Dimethoxybenzo[i]phenanthridine-12-carboxylic acid derivatives and 6H-dibenzo[c,h][2,6]naphthyridin-5-ones with potent topoisomerase I-targeting activity and cytotoxicity

Article abstract of DOI:10.1016/j.bmcl.2004.08.070

The exceptional TOP1-targeting activity and antitumor activity of ARC-111, 1, prompted studies on similarly substituted benzo[i]phenanthridine-12- carboxylic ester and amide derivatives. These studies were extended to include 6-substituted 8,9-dimethoxy-2

Full text of DOI:10.1016/j.bmcl.2004.08.070

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5585–5589
Dimethoxybenzo[i]phenanthridine-12-carboxylic acid
derivatives and 6H-dibenzo[c,h][2,6]naphthyridin-5-ones with potent
topoisomerase I-targeting activity and cytotoxicity
Alexander L. Ruchelman,a Shejin Zhu,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 27 July 2004; revised 24 August 2004; accepted 26 August 2004
Available online 28 September 2004
Abstract—The exceptional TOP1-targeting activity and antitumor activity of ARC-111, 1, prompted studies on similarly substituted
benzo[i]phenanthridine-12-carboxylic ester and amide derivatives. These studies were extended to include 6-substituted 8,9-dimeth-
oxy-2,3-methylenedioxy-dibenzo[c,h][2,6]naphthyridin-5-ones, which represent reversed lactam analogues of 1. Several of these ana-
logues retained the potent TOP1-targeting activity and cytotoxicity observed for ARC-111.
Ó 2004 Elsevier Ltd. All rights reserved.
Topoisomerase I (TOP1) is an effective molecular target
for the development of clinically useful anticancer
agents.1–4 TOP1-targeting agents, such as camptothecin,
stabilize the cleaved complex formed between the en-
zyme and DNA. This ternary complex effectively con-
verts TOP1 into a cellular poison. Extensive studies on
camptothecin and its structurally related analogues have
resulted in the development of two clinical TOP1-target-
ing drugs, topotecan (HycamptinÒ) and irinotecan
(CPT-11/CamptosarÒ). These clinical agents have the
camptothecin ring system, which includes the presence
of a d-lactone, incorporated within their structures.
Hydrolysis of this lactone results in an inactive deriva-
tive that possesses high affinity for human serum albu-
min.5–7 The metabolic instability of this lactone and
the observation that both topotecan and irinotecan are
substrates for efflux transporters associated with multi-
drug resistance8–11 have prompted further studies on
the development of novel TOP1-targeting agents.
Substituted benzo[i]phenanthridines and dibenzo[c,h]-
cinnolines can exert TOP1-targeting activity and cyto-
toxicity against several human tumor cell lines.12–16
Recent studies in our laboratory have demonstrated that
specific 5H-dibenzo[c,h][1,6]naphthyridin-6-ones and
11H-isoquino[4,3-c]cinnolin-12-ones possess exceptional
TOP1-targeting activity and cytotoxicity.17–23 The di-
benzo[c,h][1,6]naphthyridin-6-one 1 (ARC-111, topo-
vale) and the isoquino[4,3-c]cinnolin-12-one 2 (Fig. 1)
were among the analogues that exhibited potent activity.
Both compounds 1 and 2 are not substrates for either
MDR1 (p-glycoprotein) or BCRP, which are efflux
N
N
N
N
O
O
O
O
N
O
H3CO
H3CO
H3CO
H3CO
CH2CH2
N
CH2CH2
N
O
CH3
CH3
H3C
H3C
1
2
Figure 1. The structures of 8,9-dimethoxy-5-[(2-N,N-dimethylamino)-
ethyl]-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one (1) and
8,9-dimethoxy-11-[(2-N,N-dimethylamino)ethyl]-2,3-methylenedioxyiso-
quino[4,3-c]cinnolin-12-one (2).
Keywords: Topoisomerase I; Cytotoxic agents; Anticancer; Benzo[i]-
phenanthridines; Dibenzo[c,h]naphthyridines.
*
Corresponding author. Tel.: +1 732445 6274; fax: +1 732445
6312; e-mail: elavoie@rci.rutgers.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.070

