11-Substituted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine derivatives as novel topoisomerase I-targeting agents

Article abstract of DOI:10.1016/j.bmc.2008.08.018

Several 11-substituted benzo[i]phenanthridine derivatives were synthesized, and their TOP1-targeting activity and cytotoxicity were assessed. Comparative data indicate that TOP1-targeting was often the primary molecular target associated with their cytoto

Full text of DOI:10.1016/j.bmc.2008.08.018

Bioorganic & Medicinal Chemistry 16 (2008) 8598–8606
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
11-Substituted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine
derivatives as novel topoisomerase I-targeting agents
Wei Feng a, Mavurapu Satyanarayana a, Yuan-Chin Tsai b, Angela A. Liu b,
Leroy F. Liu b,c, Edmond J. LaVoie a,c,
*
a Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA
b Department of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
c The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Several 11-substituted benzo[i]phenanthridine derivatives were synthesized, and their TOP1-targeting
activity and cytotoxicity were assessed. Comparative data indicate that TOP1-targeting was often the pri-
mary molecular target associated with their cytotoxicity. Several 11-aminoalkyl derivatives, 11-amino-
carboxy derivatives as well as the 11-[(2-dimethylamino)ethyl]carboxamide of 2,3-dimethoxy-8,9-
methylenedioxybenzo[i]phenanthridine were synthesized and did exhibit considerable cytotoxicity with
IC50 values ranging from 20 to 120 nM in the human lymphoblast tumor cell line RPMI8402.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 26 March 2008
Revised 31 July 2008
Accepted 4 August 2008
Available online 7 August 2008
Keywords:
Cytotoxicity
Antitumor agents
Topoisomerase I
Benzo[i]phenanthridines
Multidrug resistance
Photocyclization
1. Introduction
these camptothecins.9–15 In view of these observations, non-cam-
ptothecin TOP1-targeting agents have been investigated for their
Topoisomerases are ubiquitous enzymes that participate in pro-
cesses such as DNA replication, repair, transcription, and recombi-
nation as well as chromosome condensation and segregation.1,2
Topoisomerase I (TOP1) is the target of several antitumor agents
based upon their ability to stabilize the enzyme–DNA cleavage
complex, which results in DNA damage and ultimately cell
death.3,4 Camptothecin (CPT) was the first compound identified
as a TOP1-targeting agent.5 Two clinical TOP1-targeting agents,
topotecan (HycamtinÒ) and irinotecan (CPT-11/CamptosarÒ), have
since been developed. The improved water-solubility of topotecan
and irinotecan relative to CPT was critical to their development
into the clinic. These agents possess the same ring structure of
camptothecin, which includes a d-lactone. This lactone moiety is
susceptible to hydrolysis, and the resulting carboxylic acid has a
high affinity for human serum albumin.6–8 In addition, it is known
that both of these clinical agents are susceptible to transporter-
mediated cellular efflux, which can limit intracellular accumula-
tion and has been associated with multidrug resistance. Specifi-
cally overexpression of MDR1 (P-glycoprotein) and breast cancer
resistance protein (BCRP) has been associated with resistance to
potential to overcome these obstacles, which could limit the effec-
tive concentration of drug within certain tumor types.
Several 5-(2-aminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones
have been identified as exceptionally active TOP1-targeting agents
with potent antitumor activity.16,17 One of the more extensively
studied of these non-camptothecin TOP1-targeting agents is 5H-
8,9-dimethoxy-5-(2-dimethylaminoethyl)-2,3-methylenedioxy-
dibenzo[c,h][1,6]-naphthyridin-6-one (ARC-111), 1 (Fig. 1).18,19
The 11-aza analog of ARC-111, 11-[2-(dimethylamino)ethyl]-2,3-
dimethoxy-8,9-methylenedioxy-11H-isoquinolin[4,3-c]cinnolin-12-
one (ARC-31), 2 (Fig. 1) and related compounds have also been
identified as potent TOP1-targeting agents.16,20 Analogs within
both of these series of compounds have proved to be active as anti-
tumor agents in vivo when administered by gavage or parenterally
to tumor-bearing mice. The presence of N-alkyl substituents within
the bay region tends to distort both of these ring systems from
planarity. It was considered advantageous for the purpose of
comparison to examine whether 11-aminoalkyl substituted
benzo[i]phenanthridines would possess similar potency as TOP1-
targeting agents and comparable cytotoxicity to structurally simi-
lar 5-(2-aminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones. Specifi-
cally targeted for synthesis and biological evaluation were a series
of 11-substituted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]
* Corresponding author. Tel.: +1 732 445 2674; fax: +1 732 445 6312.
E-mail address: elavoie@rci.rutgers.edu (E.J. LaVoie).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.018

W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 8598–8606
8599
CH3
N
O
CH3
H3C
O
N
OCH3
OCH3
H3C
R
N
OCH3
OCH3
N
OCH3
OCH3
N
N
O
O
O
O
O
O
N
N
3
1
2
Figure 1. Structure of ARC-111 (1), ARC-31 (2), and 11-substituted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridines (3).
phenanthridines, 3. In this study, we evaluated several 11-alkyl-
amino and 11-aminocarboxy derivatives together with various
11-substituted benzo[i]phenanthridine intermediates that were
utilized in their synthesis.
dimethoxybenzaldehyde via the dibromo olefin 4 using Corey’s
procedure.21,22 Hydrostannation of 5 was accomplished by Bu3SnH
in the presence of AIBN to give (Z)-vinylstannane 6 stereoselective-
ly.22 The Stille cross-coupling reaction of the resulting vinylstann-
ane 6 with bromoquinoline 723 proceeded smoothly to afford
compound 8 in good yield.24 Photocyclization of 8 in the presence
of a catalytic amount of iodine resulted in the formation of 9 in 35%
yield.25
2. Results and discussion
2.1. Chemistry
The preparation of 11-aminomethyl- and 11-N,N-dimethylami-
nomethyl-2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthri-
dine, 12 and 13 was accomplished as outlined in Scheme 2.
Reduction of the ethyl ester 9 to its hydroxymethyl derivative 10
was accomplished using lithium aluminum hydride in THF.
Oxidation of the hydroxymethyl group with MnO2 resulted in the
The key intermediate used for the preparation of 11-substituted
2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridines was
the 11-ethoxycarbonyl derivative, 9. The methodology employed
for the synthesis of this intermediate is outlined in Scheme 1. Ethyl
3-(3,4-dimethoxyphenyl)propynoate 5 was synthesized from 3,4-
Br
O
H
H
O
Br
OC2H5
b
a
H3CO
OCH3
OCH3
OCH3
OCH3
OCH3
5
4
c
O
OCH3
O
H
C2H5O
Br
N
OCH3
OCH3
d
C2H5O
O
O
OCH3
+
Bu3Sn
O
O
N
8
7
6
e
O
OCH3
OCH3
C2H5O
O
O
N
9
Scheme 1. Reagents and conditions: (a) CBr4, PPh3, CH2Cl2, 55%; (b) n-BuLi, ClCOOC2H5, THF, 96%; (c) Bu3SnH, AIBN, benzene, 66%; (d) PdCl2(PPh3)2, DMF, 57%; (e) hv, I2 (cat.),
benzene, 35%.

