Asymmetric synthesis and study of biological activity of (epi-)benzoanalogues of bioactive phenanthroquinolizidine alkaloids

Article abstract of DOI:10.1007/s00706-018-2244-5

Abstract: The increasing microbial resistance to primary active structures remains alarming and the effort to look for new antibacterial active structures is still of scientific interest. One of the attractive ways to find new active structures is derivatization of well-known natural compounds. Alkaloids are a structurally diverse group of natural products with a wide range of biological effects. Historically, an attempt to increase the antimicrobial activity of alkaloids through chemical modifications has been successful. In this work, 12 new quinolizidine derivatives were synthesized and tested for their antimicrobial activity. The asymmetric synthesis of the benzoanalogue of the phenanthroquinolizidine bioactive alkaloid (?)-cryptopleurine and the epi-benzoanalogues of (?)-(15R)-hydroxycryptopleurine were achieved in six or seven steps starting from available enantiopure (S)-2-aminoadipic acid used as source of chirality as well as nitrogen. The highest antimicrobial activity was observed in the presence of the final saturated structure, the benzoanalogue of naturally occurring plant alkaloid cryptopleurine. It features selective toxicity, and significantly inhibits the growth of G⁺ bacteria, especially Staphylococcus sp. Tested derivatives have shown only a weak antifungal activity, but partial inhibition has been observed in the case of model yeasts. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-018-2244-5

Monatshefte für Chemie - Chemical Monthly
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ORIGINAL PAPER
Asymmetric synthesis and study of biological activity of (epi-)
benzoanalogues of bioactive phenanthroquinolizidine alkaloids
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Tomas Pagac Peter Safar Stefan Marchalın Zuzana Jezıkova Barbora Balonova Miroslava Supolıkova
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Eva Novakova Jana Kubıckova Michal Soral Julius Sivy Petra Olejnıkova
Received: 17 March 2018 / Accepted: 23 May 2018
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
The increasing microbial resistance to primary active structures remains alarming and the effort to look for new
antibacterial active structures is still of scientific interest. One of the attractive ways to find new active structures is
derivatization of well-known natural compounds. Alkaloids are a structurally diverse group of natural products with a wide
range of biological effects. Historically, an attempt to increase the antimicrobial activity of alkaloids through chemical
modifications has been successful. In this work, 12 new quinolizidine derivatives were synthesized and tested for their
antimicrobial activity. The asymmetric synthesis of the benzoanalogue of the phenanthroquinolizidine bioactive alkaloid
(-)-cryptopleurine and the epi-benzoanalogues of (-)-(15R)-hydroxycryptopleurine were achieved in six or seven steps
starting from available enantiopure (S)-2-aminoadipic acid used as source of chirality as well as nitrogen. The highest
antimicrobial activity was observed in the presence of the final saturated structure, the benzoanalogue of naturally
occurring plant alkaloid cryptopleurine. It features selective toxicity, and significantly inhibits the growth of G? bacteria,
especially Staphylococcus sp. Tested derivatives have shown only a weak antifungal activity, but partial inhibition has been
observed in the case of model yeasts.
Graphical abstract
Keywords Antimicrobial activity Á Benzoanalogue of the natural phenanthroquinolizidine bioactive alkaloids Á
Quinolizidine derivatives
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-018-2244-5) contains
supplementary material, which is available to authorized
users.
4
Central Laboratories, Faculty of Chemical and Food
Technology, Slovak University of Technology in Bratislava,
81237 Bratislava, Slovak Republic
´
´
& Petra Olejnıkova
petra.olejnikova@gmail.com
5
1
Institute of Mathematics and Physics, Faculty of Mechanical
Engineering, Slovak University of Technology in Bratislava,
´
Namestie slobody 17, 812 31 Bratislava, Slovak Republic
Institute of Biochemistry and Microbiology, Faculty of
Chemical and Food Technology, Slovak University of
Technology in Bratislava, 81237 Bratislava, Slovak Republic
2
Department of Organic Chemistry, Faculty of Chemical and
Food Technology, Slovak University of Technology in
Bratislava, 81237 Bratislava, Slovak Republic
3
Department of Microbiology and Virology, Faculty of
Natural Sciences, Comenius University, 84215 Bratislava,
Slovak Republic
123

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T. Pagác et al.
Introduction
Results and discussion
Efforts to discover new antibacterial active compounds are
constantly growing, spurred by the development of
microbial resistance. The development and increase in
resistant microorganisms to the originally effective struc-
tures forces the scientists to look further for active com-
pounds [1]. The current trend favors the derivatization of
natural molecules by organic synthesis [2]. Alkaloids are a
structurally diverse group of natural products with a wide
range of biological effects [3]. Many naturally occurring
plant alkaloid derivatives have shown wide spectra of
biological properties, including cytotoxic, oxytocic, anti-
pyretic, antibacterial, antiviral, and hypoglycemic activi-
ties, as determined by in vivo pharmacological screening
[4]. Some of them are used in clinical treatment of diabetes
[5] or they act as inhibitors of testosterone-5a-reductase,
which is of great importance for treatment of various skin
diseases, for example, acne treatment, as well as excessive
hair loss treatment [6, 7]. An attempt to increase the
antimicrobial activity of alkaloids by chemical modifica-
tions has been quite successful. Interesting biological
activity has been achieved in compounds where the indole,
isoquinoline, pyrrolide, and thiazole were used as phar-
macophore [3]. Quinolizidines (QA), known as lupine
alkaloids are rare, but their biological activity is well
documented. In nature they originate from plants, mostly as
secondary metabolites of Lupinus, Leguminosaceae [8] and
are derived from the amino acid lysine (Lys) [4]. Lys is
known as the precursor of piperidine, quinolizidine, indo-
lizidine and lycopene alkaloids as compounds occurring in
the plant kingdom. Natural QA alkaloids are synthesized
by cyclization of the cadaverine unit and are further
modified by adaptation reactions (e.g., dehydrogenation,
oxygenation, or esterification) to give hundreds of struc-
turally related alkaloids [9]. Synthetically prepared
derivatives of natural lead structures possess interesting
biological effects as well even negligible antimicrobial
activity [10]. A number of synthetically prepared deriva-
tives had been tested as antimycobacterial compounds for
the potential treatment of tuberculosis [11]. An attempt to
increase the antimicrobial activity of alkaloids by chemical
modifications has been quite successful [2]. According to
antimicrobial activity of quinolizidine structures they show
greater efficacy on G? bacteria and mycobacteria [12].
Animal toxicity studies using CHO-K1 [13], HEK 293
[14], and VERO cells [12] suggest that the compounds
have selective toxicity on prokaryotic cells. In this work,
benzoanalogues and epi-benzoanalogues of bioactive
phenanthroquinolizidine alkaloids (Fig. 1) were prepared
by asymmetric synthesis and were tested for their potential
antimicrobial activity (Scheme 1).
The synthesis of the starting pyridoisoquinolinedione 4
comprises reductive amination of benzaldehyde with
available (S)-2-aminoadipic acid (1), followed by an
intramolecular N-acylation of the resulting N-benzyl
aminoadipic acid 2 to the 6-oxopipecolinic acid 3 [15].
Finally, intramolecular Friedel–Crafts acylation afforded
ketone 4 in 56% overall yield [16].
