New γ-Halo-δ-lactones and δ-Hydroxy-γ-lactones with Strong Cytotoxic Activity

Article abstract of DOI:10.3390/molecules24101875

This paper presents the synthesis of γ-halo-δ-lactones, δ-iodo-γ-lactones and δ-hydroxy-γ-lactones from readily available organic substrates such as trans-crotonaldehyde and aryl bromides. Crystal structure analysis was carried out for lactones that were obtained in crystalline form. All halo-δ-lactones and δ-hydroxy-γ-lactones were highly cytotoxic against gastric cancer AGS cells with IC₅₀ values in the range of 0.0006–0.0044 mM. Some lactones showed high bactericidal activity against E. coli ATCC 8739 and S. aureus ATCC 65389, which reduced the number of CFU/mL by 70–83% and 87% respectively.

Full text of DOI:10.3390/molecules24101875

molecules
Article
New γ-Halo-δ-lactones and δ-Hydroxy-γ-lactones
with Strong Cytotoxic Activity
Angelika Kamizela 1,* , Barbara Gawdzik 1, Mariusz Urbaniak 1, Łukasz Lechowicz 2,
Agata Białon´ska 3, Sylwia Ewa Kutniewska 4 , Weronika Gonciarz 5 and Magdalena Chmiela 5
1
´
Institute of Chemistry, Jan Kochanowski University, Swie˛tokrzyska 15 G, 25-406 Kielce, Poland;
b.gawdzik@ujk.edu.pl (B.G.); mariusz.urbaniak@ujk.edu.pl (M.U.)
Institute of Biology, Jan Kochanowski University, Swie˛tokrzyska 15 G, 25-406 Kielce, Poland;
llechowicz@ujk.edu.pl
2
3
4
5
´
Department of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wrocław, Poland;
agata.bialonska@chem.uni.wroc.pl
˙
Department of Chemistry, University of Warsaw, Zwirki i Wigury 101, 02-089 Warszawa, Poland;
sekutyla@gmail.com
Department of Immunology and Infectious Biology, University of Lodz, Banacha 12/16, 90-237 Łódz´,
Poland; weronika.gonciarz@biol.uni.lodz.pl (W.G.); magdalena.chmiela@biol.uni.lodz.pl (M.C.)
Correspondence: angelikakamizela@gmail.com; Tel.: +48-41-349-7019
*
Received: 24 April 2019; Accepted: 9 May 2019; Published: 15 May 2019
Abstract: This paper presents the synthesis of -halo- -lactones, δ-iodo-γ-lactones and δ-hydroxy-γ-
γ
δ
lactones from readily available organic substrates such as trans-crotonaldehyde and aryl bromides.
Crystal structure analysis was carried out for lactones that were obtained in crystalline form.
All halo-δ-lactones and δ-hydroxy-γ-lactones were highly cytotoxic against gastric cancer AGS
cells with IC50 values in the range of 0.0006–0.0044 mM. Some lactones showed high bactericidal
activity against E. coli ATCC 8739 and S. aureus ATCC 65389, which reduced the number of CFU/mL
by 70–83% and 87% respectively.
Keywords: halolactones; translactonization; hydroxylactones; anti-cancer properties; bactericidal
properties
1. Introduction
Lactones are compounds with unusual biological activities. The natural source of lactones are
plants [
anti-cancer activity [
and deterrent properties [17
1
,
2
] but they are also found in microorganisms [
], cytotoxic [ ], bacterio- [10
18]. This biological potential is widely used in medicine and agriculture.
3
–
] and animals [
12] and fungistatic [13
4
]. They exhibit, among others,
5,
6
7–
9
,
14], antiviral [15 16
, ]
,
Lactones also have sensory properties [19] used in the cosmetics and food industries but they can
also be toxic, such as mycotoxins produced by mold fungi [20]. The properties and application
possibilities of lactones are they reason they are still intensively studied by many groups. New
natural derivatives are still isolated, their properties are examined and new synthetic analogs with
desirable properties are sought. This interesting group of compounds also attracted our attention.
Thus far, we have developed a synthesis of
β-ayl-δ-iodo-γ-lactones [21], aryl-δ-hydroxy-γ-lactones [22],
and trans- -halo- -lactones [23], and tested their cytotoxic and bactericidal activities. Here, we
γ
δ
present stereoselective substrate-controlled synthesis of new γ-halo-δ-lactones. Some of the obtained
iodo-δ-lactones can easily undergo translactonization reactions leading to δ-hydroxy-γ-lactones with
high yields. Their biological activities were also evaluated: antimicrobial activity against Escherichia
coli strain ATCC 8739 and Staphylococcus aureus strain ATCC 65389, and cytotoxicity against L929 cell
lines (mouse fibroblasts) and human gastric cancer cell line AGS.
Molecules 2019, 24, 1875; doi:10.3390/molecules24101875
www.mdpi.com/journal/molecules

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2. Results and Discussion
2.1. Synthesis
Halolactones were obtained as a result of the four-step reaction [23] shown in Figure 1.
The first step of the synthesis was a Grignard reaction between selected aryl bromides 1a and
trans-crotonaldehyde, which gave us unsaturated allyl alcohols 2a with yields 48–62% [24].
The Johnson–Claisen rearrangement [25] of these alcohols with triethyl orthoacetate and catalytic
amount of propionic acid obtained -unsaturated ethyl esters 3a with yields 49–63%. In the third
step, the esters were subjected to alkaline hydrolysis in ethanol to obtain -unsaturated carboxylic
acids 4a . Thus obtained substrates, with suitably substituted double bonds, enforced selectivity of
–
c
–
c
γ
,
δ
–c
γ,δ
–c
halolactonization in the last step of the synthesis. The yields and products distribution are shown in
Table 1. Only two of these compounds have been described thus far in the literature [26].
Figure 1. A four-step synthesis of γ-halo-δ-lactones and δ-hydroxy-γ-lactones.
Table 1. The yields and products distribution from halolactonization of unsaturated carboxylic acids
4a–c (according to GC).
γ-Chloro-δ-lactones
γ-Bromo-δ-lactones
yield [%] trans,trans cis,trans yield [%] trans,trans cis,trans
−C10H7
−C6H5
51
82
62
5a 82%
5b 84%
5c 81%
6a 18%
6b 16%
6c 19%
72
72
78
7a 82%
7b 78%
7c 76%
8a 18%
8b 22%
8c 24%
−C6H4F
According to the literature [27
out with N-chlorosuccinimide (NCS) or N-bromosuccinimide (NBS), leads to a mixture of
-lactones, with an excess of 6-endo cyclization products. The regioselectivity of the reaction is
,28], halolactonization of
γ
,
δ
-unsaturated carboxylic acids, carried
γ
- and
δ

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influenced by steric hindrance at carbon atoms adjacent to the double bond. In the case of acids with
substituents at the C-3, nucleophilic attack at the -position is hindered and the amount of products
formed by 5-exo cyclization decreases. This is also confirmed by our results. The chlorolactonization of
unsaturated carboxylic acids 4a with N-chlorosuccinimide (NCS) in THF, only proceeded through
a 6-endo cyclization mechanism and gave new -halo- -lactones 5a and 6a . The analysis of
γ
–c
γ
δ
–
c
–c
post-reaction mixtures showed that in each case only isomers trans,trans and cis,trans were formed
(Figure 2). The thermodynamically more stable isomers trans,trans were always present in large excess,
up to 80% (Tables 1 and 2). An analogous synthesis using
methyl group in the -position led to a mixture of products. Lactonization of unsaturated carboxylic
acids with two methyl groups in the β-position have only one product, trans-γ-chloro-δ-lactone [23].
γ,δ-unsaturated carboxylic acids with a
β
Figure 2. Mechanisms of chloro- and bromolactonization of γ,δ-unsaturated carboxylic acids with NCS
and NBS.
Table 2. The yields and products distribution from iodolactonization of unsaturated carboxylic acids
4a–c (according to GC).
cis-δ-Iodo-δ-lactones
yield [%] trans cis
γ-Iodo-δ-lactones
trans,trans cis,trans
δ-Hydroxy-γ-lactones
trans
cis
−C10H7
−C6H5
-
-
12a 10%
12b 4%
12c 7%
13a 90%
13b 11%
13c 18%
75
69
-
-
11b 10%
11c 8%
9b 42%
9c 37%
10b 33%
10c 30%
−C6H4F
Bromolactones 7a
in THF with a yield of 72–78%. Mechanisms for lactonization of
–
c
and 8a
–
c
were obtained by reacting 4a
–
c
with N-bromosuccinimide (NBS)
-unsaturated carboxylic
γ,δ
acids with NCS and NBS are similar [27] (Figure 2) and lead to the same products of 6-endo
cyclization. The analysis of post-reaction mixtures showed that in bromolactonization of unsaturated
carboxylic acids 4a
of trans,trans- -bromo-
18–24%. The previously described reactions of
NaHCO3 solution, carried out under kinetic control, resulted in mixtures of
–
c
in each case only isomers trans,trans and cis,trans were formed. The yield
-lactones was 76–82% and yield of cis,trans- -bromo- -lactones was only
-unsaturated carboxylic acids with I2/KI in saturated
- and -iodolactones,
γ
δ
γ
δ
γ,δ
γ
δ
in which the products formed by 5-exo cyclization predominated. The regioselectivity-determining
step for the reaction is the attack of the negatively charged oxygen atom of the carboxyl group on the

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iodine-double bond complex. The presence of substituents in the
β
-position of unsaturated carboxylic
acids hindered nucleophilic attack at the C-4 position. This limited the formation of five-membered
lactones. The effect of steric hindrance at the C-6 negatively influenced the formation of
δ lactones and
favored γ-lactones [27].
Iodolactonization of 4a
mixture [29]. Analysis of the reaction mixtures unexpectedly showed the formation of two new isomers
of -hydroxy- -lactone 12a and 13a. The yield of reactions was in the range of 69–85%. The reaction
–c acids was carried out with I2/KI and NaHCO3 in a water/diethyl ether
δ
γ
conditions prevented the direct synthesis of these compounds because the reaction was carried out in
an alkaline solution, whereas hydroxylactonization requires an acidic environment [22]. Cis-isomers
are more thermodynamically stable than trans-isomers and predominantly formed in this reaction.
The excess of trans-isomer is usually observed in the synthesis of
initially gave -iodo- -lactones, which, through a translactonization reaction, were transformed into the
final products–hydroxylactones 12a and 13a. This was confirmed by observations of purified isomers
trans,trans and cis,trans -iodo- -lactones 9b and 10b, and 9c and 10c. These isomers underwent rapid
γ-lactones. However, the reaction
δ
γ
γ
δ
decomposition with release of iodine. The analysis of residues showed that pure isomers trans,trans or
cis,trans transformed into an equimolar mixture of isomers trans and cis hydroxylactones (Figure 3).
These results indicate that the translactonization of γ-iodo-δ-lactones involved elimination of the
iodine, which was a good leaving group and rearrangement of the six-membered lactone ring into the
five-membered one, according to the SN1 mechanism. The carbocation was stabilized by the electron
lone pairs of the oxygen atom present in the water molecule. The preparation of hydroxylactones
from iodolactones has been described thus far only for biotransformation experiments. The synthesis
described here allows obtaining these compounds without microorganisms.
Figure 3. The translactonization mechanism of γ-iodo-δ-lactones.
Iodolactonization of
previous studies (Table 1). In both reactions, five products with the lactone ring were identified.
The main products were isomers trans,trans- -iodo- -lactones 9b (yield 42%) and 9c (yield 37%).
Isomers cis,trans- -iodo- -lactones 10b,c were formed with yields of 30% and 33% and isomers
cis- -iodo- -lactones 11b,c only with yields of 10% and 8%. This result was new and unexpected,
because iodolactonization of -unsaturated carboxylic acid usually gives a mixture of -lactones as
the main product and only a trace of -lactones [27]. This reaction also produced -hydroxy- -lactones.
γ,δ-unsaturated carboxylic acids 4b and 4c also led to results different from
γ
δ
γ
δ
δ
γ
γ
,
δ
γ
δ
δ
γ
Isomers trans 12b,c were formed with yields of 5% and 7% and isomers cis 13b,c with yields of 14%
and 18%.
2.2. The Structural Analysis of Compounds
Spectroscopic and spectrometric analyses were performed for all obtained compounds. The X-ray
structural analysis was carried out for the solid products of lactonization. The 1H NMR
spectra of analogous isomers of
γ-halo-δ-lactones are similar to each other in most respects.
In cis,trans- -bromo- -lactone 8b, the H-6 proton was observed at 5.89 ppm as a doublet with coupling
γ
δ
constants 2.5 Hz (Figure 4). The signal of proton H-5 appears at 4.46 ppm as a triplet due to coupling
with protons H-4 and H-6 and the coupling constant 2.5 Hz. According to Karplus, the coupling
constant of vicinal protons depends on the dihedral angle between them. The equatorial-equatorial
and axial-equatorial coupling constants are in the range 1–5 Hz. The axial-axial coupling constant is

