Terpene conjugates of the Nigella sativa seed-oil constituent thymoquinone with enhanced efficacy in cancer cells

Article abstract of DOI:10.1002/cbdv.200900328

Thymoquinone (TQ; 1) is a weak anticancer constituent of black seed oil. Derivatives bearing terpene-terminated 6-alkyl residues were tested in cells of human HL-60 leukemia, 518A2 melanoma, multidrug-resistant KB-V1/Vbl cervix, and MCF-7/Topo breast carcinomas, as well as in non-malignant human foreskin fibroblasts. Derivatives with a short four-atom spacer between quinone and cyclic monoterpene moieties were more antiproliferative than analogues with longer spacers. 6-(Menthoxybutyryl) thymoquinone (3a) exhibited single-digit micromolar IC₅₀ (72 h) values in all four cell lines. It was seven times more active than TQ (1) in 518A2 melanoma cells and four times in KB-V1/Vbl cervix carcinoma cells, while only half as toxic in the fibroblasts. Compound 3a was also not a substrate for the Pgp and BCRP drug transporters of the resistant cancer cells. The caryophyllyl and germacryl conjugates 3e and 3f specifically inhibited the growth of the resistant MCF-7 breast carcinoma cells. Conjugation of TQ with the triterpene betulinic acid via the OH group as in 3g led to a loss in activity, while conjugation via the carboxylic acid afforded compound 4 with nanomolar IC₅₀ (72 h) activity against HL-60 cells. All anticancer-active derivatives of TQ (1) induced apoptosis associated with DNA laddering, a decrease in mitochondrial membrane potential and a slight increase in reactive oxygen species.

Full text of DOI:10.1002/cbdv.200900328

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Terpene Conjugates of the Nigella sativa Seed-Oil Constituent
Thymoquinone with Enhanced Efficacy in Cancer Cells
by Katharina Effenberger, Sandra Breyer, and Rainer Schobert*
Organisch-chemisches Laboratorium der Universitꢀt Bayreuth, Universitꢀtsstraße 30, NW 1,
D-95447 Bayreuth (fax: þ49(0)921552671; e-mail: Rainer.Schobert@uni-bayreuth.de)
Thymoquinone (TQ; 1) is a weak anticancer constituent of black seed oil. Derivatives bearing
terpene-terminated 6-alkyl residues were tested in cells of human HL-60 leukemia, 518A2 melanoma,
multidrug-resistant KB-V1/Vbl cervix, and MCF-7/Topo breast carcinomas, as well as in non-malignant
human foreskin fibroblasts. Derivatives with a short four-atom spacer between quinone and cyclic
monoterpene moieties were more antiproliferative than analogues with longer spacers. 6-(Menthoxy-
butyryl)thymoquinone (3a) exhibited single-digit micromolar IC50 (72 h) values in all four cell lines. It
was seven times more active than TQ (1) in 518A2 melanoma cells and four times in KB-V1/Vbl cervix
carcinoma cells, while only half as toxic in the fibroblasts. Compound 3a was also not a substrate for the P-
gp and BCRP drug transporters of the resistant cancer cells. The caryophyllyl and germacryl conjugates
3e and 3f specifically inhibited the growth of the resistant MCF-7 breast carcinoma cells. Conjugation of
TQ with the triterpene betulinic acid via the OH group as in 3g led to a loss in activity, while conjugation
via the carboxylic acid afforded compound 4 with nanomolar IC50 (72 h) activity against HL-60 cells. All
anticancer-active derivatives of TQ (1) induced apoptosis associated with DNA laddering, a decrease in
mitochondrial membrane potential and a slight increase in reactive oxygen species.
Introduction. – Thymoquinone (TQ; 1) is the active principle of thyme essential oils
and the essential oil of black seed (Nigella sativa), responsible for many of its
antioxidant, anti-inflammatory [1], and antineoplastic effects [2]. TQ (1) is of low
general toxicity yet showed weak antitumor effects in numerous cancer-cell studies and
animal models [3–6]. The molecular mechanism of its action is not fully understood,
yet. In certain xenograft models, it was found to delay tumor growth by induction of
cell-cycle arrest. In in vitro experiments, it induced apoptosis both in p53-dependent
and p53-independent ways [7][8]. Lately, the serine/threonine Polo-like kinases (Plk)
which are overexpressed in many types of human cancers have been identified as
targets for TQ. In HeLa cells, it effected Plk mislocalization, chromosome congression
defects, mitotic arrest, and apoptosis [9].
In a preceding article, we have shown that the cell-line specificity and the anticancer
activity of TQ can be significantly increased by covalent attachment of fatty acyl
residues of defined length and degree of unsaturation [10]. Interestingly, the most
active compounds resulted from conjugation with fatty acids that are present in the
fixed oil of black seed. In continuation of this study, we now prepared and bio-
evaluated conjugates of TQ with various monoterpenes, sesquiterpenes, and the
cytotoxic triterpene betulinic acid [11][12] to identify those structural parameters that
are associated with anticancer activity. Terpenes were also found in the essential oil of
N. sativa [13]. We attached esters of various terpene alcohols to C(6) of TQ via spacers
ꢁ 2010 Verlag Helvetica Chimica Acta AG, Zꢂrich

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of variable length. The resulting derivatives 3 were tested for growth inhibition of cells
of the human cancer cell lines HL-60 leukemia, 518A2 melanoma, KB-V1/Vbl cervix
carcinoma, and MCF-7/Topo breast adenocarcinoma, and of non-malignant foreskin
fibroblasts. Their effects on the mitochondrial membrane potential and the cellular
levels of reactive oxygen species (ROS) were also scrutinized.
Results and Discussion. – Chemistry. 3-[2-Methyl-5-(1-methylethyl)-3,6-dioxocy-
clohexa-1,4-dienyl]alkanoic acids 2 were obtained by treating mixtures of the
respective dicarboxylic acid and TQ (1) with a solution of Ag2S2O8 in H2O/MeCN
according to a general procedure [14] (Scheme). The acids 2 were then converted to the
conjugates 3 by reaction with the respective terpene alcohol, pivalic anhydride, and 4-
dimethylaminopyridine (DMAP) according to the general mixed-anhydride protocol
[15]. In this way, esters 3 were obtained of 3-(2-methyl-5-(1-methylethyl)-3,6-
dioxocyclohexa-1,4-dienyl)propanoic acid (2a) with (ꢀ)-menthol (! 3a), (ꢀ)-borneol
(! 3b), (þ)-fenchol (! 3c), (ꢀ)-carveol (! 3d), 14-hydroxycaryophyllene (! 3e),
germacrol (! 3f), and betulinic acid (! 3g). 14-Hydroxycaryophyllene was obtained
by oxidation of (ꢀ)-caryophyllene [16] with SeO2 by a known procedure [17].
