Journal of Chemical Ecology, Vol. 15, No. 12, 1989
REIMER-TIEMANN ADDUCTS AS POTENTIAL INSECT ANTIFEEDANT AGENTS Reviewing the Structure-Activity Relationship Theory of the Antifeedant, Warburganal 1
G R E G O R Y L. F R I T Z , 2 G I L E S D. M I L L S , J R . , 3 J. D A V I D W A R T H E N , J R . , 2 and R O L L A N D M. W A T E R S 2 21nsect Chemical Ecology Lab, Plant Sciences Institute 3Livestock Insects Lab, Livestock Poultry Sciences Institute U.S. Department of Agriculture, Agricultural Research Service Beltsville, Maryland 20705 (Received July 7, 1988; accepted January 19, 1989) Abstract--Structure-activity relationships of naturally occurring enedials with antifeedant activity against Spodoptera species have been extended via the synthesis and bioassay of a series of Reimer-Tiemann adducts. The activities attributed to the different chemical structures of these and other analogs interacting with the chemoreceptor site have been observed; a three-pronged mode of substrate binding via aromatic pyrrole formation, Michael addition of free sulfhydryl moieties, and van der Waals interactions of the A ring has been postulated to account for these observations. Key Words--Enedial, Spodoprera, Lepidoptera; Noctuidae, feeding deterrent, pyrrole, sulfhydryl, van der Waals interaction, Reimer-Tiemann adducts.
INTRODUCTION Interest in the protection m e c h a n i s m that plants use against pest insects p r o m p t e d the isolation and identification o f the insect antifeedant Warburgia sesquiterp e n e d i a l d e h y d e s (Barnes and L o d e r , 1962; B r o o k s and Draffen, 1969). S o m e o f these enedial c o m p o u n d s are p o l y g o d i a l (1), warburganal (2), and m u z i g a d i a l (3) ( S c h e m e 1), all o f w h i c h also exhibit o t h e r b i o l o g i c a l activity including: fish LNames of products in this paper are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA. 2607
2608
FP~TZET AL. Cl
c9
c2~
c8 c7
c3
c4
CHO
POLYGODIAL 1
c6
CHO
WARBURGANAL 2
CHO
MUZ|GADIAL 3
SCHEME 1. toxicity (Asakawa et al., 1985), hot taste to humans (Govindarajan, 1979), inhibition of alcoholic fermentation (Kubo et al., 1983), and antimicrobial activity (Kubo et al., 1984). The ability of these compounds to inhibit feeding of Spodoptera spp. at _>0.1 ppm (Kubo et al., 1977; Sodano et al., 1987) initiated interest in the syntheses of warburganal (2) (Kende and Blacklock, 1980) and cinnamodial (4) (White and Burton, 1985), as well as numerous analogs (5-12) and intermediates (Table 1). The observed biological activity led to four proposals that relate chemical structure to activity: The first suggests that when the C-8 enal moiety is allowed to react with the free sulfhydryl groups of the insect's chemoreceptor membranes, there is inhibition of stimuli transduction (Ma, 1977; Rozental et al., 1975). In support of this proposal, it was observed that o~,/3-unsaturated aldehydes inhibited papain (an enzyme containing 1 mol of active --SH) while benzaldehyde had virtually no effect. This type of reactivity is indicative of the addition of a free --SH moiety to the /3-sp 2 hybridized carbon atom as in a Michael addition (Scheme 2) (Kubo and Ganjian, 1981) and can be easily monitored by measuring the disappearance of the UV absorption band (Xmax 229 nm) of the o~,(3-unsaturated aldehyde. Kubo et al. (1976) found that addition of L-cysteine to a solution of polygodial at pH 7 or 9 (phosphate buffer) produced an absorbance change, as well as a decrease in the insect antifeedant activity. 4 Several other SH-containing compounds produced similar results under these 4When the dialdehyde was oxidized to the diacid, the corresponding methyl diester [8] with the con'ect C-9 configuration showed no feeding deterrent activity at 100 ppm, indicating that the aldehydic functionalities at C-8 and C-9 are important to the biological activity (Warthen et al., 1983).
STRUCTURE-ACTIVITYFOR ANTIFEEDANTS
2609
TABLE 1. ACTIVE AND INACTIVE ENEDIAL ANALOGS a ACTIVE
MODE OF ACTION
INACTIVE
O-D A B C D E
POLYGODIAL 1 C~
EPIPOLYGODIAL 7 II CO2Me ~ G O 2 M e
CH3 A B C D E
WARBURGANAL 2
~
ANALOG 8
O-t3
A C D E
MUZIGADIAL 3 CI4D
ANALOG 9
A C
O
CINNAMODIAL 4
ANALOG 10
A C ANALOG 5
ANALOG 11 CHO
CH~ A
~RENYL SACCALUTAL 6
B
C
~RENYL nSOSACCALUTAL 12
"(A) Insect antifeedant; (B) Fish toxicant; (C) Hot taste to humans; (D) Papain inhibitor; (E) Antimicrobial agent.
2610
FRITZ ET A L .
~
CHO
CHO CHO
§
H S-CYS
POLYGODIAL 1
NO UV ABS.
CHO +
H S CYS
NO REACTION
BENZALDEHYDE
SCHEME 2.
conditions, while no absorbance change was observed when the polygodial solution was treated with a variety of amino acids that contained no --SH moiety. However, because both the active polygodial (1) and the inactive epipolygoidal (7) showed a similar absorbance decrease upon reaction with L-cysteine; there was speculation that the stereochemistry of polygodial was an important factor influencing biological activity since polygodial differs from epipolygodial only in the configuration of the C-9 aldehyde (Scheme 3a). The absorbance decrease produced by the reaction of polygodial and L-cysteine occurs three times faster than that produced when epipolygodial is allowed to react with L-cysteine. Clearly, the configuration of the C-9 substituent plays a critical role by interacting with approaching nucleophiles and, consequently, may be important for binding of the substrate to the receptor site. Kubo et al. (1984) tested this hypothesis by examining the interactions of the C-9 methanol analogs of polygodial and epipolygodial with L-cysteine. The 9-/3 alcohol (13) showed no UV absorbance even before the addition of L-cysteine, since an intramolecular addition of the CHzOH hydroxyl moiety to the C-8 carbonyl had produced a hemiacetal furan (14). The UV absorbance (c~,/3unsaturated aldehyde) of the epipolygodial analog (15) was virtually unchanged after the addition of L-cysteine, supporting the hypothesis that the C-9 aldehydic substituents in the s-configuration inhibit addition of the free --SH moiety to the double bond of epipolygodial (Scheme 3b). However, Kubo et al. (1984) did not explain why the 9-/3 alcohol (13) formed a furan ring via intramolecular hemiacetal formation while the 9-~ alcohol (15) did not react in this fashion. Sodano et al. (1982) provided the second proposal to explain the biological activity of these enedials and the differences in activity between polygodial and epipolygodial. They postulated that pyrrole formation by reaction of the C-8, C-9 dialdehyde moieties with a primary amine of the receptor site was responsible for the antifeedant activity. Under biomimetic conditions, polygodial was allowed to react with a variety of primary amines, including: L-cysteine, L-lysine, L-alanine, and methylamine. In each case the amine caused a decrease
STRUCTURE-ACTIVITY
2611
FOR ANT1FEEDANTS
~
CH3
CH3
H
POLYGODIAL 1
EPIPOLYGODiAL 7
9- [3 CONFIGURATION
9- c~ CONFIGURATION
OH CH3
H
HSR
ALCOHOL
13
ALCOHOL
9- 13 CONFIGURATION
~
15
9- o~ CONFIGURATION
OH
NO REACTION
H
HEMIACETAL FURAN
14
SCHEME 3.
