidized with
DMP to the aldehyde 50. A HWE Wittig reaction between 50
and deprotonated 22 gave enone 51. Removal of acetonide in
51 with HCl/H2O gave the all carbon chain niphatenone B
analogue (S)-45.
BIOLOGICAL ACTIVITY OF THE NIPHATENONES
AND SYNTHETIC ANALOGUES
■
Figure 1 shows the biological activities of the natural
niphatenones [A (3) and B (4)] and a series of synthetic
analogues in an assay that measures AR transcriptional activity.
This assay consisted of transfecting LNCaP human prostate
cancer cells that express functional AR with luciferase reporter
that is regulated by AR in response to androgen. All the
compounds were tested at a concentration of 7 μM with the
exception of 32, which was limited to 3.5 μM due to poor
solubility. Niphatenone B (4) shows ≈50% inhibition at the
test concentration, while niphatenone A (3) shows weaker
activity that is not statistically different from that of the negative
control.
The synthetic enantiomers (R)-niphatenone A (7) and (R)-
niphatenone B (8) are both more active than the corresponding
natural S isomers 3 and 4, respectively, and (R)-niphatenone B
(8) is the most active compound tested in this series. (R)-
Dihydroniphatenone B (28) is less active than (R)-niphatenone
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produced a compound with roughly half the activity of 8.
Therefore, the glycerol ether linkage seems important to the
activity. Similarly, the acetonide (R)-26 and the epoxide (R)-39
are less active than 8, suggesting that the alcohol functionalities
on the glycerol moiety are important for activity. Finally, the
PEG ether (R)-41, chlorohydrin (R)-42, and alkyne ether (R)-
44 analogues are all less active than (R)-niphatenone B (8) and
comparable in activity to the natural product (S)-niphatenone
B (4). The attenuation in activity observed for analogues 41,
42, and 44 can perhaps be attributed to the loss of the glycerol
primary alcohol.
The inhibitory effects of niphatenones on the AR-driven
luciferase reporter were not due to nonspecific toxicity as
shown by comparing the proliferation of cells that are
dependent on AR for growth and proliferation to cells that
do not express functional AR, such as PC3 shown in Figure 2.
(S)-Niphatenone B (4) and (R)-niphatenone B (8) blocked
androgen-induced proliferation of LNCaP cells at comparable
concentrations to the clinical drug bicalutamide. These same
concentrations of either (S) or (R) niphatenone B had no
effect on the proliferation of PC3 cells that do not express func-
tional AR.
Figure 1. Key functional groups or structures within the niphatenone
analogues correlate with reduced endogenous AR activity at the
AR-driven promoter−luciferase reporter construct in LNCaP cells.
LNCaP cells were transiently transfected with PSA (p6.1)-luciferase
(0.5 μg/well in 12-well plates) and empty vector (1 μg/well) for 24 h
prior to a 1 h pretreatment with niphatenones at 7 μM or for 32 at
3.5 μM. Cells were then treated with R1881 (1 μM) with DMSO
vehicle (VEH) or R1881 (1 μM) with niphatenones for 48 h before
harvesting and measurement of luciferase activities and protein con-
centrations. Luciferase activity was normalized to the predicted
maximal activity induction (i.e., 100% in the absence of test com-
pounds with VEH at 0 μM niphatenone). Error bars represent the
EVIDENCE FOR COVALENT BINDING OF THE
■
NIPHATENONE B ALKYNYL ETHER ANALOGUE
44 TO THE AFI REGION OF THE AR NTD
mean
SD of three separate experiments, with each treatment
The objective of the screening program that identified the
niphatenones was to discover new antagonists of the AR NTD.
The differential effects of both (R)-8 and (S)-4 niphatenone B
on the proliferation of LNCaP prostate cancer cells versus the
proliferation of PC3 prostate cancer cells described above, and
their activity in the screening assay, were consistent with
niphatenone B being an androgen receptor antagonist. In order
to determine if niphatenone B would bind to the AR NTD, we
exposed recombinant NTD AF1 protein to the niphatenone B
analogue 44 that contains a propargyl ether. Analogue 44 was
active in the screening assay (Figure 1) indicating that it shared
the AR antagonistic properties of niphatenone B. After 50 min
of exposure at 0 °C, Click chemistry (Supporting Information
Scheme 1) was used to add a fluorescein tag, and the protein
was analyzed on a SDS−PAGE gel. Figure 3 shows that the
band corresponding to the AF1 protein is labeled with the
fluorescent tag, demonstrating that probe 44 had bound
covalently to the NTD AF1 protein.
condition performed in triplicate.
Scheme 3
CONCLUSIONS
■
The niphatenones represent a new natural product pharmaco-
phore that could serve as a lead for the development of drugs to
treat CRPC. Synthesis has shown that the unnatural (R)
analogues are more potent than the natural products.
