Milbemycin H Analogue Synthesis - ACS Symposium Series (ACS

Chapter 34

Milbemycin H Analogue Synthesis 1

S. R. Schow, M. E. Schnee, and J. J. Rauh

Downloaded by PURDUE UNIV on June 27, 2016 | http://pubs.acs.org Publication Date: December 7, 1991 | doi: 10.1021/bk-1991-0443.ch034

Biological Chemistry Group, Agricultural Products Department, Ε. I. du Pont de Nemours and Company, Wilmington, DE 19880-0402

The preparation of milbemycin H analogs is presented. The results of in vitro and in vivo biological evaluation of these compounds suggest that the strategy of molecular simplification to a minimum toxicophore is unlikely to yield a useful synthetic insecticide product in this field of chemistry.

Microbes have yielded a marvelous array of chemical entities useful to mankind. Compounds such as penicillin, tetracycline, and erythromycin became the wonder drugs of our times. These and many other microbial compounds have played a significant role in reducing human suffering and even altering the natural course of human mortality. Although the history of microbial products in agriculture has been less illustrious than in medicine, the emergence of biotechnology as a significant factor in the research programs of agribusinesses will clearly expand the future role of microbes and microbial natural products in agriculture (1). Two of the most successful new microbial compounds have come from the laboratories of Merck. Both the veterinary anthelmintic ivermectin and the recently introduced hypocholesterolemic, lovostatin, have been hailed as revolutionary new drugs (2). Although Merck has developed ivermectin primarily as an antiparasitic agent for livestock and pets, Merck also found that this compound could be used to treat the human scourge of onchocerciasis, Africanriverblindness (3). In what is one of the finest examples of corporate humanitarianism, Merck developed and now distributes ivermectin free of charge to African countries for use in the treatment of this affliction (4). Ivermectin, with its biological impact and estimated $200 to $400 million dollar market, has become the premiere example of a successful microbial product in agriculture (5). Concurrent with the development of ivermectin for the veterinary market, Merck began to report field studies demonstrating the insecticidal potential of ivermectin's parent structure, avermectin B l (abamectin) (6). Avermectin B l is currently under development for control of phytophagous mites and certain insect species such as leaf miners in crops and ornamentals (6).

1

Current address: Lederle Laboratories, Pearl River, NY 10965

0097-6156/91/0443-0436$06.00/0 © 1991 American Chemical Society

Baker et al.; Synthesis and Chemistry of Agrochemicals II ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

34. SCHOWETAL.

Milbemycin H Analogue Synthesis

437

In the mid-Seventies Sankyo reported the discovery of a series of insecticidal compounds related to the avermectins, the milbemycins ÇZ). Although the commercial development of the milbemycins has not occurred as rapidly as the avermectins, the reported levels of biological activity would suggest that the milbemycins are good candidates for veterinary medicine and agronomic crop protection (2).


Synthesis Strategy We had been following the development the avermectins for several years. However, being without fermentation capabilities, we evaluated the area only in terms that might point toward a synthetic product (&)· We were particularly interested in the milbemycins as potential insecticides. By 1985, when we initiated this program in milbemycin synthesis, there existed a substantial volume of information on the synthesis, structural chemistry, and biology of the milbemycins and avermectins. The goal of our program was to try to define the minimum structural elements within this class of compounds responsible for biological activity. We sought to use biological datafromthe literature, as well as the most efficient synthetic methodology available, to achieve our goal (2). Pictured in Figure 1 is a selected group of milbemycin and avermectin structures.

Structure Activity From surveying the reported levels of activity for various avermectins and milbemycins against nematodes, mites and insects, we were able to hypothesize what minimum structure might be responsible for these activities. The key assumption was that the neurotoxic activities of avermectins and milbemycins originatedfromthe same molecular mode of action. The key structural difference between the milbemycins and avermectins is the disaccharide moiety in avermectins. As the activity of the milbemycins and avermectins is comparable, clearly the disaccharide is unnecessary for inherent activity (£, JO, JUL, 12). Aromatic compounds such as milbemycin β-3 were reported to be inactive (11). Therefore, we focused on targets with more complex cyclohexyl acids. Milbemycin β-8, β-l and Η were all reported to have significant activity (12). Thus, we concluded that neither the perhydrofuran moiety nor the C-8' oxygen were playing a crucial role. We also knew that the C-5 keto compound had reduced activity relative to the 5-β hydroxy compound (13). The 3,4 double bond was reported by ICI to be unnecessary for high levels of activity, though Merck reported otherwise (14). Figure 2 lists three target molecules which have structures consistent with the structure activity outlined above. Two additional assumptions were tested in the design of these targets. First, the peripherial methyl groups were probably minor contributors to the activity. This was a reasonable assumption for C-25, given the wide variation in C-25 alkyl-methyl substitutions without loss of activity (8,11). The second assumption was that the spiroketal might not be necessary for activity, and thus one target lacking this functionality was included.


SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS II


438

Avermectin Β l a (abamectin) Ivermectin (22^3-dihydroavermectin Β la)

Milbemycin Milbemycin Milbemycin Milbemycin

a3 (R=ethyl, X = 0 , R'=OH) 61 (R=methyl, X = O H , H , R ' = O C H ) B8 (R=ethyl, X = H , H , R'=OCH3) H (R=isopropyl, X = H J î , R*=0)

Milbemycin B3

3

Figure 1. Naturally Occurring Avermectins and Milbemycins



34. SCHOWETAL.


439

Figure 2. Milbemycin Analog Targets In order to assess the activity of our target molecules, we needed in vitro and in vivo evaluation systems. At a molecular level, these agents appear to act as allosteric agonists at the GABA-chloride channel complex in the nervous system (1£). We established in vitro assays in order to evaluate the GABAergic effects of our target compounds. We used both binding and electrophysiological assays. We also evaluated our compounds in two insect species and in a simple nematicidal screen. Target Synthesis Our strategy for target preparation was a standard "northern-southern hemisphere" convergent synthesis (2,12), as shown in Scheme I. Scheme I

The northern hemisphere was synthesized using methodology reported by Kay (Scheme II) (11. 18). The previously described spiroketal was prepared from


440


diene IV via a cationic cyclization of the THP ether V followed by a remote photohypoiodite radical oxidation and cyclization of alcohol VII. The stereochemistry generated in these cyclization steps is a result of the chair conformation adopted by the intermediate in the cationic cyclization VI and stereoelectronic controlled axial addition of the oxygen to the cation generated in the oxidative hydrogen abstraction step. Side chain homologation to the highly hygroscopic phosphonium salt X proceeded in a straightforward manner. Only the removal of the trichloroethyl protecting group proved problematical. Deprotection protocols regularly gave significant quantities of partially reduced by-products. Sonication of this reaction helped minimize this adverse result.


Scheme Π

H0CH CC1 2

H

3

BF -0(C H5)2 3


2

34. SCHOWETAL.

441


The preparation of the southern hemisphere relied on the work of Turnbull and Danishefsky (Scheme III) (H, 12). Biacetyl XI was monoprotected and acylated to give acetoacetic ester XII. Annulation with methylvinylketone followed by hydroxyl directed reduction of the C-5 ketone and hydrolysis of the ketal yielded the previously reported ketoester XIV. Silylation of the secondary-hydroxyl group gave the desired ketol XV. Repeated attempts to add nucleophiles, such as alkyl lithium and cerium reagents, to this ketol were unsuccessful. Wittig type reagents; whether phosphonium, phosphine oxide, or silicon based, failed to yield product. The starting ketol was usually recovered, indicating possible enolate formation. Two equivalents of magnesium ethoxyl acetylide was found to add smoothly to ketol XVI. Partial hydrogénation of the aficyne followed by acidic hydrolysis gave unstable aldehyde XVII.


