Design, synthesis and anticancer activity of novel dihydrobenzofuro[4,5-b] [1,8]naphthyridin-6-one derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.08.016

On the basis of the chemical structures of psorospermin with a xanthone template and acronycine derivatives with an acridone template, rac-1 and rac-2 constructed on an 1,2-dihydrobenzofuro[4,5-b][1,8]naphthyridin-6(11H)-one scaffold were designed and synthesized as potential anticancer agents. Their anticancer activities were evaluated against five human cancer cell lines. Rac-2 showed similar anticancer activity to doxorubicin and rac-1 exhibited even higher anticancer activity against LNCaP (IC₅₀ = 0.14 μM), DU145 (IC₅₀ = 0.15 μM), PC3 (IC₅₀ = 0.30 μM) and MCF-7 (IC₅₀ = 0.26 μM) cancer lines than doxorubicin and rac-2. Also, rac-1 revealed very potent anticancer activity (IC₅₀ = 0.15 μM) against MCF-7/ADR cell (doxorubicin-resistant breast cancer cell) lines and induced G2/M phase arrest of the cell cycle in MCF-7/ADR cells.

Full text of DOI:10.1016/j.bmcl.2011.08.016

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5730–5734
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and anticancer activity of novel
dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivatives
Jin-Ah Kang a, , Zunhua Yang a, , Ji Yeon Lee a, , Umasankal De a, Tae Hyung Kim a, Ji Young Park a,
Hye Jin Lee a, Yun Jung Park a, Pusoon Chun a, Hyung Sik Kim a, Lak Shin Jeong b, Hyung Ryong Moon a,
⇑
a College of Pharmacy and Research Institute for Drug Development, Pusan National University, Busan 609-735, Republic of Korea
b College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of the chemical structures of psorospermin with a xanthone template and acronycine deriv-
atives with an acridone template, rac-1 and rac-2 constructed on an 1,2-dihydrobenzofuro[4,5-b]
[1,8]naphthyridin-6(11H)-one scaffold were designed and synthesized as potential anticancer agents.
Their anticancer activities were evaluated against five human cancer cell lines. Rac-2 showed similar anti-
cancer activity to doxorubicin and rac-1 exhibited even higher anticancer activity against LNCaP
Received 8 June 2011
Revised 1 August 2011
Accepted 3 August 2011
(IC50 = 0.14
l
M), DU145 (IC50 = 0.15
l
M), PC3 (IC50 = 0.30
l
M) and MCF-7 (IC50 = 0.26
l
M) cancer lines
M) against
Keywords:
than doxorubicin and rac-2. Also, rac-1 revealed very potent anticancer activity (IC50 = 0.15
MCF-7/ADR cell (doxorubicin-resistant breast cancer cell) lines and induced G2/M phase arrest of the cell
cycle in MCF-7/ADR cells.
l
Dihydrobenzofuro[4,5-b][1,8]naphthyridin-
6-one
Anticancer activity
Epoxide
Ó 2011 Elsevier Ltd. All rights reserved.
G2/M phase arrest
Psorospermin (Fig. 1), a natural product isolated from the trop-
ical plant Psorospermum febrifugum, consists of a xanthone scaffold1
(Fig. 2) and was reported to exhibit potent anticancer activity
against several human and murine cancer cell lines in vitro and in
vivo.2,3 Acronycine, an alkaloid first isolated from Acronychia baueri
Schott (Rutaceae) consists of an acridone scaffold and showed a
broad spectrum of activity against a variety of solid tumors includ-
ing sarcoma, myeloma, carcinoma and melanoma.4,5 However, the
poor clinical potency of this alkaloid prompted medicinal chemists
to synthesize numerous acronycine derivatives with higher clinical
anticancer activity and less cytotoxicity to normal cells than acr-
onycine. Among them, S23905-1 (Fig. 1) exhibited much higher
anticancer activities against murine P388 leukemia and colon 38
adenocarcinoma in vivo as well as L1210 cell lines in vitro than acr-
onycine itself.6
with the N-7 of guanine base8 and S23906-1 is also an alkylating
agent reacting with the N-2 of guanine base of DNA.9
Recently, we synthesized novel oxiranyl and thiiranyl phenolic
derivatives constructed on various templates such as naphthalene,
9H-carbazole, benzophenone and anthraquinone as potential
alkylating agents and the compounds exhibited better to a little
less potent anticancer activities against MDA-MB-231, LNCaP,
Du145 and PC3 cell lines than doxorubicin.10 This result implies
that introduction of electrophiles such as epoxypropyl and thioep-
oxypropyl groups into a flat scaffold may provide an ability of DNA
alkylation. The flat scaffold seems to contribute to insertion and
stacking between the base pairs of the DNA double helix.
