Synthesis and biological evaluation of cepharadiones A and B and related dioxoaporphines

Article abstract of DOI:10.1016/j.bmc.2007.06.035

Described herein is the first total synthesis and structural confirmation of cepharadione A, a naturally occurring DNA damaging agent. Also reported is the synthesis of cepharadione B, a closely related natural product, as well as the biological evaluatio

Full text of DOI:10.1016/j.bmc.2007.06.035

Bioorganic & Medicinal Chemistry 15 (2007) 6119–6125
Synthesis and biological evaluation of cepharadiones A and B
and related dioxoaporphines
Mark A. Elban,a Jean C. Chapuis,b Mei Lia and Sidney M. Hechta,b,
*
aDepartments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22904, USA
bCenter for BioEnergetics, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Received 6 May 2007; revised 11 June 2007; accepted 14 June 2007
Available online 20 June 2007
Abstract—Described herein is the first total synthesis and structural confirmation of cepharadione A, a naturally occurring DNA
damaging agent. Also reported is the synthesis of cepharadione B, a closely related natural product, as well as the biological eval-
uation of both natural products. Finally, the preparation and biological evaluation of novel dioxoaporphine analogues is described.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
none (5)7 which have been found to be DNA
topoisomerase II and topoisomerase I inhibitors, respec-
tively. Further, the oxoaporphine alkaloids oxophoe-
bine (6) and liriodenine (7) were found to be cytotoxic
to yeast deficient in DNA recombinational repair, while
lauterine (8) and 10-hydroxyliriodenine (9) inhibited the
DNA unknotting activity of topoisomerase II.8 The
Cepharadione A (2) (Fig. 1), a known 4,5-dioxoapor-
phine alkaloid, has been isolated from a number of plant
sources.1 Although originally isolated from the callus
tissue of Stephania cepharantha more than 30 years
ago,2 neither the synthesis nor any report of the biolog-
ical activity of this compound had been described. Re-
cently, we isolated cepharadione
A from Piper
caninum3 using bioassay-guided fractionation based on
DNA damaging activity in a sensitive yeast assay. Spe-
cifically, cepharadione A potently inhibited the growth
of a yeast strain lacking RAD52 (IC50 50.2 nM), which
is associated with the repair of double-strand DNA
breaks.3 In contrast with the dearth of biological activity
reported for cepharadione A, other members of the
(oxo) aporphine family of alkaloids have demonstrated
interesting biological activities. Cepharadione B (1) has
been shown to be cytotoxic to a variety of cell lines
including colon and breast cancer cell lines.4 Artabotrine
(3), a dioxoaporphine identical with cepharadione A ex-
cept for an N–OMe instead of N–Me substituent, was
also cytotoxic to repair deficient yeast (IC12 1.2 lg/
mL), as well as cultured wild-type and camptothecin-
resistant cultured P-388 cells (IC50 1.59 and 1.12 lg/
mL, respectively).5 Other bioactive oxoaporphine alka-
loid family members include dicentrine (4)6 and dicentri-
O
4
R1
R2
2
1
3
O
O
O
5
B
A
D
N
H
N
Me
R3
6
7
C
11
8
MeO
OMe
(4) dicentrine
(1) cepharadione B R1=R2= OMe, R3=Me
(2) cepharadione A R1R2 = OCH2O, R3=Me
(3) artabotrine R1R2 = OCH2O, R3=OMe
R1
R2
N
R3
O
R4
R5
(5) dicentrinone R1=H, R2R3=OCH2O, R4=R5=OMe
(6) oxophoebine R1=R2=R3=OMe, R4R5=OCH2O
(7) liriodenine R1=R4=R5=H, R2R3=OCH2O
(8) lauterine R1=R5=H, R2R3=OCH2O, R4=OMe
(9) 10-hydroxyliriodenine R1=R5=H, R2R3=OCH2O, R4=OH
Keywords: Cepharadiones A and B; Oxoaporphines; Suzuki coupling;
Cytotoxicity.
*
Corresponding author. Tel.: +1 434 924 3906; e-mail: smh9u@
virginia.edu
Figure 1. Cepharadiones A and B, and structurally related molecules.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.035

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M. A. Elban et al. / Bioorg. Med. Chem. 15 (2007) 6119–6125
methylenedioxy group could be important for the puta-
tive DNA damaging properties of 6 and 7, as structur-
ally related compounds lacking this feature were much
less cytotoxic (for tables comparing biological activities,
see Ref. 8b).
