short lecture - Faculty of Pharmacy

S1 APPLICATION OF RAMAN SPECTROSCOPY IN
DERMATOKINETIC STUDIES
VITALIS BRIEDIS 1 , DENIS NAUMENKO 2 , TOMA KEŢUTYTĖ 1 , KRISTINA
RAMANAUSKIENĖ 1 , VALENTINAS SNITKA 2
1 Lithuanian University of Health Sciences, Department of Clinical Pharmacy. A. Mickevičiaus 9, LT-
44307 Kaunas, Lithuania; Vitalis.Briedis@lsmuni.lt
2 Kaunas University of Technology, Research Centre for Microsystems and Nanotechnology. K.
Donelaičio 73, LT-44029 Kaunas, Lithuania; Valentinas.Snitka@ktu.lt
Application of Raman spectroscopy in analysis of biological objects is gaining
increasing attention because of modern technological solutions, offering new
possibilities in fast, reliable, reproducible and accurate determination of biologically
active substances in intact biological tissues. Raman spectroscopy is considered
as nondestructive and noninvasive technique which appears applicable for
qualitative and quantitative analysis of tissue structural components, externally
penetrating substances, and evaluation of processes occurring in biological
systems under normal and pathological conditions. If necessary, biological
samples used for analytical procedures are relative small and usually needs no
specific pretreatment.
Skin is considered as one of the most attractive and promising sites for application
of pharmaceuticals for local and systemic delivery of drugs. Dermis and epidermis
are main structural layers of normal skin. The outermost layer of epidermis is
stratum corneum (SC), which acts as the main barrier protecting and preventing
against external factors, controlling water flux. SC is also the main barrier on the
route of drug molecules penetrating/permeating the human skin. Therefore
thorough understanding of mechanisms of drug penetration through skin and
especially SC, the effects of drug penetration enhancers on skin structural
elements is absolutely necessary for development of efficient, safe, and stable
pharmaceuticals, for getting marketing approvals from regulatory authorities to be
used in modern medicinal therapy. Dermatokinetic studies constitute inseparable
part of such research and development projects. Similarly, determination of
dermatokinetic parameters is necessary in bioequivalence studies of topical and
transdermal pharmaceutical preparations. Today most often employed methods
include mainly in vitro and ex vivo techniques, and their results are related to
pharmacokinetic data of the drug and/or certain manifestation of therapeutic
efficacy. Therefore the techniques able to be applied for in vivo determination of
absorbed, distributed, and metabolized drug molecules and formulation excipients
should be considered at least as worth testing in research and development of
locally applied and transdermal formulations.
Nowadays Raman spectroscopy is believed to offer unique possibilities in
transdermal penetration/permeation studies. The increasing number of studies
employing Raman spectroscopy as a main investigative method confirms high
potential of this technique in performing skin penetration/permeation studies in
vivo and valuable input in development of efficient transdermal delivery systems.
Data provided by application of Raman spectroscopy confirms once again the
importance of preformulation studies and adequate selection of excipients. The
use of Raman spectroscopy in developing topical and transdermal pharmaceutical
formulations should increase the efficacy of research and provide solid ground for
thorough understanding of pharmaceutical product performance peculiarities.

S2 ASCENTIS® EXPRESS: A FUSED-CORE PARTICLE
HPLC COLUMN FOR HIGH SPEED AND HIGH
EFFICIENCY SEPARATIONS WITH LOW BACK
PRESSURES
JIŘÍ BRZOBOHATÝ
Sigma Aldrich
Ascentis Express columns provide a breakthrough in HPLC column
performance. Based on Fused-Core particle technology, Ascentis Express
provides the benefits of high speed and high efficiencies of sub-2 μm particles but
at much lower backpressure. Due to the high efficiencies at low backpressures,
Ascentis Express can benefit both conventional HPLC users as well as UHPLC or
other ultra pressure system users.
Increasing speed and resolution of HPLC analyses are drivers for
innovation in both HPLC column and hardware design. To date, reducing particle
size has been the strategy of many column manufacturers. Smaller particles result
in flatter van Deemter curves allowing for higher flow rates while still maintaining
near maximum efficiencies. The cost for the improved efficiencies is higher column
backpressures. To obtain the benefit of the sub-2 μm particles, instrumentation
beyond conventional HPLC is required. The Fused-Core particle consists of a 1.7
μm solid core and a 0.5 μm porous shell. A major benefit of the Fused-Core
particle is the small diffusion path (0.5 μm) compared to conventional fully porous
particles. The shorter diffusion path reduces axial dispersion of solutes and
minimizes peak broadening.
In fact, Ascentis Express columns are able to achieve efficiencies of
240,000 N/m, which is similar to that obtained with sub-2 μm particle columns,
even though the backpressures are only 50% of that achieved under similar
conditions with sub-2 μm particles.
This means that Ascentis Express can turn almost any HPLC system into
an extreme performance workhorse for your lab.

S3 DYNAMIC DISSOLUTION TESTING IN R&D
MARTIN CULEN, JIRI DOHNAL, JOSEF JAMPILEK, ANNA REZACOVA*
Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences,
Palackeho 1-3, 612 42 Brno, Czech Republic; e-mail: mculen@gmail.com
*Department of Methodological Development, Zentiva k.s., U kabelovny 130, 102
37 Prague 10, Czech Republic
Dissolution testing in R&D often requires more creative approach than the
classical USP 1 and 2 methods. For these purposes, our team has designed an
artificial digestive system – Golem, for biorelevant dissolution testing. The
apparatus simulates physiological human gastrointestinal conditions and it
constitutes a powerful tool for simulation of physiological dissolution of oral dosage
forms, especially when used with biorelevant dissolution media. The lecture
provides a brief description of the Golem system and an example of its use in
practice.

S4 USE OF COMPUTATIONAL METHODS IN THE STUDY
STRUCTURE – RETENTION
TATIANA DURCEKOVA a , KATARINA BORONOVA a , JAN MOCAK a ,
JOZEF LEHOTAY a , JOZEF CIZMARIK b
a Department of Chemistry, University of SS. Cyril and Methodius, J. Herdu 2, Trnava, SK-91701,
Slovak Republic, durcekova@ucm.sk, boronova@ucm.sk
b Department of Pharmaceutical Chemistry, Comenius University, Odbojarov 10, Bratislava,
SK-83232, Slovak Republic
This study compares the performance of two data mining methods - artificial
neural networks (ANN) and support vector machine (SVM), employed for the
prediction of HPLC retention factor determined for the group of esters of alkoxy
substituted phenylcarbamic acid 1,2 . To solve this problem, different molecular
descriptors were used. For their computation software packages ACDLabs and
HyperChem were used. Correlation analysis was used in the variable selection
process, by which the most informative molecular descriptors were selected
according to the value of p

S5 CALCIUM DOBESILATE IN EDUCATION AND THERAPY
OLDŘICH FARSA, MICHAL ŠABLATURA
Institute of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences Brno, Palackého 1/3 Brno, 612 42, Czech Republic, e-mail: farsao@vfu.cz
Introduction
Venotonic drugs are used in chronic venous insufficiency. They are useful also in
treatment of diabetic retinopathy. Nearly all of them are very efficient antioxidants
or reactive oxygen and nitrogen species (RONS) scavengers. They often contain
phenolic groups. Most of them are glycosides of plant origin with aglycones of
flavonoid structure, i.e. aglycones are polyhydroxylated derivatives of 2-phenyl-4Hchromen-2-one
or 2-phenyl-4H-1-benzopyran-4-one. Quercetin, which is the
aglycone of many glycosides including rutoside, can serve as a typical example.
Other glycosides, such as a saponoside escine (Reparil®) isolated from seeds of
horse chestnut Aesculus hippocastaneum, have triterpenic aglycones. (In fact,
escin is a mixture of several related compounds. The particular structure on the
Figure I express escin I which is a mixture of two geometric isomers. They differ in
E or Z configuration of 2-methylbut-2-enoyl which is attached to originally hydroxyl
oxygen in position 21 of the triterpenoid scaffold.) Flavonoid glycosides have
poor bioavailability which is due to their low lipophilicity. This problem has been
solved by etherification of phenolic groups of flavonoid aglycones with
hydroxyalkyl such as hydroxyethyl groups (troxerutin) in past. More recently,
increased bioavailability of flavonoid glycosides is reached by micronisation
(Detralex®, Daflon®) without changing their chemical structures. There is nearly
only one exception among these quite complex compounds: calcium dobesilate
(CD) (Danium ®, Doxium ®, Dexium®), which is a very simple synthetic molecule:
calcium 2,5-dihydroxybenzenesulfonate (see Figure 1)
HO
H
H
CH 3
HO
H HO
O
H
R
O H O
H
R = -H rutosid
R
O
O
HO
H HO
R = -OCH 2 CH 2 OH troxerutin
O
O
H
O
H
H
OH
free aglycone with R = -H and free
-OH in pos. 3 = quercetin
HO
HO
HO
HO
O
HO
O
HO
OH
O
OH
O
O
OH
OH
O
O
O
R
H3C HO
escin I
H
O
CH 3
H
R
H
C
H 3
CH 3
CH 3
OH
OH
O
S
O
O -
2
Calcium dobesilate
CH3 O
O
O
OH
OH
O CH3 CH 3
CH 3
Fig. 1: CD as the simplest structure among venotonic drugs.
Ca 2+

The first synthesis of 2,5-dihydroxybenzenesulfonic acid that can be found is that
in the article of Seyda, 1883. On principle it was sulfonation of hydroquinone with
diluted sulfuric acid in mild conditions; the reaction temperature did not exceed
50°C, for 3 hours. During this period, starting hydroquinone gradually dissolved in
diluted sulfuric acid. After cooling of the solution for 24 hours, crystals of 2,5dihydroxybenzenesulfonic
acid formed and could be filtered off. The filtrate was
diluted with water and under boiling saturated with barium carbonate. The
excessive barium carbonate that had not dissolved was then together with formed
barium sulfate filtered off. The filtrate was being concentrated in vacuo until a
white crystalline syrupy mass was formed. This viscous liquid was then poured on
a porous ceramic plate to be dried. Thus barium 2,5-dihydroxybenzenesulfonate
was yielded. Potassium 2,5-dihydroxybenzenesulfonate was prepared by adding
of potassium carbonate into an aqueous solution of barium 2,5dihydroxybenzenesulfonate
followed by filtering-off of formed barium carbonate
and concentration of the filtrate in vacuo into a viscous liquid that was then diluted
with double volume of absolute ethanol. A brown precipitate which had been
formed was then filtered off and all the ethanol was distilled off from the filtrate with
exclusion of air. Crystals, which had been formed from the resulted bright red
filtrate, were then recrystallized from water to give transparent octaedric crystals.
Sodium 2,5-dihydroxybenzenesulfonate was prepared similarly by adding of
sodium carbonate into a solution of the barium salt. It was formed in the
concentrated aqueous solution as a mass consisted from microscopic octaedric
crystals. Lead 2,5-dihydroxybenzesulfonate was prepared from the crude 2,5dihydroxybenzenesulfonic
acid and lead carbonate. It precipitated from a
concentrated aqueous solution as an amorphous solid which was no more soluble
in water. However, it was soluble in concentrated acetic acid. The lead salt gave
the free 2,5-dihydroxybenzenesulfonic acid by reaction with sulfane (see Figure 2).
OH
OH
H 2 SO 4
50 °C, 3h
OH
OH
PbCO 3
O
S
O
OH
H 2 S
OH
OH
-PbS 2
Ba 2 CO 3
O
S
O
O -
OH
OH
2
O
S
O
O -
2
Ba 2+
ZnSO 4
-BaSO 4
Me 2 CO 3
-Ba 2 CO 3
Me = Na, K
Fig 2.
Early syntheses of 2,5-dihydroxybenzenesulfonic acid and some its metallic salts.
Estéve-Subirana (Estéve-Subirana 1970) has developed a different synthesis of
CD which is also simple and suitable for the industrial production of it. It consists of
only one reaction of calcium hydrogensulfite with 1,4-benzoquinone. Such a
procedure could be classified as a „redox substitution― due to changes of the
oxidation states of both sulfur atom and benzene ring. However, such a synthesis
is not suitable for low scale preparations of CD including those which are
OH
OH
OH
OH
O
O
S
S
O
O
O -
O -
2
2
Zn 2+
2Me +

