Synthesis of novel isoflavene-propranolol hybrids as anti-tumor agents

Article abstract of DOI:10.1016/j.bmc.2013.01.059

Isoflavene-propranolol hybrid molecules were developed as potentially novel anti-tumour agents. Isoflavene itself has potent anti-cancer activity while propranolol can enhance anti-proliferative and anti-angiogenic properties of 5-fluorouracil and paclitaxel. The hybrids were produced via nucleophilic addition of substituted amine groups to a dioxiran intermediate, which was in turn generated from the Williamson-type reaction of isoflavene with (±)-epichlorohydrin. These analogues were tested in anti-cancer cell viability assays against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines, and were found to exhibit potent anti-proliferative activities. These compounds also displayed anti-angiogenic and anti-proliferative effects in HMEC-1 human microvascular endothelial cell lines. Notably, the most potent hybrid molecules synthesized in this work showed enhanced potency against cancer cell lines compared to either isoflavene or propranolol alone, while retaining significant selectivity for cancer cells over MRC-5 normal lung fibroblast cells.

Full text of DOI:10.1016/j.bmc.2013.01.059

Bioorganic & Medicinal Chemistry 21 (2013) 1652–1660
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel isoflavene–propranolol hybrids as anti-tumor
agents
Eugene M. H. Yee a,b, Eddy Pasquier b,c, George Iskander a, Kasey Wood a, David StC Black a,
Naresh Kumar a,
⇑
a School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia
b Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, University of NSW, Randwick, NSW 2052, Australia
c Metronomics Global Health Initiative, Marseille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoflavene–propranolol hybrid molecules were developed as potentially novel anti-tumour agents. Isofla-
vene itself has potent anti-cancer activity while propranolol can enhance anti-proliferative and anti-
angiogenic properties of 5-fluorouracil and paclitaxel. The hybrids were produced via nucleophilic
addition of substituted amine groups to a dioxiran intermediate, which was in turn generated from
the Williamson-type reaction of isoflavene with ( )-epichlorohydrin. These analogues were tested in
anti-cancer cell viability assays against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma
cell lines, and were found to exhibit potent anti-proliferative activities. These compounds also displayed
anti-angiogenic and anti-proliferative effects in HMEC-1 human microvascular endothelial cell lines.
Notably, the most potent hybrid molecules synthesized in this work showed enhanced potency against
cancer cell lines compared to either isoflavene or propranolol alone, while retaining significant selectivity
for cancer cells over MRC-5 normal lung fibroblast cells.
Received 14 December 2012
Revised 23 January 2013
Accepted 28 January 2013
Available online 5 February 2013
Keywords:
Isoflavene
Propranolol
Hybrids
Anti-cancer
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
multitarget drugs that are capable of targeting multiple sites either
directly or following metabolism.3
The most frequent forms of cancer are lung, breast, colorectal,
stomach and prostate cancer. It has been estimated that in 2012,
prostate cancer (male) and breast cancer (female) would represent
29% of all new cancer cases, and would furthermore account for 9%
and 14% of cancer-related deaths in males and females,
respectively.1
Surgery, chemotherapy, immunotherapy and radiotherapy are
some of the many treatment modalities available for cancer pa-
tients. Combinations of various therapies are commonly used to
combat different types of cancer since combination therapy can
show higher efficacies compared to monotherapy.2 Combination
chemotherapy typically involves the administration of two or more
chemotherapeutics with distinct mechanisms of action. This allows
the drugs to target different aspects of the cancer cells, as well as
minimizing chances of resistance to any single drug.
One major approach to developing multitarget drugs has been
termed ‘chemical hybridization’, whereby new compounds are
generated by combining two or more known drugs (or drug phar-
macophores) into a single hybrid molecule. Hybridization of bio-
logically active molecules may not only enhance its activity and/
or selectivity, but also may reduce adverse effects and overcome
multi-drug resistant diseases.4,5
Isoflavones are a class of phytoestrogens that are naturally pres-
ent in soy products. The dietary consumption of isoflavones has
been associated with decreased incidences of certain cancers such
as breast and lung cancer.6,7 Genistein is the most predominant
isoflavone found in soy products, and it has been shown to possess
anti-cancer and anti-oxidative properties.8 The promising biologi-
cal activities of natural isoflavones have spurred the development
of synthetic analogues with greater potency and bioavailability.
The synthetic isoflavene derivative (1) shows potent anti-prolifer-
ative activity and has been investigated for the treatment of drug-
resistant ovarian cancer and prostate cancer in clinical trials.9
Due to the rising cost of developing new pharmaceuticals, there
has been an increased interest in the repositioning of existing
drugs for the potential treatment of other diseases.10 Propranolol
(Fig. 1), a non-selective beta-blocker used primarily for the treat-
ment of cardiovascular diseases, has been recently shown to en-
hance the anti-angiogenic and anti-proliferative properties of the
Drugs are conventionally developed following a single-target
approach and this has proven to be quite successful in single-target
therapy. However, treating more complex multi-factorial diseases
such as AIDS and cancer may require the targeting of multiple sites.
This has led to an increasing interest in the development of
⇑
Corresponding author. Tel.: +61 2 9385 4698; fax: +61 2 9385 6141.
E-mail address: n.kumar@unsw.edu.au (N. Kumar).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.059

E. M. H. Yee et al. / Bioorg. Med. Chem. 21 (2013) 1652–1660
1653
a multiplet at d 2.50 ppm. The three new carbon signals at d 49.1
(2 ꢀ CH), d 23.4 (2 ꢀ CH3), and d 23.2 (2 ꢀ CH3) ppm were assigned
to the attached isopropyl groups. Further confirmation was ob-
tained from HRMS (+ESI) which showed the presence of a molecu-
lar ion signal at m/z 471.2843 [M+H]+ corresponding to the
molecular formula C26H36N2O5 (471.2853). The remaining ana-
logues showed similar chemical shift patterns for the OH and NH
protons and characteristic peaks in their aliphatic regions corre-
sponding to the respective amine substituents.
N
O
H
OH
Propranolol
Figure 1. Molecular structure of propranolol.
anti-tumor agents 5-fluorouracil and paclitaxel.11 This finding to-
gether with recent epidemiologic studies12–14 suggested that pro-
pranolol may be used in cancer treatment to enhance the efficacy
of chemotherapy.
In this paper, we discuss the synthesis, characterization and
in vitro biological activities of a series of newly synthesized isofla-
vene–propranolol hybrids. Because the anti-cancer properties of
isoflavenes are well established, this initial study focused on the
development of hybrids retaining the phenyl isoflavene substitu-
ent in preference to the naphthalene unit of propranolol.
2.2. Anti-proliferative properties of hybrid molecules
It was hypothesized that the attachment of the 1-isopropyl-
amine-2-propanol side chain of propranolol onto the isoflavene
(1) scaffold would furnish hybrid molecules with superior anti-
proliferative and anti-angiogenic properties against cancer cells
compared to either parent molecule alone. To assess the anti-
proliferative properties of the synthesized hybrid molecules, their
effects on cell proliferation were assessed in vitro using MDA-
MB-231 triple-negative breast cancer cells, SHEP neuroblastoma
cells, HMEC-1 human microvascular endothelial cells and MRC-5
human lung fibroblasts.
