Screening and identification of metabolites of two kinds of main active ingredients and hepatotoxic pyrrolizidine alkaloids in rat after lavage Farfarae Flos extract by UHPLC‐Q‐TOF‐MS mass spectrometry

Farfarae Flos, the dried flower buds of Tussilago farfara L., is usually used to treat coughs, bronchitic and asthmatic conditions as an important traditional Chinese medicine. Tussilagone and methl butyric acid tussilagin ester are seen as representatives of two kinds of active substances. In addition, the pyrrolizidine alkaloids, mainly senkirkine and senecionine, present in the herb can be hepatoxic. In this study, a rapid and sensitive ultra‐high‐performance liquid chromatography coupled with hybrid triple quadrupole time‐of‐flight mass spectrometry method was successfully applied to identify the metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine. A total of 35, 37, 18 and nine metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine in rats were tentatively identified. Hydrolysis, oxidation, reduction and demethylation were the major metabolic reactions for tussilagone and methl butyric acid tussilagin ester. The main biotransformation routes of senkirkine and senecionine were identified as demethylation, N‐methylation, oxidation and reduction. This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways might provide further understanding of the metabolic fate of the chemical constituents after oral administration of Farfarae Flos extract in vivo.     

Determination of senkirkine and senecionine in Tussilago farfara using microwave-assisted extraction and pressurized hot water extraction with liquid chromatography tandem mass spectrometry

Tussilago farfara (Kuan Donghua) is an important Chinese herbal medicine which has been shown to contain many bioactive compounds and widely used to relieve cough and resolve phlegm. However, besides therapeutic bioactive compounds, this herb has been found to contain toxic pyrrolizidine alkaloids (PAs), mainly senkirkine and traces of senecionine. In this report, conditions for microwave-assisted extraction (MAE) and pressurized hot water extraction (PHWE) were optimized for the extraction of the PAs. The results were compared against heating under reflux. It was found that the binary mixture of MeOH:H₂O (1:1) acidified using HCl to pH 2-3 was the optimal solvent for the extraction of the PAs in the plant materials. Liquid chromatography (LC) with ultra-violet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) in the positive mode was used for the determination and quantitation of senkirkine and senecionine in the botanical extract. The proposed extraction methods with LC/MS allow for the rapid detection of the major and the minor alkaloids in T. farfara in the presence of co-eluting peaks. With LC/MS, the quantitative analysis of PAs in the extract was done using internal standard calibration and the precision was found to vary from 0.6% to 5.4% on different days. The limits of detection (LODs) and limits of quantitation (LOQs) for MAE and PHWE were found to vary from 0.26 μg/g to 1.04 μg/g and 1.32 μg/g to 5.29 μg/g, respectively. The method precision of MAE and PHWE were found to vary from 3.7% to 10.4% on different days. The results showed that major and minor alkaloids extracted using MAE and PHWE were comparable to that by heating under reflux. Our data also showed that significant ion suppression was not observed in the analysis of senkirkine and senecionine in the botanical extracts with co-eluting peaks.     

Mass‐spectrometry‐directed analysis and purification of pyrrolizidine alkaloid cis/trans isomers in Gynura japonica

Pyrrolizidine alkaloids are highly hepatotoxic natural chemicals that produce irreversible chronic and acute hepatotoxic effects on human beings. Purification of large amounts of pyrrolizidine alkaloids is necessary for toxicity studies. In this study, an efficient method for targeted analysis and purification of pyrrolizidine alkaloid cis/trans isomers from herbal materials was developed for the first time. Targeted analysis of the hepatotoxic pyrrolizidine alkaloids was performed by liquid chromatography with tandem mass spectrometry (precursor ion scan and daughter ion scan), and the purification of pyrrolizidine alkaloids was achieved with a mass‐directed auto purification system. The extraction and preparative liquid chromatography conditions were optimized. The developed method was applied to analysis of Gynura japonica (Thunb.) Juel., a herbal medicine traditionally used for detumescence and relieving pain but is potentially hepatotoxic as it contains pyrrolizidine alkaloids. Twelve pyrrolizidine alkaloids (six cis/trans isomer pairs) were identified with reference compounds or characterized by liquid chromatography with tandem mass spectrometry, and five individual pyrrolizidine alkaloids, including (E)‐seneciphylline, seneciphylline, integerrimine, senecionine, and seneciphyllinine, were prepared from G. japonica roots with high efficiency. The results of this work provide a new technique for the preparation of large amounts of pyrrolizidine alkaloid reference substances, which will also benefit toxicological studies of pyrrolizidine alkaloids and treatments for pyrrolizidine alkaloid‐induced toxicity.     

