Synthesis and evaluation of apoptotic induction of human cancer cells by ester derivatives of thujone

Article abstract of DOI:10.1007/s00044-019-02481-8

Thujone (1), thujol (2), and aromatic thujol esters (3–9) were evaluated for their ability to induce cell death in human cervical (HeLa), melanoma (A375), and colon (HCT-116) cancer cell lines, using etoposide as a positive control. The compounds showed dose-dependent activity at concentrations ranging from 50–400 μg/mL. Etoposide exhibited an IC₅₀ value of 116 μg/mL in HeLa cells, and α-thujone, α/β-thujone (7:1), and thujol showed comparable activity with IC₅₀ values of 191, 198, and 136 μg/mL, respectively. All seven ester derivatives were cytotoxic to HeLa and HCT-116 cells, while a subset was cytotoxic to A375 cells. In HeLa cells, t-cinnamate (4), t-isonicotinate (5), t-nicotinate (6), and t-furoate (8) were more potent than either α-thujone or α/β-thujone. Similarly, t-furoate (8) was more potent than thujone in A375 cells, and t-isonicotinate (5) and t-nicotinate (6) were more potent against HCT-116 cells. Based on cell morphology, PARP cleavage and an increase in the caspase-3/7 levels, the esters exert their cytotoxic effects by induction of apoptosis.

Full text of DOI:10.1007/s00044-019-02481-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02481-8
ORIGINAL RESEARCH
Synthesis and evaluation of apoptotic induction of human cancer
cells by ester derivatives of thujone
1
Emily Castner1 Matthew Dickson2 Anna Mykytyn2 Navindra P. Seeram3 Geneive E. Henry
Pavithra Vivekanand2
●
●
●
●
●
Received: 9 July 2019 / Accepted: 20 November 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Thujone (1), thujol (2), and aromatic thujol esters (3–9) were evaluated for their ability to induce cell death in human cervical
(HeLa), melanoma (A375), and colon (HCT-116) cancer cell lines, using etoposide as a positive control. The compounds
showed dose-dependent activity at concentrations ranging from 50–400 μg/mL. Etoposide exhibited an IC50 value of 116 μg/
mL in HeLa cells, and α-thujone, α/β-thujone (7:1), and thujol showed comparable activity with IC50 values of 191, 198, and
136 μg/mL, respectively. All seven ester derivatives were cytotoxic to HeLa and HCT-116 cells, while a subset was
cytotoxic to A375 cells. In HeLa cells, t-cinnamate (4), t-isonicotinate (5), t-nicotinate (6), and t-furoate (8) were more potent
than either α-thujone or α/β-thujone. Similarly, t-furoate (8) was more potent than thujone in A375 cells, and t-isonicotinate
(5) and t-nicotinate (6) were more potent against HCT-116 cells. Based on cell morphology, PARP cleavage and an increase
in the caspase-3/7 levels, the esters exert their cytotoxic effects by induction of apoptosis.
●
●
●
●
●
●
●
Keywords Thujone Thujol esters HeLa cells HCT-116 cells A375 cells Anticancer Cell death induction Apoptosis
Introduction
(Sobral et al. 2014). Limonene, carvacrol, thymol, and
eugenol are known to have many pharmacological activ-
ities, and their activity against a range of human cancer cells
is well-documented. Limonene and its naturally occurring
metabolite, perillyl alcohol, have been demonstrated to
induce both cell cycle arrest and apoptosis in colon cancer,
non-small cell lung cancer and lung adenocarcinoma cell
lines (Bardon et al. 2002; Elegbede et al. 2003; Yeruva et al.
2007; Mukhtar et al. 2018). Mechanistically, they do so by
modulating the expression levels of p21, the universal CDK
inhibitor, and key apoptotic proteins (Bardon et al. 2002;
Elegbede et al. 2003; Yeruva et al. 2007). Carvacrol has
shown antiproliferative effect against human breast, cervi-
cal, liver, and oral squamous cell carcinoma cell lines, with
apoptosis as the primary mechanism of action (Özkan and
Erdoğan 2011; Yin et al. 2012; Al-Fatlawi and Rahisuddin
2014; Dai et al. 2016). The isomer of carvacrol, thymol, has
also demonstrated significant antiproliferative and apoptotic
effects against breast, liver, and gastric cancer cells lines
(Özkan and Erdoğan 2011; Kang et al. 2016; Jamali et al.
2018). Eugenol has been shown to induce apoptosis in
leukemia, melanoma, breast, and colon cancer cells. In
addition, eugenol has demonstrated the ability to reduce
tumors in animal models (Jaganathan and Supriyanto 2012).
Monoterpenes are produced by a number of plant species
and are under investigation for human use and applications
because of their varied biological properties. They are
present in high quantities in a variety of plant essential oils,
and many exert anticancer activity against human tumor cell
lines with varying selectivity and mechanisms of action
These authors contributed equally: Emily Castner, Matthew Dickson,
Anna Mykytyn
* Geneive E. Henry
henry@susqu.edu
* Pavithra Vivekanand
vivekanand@susqu.edu
1
Department of Chemistry, Susquehanna University,
Selinsgrove, PA 17870, USA
2
Department of Biology, Susquehanna University, Selinsgrove, PA
17870, USA
3
Bioactive Botanical Research Laboratory, Department of
Biomedical and Pharmaceutical Sciences, College of Pharmacy,
University of Rhode Island, Kingston, RI 02881, USA

