Efficient Synthesis of Glaziovianin A Isoflavone Series from Dill and Parsley Extracts and Their in Vitro/in Vivo Antimitotic Activity

Article abstract of DOI:10.1021/acs.jnatprod.6b00173

A concise six-step protocol for the synthesis of isoflavone glaziovianin A (GVA) and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. The reaction sequence involved an efficient conversion of the key intermediate epoxides 7 into the respective β-ketoaldehydes 8 followed by their Cu(I)-mediated cyclization into the target series 9. The biological activity of GVA and its derivatives was evaluated using a panel of seven human cancer cell lines and an in vivo sea urchin embryo assay. Both screening platforms confirmed the antimitotic effect of the parent GVA (9cg) and its alkoxy derivatives. Structure-activity relationship studies suggested that compounds 9cd and 9cf substituted with trimethoxy- and dillapiol-derived B-rings, respectively, were less active than the parent 9cg. Of the evaluated human cancer cell lines, the A375 melanoma cell line was the most sensitive to the tested molecules. Notably, the target compounds were not cytotoxic against human peripheral blood mononuclear cells up to 10 μM concentration. Phenotypic readouts from the sea urchin assay unequivocally suggest a direct microtubule-destabilizing effect of isoflavones 9cg, 9cd, and 9cf.

Full text of DOI:10.1021/acs.jnatprod.6b00173

Article
pubs.acs.org/jnp
Efficient Synthesis of Glaziovianin A Isoflavone Series from Dill and
Parsley Extracts and Their in Vitro/in Vivo Antimitotic Activity
Victor V. Semenov,*,† Dmitry V. Tsyganov,† Marina N. Semenova,‡,§ Roman N. Chuprov-Netochin,⊥
Mikhail M. Raihstat,† Leonid D. Konyushkin,† Polina B. Volynchuk,⊥ Elena I. Marusich,⊥
Vera V. Nazarenko,⊥ Sergey V. Leonov,⊥,∥ and Alex S. Kiselyov⊥
†N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991, Moscow, Russian Federation
‡Institute of Developmental Biology, RAS, Vavilov Street, 26, 119334, Moscow, Russian Federation
§Chemical Block Ltd., 3 Kyriacou Matsi, 3723, Limassol, Cyprus
⊥Life Sciences Center, Moscow Institute of Physics and Technology, Institutsky Per., 9, Dolgoprudny, Moscow Region 141700,
Russian Federation
∥Institute of Cell Biophysics, RAS, Institutskaya Street, 3, Pushchino, Moscow Region 142290, Russian Federation
S
* Supporting Information
ABSTRACT: A concise six-step protocol for the synthesis of isoflavone glaziovianin A (GVA) and its alkoxyphenyl derivatives 9
starting with readily available plant metabolites from dill and parsley seeds was developed. The reaction sequence involved an
efficient conversion of the key intermediate epoxides 7 into the respective β-ketoaldehydes 8 followed by their Cu(I)-mediated
cyclization into the target series 9. The biological activity of GVA and its derivatives was evaluated using a panel of seven human
cancer cell lines and an in vivo sea urchin embryo assay. Both screening platforms confirmed the antimitotic effect of the parent
GVA (9cg) and its alkoxy derivatives. Structure−activity relationship studies suggested that compounds 9cd and 9cf substituted
with trimethoxy- and dillapiol-derived B-rings, respectively, were less active than the parent 9cg. Of the evaluated human cancer
cell lines, the A375 melanoma cell line was the most sensitive to the tested molecules. Notably, the target compounds were not
cytotoxic against human peripheral blood mononuclear cells up to 10 μM concentration. Phenotypic readouts from the sea
urchin assay unequivocally suggest a direct microtubule-destabilizing effect of isoflavones 9cg, 9cd, and 9cf.
glaziovianin A (GVA), and less potent analogues I−III (Figure
atural compounds and their analogues account for almost
N80% of all anticancer drugs approved between 1981 and 1) were isolated from the leaves of the Brazilian tree Ateleia
glazioviana Baill. (Leguminosae).8 The leaves of this plant were
reported to induce abortions, cardiac failure, and central
nervous system injuries in live stock.8 Parent GVA and related
isoflavones I−III inhibited the growth of HL-60 human
promyelocytic leukemia cells with IC50 values of 0.29, 16, 23,
and 8.5 μM, respectively.8 Furthermore, GVA displayed high
cytotoxicity against a panel of 39 human cancer cell lines
(average IC50 = 0.66 μM),8 disrupted microtubule structure and
dynamics by binding to the colchicine site of the tubulin
2010.1 Several antimitotic agents isolated from plants, including
colchicine, combretastatins, and podophyllotoxin, as well as
their semisynthetic and synthetic analogues, contain a
trimethoxyphenyl moiety.2−6 Flavonoids are abundant in nature
and exhibit both topological and pharmacophore resemblance
to pharmacologically active combretastatin and podophyllotox-
in derivatives.7 It is reasonable to assume that properly
substituted flavonoids and their structural analogues may
show comparable antimitotic activity. Of these, isoflavones
deserve particular attention.
There is limited literature data on alkoxyisoflavones with
Received: February 28, 2016
antiproliferative activity. A potent cytotoxic compound,
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Structures of alkoxyisoflavones, combretastatins, and podophyllotoxin.
a
Scheme 1
a
Reagents and conditions:23 (a) (i) KOH, 100 °C, 40 min; (ii) O3, CHCl3−MeOH−pyridine (80:20:3 v/v), 15 °C, 1−2 h.
molecule,9 and blocked cell cycle progression in mitosis due to
mitotic spindle damage.8−10 Recently it was found that GVA
interacts with both α/β- and γ-tubulins, whereas its 7-O-benzyl
synthetic analogue, gatastatin, selectively affects γ-tubulin,
thereby inhibiting γ-tubulin-dependent microtubule nuclea-
tion.11 The isoflavones ferrugone and preferrugone (Figure 1),
which are structurally similar to GVA, were isolated from the
root bark, stem bark, and seeds of African plants of the genus
Millettia; however their biological effects were not studied.12−14
Considering these limited data, the aim of the present work was
to devise a concise synthetic route toward GVA and its
derivatives in order to expand structure−activity relationship
studies and to investigate their antimitotic effect using an in
vivo phenotypic sea urchin embryo assay.15
A common route toward the isoflavone scaffold involves
Friedel−Crafts acylation of respective alkoxyphenols with
electron-rich phenylacetic acids followed by acid-mediated
cyclization of the resulting ortho-phenol intermediate to yield
isoflavones.16,17 A noted drawback of this protocol is the rather
limited access to diverse methoxyphenylacetic acids. The
recently reported alternative approaches include (i) intra-
molecular cyclization of diarylketoaldehydes with o-benzoy-
loxy18 or o-bromo substituents19 and (ii) the Suzuki coupling
reaction.20,21 However, starting building blocks for these
synthetic protocols are not readily accessible as well. In order
to address this issue, a facile synthesis of diverse alkoxy-
substituted isoflavones starting with readily available natural
precursors isolated from plant essential oils was developed and
validated. Specifically, we focused on preparing several
structural analogues of GVA varying both the nature and
position of the alkoxyphenyl substituents in both the A- and B-
rings of the molecule. The goal was both to improve antimitotic
activity of the molecule and to study the isoflavone substitution
pattern leading to the optimal antiproliferative activity.
RESULTS AND DISCUSSION
■
The bromochalcone-mediated route was considered as the
most attractive for the synthesis of alkoxyisoflavones.
Metabolites 1 (Scheme 1) can be readily isolated from different
crop plants, namely, dill [Anetum graveolens L. (Apiaceae)],
parsley [Petroselinum sativum Hoffm. (Apiaceae)],22,23 tarragon
[Aretmisia dracunculus L. (Asteraceae)],24 and lemongrass
[Cymbopogon flexuosus W. Watson (Poaceae)].25 The technol-
ogy for planting and harvesting of these species is well
developed, and the targeted allyl derivatives 1 are scaled-up
readily. Rings A and B could be constructed from the same
aldehydes 2, accessible from allylbenzenes 1, which are the
major components (30−80%) of plant essential oils (Scheme
1).22,24 Methoxybenzaldehydes 2 were selectively o-brominated
to yield intermediates 3. These products were treated with
MeMgI followed by oxidation of the resulting o-bromobenzyl
alcohols 4 to afford bromoacetophenones 5. The subsequent
conversion of 5 to chalcones 6 followed by their epoxidation
and subsequent epoxide rearrangement of 7 resulted in
ketoaldehydes 8 in high yields (60−90%) for each step.
Cyclization of ketoaldehydes 8 in the presence of CuI19
afforded the targeted isoflavones 9 in moderate yields. The
attempted synthesis of isoflavone 9fc via cyclization of 8fc was
B
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not successful, presumably due to the instability of the 5-OMe
group of the A-ring under experimental conditions (Supporting
Information, pp S3, S4). Specifically, NMR spectra of the crude
reaction mixture revealed the presence of compounds 9fc and
9ec (13:10 ratio, respectively). Compound 9ec was successfully
isolated from the reaction mixture via chromatography. It
should be noted that this six-step sequence is considerably
shorter than the reported route,20,21 which comprises nine steps
including the synthesis of the intermediate 2,3,4-trimethox-
ybenzaldehyde.26 In addition, as opposed to our synthetic
route, which relies on readily available natural precursors 1,
protocols published earlier employed expensive 2-hydroxy-4,5-
dimethoxyacetophenone and a considerable quantity of Pd-
catalyst (342 mg to obtain 714 mg of the targeted GVA).
Combretastatin A4 disodium phosphate (CA4P) served as
reference compound. The results are summarized in Table 1.
The sea urchin embryo tests confirmed that GVA directly
affects tubulin/microtubule dynamics and structure.8−11,21 The
parent compound 9cg was the most active of the tested series.
