Regio- and Stereoselective Synthesis of 13-Substituted-milbemycins

Chapter 22

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Regio- and Stereoselective Synthesis of 13β-Substituted-milbemycins, Including the 13β-Alkyl-milbemycins Anthony C. O'Sullivan and Bruno Frei Plant Protection Division, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland

The synthesis of 13β-substituted milbemycins including 13β-alkyl milbemycins was examined in some detail. After establishing the SN reaction as the prefered method of introduction of substituents at the 13β position, it was found that the optimal synthesis of 13β-ethyl-milbemycin A 20B involves a novel syn 1,3 reaction of 15-chloro-Δ13,14-milbemycin A 22B with triethyl aluminium. Another new alkylation reaction using a nickel or cobalt catalysed reaction of dialkyl zinc with 13β-bromo-milbemycin A 14B, involving a radical intermediate, was developed. Biological results are discussed. 1

4

4

4

The milbemycins are a family of 16-membered ring macrolides, isolated by Sankyo chemists in 1973 (7), which possess potent anthelmintic, acaricidal, and insecticidal activity (2)(Table 1). The avermectins are compounds with similar structures and biological activity as the milbemycins, which were isolated later by workers at Merck, Sharp, and Dohme (J), who have commercialised a mixture of 22,23 dihydroavermectin Β la and Bib in 1981 asan antiparasitic agent under the generic name Ivermectin (4)(Table 1). The interesting chemical structures of this class have generated an enormous effort towards the total synthesis of these compounds (5), and the novel mode of action (6) and potent biological activity have attracted large industrial interest (7).

0097-6156/92/0504-0239$06.00/0 © 1992 American Chemical Society

Baker et al.; Synthesis and Chemistry of Agrochemicals III ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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SYNTHESIS AND CHEMISTRY O F A G R O C H E M I C A L S III

A few years after the introduction of Ivermectin to the market, Ciba-Geigy and Sankyo started work on a joint derivatisation program, seeking to optimise the anthelmintic activity of the milbemycins. For strategic purposes, at the beginning of the joint project, the research was divided between the two groups, with Ciba-Geigy treating the top half of the molecule, and Sankyo the bottom half, in particular the hydroxy group at C(5). Sankyo's early work led to the discovery of the heart worm activity of the C(5)-oxime, and its introduction in 1990 into the market under the trade name Interceptor. Ciba-Geigy's initial effort resulted in the derivatisation of several positions in the upper part of the molecule (e.g. position 29 (8)). We describe here the functionalisation and derivatisation of C(13), in particular the alkyl derivatives.

Table I. Structures of Milbemycins and Avermectins

R

3

H

Me

CH -CH

4

H

Et

CH2-CH2

H

i-Pr

CH -CH

OMe

s-Bu

CH=CH

i-Pr

CH=CH

5-Bu:/-Pr

CH -CH

2

80:20 j-Bu:/'-Pr

CH -CH

2

Name

1A

Milbemycin A

IB

Milbemycin A

ID

Milbemycin D

2

Avermectin B

l a

3

Avermectin B

l b

41

Ivermectin

51

X-Y

Ri

No

1

Ivermectin aglycone

J H3C

OH

OMe

Ο

Ο

2

2

2

2


2

2

2

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By compounds numbers with A,B,D, and I, we refer to derivatives of milbemycin A , A , D, and 22,23 dihydroavermectin respectively. The series differ at the C(25) alkyl group. However, this group is chemically quite inert and remote from the part of the molecule which is the subject of this paper. We have continually observed comparable results in reactions in the region of C(13) of the three milbemycins [C(25) = Me,Et, and iPr] and two ivermectins [C(25) = iPr and iBu]. The reader may thus safely ignore the lettering part of the numeration, which we are forced to adopt because no one series spans all the reactions described here. The most striking difference between the milbemycins and the avermectins is the presence of a dioleandrose unit in position 13 of the avermectins, and it was tempting therefore to ascribe the higher anthelmintic potency of the avermectins to this disaccharide, as the other structural differences are relatively small. It was of interest therefore to prepare a series of 13 substituted milbemycin derivatives in order to examine their structure-activity relationships. The biological results of several 13 substituted milbemycins have already been described by the Merck group of Fischer, Mrozik, et al. (9). In this paper we will examine the synthesis and mechanism in some detail with emphasis on the biologically more important compounds. 3

