Antitumor and antimicrobial activities of some hetero aromatic benzofurans derived from naturally occurring visnagin.

Bromination of visnaginone (1) yielded the dibromo derivative (2), which upon methylation with methyl iodide gave 1-(2,7-dibromo-4,6-dimethoxybenzofuran-5-yl) ethanone (3). Compound (3) reacted with dimethylformamide dimethylacetal to give (4). The reaction of (3) with aromatic aldehydes namely (vanillin, benzaldehyde and 3-anisaldehyde) in ammonium acetate, malononitrile and/or butyric cyanoanhydride gave the 2-amino substituted nicotinonitriles (5a-c) and the 2-hydroxyl substituted nicotinonitriles (7a-c), respectively, while in piperidine gave (E)-1-(2,7-dibromo-4,6-dimethoxybenzofuran-5-yl)-3-(substituted)prop-2-en-l-one (11a-c). (5a) was hydrolyzed with sulfuric acid on cold to give the nicotinic acid derivative (6a). When compound (3) reacted with hydrazines and aromatic amines, it gave the Schiff bases (8a,b) and (10a,b), respectively. (8b) reacted with thioglycolic acid to give the thiazolidin-4-one (9b). When (11a-c) reacted with thiourea, it gave the pyrimidine derivatives (12a-c). (11a,b) also reacted with butyric cyanoanhydride and hydroxylamine hydrochloride to give (13a,b) and (15a,b), respectively. When the carboxylate (13a) was treated with 2,4-dinitroaniline, it gave the carboxamide (14a). Compounds (11b,c) reacted with hydrazine derivatives (hydrazine hydrate and phenylhydrazine) yielding the substituted pyrazole derivatives (16b,c) and (17b,c), respectively. All the structures of the synthesized compounds were elucidated by elemental analyses and spectral data. The newly synthesized benzofuran compounds showed a strong to moderate cytotoxicity against liver HEPG2 cancer cell line compared to 5-fluorouracil and doxorubicin (the anticancer agents). Compounds (2, 6a, 13a, 14a, 16c and 17b) were the most active compounds in descending order. The synthesized compounds were also tested for their antimicrobial activity. Compound (10b) showed the highest activity against all the tested strains followed by 6, 10a, 5a, 8b and 7a in descending order.

Prevention of renal crystal deposition by an extract of Ammi visnaga L. and its constituents khellin and visnagin in hyperoxaluric rats.

In Egypt, teas prepared from the fruits of Ammi visnaga L. (syn. "Khella") are traditionally used by patients with urolithiasis. The aim of this study was to evaluate whether oral administration of an aqueous extract prepared from the fruits of A. visnaga as well as two major constituents khellin and visnagin could prevent crystal deposition in stone-forming rats. Hyperoxaluria was induced in male Sprague-Dawley rats by giving 0.75% ethylene glycol and 1% ammonium chloride via the drinking water. The Khella extract (KE; 125, 250 or 500 mg/kg) was orally administered for 14 days. The histopathological examination of the kidneys revealed that KE significantly reduced the incidence of calcium oxalate (CaOx) crystal deposition. In addition, KE significantly increased urinary excretion of citrate along with a decrease of oxalate excretion. Comparable to the extract, khellin and visnagin significantly reduced the incidence of CaOx deposition in the kidneys. However, both compounds did not affect urinary citrate or oxalate excretion indicating a mechanism of action that differs from that of the extract. For KE, a reasonably good correlation was observed between the incidence of crystal deposition, the increase in citrate excretion and urine pH suggesting a mechanisms that may interfere with citrate reabsorption. In conclusion, our data suggest that KE and its compounds, khellin and visnagin, may be beneficial in the management of kidney stone disease caused by hyperoxaluria but that it is likely that different mechanism of action are involved in mediating these effects.

Synthesis of new visnagen and khellin furochromone pyrimidine derivatives and their anti-inflammatory and analgesic activity.

