STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES

A study has been made of the growth-regulating activity of a number of phenoxy acids possessing halogen or methyl substituents in the 2:6- or 33- positions of the nucleus. The pea curvature, Avena cylinder elongation and tomato-leaf epinasty tests have been used for assessing biological activity. The results show that in general 3:s- is more closely associated with inactivity than 2:6- substitution. The introduction of a further halogen atom into the 4- position does not enhance activity in the 2:6- compounds but does so in the 3:s- derivatives. Although the 2:4:6-trichloro- and 2:4:6-tribromo-phenoxyacetic acids are almost inactive, a:4-dichloro-6-fluoro- and 2:4-dibromo-6-fluoro- phenoxyacetic acids are very highly active. When a methyl group is substituted into the side-chain of certain phenoxyacetic acids possessing 2:6- substituents, growth-promoting activity is increased. Some of these findings conflict with recent theories on mode of action of phenoxy acids which are discussed in relation to the present work.     

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 Foliar Penetration: III. THE EFFECTS OF CHLORINATION ON THE RATE OF PENETRATION OF PHENOXYACETIC ACID AND BENZOIC ACID INTO LEAVES OF PHASEOLUS VULGARIS

A comparative study has been made of the penetration into leafdisks of Phaseolus vulgaris of (a) phenoxyacetic acid and its2-, 4-, 2, 4-, 2, 6-, 3-5, 2, 4, 5-, and 2, 4, 6- chloro derivatives,and (b) benzoic acid and its 2-, 2, 4-, 2, 5-, 2, 3, 6-chloroand 3, 6-dichloro-2-methoxy derivatives. The methods of synthesisof each compound with ¹⁴C incorporated in the carboxyl groupare described. In the series of substituted phenoxyacetic acids it was establishedthat only for 2, 4, 5-T was there an appreciable loss of radioactivityfrom the system either in the light or darkness. In contrast,with the exception of 2, 4-DCBA, ¹⁴C is lost from each memberof the series of substituted benzoic acids. The level and pattern of penetration in the two series is differentiallyaffected by chlorination. In general progressive chlorinationof phenoxyacetic acid leads to an increase in the rate of penetrationin both light and darkness. There are, however, exceptions;for illuminated disks the rate of entry of 2,4,5-T is exceededby 2,4,6-T, 2, 4-D, and 2, 6-D whereas in the dark 2,4,5-T hasby far the highest rate of penetration. Progressive chlorinationof benzoic acid, however, depresses entry in both light anddarkness. Possible relationships between these findings and changes inselected physical properties of each series of compounds havebeen examined. Between members of the series of substitutedphenoxyacetic acids there is little variation in the dissociationconstant whereas for the substituted benzoic acids there isa marked lowering of the pK as the degree of chlorination increases.The rate of elution with chloroform of each compound from abuffered Silocel column gives a measure of the degree of partitioninto a lipid medium at a given pH. At pH 5.2 chlorination ofphenoxyacetic acid results in more rapid elution whereas chlorinationof benzoic acid causes a longer hold up. An apparatus was designed to enable measurements to be madeof the rate at which a compound moves from one aqueous phaseto another through a layer of lipid. Chlorination promotes therate of transfer of phenoxyacetic acid through octanol but retardsthe transfer of benzoic acid. The extent to which chlorinationdepresses the rate of transfer of benzoic acid matches the diminutionin the rate of leaf penetration. For the series of substitutedphenoxyacetic acids transfer rates and penetration rates followsimilar trends in the dark but in the light agreement is lessgood. These findings are discussed in relation to prior studies onthe rate of uptake and the metabolic fate of the ndividual compoundsin a number of plant tissues.     

Studies on Plant Growth Regulators

The auxin activities of the homologs of racemic and enantiomeric α-alkylphenylacetic acids were estimated by pea straight growth test. The α-methyl, -ethyl and -propyl acids were moderately active whereas the longer and branched alkyl chain were found to make the molecule inactive. The more active enantiomers were shown to have the same configuration as the more active enantiomers in the other series of the optical active synthetic auxins.

The auxin activities of the cyclic homologs of 1, 2, 3, 4-tetrahydro- and 3, 4-dihydro-1- naphthoic acids were determined by pea straight growth test. In the tetrahydro-acid series, it was observed that the alicyclic ring expansion from the 6-membered to the 7-membered made the molecule inactive. In the 3, 4-dihydro-acid series, on the other hand, the activity remained almost unchanged by such a structural change. Structure-activity relationships were discussed in terms of their molecular structures, in particular, the configuration of the carboxyl group.     

