Decomposition of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one in Aqueous Solutions

Cyclic hydroxamic acids present in some species of Gramineae have been reported to be important in resistance of these plants to fungi and insects. Since the nonglucosylated forms of these acids are unstable in aqueous solution, in vitro methods for the measurement of their antibiotic properties have been difficult. Kinetics of the decomposition of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), the major hydroxamate in corn (Zea mays L.) extracts, were studied in buffered aqueous solutions from pH 5 to 7.5 at temperatures from 20 to 80 C. Kinetics were apparently first order under all conditions tested; energies of activation (24 to 28 kcal/mol) were nearly pH-independent. DIMBOA decomposed rapidly (half-life = 5.3 hours at 28 C, pH 6.75) relative to the time required for many procedures which have been used to demonstrate the biological activity of DIMBOA. The rate of disappearance of inhibitory activity of DIMBOA toward Erwinia carotovora was indistinguishable from the rate of decomposition of DIMBOA. Contrary to reports, yields of 6-methoxy-2-benzoxazolinone (MBOA) were not quantitative. Gas-liquid chromatography analytical procedures were developed for quantitation of trimethylsilyl and acetyl derivatives of MBOA. As measured by ultraviolet spectroscopy and/or gas-liquid chromatography, conversion of DIMBOA to MBOA ranged from 40 to 75% of theoretical in aqueous buffers, bacterial growth medium, and ethyl acetate extracts of corn tissue resuspended in buffer. Yields varied with temperature, pH, and constituents in the medium.     

Synthesis of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole moiety: Their in vitro evaluation against PDE4B

A number of 2H-1,3-benzoxazin-4(3H)-one derivatives containing indole or benzofuran moieties were synthesized by using Pd/C–Cu mediated coupling-cyclization strategy as a key step. The o-iodoanilides or o-iodophenol were coupled with 3-{2-(prop-2-ynyloxy)ethyl}-2H-benzo[e][1,3]oxazin-4(3H)-one using 10%Pd/C–CuI–PPh₃ as a catalyst system and Et₃N as a base to give the target compounds. All the synthesized compounds were tested for their PDE4B inhibitory potential in vitro using a cell based cAMP reporter assay. Some of them showed fold increase of the cAMP level when tested at 30 μM. A representative compound showed encouraging PDE4B inhibitory properties that were supported by its docking results.     

Factors That Influence the Activity of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one on Erwinia Species in Growth Assays

Factors affecting the inhibitory activity of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) against Erwinia carotovora, a nonpathogen of Zea mays L., and against a maize pathovar of Erwinia chrysanthemi (ECZ) were examined. Most experiments were performed with DIMBOA dissolved in a bacterial growth medium containing 10 g/liter of sucrose, inorganic salts, and 1 g/liter of casamino acids at pH 6.75. When temperature and pH were held constant, inhibition of E. carotovora varied linearly with the logarithm of the initial cell population. By altering temperatures, assays with constant pH and initial cell populations were performed under conditions of varying DIMBOA stability. When E. carotovora was grown at 24, 28, 32, and 36 C in the presence of 0.1 to 0.5 mm DIMBOA, the inhibition of bacterial growth was maintained long after DIMBOA had decomposed in the medium to levels which, if added initially, would not have been inhibitory. When assays were performed at pH 5.5, the pH of aqueous maize extracts, E. carotovora was more inhibited than at pH 6.75; however, ECZ was substantially less inhibited at the lower pH.     

Potential anti-proliferative agents from 1,4-benzoxazinone-quinazolin-4(3H)-one templates

A novel synthetic protocol has been developed for the synthesis of 1,4-benzoxazinone-acetylphenylallyl quinazolin-4(3H)-one hybrids 7a–n by employing Pd-catalyzed CH arylation in presence of 5–10% phosphine ligand in good to excellent yields and evaluated for their anti-proliferative activity against three cancer cell lines such as A549 (lung), HeLa (cervical), MDA-MB-231 (breast). Compounds 7d, 7f, 7l and 7n exhibited promising anti-proliferative activity with GI₅₀ values ranging from 0.37 to 2.73?µM respectively against A549, HeLa, and MDA-MB-231, while compound 7f showed significant activity against MDA-MB-231 with GI₅₀ value 0.58?µM, 7j showed significant activity against A549 with GI₅₀ value 0.32?µM and 7l showed significant activity against HeLa with GI₅₀ value 0.37?µM. This is the first report on the synthesis and in vitro anti-proliferative evaluation of 1,4-benzoxazinone-acetylphenylallyl quinazolin-4(3H)-one hybrids.     

