Fluorescent epigenetic small molecule induces expression of the tumor suppressor ras-association domain family 1A and inhibits human prostate xenograft

Article abstract of DOI:10.1021/jm9011615

Epigenetic silencing of Ras-association domain family 1A (RASSF1A) protein in cancer cells results in a disruption of cell cycle control, genetic instability, enhanced cell motility, and apoptotic resistance. Ectopic expression of RASSF1A reverses this tumorigenic phenotype. Thus, small molecules with the ability to restore RASSF1A expression may represent a new class of therapeutic agents. Recently, we designed and synthesized a fluorescent carbazole analogue of mahanine (alkaloid from Murraya koenigii) that restored RASSF1A mRNA expression. Our fluorescent lead compound up-regulated RASSF1A in vitro, potently inhibited human prostate cancer cell proliferation, and fluoresced at a visible wavelength, allowing for the observation of intracellular distribution. The small molecule lead was not acutely toxic up to 550 mg/kg, and dosing at 10 mg/kg reduced human xenograft tumor volume by about 40%.

Full text of DOI:10.1021/jm9011615

2376 J. Med. Chem. 2010, 53, 2376–2382
DOI: 10.1021/jm9011615
Fluorescent Epigenetic Small Molecule Induces Expression of the Tumor Suppressor Ras-Association
Domain Family 1A and Inhibits Human Prostate Xenograft
Kathryn D. Sheikh,† Partha P. Banerjee,‡ Shankar Jagadeesh,‡ Scott C. Grindrod,† Li Zhang,† Mikell Paige,† and
Milton L. Brown*,†
†Drug Discovery Program, Department of Oncology, Georgetown University, 3970 Reservoir Road NW, Washington, D.C. 20057, and
‡Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road NW,
Washington, D.C. 20057
Received August 4, 2009
Epigenetic silencing of Ras-association domain family 1A (RASSF1A) protein in cancer cells results in a
disruption of cell cycle control, genetic instability, enhanced cell motility, and apoptotic resistance.
Ectopic expression of RASSF1A reverses this tumorigenic phenotype. Thus, small molecules with the
ability to restore RASSF1A expression may represent a new class of therapeutic agents. Recently, we
designed and synthesized a fluorescent carbazole analogue of mahanine (alkaloid from Murraya
koenigii) that restored RASSF1A mRNA expression. Our fluorescent lead compound up-regulated
RASSF1A in vitro, potently inhibited human prostate cancer cell proliferation, and fluoresced at a visible
wavelength, allowing for the observation of intracellular distribution. The small molecule lead was not acutely
toxic up to 550 mg/kg, and dosing at 10 mg/kg reduced human xenograft tumor volume by about 40%.
Introduction
of tumorigenic phenotype, small molecules with the ability to
restore RASSF1A expression have immense potential in the
treatment of tumors.
Ras-association domain family 1A (RASSF1Aa) is a fre-
quently inactivated protein in human cancer.1 One of the most
commonly found aberrations in human tumors is promoter
methylation of the RASSF1A gene, which results in a loss of
RASSF1A protein expression.2 This loss of protein expres-
sionhas beenlinkedto disruptions incellcycle control, genetic
instability, enhanced cell motility, and apoptotic resistance
in cancer cells, thus implicating RASSF1A as a tumor sup-
pressor protein.1,2
Located on chromosome 3p21.3, the RASSF1 gene locus
codes for seven different transcripts, with RASSF1A ex-
pressed as one of two major isoforms in normal cells. The
family of proteins share a conserved motif, the RalGDS/AF6
Ras association (RA) domain, as well as a C-terminal SAR-
AH protein-protein interaction motif. Because RASSF1A
appears to lack any enzymatic activity, it has been recently
hypothesized that it may serve as a scaffolding protein,
binding pertinent effector proteins such as K-Ras, proapop-
totic kinase MST1 and modulator of apoptosis 1 (MOAPl)
through its RA and SARAH domains.1 Ectopic expression of
RASSF1A in cancer cell lines causes G1/S arrest by reducing
cyclin D1 accumulation, promotes genetic stability by stabi-
lizing polymerized microtubules, restores cell-cell adhesion,
inhibits motility, and induces apoptosis by mediating the
proapoptotic effects of K-Ras.1,3-6 Because of this reversal
In prostate cancer, loss of function of classic tumor sup-
pressor genes such as RB1 is not frequently found; however,
the RASSF1A promoter region is methylated in 71% of
primary prostate tumors. When considering only more ag-
gressive prostate tumors (Gleason score of 7-10), the inci-
dence of RASSF1A methylation increases to nearly 85%.7,8 In
contrast, RASSF1A promoter methylation is not found in
normal primary prostate epithelial and stromal cells.9
The natural product mahanine (Chart 1) is a potent in-
hibitor of androgen dependent (LNCaP) and androgen
independent (PC-3) human prostate cancer cell proliferation.