2. Results and discussion
As shown in Figure 3, all compounds inhibited cell proliferation
in a dose-dependent manner. When compared with the parental
2.1. Synthesis of disubstituted hybrid molecules
compounds, isoflavene (GI50 = 62.9
GI50 = 8.0 M against SHEP) and propranolol (205/109
brid molecules (<6.5/<5.0 M), with the exception of 3j (229/
96.7 M), displayed improved anti-proliferative activity against
cancer cells.
l
M
against MDA-MB-231/
Williamson-type ether synthesis was utilized for the prepara-
tion of the dioxiran intermediate 2. Isoflavene (1) was stirred with
K2CO3 in acetone for 20 min to generate the phenoxide ions. ( )-
Epichlorohydrin was added and the resulting solution was heated
under reflux for 24 h (Scheme 1).
l
lM) , all hy-
l
l
When comparing the anti-proliferative activity of individual hy-
brid molecules across the two different cancer cell lines, com-
pounds 3a, 3b, 3c, 3d, and 3f, showed no significant differences.
Isoflavene, compounds 3h, 3i and 3j performed significantly better
against SHEP cells while compounds 3e performed significantly
better against MDA-MB-231 cells (Table 2).
When compared with the parental compounds, all hybrid mol-
ecules displayed improved anti-proliferative activity against
HMEC-1 as compared to propranolol but the results were more
variable when compared to isoflavene (1). Four compounds (3b,
3c, 3d, and 3e) displayed significant increased anti-proliferative
properties while four compounds (3a, 3g, 3h, and 3i) showed un-
changed efficacy and two compounds (3f and 3j) showed de-
creased activity (Table 2).
Under physiological conditions, most human cells are quiescent,
which means that they are not dividing. Chemotherapy agents
should be able to selectively target rapidly dividing cancerous cells
while leaving the normal cells unharmed. To assess the specificity
of the synthesized hybrid molecules, their toxicity against normal
quiescent cells was assessed in vitro using MRC-5 lung fibroblasts
(Fig. 2). The anti-proliferative GI50 values of the compounds against
the various cell lines are summarized in Table 2.
The crude product was purified by column chromatography to
generate the novel dioxiran compound 2 as a mixture of stereoiso-
mers in 63% yield. The 1H NMR spectrum of compound 2 (CDCl3)
showed the disappearance of the two broad singlets representing
the hydroxyl groups in isoflavene (1). The appearance of five new
multiplets with an integration of two protons each in the alkyl re-
gion indicated the successful attachment of the epoxide. 13C NMR
with DEPT-135 indicated the appropriate number of CH and CH2
resonances. Further confirmation of the structure of product 2
was performed through mass spectrometry and X-ray crystallogra-
phy of the S,R diastereoisomer which crystallised from dichloro-
methane (Fig. 1).
Nucleophilic ring-opening of the epoxides was conducted by
treating the dioxiran intermediate 2 with the corresponding
amines in a pressure tube under nitrogen at 80–90 °C for 24 h
(Scheme 2). The product was then obtained either by removal of
the solvent in vacuo followed by recrystallization from EtOAc or
CH2Cl2, or by precipitation with n-hexane followed by filtration.
The corresponding disubstituted amine hybrid molecules 3 were
obtained as a racemic mixture of diastereoisomers in moderate
to good yields (Table 1).
The structure of the isopropyl analogue 3a was established
through 1H and 13C NMR spectroscopy and HRMS data. In the 1H
NMR spectrum, the appearance of two new broad singlets at d
1.55 and 4.95 ppm with an integration of 2 protons each corre-
sponded to the OH and NH groups, respectively. Additionally,
two new doublets at d 0.90 ppm represented the four methyl
groups of the isopropyl moiety, while the CH groups appeared as
The specificity value of the compounds was calculated as a ratio
of the GI50 value of the compound against lung fibroblast cells to
that in the cell line of interest (Table 3):
GI50 of compound against normal cell
Specificity ¼
GI50 of compound against cell line of interest
O
HO
O
O
O
a
1
2
OH
O
O
Scheme 1. Reagents and conditions: (a) (i) K2CO3 (4 equiv)/acetone, rt, 20 min, and (ii) ( )-epichlorohydrin (4 equiv), reflux, 24 h.

1654
E. M. H. Yee et al. / Bioorg. Med. Chem. 21 (2013) 1652–1660
OH
O
O
O
R
O
O
a
2
O
O
R
O
OH
H
H
H
N
3a R=
3d R=
N
3g R=
3j R=
N
N
H
N
H
N
3b R=
3c R=
R=
R=
3e
3h
N
N
H
N
3f R=
N
3i R=
Scheme 2. Reagents and conditions: (a) amine, 24 h, 80–90 °C.
Table 1
Table 2
Yields of disubstituted hybrid molecules
GI50 (lM) values of compounds
R
% Yield
R
% Yield
Compounds
GI50 (lM)
3a
3b
3c
3d
3e
Isopropylamine
Benzylamine
n-Butylamine
n-Hexylamine
Ethylamine
66
51
65
63
73
3f
3g
3h
3i
Dimethylamine
Diethylamine
Pyrrolidine
Piperidine
95
96
57
80
63
HMEC-1
MDA-MB-231
SHEP
MRC-5
Isoflavene (1)
Propranolol
4.1
149
4.7
0.3
1.6
0.7
2.7
9.1
5.7
3.9
3.0
8.3
62.9
205
1.9
2.5
1.9
2.9
2.0
6.5
5.9
4.1
3.9
229
8.0
109
1.4
2.7
2.1
3.0
2.8
4.9
4.6
3.6
2.9
96.7
109
150
8.2
5.9
6.7
28.3
22.3
23.0
20.0
7.3
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
Aniline
Isoflavene (1) recorded high specificity values against HMEC-1
(specificity value = 26.4) and SHEP (13.6) cell lines, but showed lit-
tle selectivity for MDA-MB-231 cells (1.7). Compound 3j showed a
similar specificity profile to isoflavene (1), but with significantly
reduced cytotoxicity. Meanwhile compounds 3c, 3e–i (1.8–5.9)
exhibited broadly similar specificity values across the three differ-
ent cell lines. Compounds 3d (9.6, 9.6) and 3e (8.1, 11.2) showed
the best overall specificity values against SHEP and MDA-MB-231
cells, with ca. 10-fold higher potencies against the cancer cell lines
versus the normal lung fibroblasts.
16.9
449
3j
One possible explanation for the rather low anti-proliferative
activity of compound 3j against cancer cell lines may lie in the nat-
ure of its amine substituent. The aniline substituent in 3j is signif-
icantly less basic compared to the aliphatic amine moieties found
in the other hybrid molecules 3a–i. According to the ion trapping
mechanism/pH partitioning hypothesis,15 the basic hybrid mole-
cules 3a–i might be more efficiently trapped in their ionized forms
within the acidic intracellular environment of the cancer cells,
leading to accumulation of the hybrid molecules within the cell
through transmembrane equilibrium. On the other hand, the
weakly basic compound 3j would not be expected to possess the
same degree of ionization within the acidic intracellular environ-
ment of the cancer cells. Therefore, molecule 3j may readily tra-
verse the membrane and escape out of the cells, thereby
resulting in lower anti-proliferative activity for compound 3j.
