The synthesis and anticancer effects of a range of natural and unnatural hop β-acids on breast cancer cells

Article abstract of DOI:10.1016/j.phytol.2011.11.011

The β-acids derived from hops (Humulus lupulus L.) are polyphenols classified as lupulones. As part of our on-going interest in the pharmacognosy of these natural products we were keen to investigate the anticancer activity of lupulone 1 as well as individual lupulone congeners. To achieve this we undertook the synthesis of natural as well as unnatural lupulone derivatives and evaluated them for their anticancer activity against the breast cancer cell lines MCF-7 and MDA-MB-231. The results of our investigations revealed that all of the novel unnatural lupulone derivatives that were synthesised were found to be more toxic to MDA-MB-231 cell lines at 72 h than the parent lupulone 1 itself (except for the α-substituent R¹ was CH₃). Further investigations confirmed that the novel lupulone derivatives were very efficient at killing cancer cells by apoptosis but appear to do so in a time-dependant process. This outcome may be of great significance as MDA-MB-231 cell lines are characterised by an aggressive phenotype with a propensity to invade other tissue, to form metastases as well as an ability to become insensitive to chemotherapeutic agents.

Full text of DOI:10.1016/j.phytol.2011.11.011

Phytochemistry Letters 5 (2012) 144–149
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
The synthesis and anticancer effects of a range of natural and unnatural hop
b
-acids on breast cancer cells
a
a
b
a,b
Elizabeth Tyrrell a, , Roland Archer , Matt Tucknott , Kay Colston , Grisha Pirianov
,
*
Dharahana Ramanthan b, Rajdeep Dhillon b, Alex Sinclair a, G.A. Skinner a
a School of Pharmacy, Kingston University, Kingston, Surrey KT1 2EE, UK
b St George’s University of London, Cranmer Terrace, London SW 17 0RE, UK
A R T I C L E I N F O
A B S T R A C T
The -acids derived from hops (Humulus lupulus L.) are polyphenols classified as lupulones. As part of our
Article history:
b
Received 25 August 2011
on-going interest in the pharmacognosy of these natural products we were keen to investigate the
anticancer activity of lupulone 1 as well as individual lupulone congeners. To achieve this we undertook
the synthesis of natural as well as unnatural lupulone derivatives and evaluated them for their anticancer
activity against the breast cancer cell lines MCF-7 and MDA-MB-231.
Received in revised form 24 November 2011
Accepted 28 November 2011
Available online 9 December 2011
The results of our investigations revealed that all of the novel unnatural lupulone derivatives that
were synthesised were found to be more toxic to MDA-MB-231 cell lines at 72 h than the parent lupulone
Keywords:
Lupulone
1 itself (except for the
a
-substituent R1 was CH3). Further investigations confirmed that the novel
Hop acids
lupulone derivatives were very efficient at killing cancer cells by apoptosis but appear to do so in a time-
dependant process. This outcome may be of great significance as MDA-MB-231 cell lines are
characterised by an aggressive phenotype with a propensity to invade other tissue, to form metastases as
well as an ability to become insensitive to chemotherapeutic agents.
Breast cancer
Apoptosis
Cyclopropyl group
ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
effects of samples that contain mixtures of more than one congener
are reported (Lamy et al., 2007; Teotico et al., 2008; Tripp et al.,
For centuries hops and malt have been used in brewing to
provide the unique flavour and aroma associated with beer. It was
fortuitous that hop extracts not only imparted the taste to bitter
ales but were also bacteriostatic (Kulka, 1958) and thus prevented
the brew from spoiling (Simpson, 1993). When the isolates,
derived from the chloroform extraction of hops, are then further
extracted with alkali two distinct fractions may be isolated. These
are the hard resins that contain polyphenols, flavonoids and
2008; Cattor et al., 2010). Secondly acetolupuphenone 2a,
(postlupulone) propiolupuphenone 2b, and butyrolupuphenone
2c are relatively rare congeners with minimal spectral characteri-
sation (Zhang et al., 2004) and limited detection in hop extracts
(Archer, 2006; Stevens, 1966) therefore the opportunity to isolate
them from the plant source for screening purposes has not been
possible. In this paper we wish to reveal the results from the
synthesis and anticancer testing of a number of natural and
unnatural lupulone derivatives (Fig. 1).