Medicinal Chemistry Research
Metabolites and synthetically modified derivatives of
monoterpenes have also emerged as promising candidates
for use as anticancer agents, and very often are more potent
than the parent compounds. Perillic acid is a metabolite of
both limonene and perillyl alcohol. It has a similar
mechanism of action as both compounds but demonstrates
greater potency. Similarly, various synthetic derivatives of
perillyl alcohol, including esters and epoxides, showed
enhanced anticancer activity relative to the parent com-
pound (Mukhtar et al. 2018). Thiosemicarbazone deriva-
tives of limonene showed moderate to excellent activity
against a wide range of tumor cell lines (Vandresen et al.
2014). Azomethine linked hybrids containing carvacrol,
thymol, and/or eugenol have shown comparable or
enhanced activity relative to Doxorubicin in colon and
pancreatic cancer cell lines (Rajput et al. 2017).
chromatography using either a Teledyne Isco system with
Rf-Gold prepacked silica gel columns (20–40 mm), or the
traditional method using 230–400 mesh silica gel. NMR
spectra were recorded on a JEOL ECZ 400S spectrometer
(1H: 400 MHz, 13C: 100 MHz) using CDCl3 as solvent and
TMS as internal standard. FTIR data were acquired using a
Thermo Scientific Nicolet iS50 FT-IR spectrometer, and
UV data were obtained using a Varian Cary 4000 UV–Vis
spectrophotometer. Optical rotations were measured using a
Rudolph Research Analytical AUTOPOL III polarimeter.
HRESIMS data were obtained on a Waters SYNAPT G2-S
QTOFMS system, and EIMS data were measured on a
ThermoFisher Scientific ISQ™ Series Single Quadrupole
GC-MS system. The BioRad iMark Microplate absorbance
reader was used for XTT assays to determine cell viability.
Thujone is a monoterpene that is naturally present in the
essential oil extracted from Thuja occidentalis (white
cedar) and several other plant species. Extracts of Thuja
occidentalis have been reported to have several biological
properties, including bactericidal, fungicidal, insecticidal,
apoptotic, and anti-oncogenic (Naser et al. 2005; Alves
et al. 2014). Thujone exists as two diastereomers, α-thu-
jone and β-thujone, with α-thujone being primarily
responsible for mediating the above-mentioned biological
effects. The biological basis for the insecticidal activity of
α-thujone is its ability to function as a neurotoxin by
binding to and antagonizing the activity of GABA recep-
tors in the brain (Hold et al. 2000). The antineoplastic
effects of thujone or thujone-containing extracts have been
examined in some cancer cell lines, including melanoma,
cervical, and glioblastoma cells and is based on the ability
to induce apoptosis and decrease angiogenesis (Biswas
et al. 2011; Torres et al. 2016; Privitera et al. 2019). The
cinnamate derivative of thujone is known, but to our
knowledge there have been no reports on its potential as an
anticancer agent. In this study, we examined the ability of
seven aromatic ester derivatives of thujone to induce cell
death of human cervical (HeLa), melanoma (A375), and
colon (HCT-116) cancer cells, and determined their
mechanism of action.
Sodium borohydride reduction of thujone (1) to thujol (2)
A solution of α/β-thujone, 7/1 (25.01 g) in absolute EtOH
(200 mL) was cooled in an ice bath, and NaBH4 (18.68 g)
was added slowly over a 10 min period. The solution was
stirred at room temperature for 3 h, followed by removal of
the EtOH in vacuo. Saturated sodium chloride (200 mL)
was added to the residue, and 10% aqueous HCl (15 mL)
was added slowly. The resulting solution was extracted with
CH2Cl2 (3 × 75 mL). The CH2Cl2 solution was dried
(Na2SO4), filtered, and concentrated to afford thujol (2).
Thujol, 1-isopropyl-4-methylbicyclo[3.1.0]hexan-3-ol (2)
Colorless oil (98%);[α]D25 -3.4 (c 0.019, MeOH); IR (ATR)
νmax 3375 (O–H) cm−1
;
1
2A1: H NMR (CDCl3, 400 MHz): δ = 3.95 (1 H, H-3),
2.15 (1 H, H-4), 1.94/1.60 (2 H, H-2), 1.25 (1 H, H-7), 0.95
(3 H, H-8), 0.92 (3 H, H-10), 0.89 (3 H, H-9), 0.85 (1 H, H-
5), 0.35/0.22 (2 H, H-6); 13C NMR (CDCl3, 100 MHz): δ =
72.8 (CH, C-3), 37.6 (CH, C-4), 33.4 (CH, C-7), 31.3
(C, C-1), 33.3 (CH2, C-2), 28.2 (CH, C-5), 20.2 (CH3, C-9),
19.7 (CH3, C-8), 14.5 (CH3, C-10), 14.4 (CH2, C-6).
1
2A2: H NMR (CDCl3, 400 MHz): δ = 3.87 (1 H, H-3),
2.05 (1 H, H-4), 1.92/1.52 (2 H, H-2), 1.28 (1 H, H-7), 0.92
(3 H, H-8), 0.91 (3 H, H-10), 0.89 (3 H, H-9), 0.80 (1 H, H-
5), 0.41-0.38 (2 H, H-6); 13C NMR (CDCl3, 100 MHz):
δ = 80.3 (CH, C-3), 45.3 CH, (C-4), 34.5 (C, C-1), 36.0
(CH2, C-2), 33.0 (CH, C-7), 29.7 (CH, C-5), 20.2 (CH3, C-
9), 20.0 (CH3, C-8), 20.3 (CH3, C-10), 17.3 (CH2, C-6);
EIMS m/z 136 (32), 121 (82), 95 (100), 93 (84), 81 (52), 55
(61); HRESIMS m/z (pos): 155.1084 C10H19O [M + H]+
(calcd. 155.1066).
Materials and methods
Experimental
General experimental procedures
All solvents and reagents were ACS grade and were
obtained from either Fisher Scientific or Aldrich Chemical
Company. Solvents were dried according to standard pro-
cedures. Purification was performed by column
General procedure for the synthesis of thujol esters (3–9)
Acyl components: benzoyl chloride, phenacetyl chloride,
cinnamoyl chloride, nicotinoyl chloride, isonicotinoyl

