Synthesis, Characterization, Cytotoxicity, and Antibacterial Properties of trans-γ-Halo-δ-lactones

Article abstract of DOI:10.1002/open.201800110

A new four-step pathway for the synthesis of γ-halo-δ-lactones is described from simple, commercially available substrates: aryl bromides and 3-methyl crotonaldehyde. The halogenolactonization reaction of β,δ-substituted, γ,δ-unsaturated carboxylic acid 4

Full text of DOI:10.1002/open.201800110

DOI: 10.1002/open.201800110
Synthesis, Characterization, Cytotoxicity, and Antibacterial
Properties of trans-g-Halo-d-lactones
Angelika Kamizela,*[a] Barbara Gawdzik,[a] Mariusz Urbaniak,[a] Łukasz Lechowicz,[b]
[c]
[d]
[d]
´
Agata Białonska, Weronika Gonciarz, and Magdalena Chmiela
A new four-step pathway for the synthesis of g-halo-d-lactones
is described from simple, commercially available substrates:
aryl bromides and 3-methyl crotonaldehyde. The halogenolac-
tonization reaction of b,d-substituted, g,d-unsaturated carbox-
ylic acid 4a–c is regio- and stereoselective and gives only the
trans-isomers of lactones 5a–c, 6a–c, and 7a–c. The structures
of all synthesized compounds were confirmed by using spec-
troscopic methods. For bromolactone, containing a naphthyl
moiety in the structure, crystallographic analysis was also per-
formed. The lactones were tested for their cytotoxic activity
against L929 cell lines (mouse fibroblasts) and antibacterial ac-
tivity against Escherichia coli strains ATCC 8739 and Staphylo-
coccus aureus ATCC 65389. Compounds 5a, 5c, 7a, and 7b
statistically significantly inhibited the metabolic activity of
mouse fibroblasts L929. Compounds 5b and 6a were not cyto-
toxic towards L929 cells, but showed moderate bactericidal
properties.
1. Introduction
Lactones represent an important class of biologically active or-
ganic compounds. The most persistent and, thus, the most fre-
quently naturally occurring are g- and d-lactones with five- and
six-membered rings. Their rich sources are plants. Compounds
containing a lactone ring in the structure are the main group
of secondary metabolites.[1] Lactone derivatives also occur in
microorganisms and animals,[2] fulfilling a number of different
functions. The biological activities, such as cytostatic,[3–8] anti-
bacterial,[9–12] antiviral[13–17] or antifungal[18–20] activities, have ap-
plications in medicine. Sensory properties make them useful in
the production of cosmetics and in the food industry, where
they are responsible for the smell and taste of many prod-
ucts.[21] An important property of lactones is the deterrent ac-
tivity,[22–27] which can be utilized in the production of insect-
control agents.
Owing to the high costs of extracting cyclic esters from nat-
ural sources, methods for their preparation through chemical
synthesis[28–34] and by using biotechnological methods[35–39] are
still being developed. Considering the biological properties,
lactones are the focus of many research groups. In this work,
we present a new synthesis pathway for halogenolactones and
explore their cytotoxicity and bactericidal activity.
2. Results and Discussion
Numerous methods for the preparation and modification of
halogenolactones have been reported in the literature.[33,40–42]
In our previous work, we presented the synthesis pathways
and cytotoxic properties of iodine and hydroxylactones against
the tumor cell lines HL-60, U-2 OS and D-17.[40,43] Continuing
this research, we present a new four-step, regio- and stereose-
lective synthesis of halolactones 5a–c, 6a–c, and 7a–c with
aryl substituents. All of the obtained compounds were tested
for their cytotoxic activity against cell line L929 (mouse fibro-
blasts) and antibacterial activity against strains of Escherichia
coli ATCC 8739 and Staphylococcus aureus ATCC 65389. The
synthesis pathway of new d-lactones is shown in Scheme 1.
Aryl bromides 1 and 3-methyl crotonaldehyde was used as
substrates.
[a] Dr. A. Kamizela, Prof. B. Gawdzik, Dr. M. Urbaniak
Institute of Chemistry, Jan Kochanowski University
´
Swie˛tokrzyska 15 G, 25–406 Kielce (Poland)
E-mail: angelikakamizela@gmail.com
[b] Dr. Ł. Lechowicz
Institute of Biology, Jan Kochanowski University
´
Swie˛tokrzyska 15 G, 25–406 Kielce (Poland)
´
[c] Prof. A. Białonska
Department of Chemistry, University of Wroclaw
F. Joliot-Curie 14, 50–383 Wrocław (Poland)
[d] Dr. W. Gonciarz, Prof. M. Chmiela
Dept. of Immunology and Infectious Biology, University of Lódz
´
Banacha 12/16, 90–237 Łódz (Poland)
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/open.201800110.
The first step of the synthesis was a Grignard reaction be-
tween 3-methyl crotonaldehyde and a-naphthylene magnesi-
um bromide (a), phenyl magnesium bromide (b), p-fluorophen-
yl magnesium bromide (c). In this reaction, we obtained three
unsaturated secondary alcohols (2a–c) with yields of 38–65%,
already described in the literature.[44,45] The structures products
2a–c were confirmed by using IR and NMR spectroscopy as
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This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
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plex. The kinetically controlled reaction gives a mixture of g-
and d-lactones with an excess of five-membered-ring lactones.
The other important factor for iodolactonization of g,d-unsatu-
rated carboxylic acids is steric hindrance. Electrophilic attack
on the g-carbon atom is hindered by substituents at the b-po-
sition, which favors 6-endo cyclization.[47] This is also confirmed
by our results. The two methyl groups at C-3 in carboxylic
acids 4a–c prevent the formation of g-lactones via 5-exo cycli-
zation and allows formation of d-lactones. This reaction result-
ed in the selective formation of trans isomers of g-iodo-d-lac-
tones 5a--c, g-bromo-d-lactones 6a–c, and g-chloro-d-lactones
7a–c. All obtained products, except 5b,[46] have not been pre-
viously described in the literature. The structure of all products
was confirmed by using IR and NMR spectroscopy as well as
HR-MS. The yields of the halolactonization reaction are shown
in Table 1.
Scheme 1. The four-step synthesis of trans-g-halo-d-lactones 5a–c, 6a–c,
and 7a–c. The first step is the Grignard reaction of aryl magnesium bromide
with 3-methyl crotonaldehyde. The second step is a [3,3] sigmatropic rear-
rangement. The next stage is hydrolysis and the final stage of the synthesis
is halolactonization.
well as mass spectrometry (HR-MS). The presence of a hydroxyl
group in the obtained alcohols was confirmed by the broad IR
bands at 3600–3100 cmÀ1. In the 13C NMR spectrum, carbon
peaks at 68.55 ppm (2a), 70.75 ppm (2b), and 70.11 ppm (2c)
correspond to carbon atoms bonded to the hydroxyl group.
