Design, synthesis and biological evaluation of novel riccardiphenol analogs

Article abstract of DOI:10.1016/j.bmc.2005.02.010

A novel, facile, high yield, and less cumbersome synthesis of riccardiphenol analogs is described. The synthesized compounds were characterized and assessed for its in vitro activity in a panel of human cancer cell lines of differing origin: HuCCT-1, BxPC3, Panc-1, Mia-Paca, A431, Hep2, and HN006. HuCCT-1 was derived from an intrahepatic cholangiocarcinoma; BxPC3, Mia-Paca, and Panc-1 were derived from pancreatic cancers; A431 was derived from a vulvar epithelial carcinoma; and Hep2 and HN006 were derived from squamous cell carcinomas of the head and neck. The cytotoxicity of a newly developed riccardiphenol analog against human cancer cell lines was assessed. The cancer cells exhibited varying sensitivities to the compound, with IC₅₀ values from 30 to 50 μM. This susceptibility was particularly interesting in the case of lines such as Hep2 and BxPC3 that are resistant to classic cytotoxic drugs as well as some targeted agents. These results demonstrate that the novel riccardiphenol analog has effective action against human-derived cancer cell in vitro.

Full text of DOI:10.1016/j.bmc.2005.02.010

Bioorganic & Medicinal Chemistry 13 (2005) 2873–2880
Design, synthesis and biological evaluation of novel
riccardiphenol analogs
Srinivas K. Kumar,a Maria Amador,a Manuel Hidalgo,a
Sujata V. Bhatb and Saeed R. Khana,*
aDivision of Chemical Therapeutics, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA
bDepartment of Chemistry, Indian Institute of Technology Bombay, Mumbai, 400 076 India
Received 16 December 2004; revised 3 February 2005; accepted 9 February 2005
Abstract—A novel, facile, high yield, and less cumbersome synthesis of riccardiphenol analogs is described. The synthesized com-
pounds were characterized and assessed for its in vitro activity in a panel of human cancer cell lines of differing origin: HuCCT-1,
BxPC3, Panc-1, Mia-Paca, A431, Hep2, and HN006. HuCCT-1 was derived from an intrahepatic cholangiocarcinoma; BxPC3,
Mia-Paca, and Panc-1 were derived from pancreatic cancers; A431 was derived from a vulvar epithelial carcinoma; and Hep2
and HN006 were derived from squamous cell carcinomas of the head and neck. The cytotoxicity of a newly developed riccardiphe-
nol analog against human cancer cell lines was assessed. The cancer cells exhibited varying sensitivities to the compound, with IC50
values from 30 to 50 lM. This susceptibility was particularly interesting in the case of lines such as Hep2 and BxPC3 that are resis-
tant to classic cytotoxic drugs as well as some targeted agents. These results demonstrate that the novel riccardiphenol analog has
effective action against human-derived cancer cell in vitro.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Riccardiphenols, which have a seco-eudesmane skeleton
attached to phenol, have been shown to have both cyto-
toxic and anti-bacterial activity. Though such sesquiter-
pene–quinone and sesquiterpene–quinol skeletons are
rare in terrestrial plants, they are common in marine
sources6 and include zonarol,7 avarol,8 and ilimaqui-
nones.6 Tori et al.9 have reported total synthesis of ric-
cardiphenols A (1) and B (2) and have elucidated their
absolute configuration.10,11 In this approach, the seco-
eudesmane skeleton was synthesized first, and then the
benzylic component was attached.
In the course of their phytochemical study of bryo-
phytes,1–3 Toyota and Asakawa4 found that liverworts,
including the Jungermanniales, are rich sources of terp-
enoids with a variety of carbon skeletons. These Riccar-
diaceae, belonging to the order Metzgeriales,
occasionally contain aromatic compounds. The com-
pounds isolated from liverworts show very interesting
biological activities, including anti-fungal, anti-micro-
bial, and anti-tumor activity, as well as the ability to in-
hibit 5-lipoxygenase, calmodulin, and plant growth.
Here we report a novel approach to synthesize riccardi-
phenol B analogs and have tested the cytotoxic activity
of one of these analogs against a variety of cancer cell
lines.
From a Japanese collection of Riccardia crassa, Toyota
and Asakawa4 isolated riccardiphenols A (1) and B (2),
which contain a sesquiterpene portion attached to a qui-
nol ring, and later Perry et al.5 isolated riccardiphenol C
(3) from a New Zealand collection of R. crassa
(Chart 1).
Conjugated dienes are versatile building blocks in the
synthesis of organic natural products, especially as a
component of the Diels–Alder reaction in the synthesis
of six-membered cyclic compounds. 3-Methyl-3-sulfo-
lene,12,13 the isoprene–sulfur dioxide adduct, and its
derivatives are attractive as masked diene synthons, since
they generate dienes readily by thermal desulfonylation
under relatively mild conditions.14,15 The terminal CH
bonds of the original dienes are activated by the adjacent
Keywords: Anti-tumor; Riccardiphenol; Sulfolene; Diels–Alder
reaction.
