Study on the synthesis and cytotoxicity of new quinophenoxazine derivatives

Article abstract of DOI:10.1248/cpb.54.248

We have synthesized several new quinophenoxazine analogues and tested their cytotoxicity activities. The results showed that the compounds, 4a and 4b, possessing phenyl ring in the structure have almost same pharmacological capacity with A-62176. This finding suggests that the phenyl ring portion is important to this series of compounds for the activity expression.

Full text of DOI:10.1248/cpb.54.248

248
Notes
Chem. Pharm. Bull. 54(2) 248—251 (2006)
Vol. 54, No. 2
Study on the Synthesis and Cytotoxicity of New Quinophenoxazine
Derivatives
a
Youngjoo KWON and Younghwa NA*,b
a College of Pharmacy, Ewha Womans University; Seoul 120–750, Korea: and b College of Pharmacy, Catholic University
of Daegu; Gyeongsansi, Gyeongbuk, 712–702, Korea. Received August 15, 2005; accepted November 10, 2005
We have synthesized several new quinophenoxazine analogues and tested their cytotoxicity activities. The re-
sults showed that the compounds, 4a and 4b, possessing phenyl ring in the structure have almost same pharma-
cological capacity with A-62176. This finding suggests that the phenyl ring portion is important to this series of
compounds for the activity expression.
Key words fluoroquinolone; quinophenoxazine; cytotoxicity
The fluoroquinolone class analogues represented by Nor- logues. Basically the designed compounds contained diverse
floxacin (1)1) and Ciproxacin (2)2) have been clinically im- aromatic rings including pyridine, quinoline and an-
portant and widely used as antibacterial agents. These class thraquinone. Synthesis was conducted with some modifica-
drugs are known to show pharmacological activity by inhibit- tions of the published method.5,8) Chart 1 describes a repre-
ing DNA gyrase and topoisomerase IV.3,4) Because sentative synthetic pathway for the compound 4. All series of
of the excellent antibacterial efficacy, a number of fluoro- compounds were prepared with the same synthesis procedure
quinolone analogues have been prepared and tested for the with 4a. The key O-aminoaromatic alcohol compounds
development of new antimicrobial agents with improved (5a—f) were purchased or synthesized. Especially, 5b11) and
pharmacological efficacy.3,4) The similar mechanistic aspects 5f12) were prepared by the methods in the references. The in
between bacterial and eukaryotic DNA topoisomerase II at- situ coupling of compound 5a and product of ethyl 2,3,4,5-
tracted researchers to screen the possibilities of usage for an- tetrafluorobenzoyl acetate (6) and triethyl orthoformate in
titumor action of fluoroquinobenzoxazine analogues.5—7) In acetic anhydride provided 7a. Double ring cyclization under
the series of the compounds synthesized previously, A-62176 NaHCO3 basic condition in DMF generated 8a. Regioselec-
(3) prepared by Abbott researchers5) showed good pharmaco- tive nucleophilic substitution reaction at C-2 position was
logical activity against several human and murine cancer cell accomplished with 3(S)-(t-butoxycarbonylamino)pyrrolidine
lines. This compound possesses tetracyclic ring system in and 8a in pyridine solvent. Final hydrolysis of ethyl carboxy-
which another phenyl moiety is fused into the benzoxazine late and removal of protection part from amino group pro-
core of 1 and 2 (Fig. 1). Recently, University of Arizona re- duced 4a. With this procedure, six analogues including one
searchers have prepared a few A-62176 analogues, which known 4a6) and five new quinophenoxazines 4b—f were pre-
have extended aromatic ring system. The in vitro cytotoxicity pared.