New g,d-unsaturated ethyl esters (3a–c) were obtained with
yields of 46–55% as a result of the Johnson–Claisen rearrange-
ment of alcohols (2a–c). The [3,3]-sigmatropic rearrangement
reaction was carried out by using triethylorthoacetate in the
presence of a catalytic amount of propionic acid. The struc-
tures of esters were confirmed by IR and NMR spectroscopies
and HR-MS. The strong IR absorption bands at 1729 cmÀ1 (3a),
1730 cmÀ1 (3b), and 1730 cmÀ1 (3c) and signals in the 13C NMR
spectrum at 172.05 ppm (3a), 171.61 ppm (3b), and
171.56 ppm (3c) are characteristic for the carbonyl group. The
coupling constants of the olefin protons (15.8 Hz for 3a,
16.2 Hz for 3b, and 16.2 Hz for 3c) confirmed that the E con-
figuration of double bonds remained unchanged. In the next
step, esters 3a–c were hydrolyzed in ethanolic KOH solution to
the corresponding g,d-unsaturated carboxylic acids 4a–c in
yields above 90%. The products did not require further purifi-
cation. Compounds 4a and 4c are new. Compound 4b is
Table 1. The yields of the halolactonization reaction.
Compound
Yield [%]
5a
5b
5c
6a
6b
6c
7a
7b
7c
93
87
60
62
59
85
75
85
65
Iodolactones 5a–c were obtained through the reaction of
g,d-unsaturated acids 4a–c with I2/KI and NaHCO3. The prod-
ucts were purified by column chromatography using mixtures
of acetone and hexane in volume ratio of 1:10 or 1:15 as the
eluent. The reaction yields ranged from 60 to 93%. The lac-
tones, obtained from the iodolactonization reaction, occur only
in one isomeric form.
The stretching vibrations at 1723 cmÀ1 (5a), 1723 cmÀ1 (5b),
known.[46] The structure and configuration of the products 4a– and 1720 cmÀ1 (5c) as well as the signals in the 13C NMR spec-
c were also confirmed by using IR and NMR spectroscopy as
well as HR-MS spectrometry.
trum at 168.94 ppm (5a), 168.91 ppm (5b), and 168.65 ppm
(5c) are characteristic for the carbonyl group in d-lactones. The
vibrations characteristic for the g-lactones appear around
1760–1780 cmÀ1 and the chemical shifts of the carbonyl
carbon in the five-membered rings are around 176 ppm.[40] The
1H NMR spectrum of trans-5-iodo-4,4-dimethyl-6-(a-naphthyl)-
tetrahydro-2H-pyran-2-one (5a) shows signals from H-5 and H-
6 protons as two doublets at 4.71 and 6.36 ppm. The high cou-
pling constant (JH5/H6 =11.4 Hz) confirms the axial–equatorial
disposition of the protons and trans configuration of the lac-
tone. The same configuration is observed for iodolactones 5b
and 5c.
The reaction of carboxylic acids 4a–c and N-bromosuccini-
mide gives trans-g-bromo-d-lactones 6a–c with yields in the
range 59–85%. The products were purified by column chroma-
tography using mixtures of acetone and hexane in a volume
ratio of 1:10 as the eluent. The six-membered structure of bro-
molactone was confirmed by IR absorption bands at 1730–
The IR bands at 3300–2750 cmÀ1 are assigned to carboxyl
groups. In the 1H NMR spectrum, the carboxylic acid proton
signals appear as broad singlets at 11.02 ppm (4a), 11.32 ppm
(4b), and 10.41 ppm (4c). The high coupling constants of the
olefin protons: 15.8 Hz (4a), 16.2 Hz (4b), and 16.2 Hz (4c) in-
dicate a trans-configuration of double bond.
The final stage of the synthesis was halolactonization of
acids 4a–c with N-bromosuccinimide or N-chlorosuccinimide.
The reaction proceeds via either 5-exo or 6-endo cyclization. In
the case of bromolactonization, the mechanism involves an
electrophilic attack of bromine on the double bond, which re-
sults in the formation of a bromonium ion. The reaction pro-
ceeds via 6-endo cyclization. The reaction with chlorine takes
place in the same way. In the case of iodolactonization, the
key step of the reaction mechanism involves nucleophilic
attack of the carboxylate ion on the iodine double-bond com-
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1726 cmÀ1 and the chemical shifts of the carbonyl carbon
around 168 ppm. The high coupling constant (JH5/H6 =10.8 Hz)
indicates the E configuration of bromolactones. The crystallo-
graphic analysis, carried out for lactone 6a,[48] confirmed the
trans configuration between the aromatic ring and the bro-
mine atom at C6 and C5 carbon. Figure 1 shows the crystal
structure of trans-5-bromo-4,4-dimethyl-6-(a-naphthyl)tetrahy-
dro-2H-pyran-2-one (6a).
2.1. The MTT Reduction Effectiveness
The synthesized g-halogeno-d-lactones were tested for their
cytotoxic activity against the cell lines L929 (mouse fibroblasts).
Statistically significant inhibition of metabolic activity of L929
fibroblasts was observed for lactones 5a, 5c, 7a, and 7b in
the range of 50–0.1 mgmLÀ1. In the presence of these com-
pounds, 37–93% of the cells were not able to convert MTT [(3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)]
into formazan (Table 2). The ability to convert MTT loses 24–
70% of the cells when lactones 7c, 5b, and 6c were used in a
concentration range of 50–5 mgmLÀ1. Lactones 6bc were not
cytotoxic only at 0.5 mgmLÀ1, whereas 6a did not cause a sig-
nificant decrease of metabolic activity of the cells at a concen-
tration of 10 mgmLÀ1 (Table 2).
The cytotoxicity of new compounds against eukaryotic cells
may limit their medical applications related to the potential an-
timicrobial activity. In our study, the lowest IC50 values were ex-
hibited by lactones 6a, 5b, and 7c. However, the high IC50
marker can help to select highly cytotoxic compounds, which
can be screened toward cancer cells. Gładkowski et al.[49]
showed the cytotoxic effect of novel racemic iodo-, bromo-
and chlorolactones with b-phenyl-g-lactone or b-phenyl-d-lac-
tone frameworks. The highest activity versus carboplatin was
shown for cis-5-(1-iodoethyl)-4-(4’-isopropylphenyl)dihydrofur-
an-2-one towards Jurkat cell line (human leukemia). New lac-
tones have also been estimated in terms of their antifeedant
activity toward the granary weevil beetle (Sitophilus granarius
L.), the khapra beetle (Trogoderma granarium Everts) and the
confused flour beetle (Tribolium confusum Du Val.).[50] These
studies have shown the wide application possibilities of new
lactones.
