Structure-antimutagenic activity relationship study of plicatin B

Article abstract of DOI:10.1021/np980304n

A systematic structure-activity relationship study of plicatin B (1), an antimutagenic constituent of Psoralea juncea, was undertaken with a view toward elucidating its chemical mode of action and possibly optimizing its antimutagenic activity during the process. Compound 1 and its related analogues were examined for their antimutagenic activity against mutations induced by ethyl methanesulfonate, a direct acting mutagen and alkylating agent, in Salmonella typhimurium strain TA100, utilizing the modified Ames test protocol. The dihydro analogue 3 resulting from saturation of the conjugated alkene double bond of 1 was found to exhibit reduced cytotoxicity and enhanced efficacy relative to the parent compound. This result serves preliminarily to exclude a Michael acceptor role of the α,β-unsaturated carbonyl moiety in connection with its antimutagenic activity.

Full text of DOI:10.1021/np980304n

102
J . Nat. Prod. 1999, 62, 102-106
Str u ctu r e-An tim u ta gen ic Activity Rela tion sh ip Stu d y of P lica tin B1,2
Sanjay R. Menon,† Vishal K. Patel,† Lester A. Mitscher,*,† Peter Shih,‡ Segaran P. Pillai,‡ and
Delbert M. Shankel‡
Department of Medicinal Chemistry and Department of Microbiology, University of Kansas, Lawrence, Kansas 66045
Received J uly 13, 1998
A systematic structure-activity relationship study of plicatin B (1), an antimutagenic constituent of
Psoralea juncea, was undertaken with a view toward elucidating its chemical mode of action and possibly
optimizing its antimutagenic activity during the process. Compound 1 and its related analogues were
examined for their antimutagenic activity against mutations induced by ethyl methanesulfonate, a direct
acting mutagen and alkylating agent, in Salmonella typhimurium strain TA100, utilizing the modified
Ames test protocol. The dihydro analogue 3 resulting from saturation of the conjugated alkene double
bond of 1 was found to exhibit reduced cytotoxicity and enhanced efficacy relative to the parent compound.
This result serves preliminarily to exclude a Michael acceptor role of the R,â-unsaturated carbonyl moiety
in connection with its antimutagenic activity.
Cancer is one of the leading causes of death today, at
present estimated to claim annually about 550 000 lives
in the United States and approximately 6 million lives
worldwide.3,4 Epidemiological studies suggest that about
60% of cancer mortality in the United States is potentially
preventable, since it can be attributed to environmental
causes alone.5 Although early detection and subsequent
treatment of cancer constitutes an effective stratagem for
combating the disease once it has established itself,
primary prevention of cancer is optimal and desirable for
quite obvious reasons.6 The finding that the dietary con-
sumption of certain foodstuffs such as fruits, vegetables,
and tea beverages is associated with a reduced risk of
carcinogenesis eventually led to the concept of cancer
chemoprevention that encompasses the use of natural (e.g.,
phytochemicals) and synthetic compounds for preventing
the development of cancer.7-11 Some of these chemical
agents exert their cancer chemopreventive action via their
ability to operate as antimutagens, i.e., by preventing or
reversing the genetic damage caused to the organism by
environmental mutagens/carcinogens and even mutagens
arising from normal physiological processes.12,13 Depending
on their particular mode of action, antimutagenic agents
can be classified into two broad categories: desmutagens,
which operate by inactivating mutagens before they can
cause damage to cellular constituents such as DNA, and
bioantimutagens, which modulate mutagenesis at the
cellular level (i.e., within the cell), one mechanism being
the induction of cellular repair mechanisms in response to
mutagenic damage.14 It is well established that antimu-
tagenic agents may operate via a combination of these
different mechanisms.15
acting mutagen, ethyl methanesulfonate (EMS).17 Several
members of this genus are folklorically reputed to be
associated with a variety of general medicinal properties
such as diuretic, stomachic, stimulant, and tonic.18,19 Cin-
namaldehyde (2), a structurally related compound, has long
been known to be antimutagenic.20 The antimutagenic
activity of cinnamaldehyde was confirmed by us previously,
and in our hands, under conditions slightly different from
this study, cinnamaldehyde was shown to be antimutagenic
against EMS induced mutations in S. typhimurium TA100
strain.21 Although much of the focus of the scientific
literature on cinnamaldehyde-related antimutagenesis con-
cerns the biological mechanisms by which the antimu-
tagenic effects of this compound are produced,21-24 some
effort has been directed toward establishing a correlation
between the chemical structure and antimutagenic activity
of this agent. Chemical studies with cinnamaldehyde (2)
and other structurally related antimutagens with regard
to their activity against UV-induced mutations in Escheri-
chia coli have indicated that the antimutagenic activity of
these agents may be correlated with the ability of the
conjugated R,â-unsaturated carbonyl moiety to add cellular
nucleophiles such as thiol groups on proteins or possibly
DNA in a Michael acceptor sense.25-27 In light of this
evidence, we wished to examine if the same was also true
in the case of the antimutagenic activity of plicatin B (1),
particularly relating to its activity in our assay system
utilizing EMS, a DNA alkylating agent,16 as the mutagenic
insult. For this purpose, we undertook a systematic struc-
ture-activity relationship (SAR) analysis of plicatin B (1),
attempting to elucidate its chemical mode of action and
possibly optimize the antimutagenic activity of this natural
product during the process.
