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UNIVERSITY OF MEDICINE AND PHARMACY
"GRIGORE T. POPA" IASI
FACULTY OF PHARMACY
PHYTOCHEMICALS OF PAEONIA SPECIES AND
STRUCTURAL ANALOGUES OF THERAPEUTIC INTEREST
PhD THESIS ABSTRACT
Scientific coordinator,
Prof. dr. Anca Miron
PhD attendant,
Assist. Ana Maria Balan (Zbancioc)
2014
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CONTENT
REVIEW OF THE LITERATURE.............................................................. .....5
CHAPTER 1. PAEONIA L. GENUS – OVERVIEW.............................. ....5
1.1. Systematic classification................................................................... ....5
1.2. Distribution...................................................................................... ....6
1.3. Description........................................................................................ ....6
1.4. Chemical studies............................................................................... ....7
1.4.1. Terpenes isolated from various species of the genus Paeonia
L........................................................................................................... ....7
1.4.2. Polyphenols isolated from various species of the genus
Paeonia L............................................................................................ ..15
1.5. Biological studies.............................................................................. ..20
1.5.1. Antimicrobial activity................................................................ ..21
1.5.2. Antitumor activity..................................................................... ..23
1.5.3. Antioxidant activity................................................................... ..25
1.5.4. Antiplatelet and anticoagulant activity...................................... ..27
1.5.5. Anti-inflammatory activity........................................................ ..28
1.5.6. Antiallergic activity................................................................... ..28
1.5.7. Hypolipemiant activity.............................................................. . 29
1.5.8. Hypoglycemiant activity........................................................... ..29
1.5.9. Antiosteoporotic activity........................................................... . 30
1.5.10. Other biological effects......................................................... .30
CHAPTER 2. COMPOUNDS FROM PAEONIA SPECIES AND
STRUCTURAL ANALOGUES: SYNTHESIS,
CHARACTERIZATION, BIOLOGICAL EFFECTS............................ ..32
2.1. Paeonol and its structural analogues................................................. ..32
2.2. Paeonilide.......................................................................................... ..39
PERSONAL RESEARCH.............................................................................. ..44 MOTIVATION OF THE STUDY AND PROPOSED
OBJECTIVES............................................................................................. ..44
CHAPTER 3. ISOLATION AND CHARACTERIZATION OF
SOME COMPOUNDS FROM PAEONIA MLOKOSEWITSCHII
LOMAKIN................................................................................................ ..50
3.1. Paeonia mlokosewitschii Lomakin. – overview............................... ..50
3.2. Isolation and characterization of some compounds from Paeonia
mlokosewitschii Lomakin. leaves.............................................................
..50
3.2.1. Isolation and fractionation of the crude methanolic extract.... ..51
3.2.2. Isolation of some compounds from diethyl ether fraction
(FPE).................................................................................................. ..52
3.2.3. Structure elucidation of isolated compounds............................ ..56
3.2.4. Isolation of some compounds from ethyl acetate fraction
(FPA)................................................................................................... ..74
3.2.5. Structure elucidation of isolated compounds............................ ..77
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3.3. Isolation and characterization of some compounds from Paeonia
mlokosewitschii Lomakin. roots...............................................................
..85
3.3.1. Isolation and fractionation of the crude methanolic extract.... ..85
3.3.2. Isolation of some compounds from diethyl ether fraction
(RPE)................................................................................................... ..86
3.3.3. Structure elucidation of isolated compounds............................ ..89
3.3.4. Isolation of some compounds from ethyl acetate fraction
(RPA).................................................................................................. ..89
3.3.5. Structure elucidation of isolated compound.............................. ..92
3.4. Conclusions....................................................................................... ..93
CHAPTER 4. STRUCTURAL ANALOGUES WITH
ACETOPHENONE SKELETON: SYNTHESIS AND PHYSICO-
CHEMICAL CHARACTERIZATION................................................... ..94
4.1. Alkylated derivatives........................................................................ ..94
4.1.1. Synthesis and purification......................................................... ..94
4.1.2. Physical characterization........................................................... ..97
4.1.3. Structure elucidation of alkylated derivatives.......................... ..97
4.2. Brominated dialkylated derivatives.................................................. 111
4.2.1. Synthesis and purification......................................................... 111
4.2.2. Physical characterization.......................................................... 112
4.2.3. Structure elucidation of brominated dialkylated
derivatives........................................................................................... 113
4.3. Cycloimmonium salts........................................................................ 120
4.3.1. Cycloimmonium bromides........................................................ 120
4.3.1.1. Synthesis and purification................................................ 120
4.3.1.2. Physical characterization.................................................. 121
4.3.1.3. Structure elucidation of cycloimmonium bromides.......... 121
4.3.2. Cycloimmonium chlorides........................................................ 134
4.3.2.1. Synthesis and purification................................................ 134
4.3.2.2. Physical characterization.................................................. 136
4.3.2.3. Structure elucidation of cycloimmonium chlorides.......... 136
4.4. Conclusions....................................................................................... 145
CHAPTER 5. EVALUATION OF ANTIMICROBIAL ACTIVITY.... 146
5.1. Antimicrobial activity of methyl 3-(3,5-dihydroxybenzoiloxy)-
4,5-dihydroxybenzoate............................................................................. 146
5.1.1. Agar diffusion method............................................................... 146
5.2. Antimicrobial activity of synthesized structural analogues.............. 148
5.2.1. Agar diffusion method............................................................... 148
5.2.1.1. Antimicrobial activity of alkylated derivatives................ 150
5.2.1.2. Antimicrobial activity of brominated dialkylated
derivatives...................................................................................... 156
5.2.1.3. Antimicrobial activity of cycloimmonium salts............... 159
5.2.2. Broth micro dilution assay......................................................... 165
5.3. Conclusions...................................................................................... 171
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CHAPTER 6. EVALUATION OF ANTITUMOR ACTIVITY.........
173
6.1. Antitumor activity of methyl 3-(3,5-dihydroxybenzoiloxy)-
4,5-dihydroxybenzoate............................................................................. 173
6.1.1. Preparing the biological material............................................... 173
6.1.2. The effect of methyl 3-(3,5-dihydroxybenzoiloxy)-4,5-dihydroxybenzoate on HeLa cells viability (MTT method)............... 174
6.2. Antitumor activity of synthesized structural analogues.................... 175
6.2.1. The effects of synthesized structural analogues on HeLa
cells viability (MTT method) – preliminary screening..................... 175
6.2.1.1. The effects of alkylated derivatives on HeLa cells
viability......................................................................................... 175
6.2.1.2. The effects of brominated dialkylated derivatives on
HeLa cells viability....................................................................... 176
6.2.1.3. The effects of cycloimmonium bromide on HeLa cells
viability......................................................................................... 177
6.2.2. The effects of synthesized structural analogues on protein
content in HeLa cells…..................................................................... 180
6.2.2.1. The effects of alkylated derivatives on protein content in HeLa cells................................................................................. 181
6.2.2.2. The effects of brominated dialkylated derivatives on
protein content in HeLa cells........................................................ 184
6.2.2.3. The effects of cycloimmonium bromides on protein
content in HeLa cells...................................................................... 186
6.2.3. The effects of brominated derivatives on other human
tumor cell lines.................................................................................... 191
6.2.3.1. Preparing the biological material..................................... 191
6.2.3.2. The effects on cell viability (MTT method).................... 192
6.2.3.2.1. The effects on MCF-7 human breast
adenocarcinoma cells............................................................... 193
6.2.3.2.2. The effects on A549 human alveolar
adenocarcinoma cells............................................................... 194
6.2.3.2.3. The effects on Caco2 human colorectal
adenocarcinoma cells................................................................ 194
6.2.3.2.4. The effects on PC3 human prostate
adenocarcinoma cells............................................................... 195
6.2.3.3. Pro-oxidant capacity of brominated compounds.............. 196
6.2.3.3.1. Pro-oxidant activity in MCF-7 human breast
adenocarcinoma cells................................................................ 198
6.2.3.3.2. Pro-oxidant activity in A549 human alveolar
adenocarcinoma cells............................................................... 198
6.2.3.3.3. Pro-oxidant activity in Caco2 human colorectal adenocarcinoma cells................................................................ 199
6.2.3.3.4. Pro-oxidant activity in PC3 human prostate
adenocarcinoma cells...............................................................
200
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6.2.4. The effects on MCF-12F normal mammary epithelial,
immortalized cells........................................................................... 201
6.3. Conclusions....................................................................................... 203
GENERAL CONCLUSIONS. DEGREE OF ORIGINALITY.
RESEARCH PERSPECTIVES............................................................... 205
REFERENCES.......................................................................................... 213
Annex 1 Alkylated compounds – Spectral analysis...................................... 237 Annex 2 Brominated compounds – Spectral analysis.................................. 239
Annex 3 Cycloimmonium salts – Spectral analysis..................................... 247
Annex 4 ARTICLES PUBLISHED IN THESIS TOPICS.......................... 267
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MOTIVATION OF THE STUDY AND PROPOSED
OBJECTIVES
Plants provide a great variety of compounds and despite the progress in the field of chemical synthesis and semisynthesis, they
remain an important source of potential therapeutic substances (165).