*
Corresponding author. Tel.: +1 410 614 0200; fax: +1 410 614
8397; e-mail: khansa@jhmi.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.010

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S. K. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 2873–2880
OMe
O
O
OH
OH
OH
Riccardiphenol A (1)
Riccardiphenol B (2)
Riccardiphenol C (3)
Chart 1. Structures of riccardiphenols.
sulfonyl group,16 suggesting the possibility of modifying
the terminal position of the dienes.
Benzyl bromide 10 is highly unstable, and its prepara-
tion by treating the corresponding alcohol 9 with PPh3
in CCBr4 resulted in a very poor yield (9%). Therefore,
we followed CoreyÕs procedure17 for preparing benzyl
bromides from benzyl alcohol, followed by immediate
reaction with 3-methyl-3-sulfolene. Benzyl bromide 10
was synthesized from p-hydroxy anisole as shown in
Scheme 2. The phenolic hydroxyl group was protected
as a MOM derivative, followed by ortholithiation and
formylation with BuLi and DMF, to give compound
8.18,19 Compound 8, on further reduction with NaBH4,
gave the benzyl alcohol 9.
Thus, by introducing the appropriate electrophile in 3-
methyl-3-sulfolene, the required terminally substituted
conjugated dienes can be generated. We decided to use
this strategy for the construction of the substituted six-
membered ring in the synthesis of riccardiphenol B.
Since sulfolenes react only with alkyl iodides or alkyl
bromides and not with alkyl chlorides,13 we had to start
with benzyl bromide or iodide.
2. Results and discussion
2.1. Chemistry
Benzyl alcohol 9, when treated with N-bromosuccin-
amide in the presence of dimethyl sulfide in dry dichloro-
methane at 10 ꢁC, gave the benzyl bromide 10.17 Almost
all reports dealing with deprotonation/substitution reac-
tions of 3-sulfolenes describe the use of HMPA as cosol-
vent when using n-BuLi or LiHMDS as the base. Under
these conditions, the sulfolene carbanion is thought to
be stabilized by the cation-trapping action of the
HMPA. Chou and co-workers12 have reported the alkyl-
ation of 3-methyl-3-sulfolene (2.5 equiv) with geranyl
bromide (1 equiv) in the presence of HMPA (4 equiv)
at À78 ꢁC while using LiHMDS (1 equiv) as the base.
In our approach (Scheme 3) 3-methyl-3-sufolene was
first alkylated with the benzylic bromides 6 and 10 to
give 2-benzylsulfone 12. Diels–Alder reaction of the
diene 13 generated from the thermolysis of sulfolene
12 with acrolein gave benzylic-substituted six-membered
ring 14. This, upon methylation and a Wittig reaction
with isoprenyl bromide gave the skeleton of riccardiphe-
nol 2.
2,5-Dimethoxybenzyl bromide 6 was easily prepared
from 2,5-dimethoxybenzaldehyde 4 as the starting mate-
rial (Scheme 1). Aldehyde 4, on reduction with NaBH4
in methanol, gave the alcohol 5. The alcohol 5, upon
treatment with aqueous hydrobromic acid, gave 2,5-
dimethoxybenzyl bromide 6.
Following the above procedure, the 3-methyl-3-sulfo-
lene was alkylated with substituted benzyl bromide. 3-
Methyl-3-sulfolene was prepared by heating a methan-
olic solution of isoprene and liquid SO2 at 80 ꢁC for
4 h in a sealed tube. A THF solution of 3-methyl-3-sul-
folene (1 equiv), benzyl bromide (2 equiv) 10, and
HMPA (2 equiv) was cooled to À98 ꢁC (MeOH/liq. N2),
and a THF solution of LiHMDS was added at the
same temperature. The reaction mixture was allowed
to warm to 0 ꢁC over 30 min and was then quenched
with saturated aqueous NH4Cl solution. A standard
work-up, followed by silica gel chromatographic
(SGC) purification, gave a colorless, viscous liquid,
which was characterized as 2-(2-methoxymethoxy-5-
methoxybenzyl)-3-methyl-3-sulfolene 12b from spectral
data. The 1H NMR spectrum of this compound dis-
played a broad singlet at d 5.7 due to the olefinic proton
OMe
OMe
OMe
CH2Br
CHO
CH2OH
b
a
OMe
OMe
OMe
6
4
5
Scheme 1. Reagents: (a) NaBH4, MeOH; (b) aq HBr.
OH
OMOM
CH2OH
OMOM
CH2Br
OMOM
CHO
a, b
c
d
OMe
7
OMe
9
OMe
10
OMe
8
Scheme 2. Reagents: (a) NaH, MOMCl, diethylether; (b) BuLi, DMF, diethylether; (c) NaBH4, MeOH; (d) Me2S, NBS, DCM.