Figure 1. The crystal structure of trans-5-bromo-4,4-dimethyl-6-(a-naphthyl)-
tetrahydro-2H-pyran-2-one (6a).
In an analogous reaction with N-chlorosuccinimide, trans-g-
chloro-d-lactones 7a–c were obtained with yields in the range
of 65–85%. The products were purified by column chromatog-
raphy using mixtures of acetone and hexane in volume ratio of
1:10 as the eluent. The structures of all products were con-
firmed by using IR and NMR spectroscopy as well as HR-MS.
The structures of the d-lactones were confirmed by characteris-
tic bands in the IR spectra at 1730 cmÀ1 (7a), 1733 cmÀ1 (7b),
and 1730 cmÀ1 (7c). The chemical shifts of the carbonyl carbon
at 168.56 ppm (7a), 168.35 ppm (7b), and 168.17 ppm (7c)
also confirmed formation of the 6-endo cyclization products.
The signals of protons at C5 and C6 appear as two doublets at
4.37 and 6.06 ppm (7a), 3.97 and 5.25 ppm (7b), 3.92 and
5.24 ppm (7c). The high coupling constant (JH5/H6 =10.4 Hz) in-
dicates the E configuration of lactones.
2.2. Antibacterial Properties
The effect of synthesized lactones on growth of Escherichia coli
ATCC 8739 and Staphylococcus aureusin ATCC 65389 was
tested. Antibacterial properties of the compounds were tested
for the concentration 50–0.1 mgmLÀ1 of lactones. After 18 h in-
Table 2. Cytotoxic effect of investigated lactones against L929 cells.[a]
Concentration [mg/mL]
Controls
50
20
10
5
0.5
C+
C-
DMSO
Viable cells [%]
7*Æ0.010
16*Æ0.015
15*Æ0.02
45*Æ0.011
34*Æ0.011
14*Æ0.012
14*Æ0.013
26*Æ0.011
30*Æ0.015
IC50 [mg/mL]
0.39
9.26
0.46
18
4.7
10
0.49
4.7
9.26
5a
5b
5c
6a
6b
6c
7a
7b
7c
16*Æ0.018
34*Æ0.014
29*Æ0.019
55*Æ0.016
39*Æ0.010
28*Æ0.011
34*Æ0.017
34*Æ0.016
36*Æ0.013
26*Æ0.011
54*Æ0.011
32*Æ0.010
74Æ0.015
45*Æ0.018
50*Æ0.019
38*Æ0.014
45*Æ0.017
54*Æ0.015
32*Æ0.015
60*Æ0.010
45*Æ0.010
98Æ0.010
53*Æ0.012
67*Æ0.017
45*Æ0.019
53*Æ0.019
66*Æ0.019
39*Æ0.018
81Æ0.018
54*Æ0.017
99Æ0.014
66Æ0.015
70Æ0.012
51*Æ0.012
63*Æ0.014
75Æ0.015
100Æ0.015
0
79Æ0.016
[a] The cytotoxicity was assessed by the MTT reduction assay. The cell viability was calculated for four experiments, including three repeats for each com-
pound. Complete RMPI medium (cRPMI) was used as a positive control (C+) of cell viability (100% viable cells) and 0.03% H2O2 as a negative control (CÀ)
of cell viability (100% dead inactive cells). All values were expressed as the mean Æ SD. The differences positive control and tested compounds were
tested the non-parametric Mann-Whitney U test. Statistical significance: *p<0.05.
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cubation, an increase of turbidity and bacterial sediment were
observed in all examined cases, with the exception of sterility
control. In the next stage of the study, the number of living
bacterial cells in the mixtures was calculated. The tests were
carried out for the highest concentration of investigated lac-
tones (50 mgmLÀ1). The highest bactericidal activity against
both E. coli ATCC 8739 and S. aureus ATCC 65389 was found in
the case of lactone 6a. The average number of colony-forming
units (CFUs) decreased by about 50% compared to the
number of CFUs in the DMSO control mixture. Compounds 5b,
6b, and 7a exhibited moderate bactericidal properties. The
bactericidal effect of the investigated lactones against E. coli
and S. aureus is shown in Table 3.
and Staphylococcus aureus ATCC 65389 was also checked. Lac-
tones 5b and 6a showed moderate antibacterial activity. Inves-
tigated lactones have antibacterial potential. However, it is
necessary to carefully control and exclude their cytotoxic activ-
ity against eukaryotic cells in the case of future application
perspective.
Experimental Section
General Methods
The reagents used for the synthesis were purchased from Fluka
and Aldrich. The 1H and 13C NMR spectra were measured by an
NMR Bruker Avance DRX 500 MHz using chloroform-d as the sol-
vent. IR spectra were recorded on a PerkinElmer Spectrum 400
spectrometer. The ESI mass spectra were recorded on a Bruker mi-
crOTOF-Q II equipped with syringe pump. Gas chromatography
was performed on a Thermo Scientific-Trace 1310 chromatograph
equipped with a TG-5HT column (30 m ” 0.25 mm). The gas chro-
matograph was temperature programmed from 150 to 3008C at a
rate of 108CminÀ1. Melting points were determined with a Boetius
micro melting point apparatus and were uncorrected. The refrac-
tive index was determined with Atago RX-7000CX refractometer.
TLC analysis was performed on silica gel 60 F254 plates (Merck).
Chromatographic separations were carried out by using silica
gel 60 (particle size 0.040–0.063 mm, Merck). Column chromatogra-
phy was performed on silica gel (Kieselgel 60, 230–400 mesh,
Merck) using mixtures of acetone, hexane, and ethyl acetate as elu-
ents. The crystal structure was determined by single-crystal X-ray
diffraction (Xcalibur, Mo-Ka, Sapphire2 detector) and deposited
with the Cambridge Crystallographic Data Centers CCDC No.
1841378.[48]
Table 3. The bactericidal effect of the investigated compounds towards
Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 65389 in con-
centration 50 mg/mL.