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A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5585–5589
transporters associated with multi-drug resistance.19,24
Based upon its potent antitumor activity in vivo
when administered either orally or parenterally, 1 is
viewed as a promising non-camptothecin TOP1-target-
ing agent.
The methods used for the preparation of the ester and
amide derivatives of 2,3-dimethoxy-8,9-methylene-
dioxybenzo[i]phenanthridine-12-carboxylic acid are
outlined in Scheme 1. Treatment of 3,4-methylenedioxy-
aniline with methyl vinyl ketone in the presence of FeCl3
in acetic acid provided 4-methyl-6,7-methylenedi-
oxyquinoline.28
Further studies were pursued to evaluate compounds
with structural similarity to 1. As the presence of the
2-(N,N-dimethylamino)ethyl substituent was associated
with enhanced cytotoxicity and greater antitumor activ-
ity in human tumor xenograft animal models, we
initially investigated the TOP1-targeting activity of
2-aminoethyl esters or amides of 2,3-dimethoxy-8,
9-methylenedioxybenzo[i]phenanthridine-12-carboxylic
acid. As an increase in the chain length of the 5-amino-
alkyl substituent in the case of 1 reduced its activity, we
also synthesized and evaluated the homologous 3-N,N-
dimethylaminopropyl ester and amide derivatives.17,18
These studies were also extended to the reversed lactam
analogues of 1. Specifically, we synthesized the
6-[(2-N,N-dimethylamino)ethyl]- and 6-[(3-N,N-dimeth-
ylamino)propyl-8,9-dimethoxy-2,3-methylenedioxydi-
benzo[c,h][2,6]naphthyridin-6-one derivatives. These
structurally related analogues of 1 were assayed for
TOP1-targeting activity and cytotoxicity in RPMI8402
and P388 cells, as well as their camptothecin-resistant
variants, CPT-K5 and P388/CPT45 cells, respec-
tively.25,26 In addition, the relative cytotoxic activity of
these compounds as compared to the parent cell line,
KB3-1 was assessed in KBV-1 cells,27 which overexpress
MDR1 and KBH5.0 cells,19 which overexpress BCRP.
Compound 3 was prepared in 65% yield by oxidation of
this 4-methylquinoline using SeO2. Refluxing a mixture
of 3 and 429 in acetic anhydride and triethylamine pro-
vided the stilbene intermediate 5. The ethyl ester 6 was
formed by treatment with thionyl chloride in absolute
ethanol. Heck palladium cyclization of either 5 or 6
failed to cleanly provide the desired cyclized products.
Photocyclization of 6 in acetonitrile for 30min did pro-
vide the desired cyclized ester 7. This ester could be read-
ily hydrolyzed with 10% NaOH in ethanol to provide
the acid, 8. Compound 8 was used as a general interme-
diate for the preparation of both 12-carboxy ester and
amide derivatives. Using thionyl chloride, the acid chlo-
ride of 8 was prepared. Reaction of this acid chloride
with N,N-dimethylethanolamine and 3-N,N-dimethyl-
aminopropanol provided the esters 9a and 9b, respec-
tively. The amide derivatives 10a and 10b were
similarly prepared by reaction of the acid chloride of 8
with 2-N,N-dimethylethylenediamine and 3-N,N-
dimethylaminopropylamine, respectively.
The reversed lactam analogues of 1 were prepared as
outlined in Scheme 2. Intermediate 11 was prepared by
CO2H
OCH3
OCH3
CO2Et
I
OCH3
OCH3
CHO
OCH3
OCH3
a
HO2C
b
O
O
O
O
I
I
O
O
73%
99%
N
4
3
N
N
5
6
R
O
c
51%
O
C
CO2H
OCH3
OCH3
OCH3
CO2Et
d
OCH3
OCH3
e
O
O
O
87%
OCH3
O
O
O
N
N
N
9a R = CH2CH2N(CH3)2 45%
9b R = CH2CH2CH2N(CH3)2
8
7
35%
f
R
NH
O
N
OCH3
OCH3
O
O
10a R = CH2CH2N(CH3)2 52%
10b R = CH2CH2CH2N(CH3)2 45%
Scheme 1. Reagents and conditions: (a) Ac2O, TEA; (b) SOCl2, EtOH; (c) ht, MeCN; (d) 10% NaOH in EtOH; (e) SOCl2 then ROH; (f) SOCl2 then
RNH2.

