Analysis of 30 synthetic cannabinoids in serum by liquid chromatography‐electrospray ionization tandem mass spectrometry after liquid‐liquid extraction

The analysis of synthetic cannabinoids in human matrices is of particular importance in the fields of forensic and clinical toxicology since cannabis users partly shift to the consumption of ‘herbal mixtures’ as a legal alternative to cannabis products in order to circumvent drug testing. However, comprehensive methods covering the majority of synthetic cannabinoids already identified on the drug market are still lacking. In this article, we present a fully validated method for the analysis of 30 synthetic cannabinoids in human serum utilizing liquid‐liquid extraction and liquid chromatography‐electrospray ionization tandem mass spectrometry. The method proved to be suitable for the quantification of 27 substances. The limits of detection ranged from 0.01 to 2.0 ng/mL, whereas the lower limits of quantification were in the range from 0.1 to 2.0 ng/mL. The presented method was successfully applied to 833 authentic serum samples during routine analysis between August 2011 and January 2012. A total of 227 (27%) samples was tested positive for at least one of the following synthetic cannabinoids: JWH‐018, JWH‐019, JWH‐073, JWH‐081, JWH‐122, JWH‐200, JWH‐203, JWH‐210, JWH‐307, AM‐2201 and RCS‐4. The most prevalent compounds in positive samples were JWH‐210 (80%), JWH‐122 (63%) as well as AM‐2201 (29%). Median serum concentrations were all below 1.0 ng/mL. These findings demonstrate a significant shift of the market of synthetic cannabinoids towards substances featuring a higher CB₁ binding affinity and clearly emphasize that the analysis of synthetic cannabinoids in serum or blood samples requires highly sensitive analytical methods covering a wide spectrum of substances. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of the major urinary metabolites in man of seven synthetic cannabinoids of the aminoalkylindole type present as adulterants in ‘herbal mixtures’ using LC‐MS/MS techniques

Herbal mixtures, such as ‘Spice’, containing cannabimimetic compounds are easily available on the Internet and have become increasingly popular among people having to undergo urine drug testing, as these compounds are not detected by current immunochemical tests. For analysis of urine samples, knowledge of the main metabolites is necessary as the unchanged compounds are usually not found in urine after consumption. In this paper, the identification of the major metabolites of the currently most common seven synthetic cannabinoids is presented. Urine samples from patients of psychiatric facilities known to have consumed synthetic cannabinoids were screened by LC‐MS/MS and HR‐MS/MS techniques, and the major metabolites for each of the following synthetic cannabinoids were identified by their enhanced product ion spectra and accurate mass measurement: JWH‐018, JWH‐073, JWH‐081, JWH‐122, JWH‐210, JWH‐250 and RCS‐4. The major metabolic pathway is monohydroxylation either at the N‐alkyl side chain, the naphthyl moiety or the indole moiety. In addition, metabolites with carboxylated alkyl chains were identified for some of the compounds. These results facilitate the design of urine screening methods for detecting consumption of synthetic cannabinoids. Copyright © 2012 John Wiley & Sons, Ltd.     

Monitoring of herbal mixtures potentially containing synthetic cannabinoids as psychoactive compounds

Herbal mixtures like ‘Spice’ with potentially bioactive ingredients were available in many European countries since 2004 and are still widely used as a substitute for cannabis, although merchandized as ‘herbal incense’. After gaining a high degree of popularity in 2008, big quantities of these drugs were sold. In December 2008, synthetic cannabinoids were identified in the mixtures which were not declared as ingredients: the C₈ homolog of the non‐classical cannabinoid CP‐47,497 (CP‐47,497‐C8) and a cannabimimetic aminoalkylindole called JWH‐018. In February 2009, a few weeks after the German legislation put these compounds and further pharmacologically active homologs of CP‐47,497 under control, another cannabinoid appeared in ‘incense’ products: the aminoalkylindole JWH‐073. In this paper, the results of monitoring of commercially available ‘incense’ products from June 2008 to September 2009 are presented. In this period of time, more than 140 samples of herbal mixtures were analyzed for bioactive ingredients and synthetic cannabimimetic substances in particular. The results show that the composition of many products changed repeatedly over time as a reaction to prohibition and prosecution of resellers. Therefore neither the reseller nor the consumer of these mixtures can predict the actual content of the ‘incense’ products. As long as there is no possibility of generic definitions in the controlled substances legislation, further designer cannabinoids will appear on the market as soon as the next legal step has been taken. This is affirmed by the recent identification of the aminoalkylindoles JWH‐250 and JWH‐398. As further cannabinoids can be expected to occur in the near future, a continuous monitoring of these herbal mixtures is required. The identification of the synthetic opioid O‐desmethyltramadol in a herbal mixture declared to contain ‘kratom’ proves that the concept of selling apparently natural products spiked with potentially dangerous synthetic chemicals/pharmaceuticals is a continuing trend on the market of ‘legal highs’. Copyright © 2010 John Wiley & Sons, Ltd.     