4

Synthesis of intermediates. We were able to functionalise the north-west corner of the molecule through epoxidation of the 9,10 double bond and subsequent opening of the epoxide to the allylic alcohol 7B. Allylic rearrangement with Cr(VI) provided the 13β hydroxy milbemycin derivative 8B, which was epimerised at C(13) to the 13a alcohol 10B corresponding to ivermectin aglycone 51 (70)(Fig. 1). Alternatively a direct microbiological hydroxylation of the milbemycins at the 13β position was accomplished (77). It is necessary for many of the reactions described hereafter to protect the 5-OH group. The choice of protecting group is not of primary concern, but we have found the tBuMe Si (TBDMS) group to be consistently useful (72). Although only substructures are shown hereafter the protecting silyl group at position 5 should be understood to be present, except when explicitly otherwise indicated or when the biological activity is discussed. 2

Synthesis and mechanism of formation of 13-fluoro milbemycins. The synthesis of ^-fluoro-milbemycin D 10D supplied us with mechanistic information, which later proved invaluable for the synthesis of other 13 substituted milbemycins. We treated the allyl alcohols 7D, 8D, and 91 separately with Et NSF (DAST)(73) at -60°C for 5 minutes (Fig. 2). From 7D and 8D the same ^-fluoro product was formed, and from 91 a mixture of 13β and 13a fluorides 101 and 111. 2


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SYNTHESIS A N D CHEMISTRY O F A G R O C H E M I C A L S III

Figure 1. Synthesis of intermediate allylic alcohols


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7D

10D (88%)

8D

10D (100%)

91

101(20%)

111(49%)

Figure 2. Reactions of the three allylic alcohols with DAST

The reaction of allylic alcohols with DAST has been interpreted by Middleton as involving a cationic intermediate (13). Mixtures of products were formed, and there was a preponderence of 1,3 syn regioselectivity in less polar solvents. The clearest indication that the reactions described here have the same mechanism is the retention of stereochemistry observed at C(13) in 7D -* 10D, 8D -> 10D and 91 -* 111. The inversion 91 -* 101 is more ambiguous. An SN mechanism has been suggested (9), however every attempt to induce an SN reaction at positon 13 using a 13β leaving group was unsuccessful. For example treatment of the Ββ-iodo derivative 15 with thiols or cuprate reagents did not lead to 13a-substituted products. As the same transition state is involved in an SN reaction in the forward or reverse direction, we conclude that SN reactions in both directions at C(13) are slow. Therefore we attribute the inversion 91 -* 111 to an SNj reaction. All three reactions can thus be accomodated by one synthetic scheme involving the cation 12 (Fig. 3). It can be seen from these reactions, that of the four possible stereoisomers only the 13,14E 14,15E isomer 12 is formed. Having established a common mechanism for all three reactions, it can be seen that an unusually stereo- and regioselective attack on an allylic cation takes place, which would be more indiscriminate in other structural surroundings. This regio- and stereoselectivity stems from the asymmetry of the cation 12. The conformation of this cation is not amenable to study by molecular mechanics calculations. However a useful model is 13-keto-milbemycin A , which contains a similar array of planar sp centres at the pertinent 13, 14, and 15 carbons, and exhibits a similar, but less pronounced, β-selectivity of nucleophilic attack at position 13 (10). Models (10) show that the α-face of the C(13), C(14), C(15) allylic system is sterically shielded at this position by the spiroacetal subunit, hindering attack at this face and inducing the observed stereoselectivity. 2