Germacrol was prepared by LiAlH4 reduction of germacrone [18] which, in turn, was
crystallized from Zdravets oil (Geranium macrorrhizum).
Additionally, conjugates of (ꢀ)-menthol with a C6 spacer (! 3a3) or a C9 spacer (!
3a6) were prepared from 5-[2-methyl-5-(1-methylethyl)-3,6-dioxocyclohexa-1,4-dien-
yl]pentanoic acid 2b or 8-[2-methyl-5-(1-methylethyl)-3,6-dioxocyclohexa-1,4-dienyl]-
octanoic acid 2c, respectively. Three conjugates with C12 spacers were prepared from
11-(2-methyl-5-(1-methylethyl)-3,6-dioxocyclohexa-1,4-dienyl)undecanoic acid 2d and
either (ꢀ)-menthol (! 3a9), or (ꢀ)-borneol (! 3b9), or (þ)-fenchol (! 3c9).
Since esters and amides of betulinic acid were occasionally found to be more
anticancer active than the free acid [19], we also synthesised conjugate 4 by
esterification of betulinic acid with 3-(3-hydroxypropyl)-2-methyl-5-(1-methylethyl)-
cyclohexa-2,5-diene-1,4-dione, which was available by reduction of 2a with NaBH4 /I2
[20].
Growth Inhibition. The terpene conjugates 3 and 4 were tested for growth inhibition
in cells of human HL-60 leukemia, 518A2 melanoma, P-gp-rich KB-V1/Vbl cervix
carcinoma, BCRP-rich MCF-7/Topo breast carcinoma by means of the standard MTT
(¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium hydrobromide) assay.
They showed distinctly different activity profiles (Fig.). The most active derivatives
3 and 4 were also tested in non-malignant human foreskin fibroblasts (HF). The results
were compared with those of the parent compound TQ (1). The IC50 values after 72 h
exposure to the test compounds are summarized in Table 1. We checked that mixtures
of conceivable ester hydrolysis products of all included compounds had at least a
tenfold greater IC50 value.
In all tested cell lines, the monoterpene derivatives with longer side chains, i.e., 3a6,
3a9, 3b9, 3c9, were much less active than the analogues with the same terpene appendage
but shorter spacers, i.e., 3a, 3a3, 3b, 3c, and even less active than TQ (1) itself. This is
reminiscent of the structure–activity relationship we had found earlier for diamino-
platinum(II) complex–terpene conjugates [21]. Derivative 3a was the most active one
on average in this series exhibiting single-digit micromolar IC50 values after 72 h

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
131
Scheme. Thymoquinone-Terpene Conjugates 3 and 4
i) HO2Cꢀ(CH2)nꢀCO2H, AgNO3, (NH4)2S2O8, MeCN/H2O, 1008, 12 h. ii) Pivalic anhydride, 4-
(dimethylamino)pyridine (DMAP), terpene alcohol, 508, 12 h. iii) NaBH4, I2, THF, reflux, 12 h. iv)
Pivalic anhydride, DMAP, betulinic acid, 508, 12 h.
incubation in all four cancer cell lines. It was particularly efficacious in the melanoma
cells (IC50 ¼3.9ꢁ0.7 mm) and in the resistant cervix carcinoma cells (IC50 ¼7.0 ꢁ1.7 mm),
thus surpassing the parent compound TQ (1) by factors of seven and four, respectively.
Gratifyingly, in the control fibroblasts, compound 3a exerted only half the toxicity of
TQ. Its ratio of IC50 values in the HF vs. in the melanoma cells was 17.6, which is great
enough to deliberate further pharmaceutical tests.
In cells of the multidrug-resistant MCF-7/Topo breast carcinoma, most derivatives
with shorter spacers, i.e., 3a, 3a3, 3b, 3c, 3e, and 3f were active at IC50 values better than

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Figure. Cell-line specificities of thymoquinone (1), and its terpene conjugates 3 and 4 as deviation of the
log (IC50/72 h) of individual derivatives from the mean over all compounds 1, 3, and 4 log (IC50/72 h)
values for a given cell line. Negative values indicate higher, positive values lower than average activities.
Mean log (IC50/72 h): ꢀ4.7 (518A2), ꢀ4.9 (HL-60), ꢀ4.6 (KB-V1/Vbl), and ꢀ4.8 (MCF-7/Topo).
6 mm. The conjugates 3b, 3e, and 3f achieved this effect also in HL-60 leukemia cells.
The betulinic acid conjugates differed significantly in their antiproliferative activity.
The carboxylic acid 3g showed much higher IC50 (72 h) values than the alcohol 4 in all
cancer cell lines and was also less active than TQ in all cell lines except in HL-60
leukemia. In contrast, conjugate 4 was highly efficacious, especially against HL-60 cells
(IC50 ¼130ꢁ20 nm) where it surpassed TQ (1) by a factor of ca. 200.
We also tested the most interesting terpene derivatives of TQ for their potential to
overcome the multidrug resistance of KB-V1/Vbl and MCF-7/Topo cells. This was
achieved by incubating these cells with the test compounds in the presence of selective
inhibitors of their respective ABC-transporters, i.e., with 24 mm verapamil hydro-
chloride added to the P-gp overexpressing KB-V1/Vbl cervix carcinoma cells, and with
1.2 mm fumitremorgin C added to the BCRP-rich MCF-7/Topo breast cancer cells [22].