Thus, one possible hypothesis is that the nitrogen group in the
hybrid molecules has to be basic in order for the compounds to
show anti-proliferative effects against MDA-MB-231 and SHEP
cancer cell lines. Furthermore, a weak correlation was observed be-
tween the degree of substitution on the nitrogen atom and the
anti-proliferative activity of the hybrid molecules. Compounds
3a–e bearing secondary amine groups exhibited ca. twofold higher
Figure 2. ORTEP diagram of 7-(oxiran-2-ylmethoxy)-3-(4-(oxiran-2-ylmeth-
oxy)phenyl)-2H-chromene 2.
2.3. Anti-angiogenic properties of the hybrid molecules
Tumour angiogenesis, which is defined as the formation of new
blood vessels from pre-existing vasculature, is a key factor in can-
cer progression and metastasis.16 This complex and dynamic pro-
cess
requires
activation,
proliferation,
migration
and
morphological differentiation of endothelial cells. Inhibition of
one or more of these steps may dramatically alter tumour angio-
genesis and potentially slow tumour progression. To assess the
anti-angiogenic properties of the synthesized hybrid molecules,
their effects on endothelial cell proliferation and morphological
differentiation into capillary structures were assessed in vitro
using the HMEC-1 endothelial cell line.
Initially, the anti-angiogenic properties of the individual com-
pounds were analyzed at 10 lM (Fig. 4, Table 4). Isoflavene (1) in-
potencies against MDA-MB-231 (GI50 = 1.91–2.94
(1.44–2.96 M) cell lines compared to compounds 3f–i that con-
tained tertiary amine groups, which showed activities of 3.85–
6.54 and 2.86–4.85 M for MDA-MB-231 and SHEP, respectively.
lM) and SHEP
duced potent anti-angiogenic effects at this concentration (80 6%
vs untreated cells) while propranolol was observed to be inactive
under the same conditions. Interestingly, the hybrid compounds
l
l

E. M. H. Yee et al. / Bioorg. Med. Chem. 21 (2013) 1652–1660
1655
Figure 3. In vitro anti-proliferative properties of isoflavene 1, propranolol and compounds 3a–j against cancer, endothelial and normal cells. (A) Growth inhibition assay
performed on MDA-MB-231, SHEP, HMEC-1 and MRC-5 cells using Alamar Blue after 72 h incubation with a range of drug concentrations. Points, % of cell proliferation as
compared to untreated control cells, means of at least four individual experiments; bars, SE; log scale for x axis. (B) Histogram representation of the micro molar
concentration of the compounds required to inhibit 50% of MDA-MB-231, SHEP, HMEC-1, and MRC-5 cell proliferation after 72 h drug incubation (GI50). Columns, means of at
least four individual experiments; bars, SE.

1656
E. M. H. Yee et al. / Bioorg. Med. Chem. 21 (2013) 1652–1660
Table 3
angiogenic properties of the compounds are enantiospecific or are
Specificity values of hybrid compounds
affected by the interactions between stereoisomers. These interac-
tions may be insignificant or may include synergistic or antagonistic
effects. Furthermore, isoflavones are known to have relatively poor
bioavailability due to the rapid glucuronidation of the free phenolic
groups resulting in excretion of the active compound.17 These newly
synthesized hybrids do not have free phenolic groups, which might
increase their bioavailability. Although the hybrids show potent
anti-angiogenic properties, the precise mechanism of action is still
not clear. Therefore, it would be interesting to investigate the mech-
anisms of action for these compounds in terms of their effects on
particular metabolic or signaling pathways. These studies could pro-
vide a basis for the observed SAR of these hybrid molecules and pave
the way for the rational design of new hybrids. In particular hybrid
molecules with alternative or additional mechanisms of action com-
pared to isoflavene 1 could represent promising candidates for fur-
ther investigation in future in vitro and in vivo studies.
Compounds
HMEC-1
Specificity value
MDA-MB-231
SHEP
Isoflavene (1)
Propranolol
26.4
1.0
1.7
0.7
4.2
2.4
3.5
9.6
11.2
3.4
3.4
1.8
4.4
2.0
13.6
1.4
5.7
2.2
3.3
9.6
8.1
4.6
4.3
2.0
5.9
4.3
3a
3b
3c
3d
3e
3f
1.8
17.6
4.2
40.3
8.6
2.5
3g
3h
3i
3.5
1.9
5.7
3j
54.2
displayed varying anti-angiogenic properties. Compound 3d pro-
duced comparable anti-angiogenic effects to isoflavene while com-
pound 3c completely suppressed HMEC-1 cell morphological
4. Experimental section
differentiation and angiogenesis at 10 lM. The remaining com-
4.1. Materials and methods
pounds induced weak anti-angiogenic effects at the same concen-
tration. Interestingly, while compounds 3d and 3c were the second
and third-most cytotoxic hybrid molecules against HMEC-1 cells,
respectively, the most cytotoxic hybrid molecule 3b showed only
All reagents and solvents were obtained from commercial
sources and purified if necessary. Melting points were measured
using a Mel-Temp melting point apparatus and are uncorrected.
1H and 13C NMR spectra were obtained on Bruker DPX300
(300 MHz) spectrometer. Mass spectra were recorded on Micro-
mass ZQ2000 (ESI) spectrometer. Infrared spectra were recorded
with a Thermo Nicolet 370 FTIR Spectrometer using KBr discs. Col-
umn chromatography was carried out using Merck 230–400 mesh
ASTM silica gel, and preparative thin layer chromatography was
20% inhibition of angiogenesis at 10 lM. This tentatively suggests
that the anti-proliferative effects of the hybrid molecules may be
partially but not fully dependent upon their anti-angiogenic
effects.
Further dose–response studies were then carried out on isofla-
vene (1) and the two most potent anti-angiogenic compounds, 3c
and 3d. Compound 3j, which showed 27% inhibition of angiogene-
performed using Merck silica gel 7730 60GF254
.
sis activity at 10 lM, was also included due to its high anti-prolif-
erative specificity towards HMEC-1 cells.