chalcones (Stevens, 1966) and the soft resins comprising the
and -acids (Stevens, 1967). Previously we revealed our investiga-
tions into the in vitro effects of the -acid humulone, and the
a-
b
The extensive pharmacognosy of hop isolates has been
documented (Teuber and Schmalreck, 1973; Zanoli and Zavatti,
2008; Van Cleemput et al., 2009; Foster et al., 2009; Cattor et al.,
a
b-
acid lupulone 1 on both SK-MES lung cancer cell line and MDA-MB
231 breast cancer cell lines (Tyrrell et al., 2010). In this current
study we have focused our attention upon the synthesis and in
vitro testing of natural and unnatural lupulones against breast
cancer cell lines. Our rationale for this investigation is twofold. The
first is that due to the structural similarities and polarities of the
lupulone congeners they are difficult to resolve into their
individual components. As a consequence of this the screening
2010). With regard to the b-acids the effects of lupulone 1 on both
human colon cancer (Lamy et al., 2007, 2008) and HL-60 leukemia
cells as well as the derivative hexahydrocolupulone (Stephan et al.,
1998) have been reported. In addition an attempt at the elucidation
of the cellular signalling mechanism responsible for the induction
of apoptosis, via Fas and caspase cascades, has been undertaken
(Chen and Lin, 2004). An investigation into the toxicity and related
physiological activity of plant lupulones suggested that acute
toxicity in rats was slight, LD50 1800 mg/kg, which equates to a
lethal dose in man of about 250 g of lupulone 1 (Singleton et al.,
1969).
* Corresponding author. Tel.: +44 208 547 8810.
E-mail address: e.tyrrell@kingston.ac.uk (E. Tyrrell).
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2011.11.011

E. Tyrrell et al. / Phytochemistry Letters 5 (2012) 144–149
145
OH
O
O
H3C
H3C
H3C
OH
O
H3C
H3C
R1
CH3
HO
HO
O
CH3
CH3
CH3
H3C
H3C
CH3
CH3
CH3
1
2
2a
R1 = CH3 acetolupuphenone
2b
2c
2d
R1 = C2H5 propiolupuphenone (postlupulone)
R1 = C3H7 = butyrolupuphenone
R1 = CH(CH3)2 = colupulone
Fig. 1. Structures for lupulone 1 and the congeners 2.
2. Results and discussion
(acetolupuphenone), 2b (63%) R = C2H5 (postlupulone-propiolupu-
phenone), 2c (37%) R = C3H7 (butyrolupuphenone), 2d (47%)
R = CH(CH3)2 (colupulone), 2e = 1 (48%) R = CH2CH(CH3)2 (lupu-
lone), 2f (24%) R = cyclopropyl, 2g (20%) R = cyclopentyl and 2h
(40%) R = cyclohexyl. The R1 substituent used in this study were
selected as follows:
Much of our chemical understanding of hop acids is derived
from pioneering studies conducted during the 1970s (De
Keukeleire and Verzele, 1970a,b; De Keukeleire and Snatzke,
1972) and earlier (Verzele and Govaert, 1949) however the
chemistry of lupulone 1, and its congeners have presented a
synthetic challenge as they tend to undergo facile aerial oxidation
and solvent dependant keto-enol tautomerism. These have
therefore limited the developments in the chemistry of these
natural products. Previously we revealed the first X-ray structures
of the major tautomer of lupulone 1 in a polar solvent (Tyrrell et al.,
2007). This study was augmented by extensive NMR analyses and
molecular modelling experiments of both tautomers. With this
knowledge we were then able to carry out regioselective syntheses
of novel O-derivatives of lupulone 1 (Archer, 2006).
i. to investigate the effect of increasing the length of the carbon
chain linearly from C-1 to C-3.
ii. to look at the effects of a methyl substituent on the R1 carbon
chain e.g. enabling a comparison between 2b (R1 = ethyl) with
colupulone 2d R1 = CH(CH3)2 or the parent lupulone
(R1 = CH2CH(CH3)2) with 2c butyrolupuphenone (R1 = C3H7).
