A synthetic cannabinoid FDU-NNEI, two 2H-indazole isomers of synthetic cannabinoids AB-CHMINACA and NNEI indazole analog (MN-18), a phenethylamine derivative N–OH-EDMA, and a cathinone derivative dimethoxy-α-PHP, newly identified in illegal products

Six new psychoactive substances were identified together with two other substances (compounds 1–8) in illegal products by our ongoing survey in Japan between January and July 2014. A new synthetic cannabinoid, FDU-NNEI [1-(4-fluorobenzyl)-N-(naphthalen-1-yl)-1H-indole-3-carboxamide, 2], was detected with the newly distributed synthetic cannabinoid FDU-PB-22 (1). Two 2H-indazole isomers of synthetic cannabinoids, AB-CHMINACA 2H-indazole analog (3) and NNEI 2H-indazole analog (4), were newly identified with 1H-indazoles [AB-CHMINACA and NNEI indazole analog (MN-18)]. In addition, 2-methylpropyl N-(naphthalen-1-yl) carbamate (5) and isobutyl 1-pentyl-1H-indazole-3-carboxylate (6) were detected in illegal products. Compound 6 is considered to be a by-product of the preparation of NNEI indazole analog from compound 5 and 1-pentyl-1H-indazole. A phenethylamine derivative, N–OH-EDMA [N-hydroxy-3,4-ethylenedioxy-N-methylamphetamine, 7], and a cathinone derivative, dimethoxy-α-PHP (dimethoxy-α-pyrrolidinohexanophenone, 8), were newly identified in illegal products. Among them, compounds 1 and 8 have been controlled as designated substances (Shitei-Yakubutsu) under the Pharmaceutical Affairs Law in Japan since August and November 2014, respectively.


Introduction
The consumption of new psychoactive substances (NPSs) including synthetic cannabinoids and cathinone derivatives has become widespread despite regulatory control measures [1][2][3][4][5][6][7]. The EMCDDA (European Monitoring Centre for Drugs and Drug Addiction) reported that 81 NPSs were identified by the EU early warning system in 2013, with 37 NPSs reported from January to May 2014 [2]. Ninety-seven NPSs were reported to the UNODC (United Nations Office on Drugs and Crime) in 2013 alone [3].

Samples for analyses
The analyzed samples were purchased via the Internet between January and July 2014 as 241 chemical-type or herbal-type products being sold in Japan. Among them, we show the analysis data of five products (A-E) for describing the identification of compounds 1-8 in this paper. Each of the herbal-type products (A-D) contained approximately 3 g of mixed dried plants. The single powder-type product called ''fragrance powder'' consisted of 400 mg of a brown powder (E).