Ideally, a drug should not be affected by ABC-transporters and thus should be
characterized by a ratio R of its IC50 values with and without addition of specific
inhibitors close to 1. This was the case for derivatives 3a (R(KB-V1/Vbl)¼1.3;
R(MCF-7/Topo)¼1.1) and 3b (R(KB-V1/Vbl)¼0.8; R(MCF-7/Topo)¼0.8). While
3c, 3e, and 3f were good substrates for the P-gp transporter, 3a and 3b were not pumped
by P-gp. No derivative except 3g was pumped by the BCRP transporter. This could
explain the lower antiproliferative effect of 3g when compared to 4. Paradoxically,
compound 4 was even less active in the presence of the inhibitors verapamil
hydrochloride or fumitremorgin C. This effect could originate from a direct interaction
between inhibitors and conjugate. Alternatively, it is known that such inhibitors do not
only affect the efflux of anticancer drugs but also their uptake [23].
Apoptosis Induction. That compounds 3 and 4 act by inducing apoptosis in cancer
cells was first visualized by distinct DNA fragmentation (laddering) in HL-60 cells
upon treatment with TQ (1), 4, or selected derivatives 3 at 5 mm. As apoptosis

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
133
Table 1. Inhibitory Concentrationsa) IC50 [mm] of TQ (1), and Conjugates 3 and 4 When Applied for 72 h
to 518A2 Melanoma, HL-60 Leukemia, KB-V1/Vbl Cervix Carcinoma, and MCF-7/Topo Breast-
Carcinoma Cells, and Human Foreskin Fibroblasts (HF)
Compound/Cell 518A2
HL-60
KB-V1/Vbl
MCF-7/Topo
26.7 (ꢁ 5.6) 32.6 (ꢁ 20.0)
23.1 (ꢁ 2.5)
HF
TQ (1)
TQ (1)b)
3a
28.3 (ꢁ 9.2)
27.8 (ꢁ 6.0)
32.3 (ꢁ 6.0)
24.1 (ꢁ 3.5)
7.0 (ꢁ 1.7)
9.3 (ꢁ 4.8)
9.2 (ꢁ 2.5)
–
–
–
3.9 (ꢁ 0.7)
9.0 (ꢁ 2.9)
5.4 (ꢁ 1.8) 68.6 (ꢁ 4.9)
3ab)
3a3
–
–
5.8 (ꢁ 3.8)
5.5 (ꢁ 1.7)
–
–
–
–
14.7 (ꢁ 1.8)
11.7 (ꢁ 2.3)
3a6
>100
>100
35.1 (ꢁ 10.1) >100
41.3 (ꢁ 10.1) >100
3a9
>100
13.3 (ꢁ 5.6)
>100
2.3 (ꢁ 0.5)
3b
17.6 (ꢁ 3.9)
14.5 (ꢁ 3.7)
42.4 (ꢁ 8.0)
27.7 (ꢁ 5.9)
14.5 (ꢁ 3.3)
44.7 (ꢁ 8.9)
12.4 (ꢁ 1.4)
23.6 (ꢁ 1.6)
10.0 (ꢁ 4.5)
17.9 (ꢁ 1.1)
9.5 (ꢁ 5.2)
46.2 (ꢁ 3.0)
79.9 (ꢁ 2.0)
5.6 (ꢁ 0.1) 41.5 (ꢁ 6.8)
3bb)
3b9
–
–
4.6 (ꢁ 2.5)
–
–
51.7 (ꢁ 4.8) >100
>100
3c
14.1 (ꢁ 6.8)
10.8 (ꢁ 3.1)
6.1 (ꢁ 1.0) 59.0 (ꢁ 21.8)
3cb)
3c9
–
–
7.3 (ꢁ 2.7)
>100
14.0 (ꢁ 5.1)
–
–
–
60.0 (ꢁ 4.2) >100
3d
6.8 (ꢁ 0.6)
9.4 (ꢁ 1.3)
3e
13.1 (ꢁ 2.3)
4.7 (ꢁ 1.1)
3.1 (ꢁ 0.6) 23.9 (ꢁ 0.9)
3.5 (ꢁ 0.5)
2.8 (ꢁ 1.8) 48.1 (ꢁ 14.1)
4.5 (ꢁ 2.1)
33.6 (ꢁ 1.2) 46.2 (ꢁ 5.5)
13.6 (ꢁ 5.9)
10.0 (ꢁ 2.5) 24.5 (ꢁ 8.1)
44.2 (ꢁ 7.2)
3eb)
3f
–
–
–
12.2 (ꢁ 1.5)
2.5 (ꢁ 1.7)
3fb)
3g
–
–
–
53.3 (ꢁ 3.2)
13.7 (ꢁ 9.5)
3gb)
4
–
–
–
11.4 (ꢁ 2.7)
0.13 (ꢁ 0.02) 13.1 (ꢁ 3.5)
4b)
–
–
68.0 (ꢁ 6.1)
–
a) Values are derived from concentration–response curves obtained by measuring the percentual
absorbance of viable cells relative to untreated controls (100%) after 72 h exposure of 518A2 melanoma,
HL-60 leukemia, KB-V1/Vbl cervix carcinoma, and MCF-7/Topo breast-carcinoma cells, as well as
human foreskin fibroblasts (HF) to the test compounds in the MTTassay. Values represent means of four
independent experimentsꢁstandard deviation. b) With 24 mm verapamil hydrochloride added in the case
of KB-V1/Vbl cells, or with 1.2 mm fumitremorgin C added in the case of MCF-7/Topo cells.
proceeding via the intrinsic pathway is associated with a damage of the mitochondria, a
release of cytochrome c, and an activation of caspase-9, we also analyzed the changes of
the mitochondrial membrane potential DYm of 518A2 and HL-60 cells upon treatment
with the compounds 1, 3, and 4 at 5 mm for 24 and 72 h using a kit from Stratagene, which
is based on the fluorescent cationic dye JC-1 [24]. The ratio of red (JC-1 aggregates in
intact mitochondria) to green fluorescence (JC-1 monomers in the cytosol) is
decreased in apoptotic cells. In HL-60 cells, we found the most pronounced reductions
of cells with intact mitochondrial membranes upon treatment for 72 h with TQ (1)
(83%), 3a (78%), and 3b (80%), relative to untreated vital control cells (100%). In
contrast, in 518A2 cells, all tested compounds, i.e., 3a, 3a3, 3a6, 3a9, 3d, and 3e, merely
led to a small decrease of cells with intact mitochondria (90ꢁ5%).