8600
W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 8598–8606
O
OH
OCH3
OCH3
OCH3
OCH3
C2H5O
a
O
O
O
O
N
N
9
10
NH2
b
OCH3
OCH3
O
O
c
OCH3
OCH3
H
O
N
12
O
O
N
H3C
CH3
d
N
11
OCH3
OCH3
O
O
N
13
Scheme 2. Reagents and conditions: (a) LAH, THF, 57%; (b) MnO2, DMF, 62%; (c) ammonium acetate, NaBH3CN, MeOH, 47%; (d) dimethylamine, NaBH3CN, MeOH, 55%.
formation of the 11-formyl derivative 11. Reductive amination
using either ammonium acetate or dimethylamine followed by
treatment with sodium triacetoxyborohydride gave 12 and 13,
respectively.
The synthetic approach used in the preparation of 11-[3-(N,N-
dimethylamino)propyl]-2,3-dimethoxy-8,9-methylenedioxybenzo-
[i]phenanthridine 19 is outlined in Scheme 4. A solution of the for-
myl intermediate 11 in THF was added to a mixture of 2-(dimeth-
ylamino)ethyltriphenylphosphonium bromide and LiHMDS in THF.
The resulting product 18 was reduced to the propyl derivative 19
using hydrogen and 10% Pd–C.
The 11-ethoxycarbonyl derivative 9 was subjected to base
hydrolysis in aqueous ethanol, and the carboxylic acid was con-
verted to its acid chloride using thionyl chloride (Scheme 5). This
acid chloride was used without further purification for the forma-
tion of a series of 11-carboxamides. These included the primary
carboxamide, the N-methylcarboxamide, and the N,N-dimethyl-
carboxamide, 20–22 as illustrated in Scheme 5. Treatment of the
The preparation of the 11-(2-aminoethyl)- and 11-[(2-N,N-
dimethylamino)ethyl]-2,3-dimethoxy-8,9-methylenedioxybenzo-
[i]phenanthridine 16 and 17 is outlined in Scheme 3. Condensation
of the 11-formyl derivative 11 with nitromethane provided the
2-nitroethylene derivative 14, which could be reduced to the
11-(2-nitroethyl)-2,3-dimethoxy-8,9-methylenedioxybenzo[i]phe-
nanthridine 15. In the presence of zinc in acetic acid, the nitro
group was reduced to give 16. Reductive methylation using forma-
lin in the presence of sodium cyanoborohydride provided the N,N-
dimethyl derivative, 17.
O2N
O2N
OCH3
OCH3
OCH3
b
a
O
O
O
OCH3
11
O
N
N
15
14
c
CH3
N
H2N
H3C
OCH3
OCH3
d
OCH3
OCH3
O
O
O
O
N
N
16
17
Scheme 3. Reagents and conditions: (a) ammonium acetate, nitromethane, 89%; (b) NaBH4, 1,4-dioxane/EtOH, 69%; (c) Zn, AcOH, 41%; (d) formalin, NaBH3CN, MeOH, 53%.

W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 8598–8606
8601
CH3
CH3
H3C
H3C
N
H
N
b
OCH3
OCH3
OCH3
OCH3
a
11
O
O
O
O
N
N
18
19
Scheme 4. Reagents and conditions: (a) 2-(dimethylamino)ethyltriphenylphosphonium bromide, LiHMDS, THF, 47%; (b) Pd/C, H2, 44%.
R2
R1
O
N
OCH3
OCH3
O
O
c
N
Cl
20; R1,R2 = H
OCH3
OCH3
a,b
O
21; R1 = H; R2 = CH3
9
22; R1,R2 = CH3
O
O
CH3
N
c
H3C
N
NH
OCH3
OCH3
O
O
O
N
23
Scheme 5. Reagents and conditions: (a) 10% NaOH, EtOH; (b) SOCl2, CH2Cl2; (c) RNH2, Et3N, CH2Cl2, 63–75%.
acid chloride with N,N-dimethylaminoethylenediamine resulted in
the formation of N-[(2-N,N-dimethylamino)ethyl]carboxamide 23.
RPMI8402 and P388 cells, as well as their camptothecin-resistant
variants, CPT-K5 and P388/CPT-4528,29 are provided in Table 1.
2.2. Biological activity
Table 1
TOP1-targeting activity and cytotoxicity in RPMI8402 and P388 cells and their
camptothecin-resistant variants
The intrinsic TOP1-targeting activities of the various 11-substi-
tuted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridines
that were synthesized are summarized in Table 1. Several com-
pounds had comparable TOP1-targeting activity to CPT or ARC-
111. The hydroxymethyl derivative 10 and both the aminomethyl
and N,N-dimethylaminomethyl derivatives 12 and 13 had potent
TOP1-targeting activity. While the formyl derivative 11 was less
potent, it also had significant TOP1-targeting activity. As had been
previously observed with 12-carboxyester derivatives of 2,3-dime-
thoxy-8,9-methylenedioxybenzo[i]phenanthridines,26,27 9 did not
exhibit significant TOP1-targeting activity. The nitro precursor
compounds 14 and 15 proved to be much less active as TOP1-tar-
geting agents than either the primary or tertiary amino derivatives,
16 and 17, respectively. The 11-[3-(N,N-dimethylaminopropyl)]
derivative 19 was also much more potent as a TOP1-targeting
agent than its 1-propenyl precursor, 18.
Compound TOP1-mediated
cleavagea
Cytotoxicity IC50
(
l
M)
P388/
CPT45
0.001 0.23
RPMI8402 CPT-
K5
P388
1
0.3
>10
0.2
0.8
0.2
0.2
>10
>10
1.0
0.4
>10
0.8
1.1
0.8
>10
0.2
0.2
1.0
0.002
10
0.12
0.1
0.12
0.1
0.55
0.06
0.032
0.02
0.035
0.06
0.14
0.28
1.6
0.90
>10
>10
>10
2.1
9
10
11
12
4.0
0.06
0.12
>10
>10
10
0.026 0.3
13
14
15
16
2.7
0.10
0.18
0.07
0.014 0.18
0.015 0.18
0.04
0.04
0.035 >10
0.025 >10
2.0
10
>10
>10
>10
0.2
17
18
19
20
0.33
0.35
0.29
>10
>10
>10
0.63
>10
>10
0.35
0.2
21
22
23
While the primary and secondary carboxamide derivatives 20
and 21 had comparable TOP1-targeting activity, a precipitous drop
in activity was observed for the tertiary carboxamide derivative 22.
A significant increase in TOP1-targeting activity was observed
among these carboxamides in the case of the 11-[(2-N,N-dimethyl-
amino)ethylamino]carboxy derivative 23.
0.98
>10
0.035
0.004
0.21
0.015 0.26
0.004 >10
0.045 >10
CPT
Topotecan
a
Topoisomerase I cleavage values are reported as REC, relative effective con-
centration, these are concentrations relative to topotecan, whose value is arbitrarily
assumed as 1, that are able to produce 10% cleavage of the plasmid DNA in the
presence of human topoisomerase I.17
The relative cytotoxic activities of these 11-substituted
2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridines
in

8602
W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 8598–8606
The very weak cytotoxic activity observed with 9 is likely associ-
ated with its limited aqueous solubility and its potential for hydro-
lysis to the carboxylic acid. The cytotoxic activities of 10–13 in
RPMI8402 and P388 were lower than anticipated on the basis of
their TOP1-targeting activity. The cross-resistance observed in
both CPT-K5 and P388/CPT-45 for these compounds clearly indi-
cates that TOP1-targeting is the major mechanism associated with
their cytotoxic activity.