It induced sea urchin embryo spinning indicative of a
microtubule destabilizing mode of action.15 GVA analogues
9cd, 9ce, 9cf, and 9ia caused cleavage arrest and initiated the
formation of tuberculate eggs, thus providing the indirect
evidence of their microtubule-destabilizing properties.15 The B-
ring substition pattern of GVA derivatives exhibited strong
effects on activity. As suggested by the assay results, the
antimitotic activity decreased in the following order: the parent
GVA (9cg) > myristicin (9ce) ≥ 3,4,5-trimethoxyphenyl (9cd)
= 4-methoxyphenyl (9ca) > dillapiol (9cf) > 3-methoxyphenyl
(9cb) > 3,4-dimethoxyphenyl (9cc) > 2,3,4,5-tetramethox-
yphenyl (9ch) derivatives. Any definitive relationship between
the number of B-ring alkoxy groups and respective antimitotic
activity was not observed. A methylenedioxy moiety was
essential for the activity of compounds substituted with four B-
ring alkoxy groups. Thus, a compound containing an apiol
moiety (GVA, 9cg) exhibited stronger activity than the
respective dillapiol-derived analogue 9cf. In addition, the
myristicin derivative with a methylenedioxy moiety, 9ce, was
more active than isoflavonoid 9cd, with a 3,4,5-trimethoxy B-
ring. A compound with a 4-methoxy group was more potent
than the respective 3-methoxy-analogue (compare 9ca and
9cb). Dimethoxy substitution of the A-ring was also important.
The replacement of 7-OMe with a hydroxy group in 9ca
resulted in the less active isoflavone 9ia. Introduction of the
third MeO group as in 9dc led to low activity and inactivity in
compound 9ja. As opposed to GVA, iso-GVA (9gc) featuring
swapped A- and B-rings had no effect on the sea urchin
embryos up to 4 μM concentration. Isoflavone 9cc with
dimethoxy-substituted B-ring displayed low activity.
a
Scheme 2
a
Reagents and conditions: (a) NBS, DMF, rt, 8 h; (b) MeMgI, ether,
rt, 0.5 h; (c) pyridinium chlorochromate, CH2Cl2, rt, 18 h.
Literature data suggest that the 7-alkoxy substituents of the
A-ring and the 2′,5′-dimethoxy-3′,4′-methylenedioxy B-ring are
the essential features for antimitotic microtubule-modulating
properties of GVA and its analogues.10,21 Specifically, GVA
derivatives with O-allyl, O-propargyl, and O-benzyl groups at C-
7 showed the highest cytotoxicity against HeLa S3 human
cervical cancer cells and caused cell cycle arrest in the G2/M
phase.10,21 The B-ring modification effects were investigated to
a lesser extent. It was shown that both removal of 2′- and 5′-
methoxy groups and the replacement of the 3′,4′-methyl-
enedioxy moiety with two individual methoxy groups resulted
in a significant decrease of cytotoxicity.21 In the next series of
experiments the antimitotic activity of GVA and its analogues
was evaluated, using the in vivo sea urchin embryo assay.15
The inhibitory effects of isoflavones 9ca, 9cb, 9cc, 9cd, 9ce,
9ch, 9cf, 9cg, and 9gc on cell growth were evaluated using
seven human cancer cell lines, namely, A549 lung carcinoma,
A375 melanoma, DU145 and PC3 prostate cancer, HCT116
a
Scheme 3
a
Reagents and conditions: (a) NaOH, EtOH, rt, 6 h; (b) H2O2, EtOH−NaOH, rt, 54 h; (c) BF3−Et2O, CH2Cl2, rt, 3 h; (d) CuI−K2CO3, 2-picolinic
acid, DMF, 135−140 °C, 8 h.
C
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a
Table 1. Effects of GVA and its Analogues on Sea Urchin Embryos and Human Cancer Cells
a
A549: lung carcinoma; A375: melanoma; DU145 and PC3: prostate cancer; HCT116: colon cancer; MDA-MB-231: breast adenocarcinoma; SK-
b
OV-3: ovarian carcinoma; PBMCs: human peripheral blood mononuclear cells. The sea urchin embryo assay was conducted as described
previously.15 Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing concentrations of compounds. Duplicate measurements showed
c
no differences in effective threshold concentration (EC) values. IC50 values were determined by sigmoidal curve interpolation of cell survival data
after 48 h of treatment. TE: tuberculate eggs. Data in bparentheses are from ref 8, obtained for natural GVA using the SRB assay. ND: not
d
e
f
determined. IC50 values are expressed as means
compound concentration tested in quadruplicates.
SD based on dose−response curves of three independent experiments that involved each
colon cancer, MDA-MB-231 breast adenocarcinoma, and SK-
OV-3 ovarian carcinoma. The MTS cell viability assay was
employed with CA4P as a positive control. For comparison, the
cytotoxicity of compounds against nonmalignant human
peripheral blood mononuclear cells (PBMCs) was assessed
(Table 1). The maximal tested concentration of isoflavones was
10 μM due to their restricted solubility. GVA (9cg) displayed
the highest inhibitory activity, with IC50 values ranging from
0.27 (A375 cells) to 2.2 μM (MDA-MB-231 cells). The data
obtained for the six cancer cell lines by the MTS assay
correlated well with the published data acquired using the SRB
method (Table 1).8 Compounds 9cd and 9cf, containing 3,4,5-
trimethoxy and dillapiol-derived B-rings, respectively, were less
active. Isoflavones 9ca, 9cb, 9cc, 9ch, and 9gc did not affect
cancer cell growth up to 10 μM concentration. These results
correlated well with published data. GVA (9cg) was
significantly more active than 6,7,3′,4′-tetramethoxyisoflavone
9cc, exhibiting cytotoxic effects against HL-60 human leukemia
cells and HeLa S3 human cervical cancer cells with GI50 values
of 0.29 vs 23 μM and 0.59 vs 22 μM, respectively.8,21 Of the
evaluated human cancer cell lines, A375 melanoma cells were
most sensitive to the tested compounds. On the contrary,
compounds 9 showed little to no effect on A549 lung
carcinoma cells. GVA (9cg) was the only compound that
inhibited growth of A549 cells (Table 1). Importantly, none of
the synthesized isoflavones 9 demonstrated cytotoxicity in
D
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4-Bromo-6,7-dimethoxy-2H-1,3-benzodioxole-5-carbaldehyde
(3f): 4.85 g, 84% yield; colorless crystals; mp 103 °C (50% EtOH); 1H
NMR (DMSO-d6) δ 10.07 (1H, s, CHO), 6.22 (2H, s, OCH2O), 3.96
(3H, s, OCH3-6), 3.84 (3H, s, OCH3-5); EIMS m/z 290 [M + 1]
nonmalignant cells, namely, human peripheral blood mono-
nuclear cells, up to 10 μM concentration.
It should be noted that the antiproliferative effects of
isoflavones 9 observed in both the sea urchin embryo model
and human cancer cell lines correlated well. Specifically, GVA
(9cg) was found to be the most potent agent in both systems.
Swapping rings A and B as well as the replacement of the
methylenedioxy moiety with two individual methoxy groups in
GVA (9cg) resulted in inactive compounds 9gc and 9ch. Both
trimethoxy- and dillapiol-derived compounds 9cd and 9cf were
less active than the parent GVA (9cg). Compounds 9cb and
9cc were inactive in human cancer cells and showed only
marginal activity in the sea urchin embryo assay. However, the
sea urchin embryos were more sensitive to isoflavones 9ca and
9ce than the cancer cells.
In summary, GVA (9cg) and its analogues were synthesized
via a scaleable six-step reaction sequence. The synthetic route
started with readily available allylpolyalkoxybenzenes 1, which
were isolated from dill and parsley seed essential oils. The
parent GVA (9cg) and its analogues 9cd and 9cf were found to
be promising antimitotic microtubule destabilizing agents with
low toxicity against human nonmalignant cells.
+
(100), 289 [M] (25), 288 (98), 287 (14), 275 (22), 273 (22), 272
(10), 260 (13), 258 (13), 257 (11), 243 (7), 241 (5), 93 (6); anal. C
41.55; H 3.14; Br 27.64%, calcd for C10H9BrO5, C 41.67; H 3.17; Br
27.55%.
6-Bromo-4,7-dimethoxy-2H-1,3-benzodioxole-5-carbaldehyde
(3g): 4.16 g, 72% yield; colorless crystals; mp 112−113 °C (EtOH)
(lit.28 94−95 °C); H NMR (DMSO-d6) δ 10.10 (1H, s, CH(O)),
1
6.23 (2H, s, OCH2O), 3.92 (3H, s, OCH3-6), 3.87 (3H, s, OCH3-3);
EIMS m/z 290 [M + 1] (4), 288 (4), 133 (14), 131 (14), 107 (13), 95
(19), 93 (32), 92 (31), 77 (100), 76 (25); anal. C 41.55; H 3.14; Br
27.64%, calcd for C10H9BrO5, C 41.63; H 3.17; Br 27.57%.
General Procedure for the Synthesis of 2-Bromo Alcohols
4.29,30 A solution of 2-bromobenzaldehyde 3 (0.1 mmol) in THF
(140 mL) was added dropwise to MeMgBr (0.15 mol) in ether (50
mL). The reaction mixture was stirred for 2 h, evaporated to ca. 150
mL, and gently poured into aqueous NH4CI with ice (300 g). The
organic layer was separated, and the aqueous layer was extracted with
Et2O (200 mL). The combined organic extracts were washed with
H2O and brine, dried over anhydrous Na2SO4, and concentrated to
afford crude 2-bromo alcohols 4 used for the next step without further
purification.
1-(2-Bromo-4,5-dimethoxyphenyl)ethan-1-ol (4c): 16.98 g, 65%
yield; colorless crystals; mp 59−60 °C, n-hexane (lit.29 60−62 °C).