On the basis of the hypothesis that the structural combination of
two compounds possessing similar biological profiles could afford
synergistically increased pharmacological activity, replacement of
the xanthone scaffold of psorospermin with an acridone scaffold (
Fig. 2) was first considered for the target structure. However, the
existence of protonated nitrogen atoms on the structure of DNA
alkylators in biological pH might increase the binding affinity
through the electrostatic attraction between the protonated nitro-
gen atom of the DNA alkylators and the phosphate anions of the
DNA skeleton. Therefore, insertion of an additional nitrogen atom
to the acridone template is expected to enhance binding affinity
between the target compounds and the duplex DNA, which in turn
may improve selectivity of the target compounds toward DNA
as alkylators. On the basis of these findings and hypothesis, we
have finally designed and synthesized novel dihydrobenzof
On the other hand, the discovery of acronycine epoxide (Fig. 1)
isolated from several Sarcomelicope species has supported that acr-
onycine might exert its anticancer in vivo activities through bio-
transformation to the corresponding epoxide, acronycine epoxide.7
According to the previous studies, psorospermin is a selective DNA
alkylating agent and its oxirane ring acts as an electrophile reacting
⇑
Corresponding author. Address: Laboratory of Medicinal Chemistry, College
of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea.
Tel.: +82 51 510 2815; fax: +82 51 513 6754.
E-mail address: mhr108@pusan.ac.kr (H.R. Moon).
These authors equally contributed to this article.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.016

J.-A. Kang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5730–5734
5731
Figure 1. The rationale for the design of the target compounds, rac-1 and rac-2.
Figure 2. Structures of xanthone, acridone and benzofuran analogs.
uro[4,5-b][1,8]naphthyridin-6-one derivatives (Figs. 1 and 2), rac-1
and rac-2 bearing an oxiranyl substituent as a potential electro-
phile, with anticipation that the plausible protonated nitrogen
atoms at 10- and/or 11-positions of rac-1 and -2 in biological pH
might increase the binding affinity with the DNA helix. Also, anti-
cancer activities of rac-1 and rac-2 were evaluated against human
prostate cancer cell lines (LNCaP, DU145 and PC3) and breast cancer
cell lines (MCF-7 and doxorubicin-resistant MCF-7/ADR). Moreover,
we examined the effect of rac-1 and rac-2 on regulation of cell cy-
cles against MCF-7/ADR cell lines.
As shown in Scheme 1, compound 3 with a benzo[b][1,8]naph-
thyridinone template was obtained in 38% yield by heating 2-ami-
nonicotinic acid and phloroglucinol without a solvent in a sealed
tube at 230 °C. Treatment of 3 with dibromoisoprene and sodium
hydride in methanol formed fused dihydrofuran, in 42% recovered
yield, as a racemate form.11 It is worthy to note that the process of
N,O-dimethylation using dimethyl sulfate in the presence of NaH
afforded two isomeric compounds, rac-512 and rac-5a13 in an
1:1.3 ratio, separable on silica gel column chromatography
(Scheme 2). Treatment of rac-4 with sodium hydride might give
a nitrogen anion capable of acting as an ambident nucleophile at
N-10 and N-11 positions. Therefore, it is possible for two constitu-
tional isomers, rac-5 and rac-5a to be generated. Whereas the A-
ring protons of rac-5a were observed at d 8.68 (1H), 7.77 (1H)
and 6.54 (1H) ppm, respectively, those of rac-5 were observed at
d 8.64 (2H) and 7.07 (1H) ppm. Protons of A-ring of rac-5a reso-
nated at more up-field region than those of rac-5, implying that
the A-ring of rac-5a is not aromatic and rac-5 is our desired com-
pound. In addition, chemical shifts of the A-ring protons of rac-4
were similar to those of rac-5 (d 8.75, 8.49 and 7.32 ppm), suggest-
(Scheme 2) also confirms that rac-5 is the desired compound.
