6-Substituted 6H-dibenzo[c,h][2,6]naphthyridin-5-ones: Reversed lactam analogues of ARC-111 with potent topoisomerase I-targeting activity and cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2005.12.028

6-Substituted 8,9-dimethoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6] naphthyridin-5-ones were synthesized and evaluated for topoisomerase I-targeting activity and cytotoxicity. Several of these reversed lactam analogues of ARC-111 exhibited exceptional cyt

Full text of DOI:10.1016/j.bmc.2005.12.028

Bioorganic & Medicinal Chemistry 14 (2006) 3131–3143
6-Substituted 6H-dibenzo[c,h][2,6]naphthyridin-5-ones:
Reversed lactam analogues of ARC-111 with potent
topoisomerase I-targeting activity and cytotoxicity
Shejin Zhu,a Alexander L. Ruchelman,a Nai Zhou,b Angela Liu,b
Leroy F. Liub,c and Edmond J. LaVoiea,c,
*
aDepartment of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA
bDepartment of Pharmacology, The University of Medicine and Dentistry of New Jersey,
Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
cThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
Received 29 August 2005; revised 13 December 2005; accepted 16 December 2005
Available online 18 January 2006
Abstract—6-Substituted 8,9-dimethoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-ones were synthesized and evaluat-
ed for topoisomerase I-targeting activity and cytotoxicity. Several of these reversed lactam analogues of ARC-111 exhibited excep-
tional cytotoxicity with IC50 values ranging from 0.5 to 3.0 nM. In contrast to topotecan, no resistance was observed with several of
these reversed lactam analogues in tumor cell lines that overexpressed the efflux transporters MDR1 or BCRP.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Benzo[i]phenanthridines and dibenzo[c,h]cinnolines can
exert TOP1-targeting activity and cytotoxicity against
several human tumor cell lines.12–16 Specific 5H-dib-
Topoisomerases are nuclear enzymes that control the
topology of DNA and are critical for replication and
transcription. Stabilization of the cleaved complex
formed between the enzyme and DNA by TOP1-target-
ing agents such as camptothecin can be lethal to tumor
cells.1–4 Studies on camptothecin and its structurally
related analogues have resulted in the development of
two clinical anticancer agents, topotecan (Hycamtinꢁ)
and irinotecan (CPT-11/Camptosarꢁ). These clinical
agents possess a similar camptothecin ring system,
which incorporates a d-lactone. Hydrolysis of this lac-
tone results in the loss of TOP1-targeting activity. In
addition, the resulting hydroxy acid derivative has a
high binding affinity for human serum albumin.5–7 The
instability of this lactone together with data that have
identified topotecan and irinotecan as substrates for ef-
flux transporters associated with multi-drug resis-
tance8–11 have prompted further research into the
development of novel TOP1-targeting agents.
enzo[c,h][1,6]naphthyridin-6-ones
and
11H-isoqui-
no[4,3-c]cinnolin-12-ones have also been identified
that possess exceptional TOP1-targeting activity and
cytotoxicity.17–25 The dibenzo[c,h][1,6]naphthyridin-6-
one 1 (ARC-111, topovaleꢁ) and the isoquino[4,3-
c]cinnolin-12-one 2 (Fig. 1) were among the more ac-
tive compounds that were evaluated. These studies
were extended to include the synthesis and evaluation
of several 2-aminoethyl esters and amides of 2,3-dime-
thoxy-8,9-methylenedioxybenzo[i]phenanthridine-12-
carboxylic acid.26,27 In the present study, the
structure–activity trends associated with the reversed
lactam analogues of 1 were investigated. Specifically,
we synthesized several 6-substituted 8,9-dimethoxy-
2,3-methylenedioxydibenzo[c,h][2,6]naphthyridin-6-
ones. These structurally related analogues of 1 were
assayed for TOP1-targeting activity and cytotoxicity
in RPMI8402, as well as its camptothecin-resistant
variant, CPT-K5.28 In addition, the relative cytotoxic
activity of these compounds as compared to that of
the parent cell line KB3-1 was assessed in KBV-1
cells,29 which overexpress MDR1 and KBH5.0 cells,19
which overexpress BCRP. Both MDR1 and BCRP are
Keywords: Topoisomerase I; Cytotoxic; Antitumor; Dibenzo
[c,h][2,6]naphthyridin-5-ones.
*
Corresponding author. E-mail: elavoie@rci.rutgers.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.028

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S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
CH3
CH2N
O
CH3
N
HO
O
N
N
O
N
O
CH3CH2
OH
O
CH3CH2
OH
O
Camptothecin
Topotecan
N
N
N
N
O
O
O
O
N
H3CO
H3CO
H3CO
H3CO
CH2CH2
N
H3C
CH2CH2
N
H3C
O
O
CH3
CH3
2
1, ARC-111
12
N
1
2
11
O
O
10
7
9
H3CO
H3CO
3
4
N
O
8
R
Reversed Amide Analogues of ARC-111
Figure 1.
efflux transporters that have been associated with the
multi-drug resistance of tumor cells.30,31
The other chloroalkanes used to form these tertiary
amides were commercially available. Photocyclization
of 9a–e and 9g–j using 2% HCl as solvent provided
the desired compounds 10a–e and 10g–j.
2. Chemistry
Conversion of 9a to 10a employing Heck cyclization
conditions did provide the desired product together with
undesired side products that were not characterized,
Scheme 2. While chromatographic separation of 10a
proved difficult, the desired product could be separated
by trituration with small amounts of methylene chloride
wherein the by-products had greater solubility. Photo-
cyclization of the N-benzyl-substituted analogue 9j
resulted in several unidentified products with only a
5% yield of 10j and in the case of 9k little or no detect-
able amounts of the desired product 10k were formed.
In addition to compound 9a, Heck reaction conditions
were also employed for 9f, 9j, and 9k, which provided
acceptable yields of the cyclized products 10f, 10j, and
10k, Scheme 2. No effort was made to identify the side
products formed during these Heck cyclization reac-
tions. For both 10j and 10k, the benzyl groups were re-
moved in near-quantitative yield using palladium black
in acetic acid with formic acid as the hydrogen source,
Scheme 3, to provide 10l and 10m, respectively.25
Several 6H-dibenzo[c,h][2,6]naphthyridin-5-one reversed
lactam analogues of ARC-111 were prepared as detailed
in Scheme 1. 3,4-Dimethoxyaniline was converted to its
acetanilide using acetyl chloride,32 followed by treat-
ment with iodine monochloride to form the ortho iodo-
acetamide 3. Compound 4 was prepared by hydrolysis
of 3 using aqueous NaOH in ethanol. This compound
served as one of the key intermediates used to make
the 6H-dibenzo[c,h][2,6]naphthyridin-5-ones. 4-Formyl-
6,7-methylenedioxyquinoline 6 was prepared by oxida-
tion of 4-methyl-6,7-methylenedioxyquinoline27 with
SeO2 in dioxane-water. 6,7-Methylenedioxy-4-quinolin-
ecarboxylic acid 7 was prepared by treatment of 6 in
pyridine with an aqueous solution of KMnO4. Conver-
sion of 7 to the acid chloride in situ was performed using
thionyl chloride. Treatment of the acid chloride in
CH2Cl2 and TEA with freshly prepared 4 and subse-
quently allowing this mixture to reflux overnight provid-
ed the secondary amide 8. The intermediate 8 was
treated with sodium hydride to form the amide anion,
which was treated with various substituted alkyl halides
to form tertiary amide intermediates 9a–k. 4-Methyl-1-
(2-chloroethyl)piperidine and 2-(N-benzyl-N-methyla-
mino)-1-chloroethane were prepared by treatment of
their corresponding alcohols with thionyl chloride using
conditions similar to those previously described.31–33
3. Pharmacology
The relative TOP1-targeting activities of several of these
6-substituted 6H-dibenzo[c,h][2,6]naphthyridin-5-ones
are provided in Table 1. A representative gel of the

S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
3133
N
C
O
O
N
H3CO
H3CO
I
O
O
O
b
N
H
CH3
H
CH3
O
3
4
6
5
OCH3
OCH3
c
a
H
N
O
N
O
O
H3CO
H3CO
I
d
O
O
I
+
NH2
CO2H
N
8
7
e
N
O
O
N
O
O
H3CO
H3CO
I
f
9a-e, 9g-j
N
R
O
H3CO
H3CO
N
O
R
9a: R = CH2CH2N(CH3)2
10a: R = CH2CH2N(CH3)2
9b: R = CH2CH(CH3)N(CH3)2
9c: R = CH2CH2N(CH2CH3)2
10b: R = CH2CH(CH3)N(CH3)2
10c: R = CH2CH2N(CH2CH3)2
9d: R = CH2CH2N(-CH2CH2CH2CH2-)
9e: R = CH2CH2N(-CH2(CH2)3CH2-)
10d: R = CH2CH2N(-CH2CH2CH2CH2-)
10e: R = CH2CH2N(-CH2(CH2)3CH2-)
9f: R = CH2CH2N(-CH2)2CHCH3(CH2)2-)
9g: R = CH2CH2N(-CH2CH2OCH2CH2-)
9h: R = CH2CH2CH2N(CH3)2
10g: R = CH2CH2N(-CH2CH2OCH2CH2-)
10h: R = CH2CH2CH2N(CH3)2
9i: R = CH2CH2(-CHN(CH3)CH2CH2CH2-)
9j: R = CH2CH2N(Bn)2
9k: R = CH2CH2N(CH3)Bn
10i: R = CH2CH2(-CHN(CH3)CH2CH2CH2-)
10j: R = CH2CH2N(Bn)2
Scheme 1. Reagents and conditions: (a) NaOH, EtOH, H2O; (b) SeO2, dioxane, H2O; (c) KMnO4, pyr, H2O; (d) first SOCl2, then 4 in CH2Cl2, then
TEA; (e) NaH, DMF, RClÆHCl for 9a–g and 9i–k; NaNH2, PhCH3, RClÆHCl for 9h; (f) 2% HCl, hm.