S. K. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 2873–2880
2875
OMe
OMe
OR
CH2Br
a
b
c
+
S
S
O
O
O
O
OR
OR
OMe
11
13a: R = Me
13b: R = MOM
12a: R = Me
6: R = Me
12b: R = MOM
10: R = MOM
OMe
OMe
OMe
d
e
OR
OHC
CHO
OR
OR
14a: R = Me
15a: R = Me
16a: R = Me
14b: R = MOM
15b: R = MOM
16b: R = MOM
f
17: R = H
Scheme 3. Reagents: (a) LiHMDS, HMPA, THF; (b) pyridine, reflux; (c) sealed tube, toluene; (d) NaH, MeI, THF; (e) prenyl phosphonate; (f) HCl,
THF.
and four distinct singlets at d 5.16 (2H), 3.76 (3H), 3.48
(3H), and 1.71 (3H), corresponding to the methylene of
the MOM group, the methoxy group attached to the
aromatic ring, the methoxy group of MOM, and the
methyl group of the sulfolene nucleus, respectively. A
triplet and multiplet appeared at d 3.92 (1H) and 3.74
(2H), respectively, due to the methine and methylene
of the sulfolene moiety, and three aromatic protons in
the region of d 6.9 supported the formation of adduct
12b.
reochemical control of the cycloaddition, and the
predictability of the regiochemistry.
In the Diels–Alder reaction, the addition of 1-substi-
tuted isoprenes to the dienophilic carbon–carbon bond
is asymmetrical, leading to the possible formation at
least two regioisomers. However, the ortho rule predicts
that the isomer in which the carbonyl group of the
dienophile and the C-1 substituent on the diene are ortho
to each other would be preferentially formed over the
meta adduct. The Diels–Alder reaction was carried out
between the diene 13b and acrolein as the dienophile,
with a mixture of the diene 13b and acrolein in toluene
being heated in the presence of hydroquinone in a sealed
tube at 90 ꢁC for 2 h. TLC analysis of the reaction mix-
ture showed the complete disappearance of the diene
and the formation of a more polar product. Removal
of the toluene, followed by chromatographic purifica-
Takayama and co-workers13 have usually opened sulfo-
lenes by heating them in 95% ethanol in the presence of
NaHCO3 in a sealed tube at 125 ꢁC to obtain quantita-
tive yields of the corresponding dienes. A much cleaner
and milder condition for the extrusion of SO2 was re-
ported by Bhat and co-workers15 by refluxing sulfolene
in pyridine at 80 ꢁC. This method provided an optimal
reaction temperature, and the SO2 liberated was ex-
pelled and could be monitored very easily, unlike that
in a sealed tube.
1
tion of the residue, yielded the adduct 14b. H NMR
spectrum showed an aldehydic proton at d 9.36; multi-
plets at d 2.91 (2H), 2.46 (2H), and 1.8–2.2 (2H); and
a broad singlet at d 5.41 (1H), due to seven protons of
the cyclohexene ring, as well as four distinct singlets at
d 5.14 (2H), 3.74 (3H), 3.48 (3H), and 1.71 (3H) corre-
sponding to the methylene of the MOM group, the
methoxy group attached to the aromatic ring, the meth-
oxy group of MOM, and the methyl group of the cyclo-
hexene nucleus, respectively; double doublets at d 2.79
(1H) (J = 13.54 and 5.4 Hz) and 2.6 (1H) (J = 13.54
and 8.42 Hz), corresponding to benzylic protons and
three aromatic protons in the region d 6.82–7.01 (3H),
indicated structure 14b.
The adduct 12b was subjected to thermolysis by reflux-
ing in pyridine gave compound 13b. The 1H NMR spec-
trum showed typical double doublets at d 6.39 (1H)
(J = 17.39 and J = 10.8 Hz), due to the H-2 olefinic pro-
ton, as well as a triplet at d 5.64 (1H) (J = 6.96 Hz),
which was assigned to the H-4 of the olefin. Two one-
proton doublets at d 5.1 (J = 17.39 Hz) and 4.96
(J = 10.8 Hz) were ascribed to trans- and cis-protons at
C-1, and the appearance of three aromatic protons in
the region d 7.01–6.68 indicated the formation of conju-
gated diene 13b.
The compound 14b was methylated using methyl iodide
and NaH in dry THF. To the slurry of NaH in dry THF
was added compound 14b and methyl iodide at 0 ꢁC; the
reaction mixture was stirred for 4 h and quenched with
aqueous NH4Cl solution. The usual work-up and SGC
purification yielded 15b as a colorless, viscous liquid.
The use of a Diels–Alder reaction to construct an appro-
priately functionalized cyclohexyl system in a concise
manner is especially attractive, in light of the fact that
the ease of cycloaddition of the diene and dienophile,
the rapid accumulation of polyfunctionality in a rela-
tively small molecular framework, the extraordinary ste-
1
The structure of 15b was indicated from the H NMR

2876
S. K. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 2873–2880
spectrum. It displayed an absence of a multiplet at d 2.91
(1H), corresponding to the proton a to the aldehydic
group, and the appearance of a methyl group singlet
at d 1.01 (3H). The structure of compound 15b was fur-
ther confirmed from its MS analysis, which displayed a
molecular ion peak at m/z 318.
Compound 16a was similarly synthesized, and its struc-
ture was deduced.