There is a good deal of evidence for reactive oxygen species (ROS) playing a
pivotal role for the anticancer activity as well as for unwanted side effects of established
drugs featuring the p-quinone motif, e.g., doxorubicin [25]. Hence, we also assessed the
ability of the compounds 1, 3a, 3b, 3c, 3e, 3f, 3g, 3h, and 4 at 5 mm concentrations to

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initiate ROS generation in HL-60 and 518A2 cells by the colorimetric nitroblue-
tetrazolium (NBT) assay [26][27]. It is based on the selective reduction of a yellow,
water-soluble tetrazolium chloride to an insoluble violet diformazan by superoxide
.
(O2 ꢀ ). Conjugate 3a, which had the greatest growth-inhibiting effect in 518A2 cells in
the MTT assay, led, like all other test compounds, to similar ROS levels in these cells
after 72 h as TQ (1). In HL-60 leukemia cells, the pattern of ROS generation was more
heterogenous. Two compounds that were quite growth-inhibitory in HL-60 cells in the
MTT assays also led to considerable ROS levels. Compound 4 initiated ROS levels six
times that caused by TQ, and the caryophyllene derivative 3e effected three times more
ROS than TQ. So, there was some coherence between growth inhibition and ROS
initiation in case of the HL-60 cells.
Conclusions. – We prepared derivatives of the weak anticancer drug thymoquinone
(1) with covalently attached terpene residues. Some of these were far more efficacious
in certain cancer cell lines than the parent drug, while being considerably less toxic to
non-malignant fibroblasts. Generally, their efficacy in cancer cells was dependent both
on the nature of the terpene, and the length of the alkyl spacer between p-quinone and
terpene moieties. Most active were derivatives with short C3 spacers. The correspond-
ing menthol conjugate 3a exhibited single-digit micromolar IC50 (72 h) values in all four
tested cancer cell lines, and showed the greatest activity of all compounds in cells of
518A2 melanoma and multidrug-resistant KB-V1/Vbl cervix carcinoma, while being
virtually nontoxic to non-malignant human fibroblasts. It was also least affected by the
resistance-relevant ABC-transporters P-gp and BCRP. The caryophyllene and
germacrone conjugates 3e and 3f, respectively, achieved the best results of all test
compounds against multidrug-resistant MCF-7 breast carcinoma cells. The betulinic
acid derivatives 3g and 4 which differed in the mode of connection also were of quite
different antiproliferative activities. Conjugate 4 connected via its acid group was ca.
200 times more efficacious in HL-60 cells than the parent drug TQ (IC50 ¼130ꢁ20 nm).
This finding is in line with recent reports by other groups on bioactive C(17)-esters of
betulinic acid [28][29]. Further investigations such as assessment of caspase kinetics
and involvement of pro-/anti-apoptotic proteins are now underway to identify the
precise mechanism of apoptosis induced by conjugates of type 3.
This work was supported by the Deutsche Forschungsgemeinschaft (grant Scho 402/8-2). We thank
Dr. Reinhard Paschke (Biocenter of the Martin-Luther-University Halle-Wittenberg) for a gift of
betulinic acid and the Paul Kaders GmbH (Hamburg) for a batch of Zdravets oil.
Experimental Part
General. All reagent-grade chemicals were purchased from Aldrich, Merck, Acros, Alfa Aesar, and
ABCR. All solvents were dried and distilled before use. Germacrone was crystallized from Zdravets oil
according to the known procedure [30][31]. Column chromatography (CC): SiO2 60 (230–400 mesh).
Optical rotations: Perkin-Elmer polarimeter 241at 589 nm. IR Spectra: Perkin-Elmer Spectrum One FT-
IR spectrophotometer equipped with an ATR sampling unit. NMR Spectra: Bruker Avance 300
spectrometer; chemical shifts (d) are given in ppm downfield from Me4Si as internal standard for 1H and
13C. EI-MS: Varian MAT 311A. Microanalyses: Perkin-Elmer 2400 CHN elemental analyzer; correct
analyses (within ꢁ0.4% for C, H, N) were obtained for all new compounds 2, 3, and 4.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Chemistry1). Synthesis and Characterization of the Thymoquinone–Terpene Conjugates 3 (see
Table 2). Synthesis of Acids 2: Typical Procedure. 3-(2-Methyl-5-(1-methylethyl)-3,6-dioxocyclohexa-1,4-
dienyl)propanoic acid (2a). A mixture of TQ (2-methyl-5-(1-methylethyl)cyclohexa-2,5-diene-1,4-dione;
1; 500 mg, 3.05 mmol), succinic acid (288 mg, 2.44 mmol), a cat. amount of AgNO3 and MeCN/H2O, 1 : 1
(30 ml) was heated to 1008. A soln. of (NH4)2S2O8 (695 mg, 3.05 mmol) in H2O (3.05 ml) was added
dropwise, and the resulting mixture was heated under reflux overnight, then cooled to r.t., diluted with
H2O, and extracted repeatedly with Et2O. The combined Et2O extracts were washed with a sat. aq. soln.
of NaHCO3, acidified with conc. aq. HCl, extracted with CH2Cl2, and dried (Na2SO4). The volatiles were
evaporated, and the residue was purified by CC (silica gel; AcOEt/cyclohexane 1:1) to afford pure 2a
(302 mg, 52%). Yellow oil. Rf (AcOEt/cyclohexane 1:1) 0.39. IR (ATR): 2965, 1707, 1643, 1417, 1381,
1248, 1177, 1039, 892, 816, 709. 1H-NMR (300 MHz, CDCl3): 10.8–9.8 (br., 1 H); 6.42 (d, J¼1.1, 1 H);
2.95 (dsept., J¼6.9, J¼1.1, 1 H); 2.75 (t, J¼7.9, 2 H); 2.44 (t, J¼7.9, 2 H); 1.97 (s, 3 H); 1.03 (d, J¼6.9,