N
O
O
N
O
O
H3CO
H3CO
I
g
N
O
H3CO
H3CO
N
O
R
R
10a: R = N(CH3)2
10f: R = N(-CH2)2CHCH3(CH2)2-)
10j: R = N(Bn)2
9a,f,j,k
10k: R = N(CH3)Bn
Scheme 2. Reagents: (g) Pd(OAc)2, P(o-tol)3, Ag2CO3, DMF.
resulting DNA fragmentation that occurs in the pres-
ence of a few select compounds and TOP1 is illustrated
in Figure 2. We did not observe in our studies on DNA
cleavage a noteworthy difference in the pattern of DNA
fragments formed as compared to those obtained with
camptothecin. In contrast to what had been observed
with analogues of ARC-111, elongation of the alkyl
chain did not have a dramatic effect on TOP1-targeting
activity. Compound 10a, the reversed lactam of ARC-
111, and its propyl homologue 10h had comparable
intrinsic activity to each other, as well as to topotecan,
as TOP1-targeting agents. The 6-[2-(N-benzylaminoeth-
yl)] analogues 10j and 10k exhibited much less activity as
TOP1-targeting agents than almost all of the other 6H-
dibenzo[c,h][2,6]naphthyridin-5-ones evaluated in this
study. Only the 6-[2-(morphin-4-yl)ethyl] analogue 10g
had weak TOP1-targeting activity that was comparable
to that of 10k. Excluding these N-benzyl derivatives, it
was observed that the 6-[2-(N,N-dialkylamino)ethyl]
derivatives of 6H-dibenzo[c,h][2,6]naphthyridin-5-one,
such as 10c–e, that possessed more lipophilic substitu-
ents on the 2-aminoethyl group, also had substantially

3134
S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
N
O
O
N
O
O
H3CO
H3CO
H3CO
H3CO
N
R
O
h
N
R
O
N
Bn
N
H
10j: R = Bn
10k: R = CH3
10l: R = H
10m: R = CH3
Scheme 3. Reagents: (h) AcOH, formic acid, Pd black.
Table 1. TOP1-targeting activity and cytotoxicity of 6-substituted 6H-dibenzo[c,h][2,6]naphthyridin-5-ones
Compound
TOP1-mediated DNA cleavagea
Cytotoxicity IC50 (lM)
RPMI8402
CPT-K5
KB3-1 wt
KBV-1 + MDR1
KBH5.0 + BCRP
1
0.3
1.2
0.002
0.0007
0.003
0.003
0.003
0.003
0.004
0.035
0.0005
0.030
0.050
0.023
0.002
0.002
0.006
0.021
0.90
0.21
0.18
0.90
0.85
1.70
0.40
>5.0
0.20
0.30
2.10
1.90
0.30
0.40
>10
>10
0.005
0.003
0.006
0.003
0.002
0.003
0.004
0.045
0.004
0.03
0.005
0.004
0.015
0.012
0.004
0.005
0.023
0.120
0.006
0.090
0.250
0.120
0.016
0.065
0.025
0.44
0.006
0.004
0.009
0.005
0.004
0.004
0.022
0.150
0.005
0.050
0.170
0.070
0.020
0.085
0.026
0.44
10a
10b
10c
1.2
0.2
10d
10e
0.07
0.1
10f
3.0
9.0
10g
10h
10i
1.4
0.45
>100
13
10j
0.04
10k
10l
0.038
0.004
0.006
0.015
0.04
0.35
0.15
0.2
10m
CPT
Topotecan
1.0
a Topoisomerase I cleavage values are reported as REC, relative effective concentration, that is, concentrations relative to topotecan, whose value is
arbitrarily assumed as 1, that are able to produce the same cleavage on the plasmid DNA in the presence of human topoisomerase I.
10d
10f
10a
10g
CPT
.
.
.
.
.
Figure 2. Stimulation of enzyme-mediated DNA cleavage by camptothecin (CPT), 10a, 10d, 10f, and 10g, using human TOP1. The first lane is the
DNA control without enzyme. The second lane is the control with enzyme alone. The rest of the lanes contain human TOP1 and serially (10-fold
each) diluted compound from 0.001 to 1.0 lM.
greater potency as TOP1-targeting agents. These
reversed lactams were among the more potent TOP1-
targeting agents that have been synthesized in our
laboratory with potencies exceeding that of camptothe-
cin. These data suggest that the presence of lipophilic
substituents, such as the N,N-diethyl groups or the pen-
tamethylene associated with the piperidine moiety, can
significantly enhance TOP1-targeting activity. In con-
trast to the piperidin-1-yl derivative 10e appended to
the 2-ethyl side chain, it is noteworthy that the 4-meth-

S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
3135
ylpiperidin-1-yl derivative 10f and in particular the mor-
4. Experimental
pholin-4-yl derivative 10g exhibited significantly reduced
intrinsic activity as TOP1-targeting agents. These data
suggest that steric or electronic factors associated with
substituents attached to the 2-position of the ethyl group
can reduce relative TOP1-targeting activity.
Melting points were determined with a Meltemp capil-
lary melting point apparatus. Column chromatography
refers to flash chromatography conducted on SiliTech
32–63 lm (ICN Biomedicals, Eschwege, Ger.) using
the solvent systems indicated. Infrared spectral data
were obtained using a Thermo-Nicolet Avatar 360 Fou-
The relative cytotoxic activities of several of the 6-
substituted 6H-dibenzo[c,h][2,6]naphthyridin-5-ones in
RPMI8402 tumor cells and the camptothecin-resistant
variant cell line CPT-K5 are listed in Table 1.
rier transform spectrometer and are reported in cmÀ1
.
Proton (1H NMR) and carbon (13C NMR) nuclear mag-
netic resonance was recorded on a Varian Gemini-200
Fourier Transform spectrometer. NMR spectra
1
Compounds 10a and 10h were among the more cytotox-
ic analogues that were evaluated. Both of these deriva-
tives exhibited IC50 values in RPMI8402 cells that
were at or below 0.7 nM. Compounds 10b–f, 10l, and
10m had similar cytotoxic activity with IC50 values in
RPMI8402 cells ranging from 2 to 4 nM.
(200 MHz H and 50 MHz 13C) were recorded in the
deuterated solvent indicated with chemical shifts report-
ed in d units downfield from tetramethylsilane (TMS).
Coupling constants are reported in hertz (Hz). Mass
spectra were obtained from Washington University Re-
source for Biomedical and Bio-organic Mass Spectrom-
etry within the Department of Chemistry at Washington
University, St. Louis, MO. All starting materials and re-
agents were purchased from Aldrich. Solvents were pur-
chased from Fisher Scientific and were of ACS grade or
HPLC grade. Methylene chloride was freshly distilled
from calcium hydride. All other solvents were used as
provided without further purification. 2-(N,N-Dimeth-
ylamino)ethylchloride hydrochloride, 3-(N,N-dimeth-
ylamino)propylchloride hydrochloride, 2-chloro-(N,N-
dimethylpropylamine hydrochloride, 2-(N,N-diethyla-
mino)ethylchloride hydrochloride, 1-(2-chloroethyl)pyr-
rolidine hydrochloride, 1-(2-chloroethyl)piperidine
hydrochloride, 4-(2-chloroethyl)morpholine hydrochlo-
ride, 2-(2-chloroethyl)-1-methylpyrrolidine hydrochlo-
A significant loss in cytotoxic activity was observed for
10g (IC50 = 35 nM) wherein a morpholin-4-yl moiety re-
placed the piperidin-1-yl substituent of 10e attached to
the 2-position of the ethyl side chain. This is consistent
with its significantly lower intrinsic TOP1-targeting
activity. Lower cytotoxic activities were also observed
for the 2-(1-methylpyrrolidin-2-yl)ethyl derivative 10i,
and the 2-(N,N-dibenzylamino)ethyl analogue 10j, and
the 2-(N-benzyl-N-methylamino)ethyl analogue 10k,
which had IC50 values in RPMI8402 cells of 30, 50,
and 23 nM, respectively.
These reversed lactam derivatives were also evaluated in
KB3-1 cells, and in KBV-1 and KBH5.0 cells, which over-
express MDR1 and BCRP, respectively (Table 1). No
notable differences in cytotoxicity were observed for
10a, 10d, 10e, and 10h in these three cell lines. Comparison
of their relative cytotoxicity in these three cell lines indi-
cates that several reversed lactam analogues, 10b, 10c,
10g, 10i, 10k, and 10l were not substrates for these efflux
transporters, with less than a 5-fold difference observed
in cytotoxicity in the variant cell lines relative to the par-
ent. Both 10f and 10j did appear to be weak substrates for
MDR1 with a 5- to 6-fold difference observed in relative
IC50 values relative to the parent cell line. Compound
10f was also 5-fold less cytotoxic in KBH5.0 cells relative
to KB3-1 cells, indicating that it was also a weak substrate
from BCRP. Only 10m had a difference in cytotoxicity for
either KBV-1 cells or KBH5.0 cells relative to their parent
KB3-1 cell line that was at least an order of magnitude
indicating that this compound was a substrate for both
MDR1 and BCRP efflux transport.
ride,
and
N-(2-chloroethyl)dibenzylamine
were
obtained from Aldrich. 4-(2-Chloroethyl)-1-methylpi-
peridine was prepared from 4-(2-hydroxyethyl)-1-meth-
ylpiperidine33,34 and 2-(N-benzyl-N-methylamino)-1-
chloroethane hydrochloride was prepared from 2-(N-
benzyl-N-methylamino)ethanol.35
4.1. 1-(2-Hydroxyethyl)-4-methylpiperidine
To 4-methylpiperidine (27.8 g, 0.28 mol) was added
dropwise, while stirring ethylene chlorohydrin (11.26 g,
0.14 mol). After boiling the reaction mixture for 3 h, it
was cooled and stirred several times with a total of
300 mL ether. Precipitated 4-methylpiperidine hydro-
chloride was filtered off and the filtrate evaporated.