Some novel antimutagens from higher plants have been
identified previously in this laboratory with the help of a
systematic bioassay-directed fractionation methodology and
modified screening procedures.16,17 One of these agents,
plicatin B (1), a substituted cinnamyl ester isolated from
the active extract of the stem and leaf parts of Psoralea
juncea (Eastw.) Rydb. (Leguminosae), was observed to be
active in the modified Ames test against mutations induced
in Salmonella typhimurium strain TA100 by the direct-
Resu lts a n d Discu ssion
As mentioned above, inspection of the chemical structure
of cinnamaldehyde (2) and certain other structurally
related compounds in relation to their antimutagenic
activity has led to the reasonable speculation that the
* To whom correspondence should be addressed. Fax: 785-864-5326,
Tel: 785-864-4562. E-mail: lmitscher@rx.pharm.ukans.edu.
† Department of Medicinal Chemistry.
‡ Department of Microbiology.
10.1021/np980304n CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/25/1998

Structure-Antimutagenic Activity of Plicatin B
J ournal of Natural Products, 1999, Vol. 62, No. 1 103
antimutagenic activity of 1. Since an aromatic hydroxyl
group para to an R,â-unsaturated carbonyl moiety has been
observed to be essential for the antimutagenic activity of
chalcones and certain other structurally related antimu-
tagens, we sought to examine the effect of alkylation of the
phenolic group in plicatin B (1) on its antimutagenic
activity as well.28,29 For this purpose, we synthesized and
evaluated the methyl ether analogue (5) of plicatin B (1)
for its antimutagenic activity.
F igu r e 1. Putative chemical mechanism underlying the antimu-
tagenic activity of plicatin B and structurally related antimutagens.
Nu: denotes cellular nucleophiles, possibly proteins or DNA.
The synthesis of plicatin B and its related analogues is
outlined in Scheme 1. Plicatin B (1) itself was conveniently
prepared by the C-prenylation of readily accessible p-
hydroxycinnamic acid methyl ester (7) utilizing the method
of Fatope and Okogun.30 Selective reduction of the conju-
gated ester functionality in plicatin B (1) using diisobutyl-
aluminum hydride (DIBAL-H) resulted in the allylic alcohol
4.31 Catalytic hydrogenation of 7 over palladium catalyst32
followed by C-prenylation of the resulting dihydro com-
pound 8 yielded the differentially partially saturated
analogue 3. The methyl ether analogue 5 was prepared
from plicatin B (1) using standard alkylation procedures.