1
iii. see the effect of
system allowing
(R1 = C3H7) with 2f (3C-carbocyclic)
a
-carbocycles against the corresponding linear
a
comparison of 2c butyrolupuphenone
In this paper we would like to discuss the synthesis of natural as
well as novel lupulone derivatives and the results of screening
studies into their anticancer activity. The candidates were
synthesised using a modification to a literature procedure (Scheme
1) and began with the acylation of phlorogluconol 3 (Reininger and
Hartl, 1977; Drewett and Laws, 1970). Thus exposure of
phlorogluconol 3 to Friedel–Crafts acylation reaction conditions
using the relevant acyl chloride and Lewis acid, aluminium
chloride, provided the corresponding acylated compounds 4a–
4h in good yields. The yields for this reaction never equate with
similar generic acylations to simpler phenols nevertheless we were
able to obtain the following: 4a (53%) R = CH3 4b (51%) R = C2H5, 4c
(54%) R = C3H7, 4d (55%) R = CH(CH3)2, 4e (66%) R = CH2CH(CH3)2,
4f (66%) R = cyclopropyl, 4g (56%) R = cyclopentyl and 4h (36%)
R = cyclohexyl. The second step of the synthesis involved a non-
trivial C-4, C-4, C-6 triprenylation reaction. This was carried out by
the dropwise addition of a diethyl ether solution of the acylated
iv. the effects of
(C-6).
a-carbocycle ring size 2f (C3) with 2g (C-5) and 2h
With the screening compounds in hand we selected two human
breast cancer cell lines the first were MCF-7 cells. This cell-type is
responsive to the hormones oestrogen and progesterone and tend
to be less aggressive and poorly invasive. In contrast the second cell
line, MDA-MB-231, lack receptors for these hormones and are
therefore non-oestrogen dependant. In addition they have the
propensity to be an aggressive and highly invasive cell-type.
In an initial assay (Dhillon et al., 2010) the toxicity of lupulone 1
and butyrolupuphenone 2c was applied to both cell lines as a
measure of cell viability. The IC50 of lupulone 1 for MCF-7 cell lines
was 20
mM at 48 h and 9 mM at 72 h. For butyrolupuphenone 2c
˙
the IC50data was identical at both 48 h and 72 h at 9
m
; These
data show a significant difference in the toxicity of 1 over 72 h.
Both curves demonstrated a similar pattern of decline with cell
viability being consistently lower at 72 h for lupulone 1 (Fig. 2). A
similar study was carried out to determine the toxicity of the same
substrates on MDA-MB-231 cell viability (Fig. 3). The IC50 for
compound, 4a–4h, onto
a stirred sodium-dried solution of
ammonia at À78 8C maintained under an atmosphere of nitrogen
gas. This step required a significant amount of time in order to
optimise both the yields and the reproducibility of the reaction. An
initial condensation of the ammonia gas on to the sodium metal is
important to avoid any contamination from trace metals. The
liquid ammonia is then delivered to the reaction vessel by
cannulation. With practice we were able to significantly reduce the
amount of liquid ammonia needed (from 180 ml to 50 ml per gram
of phlorogluconol derivative used). An additional benefit from
using sodium-dried ammonia was that the reaction was more
facile affording a range of derivatives in acceptably workable
yields. The following yields were obtained: 2a (40%) R = CH3
lupulone 1 for MDA-MB-231 cells was 40
at 72 h. The corresponding data for 2c was 30
m
M at 48 h and 9.5
mM
m
M and 8.8 mM,
respectively. These data suggest that the growth of both MCF-7 and
MDA-MB-231 cells were inhibited in a dose and time dependent
manner by both 1 and 2c. A similar pattern of decline with cell
viability is demonstrated being consistently lower at 72 h for both
1 and 2c. The gradual decrease in viable cell numbers with time
confirms a cytotoxic/cytostatic function in these
protocol was then employed in order to investigate the toxic
effects of range of both natural and unnatural lupulone
b-acids. The same
a

146
E. Tyrrell et al. / Phytochemistry Letters 5 (2012) 144–149
CH3
O
O
OH
OH
O
OH
R1
AlCl3, R1COCl
H3C
Br
NH3 (liquid)
R1
H C
3
CH3NO2, DCM,
40oC
HO
HO
OH
HO
OH
H C
3
CH3
H3C
CH3
CH3
3
4a-h
2a-h
Scheme 1. Synthesis of lupulone 1 and congeners 2a–h.
Fig. 2. The effects of (1) graph A and (2c) graph B on MCF-7 cell viability.
derivatives 2a–g against the same breast cancer cell-lines. The IC50
data are summarised in Table 1.
associated with the bulkier methyl substituent present in
colupulone 2d. Perhaps the most surprising result to emerge
from these studies are that all of the novel derivatives 2f–2h, in
which the R1 substituent is a carbocycle, appear more toxic to
MDA cell lines than lupulone 1 however with MCF-7 cells they
appear to be less efficacious at 72 h. This result could be significant
as MDA-MB-231 cell lines are characterised by an aggressive
phenotype typically a tendency to invade other tissue, to form
metastes as well as an ability to become insensitive to
chemotherapeutic agents. A comparison in activity between the
isomeric lupulone congeners butyrolupuphenone 2c with colu-
pulone 2d and the derivative 2f reveal an interesting result.
