Comparative Effects of Heterocyclic Compounds on Inhibition of Lettuce Fruit Germination

The molecules of many biologically active plant constituentscontain heterocyclic ring systems. Inhibitory effects of a numberof heterocyclic compounds and their alicyclic and open-chainanalogues on lettuce (Lactuca sativa L. cv. Great Lakes) germinationwere therefore determined under specified conditions. The mostobvious property which correlates chemical structure with biologicalactivity is lipophilicity, as was found in previous studies.However, other less obvious factors play a part. The inhibitoryactivity of coumarin, for instance, is much greater than wouldbe expected in comparison with compounds of related structures.In general, substitution of a carbon atom in a ring structureby oxygen or nitrogen has either little effect or a loweringeffect on activity, unless the increased solubility in waterallows an inhibitory concentration to be reached which did notoccur with the carbocyclic compound. However, introduction ofunsaturation increases activity markedly, especially with someof the indole compounds. Key words: Heterocyclic compounds, inhibitory compounds, lettuce germination     

Light-controlled Cycocel Reversal of Coumarin Inhibition of Lettuce Seed Germination and Root Growth

Lettuce seed germination or lettuce root elongation after germination in water was inhibited by coumarin and these inhibitions were reversed by Cycocel. 2.53 × 10³ M Cycocel reversed the inhibition of seed germination by 6.8 × 10⁴ M coumarin. and 6.32 × lO^?4 M Cycocel reversed the inhibition of root elongation by 3.4 × 10³ M coumarin. No other analogs of Cycocel reversed these coumarin induced inhibitions of growth. Cycocel reversal of coumarin inhibition of lettuce seed germination occurred in red light but not in far-red light or darkness. The red-far-red system was photoreversible. Cycocel and kinetin appear to act similarly in reversing coumarin inhibition of germination. Gibberellin A₃ and IAA were unable to reverse these coumarin induced inhibitions. A common mechanism is suggested for Cycocel reversal of coumarin and lAA inhibition of growth.     

The Activity of Organic Acids as Germination Inhibitors and its Relation to pH

The germination inhibitory activity of organic acids relatedto coumarin was investigated. The activity was found to be unrelatedto the nature of the side chain carrying the carboxyl group.Little or no relation between structure and activity existed.pH was found to be of secondary importance on the activity ofthe acids. Synergism between buffers and the acids exists insome cases. Moreover, the use of buffers in studying germinationinhibition was found to be unnecessary, due to the powerfulbuffering action of the seeds, and impermissible due to verymarked secondary effects of the buffers themselves and theireffect on germination.     

A novel single nucleotide polymorphism altering stability and activity of CYP2a6

CYP2A6 is known as a major cytochrome P450 (CYP) responsible for the oxidation of nicotine and coumarin in humans. In this study, we explored genetic polymorphisms, which reduce CYP2A6 activity in Japanese. Two novel mutations in exon 9 of the CYP2A6 gene were found. A single nucleotide polymorphism of T1412C and G1454T resulted in Ile471Thr and Arg485Leu substitution, respectively. The frequency of the former variant allele was considerably high (15.7%), while the latter variant appeared to be a rare polymorphism. Heterologous expression of CYP2A6 using a cDNA possessing C instead of T-base at codon 471 in Escherichia coli caused remarkable reduction of the stability of holoenzyme at 37 degrees C. Furthermore, this variant enzyme almost lacked nicotine C-oxidase activity, although coumarin 7-hydroxylase activity was still observed. These data suggest that individuals homozygous for the T1412C variant allele or heterozygous for this and a defect allele such as the CYP2A6*4 may be poor metabolizer of nicotine, but not coumarin.     

Phytotoxic Effects of some Antibiotics

The toxicity of a number of antibiotics, coumarin, and 3-indolylaceticacid to wheat, clover, and mustard seedlings has been assessedby a simple germination test. The percentage germination ofwheat was not markedly affected by any of the compounds testedalthough root growth was reduced. Severe inhibition of rootgrowth of the other seeds was usually accompanied by reductionin percentage germination. None of the antibiotics tested were as inhibitory as indolylaceticacid, but most were more toxic than coumarin. The more toxicof the antibiotics were alternaric acid, glutinosin, mycophenolicacid, and gliotoxin. The least toxic were griseofulvin, penicillin,and streptomycin. Higher concentrations of the latter antibioticwere found to have an inhibiting effect on the synthesis ofchlorophyll.     

