553

DERIVATIVES

OF THE INSECT

AFFINITY

CHROMATOGRAPHY,

BIOLOGICAL Klaus-Dieter Margarethe Christoph

Received:

ANDTHEIR

Alf Hamann,

Spindler-Barth, Beckers

FOR

HORMONE

ACTIVITIES

Spindler,

Zoologisches schule

MOULTING

Annegret

and Hans

Institut

Ihne,

Emmerich

der Technischen

Hoch-

Darmstadt.

12/29/75 ABSTRACT

Several mone

have

Sepharose

derivatives

been

synthesized

4B yielding

As an indication work

esters

tested.

carboxylic lower

about

the puff

acid

were

Therefore,

moulting

coupled

ligand

capacity

with

of their

insect

gel.

for methyl

of inokosterone-C-26-

the highest

biological

the esters

and ecdysterone

for affinity

to AH

of the ligands

ester

hor-

per g wet

activity; of

hemisuccinates.

inokosterone-C-26-carboxylic

ligand

from

inducing

obtained

ecdysterone-C-6-CM-oxime

receptors

were

2 pole

The methyl

possesses

activities

be a useful

which

of the suitability

biological was

of the arthropod

acid

chromatography

should

of ecdyaone

tissues. INTRODUCTION

A common binding

feature

to cytoplasmic

molecules. established

The

of the action

(1) or membrane-bound

existence

for vertebrate

for the existence

VooZwne27, Number 4

of hormones

of such hormone hormones.

of hormone

S

binding

TREOXDl

is their

(2) receptor

receptors

In arthropods molecules

is well evidence

for the

April, 2976

S steroid

hormones

(3,

reported their

4,

hormone

to be

5,

to

(10,

receptor

is

11).

of

or

applied

the

to

tissues.

which

been

steroid

for of

14)

too.

this

paper

ligands

of

arthropod their

chromatography, biological

activities

Synthesis.

isolation

the

moul-

seems hormone

of

the

in

ecdysone of

in vertebrates Therefore

centrifugation

technique

(13,

In

about

understanding

12).

isolation

receptors

receptors

knowledge

(8,

during

applied

steroid

receptors

gradient

The

of

molecules

this

been

is known

binding

a better

lower

for

also

ecdysterone.

steroid

density

of

has

little

The

detailed

is much

arthropod

but

action

action

successfully

has

g),

or nuclear

a prerequisite

constant

filtration

8,

A more

compared

binding

7,

ecdysterone

significance.

for

mechanism As

of

6,

and

cytoplasmic

necessary

insects

the

ecdysone

physiological

ting

TDROIDS

of of

the

could we

receptors

moulting

of

derivatives.

the

ecdysone

preparation

for

to

these

for the

coupling

from

of vertebrate

useful

hormones

be

chromatography,

isolation

describe

gel

cannot

affinity

be

the

Sepharose,

affinity and

the

EXPERIMENTAL and

characterization

of

ecdysterone

derivatives Inokosterone-C-26-carboxylic catalytical from

oxidation

Rohto

Pharm.

platinum,

50

continous

stirring

the

oxidation

The

reaction

ml

of

Co., of

was

4 x at

mixture

inokosterone

Osaka, 10m3M room

checked was

acid

100

(100

mg

NaHC03)

thin

filtered,

obtained mg

freshly with

temperature. by

was

layer

inokosterone reduced

oxygen

The

by

and

progress

of

chromatography.

evaporated

to

dryness,

dissolved

in methanol

und

separated

by column

either

on silica

gel with

eluent

(see fig.

