Chemoenzymatic synthesis of enantiomeric, bicyclic δ-halo-γ-lactones with a cyclohexane ring, their biological activity and interaction with biological membranes

Article abstract of DOI:10.3390/biom10010095

Starting from 1-acetyl-1-cyclohexene, three enantiomeric pairs (ee ≥99%) of bicyclic δ-halo-γ-lactones with cyclohexane ring were obtained in five-step synthesis. The key stereochemical steps were lipase-catalyzed kinetic resolution of racemic 1-(cyclohex-1-en-1-yl) ethanol followed by transfer of chirality to ethyl 2-(2-ethylidenecyclohexyl) acetate in the Johnson–Claisen rearrangement. Synthesized halolactones exhibited antiproliferative activity towards canine B-cell leukemia cells (GL-1) and canine B-cell chronic leukemia cells (CLB70) and the most potent (IC50 18.43 ± 1.46 μg/mL against GL-1, IC50 11.40 ± 0.40 μg/mL against CLB70) comparable with the control etoposide, was (1R,6R,1’S)-1-(1’-chloroethyl)-9- oxabicyclo[4.3.0]nonan-8-one (8b). All halolactones did not have a toxic effect on erythrocytes and did not change the fluidity of membranes in the hydrophobic region of the lipid bilayer. Only weak changes in the hydrophilic area were observed, like the degree of lipid packing and associated hydration. The racemic halolactones were also tested for their antimicrobial properties and found to exhibit selectivity towards bacteria, in particular, towards Proteus mirabilis ATCC 35659.

Full text of DOI:10.3390/biom10010095

Article
Chemoenzymatic Synthesis of Enantiomeric,
Bicyclic δ‐Halo‐γ‐lactones with a Cyclohexane Ring,
Their Biological Activity and Interaction with
Biological Membranes
1,
2
3
2
4
Marcelina Mazur *, Aleksandra Włoch , Fouad Bahri , Hanna Pruchnik , Aleksandra Pawlak ,
4
5
1
Bożena Obmińska‐Mrukowicz , Gabriela Maciejewska and Witold Gładkowski
1
Department of Chemistry, Wrocław University of Environmental and Life Sciences,
Norwida 25, 50‐375 Wrocław, Poland; witold.gladkowski@upwr.edu.pl
2
Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences,
Norwida25, 50‐375 Wrocław, Poland; aleksandra.wloch@upwr.edu.pl (A.W.);
hanna.pruchnik@upwr.edu.pl (H.P.)
Laboratory of Microbiology and Plant Biology, Faculty of Natural and Life Sciences, University of Abd El
Hamid Ibn Badiss of Mostaganem, Mostaganem 27000, Algeria, bahrifouad13@gmail.com
Department of Pharmacology and Toxicology, Wrocław University of Environmental and Life Sciences,
Norwida 31, 50‐375 Wrocław, Poland; aleksandra.pawlak@upwr.edu.pl (A.P.);
bozena.obminska‐mrukowicz@upwr.edu.pl (B.O.‐M.)
3
4
5
Central Laboratory of the Instrumental Analysis, Wrocław University of Science and Technology, Wybrzeże
Wyspiańskiego 27, 50‐370 Wrocław, Poland; gabriela.maciejewska@pwr.edu.pl
* Correspondence: marcelina.mazur@upwr.edu.pl; Tel.: +48‐713205197
Received: 21 November 2019; Accepted: 28 December 2019; Published: 6 January 2020
Abstract: Starting from 1‐acetyl‐1‐cyclohexene, three enantiomeric pairs (ee ≥99%) of bicyclic
δ‐halo‐γ‐lactones with cyclohexane ring were obtained in five‐step synthesis. The key
stereochemical steps were lipase‐catalyzed kinetic resolution of racemic 1‐(cyclohex‐1‐en‐1‐yl)
ethanol followed by transfer of chirality to ethyl 2‐(2‐ethylidenecyclohexyl) acetate in the
Johnson–Claisen rearrangement. Synthesized halolactones exhibited antiproliferative activity
towards canine B‐cell leukemia cells (GL‐1) and canine B‐cell chronic leukemia cells (CLB70) and
the most potent (IC50 18.43 ± 1.46 μg/mL against GL‐1, IC50 11.40 ± 0.40 μg/mL against CLB70)
comparable
with
the
control
etoposide,
was
(1R,6R,1’S)‐1‐(1’‐chloroethyl)‐9‐
oxabicyclo[4.3.0]nonan‐8‐one (8b). All halolactones did not have a toxic effect on erythrocytes and
did not change the fluidity of membranes in the hydrophobic region of the lipid bilayer. Only weak
changes in the hydrophilic area were observed, like the degree of lipid packing and associated
hydration. The racemic halolactones were also tested for their antimicrobial properties and found
to exhibit selectivity towards bacteria, in particular, towards Proteus mirabilis ATCC 35659.
Keywords: lactones; halolactonisation; kinetic resolution ofalcohols; antimicrobial activity;
antiproliferative activity; hemolytic activity; erythrocytes; biological membranes
1. Introduction
Lactones are cyclic esters which are detected in plants and animals. The natural lactones
isolated from fruits and essential oils can be used as fragrance components, like jasmine lactone or as
a flavor in the food industry (δ‐decalactone) [1–3]. Those compounds can also be pheromones [4,5]
or antifeedants [6–8] but the largest interest is put in their antimicrobial and cytotoxic potential.
There are numerous examples presenting the antibacterial and antifungal activity of lactones [8,9].
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www.mdpi.com/journal/biomolecules

