Trapping phosphodiester-quinone methide adducts through in situ lactonization

The goal of in situ modification of DNA via phosphodiester alkylation has led to our design of quinone methide derivatives capable of alkylating dialkyl phosphates. A series of catechol derivatives were investigated to trap the phosphodiester-quinone methide alkylation adduct through in situ lactonization. The catechol derivatives were uniquely capable of characterizable p-quinone methide formation for mechanistic clarity. These investigations revealed that with a highly reactive lactonization group (phenyl ester), lactonization competed with quinone methide formation. Lactone-forming groups of lower reactivity (methyl ester, n-propyl ester, and dimethyl amide) allowed quinone methide formation followed by phosphodiester alkylation; however, they were ineffective at in situ lactonization to drain the phosphodiester alkylation equilibrium to the desired phosphotriester product. The derivatives tethered with lactone-forming functionality of intermediate reactivity (chloro-, trichloro-, and trifluoroethyl esters), allowed quinone methide formation, phosphodiester alkylation, and in situ lactonization to efficiently afford the trapped phosphotriester adduct.     

Improved alkylation and product stability in phosphotriester formation through quinone methide reactions with dialkyl phosphates

Investigating reactions of functionalized p-quinone methides continues to advance our design of a reagent being developed for controlled, in situ modification of DNA via phosphodiester alkylation. Previously reported investigations of p-quinone methides derived from catechols allowed for trapping of isolable trialkyl phosphates for characterization and mechanistic information. However, lactone formation with these derivatives required long reaction times, resulting in an unfavorable mixture of trialkyl phosphate and hydrolysis products. To enhance the rate and efficacy of trialkyl phosphate formation and trapping, a phenol derived p-quinone methide has been designed to enforce a conformation favoring lactonization of the dialkyl phosphate alkylated intermediate. The relative rates of phosphodiester alkylation and subsequent trapping of the phosphotriester adduct have been examined by UV and (1)H NMR analysis for p-quinone methide precursor 1 and the corresponding control, 1'. The incorporation of a methyl group at the meta-position of 1 (relative to 1') significantly improves the rate of lactionization to provide a much higher yield of the desired product, lactonized phosphotriester 5. The control reaction with 1' afforded only a minor amount of the corresponding lactonized trialkyl phosphate 5'.     

Alkylation of guanosine and 2'-deoxyguanosine by o-quinone alpha-(p-anisyl)methide in aqueous solution

Rates of alkylation of guanosine and 2'-deoxyguanosine with o-quinone alpha-(p-anisyl)methide were measured by flash photolysis in a series of aqueous sodium hydroxide solutions and bicarbonate ion, t-butylhydrogenphosphonate ion, and biphosphate ion buffers. The data so obtained provide rate profiles for these nucleoside plus quinone methide reactions over the range pH = 7-14, which furnish guanosine and deoxyguanosine acidity constants consistent with literature information. These profiles also provide rate constants that show the reaction of o-quinone alpha-(p-anisyl)methide with guanosine and deoxyguanosine to be fairly fast processes, considerably faster than the biologically wasteful reaction of the quinone methide with water, which is the ubiquitous medium in biological systems; that makes the quinone methide a potent guonosine and deoxyguanosine alkylator.     

Flash photolytic generation of ortho-quinone methide in aqueous solution and study of its chemistry in that medium

Flash photolysis of o-hydroxybenzyl alcohol, o-hydroxybenzyl p-cyanophenyl ether, and (o-hydroxybenzyl)trimethylammonium iodide in aqueous perchloric acid and sodium hydroxide solutions, and in acetic acid and biphosphate ion buffers, produced o-quinone methide as a short-lived transient species that underwent hydration back to benzyl alcohol in hydrogen-ion catalyzed (k(H+) = 8.4 x 10(5) M(-1) s(-1)) and hydroxide-ion catalyzed (k(HO)- = 3.0 x 10(4) M(-1) s(-1)) reactions as well as an uncatalyzed (k(UC) = 2.6 x 10(2) s(-1)) process. The hydrogen-ion catalyzed reaction gave the solvent isotope effect k(H+)/k(D)+ = 0.42, whose inverse nature indicates that this process occurs by rapid and reversible equilibrium protonation of the carbonyl oxygen atom of the quinone methide, followed by rate-determining capture of the carbocation so produced by water. The magnitude of the rate constant of the uncatalyzed reaction, on the other hand, indicates that this process occurs by simple nucleophilic addition of water to the methylene group of the quinone methide. Decay of the quinone methide is also accelerated by acetic acid buffers through both acid- and base-catalyzed pathways, and quantitative analysis of the reaction products formed in these solutions shows that this acceleration is caused by nucleophilic reactions of acetate ion rather than by acetate ion assisted hydration. Bromide and thiocyanate ions also accelerate decay of the quinone methide through both hydrogen-ion catalyzed and uncatalyzed pathways, and the inverse nature of solvent isotope effects on the hydrogen-ion catalyzed reactions shows that these reactions also occur by rapid equilibrium protonation of the quinone methide carbonyl oxygen followed by rate-determining nucleophilic capture of the ensuing carbocation. Assignment of an encounter-controlled value to the rate constant for the rate-determining step of the thiocyanate reaction leads to pK(a) = -1.7 for the acidity constant of the carbonyl-protonated quinone methide.     

