This document describes the efficient synthesis of glaziovianin A (GVA) and related isoflavones starting from readily available plant metabolites. A six-step reaction sequence involving bromination, alkylation, oxidation, condensation, epoxidation, and cyclization produced GVA and various alkoxyphenyl derivatives. Both an in vivo sea urchin embryo assay and screening of human cancer cell lines showed that GVA and some derivatives have antimitotic effects by destabilizing microtubules. Structure-activity relationship studies found that certain substituents, such as a methylenedioxy or trimethoxy group, influenced the compounds' potency. GVA was generally the most active compound, inhibiting cancer cell
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Voor de Roermondse Karin Straus (45) is deze derde dinsdag in september een bijzondere dag. Het is haar laatste Prinsjesdag als Kamerlid. Ze vertrekt na zes jaar uit de Haagse politiek.
Kiezen we dit jaar voor chique met oma’s kristallen ballen of wordt het hysterisch hip met glitters en pastel? Wie onder de lawine van knipperende ledlampjes, glanzende pieken en 1001 verschillende kerstballen zoekt, vindt een fantastisch kerstverhaal. De geboorte van de Christ-mis-boom.
La Fundación Lilly quiere hacer un guiño a la conmemoración del V Centenario de Cervantes dedicando la XI Jornada MEDES a debatir sobre la salud de nuestro idioma como vehículo de transmisión de la ciencia: a echar una mirada a sus momentos más saludables, que los tuvo en el pasado, y a diagnosticar sus achaques más o menos crónicos, que los tiene en el presente. Pero, sobre todo, este encuentro anual ha de servir para
reflexionar sobre el porvenir de nuestro idioma e identificar los mejores remedios y prácticas que en el futuro incrementen el vigor del español como lengua de intercambio científico y comunicación del conocimiento biomédico.
La opinión personal y la perspectiva de un médico radiólogo en la certificación de hospitales y radiología, en los estándares para servicios auxiliares de diagnóstico y los estándares centrados en la gestión:
“El pez muere por su boca.”
PowToon is a cloud-based software for creating animated presentations and animated explainer videos. With PowToon you can create animated presentation tool. The creators of PowToon take a look at incumbent presentation tools like Power Point, focused only on a subset of its most widely used features, and then “ kicked them into high gear”.
S. pyogenes, its virulence, antibiotic, phytochemicalsUniversité Laval
Streptococcus pyogenes (group A β-hemolytic Streptococcus (GABHS)) is the common cause of acute bacterial pharyngitis also known as strep throat or sore throat. Recorded cases of GAS bacterial pharyngitis are 15-36% in children and 5-15% in adults. School aged children in seasons of spring and winter harbor the bacterium up to 20% whereas this rate is much lower in adults. On a global scale, over 616 million new cases of GAS pharyngitis occur every year. Not only is this bacterium responsible for hundred millions of pharyngitis cases but also approximately 10,000 to 15,000 cases of invasive GAS cases are reported annually in the United States, accounting for 10% to 13% mortality rate (http://www.cdc.gov/ncidod/dbmd/abcs/forinvasiveandnoninvasivediseaseincidence). Acute infections can lead to rheumatic fever and post-streptococcal glomerulonephritis (kidney inflammation), which distress children worldwide with disability and death, if antibiotic treatment fails or if the disease is left unattended. Rheumatic fever and rheumatic heart disease are known to be the leading causes of cardiovascular death during the first five decades of life in underdeveloped countries mainly concerning children. Tissues contributing to the GAS nosocomial infections are upper respiratory tract, skin, vaginal and anal area, although latter cases are rare.
GAS has several surface proteins and produces numerous extracellular products that facilitate permeation and successive evasion of the host’s immune system. Streptococcal pharyngitis results from the proliferation of GAS in the pharynx. Virulence associated factors enable S. pyogenes to attach to host tissues, elude the immune response, and spread by penetrating the host tissue layers followed by colonization.
Carotenoids, also called tetraterpenoids, are organic pigments that are produced by plants and algae, as well as several bacteria and fungi. Carotenoids can be produced from fats and other basic organic metabolic building blocks by all these organisms.