Such substances have been isolated from various species of the genus
Paeonia L.:
paeonol (P. suffruticosa Andr.), a hydroxyacetophenone
compound, has numerous biological properties. Its antimicrobial
activity is remarkable, at low concentrations inhibiting the growth of
many microorganisms (Aspergillus spp., Staphylococcus spp., Escherichia coli) (76). Paeonol has antitumor effects, significantly
reducing the viability of HeLa (cervical adenocarcinoma), HT-29
(colorectal adenocarcinoma), Bel-7404 (hepatocellular carcinoma),
K562 (chronic myeloid leukemia), SEG-1 (esophageal adenocarcinoma), Eca-109 (esophageal squamous cell carcinoma) cells
(84, 85). It also has anti-inflammatory, antiplatelet, anticoagulant and
analgesic properties (102, 103).
paeoniflorin (P. hybrida Pall., P. lactiflora Pall., P. delavayi
Franch.), a monoterpene glycoside, has antioxidant, anticonvulsant,
lipid-lowering and antiosteoporotic effects (13, 44, 113, 166).
Moreover, paeoniflorin was reported to have antitumor effects through the induction of apoptosis in HT-29, Hef G2 and SMMC-7721 G2
(hepatocellular carcinoma) tumor cells (87, 88).
paeoninol and paeonin C (P. emodi Wall. ex Royle),
polyphenol and monoterpene glycoside, inhibit lipoxygenase (type I-B)
and have antioxidant properties (167).
Structural analogues of paeonol have been synthesized
(Schiff bases, paeonol oxime, Schiff base ligands, halogenated
derivatives); they proved to have antimicrobial, antioxidant, and antitumor effects (127, 137, 138, 144, 154).
Given the therapeutic importance of the compounds of plant
origin, both by themselves and by their use as structural prototype for obtaining biologically active analogues, the main objectives of the
researches in this Doctoral Thesis were:
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A.
isolation of pure compounds from the leaves and roots of
Paeonia mlokosewitschii Lomakin. species (this species has not been previously studied from a chemical and biological point of
view);
structural elucidation of the isolated compounds using different
spectral techniques: chemical ionization mass spectrometry
(positive mode) and nuclear magnetic resonance spectroscopy: 1H-NMR,
13C-NMR, 2D-COSY, 2D-HETCOR spectra (HMQC
and HMBC);
evaluation of some biological effects of the isolated
compounds:
antimicrobial (against Gram-positive and Gram-
negative bacteria, pathogenic fungi);
antitumor (against various tumor cell lines: HeLa
cervical adenocarcinoma cells, MCF-7 breast
adenocarcinoma cells, A549 alveolar adenocarcinoma cells, Caco2 colorectal adenocarcinoma cells, PC3
prostate adenocarcinoma cells).
B.
synthesis of some structural analogues of phenolic compounds
with acetophenone skeleton isolated from Paeonia species, with
potential antimicrobial and antitumor effects. The aim was to
synthesize new alkylated derivatives with acetophenone skeleton, brominated derivatives and cycloimmonium salts with
different heterocyclic compounds and nitrogen as heteroatom
(establishment of the methods for synthesis, optimization of
work processes);
physico-chemical and spectral characterization of the
synthesized structural analogues (melting point, elemental
analysis, and structure elucidation by infrared spectroscopy,
chemical ionization mass spectrometry (positive mode) and nuclear magnetic resonance spectroscopy:
1H-NMR,
13C-NMR,
two-dimensional 2D-COSY spectra, two-dimensional 2D-
HETCOR spectra: HMQC and HMBC);
evaluation of some biological effects of the synthesized
structural analogues:
antimicrobial (against Gram-positive and Gram-
negative bacteria, pathogenic fungi);
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antitumor (against various tumor cell lines: HeLa
cervical adenocarcinoma cells, MCF-7 breast
adenocarcinoma cells, A549 alveolar adenocarcinoma cells, Caco2 colorectal adenocarcinoma cells, PC3
prostate adenocarcinoma cells);
pro-oxidant on different tumor cell lines (MCF-7,
A549, Caco2, PC3);
cytotoxicity against a normal, immortalized cell line
(mammary epithelial cells MCF-12F).
Up to now, 15 phenolic compounds with acetophenone skeleton
have been isolated from different Paeonia species, the pharmacological
profile of paeonol being the most studied. In this study, for the synthesis of the structural analogues with acetophenone skeleton, we used: 2',4'-,
2',5'-, 2',6'-, 3',4'-, 3',5'-dihydroxyacetophenones, more readily available
and accessible in terms of purchase price than paeonol or other phenolic
compounds with acetophenone structure isolated from Paeonia species.
paeonol 2',4'-dihydroxyacetophenone 2',5'- dihydroxyacetophenone
2',6'- dihydroxyacetophenone 3',4'- dihydroxyacetophenone 3',5'- dihydroxyacetophenone
Literature data show moderate or even weak antimicrobial effects for the dihydroxyacetophenones used in the synthesis of
structural analogues (table 1) (168).
TABLE 1.
MIC (mg/mL) values of dihydroxyacetophenones
used in the synthesis of structural analogues
Microorganism Dihydroxyacetophenones
2',4'- 2',5'- 2',6'- 3',4'- 3',5'-
Staphylococcus aureus FAD-209P 1.99 > 1.99 0.49 > 1.99 1.99
Bacillus subtilis PCI-219 1.99 > 1.99 0.19 > 1.99 1.99
Micrococcus litea ATCC-1001 1.99 0.49 0.49 > 1.99 1.99
Escherichia coli O-80 0.99 > 1.99 0.19 > 1.99 1.99
Sallmonela typhi H-901 1.99 > 1.99 0.49 > 1.99 1.99
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Pseudomonas aeruginosa IFO-3080 > 1.99 > 1.99 0.99 > 1.99 1.99
Candida albicans ATCC-7491 0.49 > 1.99 0.49 > 1.99 > 4.99
Having in view the already known antibacterial and antifungal potential of nitrogen heterocyclic structures, to potentiate the
antimicrobial effects, dihydroxyacetophenones have been coupled with
various heterocycles containing nitrogen as heteroatom (pyridazine, phthalazine, chloro-p-tolyl-pyridazine, chloro-p-tolyl-pyrimidine).
Salts such as 4-(4-methylphenyl)-1-[3',4'-dihydroxyphenyl)-2-
oxo-ethyl]-pyridazin-1-ium chloride were found to be more active than
the control (chloramphenicol, 30 µg/disc) against: Staphylococcus aureus ATCC 25923, Staphylococcus saprophyticus, Sarcina lutea
ATCC 9341, Bacillus cereus, Bacillus subtilis, Escherichia coli ATCC
25922 and Pseudomonas aeruginosa (169).
4-(4-methylphenyl)-1-[3',4'-dihydroxyphenil)-2-oxo-ethyl]-pyridazin-1-ium chloride (III)
Bhuiyan et al. have tested the antimicrobial activity of some
pyrimidine derivatives by the diffusion method. Test microorganisms
were: Bacillus cereus, Listeria monocytogenes, Shigella dysenteriae, Salmonella typhi. The most active compound was found to be 4-(3,5-
dimethyl-1H-pyrazol-1-yl)-5,6-diphenylfuro [2,3-d]-pyrimidine; at a
concentration of 1%, this compound was more active than ampicillin (25 µg/disc) against Bacillus cereus and Shigella dysenteriae (170).
4-(3,5-dimethyl-1H-pyrazol-1-yl)-5,6-diphenylfuro-[2,3-d]-pyrimidine
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Synthetic derivatives with acetophenone and pyrimidine
skeleton and brominated derivatives with quinoline and pyrimidoquinoline skeleton are known for their antitumor effects (171-
173). Thus, for the synthesis of derivatives with potential antitumor
activity, various alkylation reactions with different nitrogen
heterocycles and bromination reaction have been used. Tests against human tumor cells (MCF-7 breast adenocarcinoma cells, A549 alveolar
adenocarcinoma cells, PC3 prostate adenocarcinoma cells, HT-29
colorectal adenocarcinoma cells) showed that some derivatives with acetophenone skeleton had significant antitumor effects (table 2) (174).
Compound R1
1,3-diphenylprop-2-en-1-one -H
(E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one -OH
(E)-3-phenyl-1-p-tolylprop-2-en-1-one -CH3
(E)-1-(4-methoxyphenyl)-3-phenylprop-2-en-1-one -OCH3
TABLE 2.
IC50 (µM) values of some compounds with acetophenone skeleton
Compound Tumor cell lines
MCF-7 A549 PC3 HT-29
1,3-diphenylprop-2-en-1-one 6.87 16.76 9.10 10.10
(E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one
> 100 > 100 > 100 > 100
(E)-3-phenyl-1-p-tolylprop-2-en-1-one 13.62 36.58 17.30 19.10
(E)-1-(4-methoxyphenyl)-3-phenylprop-2-en-1-one
19.15 77.04 21.13 37.28
Jin et al. have synthesized and tested some compounds with
acetophenone and pyrimidine skeleton against various human tumor cell lines: CNE2 nasopharyngeal adenocarcinoma cells, MCF-7 breast
adenocarcinoma cells and K562 leukemic cells. Some compounds were
found to be more active than 5-fluorouracil (table 3) (175).
(E)-3-(3-chloro-4-(4,6-dimethoxypyrimidin-2-
yloxy)phenyl)-1-phenylprop-2-en-1-one
(E)-3-(4-(4,6-dimethoxypyrimidin-2-yloxy)-3-
methoxyphenyl)-1-phenylprop-2-en-1-one
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TABLE 3.