An increase in RASSF1A expression was previously observed
in mahanine-treated LNCaP and PC-3 cells, as well as a
decrease in the level of cyclin D1 expression.10 This is con-
sistent with earlier findings that increased RASSF1A expres-
sion down-regulates cyclin D1, resulting in inhibited cell
growth.5 The antiproliferative activity of mahanine was asso-
ciated with its inhibition of DNA methyltransferase (DNMT)
activity in LNCaP and PC-3 cells. Presumably, inhibition of
DNMT activity by mahanine prevents the hypermethylation
and subsequent silencing of the RASSF1A gene in these
cell lines. Such epigenetic regulation of RASSF1A and other
hypermethylated genes has been observed in a variety of
human cell lines, including prostate cancer.7,11,12
In an effort to discover a novel and more potent small
molecule with a mechanistic profile similar to that of maha-
nine, a variety of synthetic analogues were designed and
synthesized, including derivatives containing a fluorescent
moiety. These mahanine analogues were evaluated for their
effects on PC-3 cell proliferation, RASSF1A expression,
cyclin D1 expression, and DNMT activity, and the lead
*To whom correspondence should be addressed. Phone: (202) 687-
8603. Fax: (202) 687-5659. E-mail: mb544@georgetown.edu.
a Abbreviations: AOT, acute oral toxicity; dansyl, 5-(dimethyl-
amino)naphthalene-1-sulfonyl; DCB, dichlorobenzene; DCM, dichloro-
methane; DNMT, DNA methyltransferase; GI50, concentration of drug
needed to inhibit cell growth by 50%; PEG, polyethylene glycol; RASSF1A,
Ras-association domain family 1A; RT-PCR, reverse transcription poly-
merase chain reaction; TEA, triethylamine.
r
pubs.acs.org/jmc
Published on Web 02/25/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2377
Chart 1. Structure of Mahanine and Template for Structural
Modifications
(i.e., 1-position versus 3-position). This was unexpected,
since the methyl group of mahanine is in the 3-position.
Compound 8, however with a methyl group in the
3-position but a deprotected hydroxyl group in the 2-posi-
tion, exhibited a GI50 value near those of compounds with a
1-position methyl (i.e., 7 and 9). It is possible that the benefit
of the free hydroxyl was due to the reduction of steric bulk at
the 2-position or the formation of a hydrogen bond. Never-
theless, it is obvious when comparing the more potent ana-
logues 5, 7, and 9 that increasing bulk at the 2-position greatly
enhances potency when the methyl is in the 1-position.
Overall, only 9 provided slightly greater antiproliferative
effects on human prostate cancer cells compared to maha-
nine. Additionally, mahanine was observed to be cytotoxic at
24 h at doses greater than 5 μM, while 9 proved to be largely
cytostatic at concentrations up to 30 μM (data not shown).
This observation was substantiated by testing performed
at the National Cancer Institute (NCI) with compound 9
against 59 cell lines, where concentrations of 9 up to 100 μM
resulted in no negative growth in 55 of the cell lines (93%)
and no instances of total cell kill in any of the cell lines
(p S2 of Supporting Information).
New Synthetic Analogues Induced a Decrease in DNA
Synthesis. To assess the levels of DNA synthesis within
prostate cancer cells treated with compounds 5-10, cultured
cells were treated for 24 h with either mahanine or com-
pounds 5-10 and then treated with 5-bromo-2-deoxyuridine
(BrdU) (Figure 1). Only three of the synthetic compounds (5,
8, and 9) paralleled mahanine in causing a decrease in DNA
synthesis. Because DNA synthesis is associated with cell
proliferation, it is not surprising that cells treated with 5
and 9 exhibited lower BrdU uptake than those treated with 6
or 10, given the respective GI50 values of the compounds.
Up-Regulation of RASSF1A and Down-Regulation of Cy-
clin D1 Was Observed in Cells treated with Mahanine Ana-
logues. It has been reported that mRNA levels of RASSF1A
correlate to its protein expression levels.4 Therefore, to
investigate the effects of the synthetic compounds on RASS-
F1A expression and cyclin D1 expression, levels of the
respective mRNAs in treated prostate cancer cells and un-
treated control cells were determined by RT-PCR (Figure 2).
Like mahanine, compounds 5 and 9 up-regulated RASS-
F1A, with 5 eliciting a response on par with mahanine and 9
increasing RASSF1A levels about 50% more than that of
mahanine treatment (Figure 3, gray bars). This occurred
with a corresponding decrease in cyclin D1 levels. A similar
profile was also observed in 7 and 8 but to a lesser extent.
Because overexpression of cyclin D1 is generally associated
with tumorigenicity and proliferation in a variety of tumors,
including prostate cancers, this down-regulation may have a
broader clinical significance.16,17 Compounds 6 and 10 show
no change in expression levels from that of untreated control.