2

2

2

2

4


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SYNTHESIS A N D CHEMISTRY O F A G R O C H E M I C A L S HI

Figure 3. The DAST reactions involve a common cationic intermediate The 13a-substituted fluoride 121 is formed with retention of configuration (Fig. 2) in a manner typical for cations reacting with a nucleophile within a solvent cage (14). The starting aglycone 101 has a propensity for reaction with retention, and it has been suggested that the cation formed from 101 is stabilised through secondary orbital overlap with the 10,11 double bond in a conformation which allows nucleophilic attack only at the α-face (9). Although this proposal may well be correct, it is perhaps also interesting to consider this behaviour as one example of a more general phenomenon. Unfunctionalised cycloalkyl bromides with ring sizes greater than 7 have greatly diminished SN, and greatly increased SN! reactivity in comparison with their open chain analogs (75). The data of Fierenz et. al. are shown below (Figure 4). The increased SNj reactivity has been convincingly explained by Prelog (76) and Brown (77), but the origin of the dependancy of SN reactivity on ring size has never been explained. It seems clear, however, that the pentacoordinate transition state is not attainable for every conformation of a larger ring, and that substituted ring systems should be similarly affected. A further albeit negative indication of lowered S N reactivity in macrolides is that in two reviews of the chemistry of macrolides (18), only one example of an S N reaction is to be found (79). We suggest that lowered SN reactivity is general for every macrolide including milbemycin, and consider the DAST reaction described here to be merely one example of this effect. Recognition of this effect will hopefully help chemists working with macrolides understand and plan their synthetic work. 2

2

2

2


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1200 τ

SN2 open chain I SN2

-SN1 open chain SN1

1 0

1 5

0

5

10

15

Figure 4. Reaction rates for SN1 and SN2 reactions plotted against ring size.

Synthesis of further 13p-substituted milbemycins. As a consequence of the mechanistic origin of the observations described above, the reaction at C(13) was successful, whereas the SN did not take place under any conditions tried. The introduction of other groups into position 13 of the molecule would then be most sensibly achieved using an strategy. In other words when the cation 12 is generated in the presence of other nucleophiles, stereoselective formation of Bp-substituted milbemycins can be expected, and, in fact, we were able to synthesise a large variety of compounds using this strategy. Chlorination, bromination and iodination (20) of the allylic alcohols 7(A,B> and/or D) led, in each case, to the 13p-substituted halides 13-15 (27)(Figure 5)(Table II). With phosphorus oxychloride, attack of chloride on cation 12 took place at position 15 in a side reaction to form 16D, but again stereoselectively on the β face. The regioselectivity of the analogous alkylation reaction will be discussed in more detail below. 2


246


13-15

16D

Figure 5

Table Π. Synthesis of 13p-halo-milbemycins

Substrate

Reagent

Product

X

Yield

7D

DAST, CH C1 , -60°

8D

F

97%

7D

SOCl , Et N, RT

13D

Cl

34%

7B

SOCl , Et N, RT

13B

Cl

47%

7A

SOCl , Et N, RT

13A

Cl

52%

7D

P O C l , pyridine

13D

Cl

28%

7D

PBr

2

2

2

3

2

3

2

3

3

7D

3

PPh ,1 , imidazole 3

2

a

14D

Br

49%

15D

I

64%

a)+16D(16%)

We applied many methods for generating the cation 12. They were chosen case for case with regard to the nucleophile to be introduced, which is present in the reaction mixture before the cation is generated. Treatment of the allylic alcohols 7 or 8 with protic or Lewis acids is probably the most straightforward example. Conversion of the hydroxyl to a leaving group such as an ester or a sulfonate and subsequent treatment with a protic or Lewis acid is also often advantageous. Alternatively, for the synthesis of the 13 ketone (70), the 13p-bromide 14D was treated with a silver salt. As nucleophiles, several alcohols (22), thiols (22), acids (25), thioacids (24), amides (23), nitriles (25), and DMSO (70) were used successfully, with variable yields, again dependent on the method used. The products were often accompanied by the regioisomeric A » E-15S-substituted products analogous to 16D, as expected from the allylic cation intermediate (Figure 6). 13

14


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247

Figure 6. Synthesis of various Bp-substituted milbemycins.

