Synthesis and Biological Evaluation of 3,5-Dimethoxystilbene Analogs

Article abstract of DOI:10.1002/cbdv.201500516

In our continuing effort to discover natural product-based pest management agents, derivatives of 3,5-dimethoxystilbene were synthesized yielding 27 new and six known compounds. Compounds 11 and 12 showed strong Aedes aegypti larvicidal activity (LC₅

Full text of DOI:10.1002/cbdv.201500516

Received Date: 29-Dec-2015
Revised Date: 09-Mar-2016
Accepted Date: 15-Mar-2016
Article Type: Full Paper
Synthesis and Biological Evaluation of 3,5-Dimethoxystilbene Analogs
Jian-Quan Weng,a),b) Abbas Ali,c) Alden Estep,d) James Becnel,d) Susan L. F. Meyer,e), David
E. Wedge,a) Melissa Jacob,c) Agnes M. Rimandoa) *
a) U.S. Department of Agriculture, Agricultural Research Service, Natural Products
Utilization Research Unit, P.O. Box 1848, University, Mississippi 38677, USA (*(phone:
+1-6629151037; fax: +1-6629151035; e-mail: agnes.rimando@ars.usda.gov)
b) Current address: College of Chemical Engineering, Zhejiang University of Technology, 18
Chaowang Rd, Hangzhou 310014, China
c) National Center for Natural Products Research, The University of Mississippi, University,
Mississippi 38677, USA
d) Center for Medical, Agricultural and Veterinary Entomology, Mosquito and Fly Research
Unit, 1600 S.W. 23rd Drive, Gainesville, FL 32608, USA
5U.S. Department of Agriculture, Agricultural Research Service, Nematology Laboratory,
Henry A. Wallace Beltsville Agricultural Research Center -West, Beltsville, MD 20705, USA
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201500516
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ABSTRACT
In our continuing effort to discover natural product-based pest management agents,
derivatives of 3,5-dimethoxystilbene were synthesized yielding 27 new and 6 known
compounds. Compounds 11 and 12 showed strong Aedes aegypti larvicidal activity (LC50
45.31 and 49.93 μM, respectively). Futhermore, 11 and 12 exhibited high effectiveness
against larvae of pesticide-susceptible and pyrethroid-resistant strains of Ae. aegypti; activity
against the adult mosquitoes was low. Compounds 6f, 6g, and 6i at either 83.3 or 166.7 μg/ml
reduced the mobility of second-stage juveniles (J2) of the root-knot nematode (Meloidogyne
incognita) that hatched from eggs immersed in the test compounds for 7 days. However, there
was little or no effect on J2 placed directly into these compounds, and none of the analogs
suppressed M. incognita egg hatch. The compounds were tested for inhibition of some
agriculturally important fungi; 6a, 7a and 7e demonstrated strong inhibition of
Colletotrichum species. Activity of the stilbenes against some human pathogens was also
explored; 11, 12, and 16 showed moderate inhibitory activity against Cryptococcus
neoformans, Staphylococcus aureus, methicillin-resistant S. aureus and Mycobacterium
intracellulare. Except for 11 and 12, which were active against mosquito larvae and some
human pathogens, no single analog demonstrated activity in all the tests, indicating specific
activities. Synthesis of the analogs and structure-activity relationships are discussed.
Keywords: 3,5-dimethoxystilbene analogs, Aedes aegypti, Meloidogyne incognita
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INTRODUCTION
Stilbenes have been widely studied for their medicinal properties [1-3]. In contrast, while
some are known as phytoalexins, studies on their pesticidal activity have been sparse. As part
of our continuing effort exploring the biological activities of stilbenes, and with the objective
to investigate their pesticidal properties, analogs of 3,5-dimethoxystilbene (Suppl. Fig. 1)
were synthesized. This was triggered by a report on 3,5-dimethoxystilbene, isolated from
Lonchocarpus chiricanus, as having potency equal to rotenone in an Aedes aegypti larvicidal
assay [4]. Ae. aegypti is the primary vector for the transmission of dengue [5]. More than
500,000 people are hospitalized each year due to dengue and about 20,000 cases lead to
severe complications resulting in death [6]. Dengue has become increasingly frequent in the
U.S.A. [7], and this trend is also occurring worldwide [8]. Ae. aegypti is also the vector for
yellow fever [9], chikungunya [10], and other tropical diseases which cause severe human
health problems throughout the world. There are no effective vaccines or drugs for the control
of these diseases, and controlling the mosquito vectors remains the major strategy for disease
prevention [11]. It was therefore of interest to determine the activity of the synthesized
analogs against Ae. aegypti.
A previous study that showed stilbenes having the 3,5-dimethoxystilbene core
structure were toxic towards Caenorhabditis elegans adults [12] likewise incited determining
the activity of the analogs against plant-parasitic nematodes. Plant-parasitic nematodes cause
crop losses of approximately ten billion dollars each year in the United States and 125 billion
globally [13]. Many of the conventional nematicides used to manage these plant pathogens
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have been deregistered due to adverse effects on health and the environment. It is therefore
necessary to develop efficacious, environmentally safe means of managing phytoparasitic
nematodes, and that includes testing of novel compounds for nematotoxicity. The current
study focused on the genus Meloidogyne (root-knot nematode), which is one of the most
economically important nematodes attacking crop plants; species in this genus are found
worldwide on numerous hosts [14]. Of specific interest is the southern root-knot nematode
Meloidogyne incognita (Kofoid and White) Chitwood, a highly destructive species in this
genus.
It was also of interest to test the activity of the analogs against some devastating fungal
plant pathogens based on reports on the activity of stilbenes against Colletotrichum, Botrytis,
Fusarium, and Phomopsis species [15], against fungi associated with esca of grapevine; i.e.,
Fomitiporia punctata, Libertella blepharis, Phaemoniella chlamydospora, Phaeoacremonium
aleophilum, and Stereum hirsutum [16], and against Cladosporium cucumerinum,
Plasmopara viticola and Pyricularia oryzae [17]. Additionally, activity of the analogs
against human and food pathogens and yeast was investigated, spurred by reports of strong
activity of stilbenes against several pathogenic bacteria, the antimicrobial activity being
similar to that of known antibiotics [18, 19].
We report the synthesis of 3,5-dimethoxystilbene analogs, most of which are novel, and
their activities against Ae. aegypti, agricultural pests and human pathogens. We also discuss
how pesticidal activity is affected by various substituents added to the core structure.
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RESULTS AND DISCUSSION
As part of our on-going efforts to discover natural products and natural product-based
pesticides, we synthesized analogs of 3,5-dimethoxystilbene, prompted by a report that this
compound was as effective as rotenone against mosquito larvae, both having MIC = 3 ppm
[4]. We prepared a series of cis- and trans- analogs of 3,5-dimethoxystilbene starting with the
aldehydes and reacting with 3,5-dimethoxybenzyl-triphenylphosphonium to the
corresponding styrenes (Schemes 1 – 4), yielding twenty-seven new and the six known (3, 6a,
6n, 7n, 11, and 17) compounds (Fig. 1). The analogs were screened for activity against
1-day-old Aedes aegypti larvae. Of the 33 analogs, only 11 and 12 exhibited high larvidical
activity, with LC50 values 45.31 and 49.93 μM, respectively (Table 1); 7f was active only at
346.29 μM, and the rest of the compounds were inactive. Compounds 11 and 12 bear a prenyl
substituent at C3′ in ring B, differing from 6n and 7n where the prenyl group is in an ether
linkage at C4′. None of the other types of substituents (phosphate, methoxy, halogen, nitro,
hydroxy) conferred larvicidal activity. Our results suggest that a prenyl group directly
attached to the benzene ring at C3′ enhances larvicidal activity. It is noted that in the study of
Ioset et al. [4], the prenylated stilbene derivatives tested for larvicidal activity had the prenyl
group(s) in ring A; these derivatives were less larvicidal than 3,5-dimethoxystilbene.
Compounds 11 and 12 were further investigated for their activity against larvae and adult
pesticide-susceptible (Orlando (ORL)) and pyrethroid-resistant (Puerto Rico (PR)) strains of
Ae. aegypti (Tables 2 and 3) Both compounds were highly effective against larvae of ORL
and PR strains. Compound 11 showed a slightly better activity than 12, and showed same
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activity as permethrin at 0.1 μg/μL concentration, indicating a trans-configuration is
preferable for larvicidal activity. Both compounds had low effectiveness against adult
mosquitoes of both strains.
Based on a report that stilbenes having a 3,5-dimethoxy substitution in ring A are
toxic towards the nematode C. elegans [12] the synthesized 3,5- dimethoxystilbene analogs
were evaluated for activity against the plant-parasitic nematode M. incognita. None of the
tested compounds suppressed hatch of M. incognita eggs. There was some suppressive effect
on mobility in J2 that had hatched from eggs immersed in solutions of 6f, 6g, 6i, and 7k
(Table 4). However, when J2 previously hatched in water were immersed directly into these
same compounds or 6b, only 6g and 7k had an effect on J2 mobility, and the suppression was
temporary and occurred in only one of two trials. The assays indicated that the test
compounds would not affect egg hatch or reliably inhibit J2 movement, and therefore may
not have practical application. In comparison, 3,5-dihydroxy- 4-isopropylstilbene (DIS)
(Suppl. Fig. 1) isolated from the culture filtrate of the bacterium Photorhabdus luminescens
has been reported to inhibit egg hatch of M. incognita [37]. The total M. incognita egg hatch
was significantly lower than in the solvent controls after treatment with 49.19 μM of DIS
over a 5-day period followed by 5 days in water. DIS differs from 6f, 6g, 6i, and 7k in having
OHs at C-3 and C-5 rather than OCH3s. The meta-hydroxy group may be important for
inhibition of M. incognita J2.