Medicinal Chemistry Research
1
chloride, 2-furoyl chloride, and thiophene-2-carbonyl
chloride.
4A1: H NMR (CDCl3, 400 MHz): δ = 7.65 (1 H, H-3′),
7.51 (2 H, H-5′/ H-9′), 7.37 (2 H, H-6′/H-7′/ H-8′), 6.42 (1
H, H-2′), 4.85 (1 H, H-3), 2.45 (1 H, H-4), 2.05/1.81 (2 H,
H-2), 1.28 (1 H, H-7), 0.98 (3 H, H-8), 0.93 (3 H, H-10),
A solution of the diastereomeric mixture of thujol
(2.00 g, 13 mmol),* triethylamine (3.6 mL, 26 mmol), and
4-dimethylaminopyridine, DMAP (0.40 g, 3.3 mmol), in
CH2Cl2 (20 mL) was cooled in an ice-bath. The acid
chloride/acid anhydride (26 mmol) was added slowly, and
the reaction mixture was allowed to warm to room tem-
perature and stirred for 3–24 h, while monitoring the pro-
gress of the reaction by TLC. The dichloromethane solution
was washed with saturated NaCl (30 mL), dried (Na2SO4),
filtered, and concentrated in vacuo. Silica gel column
chromatography using ethyl acetate-–hexanes mixtures as
eluents afforded the diastereomeric mixture of thujol esters
as colorless or pale-yellow oils.
0.91 (3 H, H-9), 0.87 (1 H, H-5), 0.44/0.30 (2 H, H-6); 13
C
NMR (CDCl3, 100 MHz): δ = 166.7 (C, C-1′), 144.7 (CH,
C-3′), 134.5 (C, C-4′), 130.3 (CH, C-7′), 128.9 (CH, C-5′,
C-9′), 128.1 (CH, C-6′, C-8′), 118.3 (CH, C-2′), 75.3 (CH,
C-3), 35.9 (CH, C-4), 33.2 (CH, C-7), 30.9 (C, C-1), 30.2
(CH2, C-2), 28.1 (CH, C-5), 20.2 (CH3, C-9), 19.8 (CH3,
C-8), 15.4 (CH3, C-10), 14.5 (CH2, C-6);
4A2: 1H NMR (CDCl3, 400 MHz): δ = 7.61 (1 H, H-3′),
7.51 (2 H, H-5′/ H-9′), 7.37 (2 H, H-6′/H-7′/ H-8′), 6.37 (1
H, H-2′), 4.89 (1 H, H-3), 2.15 (1 H, H-4), 2.20/1.72 (2 H,
H-2), 1.35 (1 H, H-7), 1.03 (3 H, H-8), 0.96 (3 H, H-10),
*1.5 g of thujol was used for some of the samples, and
reagent ratios were adjusted accordingly.
Only the NMR data for the major diastereomers (thujol
esters 3A1/3A2–9A1/9A2 are shown).
0.92 (3 H, H-9), 0.86 (1 H, H-5), 0.73/0.42 (2 H, H-6); 13
C
NMR (CDCl3, 100 MHz): δ = 166.4 (C, C-1′), 144.4 (CH,
C-3′), 134.5 (C, C-4′), 130.3 (CH, C-7′), 128.9 (CH, C-5′,
C-9′), 128.1 (CH, C-6′, C-8′), 118.9 (CH, C-2′), 82.2 (CH,
C-3), 42.8 (CH, C-4), 34.5 (C, C-1), 33.4 (CH2, C-2), 32.8
(CH, C-7), 29.6 (CH, C-5), 20.3 (CH3, C-10), 20.0 (CH3,
C-9), 19.9 (CH3, C-8), 16.0 (CH2, C-6); EIMS m/z 136
(40), 131 (100), 121 (32), 103 (33), 93 (40), 77 (20);
HRESIMS m/z (pos): 285.1874 C19H25O2 [M + H]+
(calcd. 285.1849).
Thujol benzoate, 1-isopropyl-4-methylbicyclo[3.1.0]hexan-
25
3-yl benzoate (3) Colorless oil (55%); [α]D -10 (c
0.0002, MeOH); UV (MeOH) λmax(log ε) 273(2.78) nm; IR
(ATR) νmax 1712 (C=O), 1275 (C-O) cm−1
;
1
3A1: H NMR (CDCl3, 400 MHz): δ = 8.04 (2 H, H-3′/
H-7′), 7.53 (1 H, H-5′), 7.42 (2 H, H-4′/ H-6′), 4.97 (1 H,
H-3), 2.50 (1 H, H-4), 2.12/1.86 (2 H, H-2), 1.29 (1 H, H-
7), 0.99 (3 H, H-8), 0.97 (3 H, H-10), 0.92 (3 H, H-9), 0.85
(1 H, H-5), 0.46/0.32 (2 H, H-6); 13C NMR (CDCl3,
100 MHz): δ = 166.2 (C, C-1′), 132.9 (CH, C-5′), 129.6
(CH, C-4′, C-6′), 128.4 (CH, C-3′, C-7′), 130.6 (C, C-2′),
75.7 (CH, C-3), 36.1 (CH, C-4), 33.4 (CH, C-7), 31.1 (C, C-
1), 30.3 (CH2, C-2), 28.1 (CH, C-5), 20.3 (CH3, C-9), 19.8
(CH3, C-8), 15.5 (CH3, C-10), 14.6 (CH2, C-6);
Thujol isonicotinate, 1-isopropyl-4-methylbicyclo[3.1.0]
hexan-3-yl isonicotinate (5) Colorless oil (43%); [α]D
25
-20 (c 0.0002, MeOH); UV (MeOH) λmax(log ε) 274(3.37)
nm; IR (ATR) νmax 1725 (C=O), 1279 (C–O) cm−1
;
1
5A1: H NMR (CDCl3, 400 MHz): δ = 8.74 (2 H, H-4′/
H-6′), 7.80 (2 H, H-3′/H-7′), 4.94 (1 H, H-3), 2.45 (1 H, H-
4), 2.08/1.85 (2 H, H-2), 1.25 (1 H, H-7), 0.90 (3 H, H-8),
0.89 (3 H, H-10), 0.86 (3 H, H-9), 0.80 (1 H, H-5), 0.40/
0.30 (2 H, H-6); 13C NMR (CDCl3, 100 MHz): δ = 164.6
(C, C-1′), 150.6 (CH, C-4′, C-6′), 137.6 (C, C-2′), 122.9
(CH, C-3′, C-7′), 76.7 (CH, C-3), 36.0 (CH, C-4), 33.1
(CH, C-7), 31.1 (C, C-1), 30.2 (CH2, C-2), 27.9 (CH, C-5),
20.2 (CH3, C-9), 19.7 (CH3, C-8), 15.4 (CH3, C-10), 14.6
(CH2, C-6);
3A2: 1H NMR (CDCl3, 400 MHz): δ = 7.99 (2 H, H-3′/H-
7′), 7.53 (1 H, H-5′), 7.42 (2 H, H-4′/ H-6′), 5.01 (1 H, H-
3), 2.21 (1 H, H-4), 2.24/1.78 (2 H, H-2), 1.36 (1 H, H-7),
1.10-0.85 (10 H, H-5, H-8, H-9, H-10), 0.79/0.46 (2 H,
H-6);
13C NMR (CDCl3, 100 MHz): δ = 166.0 (C, C-1′), 132.8
(CH, C-5′), 129.5 (CH, C-4′, C-6′), 128.4 (CH, C-3′, C-7′),
130.8 (C, C-2′), 82.7 (CH, C-3), 42.8 (CH, C-4), 34.5 (C,
C-1), 33.2 (CH2, C-2), 32.8 (CH, C-7), 29.6 (CH, C-5), 20.3
(CH3, C-10), 20.0 (CH3, C-9), 19.9 (CH3, C-8), 16.1 (CH2,
C-6); EIMS m/z 136 (41), 121 (34), 105 (100), 93 (49), 77
(51); HRESIMS m/z (pos): 259.1710 C17H23O2 [M + H]+
(calcd. 259.1692).
1
5A2: H NMR (CDCl3, 400 MHz): δ = 8.74 (2 H, H-4′/
H-6′), 7.80 (2 H, H-3′/H-7′), 4.98 (1 H, H-3), 2.18 (1 H,
H-4), 2.16/1.74 (2 H, H-2), 1.32 (1 H, H-7), 1.10-0.80 (10
H, H-5, H-8, H-9, H-10), 0.66/0.45 (2 H, H-6); 13C NMR
(CDCl3, 100 MHz): δ = 164.4 (C, C-1′), 150.6 (CH, C-4′,
C-6′), 137.9 (C, C-2′), 122.8 (CH, C-3′, C-7′), 83.6 (CH,
C-3), 42.8 (CH, C-4), 34.5 (C, C-1), 33.4 (CH2, C-2),
32.6 (CH, C-7), 29.5 (CH, C-5), 20.3 (CH3, C-10), 19.9
(CH3, C-9), 19.8 (CH3, C-8), 16.1 (CH3, C-6); EIMS m/z
136 (61), 121 (74), 106 (78), 93 (100), 78 (51);
HRESIMS m/z (pos): 260.1657 C16H22NO2 [M + H]+
(calcd. 260.1645).
Thujol cinnamate, 1-isopropyl-4-methylbicyclo[3.1.0]hexan-
25
3-yl cinnamate (4) Colorless oil (43%); [α]D
-15
(c 0.0002, MeOH); UV (MeOH) λmax(log ε) 278(4.25) nm;
IR (ATR) νmax 1709 (C=O), 1168 (C–O) cm−1
;