IC50 (µM) values of some compounds
with acetophenone and pyrimidine skeleton
Compound Tumor cell lines
CNE2 MCF-7 K562
(E)-3-(3-chloro-4-(4,6-dimethoxypyrimidin-2-yloxy)phenyl)-1-phenylprop-2-en-1-one
10.7 20.0 24.6
(E)-3-(4-(4,6-dimethoxypyrimidin-2-yloxy)-3-methoxyphenyl)-1-phenylprop-2-en-1-one
15.8 18.6 19.2
5-fluorouracil 13.1 10.5 > 50
Ghorab et al. have shown that a number of brominated
derivatives containing quinoline and pyrimidoquinoline skeleton have
cytotoxic effects against MCF-7 tumor cells (in vitro studies). Of all
derivatives, 2-amino-1-(4-bromophenyl)-7, 7-dimethyl-5-oxo-4-p-tolyl-1, 4, 5, 6, 7, 8-hexahydroquinoline-3-carbonitrile proved to be more
active than doxorubicin (table 4) (176).
2-amino-1-(4-bromophenyl)-
7,7-dimethyl-5-oxo-4-p-
tolyl-1,4,5,6,7,8-
hexahydroquinoline-3-
carbonitrile
4-amino-10-(4-bromophenyl)-
8,8-dimethyl-5-p-tolyl-
7,8,9,10-
tetrahydropyrimido[4,5-
b]quinolin-6(5H)-one
10-(4-bromophenyl)-8,8-
dimethyl-5-p-tolyl-7,8,9,10-
tetrahydropyrimido[4,5-
b]quinoline-4,6(3H,5H)-dione
TABLE 4.
IC50 (µM) values of brominated derivatives
containing quinoline and pyrimidoquinoline skeleton
Compound IC50 (µM)
MCF-7
2-amino-1-(4-bromophenyl)-7,7-dimethyl-5-oxo-4-p-tolyl-1,4,5,6,7,8-hexahydroquinoline-3-carbonitrile
8.5
4-amino-10-(4-bromophenyl)-8,8-dimethyl-5-p-tolyl-7,8,9,10-tetrahydropyrimido[4,5-b]quinolin-6(5H)-one
36.4
10-(4-bromophenyl)-8,8-dimethyl-5-p-tolyl-7,8,9,10-tetrahydropyrimido[4,5-b]quinoline-4,6(3H,5H)-dione
43.1
doxorubicin 32.02
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Literature also mentions the pro-oxidant effects of
acetophenone derivatives. The best known example is apocynin (4'-hydroxy-3'-methoxyacetophenone, acetovanillone), originally isolated
from Picrorhiza kurroa, which specifically inhibits NADPH oxidase
(enzyme system that catalyzes the reduction of molecular oxygen to
superoxide anion radical). Its inhibitory effect on NADPH oxidase in non-phagocytic cells is controversial. Many researchers support that
apocynin inhibits NADPH oxidase only in phagocytic cells, its action
depending on the presence of myeloperoxidase.
In the other cells, apocynin does not inhibit NADPH oxidase activity and, moreover, acts as a pro-oxidant
through different mechanisms (reduction of
intracellular glutathione, increase of H2O2 apocynin
intracellular level, activation of lipid peroxidation processes) (177, 178).
Although the ability of apocynin to act as a pro-oxidant on
tumor cells has not been reported, one of the aims of the present study was to evaluate the ability of the synthesized structural analogues with
acetophenone skeleton to reduce the viability of tumor cells by inducing
oxidative stress.
Given the increased incidence of infectious and malignant diseases, the identification of new antimicrobial and antitumor agents
with high efficiency and good tolerability is a research priority.
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PERSONAL RESEARCH
CHAPTER 3. ISOLATION AND CHARACTERIZATION OF
SOME COMPOUNDS FROM
PAEONIA MLOKOSEWITSCHII LOMAKIN.
3.2. Isolation and characterization of some compounds from
Paeonia mlokosewitschii Lomakin. leaves
3.2.1. Isolation and fractionation of the crude methanolic
extract
The crude methanolic extract from the leaves has been fractionated by liquid-liquid partition using solvents of different
polarities (diethyl ether, ethyl acetate).
3.2.2. Isolation of some compounds from diethyl ether
fraction (FPE)
Results and Discussions
From diethyl ether extractive fraction (FPE), the following
compounds were isolated in a pure state: FPE-2-1 (127 mg); FPE-3-1-1
(26 mg); FPE-3-1-2-1 (56 mg); FPE-4-1-1-1 (11 mg); FPE-4-1-2 (12 mg). TLC analysis (methods D1 and D2) showed that all isolated
compounds are pure.
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Figure 3.3. Isolation of some compounds from diethyl ether fraction (FPE)
3.2.3. Structure elucidation of isolated compounds
The structure of isolated compounds was elucidated on the
basis of NMR (1H-NMR,
13C-NMR, 2D-COSY, 2D-HETCOR: HMQC,
HMBC) and mass spectrometry (MS) analysis.
Results and Discussions
Spectral analyses showed structural identity between the
following compounds:
FPE-2-1 and FPE-3-1-1 (compound 1);
FPE-3-1-2-1 and FPE-4-1-2 (compound 2).
Compound 1
methyl gallate
C8H8O5
MW = 184 g/mol
White crystalline powder. 1H-NMR (500 MHz, CD3OD-d4, δ, ppm, J, Hz): 3.81 s, 3H:
CH3; 7.04 s, 2H: H2, H6.
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13C-NMR (100 MHz, CD3OD-d4, δ, ppm): 52.4 C2' (CH3);
110.1 C2, C6; 121.6 C1; 139.9 C4; 146.6 C3, C5; 169.1 C1'. MS (CI, m/z): 153 (35.5%), 184 (18.1%), 185 ((M+1)
+, base
peak, 100%).
Compound 2
methyl 3-(3,5-dihydroxybenzoiloxy)-
4,5-dihydroxybenzoate
C15H12O8
MW = 320 g/mol
White amorphous powder. 1H-NMR (500 MHz, CD3OD-d4, δ, ppm, J, Hz): 3.84 s, 3H:
CH3; 7.10 s, 1H: H4'; 7.26-7.27 d, 1H: H6, JH6,H2=2 Hz; 7.38-7.39 d, 1H:
H2, JH2,H6=2 Hz. 13
C-NMR (100 MHz, CD3OD-d4, δ, ppm): 52.4 C2''; 109.8 C4';
110.8 C2', C6'; 114.7 C2; 117.3 C6; 121.4 C1; 128.9 C1'; 140.1 C5; 144.3
C4; 146.5 C3', C5'; 151.7 C3; 166.5 C1''' (CO); 168.2 C1'' (CO). MS (CI, m/z): 110 (18.5%), 260 (6.7%), 288 (18.5%), 304 (base
peak, 100%), 319 ((M-1)+, 6.5%).
Compound 3
bis (2-ethyl-heptyl)
phthalate
C26H42O4
MW = 418 g/mol
Brown oil. 1H-NMR (500 MHz, CDCl3-d1, δ, ppm, J, Hz): 0.86-0.93 m,
12H: 3H (CH3 from position 7'), 3H (CH3 from position 7''), 3H (CH3 from position 9'), 3H (CH3 from position 9''); 1.25-1.42 m, 20H: 2H3',
2H3'', 2H4', 2H4'', 2H5', 2H5'', 2H6', 2H6'', 2H8', 2H8''; 1.67-1.69 m, 2H: H2',
H2''; 4.18-4.34 m, 4H: 2H1', 2H1''; 7.52-7.54 dd, 2H: H4, H5, JH4,H3=JH5,H6=3 Hz; 7.69-7.71 dd, 2H: H3, H6, JH3,H4=JH6,H5=3 Hz.
13C-NMR (100 MHz, CDCl3-d1, δ, ppm): 11.1 C9', C9''; 14.1 C7',
C7''; 23.1 C6', C6''; 23.9 C8', C8''; 29.8 C4', C4''; 30.5 C5', C5''; 32.0 C3', C3'';
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38.9 C2', C2''; 68.3 C1', C1''; 128.9 C4, C5; 131.0 C3, C6; 132.6 C1, C2;
167.9 2 × CO. MS (CI, m/z): 71 (66.3%), 127 (81%), 149 (base peak, 100%),
293 (56.8%), 307 (31.5%), 391 (17.8%), 419 ((M+1)+, 4%).
3.2.4. Isolation of some compounds from ethyl acetate
fraction (FPA)
Results and Discussions From ethyl acetate extractive fraction (FPA), the following
compounds were isolated in a pure state: FPA-2-1 (5 mg) and FPA-2-2
(10 mg). The purity of both compounds was confirmed by TLC
analysis.
Figure 3.24. Isolation of some compounds from ethyl acetate fraction (FPA)
3.2.5. Structure elucidation of isolated compounds
Results and Discussions
Spectral analyses showed structural identity between compound FPA-2-1 and compound 1.
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Compound 4
penta-O-galloyl-β-D-glucose (PGG)
C41H32O26
MW = 940 g/mol
Amorphous light cream powder. 1H-NMR (500 MHz, CD3OD-d4, δ, ppm, J, Hz): 4.36-4.42 m,
2H: H6, H7 ; 4.50-4.53 d, 1H: H7 , JH7 ,H6=11 Hz; 5.56-5.64 m, 2H: H3, H5; 5.88-5.92 t, 1H: H4, JH4,H5=JH4,H3=9.5 Hz; 6.22-6.24 d, 1H: H2, JH2,
H3=8.5 Hz; 6.90 s, 2H: H2', H6' (E); 6.95 s, 2H: H2', H6' (B); 6.98 s, 2H: H2', H6' (C); 7.05 s, 2H: H2', H6' (D); 7.11 s, 2H: H2', H6' (A).