Molecules 2019, 24, 1875
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normally large but these magnitudes are decreased (9–10 Hz) by the antiperiplanar arrangement of the
hydrogens and oxygen atom of the carbonyl group [30,31].
Figure 4.
The comparison of the NMR signals for H-6 and H-5 protons of:
(a)
trans,trans-γ-bromo-δ-lactone 7b and (b) cis,trans-γ-bromo-δ-lactone 8b.
The magnitudes of these coupling constants indicate that the protons H-5 and H-6 are in aquatorial
positions and proton H-5 is in axial position. This causes that in cis,trans- -chloro- -lactones 8b Br
γ
δ
and Ph substituents are on opposite sides of the lactone ring, in the axial position at C-5 and C-6
and CH3 substituent is in equatorial position at C-4 carbon atom. In the case of trans,trans isomer
7b, coupling constant between protons H-4, H-5 and H-6 is much larger. The signal of protons H-6
observed at 5.32 ppm as a doublet with coupling constant JH5/H6 = 10.3 Hz. The triplet observed at
3.86 ppm for H-5 proton have a coupling constant 10.2 Hz. This means that all the substituents are
in the equatorial position, alternately above and below the lactone ring plane. The pyranose chair
conformations with substituents in the equitorial position are more stable than in the axial position
due to torsional strain and 1,3-diaxial interactions. This is reflected in the composition of the reaction
mixture where trans isomers are dominant. The structure of the trans,trans-
γ-halo-δ-lactones also
have been confirmed by single crystal X-ray diffraction analysis. Figure 5a shows the crystalline
structure of trans,trans-γ-chloro-δ-lactone 5a [
32]. The dihedral angles C10-C6-C5-Cl and C7-C4-C5-Cl
being 67.01◦ and 57.24◦ respectively. Thus, the aryl substituent at C-6, chlorine atom at C-5 and
methyl group at C4 are in the equatorial positions in gauche,gauche arrangement. Hydrogen atoms H-4,
H-5 and H-6 occupy the axial position with the dihedral angles between H-4 and H-5 and between
H-5 and H-6 equal to 175.72◦ and 172.61◦, respectively. X-ray analysis of trans,trans-
trans,trans- -bromo-
analogous. Figure 5b shows the crystalline structure of trans,trans-γ-bromo-δ-lactone 7a [33].
γ
-chloro- and
γ
δ-lactones showed that the structure of the lactone rings of the same isomers is

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Figure 5. The crystalline structure of: (a) trans,trans-γ-chloro-δ-lactone 5a; and (b) trans,trans-γ-bromo-δ-
lactone 7a.
The crystalline structure of γ-hydroxylactone 13a [34], obtained as a result of iodolactonization of
acid 4a in 90% y◦ield, is shown in Figure 6a. The dihedral angles C7-C4-C5-C6 and H4-C4-C5-H5 are
◦
33.36 and 29.31 , respectively. Thus, the substituents at C-4 and C-5 are in syn-periplanar orientation,
on the same side of the lactone ring, forming the cis isomer.
The structure of
1H NMR spectra of cis-
a double doublet, due to coupling with protons H-4 and H-6 (JH4/H5 = 7.2 Hz, JH5/H6 = 3.4 Hz).
The analogous coupling constants for trans- -hydroxylactone 12a are smaller and equal to 5.5 Hz
γ
-hydroxylactones was also confirmed by NMR spectroscopic analysis. In the
γ
-hydroxylactone 13a, the signal of proton H-5 appears at = 4.86 ppm as
δ
γ
and 2.8 Hz, respectively (Figure 7). This means that the substituents are in the equatorial position in
trans-arrangement.
Figure 6. The crystalline structure of: (a) cis-δ-hydroxy-γ-lactone 13a; (b) trans-δ-hydroxy-γ-lactone
12b; and (c) trans-δ-hydroxy-γ-lactone 12c.
The structure of the lactone rings was also confirmed by 13C NMR and IR spectroscopy. The carbon
signals at : 68.97 (13a) and 72.13 ppm (12a) are characteristic of carbon atoms bonded to the hydroxyl
group. The typical chemical shift values for the ester carbon atoms in -lactones also have signals at
177.11 ppm (13a) and 175.98 ppm (12a). In the IR spectrum, the stretching bands of the C = O group
appeared at 1747 cm−1 13a) and 1774 cm−1
12a). Spectroscopic analysis showed a similar structure
of lactones 13b,c and 12b,c. The crystalline structures of -hydroxylactones 12b 35] and 12c 36] are
shown in Figure 6b,c. The dihedral angles C7-C4-C5-C6 and H4-C4-C5-H5, being 96.54◦ and 135.80◦
12b), 97.08◦ and 147.93◦ (12c), respectively, are characteristic of antiperiplanar conformers and trans
configuration of the lactone rings.
δ
γ
(
(
γ
[
[
(

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Figure 7. The comparison of the NMR signals for H-5 and H-6 protons of: (a) trans-δ-hydroxy-γ-lactone
12b; and (b) cis-δ-hydroxy-γ-lactone 13a.
The iodolactonization of γ,δ-unsaturated carboxylic acids 4b,c also gave δ-iodo-γ-lactones 11b
and 11c in low yields (7% and 8%, respectively). Their structure was determined by spectr−o1scopic
methods. In the IR spectra, the absorption bands of the C = O group were observed at 1765 cm
(
11b
)
−1
and 1760 cm
(11c). This is the typical range for
γ-lactones. The analysis of NMR spectra showed the
signal of the carbonyl carbon at
δ
176.00 ppm (11b) and 176.11 ppm (11c). The signals observed at δ:
28.38 ppm (11b) and 28.45 ppm (11c) were assigned to carbons connected to the iodine. The signal of
proton H-5 appears as a double doublet, due to coupling with protons H-4 and H-6 (JH4/H5 = 11.2 Hz,
JH5/H6 = 4.5 Hz 11b,c). This indicates the syn-periplanar orientation of the protons H-4 and H-5,
and cis configuration of γ-lactones.
2.3. Bactericidal Properties
The results of CFU counting method are shown in Table 3. The lactones 6c, 9c and 10c showed
the highest bactericidal activity against E. coli ATCC 8739 (70–78% less CFU/mL). The other lactones
exhibited moderate bactericidal properties. The highest bactericidal activity against S. aureus ATCC
65389 was found for lactone 5a which reduced the number of CFU/mL by 87%. The bactericidal
properties against tested bacterial strain also showed lactones 6b, 8c and 7a (78–83% less CFU/mL).
Lactones 9a and 12a did not show bactericidal activity against S. aureus ATCC 65389.

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Table 3. The bactericidal activity of the investigated lactones against Escherichia coli ATCC 8739 and
Staphylococcus aureus ATCC 65389.
E. coli ATCC 8739 S. aureus ATCC 65389
Number of CFU/mL (× 107 )
Compound
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
9b
9c
10b
10c
6.41
4.06
9.46
7.58
6.32
2.06
5.21
8.63
6.52
7.14
5.83
8.87
9.02
2.43
7.65
2.87
9.43
6.02
9.47
9.30
2.74
7.63
14.83
5.20
3.58
7.39
4.63
10.96
6.42
9.29
9.04
3.60
21.25
9.76
5.92
6.30
21.15
9.12
21.36
20.85
12a
13a
Control
Control with DMSO
The 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium Bromide ( Mtt) Reduction Effectiveness
All lactones inhibited metabolic activity of the cell lines in the concentration range of 0.1–50 g/mL.
µ
The ability of cells to reduce MTT to formazan decreased by 37–93% (Tables 4 and 5). The cytotoxicity
of tested lactones against normal eukaryotic cells may exclude their use as antimicrobial agents in
medicine but does not exclude their use in anti-cancer drugs. Our results showed that lactones 10b
and 6b exhibit the lowest IC50 values against L929 cells. All halo- -lactones and -hydroxy- -lactones
were highly cytotoxic against gastric cancer AGS cells with IC50 values in the range of 0.0006–0.0044 M.
These values are much lower than IC50 = 0.025 M of doxorubicin, the reference anticancer drug [37].
The differences between IC50 values for AGS and L929 cell line were evaluated by non-parametric
, 10c
δ
δ
γ
µ
µ
Mann–Whitney U test. Statistical significance (p
(p = 0.1).
≤
0.05) was observed for all tested lactones except 6b
Such cytostatic activity of lactones was reported by various research groups. Bai et al. showed the
cytotoxic effects of sesquiterpene lactones from Inula britannica against four human cancer cell lines:
COLO 205, HT 29, HL-60 and AGS [38]. The high cytotoxicity of halo lactones with
or -phenyl- -lactone framework against cancer lines Jurkat (human leukaemia) and D17 (canine
osteosarcoma) was reported by Mazur et al. [28,39,40].
β-phenyl-γ-lactone
β
δ

