Cyclopent[a]anthraquinones as DNA-intercalating agents with covalent bond formation potential: Synthesis and biological activity

Article abstract of DOI:10.1021/jm950881y

A series of mitomycin C (MMC) analogues, namely cyclopentanthraquinone derivatives, were synthesized via Diels-Alder cyclization of naphthoquinone with 1-vinylcyclopent-1-enes. These new compounds are planar structures, like MMC, and bear an aziridine rin

Full text of DOI:10.1021/jm950881y

2812
J . Med. Chem. 1996, 39, 2812-2818
Cyclop en t[a ]a n th r a qu in on es a s DNA-In ter ca la tin g Agen ts w ith Cova len t Bon d
F or m a tion P oten tia l: Syn th esis a n d Biologica l Activity
J oong Young Kim,† Tsann-Long Su,*,‡ Ting-Chao Chou,† Bernd Koehler,† Alex Scarborough,†
Ouathek Ouerfelli,† and Kyoichi A. Watanabe†
Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division of Graduate
School of Medical Sciences, Cornell University, New York, New York 10021, and Institute of Biomedical Sciences, Academia
Sinica, Taipei, Taiwan, Republic of China
Received December 4, 1995X
A series of mitomycin C (MMC) analogues, namely cyclopentanthraquinone derivatives, were
synthesized via Diels-Alder cyclization of naphthoquinone with 1-vinylcyclopent-1-enes. These
new compounds are planar structures, like MMC, and bear an aziridine ring and a methyl
carbamate side chain. After bioreduction, they are anticipated to be capable of intercalating
into double-stranded DNA and bind covalently. Structure-activity relationships were studied.
Of these compounds, 2,3-aziridino-4-[[(methylamino)carbonyl]methyl]cyclopent[a]anthracene-
6,11-dione (4) was shown to have inhibitory activity against several leukemic and solid tumor
cell lines. Mice (BDF1) bearing Lewis lung adenocarcinoma were treated with 4 and MMC (ip,
QD × 5). At a dose of 30.0 mg/kg, compound 4 was as effective as MMC (0.8 mg/kg). Compound
4 appears to be less toxic than MMC. DNA unwinding assay indicated that 4 is able to
intercalate into DNA double strands and is also a topoisomerase II inhibitor.
In tr od u ction
When searching for new drug molecules with im-
proved selectivity toward major solid tumors, identifying
the biochemical and functional differences between
normal and neoplastic cells facilitates rationale drug
design. Hypoxia, for example, has been recently rec-
ognized as a potential tool for the development of a more
selective form of anticancer therapy. Bioreductive alkyl-
ating agents activated to more cytotoxic metabolites
under hypoxic conditions may provide a physiological
means of preferential cell kill in poorly vascularized
solid tumors. The bioreductive mitomycin C (MMC, 1,
Figure 1)1 and its analogue, indoloquinone E092 (2,
Figure 1), are the prototype of compounds exploiting this
F igu r e 1.
approach. It is known that MMC can serve as a
As described above, both MMC and E09 are DNA
bifunctional alkylating agent that cross-links to DNA.3
cross-linking agents, but are not able to intercalate into
After bioreduction, MMC readily forms intermediate
DNA double strands. DNA-alkylating antitumor agents,
mitosene through elimination of methanol. The opening
in general, do not selectively inhibit cancerous cell
of the aziridine ring and elimination of the carbamate
growths. On the other hand, the DNA-intercalating
moiety of MMC generates the two reactive sites at C-1
agents cannot bind to DNA persistently. DNA-inter-
and C-10. This enables MMC to bind covalently to the
calating agents with DNA cross-linking potential are,
minor groove of double-stranded DNA. It has been
therefore, expected to be ideal anticancer drugs and
reported by Tomasz et al.4 that MMC forms interstrand
should have improved therapeutic selectivity. Several
cross-links with two diagonally opposed dG residues in
anticancer agents, linking DNA-intercalating ligands
a CpG sequence.
(such as chrysophanol, emodin, or acridine derivatives)
Indoloquinone E09, a fully synthetic bioreductive
with alkylating mustard, have been designed and
DNA cross-linking agent, was found to be at least 10
synthesized with this concept.7-9
times more cytotoxic against tumor cell lines in vitro
One of our ongoing research projects is to exploit new
under hypoxic conditions. In vivo, E09 was inactive
DNA-cross-linking agents with intercalating potential
against P388 murine leukemia, but it did exert sig-
as potent antitumor agents. Previously, we reported the
synthesis of 2,3-dihydro-1H-cyclopent[a]anthracene-6,-
11-dione (cyclopentanthraquinone), 3, bearing a mus-
nificant antiproliferative effects against several other
murine and human solid tumors, including the generally
resistant MAC mouse colon tumors and gastric, ovarian,
tard side chain at C-4 and an aziridine ring at the
and breast xemografts.5 E09 is bioactivated by the
cyclopentene moiety.9 Compound 3 can be considered
enzyme DT-diaphorase and may exhibit particularly
as a MMC analogue. The planar anthraquinone mol-
good activity against tumors rich in this enzyme.2,6
ecule of 3 is expected to intercalate into DNA double
strands and, like MMC or E09, covalently cross-link to
the macromolecule after bioreductive activation of the
aziridine function under hypoxic conditions.
† Cornell University.
