Synthesis and anti-tumor activities of novel oxazinyl isoflavonoids

Article abstract of DOI:10.1248/cpb.60.513

The design, synthesis and biological evaluation of a novel series of oxazinyl isoflavonoids is described. Several analogs were shown to exhibit growth inhibitory effects against SKOV-3, DU-145 and HL-60 human colon cancer cell lines with IC₅₀ values in the micromolar range. The cellular potency of compounds 7e and 12h were found to have greater in vitro inhibitory activities than phenoxodiol, the parental compound currently in late-stage clinical trials for the treatment of cancer. The results shown are suitable for further lead optimization.

Full text of DOI:10.1248/cpb.60.513

April 2012
Regular Article
Chem. Pharm. Bull. 60(4) 513–520 (2012)
513
Synthesis and Anti-tumor Activities of Novel Oxazinyl Isoflavonoids
,a
Dun Wang,* Like Hou,a Lirong Wu,a and Xin Yub
a College of Pharmacential Engineering, Shenyang Pharmaceutical University; Shenyang 110016, China: and
b Medical & Regulatory Affairs Department, Fresenisu Kabi (China) Co., Ltd., BD; Beijing 100007, China.
Received December 7, 2011; accepted January 16, 2012; published online February 1, 2012
The design, synthesis and biological evaluation of a novel series of oxazinyl isoflavonoids is described.
Several analogs were shown to exhibit growth inhibitory effects against SKOV-3, DU-145 and HL-60 human
colon cancer cell lines with IC50 values in the micromolar range. The cellular potency of compounds 7e and
12h were found to have greater in vitro inhibitory activities than phenoxodiol, the parental compound cur-
rently in late-stage clinical trials for the treatment of cancer. The results shown are suitable for further lead
optimization.
Key words anti-tumor activity; oxazinyl isoflavonoid compound; synthesis
With the changes in the current living environment, cancer diol was granted Fast Track status by the Food and Drug
has become the second leading cause of death in developed Administration (FDA) to be developed as a chemo-sensitizer
countries. Over 1 million cases of cancer occur in the United for platinums and taxanes in the treatments of recurrent ovar-
States annually, and cancer-related deaths are estimated to ian and hormone-refractory prostate cancers.5) Like genistein,
reach 12 million worldwide by the year 2015.1) Therefore, a phenoxodiol features a diphenolic ring structure, which may
new generation of useful anticancer chemotherapy agents is be essential for maintaining the free radical scavenging effect
becoming more urgent.
of the drug, while also being largely responsible for its ease of
Isoflavonoids, which are present in vegetables, beans, peas, oxidation when standing for a long period.
and other legumes, are one of the principal plant hormones
In this paper, our goal was to generate highly potent, novel
known to possess the potential to regulate plant cycle kinetics isoflavonoid derivatives that may have improved stabilities.
and death and may have similar effects in animals. A number We chose isoflav-3-ene (the structural skeleton of phenoxodi-
of naturally occurring plant hormones, including isoflavonoids ol), isoflavanone and isoflavane as the mother nuclei and made
and particularly daidzein and genistein, have been shown to the following modifications: (1) the integration of oxazinyl
exhibit biological activity in mammalian cells by regulating with the A rings of the mother nuclei to protect the phenolic
various functions, such as cell-cycle progression, mitotic ar- groups on the 7-positions; (2) the integration of oxazinyl with
rest and apotosis.2,3) Specifically, genistein was well tolerated, the C rings of the mother nuclei to protect the phenolic groups
with minimal toxicity, in normal tissues, and its effects were on the 4′-positions; (3) the introduction of various substituents
seen in multiple tumor types, including prostate, breast and on the N-positions of the oxazinyl rings in order to understand
pancreatic tumors. However, its limited bioavailability and the effects of the different groups on the activity; and (4) the
extensive metabolism led to difficulties in attaining adequate methylation of the phenolic groups on the 7- or 4′-positions
plasma concentrations, resulting in limited utility and dis- of the mother nuclei to understand their effects on activity.
semination in the clinic.4) For phenoxodiol (2H-1-benzopyran- Overall, thirty oxazinyl isoflavonoid compounds were synthe-
7-ol, 3-[4-hydrophenyl], Fig. 1), an analogue of genistein, sized, and their in vitro anti-tumor activities were evaluated.
a broad range of activities against human cancer cells was
found, inducing mitotic arrest (G1 phase of mitosis), terminal
Synthesis
differentiation and apoptosis. Due to these findings, phenoxo-
In this paper, four types of oxazinyl isoflavonoid com-
pounds were designed and synthesized (Fig. 2). The target
compounds were obtained as described in Charts 1–5, and the
substituents of compounds 7a–18d are listed in Table 1.
Following an established method,6) intermediates 4a–c were
synthesized by reacting resorcinol with substituted phenylace-
°
tic acid in boron trifluoride etherate (BF3/(CH3)2O) at 80 C
for 4h and then treating with N,N-dimethylformamide (DMF)
and methanesulfonyl chloride. Subsequently, treatment of
Fig. 1. The Structure of Phenoxodiol
Fig. 2. Four Type Target Compounds
*To whom correspondence should be addressed. e-mail: wangduncn@hotmail.com.
© 2012 The Pharmaceutical Society of Japan

514
Vol. 60, No. 4
°
°
°
°
Reaction conditions: (a) BF3·Et2O/80 C; (b) CH3SO2CI/DMF, 50–70 C; (c) R2NH2, 37% HCHO, 1,4-dioxane, 45–70 C; (d) 10% Pd/C, HCOONH4, CH3COOH, 110 C;
(e) (HCHO)n, KOH, CH3OH
Chart 1. The Synthesis of Oxazinyl Isoflavonoid Compounds 7a–g
Table 1. In Vitro Growth Inhibitory Effects of the Target Compounds 7a–18d in Three Human Cancer Cell Lines
In vitro IC50 (μM)
Compd.