Quadrupole‐time‐of‐flight mass spectrometry screening for synthetic cannabinoids in herbal blends

‘Legal highs’ are novel substances which are intended to elicit a psychoactive response. They are sold from ‘head shops’, the internet and from street suppliers and may be possessed without legal restriction. Several months ago, a 19‐year‐old woman came searching for medical treatment as she had health problems caused by smoking legal highs. The substances were sold as herbal blends in plastic bags under four different labels. In this work, samples of these herbal blends have been analysed to investigate the presence of psychoactive substances without any reference standard being available at the laboratory. A screening strategy for a large number of synthetic and natural cannabinoids has been applied based on the use of ultra‐high pressure liquid chromatography coupled to quadrupole‐time of flight mass spectrometry (UHPLC‐QTOF MS) under MS^E mode. A customized home‐made database containing literature‐based exact masses for parent and product ions of around 200 synthetic and natural cannabinoids was compiled. The presence of the (de)protonated molecule measured at its accurate mass was evaluated in the samples. When a peak was detected, collision‐induced dissociation fragments and characteristic isotopic ions were also evaluated and used for tentative identification. After this tentative identification, four synthetic cannabinoids (JWH‐081, JWH‐250, JWH‐203 and JWH‐019) were unequivocally confirmed by subsequent acquisition of reference standards. The presence in the herbal blends of these synthetic cannabinoids might explain the psychotic and catatonic symptoms observed in the patient, as JWH compounds could act as potent agonists of CB₁ and CB₂ receptors located in the Limbic System and Basal ganglia of the human brain. Copyright © 2013 John Wiley & Sons, Ltd.     

Direct screening of herbal blends for new synthetic cannabinoids by MALDI‐TOF MS

Since 2004, a number of herbal blends containing different synthetic compounds mimicking the pharmacological activity of cannabinoids and displaying a high toxicological potential have appeared in the market. Their availability is mainly based on the so‐called “e‐commerce”, being sold as legal alternatives to cannabis and cannabis derivatives. Although highly selective, sensitive, accurate, and quantitative methods based on GC–MS and LC–MS are available, they lack simplicity, rapidity, versatility and throughput, which are required for product monitoring. In this context, matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF MS) offers a simple and rapid operation with high throughput. Thus, the aim of the present work was to develop a MALDI‐TOF MS method for the rapid qualitative direct analysis of herbal blend preparations for synthetic cannabinoids to be used as front screening of confiscated clandestine preparations. The sample preparation was limited to herbal blend leaves finely grinding in a mortar and loading onto the MALDI plate followed by addition of 2 µl of the matrix/surfactant mixture [α‐cyano‐4‐hydroxy‐cinnamic acid/cetyltrimethylammonium bromide (CTAB)]. After drying, the sample plate was introduced into the ion source for analysis. MALDI‐TOF conditions were as follows: mass spectra were analyzed in the range m/z 150–550 by averaging the data from 50 laser shots and using an accelerating voltage of 20 kV. The described method was successfully applied to the screening of 31 commercial herbal blends, previously analyzed by GC–MS. Among the samples analyzed, 21 contained synthetic cannabinoids (namely JWH‐018, JWH‐073, JWH‐081, JWH‐250, JWH‐210, JWH‐019, and AM‐694). All the results were in agreement with GC–MS, which was used as the reference technique. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a mass spectrometric hydroxyl‐position determination method for the hydroxyindole metabolites of JWH‐018 by GC‐MS/MS