Qualitative and quantitative analysis of pyrrolizidine alkaloids for the entire process quality control from Senecio scandens to Senecio scandens‐containing preparations by high performance liquid chromatography‐tandem mass spectrometry

Senecio scandens as a commonly used traditional Chinese medicine that is used alone or in combination with other herbs in preparations such as QianBai BiYan tablets has attracted much attention because of its hepatotoxic pyrrolizidine alkaloids. Nowadays, most studies for pyrrolizidine alkaloids are only performed on herbs or a preparation, however, production of preparations is a dynamic process, control of toxic impurities for raw materials, or finished products cannot monitor the production process dynamically. Thus, in this study, qualitative and quantitative analysis of pyrrolizidine alkaloids for the entire process quality control from S. scandens to its preparations was carried out with HPLC‐MS/MS for the first time, which was more comprehensive and dynamic than the previous single‐layer analysis. First, the species of pyrrolizidine alkaloids in S. scandens were analyzed, and the characteristic fragmentation rules of pyrrolizidine alkaloids containing common parent nucleus were found, which can be used to identify these components rapidly in the future. Then, a quantitative method for S. scandens to QianBai BiYan tablets and other nine S. scandens‐containing preparations was established, and after the medication safety speculation, all of them met the relevant safety requirements. After that, in order to ensure the stability and controllable of drug quality, the limit of pyrrolizidine alkaloids in preparations was determined according to the safe dosage that is stipulated to be the same as raw materials. Finally, the factors causing the content change of pyrrolizidine alkaloids in S. scandens from different source were studies, which can provide theoretical basis for selecting suitable raw materials for production.     

高效液相色谱-串联质谱法测定蜂蜜中的5种双稠吡咯啶类生物碱

建立了强阳离子固相萃取-高效液相色谱-串联质谱法用于测定蜂蜜中野百合碱、克氏千里光宁、倒千里光碱、千里光菲啉和千里光宁等5种双稠吡咯啶类生物碱。蜂蜜样品用0.1 mol/L盐酸溶解,强阳离子交换固相萃取柱富集净化后,高效液相色谱-串联质谱进行定性和定量。以Phenomenex C₁₈柱(100 mm×4.6 mm, 2.6 μm)为分析柱,乙腈和0.1%(体积分数)甲酸-5 mmol/L乙酸铵水溶液为流动相进行梯度洗脱,在电喷雾正离子多反应监测模式下进行检测。结果表明,5种双稠吡咯啶类生物碱在1~100 μg/L范围内线性关系良好,线性相关系数均大于0.99。在1、20和50 μg/kg加标水平下,11种不同种类蜂蜜中的5种双稠吡咯啶类生物碱的平均回收率为73.1%~107.1%,相对标准偏差(RSD)为4.1%~17.0%,方法检定量限可达到1.0 μg/kg。该方法准确灵敏,可适用于不同种类进出口蜂蜜中双稠吡咯啶类生物碱的监控分析。利用该方法对来自中国8个省及自治区的洋槐蜜、葵花蜜、棉花蜜、油菜蜜、荆条蜜、枣花蜜、荞麦蜜和来自新西兰、西班牙、澳大利亚等国家的进口蜂蜜进行了筛查。结果发现,野百合碱、克氏千里光宁和倒千里光碱均未检出,而千里光菲啉和千里光宁在大多数蜂蜜中均能检出,千里光菲啉含量在11.0~31.1 μg/kg范围内,千里光宁含量在8.3~29.1 μg/kg范围内。     

Separation and determination of toxic pyrrolizidine alkaloids in traditional Chinese herbal medicines by micellar electrokinetic chromatography with organic modifier

A simple and rapid micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of four toxic pyrrolizidine alkaloids (PAs) (senkirkine, senecionine, retrorsine, and seneciphylline) in two traditional Chinese herbal medicines (Qian liguang and Kuan donghua). Separation was performed in the running buffer consisting of 20 mM borate, 30 mM SDS, and 20% methanol at pH 9.1. With the optimized separation conditions, four PAs were separated in 17 min by a single run. The calibration curves showed good linearity with correlation efficiencies (R(2)) between 0.9940 and 0.9988. RSDs in migration time and peak area were 0.31, 0.40, 0.39, 0.48% and 3.28, 3.48, 4.16, 3.42% for senkirkine, senecionine, retrorsine, and seneciphylline, respectively. Limits of detection (S/N = 3) varied from 1.19 to 2.70 microg/mL. The proposed method was applied to determine the PAs extracted from Chinese herbal medicines (Qian liguang and Kuan donghua). PA of senkirkine in Kuan donghua was detected and the amount was found to be 79.1 microg/g. The results obtained indicate that the proposed MEKC method could potentially become an effective alternative tool for qualification control and quantitative analysis of herbal medicines in pharmaceutical industry.     