R1
R2
HL60
0.123
SKOV-3
DU-145
7a
7b
OH
OH
CH2CH3
CH2CH2CH3
(CH2)5CH3
CH2CH2CH3
CH2CH3
19.505
17.131
13.206
23.155
27.04
22.693
41.585
7.706
11.86
2.185
4.645
4.809
0.763
2.195
7.11
7c
OH
7d
CH3
CH3
OCH3
OCH3
OH
20.096
16.953
9.113
7e
7f
C(CH3)3
8.132
7g
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
(CH2)5CH3
CH2CH3
16.784
23.173
21.613
31.984
60.008
67.25
21.591
30.108
23.500
14.379
39.639
39.668
27.683
14.018
9.101
12a
12b
12c
OCH3
OCH3
CH3
CH3
OCH3
OH
4.55
5.89
12d
12e
2.614
1.006
5.50
CH2CH2CH3
CH2CH3
12f
22.270
28.554
7.483
12g
12h
12i
CH2CH3
5.15
OCH3
OH
CH(CH3)2
CH(CH3)2
(CH2)5CH3
CH3
3.04
1.78
11.772
16.577
24.398
>100
8.030
12j
OH
3.16
18.395
20.932
12k
12l
OCH3
OH
1.859
8.14
p-C6H4CH3
p-C6H4CH3
p-C6H4CH3
CH2CH2CH3
CH2CH2CH3
(CH2)5CH3
CH2CH3
>100
65.856
>100
>100
>100
58.817
82.857
>100
12m
12n
13a
13b
13c
OCH3
CH3
OH
17.404
5.616
55.788
61.102
>100
93.827
>100
OCH3
OCH3
CH3
CH3
—
>100
23.81
7.432
13d
13e
52.92
70.893
>100
CH2CH2CH3
CH2CH3
>100
18a
18b
18c
3.52
1.796
1.569
3.121
12.08
35.102
34.416
35.896
39.338
25.467
32.15
27.649
22.037
23.371
11.976
28.42
—
CH2CH2CH3
CH3
—
—
18d
Cisplatin
Phenoxodiol
CH(CH3)2
30.558
21.03

April 2012
515
°
°
Reaction conditions: (a) 10% Pd/C, HCOONH4, CH3OH, 70 (b) R2NH2, 37% HCHO, 1,4-dioxane, 45–70 C; (c) NaBH4, CH3OH; (d) EtOH, HCl·EtOH; (e) (HCHO)n,
KOH, CH3OH
Chart 2. The Synthesis of Oxazinyl Isoflavonoid Compounds 12a–k
°
°
Reaction conditions: (a) (CH3)2NH, 37% HCHO, 1,4-dioxane, 45–70 C; (b) CH3COCH3, CH3Br, r.t.; (c) ArNH2, CH3COOH, EtOH, 80 ; (d) NaBH4, CH3OH; (e) EtOH,
HCl·EtOH,; (f) (HCHO)n, KOH, CH3OH
Chart 3. The Synthetic Route of Target Compounds 12l–n
compounds 4a–c with a primary amine and formaldehyde in transfer hydrogenations in methanol to obtain isoflavanones
1,4-dioxane via a Mannich reaction7) afforded compounds 5a– 8a–c,8) which were treated with a primary amine and form-
g, which were converted into their corresponding isoflavans aldehyde to obtain the Mannich products 9a–k. Next, reduc-
6a–g via transfer hydrogenations with ammonium formate, as tion of 9a–k with NaBH4 in methanol conveniently afforded
the hydrogen donator, in acetic acid. By reacting compounds isolavan-4-ols 10a–k, which were dehydrolyzed by HCl/
6a–g with paraformaldehyde in the presence of potassium hy- EtOH in ethanol at room temperature to yield compounds
droxide, compounds of type I, the chromano[8,7-e][1,3]oxazine 11a–k. Finally, treating 11a–k with paraformaldehyde and
derivatives 7a–g, were obtained, respectively (Chart 1).
potassium hydroxide gave compounds of type II, the desired
The reductions of intermediates 4a–c were performed via chromeno[8,7-e][1,3]oxazine derivatives 12a–k (Chart 2).

516
Vol. 60, No. 4
Results and Discussion
Thirty compounds were synthesized in the current study,
and we utilized the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) colorimetric assay to determine
their IC50 in vitro growth inhibitory values in a human ovarian
cancer cell line (SKOV-3) and human prostate cancer cell line
(DU-145). Additionally, the Trypan blue dye exclusion assay
was employed to evaluate the growth inhibitory effects of the
tested compounds against a human leukemic cell line (HL-
60). Cisplatin and parental compound phenoxodiol were both
used as positive controls. The ability of these new analogs to
inhibit the growth of cancer cell lines is summarized in Table
1.
Reaction conditions: (a) (HCHO)n, KOH, CH3OH
Chart 4. The Synthesis of Oxazinyl Isoflavonoid Compounds 13a–e
In general, with modifications to the A ring, both isoflavane
Unfortunately, the chromeno[8,7-e][1,3]oxazine derivatives derivatives 7a–g and isoflavene derivatives 12a–k showed
12l–n, bearing the aryl group at the N-9 position, could not increased activities compared with their parent compound,
be prepared according to the procedure described above. phenoxodiol. Compounds 7f, 12h and 12i showed the best
’
The main reason is that 11l–n can t be prepared directly by activities with IC50 values in the micromolar range (1–10μM),
Mannich Reaction. As shown in Chart 3, isolavanones 8a–c which may suggest that the increase in the steric hindrance
were instead treated with dimethylamine and formaldehyde of the aliphatic alkyl substituent on the N-position of the
to generate Mannich bases 9l–n, which were subsequently oxazinyl ring is favorable for activity. This finding was further
quaternized in acetone with CH3Br at room temperature to confirmed by 7c, which bore an n-hexyl group on the oxazinyl
achieve quaternary ammonium salts 10l–n. Next, compounds ring. The fact that compounds 12d and 12e diminished the
11l–n were prepared by reacting 10l–n with an arylamine in activity revealed that the presence of a hydroxy or methoxy
ethanol and acetic acid as the catalyst. Reducing the carbonyl group on the para-position of the C ring was essential for
groups at the C-4 positions of 11l–n, followed by dehydration optimum biological activity. This finding is consistent with the
’
and cyclization, provided target compounds 12l–n (Chart 3).