performed by students in practical classes in subjects such as medicinal or organic
chemistry. These practical educations can be met by students of fields such as
pharmacy or chemistry at appropriate schools or faculties. The main problem of
the Estéve-Subirana's procedure consists in use of calcium hydrogensulfite which
is not commercially available in solid state. Its concentrated aqueous solution is
frequently prepared by reaction of gaseous sulfur dioxide with an aqueous
suspension of calcium carbonate. This reaction can proceed in special counterstream
reaction vessels typical for industrial processes such as the procedure
developed by Rückauf and colleagues (Rückauf et al. 1990) (see Fig. 3)
CaCO 3 + SO 2 + H 2 O Ca(HSO 3 ) 2 + CO 2
O
2 + Ca(HSO 3 ) 2
O
2 Fig. 3 Preparation of
CD according to Estéve-Subirana (1970) preceded with preparation of calcium
hydrogensulfite according to Rückauf et al. (1990).
Results and discussion
To develop a simple and effective synthesis of CD which does not require calcium
hydrogensulfite, we returned to sulfonation of hydroquinone. After we had tried
several procedures using diluted sulfuric acid without significant successes
(Šablatura 2005), we came back to the original preparation of Seyda (Seyda,
1883). We modified his synthesis of barium 2,5-dihydroxybenzenesulfonate for
preparation CD in conditions of today. Hydroquinone was sulfonated with
concentrated sulfuric acid at 45°C until it dissolved; the resulted mixture was then
neutralized by short boiling with calcium carbonate. Poorly soluble calcium sulfate
precipitate was then filtered off. The filtrate was evaporated nearly to dryness. The
residuum was disperged in concentated ethanol and filtered. The ethanolic filtrate
was evaporated to dryness. Recrystallization of the residuum from the mixture
butan-1-ol / chloroform gave calcium dobesilate in the form of its monohydrate.
OH
OH
H 2 SO 4
45 °C, 3 h
OH
OH
OH
OH
O
S
O
O -
Ca 2+
(See Fig. 4.)
2
Preparation of CD proposed for practical courses in chemical disciplines
O
S
O
OH
CaCO 3
-CO 2 , -H 2 O
OH
OH
O
S
O
O -
Ca 2+
Fig. 4:
Crystallization trials from several mixtures with various ratios alcohol / water led in
most to solid solvates, e.g. calcium dobesilate : propan-2-ol 1 : 1.3 (dobesilate :
alcohol ratio in a sample was estimated from 1 H-NMR spectrum) (Šablatura 2005).

Simple confirmation of identity of the product by determination of its melting point
is impossible because of high value of its melting temperature, but it can be
identified by simple spectrophotometry in UV region according to The Czech
Pharmacopoeia 2005 Edition (The Czech Pharmacopoeia 2005) by means of a
value of specific absorption at 301 nm absorption maximum. This preparation
procedure including the identification of CD was published in Khimiya – Chemistry
(Farsa, Šablatura, 2008) and in more detailed and modified form also in an
instruction manual for practical courses in medicinal chemistry (Beneš, Farsa
2007). It is notable that, short time after its publishing, very similar procedures of
preparation of CD appeared in Chinese patents (Yao 2010, Yang 2010).
References
Beneš L., Farsa O. Medicinal Chemistry. An instruction manual for practical
courses. University of Veterinary and Pharmaceutical Sciences, Brno, 2007
Estéve-Subirana, A. Therapeutically active derivatives of p-dihydroxybenzene. US
3509207 (28. 4. 1970)
Farsa O., Šablatura M. An Alternative Synthesis of Calcium Dobesilate, a Simple
Venous Insufficiency Drug, Suitable for Use in Medicinal or Organic
Chemistry practical courses. Khimyia/Chemistry 17, 281-285 (2008)
Rückauf H., Turek F. et al. Verfahren zur Herrstellung von Hydrogensulfitlösungen.
DD 282899 (26. 9. 1990)
Seyda, A. Über Sulfonsäuren des Hydrochinons. Berichte der Deutschen
Chemischen Gesellschaft, 16, 687-694 (1883)
Šablatura, M. Development and optimization of a novel exercise for practical
courses in Medicinal Chemistry I. Master thesis. University of Veterinary
and Pharmaceutical Sciences, Brno, Czech Republic, 2006
The Czech Pharmacopoeia 2005. Grada, Prague, Czech Republic, 2005
Yang Y., Ma G., Long, H. Process for preparation of calcium dobesilate hydrate.
CN 101880248 (10. 11. 2010)
Yao C. Process for preparation of calcium dobesilate nanopowder. CN 101898980
(1. 12. 2010)

S6 SYNTHESIS OF SUBSTITUTED 2-
NAPHTHOYLANILIDES AS POTENTIAL
PHOTOSYNTHESIS INHIBITORS
GONĚC TOMÁŠ 1 , KRÁĽOVÁ KATARÍNA 2 , JAMPÍLEK JOSEF 1 , PAVLACKÁ
LENKA 1
1 Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences Brno, Palackého 1-3, 612 42 Brno, Czech Republic, tgonec@vfu.cz
2 Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina Ch-2,
842 15 Bratislava, Slovakia
Various compounds possessing an arylcarboxamide functionality
shows photosynthesis inhibition activities. A series of substituted 2naphthoylanilides
(GP01 - GP25) was prepared by two-step synthesis via 2naphthoylchloride
using method A or B (see scheme) in moderate to excellent
yields (33 – 96%).
O
OH
O
H
N
A: PCl 5
B: SOCl 2
B: toluene
Structure and purity of prepared compounds was confirmed by 1 H
NMR, 13 C NMR, IR, LC-MS spectroscopy and TLC.
Inhibition of photosynthetic electron transport in spinach chloroplasts
was determined. At higher applied concentrations the solubility of some
compounds in developing medium was limited. At least slightly soluble compounds
were tested and showed moderate to low activity in comparison with the
commercially available selective herbicide Diurone®.
R
N
H 2
A: N,N-dimethylaniline
B: TEA, methylene chloride
R = -H, -Br, -Cl, -F, -CH 3 , -OCH 3 , -CF 3 , -NO 2 , -OH
GP01 - GP25
O
R
Cl

S7 USE OF CYCLOFRUCTAN CHIRAL STATIONARY
PHASES FOR HPLC ANALYSIS OF POTENTIAL DRUG
HROBOŇOVÁ KATARÍNA 1 , LEHOTAY JOZEF 1 , ČIŢMÁRIK JOZEF 2 ,
ČIŢMÁRIKOVÁ RUŢENA 3 , ARMSTRONG DANIEL W. 4
1 Slovak University of Technology in Bratislava, Faculty Chemical and Food Technology, Institute of
Analytical Chemistry, Radlinského 9, 812 37 Bratislava, Slovak Republic;
email: katarina.hrobonova@stuba.sk
2 Comenius University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Odbojárov
10, 832 32 Bratislava, Slovak Republic
3 Comenius University, Faculty of Pharmacy, Department of Chemical Theory of Drugs, Odbojárov
10, 832 32 Bratislava, Slovak Republic
4 Department of Chemistry and Biochemistry, University of Texas at Arlington, Texas 76019, USA
Cyclofructans are a small group of macrocyclic oligosaccharides, which consist of
six or more β-linked D-fructofuranose units and represent a new class of chiral
selectors for HPLC. Their names are usually abbreviated as CF6, CF7, CF8, etc.
CF6 consists of an 18-crown-6 ether core and six fructofuranose units are
arranged in spiral fashion, either inward or outward around the crown ether
skeleton. CF6 shows a clear ―front/back‖ regionalization of hydrophilic and
hydrophobic groups. Derivatization of a native chiral selector with aliphatic or
aromatic functional groups can improve its chiral recognition capabilities.
Derivatized-cyclofructan bonded chiral stationary phases produced effective
enantiomeric separations for a variety of compounds [1-3].
Chiral potential local anaesthetic drug – derivatives of phenylcarbamic acid and
potential β-blockers of aryloxyaminopropanol type were used for HPLC study of
retention and enantioseparation on the RN-CF6 (R-naphtylethyl-carbamate CF6
chiral selector bonded on silica gel) and DMP-CF7 (dimethylphenyl-carbamate
CF7 chiral selector bonded on silica gel) chiral stationary phases in polar organic
separation mode. The results were compared with macrocyclic antibiotic and βcyclodextrin
chiral stationary phases. The mobile phase composed of methanol/
acetonitrile/ acetic acid/ triethylamine (20/80/0.3/0.2 v/v/v/v) was utilized as the
starting conditions for all tested chiral stationary phases.
The study was oriented on the testing of influence of mobile phase composition,
influence of analyte solvent nature, and influence of column temperature on
retention and enantioseparation of target analytes.
Based on the results obtained we tried to correlate the structures of the analytes
with their retention and resolution values in order to get some insight on interaction
types participating in the separation mechanism. The nature of analyte solvent
significantly influenced the retention and shape of elution peaks. The most
considerable effect was observed in the case of alcohols (methanol, ethanol,
propanol) as analyte solvent. Also the separation of different forms of studied
racemic compounds on chiral stationary phases was achieved. The effect was
observed on cyclofructan and macrocyclic antibiotic (Teicoplanin) chiral stationary
phases. This probably indicated the formation of solvatation complexes between
the solvent molecule and analyte which follows to change of retention properties of
formatted compexes. The studies with achiral potential local anaesthetic drugs
(similar structures as studied chiral derivatives) show the comparable results.

The thermodynamic study for derivatives of aryloxyaminopropanol indicated that
the resolution values of complexes not significantly decreased in the temperature
interval from 0 C to 50 C. The ratio of peak shapes of associates of studied
derivatives depended on the temperature.
References
[1] M. Kawamura, T. Uchiyama, T. Kuramoto, Y. Tamura, K. Mizutani: Formation
of a cycloinulo-oligosaccharide from inulin by an extracellular enzyme of
Bacillus circulans OKUMZ 31B. Carbohydr. Res., 192 (1989) 83-90
[2] M P. Sun, D.W. Armstrong: Effective enantiomeric separations of racemic
primary amines by the isopropyl carbamate-cyclofructan6 chiral stationary
phase.J. Chromatogr. A, 1217 (2010) 4904-4918
[3] P. Sun, C. Wang, Z.S. Breitbach, Y. Zhang, D.W. Armstrong: Development of
new HPLC chiral stationary phases based on native and derivatized
cyclofructans. Anal. Chem., 81 (2009) 10215-10226.
Acknowledgements: Work was supported by Scientific Grant Agency of the
Ministry of Education of the Slovak Republic and Slovak Academy of Sciences
(grants No. 1/0164/11, 1/0229/10, 1/266/10).

