Cytotoxicity and TOP1-targeting activity of 8- and 9-amino derivatives of 5-butyl- and 5-(2-N,N-dimethylamino)ethyl-5H-dibenzo[c,h][1,6]naphthyridin-6-ones

Article abstract of DOI:10.1016/j.ejmech.2008.09.048

Studies on substituted 5H-dibenzo[c,h][1,6]naphthyridin-6-ones and 6H-dibenzo[c,h][2,6]naphthyridin-5-ones have demonstrated that hydrophilic substituents at the 2-position of an ethyl group at the 5- and 6-positions, respectively, can enhance biological

Full text of DOI:10.1016/j.ejmech.2008.09.048

European Journal of Medicinal Chemistry 44 (2009) 1471–1476
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cytotoxicity and TOP1-targeting activity of 8- and 9-amino derivatives of 5-butyl-
and 5-(2-N,N-dimethylamino)ethyl-5H-dibenzo[c,h][1,6]naphthyridin-6-ones
,
,c,
Lisa Sharma a, Yuan-Chin Tsai b, Angela A. Liu b, Leroy F. Liu b c, Edmond J. LaVoie a
*
a Department of Pharmaceutical Chemistry, 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:
Received 1 May 2008
Received in revised form
22 September 2008
Accepted 30 September 2008
Available online 11 October 2008
Studies on substituted 5H-dibenzo[c,h][1,6]naphthyridin-6-ones and 6H-dibenzo[c,h][2,6]naphthyridin-
5-ones have demonstrated that hydrophilic substituents at the 2-position of an ethyl group at the 5- and
6-positions, respectively, can enhance biological activity. The compatibility of such hydrophilic groups at
other sites with either TOP1-targeting activity or potent cytotoxic activity has not been explored. The
present study examines the influence on biological activity of either a 2-(N,N-dimethylamino)ethyl or
a N,N-dimethylacetamide derivative of 8- or 9-amino-5H-dibenzo[c,h]naphthyridin-6-ones that have a 5-
butyl- or 5-[2-(N,N-dimethylamino)ethyl]-substituent.
Keywords:
Ó 2008 Elsevier Masson SAS. All rights reserved.
Topoisomerase I-targeting agents
5H-Dibenzo[c,h][1,6]naphthyridin-6-ones
Cytotoxicity
Multidrug resistance
1. Introduction
lactone of camptothecin analogs together with reports which
reveal that both topotecan and irinotecan are substrates for efflux
Topoisomerases are ubiquitous enzymes that participate in
processes such as DNA replication, repair, transcription, and
recombination as well as chromosome condensation and segrega-
tion [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]. One of the factors that limited its
development into the clinic was its poor water solubility. Extensive
studies on several camptothecin analogs with improved physico-
chemical properties resulted in the clinical development of two
TOP1-targeting agents, topotecan (HycamtinÒ) and irinotecan
(CPT-11/CamptosarÒ), Fig. 1. Both of these agents have incorporated
within their structure the camptothecin ring system, which
transporters that have been associated with multidrug resistance
[9–14].
Studies in our laboratory have demonstrated that benzo[i]phe-
nanthridines and dibenzo[c,h]cinnolines can exert TOP1-targeting
activityand cytotoxicityagainst several human tumorcell lines. Several
5H-dibenzo[c,h][1,6]naphthyridin-6-ones, 6H-dibenzo[c,h][2,6]naph-
thyridin-5-ones and 11H-isoquino[4,3-c]cinnolin-12-ones have been
identified to possess exceptional TOP1-targeting activity and cytotox-
icity [15–19]. The presence of a 2-(N,N-dimethylamino)ethyl substit-
uent attached to the lactam nitrogen within the core structure of these
various TOP1-targeting agents is associated with improved formula-
tion properties and enhanced efficacy in vivo.
ARC-111 and its 5-butyl analog (Fig. 1) are known to be potent
TOP1-targeting agents. The TOP1-targeting activity and cytotoxicity
associated with amino and nitro substitents on both the A- and D-
ring of 5H-dibenzo[c,h][1,6]naphthyridines-6-ones has been
investigated [20–22]. The data from these studies indicate that the
8- and 9-nitro derivatives retain similar activity to that of 2,3-
methylenedioxy-8,9-dimethoxy-5H-dibenzo[c,h]naphthyridin-6-ones.
The present study examines the influence on biological activity of
either a 2-(N,N-dimethylamino)ethyl or a N,N-dimethylacetamide
derivative of 8- or 9-amino-5H-dibenzo[c,h][1,6]naphthyridin-6-
ones that have a 5-butyl- or 5-[2-(N,N-dimethylamino)ethyl]-
substituent.
includes the presence of a d-lactone. Hydrolysis of this lactone
results in an inactive derivative that has high affinity for human
serum albumin [6–8]. Considerable interest in non-camptothecin
TOP1-targeting agents has developed in view of the instability of d-
Abbreviations: TOP1, topoisomerase I; CPT, camptothecin.