The unsaturated nitro derivative 14 was less cytotoxic than the
saturated analog 15. It is possible that the more rigid conformation
associated with the cis- and trans-isomers of 14 may be less favor-
able. Both 14 and 15 did exhibit significant cross-resistance in the
CPT-resistant cell lines despite comparatively weak intrinsic TOP1-
targeting activity. The primary and tertiary 11-[2-aminoethyl]
derivatives 16 and 17 were among the more cytotoxic 11-substi-
tuted benzo[i]phenanthridine derivatives that were evaluated.
While the 11-[3-dimethylaminoprop-1-enyl] derivative 18 was
much less potent than the 11-[3-(dimethylamino)propyl] deriva-
tive 19 as a TOP1-targeting agent, these compounds did exhibit
similar cytotoxic activity with IC50 values ranging from 35 to
60 nM with RPMI8402 and P388 cells. Significant cross-resistance
in the CPT-resistant cells CPT-K5 and P388/CPT45 was observed
for 16–20.
The conversion of the primary carboxamide 20 to its secondary
carboxamide 21 by the addition of a N-methyl substituent did not
adversely affect cytotoxic activity. Consistent with its lower TOP1-
targeting activity, the tertiary carboxamide derivative 22 was sig-
nificantly less cytotoxic than either 20 or 21. A significant increase
in activity, however, was observed with the 11-[(2-dimethyl-
amino)ethylamino]carboxy derivative 23, which is consistent with
it being among the more potent TOP1-targeting compounds in this
study.
The cytotoxic activities of these 11-substituted benzo[i]phenan-
thridines in KB3-1 cells and its variant cell lines, KBV-1 and
KBH5.0, are listed in Table 2. KBV-1 cells overexpress the efflux
transporter MDR1,30 and KBH5.0 cells overexpress the efflux trans-
porter BCRP.18 CPT-11 and topotecan are substrates for the efflux
transporters MDR1 and BCRP. Decreased cytotoxicity against
KBV-1 cells relative to the parent cell line KB3-1 is indicative of
substances that are substrates for the efflux transporter MDR1.
Similarly, resistance to the cytotoxic effects of a substance ob-
served in KBH5.0 cells relative to its parent cell line, KB3-1, is
indicative of a compound being a substrate for the BCRP efflux
transporter. The data provided in Table 2 clearly indicate that 16,
18, and 19 are substrates for the MDR1 efflux transporter with
greater than an order of magnitude difference in cytotoxicity ob-
served in KB3-1 cells relative to KBV-1 cells. The primary carbox-
amide 20 is the only compound that proved to be a very good
substrate for the BCRP efflux transporter with almost a 50-fold dif-
ferential in cytotoxicity observed for KB3-1 and KBH5.0 cells. In
addition, this compound also appears to be a weak substrate for
the MDR1 transporter. The aminomethyl derivatives 12 and 13 as
well as the 11-[(2-dimethylamino)ethylamino]carboxy derivative
23 were weak or marginal substrates for both the MDR1 and BCRP
efflux transporters. These data suggest in the case of 11-aminoalkyl
benzo[i]phenanthridine derivatives that the length of the alkyl lin-
ker as well as the degree to which the amino group is substituted
can influence their potential to serve as substrates for the MDR1
efflux transporter. In addition, changes associated with carboxam-
ide moiety of 11-aminocarboxy benzo[i]phenanthridine deriva-
tives did influence their potential to serve as substrates for the
BCRP efflux transporter.
Several of the 11-substituted 2,3-dimethoxy-8,9-meth-
ylenedioxybenzo[i]phenanthridine derivatives evaluated in this
study did exhibit potent TOP1-targeting activity, which was com-
parable to ARC-111 and CPT. These benzo[i]phenanthridine deriv-
atives, including those having the more potent TOP1-targeting
activity, were generally less cytotoxic than ARC-111, with IC50 val-
ues being more than one order of magnitude greater. Nonetheless,
several of these 11-substituted benzo[i]phenanthridines did have
cytotoxic activity comparable to topotecan in these various tumor
cell lines. The absence of a d-lactone in their structure and an abil-
ity to overcome MDR1- or BCRP-associated multidrug resistance
could prove advantageous. Evaluation of their relative efficacy in
athymic nude mice with human tumor xenografts will provide
for a more complete assessment of their potential clinical utility.
3. Experimental
Melting points were determined with a Meltemp capillary melt-
ing point apparatus. Column chromatography refers to flash chro-
matography conducted on SiliTech 32–63 lm, (ICN Biomedicals,
Eschwege, Germany) using the solvent systems indicated. Infrared
spectral data were obtained using a Thermo-Nicolet Avatar 360
Fourier transform spectrometer and are reported in cmꢀ1. Proton
(1H NMR) and carbon (13C NMR) nuclear magnetic resonance were
recorded on a Varian Gemini-200 Fourier Transform spectrometer.
NMR spectra (200 MHz 1H and 50 MHz 13C) were recorded in the
deuterated solvent indicated with chemical shifts reported in d
units downfield from tetramethylsilane (TMS). Coupling constants
are reported in Hertz (Hz). Mass spectra were obtained from Wash-
ington University Resource for Biomedical and Bio-organic Mass
Spectrometry within the Department of Chemistry at Washington
University, St. Louis, MO. All starting materials and reagents were
purchased from Aldrich. Solvents were purchased from Fisher Sci-
entific, and were ACS grade or HPLC grade. Methylene chloride was
freshly distilled from calcium hydride. All other solvents were used
as provided without further purification. Compound 7 was pre-
pared as previously described.23
Table 2
Cytotoxicity of 11-substituted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenan-
thridines in KB3-1 cells and its variant cell lines, KBV-1 and KBH5.0
Compound
Cytotoxicity IC50
KBV-1
(lM)
KB3-1
0.005
>10
0.15
KBH5.0
1
9
10
0.005
>10
0.4
0.006
>10
0.4
11
12
13
14
15
16
17
18
19
20
21
22
0.13
0.03
0.06
0.18
0.15
0.02
0.021
0.03
0.035
0.055
0.23
1.2
0.3
0.29
0.21
0.29
0.42
0.32
0.11
0.04
0.07
0.13
2.7
0.16
0.25
0.45
0.36
0.5
0.06
0.33
0.4
3.1. 1,1-Dibromo-2-(3,4-dimethoxyphenyl)ethylene (4)
0.4
0.42
4.0
0.48
3.6
A mixture of CBr4 (29.8 g, 0.09 mol) and PPh3 (47.2 g, 0.18 mol)
in dichloromethane (200 mL) was stirred under nitrogen at room
temperature for 1 h. To this mixture was added 3,4-dimethoxy-
benzaldehyde (10.0 g, 0.06 mol) at 0 °C and the mixture was stirred
for 16 h at room temperature. The resulting precipitate was filtered
23
CPT
Topotecan
Irinotecan
0.027
0.015
0.04
0.68
1.4
0.2
0.025
0.44
40.0
0.026
0.44
7.4

W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 8598–8606
8603
off, and the filtrate was concentrated under reduced pressure. The
residue was chromatographed with 10:1 hexanes/EtOAc to provide
a colorless oil in 55% yield; IR (KBr) 1600, 2834; 1H NMR (CDCl3) d
3.89 (s, 3H), 3.90 (s, 3H), 6.85 (d, 1H, J = 8.0), 7.10 (dd, 1H, J = 8.0,
2.2), 7.18 (d, 1H, J = 2.2), 7.41 (s, 1H); 13C NMR (CDCl3) d 55.0,
55.1, 86.5, 110.0, 110.4, 121.1, 127.1, 135.6, 147.8, 148.5.