antimutagenic activity of these compounds may be associ-
ated with their ability to operate as Michael acceptors
within the cell, where they could possibly effect the
alkylation of cellular nucleophiles such as DNA and/or
cellular proteins. The initial DNA damage caused by these
agents may trigger the induction of a SOS response that
can prime the cell to future exposure to other mutagens
such as EMS (Figure 1). Thus, these compounds may be
thought to be operating primarily via a bioantimutagenic
mechanism rather than a desmutagenic mechanism. Due
to the structural similarity between cinnamaldehyde (2)
and plicatin B (1), one of the obvious issues we sought to
investigate with regard to the latter was the effect of
saturation of the conjugated alkene double bond on its
antimutagenic activity. Such a modification would render
the resulting dihydro analogue 3 incapable of functioning
as a Michael acceptor, and its antimutagenic activity, or
lack thereof, could shed some light on the relevance of the
proposed chemical mode of action of cinnamaldehyde (2)
and other structurally related antimutagens to plicatin B
(1). A second, somewhat convergent approach to this
problem would be to leave the conjugated alkene double
bond in 1 intact but reduce the ester functionality. The
resulting alcohol 4, like compound 3, would be far less
capable of reacting in a Michael acceptor sense with cellular
nucleophiles such as DNA and proteins than plicatin B (1)
itself. Compound 7, lacking the C-3 alkenyl substituent of
plicatin B (1), was evaluated in the present study to
determine the contribution of this substituent to the overall
Initially, cytotoxicity assays were conducted on all the
compounds in this study to establish concentrations that
were nontoxic to the tester strain since dead cells obviously
cannot mutate. The true antimutagenic activities of these
compounds were then determined at 2-fold concentration
intervals each, ranging up to half the maximum nontoxic
dose, utilizing the modified Ames test protocol and the
direct-acting mutagen, ethyl methanesulfonate (EMS).33-35
The results of these assays, denoted by the percent inhibi-
tion of EMS-induced mutations in the tester strain S.
typhimurium TA100, by plicatin B (1) and its related
analogues are listed in Table 1.
Notably, examination of these results for plicatin B (1)
and its dihydro analogue 3 at the maximum comparative
nontoxic concentration (7.8 µM) indicates that the reduction
of the conjugated alkene double bond in plicatin B is not
detrimental to its activity, implying that the antimutagenic
activity of plicatin B is not likely to be associated with its
ability to react with cellular nucleophiles in a Michael
Sch em e 1a
a
Key: (i) MeOH, concd H2SO4; (ii) NaH, toluene; then prenyl bromide; (iii) Pd/C, H2, 1 atm, EtOH; (iv) DIBAL-H, toluene; (v) K2CO3, MeI; (vi) NaOH,
H2O.

104 J ournal of Natural Products, 1999, Vol. 62, No. 1
Menon et al.
Ta ble 1. Percent Inhibition of EMS-Induced Mutations in
Salmonella Typhimurium Strain TA100 by Plicatin B (1) and
Its Analoguesa
in the antimutagenic activity of 8 in the present assay, an
outcome that is in direct contrast to the trend observed
with plicatin B and its dihydro analogue 3. Similarly,
comparison of the antimutagenic activities of compound 7
and its acid derivative 6 with plicatin B (1) and its acid
derivative 9 implicates such a noncongruent trend as well.
These results suggest that these two distinct sets of
compounds in this study, i.e., the compounds bearing a C-3
alkenyl substituent and those lacking it, might be operating
via different mechanisms, and thus, a direct comparison
of their antimutagenic activities may not be fully justified.
Thus, the actual contribution of the C-3 alkenyl substituent
to the antimutagenic activity of plicatin B cannot be
meaningfully ascertained from this study.
In summary, the modified Ames test results from this
study, where comparable, indicate that structural modifi-
cation of plicatin B (1) generally leads to a reduction in
cytotoxicity but produces rather insignificant changes in
its antimutagenic activity with one notable exception;
reduction of the conjugated alkene double bond leads to a
compound that exhibits reduced cytotoxicity and enhanced
antimutagenic efficacy relative to plicatin B in the assay
system employed. Although plicatin B might be operating
via a combination of desmutagenic and bioantimutagenic
mechanisms of action in the present assay, this significant
finding serves to exclude the possibility of a Michael
acceptor role of the conjugated R,â-unsaturated carbonyl
moiety of plicatin B in connection with its antimutagenic
activity. The actual mechanism of antimutagenesis must
take place using different chemistry and evidence to this
point will appear subsequently. This study serves to
illustrate the point that an extension of the postulated
chemical mechanism of action of one antimutagenic agent,
such as cinnamaldehyde (2), to another, such as plicatin B
(1), based solely on structural analogy is not always fully
justifiable.
