Studies on plant growth-regulating substances. XL. 2-Chloro-3-phenylpropionitriles

The complete series of chloro-, dichloro-, methyl-, dimethyl- and chloro-methyl-ring substituted 2-chloro-3-phenylpropionitriles and several related compounds have been prepared. Their auxin activities have been assessed in the wheat coleoptile, pea segment and pea curvature tests, and the results, which are discussed in relation to molecular structure, support the suggestion by other workers that the observed activity is due to rapid uptake of the nitriles by the plant tissues, followed by metabolism to the corresponding phenylacetic acid. The high growth promoting activity of 2-chloro-3-(3-chloro-2-methyl-phenyl)propionitrile (orthonil) is confirmed but 2-chloro-3-(2,3-dichloro-phenyl)propionitrile and 2-chloro-3-(2,3-dichlorophenyl)butyronitrile are shown to be the most active members of the series.     

Prodrug-oriented molecular design of neonicotinoids: preparation of imidacloprid-related 5,5-dimethoxy-1,3-diazacyclohexane derivatives and their insecticidal activity

Prodrug-oriented molecular design was attempted for the potent acyclic neonicotinoid insecticide, clothianidin (1-(2-chloro-5-thiazolylmethyl)-3-methyl-2-nitroguanidine). Molecules bearing a CH2COCH2 bridge linking the 1,3-NH ends of clothianidin or their acetals would possibly be hydrolyzed, regenerating the mother compounds. This strategy was used to prepare seven acetals of clothianidin-based molecules that combined 2-chloro-5-thiazolylmethyl, 6-chloro-3-pyridylmethyl or 3-tetrahydrofurfuryl with a nitroimine, cyanoimine or nitromethylene group. The key intermediate, 1,3-diamino-2,2-dimethoxypropane, was prepared from the dihydroxyacetone dimer in four steps. A selected acetal showed a characteristic nerve-impulse pattern for neonicotinoids on an excised American cockroach ganglion, although the neuroblocking activity was fairly low. Some acetals were highly insecticidal against the pea aphid at 0.8-20 ppm 7 days after a spray treatment, this being in a contrast to their far weaker activity by injection into American cockroaches. The biological results suggest that the intrinsic insecticidal activities of the acetals are weak, but would exhibit enhanced activity if hydrolyzed in an external environment.     

Physiological studies on pea tendrils. XIII. Respiration is necessary for contact coiling

When 9 day old light grown pea ( Pisum sativum L. cv. Alaska 2B) plants are irrigated for 4–6 days with 50 μ M 4-chloro-5-(dimethylamino)-2-(α, α, α-trifluoro- m -totyl)-3(2H)-pyridazinone, designated San-6706, the new leaves and tendrils grow morphologically normal, but with neither chlorophyll nor the ability to carry out photosynthesis. Excised tendrils from these plants coil in response to rubbing as well as those from water irrigated controls. Tendrils from San-6706 irrigated plants, which have been dark adapted for 2 or 3 days, proceed to coil when illuminated, just as do those from water irrigated plants. Rubbing of dark adapted tendrils results in an increased respiration rate over the first hour or two, when most of the coiling response occurs. Inhibitor studies indicate that blockage of oxidative phosphorylation, but not of the alternative, cyanide-insensitive, path of respiration, results in a failure of tendrils to coil in response to mechanical perturbation. It is concluded that the normal path of respiration, perhaps via ATP production, may be necessary for thigmosensory perception leading to contact coiling in pea tendrils.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES: CHLORO-SUBSTITUTED PHENYLACETIC ACIDS

The plant growth-regulating activity of all the mono- and di- chloro-substituted phenylacetic acids, together with the 2:3:6-trichloro- derivative, has been determined in the wheat cylinder, pea segment and pea curvature tests. Each of these compounds showed activity, the 2:3-dichloro- and the 2:3:6-trichloro- derivatives being extremely active. The 3:5-dichloro- acid was inactive in the wheat test but showed moderate activity in the pea tests.
All these findings are discussed in relation to the plant growth-regulating activity of corresponding benzoic and phenoxyacetic acids.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES. XIII CHLORO- AND METHYL-SUBSTITUTED PHENOXYACETIC AND BENZOIC ACIDS