The residue obtained was then distilled under vacuum.
There was obtained 16.6 g of 1-(2-hydroxyethyl)-4-
methylpiperidine33 in 85% yield as a brown liquid, which
was used in subsequent steps without further purifica-
1
tion. H NMR (CDCl3) d 0.94 (d, 3H, J = 6.2), 1.25
(m, 3H), 1.66 (m, 2H), 2.05 (m, 2H), 2.51 (t, 2H,
J = 5.4), 2.90 (m, 3H), 3.60 (t, 2H, J = 5.4).
The in vitro data on these various 6-substituted 6H-dib-
enzo[c,h][2,6]naphthyridin-5-ones indicated that several
compounds possess exceptional TOP1-targeting activity
and cytotoxicity. In several instances, the potent phar-
macological activity observed with certain analogues,
such as 10a and 10d, exceeds that of ARC-111. ARC-
111 has proved to be efficacious after parenteral and oral
administration. Studies are in progress to assess the in vi-
vo relative antitumor efficacy of select 6-substituted 6H-
dibenzo[c,h][2,6]naphthyridin-5-ones by both these
routes of administration.
4.2. 1-(2-Chloroethyl)-4-methylpiperidine hydrochloride
4-Methyl-1-(2-hydroxyethyl)piperidine (10 g, 70 mmol)
was dissolved in 56 mL anhydrous benzene, and while
stirring, so mixed with a solution of 16.8 g thionyl chlo-
ride in 12 mL anhydrous benzene such that the temper-
ature of the mixture remained between 25 and 30 ꢂC.
After boiling for a further 30 min, the hydrochloride

3136
S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
was filtered and washed with ether to give 13.4 g of 1-(2-
chloroethyl)-4-methylpiperidine hydrochloride34 in 97%
yield of a white solid and used in subsequent steps with-
out further purification. H NMR (CDCl3) d 0.99 (d,
3H, J = 6.2), 1.62 (m, 1H), 1.88 (m, 4H), 2.85 (m, 2H),
3.38 (m, 2H), 3.59 (m, 2H), 4.08 (t, 2H, J = 6.6).
4.6. 4-Methyl-6,7-methylenedioxyquinoline (5)
Iron (III) chloride (54.2 g, 0.2 mol) was dissolved in gla-
cial acetic acid (600 mL) with warming to 60 ꢂC. 3,4-
Methylenedioxyaniline (27.4 g, 0.2 mol) was added and
the mixture was stirred for 5 min. Methyl vinyl ketone
(17.4 mL, 0.21 mol) was added dropwise over five min.
Following the completion of addition, the mixture was
heated to reflux with stirring for 1.5 h. The mixture
was cooled and the precipitate was filtered and washed
with additional acetic acid. This material was then neu-
tralized by addition to cold 30% NaOH, and the result-
ing mixture was filtered and air-dried. The crude
material was then extracted with chloroform (7·
200 mL). The combined extracts were washed with
10% K2CO3 (3· 300 mL), dried (MgSO4), and concen-
trated under vacuum. The residue was recrystallized
from ethyl ether, yielding 16.6 g of 5 as a fluffy light
1
4.3. 2-(N-Benzyl-N-methylamino)-1-chloroethane
hydrochloride
To
2-(N-benzyl-N-methylamino)ethanol
(16.5 g,
0.1 mol) was added dropwise HCl (15%) to pH < 2.
Benzene (83 mL) was added and the emulsion was re-
fluxed under Dean–Stark conditions to remove water.
The benzene solution was evaporated. The oily residue
was cooled in an ice bath and thionyl chloride was
added dropwise. The reaction mixture was then re-
fluxed for 3 h. Excess thionyl chloride was removed
under vacuum. The residue was washed with cold eth-
anol (45 mL) and dried in a vacuum oven overnight at
room temperature to give 20.6 g of 2-(N-benzyl-N-
methylamino)-1-chloroethane hydrochloride35 in 94%
yield as a white solid, which was used in subsequent
steps without further purification. 1H NMR (CDCl3)
d 2.82 (d, 3H, J = 4.8), 3.29 (m, 1H), 3.54 (m, 1H),
4.09 (t, 2H, J = 6.6), 4.35 (m, 2H), 7.48 (m, 3H),
7.63 (m, 2H).
1
beige solid, in 44% yield; mp 100.5–101.5 ꢂC; H NMR
(CDCl3) d 2.51 (s, 3H), 6.04 (s, 2H), 7.02 (d, 1H,
J = 4.4), 7.13 (s, 1H), 7.32 (s, 1H), 8.52 (d, 1H,
J = 4.4); 13C NMR (CDCl3) d 19.1, 99.3, 101.7, 106.3,
120.6, 125.0, 142.9, 146.3, 147.8, 147.9, 150.2; HRMS
calcd for C11H9O2N: 187.0633; found: 187.0627.
4.7. 4-Formyl-6,7-methylenedioxyquinoline (6)
A mixture of 5 (5.01 g, 27.0 mmol) in 30 mL dioxane
was heated to 75 ꢂC, and then a solution of SeO2 in
5:1 dioxane/H2O (36 mL) was added dropwise. The mix-
ture was heated to reflux with stirring for 4.5 h. The
cooled reaction mixture was filtered and the filtrate
evaporated. The residue was dissolved in chloroform
(50 mL), washed with water (3· 50 mL), dried (MgSO4),
and evaporated. The residue was chromatographed,
eluting with chloroform to provide 3.48 g of 6 in 65%
yield; mp 146–147.5 ꢂC; IR (CHCl3) 1702; 1H NMR
(CDCl3) d 6.18 (s, 2H), 7.45 (s, 1H), 7.63 (d, 1H,
J = 4.4), 8.41 (s, 1H), 8.96 (d, 1H, J = 4.4), 10.35 (s,
1H); 13C NMR (CDCl3) d 100.4, 102.3, 106.3, 121.4,
124.7, 135.7, 148.0, 148.3, 150.8, 151.0, 193.4; HRMS
calcd for C11H7O3N: 201.0426; found: 201.0437.
4.4. 4,5-Dimethoxy-2-iodoacetanilide (3)
A 1.0 M solution of iodine monochloride in methylene
chloride (41.7 mL) was added dropwise to a solution
of
N-(3,4-dimethyoxyphenyl)acetamide32
(7.4 g,
37.9 mmol) in methylene chloride (45 mL) and acetic
acid (7.5 mL). The mixture was stirred under nitrogen
overnight and then washed with saturated sodium
thiosulfate (2· 150 mL) and brine (150 mL). The
methylene chloride solution was dried (MgSO4) and
evaporated, and the crude residue was chromato-
graphed using 19:1 chloroform/hexanes, to provide
6.2 g of 1 as a white solid, in 52% yield; mp 140–
141.5 ꢂC; IR (CHCl3) 3397, 1687; 1H NMR (CDCl3)
d 2.25 (s, 3H), 3.86 (s, 3H), 3.90 (s, 3H), 7.17 (s,
1H), 7.26 (br, 1H), 7.86 (s, 1H); 13C NMR (CDCl3)
d 24.8, 56.1, 56.4, 77.6, 106.4, 120.4, 132.4, 146.6,
149.7, 168.4; HRMS calcd for C10H12IO3N:
320.9862; found: 320.98.61.
4.8. 6,7-Methylenedioxyquinoline-4-carboxylic acid (7)
A solution of 6 (4.8 g, 23.8 mmol) in pyridine (150 mL)
was cooled to À5 ꢂC. The mixture was maintained at
this temperature as a solution of potassium permanga-
nate (10.0 g, 63.3 mmol in 150 mL of water) was added
dropwise over the course of 1 h. The mixture was stirred
at À5 ꢂC for an additional hour and then left to stir
overnight. The mixture was filtered and the filtrate was
evaporated under vacuum. The solid on the filter was
extracted with 100 mL of water with heating to 80 ꢂC,
and the aqueous extract was added to the residue result-
ing from evaporation of the acetone solution. This mix-
ture was acidified to pH 5 using HCl. The precipitated
free acid was filtered and washed well with water, etha-
nol, ethyl acetate, and ethyl ether sequentially, and then
dried under vacuum for 2 days to provide 4.6 g of 7 in
90% yield; mp >300 ꢂC; IR (KBr) 3446, 1689; 1H
NMR (DMSO-d6) d 6.27 (s, 2H), 7.45 (s, 1H), 7.79 (d,
1H, J = 4.8), 8.08 (s, 1H), 8.79 (d, 1H, J = 4.8); 13C
4.5. 4,5-Dimethoxy-2-iodoaniline (4)
A mixture of 1 (1.0 g, 3.12 mmol) and NaOH (6.25 g,
156 mmol) in ethanol (125 mL) and water (30 mL) was
heated to reflux with stirring for 4 h. The mixture was
cooled and the solvent was removed under vacuum.
The residue was partitioned between chloroform
(100 mL) and water (100 mL). The organic phase was
washed with water (2· 100 mL), dried (MgSO4), and
evaporated under vacuum to give 810 mg of 2 in 93%
1
yield, as a light pink oil; H NMR (CDCl3) d 3.81 (s,
3H), 3.83 (s, 3H), 6.39 (s, 1H), 7.08 (s, 1H); 13C NMR
(CDCl3) d 55.9, 56.8, 71.2, 99.7, 121.7, 141.3, 142.8,
150.7; HRMS calcd for C8H10IO2N: 278.9756; found:
278.9763.

S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
3137
NMR (CDCl3 + 1 drop TFA-d) d 98.1, 102.0, 104.9,
122.2, 128.6, 139.2, 139.7, 140.1, 153.6, 156.5, 166.6;
HRMS calcd for C11H7O4N: 217.0375; found: 217.0371.
J = 4.4); 13C NMR (CDCl3) d 45.1, 45.6, 55.5, 56.1,
56.3, 88.1, 101.5, 101.9, 106.2, 114.2, 115.3, 120.8,
121.8, 135.7, 142.2, 146.7, 147.4, 148.3, 148.7, 149.2,
150.6, 168.7; HRMS calcd for C23H24IN3O5H:
550.0839; found: 550.0817.