Scheme ΙΠ

Ο

2)NaH/(CH 0) CO 3

NaOCH

Ο

2

3

ΧΠ

XI

Ç0 CH

Ο

2

Ο

OH

xm

XIV ο

Ç0 CH 2

3

3

XV ο

XVI

χνπ



442


The condensation of racemic phosphonium salt X with racemic aldehyde XVII yielded cis adduct XVIII, and the related diastereomer (Scheme IV). The highly chelating reaction conditions are extremely important for the success of this condensation. The presence of the spiroketal moiety in the phosphorane significantly interferes with the Wittig reaction, and if THF is used in place of the DME/HMPA, a dramatically reduced yield of diene is obtained. The same condensation in the synthesis of the simple pyran had a higher yield (64%) and could be carried out in THF (see Scheme V). The cis double bond was isomerized to the trans geometry using catalytic iodine or diphenyl disulfide in sunlight. The diastereomeric mixture was converted to the seco-acids XVII with hydroxide and then cyclized using Mukaiyama conditions (20). The diastereomeric mixture of seco-acids yielded the pure natural diastereomer XX. In addition to desired macrolide XX, a significant quantity of elimination product XXI was obtained. This triene appears to be selectively derived from the undesired diastereomer. Thomas has observed a similar selective cyclization in the preparation of a closely-related milbemycin analog (21). Fluoride desilylation yielded the spiroketal target II. Swern oxidation of the C-5 hydroxyl group gave milbemycin H analog III. Scheme IV

LiHMDS X + XVII

DME-HMPA (28%) + diastereomer

xvm

/TEA

l)l2/hv

or (PhS)2/hv (100%) 2)NaOH/CHOH (77%) 3

ΧΓΧ


34. SCHOWETAL.



Scheme IV continued

Simple pyran target I was prepared (Scheme V) in a manner analogous to that outlined above for the spiropyran target. As mentioned above, the Wittig coupling was high yielding in THF. In addition, macrolide formation tended to give diastereomeric mixtures rather than a single diastereomer as in the formation of spiroketal macrolide XX. It is not obvious why the respective cyclizations have different outcomes.


443

444

SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS Π Scheme V


θ ^ \

OH XXIV

I

Biological Evaluation The target compounds were evaluated in an in vitro functional assay and a binding assay. In addition, they were injected into American cockroaches and tobacco hornworms. The compounds were also added to lesion nematode (Protylenchus spp.) preparations. The crayfish stretch receptor provides a simple and rapid in vitro bioassay for drugs which have GABAergic activity (22). The stretch receptor is a single neuron which responds to a maintained tension by a constant firing of action potentials. Changes in thisfiringrate can be indicative of effects on GAB A receptors. A GABA agonist, such as ivermectin, will cause a decrease in this tonic firing rate, while a GABA antagonist will block the agonist induced decrease. Both compounds I and II were as active as GABA in reducing thefiringrate of the stretch receptor neuron. However, neither approached the potency of ivermectin. This decrease infiringrate was partially blocked by picrotoxinin (GABA antagonist) for both I (43% block) and Π (64% block) indicating the reduction in firing rate was due to an opening of chloride channels. Compound I was readily reversible in standard saline, while compound II was only partially reversible. Ivermectin produces an irreversible decrease in firing rate, which can be restored by perfusion of picrotoxinin. These results indicate that both compounds are interacting with the GABA receptor in a manner similar to ivermectin, but are substantially less potent. Radioligand binding assays provide a convenient in vitro method for assessing the degree of interaction of GABAergic molecules with the GABA-dependent chloride channel. The radiolabeled bicyclic phosphate, [35S]-t-butylbicyclophosphorothionate ([35S]-TBPS), binds specifically and with high affinity to GABA-dependent chloride channels of rat brain membranes (23) and housefly membranes (24). Allosteric displacement of [35S]-TBPSfromits binding site on these membranes was used to