‡ Academia Sinica.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
S0022-2623(95)00881-8 CCC: $12.00 © 1996 American Chemical Society

Cyclopentanthraquinones as DNA-Intercalating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2813
Sch em e 1
Sch em e 2a
The preliminary in vitro studies have showed that
compound 3 is a less potent inhibitor against both
leukemic L1210 and HL-60 cell growth than MMC. We
also found that 3 is a more potent inhibitor of topo-
isomerase II (Topo II) k-DNA decatination than MMC.9
The previous structure-activity relationship studies of
these derivatives demonstrated that both mustard side
chain and aziridine moieties play a significant role in
the drug-DNA interaction. However, while further
evaluating the antitumor activity of compound 3, we
found that after 3 months of refrigeration this agent
decomposed, probably due to the instability of the
mustard side chain. Our newly synthesized cyclopenta-
anthraquinone derivatives bearing an aziridine ring and
a carbamate side chain (e.g. compound 4) are structur-
ally even more similar to MMC, but more stable than
their precursors (e.g. compound 3). Compound 4 is,
therefore, expected to be more effective against solid
tumors under hypoxic conditions.
We here describe the synthesis and the structure-
activity relationships of the new cyclopentanthra-
quinone derivatives.
Ch em istr y
Previously, we have synthesized 4-hydroxy-substitut-
ed cyclopentanthraquinones by a Diels-Alder reaction
of 1,4-naphthoquinone with a diene Aa .9 To apply this
reaction to the synthesis of the targeted compound 4,
we needed to prepare a diene with a protected hy-
droxymethyl group (e.g. [Ab], R ) CH2OCH2C6H4(4-
OMe)). Diels-Alder cycloaddition of the diene [Ab] with
naphthoquinone should give 4-O-protected hydroxy-
methyl cyclopentanthraquinone derivative [B] as shown
in Scheme 1. Before a carbamate function to 4-CH2OH
is introduced, suitable protection of the hydroxy function
is necessary, i.e. different conditions should be used to
remove the 4-CH2OH protecting group and the di-O-
isopropylidene group at C2,3. We chose a 4-methoxy-
benzyl function to protect the 4-CH2OH group because
it can be selectively removed by treatment with di-
chlorodicyanoquinone (DDQ).10 Conversion of the
4-CH2OH into its carbamate derivative following the
construction of an aziridine ring a C2,3 should afford
the target compound [C].
As shown in the Scheme 2, the initial step was to
synthesize dienes 8a from the known aldehyde 5a .11
Treatment of 5a with [[(4-methoxybenzyl)oxy]methyl]-
lithium (readily available by transmetalation of
n-Bu3SnCH2OCH2Ph with n-BuLi)12,13 by following
previously described procedures14,15 afforded 6a . At-
tempts to oxidize 6a to the corresponding ketone 7a
resulted in a poor yield. Among the oxidizing agents
tried, we found that the best yield of the ketone 7a (78%)
was obtained when using Swern reagent [(CF3CO)]2O/
DMSO] at -50 °C. Wittig reaction of ketone 7a with
a
(i) Bu3SnCH2OCH2C6H4(4-OMe)/n-BuLi/THF, -78 °C, 1 h
(yield 40%a, 54%b); (ii) Swern oxidation, -50 °C, 1 h (78%a, 98%b);
(iii) Wittig reaction (67%a, 68%b); (iv) (1) anthraquinone/toluene,
reflux 20 h, (2) 10% Pd/C, 1-octene/toluene, reflux, 48 h (52%a,
33%b); (v) DDQ/CH2Cl2, rt, 4 h (68%a); (vi) PhOCOCl/pyridine, rt,
5 h (85%a); (vii) liquid NH3, -78 °C, 4 h (83%a); (viii) MeNCO/
pyridine, 24 h (29%a) (where superscripts a and b refer to the a
and b series, respectively).
methyltriphenylphosphonium iodide and s-BuLi in dry
THF at -78 °C afforded a diene 8a in a 67% yield.
Cycloaddition of diene 8a and naphthoquinone followed
by treatment with 10% Pd/C in refluxing 1-octene
formed cyclopentanthraquinone 9a in 51% yield. The
4-methoxybenzyl group was removed by treatment with
DDQ, and the product 10a was then directly converted
into its N-methylcarbamate 13a by treatment with
methyl isocyanate. On the other hand, treatment of 10a
with phenyl chloroformate in pyridine yielded an ester
derivative 11a . By following a procedure developed by
Islam et al.,16 compound 11a was converted into its
carabmate 12a by treatment with an excess of liquid
ammonia.
After successfully synthesizing the model compounds
(13a and 12a ), we applied the same strategy to the
preparation of cyclopentanthraquinone 4. The known
aldehyde 5b9 was converted into a mixture of diastereo-
mers 6b in a ratio of 2:3 by following the same procedure
as described above as shown in Scheme 2. Diene 8b
was than smoothly synthesized from 6b. Diel-Alders

2814 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Kim et al.
Ta ble 1. Inhibition of Cell Growth by Cyclopentanthraquinone Derivatives and MMC
inhibitory concentration (IC50
)
tumor cell lines
MMC
4
12a
13a
human promyelocytic leukemia (HL-60)
human tetratocarcinoma (833K)
human mammary adenocarcinoma (SKBr-3)
Chinese hamster lung cells (DC-3F)
Chinese hamster lung cells resistant to actinomycin D (DC-3F/
AD-II)
0.050
0.328
0.993
0.082
0.110
6.03
23.9
19.9
10.4
3.6
123.4
NDa
ND
ND
ND
273.7
ND
ND
ND
ND
a
ND: not determined.
Ta ble 2. Anticancer Activity of Compound 4 in Mice Bearing Lewis Lung Carcinomaa
average weight change (mg/kg)
average tumor volume (T/C)
compound
dose (mg/kg)
day 7
day 10
day 14
day 7
day 10
day 14
control
mitomycin C
0
-1
-0.1
-1.2
-1.8
+0.5
+0.6
+0.2
1
1
1
0.4
0.8
2.0b
10
-1.8
-2.2
-2.7
-1.5
-2.3
0.49
0.356
0.169
1.011
0.529
0.411
0.363
0.515
0.437
4
-0.2
-0.4
+2.0
+0.5
0.303
0.205
0.529
0.496
30
a
Tumor (4 × 106 cells) was inoculated subcutaneously (sc). Treatment started on day 3, ip, QD × 5. Control had four mice and each
b
dose of drugs had three mice. Tumor sizes were evaluate on days 7, 10, and 14 after the beginning of treatment. All three mice died on
day 8 or 9.
