Structure-activity relationship of karrikin germination stimulants

Article abstract of DOI:10.1021/jf101690a

Karrikins (2H-furo[2,3-c]pyran-2-ones) are potent smoke-derived germination promoters for a diverse range of plant species but, to date, their mode of action remains unknown. This paper reports the structure-activity relationship of numerous karrikin anal

Full text of DOI:10.1021/jf101690a

8612 J. Agric. Food Chem. 2010, 58, 8612–8617
DOI:10.1021/jf101690a
Structure-Activity Relationship of Karrikin Germination
Stimulants
†
G
AVIN R. FLEMATTI,*,† ADRIAN CAFFIDI,† ETHAN D. GODDARD-BORGER
S ,
†
,
C
HARLES H. HEATH,† DAVID C. NELSON,† LUCY E. COMMANDER
†
,
‡,§ ROBERT V. STICK
†
K
INGSLEY W. DIXON
,
‡,§ STEVEN M. SMITH
,
AND
EMILIO L. GHISALBERTI
†School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia,
Crawley, WA 6009, Australia, ‡Kings Park and Botanic Garden, West Perth, WA 6005, Australia, and
§School of Plant Biology, The University of Western Australia, Crawley, WA 6009, Australia
Karrikins (2H-furo[2,3-c]pyran-2-ones) are potent smoke-derived germination promoters for a
diverse range of plant species but, to date, their mode of action remains unknown. This paper
reports the structure-activity relationship of numerous karrikin analogues to increase understanding
of the key structural features of the molecule that are required for biological activity. The results
demonstrate that modification at the C5 position is preferred over modification at the C3, C4, or C7
positions for retaining the highest bioactivity.
KEYWORDS: Karrikinolide; karrikin; seed germination; seed dormancy; smoke; germination stimulant
INTRODUCTION
karrikinolide-stimulated germination. Early work demonstrated
that smoke could influence the way that seeds respond to light
and gibberellic acid (GA). For example, smoke substitutes for
light in the germination of light-sensitive lettuce seeds (Lactuca
sativa L. cv. Grand Rapids) (16), and germination is comparable
to the results obtained when seeds are treated with GA3.
Furthermore, smoke-induced germination in cv. Grand Rapids
is reduced by exposure to gibberellin biosynthesis inhibitors such
as paclobutrazol (17). Likewise, it has been shown that karriki-
nolide can act in a similar fashion to GA3 in promoting the
germination of light-sensitive Asteracea species (7) and that both
light and gibberellin biosynthesis are required for karrikinolide to
elicit germination in seeds of primary dormant Arabidopsis
thaliana (18, 19). Previous studies have suggested that smoke,
and now karrikinolide, may promote germination by stimulating
GA biosynthesis and reduce levels of abscisic acid (ABA) (20), an
inhibitor of germination (21). However, analysis of GA4 and
ABA levels in A. thaliana following treatment with karrikinolide
did not support this hypothesis. GA4 and ABA levels did not
change relative to control levels during the pregermination
period (18).
Smoke obtained from the burning of plant material promotes
the germination of seeds from at least 1200 plant species hailing
from over 80 genera (1). In 2004, the structure of the major
component of smoke responsible for promoting germination was
elucidated (2, 3). This compound was identified as 3-methyl-2H-
furo[2,3-c]pyran-2-one, 1 (Figure 1).
Recently, five analogues of 1 have been identified in smoke and
demonstrated to have similar, albeit less potent, germination-
promoting activity (4). This collection of compounds now carry
the common name “karrikin”, derived from karrik, the tradi-
tional word for smoke in the language of the Aboriginal Noongar
people, endemic to southwestern Western Australia, where this
discovery was made. The most active karrikin, compound 1, is
considered to be the parent compound and carries the name
karrikinolide or KAR1. The karrikins represent a new class of
naturally occurring seed germination stimulants that are active in
a number of species at concentrations as low as 1 nM (2).
Since the discovery of karrikinolide, there has been significant
interest in its application within the agricultural and horticultural
industries (1,5,6). Karrikinolide has proven to be highly effective
at promoting seed germination of species useful for land restora-
tion programs (7-10) and has been shown to promote the
germination of various agricultural weeds (11, 12). Furthermore,
karrikinolide can increase seedling vigor in commercially relevant
species such as maize (13) and rice (14) and may even improve
yields in tomatoes (15).