4.9. 6,7-Methylenedioxyquinoline-4-carboxylic acid
N-(2-iodo-4,5-dimethoxyphenyl)amide (8)
4.10.2. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(N,N-dimethylamino)isopropyl]-N-(2-iodo-4,5-dimethoxy-
A suspension of 7 (500 mg, 2.3 mmol) in thionyl chlo-
ride (30 mL) was heated at reflux for 2 h, during which
time the starting material completely dissolved. The
mixture was cooled and then evaporated to dryness un-
der vacuum. The acid chloride was dissolved in anhy-
drous methylene chloride (30 mL) and triethylamine
(3.0 g, 30 mmol) was added. A solution of 4 (535 mg,
1.9 mmol) in methylene chloride (15 mL) was added,
and the resulting mixture was refluxed under nitrogen
overnight. The mixture was cooled and additional meth-
ylene chloride was added, bringing the total volume up
to 100 mL. This solution was washed with saturated
sodium bicarbonate (2· 100 mL) and brine (100 mL),
dried (MgSO4) and evaporated under vacuum. The
crude residue was chromatographed in chloroform to
provide 512 mg of 8 as a very pale yellow solid, in
phenyl) amide (9b). Prepared from
8
(800 mg,
1.67 mmol), sodium iodide (375 mg, 2.50 mmol), and
2-(dimethylamino)isopropyl chloride HCl (308 mg,
1.90 mmol) in DMF (36 mL) and sodium hydride
(201 mg of a 60% mineral oil suspension, 5.01 mmol)
to provide 613 mg of 9b as a sticky semi-solid glue in
1
89% yield. IR (CHCl3) 1643 cmÀ1; H NMR (CDCl3)
d 0.97 (d, 3H, J = 6.6), 2.49 (s, 6H), 3.38 (s, 3H), 3.51
(m, 1H), 3.76 (s, 3H), 3.97 (m, 1H), 4.95 (m, 1H), 6.12
(s, 2H), 6.72 (s, 1H), 6.75 (s, 1H), 7.11 (d, 1H,
J = 4.4), 7.30 (s, 1H), 7.72 (s, 1H), 8.50 (d, 1H,
J = 4.4); 13C NMR (CDCl3) d 16.6, 45.9, 49.8, 55.5,
55.8, 56.0, 92.9, 101.6, 102.6, 106.1, 114.6, 116.3,
120.8, 121.4, 132.4, 142.1, 146.6, 147.6, 147.9, 148.4,
149.0, 150.3, 169.5; HRMS calcd for C24H26IN3O5H:
564.0996; found: 564.0984.
1
56% yield; mp 210–211 ꢂC; IR (CHCl3) 3375, 1680; H
NMR (CDCl3) d 3.91 (s, 3H), 4.00 (s, 3H), 6.17 (s,
2H), 7.25 (s, 1H), 7.47 (s, 1H), 7.55 (d, 1H, J = 4.4),
7.77 (s, 1H), 7.90 (br, 1H), 8.11 (s, 1H), 8.84 (d, 1H,
J = 4.4); 13C NMR (CDCl3) d 56.3, 56.5, 78.3, 100.9,
102.2, 106.4, 111.9, 116.9, 120.5, 121.9, 131.9, 139.9,
147.7, 147.9, 149.3, 149.8, 151.3, 165.6; HRMS calcd
for C19H15IN2O5H: 479.0104; found: 479.0081.
4.10.3. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(N,N-diethylamino)ethyl]-N-(2-iodo-4,5-dimethoxy-
phenyl) amide (9c). Prepared from
8
(350 mg,
0.73 mmol), sodium iodide (164 mg, 1.10 mmol), 2-
(diethylamino)ethyl chloride HCl (145 mg, 0.83 mmol)
in DMF (15 mL) and sodium hydride (88 mg of a 60%
mineral oil suspension, 2.19 mmol) to give 372 mg of
9c as a sticky semi-solid glue in 88% yield; IR (CHCl3)
4.10. General method for the preparation of tertiary
amide derivatives of 6,7-methylenedioxyquinoline-4-
carboxylic acid
1
1650 cmÀ1; H NMR (CDCl3) d 1.11 (t, 6H, J = 7.4),
2.79 (m, 6H), 3.27 (m, 1H), 3.34 (s, 3H), 3.76 (s, 3H),
4.88 (m, 1H), 6.11 (s, 2H), 6.71 (s, 1H), 7.06 (s, 1H),
7.23 (d, 1H, J = 4.4), 7.30 (s, 1H), 7.68 (s, 1H), 8.49
(d, 1H, J = 4.4); 13C NMR (CDCl3) d 11.1, 45.6, 46.2,
50.5, 55.6, 56.2, 88.0, 101.5, 101.9, 106.3, 114.3, 115.5,
120.9, 121.8, 136.2, 142.1, 146.7, 147.5, 148.4, 148.7,
149.1, 150.6, 168.6; HRMS calcd for C25H28IN3O5H:
578.1153; found: 578.1146.
4.10.1. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(N,N-dimethylamino)ethyl]-N-(2-iodo-4,5-dimethoxy-
phenyl) amide (9a). A mixture of 8 (350 mg, 0.73 mmol)
and 2-(dimethylamino)ethyl chloride HCl (120 mg,
0.83 mmol) in DMF (15 mL) was cooled to 0 ꢂC and
sodium hydride (160 mg of a 60% mineral oil suspen-
sion, 4.0 mmol) was added in small portions over
5 min. Cooling was removed. The mixture was allowed
to warm to room temperature with stirring for 45 min.
The reaction flask was then transferred to an oil bath
that had been preheated to 65 ꢂC, and the mixture was
stirred at this temperature for 3 h. TLC was used to
monitor the reaction. The mixture was cooled to room
temperature and quenched by addition of a few drops
of water. The solvent was removed under vacuum. The
crude product was dissolved in 1 N HCl (50 mL). The
aqueous solution was washed with chloroform (3·
50 mL), then made basic by the addition of 30% NaOH,
and extracted with chloroform (3· 75 mL). The organic
layers were combined, dried (MgSO4) and evaporated
under vacuum. The residue was chromatographed using
98:2 chloroform/methanol to provide 357 mg of 9a as a
sticky semi-solid glue in 89% yield; IR (CHCl3)
4.10.4. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(pyrrolidin-1-yl)ethyl]-N-(2-iodo-4,5-dimethoxy-
phenyl) amide (9d). A mixture of 8 (500 mg, 1.05 mmol),
sodium iodide (236 mg, 1.58 mmol), and 1-(2-chloroeth-
yl)pyrrolidine HCl (210 mg, 1.20 mmol) in DMF
(22 mL) was cooled to 0 ꢂC and sodium hydride
(126 mg of a 60% mineral oil suspension, 3.15 mmol)
was added in small portions over 5 min. The reaction
mixture was then allowed to warm to room temperature
and to stir for 45 min. The reaction flask was then trans-
ferred to an oil bath that had been preheated to 65 ꢂC
and stirred at this temperature for 3 h. TLC showed
there was still a significant amount of starting material
left. To this mixture were added more 1-(2-chloroeth-
yl)pyrrolidine HCl (210 mg, 1.20 mmol), sodium hy-
dride (126 mg of a 60% mineral oil suspension,
3.15 mmol), and sodium iodide (236 mg, 1.58 mmol) at
this temperature. The mixture was heated for another
3 h, and cooled to room temperature, and quenched
by addition of a few drops of water. The solvent was
1
1647 cmÀ1; H NMR (CDCl3) d 2.41 (s, 6H), 2.51 (m,
2H), 3.19 (m, 1H), 3.33 (s, 3H), 3.73 (s, 3H), 4.92 (m,
1H), 6.08 (s, 2H), 6.76 (s, 1H), 7.04 (s, 1H), 7.22 (d,
1H, J = 4.4), 7.27 (s, 1H), 7.66 (s, 1H), 8.47 (d, 1H,

3138
S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
removed under vacuum. The crude product was dis-
solved in 1 N HCl (100 mL). The aqueous solution
was washed with chloroform (3· 60 mL), then made ba-
sic by the addition of 30% NaOH, and extracted with
chloroform (3· 75 mL). The organic layers were com-
bined, dried (MgSO4), and evaporated under vacuum.
The residue was chromatographed using 98:2 chloro-
form/methanol to provide 550 mg of 9d as a sticky
NMR (CDCl3) d 1.01 (d, 3H, J = 5.0), 1.47 (m, 3H),
1.78 (m, 2H), 1.95 (t, 1H, J = 11.0), 2.15 (t, 1H,
J = 8.8), 2.44 (m, 2H), 2.73 (m, 1H), 3.21 (m, 1H),
3.34 (s, 3H), 3.54 (m, 1H), 3.75 (s, 3H), 5.05 (m, 1H),
6.12 (s, 2H), 6.80 (s, 1H), 7.05 (s, 1H), 7.27 (d, 1H,
J = 4.4), 7.29 (s, 1H), 7.80 (s, 1H), 8.49 (s, 1H,
J = 4.4); 13C NMR (CDCl3) d 21.9, 31.1, 34.3, 34.7,
43.7, 53.2, 55.5, 56.1, 88.0, 101.7, 101.9, 106.3, 114.2,
115.2, 120.7, 121.7, 135.2, 142.5, 146.6, 147.6, 148.3,
148.6, 149.1, 150.6, 168.8; HRMS (M+Li)+calcd for
C27H30IN3O5Li: 610.1390; found: 610.1371.
semi-solid glue in 92% yield; IR (CHCl3) 1648 cmÀ1
;
1H NMR (CDCl3) d 1.94 (m, 4H), 2.57 (m, 3H), 2.89
(m, 3H), 3.22 (m, 1H), 3.32 (s, 3H), 3.75 (s, 3H), 4.98
(m, 1H), 6.10 (s, 2H), 6.80 (s, 1H), 7.05 (s, 1H), 7.25
(d, 1H, J = 4.4), 7.29 (s, 1H), 7.70 (s, 1H), 8.49 (d, 1H,
J = 4.4); 13C NMR (CDCl3) d 23.8, 45.6, 52.5, 54.3,
55.5, 56.1, 88.2, 101.5, 101.9, 106.2, 114.2, 115.2,
120.7, 121.8, 135.4, 142.4, 146.7, 147.5, 148.3, 148.6,
149.2, 150.6, 168.7; HRMS calcd for C25H26IN3O5H:
576.0996; found: 576.1006.