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Some phthalides derivatives are active against phytopathogenic fungal strains from the genus
Fusarium [10]. Vernolide and vernodalin, identified in the extract from Vernonia colorata, exhibit
antibacterial activity against Gram‐positive bacteria Staphylococcus aureus and Bacillus subtilis [11,12].
The natural occurring lactones are proved to be cytotoxic against numerous cancer cell lines. In this
group, the most potent are the sesquiterpene lactones like costunolide, parthenolide, helenalin or
artemisinin [13–15].
On the other hand, many efforts are currently being made to obtain synthetic lactones with
simplified structure and high cytotoxic activity. This approach is reasonable in the context of low
concentrations of natural active substances in plant material and high cost and difficulties during the
isolation processes. In the last few years, our scientific interests have concentrated on the synthesis of
biologically active lactones [7,8,16]. In our previous research on antiproliferative compounds, we
developed a synthetic pathway to obtain highly active γ‐lactones with a β‐aryl substituent [17–19].
An important element of the structure of these compounds, affecting their activity, was the presence
of a halogen atom. Recently, we have also reported that some bicyclic γ‐lactones with a cyclohexane
system exhibited cytotoxicity against selected cancer cell lines [20]. Assuming that the introduction
of a halogen atom can potentially increase the cytotoxic effect exhibited by this group of compounds,
we designed the synthesis in order to obtain a series of new halogen derivatives of γ‐lactones with a
cyclohexane ring (6–8), containing iodine, bromine and chlorine atoms in the lactone structure. The
next important aspect was a comparison of biological activity of opposite enantiomers. Application
of lipase‐catalyzed kinetic resolution of their precursor, racemic allylic alcohol, allowed us to
prepare pairs of lactone enantiomers and consequently check the significance of configuration of
stereogenic centers.
The cytotoxic activity of chemotherapeutic agents is related to different mechanisms which lead
to programmed cell death. As the biological membrane is an important barrier, which protects the
internal cell structures and allows them to function properly, understanding its interaction with
cytotoxic agents is extremely important. In particular, the red blood cells (RBC) and membranes of
RBC appear to be an excellent model to evaluate toxicity of molecules by measuring cell damage and
analyzing membrane biophysical parameters.
The main goal of this research was the design and synthesis of new optically active
δ‐halo‐‐lactones with a cyclohexane ring and the evaluation of their biological activity, namely,
antimicrobial, antiproliferative and hemolytic. The second important aspect was the attempt to
explain the mechanism of interactions of the tested compounds with biological membranes.
Therefore, the effects of the synthesized lactones on erythrocyte membrane fluidity, hydration and
packing arrangement of lipids were investigated.
2. Materials and Methods
2.1. Analysis
The reactions’ progress and the purity of obtained products were analyzed by TLC
(DC‐Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany. GC analysis was registered on an
Agilent Technologies 6890N instrument (Santa Clara, CA, USA) with FID detector, the hydrogen
was used as a carrier gas. Purity of obtained compounds as well as progress of the reactions were
checked on DB‐5HT column (Agilent, Santa Clara, USA, polyimide‐coated fused silica tubing, 30 m ×
0.25 mm × 0.10 × μm). The analytical parameters: detector 300 °C, injector 250 °C, temperature
programme: 80 °C (0 min), 80–200 °C (25 °C/min), 200–300 °C (30 °C/min), 300 °C (2 min).
The enantiomeric excesses were calculated based on chiral GC analysis applying CP
Chirasil‐Dex CB column (25 m × 0.25 mm × 0.25 μm). The analytical conditions were as follows:
injector 200 °C, detector (FID) 250 °C, temperature programme: 75 °C (1 min), 75–140 °C (0.3 °C/min),
140–200 (5 °C/min), 200 °C (10 min).
The products were purified using column chromatography on silica gel (Kieselgel 60, 230–400
mesh, Merck).

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1
13
Nuclear magnetic resonance spectra ( H, C‐NMR, COSY, HMBC, and HMQC) were registered
on a Bruker Avance II 600 MHz spectrometer (Bruker, Rheinstetten, Germany) in CDCl3 solutions.
As references, signals of residual solvent (δH = 7.26, δC = 77.0) were used. High‐resolution mass
spectra (HRMS) were registered on spectrometer Waters ESI‐Q‐TOF Premier XE (Waters Corp.,
Millford, MA, USA) using electron spray ionization (ESI) technique.
IR spectra were recorded using Mattson IR 300 Thermo Nicolet spectrophotometer (Thermo
™
Scientific , Waltham, MA, USA). The melting points (uncorrected) were measured on a Boetius
apparatus.
2.2. Chemicals
1‐Acetyl‐1‐cyclohexene (≥98% purity), propionic acid (≥99.5% purity), N‐bromosuccinimide
(NBS), N‐Chlorosuccinimide (NCS) were purchased from Sigma–Aldrich. Triethylorthoacetate
(≥98% purity) were purchased from Fluka. Iodine, potassium iodide, hydrochloric acid, sodium
bicarbonate, sodium hydroxide and sodium thiosulphate were purchased from POCh (Gliwice,
Poland). All the solvents used in column chromatography, purchased from Chempur, were of
analytical grade.
2.3. Synthesis of Lactones with Cyclohexane System
Three pairs of chiral bicyclic lactones with cyclohexane system were prepared in six steps
chemoenzymatic synthesis.
2.3.1. 1‐(Cyclohex‐1‐en‐1‐yl) ethanol (2)
Known unsaturated alcohol (2) was obtained as a product of 1‐acetyl‐1‐cyclohexene reduction
(isolated yield 90%) with sodium borohydride, according to procedure describe earlier by Mazur et
al. [7]. The spectroscopic data of rac‐2 correspond to literature data [21,22].
2.3.2. Kinetic Resolution of rac‐2 by Enzymatic Transesterification
The substrate for the lipase catalysed kinetic resolution was racemic alcohol (2) (5.5 g, 40 mmol).
The reaction was performed in diisopropyl ether (50 mL) using lipase CAL‐B from Candida antarctica
(10 wt.‐% of alcohol) and vinyl propionate (6 mL, 56 mmol) as the acyl donor. After 2 h at room
temperature the reaction was finished. The lipase was separated by filtration and the organic
solvents were evaporated in vacuo. The mixture of (S)‐alkohol (2a) and (R)‐propionate (3) was
separated by column chromatography (hexane:acetone 20:1). The spectroscopic data of alcohol 2a
and propionate 3 are in accordance with literature reports [21,23].
(−)‐(S)‐1‐(cyclohex‐1‐en‐1‐yl)ethanol (2a): 2D5 = −8.9 (c = 0.55, CHCl3, ee = 98.1%), [ref. [21] 2D5 = –9.4
(c = 1.5, CHCl3, ee = 91%)];
(+)‐(R)‐1‐(cyclohex‐1‐en‐1‐yl)ethyl propionate (3): 2D5 = +58.5 (c = 0.65, CHCl3, ee = 99%);
2.3.3. (R)‐1‐(cyclohex‐1‐en‐1‐yl)ethanol (2b)
The ester 3 (3 g, 16 mmol) was refluxed in a mixture of 5% ethanolic solution of NaOH (60 mL)
and water (10 mL). When the reaction was finished (3 h, TLC), the solvent was evaporated and the
residue was resuspended with water. The alcohol 2b was extracted with dichloromethane (3 × 40
mL) and the organic phase were washed with brine and dried over anhydrous MgSO4. After solvent
evaporation (R)‐alcohol 2b was obtained (1.7 g, isolated yield 84%).
(R)‐1‐(cyclohex‐1‐en‐1‐yl)ethanol (2b): D25 = +9.7 (c = 1.25, CHCl3, ee = 98.7%), [ref. [23] D25 = +6.4 (c =
1.2, CHCl3, ee = 68%)];

