Dihydropiperazine neonicotinoid compounds. Synthesis and insecticidal activity.

Syntheses of various isomeric dihydropiperazines can be approached successfully by taking advantage of the regioselective monothionation of their respective diones. Preparation of the precursor unsymmetrical N-substituted piperazinediones from readily available diamines is key to this selectivity. The dihydropiperazine ring system, as exemplified in 1-[(6-chloropyridin-3-yl)methyl]-4-methyl-3-oxopiperazin-2-ylidenecyanamide (4) and 1-[(2-chloro-1,3-thiazol-5-yl)methyl]-4-methyl-3-oxopiperazin-2-ylidenecyanamide (25), has been shown to be a suitable bioisosteric replacement for the imidazolidine ring system contained in neonicotinoid compounds. However, placement of the cyanoimino electron-withdrawing group further removed from the pyridine ring, as in 4-[(6-chloropyridin-3-yl)methyl]-3-oxopiperazin-2-ylidenecyanamide (3a), or relocation of the carbonyl group, as in 1-[(6-chloropyridin-3-yl)methyl]-4-methyl-5-oxopiperazin-2-ylidenecyanamide (5), results in significantly decreased bioisosterism. The dihydropiperazine ring system of 4 and 25 also lends a degree of rigidity to the molecule that is not offered by the inactive acyclic counterpart 2-[(6-chloropyridin-3-yl)-methyl-(methyl)amino]-2-(cyanoimino)-N,N-dimethylacetamide (6). A pharmacophore model is proposed that qualitatively explains the results on the basis of good overlap of the key pharmacophore elements of 4 and imidacloprid (1); the less active regioisomers of 4 (3a, 5, and 6) feature a smaller degree of overlap.

N-acetylcyanamide, the major urinary metabolite of cyanamide in rat, rabbit, dog, and man.

The structure of the major urinary metabolite of cyanamide, the active component of the alcohol deterrent agents Temposil , Dipsan , and Abstem , in rats, rabbits, and dogs has been established as N- acetylcyanamide by its identity with chemically synthesized N- acetylcyanamide , and by conversion of the metabolite and the synthetic product to identical derivatives, viz. to N-benzyl-N- acetylcyanamide and to N-(p-nitrobenzyl)-N- acetylcyanamide . The latter derivatives were analyzed by pulsed positive/negative ion chemical ionization mass spectroscopy. Urine from patients receiving cyanamide as a treatment mode was shown to contain N- acetylcyanamide by chemical ionization mass spectrometric analysis of the isolated p-nitrobenzyl derivative, thereby establishing that N- acetylcyanamide is also a metabolite in man. The major portion (87%) of the first 27-hr urinary radioactivity excreted by the dog after receiving a low dose of [14C]cyanamide (0.04 mmol/kg, po) was N- acetylcyanamide , as determined by inverse isotope dilution and measurement of the specific radioactivity of its N-p-nitrobenzyl derivative. This indicates that at low doses acetylation is also a major route of biotransformation of cyanamide in the dog. Hepatic N-acetyltransferase, isolated from the rabbit and dog, catalyzed the transfer of the acetyl group from acetyl-S-CoA to [14C]cyanamide producing N-acetyl[14C]cyanamide. The enzyme isolated from the liver of a rapid acetylator phenotype rabbit was twice as effective as the dog enzyme in catalyzing this transfer. Thus, the enzyme responsible for this biotransformation of cyanamide is an acetyl-S-CoA-dependent N-acetyltransferase.

Metabolic activation of cyanamide to an inhibitor of aldehyde dehydrogenase in vitro.

The inhibition of aldehyde dehydrogenase (AIDH) by cyanamide is dependent on the conversion of the latter to an active metabolite. This accounts for the in vivo activity of cyanamide in raising ethanol-derived blood acetaldehyde levels to the mM range in the rat (ED50 for cyanamide = 0.11 mmole/kg) and its lack of inhibitory activity in vitro with purified AIDH enzymes. Liver mitochondria were shown to catalyze this activation. The Low Km mitochondrial AIDH isozyme was strongly inhibited by cyanamide when measured in intact rat liver mitochondria (I50 = 2.0 microM). Cyanamide also inhibited yeast AIDH when incubated in the presence, but not in the absence, of rat liver mitochondria (I50 = 7.8 microM). Using yeast AIDH activity as a measure of cyanamide activation, the subcellular distribution of the cyanamide-activating system was assessed. Microsomes plus an NADPH generating system were equally active as mitochondria in activating cyanamide. In the absence of NADPH, microsomal activity was about half that of mitochondria. Little or no activity was found in the cytosolic fraction. A series of cyanamide analogs and derivatives were screened for their ability to inhibit the low Km AIDH isozyme measured in intact mitochondria. Only monoalkylcyanamides exemplified by n-butylcyanamide showed significant inhibition. Other cyanamide analogs and derivatives including N-acetylcyanamide, the major urinary metabolite of cyanamide, were inactive in this system.

Studies on the cyanamide-ethanol interaction. Dimethylcyanamide as an inhibitor of aldehyde dehydrogenase in vivo.

Administration of dimethylcyanamide (DMC) to rats caused a marked elevation in ethanol-derived blood acetaldehyde (AcH) and depressed the specific activity of the low Km mitochondrial aldehyde dehydrogenase (AIDH) by 90% at 12-24 hr, coincident with depletion of hepatic glutathione levels. Comparison of the relative efficacy of DMC and cyanamide in elevating blood AcH measured at 2 hr and 1 hr post-drug treatment, respectively, indicated that DMC was at least one-fifth as active as cyanamide. However, since the comparison was not made at optimal times for DMC (12-24 hr), it is likely that its activity in vivo approaches that of cyanamide itself. DMC was essentially inactive in vitro as an inhibitor of the low Km AIDH isozyme in intact rat liver mitochondria. Although methylcyanamide, the product of N-demethylation of DMC, was too unstable to be prepared for this evaluation, the higher monoalkyl cyanamide, n-propylcyanamide, was synthesized chemically and was shown to be a good inhibitor of the mitochondrial enzyme in vitro. These results suggest that DMC must be N-demethylated before being converted to a reactive species that inhibits AIDH activity.

Mode of mutagenic action of methylnitrosocyanamide, a potent carcinogen.

A potent carcinogen, methylnitrosocyanamide was used to induce revertants in a strain of Escherichia coli carrying an amber mutation in a gene for tryptophan (trp) biosynthesis and an ochre mutation in a gene for alkaline phosphatase biosynthesis. Trp+ revertants were purified and classified into seven categories based on their ability to support the growth of particular nonsense mutants of phage lambda and on their content of alkaline phosphatase. About 90% of the Trp+ revertants induced by methylnitrosocyanamide were due to mutations in suppressor genes, and 85% of the suppressor mutations occurred in gene supE. Intragenic reversion cannot occur by a GC leads to AT base substitution mutation, whereas this is the obligate mode of mutation in gene supE. We conclude that methylnitrosocyanamide preferentially induces GC leads to AT transition mutations but that other base substitution mutations are also induced at about 10% of this frequency. N-Methyl-N-nitrosourea and, particularly, N-methyl-N'-nitro-N-nitrosoguanidine also preferentially induce GC leads to AT transition mutations.