* Corresponding author. Department of Pharmaceutical Chemistry, Rutgers, The
State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020,
USA. Tel.: þ1 732 445 2674; fax: þ1 732 445 6312.
E-mail address: elavoie@rci.rutgers.edu (E.J. LaVoie).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.048

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L. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 1471–1476
H3C
previously described [20]. Conversion of each of these nitro
compounds to their amine derivatives 8–11 was accomplished with
Raney-nickel in the presence of hydrazine, Scheme 2.
CH3
N
CH2
HO
O
O
These aryl amines were converted into their N-(2-N,N-dime-
thylamino)ethyl derivatives by treatment with (dimethylamino)-
acetaldehyde diethyl acetal in the presence of trifluoroacetic acid,
followed by reduction with sodium cyanoborohydride to provide
12–15 (Scheme 3) in modest yields that ranged from 26 to 40%.
The 2-(N,N-dimethylamino)acetamide derivatives 16–19 were
synthesized by reaction of the appropriate aryl amine with
(dimethylamino)acetyl chloride hydrochloride in the presence of
DMAP using DMF as solvent in yields of 61–65%, Scheme 3.
N
N
N
N
O
O
O
O
HO
HO
H2C
H2C
CH3
CH3
Topotecan
Camptothecin
N
CH2CH3
N
3. Results and discussion
O
O
O
N
O
N
3.1. TOP1-targeting activity
Irinotecan
HO
The TOP1-targeting activities and cytotoxic activities in
RPMI8402 and P388 cells are provided in Table 1. Only 5 and 7 had
comparable activity to camptothecin. The greater TOP1-targeting
activity and cytotoxicity observed with 5 and 7 relative to their N-
butyl analogs could be associated with the poor solubility observed
for both 4 and 6. Similarly, the significantly lower activity of 8 and
10, relative to 9 and 11 may be linked to differences in solubility.
These data do indicate that 9 and 11 had decreased TOP1-targeting
activity and cytotoxicity relative to their nitro precursors.
Derivatization of these aryl amines to increase their aqueous
solubility failed to improve either their TOP1-targeting activity or
cytotoxicity. All four of the N-(2-N,N-dimethylamino)ethyl deriva-
tives 12–15 were significantly less active as TOP1-targeting agents
than either their unsubstituted amines 9 and 11, or each of their
nitro precursor compounds, 4–7. In a comparison of the relative
activity of the 2-(N,N-dimethylamino)acetamide derivatives 16–19,
none of these derivatives exhibited significant TOP1-targeting
activity.
H2C
O
CH3
N
N
O
O
12
1
4
N
2
3
11
O
H3CO
H3CO
10
9
H3CO
H3CO
O
CH2CH2CH2CH3
8
N
CH2CH2
O
5
6
7
CH3
N
CH3
O
5-Butyl-2,3-methylenedioxy-8,9-dimethoxy
-5H-dibenzo[c,h][1,6]naphthyridin-6-one
ARC-111
Fig. 1. Structure of camptothecin, topotecan, irinotecan, ARC-111 and its 5-butyl
analog.
2. Chemistry
The synthetic methodologyemployedfor the preparation of5H-5-
butyl-2,3-methylenedioxy-9-nitrodibenzo[c,h]1,6-naphthyridin-6-
one 4 is outlined in Scheme 1. Oxidation of 2-bromo-4-nitrotoluene
with KMnO4 in the presence of aqueous pyridine provided the ben-
zoic acid 1. Treatment of 1 with thionyl chloride provided the acid
chloride, which was reacted without purification with N-butyl-4-
amino-6,7-methyenedioxyquinoline 2 to provide 3 in 96% overall
yield. Under Heck cyclization conditions 3 was converted to 4 in 53%
yield. Synthetic methods for the preparation of the 5-[2-(N,N-dime-
thylamino)ethyl]-2,3-methylenedioxy-9-nitrodibenzo[c,h]1,6-naph-
thyridine, 5, as well as the 8-nitro 2,3-methylenedioxy-8,9-
dimethoxy-5H-dibenzo[c,h]naphthyridin-6-ones, 6 and 7, have been
3.2. Cytotoxicity
Consistent with the TOP1-targeting activity, 5 and 7 exhibited
the more potent cytotoxic activity. Compounds 9 and 11 had
moderate cytotoxic activity with IC50 values ranging from 28 to
90 nM in RPMI8402 and P388 cells. Despite having no significant
TOP1-targeting activity, 15 exhibited moderate cytotoxic activity
(40–120 nM) in RPMI8402 or P388 cells. None of the compounds
Br
O2N
O2N
Br
a
CO2H
CH3
1
b,c
N
O
O
NH
2
N
N
O
O
N
N
O
O
d
O2N
O2N
Br
O
O
4
3
Scheme 1. Reagents and conditions: (a) KMnO4, Pyr, and H2O; (b) SOCl2; (c) TEA, CH2Cl2, and 2; (d) P(tolyl)3, Pd(OAc)2, Ag2CO3, DMF.

L. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 1471–1476
1473
N
N
O
O
N
N
O
O
X
Y
X
Y
R
R
O
O
4
5
6
7
X= NO2; Y = H; R = CH2CH2CH2CH3
X= NO2; Y = H; R = CH2CH2N(CH3)2
X= H; Y = NO2; R = CH2CH2CH2CH3
X= H; Y = NO2; R = CH2CH2N(CH3)2
8
X= NH2; Y = H; R = CH2CH2CH2CH3
X= NH2; Y = H; R = CH2CH2N(CH3)2
9
10 X= H; Y = NH2; R = CH2CH2CH2CH3
11 X= H; Y = NH2; R = CH2CH2N(CH3)2
Scheme 2. Reagents and conditions: Raney-Nickel, NH2NH2 at rt in EtOH for 2 h.
evaluated were found to be substrates for MDR1 or BCRP based
upon comparative cytotoxicity data (Table 2) performed in KB3-1,
KBV-1 or KBH5.0 cells.
5. Experimental
5.1. Chemistry
Melting points were determined with a Meltemp capillary
melting point apparatus. Column chromatography refers to flash
4. Conclusion
chromatography conducted on SiliTech 32-63
mm, (ICN Biomedi-
The data indicate that replacement of the 8,9-dimethoxy
substituents 5-butyl or 5-[(2-N,N-dimethylamino)ethyl]-2,3-
methylenedioxy-8,9-dimethoxydibenzo[c,h][1,6]naphthyridin-6-
ones with a N,N-dimethylaminoethylamino or a N,N-dimethyla-
minoacetamido group at either the 8- or 9-position does
improve water solubility, but results in a significant loss of
TOP1-targeting activity and cytotoxic activity. None of these
analogs have comparable TOP1-targeting activity or cytotoxicity
to ARC-111 [15,18] or the nitro derivatives 5 and 7. These data
indicate that not only is activity not enhanced by such modi-
fications in the case of the less polar and poorly soluble 5-butyl
analog of ARC-111, but also activity is diminished in the case of
those analogs that do possess a 5-[2-(N,N-dimethylamino)]ethyl
substituent. It has been recently shown that replacement of the
8,9-dimethoxyl groups of ARC-111 with diethoxy substituents
results in a dramatic loss of activity [23]. While these data
suggest that steric tolerance may be limited at these sites, steric
factors cannot explain the decreased activity of the unsub-
stituted amino derivatives 8–11 in the present study relative to
their nitro precursors. The data on these derivatives suggest
that an amino substituent at either the 8- or 9-position, at least
in the absence of a neighboring methoxy group at the available
8- or 9-position, is associated with a decrease in topoisomerase
I-targeting activity and cytotoxicity relative to its nitro
precursor.
cals, Eschwege, Ger.) 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 with chemical shifts reported in
d units down-
field from tetramethylsilane (TMS). All NMR analyses were per-
formed using CDCl3 unless otherwise noted. 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
Scientific, and were A.C.S. grade or HPLC grade. Methylene chloride
was freshly distilled from calcium hydride. All other solvents were
used as provided without further purification. Compounds 1, 2, 5–7,
and 9–11 were prepared as previously described [20].
5.1.1. 2-Bromo-N-butyl-N-(6,7-methylenedioxyquinolin-4-yl)-4-
nitrobenzamide (3)
A
solution of 2-bromo-4-nitrobenzoic acid (338 mg,
1.37 mmol) in thionyl chloride (16 mL) was refluxed for 4 h. It was
concentrated under vacuum to a semi-solid, which was placed
N
O
N(CH3)2
N
N
O
O
(H3C)2N
O
8,9
10,11
HN
N
N
H
R
O
R
O
12 R = CH2CH2CH2CH3
13 R = CH2CH2N(CH3)2
14 R = CH2CH2CH2CH3
15 R = CH2CH2N(CH3)2
N(CH3)2
N
O
O
N
N
O
O
O
(H3C)2N
O
N
HN
N
R
O
H
R
O
18 R = CH2CH2CH2CH3
19 R = CH2CH2N(CH3)2
16 R = CH2CH2CH2CH3
17 R = CH2CH2N(CH3)2
Scheme 3. Preparation of N-(2-dimethylaminoethyl) and N-(2-dimethylaminoacetyl) derivatives of 5-substituted 8- and 9-amino-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]-
naphthyridin-6-ones.

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L. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 1471–1476
Table 1
5.1.2. 5-Butyl-2,3-dimethoxy-9-nitro-5H-
dibenzo[c,h][1,6]naphthyridin-6-one (4)
Relative TOP1-targeting activity and cytotoxicity of 5-substituted 8- or 9-amino-5H-
dibenzo[c,h][1,6]naphthyridin-6-one derivatives in human lymphoblastoma cells
(RPMI8402) and a murine leukemia cell line (P388).