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Table 4. The cytotoxic activity of tested lactones against AGS cell lines.
Concentration [µg/mL]
Lactone
50
5% DMSO IC50 [µM]
20
10
5
0.5
0.1
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
9b
9c
10b
10c
12a
13a
15 ± 0.01 18 ± 0.08 27 ± 0.10 43 ± 0.09 48 ± 0.05 54 ± 0.07
8 ± 0.09 11 ± 0.07 14 ± 0.12 16 ± 0.06 20 ± 0.09 35 ± 0.06
0.0019
0.0023
0.0006
0.0019
0.0044
0.0029
0.0021
0.0036
10 ± 0.12 21 ± 0.08 34 ± 0.09 44 ± 0.10 47 ± 0.12 54 ± 0.12
8 ± 0.05
8 ± 0.06
6 ± 0.08
8 ± 0.03
8 ± 0.09
19 ± 0.01 23 ± 0.09 28 ± 0.07 35 ± 0.06
14 ± 0.07 21 ± 0.02 28 ± 0.03 35 ± 0.02 42 ± 0.02
14 ± 0.07 16 ± 0.10 28 ± 0.06 37 ± 0.05 49 ± 0.10
20 ± 0.06 21 ± 0.03 25 ± 0.10 26 ± 0.05 39 ± 0.02
10 ± 0.06 14 ± 0.03 20 ± 0.06 23 ± 0.02 25 ± 0.10 40 ± 0.10
8 ± 0.07
9 ± 0.09
18 ± 0.10 25 ± 0.12 42 ± 0.02 49 ± 0.06 63 ± 0.03
17 ± 0.07 25 ± 0.05 39 ± 0.09 47 ± 0.06 61 ± 0.10
0.0035
0.0016
85 ± 0.1
11 ± 0.05 17 ± 0.10 28 ± 0.09 31 ± 0.02 35 ± 0.10 44 ± 0.15
10 ± 0.09 11 ± 0.03 18 ± 0.07 43 ± 0.10 58 ± 0.10 69 ± 0.15
0.0019
0.0025
0.0014
0.0014
0.0018
0.0017
0.0043
0.0037
8 ± 0.09
15 ± 0.06 26 ± 0.03 43 ± 0.12 52 ± 0.15 54 ± 0.15
15 ± 0.03 18 ± 0.06 23 ± 0.02 35 ± 0.10 43 ± 0.15 51 ± 0.10
10 ± 0.06 14 ± 0.08 16 ± 0.09 37 ± 0.03 43 ± 0.02 58 ± 0.07
8 ± 0.03
9 ± 0.10
8 ± 0.08
10 ± 0.07 13 ± 0.03 20 ± 0.02 23 ± 0.15 38 ± 0.06
13 ± 0.08 18 ± 0.05 22 ± 0.09 26 ± 0.05 39 ± 0.09
10 ± 0.10 18 ± 0.12 29 ± 0.05 39 ± 0.09 44 ± 0.12
The cytotoxicity was assessed by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) reduction
assay. The cell viability was calculated for four experiments including three repeats for each compound. Complete
RPMI-1640 medium was used as a positive control (Control +) of cell viability (100% viable cells) and 0.03% H2O2
as a negative control (Control
−
) of cell viability (100% dead inactive cells). All values were expressed as the mean
percentage of viable cells SD. The differences between positive control and treated with tested compounds were
±
evaluated by non-parametric Mann–Whitney U test. Statistical significance: p < 0.05. Dimethyl sulfoxide (DMSO)
was used as a solvent; IC50, half maximal inhibitory concentration.
Table 5. The cytotoxic activity of tested lactones against L929 cell lines.
Concentration [µg/mL]
Lactone
5% DMSO IC50 [µM]
50
20
10
5
0.5
0.1
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
9b
9c
10b
10c
12a
13a
1 ± 0.01 5 ± 0.06
1 ± 0.03 4 ± 0.02
8 ± 0.07
7 ± 0.06
10 ± 0.03 15 ± 0.02 17 ± 0.09
12 ± 0.05 18 ± 0.15 20 ± 0.08
0.0058
0.0071
0.0078
0.0083
0.0041
0.0047
0.0043
0.0048
2 ± 0.12 4 ± 0.06 10 ± 0.03 14 ± 0.09 21 ± 0.15 26 ± 0.03
2 ± 0.09 6 ± 0.03
7 ± 0.07
9 ± 0.15
11 ± 0.08 15 ± 0.03
2 ± 0.05 4 ± 0.15 16 ± 0.03 25 ± 0.02 27 ± 0.12 32 ± 0.07
2 ± 0.02 4 ± 0.08
1 ± 0.12 2 ± 0.03
9 ± 0.05
6 ± 0.07
18 ± 0.12 22 ± 0.05 24 ± 0.03
12 ± 0.02 18 ± 0.12 25 ± 0.02
2 ± 0.09 5 ± 0.15 10 ± 0.05 13 ± 0.12 18 ± 0.08 25 ± 0.03
1 ± 0.03 5 ± 0.05 11 ± 0.08 17 ± 0.02 21 ± 0.08 27 ± 0.08
0.0066
0.0044
79 ± 0.07
1 ± 0.15 4 ± 0.02
1 ± 0.02 4 ± 0.08
7 ± 0.06
8 ± 0.05
14 ± 0.03 19 ± 0.03 23 ± 0.03
15 ± 0.03 17 ± 0.08 24 ± 0.03
0.0055
0.0044
0.0044
0.0068
0.0040
0.0041
0.0062
0.0081
1 ± 0.05 5 ± 0.02 11 ± 0.03 15 ± 0.03 20 ± 0.08 25 ± 0.03
1 ± 0.12 4 ± 0.15 11 ± 0.06 13 ± 0.07 18 ± 0.03 23 ± 0.03
1 ± 0.05 3 ± 0.09
1 ± 0.02 5 ± 0.06
2 ± 0.05 2 ± 0.15
2 ± 0.06 3 ± 0.03
1 ± 0.07 5 ± 0.12
6 ± 0.03
7 ± 0.15
6 ± 0.09
8 ± 0.06
6 ± 0.06
9 ± 0.06
12 ± 0.08 15 ± 0.03
15 ± 0.08 20 ± 0.03 25 ± 0.08
12 ± 0.07 18 ± 0.03 22 ± 0.05
10 ± 0.02 15 ± 0.05 19 ± 0.15
9 ± 0.03
12 ± 0.02 16 ± 0.09
The cytotoxicity was assessed by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) reduction
assay. The cell viability was calculated for four experiments including three repeats for each compound. Complete
RPMI-1640 medium was used as a positive control (Control +) of cell viability (100% viable cells) and 0.03% H2O2
as a negative control (Control
−
) of cell viability (100% dead inactive cells). All values were expressed as the mean
percentage of viable cells SD. The differences between positive control and treated with tested compounds were
±
evaluated by non-parametric Mann–Whitney U test. Statistical significance: p < 0.05. Dimethyl sulfoxide (DMSO)
was used as a solvent; IC50, half maximal inhibitory concentration.

Molecules 2019, 24, 1875
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3. Materials and Methods
All used chemicals were purchased from Sigma-Aldrich and Fluka and used without further
purification. The NMR spectra were obtained in CDCl3 on a Bruker Avance DRX 500 MHz. FTIR
spectra were recorded using an attenuated total reflection technique on a Perkin Elmer Spectrum
400 spectrometer. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were acquired
on a Bruker micrOTOF-Q II. Gas chromatography was performed using a Thermo Scientific-Trace
1310 chromatograph equipped with a TG-5HT column (30 m
×
0.25 mm). Melting points were
determined with a Boetius micro melting point apparatus and are uncorrected. The refractive index
was determined with an Abbe refractometer (Atago RX-7000 CX). TLC analysis was performed on
silica gel F254 plates (Merck). Column chromatography was performed on silica gel 60 (230–400 mesh,
Merck) using mixtures of hexane, ethyl acetate and acetone as eluents. The crystal structure was
determined by single-crystal X-ray diffraction (Xcalibur, Sapphire 2 CCD detector).
3.1. X-ray Study
The single crystal X-ray diffraction for lactones was carried out on a Rigaku Oxford Diffraction
SuperNova diffractometer equipped with a micro focus Cu X-ray source. The crystals were maintained
at 100 K by using an Oxford Cryosystems nitrogen gas-flow device. Data collection strategies
were optimized using CrysAlisPro software package [41]. The crystal structures of lactones were
solved using the charge-flipping method implemented in SUPERFLIP and refined with the JANA
package [42]. The crystal data, data collection, and refinement parameters for lactones are given in
Table 6. Crystallographic data, as CIF files, have been deposited with the Cambridge Crystallographic
Data Centre. The solid-state structure of lactone was determined by single-crystal X-ray diffraction.
The lactones 5a (Figure 8), 7a (Figure 9), and 13a (Figure 10) crystallized in the centrosymmetric space
group P21/c while lactones 12b (Figure 11) and 12c (Figure 12) in the orthorhombic space group Pbca.
Only one molecule is present in the asymmetric unit. Experimental details of the crystallographic
analysis are given in Table 5.
Figure 8. The arrangement of 5a in the unit cell box.

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Table 6. Experimental details of the crystallographic analysis for lactones 5a, 7a, 13a, 12b and 12c.
5a
7a
13a
12b
12c
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
C16H15O2Cl
274.73
293.15
C16H15O2Br
319.19
293.15
0.71073
C16H16O3
256.29
100
C12H14O3
206.24
100
1.54184
orthorhombic P
b c a
21/b21/c21/a
10.1332(2)
7.62820(10)
27.2405(2)
90
C12H13FO3
224.22
293.15
0.71073
1.54184
0.71073
Crystal system, space
group
monoclinic,
monoclinic,
monoclinic,
orthorhombic P
b c a
P21/c
P21/c
P21/c
(
P
)
a (Å)
b(Å)
c(Å)
13.670(3)
8.9220(2)
11.890(3)
111.73(3)
1347.1(5)
13.9026(9)
8.9784(4)
11.8318(9)
111.418(8)
1374.89(17)
11.4137(2)
11.46100(10)
10.7848(2)
117.359(2)
1252.982 (4)
10.042(3)
7.591(2)
28.174(3)
90
β ( ◦
)
Volume (Å3)
2105.64(5)
2147.8(9)
Z, Calculated density
4, 1.355
4, 1.542
4, 0.154
8, 0.1831
8, 1.387
(Mg/m3)
Absorption coefficient
0.278
576
2.984
648
0.126
544
0.150
880
0.110
944
(mm−1
)
F(000)
Theta range for data
collection (◦)
2.935 −28.804
2.927 −26.999
3.86 −76.29
3.24 −75.38
3.441 −28.867
−17 ≤ h ≤ 18,
−11 ≤ k ≤ 12,
−16 ≤ l ≤ 9
9623
−17≤ h≤ 17,
−11 ≤ k ≤ 11,
−12 ≤ l ≤ 15
9471
−14 ≤ h ≤ 14,
−14 ≤ k ≤ 14,
−12 ≤ l ≤ 13
17416
−142 ≤ h≤ 10,
−9 ≤ k ≤ 9,
−32 ≤ l ≤ 33
13106
−13 ≤ h≤ 13,
−8≤ k ≤ 10,
−36 ≤ l ≤ 37
14345
Index ranges
Reflections Collected
unique observed [I >
2sigma(I)]
3259
1811
2957
1923
2623
2483
2170
2017
2677
1951
Rint
0.0713
0.0959
0.0281
0.0401
0.0501
Completeness to Θ
(%)
25.242◦,
25.242◦,
76.29◦,
75.38◦,
25.242◦,
99.1
99.2
99.0
99.0
99.8
Full-matrix
least-squares
on F2
3259/0/172
0.883
R1 = 0.0569
wR2 = 0.1289
R1 = 0.1079
wR2 = 0.1433
Full-matrix
least-squares
on F2
2957/0/172
1.436
R1 = 0.0703
wR2 = 0.1686
R1 = 0.1149
wR2 = 0.2216
Full-matrix
least-squares
on F2
2623/0/259
4.030
R1 = 0.0408
wR2 = 0.1184
R1 = 0.0422
wR2 = 0.1192
Full-matrix
least-squares
on F2
2170/0/140
3.16
R1 = 0.0408
wR2 = 0.0534
R1 = 0.0429
wR2 = 0.0540
Full-matrix
least-squares
on F2
2677/0/149
1.052
R1 = 0.0485
wR2 = 0.1315
R1 = 0.0669
wR2 = 0.1384
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I >
2sigma(I)]
R indices (all data)
Figure 9. The arrangement of 7a in the unit cell box.