2. Results and discussion
The synthesis of cepharadione B (Schemes 1 and 2) be-
gan with commercially available (2-bromophenyl)acetic
acid, which was treated with thionyl chloride to form
the acid chloride and then with methylamine under ba-
sic conditions to afford N-methylamide 11 in 76% yield.
Key intermediate 10 was then obtained in quantitative
yield by Suzuki coupling15 of 11 with commercially
available 2,3-dimethoxyphenylboronic acid.16 It may
be noted that pure 10 was obtained in quantitative
yield, in contrast with an earlier finding14 in which tri-
phenylphosphine oxide hindered the purification of a
series of related compounds, resulting in poor yields.
The C and B rings were then formed sequentially with
(COCl)2 and SnCl4 according to the method of Suau
and co-workers14 to give cepharadione B in 15% yield.
In order to synthesize cepharadione A (Scheme 2)
intermediate 10 was deprotected using B-bromocate-
chol borane and BF3ÆEt2O17 to afford the unstable
diphenol 12 in 96% yield. Formation of the corre-
sponding benzodioxole 13 was accomplished in 42%
yield by treatment of diphenol 12 with BrCH2Cl and
KOH in EtOH at reflux.18 Completion of the synthesis
of cepharadione A (2) was accomplished using the
same ring closure procedure described above14 in 33%
yield. The material obtained was identical in all re-
spects to that isolated previously.1,3
Given these interesting biological results and the lack of
a synthetic route to cepharadione A (2) we focused on
this synthetic target as well as the structurally related
cepharadione B (1). Previous synthetic efforts have re-
sulted in preparation of a number of related natural
products by a variety of methods. The first reported syn-
theses of oxoaporphines involved the oxidation of the B
ring from the appropriate precursors.9 Later synthetic
efforts by Castedo et al. involved forming the C ring
as the last step. The initial synthesis of pontevedrine
was accomplished under photochemical conditions;10 la-
ter, using a radical ring closing methodology, ponteve-
drine and other 5-oxoaporphines were prepared.11
A
quite different approach employing benzynes also pro-
vided access to several 4,5-dioxoaporphines.12 Subse-
quently, Suau and co-workers used photochemistry to
form the B ring after forming rings A, C, and D.4
Perhaps the most versatile and efficient synthesis of this
class of natural products to date has been realized via
the sequential formation of the C and B rings using
(COCl)2 and SnCl4.13 This synthetic approach seemed
the best option for the synthesis of cepharadione A,
but the previously reported approach to the advanced
intermediate 10 seemed cumbersome. During our initial
synthetic studies on this class of molecules, Suau and co-
workers reported the use of a Suzuki coupling to pre-
pare a biaryl ester, which was elaborated to give the de-
sired dioxoaporphines.14 We describe herein the
application of this approach to give the advanced biaryl
amide 10 directly (Scheme 1) en route to cepharadione
B. In addition, cepharadione A has been prepared for
the first time by elaboration of the same advanced inter-
mediate. Further, we describe the screening of several
other boronic acids under the Suzuki coupling condi-
tions followed by elaboration to the corresponding diox-
oaporphines. The cytotoxic activities of these
compounds have been studied and are discussed.
In order to probe the scope of the Suzuki coupling to
give advanced biaryl amide intermediates, several boro-
nic acids were studied (Scheme 3). Gratifyingly, the 2,3-
dimethyl, 2-ethoxy, and 2-isopropoxy boronic acids all
underwent coupling to give the biaryl species 14a, c, d
in excellent yields. The low yield associated with the
preparation of 14b was due to the contamination of
the initially isolated product with triphenylphosphine
oxide. A separate purification step was required to re-
move most of this contaminant, which resulted in the
loss of significant desired material.
The subsequent formation of the corresponding dioxoa-
porphines was attempted by treating 14a–d with
(COCl)2 and SnCl4 according to the method of Suau.14
The ring closure to give 15a proceeded in low yield, pos-
sibly due to the change in electronic character of the A
ring. The methoxy and ethoxy derivatives proceeded in
good yield to give the desired dioxoaporphines 15b
and c, respectively. The final attempt using the isoprop-
oxy biaryl intermediate 14d failed to give any product,
possibly due to the increased steric bulk preventing the
ring system from aligning properly for ring closure.