Compounds
E. coli ATCC 8739 S. aureus ATCC 65389
[number of CFU/mL (”107)]
5a
5b
5c
6a
6b
6c
7a
7b
6.30
4.74
6.30
3.38
5.56
6.10
5.84
5.83
6.14
6.92
0.00
6.61
7.24
5.44
9.02
3.11
6.10
8.83
7.26
9.04
9,07
9.19
0.00
9.08
7c
Positive control
Negative control
DMSO
Grignard Reaction
The antibacterial activity of other synthetic lactones has also
been described.[50] Lactones without a halogen atom were ef-
fective towards S. auresus and Listeria momocytogenes. Antimi-
crobial activity has also been reported for halolactones and hy-
droxylactone obtained through biotransformation.[51] These
compounds inhibit growth of different bacterial species (e.g. E.
coli, S. aureus, B. subtilis), yeast (e.g. Candida albicans and Sac-
charomyces cerevisiae), as well as fungi (e.g. Fusarium linii and
Aspergillus niger). Recently, promising antibacterial properties
was also shown for bicyclic lactones with three or four methyl
groups.[39] Halolactones were also considered as potential in-
hibitors of HIV-1 non-nucleoside reverse transcriptase.[52]
The synthesis of unsaturated secondary alcohols 2a-c was carried
out following the procedure described by Gaylord and Becker.[53]
A
solution of aryl bromide (0.08 mol) in diethyl ether was added
dropwise to magnesium turnings (0.08 mol) and heated under
reflux for 4 hours. The mixture was cooled on ice and a solution of
3-methyl crotonaldehyde in diethyl ether was added dropwise and
stirred for 24 h. The mixture was dissolved in diethyl ether and
added to ice and water. The separated organic layer was dried
over anhydrous magnesium sulfate and evaporated under vacuum.
The crude products was purified by column chromatography on
silica gel [ethyl acetate/hexane, 1:8 (v/v)]. The spectral data are
given below.
3. Conclusions
Compound 2a: 3-Methyl-1-(a-naphthyl)but-2-en-1-ol
Yield 38%, yellow oil, n20D =1.5572; Rf 0.24 (EtOAc : hexane,1:8),
1H NMR (CDCl3, 500 MHz) d [ppm]: 1.73 (d, J=1.3 Hz, CH3, 3H),1.92
(d, J=1.3 Hz, CH3, 3H), 2.87 (s, OH,1H), 5.53 (m, CH=C(CH3)2, 1H),
6.10 (dd, J=8.6 Hz, J=3.9 Hz, CH(OH), 1H), 7.46–7.52 (m, HAr, 3H),
7.75 (d, J=7.1, HAr, 1H), 7.80 (d, J=8.2, HAr, 1H), 7.88 (d, J=7.8,
HAr, 1H), 8.23 (d, J=8.4, HAr, 1H). 13C NMR (125 MHz, CDCl3) d
[ppm]: 17.48, 25.02, 67.81, 123.19, 124.33, 125.27, 125.41, 125.45,
127.29, 128.52, 128.98, 130.86, 133.43, 134.05, 141.25. IR 3590-3125,
This paper describes the regio- and stereoselective synthesis of
g-halogeno-d-lactones 5a–c, 6a–c, and 7a–c from commercial-
ly available reagents. The structure of all products was con-
firmed by using IR and NMR spectroscopy as well as HR-MS.
Crystallographic analysis was carried out for lactone 5a. All
synthesized g-halogeno-d-lactones were tested for cytotoxic
activity against the cell lines L929. Statistically significant inhib-
ition of metabolic activity of L929 fibroblasts was observed for
lactones 5a, 5c, 7a, and 7b. The antimicrobial activity of ob-
tained lactones against the strains Escherichia coli ATCC 8739
1660, 1598, 1506, 1450, 1370, 1160, 1135, 1045, 970, 792, 770 cmÀ1
.
HR-MS (ESI-TOF) calculated for C15H16O, m/z [M+Na]+:
235.1098786; experimental value: 235.111019.
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calculated for C15H20O2, m/z [M+Na]+: 255.136092; experimental
Compound 2b: 3-Methyl-1-phenylbut-2-en-1-ol[45]
value: 255.137098.
Yield 42%, yellow oil, n20D =1.5543; Rf 0.27 (EtOAc : hexane, 1:8),
1H NMR (CDCl3, 500 MHz), d [ppm]: 1.75 (d, J=1.2 Hz, CH3, 3H),1.80
(d, J=1.2 Hz, CH3, 3H), 5.43–5.39 (m, CH=C(CH3)2, 1H), 5.84–5.93
(m, CH(OH), 1H), 7.27–7.36 (m, HAr, 5H). 13C NMR (125 MHz, CDCl3),
d [ppm]: 18.30, 25.83, 70.75, 126.39, 127.26, 128.45, 128.54, 137.51,
144.24. IR: 3600-3100, 1667, 1495, 1450, 1376, 1200, 1030, 1003,
863, 756, 747, 699 cmÀ1. HR-MS (ESI-TOF) calculated for C11H14O, m/
z [M+Na]+: 185.0942294; experimental value: 185.094086.
Compound 3c: Ethyl 5-(p-Fluorophenyl)-3,3-dimethylpent-4-
enoate
Yield 55%, yellow oil, n20D =1.5103; Rf 0.49 (EtOAc : hexane, 1:20),
1H NMR (CDCl3, 500 MHz), d [ppm]: 1.20 (t, J=7.1 Hz, OCH2CH3,
3H), 1.24 (s, C(CH3)2, 6H), 2.37 (s, CH2CO2CH2CH3, 2H), 4.09 (q, J=
7.1 Hz, OCH2CH3, 2H), 6.22 (d, J=16.2 Hz, CH=CHC(CH3)2, 1H), 6.30
(d, J=16.2 Hz, CH=CHC(CH3)2, 1H), 6.98 (t, J=8.7 Hz, HAr, 2H),
7.28–7.34 (m, HAr, 2H). 13C NMR (125 Hz, CDC3), d [ppm]: 14.28,
27.39, 35.81, 47.29, 60.02, 115.29, 124.98, 127.54, 138.54, 138.55,
161.99, 171.56. IR: 1730, 1505, 1365, 1220, 1160, 1030, 973, 850,
815 cmÀ1. HR-MS (ESI-TOF) calculated for C15H19FO2, m/z [M+Na]+:
273.1266706; experimental value: 273.127710.
Compound 2c: 1-(p-Fluorophenyl)-3-methylbut-2-en-1-ol[44]
Yield 65%, yellow oil, n20D =1.5266; Rf 0.25 (EtOAc : hexane, 1:8),
1H NMR (CDCl3, 500 MHz), d [ppm]: 1.76 (d, J=1.2 Hz, CH3, 3H),
1.80 (d, J=1.2 Hz, CH3, 3H), 5.35–5.40 (m, CH=C(CH3)2, 1H), 5.45 (d,
J=8.7 Hz, CH(OH), 1H), 6.94–7.07 (m, HAr, 2H), 7.28–7.43 (m, HAr,
2H). 13C NMR (125 MHz, CDCl3) d [ppm]: 18.26, 25.81, 70.20, 115.22,
127.74, 127.80, 133.53, 139.04, 162.15. IR: 3623-3087, 1605, 1501,
1459, 1370, 1221, 1150, 970, 855, 816 cmÀ1. HR-MS (ESI-TOF) calcu-
lated for C11H13FO, m/z [M+Na]+: 203.084808; experimental value:
203.085423.