Synthesis of Ββ-alkyl milbemycins. The synthesis of B-alkyl substituted milbemycins proved to be a challenge. Many variants of the cuprate addition using several different leaving groups and different cuprate reagents (26) were unsuccessful. But by applying once more the by now already established strategy of using conditions which favour an SNj mechanism, we utilised the trialkylaluminium reaction of Yamamoto (27), which is known to occur via just this mechanism. Treatment of the allylic acetate 18B, easily prepared from 7B, with triethyl- or trimethylaluminium led to the desired Ββ-alkyl milbemycins (19B and 20B) contaminated in the former case by the A E-15S-ethyl regioisomer 21B (Figure 7). 1314

American Chemical Society

Library 1155 16th St., N.W. Washington, D.C. of 20036 Baker et al.; Synthesis and Chemistry Agrochemicals III ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

248


Et Al > iBu Al to the point where the 15S-isobutyl compound becomes the major product. This bulkiness is met again in the aluminium sulfonate counterions to 12, where the camphersulfonato-aluminate anion yields more 15S-ethyl than its mesylate analog (Entries 3 and 4). As always only attack on the β face of the cation 12 is observed. 15S-Chloro-A13,14-milbemycin A 16B produced, unexpectedly but to our satisfaction, the desired compound with high stereoselectivity in ca 50% yield (Entry 8). The explanation for the regioselectivity of the reaction with the 15S-chloride requires a more detailed understanding of the mechanism of the reaction, which has much in common with the reaction of thionyl chloride with allyl alcohols (28). 4

4

3

3

3

3

4


22.

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13fi-Substituted-milbemycins

Figure 8

Table ΠΙ. Regioselectivity of the alkylation Entry

Starting Material

Reagent

Product Ratio 13:15

1

15-OAc

Et Al 3

75:25

2

13-OCHO

Et Al 3

75:25

3

15-OS0 Me

Et Al

80:20

Et Al 3

30:70

Et Al

30:70

iBu Al

40:60

2

3

4 13-OS0 — ' 2

°

4l

5

3

6

15-OS0 —' 15-OAc

7

13-C1

Et Al

70:30

8

15-C1

Et Al

95:5

2

°

3

3

3


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SYNTHESIS A N D CHEMISTRY O F A G R O C H E M I C A L S ΙΠ

When allylic esters are used as substrates, the intermediate cation 12 is generated within a solvent cage. If the ion pair reacts before the two ions diffuse from the solvent cage, then retention of stereochemistry is observed. Should escape from the solvent cage be faster than product formation, then the reaction becomes unselective. In the present case the asymmetry of the milbemycin molecule induces a selectivity, irrespective of the mechanism of the reaction, so the results can be explained by both the caged or free ion pair mechanisms. When allylic chloride 16B is used as substrate (Table ΠΙ; Entry 8), the reaction is ordered further. Restricted movement within the solvent cage influences profoundly the regioselectivity of the reaction. The trialkylaluminate anion derived from the allylic esters tumbles or rotates within the solvent cage. Both Πβ-and 15Sleaving groups then give rise to the same distribution of anion rotamers, and consequently to the same distribution of regioisomers. However, rotation of the chloroaluminate anion is slower than product formation. The sequence of events begin with complexation of triethylaluminium with the chloride. Both partners are polarised by this complexation. The alkyl groups bound to aluminium now carry a small negative charge, and the terminal atom of the double bond in the allylic system a small positive one. These two moieties are aligned towards each other by a small electrostatic interaction, which increases after bond breakage and ion pair formation. Product formation occurs before the ion tumbles out of this alignment, and a 1,3-syn facial selectivity is observed. We may regard the reaction as pseudoconcerted. Gallina observed this behaviour on reaction of an allylic benzoate (29). However, in our case a 1:1 mixture of products was observed from the 15S-benzoate, and some substituted 15S-benzoates offered no improvement. We suggest that halides have a greater predisposition for pseudoconcertedness in comparison to esters or sulfonates, because of the ring size of the intermediate reacting complex (Figure 9). Six-membered rings are known to be less strained than 8-membered ones (30). The different product ratio obtained from the 13p-chloride is characteristic for non-rotating ion pairs (Table ΙΠ; Entry 7), although full stereoselectivity was not observed.