The diastereoselective reduction of the ketone 4 with
NaBH4 reflects a preference for an axial hydride attack
from the more hindered endo face of the tricyclic system
(Scheme 2). Thus, the reduction with NaBH4 at 0 °C in
methanol gave trans-5a in 73% yield. The stereoselectivity
can be reversed using a bulkier hydride reagent that can
block the hydride axial approach observed with NaBH4.
Reduction of ketone 4 with L-Selectride solution in THF at
- 40 °C afforded alcohol cis-5b in 60% yield. The dis-
tributions of diastereomers 5a, 5b are in accordance with
those obtained in our group’s earlier investigations of
indolizidindiones containing fused thiophene and ben-
zothiophene rings [17–19] and thienoquinolizidindiones
[20]. The stereochemical assignments of these alcohols are
1
based on the analysis of the multiplet shapes of H NMR
3
signals of H11 and H11a. The J11,11a coupling constant
value served as a reliable and readily available descriptor—
its obvious difference for 5a (9.7 Hz) and 5b (1.6 Hz)
reflected the, respectively, pseudoaxial–axial and pseu-
doequatorial–axial orientations of the abovementioned pair
of hydrogens.
With the ultimate objective to prepare the enantiopure
benzoanalogues 7 and 11 of the naturally occurring (-)-
cryptopleurine and (-)-(15R)-hydroxycryptopleurine, the
reduction of the lactam group and OH function to the
corresponding alkane was envisioned.
In this perspective, alcohols trans-5a and cis-5b were
acetylated using standard conditions (Scheme 2). Their
reaction with acetic anhydride in the presence of dry tri-
ethylamine and catalytic amounts of DMAP in CH2Cl2 at
room temperature led to the acetoxy derivatives trans-
(11R,11aS)-6a and cis-(11S,11aS)-6b in yields of 70 and
75%, respectively. Ultimately, these acetyl derivatives
trans-6a and cis-6b were efficiently converted into the
expected benzoquinolizidinols trans-7a and cis-7b in good
yields (71 and 76%) using LAH reduction in refluxing THF
for 1 h according to Green’s protocol developed during the
stereoselective synthesis of the alkaloids (-)-2-epilentigi-
nosine and (?)-lentiginosine [21].
The quaternization of basic epimeric alcohols 7a, 7b
with CH3I provide the corresponding benzoquinolizinium
salts 8a, 8b in 78–81% yields. The relative configuration of
compounds 8a, 8b was deduced from the observed NOE
123

Asymmetric synthesis and study of biological activity of (epi-)benzoanalogues of bioactive…
OMe
OMe
MeO
MeO
MeO
MeO
MeO
1
OH
H
H
H
15
(R)
(R)
(R)
(R)
N
N
N
MeO
OMe
(R)-boehmeriasin A
(14aR,15R)-15-hydroxycryptopleurine
(R)-cryptopleurine
Fig. 1 Structures of representative natural phenanthroquinolizidine alkaloids
Scheme 1
1.
2.
NaOH
Ph
O
CO2H
O
H
H
CO2
1. EtOH, reflux
2. H2O, reflux
3. NaBH4
SOCl2
N
CO2H
Ph
O
N
NH2
AlCl3
Ref [12]
HO2C
HO2C
N
H
O
Ph
4
2
1
3
Scheme 2
HO
O
AcO
HO
H
H
H
H
Ac2O
NaBH4
LiAlH4
N
N
MeOH
0 °C
N
N
THF, reflux
1 h
DMAP, DCM
TEA
O
4
O
O
5a
6a
7a
HO
HO
AcO
H
H
H
LiAlH4
Ac2O
L-Selectride
THF, - 40 °C
N
N
N
THF, reflux
1 h
DMAP, DCM
TEA
O
O
5b
7b
6b
Scheme 3
123

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T. Pagác et al.
The structure of this compound was unambiguously con-
firmed by single crystal X-ray diffraction analysis (Fig. 3).
The xanthate functionality was then reductively removed
using Bu3SnH in the presence of a catalytic amount of
AIBN (toluene, reflux, 75%) forming the lactam 10.
Finally, lactam 10 was directly converted to the targeted
enantiopure (S)-2,3,4,6,11,11a-hexahydro-1H-pyrido[1,2-
b]isoquinoline (11) using LAH reduction in refluxing THF
for 1 h in a 79% yield after recrystallization from dry i-
hexane (Scheme 4).
The newly synthesized set of derivatives within this
study (Fig. 4) was tested for its antimicrobial activity
in vitro using bacteria (S. aureus, MRSA isolates—
SA21528, SAL18, S. epidermidis, P. aeruginosa, E. coli),
yeasts (C. albicans, C. parapsilosis) and filamentous fungi
(F. culmorum, M. gypseum, T. terrestre, B. cinerea, A.
alternata). The highest antibacterial activity was reached in
derivative 11 (benzoanalogue of cryptopleurine) and
derivative 9. These two derivatives were tested for their
cytotoxicity on BHK-21 and VERO cells as well.
Fig. 2 ORTEP drawing of 8a
The best antibacterial properties were observed in the
presence of derivative 11. In the case of Gram-positive
bacteria, derivative 11 was able to inhibit the growth of all
Staphylococcus sp. used in this study (see Table 1); it was
possible to obtain the MIC100 values for all tested strains of
Staphylococcus sp. The highest sensitivity was observed
for S. aureus at a concentration of 5 lg/cm3 with bacte-
riostatic effect on the cells. In addition, derivative 9 was
able to inhibit the growth of S. aureus but in higher con-
centration (100 lg/cm3) when compared to derivative 11
(Table 1). As such an important antibacterial effect was
detected on S. aureus, the growth inhibition of two hospital
MRSA isolates was attempted. Derivative 11 was able to
inhibit the growth of both isolates (MIC100 were detected)
but in ten times higher concentration as was found for S.
aureus CCM 3953 (Table 1), i.e., the MIC100 value was
observed at the concentration of 50 lg/cm3 for both iso-
lates. Sensitivity of S. epidermidis was observed as well—
its 100% growth inhibition in the presence of the derivative
11 was observed at the concentration of 10 lg/cm3 with
bacteriostatic effect on the cells.
enhancements in their 2D NOESY NMR spectra
(Scheme 3) and was secured by X-ray crystallographic
analysis of a single crystal of the methiodide salt trans-8a
(Fig. 2).
Since attempts to remove the OH function from enan-
tiopure alcohols 7a or 7b with triethylsilane in TFA failed
to provide the targeted quinolizidinone 11 we turned our
attention to the Barton–McCombie deoxygenation proto-
col. For this reason, alcohol trans-5a was converted to the
corresponding xanthate (11R,11aS)-9 in a yield of 81%.
Newly synthesized derivative 11 with the highest
antibacterial activity was tested for its potential for biofilm
eradication. The derivative was present in the medium
throughout the biofilm formation of strain S. epidermidis
CCM 7221 (recommended for biofilm assay). The effect of
derivative 11 on biofilm formation was concentration
dependent and is shown in Table 2. Significant decrease in
biofilm formation was observed in the presence of
derivative 11 at the concentration 10 and 25 lg/cm3
(Table 2). The derivative was able to reduce the biofilm
formation down to 30% when compared to the untreated
control.