1,2-Dihydroxylation of rac-5 with OsO4 in the presence of NMO
serving as a co-oxidant furnished four stereoisomers (two diaste-
reomeric racemates) inseparable on silica gel column chromatog-
raphy. A temporary protection of the primary hydroxyl group
with a TBS group allowed the diasteromeric mixture to be
separated on silica gel column chromatography, giving rac-7 and
rac-8 in 26 and 20% yields, respectively, from rac-5. The relative
stereochemistry of the obtained racemates was determined from
that of the corresponding final compounds, rac-1 and rac-2. Syn-
thesis of the target compounds, rac-1 and rac-2 from rac-7 and
rac-8 is depicted in Scheme 3. Fluoride anion from TBAF gently
removed the TBS group of each racemate to afford the correspond-
ing diol compounds, in which only the primary hydroxyl groups
were regioselectively mesylated by treatment with mesyl chloride
in pyridine to give rac-9 and rac-10, respectively. Finally,
epoxidation reactions were conducted using K2CO3 in the presence
of 18-Crown-6 to generate the target compounds, rac-1 and rac-2
in 61 and 62% yields, respectively, from rac-7 and rac-8, respec-
tively.15 Relative stereochemistry of rac-1 and rac-2 was assigned
on the basis of information from the literature16 reported by Cass-
ady and his co-workers. According to the literature, (2R,20R)-com-
pound of 2-(2-methyloxiran-2-yl)-2,3-dihydrobenzofuran (Fig. 2)
showed bigger difference in 1H NMR chemical shifts between
hydrogens of the 30-methylene than that of (2R,20S)-compound
(0.24 vs 0.11 ppm). The differences in rac-1 and rac-2 were 0.26
and 0.10 ppm, respectively, indicating that rac-117 is (2R⁄,20R⁄)-
compound and rac-218 is (2R⁄,20S⁄)-compound.
Anticancer activity19 of rac-1 and rac-2 was evaluated against
LNCaP, DU145, PC3, MCF-7/ADR and MCF-7 cancer cell lines after
24 and 48 h incubation, respectively (Table 1). Interestingly, the
final diasteroisomeric compounds, rac-1 and rac-2 exhibited the
differences in anticancer activity against several cancer cell lines.
ing that rac-5 preserves
a desired 1,2-dihydrobenzofuro[4,5-
b][1,8]naphthyridin-6(11H)-one template. Chemical shift pattern
(d 8.65, 8.53 and 7.08 ppm) of the A-ring protons of DMNO14

5732
J.-A. Kang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5730–5734
Scheme 1. Synthesis of rac-7 and rac-8 with
a
dihydrobenzofuro[4,5-b][1,8]naphthyridinone scaffold. Reagents and conditions: (a) neat, 230 °C, 38% yield; (b)
dibromoisoprene, NaH, MeOH, rt, 21% yield (recovered yield: 42%); (c) Me2SO4, NaH, DMF, rt, 25% yield for rac-5, 32% yield for rac-5a (See Scheme 2); (d) OsO4, NMO,
acetone/H2O (4:1), rt; (e) TBSCl, imidazole, DMAP, DMF, 75 °C, 26% yield (two-steps yield) for rac-7, 20% yield (two-steps yield) for rac-8 from rac-5.
Scheme 2. Plausible mechanism of formation of rac-5 and rac-5a.
Rac-2 showed 1.5- to 15-fold more potent anticancer activity than
doxorubicin used a positive control against most cancer cell lines
after 48 h incubation except DU145 and PC3 cell lines and its
between stereochemistry and anticancer activity is consistent with
the results obtained from psorospermin.15
Effects of rac-1 and rac-2 on the cell cycle progression were
IC50 values were in the range of 0.84–2.15
lM. It is notable that
examined by Flow cytometry.20 Rac-1 (0.2
lM) induced an accu-
the anticancer activity ratio of rac-2 to doxorubicin after 24 h incu-
bation is much better than that after 48 h incubation. Anticancer
activity of rac-2 against LNCaP, DU145, MCF-7/ADR and MCF-7 cell
lines were 2- to 3-fold higher than that against PC3 cell lines. Rac-1
displayed much more potent anticancer activity than rac-2 and
doxorubicin and its IC50 values revealed submicromolar concentra-
tions in all cancer cell lines tested. Especially, it is notable that
rac-1 exhibited significant anticancer activity against doxorubi-
cin-resistant breast cancer cell lines (MCF-7/ADR). Anticancer
activities of rac-1 were ⁄4- t⁄o 7-fold more potent than those of
rac-2, implying that (2R ⁄,20R⁄)-stereochemistry is better for anti-
cancer activity than (2R ,20S )-stereochemistry. This relationship
mulation of cells in the G2/M phases of the cell cycle with a con-
comitant decrease in the percentage of MCF-7/ADR cells in the
G1 phase. A total of 22% of the untreated MCF-7/ADR cells were
in the G2/M phases, compared with 42% of cells cultured with
0.2 lM rac-1. However, rac-2 (0.2 lM) did not induce G2/M phase
arrest of the cell cycle in MCF-7/ADR cells, whereas slightly in-
creased S phase of cell population in MCF-7/ADR cells (Table 2).