New Synthetic Compounds Inhibited DNA Methyltransfer-
ase Activity. By use of a DNMT assay kit from Epigentek,
the levels of DNMT activity in treated versus untreated
prostate cancer cells were measured. Significant inhibition
of DNMT activity was observed with mahanine and com-
pounds 5 and 9, with 9 causing the most potent inhibition
(Figure 3, black bars). A slight decrease in DNMT activity
was also associated with compounds 7 and 8. These reduced
DNMT activities correlate with the pattern of RASSF1A
induction described above (Figure 3, gray bars). We propose
that the inhibition of DNMT prevents hypermethylation of
the RASSF1A promoter region and consequently restores
compound was evaluated in an in vivo human prostate tumor
model.
Results and Discussion
Chemistry. Modifications of the carbazole backbone that
result from a ring-opening strategy of the chromene ring of
mahanine were selected as the primary synthetic focus
(Chart 1). The alkyl and aryl substituents detailed below
(R1 = H, Me; R2 = H, Bn, dansyl) were chosen to roughly
probe the three-dimensional space around the eastern half of
the carbazole backbone. The formation of the carbazole
moiety can be envisioned through the cyclization of a biaryl
system. To create the initial biaryl moiety (4), a direct
cross-coupling of aryl halides 2 and 3 using a substrate-
specific reaction was found to be applicable to our system
(Scheme 1).13 Contrary to what was expected from reported
observations,13 we found the nitro-substituted aryl bromide
3 to be far more reactive than the aryl iodide 2. Homocou-
pling of 3 was observed to some extent under all of our
conditions, even when used as the limiting reagent, while
homocoupling of 2 only occurred when reactions were
allowed to run for several days. No reactions occurred at
temperatures under 90 °C, while temperatures over 130 °C
resulted in little cross-coupling, yielding only the homo-
coupled products.
The biaryl 4 was cyclized to give the two possible regio-
isomers, 5 and 6, which were separable by column chroma-
tography (Scheme 1).14 The structural assignment of the two
isomers was based on the 1H NMR coupling patterns of the
aryl protons. The 1H NMR spectrum of compound 5 showed
two doublets (δ 6.91 and 7.73, J = 8.4 Hz) corresponding to
the protons on carbons 3 and 4, respectively, whereas the 1H
NMR spectrum of compound 6 showed two singlets (δ 6.89
and 7.72) corresponding to the protons on carbons 1 and 4,
respectively. Compounds 5 and 6 were produced in an ap-
proximately 1:1 ratio of regioisomers as determined by the
mass of each isolated isomer after column chromatography,
regardless of the reaction conditions. Varying the solvent (1,2-
or 1,3-dichlorobenzene) as well as the equivalents of tri-
phenylphosphine resulted in only nominal changes in the ratio
of regioisomers. A palladium-on-carbon mediated deprotec-
tion of the benzyl groups of 5 and 6 provided the alcohols 7
and 8, respectively, which were reacted with dansyl chloride to
give the fluorescent analogues 9 and 10, respectively.
Mahanine Analogues Inhibit Growth of Human Prostate
Cancer Cells without Cytotoxicity. Compounds 5-10 were
screened for their effects on growth inhibition of PC-3
human prostate cancer cells using previously reported meth-
ods (Table 1).15 Compound 5 and the related analogue 9
showed markedly improved inhibition compared to their
counterparts 6 and 10, respectively, though the two sets of
compounds differed only in the position of the methyl group

2378 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Sheikh et al.
Scheme 1a
a (a) Benzyl bromide, K2CO3, MeCN, 50 °C; (b) Pd(OAc)2, K2CO3, PEG, 120 °C; (c) PPh3, 1,3-dichlorobenzene, reflux; (d) Pd/C (5 wt %),
ammonium formate, MeOH; (e) dansyl chloride, TEA, DCM.
Table 1. GI50 Values for Mahanine and Analogues 5-10 in Human
Prostate Cancer Cellsa
compd
GI50 (μM)
mahanine
5
2.5 ( 0.18
17.6 ( 2.7
48.9 ( 1.1
18.7 ( 1.8
15.3 ( 2.8
1.5 ( 0.11
22.9 ( 4.5
6
7
8
9
10
a Concentrations required for 50% growth inhibition in PC-3 cells.
Cells were plated in triplicate wells and exposed to various concentra-
tions (0, 1, 5, 15, 30, 60 μM) of each compound. After 24 h of treatment,
viable cells (as assessed by trypan blue dye exclusion assay) were counted
using a hemocytometer. Values are the mean ( SEM of three indepen-
dent observations.