Whereas 2c and 2d both contain a three carbon acyl moiety at C-2
(with 2d bearing a bulky methyl substituent) in compound 2f the
three carbon atoms are contained in a ring structure in the form of
a cyclopropyl group (Fig. 4).
The data that emerges from Table 1 suggests that butyrolu-
puphenone 2c appears to be more potent to both cell lines than
lupulone 1 itself. A similar result emerges for the in vitro effects of
postlupulone 2b which appears to be both more efficacious and
selective for MCF-7 cells than for MDA cells. Comparing the effects
of carbon chain length (n) optimum activity is seen with n = 2 (2b)
for MCF cells. The lowest activity is seen when n = 1 (2a) however
when n = 3 (2c) the toxic effects are similar for MCF-7 cells at both
48 h and 72 h however for MDA-MB231 cells there is a significant
enhancement at 72 h. A comparison between 1 and 2c (R1 = 3-C
chain with 1 bearing a methyl substituent) suggest similar activity
at 72 h. In contrast however a comparison of 2b R1 = ethyl with 2d
R1 = isopropyl reveals a significant disparity in activity with
MCF-7 cells lines indicating a reduction in anticancer activity
Fig. 3. The effects of (1) graph C and (2c) graph D on MDA-MB231 cell viability.

E. Tyrrell et al. / Phytochemistry Letters 5 (2012) 144–149
147
Table 1
derivatives 2f and 2g all induce PARP cleavage suggesting that their
effects are mediated by apoptosis. Whilst we are unable to see
dramatic changes in cell viability after 24 h of treatment some of
the lupulone derivatives induced early PARP cleavage (2f and 2g on
MCF-7 and 2b and 2f on MDA-MB231 breast cancer cells). However
PARP cleavage was induced to a different extent by all compounds
after 48 h of treatment demonstrating their ability to activate
apoptotic signalling in a time-dependant manner. Interestingly the
most potent derivative 2f demonstrated an enhanced PARP
cleavage for MDA-MB-231 cells at 48 h. In contrast to this the
novel derivative 2g, shows no effect with MDA-MB-231 after 24 h
and only a moderate effect is seen at 48 h demonstrating the effect
of the R1 cycloalkyl ring size upon the corresponding activity. In
contrast to 2f, however, derivative 2g shows a significantly greater
effect upon MCF-7 cells at 24 h which appear even greater at 48 h.
Thus 2b, 2d as well as the novel lupulone derivatives 2f and 2g
synthesised during the course of this study are very efficient at
killing cancer cells by apoptosis and appear to do so in a time-
dependant process.
In an attempt to qualify the observation made regarding the cell
selectivities of derivatives 2f and 2g densitometry was used to
measure the intensity of the bands produced by the Western blot
for these compounds. These data provide the cleaved PARP:actin
ratio (Fig. 6). The results show that for both novel derivatives the
relative ratios increased at 48 h. Thus 2f showed an 11-fold
increase in cleaved PARP: actin ratio between 24 and 48 h for MDA-
MB-231 cells far greater than for MCF-7 cells. In contrast 2g
demonstrated a higher cleaved PARP:actin ratio for MCF-7 cells
(48 h) compared to MDA-MB-231 cells.
IC50 data for lupulone 1 (bold) and some natural and unnatural derivatives against
MDA-MB-231 and MCF-7 breast cancer cell lines.
Entry
MCF-7 IC50 MCF-7
(48 h)
MDA-MB231 MDA cells
IC50 (72 h)
IC50 (72 h) cells IC50
(48 h)
2a acetolupuphenone
2b postlupulone
propiolupuphenone
2c butyrolupuphenone
2d colupulone
1 (2e) lupulone
2f
40
6
m
M
35
8
m
M
60
36
m
m
M
M
60
22
m
m
M
M
mM
mM
˙
9.2
60
m
M
9
48
9
m
m
M
30
58
40
10
17
18
mM
mM
mM
mM
mM
mM
8 8
m
M
m
M
M
M
M
M
M
48
m
M
20
20
21
22
m
m
m
m
mM
9.5 mM
10
18
17
m
m
m
M
M
M
4.5
13
9.5
mM
M
2g
m
2h
mM
From Table 1 it is clear that at 72 h 2f is twice as potent as
butyrolupuphenone 2c against MDA-MB-231 cancer cell lines and
comparable in activity against MCF cell lines at 72 h. In contrast to
these results however colupulone 2d is consistently low in activity
against both cell lines. These data suggest that the potency
associated with the novel derivative 2f may be explained in terms
of the lower conformational freedom associated with this moiety
We are currently pursuing this line of enquiry.
