Journal of Chemical Ecology, Vol. 11, No. 12, 1985
ISOFLAVONOID FEEDING DETERRENTS FOR Costelytra
zeatandica
Structure-Activity Relationships
G E O F F R E Y A. L A N E , 1 D A V I D R. B I G G S , l G R A E M E B. R U S S E L L , 1
O L I V E R R. W. S U T H E R L A N D , 2 E. M A R E E W I L L I A M S , 2
J O H N H. M A I N D O N A L D , 3 and D E B O R A H J. D O N N E L L 3
t Applied Biochemistry Division, DS1R
Palmerston North, New Zealand
2Entomology Division
3Applied Mathematics Division, DSIR
Auckland, New Zealand
(Received January 22, 1985; accepted May 2, 1985)
Abstraet--A number of naturally occurring isoflavonoids of differing substitution patterns and oxidation states have been tested for feeding deterrent
activity in a bioassay with larvae of Costelytra zealandica White. The most
active deterrents, which reduced feeding significantly at 0.2-1.0 #g/g, arc
those compounds containing a ring B-fused cycloprenoid moiety. The least
active compounds were highly oxidized coumestans and isoflavones. The ring
B-fused cyclic isoprenoid moiety and the presence of a 2'-oxy function appear
to be structural features important for high activity. It is suggested that the
feeding deterrent activity of isoflavonoids relates to their stereochemistry and
that the most active compounds have or can adopt a similar nonplanar molecular shape with a similar arrangement of polar and lipophilic groups.
Key Words--Isoflavonoids, structure-activity relationships, insect feedingdeterrent activity, Costelytra zealandica, Coleoptera, Scarabaeidae, stereochemistry.
INTRODUCTION
R e c e n t w o r k has shown that the s u b t e r r a n e a n , r o o t - f e e d i n g larvae of the beetle
C o s t e l y t r a z e a l a n d i c a W h i t e (Coleoptera: Scarabaeidae), which inflict m a j o r
d a m a g e on N e w Z e a l a n d pastures, are d e t e r r e d f r o m f e e d i n g by several isoflavonoid constituents o f l e g u m e roots (Russell et a1.,1978; Sutherland et. al.,
1980; L a n e et. al., 1985). Several o f the m o s t active c o m p o u n d s have also been
r e c o g n i z e d as phytoalexins (antifungal m e t a b o l i t e s elicited in response to stress
17t3
098-0331/85/1200-1713504.50/0 cQ 1985 Plenum Publishing Corporation
1714
LANE ET AL.
or infection) in the aerial parts of legumes. This observation led to the proposal
that in the defense strategy of these plants, the isoflavonoids may serve two
different and perhaps independent roles, as phytoalexins and also as insect feeding deterrents (Sutherland et al., 1980). Isoflavonoids are only one of several
classes of compounds implicated in legume resistance to grass grub (Sutherland
et al., 1975, 1982a, b; Hutchins et al., 1984).
The isoflavonoids present in different legumes differ in structural type,
yield, and level of feeding deterrent activity. In order to understand and predict
the effect of the isoflavonoids of a particular legume on the feeding of C. zealandica larvae, it is important to determine whether deterrent activity can be
related quantitatively to defined structural features of the constitutive isoflavonoid molecules. It is generally accepted that molecular size and shape are important determinants for semiochemical activity (Silverstein, 1979), and studies
with insect feeding deterrents have shown that redox potential, intramolecular
hydrogen bonding capability (Norris, 1977), and functional group stereochemistry (Kubo and Ganjian, 1981 ; Kojima and Kato, 1981) can be important factors
in relating molecular structure to insect gustatory response.
In this study we have examined the effect of a range of naturally occurring
isoflavonoids, some synthetic analogs, and some simple chromenes on the feeding behavior of C. zealandica larvae in order to determine those molecular features that are important for high feeding-deterrent activity. We have drawn together data from our earlier studies on isoflavonoids (Sutherland et al., 1980;
Lane et al., 1985) and new data acquired for this study, and analyzed them with
a uniform statistical treatment.
METHODS AND MATERIALS
Feeding Deterrent Assay. Field-collected third-instar larvae of Costelytra
zealandica, which had been starved for 24 hr, were enclosed individually in 5.2cm glass Petri dishes with a 4% agar-4% cellulose powder disk (1.5 cm). Feeding-deterrent activity was assessed by comp~xing 24-hr fecal pellet counts of
larvae offered disks containing both standard feeding stimulant (0.1 M sucrose
+ 0.01 M ascorbic acid) (Sutherland, 1971) and test material with similar counts
of larvae offered disks containing feeding stimulants only (control) and of a third
group offered disks prepared with neither (blank). Twenty larvae were tested
with each medium. Blank and control treatments were run on all feeding occasions, and the difference between them was taken as the standard stimulated
response (standard).
Most isoflavonoids were tested at four concentrations in the disk, usually
2, 20, 100, and 200/~g/g. Lack of material limited the range of some compounds
to three concentrations, while the activity of a few others required a wider test
range. The required amount of each compound was dissolved in solvent, added
1715
FEEDING DETERRENTS
to a weighed sample of cellulose powder, and the solvent evaporated. Test media
were then prepared by adding an appropriate amount of hot 4 % agar solution
to each cellulose powder sample, stirring vigorously, and pouring a measured
amount into a glass Petri dish lid containing either sufficient concentrated sucrose and ascorbic acid solution to make up the desired final concentration (test
and control) or an equivalent volume of distilled water (blank). The combined
ingredients were then stirred thoroughly and set aside to cool. This procedure
was adopted to minimize oxidation of the ascorbic acid. Test disks were then
cut from the media with a cork borer.
Statistical Treatment of Data. Although the fecal pellet count is the most
accurate, reproducible, and convenient method of measuring ingestion by C.
zealandica larvae (Sutherland, 1971), the feeding behavior of individual insects
presented with the same medium at the same time often differs widely (for
example, see Figure 1). Precise quantification of the behavior and comparison
of response to often quite similar test chemicals was therefore difficult. Instead
of the mean fecal pellet count, a Hodges-Lehmann estimate (Hollander mad
Wolfe, 1973) was used as the measure of the overall level of response of all
insects tested at a particular concentration of a test compound. As used here, it
is the median of all possible pairwise averages of individual fecal pellet counts.