O
MeO
MeO
O
MeO
MeO
N
NH
We have previously evaluated cepharadione A3 in a
yeast strain (RS321 NpRAD52) which harbors a plas-
mid containing a RAD52 recombinant gene under the
control of a galactose promoter. When this strain was
grown on glucose, cepharadione A (2) inhibited the
growth of this yeast strain with an IC50 value of
50.2 nM. When the strain was grown on galactose
(i.e., under DNA repair proficient conditions), the IC50
value for 2 was 293 nM. The synthetic sample prepared
as described here produced comparable results in this as-
O
10
1
Br
MeO
MeO
NH
+
O
11
B(OH)2
Scheme 1. Retrosynthetic analysis of cepharadione B (1).

M. A. Elban et al. / Bioorg. Med. Chem. 15 (2007) 6119–6125
6121
O
1) SOCl2, benzene,
reflux
2) Et3N, MeNH2,
acetone, H2O
MeO
MeO
MeO
O
Br
Pd(PPh3)4,
NaHCO3, toluene,
EtOH, reflux
O
NH
O
(COCl)2, SnCl4,
CH2Cl2, 0oC-RT
Br
N
MeO
OH
NH
OMe
(15%)
(76%)
O
11
OMe
1
10
B(OH)2
(100%)
O
O
MeO
MeO
HO
HO
O
O
O
B
-bromocatecholborane,
(COCl)2, SnCl4,
KOH, BrCH2Cl,
EtOH, reflux
.
CH2Cl2, -30oC-RT
N
BF3 Et2O, CH2Cl2
O
NH
NH
NH
O
(96%)
(42%)
(33%)
O
O
2
13
10
12
Scheme 2. Syntheses of cepharadione A (2) and B (1).
O
O
Pd(PPh3)4,
R1
R2
R1
R2
(COCl)2, SnCl4,
CH2Cl2, 60oC
O
NH
NaHCO3, toluene,
Br
EtOH, reflux
R1
N
NH
R2
B(OH)2
11
O
14a R1=R2=CH3 (96%)
15a (13%)
15b (76%)
15c (75%)
14b R1=H, R2=OMe (61%)
14c R1=H, R2=OEt (96%)
14d R1=h, R2=OiPr (100%)
Scheme 3. Suzuki couplings and subsequent cyclization.
say. In contrast, cepharadione B (1) was essentially with-
out activity in the yeast assay.
Table 1. Human cancer cell line inhibition values (GI50) expressed in
lg/mL for 1, 2, 15a–c
Cancer cell linea
1
2
15a
15b
15c
Cepharadione B as well as 2-demethoxycepharadione B
have been tested previously using several cell lines; the
resulting IC50 values ranged from 1.9 to 6.6 lg/mL.4 In
light of this promising data, we tested cepharadiones
A and B as well as 15a–c against a different series of can-
cer cell lines (Table 1). Comparing 1 and 2 against the
cell lines tested revealed a similar trend to that of the
DNA damaging yeast assay; 2 was generally consider-
ably more active than 1. The dimethylated species 15a
also proved to be cytotoxic against several of the cell
lines tested with toxicities similar to that of cepharadi-
one A. Perhaps the most interesting biological result in-
volved the cytotoxicity of 2-demethoxycepharadione B
(15b), which was potently cytotoxic toward all six cell
lines tested with GI50 values ranging from 0.42 to
1.6 lg/mL. In particular, 15b was the only compound
tested which possessed significant activity against
BXPC-3, the pancreatic adenocarcinoma cell line. When
the methyl ether was replaced with an ethyl ether (15c),
the cytotoxicity was greatly reduced or eliminated, a re-
sult somewhat incongruous with previous findings.4
BXPC-3
MCF-7
SF-268
>10
>10
22.8
>10
>10
36.1
>10
6.3
>10
8.4
1.6
>10
>10
>10
>10
>10
>10
0.66
0.42
0.46
0.82
1.3
2.9
2.5
0.70
2.3
NCI-H460
KM20L2
DU-145
17.3
4.3
4.2
>10
a Cancer type: BXPC-3 (pancreatic adenocarcinoma); MCF-7 (breast
carcinoma); SF-268 (CNS glioblastoma); NCI-H460 (lung large cell);
KM20L2 (colon adenocarcinoma); DU-145 (prostate carcinoma).
ence of the methylenedioxy functionality enhances DNA
damage in some fashion. This observation, while sur-
prising, is entirely consistent with observations made
by Gunatilaka et al.8b for other aporphine alkaloids that
damage DNA in the same yeast assay system. Further,
these results demonstrate that substitution at C-2 of
the dioxoaporphines is not absolutely required for po-
tent biological activity. Finally, the ability of the dioxoa-
porphines to tolerate increased steric bulk at C-1 while
maintaining potent growth inhibition seems to be rather
limited.