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.     

Studies on the biosynthesis of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) from 3-hydroxyindolin-2-one in Zea mays

The ring expansion of 3-hydroxyindolin-2-one to 2-hydroxy-1,4-benzoxazin-3-one (HBOA) was investigated by labelling experiments. Action of the cytochrome P450 enzyme BX4 from maize on 3-hydroxyindolin-2-one under an 18O2 atmosphere induced production of 2-hydroxy-1,4-benzoxazin-3-one in which the ring oxygen--but not the 2-hydroxy group of HBOA--is labelled. A mechanism for this transformation is proposed.     

Synthesis of novel 1,4-benzoxazin-3-one derivatives as inhibitors against tyrosine kinases

We designed and synthesized a novel 1,4-benzoxazin-3-one derivative 4 which would have inhibitory activities against tyrosine kinases. They could be synthesized easily from various carboxylic acids 10 and commercially available amines using TFP resin without purification. In this article, we will report the design and synthesis of a novel 1,4-benzoxazin-3-one chemical library 4 and the inhibitory activities against KDR and ABL which are closely related to chronic diseases such as cancer.     

Transformation of maize by 2,4-dihydroxy-7-methoxy-2H-1,4-benzo- xazin-3(4H)-one resistant Agrobacterium strains

One of the important factors responsible for recalcitrance of maize tissue towards Agrobacterium-mediated transformation is the presence of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), an inhibitory metabolite found in maize cells. DIMBOA-resistant strains of Agrobacterium tumefaciens were used to transfer genes coding for GUS (-glucuronidase) and NPTII (neomycine phosphotransferase II) in maize shoot apical meristems derived from 20 day-old seedlings and immature embryos. GUS expression was higher (21–34%) in the apical meristem and was dependent on the type of infecting strain and explant-age. The PCR analysis of selected tissues confirmed the presence of GUS gene in the transformed cells.     

Induced accumulation of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) in maize leaves

Accumulation of 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (HDMBOA-Glc) was induced in maize leaves by treatment with CuCl2, chitopentaose, penta-N-acetylchitopentaose, or jasmonic acid (JA). The accumulation of HDMBOA-Glc was accompanied by a decrease in level of 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (DIMBOA-Glc). When the leaf segments were treated with JA in the presence of [Me-2H3]L-methionine, the label was efficiently incorporated into HDMBOA-Glc, while no incorporation into DIMBOA-Glc or HMBOA-Glc was detected, suggesting the conversion of constitutive DIMBOA-Glc to HDMBOA-Glc by methylation at the 4-position. Levels of endogenous JA and its leucine conjugate transiently increased prior to the accumulation of HDMBOA-Glc in leaf segments treated with CuCl2 and chitopentaose. The lipoxygenase inhibitor ibuprofen suppressed the accumulation of HDMBOA-Glc induced by CuCl2 treatment, and the reduced accumulation of HDMBOA-Glc was recovered by addition of JA. These findings suggested that JA functions as a signal transducer in the induction of HDMBOA-Glc accumulation.     

Determination of 2,4-dihydroxy-1,4(2h)-benzoxazin-3-one glucosides in corn (Zea mays L.)

A method has been developed for the determination of 2,4-dihydroxy-1,4(2H)-benzoxazin-3-ones, which occur in corn (Zea mays L.) plants as glucosides. The method involves freezing and thawing of corn tissue samples to allow enzyme-catalyzed cleavage of the glucosides, fragmentation, extraction of the released aglycons, removal of chlorophyll, conversion of the 2,4-dihydroxy-1,4(2H)-benzoxazin-3-ones to the more stable 2(3)-benzoxazolinones, extraction of the 2(3)-benzoxazolinones, and quantitative ir measurement of the 2(3)-benzoxazolinones in methylene chloride solution. The amount of 2(3)-benzoxazolinone calculated as 6-methoxybenzoxazolinone (MBOA) obtained from B37 × B14 single-cross corn was found to be $\text{1.56 mg}\text{50 g}$ fresh weight (10 samples; SD 0.16 mg). For synthetic samples, the precision of the method is that of the reproducibility of the spectrophotometer used. Depending upon the spectrophotometer used, a detection limit of $\text{0.03–0.06 mg MBOA}\text{50 g}$ fresh tissue was observed.     

