The Identification and Optimization of OÃ¶mycete Dihydroorotate

Chapter 28

Downloaded by PENNSYLVANIA STATE UNIV on June 16, 2012 | http://pubs.acs.org Publication Date: July 29, 2001 | doi: 10.1021/bk-2002-0800.ch028

The Identification and Optimization of Oömycete Dihydroorotate Dehydrogenase Inhibitors as Fungicides Marshall H. Parker, Greg L. Durst, Anna C . Hannum, Matthew J. Henry, L o r i K . Lawler, and A m y J. Smith Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, I N 46268-1053

The present study describes the discovery of novel antifungal compounds by using a known antifungal target from one class of fungi and exploiting this novel mode of action for the discovery of fungicides in Oömycete plant pathogens. Utilizing computational clustering for compound selection and a novel dihydroorotate dehydrogenase (DHO-DH) enzyme assay, a moderate in vitro active (I = 1.98 μΜ) was identified. Through both classical structural activity relationship (SAR) investigations and pharmacophore modeling, this new class of aza-chalcones was optimized to provide a series of nM inhibitors of Pythium aphanidermatum DHO-DH. 50

The discovery and validation of novel agrochemically relevant target sites defines the charter of most lead discovery groups across the industry. Accordingly, when a new mode of action (MOA) is discovered and the biochemical target site validated, significant resources supporting multiple approaches are activated to quickly define its potential. One such approach,

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

303

304


undertaken within Dow AgroSciences, stemmed from original investigations of dihydroorotate dehydrogenase (DHO-DH), the target site of LY214352 (7,2). This inhibitor is highly fungicidal to the Ascomycetous fungi Botrytis cinerea, Pyricularia oryzae, Venturia inaequalis and Aspergillus nidulans. In contrast, LY214352 and related 8-chloroquinoline based inhibitors are not fungicidal to Oomyceteous and Basidiomycetous plant pathogens or bacteria, presumably due to their inactivity at the Oômycete D H O - D H target site. The goal of this effort was to find new lead chemistries that inhibited Oômycete D H O - D H and provided whole plant protection.

CI

LY214352 D H O - D H is an enzyme in the pyrimidine biosynthetic pathway that is responsible for the conversion of dihydroorotate to orotate. Many 8-chloro-4phenoxyquinolines, including phenoxyquinoline LY214352, demonstrate inhibition of D H O - D H from Ascomycetous fungi such as P. oryzae and A. nidulans. Moreover, this enzyme inhibition translates to product level in vivo control of B. cinerea. The specificity of LY214352 was accredited to the assumption that the D H O - D H enzyme was not well conserved between classes of fungi. The implication was that a single compound with this M O A was unlikely to be a broad spectrum fungicide. These factors were the basis for a strategic shift in the D H O - D H target site team to re-align on an Oômycete product concept and focus on a group of taxonomically similar pathogens. To achieve this goal, several obstacles had to be overcome. First, there were no reported procedures published for the isolation of an Oômycete D H O D H enzyme and consequently no known inhibitors which were needed to establish a foundation for chemical exploitation of the target site. And finally, there were no high through-put assays in place to directly, indirectly, or virtually screen a representative collection of potential leads to establish that foundation. The following paper details the work associated with identifying and optimizing a new class of D H O - D H inhibitors, the aza-chalcones. This work was critically dependent on the development and implementation of an in vitro high through-

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

305 put screen (HTS) against Oômycete D H O - D H , the details of which have been recently patented (3).


Lead Identification One of the first tasks facing the target site team was defining the collection of chemistries to screen against the newly identified Oômycete D H O - D H target site. Initially, the Oômycete D H O - D H screen was limited to 1,000 compounds a month. A three-month window was defined for the initial screening process, setting a target library size of 3000 compounds. Toward this end, a collection of 15,000 compounds was computationally reduced to 1,500 clusters based on connectivity indices and a non-hierarchical clustering algorithm. Cluster sampling was done in two steps. First, the centroid of each cluster was selected to give 1,500 compounds. Second, a stratified random sampling of larger clusters was conducted. The combined subsets represented 2,970 compounds that were screened against the P. aphanidermatum D H O - D H enzyme. This process identified 83 (2.8%) novel actives. Clusters that represented the 83 actives were resampled based on similarity and dissimilarity to the original hit to generate a biased collection of 173 potential inhibitors. Of the 173 compounds selected through this resampling process, an additional 15 (8.7%) novel core structures were identified as active. The actives from both screens were refined through a comparison of in vitro (P. aphanidermatum) and in vivo (Plasmopara viticola) activity to rank the final selection of compounds with a bias toward greenhouse activity and a desirable absorption, distribution, metabolism, and "excretion" (ADME) profile. Representative molecules identified in this process are shown in Figure 1. Ultimately, the aza-chalcone series, represented by lead compound 1 (Figure 2), was selected for the synthetic exploitation of this "new" target site.

In vitro Lead Optimization With lead 1 identified, a similarity search of the Dow AgroSciences database provided 35 additional compounds for testing. Of these 35, the best P. aphanidermatum D H O - D H enzyme inhibitor was chalcone 2 (Figure 2) with an I50 value of 0.13 μΜ. The remaining inhibitors were used to define the scope of the initial S A R around the aza-chalcones. For analog comparison, enzyme inhibition activity has been converted to a relative activity ratio (Relac) to a standard used in each enzyme assay throughout this series (4)



306

Figure 1. Newly identified Oômycetous DHO-DH enzyme inhibitors These initial SAR studies focused on the pyridyl moiety and the orientation of the enonal linker that connected it to the benzyloxyphenol sub-unit. The orientation of the enone linker in 2 was opposite that of lead 1. Therefore, analogs with opposite tether orientations were synthesized for each of these compounds (Figure 2). Inhibitor 3 demonstrated less DHO-DH inhibition than its reverse tether counterpart 2. Interestingly, this trend did not correlate within the aza-chalcone series where 1 and 4 were equivalent inhibitors.

1 2 Relac = 0.04 a

Relac = 0.9

3 Relac = 0.007

4 Relac = 0.04

Figure 2. Initial pyridyl moiety in vitro SAR The necessity of the pyridine ring and its optimal orientation were critical to establish the scope of the chemistry early in this SAR. These questions were addressed with a series of five compounds (Figure 3). This series began with the lead compound 1 and systematically removed both the pyridyl methyl group (5) and nitrogen atom (6) independently and then simultaneously (7). Additional


307


analogs 8 and 9 probed the orientation of the pyridine nitrogen atom. At this point in the SAR, the most active inhibitor was still the dimethoxy-chalcone 2. However, a comparison of 5, 6, and 7 indicated the pyridyl nitrogen was contributing to favorable binding interactions. Additionally, analog 5 demonstrated 90% disease control against P. viticola at 400 ppm in a 1 day protectant test. Comparatively, the chalcone analogs 2, 6, and 7 were inactive in the same screen.

OBn 6 Relac = 0.01

OBn 7 Relac = 0.003

OBn 9 Relac

The Identification and Optimization of OÃ¶mycete Dihydroorotate

Recommend Documents