Studies on plant growth-regulating substances XXVI. Isatic and anthranilic acids

The plant growth-regulating activities of isatic acid and twenty-six of its derivatives, together with the twenty-seven corresponding anthranilic acids, have been assessed in the wheat cylinder, the pea segment and the pea curvature tests. Activity was sustained by substitution in the 4- and 5-positions of isatic acid but decreased by substitution in the 3- and 6-positions. In the anthranilic acid series, the parent acid was inactive but the introduction of a large grouping (bromine or iodine) into the 5-position conferred activity. The 3,6- and 5,6-dichloro and the 3,6-dibromo acids were also active; compounds substituted in the 4-position to the carboxyl group or disubstituted in the 3,5-positions, were, as expected, inactive. In metabolism experiments on wheat and pea tissues with isatic and 5-chloroisatic acids the corresponding anthranilic acid was formed, together with an unidentified non-acidic metabolite in each case. There was no evidence that the growth regulating activity of isatic acids was related to this breakdown and it is concluded that the acids possess activity per se. The results are briefly discussed in terms of recent theories relating chemical structure to plant growth-regulating activity.     

Synthesis of some newer derivatives of 2-amino benzoic acid as potent anti-inflammatory and analgesic agents

Diazotization of N-benzylidene anthranilic acids 1a-1n at pH 9 yielded N-[alpha-(phenylazo) benzylidene] anthranilic acids 2a-2n and at pH 3 yielded N-benzylidene-5-(phenylazo) anthranilic acids 3a-3n. When compounds 3a-3n were treated with thioglycolic/thiolactic acid in the presence of anhydrous ZnCl(2), 2-(4-oxo-2-phenylthiazolidin-3-yl)-5-(phenylazo) benzoic acids 4a-4n were afforded. The newly synthesized compounds were screened for their anti-inflammatory and analgesic activities and were compared with standard drugs, aspirin and phenylbutazone. Out of the compounds studied, the most active compound 4n showed more potent activity than the standard drugs at all doses tested.     

A new class of synthetic auxin transport inhibitors

Auxin transport inhibition by a new class of synthetic plant growth regulants, the 2-(3-aryl-5-pyrazolyl)benzoic acids, was examined in bean (Phaseolus vulgaris L.) using the donor-receiver agar cylinder technique. These compounds can be prepared by the dehydrogenation and ring cleavage of compounds like DPX-1840 (2-(4-methoxyphenyl)-3,3adihydro-8H-pyrazolo[5,1-a] isoindol-8-one) which was previously reported (Plant Physiol. 1972. 50: 322-327) to be a potent inhibitor of auxin transport. These new growth regulators inhibit auxin transport more than DPX-1840 does as evidenced by their consistently greater reduction of basipetal auxin transport capacity in bean when incorporated into the receiver agar cylinder or applied foliarly to intact plants. Direct comparisons of the effect of DPX-1840, its dehydrogenation product (2-(4-methoxyphenyl)-8H-pyrazolo [5,1-a]isoindol-8-one), and its open-ring form (2-(3-(4-methoxyphenyl)-5-pyrazolyl) benzoic acid) on auxin transport indicated the following order of activity: ring-open > dehydrogenated form > DPX-1840. DPX-1840-(14)C, applied at 0.5 mg/l to etiolated bean hypocotyl hooks followed by extraction and thin layer chromatography, indicated the biological conversion of DPX-1840 to its open-ring form. Collectively, these results suggest that the biologically active forms of DPX-1840-type compounds are the open-ring (2-(3-aryl-5-pyrazolyl) benzoic acids.     

Effects of plant growth regulators and other compounds on flow of latex in Hevea brasiliensis

Eighty-nine compounds have been tested for their ability to promote the flow of latex from Hevea brasiliensis after tapping. The twenty-four substances which showed significant activity were: twelve chloro- and fluoro-phenoxy alkylcarboxylic acids substituted in the 4-, 2, 4- and 2, 4, 5- positions, two chlorinated benzoic acids, α- and β-naphthaleneacetic acid, 3-indolylacetic acid, 2-methoxy-4-diethylamino-6-isopropylamino-1, 3, 5- triazine (methoxy-ipazine), N,N-diallyl-α-chloroacetamide (CDAA), 1,1′-dimethyl-4,4′-bi-pyridilium-2A (paraquat), neomycin, dichloro-m-xyienol, and phenyl mercury acetate and chloride. Despite the many replications the high variability of the yield data does not allow of a precise classification of the relative activities. The most active compounds were 2, 4-dichloro-5-fluorophenoxyacetic acid and 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T), although 2, 4-dichlorophen-oxyacetic acid (2, 4-D) gave increases in yield comparable to those of 2, 4, 5-T in some of the trials.     