compd
concn per plateb (µM)
% inhibitionc
plicatin B (1)
3.9
7.8
3.9
7.8
31.3
3.9
7.8
31.3
3.9
7.8
3.9
25.4 ( 0.95
30.2 ( 1.56
25.2 ( 2.81
38.5 ( 2.00
46.8 ( 0.25
20.2 ( 0.65
27.1 ( 0.56
36.8 ( 0.61
13.2 ( 0.30
21.5 ( 1.06
11.2 ( 0.89
13.6 ( 2.97
49.8 ( 2.10
0.0
3
4
5
6
7.8
125
3.9
7
7.8
0.9 ( 0.9
62.5
3.9-125
3.9
7.8
15.6
7.6 ( 6.0
8
9
0.0
0.3 ( 0.5
0.6 ( 0.6
12.0 ( 0.74
a
In an earlier set of experiments, cinnamaldehyde (2) at its
maximum nontoxic concentration (25 µg/mL) produced approxi-
mately 30% inhibition of EMS induced mutations in S. typhimu-
b
rium TA100 strain (see ref 21). Results of all the examined test
concentrations for each compound are not listed; rather, only the
comparative and the half-maximum nontoxic test concentrations
for each compound are given. c % inhibition ) 100 - [(no. of
induced revertants in the presence of EMS and test compound -
no. of spontaneous revertants/no. of induced revertants in the
presence of EMS - no. of spontaneous revertants) × 100].
acceptor sense. A similar conclusion has been arrived at
previously regarding the antimutagenic activity of certain
structurally related resin constituents (cinnamic acid,
cinnamyl cinnamate, and cinnamyl ricinoleate) derived
from the medicinal plant, Liquidambar orientalis and some
synthetic analogues.36 In fact, analogue 3 is observed to
be slightly more potent than plicatin B itself; furthermore,
this modification reduces the cytotoxicity of 3 toward the
tester strain so that now analogue 3 at its maximum
nontoxic dose (31.3 µM) exhibits significantly greater
efficacy in inhibiting EMS-induced mutations in the tester
strain than does plicatin B itself. Likewise, reduction of
the ester functionality in plicatin B (1) to the alcohol moiety
in 4 is also noted to have little effect on its antimutagenic
activity at the maximum comparative nontoxic concentra-
tion (7.8 µM). O-Methylation of plicatin B results in a small
reduction in the antimutagenic activity for the resulting
ether analogue 5. Since it is well-known that antimutagenic
agents can operate via a combination of desmutagenic and
bioantimutagenic mechanisms,15 this decrease in antimu-
tagenic activity for 5 in comparison to plicatin B could be
attributed to its relative inabilty to inactivate EMS directly,
possibly via the reactive phenolic hydroxyl group. An issue
of some relevance to this matter is the observation made
with certain antimutagens that phenolic hydroxyl groups
are directly involved in the scavenging of mutagens, for
example, the diol epoxide derived from benzo[a]pyrene
activation.37
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Flash column chro-
matography was carried out using silica gel 32-63 (40 micron)
purchased from Selecto, Inc., Kennesaw, GA. Proton nuclear
magnetic resonance spectra (1H NMR) and carbon nuclear
magnetic resonance spectra (13C NMR) were recorded on either
a GE QE-300 or a Bruker AM 500 spectrometer. Chemical
shifts (δ) are reported in parts per million (ppm) downfield
from tetramethylsilane (Me4Si; 0.0 ppm) for 1H NMR and
deuterated chloroform (CDCl3, 77.0 ppm) or deuterated di-
methyl sulfoxide (DMSO-d6, 39.5 ppm) for 13C NMR. Infrared
spectra (IR) were recorded on a Perkin-Elmer 1420 infrared
spectrophotometer. Electron-impact mass spectra (EIMS),
chemical-ionization mass spectra (CIMS), and high-resolution
mass spectra (HRMS) were obtained on a ZAB HS or a Nermag
R 10-10 mass spectrometer. The intensity of each peak in the
mass spectrum relative to the base peak is reported in
parentheses. Microanalyses were performed on a Perkin-Elmer
2400 CHN analyzer at the University of Kansas. Melting
points were determined in open capillaries on a Thomas-
Hoover capillary melting point apparatus and are uncorrected.
p-Hydroxycinnamic acid (6) was purchased from Aldrich
Chemical Co.
Oxoid Nutrient Broth No. 2 was commercially obtained from
Unipath, Ogdensburg, NY. Ethyl methanesulfonate (EMS) and
the other biochemicals used in this study were purchased from
Sigma.
4-Hyd r oxycin n a m ic Acid Meth yl Ester (7). A solution
of 6 (10.0 g, 60.9 mmol) in MeOH (100 mL) containing
concentrated H2SO4 (3 mL) was refluxed overnight. Solvent
was evaporated under reduced pressure, and the residual solid
was partitioned between CHCl3 (250 mL) and H2O (100 mL).