We have designed dihydrobenzofuro[4,5-b][1,8]naphthyridin-
6-one as a novel potential anticancer scaffold and synthesized its
derivatives with an oxiranyl fun⁄ctional group capable of serving
an electrophile. Rac-2 with (2R ,20S⁄)-stereochemistry exhibited
similar anticancer activity to doxorubicin against all cancer cell

J.-A. Kang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5730–5734
5733
Scheme 3. Synthesis of rac-1 and rac-2 with a dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one scaffold. Reagents and conditions: (a) (i) TBAF, THF, rt; (ii) MsCl, pyridine,
0 °C; (b) K2CO3, 18-Crown-6, acetone, 61% yield for rac-1 from rac-7, 62% yield for rac-2 from rac-8.
Table 1
Cytotoxicities of rac-1 and rac-2 against five human cancer cell lines after 24 and 48 h incubationa
Compd
IC50 (lM)
LNCaPb
24 h
DU145c
24 h
PC3d
24 h
MCF-7/ADRe
24 h
MCF-7f
24 h
48 h
0.14
0.01
0.86
0.05
1.66
0.03
48 h
0.15
0.02
0.84
0.05
0.58
0.06
48 h
0.30
0.02
2.15
0.31
1.22
0.06
48 h
0.15
0.02
0.92
0.08
13.6
0.12
48 h
0.26
0.03
1.02
0.12
1.51
0.02
rac-1
rac-2
Doxog
0.48
0.05
2.48
0.15
7.42
0.48
0.55
0.06
2.36
0.02
6.24
0.32
0.56
0.04
4.67
0.38
4.10
0.35
0.49
0.05
2.21
0.17
>20
0.66
0.07
1.84
0.11
8.94
0.54
a
b
c
d
e
f
Assay was done in triplicate.
Prostate cancer cell lines (AR-positive).
Prostate cancer cell lines.
Prostate cancer cell lines (AR-negative).
Breast cancer cell lines (adriamycin-resistant).
Breast cancer cell lines (ER-positive).
Doxorubicin.
g
Table 2
Cell cycle analysis of MCF-7/ADR cells by flow cytometry
Compounds
Cell cycle
Percentage (%)
Rac-2 (
Control
Rac-1 (
lM)
lM)
Doxo 5 lM
0.05
0.1
0.2
0.05
0.1
0.2
G1
62.15
3.14
15.34
0.98
22.51
1.50
63.48
3.29
12.37
1.42
24.15
1.87
57.41
1.82
8.97
0.15
33.68
1.09
51.23
2.14
68.37
3.65
11.16
0.62
20.47
1.43
64.64
3.97
13.60
0.97
21.76
0.63
55.16
2.23
16.55
0.85
28.30
0.97
53.26
4.21
21.92
1.81
24.62
0.96
S
7.68
0.37*
41.09
1.95*
G2/M
The MCF-7/ADR cells were treated with the indicated concentrations of test compounds for 48 h. The cells stained with propidium iodine (PI) were subjected to flow
cytometric analysis to determine the cell distributions at each phase of the cell cycle. The data is reported as the mean SEM of three independent experiments. A one-way
ANOVA with Newman–Keuls post hoc test was used to determine statistical significance.
*
p-value <0.05 was considered significant p value.
lines tested, whereas rac-1 with (2R⁄,20R⁄)-stereochemistry re-
vealed higher anticancer activity than both rac-2 and doxorubicin.