A
mixture of 3 (430 mg, 0.91 mmol), Pd(OAc)2 (41 mg,
0.18 mmol), Ag2CO3 (499 mg, 1.81 mmol), P(o-tolyl)3 (107 mg,
0.35 mmol) were placed in a flask and kept under vacuum for 3 h.
DMF (40 mL) was then added and the mixture was refluxed for
45 min. The reaction mixture was cooled to room temperature,
filtered through Celite and then washed with 50 mL of 2:1 mixture
of CH3OH:CHCl3. The solvent was removed under vacuum to
provide a dark semi-solid, which was purified by flash chroma-
tography eluting with chloroform to give 190 mg of 4ꢀa1s a yellow
Compound
TOP1-mediated DNA cleavagea
Cytotoxicity IC50
RPMI8402
(m
M)
P388
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
>10
0.1
>10
0.2
>20
0.5
>20
8.0
>20
>20
>20
>20
>20
>20
>20
>20
0.2
0.48
0.002
2.4
0.005
3.5
0.033
1.25
0.09
1.8
0.85
1.57
0.12
0.3
0.70
0.005
0.15
0.009
6.0
0.028
0.28
0.04
1.8
0.06
0.78
0.04
0.60
0.035
0.25
0.10
solid in 53% yield; mp 220–221 ꢁC; IR (KBr) 1657 cm
;
1H NMR
d
1.18 (t, 3H, J ¼ 7.4), 1.63 (m, 2H, J ¼ 7.5), 2.28 (m, 2H, J ¼ 7.6), 4.7 (t,
2H, J ¼ 7.5), 6.41 (s, 2H), 7.72 (s, 1H), 7.79 (s, 1H), 8.57 (dd, 1H,
J ¼ 10.8, J ¼ 2.2), 8.89 (d, 1H, J ¼ 10.8), 9.42 (d, 1H, J ¼ 2.2), 9.71 (s,
1H); 13C NMR
d 13.5, 20.1, 30.7, 51.1, 101.2, 102.3, 107.2, 110.7, 114.8,
117.2,121.3,128.9,130.3,133.2,142.4,143.2,148.2,148.7,150.8,151.0,
163.2; HRMS calcd for C21H17N4O5Li: 398.1328; found: 398.1314.
0.035
0.33
0.12
0.006
0.021
5.1.3. 9-Amino-5-butyl-2,3-dimethoxy-5H-
dibenzo[c,h][1,6]naphthyridin-6-one (8)
CPT
Topotecan
0.014
0.045
1.0
a
To a solution of 4 (127 mg, 0.35 mmol) in ethanol (33 mL) was
successively added Raney-Nickel (w80 mg) and hydrazine hydrate
(0.63 mL). The reaction mixture was stirred at room temperature for
2 h. The reaction mixture was filtered through Celite and the filtrate
was concentrated under vacuum to provide 85 mg of a light yellow
solid in 73% yield; mp >300 ꢁC; IR (KBr) 1659 cmꢀ1; 1H NMR (CD3OD)
Topoisomerase
I cleavage values are reported as REC, Relative Effective
Concentration, these are concentrations relative to topotecan, whose value is arbi-
trarily assumed as 1, that are able to produce 10% cleavage of the plasmid DNA in the
presence of human topoisomerase I.
under vacuum for 2 h. It was then dissolved in 10 mL of dry
CH2Cl2 and was added to
a solution of 2 [20] (244 mg,
d
0.99 (t, 3H, J ¼ 7.1), 1.43 (m, 2H, J ¼ 7.2), 2.09 (m, 2H, J ¼ 7.1), 4.46 (t,
0.99 mmol), TEA (0.5 mL) and dry CH2Cl2 (25 mL). The reaction
mixture was refluxed overnight. The mixture was allowed to cool,
the organic layer was then washed with saturated NaHCO3 solu-
tion, water, brine and then dried (anhyd. Na2SO4). Solvent was
removed under vacuum to yield 3 as a sticky brown glue
2H, J ¼ 7.5), 6.22 (s, 2H), 7.52 (s, 1H), 7.59 (s, 1H), 8.38 (dd, 1H, J ¼ 8.8,
J ¼ 1.8), 8.70 (d, 1H, J ¼ 8.8), 9.22 (d, 1H, J ¼ 1.6), 9.57 (s, 1H); 13C NMR
(CD3OD) d 13.7, 20.2, 31.0, 50.5, 101.8, 102.8, 103.2, 106.7, 112.0, 114.3,
114.8,116.3,130.4,134.6,142.2,144.2,147.9,150.3,154.3,164.2; HRMS
calcd for C21H19N3O3Li: 368.1586; found: 368.1588.