Medicinal Chemistry Research
Thujol nicotinate, 1-isopropyl-4-methylbicyclo[3.1.0]hexan-
Thujol furoate, 1-isopropyl-4-methylbicyclo[3.1.0]hexan-3-
yl furan-2-carboxylate (8) Colorless oil (73%); [α]D -15
25
25
3-yl nicotinate (6) Colorless oil (57%); [α]D
-20
(c 0.0002, MeOH); UV (MeOH) λmax(log ε) 263(3.32) nm;
(c 0.0002, MeOH); UV (MeOH) λmax(log ε) 252(3.52) nm;
IR (ATR) νmax 1719 (C=O), 1282 (C–O) cm−1
;
IR (ATR) νmax 1713 (C=O), 1295 (C–O) cm−1
;
1
1
6A1: H NMR (CDCl3, 400 MHz): δ = 9.15 (1 H, H-3′),
8.71 (1 H, H-5′), 8.20 (1 H, H-7′), 7.32 (1 H, H-6′), 4.94 (1
H, H-3), 2.46 (1 H, H-4), 2.06/1.84 (2 H, H-2), 1.13 (1 H,
H-7), 0.89 (3 H, H-8), 0.90 (3 H, H-10), 0.89 (3 H, H-9),
0.80 (IH, H-5), 0.40/0.28 (2 H, H-6); 13C NMR (CDCl3,
100 MHz): δ = 164.8 (C, C-1′), 153.4 (CH, C-3′), 150.9
(CH, C-5′), 137.0 (CH, C-7′), 126.3 (C, C-2′), 123.3 (CH,
C-6′), 76.3 (CH, C-3), 36.1 (CH, C-4), 33.1 (CH, C-7), 31.1
(C, C-1), 30.3 (CH2, C-2), 27.9 (CH, C-5), 20.2 (CH3, C-9),
19.7 (CH3, C-8), 15.4 (CH3, C-10), 14.6 (CH2, C-6);
8A1: H NMR (CDCl3, 400 MHz): δ = 7.53 (1 H, H-4′),
7.11 (1 H, H-6′), 6.44 (1 H, H-5′), 4.88 (1 H, H-3), 2.44 (1
H, H-4), 2.05/1.82 (2 H, H-2), 1.24 (1 H, H-7), 0.91 (3 H,
H-8), 0.90 (3 H, H-10), 0.89 (3 H, H-9), 0.88 (1 H, H-5),
0.40/0.26 (2 H, H-6); 13C NMR (CDCl3, 100 MHz): δ =
158.4 (C, C-1′), 144.9 (C, C-2′), 146.3 (CH, C-4′), 117.8
(CH, C-6′), 111.8 (CH, C-5′), 75.8 (CH, C-3), 36.0 (CH, C-
4), 33.2 (CH, C-7), 30.9 (C, C-1), 30.2 (CH2, C-2), 27.9
(CH, C-5), 20.2 (CH3, C-9), 19.7 (CH3, C-8), 15.3 (CH3, C-
10), 14.5 (CH2, C-6);
1
6A2: H NMR (CDCl3, 400 MHz): δ = 9.12 (1 H, H-3′),
8A2: 1H NMR (CDCl3, 400 MHz): δ = 7.53 (1 H, H-4′),
7.07 (1 H, H-6′), 6.44 (1 H, H-5′), 4.94 (1 H, H-3), 2.15 (1
H, H-4), 2.17/1.73 (2 H, H-2), 1.30 (1 H, H-7), 1.10-0.85
(9 H, H-8, H-9, H-10), 0.85 (1 H, H-5), 0.73/0.40 (2 H, H-
6); 13C NMR (CDCl3, 100 MHz): δ = 158.2 (C, C-1′),
145.2 (C, C-2′), 146.2 (CH, C-4′), 117.5 (CH, C-6′), 111.7
(CH, C-5′), 82.6 (CH, C-3), 42.7 (CH, C-4), 34.3 (C, C-1),
33.3 (CH2, C-2), 32.7 (CH, C-7), 29.4 (CH, C-5), 20.3
(CH3, C-10), 19.9 (CH3, C-9), 19.8 (CH3, C-8), 15.9 (CH2,
C-6); EIMS m/z 136 (44), 121 (52), 95 (100), 93 (69);
HRESIMS m/z (pos): 249.1512 C15H21O3 [M + H]+
(calcd. 249.1485).
8.71 (1 H, H-5′), 8.20 (1 H, H-7′), 7.32 (1 H, H-6′), 4.97 (1
H, H-3), 2.22 (1 H, H-4), 2.09/1.75 (2 H, H-2), 1.36 (1 H,
H-7), 1.10-0.80 (10 H, H-5, H-8, H-9, H-10), 0.69/0.43 (2
H, H-6); 13C NMR (CDCl3, 100 MHz): δ = 164.4 (C, C-1′),
153.3 (CH, C-3′), 150.8 (CH, C-5′), 136.9 (CH, C-7′),
126.5 (C, C-2′), 123.3 (CH, C-6′), 83.2 (CH, C-3), 42.8
(CH, C-4), 34.5 (C, C-1), 33.4 (CH2, C-2), 32.6 (CH, C-7),
29.5 (CH, C-5), 20.3 (CH3, C-10), 19.9 (CH3, C-9), 19.8
(CH3, C-8), 16.1 (CH2, C-6); EIMS m/z 136 (57), 121 (68),
106 (100), 93 (92), 78 (57); HRESIMS m/z (pos): 260.1662
C16H22NO2 [M + H]+ (calcd. 260.1645).
Thujol thiophenate, 1-isopropyl-4-methylbicyclo[3.1.0]
Thujol phenylacetate, 1-isopropyl-4-methylbicyclo[3.1.0]
hexan-3-yl 2-phenylacetate (9) Pale yellow oil (62%);
[α]D25 -30 (c 0.0002, MeOH); UV (MeOH) λmax(log ε) 258
hexan-3-yl thiophene-2-carboxylate (7) Pale yellow oil
25
(60%); [α]D -15 (c 0.0002, MeOH); UV (MeOH)
λmax(log ε) 268(3.89) nm; IR (ATR) νmax 1705 (C=O), 1259
(2.45) nm; IR (ATR) νmax 1730 (C=O), 1156 (C–O) cm−1
;
(C–O) cm−1
;
9A1: H NMR (CDCl3, 400 MHz): δ = 7.26 (5 H, H-4′-
H-8′), 4.72 (1 H, H-3), 3.58 (1 H, H-2′), 2.34 (1 H, H-4),
1.97/1.70 (2 H, H-2), 1.25 (1 H, H-7), 1.10-0.70 (10 H, H-5,
H-8, H-9, H-10), 0.35/0.25 (2 H, H-6); 13C NMR (CDCl3,
100 MHz): δ = 171.3 (C, C-1′), 134.2 (C, C-3′), 129.4 (CH,
C-4′, C-8′), 128.6 (CH, C-5′, C-7′), 127.1 (CH, C-6′), 75.7
(CH, C-3), 41.6 (CH2, C-2′), 35.7 (CH, C-4), 33.4 (CH, C-
7), 30.9 (C, C-1), 30.1 (CH2, C-2), 28.0 (CH, C-5), 20.2
(CH3, C-9), 19.7 (CH3, C-8), 15.2 (CH3, C-10), 14.4
(CH2, C-6);
1
1
7A1: H NMR (CDCl3, 400 MHz): δ = 7.77 (1 H, H-6′),
7.52 (1 H, H-4′), 7.08 (1 H, H-5′), 4.92 (1 H, H-3), 2.47 (1
H, H-4), 2.10/1.84 (2 H, H-2), 1.26 (1 H, H-7), 0.91 (3 H,
H-8), 0.90 (3 H, H-10), 0.89 (3 H, H-9), 0.87 (1 H, H-5),
0.44/0.31 (2 H, H-6); 13C NMR (CDCl3, 100 MHz): δ =
161.9 (C, C-1′), 134.2 (C, C-2′), 133.3 (CH, C-4′), 132.3
(CH, C-6′), 127.8 (CH, C-5′), 76.0 (CH, C-3), 36.2 (CH, C-
4), 33.2 (CH, C-7), 31.1 (C, C-1), 30.3 (CH2, C-2), 28.0
(CH, C-5), 20.2 (CH3, C-9), 19.7 (CH3, C-8), 15.5 (CH3,
C-10), 14.6 (CH2, C-6);
1
9A2: H NMR (CDCl3, 400 MHz): δ = 7.26 (5 H, H-4′-
1
7A2: H NMR (CDCl3, 400 MHz): δ = 7.75 (1 H, H-6′),
H-8′), 4.75 (1 H, H-3), 3.53 (1 H, H-2′), 2.10 (1 H, H-4),
2.00/1.60 (2 H, H-2), 1.31 (1 H, H-7), 1.10-0.70 (5 H, H-5,
H-8, H-9, H-10), 0.54/0.33 (2 H, H-6); 13C NMR (CDCl3,
100 MHz): δ = 171.1 (C, C-1′), 134.2 (C, C-3′), 129.3 (CH,
C-4′, C-8′), 128.6 (CH, C-5′, C-7′), 127.1 (CH, C-6′), 82.6
(CH, C-3), 42.7 (CH, C-4), 41.9 (CH2, C-2′), 34.4 (C, C-1),
33.2 (CH2, C-2), 32.7 (CH, C-7), 29.4 (CH, C-5), 20.3
(CH3, C-10), 19.9 (CH3, C-9), 19.8 (CH3, C-8), 15.8 (CH2,
C-6); EIMS m/z 136 (64), 121 (52), 93 (64), 91 (100);
HRESIMS m/z (pos): 295.1683 C18H24O2Na [M + Na]+
(calcd. 295.1668).
7.52 (1 H, H-4′), 7.08 (1 H, H-5′), 4.96 (1 H, H-3), 2.19 (1 H,
H-4), 2.21/1.78 (2 H, H-2), 1.35 (1 H, H-7), 1.10-0.85 (10 H,
H-5, H-8, H-9, H-10), 0.77/0.46 (2 H, H-6); 13C NMR
(CDCl3, 100 MHz): δ = 161.6 (C, C-1′), 134.7 (C, C-2′),
133.1 (CH, C-4′), 132.2 (CH, C-6′), 127.8 (CH, C-5′), 82.9
(CH, C-3), 42.8 (CH, C-4), 34.4 (C, C-1), 33.2 (CH2, C-2),
32.7 (CH, C-7), 29.5 (CH, C-5), 20.3 (CH3, C-10), 20.0
(CH3, C-9), 19.8 (CH3, C-8), 16.0 (CH2, C-6); EIMS m/z 136
(42), 121 (37), 110 (100), 93 (52); HRESIMS m/z (pos):
287.1076 C15H20O2NaS [M + Na]+ (calcd. for; 287.1076).