4.1.1. 7-(Oxiran-2-ylmethoxy)-3-(4-(oxiran-2-
ylmethoxy)phenyl)-2H-chromene (2)
As compound 3c induced 100% anti-angiogenic activity at
10
l
M, the concentration range for 3c was reduced to 0.1, 1, and
M to allow for the evaluation of any dose–response relationship
effects. The concentration range used for the remaining com-
pounds was 1, 5 and 10 M. As shown in Figure 4B, compound
3c exhibited significant (32%) anti-angiogenic effects from
0.1 M, while isoflavene 1 and compound 3d at 1 M significantly
inhibited angiogenesis by 18% and 21%, respectively. Compound 3j
only exhibited anti-angiogenic effects (22%) at 1 M. As expected,
Powdered K2CO3 (4.0 mmol) was added to a stirred solution of
isoflavene 1 (1.0 mmol) in acetone (15 mL) and the mixture was
stirred at room temperature for 20 minutes. ( )-Epichlorohydrin
(4.0 mmol) was then added and the reaction mixture was stirred
at reflux for 48 h. The reaction mixture was then filtered and the
filtrate was removed in vacuo to give the crude product. The crude
product was purified by column chromatography with DCM (97%)
and EtOAc (3%) to obtain the titled compound 2.
5
l
l
l
l
l
the anti-angiogenic potency of the molecules generally increased
with compound concentration.
White crystalline solid: 63%; Mp 158–160 °C decomposed; UV–
vis (MeOH): kmax 211 (
e
36859 cmꢁ1 Mꢁ1), 250 (24808), 333
(40665) nm. IR (KBr): mmax 3068, 3002, 2923, 2835, 1611, 1578,
3. Conclusions
1516, 1451, 1431, 1348, 1315, 1296, 1279, 1248, 1176, 1120,
1029, 970, 913, 865, 825, 771 cmꢁ1 1H NMR (300 MHz, CDCl3): d
.
This study has shown that the hybridization of isoflavene (1)
with the 1-(substituted amino)-2-propanol side chains based on
propranolol could generate hybrid molecules with improved
anti-angiogenic and anti-proliferative properties compared to
either parent compound alone. Significantly, the most active hy-
brid molecules showed up to 30-fold and 4-fold improved poten-
cies against MDA-MB-231 breast adenocarcinoma and SHEP
neuroblastoma cancer cell lines, respectively. However, this
improvement in anti-cancer activity was accompanied by in-
creased toxicity towards MRC-5 normal lung fibroblast cells. The
hybrid molecules 3d and 3e stand out as potentially promising
anti-cancer agents, as they show significantly higher potency
against SHEP and MDA-MB-231 cells compared to isoflavene (1)
while retaining high specificity values (ca. 10) for the two cancer
cell lines versus MRC-5.
7.35 (2H, d, J 8.87 Hz, ArH), 6.98 (1H, d, J 8.37 Hz, ArH), 6.94 (2H,
d, J 8.87 Hz, ArH), 6.68 (1H, s, HC@C), 6.49 (1H, dd, J 8.37,
2.43 Hz, ArH), 6.46 (1H, d, J 2.43 Hz, ArH), 5.10 (2H, s, CH2), 4.27–
4.17 (2H, m, CH2), 4.00–3.92 (2H, m, CH2), 3.38–3.33 (2H, m, CH),
2.93–2.89 (2H, m, CH2), 2.80–2.75 (2H, m, CH2). 13C NMR
(300 MHz, CDCl3): d 159.33 (ArC), 158.27 (ArC), 154.38 (ArC),
130.14 (ArC), 128.65 (ArC), 127.59 (C@CH), 125.94 (2 ꢀ ArCH),
118.35 (HC@C), 116.98 (ArC), 114.98 (2 ꢀ ArCH), 108.04 (ArCH),
102.30 (ArCH), 68.96 (2 ꢀ CH2), 67.38 (CH2), 50.23 (CH), 50.18
(CH), 44.87 (CH2), 44.82 (CH2).HRMS (+ESI) m/z Calcd for
C
21H20O5Na (M+Na)+ 375.1208. Found 375.1203
4.1.2. General procedure for nucleophilic ring-opening for
formation of hybrid molecule (3a–j)
Dioxiran 2 (0.30 mmol) was added to the corresponding amines
(3 mL) in a pressure tube held under a nitrogen atmosphere. The
mixture was stirred for 24 h at 80–90 °C and the product was
The current compounds synthesized and used in the biological
screening were a racemic mixtureof diastereoisomers. Further stud-
ies can be conducted to determine if the anti-proliferative and anti-

E. M. H. Yee et al. / Bioorg. Med. Chem. 21 (2013) 1652–1660
1657
Figure 4. In vitro anti-angiogenic properties of isoflavene 1, propranolol and compounds 3a–j. Representative photographs of HMEC-1 cells in Matrigel™ assays. (A) HMEC-1
cells were seeded on Matrigel™ for 5 min and then treated with 10 M of compound. Photographs were taken after 8 h of drug incubation. (B) HMEC-1 cells were seeded on
Matrigel™ and then treated with either 0.1, 1, 5 or 10 M of compound. Photographs were taken after 8 h of drug incubation. Vascular structures were imaged on a Zeiss
Axiovert 200M using a 5ꢀ objective lens. Inset, % of inhibition as compared to untreated control cells (N.S. = not statistically significant).
l
l
obtained either by removal of solvent in vacuo followed by recrystal-
lizationfrom an appropriate solvent (EtOAc or DCM/n-hexane), or by
precipitation with n-hexane followed by filtration to give the de-
sired product.
1341, 1317, 1285, 1250, 1171, 1135, 1114, 1024, 829 cmꢁ1 1H
;
NMR (300 MHz, CDCl3/DMSO-d6): d 7.46 (2H, d, J 8.87 Hz, ArH),
7.06 (1H, d, J 8.37 Hz, ArH), 6.96 (1H, d, J 8.87 Hz, ArH), 6.88 (1H,
s, HC@C), 6.50 (1H, dd, J 8.37, 2.43 Hz, ArH), 6.43 (1H, d, J
2.43 Hz ArH), 5.10 (2H, s, CH2), 4.95 (2H, br s, 2 ꢀ OH), 4.0–3.85
(6H, m, 2 ꢀ OCH2, 2 ꢀ CH), 2.72–2.54 (6H, m, 2 ꢀ NHCH2,
2 ꢀ NHCH), 1.54 (2H, br s, 2 ꢀ NH), 0.99 (6H, d, 2 ꢀ CH3), 0.97
(6H, d, 2 ꢀ CH3); 13C NMR (300 MHz, CDCl3): d 159.59 (ArC),
158.52 (ArC), 154.39 (ArC), 129.97 (ArC), 128.61 (ArCH), 127.54
(C@CH), 125.92 (2 ꢀ ArCH), 118.31 (HC@C), 116.91 (ArC), 114.91
4.1.3. 1-(4-(7-(2-Hydroxy-3-(isopropylamino)propoxy)-2H-
chromen-3-yl)phenoxy)-3-(isopropylamino)propan-2-ol (3a)
White solid Yield: 66%; Mp 129–131 °C. UV–vis (MeOH): kmax
211 (
e
23487 cmꢁ1 Mꢁ1), 250 (15573), 334 (26140) nm; IR (KBr):
mmax 3321, 2966, 2924, 2869, 1618, 1577, 1514, 1456, 1383,

1658
E. M. H. Yee et al. / Bioorg. Med. Chem. 21 (2013) 1652–1660
Table 4
(KBr): mmax 3321, 3133, 2955, 2924, 2924, 2854, 1619, 1577,
1515, 1458, 1348, 1318, 1285, 1253, 1171, 1137, 1108, 1035,
1011, 877, 832, 8112 cmꢁ1 1H NMR (300 MHz, CDCl3/DMSO-d6):
Anti-angiogenic inhibition effect of compounds
.