Fig. 3 ORTEP drawing of 9
123

Asymmetric synthesis and study of biological activity of (epi-)benzoanalogues of bioactive…
Scheme 4
Gram-negative bacteria E. coli and P. aeruginosa were
less sensitive compared to staphylococci. The growth of
E. coli was inhibited only by the derivative 11. The MIC100
value was detected at the concentration 10 lg/cm3. P.
aeruginosa was not inhibited by any of tested derivatives
(Table 1).
parapsilosis (Table 3). Both yeasts were more sensitive
when compared to filamentous fungi.
Model filamentous fungi were inhibited only in high
concentration of derivative 11. As described in previous
results such concentration was cytotoxic for tested cell
lines. Only one of the model filamentous fungi dermato-
phyte T. terrestre was inhibited by derivative 9. The MIC80
value was achieved at the concentration level of 80 lg/
cm3.
Derivatives 11 and 9 with the highest antibacterial
activity were tested for their potential cytotoxicity on
BHK-21 and VERO cells. The IC50 value of the derivative
11 for BHK-21 was observed at the concentration of 40 lg/
cm3. Concerning the VERO cells, the IC50 value of the
derivative 11 was achieved at the concentration of 50 lg/
cm3. A cytotoxic effect of derivative 9 was not observed
for BHK-21 and VERO cells in the range of tested con-
centrations. Taking these results into account, both
derivatives did not show significant toxicity on animal cell
lines in the concentration range where antimicrobial
activity was observed.
Finally, potential mutagenic activity of the effective
antibacterial derivative 11 was tested by the Ames test
using Salmonella typhimurium TA98 and S. typhimurium
TA100 without metabolic activation (Table 4).
Based on the obtained data it can be concluded that the
derivative 11 did not increase the number of revertants of
S. typhimurium TA 98 or TA 100. The number of obtained
CFU of both Salmonella strains were comparable to
observed spontaneous revertants which were significantly
lower when compared to positive mutagen control 3-(5-
nitro-2-furyl)acrylic acid (NFAA) (Table 4). It means that
the derivative 11 does not induce point nor frameshift
mutations and is therefore considered to be non-mutagenic.
Concerning the mechanism of action of the QA active
derivatives, some studies have reported that derivatives of
QA alkaloids affect nucleic acid synthesis by inhibiting the
bacterial dihydrofolate reductase (DHFR) [22]. They
indicate that DHFR binds to the N-atom/NH2 groups of its
inhibitors by a strong hydrogen bond with the amino acid
residues at the active site of the enzyme [23]. In accordance
with our findings, the highest antibacterial activity was
linked to the interaction of alkaline nitrogen in the structure
(derivative 11). It was shown that enzyme DHFR and
thymidylate synthase are inhibited by phenanthroin-
dolizidines as well as by benzoquinolizidine plant alka-
loids, namely deoxytubulosin [24]. The interaction was
tested on DHFR of Lactobacillus leichmannii. The same
type of interaction was observed for E. coli when DHFR
bound trimethoprim (TMP) frequently used in anti-infec-
tive therapy [25, 26] was also used in combination with
sulfamethoxazole.
Conclusion
We have achieved the asymmetric synthesis of benzoana-
logues of the phenanthroquinolizidine bioactive alkaloids
(-)-cryptopleurine and (-)-(15R)-hydroxycryptopleurine
in seven or six steps, respectively, starting from available
enantiopure (S)-2-aminoadipic acid. The latter was used as
source of chirality as well as nitrogen necessary for further
applications for the synthesis of these types of compounds.
The highly diastereoselective reduction of the ketone
function of the pure tricyclic keto-lactams 4 was achieved
to provide both pure trans-5a and cis-5b alcohol-lactams in
good yields. The targeted benzoquinolizidinols trans-7a
and cis-7a were reached by lactam reduction in tandem
with acetate deprotection. Starting from alcohol-lactam
trans-5a by preparing the corresponding xanthate (S)-9
followed by xanthate functionality reduction using Bu3SnH
The antifungal activity was not as high as the deter-
mined antibacterial effects. The sensitivity of model fungal
strains is shown in Table 3. Concerning the antifungal
activity of the studied set of derivatives, C. albicans was
more sensitive to the presence of 9 and 11 than C.
123

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T. Pagác et al.
O
H
N
O
4
HO
AcO
HO
H
H
H
N
N
N
O
O
5a
6a
7a
HO
AcO
HO
H
H
H
N
N
N
O
O
5b
6b
7b
HO
HO
H
H
N
N
I
I
Me
Me
8a
8b
Me
O
S
S
H
H
H
N
N
N
O
O
9
10
11
Fig. 4 Newly synthesized derivatives set assayed for antimicrobial activity
the lactam 10 was obtained. Finally, lactam 10 was directly
converted to the targeted novel benzo analogue of crypto-
pleurine—enantiopure (S)-2,3,4,6,11,11a-hexahydro-1H-
pyrido[1,2-b]isoquinoline (11) by LAH reduction. The
overall yields in this linear synthesis were 32% for 7a, 24%
for 7b, and 25% for 11. The highest antimicrobial activity
was observed in the presence of derivative 11 that signif-
icantly inhibits the growth of model bacteria especially
S. aureus. Derivatives 9 and 11 have shown only weak
antifungal activity. The most sensitive was the yeast C.
albicans. In this work, we have shown that the derivative
11 seems to be a structure with a good antibacterial activity
123

Asymmetric synthesis and study of biological activity of (epi-)benzoanalogues of bioactive…
Table 1 Antibacterial activity of tested compounds characterized by
MIC100 values (lg/cm3)
Table 4 Mutagenic activity of derivative 11
Compound
Dosage /lg PD-1
Salmonella Typhimurium
Model bacteria
Derivative
TA98
CFU SD
TA100
CFU SD
9
MIC100
11
MIC100
11
500
250
100
50
15
13
19
15
13
14
1
6
1
3
6
1
108 20
76 13
95 13
90 32
S. aureus
SA21528
SAL18
100s
–
5s
50s
50s
10s
10s
N
–
100s
S. epidermidis
E. coli
25
97
5
N
10
78 14
1175 123
90 15
P. aeruginosa
N
NFAA
SR
10
120 15
17
0
9
N not active, s bacteriostatic effect, – not assayed
PD Petri dish, SR spontaneous revertants, NFAA 3-(5-nitro-2-fur-
yl)acrylic acid—positive mutagen control
Table 2 Effect of derivative 11 on biofilm formation of S.
epidermidis
Concentration derivative 11 /lg cm-3
A570
Biofilm intensity
Experimental
0 (control)
2.6
1.7
0.8
0.7
Very strong
Very strong
Strong
Melting points were obtained using an STUART SMP 30
and are corrected. Commercial reagents were used without
further purification. All solvents were distilled before use.