Examination of a series of synthetic analogues of (R)-niphatenone
B (8) revealed that the enone functionality is not required for
activity and that the glycerol ether oxygen, glycerol alcohols, and a
reasonable length saturated alkyl chain substituent on the ketone
are important. These findings show that there is clear SAR for this
pharmacophore, and they suggest that it should be possible to
design more potent analogues of the natural products that do not
contain the undesirable enone Michael acceptor. The ability of
niphatenone B (4) and its enantiomer 8 to block androgen-
induced proliferation of LNCaP prostate cancer cells but not the
proliferation of PC3 prostate cancer cells that do not express
functional AR supports target-specific antiproliferative effects.
B (8) but only slightly less active than (S)-niphatenone B (4),
demonstrating that although the potentially reactive enone
plays a role in the potency of 8, it is not an absolute
requirement for activity. This suggests that covalent binding to
the AR is not required for the biological activity of the
niphatenones in the assay. The analogue (R)-32 with a longer
alkyl substituent on the ketone is about half as active as (R)-
niphatenone B (8) at half the dose, while the analogue (R)-35
with a one carbon alkyl substituent on the ketone has lost
essentially all activity at the test concentration. Removing the
glycerol ether oxygen atom from the linear skeleton of (R)-
niphatenone B (8) to give the all carbon chain analogue (R)-45
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Scheme 4
Scheme 5
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Scheme 6
Figure 2. Niphatenones inhibit androgen-dependent proliferation at concentrations that do not reduce proliferation of cells that do not express
function AR. (A) (S)-Niphatenone B (4) and (R)-niphatenone B (8) inhibit androgen-dependent proliferation. LNCaP cells were pretreated with
antiandrogen bicalutamide (BIC, 10 μM), (S)-niphatenone B (4) (14 μM), and (R)-niphatenone B (8) (14 μM) for 1 h prior to treatment with
0.1 nM R1881 in LNCaP cells. BrdU incorporation was measured 72 h after treatment. (B) (S)-Niphatenone B (4) and (R)-niphatenone B (8)
(14 μM) did not affect the proliferation of PC3 cells. BrdU incorporation was measured 24 h after treatment. Error bars represent the mean SD
of three separate experiments, with each treatment condition performed in 6 technical replicates. Student’s t test comparing each treatment to
R1881. * = p < 0.05.
Use of the niphatenone analogue 44 demonstrated that niphatenone
B binds covalently to the AF1 region of the AR NTD. Therefore,
niphatenone B has the desired profile of an AR NTD antagonist
drug candidate. The availability of a synthetic route to the
niphatenones has provided material for further in vitro and in
vivo biological evaluation of these promising lead compounds
and analogues in disease models of CRPC. Results from these
evaluations will be reported elsewhere.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a Jasco P-1010 Polarimeter with sodium light
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Niphatenone A (3). Isolated as a clear oil; [α]25 not possible
D
1
to measure accurately; H and 13C NMR, see Table 1; (+)-HRESIMS
[M + Na]+ m/z 379.2808 (calcd for C21H40O4Na, 379.2824).
Niphatenone B (4). Isolated as a clear oil; [α]25 not possible to
D
measure accurately; H and 13C NMR, see Table 1; (+)-HRESIMS
1
[M + Na]+ m/z 379.2811 (calcd for C21H40O4Na, 379.2824).
Synthesis of R and S Niphatenones A and B. Preparation of 11.
Sodium hydride (436 mg, 10.9 mmol, 60% suspension in oil) was
washed twice with hexanes (20.0 mL total), to which was added 45 mL
of THF, and the suspension was cooled to 0 °C. To this suspension
was added alcohol 9 (720 mg, 5.45 mmol) neat, along with 15C-5
(240 mg, 1.09 mmol). The mixture was then allowed to stir at RT for
1 h, after which bromide 10 (2.5 g, 10.9 mmol) was added dropwise.
After 4.5 h, the reaction mixture was cooled to 0 °C and quenched
with the addition of H2O, and the aqueous phase was extracted 3 times
with CH2Cl2. The organic extracts were dried with MgSO4 and con-
centrated. The crude mixture was purified with flash chromatography
(hexanes/EtOAc 9:1) to give 11 as a clear oil (623 mg, 2.22 mmol,
1
41%). [α]24 = +13.6 (c 0.12); H NMR (400 MHz, CDCl3) δ 7.33
D
(m, 4H), 7.28 (m, 1H), 4.50 (s, 2H), 4.25 (quin, J = 5.8 Hz, 1H), 4.04
(dd, J = 6.5, 1.7 Hz, 1H), 3.71 (dd, J = 6.1, 2.1 Hz, 1H), 3.56 (m, 5H),
3.42 (dd, J = 5.5, 4.4 Hz, 1H), 1.90 (quin, J = 6.1 Hz, 2H), 1.42 (s,
3H), 1.37 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 138.7, 128.5,
127.8, 127.7, 109.5, 74.9, 73.1, 72.1, 68.8, 67.3, 66.9, 30.2, 26.9, 25.6;
HRESIMS [M + H]+ calcd for C16H25O4 281.1753; found, 281.1747.