A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5585–5589
5587
OCH3
OCH3
H
N
O
CO2H
H2N
I
OCH3
OCH3
O
O
O
O
I
a
+
N
N
11
13 (56%)
12
b
OCH3
R
N
R
O
OCH3
OCH3
O
N
OCH3
O
O
O
O
c
I
N
N
15a: R = CH2CH2N(CH3)2 (34%)
15b: R = CH2CH2CH2N(CH3)2 (29%)
15c: R = CH2CH2N(CH2CH3)2 (25%)
14a: R = CH2CH2N(CH3)2 (89%
14b: R = CH2CH2CH2N(CH3)2 (88%)
14c: R = CH2CH2N(CH2CH3)2 (88%)
14d: R = CH2CH2N(-CH2(CH2)3CH2-) (84%)
15d: R = CH2CH2N(-CH2(CH2)3CH2-) (30%)
Scheme 2. Reagents and conditions: (a) SOCl2, 12, then add 11, CH2Cl2, TEA; (b) NaH, DMF, RClÆHCl for 14a, 14c–d, and NaNH2, PhCH3,
RClÆHCl for 14b; (c) 2% HCl, ht.
hydrolysis of its acetamide using aqueous NaOH in eth-
anol. Intermediate 12 was prepared in 90% yield by
treatment of 3 in pyridine with an aqueous solution of
KMnO4. Conversion of 12 to the acid chloride was per-
formed using thionyl chloride. Treatment of the acid
chloride in CH2Cl2 and triethylamine with 11 and allow-
ing this mixture to reflux overnight provided 13 in 56%
overall yield. Sodium hydride was added at 0°C to a
solution of 13 and 2-(dimethylamino)ethyl chloride
hydrochloride in DMF. The reaction mixture was then
allowed to warm to room temperature and stir for
45min. Compound 14a was isolated in 89% yield. A sim-
ilar method was employed for the preparation of 14b–d
in yields of 63%, 88%, and 84%, respectively. An im-
proved yield of 14b was obtained by treating a solution
of 13 and 3-(dimethylamino)propyl chloride hydrochlo-
ride with sodium amide in toluene. This reaction mix-
ture was refluxed for 4h to provide an 88% yield of
14b. Photolysis of 14a–d using 2% aqueous HCl through
a Vycor filter for 90min provided yields of the purified
cyclized products 15a–d ranging from 25% to 34%.
The relative TOP1-targeting activity and cytotoxicity of
these analogues in varied cell lines are provided in Table
1. The ethyl ester 7 and both alkylamino esters, 9a and
9b, were less potent as TOP1-targeting agents and cyto-
toxic agents. The propyl homologue 9b was also signif-
icantly less cytotoxic than 9a in RPMI8402, P388, and
KB3-1 cells. In light of their diminished cytotoxicity in
KBV-1 cells relative to the parent cell line, KB3-1, these
data suggest that 7 and 9a may be marginal substrates
for MDR1. In the case of the amide derivative 10a, com-
parable TOP1-targeting activity and cytotoxicity rela-
tive to 1 was observed in RPMI8402, P388, and KB3-1
Table 1. TOP1-targeting activity and cytotoxicity of 8,9-dimethoxy-2,3-methylenedioxybenzo[i]phenanthridine-12-carboxylic acid derivatives and
6-substituted 8,9-dimethoxy-2,3-methylenedioxydibenzo[c,h][2,6]naphthyridin-5-ones
Compound
TOP1-mediated
DNA cleavagea
Cytotoxicity IC50 values (lM)b
RPMI8402wt
CPT-K5 CPT-resist.
P388 wt
P388/CPT45
CPT-resist.
KB3-1
wt
KBV-1
+MDR1
KBH5.0
+BCRP
1
2.5
120.085
0.002
>10
0.03
0.90
0.001
0.23
0.11
0.020.03204.072 0.2
0.005
0.005
2.0
0.006
7
9a
05.04
0.0431.2
0.34
5
0.73
1.5
0.011
0.11
9b
7
0.22
0.003
0.05
0.14
0.50
0.6
10a
10b
15a
15b
15c
0.5
30
3
1.0
6 0.0020.2 0.001
0.03
0.002
0.10
0.03
2.05
0.21
0.20
0.90
1.5
0.34
0.20
0.020
0.03
0.23
0.045
0.002
0.003
0.003
0.006
0.015
0.04
4.5
0.50
0.004
0.004
0.0007
0.0005
0.003
0.001
0.006
0.021
0.004
0.007
0.0120.005
0.006
0.025
0.44
3
0.003
0.003
0.7
0.1
1
15d
CPT
Topotecan
0.003
0.014
0.045
0.007
0.026
0.44
>10
>10
>10
>10
5
a Topoisomerase I cleavage values are reported as REC, relative effective concentration, that is, concentrations relative to camptothecin, 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.
Topoisomerase I cleavage assays for determining the REC values were performed as previously described.18
b The origin of cell lines used in this study and the methods used to assess cytotoxicity have been detailed elsewhere.19