13C-NMR (100 MHz, CD3OD-d4, δ, ppm): 63.1 C7; 69.8 C5;
72.2 C3; 74.1 C4; 74.4 C6; 93.8 C2; 110.3 C2', C6' (A); 110.4 C2', C6' (E); 110.4 C2', C6' (B); 110.4 C2', C6' (C); 110.6 C2', C6' (D); 119.7 C1' (D);
120.2 C1' (C); 120.2 C1' (B); 120.3 C1' (E); 121.0 C1' (A); 140.0 C4' (A);
140.1 C4' (E); 140.3 C4' (B); 140.4 C4' (C); 140.8 C4' (D); 146.2 C3', C5' (E); 146.3 C3', C5' (B); 146.4 C3', C5' (C); 146.4 C3', C5' (A); 146.5 C3',
C5' (D); 166.2 CO (D); 166.9 CO (C); 167.0 CO (B); 167.3 CO (E);
167.9 CO (A).
MS (CI, m/z): 153 (41%), 170 (base peak, 100%), 184 (19.3%).
3.3. Isolation and characterization of some compounds from
Paeonia mlokosewitschii Lomakin. roots
3.3.1. Isolation and fractionation of the crude methanolic
extract
3.3.2. Isolation of some compounds from diethyl ether
fraction (RPE)
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18
Figure 3.35. Isolation of some compounds from diethyl ether fraction (RPE)
Results and Discussions From diethyl ether extractive fraction (RPE), the following
compounds were isolated in a pure state: RPE-1-3-1 (168.8 mg), RPE-
1-4-1 (61 mg) and RPE-1-4-2-1-1 (50 mg). TLC analysis showed that
all isolated compounds are pure.
3.3.3. Structure elucidation of isolated compounds
Results and Discussions
Spectral analyses showed structural identity between the
following compounds:
RPE-1-3-1, RPE-1-4-1 and compound 1;
RPE-1-4-2-1-1 and compound 2.
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19
3.3.4. Isolation of some compounds from ethyl acetate
fraction (RPA)
Figure 3.36. Isolation of some compounds from ethyl acetate fraction (RPA)
Results and Discussions From ethyl acetate extractive fraction (RPA), compound RPA-
3-3-2-2 was isolated. TLC analysis showed that this compound is pure.
3.3.5. Structure elucidation of isolated compound
Results and Discussions Spectral analyses proved the structural identity of the
compound RPA-3-3-2-2 with a compound which has been previously
isolated - compound 4.
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CHAPTER 4. STRUCTURAL ANALOGUES WITH
ACETOPHENONE SKELETON: SYNTHESIS AND
PHYSICO-CHEMICAL CHARACTERIZATION
4.1. Alkylated derivatives
4.1.1. Synthesis and purification
When the reaction occurred in a molar ratio of 1:2, dialkylated
derivatives were obtained; when the reaction occurred in a molar ratio
of 1:1, monoalkylated derivatives were obtained.
4.1.2. Physical characterization
Results and Discussions
TABLE 4.3.
Alkylated derivatives with acetophenone skeleton: aspect and melting point
Compound Aspect M.p.
(ºC) Compound Aspect
M.p.
(ºC)
3a White crystals 114-115 4a Creamy white crystals 96-97
3b Creamy white crystals 95-96 4b Yellow crystals 77-78
3c Yellow crystals 80-81 4c Yellow crystals 68-69
3d Creamy white crystals 89-90
3e White crystals 102-103
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21
4.1.3. Structure elucidation of alkylated derivatives
The structure of synthesized compounds was elucidated on the
basis of elemental, IR, NMR (1H-NMR,
13C-NMR, 2D-COSY, HMQC,
HMBC) and mass spectrometry (MS) analysis.
Compound 3a
2', 4'-bis (2-methoxy-2-oxo-ethoxy)-
acetophenone
C14H16O7
MW=296.27 g/mol
Compound 3b
2', 5'-bis (2-methoxy-2-oxo-ethoxy)-
acetophenone
C14H16O7
MW=296.27 g/mol
Compound 3c
2', 6'-bis (2-methoxy-2-oxo-ethoxy)-
acetophenone
C14H16O7
MW=296.27 g/mol
Elemental Analysis: calc: C 56.76%, H 5.44%; found: C 56.71%, H 5.39%.
IR (KBr, cm-1
): 3100, 3048, 3011 (C-H arom.), 2957, 2917 (C-
H aliph.), 1767, 1751 (C=O ester), 1697 (C=O ket.), 1599, 1470, 1441 (C=C), 1248, 1211, 1130, 1086 (C-O-C).
1H-NMR (DMSO-d6, δ, ppm, J, Hz): 2.45 s, 3H: 3 H1: CH3
from acetyl, 3.69 s, 6H: 2×CH3 from methoxy, position 2' and 6', 4.85 s,
4H: 2×CH2 from methyl acetate, position 2' and 6', 6.65 d, JH3',H4'=JH5',H4'=8.4 Hz, 2H: H3', H5', 7.25 t, JH4',H5'=JH4',H3'= 8.,4 Hz, 1H:
H4'. 13
C-NMR (DMSO-d6, δ, ppm): 31.9 C1 from acetyl, 51.8 2×CH3 from methoxy, position 2' and 6', 64.9 2×CH2 from methyl
acetate, position 2' and 6', 105.6 C3' and C5', 120.8 C1', 130.4 C4', 154.3
C2' and C6', 169.0 2 x CO ester, 200.6 C2 ket.
MS (CI, m/z): 91 (7.6%), 107 (5.7%), 195 (5.7%), 221 (9.4%), 237 (34%), 253 (20.8%), 255 (15.1%), 281 (base peak, 100%), 282
(15.1%), 296 (M+·
, 17%), 297 ((M+1)+, 11.3%).
Page 22
22
Compound 3d
3', 4'-bis (2-methoxy-2-oxo-ethoxy)-
acetophenone
C14H16O7
MW=296.27 g/mol
Compound 3e
3', 5'-bis (2-methoxy-2-oxo-ethoxy)-
acetophenone
C14H16O7
MW=296.27 g/mol
Compound 4a
2'-hydroxy-4'-(2-methoxy-2-oxo-ethoxy)-
acetophenone
C11H12O5
MW=224.21 g/mol
Compound 4b
2'-hydroxy-5'-(2-methoxy-2-oxo-ethoxy)-
acetophenone
C11H12O5
MW=224.21 g/mol
Compound 4c
2'-hydroxy-6'-(2-methoxy-2-oxo-ethoxy)-
acetophenone
C11H12O5
MW=224.21 g/mol
4.2. Brominated dialkylated derivatives
4.2.1. Synthesis and purification
H3C
O
O
OCH2
COOCH3
CH2
COOCH3
+
3a-cScheme 4.3.
2 CuBr2CHCl3/CH3COOC2H5
CH2
O
O
OCH2
COOCH3
CH2
COOCH3
5a-c
Br
Page 23
23
4.2.2. Physical characterization
Results and Discussions
TABLE 4.5.
Brominated alkylated derivatives: aspect and melting point
Compound Aspect M.p. (ºC)
5a Cream crystals 71-72
5b Yellow crystals 84-85
5c Brown crystals 57-58
5d White crystals 89-90
5e White crystals 75-76
4.2.3. Structure elucidation of brominated alkylated
derivatives
Compound 5a
2-bromo-2', 4'-bis (2-methoxy-2-oxo-
ethoxy)-acetophenone
C14H15O7Br
MW=375.17 g/mol
Elemental Analysis: calc: C 44.82%, H 4.03%; found: C
44.80%, H 4.00%. IR (KBr, cm
-1): 3093, 3072, 3018 (C-H arom.), 2965, 2918 (C-
H aliph.), 1763, 1738 (C=O ester), 1674 (C=O ket.), 1606, 1502, 1452,
1433, 1417 (C=C), 1296, 1215, 1165, 1080 (C-O-C), 608 (C-Br). 1H-NMR (CDCl3-d1, δ, ppm, J, Hz): 3.71 s, 3H: CH3 from
methoxy, position 4', 3.74 s, 3H: CH3 from methoxy, position 2', 4.91 s,
2H: CH2 from methyl acetate, position 4', 4.91 s, 2H: CH2 from methyl
acetate, position 2', 4.98 s, 2H: CH2-Br, 6.68 dd, JH5',H6'=8.4 Hz, JH5',H3'=2.0 Hz, 1H: H5', 6.74 d, JH3',H5'=2.0 Hz, 1H: H3', 7.72 d,
JH6',H5'=8.4 Hz, 1H: H6'. 13
C-NMR (CDCl3-d1, δ, ppm): 38.8 C1 from acetyl, 51.9 CH3 from methoxy, position 4', 52.0 CH3 from methoxy, position 2', 64.9
CH2 from methyl acetate, position 4', 65.8 CH2 from methyl acetate,
position 2', 100.2 C3', 108.0 C5', 118.1 C1', 132.5 C6', 158.8 C2', 163.1
C4', 168.1 CO ester, position 2’, 168.1 CO ester, position 4’ 189.8 C2 ket.