6-[(4-Methoxy/4,9-dimethoxy)-7-methylfurochromen-5-ylideneamino]-2-thioxo-2,3-dihydropyrimidin-4-ones 1a,b were prepared by reaction of 6-amino-2-thiouracil with visnagen or khellin, respectively. Reaction of 1a,b with methyl iodide afforded furochromenylideneaminomethylsulfanylpyrimidin-4-ones 2a,b. Compounds 2a,b were reacted with secondary aliphatic amines to give the corresponding furochromen-ylideneamino-2-substituted pyrimidin-4-ones 3a-d. Reaction of 3a-d with phosphorus oxychloride yielded 6-chlorofurochromenylidenepyrimidinamines 4a-d, which were reacted with secondary amines to afford furochromenylideneamino-2,6-disubstituted pyrimidin-4-ones 5a-d. In addition, reaction of 5a-d with 3-chloropentane-2,4-dione gave 3-chloro-furochromenylpyrimidopyrimidines 6a-d. The latter were reacted with piperazine and morpholine to give 1-(furochromenyl)-pyrimidopyrimidine-3,6,8-triylpiperazines or -3,6,8-triylmorpholines 7a-d. The chemical structures of the newly synthesized compound ware characterized by IR, ¹H-NMR, ¹³C-NMR and mass spectral analysis. These compounds were also screened for their analgesic and anti-inflammatory activities. Some of them, particularly 3-7, exhibited promising activities.

Synthesis of 6- and 9-alkylaminomethyl furoflavones as gastroprotective agents.

The synthesis of 9- and 6-alkylaminomethyl furoflavones 5a, b, 9a-c, 13a, b, 15a-g and 18 from the naturally occurring chromones visnagin and khellin. Gastroprotective potency of these compounds in the ethanol damage model was determined. The results indicate that, through appropriate substitution, furoflavones can be obtained that are gastroprotective.

Validated HPLC method for simultaneous estimation of khellol glucoside, khellin and visnagin in Ammi visnaga L. fruits and pharmaceutical preparations.

Tea bags including fruits of Ammi visnaga L. are used in Egypt as remedy for the treatment of kidney stones. Our study focuses on developing simple and rapid method utilising HPLC for quantitative estimation of khellol glucoside (KG), khellin (KH) and visnagin (VS) simultaneously. Their concentrations were determined in A. visnaga L. fruits at different developmental stages and in pharmaceutical formulations together with following up them during shelf life. Separation was accomplished using HPLC. Perfect resolution between KG, KH and VS was possible through using a mobile phase consisting of water:methanol:tetrahydrofuran (50:45:5, v/v/v). Peaks were detected at 245 nm. The suggested method for the determination of KG, KH and VS was successful in determining the analytes of interest without any interference of other compounds and matrix. All validation parameters were satisfactory and the procedure was relatively easy and fast as extracts are evaluated without previous steps of purification.

Active oxygen forms in photoreaction between DNA and furanochromones khellin and visnagin.

The two furanochromones khellin and visnagin react with DNA under irradiation by 365 nm light, forming photoadducts. Recently, the use of khellin as therapeutic agent for skin diseases has been proposed. It is well known that during the formation of photoadducts toxic active oxygen forms are produced. We studied therefore the behaviour of the two furanochromones as producers of 1O-2 and O-2. Our results indicate that visnagin is a strong generator of both superoxide radicals and singlet oxygen, while khellin does not exhibit strong production of OO-2, which is promptly quenched by superoxide dismutase.

Plant photosensitizers with antiviral properties.

Many antiviral compounds obtained from plants are photosensitizers, i.e., their biological properties are dependent upon or augmented by light of specific wavelengths, commonly long wave ultraviolet, UVA. Three groups of chemically distinct plant photosensitizers have been investigated in some detail in regard to antiviral properties. These are (a) thiophenes and polyacetylenes; (b) furyl compounds; (c) certain alkaloids. Some of the thiophenes and their acetylenic derivatives possess extremely potent phototoxic activities toward membrane-containing viruses. These activities are markedly affected by the chemical structures of these compounds. Inactivated virus retains its integrity, however, and penetrates cells, but does not replicate. Their mechanism of action is believed to occur via singlet-oxygen damage to the membranes, although other targets cannot be ruled out. In contrast, the antiviral activities of plant furyl compounds (such as psoralens and furanochromones) appear to depend on UVA-mediated covalent adduct formation with the viral genomes. Some of the photoactive beta-carboline alkaloids also have impressive antiviral activities, especially against viruses with single-stranded genomes. These and other types of alkaloids appear to work by mechanisms that do not require covalent bonding to nucleic acids, and may also involve other target molecules as well. Some of these compounds have potent antiviral activities at concentrations well below cytotoxic levels, and accordingly should be tested in animal models.

Vasodilator effects of visnagin in isolated rat vascular smooth muscle.