Compounds of type III, the chromeno[8,7-e][1,3]oxazin-
author s previous hypothesis.
The N-phenyl substituted isoflavene derivatives 12l–n
4-ones 13a–e, were prepared by directly reacting 9a–e with showed remarkably decreased activities, while compound 12n
paraformaldehyde and potassium hydroxide in methanol while almost lost activity completely (>100μM), which may imply
in an ice bath (Chart 4).
that an aryl substitution on the oxazinyl ring is not well toler-
Compound 8a treated with dimethyl sulfate in the pres- ated.
ence of sodium bicarbonate provided selectively methylated
Isoflavanone derivatives 13a–e were generally inactive,
compound 14. Treatment of 14 with KBH4 instead of NaBH4 while 13b and 13e had the lowest activities (IC50>100μM).
in methanol provided 7-methoxy-4′-hydroxyisoflavan-4-ol These results demonstrated that the structural skeleton of iso-
(15), which was dehydrolyzed to compound 16. The reaction flavanone had negative effects.
time had been shortened and the yields had been improved by
Compounds 18a–d with modifications on the C ring exhib-
using KBH4 instead of NaBH4 on the basis of the following ited comparable activities to phenoxodiol but were less potent
experimental result. Compound 16 underwent a Mannich reac- than 12a–k. These two types of compounds were of the same
tion, followed by condensation with paraformaldehyde to give structural skeleton, but the positions of their oxazinyl ring
compounds of type IV, 6-(chromen-3-yl)-benzo[e][1,3]oxazine were different, suggesting that integrating the oxazinyl func-
18a–d (Chart 5).
tionality with the A ring of isoflavene is better favored. Of
compounds 18a–d, 18d, whose oxazinyl ring was substituted
°
Reaction conditions: (a) (CH3)2SO4, NaHCO3, (CH3)2CO; (b) KBH4, CH3OH; (c) EtOH, HCl·EtOH; (d) R2NH2, 37% HCHO, acetic acid, 1,4-dioxane, 45–70 C; (e)
(HCHO)n, KOH, CH3OH
Chart 5. The synthesis of Oxazinyl Isoflavonoid Compounds 18a–d

April 2012
517
by an isopropyl group, displayed better potency than the other OCH2CH), 4.83 (2H, s, OCH2N), 6.38 (1H, d, J=8.4Hz, H6),
three compounds. This result was consistent with those we 6.84 (1H, d, J=8.4Hz, H5), 6.80 (2H, d, J=8.4Hz, H3′, H5′),
observed in compounds 12a–k.
7.09 (2H, d, J=8.4Hz, H2′, H6′).
7b: Yield 62%; MS [M+H]+ (m/z): 326.2; 1H-NMR
(300MHz, CDCl3) δ: 0.93 (3H, t, J=7.5Hz, CH3), 1.61 (2H,
Conclusions
In this study, a series of novel isoflavoids containing m, CH2CH3), 2.72 (2H, t, J=7.2Hz, NCH2CH2), 2.94 (2H,
protected phenolic groups with oxazinyl moieties was syn- m, PhCH2CH), 3.13 (1H, m, CH2CHCH2O), 3.93 (2H, s,
thesized, and each compound was evaluated for its in vitro PhCH2N), 3.93–4.30 (2H, m, OCH2CH), 4.82 (2H, s, OCH2N),
anti-tumor activities. These compounds showed increased 6.38 (1H, d, J=8.4Hz, H6), 6.84 (1H, d, J=8.4Hz, H5), 6.82
stabilities compared to the parental compound, phenoxo- (2H, d, J=8.4Hz, H3′, H5′), 7.10 (2H, d, J=8.4Hz, H2′, H6′).
diol. Additionally, the oxazinyl isoflavane and oxazinyl
7c: Yield 58%; MS[M+H]+ (m/z): 368.1; 1H-NMR
isoflavene derivatives exhibited potent growth inhibitory (300MHz, CDCl3) δ: 0.88 (3H, t, J=6.6Hz, CH3), 1.30–1.56
activities against the SKOV-3, DU-145 and HL-60 cell lines. (8H, m, CH3(CH2)4), 2.75 (2H, t, J=7.5Hz, NCH2CH2), 2.91
Specifically, compound 7f showed the most potent inhibition (2H, m, PhCH2CH), 3.12 (1H, m, CH2CHCH2O), 3.91 (2H, s,
activities with IC50 values of 0.763, 8.132 and 9.113μmol, re- PhCH2N), 3.91–4.31 (2H, m, OCH2CH), 4.81 (2H, s, OCH2N),
spectively. This compound was followed by compound 12h, 6.37 (1H, d, J=8.4Hz, H6), 6.85 (1H, d, J=8.4Hz, H5), 6.81
whose IC50 values were 3.04, 7.483 and 9.101μmol, respec- (2H, d, J=7.8Hz, H3′, H5′), 7.10 (2H, d, J=7.8Hz, H2′, H6′).
tively. We observed that both of the above compounds had
7d: Yield 72%; MS [M+H]+ (m/z): 324.3; 1H-NMR
aliphatic alkyl groups with steric hindrances at the N-atoms of (300MHz, CDCl3) δ: 0.93 (3H, t, J=7.2Hz, CH3), 1.59
their oxazinyl rings. Overall, the information from this study (2H, m, CH2CH3), 2.34 (3H, s, PhCH3), 2.70 (2H, t,
may be helpful for the design and synthesis of isoflavoid de- J=7.5Hz, NCH2CH2), 2.91 (2H, m, PhCH2CH), 3.14 (1H,
rivatives with stronger activities and better stabilities.
m, CH2CHCH2O), 3.92 (2H, s, PhCH2N), 3.97–4.34 (2H, m,
OCH2CH), 4.81 (2H, s, OCH2N), 6.37 (1H, d, J=8.4Hz, H6),
6.84 (1H, d, J=8.1Hz, H5), 7.12 (2H, d, J=8.4Hz, H3′, H5′),
Experimental
All the reagents and solvents were purchased from common 7.17 (2H, d, J=8.4Hz, H2′, H6′).
commercial suppliers and used without further purification.