Strained

Unstrained

Figure 9. Pseudoconcerted trialkylaluminium reactions. With the alkylation step now proceeding with an acceptable level of regioselectivity, the problem is now reduced to the synthesis of the intermediate allylic chloride 16B. It was originally obtained only as a side product in the chlorination of 7B with P0C1 (Table Π), but now a more selective method was required. The ene-like allylic chlorination of milbemycin with in situ generated HOC1 has been shown by Burckhardt to produce exclusively the chloride 22B with an exocyclic Δ · double bond (31). In an attempt to direct double bond formation towards C(13), several other reagents were examined (32), many of which led to 3

14

29


22.


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251

mixtures of 16B and 22B. The most selective was tBuOCl (33), which yielded the endocyclic compound 16B as the major product (see Fig. 10). More recently, A. Saito and colleagues at Sankyo (34) found that Me SiCl as an additive in this reaction improves the ratio of regioisomers to 8:1, from which the desired product 16B was isolated in 63% yield. 3

22B Ratio 16B:22B

Reagent NaOCl/C0

0:1

2

3:2

tBuOCl tBuOCl (+MeSiCl) 3

8:1

Figure 10. Regioselective chlorination The critical steps in the reaction scheme now seem resolved. The regioselectivity of the chlorination and subsequent alkylation steps would be adequate for further development. However a further improvement was introduced, when it was found that the tBuMe Si protecting group can be dispensed with, as both steps were sucessful with the unprotected 5-OH group. The hydroxy group reacts with triethylaluminium, but the aluminium-oxygen bond is cleaved again upon acidic work-up. The synthesis of 13p-ethyl-milbemycin A 20B from milbemycin A IB is thus reduced to two steps with yields of 63% and 54% respectively. 2

4

4

Transition Metal Catalysed Synthesis. Although the synthesis of 13p-ethyl-milbemycin A 20B was accomplished in a satisfactory manner with triethylaluminium, the synthesis of other analogs using this method in our laboratories was restricted to the series of commercially available trialkylaluminiums. In order to avoid the synthesis of these extremely air and moisture sensitive materials, and in the search for a new route to 13p-ethyl-milbemycin A 20B, a transition metal catalysed approach was explored. The well studied and understood palladium catalysed reactions used extensively by Trost and Tsuji (35) involve an SN mechanism, which has been uniformly unsuccessful at the 13 position of milbemycin. However, allylic radicals have a planar structure similar to allylic cations. A milbemycin radical intermediate may be expected to have a similar degree of conformational freedom, and thus a similar stereoselectivity in its further transformations as the allylic cation 12, which is a useful intermediate (see Figure 11). Consequentiy we counted on the propensity of transition metal catalysed reagents to undergo radical reactions (36) and were rewarded by some interesting new chemistry. 4

4

2


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SYNTHESIS AND CHEMISTRY O F A G R O C H E M I C A L S HI

Figure 11. Radical vs cation as an intermediate For the introduction of an alkyl group along these lines, an alkyl anion source and a transition metal salt or complex are necessary. Nickel was chosen as metal. The choice of alkyl anion source proved to be a matter of some importance, as demonstrated by the series of experiments depicted in Figure 12. Using the same nickel complex, the reaction took a very different course when either ethyl Grignard, ethylzinc bromide or diethylzinc was used as the ethyl source. In every case the intermediacy of the allylic radical may be assumed, which undergoes either Wurtz coupling or complexation with the nickel leading to the isolated products. No attempt will be made to explain these results, except to point out that no mechanism proposed in the literature for such alkyl-alkyl couplings (57) allows for such variability.