The physiological activity of complete series of mono- and di-substituted chloro-and methyl-phenoxyacetic and benzoic acids have been investigated using the wheat cylinder, pea segment and pea curvature tests. In the phenoxyacetic acids, high activity was associated with substitution in the 3-, 4-, 2:4-, 2:5- and 3:4-positions and in general, chlorine had a greater effect than methyl in conferring activity.
With the benzoic acids, 2:3-, 2:5- and 2:6-, disubstitution gave active compounds, the chloro-derivatives again being the more active. The 2:5-compound was the most active in the dichloro- series, all members of which were less active than the 2:3:6-trichloro- and 2:3:5:6-tetrachloro-acids. Benzoic acids substituted in the 4- position were either inactive or exhibited only weak growth-promoting activity.
The results are discussed in relation to recent theories which attempt to relate growth-promoting activity with the position of substituents in the aromatic ring.     

Studies on plant growth-regulating substances

The metabolism of certain 2,6-disubstituted phenols that possess high auxin activity in the pea segment, pea curvature and tomato-leaf epinasty tests, but are much less active in the wheat cylinder test, has been investigated in wheat, pea and tomato tissue. Metabolites were identified by thin-layer chromatography and a semi-quantitative assay method was developed. The low activity of 2,6-dihalogenophenols and inactivity of 2-halogeno-6-nitro-phenols and 3-halogeno-2-hydroxybenzonitriles in the wheat cylinder test was caused by rapid metabolic conversion of the compounds in this tissue to inactive compounds by a process involving hydroxylation of the aromatic ring in the para- position. No such inactivation occurred in pea and tomato tissues. Evidence for a novel detoxification of nitrophenols within both pea and wheat tissue was obtained; 2-bromo–6-nitrophenol was converted via 2-bromo-6-aminophenol to N-acetyl-2-bromo-6-aminophenol. Certain 3-halogeno-2-hydroxybenzaldehydes and corresponding aceto-phenones, although fulfilling the necessary structural and electronic criteria for auxin activity, are inactive. Metabolic studies indicate that this is because they are metabolized in wheat, pea and tomato tissues to compounds not possessing the structural requirements for auxin activity.     

Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases.

Four substrate analogs, 4-(2-naphthyloxy)-2-butyn-1-amine (1), 1,4-diamino-2-chloro-2-butene (2), 1,6-diamino-2,4-hexadiyne (3), and 2-chloro-5-phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper-containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO). Reactions of 1,4-diamino-2-butyne with selected amine oxidases were also examined. Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism-based inactivation. Striking differences in selectivity and rates of inactivation were observed. For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4. In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site. Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations. Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side-chain residues, consistent with the widely divergent activities. Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.     

Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2.

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-1-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50 degrees C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.     

Characterization of [3H]acifluorfen binding to purified pea etioplasts, and evidence that protoporphyrinogen oxidase specifically binds acifluorfen.

It is now generally accepted that protoporphyrinogen oxidase is the target-enzyme for diphenyl-ether-type herbicides. Recent studies [Camadro, J-M., Matringe M., Scalla, R. & Labbe, P. (1991) Biochem. J. 277, 17-21] have revealed that in maize, diphenyl ethers competitively inhibit protoporphyrinogen oxidase with respect to its substrate, protoporphyrinogen IX. In this study, we show that, in purified pea etioplast, [3H]acifluorfen specifically binds to a single class of high-affinity binding sites with an apparent dissociation constant of 6.2 +/- 1.3 nM and a maximum density of 29 +/- 5 nmol/g protein. [3H]Acifluorfen binding reaches equilibrium in about 1 min at 30 degrees C. Half dissociation occurs in less than 30 s, indicating that the binding is fully reversible. The specificity of [3H]acifluorfen binding to protoporphyrinogen oxidase is examined. [3H]Acifluorfen binding is inhibited by all the peroxidizing molecules tested. The phthalimide derivative, N-(4-chloro-2-fluoro-5-isopropoxy)phenyl-3,4,5,6-tetra hydrophthalimide, exerts a mixed-competitive inhibition on this binding. The effects of all these molecules on the binding of [3H]acifluorfen are tightly linked to their capacity to inhibit pea etioplast protoporphyrinogen oxidase activity. Furthermore, protoporphyrinogen IX, the substrate of the reaction catalyzed by protoporphyrinogen oxidase, was able to competitively inhibit the binding of [3H]acifluorfen. In contrast, protoporphyrin IX, the product of the reaction, did not inhibit this binding. All these results provide clear evidence that in pea etioplasts, [3H]acifluorfen exclusively binds to protoporphyrinogen oxidase, that the protoporphyrinogen oxidase inhibitors tested so far bind to the same region of the enzyme and that this region overlaps the catalytic site of the enzyme.     