(430 mg) in 96% yield; IR (CH2Cl2) 1658 cmꢀ1 1H NMR
; d 0.96 (t,
3H, J ¼ 7.2), 1.36–1.55 (m, 2H), 1.71–1.84 (m, 2H), 3.38–3.35 (m,
1H), 4.42–4.54 (m, 1H), 6.20 (d, 2H, J ¼ 2.2), 7.02 (d, 1H, J ¼ 8.4),
7.26 (s, 1H), 7.34 (s, 1H), 7.69 (dd, 1H, J ¼ 8.4, J ¼ 2.3), 7.94 (d, 1H,
5.2. General method for the preparation of N-(2-
dimethylaminoethyl) derivatives of 5-substituted 8- and 9-amino-
2,3-methylenedioxy-5H-dibenzo[c,h][1,6]-naphthyridin-6-ones
J ¼ 6.1), 8.18 (dd, 1H, J ¼ 2.2), 8.52 (d, 1H, J ¼ 6); 13C NMR
d 12.8,
19.2, 28.9, 48.0, 96.7, 101.6, 105.2, 118.4, 119.8, 121.0, 121.6, 127.2,
128.1, 130.6, 142.6, 143.9, 146.9, 148.9, 150.8, 165.6; HRMS calcd for
C21H18BrN3O5Li: 478.0590; found: 478.0582.
5.2.1. 5-Butyl-9-(2-dimethylaminoethylamino)-2,3-
methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one (12)
Trifluoroacetic acid (1 mL, 4.06 mmol) was added dropwise at
0 ꢁC into a mixture of 8 (111 mg, 0.30 mmol) and (dimethylami-
no)acetaldehyde diethyl acetal (0.035 mL, 0.21 mmol). The reaction
mixture was stirred for 5 min and sodium cyanoborohydride
(36 mg, 0.57 mmol) was added batchwise (added 8 mg at a 4–5 min
time interval) to the mixture. The reaction mixture was then heated
at 50–60 ꢁC for 2.5 h. A saturated solution of sodium bicarbonate
(10 mL) was added and the mixture was extracted with chloroform
(2 ꢂ15 mL). The organic layers were washed with water (30 mL),
brine (50 mL) and dried (anhyd. Na2SO4). The solvent was removed
under vacuum and the crude product was chromatographed
eluting with 2:98 CH3OH:CHCl3 to provide 34 mg of 12 as a light
Table 2
Cytotoxicity of derivatives of 5-substituted 8- or 9-amino-5H-dibenzo[c,h][1,6]-
naphthyridin-6-one in the epidermoid carcinoma cell line KB3-1 and its multidrug-
resistant variants, KBV-1 and KBH5.0.
Compound
Cytotoxicity IC50
KB3-1 (wt)
(m
M)
KBV-1 (þMDR1)
KBH5.0 (þBCRP)
4
5
6
7
0.31
0.005
0.40
0.026
5.8
0.85
0.004
0.30
0.025
2.5
0.36
0.005
0.50
0.025
3.5
yellow solid in 26% yield; mp 175–176 ꢁC; IR (KBr) 1652 cmꢀ1
NMR
;
1H
8
d
0.94 (t, 3H, J ¼ 7.2), 1.34 (m, 2H, J ¼ 7.7), 2.05 (m, 2H, J ¼ 7.5),
9
10
0.13
3.2
0.13
2.5
0.15
3.5
2.39 (s, 6H), 2.76 (t,2H, J ¼ 5.7), 3.39 (t, 2H, J ¼ 5.2), 4.40 (t, 2H,
J ¼ 7.5), 5.31 (s, 1H), 6.16 (s, 2H), 6.87 (dd, J ¼ 8.8, J ¼ 2), 7.28 (s, 1H),
7.41 (s, 1H), 7.54 (s, 1H), 8.26 (d, 1H, J ¼ 8.8), 9.34 (s, 1H); 13C NMR
11
12
0.23
3.0
0.28
4.5
0.33
3.5
13
14
0.12
1.7
0.55
3.2
0.32
1.7
d
13.7, 20.1, 29.7, 31.3, 39.9, 44.7, 45.4, 50.3, 57.4, 100.5, 101.0, 102.1,
15
16
17
18
0.05
0.9
0.036
1.5
0.41
2.0
0.19
2.6
0.3
1.9
0.12
1.6
106.8, 111.9, 114.9, 115.3, 115.8, 130.4, 134.5, 142.5, 143.9, 147.2, 149.9,
151.9, 164.5; HRMS calcd for C25H28N4O3Li: 439.2321; found:
439.2306.