duced pressure and the residue was chromatographed with 10:1
CHCl3/MeOH to provide a light yellow powder in 35% yield; mp
240–243 °C; IR (KBr) 1712; 1H NMR (CDCl3) d 1.44 (t, 3H, J = 7.0),
4.06 (s, 3H), 4.16 (s, 3H), 4.54 (q, 2H, J = 7.0), 6.15 (s, 2H), 7.29 (s,
1H), 7.47 (s, 1H), 7.55 (s, 1H), 8.09 (s, 1H), 8.18 (s, 1H), 9.89 (s,
1H); 13C NMR (CDCl3) d 13.2, 55.2, 55.3, 61.1, 101.0, 101.2, 101.8,
106.3, 107.6, 118.6, 120.6, 124.9, 125.1, 125.5, 127.0, 131.6,
142.7, 144.3, 146.7, 148.3, 149.4, 151.0, 170.0; HRMS calcd for
3.2. Ethyl 3-(3,4-dimethoxyphenyl)propynoate (5)
C23H19NO6H: 406.1273; found: 406.1273.
To a solution of 4 (4.0 g, 12.5 mmol) in dry THF was added BuLi
(1.6 M in hexane) (17.2 mL, 27.5 mmol) at ꢀ78 °C under nitrogen,
and the mixture was warmed up to room temperature with stirring
for 1 h. The reaction mixture was then cooled down to ꢀ78 °C and
ethyl chloroformate (1.62 g, 15.0 mmol) was added. The resulting
reaction mixture was stirred at room temperature for 30 min.
The reaction was quenched by addition of satd NH4Cl and ex-
tracted with EtOAc. The organic layer was washed with brine, dried
(Na2SO4), and concentrated under reduced pressure. The residue
was chromatographed with 20:1 hexanes/EtOAc to provide a color-
less oil in 96% yield; IR (KBr) 1704, 2211; 1H NMR (CDCl3) d 1.35 (t,
3H, J = 7.2), 3.88 (s, 3H), 3.91 (s, 3H), 4.29 (q, 2H, J = 7.2), 6.84 (d,
1H, J = 8.4), 7.06 (d, 1H, J = 2.0), 7.23 (dd, 1H, J = 8.4, 2.0); 13C
NMR (CDCl3) d 13.2, 55.2, 55.1, 61.0, 79.1, 86.1, 110.3, 110.6,
114.5, 126.3, 148.0, 150.7, 153.3.
3.6. 11-Hydroxymethyl-2,3-dimethoxy-8,9-methylene-
dioxybenzo[i]phenanthridine (10)
To a stirred solution of LAH (41.8 mg, 1.1 mmol) in 20 mL THF
was added a solution of 9 in THF (223.3 mg, 0.55 mmol) dropwise
at 0 °C. The resulting reaction suspension was stirred for 2 h at 0 °C,
and then carefully quenched by sequential addition of 0.05 mL
water, 0.05 mL of 15% NaOH, and 0.15 mL water. The resulting
reaction mixture was filtered and the filtrate was concentrated un-
der reduced pressure. The residue was chromatographed with 10:1
CHCl3/MeOH to provide a yellow powder in 57% yield; mp 270–
272 °C; IR (KBr) 3448; 1H NMR (DMSO-d6) d 3.95 (s, 3H), 4.07 (s,
3H), 5.08 (s, 2H), 5.88 (s, 1H), 6.25 (s, 2H), 7.54 (s, 1H), 7.56 (s,
1H), 8.19 (s, 1H), 8.37 (s, 1H), 8.57 (s, 1H), 10.13 (s, 1H); 13C
NMR (DMSO-d6) d 55.5, 55.8, 64.2, 101.7, 102.7, 104.2, 106.3,
107.7, 119.7, 121.5, 124.4, 126.3, 130.3, 132.4, 133.1, 143.1,
146.3, 147.4, 148.1, 149.6, 150.4; HRMS calcd for C21H17NO5H:
364.1185; found: 364.1196.
3.3. Ethyl (Z)-3-(3,4-dimethoxyphenyl)-2-(tributyl-
stannyl)propenoate (6)
To a solution of 5 (510 mg, 2.18 mmol) and Bu3SnH (666 mg,
2.29 mmol) in benzene was added AIBN (9 mg, 0.055 mmol) under
nitrogen, and the mixture was stirred at room temperature for
19 h. Solvent was removed and the residue was chromatographed
with 5:1 hexanes/EtOAc to provide a colorless oil in 66% yield; 1H
NMR (CDCl3) d 0.70–1.00 (m, 15H), 1.00–1.40 (m, 15H), 3.75 (s,
6H), 4.11 (q, 2H, J = 7.0), 6.69–6.75 (m, 3H), 8.21 (s, 1H); 13C
NMR (CDCl3) d 10.9, 12.6, 13.4, 26.2, 28.0, 54.9, 54.9, 59.6, 110.0,
110.6, 119.9, 130.7, 136.5, 147.8, 148.8, 152.7, 170.8.
3.7. 11-Formyl-2,3-dimethoxy-8,9-methylene-
dioxybenzo[i]phenanthridine (11)
A solution of 10 (363 mg, 1 mmol), MnO2 (870 mg, 10 mmol) in
30 mL DMF was stirred for 2 h at room temperature. The resulting
reaction mixture was filtered through a Celite bed and the filtrate
was concentrated under reduced pressure. The residue was chro-
matographed with 20:1 CH2Cl2/MeOH to provide a brown powder
in 62% yield; mp 250–255 °C; IR (KBr) 1685; 1H NMR
(CDCl3 + CD3OD) d 3.99 (s, 3H), 4.11 (s, 3H), 6.11 (s, 1H), 7.36 (s,
1H), 7.39 (s, 1H), 7.49 (s, 1H), 8.05 (s, 1H), 8.43 (s, 1H), 9.79 (s,
1H), 10.51 (s, 1H); HRMS calcd for C21H15NO5H: 362.1028; found:
362.1039.