Flash column liquid chromatography (FLC) was performed
on silica gel Kieselgel 60 (40–63 lm, 230–400 mesh) and
analytical thin-layer chromatography (TLC) was performed
on aluminum plates pre-coated with either 0.2 mm (DC-
Alufolien, Merck) or 0.25 mm silica gel 60 F254 (ALU-
GRAM-SIL G/UV254, Macherey–Nagel). The compounds
were visualized by UV fluorescence and by dipping the
plates in an aqueous H2SO4 solution of cerium sulfate/
ammonium molybdate followed by charring with a heat
gun. HPLC analyses were performed on Varian system
9012 with diode array Varian 9065 polychrom UV detec-
tor: column CC 250/3 Nucleosil 120-5 C18, 250 9 3 mm
(Macherey–Nagel). Mobile phase: solvent A: water/ace-
tonitrile/methanesulfonic acid (1000/25/1), solvent B:
water/acetonitrile/methanesulfonic acid (25/1000/1), elu-
tion mode: gradient with 5–50% solvent B, flow rate:
0.65 cm3/min, UV detection: 210 nm (DAD), 35 °C,
20 min. Optical rotations were measured with a POLAR
L-lP polarimeter (IBZ Messtechnik) with a water-jacketed
10,000 cm cell at the wavelength of sodium line D
(k = 589 nm). Specific rotations are given in units of
10-1 deg cm2 g-1 and concentrations are given in
g/100 cm3. Infrared spectra were recorded on a Nicolet
5700 FT-IR spectrometer as ATR discs (ATR) or as thin
films on ATR plates (film). NMR spectra were recorded on
a Varian VNMRS 600 MHz or INOVA 300 MHz NMR
spectrometer in CDCl3 or CD3OD. Chemical shifts (d) are
5
10
25
Strong
Table 3 Antifungal activity characterized by MIC50 or MIC80 val-
ues (lg/cm3)
Model fungi
Derivative
9
11
C. parapsilosis
MIC50
MIC80
MIC80
MIC80
MIC80
MIC80
MIC80
MIC50
50
50*
–
100
50
N
C. albicans
A. alternata
B. cinerea
25
N
N
100
75
75
100
F. culmorum
T. terrestre
M. gypseum
N
80
N
N not active, – not detected
*The maximal inhibition was 50% with increasing concentration we
did not observe increased inhibitory activity
and could be considered as a new lead structure for design
of further new antimicrobial compounds by its derivatiza-
tion. We suppose that its derivatization assisted by quan-
tum chemical docking calculations with the active center of
bacterial DHFR could lead to a group of new DHFR
inhibitors that could be used in single as well as in synergic
therapy in the future.
1
quoted in ppm with the H and 13C resonance frequencies
of tetramethylsilane (TMS) serving as standards for the
chemical shift axis calculation. The gCOSY and NOESY
123

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T. Pagác et al.
(DMAP, 0.75 mmol), and 1.53 g triethylamine (2.12 cm3).
The reaction mixture was stirred until the disappearance of
the starting material (monitored by TLC). The mixture was
diluted with 50 cm3 CH2Cl2 and quenched with a saturated
aqueous NaHCO3 solution. The aqueous layer was
extracted with 50 cm3 CH2Cl2 and the organic layers were
washed with a saturated aqueous CuSO4 solution, 25 cm3
HCl (10%), 30 cm3 Na2CO3 (25%), and water, dried over
MgSO4, and concentrated under vacuum. An oily matter,
which quickly crystallized on standing, was obtained in the
yield of 1.67 g, 86%. Recrystallization from a mixture of
AcOEt/i-hexane gave 1.36 g (70%) of 6a as colorless
crystals. Rf = 0.72 (CH2Cl2/acetone, 4:1); m.p.: 99.2–
102.4 °C; [a]2D3 = ? 22.16 (c = 1.02, THF); IR (ATR):
1
techniques were used in the assignment of H–1H rela-
tionships and the determination of relative configuration.
The gHSQC and gHMBC techniques were used for the
assignment of the 1H–13C relationships. Only apparent
splitting constants are written in the compound character-
ization data instead of coupling constants because of the
difficulty of extraction of the latter from complex multiplet
patterns. Elemental high-resolution spectrometry was per-
formed on Micromass Q-Tof Micro MS system with ESI?
ionization (measured mass represents M ? H?).
The synthesis of (S)-1,2,3,11a-tetrahydro-4H-pyr-
ido[1,2-b]isoquinoline-4,11(6H)-dione (4) and (11R,11aS)-
11-hydroxy-1,2,3,6,11,11a-hexahydro-4H-pyrido[1,2-b]-
isoquinolin-4-one (5a) is described in [16].
ꢀ
m = 1723, 1641, 1463, 1445, 1417, 1370, 1360, 1330, 1237,
(11S,11aS)-11-Hydroxy-1,2,3,6,11,11a-hexahydro-4H-pyr-
ido[1,2-b]isoquinolin-4-one (5b, C13H15NO2) The freshly
crystallized keto-lactam 4 (1.29 g, 6 mmol) was dissolved
in 120 cm3 dry THF and cooled to - 45 °C with stirring.
1 M solution of L-Selectride in THF (18 cm3, 18 mmol)
was added (30 min) dropwise via a syringe and the reaction
mixture was stirred for 2.5 h at - 40 °C, then it was
gradually quenched with 5 cm3 methanol, 5 cm3 H2O,
7.5 cm3 sodium hydroxide aqueous solution (1 M), and
7.5 cm3 hydrogen peroxide (30% in water) at - 5 °C. The
reaction mixture was then stirred 1 h at ? 5 °C, concen-
trated under vacuum (40 °C water bath and 1 mbar pres-
sure) and extracted with CH2Cl2 (3 9 50 cm3). The
combined organic layers were washed with water and brine
(2 9 20 cm3) and dried over anhydrous MgSO4. After
filtration, the filtrate was concentrated in vacuo to afford a
solid as a mixture of cis and trans diastereomers 5b and 5a
(1.1 g, 83%) in 95:5 ratio (from 1H NMR spectra).
Recrystallization of the solid from AcOEt gave 5b
(780 mg, 60%) as colorless crystals. M.p.: 178.9–182.3 °C;
[a]2D3 = ? 11.12 (c = 1.01, MeOH); Rf = 0.2 (CH2Cl2/
1187, 1174, 1030, 1016, 980, 902, 795, 759, 680, 654, 628,
544, 528, 511, 466, 438 cm-1 1H NMR (300 MHz,
;
CDCl3): d = 7.32–7.10 (m, 4H), 6.08 (d, J = 9.7, 1H), 5.50
(d, J = 17.2 Hz, 1H), 4.15 (d, J = 17.2 Hz, 1H), 3.67 (dt,
J = 9.7, 4.9 Hz, 1H), 2.51–2.37 (m, 2H), 2.23 (s, 3H),
2.10–1.91 (m, 2H), 1.90–1.73 (m, 2H) ppm; 13C NMR
(75 MHz, CDCl3): d = 171.1 (s), 169.7 (s), 133.6 (s), 133.5
(s), 128.3 (d), 127.11 (d), 126.5 (d), 126.4 (d), 70.4 (d),
57.0 (d), 44.7 (t), 32.6 (t), 24.7 (t), 21.3 (q), 17.8 (t) ppm;
HRMS: m/z calcd for C15H17NO3 [M ? H]?: 260.1281,
found 260.1272.