Significantly, compound 7, which lacks the C-3 alkenyl
moiety of plicatin B (1), is devoid of any antimutagenic
activity in this assay. One could attribute this lack of
activity for compound 7 relative to plicatin B to the absence
of the C-3 alkenyl substituent, but further inspection of
the data in Table 1 suggests a somewhat different explana-
tion. Thus, a perusal of the data in Table 1 for compound
7 and its dihydro analogue 8 reveals that reduction of the
alkene double bond in 7 does not produce an improvement

Structure-Antimutagenic Activity of Plicatin B
J ournal of Natural Products, 1999, Vol. 62, No. 1 105
1
The organic phase was separated, and the aqueous phase was
reextracted with CHCl3 (100 mL). The organic extracts were
pooled, washed successively with saturated aqueous NaHCO3
(75 mL), water (50 mL), and brine, and finally dried over
anhydrous Na2SO4. Removal of solvent under reduced pressure
followed by recrystallization of the residue from CHCl3-
hexane yielded 6.84 g (63%) of 7 as a colorless solid: mp 138-
1200 cm-1; H NMR (CDCl3, 300 MHz) δ 6.96-6.90 (2H, m,
H-2, H-6), 6.75-6.70 (1H, m, H-5), 5.35-5.25 (1H, m, H-2′′),
5.14-5.10 (1H, m, OH), 3.67 (3H, s, CO2CH3), 3.32 (2H, d, J
) 7.2 Hz, H-1′′), 2.86 (2H, t, J ) 7.8 Hz, H-1′), 2.59 (2H, t, J
) 7.8 Hz, H-2′), 1.77 (6H, s, H-4′′, H-5′′); 13C NMR (CDCl3,
500 MHz) δ 173.5, 152.7, 134.6, 132.6, 129.8, 127.1, 126.8,
121.8, 115.7, 51.6, 36.1, 30.2, 29.8, 25.8, 17.8; EIMS m/z 248
(M+, 87), 193 (100), 175 (80), 159 (65), 133 (64); anal. C 72.36%,
H 8.22%, calcd for C15H20O3, C 72.55%, H 8.12%.
139 °C; IR (KBr) νmax 3360, 1680, 1590, 1420, 1190, 1165 cm-1
;
1H NMR (CDCl3-DMSO-d6, 300 MHz) δ 8.76-8.66 (1H, b,
OH), 7.63 (1H, d, J ) 16.0 Hz, H-1′), 7.39 (2H, d, J ) 8.6 Hz,
H-2, H-6), 6.86 (2H, d, J ) 8.6 Hz, H-3, H-5), 6.27 (1H, d, J )
16.0 Hz, H-2′), 3.78 (3H, s, CO2CH3); 13C NMR (CDCl3-DMSO-
d6, 300 MHz) δ 168.4, 159.8, 145.4, 130.3, 126.5, 116.4, 114.8,
51.9; EIMS m/z 178 (M+, 53), 147 (100), 119 (30), 91 (34), 65
(28); anal. C 67.29%, H 5.80%, calcd for C10H10O3, C 67.41%,
H 5.66%.
4-Hydr oxy-3-(3-m eth yl-2-bu ten yl)cin n am yl Alcoh ol (4).
A solution of 1 (0.200 g, 0.813 mmol) in dry toluene (2 mL)
was cooled to 0 °C, and then a 1.5 M solution of DIBAL-H in
toluene (1.70 mL, 2.55 mmol) was added to it dropwise by
syringe over a period of 0.5 h. The reaction mixture was stirred
at 0 °C for an additional 4.5 h and then quenched at 0 °C by
the dropwise addition of MeOH (over a period of 20-30 min).