Its advantage is that it is unaffected by a few very large or very small counts,
which were a feature of some sets of the results.
o
o
e--
~
z
o
1
30-
-@
[]
o
c
8
D
g
s
E
~-~ 20~
8
[]
~
o
[]
[]
3
o
-~ 1 0 -"1
Z
Control
0.001
0,01
01
1
10
Blank
Phaseollin Conc.(ppm)
FIG. 1. Result of one assay of the feeding response of 20 third-instar Costelytra zealandica larvae to each of five concentrations of phaseollin and to control and blank treatments. [3 = individual fecal pellet counts; O = Hodges-Lehmann estimate; line shows
estimated dose-response curve.
1716
LANE ET AL.
The data were analyzed by a procedure analogous to probit analysis. The
Hodges-Lehmann estimates were plotted against log concentration. A straight
line was fitted to the points obtained and the FD50 was calculated as the concentration corresponding to the midpoint between the blank and the control fecal
pellet counts, i.e., that concentration at which stimulated feeding was reduced
to 50% of the standard. The slope of each line was also calculated. Figure 1
shows individual pellet counts, Hodges-Lehmann estimates, and the fitted line
for one of the trials with phaseollin (I). Providing a suitable concentration range
was used, the assumption of linear change in feeding response with log-dose
appeared adequate; the data did not allow the fitting of more complex models
such as a sigmoid curve.
In a few cases, all concentrations tested depressed feeding to a level close
to or even below that for the blank. It was then clear that the FD~o was less than
the lowest concentration tested. These experiments were repeated with a more
suitable range of concentrations.
In order to calculate standard errors for the FDso values and the slopes,
pooled standard errors of the relevant Hodges-Lehmann estimates were required. For each Hodges-Lehmann estimate, the standard error was estimated
at the 100 (1 - cr % confidence level by the method of Hollander and Wolfe
(1973). The term cr was set at 0.32 to give a confidence interval of twice the
standard error; smaller values of c~ make the interval (and hence the standard
error) sensitive to extreme data values.
For each experiment, the root mean square of the standard errors of the
Hodges-Lehmann estimates of responses at all concentrations of the test compound was calculated as an overall (pooled) estimate of the error. This allowed
calculation of standard errors for the slope and intercept of the regression line.
Following Finney (1971, p. 78) these lead to standard error limits log FDs0 d, log FDs0 + d, where standard errors of blank and control are also incorporated in d. For the FDs0, the limits are (FDs0 + c, FDso x c), where c =
antilog (d) (Tables 1-4). In order to test whether the slope of the regression line
differed from zero, it was divided by its standard error and the absolute value
taken, to give a statistic z. A value of z greater than 1.65 indicated statistical
significance, corresponding to the one-sided 5 % point for the normal distribution. Where a compound was tested twice, the requirement was at least one of
the two z values should be more than 1.95; this gives an overall test with a
nominal significance level of 5 %. For three tests, the requirement was similarly
increased.
Chemicals. The following compounds were obtained commercially.: VIII
(Aldrich) XV and XVII (ICN Pharmaceuticals Inc.), XXVII (Eastman, Kodak
Ltd.), XXXIV and XXXV (Calbiochem). L. Jurd, USDA, Albany, CA, provided compounds XXVIII-XXXI and O. Gottlieb, Instituto de Quimica, Universidade de Sao Paulo, Brazil, provided compounds XI and XXXII. The source
1717
FEE~,NO DETERRENTS
HO
H
IoL A )1 c ~L,,,,H o.
,
R=.
~"YI
[I. R=Me
Ill.
RO
V. R=Me
,
[..,I
H
Me
VH,.
VII.
gMe
~
e
IX. R'= H
x. R'=Me
XI.
of compounds I, IV, V, IX, X, XII-XIV and XVIII has been previously described (Sutherland et al., 1980). The isolation of VI, VII, XVI, and XX-XXIII
as insect feeding deterrents and antifungal components from the roots of Lupinus angustifolius is the subject of a separate report (Lane et al., 1985). The
following compounds were isolated or synthesized as described.
7-O-Methylphaseollin (I1). Phaseollin (1) was treated with ethereal diazomethane in MeOH. Crystallization from aq. MeOH gave II, mp 120-122~
Comparison of the UV, mass spectral, and [IH]NMR data with those reported
for phaseollin (I) (Perrin et al., 1972) indicated the structure II. UV (EtOH)
1718
LANE ET AL.
Xm,~207 (e 47,000), 230.5 (e 37,000), 279.5 (e 13,000), 285 sh (e 11,000), 315.5
nm (e 3,000); EIMS (probe, 70 eV) m/z (relative intensity) 336 (M +, 63), calcd
336.13615, found 336.13699, 322 (24), 321 (100), 293 (40), 160 (26); [IH]NMR
(CDC13, 80 MHz) ~ 7.43 (1 H, d, J = 8.5 Hz, H-5), 6.93 (1 H, d, J = 8.1 Hz,
H-6'), 6.62 (1 H, dd, J = 2.5, 8.5 Hz, H-6), 6.49 (1 H, d, J = 9.9 Hz, H-c0,
6.45 (1 H, d, J = 2.6 Hz, H-8), 6.32 (1 H, dd, J = 0.7, 8.0 Hz, H-5,), 5.55 (1
H, d, J = 9.9 Hz, H-r 5.48 (1 H, br d, J = 6.3 Hz, H-4), 4.2 (1 H, m, H-2),
3.77 (3 H, s, OCH3), 3.6-3.4 (2 H, m, H-2, H-3), 1.41 (3 H, s, CH3), 1.38 (3
H, s, CH3). (Numbering as for structure I).
Phaseollidin (II1) and Kievitone (XXIV). Etiolated hypocotyls of Phaseolus
vulgaris cv. Tendergreen were treated with conidial suspensions of Sclerotinia
fructicola (Wint.) Rehm, Saccardo in 0.5 ppm cycloheximide and incubated 24
hr at 25 ~ Ethanolic extracts of freeze-dried hypocotyls were chromatographed
on silica gel with CHC13-EtOH and petroleum ether-Et20.