Preparation of sample solutions
For the qualitative analyses, 10 mg of each herbal-type product was crushed into powder and extracted with 1 ml of methanol under ultrasonication for 10 min. A 2-mg portion of each powder-type product was extracted with 1 ml of methanol under ultrasonication for 10 min. After centrifugation (3,000 rpm, 5 min) of each extract, the supernatant solution was passed through a centrifugal filter (Ultrafree-MC, 0.45-lm filter unit; Millipore, Bedford, MA, USA) to serve as the sample solution for the analyses. If necessary, the solution was diluted with methanol to a suitable concentration before instrumental analyses. Each sample solution was analyzed by ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) and by gas chromatographymass spectrometry (GC-MS) in the electron ionization (EI) mode according to our previous report [11]. Two elution programs were used in the LC-MS analysis. Each analysis was carried out with a binary mobile phase consisting of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in acetonitrile). The elution program (1) used for analysis of cannabinoids was as follows: 35 % B (4-min hold), 65 % B to 75 % B (4-16 min), and up to 90 % B (16-17 min, 6-min hold) at a flow rate of 0.3 ml/ min. The elution program (2) used for the analysis of cathinone derivatives and other compounds was as follows: 5 % B to 20 % B (0-20 min), and up to 80 % B (20-30 min, 10-min hold). In this study, products A, B, and D were analyzed using program (1), and products C and E analyzed using program (2). GC-EI-MS was performed on an Agilent 6890N GC with a 5975 mass selective detector (Agilent Technologies, Santa Clara, CA, USA) using a capillary column (HP-1MS capillary, 30 m 9 0.25 mm i.d., 0.25-lm film thickness; Agilent Technologies) with helium gas as a carrier at 0.7 ml/min. The conditions were: electron energy, 70 eV; injector temperature, 220°C; injection, splitless mode for 1.0 min; oven temperature program, 80°C (1-min hold) and increase at a rate of The obtained GC mass spectra were compared to those of an EI-MS library (Mass Spectra of Designer Drugs 2013; WILEY-VCH, Weinheim, Germany). We also used our in-house EI-MS library of designer drugs obtained by our ongoing survey of illegal products and commercially available reagents for the structural elucidation.
We measured the accurate mass numbers of the target compounds by liquid chromatography-quadrupole-timeof-flight-mass spectrometry (LC-QTOF-MS) in the ESI mode according to our previous report [12].
For the isolation of each compound, we performed two preparative methods. One was recycling preparative HPLC on a JAI (Japan Analytical Industry, Tokyo, Japan) LC-9201 instrument with gel permeation JAIGEL-1H columns (JAI) and chloroform as an eluent. The other was silica gel or ODS column chromatography (CC) on a Biotage (Stockholm, Sweden) Isorela One instrument with a SNAP KP-sil column (particle size: 50 lm), SNAP Ultra column (particle size: 25 lm), or SNAP KP-C18-HS column (particle size: 50 lm) (Biotage).

Results and discussion
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.

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),   The structure of compound 2 was elucidated by NMR analysis (Fig. 4; Table 1). The analyses by 1 H and 13 C NMR, HH COSY, HMQC, HMBC, 15 N HMBC and 1D-NOE spectra of compound 2 revealed the presence of a 1-(4fluorobenzyl)-1H-indole (positions 1 0 to 7 0 a and positions 1 00 to 7 00 ) and a N-(naphthalen-1-yl)-carboxamide moieties (position 1 and positions 1 000 to 8 000 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 13 C chemical shift to determine the connection between the two moieties.
We compared the 13 C NMR spectrum of compound 2, measured in CD 3 OH, with that in CD 3 OD. The isotope shift values for the 13 C 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-1 000 , C-1, C-2 000 , and C-8 000 a of the N-(naphthalen-1-yl)carboxamide moiety. The fifth-largest deuterium shift of 0.024 ppm was attributed to the threebond deuterium isotope effect of the NH amide proton on the indole carbon (C-3 0 ). These results strongly suggested that the 1-(4-fluorobenzyl)-1H-indole moiety is connected at the 3 0 -position of the indole to the carboxamide (1-CONH).

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).
We next compared the 15 N NMR chemical shifts of compound 3 with those of 1H-indazole derivatives ( Table 3). The 15 N chemical shifts at the N-1 0 (d N -85.5) and N-2 0 (d N -153.9) of the indazole moiety in compound   (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). 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).
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 isobutylcarboxylate and N-pentyl-indazole moieties, as shown in Fig. 8d and Table 5. The 15 N HMBC correlations of N-1 0 with H-6 0 and H-2 00 (Fig. 8e) (Table 3). No HMBC correlation was observed between the two moieties at position-3 0 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.  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 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).
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 13 C NMR spectrum of compound 8 was similar to a combination of two known cathinone derivatives: an apyrrolidinohexanone moiety of a-PHP and a 3,4-  (Table 7) [13]. The observed 1 H and 13 C NMR (Table 7), HH COSY, HMQC, HMBC, and 1D-NOE correlations (Fig. 11b) suggested that the structure of compound 8 is dimethoxy-a-pyrrolidinohexanophenone (dimethoxy-a-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-a-PHP (8) has been controlled as a designated substance (Shitei-Yakubutsu) in Japan since November 2014.