The reaction mixture was then stirred at 5 °C for 15 min and
filtered through Celite, and the filter bed was washed thor-
oughly with hot MeOH. The filtrate was concentrated under
reduced pressure to yield a yellow solid, which upon flash
chromatography [20 g flash silica gel, hexanes-EtOAc (4:1)]
afforded 0.130 g (73%) of 4: mp 87-90 °C; IR (KBr) νmax 3380,
3280-3060, 2920, 1600, 1490, 1250, 1220, 1100 cm-1; 1H NMR
(CDCl3-DMSO-d6, 300 MHz) δ 8.17 (1H, s, ArOH), 7.12 (1H,
d, J ) 2.0 Hz, H-2), 7.06 (1H, dd, J ) 8.2, 2.2 Hz, H-6), 6.77
(1H, d, J ) 8.2 Hz, H-5), 6.50 (1H, d, J ) 15.9 Hz, H-1′), 6.18
(1H, dt, J ) 15.8, 5.9 Hz, H-2′), 5.38-5.29 (1H, m, H-2′′), 4.25
(2H, m, H-3′), 3.31 (2H, d, J ) 7.3 Hz, H-1′′), 2.91 (1H, t, J )
5.7 Hz, CH2OH), 1.75 (3H, s, H-4′′ or H-5′′), 1.72 (3H, s, H-4′′
or H-5′′); 13C NMR (CDCl3-DMSO-d6, 500 MHz) δ 154.4, 132.2,
130.5, 128.1, 127.7, 127.3, 125.6, 124.7, 122.2, 115.0, 63.1, 28.1,
25.5, 17.5; CIMS (NH3) m/z 218 (M+, 49), 175 (89), 163 (100),
145 (30), 133 (37); anal. C 77.00%, H 8.00%, calcd for C14H18O2,
C 77.03%, H 8.31%.
4-Meth oxy-3-(3-m eth yl-2-bu ten yl)cin n a m ic Acid Meth -
yl Ester (5) (P lica tin B Meth yl Eth er ). To a mixture of 1
(0.207 g, 0.841 mmol) and anhydrous K2CO3 (0.124 g, 0.899
mmol) in acetone (1.5 mL) was added iodomethane (0.11 mL,
1.77 mmol) in one portion at room temperature. The reaction
mixture was stirred and heated at 60-65 °C for 4 h, and
solvent was then evaporated under reduced pressure. Some
water was added to the residue, and the mixture was neutral-
ized with cold acetic acid (2 N). The aqueous phase was
extracted with EtOAc (3 6 10 mL), the combined organic
extract was dried (Na2SO4), and solvent was removed under
reduced pressure. The crude residue was subjected to flash
chromatography (7 g silica gel) with hexanes-EtOAc (5:1) as
the eluent. Rechromatography of the product fractions on flash
silica gel (20 g) using hexanes-EtOAc (7:1) as eluent afforded
0.139 g (63%) of 5 as a colorless oil: IR (neat) νmax 2940-2820,
1710, 1630, 1590, 1490, 1240 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 7.64 (1H, d, J ) 16.0 Hz, H-1′), 7.37-7.32 (2H, m, H-2, H-6),
6.83 (1H, d, J ) 8.3 Hz, H-5), 6.30 (1H, d, J ) 16.0 Hz, H-2′),
5.33-5.22 (1H, m, H-2′′), 3.86 (3H, s, ArOCH3 or CO2CH3),
3.79 (3H, s, ArOCH3 or CO2CH3), 3.30 (2H, d, J ) 7.3 Hz,
H-1′′), 1.76 (3H, s, H-4′′ or H-5′′), 1.71 (3H, s, H-4′′ or H-5′′);
13C NMR (CDCl3, 500 MHz) δ 167.9, 159.2, 145.0, 133.2, 130.7,
128.9, 127.7, 126.7, 121.7, 114.8, 110.2, 55.5, 51.5, 28.2, 25.8,
17.7; EIMS m/z 260 (M+, 100), 245 (15), 229 (16), 213 (48),
205 (26), 185 (46), 115 (35); anal. C 73.48%, H 8.00%, calcd
for C16H20O3, C 73.82%, H 7.74%.