In order to ascertain whether the reduction of cell viability was
associated with the induction of apoptosis (specific mode cell
death) we carried out Western blotting experiments to identify
specific biomarkers for apoptosis, in particular, the poly-adeno-
ribosyl polymerase 1 PARP-1 (Zeerlander, 2003). PARP is known to
play an essential role in the regulation of dynamics of chromatin,
cell cycle and DNA repair. In apoptotic cells PARP is cleaved into 2
polypeptides 89 kDa and 27 kDa and detection of PARP cleavage is
therefore considered to be a good indicator of apoptosis (Lazebnik
et al., 1996). We selected four lupulone derivatives (postlupulone
2b, butyrolupuphenone 2c, 2f and 2g) to examine their effects on
PARP-1 cleavage at 24 h and 48 h on both cancer cell-lines (Fig. 5).
These data clearly indicate that 2b, 2d, and the novel lupulone
In conclusion to this investigation we have undertaken the
synthesis of lupulone 1 and three other congeners as well as a
number of unnatural lupulones. This has allowed us to screen all of
the congeners on an individual basis and can report that
postluplone 2b, a scarce congener, proved to be particular toxic
to MCF-7 breast cancer cell lines at both 48 and 72 h.
Butyrolupuphenone, 2c, like lupulone 1 showed toxic effects on
Fig. 4.
OH
O
CH
CH
OH
O
CH
OH
O
3
3
3
CH
3
H C
CH
H C
H C
3
3
3
3
CH
3
HO
O
HO
O
HO
O
CH
CH
H C
3
H C
CH
3
3
H C
3
3
3
CH
CH
CH
CH
3
CH
3
3
CH
3
3
3
Fig. 5. PARP cleavage in breast cancer-lines MCF-7 (upper) and MDA-MB 231 (lower). The control (C) is actin.

148
E. Tyrrell et al. / Phytochemistry Letters 5 (2012) 144–149
Fig. 6. The cleaved PARP:actin ratio for 2f and 2g with MDA-MB-231 cells (left) and MCF-7 breast cancer cell-lines (right).
both cell lines at 72 h whereas colupulone 2d and acetolupuphe-
none 2a showed reduced activity to both cell-lines under
investigation. For the first time we also report the synthesis and
screening of novel, and unnatural, lupulone derivatives containing
an R1 carbocyclic moiety. These derivatives were all shown to have
considerably enhanced toxicity to MDA-MB-231 cell lines with the
most potent, 2f, containing an unusual C-2 acyl cyclopropyl
moiety. [Wilcoxon] signed ranks were used to determine statistical
differences between controls and treated cells.
added, dropwise, to sodium-dried liquid ammonia that was stirred
at À78 8C. The mixture was left to stir for 15 min whereupon a
solution of 3,3-dimethylallyl bromide (4 mol. eq.) dissolved in
anhydrous diethyl ether (15 ml) was slowly added. The tempera-
ture was maintained at À78 8C for 5 h before the evaporation of
ammonia took place slowly overnight. To the resulting crude
material was added 10% HCl solution (50 ml) and diethyl ether
(50 ml). The organic layer was washed again with 10% HCl solution,
then with water (50 ml) and dried over anhydrous sodium
sulphate. The solvent was removed in vacuo to afford the crude
product. This was purified by sequential recrystallisation firstly
from hexane and then from acetonitrile.
3. Experimental
3.1. General experimental procedures
3.5. Characterisation
HRMS were carried out by the EPSRC unit at Swansea using a
Finnigan MAT900XL-QTrap VG Autospec. IR spectra were per-
formed on a Perkin Elmer 1 FT spectrophotometer. 1H NMR spectra
were obtained at 400 MHz using a Bruker AV400 NMR spectrome-
ter and 13C NMR spectra were obtained at 100 MHz. The NMR data
was obtained using DEPT and DEPTQ experiments and the use of
2D HSQC provided proton to carbon correlation spectra.