When evaluated for activity against some agriculturally important fungi (Botrytis
cinerea, Colletotrichum acutatum, C. fragariae, C. gloeosporioides, and Fusarium
oxysporum), none of the compounds inhibited B. cinerea or F. oxysporum. Compounds 6a, 7a,
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and 7e had a strong inhibitory effect on Colletotrichum species (Table 5). The other analogs
had either low activity or were not inhibitory. Sobolev et al., 2011 [15] tested a series of
natural and synthetic stilbenoids against the same Colletotrichum species including
3,5-dimethoxystilbene as well as the analogs pterostilbene and 3,5,4-O-trimethylresveratrol
(Suppl. Fig. 1), but none had activity against Colletorichum spp. While we found three
analogs to be highly active, the structural features conferring activity for this set of
compounds could not be deduced. Pterostilbene, a stilbene having 3,5-dimethoxy substitution,
like 6a and 7a, has been reported to destroy cellular membranes by depolarization [38].
(E)-2-(4-fluorophenyl)-5-(4-(4-fluorostyryl)phenyl)-1,3,4-oxadiazole, a fluorine- containing
stilbene, like 7e, has likewise been reported to disturb mycelial cell membrane system [39].
These studies suggest that 6a, 7a, and 7e exert their effects through disruption of membrane
function.
The activity of the analogs against some human pathogens was also explored. None of
the compounds inhibited Aspergillus fumigatus, Candida albicans, C. glabrata, C. krusei,
Escherichia coli, or Pseudomonas aeruginosa. Except for 11, 12, and 16, none of the other
analogs exhibited inhibitory activity towards Cryptococcus neoformans, Mycobacterium
intracellulare, Staphylococcus aureus or methicillin- resistant S. aureus. Compounds 11, 12,
and 16 showed moderate inhibition of Cryptococcus neoformans (IC50 = 13.63, 41.77, and
5.22 μM, respectively), Mycobacterium intracellulare (IC50 = 47.17, 50.40, and 33.98 μM,
respectively), Staphylococcus aureus (IC50 = 13.11, 17.03, and 20.43 μg/mL, respectively),
and methicillin-resistant S. aureus (IC50 = 9.10, 13.02, and 43.72 μM, respectively) (Table 6).
3,5-Dimethoxystilbene has been reported to inhibit S. aureus and methicillin-resistant S.
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aureus, with a minimum inhibitory concentration >3121 μM [40]. In comparison, 11, 12,
and 16 had MIC values 42 – 344 times less than that of 3,5-dimethoxystilbene (Table 6). This
suggests that addition of OH at C-4′ enhances activity; none of the kinds of functional group
addition at C-4′ resulted in an active analog. Our results indicate that 3,5-OCH3 and 4′-OH,
the common structural features of 11, 12 and 16, must be kept intact for activity. Absence of
inhibitory effect of 17, having 3,5-OH rather than 3,5-OCH3 substituent, supports this
structural
requirement.
Interestingly,
(E)-
and
(Z)-3-(3,5-dimethoxy-4-methyl-
styryl)-6-methoxybenzene-1,2-diol (DMMD; Suppl. Fig. 1) were reported to inhibit S. aureus,
with MIC of 50-100 μg/disk [41]; this suggests that replacing 4′-OH with -OCH3 reduces
inhibitory activity. Addition of a prenyl moiety at C-3′ appears to further enhance inhibitory
activity against S. aureus and methicillin-resistant S. aureus, as observed from the MICs of 11
and 16 (Table 6).
E-DMMD also exhibited inhibition of Cryptococcus neoformans, MIC = 50-100
μg/disk [39]. E-DMMD and 16 differ in the functional groups attached in ring B. Similar to
the observation on their activities against S. aureus, substitution of the 4′-OH with -OCH3
appears to cause a reduction in activity against C. neoformans. Synthetic halostilbenes where
the C-3/5 bears either F or Cl, namely, 3-fluoro-4,4′,5-trimethoxy-3′-hydroxy-Z-stilbene;
3-fluoro-4,4′,5-trimethoxy-3′-hy-
droxy-E-stilbene;
and
3-chloro-4,4′,5-trimethoxy-3′-hydroxy-Z-stilbene (Suppl. Fig. 1) showed relatively strong
inhibition of C. neoformans, with MIC of 62 μl/mL [39]. Lack of inhibitory activity of
analogs 6e, 6f, 6i, 7e, 7f, and 7i, bearing the halogen(s) in ring B, suggests that halogenation
in ring B is not favorable. Studies on stilbenes against Mycobacterium species are scanty, and
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most report absence of or insignificant activity. (E)-3-Hydroxy-5-methoxystilbene (Suppl.
Fig. 1) was found to be effective in inhibiting the growth of Mycobacterium bovis with an
MIC of 26 μg/mL [42]. This is the first report of stilbenes having activity against M.
intracellulare. Thus, finding 11, 12 and 16 as inhibitory, albeit moderately, is quite
stimulating.
In summary, we have synthesized 33 analogs of 3,5-dimethoxystilbene, consisting of 27
new, unreported structures and 6 known compounds. The analogs were tested for activity
against mosquito larvae and adults, root-knot nematode, crop pathogens, and microorganisms
related to human diseases. No single compound exhibited activity in all the tests, indicating
activity specificity. Of interest are 11 and 12, which showed inhibition of mosquito larvae, C.
neoformans, S. aureus, methicillin-resistant S. aureus, and M. intracellulare; notably, 11
showed activity against Ae. aegytpi larvae similar to that of perrmethrin; 3.08 μM of 11 and
2.5 μM of perrmethrin had 100% mortality. Compound 11 presents a scaffold that could be
optimized to generate analogs with improved activity. Results obtained with the other analogs
provide guidance for structure-activity optimization.
ACKNOWLEDGEMENT
We are grateful for the financial support to J. Q. Weng from the Chinese Scholarship Council
(201208330199) and Science Technology Department of Zhejiang Province (2014C31127),
China. The antimicrobial screening was supported by the USDA Agricultural Research
Service Specific Cooperative Agreement No. 58-6408-2-0009. Thanks are extended to
Margaret MacDonald, Gloria Hervey, Ramona Pace, and Marsha Wright for technical
assistance in the laboratory.
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MATERIALS AND METHODS
General Methods. All solvents were redistilled prior to use. All reactions were performed in
a dry round-bottom flask and occurred under N2 atmosphere. Reactions were monitored by
thin-layer chromatography (TLC) using TLC Silica gel 60 F254 (Merck); the spots were
visualized under UV light. Purification was performed by preparative TLC on silica gel GF
plates (SORBTECH, scored, 20×20 cm, 500 μm) or flash chromatography using silica gel
(40-60 μm; Sorbent Technologies). NMR spectra were recorded on a Bruker Avance
DRX-500 MHz spectrometer in CDCl3, DMSO-d6 or Acetone-d6. ESI MS spectra were
collected using a JEOL AccuTOF JMS-T100LC mass spectrometer (JEOL USA, Inc.,
Peabody, MA). GC-MS spectra were obtained with a JEOL GCMate II spectrometer coupled
with an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA).
Preparation and Synthesis of Stilbene Analogs. (E)-4-(3,5-Dimethoxysty- ryl)phenyl
dihydrogen phosphate (3) was synthesized through reaction of pterostilbene (1) and
dibenzylphosphite using 4-dimethylaminopyridine (DMAP) and N,N-Diisopropylethylamine
(DIEA), followed by deprotection of the benzyl groups (Scheme 1), as previously reported
[20]. Pterostilbene was synthesized according to published methods [21].
3,5-Dimethoxybenzyl-triphenylphosphonium
(4b)
and
4-((tert-butyl-dimethyl-
silyl)oxy)benzaldehyde (13) were synthesized following published procedures [22] and [23],
respectively.
General Procedure for the Synthesis of Stilbene Analogs 6a-6n and 7a-7n.
n-Butyllithium (n-BuLi) (1.6 M in hexanes, 1.0 equiv) was added to a cold solution (-78 °C)
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of phosphonium salt (1.0 equiv) in THF, and the resulting red solution was stirred under
nitrogen for 2 h. A solution of aldehyde (1.0 equiv) in THF was added drop-wise over 30 min,
and the mixture was stirred for 12 h at room temperature (Scheme 2). The resulting
suspension was poured into water and extracted with dichloromethane multiple times. The
combined organic phase was washed with brine and dried over anhydrous MgSO4. After
solvent removal under reduced pressure, the crude product was purified by preparative TLC
or flash chromatography. The cis isomer eluted first followed by the trans isomer.
1,3-Dimethoxy-2-methyl-5-styrylbenzene
(6a,
7a).