Preparation of 12. To compound 11 (575 mg, 2.05 mmol)
dissolved in 2.6 mL of EtOH was added 10% Pd/C (133 mg), in a
round-bottom flask and the system flushed with H2. The reaction
mixture was then exposed to 1 atm of H2 (balloon) overnight. Upon
completion, the heterogeneous mixture was filtered and the filtrate
washed three times with EtOH (60 mL total). The organic extracts
were concentrated, and the crude mixture was purified with flash chro-
matography (hexanes/EtOAc 1:1), to give 12 as a clear oil (375 mg,
Figure 3. Binding between niphatenone analogue 44 and AR AF1
protein was detected by fluorescein labeling. Fluorescein was visualized
by an image analyzer, and the image was modified by Adobe
Photoshop to adjust the band intensity. Equal amounts of proteins
were loaded as shown on the Coomassie blue stained gel. A
representative gel is shown (n = 2).
1
(589 nm) in EtOAc. The H and 13C NMR spectra were recorded on
either a Bruker AV-400 spectrometer or a Bruker AV-600 spectrometer
with a 5 mm CPTCI cryoprobe. 1H chemical shifts are referenced to the
residual C6D6 or CDCl3 signals (δ 7.15 and 7.24 ppm, repectively),
and 13C chemical shifts are referenced to the C6D6 or CDCl3 solvent
peak (δ 128.0 and 77.0 ppm, respectively). Low and high resolution
ESI-QIT-MS were recorded on a Bruker-Hewlett-Packard 1100
Esquire−LC system mass spectrometer. Merck Type 5554 silica gel
plates and Whatman MKC18F plates were used for analytical thin
layer chromatography. Flash chromatography was carried out on
Silicycle SiliaFlash 60A. Reversed-phase HPLC purifications were
performed on a Waters 600E System Controller liquid chromato-
graph attached to a Waters 996 Photodiode Array Detector. All
solvents used for HPLC were of Fisher HPLC grade. All final
compounds tested for biological activity had a purity of >95% by
HPLC analysis.
1.97 mmol, 96%). [α]24 = +16.3 (c 0.1); 1H NMR (400 MHz,
D
CDCl3) δ 4.16 (quin, J = 5.8 Hz, 1H), 3.94 (dd, J = 8.2, 6.5 Hz, 1H),
3.62 (m, 3H), 3.54 (t, J = 6.0 Hz, 2H), 3.39 (m, 2H), 3.02 (s, 1H),
1.72 (quin, J = 6.0 Hz, 2H), 1.31 (s, 3H), 1.25 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 109.5, 74.7, 71.9, 69.9, 66.6, 60.6, 32.2, 26.7,
25.4; HRESIMS [M + Na]+ calcd for C9H18O4Na 213.1103; found,
213.1105.
Preparation of 13. To alcohol 12 (150 mg, 0.788 mmol) dissolved
in 2 mL of CH2Cl2 was added PPh3 (248 mg, 0.946 mmol), and the
solution was cooled to 0 °C. CBr4 was added to this solution, and it
was stirred for 1 h after which the mixture was concentrated under a
stream of nitrogen. The crude mixture was purified with flash chro-
matography (hexanes/EtOAc 12:1), to give 13 as a clear oil (166 mg,
0.657 mmol, 83%).1H NMR (400 MHz, CDCl3) δ 4.19 (quin, J = 6.0
Hz, 1H), 3.98 (dd, J = 6.5, 1.7 Hz, 1H), 3.65 (dd, J = 6.5, 1.7 Hz, 1H),
3.54 (td, J = 5.9, 2.6 Hz, 2H), 3.43 (m, 4H), 2.04 (td, J = 2.9, 6.2 Hz,
2H), 1.35 (s, 3H), 1.29 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
109.5, 74.7, 72.2, 69.0, 66.7, 32.8, 30.6, 26.9, 25.5; HRESIMS [M + H]+
calcd for C9H18O379Br 253.0439; found, 253.0439.
Sponge Material. Specimens of Niphates digitalis (Lamarck, 1814)
were collected by hand using SCUBA at a depth of 10−15 m on gently
sloping reefs east of Pennville, Commonwealth of Dominica on
September 3, 2000 (N 15° 37.7′, W 61° 25.5′). A voucher sample has
been deposited at the Zoological Museum of Amsterdam (ZMA POR.
19968).
Extraction of Niphates digitalis and Isolation of Niphate-
nones A (3) and B (4). Freshly collected sponge was frozen on site
and transported to UBC frozen in a portable chest freezer. The sponge
material (160 g) was cut into small pieces and immersed in and
subsequently extracted repeatedly with MeOH (3 × 100 mL) at RT.