MS (CI, m/z): 195 (9.4%), 253 (11.3%), 267 (5.7%), 281 (base
peak, 100%), 282 (15.1%), 315 (18.2%), 317 (17%), 374 ((M-1)+,
16%), 375 (M+·, 11.3%), 376 ((M+1)
+, 13.2%), 377 ((M+2)
+, 9.4%).
Page 24
24
Compound 5b
2-bromo-2', 5'-bis (2-methoxy-2-oxo-
ethoxy)-acetophenone
C14H15O7Br
MW=375.17 g/mol
Compound 5c
2-bromo-2', 6'-bis (2-methoxy-2-oxo-
ethoxy)-acetophenone
C14H15O7Br
MW=375.17 g/mol
Compound 5d
2-bromo-3', 4'-bis (2-methoxy-2-oxo-
ethoxy)-acetophenone
C14H15O7Br
MW=375.17 g/mol Compound 5e
2-bromo-3', 5'-bis (2-methoxy-2-oxo-
ethoxy)-acetophenone
C14H15O7Br
MW=375.17 g/mol
4.3. Cycloimmonium salts
In order to obtain cycloimmonium salts, different nitrogen
heterocyclic compounds have been treated with halogenated
acetophenone skeleton derivatives with increased reactivity.
4.3.1. Cycloimmonium bromides
4.3.1.1. Synthesis and purification
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25
+
CH2
O
O
OCH2
COOCH3
CH2
COOCH3
5a-eBr
N
N
6
N
N
CH2
O
O
OCH2
COOCH3
CH2COOCH3
Br
7a-eScheme 4.4.
Br
CH2
O
O
OCH2
COOCH3
CH2
COOCH3
5a-eBr
N
N
8
N
N
CH2
O
O
OCH2
COOCH3
CH2COOCH3
9a-eScheme 4.5.
+
4.3.1.2. Physical characterization
Results and Discussions
TABLE 4.7.
Cycloimmonium bromides: aspect and melting point
Compound Aspect M.p. (ºC) Compound Aspect M.p. (ºC)
7a Orange crystals 83-84 9a Brown crystals 90-91
7b Brown crystals 127-128 9b Yellow crystals 130-131
7c Brown crystals 109-110 9c Crown crystals 137-138
7d White crystals 188-189 9d Yellow crystals 151-152
7e Brown crystals 100-101 9e Creamy white crystals 165-166
4.3.1.3. Structure elucidation of cycloimmonium bromides Compound 7a
1-(2-(2',4'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide
C18H19O7N2Br
MW=455.26 g/mol
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26
Compound7b
1-(2-(2',5'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide
C18H19O7N2Br
MW=455.26 g/mol
Compound 7c
1-(2-(2',6'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide
C18H19O7N2Br
MW=455.26 g/mol
Compound 7d
1-(2-(3',4'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide
C18H19O7N2Br
MW=455.26 g/mol
Compound 7e
1-(2-(3',5'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide C18H19O7N2Br
MW=455.26 g/mol
Elemental Analysis: calc: C 47.49%, H 4.21%, N 6.15%;
found: C 47.46%, H 4.19%, N 6.10%.
IR (KBr, cm-1
): 3096, 3065, 3018 (C-H arom.), 2978, 2955, 2931 (C-H aliph.), 1749, 1732 (C=O ester), 1692 (C=O ket.), 1601,
1438, 1387 (C=C, C=N arom.), 1294, 1230, 1169 (C-O-C). 1H-NMR (DMSO-d6, δ, ppm, J, Hz): 3.72 s, 6H: 2×CH3 from
methoxy, positions 3' and 5', 4.94 s, 4H: 2×CH2 from methyl acetate,
positions 3' and 5', 6.81 s, 2H: H7, 7.00 d, J4',2'=J4',6'=1.6 Hz, 1H: H4',
7.26 d, J2',4'=J6',4'=1.6 Hz, 2H: H2', H6', 8.79 t, J4,3=4.4 Hz, J4,5=7.6 Hz,
1H: H4, 8.92 t, J5,6=6.0 Hz, J5,4=7.6 Hz, 1H: H5, 9.76 d, J3,4=4.4 Hz, 1H: H3, 9.99 d, J6,5=6.0 Hz, 1H: H6.
Page 27
27
13C-NMR (DMSO-d6, δ, ppm): 51.9 2×CH3 from methoxy,
positions 3' and 5', 65.0 2×CH2 from methyl acetate, positions 3' and 5', 70.4 C7, 107.5 C4', 107.6 C2', 107.6 C6', 135.2 C1', 136.0 C5, 137.5 C4,
151.9 C6, 154.7 C3, 159.1 C3', 159.1 C5', 168.9 2×CO ester, positions 3'
and 5', 189.9 C8 ket.
Compound 9a
2-(2-(2',4'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-
oxoethyl)phthalazin-2-ium bromide
C22H21O7N2Br
MW=505.32 g/mol
Compound 9b
2-(2-(2',5'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-
oxoethyl)phthalazin-2-ium bromide
C22H21O7N2Br
MW=505.32 g/mol
Compound 9c
2-(2-(2',6'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-
oxoethyl)phthalazin-2-ium bromide
C22H21O7N2Br
MW=505.32 g/mol
Compound 9d
2-(2-(3',4'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-
oxoethyl)phthalazin-2-ium bromide
C22H21O7N2Br
MW=505.32 g/mol
Compound 9e
2-(2-(3',5'-bis(2-methoxy-2-
oxoethoxy)phenyl)-2-
oxoethyl)phthalazin-2-ium bromide
C22H21O7N2Br
MW=505.32 g/mol
Page 28
28
CHAPTER 5. EVALUATION OF ANTIMICROBIAL ACTIVITY
5.2. Antimicrobial activity of synthesized structural
analogues
The antimicrobial activity of synthesized structural analogues was tested by the agar diffusion method and broth microdilution assay
(223, 226).
5.2.2. Broth microdilution assay
Results and Discussions Against all tested bacteria, the most active compound proved to
be the brominated derivative 5e.
Figure 5.59. MIC and MBC values against Staphyloccocus aureus ATCC 25923
(A = ampicillin)
Figure 5.60. MIC and MBC values against Sarcina lutea ATCC 9341
(A = ampicillin)
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29
Figure 5.61. MIC and MBC values against Bacillus cereus ATCC 14579
(A = ampicillin)
Figure 5.62. MIC and MBC values against Bacillus subtilis
(A = ampicillin)
Figure 5.63. MIC and MBC values against Escherichia coli ATCC 25922
(A = ampicillin)
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30
Figure 5.64. MIC and MBC values against Pseudomonas aeruginosa ATCC 27853
(A = ampicillin)
Regarding antifungal activity against Candida albicans ATCC 10231, according to MIC and MFC values, the most active derivatives
were 3a, 4b, 5a, 5b, 5d, 5e (MIC = 1.25 mg/mL, MFC = 2.5 mg/mL),
followed by 3c, 9c (MIC = 1.25 mg/mL, MFC = 5 mg/mL) and 5c, 7e
(MIC = 2.5 mg/mL, MFC = 5 mg/mL) (figure 5.65).
Figure 5.65. MIC and MFC values against Candida albicans ATCC 10231 (N = nystatin)
CHAPTER 6. EVALUATION OF ANTITUMOR ACTIVITY
6.2. Antitumor activity of synthesized structural analogues
The antitumor activity of the synthesized derivatives was evaluated in vitro on different human tumor cell lines: HeLa (cervical
adenocarcinoma), MCF-7 (breast adenocarcinoma), A549 (alveolar
adenocarcinoma), Caco2 (colorectal adenocarcinoma), PC3
(prostate adenocarcinoma). In addition, the cytotoxicity against a
normal immortalized cell line MCF-12F (human mammary
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31
epithelial cells) was evaluated. For the brominated derivatives 5a-e, the
ability to reduce the viability of tumor cells by increasing the level of intracellular ROS (pro-oxidant effect) was also investigated.
6.2.2. The effects of synthesized structural analogues on
protein content in HeLa cells
Results and Discussions
6.2.2.1. The effects of alkylated derivatives on protein
content in HeLa cells
a. Dialkylated derivatives At 500 µg/mL the most active compound was 3a (80.94 ±
3.40%), which showed a percentage of inhibition of protein synthesis
higher than methotrexate (42.26 ± 2.95%) and 5-fluorouracil (71.45 ± 2.17%). Moreover, the compound 3a showed an activity superior to
methotrexate at all tested concentrations (fig. 6.9).
Figure 6.9. The influence of dialkylated compounds 3a-e
on protein content in HeLa cells (3a-e=dialkylated derivatives; M=control; E=etoposide;
5FU=5-fluorouracil; Mx=methotrexate) (*p < 0.05; **p < 0.01; ***p < 0.001)
b. Monoalkylated derivatives
At 500 µg/mL, the activity of compounds 4a (73.52 ± 3.63) and
4c (80.25 ± 5.51) was superior to methotrexate (42.26 ± 2.95%) and 5-fluorouracil (71.45 ± 2.17%) (figure 6.11).
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32
Figure 6.11. The influence of monoalkylated compounds 4a-c
on protein content in HeLa cells (4a-c=monoalkylated derivatives; M=control; E=etoposide;
5FU=5-fluorouracil; Mx=methotrexate) (*p < 0.05; **p < 0.01; ***p < 0.001)
6.2.2.2. The effects of brominated derivatives on protein
content in HeLa cells
Because the brominated derivatives 5a-e were more active than
the dialkylated derivatives, they were tested at lower concentrations.