(3,5-Dimethoxy-4-methyl-
benzyl)triphenylphosphonium 4a (200 mg, 0.394 mmol) was reacted with benzaldehyde 5a
(42.3 mg, 0.394 mmol), then purified by preparative TLC (hexanes/ethyl acetate, 97:3) to
1
afford 6a and 7a. Compound 6a was obtained as a white solid: 22.4 mg (22.3%). H NMR
(CDCl3, 500 MHz): δ 2.13 (s, Me-C(4)), 3.89 (s, MeO-C(3,5)), 6.72 (s, H-C(2,6)), 7.09 (t, J =
16.5 Hz, H-C(7,8)), 7.27 (t, J = 7.3 Hz, H-C(4')), 7.37 (t, J = 7.7 Hz, H-C(3',5')), 7.53 (d, J =
7.3 Hz, H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ 8.3 (Me-C(4)), 55.8 (MeO-C(3,5)), 102.0
(C(2,6)), 114.6 (C(4)), 126.4 (C(2',6')), 127.5 (C(7)), 127.9(C(8)), 128.7 (C(3',5')), 129.2
(C(4')), 135.7 (C(1')), 137.4 (C(1)), 158.5 (C(3,5)). Positive ion ESI-HRMS: calcd for
C17H19O2 [M+H]+, 255.1385; found, 255.1393. Compound 7a ws a viscous liquid: 57.8 mg
1
(57.7%). H NMR (CDCl3, 500 MHz): δ 2.09 (s, Me-C(4)), 3.64 (s, MeO-C(3,5)), 6.46 (s,
H-C(2,6)), 6.57 (d, J = 12.2 Hz, H-C(7)), 6.62 (d, J = 12.2 Hz, H-C(8)), 7.19-7.22 (m,
13
H-C(4')), 7.25-7.29 (m, H-C(3',5')), 7.33 (d, J = 7.2 Hz, H-C(2,6)). C NMR (CDCl3, 125
MHz): δ 8.2 (Me-C(4)), 55.5 (MeO-C(3,5)), 104.5 (C(2,6)), 113.7 (C(4)), 127.0 (C(7)), 128.2
(C(2',6')), 128.9 (C(3',5')), 129.8 (C(8)), 130.7 (C(4')), 135.1(C(1')), 137.7 (C(1)), 157.9
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(C(3,5)). Positive ion ESI-HRMS: calcd for C17H19O2 [M+H]+, 255.1385; found, 255.1377.
1,3-Dimethoxy-5-(3-methoxystyryl)-2-methylbenzene (6b, 7b). Compound 4a (200 mg,
0.394 mmol) was reacted with 3-methoxybenzaldehyde 5b (55.3 mg, 0.394 mmol), then
purified by preparative TLC (hexanes/ethyl acetate, 95:5) to afford 6b and 7b. Compound 6b
was obained as a white solid: 34.9 mg (31.1%). 1H NMR (CDCl3): δ 2.16 (s, Me-C(4)),
3.87 (s, MeO-C(3')), 3.90 (s, MeO-C(3,5)), 6.73 (s, H-C(2,6)), 6.85 (ddd, J = 8.2, 2.5, 0.7 Hz,
H-C(4')), 7.05-7.13 (overlap, H-C(7,8,2')), 7.14-7.15 (m, H-C(6')), 7.30 (t, J = 7.9 Hz,
H-C(5')). 13C NMR (CDCl3, 125 MHz): δ 8.4 (Me-C(4)), 55.2 (MeO-C(3')), 55.8
(MeO-C(3,5)), 102.0 (C(2,6)), 111.6 (C(2')), 113.3 (C(4')), 114.7 (C(4)), 119.2 (C(6')), 127.8
(C(7)), 129.5 (C(8)), 129.6 (C(5')), 135.6 (C(1')), 138.8 (C(1)), 158.5 (C(3,5)), 159.9 (C(3')).
Positive ion ESI-HRMS: calcd for C18H21O3 [M+H]+, 285.1490; found, 285.1496. Compound
7b was a viscous liquid: 69.3 mg (61.8%). 1H NMR (CDCl3, 500 MHz): δ 2.10 (s,
Me-C(4)), 3.66 (s, MeO-C(3,5)), 3.72 (s, MeO-C(3')), 6.49 (s, H-C(2,6)), 6.57 (d, J = 12.4 Hz,
H-C(7)), 6.60 (d, J = 12.3 Hz, H-C (8)), 6.78 (ddd, J = 8.2, 2.5, 0.7 Hz, H-C (4')), 6.87-6.91
(m, H-C(2')), 6.93-6.95 (m, H-C(6')), 7.20 (t, J = 7.9 Hz, H-C(5')). 13C NMR (CDCl3, 125
MHz): δ 8.2 (Me-C(4)), 55.1 (MeO-C(3')), 55.5 (MeO-C(3,5)), 104.5 (C(2,6)), 113.0 (C(2')),
113.8 (C(4')), 114.0 (C(4)), 121.5 (C(6')), 129.2 (C(7)), 129.6 (C(8)), 130.9 (C(5')), 135.1
(C(1')), 139.0 (C(1)), 158.0 (C(3,5)), 159.5 (C(3')). Positive ion ESI-HRMS: calcd for
C18H21O3 [M+H]+, 285.1490; found, 285.1495.
1,3-Dimethoxy-5-(4-methoxystyryl)-2-methylbenzene (6c, 7c). Compound 4a (200 mg,
0.394 mmol) was reacted with 4-methoxybenzaldehyde 5c (54.8 mg, 0.394 mmol), then
purified by preparative TLC (hexanes/ethyl acetate, 95:5) to afford 6c and 7c. Compound
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6c was obtained as a white solid: 25.3 mg (22.6 %). 1H NMR (CDCl3, 500 MHz): δ 2.14 (s,
Me-C(4)), 3.86 (s, MeO-C(4')), 3.91 (s, MeO-C(3,5)), 6.71 (s, H-C(2,6)), 6.93 (d, J = 8.7 Hz,
H-C(3',5')), 6.97 (d, J = 16.0 Hz, H-C(7)), 7.05 (d, J = 16.0 Hz, H-C(8)), 7.48 (d, J = 8.7 Hz,
H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ 8.3 (Me-C(4)), 55.3 (MeO-C(4')), 55.7
(MeO-C(3,5)), 101.8 (C(2,6)), 114.1 (C(3',5')), 127.2 (C(4)), 127.4 (C(7)), 127.6 (C(2',6')),
128.4 (C(8)), 130.2 (C(1')), 136.0 (C(1)), 158.4 (C(3,5)), 159.2 (C(4')). Positive ion
ESI-HRMS: calcd for C18H21O3 [M+H]+, 285.1490; found, 285.1485. Compound 7c was a
viscous liquid: 41.5 mg (37.0 %). 1H NMR (CDCl3, 500 MHz): δ 2.08 (s, Me-C(4)), 3.66 (s,
MeO-C(3,5)), 3.79 (s, MeO-C(4')), 6.46-6.48 (overlap, H-C(2,6,7)), 6.52 (d, J = 12.3 Hz,
H-C(8)), 6.77-6.80 (m, H-C(3',5')), 7.24-7.27 (m, H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ
8.1 (Me-C(4)), 55.2 (MeO-C(4')), 55.6 (MeO-C(3,5)), 104.3 (C(2,6)), 104.4 (C(4)), 113.5
(C(3',5')), 129.2 (C(7)), 129.3 (C(8)), 129.9 (C(1')), 130.3 (C(2',6')), 135.5(C(1)), 158.0
(C(3,5)), 158.7 (C(4')). Positive ion ESI-HRMS: calcd for C18H21O3 [M+H]+, 285.1490;
found, 285.1502.
1,3-Dimethoxy-2-methyl-5-(4-nitrostyryl)benzene (6d, 7d). Compound 4a (200 mg, 0.394
mmol) was reacted with 4-nitrobenzaldehyde 5d (60.8 mg, 0.394 mmol), then purified by
preparative TLC (hexanes/ethyl acetate, 95:5) to afford 6d and 7d. Compound 6d was
1
obtained a a yellow solid: 19.0 mg (16.1 %). H NMR (CDCl3, 500 MHz): δ 2.12 (s,
Me-C(4)), 3.89 (s, MeO-C(3,5)), 6.73 (s, H-C(2,6)), 7.10 (d, J = 16.2 Hz, H-C(7)), 7.24 (d, J
= 16.23 Hz, H-C(8)), 7.62-7.64 (m, H-C(2',6')), 8.20-8.23 (m, H-C(3',5')). 13C NMR (CDCl3,
125 MHz): δ 8.4 (Me-C(4)), 55.8 (MeO-C(3,5)), 102.4 (C(2,6)), 116.1 (C(4)), 124.1 (C(3',5')),
125.4(C(7)), 126.7 (C(2',6')), 133.9 (C(8)), 134.5 (C(1)), 143.9 (C(1')), 146.6 (C(4')), 158.5
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(C(3,5)). Positive ion ESI-HRMS: calcd for C17H18NO4 [M+H]+, 300.1235; found, 300.1243.
1
Compound 7d as a yellow solid: 76.0 mg (64.4 %). H NMR (CDCl3, 500 MHz): δ 2.07 (s,
Me-C(4)), 3.63 (s, MeO-C(3,5)), 6.38 (s, H-C(2,6)), 6.57 (d, J = 12.2 Hz, H-C(7)), 6.77 (d, J
13
= 12.2 Hz, H-C(8)), 7.43-7.45 (m, H-C(2',6')), 8.08-8.10 (m, H-C(3',5')). C NMR (CDCl3,
125 MHz): δ 8.2 (Me-C(4)), 55.6 (MeO-C(3,5)), 104.2 (C(2,6)), 114.8 (C(4)), 123.4 (C(3',5')),
127.4 (C(7)), 129.8 (C(2',6')), 134.1 (C(8)), 134.4 (C(1)), 144.5 (C(1')), 146.4 (C(4')), 158.2
(C(3,5)). Positive ion ESI-HRMS: calcd for C17H18NO4 [M+H]+, 300.1235; found, 300.1237.