S8 AZAPHTHALOCYANINE DARK QUENCHER –
EVALUATION OF QUENCHING EFFICIENCY WITH
DIFFERENT FLUOROPHORES
KAMIL KOPECKÝ a , VERONIKA NOVÁKOVÁ b , MIROSLAV MILETÍN a , RADIM
KUČERA a , PETR ZIMČÍK a
Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Heyrovského 1203, 500 05
Hradec Králové, Czech Republic, kamil.kopecky@faf.cuni.cz
a Department of Pharmaceutical Chemistry and Drug Control
b Department of Biophysic and Physical Chemistry
We prepared new bifunctional derivative of azaphthalocyanine (AzaPc) which was
used for preparation of sequence of oligonucleotide. Quenching efficiency was
determined with six different commercially available fluorophores and compared to
efficiency with three commercially available dark quenchers of fluorescence, which
are routinely used in laboratories of molecular biology. DNA hybridization assays
confirmed high quenching efficiency (QE > 96%) of AzaPc quencher with six
different fluorophores ranging in emission maxima from 517 nm to 701 nm
(FAM,HEX, Cy3, Cy3.5, Cy5 and Cy5.5)
This work and its presentation was financially supported by Grant Agency of
Charles University (57810/2010) and Ministry of Education,Youth and Sports
(MSM 0021620822).
Literature:
Kopecky K, Novakova V, Miletin M, Kučera R, Zimcik P. Synthesis of new azaphthalocyanine dark
quencher and evaluation of its quenching efficiency with different fluorophores, Tetrahedron (2011),
in press, doi: 10.1016/j.tet.2011.06.038

S9 SYNTHESIS AND STUDY OF AGGREGATION
PROPERTIES AND ANTIPROTOZOAL ACTIVITIES OF
2-[DIMETHYL(OCTYL)AMMONIO]ETHYL OCTYL-
PHOSPHONATE AND ITS FLUORINATED ANALOGUE
LUKÁČ MILOŠ, 1 GARAJOVÁ MÁRIA, 2 MRVA MARTIN, 2 DEVÍNSKY
FERDINAND, 1 LACKO IVAN, 1 ONDRISKA FRANTIŠEK 3
1 Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University,
Kalinčiakova 8, 832 32 Bratislava, Slovakia; lukac@fpharm.uniba.sk; 2 Department of Zoology,
Faculty of Natural Sciences, Comenius University, Mlynská Dolina B-1, 842 15 Bratislava,
Slovakia; 3 HPL (Ltd) Department of Parasitology, Microbiological Laboratory, Istrijská 20, 841 07
Bratislava, Slovakia
Dialkylphosphocholines (DAPCs) are compounds with interesting physicochemical
properties and biological activities. They form micelles, coacervates, vesicles or gells in
dependence of their chemical structure [1]. DAPCs possess antimycotic, antiprotozoal and
antineoplastic activities [2]. The investigated compounds were prepared following the
Scheme depicted below. The micellization properties of DAPCs were determined by the
surface tension of their aqueous solutions. The antiprotozoal activites were studied against
acanthamoebae.
1)
Br
F
2)
3)
HO
F
F
F
O
O
O
O
O
Br P O
P
P
+ Br
+
O
- EtBr
O
NaOH/EtOH
O
F F
F
N +
F
F
F F
O
S
O -
O
F
F
I
Br
OH
O
P
O
+
+
O
O
O
Cl
P O
O
Bu 3 SnH
S O
+ R P
10 °C
F
- EtBr
F
F
F F
F
F
F
F
F F
pyridine O S
OH
OH
O
pyridine
TPSCl
F
F
O
P
O
O
P
O
O
O
O
O
HBr
HBr
+
F
F 3 C(CF 2 ) 5 I
F
F
HO
F F
F
F
F
F F
N
AIBN/Na 2 S 2 O 5
Scheme: Preparation of DAPCs (AIBN – azobisisobutyronitrile, TPSCl – 2,4,6-triisopropylbenzenesulfonyl
chloride, R-octyl or 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)
[1] Peresypkin AV, Menger FM: Org. Lett. 1, 1347 (1999)
[2] Lukáč M et al. Bioorg. Med. Chem Lett. 19, 6346 (2009)
This study was supported by grants: UK/243/2011, VEGA 1/0229/10, VEGA 1/0600/11
F
F
F
F
F
F F
F
F
F F
F F
O -
P O
O -
O
P O
O
N +
N +
F
F
F
HO
P
CH 3 CN
OH
P
O
OH
O
OH

S10 GA-ANN APPLIED IN THE QSAR STUDY OF
PHENYLBENZOXAZINE DIONES
PETER NEMEČEK 1 , JAN MOCÁK 1 , JOZEF LEHOTAY 1 , KAREL WAISSER 2
1
Department of Chemistry, University of Ss. Cyril and Methodius, Nám. J. Herdu 2, SK-91701
Trnava, Slovakia, peter.nemecek@ucm.sk
2
Faculty of Pharmacy, Charles University, Heyrovského 1203, CZ-50005 Hradec Králové, Czech
Republic
A combination of genetic algorithm (GA) and artificial neural networks (ANN),
considered as powerful contemporary computational techniques, was employed in
the QSAR study of a series of 64 substituted 3-phenyl-2H-1,3-benzoxazine-
2,4(3H)-diones (PBOD). This group of compounds exhibits a substantial biological
activity related to the mycobacteria growth inhibition. The same group of
compounds and similar descriptors we have already used in our previous QSRR
study for effective prediction of HPLC retention behaviour 1 .
All studied PBOD derivatives were biologically tested against three mycobacterium
strains, namely Mycobacterium tuberculosis, M. avium, and M. kansasii, which
represent a serious threat mainly for the HIV positive individuals and are the main
cause of their mortality. The data set contained 63 molecular descriptors plus
biological activity used as the target variable and expressed by the minimal
inhibition concentration (MIC) transformed into the –log(MIC) form. The set of
molecular descriptors used as the ANN input variables includes: simulated 1 H and
13 C NMR chemical shifts (in the form of the single shift values as well as the
averaged ring shifts), lipophilicity and solubility indices, further molecular
properties commonly used in QSAR (refractivity, polarizability, density, etc.) as
well as quantum chemistry descriptors (HOMO, LUMO, dipole moment, partial
charges, etc.) calculated by different software algorithms (ACDLabs, HyperChem,
Dragon, ChemDraw).
An important factor influencing a successful prediction of biological activity (MIC) is
reduction of the number of descriptors and finding an optimal set of descriptors
possessing the largest information value. For this purpose the genetic algorithm
was employed and ineffective descriptors were eliminated. The utilization of a
considerably reduced set of molecular descriptors, selected by GA, finally led to an
appropriate ANN model capable to predict the targeted biological activity in the
form of –log(MIC). The prediction was made for three series of the investigated
compounds - the training, validation and test subsets, created from the original
dataset by random selection. In addition, the –log(MIC) prediction was also
obtained for the series of promising compounds not yet synthesized.
Reference:
1. Nemecek P., Mocak J., Lehotay J., Waisser K.: J. Liq. Chrom. & Rel.
Technologies, 34 (2011) 168–181.
Acknowledgment: The support of this work by the grants VEGA 1/1005/09 and
FPPV-19-2011 is gratefully acknowledged.

S11 CHEMOENZYMATIC SYNTHESIS OF OPTICALLY
PURE ENANTIOMERS AFFECTING ADRENORECEPTORS
AND DETERMINATION OF THEIR ENANTIOMERIC
PURITY 1
OTEVŘEL JAN, GOLIANOVÁ JANA, BOBÁĽ PAVEL, CSÖLLEI JOSEF
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences
Brno, Palackého 1/3, 612 42 Brno, otevrelj@vfu.cz.
The series of compounds structurally related with arylaminoethanol family of
adrenomimetics were studied. These newly produced drugs are derived from the structures
of CL-316,243 and N-5984, the known potent and selective ß3-receptor binding agents. 2
The highly hydrophilic 1,3-benzodioxole-2,2-dicarboxylate moiety of CL-316,243
pattern is replaced with benzodioxole fragment or other ring isostere on the right-hand site
OH
NH
X
Y
R = H, 3-Me, 4-OMe,
3-Cl, 4-Cl, 3-F
R
X, Y = -O-, -CH2-
Figure: General structure of newly prepared compounds
(RHS) of the molecules.
This exchange probably
slightly lowers the ß3selectivity
but dramatically
increases the oral
bioavailability. 3 The RHS
parts of the substances
originate from piperonyl
alcohol, which was
oxidized with activated manganese (IV) dioxide to piperonal, then treated with nitroethane
in presence of ammonium acetate to get nitroolefin as a product of Henry additiondehydratation
reaction. This nitroalkene underwent Nef-type transformation with iron
powder and hydrochloric acid to
produce desired ketone.
On the left-hand site (LHS)
of the molecules there are differently
substituted phenyl rings in order to
influence their physicochemical
properties. Compounds were
prepared in enantiomerically
enriched form by convergent
HCN donor
R
R
Scheme: (R)-HNL catalysed cyanohydrin reaction
synthesis, which includes cyanohydrin reaction catalyzed by enzyme (R)-mandelonitrile
lyase (EC 4.1.2.10) from almond meal. Almond meal used in the experiments was obtained
by grinding almonds and defatting five times with ethyl acetate. Hydroxynitrile was
reduced to chiral aminoalcohol by lithium aluminium hydride and then used in reductive
amination step with appropriate ketone.
Optical purity of substances was determined by polarimetry, HPLC and NMR
spectroscopy.
1 This work was realized with financial support of IGA No. 63/2011/FaF
2 Nisoli, E.; Tonello, C.; Carruba, M. O.: ß3-Adrenergic Receptors: Really Relevant Targets for
Anti-Obesity Drugs? Curr. Med. Chem. 2003, 3, 257-273.
3 Sawa, M.; Harada, H.: Recent Developments in the Design of Orally Bioavailable ß3-Adrenergic
Receptor Agonists. Curr Med. Chem. 2006, 13, 25-37.
O
(R)-HNL
OH
N

S12 SEPARATION OF MONOSACCHARIDES AND
DISACCHARIDES BY HYDROPHILIC INTERACTION
LIQUID CHROMATOGRAPHY (HILIC)
PAZOUREK JIŘÍ, RZYMANEK MONIKA
Faculty of Pharmacy, VFU Brno, Department of chemical drugs, Palackého 1-3, CZ-612 42 Brno
Apart from the human nutrition, saccharides belong to an extremely
important part of both natural and synthetic drugs. Therefore, their analysis
represents an important branch of analytical methods. A challenge means
presence of many isomers and also absence of a suitable chromophore for a
direct UV-detection [1].
Hydrophilic interaction liquid chromatography, HILIC, is nowadays applied
to separation of non-derivatised saccharides, typically on an amino-propyl-silyl
(APS) stationary phase with a mobile phase acetonitrile:water [2].
In this case, however, separation of common monosaccharides was
obtained on a polar stationary phase Lichrosphere100 DIOL, Merck, in the HILIC
mode, detection was done with an ELSD (Evaporative Light Scattering Detector)
[3].
Very high selectivity for common mono- and disaccharides was reached
(see Figure below).
[1] Molnár-Perl, I., J. Chromatogr. A 891, 1-32 (2000).
[2] Hemström, P., Irgum, K., J. Sep. Sci. 29, 1784-1821 (2006).
[3] Pazourek, J., J. Sep. Sci. 33(6-7), 974 – 981 (2010).