19
0.18
0.04
0.6
0.44
0.24
0.44
5.2.2. 5-(2-Dimethylaminoethyl)-9-(2-dimethylethylamino)-2,3-
methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one (13)
Trifluoroacetic acid (0.5 mL, 2.03 mmol) was added dropwise
Topotecan
KB3-1 cell line is the parent cell line. KBV-1 is a variant that overexpresses MDR1, p-
glycoprotein and KBH5.0 is the variant that overexpresses the efflux transporter
BCRP.
at 0 ꢁC into
a mixture of 9 [20] (60 mg, 0.15 mmol) and

L. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 1471–1476
1475
dimethylamino
acetaldehyde
diethyl
acetal
(0.018 mL,
bicarbonate (2 ꢂ15 mL), water (20 mL) and brine (2 ꢂ 30 mL). The
organic phase was dried (anhyd. Na2SO4) and concentrated under
vacuum to provide a solid. This solid residue was subjected to
column chromatography eluting with 5:95 CH3OH:CHCl3 to
provide 30 mg of 16 as a yellow solid in 61% yield; mp 184–185 ꢁC;
0.11 mmol). The reaction mixture was stirred for 5 min and
sodium cyanoborohydride (19 mg, 0.31 mmol) was added
batchwise to the mixture. The cooled reaction was worked up
and chromatographed to provide 23 mg of 13 as a light yellow
solid in 32% yield; mp 194–195 ꢁC; IR (KBr) 1647 cmꢀ1
;
1H NMR
IR (KBr) 1648 cmꢀ1, 3256 cmꢀ1; 1H NMR
d
0.95 (t, 3H, J ¼ 7.3), 1.38
d
2.30 (s, 6H), 2.34 (s, 6H), 2.69 (t, 2H, J ¼ 5.8), 2.95 (t, 2H,
(m, 2H, J ¼ 7.6), 2.05 (m, 2H, J ¼ 7.8), 2.45 (s, 6H), 3.18 (s, 2H), 4.44 (t,
2H, J ¼ 8), 6.17 (s, 2H), 7.46 (s, 1H), 7.55 (s, 1H), 7.67 (dd, 1H, J ¼ 8.4,
J ¼ 1.8), 8.46 (d, 1H, J ¼ 8.4), 8,83 (d, 1H, J ¼ 2), 9.45 (s, 1H), 9.55 (s,
J ¼ 7.0), 3.36 (t, 2H, J ¼ 5.0), 4.58 (t, 2H, J ¼ 7.1), 5.19 (s, 1H), 6.16
(s, 2H), 6.89 (dd, 1H, J ¼ 8.8, J ¼ 2.2), 7.29 (d, 1H, J ¼ 2.2), 7.45 (s,
1H), 7.83 (s, 1H), 8.28 (d, 1H, J ¼ 8.8), 9.37 (s, 1H); 13C
1H); 13C NMR
d 13.7, 20.1, 31.1, 46.2, 50.6, 63.8, 100.8, 102.2, 107.1,
NMR
d
29.6, 40.1, 44.9, 45.4, 57.5, 57.6, 100.3, 101.0, 102.3, 106.2,
110.1, 111.7, 114.7, 119.7, 121.3, 130.1, 113.9, 142.2, 144.1, 147.4, 147.9,
148.9, 150.1, 164.0, 169.6; HRMS calcd for C25H26N4O4H: 447.2032;
found 447.2033.
112.3, 114.9, 115.1, 115.3, 130.3, 134.5, 142.4, 143.5, 147.6, 150.3,
152.4, 164.8; HRMS calcd for C25H29N5O3H: 448.2349; found
448.2348.
5.3.2. 2-(Dimethylamino)-N-(5-(2-(dimethylamino)ethyl)-2,3-
methylenedioxy-6-oxo-5H-5,6-dibenzo[c,h][1,6]naphthyridin-9-
yl)acetamide (17)
5.2.3. 5-Butyl-8-[2-(dimethylamino)ethylamino]-2,3-
methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one (14)
Trifluoroacetic acid (1 mL, 4.06 mmol) was added dropwise at
0 ꢁC into a mixture of 10 (25 mg, 0.07 mmol) and (dimethylami-
no)acetaldehyde diethyl acetal (0.012 mL, 0.07 mmol). The reac-
tion mixture was stirred for 5 min and sodium cyanoborohydride
(12 mg, 0.19 mmol) was added batchwise (added 4 mg at a 4–
5 min time interval) to the mixture. The cooled reaction was
worked up and chromatographed to provide 12 mg of 14 as a light
A mixture of (dimethylamino)acetyl chloride hydrochloride
(13 mg, 0.08 mmol),
9 (40 mg, 0.10 mmol), DMAP (42 mg,
0.30 mmol), DCC (34 mg, 0.17 mmol) was placed in a flask. DMF
(2.5 mL) was added and the reaction mixture was stirred over-
night at 40 ꢁC. The cooled reaction was worked up and chroma-
tographed to provide 32 mg of 17 as a yellow solid in 65% yield;
mp 192–194 ꢁC; IR (KBr) 1655 cmꢀ1, 3286 cmꢀ1; 1H NMR
d 2.28 (s,
yellow solid in 40% yield; mp 218–220 ꢁC; IR (KBr) 1649 cmꢀ1
NMR
;
1H
6H), 2.46 (s, 6H), 2.97 (t, 2H, J ¼ 7.0), 3.18 (s, 2H), 4.59 (t, 2H,
J ¼ 6.5), 6.17 (s, 1H), 7.67 (dd, 1H, J ¼ 8, J ¼ 1.8), 7.83 (s, 1H), 8.44 (d,
1H, J ¼ 8.8), 8.82 (d, 1H, J ¼ 1.8), 9.45 (s, 1H), 9.54 (s, 1H), 9.54 (s,
d
0.91 (t, 2H, J ¼ 7.2), 1.34 (m, 2H, J ¼ 7.6), 2.00 (m, 2H,
J ¼ 7.5), 2.39 (s, 6H), 2.75 (t,2H, J ¼ 5.6), 3.37 (t, 2H, J ¼ 5.8), 4.42 (t,
2H, J ¼ 7.5), 5.31 (s, 1H), 6.12 (s, 2H), 7.13(dd, J ¼ 8.8, J ¼ 3), 7.37(s,
1H), 7.48 (s, 1H), 7.50 (s, 1H), 8.13 (d, 1H, J ¼ 8.8), 9.29 (s, 1H); 13C
1H); 13C NMR
d 45.6, 46.1, 48.8, 63.7, 101.0, 102.3, 106.6, 110.2,
111.9, 114.7, 119.8, 121.0, 130.0, 133.9, 142.2, 142.3, 143.7, 147.5,
147.8, 150.4, 164.3, 169.8; HRMS calcd for C25H27N5O4Li: 468.2223;
found 468.2227.