5-(4-Fluorostyryl)-1,3-dimethoxy-2-methylbenzene (6e, 7e). Compound 4a (200 mg,
0.394 mmol) was reacted with 4-fluorobenzaldehyde 5e (49.9 mg, 0.394 mmol), then purified
by preparative TLC (hexanes/ethyl acetate, 98:2) to afford 6e and 7e. Compound 6e was
obtained as white solid: 21.8 mg (20.3 %). 1H NMR (CDCl3, 500 MHz): δ 2.14 (s, Me-C(4)),
3.89 (s, MeO-C(3,5)), 6.70 (s, H-C(2,6)), 6.99 (d, J = 16.3 Hz, H-C(7)), 7.03-7.08 (overlap,
H-C(8,3',5')), 7.47-7.50 (m, H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ 8.3 (Me-C(4)),
55.7 (MeO-C(3,5)), 101.9 (C(2,6)), 114.6 (C(4)), 115.6 (d, J = 21.6 Hz, (C(2',6')), 126.7 (d, J
= 0.8 Hz, C(7)), 127.9 (d, J = 7.9 Hz, C(3',5')), 129.0 (d, J = 2.4 Hz, C(8)), 133.6 (d, J = 3.3
Hz, (C(1')), 135.6 (C(1)), 158.5 (C(3,5)), 162.3 (d, J = 247.0 Hz, (C(4')). Positive ion
ESI-HRMS: calcd for C17H18FO2 [M+H]+, 273.1290; found, 273.1303. Compound 7e was a
1
viscous liquid: 29.1 mg (27.1 %). H NMR (CDCl3, 500 MHz): δ 2.14 (s, Me-C(4)), 3.89(s,
MeO-C(3,5)), 6.70 (s, H-C(2,6)), 6.54 (d, J = 12.2 Hz, H-C(7)), 6.57 (d, J = 12.2 Hz, H-C(8)),
6.93-6.98 (m, H-C(3',5')), 7.28-7.30 (m, H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ 8.2
(Me-C(4)), 55.5 (MeO-C(3,5)), 104.3 (C(2,6)), 113.9 (C(4)), 115.0 (d, J = 21.4 Hz, (C(2',6')),
128.6 (C(7)), 130.7 (d, J = 7.8 Hz, C(3',5')), 130.8 (d, J = 1.2 Hz, C(8)), 133.5 (d, J = 3.5 Hz,
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(C(1')), 134.9 (C(1)), 158.1 (C(3,5)), 161.8 (d, J = 246.6 Hz, (C(4')). Positive ion ESI-HRMS:
calcd for C17H18FO2 [M+H]+, 273.1290; found, 273.1304.
5-(4-Chlorostyryl)-1,3-dimethoxy-2-methylbenzene (6f, 7f). Compound 4a (200 mg,
0.394 mmol) was reacted with 4-chlorobenzaldehyde 5f (56.3 mg, 0.394 mmol), then purified
by preparative TLC (hexanes/ethyl acetate, 98:2) to afford 6f and 7f. Compound 6f was a
1
white solid: 41.2 mg (36.2 %). H NMR (CDCl3, 500 MHz): δ 2.14 (s, Me-C(4)), 3.89 (s,
MeO-C(3,5)), 6.70 (s, H-C(2,6)), 7.01 (d, J = 16.3 Hz, H-C(7)), 7.06 (d, J = 16.3 Hz, H-C(8)),
7.32-7.35 (m, H-C(3',5')), 7.43-7.45 (m, H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ8.4
(Me-C(4)), 55.7 (MeO-C(3,5)), 102.0 (C(2,6)), 114.9 (C(4)), 126.5 (C(7)), 127.6 (C(3',5')),
128.8 (C(2',6')), 129.9 (C(8)), 133.0 (C(4')), 135.4 (C(1')), 135.9 (C(1)), 158.5 (C(3,5)).
Positive ion ESI-HRMS: calcd for C17H18ClO2 [M+H]+, 289.0995; found, 289.1025.
Compound 7f was a viscous liquid: 36.2 mg (32.0 %). 1H NMR (CDCl3, 500 MHz): δ
2.08(s, Me-C(4)), 3.66 (s, MeO-C(3,5)), 6.42 (s, H-C(2,6)), 6.52 (d, J = 12.2 Hz, H-C(7)),
6.59 (d, J = 12.2 Hz, H-C(8)), 7.21-7.26 (m, H-C(2',3',5',6')). 13C NMR (CDCl3, 125 MHz):
δ8.2 (Me-C(4)), 55.5 (MeO-C(3,5)), 104.3 (C(2,6)), 114.0 (C(4)), 128.3 (C(3',5')), 128.4
(C(7)), 130.3 (C(2',6')), 131.4 (C(8)), 132.7 (C(4')), 134.8 (C(1')), 136.0 (C(1)), 158.1
(C(3,5)). Positive ion ESI-HRMS: calcd for C17H18ClO2 [M+H]+, 289.0995; found, 289.0971.
5-(4-Bromostyryl)-1,3-dimethoxy-2-methylbenzene (6g, 7g). Compound 4a (200 mg,
0.394 mmol) was reacted with 4-bromobenzaldehyde 5g (73.7 mg, 0.394 mmol), then
purified by preparative TLC (hexanes/ethyl acetate, 98:2) to afford 6g and 7g. Compound
1
6g was obtained as white solid: 26.2 mg (19.9 %). H NMR (CDCl3, 500 MHz): δ 2.13 (s,
Me-C(4)), 3.88 (s, MeO-C(3,5)), 6.70 (s, H-C(2,6)), 6.99 (d, J = 16.2 Hz, H-C(7)), 7.07 (d, J
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= 16.2 Hz, H-C(8)), 7.37 (d, J = 8.4 Hz, H-C(3',5')), 7.48 (d, J = 8.4 Hz, H-(2',6')). 13C NMR
(CDCl3, 125 MHz): δ8.4 (Me-C(4)), 55.8 (MeO-C(3,5)), 102.0 (C(2,6)), 114.9 (C(4)), 121.1
(C(4')), 126.6 (C(7)), 127.9 (C(3',5')), 130.0 (C(8)), 131.8 (C(2',6')), 135.3 (C(1')), 136.3
(C(1)), 158.5 (C(3,5)). Positive ion EI-HRMS: calcd for C17H18BrO2 [M]+, 332.0411; found,
1
332.0471. Compound 7g was a viscous liquid: 41.8 mg (31.8 %). H NMR (CDCl3, 500
MHz): δ 2.07(s, Me-C(4)), 3.65(s, MeO-C(3,5)), 6.41(s, H-C(2,6)), 6.49 (d, J = 12.2 Hz,
H-C(7)), 6.60 (d, J = 12.1 Hz, H-C(8)), 7.17-7.20 (m, H-C(3',5')), 7.36-7.39 (m, H-(2',6')).
13C NMR (CDCl3, 125 MHz): δ8.2 (Me-C(4)), 55.6 (MeO-C(3,5)), 104.3 (C(2,6)), 114.0
(C(4)), 120.8 (C(4')), 128.4 (C(7)), 130.7 (C(3',5')), 131.3 (C(2',6')), 131.5 (C(8)), 134.7
(C(1')), 136.4 (C(1)), 158.1 (C(3,5)). Positive ion EI-HRMS: calcd for C17H18BrO2 [M]+,
332.0411; found, 3320471.
1,3-Dimethoxy-2-methyl-5-(4-(trifluoromethyl)styryl)benzene (6h, 7h). Compound 4a
(200 mg, 0.394 mmol) was reacted with 4-(trifluoromethyl)ben- zaldehyde 5h (72.3 mg,
0.394 mmol), then purified by preparative TLC (hexanes/acetone, 95:5) to afford 6h and 7h.
Compound 6h was obtained as white solid: 49.4 mg (38.9 %). 1H NMR (CDCl3, 500 MHz): δ
2.15 (s, Me-C(4)), 3.90 (s, MeO-C(3,5)), 6.73 (s, H-C(2,6)), 7.08 (d, J = 16.3 Hz, H-C(7)),
7.17 (d, J = 16.3 Hz, H-C(8)), 7.59~7.63 (overlap, H-C(2',3',5',6')). 13C NMR (CDCl3, 125
MHz): δ 8.4 (Me-C(4)), 55.7 (MeO-C(3,5)), 102.2 (C(2,6)), 115.4 (C(4)), 124.26 (q, J =
271.7 Hz, CF3), 125.6 (q, J = 3.8 Hz, C(3',5')), 126.2 (C(7)), 126.5 (C(2',6')), 129.1 (q, J =
32.4 Hz, C(4')), 131.7 (C(8)), 135.0 (C(1)), 140.9 (q, J = 1.4 Hz, C(1')), 158.5 (C(3,5)).
Positive ion ESI-HRMS: calcd for C18H18F3O2 [M+H]+, 323.1258; found, 323.1278.
Compound 7h was a viscous liquid: 51.8 mg (40.7 %). 1H NMR (CDCl3, 500 MHz): δ 2.08 (s,
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Me-C(4)), 3.62 (s, MeO-C(3,5)), 6.38 (s, H-C(2,6)), 6.60 (d, J = 12.2 Hz, H-C(7)), 6.68 (d, J
= 12.2 Hz, H-C(8)), 7.41 (d, J = 8.2 Hz, H-C(3',5')), 7.52 (d, J = 8.2 Hz, H-C(2',6')). 13C
NMR (CDCl3, 125 MHz): δ 8.1 (Me-C(4)), 55.4 (MeO-C(3,5)), 104.4 (C(2,6)), 114.3 (C(4)),
124.2 (q, J = 271.8 Hz, CF3), 125.1 (q, J = 3.8 Hz, C(3',5')), 128.2 (C(7)), 128.9 (q, J = 32.4
Hz, C(4')), 129.3 (C(2',6')), 132.7 (C(8)), 134.4 (C(1)), 141.4 (q, J = 1.3 Hz, C(1')), 158.1
(C(3,5)). Positive ion ESI-HRMS: calcd for C18H18F3O2 [M+H]+, 323.1258; found, 323.1263.