Phaseollidin (III), purified by repeated chromatography on Sephadex LH20 with CHC13 and CHCI3-EtOH (19 : 1) gave UV, EIMS, and [IH]NMR spectra in agreement with reported data (Perrin et al., 1972).
Kievitone (XXIV) was purified by repeated chromatography on Sephadex
LH-20 with CHC13-petroleum ether-EtOH (10 : 10 : 5). The UV, ELMS, and
[~HINMR were in agreement with data reported by Smith et al. (1973).
2 '-Hydroxyformononetin (XIX). This was synthesized from 2,4-dihydroxyacetophenone and 2-hydroxy-4-methyoxybenzaldehyde as described by Farkas
et al. (1974). The mp 220-222~ (lit. 220-221~ Farkas et al., 1974), UV,
and [~H]NMR data were in agreement with reported values (Dewick, 1977; Braz
Filho et al., 1977).
( + )-Medicarpin (Racemic XII) and ( + )-Vestitol (Racemic IX). Catalytic
hydrogenation of XIX in HOAc at room temperature and atmospheric pressure
over 10% Pd/carbon and column chromatography on silica gel with petroleum
ether-Et20 afforded (+)-medicarpin (racemic XII), mp 198-200~ (lit. 194195~ Cocker et al., 1965), and (+)-vestitol (racemic IX), mp 173-175~ (lit.
173-175~ Farkas et al., 1974). The UV, MS, and [1H]NMR spectra were
identical to those for (-)-medicarpin (XII) and (-)-vestitol (IX) respectively.
(+_)-Methylequol (XXIO. Catalytic hydrogenation of XVIII in HOAc over
10% Pd/carbon (Nottle and Beck, 1974) gave XXV, mp 160-161 ~ (lit. 160~
Nottle and Beck, 1974). The MS and [IH]NMR data were in accord with those
reported by Breytenbach et al. (1981).
Dihydroformononetin (XXV1). Catalytic hydrogenation of the acetate of
XVIII in acetone at room temperature and atmospheric pressure over 10% Pd/
carbon and mild hydrolysis with NaHCO3-MeOH gave XXVI, nap 197-198~
(lit. 197-199~ Breytenbach et al., 1981). The MS and [IH]NMR data were
in agreement with those reported by Breytenbach et al. (1981).
Epirotenone (XXXIll). Rotenone (VIII) was converted to the crystalline
1719
FEEDING DETERRENTS
Xtl.
XIII. R'= R"=H
XIV. R'=Me, R"=OH
XV. R'=R"=H
XVI. R'=OH, R"=H
XVII. R'=H, R"=Me
xx.:.
XXI. R=OH
XXIII.
XVlll. R=H
XIX. R=OH
xx
XXIV.
epimeric mixture "mutarotenone" by treatment with NaOAc in EtOH as described by Crombie et al. (1961). The mixture was separated by preparative
recycle HPLC on a silica gel column with petroleum ether-EtOAc (5 : 2) using
a Waters Prep-500A chromatograph, with refractive index detection. The separation was monitored by analytical HPLC on a 25-cm, 10#m silica gel column
with hexane-EtOAc (5 : 1) with UV detection at 280 nm, and rotenone could
not be detected in the purified fraction ( < 1%). Crystallization from MeOH
gave XXXIII mp 88-91~
[cq D + 60 ~ (c 1.1, benzene) [lit. mp 90~ [c~]D +
75.6 ~ (c 0.6), Crombie et al., 1961]. The [~3C]NMR (CDCI~, 20 MHz) data for
1720
L A N E ET
AL.
HO"~/j....OMe
xxv. R=H~
XXVI. FI=O
XXVII. R'=OH, R'~
XXVIII. R'=H, R"=OH
XXXI.
XXIX. R=H
XXX. R=OH
HO.~O
XXXII.
L
H
HO/ ~ /
--"
0....//~-.....~ 'i
"OMe
XXXIII.
"~/'"OMe
6Me
Me
XXXIV. R=H
XXXV. R=OMe
MeC
XXXVl.
XXXVll,
XXXIII are closely similar to those for rotenone (VIII) recorded under identical
conditions: epirotenone (XXXIII), 6 188.9 (C-4), 167.3 (C-7), 157.9 (C-9), 149.5
(C-4'), t47.4 (C-2'), 143.9 (C-5'), 143.0 (-C=), 129.9 (C-5), 113.4 (C-10), 112.9
(C-8), 112.4 (=CH2), 110.5 (C-6'), 104.9 (C-6), 100.9 (C-3'), 87.9 (furanoid
CH-O), 72.2 (C-2), 66.3 (CH2-O), 56.3 (OCH3), 55.8 (OCH3), 44.5 (C-3), 31.1
(Ar-CH2), 17.1 (-CH3); rotenone (VIII), 6 188.8, 167.3, 157.8, 149.5, 147.3,
143.8, 143.0, 129.8, 113.2, 112.9, 112.4, 110.5, 104.8, 100.9, 87.7, 72.2, 66.2,
56.3, 55.7, 44.5, 31.2, 17.0. The assignments for XXXIII are based on those
reported by Crombie et al. (1975) for VIII, and the numbering used is that shown
for I and VIII. The assignments are supported by multiplicities determined using
a DEPT pulse sequence (Doddrell et al., 1982).
The [IH]NMR (CDCI3, 80 MHz) data for XXXIII are also very similar to
those reported for VIII (Carlson et al., 1973). 6 7.81 (1 H, d, J = 8.5 Hz, 5H), 6.77 (1 H, d, J = 0.6 Hz, 6'-H), 6.47 (1 H, d, J = 8.6 Hz, 6-H), 6.42 (1
H, s, 3'-H), 5.26 (1 H, br t, J = 8.9 Hz, furanoid CH-O), 5.01 (1 H, br s,
FEEDING DETERRENTS
172!