6 H). 13C-NMR (75.5 MHz, CDCl3): 187.9; 186.5; 178.2; 154.6; 142.6; 141.1; 129.9; 32.4; 29.0; 26.6; 21.9;
21.2. EI-MS: 236 (4, Mþ ), 155 (7), 138 (100), 101 (47), 95 (100), 55 (34), 41 (26).
Synthesis of Thymoquinone–Terpene Esters 3. Typical Procedure. (ꢀ)-Menthyl 3-(2-Methyl-5-(1-
methylethyl)-3,6-dioxocyclohexa-1,4-dienyl)propanoate (3a). Acid 2a (130 mg, 0.55 mmol), (ꢀ)-menthol
(77 mg, 0.49 mmol), pivalic anhydride (0.11 ml, 102 mg, 0.55 mmol), and a cat. amount of DMAP were
dissolved in CHCl3 (30 ml), and the resulting mixture was heated at 508 overnight. H2O was added, and
the solvent was evaporated. The residue was dissolved in Et2O, the resulting mixture was washed with a
sat. aq. NaHCO3 and brine, dried (Na2SO4), and concentrated in vacuum. The residue thus obtained was
purified by CC (silica gel; AcOEt/cyclohexane 1:10) to afford pure 3a (110 mg, 60%). Yellow oil. Rf
(AcOEt/cyclohexane 1:1) 0.83. [a]2D4 ¼ ꢀ39.9 (c¼0.5, CHCl3). IR (ATR): 2956, 2928, 2871, 1729, 1702,
1647, 1456, 1369, 1248, 1173, 1149, 982. 1H-NMR (300 MHz, CDCl3): 6.43 (d, J¼1.2, 1 H); 4.62 (ddd, J¼
4.5, J¼10.8, J ¼11.8, 1 H); 2.99 (dsept., J¼6.9, J¼1.2, 1 H); 2.38 (m, 4 H); 2.00 (s, 3 H); 1.9–1.2 (m,
9 H); 1.06 (d, J¼6.9, 6 H); 0.82 (dd, J¼7.0, J¼2.5, 6 H); 0.66 (d, J¼6.9, 3 H). 13C-NMR (75.5 MHz,
CDCl3): 188.0; 186.7; 171.9; 154.6; 143.1; 141.1; 130.0; 74.4; 46.9; 34.2; 33.8; 33.1; 31.3; 26.3; 23.5; 22.6;
21.9; 21.4; 20.7; 16.3. EI-MS: 374 (3, Mþ ), 238 (100), 236 (46), 220 (59), 175 (19), 138 (100), 83 (100), 57
(44).
9-{3-[2-Methyl-5-(1-methylethyl)-3,6-dioxocyclohexa-1,4-dienyl]propanoyloxy}-5b,8,8,11a,11b-pen-
tamethyl-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-3a-carboxylic Acid (3g). Acid 2a
(30 mg, 0.12 mmol), pivalic anhydride (0.02 ml, 22 mg, 0.12 mmol), and a cat. amount of DMAP were
dissolved in CHCl3 (25 ml), and the resulting mixture was stirred at 508 for 2 h. Betulinic acid (50 mg,
0.11 mmol) was added, and stirring at 508 was continued overnight. H2O was added, and the volatiles
were evaporated. The remainder was dissolved in Et2O, the resulting mixture was washed with sat. aq.
NaHCO3 and brine, dried (Na2SO4), and concentrated in vacuum. The residue was purified by CC (silica
gel; AcOEt/cyclohexane 1:10) to afford pure 3g (29 mg, 36%). Yellow oil. Rf (AcOEt/cyclohexane 1:1)
0.77. [a]2D4 ¼ þ8.7 (c¼0.5, CHCl3). IR (ATR): 3407, 2962, 2929, 2855, 1719, 1649, 1455, 1379, 1285, 1262,
1160, 1107, 1057, 951, 739. 1H-NMR (300 MHz, CDCl3): 9.64 (br., 1 H); 6.46 (d, J¼1.1, 1 H); 4.71 (s, 1 H);
4.49 (s, 1 H); 4.43 (m, 1 H); 3.01 (dsept., J¼6.9, J¼1.1, 1 H); 2.81 (br., 1 H); 2.79 (t, J¼8.5, 2 H); 2.41 (t,
J¼8.5, 2 H); 2.0–1.1 (m, 22 H); 1.21 (s, 3 H); 1.08 (d, J¼6.9, 6 H); 0.94 (m, 6 H); 0.81 (m, 3 H); 0.77 (m,
3 H). 13C-NMR (75.5 MHz, CDCl3): 188.1; 186.7; 181.3; 172.3; 154.7; 149.9; 143.2; 141.1; 130.1; 109.9;
81.4; 56.3; 55.4; 50.3; 49.2; 46.5; 42.4; 40.7; 38.4; 37.9; 37.8; 37.0; 34.2; 33.9; 33.2; 31.7; 30.2; 29.7; 26.9; 26.5;
23.7; 22.6; 21.4; 20.9; 19.3; 18.2; 16.5; 16.2; 15.9; 14.6. EI-MS: 674 (2, Mþ ), 628 (3), 439 (27), 395 (11), 274
(19), 238 (100), 189 (63), 121 (26), 57 (48).
Synthesis of 3-(3-Hydroxypropyl)-2-methyl-5-(1-methylethyl)-cyclohexa-2,5-diene-1,4-dione. A soln.
of I2 (107 mg, 0.85 mmol) in THF (3 ml) was added dropwise during 30 min to a soln. of acid 2a (200 mg,
0.85 mmol) and NaBH4 (77 mg, 2.04 mmol) in THF (20 ml). After gas evolution had ceased, the mixture
was heated under reflux overnight, then cooled to r.t., and the reaction was quenched with MeOH. The
volatiles were evaporated, and the remainder was taken up in aq. KOH (20%) and repeatedly extracted
with Et2O. The combined org. layers were washed with brine, dried (Na2SO4), and concentrated in
1
)
Synthetic details and characterizations of all new compounds are available, free of charge, from the
authors.