3.4. 2-(6,7-Methylenedioxyquinolin-4-yl)-3-(4,5-dimethoxy-
phenyl)acrylic acid ethyl ester (8)
A mixture of 6 (500 mg, 0.95 mmol), 4-bromo-6,7-methylenedi-
oxyquinoline, 7 (240 mg, 0.95 mmol), and PdCl2(PPh3)2 (33.4 mg,
0.048 mmol) in DMF (15 mL) was stirred under nitrogen at 60 °C
for 5 h. A solution of aq KF (276.1 mg, 4.76 mmol) was added, and
the reaction mixture was stirred for 1 h. The insoluble material
was filtered off and the filtrate was extracted with dichlorometh-
ane. The organic layer was washed with brine, dried, and concen-
trated under reduced pressure. The residue was chromatographed
with 1:1 hexanes/EtOAc to provide a light yellow sticky gum in
57% yield; IR (KBr) 1705; 1H NMR (CDCl3) d 1.23 (t, 3H, J = 7.0),
3.18 (s, 3H), 3.80 (s, 3H), 4.26 (q, 2H, J = 7.0), 6.06 (m, 2H), 6.22 (d,
1H, J = 2.2), 6.68 (d, 1H, J = 8.4), 6.82 (dd, 1H, J = 8.4, 2.2), 7.06 (s,
1H), 7.19 (d, 1H, J = 4.8), 7.44 (s, 1H), 8.05 (s, 1H), 8.76 (d, 1H,
J = 4.8); 13C NMR (CDCl3) d 13.3, 54.0, 54.9, 60.4, 99.4, 100.9,
105.3, 109.8, 111.1, 119.7, 123.3, 124.86 125.1, 125.6, 141.2,
141.8, 146.2, 147.3, 147.5, 147.6, 149.7, 150.0, 166.0; HRMS calcd
for C23H21NO6H: 408.1447; found: 408.1437.
3.8. 11-Aminomethyl-2,3-dimethoxy-8,9-methylene-
dioxybenzo[i]phenanthridine (12)
To a solution of 11 (7.2 mg, 0.02 mmol) and ammonium acetate
(15.4 mg, 0.2 mmol) in 3 mL MeOH was added a solution of
NaBH3CN (9.4 mg, 0.15 mmol in 0.1 mL MeOH) at 0 °C. The result-
ing reaction solution was stirred for an additional 30 min at 0 °C,
and then two drops of AcOH were added. The reaction mixture
was warmed up to 40 °C with stirring for another 2 h. The resulting
mixture was quenched by 0.1 mL of 1 N NaOH solution, concen-
trated, and extracted using CHCl3 (2ꢁ 15 mL). The organic layer
was concentrated and the residue was chromatographed with
15:1 CH2Cl2/MeOH to provide a yellow powder in 47% yield; mp
189–194 °C; 1H NMR (CDCl3) d 4.07 (s, 3H), 4.15 (s, 3H), 4.61 (s,
2H), 6.17 (s, 2H), 7.26 (s, 1H), 7.60 (s, 1H), 8.05 (s, 1H), 8.12 (s,
1H), 8.56 (s, 1H), 9.94 (s, 1H); HRMS calcd for C21H18N2O4H:
363.1339; found: 363.1342.
3.5. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenan-
thridine-11-carboxylic acid ethyl ester (9)
3.9. 11-Dimethylaminomethyl-2,3-dimethoxy-8,9-methylene-
A solution of 8 (100 mg, 0.245 mmol) and iodine (2 mg) in ben-
zene (250 mL) was irradiated by a Hanovia 450 W medium-pres-
sure lamp through a Pyrex filter for 6 h, with air bubbling
through the reaction mixture. The solvent was removed under re-
dioxybenzo[i]phenanthridine (13)
To a solution of 11 (7.2 mg, 0.02 mmol) and dimethylamine
(0.2 mmol, 2 M in THF) in 3 mL MeOH was added a solution of

8604
W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 8598–8606
NaBH3CN (9.4 mg, 0.15 mmol in 0.1 mL MeOH) at 0 °C. The result-
ing reaction solution was stirred for an additional 30 min at 0 °C,
and then two drops of AcOH were added. The reaction mixture
was warmed up to 40 °C with stirring for another 2 h. The resulting
mixture was quenched by 0.1 mL of 1 N NaOH solution, concen-
trated, and extracted using CHCl3 (2ꢁ 15 mL). The organic layer
was concentrated and the residue was chromatographed with
15:1 CH2Cl2/MeOH to provide a yellow powder in 55% yield; mp
220–222 °C; 1H NMR (CDCl3) d 2.48 (s, 6H), 3.93 (s, 2H), 4.08 (s,
3H), 4.15 (s, 3H), 6.16 (s, 2H), 7.28 (s, 1H), 7.58 (s, 1H), 7.92 (s,
1H), 8.14 (s, 1H), 8.93 (s, 1H), 9.93 (s, 1H); HRMS calcd for
of NaBH3CN (9.4 mg, 0.15 mmol in 0.1 mL MeOH) at 0 °C. The
resulting reaction solution was stirred for an additional 30 min at
0 °C, and then two drops of AcOH were added. The reaction mix-
ture was warmed up to room temperature with stirring for another
2 h. The resulting mixture was quenched by 0.1 mL 1 N NaOH solu-
tion, concentrated, and extracted by CHCl3 (2ꢁ 15 mL). The organ-
ic layer was concentrated and the residue was chromatographed
with 15:1 CH2Cl2/MeOH to provide a yellow powder in 53% yield;
mp 199–201 °C; 1H NMR (CDCl3 + CD3OD) d 2.46 (s, 6H), 2.85 (t,
2H, J = 7.8), 3.65 (t, 2H, J = 7.8), 4.07 (s, 3H), 4.14 (s, 3H), 6.17 (s,
2H), 7.28 (s, 1H), 7.60 (s, 1H), 7.89 (s, 1H), 8.10 (s, 1H), 8.18 (s,
1H), 9.94 (s, 1H); HRMS calcd for C24H24N2O4H: 405.1814; found:
405.1809.
C23H22N2O4H: 391.1652; found: 391.1654.
3.10. 11-(2-Nitrovinyl)-2,3-dimethoxy-8,9-methylene-
dioxybenzo[i]phenanthridine (14)
3.14. 11-[(3-Dimethylamino)prop-1-enyl]-2,3-dimethoxy-8,9-
methylenedioxybenzo[i]phenanthridine (18)
A suspension of 11 (36 mg, 0.1 mmol), ammonium acetate
(38.6 mg, 0.5 mmol) in 2 mL nitromethane was stirred overnight
at 80 °C. The resulting reaction mixture was triturated in the pres-
ence of a small amount of CH2Cl2, and the residue was pure enough
and used for the next reaction without further purification; yield:
89%; mp 241–245 °C; IR (KBr) 1509; 1H NMR (CDCl3 + CD3OD) d
4.08 (s, 3H), 4.18 (s, 3H), 6.19 (s, 2H), 7.33 (s, 1H), 7.63 (s, 1H),
7.67 (s, 1H), 7.76 (d, 1H, J = 13.2), 8.06 (s, 1H), 8.12 (s, 1H), 8.80
(d, 1H, J = 13.2), 9.91 (s, 1H); HRMS calcd for C22H16N2O6H:
405.1087; found: 405.1089.