The combined methanolic extracts were concentrated in vacuo, and
the resultant extract was then partitioned between EtOAc (3 × 30 mL)
and H2O (100 mL). The combined EtOAc extract was evaporated to
dryness, and the resulting oil was fractionated using reversed-phase Si
gel flash chromatography, employing a step gradient from 1:1 MeOH/
H2O to MeOH with a final CH2Cl2 wash. A 30.2 mg fraction, eluting
with 9:1 MeOH/H2O, elicited activity and was chromatographed on
Sephadex LH-20 with MeOH as eluent. Purification of the resulting
active fraction via C18 reversed-phase HPLC using a CSC-Inertsil
150A/ODS2, 5 μm 25 × 0.94 cm column, with a linear gradient of
65:35 MeCN/H2O to MeCN over 60 min, gave pure samples of the
two minor compounds niphatenone A (3) (∼0.1 mg) and
niphatenone B (4) (∼0.1 mg) (with retention times of 37.5 and
36.5 min, respectively).
Preparation of 17. Phosphorane 14 (248 mg, 0.78 mmol) was
dissolved in 5 mL of THF, and the solution was cooled to −78 °C. To
this solution was added 1.6 M n-Buli (0.53 mL, 0.86 mmol), and it was
stirred for 20 min. To this solution, bromide 13 (166 mg, 0.66 mmol)
dissolved in 0.5 mL CH2Cl2 was added dropwise, and the mixture was
allowed to warm from 0 °C to RT overnight. The reaction mixture was
then diluted with H2O and extracted 3 times with CH2Cl2. The
organic extracts were dried with MgSO4 and concentrated. The crude
phosphorane was then used immediately in the follow up Wittig
reaction by dissolving it in 1 mL of CH2Cl2 and adding aldehyde 16
(121 mg, 0.657 mmol) before stirring at RT overnight. The reaction
mixture was concentrated under a stream of N2. The crude mixture
was purified with flash chromatography (hexanes/EtOAc 7:1), to give
17 as a clear oil (165 mg, 0.415 mmol, 63% over 2 steps). [α]24
=
D
1
+11.4 (c 0.09); H NMR (400 MHz, CDCl3) δ 4.19 (dt, J = 16.0,
7.2 Hz, 1H), 6.01 (d, J = 16.0 Hz, 1H), 4.19 (quin, J = 6.1 Hz, 1H), 3.99
(dd, J = 8.2, 6.5 Hz, 1H), 3.66 (dd, J = 6.5, 1.7 Hz, 1H), 3.42 (m, 4H),
2.50 (t, J = 7.2 Hz, 2H), 2.15 (q, J = 6.8 Hz, 2H), 1.57 (m, 4H), 1.40
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(m, 1H), 1.35 (s, 3H), 1.30 (s, 3H), 1.21 (m, 17H), 0.82 (t, J = 6.8 Hz,
3H); 13C NMR (100 MHz, CDCl3) δ 200.4, 147.5, 130.3, 109.4, 74.8,
71.9, 71.5, 66.9, 39.7, 32.5, 32.0, 29.7 (2C), 29.6, 29.5, 29.4, 29.3, 29.2,
28.2, 26.9, 25.5, 22.8, 20.9, 14.2; HRESIMS [M + H]+ calcd for
C24H45O4 397.3318; found, 397.3310.
Preparation of 7. To acetonide 26 (45 mg, 0.11 mmol), dissolved
in 1.65 mL of THF/H2O (5:1), was added 12.4 M HCl (0.045 mL,
0.56 mmol), and the solution was stirred at RT for 30 min. The
reaction mixture was then quenched with the addition of saturated
NaHCO3, and the aqueous phase was extracted 3 times with CH2Cl2.
The organic extracts were dried with MgSO4 and concentrated. The
crude mixture was purified with flash chromatography (4:1 EtOAc/
3.39 (m, 1H), 2.49 (td, J = 7.2, 1.2 Hz, 2H), 1.89 (quin, J = 6.8 Hz,
2H), 1.39 (s, 3H), 1.33 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
202.3, 109.5, 74.8, 71.9, 70.6, 66.8, 40.9, 26.8, 25.5, 22.5; HRESIMS
[M + Na]+ calcd for C10H18O4Na 225.1103; found, 225.1098.
