Identification of an unknown peak 1

An unknown peak 1 was detected with an NMDA receptor channel blocker, diphenidine (Fig. 1b) [13] in the LC–MS and GC–MS for product A (Fig. 2a, b, e). Based on the LC–MS and GC–MS data, peak 1 was finally identified as a synthetic cannabinoid FDU-PB-22 (Figs. 1a, 2c, f) by direct comparison of the data to those of the purchased authentic compound (Fig. 2d, g). Compound 1 was thus detected as a newly distributed NPS in Japan. In addition, FDU-PB-22 (1) has been controlled as a designated substance (Shitei-Yakubutsu) in Japan since August 2014.

Fig. 2 Liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) analyses of product A. The liquid chromatography–ultraviolet-photodiode array (LC–UV-PDA) chromatogram (a), total ion chromatogram (TIC) (b), and electrospray ionization (ESI) mass and ultraviolet (UV) spectra of peaks 1 (c) and the authentic FDU-PB-22 (d) are shown. TIC (e), electron ionization (EI) mass spectra of peaks 1 (f) and the authentic FDU-PB-22 (g) obtained by GC–MS are also shown Full size image

Identification of an unknown peak 2

In the LC–MS and GC–MS analyses, an unknown peak 2 was detected with a synthetic cannabinoid (FUB-PB-22), its synthetic component 8-quinolinol, and a cathinone derivative (4-methylbuphedrone) in product B (Figs. 1b, 3a, b, d, f). By GC–MS analysis, peak 2 showed a molecular ion at m/z 394 (Fig. 3g). The major fragment ions of peak 2 (m/z 109 and 252) are the same as those of FDU-PB-22 (1) except for the molecular ion of compound 1 at m/z 395 (Fig. 2g). In the LC–MS analysis, peak 2 showed the protonated molecular ion at m/z 395 ([M + H]+) (Fig. 3c, e). However, the major fragment ion at m/z 252 of FDU-PB-22 (1) given by cleavage of an ester group (Fig. 2d) was not observed in the mass spectrum of peak 2 (Fig. 3e). The accurate mass spectrum obtained by LC–QTOF–MS gave an ion peak at m/z 395.1565, suggesting that the protonated molecular formula of compound 2 was C 26 H 20 N 2 OF (calcd. 395.1560).

Fig. 3 LC-MS and GC–MS analyses of product B. LC–UV-PDA chromatogram (a), TIC (b), extracted-ion chromatograms at m/z 395 (c) and 397 (d), and ESI mass and UV spectra of peak 2 (e) are shown. TIC (f) and EI mass spectrum of peak 2 (g) obtained by GC–MS are also presented Full size image

The structure of compound 2 was elucidated by NMR analysis (Fig. 4; Table 1). The analyses by 1H and 13C NMR, HH COSY, HMQC, HMBC, 15N HMBC and 1D-NOE spectra of compound 2 revealed the presence of a 1-(4-fluorobenzyl)-1H-indole (positions 1′ to 7′a and positions 1″ to 7″) and a N-(naphthalen-1-yl)-carboxamide moieties (position 1 and positions 1″′ to 8″′a) as shown in Fig. 4a, b. However, no HBMC correlation between the two moieties was observed. We, therefore, measured the deuterium isotope effect on the NH amide proton on the 13C chemical shift to determine the connection between the two moieties.

Fig. 4 Results of HH correlation spectroscopy (HH COSY), heteronuclear multiple-bond correlation (HMBC), selected nuclear Overhauser effect (NOE) correlations (a), 1H-15N HMBC correlations (b) and deuterium-induced isotope shift of NH protons for the 13C nuclear magnetic resonance (NMR) signals of compound 2 (FDU-NNEI) in CD 3 OD (c) Full size image

Table 1 NMR data of compound 2 Full size table

We compared the 13C NMR spectrum of compound 2, measured in CD 3 OH, with that in CD 3 OD. The isotope shift values for the 13C NMR signals of this compound are shown in Fig. 4c. The first- to fourth-largest deuterium shifts (0.115, 0.081, 0.043, 0.034 ppm) were observed at the positions of C-1″′, C-1, C-2″′, and C-8″′a of the N-(naphthalen-1-yl)carboxamide moiety. The fifth-largest deuterium shift of 0.024 ppm was attributed to the three-bond deuterium isotope effect of the NH amide proton on the indole carbon (C-3′). These results strongly suggested that the 1-(4-fluorobenzyl)-1H-indole moiety is connected at the 3′-position of the indole to the carboxamide (1-CONH).

