Screening Mixtures: An Experiment in Pesticide Lead Generation

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Screening Mixtures: An Experiment in Pesticide Lead Generation 1,2

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Karl J. Fisher , Ray A. Felix , and Robert M. Oliver 1

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Zeneca Agrochemicals, 1200 South 47 Street, Richmond, CA 94804 Current address: Cambridge Discovery Chemistry, 1391 South 47 Street, Richmond, CA 94804 Current address: Pfizer Global Research and Development, Pfizer, Inc., Groton, CT 06340 th

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The screening of mixtures was evaluated as a way of improving the rate of new lead generation, one of the greatest challenges facing modern agricultural chemists. We have observed mixture hits on our herbicide screens; when we have deconvoluted these mixture hits by making all of the mixture components we have, in every case, observed activity from single compounds. In some cases, the activity is cumulative with activity found for more than one component of the mixture. In other cases, all of the activity comes from a single component of the mixture.

The discovery of new chemical classes with novel modes of action poses one of the most challenging problems faced by scientists doing agrichemical research " . One typically has to screen thousands of compounds to find a hit of sufficient activity and novelty to warrant follow up chemistry by an optimization 4

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© 2002 American Chemical Society In Synthesis and Chemistry of Agrochemicals VI; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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10 team. In order to increase the number of compounds tested in our primary screens without increasing staff, we decided to make compounds as mixtures by reacting mixtures of alkyl halides with a variety of single nucleophiles. We chose to make mixtures of ten compounds rather than larger mixtures to simplify the identification of any active compounds: Ten halides (RX) + one nucleophile N u H forms ten products (lONuR) + H X , where X = halogen. Any active mixture would be deconvoluted by simply preparing the ten expected reaction products and screening them individually. The mixtures of ten alkyl halide regents were made up as equimolar reagents in acetonitrile (0.10M per component, LOOM total) and heated with one equivalent of nucleophile, 1-1.5 equivalents of K I and 1.1 equivalents of base (usually sodium hydride or potassium t-butoxide). Prior to producing mixtures, candidate nucleophiles were checked for suitability by heating with 4-(4-methylphenoxy)-l-chlorobutane, an unactivated alkyl halide. Nucleophiles which did not react cleanly with this halide were not used to make mixtures. A typical mixture of alkyl halides is shown in Figure 1 :

F Figure 1: A typical mixture

In Synthesis and Chemistry of Agrochemicals VI; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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Synthetic Studies and Screening Strategy Our initial studies focused on the alkylation of 4-substituted pyrazoles. We also alkylated other nucleophiles successfully including mercaptans, heterophenols, phenols, select alcohols, thioureas and imides. A s a gérerai guiding principal, we tried to incorporate at least three potential binding groups per molecule. Examples of binding groups are aromatic rings, carboxamides, esters, sulfonamides or heterocycles. We assumed that molecules with only two potential binding groups were too simple and, i f active, were only too likely to have precedents in the literature. In the first year using the mixture strategy 700 mixtures (7000 compounds, theoretically) were prepared by one chemistry team of two people. We agreed on a screening rate of 101b per acre, l i b per component based on our knowledge that, historically, individual compounds were screened at rates up 201b without getting too high a hit rate and modern screening rates are in the range of l-41b per acre. When components from hits were tested, they were tested at both a l i b rate and at a 41b rate. Without testing components at the 41b rate, the activity of cumulative hits (hits where more than one component had activity) could never have been assigned to individual components. From 700 mixtures, thirteen types of herbicide hits were obtained. The first hit obtained was deconvoluted by preparing the components which were screened individually. Six of the components were inactive. CI

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R Figure 2: Reaction mixture 1, our first hit contained compounds I-IV The active compounds I-IV from this mixture had the order of activity R=-CH COOEt > -(CH ) COOEt > CH(CH )COOEt > (CH ) COOEt and all four compounds had 2,4 D like symptomology. This type of auxin activity is 2

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The second active mixture we obtained was prepared by alkylating ethyl 2mercapto-5-methyl-4-imidazolecarboxylate. When we tried to make the ten components only seven formed; upon screening these seven compounds we found that all of the activity came from a single component, compound V :

Figure 3: Compound V Compound V had activity only on broadleaf weeds and, while interesting, was not active enough to encourage further synthesis. This compound had a rapid leaf burn effect similar to a PPGO herbicide ' or PSII inhibitor ' . We also got several hits which upon inspection were closely related to known series of herbicides which inhibit phytoene desaturase or were covered in previous patents. 10 11

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We also found some herbicidal activity from two novel types of mercaptotriazoles. Compounds V I and V I I were prepared by alkylating the appropriate mercaptotriazole with commercially available benzyl chlorides: N - N

N - N

Figure 4: Compounds V I and V I I


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Both of these compounds were present in mixtures which had cumulative activity. The N-methyl triazole compound V I had preemergence activity on some grasses with a rapid leaf burn effect. The N-phenyl triazole compound V I I caused a bleaching effect similar to phytoene desaturase inhibitors. However, it was not active when tested in this assay . 15


The most interesting herbicide we found is this previously unknown triazolylthioacetamide, compound VIII:

Figure 5: Compound VIII This compound was prepared by alkylating the mercaptotriazole with N (2,4-dichlorophenyl) 2-bromoisobutyramide. None of the other components of this mixture had any activity. This compound controls both grasses and broadleaf weeds at 1 pound per acre and controls some of the more susceptible weeds at a quarter pound per acre. As far as we were able to determine this compound has a new mode of action. Since this compound has significant activity and is reasonably easy to prepare, it became a starting point for a lead optimization effort.

Conclusions Without any laboratory automation, we were able to prepare large numbers of compounds and identify some new herbicidally active chemicals reasonably quickly using our mixture strategy. Screening mixtures can be an effective strategy, particularly when screening capacity is limited and false positives are rare.

Acknowledgments We would like to thank Derek Dagarin, Nick Polge, Sean Hanser and Chris Knudsen, our good friends in Weed Science, for testing all of these mixtures and accommodating all of our requests. Without their spirited efforts this work would never have been accomplished.


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Canel, Camilo. D N A microarrays: New tools for the discovery of Agriculturally useful phytochemicals. Rev. Latinoam. Quim. (2000), 28(1), pp21-26.

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Matringe, Michel; Camadro, Jean Michel; Labbe, Pierre; Scalla, Rene. Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides. Biochem. J. (1989), 260(1), pp231-5.


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Scalla, Rene; Matringe, Michel. Inhibitors of protoporphyrinogen oxidase as herbicides: Diphenyl ethers and related photobleaching molecules. Rev. Weed Sci. (1994), 6 pp103-32.


12. Trebst, A . Inhibitors of electron flow tools for the functional and structural localization of carriers and energy conservation sites, MethodsEnzymol., 69, pp675, 1980. 13. Moorland, D . E., Mechanisms of actions of herbicides, Annu, Rev. Plant Physiol., 31, pp597, 1980. 14. Renger, G., Herbicide interaction with photosystem developments, Physiol. Vég., 24, pp509, 1986.

II: recent

15. Dr. Tom Cromartie, personal communication.


Screening Mixtures: An Experiment in Pesticide Lead Generation

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