Chromatographic determination of itopride hydrochloride in the presence of its degradation products

Two sensitive and reproducible methods are described for the quantitative determination of itopride hydrochloride (IH) in the presence of its degradation products. The first method is based on HPLC separation on a reversed phase Kromasil column [C18 (5-microm, 25 cm x 4.6 mm, ID)] at ambient temperature using a mobile phase consisting of methanol and water (70:30, v/v) adjusted to pH 4.0 with orthophosphoric acid with UV detection at 258 nm. The flow rate was 1.0 mL per min with an average operating pressure of 180 kg/cm2. The second method is based on HPTLC separation on silica gel 60 F254 using toluene:methanol:chloroform:10% ammonia (5.0:3.0:6.0:0.1, v/v/v/v) as mobile phase at 270 nm. The analysis of variance (ANOVA) and Student's t-test were applied to correlate the results of IH determination in dosage form by means of HPLC and HPTLC methods. The drug was subjected to acid and alkali hydrolysis, oxidation, dry heat, wet heat treatment, UV, and photodegradation. The proposed HPLC method was utilized to investigate the kinetics of the acidic, alkaline, and oxidative degradation processes at different temperatures and the apparent pseudo-first-order rate constant, half-life, and activation energy were calculated. In addition the pH-rate profile of degradation of IH in constant ionic strength buffer solutions in the pH range 2-11 was studied.     

Flash photolytic generation and study of p-quinone methide in aqueous solution. An estimate of rate and equilibrium constants for heterolysis of the carbon-bromine bond in p-hydroxybenzyl bromide

Flash photolysis of p-hydroxybenzyl acetate in aqueous perchloric acid solution and formic acid, acetic acid, biphosphate ion, and tris(hydroxymethyl)methylammonium ion buffers produced p-quinone methide as a short-lived species that underwent hydration to p-hydroxybenzyl alcohol in hydronium ion catalyzed (k(H(+)) = 5.28 x 10(4) M(-1) s(-1)) and uncatalyzed (k(uc) = 3.33 s(-1)) processes. The inverse nature of the solvent isotope effect on the hydronium ion-catalyzed reaction, k(H(+))/k(D(+)) = 0.41, indicates that this process occurs by rapid and reversible protonation of the quinone methide on its carbonyl carbon atom, followed by rate-determining capture of the p-hydroxybenzyl carbocation so produced by water, while the magnitude of the rate constant on the uncatalyzed process indicates that this reaction occurs by simple nucleophilic addition of water to the methylene group of the quinone methide. p-Quinone methide also underwent hydronium ion-catalyzed and uncatalyzed nucleophilic addition reactions with chloride ion, bromide ion, thiocyanate ion, and thiourea. The solvent isotope effects on the hydronium ion-catalyzed processes again indicate that these reactions occurred by preequilibrium mechanisms involving a p-hydroxybenzyl carbocation intermediate, and assignment of a diffusion-controlled value to the rate constant for reaction of this cation with thiocyanate ion led to K(SH) = 110 M as the acidity constant of oxygen-protonated p-quinone methide. In a certain perchloric acid concentration range, the bromide ion reaction became biphasic, and least-squares analysis of the kinetic data using a double-exponential function provided k(Br(-)) = 3.8 x 10(8) M(-1) s(-1) as the rate constant for nucleophilic capture of the p-hydroxybenzyl carbocation by bromide ion, k(ionz) = 8.5 x 10(2) s(-1) for ionization of the carbon-bromine bond of p-hydroxybenzyl bromide, and K = 4.5 x 10(5) M(-1) as the equilibrium constant for the carbocation-bromide ion combination reaction, all in aqueous solution at 25 degrees C. Comparisons are made of the reactivity of p-quinone methide with p-quinone alpha,alpha-bis(trifluoromethyl)methide as well as p-quinone methide with o-quinone methide.     