test results showed some of these compounds exhibited better
In order to see the anticancer effect, we tested all the com-
antitumor activity than A-62176 (3).8—10) These information pounds against several human cancer cell lines. The data are
suggested that modification of the phenyl ring moiety of 3 summarized in Table 1. In these results we could observe the
can modulate the biological capacity of this compound. In structure–activity relationship. Apparently, no compounds
order to prepare new quinophenoxazine analogues of 3 pos- showed better biological efficacy compared to A-62176 (3)
sessing enhanced pharmacological profile, we have synthe- used as reference (values shown in the parenthesis in Table
sized six analogues (Fig. 2), one known and five new ones, 1). The compounds, 4a and b, possessing phenyl ring moiety
and tested their cytotoxicity activities in vitro against several like A-62176 exhibited almost same antitumor activity
human cancer cell lines.
against cell lines tested regardless of the substituents on the
Results and Discussion
According to the previous model study of the complex, 3
and DNA, the intercalation of tetracyclic ring of 3 into the
DNA base pairs is important. In addition, the chelation of
Mg2ꢀ and b-keto acid and the amino group of the side chain
on 3 provides more stabilization.8) Based on this information
we have designed and synthesized six quinophenoxazine ana-
Fig. 2. Structures of Target Compounds 4a—f
Fig. 1. Structures of Norfloxacin (1), Ciproxacin (2), and A-62176 (3)
∗ To whom correspondence should be addressed. e-mail: yna7315@cu.ac.kr
© 2006 Pharmaceutical Society of Japan

February 2006
249
Chart 1. Preparation of Target Compounds 4a—f
studies should be investigated for better understanding and
preparing new potential compounds as new drug candidates.
Table 1. Cytotoxicity (IC50, mM) of Compounds 4a—f against Cancer
Cells
Experimental
Compound/Cell line HT29-colon Du145-prostate MiaPaCa-pancreatic
General Method for Chemistry 1H-NMR spectra were taken on
Bruker AMX 500 at University of Arizona Cancer Center. Chemical shifts
(d) are in parts per million (ppm) relative to tetramethylsilane as an internal
standard and coupling constants (J values) are in hertz (Hz). Low-resolution
and high-resolution mass spectral data were recorded at mass spectroscopy
center in the University of Arizona. The solvents and reagents used for syn-
thesis were of the best commercial grade available and were used without
further purification unless noticed.
4a
4b
4c
4d
4e
4f
0.03 (0.03a)) 0.02 (0.02)
0.01 (0.02)
0.02 (0.02)
ꢂ100 (0.64)
0.05 (0.03)
40.0 (0.29)
2.20 (0.84)
NDb)
0.04 (0.02)
12.8 (0.84)
2.49 (0.58)
ND
2.31 (0.85)
ND
ND
ND
ND
a) The values in the parenthesis indicate one corresponding to A-62176. b) ND
represents not determined.
General Procedure for the Preparation of Compounds 7a—f A solu-
tion of ethyl 2,3,4,5-tetrafluorobenzoyl acetate (6) (1 eq) in triethyl orthofor-
mate (3 eq) and acetic anhydride (8.4 eq) was stirred at 130 °C (4 h). The
solvent was removed in vacuo. The oil residue was dissolved in CH2Cl2
and corresponding O-aminophenol compounds (5a—f) (1.0—1.2 eq) in
CH2Cl2/pyridine was added. After stirring at room temperature (17 h), sol-
vent was removed under reduced pressure. The residue was purified by silica
gel column chromatography to give products as isomeric mixture; the values
in the parenthesis indicate another set of isomer.
phenyl ring. But replacement of phenyl ring of A-62176 with
other components, anthraquinone, pyridine, and quinoline,
significantly reduced or abolished antitumor capacity. It is in-
teresting to find the reduction on activity of 4d. One possible
explanation of this phenomenon could be that the introduc-
tion of nitrogen atom in the phenyl ring might alter the p–p
stacking interaction between DNA and 4d.8) The result ob-
tained from 4e also suggested that the location of nitrogen
atom would be important factor for the activity expression.