2.2. Biology
A panel of seven different human cancer cell lines from
different origins was used for this study. The cell lines in-
cluded: HuCCT-1 cell line obtained from the Health Sci-
ence Research Resources Bank (Japan); BxPC3, Panc-1,
Mia-Paca, A431, and Hep2 cell lines were purchased to
the American Tissue Culture Collection (Manassas,
VA), and HN006 was obtained from Dr. David Sidran-
skyÕs laboratory at Johns Hopkins. Of these, HuCCT-1
was derived from an intrahepatic cholangiocarcinoma;
BxPC3, Mia-Paca, and Panc-1 were derived from pan-
creatic cancers; A431 was derived from a vulvar epithe-
lial carcinoma; and Hep2 and HN006 were derived from
squamous cell carcinomas of the head and neck. Cells
were grown in monolayer culture in different media (Ea-
gleÕs minimum essential medium for Mia Paca, Dulbelc-
cosÕs modified EagleÕs medium for A431 and Panc 1; and
RPMI 1640 for SNU 308, HuCCT-1, HN006, and
BxPC3 cell lines). Media were supplemented with 10%
FBS and 50UI Penicillin/Streptomycin. Cell cultures
were maintained at 37 ꢁC in an humified atmosphere
of 5% CO2 in air. In all assays, cells were plated for
24 h before drug administration. Stock solutions were
prepared for each compound in dimethyl sulfoxide
(DMSO) and stored at À20 ꢁC.
As is well known, in the Wittig reaction an aldehyde or
ketone is treated with phosphorane ylide (also called a
phosphorane) to give an olefin and phosphine oxide.
The Horner–Emmons modification involves the use of
other ylides, the most important being prepared from
phosphonates; this modification has several advantages
over the use of phosphoranes: these ylides are more
reactive than the corresponding phosphoranes, and the
compounds often react with ketones that are inert to
phosphoranes. In addition, the product is a water-solu-
ble phosphate ester, unlike Ph3PO, which makes it easy
to separate it from the olefin product. Moreover, phos-
phonates are less expensive than the phosphonium salt
and can be easily prepared by the Arbuzov reaction.
Initially, the reaction of aldehyde 15b with the Wittig
salt of isoprenyl bromide using NaH/BuLi as the base
gave back the starting material: the reaction did not pro-
ceed, probably because of the bulkiness of the tri-phenyl
phosphine groups of the Wittig salt. Hence, the Horner–
Emmons method was used. First, the prenyl phospho-
nate 20 was prepared by refluxing prenyl bromide 19
with triethyl phosphite in toluene for 12 h20 (Scheme
4). The required prenyl bromide was obtained from iso-
prene using the reported procedure.21 The reaction of
phosphonate 20 with the LDA in THF at À23 ꢁC gave
the corresponding ylide, which was treated with alde-
hyde 15b to give the required product 16b in 17% yield.
The 1H NMR spectrum also displayed the absence of an
aldehydic proton and showed the presence of six distinct
singlets at d 5.12 (2H), 3.75 (3H), 3.48 (3H), 1.8 (3H),
1.75 (3H), 1.74 (3H), and 0.98 (3H), corresponding to
the methylene of MOM, the methoxy group attached
to the aromatic ring, the methoxy group of MOM, the
methyl group attached to the olefin of the cyclohexene
ring, the gem-dimethyls and the methyl group attached
to the cyclohexene nucleus, respectively. Double dou-
blets at d 6.15 (J = 15.7 and 10.2 Hz) due to the proton
at C-2, and two doublets at d 5.75 (J = 10.25 Hz) and
5.53 (J = 15.7 Hz) due to C-1 and C-3 of the 1,3-penta-
dienyl unit indicated the formation of the olefin in the
trans confirmation. Two double doublets of the benzylic
proton at d 2.77 (J = 13.5 and 5.5 Hz) and 2.38 (J = 13.5
and 5.5 Hz) and multiplets at d 6.85–7.02 due to aro-
matic protons indicated the structure of compound
16b. The structure of compound 16b was further sup-
ported by the MS analysis, which showed a molecular
ion peak at m/z 370. Further, the deprotection of
MOM with HCl in THF gave compound 17.
2.2.1. Growth inhibition studies. In vitro drug sensitivity
was assessed using the tetrazolium (MTT) dye conver-
sion method. The MTT assay is a colorimetric method22
that quantify viable cells through their ability to reduce
a salt, a process that requires active mitochondrial func-
tion. The intensity of the dye is proportional to the num-
ber of cells. Briefly, cells were trypsinized, seeded at
5 · 103 cells/well in 96-well plate and allowed to grow
for 24 h before the treatment with exponential increas-
ing concentrations of the drugs in the presence of 10%
FBS. After a 96-h period of treatment, 20 lL of MTT
solution (5 mg/mL in PBS) (Sigma, St. Louis, MO) were
added to each well, and the plates were then incubated
for 3 h at 37 ꢁC. Medium was then replaced with
100 lL of DMSO per well. Plates were shaken and the
optical density was measured at 570 nm using a multi-
well plate reader (Bio-Rad, Model 550, bio-Rad Inc.,
Hercules, CA). Each experiment were performed in trip-
licate for each drug concentration and was carried out
independently at least three times. The IC50 value was
defined as the concentration needed for a 50% reduction
in the absorbance calculated based on the survival
curves. Response to drug treatment was assessed by
standardizing treatment groups to untreated controls.