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2.3.4. Ethyl 2‐(2‐ethylidenecyclohexyl) Acetates (4a and 4b)
General Procedure
A solution of alcohol 2 (17.5 mmol) in triethylorthoacetate (15 mL) and drop of propionic acid
was heated in 138 °C with continuous distilling off the ethanol. After 24 h the crude product was
purified by column chromatography (silica gel, hexane:acetone 10:1) and the pure ester 4 was
obtained.
(−)‐(1S,2E)‐Ethyl 2‐(2‐ethylidenecyclohexyl) acetate (4a), Obtained from alcohol 2a (2.2 g, 17.5
mmol), yield 2.8 g (82%); Rt = 84 min, 2D5 = −13 (c = 0.75, CHCl3, ee = 98%); HRMS: (ESI‐TOF) m/z [M
+
+ Na] calcd for C12H20O2Na 219.1361; found 219.1355, other spectroscopic data consistent with
literature reports [24,25].
(+)‐(1R,2E)‐Ethyl 2‐(2‐ethylidenecyclohexyl)acetate (4b), Obtained from alcohol 2b (1.7 g, 13.6
mmol), yield 2.1 g (87%); Rt = 81 min, 2D5 = +14 (c = 0.8, CHCl3, ee = 99%); spectroscopic data identical
to the values reported for (−)‐(1S,2E)‐4a
2.3.5. (E)‐2‐(2‐Ethylidenecyclohexyl) Acetic acid (5a and 5b)
General Procedure
The ester 4 was refluxed in 2.5% a ethanolic solution of KOH (50 mL) for 5 h. When the reaction
was finished, solvent was evaporated, the residue was resuspended in water and then extracted
three times with diethyl ether (Et2O). The aqueous layer was acidified (1 M HCl) and the carboxylic
acid was extracted three times with Et2O. The combined organic fractions were washed with NaCl
and dried over anhydrous magnesium sulphate. After evaporating the solvent, a pure acid was
obtained.
(−)‐(S, E)‐2‐(2‐Ethylidenecyclohexyl) acetic acid (5a), Obtained from ester 4a (2.8 g, 14.4 mmol),
yield 2.2 g (94%); Rt = 186 min, 2D5 = −18 (c = 0.45, CHCl3, ee > 99%); HRMS: (ESI‐TOF) m/z [M − H]
−
calcd for C10H16O2 241.1046; found 241.1050. The spectroscopic data are in accordance with literature
reports [26].
(+)‐(R, E)‐2‐(2‐Ethylidenecyclohexyl) acetic acid (5b), Obtained from ester 4b (2.1 g, 10.6 mmol),
yield 1.3 g (75%); Rt = 184 min, 2D5 = +18 (c = 0.45, CHCl3, ee > 99%); spectroscopic data identical to
the values reported for (−)‐(S, E)‐5a
2.3.6. 1‐(1’‐Iodoethyl)‐9‐Oxabicyclo[4.3.0]nonan‐8‐one (6a and 6b)
General Procedure
The mixture of acid 5, 25 mL in Et2O with addition of saturated sodium bicarbonate (25 mL) was
stirred at room temperature for 1 h. Then, a mixture of iodine (1.4 g) and potassium iodide (2.8 g) in
H2O was added dropwise to the stirred solution until a stable brown color was maintained. When
the reaction was completed, the mixture was diluted with Et2O and washed with Na2S2O3. The
organic layer was washed with saturated sodium bicarbonate, brine and dried over MgSO4. The
crude
product
was
purified
on
silica
gel
(hexane:acetone,
20:1).
New
1‐(1‐iodoethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one was obtained as the product. The physical and
spectral data of the synthesized iodolactone 6 are given below.
(−)‐(1S,6S,1’R)‐1‐(1’‐Iodoethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one (6a), Obtained from acid 5a (0.5
g, 3 mmol), yield 0.6 g (72%); Rt = 189 min, 2D5 = −54 (c = 0.45, CHCl3, ee > 99%); mp 104–107 °C;
1
H‐NMR (600 MHz, CDCl3) δ: 1.26 (m, 1H,one of CH2‐5), 1.35 (m, 1H, one of CH2‐4), 1.46 (m, 1H, one
of CH2‐3), 1.57–1.67 (m, 2H, one of CH2‐3 and one of CH2‐4), 1.81(m, 1H, one of CH2‐5), 1.94 (d, 3H, J
= 7.0, CH3‐2’), 2.01 (dt, 1H, J = 14.8, 4.4 Hz, one of CH2‐2), 2.13 (ddd, 1H, J = 14.8, 11.5, 4.7 Hz, one of
CH2‐2), 2.25 (dd, 1H, J = 17.5, 3.0 Hz, one of CH2‐7), 2.78 (dd, 1H, J = 17.5, 7.8 Hz, one of CH2‐7), 2.84
13
(m, 1H, H‐6), 4.36 (q, 1H, J = 7.0 Hz, H‐1’); C‐NMR (600 MHz, CDCl3) δ: 20.2 (C‐3), 21.02 (C‐4), 22.8