Sch em e 3a
displaced the benzylic 3-mesyloxy function giving an
approximate 80% yield of the 3-azido-substituted prod-
uct 18. Upon treatment of 18 with triethylamine and
triphenylphosphine in a mixture of THF and water, the
target compound aziridine derivative 4 was prepared
in moderate yield.
Biologica l Activity
The preliminary in vitro cytotoxicity evaluation of
compounds 4, 12a , and 13a against various cancer cells,
such as human promyelocytic leukemic (HL-60), human
teratocarcinoma (833K), human mammary adeno-
carcinoma (SKBr-3), Chinese hamster lung cells (DC-
3F), and Chinese hamster lung cells resistant to actino-
mycin D (DC-3F/AD-II), was compared with MMC. It
was shown that the cyclopentanthraquinone derivatives
were less potent than MMC (Table 1). Among cyclo-
pentanthraquinones, compounds 12a and 13a without
the aziridine ring were less cytotoxic than compound 4
with this functionality (Table 1). The same observation
has been noted previously.9 It is worthwhile to note that
compound 4 was more potent to Chinese hamster lung
cells resistant to actinomycin D (DC-3F/AD-II) (drug
concentration required to reduce the cell density to 50%
of control value, IC50 ) 3.6 µM) than that of the sensitive
cell line (DC-3F) (IC50 ) 10.4 µM). On the contrary,
MMC appeared to be less cytotoxic to the resistant cell
line than to the sensitive one.
Mice (BDF1) bearing Lewis lung adenocarcinoma were
treated with compounds 4 and MMC by intraperitoneal
injection, single dose for 5 days (ip, QD × 5). At a dose
of 30 mg/kg compound 4 was as effective as MMC (0.8
mg/kg) (Table 2). All three mice died on day 8 or day 9
after treatment with MMC at dose of 2.0 mg/kg. On
the basis of these this preliminary study, compound 4
appears to be less toxic than MMC.
As described above, the cyclopentanthraquinone de-
rivative 4 was designed as a DNA-intercalating agent
with covalent bond formation potential. We have
proved that 4 exhibits DNA-intercalating property as
shown by the DNA unwinding assay.18 The ability of
compound 4 to form covalent bonds with macromolecu-
a
(i) Pyridinium toluenesulfonate/MeOH, reflux, 4 h (44%); (ii)
MeSO2)2O/pyridine (91%); (iii) DDQ/CH2Cl2, rt, 4 h (56%); (iv)
MeNCO/CH2Cl2, rt, 24 h (32%); (v) NaN3/DMF, 0 °C, 2 h (79%);
(vi) Et3N/THF/H2O, Ph3P, rt, 1.5 h (49%).
reaction of diene 8b with 1,4-naphthoquinone followed
by treatment with 10% Pd/C in refluxing 1-octene
afforded cyclopentanthraquinone 9b in good yield. Usu-
ally, removal of the isopropylidene protecting group can
be easily achieved by treatment with trifluoroacetic acid
at room temperature. However, we found that this
reagent cleaved both O-(4-methoxybenzyl) and 2,3-
isopropylidenedioxy groups of 9b simultaneously under
various conditions. After several trials, we found that
by using pyridinium p-toluenesulfonate (PPPT),17 the
2,3-di-O-isopropylidene group of 9b could be selectively
removed to give 14 (Scheme 3). Compound 14 was then
allowed to react with methanesulfonic anhydride in
pyridine, and the product, 2,3-cis-bis(mesyloxy) deriva-
tive 15, was obtained in almost quantitative yield.
Compound 15 was treated with DDQ in a mixture of
CH2Cl2/H2O at room temperature afforded 4-hydroxy-
methyl derivative 16, which was then further converted
into N-methylcarbamate 17 in rather low yield (32%)
by treatment with an excess of methyl isocyanate.
Treatment of 17 with NaN3 in DMF at 0 °C selectively

Cyclopentanthraquinones as DNA-Intercalating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2815
and evaporated in vacuo to dryness. The residue was chro-
matographed on a silica gel column (3 × 30 cm) using toluene/
EtOAc (10:1 v/v) as the eluent. The main fraction was collected
and evaporated in vacuo to dryness to give 3.1 g (40%) of 6a
as syrup: 1H-NMR (CDCl3) δ 1.83-1.91 (2H, m, CH2), 2.23-
2.35 (4H, m, CH2), 2.56 (1H, br, exchangeable, OH), 3.35-3.62
(2H, m, 2-CH2), 3.80 (3H, s, OMe), 4.44 (1H, d, J ) 7.73 Hz,
CH), 4.50 (2H, s, PhCH2), 5.67 (1H, s, CHd), 6.88 and 7.26
(each 2H, d, J ) 8.48 Hz, ArH); MS m/e 266 (MNH4+), 246 (M
- 2). Anal. (C21H20O3) C, H.