MTT assay in a panel of seven human-derived cancer
cell lines from different origins. Relative growth after
exposure to increasing concentrations of compounds.
The results indicate a susceptibility of some of the cell
lines in our panel to the compound. This finding is espe-
cially interesting in the case of some of these cell lines,
such as Hep2 and BxPC3, which are known to be resis-
tant to classic cytotoxic drugs and to some targeted
O
a
b
P(OEt)2
Br
Scheme 4. Reagents: (a) 48% HBr, CuBr; (b) P(OEt)3, toluene.

S. K. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 2873–2880
2877
agents. These results suggest that at least one of the
compounds discussed in this paper has activity against
certain human-derived cancer cell lines. Compounds
16a and 16b showed mild anti-tumor activity against
these cancer cell lines.
mesh) using a petroleum ether–EtOAc gradient gave
2,5-dimethoxybenzyl alcohol (5) in 95% yield. IR (neat):
mmax 3400, 2940, 2900, 1590, 1450 cmÀ1 1H NMR
;
(400 MHz, CDCl3): d 6.85 (3H, m), 4.2 (2H, s), 3.8
(6H, s). Anal. (C9H12O3) C, H, N. Mass spectrum: m/z
168 (M+).
3. Conclusions
4.2. Synthesis of 2-(bromomethyl)-1,4-dimethoxybenzene
(6)
A novel, facile, high yield, and less cumbersome synthesis
of riccardiphenol analogs, 16a, 16b, and 17 is described.
The synthesized compounds were characterized and as-
sessed for its in vitro activity in a panel of human cancer
cell lines of differing origin. In this study, we demonstrate
that one of the leading compound, 17, significantly inhib-
its the growth of different human cancer cells (Fig. 1).
The fact that some of these cell lines, including Hep2
and BxPC3 are known to be resistant to classic cytotoxic
drugs, as well as some targeted agents, makes this sensi-
tivity to the riccardiphenol analog particularly intrigu-
ing. These results demonstrate that the novel
riccardiphenol analog has effective action against hu-
man-derived cancer cell in vitro. Further investigations
on structural activity relationship of the diastereomers
are underway and will be reported in due course.
Hydrobromic acid (40%, 20 mL) was added to the 2,5-
dimethoxybenzylalcohol (5) (7 g, 0.042 mol) at 0–5 ꢁC,
and the reaction mixture was stirred for 30 min. The
reaction mixture was extracted with dichloromethane
(3 · 50 mL), and the organic layer was washed with
NaHCO3, water, and brine and dried over anhydrous
Na2SO4. Removal of the solvent under reduced pressure
and column chromatography of the residue over silica
gel (100–200 mesh) using a petroleum ether–EtOAc gra-
dient gave compound (6) in 96% yield. IR (neat): mmax
2940, 2900, 1590, 1450, 1420 cmÀ1
;
1H NMR
(400 MHz, CDCl3): d 6.83 (3H, m), 4.1 (2H, s), 3.8
(6H, s). Anal. (C9H11BrO2) C, H, N. Mass spectrum:
m/z 230 and 232 (M+).
4.3. Synthesis of 5-methoxy-2-(methoxymethoxy)benzal-
dehyde (8)
4. Experimental section
N-butyl lithium (15%, 4.13 g, 0.06 mol) was added to the
4-methoxymethoxy anisole (5.56 g, 0.034 mol) in dry
diethylether (30 mL) under argon at room temperature
and stirred for 3 h. Dimethylformide (DMF) (7.30 g,
0.09 mol) was added to this reaction mixture and stirred
for a further 30 min. The reaction mixture was quenched
with saturated NH4Cl solution, and the residual mixture
was extracted with diethyl ether (3 · 30 mL). The organ-
ic layer was washed with water and brine and dried over
anhydrous Na2SO4. Removal of the solvent under re-
duced pressure yielded compound (8) in 80% yield
(crude). IR (neat): mmax 2940, 2900, 2730, 1686, 1607,
4.1. Synthesis of (2,5-dimethoxyphenyl)methan-1-ol (5)
2,5-Dimethoxybenzaldehyde (4) (7 g, 0.042 mol) in
methanol (100 mL) was placed in a round bottom flask
and cooled to 0 to À10 ꢁC. To this, sodium borohydride
(1.9 g, 0.05 mol) was added in portions, and the reaction
mixture was allowed to stir for 3 h at room temperature.
The reaction mixture was quenched with water, and the
methanol was removed under vacuum. The residual
mixture was then extracted with diethyl ether
(3 · 50 mL), and the organic layer was washed with
water and brine and dried over anhydrous Na2SO4. Re-
moval of the solvent under reduced pressure and column
chromatography of the residue over silica gel (100–200
1
1512, 1450 cmÀ1; H NMR (400 MHz, CDCl3): d 10.4
(1H, s), 7.0 (2H, s), 7.2 (1H, s), 5.2 (2H, s), 3.8 (3H,
s), 3.4 (3H, s). Anal. (C10H12O4) C, H, N. Mass spec-
trum: m/z 196 (M+).