When nitrogen is moved to one more carbon away from
quinobenzoxazine core, the biological capacity completely
disappeared. We also expected that introduction of intercala-
tion property of anthraquinone group could increase the bio-
Ethyl 3-(2-Hydroxy-5-methoxyphenylamino)-2-(2,3,4,5-tetrafluoroben-
1
zoyl)acrylate (7a): Orange solid (81%); Rf: 0.67 (40% EtOAc/hexane); H-
NMR (CDCl3, 500 MHz) d: 1.16 (1.05) (3H, t, Jꢁ6.4 Hz), 3.82 (3H, s), 4.15
(4.40) (2H, q, Jꢁ6.4 Hz), 6.64 (6.84) (1H, m), 6.91 (1H, m), 7.09 (7.06)
(1H, m), 8.62 (8.56) (1H, d, Jꢁ13.5 Hz), 12.68 (1H, d, Jꢁ16.3 Hz).
Ethyl
3-(2-Hydroxy-5-allyloxyphenylamino)-2-(2,3,4,5-tetrafluoroben-
1
zoyl)acrylate (7b): Yellow solid (84%); Rf: 0.53 (30% EtOAc/hexane); H-
NMR (CDCl3, 500 MHz) d: 1.17 (1.04) (3H, t, Jꢁ7.2 Hz), 4.16 (2H, q,
Jꢁ7.2 Hz), 4.53 (4.52) (2H, d, Jꢁ5.4 Hz), 5.32 (5.30) (1H, d, Jꢁ10.1 Hz),
5.43 (1H, d, Jꢁ17.3 Hz), 6.07 (1H, m), 6.66 (6.63) (1H, dd, Jꢁ8.6, 2.1 Hz),
logical efficacy. In contrast to our expectation, anthraquinone 6.79 (1H, d, Jꢁ8.6 Hz), 6.93 (1H, d, Jꢁ2.1 Hz), 7.10 (7.20) (1H, dd,
Jꢁ12.9, 8.0 Hz), 8.62 (8.56) (1H, d, Jꢁ13.9 Hz).
moiety did not give positive substitution effect.
Ethyl
3-(3-Hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-ylamino)-2-
With the preliminary results of the cytotoxicity test on
small numbers of quinophenoxazine analogues, it is likely
the phenyl ring moiety of A-62176 is important component
for the biological efficacy. More biological and structural
(2,3,4,5-tetrafluorobenzoyl)acrylate (7c): Orange solid (70%); Rf: 0.36
(10%/10% MeOH/CHCl3/hexane); 1H-NMR (CDCl3, 500 MHz) d: 1.24
(1.04) (3H, t, Jꢁ7.2 Hz), 4.24 (4.17) (2H, q, Jꢁ7.2 Hz), 7.14 (7.26) (1H, m),
7.85 (2H, m), 8.35 (2H, m), 8.36 (8.23) (1H, s), 8.31 (8.20) (1H, s), 8.77

250
Vol. 54, No. 2
(1H, d, Jꢁ13.0 Hz), 12.75 (11.67) (1H,d, Jꢁ12.9 Hz).
4.48 (2H, q, Jꢁ7.1 Hz), 7.63 (1H, d, Jꢁ13.6 Hz), 7.83 (1H, s), 7.85 (2H, m),
Ethyl
3-(3-Hydroxypyridin-2-yl-amino)-2-(2,3,4,5-tetrafluorobenzoyl)- 8.26 (1H, s), 8.34 (2H, m), 8.94 (1H, s).
acrylate (7d): Yellow solid (96%); 1H-NMR (CDCl3, 500 MHz) d: 1.24
(1.09) (3H, t, Jꢁ7.0 Hz), 4.20 (2H, q, Jꢁ7.0 Hz), 7.04 (6.99) (1H, dd,
Jꢁ7.5, 4.4 Hz), 7.17 (7.12) (1H, d, Jꢁ7.5 Hz), 7.99 (7.92) (1H, d,
Jꢁ4.4 Hz), 9.33 (9.16) (1H, d, Jꢁ13.3 Hz), 12.71 (11.48) (1H, d,
Jꢁ13.3 Hz).