On the basis of the above data, compound 2 was finally identified as 1-(4-fluorobenzyl)-N-(naphthalen-1-yl)-1H-indole-3-carboxamide (Fig. 1a). Compound 2 is a novel compound, and therefore, we named it FDU-NNEI because the structure of compound 2 consists of a [1-(4-fluorobenzyl)-1H-indolyl]carbonyl moiety and a (naphthalen-1-yl)amino moiety, which are characteristic parts of two synthetic cannabinoids, FDU-PB-22 (1) and NNEI, respectively (Fig. 1a–c) [10].

Identification of an unknown peak 3

We detected an unknown peak 3 together with seven peaks of five known synthetic cannabinoids (AB-CHMINACA, 5-fluoro-AMB, FUB-PB-22, AM-2201 indazole analog and NNEI indazole analog), a known cathinone derivative DL-4662, and 8-quinolinol in the LC–MS and GC–MS chromatograms for product C (Fig. 5a, b, e). In the LC–MS and GC–MS analysis, the unknown peak 3 showed a protonated molecular ion signal at m/z 357 [M + H+] (Fig. 5c) and a molecular ion signal at m/z 356 [M+] (Fig. 5f). The accurate mass spectrum obtained by LC–QTOF–MS gave an ion signal at m/z 357.2282, suggesting that the protonated molecular formula of compound 3 was C 20 H 29 N 4 O 2 (calcd. 357.2291). The presumed molecular formula of compound 3 (C 20 H 28 N 4 O 2 : 356) was thus the same as that of AB-CHMINACA (Fig. 1b). However, the LC–MS and GC–MS spectra patterns, in addition to each retention time, were different (Fig. 5c, d, f, g).

Fig. 5 LC–MS and GC–MS analyses of product C. The LC–UV-PDA chromatogram (a), TIC (b), and ESI mass and UV spectra of peak 3 (c) and the authentic AB-CHMINACA (d) are shown. TIC (e), and EI mass spectra of peaks 3 (f) and the authentic AB-CHMINACA (g) obtained by the GC–MS analysis are also shown Full size image

The observed 1H and 13C NMR spectra (Table 2), HH COSY, HMQC, HMBC and 15N HMBC correlations for compound 3 indicated the presence of N-cyclohexylmethyl-indazole and N-(1-amino-3-methyl-1-oxobutan-2-yl)-carboxamide moieties (Fig. 6a, b). Additionally, 2D-INADEQUATE correlation revealed that the cyclohexylmethyl-indazole moiety is connected at the 3′-position of the indazole to the carboxamide (C-1″′). Nevertheless, the 13C NMR chemical shifts of compound 3 were significantly different at the C-3′ (δc 129.1), C-7′ (δc 117.4) and C-7′a (δc 146.4) positions from those of AB-CHMINACA [C-3′ (δc 136.4), C-7′ (δc 110.6) and C-7′a (δc 141.2) as shown in Table 2.

Table 2 NMR data of compound 3 and AB-CHMINACA Full size table

Fig. 6 Results of incredible natural abundance double-quantum transfer experiment (INADEQUATE), HMBC, selected NOE correlations (a) and 1H-15N HMBC correlations (b) for compound 3 (AB-CHMINACA 2H-indazole analog) Full size image

We next compared the 15N NMR chemical shifts of compound 3 with those of 1H-indazole derivatives (Table 3). The 15N chemical shifts at the N-1′ (δ N −85.5) and N-2′ (δ N −153.9) of the indazole moiety in compound 3 were different from those of the 1H-indazole moiety in 1-methyl-1H-indazole [N-1′ (δ N −202.8) and N-2′ (δ N −56.6)] and AB-CHMINACA [N-1′ (δ N −191.4) and N-2′ (δ N −62.8)], respectively (Table 3; Fig. 1c). However, their shifts of compound 3 were similar to those of 2-methyl-2H-indazole [N-1′ (δ N −91.2) and N-2′ (δ N −161.0)] (Table 3; Fig. 1c) [14]. Therefore, the structure of compound 3 was determined as AB-CHMINACA 2H-indazole analog [N-(1-amino-3-methyl-1-oxobutan-2-yl)-2-(cyclohexylmethyl)-2H-indazole-3-carboxamide] (Fig. 1a).