To a suspension of 2-(dimethylamino)ethyltriphenylphosphoni-
um bromide (40 mg, 0.095 mmol) in 5 mL THF was added LiHMDS
(1.0 M in THF, 0.1 mmol) dropwise at room temperature. A solu-
tion of 11 (0.08 mmol) in THF was added to the reaction mixture
dropwise, and the resulting solution was stirred for 1.5 h,
quenched by 0.1 mL water, concentrated, and partitioned into
CH2Cl2/water. The organic layer was again concentrated under re-
duced pressure and the residue was chromatographed with 20:1
CH2Cl2/MeOH to provide a yellow powder in 47% yield; two insep-
arable isomers were obtained. The major isomer: mp 220–225 °C;
1H NMR (CDCl3 + CD3OD) d 2.47 (s, 6H), 3.29 (d, 2H, J = 6.6), 4.08 (s,
3H), 4.16 (s, 3H), 6.18 (s, 2H), 6.35 (dt, 1H, J = 15.8, 6.6), 7.27 (s, 1H),
7.29 (d, 1H, J = 15.8), 7.58 (s, 1H), 7.71 (s, 1H), 8.13 (s, 1H), 8.62 (s,
1H), 9.94 (s, 1H); HRMS calcd for C25H24N2O4H: 417.1814; found:
407.1801.
3.11. 11-(2-Nitroethyl)-2,3-dimethoxy-8,9-methylene-
dioxybenzo[i]phenanthridine (15)
To a stirred suspension of NaBH4 (38 mg, 1 mmol) in 10 mL 1,4-
dioxane/EtOH (2:1) was added a solution of 14 (80.8 mg, 0.2 mmol)
in 1,4-dioxane (5 mL) dropwise at 0 °C. After this addition, the
reaction mixture was stirred for an additional 30 min. The result-
ing reaction mixture was diluted with ice-water and quenched
with 50% aqueous AcOH. The resulting suspension was concen-
trated and then partitioned between sat NaHCO3 and CH2Cl2. The
organic layer was again concentrated and the residue was chro-
matographed with 10:1 CH2Cl2/MeOH to provide a yellow powder
in 69% yield; mp 277–280 °C; IR (KBr) 1554; 1H NMR
(CDCl3 + CD3OD) d 3.99 (s, 3H), 4.08 (s, 3H), 4.16 (t, 2H, J = 7.6),
4.79 (t, 2H, J = 7.6), 6.13 (s, 2H), 7.19 (s, 1H), 7.52 (s, 1H), 7.82 (s,
1H), 7.94 (s, 1H), 8.04 (s, 1H), 9.82 (s, 1H); HRMS calcd for
3.15. 11-[(3-Dimethylamino)propyl]-2,3-dimethoxy-8,9-
methylenedioxybenzo[i]phenanthridine (19)
A suspension of 18 (15 mg, 0.04 mmol) and Pd–C (10 mg) in
10 mL ethanol was shaken under hydrogen (40 psi) for 24 h. The
mixture was filtered through Celite and concentrated under re-
duced pressure. The residue was chromatographed with 20:1
CH2Cl2/MeOH to provide a yellow powder in 44% yield; mp 204–
209 °C; 1H NMR (CDCl3) d 2.10 (m, 2H), 2.34 (s, 6H), 2.54 (t, 2H,
J = 7.8), 3.49 (t, 2H, J = 7.8), 4.07 (s, 3H), 4.14 (s, 3H), 6.17 (s, 2H),
7.26 (s, 1H), 7.60 (s, 1H), 7.89 (s, 1H), 8.12 (s, 1H), 8.14 (s, 1H),
9.95 (s, 1H); HRMS calcd for C25H26N2O4H: 419.1971; found:
419.1978.
C22H18N2O6H: 407.1243; found: 407.1231.
3.12. 11-(2-Aminoethyl)-2,3-dimethoxy-8,9-methylene-
dioxybenzo[i]phenanthridine (16)
3.16. 11-Aminocarbonylmethyl-2,3-dimethoxy-8,9-methylene-
To a stirred suspension of 15 (50 mg, 0.12 mmol) in 1.0 mL ace-
tic acid was added Zn power (15.7 mg, 0.24 mmol) portionwise at
room temperature. The reaction mixture was stirred for an addi-
tional 3 h, diluted with saturated sodium bicarbonate, and then fil-
tered. The filtrate was concentrated, and partitioned between satd
NaHCO3 and CH2Cl2. The organic layer was again concentrated and
the residue was chromatographed with 10:1:0.1 CH2Cl2/MeOH/
TEA to provide a yellow-green powder in 41% yield; mp 192–
198 °C; 1H NMR (CDCl3 + CD3OD) d 3.27 (t, 2H, J = 6.6), 3.62 (t,
2H, J = 6.6), 4.07 (s, 3H), 4.14 (s, 3H), 6.17 (s, 2H), 7.24 (s, 1H),
7.60 (s, 1H), 7.87 (s, 1H), 8.11 (s, 1H), 8.13 (s, 1H), 9.94 (s, 1H);
HRMS calcd for C22H20N2O4H: 377.1501; found: 377.1497.
dioxybenzo[i]phenanthridine (20)
A mixture of 9 (41 mg, 0.1 mmol) in 10% NaOH (5 mL) and eth-
anol (10 mL) was heated to reflux with stirring for 1 h. The reaction
mixture was acidified with 2 N HCl to pH 4, and then evaporated to
dryness. The residue was suspended in 10 mL dichloromethane
and 0.5 mL thionyl chloride was added. The resulting reaction mix-
ture was refluxed for 2 h and then concentrated. The reaction res-
idue was again suspended in dichloromethane and 0.5 mL
triethylamine was added. After 15 min, 0.5 mL ammonia solution
(2.0 M in THF) was added, and the resulting reaction mixture
was refluxed for 1 h. The organic solvent and excess amine were
removed under reduced pressure, and the residue was chromato-
graphed with 20:1 CH2Cl2/MeOH to provide an off-white powder
in 65% yield; mp 265–269 °C; IR (KBr) 1647; 1H NMR (CD3COOD)
d 3.95 (s, 3H), 4.09 (s, 3H), 6.25 (s, 2H), 7.56 (s, 1H), 7.64 (s, 1H),
7.94 (br, 1H), 8.08 (s, 2H), 8.36 (br, 1H), 8.42 (s, 1H), 10.18 (s,
1H); 13C NMR (CD3COOD) d 55.5, 55.9, 101.9, 102.0, 102.7, 106.4,
3.13. 12-(2-Dimethylamino)ethyl-2,3-dimethoxy-8,9-
methylenedioxybenzo[i]phenanthridine (17)
To a solution of 16 (8 mg, 0.02 mmol) and formaldehyde
(0.1 mL, 37% solution in H2O) in 3 mL MeOH was added a solution

W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 8598–8606
8605
108.2, 118.8, 120.9, 124.7, 125.6, 130.6, 142.7, 146.1, 147.2, 148.5,
149.8, 150.9, 173.0; HRMS calcd for C21H16N2O5H: 377.1132;
found: 377.1134.
2.64 (t, 2H, J = 6.0), 3.71 (m, 2H), 4.03 (s, 3H), 4.11 (s, 3H), 6.09
(s, 2H), 7.15 (br, 1H), 7.18 (s, 1H), 7.38 (s, 1H), 7.66 (s, 1H), 7.85
(s, 1H), 7.94 (s, 1H), 9.61 (s, 1H); 13C NMR (CDCl3) d 36.9, 44.2,
55.1, 55.2, 56.7, 100.9, 101.1, 101.7, 106.1, 107.1, 118.4, 120.4,
124.8, 125.1, 126.5, 129.3, 129.9, 142.4, 144.3, 146.8, 148.0,
149.3, 150.4, 171.0; HRMS calcd for C25H25N3O5H: 448.1872;
found: 448.1870.