Compounds
Anti-angiogenic inhibition
effect at 10
lM (%)
d 7.46 (2H, d, J 8.87 Hz, ArH), 7.06 (1H, d, J 8.37 Hz, ArH), 6.96
(1H, d, J 8.87 Hz, ArH), 6.88 (1H, s, HC@C), 6.50 (1H, dd, J 8.37,
2.43 Hz, ArH), 6.43 (1H, d, J 2.43 Hz ArH) 5.10 (2H, s, CH2), 4.94
(2H, br s, 2 ꢀ OH), 4.00–3.90 (2H, m, 2 ꢀ CH), 3.89–3.82 (4H, m,
2 ꢀ OCH2), 2.68–2.55 (4H, m, 2 ꢀ CH2), 2.54–2.45 (4H, m,
2 ꢀ CH2), 1.71 (2H, br s, 2 ꢀ NH), 1.41–1.35 (4H, m, 2 ꢀ CH2),
1.28–1.25 (12H, m, 6ꢀCH2), 0.86 (6H, t, 2 ꢀ CH3). 13C NMR
(300 MHz, CDCl3): d 159.60 (ArC), 158.53 (ArC), 154.39 (ArC),
129.97 (ArC), 128.61 (ArCH), 127.54 (C@CH), 125.91 (2 ꢀ ArCH),
118.31 (HC@C), 116.84 (ArC), 114.91 (2 ꢀ ArCH), 108.00 (ArCH),
102.27 (ArCH), 70.78 (CH2), 70.74 (CH2), 68.29 (2 ꢀ CH), 67.41
(CH2), 51.81 (2 ꢀ CH2), 50.06 (2 ꢀ CH2), 31.90 (2 ꢀ CH2), 30.34
(2 ꢀ CH2), 27.08 (2 ꢀ CH2), 22.77 (2 ꢀ CH2), 14.20 (2 ꢀ CH3). HRMS
(+ESI) m/z Calcd for (M+H)+ C32H48N2O5 555.3793. Found 555.3779.
Isoflavene (1)
Propranolol
80
Not significant
26
20
100
70
18
24
19
3a
3b
3c
3d
3e
3f
3g
3h
3i
23
Not significant
27
3j
(2 ꢀ ArCH), 107.99 (ArCH), 102.27 (ArCH), 70.79 (2 ꢀ CH2), 68.62
(2 ꢀ CH), 67.42 (CH2), 49.32 (2 ꢀ CH2), 49.06 (2 ꢀ CH), 23.36
(2 ꢀ CH3), 23.22 (2 ꢀ CH3); HRMS (+ESI) m/z Calcd For
4.1.7. 1-(Ethylamino)-3-(4-(7-(3-(ethylamino)-2-
hydroxypropoxy)-2H-chromen-3-yl)phenoxy)propan-2-ol (3e)
White powder, Yield: 76%. Mp 144–146 °C. UV–vis (MeOH):
C
26H36N2O5 (M+H)+ 471.2853. Found 471.2843.
kmax 201 (e
21081 cmꢁ1 Mꢁ1), 251 (11305), 334 (17420) nm. IR
4.1.4. 1-(Benzylamino)-3-(4-(7-(3-(benzylamino)-2-
(KBr): mmax 3319, 2963, 2928, 2863, 1618, 1577, 1514, 1457,
hydroxypropoxy)-2H-chromen-3-yl)phenoxy)propan-2-ol (3b)
White powder, Yield: 51%. Mp 164–166 °C. UV–vis (MeOH):
1386, 1347, 1317, 1284, 1251, 1170, 1135, 1042, 1012, 874, 830,
809 cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 7.46 (2H, d, J 8.87 Hz,
.
kmax 208 (e
25218 cmꢁ1 Mꢁ1), 251 (10861), 335 (18701) nm. IR
ArH), 7.06 (1H, d, J 8.37 Hz, ArH), 6.96 (1H, d, J 8.87 Hz, ArH),
6.88 (1H, s, HC@C), 6.50 (1H, dd, J 8.37, 2.43 Hz, ArH), 6.43 (1H,
d, J 2.43 Hz ArH), 5.10 (2H, s, CH2), 4.95 (2H, br s, 2 ꢀ OH), 4.00–
3.83 (6H, m, 2 ꢀ OCH2, 2 ꢀ CH), 2.65–2.54 (8H, m, 4ꢀCH2), 1.87
(2H, br s, 2 ꢀ NH), 1.02 (6H, t, 2 ꢀ CH3). 13C NMR (300 MHz, CDCl3):
d 159.58 (ArC), 158.51 (ArCH), 154.40 (ArC), 129.97 (ArC), 128.61
(ArC), 127.55 (C@CH), 125.91 (2 ꢀ ArCH), 118.40 (HC@C), 116.85
(ArC), 114.91 (2 ꢀ ArCH), 107.99 (ArCH), 102.27 (ArCH), 70.79
(CH2), 70.74 (CH2), 68.37 (2 ꢀ CH), 67.39 (CH2), 51.66 (2 ꢀ CH2),
44.25 (2 ꢀ CH2), 15.56 (2 ꢀ CH3). HRMS (+ESI) m/z Calcd for
(M+H)+ C25H35N2O5 443.2540. Found 443.2529.
(KBr): mmax 3416, 3061, 3029, 2922, 2867, 1617, 1578, 1514,
1455, 1346, 1318, 1284, 1250, 1171, 1134, 1047, 1026, 831, 746,
697 cmꢁ1 1H NMR (300 MHz, CDCl3/DMSO-d6): d 7.46 (2H, d, J
.
8.87 Hz, ArH), 7.35–7.19 (10H, m, ArH), 7.06 (1H, d, J 8.37 Hz,
ArH), 6.96 (1H, d, J 8.87 Hz, ArH), 6.88 (1H, s, HC@C), 6.50 (1H,
dd, J 8.37, 2.43 Hz, ArH), 6.43 (1H, d, J 2.43 Hz ArH) 5.10 (2H, s,
CH2), 4.98 (2H, bt, 2 ꢀ OH), 4.01–3.94 (2H, m, 2 ꢀ CH), 3.93–3.84
(4H, m, 2 ꢀ OCH2), 3.72 (4H, s, 2 ꢀ BnCH2), 2.73–2.54 (4H, m,
2 ꢀ NHCH2), 2.16 (2H, br s, 2 ꢀ NH). 13C NMR (300 MHz, CDCl3):
d 159.53 (ArC), 158.47 (ArC), 154.39 (ArC), 140.11 (2 ꢀ ArC),
130.01 (ArC), 128.66 (4ꢀArCH), 128.24 (6ꢀArCH), 127.55 (C@CH),
127.32 (2 ꢀ ArCH), 125.92 (ArC), 118.32 (HC@C), 114.92
(2 ꢀ ArCH), 107.99 (ArCH), 102.28 (ArCH), 70.66 (2 ꢀ CH2), 68.52
(2 ꢀ CH), 67.41 (CH2), 53.98 (2 ꢀ CH2), 51.27 (2 ꢀ CH2). HRMS
(+ESI) m/z Calcd for C34H38N2O5 (M+H)+ 567.2853. Found 567.2839.