Table 3 15N NMR data of detected and related compounds Full size table

It was reported that different forms of tautomerism are very common in nitrogen compounds. For indazoles, in most cases the 1H-tautomer is the most stable; however, sometimes several indazoles of the 2H-tautomer can be more stable than 1H-tautomer [14]. The differences in energy between the 1H- and 2H-tautomers were interpreted in terms of substituent effects [14], and we have assumed that the minor component AB-CHMINACA 2H-indazole analog (3) is generated via tautomerization from AB-CHMINACA (1H-indazole). This is the first report of the identification of 2H-indazole isomers of synthetic cannabinoids in illegal products to our knowledge. The chemical characterization, pharmacological activity and toxicological activity of 2H-indazole isomers of synthetic cannabinoids as NPSs have never been reported before.

Identification of unknown peaks 4–6

Three unknown peaks 4, 5, and 6 were detected along with a synthetic cannabinoid NNEI indazole analog in the LC–MS and GC–MS chromatograms for product D (Figs. 7a, b, i, 1b). In the LC–MS and GC–MS analysis, the unknown peak 4 showed a protonated molecular ion signal at m/z 358 [M + H+] (Fig. 7c, d) and a molecular ion signal at m/z 357 [M+] (Fig. 7j). The accurate mass spectrum obtained by LC–QTOF–MS gave an ion signal at m/z 358.1916, suggesting that the protonated molecular formula of compound 4 was C 23 H 24 N 3 O (calcd. 358.1919). Hence, the presumed molecular formula of compound 4 (C 23 H 23 N 3 O: 357) was the same as that of NNEI indazole analog. However, the GC–MS and LC–MS spectra patterns and each retention time of both compounds were different (Fig. 7d, e, j, k).

Fig. 7 LC–MS and GC–MS analyses of product D. The LC–UV-PDA chromatogram (a), TIC (b) and an extracted-ion chromatogram at m/z 358 (c) are shown, along with the ESI mass and UV spectra of peaks 4 (d), 5 (f), 6 (h), the authentic NNEI indazole analog (e) and the authentic 2-methylpropyl N-(naphthalen-1-yl) carbamate (g). TIC (i) and EI mass spectra of peaks 4 (j), 5 (l), 6 (n), the authentic NNEI indazole analog (k) and the authentic 2-methylpropyl N-(naphthalen-1-yl) carbamate (m) obtained by the GC–MS analysis are also indicated Full size image

The 1H and 13C NMR spectra (Table 4), HH COSY, HMQC, HMBC and 15N HMBC correlations of compound 4 suggested the presence of N-pentyl-indazole and N-(naphthalen-1-yl)carboxamide moieties (Fig. 8a, b). Therefore, the connectivity of the two moieties (positions 1 and 3′) was not observed. On the other hand, the 13C NMR chemical shifts of compound 4 at the C-3′ (δc 127.1), C-7′ (δc 119.0) and C-7′a (δc 147.3) positions were different from those of NNEI indazole analog [C-3′ (δc 137.3), C-7′ (δc 109.4), and C-7′a (δc 141.1)] (Table 4). Comparing the 15N NMR chemical shifts between the two compounds showed that the 15N chemical shifts at the N-1′ (δ N −82.7) and N-2′ (δ N −147.4) of the indazole moiety in compound 4 were different from those of the 1H-indazole moiety in NNEI indazole analog [N-1′ (δ N −187.8) and N-2′ (δ N −62.0)] (Table 3). However, their shifts of compound 4 were similar to those of 2-methyl-2H-indazole [N-1′ (δ N −91.2) and N-2′ (δ N −161.0)] (Table 3) [14]. Therefore, it was revealed that compound 4 has a 2-pentyl-2H-indazole moiety.

