Flavonolignan and flavone inhibitors of a Staphylococcus aureus multidrug resistance pump: Structure-activity relationships

Article abstract of DOI:10.1021/jm0004190

Although some progress has been reported on structure-activity relationships (SARs) for inhibitors of mammalian P-glycoprotein MDR efflux pumps, there is almost nothing in the literature regarding SARs for inhibitors of any bacterial efflux pump. Indeed, only a few of these have been described. Our discovery of a potent naturally occurring flavonolignan inhibitor of the NorA MDR pump of Staphylococcus aureus provided a structural foundation upon which SARs could be assessed via synthetic analogues. Several flavonolignans were prepared which proved to have greater potency than the natural isolate, 5′-methoxyhydnocarpin-D, while others showed decreased potency. Surprisingly, some simple alkylated flavones also were quite active MDR pump inhibitors. Variability of activity among the compounds tested was sufficient so that at least some SARs could be postulated and compared with those known for P-glycoprotein.

Full text of DOI:10.1021/jm0004190

J . Med. Chem. 2001, 44, 261-268
261
F la von olign a n a n d F la von e In h ibitor s of a Sta ph ylococcu s a u r eu s Mu ltid r u g
Resista n ce P u m p : Str u ctu r e-Activity Rela tion sh ip s
Nathan R. Guz,† Frank R. Stermitz,*,† J effrey B. J ohnson,† Teresa D. Beeson,† Seth Willen,‡
J en-Fang Hsiang,‡ and Kim Lewis*,‡
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, and Biotechnology Center,
Tufts University, Medford, Massachusetts 02155
Received September 29, 2000
Although some progress has been reported on structure-activity relationships (SARs) for
inhibitors of mammalian P-glycoprotein MDR efflux pumps, there is almost nothing in the
literature regarding SARs for inhibitors of any bacterial efflux pump. Indeed, only a few of
these have been described. Our discovery of a potent naturally occurring flavonolignan inhibitor
of the NorA MDR pump of Staphylococcus aureus provided a structural foundation upon which
SARs could be assessed via synthetic analogues. Several flavonolignans were prepared which
proved to have greater potency than the natural isolate, 5′-methoxyhydnocarpin-D, while others
showed decreased potency. Surprisingly, some simple alkylated flavones also were quite active
MDR pump inhibitors. Variability of activity among the compounds tested was sufficient so
that at least some SARs could be postulated and compared with those known for P-glycoprotein.
In tr od u ction
Multidrug resistance (MDR) represents an increasing
problem in the treatment of bacterial infections and
cancer. It often appears after prolonged exposure of cells
to a single drug and is often characterized by resistance
to a series of structurally unrelated compounds.1 One
important resistance mechanism involves drug deple-
tion in cells by membrane efflux proteins, for example
P-glycoprotein (P-gp) in mammalian cells,2 Bmr in
Bacillus subtilis, and NorA in Staphylococcus aureus.3
Although numerous small-molecule inhibitors of P-gp
have been reported as well as some structure-activity
relationships (SARs) postulated for these inhibitors,
only a few inhibitors of bacterial MDR pumps are
known. The alkaloid reserpine and some amide ana-
logues were found to inhibit NorA in S. aureus.4,5 Some
tetracycline analogues can inhibit Tet protein efflux
activity in Escherichia coli.6 Diamide amines structur-
ally reminiscent of those active against NorA5 inhibited
Pseudomonas aeruginosa efflux pumps.7 We recently
found two plant-derived compounds, the flavonolignan
5′-methoxyhydnocarpin-D8 (1) and the porphyrin pheo-
phorbide a,9 to be potent inhibitors of the NorA MDR
efflux pump in S. aureus. While exhibiting no activity
themselves, 1 and pheophorbide a potentiated growth
inhibitory activity of the natural antibacterial alkaloid
berberine (2), the fluoroquinolone norfloxacin (3), and
the antiseptic benzalkonium chloride (4) against resis-
tant S. aureus. A number of hydnocarpin-type flavon-
olignans were synthesized10 for structure determination
purposes, and these were also found to exhibit a range
of activity as potentiators of berberine activity. We
report here the inhibition activities of those synthetics
(Figure 1) as well as those of an extended series of
related compounds and simple flavones (Figure 2).
Ch em istr y
Synthetic flavonolignans (1, 5, 6, 8-12, 14) were
prepared by reaction of a commercial catecholic flavone
with a lignan (coniferyl alcohol or an analogue) via
radical coupling.10-12 Syntheses of 1, 5, and 6 were
reported previously.10 One of the couplings was initiated
with horseradish peroxide (HRP) and the others with
Ag2CO3. These methods typically gave different regio-
selectivities as described10 for the preparation of 1 and
5. The Ag2CO3 reactions yielded a mixture having the
major product with the D ring in the “down” position
(e.g. 1) and the HRP reactions with that ring in the “up”
position (e.g. 5). Yields were not optimized in any of the
reactions.
Syntheses of 7, 8, and 18 are exemplary (Scheme 1).
A flavonolignan isolated from the plant Onopordon
corymbosum was assigned structure 8 and given the
name 5′′-methoxyhydnocarpin.13 There were no data
presented which would distinguish 8 from its regioiso-
mer 7. Hydnocarpin itself had actually earlier been
shown14 to be the regioisomer 5 and not 6, so the name
* To whom correspondence should be addressed. F.R.S.: tel, 970-
491-5158; fax, 970-491-5610; e-mail, frslab@lamar.colostate.edu.
K.L.: tel, 617-627-3731; fax, 617-627-3991; e-mail, klewis@tufts.edu.
† Colorado State University.
‡ Tufts University.
10.1021/jm0004190 CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/22/2000

262 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2
Guz et al.
F igu r e 1. Flavonolignans tested as inhibitors of the S. aureus MDR efflux pump NorA.
Flavonolignan 13 (sinaiticin) had been isolated from
Verbascum sinaiticum and its structure confirmed by
INEPT NMR.15 In our hands, a regiopure sample of 13
could not be obtained via HRP initiation, but only a 3:2
13/14 mixture. Regiopure 14 was obtained via Ag2CO3
initiation and the regiochemistry verified by HMBC
NMR.10 A synthesis of sinaiticin (13) was recently
reported,16 but no evidence was given which would
distinguish the product from isomer 14 and a request
for a sample was not answered.
The coupling of coniferyl alcohol to an A ring cat-
echolic flavone proceeded with regiospecificity, unlike
those exemplified by Scheme 1. The 5-OH analogues of
18 and 19 are known as scutellaprostin A and B,
respectively, and were isolates from Scutellaria pros-
trata.17 Both scutellaprostins were also reportedly syn-
thesized as pure regioisomers via similar coupling
reactions and Ag2O.17
A few flavanolignans are also known as natural
products. For example, (-)-silandrin (also known as
3-deoxyisosilybin) was isolated from Silybum marianum
and was also synthesized.18 We prepared the D ring
“down” isomer, rac-silandrin-D (15) for testing, while
the corresponding 5′-methoxy derivative 16 was avail-
able from previous synthetic work.10 An extract of
Silibum marianum, known as silymarin, is commeri-
cally available and was recrystallized from methanol to
yield an approximately 50:50 mixture of silybin diaster-
eomers 17 (the other diastereomer has the alternate
trans stereochemistry of substituents on the benzodiox-
ane ring). The chalcone 20 and its precursor 21,
intermediates in the synthesis10 of 15 and 1, were also
available.