4.4. Synthesis of (5-methoxy-2-(methoxymethoxy)phenyl)-
methan-1-ol (9)
120%
100%
80%
60%
40%
20%
0%
HN006
HuCCT-1
BxPC3
Panc-1
A431
2-Methoxymethoxy-5-methoxybenzaldehyde (8) (3 g,
0.015 mol) in methanol (100 mL) was placed in a round
bottom flask and cooled to 0 to À10 ꢁC. Sodium borohy-
dride (1 g, 0.03 mol) was added in portions, and the reac-
tion mixture was allowed to stir for 3 h at room
temperature. The reaction mixture was quenched with
water, and methanol was removed under vacuum. The
residual mixture was extracted with diethyl ether
(3 · 50 mL), and the organic layer was washed with
water and brine and dried over anhydrous Na2SO4. Re-
moval of the solvent under reduced pressure and column
chromatography of the residue over silica gel (100–200
mesh) using a petroleum ether–EtOAc gradient gave
compound (9) in 95% yield. IR (neat): mmax 3400, 2940,
MiaPaca
Hep2
3 miliM
0 microM 3 microM
30 microM
300 microM
Drug Concentration
Figure 1. MTT assay in a panel of seven human-derived cancer cell
lines from different origins. Relative growth after exposure to
increasing concentrations of compound 17.
1
2900, 1590, 1450 cmÀ1; H NMR (400 MHz, CDCl3): d

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S. K. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 2873–2880
6.83 (3H, m), 5.2 (2H, s), 4.2 (2H, s) 3.8 (3H, s), 3.4 (3H,
s). Anal. (C10H14O4) C, H, N. Mass spectrum: m/z 198
(M+).
3 h. Pyridine was removed under reduced pressure,
and flash chromatography of the residue over silica gel
(100–200 mesh) using a petroleum ether–EtOAc gradi-
ent gave compound 13a–b.
4.5. Synthesis of 2-(bromomethyl)-4-methoxy-1-(meth-
oxymethoxy)benzene (10)
4.7.1. 1,4-Dimethoxy-2-(3-methylpenta-2,4-dienyl)ben-
zene (13a). 90%; IR (neat): mmax 2950, 2880, 1650,
1
Dimethyl sulfide (1.9 g, 0.03 mol) was added to a sus-
pension of NBS (4.56 g, 0.025 mol) in dry dichlorometh-
ane (10 mL) at 0 ꢁC under argon and stirred for 10 min.
The reaction mixture was cooled to À20 ꢁC; alcohol (9)
(0.017 mol) in dichloromethane (10 mL) was added, and
the mixture was further stirred at 0 ꢁC for 4 h. The reac-
tion mixture was poured into ice-cold water (10 mL) and
extracted with dichloromethane (3 · 50 mL). The organic
layer was washed with saturated NaHCO3, water, and
brine and dried over anhydrous Na2SO4. Removal of
the solvent under reduced pressure gave compound
(10) in 90% yield. IR (neat): mmax 2938, 2900, 1588,
1610, 1590, 1420 cmÀ1; H NMR (400 MHz, CDCl3):
d 6.76 (3H, m), 6.4 (1H, dd, J = 10.62 and 17.57 Hz),
5.63 (1H, t, J = 7.34 Hz), 5.11 (1H, d, J = 17.57 Hz),
4.95 (1H, d, J = 10.62 Hz), 3.78 (3H, s), 3.75 (3H, s),
3.45 (2H, d, J = 7.35 Hz), 1.83 (3H, s). Anal.
(C14H18O2) C, H, N. Mass spectrum: m/z 218 (M+).
4.7.2. 4-Methoxy-1-(methoxymethoxy)-2-(3-methylpenta-
2,4-dienyl)benzene (13b). 86%; IR (neat): mmax 2950,
2880, 1650, 1610, 1590, 1420 cmÀ1
;
1H NMR
(400 MHz, CDCl3): d 7.01 (1H, d), 6.68 (2H, m), 6.39
(1H, dd, J = 10.8 and 17.39 Hz), 5.64 (1H, t,
J = 6.96 Hz), 5.12 (2H, s), 5.1 (1H, d, J = 17.38 Hz),
4.96 (1H, d, J = 10.8 Hz), 3.75 (3H, s), 3.47 (5H, br s),
1.84 (3H, s). Anal. (C15H20O3) C, H, N. Mass spectrum:
m/z 248 (M+).
1
1450, 1420 cmÀ1; H NMR (400 MHz, CDCl3): d 6.8
(3H, m), 5.2 (2H, s), 4.1 (2H, s) 3.8 (3H, s), 3.3 (3H,
s). Anal. (C10H13BrO3) C, H, N. Mass spectrum: m/z
260 and 262 (M+).
4.6. General procedure for the preparation of sulfones
12a–b
4.8. General procedure for the preparation of the Diels–
Alder adduct 14a–b
3-Methyl-3-sulfolene
(4.5 g,
0.057 mol), HMPA
The diene 13a–b (9.1 mmol), acrolein (1 g, 17 mmol),
and hydroquinone (1 mmol) were taken in toluene
(8 mL) was placed in a sealed tube and heated at
90 ꢁC for 2 h. After the reaction, the toluene was re-
moved under vacuum, and silica gel (100–200 mesh) col-
umn chromatography of the residual mixture, with
petroleum ether and ethyl acetate as eluent, gave com-
pounds 14a–b.