4.10.7. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(morpholin-4-yl)ethyl]-N-(2-iodo-4,5-dimethoxyphe-
nyl) amide (9g). Prepared from 8 (500 mg, 1.05 mmol),
sodium iodide (236 mg, 1.58 mmol), 2-(morpholin-4-
yl)ethyl chloride HCl (224 mg, 1.20 mmol) in DMF
(22 mL), and sodium hydride (126 mg of a 60% mineral
oil suspension, 3.15 mmol). The reaction flask was then
transferred to an oil bath that had been preheated 60 ꢂC
and stirred at this temperature for 3 h. TLC showed
there was still a significant amount of starting material
left. To this mixture was added more 2-(morpholin-4-
yl)ethyl chloride HCl (224 mg, 1.20 mmol), sodium hy-
dride (126 mg of a 60% mineral oil suspension,
3.15 mmol), and sodium iodide (236 mg, 1.58 mmol) at
this temperature. The mixture was heated for another
3 h, cooled to room temperature, and quenched by addi-
tion of a few drops of water. The solvent was removed
under vacuum. The crude product was dissolved in
1 N HCl (100 mL). The aqueous solution was washed
with chloroform (3· 60 mL), then made basic by the
addition of 30% NaOH, and extracted with chloroform
(3· 75 mL). The organic layers were combined, dried
(MgSO4), and evaporated under vacuum. The residue
was chromatographed using 98.5:1.5 chloroform/metha-
nol to provide 500 mg of 9g as a sticky semi-solid glue in
4.10.5. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(piperidin-1-yl)ethyl]-N-(2-iodo-4,5-dimethoxyphen-
yl) amide (9e). Prepared from 8 (500 mg, 1.05 mmol),
sodium iodide (236 mg, 1.58 mmol), 1-(2-chloroethyl)pi-
peridine HCl (225 mg, 1.20 mmol) in DMF (22 mL),
and sodium hydride (126 mg of a 60% mineral oil sus-
pension, 3.15 mmol) The residue was chromatographed
using 97.5:2.5 chloroform/methanol to provide 520 mg
of 9e as a sticky semi-solid glue in 84% yield; IR
1
(CHCl3) 1650 cmÀ1; H NMR (CDCl3) d 1.55 (m, 2H),
1.74 (m, 4H), 2.37 (m, 4H), 2.79 (m, 2H), 3.19 (m,
1H), 3.34 (s, 3H), 3.74 (s, 3H), 5.00 (m, 1H), 6.10 (s,
2H), 6.80 (s, 1H), 7.03 (s, 1H), 7.26 (d, 1H, J = 4.4),
7.27 (s, 1H), 7.81 (s, 1H), 8.49 (d, 1H, J = 4.4); 13C
NMR (CDCl3) d 24.6, 26.1, 43.8, 54.9, 55.5, 55.9, 56.1,
88.0, 101.7, 101.9, 106.2, 114.3, 115.3, 120.8, 121.7,
135.4, 142.4, 146.7, 147.5, 148.3, 148.6, 149.2, 150.6,
168.7; HRMS calcd for C26H28IN3O5H: 590.1153;
found: 590.1148.
1
81% yield; IR (CHCl3) 1650 cmÀ1; H NMR (CDCl3) d
2.50 (m, 4H), 2.94 (m, 2H), 3.22 (m, 1H), 3.31 (s, 3H),
3.75 (s, 3H), 3.91 (t, 4H, J = 4.4), 5.05 (m, 1H), 6.12
(s, 2H), 6.73 (s, 1H), 7.07 (s, 1H), 7.26 (d, 1H,
J = 4.4), 7.30 (s, 1H), 7.82 (s, 1H), 8.50 (d, 1H,
J = 4.4); 13C NMR (CDCl3) d 43.2, 54.0, 55.6, 55.8,
56.2, 67.0, 88.1, 101.4, 102.0, 106.4, 114.1, 115.2,
121.0, 121.7, 135.2, 142.2, 146.6, 147.6, 148.4, 148.7,
149.3, 150.7, 168.8; HRMS calcd for C25H26IN3O6H:
592.0945; found: 592.0933.
4.10.6. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(4-methylpiperidin-1-yl)ethyl]-N-(2-iodo-4,5-dimeth-
oxyphenyl) amide (9f). Prepared from 8 (500 mg,
1.0 mmol), sodium iodide (235 mg, 1.5 mmol), 4-meth-
yl-1-(2-chloroethyl)piperidine hydrochloride (224 mg,
1.14 mmol) in DMF (20 mL), and sodium hydride
(125 mg of a 60% suspension, 3.0 mmol). The reaction
flask was then transferred to an oil bath that had been
preheated to 60 ꢂC, and was stirred at this temperature
for 3 h. TLC showed there was still starting material left.
More 4-methyl-1-(2-chloroethyl)piperidine hydrochlo-
ride (75 mg, 0.38 mmol) and sodium hydride (42 mg of
a 60% suspension, 1.05 mmol) were added. The mixture
was heated for another 1.5 h, and was cooled to room
temperature, and quenched by addition of a few drops
of water. The solvent was removed under vacuum and
the crude product was dissolved in dilute 1 N HCl
(70 mL) and was washed with chloroform (3· 60 mL),
and then made basic by the addition of 30% NaOH.
The resulting mixture was extracted into chloroform
(3· 75 mL), dried (MgSO4), and evaporated under vac-
uum. The residue was chromatographed using 98:2–96:4
chloroform/methanol to provide 550 mg of 9f as a sticky
semi-solid glue, in 91% yield. IR (CHCl3) 1649; 1H
4.10.8. 6,7-Methylenedioxyquinoline-4-carboxylic acid N-
[3-(N,N-dimethylamino)propyl]-N-(2-iodo-4,5-dimethoxy-
phenyl) amide (9h). Prepared from 8 (478 mg, 1.0 mmol),
3-(dimethylamino)propyl chloride HCl (190 mg,
1.2 mmol), and sodium amide (156 mg, 4.0 mmol) in
toluene (25 mL) heated to reflux with stirring under
nitrogen for 4 h. At this time, an equivalent amount of
3-(dimethylamino)propyl chloride HCl and sodium
amide was added, and stirring was continued for an
additional 3 h. The mixture was cooled to room temper-
ature and quenched by addition of a few drops of water.
The solvent was removed under vacuum, and the crude
product was dissolved in 1 N HCl (50 mL). This aque-
ous solution was washed with chloroform (3· 50 mL),
then made basic by the addition of 30% NaOH, and

S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
3139
extracted with chloroform (3· 75 mL). The organic lay-
ers were combined, dried (MgSO4), and evaporated un-
der vacuum. The residue was chromatographed using
97:3 chloroform/methanol to provide 498 mg of 9h as
a sticky semi-solid glue in 88% yield; IR (CHCl3)
(333 mg, 1.10 mmol), sodium hydride (116 mg of a
60% mineral oil suspension, 2.89 mmol), and sodium io-
dide (217 mg, 1.45 mmol) at this temperature. The mix-
ture was then heated for another 3 h, cooled to room
temperature, and quenched by addition of a few drops
of water. The solvent was removed under vacuum. The
crude product was dissolved in 1 N HCl (100 mL). The
aqueous solution was washed with chloroform (3·
60 mL), then made basic by the addition of 30% NaOH,
and extracted with chloroform (3· 75 mL). The organic
layers were combined, dried (MgSO4), and evaporated
under vacuum. The residue was chromatographed using
99:1 chloroform/methanol to provide 677 mg of 9j as a
sticky semi-solid glue in 85% yield; IR (CHCl3)
1
1648 cmÀ1; H NMR (CDCl3) d 2.27 (s, 6H), 2.45 (m,
4H), 3.37 (s, 3H), 3.74 (s, 3H), 3.93 (m, 1H), 4.55 (m,
1H), 6.10 (s, 2H), 6.44 (s, 1H), 7.07 (s, 1H), 7.18 (d,
1H, J = 4.6), 7.29 (s, 1H), 7.39 (s, 1H), 8.46 (d, 1H,
J = 4.6); 13C NMR (CDCl3) d 26.0, 45.7, 47.6, 55.8,
56.2, 57.4, 87.8, 100.8, 102.0, 106.4, 113.7, 116.03,
121.4, 121.8, 136.5, 141.4, 146.9, 147.3, 148.5, 149.1,
149.2, 150.6, 168.3; HRMS calcd for C24H26IN3O5H:
564.0995; found: 564.0997.
1
1649 cmÀ1; H NMR (CDCl3) d 2.93 (m, 2H), 3.19 (s,
4.10.9. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(1-methylpyrrolidin-2-yl)ethyl]-N-(2-iodo-4,5-dimeth-
oxyphenyl) amide (9i). Prepared from 8 (250 mg,
0.52 mmol), sodium iodide (118 mg, 0.79 mmol), 2-(2-
3H), 3.55 (m, 1H), 3.75 (m, 7H), 4.88 (m, 1H), 6.13 (s,
2H), 6.26 (s, 1H), 7.05 (s, 1H), 7.14 (d, 1H, J = 4.4),
7.34 (m, 11H), 7.42 (s, 1H), 8.46 (d, 1H, J = 4.4); 13C
NMR (CDCl3) d 46.4, 51.1, 55.9, 56.2, 56.3, 58.2, 87.6,
101.1, 102.0, 106.4, 114.1, 116.1, 121.2, 121.8, 127.2,
128.3, 128.5, 129.3, 136.6, 138.8, 141.3, 146.9, 147.2,
148.5, 148.9, 149.1, 150.7, 168.4; HRMS calcd for
C35H32IN3O5Li: 708.1547; found: 708.1568.
chloroethyl)-1-methylpyrrolidine
HCl
(111 mg,
0.60 mmol) in DMF (11 mL), and sodium hydride
(63 mg of a 60% mineral oil suspension, 1.58 mmol).