Optimization of sulfonamide derivatives as highly selective EP1 receptor antagonists

A series of 4-[(2-{isobutyl[(5-methyl-2-furyl)sulfonyl]amino}phenoxy)methyl]benzoic acids and 4-({2-[isobutyl(1,3-thiazol-2-ylsulfonyl)amino]phenoxy}methyl)benzoic acids were synthesized and evaluated for their EP receptor affinities and EP1 receptor antagonist activities. Further structural optimization was carried out to reduce inhibitory activity against hepatic cytochrome P450 isozymes, which could represent a harmful potential drug interaction. Selected compounds were also evaluated for their binding affinities to hTP, hDP, mFP, and hIP, and for their hEP1 receptor antagonist activities. The results of structure-activity relationship studies are also presented.     

Anaerobic degradation of halogenated benzoic acids coupled to denitrification observed in a variety of sediment and soil samples

Abstract Denitrifying enrichment cultures utilizing monochlorinated benzoic acids as a carbon source were established using sediments and soils from a variety of sources as inocula. Enrichment cultures from most of the sites readily degraded 3- and 4chlorobenzoate within 2–4 weeks. Upon refeeding, 3- and 4-chlorobenzoate were rapidly depleted, and stable denitrifying cultures were obtained by repeated dilution and refeeding of the substrates. 2-Chlorobenzoate, however, was only slowly metabolized and this activity was only observed in a few sites. Denitrifying consortia were maintained on either 3- or 4chlorobenzoate as the sole source of carbon and energy and chlorobenzoate utilization was dependent on denitrification. These cultures were also capable of utilizing the corresponding brominated and iodinated benzoic acids, but the activity was specific to the position of the halogen substituent. Removal of halogen was stoichiometric, indicating that dehalogenation occurred at some step in metabolism.     

Cold-adapted and rhizosphere-competent strain of Rahnella sp. with broad-spectrum plant growth-promotion potential

A phosphate-solubilizing bacterial strain isolated from Hippophae rhamnoides rhizosphere was identified as Rahnella sp. based on its phenotypic features and 16S rRNA gene sequence. The bacterial strain showed the growth characteristics of a cold-adapted psychrotroph, with the multiple plant growth-promoting traits of inorganic and organic phosphate solubilization, 1-aminocyclopropane-1- carboxylate-deaminase activity, ammonia generation, and siderophore production. The strain also produced indole- 3-acetic acid, indole-3-acetaldehyde, indole-3-acetamide, indole-3-acetonitrile, indole-3-lactic acid, and indole-3- pyruvic acid in tryptophan-supplemented nutrient broth. Gluconic, citric and isocitric acids were the major organic acids detected during tricalcium phosphate solubilization. A rifampicin-resistant mutant of the strain exhibited high rhizosphere competence without disturbance to the resident microbial populations in pea rhizosphere. Seed bacterization with a charcoal-based inoculum significantly increased growth in barley, chickpea, pea, and maize under the controlled environment. Microplot testing of the inoculum at two different locations in pea also showed significant increase in growth and yield. The attributes of coldtolerance, high rhizosphere competence, and broad-spectrum plant growth-promoting activity exhibited the potential of Rahnella sp. BIHB 783 for increasing agriculture productivity.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES

Fifteen series of aryloxyacetic, α-aryloxy-propionic, -butyric and -isobutyric acids and the corresponding arylthio-analogues have been synthesized and their growth-regulating activity has been assessed in the tomato-leaf epinasty, wheat cylinder and the pea curvature tests. It is shown that in general arylthio-derivatives are less active than their aryloxy-analogues. Although for a cell elongation response the presence in the molecule of an a-hydrogen atom is usually necessary, three exceptions to this generalization have now been found. A lower activity in the 3:5-dichloro- compared with the 2:6-dichloro-compounds has been demonstrated. Evidence is presented to show that an ortho hydrogen atom in the nucleus is not an essential requirement for cell elongating activity as is postulated by other workers.     

STUDIES ON PLANT GROWTH-REGULATING SUBSTANCES XI. AUXIN ANTAGONISM IN RELATION TO A THEORY ON MODE OF ACTION OF ARYL- AND ARYLOXY-ALKANECARBOXYLIC ACIDS

A study has been made of the antagonistic effects of a range of phenoxy and other acids on the growth-promoting activity of a number of auxins as assessed by the Avena cylinder and pea curvature tests. The compounds examined as inhibitors were selected on a logical basis, these molecules not possessing the structural requirements considered necessary for growth-regulating activity, yet possessing groupings which might allow them to accumulate at a site of action from which the growth response by auxins might be initiated. Our previous demonstrations that inactive stereoisomers of x-aryloxy propionic acids can competitively inhibit the activity of their active enantiomorph has been confirmed and a similar antagonism has been found to operate with other inactive aryloxy acids. Such competitive antagonism is discussed in relation to a 'three-point' contact theory on mode of action.     