in UV absorbance associated with the ~,/3-unsaturated aldehyde. Sodano et al. (1982) used Drieding models to calculate the intramolecular distances between the aldehyde carbonyls of polygodial and epipolygodial, respectively (Scheme 4); they concluded that the 9-/3 isomer's carbonyl groups were close enough to allow for intramolecular ring closure while the 9-c~ isomer's carbonyls were too far apart for this to occur, thereby showing that the configuration of the C-9 aldehyde plays a critical role in the binding of the substrate to the chemoreceptor site. Additional evidence was obtained to support this hypothesis when two new
2612
FRITZ ET AL.
~
-
C
H
3
~
C
H
3
H POLYGODIAL1 DISTANCE= 2.7 A 9- [~ CONFIGURATION
EPIPOLYGODIAL7 DISTANCE = 3.0 A 9- o~ CONFIGURATION SCHEME 4.
intermediates (16 and 17) were identified from the various reactions of primary amines on the ~,3-unsaturated aldehydic carbonyl moiety of polygodial (Scheme 5) (Sodano et al., 1984). The pyrrole formation was only observed for molecules that had the appropriate stereochemical configuration (i.e., the 9-3 of the trans-fused decalins) to allow intramolecular cyclization. Sodano et al, (1984) suggested that this information confirmed that pyrrole formation was responsible for the observed biological activity. The third proposal relating the chemical structure of the enedials to biological activity was also made by Sodano et al. (1987), when it was observed that the steroidal analogs (18 and 19) of polygodial, having the identical C-9 polygodial stereochemical configuration (Scheme 6), were inactive. These authors claimed that the large steroid ring system inhibited the molecule from reaching the active site, thereby preventing any reaction with the --NH2. Thus, there was a molecular size limitation even though the polygodial functionality and stereochemical backbone were present. The fourth proposal by these same authors originated because the addition of a prenyl substituent to the ~-C-4 methyl group of polygodial (saccalutal, 6) and epipolygodial (isosaccalutal, 12) had no effect on the activity of either molecule. Thus, it seems as though a change of the alkyl substituent on C-4 of polygodial has no effect on insect antifeedant activity. This paper reports new information relating the chemical structure of enedial analogs to insect antifeedant activity.
OHC
NR
N* R
PRODUCT 16
SCHZME5.
NR
PRODUCT 17
2613
STRUCTURE-ACTIVITY FOR ANTIFEEDANTS
AcO
ANALOG
O-13
CI-D
18
ANALOG
19
SCHEME6.
METHODS
AND MATERIALS
Gas-liquid chromatography (GLC) analyses were performed on a Hewlett Packard 5830A instrument equipped with a split injector (flame ionization detector, He cartier gas). A DURAWAX-DX1 (J&W Scientific, Inc.) capillary column (0.25 mm x 15 m) was operated at the temperature ranges indicated. Infrared (IR) spectra were obtained with a Nicolet 60SX Fourier Transform IR spectrometer using neat sample smears between KC1 plates. 1H nuclear magnetic resonance (NMR) spectra, unless otherwise stated, were obtained with a Nicolet (General Electric) QE 300 MHz instrument. All spectra were obtained using deuterochloroform (CDC13) solutions with tetramethylsilane (TMS) as an internal standard. Flash chromatography by the procedure of Still (1984) was performed using Bio-Rad silicic acid-HA (-325 mesh). Analytical thin-layer chromatography (TLC) was performed on prescored silica gel GF (250-1zm) plates (Analtech, Inc). Hydrogenations were performed using a Pressure Reaction. Apparatus (Parr Instrument Company, Inc.). All commercially obtained chemicals were used without further purification.
Chemicals 6-Dichloromethyl-6-methyl-3- (2-propyl)-2, 4-cyclohexadienone (20). The dienone (Scheme 7) was prepared in 8 % yield and had physical constants identical to those first reported by Wenkert (1970), who had prepared this compound in a similar yield by refluxing a two-phase mixture containing a chloroform solution of the appropriate phenol, carvacrol, and - 1 0 % aq. NaOH solution. The pure sample (flash chromatography, hexane) was subjected to GLC analysis with an oven temperature at 70~ for 1 min and then programmed at 5~ rain; 98% purity, Rt = 17.36 min. IR (neat) 1662 ( C = O ) , 1642 cm -1 ( C = C ) . 6-Dichloromethyl-2,6-dimethyl-2, 4-cyclohexadienone (21). The dienone was prepared in 17% yield as described for compound 20 and was purified by flash chromatography (hexane). The pure sample was subjected to GLC analysis with an oven temperature at 90~ for 1 rain and then programmed at 5~
2614
FRITZET AL. OH
0
H , ~
: C CIz
H
~ HCI2
COMPOUND
OH
20
: C el=
~
C HCI
H
H COMPOUND
21
SCHEME 7.
min; 99% purity, R t = 7.16 min. IR (neat) 1662 ( C = O ) , 1650 cm -1 (C----C); [IH]NMR (CDC13) 6 1.30 (s, 3H, 6--CH3), 1.92 (bs, 3H, CH3C~C), 6.09 (s, 1H, --CClzH), 6.40 (dd, 1H, J --- 7.5, 11 Hz, C - - 4 H), 6.58 (d, 1H, J = 11 Hz, C--5 H), 6.90 (bd, 1H, J = 7.5 Hz, C - - 3 H). cis- and trans-2-Dichloromethyl-2-methyI-5-(2-propyl)cyclohexanone (22). An ethanol solution (50 ml) of dienone 20 (2.00 g, 8.58 retool) was hydrogenated over 10% Pd/C at 50 psi for 24 hr and filtered over Celite. The solvent was removed in vacuo; the residue was purified by flash chromatography (petroleum ether, 35-60 ~ to give a 57 % yield of product (1.16 g, Scheme 8). GLC O H--...~
HCI2
,//
H2
// H3
~'~
CHCI2
+
CHCI2
COMPOUND
20
TRANS
C H3
COMPOUND 22
MAJOR ISOMER
"CIS COMPOUND 22 3
: 1
MINOR ISOMER
p
0
CHCI2
o
O
H~
C HCI2 COMPOUND
21
TRANS COMPOUND 23 MAJOR ISOMER
SCHEME 8.