6-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors

Investigation of a series 6-[2-(4-aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones has led to the discovery of potent 5-HT(1A/1B/1D) receptor antagonists with and without additional SerT affinity. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.     

Synthesis of novel 3-(1-(1-substituted piperidin-4-yl)-1H-1,2,3-triazol-4-yl)-1,2,4-oxadiazol-5(4H)-one as antifungal agents

A novel series of 1,2,3 triazole compounds possessing 1,2,4 oxadiazole ring were efficiently synthesized. Synthesized compounds were evaluated for their in vitro antifungal activities using standard cup plate method. SAR for the series has been developed by comparing their MIC values with miconazole and fluconazole. Compound 11a from the series was more potent than miconazole against Candida albicans (MIC-20) and Aspergillus flavus (MIC-10) whereas equipotent with miconazole against Fusarium oxysporum (MIC-25) and Aspergillus niger (MIC-12.5). Also compound 11h was more potent than miconazole against Candida albicans (MIC-20) and Aspergillus niger (MIC-10) and equipotent with miconazole against Fusarium oxysporum. Compound 11h was equipotent with fluconazole against Aspergillus niger (MIC-10).     

2-(4-Fluorophenyl)-quinazolin-4(3H)-one as a novel tyrosinase inhibitor: Synthesis,inhibitory activity,and mechanism

2-(4-Fluorophenyl)-quinazolin-4(3H)-one (FQ) was synthesized, and its structure was identified with ¹H nuclear magnetic resonance (¹H NMR), ¹³C nuclear magnetic resonance (¹³C NMR), fourier transform infrared spectroscopy (FTIR), and high resolution mass spectrometry (HRMS). From the enzyme analysis, the results showed that it could inhibit the diphenolase activity of tyrosinase (IC₅₀ = 120 ± 2 μM). Furthermore, the results of kinetic studies showed that the compound was a reversible mixed-type inhibitor, and that the inhibition constants were determined to be 703.2 (K_I) and 222.1 μM (K_IS). The results of fluorescence quenching experiment showed that the compound could interact with tyrosinase and the substrates (tyrosine and l-DOPA). Molecular docking analysis revealed that the mass transfer rate was affected by FQ blocking the enzyme catalytic center. In brief, current study identified a novel tyrosinase inhibitor which deserved further study for hyperpigmentation drugs.     

Degradation of diazinon by 2,4-dihydroxy-7-methoxy-2h-1, 4-benzoxazin-3(4h)-one in maize

A cyclic hydroxamate, 2,4-dihydroxy-7-methoxy-2H- 1,4-benzoxazin-3(4H)-one (DIMBOA), was isolated and identified from shoots of 6-day-old corn seedlings grown in the dark. From 100 g of plant tissue 100 mg of DIMBOA were isolated. This hydroxamate was very effective in catalysing the hydrolysis of the pyrimidinyl organophosphate insecticide, diazinon (O, O-diethyl- O-[6- methyl-2-(1-methylethyl)-4-pyrimidinyl] phosphorothioate) to 6- methyl-2-(1-methylethyl)-4-hydroxypyrimidine and diethyl phosphorothioic acid. The optimum pH for hydrolytic activity was 5 and at pH values equal to or higher than the pK_a of the hydroxamic group (6.95) most of the activity was lost.     

2-Phenylquinazolin-4(3H)-one,a class of potent PDE5 inhibitors with high selectivity versus PDE6

In our efforts to minimize the side effects associated with low selectivity against the other PDE isozymes, a novel class of 2-phenylquinazolin-4(3H)-one derivatives were designed and prepared as potent PDE5 inhibitors with high selectivity against PDE6. The syntheses and SAR studies of such molecules were reported.     

Synthesis and anti-HIV activity of 1,3,4,5-tetrahydro-2H-1,4-benzodiazepin-2-one (TBO) derivatives. Truncated 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2( 1H)-on es (TIBO) analogues

4,5,6,7-Tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)-ones (TIBO), 1, have been shown to significantly inhibit HIV-1 replication, as reported in detail in our prior publications. Since our earlier reports, we have modified the TIBO structures 1 by removing the 5-membered ring of 1, generating 1,3,4,5-tetrahydro-2H-1,4-benzodiazepin-2-ones (TBO), 4, a bicyclic series of compounds. Although compounds 4 possess modest activity when compared to TIBO analogues 1, they clearly demonstrated significant anti-HIV-1 activity.     