4.1.8. 1-(Dimethylamino)-3-(4-(7-(3-(dimethylamino)-2-
hydroxypropoxy)-2H-chromen-3-yl)phenoxy)propan-2-ol (3f)
White powder, Yield: 95%. Mp 109–111 °C. UV–vis (MeOH):
kmax 211 (e
29289 cmꢁ1 Mꢁ1), 250 (18587), 334 (31340) nm. IR
(KBr): mmax 3384, 2936, 2826, 2779, 1616, 1577, 1514, 1460,
4.1.5. 1-(Butylamino)-3-(4-(7-(3-(butylamino)-2-
1387, 1346, 1317, 1284, 1251, 1170, 1135, 1117, 1042, 1009,
hydroxypropoxy)-2H-chromen-3-yl)phenoxy)propan-2-ol (3c)
White powder, Yield: 65%. Mp 163–165 °C. UV–vis (MeOH): kmax
974, 876, 828 cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 7.46 (2H, d,
.
J 8.87 Hz, ArH), 7.06 (1H, d, J 8.37 Hz, ArH), 6.96 (1H, d, J 8.87 Hz,
ArH), 6.88 (1H, s, HC@C), 6.50 (1H, dd, J 8.37, 2.43 Hz, ArH), 6.43
(1H, d, J 2.43 Hz ArH), 5.22 (2H, br s, 2 ꢀ OH), 5.10 (2H, s, CH2),
4.00–3.83 (6H, m, 2 ꢀ OCH2, 2 ꢀ CH), 2.47–2.41 (2H, m, CH2),
2.40–2.36 (2H, m, CH2), 2.35 (12H, s, 4ꢀCH3). 13C NMR (300 MHz,
DMSO-d6): d 159.37 (ArC), 158.37 (ArC), 153.75 (ArC), 128.51
(ArC), 127.90 (ArCH), 127.16 (C@CH), 125.28 (2 ꢀ ArCH), 117.60
(HC@C), 116.07 (ArC), 114.31 (2 ꢀ ArCH), 107.39 (ArCH), 101.70
(ArCH), 71.01 (CH2), 70.94 (CH2), 66.56 (CH2), 66.40 (CH), 66.35
(CH), 46.15 (CH3), 45.77 (CH3), 45.59 (CH3), 45.56 (CH3). HRMS
(+ESI) m/z Calcd for C25H35N2O5 (M+H)+ 443.2541. Found 443.2532.
202 (
3318, 2957, 2926, 2872, 1618, 1577, 1514, 1457, 1380, 1347, 1318,
1284, 1250, 1171, 1136, 1040, 1013, 876, 830, 811 cmꢁ1 1H NMR
e
16705 cmꢁ1 Mꢁ1), 250 (8477), 333 (14212) nm. IR (KBr): mmax
.
(300 MHz, CDCl3/DMSO-d6): d 7.46 (2H, d, J 8.87 Hz, ArH), 7.06
(1H, d, J 8.37 Hz, ArH), 6.96 (1H, d, J 8.87 Hz, ArH), 6.88 (1H, s, HC@C),
6.50 (1H, dd, J 8.37, 2.43 Hz, ArH), 6.43 (1H, d, J 2.43 Hz ArH), 5.10
(2H, s, CH2), 4.01–3.82 (6H, m, 2 ꢀ OCH2, 2 ꢀ CH), 2.73–2.66 (2H,
m, CH2), 2.64–2.60 (2H, m, CH2), 2.56 (4H, t, 2 ꢀ CH2), 1.40 (4H, sex-
tet, 2 ꢀ CH2), 1.30 (4H, sextet, 2 ꢀ CH2), 0.88 (6H, t, 2 ꢀ CH3). 13C
NMR (300 MHz, CDCl3): d 159.59 (ArC), 158.53 (ArC), 154.39 (ArC),
129.97 (ArC), 128.61 (ArCH), 127.54 (C@CH), 125.92 (2 ꢀ ArCH),
118.31 (HC@C), 116.85 (ArC), 114.91 (2 ꢀ ArCH), 108.00 (ArCH),
102.27 (ArCH), 70.79 (2 ꢀ CH2), 68.28 (2 ꢀ CH), 67.42 (CH2), 51.83
(2 ꢀ CH2), 49.72 (2 ꢀ CH2), 32.49 (2 ꢀ CH2), 20.52 (2 ꢀ CH2), 14.14
(2 ꢀ CH3). HRMS (+ESI) m/z Calcd for (M+H)+ C29H43N2O5
499.3166. Found 499.3155.
4.1.9. 1-(Diethylamino)-3-(4-(7-(3-(diethylamino)-2-
hydroxypropoxy)-2H-chromen-3-yl)phenoxy)propan-2-ol (3g)
Light brown powder, Yield: 96%. Mp 53–55 °C. UV–vis (MeOH):
kmax 201 (e
23487 cmꢁ1 Mꢁ1), 250 (12666), 335 (20644) nm. IR
(KBr): mmax 3406, 2967, 2923, 2863, 1616, 1577, 1514, 1456,
1385, 1346, 1317, 1284, 1250, 1203, 1166, 1133, 1087, 1034,
979, 872, 828, 806 cmꢁ1 1H NMR (300 MHz, CDCl3/DMSO-d6): d
.
4.1.6. 1-(Hexylamino)-3-(4-(7-(3-(hexylamino)-2-
hydroxypropoxy)-2H-chromen-3-yl)phenoxy)propan-2-ol (3d)
White powder, Yield: 63%. Mp 162–164 °C. UV–vis (MeOH):
7.46 (2H, d, J 8.87 Hz, ArH), 7.06 (1H, d, J 8.37 Hz, ArH), 6.96 (1H,
d, J 8.87 Hz, ArH), 6.88 (1H, s, HC@C), 6.50 (1H, dd, J 8.37,
2.43 Hz, ArH), 6.43 (1H, d, J 2.43 Hz ArH), 5.10 (2H, s, CH2), 4.76
kmax 212 (e
15811 cmꢁ1 Mꢁ1), 250 (10984), 333 (18806) nm. IR

E. M. H. Yee et al. / Bioorg. Med. Chem. 21 (2013) 1652–1660
1659
(2H, br s, 2 ꢀ OH), 4.03–3.93 (6H, m, 2 ꢀ OCH2, 2 ꢀ CH), 2.72–2.49
(12H, m, 6ꢀCH2), 1.06 (12H, t, 4ꢀCH3). 13C NMR (300 MHz, CDCl3):
d 159.74 (ArC), 158.66 (ArC), 154.34 (ArC), 129.85 (ArC), 128.56
(ArCH), 127.49 (C@CH), 125.85 (2 ꢀ ArCH) 118.27 (HC@C), 116.74
(ArC), 114.90 (2 ꢀ ArCH), 108.04 (ArCH), 102.22 (ArCH), 70.77
(CH2), 70.73 (CH2), 67.42 (CH2), 65.96 (CH), 65.95 (CH), 55.99
(2 ꢀ CH2), 47.32 (4ꢀCH2), 12.12 (4ꢀCH3). HRMS (+ESI) m/z Calcd
for C29H43N2O5 (M+H)+ 499.3172. Found 499.3156.