Preparation of 26. To phosphonate 22 (353 mg, 1.0 mmol)
dissolved in 3.0 mL of THF was added Ba(OH)2 (254 mg,
0.80 mmol), and the suspension was stirred at RT for 30 min. To the
suspension was added aldehyde 21 (205 mg, 1.0 mmol), dissolved in
5.2 mL of THF/H2O (40:1), and it was stirred at RT for 1 h. The
reaction mixture was then diluted with H2O, and the aqueous phase
was extracted 3 times with CH2Cl2. The organic extracts were dried
with MgSO4 and concentrated. The crude mixture was purified
hexanes) to give 27 as a white solid (35 mg, 0.099 mmol, 90%).
with flash chromatography (hexanes/EtOAc 6:1) to give 26 as a clear
1
[α]24 = +2.8 (c 0.07); H NMR (600 MHz, C6D6) δ 6.70 (dt, J =
1
oil (326 mg, 0.82 mmol, 81%). [α]24 = +10 (c 0.01); H NMR
D
D
15.6, 7.2 Hz, 1H), 6.03 (d, J = 15.6 Hz, 1H), 3.91 (bs, 1H), 3.81 (bs,
1H), 3.73 (m, 1H), 3.66 (m, 1H), 3.41 (m, 2H), 3.26 (m, 2H), 3.28
(bs, 1H), 2.29 (t, J = 7.2 Hz, 2H), 1.90 (q, J = 6.6 Hz, 2H), 1.67 (quin,
J = 7.2 Hz, 2H), 1.48 (quin, J = 6.6 Hz, 2H), 1.28 (m, 18H), 0.91
(t, J = 4.8 Hz, 3H); 13C NMR (150 MHz, C6D6) δ 199.2, 146.6, 130.6,
72.6, 71.3(2C), 64.4, 39.8, 32.5, 32.3, 30.0, 30.0, 29.9, 29.8, 29.8,
29.6, 29.4, 28.4, 23.1, 21.0, 14.3; HRESIMS [M + Na]+ calcd for
C21H40O4Na 379.2824; found, 379.2814.
(400 MHz, C6D6) δ 6.65 (dt, J = 15.6, 7.2 Hz, 1H), 6.02 (d, J = 15.6
Hz, 1H), 4.12 (quin, J = 5.2 Hz, 1H), 3.83 (dd, J = 6.4, 1.6 Hz, 1H),
3.67 (dd, J = 6.4, 2.0 Hz, 1H), 3.32 (dd, J = 4.8, 4.8 Hz, 1H), 3.19 (dd,
J = 6.0, 3.6 Hz, 1H), 3.11 (t, J = 6.8 Hz, 2H), 2.27 (t, J = 7.3 Hz, 2H),
1.96 (q, J = 7.6 Hz, 2H), 1.66 (m, 2H), 1.42 (s, 3H), 1.38 (m, 2H),
1.30 (s, 3H), 1.28 (m, 18H), 0.90 (t, J = 6.8 Hz, 3H); 13C NMR (100
MHz, C6D6) δ 198.5, 145.2, 130.8, 109.3, 75.1, 72.1, 70.5, 67.1, 40.4,
32.3, 30.0, 30.0, 29.9, 29.9, 29.7, 29.7, 29.7, 29.1, 28.5, 27.1, 25.6, 24.4,
23.0, 14.3; HRESIMS [M + Na]+ calcd for C24H44O4Na 419.3124;
found, 419.3130.
Preparation of 8. To acetonide 26 (218 mg, 0.55 mmol) dissolved
in 8 mL of THF/H2O (5:1) was added 12.4 M HCl (0.22 mL, 2.7 mmol)
and the solution was stirred at RT for 30 min. The reaction mixture
was then quenched with the addition of saturated NaHCO3, and the
aqueous phase was extracted 3 times with CH2Cl2. The organic
extracts were dried with MgSO4 and concentrated. The crude mixture
Preparation of 3. Procedures identical to those used to prepare 7
were used but only using (R)-4-hydroxymethyl-2,2-dimethyl-1,3-
dioxolane as the starting material. Synthetic 3: [α]24 = −2.5 (c
D
0.09); HRESIMS [M + Na]+ calcd for C21H40O4Na 379.2824; found,
379.2813.
Preparation of 19. NaH (330 mg, 8.2 mmol, 60% suspension in
oil) was washed twice with hexanes (20 mL total) and added to 4 mL
of THF to give a suspension that was cooled to 0 °C. To this sus-
pension was added alcohol 9 (271 mg, 2.1 mmol) dissolved in 1 mL
of THF along with 15C-5 (90 mg, 0.41 mmol). The mixture was
then allowed to stir at RT for 30 min after which bromide 18 (1.0 g,
4.1 mmol) was added dropwise. After 4.5 h, the reaction mixture was
cooled to 0 °C and quenched with the addition of H2O, and the
aqueous phase was extracted 3 times with CH2Cl2. The organic
extracts were dried with MgSO4 and concentrated. The crude mixture
was purified with flash chromatography (hexanes/EtOAc 9:1), to give
19 as a clear oil (350 mg, 1.18 mmol, 58%). [α]24D = +9.9 (c 0.35); 1H
NMR (400 MHz, CDCl3) δ 7.32 (m, 2H), 7.31 (m, 2H), 7.25 (m,
1H), 4.47 (s, 2H), 4.23 (quin, J = 6.0 Hz, 1H), 4.01 (dd, J = 8.2, 6.5 Hz,
1H), 3.70 (dd, J = 8.2, 6.5 Hz 1H), 3.45 (m, 5H), 3.39 (m, 1H), 1.66
(m, 4H), 1.41 (s, 3H), 1.34 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
138.7, 128.5, 127.7, 127.6, 109.4, 74.9, 72.9, 71.9, 71.6, 70.2, 67.0, 26.9
(2C), 26.5 (2C), 25.5; HRESIMS [M + Na]+ calcd for C17H26O4Na
317.1729; found, 317.1733.