C = C H ) , 4.9 (2 H, m, C = C H , 2-H), 4.59 (1 H, dd, J
3.0, 11.9 Hz, 2a-H),
11.6 Hz, 2a-H), 3.81 (1 H, br d, J
4.1 Hz, 3-H), 3.77
4.14 (1 H, br d, J
(3 H, s, OCH3), 3.74 (3 H, s, OCH3), 3.29 (1 H, dd, J = 15.8, 9.6 Hz, Ar-CH),
2.92 (1 H, dd, J = 15.9, 8.3 Hz, Ar-CH), 1.70 (3 H, s, CH3). The observed
couplings are almost identical to those reported for VIII by Carlson et al. (1973),
and any chemical shift differences are slight. The coupling of 4.1 Hz for 3-H,
obscured by the OCH 3 signals in CDC13, was determined from a spectrum in
pyridine-d 5 (Carlson et al., 1973). Homonuclear shift-correlated two-dimensional NMR experiments (COSY; Bax and Freeman, 1981) showed an identical
pattern of connectivities for XXXIII and VIII, and closely similar resolved subspectra for the protons in the furanoid ring and those about the C/D ring junction. These data indicate the heterocyclic ring conformations of XXXIII and
VIII are closely similar, despite the different relative configurations in the two
compounds.
Dihydroprecocene II (XXXV1). Catalytic hydrogenation of XXXV in HOAc
at room temperature and atmospheric pressure over 10% Pd/carbon gave
XXXVI, mp 62~ [lit 61-62~ Ahluwalia et al. (1982)] with [tH]NMR data
in agreement with that of Ahluwalia et al. (1982).
6-Acetyl-5-hydroxy-2,2-dimethylchromene (XXXVII). This was prepared
from 2,4-dihydroxyacetophenone and 3-hydroxy-3-methylbutanal dimethyl acetal in pyridine as described by Bandaranayake et al. (1971). The mp and
[~H]NMR data were in agreement with those reported by Bandaranayake et al.
(1971).
=
=
-----
RESULTS
Table 1 gives FDso values and slopes of dose-response lines, together with
the standard errors, for compounds giving a significant correlation between dose
and feeding response. The criteria for inclusion in Table 1 are that on at least
one occasion: (1) the dose-response gave an FDs0 not more than 1000 ~g/g (five
times the maximum concentration tested), and (2) the slope of the regression
line was negative and significantly different from zero at the 5 % level. Different
sets of values for a compound correspond to measurements on different days.
The active feeding deterrents in Table 1 are listed in decreasing order of
activity (as measured by the FDso), although the wide error limits of the FDso
values imply that the ranking is approximate. No account has been taken of
differences in the slopes of the dose-response lines. While these isoflavonoids,
as a group, all show feeding-deterrent activity, the table spans a very wide range
of activities. With FDso values of 0.02 #g/g and 0.03 #g/g, phaseollinisoflavan
(IV) and phaseollin (I) are among the most active insect feeding deterrents yet
recorded. The variation between the FDso values of compounds tested on dif-
1722
LANE ET AL,
TABLE 1. EFFECT OF ISOFLAVONOIDS ON FEEDING OF Costelytra zealandica LARVAE:
SIGNIFICANT DOSE-REsPONSE CORRELATION
Feeding response
Compound
( -)-Phaseollinisoflavan (IV)
(-)-Phaseollin (I)
(-)-Rotenone (VIII)
(+)-2'-O-Methylphaseollin
isoflavan (V)
(-)-7-O-Methylphaseollin (II)
LicoisoflavoneB (VI)
(-)-Phaseollidin (III)
(-)-Clausequinone (XI)
(-)-Vestitol (IX)
2'-Hydroxyformononetin(XIX)
2'-Hydroxygenistein(XVI)
(-)-Medicarpin (XII)
(-)-Maackiain (XIII)
(+)-Pisatin (XIV)
Kievitone(XXIV)
Luteone (XXI)
FDs0
(t~g/g)
c (•
+)4
Slope of doseresponse line
0.02
0.03
0.06
4.7
2.2
3.3
-3.8
-6.3
-4.3
0.21
0.35
5.7
4.1
-3.1
-2.1
1.2
2.9
1.6
6.5
16
9.3
3.4
14
20
27
42
43
49
50
55
120
260
2.8
2.2
2.2
2.7
4.7
10
7.1
3.6
1.8
9.6
2.2
1.8
2.1
3.6
8.1
-3.7
-3.9
-5.8
-6.5
-6.0 NSb
-4,7
-1.1
-3.8 NS
-2.7
-10.2
-3.4 NS
- 10
- 12
-7.8
-3.6
- 3.9
"The error limits are (FDso + c, FDs0 x c).
bNS: slope not significantlydifferentfrom zero at 5 % level. All other slopes are significant.
ferent occasions (compare Tables 1 and 3) reflects the inherent scatter of data
in such a behavioral bioassay.
The slopes of the regression lines given in Table 1 are given as fecal pellet
count per log concentration. On average, the standard stimulated response was
approximately 15 pellets. The slope values listed correspond to a reduction in
feeding of between 4 and 20% of this average when the concentration of the
compound is doubled.
Isoflavonoids which failed the FDso or the slope criteria for feeding-deterrent activity over the concentration range are listed in Table 2 together with their
feeding responses at the highest concentration (200 /~g/g). Compounds VII,
XXII, and XXXI showed significant reduction in feeding at this concentration
by the Wilcoxon's Rank sum test, and XXIII and X showed significant reduction
in feeding at a lower concentration (Lane et al., 1985). These compounds, while
FEEDING DETERRENTS
1723
TABLE 2. EFFECT OF [SOFLAVONOIDS ON FEEDING OF Costelytra zealandica LARVAE:
NO SIGNIFICANT DOSE-RESPONSE CORRELATION
Compound
Feeding response" (%),
200 ~zg/g
Licoisoflavone A (VII)
(-)-Sativan (X)
Genistein (XV)
Biochanin A (XVII)
Formononetin (XVIII)
Wighteone (XX)
Angustone A (XXII)
Angustone B (XXIII)
(+)-Methylequol (XXV)
Dihydroformonorletin (XXVI)
Coumestrol (XXVII)
7-Deoxy-5'-hydroxycoumestrol(XXVIII)
Osajin (XXIX)
Pomiferin (XXX)
4-Hydroxy-3-aryleoumarylquinone (XXXI)
67b
84
80
76
79
69
31b
74
98
175
86
77
76
110
48h
~Based on Hodges-Lehmann estimates, standard = 100.
bSignificant reduction in feeding: P < 0.05 Wilcoxon's rank sum test.
probably active, do not show the steady reduction in feeding with increasing
dose over the range tested that is characteristic of the compounds in Table 1.