5-(3,4-Dichlorostyryl)-1,3-dimethoxy-2-methylbenzene (6i, 7i). Compound 4a (200 mg,
0.394 mmol) was reacted with 3,4-dichlorobenzaldehyde 5i (70.4 mg, 0.394 mmol), then
purified by preparative TLC (hexanes/acetone, 95:5) to afford 6i and 7i. Compound 6i was a
1
white solid: 43.0 mg (33.7 %). H NMR (CDCl3, 500 MHz): δ 2.12 (s, Me-C(4)), 3.88 (s,
MeO-C(3,5)), 6.68 (s, H-C(2,6)), 6.94 (d, J = 16.2 Hz, H-C(7)), 7.06 (d, J = 16.2 Hz, H-C(8)),
7.32 (dd, J = 8.3, 2.0 Hz, H-C(6')), 7.41 (d, J = 8.3 Hz, H-C(5')), 7.59 (d, J = 1.9 Hz, H-C(2')).
13C NMR (CDCl3, 125 MHz): δ 8.4 (Me-C(4)), 55.8 (MeO-C(3,5)), 102.1 (C(2,6)), 115.3
(C(4)), 125.3 (C(6')), 125.5 (C(7)), 127.9 (C(8)), 130.5 (C(2')), 130.9 (C(5')), 131.1 (C(4')),
132.8 (C(3')), 134.9 (C(1')), 137.6 (C(1)), 158.5 (C(3,5)). Positive ion ESI-HRMS: calcd for
C17H17Cl2O2 [M+H]+, 323.0606; found, 323.0543. Compound 7i was a viscous liquid: 46.8
mg (36.7 %). 1H NMR (CDCl3, 500 MHz): δ 2.08 (s, Me-C(4)), 3.67 (s, MeO-C(3,5)), 6.43 (s,
H-C(2,6)), 6.44 (d, J = 12.2 Hz, H-C(7)), 6.62 (d, J = 12.2 Hz, H-C(8)), 7.13 (dd, J = 8.3, 1.6
13
Hz, H-C(6')), 7.30 (d, J = 8.3 Hz, H-C(5')), 7.44 (d, J = 1.8 Hz, H-C(2')). C NMR (CDCl3,
125 MHz): δ 8.2 (Me-C(4)), 55.6 (MeO-C(3,5)), 104.3 (C(2,6)), 114.5 (C(4)), 127.0 (C(6')),
128.4 (C(7')), 130.0 (C(8')), 130.7 (C(2',5')), 132.1 (C(4')), 132.5 (C(3')), 134.2 (C(1')), 137.6
(C(1)), 158.1 (C(3,5)). Positive ion ESI-HRMS: calcd for C17H17Cl2O2 [M+H]+, 323.0605;
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found, 323.0601.
5-(2,4-Dimethoxystyryl)-1,3-dimethoxy-2-methylbenzene (6j, 7j). Compound 4a (200 mg,
0.394 mmol) was reacted with 2,4-dimethoxybenzaldehyde 5j (66.8 mg, 0.394 mmol), then
purified by preparative TLC (hexanes/ethyl acetate, 90:10) to afford 6j and 7j. Compound 6j
was obtained as white solid: 31.2 mg (25.2 %). 1H NMR (CDCl3, 500 MHz): δ 2.13 (s,
Me-C(4)), 3.84 (s, MeO-C(4')), 3.89 (s, MeO-C(3,5)), 3.89 (s, MeO-C(2')), 6.50 (d, J = 2.4
Hz, H-C(3')), 6.54 (dd, J = 8.5, 2.4 Hz, H-C(5')), 6.72 (s, H-C(2,6)), 7.01 (d, J = 16.4 Hz,
H-C(7)), 7.35 (d, J = 16.4 Hz, H-C(8)), 7.52 (d, J = 8.5 Hz, H-C(6')). 13C NMR (CDCl3,
125 MHz): δ 8.3 (Me-C(4)), 55.4 (MeO-C(4')), 55.5 (MeO-C(2')), 55.8 (MeO-C(3,5)), 98.5
(C(3')), 101.9 (C(2,6)), 105.0 (C(5')), 113.8 (C(1')), 119.6 (C(4)), 122.5 (C(8)), 127.3 (C(7)),
127.7 (C(6')), 136.7 (C(1)), 158.0 (C(2')), 158.4 (C(3,5)), 160.5 (C(4')). Positive ion
ESI-HRMS: calcd for C19H23O4 [M+H]+, 315.1596; found, 315.1601. Compound 7j was
1
obatained as a white solid: 46.0 mg (37.1 %). H NMR (CDCl3, 500 MHz): δ 2.07 (s,
Me-C(4)), 3.65 (s, MeO-C(3,5)), 3.79 (s, MeO-C(4')), 3.83 (s, MeO-C(2')), 6.35 (dd, J = 8.5,
2.4 Hz, H-C(5')), 6.48 (d, J = 2.4 Hz, H-C(3')), 6.49 (s, H-C(2,6)), 6.52 (d, J = 12.1 Hz,
H-C(7)), 6.61 (d, J = 12.1 Hz, H-C(8)), 7.22 (d, J = 8.5 Hz, H-C(6')). 13C NMR (CDCl3, 125
MHz): δ8.1 (Me-C(4)), 55.4 (MeO-C(4')), 55.5 (MeO-C(2')), 55.5 (MeO-C(3,5)), 98.2 (C(3')),
104.2 (C(5')), 104.4 (C(2,6)), 113.3 (C(1')), 119.0 (C(4)), 124.8 (C(8)), 129.5 (C(7)), 130.8
(C(6')), 135.6 (C(1)), 157.8 (C(3,5)), 158.3 (C(2')), 160.3 (C(4')). Positive ion ESI-HRMS:
calcd for C19H23O4 [M+H]+, 315.1596; found, 315.1600.
5-(3,4-Dimethoxystyryl)-1,3-dimethoxy-2-methylbenzene (6k, 7k). Compound 4a (200
mg, 0.394 mmol) was reacted with 3,4-dimethoxybenzaldehyde 5k (66.2 mg, 0.394 mmol),
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then purified by preparative TLC (hexanes/ethyl acetate, 80:20) to afford 6k and 7k.
Compound 6k was obtained as white solid: 48.3 mg (38.9 %). 1H NMR (CDCl3, 500 MHz):
δ 2.11 (s, Me-C(4)), 3.88 (s, MeO-C(3,5)), 3.90 (s, MeO-C(3')), 3.95 (s, MeO-C(4')), 6.69 (s,
H-C(2,6)), 6.86 (d, J = 8.2 Hz, H-C(5')), 6.95 (d, J = 16.2 Hz, H-C(7)), 7.02 (d, J = 16.2 Hz,
H-C(8)), 7.06 (dd, J = 8.3, 1.9 Hz, H-C(6')), 7.08 (d, J = 1.7 Hz, H-C(2')). 13C NMR (CDCl3,
125 MHz): δ8.3 (Me-C(4)), 55.7 (MeO-C(3,5)), 55.8 (MeO-C(3')), 55.9 (MeO-C(4')), 101.8
(C(2,6)), 108.7 (C(2')), 111.3 (C(5')), 114.2 (C(4)), 119.8 (C(6')), 127.4 (C(8)), 127.6 (C(7)),
130.5 (C(1')), 135.9 (C(1)), 148.9 (C(4')), 149.1 (C(3')), 158.4 (C(3,5)). Positive ion
ESI-HRMS: calcd for C19H23O4 [M+H]+, 315.1596; found, 315.1595. Compound 7k was
obtained as white solid: 60.2 mg (48.6 %). 1H NMR (CDCl3, 500 MHz): δ 2.06 (s, Me-C(4)),
3.66 (s, MeO-C(3,5)), 3.67 (s, MeO-C(3')), 3.85 (s, MeO-C(4')), 6.49 (s, H-C(2,6)), 6.50 (s,
H-C(7,8)), 6.76 (d, J = 8.2 Hz, H-C(5')), 6.85 (d, J = 1.9 Hz, H-C(2')), 6.87 (dd, J = 8.2, 2.0
Hz, H-C(6')). 13C NMR (CDCl3, 125 MHz): δ8.1 (Me-C(4)), 55.6 (MeO-C(3,5)), 55.6
(MeO-C(3')), 55.8 (MeO-C(4')), 104.3 (C(2,6)), 110.8 (C(2')), 111.9 (C(5')), 113.5 (C(4)),
121.9 (C(6')), 129.4 (C(8)), 129.5 (C(7)), 130.1 (C(1')), 135.5 (C(1)), 148.1 (C(4')), 148.4
(C(3')), 158.0 (C(3,5)). Positive ion ESI-HRMS: calcd for C19H23O4 [M+H]+, 315.1596;
found, 315.1602.
5-(3,5-Dimethoxystyryl)-1,3-dimethoxy-2-methylbenzene (6l, 7l). Compound 4a (200 mg,
0.394 mmol) was reacted with 3,5-dimethoxybenzaldehyde 5l (65.5 mg, 0.394 mmol), then
purified by preparative TLC (hexanes/ethyl acetate, 90:10) to afford 6l and 7l. Compound 6l
as a white solid: 26.0 mg (20.9 %). 1H NMR (CDCl3, 500 MHz): δ 2.13 (s, Me-C(4)), 3.84 (s,
MeO-C(3,5)), 3.89 (s, MeO-C(3',5')), 6.41 (t, J = 2.2 Hz, H-C(4')), 6.70 (d, J = 2.2 Hz,
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H-C(2',6')), 6.71 (s, H-C(2,6)), 7.01 (d, J = 16.2 Hz, H-C(7)), 7.08 (d, J = 16.2 Hz, H-C(8)).