CIS COMPOUND 23 15
:
1
MINOR ISOMER
STRUCTURE-ACTIVITY FOR ANTIFEEDANTS
2615
analysis with an oven temperature at 70~ for 1 rain and then programmed at 5~ revealed the presence of a 3 : 1 mixture of two diastereomers; R, = 17.34 and 17.67 rain, respectively. No attempt was made to isolate the individual isomers. Wenkert (1970) obtained a 4 : 1 ratio of these isomers; the isomer in the greater quantity (Scheme 8) had the isopropyl and methyl groups trans to each other. In our mixture the isomer in the greater quantity was identical to the trans isomer isolated by Wenkert (1970). IR (neat) 1716 ( C = O ) , 762 cm -1 (C--C1); [IH]NMR (CDC13) 6 0.92 [m, 6H, (CH3)2CH--], 1.27 and 1.32 (s, 3H, trans isomer - - C - - C H 3 and cis isomer - - C - - C H 3, respectively), 1.32-2.49 (m, 8H), 6.19 and 6.29 (s, 1H, cis isomer --CC12H and trans isomer --CC12H, respectively). cis- and trans-2-dichloromethyl-2,6-dimethylcyclohexanone (23). Dienone 21 (4.83 g, 19 mmol) was hydrogenated as described for compound 22 to give a 91% yield of 23. GLC analysis with an oven temperature of 90~ for 1 rain and then programmed at 5~ revealed a 1 : 15 ratio 5 of isomers; R, = 7.69 and 7.73 rain, respectively. IR (neat) 1702 cm 1 ( C = O ) ; [IH]NMR (CDC13) 6 1.05 (d, 3H, J = 6.0 Hz, C H_3CH--), 1.37 (s, 3H, - - C - - C H 3 ) , 1.30-2.20 (m, 6H, --C--(CHH2)3--CH--), 2.56 (m, 1H, CH3C__HCH2--), 6.20 ppm (s, 1H, --CClzH ). Bioassay Fall armyworm larvae, Spodopterafrugiperda (J.E. Smith), from our stock culture, reared on an artificial diet (Redfern and Raulston, 1970), were used for antifeedant tests. The samples were dissolved at an appropriate concentration in a 50:50 acetone-dimethyl sulfoxide solvent mixture; the resultant solution was added to a hot diet and mixed by using a 1/2-pint (720 ml) jelly jar fitted with an Osterizer blender cutter head. After the treated diet was blended for approximately 1 min, it was poured into 1-oz clear plastic cups ( - 8 g/cup) and allowed to cool to room temperature. Control diets were only treated with the neat solvent. One first-instar larva (newly emerged) was placed in each cup (10 cups for each sample concentration). The cups were then capped with prepunched lids and placed in a holding room at 27 ~ _+ 1 ~ with 50 + 5 % relative humidity. The larvae were observed for mortality, and the live larvae weighed on the sixth or seventh day after introduction into the cups. The sum of weights for the 10 repetitions per sample, as well as the control, were then divided by
5The isomerization of the 15:1 mixture was accomplished by the addition of lithium diisopropylamide to a THF solution ( - 7 8 ~ of 23, stirring for 2 hr as the temperature rose to 26~ and then acidifying with 10% (v/v) aq. HCI. GLC revealed the presence of a 50:50 mixture. Repetitive flash chromatography (petroleum ether, 35-60~ gave a fraction in which the new isomer predominated in a 60:40 ratio. IR (neat) 1702 cm ~ ( C = O ) ; [tH]NMR (CDCI3) 6 1.05 (d, 3H, J = 6.0 Hz, CH3CH), 1.27 (s, 3H, 2-CH3), 1.32-2.19 (m, 5H), 2.30 (ddd, 1H, J = 3.9, 6.6, 14 Hz; C-4 Hax), 2.59 (m, 1H, CH3CHCH2--), 6.40 (s, 1H, --CC12H).
2616
FRITZ ET AL.
the number of live larvae weighed in order to obtain an average larval weight for each sample. The ratio of these average larval weights (treated/control) was then used to assess the antifeedant activity of the various samples (Table 2). RESULTS AND DISCUSSION
The work of Kubo and Sodano has been very thorough. However, there are still several questions to be addressed that relate the structures of the enedials to their antifeedant activity, namely: (1) are both the C-8 and C-9 aldehydic oxidation states necessary for antifeedant activity at low sample concentrations ( _ 10 ppm); (2) if pyrrole formation is the most important reaction for activity, does the --SH moiety react in conjunction with the NH2 to enhance the activity; (3) why does the addition of the c~-OH at C-9 of warburganal enhance the antifeedant activity compared to polygodia~ (no c~-OH, Scheme 1); (4) if steric bulk inhibits the approach of the substrate tO the receptor site, what effect will a truncated natural product analog have; and (5) what effect do the alkyl substituents have on the antifeedant activity? The first conclusion that can be drawn from the data in Table 1 is that the C-8 and C-9 carbon atoms must have their substituents in the aldehydic oxidation state in order to demonstrate potent antifeedant activity. This is especially apparent when comparing the structural differences of polygodial (1) and the diester analog (8) (Scheme 9). Both molecules would be expected to form a cyclic intermediate, since both corresponding/3-C-9 carbonyl moieties should be in close enough proximity to the C-8 carbonyl to allow the intramolecular cyclization (see above). Once cyclization has occurred in both systems, only the intermediate arising from the dialdehyde can proceed to form the aromatic pyrrole (24). At this point it becomes necessary to interject our viewpoint that the sulfhydryl attack on the /3-carbon of the enal, as proposed by Kubo and TABLE 2. ANTIFEEDANT ACTIVITY OF SYNTHETIC COMPOUNDS 2 0 - 2 3 a
Sample Compound Compound Compound Compound Control
Conc. (ppm) 20 21 22 b 23 c
1000 1000 1000 1000 1000
Mortality 1 of 0 of 1 of 1 of 1 of
10 10 10 10 10
% wt. of control 103 21 99 28 100
~No antifeedant activity was observed for any sample at 100 ppm. Percent weight was calculated as described in Methods and Materials. b 1 : 3 mixture of cis and trans isomers. c 1 : 15 mixture of cis and trans isomers.