136
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 2. NMR Data (in CDCl3) and Optical Rotations (c¼0.5, CHCl3) of All New Thymoquinone-
Terpene Conjugates 3 and Thymoquinone Mono Acids 2. Chemical shifts (d) are given in ppm downfield
from Me4Si as internal standard for 1H (300 MHz) and 13C (75.5 MHz).
d(H)
d(C)
[a]2D4
2b 6.46 (d, J¼1.1, 1 H); 3.01 (dsept., J¼6.9,
J¼1.1, 1 H); 2.49 (t, J¼7.9, 2 H);
188.3; 186.9; 179.2; 154.6; 144.6;
134.2; 129.9; 33.6; 26.7; 26.3;
25.6; 24.7; 21.4; 11.7
–
2.37 (t, J¼7.5, 2 H); 1.99 (s, 3 H);
1.8 – 1.4 (m, 4 H); 1.08 (d, J¼6.9, 6 H)
2c 6.45 (d, J¼1.2, 1 H); 3.01 (dsept.,3J¼6.9,
J¼1.2, 1 H); 2.44 (t, J¼7.8, 2 H);
188.2; 186.0; 179.8; 154.5; 145.6;
136.9; 129.9; 33.9; 29.6; 28.9; 28.7;
28.5; 26.6; 26.1; 24.5; 21.1; 13.4
–
–
2.32 (t, J¼7.4, 2 H); 1.98 (s, 3 H);
1.7 – 1.2 (m, 10 H); 1.08 (d, J¼6.9, 6 H)
2d 6.44 (d, J¼1.1, 1 H); 3.01 (dsept., J¼6.9,
188.2; 187.6; 180.1; 154.7; 145.3;
139.3; 130.0; 34.1; 29.9; 29.8; 29.3;
29.2; 29.0; 28.8; 26.7 24.6; 21.2; 12.0
J¼1.1, 1 H); 2.30 (t, J¼7.4, 2 H);
2.30 (t, J¼7.6, 2 H); 1.95 (s, 3 H);
1.3–1.1 (br., 16 H); 1.07 (d, J¼6.9, 6 H)
3a3 6.45 (d, J¼1.1, 1 H); 4.64 (ddd, J¼4.4,
J¼10.9, J¼11.9, 1 H); 3.01 (dsept., J¼6.9,
J¼1.1, 1 H); 2.48 (t, J¼7.5, 2 H);
2.29 (t, J¼7.5, 2 H); 1.98 (s, 3 H);
1.9 – 1.3 (m, 13 H); 1.08 (d, J¼6.9, 6 H);
0.86 (dd, J¼7.0, J¼2.8, 6 H);
188.3; 186.8; 172.9; 154.5; 144.7;
140.1; 129.9; 74.0; 46.9; 34.4; 34.3;
31.3; 29.1; 28.1; 26.4; 26.3; 25.2;
23.4; 21.9; 21.4; 20.7; 16.3
ꢀ35.8
0.72 (d, J¼6.9, 3 H)
3a6 6.44 (d, J¼1.2, 1 H); 4.64 (ddd, J¼4.3,
J¼10.9, J¼11.5, 1 H); 2.99 (dsept., J¼6.9,
J¼1.2, 1 H); 2.24 (t, J¼7.7, 2 H);
1.95 (t, J¼7.7, 2 H); 1.94 (s, 3 H);
1.08 (d, J¼6.9, 6 H); 1.9–1.0 (m, 19 H);
0.87 (dd, J¼6.5, J¼2.4, 6 H);
187.9; 186.8; 172.8; 154.5; 144.6; 140.0;
129.7; 73.9; 46.9; 40.9; 34.7; 34.3; 31.3;
29.0; 28.9; 28.8; 26.6; 26.5; 26.2; 25.0;
23.4; 21.9; 20.7; 16.3; 13.0
ꢀ41.3
0.72 (d, J¼6.9, 3 H)
3a9 6.43 (d, J¼1.2, 1 H); 4.64 (ddd, J¼4.3,
J¼10.9, J¼11.5, 1 H); 3.02 (dsept., J¼6.9,
J¼1.2, 1 H); 2.25 (t, J¼7.4, 2 H);
2.24 (t, J¼7.7, 2 H); 1.98 (s, 3 H);
1.9 – 1.2 (m, 25 H);
188.3; 187.0; 173.4; 152.2; 143.8; 139.2;
129.9; 73.8; 47.0; 40.9; 34.7; 34.3; 31.4;
29.4; 29.3; 29.2; 29.1; 27.1; 26.7; 26.5;
26.2; 23.4; 23.3; 21.9; 20.7; 16.3; 161; 14.2
ꢀ23.1
ꢀ11.2
ꢀ4.4
1.08 (d, J¼6.9, 6 H); 0.86 (d, J¼6.5, 6 H)
3b 6.47 (d, J¼1.2, 1 H); 4.84 (m, 1 H);
3.03 (dsept., J¼6.9, J¼1.2, 1 H);
2.81 (t, J¼7.3, 2 H);
188.1; 186.7; 172.9; 154.7; 143.2; 141.1;
130.2; 80.2; 48.7; 47.8; 44.8; 36.8; 33.1,
28.0; 27.1; 26.7; 22.6; 21.4; 19.7; 18.8; 13.5
2.45 (t, J¼7.3, 2 H); 2.04 (s, 3 H);
1.4 – 1.2 (m, 7 H); 1.09 (d, J¼6.9, 6 H);
0.87 (s, 3 H); 0.84 (s, 3 H); 0.78 (s, 3 H)
3b9 6.44 (d, J¼1.1, 1 H); 4.83 (tq, J¼9.9,
188.2; 187.4; 173.9; 154.5; 143.8; 139.5;
J¼2.1, 1 H); 3.02 (dsept., J¼6.9, J¼1.1, 1 H); 130.0; 78.9; 50.5; 48.7; 44.9; 34.4; 30.0;
2.25 (t, J¼7.6, 2 H); 2.5–2.3 (m, 3 H);
1.98 (s, 3 H); 2.0–1.2 (m, 20 H);
1.08 (d, J¼6.9, 6 H); 0.87 (s, 3 H);
0.79 (s, 6 H)
29.8; 29.7; 29.3; 29.1; 28.7; 28.1; 26.7;
26.6; 26.5; 24.9; 21.4; 21.2; 14.3; 13.5

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
137
Table 2 (cont.)