7e: Yield 76%; MS [M+H]+ (m/z): 310.1; 1H-NMR
Nuclear magnetic resonance spectra were recorded in CDCl3 (300MHz, CDCl3) δ: 1.19 (3H, t, J=7.2Hz, CH3), 2.35 (3H,
or d-DMSO solutions on a Bruker AX 300 spectrometer. s, PhCH3), 2.80 (2H, q, J=7.2Hz, NCH2CH3), 2.94 (2H,
Chemical shifts were reported in δ (ppm) relative to the in- m, PhCH2CH), 3.15 (1H, m, CH2CHCH2O), 3.93 (2H, s,
ternal standard of tetramethylsilane (TMS), and J values were PhCH2N), 3.98–4.33 (2H, m, OCH2CH), 4.83 (2H, s, OCH2N),
reported in Hz. Mass spectra were taken on an Agilent 1100 6.37 (1H, d, J=8.4Hz, H6), 6.85 (1H, d, J=8.4Hz, H5), 7.13
LC-MS electrospray/ion trap instrument in positive and nega- (2H, d, J=8.4Hz, H3′, H5′), 7.17 (2H, d, J=8.1Hz, H2′, H6′).
tive ion modes. Compounds 3a–c, 4a–c, and 8a–c were syn-
7f: Yield 70%; MS [M+H]+ (m/z): 354.1; 1H-NMR
(300MHz, CDCl3) δ: 1.22 (9H, s, C(CH3)3), 2.92 (2H, m,
thesized according to the literature.6,8,9)
.
3-(4-R1-Phenyl)-9-R2-2,3,4,8,9,10-hexahydrochromeno- PhCH2CH), 3.16 (1H, m, CH2CHCH2O), 3.81 (3H, s, PhOCH3),
[8,7-e][1,3]oxazines (7a–g) A primary amine (20.7mmol) 4.00 (2H, s, PhCH2N), 3.95–4.34 (2H, m, OCH2CH), 4.93 (2H,
and a 37% formaldehyde solution (1.4g, 16.6mmol) were dis- s, OCH2N), 6.35 (1H, d, J=8.1Hz, H6), 6.82 (1H, d, J=8.4Hz,
°
solved in 1,4-dioxane (1mL) at 45 C and stirred for 15min, H5), 6.89 (2H, d, J=8.7Hz, H3′, H5′), 7.17 (2H, d, J=8.7Hz,
and then the resulting mixture was added to a solution mix- H2′, H6′).
ture of 4a–c (13.8mmol) in 1,4-dioxane (35mL). The mixture
7g: Yield 75%; MS [M+H]+ (m/z): 325.9; 1H-NMR
°
was heated at 70 C for 2–4h and then concentrated in vacuo. (300MHz, CDCl3) δ: 0.93 (3H, t, J=7.5Hz, CH3), 1.60 (2H,
The residue was collected under suction filtration and dried to m, CH2CH3), 2.71 (2H, t, J=7.5Hz, NCH2CH2), 2.93 (2H, m,
afford compounds 5a–g.
PhCH2CH), 3.14 (1H, m, CH2CHCH2O), 3.81 (3H, s, PhOCH3),
Next, ammonium formate (3.97g, 63mmol) and 10% Pd/C 3.91 (2H, s, PhCH2N), 3.95–4.32 (2H, m, OCH2CH), 4.81 (2H,
(1.5g), followed by solids 5a–g (10.0mmol), were added to s, OCH2N), 6.37 (1H, d, J=8.1Hz, H6), 6.84 (1H, d, J=8.4Hz,
°
acetic acid (45mL). The suspension was heated to 110 C for H5), 6.89 (2H, d, J=8.7Hz, H3′, H5′), 7.16 (2H, d, J=8.7Hz,
45min. After filtering, the filtrate was concentrated to remove H2′, H6′).
a majority of the solvent, and subsequently poured into ice-
3-(4-R1-Phenyl)-9-R2-2,8,9,10-tetrahydrochromeno[8,7-
cold water (100mL) and extracted with chloroform (2×60mL). e][1,3]oxazines (12a–n) Compounds 9a–k were obtained
The collected organic layers were dried over Na2SO4, and the using compounds 8a–c as the starting materials in the
“ ”
Mannich reaction described in Section Synthesis . Next, a
solvent was removed under reduced pressure to give 6a–g.
Finally, compounds 6a–g (4.0mmol) were dissolved in solid mixture of compounds 9a–k (8.0mmol) was dissolved
methanol (2.0mL), and the methanolic solution was added to in methanol (160mL) and treated with NaBH4 (3.1g, 80mmol)
paraformaldehyde (0.36g, 12.0mmol) and potassium hydrox- for 48h at room temperature. The reaction was quenched with
ide (0.02g, 0.4mmol), which were stirred at room temperature. a saturated ammonium chloride solution (12.5mL) and then
After stirring, a white precipitate was collected to afford com- was poured into ice water (500mL). The solution was extract-
pounds 7a–g.
ed with ethyl acetate (2×100mL), and the ethyl acetate layers
7a: Yield 56%; MS [M+H]+ (m/z): 312.1; 1H-NMR were dried with Na2SO4 and concentrated under reduced pres-
(300MHz, CDCl3) δ: 1.20 (3H, t, J=7.2Hz, CH3), 2.81 (2H, sure to give solids 10a–k. Compounds 10a–k (3.0mmol) were
q, J=7.2Hz, NCH2CH3), 2.91 (2H, m, PhCH2CH), 3.11 (1H, dehydrated by treating with HCl/EtOH (5.0mL, 4.5mmol),
m, CH2CHCH2O), 3.94 (2H, s, PhCH2N), 3.94–4.30 (2H, m, and thus, a faint yellow precipitate, containing compounds

518
Vol. 60, No. 4
11a–k, was collected under suction filtration. Title compounds (1H, m, NCH(CH3)2), 4.02 (2H, s, PhCH2N), 4.93 (2H, s,
12a–k were prepared by reacting 11a–k (1.5mmol) with para- OCH2C), 5.11 (2H, s, OCH2N), 6.36 (1H, d, J=8.1Hz, H6),
formaldehyde (0.14g, 4.5mmol) in the presence of potassium 6.63 (1H, s, PhCH=C), 6.83 (2H, d, J=8.7Hz, H3′, H5′), 6.83
hydroxide at room temperature in methanol.
