UNIVERSITY OF MEDICINE AND PHARMACY … OF MEDICINE AND PHARMACY "GRIGORE T. POPA" IASI ... Ana Maria Balan ... Escherichia coli O-80 0.99 > 1.99 0.19 > 1.99 1.99

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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


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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


3.3.4. 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


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


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


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.


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.

15

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'';

16

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)



Spectral analyses showed structural identity between compound FPA-2-1 and compound 1.

17

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)

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.



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.

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.

20

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


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

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


acetophenone

C14H16O7

MW=296.27 g/mol

Compound 3c


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%).

22

Compound 3d


acetophenone

C14H16O7

MW=296.27 g/mol

Compound 3e


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


acetophenone

C11H12O5

MW=224.21 g/mol

Compound 4c


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

23

4.2.2. Physical characterization


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%).

24

Compound 5b



C14H15O7Br

MW=375.17 g/mol

Compound 5c



C14H15O7Br

MW=375.17 g/mol

Compound 5d



C14H15O7Br

MW=375.17 g/mol Compound 5e



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

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


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

26

Compound7b

1-(2-(2',5'-bis(2-methoxy-2-


1-ium bromide

C18H19O7N2Br

MW=455.26 g/mol

Compound 7c

1-(2-(2',6'-bis(2-methoxy-2-


1-ium bromide

C18H19O7N2Br

MW=455.26 g/mol

Compound 7d

1-(2-(3',4'-bis(2-methoxy-2-


1-ium bromide

C18H19O7N2Br

MW=455.26 g/mol

Compound 7e

1-(2-(3',5'-bis(2-methoxy-2-


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.

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-



C22H21O7N2Br

MW=505.32 g/mol

Compound 9c

2-(2-(2',6'-bis(2-methoxy-2-



C22H21O7N2Br

MW=505.32 g/mol

Compound 9d

2-(2-(3',4'-bis(2-methoxy-2-



C22H21O7N2Br

MW=505.32 g/mol

Compound 9e

2-(2-(3',5'-bis(2-methoxy-2-



C22H21O7N2Br

MW=505.32 g/mol

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)

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)

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

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


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).

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;


6.2.2.2. The effects of brominated derivatives on protein


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;


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


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)

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


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)

35

6.2.3.2.2. The effects on A549 human alveolar


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


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)

36

6.2.3.2.4. The effects on PC3 human prostate


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


6.2.3.3.1. Pro-oxidant activity in MCF-7 human breast


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)

37

6.2.3.3.2. Pro-oxidant activity in A549 human alveolar


In A549 tumor cells, the brominated derivatives 5a-e increased

intracellular ROS levels, the most active being 5e.


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


All brominated compounds showed a remarkable pro-oxidant

activity in Caco2 cells.


in Caco2 tumor cells (*p < 0.05; **p < 0.01; ***p < 0.001)

38

6.2.3.3.4. Pro-oxidant activity in PC3 human prostate


On PC3 tumor cells, the pro-oxidant effects of brominated

derivatives 5a-e were insignificant.


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


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)

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).

40

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

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).

42

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-ium bromide (9c); - 2-(2-(3',4'-bis(2-methoxy-2-oxoethoxy)phenyl)-2-oxoethyl)phthalazin-

2-ium bromide (9d);


2-ium bromide (9e).

43

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).

44

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:

45

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:


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:


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.

46

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.

47

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%).

48

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

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.

50

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54

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.

Documents

UNIVERSITY OF MEDICINE AND PHARMACY … OF MEDICINE AND PHARMACY "GRIGORE T. POPA" IASI ... Ana Maria Balan ... Escherichia coli O-80 0.99 > 1.99 0.19 > 1.99 1.99