Synthesis of haptens and potential radioligands and development of antibodies to insect growth regulators diflubenzuron and BAY SIR 8514

Abstract

A variety of synthetic approaches were undertaken, leading to potential haptens and radioligands for the benzoylphenylurea insect growth regulators diflubenzuron and BAY SIR 8514. One successful approach involved derivatization of the aniline nitrogen by ethyl 4-bromobutyrate followed by reaction with an appropriate isocyanate and cleavage of the ethyl ester to yield a free carboxypropyl "handle".Useful haptens were also synthesized by using a 3'-phenolic metabolite of diflubenzuron as well as acetate and amine functionalities in the 4' position while the N-sulfenyl bond proved too unstable for use as an antigen. With the exception of the sulfenylated derivatives, the haptens lacked significant biological activity on three insect species. Following protein coupling by the active ester or water-soluble diimide method, antibodies were raised to two diflubenzuron haptens in each of seven rabbits immunized as demonstrated by radioimmunoassay using ["C]diflubenzuron, Ouchterlony gel diffusion, and immunoelectrophoresis.

Development of enzyme-linked immunosorbent assays for residue analysis of diflubenzuron and BAY SIR 8514

“…All sera gave strong binding to the IV-(carboxypropyl)diflubenzuron coating antigen except sera F and G, which were obtained from rabbits immunized with diflubenzuron phenoxyacetate-KLH. The binding of these antibodies to diflubenzuron phenylacetate-OA coated cuvettes was also significant, and their titers were consistent with the results obtained in radioimmunoassay as described in the companion paper (Wie et al, 1982). Hence, sera from rabbits C and D exhibited strong affinity to both [uC]diflubenzuron and diflubenzuron phenylacetate-OA, and they were used in the following experiments.…”

“…Antisera. The anti-diflubenzuron antibodies were obtained from New Zealand white rabbits as described in the preceding paper (Wie et al, 1982). Thus, sera A and B were obtained from rabbits immunized with JV-(carboxypropyl)diflubenzuron-KLH prepared by the active ester method (Figure 1).…”

“…In the preceding paper (Wie et al, 1982), the synthesis of haptens for the insect growth regulators (IGRs) diflubenzuron [Dimilin,amino]carbonyl]-2,6-difluorobenzamide], BAY SIR 8514 [N-[[[4-(trifluoromethoxy) phenyl] amino] carbonyl]-2-chlorobenzamide], and their protein conjugates were described, and high titers of specific antibodies to diflubenzuron were produced in rabbits. In the present study enzyme-linked immunosorbent assays (ELISA's) for both diflubenzuron and BAY SIR 8514 were developed and shown to be highly specific and of adequate sensitivity for residue work.…”

A Highly Sensitive and Rapid ELISA for the Arylurea Herbicides Diuron, Monuron, and Linuron

“…amino] hexanoate was obtained following the procedure of Wie et al (1982) whereby 5.10 g (40.0 mmol) of 4-chloroaniline, 6.00 g (44.0 mmmol) of sodium acetate trihydrate, 9.82 g (44.0 mmol) of ethyl 6-bromohexanoate, and 10 mL of ethanol were heated under reflux for 11 h. The hot solution was filtered, and the filtrate was cooled slowly to obtain 6.52 g (60%) of the crude product as white crystals, mp 67.0-69.0 °C. Recrystallization from ethanol-water (2:1, v/v) produced white platelets: mp 67.0-68.0 °C; TLC Rf 0.67 (solvent A); IR (KBr) 3391 (vs, NH), 1721 (vs, C=0, ester), 1185 (s, CO) cm*1; NMR (DMSO-dg) 7.06 (d, =7 = 8.8 Hz, 2 H, Ar-Hg,g), 6.53 (d, =7 = 8.8 Hz, 2 H, Ar-H26), 5.75 (t, =7 = 5.3 Hz, 1 , NH), 4.04 (q, J = 7.1 Hz, 2 H, CH20), 2.95 (dt, =7 = 6, 6.9 Hz, 2 H, CH2N), 2.28 (t, =7 = 7.3 Hz, 2 H, CH2C02), 1.5 (m, 4 H, 2 CH2), 1.4 (m, 2 H, CH2), 1.17 (t, =7 = 7.1 Hz, 3 H, CHg) (the 5.75 ppm peak disappeared, and the 2.95 ppm dt became a single triplet with added D20); 13C NMR (DMSOdg) 173.0 (ester C=0,Ci), 148.1 (Ar-Ci), 128.7 (Ar-Cgg), 118.6 (Ar-C4), 113.3 (Ar-C2,6), 59.8 (CH20), 42.9 (C6), 33.7 (C2), 28.4 (Cg), 26.3 (C4), 24.5 (C3), 14.3 (CHg).…”

Development of a Compound-Specific ELISA for Flufenoxuron and an Improved Class-Specific Assay for Benzoylphenylurea Insect Growth Regulators

“…1 [2,6-difluoro-3-(2-ethoxycarbonylethyl)benzoyl]urea (6). Compound 4 was reacted with freshly distilled oxalyl chloride to make 2,6-difluoro-[2-(ethyoxycarbonyl)ethyl]benzoyl isocyanate according to the method of Wie et al (1982). Next, a solution of the crude isocyanate in dry benzene was added directly to a stirred solution of compound 5 in dry benzene.…”