(1H, d, J=8.1Hz, H5), 7.29(2H, d, J=8.7Hz, H2′, H6′).
12a: Yield 87%; MS [M+H]+ (m/z): 323.9; 1H-NMR
12j: Yield 87%; MS [M+H]+ (m/z): 365.8; 1H-NMR
(300MHz, CDCl3) δ: 0.94 (3H, t, J=7.2Hz, CH3), 1.63 (2H, (300MHz, CDCl3) δ: 0.87 (3H, t, J=6.6Hz, CH3), 1.28–1.58
m, CH2CH3), 2.72 (2H, t, J=7.4Hz, NCH2CH2), 3.96 (2H, s, (8H, m, CH3(CH2)4), 2.73 (2H, t, J=7.5Hz, NCH2CH2), 3.95
PhCH2N), 4.84 (2H, s, OCH2C), 5.10 (2H, s, OCH2N), 6.37 (2H, s, PhCH2N), 4.84 (2H, s, OCH2C), 5.11 (2H, s, OCH2N),
(1H, d, J=8.1Hz, H6), 6.63 (1H, s, PhCH=C), 6.84 (2H, d, 6.37 (1H, d, J=8.1Hz, H6), 6.64 (1H, s, PhCH=C), 6.84 (2H,
J=8.7Hz, H3′, H5′), 6.85 (1H, d, J=8.1Hz, H5), 7.28 (2H, d, d, J=8.4Hz, H3′, H5′), 6.85 (1H, d, J=8.4Hz, H5), 7.29 (2H,
J=8.7Hz, H2′, H6′).
d, J=8.7Hz, H2′, H6′).
12b: Yield 89%; MS [M+H]+ (m/z): 338.1; 1H-NMR
12k: Yield 88%; MS [M+H]+ (m/z): 310.2; 1H-NMR
(300MHz, CDCl3) δ: 0.94 (3H, t, J=7.35Hz, CH3), 1.60 (2H, (300MHz, CDCl3) δ: 2.62 (3H, s, NCH3), 3.83 (3H, s,
m, CH2CH3), 2.71 (2H, t, J=7.5Hz, NCH2CH2), 3.83 (3H, PhOCH3), 3.93 (2H, s, PhCH2N), 4.77 (2H, s, OCH2C), 5.12
s, PhOCH3), 3.95 (2H, s, PhCH2N), 4.84 (2H, s, OCH2C), (2H, s, OCH2N), 6.39 (1H, d, J=8.4Hz, H6), 6.65 (1H, s,
5.12 (2H, s, OCH2N), 6.37 (1H, d, J=8.28Hz, H6), 6.65 PhCH=C), 6.87 (1H, d, J=8.4Hz, H5), 6.93 (2H, d, J=8.8Hz,
(1H, s, PhCH=C), 6.85 (1H, d, J=8.31Hz, H5), 6.91 (2H, d, H3′, H5′), 7.34 (2H, d, J=8.8Hz, H2′, H6′).
J=8.73Hz, H3′, H5′), 7.34 (2H, d, J=8.73Hz, H2′, H6′).
Dimethylamine (24mmol) and a 37% formaldehyde solution
12c: Yield 90%; MS [M+H]+ (m/z): 380.2; 1H-NMR (1.56g, 19.0mmol) were dissolved in 1,4-dioxane (1mL) and
°
(300MHz, CDCl3) δ: 0.89 (3H, t, J=6.6Hz, CH3), 1.30–1.56 stirred at 45 C for 15min, and then the reaction was added
(8H, m, CH3(CH2)4), 2.73 (2H, t, J=7.5Hz, NCH2CH2), 3.83 to a solution mixture of compounds 8a–c (16mmol) in 1,4-di-
(3H, s, PhOCH3), 3.95 (2H, s, PhCH2N), 4.84 (2H, s, OCH2C), oxane (40mL). The resulting mixture was stirred and heated
°
5.12 (2H, s, OCH2N), 6.37 (1H, d, J=8.4Hz, H6), 6.65 (1H, s, at 70 C for 30min, and then it was distilled under reduced
PhCH=C), 6.86 (1H, d, J=8.1Hz, H5), 6.92 (2H, d, J=9.0Hz, pressure until crystals began to form. Compounds 9l–n were
H3′, H5′), 7.34 (2H, d, J=8.8Hz, H2′, H6′).
collected on a filter and subsequently quaternized by CH3Br
12d: Yield 90%; MS [M+H]+ (m/z): 308.1; 1H-NMR in acetone to give compounds 10l–n. Next, compounds 11l–n
(300MHz, CDCl3) δ: 1.19 (3H, t, J=7.2Hz, CH3), 2.36 (3H, were prepared by treating the solid containing compounds
s, PhCH3), 2.81 (2H, q, J=6.9Hz, NCH2CH3), 3.97 (2H, s, 10l–n (11.7mmol) with an arylamine (18mmol) in ethanol and
PhCH2N), 4.85 (2H, s, OCH2C), 5.14 (2H, s, OCH2N), 6.37 using acetic acid as the catalyst. Compounds 11l–n (7.4mmol)
(1H, d, J=8.4Hz, H6), 6.70 (1H, s, PhCH=C), 6.86 (1H, d, in methanol (60mL) were reduced to isoflavan-4-ola 11′l–n
J=8.4Hz, H5), 7.18 (2H, d, J=8.1Hz, H3′, H5′), 7.30 (2H, d, when treated with NaBH4 (3.1g, 80mmol) for 48h at room
J=7.8Hz, H2′, H6′).