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−1
(C‐2’), 28.0 (C‐2), 28.7 (C‐5), 33.0 (C‐1’), 36.6 (C‐7), 37.8 (C‐6), 87.8 (C‐1), 175.9 (C‐8); IR (KBr, cm ):
+
1774, 1173, 934; HRMS: (ESI‐TOF) m/z [M + Na] calcd for C10H15IO2Na 317.0015; found 317.0018.
(+)‐(1R,6R,1’S)‐1‐(1’‐Iodoethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one (6b), Obtained from acid 5b
(0.25 g, 1.5 mmol), yield 0.3 g (64%); Rt = 190 min, 2D5 = +58 (c = 0.45, CHCl3, ee > 99%); spectroscopic
data identical to the values reported for (−)‐(1S,6S,1’R)‐6a
2.3.7. 1‐(1’‐Bromoethyl)‐9‐Oxabicyclo[4.3.0]nonan‐8‐one (7a and 7b)
General Procedure
To the unsaturated acid 5 dissolved in tetrahydrofuran, the solution of N‐bromosuccinimide (1
g, 5.6 mmol) in tetrahydrofuran and a 0.1 mL of acetic acid were added. The reaction was stirred for
two days at room temperature. Then, Et2O was added to the reaction and the mixture was washed
with sodium bicarbonate and NaCl. The organic layer was dried over anhydrous MgSO4 and
evaporated. The reaction product was purified on silica gel (hexane:acetone, 20:1). New
1‐(1‐bromoethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one was obtained as the product. The physical and
spectral data of synthesized bromolactone 7 are as follows:
(−)‐(1S,6S,1’R)‐1‐(1’‐Bromoethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one (7a), Obtained from acid 5a
(0.5 g, 3 mmol), yield 0.5 g (67%); Rt = 186 min, 2D5 = −45 (c = 0.65, CHCl3, ee > 99%); mp 44–48 °C;
1
H‐NMR (600 MHz, CDCl3) δ: 1.26 (m, 1H, one of CH2‐5), 1.34 (m, 1H, J = 13.9, 10.2, 3.6 Hz, one of
CH2‐4), 1.46 (dtt, 1H, J = 13.3, 11.2, 4.1 Hz, one of CH2‐3), 1.60 (m, 1H, one of CH2‐4), 1.66 (m, 1H, one
of CH2‐3), 1.72 (d, 3H, J = 6.8 Hz, CH3‐2’), 1.83 (m, 1H, one of CH2‐5), 1.95 (dt, 1H, J = 14.7, 4.5 Hz, one
of CH2‐2), 2.05 (ddd, 1H, J = 14.7, 11.6, 4.9 Hz, one of CH2‐2), 2.25 (dd, 1H, J = 14.1, 2.5 Hz, one of
CH2‐7), 2.79 (dd, 1H, J = 14.1, 7.2 Hz, one of CH2‐7), 2.81 (m, 1H,H‐6), 4.19 (q, 1H, J = 6.8 Hz, H‐1’);
13
CNMR (600 MHz, CDCl3) δ: 20.0 (C‐2’), 20.1 (C‐3), 21.1 (C‐4), 26.4 (C‐2), 28.7 (C‐5), 36.3 (C‐6), 36.4
−1
(C‐7), 52.6 (C‐1’), 87.9 (C‐1), 176.1 (C‐8); IR (KBr, cm ): 2936, 1780, 1170, 974, 938, 641; HRMS:
(ESI‐TOF) m/z [M + H] calcd for C10H15BrO2 247.0334; found 247.0335.
+
(+)‐(1R,6R,1’S)‐1‐(1’‐Bromoethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one (7b), Obtained from acid 5b
(0.25 g, 1.5 mmol), yield 0.2 g (60%); Rt = 182 min, 2D5 = + 44 (c = 0.7, CHCl3, ee = 99%); spectroscopic
data identical to the values reported for (−)‐(1S,6S,1’R)‐7a
2.3.8. 1‐(1’‐Chloroethyl)‐9‐Oxabicyclo[4.3.0]nonan‐8‐one (8a and 8b)
General Procedure
To the unsaturated acid 5 dissolved in tetrahydrofuran, the N‐Chlorosuccinimide (0.78 g, 5.8
mmol) and 0,1 mL of acetic acid were added. The reaction was stirred for two days at room
temperature. Afterwards, diethyl ether was added to the reaction and the mixture was washed with
sodium bicarbonate and NaCl. The ether fraction was dried over anhydrous MgSO4 and evaporated.
New 1‐(1‐chloroethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one was obtained as the product. The physical
and spectral data of synthesized chlorolactone 8 are as follows:
(−)‐(1S,6S,1’R)‐1‐(1’‐Chloroethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one (8a), Obtained from acid 5a
(0.5 g, 3 mmol), yield 0.4 g (65%); Rt = 162 min, 2D5 = −41 (c = 0.4, CHCl3, ee = 99%); mp 37–40 °C;
1
H‐NMR (600 MHz, CDCl3) δ: 1.27 (m, 1H, one of CH2‐5), 1.34 (m, 1H, one of CH2‐4), 1.46 (m, 1H, one
of CH2‐3), 1.54 (d, 3H, J = 6.7 Hz, CH3‐2’), 1.60 (m, 1H, one of CH2‐4), 1.66 (m, 1H, one of CH2‐3), 1.84
(m, 1H, one of CH2‐5), 1.90 (dt, 1H, J = 14.8, 4.4 Hz, one of CH2‐2), 1.97 (ddd, 1H, J = 14.8, 11.4, 4.9 Hz,
one of CH2‐2), 2.25 (dd, 1H, J = 17.2, 2.7 Hz, one of CH2‐7), 2.74 (m, 1H, H‐6), 2.79 (dd, 1H,J = 17.2, 7.8
13
Hz, one of CH2‐7), 4.07 (q, 1H, J = 6.7 Hz, H‐1’); C‐NMR (600 MHz, CDCl3) δ: 19.3 (C‐2’), 20.0 (C‐3),
21.0 (C‐4), 25.7 (C‐2), 28.6 (C‐5), 35.5 (C‐6), 36.3 (C‐7), 59.9 (C‐1’), 88.0 (C‐1), 176.1 (C‐8); IR (KBr,
−1
+
cm ): 1783, 1172, 976, 942; HRMS: (ESI‐TOF) m/z [M + Na] calcd for C10H15ClO2Na 225.0658; found
225.0661.

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(+)‐(1R,6R,1’S)‐1‐(1’‐Chloroethyl)‐9‐oxabicyclo[4.3.0]nonan‐8‐one (8b), Obtained from acid 5b
(0.25 g, 1.5 mmol), yield 0.2 g (63%); Rt = 159 min, 2D5 = +41 (c = 0.45, CHCl3, ee > 99%); spectroscopic
data identical to the values reported for (−)‐(1S,6S,1’R)‐8a
2.4. Antimicrobial Activity of Lactones
The antimicrobial activity of racemic halolactones were tested towards three bacterial strains
classified as Gram‐negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853,
Proteus mirabilis ATCC 6380) and Gram‐positive (Bacillus cereus ATCC 10876) and two fungal strains
(Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404). The microorganisms were
supplied by Pasteur Institute of Algiers (Algeria).
The tests were carried out using the disc diffusion method. Briefly, overnight microbial cultures
were adjusted to 0.5 MacFarland turbidity standards and spread in Petri dishes containing
Mueller–Hinton and Sabouraud media for bacteria and fungi, respectively. The paper discs (6 mm
diameter) was sterilized and impregnated with lactone dissolved in DMSO. Then, the discs were
placed on the inoculated agar. The negative controls were discs immersed in pure DMSO. The plates
were incubated for 24 h at 37 °C (bacteria) and for 48 h at 25 °C (yeast) and 5–7 days for fungi. Then,
the inhibition zone diameters were determined in millimeters (including disc diameter of 6 mm).
The test was carried out in triplicates.
2.5. Antiproliferative Activity
The GL‐1 (canine B‐cell leukemia) was obtained from Yasuhito Fujino and Hajime Tsujimoto
from the Department of Veterinary Internal Medicine University of Tokyo [27], the CLB70 (canine
B‐cell chronic leukemia) was described earlier by Pawlak et al. [28]. The GL‐1 cells were maintained
in RPMI 1640 medium with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 U/mL
penicillin and 2 mM L‐glutamine (Sigma‐Aldrich, Stainheim, Germany). The CLB70 cell line was
maintained in Advanced RPMI (Gibco) medium with 100 μg/mL streptomycin, 100 U/mL penicillin
and 20% heat‐inactivated FBS. Cells were cultured at 37 °C in the incubator with 95% humidified
atmosphere and 5% CO2. The antiproliferative activity was tested according to the procedure
described previously [17]. The tested lactones were prepared within a concentration range of 6.25–50
μg/mL in the culture medium (DMSO concentration was less than 1% in each dilution). The activity
curves used to calculate IC50 are presented in the Supplementary information (Figures S7–S18). The
optical density of formed formazan in untreated control cells was assigned as 100%. Viability of test
samples was calculated as: % Viability = (average OD for test group/average OD for control group) ×
100. The results were determined from more than 3 independent experiments (four wells each) and
presented as mean IC50 value ± SD
2.6. Hemolytic Activity
The hemolytic activity test was performed using pig red blood cells (RBCs) and their
membranes (RBCMs). The method has been described previously by Pruchnik et al. [29]. The
hemolytic activity of the lactones was tested as DMSO solutions (10, 20, 40, 60, 80, and 100 μM
concentrations). The control contained only DMSO in the same volume as the tested samples. The
final concentration of erythrocytes in the sample was 1.2% hematocrit. The measurement was
recorded at 540 nm using a UV–Vis spectrophotometer (Specord 40, Analytik Jena, Jena, Germany).
The percentage of hemolysis caused by the compounds was calculated based on the hemoglobin
concentration released from red blood cells, according the following formula:
A
s
H %