34. SCHOW ET AL.


445

quantitate the extent of interaction of avermectin, ivermectin, and compounds I and Π with the chloride channel. In rat brain membranes, ivermectin was found to be the most potent displacer of [35S]-TBPS. It had an IC50 value (concentration of compound which displaced 50% of the radioligand) of 2 μΜ. Avermectin was approximately 12-fold less active (IC50 = 25 μΜ) than ivermectin. Compound II had an IC50 of just over 100 μΜ and compound I was virtually inactive. These results indicate that compounds I and II have significantly weaker binding interactions (at least 50-fold) than ivermectin at the [35S]-TBPS binding site on the chloride channel. Neither I nor II was toxic to American cockroaches or tobacco hornworms when up to 300 ppm of compound was injected. However, at 10 ppm, both I and II caused an immediate, but temporary paralysis of lesion nematodes which lasted for 24 hours. Unfortunately, within 48 hours the nematodes recovered and movement was normal for the remaining three days of observation. In additional tests of compound II, it was shown that II was inactive in a standard mite screen (50 ppm) and that Π was lethal to crayfish at 100 ppm. The crayfish showed neurotoxic symptoms to II at concentrations as low as 30 ppm. It is clear that although the target structures possess a hint of the in vitro and in vivo activity seen in avermectin, the potency is dramatically reduced. Milbemycin Η analog ΙΠ was inactive in the in vitro assays, as well as in the whole animal tests. Conclusion Three partial structures of milbemycin Η and related natural milbemycins have been prepared, yielding new information about structure-activity relationships for this new class of insecticides. Structure I was shown to be a simple toxicophore retaining the characteristic activity of the milbemycins. However, with the minimum structure, minimum activity is observed. This raises interesting philosophical questions with respect to the strategy of looking for a potent simple core molecule imbedded within a potent complex molecule. Such a strategy has clearly been successful for morphine simplification (e.g. meperidine). However, morphine may be more the exception than the rule. Frequentiy, a seemingly insignificant piece of molecular architecture is removed, only tofindthat the activity seen in a very potent parent molecule is dramatically reduced or lost altogether in the analog. It appears that the evolutionarytidesthat push an organism to produce and refine complex natural products may lead to structures that are exquisitely fine tuned to a desirable activity. Slight modification of such structures quickly erodes activity. In such cases, only by adding complexity can the activity in the parent be significantly increased. The work presented here suggests the more prudent strategy for product discovery in the milbemycin class of insecticides is modification of the natural product However, it should be noted that the Upjohn group has reported that certain spiroketal northern fragments possess anthelmintic activity, thus keeping the idea of a simple avermectin toxicophore alive (25). In general, it may be wiser to start a discovery program with an active simple molecule and to try to optimize activity by increasing complexity of the analogs. Such a strategy allows the chemist quickly to survey a large number of modifications, as well as to learn the lessons that evolution hides in the complexity of the natural product.


446

SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS Π

Acknowledgments The authors gratefully acknowledge the technical assistance of James Krywko, Dr. M J . Fielding (nematode assay), Dr. Timothy Halls (C-l3 spectra), and Dr. George Furst (500 MHz proton spectrum, University of Pennsylvania, NSF Regional NMR Center). Literature Cited 1.


2. 3. 4. 5. 6.

7.

8.

9. 10. 11. 12. 13.

Biotechnol. News 1989, 9, 4-5; Farm Chem. 1989, 152, 23-24, 28; Ratner, M. Biotechnology 1989, 1, 337, 339-341. Business Week November 26, 1984, 114, 118; Time February 22, 1988, 44-45. Campbell, W.; Fisher, M.; Stapley, E.; Albers-Schonberg, G.; Jacob, T. Science 1983, 221, 823-828; Campbell, W. New Zealand Veterinary Journal 1981, 29, 174-178. The New York Times Magazine January 8, 1989, 20-27, 58-59; Nature 1987, 329, 752; Business Week April 26, 1984, 168, 172-173; Science 1987, 238, 610; Scrip December 21, 1988, 1371, 11. Wall Street Journal August 2, 1984, 1; P.R. Newswire July 16, 1987; Stinson, S. Chemical and Engineering News October 5, 1987, 51-67. Strong, L.; Brown, T. Bull. ent. Res. 1987, 77, 357-389; Valiulis, D. Agrichemical Age January, 1985, 28, 40; Roush, R.; Wright, J. J. Economic Entomology 1986, 79, 562-564; Dybas, R.; Babu, J. Proc. Brit. Crop. Prot. Conf. - Pests and Diseases 1988, Vol. 1, pp. 57-64. Mishima, H.; Kurabayashi, M.; Tamura, C.; Sato, S.; Kuwano, H.; Saito, A. Tetrahedron Letters 1975, 711-714; Mishima, H. In Pestic. Chem.; Hum. Welfare Environ. Proc. Int. Congr. Pestic. Chem., 5th, 1982; Miyamoto, J., Ed.; Pergamon Press: NY, 1983, Vol. 2, pp. 129-134; Takiguchi, Y.; Mishima, H.; Okuda, M.; Terao, M.; Aoki, Α.; Fukuda, R. J. Antibiotics 1980, 33, 1120-1127; Okazoki, T.; Ono, M.; Aoki, Α.; Fukuda, R. J. Antibiotics 1983, 36, 438-441; Takiguchi, Y.; Ono, M.; Muramatsu, S.; Ide, J.; Mishima, H.; Terao, M. J. Antibiotics 1983, 36, 502-508; Ono, M.; Mishima, H.; Takiguchi, Y.; Terao, M. J. Antibiotics 1983, 36, 509-515; Mishima, H.; Ide, J.; Muramatsu, S.; Ono, M. J. Antibiotics 1983, 36, 980-990. Fisher, M; Mrozik, H. In Macrolide Antibiotics; Omura, S., Ed.; Academic Press: NY, 1984, pp. 553-606; b). Fisher, M. In Recent Advances in the Chemistry of Insect Control; Janes, N., Ed.; The Royal Society of Chemistry Special Publication no. 53: London, 1985, pp. 53-72. Crimmins, M.; Hollis, W., Jr.; O'Mahony, R. In Studies in Natural Products Chemistry, Vol. 1: Rahman, Α., Ed.; Elsevier: Amsterdam, 1988, pp. 435-495. Mrozik, H.; Linn, B.; Eskola, P.; Lusi, Α.; Matzuk, Α.; Preiser, F.; Ostlind, D.; Schaeffer, J.; Fisher, M. J. Med. Chem. 1989, 32, 375-381. Kay, I; Turnbull, M. In Recent Advances in the Chemistry of Insect Control; Janes, N., Ed.; The Royal Society of Chemistry Special Publication no. 53: London, 1985, pp. 229-244. Sankyo, Japanese Patent 57 136 585, 1982; Sankyo, Japanese Patent 62 155 281, 1987; Sankyo, Japanese Patent 63 227 590, 1988; Goegelman, R.; Australian Patent 86 570 88, 1986. Fisher, M.; Verbal comments during the symposium on "Recent Advances in the Chemistry of Insect Control"; Cambridge, England, September 25-27, 1984; Jaglan, P.; Arnold, T.; Conders, G.; Gemrich, E. Third Chemical Congress of North America, Toronto, Canada, June 5-10, 1988; Agrochemical Abstracts 160.