Table 4 NMR data of compound 4 and NNEI indazole analog Full size table

Fig. 8 Results of HH COSY, HMBC, selected NOE correlations (a), 1H-15N HMBC correlations (b) and deuterium-induced isotope shift of NH protons for the 13C NMR signals of compound 4 (NNEI 2H-indazole analog) in CD 3 OD (c). HH COSY and HMBC correlations (d) and 1H-15N HMBC correlations (e) for compound 6 (isobutyl 1-pentyl-1H-indazole-3-carboxylate), are also shown Full size image

We measured the deuterium isotope effect in compound 4 in the same way as that used for compound 2 (Fig. 4c) to determine the connection between the two moieties. The isotope shift values for the 13C NMR signals of this compound are shown in Fig. 8c. The first- to fourth-largest deuterium shifts (0.115, 0.087, 0.048, 0.038 ppm) were observed at the C-1″′, C-1, C-2″′, and C-8″′a positions of the N-(naphthalen-1-yl)-carboxamide moiety. The fifth-largest deuterium shift of 0.029 ppm was attributed to the three-bond deuterium isotope effect of the NH amide proton on the indazole carbon (C-3′). These results suggested that the 2-pentyl-2H-indazole moiety is connected at the 3′-position of the 2H-indazole to the carboxamide (1-CONH). Finally, the structure of compound 4 was determined as NNEI 2H-indazole analog [N-(naphthalen-1-yl)-2-pentyl-2H-indazole-3-carboxamide] (Fig. 1a).

The unknown peak 5 was identified as a 2-methylpropyl N-(naphthalen-1-yl) carbamate (Fig. 7f, l) by direct comparison of the GC–MS and LC–MS data to those of the purchased authentic compound (Fig. 7g, m). This compound has not been reported as any cannabimimetic- or cannabinoid-related substance.

The GC–MS and LC–MS spectra of the unknown peak 6 are shown in Fig. 7h, n. A molecular ion signal of compound 6 was observed at m/z 288 in the GC–MS analysis (Fig. 7n). The accurate mass spectrum obtained by LC–QTOF–MS gave an ion peak at m/z 289.1906, suggesting that the protonated molecular formula of compound 6 was C 17 H 25 N 2 O 2 (calcd. 289.1916). One-dimensional (1D)- and 2D-NMR analyses revealed that compound 6 has isobutyl-carboxylate and N-pentyl-indazole moieties, as shown in Fig. 8d and Table 5. The 15N HMBC correlations of N-1′ with H-6′ and H-2″ (Fig. 8e) and the 15N chemical shifts of compound 6 at the N-1′ (δ N −185.5) and N-2′ (δ N −53.0) of the indazole moiety, which were similar to those of the 1H-indazole moiety in NNEI indazole analog [N-1′ (δ N −187.8) and N-2′ (δ N −62.0)], suggested the presence of 1H-indazole (Table 3). No HMBC correlation was observed between the two moieties at position-3′ and ester group of compound 6. However, the major GC–MS fragment ion signal at m/z 187 was probably caused by the cleavage of a bond between the 1H-indazole and the ester group (Fig. 7n). In addition, another fragment ion signal at m/z 215 was probably caused by the cleavage of a bond of the ester group (Fig. 7n). Therefore, the structure of compound 6 was elucidated as isobutyl 1-pentyl-1H-indazole-3-carboxylate (Fig. 1a). Compound 6, which is a novel substance, was not reported to have any pharmacological and toxicological activity.

Table 5 NMR data of compound 6 Full size table

As a result, two major components, i.e., NNEI indazole analog and 2-methylpropyl N-(naphthalene-1-yl) carbamate (5), and three minor components, i.e., isobutyl 1-pentyl-1H-indazole-3-carboxylate (6), NNEI 2H-indazole analog (4) and a presumed 1-pentyl-1H-indazole (elucidated by GC–MS, data not shown) were detected in product D. On the basis of these minor components, we expected the following reaction mechanism for the preparation of the major NNEI indazole analog; compound 5 is likely to react with 1-pentyl-1H-indazole to yield the major component NNEI indazole analog (path a) and the minor component 6 (path b), as shown in Fig. 9. In addition, the 1H- and 2H-indazole tautomerism of the starting material or the reaction product accounted for the existence of the minor 2H-indazole product (4).