1) or on XAD 2 with

gradient done

according

to Hori

by preparative The acetates

were

prepared

by thin

layer

dichloromethane

(3H) acetic

anhydride

(Merck,

on silica

rate

in order

synthesized

to allow

using

were

Thin

layer

scanned

the esti-

of ecdysterone to (19).

chromatography

a fluorescent

dichloromethane

system.

material

with

was

efficiency.

according

thin layer

indicator

- methanol

chromatograms

with

a Berthold

8:2 or with

Thin

Scanner. Inokosterone-C-26-carboxylic

CM-oxime

and ecdysterone

in methanolic thane

61 Ci/mmole)

and the coupling

gel plates

isolated

(18). A small

to (17) and

and analytical

as the solvent

Layer

synthesized,

(spec.act.

were

Darmstadt),

radioactive

acid

(16) and purified

of 2B- and 3Ghemisuccinates

(3H)oc-ecdysone

was done

was

mixture

of the reaction

Preparative

7r3

according

to the reaction

Mixtures and

were

chromatography.

of (3H)o(-ecdysone

mation

- methanol

purifications

of inokoaterone-C-26-carboxylic

with

and characterized

added

a water

8r2 as

chromatography.

Ecdyeterone-C-6-CM-oxime

amount

- methanol

(15). Further

thin layer

chromatography

solution

acid,

hemisuccinates with

freshly

ecdysterone-C-6were

methylated

distilled

(14C)

diazome-

(NEN, Dreieichenhain). The derivatives

spectroscopy, latter

were

further

IR-spectroscopy

Coupling

with

Sepharose

of the ligands

and ecdysterone

as

to the Seoharose acid,

hemisuccinate

4B. 'The gel was prepared

manufacturer

the

tetramethylsilane

standard.

Inokosterone-C-26-caboxylic CM-oxime

by UV-

and NMR-spectroscopy,

in pentadeuteropyridine

an internal

characterized

(20). For each

ecdysterone-C-6-

were

coupled

as described

coupling

to AH

by the

2 mg steroid

and

S 20

mg

I-ethyl-3-(3-dimethylaminopropyl)

were In gel

dissolved

in

dist.

order to determine 6 10 -5 -3 x 10 dpm

responding

ligands 100

at room

temperature

wet

After

- 6.0,

times

mg

water the

of

gel

and

preincubated of

added was

to

added

An aliquot

(Zinsser,

Frankfurt/M.)

of

solution.

and

the

to

slurry

the

the To

cor-

this

stirred

The pH was adjusted

the gel was washed

no radioactivity

30 min.

ligands

the

for 24 hours.

the wash.

the

for

(3H)-d erivatives

coupling,

(20) until

carbodiimide-HCl

coupling

the

were

mixture

4.5

T~EOXDS

could

be detected

of the wet gel was in a Berthold

counted

Liquid

to

several in

in Unisolve

Scintillation

Counter. Bioassay

of the ecdysterone

The biological were

tested

instar

salivary medium gland

one gland

orcein value

After

derivative.

- acetic

the early puffed

ecdysone

region

and

mid-third

each

pair

size

and

the sister

moulting

incubation

acid

of

in Schneider's

the desired

one hour

Puff

acid.

representing

From

ligands

from

as a control

- acetic

in ethanol

isolated

was incubated

medium

derivatives

of the different

hydei.

Detroit)

Lab.,

in Schneider's

fixed

glands

of Drosophila

glands (Difco

hormone were

on salivary

larvae

and inokosterone

activities

3:1

the glands

and stained

is given

with

as a relative

the quotient

of the diameter

of one of

puffs

the diameter

the non-

78B

to

of

77CD.

RESULTS The

oxidation

of inokosterone

different

products

pure

a compound

rative is

form

thin

layer

approximatively

a 3f3-hydroxysteroid

(Fig.

1). Peak

which

was

chromatography. 10s. by

The

gives

IV contains

further The

compound

staining

a mixture

with

purified yield

was

of

of

in nearly by prepathis

characterized

vanillin-sulphuric

product as

s acid

and by the method

absorption The

maximum

compound

as shown

E254

of Eberlein

at 242 nm like

is much

by paper

557

TEROXDrn

more

polar

(21). It possesses inokosterone

than

electrophoresia

an

and ecdysone.

inokosterone

(table

1);

(fig. 2) the compound

nm I IV .

.