Molecularly imprinted polymer solid‐phase microextraction coupled with ultra high performance liquid chromatography and tandem mass spectrometry for rapid analysis of pyrrolizidine alkaloids in herbal medicine

Pyrrolizidine alkaloids are the most widely distributed natural toxins, and pyrrolizidine alkaloid‐containing herbal medicines are probably the most common poisonous plants affecting humans. We reported pyrrolizidine alkaloid‐molecularly imprinted polymer solid‐phase microextraction for the selective adsorption of toxic pyrrolizidine alkaloids from herbal medicine. A sulfonic compound, sodium allylsulfonate, was chosen as the functional monomer to interact with pyrrolizidine alkaloids through strong ionic interaction. To avoid template leakage and for the aim of cost saving, a relatively cheap dummy template was used for the fabrication of molecularly imprinted polymer‐solid‐phase microextraction fibers. The obtained fibers showed selective adsorption ability for four pyrrolizidine alkaloids, including europine, echimidine, lasiocarpine, and heliotrine. The extraction parameters, such as extraction time, extraction temperature, shaking speed, elution solvent and elution time, were optimized. Then ultra high performance liquid chromatography with mass spectrometry coupled with molecularly imprinted polymer‐solid‐phase microextraction method was developed for the fast and efficient analysis of four pyrrolizidine alkaloids from the model herbal plant Farfarae Flos. The established method was validated and exhibited satisfactory accuracy and precision. The present method provides an innovative and fast analytical strategy for the determination of trace toxic pyrrolizidine alkaloids in complicated samples.     

Determination of Hepatotoxic Pyrrolizidine Alkaloids in Gynura segetum by MEKC

A novel, rapid and sensitive micellar electrokinetic capillary chromatography method was developed for the separation and determination of two hepatotoxic pyrrolizidine alkaloids in Gynura segetum (Lour.) Merr. (Jusanqi) within 8 min. The method was successfully applied to the simultaneous determination of seneciphylline and senecionine in a Jusanqi sample.     

Stereocomplementary Routes to Hydroxylated Nitrogen Heterocycles: Total Syntheses of Casuarine,Australine, and 7‐epi‐Australine

Addition of lithiated 1‐benzyloxyallene to a D ‐arabinose‐derived cyclic nitrone occurred with perfect diastereoselectivity furnishing a bicyclic 1,2‐oxazine derivative, which is an excellent precursor for pyrrolizidine alkaloids hydroxylated at C‐7 with optional configuration at this stereogenic center. Depending on the stage of the N? O bond cleavage and ring re‐closure, 7‐hydroxypyrrolizidines with 7R or 7S configuration were obtained, as a result of completely selective addition reactions occurring complementarily at the bottom or top face of the endocyclic C? C double bond in six‐ and five‐membered B rings, respectively. Applicability of these stereodivergent routes to obtain polyhydroxy pyrrolizidine alkaloids is demonstrated by the efficient syntheses of casuarine and australine as examples of the two classes of diversely configured 7‐hydroxypyrrolizidine alkaloids. An alternative synthesis of australine and two strategies for the preparation of 7‐epi‐australine are also reported, which demonstrate that the stereoselectivity of hydride reduction of an exocyclic C? O double bond is independent of the ring size, occurring preferentially from the top face either in a six‐ or five‐membered ring.     

Determination of total retronecine esters‐type hepatotoxic pyrrolizidine alkaloids in plant materials by pre‐column derivatization high‐performance liquid chromatography