These results indicate that the replacement of the 1,2-di-
methyl ethers with a methylenedioxy group (1 vs 2) in-
creases the cytotoxic potency of this dioxoaporphine.
Since the assay depends explicitly on differences in
DNA repair capability between two yeast strains that
are otherwise isogenic, it is logical to think that the pres-
3. Conclusions
In summary, we have described the first total synthesis
of cepharadione A, permitting confirmation of the as-
signed structure as well as verification of its biological

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M. A. Elban et al. / Bioorg. Med. Chem. 15 (2007) 6119–6125
activity. The synthesis was accomplished via the key bia-
ryl amide intermediate 10, which was prepared utilizing
a Suzuki coupling. The preparation of additional biaryl
intermediates was also accomplished using the same
conditions, further demonstrating the utility of this ap-
proach for preparing congeners of the dioxoaporphine
family of natural products. Finally, the biological activ-
ity of several new dioxoaporphine congeners is reported,
further establishing the biological profile of this interest-
ing class of natural products.
spectrum (FAB) m/z 228.0025 (M+H)+ (C9H11NOBr re-
quires 228.0024).
4.2.2. 2-(20,30-Dimethoxybiphenyl-2-yl)-N-methylaceta-
mide (10). To a solution containing 1.00 g (4.38 mmol)
of 11 in 20 mL of toluene were added 0.30 g (0.26 mmol)
of Pd(PPh3)4 and 8.76 mL (17.5 mmol) of 2.0 M aq
Na2CO3. The reaction mixture was heated to reflux
while degassing with N2. At this point a degassed solu-
tion containing 1.59 g (8.77 mmol) of 2,3-dim-
ethoxyphenylboronic acid in 10 mL of EtOH was
added. The reaction mixture was maintained at reflux
for 16 h at which time it was cooled and washed with
two 50-mL portions of 2.0 M aq Na2CO3. The organic
phase was dried over anhydrous MgSO4, filtered, and
concentrated under diminished pressure to give a crude
dark oil. Chromatography on a flash silica gel column
(30 · 4 cm) using 1:2 ethyl acetate–hexanes ! 100%
ethyl acetate as eluant gave 10 as a colorless solid: yield
1.25 g (100%); mp 116–118 ꢁC, lit.13b 93–96 ꢁC; silica gel
TLC Rf 0.47 (10% MeOH in CH2Cl2); 1H NMR
(CDCl3) d 2.65 (d, 3H, J = 4.8 Hz), 3.40 (s, 2H), 3.56
(s, 3H), 3.91 (s, 3H), 5.84 (br, 1H), 6.71 (dd, 1H,
J = 7.8 Hz, 1.8 Hz), 6.93 (dd, 1H, J = 8.1 Hz, 1.4 Hz),
7.10 (t, 1H, J = 7.8 Hz) and 7.35 (m, 4H); 13C NMR
(CDCl3) d 26.39, 41.19, 55.77, 60.80, 111.86, 122.75,
124.32, 126.72, 128.07, 129.60, 130.22, 134.07, 135.32,
138.13, 145.75, 152.75 and 171.89; mass spectrum
(FAB) m/z 285.1442 (M+H)+ (C17H20NO3 requires
285.1443).
4. Experimental
4.1. General experimental procedures
Reagents and solvents were of reagent grade and used
without further purification. Anhydrous grade solvents
were purchased from VWR. All reactions involving air
or moisture sensitive reagents or intermediates were per-
formed under a nitrogen or argon atmosphere. Flash
chromatography was performed using Silicycle 40–60
mesh silica gel. Analytical TLC was performed using
0.25 mm EM silica gel 60 F250 plates that were visualized
by irradiation (254 nm) or by staining with Hanessian’s
stain (cerium molybdate). 1H and 13C NMR spectra
were obtained using 300 and 500 MHz Varian instru-
ments. Chemical shifts are reported in parts per million
1
(ppm d) referenced to the residual H resonance of the
solvent (CDCl3, 7.26 ppm). 13C spectra were referenced
to the residual 13C resonance of the solvent (CDCl3,
77.0 ppm). Splitting patterns are designated as follows:
s, singlet; br, broad; d, doublet; dd, doublet of doublets;
t, triplet; q, quartet; m, multiplet. High resolution mass
spectra were obtained at the Michigan State University–
NIH Mass Spectrometry Facility.