Utilization of 3-chloro-2-methylbenzoic acid by Pseudomonas cepacia MB2 through the meta fission pathway.

Pseudomonas cepacia MB2 grew on 3-chloro-2-methylbenzoate as a sole carbon source by metabolism through the meta fission pathway with the subsequent liberation of chloride. meta pyrocatechase activity in cell extracts was induced strongly by 3-chloro-2-methylbenzoate, but not by nongrowth analogs 4- or 5-chloro-2-methylbenzoate. Although rapid turnover of metabolites precluded direct identification, a mutant strain MB2-G5 lacking meta pyrocatechase activity produced 4-chloro-3-methylcatechol when incubated with 3-chloro-2-methylbenzoate. The catecholic product, confirmed by nuclear magnetic resonance and mass spectral analyses, produced a transient meta fission product (lambda max = 391 nm) from cell extracts of the wild-type MB2 strain. Further confirmation of meta pyrocatechase activity was noted by conversion of 4-chlorocatechol to 2-hydroxy-5-chloromuconic semialdehyde, which was not further metabolized. In contrast to 3-chlorocatechol, which was not metabolized and is known to generate suicidal products, 4-chlorocatechols do not generate acyl halides. Thus, further metabolism of the ring fission products is governed in strain MB2 by their suitability as substrates for the hydrolase.     

Utilization of 3-chloro-2-methylbenzoic acid by Pseudomonas cepacia MB2 through the meta fission pathway.

Pseudomonas cepacia MB2 grew on 3-chloro-2-methylbenzoate as a sole carbon source by metabolism through the meta fission pathway with the subsequent liberation of chloride. meta pyrocatechase activity in cell extracts was induced strongly by 3-chloro-2-methylbenzoate, but not by nongrowth analogs 4- or 5-chloro-2-methylbenzoate. Although rapid turnover of metabolites precluded direct identification, a mutant strain MB2-G5 lacking meta pyrocatechase activity produced 4-chloro-3-methylcatechol when incubated with 3-chloro-2-methylbenzoate. The catecholic product, confirmed by nuclear magnetic resonance and mass spectral analyses, produced a transient meta fission product (lambda max = 391 nm) from cell extracts of the wild-type MB2 strain. Further confirmation of meta pyrocatechase activity was noted by conversion of 4-chlorocatechol to 2-hydroxy-5-chloromuconic semialdehyde, which was not further metabolized. In contrast to 3-chlorocatechol, which was not metabolized and is known to generate suicidal products, 4-chlorocatechols do not generate acyl halides. Thus, further metabolism of the ring fission products is governed in strain MB2 by their suitability as substrates for the hydrolase.     

R-stereoselective amidase from Rhodococcus erythropolis No. 7 acting on 4-chloro-3-hydroxybutyramide

Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolis strain (No. 7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at 18 degrees . The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with midchain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 3.5.1.4). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.     

Herbicidal Activity of Some Pyridazine Derivatives

Herbicidal activity of some 50 pyridazyl ethers was evaluated. Among them, 3-chloro-6-(2-phenylphenoxy)-, 3-chloro-6-(2,6-dichlorophenoxy)-, 3-chloro-6-(3-methoxyphenoxy)- and 3-chloro-6-(4-methoxyphenoxy)-pyridazines showed conspicuous effectiveness on radish and millet in pre-emergence test, whereas postemergent activity of the compounds tested was not so remarkable in general. Relationships between herbicidal activity and chemical structure are discussed.     

Phytohormonal regulation of S-adenosylmethionine synthetase and S-adenosylmethionine levels in dwarf pea epicotyls.