EXPERIMENTAL SECTION
■
1-(2-Bromo-3,4,5-trimethoxyphenyl)ethan-1-ol (4d): 2.1 g, 72%
30
General Experimental Procedures. Melting points were
measured on a Boetius melting point apparatus and were uncorrected.
1H NMR spectra were recorded on a Bruker DRX-500 (500.13 MHz)
1
1
yield; colorless oil; H NMR spectra as in lit. ; H NMR (CDCl3) δ
6.99 (1H, s, H-6), 5.22−5.25 (1H, dd,J = 6.3 Hz, CH), 3.87 (3H, s,
OCH3), 3.88 (3H, s, OCH3), 3.89 (3H, s, OCH3), 2.09 (1H, s, OH),
1.46 (3H, d, J = 6.3 Hz, CH3).
instrument. Chemical shifts are reported in parts per million (ppm)
and referenced to the appropriate NMR solvent peak(s). 2D NMR
experiments {1H−1H} NOESY, {1H−13C} HMBC-qs, and {1H−13C}
HSQC were used where necessary in assigning NMR spectra. Spin−
spin coupling constants (J) are reported in hertz (Hz). 13C NMR
spectra were recorded on a Bruker DRX-500 (75.47 MHz) instrument.
Chemical shifts are reported in ppm and referenced to the appropriate
solvent peak(s) and were assigned C, CH, CH2, and CH3 as
determined using HSQC and HMBC experiments. NMR spectra
(Supporting Information) were produced using original software
designed at the N. D. Zelinsky Institute of Organic Chemistry RAS
(Moscow, Russian Federation) (http://nmr.ioc.ac.ru:8080/SDF2PDF.
kl1). Low-resolution mass spectra (m/z) were recorded on a Finnigan
MAT/INCOS 50 mass spectrometer at 70 eV using direct probe
injection. Elemental analysis was performed on the automated
PerkinElmer 2400 CHN microanalyzer. Flash chromatography was
carried out on silica gel (Acros 0.035−0.070 mm, 60 Å). TLC was
performed on Merck 60 F254 plates.
Nonanhydrous solvents and reagents were purchased at the highest
commercial quality and used as received. The starting materials, 4-
methoxybenzaldehyde (2a), 3-methoxybenzaldehyde (2b), 3,4-dime-
thoxybenzaldehyde (2c), 3,4,5-trimethoxybenzaldehyde (2d), and 2-
bromo-4,5-dimethoxybenzaldehyde (3c), were purchased from Acros
Organics (Belgium). Allylmethoxybenzenes 1d−h with 98−99% purity
were obtained by high-efficiency distillation of CO2 extracts of parsley
and dill seeds using a pilot plant device at the N. D. Zelinsky Institute
of Organic Chemistry RAS (Moscow, Russian Federation).22
General Procedure for the Synthesis of 2-Bromobenzalde-
hydes 3. Freshly recrystallized and free from bromine N-
bromosuccinimide (0.02 mol) was added to a solution of the
corresponding benzaldehyde 2 (0.02 mol) in DMF (30 mL) cooled
with ice water. The reaction mixture was stirred at room temperature
for 8 h and diluted with ice water (150 mL). The precipitate was
filtered, washed with water (3 × 10 mL), and dried to afford 2-
bromobenzaldehyde 3.
1-(2-Bromo-5,6-dimethoxy-3,4-methylendioxyphenyl)ethan-1-ol
(4f): 2.32 g, 76% yield; colorless oil; 1H NMR (CDCl3) δ 5.99 (2H, s,
OCH2O), 5.17 (1H, m, CH), 3.78 (1H, d, J = 6.2 Hz, OH), 3.83 (3H,
s, OCH3), 3.82 (3H, s, OCH3), 1.53 (3H, d, J = 6.7 Hz, CH3); anal. C
43.33; H 4.27; Br 26.16%, calcd for C11H13BrO5, C 43.40; H 4.32; Br
26.09%.
1-(6-Bromo-4,7-dimethoxy-2H-1,3-benzodioxol-5-yl)ethan-1-ol
(4g): 1.95 g, yield 64%; colorless crystals; mp 98−100 °C (EtOAc−n-
hexane, 1:3); 1H NMR (DMSO-d6) δ 6.09 (1H, s, OCH2O) and 6.07
(1H, s, OCH2O), 5.20 (1H, quint, J = 6.4, J = 6.6 Hz, CH), 4.89 (1H,
d, J = 6.0 Hz, OH), 3.83 (3H, s, OCH3), 3.82 (3H, s, OCH3), 1.40
(3H, d, J = 6.6 Hz, CH3); EIMS m/z 306 [M + 1] (31), 305 [M]+ (4),
304 (31), 292 (11), 291 (98), 290 (11), 289 (100), 276 (9), 274 (9),
261 (6), 259 (6), 210 (6), 182 (16); anal. C 43.30; H 4.29; Br 26.19%,
calcd for C11H13BrO5, C 43.40; H 4.32; Br 26.09%.
General Procedure for the Synthesis of Bromoacetophe-
nones 5. The crude bromo alcohol 4 (0.01 mol) dissolved in CH2Cl2
(20 mL) was added rapidly to a suspension of pyridinium
chlorochromate (3.23 g, 0.015 mol) in CH2Cl2 (10 mL). The
resulting mixture was stirred for 18 h, diluted with 15 mL of CH2Cl2,
and filtered through Celite. The residual gum was treated with boiling
CH2Cl2 (3 × 25 mL). The combined organic extracts were washed
with 15% aqueous KOH, aqueous NH4I, and brine. The extracts were
dried over anhydrous Na2SO4, concentrated, and crystallized or
separated by column chromatography (silica gel 0.035−0.070 mm, 60
Å) to yield the corresponding bromoacetophenones 5.
1-(2-Bromo-4,5-dimethoxyphenyl)ethan-1-one (5c): 16.8 g, 65%
yield; colorless crystals; mp 75−77 °C (EtOAc−n-hexane, 1:1) (lit.29
73−75 °C); 1H NMR (DMSO-d6) δ 7.28 (1H, s, H-6), 7.21 (1H, s, H-
3), 3.83 (3H, s, OCH3), 3.81 (3H, s, OCH3), 2.57 (3H, s, CH3); EIMS
m/z 260 [M + 1] (36), 258 (36), 245 (97), 243 (100), 108 (16), 93
(15), 77 (12).
1-(2-Bromo-3,4,5-trimethoxyphenyl)ethan-1-one (5d): 2.20 g,
76% yield; colorless crystals; mp 33−34 °C (Et2O−n-hexane) (lit.30
1
2-Bromo-3,4,5-trimethoxybenzaldehyde (3d): 4.73 g, 86% yield;
colorless crystals; mp 68−70 °C (50% EtOH) (lit.27 70.5−71.5 °C);
1H NMR (DMSO-d6) δ 10.17 (1H, s, CHO), 7.28 (1H, s, H-6), 3.90
34.5−35 °C); H NMR (CDCl3) δ 6.82 (1H, s, H-6), 33.92 (3H, s,
OCH3), 3.90 (3H, s, OCH3), 3.88 (3H, s, OCH3), 2.65 (3H, s, CH3);
EIMS m/z 290 [M + 1] (27), 288 (27), 275 (43), 273 (43), 230 (10),
123 (10), 93 (20), 77 (21), 43 (100).
(3H, s, OCH3), 3.88 (3H, s, OCH3), 3.83 (3H, s, OCH3).
E
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11-(4-Bromo-6,7-dimethoxy-2H-1,3-benzodioxol-5-yl)ethan-1-
Hz, H-2), 7.24 (1H, s, H-6′), 7.18 (1H, d, J = 16.0 Hz, H-3), 7.16 (1H,
s, H-3′), 7.10 (2H, s, H-4″,6″), 6.07 (2H, s, OCH2O), 3.85 (6H, s,
OCH3), 3.80 (3H, s, OCH3); EIMS m/z 423 [M + 2] (20), 422 [M +
1] (100), 421 [M]+ (31), 420 (97), 419 (10), 391 (16), 389 (16), 342
(9), 341 (29), 313 (8), 311 (12), 298 (7), 284 (5), 283 (28), 245 (11),
243 (11), 205 (9); anal. C 54.17; H 4.07; Br 18.97%, calcd for
C19H17BrO6, C 54.25; H 4.09; Br 19.07%.
(2E)-1-(2-Bromo-4,5-dimethoxyphenyl)-3-(6,7-dimethoxy-2H-1,3-
benzodioxol-5-yl)prop-2-en-1-one (6cf): 4.01 g, 89% yield; yellow
solid; mp 124−126 °C; 1H NMR (DMSO-d6) δ 7.58 (1H, d, J = 16.1
Hz, H-2), 7.25 (1H, s, H-6′), 7.23 (1H, s, H-3′), 7.17 (1H, d, J = 16.1
Hz, H-3), 7.13 (1H, s, H-4″), 6.08 (2H, s, OCH2O), 3.94 (3H, s,
OCH3-6″), 3.85 (3H, s, OCH3), 3.80 (3H, s, OCH3), 3.70 (3H, s,
OCH3-7″); EIMS m/z 452 [M + 1] (13), 450 (13), 422 (21), 421
(100), 420 (21), 419 (97), 405 (5), 404 (5), 245 (6), 243 (6), 209
(9); anal. C 53.23; H 4.24; Br 17.71%, calcd for C20H19BrO7, C 53.27;
H 4.27; Br 17.66%.