A pre‐column derivatization high‐performance liquid chromatography method with diode array detection was developed and validated to determine the total retronecine esters‐type hepatotoxic pyrrolizidine alkaloids (RET‐HPAs) in herbs. The RET‐HPAs reacted with o‐chloranil in methanolic solution heated for 3 h, and an oxidative derivative was produced that could be detected at a maximal absorption of 223 nm. The analysis was performed using a C₁₈ column with an isocratic elution of methanol and aqueous 0.01% triethylamine (adjusted to pH 4 with formic acid), and the detection was carried out with DAD at 223 nm. The validation of the method included linearity, sensitivity, recovery and stability. It showed a good linear regression (r² > 0.9900) in the range of 2.5–250 µm with a limit of detection (S/N = 3) of 0.5 µm . The method provided desirable repeatability with overall intra‐ and inter‐day variations of less than 4.6%. The obtained recoveries for both of the extraction and derivatization process were between 94.6 and 100.7% (n = 3). Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of dihydroindole‐type alkaloids in Strychnos nux‐vomica unprocessed and processed seeds by high‐performance liquid chromatography coupled with diode array detection and mass spectrometry

The ripened seeds of Strychnos nux‐vomica L. have been extensively used as herbal medicines in Asian countries. Dihydroindole‐type alkaloids are not only the active constituents but also the toxicants in Strychnos. However, the simultaneous determination of these alkaloids in both crude and processed Semen Strychni is still lacking. The present study represents the first quantitation and relative quantitation assay of 12 dihydroindole‐type alkaloids in Strychnos nux‐vomica unprocessed and sand‐processed seeds using high‐performance liquid chromatography coupled with diode array detection and mass spectrometry. The relative concentration of ten alkaloids was calculated by semi‐quantification using the internal standard and their amounts in unprocessed and detoxified Semen Strychni were compared. We report here for the first time the significant increase of the two alkaloids, 19‐N‐methyl‐strychnine, and 2,3‐dimethoxy‐19‐N‐methyl‐strychnine, during the processing of Semen Strychni. Our study provides new insight into the true complexity of seed processing procedure and valuable information for assessing the efficacy and safety for clinical applications of Semen Strychni‐containing drugs.     

Simultaneous characterization of pyrrolizidine alkaloids and N‐oxides in Gynura segetum by liquid chromatography/ion trap mass spectrometry

Gynura segetum (Lour.) Merr. (Jusanqi) is a traditional herbal product used for hemostasis and detumescence in Chinese folk medicine. However, its hepatic toxicity should not be ignored. In this study, pyrrolizidine alkaloids (PAs) and their corresponding N‐oxides (PANOs) were extracted from the whole plant of G. segetum and analyzed by high‐performance liquid chromatography coupled to ion trap mass spectrometry (ITMS). Identification of eluted peaks as PANOs was indicated by virtue of their MS and MSⁿ analysis, in addition to the [M+H]⁺ adduct ion, characteristically showed a significant (usually 100% abundance) dimer adduct [2M+H]⁺ that is not observed in the MS of the parent PAs. A total of 20 compounds were identified or tentatively characterized based on their mass spectra and possible biosynthetic pathways, of which three PAs and one PANO, namely seneciphylline, senecionine, seneciphylline and seneciphyllinine N‐oxide, were unequivocally characterized, while other PAs and PANOs were tentatively assigned. Sixteen constituents were reported for the first time from G. segetum and tetrahydrosenecionine has not been previously reported as a natural product. Our results are the first comprehensive analysis of PAs and PANOs in G. segetum constituents and will be helpful for the quality control of the herb of G. segetum and its related preparations. Copyright © 2008 John Wiley & Sons, Ltd.     

pH‐Zone‐refining counter‐current chromatography for two new lipo‐alkaloids separated from refined alkaline extraction of Kusnezoff monkshood root

An efficient and refined method for the separation of six aconitine‐type alkaloids from the alkaline prepared “Kusnezoff monkshood root” was established. It is the first study that two new lipo‐alkaloids were successfully isolated from refined sample by pH‐zone‐refining counter‐current chromatography rather than synthetic method. It was of interest that a great deal of lipo‐alkaloids was produced in crude extract from the alkalization of “Kusnezoff monkshood root.” A refined sample method was proposed to enrich two types of alkaloids by liquid–liquid extraction, i.e. lipo‐alkaloids and monoester‐diterpenoid alkaloids. The pH‐zone‐refining counter‐current chromatography was performed with an optimized two‐phase solvent system composed of n‐hexane‐ethyl acetate–methanol–water (3:5:4:5, v/v), where upper organic phase was added to 3 mmol/L triethylamine as a retainer and lower aqueous mobile phase was added to 3 mmol/L hydrochloric acid as an eluter. As a result, six aconitum alkaloids, including two lipo‐alkaloids (8‐lino‐14‐benzoylaconine, 8‐pal‐14‐benzoylaconine), three monoester‐diterpenoid alkaloids (14‐benzoylmesaconine, 14‐benzoylaconine, beyzoyldeoxyaconine), and one aconine alkaloid (neoline) were acquired from the plant at the same time. The anti‐inflammatory activities of the two new lipo‐alkaloids were compared to the six alkaloids in vitro, in cyclo‐oxygen‐ase‐2 inhibition assays. The separation mechanism of six alkaloids by pH‐zone‐refining counter‐current chromatography was illustrated.     