Basic Hydrolysis
g,d-Unsaturated carboxylic acids were obtained by carrying out al-
kaline hydrolysis of esters 3a–c. The ester was dissolved in ethanol-
ic KOH and heated for 2 h under reflux. When the reaction was
complete, the ethanol was evaporated and the mixture was acidi-
fied with 0.1m hydrochloric acid. The product was extracted with
diethyl ether and dried over anhydrous magnesium sulfate. The
product did not require purification. Spectral data are given below.
Claisen Rearrangement
g,d-Unsaturated ethyl esters 3a–c were obtained from a Claisen re-
arrangement.[54] A mixture of the alcohol (0.02 mol) and triethyl or-
thoacetate (0.15 mol) with a drop of propionic acid was heated at
1388C for 5 h while distilling ethanol. When the reaction was com-
pleted (as monitored by TLC), the excess triethyl orthoacetate was
distilled off and the crude product was purified by column chroma-
tography on silica gel [ethyl acetate/hexane 1:80 (v/v)]. Spectral
data are given below.
Compound 4a: 3,3-Dimethyl-5-(a-naphthyl)pent-4-enoic Acid
Yield 97%, yellow oil, n20D =1.5830; Rf 0.24 (EtOAc : hexane, 1:5),
1H NMR (CDCl3, 500 MHz), d [ppm]: 1.37 (s, C(CH3)2, 6H),2.52 (s,
CH2COOH, 2H), 6.29 (d, J=15.8 Hz, ArCH=CH, 1H), 7.10 (d, J=
15.8 Hz, ArCH=CH, 1H), 7.38–7.53 (m, HAr, 4H), 7.73 (d, J=8.1 Hz,
HAr, 1H), 7.82 (dd, J=7.42 Hz, J=2.0 Hz, HAr, 1H), 8.07 (d, J=
7.9 Hz, HAr, 1H), 11.02 (s, COOH, 1H). 13C NMR (125 Hz, CDCl3), d
[ppm]: 27.54, 36.16, 46.90, 123.84, 123.92, 124.00, 125.64, 125.82,
127.45, 127.76, 128.42, 131.28, 133.53, 135.55, 141.76, 177.51. IR:
Compound 3a: Ethyl 3,3-Dimethyl-5-(a-naphthyl)pen-4-enoate
Yield 54%, yellow oil, n20D =1.5753; Rf 0.46 (EtOAc : hexane, 1:20),
1H NMR (CDCl3, 500 MHz), d [ppm]: 1.17 (t, J=7.2 Hz, OCH2CH3,
3H), 1.38 (s, C(CH3)2, 6H), 2.50 (s, CH2COOCH2CH3, 2H) 4.16 (q, J=
7.2 Hz, OCH2CH3, 2H), 6.33 (d, J=15.8 Hz, CH=CH, 1H), 7.11 (d, J=
15.8 Hz, Ar-CH=CH, 1H), 7.43–7.46 (m, HAr, 1H), 7.47–7.53 (m, HAr,
2H), 7.56 (d, J=8.1 Hz, HAr, 1H), 7.76 (d, J=8.1 Hz, HAr, 1H), 7.85
(d, J=8.1 Hz, HAr, 1H), 8.11 (d, J=8.1 Hz, HAr, 1H). 13C NMR
(125 Hz, CDCl3) d [ppm]: 14.34, 27.60, 36.37, 47.40, 60.15, 123.57,
123.80, 123.95, 125.68, 125.84, 127.43, 128.48, 128.98, 131.32,
133.62, 135.67, 142.27, 171.72. IR: 1729, 1507, 1394, 1367, 1235,
1120, 1035, 970, 789, 777 cmÀ1. HR-MS (ESI-TOF) calculated for
C18H20O2, m/z [M+Na]+: 291.136092; experimental value:
291.136897.
3300-2745, 1701, 1507, 1394, 1288, 1168, 1115, 968, 794, 778 cmÀ1
HR-MS (ESI-TOF) calculated for C17H18O2, m/z [M+Na]+:
.
277.1204428; experimental value: 277.127429.
Compound 4b: 3,3-Dimethyl-5-phenylpent-4-enoic Acid[46]
Yield 95%, yellow oil, n20D =1.5830; Rf 0.33 (EtOAc : hexane, 1:5),
1H NMR (CDCl3, 500 MHz), d [ppm]: 1.26 (s, C(CH3)2, 6H), 2.43 (s,
CH2COOH, 2H), 6.30 (d, J=16.2 Hz, ArCH=CH, 1H), 6.32 (d, J=
16.2 Hz, ArCH=CH, 1H), 7.18–7.22 (m, HAr, 1H), 7.18–7.22 (m, HAr,
1H), 7.34–7.36 (m, HAr, 2H), 11.32 (s, COOH, 1H). 13C NMR (125 Hz,
CDCl3), d [ppm]: 27.36, 35.67, 46.88, 126.22, 126.44, 127.12, 128.50,
137.52, 138.42, 177.77. IR: 3280-2760, 1697, 1488, 1445, 1409, 1318,
1270, 965, 750, 670 cmÀ1. HR-MS (ESI-TOF) calculated for C13H16O2,
m/z [M+Na]+: 227.1047936; experimental value: 227.103997.