Medicinal Chemistry Research
R
Thujol ester
3 α-OCOR
4 α-CH3
Neothujol ester
3 β-OCOR
4 α-CH3
Isothujol ester
3 α-OCOR
4 β-CH3
Neoisothujol ester
3 β-OCOR
4 β-CH3
3A1
4A1
5A1
6A1
7A1
8A1
9A1
3A2
4A2
5A2
6A2
7A2
8A2
9A2
3B1
4B1
5B1
6B1
7B1
8B1
9B1
3B2
4B2
5B2
6B2
7B2
8B2
9B2
Fig. 1 Synthesis of ester derivatives of thujone
Cell culture materials
Cell viability assay
Human cervical cancer cells HeLa (CCL-2), A375 mela-
noma cells (CRL-1619), and HCT-116 colon cancer cells
(CCL-247) were obtained from American Type Culture
Collection. HeLa and A375 cells were cultured in Dulbec-
co's modified Eagles medium (DMEM) while HCT-116
cells were cultured in McCoy's medium. DMEM and
McCoy’s media were supplemented with 10% FBS, peni-
cillin, and streptomycin and maintained in a humidified
incubator at 5% CO2 and 37 °C.
HeLa, A375 and HCT-116 cells were plated in 96-well
plates at a density of 1 × 105 cells/mL, 2 × 105 cells/mL and
5 × 105 cells/mL, respectively. After 24 h of incubation,
cells were treated with concentrations of test compounds
ranging from 50 to 400 μg/mL. The control was treated with
10% DMSO. After incubation for 24 h in the presence of the
compounds, 50 μL of XTT solution (0.3 mg/mL) was added
to each well. Cells were incubated with XTT reagent for 4 h,
and the absorbance at 495 nm was measured using an

Medicinal Chemistry Research
120
100
80
60
40
20
0
0
g/mL
50 g/mL
100 g/mL
200 g/mL
300 g/mL
400 g/mL
Fig. 2 Treatment of HeLa cells with thujone results in dose dependent
increase in cytotoxicity. HeLa cells were treated with 50–400 μg/mL of
the different compounds for 24 h. Data are average +/− SD of two
independent experiments, each done in duplicate
140
120
100
80
0
g/mL
60
ELISA plate reader. The average absorbance of the media
was subtracted, and the percent of cell viability was cal-
culated as follows:
50 g/mL
100 g/mL
200 g/mL
300 g/mL
400 g/mL
40
20
ꢀ
ꢁ
Absorbance of drug À treated cells
0
Percent cell viability ¼
 100
Absorbance of control cells
IC50 values were determined using Graphpad Prism
software.
Fig. 3 Ester derivatives of thujone are more potent than α-thujone or α/
β-thujone in inducing death of HeLa cells. HeLa cells were treated
with 50–400 μg/mL of the different compounds for 24 h. Data are
average +/− SD of two independent experiments, each done in
duplicate
Cell morphology
HeLa cells were plated in a 24-well plate, allowed to
become 95% confluent and treated with 100 μg/mL of the
different compounds for 24 h, following which images were
captured using Nikon Eclipse TE300.
Activated caspase 3/7 detection
Cells were treated with either 1 μM of Staurosporine or
100 μg/mL of the ester derivatives of thujone for 3 h, fol-
lowing which they were stained with Cell Event Caspase 3/
7 (Invitrogen #R37111) for 60 min according to the man-
ufacturer's instructions. Cells were fixed with 3.7% for-
maldehyde for 15 min and imaged using Nikon confocal
microscope.
Western blotting
Cells were induced with either 1 μM of Staurosporine or
100 μg/mL of the ester derivatives of thujone and were
harvested 3 h after drug addition in RIPA lysis buffer
supplemented with protease inhibitors. The lysates were
sonicated 2 × 30 sec and centrifuged at 14,000 rpm for
10 min at 4 °C. Proteins were resolved using 10% SDS-
PAGE and transferred to nitrocellulose membrane. PARP
protein was detected using Rabbit anti-PARP antibody
(#9542 Cell Signaling Technology) at 1:1000 and anti-
Rabbit-HRP (Jackson ImmunoResearch) at 1:5000. Z-
VAD-FMK was added at a final concentration of 20 μM 1 h
before the addition of the ester derivatives. The cells were
harvested for western blotting 2 h after addition of the ester
derivatives. For the hydrolysis experiments cells were
treated with 250 μM of various compounds and harvested
4 h after drug addition in RIPA lysis buffer supplemented
with protease inhibitors.
Chemistry
A 7:1 mixture of α-(−)-thujone and β-(+)-thujone (iso-
thujone) was reduced using sodium borohydride in abso-
lute ethanol to obtain
a mixture of four thujol
diastereomers in near quantitative yield (Fig. 1). The NMR
data indicated 49%, 31%, 14% and 6% of (−)-thujol
(2A1), (−)-neothujol (2A2), (−)-isothujol (2B1), and
(+)-neoisothujol (2B2), respectively. The structures were
confirmed by comparison of the NMR data with literature
values (Bergqvist and Norin 1963; Bohlman and Zeisberg
1975). The approximate ratio of 1.5:1 for thujol:neothujol

