Metabolism of the juvenile hormone analogue methyl farnesoate 10, 11-epoxide in two insect species

Pergamoa Prose

Life 8ciencea Yol. 11, Part II, pp . 201-210, 1972 . Printed in Great Hritaia

METABOLISM OF THE JIIVENILE HORMONE ANALOGUE METHYL FARNESOATE 10,11 EPO%IDE IN TWO INSECT SPECIES A .F . White Agricultural Research Council, IInit of Invertebrate Chemistry and Physiology, The Chemical Laboratory, IIniveraity of Sussex, Brighton BN1 9QJ, Sussex . (Received in final form iS January 1972) ~17ttIT1A T4

The metabolism of methyl farneaoate 10,11epoxide in Schiatocerca gre¢aria and Rhodniua ~rolixus was shown to occur by eater hydrolysis and epoxide hydration . The metabolites were found to be inactive when tested for juvenile hormone activity in both insect species . Conjugate~ of the metabolites were also detected in Sch atocerca . Many compounds have been shown to mimic the activity oP the juvenile hormones (I and II) Prom Hyelo~hora cecronia (1 ),

The

closely related analogue methyl farneaoate 10,11-epoxide (III) also showed high juvenile hormone (JH) activity (2) in Teneb io molitor,

COaCHa

I II III

R1 = R~ = CaH6 R1 = CaH6, Ra = CHa Rl = Ra = CHa

A study of the metabolism of JH analogues is of prime importance in assessing their effectiveness ae potential insect control agents .

This report describes the metabolic inactivation of 201

Metabolism oaf Juvenile Hormone Analogue

202

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[2- 3H]-traps traps methyl farnesoate 1011-epoxide in the locust ( Schistocerca gregaria ) and Rhodnius prolixus . FXPFRTMFTTTAT,

Preparation of j2- 3 H -tram tram me tal farnesoate 1p111-epoxide Geranyl acetone was prepared from linalool (3) and the ç~,q and tram mixture of isomers was separated by silver nitratesilica gel preparative thin layer chromatography (t .l .c .),[2-3H] Methyl farnesoate was synthesised by a modified Wittig

reaction

(4) from tr~ga_ geranyl acetone and [2-3H]methyl bromoacetate

(71 .5 m01/m.mole) .

The product wsa chromatographically identical

to methyl farnesoate prepared by oxidative esterification of farnesol by the manganese dioxide - cyanide-methanol procedure (5) .

The labelled ester was purified by silica gel t .l .c, using

benzene-light petroleum B .pt . 60°-80° (50 :50) as solvent, which enabled separation of the c~gi isomers .

r

q (20%) and traps traps ( 8 090 Pure traps traps [2-3H]methyl farnesoate (7 .1 x 109

d .p .m ./m .mole) was treated with N-bromosuccinimide in aqueous dimethoxyethane and the resulting bromohydrin converted to the required labelled epoxy eater in i~pg yield by methanolic sodium iso-propoxide solution (6) . The radiochemical purity of [2-3H]-traps traps methyl farnesoate 10,11-epoxide was checked and after dilution with inactive material gave a specific activity of 2 .35 x 10 9 d .p .m ./ m .mole . Preparation of reference cot~ounda Methyl farnesoate 10,11-epoxide was treated with 0 .01N perchloric acid in aqueous tetrahydrofuran at 23 ° for 20 hours to form methyl farnesoate 10,11-diol (IV) in 8 5~ yield,

Hydrolysis

of this diol ester with O .SN sodium hydroxide in 50~ aqueous

aos

Metabolism a~ Juvenile Hormone Analogue

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ethanol at 23° for 2!E hours (7) gave farneaenic acid 1011-diol (VI) in 80~ yield .

Farneaenic acid 10,11-epoxide (V) was pre-

pared under similar hydrolytic conditions in 72~ yield or by epoxidation of farneaenic acid in lower yield .