3.17. 11-(N-Methylamino)carbonylmethyl-2,3-dimethoxy-8,9-
methylenedioxybenzo[i]phenanthridine (21)
A mixture of 9 (41 mg, 0.1 mmol) in 10% NaOH (5 mL) and eth-
anol (10 mL) was heated to reflux with stirring for 1 h. The reaction
mixture was acidified with 2 N HCl to pH 4, and then evaporated to
dryness. The residue was suspended in 10 mL dichloromethane
and 0.5 mL thionyl chloride was added. The resulting reaction mix-
ture was refluxed for 2 h and then concentrated. The reaction res-
idue was again suspended in dichloromethane and 0.5 mL
triethylamine was added. After 15 min, 0.5 mL methylamine
(2.0 M in THF) was added and the resulting reaction mixture was
refluxed for 1 h. The organic solvent and excess amine were re-
moved under reduced pressure, and the residue was chromato-
graphed with 20:1 CH2Cl2/MeOH to provide an off-white powder
in 67% yield; mp 271–273 °C; IR (KBr) 1653; 1H NMR (DMSO-d6)
d 2.91 (d, 3H, J = 4.4), 4.08 (s, 3H), 4.08 (s, 3H), 6.24 (m, 2H), 7.55
(s, 1H), 7.63 (s, 1H), 7.73 (s, 1H), 8.06 (s, 1H), 8.42 (s, 1H), 9.00
(br, 1H), 10.19 (s, 1H); 13C NMR (CDCl3) d 26.2, 55.5, 55.9, 101.6,
101.9, 102.7, 106.5, 108.1, 118.7, 120.8, 124.8, 125.6, 130.1,
130.3, 142.8, 146.1, 146.1, 147.2, 148.5, 149.8, 151.0, 171.3; HRMS
calcd for C22H18N2O5H: 391.1288; found: 391.1290.
3.20. Topoisomerase-mediated DNA cleavage assays
Human topoisomerase I was expressed in Escherichia coli and
isolated as a recombinant fusion protein using a T7 expression
system as described previously.31 Plasmid YepG was also puri-
fied by the alkali lysis method followed by phenol deprotein-
ation and CsCl/ethidium isopycnic centrifugation method as
described.32 The 30-end labeling of the plasmid was accom-
plished by digestion with a restriction enzyme followed by end
filling with Klenow polymerase as previously described.33 The
cleavage assays were performed as previously reported.34,35 The
drug and the DNA in the presence of topoisomerase I was incu-
bated for 30 min at room temperature. The reactions were termi-
nated by the addition of 5 mL of 5% SDS and 1 mg/mL protein
kinase K with an additional 1 h of incubation at 37 °C. Samples
were then alkali denatured by the addition of NaOH, EDTA, su-
crose, and bromophenol blue to final concentrations of 75 mM,
2.5%, and 0.05 mg/mL, respectively, prior to loading onto a neu-
tral agarose gel. After development of the gels, typically 24-h
exposure was used to obtain autoradiograms outlining the ex-
tent of DNA fragmentation. Topoisomerase I-mediated DNA
cleavage values are reported as relative effective concentration
(REC), that is, concentrations relative to camptothecin, whose va-
lue is arbitrarily assumed as 0.2, that are able to produce the
same 10% cleavage on the plasmid DNA in the presence of hu-
man topoisomerase I.
3.18. 11-(N,N-Dimethylamino)carbonylmethyl-2,3-dimethoxy-
8,9-methylenedioxybenzo[i]phenanthridine (22)
A mixture of 9 (41 mg, 0.1 mmol) in 10% NaOH (5 mL) and eth-
anol (10 mL) was heated to reflux with stirring for 1 h. The reaction
mixture was acidified with 2 N HCl to pH 4, and then evaporated to
dryness. The residue was suspended in 10 mL dichloromethane
and 0.5 mL thionyl chloride was added. The resulting reaction mix-
ture was refluxed for 2 h and then concentrated. The reaction res-
idue was again suspended in dichloromethane and 0.5 mL
triethylamine was added. After 15 min, 0.5 mL dimethylamine
(2.0 M in tetrahydrofuran) was added, and the resulting reaction
mixture was refluxed for 1 h. The organic solvent and the excess
amine were removed under reduced pressure, and the residue
was chromatographed with 20:1 CH2Cl2/MeOH to provide an off-
white powder in 75% yield; mp 271–275 °C; IR (KBr) 1621; 1H
NMR (CDCl3) d 2.68 (s, 3H), 3.31 (s, 3H), 4.06 (s, 3H), 4.17 (s, 3H),
6.15 (m, 2H), 7.27 (s, 1H), 7.58 (s, 1H), 7.72 (s, 1H), 7.89 (s, 1H),
8.15 (s, 1H), 9.97 (s, 1H); 13C NMR (CDCl3) d 34.4, 37.5, 55.2,
55.3, 100.2, 101.0, 101.3, 106.7, 107.1, 118.9, 120.3, 124.7, 125.6,
126.5, 128.5, 128.8, 142.7, 144.8, 147.8, 148.3, 149.5, 150.5,
171.8; HRMS calcd for C23H20N2O5H: 405.1445; found: 405.1447.
3.21. Cytotoxicity assays
The cytotoxicity was determined using the MTT-microtiter
plate tetrazolinium cytotoxicity assay (MTA). The human lym-
phoblast RPMI 8402 and its camptothecin-resistant variant cell
line CPT-K5 were provided by Dr. Toshiwo Andoh (Aichi Cancer
Center Research Institute, Nagoya, Japan).28 The P388 mouse leu-
kemia cell line and its CPT-resistant TOP1-deficient variant P388/
CPT4529 were obtained from Michael R. Mattern and Randal K.
Johnson (GlaxoSmithKline, King of Prussia, PA). The KB3-1 cell
line and its multidrug-resistant variant KBV-130 were obtained
from K.V. Chin (The Cancer Institute of New Jersey, New Bruns-
wick, NJ). The KBH5.0 cell line as noted previously18 was derived
from KB3-1 by stepwise selection against Hoechst 33342. The
cytotoxicity assay was performed using 96-well microtiter
plates. Cells were grown in suspension at 37 °C in 5% CO2 and
maintained by regular passage in RPMI medium supplemented
3.19. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenan-
thridine-11-carboxylic acid 2-(dimethylamino)-ethylamide (23)
with 10% heat-inactivated fetal bovine serum,
L-glutamine
A mixture of 9 (41 mg, 0.1 mmol) in 10% NaOH (5 mL) and eth-
anol (10 mL) was heated to reflux with stirring for 1 h. The reaction
mixture was acidified with 2 N HCl to pH 4, and then evaporated to
dryness. The residue was suspended in 10 mL dichloromethane
and 0.5 mL thionyl chloride was added. The resulting reaction mix-
ture was refluxed for 2 h and then concentrated. The reaction res-
idue was again suspended in dichloromethane and 0.5 mL
triethylamine was added. After 15 min, 0.5 mL N,N-dimethylethyl-
enediamine was added, and the resulting reaction mixture was re-
fluxed for 1 h. The organic solvent and excess amine were removed
under reduced pressure, and the residue was chromatographed in
20:1 CH2Cl2/MeOH to provide an off-white powder in 63% yield;
mp 221–225 °C; IR (KBr) 1635; 1H NMR (CDCl3) d 2.26 (s, 6H),
(2 mM), penicillin (100 U/mL), and streptomycin (0.1 mg/mL).