S13 MOLECULAR DYNAMICS OF THE INACTIVE AND
ACTIVE CDK2 AND ITS STUDIES WITH INHIBITORS
MONIKA PĚNTÁKOVÁ
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences, Palackého 1/3, 612 42 Brno, Czech Republic
Cyclin-dependent kinases (CDKs) control the cell division cycle. Because deregulation of
CDKs has been proved in cancer series, these enzymes are promising biological targets for
designing of new inhibitors in human medicine [1]. Drug design is usually carried out on
inactive forms of CDKs. Herein the purine-based inhibitor roscovitine is chosen as a model
inhibitor [2].
Two complexes of both the inactive and active forms of CDK2 with roscovitine were
studied by means of nanoseconds-long molecular dynamics simulations (MD). Roscovitine
binds to the active site in a similar fashion as the native substrate (ATP) does, which means
that it is a competitive inhibitor of CDK2. Interactions of roscovitine in the active site were
analyzed in both forms of CDK2. Differences in CDK2 conformation were observed not
only in the flexible parts of CDK2 but also in the domains known to change their
conformation and position during activation, such as G-loop, PSTAIRE motif, CGMC
insert and T-loop (activation segment). Stability of the MD trajectories of both CDK2
complexes was verified on the basis of the root-mean-square deviation (RMSD), total
energy and secondary structural elements. The interaction energy of roscovitine with the
residues in the active site of CDK2 was calculated to assess the strength of binding. While
no significant differences were found for the van der Waals interactions, important
differences were observed in the electrostatic interaction term, especially for the Glu 12,
Lys 33, Glu 51 and Gln 131 residues. Other minor differences originated from the Hbonded
interactions. Nevertheless, the total interaction energy of roscovitine with CDK2
was very similar in both the active and inactive forms. The established facts support the
assumption that use of the inactive form as a model for drug design is adequate.
[1] Morgan D.O. The cell cycle: Principles of control. New Science Press:
London, UK, 2007.
[2] Meijer L., Borgne A., Mulner O., Chong J.P.J., Blow J.J., Inagaki N., Inagaki
M., Delcros J.-G., Moulinoux, J.-P. Biochemical and cellular effects of
roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases
cdc2, cdk2 and cdk5. Eur. J. Biochem. 1997; 243: 527–536.

S14 PRAGOLAB PROVIDES COMPLEX SOLUTION FOR
DRUG ANALYSIS AND DISCOVERY.
LUKÁŠ PLAČEK, PAVEL JANDERKA
Pragolab, s.r.o., Nad Krocínkou 55, Praha 9, www.pragolab.eu, pragolab@pragolab.cz
Mass spectrometry, high performance chromatography, capillary electrophoresis,
trace elemental analysis, atomic absorption spectroscopy, inductively coupled
plasma spectroscopy, UV-VIS spectroscopy, X-ray photoelectron spectroscopy,
etc. are the most powerful techniques used in drug discovery or drug analysis.
Pragolab covers all these techniques and provides the complex solution for
scientists, chemists, analysts or formulation engineers.
The Thermo Scientific TSQ Quantum XLS Ultra is the highest performing GC-
MS/MS instrument available. It features best in class target compound selectivity,
analytical performance and lab productivity – it’s the new ―Gold Standard‖ from the
technology leader GC-MS/MS with HyperQuad resolution FWHM 0.1 Da.
Orbitrap Elite mass spectrometer can analyze complex samples at a greater depth
than ever before, helping scientists meet the most demanding challenges in drug
discovery, proteomics, metabolomics or metabolism.
Figure 1: SRM chromatogram of IAA and glucose at the level of 50 pmol injected amount (TSQ Quantum).

Figure 2: 47+ charge state of yeast enolase showing resolution > 100.000 FWHM at 1,000 Da (Orbitrap).
The Thermo Scientific XPS (X-ray Photoelectron Spectroscopy) instruments such
as K-Alpha XPS can produce high-resolution, chemical-state images of complex
surfaces and nanosurfaces.
Figure 3: XPS spectra from three areas of a drug blister pack (K-Alpha).

Figure 4: XPS spectrometer K-Alpha.
Figure 5: Distribution maps of the whole Paracetamol
tablet surface (K-Alpha).

S15 In silico analysis of the interactions of β-blockers in
the β1-adrenergic receptor
POLAKOVIČOVÁ MÁJA 1 , GARAJ VLADIMÍR 2 , DEVÍNSKY FERDINAND 1 ,
ČIŢMÁRIKOVÁ RUŢENA 1
1
Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University,
Odbojárov 10, 832 32 Bratislava, Slovakia
2
Department of Medicinal Chemistry, Faculty of Pharmacy, Comenius University, Odbojárov 10,
832 32 Bratislava, Slovakia
polakovicova@fpharm.uniba.sk
β1-adrenergic receptors are members of a large family of G-protein coupled
receptors (GPCRs). Activation by endogenous catecholamines leads to the
coupling with G-protein and modulation of downstream effector proteins.
Drugs, β-blockers inhibiting the stimulatory effect of endogenous catecholamines
are widely used in treatment of cardiovascular diseases.
For better understanding of mechanism of action and rational drug design high
resolution 3D-structure is important.
GPCRs are very flexible and conformationally unstable. Obtaining diffracting
crystals has been exceptionally difficult. First image of GPCR crystal structure was
rhodopsine reported in 2000. To date only few GPCR structures has been solved.
In 2008 crystal structure of β1-adrenergic receptor complexed with cyanopindolol
was published [1].
Molecular docking and 3D-QSAR analysis
were performed to indentify important
interactions of selective β-blockers celiprolol
and metoprolol and phenoxyaminopropanol
type compounds prepared in our department
within the binding site of the β1-adrenergic
receptor.
Binding mode of selective β-blockers should
be slightly different comparing to the binding
mode of co-crystallized non-selective ligand in
the β1-adrenergic receptor. Substituent in
para-position interacts with amino acid
residues from extracellular loop 2 of the
receptor. Interactions in this non-conserved
region of GPCRs are supposed to be involved
in subtype selectivity. The extracellular loops
therefore could provide a diverse site for the
development of subtype selective drugs [2].
[1] Kobilka B.K.: Trends Pharmacol. Sci. 32, (2011), 213-218
[2] Peeters M.C. et al.: Trends Pharmacol. Sci. 32, (2011), 35-42
This contribution is supported by project 1/0229/10 of Research and Development Operation
programe funded by the ERDF

S16 SYNTHESIS AND EVALUATION OF ANTIMICROBIAL
EFFICACY OF NEW SINGLE CHAIN QUATERNARY
AMMONIUM SALTS
PUPÁK MATÚŠ 1 , LACKO IVAN 1 , DEVÍNSKY FERDINAND 1 , BUKOVSKÝ
MARIÁN 2 , KARLOVSKÁ JANKA 3
1
Department of Chemical Theory of Drugs, Faculy of Pharmacy, Kalinčiakova 8, SK-832 32
Bratislava; matus.pupak@gmail.com
2
Department of Cell and Molecuar Biology of Drugs, , Faculy of Pharmacy, Kalinčiakova 8, SK-832
32 Bratislava;
3
Department of Physical Chemistry of Drugs, , Faculy of Pharmacy, Kalinčiakova 8, SK-832 32
Bratislava
The work deals with the preparation and antimicrobial susceptibility testing of new
series of monomeric quaternary ammonium bromides (QUAT), derived from 2-
(dimethylamino)ethyl cyclohexanecarboxylate, with variable length of the side alkyl
chain (Fig. 1, R-C8H17 – C16H33). The antimicrobial efficacy of the QUATs prepared
expressed as the minimum microbicidal concentration (MMC) was determined
using two bacterial strains (Staphylococcus aureus ATCC 6538, Escherichia coli
ATCC 11229) and one yeast strain Candida albicans CCM 8186.
Fig.1. Structure of the compounds studied I – V (R-octyl, decyl, dodecyl,
tetradecyl, hexadecyl).
The MMCs values were obtained using micro-dilution test method based on the growth
inhibition of the microorganism by tested compound [1, 2]. All the QUATs prepared
showed antimicrobial efficacy in a different manner; the MMCs found were in the range
1280 μmol.l -1 – 8 μmol.l -1 . The compound IV (R = 14) was found to be the most efficient
when applied on the bacterial strains mentioned above and the compound V (R = 16)
showed the best antifungal activity. The relationships between chemical structure,
physicochemical properties and antimicrobial activity of prepared QUATs are discussed
using Kubinyi’s bilinear model [3].
[1] Lacko, I., Devínsky, F., Mlynarčík, D., Krasnec, Ľ. Acta Facult. Pharm. Univ.
Comenianae 30: 109-117 (1977).
[2] Lukáč, M., Lacko, I., Bukovský, M., Kyselová, Z., Karlovská, J., Horváth, B.,
Devínsky, F. Cent. Eur. J. Chem. 8(1): 194-201 (2010).
[3] Devínsky, F. et al. QSAR in Design of Bioactive Compounds, Ed. J.R. Prous,
Barcelona, s. 233 – 247 (1992).
Acknowledgement – this work was supported by Grants No. VEGA 1/0164/08, UK
/248/2011 and APVV 20-030804.

S17 5-SUBSTITUTED TETRAZOLES AS CARBOXYLIC
ACID BIOISOSTERES
JAROSLAV ROH, GALINA KARABANOVICH, KATEŘINA VÁVROVÁ,
ALEXANDR HRABÁLEK
Charles University in Prague, Faculty of Pharmacy, Department of Inorganic and Organic
Chemistry, Heyrovského 1203, 50005 Hradec Králové, Czech Republic; jaroslav.roh@faf.cuni.cz
Tetrazole is a synthetic heterocycle, with the highest content of nitrogen among
the stable heterocycles. Tetrazole-containing compounds are used in coordination
chemistry as the ligands, in photographic industry or as components of some
explosives [1]. Moreover, tetrazole ring is an important intermediate in the
synthesis of other heterocycles via various rearrangements [2]. However, the
essential usage of tetrazole can be found in medicinal chemistry. Thanks to its
resistance to metabolic degradation, variously substituted tetrazoles are often
used as bioisosteric surrogates for other functional groups [3].
The most remarkable compounds, which contain tetrazole ring, are 5-substituted
tetrazoles. It is because they represent a nonclassical bioisosteric replacement for
a carboxylic acid group. Although these two functional groups are structurally
different, they even have different number of atoms, thanks to their close physicochemical
properties they exhibit a similar type of biological activity [4].
The current approaches to 5-substituted tetrazoles synthesis, regioselective
functionalization and their usage in the medicinal chemistry will be presented.
This work was supported by Ministry of Education of the Czech Republic (MSM
0021620822) and Grant agency of Charles University (Project No. 55610/2010).
[1] Koldobskii, G. I.; Ostrovskii, V. A. Usp. Khim. 1994, 63, 847-865
[2] Moderhack, D. J. Prakt. Chem. 1998, 340, 687-709
[3] Myznikov, L. V.; Hrabalek, A.; Koldobskii G. I. Chem. Heterocycl. Comp. 2007,
43, 3-14
[4] Herr, R. Bioorg. Med. Chem. 2002, 10, 3379-3393