Hydroxylated 2-amino-3H-phenoxazin-3-one derivatives as products of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) biotransformation by Chaetosphaeria sp., an endophytic fungus from Aphelandra tetragona

The biotransformation of the phytoanticipin HBOA and its major degradation metabolites 2-hydroxy-N-(2-hydroxyphenyl)acetamide (7) and N-(2-hydroxyphenyl)acetamide (8) by Chaetosphaeria sp., an endophytic fungus isolated from Aphelandra tetragona, was studied. Three new metabolites could be identified as 2-amino-7-hydroxy-3H-phenoxazin-3-one (12), 2-acetylamino-7-hydroxy-3H-phenoxazin-3-one (13) and 7-hydroxy-2-(2-hydroxyacetyl)-amino-3H-phenoxazin-3-one (14). Structure elucidation of 12 and 13 was performed by MS, 1H, 13C NMR and 2D NMR techniques and confirmed by chemical transformation.     

3-(1H-imidazol-4-yl)propyl]guanidines containing furoxan moieties: a new class of H3-antagonists endowed with NO-donor properties

Synthesis and pharmacological characterisation of a series of products obtained by coupling the H(3)-antagonist SKF 91486 through appropriate spacers with the NO-donor 3-phenylfuroxan-4-yloxy and 3-benzenesulfonylfuroxan-4-yloxy moieties, as well as with the corresponding furazan substructures, devoid of NO-donating properties, are reported. All the products were tested for their H(3)-antagonistic and H(2)-agonistic properties on electrically-stimulated guinea-pig ileum segments and guinea-pig papillary muscle, respectively. The whole series of compounds displayed good H(3)-antagonist behaviour and feeble partial H(2)-agonist activity. Among furoxan derivatives, the benzenesulfonyl hybrid 28, a good NO-donor, triggered a dual NO-dependent muscle relaxation and H(3)-antagonistic effect on guinea-pig intestine.     

1,4-benzoxazin-3-one, 2-benzoxazolinone and gallic acid from Calceolaria thyrsiflora Graham and their antibacterial activity

Secondary metabolites, DIBOA, HBOA, 7-OH-HBOA, BOA and gallic acid, were isolated and quantified from Calceolaria thyrsiflora Graham, a native medicinal plant of Chile belonging to the Scrophulariaceae family. The highest DIBOA contents were determined in leaves (145 mmol kg(-1) dry wt) and flowers (161 mmol kg(-1) dry wt). Antibacterial activities of DIBOA, HBOA, BOA, gallic acid and infusions of flowers and leaves were determined. The phytomedicinal properties attributed to C. thyrsiflora Graham could be understood on the basis of its antibacterial activity.     

The aglycone specificity-determining sites are different in 2, 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (Maize beta -glucosidase) and dhurrinase (Sorghum beta -glucosidase)

The maize beta-glucosidase isozyme Glu1 hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc (2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-on e), whereas the sorghum beta-glucosidase isozyme Dhr1 hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-d-glucose). To study the mechanism of substrate specificity further, eight chimeric beta-glucosidases were constructed by replacing peptide sequences within the C-terminal region of Glu1 with the homologous peptide sequences of Dhr1 or vice versa, where the two enzymes differ by 4 to 22 amino acid substitutions, depending on the length of the swapped regions. Five Glu1/Dhr1 chimeras hydrolyzed substrates that are hydrolyzed by both parental enzymes, including dhurrin, which is not hydrolyzed by Glu1. In contrast, three Dhr1/Glu1 chimeras hydrolyzed only dhurrin but with lower catalytic efficiency than Dhr1. Additional domain-swapping within the C-terminal domain of Glu1 showed that replacing the peptide (466)FAGFTERY(473) of Glu1 with the homologous peptide (462)SSGYTERF(469) of Dhr1 or replacing the peptide (481)NNNCTRYMKE(490) in Glu1 with the homologous peptide (477)ENGCERTMKR(486) of Dhr1 was sufficient to confer to Glu1 the ability to hydrolyze dhurrin. Data from various reciprocal chimeras, sequence comparisons, and homology modeling suggest that the Dhr1-specific Ser-462-Ser-463 and Phe-469 play a key role in dhurrin hydrolysis. Similar data suggest that DIMBOAGlc hydrolysis determinants are not located within the extreme 47-amino acid-long C-terminal domain of Glu1.