4.2.3. 1,2-Dimethoxy-6-methyl-6H-dibenzo[de,g]quino-
line-4,5-dione (cepharadione B) (1).13 To a solution con-
taining 0.08 g (0.28 mmol) of 10 in 1 mL of CH2Cl2 at
0 ꢁC was added 0.84 mL (1.68 mmol) of (COCl)2. The
reaction mixture was stirred for 15 min at which time
1.72 mL (1.68 mmol) of SnCl4 was added. The reaction
mixture was stirred at room temperature for 72 h and
then quenched with 50 mL of 1 N HCl. The mixture
was extracted with three 100-mL portions of ethyl ace-
tate. The combined organic phase was washed with
100 mL of H2O, dried over anhydrous MgSO4, filtered,
and concentrated under diminished pressure to give a
dark red oil. Chromatography on flash silica gel
(30 · 4 cm) using 20% MeOH in 1:1 ethyl acetate–hex-
anes as eluant gave 1 as an orange solid: yield 0.016 g
(18%); mp 265–266 ꢁC, lit.2 267–268 ꢁC; silica gel TLC
4.2. Specific procedures
4.2.1. 2-(2-Bromophenyl)-N-methylacetamide (11). To a
solution containing 1.00 g (4.65 mmol) of (2-bromophe-
nyl)acetic acid in 15 mL of benzene was added 0.83 g
(6.90 mmol) of thionyl chloride. The reaction mixture
was stirred at reflux for 3 h at which time excess solvent
was removed under diminished pressure. The resulting
residue was dissolved in 5 mL of acetone and added
slowly to a solution containing 3.60 mL (4.65 mmol) of
methylamine (40% in water), 1.85 mL (14.0 mmol) of
triethylamine, and 3 mL of H2O chilled to 0 ꢁC. The
reaction mixture was stirred at room temperature for
1 h at which time it was poured into 150 mL of benzene.
The organic phase was washed with 25 mL of brine,
25 mL of 1 N HCl, and 25 mL of brine. The organic
phase was concentrated under diminished pressure to
give a crude oil. The residue was purified by flash chro-
matography on a silica gel column (30 · 2 cm). Elution
with 20% MeOH in CH2Cl2 gave 11 as a colorless solid:
yield 0.80 g (76%); mp 102–104 ꢁC; silica gel TLC Rf
1
Rf 0.51 (10% MeOH in CH2Cl2); H NMR (CDCl3) d
3.84 (s, 3H), 4.10 (s, 3H), 4.11 (s, 3H), 7.48 (s, 1H),
7.65 (m, 2H), 7.85 (m, 1H), 8.23 (s, 1H) and 9.48 (m,
1H); 13C NMR (CDCl3) d 30.48, 56.47, 60.43, 112.54,
114.30, 119.45, 123.60, 124.51, 126.84, 127.58, 127.64,
128.01, 129.03, 131.69, 132.23, 152.79, 154.90, 156.23
and 175.45; mass spectrum (ESI) m/z 321.4 (M)+ (theo-
retical m/z 321.10); mass spectrum (FAB) m/z 322.1080
(M+H)+ (C19H16NO4 requires 322.1079).
4.2.4. 2-(20,30-Dihydroxybiphenyl-2-yl)-N-methylaceta-
mide (12). To a solution containing 0.50 g (1.75 mmol)
of 10 in 10 mL of CH2Cl2 were added 2.89 g
(10.5 mmol) of B-bromocatechol borane and 0.28 mL
(1.75 mmol) of BF3ÆEt2O. The reaction mixture was stir-
red at room temperature for 5 h at which time it was
1
0.38 (1:1 ethyl acetate–hexanes); H NMR (CDCl3) d
2.74 (d, 3H, J = 4.8 Hz), 3.68 (s, 2H), 5.61 (br, 1H),
7.14 (m, 1H), 7.31 (m, 2H) and 7.55 (d, 1H,
J = 7.8 Hz); 13C NMR (CDCl3) d 26.48, 43.78, 124.92,
127.91, 129.02, 131.63, 133.02, 134.82 and 170.11; mass

M. A. Elban et al. / Bioorg. Med. Chem. 15 (2007) 6119–6125
6123
poured into 50 mL of 2 N HCl and extracted with two
50-mL portions of ethyl acetate. The combined organic
phase was dried over anhydrous MgSO4, filtered, and
concentrated under diminished pressure to give a crude
brown oil. Chromatography on flash silica gel
(30 · 4 cm) using 78:20:2 CH2Cl2–MeOH–AcOH as elu-
ant gave 12 as a brown foam which was used immedi-
ately in the subsequent step to avoid decomposition:
yield 0.43 g (96%); silica gel TLC Rf 0.23 (10% MeOH
and 1.75 mL (3.50 mmol) of 2.0 M aq Na2CO3. The
reaction mixture was degassed with N2 for 15 min at
which time a degassed solution containing 1.75 mmol
of the substituted boronic acid in 10 mL of EtOH was
added. The reaction mixture was heated at reflux for
16 h. The cooled reaction mixture was extracted with
two 100-mL portions of ethyl acetate and the organic
extract was washed with two 50-mL portions of 2.0 M
aq Na2CO3. The organic phase was dried over anhy-
drous MgSO4, filtered, and concentrated under dimin-
ished pressure to give a crude oil. The product was
purified by flash chromatography on silica gel to give
the desired product.