NMR
d 13.6, 20.0, 31.0, 40.2, 50.5, 57.5, 100.4, 102.1, 106.4, 107.1,
112.8, 115.1, 121.2, 122.4, 122.6, 126.6, 139.2, 143.1, 145.9, 147.5,
148.5, 149.5, 168.8; HRMS calcd for C25H28N4O3H: 433.2161; found
433.2222.
5.3.3. N-(5-Butyl-2,3-methylenedioxy-6-oxo-5H-
dibenzo[c,h][1,6]naphthyridin-8-yl)-2-(dimethylamino)acetamide (18)
A mixture of (dimethylamino)acetyl chloride hydrochloride
(12 mg, 0.08 mmol), 10 (24 mg, 0.07 mmol), DMAP (32 mg,
0.23 mmol) and DCC (23 mg, 0.10 mmol). DMF (2 mL) was added
and the reaction mixture was stirred overnight at 40 ꢁC. The cooled
reaction was worked up and chromatographed to provide 19.4 mg
of 18 as a yellow solid in 63% yield; mp 214–216 ꢁC; IR (KBr)
5.2.4. 5-(2-(Dimethylamino)ethyl)-8-(2-
(dimethylamino)ethylamino)-2,3-methylenedioxy-5H-
dibenzo[c,h][1,6]naphthyridin-6-one (15)
Trifluoroacetic acid (1 mL, 4.06 mmol) was added dropwise at
0 ꢁC into a mixture of 11 (120 mg, 0.316 mmol) and (dimethyla-
mino)acetaldehyde diethyl acetal (0.036 mL, 0.21 mmol). The
reaction mixture was stirred for 5 min and sodium cyanobor-
ohydride (38 mg, 0.63 mmol) was added batchwise (added 10 mg
at a 4–5 min time interval) to the mixture. The cooled reaction
was worked up and chromatographed to provide 23 mg of 15 as
a light yellow solid in 35% yield; mp 231–232 ꢁC; IR (KBr)
1646 cmꢀ1, 3220 cmꢀ1
;
1H NMR
d
0.95 (t, 3H, J ¼ 6.7), 1.36 (m, 2H,
J ¼ 7.4), 2.02 (m, 2H, J ¼ 7.2), 2.45 (s, 6H), 3.17 (s, 2H), 4.48 (t, 2H,
J ¼ 7.5), 6.18 (s, 2H), 7.45 (s,1H), 7.54 (s,1H), 8.28 (d,1H, J ¼ 2.2), 8.33
(d, 1H, J ¼ 8.8), 8.53 (dd, 1H, J ¼ 8.8, J ¼ 2.2), 9.43 (s, 1H), 9.56 (s, 1H);
13C NMR
d 13.7, 20.1, 30.9, 46.1, 50.6, 63.6, 100.6, 102.2, 107.1, 111.8,
1637 cmꢀ1
;
1H NMR
d
2.32 (s, 6H), 2.34 (s, 6H), 2.69 (t, 2H,
114.9, 117.5, 122.5, 125.1, 126.0, 128.4, 138.2, 140.9, 143.6, 147.4, 147.5,
149.9, 164.1, 169.2; HRMS calcd for C25H26N4O4Li: 453.2114; found
453.2103.