For determination of IC50, cells were exposed continuously for
four days to varying concentrations of drug, and MTT assays
were performed at the end of the fourth day. Each assay was
performed with a control that did not contain any drug. All as-
says were performed at least twice in six replicate wells.
Acknowledgments
This study was supported by the National Cancer Institute,
Grants CA098127 (E.J.L.) and CA39662 (L.F.L.), and Genzyme
Corporation.

8606
W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 8598–8606
19. Ruchelman, A. L.; Zhou, N.; Liu, A.; Liu, L. F.; LaVoie, E. J. J. Med. Chem. 2005, 48,
References and notes
792.
20. Ruchelman, A. L.; Singh, S. K.; Ray, A.; Wu, X.; Yang, J.-M.; Zhou, N.; Liu, A.; Liu,
L. F.; LaVoie, E. J. Bioorg. Med. Chem. 2004, 12, 795.
21. Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 13, 3769.
22. Sai, H.; Ogiku, T.; Nishitani, T.; Hiramatsu, H.; Horikawa, H.; Iwasaki, T.
Synthesis 1995, 582.
23. Margolis, B. J.; Long, K. A.; Laird, D. L.; Ruble, J. C.; Pulley, S. R. J. Org. Chem. 2007,
72, 2232.
24. Stille, J. K. Angew. Chem. Int. Ed. 1986, 25, 508.
25. Amin, S.; Hecht, S. S.; LaVoie, E.; Hoffmann, D. J. Med. Chem. 1979, 22, 1336.
26. Ruchelman, A. L.; Zhu, S.; Zhou, N.; Liu, A.; Liu, L. F.; LaVoie, E. J. Bioorg. Med.
Chem. Lett. 2005, 15, 5585.
27. Zhu, S.; Ruchelman, A. L.; Zhou, N.; Liu, A.; Liu, L. F.; LaVoie, E. J. Bioorg. Med.
Chem. 2005, 13, 6782.
28. Andoh, T.; Ishii, K.; Suzuki, Y.; Ikegami, Y.; Kusunoki, Y.; Takemoto, Y.; Okada,
K. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5565.
29. Woessner, R. D.; Eng, W. K.; Hofmann, G. A.; Rieman, D. J.; McCabe, F. L.;
Hertzberg, R. P.; Mattern, M. R.; Tan, K. B.; Johnson, R. K. Oncol. Res. 1992, 4,
481.
30. Gervasoni, J. E., Jr.; Fields, S. Z.; Krishna, S.; Baker, M. A.; Rosado, M.;
Thuraisamy, K.; Hindenburg, A. A.; Taub, R. N. Cancer Res. 1991, 51, 4955.
31. Makhey, D.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J. Bioorg. Med. Chem. 2000, 8,
1171.
1. Champoux, J. J. Annu. Rev. Biochem. 2001, 70, 369.
2. Wang, J. C. Nat. Rev. Mol. Cell Biol. 2002, 3, 430.
3. Li, T. K.; Liu, L. F. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 53.
4. Pommier, Y. Nat. Rev. Cancer 2006, 6, 789.
5. Hsaing, Y. H.; Hertsberg, R.; Hecht, S.; Liu, L. F. J. Biol. Chem. 1985, 260, 14873.
6. Burke, T. G.; Mi, Z. J. Med. Chem. 1993, 36, 2580.
7. Burke, T. G.; Mi, Z. Anal. Biochem. 1993, 212, 285.
8. Bom, D.; Curran, D. P.; Chavan, A. J.; Kruszewski, S.; Zimmer, S.; Fraley, K. A.;
Burke, T. G. J. Med. Chem. 1999, 42, 3018.
9. Chu, X.; Kato, Y.; Sugiyama, Y. Cancer Res. 1997, 57, 1934.
10. Kawabata, S.; Oka, M.; Shiozawa, K.; Tsukamoto, K.; Nakatomi, K.; Soda, H.;
Fududa, M.; Ikegami, Y.; Sugahara, K.; Yamada, Y.; Kamihira, S.; Doyle, L. A.;
Ross, D. D.; Kohno, S. Biochem. Biophys. Res. Commun. 2001, 280, 1216.
11. Robey, R. W.; Medina-Perez, W. Y.; Nishiyama, K.; Lahusen, T.; Miyake, K.;
Litman, T.; Senderowicz, A. M.; Ross, D. D.; Bates, S. E. Clin. Cancer Res. 2001, 7,
145.
12. Mattern, M. R.; Hofmann, G. A.; Polsky, R. M.; Funk, L. R.; McCabe, F. L.; Johnson,
R. K. Oncol. Res. 1993, 5, 467.
13. Erlichman, C.; Boerner, S. A.; Hallgren, C. G.; Spieker, R.; Wang, X.-Y.; James, C.
D.; Scheffer, G. L.; Maliepaard, M.; Ross, D. D.; Bible, K. C.; Kaufmann, S. H.
Cancer Res. 2001, 61, 739.
14. Yang, C.-H.; Schneider, E.; Kuo, M.-L.; Volk, E. L.; Rocchi, E.; Chen, T.-C. Biochem.
Pharmacol. 2000, 60, 831.
15. Ross, D. D. Leukemia 2000, 14, 467–473.
16. Ruchelman, A. L.; Singh, S. K.; Wu, X. H.; Ray, A.; Yang, J.-M.; Li, T.-K.; Liu, A.;
Liu, L. F.; LaVoie, E. J. Bioorg. Med. Chem. Lett. 2002, 12, 3333.
17. Ruchelman, A. L.; Singh, S. K.; Ray, A.; Wu, X.; Yang, J.-M.; Li, T.-K.; Liu, A.; Liu, L.
F.; LaVoie, E. J. Bioorg. Med. Chem. 2003, 11, 2061.
18. Li, T.-K.; Houghton, P. J.; Desai, S. D.; Daroui, P.; Liu, A. A.; Hars, E. S.;
Ruchelman, A. L.; LaVoie, E. J.; Liu, L. F. Cancer Res. 2003, 63, 8400.
32. Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular Cloning, a Laboratory Manual;
Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 2000. pp 149–185.
33. Tewey, K. M.; Rowe, T. C.; Yang, L.; Hallogan, B. C.; Liu, L. F. Science 1984, 226,
466.
34. Gatto, B.; Sanders, M. M.; Yu, C.; Wu, H.-Y.; Makhey, D.; LaVoie, E. J.; Liu, L. F.
Cancer Res. 1996, 56, 2795.
35. Wang, H.; Mao, Y.; Chen, A.; Zhou, N.; LaVoie, E. J.; Liu, L. F. Biochemistry 2001,
40, 3316.