Figure 1. Mahanine analogues decrease DNA synthesis in PC-3
cells. PC-3 cells were plated in 96-well plates and treated with 5 μM
mahanine (M), synthetic analogue (number), or DMSO (C). After
24 h, the cells were assayed for BrdU labeling using the cell
proliferation ELISA BrdU kit from Roche. Background BrdU
incorporation levels (absent anti-BrdU-peroxidase solution) were
subtracted, and values were normalized to those of control
untreated cells. Values are presented as the mean ( SEM.
RASSF1A protein, ultimately decreasing cyclin D1 levels
and causing the observed human prostate cancer cell growth
inhibition.
Fluorescent Analogue 9 Confirmed Intracellular Delivery
and Revealed Cellular Localization. The dansyl moiety pre-
sent in compound 9 allows it to visibly fluoresce at 552 nm
when excited at 370 nm (Figure 4A). After treatment of PC-3
cells with 9, a multiphoton laser was used to excite the
compound, and its emission from within the cells was
observed by confocal microscopy (Figure 4B-D). With
propidium iodide (blue) staining the nuclei of the cells, it
was apparent that 9 (green) was present within the cytoplasm
of the cells but not in the nuclei (Figure 4B,C). To test the
hypothesis that 9 interaction with DNMT is a crucial factor
in the ultimate up-regulation of RASSF1A, the cell samples
were stained with antibodies for the DNMT isoforms known
to shuttle between the nucleus and the cytoplasm, i.e.,
DNMT3a and DNMT3b.18,19 Interestingly, while DNMT3b
(red) was present in both the cytoplasm and nuclei of the
control cells (Figure 4D), after 1 h there was a population of
treated cells that had DNMT3b only in the cytoplasm
(Figure 4B, arrows). By 6 h this effect was very pronounced,
with the majority of treated cells having DNMT3b only in
the cytoplasm (Figure 4C). No such corresponding effect
was seen with DNMT3a (data not shown), suggesting that
this effect was not global inhibition of nuclear access. This
cytoplasmic sequestering could be indicative of a mode of
action for 9, but further studies are required.
Figure 2. Mahanine analogues induce RASSF1A expression and
down-regulate cyclin D1. PC-3 cells were treated 72 h with either
5 μM compound (mahanine (M), 9) or 15 μM compound (5, 6, 7, 8,
10, DMSO (C)) before RNA extraction and RT-PCR amplification.
Preliminary in Vivo Studies Indicated a Large Therapeutic
Index and Significant Volume Reduction of Human Prostate
Xenograft Tumors in Mice Treated with the Lead Compound
9. By use of the acute oral toxicity (AOT) up-and-down
procedure, no toxicity was observed for compound 9 in wild-
type Balb/c mice dosed up to 550 mg/kg.20 This provided an
estimated therapeutic index of >55 at a starting efficacy dose
of 10 mg/kg. Athymic Balb/c nude mice with human prostate
tumor xenografts were dosed via intraperitoneal injection
with 10 mg/kg 9 once a day every other day for 28 days.
Control mice were dosed with vehicle alone. Compound 9
reduced tumor volume by about 40% compared to control

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2379
over 28 days (Figure 5A), with statistically significant T/C
ratios21 for all but two time points (Figure 5B). Although
dose-escalating studies have yet to be performed to find the
optimum dose and schedule, these results demonstrate the
potential use of this compound for reducing human prostate
tumor volume.
Conclusions
Over the past 5 years, epigenetic regulation in cancer has
quickly risen to the forefront of cancer research.22,23 By
Figure 5. Treatment with compound 9 reduces the volume of
human prostate xenograft tumors. (A) Male athymic Balb/c nude
mice were injected with PC-3 cells and the resulting tumors
allowed to grow for a week before dosing ip once a day every
other day with 10 mg/kg compound 9 or DMSO control in
1:1 PEG/PBS. Data are presented as the mean ( SEM (n = 4).
(B) T/C is defined as the relative sizes of treated (t) and control
(c) tumors and is calculated using T/C = (t RTV)/(c RTV),
where RTV = Vt/V0. V0 represents the volume at time zero, and
Vt is the volume at the given time point. T/C > 0.60 is defined as
null, and T/C < 0.60 is defined as effective (statistically signif-
icant).
Figure 3. Mahanine analogues that up-regulate RASSF1A also
inhibit DNA methyltransferase activity. Black bars: PC-3 cells were
treated 72 h with either 2 μM compound (mahanine, 9) or 15 μM
compound (5, 6, 7, 8, 10) and then assayed for DNMT activity using
the DNMT activity kit from Epigentek. Data are the mean of three
independent experiments ( SEM. Gray bars: Densitometry from
RT-PCR blot is shown where the amount of RASSF1A mRNA
re-expressed by mahanine treatment is normalized to 100%.