Rac-1 was found to induce the accumulation of cells in the G2/M
phases of the cell cycle of MCF-7/ADR cells. Further studies on
the inhibition mechanism of rac-1 and rac-2 in molecular levels
and their possibility capable of acting as topoisomerase inhibitors
are in progress in our laboratory and the results will be reported in
due course. Further studies on the separation of the two enantio-
mers rac-1 and rac-2, their cytotoxicity assays, their inhibition
mechanism in molecular levels and their possibility capable of act-
ing as topoisomerase inhibitors are in progress and the results will
be reported in due course.
Acknowledgments
This work was supported by the Bio-Scientific Research Grant
funded by the Pusan National University (PNU, Bio-Scientific
Research Grant) (PNU-2008-101-210).

5734
J.-A. Kang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5730–5734
CDCl3) d 8.62 (m, 2H, 7-H, 9-H), 7.15 (dd, 1H, J = 7.6, 4.8 Hz, 8-H), 6.30 (s, 1H,
References and notes
4-H), 4.73 (dd, 1H, J = 10.0, 7.6 Hz, 2-H), 4.12 (s, 3H, NCH3), 3.94 (s, 3H, OCH3),
3.80 (dd, 1H, J = 14.4, 10.0 Hz, 1-Ha), 3.64 (dd, 1H, J = 14.4, 7.6 Hz, 1-Hb), 2.97
(d, 1H, J = 4.8 Hz, 30-Ha), 2.71 (d, 1H, J = 4.8 Hz, 30-Hb), 1.42 (s, 3H, 10-H); 13C
1. Peres, V.; Nagem, T. J.; de Oliveira, F. F. Phytochemistry 2000, 55, 683.
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7. Brum-Bousquet, M.; Mitaku, S.; Skaltsounis, A.-L.; Tillequin, F.; Koch, M. Planta
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NMR (100 MHz, CDCl3)
d 177.46, 165.88, 164.22, 152.15, 151.84, 144.66,
136.60, 119.27, 117.80, 109.82, 102.22, 89.48, 84.90, 57.78, 56.56, 51.24, 35.58,
35.23, 17.17; HRMS (FAB+) calcd for C19H19N2O4 (M+H)+: 339.1345, found:
339.1351.
18. (2R⁄)-5-Methoxy-11-methyl-2-((2S⁄)-2-methyloxiran-2-yl)-1,2-
dihydrobenzofuro[4,5-b][1,8]naphthyridin-6(11H)-one: 1H NMR (400 MHz,
CDCl3) d 8.61 (m, 2H, 7-H, 9-H), 7.15 (dd, 1H, J = 7.2, 4.8 Hz, 8-H), 6.30 (s, 1H,
4-H), 4.70 (dd, 1H, J = 9.6, 7.6 Hz, 2-H), 4.11 (s, 3H, NCH3), 3.93 (s, 3H, OCH3),
3.80 (dd, 1H, J = 14.8, 9.6 Hz, 1-Ha), 3.62 (dd, 1H, J = 14.8, 7.6 Hz, 1-Hb), 2.84 (d,
1H, J = 4.4 Hz, 30-Ha), 2.74 (d, 1H, J = 4.4 Hz, 30-Hb), 1.42 (s, 3H, 10-H); 13C NMR
(100 MHz, CDCl3) d 177.47, 165.58, 164.17, 152.17, 151.81, 144.68, 136.58,
119.24, 117.91, 109.82, 102.56, 89.47, 85.00, 56.90, 56.56, 52.71, 35.59, 34.65,
16.81; HRMS (FAB+) calcd for C19H19N2O4 (M+H)+: 339.1345, found: 339.1336.
19. Cell culture and cytotoxicity test. The human cancer cell lines were purchased
from American Type Culture Collection (Rockville, MD) and maintained in a
humidified atmosphere at 37 °C in 5% CO2. The cells was grown in RPMI-1640
(Gibco, Rockville, MD) media containing 10% heat-inactivated fetal bovine
serum (FBS), 1.25 mM HEPES and 1% penicillin/streptomycin. Cytotoxicity was
determined by the MTT (Promega, Madison, WI) assay according to the
manufacturer’s protocol. Briefly, cells were seeded in 96-well microtiter plates
at a density of 2 ꢀ 103 cells per well. After 48 h incubation, cells were treated
with various concentrations of rac-1 and rac-2, cultured for for 24 and 48 h,
8. Kwok, Y.; Zeng, Q.; Hurley, L. H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13531.
9. David-Cordonnier, M.-H.; Laine, W.; Lansiaux, A.; Kouach, M.; Briand, G.; Pierre,
A.; Hickman, J. A.; Bailly, C. Biochemistry 2002, 41, 9911.
