Antitumor agents. 289. Design, synthesis, and anti-breast cancer activity in vivo of 4-amino-2 H -benzo[ h ]chromen-2-one and 4-amino-7,8,9,10-tetrahydro- 2 h -benzo[ h ]chromen-2-one analogues with improved water solubility

Article abstract of DOI:10.1021/np2007878

Previously, we reported that 4-amino-2H-benzo[h]chromen-2-one (ABO) and 4-amino-7,8,9,10-tetrahydro-2H-benzo[h]chromen-2-one (ATBO) analogues, which were developed from the lead natural product neo-tanshinlactone, are potent cytotoxic agents. In order to improve on their water solubility, the diamino analogues and related salts were designed. All synthesized compounds were assayed for cytotoxicity, and selected compounds were evaluated for in vivo anti-mammary epithelial proliferation activity in wild-type mice and mice predisposed for mammary tumors due to Brca1/p53 mutations. The new derivatives 10, 16 (ABO), 22, and 27 (ATBO) were the most active analogues, with IC ₅₀ values of 0.038-0.085 μM in the cytotoxicity assay. Analogue 10 showed around 50-fold improved water solubility compared with the prior lead ABO compound 4-[(4′-methoxyphenyl)amino]-2H-benzo[h]chromen-2-one (3). Compounds 3, 4, 10, and 22 significantly reduced overall numbers of mammary cells, as indicated by the reduction of mammary gland branching in mutant mice. A one-week treatment with 10 resulted in 80% reduction in BrdU-positive cells in the cancer prone mammary gland. These four compounds had differential effects on cellular proliferation and apoptosis in wild-type mouse and a mouse model of human breast cancers. Compound 10 merits further development as a promising anticancer clinical trial candidate.

Full text of DOI:10.1021/np2007878

Article
pubs.acs.org/jnp
Antitumor Agents. 289. Design, Synthesis, and Anti-Breast Cancer
Activity in Vivo of 4-Amino-2H-benzo[h]chromen-2-one and 4-
Amino-7,8,9,10-tetrahydro-2H-benzo[h]chromen-2-one Analogues
with Improved Water Solubility
Yizhou Dong,† Kyoko Nakagawa-Goto,† Chin-Yu Lai,† Susan L. Morris-Natschke,† Kenneth F. Bastow,‡
Yoon Kim,§ Eva Y.-H. P. Lee,§ and Kuo-Hsiung Lee*,†,⊥
†Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North
Carolina 27599-7568, United States
‡Division of Medicinal Chemistry and Natural Products, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel
Hill, North Carolina 27599-7568, United States
§Department of Biological Chemistry, College of Medicine, University of California, Irvine, California 92697, United States
⊥Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan, Republic of China
ABSTRACT: Previously, we reported that 4-amino-2H-
benzo[h]chromen-2-one (ABO) and 4-amino-7,8,9,10-tetrahy-
dro-2H-benzo[h]chromen-2-one (ATBO) analogues, which
were developed from the lead natural product neo-
tanshinlactone, are potent cytotoxic agents. In order to
improve on their water solubility, the diamino analogues and
related salts were designed. All synthesized compounds were
assayed for cytotoxicity, and selected compounds were
evaluated for in vivo anti-mammary epithelial proliferation
activity in wild-type mice and mice predisposed for mammary
tumors due to Brca1/p53 mutations. The new derivatives 10, 16 (ABO), 22, and 27 (ATBO) were the most active analogues,
with IC50 values of 0.038−0.085 μM in the cytotoxicity assay. Analogue 10 showed around 50-fold improved water solubility
compared with the prior lead ABO compound 4-[(4′-methoxyphenyl)amino]-2H-benzo[h]chromen-2-one (3). Compounds 3, 4,
10, and 22 significantly reduced overall numbers of mammary cells, as indicated by the reduction of mammary gland branching in
mutant mice. A one-week treatment with 10 resulted in 80% reduction in BrdU-positive cells in the cancer prone mammary
gland. These four compounds had differential effects on cellular proliferation and apoptosis in wild-type mouse and a mouse
model of human breast cancers. Compound 10 merits further development as a promising anticancer clinical trial candidate.
4-Amino-2H-benzo[h]chromen-2-one (ABO, 1) and 4-
amino-7,8,9,10-tetrahydro-2H-benzo[h]chromen-2-one
(ATBO, 2) analogues were previously reported as potent
cytotoxic agents. These compounds were developed based on
neo-tanshinlactone, a natural product isolated from the Chinese
drug “Tanshen” (Salvia miltiorrhiza Bunge; Lamiaceae) (Figure
1).8,9 The specific compounds 3 and 4 were extremely potent
against a panel of human tumor cell lines, with nanomolar IC50
values. Moreover, the synthetic pathway to the analogues was
quite efficient, which greatly increases their chemical
availability. However, these prior inhibitory compounds have
limited water solubility, which could adversely affect their in
vivo efficacy and make formulation difficult. Previous studies
also suggested that compounds with an amino functional group,
which can impart higher polarity and be converted to a salt
ater solubility of a drug and its behavior in water are
Wcritical to the bioavailability of pharmacological
preparations.1 Drugs administered orally as a solid must first
dissolve in the aqueous gastric fluid and then can be absorbed
and transported through the systemic circulation to their site of
action.2 Even when drugs enter the circulatory system, their
water solubility not only can influence subsequent bioavail-
ability but also may lead to side effects such as crystallization in
the kidney, possibly resulting in kidney damage.3 Therefore,
water-soluble derivatives could have superior pharmacokinetic
properties compared with poorly soluble ones. Due to the
importance of this issue, new analogues with a reasonable
degree of water solubility must be designed at an early stage of
drug development. Since water solubility depends on a
compound’s chemical structure, the structures and incorporated
functional groups may be modified to improve the water
solubility.4,5 A useful approach is to install polar functional
groups, such as acidic or basic groups, which can also be
converted to salts that have better water solubility.6,7
Special Issue: Special Issue in Honor of Gordon M. Cragg
Received: September 28, 2011
Published: February 3, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
370
dx.doi.org/10.1021/np2007878 | J. Nat. Prod. 2012, 75, 370−377

Journal of Natural Products
Article
were designed to increase overall polarity and allow further
conversion to the related salts. Aniline, piperidine, and
aminonaphthylene groups were incorporated to study the
effect of amino position and group size. All target compounds,
7−12 and 19−23, as well as their salts, 13−18 and 24−28,
were synthesized from chlorides 5 and 6 according to methods
reported before (Scheme 1).8 Treatment of 5 and 6 with
various amines afforded the related diamino analogues 7−12
and 19−23, respectively, which were converted to their salt
forms, 13−18 and 24−28
̧, respectively, with 3 N HCl in
methanol.