Figure 4. Fluorescent analogue 9 is seen exclusively in the cytoplasm of PC-3 cells and appears to sequester DNMT3b in the cytoplasm. (A)
Fluorescent spectrum of 9 is shown. (B) PC-3 cells were treated with a 5 μM solution of 9 for 1 h before being fixed and stained with propidium
iodide and DNMT3b antibody. (C) Cells were treated with a 5 μM solution of 9 for 6 h before fixation. (D) Cells were treated with a 5 μM
solution of DMSO for 6 h before fixation. The frames in each set of images are as follows, from left to right and from top to bottom: propidium
iodide (blue); clear field; DNA methyltransferase 3b (DNMT3b) (red); compound 9 (green) or vehicle; and merged. Compound 9 was excited at
725 nm with a multiphoton laser and imaged with a 500-550 nm filter. Arrows indicate the subcellular localization of DNMT3b.

2380 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Sheikh et al.
synthesizing and testing analogues of the natural product
mahanine, we have found a compound, 9, that possesses
several advantages as a possible epigenetic prostate cancer
therapeutic. Compound 9 inhibited human prostate cancer
cell proliferation at 1.5 μM while avoiding cytotoxic effects
seen with mahanine treatment, and it fluoresced, providing
visual evidence of the compound entering human prostate
cancer cells. Like mahanine, compound 9 up-regulated tumor
suppressor protein RASSF1A and down-regulated cyclin D1.
It also inhibited DNMT activity, proposed to be the event
leading to the restoration of RASSF1A expression. Indeed,
depletion of one of the three active DNMT isoforms,
DNMT3b, has been shown to cause potent antiproliferation
and restoration of RASSF1A expression in cancer cell lines.11
Preliminary data suggest that 9 may have some effect on
DNMT3b, though further investigation remains to determine
the precise mechanism of action. Regardless of DNMT iso-
form specificity, we predict 9 will possess an improved ther-
apeutic index compared to current DNMT inhibitors, since it
isthe irreversible DNMT-bindingaction ofcurrent nucleoside
DNMT inhibitors that is associated with significant cytotoxi-
city.22 Preliminary in vivo studies of the RASSF1A up-
regulator 9 lend credence to this prediction and justify study-
ing compound 9 in more advanced preclinical models of
prostate cancer.
4 (0.50 g, 1.4 mmol) was dissolved in 1,3-dicholorobenzene (1,3-
DCB) (2 mL). The solution was purged with nitrogen and kept
under a nitrogen atmosphere. TEA triphenylphosphine (0.94 g,
3.6 mmol) was added, and the mixture was refluxed for 48 h. The
1,3-DCB was removed under vacuum reduced pressure, and the
residue was taken up in CHCl3, impregnated on silica gel, and
purified via column chromatography (3:1 hexanes/EtOAc) to
yield both cyclization products 5 and 6 as solids (92%). Com-
pound 5: Rf = 0.56; yellowish solid (0.22 g); mp 167-168 °C. 1H
NMR (400 MHz, CDCl3): δ 7.84 (d, J = 8.4 Hz, 1H), 7.76 (s,
1H), 7.73 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 7.6 Hz, 2H), 7.41 (t, J
= 7.6 Hz, 2H), 7.34 (m, 1H), 6.93 (d, J = 2.0 Hz, 1H), 6.91 (d, J
= 8.4 Hz, 1H), 6.83 (dd, JS = 2.0 Hz, JL = 8.4 Hz, 1H), 5.18 (s,
2H), 3.90 (s, 3H), 2.44 (s, 3H). 13C NMR (100.6 MHz, CDCl3): δ
158.21, 154.40, 141.09, 140.27, 137.76, 128.46, 127.71, 127.29,
120.33, 117.90, 117.67, 116.88, 107.96, 107.69, 106.14, 95.00,
71.33, 55.60, 10.12. Compound 6: Rf = 0.45; light-tan solid (0.20
g); mp 250-251 °C. 1H NMR (400 MHz, CDCl3): δ 7.80 (d, J =
8.4 Hz, 1H), 7.76 (s, 1H), 7.72 (s, 1H), 7.49 (d, J = 7.2 Hz, 2H),
7.41 (t, J = 7.2 Hz, 2H), 7.33 (m, 1H), 6.89 (s, 1H), 6.87 (d, J =
2.4 Hz, 1H), 6.81 (dd, JS = 2.4 Hz, JL = 8.4 Hz, 1H), 5.16 (s,
2H), 3.88 (s, 3H), 2.42 (s, 3H). 13C NMR (100.6 MHz, DMSO-
d6): δ 157.25, 154.53, 140.69, 139.13, 137.56, 128.33, 127.51,
127.11, 120.38, 119.64, 117.34, 116.30, 115.66, 107.05, 94.47,
94.26, 69.20, 55.10, 16.59.