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A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5585–5589
cells. The increased cytotoxic activity of 10a relative to
the ester 9a could also be partly attributed to the anti-
cipated increased stability of 10a relative to 9a in cell
culture media. The propyl homologue, 10b, had signifi-
cantly reduced TOP1-targeting activity and cytotoxicity
in these cell lines. It was also evident that both 10a and
10b, unlike 1, were substrates for the efflux transporters
MDR1 and BCRP, based upon their significantly
reduced toxicity in both KBV-1 and KBH5.0 cells rela-
tive to their parent cell line, KB3-1. As anticipated for
compounds that principally exert their cytotoxic activity
by targeting TOP1, these amide derivatives exhibited
decreased cytotoxic activity in CPT-K5 and P388/
CPT45 relative to their parent cell lines, RPMI8402
and P388, respectively.
Acknowledgements
This study was supported by Grant CA098127 (E.J.L.)
and Grant CA39662(L.F.L.) and Grant CA077433
(L.F.L.) from the National Cancer Institute.
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The series of reversed lactams, 15a–d, provided a very
different profile of biological activity. Compounds 15a
and 15b have comparable activity to 1 as TOP1-target-
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times more cytotoxic in RPMI8402than 1, but exhibited
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or BCRP. Compound 15c is 3 to 4-fold more potent
than 1, 15a, or 15b as a TOP1-targeting agent. In terms
of intrinsic activity as a TOP1-targeting agent, it is
evident that the reversed lactam 15d is the most
potent derivative with 10-fold greater activity than
camptothecin. While the difference between the ethyl
and propyl homologue was negligible, these data suggest
that the presence of the more hydrophobic 2-(N,N-
diethylamino) substituent or 2-(piperidin-1-yl) group in
place of the 2-(N,N-dimethylamino) substituent on the
6-ethyl moiety substantially enhances intrinsic TOP1-
targeting activity. Compounds 15c and 15d had compa-
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KB3-1 cells.
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have extraordinary potency as TOP1-targeting agents
and possess cytotoxic activity greater than 1 or campto-
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