(2 equiv), and benzyl bromide 6/10 (0.038 mol) in dry
THF (75 mL) were placed in a three-necked round bot-
tom flask and cooled to À92 ꢁC. To this mixture
LiHMDS (0.038 mol) was added and stirred for
25 min at the same temperature. The reaction mixture
was quenched with saturated NH4Cl solution, and
THF was removed under vacuum. The residual mixture
was extracted with diethyl ether (3 · 30 mL), and the or-
ganic layer was washed with water and brine and dried
over anhydrous Na2SO4. Removal of the solvent under
reduced pressure and silica gel (100–200 mesh) column
chromatography of the residue using a petroleum
ether–EtOAc gradient gave compound 12a–b.
4.8.1. 2-((2,5-Dimethoxyphenyl)methyl)-3-methylcyclo-
hex-3-enecarbaldehyde (14a). 80%; IR (neat): mmax
2940, 2897, 1722, 1592, 1500, 1461 cmÀ1 1H NMR
;
(400 MHz, CDCl3): d 9.46 (1H, s), 6.75 (3H, m), 5.41
(1H, s), 3.76 (3H, s) 3.74 (3H, s), 2.9 (1H, m), 2.78
(1H, dd, J = 13.54 and 5.12 Hz), 2.65 (1H, dd,
J = 13.54 and 8.42 Hz), 2.43 (1H, m), 2.0 (5H, m), 1.73
(3H, s). Anal. (C17H22O3) C, H, N.
4.6.1. 2-((2,5-Dimethoxyphenyl)methyl)-3-methylthiolene-
1,1-dione (12a). 73%; IR (neat): mmax 2942, 2900, 1592,
1450, 1420, 1310, 1130 cmÀ1 1H NMR (400 MHz,
;
CDCl3): d 6.85 (3H, m), 5.7 (1H, s), 3.94 (3H, s), 3.83
(3H, s) 3.75 (2H, m), 3.1 (3H, m), 1.7 (3H, s). Anal.
(C14H18O4S) C, H, S.
4.8.2. 2-((5-Methoxy-2-(methoxymethoxy)phenyl)methyl)-
3-methylcyclohex-3-enecarbaldehyde (14b). 78%; IR
(neat): mmax 2940, 2897, 1722, 1592, 1500, 1461 cmÀ1
;
1H NMR (400 MHz, CDCl3): d 9.48 (1H, s), 7.01 (1H,
d, J = 8.77 Hz), 6.82 (2H, m), 5.41 (1H, s), 5.14 (2H,
s), 3.74 (3H, s), 3.48 (3H, s), 2.91 (1H, m), 2.79 (1H,
dd, J = 5.4 and 13.54 Hz), 2.66 (1H, dd, J = 8.4 and
13.54 Hz), 2.0 (5H, m), 1.71 (3H, s); Anal. (C18H24O4):
C, H, N.
4.6.2. 2-((5-Methoxy-2-methoxymethoxyphenyl)methyl)-
3-methylthiolene-1,1-dione (12b). 70%; IR (neat):
mmax 2942, 2900, 1592, 1450, 1420, 1310, 1130 cmÀ1
;
7.02 (1H, d,
1H NMR (400 MHz, CDCl3):
d
J = 8.97 Hz), 6.86 (1H, d, J = 3.11 Hz), 6.74 (1H, q,
J = 8.97 and 3.11 Hz), 5.67 (1H, s), 5.16 (2H, s), 3.92
(1H, t), 3.76 (3H, s), 3.74 (2H, m), 3.48 (3H, s), 3.28
(1H, dd, J = 7.14 and 14.28 Hz), 2.92 (1H, dd, J = 6.92
and 14.28 Hz), 1.71 (3H, s). Anal. (C15H20O5S) C, H, S.
4.9. General procedure for the preparation of compounds
15a–b
NaH (50%, 0.5 g, 10.5 mmol) was placed in a three-neck
round bottom flask and washed with dry petroleum
ether. Dry THF (30 mL) was added and the mixture
cooled to 0 ꢁC. To this was added compound 14a–b
(5 mmol) and methyl iodide (1.5 g, 10.5 mmol) in THF
4.7. General procedure for the preparation of diene 13a–b
Compound 12a–b (27.3 mmol) in dry pyridine (15 mL)
was placed in a round bottom flask and refluxed for

S. K. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 2873–2880
2879
(20 mL), and the reaction mixture was allowed to stir for
4 h at room temperature. The reaction mixture was
quenched with saturated NH4Cl solution, and THF
was removed under vacuum, then the residual mixture
was extracted with diethyl ether (3 · 30 mL), and the or-
ganic layer was washed with water and brine and dried
over anhydrous Na2SO4. Removal of the solvent under
reduced pressure and column chromatography of the
residue over silica gel (100–200 mesh) with petroleum
ether and ethyl acetate as eluent gave compound 15a–b.