To further explore the mode of action of karrikinolide, a
number of techniques could be used. The use of various probes
to label molecules of biological interest has become a popular
At present, the mechanism by which karrikinolide promotes
seed germination is unknown, although a number of physiologi-
cal studies have been conducted to investigate smoke- and/or
*Corresponding author (phone þ61 8 6488 4461; fax þ61 8 6488
7330; e-mail gavin.flematti@uwa.edu.au).
Figure 1. Structure of karrikinolide (1) and various C3-substituted karrikins.
pubs.acs.org/JAFC
Published on Web 07/09/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 15, 2010 8613
approach in chemical biology. However, labeling of 1 would be
useful only if the resulting compound retained activity as a
germination promoter. Thus, some knowledge of structure-
activity relationships for the karrikins would aid in further mode
of action studies. Up to this point, only a small number of
karrikinolide derivatives have been prepared and tested as ger-
mination stimulants (22). Here, the germination activity of a
range of new and previously prepared karrikin analogues is
described, and some emerging structure-activity relationships
are outlined.
afforded the butenolide 20 as pale yellow needles (50 mg, 4%): 111-
113 °C; δH (600 MHz) 7.45 (s, 1H, H7), 7.11 (s, 1H, H5), 2.60 (q, J8,9
=
7.3 Hz, 2H, H8), 2.10 (s, 3H), 1.25 (t, 3H, H9); δC (150.9 MHz) 171.7 (C2),
144.1 (C5), 141.5, 139.8 (C3a, C7a), 126.5 (C7), 120.1 (C4), 100.1
(C3), 21.5 (C8), 13.8 (C9), 8.9 (C3b); m/z (EI) 178.0636, (M)þ requires
178.0630.
Dihydro-5-ethyl-6-isopropoxy-2H-pyran-3-(4H)-one (21). Tin(IV) chloride
(1.0 M in dichloromethane, 300 μL, 0.3 mmol) and isopropanol (3.0 mL)
were added to the acetate 19 (23) (1.0 g, 5.4 mmol) in dichloromethane
(50 mL), and the resulting solution was left to stand (3 h). The reaction was
quenched with a saturated sodium bicarbonate solution (50 mL) followed
by the usual workup (ethyl acetate) and flash chromatography (10% ethyl
acetate-hexane) to yield 21 as a colorless oil (500 mg, 50%, diastereo-
MATERIALS AND METHODS
mixture 5:7): δH (600 0MHz) 4.92 (d, J5,6 = 2.8 Hz, 1H, H6), 4.77 (d,
General. 1H and 13C nuclear magnetic resonance (NMR) spectra were
obtained on a Bruker ARX500 (500 MHz for δH and 125.7 MHz for δC) or
on a Bruker AV600 (600 MHz for δH and 150.9 MHz for δC) spectrometer.
Unless otherwise stated, deuterochloroform (CDCl3) was used as the
solvent with residual CHCl3 (δH 7.26) or CDCl3 (δC 77.0) being employed
as internal standard. Spectra run in hexadeuteroacetone ((CD3)2CO) used
residual (CH3)2CO (δH 2.04) or (CD3)2CO (δC 29.8) as internal standard.
Melting points (mp) were determined on a Reichert hot-stage apparatus.
High-resolution mass spectra (HR-MS) were recorded with a VG-Auto-
spec spectrometer using the fast atom bombardment (FAB) technique or
electron impact (70 eV) ionization (EI).
All experiments were carried out under an inert atmosphere, and all
solvents were dried prior to use. A “usual workup” refers to dilution with
water, repeated extraction into an organic solvent, and sequential washing
of the combined extracts with hydrochloric acid (1 M, where appropriate),
saturated sodium bicarbonate, and brine solutions, followed by drying
over anhydrous magnesium sulfate, filtration, and evaporation of the
solvent by means of a rotary evaporator under reduced pressure. Flash
chromatography was performed on Merck silica gel 60 with the specified
solvents. Thin-layer chromatography (TLC) was effected on Merck
silica gel 60 F254 aluminum-backed plates that were stained by heating
(>200 °C) with 0.25 M ceric sulfate in 2 M sulfuric acid. Percentage yields
for chemical reactions as described are quoted for only those compounds
that were purified by recrystallization or by chromatography and for
which the purity was assessed by TLC or NMR spectroscopy.