1
in 1:1 ethyl acetate–hexanes); H NMR (CDCl3) d 2.56
(d, 3H, J = 4.8 Hz), 3.37 (s, 2H), 6.52 (dd, 1H,
J = 7.5 Hz, 1.5 Hz), 6.77 (t, 1H, J = 7.8 Hz), 6.84 (dd,
1H, J = 8.1 Hz, 1.5 Hz) and 7.24 (m, 4H); 13C NMR
(CDCl3) d 26.41, 40.53, 114.64, 120.56, 121.67, 127.32,
128.00, 128.89, 129.42, 130.75, 133.44, 138.47, 141.32,
145.48 and 173.74; mass spectrum (ESI) m/z 257.3
(M)+ (theoretical m/z 257.11).
4.2.7.1. 2-(20,30-Dimethylbiphenyl-2-yl)-N-methylace-
tamide (14a). Flash chromatography on a silica gel col-
umn
(20 · 2 cm)
using
2:1
hexanes–ethyl
acetate ! 100% ethyl acetate afforded 14a as a colorless
solid: yield 0.212 g (96%); mp 126–128 ꢁC; silica gel TLC
Rf 0.30 (2:1 hexanes–ethyl acetate); 1H NMR (CDCl3) d
1.93 (s, 3H), 2.31 (s, 3H), 2.59 (d, 3H, J = 4.8 Hz), 3.26
(dd, 2H, J = 24.3 Hz, 15.3 Hz), 5.35 (br, 1H), 6.14 (m,
1H), 7.14 (m, 3H) and 7.36 (m, 3H); 13C NMR (CDCl3)
d 16.32, 20.37, 26.11, 40.84, 125.04, 126.94, 127.49,
128.99, 129.92, 130.00, 132.99, 134.16, 136.94, 140.11,
142.22 and 171.18; mass spectrum (FAB) m/z 254.1546
(M+H)+ (C17H20NO requires 254.1545).
4.2.5. 2-(2-Benzo[1,3]dioxol-4-yl-phenyl)-N-methylaceta-
mide (13). To a solution containing 0.10 g (0.39 mmol)
of 12 in 3 mL of EtOH were added 0.03 mL (0.43 mmol)
of CH2BrCl and 0.04 g (0.78 mmol) of KOH. The reac-
tion was maintained at reflux for 48 h at which time the
reaction mixture was concentrated under diminished
pressure. Chromatography on a flash silica gel column
(30 · 4 cm) using 10% MeOH in 1:1 ethyl acetate–hex-
anes as eluant gave 13 as a colorless oil: yield 0.040 g
(42%); silica gel TLC Rf 0.44 (20% MeOH in 1:1 ethyl
acetate–hexanes); 1H NMR (CDCl3) d 2.62 (d, 3H,
J = 4.8 Hz), 3.52 (s, 2H), 5.61 (br, 1H), 5.87 (s, 2H),
6.69 (dd, 1H, J = 7.2 Hz, 1.5 Hz), 6.82 (m, 2H), and
7.34 (m, 4H); 13C NMR (CDCl3) d 26.36, 41.27,
100.64, 108.10, 121.94, 123.11, 127.51, 128.58, 130.50,
130.72, 132.00, 133.39, 136.38, 144.48, 147.16 and
171.57; mass spectrum (ESI) m/z 269.3 (M)+ (theoretical
m/z 269.1); mass spectrum (FAB) m/z 270.1129 (M+H)+
(C16H16NO3 requires 270.1130).