(11S,11aS)-4-Oxo-1,3,4,6,11,11a-hexahydro-2H-pyrido-
[1,2-b]isoquinolin-11-yl acetate (6b, C15H17NO3) This
product was obtained from 1.52 g freshly crystallized cis-
5b (7.0 mmol), 45 cm3 CH2Cl2, 1.43 g acetic anhydride
(1.31 cm3, 14 mmol), 85 mg 4-(dimethylamino)pyridine
(DMAP, 0.7 mmol), and 1.42 g triethylamine (1.95 cm3)
in the same way as for 6a. Yield 1.34 g (74.1%); colorless
crystals; Rf = 0.67 (CH2Cl2/acetone, 4:1); m.p.: 144.3–
152.4 °C; [a]D = ? 28.11 (c = 1.02, THF); IR (ATR):
ꢀ
ꢀ
m = 2952, 1735, 1637, 1591, 1443, 1417, 1369, 1224, 1190,
acetone, 8:1); IR (ATR): m = 3334, 1614, 1582, 1481,
1444, 1364, 1344, 1333, 1242, 1190, 1172, 1118, 1108,
1163, 1043, 1032, 1019, 976, 945, 898, 764, 743, 548, 518,
1
463 cm-1; H NMR (300 MHz, CD3OD): d = 7.43–7.31
1077, 1037, 1008, 982, 952, 896, 797, 749, 721, 663, 625,
1
564, 514, 513, 481, 470, 439 cm-1; H NMR (300 MHz,
(m, 2H), 7.28–7.22 (m, 2H), 5.92 (d, J = 1.7 Hz, 1H), 5.12
(d, J = 17.9 Hz, 1H), 4.40 (d, J = 17.9 Hz, 1H), 3.94-3.86
(m, 1H), 2.51–2.40 (m, 2H), 2.20–1.94 (m, 2H), 1.92–1.70
(m, 2H) ppm; 13C NMR (75 MHz, CD3OD): d = 173.4 (s),
172.1 (s), 134.6 (s), 133.8 (s), 1230.7 (d), 130.6 (d), 128.0
(d), 1267.8 (d), 72.3 (d), 57.5 (d), 45.8 (t), 33.3 (t), 26.6 (t),
21.0 (q), 20.0(t) ppm; HRMS: m/z calcd for C15H17NO3
(259.31) [M ? H]?: 260.1281, found 260.1276.
CD3OD): d = 7.35–7.19 (m, 4H), 5.18 (d, J = 17.8 Hz,
1H), 4.50 (d, J = 1.6 Hz, 1H), 4.30 (d, J = 17.8 Hz, 1H),
3.72–3.64 (m, 1H), 2.47–2.37 (m, 2H), 2.24–2.00 (m, 3H),
1.84–1.66 (m, 1H) ppm; 13C NMR (75 MHz, CD3OD):
d = 173.7 (s), 137.6 (s), 133.8 (s), 130.2 (d), 129.7 (d),
127.9 (d), 127.6 (s), 71.1 (d), 58.9 (d), 45.8 (t), 33.4 (t),
26.5 (t), 20.1 (t) ppm; HRMS: m/z calcd for C13H15NO2
[M ? H]?: 218.1176, found 218.1165.
(11R,11aS)-1,3,4,6,11,11a-Hexahydro-2H-pyrido[1,2-b]iso-
quinolin-11-ol (7a, C13H17NO) Lithium aluminum hydride
(584 mg, 15.4 mmol) was added to a solution of 800 mg
freshly crystallized acetyl derivative 6a (3.1 mmol) in
25 cm3 dry THF at room temperature and the mixture was
then heated under reflux for 1.5 h. The slurry was then
(11R,11aS)-4-Oxo-1,3,4,6,11,11a-hexahydro-2H-pyrido[1,2-
b]isoquinolin-11-yl acetate (6a, C15H17NO3) To a solution
of 1.63 g freshly crystallized trans-5a (7.5 mmol) in
45 cm3 dry CH2Cl2 was added 1.53 g acetic anhydride
(1.41 cm3, 22.5 mmol), 92 mg 4-(dimethylamino)pyridine
123

Asymmetric synthesis and study of biological activity of (epi-)benzoanalogues of bioactive…
warmed to ambient temperature and after additional
40 min was carefully quenched with 2:1 (w/w) Na2-
SO4.10H2O:Celite (15 g, gas evolution!). Dry diethyl ether
(20 cm3) was then added and after 30 min. The white
suspension was filtered, dried over MgSO4, and concen-
trated in vacuo to give a light yellow solid (548 mg, 90%).
Recrystallization of the solid from n-hexane gave pure
alcohol 7a as pale cream-colored crystals (433 mg, 68.7%).
M.p.: 137.2–139.1 °C; [a]2D3 = ? 64.51 (c = 1.01, MeOH);
off and washed with 2 cm3 dry acetone to provide pure
isoquinolinium iodide 8 (280 mg, 81%). M.p.: 237.4–
239.6 °C (methanol/diethyl ether); [a]2D2 = ? 24.97
(c = 1.02, MeOH); IR (ATR): mꢀ = 3228, 1495, 1469, 1451,
1435, 1415, 1357, 1268, 1168, 1090, 1039, 1003, 979, 957,
1
929, 918, 873, 793, 749, 638, 549, 523, 495 cm-1; H
NMR (600 MHz, D2O): d = 7.64 (d, J = 7.8 Hz, 1H), 7.54
(t, J = 7.6 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.27 (d,
J = 7.7 Hz, 1H), 4.79 (d, J = 15.2 Hz, 1H), 4.75 (d,
J = 9.8 Hz, 1H), 4.55 (d, J = 15.2 Hz, 1H), 3.72–3.67 (m,
1H), 3.61–3.54 (m, 2H), 3.03 (s, 3H), 2.48–2.40 (m, 1H),
2.00–1.92 (m, 1H), 1.90–1.80 (m, 2H), 1.69 (tq, J = 4.2,
13.4 Hz, 1H) ppm; 13C NMR (151 MHz, D2O): d = 135.9
(s), 132.1 (d), 131.9 (d), 130.7 (d), 129.3 (d), 128.8 (s), 73.1
(d), 69.8 (d), 69.0 (t), 68.8 (t), 42.7 (q), 25.9 (t), 24.1 (t),
22.3 (t) ppm.
ꢀ
Rf = 0.39 (CH2Cl2/acetone, 1:1); IR (ATR): m = 3332,
2929, 1490, 1453, 1431, 1349, 1270, 1124, 1047, 1028,
1
877, 796, 743, 714, 637, 552, 461, 450 cm-1; H NMR
(300 MHz, CDCl3): d = 7.47–7.42 (m, 1H), 7.21 (dt,
J = 1.6, 7.3 Hz, 1H), 7.17 (dt, J = 1.6, 7.3 Hz, 1H), 7.01-
6.95 (m, 1H), 4.30 (d, J = 8.4 Hz, 1H), 3.74 (d,
J = 15.2 Hz, 1H), 3.32 (d, J = 15.2 Hz, 1H), 2.99 (dtd,
J = 11.4, 3.3, 1.7 Hz, 1H), 2.29–2.20 (m, 1H), 2.11 (dt,
J = 11.3, 4.2 Hz, 1H), 1.95 (dt, J = 3.4, 8.7 Hz, 1H),
1.86–1.77 (m, 1H), 1.72–1.54 (m, 2H), 1.38–1.16 (m, 2H)
ppm; 13C NMR (75 MHz, CDCl3): d = 137.3 (s), 134.1 (s),
127.4 (d), 127.0 (d), 126.9 (d), 125.6 (d), 73.2 (d), 65.9 (d),
58.1 (t), 56.2 (t), 30.4 (t), 25.4 (t), 24.0 (t) ppm; HRMS: m/z
calcd for C13H17NO (203.29) [M ? H]?: 204.1383, found
204.1369.