2617
STRUCTURE-ACTIVITY FOR ANT1FEEDANTS
POLYGODIAL 1
HO
DIESTER 8
NA *
O~..._._NR 0
I HSR
l
HSR
REACTION R PYRROLE 24 SCHEME 9.
Ganjian (1981), and the pyrrole formation by NH2 attack on the aldehyde carbonyls, as proposed by Sodano et al. (198'2), are not necessarily independent reactions. One can hypothesize that both the L-lysine --NH2 and the L-cysteine --SH moieties of the chemoreceptor site complex with the substrate in accordance with both the Sodano and Kubo mechanisms in order to produce the biological activity (Scheme 10a). Initial imine formation at the C-8 carbonyl followed by fl-addition of the SH and subsequent ring closure and pyrrole aromatization would produce a product similar to compound 24. This mechanism could explain why warburganal is a more potent antifeedant than the simple polygodial. For example, if the c~-C-9 hydroxide were displaced by an SN2' attack of the SH moiety on the fl-carbon of the enal, the resultant alkene would force the C-8 and C-9 substituents into an even closer proximity; thereby enhancing the pyrrole formation (Scheme 10b).
2618
FRITZ ET AL.
~
CHO CHO
l - H~O (31-13
N --LYS
/
~
-s--cYs
CHO ICHO
f
v
T CI-O
"~S-- CYS
"
HO
N --LYS
SCHEME 10.
There are other mechanisms by which SH moieties are known to react in vivo. One of the more common reactions is the oxidative decarboxylation reaction of the acid dehydrogenase (Scheme 1 la) (Lehninger, 1975). This type of reaction could occur with compounds like warburganal (Scheme 1 lb) to generate an intermediate (25), which would then induce the antifeedant activity; our synthetic compounds, 20 and 21, would be expected to react in a similar fashion with the sulfhydryl moiety via Michael addition to the alkene. The synthetic compounds 20-23 would also be expected to form an imine via coupling of the substrate's dichloromethyl moiety (carbon in the aldehydic oxidation state) and amine groups of the active site in a manner identical to that expected of the aldehyde moiety of the warburganal metabolite, 25. Compounds 20-23 would
STRUCTURE-ACTIVITY
2619
FOR ANTIFEEDANTS
C (Me) 0 J
C=O
+
HS--CoA
+
,'
NAD+
c~
+
Ac S--Co A
+
NADH
OH
~
CHO CHO
HS-ENZ
9
RS--ENZ
NAD+
9 NADH *
WARBURGANAL
~
0 CHO
25
0
...... I~ 20
22
O
O
C HOlz
21
"
~
ANTIFEEDANT
ACTIVITY
C
HCI~
23
SCHEME
1 1.
therefore be expected to be as active as warburganal since the chemical reactivity is similar to that proposed for the intermediate (25) and other quinones that are known to elicit antifeedant activity (Rozental et al., 1975). Unfortunately, all the synthetic compounds were inactive at 100 ppm, and this mechanistic pathway was therefore discarded in favor of the one shown in Schemes 10a and 10b. Obviously, these cyclohexane molecules are not inactive at the 100-ppm level because their steric bulk prevents binding with the chemoreceptor site, as in the case of the steroidal analogs of polygodial. Since the synthetic samples are not sterically prohibited from reaching the receptor site, it must be the inability of compounds 20-23 to form the pyrrole ring that accounts for their inactivity at low sample concentration. This observation supports the idea that the C-8 and C-9 aldehydes are necessary for antifeedant activity at lower concentrations. However, the 1000-ppm test results indicate that some dramatic differences exist between the inactive compounds, 20 and 22, and the active compounds, 21 and 23.
FRITZET AL.
2620 0
O HCI2 RS-~ ~ 2 0 /
NO
O CHClz
RS-H
RS-H
21
23
l
0
NO REACTION
REACTION R s ~ C
CHCI2
HCla
SCHEME 12.
Compounds 20 and 21 differ only in the alkyl substitution pattern (Scheme 12). Our reasoning used to explain the difference in activity was that the two methyl groups of the C-3 isopropyl substituent on compound 20 were sterically blocking the SH from adding to the/3-carbon of the c~,/3-unsaturated ketone in the same manner that the 9-c~ CHzOH prevented sulfhydryl coupling in alcohol 15 (Scheme 3b). This could not explain why the saturated analog 23 was nearly as active as compound 21 at 1000 ppm (72 % and 79 % weight reduction, respectively) since 23 had no alkene with which the SH could react. In hindsight, it was no surprise that compounds 20 and 22 were inactive, since it had previously been demonstrated that the alkyl substitution changes that produced saccalutal (6) from polygodial (1) and isosaccalutal (12) from epipolygodial (7) had had virtually no effect on the activity of the compounds. Apparently then, it is the C-2 methyl group of compound 21 and the corresponding C-6 methyl of compound 23 that was responsible for the difference in activity between these two active compounds and their inactive counterparts, 20 and 22. We noted that the bridge-head methyl group had been reported to exist exclusively in the axial position in all of the naturally occurring antifeedant compounds 1-4, and 6 (Table 1), while the C-2 methyl group of compound 21 and the C-6 methyl group of compound 23 were both equatorial (the C-2 methyl of compound 21 lies in the plane of the cyclohexadiene ring and can therefore be formally considered equatorial, Schemes 7 and 8). Epimerization of compound 23 produced only equatorial methyl groups, as determined by proton NMR analysis (Scheme 13). Alkyl groups have been known to effectively bind to chemoreceptor sites via dispersion and van der Waals forces that require tight fits to the active site since the magnitude of these attractive forces decreases rapidly with distance (Liljefors and Thelin, 1985). There is, therefore, precedence to expect that an
-~
23
CH3
'5 = 1.37
HCI2~C % O
TRANS
'5 = 6.20
H
'5 = 1.32 - 2.20
8 =
.05
1 2)
H+
- - t b . -
1) LDA
SCHEME 13.
HClz C ~ 0
,5
,5
H
= 2.32
CIS
= 1.27
23
H)C%o
~-~CH3
8 = 6.40
'5 = 1.05
i'-o o'-, i-o
2:
['11
> Z ,.q
0
,..