4.2.7.2. 2-(20-Methoxybiphenyl-2-yl)-N-methylaceta-
mide (14b). In order to remove triphenylphosphine oxide
(which comigrated with the product on TLC), the crude
oil was dissolved in cold hexanes, which deposited a
white precipitate. The suspension was filtered through
a silica gel pad and washed with 200 mL of ether. The
filtrate was concentrated under diminished pressure to
afford a crude oil. Flash chromatography on a silica
gel column (20 · 2 cm) using 2:1 hexanes–ethyl ace-
tate ! 100% ethyl acetate afforded 14b as a colorless so-
lid: yield 0.135 g (61%); mp 126–128 ꢁC, lit.13b 125–
126 ꢁC; silica gel TLC Rf 0.13 (2:1 hexanes–ethyl ace-
tate); 1H NMR (CDCl3) d 2.65 (d, 3H, J = 4.8 Hz),
3.42 (s, 2H), 3.72 (s, 3H), 5.56 (br, 1H), 7.00 (m, 2H),
7.11 (m, 1H), 7.23 (m, 1H) and 7.34 (m, 4H); 13C
NMR (CDCl3) d 26.23, 41.15, 55.19, 110.60, 120.67,
127.07, 128.86, 129.08, 129.26, 129.93, 130.76, 130.90,
133.71, 138.85, 155.395 and 171.68; mass spectrum
(FAB) m/z 256.1339 (M+H)+ (C16H18NO2 requires
256.1338).
4.2.6. 1,2-Benzodioxol-6-methyl-6H-dibenzo[de,g]quino-
line-4,5-dione (cepharadione A) (2). To a solution con-
taining 0.14 g (0.54 mmol) of 13 in 5 mL of CH2Cl2 at
ꢀ30 ꢁC was added 0.52 mL (5.94 mmol) of (COCl)2.
The reaction mixture was stirred for 15 min at which
time 0.58 mL (4.95 mmol) of SnCl4 was added. The
reaction mixture was stirred at room temperature for
72 h and then quenched with 50 mL of 1 N HCl. The
mixture was extracted with three 100-mL portions of
ethyl acetate. The combined organic phase was washed
with 100 mL of H2O, dried over anhydrous MgSO4, fil-
tered, and concentrated under diminished pressure to
give a dark red oil. Chromatography on flash silica gel
column (30 · 4 cm) using 5% MeOH in CHCl3 as eluant
gave 2 as an orange solid: yield 0.050 g (33%); mp 338–
340 ꢁC, lit.1a 340–342 ꢁC; silica gel TLC Rf 0.70 (ethyl
acetate); 1H NMR (CDCl3) d 3.85 (s, 3H), 6.46 (s,
2H), 7.52 (s, 1H), 7.66 (m, 2H), 7.89 (m, 1H), 8.14 (s,
1H) and 8.98 (m, 1H); mass spectrum (ESI) m/z 305.0
(M)+ (theoretical m/z 305.3); mass spectrum (FAB) m/z
306.0765 (M+H)+ (C18H12NO4 requires 306.0766).
4.2.7.3.
2-(20-Ethoxybiphenyl-2-yl)-N-methylaceta-
mide (14c). Flash chromatography on a silica gel column
(20 · 2 cm) using 2:1 hexanes–ethyl acetate ! 100%
ethyl acetate afforded 14c as a colorless solid: yield
0.225 g (96%); mp 112–116 ꢁC; silica gel TLC Rf 0.40
1
(2:1 hexanes–ethyl acetate); H NMR (CDCl3) d 1.22
(t, 3H, J = 6.9 Hz), 2.61 (d, 3H, J = 5.1 Hz), 3.44 (d,
2H, J = 4.2 Hz), 3.99 (m, 2H), 5.58 (br, 1H), 6.98 (m,
2H), 7.08 (m, 1H), 7.21 (m, 1H) and 7.32 (m, 4H); 13C
NMR (CDCl3) d 14.55, 26.11, 41.16, 63.84, 112.27,
120.68, 126.87, 127.71, 128.96, 129.74, 130.77, 130.93,
133.68, 138.87, 155.32 and 171.75; mass spectrum
(FAB) m/z 270.1493 (M+H)+ (C17H20NO2 requires
270.1494).