F igu r e 2. Flavones tested as inhibitors of the S. aureus MDR
efflux pump NorA.
5′′-methoxyhydnocarpin would not have been valid for
8. Under our suggested nomenclature system,10 7 would
be 5′′-methoxyhydnocarpin and 8 would be 5′′-methoxy-
hydnocarpin-D. Which structure represented the actual
plant isolate13 is not determinable without an authentic
sample, which was unavailable. Our structures for 7 and
8 were verified by HMBC NMR.10

Flavonolignan/ Flavone Inhibitors of a S.a. MDR Pump
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2 263
Sch em e 1. Examples of Flavonolignan Syntheses
Ta ble 1. Minimum Inhibitory Concentration of Flavonolignans
That Inhibit Growth of S. aureus in the Presence of
Subinhibitory (30 µg/mL) Berberine, 2
Some commercially available flavones were tested
with varying results. Activity in a few, combined with
the results on some ring A deoxy compounds, suggested
the preparation and testing of 4′-hydroxyflavone (33)
in comparison with some alkylated derivatives 22 and
34-40, simply prepared from base-catalyzed reaction
of 33 with several alkyl bromides.
MIC
MIC
MIC
compd
(µg/mL)
compd
(µg/mL)
compd
(µg/mL)
1
5
6
1-2
3.1
0.1
10
10a
11
0.8
1.6
163
16
17
18
1.9
12.5
1.9
6a
7
8
0.1
7.8
4-8
0.08
12, 12a
14
15
inactive
inactive
3.1
19
20, 21
0.6
inactive
Biologica l Assa ys
Cell Cu ltu r in g an d Su sceptibility Testin g. Growth
of bacteria and susceptibility measurements were per-
formed according to the National Center for Clinical
Laboratory Standards recommendations. S. aureus
RN42228 were cultured in Mueller-Hinton (MH) broth
overnight with aeration at 37 °C. Cells were then
inoculated into fresh MH medium at a 1:10 dilution and
were allowed to grow for 1 h. This suspension was
diluted 1:2000 into MH broth and 0.05 mL was dis-
pensed per well of microtiter plates. For measurements
of direct antimicrobial activity, test substances were
serially diluted 2-fold in the wells. The final volume of
a well was 0.2 mL, and the cell concentration was 105/
mL. MIC was defined as a concentration of an antimicro-
bial that completely prevented cell growth during an
18-h incubation at 37 °C. Growth was assayed with a
microtiter plate reader (Biorad) by absorption at 600
nm. Tests for MDR inhibitory activity were done simi-
larly, but with berberine present at a subinhibitory
concentration (30 µg/mL, 1/8 MIC) throughout. The test
substance was then serially diluted 2-fold, and MIC for
test substances was then defined as their minimal
concentration that completely inhibited cell growth in
the presence of 30 µg/mL berberine. MIC values (Tables
1 and 2) could vary within a factor of 2.
9
15a
1.6
Ta ble 2. Minimum Inhibitory Concentration of Flavones That
Inhibit Growth of S. aureus in the Presence of Subinhibitory
(30 µg/mL) Berberine, 2
MIC
MIC
MIC
compd
22
(µg/mL)
compd
(µg/mL)
compd
(µg/mL)
6.3
30
25
37
6.3
23
24
6.3
15
31-33
34
inactive
0.4
38
39, 40
25
inactive
25-28a
29
inactive
25
35
36
1.9
1.6
showed independent activity (with no berberine present)
against S. aureus, except flavonolignan 18 and flavone
34, which were only very weakly active (MIC ) 125 µg/
mL). Many compounds were, however, synergistic (some
very potently so) with a subinhibitory concentration of
berberine (2). A number of the tested flavonolignans (6,
6a , 9, 10, 10a , 18, 19) were as potent as or more potent
(<2 µg/mL) than the natural product 1. Flavonolignans
with and without free phenolic groups at the 5 and 7
positions were comparably active (6, 9, 10, 18, 19),
although the most potent, 9, completely lacks A ring OH
groups. The potency of peracetate derivatives (6a , 10a ,
15a ) was approximately the same as that of their parent
compounds. These peracetates could be inherently active
or, perhaps less likely, deacetylated by S. aureus and
hence considered “prodrugs.” The presence of a 3-hy-
droxy group resulted in markedly decreased activity (11)
Resu lts a n d Discu ssion
Bioassay results are summarized for flavonolignans
(Table 1) and flavones (Table 2). None of compounds

264 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2
Guz et al.
or no activity (12, 12a ) in flavone-derived systems. In
the case of the one 3-hydroxyflavanone we tested, silybin
(17), potency was, however, still fairly high.
particular series of compounds”. This seems to be true
for S. aureus NorA inhibitors as well since the data
(Tables 1 and 2) show very clear SARs among fairly
closely related compounds, even though the other
natural inhibitor we discovered9 was the structurally
completely unrelated porphyrin pheophorbide a. A cy-
anobacterium porphinoid was also reported to be a P-gp
inhibitor.21 It is possible that these effects in P-gp stem
from the fact that P-gp has both ATP-binding and
steroid-interacting sites,22,23 or one large binding site
with a number of distinct subpockets.24 These sites or
subpockets in P-gp and NorA could interact differen-
tially with structurally very diverse inhibitors. A previ-
ously known naturally occurring inhibitor of NorA was
the indole alkaloid reserpine, while an active nitroin-
dole, a biarylurea, and other inhibitors were found by
chemical library screening.5 One study25 has genetically
compared a bacterial-resistance protein (LmrA in Lac-
tobacillus lactis) with the P-gp gene. In one case where
some diaryl peptide bacterial efflux pump inhibitors
were tested for P-gp inhibitory activity, they proved
inactive.7
In contrast to the case of A ring substitution, some
changes on the D ring played an important role. The
majority of flavonolignans had 3-methoxy-4-hydroxy
substitution and were quite active, while 3,5-dimethoxy-
4-hydroxy D ring substituted compounds (7, 8) were
slightly less active. On the other hand, 14, with only
4-hydroxy substitution on ring D was, perhaps remark-
ably, completely inactive. The regiochemistry of the D
ring benzodioxane ring fusion in the flavonolignans was
important in one case where we were able to compare
isomers (5 vs 6), but not in another case (7 vs 8). The
two scutellaprostin analogues 18 and 19 were both quite
active, so in this system H vs OH in ring B is not critical.
Two synthetic intermediates, chalcone 20 and its pre-
cursor 21, were inactive.