4.10.2. 2-((2,6-Dimethyl-6-(4-methylpenta-1,3-dienyl)cyclo-
hex-2-enyl)methyl)-4-methoxymethoxybenzene (16b). 19%;
1
IR (neat): mmax 2940, 2895, 1670, 1620, 1510 cmÀ1; H
NMR (400 MHz, CDCl3): d 6.75 (3H, m), 6.15 (1H,
dd, J = 15.7 and 10.2 Hz), 5.75 (1H, d, J = 10.25 Hz),
5.53 (1H, d, J = 15.7 Hz), 5.4 (1H, s), 5.1 (2H, s), 3.7
(3H, s), 3.4 (3H, s), 2.38 (1H, dd, J = 13.5 and 5.5 Hz),
2.77 (1H, dd, J = 13.5 and 7.23 Hz), 2.07 (1H, m), 1.8
(3H, s), 1.74 (3H, s), 1.75 (3H, s), 0.98 (3H, s); 13C
NMR (CDCl3): d 19.5, 21.2, 23.1, 23.5, 23.9, 25.8, 37.2,
41.6, 51.8, 56.0, 58.3, 112.3, 114.9, 114.5, 122.1, 124.3,
126.2, 135.3, 141.1, 141.7, 154.1, 154.4, 171.2. Anal.
(C24H34O3) C, H, N. MS: m/z 370 (M+).
4.9.1. 2-((2,5-Dimethoxyphenyl)methyl)-1,3-dimethylcy-
clohex-3-enecarbaldehyde (15a). 50%; IR (neat): mmax
2940, 2895, 2707, 1722, 1592, 1500, 1461 cmÀ1 1H
;
NMR (400 MHz, CDCl3): d 9.43 (1H, s), 6.73 (3H,
m), 5.35 (1H, br s), 3.78 (3H, s), 3.74 (3H, s), 2.63
(1H, dd, J = 14 and 6.59 Hz), 2.56 (1H, dd, J = 14 and
6.77 Hz), 2.39 (1H, t), 2.0 (4H, m), 1.6 (3H, s), 1.0
(3H, s). Anal. (C18H24O3) C, H, N. MS: m/z 288 (M+).
4.11. 2-[2,6-Dimethyl-6-(4-methyl-penta-1,3-dienyl)-
cyclohex-2-enylmethyl]-4-methoxy-phenol (17)
Compound 16b (25 mg; 67 lM) was treated with
THF:3 M HCl = 1:1 (3 mL) at 50 ꢁC for 3 h. The solvent
was evaporated, and the residue was extracted with
ether. The organic phase was washed with water, dried
(NaSO4), and evaporated. The residue was purified
by HPLC to give 17(8 mg) (yield; 30%). 1H NMR
(400 MHz, CDCl3): d 6.57 (1H, d, J = 8 Hz), 6.46 (1H,
s), 6.42 (1H, d, J = 8 Hz), 6.05 (1H, dd, J = 15.7 and
10.2 Hz), 5.72 (1H, d, J = 10.25 Hz), 5.51 (1H, d,
J = 15.7 Hz), 5.38 (1H, s), 3.73 (3H, s), 2.36 (1H, dd,
J = 13.5 and 5.5 Hz), 2.75 (1H, dd, J = 13.5 and
7.23 Hz), 2.06 (1H, m), 1.82 (3H, s), 1.73 (3H, s), 1.75
(3H, s), 0.98 (3H, s). Anal. (C22H30O2) C, H, N. MS:
m/z 326 (M+).
4.9.2. 2-((5-Methoxy-2-(methoxymethoxy)phenyl)methyl)-
1,3-dimethylcyclohex-3-enecarbaldehyde (15b). 55%; IR
(neat): mmax 2940, 2895, 2707, 1722, 1592, 1500,
1
1461 cmÀ1; H NMR (400 MHz, CDCl3): d 9.36 (1H,
s), 6.98 (1H, d, J = 8.42 Hz), 6.65 (2H, m), 5.36 (1H,
s), 5.12 (2H, s), 3.75 (3H, s), 3.48 (3H, s), 2.64 (1H,
dd, J = 13.8 and 6.408 Hz), 2.6 (1H, dd, J = 13.8
and 6.591 Hz), 2.41 (1H, m), 2.0 (4H, m), 1.57 (3H, s),
1.02 (3H, s). Anal. (C19H26O4) C, H, N. MS: m/z 318
(M+).
4.10. General procedure for the preparation of compounds
16a–b
Acknowledgements
LDA (0.20 mmol) was generated in THF at À23 ꢁC, and
prenyl phosphite (0.20 mmol), obtained by refluxing
prenyl bromide and triethlyphosphite in toluene
(5 mL) for 12 h, was added and stirred for 30 min. To
this mixture aldehyde 15a–b (50 mg, 0.17 mmol) was
added and stirred for 4 h. The reaction mixture was
quenched with saturated NH4Cl, and THF was removed
under vacuum. The residual mixture was extracted with
diethyl ether (3 · 20 mL), and the organic layer was
washed with water and brine and dried over anhydrous
Na2SO4. Removal of the solvent under reduced pressure
and column chromatography of the residue over silica
gel (100–200 mesh) with petroleum ether and ethyl ace-
tate as eluent gave compound 16a–b.
We gratefully acknowledge the financial support of
grant K01 CA89263 from NCI and FAMRI.
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;
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