The reaction flask was then transferred to an oil bath
that had been preheated to 60 ꢂC and stirred at this tem-
perature for 3 h. TLC showed there was a significant
amount of starting material left. To this mixture were
then added more 2-(2-chloroethyl)-1-methylpyrrolidine
HCl (28 mg, 0.15 mmol), sodium hydride (16 mg of a
60% mineral oil suspension, 0.4 mmol) and sodium io-
dide (30 mg, 0.2 mmol) at this temperature. The mixture
were then heated for another 3 h, cooled to room tem-
perature, and quenched by addition of a few drops of
water. The solvent was removed under vacuum. The
crude product was dissolved in 1 N HCl (50 mL). The
aqueous solution was washed with chloroform (3·
30 mL), then made basic by the addition of 30% NaOH,
and extracted with chloroform (3· 40 mL). The organic
layers were combined, dried (MgSO4), and evaporated
under vacuum. The residue was chromatographed using
95.5:4.5 chloroform/methanol to provide 230 mg of 9i as
a sticky semi-solid glue in 75% yield; IR (CHCl3)
4.10.11. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(N-benzyl-N-methylamino)ethyl]-N-(2-iodo-4,5-di-
methoxyphenyl) amide (9k). Prepared from 8 (500 mg,
1.0 mmol), sodium iodide (235 mg, 1.5 mmol), N-
benzyl-N-methyl-2-chloroethylamine
hydrochloride
(261 mg, 1.14 mmol) in DMF (20 mL), and sodium hy-
dride (125 mg of a 60% suspension, 3.0 mmol). The res-
idue was chromatographed in 98:2 to 96:4 chloroform/
methanol providing 550 mg of 9k as a sticky semi-solid
glue, in 84% yield; IR (CHCl3) 1649; 1H NMR (CDCl3)
d 2.28 (s, 3H), 2.76 (m, 2H), 3.22 (s, 3H), 3.36 (m, 1H),
3.61 (s, 2H), 3.75 (s, 3H), 4.95 (m, 1H), 6.14 (s, 2H), 6.70
(s, 1H), 7.05 (s, 1H), 7.31 (m, 6H), 7.59 (s, 1H), 7.02 (s,
1H), 8.49 (d, 1H, J = 4.8); 13C NMR (CDCl3) d 41.7,
45.6, 55.2, 55.7, 56.2, 62.9, 87.8, 101.2, 102.0, 106.4,
114.3, 115.8, 120.9, 121.8, 127.2, 128.4, 129.3, 136.1,
138.3, 141.8, 146.8, 147.4, 148.4, 148.8, 149.2, 150.6,
168.6; HRMS (M+Li)+ calcd for C29H28IN3O5Li:
632.1234; found: 632.1238.
1
1649 cmÀ1, H NMR (CDCl3) d 1.94 (m, 4H), 2.17 (m,
2H), 2.59 (m, 6H), 3.47 (s, 3H), 3.76 (s, 3H), 3.93 (m,
1H), 4.55 (m, 1H), 6.13 (s, 2H), 6.52 (s, 1H), 7.06 (s,
1H), 7.20 (d, 1H, J = 4.4), 7.31 (s, 1H), 7.37 (s, 1H),
8.48 (d, 1H, J = 4.4); 13C NMR (CDCl3) d 21.9, 22.2,
29.8, 30.3, 30.5, 40.0, 46.8, 56.1, 56.6, 87.8, 100.7,
102.1, 106.5, 113.7, 116.2, 121.4, 121.8, 136.3, 140.8,
146.9, 147.2, 148.6, 149.3, 149.4, 150.8, 168.5; HRMS
calcd for C26H28IN3O5Li: 596.1234; found: 596.1212.
4.11. Methods for the formation of 6-substituted 8,9-dime
thoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyri-
din-5-ones
4.11.1. 6-[2-(N,N-Dimethylamino)ethyl]-8,9-dimethoxy-
2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-
one (10a). A solution of 9a (275 mg, 0.5 mmol) in
950 mL of 2% HCl was transferred to the photoreactor
apparatus and degassed by nitrogen purge for 30 min.
The solution was irradiated through a Vycor filter for
90 min. The mixture was basified (30% NaOH) and
extracted with ethyl acetate (4· 50 mL). The combined
organic extracts were evaporated and the residue was
chromatographed on silica eluting with 98:2 chloro-
form/methanol to provide 70 mg of 10a as a yellow solid
4.10.10. 6,7-Methylenedioxyquinoline-4-carboxylic acid,
N-[2-(N,N-dibenzylamino)ethyl]-N-(2-iodo-4,5-dimethoxy-
phenyl) amide (9j). Prepared from
0.96 mmol), sodium iodide (217 mg, 1.45 mmol), and
N-(2-chloroethyl)dibenzylamine HCl (333 mg,
8
(461 mg,
1.10 mmol) in DMF (20 mL) and sodium hydride
(116 mg of a 60% mineral oil suspension, 2.89 mmol).
The reaction flask was then transferred to an oil bath
that had been preheated to 60 ꢂC, and was stirred at this
temperature for 3 h. TLC showed there was still a signif-
icant amount of starting material left. To this mixture
were added more N-(2-chloroethyl)dibenzylamine HCl
in 34% yield; mp 253–255 ꢂC; IR (CHCl3) 1639 cmÀ1
;
1H NMR (CDCl3 + 1 drop CD3OD) d 2.49 (s, 6H),
2.79 (t, 2H, J = 7.9), 3.97 (s, 3H), 3.99 (s, 3H), 4.57 (t,
2H, J = 7.9), 6.08 (s, 2H), 7.01 (s, 1H), 7.32 (s, 1H),

3140
S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
7.73 (s, 1H), 9.32 (s, 1H), 9.44 (s, 1H); 13C NMR
(CDCl3 + 1 drop CD3OD) d 41.0, 45.0, 55.0, 56.3,
56.4, 98.3, 102.2, 103.5, 104.8, 105.7, 110.3, 121.9,
122.3, 125.9, 132.6, 143.3, 144.4, 145.9, 149.9, 150.1,
152.2, 161.1; HRMS calcd for C23H23N3O5H:
422.1716; found: 422.1703.
2H), 6.14 (s, 2H), 7.14 (s, 1H), 7.42 (s, 1H), 7.79 (s,
1H), 9.44 (s, 1H), 9.52 (s, 1H); 13C NMR (CDCl3 + 1
drop CD3OD) d 23.9, 25.6, 41.1, 54.8, 56.5, 98.6,
102.2, 103.7, 104.8, 106.0, 110.4, 122.4, 125.9, 132.9,
143.4, 144.6, 145.9, 149.9, 150.1, 152.2, 161.2; HRMS
calcd for C26H27N3O5H: 462.2030; found: 462.2026.
4.11.2.
6-[2-(N,N-Dimethylamino)isopropyl]-8,9-dime-
4.11.6.
6-[2-(4-Methylpiperidin-1-yl)ethyl]-8,9-dime-
thoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyri-
din-5-one (10b). Prepared from 9b (200 mg, 0.36 mmol)
in 900 mL of 2% HCl using a photoreactor apparatus
to provide 51 mg of 10b as a yellow solid in 29% yield;
thoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyri-
din-5-one (10f). A mixture of 9f (500 mg, 0.83 mmol),
Pd(PPh3)4 (195 mg, 0.166 mmol), and Ag2CO3
(457 mg, 1.66 mmol) in DMF (36 mL) was heated to re-
flux for 45 min. The mixture was cooled, diluted with
CHCl3, and filtered through Celite. The filtrate was
evaporated, triturated with 15 mL of 50% CH2Cl2 in
ether, and filtered to give 161 mg of 10f in 41% yield
as a yellow solid. mp (dec.): 245.5–246.5 ꢂC; IR (KBr)
mp: 262–264 ꢂC; IR (KBr) 1642 cmÀ1
;
1H NMR
(CDCl3 + 3 drops CD3OD) d 1.07 (d, 3H, J = 6.6),
2.46 (s, 6H), 3.23 (m, 1H), 4.07 (s, 6H), 4.24 (m, 1H),
5.03 (m, 1H), 6.18 (s, 2H), 7.17 (s, 1H), 7.49 (s, 1H),
7.84 (s, 1H), 9.52 (s, 1H), 9.60 (s, 1H); 13C NMR
(CDCl3 + 1 drop CD3OD) d 10.8, 40.7, 45.9, 56.2,
56.4, 57.4, 99.0, 102.2, 103.8, 104.7, 105.7, 110.4,
122.1, 122.5, 125.9, 133.0, 143.3, 144.5, 145.8, 149.9,
150.1, 151.8, 161.5; HRMS calcd for C24H25N3O5H:
436.1873; found: 436.1852.
1
1637; H NMR (CDCl3) d 0.94 (d, 3H, J = 5.8), 1.32
(m, 3H), 1.70 (m, 2H), 2.53 (m, 2H), 2.72 (m, 2H),
3.07 (m, 2H), 4.04 (s, 6H), 4.58 (m, 2H), 6.14 (s, 2H),
7.10 (s, 1H), 7.41 (s, 1H), 7.77 (s, 1H), 9.44 (s, 1H),
9.50 (s, 1H); 13C NMR (CDCl3) d 21.8, 30.5, 34.2,
41.4, 54.4, 54.9, 56.4, 98.5, 102.2, 103.8, 104.6, 106.0,
110.3, 121.9, 122.3, 125.8, 132.9, 143.4, 144.6, 145.8,
149.8, 150.1, 152.0, 161.1; HRMS calcd HRMS
(M+Li)+ calcd for C27H29N3O5Li: 482.2267; found:
482.2265.