3.5.1. Synthesis of propiolupuphenone (postlupululone) 2b 3,5-
dihydroxy-4,6,6-tris-(3-methyl-but-2-enyl)-2-(3-propionyl)-
cyclohexa-2,4-dienone
Mp 101–102.4 8C (lit. 101 8C) (Stevens, R. 1966); IR nmax (ATR)
3161, 2923, 2854, 1653, 1584, 1519, 1358, 1230, 1172, 1106 cmÀ1
;
1H NMR (MeOD):
d 1.09 (t, J = 7.32 Hz, 3H, CH3 19), 1.53 (s, 6H, CH3
11); 1.56 (s, 6H, CH3 12); 1.66 (s, 3H, CH3 16); 1.72 (s, 3H, CH3 17);
2.59 (m, 4H, CH2 8); 2.96 (q, J = 7.32 Hz, 2H, CH2 18); 3.08 (d,
J = 6.77 Hz, 2H, CH2 13); 4.75 (t, J = 7.60 Hz, 2H, CH 9); 5.00 (t,
3.2. Inhibition of cancer cell proliferation assay
MCF-7 cells and MDA-MB-231 cells (10,000 cells/well in a 96
well plate) were incubated in 2% FCS up to 72 h in the presence or
absence of lupulone 1 and the derivatives 2a–2h at concentrations
J = 6.50 Hz, 1H, CH 14); 13C NMR (MeOD):
d = 16.8 (CH3 11); 20.3
(CH2 13); 22.9 (CH3 19); 24.7 (CH3 12, 17); 33.9 (CH2 18); 37.5 (CH2
8); 57.5 (C 6) 110.7 (C 4); 117.9 (C 9); 121.9 (CH 14); 131.2 (C 10);
134.3 (C 15); 173.2 (C 5); 189.3 (C 3); 196.9 (C 7); 204.1 (C 1). LRMS
(EI): m/z 386 [M]+ HRMS (ES): calcd for C24H34O4 [M]+ 386.2452
found [386.2450].
(between 1 and 100
mM). Cell viability was measured by MTT
assay and expressed as % of untreated control accepted as 100% cell
viability. Difference between non-treated and treated MCF-7 cells
(5
m
M) was found to be significant at P-value = 9.75 Â 10À7 using
student t-test. Difference between non-treated and treated MDA-
3.5.2. Synthesis of butyrolupuphenone 2c-3,5-dihydroxy-4,6,6-tris-
MB-231 cells (5
m
M) was found to be significant at P-
(3-methyl-but-2-enyl)-2-(butyryl)-cyclohexa-2,4-dienone
value = 1.60 Â 1À8 using student t-test.
Mp 107.2–109.7 8C (lit. 107 8C) (Stevens, R. 1966); IR
2924, 2854, 1652, 1584, 1375, 1217, 1160, 1107 cmÀ1
(MeOD): d 0.96 (t, J = 7.37 Hz, 3H, CH3 20); 1.54 (s, 6H, CH3 12); 1.56
n
;
max (ATR)
1H NMR
3.3. Western blotting experiment
(s, 6H, CH3 11); 1.58–1.64 (m, 2H, CH2 19); 1.66 (s, 3H, CH55C-
(cis)CH3 16); 1.72 (s, 3H, CH3 17); 2.58 (sept, J = 7.58 Hz, 2H, CH2 8);
2.91 (t, J = 7.42, 2H, CH2 18); 3.08 (d, J = 6.87 Hz, 2H, CH2 13); 4.75
(tt, J = 1.01 and 7.85 Hz, 2H, CH 9); 4.99–5.02 (m, 1H, CH 14); 13C
MDA-MB-231 cells were cultured in 75 cm2 flasks and were
treated with 15
were isolated and measuring by a bicinchoninic acid assay (BCA
assay). 50 g of cell lysate were analysed for PARP cleavage by
mM of a lupulone derivative up to 48 h. Proteins
m
NMR (MeOD): d = 14.6 (CH3 20); 18.1 (CH3 16); 18.23 (CH3 11);
Western blotting and densitometry analysis. In addition actin was
used as a loading control. The MCF-7 and MDA-MB-231 cells were
incubated in 2% FCS for up to 48 h in the presence or absence of
19.9 (CH2 19); 21.74 (CH2 13); 26.2 (CH3 12, 17); 38.9 (CH2 8); 43.7
(CH2 18); 59.0 (C 6); 109.2 (C 2); 112.4 (C 4); 119.4 (C 9); 123.4 (CH
14); 132.7 (C 10); 135.8 (C 15); 174.6 (C-OH 5);191.0 (C 3); 198.3 (C
7) 204.6 (C 1); LRMS (EI): m/z 400 [M]+ HRMS (ES): calcd for
lupolone derivatives (2a, 2b 2f and 2g) at concentrations 10
mM.