Plant distribution and pharmacological activity of flavonoidsLucyPi1
Abstract Flavonoids are natural organic compounds that are widely found in nature, their structural types are complex, and they mainly include flavonoids, flavonols, dihydroflavonols, isoflavones, dihydroisoflavones, chalcones, orange ketones, flavanoids, anthocyanidins, and biflavonoids. This review covers the plant distribution and pharmacological activities of flavonoids. Flavonoids are mainly distributed in angiosperms and gymnosperms, and they are abundant in plants such as Rutaceae, Labiatae, Zingiberaceae, Scrophulariaceae, and Leguminosae. Because of their wide distribution and variety, researchers have found that flavonoids have diverse biological activities, mainly focusing on anti-inflammatory, antibacterial and antitumor activities. Mechanistically, the anti-inflammatory effects are mainly related to the NF-κB and MAPK (mitogen-activated protein kinase) signaling pathway and then the inhibition of the production of inflammatory cytokines and mediators. The antibacterial activity is mainly manifested as inhibitory effects on many strains, including Escherichia coli, Cryptococcus neoformans, and Pseudomonas aeruginosa, via destroying the stability of the microbial membrane, inhibiting the invasion of virulent bacteria into host cells, promoting the apoptosis of bacteria, inhibiting bacterial fatty acid synthesis, etc. The antitumor activity of flavonoids is related to their inhibition of cell proliferation and induction of apoptosis via the mitochondria-mediated, endoplasmic reticulum-mediated, and death factor and its receptor-mediated signal transduction pathways. Understanding the plant distribution and pharmacological activity of flavonoids not only reveals the importance of identifying such valuable flavonoids in another genus or family but also provides a basis for fully exploiting the therapeutic potential of flavonoids.
Introduction
Classification
Therapeutic values of peptidomimetics
Design of peptidomimetics by manipulation of amino acids
Modification of peptide backbone
Chemistry of prostaglandins, leukotrienes and thromboxanes
The use of agrochemicals has increased considerably in recent years, and consequently, there has been increased exposure of ecosystems and human populations to these highly toxic compounds. The study and development of methodologies to detect these substances with greater sensitivity has become extremely relevant. This article describes, for the first time, the use of atomic force spectroscopy (AFS) in the detection of enzyme-inhibiting herbicides. A nanobiosensor based on an atomic force microscopy (AFM) tip functionalised with the acetolactate synthase (ALS) enzyme was developed and characterised. The herbicide metsulfuron-methyl, an ALS inhibitor, was successfully detected through the acquisition of force curves using this biosensor. The adhesion force values were considerably higher when the biosensor was used. An increase of ~250% was achieved relative to the adhesion force using an unfunctionalised AFM tip. This considerable increase was the result of a specific interaction between the enzyme and the herbicide, which was primarily responsible for the efficiency of the nanobiosensor. These results indicate that this methodology is promising for the detection of herbicides, pesticides, and other environmental contaminants.
In vitro experiments of prokaryotic and eukaryotic antimicrobial peptide cyto...AI Publications
These proteinaceous molecules, called antimicrobial peptides (AMPs), are a varied collection of antimicrobial peptides. The ability of AMPs to combat gut infections necessitates further study of the AMP-GI tract interaction. These peptides need to be tested in vitro for cytotoxicity before they may be considered for use in clinical infections. Using the MTT conversion assay, neutral red dye absorption assay, and a comparison to vancomycin, researchers examined the cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin in two gastrointestinal cell types (HT29 and Caco-2). Sheep erythrocyte hemolytic activity was also studied, and the influence of AMPs on paracellular permeability was assessed using transepithelial resistance (TEER) and TEM. Gallidermin, nisin A, magainin I, magainin II, and melittin were the least cytotoxic AMPs. To our knowledge, only Melittin and NIS caused considerable hemolysis. There are two distinct ways that melittin and nisin differ in their ability to kill bacteria. It was the only AMP that had an effect on the permeability of the paracellular space. Intestinal tight junctions and cell–cell adhesion were destroyed by long-term melittin therapy, as were microvilli, cell debris, and cell–cell adhesion. Antimicrobial activity and low cytotoxicity make Gallidermin a promising therapeutic drug. The antibacterial properties of Melittin are limited, but its ability to transport poorly bioavailable medicines may be useful.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
2. 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
Figure 1. Structures of alkoxyisoflavones, combretastatins, and podophyllotoxin.
Scheme 1a
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.
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.6b00173
J. Nat. Prod. XXXX, XXX, XXX−XXX
B
3. 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).
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
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.
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
Scheme 2a
a
Reagents and conditions: (a) NBS, DMF, rt, 8 h; (b) MeMgI, ether,
rt, 0.5 h; (c) pyridinium chlorochromate, CH2Cl2, rt, 18 h.
Scheme 3a
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.