(2E)-1-(2-Bromo-4,5-dimethoxyphenyl)-3-(4,7-dimethoxy-2H-1,3-
benzodioxol-5-yl)prop-2-en-1-one (6cg): 3.88 g, 86% yield; yellow
solid; mp 158−161 °C; 1H NMR (DMSO-d6) δ 7.58 (1H, d, J = 16.0
Hz, H-2), 7.26 (1H, d, J = 16.0 Hz, H-3), 7.25 (1H, s, H-6′), 7.13 (1H,
s, H-3′), 7.11 (1H, s, H-6″), 6.10 (2H, s, OCH2O), 3.87 (3H, s,
OCH3), 3.85 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.80 (3H, s,
OCH3); EIMS m/z 452 [M + 1] (10), 450 (10), 422 (13), 421 (65),
420 (14), 419 (75), 306 (23), 292 (25), 245 (60), 243 (60), 235 (26),
220 (39), 206 (16), 205 (18), 192 (39), 191 (28), 181 (68), 177 (34),
175 (20), 165 (34), 164 (35), 157 (41), 149 (40), 147 (54), 135 (33),
121 (40), 119 (29), 108 (43), 107 (36), 106 (27), 105 (25), 93 (70),
92 (30), 91 (53), 90 (22), 79 (100), 78 (58), 77 (76), 75 (43); anal. C
53.23, H 4.24, Br 17.71%; calcd for C20H19BrO7; C 53.29; H 4.28; Br
17.67%.
1
one (5f): 2.27 g, 75% yield; colorless oil; H NMR (CDCl3) δ 6.02
(2H, s, OCH2O), 4.02 (3H, s, OCH3-6), 3.77 (3H, s, OCH3-5), 2.50
(3H, s, CH3); EIMS m/z 304 [M + 1] (29), 302 (29), 289 (53), 287
(53), 92 (18), 77 (15), 43 (100); anal. C 43.59; H 3.66; Br 26.36%,
calcd for C11H11BrO5, C 3.68; H 3.70; Br 26.27%.
1-(6-Bromo-4,7-dimethoxy-2H-1,3-benzodioxol-5-yl)ethan-1-one
(5g): 2.58 g, 85% yield; colorless crystals; mp 55−57 °C (EtOAc−n-
1
hexane, 1:1); H NMR (DMSO-d6) δ 6.14 (2H, s, OCH2O), 3.86
(6H, s, OCH3-3,6), 2.40 (3H, s, CH3); EIMS m/z 304 [M + 1] (51),
302 (51), 289 (100), 288 (11), 287 (100), 274 (13), 272 (13), 43
(19); anal. C 43.59; H 3.66; Br 26.36%, calcd for C11H11BrO5, C
43.65; H 3.68; Br 26.29%.
General Procedure for the Synthesis of Chalcones 6. NaOH
(1.2 g, 30 mmol) was added to a vigorously stirred solution containing
2-bromoacetophenones 5 (10 mmol) and benzaldehyde 2 (10 mmol)
in EtOH (30 mL) at 20 °C. The reaction mixture was stirred at room
temperature for 6 h, kept overnight, acidified with 10% HCl to pH 3−
4, and strirred for 1 h. The residue was filtered, washed with H2O (3 ×
20 mL), and crystallized from EtOAc to afford chalcones 6.
(2E)-1-(2-Bromo-4,5-dimethoxyphenyl)-3-(4-methoxyphenyl)-
prop-2-en-1-one (6ca): 3.13 g, 83% yield; light yellow solid; mp 171−
1
173 °C; H NMR (DMSO-d6) δ 7.73 (2H, d, J = 8.8 Hz, H-2″,6″),
7.41 (1H, d, J = 16.0 Hz, H-2), 7.24 (1H, s, H-6′), 7.14 (1H, d, J =
16.0 Hz, H-3), 7.11 (1H, s, H-3′), 7.00 (2H, d, J = 8.8 Hz, H-3″,5″),
3.85 (3H, s, OCH3), 3.81 (3H, s, OCH3), 3.80 (3H, s, OCH3); EIMS
m/z 379 [M + 2] (15), 378 [M + 1] (80), 377 [M]+ (30), 376 375
(14), 363 (12), 361 (12), 347 (13), 345 (13), 297 (34), 269 (11), 254
(14), 245 (19), 243 (19), 238 (14), 161 (100), 159 (10), 157 (10),
133 (45), 118 (23), 90 (27), 89 (30), 77 (29); anal. C 57.31; H 4.54;
Br 21.18%, calcd for C18H17BrO4, C 57.24; H 4.51; Br 21.25%.
(2E)-1-(2-Bromo-4,5-dimethoxyphenyl)-3-(3-methoxyphenyl)-
prop-2-en-1-one (6cb): 3.17 g, 84% yield; light yellow solid; mp 139−
141 °C; 1H NMR (DMSO-d6) δ 7.43 (1H, d, J = 16.0 Hz, H-2), 7.36−
7.34 (3H, m, H-2″,5″,6″), 7.31 (1H, d, J = 16.0 Hz, H-3), 7.26 (1H, s,
H-6′), 7.14 (1H, s, H-3′), 7.04−7.01 (1H, m, H-4″), 3.85, 3.81, 3.80
(9H, 3s, 3OCH3-4′,5′,3″); EIMS m/z 379 [M + 2] (13), 378 [M + 1]
(73), 377 [M]+ (34), 376 (73), 375 (20), 347 (36), 345 (36), 297
(44), 269 (71), 255 (19), 254 (47), 245 (48), 243 (50), 239 (25), 238
(57), 211 (21), 161 (100), 159 (26), 157 (26), 152 (25), 139 (22),
133 (46), 118 (68), 103 (23), 102 (33), 90 (54), 89 (44), 77 (53);
anal. C 57.31; H 4.54; Br 21.18%, calcd for C18H17BrO4, C 57.22; H
4.50; Br 21.27%.
(2E)-1-(2-Bromo-4,5-dimethoxyphenyl)-3-(2,3,4,5-
tetramethoxyphenyl)prop-2-en-1-one (6ch): 3.88 g, 83% yield;
1
yellow solid; mp 94−96 °C; H NMR (DMSO-d6) δ 7.58 (1H, d, J
= 16.1 Hz, H-2), 7.29 (1H, d, J = 16.1 Hz, H-3), 7.26 (1H, s, H-6′),
7.22 (1H, s, H-3′), 7.12 (1H, s, H-6″), 3.86 (3H, s, OCH3), 3.84 (3H,
s, OCH3), 3.82 (3H, s, OCH3), 3.81 (3H, s, OCH3), 3.80 (3H, s,
OCH3), 3.71 (3H, s, OCH3); EIMS m/z 468 [M + 1] (11), 466 (11),
438 (23), 437 (100), 436 (31), 435 (98), 245 (26), 243 (31), 215
(10), 193 (10), 172 (14), 165 (13), 164 (17), 157 (12), 149 (6), 93
(11); anal. C 53.97; H 4.96; Br 17.10%, calcd for C21H23BrO7, C
53.84; H 4.90; Br 17.19%.
(2E)-1-(2-Bromo-3, 4, 5-trimethoxyphenyl)-3-(3, 4-
dimethoxyphenyl)prop-2-en-1-one (6dc): 2.71 g, 62% yield; light
yellow solid; mp 94−96 °C; 1H NMR (DMSO-d6) δ 7.39 (1H, d, J =
1.9 Hz, H-2″), 7.35 (1H, d, J = 16.0 Hz, H-2), 7.30 (1H, dd, J = 8.4
Hz, J = 1.9 Hz, H-6″), 7.11 (1H, d, J = 16.0 Hz, H-3), 7.00 (1H, d, J =
8.4 Hz, H-5″), 6.95 (1H, s, H-6′), 3.84 (9H, s, OCH3), 3.80 (6H, s,
OCH3); EIMS m/z 439 [M + 2] (13), 438 [M + 1] (69), 437 [M]+
(20), 436 (69), 423 (14), 421 (14), 407 (9), 405 (9), 358 (7), 357
(24), 315 (11), 299 (20), 298 (32), 191 (100), 163 (20), 157 (23),
149 (8), 133 (11), 132 (10), 119 (12), 118 (10), 91 (13), 77 (19);
anal. C 54.93; H 4.84; Br 18.27%, calcd for C20H21BrO6, C 54.87; H
4.83; Br 18.32%.
(2E)-1-(4-Bromo-6,7-dimethoxy-2H-1,3-benzodioxol-5-yl)-3-(3,4-
dimethoxyphenyl)prop-2-en-1-one (6fc): 3.88 g, 86% yield; light
yellow solid; mp 146−148 °C; 1H NMR (DMSO-d6) δ 7.37 (1H, d, J
= 2.0 Hz, H-2″), 7.28 (1H, dd, J = 8.4 Hz, J = 2.0 Hz, H-6″), 7.24 (1H,
d, J = 16.1 Hz, H-2), 6.99 (1H, d, J = 16.1 Hz, H-3), 6.98 (1H, d, J =
8.4 Hz, H-5″), 6.17 (2H, s, OCH2O), 3.98 (3H, s, OCH3-6′), 3.81
(3H, s, OCH3), 3.80 (3H, s, OCH3),3.63 (3H, s, OCH3-7′); EIMS m/
z 453 [M + 2] (10), 452 [M + 1] (47), 451 [M]+ (10), 450 (47), 435
(7), 371 (11), 328 (13), 313 (24), 312 (58), 285 (18), 191 (100), 164
(41), 163 (32), 151 (46), 148 (19), 147 (18), 133 (27), 132 (19), 119
(25), 118 (20), 92 (29), 91 (32), 77 (50); anal. C 53.23; H 4.24; Br
17.71%, calcd for C20H19BrO7, C 53.28; H 4.27; Br 17.65%.