All synthesized analogues, 7−28, were tested for in vitro
cytotoxic activity against a panel of human tumor cell lines
according to previously published methods (Table 1).11 Cell
lines included KB (nasopharyngeal carcinoma), KB-vin
(vincristine-resistant MDR KB subline), A549 (non-small-cell
lung cancer), DU145 (prostate cancer cell line), and SK-BR-3
(estrogen receptor negative, HER2 overexpressing breast
cancer).
Figure 1. Structures of natural product neo-tanshinlactone, ABO (1)/
ATBO (2) scaffolds, and lead compounds 3 and 4.
Among the ABO analogues, the 4′-aniline analogue 10 was
the most potent compound, with IC50 values of 0.038−0.055
μM. It was about 10-fold more active than 7, suggesting that
the presence of an unsubstituted primary amine is preferable to
a dimethyl-substituted tertiary amine. Analogue 10 was also
about 2-fold more active than 9 and significantly more active
than 8. Thus, the rank order of activity based on position of the
NH2 group was 4′- (10) > 3′- (9) ≫ 2′- (8) anilino. The
piperidine analogue 11 showed only moderate activity, while
the aminonaphthalene analogue 12 was equally potent to the
3′-anilino analogue 9. These results indicated that the aromatic
ring connected directly at the 4-amino position led to better
activity. The salt forms 13−18 also displayed potent cytotoxic
activity, comparable to that of their free bases 7−12. The
ATBO analogues, 19−23, and the salt forms, 24−28, exhibited
similar SAR to the ABO analogues. With IC50 values of 0.053−
0.074 μM, 22 and its salt form 27 were the most potent ATBO
analogues and showed similar or slightly lower activity
compared with the corresponding ABO analogues 10 and 16.
This finding suggested that the aromaticity of ring A was not
strongly correlated with the activity.
form, exhibited improved water solubility without any loss in
inhibitory potency.7 Motivated by these results, a series of
diamino analogues was designed to improve water solubility
and explore the structure−activity relationships (SAR). The
anti-mammary epithelial proliferation and apoptosis-promoting
activities of the lead compounds were examined with in vivo
wild-type and Brca1/p53 mouse mammary models of human
breast cancer. Individuals who inherit a mutation of either the
BRCA1 or p53 gene have an increased risk of breast cancer.
Brca1/p53 mutant mice are predisposed to mammary tumor.
The mammary gland of Brca1/p53 mutant mice undergoes
extensive proliferation and ductal branching.10 Herein, the
design and synthesis of new diamino analogues, the effect of
amino groups on water solubility, and the antitumor activity of
lead compounds both in vitro and in vivo are reported.
On the basis of previous experience, the R group in Scheme 1
can accommodate various substituents without dramatic drops
in antitumor potency. Therefore, analogues 7−12 and 19−23
a
Scheme 1
a
Reagents and conditions: (a) amine, EtOH, reflux (ethylene glycol for 11 and 12, 160 °C); (b) 3 N HCl, MeOH.
371
dx.doi.org/10.1021/np2007878 | J. Nat. Prod. 2012, 75, 370−377

Journal of Natural Products
Article
a
Table 1. Cytotoxicity of 3, 4, and 7−28 against a Human Tumor Cell Line Panel
compd
KB
KB-vin
A549
DU145
SKBR-3
3
4
0.11 0.01
0.13 0.02
0.17 0.03
0.11 0.02
0.13 0.01
0.037 0.005
0.046 0.002
0.049 0.005
0.038 0.005
0.064 0.027
ABO free bases
7
0.60 0.13
>10
0.35 0.07
>10
0.43 0.10
>10
0.36 0.07
>10
0.30 0.09
>10
8
9
0.12 0.009
0.054 0.013
8.3 2.3
0.16 0.006
0.044 0.004
7.6 2.5
0.19 0.02
0.055 0.004
>10
0.15 0.02
0.040 0.005
>10
0.15 0.01
0.038 0.007
>10
10
11
12
0.16 0.01
0.15 0.004
0.16 0.002
0.15 0.02
0.20 0.02
ABO HCl salts
13
0.47 0.17
8.2 3.3
0.41 0.19
9.0 2.7
0.59 0.06
9.1 2.1
0.30 0.11
8.7 2.3
0.27 0.09
9.5 2.0
14
15
0.17 0.03
0.048 0.005
>10
0.15 0.01
0.047 0.002
9.4 1.0
0.15 0.03
0.054 0.003
>10
0.18 0.01
0.049 0.004
>10
0.24 0.06
0.053 0.011
>10
16
17
18
0.21 0.04
0.17 0.01
0.24 0.06
0.18 0.01
0.24 0.06
ATBO free bases
19
0.72 0.11
4.9 0.50
0.54 0.04
5.2 0.14
0.54 0.14
5.0 0.35
0.47 0.02
5.0 0.23
0.77 0.20
5.2 0.27
20
21
0.44 0.08
0.056 0.004
6.5 1.6
0.47 0.03
0.053 0.001
6.2 1.4
0.40 0.04
0.058 0.001
7.0 2.1
0.44 0.05
0.053 0.006
5.8 1.9
0.48 0.05
0.074 0.016
7.7 1.4
22
23
ATBO HCl salts
24
25
26
27
28
0.38 0.02
7.4 0.21
0.39 0.03
7.0 0.10
0.44 0.04
8.0 6.4
0.33 0.15
>10
0.49 0.07
8.7 1.8
0.48 0.03
0.069 0.011
9.1 1.1
0.45 0.03
0.065 0.014
8.3 1.1
0.50 0.04
0.062 0.006
>10
0.34 0.10
0.066 0.011
8.3 1.6
0.56 0.03
0.085 0.023
9.3 1.3
a
Mean IC50 standard error (μM), from three or more independent tests. For explanation of cell lines, see text.