Most of the commercially available simple flavones
(Figure 2 and Table 2), especially those with free
phenolic groups or as peracetates, had little or no
activity (26, 26a , 27, 28, 28a , 29, 32). Luteolin (26),
which was the flavone most often used for preparation
of flavonolignans (Scheme 1), was not active, nor was
coniferyl alcohol, often used as the coupling partner
(Scheme 1) with the catecholic flavones. Flavones devoid
of B ring substitution (chrysin (29) and its dimethyl
ether 30) as well as two devoid of substitution on the A
ring (22 and 23) had moderate activity. Diosmetin (24),
which is the 4′ methyl ether of the inactive luteolin, was
surprisingly active (15 µg/mL). The relatively high
activity of 22-24 suggested that 4′-alkylation might be
of importance and led to the synthesis of various ethers
of 4′-hydroxyflavone (33). While 33 was inactive, deriva-
tives with small lipophilic alkylated side chains (34-
38) were quite active. As lipophilicity was increased,
however, activity was completely lost (39, 40). The
difference between 33 and 34 is particularly striking.
The importance of maintaining lipophilicity at the 4′
carbon was also demonstrated by the decreased activity
for 38 as compared to 34. Because of the potency of 34-
36 and their simplified structures as compared to the
flavonolignans, they were also tested against a mutant
S. aureus strain lacking NorA. They did not potentiate
the activity of norfloxacin against the mutant, as was
similarly the case for the flavonolignan 1, thus indicat-
ing that the activities for 34-36 against the wild-type
S. aureus were indeed due to NorA inhibition.
There have been a few SAR studies on P-gp inhibitors
which can be compared with the present results. Most
closely related to the present work were studies on some
natural flavonoids,26 some synthetically modified fla-
vonoid derivatives,27 and some halogenated chalcones.28
These papers also provide an entrance into extensive
literature in this area. Among the natural flavonoids,
kaempferide (3,5,7-trihydroxy-4′-methoxyflavone) was
found to be most active, while kaempferol (3,5,7,4′-
tetrahydroxyflavone) and chrysin (29) were less so.26
The reverse occurred with NorA inhibition (Table 2),
with chrysin being the most active followed by kaempfer-
ide (100 µg/mL). The latter two flavones have 3-OH
substituents, which were suggested26 to be valuable for
P-gp inhibition, while we found such substitution to be
detrimental to NorA inhibition (11, 12). Active MDR
modulators of human leukemia cells were reported29 to
display two or more lipophilic ring systems separated
by a linker containing a basic nitrogen. As a result of
studies on some verapamil analogues, it was concluded
that besides overall lipophilicity, weak polar interactions
provided by overlapping of aromatic rings were valuable
for binding MDR-reverting agents to P-gp.30 The im-
portance of a basic nitrogen atom for best P-gp-mediated
efflux was also affirmed by some studies on active
tetrahydroisoquinolines, but here high molar refractiv-
ity values were considered to be more important than
lipophilicity.31 Some natural flavones were modified by
attaching substituted N-benzylpiperazinyl side chains
to the 5- and 7-OH groups.27 Several of these were quite
potent, but it was also found that activity depended not
only on these substituents but also on the type of
flavonoid moiety and the nature of substituents on other
parts of the flavonoid. Acylation of the 7-OH was very
important since derivatives with a free 7-OH were
devoid of activity, as was the parent diosmetin (24). In
the case of NorA inhibition, however, presence of a 7-OH
did not negate activity, although the most potent
compounds in both the flavonolignan and flavone series
lacked any OH groups in ring A. Diosmetin itself was
also quite active against NorA. Replacement of the
flavone ring by a flavanone did not markedly alter MDR-
It is of interest to compare these S. aureus NorA
inhibitors with some inhibitors of the mammalian MDR
P-gp efflux proteins. Any inhibitor of a microbial MDR
pump developed for therapeutic use should not affect
P-gp, which plays a role in xenobiotic efflux of normal
tissues. SARs of existing inhibitors will help in design-
ing therapeutic compounds that specifically inhibit
microbial MDR pumps but not P-gp. A comparison of
SARs of these two different proteins, NorA belonging
to the major facilitator family of transporters and P-gp
being a member of the ABC family, might shed light on
some general principles of drug binding and recognition
by multidrug pumps. An extremely wide variety of
structurally unrelated P-gp inhibitors is known,19 but
as has been pointed out,20 P-gp nevertheless “displays
a significant sensitivity to structural changes within a

Flavonolignan/ Flavone Inhibitors of a S.a. MDR Pump
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2 265
modulating efficiency,27 and this was true in our fla-
vonolignan series as well (15-17). The one chalcone we
tested, 20, was inactive against NorA, while several
were found28 to have high-affinity binding to P-gp. The
most active in that case was 2′,4′,6′-trihydroxy-4-iodo-
chalcone. The work in general showed an importance
for lipophilicity at C-4, with a 4-OMe group conferring
higher activity than a 4-OH group and increasing
activity in the sequence 4-Cl/4-Br/4-I. This is partially
mirrored by our results (Table 2) with simple flavones,
although here presence of a free 4′-OH (which corre-
sponds to the 4-OH in chalcones) completely negates
MDR inhibition activity. In a synthesis of a complex
natural product, ningalin B (which contains four phe-
nolic groups), a synthetic tetramethoxy intermediate
proved to have activity against P-gp although ningalin
B did not.32 An intermediate bearing a carboxylic acid
was inactive, but the corresponding methyl ester was
active. The presence/absence of acidic functions (either
phenolic or as a carboxylic acid) thus seems to some-
times have marked influence on the potency of MDR
inhibitors in both the bacterial NorA and mammalian
P-gp systems. In summary, we have found both overall
structural similarities between some inhibitors of these
two efflux pump proteins but also differences in effects
related to changes of functional groups. The flavonolig-
nan 1 was also tested as an inhibitor against the Gram-
negative bacteria E. coli and P. aeruginosa but was
ineffective.
cooled to 0 °C. Two drops of 30% H2O2 and 1 mL of a
horseradish peroxidase solution (1.5 mg HRP (1100 U/mg)/3
mL water) were added. The HRP solution (1 mL) was added
every 15 min thereafter, and the reaction then allowed to stir
at 0 °C for 7 h. The reaction solution was allowed to warm to
room temperature, washed with brine, and extracted with
EtOAc. The EtOAc was dried with anhydrous MgSO4, filtered,
and removed by rotary evaporation to yield a brown-orange
solid. The solid was subjected to CC using 92:8 CHCl3/MeOH
to yield 0.192 g (37%) of a yellow-orange solid, 3:2 ratio of 7/8
(by 1H NMR) with minor impurities present. This solid was
recrystallized from 9:1 MeOH/H2O to give 0.025 g of regiopure
7: yellow crystals, mp 260 °C dec; 1H NMR (75 °C, DMSO-d6)
δ 3.46 (m), 3.63 (dd, J ) 12.6, 2.8 Hz), 4.29 (m), 4.77 (t, CH2-
OH), 5.03 (d, J ) 7.8 Hz), 6.22 (d, J ) 2.0 Hz), 6.51 (d, J ) 2.0
Hz), 6.76 (s), 6.79 (s), 7.09 (d, J ) 8.4 Hz), 7.58 (d, J ) 8.6, 2.0
Hz), 7.63 (d, J ) 2.0 Hz); 13C NMR (25 °C, DMSO-d6) δ 56.1,
60.1, 76.7, 77.9, 94.1, 98.9, 103.8, 103.9, 105.4, 114.8, 117.6,
119.9, 123.7, 126.0, 136.1, 143.7, 147.1, 148.0, 157.3, 161.4,
162.9, 164.3, 181.8. Anal. (C26H22O10‚3H2O) C, H.