Seed Bioassay. All germination experiments were performed using
Solanum orbiculatum seeds collected in the Shark Bay region (Western
Australia) and stored at -80 °C until use. All assays were conducted using
Millipore (MP) water obtained by filtration through a Milli-Q ultrapure
water system (Millipore, Australia). A 1% acetone solution was used as a
control. Stock solutions of 100 ppm were prepared by dissolving 1.0 mg of
compound in 100 μL of acetone prior to the addition of 9.9 mL of MP
water. Subsequent dilutions with Milli-Q water gave concentrations of
10 ppm, 1 ppm, 100 ppb, 10 ppb, and 1 ppb. The solutions were tested for
germination activity by adding 2.5 mL to two layers of Whatman no. 1
filter paper (7.0 cm) in plastic Petri dishes (9.0 cm) followed by approxi-
mately 20-30 seeds. The Petri dishes were sealed with a layer of plastic
wrap and stored in a light-proof container for 6 days at 20 ( 1 °C. All
experiments were conducted in triplicate.
Synthesis. 4-Ethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (20). Titanium
tetrachloride (1.7 mL, 15 mmol) was added to a stirred solution of 21
(1.3 g, 7.0 mmol), tributylamine (3.9 mL, 21 mmol), and methyl pyruvate
(1.4 g, 14 mmol) in dichloromethane (20 mL) at -60 °C, and the resulting
solution was stirred under an atmosphere of argon (1.5 h). The usual
workup (ethyl acetate) followed by flash chromatography (10% ethyl
acetate-hexane) gave a pale yellow oil (1.0 g). This oil was dissolved in
acetonitrile (15 mL) followed by the addition of triethylamine (590 mg, 5.8
mmol), trifluoroacetic anhydride (900 μL, 6.5 mmol), and 4-dimethyla-
minopyridine (20 mg, 0.2 mmol) at 0 °C. The solution was stirred (3 h)
before being subjected to the usual workup (ethyl acetate) to give a
yellow residue that was dissolved in acetonitrile (20 mL) and treated with
1,8-diazabicycloundec-7-ene (800 mg, 5.3 mmol) at 0 °C. This mixture was
stirred (2 h) followed by the usual workup to return a brown residue.
p-Toluenesulfonic acid (870 mg, 4.6 mmol) was added to the brown residue
in toluene (25 mL) and the resulting mixture refluxed under an atmosphere
of argon (2 h). The reaction was cooled followed by the usual workup
(ethyl acetate), and flash chromatography (20% ethyl acetate-hexane)
0
0
0
0
0
J5 ,6 = 2.8 Hz, 1H, H6 ), 4.18 (d, J2 ,2 = 17.1 Hz, 1H, H2 ), 4.14 (d, J2,2
=
16.3 Hz, 1H, H2), 4.02-3.94 (m, 2H, H7, H70), 3.91 (d, 1H, H2), 3.85 (d,
1H, H20), 2.54 (dd, J4 ,4 = 15.8, J4 ,5 = 4.9 Hz, 1H, H40), 2.47-2.36 (m,
0
0
0
0
2H, H4, H4), 2.29 (dd, J4 ,5 = 9.8 Hz, 1H, H40), 2.02-2.10 (m, H5),
0
0
1.83-1.90 (m, H50), 1.85-1.25 (m, 4H, H9, H9,0H90, H90), 1.24 (d, J7,8
=
0
0
0
0
6.2 Hz, 3H, H8), 1.23 (d, J7 ,8 = 6.2 Hz, 3H, H8 ), 1.19 (d, J7 ,8 = 6.2 Hz,
3H, H80), 1.18 (d, J7,8 = 6.2 Hz, 3H, H8), 0.95 (t, J9 ,10 = 7.4 Hz, 3H,
H100), 0.90 (t, J9,10 = 7.5 Hz, 3H, H10); δC (150.9 MHz) 210.6 (C30), 207.8
(C3), 99.3 (C60), 95.6 (C6), 69.2 (C70), 69.2 (C7), 67.2 (C2), 66.8 (C20), 41.4
(C5), 40.8 (C50), 39.8 (C4), 39.6 (C40), 25.7 (C90), 24.4 (C9), 23.4 (C8), 23.1
(C80), 21.5 (C80), 21.4 (C8), 11.2 (C100), 11.0 (C10); m/z (FAB) 127.0761,
(M þ H)þ requires 127.0759.