13C NMR (CDCl3, 125 MHz): δ 8.3 (Me-C(4)), 55.4 (MeO-C(3,5)), 55.7 (MeO-C(3',5')),
100.0 (C(4')), 102.0 (C(2',6')), 104.5 (C(2,6)), 114.7 (C(4)), 127.8 (C(7)), 129.7 (C(8)), 135.5
(C(1)), 139.4 (C(1')), 158.4 (C(3,5)), 161.0 (C(3',5')). Positive ion ESI-HRMS: calcd for
C19H23O4 [M+H]+, 315.1596; found, 315.1595. Compound 7l was a white solid: 46.0 mg
1
(37.1 %). H NMR (CDCl3, 500 MHz): δ 2.07 (s, Me-C(4)), 3.66 (s, MeO-C(3,5)), 3.69 (s,
MeO-C(3',5')), 6.34 (t, J = 2.3 Hz, H-C(4')), 6.49 (d, J = 2.4 Hz, H-C(2,6,2',6')), 6.53 (d, J =
12.3 Hz, H-C(7)), 6.56 (d, J = 12.2 Hz, H-C(8)). 13C NMR (CDCl3, 125 MHz): δ 8.1
(Me-C(4)), 55.2 (MeO-C(3,5)), 55.5 (MeO-C(3',5')), 99.7 (C(4')), 104.5 (C(2',6')), 106.7
(C(2,6)), 113.8 (C(4)), 129.7 (C(7)), 131.0 (C(8)), 135.0 (C(1)), 139.5 (C(1')), 157.9 (C(3,5)),
160.6 (C(3',5')). Positive ion ESI-HRMS: calcd for C19H23O4 [M+H]+, 315.1596; found,
315.1599.
5-(2,6-Dimethoxy-4-methylstyryl)-1,3-dimethoxy-2-methylbenzene (6m, 7m). Compound
4a (200 mg, 0.394 mmol) was reacted with 2,6-dimethoxy-4- methylbenzaldehyde 5m (73.2
mg, 0.394 mmol), then purified by preparative TLC (hexanes/acetone, 90:10) to afford 6m
1
and 7m. Compound 6m was obtained as white solid: 20.5 mg (15.8 %). H NMR (CDCl3,
500 MHz): δ 2.12 (s, Me-C(4)), 2.38 (s, Me-C(4')), 3.89 (s, MeO-C(3,5)), 3.90 (s,
MeO-C(2',6')), 6.43 (s, H-C(2,6)), 6.72 (s, H-C(3',5')), 7.38 (d, J = 16.6 Hz, H-C(7)), 7.51 (d,
J = 16.6 Hz, H-C(8)). 13C NMR (CDCl3, 125 MHz): δ 8.3 (Me-C(4)), 22.2 (Me-C(4')), 55.8
(MeO-C(3,5)), 55.8 (MeO-C(2',6')), 101.9 (C(2,6)), 105.0 (C(3',5')), 112.1 (C(1')), 113.5
(C(4)), 119.2 (C(7)), 132.0 (C(8)), 137.9 (C(1)), 138.4 (C(4')), 158.3 (C(3,5)), 158.4 (C(2',6')).
Positive ion ESI-HRMS: calcd for C20H25O4 [M+H]+, 329.17528; found, 329.17524.
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Compound 7m was a white solid: 73.5 mg (56.8 %). 1H NMR (CDCl3, 500 MHz): δ 2.05 (s,
Me-C(4)), 2.36 (s, Me-C(4')), 3.59 (s, MeO-C(3,5)), 3.65 (s, MeO-C(2',6')), 6.38 (s,
H-C(2,6)), 6.40 (s, H-C(3',5')), 6.42 (d, J = 12.0 Hz, H-C(7)), 6.67 (d, J = 12.0 Hz, H-C(8)).
13C NMR (CDCl3, 125 MHz): δ 8.1 (Me-C(4)), 22.1 (Me-C(4')), 55.3 (MeO-C(3,5)), 55.6
(MeO-C(2',6')), 103.6 (C(2,6)), 104.8 (C(3',5')), 112.8 (C(1')), 113.1 (C(4)), 121.0 (C(7)),
132.0 (C(8)), 136.7 (C(1)), 138.8 (C(4')), 157.5 (C(3,5)), 157.6 (C(2',6')). Positive ion
ESI-HRMS: calcd for C20H25O4 [M+H]+, 329.1752; found, 329.1768.
1,3-Dimethoxy-5-(4-((3-methylbut-2-en-1-yl)oxy)styryl)benzene
(6n,
7n).
(3,5-
Dimethoxybenzyl)triphenylphosphonium 4b (379.5 mg, 0.769 mmol) was reacted with
4-((3-methylbut-2-en-1-yl)oxy)benzaldehyde 5n (154.0 mg, 0.769 mmol), then purified by
preparative TLC (hexanes/ethyl acetate, 97:3) to afford 6n and 7n. Compound 6n was a white
1
solid: 56.4 mg (22.6 %). H NMR (CDCl3, 500 MHz): δ 1.76 (s, H-C(5'')), 1.81 (s, H-C(4'')),
3.83 (s, MeO-C(3,5)), 4.53 (d, J = 6.7 Hz, H-C(1'')), 5.49-5.53 (m, H-C(2'')), 6.38 (t, J = 2.2
Hz, H-C(4)), 6.65 (d, J = 2.2 Hz, H-C(2,6)), 6.89-6.92 (overlap, H-C(7,3',5')), 7.04 (d, J =
16.2 Hz, H-C(8)), 7.44 (d, J = 8.7 Hz, H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ 18.2
(C(5'')), 25.8 (C(4'')), 55.3 (MeO-C(3,5)), 64.8 (C(1'')), 99.6 (C(4)), 104.3 (C(2,6)), 114.9
(C(3',5')), 119.6(C(2'')), 126.5 (C(7)), 127.8 (C(2',6')), 128.8 (C(8)), 129.8 (C(1')), 138.3
(C(3'')), 139.7 (C(1)), 158.7 (C(4')), 161.0 (C(3,5)). Positive ion ESI-HRMS: calcd for
C21H25O3 [M+H]+, 325.1803; found, 325.1842. Compound 7n was a viscous liquid: 163.2 mg
1
(65.4 %). H NMR (CDCl3, 500 MHz): δ 1.65 (s, H-C(5'')), 1.71 (s, H-C(4'')), 3.58 (s,
MeO-C(3,5)), 4.40 (d, J = 6.7 Hz, H-C(1'')), 5.38-5.40 (m, H-C(2'')), 6.24 (t, J = 2.0 Hz,
H-C(4)), 6.34-6.37 (overlap, H-C(2,6,7)), 6.44 (d, J = 12.3 Hz, H-C(8)), 6.70 (d, J = 8.6 Hz,
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H-C(3',5')), 7.13 (d, J = 8.7 Hz, H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ 18.1 (C(5'')),
25.7 (C(4'')), 55.1 (MeO-C(3,5)), 64.6 (C(1'')), 99.6 (C(4)), 106.5 (C(2,6)), 114.2 (C(3',5')),
119.6 (C(2'')), 128.5 (C(7)), 129.4 (C(8)), 130.1 (C(1')), 130.1 (C(2',6')), 138.0 (C(3'')), 139.4
(C(1)), 158.0 (C(4')), 160.5 (C(3,5)). Positive ion ESI-HRMS: calcd for C21H25O3 [M+H]+,
325.1803; found, 325.1804.