Molecules 2019, 24, 1875
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Figure 10. The arrangement of 13a in the unit cell box.
Figure 11. The arrangement of 12b in the unit cell box.
Figure 12. The arrangement of 12c in the unit cell box.
3.2. Claisen Rearrangement
The γ,δ-unsaturated ethyl esters 3a–c were obtained by Claisen rearrangement [25]. A mixture
of triethyl orthoacetate (0.15 mol), ethanol (0.02 mol) and a catalytic amount (one drop) of propionic
acid was heated at 138 ◦C for 5 h while distilling off alcohol. When the reaction was completed (TLC),
the excess of triethyl orthoacetate was removed by evaporation. The crude product was purified by
column chromatography on silica gel using a mixture of ethyl acetate and hexane (1:80). Spectral data
are given below.
Ethyl 3-methyl-5-(naphthalen-1-yl)pent-4-enoate (3a): The product was obtained as a yellow oil with a
yield of 62.75%; n2D0 = 1.5641; Rf = 0.38 (EtOAc:hexane 1:20); 1H NMR (CDCl3, 600 MHz),
δ [ppm]: 1.28
(d, 3H, J = 6.8 Hz, CH(CH3)), 1.29 (t, 3H, J = 7.1 Hz, OCH2CH3), 2.48 (dd, 1H, J1 = 14.7 Hz, J2 = 7.1 Hz,
CHaHb), 2.55 (dd, 1H, J1 = 14.7 Hz, J2 = 7.6 Hz, CHaHb), 2.99–3.10 (m, 1H, CH(CH3)), 4.12 (dd, 2H, J1
= 7.1 Hz, J2 = 1.7 Hz, CH2CH3), 6.19 (dd, 1H, J1 = 15.6 Hz, J2 = 7.6 Hz, CH = CH), 7.19 (d, 1H, J = 15.6
Hz, CH = CH), 7.45–7.60 (m, 4H, HAr), 7.79 (d, 1H, J = 8.2 Hz, HAr), 7.87 (d, 1H, J = 7.7 Hz, HAr), 8.13
(d, 1H, J = 8.2 Hz, HAr); 13C NMR (151 MHz, CDCl3),
34.57 (CH(CH3 2
δ
[ppm]: 14.34 (OCH2CH3), 20.39 (CH(CH3)),
)
), 41.93 (CH2), 60.38 (OCH2CH3), 125.90 (CH = CH), 133.62 (CH = CH), 172.50 (C =
O), CAr: 123.74, 123.92, 125.65, 125.71, 125.90, 126.23, 127.55, 128.50, 131.21, 135.33, 137.66; IR (cm−1):
1727, 1589, 1507, 1455, 1392, 1365, 1345, 1268, 1239, 1164, 1094, 1028, 968, 861, 791, 775, 732; HR-MS
(ESI-TOF) calculated for C18H20O2, m/z [M + Na]+: 291.136092; experimental value: 291.137301.
Eethyl 3-methyl-5-phenylpent-4-enoate (3b): The product was obtained as a yellow oil with a yield
1
of 59.26%; n2D0 = 1.5201; Rf = 0.37 (EtOAc:hexane 1:20); H NMR (CDCl3, 500 MHz),
δ [ppm]: 1.15
(d, 3H, J = 6.8 Hz, CH(CH3)), 1.23 (t, 3H, J = 7.1 Hz, OCH2CH3), 2.35 (dd, 1H, J1 = 14.8 Hz, J2 = 7.3
Hz, CHaHb), 2.42 (dd, 1H, J1 = 14.8 Hz, J2 = 7.3 Hz, CHaHb), 2.80–2.92 (m, 1H, CH(CH3)), 4.12 (q,

Molecules 2019, 24, 1875
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2H, J = 7.1 Hz, OCH2CH3), 6.14 (dd, 1H,
J
1 = 15.9 Hz,
J
2 = 7.6 Hz, CH = CH), 6.40 (d, 1H, J = 15.9 Hz,
CH = CH), 7.17–7.39 (m, 5H, HAr); 13C NMR (125 MHz, CDCl3),
δ [ppm]: 13.82 (OCH2CH3), 19.74
(
CH(CH3)), 33.62 (C(CH3)2), 41.30 (CH2), 59.79 (OCH2CH3), 125.63 (CH = CH), 133.79 (CH = CH),
171.91 (C = O), CAr: 126.08, 126.63, 128.01, 136.95; IR (cm−1): 1731, 1597, 1494, 1450, 1368, 1348, 1298,
1285, 1239, 1162, 1100, 1079, 1071, 1030, 964, 929, 909, 842, 747, 690; HR-MS (ESI-TOF) calculated for
C14H18O2, m/z [M + Na]+: 241.120443; experimental value: 241.121304.
Ethyl 5-(4-fluorophenyl)-3-methylpent-4-enoate (3c): The product was obtained as a yellow oil with a
yield of 48.50%; n2D0 = 1.5201, Rf = 0.38 (EtOAc:hexane 1:20); 1H NMR (CDCl3, 500 MHz),
δ [ppm]: 1.15
(d, 3H, J = 6.8 Hz, CH(CH3)), 1.23 (t, 3H, J = 7.1 Hz, OCH2CH3), 2.35 (dd, 1H, J1 = 14.8 Hz, J2 = 7.2 Hz,
CHaHb), 2.41 (dd, 1H, J1 = 14.8 Hz, J2 = 7.4 Hz, CHaHb), 2.78–32.92 (m, 1H, CH(CH3)), 4.13 (q, 2H, J =
7.1 Hz, OCH2CH3)), 6.05 (dd, 1H,
J
1 = 15.9 Hz,
J
2 = 7.6 Hz, CH = CH), 6.36 (d, 1H, J = 15.9 Hz, CH =
CH), 6.98 (t, 2H, J = 8.7 Hz, HAr), 7.24–37.32 (m, 2H, HAr); 13C NMR (125 MHz, CDCl3),
δ
[ppm]: 14.27
(OCH2CH3), 20.19 (CH(CH3)), 34.04 (CH(CH3)), 41.74 (CH2), 60.26 (OCH2CH3), 127.68 (CH = CH),
133.99 (CH = CH), 172.32 (C = O), CAr: 115.32, 127.53, 133.55, 162.03; IR (cm−1): 1732, 1603, 1506, 1459,
1415, 1370, 1348, 1298, 1283, 1225, 1207, 1174, 1156, 1095, 1079, 1028, 970, 935, 851, 814, 772; HR-MS
(ESI-TOF) calculated for C14H17FO2, m/z [M + Na]+: 259.111021; experimental value: 259.111099.
3.3. Basic Hydrolysis of Esters
The
γ,δ-unsaturated carboxylic acids were obtained by alkaline hydrolysis of esters 3a–c.
The reaction occurred in ethanolic KOH under reflux conditions (2 h). The excess of ethanol was
evaporated and the residue was acidified with 0.1 M hydrochloric acid. The product was extracted
with diethyl ether and dried over anhydrous magnesium sulfate. Spectral data are given below.
3-Methyl-5-(naphthalen-1-yl)pent-4-enoic acid (4a): The product was obtained as a colorless oil with a
yield of 98%; n2D0 = 1.5940; Rf = 0.16 (EtOAc:hexane 1:5); 1H NMR (CDCl3, 500 MHz),
δ
[ppm]: 1.23
(d, 3H, J = 6.8 Hz, CH(CH3)), 2.46 (dd, 1H,
J
1 = 15.2 Hz, J2 = 7.1 Hz, CHaHb), 2.54 (dd, 1H, 1 = 15.2
J
Hz, J2 = 7.4 Hz, CHaHb), 2.92–3.03 (m, 1H, CH(CH3)), 6.13 (dd, 1H, J1 = 15.6 Hz, J2 = 7.5 Hz, CH =
CH), 7.15 (d, 1H, J = 15.6 Hz, CH = CH), 7.53–7.37 (m, 5H, HAr), 7.72 (d, 1H, J = 8.2 Hz, HAr), 7.81 (d,
1H, J = 8.7 Hz, HAr), 8.06 (d, 1H, J = 8.5 Hz, HAr), 9.28 (s, 1H, COOH); 13C NMR (125 MHz, CDCl3),
δ
[ppm]: 20.25 (CH(CH3)), 34.14 (CH(CH3)), 41.50 (CH2), 125.86 (CH = CH), 137.16 (CH = CH), 178.47
(C = O), CAr: 123.70, 123.84, 125.57, 125.65, 126.46, 127.54, 128.41, 131.13, 133.52, 135.15; IR (cm−1):
2600, 1704, 1591, 1507, 1451, 1406, 1394, 1374, 1288, 1230, 1170, 1140, 1089, 1069, 1013, 965, 911, 857, 791,
773, 730; HR-MS (ESI-TOF) calculated for C16H16O2, m/z [M + Na]+: 263.104794; experimental value:
263.105649.
3-methyl-5-phenylpent-4-enoic acid (4b): The product was obtained as a colorless oil with a yield of
96.5%; n2D0 = 1.5434; Rf = 0.15 (EtOAc:hexane 1:5); 1H NMR (CDCl3, 500 MHz),
δ
[ppm]: 1.17 (d, 3H, J
= 6.8 Hz, CH(CH3)), 2.39 (dd, 1H, J1 = 15.3 Hz, J2 = 7.3 Hz, CHaHb), 2.48 (dd, 1H, J1 = 15.3 Hz, J2 = 7.2
Hz, CHaHb), 2.80–2.91 (m, 1H, CH(CH3)), 6.14 (dd, 1H, 1 = 15.9 Hz, 2 = 7.5 Hz, CH = CH), 6.41 (d,
1H, J = 15.9 Hz, CH = CH), 7.16–7.37 (m, 5H, HAr), 10.14 (s, 1H, COOH); 13C NMR (125 MHz, CDCl3),
J
J
δ
[ppm]: 20.17 (CH(CH3)), 33.66 (CH(CH3)), 41.39 (CH2), 127.09 (CH = CH), 133.84 (CH = CH), 178.68
(C = O), CAr: 126.14, 128.50, 129.05, 137.30; IR (cm−1): 2630, 1702, 1598, 1494, 1446, 1408, 1374, 1347,
1288, 1250, 1218, 1173, 1144, 1096, 1074, 1024, 965, 931, 909, 859, 841, 746, 689, 664; HR-MS (ESI-TOF)
calculated for C12H14O2, m/z [M + Na]+: 213.089144; experimental value: 213.088930.
5-(4-fluorophenyl)-3-methylpent-4-enoic acid (4c): The product was obtained as a colorless oil with a
yield of 98.0%; n2D0 = 1.5205, Rf = 0.16 (EtOAc:hexane, 1:5); 1H NMR (CDCl3, 500 MHz),
δ
[ppm]: 1.17
(d, 3H, J = 6.8 Hz, CH(CH3)), 2.40 (dd, 1H,
J
1 = 15.3 Hz, J2 = 7.2 Hz, CHaHb), 2.47 (dd, 1H, 1 = 15.3
J
Hz, J2 = 7.2 Hz, CHaHb), 2.79–2.90 (m, 1H, CH(CH3)), 6.05 (dd, 1H, J1 = 15.9 Hz, J2 = 7.5 Hz, CH =
CH), 6.38 (d, 1H, J = 15.9 Hz, CH = CH), 6.93–7.02 (m, 2H, HAr), 7.27–7.32 (m, 2H, HAr), 11.06 (s, 1H,
COOH); 13C NMR (125 MHz, CDCl3),
δ [ppm]: 20.19 (CH(CH3)), 33.65 (CH(CH3)), 41.36 (CH2), 127.97