d(H)
d(C)
[a]2D4
3c 6.47 (d, J¼1.2, 1 H); 4.34 (d, J¼1.9, 1 H);
3.03 (dsept., J¼6.9, J¼1.2, 1 H);
2.81 (t, J¼7.3, 2 H); 2.46 (t, J¼7.3, 2 H);
2.04 (s, 3 H); 1.09 (d, J¼6.9, 6 H);
1.7 – 1.0 (m, 7 H); 1.07 (s, 3 H); 0.99 (s, 3 H);
0.73 (s, 3 H)
188.1; 186.7; 172.8; 154.7; 143.2; 134.5;
130.1; 86.5; 48.3; 41.3; 39.4; 33.7; 32.8;
29.7; 26.6; 22.6; 20.1; 19.4; 15.6; 15.2;
11.8
13.5
3c9 6.45 (d, J¼1.2, 1 H); 4.33 (br, 1 H);
3.02 (dsept., J¼6.9, J¼1.2, 1 H);
188.2; 187.4; 173.9; 154.5; 143.8; 139.5;
130.0; 91.6; 47.9; 43.1; 35.2; 34.4; 30.0;
29.9; 29.8; 29.5; 29.4; 29.3; 29.2; 29.1;
27.1; 25.7; 25.0; 22.9; 17.1; 14.3
1.5
2.44 (m, 2 H); 2.26 (t, J¼7.7, 2 H);
1.99 (s, 3 H); 1.8–1.2 (m, 23 H); 1.24 (s, 3 H);
1.23 (s, 6 H); 1.08 (d, J¼6.9, 6 H)
3d 6.43 (d, J¼1.2, 1 H); 5.54 (m, 1 H);
4.68 (m, 1 H); 4.67 (m, 1 H); 4.64 (m, 1 H);
2.98 (dsept., J¼6.8, J¼1.2, 1 H);
187.9; 186.6; 172.1; 154.5; 143.0; 140.9; ꢀ46.1
132.6; 130.7; 129.9; 125.9; 109.2; 70.9;
35.7; 33.5; 33.0; 32.9; 26.4; 22.5; 21.3;
20.7; 20.3; 18.7
2.78 (t, J¼7.8, 2 H); 2.43 (t, J¼7.8, 2 H);
2.00 (s, 3 H); 1.66 (br, 3 H); 1.58 (m, 3 H);
2.4–1.4 (m, 5 H); 1.05 (d, J¼6.8, 6 H)
3e 6.47 (d, J¼1.2, 1 H); 5.54 (t, J¼8.1, 1 H);
187.9; 186.7; 172.3; 155.2; 154.7; 143.0; ꢀ8.0
4.81 (d, J¼1.2, 1 H); 4.73 (d, J¼1.5, 1 H);
141.1; 137.9; 130.6; 129.0; 110.8; 68.7;
4.46 (br., 1 H); 3.02 (dsept., J¼6.9, J¼1.2, 1 H); 51.6; 40.3; 40.1; 34.7; 33.2; 32.9; 29.9;
2.81 (t, J¼7.9, 2 H); 2.45 (t, J¼7.9, 2 H);
2.03 (s, 3 H); 2.3–1.4 (m, 10 H);
29.2; 27.4; 26.7; 26.2; 25.2; 22.9;
21.4; 11.8
1.09 (d, J¼6.9, 6 H); 0.98 (s, 3 H); 0.95 (s, 3 H)
3f 6.47 (d, J¼1.1, 1 H); 4.71 (m, 2 H);
188.1; 186.7; 171.6; 154.7; 143.1; 137.8;
8.7
3
4.47 (m, 1 H); 3.02 (dsept., J¼6.9, J¼1.1, 1 H); 134.3; 133.1; 130.6; 130.4; 130.1; 127.0;
2.79 (br., 4 H); 2.61 (br., 2 H); 2.40 (br., 2 H);
2.02 (s, 3 H); 2.0–1.4 (m, 4 H); 1.40 (s, 6 H);
1.36 (br., 3 H); 1.26 (s, 3 H); 1.09 (d, J¼6.9, 6 H)
68.1; 47.0; 38.7; 33.7; 32.9; 26.7; 22.6;
21.4; 21.3; 21.1; 19.1; 15.6; 11.8
vacuum. The residue was purified by CC (silica gel; AcOEt/cyclohexane 1:1) to afford pure dione
(110 mg, 58%). Yellow oil. Rf (AcOEt/cyclohexane 1:1) 0.44. IR (ATR): 3384, 2963, 2874, 1644, 1612,
1456, 1384, 1307, 1248, 1191, 1147, 1058, 945, 892, 708. 1H-NMR (300 MHz, CDCl3): 6.43 (d, J¼1.2, 1 H);
3.55 (t, J¼6.0, 2 H); 2.98 (dsept., J¼6.9, J¼1.2, 1 H); 2.54 (t, J¼7.3, 2 H); 1.97 (s, 3 H); 1.63 (tt, J¼6.0,
J¼7.3, 2 H); 1.05 (d, J¼6.9, 6 H). 13C-NMR (75.5 MHz, CDCl3): 188.1; 187.4; 154.5; 144.5; 140.6; 130.1;
61.6; 31.2; 29.2; 26.7; 22.7; 21.3. EI-MS: 222 (100, Mþ ), 204 (33), 189 (74), 179 (80), 161 (84), 151 (65),
135 (42), 121 (18), 105 (36), 91 (39).
Synthesis of 3-[2-Methyl-5-(1-methylethyl)-3,6-dioxocyclohexa-1,4-dienyl)propyl Betulinate (4). A
mixture of betulinic acid (50 mg, 0.11 mmol), pivalic anhydride (0.05 ml, 42 mg, 0.23 mmol), a cat.