Figure 12. Dependence of product on ethyl anion source


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The radical route is thus successful using diethylzinc, but again a mixture of 13P:15S regioisomers (20B and 21B) was formed. A variety of other transition metal catalysts was screened using diethylzinc under conditions identical to those used above (Fig. 13 Table IV). Only nickel and cobalt complexes led to the desired products. The choice of ligand is important too. A typical result with many complexes was the formation of milbemycin dimers via Wurtz coupling. Palladium chloride complexed with bisdiphenylphosphinoferrocine (dppf) led to the formation of 5-OtBuMe Si-milbemycin A 4 together with its Δ* · isomer. However nickel dichloride complexed with 1,3-bisdiphenylphosphinopropane (dppp) or 1,4-bisdiphenylphosphinobutane (dppb) provided compounds 20B and 21B in good yields, again in approximately equal amounts. The best result was obtained with nickel chloride alone, without organic ligands, which led to a 2.5:1 ratio of the 13p:15S ethyl-milbemycin regioisomers, from which the desired isomer could be isolated in ca 40% yield. A further improvement in this route was achieved with the discovery that the Δ · 15S-chloro compound 16B used for the alkyl aluminium reaction could also be used here. In addition the protection of the 5-hydroxy group could be dispensed with, although separation of the isomers was then more difficult. Overall from milbemycin A IB, the synthesis then involved two steps of 63% and 40% yields respectively. 3

14

2

13

14

4

CH,

CH Ε

Et Zn 2

ι

E 3

ν Λ ^

T

-

/

15

CH^

Catalyst 14B

21B

20B Figure 13.

Table IV. Optimisation of the Catalyst Catalyst

Ratio 20B:21B

(Ph P) NiCl 3

2

dppe.NiCl

1:1

2

Slow Reaction

2

dppb.NiCl

2

(cyclohex P) NiCl 3

2

triphos.NiClBF

2

50%

1:1

76% Dimers

2

Dimers

4

Slow Reaction

2

dppf.PdCl NiCl

1.7:1

2

(Ph P) CoBr 3

Other Products

70%

2

dppp.NiCl

dppf.NiCl

Yield

2

1:1

40%

2.5:1

54%

Reduction(13 and 15H)

2


254


As a process for the synthesis of Ββ-ethyl milbemycin A 20B, the nickel catalysed organozinc reaction is not competitive with the triethylaluminium reaction described above. However, it was possible to synthesise products, which would have been obtainable only with difficulty from the corresponding trialkylaluminium reagent (Fig. 14; Table V). Of course this new process has its own limitations. The most serious one is due to the tendency of organo transition metal compounds to undergo β-Η elimination to the metal hydride. This results, in the present case, in the reduction of 15S-chloro-millbemycin A 16B or Ββ-^οπιο-ιηίΙΙ^Γηναη A 14B to milbemycin A IB when diisopropylzinc or di-3-phenylpropylzinc was used. Alkyl groups without hydrogens in the β position do not suffer from this problem, but use of dineopentylzinc and its phenyl analog resulted in a new β-elimination process, this time in the milbemycin ring affording a Δ12,13 milbemycin derivative, which was competitive with the alkyl-alkyl coupling. Diphenylzinc was unproblematic in this respect and produced smoothly Ββ-ρΐιβηνί-ππ^πινΰΐη A 24B. This compound showed an interesting biological profile, and served as a lead for other 13β-aryl derivatives (59). Several were synthesised from the corresponding diarylzincs, which were prepared either in salt-free form using the centrifugation method of van Koten (40), or more often but with a decrease in yield, from reaction of the Grignard reagent with zinc chloride and coupling in situ. 4

4

4

4

4

Figure 14.

Table V. Variation of the alkyl group R Group

H C

Ratio 13R:15R

23B

2:1

Yield

25%

Other Products

1 2

Δ ·

1 3

(3%)

3

Ph Δ

12.13

(23%)

H C 3

Ph

13H(reduction) (65%)

13H(reduction) (35%)