0
0
General Procedure for the Preparation of Ether Derivatives of 5-
Hydroxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (26) and 7-Hydroxy-
methyl-3-methyl-2H-furo[2,3-c]pyran-2-one (33). Sodium hydride (60%
dispersion in mineral oil, 2 mmol) was added to the alcohol (1 mmol) and a
bromide (1-10 mmol) in dimethylformamide (15 mL) at 0 °C. The
mixture was allowed to warm to room temperature and stirred (0.5-4.5
h). The mixture was cooled to 0 °C and quenched by the dropwise addition
of 2 M HCl. The usual workup (dichloromethane) followed by flash
chromatography afforded the appropriate ether.
5-Butoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (27). The reaction
using 26 (24) (18 mg, 0.10 mmol) and 1-bromobutane (0.1 mL, 0.9 mmol)
was carried out according to the general procedure (4.5 h). Flash
chromatography (20% ethyl acetate-hexane) yielded the ether 27 as a
light yellow wax (7 mg, 35%): δH (500 MHz, (CD3)2CO) 7.77 (s, 1H, H7)
6.80 (s, 1H, H4), 4.35 (s, 2H, H8), 3.55 (t, J = 6.5 Hz, 2H, H10), 1.88 (s,
3H, H3b), 1.62-1.57 (m, 2H, H11), 1.41 (m, 2H, H12), 0.91 (t, J = 7.4 Hz,
3H, H13); δC (125.8 MHz, (CD3)2CO) 171.3 (C2), 159.3 (C5), 142.6, 141.5
(C3a, C7a), 127.5 (C7), 101.1 (C4), 100.2 (C3), 71.5 (C8), 69.4 (C10), 32.4
(C11), 19.9 (C12), 14.1 (C13), 7.6 (C3b); m/z (EI) 236.1049, (M)þ requires
236.1049.
5-Heptoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (28). The reac-
tion using 26 (24) (35 mg, 0.20 mmol) and 1-bromoheptane (0.2 mL, 1.3
mmol) was carried out according to the general procedure (0.5 h). Flash
chromatography (20% ethyl acetate-hexane) furnished the ether 28 as a
colorless wax (12 mg, 22%): δH (500 MHz) 7.41 (s, 1H, H7), 6.55 (s, 1H,
H4), 4.28 (s, 2H, H8), 3.55 (t, J = 6.6 Hz, 2H, H10), 1.89 (s, 3H, H3b), 1.61
(tt, J10,11, J11,12 = 6.6 Hz, 2H, H11), 1.41-1.25 (m, 8H, H12, H13, H14,
H15), 0.90 (m, 3H, H16); δC (125.8 MHz) 171.6 (C2), 157.9 (C5), 142.0,
141.0 (C3a, C7a), 126.4 (C7), 100.5, 100.5 (C3, C4), 72.0 (C8), 69.0 (C10),
31.9, 29.7, 29.2, 26.2, 22.7, 14.2 (C11, C12, C13, C14, C15, C16), 7.9 (C3b);
m/z (FAB) 279.1592, (M þ H)þ requires 279.1596.
5-Dodecoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (29). The reac-
tion using 26 (24) (27 mg, 0.15 mmol) and 1-bromododecane (0.2 mL,
0.8 mmol) was carried out according to the general procedure (0.5 h).