Coumarin-3-carboxylic acid derivatives as potentiators and inhibitors of recombinant and native N-methyl-d-aspartate receptors

N-Methyl-d-aspartate receptors (NMDARs) are known to be involved in a range of neurological and neurodegenerative disorders and consequently the development of compounds that modulate the function of these receptors has been the subject of intense interest. We have recently reported that 6-bromocoumarin-3-carboxylic acid (UBP608) is a negative allosteric modulator with weak selectivity for GluN2A-containing NMDARs. In the present study, a series of commercially available and newly synthesized coumarin derivatives have been evaluated in a structure-activity relationship (SAR) study as modulators of recombinant NMDAR activity. The main conclusions from this SAR study were that substituents as large as iodo were accommodated at the 6-position and that 6,8-dibromo or 6,8-diiodo substitution of the coumarin ring enhanced the inhibitory activity at NMDARs. These coumarin derivatives are therefore excellent starting points for the development of more potent and GluN2 subunit selective inhibitors, which may have application in the treatment of a range of neurological disorders such as neuropathic pain, epilepsy and depression. Surprisingly, 4-methyl substitution of UBP608 to give UBP714, led to conversion of the inhibitory activity of UBP608 into potentiating activity at recombinant GluN1/GluN2 receptors. UBP714 also enhanced NMDAR mediated field EPSPs in the CA1 region of the hippocampus. UBP714 is therefore a novel template for the development of potent and subunit selective NMDAR potentiators that may have therapeutic applicability in the treatment of patients with cognitive deficits or schizophrenia.     

Pollen morphology and germination inNarcissus

Pollen grains of several species and varieties ofNarcissus were examined with a light microscope and a scanning electron microscope. Normal pollen grains were kidney- or spindle-shaped with a germination furrow and a reticulate structure similar to that of the pollen grains ofAmaryllis. Pollen grains germinated within 2 to 3 hr. Percentage of germination was dependent upon temperature and treatment. Pollen tubes grew in length up to 1,000 μm and branched occasionally or behaved in strange fashion. Fresh pollen grains germinated more in distilled water at lower temperature than in sucrose-aqueous medium. Both in the presence and in the absence of stigmatic exudate calcium increased the percentage of germination. Gibberellic acid, abscisic acid and coumarin inhibited the pollen germination. Plasmoptysis occurred in all species and in all media tested except in a medium containing coumarin without stigmatic exudates. Plasmoptysis did not seem to be induced by hypotonic medium alone. Pollen of high germination capacity showed a high percentage of plasmoptysis. Based on the results obtained, evolution and sterility of theNarcissus plant was discussed.     

Oxidative Phosphorylation in Germinating Lettuce Seeds

The effect of thiourea and coumarin, in vivo and in vitro, onthe phosphorylating activity of lettuce mitochondria was investigated,as well as the effect of coumarin on the P/O ratio. It was shownthat both substances in vitro inhibit phosphorylation; whilein vivo coumarin inhibits but thiourea under certain circumstancesstimulates. Coumarin was also shown to decrease the P/O ratioand therefore may be considered as an uncoupler. The difficulties in considering these effects of the substancesas a primary mechanism controlling germination are pointed outand discussed.     

Structural function of residue-209 in coumarin 7-hydroxylase (P450coh). Enzyme-kinetic studies and site-directed mutagenesis

Residue-209 plays a critical role in determining the substrate and product specificity of cytochrome P450coh. In order to investigate further the structural function of residue-209 in coumarin 7-hydroxylase reaction, we measured the enzyme-kinetic properties of wild-type P450coh and its mutants in which residue-209 was substituted with various amino acids. In general, the Km and Vmax values for coumarin increased as the size of residue-209 became smaller and Vmax values decreased. The size of residue-209, therefore, was a principle factor determining Km, Kd, and Vmax values of P450coh. Although the polarity and charge also increased the Km value consistently, they altered Vmax and Kd values in an irregular manner. The substitution of serine for residue-209 increased the Vmax, while the substitution of lysine decreased it. Coumarin 7-hydroxylase activity was inhibited weakly by indan, but competitively and strongly by 2-coumaranone. Moreover, Ki values for the inhibitor were similar to Km values of the corresponding, mutated P450s. The results indicate, therefore, that residue-209 is localized in a proposed substrate-binding sequence 1 which binds to the 2-keto group of coumarin and directs its 7-position toward the sixth ligand of heme. Consequently, the identity of residue-209 determines not only the binding of coumarin in P450coh, but also the other reaction step(s) of coumarin 7-hydroxylation.     