S18 CHARACTERIZATION OF IN VITRO METABOLITES
OF THE NOVEL THIOSEMICARBAZONE ANTI-TUMOUR
AGENT - Dp4cyclo4mT USING UPLC-QTOF
JAN STARIAT (1), PETRA KOVARIKOVA (1), JIRI KLIMES (1), RAIMO A.
KETOLA (2), DES R. RICHARDSON (3)
(1) Department of Pharmaceutical Chemistry and Drug Control, Faculty of
Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203,
500 05 Hradec Kralove, Czech Republic. jan.stariat@faf.cuni.cz
(2) Centre for Drug Research, Faculty of Pharmacy, University of Helsinki,
Helsinki, P.O. Box 56, FI-00014 Finland. raimo.ketola@helsinki.fi
(3) Iron Metabolism and Chelation Program, Department of Pathology and Bosch
Institute, University of Sydney, New South Wales 2006, Sydney, Australia.
d.richardson@sydney.edu.au
Selective targeting of the essential nutrient iron (Fe) using Fe chelators is a
promising new strategy for anti-cancer therapy, which can overcome resistance to
established therapeutical regimes.
Fe is a vital element for cell growth and metabolism, forming a crucial
component of the active site of ribonucleotide reductase, the rate-limiting enzyme
in DNA synthesis. Thanks to the extensive metabolic activity and grow, cancer
cells are more sensitive to Fe deprivation in comparison to normal cells.
Thiosemicarbazone iron chelators are among intensively studied anticancer drugs.
The novel thiosemicarbazones have been demonstrated to possess a complex
mechanism of anti-tumour action. Besides their ability to selectively bind iron and
thus inhibit grow and proliferation of cancer cells, their Fe complexes formed
inside the cancer cells, are able to produce cytotoxic reactive oxygen species
(ROS) that also contribute to anti-proliferative effect of these compounds.
Di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone
(Dp4cyclo4mT) is one of the most promising anti-proliferative agents currently
under development. Investigation of the fate of this drug in an organism is
essential for further progress in the development of this compound. Data about a
chemical structure of metabolites are essential to study biotransformation
pathways as well as to modify the chemical structure of a drug in order to improve
its pharmacokinetic/toxicological profile.
The aim of this project was to identify in vitro phase I. metabolites of
Dp4cyclo4mT using UPLC-QTOF.
The compound was incubated in vitro with human liver microsomes and
after simple treatment the samples were analysed on Acquity UPLC BEH C18
column (2.1×100 mm, 1.7 µm, Waters). 2 mM ammonium acetate pH 6 and ACN
were used as the mobile phase and the following gradient was applied: 0. min
10% B, 7. min 95% B, 9.5 min 95% B, 9.7 min 10% B, 12. min 10% B. The flow
rate of 0.3 mL/min, the column temperature of 25 ˚C and the injection of 7 µL were
employed. Xevo QTOF mass spectrometer with lock mass correction was utilised
as a detection technique. Data were processed manually as well as automatically
using MetaboLynx software. Structures of the putative metabolites were proposed

using accurate mass measurement as well as MS/MS fragmentation.
Several metabolic reactions including various combinations of the oxidative
substitution of sulphure by oxygen, hydroxylation, dealkylation, desaturation,
hydrolysis and ring opening by water addition were observed.
Further effort will be focused on the investigation of possible in vitro
phase II. conjugates. And finally, pilot in vivo experiments in rats will be performed
in order to confirm the relevance of our in vitro findings.
N
N
N
H
N N
This work was supported by the Internal Grant Agency of the Ministry of
Health of the Czech Republic (NT 12403-3/2011), by the Charles University in
Prague (SVV 263 001) and by the Centre for Drug Research (University of
Helsinki, Finland).
S
Dp4cyclo4mT
CH 3

S19 SYNTHESIS OF SHORT-CHAIN CERAMIDES AND THEIR
EFFECTS ON THE SKIN BARRIER FUNCTION
BARBORA ŠKOLOVÁ, KATEŘINA VÁVROVÁ, JAKUB NOVOTNÝ
Charles University in Prague, Faculty of Pharmacy, Department of Inorganic and Organic
Chemistry, Heyrovského 1203, 500 05 Hradec Králové; skolb5aa@faf.cuni.cz
Ceramides are a complex group of lipids, naturally occurring in the
uppermost layer of the epidermis. They constitute a major component of
extracellular matrix and they are responsible for the skin barrier properties.
Diseases such as atopic dermatitis or psoriasis are associated with the decline in
content and changes in the composition of ceramides.
Although the importance of ceramides is known, the relationship between
their structure and effect on the barrier function is not yet fully elucidated. Earlier
studies indicate that the length of ceramide acyl chain affects the skin
permeability. It appears that ceramides with short acyl lose the protective
properties.
We have prepared a series of analogues of ceramides with fifteen, twelve and
eight carbon atoms chain in the sphingosine part and the acyl part of a length of 2,
4 and 6 carbons. Starting substance of the synthesis was N-protected L-serine
methyl ester, which was further protected by the formation and subjected to the
reduction. The resulting aldehyde reacted with 1-alcynide, the triple bond was
subsequently reduced to a trans-double bond. After deprotection of the functional
groups, the sphingosine analogue was acylated using succinimidyl esters of the
pertinent acids to yield the target ceramides.
The effects of the prepared ceramides on the skin permeability were
evaluated on pig skin in Franz diffusion cells using two model drugs.
The results confirmed our hypothesis that short chain ceramides
decrease the barrier function of skin. Maximum increase in permeability was
achieved by applying the analogue with 4-carbon acyl chain, the highest
concentration of the drug in the skin was observed after the application of
ceramide with 6-carbons acyl chain. Surprisingly, these ceramides containing 15carbon
sphingosine generally caused greater permeability increase than those
with 12 and 18-carbon sphingosine. Nevertheless, the role of the sphingosine
chain length, double bond geometry and other structural features in the ceramide
molecule warrants further investigation.
This work was supported by the Charles University in Prague (Project
SVV 263 001) and the Czech Science Foundation (GACR 207/11/0365).

S20 PRENYL FLAVONOIDS AS PROMISING
BIOLOGICALLY ACTIVE COMPOUNDS
KAREL ŠMEJKAL 1 , STEFANO DALL´ACQUA 2 , ZUZANA HANÁKOVÁ 1 , ALICE NAVRÁTILOVÁ 1 ,
ONDŔEJ NEŚUTA 1 , JAN HOŠEK 1 , PETR KOLLÁR 3 , PETR BABULA 1
1 Department of Natural Drugs, Faculty of Pharmacy, UVPS Brno, Palackého 1-3, 612 42, Brno,
Czech Republic; karel.mejkal@post.cz, zuzana.hanakova@email.cz,
alousek.navratilova@seznam.cz, helezombie@seznam.cz, hosekj@vfu.cz, babulap@vfu.cz
2 Università degli Studi di Padova, Dipartimento di Scienze Farmaceutiche, Natural Product Lab
(NPL), Via Marzolo, 5 - 35100 Padova, Italy; stefano.dallacqua@unipd.it
3 Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, UVPS Brno,
Palackého 1-3, 612 42, Brno, Czech Republic; kollarp@vfu.cz
The biological activity of polyphenols varies and is often modified by presence of
different substitution on basic skeleton. One of the possible structural
modifications is substitution by prenyl side chain. The prenyl flavonoids incurred by
interference of phenolic and terpenoid metabolic pathway are often in focus of
phytochemists and experimental biologist because of their interesting chemical
properties and biological activities. The chemical character of prenyl flavonoids
combines the lipophilic properties of prenyl or geranyl side chains with
hydrophilicity of phenolic skeleton. The type of prenyl substitution and its
modification affects the biological activity of modified polyphenolic compounds.
Prenylated polyphenols can exhibit broad spectrum of biological effects, including
antioxidative, antiphlogistic, anticancerogenic. From wide spectrum of
pharmacological activities, the ability to inhibit growth or induce cell death of
bacteria and cancer cells significantly arises from above mentioned.
As a part of our effort to discover new natural compounds with potent biological
activity, we have isolated several prenylated and geranylated flavonoids from
Morus alba roots and Paulownia tomentosa fruits. These compounds have been
isolated by extensive chromatographic separation including reversed-phase
HPLC. Compounds were identified by means of UV, IR, HRMS, 1 H and 13 C NMR
and CD. Several of them have been isolated from the natural source for the first
time.
Compounds isolated were further used to elucidate their potential biological
activity. According to the literature search, prenylated and geranylated compounds
were subjected to assays of antibacterial, antileishmanial, cytotoxic and antiinflammatory
activity. Several interesting effects of compounds tested have been
identified. Compounds proved their ability both to kill leishmanias and Grampositive
bacteria in concentrations closed to concentration of standards.
Furthermore, several geranylated flavanones have shown interesting cytotoxic
activity based on specific structural parameters. The mechanism of action of
tomentodiplacone was elucidated. As a part of our latest studies, we also identified
potent anti-inflammatory activity of major flavonoid from M. alba root cudraflavone
B.
As can be seen for our work, we proved the promising bioactive potential of
prenylated and geranylated flavonoids. Our research on this topic will continue.
The project was supported by grant of IGA VFU 54/2011/FaF and grant of IGA VFU 7/2010/FaF.

S21 AN ANTI-INFLAMMATORY COMPONENT IN A
LEBANESE ENDEMIC PLANT, COTA PALESTINA,
EXHIBITS PARTHENOLIDE ANALOGOUS PREDICTED
BINDING AND ANTI-PROLIFERATIVE ACTIVITIES
RABIH TALHOUK 1,3 *, BILAL NASR 1,3 , MOHAMMAD-BILAL FARES 1,3 , BUSHRA
AJEEB 2,3 , TARK GHADDAR 2,3 and NAJAT SALIBA 2,3
1 Departments of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut,
Lebanon
2 Departments of Chemistry, Faculty of Arts and Sciences, American University of Beirut, Beirut,
Lebanon
3 IBSAR Center for Biodiversity, American University of Beirut, Beirut, Lebanon
* E-mail for correspondence: rtalhouk@aub.edu.lb
K100, an anti-inflammatory sesquiterpene lactone isolated from a Lebanese
endemic plant, Cota palestina is analogous to the feverfew-extracted parthenolide
(PT) that is known to inhibit proliferation and apoptosis in human cancer cell lines
and its analog DMAPT is currently in clinical trials. K100 is a germacranolide
1β,10α-Epoxy-6α-hydroxy-1,10H-inunolide and its potent activity could be
attributed to three functional groups: i) the exocyclic α-methylene-γ-lactone, ii) an
OH group adjacent to the α-methylene, and iii) an epoxide. K100, was identified
through bioactive guided fractionation using column chromatography and
spectroscopic techniques. Cytotoxicity of K100 was assessed using LDH and
trypan blue exclusion assays. IL-6 levels were monitored by ELISA to assess
K100 ability to suppress IL-6 in ET-treated SCp2 cells at non-cytotoxic
concentrations. K100 showed less cytotoxicity than PT, and inhibited the growth of
two mammary epithelial tumor cell lines (MDA-MB-231 and MCF-7) in a dose
dependent manner, at concentrations tolerable by the normal human mammary
epithelial cell line MCF10-A. Moreover, when compared to PT, K100 exhibited a
wider differential between concentrations affecting cancerous but not normal cells.
Molecular docking in silico of one K100 isomer and DMAPT against several target
proteins of PT, predicted that K100 can bind to all tested targets at similar
positions as PT, and with comparable binding affinities. In conclusion, we suggest
that K100 mediates its anti-inflammatory and anti-proliferative effects via pathways
similar to those utilized by PT.