)I

II .

III i

p

f

Ji

J\ ! i

.I i

t

I ‘!

l

i

,F

•--.-.-._._._.~*

v

100

; /

t

150

200

250

300

350

400

fraction Fig.

1.

450 number

Separation of the oxidation products of inokosterone by column chromatography on silica gel. Column: 100.0 x 5.6

cm, elution

with

dist.

= 80:20:2.5

(v/v/v),

with

water

dichloromethanermethanol:

dichloromethane:methanol:dist.

(v/v/v).

volume:

Elution about

C-26-carboxylic

rate:

17 ml.

acid.

beginning water

4 ml per minute.

Peak IV contains

at 1 = 65r35r2.5

Fraction

inokosterone-

S migrates

to the anode

function.

The

spectroscopy compound methyl 1565cm

an ester.

with

a signal

This

are

has

compound

with

are differences

chain

(table

does

proton

-1

not

(fig.

3). The

as compared

with

in the methyl

to a

the signal -1

at

indicating

characterized

signals

by IR

leading

show

at 1725cm

was further

identical

there

be demonstrated

diazomethane

a signal

The methyl

the carboxylic

at 1565 cm

derivative

but instead The

indicating

ion could

can be esterified

spectroscopy. skeleton

thus

carboxylic

ester. -1

TDEOXD-

by NMR

of the steroid inokosterone,

resonances

but

of the side

1).

11. h E

C % u-l

a

3

1'1

3

5

7

Cd

start

Figo

2, Paper

electrophoresis

0.1 M, pH 8.8,

(sodium

250 V, 7 mA,

borate/He1

3 hours).

I, and inokosterone-C-26-carboxylic bromphenolblue. stained

After

according

acid=

electrophoresis

to (21) and scanned

buffer

Inokosterone= II

the strip at 540 nm.

I= was

S

TBEOXDI

559

frequency 4000

Fig.

carboxylic

1800

2400

3200

3. IR-spectra

[cm-l]

of inokosterone acid

Table

1: Physical

characteristics

of the insect Rf-

(a), inokosterone-C-26-

(b) and the methyl

kosterone-C-26-carboxylic

moulting

chemical

1000

1400

acid

ester

of ino-

(c).

of the C-26-derivatives hormones

shifts

of methyl

resonances c 26/27

value

c 18

C 19

c 21

ecdysterone

0.35

1.23

1.09

1.60

1.39

inokosterone

0.35

1.24

1.09

1.44

0.94/0.88

0.10

1.24

1.07

1.40

0.94

inokosterone-C-26 carboxylic methyl c-26

ester acid

acid of

0.67

-

-

-

-

6

S

560

Based

on these

data

T=R.OIDS

the compound

of peak

as inokosterone-C-26-carboxylic

acid

IV was

(Fig.

identified

4, compound

2):

2 Fig.

4. Conversion acid

This

was

could

ligand

calculated

carboxylic

(1) to its C-26-carboxylic

(2)

substance

of 2 ~01s

of inokosterone

be coupled

per g wet gel.

by adding

acid

to AH-Sephsrose The

(3H) acetates

to the reaction

active

capacities

whereas

as inokosterone

(fig.

a more

smaller

puff

sizes

in the lower

tration

ranges.

This

compound

acid

in inducing

methylated, capacities

sigmoid

is as inactive puffs,

the resulting (fig.

ester

5b).

slight

is nearly

curve

and in the higher

extent

puff as

to ecdysterone,

dose-response

Ecdysterone-C-6-CM-oxime to the same

only

5a). As compared

shows

to AH-Sepharose

efficiency

of inokosterone-C-26-

acid has

its methyl

inokosterone

a rate

mixture.