In order to investigate the water solubility of the new
compounds also reduced branching in the wild-type mice, but
to lesser degrees, ranging from 15−46%.
analogues, lead compound 10 was selected and its water
solubility was measured using an HPLC assay,12 with 3 and 1-
naphthol as controls. As shown in Table 2, 10 (33.9 mg/L) was
Interestingly, BrdU-positive populations, which are indicative
of cells undergoing DNA synthesis, were significantly reduced
by 55% and 81% in mutant mice treated with compounds 3 and
10, respectively, relative to vehicle-treated mice. The effects
were not pronounced in the mammary gland of wild-type mice
(Figure 3). However, the BrdU-positive populations in mutant
mice increased approximately 90% upon treatment with 4, but
did not change appreciably upon treatment with 22. These
latter results indicate that compound 4 could enhance cell
proliferation and might not be best for the prevention or
treatment of mammary tumor.
Table 2. Water Solubility of 3, 10, and 1-Naphthol
compd
water solubility (mg/L)
3
0.69
10
33.9
1-naphthol
1050 (1350)
about 50-fold more water-soluble than 3 (0.69 mg/L). The
measured water solubility of 1-naphthol was 1050 mg/L
compared with 1350 mg/L reported in the literature.13
The total cell numbers are a net result of cell proliferation
and apoptosis. Activated cleaved caspase-3 is required for the
execution of apoptosis.14,15 Immunohistochemical staining with
antibodies recognizing cleaved caspase-3 was performed using
the paraffin-embedded mammary gland.16 Compounds 10 and
22 induced 4.8- and 4.5-fold increases, respectively, in the
numbers of apoptotic cells in the Brca1/p53 mutant gland
compared to vehicle-treated glands (Figure 4). In the wild-type
mice, 2.1- and 4.0-fold increases in apoptosis were seen after
treatment with compounds 10 and 22, respectively. No
conclusive data were obtained from 3- and 4-treated mice
due to the high background staining (data not shown). While
the dosage responses and the effects on tumors remain to be
studied, the in vivo data show that both compounds 10 and 22
reduced branching and increased apoptosis (Figures 2 and 4);
however, only compound 10 reduced cell proliferation (Figure
3). Compound 22 significantly increased apoptosis in the
Subsequently, lead compounds 3, 4, 10, and 22 were selected
for evaluation of in vivo antiproliferative activity. In addition to
studying the effects of these compounds on wild-type
mammary glands, the effects on Brca1/p53-mutated glands,
which have extensive proliferation and ductal branching and are
cancer-prone,10,11 were investigated for comparison. The
vehicle-treated Brca1/p53-mutated glands had significantly
more branching, a phenotype similar to the untreated Brca1/
p53 mutant mouse.10 All four tested compounds resulted in
considerable reduction of branching in the mutant mouse.
Specifically, lead compounds 3, 4, 10, and 22 reduced
mammary gland branching in the Brca1/p53-mutated glands
by 75%, 65%, 69%, and 70%, respectively, after 10 days of daily
injection of 0.1 mg of the compound (Figure 2). These
372
dx.doi.org/10.1021/np2007878 | J. Nat. Prod. 2012, 75, 370−377

Journal of Natural Products
Article
Figure 2. Mammary gland whole mounts (left) of vehicle and 3-, 4-, 10-, and 22-treated 3-month-old mice and number of branching points (right)
in wild-type (wt) and Brca1f11/f11p53f5&6/f5&6Crec mammary glands after treatment with 0.1 mg of compound daily for 10 days. Compounds 3, 4, 10,
and 22 decreased mammary ductal branching, especially in Brca1f11/f11p53f5&6/f5&6Crec mice.
from Aldrich, Inc., and Fisher, Inc. All compounds were >95% pure on
the basis of HPLC conditions.
mammary glands of both wild-type and mutant mice. Taken
together, further studies using compound 10 are warranted.
Spectroscopic and Analytical Data for New Com-
pounds. 4-{[4′-(Dimethylamino)phenyl]amino}-2H-benzo[h]-
1
EXPERIMENTAL SECTION
chromen-2-one (7): H NMR (400 MHz, DMSO-d6) δ 9.27 (s, 1H,
■
NH), 8.38 (d, 1H, J = 6.8 Hz, Ar-H), 8.25 (d, 1H, J = 8.8 Hz, Ar-H),
8.04−8.06 (m, 1H, Ar-H), 7.88 (d, 1H, J = 9.2 Hz, Ar-H), 7.71−7.74
(m, 2H, Ar-H), 7.20 (d, 2H, J = 9.2 Hz, Ar-H), 6.83 (d, 2H, J = 9.2 Hz,
Ar-H), 5.14 (s, 1H, 3-H), 2.95 (s, 6H, N(CH3)2); HRMS m/z [M+ +
1] calcd for C21H18N2O2, 331.1446, found 331.1451; HPLC (80%
ACN) 99.8%; tR 1.944 min.