Figure 6.13. The influence of brominated compounds 5a-e (200-500 µg/mL)
on protein content in HeLa cells (5a-e=brominated derivatives; M=control; E=etoposide;
5FU=5-fluorouracil; Mx=methotrexate) (*p < 0.05; **p < 0.01; ***p < 0.001)
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33
Figure 6.15. The influence of brominated compounds 5a-e (25-100 µg/mL)
on protein content in HeLa cells
(5a-e=brominated derivatives; M=control; E=etoposide; 5FU=5-fluorouracil; Mx=methotrexate) (*p < 0.05; **p < 0.01; ***p < 0.001)
6.2.2.3. The effects of cycloimmonium bromides on protein
content in HeLa cells
a. Pyridazinium salts The pyridazinium salts were less active than dialkylated and
brominated derivatives.
Figure 6.17. The influence of pyridazinium salts 7a-e on protein content in HeLa cells
(7a-e=pyridazinium salts; M=control; E=etoposide; 5FU=5-fluorouracil; Mx=methotrexate) (*p < 0.05; **p < 0.01; ***p < 0.001)
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34
b. Phtalazinium salts
The phtalazinium salts were more active than pyridazinium salts.
Figure 6.19. The influence of phtalazinium salts 9a-e on protein content in HeLa cells
(9a-e=phtalazinium salts; M=control; E=etoposide; 5FU=5-fluorouracil; Mx=methotrexate) (*p < 0.05; **p < 0.01; ***p < 0.001)
6.2.3. The effects of brominated derivatives on other human
tumor cell lines
Results and Discussions
6.2.3.2.1. The effects on MCF-7 human breast
adenocarcinoma cells
At 10 µg/mL, compound 5c was the most active with a cytotoxicity of 65.87 ± 3.57%, followed by 5d (33.58 ± 2.31%), 5b
(8.99 ± 5.13%), 5e (7.06 ± 2.30%) and 5a (4.74 ± 2.91%).
Figure 6.21. Cytotoxicity of brominated derivatives 5a-e on MCF-7 tumor cells
(*p < 0.05; **p < 0.01; ***p < 0.001)
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35
6.2.3.2.2. The effects on A549 human alveolar
adenocarcinoma cells
At 10 µg/mL, the most active was 5c with a cytotoxicity of
42.63 ± 4.99%.
Figure 6.22. Cytotoxicity of brominated derivatives 5a-e on A549 tumor cells
(*p < 0.05; **p < 0.01; ***p < 0.001)
6.2.3.2.3. The effects on Caco2 human colorectal
adenocarcinoma cells
At 10, 25 and 50 µg/mL the most active was 5c with a
cytotoxicity of 35.05 ± 3.72%, 59.72 ± 4.40% and 67.88 ± 5.03%, respectively.
Figure 6.23. Cytotoxicity of brominated derivatives 5a-e on PC3 tumor cells
(*p < 0.05; **p < 0.01; ***p < 0.001)
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36
6.2.3.2.4. The effects on PC3 human prostate
adenocarcinoma cells
On PC3 tumor cells, all brominated derivatives exhibited
remarkable cytotoxic effects. At 10 µg/mL, all derivatives exhibited
cytotoxic effects over 83%; at 100 µg/mL, the cytotoxic effects were higher than 90% (figure 6.24).
Figure 6.24. Cytotoxicity of brominated derivatives 5a-e on PC3 tumor cells
(*p < 0.05; **p < 0.01; ***p < 0.001)
6.2.3.3. Pro-oxidant capacity of brominated derivatives
Results and Discussions
6.2.3.3.1. Pro-oxidant activity in MCF-7 human breast
adenocarcinoma cells
The results suggested that the cytotoxic effects on MCF-7 cells are not due to an increase of intracellular oxidative stress.
Figure 6.26. Pro-oxidant activity of brominated derivatives 5a-e
in MCF-7 tumor cells (*p < 0.05; **p < 0.01; ***p < 0.001)
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37
6.2.3.3.2. Pro-oxidant activity in A549 human alveolar
adenocarcinoma cells
In A549 tumor cells, the brominated derivatives 5a-e increased
intracellular ROS levels, the most active being 5e.
Figure 6.27. Pro-oxidant activity of brominated derivatives 5a-e
in A549 tumor cells (*p < 0.05; **p < 0.01; ***p < 0.001)
6.2.3.3.3. Pro-oxidant activity in Caco2 human colorectal
adenocarcinoma cells
All brominated compounds showed a remarkable pro-oxidant
activity in Caco2 cells.
Figure 6.28. Pro-oxidant activity of brominated derivatives 5a-e
in Caco2 tumor cells (*p < 0.05; **p < 0.01; ***p < 0.001)
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38
6.2.3.3.4. Pro-oxidant activity in PC3 human prostate
adenocarcinoma cells
On PC3 tumor cells, the pro-oxidant effects of brominated
derivatives 5a-e were insignificant.
Figure 6.29. Pro-oxidant activity of brominated derivatives 5a-e
in PC3 tumor cells (*p < 0.05; **p < 0.01; ***p < 0.001)
6.2.4. The effects on MCF-12F normal mammary epithelial,
immortalized cells
Results and Discussions
On MCF-12F normal, immortalized cells, at 10 µg/mL, the
brominated derivatives 5a-e exhibited low cytotoxic effects (2.59 ±
1.84% - 11.94 ± 3.03%).
Figure 6.30. Cytotoxicity of brominated derivatives 5a-e on MCF-12F cells
(*p < 0.05; **p < 0.01; ***p < 0.001)
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39
The cytotoxic effects of brominated derivatives 5a-e on MCF-7,
A549, Caco2, PC3 and MCF-12F cells, expressed by IC50 values (µg/mL), are shown in figure 6.31.
Figure 6.31. The cytotoxic effects of the brominated derivatives 5a-e
*IC50 < 10 µg/mL;** IC50 > 100 µg/mL
All brominated derivatives 5a-e were very active against
prostate adenocarcinoma PC3 cells (IC50 < 10 µg/mL). Compound 5c
was strongly cytotoxic against all tested tumor cell lines (IC50 < 18.4 µg/mL). The most pronounced cytotoxic effects (IC50 <10 mg/mL) were
determined on MCF-7 and PC3 tumor cells. It is worthy to note that this
derivative showed a low cytotoxicity on MCF-12F normal cells (IC50 > 100 µg/mL).
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GENERAL CONCLUSIONS. DEGREE OF ORIGINALITY.
RESEARCH PERSPECTIVES
Based on the results of this research, the following conclusions
might be drawn:
Fractionation of methanolic extracts from the leaves and roots of Paeonia mlokosewitschii Lomakin. species by different
chromatographic methods (open column chromatography, low pressure
column chromatography, preparative thin-layer chromatography, semipreparative high-performance liquid chromatography) resulted in
the isolation of four pure compounds:
methyl gallate (compound 1)
methyl 3-(3,5-dihydroxybenzoiloxy)-4,5-dihydroxybenzoate (compound 2)
bis (2-ethyl-heptyl) phthalate
(compound 3) penta-O-galloyl-β-D-glucose
(compound 4)
The structures of these compounds were established by nuclear
magnetic resonance spectroscopy: 1H-NMR,
13C-NMR, 2D-COSY, 2D-
HETCOR spectra (HMQC and HMBC) and chemical ionization mass spectrometry (positive mode).
Methyl gallate and penta-O-galloyl-β-D-glucose have been
previously isolated from other plant species while bis (2-ethyl-heptyl)
phthalate was isolated from the marine organism Hippocampus kuda
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41
Bleeler. This study is the first to report the isolation of these three
compounds from Paeonia mlokosewitschii Lomakin. species. In the reviewed literature no information on the presence of
compound 2 - methyl 3-(3,5-dihydroxybenzoiloxy)-4,5-dihydroxy-
benzoate) in the studied species or other plant species was found.
31 structural analogues with acetophenone skeleton were synthesized:
alkylated derivatives
dialkylated derivatives 3a-e
- 2', 4'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (3a); - 2', 5'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (3b);
- 2', 6'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (3c);
- 3', 4'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (3d); - 3', 5'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (3e).
monoalkylated derivatives 4a-c
- 2'-hydroxy-4'-(2-methoxy-2-oxo-ethoxy)-acetophenone (4a);
- 2'-hydroxy-5'-(2-methoxy-2-oxo-ethoxy)-acetophenone (4b);
- 2'-hydroxy-6'-(2-methoxy-2-oxo-ethoxy)-acetophenone (4c).
brominated dialkylated derivatives 5a-e
- 2-bromo-2', 4'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (5a);
- 2-bromo-2', 5'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (5b);
- 2-bromo-2', 6'-bis (2-mehtoxy-2-oxo-ethoxy)-acetophenone (5c); - 2-bromo-3', 4'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (5d);
- 2-bromo-3', 5'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone (5e).
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cycloimmonium bromides
pyridazinium bromides 7a-e
- 1-(2-(2',4'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide (7a); - 1-(2-(2',5'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide (7b);
- 1-(2-(2',6'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide (7c); - 1-(2-(3',4'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)pyridazin-
1-ium bromide (7d);
- 1-(2-(3',5'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)pyridazin-1-ium bromide (7e).
phtalazinium bromides 9a-e
- 2-(2-(2',4'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)phthalazin-
2-ium bromide (9a);
- 2-(2-(2',5'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)phthalazin-2-ium bromide (9b);
- 2-(2-(2',6'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)phthalazin-
2-ium bromide (9c); - 2-(2-(3',4'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)phthalazin-
2-ium bromide (9d);
- 2-(2-(3',5'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)phthalazin-
2-ium bromide (9e).
