Recent Progress in Avermectin Research - ACS Symposium Series

Chapter 12

Recent Progress in Avermectin Research M. H. Fisher

Downloaded by UNIV QUEENSLAND on September 30, 2013 | http://pubs.acs.org Publication Date: March 12, 1993 | doi: 10.1021/bk-1993-0524.ch012

Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065

In 1976 scientists at Merck & Co. Inc. discovered a complex of eight closely related natural products, subsequently named avermectins, in a culture of Streptomyces avermitilis MA-4680 (NRRL8165) originating from an isolate by the Kitasato Institute from a soil sample collected at Kawana, Ito City, Shizuoka Prefecture, Japan. They are among the most potent anthelmintic, insecticidal and acaricidal compounds known. Avermectin B1, under the non-proprietory name abamectin, is widely used as an agricultural miticide and its 22,23-dihydro derivative, ivermectin is used world wide as a broad spectrum endectocide in animals and in man. Recently, an intensive program on avermectin derivatives discovered 4"-deoxy-4"-epimethylamino avermectin B benzoate to be the most potent lepidopteracide known. It is being developed under the non-proprietory name emamectin. Mode of action studies, have revealed a high affinity binding protein in membrane fractions from Caenorhabditis elegans and Drosophila melanogaster. The use of an affinity probe enabled the isolation of three proteins having molecular weights 8,47 and 53 kD. 1

In 1976 scientists at Merck & Co. Inc. discovered a complex of eight closely related natural products, subsequently named avermectins, in a culture of Streptomyces avermitilis MA-4680 (NRRL8165) originating from an isolate by the Kitasato Institute from a soil sample collected at Kawana, Ito City, Shizuoka Prefecture, Japan. Their structures are shown in Figure 1 (7). They are among the most potent anthelmintic, insecticidal and acaricidal compounds known. The avermectins (Figure 1) are closely related to another group of pesticidal natural products, the milbemycins, the first examples of which were described by Japanese workers, but later were found to be more abundant in nature than die avermectins (2-7). Milbemycin structures are shown in Figure 2. Interestingly, the milbemycins were first discovered as miticides. Their anthelmintic properties were found only after the anthelmintic activity of the avermectins was demonstrated. To date, two avermectins, ivermectin and abamectin, have been introduced to date for use in animal and human health. Doramectin (8), prepared 0097-6156/93/0524-0169$06.00/0 © 1993 American Chemical Society In Pest Control with Enhanced Environmental Safety; Duke, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


170

PEST CONTROL WITH ENHANCED ENVIRONMENTAL SAFETY

AVERMECTIN

A

:

R

1

:

X = -CH=CH-

5

= OCH3

Rc = OH OH

R

IVERMECTIN

: *5 =

-CHo-CH-

25 =

0

H

x

r

PRODUCT

Ph P=CHCH CH OSi(Me )(t-Bu) 3

2

2

2


3

2

2

2

3

6

(R,S)-Ph P^CHCH CH(OSiMe )C H 3

2

R =Me,R =H

40%

R =H, R =Ph

41%

R = H , R ^=cyclohexyl

39%

R = H , R ^=cyclopentyl

40%

2 5

2 4

3

(R,S)-Ph P=CHCH CH(OSiMe )C H j 3

4%

2 5

2 4

3

(S)-Ph P=CHCH CHPH(OsiMe ) 3

R =H, R =H 2 4

(R or S)-Fh P=CHCH(Me)CH OSiMe

3

5

9

{

2 4

2 4

25

2

2

45%

(R or S)-Ph P=CHCH CH(OSiMe )Me 3

2

YIELD

3

Ph P=CHCH(Et)CH(OSiMe )(sec-Bu)

R =Et, R =sec-Bu

33%

Ph P=CHCH(Me)CH(OSiMe )(sec-Bu)