Molecules 2019, 24, 1875
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(CH = CH), 133.56 (CH = CH), 178.60 (C = O), CAr: 115.37 (d, 2 JF−C = 21.6 Hz), 127.61 (d, 3 JF−C = 7.8
Hz), 133.44 (d, 4 JF−C = 3.3 Hz), 162.11 (d, JF−C = 246.20 Hz); IR (cm−1): 2622, 1698, 1593, 1508, 1461,
1431, 1411, 1378, 1354, 1314, 1261, 1221, 1190, 1160, 1128, 1100, 1079, 1012, 974, 960, 952, 935, 855, 812,
+
770, 703; HR-MS (ESI-TOF) calculated for C12H13FO2, m/z [M + Na] : 231.079723; experimental value:
231.080843.
3.4. The Chlorolactonization of Unsaturated Carboxylic Acids 4a–c
The mixture of an unsaturated carboxylic acid (0.005 mol) and N-chlorosuccinimide (0.009 mol)
was dissolved in 60 mL of THF. Acetic acid was added dropwise and the reaction mixture was stirred
at room temperature for 48 h. When the reaction was completed, the mixture was dissolved in diethyl
ether, washed with saturated NaHCO3 and dried over anhydrous magnesium sulfate. The crude
product was purified by column chromatography on silica gel using a mixture of acetone and hexane
(1:10) [28]. Spectral data are given below. The chlorolactonization of 4a gave chlorolactones 5a and 6a
with a total yield of 51.19% (5a
:6a 81.55:18.45). The chlorolactonization of 4b gave chlorolactones 5b and
6b with a total yield of 82.21% (5b
:6b 84.42:15.58). The chlorolactonization of 4c gave chlorolactones 5c
and 6c with a total yield of 61.67% (5c:6c 81.02:18.98).
Trans,trans-5-chloro-tetrahydro-4-methyl-6-(n◦aphthalen-1-yl)pyran-2-one (5a): The product was
1
obtained as a colorless solid; mp = 157–158 C; Rf = 0.19 (acetone:hexane 1:7); H NMR (CDCl3,
500 MHz), δ [ppm]: 1.29 (d, 3H, J = 6.4 Hz, CH(CH3)), 2.47–2.61 (m, 2H, CH(CH3), CHaHb), 3.07 (dd,
1H, J1 = 18.5 Hz, J2 = 10.0 Hz, CHaHb), 4.18 (t, 1H, J = 9.7 Hz , CHCl), 5.99 (d, 1H, J = 10.0 Hz, CHAr),
7.47–7.61 (m, 4H, HAr), 7.90 (d, 2H, J = 8.3 Hz, HAr), 8.09 (d, 1H, J = 8.5 Hz, HAr); 13C NMR (125 MHz,
CDCl3), δ [ppm]: 19.70 (CH(CH3)), 35.98 (CH(CH3)), 37.15 (CH2), 62.72 (CHCl), 81.97 (CHAr), 169.03
(C = O), CAr: 123.01, 125.00, 125.85, 125.97, 126.62, 129.09, 130.10, 131.18, 131.85, 133.87; IR (cm−1):
1727, 1596, 1512, 1453, 1408, 1375, 1350, 1324, 1286, 1263, 1239, 1216, 1175, 1105, 1085, 1062, 1022, 997,
950, 900, 860, 834, 805, 775, 732, 674, 640; HR-MS (ESI-TOF) calculated for C16H15ClO2, m/z [M + K]+:
313.039760; experimental value: 313.041132.
Cis,trans-5-chloro-tetrahydro-4-methyl-6-(naphthalen-1-yl)pyran-2-one (6a): The product was obtained
as a colorless solid; mp = 98–99.5 ◦C; Rf = 0.28 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
[ppm]: 0.98 (d, 3H, J = 6.5 Hz, CH(CH3)), 2.03–2.11 (m, 1H, CH(CH3)), 2.67 (dd, 1H, J1 = 18.7 Hz, J2
δ
=
11.4 Hz, CHaHb), 2.75 (dd, 1H, J1 = 18.7 Hz, J2 = 6.3 Hz, CHaHb), 4.51 (t, 1H, J = 1.9 Hz, CHCl), 6.55 (d,
1H, J = 1.9 Hz, CHAr), 7.44–7.59 (m, 4H, HAr), 7.89 (d, 2H, J = 8.2 Hz, HAr), 8.07 (d, 1H, J = 8.4 Hz,
HAr); 13C NMR (125 MHz, CDCl3),
δ [ppm]: 17.81 (CH(CH3)), 27.12 (CH(CH3)), 33.06 (CH2), 62.19
(CHCl), 84.33 (CHAr), 169.24 (C = O), CAr: 121.76, 123.56, 125.22, 126.30, 127.35, 129.46, 129.51, 130.12,
132.99, 133.83; IR (cm−1): 1733, 1598, 1507, 1455, 1393, 1342, 1281, 1218, 1169, 1112, 1074, 1038, 997, 945,
909, 886, 863, 802, 777, 737, 703, 615; HR-MS (ESI-TOF) calculated for C16H15ClO2, m/z [M + Na]+:
297.065822; experimental value: 297.067366.
Trans,trans-5-chloro-tetrahydro-4-methyl-6-phenylpyran-2-one (5b): The product was obtained as a
◦
1
colorless solid; mp = 105–106.5 C, Rf = 0.18 (acetone:hexane 1:7); H NMR (CDCl3, 500 MHz),
[ppm]: 1.25 (d, 3H, J = 6.4 Hz, CH(CH3)), 2.35–2.47 (m, 2H, J = 17.2 Hz, CHaHb), 2.92–3.01 (m, 1H,
CHaHb), 3.76 (t, 1H, J = 9.8Hz, CHCl), 5.18 (d, 1H, J = 10.0 Hz, CHAr), 7.33–7.43 (m, 5H, HAr); 13
NMR (125 MHz, CDCl3), [ppm]: 19.62 (CH(CH3)), 35.22 (CH(CH3 2), 37.10 (CH2), 63.27 (CHCl),
δ
C
δ
)
85.05 (CHAr), 168.95 (C = O), CAr: 127.51, 128.52, 129.29, 136.44; IR (cm−1): 1730, 1496, 1460, 1401,
1376, 1345, 1288, 1240, 1218, 1184, 1107, 1074, 1055, 1031, 1020, 995, 930, 906, 893, 850, 800, 757, 670, 651,
618; HR-MS (ESI-TOF) calculated for C12H13ClO2, m/z [M + Na]+: 247.050173; experimental value:
247.051037.
Cis,trans-5-chloro-tetrahydro-4-methyl-6-phenylpyran-2-one (6b): The product was obtained as a
colorless oil; n2D0 = 1.5453; Rf = 0.27 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
δ
[ppm]: 1.05
(d, 3H, J = 6.6 Hz, CH(CH3)), 2.24–2.36 (m, 1H, CH(CH3)), 2.59 (dd, 1H, J1 = 18.4 Hz, J2 = 10.3 Hz,

Molecules 2019, 24, 1875
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CHaHb), 2.64 (dd, 1H, J1 = 18.4 Hz, J2 = 6.9 Hz, CHaHb), 4.33 (t, 1H, J = 2.5 Hz, CHCl), 5.79 (d, 1H,
J = 2.5 Hz, CHAr), 7.25–7.28 (m, 2H, HAr), 7.32–7.47 (m, 3H, HAr); 13C NMR (125 MHz, CDCl3),
δ
[ppm]: 17.65 (CH(CH3)), 27.07 (CH(CH3)2), 33.54 (CH2), 63.32 (CHCl), 85.20 (CHAr), 168.97 (C = O ),
CAr: 125.30, 128.67, 129.00, 137.80; IR (cm−1): 1736, 1949, 1453, 1415, 1370, 1333, 1286, 1243, 1220, 1167,
1112, 1071, 1031, 1004, 949, 909, 881, 860, 830, 802, 757, 710, 699, 606; HR-MS (ESI-TOF) calculated for
C12H13ClO2, m/z [M + Na]+: 247.050173; experimental value: 247.050609.
Trans,trans-5-chloro-6-(4-fluorophenyl)-tetrahydro-4-methylpyran-2-one (5c): The product was
obtained as a colorless solid; mp = 80–81 ◦C; Rf = 0.16 (acetone:hexane 1:7); 1H NMR (CDCl3, 500
MHz), δ [ppm]: 1.25 (d, 3H, J = 6.3 Hz, CH(CH3)), 2.34–2.49 (m, 2H, CHaHb, CH(CH3)), 2.94–3.05 (m,
1H, CHaHb), 3.71 (t, 1H, J = 9.8 Hz, CHCl), 5.17 (d, 1H, J = 10.0 Hz, CHAr), 7.03–7.13 (m, 2H, HAr),
7.32–7.42 (m, 2H, HAr); 13C NMR (125 MHz, CDCl3),
δ [ppm]: 19.62 (CH(CH3)), 35.51 (CH(CH3)2),
37.04 (CH2), 63.25 (CHCl), 84.32 (CHAr), 168.71 (C = O), CAr: 115.53 (d, 2 JF−C = 21.9 Hz), 129.35 (d,
3 JF−C = 8.4 Hz), 132.33 (d, 4 JF−C = 3.2 Hz), 163.10 (d, JF−C = 248.4 Hz); IR (cm−1): 1731, 1609, 1514,
1453, 1435, 1427, 1381, 1352, 1334, 1260, 1221, 1192, 1160, 1105, 1083, 1065, 1035, 1010, 960, 919, 883,
+
844, 825, 810, 786, 719, 687; HR-MS (ESI-TOF) calculated for C12H12ClFO2, m/z [M + Na] : 265.040751;
experimental value: 265.041057.
Cis,trans-5-chloro-6-(4-fluorophenyl)-tetrahydro-4-methylpyran-2-one (6c): The product was obtained
as a colorless oil; n2D0 = 1.5453, Rf = 0.27 (acetone:heksane 1:7); 1H NMR (CDCl3, 500 MHz),
1.10 (d, 3H, J = 6.6 Hz, CH(CH3)), 2.27–2.37 (m, 1H, CH(CH3)), 2.62 (dd, 1H, J1 = 18.4 Hz, J
δ
[ppm]:
2 = 9.7 Hz,
CHaHb), 2.66 (dd, 1H, J1 = 18.4 Hz, J2 = 7.0 Hz, CHaHb), 4.30 (t, 1H, J = 2.8 Hz, CHCl), 5.75 (d, 1H,
J = 2.7 Hz, CHAr), 7.09–7.15 (m, 2H, HAr), 7.23–7.33 (m, 2H, HAr); 13C NMR (125 MHz, CDCl3),
[ppm]: 17.42 (CH(CH3)), 27.41 (CH(CH3 2), 33.71 (CH2), 63.02 (CHCl), 84.46 (CHAr), 168.69 (C = O),
δ
)
CAr: 116.02 (d, 2 JF−C = 21.9 Hz), 127.29 (d, 3 JF−C = 8.3 Hz), 133.56 (d, 4 JF−C = 3.1 Hz), 163.65 (d, JF−C
= 248.4 Hz); IR (cm−1): 1737, 1605, 1510, 1453, 1414, 1372, 1358, 1340, 1286, 1272, 1218, 1176, 1155, 1110,
1096, 1078, 1060, 1040, 1000, 950, 911, 888, 860, 834, 796, 745, 723, 692, 631; HR-MS (ESI-TOF) calculated
for C12H12ClFO2, m/z [M + Na]+: 265.040751; experimental value: 265.041871.
The bromolactonization of unsaturated carboxylic acids 4a–c: The mixture of an unsaturated
carboxylic acid (0.005 mol) and N-bromosuccinimide (0.01 mol) was dissolved in 60 mL of THF.
Acetic acid was added dropwise and reaction mixture was stirred at room temperature for 48 h.
When the reaction was completed, the mixture was dissolved in diethyl ether, washed with saturated
NaHCO3 and dried over anhydrous magnesium sulfate. The crude product was purified by column
chromatography on silica gel using a mixture of acetone and hexane (1:10) [28]. Spectral data are given
below. The bromolactonization of 4a gave bromolactones 7a and 8a with a total yield of 72.23% (7a
82.30:17.70). The bromolactonization of 4b gave bromolactones 7b and 8b with a total yield of 71.94%
7b 8b 78.15:21.85). The bromolactonization of 4c gave bromolactones 7c and 8c with a total yield of
:8a
(
:
77.72% (7c:8c 76.27:23.73).
Trans,trans-5-bromo-tetrahydro-4-methyl-6-(naphthalen-1-yl)pyran-2-one (7a): The product was
◦
1
obtained as a colorless solid; mp = 197–198 C; Rf = 0.13 (acetone:hexane 1:7); H NMR (CDCl3,
500 MHz), [ppm]: 1.33 (d, 3H, J = 6.6 Hz, CH(CH3)), 2.55 (dd, 1H, J1 = 17.4 Hz, J2 = 10.1 Hz, CHaHb),
2.59–2.72 (m, 1H, CH(CH3)), 3.08 (dd, 1H, J1 = 17.4 Hz, J2 = 5.7 Hz, CHaHb), 4.28 (dd, 1H, J1 = 10.3 Hz,
2 = 9.6 Hz, CHBr), 6.12 (d, 1H, J = 10.3 Hz, CHAr), 7.49–7.53 (m, 2H, HAr), 7.53–7.59 (m, 4H, HAr),
7.88–7.93 (m, 2H, HAr), 8.10 (d, 1H, J = 8.6 Hz, HAr); 13C NMR (125 MHz, CDCl3),
[ppm]: 21.13
δ
J
δ
(CH(CH3)), 36.34 (CH(CH3)), 37.49 (CH2), 54.75 (CHBr), 82.12 (CHAr), 169.22 (C = O), CAr: 123.06,
125.01, 125.89, 126.08, 126.65, 129.14, 130.19, 131.14, 132.24, 133.90; IR (cm−1): 1724, 1596, 1514, 1453,
1413, 1400, 1376, 1345, 1324, 1285, 1236, 1187, 1170, 1105, 1062, 1013, 994, 947, 906, 893, 860, 834, 800, 773,
+
735, 660, 631; HR-MS (ESI-TOF) calculated for C16H15BrO2, m/z [M + Na] : 341.015305; experimental
value: 341.016902.