Flash chromatography (10% ethyl acetate-hexane) returned the ether 29
as a colorless wax (9 mg, 17%): δH (500 MHz) 7.41 (s, 1H, H7), 6.55 (s, 1H,
H4), 4.28 (s, 2H, H8), 3.55 (t, J10,11 = 6.7 Hz, 2H, H10), 1.89 (s, 3H, H3b),
1.65 (tt, J11,12 = 6.7 Hz, 2H, H11), 1.40-1.22 (m, 18H), 0.90 (t, J20,21
=
7.0 Hz, 3H, H21); δC (125.8 MHz) 171.6 (C2), 157.9 (C5), 142.0, 140.8
(C3a, C7a), 126.4 (C7), 100.5 (C3, C4), 72.0 (C8), 69.0 (C10), 32.1, 29.8,
29.7, 29.6, 29.5, 14.3 (C11-21), 7.9 (C3b); m/z (FAB) 349.2344, (M þ H)þ
requires 349.2379.
5-Benzyloxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (30). The re-
action using 26 (24) (40 mg, 0.22 mmol) and benzyl bromide (0.2 mL,

8614 J. Agric. Food Chem., Vol. 58, No. 15, 2010
Flematti et al.
1.7 mmol) was carried out according to the general procedure (0.5 h).
Flash chromatography (30% ethyl acetate-hexane) yielded the ether 30 as
a light orange oil (26 mg, 42%): δH (500 MHz, (CD3)2CO) 7.76 (s, 1H, H7)
7.25-7.45 (m, 5H, Ar), 6.85 (s, 1H, H4), 4.67 (s, 2H, H8/H10), 4.42 (s, 2H,
H10/H8), 1.92 (s, 3H, H3b); δC (125.8 MHz, (CD3)2CO) 171.3 (C2), 158.9
(C5), 142.6, 141.4 (C3a, C7a), 138.8 (C11) 129.2, 128.7, 128.6, 127.6 (C7,
Ar), 101.5 (C4), 100.3 (C3), 73.5, 68.9 (C8, C10), 7.7 (C3b); m/z (EI)
270.0890, (M)þ requires 270.0892.
C3-Substituted Analogues and Their Germination Activity. Two
recent synthetic routes have been reported that provide the
desmethyl analogue 2 directly in gram quantities (24, 25), a
substantial improvement on the original synthesis from pyrome-
conic acid (26). It has been demonstrated that the C3 position is
activated toward electrophilic substitution (24), which opens the
door to a number of 3-substituted analogues. These analogues
can be further modified to yield a small suite of compounds with
varied steric and electronic effects.
Previous work had demonstrated that removal of the methyl
group of 1 at C3 reduces germination activity by around 2
orders of magnitude (22). Replacing the methyl group at C3
with larger alkyl substituents similarly reduced the potency of
the resulting compounds, as observed for the ethyl 3 (24) and
propyl 4 (24) derivatives (Figures 1 and 2). The ethyl analogue
3 appeared to be an order of magnitude more active than the
slightly larger propyl derivative 4, but was still at least 100
times less potent than the methyl-bearing karrikinolide (1).
Analogues oxygenated at C3, such as the hydroxymethyl 5 (24)
and methoxymethyl 6 (24), also possessed reduced activity
relative to karrikinolide (Figure 2).
Compounds with electron-withdrawing groups at C3 have
greatly diminished germination activity. The aldehyde 7 (24),
methyl ketone 8 (24), oxime 9 (24), and difluoromethyl analogue
10 (24) are far less active than karrikinolide, promoting germina-
tion only at the highest concentration tested (Figure 2). Given the
similar van der Waals radii of fluorine and hydrogen, the
relatively poor activity of the difluoromethyl analogue 10 (24)
with respect to karrikinolide is particularly telling of this apparent
electronic effect.
5-(3-Phenoxypropoxy)methyl-3-methyl-2H-furo[2,3-c]pyran-2-one (31).