Medicinal Chemistry Research
140
120
100
80
120
100
80
60
40
20
0
0
g/mL
0
g/mL
50 g/mL
50 g/mL
100 g/mL
200 g/mL
300 g/mL
400 g/mL
60
100 g/mL
200 g/mL
300 g/mL
400 g/mL
40
20
0
Fig. 5 Ester derivatives of thujone affect cell viability of HCT-116
colon cancer cells. HCT-116 cells were treated with 50–400 μg/mL of
the different compounds for 24 h. Data are average +/− SD of two
independent experiments, each done in duplicate
Fig. 4 A subset of thujone ester derivatives are cytotoxic to A375
cells. Cells were treated with 50–400 μg/mL of the different com-
pounds for 24 h. Data are average +/− SD of two independent
experiments, each done in duplicate
is in good agreement with the study by Banthorpe, which
showed a similar ratio of products for the reduction of pure
α-thujone (Banthorpe and Davies 1968). The ratio of
products reported for the reduction of β-thujone in the
same study is 1.2:1 neoisothujol:isothujol. However, in
our study the amount of isothujol was greater than that of
neoisothujol by factor of 2.3:1. The higher percentage of
2A1 relative to 2A2 can be rationalized based on the
configuration of the isopropyl and methyl groups at posi-
tions C-1 and C-4. Since both groups are α-oriented, the
hydride preferentially attacks the carbonyl group from the
β-face of the molecule, resulting in the α-hydroxyl group
at C-323. Although β-thujone has the C-4 methyl and the
C-6 methylene groups in the β-orientation, and only the C-
1 isopropyl group in the α-orientation, based on our results
there is still a preference for hydride attack from the β-face
of the molecule. It can therefore be concluded that the
isopropyl group has a greater impact on the stereochemical
outcome of the sodium borohydride reduction of thujone
than either the C-4 methyl or the C-6 methylene group.
Surprisingly, the approximate ratio of alcohol products
derived from α-thujone to those from β-thujone is 4:1. This
would indicate that there was slight epimerization of α-
thujone to β-thujone under the reaction conditions. It has
been reported by Hach that significant epimerization of pure
α-thujone and pure β-thujone occur when the reduction is
carried out in anhydrous solvents (Hach et al. 1971), but this
was not observed by us.
the acyl component. The esters were obtained in 43–73%
yields after workup and purification, and the structures were
confirmed by NMR, IR, and MS analyses.
Based on NMR integration, the ratios of the ester dia-
stereomers were similar to those observed for the thujols.
As with the mixture of thujols, we encountered difficulty
trying to separate the diastereomeric mixture of esters by
silica gel column chromatography. Attempts to separate the
mixtures by reversed phase HPLC using an achiral column
were similarly unsuccessful.
Results and discussion
Anticancer studies
Thujone has been shown to induce apoptosis of melanoma
and glioblastoma cells (Biswas et al. 2011; Torres et al.
2016). In addition, an essential oil preparation containing
thujone has been shown to decrease cell viability of HeLa
cells (Privitera et al. 2019). However, thujone has been
shown to have no effect on the proliferation of colon cancer
cells (Zhou et al. 2016). In this study, we synthesized a
series of ester derivatives of thujol and evaluated them for
cytotoxic activity against human cervical (HeLa), mela-
noma (A375), and colon (HCT-116) cells, together with
thujone, α/β-thujone, and thujol.
HeLa cells were treated with increasing concentrations of
α-thujone, α/β-thujone (7:1), and thujol. As a positive
control, we examined the ability of etoposide, a well-known
chemotherapeutic drug, to induce cell death compared with
α-thujone, α/β-thujone, and thujol. Treatment of HeLa cells
with 50 μg/mL of etoposide resulted in reduction of cell
viability to 59%, but treatment with either 50 or 100 μg/mL
of α-thujone, α/β-thujone, and thujol did not have any effect
The four thujol diastereomers were not readily separated
by silica gel column chromatography, and the mixture was
used without further purification. Seven thujol esters con-
taining aromatic moieties (3–9) were prepared by reacting the
mixture of thujols with benzoyl chloride, phenacetyl chloride,
cinnamoyl chloride, nicotinoyl chloride, isonicotinoyl chlor-
ide, 2-furoyl chloride, and thiophene-2-carbonyl chloride as

Medicinal Chemistry Research
Fig. 6 HeLa cells exposed to
ester derivatives exhibit
apoptotic morphology.
Morphology of HeLa cells
treated with 100 μg/mL of α-
thujone, α/β− thujone (a) or
seven ester derivatives for 24 h
(b)

Medicinal Chemistry Research
α/β-thujone were able to elicit the death of HeLa cells.
Cell viability was reduced to 50% when treated with
300 μg/mL and 400 μg/mL concentrations of α/β-thujone
(7:1), for U87MG and C6 cells (Torres et al. 2016),
respectively, while 200 μg/mL of α-thujone, and α/
β-thujone resulted in 41 and 47% of HeLa cells being
viable. Therefore, lower concentrations of both α-thujone,
and α/β-thujone are effective in inducing the death of
HeLa cells when compared with their ability to induce
death of glioblastoma cells.
We subsequently examined the ability of the ester
derivatives of thujone (3–9) to induce HeLa cell death, and
evaluated whether there were direct structure–activity
correlations. Of the seven ester derivatives examined, t-
isonicotinate (5) and t-furoate (8) are the most effective at
inducing cell death, as treatment of HeLa cells with the
lowest tested concentration of 50 μg/mL resulted in
reduction of cell viability to 63% and 34%, respectively
(Fig. 3). Increasing the dosage to 100 μg/mL further
decreased cell viability to <5% for both of these deriva-
tives. While treatment of HeLa cells with 50 μg/mL of t-
cinnamate (4) and t-nicotinate (6) produced only a modest
effect on cell viability, treatment with the next highest
concentration of 100 μg/mL reduced cell viability to 46%
and 45%, respectively. Further treatment with higher
concentrations resulted in 100% cell death. No appreciable
cell death was observed when HeLa cells were treated with
50 or 100 μg/mL of the remaining derivatives, t-benzoate
(3), t-thiophenate (7), and t-phenylacetate (9). However,
exposure of cells to the next highest concentration of
200 μg/mL reduced cell viability to 8%, 0%, and 8%,
respectively (Fig. 3). Based on these results, it can be
concluded that the structure of the thujone ester has an
effect on the viability of HeLa cells.
Similar to HeLa cells, A375 cells treated with α-thu-
jone and α/β-thujone exhibit a dose-dependent decrease
in cell viability, with viability being reduced to 46% and
56%, when exposed to 200 μg/mL of the respective
derivative (Fig. 4). This is comparable with what was
observed previously, with viability decreasing to 58%
and 41% when A375 cells were treated with 100 and
200 μg/mL concentrations of thujone rich fractions,
respectively (Biswas et al. 2011). Of the seven ester
derivatives, only t-cinnamate (4), t-isonicotinate (5), t-
nicotinate (6), and t-furoate (8) had an effect on the
viability of A375 cells, and their activity was lower than
observed in HeLa cells at similar concentrations. The
viability of A375 cells treated with 100 μg/mL of
t-furoate decreased to 33%, with higher concentrations
resulting in 100% cell death. While treatment of cells
with either 50 or 100 μg/mL of t-isonicotinate and
t-nicotinate had no effect, treatment with 200 μg/mL or
higher concentrations resulted in 0% viability (Fig. 4). As
Fig. 7 PARP cleavage is detected in HeLa cells treated with ester
derivatives of thujone. a HeLa cells were treated for 3 h with either
1 µM of Staurosporine (lane 2) or 100 µg/mL of t-furoate (lane 3),
t-isonicotinate (lane 4), t-cinnamate (lane 5), and t-nicotinate (lane
6). SDS-PAGE and western blotting were performed to detect
PARP in the lysates of control (lane 1) and treated cells. Full-length
PARP is detected in control cells (lane 1) while the 89 kDa cleaved
fragment is present in treated cells (lanes 2–6). Std. refers to protein
standard. b PARP cleavage was examined in cells treated in the
presence or absence of Z-VAD-FMK (20 µM), which was added an
hour before addition of the apoptotic inducers. Addition of Z-VAD-
FMK significantly inhibits the induction of PARP cleavage by
t-furoate and t-isonicotinate. Std. refers to protein standard, Lane
1: control, Lane 2: Z-VAD-FMK only, Lane 3: t-furoate, Lane 4:
t-furoate + Z-VAD-FMK, Lane 5: t-isonicotinate, Lane 6: t-iso-
nicotinate + Z-VAD-FMK
on cell viability (Fig. 2). However, treatment of cells with
200 μg/mL of α-thujone, α/β-thujone, and etoposide resul-
ted in comparable reduction in viability to 41%, 47% and
50%, respectively. Moreover, at higher concentrations, α-
thujone, and α/β-thujone appear to be more effective than
etoposide at inducing cell death, as treatment with 400 μg/
mL of α-thujone, α/β-thujone, and etoposide, the highest
concentration tested, reduced cell viability to 2%, 7%, and
39%, respectively (Fig. 2). Interestingly, while HeLa cells
exposed to either α-thujone, α/β-thujone, or etoposide
exhibited a dose-dependent decrease in cell viability,
treatment with 200 μg/mL of thujol lead to a dramatic
decrease in cell viability with only 2% of cells being viable.
The IC50 values for α-thujone, α/β-thujone, and thujol were
191, 198, and 136 μg/mL, respectively, while etoposide
exhibited an IC50 value of 116 μg/mL (197 μM). The IC50
values for etoposide vary depending on the cell line tested,
but for HeLa cells it has been reported to be 209 μM (Xiao
et al. 2014).
Interestingly, while treatment with α/β-thujone reduced
the viability of human (U87MG) and rat (C6) glio-
blastoma cells, α-thujone was ineffective at inducing cell
death (Torres et al. 2016). However, both α-thujone, and

