Effect of citral and citral related compounds on viability of pancreatic and human B-lymphoma cell lines

Article abstract of DOI:10.1007/s00044-017-1779-z

Citral, 3,7-dimethyl-2,6-octadienal, is a key component of the essential oils extracted from several lemon-scented herbal plants. It has been demonstrated to be an effective antifungal agent and in recent years, to have antiproliferative effect in certain tumor cell lines: thus citral could be a potential anticancer drug. In this study, we investigated the effect of citral on the proliferation of pancreatic cell tumor lines (MIA PaCa-2 cells) and human B-lymphoma (DeFew cells). To analyze which part of the molecule is pivotal for the activity, we also studied the effect of several citral related compounds, in particular focusing on some chemical aspects of the terpenic scaffold. The in vitro studies revealed the significant cytotoxic activity of some of the tested compounds.

Full text of DOI:10.1007/s00044-017-1779-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1779-z
ORIGINAL RESEARCH
Effect of citral and citral related compounds on viability of
pancreatic and human B-lymphoma cell lines
Antonia Di Mola1 Antonio Massa2 Vincenzo De Feo1 Anna Basile3
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Maria Pascale1 Rita Patrizia Aquino1 Paolo De Caprariis1
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Received: 16 July 2016 / Accepted: 1 January 2017
© Springer Science+Business Media New York 2017
Abstract Citral, 3,7-dimethyl-2,6-octadienal, is a key
component of the essential oils extracted from several
lemon-scented herbal plants. It has been demonstrated to be
an effective antifungal agent and in recent years, to have
antiproliferative effect in certain tumor cell lines: thus citral
could be a potential anticancer drug. In this study, we
investigated the effect of citral on the proliferation of pan-
creatic cell tumor lines (MIA PaCa-2 cells) and human B-
lymphoma (DeFew cells). To analyze which part of the
molecule is pivotal for the activity, we also studied the
effect of several citral related compounds, in particular
focusing on some chemical aspects of the terpenic scaffold.
The in vitro studies revealed the significant cytotoxic
activity of some of the tested compounds.
Introduction
Nowadays, one of the fastest growing areas of research is
exploring natural plant products as an option to find new
chemical entities as anticancer agents (Lee 1999). Citral 1
(3,7-dimethyl-2,6-octadienal), a key component of essential
oils extracted among others, from lemongrass (Cymbopo-
gon citratus), lemon balm (Melissa officinalis), and vervain
(Verbena officinalis). Citral is a mixture of two isomeric
acyclic monoterpene aldehydes used in foods and cosmetics
(Kumar et al. 2000; Nakamura et al. 2003; Dudai et al.
2005): geranial (trans isomer) has a strong lemon odor,
neral (cis isomer) has a lemon odor that is less intense and
sweeter than geranial (Fig. 1).
The toxicity studies indicate that citral is devoid of major
toxicity and carcinogenic potential in both mice and rats
(Rabbani et al. 2005; Dieter et al. 1993); citral is also devoid
of mutagenic effect on in vitro models (Vinitketkumnuen
et al. 1994). Citral possesses many bioactivities such as
antispasmodic (Ganjewala 2009; Devi et al. 2011), anti-
inflammatory (Katsukawa et al. 2011), antipyretic, diuretic,
and sedative. It also has strong antimicrobial properties
(Onawunmi 1989) and pheromonal effects in insects
(Kuwahara et al. 1983). Citral is also used in the synthesis
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Keywords Citral Viability Mia PaCa-2 cells DeFew
cells
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1779-z) contains supplementary material,
which is available to authorized users.