S22 MODEL SKIN LIPID MEMBRANES: RELATIONSHIPS
BETWEEN CERAMIDE STRUCTURE, PERMEABILITY
AND BIOPHYSICS
BARBORA ŠKOLOVÁ, BARBORA JANŮŠOVÁ, JARMILA ZBYTOVSKÁ,
JAROSLAV ROH, KAREL PALÁT, ALEXANDR HRABÁLEK, KATEŘINA
VÁVROVÁ
Charles University in Prague, Faculty of Pharmacy, Heyrovského 1203, 50005 Hradec Králové, Czech
Republic; katerina.vavrova@faf.cuni.cz
Stratum corneum ceramides (Cer) play an essential role in the barrier properties of skin.
Our long-term aim is to study the structural requirements for Cer function in a competent
skin barrier because little is known about the role of the individual Cer species and their
structure-activity relationships. In this work, we investigated the effects of Cer acyl chain
length and polar head structure on the permeability of the skin and stratum corneum model
lipid membranes composed of Cer/lignoceric acid/cholesterol/cholesterol sulfate and their
thermotropic phase behavior. The long- to medium-chain ceramides (8-24C) did not
change the skin/membrane permeability. In contrast, the short-chain Cer decreased skin
electrical impedance and increased both skin and membrane permeability for two marker
permeants with maxima in the 4-6C acyl Cer. The infrared spectroscopy of the lipid
membranes revealed marked differences between the long and short-chain lipids: those
ceramides that had the greatest impact on the skin barrier properties displayed the lowest
phase transitions and formed the least dense, phase separated stratum corneum lipid
membranes at 32°C. In conclusion, the long hydrophobic chains in Cer are essential for
maintaining the skin barrier.
This work was supported by the Czech Science Foundation (207/11/0365) and Charles
University (SVV 263 001).

S23 WAY TO HIGHLY ACTIVE POTENTIAL
ANTITUBERCULOTICS.
KAREL WAISSER
Department of Inorganic and Organic Chemistry, Charles University in Prague, Faculty of
Pharmacy in Hradec Králové, Heyrovského 1203, CZ-50003 Hradec Králové, Czech Republic,
E-mail: Waisser@faf.cuni.cz
The musical composer and singer Jiří Zmoţek in a song of his once stated that
a rather thorny path leads to something that should become a hit. In his song
―Write a Song‖ he wrote that a hit is not just a bow. It is a hard work and a little
bit of luck is needed as well. Also pharmaceutical chemists working in drug
development long for a hit, i.e. a substance that will exceed the existing
pharmaceuticals. But it is also a hard work which requires a little bit of luck.
Recently the interest of the medical community was aroused by salicylanilides,
which can have the mechanisms of effect different from other antituberculotics,
being the inhibitors of the two-component system in bacteria. These systems,
consisting of histidine-proteinkinase and the regulator of the response, are the
signal transinductor systems, making it possible for bacteria to perceive
changes in their environment and to quickly respond by activating or repressing
the genes. They occur in all bacterial strains (including mycobacteria) and in
some fungi, but not in other eukaryotic organisms. The substances that inhibit
these systems are thus able to block important bacterial communication paths,
which can finally result in the death of the bacterial cell -3 . The pivotal world
scientific journals accept papers dealing with the studies of antimycobacterial
salicylanilides. In our laboratory we have also worked in this direction. We have
been searching for the substances with new mechanisms of effect, different
from the currently used drugs. Our approach aimed to prepare large groups of
substances which could be analyzed by the QSAR methods. In a paper 4
focused on the derivatives of salicylanilides substituted in position 5 in the
salicyl moiety of the molecule altogether 48 compounds were prepared. The
substances showed antimycobacterial effects. All compounds were active
against INH-resistant strains of M. kansasii and M. avium. A similar situation
was found also in other groups of substances reported in the paper. However,
we wanted to find substances whose activities could exceed INH even against
M. tuberculosis. QSAR analysis showed that the substitution in position 5 with
chlorine and bromine is advantageous. The synthesis was simple, consisting in
heating a mixture of salicylic acid (or substituted salicylic acid) in pyridine in the
presence of phosphorus trichloride with aniline derivatives.

In the following study, 143 compounds were prepared in a similar manner. The
group also included the derivatives substituted in position 4 in the salicyl moiety.
The study also showed a positive influence of electron-acceptor substituents. 5
The equations of Hansch type were also searched for to examine the influence
of lipophilicity. An increase in lipophilicity favourably influenced the activity.
Lipophilicity, however, had to be attributed to the substituents and not the
molecule as a whole. The activities of the most active substances were close to
that of INH against M. tuberculosis, but they did not reach the values of INH. To
obtain a more complex view, we began to monitor the isosteres of
salicylanilides. Besides salicylanilides, they included the isosteric derivatives of
3-hydroxypicolinic acid, 2-sulfanylbenzoic acid and the corresponding N-benzyl
derivatives of the amides of these acids. The relationships between the
structure and activity was analyzed using the Free-Wilson method. 6,7
The study resulted in a recommendation to examine the N-benzyl derivatives of
the corresponding amides. 6 Therefore we focused on salicyl-Nbenzylamides.
8.19 Later, in cooperation with the University in Ghent, the QSAR
methods, based on quantum-chemical approaches, showed that it was
necessary to return to salicylanilides. 11 Another intervention into the molecule of
antimycobacterial salicylanilides was the introduction of heterocycles instead of
phenyl to the molecule. 12-14 In this case we had to change the principle of
synthesis, because the previous simple procedure failed. We had to prepare
phenyl esters of salicylic acid (i.e. salols which we then allowed to react
with heterocyclic aromatic amines.) 14

The R 2 substituent was mainly hydrogen. To increase reactivity, in some cases
it was trifluoromethyl. Heterocyclic aryls:
Nevertheless, the activity against M. tuberculosis of none of the new
substances exceeded that of INH, the most effective being the derivatives of 5methyloxazol.
Due to its easy thermic decomposition, 4´-trifluomethylsalols had
to be used. The use of an analogical nitro derivative failed due to oxidative
reactions. Two review papers were written about the antibacterial activity of
salicylanilides 15,16 . On the basis of previous QSAR studies, attention was paid to
salicylanilides substituted with a halogen on the aryl moiety and the phenyl
in position 4 substituted with an alkyl. 17 We also invited to cooperate, besides
others, Prof. Míko from the Slovak Technical University in Bratislava. The most
active of the substances of the study was 4-chloro-4´-propylsalicylanilide,
whose activity against M. tuberculosis got closely near to that of INH and which
was active against IHN-resistant strains of M. kansasii and M. avium (moreover,
as all salicylanilides). The most valuable finding was that the mechanism of
activity was apparently different from what had been assumed yet. The case
was the inhibitors of ATP synthesis. The final study of antimycobacterial
salicylanilides was based on a new hypothesis of the mechanism of effect and it
demonstrated that also the methyl derivatives of salicylanilides were of
importance. 18 The most recent paper, published this year, examines pyridine
analogues of N-benzylsalicylamides. The paper calculated the geometry of the
molecules and verified it by hydrogen bonds in the IR spectra. Lipophilicity was
evaluated both by calculation and on thin layers in the reverse phase on
octadecylsilanylized silica gel. 19
In several recent years the study of these substances was the focus of research
by Associate Professor Jarmila Vinšová. If it were possible to bind a carrier to
the hydroxyl group, resorption from the digestive tract would be increased. Such
a carrier could be amino acids. The process, however, took its course in a
different way. Unexpected cyclizations occurred, interesting from the viewpoint
of organic chemistry. 20,21 Nevertheless, Prof. Vinšová managed to bind the
amino acid with a protected amino group to the hydroxyl group. The prepared

substances were also active towards mycobacteria. 22-24 If a substituted
carbamic group was bound to the salicyl hydroxyl group, activity against
multiresistant strains of M. tuberculosis was obtained. 25 Acylation of the
hydroxyl group of salicylanilides can thus be considered a fortunate choice in
the development of new active derivatives of salicylanilides. 26
Another stage in the development represented the antituberculotics derived
from salicylanilides, 3-phenyl-2H-1,3-benzoxazin-2,4(3H)-diones. They were
prepared by the reaction of salicylanilides with methyl-chloroformiate. The
reactions took place in lipophilic solvents, e.g. in toluene or chlorobenzene. The
products ranked among antimycobacterial substances. 27
When oxygen was replaced with nitrogen (it means that isosteric chinazoline
derivatives developed), antimycobacterial activity disappeared. For the QSAR
study 81 derivatives were prepared. 28 Activity was favourably influenced by
substitution with chlorine in position 7. There was a certain parallel between
antimycobacterial activity against salicylanilides and 3-phenyl-2H-1,3benzoxazin-2,4(3H)-diones.
30 Further procedures were aimed at the expansion
of the substances under study. Similarly as in salicylanilides, the benzyl group
was introduced as the substituent in position 3. 31 As substitution with halogens
increased activity, these substances were paid increased attention to. 32
The most active substances were obtained by replacing one or both oxo groups
in 3-phenyl-2H-1,3-benzoxazin-2,4(3H)-diones with a thioxo group. 33-37 The
path was not straightforward here, as we had to find the best substitution
analogies. 38,39 An introduction of the first thioxy group strongly increased
antimycobacterial activity of the substances. The second thioxo group did not
achieve a greater increase in the activity. The third thioxo group, which was
introduced into the substituents, strongly decreased the activity. 40 Macháček
and Skála made an attempt to analyze 3-phenyl-1,2,3-benzotriazin-4(3H)thiones.
41 The most important QSAR study of 3-phenyl-2H-1,3-benzoxazin-
2,4(3H)-diones containing one or two replacements of oxo groups with thioxo
groups can be considered the study which was published this year. 42 The
analysis revealed that not only substitutions with halogens is important, but also
the substitution with a methyl group in position 6. In the substitution on the
phenyl in position 4 an alkyl was of importance. This gave rise to the paper
proudly called highly active antimycobacterial derivates of benzoxazin. 43 The
substances are at present studied at the Department of Biochemical Sciences.
The activities of the most effective substances exceed that of INH. A preliminary
study has revealed that their toxicity is very low. When the methyl group is on
the benzoxazine cycle in position 7, the substances possess a high activity
against INH-resistant strains of M. avium. 44 Highly effective substances against
M. avium have their phenyl either unsubstituted or substituted with a methyl or
chlorine or bromine and also one or both oxo groups replaced by thioxy groups.
A high antimycobacterial activity was observed also in salicylthioamides 45 and