Licoisoflavone A (VII) has consistently shown a mild depression in feeding at
all concentrations, while for XXII and XXXI the highest dose may be at the
beginning of the response range.
In order to compare directly the effect of certain structural differences on
the feeding response of C. zealandica larvae, sets of two or three homologous
isoflavonoids were tested on the same day under the same conditions. The FDso
values and slopes of dose-response lines for these comparative tests are given
in Table 3. For comparison of active compounds in these sets, a parallel line
analysis was used to check whether there were significantly different feeding
responses and to provide a numerical measure of the difference. Parallel response lines were fitted to the Hodges-Lehmann estimates of the pellet counts
for the compounds to be compared. The common slope was then checked for
statistical significance. The Y-axis intercepts were compared using Fisher's LSD
test (5% level), and the ratio of FDso values was calculated to give a measure
of the relative activity. This analysis was not appropriate for comparisons involving compounds with an FDso > 1000 and/or a positive or nonsignificant
slope of the dose-response line.
All the compounds tested on this occasion (Table 3) gave similar results,
TABLE 3. COMPARATIVE EFFECT OF SELECTED ISOFLAVONOIDS ON FEEDING OF
Costelytra zealandica
LARVAE
Feeding response
Compound
FDs0 (/~g/g)
( - ) - M e d i c a r p i n (XII)
( - )-Phaseollidin ( l i d
( - ) - P h a s e o l l i n (1)
76
5.3
0.050
2'-Hydroxygenistein (XV1)
Licoisoflavone A (VII)
Lieoisoflavone B (VI)
Genistein (XV)
2'-Hydroxygenistein (XVI)
Formononetin (XVIII)
2'-Hydroxyformononetin (XIX)
(•
(XXV)
(+_)-Vestitol (rac. IX)
(_,•
(rac. XII)
( + ) - V e s t i t o l (XXXII)
(+)-Vestitol (rac. IX)
( - ) - V e s t i t o l (IX)
C (•
+)
Relative
activity"
C ( x , +)
4.7
4.0
2.3
- 4 . 4 NS'
- 5.1
- 8.1
-1
75
2.0
4.9
44
1.9
-5.1
- 3 . 4 NS
-7.8
1
-68
2.2
-2.3
- 2 . 5 NS
-6.0
---
-390
+ 2 . 1 NS
- 1.2 NS
---
> 1000
370
NO
-17
--
- 6.2
- 2 . 8 NS
+ 1.2 NS
----
460
35
43
23
3.1
2.5
- 3 . 2 NS
-6.4
-7,6
-1.6 NS
1
- 5,4
-8.5
380
320
2.7
> 1000
97
NO b
18
Epirotenone (XXXIII)
Rotenone (VIII)
56
0,44
5.0
2.0
Epirotenone (XXXIII)
Rotenone (VIII)
2,1
0.42
2.3
1.8
4.6
3.6
Epirotenone (XXXIII)
Rotenone (VIII)
Slope of d o s e response line
120
5.0
2.2
1
86
2.9
- 7.8
- 11
1
8.4
1.5
-3,8
-3.1
1
34
3.1
~Inverse ratio of FDs0 values for pairs of active compounds as determined by the parallel line analysis, expressed as i : n.
bNO: not obtainable, FD50 not possible with compounds of positive slope.
' N S : not significant, slope not significantly different from zero at 5% tevel; activity difference nol significant at 5% level,
z
FEEDING DETERRENTS
1725
TABLE 4. EFFECT OF SIMPLE CHROMENES ON FEEDING OF
Costelytra zealandica LARVAE
Feeding response
Compound
FDs0 (#g/g)
C ( x , +)
PrecoceneII (XXXV)
Precocene I (XXXIV)
6-Acetyl-5-hydroxy-2'-2'-dimethylchromene(XXXVII)
DihydroprecoceneII (XXXVI)
2.4
6.9
8.4
47
1.7
3.8
2.1
2.1
within the limits of error, to those obtained previously (Table 1 and 2). Thus,
for example, phaseollidin (III) and phaseollin (I) gave comparable FDs0 and
slope values, and (-)-medicarpin (XII) again gave a nonsignificant slope.
Table 4 records the FDso values and slopes of three simple chromenes and
a chroman. In all cases, the data showed these compounds to have feedingdeterrent activity.
To simplify the discussion of each of the isoflavonoids, the isoflavone numbering system (see structure I) has been used throughout. Unless specifically
indicated in the diagrams or in the text, the isoflavans and pterocarpans (reduced
ring C compounds) have the same stereochemistry as phaseollin (I). The exceptions are (+)-pisatin (XIV) which has the opposite configuration to phaseollin at C-3 and C-4 and (+)-methylequol (XXV) which is a racemate. The
isoflavanones, dihydroformononetin (XXVI) and kievitone (XXIV) Were also
obtained as racemates (Wong, 1975).
DISCUSSION
The data included in this paper are the quantitative measures of a behavioral
response of field-collected insects. In interpreting them as a whole, we have
sought to identify trends. Some have emerged which are clearly supported by
the statistical tests. Other apparent trends are less strongly supported by the
analysis but still suggest a particular interpretation. We have attempted to find
a common molecular basis for this pattern of results and consider a number of
hypotheses in turn.
Structural Correlations. Of 36 isoflavonoids, including optical isomers,
tested for feeding-deterrent activity towards Costelytra zealandica larvae, 18
were active. Phaseollin (I) and related compounds with a cyclic isoprenoid unit
fused to ring B, and rotenone (VIII) are particularly active with a significant
effect on feeding at concentrations below 1/zg/g. The feeding deterrent activity
of rotenone (VIII) is of interest as it is well known as an insect toxin. Feeding
1726
LANE ET AL.
deterrent activity is not restricted to a particular isoflavonoid class. While all
the pterocarpans tested (I-III and XII-XIV) showed feeding deterrent activity
and neither of the coumestans tested (XXVII, XXVIII) showed activity, isoftavans, isoflavones, and isoflavanones occurred in both the "active" (Table 1) and
"inactive," (Table 2) categories.