107.93 (ArCH), 102.33 (ArCH), 70.39 (CH2), 70.35 (CH2), 68.95 (CH),
68.91 (CH), 67.42 (CH2), 46.74 (2 ꢀ CH2). HRMS (+ESI) m/z Calcd for
C
33H34N2O5Na (M+Na)+ 561.2360. Found 561.2346.
4.2. Biological experiments
4.2.1. Cell biology techniques
The human microvascular endothelial cell line HMEC-1 was cul-
tured on 0.1% gelatin-coated flask using MCDB-131 medium (Invit-
rogen, Mount Waverley, Australia) supplemented with 10% fetal
4.1.10. 1-(4-(7-(2-Hydroxy-3-(pyrrolidin-1-yl)propoxy)-2H-
chromen-3-yl)phenoxy)-3-(pyrrolidin-1-yl)propan-2-ol (3h)
Light brown powder, Yield: 57%. Mp 130–132 °C. UV–vis
calf serum (FCS), 1% penicillin and streptomycin and 2 mM
L-gluta-
mine (PSG), g/mL hydrocortisone and 10 ng/mL epithelial
1
l
(MeOH): kmax 212 (
e
12959 cmꢁ1 Mꢁ1), 250 (8013), 335 (13948)
growth factor (BioScientific, Gymea, Australia). The human
neuroblastoma cell line, SHEP, and human breast cancer cell line,
MDA-MB-231, were cultured in DMEM medium (Invitrogen)
supplemented with 10% FCS, 1% PSG. Human lung fibroblasts,
MRC-5, were cultured in MEM medium (Invitrogen) supplemented
with 10% FCS, 1% PSG, 2% sodium bicarbonate, 1% non-essential
amino acids and 1% sodium pyruvate. All cell lines were maintained
at 37 °C in 5% CO2 as an adherent monolayer and were passaged
upon reaching confluence by standard cell culture techniques.
nm. IR (KBr): mmax 3375, 2963, 2872, 2801, 1615, 1577, 1514,
1485, 1348, 1317, 1284, 1250, 1170, 1135, 1116, 1085, 1040,
889, 829 cmꢁ1 1H NMR (300 MHz, CDCl3/DMSO-d6): d 7.46 (2H,
.
d, J 8.87 Hz, ArH), 7.06 (1H, d, J 8.37 Hz, ArH), 6.96 (1H, d, J
8.87 Hz, ArH), 6.88 (1H, s, HC@C), 6.50 (1H, dd, J 8.37, 2.43 Hz,
ArH), 6.43 (1H, d, J 2.43 Hz ArH) 5.10 (2H, s, CH2), 4.90 (2H, br s,
2 ꢀ OH), 4.12–4.06 (2H, m, 2 ꢀ CH), 3.99 (4H, d, 2 ꢀ CH2), 2.88–
2.81 (2H, m, CH2), 2.79–2.73 (4H, m, 2 ꢀ CH2), 2.59–2.53 (6H, m,
3ꢀCH2), 1.82–1.80 (8H, m, 4ꢀCH2). 13C NMR (300 MHz, CDCl3): d
159.65 (ArC), 158.58 (ArC), 154.37 (ArC), 129.93 (ArC), 128.60
(ArCH), 127.52 (C@CH), 125.89 (2 ꢀ ArCH), 118.30 (HC@C),
116.81 (ArC), 114.92 (2 ꢀ ArCH), 108.04 (ArCH), 102.26 (ArCH),
70.69 (CH2), 70.64 (CH2), 67.42 (CH2), 67.37 (2 ꢀ CH), 58.62
(2 ꢀ CH2), 54.38 (4ꢀCH2), 23.80 (4ꢀCH2). HRMS (+ESI) m/z Calcd
for C28H40N2O5 (M+H)+ 495.2854. Found 495.2840.
4.2.2. Cell viability assays
HMEC-1, SHEP and MDA-MB-231 cells were seeded at 3750,
6000 and 6000 cells per well in 96-well plates respectively to en-
sure sustained exponential growth for 4 days. MRC-5 cells were
seeded at 30,000 cells per well in 96-well plates to ensure full con-
fluence (quiescence). Cells were treated 24 h after seeding with a
range of concentrations from 0.1 to 1000 lM of compounds 3a–j.
4.1.11. 1-(4-(7-(2-Hydroxy-3-(piperidin-1-yl)propoxy)-2H-
chromen-3-yl)phenoxy)-3-(piperidin-1-yl)propan-2-ol (3i)
White powder, Yield: 80%. Mp 151–153 °C. UV–vis (MeOH):
After 72 h drug incubation, the metabolic activity was detected
by spectrophotometric analysis by assessing the absorbance of Ala-
mar blue as previously described by Pasquier et. al.11 Cell prolifer-
ation was determined and expressed as a percentage of untreated
control cells. The determination of GI50 values was performed
using GraphPad Prism 5 (San Diego, CA, USA).
kmax 202 (e
38939 cmꢁ1 Mꢁ1), 250 (21996), 333 (36326) nm. IR
(KBr): mmax 3412, 2933, 2852, 2802, 1615, 1577, 1514, 1455,
1440, 1385, 1317, 1283, 1250, 1169, 1116, 1039, 990, 875, 828,
809 cmꢁ1 1H NMR (300 MHz, CDCl3/DMSO-d6): d 7.46 (2H, d, J
.