REIMER-TIEMANN ADDUCTS AS POTENTIAL INSECT ANTIFEEDANT AGENTS Reviewing the Structure-Activity Relationship Theory of the Antifeedant, Warburganal 1
G R E G O R Y L. F R I T Z , 2 G I L E S D. M I L L S , J R . , 3 J. D A V I D W A R T H E N , J R . , 2 and R O L L A N D M. W A T E R S 2 21nsect Chemical Ecology Lab, Plant Sciences Institute 3Livestock Insects Lab, Livestock Poultry Sciences Institute U.S. Department of Agriculture, Agricultural Research Service Beltsville, Maryland 20705 (Received July 7, 1988; accepted January 19, 1989) Abstract--Structure-activity relationships of naturally occurring enedials with antifeedant activity against Spodoptera species have been extended via the synthesis and bioassay of a series of Reimer-Tiemann adducts. The activities attributed to the different chemical structures of these and other analogs interacting with the chemoreceptor site have been observed; a three-pronged mode of substrate binding via aromatic pyrrole formation, Michael addition of free sulfhydryl moieties, and van der Waals interactions of the A ring has been postulated to account for these observations. Key Words--Enedial, Spodoprera, Lepidoptera; Noctuidae, feeding deterrent, pyrrole, sulfhydryl, van der Waals interaction, Reimer-Tiemann adducts.
INTRODUCTION Interest in the protection m e c h a n i s m that plants use against pest insects p r o m p t e d the isolation and identification o f the insect antifeedant Warburgia sesquiterp e n e d i a l d e h y d e s (Barnes and L o d e r , 1962; B r o o k s and Draffen, 1969). S o m e o f these enedial c o m p o u n d s are p o l y g o d i a l (1), warburganal (2), and m u z i g a d i a l (3) ( S c h e m e 1), all o f w h i c h also exhibit o t h e r b i o l o g i c a l activity including: fish LNames of products in this paper are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA. 2607
2608
FP~TZET AL. Cl
c9
c2~
c8 c7
c3
c4
CHO
POLYGODIAL 1
c6
CHO
WARBURGANAL 2
CHO
MUZ|GADIAL 3
SCHEME 1. toxicity (Asakawa et al., 1985), hot taste to humans (Govindarajan, 1979), inhibition of alcoholic fermentation (Kubo et al., 1983), and antimicrobial activity (Kubo et al., 1984). The ability of these compounds to inhibit feeding of Spodoptera spp. at _>0.1 ppm (Kubo et al., 1977; Sodano et al., 1987) initiated interest in the syntheses of warburganal (2) (Kende and Blacklock, 1980) and cinnamodial (4) (White and Burton, 1985), as well as numerous analogs (5-12) and intermediates (Table 1). The observed biological activity led to four proposals that relate chemical structure to activity: The first suggests that when the C-8 enal moiety is allowed to react with the free sulfhydryl groups of the insect's chemoreceptor membranes, there is inhibition of stimuli transduction (Ma, 1977; Rozental et al., 1975). In support of this proposal, it was observed that o~,/3-unsaturated aldehydes inhibited papain (an enzyme containing 1 mol of active --SH) while benzaldehyde had virtually no effect. This type of reactivity is indicative of the addition of a free --SH moiety to the /3-sp 2 hybridized carbon atom as in a Michael addition (Scheme 2) (Kubo and Ganjian, 1981) and can be easily monitored by measuring the disappearance of the UV absorption band (Xmax 229 nm) of the o~,(3-unsaturated aldehyde. Kubo et al. (1976) found that addition of L-cysteine to a solution of polygodial at pH 7 or 9 (phosphate buffer) produced an absorbance change, as well as a decrease in the insect antifeedant activity. 4 Several other SH-containing compounds produced similar results under these 4When the dialdehyde was oxidized to the diacid, the corresponding methyl diester [8] with the con'ect C-9 configuration showed no feeding deterrent activity at 100 ppm, indicating that the aldehydic functionalities at C-8 and C-9 are important to the biological activity (Warthen et al., 1983).
STRUCTURE-ACTIVITYFOR ANTIFEEDANTS
2609
TABLE 1. ACTIVE AND INACTIVE ENEDIAL ANALOGS a ACTIVE
MODE OF ACTION
INACTIVE
O-D A B C D E
POLYGODIAL 1 C~
EPIPOLYGODIAL 7 II CO2Me ~ G O 2 M e
CH3 A B C D E
WARBURGANAL 2
~
ANALOG 8
O-t3
A C D E
MUZIGADIAL 3 CI4D
ANALOG 9
A C
O
CINNAMODIAL 4
ANALOG 10
A C ANALOG 5
ANALOG 11 CHO
CH~ A
~RENYL SACCALUTAL 6
B
C
~RENYL nSOSACCALUTAL 12
"(A) Insect antifeedant; (B) Fish toxicant; (C) Hot taste to humans; (D) Papain inhibitor; (E) Antimicrobial agent.
2610
FRITZ ET A L .
~
CHO
CHO CHO
§
H S-CYS
POLYGODIAL 1
NO UV ABS.
CHO +
H S CYS
NO REACTION
BENZALDEHYDE
SCHEME 2.
conditions, while no absorbance change was observed when the polygodial solution was treated with a variety of amino acids that contained no --SH moiety. However, because both the active polygodial (1) and the inactive epipolygoidal (7) showed a similar absorbance decrease upon reaction with L-cysteine; there was speculation that the stereochemistry of polygodial was an important factor influencing biological activity since polygodial differs from epipolygodial only in the configuration of the C-9 aldehyde (Scheme 3a). The absorbance decrease produced by the reaction of polygodial and L-cysteine occurs three times faster than that produced when epipolygodial is allowed to react with L-cysteine. Clearly, the configuration of the C-9 substituent plays a critical role by interacting with approaching nucleophiles and, consequently, may be important for binding of the substrate to the receptor site. Kubo et al. (1984) tested this hypothesis by examining the interactions of the C-9 methanol analogs of polygodial and epipolygodial with L-cysteine. The 9-/3 alcohol (13) showed no UV absorbance even before the addition of L-cysteine, since an intramolecular addition of the CHzOH hydroxyl moiety to the C-8 carbonyl had produced a hemiacetal furan (14). The UV absorbance (c~,/3unsaturated aldehyde) of the epipolygodial analog (15) was virtually unchanged after the addition of L-cysteine, supporting the hypothesis that the C-9 aldehydic substituents in the s-configuration inhibit addition of the free --SH moiety to the double bond of epipolygodial (Scheme 3b). However, Kubo et al. (1984) did not explain why the 9-/3 alcohol (13) formed a furan ring via intramolecular hemiacetal formation while the 9-~ alcohol (15) did not react in this fashion. Sodano et al. (1982) provided the second proposal to explain the biological activity of these enedials and the differences in activity between polygodial and epipolygodial. They postulated that pyrrole formation by reaction of the C-8, C-9 dialdehyde moieties with a primary amine of the receptor site was responsible for the antifeedant activity. Under biomimetic conditions, polygodial was allowed to react with a variety of primary amines, including: L-cysteine, L-lysine, L-alanine, and methylamine. In each case the amine caused a decrease
STRUCTURE-ACTIVITY
2611
FOR ANT1FEEDANTS
~
CH3
CH3
H
POLYGODIAL 1
EPIPOLYGODiAL 7
9- [3 CONFIGURATION
9- c~ CONFIGURATION
OH CH3
H
HSR
ALCOHOL
13
ALCOHOL
9- 13 CONFIGURATION
~
15
9- o~ CONFIGURATION
OH
NO REACTION
H
HEMIACETAL FURAN
14
SCHEME 3.