PROPERTIES AND CHANGES OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE IN GERMINATING LETTUCE SEEDS

Glucose-6-phosphate dehydrogenase is present in lettuce seedsand seedlings in a particulate, 20,000 g fraction and in a solublefraction of the cells. In both fractions the enzyme activityoccurs in the presence of either NAD or NADP. It seems probablethat an enzyme with two active sites is involved. Total activity does not change during 48 hours germination inwater. It is depressed by coumarin or thiourea treatment. Specificactivity rises in the soluble fraction when germination occurs,but not in the mitochondrial one. Coumarin treatment largelydepresses the increase in specific activity. In vitro 100 ppmcoumarin totally inhibit enzyme activity. 1,250 ppm thioureado not affect enzyme activity. The results are analysed and it is concluded that glucose-6-phosphatedehydrogenase is important, if at all, only in the very earlystages of germination. (Received August 13, 1965; )     

Discovery of heterocyclic replacements for the coumarin core of anti-tubercular FadD32 inhibitors

Previous work established a coumarin scaffold as a starting point for inhibition of Mycobacterium tuberculosis (Mtb) FadD32 enzymatic activity. After further profiling of the coumarin inhibitor 4 revealed chemical instability, we discovered that a quinoline ring circumvented this instability and had the advantage of offering additional substitution vectors to further optimize. Ensuing SAR studies gave rise to quinoline-2-carboxamides with potent anti-tubercular activity. Further optimization of ADME/PK properties culminated in 21b that exhibited compelling in vivo efficacy in a mouse model of Mtb infection.     

A structure-activity relationship study on a natural germination inhibitor, 2-methoxy-4-vinylphenol (MVP), in wheat seeds to evaluate its mode of action

The first aim of the present study was to evaluate which structural elements of the 2-methoxy-4-vinylphenol (MVP) molecule (1) are responsible for its observed activity as germination inhibitor in wheat seeds. To find its mode of action, a series of compounds with varying functional moieties and substitution patterns were prepared and evaluated for their inhibitory activity. This systematic competitive inhibition study characterized two criteria for the effective increase of the inhibiting ability: (i) ortho substitution to each of the hydroxy and methoxy groups; (ii) alkene moiety on the ring. Understanding how the structure of natural compounds relates to their inhibition function is fundamentally important and may help to facilitate their application as novel inhibitors to restrain preharvest sprouting (PHS) in wheat fields. In this regard, in MVP and its natural analogues 8 and 9 as the most active inhibitors, the ortho substitution of hydroxy and methoxy groups plays a key role in their activity and, as well, the alkene moiety influences the activity significantly.     

Effect of Endogenous Coumarin on the Germination of Seeds of Coumarouna odorta Aublet

Seeds of Coumarouna germinate readily at 33?C in darkness orlight in spite of high levels of endogenous coumarin. The concentrationof coumarin on a dry-weight basis remained constant in the cotyledonsand increased in the embryos during germination. Applied coumarin(0.4 and 4 mM) did not inhibit germination of Coumarouna butenhanced the growth of embryos; yet a single seed of Coumarouna,placed at the centre of a Petri-dish, drastically inhibitedgermination of other neighbouring seeds (lettuce, radish, andtomato). The presence of coumarin in the soil of potted seedlingssuggest that coumarin exuded by the radicle can inhibit germinationand growth of other species in its vicinity, thus eliminatingcompetition during the first stages of growth.     