Visnagin (4-methoxy-7-methyl-5H-furo [3,2-g][1]-benzopyran-5-one) is an active principle of the fruit of Ammi visnaga, a plant traditionally used in cardiovascular disorders. We have studied its vasodilator effects in rat vascular smooth muscle. The results demonstrated that visnagin inhibited the contractile responses induced in rat aortic rings by: (a) KCl or increases of extracellullar Ca2+ in KCl depolarized aortic rings, its effects being more potent against low (20 mM) than high (80 mM) KCl-induced contractions, (b) noradrenaline in Ca(2+)-containing solution and less effectively those in Ca(2+)-free solution and (c) phorbol 12-myristate 13-acetate (PMA) in a Ca(2+)-containing and with a lower potency in Ca(2+)-free medium. The relaxation induced by visnagin in aorta precontracted with noradrenaline was not affected by endothelium removal. Additionally, visnagin inhibited the spontaneous myogenic contractions of portal veins. The results showed that visnagin inhibited vascular smooth muscle contractility by acting at multiple sites. In the range of 10(-6) M to 5 x 10(-5) M visnagin appears to inhibit only the contractions mediated by Ca2+ entry through pathways with low sensitivity to classical Ca(2+)-entry blockers, i.e. agonist-, PMA- or mild depolarization-induced Ca2+ entry. Therefore, the vasodilator profile of visnagin, is not that of typical Ca(2+)-entry blockers which preferentially inhibit the contractions induced by strong depolarizations. At higher concentrations (> 5 x 10(-5) M) visnagin causes non-specific inhibition of vascular smooth muscle contractility.

The mechanisms of interaction between furanochromones and DNA. A heteronuclear Overhauser effect study on the khellin-thymidine model system.

The furanochromones khellin and visnagin have been characterised by 1H and 13C mono- and bidimensional NMR spectroscopies. Their strong affinity with DNA was experimentally confirmed by the complete disappearance of the furano-chromones' NMR signals upon additions of DNA. An intermolecular interaction between furanochromones and the thymidyl moieties of DNA, stabilized by the formation of a hydrogen bond between the thymidyl NH hydrogen and the C = O group of khellin or visnagin, is here proposed. This is suggested by the strong donor-acceptor behavior of these two molecular moieties, as pointed out by a selective 1H-13C Overhauser effect study of the khellin-thymidine model system.

Furanochromone radical cations: generation, characterization and interaction with DNA.

The radical cations of naturally occurring furanochromones visnagin (VI) and khellin (KH) have been generated and identified for the first time by use of laser flash photolysis and pulse radiolysis techniques. The lifetimes of VI(.+) and KH(.+) are determined as approximately 6 and approximately 35 micros under these conditions, respectively. Direct 308-nm excitation of VI in aqueous buffer at physiological pH results in monophotonic photoionization to generate VI(.+), with a quantum yield of 0.075, which is much higher than that of 8-methoxypsoralen and KH under identical conditions. Though VI(.+) is a more powerful oxidant than KH(.+), both of them react with guanosine mononucleotide (k=1.2x10(9) and 3.8x10(7) dm(3) mol(-1) s(-1), respectively) via electron transfer to give the guanine radical cation. Furthermore, selective oxidation of guanine in single and double strand DNA by VI(.+) was also observed. These novel findings suggest that electron transfer reactions involving furanochromone radical cations may be of considerable importance in furanochromone photochemotherapy.

Photophysical properties and photobiological activity of the furanochromones visnagin and khellin.

The larger photobiological activity of visnagin (VI) versus khellin (KH) toward several living organisms, including fungi, viruses, yeasts and bacteria, induced a detailed investigation of the photophysical properties of these naturally occurring furanochromones, using laser-flash-photolysis, photoacoustic calorimetry and fluorescence (steady-state and time-resolved) techniques in solvents with different polarity and content of water, including micelles and vesicles. The results have shown that the magnitude of all the three rate constants out of S1 (radiative, kf; internal conversion, kic and intersystem crossing, kisc) for VI and KH strongly depend on the solvent, namely on its hydrogen bonding ability and polarity. The changes of kf and kisc are due to the solvent-assisted mixing and/or inversion of the two first singlet excited states (1n, pi and 1 pi, pi), while kic increases with a decrease of the S0-S1 energy gap. As a consequence, the quantum yield of triplet formation (phi T) strongly decreases from values of approximately 0.8 in dioxane to < 0.05 in water for both compounds. The magnitude of solvent polarity/hydrogen bonding ability required, at which the state order is inverted and phi T starts to decrease, is greater for VI than for KH and consequently phi T (VI) >> phi T (KH) over a broad range of water content including that appropriate to the environment of the compounds in a living system. These facts account for the larger photobiological activity of VI with respect to KH, regarding both the fungus Fusarium culmorum L. and the wild strain of Escherichia coli, studied by us.