Compound 3b: Ethyl 3,3-Dimethyl-5-phenylpent-4-enoate
Yield 46%, yellow oil, n20D =1.5201; Rf 0.51 (EtOAc : hexane, 1:20),
1H NMR (CDCl3, 500 MHz), d [ppm]: 1.21 (t, J=7.1 Hz, OCH2CH3, 3H)
1.25 (s, C(CH3)2, 6H), 2.38(s, CH2COOCH2CH3, 6H), 4.09 (q, J=7.1 Hz,
OCH2CH3, 2H), 6.30 (d, J=16.2 Hz, CH=CHC(CH3)2, 1H), 6.34 (d, J=
16.2 Hz, CH=CHC(CH3)2, 1H), 7.19 (t, J=7.3 Hz, HAr, 1H), 7.29 (t, J=
7.7 Hz, HAr, 2H), 7.35 (d, J=7.2 Hz, HAr, 2H). 13C NMR (125 Hz,
CDCl3) d [ppm]: 14.29, 27.39, 29.26, 47.29, 60.01, 126.10, 126.14,
126.99, 128.45, 137.62, 138.80, 171.61. IR: 1730, 1463, 1447, 1360,
1230, 1200, 1150, 1120, 1030, 960, 750, 960 cmÀ1. HR-MS (ESI-TOF)
Compound 4c: 5-(p-Fluorophenyl)-3,3-dimethylpent-4-enoic
Acid
Yield 97%, yellow oil, n20D =1.5205; Rf 0.30 (EtOAc : hexane, 1:5),
1H NMR (CDCl3, 500 MHz), d [ppm]:1.26 (s, C(CH3)2, 6H), 2.42 (s,
CH2COOH, 2H), 6.22 (d, J=16.2 Hz, ArCH=CH, 1H), 6.32 (d, J=
16.2 Hz, ArCH=CH, 1H), 6.93–7.02 (m, HAr, 1H), 7.28–7.34 (m, HAr,
2H), 10.41 (s, COOH, 1H). 13C NMR (125 Hz, CDCl3), d [ppm]: 27.36,
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35.63, 46.78, 115.32, 125.34, 127.63, 133.61, 138.10, 177.09, 162.05.
Procedure of Bromolactonization
IR: 3280-2780, 1697, 1506, 1315, 1275, 1230, 1160, 967, 811 cmÀ1
.
Bromolactones 6a–c were obtained according to the procedure
described in Ref. [50]. The mixture of the above-mentioned acid
(0.005 mol) and N-bromosuccinimide (0.01 mol) was dissolved in
60 mL of tetrahydrofuran (THF). Acetic acid was added dropwise
and mixture was stirred at room temperature for 48 h. When the
reaction was complete, the mixture was dissolved in diethyl ether,
washed with saturated NaHCO3 solution, and dried over anhydrous
magnesium sulfate. The crude products were purified by column
chromatography on silica gel [acetone/hexane, 1:10 (v/v)]. Spectral
data are given below.
HR-MS (ESI-TOF) calculated for C13H15FO2, m/z [M+Na]+:
245.0953722; experimental value: 245.096365.
Procedure of Iodolactonization
Iodolactones 5a–c were prepared according to the procedure de-
scribed in Ref. [55]. The mixed solution of the above-mentioned
acid in diethyl ether and saturated aqueous NaHCO3 was stirred for
30 min. A solution of iodine in potassium iodide was added drop-
wise and the reaction mixture was heated under reflux for 24 h.
When the reaction was complete, the mixture was dissolved in di-
ethyl ether and washed with saturated Na2S2O3 solution. The or-
ganic layer was washed with saturated NaHCO3 solution and dried
over anhydrous magnesium sulfate. The crude products were puri-
fied by column chromatography on silica gel [acetone/hexane,
1:15 (v/v)]. Spectral data are given below.
Compound 6a: trans-5-Bromo-4,4-dimethyl-6-(a-naphthyl)-
tetrahydro-2H-pyran-2-one
Yield 62%, colorless crystals, m.p.=197–1988C, Rf 0.20 (EtOAc :
hexane, 1:7), 1H NMR (CDCl3, 500 MHz), d [ppm]: 1.29 (s, C(CH3)2,
3H), 1.42 (s, C(CH3)2, 3H), 2.76 (d, J=17.4 Hz, CH2, 1H), 2.94 (d, J=
17.4 Hz, CH2, 1H), 4.56 (d, J=10.7 Hz, CHBr, 1H), 6.25 (d, J=
10.7 Hz, CHAr, 1H), 7.47–7.53 (m, HAr, 2H), 7.53–7.59 (m, HAr, 2H),
7.89 (d, J=8.2 Hz, HAr, 2H), 8.08 (d, J=8.6 Hz, HAr, 1H). 13C NMR
(125 Hz, CDCl3), d [ppm]: 21.74, 29.68, 35.65, 44.18, 60.51, 79.69,
122.93, 125.00, 125.85, 125.95, 126.60, 129.12, 130.12, 131.22,
132.53, 133.88, 168.59. IR: 1730 (carbonyl band), 1460, 1355, 1240,
1160, 1100, 1020, 1007, 908, 802, 780, 740, 660, 630 cmÀ1. HR-MS
(ESI-TOF) calculated for C17H17BrO2, m/z [M+K]+: 371.0048922; ex-
perimental value: 371.004088.
Compound 5a: trans-5-Iodo-4,4-dimethyl-6-(a-naphthly)tetra-
hydro-2H-pyran-2-one
Yield 93%, yellow crystals, m.p.=118–1198C, Rf 0.16 (EtOAc :
hexane, 1:7), 1H NMR (CDCl3, 500 MHz), d [ppm]: 1.26 (s, C(CH3)2,
3H), 1.40 (s, C(CH3)2, 3H), 2.78 (d, J=17.3 Hz, CH2, 1H), 2.96 (d, J=
17.3 Hz, CH2, 1H), 4.71 (d, J=11.4 Hz, CHI, 1H), 6.36 (d, J=11.4 Hz,
CHAr, 1H), 7.48–7.55 (m, HAr, 2H), 7.56–7.61 (m, HAr, 2H), 7.90 (d,
J=8.2 Hz, HAr, 2H), 8.10 (d, J=8.6 Hz, HAr, 1H). 13C NMR (125 Hz,
CDCl3), d [ppm]: 23.60, 32.58, 35.75, 42.59, 43.57, 77.23, 122.95,
124.98, 125.90, 125.95, 126.60, 129.16, 130.24, 131.24, 133.13,
133.92, 168.94. IR: 1723 (carbonyl band), 1352, 1235, 1155, 1020,
1002, 800, 775, 735, 650, 620, 610 cmÀ1. HR-MS (ESI-TOF) calculated
for C17H17IO2, m/z [M+Na]+: 403.0170952; experimental value:
403.017215.