amount of DMAP and CHCl3 (30 ml) was stirred at 508 for 2 h. A soln. of 3-(3-hydroxypropyl)-2-methyl-
5-(1-methylethyl)-cyclohexa-2,5-diene-1,4-dione (100 mg, 0.45 mmol) in CHCl3 (10 ml) was slowly
added, and stirring at 508 was continued overnight. H2O was added and the solvent was evaporated. The
residue was dissolved in Et2O and washed with sat. aq. NaHCO3 and brine, dried (Na2SO4), and
concentrated in vacuum. The residue was purified by CC (silica gel; AcOEt/cyclohexane 1:4) to afford
pure 4 (27 mg, 39%). Yellow oil. Rf (AcOEt/cyclohexane 1:1) 0.47. [a]2D4 ¼ þ2.1 (c¼0.5, CHCl3). IR
1
(ATR): 3376, 2960, 2925, 2854, 1710, 1646, 1456, 1378, 1284, 1259, 1161, 1107, 1057, 952, 738. H-NMR
(300 MHz, CDCl3): 6.44 (d, J¼1.6, 1 H); 6.39 (br., 1 H); 4.72 (br., 1 H); 4.59 (br., 1 H); 4.06 (t, J¼6.2,
2 H); 3.3–3.1 (m, 1 H); 3.00 (dsept., J¼7.0, J¼1.6, 1 H); 2.9–2.7 (m, 1 H); 2.59 (t, J¼6.9 Hz, 2 H); 1.98

138
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
(s, 3 H); 1.67 (br., 5 H); 1.15 (d, J¼7.0, 6 H); 2.3–1.0 (m, 24 H); 0.95 (s, 3 H); 0.94 (s, 3 H); 0.91 (s, 3 H);
0.79 (s, 3 H); 0.73 (s, 3 H). 13C-NMR (75.5 MHz, CDCl3): 188.0; 187.5; 174.1; 154.5; 144.6; 140.5; 130.0;
109.7; 78.9; 65.9; 56.3; 55.3; 50.5; 49.2; 46.9; 42.4; 40.7; 38.8; 38.4; 37.2; 37.0; 34.3; 32.1; 30.5; 29.7; 27.9;
27.4; 25.5; 24.8; 21.8; 20.8; 19.4; 18.3; 16.1; 16.0; 15.3; 14.7. EI-MS: 660 (2, Mþ ), 410 (13), 395 (5), 233
(17), 189 (28), 135 (18), 69 (31), 57 (100).
Cell Lines and Culture Conditions. The human HL-60 leukemia cells were obtained from the
German National Resource Center for Biological Material (DSMZ), Braunschweig, and incubated in
RPMI (Roswell Park Memorial Institute) Medium 1640 with 10% FCS (fetal calf serum), 100 IU/ml
penicillin G, 100 mg/ml streptomycin sulfate, 0.25 mg/ml amphotericin B, and 250 mg/ml gentamycine (all
from Gibco, D-Eggenstein). The human 518A2 melanoma cells were a gift from the Department of
Oncology and Hematology of the Martin-Luther-University Halle-Wittenberg, Germany, the human
KB-V1/Vbl cervix carcinoma and the human MCF-7/Topo breast adenocarcinoma cells were obtained
from the Institute of Pharmacy of the University Regensburg, Germany, and the human foreskin (HF)
fibroblasts from the University Hospital Erlangen, Germany. The 518A2, the KB-V1/Vbl, and the HF
cells were cultured in DMEM (Dulbeccoꢃs modified eagle medium; Gibco) containing the same
additions as the RPMI medium above. The MCF-7/Topo cells were grown in Eagleꢃs minimum essential
medium (E-MEM, Sigma) supplemented with 2.2 g/l NaHCO3, 110 mg/l sodium pyruvate, and 5% FCS.
The cells were maintained in a moisture-saturated atmosphere (5% CO2) at 378.
Inhibition of Cell Growth and Metabolic Activity (MTT Assay). MTT (¼ 3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyl-2H-tetrazolium hydrobromide; ABCR) was used to identify the metabolic activity of
cells, which are capable of reducing it by their mitochondrial dehydrogenases to a violet formazan
product. HL-60 Leukemia cells (5ꢂ105/ml), and cells (5ꢂ104/ml) of 518A2 melanoma, KB-V1/Vbl
cervix carcinoma, MCF-7/Topo breast carcinoma, and foreskin fibroblasts (HF) were seeded, and
cultured for 24 h in 96-well microplates [32]. Inhibitors of P-pg or BCRP were optionally added.
Incubation (5% CO2, 95% humidity, 378) of cells following treatment with the test compounds was
continued for 72 h. Blank and solvent controls were treated identically. Then, a 5 mg/ml stock soln. of
MTT in phosphate-buffered saline (PBS) was added to a final MTT concentration of 0.05% (HL-60,
518A2, and HF) or 0.1% (KB-V1/Vbl, and MCF-7/Topo). After 2 h, the precipitate of formazan crystals
was redissolved in a 10% soln. of sodium dodecylsulfate (SDS) in DMSO containing 0.6% AcOH in the
case of the HL-60 cells. For the adherent 518A2, KB-V1/Vbl, MCF-7/Topo, and HF cells, the microplates
were swiftly turned, flicked, and blotted to discard the medium before adding the solvent mixture. The
microplates were gently shaken in the dark for 30 min, and absorbance at 570 and 630 nm (background)
was measured with an ELISA plate reader. All experiments were carried out in quadruplicate, and the
percentage of viable cells was calculated as the meanꢁSD relative to the controls (100%).
Mitochondrial Membrane Potential. Changes in mitochondrial membrane potentials were deter-
mined by the Mitochondrial Membrane Detection Kit (Stratagene, La Jolla, CA, USA) according to the
manufacturerꢃs procedure. Following treatment, cell samples were centrifuged at 400g for 5 min. The
pellets were resuspended in 500 ml of diluted JC-1 soln. (0.1ꢂ), incubated at 378 for 15 min (HL-60) or
35 min (518A2), and then centrifuged again for 5 min at 400g. After washing, the pellets were
resuspended in 100 ml of PBS and transferred into the wells of a black 96-well plate. The red (lex
585 nm, lem ¼590 nm) and green (lex ¼510 nm, lem ¼527 nm) fluorescence intensities were measured,
and their ratio was calculated [26].
¼
Generation of ROS (NBTAssay). HL-60 Cells (0.5ꢂ106/ml) were plated in 96-well microplates, and
test compounds were added after 24 h incubation at 378 to achieve a final concentration of 5 mm.
Incubation (5% CO2, 95% humidity, 378) of cells following treatment with the test compounds was
continued for 72 h. After removal of the cell medium by centrifugation, the cells in each well were
resuspended in 100 ml 0.1% NBT, and the plates were placed in the incubator for 1 h. The reduced NBT
was solubilized with 100 ml of 2m KOH and 130 ml of DMSO for 30 min. The absorbance was measured
for each well at 630 and 405 nm (background) using an ELISA plate reader. The adherent 518A2 cells
(0.5ꢂ104/ml) were seeded after trypsinization and incubated in 96-well microplates for 24 h at 378 to
allow attachment, then treated similarly, only that the medium was removed prior to incubation with
NBT for 4 h. All experiments were carried out in quadruplicate [27][28].

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Received October 9, 2009