General Experimental Procedures. 1H NMR spectra were
measured on a 300 or 400 MHz Varian Gemini 2000 spectrometer
using TMS as internal standard. The solvent used was CDCl3 unless
indicated. Mass spectra were measured on a Shimadzu LC-MS2010
instrument. Thin-layer chromatography (TLC) and preparative TLC
were performed on precoated silica gel GF plates purchased from
Merck, Inc. Isco Companion systems were used for flash
chromatography. Silica gel (200−400 mesh) from Aldrich, Inc., was
used for column chromatography. All other chemicals were obtained
4-[(2′-Aminophenyl)amino]-2H-benzo[h]chromen-2-one (8): 1H
NMR (400 MHz, CD3OD) δ 8.52 (d, 1H, J = 6.8 Hz, Ar-H), 8.12 (d,
1H, J = 8.8 Hz, Ar-H), 8.00 (d, 1H, J = 8.0 Hz, Ar-H), 7.86 (d, 1H, J =
8.8 Hz, Ar-H), 7.69−7.71 (m, 2H, Ar-H), 7.13−7.20 (m, 2H, Ar-H),
373
dx.doi.org/10.1021/np2007878 | J. Nat. Prod. 2012, 75, 370−377

Journal of Natural Products
Article
Figure 3. Effects of compounds 3, 4, 10, and 22 on mammary epithelial proliferation. BrdU-containing drinking water was provided during the last
three days of treatment. Cells that took up BrdU, indicative of DNA synthesis, were detected by immunostaining (left). BrdU-positive cells in 15
mammary ducts were quantified as the average numbers of BrdU-positive cells in vehicle and 3-, 4-, 10-, and 22-treated wild-type (wt) and
Brca1f11/f11p53f5&6/f5&6Crec mice (right).
6.92 (d, 1H, J = 8.0 Hz, Ar-H), 6.78 (t, 1H, J = 7.6 Hz, Ar-H), 5.08 (s,
1H, 3-H); HRMS m/z [M+ − 1] calcd for C19H14N2O2, 301.0977,
found 301.0985; HPLC (70% ACN) 99.9%; tR 1.817 min.
NH), 5.09 (s, 1H, 3-H); HRMS m/z [M+ − 1] calcd for C19H14N2O2,
301.0977, found 301.0985. HPLC (70% ACN) 98.7%; tR 1.727 min.
4-[(1′-Benzylpiperidin-4′-yl)amino]-2H-benzo[h]chromen-2-one
(11): 1H NMR (400 MHz, CDCl3) δ 8.57−8.60 m, 1H, Ar-H), 7.83−
7.85 (m, 1H, Ar-H), 7.61−7.68 (m, 3H, Ar-H), 7.41 (d, 1H, J = 8.4
Hz, Ar-H), 7.26−7.34 (m, 5H, Ar-H), 5.43 (s, 1H, 3-H), 5.18 (d, 1H, J
= 7.2 Hz, NH), 3.55 (s, 2H, 4′-NCH2), 3.49−3.52 (m, 1H, 1′-H), 2.91
(d, 2H, J = 12 Hz, 3′ and 5′-H), 2.13−2.21 (m, 4H, 2′, 3′, 5′, and 6′-H),
1.66−1.72 (m, 2H, J = 12 Hz, 2′ and 6′-H); HRMS m/z [M+ + 1] calcd
for C25H24N2O2, 385.1916, found 385.1919; HPLC (80% ACN)
98.2%; tR 2.167 min.
4-[(3′-Aminophenyl)amino]-2H-benzo[h]chromen-2-one (9): 1H
NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H, NH), 8.39 (d, 1H, J = 6.4
Hz, Ar-H), 8.25 (d, 1H, J = 8.8 Hz, Ar-H), 8.05 (d, 1H, J = 6.4 Hz, Ar-
H), 7.89 (d, 1H, J = 8.8 Hz, Ar-H), 7.73 (s, 2H, Ar-H), 7.13 (t, 1H, J =
8.0 Hz, Ar-H), 6.60 (s, 1H, Ar-H), 6.51 (s, 2H, Ar-H), 5.43 (s, 1H, 3-
H), 5.33 (s, 2H, NH2); HRMS m/z [M+ − 1] calcd for C19H14N2O2,
301.0977, found 301.0983; HPLC (70% ACN) 99.6%; tR 1.776 min.
4-[(4′-Aminophenyl)amino]-2H-benzo[h]chromen-2-one (10): 1H
NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H, NH), 8.37 (d, 1H, J = 7.6
Hz, Ar-H), 8.24 (d, 1H, J = 9.2 Hz, Ar-H), 8.04 (d, 1H, J = 6.8 Hz, Ar-
H), 7.87 (d, 1H, J = 8.4 Hz, Ar-H), 7.71−7.75 (m, 2H, Ar-H), 7.03 (d,
1H, J = 8.0 Hz, Ar-H), 6.67 (d, 1H, J = 8.0 Hz, Ar-H), 5.27 (s, 1H,
4-[(5′-Aminonaphthalen-1-yl)amino]-2H-benzo[h]chromen-2-
one (12): 1H NMR (400 MHz, CD3OD) δ 8.53 (d, 1H, J = 8.8 Hz, Ar-
H), 8.25 (d, 1H, J = 8.8 Hz, Ar-H), 8.12 (d, 1H, J = 7.2 Hz, Ar-H),
8.03 (d, 1H, J = 7.2 Hz, Ar-H), 7.91 (d, 1H, J = 8.4 Hz, Ar-H), 7.64−
374
dx.doi.org/10.1021/np2007878 | J. Nat. Prod. 2012, 75, 370−377

Journal of Natural Products
Article
Figure 4. Immunohistochemical staining of paraffin-embedded mammary gland sections treated with compounds 10 and 22 (left) showing
cytoplasmic and perinuclear localization of cleaved caspase-3. The compounds had a significant effect on mammary apoptosis as indicated by the
increased percentages of activated caspase-3-positive cells/duct (right).