100%


A
100%
where As was absorbance of hemoglobin released to supernatant in probes, A100% was defined as
absorbance of hemoglobin released to supernatant in probes where RBCs were lysed in distilled
water in 100%.

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2.7. Fluorescence Spectroscopy
In the fluorometric method, the impact of six lactones (6a,b, 7a,b, 8a and 8b) on red blood cell
membranes (RBCMs) was investigated, especially, fluidity, hydration and packing arrangement of
lipids. This analysis has been described precisely by Pruchnik et al. [29]. The RBCMs were isolated
following the method presented earlier by Dodge et al. [30]. For fluorometric analysis, three
fluorescent
probes
were
applied:
MC540
(Merocyanine
540),
Laurdan
(6‐dodecanoyl‐2‐dimethylaminonaphthalene) and DPH (1,6‐diphenyl‐1,3,5‐hexatriene) at the
concentration in the samples of 1 μM. The excitation and emission wavelengths were as follows: for
DPH, λexc = 360 nm and λem = 425 nm; for Laurdan, λexc = 360 nm and the emission wavelengths were
λem = 440 nm and λem = 490 nm; and for MC540, λexc = 540 nm and λem = 585 nm.
The compounds were tested in the same concentrations as in the hemolytic activity test (10, 20,
40, 60, 80 and 100 μM). The control contained DMSO of the same volume as the tested samples.
Measurements were carried out after 1 h of incubation at 37 °C, on a VARIAN CARY Eclipse
fluorimeter, and performed in three independent experiments. The Statistica 13.0 was used for all
statistical calculations. The results were analyzed by one‐way ANOVA followed by Duncan test.
Data are shown as mean values ± standard deviation (SD). p values ˂ 0.05 were considered
statistically significant.
2.8. Fourier Transform Infrared Spectroscopy Method (FTIR)
The molecular interactions of the tested lactones with specific functional groups of lipid were
investigated using the FTIR method. Lipid membranes have been used from RBCMs obtained from
erythrocytes to serve as a model. The samples contained RBCMs and DMSO solution of lactones at
final concentration 100 μM. The control samples contained the same amount of RBCMs as the tested
samples and the addition of DMSO instead of lactones. After mixing, each sample was separately
applied at the ZnSn plate. The spectrum of samples (with water) were taken at 37 °C using a Thermo
Nicolet 6700 MCT (Thermo Fisher Scientific, Waltham, MA, USA). To remove the water, the plate
was incubated for 24 h at 37 °C. After drying the samples, measurements were repeated. In the
spectrum of the tested samples, we considered the RBCMs’ characteristic bands.
3. Results and Discussion
3.1. Synthesis of Halolactones
The halolactones (6–8) were obtained in five steps of chemical synthesis (Schemes 1–3) and
together with all intermediates (2–5) their structures were established on the basis of spectroscopic
1
13
data (IR, H NMR, C NMR, COSY, HMQC, HMBC). Starting from 1‐acetylcyclohexene (1), the
standard reduction with sodium borohydride in methanol was performed to obtain the racemic
allylic alcohols (2). The next step was kinetic resolution of alcohol 2. The reaction was performed in
diisopropyl ether, catalysed by lipase CAL‐B from Candida antarctica; vinyl propionate was used as a
acyl donor. As a result, the mixture of (S)‐alkohol (2a) and (R)‐enantiomer of propionate (3) was
afforded. The configuration of stereogenic centres was assigned on the basis of specific optical
rotation data presented in the literature [21,22]. The obtained results are in accordance with our
previous findings for 4‐arylbut‐3‐en‐2‐ols and the Kazlauskas’ rule, which assume the
enantiopreference of most lipases for transersryfication of (R)‐enantiomer of alcohols [31,32]. A
prerequisite is a significant difference in size of substituent at the stereogenic centre and the
following sequence of substituents: OH > large substituent > medium substituent. After kinetic
resolution, the products were separated on column chromatography and the (R)‐enantiomer of
propionate was hydrolysed in basic conditions to (R)‐alkohol (2b).

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Scheme 1. Synthesis of enantiomeric 1‐(cyclohex‐1‐en‐1‐yl) ethanol (2a and 2b).
Two enantiomers of alcohol 2 were subjected to Johnson–Claisen rearrangement which
provides transfer of chirality to the corresponding esters as shown on Scheme 2 for rearrangement of
(S)‐alkohol 2a. The careful analysis of the mechanism of this rearrangement allowed as to study the
configurations of stereogenic centers in esters (4a and 4b). One can see that the 1,3‐diaxial interaction
in chairlike transition state disfavor the formation of conformer B. Favorable formation of the
conformer A leads to ester 4a with S‐configuration on the new stereogenic center. This is in
accordance with previous findings that for the alcohols with the E‐configuration of double bond the
retention of configuration is observed [33,34]. Therefore, from (E,S)‐alcohol 2a the (E,S)‐ester 4a was
formed and from (E,R)‐alcohol 2b the (E,R)‐ester 4b was obtained. Afterwards, the esters were
hydrolyzed in ethanolic NaOH solutions. The alkaline hydrolysis does not affect configuration of
stereogenic centers and hence, the (S)‐acid 5a and (R)‐acid 5b were obtained, respectively, from ester
4a and 4b.
Scheme 2. The mechanism of Johnson–Claisen rearrangement of (S)‐1‐(cyclohex‐1‐en‐1‐yl) ethanol
2a.
The incorporation of different halogen atoms was possible with the application of different
halolactonization strategies. Starting from enantiomers of unsaturated acids (5a and 5b), iodo‐
bromo‐ and chlorolactonisation were performed. For the iodolactonization reaction, the iodine and
potassium iodide in the biphasic Et2O/NaHCO3 system were used. For bromo‐ and
chlorolactonisation, the process was carried out in tetrahydrofuran with corresponding
N‐bromosuccinimide or N‐Chlorosuccinimide, respectively, and in the presence of a catalytic
amount of acetic acid. In each halolactonization reaction the corresponding δ‐halo‐‐lactone was
obtained as the only product (Scheme 3).