the most effective ones of them are also studied at the Department of
Biochemistry.
The preparation of the above-mentioned sulphur-substituted substances was
most frequently prepared by melting with phosphorus pentasulphide. However,
it was necessary to study the conditions. 46 Salicylthioamides were prepared via
a complex of salicylamides 45 with se phosphorus pentasulphide and subsequent
decomposition of the complex with hydrochloric acid.
At the end of the way to highly effective substances we hope that we have
found a hit. And, maybe, also some compounds produced by Assoc. Prof.
Jarmila Vinšová may become hits.
This work is financially supported by project no. MSM 0021620822 of the
Ministry of Education of the Czech Republic.
References
1. Macielag, M. J., Demers, J. P., Fraga-Spano, S. A., Hlasta, D. J., Johnson, S.
G., Kanojia, R. M., Russel, R. K., Sui ,Z., Weidner-Wells, M. A., Werblood, H.,
Foleno, B. D., Goldschmidt,R. M., Loeloff, M. J., Webb, G. C., Barrett. J.:
Substituted salicylanilides as inhibitors of two-component regulatory systems
,35in bakteria. J.Med.Chem., 41, 1998, 2939—2945.
2. Lu Guo, Qing-Li, W., Quian-Quian, J., Qiu-Ju, J., Yun-BaoJ.: Anion-trirrered
substituent-dependent conformational switching of salicylanilides, new hints for
understanding the inhibitory mechanism of salicylanilides. J. Org. Chem. 77,
2007, 9947—9953.
3. Hlasta, D..J., Demers, J. P., Foleno, B. D., Fraga-Spano, S. A., Guan, J.,
Gillard, J. J., Macielag, M.J., Ohemeng, K. A. , Shepard, Ch. M., Sui ,Z., Webb,
G. C.: ovel inhibitors of bacterial two-component systems with Gramm-positive
antibacterial aktivity: Pharmacophore identification based on the screning hit
closantel. Bioorg. and Med. Med. Chem. Letters, 8, 1998, 1923—1928.
4. Waisser, K., Hladůvková, J., Kuneš, J., Kubicová, L., Klimešová, V., Karajannis,
P., Kaustová, J.: Synthesis and antimycobacterial activity of salicylanilides
substituted in position 5. Chem. Pap. 55, 2001, 121-129.
5. Waisser, K., Bureš, O., Holý, P., Kuneš, J., Oswald, R., Jirásková, L., Pour, M.,
Klimešová, V., Kubicová, L., Kaustová, J.: Relationships between structure and
antimycobacterial activity of substituted salicylanides. Arch. Pharm. 336, 2003,
53-71.
6. Waisser, K., Peřina, M., Holý, P., Pour, M., Bureš, O., Kuneš, J., Klimešová, V.,
Buchta, Vl., Kubanová, P., Kaustová, J.: Antimycobacterial and antifungal
isosters of salicylamides. Arch. Pharm. 336, 2003, 322-335.

7. Waisser, K.,. Draţková, K, Matyk, J., Palát, K.: QSAR analysis of
antimycobacterial N-pyridinylsalicylamides. Folia Pharmaceutica Univ. Carol 33,
2005, 7-11.
8. Waisser, K., Peřina, M., Klimešová, V., Kaustová, J.: On the relationship
between the structure and antimycobacterial activity substituted Nbenzylsalicylamides.
Collect. Czech, Chem. Commun 68, 2003, 1275-1294.
9. Waisser, K., Peřina, M., Boudová, I., Kuneš, J., Kaustová, J.: Nová skupina
potenciálních antituberkulotik: Antimykobakteriální N-benzylsalicylamidy. (A
new group of potential antituberculotic agents. Antimycabacterial Nbenzylsalicylamides).
Česk. Slov. Farm. 52, 2003, 291-294.
10. Waisser, K., Draţková, K., Kuneš, J., Klimešová, V., Kaustová, J.:
Antimycobacterial N-pyridinylsalicylamides, isosters of salicylamides. Farmaco,
59, 2004, 615-625.
11. Doleţal R. Van Damme, S. Bultinck, P., Waisser, K.: QSAR analysis of
salicylamide isosteres with the use of quantum chemical molecular descriptors,
Eur. J. Med. Chem. 41, 2009, 869-876.
12. Waisser, K., Draţková, K., Matyk, J., Kuneš, J., Kaustová, J.: Heterocyclic
Isosters of Antimycobacterial 5-Methylsalicylanilides with Other Hetarocyclyc
Moiety as Pyridine. Folia Pharm. Univ. Carol. 31-32, 2004, 47-52.
13. WAISSER, K., DRAŢKOVÁ, K., MATYK, J., KUNEŠ, J., KAUSTOVÁ, J.: Heterocyclic
Isosters of Antimycobacterial Salicylanilides: N-Pyridyl-salicylamide with Steric
Hindrance of Amide Group. Folia Pharm. Univ. Carol. 31-32, 2004, 41-46.
14. Matyk, J., Waisser, K. , Draţková, K., Kuneš, J., Klimešová, V., Palát, K, Jr.,
Kaustová ,J.:Heterocyclic isosters of antimycobacterial salicylanilides. Farmaco
60, 2005, 399-408.
15. Kubicová L., Waisser K.: Biologická aktivita salicylanilidů. (Biological activity of
salicylanilides). Česk. Farm. 41, 1992, 208-216.
16. Vinšová J., Imramovský A.: Salicylanilidy - stále aktuální skupina s potenciální
antibakteriální aktivitou.Česk. Slov. Farm. 53, 2004, 294-299.
17. Waisser, K., Matyk, J., Divišová, H., Husáková, P., Kuneš, J., Klimešová, V.,
Möllmann, U., Dahse, H. M., MIKO, M.: The oriented development of
antituberculotics: Salicylamides. Arch. Pharm. Chem. Life Sci. 339,2006, 616-
620.
18. Waisser, K., Petrlíková, E., Kuneš, J., Vrabcová, P., Kolář, K., Stolaříková, J.:
The antimycobacterial salicylanilides, potential inhibitors of ATP synthesis. Folia
Pharm. Univ. Carol 39, 2010, 17-21.
19. Petrlíková, E., Waisser, K., Palát, K., Kuneš, J., Kaustová, J.: New Group of
Potential Antituberculotics: N-(2-Pyridylmethyl)salicylamides and N-(3-
Pyridylmethyl)salicylamides. Chem. Pap. , 65,. 201152–59.
20. Vinšová, J., Imramovský, A., Krátký, M., Férriz, J. M., Palát, K., Lyčka, A.,
Růţička, A.: An uprecedented rearrangement of salicylanilide deritive.
Imidazolinone inermediate formation. Tetrahedron Letters 51, 2010, 27-28.
21. Imramovský, A., Vinšová, J., Férriz, J. M., Kuneš, J., Pour, M., Doleţal, M.:
Salicalanilide esterifiction: unexpected formation of novel seven-mambered
rings. Tetrahedron Letters 47, 2006, 5007-5011.
22. Imramovský, A., Vinšová, J., Férriz, J. M., Buchta, V., Jampílek, J.:
Salicylanilide esters of N-protected amino acids as novel antimicrobial agents.
Bioorg. And Medicinal Chemisstry Letters, 18. 2009, 348-351.

23. Imramovský, A., Vinšová, J., Férriz, J. M., Doleţal , R., Jampílek, J., Kaustová,
J., Kunc.F.: New antituberculotics originated from salicylanilide with promising in
vivo activity against atypical mycobacterium strains. Bioorg. and Medicinal
Chemisstry 17. 2009, 3572-3579.
24. Krátký, M., Vinšová, J., Buchta, V., Horvati, K., Bösze, S., Stolaříková, J.: New
aminoacids esters of salicylanilides active against MDR-TB and other microbes.
Eur. J. Med. Chem. 45, 2010, 6106-6112.
25. Férriz, J. M., Vávrová, K., Kunc, F., Imramovský, A.,, Stolaříková, J., Vavříková,
E., Vinšová, J.:Salicyl carbamates. Antitubercular agensts active against
multidrug-resistant Mycobacterium tuberculosis strains. . Bioorg. and Medicinal
Chemisstry 18. 2010, 1054-1061..
26. Vinšová, J., Imramovský, A., Buchta, V., Cecková, M., Doleţal, M., Staud, F.,
Jampílek, J., Kaustová, J.: Salicylanilide acetates: Synthesis and antibacterial
evaluation. Molecules 12, 2007, 12-12.
27. Waisser, K., Hladůvková, J., Holý, P., Macháček, M., Karajannis, P., Kubicová,
L., Klimešová, V., Kuneš, J., Kaustová, J.: 2H-1,3-Benzoxazine-2,4(3H)-diones
substituted in position 6 as antimycobacterial agents. Chem. Pap. 55, 2001,
323-334.
28. Waisser, K., Gregor, J., Dostál, H., Kuneš, J., Kubicová, L., Klimešová, V.,
Kaustová, J.: Influence of the replacement of the oxo function with the thioxo
group an the antimycobacterial activity of 3-aryl-6,8.dichloro-2H-1,3benzoxazine-2,4(3H)-diones
and 3-arylquinazoline-2,4(1H, 3H)-diones.
Farmaco, 56, 2001, 803-807.
29. Waisser, K., Bureš, O., Holý, P., Kuneš, J., Oswald, R., Jirásková, L., Pour, M.,
Palát, K., Kaustová, J., Danse, H.-M., Möllmann, U.: Antimycobacterial 3-aryl-
2H-1,3-benzoxazine-2,4(3H)-diones. Pharmazie 58, 2003, 83-94.
30. Waisser, K., Draţková, K., Holý, P., Palát, K. Jr. Kaustová, J.: Correlation of
antimycobacterial activity of substituted salicylanilides and 3-aryl-2H-1,3benzoxazine-2,4(3H)-diones.
Folia Pharm. Univ. Carrol. 29-30, 2003, 17-20.
31. Waisser, K., Peřina, M., Kuneš, J., Klimešová, V., Kaustová, J.: 3-Benzyl-2H-
1,3-benzoxazine-2,4(3H)-diones, new group of antimycobacterial compounds
against potentially pathogenic strains. Farmaco 58, 2003, 1137-1149.
32. Waisser, K., Matyk, J., Divišová, H., Husáková, P., Kuneš, J., Klimešová, V.,
Kaustová, J.: The Oriented Development of Antituberculotics:(part 2.)
Halogenated 3-(4-Alkylphenyl)benzoxazine-2,4(3H)-diones. Arch. Pharm. Life
Sci., 340, 2007, 264-267.
33. Waisser, K., Holý, P., Bureš, O., Kuneš, J., Kaustová, J.: Nové skupiny
potenciálních antituberkulotik: 3-(4-ethoxythiokarbonylfenyl)-2H-benzoxazin-
2,4(3H)-dithiony a 3-(4-ethoxythiokarbonylfenyl)-4-thioxy-2H-benzoxazin-2(3H)ony.
- New groups of potential antituberculotics: 3-(4-ethoxythiocarbonylphenyl)-
2H-benzixazin-2,4(3H)-dithiones and 3(4-ethoxythiocarbonylphenyl)-4-thioxo-
2H-benzoxazin-2,4(3H)-ones. Česk. Slov. Farm. 52, 2003, 42-47.
34. Waisser, K., Gregor, J., Dostál, H., Kuneš, J., Kubicová, L., Klimešová, V.,
Kaustová, J.: Influence of the replacement of the oxo function with the thioxo
group an the antimycobacterial activity of 3-aryl-6,8.dichloro-2H-1,3benzoxazine-2,4(3H)-diones
and 3-arylquinazoline-2,4(1H, 3H)-diones.
Farmaco, 56, 2001, 803-807.