1-[4,5-(Isop r op ylid en ed ioxy)-1-cyclop en ten -1-yl]-2-[(4-
m eth oxyben zyl)oxy]eth a n ol (6b). By following the same
procedure as described above, 6b (7.48 g, 54%) was prepared
from 4,5-(isopropylidenedioxy)-1-cyclopentenecarbaldehyde (5b,
7.58 g, 43.2 mmol)5 as a syrup (a pair of diastereomers were
1
observed in a ratio of 3:2 from its H NMR): 1H-NMR (CDCl3)
δ 1.33 and 1.39 (each 3H, s, 2 × Me), 2.49-2.55 (2H, m, CH2),
2.93 (1/3H, brs, exchangeable, OH), 3.02 (2/3H, brs, exchange-
able, OH), 3.48 (2/3H, dd, J ) 7.51, J ) 9.68 Hz, 2-CH2), 3.59
(4/3H, d, J ) 5.42 Hz, 2-CH2), 3.72 (1H, dd, J ) 1.92, J ) 3.72
Hz, 1-CH), 3.79 (3H, s, OMe), 4.50 (2/3H, s, PhCH2), 4.51 (4/3H,
s, PhCH2), 4.43-4.76 (1H, m, CH), 5.03 (1/3H, d, J ) 5.93 Hz,
CH), 5.08 (2/3H, d, J ) 5.91 Hz, 5′-CH), 5.71 (1H, d, J ) 0.56
Hz, CHd), 6.86 and 7.25 (each 2H, d, J ) 8.49 Hz, ArH); MS
m/e 338 (MNH4+), 319 (M - 1). Anal. (C18H24O5) C, H.
1-Cyclop en ten -1-yl [(4-Meth oxyben zyl)oxy]m eth yl Ke-
ton e (7a ). To a cooled (ca. -55 °C) solution of trifluoroacetic
anhydride (2.7 mL, 14.8 mmol) in dry CH2Cl2 (100 mL) was
added slowly 2.1 mL (30 mmol) of DMSO. After being stirred
for 5 min, 6a (2.82 g, 11.4 mmol) in CH2Cl2 (20 mL) was added
dropwise into the mixture. The reaction mixture was stirred
at the same temperature for additional 30 min and then
quenched with triethylamine (8 mL). The mixture was
warmed to room temperature, washed with ice-water (10 mL
× 3), dried (MgSO4), and evaporated in vacuo to dryness. The
residue was chromatographed on silica gel (3 × 25 cm) using
toluene/EtOAc (9:1 v/v) as the eluent to give 2.17 g (78%) of
7a as a pale yellow syrup: 1H NMR (CDCl3) δ 1.86-1.94 (2H,
m, CH2), 2.52-2.60 (4H, m, CH2), 3.80 (3H, s, OMe), 4.39 (2H,
s, COCH2), 4.55 (2H, s, PhCH2), 6.78 (1H, dd, J ) 1.72, J )
1.93 Hz, CHd), 6.88 and 7.30 (each 2H, d, J ) 8.70 Hz, ArH);
MS m/e 264 (MNH4+), 245 (M - 1). Anal. (C15H18O3) C, H.
4,5-(Isop r op ylid en ed ioxy)-1-cyclop en ten -1-yl [(4-Meth -
oxyben yzyl)oxy]m eth yl Keton e (7b). Compound 7b, 7.38
g (99%), was prepared from 6b (7.48 g, 23.4 mmol) in a similar
manner: 1H NMR (CDCl3) δ 1.36 and 1.38 (each 3H, s, Me),
2.70-2.74 (2H, m, CH2), 3.81 (3H, s, OMe), 4.39 (2H, dd, J )
6.54 Hz, COCH2), 4.56 (2H, s, PhCH2), 4.79-4.80 (1H, m, CH),
5.33-5.34 (1H, m, 5′-CH), 6.85 (1H, m, CHd), 6.88 and 7.29
(each, 2H, J ) 8.66 Hz, ArH); MS m/e 336 (MNH4+), 317 (M -
1). Anal. (C18H22O5) C, H.
2-(1-Cyclopen ten -1-yl)-3-[(4-m eth oxyben zyl)oxy]-1-pr o-
p en e (8a ). To a mixture of methyltriphenylphosphonium
iodide (6.79 g, 16.8 mmol) in dry THF (100 mL) was added
slowly 12.3 mL (16.8 mmol) of s-BuLi (1.3 M in hexane) at
-78 °C. After the solution was stirred for 30 min, a solution
of 7a (1.97 g, 8 mmol) in dry THF (10 mL) was added to the
mixture. The mixture was stirred at 0 °C for 1 h and then
was stirred at room temperature for an additional 3 h. The
mixture was treated with saturated aqueous NH4Cl solution
(20 mL) and extracted with Et2O (100 mL × 4). The combined
organic extracts were washed with water, dried (MgSO4), and
evaporated in vacuo to dryness. The product 8a (1.32 g, 67%),
as syrup, was purified by column chromatography (silica gel,
toluene/EtOAc, 10:1 v/v): 1H NMR (CDCl3) δ 1.84-1.92 (2H,
m, CH2), 2.42-2.51 (4H, m, CH2), 3.77 (3H, s, OMe), 4.17 (2H,
d, J ) 0.40 Hz, 1-CH2), 4.46 (2H, s, PhCH2), 5.07 and 5.20
(each 1H, s, CH2d), 5.82-5.84 (1H, m, CHd), 6.87 and 7.26
(each 2H, d, J ) 8.67 Hz, ArH); MS m/e 262 (MNH4+), 245 (M
+ 1). Anal. (C16H20O2) C, H.
F igu r e 2. DNA unwinding assay; compound 4 was compared
with known DNA-intercalating agents (m-AMSA and AHMA)
and with DNA-nonintercalating agents (VP-16 and MMC).
lar DNA will be examined later. Compound 4 was
compared with known DNA-intercalating agents [amsa-
crin (m-AMSA)19 and 3-(acridin-9-ylamino)-5-(hydroxy-
methyl)aniline (AHMA, a potential antitumor DNA
intercalator)20] and with nonintercalating DNA-reactive
agents (VP-16 and MMC) as shown in Figure 2. Gel
electrophoresis showed that the band pattern of com-
pound 4 was quite similar to that of m-AMSA and
AHMA and was different from the patterns of VP-16
and MMC. Thus, the DNA unwinding assay indicated
that compound 4, like m-AMSA and AHMA, was able
to intercalate into DNA double strands.