(5S,11S,11aS)-11-Hydroxy-5-methyl-1,2,3,4,5,6,11,11a-oc-
tahydropyrido[1,2-b]isoquinolin-5-ium iodide (8b, C14H20-
INO) This product was obtained from 203 mg freshly
crystallized cis-7b (1 mmol), 213 mg methyl iodide
(0.095 cm3, 1.5 mmol), and 5 cm3 dry acetone in the same
way as for 8a. Yield 270 mg (78%); colorless crystals;
m.p.: 237.4–239.6 °C (methanol/diethyl ether); [a]D21-
ꢀ
= ? 10.75 (c = 1.01, MeOH); IR (ATR): m = 3311, 2938,
(11S,11aS)-1,3,4,6,11,11a-Hexahydro-2H-pyrido[1,2-b]iso-
quinolin-11-ol (7b, C13H17NO) This product was obtained
from 800 mg freshly crystallized acetyl derivative 6b
(3.1 mmol) and 584 mg lithium aluminum hydride
(15.4 mmol) in 30 cm3 dry THF in the same way as for 7a.
Yield 463 mg (73.5%), colorless crystals. M.p.: 137.2–
139.1 °C (i-hexane); Rf = 0.17 (CH2Cl2/acetone, 1:1);
1495, 1445, 1303, 1277, 1174, 1094, 1017, 970, 935, 911,
1
865, 797, 747, 667, 624, 580, 504, 451 cm-1; H NMR
(600 MHz, CD3OD): d = 7.55 (dd, J = 1.3, 7.7 Hz, 1H),
7.47 (tt, J = 1.1, 7.7 Hz, 1H), 7.42 (td, J = 1.4, 7.5 Hz,
1H), 7.25 (d, J = 7.7 Hz, 1H), 4.82 (d, J = 1.7 Hz, 1H),
4.70 (d, J = 15.2 Hz, 1H), 4.62 (d, J = 15.2 Hz, 1H), 3.90
(td, J = 3.4, 12.4 Hz, 1H), 3.73–3.68 (m, 1H), 3.59 (dt,
J = 3.2, 13.4 Hz, 1H), 3.13 (s, 3H), 2.38–2.29 (m, 1H),
2.20–2.11 (m, 1H), 2.10-2.03 (m, 1H), 1.98–1.90 (m, 2H),
1.80 (tq, J = 13.4, 4.2 Hz, 1H) ppm; 13C NMR (151 MHz,
CD3OD): d = 135.9 (s), 131.3 (d), 130.4 (d), 130.2 (d),
127.7 (d), 127.0 (s), 71.3 (d), 67.7 (t), 67.6 (t), 66.9 (d),
43.1 (q), 23.9 (t), 23.3 (t), 21.4 (t) ppm.
[a]2D2 = ? 52.66 (c = 1.01, MeOH); IR (ATR): m = 3176,
ꢀ
2923, 2761, 1490, 1455, 1435, 1413, 1350, 1289, 1274,
1109, 1088, 1067, 1041, 988, 941, 921, 879, 839, 801, 752,
1
730, 702, 627, 584, 445 cm-1; H NMR (600 MHz, CD3-
OD): d = 7.36–7.32 (m, 1H), 7.25–7.20 (m, 2H), 7.10–7.05
(m, 1H), 4.33 (d, J = 2.8 Hz, 1H), 3.85 (d, J = 15.5 Hz,
1H), 3.30 (d, J = 15.5 Hz, 1H), 3.13–3.07 (m, 1H), 2.30
(td, J = 11.3, 2.9 Hz, 1H), 2.16 (dt, J = 3.0, 12.0 Hz, 1H),
2.00–1.91 (m, 1H), 1.73–1.67 (m, 2H), 11.65–1.57 (m,
1H), 1.63 (tq, J = 3.7, 12.8 Hz, 1H), 1.42 (qt, J = 3.8,
13.0 Hz, 1H) ppm; 13C NMR (151 MHz, CD3OD):
d = 137.6 (s), 135.1 (s), 130.9 (d), 128.9 (d), 127.8 (d),
126.9 (d), 70.2 (d), 64.4 (d), 59.2 (t), 57.4 (t), 28.5 (t), 26.5
(t), 25.3 (t) ppm; HRMS: m/z calcd for C13H17NO (203.29)
[M ? H]?: 204.1383, found 204.1371.
S-Methyl O-[(11R,11aS)-4-oxo-1,3,4,6,11,11a-hexahydro-2H-
pyrido[1,2-b]isoquinolin-11-yl]carbonodithioate (9, C15H17-
NO2S2) A mixture of 1.31 g freshly crystallized alcohol
trans-5a (6 mmol), 228 mg NaH (60% in oil, 12 mmol)
and 25 mg imidazole in 55 cm3 dry THF was refluxed with
stirring for 30 min under nitrogen. CS2 (3.6 cm3, 60 mmol)
in 2 cm3 THF was then added to the reaction mixture. After
refluxing for further 45 min, 4.4 cm3 MeI (70 mmol) was
added and the reflux continued for another 30 min. The
reaction mixture was brought to room temperature and
quenched with iced water containing acetic acid (0.3 cm3),
diluted with water and extracted with CH2Cl2
(3 9 50 cm3). The combined organic layer was washed
with 15% Na2CO3 solution and brine and dried. The sol-
vent was removed in vacuo, and the residue was
(5S,11R,11aS)-11-Hydroxy-5-methyl-1,2,3,4,5,6,11,11a-oc-
tahydropyrido[1,2-b]isoquinolinium iodide (8a, C14H20-
INO) Methyl iodide (213 mg, 0.095 cm3, 1.5 mmol) was
added to a solution of 203 mg 7a (1 mmol) in 5 cm3 dry
acetone and the mixture was allowed to stand at room
temperature for 12 h. The colorless crystals were filtered
123

ˇ
T. Pagác et al.
chromatographed (100 g, 35 9 750 mm, CH2Cl2 as elu-
ent) on silica gel to give the thiocarbamate as pale pink
solid (1.65 g, 89%) which after recrystallization from n-
heptane gave 9 as a colorless crystals (1.50 g, 81%). M.p.:
171.2–171.6 °C; [a]2D2 = - 28.8 (c = 1.05, MeOH);
(S)-1,3,4,6,11,11a-Hexahydro-2H-pyrido[1,2-b]isoquinoline
(11) Lithium aluminum hydride (750 mg, 20 mmol) was
added to a solution of 550 mg lactam 10 (2.73 mmol) in
30 cm3 dry THF at room temperature and the mixture was
then heated under reflux for 2 h. The resulting mixture was
cooled, NH4Cl and water were added cautiously until the
lithium complex was decomposed. The mixture was then
diluted with 10 cm3 water and 50 cm3 dichloromethane.