7-Methoxy-1-methyl-9H-carbazol-2-ol (7). Compound
5
(0.33 g, 1.0 mmol) was dissolved in MeOH (15 mL). The solution
was purged with nitrogen and kept under a nitrogen atmo-
sphere. Ammonium formate (0.65 g, 10.3 mmol) was added with
a catalytic amount of 5% Pd/C. After the mixture was stirred for
3 h, the solution was filtered through a plug of Celite, washing
with MeOH and EtOAc. The solvent was removed under
reduced pressure and the resulting residue taken up in EtOAc
(20 mL) and washed with H2O (3 ꢀ 20 mL). The organic layer
was dried over MgSO4, and the solvent was removed under
reduced pressure. Column chromatography (3:1 hexanes/
EtOAc) provided 7 as a yellowish tan solid (0.22 g, 94%); mp
200-202 °C (dec). 1H NMR (400 MHz, DMSO-d6): δ 10.73 (s,
1H), 9.08 (s, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 8.4 Hz,
1H), 6.89 (d, J = 2.4 Hz, 1H), 6.69 (dd, JS = 2.4 Hz, JL = 8.4 Hz,
1H), 6.65 (d, J = 8.4 Hz, 1H), 3.80 (s, 3H), 2.29 (s, 3H). 13C NMR
(100.6 MHz, DMSO-d6): δ 157.05, 152.55, 141.03, 119.47,
117.29, 116.47, 115.10, 113.60, 107.93, 106.61, 104.74, 94.58,
55.19, 10.22.
Experimental Section
All reagents and solvents were purchased from commercial
suppliers and used as received unless noted otherwise. Flash
column chromatography separations were done on a Biotage
SP1 system monitoring at 254 and 310 nm. NMR spectra were
recorded on a Varian 400 spectrometer at 22.5 °C, operating at
400 MHz for 1H and 100 MHz for 13C NMR. The chemical shifts
are expressed in ppm downfield from TMS as an internal
standard (CDCl3 or DMSO-d6 solution). Melting points were
determined in open capillary tubes on an Electrothermal melting
point apparatus and are uncorrected. Compound purity of at
least 95% was established by combustion analysis and confirmed
by HPLC and HRMS when deemed appropriate. Elemental
analyses were performed by Atlantic Microlab, Inc. (Norcross,
GA). HPLC traces were recorded with a Shimadzu LC20-series
LCMS. Quadrapole-time-of-flight tandem mass spectrometry
was performed on a QSTAR Elite (Applied Biosystems).
40-(Benzyloxy)-4-methoxy-30-methyl-2-nitrobiphenyl (4). Un-
der vacuum reduced pressure, PEG 4600 (29.8 g, 6.48 mmol) was
heated at 120 °C for 2 h, then flushed with nitrogen. Compound
3 (5.00 g, 15.4 mmol), 4-bromo-3-nitroanisole (2.39 g, 10.3
mmol), K2CO3 (2.84 g, 20.6 mmol), and Pd(OAc)2 (0.12 g,
0.51 mmol) were added in this order, and the mixture was stirred
at 120 °C for 48 h. The mixture was cooled and the resulting solid
was dissolved in the minimum amount of DCM required for
complete dissolution. Ether was slowly added to the mixture
until the PEG precipitated out of solution, and the resulting
suspension was filtered. This process was repeated twice with the
residual PEG. The solvent was removed under reduced pressure
and the resulting yellow residue was purified by column chro-
matography (19:1 hexanes/EtOAc) to give 4 as bright-yellow
crystals (1.86 g, 52%); mp 119 °C. 1H NMR (400 MHz, CDCl3):
δ 7.46 (d, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.35-7.31
(m, 3H), 7.12 (dd, JS = 2.8 Hz, JL = 8.4 Hz, 1H), 7.10 (d, J =
2.0 Hz, 1H), 7.06 (dd, JS = 2.0 Hz, JL = 8.4 Hz, 1H), 6.90 (d,
J = 8.0 Hz, 1H), 5.11 (s, 2H), 3.89 (s, 3H), 2.30 (s, 3H). 13C
NMR (100.6 MHz, CDCl3): δ 158.90, 156.98, 149.87, 137.44,
132.99, 130.57, 129.47, 128.76, 128.60, 128.05, 127.66, 127.36,
126.58, 118.79, 111.53, 109.03, 70.10, 56.11, 16.72.
7-Methoxy-3-methyl-9H-carbazol-2-ol (8). The above proce-
dure was followed using compound 6 (0.38 g, 1.2 mmol) as the
substrate. The product was purified by column chromatography
(3:1 hexanes/EtOAc) to yield 8 as an off-whitechalky solid (0.26 g,
1
97%); mp 238-240 °C (dec). H NMR (400 MHz, DMSO-d6):
δ 10.66 (s, 1H), 9.18 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.60 (s, 1H),
6.84 (d, J = 2.4 Hz, 1H), 6.82 (s, 1H), 6.66 (dd, JS = 2.4 Hz, JL =
8.4 Hz, 1H), 3.79 (s, 3H), 2.22 (s, 3H). 13C NMR (100.6 MHz,
DMSO-d6): δ 156.96, 153.45, 140.54, 139.45, 120.27, 119.23,
116.68, 115.79, 114.97, 106.58, 96.02, 94.42, 55.10, 16.40.