Topo II-mediated cleavage of double-stranded DNA
is a common mechanism of DNA-intercalating agents.
When comparing compound 4 with m-AMSA and MMC,
we studied the inhibitory effect of these compounds on
Topo II as detected by an assay based on decatenation
of k-DNA by Topo II.21 Figure 3 shows that compound
4 and m-AMSA are potent inhibitors of Topo II, while
MMC is a weaker inhibitor.
Exp er im en ta l Section
Melting points are uncorrected and were determined on a
Thomas-Hoover capillary apparatus. Column chromatography
was performed on silica gel G60 (70-230 mesh, ASTM, Merck).
Thin-layer chromatography was performed on Analtech Uni-
plates and visualized with short-wavelength UV light. Ele-
mentary analyses were carried out by MHW Laboratories,
Phoenix, AZ. 1H-NMR spectra were recorded on Bruker ACF-
250 or Bruker AMX-400 spectrometer with Me4Si as the
internal standard. Values reported for coupling constants are
first order. Mass spectra were recorded on Delsi Nermag
Instruments GC/MS spectrometer with ammonia as the car-
rier.
1-(1-Cyclop en ten -1-yl)-2-[(4-m eth oxyben zyl)oxy]eth a -
n ol (6a ). To a solution of tributyl[(4-methoxybenzyl)oxy]-
methyltin11 (16.5 g, 37.5 mmol) in dry THF (250 mL) at -78
°C was added dropwise 15.5 mL of n-BuLi (2.5 M in hexane,
37.5 mmol). The resulting yellow solution was stirred at -78
°C for 10 min, followed by addition of 1-cyclopentenecarbal-
dehyde (5a )12 (3 g, 31.3 mmol) in 20 mL of dry THF. After 1
h of stirring, the mixture was quenched with saturated NH4Cl
solution, extracted with ether (100 mL × 3), dried over MgSO4,
2-[4,5-(Isop r op ylid en ed ioxy)-1-cyclop en ten -1-yl]-3-[(4-
m eth oxyben zyl)oxy]-1-p r op en e (8b). By following the
same procedure, 8b (5.02 g, 68%) was prepared from 7b (7.38
g, 23.1 mmol) as a colorless syrup: 1H NMR (CDCl3) δ 1.38
and 1.43 (each 3H, s, Me), 2.60-2.66 (2H, m, CH2), 3.80 (3H,
s, OMe), 4.18 (2H, dd, J ) 4.84 Hz, 1-CH2), 4.15 (2H, s, PhCH2),

2816 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Kim et al.
F igu r e 3. Inhibition of Topo II-mediated k-DNA decatenation by (a) MMC, compounds 4, 13a , and 12a ; (b) m-AMSA, 4, and
MMC, using purified Topo II from calf thymus.
4.78 (1H, t, J ) 5.60 Hz, CH), 5.25 (1H, d, J ) 5.60 Hz, CH),
5.33 and 5.51 (each 1H, s, CH2d), 5.84 (1H, t, J ) 0.50 Hz,
CHd), 6.88 and 7.26 (each 2H, d, J ) 8.70 Hz, ArH); MS m/e
334 (MNH4+), 316 (M). Anal. (C19H24O4) C, H.
at room temperature for 30 min. The mixture was concen-
trated in vacuo, and the residue was coevaporated several
times with toluene to remove all traces of pyridine. The
residue was chromatographed on a silica gel column (3 × 20
cm) using CH2Cl2/MeOH (100:1 v/v) as the eluent to give 1.43
g (91%) of 15 as yellow crystals: mp 73-74 °C (EtOH); 1H
NMR (CDCl3) δ 3.15 and 3.20 (each 3H, s, OMs), 3.76 (1H,
dd, J 1a,2 ) 7.15, J 1a,1b ) 18.0 Hz, 1-Ha), 3.81 (3H, s, OMe),
4.11 (1H, dd, J 1b,2 ) 7.01, J 1a,1b ) 18.0 Hz, 1-Hb), 4.62 (2H,
dd, J ) 11.3 Hz, PhCH2), 4.79 (2H, dd, J ) 12.8 Hz, CH2),
5.29-5.37 (1H, m, 2-H), 6.32 (1H, d, J ) 5.17 Hz, 3-H), 6.90
and 7.33 (each 2H, d, J ) 8.46 Hz, ArH), 7.80-7.82 (2H, m,
ArH), 8.23-8.30 (2H, m, ArH), 8.33 (1H, s, 5-H). Anal.
(C28H26O10S2) C, H, S.
2,3-D ih y d r o -4-(h y d r o x y m e t h y l)-1H -c y c lo p e n t [a ]-
a n th r a cen e-6,11-d ion e (10). To a vigorously stirred mixture
of 9a (800 mg, 2.0 mmol), CH2Cl2 (60 mL), and H2O (3 mL)
was added DDQ (676 mg, 3.0 mmol) at room temperature.
After being stirred for 4 h, the mixture was washed with
saturated aqueous solution of NaHCO3, followed with water,
dried (MgSO4), and evaporated in vacuo to dryness. The
residue was chromatographed on a silica gel column (3 × 25
cm) using CH2Cl2/MeOH (200:1 v/v) as the eluent to give 406
mg (75%) of 10: mp 191-192 °C (hexane/MeOH); 1H NMR
(CDCl3 + DMSO-d6) δ 2.17-2.25 (2H, m, 2-H), 2.92 (2H, t, J
) 7.66 Hz, 3-H), 3.47 (2H, t, 1-H), 4.72 (2H, d, J ) 2.18 Hz,
CH2OH), 5.07 (1H, brs, exchangeable, OH), 7.77-7.80 (2H, m,
ArH), 8.22-8.31 (2H, m, ArH), 8.31 (1H, s, 5-H). Anal.