The reaction using 26 (24) (44 mg, 0.24 mmol) and 3-phenoxypropyl
bromide (0.2 mL, 1.3 mmol) was carried out according to the general
procedure (4 h). Flash chromatography (10% diethyl ether-toluene)
afforded the ether 31 as a pale yellow wax (12 mg, 16%): δH (600 MHz)
7.36 (s, 1H, H7), 7.30-7.26, 6.96-6.87 (2 m, 5H, Ar), 6.53 (d, J4,8 = 0.6
Hz, 1H, H4), 4.30 (d, 2H, H8), 4.10 (t, J11,12 = 6.0 Hz, 2H, H12), 3.75 (t,
J10,11 = 6.1 Hz, 2H, H10), 2.11 (tt, 2H, H11), 1.89 (s, 3H, H3b); δC (125.8
MHz) 171.6 (C2), 158.9, 157.5 (C5, C14), 141.9, 140.7 (C3a, C7a), 129.6,
121.0, 114.5 (Ar), 126.4 (C7), 100.7, 100.5 (C3, C4) 69.3 (C8), 68.0 (C10),
64.2 (C12), 29.8 (C11), 7.8 (C3b); m/z (FAB) 315.1250, (M þ H)þ requires
315.1232.
7-Hydroxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (33). Aluminum-
(III) chloride (480 mg, 3.6 mmol) was added to the ester 32 (25) (240 mg,
1.2 mmol) and tert-butylamine borane (640 mg, 7.2 mmol) in dichloro-
methane (25 mL), and the mixture was stirred at 40 °C (15 min).
The reaction was poured into ice-cold 1 M HCl (50 mL) followed by the
usual workup (dichloromethane) and flash chromatography (50-70%
ethyl acetate-hexane) to afford the alcohol 33 as light tan needles (180
mg, 87%): 160-162 °C; δH (600 MHz) 7.35 (d, J4,5 = 5.5 Hz, 1H, H5),
6.48 (d, 1H, H4), 4.68 (s, 2H, H8), 1.94 (s, 3H, H3b); δC (150.9 MHz)
170.9 (C2), 147.5 (C5), 139.9, 138.5, 136.5 (C3a, C7, C7a), 103.1, 101.2
(C3, C4), 56.8 (C8), 7.7 (C3b); m/z (EI) 180.0422, (M)þ requires
180.0423.
7-Methoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (34). The reac-
tion using 33 (50 mg, 0.26 mmol) and iodomethane (0.1 mL, 1.6 mmol) was
carried out according to the general procedure (1 h). Flash chromatogra-
phy (30% ethyl acetate-hexane) yielded the ether 34 as a tan powder (37
mg, 68%): mp 86-88 °C; δH (600 MHz) 7.35 (d, J4,5 = 5.5 Hz, 1H, H5)
6.50 (d, 1H, H4), 4.47 (s, 2H, H8), 3.42 (s, 3H, H10), 2.01 (s, 3H, H3b); δC
(150.9 MHz) 170.9 (C2), 148.0 (C5), 140.0, 139.7 (C3a, C7a), 134.4 (C7),
103.0 (C4), 101.3 (C3), 65.4 (C8), 58.6 (C10), 7.8 (C3b); m/z (EI) 194.0585,
(M)þ requires 194.0579.
The phenyl 11 (25), cyano 12 (24), and nitro 13 (24) analogues
are inactive at the concentrations tested (Figure 2). Compound 11
is probably inactive because the phenyl group is simply too
“bulky”, whereas the strongly electron-withdrawing groups of
the cyano 12 and nitro 13 analogues appear to abolish biological
activity. The chloride 14 (25) has appreciable germination-pro-
moting activity. However, substitution in line with the periodical
halogen atom order (Cl, Br, and I) at C3 results in progressively
weaker biological activity, as observed for the bromide 15 (25)
and iodide 16 (25) (Figure 2).
7-Butoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (35). The reac-
tion using 33 (50 mg, 0.26 mmol) and 1-bromobutane (0.2 mL, 1.8 mmol)
was carried out according to the general procedure (0.5 h). Flash
chromatography (20% ethyl acetate-hexane) yielded the ether 35 as a
colorless gum (17 mg, 26%): δH (600 MHz, (CD3)2CO) 7.64 (d, J4,5 = 5.5
Hz, 1H, H5) 6.77 (d, 1H, H4), 4.47 (s, 2H, H8), 3.52 (t, J10,11 = 6.5 Hz, 2H,
H10), 1.88 (s, 3H, H3b), 1.57-1.52, 1.39-1.34 (2m, 4H, H11, H12), 0.90 (t,
J12,13 = 7.5 Hz, 3H, H13); δC (150.9 MHz, (CD3)2CO) 169.9 (C2), 148.8
(C5), 139.9, 139.5 (C3a, C7a), 135.1 (C7), 102.8 (C4), 100.0 (C3), 70.1 (C8),
63.5 (C10), 31.4, 18.8, 13.1 (C11, C12, C13), 6.8 (C3b); m/z (EI) 236.1052,
(M)þ requires 236.1049.