35. Waisser, K., Gregor, J., Kaustová, J.: High aktive potential antituberculotics with
thioxo group: 3-aryl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-aryl-2H-
1,3-benzoxazine-2,4(3H)-dithiones: Folia Pharm. Univ. Carol. 38, 2009, 43-46.
36. Skála, P., Macháček, M., Vejsová, M., Kubicová, L., Kuneš, J., Waissser, K.:
Syntnesis and atnitungal evaluation of hydroxy 3-phenyl-2-1,3-benzoxazine-
2,4(3H¨-diones and thein thioanalogs. J. Het.2009, 46, 873-880.
37. Waisser, K., Petrlíková,E., Peřina, M., Klimešová, , V., Kuneš , J., Kaustová J.,
Dahse, H.—M., Möllmann. U.:Note to biological activity of benzoxazine
derivatives containing thioxo group, Eur. J. Med. Chem. 2010, 45, 2719-2725
38. Gallegos, A., Carbó-Dorca, R., Ponec, R., Waisser, K.: Similarity approach to
QSAR. Application to antimycobacterial benzoxazines. Int. J. Pharmaceutics
269, 2004, 51-60.
39. Němeček, P., Mocák, J., Lehotay, J., Waisser, K.: QSAR study of potential
antituberulotic agents. Chem. Listy 2010, 104, 470-474.
40. Waisser, K., Čiţmárik, J., Holý, P., Petrlíková, E., Kuneš, J., Kaustová, J.:
Antimycobacterial fenyl-4-thioxo-2H-1,3-benzoxazine-2(3H)-ones and 3-(4ethoxythiocarbonylphenyl)-2H-1,3-benzoxazine-2,4(3H)-dithiones.
Acta Fac.
Pharm. Univ. Comen. 2009. 56. 171-179.
41. Skála, P., Macháček, M., Kubicová, L., Kaustová, J., Buchta, Vl., Waisser, K.:
Synthesis of 3-phenyl-1,2,3-benzotriazine-4(3H)-thiones, their antifungal and
antimycobacterial activities. : Folia Pharm. Univ. Carol. 38, 2009.
42. Petrlíková, E., Waisser, K., Doleţal, R., Holý, P., Gregor, J., Kuneš, J.,
Kaustová, J.: Antimycobacterial 3-phenyl-4-thioxo-2H-1,3-benzoxazine-
2(3H)ones and 3-phenyl-2H-1,3-benzoxazine-2,4(3H)dithiones substituted on
phenyl and on benzoxazine moiety in position 6, Chem. Pap. 2011,65,352-366.
43. Petrlíková, E., Waisser, K., Divišová, H., Husáková, P., Vrabcová, P., Kuneš, J.,
Kolář, K., Stolaříková, J.: Highly active antimycobacterial derivatives of
benzoxazine, Bioorg. Med. Chem. 2010, 18, 8178-8187.
44. Waisser, hitherto unpublished results.
45. Doleţal, R., Waisser, K., Petrlíková, E., Kuneš, J., Kubicová, L., Macháček
M.,.Kaustová, J., Dahse, H. M.: N-Benzylsalicylthioamides: highly active
potential antituberculotics. Arch. Pharm. Life Sci 342, 2009, 113-116.
46. Waisser, K., Petrlíková,E., Peřina, M., Klimešová, , V., Kuneš , J., Kaustová J.,
Dahse, H.—M., Möllmann. U.:Note to biological activity of benzoxazine
derivatives containing thioxo group, Eur. J. Med. Chem. 2010, 45, 2719-2725.

S24 MAGNESIUM AZAPHTHALOCYANINES – NEW
STRONG RED-EMITTING FLUOROPHORES
ZIMČÍK PETR, NOVÁKOVÁ VERONIKA, MILETÍN MIROSLAV, ŠVANDRLÍKOVÁ
EVA
Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec
Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05, Hradec Kralove, Czech
Republic, petr.zimcik@faf.cuni.cz
Azaphthalocyanines (AzaPc) are well-known aza-analogues of synthetic dyes
phthalocyanines (Pc) with very promising photophysical and photochemical
properties. They have been appreciated in many areas including medicinal
application in photodynamic therapy [1] or recently as fluorophores in DNA
hybridization probes [2] . However, the applications in fluorescent sensing utilized
only zinc Pc that are not optimal for this application from the point of view of
photophysical properties. Our research showed that simple exchange of central
metal may lead to changes in relaxation pathways of excited state and thus the
properties of final complexes may be simply optimized for different applications.
This work concerns with strong fluorescent properties of magnesium AzaPc that
showed generally fluorescence quantum yields over 0.70. Different synthetic
approaches leading to magnesium complexes were investigated and will be
discussed in the lecture. They involved both well-known magnesium butoxide
approach and incorporation method that has not yet been described for
magnesium complexes (see Figure). Optimization of the latter method was
performed.
Photophysical and photochemical properties of magnesium complexes were
studied on the series of Pc and AzaPc with different peripheral pattern (see
Figure) and compared with corresponding zinc complexes. Alkylsulfanyl
magnesium AzaPc showed the best photophysical properties, increased
photostability and well suited absorption and emission spectra that overlapped the
spectra of red-emitting standard Cy5 ® . In all parameters, this new structural type of
fluorophores exceeded the established standard and showed high promise for
future development.
R
R
R
X
X
R
N
X
X
N
NH
N
N
HN
N
X
X
N
R
R
X
X
R
R
Mg(CH 3COO) 2, pyridin,
reflux, 30 min
R
R
R
X
X
R
N
X
X
N
N
N
Mg
N
N
N
X
X
N
R
X
X
R
R
R
X = N, CH
[1] Zimcik P., Miletin M., Radilova H., Novakova V., Kopecky K., Svec J., Rudolf E.
Photochem. Photobiol., 2010, 86, 168-175.
[2] Nesterova I. V., Erdem S. S., Pakhomov S., Hammer R. P., Soper S. A. J. Am.
Chem. Soc., 2009, 131, 2432-2433.
R =
O
S
N

S25 SYNTHESIS AND ANTIMYCOBACTERIAL
PROPERTIES OF NEW PYRAZINAMIDE ANALOGUES
JAN ZITKO 1 , MARCELA VEJSOVÁ 1 , MICHAELA SVOBODOVÁ 2 , MARTIN
DOLEŢAL 1
1 Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, Hradec Kralové,
500 05, Czech Republic, jan.zitko@faf.cuni.cz
2 Department of Microbiology, Regional Hospital, Kyjevská 44, Pardubice, 532 03, Czech Republic
Pyrazinamide (PZA) is one of the most important antitubercular drugs. Along with
rifampicin, it’s the only used active substance to posses so called sterilizing activity
– the ability to kill the dormant non-growing tubercle bacilli of low metabolism
activity. The aim of our project is to synthesize various compounds derived from
PZA and test them for in vitro antimycobacterial activity (Mycobacterium
tuberculosis H37Rv, M. avium and M. kansasii). As a complementary test, all the
prepared compounds are tested for antifungal and antibacterial activity against
selected species.
6-chloro-5-cyanopyrazine-2-carboxamide (I) treated with various non-aromatic
amines yielded N-substituted 6-amino-5-cyanopyrazine-2-carboxamides (II) 1 .
Similarly, 3-chloropyrazine-2,5-dicarbonitile (III) yielded N-substituted
3-aminopyrazine-2,5-dicarbonitriles (IV). In this series, both chlorinated
compounds I and III exhibited interesting antimycobacterial activity comparable
with PZA against M. tuberculosis (MIC = 12.5-25 μg/mL). More importantly, they
were also active against non-typical mycobacteria (M. kansasii, M. avium), which
are completely resistant to PZA itself. Lipophilicity was one of the determinants of
antimycobacterial activity and in series II and IV the activity culminated in
heptylamino substituted compounds (MIC = 12.5 μg/mL for M. tuberculosis).
To further examine the effect of increased lipophilicity, we prepared similar series
with benzylamine substitution (V, VI). Preliminary results suggest similar level of
activity against M. tuberculosis (MIC = 6,25 - 12,5 μg/mL).
5-Chloropyrazinamide is well known to inhibit the FAS I (Fatty Acid Synthase I)
pathway 2,3 , impairing the building of normal mycobacterial cell wall. Inspired by
this, we synthesized a series of 5-chloro-N-phenylpyrazine-2-carboxamides
(VII). The substituents in the aniline part of the molecule were chosen upon our
previous experience (e.g. -CH3, 3-Cl, 3,4-Cl2, 3-I -4-CH3, 3-CF3). The activity is
being evaluated.
This study was supported by the Grant Agency of Charles University in Prague
(B-CH/120509) and by the Charles University in Prague (SVV-2011-263-001).

References:
1. Zitko J.; Dolezal, M.; Svobodova, M.; Vejsova, M.; Kunes, J.; Kucera, R.; Jilek, P. Bioorg. Med.
Chem. 2011, 19, 1471-1476.
2. Zimhony, O.; Cox, J. S.; Welch, J. T.; Vilcheze, C.; Jacobs, W. R., Jr. Nat. Med. 2000, 6, 1043–
1047.
3. Ngo, S. C.; Zimhony, O.; Chung, W. J.; Sayahi, H.; Jacobs, W. R., Jr.; Welch, J. T. Antimicrob.
Agents Chemother. 2007, 51, 2430-2435.

S26 SECONDARY METABOLITES FROM TURKISH GLOBULARIA SPECIES
AND THEIR CHEMOTAXONOMIC IMPORTANCE
KIRMIZIBEKMEZ H 1 AND CALIS I 2
1 Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, TR-34755, Kayisdagi, Istanbul, Turkey
2 Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Nicosia, Turkish Republic of Northern Cyprus
The genus Globularia (formerly Globulariaceae, now “new” Plantaginaceae) with its around 22 species is
distributed to mainly Europe, and Mediterranean region. In the flora of Turkey, nine species are growing wild
three of which are endemic 1,2 . Some of these species are used as diuretic, laxative, stomachic, tonic and for
the treatment of haemorrhoids in Anatolian folk medicine 3,4 . Among these species, G. alypum is widely used in
indigenous systems of medicine in some Mediterrean countries, especially in Morocco for a variety of
purposes such as hypoglycaemic agent, laxative, cholagogue, stomachic and purgative as well as
cardiovascular diseases 5 . As a part of our ongoing research on Turkish medicinal plants we have investigated
the secondary metabolites of eight Globularia species namely, G. trichosantha, G. orientalis, G. cordifolia, G.
dumulosa, G. davisiana, G. sintenisii, G. alypum and G. aphyllanthes. Totally, 69 diverse compounds, which
can be categorized under eight chemical classes; iridoids (32), phenylethanoid glycosides (17), flavone
glycosides (9), lignan glycosides (4), sugar esters (3), sterols (2), acetophenone glycoside (1), and
phenylpropanoid glycoside (1) were obtained from the MeOH extracts by utilizing VLC, C18-MPLC and OCC
(SiO2 and Sephadex LH-20) techniques. Identification of the isolates was accomplished by using
spectroscopic analysis including 1D and 2D NMR and MS experiments as well as chemical methods. Among
the isolated metabolites 19 were reported for the first time by this work, while many of them were new to the
genus Globularia. The occurrence of such diverse compounds in Globularia possesses chemotaxonomical
importance at both the genus and family level. A recent phylogenetic study 6 based on the DNA sequence of
the genus Globularia placed the genus under the “new” Plantaginaceae family, which was in good accordance
with our results.
References: 1. Edmondson, J.R. (1982) Globularia L., in: Flora of Turkey and the East Aegean Islands. Vol. 7 (Ed. Davis P.H.),
University Press, Edinburgh. 2. Duman, H. (2001) Bot. J. Linn. Soc. 137: 425-428. 3. Baytop, T. (1999) Therapy with Medicinal Plants
in Turkey (Past and Present), Nobel Tip Kitapevleri, Istanbul, p. 371. 4. Sezik et al. (1991) J. Ethnopharmacol. 35: 191-196. 5.
Bellakhdar, J. et al. (1991) J. Ethnopharmacol, 35: 123-143. 6. Albach, D.C. et al. (2005) Am. J. Bot. 92: 297-315.