Soluble proteins were extracted and analysed for PARP cleavage
using Western blotting analysis.
C
25H36O4 [M]+ 400.2608 found [400.2607].
3.5.3. Synthesis of co-lupulone 2d-3,5-dihydroxy-4,6,6-tris-(3-
3.4. General procedure for the triprenylation reaction
methyl-but-2-enyl)-2-(isopropyl)-cyclohexa-2,4-dienone
Mpt = 92.2–92.8 8C (lit. 93 8C) (Stevens, R. 1966); IR
3401, 2965, 2914, 1655, 1584, 1436, 1372, 1227, 1196, 1099 cmÀ1
1H NMR (MeOD): 1.09 (d, J = 6.78 Hz, 6H, CH3 19); 1.55 (s, 6H, CH3
nmax (ATR)
Under an atmosphere of dry nitrogen gas acylphloroglucinol
(1 mol. eq.), dissolved in anhydrous diethyl ether (15 ml), was
;

E. Tyrrell et al. / Phytochemistry Letters 5 (2012) 144–149
149
References
12); 1.57 (s, 6H, CH3 11); 1.67 (s, 3H, CH3 16); 1.73 (s, 3H, CH3 17);
2.53–2.66 (m, 4H, CH2 8); 3.10 (d, J = 6.67 Hz, 2H, CH2 13); 3.98
(pent, J = 6.84 Hz, 1H, CH 18); 4.77 (t, J = 7.40 Hz, 2H, CH 9); 5.02 (t,
Archer, R., 2006. Ph.D. Thesis. Kingston University.
Cattor, K., Bracke, M., Deforce, D., De Keukeleire, D., Hayerick, A., 2010. J. Agric. Food
Chem. 58, 4132–4140.
J = 6.67 Hz 1H, CH 14); 13C NMR (MeOD):
d = 18.1 (CH3 16); 18.2
Chen, W.-J., Lin, J.-K., 2004. J. Agric. Food Chem. 52, 55–64.
De Keukeleire, D., Verzele, M., 1970a. J. Inst. Brew. 76, 265–266.
De Keukeleire, D., Verzele, M., 1970b. Tetrahedron 26, 385–393.
De Keukeleire, D., Snatzke, G., 1972. Tetrahedron 28, 2011–2018.
Dhillon, R., Ramanthan, D., 2010. The biological screening data were obtained by
Dhillon and Ramanthan as part of an M.Sc. project. MDA-MB-231 cell lines were
shown to undergo programmed cell death in an earlier study using lupulone 1
and so with a wider range of drug candidates we focussed upon breast cancer
cell-lines. The results obtained from these studies complement the chemistry
component of this paper. M.Sc. Project. St George’s, London.
Drewett, K.G., Laws, D.D., 1970. J. Inst. Brew. 76, 188–190.
Foster, B.C., Kearns, N., Arnason, J.T., Saleem, A., Ogrodowczyk, C., Desjardins, S.,
2009. J. Agric. Food Chem. 57, 5100–5105.
Kulka, D., 1958. J. Inst. Brew. 64, 331–337.
Lamy, A.V., Roussi, S., Chaabi, M., Gosse, F., Schall, N., Lobstein Raul, F., 2007.
Carcinogenesis 28, 1575–1581.
Lamy, A.V., Roussi, S., Chaabi, M., Gosse, F., Schall, N., Lobstein Raul, F., 2008.
Apoptosis 13, 1232–1242.
Lazebnik, Y.A., Kaufman, S.H., Desnoyers, S., Poirer, G.G., Earnshaw, W.C., 1996.
Nature 371, 346–347.