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH‐018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography‐electron ionization‐tandem mass spectrometry (GC‐EI‐MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole‐type SC JWH‐081, and speculated that the same approach could be used for the metabolite isomers. Using JWH‐018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC‐MS/MS. Standard compounds of JWH‐018 and its hydroxyindole metabolite positional isomers were first analyzed by GC‐EI‐MS in full scan mode, which was only able to differentiate the 4‐hydroxyindole isomer. Further GC‐MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH‐018 administered mice and determined the hydroxyl positions to be at the 6‐position on the indole ring. GC‐EI‐MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH‐018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.     

A comprehensive library‐based,automated screening procedure for 46 synthetic cannabinoids in serum employing liquid chromatography‐quadrupole ion trap mass spectrometry with high‐temperature electrospray ionization

Considering the vast variety of synthetic cannabinoids and herbal mixtures – commonly known as ‘Spice’ or ‘K2’ – on the market and the resulting increase of severe intoxications related to their consumption, there is a need in clinical and forensic toxicology for comprehensive up‐to‐date screening methods. The focus of this project aimed at developing and implementing an automated screening procedure for the detection of synthetic cannabinoids in serum using a liquid chromatography‐ion trap‐MS (LC‐MSⁿ) system and a spectra library‐based approach, currently including 46 synthetic cannabinoids and 8 isotope labelled analogues. In the process of method development, a high‐temperature ESI source (IonBooster^TM, Bruker Daltonik) and its effects on the ionization efficiency of the investigated synthetic cannabinoids were evaluated and compared to a conventional ESI source. Despite their structural diversity, all investigated synthetic cannabinoids benefitted from high‐temperature ionization by showing remarkably higher MS intensities compared to conventional ESI. The employed search algorithm matches retention time, MS and MS²/MS³ spectra. With the utilization of the ionBooster source, limits for the automated detection comparable to cut‐off values of routine MRM methods were achieved for the majority of analytes. Even compounds not identified when using a conventional ESI source were detected using the ionBooster‐source. LODs in serum range from 0.1 ng/ml to 0.5 ng/ml. The use of parent compounds as analytical targets offers the possibility of instantly adding new emerging compounds to the library and immediately applying the updated method to serum samples, allowing the rapid adaptation of the screening method to ongoing forensic or clinical requirements. The presented approach can also be applied to other specimens, such as oral fluid or hair, and herbal mixtures and was successfully applied to authentic serum samples. Quantitative MRM results of samples with analyte concentrations above the determined LOD were confirmed as positive findings by the presented method. Copyright © 2014 John Wiley & Sons, Ltd.     

Positional isomer differentiation of synthetic cannabinoid JWH‐081 by GC‐MS/MS

Like many new designer drugs of abuse, synthetic cannabinoids (SC) have structural or positional isomers which may or may not all be regulated under law. Differences in acute toxicity may exist between isomers which impose further burden in the fields of forensic toxicology, medicine and legislation. Isomer differentiation therefore becomes crucial from these standpoints as new designer drugs continuously emerge with just minor positional modifications to their preexisting analogs. The aim of this study was to differentiate the positional isomers of JWH‐081. Purchased standard compounds of JWH‐081 and its positional isomers were analyzed by gas chromatography‐electron ionization‐mass spectrometry (GC‐EI‐MS) first in scan mode to investigate those isomers who could be differentiated by EI scan spectra. Isomers with identical or near‐identical EI spectra were further subjected to GC‐tandem mass spectrometry (MS/MS) analysis with appropriate precursor ions. EI scan was able to distinguish 3 of the 7 isomers: 2‐methoxy, 7‐methoxy and 8‐methoxy. The remaining isomers exhibited near‐identical spectra; hence, MS/MS was performed by selecting m/z 185 and 157 as precursor ions. 3‐Methoxy and 5‐methoxy isomers produced characteristic product ions that enabled the differentiation between them. Product ion spectrum of 6‐methoxy isomer resembled that of JWH‐081; however, the relative ion intensities were clearly different from one another. The combination of EI scan and MS/MS allowed for the regioisomeric differentiation of the targeted compounds in this study. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of naphthoylindoles acting on cannabinoid receptors based on their fragmentation patterns under ESI‐QTOFMS