Ethyl 1-(3(S)-(tert-Butoxycarbonylamino)pyrrolidin-1-yl)-2-fluoro-4-oxo-
4H-pyrido[3,2,1-kl]-7-azaphenoxazine-5-carboxylate (9d): Yellow solid
(97%); Rf: 0.73 (12.5% MeOH/EtOAc); 1H-NMR (CDCl3, 500 MHz) d:
1.45 (3H, t, Jꢁ7.1 Hz), 1.48 (9H, s), 1.94 (1H, m), 2.24 (1H, m), 3.51 (1H,
m), 3.63 (1H, m), 3.84 (1H, m), 3.92 (1H, m), 4.3 (1H, m), 4.41 (2H, q,
Ethyl 3-(8-Hydroxyquinolin-7-yl-amino)-2-(2,3,4,5-tetrafluorobenzoyl)- Jꢁ7.1 Hz), 7.17 (1H, dd, Jꢁ7.6, 4.1 Hz), 7.34 (1H, d, Jꢁ7.6 Hz), 7.62 (1H,
1
acrylate (7f): Red solid (64%); H-NMR (CDCl3, 500 MHz) d: 1.09 (1.19)
(3H, t, Jꢁ7.2 Hz), 4.19 (2H, q, Jꢁ7.2 Hz), 7.23 (7.14) (1H, dd, Jꢁ7.6,
d, Jꢁ13.6 Hz), 8.09 (1H, d, Jꢁ4.1 Hz), 9.58 (1H, s).
Ethyl 1-(3(S)-(tert-Butoxycarbonylamino)pyrrolidin-1-yl)-2-fluoro-4-oxo-
1.8 Hz), 7.46 (7.61) (1H, dd, Jꢁ8.0, 4.3 Hz), 7.48 (7.62) (1H, dd, Jꢁ8.8, 4H-pyrido[3,2,1-kl]-8-azaphenoxazine-5-carboxylate (9e): Yellow solid
1.8 Hz), 8.20 (1H, dd, Jꢁ7.1, 1.2 Hz), 8.77 (8.86) (1H, d, Jꢁ14.2 Hz), 8.84
(1H, dd, Jꢁ11.2, 1.2 Hz), 11.68 (12.98) (1H, d, Jꢁ14.0 Hz).
(80%); Rf: 0.35 (6% MeOH/CHCl3); 1H-NMR (CDCl3, 500 MHz) d: 1.39
(3H, t, Jꢁ7.1 Hz), 1.45 (9H, s), 1.95 (1H, m), 2.18 (1H, m), 3.46 (1H, m),
General Procedure for the Preparation of Compounds 8a—f Com- 3.63 (1H, m), 3.81 (1H, m), 3.90 (1H, m), 4.29 (1H, m), 4.36 (2H, q,
pounds 7a—f (1 eq) and NaHCO3 (5 eq) were placed in DMF under nitro- Jꢁ7.1 Hz), 6.93 (1H, d, Jꢁ5.4 Hz), 7.49 (1H, d, Jꢁ13.7 Hz), 8.36 (1H, d,
gen and the mixture was refluxed at 110 °C (2 h). Solvent was removed
under reduced pressure and the residue was dissolved with CHCl3 and water.
Jꢁ5.4 Hz), 8.63 (1H, s), 8.73 (1H, s).