(CH3 11); 19.4 (CH3 19); 21.8 (CH2 13); 26.2 (CH3 12, 17); 37.3 (CH
18); 39.0 (CH2 8); 59.1 (C 6); 108.2 (C 2); 112.4 (C 4); 119.4 (CH 9);
123.4 (CH 14); 132.7 (C 10); 135.8 (C 15); 174.6 (C 5); 191.2 (C 3);
198.1 (C 7); 209.0 (C 1); LRMS (EI): m/z 400 [M]+ HRMS (ES): calcd
for C25H36O4 [M]+ 400.2608 found [400.2602].
3.5.4. Synthesis of (2f) 3,5-dihydroxy-4,6,6-tris-(3-methyl-but-2-
enyl)-2-(3-cyclopropyl)-cyclohexa-2,4-dienone (2f)
Mpt = 98.2–99.1 8C; IR
1517, 1443, 1365, 1218, 1160, 1106, 921, 826 cmÀ1
(MeOD): = 0.99–1.07 (m, 2H, CH 19 Hb); 1.16–1.23 (m, 2H, CH 19
n
max (ATR) 3583, 3271, 2923, 1645, 1578,
;
1H NMR
d
Ha); 1.59 (s, 6H, CH3 12); 1.61 (d, J = 0.34 Hz, 6H, CH3 11); 1.69 (s, 3H,
CH3 16); 1.75 (s, 3H, CH3 17); 2.54–2.76 (m, 4H, CH2 8); 3.11 (d,
J = 7.03 Hz, 2H, CH2 13); 3.57–3.68 (m, CH 18); 4.83 (t, J = 7.47 Hz,
2H, CH 9); 5.03 (t, J = 7.02 Hz 1H, CH 14); 13C NMR (MeOD):
d = 11.1
(CH2 19); 16.6 (CH3 16); 16.7 (CH3 11); 20.2 (CH2 13); 22.9 (CH 18);
24.6 (CH3 17); 24.7 (CH3 12); 37.4 (CH2 8); 56.6 (C, 6); 108.16 (C 2);
110.45 (C 4); 117.9 (CH 9); 121.9 (CH 14); 131.1 (C 10); 134.3 (C 15);
172.6 (C 5); 188.6 (C 3); 197.3 (C 7); 202.6 (C 1); LRMS (EI): m/z 398
[M]+ HRMS (ES): calcd for C25H34O4 [M]+ 398.2452 found
[398.2450].
Reininger, W., Hartl, A., 1977. U.S. Patent 4053517.
Simpson, W.J., 1993. J. Inst. Brew. 99, 405–411.
Singleton, V.L., Kratzer, F.H., 1969. J. Agric. Food Chem. 17, 497–511.
Stephan, T.E., Ngo, E.O., Nutter, L.M., 1998. Biochem. Pharmacol. 55, 505–514.
Stevens, R., 1966. Brew. Indus. Res. Found. 19–71.
Stevens, R., 1967. Chem. Rev. 67, 19–31.
Teotico, D.G., Bischof, J.J., Peng, L., Kliewer, S.A., Redinbo, M.R., 2008. Mol. Pharma-
col. 74, 1512–1520.
Teuber, M., Schmalreck, A.F., 1973. Arch. Microbiol. 94, 159–171.
Tripp, M.L., et al., 2008. U.S. Patent No: 2008/0031894 A1.
Tyrrell, E., Archer, R.P., Singh, K., 2007. Acta Crystallogr. Sect. E 1511–1512.
Tyrrell, E., Archer, R., Skinner, G.A., Singh, K., Colston, K., Driver, C., 2010. Phytochem.
Lett. 3, 17–23.
Verzele, M., Govaert, F., 1949. Bull. Soc. Chim. Belges. 58 (43) 2–438.
Wilcoxon signed ranks are the non-parametric equivalent of paired t-tests
Van Cleemput, M., Cattoor Ko De Bosscher, K., Haegeman, G., De Keukeleire Denis
Heyerick, A., 2009. J. Nat. Prod. 72, 1220–1230.
Acknowledgements
We acknowledge the generosity of the BPSRG at Kingston
University for financial support to MT, colleagues at St Georges
Hospital for their continued interest in this project and to the
EPSRC Mass Spectroscopy service at the University of Swansea for
providing an outstanding MS service. In addition we are grateful to
Leon Whiteman for his continued interest in the chemistry
associated with this project.
Zanoli., P., Zavatti, M., 2008. J. Ethnopharmacol. 116, 383–396.
Zeerlander, S., 2003. Biochemica 1, 14–15.
Zhang, X., Liang, X., Xiao, H., Xu, Q., 2004. J. Am. Soc. Mass Spectrom. 15, 180–187.