* Antonia Di Mola
toniadimola@libero.it
* Paolo De Caprariis
pdecaprariis@unisa.it
CHO
1
Dipartimento di Farmacia, Università di Salerno, Via Giovanni
CHO
Paolo II, 132, Fisciano, SA 84084, Italy
2
Dipartimento di Chimica e Biologia, Università di Salerno, Via
Giovanni Paolo II, 132, Fisciano, SA 84084, Italy
3
Dipartimento di Medicina, Chirurgia e Odontoiatria “Scuola
Medica Salernitana”, Università di Salerno, Via Allende,
Baronissi, SA 84084, Italy
geranial
Fig. 1 Citral structure
neral

Med Chem Res
Fig. 2 Chemical modifications
carried out on the terpenic
scaffold
H
O
O
H
isomers
H
H
H
R
O
O
O
O
O
H
C1 oxidation state
O
O
O
citral
H
*
O
H
epoxides
*
O
double bonds reduction
2,3 double bond reduction
of vitamin A and to mask the smell of smoke (Lewinsohn
et al. 1998). Citral is a potent inducer of glutathione-s-
transferase class of enzymes, involved into protection of
healthy hepatocytes against apoptosis during chemotherapy
of liver cancers (Dudai et al. 2005). A good superoxide
scavenging activity (EC50 = 19 μg/ml) was reported in
Swiss albino mice in citral treated groups, suggesting that
the antioxidant action could be responsible for the anti-
clastogenic effect of citral against nickel chloride (Rabbani
et al. 2006). Citral also disrupts animal microtubules and
inhibits polymerization of microtubules in vitro (Chaimo-
vitsh et al. 2012) and at a concentration comparable to that
found in a cup of tea brewed with 1 g of lemongrass, citral
was found to induce apoptosis in cancer cells. Apoptosis
was accompanied by DNA fragmentation and caspase-3
catalytic induction (Rabbani et al. 2005). Citral treatment
also caused inhibition of breast cancer MCF-7 cell growth
(IC50 48 h: 180 μM) with a cycle arrest in G2/M phase and
citral related compounds, in particular focusing on four
chemical aspects of the terpenic scaffold (Fig. 2):
a) geometry of double bond in 2,3 position (as already
mentioned citral is a mixture of two isomeric acyclic
monoterpene aldehydes which are double bond
isomers)
b) oxidation state of carbon in C1 position
c) oxidation of double bonds into epoxides
d) removal of double bonds
Our initial experiments focused on the effect of citral 1
on cell growth and proliferative potential of MIA PaCa-2
cells and DeFew cell lines at a concentration range between
12.5 and 400 μM in DMSO (Dimethyl sulphoxide). Cancer
cells were exposed to the compound 1 day after seeding and
their viability was followed for 3 days using the MTT (3-
(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bro-
mide) cell proliferation assay and the results are presented
as percentage of viability. Figure 3 shows the effect of citral
on the viability of MIA PaCa-2 cells and DeFew cell lines
estimated by MTT assay, in particular the exposure to citral
at a range between 50 and 400 μM strongly inhibited the
growth of the cell line by 10–90 % indicating that the
compounds affect cell viability in a dose-dependent manner.
IC50 values were calculated to be 238 μM on MIA PaCa-2
cells and 300 μM on DeFew cells. DMSO concentration did
not affected cell viability.
apoptosis induction suggesting
a
potential chemo-
preventive effect (Chaouki et al. 2009). Pro-apoptotic
activity of citral in patients with chronic lymphocytic leu-
kemia as well as its effect on the acute promyelocytic leu-
kemia cell line NB4 were also reported (De Martino et al.
2009; Hailong et al. 2013). Citral is also capable to induce
cancer cell death by decreasing cell proliferation, altering
mitochondrial membrane potential, and initiating apoptosis
in HeLa and ME-180 cell lines (Ghosh 2013). Recently it
has been demonstrated that citral potentiated cytotoxicity of
doxorubicin by increasing apoptotic effects in human lym-
phoma Ramos cells (Dangkong and Limpanasithikul 2015).
Taking into account these published reports, the aim of
the present study was to evaluate the effect of citral 1 on
viability of malignant cells such as pancreatic cell tumor
lines (MIA PaCa-2 cells) and human B-lymphoma cell lines
(DeFew cells). To analyze which part of the molecule is
pivotal for the activity, we also studied the effect of several
Materials and methods
Chemistry
Citral, citronellol and citronellal were purchased from
Sigma Aldrich (Germany). Geraniol, nerol and geranic acid
were purchased from Alfa Aesar (Germany). Citral isomers

Med Chem Res
Fig. 3 a. Cell viability of MIA
PaCa 2-cells treated with citral.
b. Cell viability of DeFew cells
treated with citral. MIA PaCa-2
and DeFew cells were treated
with citral (12.5., 25, 50, 100,
200, 400 µM) for 72 h. Their
viability was evaluated using the
MTT assay as described in the
materials and methods Section.