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Cis,trans-5-bromo-tetrahydro-4-methyl-6-(naphthalen-1-yl)pyran-2-one (8a): The product was obtained
as a yellow oil, n2D0 = 1.5140, Rf = 0.22 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
[ppm]: 0.96
δ
(d, 3H, J = 6.4 Hz, CH(CH3)), 2.04–2.12 (m, 1H, CH(CH3)), 2.66 (dd, 1H, J1 = 18.5 Hz, J2 = 11.4 Hz,
CHaHb), 2.72 (ddd, 1H, J1 = 18.5 Hz, J2 = 6.3 Hz, J3 = 1.0 Hz, CHaHb), 4.62 (m, 1H, CHBr), 6.65 (d, 1H,
J = 1.7 Hz, CHAr), 7.33–7.71 (m, 4H, HAr), 7.83 (d, 1H, J = 8.2 Hz, HAr), 7.88 (d, 1H, J = 8.2 Hz, HAr),
7.92–7.96 (m, 1H, HAr); 13C NMR (125 MHz, CDCl3),
δ [ppm]: 19.26 (CH(CH3)), 27.13 (CH(CH3)),
34.27 (CH2), 56.19 (CHBr), 84.74 (CHAr), 169.10 (C = O), CAr: 121.81, 123.52, 125.22, 126.32, 127.41,
129.15, 129.47, 129.52, 133.60, 133.85; IR (cm−1): 1740, 1596, 1507, 1453, 1390, 1340, 1270, 1236, 1200,
1153, 1107, 1076, 1037, 994, 947, 904, 886, 861, 800, 780, 746, 683, 606; HR-MS (ESI-TOF) calculated for
C16H15BrO2, m/z [M + K]+: 356.989243; experimental value: 356.990538.
Trans,trans-5-bromo-tetrahyd◦ro-4-methyl-6-phenylpyran-2-one (7b): The product was obtained as a
colorless solid; mp = 111–112 C; Rf = 0.16 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
δ [ppm]:
1.28 (d, 3H, J = 6.5 Hz, CH(CH3)), 2.44 (dd, 1H,
CH(CH3)), 2.99 (dd, 1H, J1 = 17.2 Hz, J2 = 5.6 Hz, CHaHb), 3.86 (dd, 1H, J1 = 10.2 Hz, J2 = 9.7 Hz,
CHBr), 5.32 (d, 1H, J = 10.3 Hz, CHAr), 7.34–7.44 (m, 5H, HAr); 13C NMR (125 MHz, CDCl3),
[ppm]:
21.04 (CH(CH3)), 35.84 (CH(CH3 2), 37.39 (CH2), 55.44 (CHBr), 85.36 (CHAr), 169.19 (C = O); CAr:
J1 = 17.2 Hz, J2 = 10.0 Hz, CHaHb), 2.47–2.59 (m, 1H,
δ
)
127.59, 128.55, 129.36, 136.70; IR (cm−1): 1720, 1497, 1458, 1372, 1345, 1284, 1248, 1184, 1170, 1103, 1061,
1006, 956, 906, 889, 845, 775, 753, 696, 631, 617; HR-MS (ESI-TOF) calculated for C12H13BrO2, m/z [M +
Na]+: 290.999656; experimental value: 291.000717.
Cis,trans-5-bromo-tetrahydro-4-methyl-6-phenylpyran-2-one (8b): The product was obtained as a
1
yellow oil; n2D0 = 1.5140, Rf = 0.23 (acetone:hexane 1:7); H NMR (CDCl3, 500 MHz),
δ [ppm]: 1.04
(d, 3H, J = 6.5 Hz, CH(CH3)), 2.05–2.14 (m, 1H, CH(CH3)), 2.59 (dd, 1H, J1 = 18.4 Hz, J2 = 10.8 Hz,
CHaHb), 2.66 (dd, 1H, J1 = 18.4 Hz, J2 = 6.0 Hz, CHaHb), 4.46 (t, 1H, J = 2.5 Hz, CHBr), 5.89 (d, 1H,
J = 2.5 Hz, CHAr), 7.27–7.45 (m, 5H, HAr); 13C NMR (125 MHz, CDCl3),
δ
[ppm]: 19.07 (CH(CH3)),
27.18 (CH(CH3 2
) ), 34.69 (CH2), 57.01 (CHBr), 85.53 (CHAr), 168.80 (C = O), CAr: 125.35, 128.67, 129.01,
138.31; IR (cm−1): 1742, 1598, 1451, 1370, 1338, 1281, 1270, 1239, 1162, 1105, 1067, 1035, 1001, 954, 920,
850, 800, 764, 740, 701, 683; HR-MS (ESI-TOF) calculated for C12H13BrO2, m/z [M + K]+: 306.973594;
experimental value: 306.974724.
Trans,trans-5-bromo-6-(4-fluorophenyl)-tetrahydro-4-methylpyran-2-one (7c): The product was
◦
1
obtained as a colorless solid; mp = 123–124 C; Rf = 0.12 (acetone:hexane 1:7); H NMR (CDCl3,
500 MHz), [ppm]: 1.28 (d, 3H, J = 6.5 Hz, CH(CH3)), 2.44 (dd, 1H, J1 = 17.2 Hz, J2 = 10.0 Hz, CHaHb),
2.47–2.61 (m, 1H, CH(CH3)), 2.99 (dd, 1H, J1 = 17.2 Hz, J2 = 5.6 Hz, CHaHb), 3.80 (dd, 1H, J1 = 10.4 Hz,
2 = 9.7 Hz, CHBr), 5.31 (d, 1H, J = 10.4 Hz, CHAr), 7.04–7.16 (m, 2H, HAr), 7.32–7.42 (m, 2H, HAr);
13C NMR (125 MHz, CDCl3),
[ppm]: 21.07 (CH(CH3)), 35.88 (CH(CH3 2), 37.33 (CH2), 55.53 (CHBr),
δ
J
δ
)
84.64 (CHAr), 168.93 (C = O), CAr: 115.56 (d, 2 JF−C = 21.8 Hz), 129.48 (d, 3 JF−C = 8.5 Hz), 132.77 (d,
4 JF−C = 3.3 Hz), 163.14 (d, JF−C = 248.6 Hz); IR (cm−1): 1729, 1607, 1512, 1454, 1424, 1372, 1331, 1261,
1220, 1190, 1160, 1035, 1004, 850, 918, 877, 840, 823, 790, 773, 719, 660; HR-MS (ESI-TOF) calculated for
C12H12BrO2, m/z [M + Na]+: 308.990234; experimental value: 308.991651.
Cis,trans-5-bromo-6-(4-fluorophenyl)-tetrahydro-4-methylpyran-2-one (8c): The product was obtained
as a colorless oil; n2D0 = 1.5140, Rf = 0.21 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
δ [ppm]:
1.07 (d, 3H, J = 6.5 Hz, CH(CH3)), 2.08–2.12 (m, 1H, CH(CH3)), 2.60 (dd, 1H, J1 = 18.4 Hz, J2 = 10.3
Hz, CHaHb), 2.67 (ddd, 1H, J1 = 18.4 Hz, J2 = 6.1 Hz, J3 = 0.9 Hz, CHaHb), 4.41 (td, 1H, J1 = 3.1 Hz,
J2 = 0.8 Hz, CHBr), 5.84 (dd, 1H, J1 = 3.1 Hz, J2 = 0.5 Hz CHAr), 7.08–7.13 (m, 2H, HAr), 7.24–7.29
(m, 2H, HAr); 13C NMR (125Hz, CDCl3),
δ[ppm]: 18.84 (CH(CH3)), 27.54 (CH(CH3)2), 34.84 (CH2),
56.58 (CHBr), 84.80 (CHAr), 168.63 (C = O), CAr: 116.06 (d, 2 JF−C = 21.9 Hz), 127.38 (d, 3 JF−C = 8.3
Hz), 134.02 (d, 4 JF−C = 3.2 Hz), 162.67 (d, JF−C = 248.6 Hz); IR (cm−1): 1740, 1602, 1507, 1453, 1413,
1370, 1359, 1338, 1273, 1227, 1190, 1160, 1115, 1096, 1074, 1062, 1033, 1004, 951, 911, 855, 832, 802, 790,

Molecules 2019, 24, 1875
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+
750, 716, 670; HR-MS (ESI-TOF) calculated for C12H12BrO2, m/z [M + Na] : 308.990234; experimental
value: 308.991301.
The iodolactonization of unsaturated carboxylic acids 4a–c: The mixture of an unsaturated
carboxylic acid (0.005 mol), diethyl ether and saturated aqueous NaHCO3 was stirred for 30 min
and a solution of iodine in potassium iodide was dropwise added. The reaction mixture was
refluxed for 24 h. When the reaction was completed, the mixture was dissolved in diethyl ether
and washed with saturated Na2S2O3 solution. The organic layer was washed with saturated
NaHCO3 and dried over anhydrous magnesium sulfate. The crude product was purified by column
chromatography on silica gel using a mixture of acetone and hexane (1:15) [29]. Spectral data are given
below. The iodolactonization of 4a gave
12a 13a 10.24:89.76). The iodolactonization of 4b gave
-hydroxylactone 12b 13b with total yield of 75.28% (9b
The iodolactonization of 4c gave -iodolactones 9c 10c
13c with total yield of 68.55% (9c:10c:11c:12c:13c 30.26:37.42:8.00:7.23:18.08).
γ
-hydroxylactones 12a and 13a with a total yield of 84.59%
-iodolactones 9b 10b -iodoactone 11b and
10b 11b 12b 13b 41.71:33.19:7.00:13.79:4.65).
-iodoactone 11c and -hydroxylactone 12c
(
γ
:
δ
,
, γ
,
:
γ
:
:
:
δ
,
,
γ
,
Trans-dihydro-5-((S)-hydroxy(naphthalen-1-yl)methyl)-4-methylfuran-2(3H)-one (12a): The product
was obtained as a yellow oil; n2D0 = 1.427; Rf = 0.08 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
δ
[ppm]: 0.56 (d, 3H, J = 6.8 Hz, CH3), 2.11 (dd, 1H, J1 = 17.5 Hz, J2 = 6.6 Hz, CHaHb), 2.72–2.79 (m,
1H, CH(CH3)), 2.85 (dd, 1H, J1 = 17.5 Hz, J2 = 9.3 Hz, CHaHb), 4.53 (dd, 1H, J1 = 5.5 Hz, J2 = 2.8
Hz , COCH), 5.92 (d, 1H, J = 2.3 Hz, CHOH), 7.50–7.59 (m, 3H, HAr), 7.80–7.86 (m, 2H, HAr), 7.90
(d, 1H, J = 8.1 Hz, HAr), 7.96 (d, 1H, J = 8.1 Hz, HAr); 13C NMR (125 MHz, CDCl3),
δ [ppm]: 19.74
(CH3), 28.38 (CH(CH3)), 37.34 (CH2), 69.97 (CHOH), 88.20 (COCH), 175.91 (C = O), CAr: 122.11, 123.65,
125.49, 125.82, 126.64, 128.69, 129.18, 129.88, 133.56, 133.69; IR (cm−1): 3422, 1774, 1593, 1510, 1458, 1417,
1379, 1359, 1327, 1291, 1254, 1209, 1166, 1112, 1092, 1078, 1037, 1008, 975, 936, 920, 881, 854, 823, 802,
782, 741, 694, 660, 628, 615; HR-MS (ESI-TOF) calculated for C16H16O3, m/z [M + Na]+: 279.099709;
experimental value: 279.100786.
Cis-dihydro-5-((S)-hydroxy(naphthalen-1-yl)methyl)-4-methylfuran-2(3H)-one (13a): The product was
obtained as a brown solid; mp = 123–124 ◦C; Rf = 0.06 (acetone:hexane 1:7); 1H NMR (CDCl3, 500
MHz),
CHaHb), 2.64 (dd, 1H, J1 = 17.0 Hz, J2 = 8.6 Hz, CHaHb), 2.72–2.83 (m, 1H, CH(CH3)), 4.86 (dd, 1H, J1
= 7.2 Hz, 2 = 3.4 Hz, COCH), 5.72 (t, 1H, J = 3.5 Hz, CHOH), 7.47–7.58 (m, 3H, HAr), 7.71 (d, 1H, J =
6.9 Hz, HAr), 7.84 (d, 1H, J = 8.2 Hz, HAr), 7.90 (d, 1H, J = 8.0 Hz, HAr), 8.02 (d, 1H, J = 7.9 Hz, HAr);
13C NMR (125 MHz, CDCl3),
[ppm]: 14.30 (CH3), 33.02 (CH(CH3)), 36.87 (CH2), 68.97 (CHOH),
δ [ppm]: 1.31 (d, 3H, J = 7.0 Hz, CH3), 1.57 (s, 1H, OH), 2.57 (dd, 1H, J1 = 17.0 Hz, J2 = 8.8 Hz,
J
δ
83.96 (COCH), 177.11 (C = O), CAr: 122.28, 122.95, 125.49, 125.76, 126.58, 129.02, 129.24, 130.36, 133.88,
134.90; IR (cm−1): 3492, 1747, 1593, 1507, 1453, 1415, 1374, 1350, 1327, 1286, 1275, 1257, 1214, 1189, 1173,
1153, 1110, 1096, 1037, 1026, 1000, 942, 918, 852, 834, 787, 773, 755, 730, 655, 644, 622; HR-MS (ESI-TOF)
calculated for C16H16O3, m/z [M + K]+: 295.073647; experimental value: 295.074508.
Trans,trans-tetrahydro-5-iodo◦-4-methyl-6-phenylpyran-2-one (9b): The product was obtained as a
colorless solid; mp = 107–108 C; Rf = 0.15 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
δ [ppm]:
1.29 (d, 3H, J = 6.6 Hz, CH(CH3)), 2.41 (dd, 1H, J1 = 17.4 Hz, J2 = 9.8 Hz, CHaHb), 2.48–2.61 (m, 1H,
CH(CH3)), 2.93 (dd, 1H, J1 = 17.4 Hz, J2 = 5.9 Hz, CHaHb), 3.97 (dd, 1H, J1 = 10.9 Hz, J2 = 10.0 Hz,
CHI), 5.45 (d, 1H, J = 10.9 Hz, CHAr), 7.32–7.44 (m, 5H, HAr); 13C NMR (125 MHz, CDCl3),
δ
[ppm]:
23.57 (CH(CH3)), 36.00 (CH(CH3 2
) ), 37.12 (CH2), 38.88 (CHI), 86.82 (CHAr), 169.49 (C = O), CAr:
127.74, 128.50, 129.43, 137.53; IR (cm−1): 1719, 1499, 1453, 1413, 1372, 1340, 1290, 1245, 1211, 1140, 1100,
1070, 996, 950, 933, 907, 887, 850, 769, 743, 695; HR-MS (ESI-TOF) calculated for C12H13IO2, m/z [M +
Na]+: 338.985797 experimental value: 338.986432.
Cis,trans-tetrahydro-5-iodo-4-methyl-6-phenylpyran-2-one (10b): The product was obtained as a
1
colorless oil, n2D0 = 1.5227, Rf = 0.19 (acetone:hexane 1:7); H NMR (CDCl3, 500 MHz),
δ [ppm]:
0.97 (d, 3H, J = 6.4 Hz, CH(CH3)), 1.27–1.31 (m, 1H, CH(CH3)), 2.49 (dd, 1H, J1 = 18.5 Hz, J2 = 10.5 Hz,