4-(3,5-Dimethoxystyryl)-2-(3-methylbut-2-en-1-yl)phenol (11, 12). Compound 4b (453.9
mg,
0.92
mmol)
was
reacted
with
4-((tert-butyldimethylsilyl)oxy)-3-
(3-methylbut-2-en-1-yl)benzaldehyde 9 (280 mg, 0.92 mmol), then purified by flash
chromatography (hexanes/ethyl acetate, 97:3) to afford 270 mg (66.9 % yield) of a mixture of
cis and trans stilbenes 10 (Scheme 3). Due to difficulty encountered in separating the two
isomers, deprotection of TBS group was performed without isolation of the isomers. To a
solution of 10 (270 mg, 0.61 mmol) in anhydrous THF was added tetrabutylammnonium
fluoride (TBAF) (800 μL, 0.80 mmol). The solution was stirred for 45 min, poured into water,
extracted with dichloromethane and dried over anhydrous MgSO4. The solvent was removed
under reduced pressure, and the crude product was purified by preparative TLC
(hexanes/ethyl acetate, 80:20) to afford to afford 11 and 12. Compound 11 was obtained as a
1
viscous liquid: 84.7 mg (42.4 %). H NMR (CDCl3, 500 MHz): δ 1.80 (s, H-C(5'')), 1.81 (s,
H-C(4'')), 3.39 (d, J = 7.2 Hz, H-C(1'')), 3.83 (s, MeO-C(3,5)), 5.31 (brs, OH), 5.33-5.37 (m,
H-C(2'')), 6.39 (t, J = 2.2 Hz, H-C(4)), 6.66 (d, J = 2.2 Hz, H-C(2,6)), 6.80 (d, J = 8.0 Hz,
H-C(5')), 6.89 (d, J = 16.2 Hz, H-C(7)), 7.02 (d, J = 16.2 Hz, H-C(8)), 7.26-7.29 (overlap,
H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ 17.9 (C(5'')), 25.8 (C(4'')), 29.8 (C(1'')), 55.4
(MeO-C(3,5)), 99.6 (C(4)), 104.3 (C(2,6)), 116.0 (C(5')), 121.6 (C(2'')), 125.8 (C(6')), 126.3
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(C(7)), 127.1 (C(2')), 128.4 (C(3')), 129.0 (C(8)), 130.0 (C(1')), 134.9 (C(3'')), 139.8 (C(1)),
154.3 (C(4')), 160.9 (C(3,5)). Positive ion ESI-HRMS: calcd for C21H25O3 [M+H]+, 325.1803;
1
found, 325.1800. Compound 12 was a viscous liquid: 35.8 mg (17.9 %). H NMR (CDCl3,
500 MHz): δ 1.72 (s, H-C(5'')), 1.74 (s, H-C(4'')), 3.26 (d, J = 7.2 Hz, H-C(1'')), 3.68 (s,
MeO-C(3,5)), 5.21-5.25 (m, H-C(2'')), 5.27 (s, OH), 6.34 (t, J = 2.3 Hz, H-C(4)), 6.44 (d, J =
12.2 Hz, H-C(7)), 6.47 (d, J = 2.2 Hz, H-C(2,6)), 6.51 (d, J = 12.2 Hz, H-C(8)), 6.66 (d, J =
8.1 Hz, H-C(5')), 7.03-7.05 (overlap, H-C(2',6')). 13C NMR (CDCl3, 125 MHz): δ 17.8
(C(5'')), 25.7 (C(4'')), 29.3 (C(1'')), 55.2 (MeO-C(3,5)), 99.7 (C(4)), 106.6 (C(2,6)), 115.3
(C(5')), 121.6 (C(2'')), 126.6 (C(6')), 128.2 (C(7)), 128.5 (C(2')), 129.6 (C(3')), 130.4 (C(8)),
130.8 (C(1')), 134.4 (C(3'')), 139.6 (C(1)), 153.4 (C(4')), 160.5 (C(3,5)). Positive ion
ESI-HRMS: calcd for C21H25O3 [M+H]+, 325.1803; found, 325.1802.
(E)-4-(3,5-dimethoxy-4-methylstyryl)phenol (16). Compound 4a (326.5 mg, 0.643 mmol)
was reacted with 4-((tert-butyldimethylsilyl)oxy)benzaldehyde 13 (152.0 mg, 0.643 mmol),
then purified by flash chromatography (hexanes/ethyl acetate, 97:3) to afford 185.7 mg
(75.0 % yield) of a mixture of cis and trans stilbenes 14 (Scheme 4). To a solution of 14
(185.7 mg, 0.483 mmol) in THF was added TBAF (628 μl, 0.628 mmol). The solution was
stirred for 45 min, poured into water, and extracted with dichloromethane. The solvent was
removed under reduced pressure, and the crude product was purified by preparative TLCs
(hexanes/ethyl acetate, 80:20) to afford (E)-4-(3,5-dimethoxy-4-methylstyryl)phenol 16 as a
light-yellow solid (98.2 mg, 75.2 %). 1H NMR (CDCl3, 500 MHz): δ 2.11 (s, Me-C(4)),
3.88 (s, MeO-C(3,5)), 5.11 (brs, OH), 6.68 (s, H-C(2,6)), 6.83 (dd, J = 8.6, 2.0 Hz, H-C(3',5')),
6.93 (d, J = 16.2 Hz, H-C(7)), 7.01 (d, J = 16.2 Hz, H-C(8)), 7.41 (d, J = 8.5 Hz, H-C(2',6')).
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13C NMR (CDCl3, 125 MHz): δ 8.3 (Me-C(4)), 55.8 (MeO-C(3,5)), 101.8 (C(2,6)), 114.2
(C(4)), 115.6 (C(3',5')), 127.2 (C(7)), 127.4 (C(8)), 127.8 (C(2',6')), 130.3 (C(1')), 136.0
(C(1)), 155.2 (C(4')), 158.4 (C(3,5)). Negative ion ESI-HRMS: calcd for C17H17O3 [M–H]–,
269.1177; found, 269.1197.
(E)-5-(4-hydroxystyryl)-2-methylbenzene-1,3-diol (17). To a cold solution (-20 °C) of 16
(30 mg, 0.111 mmol) in anhydrous CH2Cl2 was added, dropwise, BBr3 (153 mg, 0.611 mmol)
(Scheme 4). The mixture was allowed to warm to room temperature, and was stirred for 10 h.
The reaction was quenched by ice-water, and extracted with ethyl acetate. The organic phase
was washed with water and dried over anhydrous Na2SO4. After removal of the solvent under
reduced pressure, the crude product was purified by preparative TLC (hexanes/ethyl acetate,
60:40) to afford (E)-5-(4-hydroxystyryl)-2-methylbenzene-1,3-diol 17 (9.0 mg, 33.5 % ) as a
white solid. 1H NMR (Acetone-d6, 500 MHz): δ 2.09 (s, Me-C(4)), 6.60 (s, H-C(2,6)), 6.83
(d, J = 8.6 Hz, H-C(3',5')), 6.83 (d, J = 16.3 Hz, H-C(7)), 6.92 (d, J = 16.3 Hz, H-C(8)), 7.38
13
(d, J = 8.6 Hz, H-C(2',6')), 8.09 (s, OH-C(3,5)), 8.46 (s, OH-C(4')). C NMR (Acetone-d6,
125 MHz): δ 7.8 (Me-C(4)), 104.6 (C(2,6)), 110.2 (C(4)), 115.5 (C(3',5')), 126.1 (C(7)), 127.1
(C(8)), 127.6 (C(2',6')), 129.2 (C(1')), 136.1 (C(1)), 156.4 (C(3,5)), 157.1(C(4')). Positive ion
ESI-HRMS: calcd for C15H15O3 [M+H]+, 243.1021; found, 243.1028.
4-((Tert-butyldimethylsilyl)oxy)-3-(3-methylbut-2-en-1-yl)benzaldehyde (9). To
a
solution of 4-hydroxy-3-(3-methylbut-2-en-1-yl)benzaldehyde 8 (200 mg, 1.0 mmol), was
added imidazole (96.3 mg, 1.4 mmol) in DMF (10 ml) and tertbutyldimethylsilyl chloride
(202 mg, 1.3 mmol). The solution was left stirring for 20 h at room temperature, and then the
mixture was poured into water and extracted with ethyl acetate. The organic phase was
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combined and dried over anhydrous MgSO4, and the solvent was removed under reduced
pressure. The crude product was purified by flash chromatography (hexanes/ethyl acetate,
95:5) to give 9 (283.2 mg, 90.3%) as a viscous liquid. 1H NMR (CDCl3, 500 MHz): δ 0.29 (s,
Me-Si), 1.02 (s, (CH3)3C-Si), 1.70 (s, H-C(5')), 1.77 (s, H-C(4')), 3.34 (d, J = 7.2 Hz, H-C(1')),
5.29-5.33 (m, H-C(2')), 6.88 (d, J = 8.3 Hz, H-C(5)), 7.62 (dd, J = 8.3, 2.2 Hz, H-C(6)), 7.67
13
(d, J = 2.1 Hz, H-C(2)), 9.85 (s, CHO). C NMR (CDCl3, 125 MHz): δ 14.1 (Me-Si), 17.9
(C(5')), 18.3 (C(4')), 25.7 ((CH3)3), 25.8 (C(1')), 28.4 (C-Si), 118.4 (C(5)), 121.5 (C(2')),
129.5 (C(6)), 130.1 (C(3')), 131.4 (C(1)), 133.3 (C(2)), 133.6 (C(3)), 159.3 (C(4)), 191.2
(CHO). Positive ion ESI-HRMS: calcd for C18H29O2Si [M+H]+, 305.1936; found, 305.1895.
(3,5-Dimethoxy-4-methylbenzyl)triphenylphosphonium Bromide (4a). To a solution of
5-(bromomethyl)-1,3-dimethoxy-2-methylbenzene (500 mg, 1.938 mmol) in toluene (10 ml)
was added triphenylphosphine (565.4 mg, 2.134 mmol). The solution was heated at reflux for
6 h. The resulting precipitate was collected and recrystallized from ethanol as a white solid
1
(930.0 mg, 94.5%). H NMR (DMSO-d6, 500 MHz): δ 1.89 (s, Me-C(4)), 3.39 (s,
MeO-C(3,5)), 5.12 (d, J = 15.4 Hz, CH2-C(1)), 6.22 (s, H-C(2,6)), 7.67-7.76 (m,
H-C(2',3',5',6',2'',3'',5'',6'',2''',3''',5''',6''')), 7.89 (t, J = 7.3 Hz, H-C(4',4'',4''')). 13C NMR
(DMSO-d6, 125 MHz): δ 8.5 (d, J = 1.3 Hz, Me-C(4)), 29.4 (d, J = 46.8 Hz, CH2), 55.8
(MeO-C(3,5)), 107.0 (d, J = 5.7 Hz, C(2,6)), 113.4 (d, J = 4.3 Hz, C(4)), 118.4 (d, J = 85.5
Hz, C(4',4'',4''')), 126.5 (d, J = 8.7 Hz, C(1)), 130.5 (d, J = 12.4 Hz, C(3',5',3'',5'',3''',5''')),
134.6 (d, J = 9.8 Hz, C(2',6',2'',6'',2''',6''')), 135.5 (d, J = 2.9 Hz, C(1',1'',1''')), 157.9 (d, J = 3.5
Hz, C(3,5)). Positive ion ESI-HRMS: calcd for C28H28O2P [M–Br]+, 427.1826; found,
427.1913.