Crystal Data for 6a
C17H17BrO2, M=333.21, colorless block, orthorhombic, space group
P212121, a=6.2726(10) Š, b=7.6137(16) Š, c=29.901(4) Š, b=
908,V=1428.0(4) Š3, Z=4, 1cald =1.5497, MgmÀ3, T=298(2) K, R=
0.1065, wR=0.2092 [2893 reflections with I>2 sigma(I)] for 181
variables).[48]
Compound 5b: trans-5-Iodo-4,4-dimethyl-6-phenylo-tetra-
hydro-2H-pyran-2-one[46]
Compound 6b: trans-5-Bromo-4,4-dimethyl-6-phenyl-tetra-
hydro-2H-pyran-2-one
Yield 87%, yellow crystals, m.p.=72–738C, Rf 0.19 (EtOAc : hexane,
1
1:7), H NMR (CDCl3, 500 MHz), d [ppm]: 1.21 (s, C(CH3)2, 3H), 1.29
Yield 59%, colorless crystals, m.p.=107–1088C, Rf 0.25 (EtOAc :
hexane, 1:7), 1H NMR (CDCl3, 500 MHz), d [ppm]: 1.20 (s, C(CH3)2,
3H), 1.28 (s, C(CH3)2, 3H), 2.61 (d, J=17.4 Hz, CH2, 1H), 2.81 (d, J=
17.4 Hz, CH2, 1H), 4.11 (d, J=10.8 Hz, CHBr, 1H), 5.40 (d, J=
10.8 Hz, CHAr, 1H), 7.35–7.42 (m, HAr, 5H). 13C NMR (125 Hz, CDCl3),
d [ppm]: 21.40, 29.64, 35.53, 44.02, 61.33, 83.39, 168.40, 127.59,
128.50, 129.25, 137.19. IR: 1730 (carbonyl band), 1462, 1340, 1235,
1180, 1017, 923, 860, 806, 755, 670, 640 cmÀ1. HR-MS (ESI-TOF) cal-
culated for C13H15BrO2, m/z [M+Na]+: 305.015305; experimental
value: 305.013815.
(s, C(CH3)2, 3H), 2.65 (d, J=17.3 Hz, CH2, 1H), 2.87 (d, J=17.3 Hz,
CH2, 1H), 4.28 (d, J=11.3 Hz, CHBr, 1H), 5.56 (d, J=11.3 Hz, CHAr,
1H), 7.34–7.48 (m, HAr, 5H). 13C NMR (125 Hz, CDCl3), d [ppm]:
23.28, 32.57, 35.44, 42.38, 44.97, 84.98, 127.78, 128.53, 129.40,
137.75, 168.91. IR: 1723 (carbonyl band), 1460, 1350, 1240, 1160,
1070, 1005, 925, 851, 750, 700, 620, 609 cmÀ1. HR-MS (ESI-TOF) cal-
culated for C13H15IO2, m/z [M+Na]+: 353.003446; experimental
value: 353.004945.
Compound 5c: trans-6-(p-Fluorophenyl)-5-iodo-4,4-dimethyl-
tetrahydro-2H-pyran-2-one
Compound 6c: trans-5-Bromo-6-(p-fluorophenyl)-4,4-di-
methyltetrahydro-2H-pyran-2-one
Yield 60%, yellow crystals, m.p.=81–828C, Rf 0.18 (EtOAc : hexane,
1
1:7), H NMR (CDCl3, 500 MHz) d [ppm]: 1.20 (s, C(CH3)2, 3H), 1.28
(s, C(CH3)2, 3H), 2.64 (d, J=17.3 Hz, CH2, 1H), 2.76 (d, J=17.3 Hz,
CH2, 1H), 4.22 (d, J=11.4 Hz, CHI, 1H), 5.54 (d, J=11.4 Hz, CHAr,
1H), 7.04–7.14 (m, HAr, 2H), 7.32–7.35 (m, HAr, 2H). 13C NMR
(125 Hz, CDCl3), d [ppm]: 23.26, 32.56, 35.43, 42.31, 45.01, 84.16,
115.54, 129.59, 133.77, 163.09, 168.65. IR: 1720 (carbonyl band),
1605, 1515, 1368, 1342, 1300, 1220, 1154, 1078, 1009, 904, 850,
805, 615 cmÀ1. HR-MS (ESI-TOF) calculated for C13H14FIO2, m/z [M+
Na]+: 370.9920246; experimental value: 371.001004.
Yield 85%, colorless crystals, m.p.=102–1038C, Rf 0.23 (EtOAc :
hexane, 1:7), 1H NMR (CDCl3, 500 MHz), d [ppm]: 1.21 (s, C(CH3)2,
3H), 1.28 (s, C(CH3)2, 3H), 2.60 (d, J=17.4 Hz, CH2, 1H), 2.82 (d, J=
17.4 Hz, CH2, 1H), 4.05 (d, J=10.8 Hz, CHBr, 1H), 5.39 (d, J=
10.8 Hz, CHAr, 1H), 7.03–7.12 (m, HAr, 2H), 7.32–7.39 (m, HAr, 2H).
13C NMR (125 Hz, CDCl3), d[ppm]: 21.38, 29.55, 35.34, 44.01, 61.35,
82.61, 115.52, 129.42, 133.12, 163.05, 168.25. IR: 1726 (carbonyl
band), 1510, 1360, 1230, 1160, 1020, 905, 855, 805, 650, 620 cmÀ1
.
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HR-MS (ESI-TOF) calculated for C13H14BrFO2, m/z [M+Na]+:
In Vitro Cell Study
323.0058836; experimental value: 323.008034.
The L929 mouse fibroblasts (LGC Standards, Middlesex, UK) were
used for in vitro cytotoxicity testing. The cells were maintained
under standard conditions (378C, 5% CO2) in 25 mL tissue culture
flasks in RPM 1640 medium supplemented with 10% fetal bovine
serum (FBS) and antibiotics: 100 UmLÀ1 penicillin and 100 mgmLÀ1
streptomycin. Cell suspension for cytotoxicity assay or to start a
new culture was obtained by treatment of confluent monolayers
with 0.25% trypsin solution washed and subcultured at the cell
density 108 cellsmLÀ1. Cell cultures were supplemented with fresh
medium two or three times per week to maintain them in log
phase. The viability of the cells was assessed by exclusion of
trypan blue dye and was in the range 93–95%.
Procedure of Chlorolactonization
Chlorolactones 7a–c were obtained according to the procedure
described in Ref. [50]. The mixture of the above-mentioned acid
(0.005 mol) and N-chlorosuccinimide (0.009 mol) was dissolved in
60 mL of THF. Acetic acid was added dropwise and mixture was
stirred at room temperature for 48 h. When the reaction was com-
plete, the mixture was dissolved in diethyl ether, washed with satu-
rated NaHCO3 solution, and dried over anhydrous magnesium sul-
fate. The crude products was purified by column chromatography
on silica gel [acetone/hexane, 1:10 (v/v)]. Spectral data are given
below.
Measurements of Cellular Metabolic Activity and Global
Growth Inhibition
Lactones 5a–c, 6a–c, and 7a–c were used for the assessment of
L929 cells metabolic activity. Cells in culture medium were seeded
in the 96-well plates (2”105 cells/well) for 24 h, at 378C, 5% CO2.