7.76 (m, 2H, Ar-H), 7.53−7.54 (m, 2H, Ar-H), 7.28 (s, 2H, Ar-H),
6.87−6.89 (m, 1H, Ar-H); HRMS m/z [M+ − 1] calcd for
C23H16N2O2, 351.1133, found 351.1148; HPLC (70% ACN) 99.8%;
tR 1.940 min.
NH), 8.35−8.37 (m, 1H, Ar-H) 8.20 (d, 1H, J = 9.2 Hz, Ar-H), 8.01−
8.04 (m, 1H, Ar-H), 7.83 (d, 1H, J = 9.2 Hz, Ar-H), 7.67−7.74 (m,
2H, Ar-H), 7.49−7.61 (m, 5H, Ar-H), 5.49 (s, 1H, 3-H), 4.31 (d, 2H, J
= 4.8 Hz, 4′-NCH2), 3.79−3.81 (m, 1H, 1′-H), 3.46 (d, 2H, J = 11.6
Hz, 3′ and 5′-H), 3.10 (dd, 2H, J = 10.4, 23.2 Hz, 3′ and 5′-H), 2.18 (d,
2H, J = 12.8 Hz, 2′ and 6′-H), 1.97 (dd, 2H, J = 12.4, 24.8 Hz, 2′ and
6′-H).
4-{[4′-(Dimethylamino)phenyl]amino}-2H-benzo[h]chromen-2-
1
one hydrochloride (13): H NMR (400 MHz, DMSO-d6) δ 9.32 (s,
1H, NH, D2O exchanged), 8.38 (d, 1H, J = 8.0 Hz, Ar-H), 8.26 (d, 1H,
J = 9.2 Hz, Ar-H), 7.91−8.06 (m, 1H, Ar-H), 7.87 (d, 1H, J = 8.8 Hz,
Ar-H), 7.69−7.76 (m, 2H, Ar-H), 7.27 (d, 2H, J = 8.0 Hz, Ar-H), 7.00
(s, 2H, Ar-H), 5.20 (s, 1H, 3-H, D2O exchanged), 2.99 [s, 6H,
N(CH3)2].
4-[(5′-Aminonaphthalen-1′-yl)amino]-2H-benzo[h]chromen-2-
1
one hydrochloride (18): H NMR (400 MHz, DMSO-d6) δ 9.72 (s,
1H, NH, D2O exchanged), 8.38−8.43 (m, 2H, Ar-H), 8.16−8.18 (m,
1H, Ar-H), 8.10 (d, 1H, J = 8.0 Hz, Ar-H), 7.96 (d, 1H, J = 8.8 Hz, Ar-
H), 7.12−7.77 (m, 2H, Ar-H), 7.56−7.58 (m, 2H, Ar-H), 7.27−7.32
(m, 2H, Ar-H), 6.96 (s, 1H, Ar-H), 4.62 (s, 1H, 3-H, D2O exchanged).
4-{[4′-(Dimethylamino)phenyl]amino}-7,8,9,10-tetrahydro-2H-
4-[(2′-Aminophenyl)amino]-2H-benzo[h]chromen-2-one hydro-
1
chloride (14): H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H, NH,
D2O exchanged), 8.37−8.39 (m, 1H, Ar-H), 8.29 (d, 1H, J = 9.2 Hz,
Ar-H), 8.05−8.07 (m, 1H, Ar-H), 7.89 (d, 1H, J = 8.8 Hz, Ar-H),
7.70−7.76 (m, 2H, Ar-H), 7.15−7.20 (m, 2H, Ar-H), 6.96 (d, 1H, J =
7.6 Hz, Ar-H), 6.80 (t, 1H, J = 7.2 Hz, Ar-H), 4.80 (s, 1H, 3-H, D2O
exchanged).
1
benzo[h]chromen-2-one (19): H NMR (400 MHz, DMSO-d6) δ
9.02 (s, 1H, NH), 7.94 (d, 1H, J = 8.4 Hz, Ar-H), 7.14 (d, 2H, J = 8.8
Hz, Ar-H), 7.09 (d, 1H, J = 8.4 Hz, Ar-H), 6.80 (d, 2H, J = 9.2 Hz, Ar-
H), 5.01 (s, 1H, 3-H), 2.94 (s, 6H, N(CH3)2), 2.82 (t, 2H, J = 5.6 Hz,
10-H), 2.76 (t, 2H, J = 6.0 Hz, 7-H), 1.75−1.81 (m, 4H, 8 and 9-H);
HRMS m/z [M+ + 1] calcd for C21H22N2O2, 335.1760, found
335.1762; HPLC (70% ACN) 97.6%; tR 2.656 min.