temperature, which were dehydrated by reacting with HCl/
12e: Yield 89%; MS [M+H]+ (m/z): 321.9; 1H-NMR EtOH (5.0mL, 4.5mmol, 0.9mol/L) to yield a solid containing
(300MHz, CDCl3) δ: 0.94 (3H, t, J=7.2Hz, CH3), 1.61 (2H, compounds 12′l–n. Lastly, compounds 12′l–n were condensed
m, CH2CH3), 2.36 (3H, s, PhCH3), 2.71 (2H, q, J=7.5Hz, with paraformaldehyde (0.14g, 4.5mmol) with potassium hy-
NCH2CH2), 3.95 (2H, s, PhCH2N), 4.84 (2H, s, OCH2C), 5.13 droxide as a catalyst to give title compounds 12l–n.
(2H, s, OCH2N), 6.37 (1H, d, J=8.4Hz, H6), 6.71 (1H, s,
PhCH=C), 6.86 (1H, d, J=8.1Hz, H5), 7.18 (2H, d, J=8.1Hz, (300MHz, CDCl3) δ: 2.19 (3H, s, PhCH3), 4.51 (2H, s,
12l: Yield 86%; MS [M+H]+ (m/z): 372.2; 1H-NMR
H3′, H5′), 7.30 (2H, d, J=8.1Hz, H2′, H6′).
PhCH2N), 5.13 (2H, s, OCH2C), 5.36 (2H, s, OCH2N), 6.31
12f: Yield 87%; MS [M+H]+ (m/z): 323.9; 1H-NMR (1H, d, J=8.1Hz, H6), 6.77 (1H, s, PhCH=C), 6.80 (2H, d,
(300MHz, CDCl3) δ: 1.19 (3H, t, J=7.2Hz, CH3), 2.81 (2H, J=8.1Hz, H3′, H5′), 6.89 (1H, d, J=8.4Hz, H5), 6.99–7.07
q, J=7.2Hz, NCH2CH3), 3.83 (3H, s, PhOCH3), 3.97 (2H, s, (4H, d×2, NPh), 7.35 (2H, d, J=8.4Hz, H2′, H6′).
PhCH2N), 4.86 (2H, s, OCH2C), 5.12 (2H, s, OCH2N), 6.37
12m: Yield 88%; MS [M+H]+ (m/z): 386.1; 1H-NMR
(1H, d, J=8.4Hz, H6), 6.65 (1H, s, PhCH=C), 6.86 (1H, d, (300MHz, CDCl3) δ: 2.26 (3H, s, PhCH3), 3.83 (3H, s,
J=8.4Hz, H5), 6.92 (2H, d, J=9.0Hz, H3′, H5′), 7.35 (2H, d, PhOCH3), 4.54 (2H, s, PhCH2N), 5.13 (2H, s, OCH2C), 5.30
J=8.8Hz, H2′, H6′).
(2H, s, OCH2N), 6.38 (1H, d, J=8.1Hz, H6), 6.63 (1H, s,
12g: Yield 86%; MS [M+H]+ (m/z): 310.1; 1H-NMR PhCH=C), 6.84 (1H, d, J=7.8Hz, H5), 6.91 (2H, d, J=8.7Hz,
(300MHz, CDCl3) δ: 1.08 (3H, t, J=7.2Hz, CH3), 2.68 (2H, H3′, H5′), 7.02–7.09 (4H, NPh), 7.34 (2H, d, J=8.7Hz, H2′,
q, J=7.2Hz, NCH2CH3), 3.84 (2H, s, PhCH2N), 4.80 (2H, s, H6′).
OCH2C), 5.10 (2H, s, OCH2N), 6.31 (1H, d, J=8.4Hz, H6),
6.76 (1H, s, PhCH=C), 6.78 (2H, d, J=8.7Hz, H3′, H5′), 6.89 (300MHz, CDCl3) δ: 2.26 (3H, s, PhCH3), 2.34 (3H, s,
12n: Yield 86%; MS [M+H]+ (m/z): 369.8; 1H-NMR
(1H, d, J=8.1Hz, H5), 7.35 (2H, d, J=8.7Hz, H2′, H6′).
PhCH3), 4.55 (2H, s, PhCH2N), 5.14 (2H, s, OCH2C), 5.30
12h: Yield 87%; MS [M+H]+ (m/z): 338.2; 1H-NMR (2H, s, OCH2N), 6.38 (1H, d, J=8.4Hz, H6), 6.69 (1H, s,
(300MHz, CDCl3) δ: 1.17 (6H, d, J=6.6Hz, CH(CH3)2), PhCH=C), 6.85 (1H, d, J=8.4Hz, H5), 7.02–7.09 (4H, d×2,
3.11 (H, m, NCH(CH3)2), 3.83 (3H, s, PhOCH3), 4.01 (2H, s, NPh), 7.15 (2H, d, J=8.1Hz, H3′, H5′), 7.30 (2H, d, J=8.1Hz,
PhCH2N), 4.93 (2H, s, OCH2C), 5.12 (2H, s, OCH2N), 6.35 H2′, H6′).