The optical densities (OD 620)
of citral-treated cells were
compared with the OD of the
control wells (untreated cells).
Each percent value is the mean
SD of three independent
experiments
O
O
a
O
d
H
H
OH
Geraniol 5
Geranial 2 yield: 92%
9
yield: 85%
Nerol
6
Nerale 3
yield: 85%
10 yield: 91%
e
b
O
O
OH
O
23, 24
OH
21, 22
c
c
O
O
O
O
O
H
H
7 yield: 96%
8 yield: 94%
11 yield: 86%
12 yield: 81%
Scheme 1 Reagents and conditions: a MnO2, benzene, r.t; b tert-butyl hydroperoxide (TBHP), vanadyl acetyl acetonate, CH2Cl2, r.t; c Py-SO3,
CH2Cl2, DMSO, Et3N, r.t; d MCPBA 77% (1.1 equiv.), CH2Cl2, 0 °C; e MCPBA 77% (2.2 equiv), CH2Cl2, 0 °C
2 and 3 were prepared according to the Scheme 1, by oxi-
dation of commercially available geraniol and nerol using
manganese dioxide in benzene (Surendra and Corey 2008).
The 2,3 Citral epoxides 7,8 were synthesized by oxidation
of 2,3 double bond in the presence of tert-butyl
hydroperoxide and vanadylacetyl acetonate and following
oxidation of the alcohols 21, 22 to aldehydes with pyr-
idinium SO3 complex (Rose et al. 2006). The 6,7 Citral
epoxides 8, 9 were synthesized by oxidation of 6,7 double
bond in the presence of 3-chloroperoxybenzoic acid (77%)

Med Chem Res
O
Table 1 IC50 values of citral and cis/trans isomers
O
a
O
compounds
IC50 μM MIA IC50 μM
H
H
*
*
PaCa 2-cell
DeFew cells
(S)-Citronellal 14
(R)-Citronellal 15
(S)-16
(R)- 17
238
300
H
H
b
O
O
O
citral 1
*
H
270
280
320
250
(S)- 18
(R)- 19
Scheme 2 Reagents and conditions: a MCPBA 77%, CH2Cl2, 0 °C.; b
H2, 5% Pd/C, ethyl acetate, r.t
geranial 2
in dichloromethane (Elgendy and Khayyat 2008; Yarovaya
et al. 2002). Epoxides were recovered in very good yields.
Diepoxides 11, 12 were prepared treating geraniol and nerol
with an excess of 3-chloroperoxybenzoic acid
(MCPBA77%) in dichloromethane affording the corre-
sponding alcohols diepoxides which were converted into
aldehydes with pyridinium SO3 complex.
Citronellal derivatives were prepared as reported in
Scheme 2. (S)-citronellal 14 and (R)-citronellal 15 were
treated with 3-chloroperoxybenzoic acid (77%) in dichlor-
omethane affording compounds 16 and 17. Compound 18
and 19 were obtained after reduction of both double bonds
of citronellal using 5% Pd/C in ethyl acetate under hydro-
gen atmosphere (Mikami et al. 2011).
O
H
neral 3
investigated the anticancer activity of geraniol finding a
significant (60–90%) inhibition of the anchorage-
independent growth of human MIA PaCa-2 cells (Burke
et al. 1997). When we consider the effect on DeFew cells
survival, oxidation of citral aldehyde to geranic acid 6 acid
strongly affect viability, confirming the results reported by
Dudai for similar leukemia lines (Dudai et al. 2005).
Next, we focused on double bonds oxidation considering
selective 2,3 epoxidation, 6,7 epoxidation and diepoxida-
tion as reported in Table 3. Epoxidation of double bond in
position 2,3 increased three fold times cytotoxic activity in
both malignant tested cell lines, compared with citral.
Compound 7 was already reported as active to inhibit cell
growth in various human cancer cell lines in culture (Rose
et al. 2006). In particular, on DeFew cells, epoxide 7 was
very active if compared with epoxide 8 suggesting the
importance of trans epoxide geometry for the reduction of
cell viability in human B-lymphoma cells. When we con-
sidered epoxidation of double bond in position 6,7 (com-
pounds 9 and 10) and diepoxidation (compounds 11 and
12), a complete loss of cytotoxic effect was observed,
highlighting the necessary presence of double bond in 6,7
position for optimal activity.