Inokosterone-C-26-carboxylic inducing

coupling

with

could

with

concen-

be coupled

as the former

compound.

as inokosterone-C-26-carboxylic

but when ester

the carboxylate has

some

puff

ion is

inducing

561

78 6177 CD

23 2,l

1,9

fig.5

1.7

a)

1 .5 1,3 l,l

10-g

10-8

1o-7

10-6

10-S

10-4

1O-3 M

I 217 2.3-

I/

2. l-

A

b

b) .l

f 10-9

1 f/;/y 10-8

10-7

lo4

10-5

1O-4 ,271

M

S

562

Mixtures be

of

2B-

coupled

ligand

per

esters

have

3Ghemisuccinates

to AH

Sepharose

g wet

gel.

about

response

curves

range

for

as

and

TEIROXD~

The

the for

the

with

of

an

ecdysterone

efficiency

hemisuccinates

same

and

biological

these

two

of

0.6

their

activities.

substances

could

are

pmole

methyl The

in

the

ecdysterone-C-6-CM-oxime-ester

dose-

same

(fig.

5~).

DISCUSSION Application of

steroid

of

receptors

resulted

in

moulting

hormone.

small

the

amounts

of

This

ecdysones

to

the

and

derivatives the

affinity Fig.

5.

the

(22,

end

of

gel

the

in

23), side

chain

all

curves

for

salivary (A),

(A),

methyl

ester

ecdysterone

(A), ester

(0)

and

its

for

arthropod the hormone

the

gel

a good

groups of

of the

Drosophila

(X),

is

useful

to

induction

of

It

moulting

in

of

the

constant

hormone

inokosterone acid

for

between

functional the

relatively

from

result

yet

arthropod

prove

of

glands

not

tissues.

far

the

should

important

isolated

so

of

the

binding

activities

coupling

has

the

proteins

known

biological

methyl

lower

binding

isolation

accessible

will

C-26-carboxylic

its

to

chromatography

ecdysterone

its

due

target

relations

ecdysterone

for

the

Dose-response 78B

in

the

tissues

receptors

the

receptors

since

for

material

to

of hormone

F'rom the

structure

of

is mainly

and

affinity

enrichment

tissues.

at

the

methods

vertebrate

biological

procedure

that

from

isolation

isolation

likely

classical

puff hydei.

inokosterone-

methyl

ester

ecdysterone-C-6-CM-oxime

(m)

(Cl). succinyl (*).

ecdysterone

(0)

and

(0). and

S the hormone receptor.

are accessible

Cur results

capacities We chose esters

frssmofmm

for the interaction

concerning

of the different to measure

ligands

ligands

activity

found

the C-26-derivative

nearly

total

containing

absence

somewhat

by generation

poor gland with

cell

of the acidic by methylating slow hydrolysis amount

affinity. expect

Possibly

the incubation

whether by

the salivary

of these

is not

with

already

demonstrated

If this

biological

correlation

that affinity

isolation

5~).

is removed

is caused

explain

studies

between

C-26-carboxylic

which

can

derivatives

the activity further

this

giving

improved

is due to rise

to a

of ecdysterone.

(8) have

correlation

interactions

is the

in derivatives

It is not known

into

We

activity

charge

molecules

the

activity.

tests

activities

can we then

(Fig.

during

Preliminary nogaster

induction

ester.

hemisuccinates them?

mimic

to the gel.

of the carboxylafe

or by the weak How

of the methyl

the highest

the negative

of charged

the receptor.

certain

when

the

this hypothesis.

ion. The biological

inactivity

penetration

has

of biological

of its methyl

the relative

coupled

of the puff

a carboxylate

be restored

confirm activities

of the ligand

result

with

inducing

for they should

biological

An interesting

the puff

the biological

of different

that

563

acid

imaginal

activity

as a ligand

and receptor

using

we should

inokosterone-

is suitable

receptors.

of 2. mela-

a possible

is significant

chromatography

of the ecdysone

discs

for the

The

TDROIDS

S

564

work

schaft

was

supported

through

by

a grant

the

to H.

K.

Deutsche E.

The

Wortelkamp

assistance

of Mrs.

gratefully

acknowledged.

Forschungsgemein-

skillful

and

T.

technical

Schneider

is

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