Many of the isoflavonoids showing insect feeding deterrent activity are also
antifungal (Sutherland et al., 1980, Hart et al., 1983). However, a study of lupin
isoflavones (Lane et al., 1985) has shown that the two activities do not always
coincide and appear to have different structural requirements. Thus, 2'-hydroxygenistein (XVI) shows insect feeding-deterrent activity but is not antifungal,
while the antifungal prenylisoflavone, wighteone (XX), does not show insect
feeding-deterrent activity. Perrin and Cruickshank (1969) suggested a stereochemical basis for the antifungal activity of pterocarpans, but this was later
rejected by Van Etten (1976), who found the antifungal activity of 17 isoflavonoids tested could not be correlated with a common three-dimensional shape.
Whatever the structural requirements for the antifungal activity of isoflavonoids,
further evidence that they are not identical with those responsible for insect feeding-deterrent activity is provided by the results reported here for 7-O-methylphaseollin (II) and rotenone (VIII). Both of these compounds show very high
feeding-deterrent activity (Table 1), but both lack antifungal activity (R.A.
Skipp, personal communication; Georgopoulos, 1977).
Two structural features that are characteristic of most of the highly active
feeding deterrents are: (1) the presence of a 2'-oxy function, and (2) the occurrence of a 2.2-dimethyl-l,H-pyran (cyclic isoprenoid group) fused to ring B (I.
II, IV, V). All the active isoflavonoids in Table 1 contain an oxy function at the
2'-position of ring B. Conversely, none of the 2'-deoxy compounds tested was
active (Table 2). When pairs of homologous compounds were compared, the 2'deoxy compounds, formononetin (XVIII), genistem (XV), methylequol (XXV),
and wighteone (XX) were inacnve (Table 2), whereas their 2'-hydroxy analogs,
XIX, XVI, IX, and XXI, were active (Table 1). Direct comparative assays of
several of these pairs proved less clear-cut (Table 3). The 2'-deoxy compounds
genistein (XV), formononetin (XVIII), and methylequol (XXV) were again
clearly inactive, and the difference between genistein (XVt and its 2'--hydroxy
homolog (XVI) was confirmed. However, a difference between formononetin
(XVIII) and 2'-hydroxyformononetin (XIX) and between (_+)-methylequol
(XXV) and (_+)-vestitol (racemic IX) could not be unequivocally established as
the 2'-hydroxy compounds on this occasion did not show a statistically significant dose-response slope. The cyclic ether homolog of (:t:_)-methylequol (XXV),
(+)-medicarpin (racernic XII) was also inactive, although (-)-medicarpin (XII)
is a marginally active feeding deterrent (Table 1). These results highlight the
difficulty of comparing inactive compounds with those of marginal activity, but
the balance of evidence supports the view that the difference in activity between
2'-deoxy compounds and their 2'-oxy counterparts is real. Several reactive or
FEEDING DETERRENTS
1727
marginally active compounds do contain a 2'-oxy moiety (X, XXVII, XXVIII,
XXII, XXIII, XXX1, VII), and the contrast between the inactivity of the coumestans (XXVII, XXVIII) and the active pterocarpans has already been noted.
Thus, while 2'-oxygenation is a feature of all the active feeding deterrents, ,its
occurrence does not always correlate with activity.
The second important structural feature is the presence of a ring B-fused
cyclic isoprenoid moiety (2,2-dimethyl-l,H-pyran). The significance of this feature is highlighted by the high absolute activity of phaseollin (I) and its homologs and by the high relative activity of compounds I and VI compared to compounds III, XII and VII, XVI respectively (Table 3). Phaseollin with a cyclic
isoprenoid group fused to ring B is much more active than (-)-medicarpin (XII)
and 75 times more active than phaseollidin (III) with a noncyclic isoprenyl group.
A similar tendency is seen when 2'-hydroxygenistein (XVI) is compared with
the complex isoflavones containing isoprenyl (VII) and cyclic isoprenoid (VI)
groups. Licoisoflavone B (VI) containing a ring B cyclic isoprenoid moiety is
68 times more active than 2'-hydroxygenistein (XVI) and the analog with a
noncyclic isoprenyl group (VII) gave a nonsignificant dose response.
The occurrence of a cyclic isoprenoid moiety on the isoflavonoid molecule
is not always associated with feeding-deterrent activity. Thus, angustone B
(XXIII) shows only marginal signs of activity, and the ring A isoprenoids, osajin
(XXIX) and pomiferin (XXX) are both inactive. On the other hand, of the highly
active compounds, only rotenone (VIII) does not have a cyclic isoprenoid moiety
fused to ring B, but rather, it contains an isoprenoid-derived furano group attached to ring A. The activity of this complex isoflavonoid is discussed below.
The high activity of the complex isoflavonoids prompted us to investigate
the feeding deterrent activity of some simple 2,2-dimethylchromenes (Table 4).
These compounds can be considered as partial structures to the ring B cyclic
isoprenoid isoflavonoids. Precocene I (XXXIV) and precocene II (XXXV) are
of particular interest as they have been found to act as antijuvenile hormones
against a range of insects by cytotoxic action on the corpora allata (Bowel"s,
1976, 1981). The chromenes XXXIV, XXXV, and XXXVII all showed appreciable feeding deterrent activity, while the chroman XXXVI was less active.
The chromene encecalin (analog of XXXIV, XXXV with R = COCH3) has
been reported as a feeding deterrent to Heliothis zea (Proksch and Rodriguez,
1983). The feeding-deterrent activity of these compounds may reflect structural
similarities to the cyclic isoprenoid isoflavonoids or may have an independent
basis.
Modes o f Action: Reactive Centers. In considering and interpreting the
pattern of results discussed thus far, we have not found any direct parallels with
previous studies of insect feeding deterrents. Oxygen functionality and oxidation state have been found to be significant factors in studies of the insect feeding-deterrent activity of phenolics. Elliger et al. (1980) found the inhibition of
growth of Heliothis zea larvae caused by the presence of a range of flavonoids
1728
LANEET AL.
in their food was associated with ring B ortho-dihydroxylation. Norris (1977)
found that the presence of a carbonyl and an adjacent hydroxyl in compounds
such as 5-hydroxynaphthoquinone, quercetin, and kaempferol gave high feeding
inhibition toward Scolytus multistriatus. He concluded that there was a strong
positive correlation of antifeeding activity with the degree of oxidation state of
ring C of the flavonoids and also with the intramolecular hydrogen-bonding capability of the molecule. Any correlation between the oxidation state of the isoflavonoids and the feeding response of Costelytra zealandica does not follow
the same pattern, since it is the reduced isoflavans and pterocarpans which are
more active than the highly oxidized isoflavones, coumestans, and quinones.