J ¼ 5.6), 2.97 (t, 2H, J ¼ 7.2), 3.33 (t, 2H, J ¼ 5.5), 4.63 (t, 2H, J ¼ 7),
4.59 (s, 1H), 6.15 (s, 2H), 7.17 (dd, 1H, J ¼ 9, J ¼ 3), 7.44 (s, 1H), 7.56
(d, 1H, J ¼ 2.6), 7.8 (s, 1H), 8.17 (d, 1H, J ¼ 8.8), 9.39 (s, 1H); 13C
NMR
d
29.7, 40.78, 45.1, 49.0, 57.7, 100.9, 102.0, 107.1, 107.6, 112.8,
5.3.4. 5H-2-(Dimethylamino)-N-(5-(2-(dimethylamino)ethyl)-2,3-
methylenedioxy-6-oxo-5H-dibenzo[c,h][1,6]naphthyridin-8-
yl)acetamide (19)
115.0, 121.1, 122.5, 122.6, 126.7, 139.0, 143.4, 146.5, 147.5, 148.7,
149.3, 164.8; HRMS calcd for C25H29N5O3H: 448.2349; found
448.2349.
A mixture of (dimethylamino)acetyl chloride hydrochloride
(13 mg, 0.08 mmol), 11 (30 mg, 0.08 mmol), DMAP (32 mg,
0.26 mmol) and DCC (26 mg, 0.12 mmol). DMF (2 mL) was
added and the reaction mixture was stirred overnight at 40 ꢁC.
The cooled reaction was worked up and chromatographed to
provide 22 mg of 19 as a yellow solid in 61% yield; mp 199–
5.3. General method for the preparation of N-(2-
dimethylaminoacetyl) derivatives of 5-substituted 8- and 9-amino-
2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-ones
5.3.1. N-(5-Butyl-2,3-methylenedioxy-6-oxo-5H-
201 ꢁC; IR (KBr) 1655 cmꢀ1, 3290 cmꢀ1 1H NMR
; d 2.28 (s, 6H),
dibenzo[c,h][1,6]naphthyridin-9-yl)-2-(dimethylamino)acetamide (16)
A mixture of (dimethylamino)acetyl chloride hydrochloride
2.44 (s, 6H), 2.91 (t, 2H, J ¼ 7.0), 3.15 (s, 2H), 4.65 (t, 2H, J ¼ 7.2),
6.17 (s, 1H), 7.46 (s, 1H), 7.82 (s, 1H), 8.28 (d, 1H, J ¼ 2.2), 8.35
(d, 1H, J ¼ 8.8), 8.54 (dd, 1H, J ¼ 8.8, J ¼ 2.2), 9.44 (s, 1H), 9.54 (s,
(17.8 mg, 0.12 mmol),
8 (40 mg, 0.11 mmol), DMAP (50 mg,
0.36 mmol), DCC (35.6 mg, 0.17 mmol) were placed in a flask. DMF
(2.5 mL) was added and the reaction mixture was stirred overnight
at 40 ꢁC. After the reaction was complete, 10 mL chloroform was
added. The organic layer was then washed with sodium
1H); 13C NMR
d 45.7, 46.1, 49.1, 57.5, 63.6, 101.0, 102.2, 111.9,
114.9, 117.5, 122.5, 125.2, 125.9, 128.6, 138.2, 140.8, 143.6, 147.5,
150.0, 164.3, 169.2; HRMS calcd for C25H27N5O4Li: 468.2223;
found: 468.2225.

1476
L. Sharma et al. / European Journal of Medicinal Chemistry 44 (2009) 1471–1476
5.4. Topoisomerase-mediated DNA cleavage assays
Acknowledgments
Human topoisomerase I was expressed in Escherichia coli and
isolated as a recombinant fusion protein using a T7 expression
system as described previously [24]. Plasmid YepG was also
purified by the alkali lysis method followed by phenol deprotei-
nation and CsCl/ethidium isopycnic centrifugation method as
described [25]. The 30 endlabeling of the plasmid was accom-
plished by digestion with a restriction enzyme followed by end
filling with Klenow polymerase as previously described [26]. The
cleavage assays were performed as previously reported [27,28].
The drug and the DNA in the presence of topoisomerase I was
incubated for 30 min at room temperature. The reactions were
This study was supported by Grant CA098127 (E.J.L.), Grant
CA39662 (L.F.L.) from the National Cancer Institute and the Gen-
zyme Corporation.
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The cytotoxicity was determined using the MTT-microtiter plate
tetrazolinium cytotoxicity assay (MTA). The human lymphoblast
RPMI8402 was provided by Dr. Toshiwo Andoh (Aichi Cancer
Center Research Institute, Nagoya, Japan) [29]. The P388 mouse
leukemia cell line [30] was obtained from Michael R. Mattern and
Randal K. Johnson (GlaxoSmithKline, King of Prussia, PA). The KB3-
1 cell line and its multidrug-resistant (MDR1, p-glycoprotein)
variant KBV-1 [31] was obtained from K.V. Chin (The Cancer Insti-
tute of New Jersey, New Brunswick, NJ). The KBH5.0 cell line as
noted previously [17] was derived from KB3-1 by stepwise selec-
tion 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 with 10% heat inactivated fetal bovine
serum, L-glutamine (2 mM), penicillin (100 U/mL), and strepto-
mycin (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
assays were performed at least twice in six replicate wells.