5′′-Meth oxyh yd n oca r p in -D (8). To a 100-mL three-neck
round-bottom flask were added 0.160 g (0.559 mmol) of
luteolin, 0.118 g (0.559 mmol) of sinapyl alcohol, 40 mL
benzene and 20 mL of acetone. The reaction vessel was placed
in a 60 °C oil bath and let stir for 10 min. Next, 0.154 g (0.559
mmol) of Ag2CO3 was added and the reaction solution stirred
vigorously for 10 h. The reaction was then allowed to cool,
filtered through a Buchner funnel, and the solvent removed
by rotary evaporation to yield a pale yellow powder. The
powder was subjected to CC using 4:1 CH2Cl2/(CH3)2CO to
yield 0.052 g of pure 8 (19%, pale yellow powder, 6:1 8/7). The
purified material was recrystallized from 1:1 H2O/MeOH to
give 0.033 g of regiopure 8: pale yellow white crystals, mp
213-214 °C; 1H NMR (DMSO-d6) δ 3.38 (dd, J ) 12.6, 4.4 Hz),
3.57 (dd, J ) 12.6, 2.8 Hz), 3.78 (s, OMe), 4.33 (m), 4.95 (d, J
) 8.0 Hz), 6.19 (d, J ) 2.0 Hz), 6.50 (d, J ) 2.0 Hz), 6.77 (s),
6.87 (s), 7.12 (d, J ) 8.4 Hz), 7.63 (dd, J ) 8.4, 2.0 Hz), 7.67
(d, J ) 2.0 Hz) 12.91 (s); 13C NMR (DMSO-d6) δ 56.1, 60.0,
76.2, 78.5, 94.1, 98.9, 103.8, 103.9, 105.4, 115.1, 117.4, 120.1,
123.4, 126.1, 136.1, 144.0, 146.9, 148.0, 157.3, 161.4, 162.9,
164.3, 181.8. Anal. (C26H22O10‚2.5H2O), C, H.
Con clu sion s
Some potent potentiators of antimicrobial activity,
which apparently act by inhibition of the S. aureus MDR
efflux pump protein NorA, have been found. The range
of activites discovered have provided SARs which, in
comparison with some SARs known for P-gp inhibitors,
provide information which could be useful for under-
standing inhibitor/NorA interactions. Further system-
atic studies of bacterial MDR efflux pump inhibitors
should prove valuable in helping to understand and
combat antibiotic resistance.33
5,7-Deoxyh yd n oca r p in -D (9). The title compound was
synthesized in the same manner as 8 using 0.291 g (1.15 mmol)
of 3,4-dihydroxyflavone, 0.207 g (1.15 mmol) of coniferyl
alcohol, 25 mL of benzene, 12.5 mL of acetone, and 0.317 g
(1.15 mmol) of Ag2CO3 to yield a dark yellow solid. This solid
was subjected to CC using 93:7 CHCl3/MeOH to yield 0.129 g
of a 7:1 down/up mixture of regioisomers and minor impurities.
A portion (0.016 g) of the purified product 9 was subjected to
acetylation to give 0.014 g of acetylated product that was
further purified by CC (1:1 hexanes/EtOAc) to give 12 fractions
of pure 9a , in which 3 fractions (0.006 g) contained a 15:1
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. 1H and 13C NMR
spectra were recorded at 25 °C on a Varian Inova spectrometer
at 400 and 100 MHz, respectively, using CDCl3, acetone-d6,
methanol-d4, or DMSO-d6 as the solvent and internal refer-
ence. Melting points were determined on a Laboratory Device’s
Mel-Temp and are uncorrected. All solvents were distilled prior
to use. THF was freshly distilled from benzophenone-ketyl and
benzene was freshly distilled from CaH2. ACS acetone was
stored over 4-Å molecular sieves. All nonaqueous reactions
were performed in dry glassware under an argon atmosphere.
All starting materials were used as received. Acetylations were
performed using standard acetic anhydride/pyridine condi-
tions. All flavonoids except quercetin dihydrate were pur-
chased from Indofine Chemical Co. and all other reagents
purchased from Aldrich Chemical Co. All column chromatog-
raphy separations (CC) were performed with normal-phase
silica gel (Scientific Adsorbents Inc.; 32-63-µm particle size,
60-Å pore size). Coniferyl, sinapyl, and p-coumaryl alcohols
were prepared by a DIBAL reduction (THF, 0 °C, 12 h, >75%
yield) of the corresponding aldehyde.
1
down/up mixture (by H NMR) of regioisomers: white micro-
crystalline solid, mp 172-174 °C. Peracetate: 1H NMR (CDCl3)
δ 2.09 (s), 2.35 (s), 3.89 (s, OMe), 4.04 (dd, J ) 12.4, 4.4 Hz),
4.36 (m), 4.42 (dd, J ) 12.4, 3.2 Hz), 5.01 (d, J ) 8.0 Hz), 6.76
(s), 7.00 (d, J ) 2.0 Hz), 7.02 (m), 7.12 (J ) 8.0 Hz), 7.12 (d, J
) 8.0 Hz), 7.42 (ddd, J ) 8.2, 6.8, 0.8 Hz), 7.52 (d, J ) 2.0
Hz), 7.54 (d, J ) 2.0 Hz), 7.54 (dd, J ) 8.4, 1.6 Hz), 7.61 (d, J
) 2.0 Hz), 7.63 (dd, J ) 8.0, 2.0 Hz), 7.70 (ddd, J ) 8.6, 7.0,
1.6 Hz), 8.23 (dd, J ) 8.4, 1.6 Hz); 13C NMR (CDCl3) δ 20.7,
20.7, 56.0, 62.5, 75.9, 76.4, 106.7, 111.0, 115.5, 117.8, 118.0,
119.8, 120.4, 123.4, 123.9, 125.2, 125.4, 125.7, 133.7, 134.0,
140.7, 143.7, 145.9, 151.7, 156.2, 163.0, 168.7, 170.4, 178.3.
Anal. (C25H20O7‚0.5H2O) C, H.