Cytotoxic activity evaluation
To analyze which part of the citral molecule is pivotal for
the activity, we also considered some chemical structural
modifications of the terpenic scaffold, focused on the effect
of several citral related compounds on cell growth and
proliferative potential of same cells. IC50 values of tested
compounds are reported in Tables 1–4. As already men-
tioned, citral is a mixture of two isomeric acyclic mono-
terpene aldehydes that are double bond isomers, but few
studies have directly compared their antitumor properties
(Zeng et al. 2015). Thus first of all, we considered if the
geometry of double bond in 2,3 position could affect the
citral antiproliferactive effect. As reported in Table 1 no
substantial difference was observed for the trans/cis isomers
2, 3 in comparison with citral mixture on MIA PaCa-2 cells
proliferation and DeFew cells proliferation.
Other natural monoterpenes structurally related to citral
are citronellal and citronellol, also present in the essential
oils extracted from several lemon-scented herbal plants. It
has been demonstrated that a number of monoterpenes have
antitumoral activity that can prevent the formation or pro-
gress of cancer and cause tumor regression (Zhaung et al.
2009).
Considering C-1 oxidation state, both reduction of
aldehyde to alcohol or oxidation to carboxylic acid did not
seem to affect cytotoxic activity of citral on MIA PaCa-2
cells lines, except in the case of nerol 5 which showed
higher IC50 than citral (Table 2). IC50 value of geraniol was
found to be in according to that reported by Burke, who
Citronellal (in which 2,3 double bond is missing) showed
antiproliferactive effect similar to citral, on MIA PaCa-2

Med Chem Res
Table 2 IC50 values of citral and related C1 oxidation state
Table 3 IC50 values of citral and related epoxides
compounds
compound
IC50 μM
MIA PaCa
2-cells
IC50 μM
DeFew cells
compounds
IC50 μM
IC50 μM
MIA PaCa DeFew
2-cells
238
cells
300
238
88.7
94
300
H
H
H
O
O
O
citral 1
citral 1
45
257
392
280
323
O
OH
7
geraniol 4
89
O
O
O
O
H
OH
8
nerol 5
–
–
–
–
263
580
H
O
OH
O
O
9
geranic acid 6
O
cells, when we consider the racemic mixture 13 (Table 4),
suggesting that removal of 2,3 double bond in not crucial
for activity.
H
10
–
–
–
–
When testing the two enantiomers, a crucial role of ste-
reochemistry was observed since R-citronellal 15 was found
to be two fold times more potent than racemic citronellal
and six fold times than S-citronellal 14. Proliferation mea-
surement showed a different trend on DeFew cells, because
in this case S-citronellal was four fold times more potent
than R-citronellal and two fold times than racemic mixture.
Epoxidation of 6,7 double bond (compounds 16 and 17) or
removal of 6,7 double bond (compounds 18 and 19) led to a
complete loss of cytotoxic effect, highlighting also in this
case the necessary presence of double bond in 6,7 position
for optimal activity. Citronellol showed a good cell viability
reduction effect only on human B-lymphoma, in particular
S-citronellol 21 was almost seven fold times more active
than R-citronellol and two fold times than citronellal. Also
Zhuang reported that citronellol has anticancer and anti-
inflammatory properties (Zhuang et al. 2009). No effect was
observed on MIA PaCa-2 cell lines.
H
O
O
O
11
O
O
H
12
S-citronellol 21), in vitro studies were also carried out on
HUVEC (Human Umbilical Vein Endothelial cells) to
evaluate eventual toxicity (Table 5). As already reported in
literature, citral was found to be devoid of toxicity as well as
the other natural monoterpenes. Indeed synthetic epoxides
7–8 affect the survival of HUVEC cells with calculated IC50
values as reported in Table 5, however very high value
when compared to IC50 values on the tumor cell lines.