Studies on plant growth-regulating substances: XXXIII. The influence of ring substituents on the plant growth-regulating activity of phenylacetic acid

A comprehensive range of phenylacetic acids substituted with nitro, halogen, methyl, amino, hydroxyl and N-acetylamino groups have been synthesized and their growth-regulating activities assessed in the wheat cylinder, pea curvature and pea segment tests. The influence of substituents on molecular shape is shown to be more important in determining activity than their effects on electron distribution. Studies with 2,6-disubstituted phenylacetic acids have indicated that the most active compounds can attain a certain spatial configuration in which one surface of the molecule, including the plane of the ring system, is flat and the carboxyl group is above with its axis of rotation perpendicular to this surface. Positional requirements for growth-regulating activity in phenylacetic acids are shown to be less important than in the phenoxyacetic and benzoic acids.     

Synthesis of some newer derivatives of substituted quinazolinonyl-2-oxo/thiobarbituric acid as potent anticonvulsant agents

5-[1'-[3"-Aminoacetyl-2"-methyl-6",8"-dihalosubstitutedquinazolin-4"(3"H)-onyl]-thiosemicarbazido]-2-oxo/thiobarbituric acids 3a-3h and 5-[2'-amino-5'-[3"-aminomethylene-2"-methyl-6",8"-dihalosubstitutedquinazolin-4"(3"H)-onyl]-1',3',4'-thiadiazol-2'-yl]-2-oxo/thiobarbituric acid 5a-5h were prepared by incorporating 1-[3'-aminoacetyl-2'-methyl-6",8"-dihalosubstituted-quinazolin-4'(3'H)-onyl]-thiosemicarbazides 2a-2d and 2-amino-5-[3'-aminomethylene-2'-methyl-6',8'-dihalosubstituted-quinazolin-4'(3'H)-onyl]-1,3,4-thiadiazoles 4a-4 h respectively at 5(th) position of 2-oxo/thiobarbituric acids (via Mannich reaction). All the newly synthesized compounds were screened for their anti-convulsant activity in MES and PTZ models and were compared with standard drugs phenytoin sodium and sodium valproate. Interestingly, these compounds were found to be devoid of sedative and hypnotic activities when tested. Out of the compounds studied, the most active compound 5h, that is 5-[2'-amino-5'-[3"-aminomethylene-2"-methyl-6",8"-dibromoquinazolin-4"(3"H)-onyl]-1',3',4'-thiadiazol-2'-yl]-2-thiobarbituric acid showed activity (90%) more potent than the standard drug.     

Studies on plant growth-regulating substances XXVIII. Halogen-substituted benzoic acids

The plant growth-regulating activities of chloro-, bromo- and iodo-isomers in 2,5-, 2,3,5- and 2,3,6-substituted benzoic acids were assessed in the wheat cylinder, pea segment and pea curvature tests. Their effects in the tomato-leaf epinasty test were also investigated. Replacing an ortho-chloro atom by bromine had little or no effect on activity. An ortho-iodo substituent, however, reduced activity in the 2,3,6-substituted series and almost completely inhibited it in the 2,5- substituted series. This decreased activity of ortho-iodo-substituted isomers is visualized as being due to a steric inhibition of the attachment of the carboxyl group to its receptor unit prior to the initiation of the growth response.     

Tissue culture media; the essential non-dialyzable factors in human placental cord serum

1. Human placental cord serum contains a factor or factors necessary for active growth of fibroblasts in tissue culture and a factor which prevents the appearance of a characteristic toxicity. 2. The growth-promoting factor(s) is stable to heating to 60°C. for 30 minutes and is associated with the euglobulin fraction of the serum. 3. Extraction of dried cord serum with benzene, chloroform, and ether does not cause any decrease in growth-promoting activity. 4. Sera from hypophysectomized, adrenalectomized, and thyroid-parathyroidectomized rats show as much growth-promoting activity as normal rat sera. 5. Bovine gamma globulins, anterior pituitary growth hormone, and insulin do not replace the serum growth factor. 6. The toxicity-preventing factor is serum albumin. 7. Evidence is presented that the toxicity is due to free fatty acids in the medium and that serum albumin acts by binding the fatty acids.     