5-Deoxyh yd n oca r p in -D (10). To a 250-mL three-neck
round-bottom flask were added 0.400 g (1.48 mmol) of 7,3′,4′-
trihydroxyflavone, 0.267 g (1.48 mmol) of coniferyl alcohol, 70
mL of benzene, and 35 mL of acetone. The reaction vessel was
placed in a 60 °C oil bath and let stir for 10 min. Next, 0.408
g (1.48 mmol) of Ag2CO3 was added and the reaction solution
stirred vigorously for 36 h. The reaction was then allowed to
cool, filtered through a Buchner funnel, and the solvent
removed by rotary evaporation to yield a yellow powder. The
F la von olign a n s. 5′′-Meth oxyh yd n oca r p in (7). To a 100-
mL three-neck round-bottom flask were added 0.300 g (1.05
mmol) of luteolin and 0.220 g (1.05 mmol) of sinapyl alcohol.
Next, 20 mL of acetone and 5 mL of a 0.2 M citric acid/
phosphate buffer were added to the reaction flask and the flask

266 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2
Guz et al.
yellow powder was subjected to CC using 95:5 CHCl3/MeOH
to yield 0.172 g of a pale yellow powder that contained a 6:1
down/up (by H NMR) mixture of regioisomers and dehydrodi-
120.0, 122.6, 123.0, 123.5, 133.7, 134.2, 140.9, 143.7, 146.0,
150.5, 151.9, 154.3, 154.9, 157.0, 168.0, 168.1, 168.9, 169.5,
170.3, 170.6. Anal. (C35H30O15) C, H.
1
coniferyl alcohol. VLC (vacuum liquid chromatography) using
C-18 silica gel (4:1 H2O/MeOH to 100% MeOH) yielded 0.073
g of pure 10 (11%, pale yellow powder, 6:1 down/up regioiso-
mers). A portion (0.035 g) of the purified flavonolignan was
acetylated (0.039 g, 89%) and recrystallized from 1:1 H2O/
MeOH to yield 0.025 g (64% from acetylated product) of
regiopure 10a . Peracetate: white microcrystalline sold, mp 212
°C; 1H NMR (CDCl3) δ 2.09 (s), 2.34 (s), 2.37(s), 3.88 (s, OMe),
4.05 (dd, J ) 12.3, 4.0 Hz), 4.35 (m), 4.42 (dd, J ) 12.3, 3.2
Hz), 6.75 (s), 7.01 (m, 2H), 7.11 (d, J ) 8.8 Hz), 7.12 (d, J )
8.0 Hz), 7.16 (dd, J ) 8.0, 2.2 Hz), 7.37 (d, J ) 2.2 Hz), 7.50
(dd, J ) 8.4, 2.2 Hz), 7.57 (d, J ) 2.2 Hz), 8.24 (d, J ) 8.8 Hz);
13C NMR (CDCl3) δ 20.6, 20.7, 21.2, 56.0, 62.4, 75.9, 76.3, 106.6,
110.9, 111.0, 115.4, 117.9, 119.3, 119.8, 120.4, 121.6, 123.3,
125.0, 127.1, 134.0, 140.7, 143.7, 146.1, 151.7, 154.5, 156.6,
163.2, 168.5, 168.7, 170.3, 177.6. Anal. (C31H26O11) C, H.
Sin a iticin -D (14). The title compound was synthesized in
the same manner as 8 using 0.200 g (0.699 mmol) of luteolin,
0.105 g (0.699 mmol) of p-coumaryl alcohol, 25 mL of benzene,
12.5 mL of acetone, and 0.193 g (0.699 mmol) of Ag2CO3 to
yield a brown-orange solid. The solid was subjected to CC using
91:9 CHCl3/MeOH to yield 0.146 g (48%) of a 1:2 mixture of
14/13 and minor impurities. The solid was recrystallized from
MeOH to yield 0.038 g of regiopure 14: 13%, yellow crystals,
mp 269 °C; 1H NMR (DMSO-d6) δ 3.34 (dd, J ) 12.0, 4.0 Hz),
3.58 (dd, J ) 12.0, 2.4 Hz), 4.26 (m), 4.98 (d, J ) 8.0 Hz), 6.19
(d, J ) 1.8 Hz), 6.50 (d, J ) 1.8 Hz), 6.81 (d, J ) 8.8 Hz), 6.87
(s), 7.11 (d, J ) 8.8 Hz), 7.29 (d, J ) 8.8 Hz), 7.62 (d, J ) 8.8,
2.0 Hz), 7.65 (d, J ) 2.0 Hz), 12.90 (s); 13C NMR (DMSO-d6) δ
60.0, 75.7, 78.6, 94.1, 98.9, 103.7, 103.9, 115.0, 115.3, 117.4,
120.1, 123.5, 126.5, 129.2, 144.0, 146.9, 157.3, 157.9,161.4,
162.9, 164.5, 181.8. Anal. (C24H18O8) C, H.
5-Deoxy-3-h yd r oxyh yd oca r p in -D (11). The title com-
pound was synthesized in the same manner as 10 using 0.200
g (0.699 mmol) of fisetin, 0.126 g (0.699 mmol) of coniferyl
alcohol, 50 mL of benzene, 25 mL of acetone and 0.193 g (0.699
mmol) of Ag2CO3 for 36 h to yield a yellow powder. The yellow
powder was subjected to CC using 95:5 CHCl3/MeOH to yield
0.172 g of a pale yellow powder that contained a 14:1 down/
up (by 1H NMR) mixture of regioisomers and dehydrodico-
niferyl alcohol. VLC using C-18 silica gel (4:1 H2O/MeOH to
100% MeOH) yielded 0.086 of pure 12: 27%, pale yellow
powder; 1H NMR (acetone-d6) δ 3.55 (dd, J ) 12.3, 4.0 Hz),
3.79 (dd, J ) 12.3, 2.5 Hz), 3.89 (s, OMe), 4.24 (m), 5.06 (d, J
) 8.4 Hz), 6.90 (d, J ) 8.1 Hz), 6.99 (dd, J ) 8.7, 2.1 Hz), 7.01
(dd, J ) 8.1, 1.8 Hz), 7.08 (d, J ) 9.0 Hz), 7.09, (d, J ) 2.1
Hz), 7.18 (d, J ) 1.8 Hz), 7.86 (m, 2H), 8.02 (d, J ) 8.7 Hz).