Molecules 2019, 24, 1875
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CHaHb), 2.67 (ddd, 1H, J1 = 18.5 Hz, J2 = 5.6 Hz, J3 = 1.1 Hz, CHaHb), 4.60 (td, 1H, J1 = 3.1 Hz, J2
1.1 Hz, CHI), 5.91 (d, 1H, J = 3.1 Hz, CHAr), 7.25–7.43 (m, 5H, HAr); 13C NMR (125 MHz, CDCl3),
=
δ
[ppm]: 21.03 (CH(CH3)), 27.34 (CH(CH3)2), 36.36 (CH2), 38.08 (CHBr), 86.32 (CHAr), 168.32 (C = O),
CAr: 125.06, 128.18, 128.51, 138.42; IR (cm−1): 1735, 1495, 1451, 1407, 1380, 1368, 1354, 1334, 1322, 1285,
1265, 1233, 1190, 1132, 1103, 1061, 1030, 998, 950, 903, 883, 852, 796, 763, 735, 695, 664; HR-MS (ESI-TOF)
calculated for C12H13IO2, m/z [M + K]+: 354.959735 experimental value: 354.958974.
Cis-dihydro-5-(iodo(phenyl)methyl)-4-methylfuran-2(3H)-one (11b): The product was obtained as a
colorless solid, mp = 86–87 ◦C; Rf = 0.15 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
δ [ppm]:
1.17 (d, 3H, J = 7.0 Hz, CH3), 2.32 (dd, 1H, J1 = 17.0 Hz, J2 = 0.5 Hz, CHaHb), 2.87 (dd, 1H, J1 = 17.0 Hz,
J2 = 7.4 Hz, CHaHb), 2.96–3.03 (m, 1H, CH(CH3)), 4.96 (d, 1H, J = 11.2 Hz, CHI), 5.11 (dd, 1H, J1 = 11.2
Hz, J2 = 4.5 Hz, COCH), 7.34–7.39 (m, 3H, HAr), 7.39–7.44 (m, 2H, HAr); 13C NMR (125 MHz, CDCl3),
δ
[ppm]: 12.82 (CH3), 28.38 (CHI), 34.00 (CH(CH3)), 38.88 (CH2), 84.45 (COCH), 176.00 (C = O), CAr:
127.79, 128.61, 128.85, 140.09; IR (cm−1): 1765, 1500, 1452, 1414, 1372, 1341, 1290, 1247, 1212, 1141, 1100,
1071, 997, 950, 932, 908, 886, 851, 769, 742, 695; HR-MS (ESI-TOF) calculated for C12H13IO2, m/z [M +
Na]+: 338.985797 experimental value: 338.986737.
Trans-dihydro-5-(hydroxy(phenyl◦)methyl)-4-methylfuran-2(3H)-one (12b): The product was obtained
1
as a colorless solid, mp = 86–87 C; Rf = 0.11 (acetone:hexane 1:7); H NMR (CDCl3, 500 MHz),
δ
[ppm]: 0.81 (d, 3H, J = 6.9 Hz, CH3), 2.10 (dd, 1H, J1 = 17.6 Hz, J2 = 6.7 Hz, CHaHb), 2.57–2.76 (m,
1H, CH(CH3)), 2.73 (dd, 1H, 1 = 17.6 Hz, J2 = 9.2 Hz, CHaHb), 4.30 (dd, 1H, 1 = 5.5 Hz, 2 = 3.3 Hz,
COCH), 5.09 (t, 1H, J = 3.6 Hz, CHOH), 7.29–7.34 (m, 3H, HAr), 7.36–7.42 (m, 2H, HAr); 13C NMR
(125 MHz, CDCl3), [ppm]: 19.77 (CH3), 28.80 (CH(CH3)), 37.06 (CH2), 72.29 (CHOH), 89.74 (COCH),
J
J
J
δ
176.99 (C = O), CAr: 126.00, 128.12, 128.63, 138.34; IR (cm−1): 3442, 1767, 1600, 1496, 1451, 1415, 1379,
1356, 1324, 1275, 1225, 1214, 1202, 1162, 1103, 1085, 1060, 1008, 983, 936, 915, 879, 859, 818, 762, 707, 694,
676, 617; HR-MS (ESI-TOF) calculated for C12H14O3, m/z [M + Na]+: 229.084059 experimental value:
229.084576.
Cis-dihydro-5-(hydroxy(phenyl)methyl)-4-methylfuran-2(3H)-one (13b): The product was obtained as
a colorless solid, mp = 89–91 ◦C; Rf = 0.11 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
δ
[ppm]:
1.32 (d, 3H, J = 6.9 Hz, CH3), 1.58 (s, 1H, OH), 2.20 (dd, 1H, J1 = 17.2 Hz, J2 = 4.8 Hz, CHaHb), 2.77 (dd,
1H, 1 = 17.3 Hz, J2 = 8.4 Hz, CHaHb), 2.80–2.89 (m, 1H, CH(CH3)), 4.85 (dd, 1H, 1 = 7.2 Hz, 2 = 4.0
Hz, COCH), 5.11 (d, 1H, J = 3.9 Hz, CHOH), 7.27–7.35 (m, 2H, HAr), 7.37–7.48 (m, 2H, HAr); 13C NMR
J
J
J
(125 MHz, CDCl3), δ [ppm]: 19.20 (CH3), 32.85 (CH(CH3)), 35.03 (CH2), 69.70 (CHOH), 84.59 (COCH),
175.49 (C = O), CAr: 125.67, 128.29, 128.78, 138.37; IR (cm−1): 3419, 1751, 1587, 1497, 1434, 1403, 1387,
1361, 1301, 1255, 1243, 1202, 1167, 1075, 1067, 1054, 1011, 983, 933, 887, 828, 785, 752, 732, 697; HR-MS
(ESI-TOF) calculated for C12H14O3, m/z [M + Na]+: 229.084059 experimental value: 229.084781.
Trans,trans-6-(4-fluorophenyl)-tetrahydro-5-iodo-4-methylpyran-2-one (9c): The product was obtained
as a colorless solid, mp = 136–138 ◦C, Rf = 0.16 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
δ
[ppm]: 1.28 (d, 3H, J = 6.5 Hz, CH(CH3)), 2.36 (dd, 1H, J1 = 17.2 Hz, J2 = 10.0 Hz, CHaHb), 2.51–2.57 (m,
1H, CH(CH3)), 2.92 (dd, 1H, J1 = 17.5 Hz, J2 = 6.0 Hz, CHaHb), 3.90 (dd, 1H, J1 = 10.8 Hz, J2 = 10.1 Hz,
CHI), 5.42 (d, 1H, J = 10.8 Hz, CHAr), 7.05–7.09 (m, 2H, HAr), 7.32–7.36 (m, 2H, HAr); 13C NMR (125
MHz, CDCl3), δ [ppm]: 23.6 (CH(CH3)), 27.03 (CH(CH3)2), 31.66 (CH2), 37.69 (CHI), 86.43 (CHAr),
2
3
4
169.68 (C = O), CAr: 115.53 (d, JF−C = 21.7 Hz), 129.58 (d, JF−C = 8.4 Hz), 133.78 (d, JF−C = 3.3
Hz), 163.09 (d, JF−C = 248.60 Hz); IR (cm−1): 1720, 1601, 1515, 1459, 1421, 1411, 1366, 1334, 1300, 1285,
1223, 1207, 1176, 1162, 1110, 1090, 1071, 1053, 1004, 952, 909, 896, 858, 844, 822, 778, 775, 717; HR-MS
(ESI-TOF) calculated for C12H12FIO2, m/z [M + Na]+: 356.976375 experimental value: 356.977702.
Cis,trans-6-(4-fluorophenyl)-tetrahydro-5-iodo-4-methylpyran-2-one (10c): The product was obtained
1
as a yellow oil; n2D0 = 1.5227, Rf = 0.24 (acetone:hexane 1:7); H NMR (CDCl3, 500 MHz),
δ [ppm]:
1.04 (d, 3H, J = 6.5 Hz, CH(CH3)), 1.39–1.48 (m, 1H, CH(CH3)), 2.52 (dd, 1H, J1 = 18.4 Hz, J2 = 9.8
Hz, CHaHb), 2.71 (ddd, 1H, J1 = 18.4 Hz, J2 = 5.5 Hz, J3 = 1.0 Hz, CHaHb), 4.55 (td, 1H, J1 = 3.9 Hz,

