Development of Broad-Spectrum Insecticide ... - ACS Publications

Chapter 15

Downloaded by UNIV OF GUELPH LIBRARY on June 19, 2012 | http://pubs.acs.org Publication Date: May 14, 1998 | doi: 10.1021/bk-1998-0686.ch015

Development of Broad-Spectrum Insecticide Activity from a Miticide Ronald E. Hackler, C. J. Hatton,M.B.Hertlein, Peter L. Johnson, J. M. Owen, J. M. Renga, Joel J. Sheets, T. C. Sparks, and R. G. Suhr Discovery Research Center, DowElanco, 9330 Zionsville Road, Indianapolis, IN 46268-1053 A series of quinazolines which are insecticidal by virtue of their inhibition of mitochondrial electron transport were examined for their spectrum of activity against mites and several insects. It was shown that several factors could account for the differences between compounds which were primarily acaricidal and those which had broad-spectrum insecticidal activity. Although mitochondria could not be tested from each insect, there is some evidence of differences in the target sites between insects. A major factor in insect selectivity is shown to be differences in metabolism. Examination of penetration of the insect cuticle did not reveal useful data. Additional pharmocokinetic factors such as internal tissue partitioning, protein binding, or spiracle entry and insect behaviour were not considered in this analysis. For synthesis chemists who seek to understand structure-activity relationships for in vivo data, selectivity among biological species can be a great mystery. Depending upon the biological targets, a wide variety of issues may affect the biological results. If the mode of action is understood and in vitro data are available, it is not uncommon for the in vivo data to diverge widely from the intrinsic data. This paper addresses one such scenario for one series of insecticides, and it is possible that it may have application to other series. This publication is not intended to be a comprehensive review of the subject, but rather it is the results of our examination of selected issues, notably intrinsic activity, metabolism, and penetration as they affected the in vivo results against mites and tobacco budworm. We know that insect behavior also has a profound effect on selectivity. Whether a compound is active on sucking insects or chewing insects may depend upon distribution of the compound upon and within the plant. Where the insect feeds may make the difference in whether or not it is exposed to a toxic dose or even to no compound at all. We do not attempt to address these behavioral issues in this paper, but have restricted our discussion to limited observations which we feel shed some light on some common reasons for selectivity. ©1998 American Chemical Society

In Synthesis and Chemistry of Agrochemicals V; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

147

148


Fenazaquin is a commercial miticide marketed by DowElanco. References to the synthesis of fenazaquin may be found elsewhere (1). At about the same time as fenazaquin was being developed, several other miticides were reported that were subsequently determined to operate by the same mode of action as fenazaquin (2). This mode of action is the same as rotenone - inhibition at what is usually referred to as site I of the electron transport chain (NADH: ubiquinone oxidoreductase). All of the compounds shown (Figure 1) also exhibit a similar potency for inhibition of this metabolic process. Fenazaquin exhibits target site binding characteristics similar to rotenone as well.

tebufenpyrad Figure 1. Inhibitors at Site I of the Electron Transport Chain. Rotenone is as well known for its level of toxicity to fish as for its toxicity to insects. In our in-house tests it had an LC50 of about 10 ppb against Japanese carp, and exhibited even higher toxicity to trout. Fenazaquin is more selective against carp than rotenone, but fish toxicity was a serious issue with our series of quinazolines. In fact we examined several series of mitochondrial electron transport inhibitors and found many derivatives which demonstrated very low LC50's against trout and carp. The compounds which were most toxic to fish were the compounds which


149 demonstrated a broader spectrum against insects. This paper will focus on two such compounds, XR-100 and Compound 11.


Synthesis Because the previous paper (1) discusses the fenazaquin synthesis, we shall discuss only some of the unique aspects of the synthesis of XR-100 and 11. As shown in Figure 2, the XR-100 side chain is made by reaction of 4-bromobenzotrifluoride with the sodium salt of 4-bromophenol using cuprous chloride in pyridine. This Ullmann reaction would not work with the 4-chlorobenzotrifluoride, but a displacement in DMSO was successful. The diphenyl ether product is then lithiated and the aryl lithium used to open ethylene oxide to give the phenethyl alcohol, which is coupled with 4-chloroquinazoline to give XR-100. ONa

Br

Figure 2. Synthesis of XR-100. The side chain which is used for Compound 11 was originally made from the hydroxynicotinic acid by a series of classical steps shown in Figure 3. This is typical of the manner in which we made many phenethyl alcohols and phenethyl amines for


150 this series. This chain is elongated through the benzyl alcohol, benzyl chloride, and benzyl cyanide. This was a fairly messy route which gave reasonable yields in each step, but the overall yield was only moderate. This route was used for many compounds starting from various commercial materials. The nitriles may also be hydrolyzed to the acids and these reduced to the phenethyl alcohols. CF CH20H 3


NaH, DMF 1. DD3AL-H 2. NaBH 4

CFgCRjO

Ν

92%

3. HC1 4. NaCN

CF CH20 3

Ν

81% (4 steps)

Figure 3. Original Route to Compound 11 Side Chain. An improved route to this side chain is shown in Figure 4. The key to this route is a modified Heck reaction in which the additional carbons come from the allyl alcohol, and the pyridine unit is derived from 2,5-dibromopyridine. By formation of the oxime of the resulting butanone and rearrangement, the acetamide is made as the major product with a by-product of the propionamide. Hydrolysis of the acetamide gives the desired side chain which can be easily separated from the acid which is derived from the propionamide. We found this separation to be conveniently achieved by formation of the carbon dioxide adduct, something which is observed with many phenethyl amines. Exposure to the air for only a few minutes results in a solid being formed on the surface of the liquid amines. Compound 11 is formed in 90% yield by reaction of this amine with the 4-chloroquinazoline in 1,2dichloroethane using triethylamine to scavenge the acid.