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cycloimmonium chlorides
imidazolium chlorides 13a,b, 15a,b
-3-(2-(3',4'-bis(2-chloroacetoxy)phenyl)-2-oxoethyl)-1-(2-cyanoethyl)-
1H-imidazol-3-ium chloride (13a);
-3-(2-(3',4'-bis(4-chlorobutanoyloxy)phenyl)-2-oxoethyl)-1-(2-cyanoethyl)-1H-imidazol-3-ium chloride (13b);
- 3-(2-(3',4'-bis(2-chloroacetoxy)phenyl)-2-oxoethyl)-1-(2-cyanoethyl)-
1H-benzo[d]imidazol-3-ium chloride (15a); -3-(2-(3',4'-bis(4-chlorobutanoyloxy)phenyl)-2-oxoethyl)-1-(2-
cyanoethyl)-1H-benzo[d]imidazol-3-ium chloride (15b).
pyridazinium chlorides 17a,b
-1-(2-(3',4'-bis(2-chloroacetoxy)phenyl)-2-oxoethyl)-3-(4-chloro
phenyl) pyridazin-1-ium chloride (17a);
-1-(2-(3',4'-bis(4-chlorobutanoyloxy)phenyl)-2-oxoethyl)-3-(4-
chlorophenyl)pyridazin-1-ium chloride (17b).
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pyrimidinium chlorides 19a,b
-1-(2-(3',4'-bis(2-chloroacetoxy)phenyl)-2-oxoethyl)-3-(4-chloro phenyl)pyrymidin-1-ium chloride (19a);
-1-(2-(3',4'-bis(4-chlorobutanoyloxy)phenyl)-2-oxoethyl)-3-(4-
chlorophenyl) pyrymidin-1-ium chloride (19b).
Except salt 15b, all synthesized compounds are original
substances; their synthesis has not been previously reported in literature.
The structures of the synthesized derivatives were elucidated by
elemental and spectral analyses: infrared spectroscopy, chemical
ionization mass spectrometry (positive mode) and nuclear magnetic resonance spectroscopy:
1H-NMR,
13C-NMR, 2D-COSY, 2D-HETCOR
spectra: HMQC and HMBC. These analyses confirmed the proposed
structures.
The antimicrobial activity of compound 2 (methyl 3-(3,5-
dihydroxybenzoiloxy)-4,5-dihydroxybenzoate), isolated from the
leaves and roots of Paeonia mlokosewitschii Lomakin. species, was
evaluated only qualitatively by agar diffusion method. This compound proved to be inactive against Gram-positive bacteria and pathogenic
yeasts. In contrast, it showed good antibacterial activity against Gram-
negative bacteria Escherichia coli ATCC 25922 (d = 15 ± 0 mm) and Pseudomonas aeruginosa ATCC 27853 (d = 14.66 ± 0.57 mm).
The antimicrobial activity of the synthesized structural
analogues with acetophenone skeleton was assessed both qualitatively by agar diffusion method and quantitatively by broth microdilution
method. They showed, in general, a good antibacterial activity against
both Gram-positive and Gram-negative bacteria, which varied as
follows:
against Staphylococcus aureus ATCC 25923:
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5e (MIC = 0.31 mg/mL; MBC = 0.62 mg/mL) > 5a (MIC =
0.62 mg/mL; MBC = 2.5 mg/mL) > 3c, 4b, 5b, 5c, 5d, 7e (MIC = 1.25 mg/mL; MBC = 2.5 mg/mL) > 3a, 9c (MIC = 2.5
mg/mL; MBC = 5 mg/mL);
against Sarcina lutea ATCC 9341:
5e (MIC = 0.31 mg/mL; MBC = 0.62 mg /mL) > 3a, 3c, 4b, 5c,
5d, 7e, 9c (MIC = 0.64 mg/mL; MBC = 1.25 mg/mL) > 5a, 5b (MIC = 0.64 mg/mL; MBC = 2.5 mg/mL);
against Bacillus cereus ATCC 14579:
5e (MIC = 0.31 mg/mL; MBC = 0.62 mg/mL) > 5b, 5d (MIC =
0.62 mg/mL; MBC = 1.25 mg/mL) > 4b, 5a, 7e (MIC = 1.25 mg/mL; MBC = 2.5 mg/mL) > 3a, 9c (MIC = 1.25 mg/mL;
MBC = 5 mg/mL) > 3c, 5c (MIC = 2.5 mg/mL; MBC = 5
mg/mL);
against Bacillus subtilis:
5e (MIC = 0.62 mg/mL; MBC = 0.62 mg/mL) > 5d (MIC = 0.62 mg/mL; MBC = 1.25 mg/mL) > 3a, 4b, 5a, 5b, 5c (MIC =
1.25 mg/mL; MBC = 2.5 mg/mL) > 9c (MIC = 2.5 mg/mL;
MBC = 5 mg/mL);
against Escherichia coli ATCC 25922:
5e (MIC = 0.62 mg/mL; MBC = 0.62 mg/mL) > 3a (MIC =
1.25 mg/mL; MBC = 2.5 mg /mL) > 4b, 5a, 5b, 5c, 5d (MIC =
1.25 mg/mL; MBC = 1.25 mg/mL) > 3c, 7e, 9c (MIC = 2.5 mg/mL; MBC = 5 mg/mL);
against Pseudomonas aeruginosa ATCC 27853:
5e (MIC = 0.62 mg/mL; MBC = 0.62 mg/mL) > 3a (MIC =
1.25 mg/mL; MBC = 2.5 mg/mL) > 3c, 5a, 5b, 5c, 5d, 7e (MIC
= 2.5 mg/mL; MBC = 5 mg/mL) > 4b, 9c (MIC = 2.5 mg/mL; MBC = 10 mg/mL).
Among the synthesized compounds, 5e proved to be the most
active against all tested microorganisms. Its antibacterial effects against Gram-negative bacteria (Escherichia coli ATCC 25922 and
Pseudomonas aeruginosa ATCC 27853) are worthy to note as Gram-
negative bacteria are resistant to antibiotics due to the presence of cell wall lipopolysaccharides.
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Dialkylated compound 3a showed a good antibacterial activity
against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, but also against Sarcina lutea ATCC 9341.
The synthesized derivatives showed a low antifungal activity
against Candida albicans ATCC 10231 which varied as follows:
3a, 4b, 5a, 5b, 5d, 5e (MIC = 1.25 mg/mL; MFC = 2.5 mg/mL) > 3c, 9c (MIC = 1.25 mg/mL; MFC = 5 mg/mL) > 5c, 7e (MIC = 2.5
mg/mL; MFC = 5 mg/mL).
The antitumor potential of compound 2 (methyl 3-(3,5-
dihydroxybenzoiloxy)-4,5-dihydroxybenzoate), isolated from the
leaves and roots of Paeonia mlokosewitschii Lomakin. species, was
assessed by studying its influence on the viability of HeLa cells
(cervical adenocarcinoma) (MTT assay). Compound 2 showed no cytotoxic activity.
The antitumor potential of the synthesized structural
analogues with acetophenone skeleton was assessed by studying their effects on protein synthesis in HeLa cells, viability of HeLa, MCF-7
(breast adenocarcinoma), A549 (alveolar adenocarcinoma), Caco2
(colorectal adenocarcinoma), and PC3 (prostate adenocarcinoma) cells, and oxidative stress in MCF-7, A549, Caco2, and PC3 cells. Their
cytotoxicity against normal and immortalized MCF-12F cells was
also tested.
All the synthesized compounds inhibited protein synthesis in HeLa cells in a concentration dependent-manner. The activity of protein
synthesis inhibition varied as follows:
5-fluorouracil (IC50 = 17.2 ± 3.8 µg/mL) > etoposide (IC50 = 19.3 ± 2.1 µg/mL) > 5c (IC50 = 30.9 ± 7.2 µg/mL) > 5a (IC50 = 41.5 ±
1.3 µg/mL) > 5e (IC50 = 64.6 ± 8.0 µg/mL) > 5b (IC50 = 101.8 ± 9.1
µg/mL) > 5d (IC50 = 203.4 ± 11.6 µg/mL) > 4c (IC50 = 221.7 ± 48.4 µg/mL) > 9c (IC50 = 343.1 ± 10.2 µg/mL) > 3a (IC50 = 364.3 ± 17.5
µg/mL) > 3e (IC50 = 380.6 ± 15.3 µg/mL) > 3b (IC50 = 380.8 ± 16.7
µg/mL) > 4a (IC50 = 412.8 ± 15.2 µg/mL) > 9d (IC50 = 432.0 ± 7.3
µg/mL) > 3c (IC50 = 442.4 ± 27.7 µg/mL) > 9a (IC50 = 459.3 ± 5.5 µg/mL) > 4b (IC50 = 462.6 ± 15.5 µg/mL) > 9e (IC50 = 468.9 ± 13.7
µg/mL) > 3d (IC50 = 486.7 ± 18.0 µg/mL).
As brominated derivatives 5a-e were the most active as protein synthesis inhibitors in HeLa cells (IC50 = 30.9 ± 7.2 to 203.4 ± 11.6
µg/mL), their cytotoxicity was also tested against other human tumor
cell lines as well as against a normal cell line.