Taking into account these data which revealed a sig-
nificant cytotoxic activity of some of the tested compounds
(citral 1, epoxides 7–8, citronellal enantiomers 14–15, and

Med Chem Res
Table 4 IC50 values of citral , citronellal , citronellol and related
Table 5 IC50 values of most promising compounds on HUVEC cells
compounds
proliferation
compounds
IC50 μM MIA IC50 μM
compounds
IC50 μM HUVEC cells
PaCa 2-cells
DeFew cells
Citral 1
–
Citral 1
238
231
300
61
200
H
O
H
O
O
7
8
(RS)-Citronellal 13
(S)-Citronellal 14
162
668
128
31
O
H
O
O
H
129
H
–
H
O
O
(R)-Citronellal 15
(S)-Citronellal 14
(R)-Citronellal 15
–
–
–
–
–
–
H
–
–
O
H
O
(S)- 16
O
H
O
O
(R)- 17
(S)- 18
OH
(S)-Citronellol 21
H
O
–
–
Conclusions
H
Results presented in the current study showed a remark-
able effect of citral, citronellal, and citronellol on viability
of pancreatic cell tumor lines (MIA PaCa-2 cells) and
human B-lymphoma lines (DeFew cells), such that they
should be considered good candidates for chemopreven-
tion and chemotherapy. In addition a careful structure-
activity relationship study carried out on the terpenic
scaffold to analyze which part of the citral molecule is
pivotal for the cell viability reduction, highlighted
substantial no role of 2,3 double bond and crucial
importance of 6,7 double bond. In addition some citral
related compounds like 2,3 epoxides, which are very easy
to synthesize, strongly reduced cell viability in both
malignant cell lines. Thus, current studies are in course to
identify and to understand well the mechanisms (apopto-
sis or cell cycle arrest or both of these phenomena)
involved in the effect induced by the most potent
compounds.
O
(R)- 19
567
463
–
252
95
OH
OH
(RS)-Citronellol 20
(S)-Citronellol 21
(R)-Citronellol 22
679
OH

Med Chem Res
Experimental Section
0,882 mmol) in DMSO (22 mmol, 460 μl ) was added
droppwise and the reaction mixture was allowed to stir
overnight. Purification by chromatography (Petroleum
ether/ethyl acetate 9:1) gave a yellow oil.
All reactions were performed using commercially available
compounds without further purification. Column chroma-
tographic purification of products was carried out using
silica gel 60 (70–230 mesh, Merck). The NMR spectra were
recorded on Bruker DRX 400, 300, 250 spectrometers (400,
300, 250 MHz, 1H). Spectra were referenced to residual
CHCl3 (7.26 ppm, 1H, 77.23 ppm, 13C). Coupling constants
J are reported in Hz. Yields are given for isolated products
showing one spot on a TLC plate and no impurities
detectable in the NMR spectrum. Mass spectral analyses
were carried out using an electrospray spectrometer Waters
4 micro quadrupole or Agilent GC-MSD 5975C with triple
axis detector. Elemental analyses were performed with
FLASHEA 1112 series-Thermo Scientific for CHNS-O
apparatus.
(E)- 3-methyl-3-(4-methylpent-3-enyl)oxiran-2-
carbaldehyde 7
Yellow oil. Yield: 96%. ESI (m/z): 191 [M+ + 23]. Anal.
calcd for C10H16O2: C 71.39, H 9.59. Found: C 71.19, H
9.49. Spectroscopic data are in agreement with literature
(Elgendy and Khayyat 2008).
(Z)- 3-methyl-3-(4-methylpent-3-enyl)oxiran-2-
carbaldehyde 8
Yellow oil. Yield: 94%. ESI (m/z): 191 [M+ + 23]. Anal.
calcd for C10H16O2 : C 71.39, H 9.59. Found C 71.19, H
9.49. Spectroscopic data are in agreement with literature
(Leal et al. 1898).
General procedure for the synthesis of trans/cis isomers
2 and 3
To a solution of geraniol or nerol 300 mg (1.94 mmol) in
benzene (6 ml), manganese dioxide (4 equiv.) was added
and the mixture was allowed to stir for 6 h. Filtration on
celite and evaporation of the solvent gave a pale oil which
was purified by chromatography (Petroleum ether/ diethyl
ether 9:1).
General procedure for the synthesis of 6,7 epoxides 9,10
To a solution of geraniol or nerol (50 mg, 0.328 mmol ) in
CH2Cl2 (2 ml) , MCPBA 77% (92 mg, 1.76 mmol), was
added keeping the mixture at 0 °C for 1 h. The organic layer
was washed three times with NaHCO3 and brine; purifica-
tion by chromatography (Petroleum ether/diethyl ether 7:2)
gave a yellow oil.