P lica tin B (1). To a suspension of 7 (3.31 g, 18.6 mmol) in
dry toluene (50 mL) was added NaH (60%, 0.820 g, 20.5 mmol)
in one portion at ambient temperature, and subsequently the
reaction mixture was heated at 60-65 °C for 3.5 h. The
reaction mixture was cooled to 35 °C, 4-bromo-2-methyl-2-
butene (prenyl bromide; 2.95 mL, 25.6 mmol) was added
dropwise (15-20 min) by syringe to the reaction mixture at
35 °C, and stirring was continued at this temperature for 72
h. At this time, the reaction mixture was quenched by pouring
it into 300 mL of ice-water. The aqueous layer was neutral-
ized, with ice cooling, with cold 2 N acetic acid and then
extracted with Et2O (3 6 200 mL). The organic extract was
washed with H2O (100 mL) and brine, and then dried
(anhydrous Na2SO4). Evaporation of the solvent under reduced
pressure and subsequent flash chromatography (300 g silica
gel) on the residue utilizing hexanes-EtOAc (5:1) as the eluent
yielded 1.84 g (40%) of 1. An analytical sample of 1 was
recrystallized from Et2O-petroleum ether as a pale yellow
solid: mp 78-82 °C (lit.38 mp 86-87 °C, lit.39 mp 70-72 °C);
IR (KBr) νmax 3220, 1660, 1580, 1260, 1230, 1160 cm-1 1H
;
NMR (CDCl3, 300 MHz) δ 7.63 (1H, d, J ) 16.0 Hz, H-1′),
7.31-7.26 (2H, m, H-2, H-6), 6.82 (1H, d, J ) 8.9 Hz, H-5),
6.29 (1H, d, J ) 16.0 Hz, H-2′), 5.92 (1H, s, OH), 5.35-5.29
(1H, m, H-2′′), 3.80 (3H, s, CO2CH3), 3.36 (2H, d, J ) 7.2 Hz,
H-1′′), 1.81-1.77 (6H, m, H-4′′, H-5′′); 13C NMR (CDCl3, 300
MHz) δ 168.4, 156.9, 145.4, 135.5, 130.5, 128.1, 128.0, 127.6,
121.6, 116.5, 115.3, 51.9, 29.8, 26.1, 18.2; EIMS m/z 246 (M+,
87), 191 (100), 171 (26), 131 (31), 115 (25); anal. C 72.83%, H
7.75%, calcd for C15H18O3, C 73.15%, H 7.36%.
4-Hyd r oxyh yd r ocin n a m ic Acid Meth yl Ester (8). A
mixture of 7 (3.25 g, 18.3 mmol) and 5% Pd/C catalyst (0.325
g) in EtOH (65 mL) was hydrogenated at room temperature
and 1 atm for 24 h. The reaction mixture was filtered through
Celite, and the filter bed was washed with MeOH. The filtrate
was evaporated under reduced pressure, and the residual oil
was distilled bulb to bulb (145 °C, 3.5 mmHg) to afford 3.27 g
(99%) of 8 as a colorless oil: 1H NMR (CDCl3, 300 MHz) δ 7.05
(2H, d, J ) 8.5 Hz, H-2, H-6), 6.75 (2H, d, J ) 8.5 Hz, H-3,
H-5), 5.27 (1H, s, OH), 3.67 (3H, s, CO2CH3), 2.88 (2H, t, J )
7.7 Hz, H-1′), 2.60 (2H, t, J ) 7.7 Hz, H-2′); CIMS (NH3) m/z
180 (M+, 70), 149 (14), 120 (65), 107 (100); anal. C 66.48%, H
7.00%, calcd for C10H12O3, C 66.65%; H 6.71%.
4-Hyd r oxy-3-(3-m eth yl-2-bu ten yl)h yd r ocin n a m ic Acid
Meth yl Ester (3) (Dih yd r op lica tin B). To a solution of 8
(1.40 g, 7.78 mmol) in dry toluene (20 mL) was added NaH
(60%, 0.343 g, 8.58 mmol) in one portion at room temperature.
The reaction mixture was heated at 65 °C for 3.5 h and cooled
to 35 °C, and then prenyl bromide (1.30 mL, 11.3 mmol) was
added dropwise by syringe over a period of 10 min. The
reaction mixture was stirred and heated at this temperature
for 3 days and then quenched by pouring it into 150 mL of
ice-water. After neutralization with cold 2 N acetic acid, the
aqueous phase was extracted with Et2O (3 × 100 mL). The
organic extract was washed with H2O (100 mL) and brine and
then dried (anhydrous Na2SO4). Solvent was evaporated under
reduced pressure and the residue was subjected to flash
chromatography (200 g silica gel) with hexanes-EtOAc (10:
1) as eluent to ultimately afford 0.638 g (33%) of 3 as an oil:
IR (neat) νmax 3420, 2940, 2900, 1715, 1500, 1430, 1340, 1250,
Dr u p a n in (9). A mixture of 1 (0.333 g, 1.35 mmol) and
NaOH (0.125 g, 3.12 mmol) in H2O (0.5 mL) was heated at
reflux for 2.5 h. The black solution was allowed to cool to room
temperature and then extracted with Et2O (3 × 1 mL). The
aqueous phase was cooled (ice-water bath), acidified with cold
AcOH (2 N), and then reextracted with Et2O (3 × 5 mL). The
combined organic extract was washed with H2O (5 mL) and
then brine and finally dried over anhydrous Na2SO4. Solvent
was evaporated under reduced pressure to yield 0.274 g (87%)
of 9. An analytical sample of 9 was recrystallized from Et2O-
hexane: mp 145-147 °C (lit.38 mp 146-147 °C); IR (KBr) νmax
3300, 3040-2840, 1640, 1580, 1420, 1300, 1250, 1220, 1150,
1100 cm-1; 1H NMR (CDCl3-DMSO-d6, 300 MHz) δ 9.20-8.90
(1H, b, CO2H), 7.58 (1H, d, J ) 15.9 Hz, H-1′), 7.26 (1H, d, J

106 J ournal of Natural Products, 1999, Vol. 62, No. 1
Menon et al.