4-[(2′-Aminophenyl)amino]-7,8,9,10-tetrahydro-2H-benzo[h]-
chromen-2-one (20): 1H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1H,
NH). 7.95 (d, 1H, J = 8.4 Hz, Ar-H), 7.06−7.10 (m, 2H, Ar-H), 7.00
(dd, 1H, J = 8.0, 1.2 Hz, Ar-H), 6.82 (dd, 1H, J = 8.0, 1.2 Hz, Ar-H),
6.61−6.65 (m, 1H, Ar-H), 5.02 (s, 2H, NH2), 4.62 (s, 1H, 3-H), 2.82
(t, 2H, J = 6.0 Hz, 10-H), 2.76 (t, 2H, J = 6.0 Hz, 7-H), 1.74−1.81 (m,
4H, 8 and 9-H); HRMS m/z [M+ − 1] calcd for C19H18N2O2,
305.1290, found 305.1297; HPLC (85% ACN) 99.9%; tR 1.690 min.
4-[(3′-Aminophenyl)amino]-7,8,9,10-tetrahydro-2H-benzo[h]-
chromen-2-one (21): 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H,
NH), 7.95 (d, 1H, J = 8.4 Hz, Ar-H), 7.06−7.10 (m, 2H, Ar-H), 6.53−
6.55 (m, 1H, Ar-H), 6.44−6.48 (m, 2H, Ar-H), 5.31 (s, 1H, 3-H), 5.28
4-[(3′-Aminophenyl)amino]-2H-benzo[h]chromen-2-one hydro-
1
chloride (15): H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H, NH),
8.38−8.40 (m, 1H, Ar-H), 8.27 (d, 1H, J = 8.8 Hz, Ar-H), 8.05−8.08
(m, 1H, Ar-H), 7.90 (d, 1H, J = 9.2 Hz, Ar-H), 7.70−7.77 (m, 2H, Ar-
H), 7.26 (t, 1H, J = 8.0 Hz, Ar-H), 7.70−7.27 (m, 2H, Ar-H), 6.74−
6.85 (m, 3H, Ar and NH2), 6.51 (s, 1H, Ar-H), 5.49 (s, 1H, 3-H).
4-[(4′-Aminophenyl)amino]-2H-benzo[h]chromen-2-one hydro-
1
chloride (16): H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H, NH,
D2O exchanged), 8.38(d, 1H, J = 8.0 Hz, Ar-H), 8.26 (d, 1H, J = 8.8
Hz, Ar-H), 8.06−8.08 (m, 1H, Ar-H), 7.82 (d, 1H, J = 8.8 Hz, Ar-H),
7.71−7.76(m, 2H, Ar-H), 7.38 (d, 1H, J = 8.4 Hz, Ar-H), 7.21 (d, 1H,
J = 8.0 Hz, Ar-H), 6.88 (s, 1H, NH, D2O exchanged), 5.33 (s, 1H, 3-H,
D2O exchanged).
4-[(1′-Benzylpiperidin-4′-yl)amino]-2H-benzo[h]chromen-2-one
1
hydrochloride (17): H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H,
375
dx.doi.org/10.1021/np2007878 | J. Nat. Prod. 2012, 75, 370−377

Journal of Natural Products
Article
(s, 2H, NH2), 2.82 (t, 2H, J = 6.0 Hz, 10-H), 2.76 (t, 2H, J = 6.0 Hz, 7-
H), 1.74−1.81 (m, 4H, 8 and 9-H); HRMS m/z [M+ − 1] calcd for
C19H18N2O2, 305.1290, found 305.1298; HPLC (70% ACN) 99.2%; tR
1.946 min.
4-[(4′-Aminophenyl)amino]-7,8,9,10-tetrahydro-2H-benzo[h]-
chromen-2-one (22): 1H NMR (400 MHz, CD3OD) δ 7.78 (d, 1H, J
= 8.0 Hz, Ar-H), 7.10 (d, 1H, J = 8.4 Hz, Ar-H), 7.04−7.06 (m, 2H,
Ar-H), 6.79−6.82 (m, 2H, Ar-H), 5.23 (s, 1H, 3-H), 2.88 (t, 4H, J =
6.0 Hz, 7 and 10-H), 1.85−1.87 (m, 4H, 8 and 9-H); HRMS m/z [M+
− 1] calcd for C19H18N2O2, 305.1290, found 305.1298; HPLC (70%
ACN) 99.9%; tR 1.904 min.
Histology and Immunohistochemistry. The fourth pair glands
were dissected and spread on a glass slide. After fixation with Carnoy’s
fixative for 3 h, the tissues were hydrated and stained in Carmine alum
overnight as described (http://mammary.nih.gov/tools/histological/
Histology/index.html#a1). Branching points in three random areas
totaling approximately 2 mm2 were counted. For histological section,
tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis,
MO, USA) at 4 °C overnight followed by paraffin embedding. Paraffin
sections were stained with hematoxylin and eosin and examined by
light microscopy. Immunostaining was performed following the
protocol described in the Vectastain Elite ABC kit (Vector
Laboratories, Burlingame, CA, USA). To retrieve the antigen, slides
were heated for 20 min in 10 mM citrate buffer, pH 6.0, in a
microwave oven. BrdU monoclonal antibody (GeneTex Inc., Irvine,
CA, USA) at 1:1000 dilution and cyclin D1 polyclonal rat antibody
(NeoMarkers/Thermo Fisher Scientific, Fremont, CA, USA) at 1:500
dilution, respectively, were used for immunostaining.