Medicinal Chemistry Research
Fig. 8 The effector Caspase 3/7 is activated at high levels in t-furoate
and t-isonicotinate treated cells. HeLa cells were treated with either
1 µM of Staurosporine (d–f), 100 µg/mL of t-furoate (G-H), or 100 µg/
mL of t-isonicotinate (j–l) for 3 h and stained with Cell Event Caspase
3/7 Green to detect activated caspase 3/7 (a, d, g, j), Hoecsht to detect
DNA (b, e, h, k) and merged (c, f, i, l). Arrows point to cells that show
very low levels of activated caspase in the control
was observed for the parent compounds (α-thujone, and
α/β-thujone), treatment with t-cinnamate also produced a
dose-dependent cytotoxicity, but only treatment with the
highest concentration of 400 μg/mL reduced the viability
significantly to 54% (Fig. 4). The remaining derivatives
had no appreciable effect on the viability of A375 cells.
Interestingly, in contrast with HeLa cells, t-isonicotinate
and t-nicotinate showed similar activity profile in A375
cells, with no effect in cell viability up to 100 μg/mL, but
a dramatic reduction to <5% at 200 μg/mL.
Lastly, we examined whether the ester derivatives of
thujone exhibited cytotoxicity against the HCT-116 colon
cancer cell line. All seven ester derivatives affected cell
viability of HCT-116 cells to varying degrees, with treat-
ment of cells with 50 μg/mL of t-cinnamate (4),
t-isonicotinate (5), and t-nicotinate (6) resulting in reduction
of viability to 55%, 48% and 70%, respectively (Fig. 5).
Neither 50 or 100 μg/mL of t-benzoate (3), t-phenylacetate
(7), or t-thiophenate (9) has an appreciable effect on via-
bility. However, while t-thiophenate (9) exhibited a dose

Medicinal Chemistry Research
cells were treated with Staurosporine, a protein kinase
inhibitor that has been shown to potently induce apoptosis
of several human cancer cells (Tamaoki et al. 1986; Bruno
et al. 1992). As expected, PARP cleavage was detected in
HeLa cells treated with Staurosporine (Fig. 7a, lane 2).
Similarly, treatment of HeLa cells with 100 μg/mL of
t-cinnamate (4), t-isonicotinate (5), t-nicotinate (6), and
t-furoate (8) resulted in PARP cleavage, which is consistent
with caspase mediated apoptotic cell death (Fig. 7a, lanes
3–6). The role of caspases in mediating the death of HeLa
cells treated with the ester derivatives was further supported
by the ability of the pan caspase inhibitor Z-VAD-FMK to
significantly reduce PARP cleavage in t-furoate and
t-isonicotinate treated cells (Fig. 7b, lane 3 vs 4 and 5 vs 6).
In addition, we examined whether we could detect the
presence of activated effector caspase 3/7 in HeLa cells
treated with t-furoate and t-isonicotinate. While very few
control cells exhibited the presence of very low levels of
activated caspase 3/7 (Fig. 8a), nearly 100% of t-furoate and
t-isonicotinate treated cells exhibit very high levels of
activated caspase 3/7 (Fig. 8g, j). The fluorescence signal
detected in these cells is much brighter than seen in
Staurosporine treated cells (Fig. 8d vs g/j), which is con-
sistent with the degree of PARP cleavage. The ester deri-
vatives of thujone therefore potently induce apoptosis of
HeLa cells.
Fig. 9 The ester derivatives of thujone and not the hydrolysis products
are responsible for the cytotoxic effects. HeLa cells were treated with
250 µM of thujol, t-furoate, t-isonicotinate, Furoic acid (FA), Iso-
nicotinic acid (IsnA) for 24 h
dependent effect with increasing concentrations, treatment
of cells with 200 μg/mL of t-benzoate (3), and
t-phenylacetate resulted in viability being reduced to
61–65% with no further decrease in viability at higher
concentrations. T-isonicotinate (5) and t-nicotinate (6) were
the most cytotoxic to HCT-116 cells as viability decreased
to 6% (Fig. 5).
As the cytotoxic effects could potentially be mediated
by the hydrolysis of ester derivatives, we compared the
apoptosis inducing ability of t-isonicotinate (5) and
t-furoate (8), with their corresponding acids alone and in
combination with thujol. While treatment with 250 µM of
either t-furoate or t-isonicotinate reduced cell viability to
4% and 15%, respectively, treatment with neither their
corresponding acids alone nor in combination with thujol
had a significant effect on viability (Fig. 9). To further
confirm that the cytotoxic effects were not being mediated
by the hydrolysis products of the ester derivatives, we
examined the status of PARP cleavage (Fig. 10). PARP
cleavage was observed only in cells treated with t-furoate
(lane 3) and t-isonicotinate (lane 6) and not in cells treated
with either of their corresponding acids alone (lanes 4 and
7), or in cells treated with thujol and the acids combined
(lanes 5 and 8).
Ester derivatives of thujone induce apoptosis
To determine whether the cytotoxic effects were as a result
of apoptotic induction, we first examined the morphology
of HeLa cells after exposure to 100 μg/mL of thujone,
thujol, and the ester derivatives. Cells treated with α-thu-
jone, α/β-thujone (7:1), thujol (Fig. 6a) were mostly nor-
mal which is consistent with the cell viability data. Cells
treated with the ester derivatives of thujone (Fig. 6b)
exhibited morphology that is consistent with the cells
undergoing apoptosis. The most dramatic changes were
observed when cells were treated with t-cinnamate (4), t-
isonicotinate (5), t-nicotinate (6), and t-furoate (8), with a
significant proportion of cells appearing rounded and
detached from the substratum. This is consistent with the
cell viability data, as these four derivatives were able to
induce significant cytotoxicity when used at 50 or 100 μg/mL
concentrations.
As the morphology of the treated cells is consistent with
apoptotic cell death, we examined whether we could detect
PARP cleavage when cells were treated with the four
derivatives that exhibited the strongest effect on HeLa cell
viability. PARP, a 116 kDa nuclear protein is involved in
DNA repair and its proteolytic cleavage is a characteristic
biochemical feature of Caspase mediated apoptotic cell
death (Kaufmann et al. 1993). As a positive control, HeLa
Conclusions
We have examined seven ester derivatives of thujone and
compared their ability to induce the death of HeLa, A375, and
HCT-116 cells with that of thujone and thujol. In HeLa cells,
t-Cinnamate (4), t-isonicotinate (5), t-nicotinate (6), and t-
furoate (8) are more potent than the parent compounds, α-
thujone, α/β-thujone, and thujol in eliciting cell death at lower