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Cytotoxic activity of brominated compounds 5a-e varied as
follows:
on MCF-7 tumor cells:
5c (IC50 < 10 µg/mL) > 5d (IC50 = 29.66 ± 2.24 µg/mL) > 5b
(IC50 = 33.20 ± 1.22 µg/mL) > 5a (IC50 = 52.33 ± 3.64 µg/mL) > 5e
(IC50 = 67.03 ± 1.38 µg/mL);
on A549 tumor cells:
5c (IC50 = 11.80 ± 0.89 µg/mL > 5d (IC50 = 36.30 ± 1.28 µg/mL) > 5b (IC50 = 41.50 ± 1.55 µg/mL) > 5e (IC50 = 59.86 ± 2.45
µg/mL) > 5a (IC50 = 60.93 ± 1.30 µg/mL);
on Caco2 tumor cells:
5c (IC50 = 18.40 ± 4.70 µg/mL) > 5e (IC50 = 64.50 ± 6.69 µg/mL) > 5d (IC50 = 69.46 ± 7.43 µg/mL) > 5b (IC50 = 76.16 ± 1.88
µg/mL) > 5a (IC50 = 84.50 ± 1.14 µg/mL);
On PC3 tumor cells, all brominated derivatives tested exhibited
remarkable cytotoxic effects over 80% at 10 µg/mL.
Since the pro-oxidant effects can explained, at least in part, the
cytotoxic effects of these substances, the pro-oxidant effects of
brominated derivatives 5a-e were evaluated in MCF-7, A549, Caco2 and PC3 tumor cells. Pro-oxidant activity of brominated derivatives 5a-
e (100 µg/mL), after three hours of incubation, varied as follows:
on MCF-7 tumor cells:
5e (24.24 ± 2.00%) > 5d (11.36 ± 1.50%) > 5b (9.21 ± 0.90%) > 5c (7.07 ± 0.36%) > 5a (4.41 ± 1.35%);
on A549 tumor cells:
5e (69.62 ± 4.13%) > 5d (52.26 ± 3.12%) > 5b (33.21 ± 3.13%)
> 5a (21.95 ± 2.76%) > 5c (10.32 ± 1.15%);
on Caco2 tumor cells:
5d (67.89 ± 2.17%) > 5e (58.89 ± 3.11%) > 5b (47.79 ± 3.78%) > 5a (45.27 ± 2.11%) > 5c (31.08 ± 0.90%);
on PC3 tumor cells:
5e (22.05 ± 1.18%) > 5d (15.58 ± 2.07%) > 5b (11.64 ± 2.01%)
> 5a (7.25 ± 0.87%) > 5c (4.04 ± 0.03%).
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It is obvious that in the case of Caco2 cells one of the
mechanisms by which the brominated derivatives 5a-e decrease cell viability is the increase of intracellular oxidative stress (31.08 ± 0.90% -
67.89 ± 2.17%). A similar effect was observed for brominated
derivatives 5d and 5e in A549 cells (52.26 ± 3.12% and 69.62 ± 4.13%,
respectively). The cytotoxicity of brominated derivative 5a-e against MCF-7 and PC3 cells as well as of derivatives 5a-c against A549 cells
is mainly due to other mechanisms than the increase of intracellular
oxidative stress.
Against normal and immortalized MCF-12F cells, the least
harmful proved to be 5c which, at 100 µg/mL, showed only 10.77 ±
2.14% cytotoxicity.
The brominated derivative 5c is a promising candidate for in vivo studies; it showed a high cytotoxicity against tumor cells (IC50 <
10 µg/mL against MCF-7 and PC3 tumor cells; IC50 = 11.80 ± 0.89
µg/mL against A549 tumor cells; IC50 = 18.4 ± 4.7 µg/mL against Caco2 tumor cells) being less toxic against normal and immortalized
MCF-12F cells (only 10,77 ± 2,14% cytotoxic activity at 100 µg/mL;
IC50 > 100 µg/mL).
It is worthy to note derivative 5d which showed a high
cytotoxicity against tumor cells (IC50 < 10 µg/mL against PC3 tumor
cells; IC50 = 29.66 ± 2.24 µg/mL against MCF-7 tumor cells; IC50 =
36.30 ± 1.26 µg/mL against A549 tumor cells; IC50 = 69.46 ± 7.43 µg/mL against Caco2 tumor cells) affecting, to a smaller extent, the
viability of normal cells MCF-12F (IC50 = 95.30 ± 2.08 µg/mL).
Degree of originality. Research perspectives Degree of originality consists in:
chemical and biological investigation of Paeonia
mlokosewitschii Lomakin., a species which has not been studied
so far and, therefore, is not yet valued in therapy;
isolation of pure compounds from the leaves and roots of this
species; the isolated compounds were not mentioned before in
this species; the structure elucidation of isolated compounds by
chemical ionization mass spectrometry (positive mode) and
nuclear magnetic resonance spectroscopy: 1H-RMN,
13C-RMN,
2D-COSY and 2D-HETCOR;
synthesis of 31 structural analogues with acetophenone
skeleton, including 30 new compounds whose synthesis hasn’t
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49
been reported in literature; the physico-chemical and spectral
characterization of synthesized compounds (elemental analysis, and structure elucidation by infrared spectroscopy, chemical
ionization mass spectrometry (positive mode) and nuclear
magnetic resonance spectroscopy: 1H-NMR,
13C-NMR, 2D-
COSY and 2D-HETCOR spectra: HMQC and HMBC));
highlighting antibacterial effects against Gram-negative
bacteria Escherichia coli ATCC 25922 and Pseudomonas
aeruginosa ATCC 27853 for the brominated compound 5e (2-
bromo-3', 5'-bis (2-methoxy-2-oxo-ethoxy)-acetophenone);
highlighting cytostatic/cytotoxic effects against various human
tumor cell lines (HeLa, MCF-7, A549, Caco2, PC3)
accompanied by a low toxicity against MCF-12F human normal
cells for the brominated compound 5c (2-bromo-2', 6'-bis (2-
mehtoxy-2-oxo-ethoxy)-acetophenone).
Research perspectives The results justify further studies on this species in the following
directions:
in vitro study of the cytotoxicity of the brominated derivatives
5a-e against other human tumor cell lines;
elucidation of the mechanism of the antitumor activity for the
brominated compounds 5a-e; investigation of their ability to induce apoptosis in various human tumor cell lines;
study of the antitumor activity and toxicity of brominated
derivatives 5a-e, particularly derivatives 5c and 5d
(experimental animal models);
evaluation of the antimicrobial potential and toxicity for
derivative 5e (experimental animal models);
isolation of other pure compounds from the leaves and roots of
Paeonia mlokosewitschii Lomakin. species, but also from other
plant parts;
evaluation, depending on the structural features, of other
biological effects of the isolated compounds and elucidation of
the mechanisms of activity;
synthesis of some structural analogues of the isolated
compounds with higher activity against tumor cells and of less
cytotoxicity on normal cells.
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SCIENTIFIC PAPERS
ISI publications
1. Zbancioc Ana Maria, Miron Anca, Tuchiluş Cristina,
Rotinberg Pincu, Mihai Cosmin Teodor, Mangalagiu Ionel,
Zbancioc Gheorghiţă. Synthesis and in vitro analysis of novel dihydroxiacetophenone derivatives with antimicrobial and
antitumoral activities. Med Chem 2014; 10, accepted for
publication. (IF=1.373)
2. Zbancioc Ana Maria, Miron Anca, Moldoveanu Costel, Zbancioc Gheorghiţă. Imidazolium Salts with
Dihydroxyacetophenone Skeleton with Anticipated Anticancer
Activity. Part II. Rev Chim 2013; 64 (6): 584-586. (IF=0.538)
3. Zbancioc Ana Maria, Zbancioc Gheorghiţă, Tănase Cătălin,
Miron Anca, Mangalagiu Ionel. Design, synthesis and in vitro
anticancer activity of a new class of dual DNA intercalators. Lett Drug Des Discov 2010; 7: 644-649. (IF=0.668)
4. Zbancioc Gheorghiţă, Zbancioc Ana-Maria, Mantu Dorina,
Miron Anca, Tanase Cătălin, Mangalagiu Ionel. Ultrasounds assisted synthesis of highly functionalized acetophenone
derivatives in heterogeneus catalysis. Rev Roum Chim 2010; 55
(11-12): 983-987. (IF=0.311)
Poster presentations
1. Zbancioc Ana Maria, Tătărînga Gabriela, Jităreanu Alexandra, Rotinberg Pincu, Cosmin Teodor Mihai, Zbancioc Gheorghiţă,
Mangalagiu Ionel, Miron Anca. New compounds with
acetophenone skeleton: synthesis and anticancer activity. Proceedings of the 13
th Panhellenic Pharmaceutical Congress,
Athens, Greece, 2013.
2. Zbancioc Ana Maria, Tătărîngă Gabriela, Jităreanu Alexandra, Miron Anca, Mangalagiu Ionel. Noi derivaţi de acetofenonă cu
citotoxicitate selectivă/New acetophenone derivatives with
selective cytotoxicity, 50 de Ani de Învăţământ Universitar
Farmaceutic în Iaşi/50 Years of Pharmaceutical Academic Education in Iaşi, Ed. Grigore T. Popa, 2011; 172-174.