Stability-indicating validated HPLC method for simultaneous determination of oral antidiabetic drugs from thiazolidinedione and sulfonylurea groups in combined dosage forms

For type 2 diabetes treatment, combinations of drugs from the thiazolidinedione and sulfonylurea groups are now available in the same tablet or capsule. Therefore, a stability-indicating and validated HPLC method was developed for simultaneous determination of pioglitazone, rosiglitazone, and glipizide in combined dosage forms. The examined drugs were subjected to different conditions such as acid and base, temperature, and UV light, and degradation of pioglitazone and glipizide was observed under thermal and acidic stress. However, selectivity of the presented method for pioglitazone, rosiglitazone, and glipizide assay against their degradation products was confirmed. It was also demonstrated to be robust, resisting small deliberate changes in pH of the buffer, flow rate, and percentage of acetonitrile in the mobile phase. The presented method utilizes a LiChrospher RP18 column (125 x 4.0 mm), acetonitrile in phosphate buffer at pH 4.3 (40 + 60, v/v) as the mobile phase, and UV detection at 225 nm for pioglitazonel glipizide or 245 nm for rosiglitazone/glipizide. The method was validated with respect to linearity, precision, and accuracy. Finally, the elaborated procedure was applied for the QC of pioglitazone/glipizide and rosiglitazone/glipizide mixtures.     

Kinetic Study of the Hydrolysis of Schiff Bases Derived from 2-Aminothiophenol

The kinetics of the hydrolysis of Schiff bases derived from 2-aminothiophenol have been studied in aqueous sodium hydroxide media containing 40?% (v/v) methanol in the temperature range 22?C45?°C. The Schiff base molecular structure-hydrolysis reactivity relationship has been investigated and discussed. Suitable reaction mechanisms have been suggested. From the effect of temperature on the rate constant, various activation parameters have been evaluated. The work has been extended to study the hydrolysis mechanism in buffer solutions of pH?=?2?C13 at 22?°C for Schiff base I (H). A rate profile diagram of pH-rate constant has been proposed.     

Ion chromatographic determination of some organic acids, chloride and phosphate in coffee and tea

An ion chromatographic method for the simultaneous determination of organic acids and inorganic ions is described. Acetic, malic, ascorbic, citric, malic and succinic acids, chloride and phosphate were determined in coffee and tea samples. The separation is performed on an anion-exchange column operated at 40 °C within 25 min by an isocratic elution with 0.6 mM aqueous potassium hydrogenphthalate (pH 4.0) solution containing 4% (v/v) acetonitrile as eluent and determination by conductivity detection. The method does not need a special sample treatment and was successfully applied to the analysis of black, green and oolong tea samples. Also, green and roasted coffee samples from the varieties arabica and robusta were analyzed.     

Determination of pKa values of organic bases in aqueous acetonitrile solutions using capillary electrophoresis

Capillary electrophoresis (CE) was used for the determination of ionisation constants (pKa) of a variety of organic bases in aqueous acetonitrile solutions over the range 0-60% (v/v) acetonitrile. These bases are used as test compounds in HPLC column evaluation, thus knowledge of their pKa in hydro-organic solutions is useful. The base pKa decreased with acetonitrile concentration and significant shifts from the aqueous pKa (up to -0.8) were found using 60% acetonitrile. The CE application was confirmed to be very suitable for fast and accurate pKa measurement in aqueous organic solutions.     

Probing potential medium effects on phosphate ester bonds using 18O isotope shifts on 31P NMR

Dipolar aprotic cosolvents, such as DMSO and acetonitrile, accelerate the rates of hydrolysis of phosphate monoester dianions. It has been speculated that the rate acceleration arises from the disruption of hydrogen bonding to the phosphoryl group. An aqueous solvation shell can stabilize the dianionic phosphoryl group by forming hydrogen bonds to the phosphoryl oxygens, whereas solvents such as DMSO are incapable of forming such bonds. It has been proposed that the loss of stabilization could result in a weakened P-OR ester bond, contributing to the observed faster rate of hydrolysis. Computational results support this notion. We have used the 18O-induced perturbation to the 31P chemical shift to ascertain whether solvent changes result in alterations to the P-O(R) bond. We have studied 16O18O-labeled methyl, ethyl, phenyl, p-nitrophenyl, diethyl p-nitrophenyl, triphenyl, and di-tert-butyl ethyl phosphate in the solvents water, methanol, chloroform, acetonitrile, dioxane, and DMSO. The results suggest no significant solvent-induced weakening of the phosphate ester bonds in any of the solvents tested, and this is unlikely to be a significant source for the acceleration of hydrolysis in mixed solvents.     