Determination of khellin and visnagin in Ammi visnaga fruits by capillary electrophoresis.

A new, simple and rapid capillary electrophoresis method was developed for the identification and quantitative determination of two medically active constituents-khellin and visnagin-in the extracts of Ammi visnaga fruits. Micellar electrochromatographic separation of khellin and visnagin was carried out using 10 mmol/l borate, 50 mmol/l sodium dodecylsulfate, 25% (v/v) acetonitrile at pH 9 as running buffer. Ammi visnaga fruits were extracted with methanol and the extracts were directly injected without any purification and pre-separation processes. Coumarin was used as internal standard for quantitation and the limits of detection for khellin and visnagin were 2.36 and 1.97 microg/ml, respectively using UV detection at 245 nm.

Determination of furanochromones and pyranocoumarins in drugs and Ammi visnaga fruits by combined solid-phase extraction-high-performance liquid chromatography and thin-layer chromatography-high-performance liquid chromatography.

A new, simple and rapid solid-phase extraction method for the determination of furanochromones and pyranocoumarins in Ammi visnaga L. fruits and pharmaceuticals by reversed-phase high-performance liquid chromatography (RP-HPLC) was developed. The isolation of compounds examined was carried out on octadecyl BakerBond SPE columns using various concentrations of methanol, acetonitrile and tetrahydrofuran in water. High and reproducible recoveries were obtained. To compare the results of quantitative analysis a preparative TLC procedure was also elaborated and carried out.

Spectrophotometric determinations of 3-dimethylaminomethylkhellin hydrochloride and khellin.

Spectrophotometric assays are proposed for the determination of 3-dimethylaminomethylkhellin hydrochloride and khellin in bulk chemical and dosage forms. The acid dye method, using methyl orange at pH5, is applied to assay the amine in the form of an ion-pair extractable in chloroform with maximum abosrbance at 420 nm. The perchloric acid method, depending on formation and extraction of the oxonium salts of both compounds, is used to assay the amine and khellin at 333 or 430 nm and at 325 or 410 nm, respectively. The reineckate method can be used to assay the amine as the reineckate derivative in acetone with maximum absorbance at 530 nm. However, small amounts of the amine (1.5--3 mg) can be determined as the reineckate in methanol with maximum absorbance at 245 nm. Stability determination of the two compounds can be done by the acid dye and perchloric acid methods. The three methods are sufficiently accurate, sensitive, and precise.

Improved high-performance liquid chromatographic determination of khellin and visnagin in Ammi visnaga fruits and pharmaceutical formulations.

An improved, simple, selective, and sensitive reversed-phase high-performance liquid chromatographic (HPLC) assay for khellin and visnagin in Ammi visnaga L. fruits was developed by using an internal standardized technique. The HPLC column was a reversed-phase microBondapack C18 column, the mobile phase was water: methanol:acetonitrile (49:49:2), and the flow rate was 1.5 mL/min. Khellin and visnagin were detected and analyzed with a spectrophotometer set at 250 nm. Results of the HPLC analysis indicate a relative standard deviation of less than 0.04%. The analytical procedure was used for the quantification of khellin in various pharmaceutical dosage forms, such as ampules, tablets, and suppositories, with relative standard deviations of 1.2, 1.4, and 1.7%, respectively. As little as 10 ng of khellin or visnagin could be detected accurately in less than 13 min.

Photosensitized cleavage and cross-linking of pBR322 DNA with khellin and visnagin.

The naturally occurring furanochromones khellin and visnagin have received considerable attention, largely because of their vasodilatory properties and of their ability (particularly that of khellin) to induce skin pigmentation upon ultraviolet light treatment of patients suffering from vitiligo. There are conflicting statements in the literature on whether or not they are capable of cross-linking DNA photochemically. Supercoiled and linear pBR322 DNA was used to probe this reaction. The results showed that both khellin and, to a greater extent, visnagin photosensitized DNA cross-linking. In addition, both photosensitizers induced extensive DNA cleavage.

Photosensitization of DNA of defined sequence by furochromones, khellin and visnagin.

