Discovery of Insecticidal Cyanine Dyes - American Chemical Society

Chapter 7

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Discovery of Insecticidal Cyanine Dyes Robert N. Henrie II, Thomas Cullen, Benjamin Dugan, Larry Zhang, Yanli Deng, Sandra F. Simpson, Bruce Black, Franz Schuler, and Seong Jae Yu Discovery Research Department, Agricultural Products Group, F M C Corporation, P.O. Box 8, Princeton, NJ 08543-0008

Inhibition of chitin synthase (CS, E C 2.4.1.16) is widely recognized as a largely untapped, intrinsically mammaliansafe, arthropodicidal mechanism of action. Certain classes of cyanine dyes were observed as hits in a high-throughput Lepidopteran (tobacco budworm, Heliothis virescens) chitin synthase assay. Optimization produced in vivo-active analogs; however, ultimately these were shown to be off-target, active instead as mitochondrial electron transport inhibitors (METI) at Complex 1. A n overview of the chitin synthase project and chemistry is presented.

Chitin synthase (CS), Enzyme Commission number 2.4.1.16, is the enzyme that converts uridine diphosphoryl-N-acetylglucosamine (UDP-GlcNAc) into chitin, the β-l,4-linked polymer of N-acetylglucosamine. CS is a member of the class of enzymes known as polymerizing glycosyltransferases which are responsible for the synthesis of critical structural biopolymers, including cellulose as well as chitin. It is a processive enzyme, adding successive GlcNAc © 2007 American Chemical Society In Synthesis and Chemistry of Agrochemicals VII; Lyga, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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units onto a primer, cf. Scheme 1. Inhibition of chitin synthase is widely recognized as a largely untapped, intrinsically mammalian-safe, arthropodicidal mechanism of action (7). Since the exoskeleton of insects consists mostly of chitin, disrupting the formation, deposition, and/or cross-linking of chitin should lead to insect death, especially during molting. One of the attractive features of chitin synthase as an insecticidal target site is its absence in mammals, although its presence in crustaceans such as Daphnia could cause potential ecotoxicology issues. There are no known commercial insecticides that are bona fide inhibitors of chitin synthase, nor are there any in development to our knowledge.

Scheme 7. Polymerization ofN-acetylglucosamine

by Chitin Synthase

The well-known insecticidal benzoyl phenyl ureas (BPUs), exemplified by diflubenzuron, are chitin biosynthesis disrupters, but at an uncharacterized site distinct from chitin synthase (2), Figure 1. The related bridge-cyclized oxazoline chemistries from DuPont and Yashima, represented by etoxazole, are also commercial or near-commercial insecticides with the same mechanism of action (J), Figure 1. F

Uniroyal Diflubenzuron

Dupont experimental (3)

Yashima experimental (3)

Yashima Etoxazole

Figure 7. Chitin Biosynthesis Disruptors

In Synthesis and Chemistry of Agrochemicals VII; Lyga, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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The known bona fide CS enzyme inhibitors are natural product-derived substrate analogs, eg. polyoxin D, tunicamycin, and nikkomycin Z, Figure 2. These complex natural products inhibit the enzyme well, but express little in vivo insecticidal activity, presumably due to unfavorable A D M E attributes.

Nikkomycin Z

Figure 2. Bona Fide Chitin Synthase Inhibitors

Lead Identification We have developed a proprietary CS assay using tobacco budworm (TBW, heliothis virescens) tissue, and automated the protocol for high-throughput screening in 96-well microtiter plates (4). During the screening phase of the CS project we defined a hit as a compound showing >30% inhibition of T B W CS at I O J I M . Initial hits were confirmed in a second screen (overall confirmed hit rate = 0.55%), and the 366 confirmed hits were run through both the CS rate series and an in vivo surface-applied diet screen (5). Simultaneously, the chemical structures were confirmed using mass spectrometry and, i f necessary, H N M R . High-throughput screening required three months to process 67,000 compounds. A total of 210 compounds passed both the CS enzyme rate series test and structure confirmation. These actives were clustered into 30 chemical classes using HCA/Ward clustering on Daylight fingerprints. Thirty was the number of clusters observed to give good separation of scaffolds into chemically reasonable classes, yet still be manageable for lead optimization. These 30 chemical classes were prioritized, with 18 generics identified for synthesis; 3 out of the 18 were cyanine dye-related structures, Table I. A fourth symmetrical generic was also added. Prioritization criteria included preliminary literature and patent searches, level of biological activity, scaffold diversity, A D M E properties, and perceived amenability to synthesis. l


86 Table I. Cyanine Chitin Synthase Projects Proj Code

Generic Structure

Lead Structure

19

X~


21B

R6

X

/=K

R7

R3

4.0-5.4

3.6 - 5.6 3.6-4.1

4.0-5.6

3.8-6.6 3.6-5.7

4.0-5.7

NM - 5.7 NM-4.5

R1

40A

R

40B

SRTD Range piso pLDso 4.0-5.9 3.6-5.1

R1

" R4

R

CS Range piso 4.0-5.0

R

R4

R3

R6

R

designed

R1

Optimization We first defined the core. Both enzyme (CS) and in vivo (SRTD) data were generated for the analog sets in parallel biological testing. S A R was developed for each chemical class, attempting to correlate in vitro to in vivo activity. Our standard Discovery paradigm was followed. Scaffold probing was done with CI, C H , and O C H groups. Isosteric replacements were investigated simultaneously; eg., benzoxazoles as well as benzothiazoles were prepared. Specific sets of analogs were designed and prepared to answer questions and generate data for Q S A R analysis. Anions explored included halides (CI", I") and alkyl sulfates ('OS03R, with R = C H , C H ) . Classical Hansch 2D Q S A R and Sequential Simplex Optimization (SSO) were utilized for optimization of the trimethine-bridged analogs, project 40A. Free-Wilson analysis of monomethine-bridged analogs showed the very positive effect of a 6-OEt on SRTD weight inhibition (piso). Results of the Hansch analysis are summarized in Figure 3. There appeared to be an optimum in lipophilicity ( 7 1 ) . Electronically, the sum of the Swain and Lupton Resonance prarmeter, R, should be negative, making the substituents electron-donating. 3

3

3

2

5


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Steric factors also appeared to be important around the aromatic rings, although there was cross correlation between the Sterimol parameters L , B l , and B5 and 7 1 . Also, steric and/or lipophilicity factors were important for the R/R' alkyls, with the most active being small (methyl or ethyl) groups.

Bisbenzothiazoles: Bisbenzoxazoles: QSAR Summary: XW: TCsuMOpt: 0.92.0 R S U M < - 0 . 6 (edonating) 4:B1 2.0 5:B1

Discovery of Insecticidal Cyanine Dyes - American Chemical Society

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