(E)-3,7-dimethylocta-2,6-dienale (Geranial) 2
Pale oil. Yield: 92%. ESI (m/z): 153 [M + H]+. Anal. calcd
for C10H16O: C 78.90, H 10.59. Found: C 78.80, H 10.69.
Spectroscopic data are in agreement with literature (Elgendy
and Khayyat 2008; Yarovaya et al. 2002)
(E)-3-methyl-5-(3,3-dimethyloxiran-2-yl)pent-2-enal 9
Yellow oil. Yield: 85%. ESI (m/z): 169 [M + H]+ Anal.
calcd for C10H16O2: C 71.39, H 9.59. Found C 71.43, H
9.56. Spectroscopic data are in agreement with literature
(Elgendy and Khayyat 2008; Yarovaya et al. 2002).
(Z)-3,7-dimethylocta-2,6-dienale (Neral) 3
Pale oil. Yield: 85%. ESI (m/z): 153 [M + H]+. Anal. calcd
for C10H16O: C 78.90, H 10.59. Found: C 78.83, H 10.63.
Spectroscopic data are in agreement with literature (Elgendy
and Khayyat 2008; Yarovaya et al. 2002)
(Z)-3-methyl-5-(3,3-dimethyloxiran-2-yl)pent-2-enal 10
Yellow oil. Yield: 91%. ESI (m/z): 169 [M + H]+. Anal.
calcd for C10H16O2: C 71.39, H 9.59. Found C 71.44, H
9.58. Spectroscopic data are in agreement with literature
(Elgendy and Khayyat 2008; Yarovaya et al. 2002).
General procedure for the synthesis of 2,3 epoxides 7, 8
To a solution of geraniol or nerol (300 mg, 1.94 mmol) in
CH2Cl2 (10 ml) keeping the mixture at 0 °C, TBHP (5.5M
in decano, 3.5 mmol, 635 μL) and vanadylacetylacetonate
(10.3 mg, 3.5 mmol) were added and the mithure was stirred
till starting material disappeared. Purification by chroma-
tography (Petroleum ether/ diethyl ether 8:2), gave pure
products which were solubilised (50 mg, 0.294 mmol) in
CH2Cl2 (1 ml) and under nitrogen atmosphere Et3N (1.47
mmol, 204 μl) was added. A solution of Py-SO3 ( 140 mg,
(E)-3-methyl-3-(2-(3,3-dimethyloxiran-2-yl)ethyl)
oxirane-2-carbaldehyde 11
Prepared as described for 6,7 epoxides using an excess of
MCPBA 77% and following oxidation of alcohol with a
solution of Py-SO3 in DMSO. Yellow oil. Yield: 86%. ESI
(m/z): 184 [M + H]+. Anal. calcd for C10H16O3. C 65.19, H
8.75. Found C 65.27, H 8.72. Spectroscopic data are in
agreement with literature (Escande et al. 2015)

Med Chem Res
(Z)-3-methyl-3-(2-(3,3-dimethyloxiran-2-yl)ethyl)
oxirane-2-carbaldehyde 12
USA). MIA PaCa-2 cells were cultured in Dulbecco’s
Modified Eagle’s Medium supplemented with 10% heat-
inactivated fetal bovine serum (GIBCO, Life Technologies,
Grand Island, NY, USA), penicillin and streptomycin (P/S,
100 U ml−1) (Lonza, Walkersville, MD, USA) and 2.5% of
horse serum (Gibco). The DeFew cells were cultured in
RPMI-1640 medium supplemented with 10% FBS and 1%
P/S. HUVECs were purchased from Promocell (Heidelberg,
Germany). All experiments were performed on low-passage
cell cultures. Cells were grown in endothelial growth
medium-2 (EBM-2, FBS 2%, VEGF, R3-IGF-1, hEGF,
hFGF, hydrocortisone, ascorbic acid, heparin, and GA-
1000) (Clonetics, Cambrex Bio Science Walkersville, Inc.,
Walkersville, MD, USA). All cell lines were grown at 37 °C
in a 5% CO2 atmosphere.