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Scheme 3. Synthesis of enantiomeric pairs of halolactones 6, 7, 8 from (S)‐1‐(cyclohex‐1‐en‐1‐yl)
ethanol 2a and (R)‐1‐(cyclohex‐1‐en‐1‐yl)ethanol 2b.
The mechanisms of different halolactonisations reactions involves the formation of positively
charged halonium cations, as well as the activation of the carboxyl group to a carboxylate anion. The
nucleophilic attack of an oxygen atom from carboxylate ion is highly stereospecific and goes from
opposite sides of halonium ion. The steric consequences are the antiperiplanar orientations of C‐X
and C‐O bonds [17,19,35,36] that determine configurations of two new stereogenic centers at C‐1 and
C‐1’ in the formed halolactones. The rigid structure of the cyclohexane ring as well as the presented
lactonisation mechanisms allowed us to assign (1S,6S,1’R) configuration for halolactones 6a, 7a and
8a obtained from (S)‐acid 5a (Scheme 4). Analogically, the lactonisation of (R)‐enantiomer of acid 5b
results in the formation of opposite enantiomers 6b, 7b, 8b with R‐configuration on C‐1 and C‐6 and
S‐configuration on C‐1’ carbon atom.
O
O
O
8
OH
O
(S)
(S)
halolactonisation
X = I or Br or Cl
(S)7
O-
1
5
(S)
H
6
3
(E)
H
+
X
1'
H3C
4
2'
2
(R)
CH3
X
Scheme 4. The mechanism of halolactonisation of (S)‐acid 5a and the resulting configuration of
stereogenic centers in halolactones 6a, 7a and 8a.
The IR spectra of iodolactones (6a and 6b) showed the presence of absorption bands at 1774
−1
1
cm characteristic for a ‐lactone ring. In the H‐NMR spectrum quartet (J = 7.0 Hz), the H‐1’ proton
is present at 4.36 ppm. The same coupling constant can be found in the doublet from three protons at
1.94 ppm. The shift of those signals towards the lower field is caused by the deshielding effect of a
iodine atom. The protons of the methylene group CH2‐7 (2.25 and 2.78 ppm) are shown as pair of
doublets of doublets coupled with a large geminal coupling constant (J = 17.5 Hz). The signals
13
present on C NMR spectrum also confirm the formation of ‐lactone witch iodoethyl substituent
(6a and 6b). The signal from the carbon atom C‐1’ is present at 33.0 ppm and from C‐1 at 87.8 ppm.
1
The COSY, HSQC and HMBC correlations were useful tool for assignment the signals on H‐NMR
spectrum to the corresponding methylene protons in cyclohexane ring. Table 1 present the sequence

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of correlation observed on HMBC spectrum. The crucial ones were coupling between C‐1’ and CH2‐2
and between C‐6 and CH2‐4 and CH2‐5.
Table 1. Heteronuclear multiple‐bond correlation—spectroscopy data of iodolactone 6.
CH2‐2 CH2‐3 CH2‐4 CH2‐5 H‐6 CH2‐7 H‐1’ CH3‐2’
C‐1
C‐2
C‐3
C‐4
C‐5
C‐6
C‐7
C‐8
C‐1’
C‐2’
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
1
The H NMR spectra of bromo‐ and chlorolactones are almost identical compared to
iodolactone in terms of the signal shape and chemical shift value. The main differences relate to
13
C‐NMR, where the complex effect on chemical shifts caused by halogen atoms can be observed.
13
The correlation between halide electronegativity and C shift for the α‐carbons is clearly seen on the
spectra of three lactones. Usually, the effects of halogen atoms directly bonded to carbon follow the
electronegativities of the elements in series chlorine to bromine. The iodine atom is an exception due
to the “heavy atom effect” related to increased diamagnetic shielding caused by the large number of
electrons derived from iodine. This tendency can be seen for C‐1’ carbon atom of iodolactone 6a,b
shifted to upper field (33.0 ppm) in comparison to bromo‐ and chlorolactone (52.6 and 59.9 ppm
respectively). The bond polarization due to the electronegativity of halogens should propagate along
the carbon chain but there is an opposite correlation between the substituent electronegativity and
chemical shifts of C‐2’ carbon. As the inductive effects play a predominant role, the β‐carbons are
slightly shifted downfield on all halolactones spectra [37].
3.2. The Biological Activity
The screening of antimicrobial properties of synthesized halolactones was performed on four
bacterial strains, a fungal strain and a yeast strain. The diffusion test aimed at pre‐screening
antimicrobial properties and was performed on racemic compounds 6, 7, 8. If the compounds
showed high activity; we would plan to determine it also for enantiomers. However, the activity
towards bacterial strains was not significant, therefore, further in‐depth study seemed to be
unfounded. The tested halolactones was not active against yeast and fungal strains Candida albicans
ATCC 10231 and Aspergillus brasiliensis ATCC 16404 as well as the Gram‐negative bacteria
Pseudomonas aeruginosa ATCC 27853. The tested iodo‐ and bromolactones (rac‐6 and rac‐7) exhibited
rather moderate antibacterial activity towards Gram‐positive bacteria Bacillus cereus ATCC 10876
(inhibition zone 12 ± 0.00 and 8 ± 0.00 mm respectively) and Gram‐negative Escherichia coli ATCC
25922 (inhibition zone 7 ± 0.00 and 9 ± 0.00 mm respectively). The most sensitive strain was
Gram‐negative Proteus mirabilis ATCC 35659, the diameter of inhibition zone was 11 ± 0.00 mm for
iodolactone 6, 12 ± 0.35 mm for bromolactone 7 and 12 ± 0.00 mm for chlorolactone 8. A similar
tendency, have also been noticed by Grabarczyk and coworkers [38–40]. In previous research,
‐lactones with similar carbon skeleton exhibited higher selectivity towards bacterial strains than
towards yeast or fungal strains. In general, the most responsive were the Gram‐positive bacteria like
B. cereus and Gram‐negative E. coli.
The antyproliferative activity of lactone enantiomers was tested on two canine cell lines CLB70
and GL‐1 (Table 2). The choice of these specific cell lines was due to the possibility of comparing the
results to previous experiments [18–20]. All tested substances exhibited significant activity. The GL‐1
cell line was more sensitive to all tested compounds including the control etoposide. For the CLB70
cell line, the small differences in activity can be observed between the enantiomer activities,
especially comparing the more potent (−)‐enantiomer of bromolactone (7a) to less active