7-Methoxy-1-methyl-9H-carbazol-2-yl 5-(dimethylamino)na-
phthalene-1-sulfonate (9). Compound 7 (0.15 g, 0.66 mmol)
was dissolved in DCM. Then TEA (0.18 mL, 1.3 mmol) was
added, and reaction mixture was stirred for 10 min. Dansyl
chloride (0.18 g, 0.66 mmol) was added, and the reaction
mixture was stirred for 3 h. Solvent was removed under reduced
pressure, the resulting residue impregnated onto silica gel,
and the product was purified by column chromatography (3:1
hexanes/EtOAc) to yield 9 as a fluffy yellow solid (0.30 g, 99%);
mp 174 °C. 1H NMR (400 MHz, CDCl3): δ 8.62 (d, J = 8.4 Hz,
1H), 8.55 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.95 (s,
1H), 7.75 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.45 (m,
2H), 7.24 (m, 1H), 6.89 (s, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.33 (d,
J = 8.4 Hz, 1H), 3.85 (s, 3H), 2.91 (s, 6H), 2.40 (s, 3H). 13C
NMR (100.6 MHz, CDCl3): δ 159.02, 151.82, 145.35, 141.32,
139.32, 132.00, 131.77, 130.91, 130.14, 129.83, 128.93, 123.02,
2-(Benzyloxy)-7-methoxy-1-methyl-9H-carbazole (5) and 2-
(Benzyloxy)-7-methoxy-3-methyl-9H-carbazole (6). Compound

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2381
121.58, 121.08, 119.74, 117.02, 116.81, 115.59, 113.87, 113.57,
108.68, 94.78, 55.60, 45.43, 11.30.
Multiphoton Laser Imaging. After incubation of slides with a
fibronectin/cortactin solution for 30 min, PC-3 cells were plated
onto the slides and incubated overnight. The cells were then
treated with a 5 μM solution of compound 9 or DMSO control
and incubated for 1-6 h before washing with PBS. The slides
were fixed with a 4% formaldehyde solution, washed with PBS,
then treated with propidium iodide (nuclear localization) and
DNMT3a or DNMT3b. The compound was excited at 725 nm
with a multiphoton laser and imaged with a 500-550 nm filter.
Animals. Balb/c mice and athymic Balb/c nude mice were
purchased from the National Cancer Institute (NCI). Animals
were housed 4-6 per cage with microisolater tops and provided
food (Furina mice chow) and water ad libitum. The light cycle
was regulated automatically (12 h light/dark cycle), and tem-
perature was maintained at 23 ( 1 °C. All animals were allowed
to acclimate to this environment for 1 week prior to experi-
mental manipulations. The Georgetown University Animal
Care and Use Committee approved all animal studies in accor-
dance with the guidelines adopted by the National Institutes of
Health.
7-Methoxy-3-methyl-9H-carbazol-2-yl 5-(dimethylamino)na-
phthalene-1-sulfonate (10). The above procedure was followed
using compound 8 (0.14 g, 0.60 mmol) as the substrate. The
product was purified by column chromatography (3:1 hexanes/
EtOAc), providing 10 as a yellow solid (0.24 g, 86%); mp 242 °C.
1H NMR (400 MHz, DMSO-d6): δ 10.96 (s, 1H), 8.66 (d, J = 8.4
Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.89
(d, J = 8.4 Hz, 1H), 7.85 (s, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.66
(t, J = 8.0 Hz, 1H), 7.37 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 2.4 Hz,
1H), 6.74 (dd, JS = 2.4 Hz, JL = 8.4 Hz, 1H), 6.58 (s, 1H), 3.80 (s,
3H), 2.90 (s, 6H), 2.23 (s, 3H). 13C NMR (100.6MHz, DMSO-d6):
δ 158.52, 151.58, 145.06, 141.71, 137.73, 131.83, 131.26, 130.60,
129.18, 129.06, 128.99, 123.46, 121.46, 120.90, 120.81, 120.72,
118.44, 115.54, 115.07, 107.98, 103.34, 94.29, 55.07, 44.97, 16.39.
Cell Line and Cell Growth Assay. Human prostate cancer cell
line PC-3 was procured from the American Type Cell Culture
Collection. Cells were grown in IMEM without phenol red
(Invitrogen) with 10% fetal bovine serum, 2 mM glutamine,
100 U/mL penicillin G sodium, and 100 μg/mL streptomycin
sulfate (Sigma) in the presence of 5% CO2 at 37 °C. For the cell
growth experiment, PC-3 cells were seeded in six-well plates in
triplicate with an initial density of 1.5 ꢀ 104 cells per well.