Sites for intramolecular hydrogen bonding are available around the carbonyl region in isoflavones, and it could be argued that this factor accounts for the higher
activity of the 2'-hydroxyisofiavones (VI, XVI, XIX, XXI). However, such a
mechanism cannot account for the activity of the 2'-hydroxyisoflavans (IV, IX)
and pterocarpans (I-III, XII, XIII). Moreover, the presence of a strongly intramolecularly hydrogen-bonded 5-hydroxy group in genistein (XV) and its homologs (VI, VII, XVI, XVII, XX-XXIII) does not seem to confer any special
increase in feeding-deterrent activity. Therefore, we conclude that our results
with C. zealandica feeding are not explained by the relative location of oxygen
functionality alone.
Reactive centers of the electron donor-acceptor type in close proximity, as
typified by the intramolecular hydrogen-bonding sites discussed above, have been
identified as the key structural feature for the activity of quinoid and flavonoid
(Norris, 1977) and isodon diterpenoid (Kubo and Genjian, 1981) feeding deterrents. The mode of action of such compounds as juglone, polygodial, and warburganal has been attributed to the interaction of the reactive electrophilic center
with the sulfhydryl site of a receptor macromolecule (Norris, 1976; Singer et
al., 1975, Ma, 1977). If such binding to sulfhydryl sites were important in the
gustatory response of C. zeaIandica, then the quinone isoflavonoids (XI, XXXI)
would be expected to be especially deterrent, but this was not observed. Further,
such structural features are not evident in the most highly active isoflavonoids
(I, II, IV-VI, VIII).
In Situ Activation. If isoflavonoid feeding deterrents are not characterized
by the presence of reactive electrophilic centers, they may be precursors of
reactive species generated in situ which could react with a receptor macrotmolecule. A number of possibilities are suggested by the literature. Recently,
!Bakker et al. (1983) have reported the photoactivation of several pterocarpan
~pbytoalexins to free-radical species which deactivated the enzyme glucose-6phosphate dehydrogenase in an in vitro assay system. Other photoactive compounds such as furocoumarins have been reported as insect feeding deterrents
(Yajima et al., 1977). The pertinence of a photoactivation mechanism to resistance to root-feeding larvae is not clear. In any case, the pattern of photoactiv-
FEEDING DETERRENTS
1729
ities reported by Bakker et al. (1983) [pisatin (XIV) > phaseollin (I); medicarpin (XII) inactive] differs from the pattern of feeding deterrent activity for these
compounds with C. zealandica (Table 1), and there was no exposure of materials to direct sunlight in the feeding assay procedure. While a photoactivation
mechanism seems unlikely, the involvement of free radical species in the insect
response to isoflavonoids cannot be excluded.
The reported oxidation of the cyclic isoprenoid moiety of precocene
(XXXIV) to a reactive epoxide in insect corpora allata (Pratt et al., 1980) indicates another mode of bioactivation of these molecules. The reduced activity
of dihydroprecocene II (XXXVI) compared to precocene II (XXXV) (Table 4)
suggests that the presence of the reactive double bond favors feeding-deterrent
activity, but this feature is clearly not essential for activity.
The oxidative formation of reactive ortho-quinone-methide species has been
invoked by Jurd et al. (1979) to explain the pattern of insect toxicity and chemosterilant activity of a range of benzylphenols. A similar process has been
suggested to account for the anti-juvenile hormone activity of a prenyl phenol
analogous to precocene II (XXXV) (Bowers et al., 1982), and valence isomerization to the corresponding quinone-methide has been suggested as a mode of
activation of the precocenes (XXXIV, XXXV) (Bowers, 1981). Chemical evidence has recently been obtained for the formation of para-quinone-methides
from 7-hydroxyflavans (Attwood et al., 1984). Of the isoflavonoids studied here,
those with a benzylic proton ortho or para to a phenolic hydroxyl, or with a
fused 2,2-dimethyl-l,4-pyranyl ring, can be considered as quinone-methide precursors. However, the presence or absence of these structural features alone
does not correlate with the observed pattern of feeding-deterrent activity (e.g.,
compare XVI, Table 1; and XX, Table 2).
Stereochemistry. From this discussion it is clear that reactivity factors
such as intramolecular hydrogen bonding, redox potentials, and the formation
of reactive species could be envisaged as accounting for the feeding-deterrent
activity of some of the active isoflavonoids but not in a consistent way. If we
make the assumption that there is a common basis to the observed pattern of
structure-activity relationships, it cannot be found in such reactivity considerations alone. This has led us to consider the stereochemical similarities of the
compounds, in particular phaseollin (I) and rotenone (VIII). These are both
highly active deterrents, have few reactive centers, and have rather rigid polycyclic structures with relatively few stable conformations. However, rotenone
(VIII) does not contain a ring B cyclic isoprenoid moiety characteristic of the
other highly active feeding deterrents, and the alignment of the 2'-oxygen is
quite different from that in the pterocarpans. Further, the absolute stereochemistry at C-3 is opposite to that for (-)-pterocarpans such as phaseollin (I) (Wong,
1975).
In a study of the bifunctional binding of rotenone with mitochondrial NADH
1730
LANE ET AL.
dehydrogenase, Yaguzhinskii and Kolesova (1975) concluded that the polar ring
B and the carbonyl group determine the interaction of the molecule with the
binding site. That this binding is stereospecific is indicated by the fact that
epimeric compounds are inactive, which the authors attributed to the inability
of the carbonyl to lie in the proper region at the phase boundary. The nature of
the furanoid moiety on ring A seemed to be less specific. Rotenone can thus be
considered as a nonplanar molecule with lipophilic and polar ends and containing a central dipole (Figure 2). This stereospecific enzyme binding model raises
the possibility that there might be a stereochemical basis for the observed pattern
of feeding deterrent activity, albeit less specific than for NADH dehydrogenase
inhibition.