The CH2Cl2 layer was separated and the aqueous layer
extracted with CH2Cl2 (2 9 20 cm3). The combined
extracts were washed with water, brine, dried over MgSO4,
and concentrated in vacuo to give a pale yellow, low-
melting solid (449 mg, 87.7%). Distillation on Kugelrohr
(135 °C/10.8 Pa) gave pure amine 11 as colorless crystals
(404 mg, 78.9%); m.p.: 44.1–45.2 °C. Analytically pure
material was obtained by recrystallization from dry n-
hexane. M.p.: 44.8–45.3 °C; [a]2D0 = ? 42.26 (c = 0.5,
CH2Cl2); Rf = 0.53 (AcOEt—petroleum ether—Et3N,
8:2:1); [27] [a]D20 = ? 26.0 (c = 0.5, CH2Cl2); Rf = 0.25
(CHCl3—MeOH, 10:1), yellow oil; or [28] [a]2D0 = ? 29.3
ꢀ
Rf = 0.56 (CH2Cl2/i-PrOH; 10:1); IR (ATR): m = 3261,
3057, 2945, 2920, 1650, 1498, 1461, 1437, 1419, 1359,
1331, 1244, 1198, 1168, 1099, 1049, 1019, 992, 955, 941,
908, 894, 877, 849, 814, 748, 728, 667, 630, 586, 534, 464,
439, 407 cm-1; 1H NMR (600 MHz, CDCl3): d = 7.29 (td,
J = 7.4, 1.5 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.20 (d,
J = 7.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.08 (d,
J = 9.8 Hz, 1H), 5.55 (d, J = 17.2 Hz, 1H), 4.16 (d,
J = 17.2 Hz, 1H), 3.86 (dt, J = 9.5, 4.5 Hz, 1H), 2.66 (s,
3H), 2.57–2.37 (m, 2H), 2.06–1.96 (m, 2H), 1.95–1.89 (m,
1H), 1.84–1.75 (m, 1H) ppm; 13C NMR (151 MHz,
CDCl3): d = 217.4 (s), 169.5 (s), 133.3 (s), 132.6 (s), 128.4
(d), 126.9 (d), 126.4 (d), 126.2 (d), 78.3 (d), 56.7 (d), 44.4
(t), 32.3 (t), 24.0 (t), 19.5 (q), 17.8 (t) ppm; HRMS: m/z
calcd for C15H17NO2S2 (307.43) [M ? H]?: 308.0701
found 308.0691.
ꢀ
(c = 0.28); m.p.: 42.0–47.0 °C; IR (ATR): m = 3062, 3026,
2933, 2850, 2750, 2675, 1493, 1454, 1439, 1348, 1296,
(S)-1,2,3,6,11,11a-Hexahydro-4H-pyrido[1,2-b]isoquinolin-4-
one (10, C13H15NO) To a boiling solution of 1.5 g thio-
carbamate 9 (4.88 mmol) in 50 cm3 toluene containing
2 cm3 AIBN (0.2 M solution in toluene) was added 2 cm3
tributyltin hydride (7.31 mmol) in an argon atmosphere,
and the reaction mixture was refluxed for 1 h. The reaction
mixture was brought to room temperature, the solvent
removed, and the crude product chromatographed on silica
gel (80 g, 35 9 750 mm). Elution with CH2Cl2/cyclohex-
ane (1:3) removed the organotin impurities. Further elution
with CH2Cl2/MeOH (80:1) furnished the amide (864 mg,
88%) as colorless oil, which quickly crystallized on
standing. Recrystallization from i-hexane provided 10 as
colorless crystals (737 mg, 75%). M.p.: 78.9–79.7 °C;
[a]2D2 = ? 75.2 (c = 1.06, MeOH); Rf = 0.50 (CH2Cl2/i-
1250, 1174, 1116, 1063, 937, 874, 734, 638, 575, 542,
430 cm-1 1H NMR (600 MHz, CDCl3): d = 7.12–7.06
;
(m, 2H), 7.04 (d, J = 7.1 Hz, 1H), 7.00 (d, J = 7.2 Hz,
1H), 3.85 (d, J = 15.1 Hz, 1H), 3.38 (d, J = 15.1 Hz, 1H),
3.07 (d, J = 11.3 Hz, 1H), 2.77 (dd, J = 16.5, 3.4 Hz, 1H),
2.70 (dd, J = 10.5, 16.5 Hz, 1H), 2.27–2.18 (m, 1H), 2.11
(td, J = 3.0, 11.3 Hz, 1H), 1.88–1.75 (m, 2H), 1.74–1.62
(m, 2H), 1.40–1.28 (m, 2H) ppm; 13C NMR (151 MHz,
CDCl3): d = 134.3 (s), 133.9 (s), 127.9 (d), 126.1 (d), 125.9
(d), 125.5 (d), 58.4 (t), 58.2 (d), 56.1 (t), 36.8 (t), 33.7 (t),
25.9 (t), 24.3 (t) ppm; HRMS: m/z calcd for C13H17N
(187.29) [M ? H]?: 188.1434 found 188.1428.
X-ray diffraction
ꢀ
PrOH; 10:1); IR (ATR): m = 3246, 3030, 2945, 2839, 1628,
1582, 1463, 1440, 1415, 1361, 1349, 1332, 1246, 1183,
The X-ray diffraction data for the crystals 8a and 9 were
collected at 298 K on a four-circle diffractometer Xcalibur,
Ruby, Gemini using graphite-monochromated radiation.
The structures were solved by direct (heavy) methods and
refined by full-matrix least-squares using the SHELXL
[29], ShelXle [30], and OLEX [31] programs. All the non-
hydrogen atoms in the molecules were refined with ani-
sotropic atomic displacement parameters. All figures were
made using the program DIAMOND [32].
1171, 1160, 1104, 1039, 991, 926, 891, 849, 749, 710, 673,
1
633, 606, 524, 515, 448, 435 cm-1; H NMR (600 MHz,
CDCl3): d = 7.21–7.12 (m, 3H), 7.09 (dd, J = 7.1, 1.9 Hz,
1H), 5.26 (d, J = 17.5 Hz, 1H), 4.29 (d, J = 17.5 Hz, 1H),
3.66–3.59 (m, 1H), 2.88 (dd, J = 15.8, 11.1 Hz, 1H), 2.78
(dd, J = 15.7, 3.7 Hz, 1H), 2.52–2.42 (m, 2H), 2.17–2.09
(m, 1H), 2.00–1.85 (m, 1H), 1.82–1.75 (m, 1H), 1.75-1.67
(m, 1H) ppm; 13C NMR (151 MHz, CDCl3): d = 169.8 (s),
133.9 (s), 133.0 (d), 128.3 (d), 126.8 (d), 126.7 (d), 126.6
(d), 53.7 (d), 45.1 (t), 37.0 (t), 32.9 (t), 29.5 (t), 18.9
(t) ppm; HRMS: m/z calcd for C13H15NO (201.27)
[M ? H]?: 202.1226 found 202.1219.