Fig. 9 Possible reaction mechanisms for the preparation of NNEI indazole analog and compound 6 Full size image

Identification of unknown peaks 7 and 8

Unknown peaks 7 and 8 were detected in the LC–MS chromatogram for product E (Fig. 10a, b, e). In the LC–MS chromatogram, the unknown peak 7 at 12.2 min showed a protonated molecular ion signal at m/z 224 ([M + H]+) (Fig. 10c). The accurate mass spectrum obtained by LC–QTOF–MS gave an ion peak at m/z 224.1280, suggesting that the protonated molecular formula of compound 7 was C 12 H 18 NO 3 (calcd. 224.1287).

Fig. 10 LC–MS and GC–MS analyses of product E. The LC–UV-PDA chromatogram (a), TIC (b), ESI mass and UV spectra of peaks 7 (c) and 8 (d) are shown. TIC (e) and EI mass spectra of peaks of decomposed compound [3,4-ethylenedioxy-N-methylamphetamine (3,4-EDMA)] (f), compound 8 (h) and the authentic 3,4-EDMA (g) obtained by GC–MS analysis are also presented Full size image

The observed 1H and 13C NMR (Table 6), HH COSY, HMQC, HMBC and 1D-NOE revealed that the structure of compound 7 was N-hydroxy-3,4-ethylenedioxy-N-methylamphetamine (N-OH-EDMA), as shown in Fig. 11a. In addition, Noggle et al. [15] reported that N-hydroxy-3,4-methylenedioxyamphetamine (N-OH-MDA), an analog of N-OH-EDMA (7), was unstable at high temperatures in a GC–MS analysis, and that N-OH-MDA (Fig. 1c) decomposed to MDA (Fig. 1c) [15]. In the present GC–MS TIC chromatogram, peak 7 (N-OH-EDMA) was not observed (Fig. 10e). However, the peak of its decomposed compound, which was finally identified as 3,4-ethylenedioxy-N-methylamphetamine (3,4-EDMA, Fig. 1b) by direct comparison of the GC–MS data to those of the purchased authentic compound (Fig. 10g), was detected (Fig. 10e, f). No pharmacological information is available for N-OH-EDMA (7); however, 3,4-EDMA has been reported to stimulate the release of serotonin and dopamine from rat brain synaptosomes at 1 μM [16].

Table 6 NMR data of compound 7 Full size table

Fig. 11 HH COSY, HMBC, and selected NOE correlations for compound 7 (N-OH-EDMA, a) and for compound 8 (dimethoxy-α-PHP, b) Full size image

The proposed fragment pattern and the presumed structure of peak 8 obtained by the GC–MS analysis are shown in Fig. 10h. The LC–MS data revealed that peak 8 gave a protonated ion signal at m/z 306 ([M + H]+) (Fig. 10d). The accurate mass spectrum obtained by LC–QTOF–MS gave an ion peak at m/z 306.2072, suggesting that the protonated molecular formula of compound 8 was C 18 H 28 NO 3 (calcd. 306.2069).

The 13C NMR spectrum of compound 8 was similar to a combination of two known cathinone derivatives: an α-pyrrolidinohexanone moiety of α-PHP and a 3,4-dimethoxyphenyl moiety of DL-4662 (Table 7) [13]. The observed 1H and 13C NMR (Table 7), HH COSY, HMQC, HMBC, and 1D-NOE correlations (Fig. 11b) suggested that the structure of compound 8 is dimethoxy-α-pyrrolidinohexanophenone (dimethoxy-α-PHP), as shown in Fig. 1a. The fragment ions at m/z 140 and 165 of compound 8 in the GC–MS spectrum corroborated the structure (Fig. 10h). Compound 8 was detected as a newly distributed designer drug, and its chemical and pharmaceutical data have not been reported. Dimethoxy-α-PHP (8) has been controlled as a designated substance (Shitei-Yakubutsu) in Japan since November 2014.