4.11.3.
6-[2-(N,N-Diethylamino)ethyl]-8,9-dimethoxy-
2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-
one (10c). Prepared from 9c (200 mg, 0.36 mmol) in
900 mL of 2% HCl using a photoreactor apparatus.
The residue was chromatographed on silica eluting with
98.5:1.5 chloroform/methanol to provide 40 mg of 10c
as a yellow solid in 25% yield; mp: 218–220 ꢂC; IR
(KBr) 1640 cmÀ1; 1H NMR (CDCl3 + 3 drops CD3OD)
d 1.20 (t, 6H, J = 6.6), 2.82 (m, 6H), 4.05 (s, 3H), 4.07 (s,
3H), 4.64 (m, 2H), 6.16 (s, 2H), 7.16 (s, 1H), 7.46 (s, 1H),
7.83 (s, 1H), 9.48 (s, 1H), 9.57 (s, 1H); 13C NMR
(CDCl3 + 3 drops CD3OD) d 11.3, 41.7, 47.5, 49.2,
56.5, 98.5, 102.2, 103.8, 104.8, 106.1, 110.4, 122.0,
122.3, 125.9, 132.9, 143.4, 144.7, 145.9, 149.8, 150.1,
152.1, 161.2; HRMS calcd for C25H27N3O5H:
450.2030; found: 450.2009.
4.11.7.
6-[2-(Morpholin-4-yl)ethyl]-8,9-dimethoxy-2,3-
methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-one
(10g). Prepared from 9g (450 mg, 0.76 mmol) in 900 mL
of 2% HCl using a photoreactor to provide 100 mg of
10g as a yellow solid in 31% yield; mp 273–275 ꢂC; IR
(KBr) 1636 cmÀ1; 1H NMR (CDCl3 + 3 drops CD3OD)
d 2.66 (m, 4H), 3.73 (m, 4H), 4.00 (m, 8H), 4.55 (m, 2H),
6.08 (s, 2H), 7.03 (s, 1H), 7.32 (s, 1H), 7.74 (s, 1H), 9.31
(s, 1H), 9.44 (s, 1H); 13C NMR (CDCl3 + 3 drops
CD3OD) d 41.1, 53.8, 55.1, 56.4, 66.8, 98.4, 102.2,
103.6, 104.8, 105.8, 110.4, 122.3, 125.9, 132.7, 143.3,
144.5, 145.9, 149.9, 150.1, 152.0, 161.2; HRMS calcd
for C25H25N3O6H: 464.1823; found: 464.1821.
4.11.4.
6-[2-(Pyrrolidin-1-yl)ethyl]-8,9-dimethoxy-2,3-
methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-one
(10d). Prepared from 9d (450 mg, 0.78 mmol) in 900 mL
of 2% HCl using a photoreactor apparatus to provide
110 mg of 10d as a yellow solid in 34% yield; mp: 240–
4.11.8. 6-[3-(N,N-Dimethylamino)propyl]-8,9-dimethoxy-
2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-
one (10h). Prepared from 9h (200 mg, 0.42 mmol) in
950 mL of 2% HCl using a photoreactor to provide
51 mg of 10h as a yellow solid in 29% yield; mp 238–
242 ꢂC; IR (KBr) 1638 cmÀ1
;
1H NMR (CDCl3 + 3
drops CD3OD) d 1.92 (m, 4H), 2.87 (m, 6H), 4.04 (s,
3H), 4.08 (s, 3H), 4.67 (m, 2H), 6.15 (s, 2H), 7.16 (s,
1H), 7.44 (s, 1H), 7.82 (s, 1H), 9.45 (s, 1H), 9.55 (s,
1H); 13C NMR (CDCl3 + 3 drops CD3OD) 23.5, 42.1,
52.2, 54.3, 56.5, 98.5, 102.2, 103.7, 104.8, 105.9, 110.3,
122.0, 122.3, 125.9, 132.8, 143.4, 144.5, 145.9, 149.8,
150.1, 152.2, 161.1; HRMS calcd for C25H25N3O5H:
448.1873; found: 448.1872.
1
240 ꢂC; IR (CHCl3) 1638 cmÀ1; H NMR (CDCl3 + 1
drop CD3OD) d 2.07 (m, 2H), 2.38 (s, 6H), 2.65 (t,
2H, J = 7.3), 4.04 (s, 6H), 4.48 (t, 2H, J = 7.5), 6.16 (s,
2H), 7.02 (s, 1H), 7.44 (s, 1H), 7.81 (s, 1H), 9.46 (s,
1H), 9.54 (s, 1H); 13C NMR (CDCl3 + 1 drop CD3OD)
d 25.1, 41.9, 45.1, 56.3, 56.5, 56.8, 98.4, 102.2, 103.8,
104.8, 106.1, 110.5, 122.2, 122.4, 125.9, 132.8, 143.4,
144.7 145.9, 149.8, 150.1, 152.0, 161.3; HRMS calcd
for C24H25N3O5H: 436.1872; found: 436.1883.
4.11.5.
6-[2-(Piperidin-1-yl)ethyl]-8,9-dimethoxy-2,3-
methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-one
(10e). Prepared from 9e (480 mg, 0.82 mmol) in 900 mL
of 2% HCl using a photoreactor apparatus to provide
110 mg of 10e as a yellow solid in 30% yield; mp:
4.11.9.
6-[2-(1-Methylpyrrolidine-2-yl)ethyl]-8,9-dime-
thoxy-2,3- methylenedioxy-6H-dibenzo[c,h][2,6]naph-
thyridin-5-one (10i). Prepared from 9i (200 mg,
0.34 mmol) in 900 mL of 2% HCl using a photoreactor
apparatus to provide 38 mg of 10i as a beige solid in
247.5–249.5 ꢂC; IR (KBr) 1638 cmÀ1
;
1H NMR
(CDCl3 + 1 drop CD3OD) d 1.51 (m, 2H), 1.69 (m,
4H), 2.65 (m, 6H), 4.03 (s, 3H), 4.06 (s, 3H), 4.62 (m,
24% yield; mp: 272–274 ꢂC; IR (KBr) 1637 cmÀ1; H
1

S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
3141
NMR (CDCl3 + 3 drops CD3OD) d 2.07 (m, 4H), 2.52
(m, 3H), 2.84 (m, 2H), 3.42 (m, 1H), 3.82 (m, 2H),
4.03 (s, 3H), 4.10 (s, 3H), 4.59 (m, 2H), 6.15 (s, 2H),
6.95 (s, 1H), 7.42 (s, 1H), 7.77 (s, 1H), 9.34 (s, 1H),
9.51 (s, 1H); 13C NMR (CDCl3 + 3 drops CD3OD) d
21.7, 29.2, 30.2, 39.6, 40.6, 56.2, 56.5, 56.8, 68.0, 98.3,
102.3, 103.5, 104.9, 106.2, 110.3, 122.2, 126.0, 132.3,
143.4, 144.6, 146.2, 149.8, 150.1, 152.4, 161.7; HRMS
calcd for C26H27N3O5H: 462.2029; found: 462.2017.
7.11 (m, 5H), 7.32 (s, 1H), 7.72 (s, 1H), 9.35 (s, 1H),
9.44 (s, 1H); HRMS (M+Li)+ calcd for C29H27IN3O5Li:
504.2111; found: 504.2108.
4.11.13.
6-[2-(Amino)ethyl]-8,9-dimethoxy-2,3-methyl-
enedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-one (10l).
Compound 10j (50 mg, 0.09 mmol) was dissolved in ace-
tic acid (46 mL). Formic acid (2.6 mL) and palladium
black (93 mg) were then added while stirring. After
30 min, more palladium black (70 mg) was added. The
whole mixture was stirred for another 30 min, at which
time additional palladium black (50 mg) was then added
and the reaction mixture was allowed to stir for another
30 min. The reaction mixture was diluted with chloro-
form (30 mL), filtered through Celite, and evaporated.
The residue was partitioned between chloroform
(50 mL) and 10% NaOH (20 mL). The aqueous phase
was washed by chloroform three times. Combined
organic phases were washed with water (2 · 20 mL),
and brine (20 mL), evaporated, and chromatographed
with chloroform/methanol/triethylamine (96:4:0.2) pro-
viding 28 mg of 10l in 82 % yield as a light orange solid;
mp (dec.): 218.5–219.5 ꢂC; IR (KBr) 1637; 1H NMR
(CDCl3) d 3.11 (m, 2H), 4.01 (s, 6H), 4.50 (m, 2H),
6.12 (s, 2H), 6.95 (s, 1H), 7.37 (s, 1H), 7.76 (s, 1H),
9.37 (s, 1H), 9.48 (s, 1H); HRMS (M+Li)+ calcd for
C21H19N3O5Li: 400.1485; found: 400.1480.
4.11.10.
6-[(2-N,N-Dibenzylamino)propyl]-8,9-dime-
thoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyri-
din-5-one (10j). Prepared from 9j (200 mg, 0.28 mmol) in
900 mL of 2% HCl using a photoreactor to provide 8 mg
of 10j as a yellow solid in 5% yield.
4.11.11.
6-[(2-N,N-Dibenzylamino)propyl]-8,9-dime-
thoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyri-
din-5-one (10j). A mixture of 9j (560 mg, 0.8 mmol),
Pd(PPh3)4 (188 mg, 0.16 mmol), and Ag2CO3 (442 mg,
1.6 mmol) in DMF (35 mL) was heated to reflux for
1 h. The mixture was cooled, diluted with CHCl3, and
filtered through Celite. The filtrate was evaporated, trit-
urated with 15 mL of 30% CH2Cl2 in ether, and filtered
to give 65 mg of 10j as a yellow solid. The filtrate was
evaporated. To the residue added Pd(PPh3)4 (188 mg,
0.16 mmol), Ag2CO3 (442 mg, 1.6 mmol), and DMF
(35 mL). The reaction mixture was heated to reflux for
1 h, cooled, diluted with chloroform, and filtered
through Celite. The filtrate was evaporated, triturated
with 15 mL of 30% methylene chloride in ether, and fil-
tered to give another 40 mg of 10j, total 105 mg of 10j in
23% yield as a yellow solid; mp: 215–217 ꢂC; IR (KBr)
4.11.14. 6-[2-(N-Methylamino)ethyl]-8,9-dimethoxy-2,3-
methylenedioxy-6H-dibenzo[c,h][2,6]naphthyridin-5-one
(10m). Formic acid (1.06 mL) and palladium black
(24 mg) were added with stirring to a solution of 10k
(40 mg, 0.08 mmol) in acetic acid (27 mL). After
30 min, 20 mg of palladium black (26 mg) was added.