Overall, the natural stimulant karrikinolide is at least an order
of magnitude more potent than any C3-modified compound
tested here. Thus, it would appear that a methyl group at this
position is optimal for germination-promoting activity.
C4-Substituted Analogues and Their Germination Activity. The
only analogue prepared so far with substitution at C4 is the
4-bromo-3,7-dimethyl derivative 17 (22) (Figure 3). The germina-
tion activity of 17 was found to be comparable to that of the 3,7-
dimethyl analogue 18 (22) (Figure 3); thus, investigation into
other C4-substituted analogues seemed to be appropriate. The
synthesis of karrikinolide recently developed by Nagase et al. (27)
provided a convenient route to C4-substituted analogues by modi-
fying the starting pyranone. The use of the known acetate 19 (23)
provided the 4-ethyl derivative 20, via the acetal 21 (Figure 4).
Surprisingly, compound 20 was found to have very poor
germination-promoting activity (Figure 5). With such a simple
modification resulting in such a dramatic loss in activity, no
further C4-substituted analogues were investigated.
C5-Substituted Analogues and Their Germination Activity. Two
C5-substituted compounds, the 3,5-dimethyl 22 (22) and 5-meth-
oxymethyl-3-methyl 23 (22) analogues (Figure 6), have previously
demonstrated good germination-promoting activity (22).
Recently, an efficient synthetic route to the 5-methoxycarbonyl
analogue 24 (24) was reported (Figure 7A). Whereas the activity
of 24 (24) itself is poor (Figure 5), which is not surprising given the
7-Benzyloxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (36). The re-
action using 33 (30 mg, 0.2 mmol) and benzyl bromide (0.1 mL, 0.8 mmol)
was carried out according to the general procedure (0.5 h). Flash
chromatography (30% diethyl ether-hexane) yielded the ether 36 as a
colorless gum (12 mg, 30%): δH (600 MHz) 7.35-7.26 (m, 6H, H7, Ar),
6.74 (d, J4,5 = 5.5 Hz, 1H, H4), 4.59, 4.55 (2s, 4H, H8, H10), 1.92 (s, 3H,
H3b); δC (150.9 MHz) 170.9 (C2), 147.9 (C5), 139.8, 137.0, 134.7 (C3a, C7,
C7a), 139.8, 128.4, 127.9, 127.8 (Ar), 103.0, 101.2 (C3, C4), 73.0, 63.2 (C8,
C10), 7.7 (C3b); m/z (EI) 270.0893, (M)þ requires 270.0892.
RESULTS AND DISCUSSION
The compounds described herein were evaluated as germination
stimulants using the seeds of Solanum orbiculatum (Solanaceae,
Dunal ex Poir.). S. orbiculatum is a smoke-responsive species native
to Western Australia (8) and provides an ideal species for testing
analogues as it is highly sensitive to the stimulatory effects of 1 and
provides a very low control germination yield (22).

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J. Agric. Food Chem., Vol. 58, No. 15, 2010 8615
Figure 2. Germination activity of C3-substituted karrikins.
Figure 3. Structure of the 4-bromo-3,7-dimethyl analogue 17 and 3,7-
dimethyl analogue 18.
Figure 4. Synthesis of C4-substituted karrikins: (a) SnCl4, PriOH, CH2Cl2;
(b) (i) TiCl4, Bu3N, methyl pyruvate, CH2Cl2, (ii) (CF3CO)2O, Et3N, DMAP,
CH3CN, (iii) DBU, CH3CN, (iv) p-TsOH, PhCH3.
absence of the methyl group at C3, it has been demonstrated to
be an excellent synthon for the preparation of C5-substituted
analogues (24).