Ethyl 1-(3(S)-(tert-butoxycarbonylamino)pyrrolidin-1-yl)-2-fluoro-4-oxo-
Undissolved material was filtered and the organic layer was separated. After 4H-pyrido[3,2,1-kl]pyridino[3,2-c]phenoxazine-5-carboxylate (9f): Yellow
1
removing the solvent, the residue was purified by silica gel column chro-
matography to give products: Compound 8b was directly used for next reac- 1.46 (3H, t, Jꢁ7.1 Hz), 1.50 (9H, s), 2.17 (1H, m), 2.38 (1H, m), 3.64 (1H,
tion without spectroscopic identification. m), 3.77 (2H, m), 4.07 (1H, m), 4.32 (1H, m), 4.46 (2H, q, Jꢁ7.1 Hz), 7.51
solid (99%); Rf; 0.21 (2% MeOH/CHCl3); H-NMR (CDCl3, 500 MHz) d:
Ethyl 1,2-Difluoro-8-methoxy-4-oxo-4H-pyrido[3,2,1-kl]phenoxazine-5- (1H, dd, Jꢁ7.7, 4.1 Hz), 7.64 (1H, d, Jꢁ12.8 Hz), 7.67 (2H, s), 8.17 (1H, d,
1
carboxylate (8a): Orange solid (90%); H-NMR (CDCl3, 500 MHz) d: 1.44 Jꢁ7.7 Hz), 8.95 (1H, s), 9.10 (1H, br s).
(3H, t, Jꢁ6.4 Hz), 3.88 (3H, s), 4.46 (2H, q, Jꢁ7.1 Hz), 6.81 (1H, dd,
Jꢁ8.9, 2.4 Hz), 7.01 (1H, d, Jꢁ2.4 Hz), 7.16 (1H, d, Jꢁ8.9 Hz), 7.79 (1H,
dd, Jꢁ10.0, 7.8 Hz), 8.92 (1H, s).
General Procedure for the Preparation of Compounds 4a—f Com-
pounds 9a—f (1 eq) was mixed with aqueous 1 N KOH in ethanol (1 : 2) and
refluxed at 80 °C (30 min). And then aqueous 2 N HCl (0.5 ml) in ethanol
Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-(1,4-dioxonaphtho)[2,3-c]- (3 : 2) was added into the reaction mixture. After 3 h more refluxing, the re-
phenoxazine-5-carboxylate (8c): Orange solid (recrystallized from CHCl3) action mixture was cooled and solid precipitated was filtered. Ethanol was
(73%); Rf: 0.70 (5% MeOH/CHCl3); 1H-NMR (CDCl3, 500 MHz) d: 1.48 added to the solid and the mixture was heated. The solid collected after fil-
(3H, t, Jꢁ7.1 Hz), 4.50 (2H, q, Jꢁ7.1 Hz), 7.84 (1H, dd, Jꢁ9.9, 8.8 Hz),
7.88 (1H, d, Jꢁ13.4 Hz), 7.87 (1H, m), 8.05 (1H, s), 8.34 (2H, m), 8.38 (1H,
s), 9.11 (1H, s).
tration was dried in vacuo to give products.
1-(3(S)-Pyrrolidin-1-yl)-2-fluoro-8-methoxy-4-oxo-4H-pyrido[3,2,1-kl]-
phenoxazine-5-carboxylic Acid HCl (4a): Orange solid (97%). 1H-NMR
Ethyl
1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-7-azaphenoxazine-5-car- (CD3ODꢀDMSO-d6, 500 MHz) d: 2.10 (1H, m), 2.37 (1H, m), 3.78 (2H,
boxylate (8d): Light yellow solid (78%); Rf; 0.34 (50% EtOAc/hexane); 1H-
NMR (CDCl3, 500 MHz) d: 1.45 (3H, t, Jꢁ7.0 Hz), 4.43 (2H, q, Jꢁ7.0 Hz),
7.27 (1H, dd, Jꢁ8.0, 4.3 Hz), 7.49 (1H, d, Jꢁ8.0 Hz), 7.81 (1H, dd, Jꢁ9.7,
8.0 Hz), 8.19 (1H, d, Jꢁ4.3 Hz), 9.71 (1H, s).
m), 3.89 (3H, s), 3.94 (2H, m), 3.98 (1H, m), 6.97 (1H, dd, Jꢁ8.8, 2.1 Hz),
7.23 (1H, d, Jꢁ8.8 Hz), 7.36 (1H, d, Jꢁ2.1 Hz), 7.53 (1H, d, Jꢁ13.5 Hz),
9.12 (1H, s). FAB-MS m/z: 412.1305 (Calcd for C21H19N3O5F: 412.1309).