‘Herbal highs’ have been advertised as legal and natural substitutes to cannabis, but a detailed examination of these products has revealed that the herbal matrix is laced with synthetic substances that mimic the effects of marijuana. Producers select the ingredients based on the results of scientific studies on the affinities of different chemicals to cannabinoid receptors. Naphthoylindoles have turned out to be the most popular class of substances identified in the products. Legal actions taken in order to tackle the problem of uncontrolled access to one substance have usually resulted in the marketing of derivatives or analogues. In the study, the mass spectral behavior of twelve synthetic cannabinoids from the naphthoylindole family under electrospray ionization (ESI) was investigated. LC‐QTOFMS experiments were performed in three modes (low fragmentor voltage, high fragmentor voltage with/without collision energy), and they enabled the identification of protonated molecules and main ions. A general fragmentation pattern under this ionization method was proposed, and mechanisms of ion formation were discussed. The developed procedure allowed the determination of substituent groups of the core naphthoylindole structure and distinction between positional isomers. The obtained results were used for the prediction of the ESI‐MS spectra for many naphthoylindoles with a high affinity to cannabinoid receptors. Similarities and differences between ESI‐MS and electron impact‐MS spectra of naphthoylindoles were discussed. The developed identification process was presented on an example of an analysis of an unknown herbal material, in which JWH‐007 was finally identified. Knowledge of the fragmentation mechanisms of naphthoylindoles could also be used by other researchers for identification of unknown substances in this chemical family. Copyright © 2012 John Wiley & Sons, Ltd.     

A solid‐phase microextraction gas chromatography/ion trap tandem mass spectrometry method for simultaneous determination of ‘foxy smelling compounds’ and 3‐alkyl‐2‐methoxypyrazines in grape juice

2′‐Aminoacetophenone (o‐AAP) was identified as the main cause of the aging note called ‘hybrid note’ or ‘foxy smell’ which is typical of non‐Vitis vinifera grapes. Together with methyl anthranilate (MA), this compound contributes to the typical foxy taint of wines made with non‐Vitis vinifera grapes. 3‐Alkyl‐2‐methoxypyrazines in grapes, with their herbaceous note and very low sensory thresholds, contribute to the aroma of several wines. In this study, a solid‐phase microextraction gas chromatography/ion trap tandem mass spectrometry (SPME‐GC/IT‐MS/MS) method for simultaneous detection of o‐AAP, MA and ethyl‐, isopropyl‐, sec‐butyl‐ and isobutylmethoxypyrazine in grape juice was developed. The method was applied to the study of several grape juices: the time required for analysis was less than 24 min, and the method was considered to be suitable for analysis of ‘foxy compounds’ and methoxypyrazines in grape juice. The high levels of MA and o‐AAP found in Clinton and Siebel grapes confirmed the ‘hybrid’ character of these varieties. Copyright © 2010 John Wiley & Sons, Ltd.     

New cathinone‐derived designer drugs 3‐bromomethcathinone and 3‐fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS and their detectability in urine

3‐Bromomethcathinone (3‐BMC) and 3‐Fluoromethcathinone (3‐FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so‐called “bath salts” or “plant feeders.” The aim of the present study was to identify for the first time the 3‐BMC and 3‐FMC Phase I and II metabolites in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS (HR‐MS) and to test for their detectability by established urine screening approaches using GC–MS or LC–MS. Furthermore, the human cytochrome‐P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drug–drug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GC–MS and by LC–HR‐MS. The main metabolic steps were N‐demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GC–MS could be confirmed by LC–HR‐MS. Furthermore, corresponding Phase II metabolites were identified using the LC–HR‐MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GC–MS and LC–MSⁿ after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N‐demethylation of 3‐BMC and 3‐FMC after in vitro–in vivo extrapolation. Copyright © 2012 John Wiley & Sons, Ltd.     