was purified with flash chromatography (EtOAc) to give 8 as a white
1
solid (168 mg, 0.47 mmol, 86%). [α]24 = +2.9 (c 0.17); H NMR
D
(600 MHz, C6D6) δ 6.69 (dt, J = 14.4, 7.2 Hz, 1H), 6.02 (d, J = 15.6
Hz, 1H), 3.87 (m, 1H), 3.70 (m, 1H), 3.63 (m, 2H), 3.33 (m, 3H),
3.16 (m, 2H), 2.31 (t, J = 6.6 Hz, 2H), 1.99 (q, J = 6.6 Hz, 2H), 1.65
(m, 2H), 1.45 (quin, J = 7.2 Hz, 2H), 1.28 (s, 18H), 0.91 (t, J = 7.2
Hz, 3H); 13C NMR (150 MHz, C6D6) δ 199.3, 145.8, 130.7, 72.5,
71.2, 70.5, 64.3, 40.4, 32.3, 30.1, 30.1, 30.0, 30.0, 29.9, 29.8, 29.7, 29.2,
28.3, 24.4, 23.0, 14.3; HRESIMS [M + Na]+ calcd for C21H40O4Na
379.2835; found, 379.2830.
Preparation of 4. Procedures identical to those used to prepare 8
were employed but only using (R)-4-hydroxymethyl-2,2-dimethyl-1,3-
dioxolane as the starting material. Synthetic 4: [α]24D = −2.7 (c 0.05);
HRESIMS [M + Na]+ calcd for C21H40O4Na 379.2824; found,
379.2816.
Chiral HPLC Analysis of the Sponge-Derived Niphatenones
A (3) and B (4). The samples of niphatenones A (3) and B (4)
isolated from the sponge Niphates digitalis were analyzed by chiral
HPLC using a Daicel Chemical Industries Ltd., Chiralcel OD-H 5 μm,
4.6 × 250 mm column. For niphatenone A (3) 3:97 isopropanol/
hexanes and for niphatenone B (4) 1:19 isopropanol/hexanes were
used as eluent (flow rate 1 mL/min), with UV detection at 224 nm.
The retention times of the peaks in the chromatographic trace were
compared to the retention times and photodiode array UV spectra of
the synthetically derived R and S versions of niphatenones A (3) and B
(4) and by coinjection. Retention times (min) are given in
parentheses: (R)-niphatenone A (7) (31.62); (S)-niphatenone A (3)
(29.75); (R)-niphatenone B (8) (19.77); (S)-niphatenone B (4)
(18.30). The results of the analysis indicated that the sponge derived
niphatenones A (3) and B (4) both had the S configuration.
Preparation of 24. To epoxide 23 (2.55 g, 11.9 mmol) dissolved in
150 mL of MeCN was added NaBr (1.45 g, 14.1 mmol) followed by
cerium(III) chloride heptahydrate (5.26 g, 14.1 mmol), and the
solution was stirred at RT for 24 h. The reaction mixture was then
concentrated and extracted three times with EtOAc (150 mL total).
The EtOAc fractions we combined, concentrated in vacuo, and
purified with flash chromatography (hexanes/EtOAc gradient 15:1,
10:1, and 7:1) to give bromohydrin 24 as a white solid (3.19 g, 10.9
mmol, 92%). 1H NMR (400 MHz, CDCl3) δ 3.78 (bs, 1H), 3.53 (dd,
J = 6.8, 3.2 Hz, 1H), 3.38 (dd, J = 6.8, 3.2 Hz, 1H), 2.26 (m, 1H), 1.53
Preparation of 20. To compound 19 (760 mg, 2.6 mmol) dissolved
in 4 mL of EtOH in a round-bottom flask was added 10% Pd/C
(200 mg), and the system was flushed with H2. The reaction mixture
was then exposed to 1 atm of H2 (balloon) overnight. Upon comple-
tion, the heterogeneous mixture was filtered and the filtrate washed
three times with EtOH (60 mL total). The organic extracts were
concentrated, and the crude mixture was purified with flash chro-
matography (hexanes/EtOAc 2:1), to give 20 as a clear oil (515 mg,
2.52 mmol, 98%). [α]24 = +12 (c 0.09); 1H NMR (400 MHz,
D
CDCl3) δ 4.20 (quin, J = 5.8 Hz, 1H), 3.99 (dd, J = 8.2, 6.5 Hz, 1H),
3.66 (dd, J = 8.4, 6.3 Hz, 1H), 3.57 (t, J = 5.8 Hz, 2H), 3.46 (m, 3H),
3.39 (m, 1H), 2.59 (bs, 1H), 1.60 (m, 4H), 1.36 (s, 3H), 1.31
(m, 3H); 13C NMR (100 MHz, CDCl3) δ 109.5, 74.7, 71.9, 71.7,
66.9, 62.5, 29.9, 26.8, 26.4, 25.5; HRESIMS [M + Na]+ calcd for
C10H20O4Na 227.1259; found, 227.1265.