The low activity of the hydroxymethyl derivative 26 (Figure 5),
when contrasted against the 5-methyl and 5-alkoxymethyl deri-
vatives, suggests that germination activity is reduced when a
hydrophilic substituent is present at the C5 position. Also
noteworthy is the fact that the n-butoxymethyl derivative 27 is
significantly more active than the previously prepared 5-methoxy
methyl derivative 23 (22), indicating lipophilic substituents are
well tolerated at this position. Despite the fact that a reduction in
germination activity is observed when large alkyl groups are
installed at C5, this position can clearly tolerate modification
better than C3 and C4.
The alcohol 25 (24) and the 5-hydroxymethyl-3-methyl analo-
gue 26 (24) (Figure 6), both prepared from 24, exhibited limited
germination-promoting activity (Figure 5). Given that the pre-
viously tested methoxymethyl analogue 23 exhibited high germina-
tion activity (22), additional analogues were prepared by conver-
ting 26 into a series of simple ether derivatives (Figure 7A). The
n-butoxymethyl analogue 27 was found to be highly active and
comparable to karrikinolide (Figure 5). Extension of the alkyl
chain length reduced activity significantly, as observed for the
n-heptoxymethyl 28 and n-dodecoxymethyl 29 derivatives (Figure 5).
The benzyloxymethyl derivative 30 exhibited good activity
(Figure 5) and demonstrated that some steric bulk is tolerated at
the C5 position, although this result is clouded by the fact that the
phenoxypropyl ether 31 showed no activity at the concentrations
tested (Figure 5).
C7-Substituted Analogues and Their Germination Activity. Ear-
lier work demonstrated that analogues substituted with a methyl
group at C7 had significantly reduced germination activity
compared to 1 (22). Further investigations were conducted.

8616 J. Agric. Food Chem., Vol. 58, No. 15, 2010
Flematti et al.
Figure 5. Germination activity of C4- and C5-substituted karrikins.
Surprisingly, the 7-trimethylsilyl derivative 37 (25) (Figure 7B)
showed high germination-promoting activity that was compar-
able to that of karrikinolide (1) (Figure 8). However, it seems
likely that compound 37 may undergo spontaneous hydrolysis to
liberate karrikinolide under the conditions of the assay (28),
which may explain its potent germination activity.
Figure 6. Structure of C5-substituted karrikins.
Knowledge of karrikin structure-activity relationships is
valuable for the design of karrikin-based molecular probes for
mode of action studies. To this end, the structure of karriki-
nolide provides only four carbons that can be modified. The
results presented here demonstrate that karrikins modified at
the C3 position have reduced activity compared with the
methyl-bearing karrikinolide, indicating that the methyl sub-
stituent at C3 is important for biological activity. Compounds
substituted at the C4 position showed significantly impaired
bioactivity. However, modifications at C5 and C7 were better
tolerated, with compounds modified at C5 found to retain the
highest bioactivity. In terms of labeling, compound 26 pro-
vides an excellent scaffold for preparing analogues to inves-
tigate the karrikinolide mode of action with C5 derivatives
reported here demonstrating a high level of bioactivity with
S. orbiculatum.
The 7-methylester 32 (25) showed good activity, whereas the
7-hydroxymethyl analogue 33, derived from 32 (Figure 7B), showed
only activity at the highest concentration tested (Figure 8). This
result is analogous to that seen at C5, where the hydroxymethyl
group was also poorly tolerated.
Several ethers were prepared from the alcohol 33, with the
expectation that some activity may be reclaimed, as observed
in the case of the C5-substituted compounds. Thus, the
alcohol 33 was converted into the 7-methoxymethyl 34, n-
butoxymethyl 35, and benzyloxymethyl 36 analogues in good
yield (Figure 7B). The n-butoxymethyl 35 and the benzylox-
ymethyl 36 proved to be an order of magnitude less potent
than the corresponding C5 analogues, compounds 27 and 30,
respectively (Figure 8).
Figure 7. Synthesisof (A) C5-substitutedkarrikins and (B) C7-substitutedkarrikins:(a) NaH, RX, DMF; (b) tBuNH2 BH3, AlCl3, CH2Cl2; (c) NaH, R0X, DMF.
3

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J. Agric. Food Chem., Vol. 58, No. 15, 2010 8617
Figure 8. Germination activity of C7-substituted karrikins.
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