ANION AND SOLVENT EFFECTS ON THE RATE OF REDUCTION OF TRIPLET EXCITED THIAZINE DYES BY FERROUS IONS

Abstract— The lifetimes of the triplet excited states of thionine and methylene blue were measured in aqueous and 50 v/v% aqueous acetonitrile solutions acidified with 0.01 N sulfuric or trifluoromethyl-sulfonic acid. The rate constants for reaction of the triplet excited dyes with ferrous ions were measured in the same solutions. The triplet lifetimes in the absence of added quenchers were insensitive to a change in acid from trifluoromethylsulfonic to sulfuric or to a change in solvent from water to 50v/v% aqueous acetonitrile (τ for triplet thionine ˜7.5 μs, τ for triplet methylene blue ˜4.5 μs). In contrast, the rate constant for reaction of the triplet dyes with ferrous ions increased by nearly a factor of 10 with a change in acid from trifluoromethylsulfonic to sulfuric. In solutions containing sulfate ions this reaction rate constant increased with increasing sulfate concentration and with a change in solvent from water to 50 v/v% aqueous acetonitrile. The results are discussed in terms of the possibility of association of the positively charged reactive ions with sulfate anions. Quenching of the triplet excited dyes by ferric ions or by ground state dye molecules was shown to be negligible at the concentration used for the ferrous ion quenching study.     

高效液相色谱手性流动相添加剂法分离3种黄酮类化合物对映体

采用反相高效液相色谱法(RP-HPLC),以磺丁基醚-β-环糊精(SBE-β-CD)作为手性流动相添加剂,建立了二氢黄豆苷原(dihydrodaidzein)、雌马酚(equol)和山姜素(alpinetin)3种黄酮类化合物的手性拆分方法。考察了环糊精的种类和浓度、有机相的种类和比例、缓冲盐的种类和浓度以及pH对3种化合物手性拆分效果的影响。结果表明:采用Kromasil 100-5C₁₈(250 mm×4.6 mm, 5 μm)色谱柱,流动相为乙腈-10 mmol/L SBE-β-CD水溶液(含20 mmol/L KH₂PO₄, pH值到4.0)(体积比为20:80)的条件下,二氢黄豆苷原、雌马酚和山姜素的对映体都达到了基线分离,分离度分别为1.8, 1.9和1.4。该方法简便,分离效果好,对黄酮类化合物的拆分具有应用价值。     

High-Performance Liquid Chromatographic Determination of SAZ-VII-23, A Novel Antiarrhythmic Agent,in Dog Plasma and Urine

Abstract

A HPLC method has been developed to determine the concentrations of SAZ-VII-23 (3-benzoyl-7-isopropyl-3,7-diazabicyclo[3.3.1]nonane HClO₄), a novel antiarrhythmic agent, in dog plasma and urine. Plasma treated with acetonitrile and alkalinized urine were extracted with chloroform- propanol (9:1). An aliquot was injected on to HPLC system using a C₆ reversed-phase column and acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8 (28.5:28.5:43 v/v) containing 4.0 mM triethylamine as mobile phase. Detection wavelength was 255 nm. The linear range were 0.04–8 μg/ml, and the lower limit of quantitation was 0.04 μg/ml in plasma and urine, respectively. The method was applied to determine plasma and urine concentrations and preliminary pharmacokinetic profiles of SAZ-VII-23 in a dog.     

Selective Alkylation of C‐Rich Bulge Motifs in Nucleic Acids by Quinone Methide Derivatives

A quinone methide precursor featuring a bis‐cyclen anchoring moiety has been synthesized and its capacity to alkylate oligonucleotide targets quantified in the presence and absence of divalent metal ions (Zn²⁺, Ni²⁺ and Cd²⁺). The oligonucleotides were designed for testing the sequence and secondary structure specificity of the reaction. Gel electrophoretic analysis revealed predominant alkylation of C‐rich bulges, regardless of the presence of divalent metal ions or even the bis‐cyclen anchor. This C‐selectivity appears to be an intrinsic property of the quinone methide electrophile as reflected by its reaction with an equimolar mixture of the 2′‐deoxynucleosides. Only dA‐N1 and dC‐N3 alkylation products were detected initially and only the dC adduct persisted for detection under conditions of the gel electrophoretic analysis.     