OH


151

r

l.NH OH/MeOH 2

2. H S0 2

CFJCHJO

4

from anti-oxime (favored 6:1)

0

Ν

KOH CF CH20 Downloaded by UNIV OF GUELPH LIBRARY on June 19, 2012 | http://pubs.acs.org Publication Date: May 14, 1998 | doi: 10.1021/bk-1998-0686.ch015

3

Œ^np

Ν

from syn-oxime Ο OH

Ν CF CKp

38% overall yield as C 0 adduct

3

Ν

7%

2

Et N 3

C1CH CH C1 2

2

Compound 11 90%

Figure 4. Synthesis of Compound 11. Biological Activity The commercial compounds shown previously are all primarily miticides and consist of a basic heterocycle, a bridge, and a phenyl group substituted with either a t-butyl or a t-butoxycarbonyl. Within our series of quinazolines, a t-butyl group on the phenyl ring was one of the best substituents for mite toxicity. In our tests rotenone has very poor mite toxicity with an LC50 more than 800 times that of fenazaquin. Table I shows LC50's for a group of quinazolines against two-spotted spider mite. These results were obtained by spraying squash plants which were previously infested with a mixed age population of two-spotted spider mites until the spray solution ran off the plants. A post-infest spray will give results typically three times greater, and higher L C 5 0 values will be obtained on cotton or bean plants. The table illustrates that a variety of substituents impart miticidal activity to this series of molecules. Parasubstitution on the phenyl ring is preferred, with ortho or meta substitution as in compounds 7-9 greatly diminishing activity. There is an optimal length for alkyl or alkoxyl groups on the phenylringof about 5 to 6 atoms, illustrated with compound 5, and consistent with other site I inhibitors of mitochondrial electron transport such as the piericidins (3). A phenyl or


152 Table I. Two-Spotted Spider Mite LC50's for a series of quinazolines


Ν Compound fenazaquin XR-100 2 3 4 5 6 7 8 9 10

TSSM (nom) 1.0 400 93 43 1.9 55

C A = Cotton Aphid LC50 (ppm) MOSQ = Mosquito Larvae LC50 in a well test (ppm) C L = Cabbage Looper topical LC50 ^g/g) NEM WELL = Free living nematode LC50 in a well test (ppm) N E M = Root Knot Nematode LC50 in a sand/soil test (ppm) SCRW = Southern Corn Rootworm LC50 in a soil test (ppm) TBW CON = Tobacco Budworm LC50 in a petri dish contact test (ppm) TBW L F = Tobacco Budworm LC50 in a leaf spray test (ppm)

virescens). This species is easier to study than many other insects, and it also represents a major commercial market. Fenazaquin is nearly inactive against this organism, XR-100 is much more active, and Compound 11 is one of the most active quinazolines against this species. Let us focus first on the intrinsic activity of these molecules as it may relate to this in vivo data. Can there be a difference between the binding sites for different species which explains the selectivity which we observe? It is not practical to isolate mitochondria from many insects, and in fact our initial measurements of inhibition of electron transport were done with bovine heart tissue. Later assays were developed using tissue from housefly thorax and a cabbage looper cell line. It was hoped that the housefly and cabbage looper assays would be more representative of insects in general, and the cabbage looper assay might better represent binding in lepidoptera. It is impossible to review all of the data from these tests, so some general observations will be made. Examining data from hundreds of compounds led to the observation that the bovine heart tissue data correlated both with the housefly and the cabbage looper data, but there was no correlation between the housefly and the cabbage looper data. Ail of the data demonstrated some correlation with toxicity against tobacco budworm as shown in Table Π. That is to say that the better the in vivo activity against tobacco budworm, the better the level of intrinsic activity in these assays. There was an especially good correlation between in vivo tobacco budworm activity and intrinsic data from the cabbage looper cell line. Data for our three selected compounds are shown in Table ΙΠ. A broader look including many compounds suggests that the picture is much more complicated than this, and there is not always a simple correlation between intrinsic activity and whole insect activity. High intrinsic activity, usually with an


154 Table III. Intrinsic assays for inhibition of mitochondrial electron transport as LC50's.


Compound fenazaquin XR-100 11

Bovine 6.9 nM 2.6 1.2

Housefly 1.0 nM 0.6 0.5

Cabbage Looper 1.1 nM 0.4 0.012

LC50 less than 10 nM, is necessary for good activity against any insect, but some compounds with high intrinsic activity were inactive in whole insect assays or showed a very narrow spectrum. Many more compounds were strictly acaricidal than were broadly insecticidal. The most active compounds against tobacco budworm or other lepidoptera also had high intrinsic activity as measured in tissue from the cabbage looper cell line. While the cabbage looper data does show some moderate correlation with activity against lepidoptera, it still explains less than 50% of the biological activity. This suggests that intrinsic activity has some ability to predict whole insect activity for specific insects, and thus, by implication differences between active sites in these species. In other words, the activity of Compound 11 is partly explained by a heightened specificity for the tobacco budworm active site, but we must look at additional factors for a more complete explanation. Metabolism We believed at a very early stage that metabolism was playing a major role in selectivity, and a series of studies were initiated which confirmed this suspicion. Studies were conducted with tobacco budworm (TBW) midgut microsomes, rat liver microsomes, and trout liver microsomes using radiolabeled fenazaquin, XR-100, and Compound 11. Dramatically different rates of metabolism for these compounds were observed in all three species as shown in Table IV. Table IV. Rates of metabolism in nmoles/min/mg protein Compound fenazaquin XR-100 11

TBW 0.91 0.03 0.07

Rat 0.97 0.14

Trout 0.11

Development of Broad-Spectrum Insecticide ... - ACS Publications

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