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(+)‐enantiomer 7b. Overall, there were no significant differences in lactone activity regardless of
halogen type and configuration of stereogenic centers. It is worth to point that the activity of tested
compounds was comparable with the control and was much higher than antiproliferative activity of
bicyclic lactones with the cyclohexane system reported earlier [20,41].
Table 2. The antiproliferative activity of halolactones 6a,b, 7a,b, 8a and 8b and control—Etoposide
against the selected cancer cell lines expressed as IC50.
Compound (IC50 μg/mL)
Cell
Line
6a
7a
8a
6b
7b
8b
Etoposide
14.31 ± 2.83
4.4 ± 1.14
CLB70
GL‐1
19.59 ± 0.51
13.76 ± 1.08
19.40 ± 0.35
15.55 ± 0.79
23.93 ± 5.14
17.17 ± 3.62
19.93 ± 0.07
14.53 ± 2.38
28.57 ± 4.89
14.95 ± 1.93
18.43 ± 1.46
11.40 ± 0.40
The cytotoxicity of compounds was evaluated on the basis of hemolytic test. The results
presented as the percentage of hemolysis are summarized on Figure 1. The percentage of hemolysis
registered for all lactones was not significantly different from the percentage of hemolysis in control
samples. Even at much higher concentrations (1 mM), the compounds showed no hemolytic activity
(Table S1). Therefore, the compounds did not induce an increase in hemolysis in tested
concentrations. According to toxicity classification, a compound is nontoxic if the hemolysis does
not exceed 9% [42]. In this regard all tested compounds did not have a toxic effect on red blood cells
(RBCs). These results are consistent with the results obtained for bromolactones with a
2,5‐dimethylfenyl substituent [19].
Figure 1. Hemolysis of RBCs dependent on concentration of compounds.
3.3. Biophysical Research
The interactions of lactones with red blood cell membranes (RBCMs) were studied using
fluorescence spectroscopy and Fourier transform infrared spectroscopy (FTIR) methods. The
fluorescence spectroscopy allowed us to measure effects of tested compounds on physicochemical
properties of RBCMs, especially fluidity, hydration and packing arrangement of lipids in
membranes. Three fluorescent probes (DPH, Laurdan and MC540) were used in those experiments.
Each of these probes is located in a different area of the membrane. The DPH probes are located in
hydrophobic region of the membrane. Laurdan is incorporated at the level of the phospholipid
glycerol backbone (hydrophilic‐hydrophobic regions) and MC540 is attached above the glycerol
skeleton.
The DPH results are presented as the changes in fluorescence anisotropy (A) and provide
information about fluidity of the hydrocarbon chains in the phospholipid bilayers (Figure 2). The

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decrease in the value of anisotropy determines increased fluidity of the membrane and the increased
anisotropy indicates increased organisation of hydrocarbon chains and, hence, the rigidity of the
membrane. The results showed that the presence of compounds does not change the value of
anisotropy. This suggests that all bicyclic δ‐halo‐γ‐lactones did not have an impact on fluidity in the
hydrophobic region of the lipid bilayers.
Figure 2. Values of anisotropy (A) of DPH probe for RBCMs (control) and RBCMs with addition of
compounds 6a,b, 7a,b, 8a,b at 37 °C. Values are mean ± SEM. Values are not significantly (p ˂ 0.05)
different from control.
Laurdan locates in the hydrophilic–hydrophobic interface of the bilayer. This fluorescent probe
is sensitive to polarity modifications and reflects changes in hydration and packing order in the head
groups of lipids in RBCMs [43,44]. The interactions of lactones with membranes were determined on
the basis of generalized polarization (GP). The results indicate small changes in GP values for all
bicyclic δ‐halo‐γ‐lactones in the highest concentrations (80 and 100 μM). For two enantiomeric pairs
(6a and 6b, 8a and 8b), a slight increase of GP values can be observed (Figure 3). In both cases, the
higher values were registered for (+)‐enantiomers (6b and 8b). It was probably indicated by an
increase in the organization in hydrophobic–hydrophilic regions of the phospholipid bilayers which
was associated with a decrease in water content in the area of glycerol backbone. Interestingly, for 7a
and 7b, the effect was the opposite. The results have shown a small decrease in GP values at 80 and
100 μM concentrations. It indicated that more water molecules were incorporated at the level of the
glycerol backbone, consequently, decreasing the order in the polar group of lipids. These results
suggest that bromolactones 7a and 7b are probably located slightly deeper in the hydrophilic area
than iodo‐ and chlorolactones.

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Figure 3. Values of generalized polarization (GP) of Laurdan probe for the RBCMs (control) and
RBCMs with addition of compounds 6a,b, 7a,b, 8a,b at 37 °C. Values are mean ± SEM. Means labeled
with asterisk (*) are significantly (p ˂ 0.05) different from control.
The MC540 probe is located slightly higher in the membrane than Laurdan. This probe is very
sensitive to lipid packing in the membrane and aqueous environments [45–47]. The fluorescence
intensity of MC540 strongly increases in the liquid phase of lipid bilayers and decreases in the gel
phase. In the presented experiments, the maximum of fluorescence intensity (585 nm) was recorded
for each sample and compared to the control (Figure 4). The results have shown a small decrease in
fluorescence intensity registered in experiments with all bicyclic δ‐halo‐γ‐lactones. The largest
decrease was observed for the chlorolactone 8b. The results suggest that in the presence of 8b, the
organization of the lipids in bilayers probably increases.
Figure 4. Changes of intensity of MC540 probe measured at 585 nm for RBCMs (control) and RBCMs
with addition of tested compounds. The measurement was carried out at 37 °C. Values are mean ±
SEM. Means labeled with asterisk (*) are significantly (p ˂ 0.05) different from control.
Generally, fluorescence spectroscopy results have shown that the compounds are weakly
incorporated into the membrane. Only weak changes in the hydrophilic area were observed.
Spectroscopic studies in the near infrared band allowed to register absorption spectra for
isolated red blood cell membranes (RBCMs). In the IR spectra, the characteristic frequency bands of
the protein and lipid components were analyzed (Figure 5). We identified the distinguished
erythrocyte frequency bands for individual membrane components: methyl groups and methylene