A significant stimulation (2- to 2.5-fold) of AdoMet synthetase was witnessed in glibberellicd acid (GA3, 1 microM)-treated epicotyls of the dwarf pea (Pisum sativum). This was accompanied by a 2.4-fold increase in the endogenous pool of S-adenosylmethionine. Both abscisic acid (10 microM) and cycloheximide (20 micrograms/ml) inhibited the GA3-mediated enhancement of AdoMet synthetase activity. Three isozymes of AdoMet synthetase were detected in GA3-treated epicotyls, whereas a single activity peak was observed in controls. Thus, GA3 seems to control the induction of two new isozymes of AdoMet synthetase in the dwarf pea. By contrast, the tall pea exhibited three isozymes of AdoMet synthetase even in the absence of GA3 treatment. High concentration of L-methionine (2 mM) mimicked the GA3-elicited induction of two new isozymes of AdoMet synthetase in dwarf pea epicotyls.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation.

The effects of three tetrachlorobiphenylols [2',3',4',5'-tetrachloro-2-biphenylol (1); 2',3',4',5'-tetrachloro-4- biphenylol (2); and 2',3',4',5'-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3',5,5'-tetrachloro-4,4'-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase) activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1-3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5-7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (greater than or equal to 0.4 microM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1-7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

Phytohormones,Rhizobium mutants, and nodulation in legumes. IV. Auxin metabolites in pea root nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied directly to root nodules of intact pea plants. After 24 h, radioactivity was detected in all plant tissues. In nodule and root tissue, only 2–3% of³H remained as IAA, and analysis by thin layer chromatography suggested that indole-3-acetyl-L-aspartic acid (IAAsp) was a major metabolite. The occurrence of IAAsp in pea root and nodule tissue was confirmed unequivocally by gas chromatography-mass spectrometry (GC-MS). The following endogenous indole compounds were also unequivocally identified in pea root nodules by GC-MS: IAA, indole-3-pyruvic acid, indole-3-lactic acid, indole-3-propionic acid, indole-3-butyric acid, and indole-3-carboxylic acid. Evidence of the occurrence of indole-3-methanol was also obtained. With the exception of IAA and indole-3-propionic acid, these compounds have not previously been unequivocally identified in a higher plant tissue.     

Synthesis,structural characterization and in vitro antitumor activity of novel 6-chloro-1,1-dioxo-1,4,2-benzodithiazie derivatives

A series of nonconventional aminium N-(6-chloro-7-R-1,1-dioxo-1,4,2-benzodithiazin-3-yl)arylsulfonamidates 7-15 have been synthesized by the reactions of 6-chloro-7-R-3-methylthio-1,4,2-benzodithiazine 1,1-dioxides with 4-dimethylaminopyridine or Et(3)N and some arylsulfonamides. The free N-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodithiazin-3-yl)benzenesulfonamides 16-18 were obtained by treatment of their aminium salts with H(2)SO(4) in boiling acetic acid. The in vitro antitumor activity of the compounds 9, 11-14 and 16-18 has been tested in the antitumor screening of the National Cancer Institute (NCI), and relationships between structure and antitumor activity are discussed. 4-Dimethylaminopyridinium 4-chloro-N-(6-chloro-7-methyl-1,1-dioxo-1,4,2-benzodithiazin-3-yl)benzenesulfonamidate 9 is the prominent of the compounds due to its remarkable activity (log GI(50)<-8.00, log TGI=-5.50) and selectivity for the leukemia SR cell line. For that reason experimental and theoretical analysis of the geometric and electronic properties of 9 was carried out.     

The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.     

Synthesis and biological evaluation of 2-chloro-3-[(thiazol-2-yl)amino]-1,4-naphthoquinones

A series of novel, substituted 2-chloro-3-[(thiazol-2-yl)amino]-1,4-naphthoquinones have been prepared and shown to exhibit promising concentration-dependent activity against human SH-SY5Y cells, Plasmodium falciparum, Mycobacterium tuberculosis and P. aeruginosa. Substituent effects on observed bioactivity have been explored; the para-fluorophenyl derivative 3d exhibited activity across the range of the bioassays employed, indicating the potential of the 2-chloro-3-[(4-arylthiazol-2-yl)amino]-1,4-naphthoquinone scaffold in the development of novel, broad spectrum therapeutics.