All three

reference compounds were purified by preparative t,l,c,

(using

chloroform-methanol [90 :10] as solvent for which satisfactory infra-red and nuclear magnetic resonance spectra were obtained, Metabolic Studies The labelled epoxy-ester (50 fig . in 5 ul, of peanut oil) was injected into the soft membrane

at the base of the mesothoracic

trochanter of the Fifth instar of Schistocerca ,

Fifth instar

larvae of $hodnius were injected with 5 4~g . in 1 ~1, of oil into the back leg .

The wounds were sealed with molten wax,

After time intervals of up to 20 hours the insects were killed by freezing, homogenised and extracted with diethyl ether, and the residue extracted with water,

The frase was collected

and extracted separately in a similar manner .

Aliquots of the

aqueous extracts were incubated at 35° for 60 hours with ~omatia digestive juice,

Hem

Other aliquote were adjusted to pH 5,2

with 0 .05 M sodium acetate buffer and incubated at 35° for 6 hours with ß-glucoaidase,

Samples were then extracted with ether .

After concentration, aliquots of the ether extracts were examined on t,l,c, plates coated with 0 .5 mm, silica gel GFßss " The solvent sygtema employed were diethyl ether - light petroleum

(20 :80) (wystem A), diethyl ether - light petroleum (75 :25) (system B), methanol - chloroform (10 :90) (system C) and acetic acid - di-iso-propyl ether (5 :95) (system D),

Initially radio-

active arena were scraped off the plates and analysed for radio activity by means of a liquid scintillation counter,

Those

radioactive areas which co-chromatographed on t,l,c, with the .diol

Metabolism ad Juvenile Hormone Analogue

204 eater,

Yol . 11, No. 4

epoxy acid and diol acid reference metabolites were

examined, as their trimethylsilyl (TMS) ethers, by gas-liquid chromatography (g .l .c .) (at 135 0 on a 92 cm, x 0 .1

cm, i .d, glass

column of 8p-100 mesh Chromosorb Q carrying 59ß OV 210 with Nß carrier gas at 2p ml/min .) . Dilution analysis was then achieved by adding 10 mg, of each of the three reference metabolites to aliquots of the ether extracts .

Each metabolite was purif ied to constant specif is

activity by repeated chromatography in the different solvent systems . RESIILTS AND DISCIISSION Methyl farneaoate 10,11-epoxide showed high JH activity when assayed in Rhodnius (8) where it produced a a core of 1p (ie . half juvenilised) with 5 4~g, applied topically .

This activity was

slightly leas than that for .tram traps farnesyl methyl ether and synthetic racemic çecrouia hormone (I) mixed isomers .

However,

in Schistocerca methyl farneaoate 10,11-epoxide showed only moderate activity and it required ten times as much to produce the same degree of juvenilieation . In a Hemimetabolous insect the adult character of the final moult is determined during a prior exposure to moulting hormone in the absence of JH .

During this period in which the adult

character is being determined, when the animal s own corpus allatum is not releasing JH, we would expect there to be an appreciable competence to destroy JH so that any hormone remaining Prom the previous juvenile determination can be destroyed .

In

Rhn~dn~~ the period of determination is between 0 to L~ days after being fed sa a fifth motor .

In 8chistocerca the period of

determination conincidea with the moult Pram Lath to 5th motor . However,

in locusts JH also controls at leagt some aspects of the

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Metabolism od Juvenile Hormone Analogue

x05

The locusts

phase status occupies much of the intermoult (9) .

employed in these observations were of the ~ragaria phase .=and accordingly they maintain little or no circulating JH during the intermoult .

For these reasons it is to be expected that there

will be a considerable competence to inactivate JH during the whole of the 5th instar in Schistocerca .