Crystallographic data for the structures in this paper
have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication nos. CCDC
123

Asymmetric synthesis and study of biological activity of (epi-)benzoanalogues of bioactive…
1815854 for 8a and 1815853 for 9. Copies of the data can
be obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK, fax: ? 44-
(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk.
shaking (200 rpm). Cultures were diluted to a cell density of
107/cm3 into MHB. 198 mm3 aliquots of fresh diluted cells
were added into wells of the 96 flat bottom well polystyrene
microplate (Sarstedt, Nu¨mbrecht, Germany). For biofilm
inhibition assay 2 mm3 of appropriate concentrated (100
times) derivative 11 solution (diluted in DMSO) was added to
each well of the plate. As controls, 10 wells of each microtiter
plate were handled in an identical fashion, except that no
derivative solution was added to the cells. Cells were culti-
vated without shaking at 37 °C for the biofilm formation.
After that, plates were incubated without shaking at 37 °C for
24 or 48 h. Formed biofilm was evaluated by the crystal violet
staining [36]. Biofilm intensity was evaluated according to the
observed absorbance values as weak, medium or strong
(A570 \ 0.1–0.29[ weak biofilm formation; A570\ 0.3–
0.5 [ medium biofilm formation; A570\ 0.5–0.99[
strong biofilm formation; A570[ 1.0). All experiments were
performed in six parallel measurements.
Antimicrobial activity in vitro of newly
synthesized derivatives
Antimicrobial activity of newly synthesized derivatives
was assayed on Staphylococcus epidermidis CCM 7221,
Staphylococcus aureus CCM 3953, two S. aureus isolates
described as methicillin resistant (MRSA) SA21528 and
SAL18 (hospital isolates), and on c-Proteobacteria
Escherichia coli CCM 3988, Pseudomonas aeruginosa
CCM 1959 (Czech Collection of Microorganisms, Masaryk
University, Brno, Czech Republic). Antibacterial and
antifungal activities on model yeast Candida albicans SC
5314 and Candida parapsilosis ATCC 22019 were evalu-
ated by the micro-dilution method [33, 34]. The sensitivity
of filamentous fungi Fusarium culmorum CCM F-21,
Botrytis cinerea CCM F-16 (Czech Collection of
Microorganisms, Masaryk University, Brno, Czech
Republic), Alternaria alternata (Collection of Microor-
ganisms of Department of Biochemistry and Microbiology,
Faculty of Chemical and Food Technology STU, Bra-
tislava, Slovakia) Microsporum gypseum and Trichophyton
terrestre (Laboratory of Medical Mycology, Postgraduate
Medical Institute, Bratislava, Slovakia) was studied by the
macrodilution method [35]. Subcultures of microorganisms
were prepared separately in Petri dishes containing
appropriate agarized growth medium and incubated at
37 °C for 24 h (bacteria); 48 h (model yeasts) and at 25 °C
for 96 h for filamentous fungi except M. gypseum and T.
terrestre—their cultivation time was prolonged for
2 weeks according to their growth rate. The assessment of
antibacterial and antifungal activities was expressed as the
concentration of the derivative inhibiting the growth of
bacteria by 50% (MIC50), 80% MIC80 resp. 100% MIC100
values. The MIC50, MIC80 and MIC100 values were derived
from the dosage response curves. Chromatographically
pure compounds were dissolved in dimethyl sulfoxide
(DMSO) Sigma-Aldrich Ltd; (Steinheim, Germany); its
final concentration never exceeded 1.0 vol% either in
control or in treated samples.
Ames test for evaluation on potential
mutagenicity
Assessment of potential mutagenicity was performed using
classical plate incorporation method [37] without meta-
bolic activation using Salmonella Typhimurium TA 98 and
TA100. As positive mutagen 3-(5-nitro-2-furyl)acrylic acid
(NFAA) was used. Positive response was defined as a
reproducible twofold increase of revertants with dose
response relationship.
The cytotoxicity assay on BHK-21 and VERO
cell lines
The cytotoxicity of the derivatives 9 and 11 was tested on
BHK-21 (BHK-21 stabilized adherent cell line derived in
1962 from the kidney of day-old hamster, fibroblast cells)
and VERO (VERO-stabilized adherent cell line derived from
kidney of Cercopithecus aethiops, fibroblast cells) cell lines
in 96-well microplates plates at the density of 1.8 9 104
cells/well in 100 mm3 of DMEM growth media supple-
mented with 10% FBS, 1% penicillin/streptomycin, 1% L-
glutamine and 0.1% gentamicin. Cells were treated with
1 mm3 of derivative 9 or 11 dissolved in DMSO (in con-
centration range 1-100 lg/cm3) at 37 °C for 24 h in 5% CO2
atmosphere. After 24 h of incubation at 37 °C, 100 mm3 of
MTT (3-[4,5-dimethylazol-2-yl]-2,5-diphenyltetrazolium
bromide) 5 mg/cm3 in PBS was added. The cells were further
incubated for 4 h and the reaction was stopped with
200 mm3 DMSO that was added to each well. Subsequently,
absorbance values at the wavelength of 570 nm were mea-
sured and cell viability was compared to untreated
Bacterial biofilm formation in vitro
Biofilm formation was assayed after crystal violet staining in
96-well polystyrene microtiter plates for 24 h. Briefly, S.
epidermidis CCM 7221 was cultivated for 16 h in Mueller–
Hinton growth medium (MHB) (Biolife, Milano, Italy) under
123

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T. Pagác et al.
14. Kumar NS, Dullaghan EM, Finlay BB, Gong H, Reiner NE,
Selvam JJP, Zoraghi R (2014) Bioorg Med Chem 22:1708
´
controls—cells incubated with 1% DMSO (derivatives sol-
vent). Finally, cells’ survival rate (%) was calculated
according the formula
ˇ
15. Sivy J, Vrabel V, Marchalın S, Safar P (2015) Asian J Chem
ˇ
´
27:2635
´
´
´ˇ
À
Á
ˇ
16. Safar P, Marchalın S, Soral M, Moncol J, Daıch A (2017) Org
ˇ ˇ
´ˇ
´
¨
% Survival rate ¼ A570sample À A570blank
=
Lett 19:4742
ˇ
ðA570controlÀA570blankÞ Â 100 %;
and IC50 values were evaluated based on the cell survival
´
17. Marchalın S, Szemes F, Bar N, Decroix B (1999) Heterocycles
50:445
ˇ
18. Safar P, Zuziova J, Bobosikova M, Marchalın S, Pronayova N,
ˇ
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´
´ˇ
´ˇ
´
ˇ
Comesse S (2009) Tetrahedron Asymmetry 20:2137
´
´
curve.
ˇ
19. Safar P, Zuziova J, Marchalın S, Tothova E, Pronayova N, Svorc
ˇ
ˇ
ˇ
´ˇ
´ˇ
´
´
´
´
L’, Vrabel V, Daıch A (2009) Tetrahedron Asymmetry 20:626
´
´
¨
Acknowledgements This work was supported by the Grant Agency of
the Slovak Republic (Grant no. 1/0371/16). This contribution is also
the result of the project: Research Center for Industrial Synthesis of
Drugs, ITMS 26240220061, supported by the Research & Develop-
ment Operational Programme funded by the ERDF.
ˇ
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20. Safar P, Marchalın S, Pronayova N, Vrabel V, Lawson AM,
Othman M, Daich A (2016) Tetrahedron 72:3221
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66:5438
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FEA, Smith DR, Stammers DK (1981) FEBS Lett 126:49
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