MS m/z: 412 [MꢀH]ꢀ.
Ethyl
1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-azaphenoxazine-5-car-
1-(3(S)-Pyrrolidin-1-yl)-2-fluoro-8-allyloxy-4-oxo-4H-pyrido[3,2,1-kl]-
1
boxylate (8e): Light yellow solid (18%); Rf: 0.58 (6% MeOH/CHCl3); H- phenoxazine-5-carboxylic Acid HCl (4b): Yellow solid (68%). 1H-NMR
NMR (CDCl3+CD3OD, 500 MHz) d: 1.27 (3H, t, Jꢁ7.1 Hz), 4.24 (2H, q, (DMSO-d6, 500 MHz) d: 2.00 (1H, m), 2.26 (1H, m), 3.74 (2H, m), 3.87
Jꢁ7.1 Hz), 7.05 (1H, d, Jꢁ5.4 Hz), 7.62 (1H, dd, Jꢁ9.9, 9.9 Hz), 8.32 (1H,
d, Jꢁ5.4 Hz), 8.74 (1H, s), 8.94 (1H, s).
(2H, m), 3.93 (1H, m), 4.69 (2H, d, Jꢁ5.0 Hz), 5.29 (1H, dd, Jꢁ10.5,
1.0 Hz), 5.44 (1H, dd, Jꢁ17.2, 1.0 Hz), 6.05 (1H, m), 7.00 (1H, dd, Jꢁ9.0,
Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]pyridino[3,2-c]phenoxazine- 2.3 Hz), 7.27 (1H, d, Jꢁ9.0 Hz), 7.51 (1H, d, Jꢁ13.5 Hz), 7.56 (1H, d,
5-carboxylate (8f): Gray solid (95%); Rf: 0.35 (1% MeOH/CHCl3); 1H-
NMR (CDCl3, 500 MHz) d: 1.47 (3H, t, Jꢁ7.1 Hz), 4.48 (2H, q, Jꢁ7.1 Hz),
7.54 (1H, dd, Jꢁ8.2, 4.2 Hz), 7.72 (1H, d, Jꢁ9.2 Hz), 7.75 (1H, d,
Jꢁ9.2 Hz), 7.82 (1H, dd, Jꢁ10.0, 7.6 Hz), 8.19 (1H, dd, Jꢁ8.2, 1.2 Hz),
9.07 (1H, s), 9.10 (1H, dd, Jꢁ4.2, 1.2 Hz).
Jꢁ2.3 Hz), 9.18 (1H, s). FAB-MS m/z: 438.1469 (Calcd for C23H21N3O5F:
438.1465). MS m/z: 438 [MꢀH]ꢀ.
1-(3(S)-Pyrrolidin-1-yl)-2-fluoro-4-oxo-4H-pyrido[3,2,1-kl]-1,4-dioxon-
aphtho[2,3-c]phenoxazine-5-carboxylic Acid HCl (4c): Brown solid (87%).
1H-NMR (CD3ODꢀDMSO-d6, 500 MHz) d: 2.25 (1H, m), 2.50 (1H, m),
General Procedure for the Preparation of Compounds 9a—f Com- 3.91 (1H, m), 3.97 (1H, m), 4.09 (2H, m), 4.15 (1H, m), 7.51 (1H, d,
pounds 8a—f (1 eq) and 3(S)-(tert-butoxycarbonylamino)pyrrolidine (3 eq) Jꢁ13.5 Hz), 7.91 (1H, s), 7.93 (2H, m), 8.23 (1H, d, Jꢁ7.2 Hz), 8.26 (1H, d,
were dissolved in anhydrous pyridine and the mixture was stirred under ni- Jꢁ7.2 Hz), 8.45 (1H, s), 8.94 (1H, s). MS m/z: 512 [MꢀH]ꢀ.
trogen at 110 °C (24—40 h). The solvent was removed under reduced pres-
sure and the residue was purified by silica gel column chromatography to
give products.