Peracetate 11a : white microcrystalline solid, mp 199-200 °C;
1H NMR (CDCl3) δ 2.09 (s), 2.34 (s), 2.37 (s), 2.38 (s), 3.88 (s,
OMe), 4.03 (dd, J ) 12.3, 4.0 Hz), 4.34 (m), 4.42 (dd, J ) 12.3,
3.2 Hz), 5.01 (d, 8.0 Hz), 7.00 (dd, J ) 8.4, 1.6 Hz), 7.01 (d, J
) 1.6 Hz), 7.11 (d, J ) 8.4 Hz), 7.12 (d, J ) 8.4 Hz), 7.17 (dd,
J ) 8.8, 2.4 Hz), 7.39 (d, J ) 2.4 Hz), 7.51 (J ) 8.4, 2.4 Hz),
7.57 (d, J ) 2.4 Hz), 8.25 (d, J ) 8.8 Hz); 13C NMR (CDCl3) δ
20.6, 20.6, 20.7, 21.2, 56.0, 62.5, 75.9, 76.3, 110.9, 111.0, 117.5,
119.4, 119.8, 121.3, 122.4, 123.2, 123.3, 127.4, 133.4, 134.0,
140.6, 143.5, 145.7, 151.7, 154.7, 155.7, 155.9, 168.0, 168.4,
168.7, 170.4, 171.5. Anal. (C33H28O13) C, H.
r a c-Sila n d r in -D (15). The title compound was synthesized
in the same manner as 10 using 0.400 g (1.39 mmol) of
eriodictyol, 0.250 g (1.39 mmol) of coniferyl alcohol, 60 mL of
benzene, 30 mL of acetone, and 0.383 g (1.39 mmol) of Ag2-
CO3 to yield a brown-white solid. This solid was subjected to
CC 95:5 CHCl3/MeOH to yield 0.262 g of a diastereomeric
mixture of near pure 15 with only the down regioisomer
detectible by 1H NMR. This sample was further purfied using
CC (7:3 hexanes/EtOAc) to yield 0.208 g of a regiopure
diastereomeric mixture of 15: 24%, white microcrystalline
solid; key 1H NMR resonances (acetone-d6) δ 2.78 (m), 3.18
(dd, J ) 17.0, 12.8 Hz), 3.52 (dd, J ) 12.4, 4.0 Hz), 3.75 (dd,
J ) 12.4, 2.5 Hz), 3.87 (s, OMe), 4.14 (m), 5.00 (d, J ) 8.0 Hz),
5.48 (m), 5,96 (m), 6.84-7.14 (m), 12.17 (s), 12.18 (s) (59:41
mixture of diastereomers by 1H NMR). Key peracetate 1H NMR
resonances (CDCl3): δ 2.07 (s), 2.08 (s), 2.30 (s), 2.31 (s), 2.33
(s), 2.39 (s), 2.39 (s), 2.76 (br d, J ) 16.8 Hz), 3.05 (br d, J )
16.8 Hz), 3.86 (s, OMe), 4.00 (dd, J ) 12.0, 4.0 Hz), 4.26 (m),
4.37 (dd, J ) 12.0, 2.5 Hz), 4.96 (d, J ) 8.0 Hz), 5.42 (br d, J
) 13.2 Hz), 6.54 (m), 6.79 (m), 6.95-7.10 (m). Anal. (C33H30O13
)
C, H.
5-Deoxyscu tella p r ostin -A (18). To a 250-mL three-neck
round-bottom flask were added 0.400 g (0.157 mmol) of 7,8-
dihydroxyflavone, 0.284 g (0.157 mmol) of coniferyl alcohol,
130 mL of benzene, and 65 mL of acetone. The reaction vessel
was placed in a 60 °C oil bath and let stir for 10 min. Next,
0.758 g (2.75 mmol) of Ag2CO3 was added and the reaction
vigorously stirred for 24 h. The reaction was then allowed to
cool, filtered through a Buchner funnel, and the solvent
removed by rotary evaporation to yield an orange solid. The
solid was subjected to CC using 95:5 CHCl3/MeOH to yield
0.270 g of near pure sample of 18. This sample was subjected
to VLC using C-18 silica gel (1:1 H2O/MeOH to 3:7 H2O/MeOH)
to yield 0.142 g of pure 18: 21%, yellow microcrystals, mp
233-234 °C; 1H NMR (35 °C, DMSO-d6) δ 3.48 (br d, J ) 12.4
Hz), 3.74 (br d, J ) 12.4 Hz), 3.80 (s, OMe), 4.40 (m), 5.08 (t,
CH2-OH), 5.14 (d, J ) 7.6 Hz), 6.83 (d, J ) 8.0 Hz), 6.92 (dd,
J ) 8.0, 2.0 Hz), 7.01 (s), 7.07 (d, J ) 2.0 Hz), 7.09 (d, J ) 8.8
Hz), 7.54 (d, J ) 8.8 Hz), 7.61 (m), 8.13 (m), 9.17 (s); 13C NMR
(35 °C, DMSO-d6) δ 55.7, 60.0, 76.3. 78.0, 106.6, 111.9, 114.8,
115.4, 115.9, 117.9, 120.6, 126.2, 126.7, 129.1, 131.2, 131.7,
132.2, 145.8, 147.2, 147.6, 161.8, 176.4. Anal. (C25H20O7) C,
H.
5-Deoxyscu tella p r ostin -B (19). The title compound was
synthesized in the same manner as 18 using 0.350 g (1.30
mmol) of 7,8,4′-trihydroxyflavone, 0.233 g (1.30 mmol) of
coniferyl alcohol, 110 mL of benzene, and 55 mL of acetone
and 0.625 g (2.28 mmol) of Ag2CO3 to yield an orange oil. The
oil was subjected to column chromatography (CC) using 95:5
CHCl3/MeOH to yield 0.145 g of a regiopure sample of 19 and
dehydrodiconiferyl alcohol. VLC using C-18 silica gel (1:1 H2O/
MeOH to 1:4 H2O/MeOH) yielded 0.050 g of pure 19: 12%,
white powder, mp 254 °C; 1H NMR (DMSO-d6) δ 3.45 (br d, J
) 12.8 Hz), 3.71 (br d, J ) 12.8 Hz), 3.79 (s, OMe), 4.39 (m),
5.10 (t, CH2-OH), 5.11 (d, J ) 7.6 Hz), 6.82 (d, J ) 8.0 Hz),
3-Hyd r oxyh yd n oca r p in -D (12). The title compound was
synthesized in the same manner as 10 using 0.600 g (1.77
mmol) of quercetin dihydrate, 0.320 g (1.77 mmol) of coniferyl
alcohol, 90 mL of benzene and 45 mL of acetone, and 0.489 g
(1.77 mmol) of Ag2CO3 to yield a yellow powder. The yellow
powder was subjected to CC using 95:5 CHCl3/MeOH to yield
0.341 g of a yellow powder that contained >30:1 down/up ratio
of regioisomers (by 1H NMR) plus minor impurities. In the
more concentrated CC fractions, the product crystallized out
to give regiopure samples of pure 12 (0.055 g). The remaining