34.

SCHOWETAL.


447

14. Ref. 11 pp. 238-240; Chabala, J.; Mrozik, H.; Tolman, R.; Eskola, P.; Lusi, Α.; Peterson, L.; Woods, M.; Fisher, M. J. Med. Chem. 1980, 23, 11341136. 15. Yamamoto, J.; Nishida, Α.; Aoki, A. Jpn. J. Appl. Entomol. 1981, 25, 182. 16. Pong, S.; Wang, C. J. Neurochemistrv 1982, 38, 375-379; Wann, K. Phytotherapy Research 1987, 1, 143-149; Wright, D. In Neuropharmacol. Pestic Action 1985: Ford, M., Ed.; Horwood: Chichester, UK, 1986, pp. 174202. Graham, D.; Pfeiffer, F.; Betz, H. Neuroscience Letters 1982, 29, 173176. 17. Schow, S.; Bloom, J.; Thompson, Α.; Winzenberg, K.; Smith, Α., III, J. Am. Chem. Soc. 1986,108,2662-2674. 18. Kay, I.; Williams, E. Tetrahedron Letters 1983, 24, 5915-5918; Concepcion, J.; Francisco, C.; Hernandez, R.; Salazar, J.; Suarez, E. Tetrahedron Letters 1984, 25, 1953-1956; Kay, I.; Bartholomew, D. Tetrahedron Letters 1984, 25, 2035-2038. 19. Turnbull, M.; Hatter, G.; Ledgerwood, D. Tetrahedron Letters 1984, 25, 5449-5452; Danishefsky, S.; Etheredge, S. J. Org. Chem. 1982, 47, 47914793. 20. Mukaiyama, T.; Usui, M.; Saigo, K. Chemistry Letters 1976, 49-50. 21. Hughes, M.; Thomas, E.; Turnbull, M.; Jones, R.; Warner, R. J. Chem. Soc., Chem. Commun. 1985, 755-758. 22. Edwards, C.; Kuffler, S. J. Neurochem. 1959, 4, 19-30. 23. Squires, R.; Casida, J.; Richardson, M.; Saederup, E. Mol. Pharmacol. 1983, 23, 326-336. 24. Olsen, R.; Szamraj, O.; Miller, T. J. Neurochem. 1989, 52, 1311-1318. 25. Nelson, S. U.S. Patent 4 686 297, 1987. RECEIVED August 2, 1990


Milbemycin H Analogue Synthesis - ACS Symposium Series (ACS

Recommend Documents