All tested compounds were diluted in RPM-1640 medium in con-
centrations of 50, 20, 10, 5, 0.5, and 0.1 mgmLÀ1, added to the cells
(100 mL/well), and incubated under standard conditions for 24 h.
Following incubation, the cell monolayers were carefully screened
using light microscopy, as recommended by ISO norm 10993-5 to
evaluate the morphology of cells.[56] They were estimated by meas-
uring the ability of the cells to reduce MTT [(3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide)], which is one of the tests
recommended by the Food and Drug Administration (FDA) and
the International Organization for Standardization (IOS). Fresh MTT
solution (5 mgmLÀ1 in sterile PBS) was added to each well and in-
cubated for 4 h at 378C. Formazan crystals were dissolved with
acidic isopropanol (0.1m HCl in absolute isopropanol). Optical den-
sity values were measured at 570 nm with a plate reader Victor2.
The results are presented as the average number of cells able to
convert MTT obtained from four independent experiments, includ-
ing three repeats for each compound.
Compound 7a: trans-5-Chloro-4,4-dimethyl-6-(a-naphthyl)-
tetrahydro-2H-pyran-2-one
Yield 75%, colorless crystals, m.p.=192–1938C, Rf 0.20 (EtOAc :
hexane, 1:7), 1H NMR (CDCl3, 500 MHz), d [ppm]: 1.23 (s, C(CH3)2,
3H), 1.35 (s, C(CH3)2, 3H), 2.68 (d, J=17.4 Hz, CH2, 1H), 2.84 (d, J=
17.4 Hz, CH2, 1H), 4.37 (d, J=10.4 Hz, CHCl, 1H), 6.06 (d, J=
10.4 Hz, CHAr, 1H), 7.47–7.60 (m, HAr, 4H), 7.88 (d, J=7.8 Hz, HAr,
2H), 8.05 (d, J=8.5 Hz, HAr, 1H). 13C NMR (125 Hz, CDCl3), d [ppm]:
20.83, 29.54, 35.72, 44.35, 66.68, 79.59, 122.92, 125.01, 125.84,
125.91 126.61, 129.10, 130.06, 131.24, 132.24, 133.87, 168.56. IR:
1730 (carbonyl band), 1350, 1240, 1216, 1170, 1140, 1035, 930, 910,
850, 790, 775, 730, 675, 640 cmÀ1. HR-MS (ESI-TOF) calculated for
C17H17ClO2, m/z [M+Na]+: 311.0814712; experimental value:
311.080645.
Compound 7b: trans-5-Chloro-4,4-dimethyl-6-phenylotetra-
hydro-2H-pyran-2-one
Yield 85%, colorless crystals, m.p.=96–978C, Rf 0.24 (EtOAc :
hexane, 1:7), 1H NMR (CDCl3, 500 MHz), d [ppm]: 1.19 (s, C(CH3)2,
3H), 1.26 (s, C(CH3)2, 3H), 2.57 (d, J=17.4 Hz, CH2, 1H), 2.76 (d, J=
17.4 Hz, CH2, 1H), 3.97 (d, J=10.4 Hz, CHCl, 1H), 5.25 (d, J=
10.4 Hz, CHAr, 1H), 7.33–7.44 (m, HAr, 5H). 13C NMR (125 Hz, CDCl3),
d [ppm]: 20.45, 28.22, 35.39, 44.26, 67.26, 83.01, 127.48, 128.53,
129.22, 136.92, 168.35. IR: 1733 (carbonyl band), 1460, 1350, 1250,
1215, 1174, 1143, 1020, 930, 910, 863, 830, 760, 700, 660 cmÀ1. HR-
MS (ESI-TOF) calculated for C13H15ClO2, m/z [M+Na]+: 261.065822;
experimental value: 261.065139.
Assessment of Antibacterial Activity
The study of the antibacterial properties of chemical compounds
was carried out by using spectrophotometric methods and count-
ing of bacterial colonies.[57] The cultures of E. coli ATCC 8739 and
S.aureus ATCC 65389 were grown on LB Agar (Biocorp) for 18 h at
378C. A single colony was used to prepare a cell suspension in
saline (0.85%), which was adjusted to 0.5 on the McFarland scale.
Added to the wells of the microtiter plate were: 180 mL of sterile
enriched broth (BTL), 10 mL of the tested compound dissolved in
DMSO, and 10 mL of the bacterial suspension. Control solutions
were also prepared: a) 180 mL of BTL and 20 mL saline (sterility con-
trol); b) 80 mL of BTL, 10 mL of DMSO and 10 mL bacterial suspen-
sion (control of antibacterial activity of DMSO); c) 180 mlBTL 10 mL
saline and 10 mL bacterial suspension (bacterial growth controls).
The microtiter plate was incubated for 18 h at 378C. The experi-
ment was repeated three times. The bacteriostatic effect of lac-
tones was determined by measuring of bacterial suspension tur-
bidity with Infinite PRO Tecanmicroplate reader at 600 nm. The
CFU method was used to indicate the number of living bacterial
cells after the exposition at the highest amine concentration.
Compound 7c: trans-5-Chloro-6-(p-fluorophenyl)-4,4-di-
methyltetrahydro-2H-pyran-2-one
Yield 65%, colorless crystals, m.p.=89–908C, Rf 0.22 (EtOAc :
hexane, 1:7), 1H NMR (CDCl3, 500 MHz), d [ppm]: 1.19 (s, C(CH3)2,
3H), 1.26 (s, C(CH3)2, 3H), 2.56 (d, J=17.4 Hz, CH2, 1H), 2.76 (d, J=
17.4 Hz, CH2, 1H), 3.92 (d, J=10.5 Hz, CHCl, 1H), 5.24 (d, J=
10.5 Hz, CHAr, 1H), 6.98–7.14 (m, HAr, 2H), 7.31–7.42 (m, HAr, 2H).
13C NMR (125 Hz, CDCl3), d [ppm]: 20.43, 28.22, 35.38, 44.24, 67.28,
82.23, 115.53, 129.30, 132.80, 163.05, 168.17. IR: 1730 (carbonyl
band), 1605, 1515, 1370, 1340, 1250, 1230, 1160, 1025, 913, 850,
805, 722, 660, 625 cmÀ1
.
HR-MS (ESI-TOF) calculated for
Microbial media: LB Agar composition: tryptone À10 glÀ, sodium
chloride À5 glÀ, yeast extract À5 glÀ, agar À15 glÀ; enriched
broth (BTL), composition: beef extract À0.4 glÀ, peptone À4 glÀ,
C13H14ClFO2, m/z [M+Na]+: 279.0564006; experimental value:
279.057500.
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Received: June 12, 2018
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