cis‐trans Peptide‐Bond Isomerization in α‐Methylproline Derivatives

α‐Methyl‐L ‐proline is an α‐substituted analog of proline that has been previously employed to constrain prolyl peptide bonds in a trans conformation. Here, we revisit the cis‐trans prolyl peptide bond equilibrium in derivatives of α‐methyl‐L ‐proline, such as N‐Boc‐protected α‐methyl‐L ‐proline and the hexapeptide H‐Ala‐Tyr‐αMePro‐Tyr‐Asp‐Val‐OH. In Boc‐α‐methyl‐L ‐proline, we found that both cis and trans conformers were populated, whereas, in the short peptide, only the trans conformer was detected. The energy barrier for the cis‐trans isomerization in Boc‐α‐methyl‐L ‐proline was determined by line‐shape analysis of NMR spectra obtained at different temperatures and found to be 1.24 kcal/mol (at 298 K) higher than the corresponding value for Boc‐L ‐proline. These findings further illuminate the conformationally constraining properties of α‐methyl‐L ‐proline.     

A biochemometrics strategy combining quantitative determination,bioactivity evaluation and relationship analysis for identification of analgesic alkaloids of raw and vinegar‐processed Corydalis turtschaninovii

A biochemometrics strategy combining quantitative determination, bioactivity evaluation, and relationship analysis was proposed for identification of analgesic components of herbs. First, a robust liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of nine major alkaloids in crude and vinegar‐processed Corydalis turtschaninovii. Nine alkaloids were separated on a BEH C₁₈ column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid and then detected by multiple reactions monitoring in the positive ion mode. Nitidine chloride was employed as the internal standard. The method displayed good linearity and the precisions of intra‐day and inter‐day were all within 3.0%. The recovery rates of each alkaloid ranged from 97.1 to 102.9%. The method was successfully applied for quantitative analysis of nine alkaloids in ten batches of crude and vinegar‐processed Corydalis turtschaninovii. Second, the analgesic effects of crude and vinegar‐processed Corydalis turtschaninovii were evaluated in mice. Third, principle component analysis, canonical correlation analysis, and partial least squares regression were used to analysis the relationship between the contents of nine major alkaloids and the analgesic effect of different crude and vinegar‐processed samples. Tetrahydropalmatine, coptisine, and dehydrocorydaline have a close positive correlation with the analgesic effect.     

Single‐drop microextraction followed by in‐syringe derivatization and GC‐MS detection for the determination of parabens in water and cosmetic products

A single‐drop microextraction (SDME) method followed by in‐syringe derivatization and GC‐MS determination has been developed for analysis of five parabens, including methyl, ethyl, isopropyl, n‐propyl and n‐butyl paraben in water samples and cosmetic products. N,O‐Bis(trimethylsilyl)acetamide (BSA) was used as derivatization reagent. Derivatization reaction was performed inside the syringe barrel using 0.4 μL of BSA. Parameters that affect the derivatization yield such as temperature and time of the reaction were studied. In addition, experimental SDME parameters such as selection of organic solvent, addition of salt, extraction time and extraction temperature were investigated and optimized. The RSD of the method for aqueous samples varied from 8.1 to 13%. The LODs ranged from 0.001 (n‐butyl paraben) to 0.015 (methyl paraben) μg/L, and the enrichment factors were between 23 and 150.     

The in‐capillary DPPH‐capillary electrophoresis‐the diode array detector combined with reversed‐electrode polarity stacking mode for screening and quantifying major antioxidants in Cuscuta chinensis Lam

An in‐capillary 2, 2‐diphenyl‐1‐picrylhydrazyl (DPPH)‐CE‐the DAD (in‐capillary DPPH‐CE‐DAD) combined with reversed‐electrode polarity stacking mode has been developed to screen and quantify the active antioxidant components of Cuscuta chinensis Lam. The operation parameters were optimized with regard to the pH and concentration of buffer solution, SDS, β‐CDs, organic modifier, as well as separation voltage and temperature. Six antioxidants including chlorogenic acid, p‐coumaric acid, rutin, hyperin, isoquercitrin, and astragalin were screened and the total antioxidant activity of the complex matrix was successfully evaluated based on the decreased peak area of DPPH by the established DPPH‐CE‐DAD method. Sensitivity was enhanced under reversed‐electrode polarity stacking mode and 10‐ to 31‐fold of magnitude improvement in detection sensitivity for each analyte was attained. The results demonstrated that the newly established in‐capillary DPPH‐CE‐DAD method combined with reversed‐electrode polarity stacking mode could integrate sample concentration, the oxidizing reaction, separation, and detection into one capillary to fully automate the system. It was considered a suitable technique for the separation, screening, and determination of trace antioxidants in natural products.     