fractions were subjected to VLC using C-18 silica gel (3:2 H2O/
MeOH to 1:4 H2O/MeOH) to give 0.142 g of a regiopure sample
of pure 12: 23% combined, yellow powder, mp 279-280 °C;
1H NMR (acetone-d6) δ 3.54 (dd, J ) 12.4, 4.0 Hz), 3.78 (dd, J
) 12.4, 2.4 Hz), 3.89 (s, OMe), 4.25 (m), 5.06 (d, J ) 8.4 Hz),
6.27 (d, J ) 2.0 Hz), 6.59 (d, J ) 2.0 Hz), 6.90 (d, J ) 8.0 Hz),
7.01 (dd, J ) 8.4, 2.0 Hz), 7.10 (d, J ) 8.4 Hz), 7.17 (d, J ) 2.0
Hz), 7.85 (dd, J ) 8.4, 2.0 Hz), 7.86 (d, J ) 2.0 Hz), 12.14 (s);
13C NMR (acetone-d6) δ 56.3, 61.7, 77.2, 80.0, 94.6, 99.2, 104.2,
111.9, 115.8, 117.2, 117.7, 121.7, 122.2, 124.9, 128.9, 137.1,
144.8, 146.1, 146.5, 148.1, 148.5, 157.8, 162.3, 165.1, 172.8,
176.6. Peracetate 13a : pale yellow microcrystals, mp 176-
1
178 °C; H NMR (CDCl3) δ 2.09 (s), 2.34 (s), 2.35 (s), 2.35 (s),
2.44 (s), 3.88 (s, OMe), 4.02 (dd, J ) 12.0, 4.0 Hz), 4.34 (m),
4.41 (dd, J ) 12.0, 3.2 Hz), 5.00 (d, J ) 8.0 Hz), 6.99 (dd, J )
8.4, 2.0 Hz), 7.01 (d, J ) 2.0 Hz), 7.09 (d, J ) 8.4 Hz), 7.12 (d,
J ) 8.4 Hz), 7.32 (d, J ) 2.0 Hz), 7.47 (dd, J ) 8.4, 2.0 Hz),
7.53 (d, J ) 2.0 Hz); 13C NMR (CDCl3) δ 20.8, 20.9, 20.9, 21.3,
21.4, 56.2, 62.7, 76.1, 76.5, 109.1, 111.2, 113.9, 117.6, 117.7,

Flavonolignan/ Flavone Inhibitors of a S.a. MDR Pump
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2 267
6.83 (s), 6.91 (d, J ) 8.0, 2.0 Hz), 6.94 (d, J ) 8.8 Hz), 7.06 (d,
J ) 8.8 Hz), 7.07 (d, J ) 2.0 Hz), 7.50 (d, A of A2B2), 8.13 (d,
B of A2B2), 9.22 (bs), 10.32 (bs); 13C NMR (DMSO-d6) δ 55.7,
60.0, 76.4, 78.1, 104.4, 111.8, 114.5, 115.4, 115.8, 115.9, 117.9,
120.6, 121.6, 126.8, 128.2, 132.2, 145.7, 147.2, 147.5, 147.7,
160.9, 162.3, 176.3. Anal. (C25H20O8) HRFAB+.
7.02 (A of A2B2), 7.42 (ddd, J ) 8.0, 7.6, 1.2 Hz), 7.56 (dd, J )
7.6, 1.2 Hz), 7.70 (ddd, J ) 8.6, 7.0, 1.4 Hz), 7.89 (B of A2B2),
8.24 (dd, J ) 8.0, 1.6 Hz); 13C NMR (CDCl3) δ 14.7, 63.8, 106.2,
114.9, 123.9, 124.0, 125.0, 125.7, 128.0, 133.5, 156.2, 161.8,
163.5, 178.4. Anal. (C17H14O3) C, H.
4′-Allyloxyfla von e (37). The title compound was synthe-
sized in the same manner as 40 using 0.075 g (0.315 mmol) of
33, 25 mL of dry acetone, 0.076 g (0.630 mmol) of allyl bromide,
and 0.218 g (1.58 mmol) of anhydrous K2CO3 heating at reflux
for 18 h to yield a brown-white powder. The powder was
subjected to CC using 7:3 hexanes/EtOAc to yield 0.065 g of
near pure 37. This sample was recrystallized from hot 99:1
hexanes/EtOAc to yield 0.050 g of pure 37: 57%, white fibrous
crystals, mp 108-109 °C; 1H NMR (CDCl3) δ 4.64 (dd, J )
5.2, 1.6 Hz), 4.65 (dd, J ) 3.2, 1.6 Hz), 5.35 (dd, J ) 10.2, 1.6
Hz), 5.46 (dd, J ) 17.2, 1.6 Hz), 6.08 (dddd, J ) 17.2, 10.2,
5.2, 3.2 Hz), 6.76 (s), 7.05 (A of A2B2), 7.42 (ddd, J ) 8.4, 7.0,
0.8 Hz), 7.56 (dd, J ) 8.4, 0.8 Hz), 7.70, (ddd, J ) 8.4, 7.0, 2.0
Hz), 7.89 (B of A2B2), 8.24 (dd, J ) 7.6, 1.6 Hz); 13C NMR
(CDCl3) δ 69.0, 106.3, 115.2, 118.0, 118.3, 124.0, 124.2, 125.1,
125.7, 128.0, 132.5, 133.6, 156.2, 161.4, 163.3, 178.4. Anal.
(C18H14O3) C, H.
F la von es. 4′-Ben zyloxyfla von e (40). To a 100-mL three-
neck round-bottom flask were added 0.075 g (0.315 mmol) of
33, 25 mL of dry acetone, 0.060 g (0.351 mmol) of benzyl
bromide, and 0.218 g (1.58 mmol) of anhydrous K2CO3. The
reaction solution was heated at reflux for 9 h, cooled to room
temperature, poured into a solution of brine, the acetone
removed in vacuo, and the organics extracted with EtOAc. The
EtOAc was dried with MgSO4, filtered, and removed in vacuo
to yield a white powder. The sample was recrystallized from
MeOH to yield 0.086 g of pure 40: 84%, white powder, mp
1
192 °C; H NMR (CDCl3) δ 5.17 (s), 7.10 (A of A2B2), 7.42 (m,
6H), 7.55 (d, J ) 8.4, 0.8 Hz), 7.70 (ddd, J ) 8.4, 7.0, 2.0 Hz)
7.90 (B of A2B2), 8.24 (J ) 8.0, 2.0 Hz); 13C NMR (CDCl3) δ
70.2, 106.2, 115.3, 117.9, 123.9, 124.3, 125.1, 125.7, 127.5,
128.0, 128.3, 128.7, 133.6, 136.2, 156.2, 161.5, 163.3, 178.4.
Anal. (C22H16O3) C, H.
4′-(2′′-Hyd r oxyeth yl)fla von e (38). To a 100-mL three-neck
round-bottom flask were added 0.075 g (0.315 mmol) of 33, 25
mL of dry acetone, 0.075 g (0.315 mmol) of (2-bromoethoxy)-
tert-butyldimethylsilane, and 0.218 g (1.58 mmol) of anhydrous
K2CO3. The reaction solution was heated at reflux for a total
of 9 h with 2 separate additions of 1 equiv of (2-bromoethoxy)-
tert-butyldimethylsilane added at 2 and 5 h. The reaction
solution was then cooled to room temperature, poured into a
solution of brine, the acetone removed in vacuo, and the
organics extracted with EtOAc. The EtOAc was dried with
MgSO4, filtered, and removed in vacuo to yield a white powder.