Preparation of 21. To Dess−Martin periodinane (622 mg,
1.46 mmol) was added 9 mL of CH2Cl2, followed by pyridine (387 mg,
4.9 mmol). To this mixture was added alcohol 20 (190 mg, 0.97 mmol)
dissolved on 0.5 mL of CH2Cl2, and the solution was stirred at RT for
30 min. The crude mixture was then concentrated and purified with
flash chromatography (hexanes/EtOAc 2:1), to give 21 as a clear
1
oil (150 mg, 0.74 mmol, 80%). [α]24 = +13 (c 0.03); H NMR
D
(400 MHz, CDCl3) δ 9.75 (s, 1H), 4.21 (quin, J = 6.0 Hz, 1H), 4.02
(dd, J = 8.4, 6.7 Hz, 1H), 3.66 (dd, J = 8.2, 6.5 Hz, 1H), 3.48 (m, 3H),
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(m, 2H), 1.26 (m, 20H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ 71.2, 40.8, 35.3, 32.1, 29.83/29.80/29.72/29.67/29.52 (all
five signals account for a total of 7C), 25.7, 22.8, 14.3. Elemental
analysis: theoretical, 57.33% C and 9.97% H; found, 57.51% C and
10.04% H.
and 10 μM compound 44) for fluorescein labeling on compound.
Labeling was done by a Click chemistry reaction with 10 μM fluo-
rescein azide, 0.1 mM ascorbic acid, and 0.1 mM copper(II)−TBTA
complex (Lumiprobe) and incubated at RT for 30−40 min. Samples
were separated on 12.5% SDS−PAGE. Fluorescein was detected by an
image analyzer (Fujifilm FLA-7000, GE Healthcare). The same gel was
stained with Coomassie blue.
Preparation of 25. To bromohydrin 24 (158 mg, 0.54 mmol)
dissolved in 6 mL of CH2Cl2 at RT was added Dess−Martin
periodinane (455 mg, 1.07 mmol), and the mixture was stirred for 1 h.
The reaction mixture was then quenched with the addition of
saturated NaHCO3, and the aqueous phase was extracted 3 times with
CH2Cl2. The organic extracts were dried with MgSO4 and
concentrated in vacuo. The crude mixture was purified with flash
chromatography (hexanes/EtOAc 30:1), to give 25 as a white solid
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for the synthesis of the niphatenone
analogues and NMR spectra for natural 3 and 4 and all
synthetic compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
(126 mg, 0.43 mmol, 100%). H NMR (300 MHz, CDCl3) δ 3.89 (s,
2H), 2.64 (t, J = 7.4 Hz, 2H), 1.60 (m, 2H), 1.26 (m, 18H), 0.88 (t, J =
6.7 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 202.2, 39.9, 34.4, 32.0,
31.7, 29.8, 29.7, 29.6(2C), 29.5, 29.2, 24.0, 22.9, 14.2; HRESIMS
[M + Na]+ calcd for C14H27ONa79Br 313.1143; found, 313.1136.
Preparation of 22. To bromide 25 (80 mg, 0.27 mmol) dissolved
in 1 mL of toluene was added triethylphosphite (228 mg, 1.4 mmol).
The reaction mixture was refluxed for 24 h, after which it was cooled
to RT and concentrated under a stream of N2. The crude mixture was
purified with flash chromatography (hexanes/EtOAc 1:1), to give 22
as a clear oil (89 mg, 0.25 mmol, 95%). 1H NMR (400 MHz, CDCl3)
δ 4.09 (t, J = 7.2 Hz, 4H), 3.02 (m, 2H), 2.56 (t, J = 7.2 Hz, 2H), 1.53
(m, 2H), 1.28 (t, J = 7.0 Hz, 6H), 1.19 (m, 18H), 0.82 (t, J = 6.0 Hz,
3H); 13C NMR (100 MHz, CDCl3) δ 202.3, 62.6, 44.2, 43.1, 41.8,
32.0, 29.7, 29.6(2C), 29.5, 29.48, 29.43, 29.1, 23.5, 22.8, 16.4, 16.3,
14.2; HRESIMS [M + Na]+ calcd for C18H38O4P 349.2508; found,
349.2517.