Twenty-four hours after seeding, the attached cells were treated
with vehicle (DMSO) or 1, 5, 15, 30, and 60 μM compound.
After another period of 24 h, the cells were washed with 1 ꢀ PBS,
trypsinized, and resuspended in complete growth medium.
Trypan blue (0.4%) was added to the cell suspension, and both
live and dead cells were counted using a hemocytometer.
BrdU Labeling. For the BrdU experiment, PC-3 cells were
seeded in 96-well plates at a density of 2 ꢀ 104 cells/well, treated
with 5 μM compound for 24 h and assayed for BrdU using the
cell proliferation ELISA BrdU (chemiluminescence) (Roche
Diagnostics) kit according to the manufacturer’s protocol.
Light emission was measured using a microplate luminometer
(Harta Instruments, Inc.).
Reverse Transcription Polymerase Chain Reaction (RT-PCR).
From PC-3 cells incubated 3 days with either 15 μM compound
(5-10) or 5 μM (mahanine, 9), RNA was extracted with TRIzol
solution (Invitrogen) and genes of interest were amplified using
500 ng of total RNA reverse-transcribed to cDNA using a
Superscript II kit (Invitrogen) with random hexamers. Hu-
man-specific primers were designed using the Primer Quest
program and purchased from Integrated DNA Technologies,
Inc. Their sequences and product band sizes are the following:
cyclin D1 forward primer 50-CACACGGACTACAGGG-
GAGT-30, cyclin D1 reverse primer 50-AGGAAGCGGTC-
CAGGTAGTT-30 (475 bp), and GAPDH forward primer: 50-
CCA CCCATGGCAAATTCCATGGCA-30. GAPDH reverse
primer: 50-TCTAGACGGCAG GTCAGGTCCACC-30 (598
bp). PCRs were initiated at 94 °C for 2 min, followed by 28
cycles of 94 °C for 1 min, 1 min at annealing temperature, 72 °C
for 1 min, and final extension at 72 °C for 5 min. The annealing
temperature for cyclin D1 and GAPDH was 60 °C. Primers
and PCR conditions for RASSF1A were used as previously
described.4 After amplification, PCR products were separated
on 1.5% agarose gels and visualized by ethidium bromide
fluorescence using the Fuji LAS-1000 Imager. Images were
captured and imported to Adobe Photoshop. Band intensities
were quantified by using ImageJ software (NIH).
Cell Culture for Xenograft. PC-3 cell line (ATCC, Manassas,
VA) was cultured in RPMI-1640 with L-glutamine (Mediatech
Inc., Herdon, VA) containing 5% fetal bovine serum (FBS),
2.5 mM L-glutamine at 37 °C with 5% CO2.
Xenograft Study. Male athymic Balb/c nude mice (18-22 g)
were injected with 3 ꢀ 106 (0.3 mL) human prostate cancer cells
(PC-3). The human prostate cancer cells were injected in the
subcutaneous tissue of the right axillary region of the mice. One
week after the injection, the mice were randomly sorted into two
groups with four mice per group. A stock solution of compound
9 was obtained by dissolving 1 mg of compound in 1 μL of
DMSO. The stock was added to polyethylene glycol 400 (PEG)
(Hampton) and PBS in a 1:1 ratio. The test concentrations were
obtained by diluting with PEG/PBS. The tumor-bearing mice
received an intraperitoneal injection (ip) with either 10 mg/kg 9
or vehicle control once every other day for 28 days. At the same
time, the tumor size of each mouse was measured by caliper and
calculated by the formula length ꢀ width ꢀ height/2.
Statistical Analyses. Cell data were derived from at least three
independent experiments, and animal data were derived from
the xenograft study described above. Statistical analyses were
conducted using Prism 4 GraphPad software, referencing Li and
Yuan21 for animal evaluation methods. Values are presented as
the mean ( SEM.
Acknowledgment. We thank the Lombardi shared re-
sources for technical support and the Georgetown Drug
Discovery Program and Lombardi Comprehensive Cancer
Center for financial assistance. We also thank Dr. Samir
Bhattacharya and Dr. Bikas C. Pal of the Indian Institute of
Chemical Biology, Kolkata, India, for providing us with
purified mahanine. A patent application has been filed by
Georgetown University on the behalf of the inventors that are
listed as authors in this article.
Supporting Information Available: NCI data for compound 9
and elemental analysis and HRMS data for compounds 5-10.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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according to manufacturer’s protocol (nuclear extraction kit,
Epigentek). DNMT activity was measured using an EpiQuik
DNA methyltransferase activity assay kit (Epigentek). Results
were expressed as percent DNMT activity compared to the
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