4.2.7. General procedure for preparation of 14a–d. To a
solution containing 0.20 g (0.88 mmol) of 11 in 20 mL
of toluene were added 0.06 g (0.05 mmol) of Pd(PPh3)4

6124
M. A. Elban et al. / Bioorg. Med. Chem. 15 (2007) 6119–6125
4.2.7.4. 2-(20-Isopropoxybiphenyl-2-yl)-N-methylace-
3H, J = 6.9 Hz), 3.76 (s, 3H), 4.34 (q, 2H, J = 7.2 Hz),
7.30 (d, 1H, J = 8.7 Hz), 7.50 (s, 1H), 7.65 (m, 2H),
7.86 (m, 1H), 8.57 (d, 1H, J = 8.7 Hz) and 9.56 (m,
1H); 13C NMR (CDCl3) d 14.79, 30.61, 65.79, 111.02,
115.67, 119.83, 120.87, 124.75, 127.26, 127.32, 128.04,
128.96, 131.60, 131.81, 131.86, 156.76, 164.51, and
175.15; mass spectrum (FAB) m/z 306.1132 (M+H)+
(C19H16NO3 requires 306.1130).
tamide (14d). Flash chromatography on a silica gel col-
umn (20 · 2 cm) using 2:1 hexanes–ethyl acetate !
100% ethyl acetate afforded product as a colorless solid:
yield 0.247 g (100%); mp 114–116 ꢁC; silica gel TLC Rf
1
0.36 (2:1 hexanes–ethyl acetate); H NMR (CDCl3) d
1.05 (d, 3H, J = 6.0 Hz), 1.20 (d, 3H, J = 6.0 Hz), 2.62
(d, 3H, J = 5.1 Hz), 3.42 (s, 2H), 4.40 (dq, 1H, J = 6.0,
6.0 Hz), 5.71 (br, 1H), 7.00 (m, 2H), 7.09 (m, 1H), 7.19
(m, 1H) and 7.32 (m, 4H); 13C NMR (CDCl3) d 21.75,
22.12, 26.09, 41.15, 71.53, 115.40, 121.15, 126.67,
127.66, 128.84, 129.49, 130.67, 131.09, 131.46, 133.88,
138.95, 154.52, and 171.87; mass spectrum (FAB) m/z
284.1649 (M+H)+ (C18H22NO2 requires 284.1650).
4.3. Biological evaluation
4.3.1. SRB assay experimental procedure. Inhibition of
human cancer cell growth was assessed using the Na-
tional Cancer Institute’s standard sulforhodamine B as-
say as previously described.19 Briefly, cells in a 5% fetal
bovine serum/RPMI1640 medium solution were inocu-
lated in 96-well plates and incubated for 24 h. Serial
dilutions of the compounds were then added. After
48 h, the plates were fixed with trichloroacetic acid,
stained with sulforhodamine B, and read with an auto-
mated microplate reader. A growth inhibition of 50%
(GI50 or the drug concentration causing a 50% reduction
in the net protein increase) was calculated from optical
density data with Immunosoft software.
4.2.8. Procedures for preparation of 15a–c.
4.2.8.1. 1,2-Dimethyl-6-methyl-6H-dibenzo[de,g]quino-
line-4,5-dione (15a). To a degassed solution containing
0.065 g (0.26 mmol) of 14a in 3 mL of CH2Cl2 was added
0.056 mL (0.64 mmol) of (COCl)2. The reaction mixture
was stirred for 5 min and 0.075 mL (0.64 mmol) of SnCl4
was added dropwise. The reaction mixture was sealed
tightly with a septum and stirred at 60 ꢁC for 18 h. The
reaction mixture was quenched with 50 mL of 2 N HCl,
extracted with two 100-mL portions of CH2Cl2, dried
over anhydrous MgSO4, filtered, and concentrated under
diminished pressure to give a dark oil. Step gradient elu-
tion chromatography on a flash silica gel column
(15 · 2 cm) using 2% MeOH in 1:1 CHCl3–hexanes ! 2%
MeOH in CHCl3 as eluant gave 15a as a yellow solid: yield
0.010 g (13%); mp 255–257 ꢁC; silica gel TLC Rf 0.67 (10%
MeOH in CHCl3); 1H NMR (CDCl3) d 2.63 (s, 3H), 3.03
(s, 3H), 3.83 (s, 3H), 7.50 (s, 1H), 7.62 (m, 2H), 7.93 (m,
1H), 8.43 (s, 1H) and 8.54 (m, 1H); 13C NMR (CDCl3) d
21.57, 23.17, 30.60, 114.02, 122.21, 125.05, 125.72,
127.44, 127.83, 128.14, 128.96, 130.13, 131.09, 132.29,
132.95, 139.12, 144.22, 156.64 and 176.46; mass spectrum
(FAB) m/z 290.1182 (M+H)+ (C19H16NO2 requires
290.1181).
Acknowledgment
This work was supported by NIH Research Grant
CA50771, awarded by the National Cancer Institute.
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