After an additional 30 min, 20 mg of palladium black
was added and the reaction mixture was then allowed
to stir for another 30 min. The reaction mixture was
diluted with chloroform (30 mL), filtered through Celite,
and evaporated. The residue was partitioned between
chloroform (50 mL) and 10% NaOH (20 mL). The aque-
ous phase was extracted with chloroform (3· 50 mL).
The combined organic phases were washed with water
(2· 20 mL), and brine (20 mL), evaporated, and the res-
idue was triturated with ethyl ether to give 28 mg of 10m
in 88% yield as a yellow solid; mp (dec.): 221–222.5 ꢂC;
IR (KBr) 1635; 1H NMR (CDCl3) d 2.50 (s, 3H), 2.99 (t,
2H, J = 6.8), 4.02 (s, 6H), 4.53 (t, 2H, J = 6.8), 6.12 (s,
2H), 6.98 (s, 1H), 7.37 (s, 1H), 7.74 (s, 1H), 9.37 (s,
1H), 9.46 (s, 1H); 13C NMR (CDCl3) d 29.7, 36.1,
42.9, 56.3, 56.4, 98.2, 102.3, 103.6, 104.6, 105.9, 110.3,
121.9, 122.3, 125.8, 132.7, 143.3, 144.5, 145.8, 149.8,
150.1, 151.9, 161.4; HRMS (M+Li)+calcd for
C22H21N3O5Li: 414.1641; found: 414.1649.
1640 cmÀ1 1H NMR (CDCl3 + 3 drops CD3OD) d
;
2.90 (m, 2H), 3.54 (s, 4H), 3.79 (s, 3H), 4.01 (s, 3H),
4.57 (m, 2H), 6.15 (s, 2H), 6.50 (s, 1H), 7.24 (m, 11H),
7.68 (s, 1H), 9.43 (s, 1H), 9.46 (s, 1H); 13C NMR
(CDCl3 + 3 drops CD3OD) d 41.9, 50.4, 55.8, 56.5,
59.0, 98.1, 102.1, 104.0, 104.7, 106.6, 110.2, 122.3,
127.1, 128.4, 128.8, 129.2, 139.6, 143.5, 145.1, 145.6,
149.7, 150.0, 161.2; HRMS calcd for C35H31N3O5Li:
580.2424; found: 580.2409.
4.11.12. 6-[2-(N-Benzyl-N-methylamino)ethyl]-8,9-dime-
thoxy-2,3-methylenedioxy-6H-dibenzo[c,h][2,6]naphthyri-
din-5-one (10k). A mixture of 9k (500 mg, 0.8 mmol),
Pd(PPh3)4 (188 mg, 0.16 mmol), and Ag2CO3 (442 mg,
1.6 mmol) in DMF (35 mL) was heated to reflux for
1 h. The mixture was cooled, diluted with chloroform,
and filtered through Celite. The filtrate was evaporated,
triturated with 15 mL of 30% methylene chloride in
ether, and filtered to give 50 mg of 10k as a yellow solid.
The filtrate was evaporated. To the residue were added
Pd(PPh3)4 (188 mg, 0.16 mmol), Ag2CO3 (442 mg,
1.6 mmol), and DMF (35 mL). The reaction mixture
was heated to reflux for 1 h, cooled, diluted with chloro-
form, and filtered through Celite. The filtrate was evap-
orated, triturated with 15 mL of 30% methylene chloride
in ether, and filtered to give another 35 mg of 10k, total
85 mg of 10k in 21% yield as a yellow solid; mp (dec.):
4.12. 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.36 Plas-
mid YepG was purified by the alkali lysis method fol-
lowed by phenol deproteination and CsCl/ethidium
bromide isopycnic centrifugation method as de-
scribed.37 The end-labeling of the plasmid was accom-
1
222.5–223.5 ꢂC; IR (CHCl3) 1638; H NMR (CDCl3) d
2.42 (s, 3H), 2.73 (m, 2H), 3.57 (s, 2H), 3.74 (s, 3H),
3.93 (s, 3H), 4.51 (m, 2H), 6.06 (s, 2H), 6.80 (s, 1H),

3142
S. Zhu et al. / Bioorg. Med. Chem. 14 (2006) 3131–3143
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. Yang, C. H.; Schneider, E.; Kuo, M. L.; Volk, E. L.;
Roccchi, E.; Chen, Y. C. Biochem. Pharmacol. 2000, 60,
831.
12. Makhey, D.; Li, D.; Zhao, B.; Sim, S.-P.; Li, T.-K.; Liu,
A.; Liu, L. F.; LaVoie, E. J. Bioorg. Med. Chem. 2003, 11,
1809.
13. Li, D.; Zhao, B.; Sim, S.-P.; Li, T.-K.; Liu, A.; Liu, L. F.;
LaVoie, E. J. Bioorg. Med. Chem. 2003, 11, 521.
14. Li, D.; Zhao, B.; Sim, S.-P.; Li, T.-K.; Liu, A.; Liu, L. F.;
LaVoie, E. J. Bioorg. Med. Chem. 2003, 11, 3795.
15. Yu, Y.; Singh, S. K.; Liu, A.; Li, T.-K.; Liu, L. F.; LaVoie,
E. J. Bioorg. Med. Chem. 2003, 11, 1474.
plished by digestion with a restriction enzyme followed
by end-filling with Klenow polymerase as previously
described.38 The cleavage assays were performed as
previously reported.36,39 The drug and the DNA in
the presence of topoisomerase I were incubated for
30 min at 37 ꢂC. After development of the gels, typical-
ly 24 h exposure was used to obtain autoradiograms
outlining the extent of DNA fragmentation. Topoiso-
merase I-mediated DNA cleavage values are reported
as REC, Relative Effective Concentration, that is,
concentrations relative to topotecan, whose value is
arbitrarily assumed as 1.0, that are able to produce
the same cleavage on the plasmid DNA in the presence
of human topoisomerase I.
4.13. Cytotoxicity assays
16. Singh, S. K.; Ruchelman, A. L.; Zhou, N.; Li, T.-K.;
Liu, A.; Liu, L. F.; LaVoie, E. J. Med. Chem. Res.
2003, 12, 1.
The cytotoxicity was determined using the MTT-microti-
ter plate tetrazolinium cytotoxicity assay (MTA).40–42
The human lymphoblast RPMI 8402 and its camptothe-
cin-resistant variant cell line, CPT-K5 were provided by
Dr. Toshiwo Andoh (Aichi Cancer Center Research Insti-
tute, Nagoya, Japan).28 The KB3-1 cell line and its multi-
drug-resistant variant KBV-1 were obtained from K.V.
Chin (The Cancer Institute of New Jersey, NJ).29 The
KBH5.0 cell line was derived from KB3-1 by stepwise
selection against Hoechst 33342.19 The cytotoxicity assay
was performed using 96-well microtiter plates. Cells were
grown in suspension at 37 ꢂC in 5% CO2 and maintained
by regular passage in RPMI medium supplemented with
10% heat-inactivated fetal bovine serum, L-glutamine
(2 mM), penicillin (100 U/mL), and streptomycin
(0.1 mg/mL). For determination of IC50, cells were ex-
posed continuously for four days to varying concentra-
tions of the 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 assays were
performed at least twice in six replicate wells.
17. Ruchelman, A. L.; Singh, S. K.; Wu, X.; Ray, A.; Yang,
J.-M.; Li, T.-K.; Liu, A.; Liu, L. F.; LaVoie, E. J. Bioorg.
Med. Chem. Lett. 2002, 12, 3333.
18. 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.
19. 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.
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. Singh, S. K.; Ruchelman, A. L.; Li, T.-K.; Liu, A.; Liu, L.
F.; LaVoie, E. J. J. Med. Chem. 2003, 46, 2254.
22. Kerrigan, J. E.; Pilch, D. S.; Ruchelman, A. L.; Li, T.-K.;
Liu, A.; Liu, L. F.; LaVoie, E. J. Bioorg. Med. Chem. Lett.
2003, 13, 3395.
23. Ruchelman, A. L.; Li, T.-K.; Liu, A.; Liu, L. F.; LaVoie,
E. J. Bioorg. Med. Chem. 2004, 12, 3731.
24. Ruchelman, A. L.; Liu, A.; Zhou, N.; Liu, L. F.; LaVoie,
E. J. Lett. Drug Des. Discovery 2004, 1, 198.
25. Ruchelman, A. L.; Houghton, P. J.; Liu, A.; Zhou, N.;
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LaVoie, E. J. Bioorg. Med. Chem. 2005, 13, 6782.
28. Tamura, H.; Kohchi, C.; Yamada, R.; Ikeda, T.; Koiwai,
O.; Patterson, E.; Keene, J. D.; Okada, K.; Kjeldsen, E.;
Nishikawa, K.; Andoh, T. Nucleic Acids Res. 1990, 19, 69.
29. Shen, D. W.; Fojo, A.; Chin, J. E.; Roninson, I. B.;
Richert, N.; Pastan, I.; Gottesman, M. M. Science 1986,
232, 643.
Acknowledgments
This study was supported by Grant CA098127 (E.J.L.),
Grant CA39662 (L.F.L.), and Grant CA077433 (L.F.L.)
from the National Cancer Institute.
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