Elucidation of the absolute configuration of a tadalafil analogue found as adulterant in a health supplement by mass spectrometry, chiroptical methods and NMR spectroscopy

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Highlights

  • The cis-(6R, 12aR) configuration of dipropylaminopretadalafil has been revealed by chiroptical methods and NMR.

  • Possibility of misidentification of tadalafil-like stereoisomer due to limitation of achiral separation and MS.

  • NOESY is a promising method to identify the position of stereogenic protons.

Abstract

The chirality of a dipropylaminopretadalafil stereoisomer isolated from a health supplement has been studied. Under high resolution mass spectrometry (HRMS) study, this unknown compound seems to be one of the trans configuration tadalafil analogues i.e. (6S, 12aR) or (6R, 12aS), owing to the same precursor ion at m/z 492 with mass errors within ±2 ppm tolerance and very close retention times. Moreover, the MS2 fragmentation pattern is also very similar to the two trans isomers. Fortunately, the unknown compound can be distinguished from the two trans isomers by enantioselective separation with the use of a chiral column. Further comparison studies with a series of homologous compounds without a diketopiperazine ring on ellipticity and optical rotation support the unknown compound to be in the cis-(6R, 12aR) configuration. The nuclear magnetic resonance (NMR) in one dimensional (1D) and nuclear overhauser effect spectroscopy (NOESY) have affirmed the abovementioned configuration.

Introduction

In the drug discovery of tadalafil, various cis and trans isomers can be formed in the tetrahydro β-carboline systems. For instance, the use of racemic tryptophan methyl ester as starting material has resulted two cis (6R, 12aR/6S, 12aS) and two trans (6R, 12aS/6S, 12aR) stereroisomers [1]. This has also been demonstrated in the synthesis of 4 stereoisomers of aminotadalafil [2]. Amongst these four stereoisomers, only cis-(6R, 12aR) configuration tadalafil and its homologous diastereoisomers have shown the promising PDE-5 inhibition activities [1].

Thus, in order to obtain this configuration, researchers have optimized the yield of therapeutically active cis-oriented stereoisomer with the use of D-tryptophan methyl ester as the starting material [2,3]. Results have shown that both cis and trans products can be yielded through the epimerization process [1,3]. For instance, when D-tryptophan methyl ester is used to react with 1,3-benzodioxo-5-carboxylaldehye under the modified Piectet-Spengler reaction, only 47% of cis-(1R, 3R)-methyl (1,3-benzodioxol-5-yl)-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylate has been achieved. A smaller fraction of trans isomer (17%), has also been obtained [1]. It is worthwhile pointing out that the epimerization also occurred at different stereogenic centers under thermodynamically favoured conditions, resulting in quantitative yield of trans-(6S, 12aR) N-benzyl nortadalafil between cis-(6R, 12aR) nortadalafil and iodobenzene at 110 °C. Only by lowering the reaction temperature to 14 °C and increasing the catalyst equivalent, the ratio of desired cis-(6R, 12aR)/trans-(6S, 12aR) arylated nortadalafil compounds to increases [3].

With the increase in the detection of tadalafil analogues as adulterants in health supplements and as the structures are getting more complex, there is an urgent need to detect them in a more reliable manner [4]. Up-to-date, 23 tadalafil analogues have been reported, including 21 analogues summarized in a review article [4] and two recently discovered analogues, N-3-hydroxypropylnortadalafil [5], and a new type tadalafil analogue with a hydantoin ring [6]. Even though the chirality of tadalafil and related compounds has been thoroughly investigated in the drug discovery, the trend of analysis of tadalafil analogues found as adulterants in dietary supplements, however, are mainly relied on mass spectrometry and NMR spectroscopy [4,7,8]. One plausible reason is that the achiral chromatography used for drug screening is not ideal to identify these stereoisomers. Only compounds with distinctive retentivity like trans-tadalafil [9] and trans-cylcopentyl tadalafil have been described [10].

Two dimeric tadalafil analogues, trans-bisprehomotadalafil [11] trans-bisprecyclopentyltadalafil [10] have been reported in 2015 and 2016 respectively. According to researchers, the stereochemistry of trans-bisprehomotadalafil and trans-bisprecyclopentyltadalafil has been assigned by observing no nuclear overhauser effect (NOE) correlation between protons at the stereogenic centers (positions 6 and 12a) and also close proximity of the proton resonance signals with trans-tadalafil. In 2017, Lee and workers revised these two analogues to cis-(6R, 12aR) configuration with a series of synthetic work after realizing that the synthesized trans-oriented analogues had different HPLC retention times from the isolated compounds [12]. They only observed the strong NOE-correlation when deuterated acetone (acetone-d6) was used as solvent, which did not happen for the experiments using deuterated chloroform (CDCl3) [10,11]. Therefore, it is important to elucidate the absolute configuration of new emerging tadalafil analogues with extensive effort because the synthetic routes of active substance and manufacturing processes of illegal sexual enhancement products are poorly controlled [13].

In this study, we have characterized a tadalafil analogue found in a health supplement using HRMS, circular dichroism, optical rotation and NMR spectroscopy. The high resolution mass data (MS and MS2) support the unknown compound has the same skeleton as dipropylaminopretadalafil reported in 2016 [14]. Meanwhile, both chiroptical methods have suggested it to be in cis-(6R, 12aR) orientation. Ultimately, the NOESY study has clearly shown that the two stereogenic protons are aligned in the cis orientation.

Section snippets

Materials and chemicals

One health supplement was received from a commercial source. The product was capsules containing brown powder. Tadalafil impurity 9 or trans-(6R, 12aS) dipropylaminopretadalafil (99.4% purity), tadalafil N-dipropyl impurity or trans-(6S, 12aR) dipropylaminopretadalafil (100% purity) and tadalafil related compound 1 or cis-(6R, 12aR) diethylaminopretadalafil (97.8%) were purchased from TLC Pharmaceutical Standards Ltd. (Aurora, Ontario, Canada). Tadalafil (99.9% purity) was purchased from United

HPLC-UV screening

The methanolic extracted sample solution showed an overloading peak in the time range of 19.5–24.5 minutes. The sample solution has been further diluted to produce an intense peak at 24.6 min, showing very similar UV profile like diethylaminopretadalafil (Fig. 1). However, the retention time of the diluted sample solution in database is 20.8 min, indicating the unknown compound has much higher hydrophobicity than diethylaminopretadalafil. Given the “clean” chromatogram with solely a major peak,

Conclusion

The identity of the unknown compound has been unveiled by chiroptical methods and NMR spectroscopy studies. Obviously, achiral separation and mass spectrometry have their own limitations for the detection of tadalafil analogues. The specific optical rotation of the homologous tadalafil compounds can be a very useful measurement to gauge the type of stereoisomer. However, more conclusive results can be only achieved with proper methodology. In this correlative study, NOESY has provided us with

Acknowledgements

The authors would like to thank TLC Pharmaceutical Standards Ltd. for providing us the specific optical rotation values for tadalafil related compound 1, tadalafil related compound 2, tadalafil dichloro impurity, chloropretadalafil, tadalafil N-dipropyl impurity and tadalafil impurity 9. They also would like to thank Dr. Lingkai Wong from Chemical Metrology Laboratory, Health Sciences Authority for his assistance in the NMR experiments.

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