(C18H14O3) C, H.
2,3-Dih yd r o-2,3-cis-bis(m esyloxy)-4-(h yd r oxym eth yl)-
1H-cyclop en t[a ]a n th r a cen e-6,11-d ion e (16). By following
the same procedure for the synthesis of 10, compound 16 was
prepared from 15 (1.40 g, 2.38 mmol): yield 0.63 g (56%); mp
137-138 °C (EtOH); 1H NMR (CDCl3) δ 3.15 and 3.18 (3H, s,
OMs), 3.67 (1H, dd, J 1a,2 ) 6.76, J 1a,1b ) 18.0 Hz, 1-Ha), 3.99
(1H, dd, J 1b,2 ) 6.99, J 1a,1b ) 18.0 Hz, 1-Hb), 4.56 (1H, brs,
exchangeable, OH), 4.90 (2H, dd, J ) 4.25 Hz, CH2OH), 5.35
(1H, dt, J 1a,2 ) 6.76, J 1b,2 ) 6.99, J 2,3 ) 5.66 Hz, 2-H), 6.35
(1H, d, J 2,3 ) 5.22 Hz, 3-H), 7.73-7.76 (2H, m, ArH), 8.13-
8.21 (2H, m, ArH), 8.22 (1H, s, 5-H). Anal. (C20H18O9S2) C,
H, S.
2,3-Dih yd r o-4-[[(4-m eth oxyben zyl)oxy]m eth yl]-1H-cy-
clop en t[a ]a n th r a cen e-6,11-d ion e (9a ). A mixture of 8a
(1.25 g, 5 mmol) and 1,4-naphthoquinone (0.87 g, 5.5 mmol)
in toluene (30 mL) was heated under reflux. The reaction was
monitored by TLC (silica gel, Et2O/hexane, 1:5 v/v), which
indicated that the starting materials were consumed and one
major product was formed after refluxing for 20 h. To the
mixture was then added 10% Pd/C (400 mg, 100 mg per 12 h)
and 1-octene (10 mL), and refluxing continued for an additional
48 h. The mixture was allowed to cool to room temperature
and filtered through a bed of Celite, and the filter cake was
washed with ether. The combined filtrate and washings were
evaporated in vacuo to dryness. The residue was crystallized
from ethanol to give 9a (945 mg, 51%) as yellow crystals: mp
166-167 °C; 1H NMR (CDCl3) δ 2.17-2.24 (2H, m, 2-H), 2.94
(2H, t, J ) 7.70 Hz, 3-H), 3.51 (2H, t, J ) 7.70 Hz, 1-H), 4.82
(3H, s, OMe), 4.56 and 4.59 (each 2H, s, CH2), 6.91 and 7.32
(each 2H, d, J ) 8.55 Hz, ArH), 7.74-7.85 (2H, m, ArH), 8.25
(1H, s, 5-H), 8.26-8.30 (2H, m, ArH). Anal. (C26H22O4) C, H.
2,3-Dih yd r o-2,3-(isop r op ylid en ed ioxy)-4-[[(4-m eth oxy-
ben zyl)oxy]m eth yl]-1H-cyclop en t[a ]a n th r a cen e-6,11-d i-
on e (9b). In a similar manner, 9b was prepared from 8b (5.0
g, 15.8 mmol): yield 3.1 g (33%) as yellow needles; mp 119-
120 °C (EtOH); 1H NMR (CDCl3) δ 1.13 and 1.36 (each 3H, s,
Me), 3.50 (1H, dd, J 1a,2 ) 5.63, J 1a,1b ) 19.4 Hz, 1-Ha), 3.79
(3H, s, OMe), 3.79 (1H, d, J 1a,1b ) 19.4 Hz, 1-Hb), 4.55 (2H, s,
PhCH2), 4.78 (2H, dd, J ) 13.4 Hz, CH2), 5.01 (1H, t, J ) 5.60
Hz, 2-H), 5.57 (1H, d, J ) 5.60 Hz, 3-H), 6.84 and 7.28 (each
2H, d, J ) 8.60 Hz, ArH), 7.70-7.73 (2H, m, ArH), 8.18-8.24
(2H, m, ArH), 8.33 (1H, s, 5-H). Anal. (C29H26O6) C, H.
2,3-Dih yd r o-2,3-d ih yd r oxy-4-[[(4-m eth oxyben zyl)oxy]-
m eth yl]-1H-cyclop en t[a ]a n th r a cen e-6,11-d ion e (14).
A
mixture of 9b (2.48 g, 6.0 mmol) and pyridinium p-toluene-
sulfonate (1.52 g, 6.0 mmol) in dry MeOH (500 mL) was heated
at reflux for 4 h. The solvent was removed by vacuum
distillation, and the residue was chromatographed on a silica
gel column (3 × 25 cm) using CH2Cl2/MeOH (200:1 v/v) as the
eluent to give 1.15 g (44%) of 14: mp 165-166 °C (EtOH); 1H
NMR (CDCl3) δ 3.56-3.66 (2H, m, 1-H), 3.81 (3H, s, OMe),
4.59 (2H, s, PhCH2), 4.57-4.61 (1H, m, 2-H), 4.80 (2H, dd, J
) 13.5 Hz, CH2), 5.15 (2H, d, J ) 5.36 Hz, 3-H), 6.90 and 7.29
(each 2H, d, J ) 8.60 Hz, ArH), 7.75-7.79 (2H, m, ArH), 8.13
(1H, s, 5-H), 8.19-8.25 (2H, m, ArH). Anal. (C26H22O6) C, H.