We suggest that the common feature for the active feeding deterrents is that
they have, or can adopt, a similar nonplanar molecular shape with a similar
arrangement of polar and lipophilic groups. The molecular structures of phaseollin (I) and rotenone (VIII) have been the subject of both X-ray crystallographic
(DeMartinis et al., 1978; Arora et al., 1975) and NMR studies (Pachler and
Underwood, 1967; Perrin et al., 1972; Carlson et al., 1973) which indicated a
consistent shape in both the solid and solution phases. Both Dreiding models
and computer generated plots (PLUTO; Motherwell, 1976) (Figure 2) suggest
that when the molecules are appropriately aligned their stereochemistry is cornRotenone
FIG. 2. Projections of molecular structures of rotenone and phaseollin from X-ray crystal
coordinates.
FEEDING DETERRENTS
1731
parable. The ring A of phaseollin can be aligned with the ring B of rotenone so
that the dihydrofuran oxygen (2'-oxygen) occurs within the same region as the
important carbonyl of rotenone. The cyclic isoprenoid group is then in a similar
region to the dihydrofuran ring of rotenone and the phenolic hydroxyl of
phaseollin in a similar region to the two methoxyl groups of rotenone. This
comparison suggests a basis for the importance of the isoflavonoid 2'-oxy function, which could serve a similar role to the rotenone carbonyl in binding to a
macromolecule, and the importance of the cyclic isoprenoid group on ring B in
defining a lipophilic region of the molecule. Of the active chiral isoflavonoids
initially tested (Table 1), all the isoflavans and pterocarpans, with the exception
of ( +)-pisatin (XIV) are of the same absolute stereochemistry as ( -)-phaseollin
and are able to adopt a similar relative arrangement of 2'-oxygen and aromatic
rings. (+)-Pisatin (XIV) can be aligned similarly to rotenone with the angular
hydroxyl (on C-3) in the vicinity of the carbonyl group.
The inactive 2'-deoxyisoflavones (XV, XVII, XVIII, XX) and isoflavan
(XXV) cannot be aligned with a similar arrangement of groups, nor can the
planar coumestans (XXVII, XXVIII). The 2'-hydroxyisoflavones can be aligned
to some extent by rotation of ring B out of the plane of rings A and C, and most
of these compounds are active. The high activity of licoisoflavone B (VI) may
be associated with the possibility of aligning both the 2'-hydroxy and the cyclic
isoprenoid group similarly to phaseollin. In the inactive prenyl homolog (XXIII),
the molecule no longer has defined polar and lipophilic ends. The contrast between the activity of vestitol (IX) and the inactivity of sativan (X) may be associated with the steric effect of the 2'-O-methyl, as 2'-O-methyl phaseollin
isoflavan (V) also appears to be appreciably less active than its 2'-hydroxy counterpart (IV) (Table t). The activity of the simple chromenes (XXXIV, XXXV,
XXXVII) can be rationalized if they are considered to be partial phaseollin-like
structures corresponding to the ring B and cyclic isoprenoid regions. Thus, the
stereochemieal binding model does appear to be consistent with the observed
pattern of results discussed so far.
Some feeding assays were carried out, using available stereoisomers, to
investigate the importance of stereochemistry to feeding-deterrent activity of C.
zealandica larvae. The activity of rotenone was compared on several occasions
with that of epirotenone (XXXIII) which has the opposite configuration at the
junction of tings C-D. The NMR data (Methods) indicate an essentially enantiomeric relationship between rings C and D of rotenone and epirotenone and
hence an opposite sense of folding. The results (Table 3) show that epirotenone
is significantly less active than rotenone, although the difference is considerably
less than for enzyme inhibition (epirotenone 1000-fold less active, Yaguzhinskii
and Kolesova, 1975). Further, (+)-vestitol (XXXII) appeared to be less active
in a comparative assay with (-~-vestitol (IX) and synthetic racemic (+)-vestitol, although this difference was statistically not significant. Thus stereochemical considerations appear to be a factor in isoflavonoid feeding deterrent activ-
1732
LANE ET AL.
ity, and the relative activity of these stereoisomers can be related to how closely
they approximate a phaseollin-like shape.
CONCLUSIONS
Kojima and Kato (1981) concluded in their study of clerodin feeding deterrents that a definite steric environment around an active binding center is
required for high activity. Similarly, the active isoflavonoids (including rotenone)
can be considered as defining, in terms of stereochemistry, polarity, and lipophilicity, the environment of the key oxygen. Further elucidation of the stereochemical and possible reactivity factors involved in the feeding-deterrent activity
of isoflavonoids awaits the availability of suitable synthetic analogues.
The sensitivity of C. zealandica larvae to phaseollin, its analogs, and rotenone is remarkable. While our results suggest a common stereochemicat basis
for this phenomenon, the biochemical mechanism of this behavioral response
remains to be established. The ability of these root-feeding larvae to detect and
avoid low concentrations of the insect toxin, rotenone, normally found in roots
of several tropical legumes (e.g., Dalbergia and Tephrosia spp.) is intriguing
for its evolutionary implications. C. zealandica is indigneous to New Zealand
flora, although pterocarpans and isoflavans are present (Briggs et al., 1975;
Ingham, 1983).
The wide differences in activity we have found for isoflavonoid feeding
deterrents of the same structural class emphasize the need for quantitative methods and detailed analysis in determining the ecological role of plant secondary
metabolites. Thus, phaseollin is likely to play a significant role in defense against
insect attack at much lower plant concentrations than the simple pterocarpan,
medicarpin, and the insect resistance of legumes may relate to the structures of
the endogenous isoflavonoids. This raises the possibility of modifying the isoflavonoid biosynthetic pathway to enhance the resistance of a susceptible legume
toward the feeding of C. zealandica larvae.
Acknowledgments--We would like to thank Dr. L. Jurd and Professor O. Gottlieb for the gift
of samples, Mrs. C. Ford for the collection of grass grubs, and H. Wright, D. Gardiner, R. M. P,
Beeler, P. Dittmer, W. J. Greenfield, and A. Ngan for technical help.
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