Brassinolide activities of 2alpha,3alpha-diols versus 3alpha,4alpha-diols in the bean second internode bioassay: explanation by molecular modeling methods

In general, the structural requirements postulated for a high brassinolide activity are: 2alpha,3alpha-diol, 6-ketone or better 7-oxalactone in B-ring, A/B trans fused ring junction, a cis C-22,C-23-diol preferentially with RR configurations, and a C-24 methyl or ethyl substituent [Takatsuto S, Yazawa N, Ikekawa N, Takematsu T, Takeuchi Y, Koguchi M. Structure-activity relationship of brassinosteroids. Phytochemistry 1983;22:2437-41; Thompson MJ, Meudt WJ, Mandava NB, Dutky SR, Lusby WR, Spaulding DW. Synthesis of brassinosteroids and relationship of structure to plant growth-promoting effects. Steroids 1982;39:89-105]. We found that the 3alpha,4alpha-diols 4, 6 and 8 are more active than the 2alpha,3alpha-diols 3, 5 and 7 [Sísa M, Budesínsky M, Kohout L. Synthesis of 7a-homo and 7a,7b-dihomo-5alpha-cholestane analogues of brassinolide. Collect Czech Chem Commun 2003;68:2171-89]. This fact is in strong contrast with the structure requirements mentioned above. Our hypothesis suggests that the lower activity of 2alpha,3alpha-diols and/or the higher activity of 3alpha,4alpha-diols could be explained by twisting and distortion of the molecule due to the seven- or eight-membered B-ring and also by the position of a carbonyl group relative to the A-ring diol. 3D-SAR computer methodologies as alignments and overlaps of GRID maps and 3D-QSAR analysis GRID-GOLPE (CoMFA-like) were used as an effort to explain the higher bioactivity of 3alpha,4alpha-diols 4, 6 and 8 in comparison with the 2alpha,3alpha-diols 3, 5 and 7 of B-ring enlarged brassinosteroids.     

Cytokinins: Synthesis of 2-, 8-, and 2,8-substituted 6-(3-methyl-2-butenylamino)purines and their relative activities in promoting cell growth

We have synthesized 2- and 8-monosubstituted and 2,8-disubstituted derivatives of the cytokinin 6-(3-methyl-2-butenylamino)purine (N⁶-isopentenyladenine) and have shown the dependence of growth-promoting activity in the tobacco bioassay upon the position, number, and type of substituent. The representative substituent groups were MeS, Me, MeSO₂, C₆H₅CH₂S, HS and Cl. The 8-methyl derivative was exceptional in being more active than the unsubstituted parent compound. In general, substitution in the 8-position decreases activity less than substitution in the 2-position, with the exception of the electron-attracting methylsulfonyl. Substitution in both the 2- and 8-positions lowers the activity more than substitution at either single position on the adenine nucleus, with the exception of the 2,8-dimethyl derivative. The chloro and methylthio derivatives show activity in the same range as the methyl derivatives, and the mercapto compounds, which exist mainly as CS tautomers, show somewhat less activity than the corresponding methylthio compounds. Bulky (C₆H₅CH₂S and MeSO₂) and strongly electron-attracting (MeSO₂) substituents cause relatively great reduction in cytokinin activity.     

Effects of cholesterol on viability and (n - 6) fatty acid metabolism in cultured human monocyte-like cells (U937).

Effects of supplementation of growth-promoting cholesterol on metabolism of the cytotoxic (n - 6) polyunsaturated fatty acids in cultured human monocyte-like cells (U937) have been examined. U937 cells were incubated in 5% delipidated fetal bovine serum containing 0 or 38.7 microM cholesterol. The rate of uptake and the distribution of metabolites of (n - 6) fatty acids (such as 18:2(n - 6), 18:3(n - 6), and 20:3(n - 6), and 20:4(n - 6)) were examined by adding radiolabelled fatty acid at a level of 1 microgram/mL (3.3 microM for 20-carbon fatty acids and 3.6 microM for 18-carbon-fatty acids). For assessing the cytotoxicity, (n - 6) fatty acids were added to medium at a concentration of 5 micrograms/mL (16.4 microM for 20-carbon fatty acids and 17.9 microM for 18-carbon fatty acids). Cholesterol supplementation suppressed the uptake of all (n - 6) fatty acids and reduced the cytotoxic effects of 18:2(n - 6), 20:3(n - 6), and 20:4(n - 6), but not 18:3(n - 6). In addition, cholesterol supplementation increased peroxide production and metabolism of (n - 6) fatty acids in U937 cells. Thus, the differential suppressive effect of cholesterol on the cytotoxicity of different fatty acids could not be attributed to an inhibitory effect on fatty acid delta 6- and delta 5-desaturation, or to an antioxidant effect on peroxide formation.