Prepared as described for 6,7 epoxides using an excess of
MCPBA 77% and following oxidation of alcohol with a
solution of Py-SO3 in DMSO. Yield: 81%. ESI (m/z): 184
[M + H]+. Anal. calcd for C10H16O3. C 65.19, H 8.75.
Found C 65.25, H 8.74. Spectroscopic data are in agreement
with literature (Escande et al. 2015).
(S)-3-methyl-5-(3,3-dimethyloxiran-2-yl)pentanal 16
Prepared as described for 6,7 epoxides. Yield: 95%. ESI
(m/z): 171 [M+H]+. [α]D20: +45(c 0.6, CHCl3). Spectro-
scopic data are in agreement with literature (Yarovaya et al.
2002).
Determination of cell viability
(R)-3-methyl-5-(3,3-dimethyloxiran-2-yl)pentanal 17
The viability of cells was determined by MTT test to detect
functional mitochondria in living cells (Mosmann 1983).
Functional mitochondria can transform MTT to formazan
salts, which can be measured with a spectrophotometer. The
proportion of viable cells in treated samples was compared
with control samples. MIA PaCa-2 cells (5 × 103/well) and
DeFew (20 × 104/well) cells were plated on 96-well
microtiter plates to a final volume of 200 µl. After 72 h of
incubation with different concentrations of tested com-
pounds (12.5, 25, 50, 100, 200, 400 µM) at 37 °C, 25 µl of
MTT (5 mg/ml) were added to each well and the cells were
incubated for an additional 3 h. Thereafter, cells were lysed
with 100 µl of a solution containing 50% (v/v) N,N-dime-
thylformamide, 20% (w/v) SDS (pH 4.5) to allow solubi-
lization of dark blue crystals. The optical density of each
well was measured with a microplate spectrophotometer
(TitertekMultiskan MCC/340, Titertek Berthold, Huntsville,
AL) equipped with a 620 nm filter. Cells viability in
response to treatment with compounds was calculated as %
viable cells = (OD treated/OD control)×100.
Prepared as described for 6,7 epoxides. Yield: 96%. ESI (m/
z): 171 [M + H]+. [α]D20: −43 (c 0.6, CHCl3). Spectro-
scopic data are in agreement with literature (Yarovaya et al.
2002).
General procedure for the synthesis of compounds 18
and 19
A mixture of 5% Pd/C (15.6 mg, 5 mol %), citronellal
(151.2 g, 0.98 mmol), and ethyl acetate (12 mL) was stirred
at room temperature for 2 h under an H2 atmosphere. The
reaction mixture was filtered through celite and the filtrate
was concentrated under vacuum to afford 20 as colorless
liquid.
(S)-3,7-Dimethyloctanal 18
Yield: 99%. ESI (m/z): 157 [M + H]+. [α]D20: −74 (c 0.2,
CHCl3). Anal. calcd for C 10H20O. C 76.86, H 12.90.
Found: C 76.63, H 12.66. Spectroscopic data are in agree-
ment with literature (Mikami et al. 2011).
Determination of half maximal inhibitory concentration
(IC50) of drugs
(R)-3,7-Dimethyloctanal 19
The half maximal inhibitory concentration is a measure of
the effectiveness of a compound in inhibiting biochemical
processes and biological functions. According to the in vitro
MTT assay, the IC50 represents the concentration of the
tested drugs that is required for 50% inhibition of the cell
viability. Based on the obtained data using in vitro MTT
assay, the IC50 values was calculated separately at 72 h after
that MIA PaCa-2 cells, DeFew and HUVECs cells were
exposed to the agents. To determine the IC50 values, the
concentration range used of each compounds was from
12.5–400 µM. IC50 values were then calculated from cell
Yield: 99%. ESI (m/z): 157 [M + H]+. [α]D20: +76 (c 0.2,
CHCl3). Anal. calcd for C 10H20O. C 76.86, H 12.90.
Found: C 76.63, H 12.60. Spectroscopic data are in agree-
ment with literature (Mikami et al. 2011).
Cell line, cell culture, and treatment
The human pancreatic cancer MIA PaCa-2 cell line and
human B-lymphoma DeFew cells were purchased from the
American Type Culture Collection (ATCC, Manassas, VA,

Med Chem Res
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Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict
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