1-(3(S)-Pyrrolidin-1-yl)-2-fluoro-4-oxo-4H-pyrido[3,2,1-kl]-7-azaphenox-
azine-5-carboxylic Acid 2HCl (4d): Yellow solid (69%). H-NMR (DMSO-
d6, 500 MHz) d: 2.03 (1H, m), 2.35 (1H, m), 3.74 (3H, m), 3.88 (1H, m),
1
Ethyl
1-(3(S)-(tert-Butoxycarbonylamino)pyrrolidin-1-yl)-2-fluoro-8- 3.95 (1H, m), 7.49 (1H, dd, Jꢁ7.7, 4.2 Hz), 7.52 (1H, d, Jꢁ13.6 Hz), 7.76
methoxy-4-oxo-4H-pyrido[3,2,1-kl]phenoxazine-5-carboxylate (9a): Orange (1H, d, Jꢁ7.7 Hz), 8.24 (1H, d, Jꢁ4.2 Hz), 9.54 (1H, s). FAB-MS m/z:
1
solid (88%); Rf: 0.43 (20% MeOH/CHCl3); H-NMR (CDCl3, 500 MHz) d: 383.1151 (Calcd for C19H16N4O4F: 383.1156). MS m/z: 383 [MꢀH]ꢀ.
1.43 (3H, t, Jꢁ6.9 Hz), 1.48 (9H, s), 1.96 (1H, m), 2.23 (1H, m), 3.53 (1H,
1-(3(S)-Pyrrolidin-1-yl)-2-fluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-azaphenox-
m), 3.61 (1H, m), 3.84 (3H, s), 3.88 (2H, m), 4.35 (1H, m), 4.41 (2H, q, azine-5-carboxylic Acid 2HCl (4e): Yellow solid (70%). 1H-NMR
Jꢁ6.9 Hz), 6.70 (1H, dd, Jꢁ8.8, 1.7 Hz), 6.85 (1H, d, Jꢁ1.7 Hz), 6.98 (1H,
d, Jꢁ8.8 Hz), 7.56 (1H, d, Jꢁ13.5 Hz), 8.70 (1H, s).
(CD3ODꢀDMSO-d6, 500 MHz) d: 2.03 (1H, m), 2.49 (1H, m), 3.14 (1H,
m), 3.21 (1H, m), 3.38 (1H, m), 3.63 (1H, m), 3.98 (1H, m), 6.63 (1H, dd,
Ethyl 1-(3(S)-(tert-Butoxycarbonylamino)pyrrolidin-1-yl)-2-fluoro-8-al- Jꢁ6.8, 1.8 Hz), 7.70 (1H, d, Jꢁ12.1 Hz), 7.96 (1H, d, Jꢁ6.8 Hz), 8.32 (1H,
lyloxy-4-oxo-4H-pyrido[3,2,1-kl]phenoxazine-5-carboxylate (9b): Yellow s), 8.56 (1H, d, Jꢁ5.4 Hz). FAB-MS m/z: 383.1143 (Calcd for C19H16N4O4F:
solid (77%); 1H-NMR (CDCl3, 500 MHz) d: 1.43 (3H, t, Jꢁ7.1 Hz), 1.46
383.1156). MS m/z: 383 [MꢀH]ꢀ.