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P.; Jeong, L. S.; Moon, H. R. Bull. Korean Chem. Soc. 2009, 30, 1463.
11. Kim, M.-Y.; Na, Y.; Vankayalapati, H.; Gleason-Guzman, M.; Hurley, L. H. J. Med.
Chem. 2003, 46, 2958.
12. 1H NMR (500 MHz, CDCl3) d 8.66–8.62 (m, 2H), 7.17 (dd, 1H, J = 4.5, 7.5 Hz),
6.35 (s, 1H), 5.24 (t, 1H, J = 9.0 Hz), 5.13 (s, 1H), 4.98 (s, 1H), 4.12 (s, 3H), 3.97 (s,
3H), 3.93 (dd, 1H, J = 9.5, 14.5 Hz), 3.55 (dd, 1H, J = 8.0, 14.5 Hz), 1.81 (s, 3H);
13C NMR (100 MHz, CDCl3) d 177.46, 166.11, 164.14, 152.10, 152.07, 143.38,
141.12, 136.57, 117.81, 113.78, 113.16, 109.64, 102.91, 89.49, 86.40, 56.53,
38.11, 35.62, 17.34.
respectively. At the end of the treatment period, 15
reagent was added to each well. After 4 h incubation at 37 °C, the supernatant
was aspirated, and formazan crystals were dissolved in 100 L DMSO at 37 °C
lL of the MTT (5 mg/mL)
13. 1H NMR (400 MHz, CDCl3) d 8.68 (dd, 1H, J = 2.0, 7.2 Hz), 7.77 (dd, 1H, J = 1.6,
6.4 Hz), 6.54 (t, 1H, J = 6.8 Hz), 6.24 (s, 1H), 5.28 (t, 1H, J = 8.8 Hz), 5.08 (s, 1H),
4.89 (s, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.43 (dd, 1H, J = 10.0, 15.2 Hz), 3.41 (dd,
1H, J = 8.0, 15.2 Hz), 1.77 (s, 3H); 13C NMR (100 MHz, CDCl3) d 176.57, 164.70,
163.77, 150.99, 149.69, 144.38, 143.24, 143.19, 141.49, 123.62, 112.04, 111.37,
108.45, 88.43, 87.74, 56.33, 40.52, 33.24, 17.43.
l
for 10 min with gentle agitation. The absorbance per well was measured at
540 nm with a VERS Amax Microplate Reader (Molecular Devices Corp.). Assay
was done in triplicate. The IC50 values were then determined for each drugs
from a plot of log (drug concentration) versus percentage of loss of viability.
20. Flow cytometry. Cancer cells were exposed to rac-1 and rac-2 for 48 h. Total
number of cells was collected, washed, and suspended in cold PBS. The cells
were fixed in chilled 75% methanol and stained with propidium iodine (PI)
14. Reisch, J.; Mester, I.; El-Moghazy Aly, S. M. J. Chem. Soc., Perkin Trans. 1 1983,
219.
15. Fellows, I. M.; Schwaebe, M.; Dexheimer, T. S.; Vankayalapati, H.; Gleason-
Guzman, M.; Whitten, J. P.; Hurley, L. H. Mol. Cancer Ther. 2005, 4, 1729.
16. Habib, A. M.; Ho, D. K.; Masuda, S.; McCloud, T.; Reddy, K. S.; Aboushoer, M.;
McKenzie, A.; Byrn, S. R.; Chang, C.-J.; Cassady, J. M. J. Org. Chem. 1987, 52, 412.
17. (2R⁄)-5-Methoxy-11-methyl-2-((2R⁄)-2-methyloxiran-2-yl)-1,2-
dihydrobenzofuro[4,5-b][1,8]naphthyridin-6(11H)-one: 1H NMR (400 MHz,
solution (100 lg/mL RNase and 10 lg/mL PI in PBS). Data acquisition and
analysis was performed using a flow cytometry system (Becton Dickinson, San
Jose, CA).