(2E)-1-(6-Bromo-7-methoxy-4-methyl-2H-1,3-benzodioxol-5-yl)-
3-(3,4-dimethoxyphenyl)prop-2-en-1-one (6gc): 3.52 g, 78% yield;
yellow solid; mp 125−127 °C; 1H NMR (DMSO-d6) δ 7.37 (1H, d, J
= 2.0 Hz, H-2″), 7.28 (1H, dd, J = 8.4 Hz, J = 2.0 Hz, H-6″), 7.23 (1H,
(2E)-1-(2-Bromo-4,5-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-
prop-2-en-1-one (6cc): 3.51g, 86% yield; light yellow solid; mp 146−
1
148 °C; H NMR (DMSO-d6) δ 7.39 (1H, s, H-2″), 7.36 (1H, d, J =
16.0 Hz, H-2), 7.30 (1H, d, J = 8.4 Hz, H-6″), 7.24 (1H, s, H-6′), 7.17
(1H, d, J = 16.0 Hz, H-3), 7.09 (1H, s, H-3′), 7.00 (1H, d, J = 8.4 Hz,
H-5″), 3.85 (3H, s, OCH3), 3.81 (6H, s, OCH3), 3.80 (3H, s, OCH3);
EIMS m/z 409 [M + 2] (21), 408 [M + 1] (100), 407 [M]+ (37), 406
(98), 393 (15), 391 (15), 377 (14), 375 (14), 327 (26), 299 (15), 284
(15), 269 (14), 268 (29), 245 (15), 243 (15), 241 (15), 225 (10), 191
(79), 165 (15), 163 (23), 159 (12), 157 (12), 152 (10), 150 (20), 139
(22), 119 (21), 118 (16), 105 (15), 102 (13), 93 (15), 91 (26), 89
(23), 77 (41); anal. C 56.04; H 4.70; Br 19.62%, calcd for C19H19BrO5,
C 55.98; H 4.67; Br 19.67%.
( 2E )-1 -( 2-Br omo - 4, 5 -d i metho xyp heny l) -3 -(3 , 4, 5 -
trimethoxyphenyl)prop-2-en-1-one (6cd): 3.63 g, 83% yield; yellow
solid; mp 148−150 °C; 1H NMR (DMSO-d6) δ 7.35 (1H, d, J = 16.0
Hz, H-2), 7.25 (1H, s, H-6′), 7.24 (1H, d, J = 16.0 Hz, H-3), 7.11 (2H,
s, H-2″,6″), 7.09 (1H, s, H-3′), 3.85 (3H, s, OCH3), 3.82 (6H, s,
OCH3), 3.80 (3H, s, OCH3), 3.70 (3H, s, OCH3); EIMS m/z 439 [M
+ 2] (21), 438 [M + 1] (97), 437 [M]+ (28), 436 (100), 423 (14), 421
(14), 407 (30), 405 (30), 358 (18), 357 (62), 314 (10), 299 (22), 298
(40), 283 (11), 271 (15), 245 (10), 243 (10), 221 (11), 157 (5), 149
(5); anal. C 54.93; H 4.84; Br 18.27%, calcd for C20H21BrO6, C 54.84;
H 4.80; Br 18.33%.
(2E)-1-(2-Bromo-4,5-dimethoxyphenyl)-3-(7-methoxy-2H-1,3-
benzodioxol-5-yl)prop-2-en-1-one (6ce): 2.62 g, 62% yield; yellow
solid; mp 179−181 °C; 1H NMR (DMSO-d6) δ 7.35 (1H, d, J = 16.0
F
DOI: 10.1021/acs.jnatprod.6b00173
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
d, J = 16.1 Hz, H-2), 6.98 (1H, d, J = 8.4 Hz, H-5″), 6.97 (1H, d, J =
16.1 Hz, H-3), 6.17 (2H, s, OCH2O), 3.90 (3H, s, OCH3), 3.81 (3H,
3s, OCH3), 3.80 (3H, s, OCH3), 3.77 (3H, s, OCH3); EIMS m/z 453
[M + 2] (22), 452 [M + 1] (96), 451 [M]+ (31), 450 (96), 437 (10),
435 (10), 421 (10), 419 (10), 371 (45), 341 (17), 328 (22), 313 (58),
312 (79), 287 (16), 285 (16), 191 (100), 164 (27), 163 (21), 151
(15), 148 (10), 147 (10), 133 (11), 132 (8), 119 (8), 118 (7), 77 (7);
anal. C 53.23; H 4.24; Br 17.71%, calcd for C20H19BrO7, C 53.30; H
4.27; Br 17.63%.
OCH3); EIMS m/z 439 [M + 2] (1), 438 [M + 1] (8), 437 [M]+ (1),
436 (8), 300 (24), 299 (16), 285 (6), 245 (96), 244 (10), 243 (100),
215 (10), 193 (19), 179 (21), 165 (42), 157 (11), 149 (25), 135 (11),
93 (12), 79 (12), 77 (15); anal. C 52.19, H 3.92, Br 18.27%, calcd for
C19H17BrO7, C 52.27; H 3.96; Br 18.21%.
(2-Bromo-4,5-dimethoxyphenyl)[3-(6,7-dimethoxy-1,3-benzo-
dioxol-5-yl)oxiran-2-yl]methanone (7cf): 1.66 g, 89% yield; yellow
1
solid; mp 179−181 °C; H NMR (DMSO-d6) δ 7.32 (1H, s, H-6′),
7.27 (1H, s, H-3′), 6.42 (1H, s, H-4″), 6.02 (1H, s, OCH2O), 6.01
(1H, s, OCH2O), 4.44 (1H, d, J = 1.9 Hz, H-2), 4.15 (1H, d, J = 1.9
Hz, H-3), 3.94 (3H, s, OCH3-6″), 3.85 (3H, s, OCH3), 3.80 (3H, s,
OCH3), 3.71 (3H, s, OCH3-7″); EIMS m/z 469 [M + 2] (8), 468 [M
+ 1] (36), 467 [M]+ (8), 466 (36), 421 (21), 419 (21), 411 (18), 409
(18), 245 (97), 244 (11), 243 (100), 223 (28), 195 (68), 180 (19),
165 (14), 157 (11), 149 (25), 121 (11), 93 (16), 79 (16), 77 (15);
anal. C 51.41; H 4.10; Br 17.10%; calcd for C20H19BrO8, C 51.44; H
4.12; Br 17.02%.
General Procedure for the Synthesis of Epoxides 7.31 H2O2
(30%, 0.6 mL) was added to a vigorously stirred suspension of
chalcone 6 (4 mmol) in EtOH (15 mL) and NaOH (1N, 1.9 mL) at
room temperature. The reaction mixture was stirred at 30 °C for 3 h
and left for 24 h at room temperature; then the second portion of
NaOH (1N, 1.9 mL) and H2O2 (30%, 0.6 mL) was added, and the
mixture was stirred for 6 h at room temperature. The third portion of
NaOH (1N, 1.9 mL) and H2O2 (30%, 0.6 mL) was added and stirred
for 24 h at room temperature. The residue was filtered, washed with
EtOH and H2O, and dried at reduced pressure to afford
epoxychalcones 7.
(2-Bromo-4,5-dimethoxyphenyl)[3-(4,7-dimethoxy-1,3-benzo-
dioxol-5-yl)oxiran-2-yl]methanone (7cg): 1.44 g, 77% yield; yellow
1
solid; mp 152−155 °C; H NMR (DMSO-d6) δ 7.30 (1H, s, H-6′),
7.27 (1H, s, H-3′), 6.44 (1H, s, H-6″), 6.05 (1H, s, OCH2O), 6.04
(1H, s, OCH2O), 4.45 (1H, d, J = 1.9 Hz, H-2), 4.16 (1H, d, J = 1.9
Hz, H-3), 3.85 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.81 (3H, s,
OCH3), 3.76 (3H, s, OCH3); EIMS m/z 469 [M + 2] (1), 468 [M +
1] (11), 467 [M]+ (1), 466 (11), 421 (3), 419 (3), 330 (12), 245 (97),
244 (10), 243 (100), 223 (45), 195 (72), 180 (11), 165 (14), 157 (8),
149 (20), 135 (15), 121 (10), 93 (28), 79 (20), 77 (24); anal. C 51.41;
H 4.10; Br 17.10%, calcd for C20H19BrO8, C 51.46; H 4.11; Br 17.04%.
(2-Bromo-3,4,5-trimethoxyphenyl)[3-(3,4-dimethoxyphenyl)-
oxiran-2-yl]methanone (7dc): 1.41 g, 78% yield; light yellow solid;
(2-Bromo-4,5-dimethoxyphenyl)[3-(4-methoxyphenyl)oxiran-2-
yl]methanone (7ca): 1.23 g, 78% yield; light yellow solid; mp 121−
1
122 °C; H NMR (DMSO-d6) δ 7.36 (2H, d, J = 8.7 Hz, H-2″,6″),
7.27 (1H, s, H-6′), 7.25 (1H, s, H-3′), 6.96 (2H, d, J = 8.7 Hz, H-
3″,5″), 4.45 (1H, d, J = 1.8 Hz, H-2), 4.08 (1H, d, J = 1.6 Hz, H-3),
3.85 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.77 (3H, s, OCH3); EIMS
m/z 395 [M + 2] (4), 394 [M + 1] (18), 393 [M]+ (4), 392 (18), 337
(31), 335 (31), 246 (10), 245 (97), 244 (10), 243 (100), 159 (12),
157 (17), 135 (28), 121 (88), 119 (16), 93 (24), 91 (38), 77 (61);
anal. C 54.98; H 4.36; Br 20.32%, calcd for C18H17BrO5; C 54.92; H
4.32; Br 20.38%.
1
mp 128−130 °C; H NMR (DMSO-d6) δ 7.12 (1H, s, H-6′), 7.00
(1H, dd, J = 1.7 Hz, J = 8.3 Hz, H-6″), 6.96 (1H, d, J = 8.3 Hz, H-5″),
6.94 (1H, d, J = 1.7 Hz, H-2″), 4.40 (1H, d, J = 1.6 Hz, H-2), 4.10
(1H, d, J = 1.6 Hz, H-3), 3.85 (3H, s, OCH3), 3.83 (3H, s, OCH3),
3.81 (3H, s, OCH3), 3.76 (6H, s, OCH3); EIMS m/z 455 [M + 2] (2),
454 [M + 1] (18), 453 [M]+ (2), 452 (18), 397 (14), 395 (15), 345
(22), 316 (22), 275 (96), 273 (100), 230 (21), 179 (24), 165 (22),
151 (83), 149 (13), 135 (12), 121 (11), 119 (5), 107 (20), 93 (18), 79
(21), 77 (36); anal. C 53.00; H 4.67; Br 17.63%, calcd for C20H21BrO7,
C 52.88; H 4.62; Br 17.70%.