反相高效液相色谱法测定阿德福韦酯及其降解产物

建立了一快速、简单地测定阿德福韦酯及其降解产物阿德福韦单特戊酸甲基酯、阿德福韦的反相高效液相色谱方法。以Inertsil CN-3化学键合硅胶为固定相,以乙腈-25 mmol/L磷酸盐缓冲液(pH 4.0)(体积比为33∶67)为流动相,流速1.0 mL/min,检测波长260 nm。阿德福韦酯、阿德福韦的质量浓度分别为1.861~181.7 mg/L和2.018~197.2 mg/L时与峰面积呈良好的线性关系(r分别为0.9999和0.9998);阿德福韦酯及阿德福韦平均加样回收率分别为99.5%~10     

Evaluation of astemizole purity by HPLC

A liquid chromatographic method is described for the evaluation of Astemizole purity. The method is also applicable for the analysis of pharmaceutical dosage forms of Astemizole using indole as internal standard. Separation was achieved with a micro bondapak C(18) column and phosphate buffer (pH 6.0):acetonitrile (50:50 v/v) as eluent, at a flow rate of 1.5 ml/min with UV detection at 280 nm. The method is suitable for estimating the possible impurities if any present in Astemizole up to 0.05% for 1a and 0.25% for 1b. The method is simple, rapid and accurate for the analysis of Astemizole.     

Flash photolytic generation of o-quinone alpha-phenylmethide and o-quinone alpha-(p-anisyl)methide in aqueous solution and investigation of their reactions in that medium. Saturation of acid-catalyzed hydration.

o-quinone alpha-phenylmethide was generated as a short-lived transient species in aqueous solution by flash photolysis of o-hydroxy-alpha-phenylbenzyl alcohol, and its rate of decay was measured in HClO4 and NaOH solutions as well as in CH3CO2H, H2PO4-, and HCO3- buffers. These data show that hydration of this quinone methide back to its benzyl alcohol precursor occurs by acid-, base-, and uncatalyzed routes. The acid-catalyzed reaction gives the solvent isotope effect kH+/kD+ = 0.34, whose inverse nature indicates that this reaction occurs via rapid preequilibrium protonation of the quinone methide on its carbonyl oxygen atom followed by rate-determining capture of the ensuing carbocationic intermediate by water, a conclusion supported by the saturation of acid catalysis in concentrated HClO4 solution. o-quinone alpha-(p-anisyl)methide was also generated by flash photolysis of the corresponding benzyl alcohol and of the p-cyanophenol ether of this alcohol as well, and its rate of decay was measured in HClO4 and NaOH solutions and in HCO2H, CH3CO2H, HN3, CF3CH2NH3+, imidazolium ion, H2PO4-, (CH2OH)3CNH3+, (CH3)3CPO3H-, and HCO3- buffers. Acid-, base-, and uncatalyzed hydration reaction routes were again found, and solvent isotope effects as well as saturation of acid catalysis, this time in dilute HClO4, confirmed a preequilibrium mechanism for the acid-catalyzed reaction. Analysis of the buffer data gave buffer-base rate constants that did not conform to the Br?nsted relation, consistent with the expected nucleophilic nature of the buffer reactions.     

Indirect resolution of baclofen enantiomers from pharmaceutical dosage form by reversed-phase liquid chromatography after derivatization with Marfey's reagent and its structural variants

A high-performance liquid chromatographic (HPLC) method was developed for chiral assay of baclofen enantiomers in pharmaceutical formulations using an indirect approach. Baclofen enantiomers were derivatized with Marfey's reagent (FDNP-L-Ala-NH2) and its structural variants FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2. The resultant diastereomers were separated on RP-TLC [triethylammonium phosphate buffer (pH 4.0, 50 mm)-acetonitrile, 50:50] and on a C18 column using a linear gradient (45 min) of acetonitrile and 0.01% aqueous trifluoroacetic acid (TFA) with UV detection at 340 nm. The differences in the retention times (Delta t R) of diastereomers due to the five chiral reagents were compared. The maximum and minimum difference in retention times between separated diastereomers was for FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2, respectively. The effect of flow rate, acetonitrile content and TFA concentration on resolution was studied. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

Binol quinone methides as bisalkylating and DNA cross-linking agents

The photogeneration and detection of new binol quinone methides undergoing mono- and bisalkylation of free nucleophiles was investigated by product distribution analysis and laser flash photolysis in water solution using binol quaternary ammonium derivatives 2 and 12 as photoactivated precursors. The alkylation processes of N and S nucleophiles are strongly competitive with the hydration reaction. DNA cross-linking potency of the water-soluble binol quaternary ammonium salt 2 was investigated as a pH function and compared to that of other quaternary ammonium salts capable of benzo-QM (QM = quinone methide) photogeneration by gel electrophoresis. DFT calculations in the gas phase and in water bulk on the binol and benzo quaternary ammonium salts 2 and 4 evidence structural and electrostatic features of the binol derivative which might offer a rationalization of its promising high photo-cross-linking efficiency.