R =Me,R ^=sec-Bu

49%

3

3

3

3

24

24

25

2

With a method in hand for the synthesis of novel 24,25-substituted avermectins, we turned our attention to the replacement of the 6,6-spiroketal with a 6,5-system. Starting with the 22,23-hydroxy ketone, oxidation with lead tetraacetate, followed by Horner-Emmons olefination of the adehyde gave an enone which by conjugate reduction of the olefin, followed by reduction to an alcohol, gave an intermediate which could be cyclized to the desired 6,5spiroketals by acid catalysis under thermodynamic control (72). Figure 10 shows the sequence of reactions. Products were obtained in excellent yields as shown in Table IV. The biological activities of some of the novel 24,25-substituted avermectins and the derivatives containing 6,5-spiroketal systems, as measured by their lethality to brine shrimp, are shown in Tables V and VI. The mode of action of the avermectins is still incompletely understood. Some pharmacological effects are shown in Table VII. It appears to bind specifically to, and cause opening of, a number of chloride channels at picomolar concentrations (13). A high affinity ivermectin binding site has been found in a membrane fraction from C. elegans, the membrane-bound receptor has a kD of 0.14 nm bmax 0.57 p. mol/mg of protein whereas the l-O-n-octyl-fJ-D-glucopyranoside (NOG) solubilized receptor has kD 0.18 nm, bmax 0.82 pmol/mg protein. Binding to a similar membrane fraction from rat brain membrane fraction is much less efficient, kD 22 nm which may help to explain the wide margin of safety in mammals (14). In attempt to locate this receptor, an affinity probe, the structure of which is shown in Figure 11 was designed and synthesized (15).

In Pest Control with Enhanced Environmental Safety; Duke, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

Recent Progress in Avermectin Research

FISHER

177


12.

a) Pb(OAc) , MeOH, pyr; b) (MeO) P(0)CH C(0)R, LiCI, DIEA; c) Ph PCHC(0)R; d) 9 eq N a S 0 , 1 8 eq NaHC0 ; 1:1 PhH:H 0, reflux; e) Pd(PPh ) , nBu SnH; f) NaBH ; g) RMgBr; h) BH .SMe , oxazaborolidine, 0° C; i) 4:1 PPTS:TsOH; j) HF.pyr 4

2

4

2

2

4

3

3

2

2

3

3

4

3

2

Figure 10. Synthesis of 25-NOR-6, 5-Spiroketal Avermectins


178


Table IV.


R

25-OR-24-Substituted Avermectins % Yield

R

% Yield

H

62

CKfcOme

57

Nfe

42

CH20Ph

43

z-pr

68

Ph

38

t-Bu

76

(p-F)Ph

30

n-C Hi7

41

MeO)Ph

42

0C6H17

59

2-Furyl

25

CH OH

48

OMe

56

6

2

Table V.

( r

Activity of Novel 24,25-Substituted Avermectins Brine Shrimp 24

25

H

H

13,900

CH3

H

6,930

H

C H

CH3

H CH

3

Et

3

CH

3

100 "

870 220

sec-Bu

220

sec-Bu

870

cyclohexyl

CH

phenyl

n

1,730

sec-Bu

CH, 3

L C

1,730 870


g

/

m

l

12.

FISHER


179

Table VI. Activity of 6,5-Spiroketals Brine Shrimp


24-R

L C

100 "

n

H

55,500

Methyl

55,500

t-Butyl

870

Phenyl

1,730

p-Fluorophenyl

1,730

p-Methoxyphenyl

6,930

2-Furyl

1,730

Table VII.

g

/

m

l

Pharmacological Effects of Avermectins

Nematodes paralyzed rapidly without causing hypercontraction or flaccid paralysis. Blockage of signal transmission from ventral interneurons to excitatory motorneurons of Ascaris. Reversible increase of chloride ion permeability of G A B A sensitive fibers of the extensor tibiae muscle of the locust Schistocerca gregaria at nanamolar concentrations. Irreversible inhibition of G A B A sensitive and insensitive muscle fibers of Schistocerca gregaria at micromolar concentrations. Reversible opening of crayfish stomach chloride channels at subpicomolar concentrations. Irreversible opening of crayfish stomach chloride channels at lOpmol or higher. Binds specifically to a number of chloride channel proteins but its binding site is distinct from that of all other effector molecules.