(2-Bromo-4,5-dimethoxyphenyl)[3-(3-methoxyphenyl)oxiran-2-
yl]methanone (7cb): 1.35 g, 86% yield; light yellow solid; mp 133−
1
136 °C; H NMR (DMSO-d6) δ 7.32 (1H, t, J = 7.9 Hz, H-5″), 7.28
(1H, s, H-6′), 7.26 (1H, s, H-3′), 7.01 (1H, d, J = 7.9 Hz, H-6″), 6.97
(1H, br s, H-2″), 6.94 (1H, dd, J = 8.2 Hz, J = 2.4 Hz, H-4″), 4.46
(1H, d, J = 1.6 Hz, H-2), 4.13 (1H, d, J = 1.6 Hz, H-3), 3.85 (3H, s,
OCH3), 3.79 (3H, s, OCH3), 3.76 (3H, s, OCH3); EIMS m/z 394 [M
+ 1] (20), 392 (20), 313 (6), 256 (21), 246 (10), 245 (96), 244 (10),
243 (100), 149 (19), 135 (10), 121 (10), 119 (5), 93 (6), 91 (10), 77
(14); anal. C 54.98; H 4.36; Br 20.32%, calcd for C18H17BrO5, C
54.90; H 4.33; Br 20.40%.
(4-Bromo-6,7-dimethoxy-2H-1,3-benzodioxol-5-yl)[3-(3,4-
dimethoxyphenyl)oxiran-2-yl]methanone (7fc): 1.46 g, 78% yield;
1
light yellow solid; mp 89−91 °C; H NMR (DMSO-d6) δ 6.98 (1H,
(2-Bromo-4,5-dimethoxyphenyl)[3-(3,4-dimethoxyphenyl)oxiran-
dd, J = 8.3 Hz, J = 1.9 Hz, H-6″), 6.94 (1H, d, J = 8.3 Hz, H-5″), 6.88
(1H, d, J = 1.9 Hz, H-2″), 6.17 (1H, s, OCH2O), 6.16 (1H, s,
OCH2O), 4.23 (1H, d, J = 1.8 Hz, H-2), 4.00 (1H, d, J = 1.8 Hz, H-3),
3.94 (3H, s, OCH3-6′), 3.75 (6H, s, OCH3-3″,4″), 3.70 (3H, s, OCH3-
7′); EIMS m/z 469 [M + 2] (7), 468 [M + 1] (34), 467 [M]+ (7), 466
(34), 412 (11), 411 (56), 410 (11), 409 (56), 330 (29), 329 (28), 289
(96), 287 (100), 285 (19), 192 (46), 165 (25), 151 (96), 135 (14),
133 (16), 121 (11), 119 (11), 107 (31), 105 (17), 93 (18), 92 (40), 91
(18), 79 (33), 77 (62); anal. C 51.41; H 4.10; Br 17.10%, calcd for
C20H19BrO8, C 51.47; H 4.13; Br 17.00%.
2-yl]methanone (7cc): 1.10 g, 65% yield; light yellow solid; mp 108−
1
110 °C; H NMR (DMSO-d6) δ 7.27 (1H, s, H-6′), 7.26 (1H, s, H-
3′), 7.00 (1H, d, J = 8.2 Hz, H-6″), 6.96 (1H, d, J = 8.2 Hz, H-5″),
6.96 (1H, br s, H-2″), 4.48 (1H, d, J = 1.5 Hz, H-2), 4.07 (1H, d, J =
1.5 Hz, H-3), 3.85 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.76 (3H, s,
OCH3); EIMS m/z 425 [M + 2] (4), 424 [M + 1] (20), 423 [M]+ (4),
422 (20), 367 (19), 365 (19), 286 (13), 245 (97), 244 (12), 243
(100), 241 (11), 179 (13), 165 (24), 151 (78), 149 (39), 121 (13),
119 (15), 107 (24), 93 (22), 92 (20), 79 (27), 77 (37); anal. C 53.92;
H 4.52; Br 18.88%, calcd for C19H19BrO6, C 53.87; H 4.50; Br 18.94%.
(2-Bromo-4,5-dimethoxyphenyl)[3-(3,4,5-trimethoxyphenyl)-
oxiran-2-yl]methanone (7cd): 1.41 g, 78% yield; yellow solid; mp
144−146 °C; 1H NMR (DMSO-d6) δ 7.27 (1H, s, H-6′), 7.26 (1H, s,
H-3′), 6.74 (2H, s, H-2″,6″), 4.48 (1H, d, J = 1.6 Hz, H-2), 4.09 (1H,
d, J = 1.6 Hz, H-3), 3.85 (3H, s, OCH3), 3.80 (3H, s, OCH3), 3.78
(6H, 3s, OCH3), 3.66 (3H, s, OCH3); EIMS m/z 455 [M + 2] (2),
454 [M + 1] (12), 453 [M] + (2), 452 (12), 316 (15), 315 (13), 285
(14), 245 (95), 244 (11), 243 (100), 215 (10), 209 (34), 181 (66),
165 (18), 157 (11), 149 (25), 135 (10), 108 (24), 93 (13), 79 (14), 77
(11); anal. C 53.00; H 4.67; Br 17.63%, calcd for C20H21BrO7, C
52.87; H 4.63; Br 17.73%.
(6-Bromo-4,7-dimethoxy-2H-1,3-benzodioxol-5-yl)[3-(3,4-
dimethoxyphenyl)oxiran-2-yl]methanone (7gc): 1.61 g, 86% yield;
yellow solid; mp 124−126 °C; 1H NMR (DMSO-d6) δ 6.98 (1H, dd, J
= 8.3 Hz, J = 1.8 Hz, H-6″), 6.94 (1H, d, J = 8.3 Hz, H-5″), 6.87 (1H,
d, J = 1.8 Hz, H-2″), 6.16 (1H, s, OCH2O), 6.15 (1H, s, OCH2O),
4.20 (1H, d, J = 1.8 Hz, H-2), 3.99 (1H, d, J = 1.8 Hz, H-3), 3.76 (6H,
s, OCH3), 3.75 (6H, s, OCH3); EIMS m/z 469 [M + 2] (7), 468 [M +
1] (32), 467 [M]+ (7), 466 (32), 411 (25), 409 (25), 387 (17), 330
(56), 329 (48), 289 (97), 287 (100), 285 (15), 274 (16), 272 (16),
262 (15), 260 (15), 206 (29), 192 (16), 178 (18), 165 (22), 151 (31),
135 (10), 133 (9), 107 (14), 92 (10), 77 (12); anal. C 51.41; H 4.10;
Br 17.10%, calcd for C20H19BrO8, C 51.49; H 4.12; Br 16.97%.
General Rearrangement Procedure for the Synthesis of 3-
Oxo-2,3-diphenyl Propanals (8).32 BF3·OEt2 (0.39 mL, 3 mmol)
was added dropwise to an ice-cooled solution of chalcone epoxide 7 (3
mmol) in absolute CH2Cl2 (15 mL) in a flask filled with Ar and stirred
for 3 h at 20 °C. The reaction was then quenched with a 5% aqueous
(2-Bromo-4,5-dimethoxyphenyl)[3-(7-methoxy-1,3-benzodioxol-
5-yl)oxiran-2-yl]methanone (7ce): 1.21 g, 69% yield; yellow solid; mp
148−150 °C; 1H NMR (DMSO-d6) δ 7.27 (1H, s, H-6′), 7.26 (1H, s,
H-3′), 6.78 (1H, d, J = 1.3 Hz, H-4″), 6.65 (1H, d, J = 1.3 Hz, H-6″),
6.01 (2H, br s, OCH2O), 4.47 (1H, d, J = 1.9 Hz, H-2), 4.07 (1H, d, J
= 1.9 Hz, H-3), 3.85 (3H, s, OCH3), 3.83 (3H, s, OCH3), 3.80 (3H, s,
G
DOI: 10.1021/acs.jnatprod.6b00173
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Journal of Natural Products
Article
NaHCO3 solution (15 mL) and extracted with CHCl3 (2 × 15 mL).
The organic layer was washed with H2O (2 × 15 mL) and dried over
anhydrous Na2SO4. Evaporation of the solvent afforded the crude
ketoaldehydes 8 as oils. The products were roughly purified by column
chromatography from CH2Cl2 extract (EtOAc−n-hexane, 1:4, Rf =
0.6). Yields: 8ca (92%), 8cb (89%), 8cc (86%), 8cd (86%), 8ce
(92%), 8cf (95%), 8cg (21%), 8ch (55%), 8dc (95%), 8fc (92%), 8gc
6,7-Dimethoxy-3-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
1
(9cd): 0.71 g, 38% yield; yellowish solid; mp 172−174 °C; H NMR
(CDCl3) δ 7.99 (1H, s, H-2), 7.64 (1H, s, H-5), 6.90 (1H, s, H-8),
6.82 (2H, s, H-2′,6′), 4.01 (3H, s, OCH3), 3.99 (3H, s, OCH3), 3.91
(6H, s, OCH3-3′,5′), 3.89 (3H, s, OCH3-4′); 13C NMR (CDCl3) δ
175.3, 154.4, 153.2, 152.3, 152.2, 147.8, 138.1, 127.6, 124.5, 117.8,
106.3, 104.7, 99.5, 60.8, 56.4, 56.3, 56.2; EIMS m/z 373 [M + 1] (25),
372 [M]+ (100), 358 (10), 357 (48), 329 (10), 271 (20), 171 (14),
149 (17), 134 (10); anal. C 64.51; H 5.41%, calcd for C20H20O7, C
64.42; H 5.36%.