1-(3(S)-Pyrrolidin-1-yl)-2-fluoro-4-oxo-4H-pyrido[3,2,1-kl]pyridino[3,2-
(9H, s), 1.95 (1H, m), 2.23 (1H, m), 3.52 (1H, m), 3.61 (1H, m), 3.83 (1H,
1
m), 3.88 (1H, m), 4.34 (1H, m), 4.42 (2H, q, Jꢁ7.1 Hz), 4.55 (2H, d, c]phenoxazine-5-carboxylic Acid 2HCl (4f): Yellow solid (73%). H-NMR
Jꢁ5.2 Hz), 5.34 (1H, d, Jꢁ10.6 Hz), 5.45 (1H, d, Jꢁ17.2 Hz), 6.04 (1H, m),
6.70 (1H, dd, Jꢁ9.0, 2.4 Hz), 6.90 (1H, d, Jꢁ2.4 Hz), 6.96 (1H, d, m), 4.01 (1H, m), 4.15 (1H, m), 4.33 (1H, m), 7.56 (1H, d, Jꢁ12.7 Hz), 7.70
Jꢁ9.0 Hz), 7.55 (1H, d, Jꢁ13.5 Hz), 8.69 (1H, s). (1H, dd, Jꢁ8.3, 4.2 Hz), 7.85 (1H, d, Jꢁ8.3 Hz), 8.43 (1H, d, Jꢁ8.3 Hz),
(CD3OD, 500 MHz) d: 2.25 (1H, m), 2.54 (1H, m), 3.82 (1H, m), 3.87 (1H,
Ethyl 1-(3(S)-(tert-Butoxycarbonylamino)pyrrolidin-1-yl)-2-fluoro-4-oxo- 9.04 (1H, d, Jꢁ4.2 Hz), 9.22 (1H, s). FAB-MS m/z: 433.1307 (Calcd for
4H-pyrido[3,2,1-kl]-(1,4-dioxonaphtho)[2,3-c]phenoxazine-5-carboxylate C23H18N4O4F: 433.1312). MS m/z: 433 [MꢀH]ꢀ.
(9c): Red solid (80%); Rf: 0.37 (2% MeOH/CHCl3); 1H-NMR (CDCl3,
500 MHz) d: 1.47 (3H, t, Jꢁ7.1 Hz), 1.50 (9H, s), 1.98 (1H, m), 2.27 (1H,
m), 3.56 (1H, m), 3.74 (1H, m), 3.92 (1H, m), 4.01 (1H, m), 4.37 (1H, m),
Cytotoxicity Test13) Cancer cells were purchased from the American
Tissue Culture Collection (Rockville, MD, U.S.A.) and cultured by the sup-
plier’s instructions. Exponentially growing cells ((1ꢃ2)ꢄ103 cells) in 0.1 ml

February 2006
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of medium were seeded on day 0 in a 96-well microtiter plate. On day 1,
0.1 ml aliquots of medium containing graded concentrations of compound
were added to the cell plates. On day 4, the cell cultures were incubated with
50 ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(1 mg/ml in Dulbecco’s phosphate buffered saline) for 4 h at 37 °C. The re-
sulting formazan precipitate was dissolved with 200 ml of 0.04 M HCl in iso-
propyl alcohol. For determination of the IC50 values, the absorbance read-
ings at 570 nm were fitted to the four-parameters logistic equation.
6) Chu. D. T. W., Hallas R., Clement J. J., Alder J., McDonald E., Drugs
Exp. Clin. Res., 18, 275—282 (1992).
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9) Duan W., Rangan A., Vankayalapati H., Kim M.-Y., Zeng Q., Sun D.,
Han H., Federoff O. Y., Nishioka D., Rha S. Y., Izbicka E., Von Hoff
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Acknowledgments This work was financially supported by Catholic
University of Daegu, Korea. The authors express sincere gratitude to Profes-
sor Laurence Hurley at University of Arizona, U.S.A., for his generous help
for this research. The authors give thanks to Mary Gleason-Guzman for con-
ducting cytotoxicity test.
11) Synthesis of 4-allyloxy-2-aminophenol (5b)
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