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Aedes aegypti larvicidal assay. All of the analogs were screened for activity against Ae.
aegypti larvae following a bioassay system described in Ali et al., 2014 [24]. Ae. aegypti
larvae used in these studies were from a laboratory colony maintained at the Mosquito and
Fly Research Unit at the Center for Medical, Agricultural and Veterinary Entomology,
USDA-ARS, Gainesville, Florida. For larval bioassays, the eggs were hatched and the larvae
were maintained at a temperature of 27 2˚C and 60 10 % RH with a photoperiod regimen
of 12:12 h (L: D). Five 1-d-old Ae. aegypti larvae were added in a droplet of water to each
well of 24-well plates (BD Labware, Franklin Lakes, NJ) by use of a disposable 22.5 cm
Pasteur pipette. Fifty microliters of larval diet (2% slurry of 3:2 beef liver powder (Now
Foods, Bloomingdale, Illinois) and Brewer’s yeast (Lewis Laboratories Ltd., Westport, CT)
was added to each well by using a Finnpipette stepper (Thermo Fisher, Vantaa, Finland). All
chemicals tested were diluted in dimethyl sulfoxide (DMSO). Eleven microliters of the test
chemical was added to the labeled wells, while 11 µL of DMSO was added to control
treatments. After the treatment application, the plates were swirled in clockwise and
counterclockwise motions and front and back and side to side five times to ensure even
mixing of the chemicals. Larval mortality was recorded 24-h post treatment. Larvae that
showed no movement in the well after manual disturbance of the water were recorded as dead.
Three dosages, 100, 50 and 25 ppm, were used in the screening bioassay to determine the
larvicidal activity and each treatment was replicated twice. A series of 4 dosages ranging
between 50 and 6.25 ppm was used to determine the dose-response of 11 and 12, which
showed high activity in the screening bioassay. Each treatment was replicated 10 times.
Statistical Analyses. LC50 values for larvicidal data were calculated by using SAS, Proc
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Probit [25]. Control mortality was corrected by using Abbott’s formula [16].
Testing for activity against larvae and adult pyrethroid-resistant and susceptible
strains of Aedes aeypti. Further tests were performed on 11 and 12 to determine their effect
on larvae and adult mosquitoes. The compounds were tested in the standard bioassays using
the Orlando (ORL), pesticide-susceptible strain and the Puerto Rico (PR), pyrethroid-resistant
strain of Ae. aegypti. The Orlando strain has been in continuous colony since 1952 with no
pesticide exposure while the resistant PR strain was colonized from egg papers collected near
San Juan, PR in June, 2012. The PR strain is available as a resistant reference strain through
BEIResources/CDC [27]. Mortality was determined in the larval assays at four different
concentrations (1, 0.5, 0.25, and 0.1μg/μl) and three concentrations for adults (6.25%,
3.125% and 1.56%). Mortality was recorded 24 hours post application and in all assays a
negative solvent and a positive control using permethrin were utilized, tests were done I
triplicate (Tables 2 and 3). Assays were conducted according to published procedures [28].
Testing for activity against Meloidogyne incognita. Two types of assays were used to
investigate activity against M. incognita: A) eggs were immersed in solutions of the test
compounds, and B) second-stage juveniles (J2) previously hatched from eggs immersed in
the test solutions. These assays were conducted with procedures similar to those in Meyer et
al. [29]. Meloidogyne incognita race 1 (originally isolated in 2013 from a field in Maryland)
was grown in the greenhouse on pepper (Capsicum annuum L.) cultivar PA-136. Egg
masses were hand-picked from plant roots, rinsed three times with sterile distilled water, and
agitated in 0.6% sodium hypochlorite for 3½ min to separate and surface-sterilize eggs. The
eggs were rinsed in sterile distilled water and stored overnight at 4°C prior to use in egg
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immersion assays. To collect J2 for direct immersion into the test compounds, sterilized
eggs were placed into a hatching chamber comprised of a Spectra/Mesh Nylon Filter
(openings 25 µm in diameter; Spectrum Laboratories Inc., Rancho Dominguez, CA) in an
autoclaved dish. J2 that passed through the filter within 3 d were used immediately for
assays.
The assays were conducted in 96-well polystyrene plates. For the assays with
immersed eggs, each well received ca. 50 eggs in 35 μl sterile deionized water (SDW). For
assays with previously hatched J2, a suspension of ca. 50 J2 in 35 μL SDW was placed into
each well. Each well then received 165 μl of treatment or control. The solvent used to
dissolve the test chemicals was a 1:1:1 mixture referred to herein as CTD: comprised of equal
parts Cremophor® EL Castor Oil (BASF Corporation, Vandalia, IL), Tween 80®
(Sigma-Aldrich, St. Louis, MO), and dimethyl sulfoxide (DMSO; Sigma-Aldrich). A high
and low rate of each compound was tested. After addition to the nematode suspensions in the
wells, the low and high rates were 83.3 μg/mL and 166.7 μg/mL of each test compound,
respectively, dissolved in 0.5% and 1.0% of each of the three combined solvents. Controls
were CTD equivalent to the low and high rates, and SDW. Because of the large number of
treatments, the test compounds were divided into two groups for the egg immersion assays.
The compounds tested in egg immersion Assay 1 were 3, 6a, 6e, 6f, 6g, 6n, 7a, 7c, , 7e, 7f,
7n, 11, 12, 16, and 17. The compounds tested in egg immersion Assay 2 were 6b, 6h, 6i, 6j,
6k, 6l, 7b, 7g, 7h, 7i, 7j, 7k, 7l, and 7m. Test compounds used for assays with previously
hatched J2 were selected based on results with the egg assays. The five compounds tested
were 6b, 6f, 6g, 6i, and 7k, high and low rates. Assays were then repeated with 6g and 7k,
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high rates.
Each polystyrene culture plate was covered with a plastic adhesive sheet (Excel
Scientific, Inc., Victorville CA) and incubated at 25°C. Ten wells (water controls) or five
wells (all other treatments) were used per treatment in each assay. In the egg immersion
assays, total numbers of hatched J2, and numbers of mobile and immobile J2, were counted
after 2 d and 7 d incubation in the test compounds. In the first assay with previously
hatched J2, the numbers of mobile and immobile J2 were counted after 1 and 2 d incubation,
the treatments removed and replaced with a SDW rinse, and 2 d later the mobile vs. immobile
J2 counted again. In the assays with 6g and 7k, high rates, counts were made on Days 1, 2,
4 and 6, without a SDW rinse.
Statistical methods. Data from the M. incognita assays were analyzed with the
statistical package JMP 11.2.0 (SAS Institute, Cary, NC). Differences among numbers of
hatched J2 in each treatment, and among percentage of mobile J2 per treatment (number
mobile J2/total J2 × 100), were determined by ANOVA, and means were compared using
Tukey Kramer’s adjustment for multiple comparisons (P < 0.05). For percent mobile J2 on
Day 2 in egg immersion Assays 1 and 2, and Day 4 with the five compounds in the J2
immersion assay, data were log10(x+1)-transformed prior to analysis. Data presented are
non-transformed means.
Assay against plant-pathogenic fungi. The analogs were tested for activity against
Botrytis cinerea Pers.:Fr, Colletotrichum acutatum Simmonds, C. fragariae Brooks, C.
gloeosporioides (Penz.) Penz. & Sacc. in Penz., and Fusarium oxysporum Schlechtend:Fr
using a direct bioautography method as previously described [30, 31]. Technicial grade
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commercial fungicide standards benomyl, cyprodinil, azoxystrobin, and captan were used at
0.9 to 1.61 µg/µl concentrations in 95% ethanol. After sample application, each TLC plate
was subsequently sprayed with a spore suspension (3.0 x 105 spores/ml) of the fungus of
interest and incubated in a moisture chamber for 4 days at 26°C with a 12 h photoperiod.
Clear zones of fungal growth inhibition on the TLC plate indicated the presence of antifungal
constituents in each extract or pure compound. The test compounds were prepared at 2 mM
solution in 95% ethanol, and 4 µl were spotted on the plates (2 µl of the positive controls).
Assays againt human-associated microorganisms. All organisms were obtained from
the American Type Culture Collection (Manassas, VA) and included the fungi Candida
albicans ATCC 90028, C. glabrata ATCC 90030, C. krusei ATCC 625), Cryptococcus
neoformans ATCC 90113, and Aspergillus fumigatus ATCC 204305, and the bacteria
Staphylococcus aureus ATCC 29213, methicillin-resistant S. aureus ATCC 33591 (MRSA),
Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, and Mycobacterium
intracellulare ATCC 23068. All organisms were tested using modified versions of the CLSI
(formerly NCCLS) methods. For all organisms excluding M. intracellulare and A. fumigatus,
optical density is used to monitor growth [32], [33]. Medium supplemented with 5% Alamar
Blue™ (BioSource International, Camarillo, CA) was utilized for growth detection of M.
intracellulare [34], [35] and A. fumigatus [36]. Samples (dissolved in DMSO) were
serially-diluted in 20% DMSO/saline and transferred (10µL) in duplicate to 96 well
flat-bottom microplates. Inocula were prepared by correcting the OD630 of microbe
suspensions in incubation broth [RPMI 1640/0.2% dextrose/0.03% glutamine/MOPS @ pH
6.0 (Cellgro) for Candida spp., Sabouraud Dextrose for C. neoformans, cation-adjusted
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