The sample was subjected to CC using 1:1 hexanes/EtOAc to
yield 0.030 g of the pure ether (24%, white microcrystalline
powder). The ether and 15 mL of THF were added to a 50-mL
three-neck round-bottom flask and the reaction solution cooled
to 0 °C by an ice bath. After stirring 10 min, 0.2 mL of
tetrabutylammonium fluoride (TBAF, 1.0 M in THF) was
added and the reaction stirred for 30 min at 0 °C. The contents
of the flask were then poured into water, THF removed in
vacuo, and the organics extracted with EtOAc. The EtOAc was
dried with MgSO4, filtered, and removed in vacuo to yield a
white powder. The powder was subjected to CC using 3:7
hexanes/EtOAc to yield 0.018 g of pure 38: 86%, white
microcrystalline powder, mp 142-143 °C; 1H NMR (methanol-
d4) δ 3.93 (m, 2H), 4.15 (m, 2H), 6.84 (s), 7.15 (A of A2B2), 7.50
(ddd, J ) 8.4, 7.2, 0.8 Hz), 7.72 (dd, J ) 8.4, 0.8 Hz), 7.82 (ddd,
J ) 8.4, 7.0, Hz), 8.02 (B of A2B2), 8.14 (dd, J ) 8.4, 1.6 Hz);
13C NMR (methanol-d4) δ 61.7, 71.1, 106.3, 116.4, 119.5, 124.7,
125.0, 126.3, 126.8, 129.6, 135.7, 157.9, 163.9, 166.2, 180.7.
Anal. (C17H14O4), C, H.
4′-Non oxyfla von e (39). The title compound was synthe-
sized in the same manner as 40 using 0.075 g (0.315 mmol) of
33, 25 mL of dry acetone, 0.065 g (0.315 mmol) of 1-bromo-
nonane, and 0.218 g (1.58 mmol) of anhydrous K2CO3 heating
at reflux for 12 h to yield an off-white powder. The powder
was subjected to CC using 7:3 hexanes/EtOAc to yield 0.048 g
1
of pure 39: 45%, white powder, mp 102 °C; H NMR (CDCl3)
δ 0.90 (t), 1.34 (m, 10 H), 1.49 (m, 2H), 1.83 (m, 2H), 4.04 (t),
6.76 (s), 7.02 (A of A2B2), 7.42 (ddd, J ) 8.4, 7.0, 1.2 Hz), 7.56
(dd, J ) 8.4, 1.2 Hz), 7.69 (ddd, J ) 8.4, 7.0, 1.2 Hz), 7.88 (B
of A2B2), 8.23 (dd, J ) 8.0, 1.2); 13C NMR (CDCl3) δ 14.1, 22.7,
26.0, 29.1, 29.2, 29.3, 29.5, 31.8, 68.3, 106.1, 114.9, 117.9, 123.7,
123.9, 125.0, 125.6, 127.9, 133.5, 156.2, 162.0, 163.5, 178.4.
Anal. (C24H28O3) C, H.
4′-n -P r op oxyfla von e (34). The title compound was syn-
thesized in the same manner as 40 using 0.074 g (0.311 mmol)
of 33, 25 mL of dry acetone, 0.076 g (0.622 mmol) of 1-bro-
mopropane, and 0.215 g (1.56 mmol) of anhydrous K2CO3
heating at reflux for 12 h to yield a white powder. The white
powder was subjected to CC using 7:3 hexanes/EtOAc to yield
1
0.071 g of pure 34: 82%, white powder, mp 121-122 °C; H
NMR (CDCl3) δ 1.08 (t), 1.86 (m), 4.01 (t), 6.76 (s), 7.02 (A of
A2B2), 7.42 (ddd, J ) 8.4, 7.0, 0.8 Hz), 7.56 (dd, J ) 8.0, 1.2
Hz), 7.69 (ddd, J ) 8.4, 7.0, 1.2 Hz), 7.88 (B of A2B2), 8.23 (dd,
J ) 8.0, 1.2 Hz); 13C NMR (CDCl3) δ 10.5, 22.4, 69.8, 106.1,
114.9, 117.9, 123.7, 123.9, 125.0, 125.6, 128.0, 133.5, 156.2,
162.0, 163.5, 178.4. Anal. (C18H16O3) C, H.
4′-Isop r op oxyfla von e (35). The title compound was syn-
thesized in the same manner as 40 using 0.075 g (0.315 mmol)
of 33, 25 mL of dry acetone, 0.078 g (0.630 mmol) of 2-bromo-
propane, and 0.218 g (1.58 mmol) of anhydrous K2CO3 heating
at reflux for 12 h to give an off-white powder. The powder was
subjected to CC using 7:3 hexanes/EtOAc to yield 0.041 g of
Ack n ow led gm en t. This work was supported by
National Science Foundation (NSF) Grant CHE9619213
and Colorado State University Agricultural Experiment
Station Project 15-271 to F.R.S. and by National Insti-
tutes of Health Grant GM54412 to K.L. Support for J .
J . was from an NSF Research Experience for Under-
graduate grant, and T.B. was supported by a Camille
and Henry Dreyfus Foundation Senior Scientist Mentor
Award to F.R.S.
1
pure 35: 47%, white powder, mp 102 °C; H NMR (CDCl3) δ
1.39 (s), 1.40 (s), 4.67 (m), 6.75 (s), 7.00 (A of A2B2), 7.41 (ddd,
J ) 8.4, 7.0, 0.8 Hz), 7.55 (dd, J ) 8.4, 1.2 Hz), 7.69 (ddd, J )
8.4, 7.0, 1.2 Hz), 7.87 (B of A2B2), 8.23 (dd, J ) 8.0, 1.2 Hz);
13C NMR (CDCl3) δ 21.9, 70.2, 106.0, 115.9, 117.9, 132.5, 123.9,
125.0, 125.6, 128.0, 133.5, 156.2, 160.9, 163.5, 178.4. Anal.
(C18H16O3) C, H.
4′-Eth oxyfla von e (36). To a 100-mL three-neck round-
bottom flask were added 0.078 g (0.327 mmol) of 33, 25 mL
dry acetone, 0.237 g (1.31 mmol) of bromoethane, and 0.226 g
(1.63 mmol) of anhydrous K2CO3. The reaction flask was placed
in a 37 °C oil bath and stirred for 17 h. The reaction was then
allowed to cool to room temperature, poured into a solution of
brine, the acetone removed in vacuo, and the organics ex-
tracted with EtOAc. The EtOAc was dried with MgSO4,
filtered, and removed in vacuo to yield a white powder of near
pure 36. The powder was subjected to CC using 7:3 hexanes/
EtOAc to yield 0.076 g of pure 36: 87%, white amorphous solid,
mp 122-123 °C; 1H NMR (CDCl3) δ 1.47 (t), 4.13 (q), 6.76 (s),
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