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.6b00173
J. Nat. Prod. XXXX, XXX, XXX−XXX
C
4. 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
Table 1. Effects of GVA and its Analogues on Sea Urchin Embryos and Human Cancer Cellsa
a
A549: lung carcinoma; A375: melanoma; DU145 and PC3: prostate cancer; HCT116: colon cancer; MDA-MB-231: breast adenocarcinoma; SK-
OV-3: ovarian carcinoma; PBMCs: human peripheral blood mononuclear cells. b
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
no differences in effective threshold concentration (EC) values. c
IC50 values were determined by sigmoidal curve interpolation of cell survival data
after 48 h of treatment. d
TE: tuberculate eggs. e
Data in bparentheses are from ref 8, obtained for natural GVA using the SRB assay. f
ND: not
determined. IC50 values are expressed as means ± SD based on dose−response curves of three independent experiments that involved each
compound concentration tested in quadruplicates.
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.6b00173
J. Nat. Prod. XXXX, XXX, XXX−XXX
D
5. 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.
■ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
measured on a Boetius melting point apparatus and were uncorrected.
1
H NMR spectra were recorded on a Bruker DRX-500 (500.13 MHz)
instrument. Chemical shifts are reported in parts per million (ppm)
and referenced to the appropriate NMR solvent peak(s). 2D NMR
experiments {1
H−1
H} NOESY, {1
H−13
C} HMBC-qs, and {1
H−13
C}
HSQC were used where necessary in assigning NMR spectra. Spin−
spin coupling constants (J) are reported in hertz (Hz). 13
C 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.
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);
1
H NMR (DMSO-d6) δ 10.17 (1H, s, CHO), 7.28 (1H, s, H-6), 3.90
(3H, s, OCH3), 3.88 (3H, s, OCH3), 3.83 (3H, s, OCH3).
4-Bromo-6,7-dimethoxy-2H-1,3-benzodioxole-5-carbaldehyde
(3f): 4.85 g, 84% yield; colorless crystals; mp 103 °C (50% EtOH); 1
H
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]
(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); 1
H NMR (DMSO-d6) δ 10.10 (1H, s, CH(O)),
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).
1-(2-Bromo-3,4,5-trimethoxyphenyl)ethan-1-ol (4d): 2.1 g, 72%
yield; colorless oil; 1
H NMR spectra as in lit.30
; 1
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).
1-(2-Bromo-5,6-dimethoxy-3,4-methylendioxyphenyl)ethan-1-ol
(4f): 2.32 g, 76% yield; colorless oil; 1
H 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); 1
H 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); 1
H 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
34.5−35 °C); 1
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).
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.6b00173
J. Nat. Prod. XXXX, XXX, XXX−XXX
E
9. 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%.
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.
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.
All cancer cell cultures were performed in media contained 10%
fetal bovine serum, penicillin (100 units/mL), and streptomycin (100
μg/mL) at 37 °C under a 5% CO2 humidified atmosphere.
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.
■ ASSOCIATED CONTENT
*S Supporting Information
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; 1
H NMR and 13
C 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).
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
(1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−325.
(2) Hamel, E. Med. Res. Rev. 1996, 16, 207−231.
(3) Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med.
Res. Rev. 1998, 18, 259−296.
(4) Desbene, S.; Giorgi-Renault, S. Curr. Med. Chem.: Anti-Cancer
Agents 2002, 2, 71−90.
(5) Nam, N.-H. Curr. Med. Chem. 2003, 10, 1697−1722.
(6) Brancale, A.; Silvestri, R. Med. Res. Rev. 2007, 27, 209−238.
(7) Semenov, V. V.; Semenova, M. N. Russ. Chem. Rev. 2015, 84,
134−158.
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.6b00173
J. Nat. Prod. XXXX, XXX, XXX−XXX
I
10. (8) Yokosuka, A.; Haraguchi, M.; Usui, T.; Kazami, S.; Osada, H.;
Yamori, T.; Mimaki, Y. Bioorg. Med. Chem. Lett. 2007, 17, 3091−3094.
(9) Chinen, T.; Kazami, S.; Nagumo, Y.; Hayakawa, I.; Ikedo, A.;
Takagi, M.; Yokosuka, A.; Imamoto, N.; Mimaki, Y.; Kigoshi, H.;
Osada, H.; Usui, T. ACS Chem. Biol. 2013, 8, 884−889.
(10) Ikedo, A.; Hayakawa, I.; Usui, T.; Kazami, S.; Osada, H.;
Kigoshi, H. Bioorg. Med. Chem. Lett. 2010, 20, 5402−5404.