Hydrogen‐bonded sheet structures in methyl 4‐(4‐chloroanilino)‐3‐nitrobenzoate and methyl 1‐benzyl‐2‐(4‐chlorophenyl)‐1H‐benzimidazole‐5‐carboxylate

In methyl 4‐(4‐chloroanilino)‐3‐nitrobenzoate, C₁₄H₁₁ClN₂O₄, (I), there is an intramolecular N—H...O hydrogen bond and the intramolecular distances provide evidence for electronic polarization of the o‐quinonoid type. The molecules are linked into sheets built from N—H...O, C—H...O and C—H...π(arene) hydrogen bonds, together with an aromatic π–π stacking interaction. The molecules of methyl 1‐benzyl‐2‐(4‐chlorophenyl)‐1H‐benzimidazole‐5‐carboxylate, C₂₂H₁₇ClN₂O₂, (II), are also linked into sheets, this time by a combination of C—H...π(arene) hydrogen bonds and aromatic π–π stacking interactions.     

fac‐Tri­carbonyl(4,4′‐di­methyl‐2,2′‐bi­pyridine)­tri­phenyl­tin(II)­rhenium(I)

The title organometallic compound, fac‐tri­carbonyl‐2κ³C‐(4,4′‐di­methyl‐2,2′‐bi­pyridine)‐2κ²N,N′‐tri­phenyl‐1κ³C¹‐tin(II)­rhenium(I)(Sn—Re), [ReSn(C₆H₅)₃(C₁₂H₁₂N₂)(CO)₃], con­tains three unique π–π stacking interactions. The result is an infinite chain of uninterrupted alternating intra‐ and intermolecular offset π–π stacking interactions throughout the crystal lattice. This extended π–π stacking arrangement, and an additional isolated intramolecular π–π interaction between the remaining 4,4′‐di­methyl‐2,2′‐bi­pyridine ring and a second phenyl group, impose geometric constraints on the Re and Sn atoms, yielding distorted octahedral and tetrahedral coordinations, respectively, for the metal centers.     

Supramolecular aggregation in three 4‐aryl‐6‐(1H‐indol‐3‐yl)‐3‐methyl‐1‐phenyl‐1H‐pyrazolo[3,4‐b]pyridine‐5‐carbonitriles

Both 6‐(1H‐indol‐3‐yl)‐3‐methyl‐4‐(4‐methylphenyl)‐1‐phenyl‐1H‐pyrazolo[3,4‐b]pyridine‐5‐carbonitrile and 6‐(1H‐indol‐3‐yl)‐3‐methyl‐4‐(4‐methoxyphenyl)‐1‐phenyl‐1H‐pyrazolo[3,4‐b]pyridine‐5‐carbonitrile crystallize from dimethylformamide solutions as stoichiometric 1:1 solvates, viz. C₂₉H₂₁N₅·C₃H₇NO, (I), and C₂₉H₂₁N₅O·C₃H₇NO, (II), respectively; however, 6‐(1H‐indol‐3‐yl)‐3‐methyl‐1‐phenyl‐4‐(3,4,5‐trimethoxyphenyl)‐1H‐pyrazolo[3,4‐b]pyridine‐5‐carbonitrile, C₃₁H₂₅N₅O₃, (III), crystallizes in the unsolvated form. The heterocyclic components of (I) are linked by C—H...π(arene) hydrogen bonds to form cyclic centrosymmetric dimers, from which the solvent molecules are pendent, linked by N—H...O hydrogen bonds. In (II), the heterocyclic components are linked by a combination of C—H...N and C—H...π(arene) hydrogen bonds into chains containing two types of centrosymmetric ring, and the pendent solvent molecules are linked to these chains by N—H...O hydrogen bonds. Molecules of (III) are linked into simple C(12) chains by an N—H...O hydrogen bond, and these chains are weakly linked into pairs by an aromatic π–π stacking interaction.