iPr Ph

24B

4:1

40%


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Biological Activity. There are two ways of looking at the biological activity of this series of compounds. Firstly in vivo by measuring activity against a parasite isolated from, or in relation with, its host. Secondly in vitro by measuring the binding of the milbemycin analog to its receptor, which triggers a chain of physiological events which culminates in the death of the parasite. The receptor binding assay has been described by Schaeffer et.al.(4V). Several 13-substituted milbemycin derivatives were tested in the receptor assay and all bound well to the receptor (42), however this was by no means reflected in vivo. Each molecule is a separate entity and is prone to the vagaries of the permeability of the various membranes and other bodily barriers, the metabolism in the host and parasite, and other factors, which are loosely termed transport phenomena. The transport factors dominate the in vivo results to such an extent that the tentative structure-activity relationships we made in the early stages of the work without the benefit of the results from the receptor tests were invalidated later by newer results. Because the in vivo results did not correlate well with the in vitro results or the results of tests in model animals, we tested all new compounds for anthelmintic activity directly in a sheep test, involving a mixed infection of Haemonchus contortus and Trichostrongulus colubriformis. Amblyomma hebraeum larvae on chicks were used as a test for tick activity. Mange activity was screened using a mouse model. The anthelmintic activity of one of the first Hp-substituted milbemycins 13P-fluoro-milbemycin D 10D - was about the same as that of milbemycin D ID itself in the sheep test, but the activity against mange on mouse and A. hebraeum ticks on chicks was improved, which encouraged us to synthesise further ^-substituted milbemycins. Of the many compounds tested by us up to the synthesis of the 13-alkyl derivatives, the 13p-methyl- (19) and ethyl- (20) milbemycins showed very high activities in the sheep test described above. Whereas milbemycin A IB does not control the mixed infection sufficiently at a dose of 0.2 mg/Kg, the 13P-ethyl derivative, brought about a 100% reduction of eggs/g feces at a dose of 0.025 mg/kg. The other parasites tested in sheep were controlled well by a dose of 0.1 mg/kg, and overall the compound appeared at least as useful as Ivermectin 41 as an anthelmintic in sheep. Its disadvantages were firstly a weak activity against ectoparasites, and, more crucially for our purposes at the time, a weakness when used as an anthelmintic in cattle. One particularly critical example was a trial against Ostertagia ostertagii arrested larvae in cattle, normally controlled well by a dose of 0.2 mg/kg Ivermectin, which were not fully controlled by a dose of 0.5 mg/kg Ββ-ethyl milbemycin A 20B. In the n-alkyl series, ethyl 20B was slightly better than methyl 19B, which in turn was much better than η-propyl. The longer straight chain Ββ-alkyl milbemycins tested were virtually inactive in the sheep test. ^-Neopentyl-milbemycin 23B was a weak anthelmintic but was much more active than milbemycin A IB against ectoparasites. Against A. hebraeum on chicks it was fully active at 0.5 mg/kg. In comparison milbemycin A IB is inactive at 12 mg/kg, whereas ivermectin 41 is fully active at 1.4 mg/kg. 13(î-Phenyl-milbemycin A 24B shows both endo and ecto parasitic activity. It is similar in activity to ^-ethyl-milbemycin A 20B against intestinal helminths both in sheep and cattle, again with about the same weakness against inhibited Ostertagia larvae in cattle. It is fully active against A. hebraeum in chicks at 1.5 mg/kg. However none of its analogs tested were substantially more active than ^-phenyl-milbemycin A 20B itself. 4

4

4

4

4

4

4


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Conclusion. In summary we have shown how to functionalise the north-west corner of milbemycin and introduce various substituents into the 13 position. The consequences of using allylic cations and radicals as intermediates were evident in the mixtures of regioisomers which were formed in these reactions, and the problem posed by the regioisomeric mixture was solved in the case of Ββ-ethyl-milbemycin A 20B, a potent sheep anthelmintic. Some interesting new organometallic chemistry was developed during this optimisation, which resulted in two two-step syntheses of Ββ-ethyl-milbemycin A 20B. The large differences in in vivo biological activity between structurally very similar compounds make the correlation of structure with activity precarious. After testing several Bp-substituted milbemycins in the receptor binding assay, we conclude that all Ββ-substituted milbemycins are potentially active on the receptor. However due to the dominant effect of transport phenomena on the biological activity of the milbemycins, the testing of derivatives in vivo will always be important, with no rational way of avoiding the hard work involved in the synthesis and testing of a large number of derivatives, in order to find one with the required level and breadth of biological performance. 4

4

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RECEIVED July 5, 1992


Regio- and Stereoselective Synthesis of 13-Substituted-milbemycins

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