8.87 Hz, ArH), 7.06 (1H, d, J 8.37 Hz, ArH), 6.96 (1H, d, J 8.87 Hz,
ArH), 6.88 (1H, s, HC@C), 6.50 (1H, dd, J 8.37, 2.43 Hz, ArH), 6.43
(1H, d, J 2.43 Hz ArH) 5.10 (2H, s, CH2), 4.90 (2H, bt, 2 ꢀ OH),
4.10–4.04 (2H, m, CH), 3.97 (4H, d, 2 ꢀ CH2), 2.62–2.50 (4H, m,
2 ꢀ CH2), 2.49–2.47 (4H, m, 2 ꢀ CH2), 2.39–2.38 (4H, m, 2 ꢀ CH2),
1.62–1.56 (8H, m, 4ꢀCH2), 1.49–1.44 (4H, m, 2 ꢀ CH2). 13C NMR
(300 MHz, CDCl3): d 159.73 (ArC), 158.65 (ArC), 154.35 (ArC),
129.88 (ArC), 128.58 (ArCH), 127.50 (C@CH), 125.87 (2 ꢀ ArCH),
118.29 (HC@C), 116.76 (ArC), 114.92 (2 ꢀ ArCH), 108.07 (ArCH),
102.24 (ArCH), 70.74 (CH2), 70.69 (CH2), 67.42 (CH2), 65.42
(2 ꢀ CH), 61.21 (2 ꢀ CH2), 54.87 (4ꢀCH2), 26.26 (4ꢀCH2), 24.38
(2 ꢀ CH2). HRMS (+ESI) m/z Calcd for C31H43N2O5 (M+H)+
523.3166. Found 523.3147.
4.2.3. Anti-angiogensis assay using Matrigel™
The anti-angiogenic properties of the compounds were deter-
mined by using the Matrigel™ assay, as previously described by
Pasquier et. al.11,18 Briefly, 24-well plates were coated at 4 °C with
250 lL of Matrigel™ solution, which was then allowed to solidify
at 37 °C for 1 h before cell seeding. HMEC-1 cells were seeded at
100,000 cells per well and allowed to adhere for 5 min before treat-
ment was initiated. Cells were treated with 0.1 to 10 lM of the
compounds. Photographs were taken after 8 h of drug incubation
using the 5ꢀ objective of an Axiovert 200 M fluorescent micro-
scope coupled to an AxioCamMR3 camera driven by AxioVision
4.8 software (Carl Zeiss, North Ryde, Australia). The total surface
area of capillary tubes formed was measured in 5 view fields per
well using AxioVision 4.8 software to quantitatively evaluate the
anti-angiogenic properties of the compounds.
4.1.12. 1-(4-(7-(2-Hydroxy-3-(phenylamino)propoxy)-2H-
chromen-3-yl)phenoxy)-3-(phenylamino)propan-2-ol (3j)
Light grey powder, Yield: 63%. Mp 126–128 °C. UV–vis (MeOH):
4.2.4. Statistical analysis
kmax 202 (
e
100724 cmꢁ1 Mꢁ1), 247 (59070), 333 (43809) nm. IR
All in vitro experiments were performed at least in quadrupli-
cate and statistical significance was determined using the two-
sided Student’s t-test. All statistical analyses were performed using
GraphPad Prism 5 (GraphPad Software, Inc).
(KBr): mmax 3396, 3050, 2926, 2869, 1604, 1578, 1511, 1456,
1434, 1316, 1275, 1248, 1169, 1132, 1115, 1032, 827, 751,
694 cmꢁ1 1H NMR (300 MHz, CDCl3/DMSO-d6): d 7.46 (2H, d, J
.
8.87 Hz, ArH), 7.09–7.03 (5H, m, ArH), 6.98 (1H, d, J 8.87 Hz,
ArH), 6.88 (1H, s, HC@C), 6.61 (4H, dt, J 8.8 Hz, ArH), 6.54–6.50
(3H, m, ArH), 6.43 (1H, d, J 2.43 Hz ArH), 5.55 (2H, bq, 2 ꢀ NH),
5.16 (2H, bt, 2 ꢀ OH), 5.10 (2H, s, CH2), 4.01–3.91 (6H, m,
2 ꢀ CH2, 2 ꢀ CH), 3.26–3.17 (2H, m, CH2), 3.12–3.04 (2H, m, CH2).
13C NMR (300 MHz, CDCl3): d 159.31 (ArC), 158.26 (ArC), 154.47
(ArC), 148.19 (ArC), 130.23 (ArC), 129.51 (4ꢀArCH), 128.69 (ArCH),
127.66 (C@CH), 126.03 (2 ꢀ ArCH), 118.40 (ArCH), 118.29 (HC@C),
118.23 (ArCH), 117.08 (ArC), 114.93 (2 ꢀ ArCH), 113.44 (4ꢀArCH),
Acknowledgments
We thank the University of New South Wales, and the Austra-
lian Research Council (ARC) for financial support. E.P. was sup-
ported by
a Grant from the Balnaves Foundation. Mass
spectrometric analysis for this work was carried out at the Bioan-
alytical Mass Spectrometry Facility, UNSW and was supported in
part by infrastructure funding from the New South Wales Govern-

1660
E. M. H. Yee et al. / Bioorg. Med. Chem. 21 (2013) 1652–1660
9. Lund, T. D.; Blake, C.; Bu, L.; Hamaker, A. N.; Lephart, E. D. Reprod. Biol.
Endocrinol. 2011, 9, 4.
10. Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.;
Lindborg, S. R.; Schacht, A. L. Nat. Rev. 2010, 9, 203.
ment as part of its coinvestment in the National Collaborative Re-
search Infrastructure Strategy.
11. Pasquier, E.; Ciccolini, J.; Carre, M.; Giacometti, S.; Fanciullino, R.; Pouchy, C.;
Montero, M. P.; Serdjebi, C.; Kavallaris, M.; André, N. Oncotarget 2011, 2, 797.
12. Powe, D. G.; Voss, M. J.; Zänker, K. S.; Habashy, H. O.; Green, A. R.; Ellis, I. O.;
Entschladen, F. Oncotarget 2010, 1, 628.
13. Barron, T. I.; Connolly, R. M.; Sharp, L.; Bennett, K.; Visvanathan, K. J. Clin. Oncol.
2011, 29, 2635.
14. Melhem-Bertrandt, A.; Chavez-Macgregor, M.; Lei, X.; Brown, E. N.; Lee, R. T.;
Meric-Bernstam, F.; Sood, A. K.; Conzen, S. D.; Hortobagyi, G. N.; Gonzalez-
Angulo, A. M. J. Clin. Oncol. 2011, 29, 2645.
15. Ndolo, R. A.; Jacobs, D. T.; Lairdforrest, M.; Krise, J. P. J. Pharmacol. Exp. 2010,
333, 120.
References and notes
1. American Cancer Society. Cancer Facts
& Figures. Available at: http://
www.cancer.org, 2012 [accessed April 1, 2012].
2. World Health Organization. Media Centre. Available at: http://www.who.int,
Feb 2012 [accessed April 1, 2012].
3. Gasco, A.; Boschi, D.; Chegaev, K.; Cena, C.; Stilo, A. D.; Fruttero, R.; Lazzarato,
L.; Rolando, B.; Tosco, P. Pure Appl. Chem. 2008, 80(8), 1693.
4. Morphy, R.; Kay, C.; Rankovic, Z. Drug Discovery Today 2004, 9, 641.
5. Morphy, R.; Rankovic, Z. J. Med. Chem. 2005, 48, 6523.
6. Lu, L. J.; Anderson, K. E.; Grady, J. J.; Kohen, F.; Nagamani, M. Cancer Res. 2000,
60, 4112.
16. Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249.
17. Scalbert, A.; Williamson, G. J. Nutr. 2000, 130, 2073S.
18. Pasquier, E.; Sinnappan, S.; Munoz, M. A.; Kavallaris, M. Mol. Cancer Ther. 2010,
9, 1408.
7. Yang, W. S.; Va, P.; Wong, M. Y.; Zhang, H. L.; Xiang, Y. B. Am. J. Clin. Nutr. 2011,
94, 1575.
8. Banerjee, S.; Li, Y.; Wang, Z.; Sarkar, F. H. Cancer Lett. 2008, 269, 226.