AR Transcriptional Activity. AR transcriptional activity was
measured using the PSA (6.1 kb)−luciferase reporter gene construct
transiently transfected into LNCaP human prostate cancer cells which
express functional AR. This reporter contains several well-
characterized androgen response elements to which the AR specifically
binds to increase luciferase activity in response to androgen such as
R1881. LNCaP (2.5 × 104 cell/well) cells were seeded on 24-well
plates overnight before transfection with PSA (6.1 kb)-luc, (0.5 μg/
well) in serum-free, phenol red-free media using lipofectin
(Invitrogen) according to published methods.4b For SAR analysis,
LNCaP cells in 12-well plates were pretreated 1 h with (S)-
niphatenone B (4), (R)-niphatenone B (8), or their SAR analogues
(all added at 7 μM, with the exception of 32, which was tested at
3.5 μM due to solubility limitations) prior to the addition of 1 nM
R1881. After 48 h of exposure, cells were harvested, luciferase activity
measured, and normalized to protein concentration determined by the
Bradford assay.
Proliferation Assays. Experiments using LNCaP human prostate
cancer cells were done in phenol red-free RPMI 1640 medium with
0.5% (v/v) fetal bovine serum, while for PC3 human prostate cancer
cells, they were done in phenol red DMEM medium with 5% (v/v)
fetal bovine serum. Both media were supplemented with 100 units/mL
penicillin and 100 mg/mL streptomycin. Cells were seeded in 96-well
plates for 24 h before pretreatment for 1 h with bicalutamide (10 μM),
(S)-niphatenone B (4), and (R)-niphatenone B (8) (∼14 μM) before
treatment with 0.1 nM R1881 for LNCaP cells. LNCaP cells were
incubated for 72 h with R1881, while the duration of the experiment
was 24 h for PC3 cells. BrdU was added to the cells for an additional
2 h. Cells were fixed prior to incubation for 1.5 h with the anti-BrdU-
POD antibody (Roche). BrdU incorporation was measured at 570 nm
via VersaMax ELISA Microplate Reader (Molecular Devices).
Expression and Purification of Recombinant Protein. AR AF1
recombinant protein was expressed and purified as described
previously.3,16 The Ni2+-agarose affinity chromatography purified
recombinant AR AF1 protein was further purified by size exclusion
chromatography.
AUTHOR INFORMATION
Corresponding Author
■
*(M.D.S.) Phone: 604 675 8000. Fax: 604 675 8178. E-mail:
msadar@bcgsc.ca. (R.J.A.) Phone: 604 822 4511. Fax: 604 822
6091. E-mail: raymond.andersen@ubc.ca
ACKNOWLEDGMENTS
■
Financial Support was provided by the Prostate Cancer
Foundation (to M.D.S.), NSERC (to R.J.A.), and CCSRI (to
R.J.A.; grant number 017289).
ABBREVIATIONS USED
■
AR, androgen receptor; CRPC, castration recurrent prostate
cancer; PC3, androgen independent human prostate cancer cell
line; LNCaP, androgen-sensitive human prostate cancer cell
line; SAR, structure−activity relationship; PSA, prostate-specific
antigen; LBD, ligand binding domain; DBD, DNA binding
domain; NTD, N-terminus domain; AF-1, activation function-1;
POP, proof of principle; MeOH, methanol; EtOH, ethanol;
EtOAc, ethyl acetate; CH2Cl2, dichloromethane; MeCN,
acetonitrile; SCUBA, self-contained underwater breathing
apparatus; IL6, interleukin-6; HPLC, high peformance liquid
chromatography; HRESIMS, high resolution electrospray mass
spectrometry; NMR, nuclear magnetic resonance; COSY,
correlation spectroscopy; HSQC, heteronuclear single-quantum
correlation; HMBC, heteronuclear multiple bond correlation;
THF, tetrahydrofuran; HWE, Horner−Wadsworth−Emmons;
DMP, Dess−Martin periodinane; PEG, polyethylene glycol;
RT, room temperature; h, hour; min, minutes; PKA, protein
kinase A; ELISA, enzyme-linked immunosorbent assay; BrdU,
bromodeoxyuridine; DMEM, Dulbecco’s modified Eagle's
medium; RPMI, Roswell Park Memorial Institute; SDS−PAGE,
sodium dodecyl sulfate−polyacrylamide gel electrophoresis;
TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; 15c5,
1,4,7,10,13-pentaoxacyclopentadecane
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NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published online December 28, 2011, a
spelling error was corrected in the name of author Nicole J. de
Voogd. The revised version was published January 4, 2012.
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