The sequence specificity in the in vitro DNA photobinding of khellin and visnagin, two naturally occurring furochromones proposed for chemotherapy of vitiligo, was investigated by using DNA sequencing methodology. The 3'-5' exonuclease associated with the T4 DNA polymerase served as a tool for determining photoadducts distribution on DNA fragments of the lac I gene of Escherichia coli. The photoadduct distribution of psoralen is also studied for comparison. Upon UVA irradiation, visnagin mainly forms monoadducts with thymine and to a lower extent with cytosine. Alternating (A-T)n sequences are hot spots for visnagin photoaddition. This is a property shared with furocoumarins. TTT sites are also quite reactive to visnagin, as they are to methylated angelicins. In contrast, with psoralen derivatives, there is no preferential photobinding in 5'-TpA sites, and 5'-ApT sites react as well. Furthermore, many sites such as T in the GC context, and C in any context, react, although weakly. The visnagin photoadduct distribution resembles very much the photoadduct distribution of methylated angelicins as described by Miolo et al. The photoreaction of these two series of compounds is less sequence dependent than the photobinding of psoralen derivatives as described by Sage and Moustacchi and by Boyer et al. The sequence specificity in khellin-DNA photobinding is the same as for visnagin, even though it forms much fewer photoadducts. The absence of photo-oxidation of DNA after treatment with visnagin or khellin plus UVA suggests that furochromones do not present any photodynamic effect on DNA.

High performance liquid chromatography analysis of the furanochromones khellin and visnagin in various organs of Ammi visnaga (L.) Lam. at different developmental stages.

The content of the furanochromones khellin and visnagin, in the organs of Ammi visnaga (L.) Lam. at different developmental stages, has been examined. Determinations have been performed by means of a high performance liquid chromatography (HPLC) technique which allows both the separation and the quantitative determination of these chromones. Unripe fruits are the richest in both chromones, but the collection of ripe dry fruits--as it occurs in Egyptian folk-medicine--seems more reasonable because they might not undergo degradation processes during desiccation and storage.

Photosynthesis of furocoumarin- and furochromone-types potential intercalative alkylating and oxidizing agents of DNA through photooxidations using gamma-ray.

The photooxygenation of imperatorin (1a) under gamma-ray irradiation afforded the hydroperoxides 2a and 3a. Similarly, the photooxygenation of alloimperatorin (1b) gave the hydroperoxide (2b). Visnagin (1c) was also photooxygenated to give the hydroperoxide (2c) as sole product. On the other hand, the photooxygenation of khellin (1d) gave the endoperoxide (2d) as a sole product. The epoxidation of imperatorin (1a) using hydrogen peroxide under gamma-ray irradiation afforded the epoxide 5a. Similarly visnagin (1c) and khellin (1d) were epoxidized to give the epoxides 5c and 5d.

Nonlinear pharmacokinetics of visnagin in rats after intravenous bolus administration.

Ammi visnaga L. (syn. Khella, Apiaceae) preparations have traditionally been used in the Middle East for the treatment of kidney stone disease. Visnagin, a furanocoumarin derivative, is one of the main compounds of Ammi visnaga with potential effects on kidney stone prevention. To date, no information is available about the pharmacokinetic (PK) properties of visnagin. It was the aim of the study to characterize the PK properties of visnagin after intravenous (i.v.) bolus administration in rats and to develop an adequate model for the description of the observed data, including model parameter estimates. Therefore, three doses of visnagin (1.25, 2.5, and 5mg/kg) solubilized in 25% Captisol® were administered by i.v. bolus injection to male Sprague-Dawley rats. Plasma samples were extracted and subsequently analyzed using a validated LC-MS/MS method. Both non-compartmental and compartmental PK analyses were performed. A stepwise model building approach was applied including nonlinear mixed effect modeling for final model selection and to obtain final model estimates in NONMEM VI. The average areas under the curve (AUC(0-last)) after doses of 1.25, 2.5, and 5mg/kg were 1.03, 3.61, and 12.6 mg *h/l, respectively. The shape of the plasma concentration-time profiles and the observed disproportionate increase in AUC(0-last) with increasing dose suggested nonlinearity in the elimination of visnagin. A two-compartment Michaelis-Menten model provided the best fit with following typical values of the parameter estimates: 2.09 mg/(l*h) (V(max)), 0.08 mg/l (K(M)), 0.175 l (V(C)), 1.0 h⁻¹ (k12), and 1.22 h⁻¹ (k21). Associated inter-subject variability estimates (% CV) for V(max), K(M) and V(C) were 21.8, 70.9, and 9.2, respectively. Intra-subject variability (constant CV error model) was estimated to be 7.0%. The results suggest the involvement of a saturable process in the elimination of visnagin, possibly an enzyme or transporter system.