(8) Hayatsu, H.; Arimoto, S.; Negishi, T. Mutation Res. 1988, 202, 429-
446.
(9) J ang, M.; Cai, L.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.;
Beecher, C. W. W.; Fong, H. H. S.; Farnsworth, N. R.; Kinghorn, A.
D.; Mehta, R. G.; Moon, R. C.; Pezzuto, J . M. Science 1997, 275, 218-
220.
) 2.0 Hz, H-2), 7.21 (1H, dd, J ) 8.3, 2.2 Hz, H-6), 6.84 (1H,
d, J ) 8.2 Hz, H-5), 6.22 (1H, d, J ) 15.9 Hz, H-2′), 5.35-5.29
(1H, m, H-2′′), 3.30 (2H, d, J ) 7.3 Hz, H-1′′), 1.76 (3H, s, H-4′′
or H-5′′), 1.71 (3H, s, H-4′′ or H-5′′); 13C NMR (CDCl3-DMSO-
d6, 500 MHz) δ 168.9, 157.0, 144.8, 132.4, 129.1, 128.2, 126.8,
125.4, 121.6, 115.0, 114.4, 27.7, 25.4, 17.4; CIMS (NH3) m/z
233 (M + 1, 74), 215 (37), 189 (100), 177 (33), 133 (36); anal.
C 72.30%, H 7.30%, calcd for C14H16O3, C 72.39%, H 6.94%.
Cytotoxicity Assa ys. Cytotoxicity assays were performed
using S. typhimurium strain TA100 grown in Oxoid Nutrient
Broth No. 2 using a 96 well ELISA plate containing varying
concentrations of test compounds incubated for 15 h at 37 °C.
After incubation, the plates were subjected to readings at
absorbance 570 nm using a Cambridge Technology, Inc., plate
solver ver. 4.00.
Mod ified Am es Tests. The modified Ames tests were
performed based on the protocol described by Maron and
Ames33 using S. typhimurium strain TA100. Each experiment
was performed in triplicate. The cells were grown for 15 h in
Oxoid Nutrient Broth No. 2 at 37 °C with shaking to an
approximate density of (1-2) × 108 cells/mL prior to each
assay.
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To induce His+ revertants, 0.1 mL of the culture was added
to test tubes containing 0.5 µL/mL of EMS in the presence or
absence of varying concentrations of the test compounds and
incubated at 37 °C for 30 min. Following this incubation, 2
mL of Ames top agar supplemented with 50 µg/mL of L-
histidine and 0.74 µg/mL of D-biotin was added to the above
compound, and the mixture was then plated on VogelBonner
E Medium using the top agar layer method as described by
Maron and Ames.33 After incubation at 37 °C for 2 days, the
plates were scored for His+ revertant colonies using a Dynatech
Autocount colony counter.
Ack n ow led gm en t. The Salmonella typhimurium strain
TA100 was generously provided by Dr. Bruce Ames, University
of California (Berkeley). This research was supported by a
grant from the NIH, Bethesda, MD.
Refer en ces a n d Notes
(1) Undergraduate research participant in Medicinal Chemistry, 1995-
1996.
(2) Taken in part from: Shih, P. Antimutagenesis and Antioxidation by
Phenolic Compounds. Ph.D. Thesis, University of Kansas, Lawrence,
KS, 1997.
(3) Anonymous. Health, United States, 1996-97 and Injury Chartbook;
National Center for Health Statistics: Hyattsville, MD, 1997.
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(6) Wattenberg, L. W. Cancer Res. 1993, 53, 5890-5896.
(7) Greenwald, P. Sci. Am. 1996, 275, 96-99.
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