in UV absorbance associated with the ~,/3-unsaturated aldehyde. Sodano et al. (1982) used Drieding models to calculate the intramolecular distances between the aldehyde carbonyls of polygodial and epipolygodial, respectively (Scheme 4); they concluded that the 9-/3 isomer's carbonyl groups were close enough to allow for intramolecular ring closure while the 9-c~ isomer's carbonyls were too far apart for this to occur, thereby showing that the configuration of the C-9 aldehyde plays a critical role in the binding of the substrate to the chemoreceptor site. Additional evidence was obtained to support this hypothesis when two new
2612
FRITZ ET AL.
~
-
C
H
3
~
C
H
3
H POLYGODIAL1 DISTANCE= 2.7 A 9- [~ CONFIGURATION
EPIPOLYGODIAL7 DISTANCE = 3.0 A 9- o~ CONFIGURATION SCHEME 4.
intermediates (16 and 17) were identified from the various reactions of primary amines on the ~,3-unsaturated aldehydic carbonyl moiety of polygodial (Scheme 5) (Sodano et al., 1984). The pyrrole formation was only observed for molecules that had the appropriate stereochemical configuration (i.e., the 9-3 of the trans-fused decalins) to allow intramolecular cyclization. Sodano et al, (1984) suggested that this information confirmed that pyrrole formation was responsible for the observed biological activity. The third proposal relating the chemical structure of the enedials to biological activity was also made by Sodano et al. (1987), when it was observed that the steroidal analogs (18 and 19) of polygodial, having the identical C-9 polygodial stereochemical configuration (Scheme 6), were inactive. These authors claimed that the large steroid ring system inhibited the molecule from reaching the active site, thereby preventing any reaction with the --NH2. Thus, there was a molecular size limitation even though the polygodial functionality and stereochemical backbone were present. The fourth proposal by these same authors originated because the addition of a prenyl substituent to the ~-C-4 methyl group of polygodial (saccalutal, 6) and epipolygodial (isosaccalutal, 12) had no effect on the activity of either molecule. Thus, it seems as though a change of the alkyl substituent on C-4 of polygodial has no effect on insect antifeedant activity. This paper reports new information relating the chemical structure of enedial analogs to insect antifeedant activity.
OHC
NR
N* R
PRODUCT 16
SCHZME5.
NR
PRODUCT 17
2613
STRUCTURE-ACTIVITY FOR ANTIFEEDANTS
AcO
ANALOG
O-13
CI-D
18
ANALOG
19
SCHEME6.
METHODS
AND MATERIALS
Gas-liquid chromatography (GLC) analyses were performed on a Hewlett Packard 5830A instrument equipped with a split injector (flame ionization detector, He cartier gas). A DURAWAX-DX1 (J&W Scientific, Inc.) capillary column (0.25 mm x 15 m) was operated at the temperature ranges indicated. Infrared (IR) spectra were obtained with a Nicolet 60SX Fourier Transform IR spectrometer using neat sample smears between KC1 plates. 1H nuclear magnetic resonance (NMR) spectra, unless otherwise stated, were obtained with a Nicolet (General Electric) QE 300 MHz instrument. All spectra were obtained using deuterochloroform (CDC13) solutions with tetramethylsilane (TMS) as an internal standard. Flash chromatography by the procedure of Still (1984) was performed using Bio-Rad silicic acid-HA (-325 mesh). Analytical thin-layer chromatography (TLC) was performed on prescored silica gel GF (250-1zm) plates (Analtech, Inc). Hydrogenations were performed using a Pressure Reaction. Apparatus (Parr Instrument Company, Inc.). All commercially obtained chemicals were used without further purification.
Chemicals 6-Dichloromethyl-6-methyl-3- (2-propyl)-2, 4-cyclohexadienone (20). The dienone (Scheme 7) was prepared in 8 % yield and had physical constants identical to those first reported by Wenkert (1970), who had prepared this compound in a similar yield by refluxing a two-phase mixture containing a chloroform solution of the appropriate phenol, carvacrol, and - 1 0 % aq. NaOH solution. The pure sample (flash chromatography, hexane) was subjected to GLC analysis with an oven temperature at 70~ for 1 min and then programmed at 5~ rain; 98% purity, Rt = 17.36 min. IR (neat) 1662 ( C = O ) , 1642 cm -1 ( C = C ) . 6-Dichloromethyl-2,6-dimethyl-2, 4-cyclohexadienone (21). The dienone was prepared in 17% yield as described for compound 20 and was purified by flash chromatography (hexane). The pure sample was subjected to GLC analysis with an oven temperature at 90~ for 1 rain and then programmed at 5~
2614
FRITZET AL. OH
0
H , ~
: C CIz
H
~ HCI2
COMPOUND
OH
20
: C el=
~
C HCI
H
H COMPOUND
21
SCHEME 7.
min; 99% purity, R t = 7.16 min. IR (neat) 1662 ( C = O ) , 1650 cm -1 (C----C); [IH]NMR (CDC13) 6 1.30 (s, 3H, 6--CH3), 1.92 (bs, 3H, CH3C~C), 6.09 (s, 1H, --CClzH), 6.40 (dd, 1H, J --- 7.5, 11 Hz, C - - 4 H), 6.58 (d, 1H, J = 11 Hz, C--5 H), 6.90 (bd, 1H, J = 7.5 Hz, C - - 3 H). cis- and trans-2-Dichloromethyl-2-methyI-5-(2-propyl)cyclohexanone (22). An ethanol solution (50 ml) of dienone 20 (2.00 g, 8.58 retool) was hydrogenated over 10% Pd/C at 50 psi for 24 hr and filtered over Celite. The solvent was removed in vacuo; the residue was purified by flash chromatography (petroleum ether, 35-60 ~ to give a 57 % yield of product (1.16 g, Scheme 8). GLC O H--...~
HCI2
,//
H2
// H3
~'~
CHCI2
+
CHCI2
COMPOUND
20
TRANS
C H3
COMPOUND 22
MAJOR ISOMER
"CIS COMPOUND 22 3
: 1
MINOR ISOMER
p
0
CHCI2
o
O
H~
C HCI2 COMPOUND
21
TRANS COMPOUND 23 MAJOR ISOMER
SCHEME 8.