Medicinal Chemistry Research
Bergqvist MS, Norin T (1963) Steric relationships in the thujane
series. Ark Kemi 22:137–149
Biswas R, Mandal SK, Dutta S, Bhattacharyya SS, Boujedaini N,
Khuda-Bukhsh AR (2011) Thujone-rich fraction of Thuja occi-
dentalis demonstrates major anti-cancer potentials: evidences
from in vitro studies on A375 cells. Evid Based Complement
Altern Med 2011:1–16
Bohlman F, Zeisberg R (1975) 13C NMR spektren von monoterpenen.
Org Magn Reson 7:426–432
Bruno S, Ardelt B, Skierski JS, Traganos F, Darzynkiewicz Z (1992)
Different effects of staurosporine, an inhibitor of protein kinases,
on the cell cycle and chromatin structure of normal and leukemic
lymphocytes. Cancer Res 52:470–473
Dai W, Sun C, Huang S, Zhou Q (2016) Carvacrol suppresses pro-
liferation and invasion in human oral squamous cell carcinoma.
Onco Targets Ther 9:2297–2304
Fig. 10 PARP cleavage is detected only in cells treated with the ester
derivatives. HeLa cells were treated with 250 µM of thujol (lane 2),
t-furoate (lane 3), Furoic acid (FA: lane 4), FA + Thujol (lane 5),
t-isonicotinate (lane 6), Isonicotinic acid (IsnA: lane 7), and IsnA +
Thujol (lane 8) for 4 h. SDS-PAGE and western blotting were per-
formed to detect PARP in the lysates of control and treated cells. Full-
length PARP is detected in lanes 1, 2, 4, 5, 7, and 8 while the 89 kDa
cleaved product is detected in lanes 3 and 5. Std. refers to protein
standard
Elegbede JA, Flores R, Wang RC (2003) Perillyl alcohol and peri-
llaldehyde induced cell cycle arrest and cell death in BroTo and
A549 cells cultured in vitro. Life Sci 73:2831–2840
Hach V, Fryberg EC, McDonald E (1971) Epimerization in the NaBH4
reduction of asymmetric ketones. Tetrahedron Lett 28:2629–2632
Hold KM, Sirisoma NS, Ikeda T, Narahashi T, Casida JE (2000)
Alpha-thujone (the active component of absinthe): gamma-
aminobutyric acid type A receptor modulation and metabolic
detoxification. Proc Natl Acad Sci USA 97:3826–3831
Jaganathan SK, Supriyanto E (2012) Antiproliferative and molecular
mechanism of eugenol-induced apoptosis in cancer cells. Mole-
cules 17:6290–6304
Jamali T, Kavoosi G, Safavi M, Ardestani SK (2018) In-vitro evaluation
of apoptotic effect of OEO and thymol in 2D and 3D cell cultures
and the study of their interaction mode with DNA. Sci Rep. 8:1–19
Kang SH, Kim YS, Kim EK, Hwang JW, Jeong JH, Dong X, Lee JW,
Moon SH, Jeon BT, Park PJ (2016) Anticancer effect of thymol
on AGS human gastric carcinoma cells. J Microbiol Biotechnol
26:28–37
concentrations. The same four derivatives were also cytotoxic
to A375 and HCT-116 cells, with t-furoate (8) being the most
effective against A375 cells, while t-isonicotinate (5) and t-
nicotinate (6) were the most effective in HCT-116 cells. We
also demonstrate that the derivatives induce caspase-mediated
apoptosis as evidenced by PARP cleavage and presence of
high levels of activated effector caspases.
Acknowledgements We are grateful to Susquehanna University for
financial support of this project. The Waters SYNAPT G2-S QTOFMS
system used for HRMS data was obtained by
a
grant
(# P20GM103430) from National Center for Research Resources
(NCRR). The JEOL ECZ 400S NMR spectrometer was acquired by a
grant to Susquehanna University from the National Science Founda-
tion (NSF:MRI CHE 1625340). The Thermo Scientific Nicolet iS50
FT-IR spectrometer was funded by the Sherman Fairchild Foundation.
Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG
(1993) Specific proteolytic cleavage of poly(ADP-ribose) poly-
merase: an early marker of chemotherapy-induced apoptosis.
Cancer Res 53:3976–3985
Mukhtar YM, Adu-Frimpong M, Xu X, Yu J (2018) Biochemical
significance of limonene and its metabolites: future prospects for
designing and developing highly potent anticancer drugs. Biosci
Rep 38:1–12.
Naser B, Bodinet C, Tegtmeier M, Lindequist U (2005) Thuja occi-
dentalis (Arbor vitae): a review of its pharmaceutical, pharma-
cological and clinical properties. Evid Based Complement Altern
Med 2:69–78
Özkan A, Erdoğan A (2011) A comparative evaluation of antioxidant
and anticancer activity of essential oil from Origanum onites
(Lamiaceae) and its two major phenolic components. Turk J Biol
35:735–742
Privitera G, Luca T, Castorina S, Passanisi R, Ruberto G, Napoli E
(2019) Anticancer activity of Salvia officinalis essential oil and its
principal constituents against hormone-dependent tumour cells.
Asian Pac J Trop Biomed 9:24–28
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher′s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
Al-Fatlawi AA, Rahisuddin AA (2014) Cytotoxicity and pro-apoptotic
activity of carvacrol on human breast cancer cell line MCF-7.
World J PharmSci 2:1218–1223
Rajput JD, Bagul SD, Hosamani AA, Patil MM, Bendre RS (2017)
Synthesis, characterizations, biological activities and docking
studies of novel dihydroxy derivatives of natural phenolic
monoterpenoids containing azomethine linkage. Res Chem
Intermed 43:5377–5393
Alves LDS, Figueirêdo CBM, Silva CCAR, Marques GS, Ferreira PA,
Soares MFR, Silva RMF, Rolim-Neto PJ (2014) Thuja occi-
dentalis L. (Cupressaceae): review of botanical, phytochemical,
pharmacological and toxicological aspects. Int J Pharm Sci Res
9:1163–1177
Sobral MV, Xavier AL, Lima TC, de Sousa DP (2014) Antitumor
activity of monoterpenes found in essential oils. Sci World J
2014:1–35
Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F
(1986) Staurosporine, a potent inhibitor of phospholipid/Ca+
Banthorpe DV, Davies HfS (1968) Stereochemistry of reduction of
(−)-thujone and (−)-isothujone. J Chem Soc (B): 1356–1360
Bardon S, Foussard V, Fournel S, Loubat A (2002) Monoterpenes
inhibit proliferation of human colon cancer cells by modulating
cell cycle-related protein expression. Cancer Lett 181:187–194

Medicinal Chemistry Research
+dependent protein kinase. Biochem Biophys Res Commun
135:397–402
4′-demethylepipodophyllotoxin derivates exhibiting anti-
tumor activity by activating ATM/ATR signaling pathways.
Eur J Med Chem 80:267–277
Torres A, Vargas Y, Uribe D, Carrasco C, Torres C, Rocha R, Oyarzun
C, San Martin R, Quezada C (2016) Pro-apoptotic and anti-
angiogenic properties of the alpha/beta-thujone fraction from Thuja
occidentalis on glioblastoma cells. J Neurooncol 128:9–19
Vandresen F, Falzirolli H, Almeida Batista SA, da Silva-Giardini AP,
de Oliveira DN, Catharino RR, Ruiz AL, de Carvalho JE, Foglio
MA, da Silva CC (2014) Novel R-(+)-limonene-based thiose-
micarbazones and their antitumor activity against human tumor
cell lines. Eur J Med Chem 79:110–116
Yeruva L, Pierre KJ, Elegbede A, Wang RC, Carper SW (2007)
Perillyl alcohol and perillic acid induced cell cycle arrest and
apoptosis in non small cell lung cancer cells. Cancer Lett
257:216–226
Yin QH, Yan FX, Zu XY, Wu YH, Wu XP, Liao MC, Deng SW, Yin
LL, Zhuang YZ (2012) Anti-proliferative and pro-apoptotic effect
of carvacrol on human hepatocellular carcinoma cell line HepG-
2. Cytotechnology 64:43–51
Xiao L, Zhao W, Li HM, Wan DJ, Li DS, Chen T, Tang YJ (2014)
Design and synthesis of the novel DNA topoisomerase
II inhibitors: esterification and amination substituted
Zhou Y, Liu JQ, Zhou ZH, Lv XT, Chen YQ, Sun LQ, Chen FX
(2016) Enhancement of CD3AK cell proliferation and killing
ability by alpha-Thujone. Int Immunopharmacol 30:57–61