1
(88%). The H NMR spectra of these compounds were complicated
due to keto−enol tautomerism and the presence of some impurities.
The NMR data of compound 8ch are shown below. Compounds 8
were used for the next step without further purification.
3-(2-Bromo-4,5-dimethoxyphenyl)-3-oxo-2-(2,3,4,5-
tetramethoxyphenyl)propanal (8ch). During the epoxidation proce-
dure, chalcone 6ch spontaneously rearranged to ketoaldehyde 8ch.
6,7-Dimethoxy-3-(7-methoxy-1,3-benzodioxol-5-yl)-4H-chro-
men-4-one (9ce): 0.46 g, 26% yield; white solid; mp 198−201 °C; 1H
NMR (CDCl3) δ 7.94 (1H, s, H-2), 7.62 (1H, s, H-5), 6.88 (1H, s, H-
8), 6.84 (1H, s, H-6′), 6.72 (1H, s, H-4′), 6.00 (2H, s, OCH2O), 3.99
(6H, s, OCH3-6,7), 3.94 (3H, s, OCH3-7′); 13C NMR (CDCl3) δ
175.2, 154.4, 152.1, 148.8, 147.7, 143.5, 135.3, 126.2, 124.4, 117.8,
108.8, 104.8, 103.1, 101.5, 99.5, 56.6, 56.4, 56.3; EIMS m/z 357 [M +
1] (23), 356 [M]+ (100), 355 (21), 341 (1), 327 (3), 326 (2), 176
(5); anal. C 64.04; H 4.53%, calcd for C19H16O7, C 63.96; H 4.48%.
6,7-Dimethoxy-3-(6,7-dimethoxy-1,3-benzodioxol-5-yl)-4H-chro-
men-4-one (9cf): 0.46 g, 24% yield; yellow-greenish solid; mp 204−
1
0.96 g, 55% yield; yellow oil; H NMR (DMSO-d6) δ 9.82 (1H, s,
1
206 °C; H NMR (CDCl3) δ 7.93 (1H, s, H-2), 7.62 (1H, s, H-5),
CH(O)), 7.74 (1H, s, H-2), 7.26 (1H, s, H-6″), 7.12 (1H, s, H-3″),
6.26 (1H, s, H-6′), 3.86 (3H, s, OCH3), 3.78 (3H, s, OCH3), 3.77
(3H, s, OCH3), 3.76 (6H, s, OCH3), 3.53 (3H, s, OCH3); EIMS m/z
485 [M + 2] (9), 484 [M + 1] (39), 438 [M]+ (9), 482 (39), 456 (4),
454 (4), 453 (1), 451 (1), 246 (9), 245 (98), 244 (9), 243 (98), 238
(16), 211 (100), 196 (30), 181 (14), 165 (13), 157 (5), 153 (11), 135
(10), 93 (9); anal. C 52.19; H 4.80; Br 16.53%, calcd for C21H23BrO8,
C 52.29; H 4.84; Br 16.43%.
6.89 (1H, s, H-8), 6.56 (1H, s, H-4′), 5.96 (2H, s, OCH2O), 4.05 (3H,
s, OCH3-6′), 4.00 (3H, s, OCH3), 3.99 (3H, s, OCH3), 3.68 (3H, s,
OCH3-7′); 13C NMR (CDCl3) δ 175.4, 154.3, 153.8, 152.3, 147.6,
145.2, 144.6, 137.9, 137.7, 121.3, 118.3, 117.9, 104.9, 104.5, 101.5,
99.6, 61.2, 60.0, 56.4, 56.3; EIMS m/z 387 [M + 1] (23), 386 [M]+
(90), 371 (7), 357 (8), 356 (29), 355 (100), 343 (5), 340 (9), 313 (5),
181 (16), 178 (9); anal. C 62.18; H 4.70%, calcd for C20H18O8, C
62.06; H 4.65%.
General Procedure for the Synthesis of Isoflavones 9.19
A
6,7-Dimethoxy-3-(4,7-dimethoxy-1,3-benzodioxol-5-yl)-4H-chro-
men-4-one (9cg, glaziovianin A): 0.62 g, 32% yield; yellowish solid;
mp 139−141 °C; 1H NMR (CDCl3) δ 7.91 (1H, s, H-2), 7.62 (1H, s,
H-5), 6.89 (1H, s, H-8), 6.53 (1H, s, H-6′), 6.03 (2H, s, OCH2O),
4.00 (3H, s, OCH3), 3.99 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.86
(3H, s, OCH3); 13C NMR (CDCl3) δ 175.4, 154.3, 153.4, 152.3,
147.6, 139.1, 139.0, 137.0, 136.8, 121.7, 118.0, 117.8, 110.1, 104.9,
101.8, 99.5, 60.1, 56.8, 56.4, 56.3; EIMS m/z 387 [M + 1] (24), 386
[M]+ (100), 371 (7), 357 (7), 356 (12), 355 (49), 313 (10), 206 (12),
205 (15), 181 (50), 137 (10); anal. C 62.18; H 4.70%, calcd for
C20H18O8, C 62.08; H 4.66%.
6,7-Dimethoxy-3-(2,3,4,5-tetramethoxyphenyl)-4H-chromen-4-
one (9ch): 0.68 g, 34% yield; yellow solid; mp 204−206 °C; 1H NMR
(CDCl3) δ 7.61 (1H, s, H-2), 7.24 (1H, s, H-5), H-2′), 7.20 (1H, s, H-
8), 6.75 (1H, s, H-2′), 4.03 (3H, s, OCH3), 3.97 (3H, s, OCH3), 3.96
(3H, s, OCH3), 3.94 (3H, s, OCH3), 3.92 (3H, s, OCH3), 3.91 (3H, s,
OCH3); 13C NMR (CDCl3) δ 183.2, 163.0, 157.5, 149.5, 148.7, 147.8,
146.9, 146.6, 145.2, 120.9, 113.3, 109.1, 106.2, 104.2, 95.5, 62.2, 61.3,
61.2, 56.7, 56.5, 56.4; EIMS m/z 403 [M + 1] (10), 402 [M]+ (46),
373 (9), 372 (48), 371 (100), 356 (6), 355 (6), 341 (17), 313 (11),
230 (12), 186 (19), 149 (12), 137 (11), 136 (10), 93 (10); anal. C
62.68; H 5.51%, calcd for C21H22O8, C 62.57; H 5.46%.
mixture of ketoaldehyde 8 (5 mmol), CuI (95 mg, 0.5 mmol), K2CO3
(1.38 g, 10 mmol), and 2-picolinic acid (123 mg, 1 mmol) in dry DMF
(30 mL) in a flask filled with Ar was stirred at 135−140 °C for 8 h.
The mixture was separated between EtOAc (3 × 50 mL) and H2O
(100 mL). The organic layer was dried over anhydrous Na2SO4,
filtered, evaporated under vacuum, and purified by column
chromatography (EtOAc−n-hexane, 3:1, Rf = 0.3−0.4) to afford
target izoflavones 9.
6,7-Dimethoxy-3-(4-methoxyphenyl)-4H-chromen-4-one (9ca):
0.72 g, 46% yield; dark yellow solid; mp 174−176 °C; 1H NMR
(CDCl3) δ 7.94 (1H, s, H-2), 7.63 (1H, s, H-5), 7.51 (2H, d, J = 8.6
Hz, H-2′,6′), 6.97 (2H, d, J = 8.6 Hz, H-3′,5′), 6.88 (1H, s, H-8), 3.99
(6H, s, OCH3-6,7), 3.85 (3H, s, OCH3-4′); 13C NMR (CDCl3) δ
175.5, 159.5, 154.3, 152.2, 151.8, 147.6, 130.1, 124.4, 124.3, 117.9,
113.9, 104.8, 99.5, 56.4, 56.3, 55.3; EIMS m/z 313 [M + 1] (24), 312
[M]+ (100), 311 (33), 297 (8), 281 (5), 180 (15), 165 (7), 156 (6),
132 (7), 89 (7); anal. C 69.22; H 5.16%, calcd for C18H16O5; C 69.16;
H 5.12%.
6,7-Dimethoxy-3-(3-methoxyphenyl)-4H-chromen-4-one (9cb):
0.37 g, 24% yield; yellowish solid; mp 182−184 °C; 1H NMR
(CDCl3) δ 7.98 (1H, s, H-2), 7.64 (1H, s, H-5), 7.35 (1H, t, J = 8.0
Hz, H-5′), 7.19 (1H, t, J = 2.0 Hz, H-2′), 7.12 (1H, d, J = 7.6 Hz, H-
6′), 6.93 (1H, dd, J = 2.6 Hz, J = 8.2 Hz, H-4′), 6.89 (1H, s, H-8), 4.00
(3H, s, OCH3), 3.99 (3H, s, OCH3), 3.85, (3H, s, OCH3-3′); 13C
NMR (CDCl3) δ 175.2, 159.5, 154.4, 152.4, 152.2, 147.7, 133.5, 129.4,
124.5, 121.1, 117.9, 114.4, 114.0, 104.8, 99.5, 56.4, 56.3, 55.3; EIMS
m/z 313 [M + 1] (20), 312 [M]+ (100), 311 (87), 297 (8), 283 (14),
282 (20), 281 (9), 266 (5), 253 (5), 180 (10), 156 (9), 137 (13), 89
(7). anal. C 69.22; H 5.16%, calcd for C18H16O5, C 69.28; H 5.29%.