Exploring the chemical profile of designer drugs by ESI(+) and PSI(+) mass spectrometry—An approach on the fragmentation mechanisms and chemometric analysis

The consumption of design drugs, frequently known as new psychoactive substances (NPS), has increased considerably worldwide, becoming a severe issue for the responsible governmental agencies. These illicit substances can be defined as synthetic compounds produced in clandestine laboratories in order to act as analogs of schedule drugs mimetizing its chemical structure and improving its pharmacological effects while hampering the control and making regulation more complicated. In this way, the development of new methodologies for chemical analysis of NPS drugs is indispensable to determine a novel class of drugs arising from the underground market. Therefore, this work shows the use of high‐resolution mass spectrometry Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS) applying different ionization sources such as paper spray ionization (PSI) and electrospray ionization (ESI) in the evaluation of miscellaneous of seized drugs samples as blotter paper (n = 79) and tablet (n = 100). Also, an elucidative analysis was performed by ESI(+)MS/MS experiments, and fragmentation mechanisms were proposed to confirm the chemical structure of compounds identified. Besides, the results of ESI(+) and PSI(+)‐FT‐ICR MS were compared with those of GC–MS, revealing that ESI(+)MS showed greater detection efficiency among the methodologies employed in this study. Moreover, this study stands out as a guide for the chemical analysis of NPS drugs, highlighting the differences between the techniques of ESI(+)‐FT‐ICR MS, PSI(+)‐FT‐ICR MS, and GC–MS.     

‘Click Synthesis’ of Some Novel O‐Substituted Oximes Containing 1,2,3‐Triazole‐1,4‐diyl Residues as New Analogs of β‐Adrenoceptor Antagonists

The ‘click synthesis’ of some novel O‐substituted oximes, 7a – 7t , which contain 1,2,3‐triazolediyl residues, as new analogs of β‐adrenoceptor antagonists is described (Schemes 1–4). The synthesis of these compounds was achieved in four to five steps. The formation of oximes of 9H‐fluoren‐9‐one and benzophenone, i.e., 9a and 9b , respectively, followed by their reaction with propargyl bromide, afforded O‐propargyl oximes 10a and 10b , respectively, which by a subsequent CuI‐catalyzed Huisgen cycloaddition with prepared β‐azido alcohols 11a – 11j (Schemes 2 and 3), led to the target compounds 7a – 7t in good yields.     

Sensor Platform with a Custom‐tailored Aptamer for Diagnosis of Synthetic Cannabinoids

Synthetic cannabinoids (SCs) are the large group of abused drugs and detection of them is still a challenge. Hence, new methods for analysis of SCs are being investigated. We aimed to develop a novel system for selective analysis of SCs. First, various custom‐tailored aptamers against the target SCs were selected through GO‐SELEX process. Toggling between different SC analytes during successive rounds of selection was performed to generate cross‐reactive aptamers. Then, the amino‐capped aptamers were synthesized and easily attached to the cysteamine‐covered gold electrodes. Analytical parameters and selectivity of the aptasensors were compared by using electrochemical techniques. After comparison of the analytical features and selectivity towards target analytes, one of the aptamers designated as Apta‐1 was chosen for further measurements. The aptasensor was tested by using differential pulse voltammetry technique against JWH‐018 (5‐pentanoic acid), selected as a model for SCs. The linearity and limit of detection were determined as 0.01–1.0 ng/mL and 0.036 ng/mL. Finally, sample application in synthetic urine samples was successfully performed with standard addition method, as confirmed by LC‐QTOF/MS. JWH‐018 (4‐hydroxypentyl), JWH‐073 (3‐hydroxybutyl), JWH‐250 (5‐hidroxypentyl) and HU‐210 were used to test the selectivity of the aptasensor and the system was shown to recognize all these SCs. Also other illegal drugs did not significantly interfere with the signal responses.     