CIS COMPOUND 23 15
:
1
MINOR ISOMER
STRUCTURE-ACTIVITY FOR ANTIFEEDANTS
2615
analysis with an oven temperature at 70~ for 1 rain and then programmed at 5~ revealed the presence of a 3 : 1 mixture of two diastereomers; R, = 17.34 and 17.67 rain, respectively. No attempt was made to isolate the individual isomers. Wenkert (1970) obtained a 4 : 1 ratio of these isomers; the isomer in the greater quantity (Scheme 8) had the isopropyl and methyl groups trans to each other. In our mixture the isomer in the greater quantity was identical to the trans isomer isolated by Wenkert (1970). IR (neat) 1716 ( C = O ) , 762 cm -1 (C--C1); [IH]NMR (CDC13) 6 0.92 [m, 6H, (CH3)2CH--], 1.27 and 1.32 (s, 3H, trans isomer - - C - - C H 3 and cis isomer - - C - - C H 3, respectively), 1.32-2.49 (m, 8H), 6.19 and 6.29 (s, 1H, cis isomer --CC12H and trans isomer --CC12H, respectively). cis- and trans-2-dichloromethyl-2,6-dimethylcyclohexanone (23). Dienone 21 (4.83 g, 19 mmol) was hydrogenated as described for compound 22 to give a 91% yield of 23. GLC analysis with an oven temperature of 90~ for 1 rain and then programmed at 5~ revealed a 1 : 15 ratio 5 of isomers; R, = 7.69 and 7.73 rain, respectively. IR (neat) 1702 cm 1 ( C = O ) ; [IH]NMR (CDC13) 6 1.05 (d, 3H, J = 6.0 Hz, C H_3CH--), 1.37 (s, 3H, - - C - - C H 3 ) , 1.30-2.20 (m, 6H, --C--(CHH2)3--CH--), 2.56 (m, 1H, CH3C__HCH2--), 6.20 ppm (s, 1H, --CClzH ). Bioassay Fall armyworm larvae, Spodopterafrugiperda (J.E. Smith), from our stock culture, reared on an artificial diet (Redfern and Raulston, 1970), were used for antifeedant tests. The samples were dissolved at an appropriate concentration in a 50:50 acetone-dimethyl sulfoxide solvent mixture; the resultant solution was added to a hot diet and mixed by using a 1/2-pint (720 ml) jelly jar fitted with an Osterizer blender cutter head. After the treated diet was blended for approximately 1 min, it was poured into 1-oz clear plastic cups ( - 8 g/cup) and allowed to cool to room temperature. Control diets were only treated with the neat solvent. One first-instar larva (newly emerged) was placed in each cup (10 cups for each sample concentration). The cups were then capped with prepunched lids and placed in a holding room at 27 ~ _+ 1 ~ with 50 + 5 % relative humidity. The larvae were observed for mortality, and the live larvae weighed on the sixth or seventh day after introduction into the cups. The sum of weights for the 10 repetitions per sample, as well as the control, were then divided by
5The isomerization of the 15:1 mixture was accomplished by the addition of lithium diisopropylamide to a THF solution ( - 7 8 ~ of 23, stirring for 2 hr as the temperature rose to 26~ and then acidifying with 10% (v/v) aq. HCI. GLC revealed the presence of a 50:50 mixture. Repetitive flash chromatography (petroleum ether, 35-60~ gave a fraction in which the new isomer predominated in a 60:40 ratio. IR (neat) 1702 cm ~ ( C = O ) ; [tH]NMR (CDCI3) 6 1.05 (d, 3H, J = 6.0 Hz, CH3CH), 1.27 (s, 3H, 2-CH3), 1.32-2.19 (m, 5H), 2.30 (ddd, 1H, J = 3.9, 6.6, 14 Hz; C-4 Hax), 2.59 (m, 1H, CH3CHCH2--), 6.40 (s, 1H, --CC12H).
2616
FRITZ ET AL.
the number of live larvae weighed in order to obtain an average larval weight for each sample. The ratio of these average larval weights (treated/control) was then used to assess the antifeedant activity of the various samples (Table 2). RESULTS AND DISCUSSION
The work of Kubo and Sodano has been very thorough. However, there are still several questions to be addressed that relate the structures of the enedials to their antifeedant activity, namely: (1) are both the C-8 and C-9 aldehydic oxidation states necessary for antifeedant activity at low sample concentrations ( _ 10 ppm); (2) if pyrrole formation is the most important reaction for activity, does the --SH moiety react in conjunction with the NH2 to enhance the activity; (3) why does the addition of the c~-OH at C-9 of warburganal enhance the antifeedant activity compared to polygodia~ (no c~-OH, Scheme 1); (4) if steric bulk inhibits the approach of the substrate tO the receptor site, what effect will a truncated natural product analog have; and (5) what effect do the alkyl substituents have on the antifeedant activity? The first conclusion that can be drawn from the data in Table 1 is that the C-8 and C-9 carbon atoms must have their substituents in the aldehydic oxidation state in order to demonstrate potent antifeedant activity. This is especially apparent when comparing the structural differences of polygodial (1) and the diester analog (8) (Scheme 9). Both molecules would be expected to form a cyclic intermediate, since both corresponding/3-C-9 carbonyl moieties should be in close enough proximity to the C-8 carbonyl to allow the intramolecular cyclization (see above). Once cyclization has occurred in both systems, only the intermediate arising from the dialdehyde can proceed to form the aromatic pyrrole (24). At this point it becomes necessary to interject our viewpoint that the sulfhydryl attack on the /3-carbon of the enal, as proposed by Kubo and TABLE 2. ANTIFEEDANT ACTIVITY OF SYNTHETIC COMPOUNDS 2 0 - 2 3 a
Sample Compound Compound Compound Compound Control
Conc. (ppm) 20 21 22 b 23 c
1000 1000 1000 1000 1000
Mortality 1 of 0 of 1 of 1 of 1 of
10 10 10 10 10
% wt. of control 103 21 99 28 100
~No antifeedant activity was observed for any sample at 100 ppm. Percent weight was calculated as described in Methods and Materials. b 1 : 3 mixture of cis and trans isomers. c 1 : 15 mixture of cis and trans isomers.