(11) Chinen, T.; Liu, P.; Shioda, S.; Pagel, J.; Cerikan, B.; Lin, T.-C.;
Gruss, O.; Hayashi, Y.; Takeno, H.; Shima, T.; Okada, Y.; Hayakawa,
I.; Hayashi, Y.; Kigoshi, H.; Usui, T.; Schiebel, E. Nat. Commun. 2015,
6, 8722.
(12) Dagne, E.; Bekele, A.; Waterman, P. G. Phytochemistry 1989, 28,
1897−1900.
(13) Dagne, E.; Bekele, A. Phytochemistry 1990, 29, 2679−2682.
(14) Dagne, E.; Mammo, W.; Bekele, A. Bull. Chem. Soc. Ethiop.
1991, 5, 81−86.
(15) Semenova, M. N.; Kiselyov, A. S.; Semenov, V. V. BioTechniques
2006, 40, 765−774.
(16) Shaw, S.; Mandal, S. P.; Azad, R. Indian J. Chem. 1983, 228, 488.
(17) Zaninetti, R.; Cortese, S. V.; Aprile, S.; Massarotti, A.; Canonico,
P. L.; Sorba, G.; Grosa, G.; Genazzani, A. A.; Pirali, T. ChemMedChem
2013, 8, 633−643.
(18) Lewin, G.; Aubert, G.; Thoret, S.; Dubois, J.; Cresteil, T. Bioorg.
Med. Chem. 2012, 20, 1231−1239.
(19) Li, Q.-L.; Liu, Q.-L.; Ge, Z.-Y.; Zhu, Y.-M. Helv. Chim. Acta
2011, 94, 1304−1309.
(20) Hayakawa, I.; Ikedo, A.; Kigoshi, H. Chem. Lett. 2007, 36,
1382−1383.
(21) Hayakawa, I.; Ikedo, A.; Chinen, T.; Usui, T.; Kigoshi, H. Bioorg.
Med. Chem. 2012, 20, 5745−5756.
(22) Semenov, V. V.; Rusak, V. A.; Chartov, E. M.; Zaretsky, M. I.;
Konyushkin, L. D.; Firgang, S. I.; Chizhov, A. O.; Elkin, V. V.; Latin,
N. N.; Bonashek, V. M.; Stas’eva, O. N. Russ. Chem. Bull. 2007, 56,
2448−2455.
(23) Semenov, V. V.; Kiselyov, A. S.; Titov, I. Y.; Sagamanova, I. K.;
Ikizalp, N. N.; Chernysheva, N. B.; Tsyganov, D. V.; Konyushkin, L.
D.; Firgang, S. I.; Semenov, R. V.; Karmanova, I. B.; Raihstat, M. M.;
Semenova, M. N. J. Nat. Prod. 2010, 73, 1796−1802.
(24) De Vincenzi, M.; Silano, M.; Maialetti, F.; Scazzocchio, B.
Fitoterapia 2000, 71, 725−729.
(25) Public Statement on the Use of Herbal Medicinal Products
Containing Methyleugenol. www.ema.europa.eu/ema/pages/includes/
document/open_document.jsp?webContentId=WC500089961.
(26) Rizzacasa, M.; Sargent, M. J. Chem. Soc., Perkin Trans. 1 1987,
2017−2022.
(27) Gutsche, C. D.; Jason, E. F.; Coffey, R. S.; Johnson, H. E. J. Am.
Chem. Soc. 1958, 80, 5756−5767.
(28) Dallacker, F.; Schleuter, H.-J.; Schneider, P. Z. Naturforsch. B
1986, 41, 1273−1280.
(29) Sanceau, J.-Y.; Dhal, R.; Brown, E. Tetrahedron 1994, 50, 3363−
3380.
(30) Ziegler, F. E.; Chliwner, I.; Fowler, K. W.; Kanfer, S. J.; Kuo, S.
J.; Sinha, N. D. J. Am. Chem. Soc. 1980, 102, 790−798.
(31) Sun, L.; Borella, Ch.; Li, H.; Jiang, J.; Chen, Sh.; Koya, K.;
Inoue, T.; Du, Z.; Foley, K.; Wu, Y.; Zhang, M.; Ying, W. WIPO
Patent WO2006/089177, 2006.
(32) Mathew, P.; Mathew, D.; Asokan, C. V. Synth. Commun. 2007,
37, 661−665.
(33) https://worldwide.promega.com/products/cell-health-and-
metabolism/cell-viability-assays/celltiter-96-aqueous-one-solution-cell-
proliferation-assay-_mts_/.
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.6b00173
J. Nat. Prod. XXXX, XXX, XXX−XXX
J