(1H, d, J=8.25Hz, H6), 6.65 (1H, s, PhCH=C), 6.84 (1H, d,
3-(4-Methoxyphenyl)-9-methyl-2,3,9,10-tetrahydro-
J=8.27Hz, H5), 6.91 (2H, d, J=8.8Hz, H3′, H5′), 7.35 (2H, d, chromeno[8,7-e][1,3]oxazin-4(8H)-on-es (13a–e) A metha-
J=8.8Hz, H2′, H6′).
nolic solution of paraformaldehyde (0.72g, 24.0mmol) and
12i: Yield 86%; MS [M+H]+ (m/z): 324.2; 1H-NMR potassium hydroxide platelet was added to the solution mix-
(300MHz, CDCl3) δ: 1.18 (6H, d, J=6.4Hz, CH(CH3)2), 3.10 ture of compounds 9a–e (8.0mmol) dissolved in methanol

April 2012
519
(2.0mL). Target compounds 13a–e were separated, filtered pounds 18a–d.
and dried under vacuum filtration.
18a: Yield 70%; MS [M+H]+ (m/z): 324.2; 1H-NMR
13a: Yield 87%; MS [M+H]+ (m/z): 340.1; 1H-NMR (300MHz, CDCl3) δ: 1.18 (3H, t, J=7.2Hz, CH3), 2.81 (2H,
(300MHz, CDCl3) δ: 0.94 (3H, t, J=7.2Hz, CH3), 1.60 (2H, q, J=7.2Hz, NCH2CH3), 3.79 (3H, s, PhOCH3), 4.02 (2H, s,
m, CH2CH3), 2.69 (2H, t, J=7.2Hz, NCH2CH2), 3.95 (2H, s, PhCH2N), 4.90 (2H, s, OCH2C), 5.08 (2H, s, OCH2N), 6.43
PhCH2N), 3.87 (1H, m, PhCH), 4.60 (2H, m, OCH2C), 4.91 (1H, s, H8′), 6.47 (1H, dd, J=8.1, 2.7Hz, H6′), 6.65 (1H, s,
(2H, s, OCH2N), 6.50 (1H, d, J=8.7Hz, H6), 6.72 (2H, d, H4′), 6.77 (H, d, J=8.4Hz, H5′), 6.96 (1H, s, H5), 7.00 (1H, d,
J=8.4Hz, H3′, H5′), 7.06 (2H, d, J=8.1Hz, H2′, H6′), 7.76 J=8.7Hz, H8), 7.19 (1H, dd, J=8.7, 2.1Hz, H7).
(1H, d, J=8.7Hz, H5).
18b: Yield 69%; MS [M+H]+ (m/z): 338.1; 1H-NMR
13b: Yield 90%; MS [M+H]+ (m/z): 354.2; 1H-NMR (300MHz, CDCl3) δ: 0.93 (3H, t, J=7.8Hz, CH3), 1.59 (2H,
(300MHz, CDCl3) δ: 0.95 (3H, t, J=7.2Hz, CH3), 1.61 (2H, m, CH2CH3), 2.72 (2H, t, J=7.8Hz, NCH2CH2), 3.79 (3H,
m, CH3CH2), 2.71 (2H, t, J=7.2Hz, NCH2CH2), 3.80 (3H, s, s, PhOCH3), 4.01 (2H, s, PhCH2N), 4.89 (2H, s, OCH2C),
PhOCH3), 3.89 (1H, m, PhCH), 3.97 (2H, s, PhCH2N), 4.62 5.09 (2H, s, OCH2N), 6.44 (1H, s, H8′), 6.47 (1H, dd, J=8.4,
(2H, m, OCH2C), 4.91 (2H, s, OCH2N), 6.50 (1H, d, J=8.7Hz, 2.4Hz, H6′), 6.66 (1H, s, H4′), 6.78 (H, d, J=8.4Hz, H5′),
H6), 6.89 (2H, d, J=8.7Hz, H3′, H5′), 7.19 (2H, d, J=8.7Hz, 6.97 (1H, d, J=8.4Hz, H8), 7.00 (1H, s, H5), 7.19 (1H, dd,
H2′, H6′), 7.77 (1H, d, J=9.0Hz, H5).
J=8.4, 1.2Hz, H7).
13c: Yield 89%; MS [M+H]+ (m/z): 395.9; 1H-NMR
18c: Yield 75%; MS [M+H]+ (m/z): 309.9; 1H-NMR
(300MHz, CDCl3) δ: 0.88 (3H, t, J=6.9Hz, CH3), 1.31–1.58 (300MHz, CDCl3) δ: 2.62 (1H, s, NCH3), 3.79 (3H, s,
(8H, m, CH3(CH2)4), 2.72 (2H, t, J=6.9Hz, NCH2CH2), 3.79 PhOCH3), 3.98 (2H, s, PhCH2N), 4.82 (2H, s, OCH2C), 5.09
(3H, s, PhOCH3), 3.88 (1H, m, PhCH), 3.95 (2H, s, PhCH2N), (2H, s, OCH2N), 6.44 (1H, d, J=2.1Hz, H8′), 6.47 (1H, dd,
4.62 (2H, m, OCH2C), 4.90 (2H, s, OCH2N), 6.50 (1H, d, J=8.4, 2.4Hz, H6′), 6.66 (1H, s, H4′), 6.80 (H, d, J=8.7Hz,
J=8.7Hz, H6), 6.89 (2H, d, J=8.7Hz, H3′, H5′), 7.19 (2H, d, H8), 6.98 (1H, d, J=8.1Hz, H5′), 7.01 (1H, d, J=1.8Hz, H5),
J=8.7Hz, H2′, H6′), 7.77 (1H, d, J=9.0Hz, H5).
7.21 (1H, dd, J=8.4, 2.1Hz, H7).