2,3-Dih yd r o-2,3-cis-bis(m esyloxy)-4-[[(4-m eth oxyben -
zyl)oxy]m eth yl]-1H-cyclop en t[a ]a n th r a cen e-6,11-d ion e
(15). A mixture of 14 (1.15 g, 2.66 mmol) and methanesulfonic
anhydride (6.96 g, 40.0 mmol) in pyridine (70 mL) was stirred
4-[[(P h en oxyca r b on yl)oxy]m et h yl]-1H -cyclop en t [a ]-
a n th r a cen e-6,11-d ion e (11). A mixture of 10 (200 mg, 0.72
mmol) and phenyl chloroformate (0.8 mL) in dry pyridine (10
mL) was stirred in an ice bath for 30 min and then at room
temperature for 2 h and then evaporated in vacuo to dryness.
The residue was dissolved in CHCl3 (50 mL), washed with
water (20 mL × 2), dried (MgSO4), and evaporated in vacuo
to dryness. The residue was crystallized from hexane/CHCl3/
1
MeOH to give 11: 152 mg (53%); mp 155-156 °C; H NMR
(CDCl3) δ 2.23-2.31 (2H, m, 2-H), 3.06 and 3.57 (each 2H, t,

Cyclopentanthraquinones as DNA-Intercalating Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2817
J ) 7.70 Hz, 3-H and 1-H), 5.38 (2H, s, CH2), 7.20-7.38 (3H,
m, ArH), 7.38-7.42 (2H, m, ArH), 7.80-7.82 (2H, m, ArH),
8.31 (1H, s, 5-H), 8.27-8.32 (2H, m, ArH). Anal. (C25H18O5)
C, H.
to actinomycin D (DC-3F/AD-II). For HL-60 cells, assays were
carried out by XTT-tetrazolium microculture assay after 72
h
of incubation as described by Scudiero et al.22 After
incubation with phenazine methosulfate-XTT solution at 37
°C for 6 h, absorbance at 450 and 630 nm was detected with
microplate reader (EL 340; Bio-Tek Instruments Inc., Wi-
nooski, VT). Other solid tumor cells in monolayers were
assayed using SRB protein-staining methods as described by
Skehan et al.23 Six to seven concentrations of each compound
were used. The IC50 and dose-effect relationships of com-
pounds for antitumor activity were calculated by a median-
effect plot.24,25
4-[(Ca r ba m oyloxy)m eth yl]-2,3-d ih yd r o-1H-cyclop en t-
[a ]a n th r a cen e-6,11-d ion e (12). To a solution of 11 (100 mg,
0.25 mmol) in a mixture of CH2Cl2/MeOH (9:1 v/v) was added
liquid NH3 (10 mL) in a dry ice/acetone bath. After being
stirred for 30 min, the temperature was allowed to warm to
room temperature over a period of 3 h. The solvent was
evaporated and the solid residue crystallized from a mixture
of hexane/CH2Cl2 to give 13: 66 mg (83%); mp 229-230 °C;
1H NMR (DMSO-d6) δ 2.19-2.27 (2H, m, 2-H), 3.02 (2H, t, J
) 7.70 Hz, 3-H), 3.47 (2H, t, J ) 7.70 Hz, 1-H), 5.19 (2H, s,
CH2), 7.96-8.01 (2H, m, ArH), 8.10 (1H, s, 5-H), 8.22-8.26
(2H, m, ArH). Anal. (C19H15NO4) C, H, N.
2,3-Dih yd r o-4-[[(m eth ylca r ba m oyl)oxy]m eth yl-1H-cy-
clop en t[a ]a n th r a cen e-6,11-d ion e (13). A mixture of 10
(100 mg, 0.36 mmol) and excess of MeNCO (2 mL) in acetone
(20 mL) was stirred at room temperature for 1 day. The
solvent was removed in vacuo, and the residue was chromato-
graphed on a silica gel column (2 × 25 cm) using CH2Cl2/MeOH
(200:1 v/v) as the eluent to give 13: 32 mg (27%); mp 232-
233 °C (CH2Cl2/EtOH): 1H NMR (CDCl3) δ 2.20-2.27 (2H, m,
2-H), 2.86 (3H, d, J ) 4.85 Hz, NHMe), 2.98 (2H, t, J ) 7.60
Hz, 3-H), 3.53 (2H, t, J ) 7.50 Hz, 1-H), 4.85 (1H, brs,
exchangeable, NH), 5.20 (2H, s, CH2), 7.75-7.80 (2H, m, ArH),
8.19 (1H, s, 5-H), 8.25-8.57 (2H, m, ArH). Anal. (C20H17NO4)
C, H, N.
In a similar manner, compound 17 was synthesized.
2,3-cis-Bis(m esyloxy)-2,3-d ih yd r o-4-[[(m eth ylca r ba m -
oyl)oxy]m e t h yl]-1H -cyclop e n t [a ]a n t h r a ce n e -6,11-d i-
on e (17) was prepared from 16 (400 mg, 0.85 mmol): yield
142 mg (32%); mp 161-162 °C (EtOH); 1H NMR (CDCl3) δ
2.80 (3H, d, J ) 4.80 Hz, NHMe), 3.26 and 3.32 (each 3H, s,
OMs), 3.84 (1H, dd, J 1a,2 ) 6.71, J 1a,1b ) 18.5 Hz, 1-Ha), 4.13
(1H, dd, J 1b,2 ) 7.70 Hz, J 1a,1b ) 18.5 Hz, 1-Hb), 5.14 (1H, brs,
exchangeable, NH), 5.37 (2H, dd, J ) 14.5 Hz, CH2), 5.40-
5.45 (1H, m, 2-H), 6.38 (1H, d, J 2,3 ) 5.15 Hz, 3-H), 7.82-7.86
(2H, m, ArH), 8.26-8.32 (2H, m, ArH), 8.34 (1H, s, 5-H). Anal.