The Interaction of Coumarin and Temperature in the Germination of Lettuce Seed

Dormancy was induced in lettuce seed by exposing to coumarin during imbibition. The level of dormancy which is imposed is dependent on the temperature during germination, coumarin being ineffective below 20°C. At 25°C there is marked interaction between coumarin and this temperature, and the response of the seed. It was shown by exposing treated seeds to temperature regimes involving periods at 25°C and 10°C. that the action of coumarin can be explained more readily by concluding that the events which are affected are cell elongation and division. There is also evidence that the coumarin is metabolised more rapidly at 25°C, and that the effectiveness of the coumarin in the experiments cited depends on its presence at the time when embryo growth is initiated. The mode of action of coumarin is unknown.     

Anti-influenza activity of monoterpene-containing substituted coumarins

Compounds simultaneously carrying the monoterpene and coumarin moieties have been tested for cytotoxicity and inhibition of activity against influenza virus A/California/07/09 (H1N1)pdm09. The structure of substituents in the coumarin framework, as well as the structure and the absolute configuration of the monoterpenoid moiety, are shown to significantly influence the anti-influenza activity and cytotoxicity of the compounds under study. The compounds with a bicyclic pinane framework exhibit the highest selectivity indices (the ratios between the cytotoxicity and the active dose). The derivative of (?)-myrtenol 15c, which is characterized by promising activity, low cytotoxicity, and synthetic accessibility, has the greatest potential among this group of compounds. It exhibited the highest activity when added to the infected cell culture at early stages of viral reproduction.     

Effect of phenolic compounds on abscisic acid-induced stomatal movement: Structure – activity relationship

Application of abscisic acid (ABA) brings about stomatal closure within 30 min in epidermal peels of Vicia faba . A number of phenolic compounds antagonise the effect of ABA. Derivatives of benzoic acid, cinnamic acid, coumarin and flavonoids have been studied in order to establish structure – activity relationship. Derivatives of benzoic acid reverse the ABA effects. Coumarin, esculetin and three hydro derivatives of cinnamic acid fail to show the anti-ABA activity. Thus, the presence of parahydroxyl group and double bond in the side chain is necessary for anti-ABA activity.     

Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis

Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k(cat) and Km) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (k(cat)/Km) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities (and also higher Km values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822-823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations.     

Comparative Effects of Aromatic Compounds on Inhibition of Lettuce Fruit Germination

Germination of lettuce (Lactuca sativa L. cv. Great Lakes) wasinhibited by aromatic alcohols, aldehydes and carboxylic acidsin a way similar to that by aliphatic members. Increased lipophilicityusually leads to increased inhibitory activity. Thus methylationincreased activity while hydroxylation decreased it. Exceptionswere seen with ortho-hydroxylated compounds and other exceptionsindicated the presence of unrecognized factors. Many of thephenolic compounds often quoted as inhibitors were shown tobe not particularly activeagainst lettuce germination when comparedwith, for instance, abscisic acid or coumarin.     

TRANS-CINNAMIC ACID AND SOME RELATED COMPOUND EFFECTS ON ORNITHOGALUM CAUDATUM POLLEN

Fresh pollen of Ornithogalum caudatum was germinated in the absence or presence of trans-cinnamic acid (t-CA), trans-cinnamaldehyde (t-CAld) and related compounds, and germination percentage studied. Hydrocinnamic acid, t-CA, and t-CAld reduced germination to 50% of the control at 20–40 mg/liter. Hydrocinnamaldehyde, 3-phenyl-1-propanol and phenylacetic acid gave 50% reduction at 70–80 mg/liter, and caffeic acid, ferulic acid, phenethyl alcohol, coumarin, styrene, phenylalanine or ethyl cinnamate required 200 mg/liter or more for similar reduction in germination percentage. The inhibition of germination by t-CA could not be overcome by indole acetic acid and vice versa, indicating a lack of antiauxin activity.     

Antibacterial activity of coumarins

The antibacterial activity of coumarin per se and other 45 coumarin derivatives was tested against strains of Bacillus cereus MIP 96016, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923. The inhibitory effects of coumarins were affected by their substitution patterns. Osthenol (44) showed the most effective antibacterial activity against Gram-positive bacteria with MIC values ranging between 125 and 62.5 microg/ml. These results suggested that the prenyl chain of 44 at position 8 and the presence of OH at position 7 of the benzenic ring are required for the antibacterial activity against these strains.