6,7-Dimethoxy-3-(3,4-dimethoxyphenyl)-4H-chromen-4-one
(9cc, II (Figure 1): 0.41 g, 24% yield; dark yellow solid; mp 172−174
3-(3,4-Dimethoxyphenyl)-6,7,8-trimethoxy-4H-chromen-4-one
1
(9dc): 0.70 g, 36% yield, beige-orange solid; mp 132−134 °C; H
NMR (CDCl3) δ 8.06 (1H, s, H-2), 7.47 (1H, s, H-5), 7.25 (1H, d, J =
2.0 Hz, H-2′), 7.06 (1H, dd, J = 2.0 Hz, J = 8.3 Hz, H-6′), 6.94 (1H, d,
J = 8.3 Hz, H-5′), 4.05 (3H, s, OCH3), 4.04 (3H, s, OCH3), 3.96 (3H,
s, OCH3), 3.93 (3H, s, OCH3), 3.92 (3H, s, OCH3); 13C NMR
(CDCl3) δ 175.6, 152.2, 151.3, 149.1, 148.7, 147.2, 145.8, 141.7, 124.6,
124.2, 120.9, 120.3, 112.5, 111.1, 100.3, 62.0, 61.4, 56.2, 55.9; EIMS
m/z 373 [M + 1] (29), 372 [M]+ (100), 371 (10), 358 (5), 357 (23),
329 (7), 327 (5), 326 (7), 186 (10), 171 (7), 157 (6), 119 (11), 91
(10); anal. C 64.51; H 5.41%, calcd for C20H20O7, C 64.41; H 5.36%.
7-(3,4-Dimethoxyphenyl)-4,9-dimethoxy-8H-[1,3]dioxolo[4,5-g]-
chromen-8-one (9gc, iso-glaziovianin A): 0.95 g, 49% yield; light
brown solid; mp 176−178 °C; 1H NMR (DMSO-d6) δ 7.15 (1H, s, H-
2), 6.93 (1H, d, J = 8.2 Hz, H-5′), 6.89 (1H, d, J = 2.0 Hz, H-2′), 6.81
(1H, dd, J = 2.0 Hz, J = 8.1 Hz, H-6′), 6.22 (2H, s, OCH2O), 3.89
(3H, s, OCH3-9), 3.77 (3H, s, OCH3), 3.73 (3H, s, OCH3), 3.25 (3H,
s, OCH3-4); 13C NMR (DMSO-d6) δ 157.2, 148.1, 147.8, 146.1,
1
°C; H NMR (CDCl3) δ 7.98 (1H, s, H-2), 7.64 (1H, s, H-5), 7.25
(1H, d, J = 2.0 Hz, H-2′), 7.06 (1H, dd, J = 2.0 Hz, J = 8.3 Hz, H-6′),
6.93 (1H, d, J = 8.3 Hz, H-5′), 6.89 (1H, s, H-8), 4.00 (3H, s, OCH3),
3.99 (3H, s, OCH3), 3.94 (3H, s, OCH3), 3.92 (3H, s, OCH3); 13C
NMR (CDCl3) δ 175.5, 154.4, 152.2, 152.0, 149.0, 148.7, 147.7, 124.8,
124.3, 120.9, 117.9, 112.5, 111.1, 104.8, 99.5, 56.4, 56.3, 55.9; EIMS
m/z 343 [M + 1] (19), 342 [M]+ (100), 327 (13), 299 (6), 296 (5),
256 (7), 171 (6), 156 (5), 119 (9), 91 (8); anal. C 66.66; H 5.30%,
calcd for C19H18O6, C 66.71; H 5.33%.
H
DOI: 10.1021/acs.jnatprod.6b00173
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
142.5, 140.4, 140.2, 134.3, 129.0, 127.2, 121.3, 117.1, 113.6, 110.9,
108.6, 103.4, 60.9, 60.0, 55.6; EIMS m/z 387 [M + 1] (22), 386 [M]+
(100), 371 (16), 358 (34), 357 (10), 356 (12), 344 (8), 343 (54), 328
(6), 193 (7), 135 (12), 128 (10), 127 (10); anal. C 62.18; H 4.70%,
calcd for C20H18O8, C 62.12; H 4.67%.
Preparation and Culture of Human PBMCs. Peripheral blood
mononuclear cells were obtained from healthy volunteers by the
standard Ficoll-Paque gradient density sedimentation procedure to
yield mononuclear cells. PBMCs were washed three times in RPMI-
1640 medium before further processing. The purified PBMCs (1 × 106
cells/mL) were seeded in RPMI 1640 medium supplemented with
heat-inactivated 10% fetal bovine serum, penicillin (100 units/mL),
and streptomycin (100 μg/mL) at 37 °C in a 5% CO2 humidified
atmosphere.
Cell Toxicity Assay. Cells were seeded in 96-well plates at a
density of 1 × 104 cells per well. Stock solutions of test compounds
were prepared in DMSO. Cells were treated for 48 h with compounds
9ca, 9cb, 9cc, 9cd, 9ce, 9ch, 9cf, 9cg, and 9gc at 0.01−100 μM or with
CA4P at 0.001−10 μM as a positive control (four wells per each
concentration value). DMSO (0.1%) served as a control. The number
of surviving cells was determined by the colorimetric CellTiter 96
AQueous One Solution cell proliferation assay (MTS).33 A 20 μL
amount of the CellTiter 96 AQueous One Solution reagent was added
to each well. The plate was incubated at 37 °C for 1−4 h under a 5%
CO2 humidified atmosphere. The absorbance was recorded at 490 nm
using a CLARIOstar microplate reader (BMG LABTECH, Ortenberg,
Germany). Experiments for all compounds were repeated three times.
IC50 values were determined by sigmoidal curve fitting using
GraphPad Prism software.
Phenotypic Sea Urchin Embryo Assay.15 Adult sea urchins,
Paracentrotus lividus L. (Echinidae), were collected from the
Mediterranean Sea on the Cyprus coast in March 2013 and December
2014 and kept in an aerated seawater tank. Gametes were obtained by
intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered
seawater and fertilized by adding drops of diluted sperm. Embryos
were cultured at room temperature under gentle agitation with a
motor-driven plastic paddle (60 rpm) in filtered seawater. The
embryos were observed with a Biolam light microscope (LOMO, St.
Petersburg, Russian Federation). For treatment with the test
compounds, 5 mL aliquots of embryo suspension were transferred
to six-well plates and incubated as a monolayer at a concentration up
to 2000 embryos/mL. Stock solutions of isoflavones 9 were prepared
in DMSO at 10 mM concentration followed by a 10-fold dilution with
96% EtOH. This procedure enhanced the solubility of the test
compounds in the salt-containing medium (seawater), as evidenced by
microscopic examination of the samples. The maximal tolerated
concentrations of DMSO and EtOH in the in vivo assay were
determined to be 0.05% and 1%, respectively. Higher concentrations
of either DMSO (>0.1%) or EtOH (>1%) caused nonspecific
alterations and retardation of the sea urchin embryo development
independent of the treatment stage. A stock solution of the reference
compound, combretastatin A4 disodium phosphate (OXiGENE), was
prepared as 5 mM in distilled H2O. The antimitotic activity was
assessed by exposing fertilized eggs (8−15 min postfertilization, 45−
55 min before the first mitotic cycle completion) to 2-fold decreasing
concentrations of the compound. Cleavage alteration and arrest were
clearly detected at 2.5−5.5 h after fertilization. The effects were
estimated quantitatively as an effective threshold concentration,
resulting in cleavage alteration and embryo death before hatching or
full mitotic arrest. At these concentrations, all tested compounds
caused 100% cleavage alteration and embryo death before hatching,
whereas at 2-fold lower concentrations the compounds failed to
produce any effect.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00173.
Physical and spectroscopic data of compounds 9ec, 9fc,
9ia, and 9ja; 1H NMR and 13C NMR spectra of
compounds 3d, 3f, 3g, 4d, 4f, 4g, 5c, 5d, 5f, 5g, 6−9, and
8cg-acetal (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +7 916 620 9584. Fax: +7 499 137 2966. E-mail: vs@
zelinsky.ru (V. V. Semenov).
■
For microtubule-destabilizing activity, the compounds were tested
on free-swimming blastulae just after hatching (8−9 h postfertiliza-
tion), which originated from the same embryo culture. Embryos were
observed until the four-arm pluteus stage (34−36 h postfertilization).
The specific changes in swimming pattern, namely, both spinning on
the bottom of the well and lack of forward movement, were
interpreted to be the result of the microtubule-destabilizing activity
of a molecule. The sea urchin embryo assay data are available free of
charge via the Internet at http://www.zelinsky.ru. Experiments with
the sea urchin embryos fulfill the requirements of biological ethics.
The artificial spawning does not cause animal death, embryos develop
outside the female organism, and both postspawned adult sea urchins
and the excess of intact embryos are returned to the sea, their natural
habitat.
Human Cancer Cell Culture. A549 human lung epithelial
carcinoma cells (CCL-185) and A375 human skin epithelial malignant
melanoma cells (CRL-1619) were cultured with Dulbecco’s modified
Eagle medium. DU145 human prostate epithelial carcinoma cells
derived from brain metastatic site (HTB-81) were cultured with
Eagle’s minimum essential medium. PC3 human prostate epithelial
adenocarcinoma grade IVcells, derived from bone metastatic site
(CRL-1435), were cultured with Ham’s F-12K (Kaighn’s) medium.
HCT116 human colon epithelial colorectal carcinoma cells (CCL-
247) and SK-OV-3 human ovary ascites epithelial adenocarcinoma
cells (HTB-77) were cultured with McCoy’s 5a modified medium.
MDA-MB-231 human mammary gland/breast adenocarcinoma cells,
derived from pleural effusion metastatic site (HTB-26), were cultured
with Leibovitz’s L-15 medium.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was carried out with the financial support of the
Ministry of Education and Science of Russian Federation
(Agreement No. 02.A03.21.0003 dated August 28, 2013) and
Russian Foundation for Basic Research (Grant No. 16-03-
00648 dated February 15, 2016). The authors greatly appreciate
the financial and technical support from Chemical Block Ltd.,
/www.chemblock.com/. The authors thanks Dr. Ju. A.
Strelenko from N. D. Zelinsky Institute of Organic Chemistry
RAS for the development of software for NMR spectra
presentation.
REFERENCES
■
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