(C22H21NO10S2) C, H, N, S.
In h ibition of k -DNA Deca ten a tion . k-DNA was isolated
from C. fasciculate according to previously published meth-
ods.21 The standard reaction mixture for k-DNA decatenation
assay contained 50 mM Tris‚HCl, pH 7.5, 5 mM MgCl2, 50
mM KCl, 0.5 mM DTT, 30 µg/mL BSA, 5 mM ATP, 1.5 µg of
k-DNA, and 5 µg of protein of nuclear extracts in 50 µL of 10%
SDS and 1 mg/mL proteinase K, and the mixture was further
incubated at 37 °C for 30 min.25 Reaction was terminated by
the addition of 5 µL of 10% SDS and 1 mg/mL proteinase K,
and the mixture was further incubated at 37 °C for 30 min,
followed by electrophoresis on 1% agarose with TBE buffer.
After ethidium bromide staining (5 µg/mL), gels were photo-
graphed under UV illumination, using Polaroid type 55 film.
DNA Un w in d in g Assa y. To measure the DNA intercalat-
ing activity, DNA unwinding assay was carried out using the
method described by Fisher et al.18 Supercoiled pBR322
(Boehringer Mannheim) (0.5 µg) was relaxed with 2.2 units of
calf thymus DNA Topo I (Bethesda Research Labs) in 100 µL
of relaxation buffer (10 mM Tris, 50 mM KCl, 100 mM MgCl2,
0.5 mM DTT, 0.5 mM EDTA, and 30 µg/mL bovine serum
albumin, pH 7.9) for 1.5 h at 37 °C. Various concentrations
of drugs in DMSO were added, and the solution was mixed
and incubated at 37 °C for 30 min. Rections were stopped by
adding 5 µL of 20% SDS. Samples were extracted twice with
equal volumes of equilibrated phenol and once with chloroform.
Five microliters of gel-loading buffer (0.5% bromophenol blue,
0.25% xylene cyanol, 1.5% ficol type 400) was added to 20 µL
of the sample. Electrophoresis was carried out through 1%
agarose in 40 mM Tris base and 1 mM EDTA and titrated to
pH 7.2 with acetic acid at 2 V/cm.
3-Azid o-2,3-tr a n s-d ih yd r o-2-(m esyloxy)-4-[[(m et h yl-
car bam oyl]oxy]m eth yl]-1H-cyclopen t[a ]an th r acen e-6,11-
d ion e (18). A mixture of 17 (190 mg 0.36 mmol) and NaN3
(24 mg, 0.36 mmol) in DMF (15 mL) was stirred at 0 °C for 2
h. The mixture was evaporated in vacuo to dryness, and the
residue was chromatographed on a silica gel column (2 × 25
cm) using CH2Cl2/MeOH (200:1 v/v) as the eluent to give 134
mg (70%) of 18 as syrup: 1H NMR (CDCl3) δ 2.81 (3H, d, J )
4.83 Hz, NHMe), 3.84 (1H, d, J 1a,1b ) 16.0 Hz, 1-Ha), 4.01 (1H,
dd, J 1b,2 ) 6.10, J 1a,1b ) 16.0 Hz, 1-Hb), 5.31 (1H, s, 3-H), 5.33
(2H, d, J ) 13.8 Hz, CH2), 5.45 (1H, d, J ) 6.10 Hz, 2-H), 7.78-
7.81 (2H, m, ArH), 8.23-8.29 (2H, m, ArH), 8.32 (1H, s, 5-H);
MS m/e 488 (MNH4+). Anal. (C21H18N4O7S) C, H, N, S.
2,3-Im in o-2,3-d ih yd r o-4-[[(m e t h ylca r b a m oyl)oxy]-
m eth yl]-1H-cyclop en t[a ]a n th r a cen e-6,11-d ion e (4). To a
mixture of 18 (134 mg, 0.284 mmol) and Et3N (0.5 mL) in THF
(10 mL) containing H2O (0.5 mL) was added triphenylphos-
phine (118 mg, 0.446 mmol). The mixture was stirred at room
temperature for 1.5 h and then diluted with EtOAc (30 mL).
The solution was washed with H2O (15 mL × 3), dried
(MgSO4), and evaporated in vacuo to dryness. The residue
was chromatographed on a silica gel column (2 × 25 cm) using
CH2Cl2/MeOH (200:1 v/v) as the eluent to give 4: 51 mg (49%);
An titu m or Activity in Tu m or -Bea r in g Mice. BDF1
mice bearing E0771 mammary adenocarcinoma or Lewis lung
carcinoma were injected (ip) daily for 5 days beginning day 3
after tumor inoculation (subcutaneous implant with 0.2 mL
brei). Body weight changes, mortality, and average tumor
volume were recorded on days 7, 10, and 14. Day 14 results
are reported. Drugs were dissolved in dimethyl sulfoxide
(DMSO). Untreated controls were injected with DMSO alone.
Two Topo II inhibitors, etoposide (VP-16) and m-AMSA, were
also dissolved in DMSO and served as treated controls for
comparing the therapeutic efficacy.
Ack n ow led gm en t . This investigation was sup-
ported in parts by funds from the National Cancer
Institute, National Institutes of Health (Grant Nos.
PO1-CA18856, RO1-AI 32350), Elsa U. Pardee founda-
tion, MSKCC Preclinical Pharmacology Core Facility,
U.S.A., and Institute of Biomedical Sciences, Academia
Sinica, Taiwan, R.O.C.
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J M950881Y