2617
STRUCTURE-ACTIVITY FOR ANT1FEEDANTS
POLYGODIAL 1
HO
DIESTER 8
NA *
O~..._._NR 0
I HSR
l
HSR
REACTION R PYRROLE 24 SCHEME 9.
Ganjian (1981), and the pyrrole formation by NH2 attack on the aldehyde carbonyls, as proposed by Sodano et al. (198'2), are not necessarily independent reactions. One can hypothesize that both the L-lysine --NH2 and the L-cysteine --SH moieties of the chemoreceptor site complex with the substrate in accordance with both the Sodano and Kubo mechanisms in order to produce the biological activity (Scheme 10a). Initial imine formation at the C-8 carbonyl followed by fl-addition of the SH and subsequent ring closure and pyrrole aromatization would produce a product similar to compound 24. This mechanism could explain why warburganal is a more potent antifeedant than the simple polygodial. For example, if the c~-C-9 hydroxide were displaced by an SN2' attack of the SH moiety on the fl-carbon of the enal, the resultant alkene would force the C-8 and C-9 substituents into an even closer proximity; thereby enhancing the pyrrole formation (Scheme 10b).
2618
FRITZ ET AL.
~
CHO CHO
l - H~O (31-13
N --LYS
/
~
-s--cYs
CHO ICHO
f
v
T CI-O
"~S-- CYS
"
HO
N --LYS
SCHEME 10.
There are other mechanisms by which SH moieties are known to react in vivo. One of the more common reactions is the oxidative decarboxylation reaction of the acid dehydrogenase (Scheme 1 la) (Lehninger, 1975). This type of reaction could occur with compounds like warburganal (Scheme 1 lb) to generate an intermediate (25), which would then induce the antifeedant activity; our synthetic compounds, 20 and 21, would be expected to react in a similar fashion with the sulfhydryl moiety via Michael addition to the alkene. The synthetic compounds 20-23 would also be expected to form an imine via coupling of the substrate's dichloromethyl moiety (carbon in the aldehydic oxidation state) and amine groups of the active site in a manner identical to that expected of the aldehyde moiety of the warburganal metabolite, 25. Compounds 20-23 would
STRUCTURE-ACTIVITY
2619
FOR ANTIFEEDANTS
C (Me) 0 J
C=O
+
HS--CoA
+
,'
NAD+
c~
+
Ac S--Co A
+
NADH
OH
~
CHO CHO
HS-ENZ
9
RS--ENZ
NAD+
9 NADH *
WARBURGANAL
~
0 CHO
25
0
...... I~ 20
22
O
O
C HOlz
21
"
~
ANTIFEEDANT
ACTIVITY
C
HCI~
23
SCHEME
1 1.
therefore be expected to be as active as warburganal since the chemical reactivity is similar to that proposed for the intermediate (25) and other quinones that are known to elicit antifeedant activity (Rozental et al., 1975). Unfortunately, all the synthetic compounds were inactive at 100 ppm, and this mechanistic pathway was therefore discarded in favor of the one shown in Schemes 10a and 10b. Obviously, these cyclohexane molecules are not inactive at the 100-ppm level because their steric bulk prevents binding with the chemoreceptor site, as in the case of the steroidal analogs of polygodial. Since the synthetic samples are not sterically prohibited from reaching the receptor site, it must be the inability of compounds 20-23 to form the pyrrole ring that accounts for their inactivity at low sample concentration. This observation supports the idea that the C-8 and C-9 aldehydes are necessary for antifeedant activity at lower concentrations. However, the 1000-ppm test results indicate that some dramatic differences exist between the inactive compounds, 20 and 22, and the active compounds, 21 and 23.
FRITZET AL.
2620 0
O HCI2 RS-~ ~ 2 0 /
NO
O CHClz
RS-H
RS-H
21
23
l
0
NO REACTION
REACTION R s ~ C
CHCI2
HCla
SCHEME 12.
Compounds 20 and 21 differ only in the alkyl substitution pattern (Scheme 12). Our reasoning used to explain the difference in activity was that the two methyl groups of the C-3 isopropyl substituent on compound 20 were sterically blocking the SH from adding to the/3-carbon of the c~,/3-unsaturated ketone in the same manner that the 9-c~ CHzOH prevented sulfhydryl coupling in alcohol 15 (Scheme 3b). This could not explain why the saturated analog 23 was nearly as active as compound 21 at 1000 ppm (72 % and 79 % weight reduction, respectively) since 23 had no alkene with which the SH could react. In hindsight, it was no surprise that compounds 20 and 22 were inactive, since it had previously been demonstrated that the alkyl substitution changes that produced saccalutal (6) from polygodial (1) and isosaccalutal (12) from epipolygodial (7) had had virtually no effect on the activity of the compounds. Apparently then, it is the C-2 methyl group of compound 21 and the corresponding C-6 methyl of compound 23 that was responsible for the difference in activity between these two active compounds and their inactive counterparts, 20 and 22. We noted that the bridge-head methyl group had been reported to exist exclusively in the axial position in all of the naturally occurring antifeedant compounds 1-4, and 6 (Table 1), while the C-2 methyl group of compound 21 and the C-6 methyl group of compound 23 were both equatorial (the C-2 methyl of compound 21 lies in the plane of the cyclohexadiene ring and can therefore be formally considered equatorial, Schemes 7 and 8). Epimerization of compound 23 produced only equatorial methyl groups, as determined by proton NMR analysis (Scheme 13). Alkyl groups have been known to effectively bind to chemoreceptor sites via dispersion and van der Waals forces that require tight fits to the active site since the magnitude of these attractive forces decreases rapidly with distance (Liljefors and Thelin, 1985). There is, therefore, precedence to expect that an
-~
23
CH3
'5 = 1.37
HCI2~C % O
TRANS
'5 = 6.20
H
'5 = 1.32 - 2.20
8 =
.05
1 2)
H+
- - t b . -
1) LDA
SCHEME 13.
HClz C ~ 0
,5
,5
H
= 2.32
CIS
= 1.27
23
H)C%o
~-~CH3
8 = 6.40
'5 = 1.05
i'-o o'-, i-o
2:
['11
> Z ,.q
0
,..