180



OMe

R

=

HO

=

R

Avermectin B

1

=

Ri

=

H 1 2 5

=

| =

1 a

Azido-AVM 1 2 5

|-Azido-AVM

Figure 11. An Affinity Probe for the Avermectin Binding Protein To demonstrate that azido-AVM retained anthelmintic activity, it was compared to ivermectin in the C. elegans motility assay (16). The L D ^ ' s were azido-AVM lOng/ml and ivermectin 3 ng/ml. A z i d o - A V M was then shown to bind to triton-solubilized C. elegans membrane proteins with IC^Q 0.3 nM, compared to 0.2 n M for ivermectin. The biologically inactive derivative octahydroavermectin which has been used routinely to uncover non-specific binding, did not inhibit the specific binding of either ivermectin or azido-AVM at concentrations up to 100 n M Furthermore, in a competitive binding study, azido-AVM was shown to be a competitive inhibitor of ivermectin with K . = 0.2 nM. 125 Similar high-affinity binding was shown for I-Azido A V M which bound with kD = 0.48 n M and bmax = 0.38 pmol/mg of protein. 125 With the assurance that I-azido-AVM bound specifically and with high affinity to the ivermectin binding site, labelling studies were undertaken using Triton X-100-solubilized C. elegans membrane proteins. Figure 12 is an autoradiogram of the result obtained when these solubilized 125 proteins were incubated with I-azido-AVM in the presence or absence of increasing concentrations of unlabeled ivermectin and then cross-linked with U V tight. Three major proteins having molecular weights approximately 8, 47 and 53 kD were revealed. As the concentration of ivermectin during the incubation was increased, the intensity of labelling diminished indicating that the bands were related to the high affinity binding site. Figure 13 shows a coomassie-stained gel of a similar solubilizedmembrane preparation on a 5-20% SDS-polyacrylamide gradient gel. The arrows show where the affinity labelled bands line up with the coomassie-stained bands. In Pest Control with Enhanced Environmental Safety; Duke, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


FISHER


Figure 12. Photoaffinity Labelling of the C. Elegans Avermectin Receptor

Figure 13. Coomassie Stained Gel of C. Elegans Membrane Proteins


182


In a similar experiment using Drosophila head membranes, a single protein with a molecular weight of approximately 47 kD was labelled (77). Although the mode of action of the avermectins is still not completely understood, isolation and sequencing of the 53,47 and 8 kD binding proteins from C. elegans and the 47 kD protein from Drosophila melanogaster, which may turn out to be degradation products of a single protein, takes us one step closer to an understanding.

Literature Cited


1.

Albers-Schonberg, G., Arison, B.H., Chabala, J.C. Douglas A. W., Eskola, P., Fisher, M.H., Lusi, A., Mrozik, H., Smith J.L., Tolman, R.L. J. Am. Chem. Soc. 1981, 103, 4216. 2. Takiguchi, Y., Mishima, H., Okuda, M., Terao, M., Aoki, A., Fukuda, R. J. Antibiotics 1980,33,1120. 3. Mishima, H., Ide, J., Muramatsu, S., Ono, M. J. Antiobiot. 1983,36,980. 4. Wood, I.B., Panksvich, J.A., Carter, G.T., Torrey, M.J., Greenstein, M . European Patent Application EP 170006, 1986. 5. Ward, J.B., Noble, H.M., Porter, N., Flatton, R.A., Noble D. UK Patent Application GB 2166436A, 1986. 6. Carter, G.T., Nietsche, J.A., Borders, D.B. J. Chem Soc. Commun., 1987, 402. 7. Ramsay, M.J.V., Roberts, S.M., Russell, J.C., Shingler, A.H., Slawin, A.M.Z., Sutherland, D.R., Tiley, E.P., Williams, D.J. Tet. Lett. 1987, 28, 5353. 8. USAN and the USP Dictionary of Drug Names, J.A. Halperin Ed., USP Convention Inc., Rockville, MD, 1991 9. Mrozik, H., Eskola P., Linn B.O., Lusi, A., Tischler, M., Waksmunski, F.S, Wyvratt, M.J., Hilton N.J., Anderson T.E., Babu, J.R., Dybas, R.A., Preiser, F.A., Fisher, M.H., Experientia, 1989, 45, 315. 10. Shih, T.L., Mrozik, H., Holmes, M., Fisher, M.H. Tet. Lett. 1990, 31, 3525. 11. Shih, T.L., Mrozik, H., Holmes, M., Fisher, M.H. Tet. Lett. 1990, 31, 3529. 12. Meinke, P.T., O'Connor, S.P., Mrozik, H. Fisher, M.H. Tet. Lett. 1992, 33, 1203. 13. Arena, J.P., Liu, K.K., Paress, P.S., Cully, D.F., in press. 14. Cully, D.F., Paress, P.S. Mol. Pharmacol. 1991,40,326. 15. Meinke, P.T., Rohrer, S.P., Hayes, E.C., Shaeffer, J.M., Fisher, M.H., Mrozik, H. J. Med. Chem, in press. 16. Schaeffer, J.M., Haines, H.W. Biochem. Parasitol. 1989, 38, 2329. 17. Rohrer, S.P., Meinke, P.K., Hayes, E.C., Mrozik, H. Schaeffer, J. Proc. Nat. Acad. Sci., 1992,89,4168. RECEIVED July 29, 1992


Recent Progress in Avermectin Research - ACS Symposium Series

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