13d: Yield 88%; MS [M+H]+ (m/z): 323.8; 1H-NMR
18d: Yield 65%; MS [M+H]+ (m/z): 337.8; 1H-NMR
(300MHz, CDCl3) δ: 1.19 (3H, t, J=7.2Hz, CH3), 2.32 (3H, (300MHz, CDCl3) δ: 1.17 (6H, d, J=6.3Hz, CH(CH3)2), 3.08–
s, PhCH3), 2.79 (2H, q, J=7.2Hz, NCH2CH3), 3.80 (3H, s, 3.16 (1H, m, J=6.3Hz, NCH(CH3)2), 3.79 (3H, s, PhOCH3),
PhOCH3), 3.89 (1H, m, PhCH), 3.97 (2H, s, PhCH2N), 4.62 4.09 (2H, s, PhCH2N), 4.98 (2H, s, OCH2C), 5.09 (2H, s,
(2H, m, OCH2C), 4.91 (2H, s, OCH2N), 6.50 (1H, d, J=8.7Hz, OCH2N), 6.43 (1H, d, J=2.1Hz, H8′), 6.47 (1H, dd, J=8.1,
H6), 6.89 (2H, d, J=8.7Hz, H3′, H5′), 7.19 (2H, d, J=8.7Hz, 2.4Hz, H6′), 6.66 (1H, s, H4′), 6.76 (H, d, J=8.7Hz, H8), 6.97
H2′, H6′), 7.77 (1H, d, J=9.0Hz, H5).
(1H, d, J=8.1Hz, H5′), 7.01 (1H, d, J=2.1Hz, H5), 7.17 (1H,
13e: Yield 90%; MS [M+H]+ (m/z): 386.2; 1H-NMR dd, J=8.4, 2.1Hz, H7).
(300MHz, CDCl3) δ: 0.94 (3H, t, J=7.35Hz, CH3), 1.62 (2H,
Determination of in Vitro Anticancer Activities against
m, CH2CH3), 2.33 (3H, s, PhCH3), 2.69 (2H, t, J=7.5Hz, SKOV-3 and DU-145 Cell Lines Using a MTT Assay The
NCH2CH2), 3.80 (3H, s, PhOCH3), 3.89 (1H, m, PhCH), 4.00 three cell lines used in this study were cultured in RPMI1640
(2H, s, PhCH2N), 4.64 (2H, m, OCH2C), 4.99 (2H, s, OCH2N), medium containing sodium bicarbonate (2.0g/L) supplement-
°
6.47 (1H, d, J=8.7Hz, H6), 7.15 (4H, s, H3′, H5′, H2′, H6′), ed with 10% (v/v) heat-inactivated (57 C, 40min) fetal calf
7.77 (1H, d, J=8.7Hz, H5). serum (FCS), streptomycin (100μg/mL), penicillin (100IU/
6-(7-Methoxy-2H-chromen-3-yl)-3-R2-3,4-dihydro-2H- mL), and L-glutamine (4μmol/mL); all of these substances
benzo[e][1,3]oxazines (18a–d) Dihydrodaidzein was syn- were obtained from Serva (Heidelberg, FRG). All of the cell
thesized according to patent WO9808503A1. Briefly, to a lines were free of pathogenic contaminations during the ex-
mixture of dihydrodaidzein (4.0g, 15.7mmol) and sodium periments and were grown as monolayers in a humidified
°
bicarbonate (6.0g) in acetone (100mL) was added dimethyl atmosphere (5% CO2/95% air) at 37 C. Exponentially grown
sulfate (2.02g, 16mmol) dropwise under stirring at 60 C, cells were plated at 5×104 cells/cm−2 into 96-well plates and
°
and then the reaction was refluxed for 20h. After filtration, incubated for 24h. Next, the cells were treated with serial
the filtrate was poured into water (400mL) with stirring to dilutions of the tested compounds at the following final con-
give 3-(4-hydroxyphenyl)-7-methoxychroman-4-one (3.78g, centrations: 100, 30, 10, 3 and 1μM. Stock solutions of the
14mmol) as a white solid, which was dissolved in methanol compounds were initially dissolved in 20% dimethyl sulfoxide
(50mL) and treated with KBH4 (3.02g, 56mmol) at room (DMSO) and further diluted with fresh complete medium.
°
temperature for 30min. The reaction was quenched with a After 72h of incubation at 37 C, the medium was removed
saturated ammonium chloride solution (20mL) and then ice and 20μL of MTT reagent (5mg/mL) in serum-free medium
°
water (250mL); compound 15 (3.10g, 11mmol) precipitated was added to each well. The plates were incubated at 37 C for
as a white solid. Compound 15 was treated with HCl/EtOH 4h. At the end of the incubation period, the medium was re-
(34.1mL, 0.5mol/L) to make the hydroxide radical at the C-4 moved and pure DMSO (150μL) was added to each well. The
position. The reaction mixture was left in the refrigerator metabolized MTT products dissolved in DMSO were quanti-
overnight to crystallize, and compound 16 (2.02g, 8mmol) fied by reading their absorbances at 570nm using a spectro-
was collected under suction filtration. Next, treatment of com- photometer. IC50 values (the concentrations of the compounds
pound 16 with a primary amine (12mmol) and a 37% form- that caused 50% cell growth inhibition) were calculated with
aldehyde solution (0.78g, 9.6mmol) in 1,4-dioxane yielded data processing software (SPSS).
compounds 17a–d, which were reacted with paraformaldehyde
Determinations of in Vitro Anticancer Activities against
(0.47g, 16mmol) and potassium hydroxide (0.04g, 0.8mmol) the HL-60 Cell Line by the Trypan Blue Exclusion Assay
in methanol (2mL) at room temperature to obtain title com- Exponentially grown cells were plated at 4×104 cells/cm−2 into

520
Vol. 60, No. 4
24-well plates, incubated for 24h, and then the compounds
were added to obtain the following final concentrations: 100,
30, 10, 3 and 1μM. The vehicle cells were exposed to culture
medium containing 0.1% DMSO. After incubation for 72h at
References
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9072 (2005).
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Shigemasa Y., Tetrahedron Lett., 42, 7273–7275 (2001).
°
37 C, the cells were collected and suspended in 0.4% Trypan
blue. The number of viable cells was counted using a hemocy-
tometer, and IC50 values were calculated using data processing
software (SPSS).
’
Acknowledgements The authors thank Dr. Englong Ma s
research group at the Shenyang Pharmaceutical University for
8) Wähälä K., Hase T. A., Heterocycles, 28, 183–186 (1989).
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the use of their in vitro anti-tumor assay.