4-[(1′-Benzylpiperidin-4-yl)amino]-7,8,9,10-tetrahydro-2H-
1
benzo[h]chromen-2-one (23): H NMR (400 MHz, CD3OD) δ 7.71
(d, 1H, J = 8.4 Hz, Ar-H), 7.27−7.35 (m, 5H, Ar-H), 7.04 (d, 1H, J =
8.4 Hz, Ar-H), 5.27 (s, 1H, 3-H), 3.57 (s, 2H, 4′-NCH2), 3.52−3.55
(m, 1H, 1′-H), 2.98 (d, 2H, J = 12 Hz, 3′ and 5′-H), 2.85 (dd, 4H, J =
11.2, 5.2 Hz, 7 and 10-H, 2′ and 6′-H), 2.19−2.25 (m, 2H, 2′ and 6′-H),
2.03 (d, 2H, J = 12.8 Hz, 3′ and 5′-H), 1.72−1.87 (m, 6H, 8, 9, 2′ and
6′-H); HRMS m/z [M+ − 1] calcd for C25H28N2O2, 387.2072, found
387.2097; HPLC (60% ACN) 99.2%; tR 4.439 min.
AUTHOR INFORMATION
■
Corresponding Author
4-{[4′-(Dimethylamino)phenyl]amino}-7,8,9,10-tetrahydro-2H-
1
benzo[h]chromen-2-one hydrochloride (24): H NMR (400 MHz,
*Tel: +1-919-962-0066. Fax: +1-919-966-3893. E-mail: khlee@
unc.edu.
DMSO-d6) δ 9.09 (s, 1H, NH, D2O exchanged), 7.94 (d, 1H, J = 8.4
Hz, Ar-H), 7.22 (d, 2H, J = 6.8 Hz, Ar-H), 7.09 (d, 3H, J = 8.4 Hz, Ar-
H), 7.01 (s, 2H, NH2, D2O exchanged), 5.08 (s, 1H, 3-H, D2O
exchanged), 2.99 [s, 6H, N(CH3)2], 2.82 (t, 2H, J = 5.6 Hz, 10-H),
2.76 (t, 2H, J = 6.0 Hz, 7-H), 1.75−1.81 (m, 4H, 8 and 9-H).
4-[(2′-Aminophenyl)amino]-7,8,9,10-tetrahydro-2H-benzo[h]-
chromen-2-one hydrochloride (25): 1H NMR (400 MHz, DMSO-d6)
δ 8.92 (s, 1H, NH, D2O exchanged), 7.99 (d, 1H, J = 8.4 Hz, Ar-H),
7.09−7.19 (m, 3H, Ar-H), 6.98 (t, 1H, J = 8.0 Hz, Ar-H), 6.82 (t, 1H, J
= 8.0 Hz, Ar-H), 4.68 (s, 1H, 3-H, D2O exchanged), 2.83 (t, 2H, J =
6.0 Hz, 10-H), 2.77 (t, 2H, J = 6.0 Hz, 7-H), 1.75−1.81 (m, 4H, 8 and
9-H).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are thankful to Dr. X. S. Wang and H. Tang of the
University of North Carolina at Chapel Hill for valuable
discussions. This work was supported by NIH grant CA-17625-
32 from the National Cancer Institute, awarded to K.H.L. This
study was also supported in part by the Taiwan Department of
Health, China Medical University Hospital Cancer Research
Center of Excellence (DOH100-TD-C-111-005).
4-[(3′-Aminophenyl)amino]-7,8,9,10-tetrahydro-2H-benzo[h]-
chromen-2-one hydrochloride (26): 1H NMR (400 MHz, DMSO-d6)
δ 9.23 (s, 1H, NH, D2O exchanged), 7.97 (d, 1H, J = 8.4 Hz, Ar-H),
7.38 (t, 1H, J = 8.0 Hz, Ar-H), 7.12 (d, 1H, J = 8.4 Hz, Ar-H), 7.05 (s,
2H, Ar-H), 5.42 (s, 1H, 3-H, D2O exchanged), 2.83 (t, 2H, J = 6.0 Hz,
10-H), 2.77 (t, 2H, J = 6.0 Hz, 7-H), 1.74−1.81 (m, 4H, 8 and 9-H).
4-[(4′-Aminophenyl)amino]-7,8,9,10-tetrahydro-2H-benzo[h]-
chromen-2-one hydrochloride (27): 1H NMR (400 MHz, DMSO-d6)
δ 9.23 (s, 1H, NH, D2O exchanged), 7.96 (d, 1H, J = 8.4 Hz, Ar-H),
7.37 (d, 2H, J = 8.4 Hz, Ar-H), 7.27 (d, 2H, J = 8.4 Hz, Ar-H), 7.12 (d,
1H, J = 8.4 Hz, Ar-H), 6.93 (s, 1H, NH2), 5.25 (s, 1H, 3-H, D2O
exchanged), 2.83 (t, 2H, J = 6.0 Hz, 10-H), 2.77 (t, 2H, J = 6.0 Hz, 7-
H), 1.74−1.81 (m, 4H, 8 and 9-H).
DEDICATION
■
Dedicated to Dr. Gordon M. Cragg, formerly Chief, Natural
Products Branch, National Cancer Institute, for his pioneering
work on the development of natural product anticancer agents.
REFERENCES
■
(1) Thomas, G. Medicinal Chemistry: An Introduction, 2nd ed.; John
Wiley & Sons, Ltd.: West Sussex, U.K., 2008; p 47.
(2) Orme, M. Br. J. Anaesth. 1984, 56, 59−67.
(3) Izzedine, H.; Launay-Vacher, V.; Deray, G. Am. J. Kidney Dis.
2005, 45, 804−817.