Novel Approach for Rapid and Efficient Synthesis of 2‐(3‐(4‐Aryl)‐1‐isonicotinoyl‐4,5‐dihydro‐1H‐pyrazol‐4‐yl)‐3‐phenylthiazolidin‐4‐one Derivatives and Screened Their Antimicrobial Activity

A series of compounds, viz. 2‐(3‐(4‐aryl)‐1‐isonicotinoyl‐4,5‐dihydro‐1H‐pyrazol‐4‐yl)‐3‐phenylthiazolidin‐4‐one 4 ( a – n ), have been synthesized by reaction of 3 ( a – n ) with thioglycolic acid in the presence of zinc chloride. Compounds 3 ( a – n ) have been synthesized by amination of formylated pyrazoles 2 ( A – B ), which were synthesized by formylation of 1 ( A – B ) by Vilsmeier–Haack reagent (POCl₃/DMF). Compounds 1 ( A – B ) were synthesized by condensation of hydrazide and substituted acetophenones under conventional method and microwave irradiation method. These compounds were identified on the basis of melting point range, Rf values, infrared, ¹H NMR, and mass spectral analysis. These compounds were evaluated for their in vitro antimicrobial activity, and their minimum inhibitory concentration was determined. Among them, compound 4b and compound 4l possess appreciable antimicrobial and antifungal activities. Antibacterial activity results showed that compounds containing electron‐withdrawing groups were more active than compounds containing electron‐releasing groups.     

Classical Benzotriazole‐Mediated α‐Aminoalkylations of Alkynes: Synthesis and Characterization of Alk‐2‐yn‐1‐amines as Amphiphilic Materials

Reactions of readily available and stable benzotriazolemethanamines 1a – l , obtained from aldehydes and secondary amines (Scheme 2), gave the expected alk‐2‐yn‐1‐amines 3a – t (Scheme 3). The amphiphilic character of the synthesized products was responsible for physicochemical measurements. Specific aggregation properties of the obtained compounds make them useful as electroactive materials in the Langmuir–Blodgett technique.     

Diastereoselective Electrophilic Amination of Chiral 1‐Benzoyl‐2,3,5,6‐tetrahydro‐3‐methyl‐2‐(1‐methylethyl)pyrimidin‐4(1H)‐one for the Asymmetric Syntheses of α‐Substituted α,β‐Diaminopropanoic Acids

The chiral compounds (R)‐ and (S)‐1‐benzoyl‐2,3,5,6‐tetrahydro‐3‐methyl‐2‐(1‐methylethyl)pyrimidin‐4(1H)‐one ((R)‐ and (S)‐ 1 ), derived from (R)‐ and (S)‐asparagine, respectively, were used as convenient starting materials for the preparation of the enantiomerically pure α‐alkylated (alkyl=Me, Et, Bn) α,β‐diamino acids (R)‐ and (S)‐ 11 – 13 . The chiral lithium enolates of (R)‐ and (S)‐ 1 were first alkylated, and the resulting diasteroisomeric products 5 – 7 were aminated with ‘di(tert‐butyl) azodicarboxylate’ (DBAD), giving rise to the diastereoisomerically pure (≥98%) compounds 8 – 10 . The target compounds (R)‐ and (S)‐ 11 – 13 could then be obtained in good yields and high purities by a hydrolysis/hydrogenolysis/hydrolysis sequence.     

Facile Synthesis and Antimicrobial Activity of 5‐Amino‐3‐methyl‐1‐phenyl‐1H‐thieno[3,2‐c]pyrazole‐6‐carbonitrile and Their Derivatives

5‐Amino‐thieno[3,2‐c]pyrazole derivative 2 was prepared by Gewald reaction in a one‐pot procedure. The amino group of compound 2 like primary aromatic amine formed the diazonium salt when treated with NaNO₂/HCl, followed by coupling with different nucleophiles to yield the azo coupling products 3a – d . The reactivity of 5‐amino‐thienopyrazole 2 has been investigated towards different electrophilic reagents such as aromatic aldehydes, alkyl halide, acid chloride, acid anhydride, phenyl isothiocyanate, carbon disulfide, ethyl glycinate, and thioacetamide, which afforded the reaction products 4 – 14 , respectively.