Accordingly, the

metabolism oP methyl farneaoate 1011-epoxide was studied during the 5th instar of both Rhodniua and Schistocerca . Fsxamination by radio-t,l .c . of the ,g,chistocerca ether extract 20 hours after injection of labelled methyl farneaoate 10,11-

2000 ~

1000 s

~d

a

1000 ~

b

1', 0

FIGIIRE 1 Chromatography oP [2 3H] methyl farneaoate 1011-epoxi$a metabolites in Sch ~tQçerca , $ara indicate popi,tions oP co-chromatographed non-radioactive metabolites . 1~ tarneaenic acid 1011-diol ; ~, farneaenic acid 10~11époxide; ~, methyl Parneaoate 1p~11-diol ; L~, methyl Parneeoate 1011-epoxide,

206

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epoxide showed (Figure 1a) two bands of radioactivity; a major band (Rf . 0 .3 in system A) which corresponded to unchanged epoxy ester and the more polar band which remained at the origin . G .l .c, analysis confirmed the presence of unchanged epoxy ester (retention time = 3 minutes) .

The more polar band was resolved by

t .l .c . (figure 1b) into three components (1

- 3) using system C .

The moat polar component (Rf . 0 .1) co-chrometographed with fernesenic acid 1p,11 diol (VI) .

Farnesenic acid 10,11-epoxide

(V) end methyl ferneaoate 10,11-diol (IV) co-chromatographed with the two components of RF .

0 .5

and Rf . 0 .6, respectively .

This

result was conf irmed by g .l .c, analysis of the three components as their TMS ethers .

Retention times relative to trees traps methyl

fe.rneaoate 10,11-epoxide (=1) were 1 .6, 1 .7 and 3 .3 For the epoxy acid, diol ester and diol acid TMS ethers respectively . separation of ~chiatocerca frasa ether extract

5

T .l .c .

hours after in-

jection of labelled methyl farnesoate 10,11-epoxide showed a similar pattern of metabolites .

In Rhodnius, ell three metabo-

lites were again identified by t .l .c, co-chromatography .

Here,

both the whole insect and frasa ether extracts obtained 2p hours after injection contained the diol ester as the major metabolite . lamination by radio-t .l .c . of ,~chiatocerca whole insect end Prass aqueous extracts showed none of the metabolites obtained above .

All the radioactivity remained at the origin oP the t .l .c .

plate in the solvent systems tried .

The possibility that these

were polar conjugates of the eater (IV) and the acids (V and VI) was explored by enzymatic hydrolysis . extracts with

Incubation of the aqueous

aliz ~omatia digestive juice, which hydrolyses

ethereal sulphates,

glucoaidea, glucosiduronates and phosphates,

rendered up to 100$ of the radioactive metabolites ether soluble . Co-chromatography oP these ether extracts revealed the three

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Metabolism ad Juvenile Hormone Analogue

metabolites, but no methyl farneaoate 10,11-epoxide .

207

Thus all

three metabolites were present as xater soluble ca~njugatea in the extracts .

Incubation of the aqueous extracts with ß-glucoaidase

showed up to

40yß

of these water soluble conjugates were present

as glucoaides . It is probable from structure-activity relationship atudiea (8) with numerous JH analogues that these metabolites have less JH activity than methyl Paraeaoate 10,11-epoxide itself .

All

TABLE Metabolic products after injection [2 3H]-trace traps methyl farnesoate 1p~11-epoxide in and R~n~~ (d .p .m, x 10 -~/m .mole metabolite insect) SCHISTOCERCA

Dial Ester (IV)

Epo acid ~V)

Diol acid (VI)

2 hours ether extract

10 .50

1 .90

26 .00

5 hours ether extract

8 .35

7 .90

10 .50

20 hours ether extract

1 .LEO

2 .10

3 .40

5 houre a aqueous extract

0 .25

1,80

2 .20

5 hours aqueous a Prosa extract

2,20

0 .70

16 .00

5 hours aqueouab Praea extract

2 .00

0 .45

4.95

20 haare ether extract

1 .70

0 .06

0 .09

20 hours ether frass extract

0 .45

0 .13

0 .08

RHODNI[TS

c after incubation with Helix pomatia digestive juice haftet incubation with ß-glucoaidas .e

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three metabolites were tested Por JH activity in 3chistocerca and Rhodnius ,

No activity was detected with up to 200 I~g ./

Schietocerca of either metabolite .