Biomolecules 2020, 10, 95
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−1
−1
hydrocarbon chains (2980–2830 cm ), carbonyl groups of lipids (1760–1710 cm ), the amide band I
−1
(80% C=O stretch, near 1650 cm ), amide band II (60% N‐H bend and 40% C‐N stretch, near 1550
cm ), amide band III (40% C‐N stretch, 30% N‐H bend, near 1300 cm ), phosphate band (1280–1020
cm ) and choline band (1000–940 cm ) [48,49].
−1
−1
−1
−1
Figure 5. FT‐IR spectra of erythrocyte membrane RBCMs (black lines) and of RBCMs modified with
lactones (blue lines—6a and 6b, green lines—7a and 7b, red lines—8a and 8b); (a) phosphate band I
(asymmetric vibration), (b) phosphate band II (symmetric vibration), (c) carbonyl band.
Figure 5 shows the infrared spectra of RBCMs and RBCMs in the presence of 100 μM of 6a, 6b,
7a, 7b, 8a and 8b compounds. Analysis of the IR spectra of RBCMs treated with six lactones showed
no significant changes in the hydrophobic part of the membrane because none of the tested
compounds change the frequency of signal for hydrocarbon chains (Figure S19). There were also no
changes in the vibrational bands of choline groups of lipids, but they appeared within the bands of
carbonyl and phosphate groups (Table S2). The carbonyl and phosphate groups form hydrogen
bonds with water. Therefore, changes observed in these bands testify to changes in the degree of
hydration of the carbonyl and phosphate groups [50,51]. A slight frequency shifts of the ester groups
we observed the presence of lactones, in particular for 7b, 8a and 8b, which suggests that these
compounds change hydration in the area of lipid carbonyl groups (Figure 5c). Similar changes were
observed in the polar regions of the lipid bilayers. The interaction between lactones and the head
group of RBCMs was monitored by analyzing the symmetric and asymmetric phosphate bands. An
increase in the wave number of the maximum of this band is indicative of the location of the lipid
phosphate polar head group in less polar environments and a decrease in head group hydration (or
hydrogen bonding) [49,52,53]. The tested compounds shifted the maximum of the phosphate band
−
very slightly towards lower values of wave numbers, especially 7b, 8a and 8b for νs(PO2 ) (Figure 5b)
−
and 6a, 7b, 8a and 8b for νas(PO2 ) (Figure 5a). Additionally, in the presence of lactones the band of
the phosphate group is slightly broader than for pure RBCMs, indicating that there might be an

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interaction between the investigated compound and the lipid [29]. In addition, preliminary analysis
of the amide bands indicates that the tested compounds slightly affect the structure of proteins
present in the membrane, which is the subject of our next work.
The results obtained from IR tests confirm the changes observed in the fluorimetric method.
The tested compounds do not change the fluidity of RBCMs in the hydrophobic region of the lipid
bilayer; they slightly affect the degree of lipid packing and the associated hydration of the RBCMs’
polar membrane. Interestingly, there are slight differences in the impact on these parameters
depending on the type of halogen in the lactone structure. Larger changes in RBCMs are observed
for chloro‐ and bromolactones, probably these compounds interact more strongly with the polar part
of the lipid bilayer. However, we do not observe significant changes within the enantiomeric pairs of
bicyclic δ‐halo‐γ‐lactones.
4. Conclusions
The chemoenzymatic synthesis was applied to obtain new biologically active δ‐halo‐γ‐lactones.
The lipase CAL‐B‐catalyzed kinetic resolution was the crucial step leading to separation of
1‐(cyclohex‐1‐en‐1‐yl) ethanol enantiomers and consequently allowed us to obtain opposite
stereoisomers of lactones. Analyzing the mechanism of Johnson–Claisen rearrangement and the
halolactonization, we were able to predict the configurations of stereogenic centers in the obtained
compounds. As a result, pairs of enantiomerically pure (ee ≥ 99%) halolactones containing iodine,
bromine or chlorine atom were synthesized. All tested compounds exhibited significant
antiproliferative activity, being slightly more active against acute leukemia (GL‐1) than a chronic
leukemia (CLB70) cell line. Importantly, we did not observed much influence of halogens and
configuration of chiral centers on this activity. The studies on interactions of lactones with red blood
cell membranes revealed that tested compounds cause weak changes in the hydrophilic area of
bilayer whereas no influence on fluidity in the hydrophobic region was observed. The presented
results demonstrated that the tested lactones have a tendency for weak binding into the membrane
but there is no evidence on increasing membrane permeability. Increased permeability may lead to
cell lysis or, alternatively, may provide small changes in the lipid bilayer function that entail cell
death. Taking into consideration that halolactones were non‐toxic to erythrocytes in the hemolysis
test, more in‐depth studies are required to explain the mechanism of their promising
antiproliferative activity.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: 1H‐NMR of
1
lactone 6, Figure S2: 13C‐NMR of lactone 6, Figure S3: H‐NMR of lactone 7, Figure S4: 13C‐NMR of lactone 7,
1
Figure S5: H‐NMR of lactone 8, Figure S6: 13C‐NMR of lactone 8, Figure S7: Dose‐response curve used to
calculate IC50 for lactone 6a and CBL70 cell line, Figure S8: Dose‐response curve used to calculate IC50 for lactone
6a and GL‐1 cell line, Figure S9: Dose‐response curve used to calculate IC50 for lactone 7a and CBL70 cell line,
Figure S10: Dose‐response curve used to calculate IC50 for lactone 7a and GL‐1 cell line, Figure S11:
Dose‐response curve used to calculate IC50 for lactone 8a and CBL70 cell line, Figure S12 Dose‐response curve
used to calculate IC50 for lactone 8a and GL‐1 cell line, Figure S13: Dose‐response curve used to calculate IC50 for
lactone 6b and CBL70 cell line, Figure S14: Dose‐response curve used to calculate IC50 for lactone 6b and GL‐1
cell line, Figure S15: Dose‐response curve used to calculate IC50 for lactone 7b and CBL70 cell line, Figure S16:
Dose‐response curve used to calculate IC50 for lactone 7b and GL‐1 cell line, Figure S17: Dose‐response curve
used to calculate IC50 for lactone 8b and CBL70 cell line, Figure S18: Dose‐response curve used to calculate IC50
for lactone 8b and GL‐1 cell line, Figure S19: FT‐IR spectra of erythrocyte membrane RBCMs (black lines) and of
RBCMs modified with lactones (blue lines—6a and 6b, green lines—7a and 7b, red lines—8a and 8b) for
symmetric and asymmetric CH2 stretching band, Table S1: Hemolysis of RBCs dependent on concentration of
compounds, Table S2: Selected bands (wavenumbers cm−1) of IR spectra of RBCM and RBCM+compounds (100
μM).
Author Contributions: Conceptualization, M.M.; Methodology M.M., A.W., B.F. and A.P.; Investigation, M.M.,
A.W., B.F., H.P., A.P. and G.M.; Project Administration, M.M.; Validation, B.O.‐M. and W.G.; Writing‐original
draft, M.M. and A.W.; Writing—Review & Editing, M.M., A.W., H.P. and W.G. All authors have read and
agreed to the published version of the manuscript.

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Funding: This research was funded by the statutory activities of the Department of Chemistry, Wrocław
University of Environmental and Life Sciences.
Conflicts of Interest: The authors declare no conflict of interest.
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