Molecules 2019, 24, 1875
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J2 = 0.9 Hz , CHI), 5.86 (d, 1H, J = 3.9 Hz, CHAr), 7.04–7.13 (m, 2H, HAr), 7.22–7.29 (m, 2H, HAr);
13C NMR (125 MHz, CDCl3),
δ [ppm]: 21.13 (CH(CH3)), 28.37 (CH(CH3)2), 30.90 (CH2), 36.88 (CHI),
85.92 (CHAr), 168.53 (C = O), CAr: 115.96 (d, 2 JF−C = 21.8 Hz), 127.56 (d, 3 JF−C = 8.3 Hz), 134.62 (d,
4 JF−C = 3.0 Hz), 162.62 (d, JF−C = 248.8 Hz); IR (cm−1): 1735, 1605, 1508, 1453, 1411, 1384, 1366, 1354,
1340, 1328, 1283, 1263, 1225, 1184, 1157, 1133, 1095, 1073, 1056, 998, 946, 907, 890, 851, 829, 802, 782,
+
750, 714; HR-MS (ESI-TOF) calculated for C12H12FIO2, m/z [M + Na] : 356.976375 experimental value:
356.977461.
Cis-5-((4-fluorophenyl)iodomethyl)-dihydro-4-methylfuran-2(3H)-one (11c): The product was obtained
as a colorless solid, mp = 118–119 ◦C; Rf = 0.16 (acetone:hexane 1:7); 1H NMR (CDCl3, 500 MHz),
[ppm]: 1.16 (d, 3H, J = 7.1 Hz, CH3), 2.33 (dd, 1H, J1 = 17.1 Hz, J2 = 0.5 Hz, CHaHb), 2.87 (dd, 1H, J1
δ
=
17.1 Hz, J2 = 7.3 Hz, CHaHb), 2.95–3.00 (m, 1H, CH(CH3)), 4.94 (d, 1H, J1 = 11.2 Hz, CHI), 5.04 (dd,
1H, 1 = 11.2 Hz,
2 = 4.5 Hz, COCH), 6.97–7.10 (m, 2H, HAr), 7.32–7.43 (m, 2H, HAr); 13C NMR (125
MHz, CDCl3), [ppm]: 12.85 (CH3), 28.45 (CHI), 34.05 (CH(CH3)), 38.91 (CH2), 84.30 (COCH), 176.11
J
J
δ
(C = O), CAr: 116.24 (d, 2 JF−C = 22.1 Hz), 127.64 (d, 3 JF−C = 8.0 Hz), 133.62 (d, 4 JF−C = 3.0 Hz), 162.38
(d, JF−C = 246.3 Hz). IR (cm−1): 1760, 1600, 1512, 1460, 1420, 1412, 1361, 1335, 1301, 1284, 1226, 1203,
1174, 1161, 1108, 1091, 1070, 1052, 1001, 950, 906, 894, 857, 842, 821, 779, 776, 714; HR-MS (ESI-TOF)
calculated for C12H12FIO2, m/z [M + Na]+: 356.976375 experimental value: 356.976932.
Trans-5-((4-fluorophenyl)(hydroxy)methyl)-dihydro-4-methylfuran-2(3H)-one (12c): The product was
obtained as a colorless solid, mp = 118–119 ◦C, Rf = 0.10 (acetone:hexane 1:7); 1H NMR (CDCl3, 500
MHz),
(m, 1H, CH(CH3)), 2.74 (dd, 1H,
Hz, COCH), 5.07 (d, 1H, J = 3.2 Hz, CHOH), 6.98–7.12 (m, 2H, HAr), 7.33–7.43 (m, 2H, HAr); 13C NMR
(125 MHz, CDCl3), [ppm]: 19.74 (CH3), 28.84 (CH(CH3)), 37.03 (CH2), 72.70 (CHOH), 89.65 (COCH),
δ
[ppm]: 0.83 (d, 3H, J = 6.9 Hz, CH3), 2.11 (dd, 1H, J1 = 17.7 Hz, J2 = 6.9 Hz, CHaHb), 2.53–2.64
J
1 = 17.7 Hz, J2 = 9.2 Hz, CHaHb), 4.26 (dd, 1H, 1 = 5.3 Hz, 2 = 3.4
J
J
δ
176.93 (C = O), CAr: 115.57 (d, 2 JF−C = 21.5 Hz), 127.72 (d, 3 JF−C = 8.1 Hz), 134.17 (d, 4 JF−C = 3.1 Hz),
163.46 (d, JF−C = 246.6 Hz); IR (cm−1): 3431, 1772, 1593, 1509, 1456, 1412, 1378, 1361, 1329, 1287, 1257,
1215, 1169, 1114, 1093, 1075, 1041, 1008, 975, 936, 920, 881, 854, 826, 802, 782, 749, 715; HR-MS (ESI-TOF)
calculated for C12H13FO3, m/z [M + Na]+: 247.074638 experimental value: 247.075208.
Cis-5-((4-fluorophenyl)(hydroxy)methyl)-dihydro-4-methylfuran-2(3H)-one (13c): The product was
obtained as a colorless solid, mp = 118–119 ◦C; Rf = 0.08 (acetone:hexane 1:7); 1H NMR (CDCl3, 500
MHz),
CHaHb), 2.78 (dd, 1H, J1 = 17.3 Hz, J2 = 8.5 Hz, CHaHb), 2.81–2.89 (m, 1H, CH(CH3)), 4.81 (dd, 1H, J1
= 7.3 Hz, 2 = 4.1 Hz, COCH), 5.09 (d, 1H, J = 4.1 Hz, CHOH), 7.14–7.24 (m, 2H, HAr), 7.29–7.38 (m, 2H,
HAr); 13C NMR (125 MHz, CDCl3),
[ppm]: 19.22 (CH3), 32.86 (CH(CH3)), 35.05 (CH2), 69.72 (CHOH),
δ [ppm]: 1.34 (d, 3H, J = 6.8 Hz, CH3), 1.59 (s, 1H, OH), 2.24 (dd, 1H, J1 = 17.3 Hz, J2 = 4.8 Hz,
J
δ
84.57 (COCH), 175.47 (C = O), CAr: 116.26 (d, 2 JF−C = 22.1 Hz), 127.69 (d, 3 JF−C = 8.0 Hz), 133.63 (d,
4 JF−C = 3.0 Hz), 162.34 (d, JF−C = 246.4 Hz); IR (cm−1): 3439, 1751, 1592, 1507, 1459, 1410, 1380, 1365,
1327, 1285, 1259, 1216, 1169, 1115, 1094, 1077, 1044, 1010, 973, 937, 920, 882, 853, 827, 801, 782, 748, 716;
HR-MS. HR-MS (ESI-TOF) calculated for C12H13FO3, m/z [M + Na]+: 247.074638 experimental value:
247.075284.
3.5. Cytotoxic Activity
Cell cultures: The L929 mouse fibroblasts and human gastric adenocarcinoma cell line AGS were
used for in vitro cytotoxicity assays. The cells were maintained under standard conditions (37 ◦C,
5% CO2) in 25 cm2 tissue culture flasks in RPMI-1640 medium supplemented with 10% fetal bovine
serum (FBS) and antibiotics: 100 U/mL penicillin and 100 µg/mL streptomycin. The cell suspension
containing 108 cells in 1 mL, was obtained by treatment of confluent monolayers with 0.25% trypsin
solution, washed and subcultured. Cell cultures were supplemented with medium to maintain them
in log phase growth. The viability of the cells was assessed by exclusion of trypan blue dye.
Measurements of cellular metabolic activity and global growth inhibition: L929 cells were seeded
into 96-well plates (2 –c, 6a–c, 7a–c,
105 cells/well) for 24 h, at 37 ◦C, 5% CO2. Tested compounds 5a
×

Molecules 2019, 24, 1875
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8a
–
c
,
9b,c
,
10b,c
,
µ
12a and 13a were diluted serially in RPMI-1640 medium in concentrations of 50, 20,
g/mL, added to the cells (100 l/well), and incubated under standard conditions for
10, 5, 0.5 and 0.1
µ
24 h [43]. The potential harmful effect of lactones on L929 and AGS cells was assessed by the ability of
the cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan.
The solution of MTT (5 mg/mL in PBS), was added to each well and the plates were incubated for 4h at
37 ◦C. Formazan crystals were dissolved with acidic isopropanol (0.1 M HCl in absolute isopropanol)
and the plates were read in a plate reader (Victor 2) at 570 nm. The percentage of MTT reduction
was calculated by comparing the absorbance of treated cells to that of untreated control cells. Results
are expressed as the mean percentage of viable cells
experiments. Statistical significance was accepted at a p-value < 0.05.
±
standard deviation (SD) of four independent
3.6. Bactericidal Activity
The bacterial cultures: Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 65389 were
grown on Luria–Bertani Agar for 18 h at 37 ◦C. The strain suspension was prepared in 0.85% saline,
with an optical density equivalent to a 0.5 McFarland standard. Next, the following mixtures were
prepared: 180
tested compound (range 0.1–50
was added. As a sterility control, 180
µ
L of sterile enriched broth (BTL), 10
g/mL) solubilized in DMSO. As a negative control, only DMSO
L of BTL and 20 L saline were used. The microtiter plate
µL of the bacterial suspension and 10 µL of the
µ
µ
µ
was incubated at 37 ◦C for 18 h. The colony forming unit (CFU) method was used to evaluate the
microbial population. The experiment was repeated three times. Gentamicin was used as the reference
antibiotic [23]
4. Conclusions
A four-step synthesis gave sixteen new
-hydroxy- -lactones. The stereoselectivity of the iodolactonization reaction is significant. The yield
of trans,trans chloro- and bromo- -lactone was in the range of 76–82%. The iodolactonization of
unsaturated carboxylic acids gave cis -hydroxy- -lactones with a yield of 90%. All obtained lactones
were tested for antimicrobial activity against Escherichia coli strains ATCC 8739 and Staphylococcus aureus
ATCC 65389. The -iodolactone 10c and -hydroxy- -lactones 12a and 13a showed high bactericidal
γ-halo-δ-lactones, two γ-iodo-δ-lactones and unexpectedly
six
δ
γ
δ
δ
γ
δ
δ
γ
activity against E. coli.. The highest bactericidal activity against S. aureus was observed for lactone
5a, which reduced the number of CFU by 87%. Significant bactericidal activity was observed for
cis,trans-γ-chloro-δ-lactone 6c (83%) and cis,trans-γ-bromo-δ-lactone 8a (83%). Synthesized lactones
were also tested for cytotoxic activity against two cell lines, mouse fibroblasts L929 and gastric
cancer cells AGS. The strongest cell growth inhibition against L929 fibroblasts was observed for
cis,trans-
sensitive to trans,trans-
γ
-iodo-
δ
-lactones 10b,c and cis,trans-
γ
-chloro-
δ
-lactone 6b. The AGS cells were highly
-iodo- -lactones 9b,c. Their cytotoxic
γ
-chloro- -lactone 5c, and trans,trans-
δ
γ
δ
activity against gastric cancer AGS cells was much higher than doxorubicin, thus these lactones could
be promising candidates for anticancer formulations.
Author Contributions: Conceptualization, investigation and writing—original draft preparation, A.K.;
supervision and project administration, B.G.; investigation, Ł.L., A.B., S.E.K., W.G., and M.C.; and data curation,
writing—review and editing, M.U.
Funding: This research received no external funding.
Acknowledgments: Special thanks to Division of Biophysics, Faculty of Physics, University of Warsaw for the
X-ray diffraction measurements. The Rigaku Oxford Diffraction SuperNova diffractometer was cofinanced by the
European Regional Development Fund (POIG.02.01.00-14-122/09).
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 1875
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Abbreviations
The following abbreviations are used in this manuscript:
NBS
NCS
CFU
MTT
SD
N-bromosuccinimide
N-chlorosuccinimide
the colony forming unit
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
standart deviations
IC50
NMR
FTIR
the half maximal inhibitory concentration
nuclear magnetic resonance
Fourier-transform infrared spectroscopy
HR-MS-MS high-resolution electrospray ionization mass spectra
TLC
thin layer chromatography
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