The epoxy acid showed very

low activity (score 1) at 250 I~g ./$hod~~ i but no activity wa.s observed with the dSol eater and acid, Further evidence towards establishing the identity of the metabolic products of methyl farneaoate 1011-epoxide was achieved by dilution analysis and t .l .c, purification to constant specific activity .

These are illustrated in the table,

Solvent systems B

and C were used to purify the diol ester and epoxy acid and systems B~

C,

and D.f or the diol acid . In iSchiatocerca the epecif is activities of the diol ester

and acid declined during 2 to 20 hours, whereas the epoxy acid showed a delayed rise followed by a decline in specific activity . Methylation of the epoxy acid (7 .9 x 1p 4 d .p .m,/m,mole) with ethereal diazomethane gave the original methyl ester (7 .9 x 10 4 d,p,m ./m,mole) with no lose of radioactivity after purification in system A,

Clearly, rapid metabolism of the epoxy ester was

accompanied by conjugation and the metabolites were excreted ss such or ae conjugates .

Rome of the conjugates were present ea

glucoeides, the majority of which were formed Prom the diol eater and acid, The present findings indicate that in Schiatocerca end Rhodnius the metabolic inactivation of methyl faxmeaoate 10,11epoxide occurs by ester hydrolysis and epoxide hydration,

The

occurrence of two intermediate metabolites suggests that a possible pathway can be outlined as in figure 2~ which is Followed by conjugation oP each metabolite, These findings differ from those reported (10) for the metabolism of Cecro~ia JH (I) in the Lepidopteren j+j~çg aexta

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Metabolism a~ Juvenile Hormone Analogue

209

OsH

FIGURE 2

Pathwe,y for metabolism of methyl farnesoate 10,11-epoxide in which eater hydrolysis preceded epoxide and no diol éster was deteoted, Clearly,

~pecieA variationA exipt and the presence of

epoxide hydrsae and esterase enz9tneA will def ins the pathway of metabolism,

Interference with the pathway by using enzyme in

hibitors may well potentiate the effectiveness of a JH analogue where metabolism proceeds rapidly, ACSNOWLEDGEMENT I thank Dr . G . Ellis Pratt for advice and help with the injections . REF'EREN CES 1 ,

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2,

W.S . BOWERS, M .J . THOMPSON and E.C . IIEBEL, Life flci , ~, 2323, ' (1965) .

3.

0 . ISLER, R . RIIEGG, L . CHOPARD-DIT-JEAN H . WAGNER and K . BERNARD, Helv_ Chim_ Acta ,, ~, 897, (156) .

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I . OGONI and I . IIRITANI, Aar . Biol_ Chem ,, ~,

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1650 .,

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C~1R~~)N .W . GILMAN and B .E . GANEM, J_ Am . Chem_ soc ., ~,

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E .J, COREY, J .A . HATZEDTELLENBOGEN, N .W . GILMAN S .A . ROMAN and H .W . ERICSSON, J . Am_ Chem . soc . , ~, 5618, (168) .

7.

G . POPJAg and R .H, CORNFORTH, J . Chromstoa . , ~, 214,

(1960) .

210

Metabolism ad Juvenile Hormone Analogue

8.

V .B . WIGGLESWORTH, .7 . Tne- Phvsiol ., ~, 73,

9.

G . ELLIS PRATT, unpublished results .

70 .

Vol. 11, No . 4

(~969) .

J .B . SIDDALL, R .J . ANDERSON, B .J . BERGOT, L .L . DIINHAM, W . EAF'F'ERL, C .A . HENRICg and D .S . GING, X7CII Int ornat ional C ongrenn of Pure and Applied Chemistry, Bonton, II .S .A ., July 25-30, (~97~ ) .