4-[(1′-Benzylpiperidin-4-yl)amino]-7,8,9,10-tetrahydro-2H-
1
benzo[h]chromen-2-one hydrochloride (28): H NMR (400 MHz,
DMSO-d6) δ 10.35 (s, 1H, NH), 7.88 (d, 1H, J = 8.4 Hz, Ar-H), 7.58−
7.61 (m, 2H, Ar-H), 7.48−7.50 (m, 3H, Ar-H), 7.31 (d, 1H, J = 7.6
Hz, NH), 7.02 (d, 1H, J = 8.4 Hz, Ar-H), 5.31 (s, 1H, 3-H), 4.28 (d,
2H, J = 5.2 Hz, 4′-NCH2), 3.70−3.72 (m, 1H, 1′-H), 3.26−3.77 (m,
2H, 3′ and 5′-H), 3.07 (dd, 2H, J = 10.4, 23.2 Hz, 3′ and 5′-H), 2.79 (t,
2H, J = 6.0 Hz, 10-H), 2.73 (t, 2H, J = 6.0 Hz, 7-H), 2.12 (d, 2H, J =
14.0 Hz, 2′ and 6′-H), 1.93 (dd, 2H, J = 11.2, 23.6 Hz, 2′ and 6′-H),
1.73−1.78 (m, 4H, 8 and 9-H).
(4) Kim, I.-H.; Heirtzler, F. R.; Morisseau, C.; Nishi, K.; Tsai, H.-J.;
Hammock, B. D. J. Med. Chem. 2005, 48, 3621−3629.
(5) Ionov, M.; Gordiyenko, N.; Olchowik, E.; Baram, N.; Zijaev, K.;
Salakhutdinov, B.; Bryszewska, M.; Zamaraeva, M. J. Med. Chem. 2009,
52, 4119−4125.
(6) Berge, S. M.; Bighley, L. D.; Monkhouse, D. C. J. Pharm. Sci.
1977, 66, 1−19.
Brca1f p / f p p53f p / f p Cre Mutant Mice. Generation of
Brca1fp/fpp53fp/fpCre and p53fp/fpCre mice has been described
previously.10,17 The mice were in a C57BL/6 and 129/Sv, mixed
background. All animal experiments were performed in accordance
with guidelines of Federal and Institutional Animal Care and Use
Committee at the University of California, Irvine.
(7) Wei, L.; Shi, Q.; Bastow, K. F.; Brossi, A.; Morris-Natschke, S. L.;
Nakagawa-Goto, K.; Wu, T.-S.; Pan, S.-L.; Teng, C.-M.; Lee, K.-H. J.
Med. Chem. 2007, 50, 3674−3680.
(8) Dong, Y.; Nakagawa-Goto, K.; Lai, C. Y.; Morris-Natschke, S. L.;
Bastow, K. F.; Lee, K. H. Bioorg. Med. Chem. Lett. 2010, 20, 4085−
4087.
Treatment with 3, 4, 10, and 22. Three-month-old mice were
treated with 0.1 mg of 3 or 4, 10, 22, or vehicle daily for 10 days. The
concentration of stock solution of 3 or 4, 10, and 22 was 1 mg/mL in
dimethylsulfoxide. A mixture of 10 μL of stock solution, 30 μL of 40%
polyethylene glycol, and 60 μL of 0.9% NaCl solution was prepared at
the time of treatment. Vehicle includes all solution except 3, 4, 10, or
22. Vehicle or compound was administered ip every day for 11 days.
(9) Wang, X.; Bastow, K. F.; Sun, C. M.; Lin, Y. L.; Yu, H. J.; Don, M.
J.; Wu, T. S.; Nakamura, S.; Lee, K. H. J. Med. Chem. 2004, 47, 5816−
5819.
(10) Poole, A.; Li, Y.; Kim, Y.; Lin, S.-C. J.; Lee, W.-H.; Lee, E. Y.-H.
P. Science 2006, 314, 1467−1470.
(11) Dong, Y.; Shi, Q.; Pai, H.-C.; Peng, C.-Y.; Pan, S.-L.; Teng, C.-
M.; Nakagawa-Goto, K.; Yu, D.; Liu, Y.-N.; Wu, P.-C.; Bastow, K. F.;
376
dx.doi.org/10.1021/np2007878 | J. Nat. Prod. 2012, 75, 370−377

Journal of Natural Products
Article
Morris-Natschke, S. L.; Brossi, A.; Lang, J.-Y.; Hsu, J. L.; Hung, M.-C.;
Lee, E. Y. H. P.; Lee, K.-H. J. Med. Chem. 2010, 53, 2299−2308.
(12) Zhou, T.; Shi, Q.; Chen, C. H.; Zhu, H.; Huang, L.; Ho, P.; Lee,
K. H. Bioorg. Med. Chem. 2010, 18, 6678−6689.
(13) Guiseppi-Elie, A.; Maharajh, D. M. Thermochim. Acta 1984, 73,
187−191.
(14) Fernandes-Alnemri, T.; Litwack, G; Alnemri, E. S. J. Biol. Chem.
1994, 269, 30761−30764.
(15) Nicholson, D. W.; Ali, A.; Thornberry, N. A.; Vaillancourt, J. P.;
Ding, C. K.; Gallant, M.; Gareau, Y.; Griffin, P. R.; Labelle, M.;
Lazebnik, Y. A.; Munday, N. A.; Raju, S. M.; Smulson, M. E.; Yamin, T.
T.; Yu, V. L.; Miller, D. K. Nature 1995, 376, 37−43.
(16) Details on procedures: Rabbit polyclonal antibodies against
cleaved caspase-3 [Gene Tex (cat. #GTX86952)] were used.
Mammary tissue sections were paraffin embedded and cut into 5
μm sections. Antigen retrieval was performed with citrate buffer (pH
6.0) followed by boiling.
(17) Shafee, N.; Smith, C. R.; Wei, S.; Kim, Y.; Mills, G. B.;
Hortobagyi, G. N.; Stanbridge, E. J.; Lee, E. Y. Cancer Res. 2008, 68,
3243−3250.
377
dx.doi.org/10.1021/np2007878 | J. Nat. Prod. 2012, 75, 370−377