Two thiazolylindoles and a benzimidazole: Novel compounds on the designer drug market with potential cannabinoid receptor activity

Accepted Manuscript Title: Two thiazolylindoles and a benzimidazole: novel compounds on the designer drug market with potential cannabinoid receptor activity Author: Folker Westphal Frank D. S¨onnichsen Siegfried Knecht Volker Auw¨arter Laura Huppertz PII: DOI: Reference:

S0379-0738(15)00028-6 http://dx.doi.org/doi:10.1016/j.forsciint.2015.01.014 FSI 7877

To appear in:

FSI

Received date: Revised date: Accepted date:

21-9-2014 27-12-2014 22-1-2015

Please cite this article as: F. Westphal, F.D. S¨onnichsen, S. Knecht, V. Auw¨arter, L. Huppertz, Two thiazolylindoles and a benzimidazole: novel compounds on the designer drug market with potential cannabinoid receptor activity, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.01.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights Two 3-([1,3]-thiazol-2-yl)indoles and one benzimidazole seized as pure

ip t

compounds

cr

New compounds on the designerdrug market with potential cannabinoid

us

receptor activity

an

Mass spectrometric, infrared spectroscopic and NMR spectroscopic data

uncommon

compared

to

already

established

Ac ce p

te

cannabimimetics

compounds

d

Rather

M

presented

Page 1 of 61

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Two thiazolylindoles and a benzimidazole: novel compounds on the designer drug market with potential cannabinoid receptor activity Folker Westphal1*, Frank D. Sönnichsen2**, Siegfried Knecht3, Volker

Bureau

of

Criminal

Investigation

cr

State

Schleswig-Holstein,

Section

us

1

ip t

Auwärter4, Laura Huppertz4

Narcotics/Toxicology, Mühlenweg 166, D-24116 Kiel, Germany, E-Mail: Dr.-

an

[email protected], Tel.: 0049 (0)431/160-4724, Fax: 0049 (0)431/160-4444

Otto-Diels-Institute of Organic Chemistry, Christian-Albrechts-University

M

2

Center for Education and Science of the Federal Finance Administration,

te

3

d

Kiel, Olshausenstr. 40, 24098 Kiel, Germany

4

Ac ce p

Laboratory Frankfurt, Gutleutstr. 185, 60327 Frankfurt a.M., Germany Institute of Forensic Medicine, Department of Forensic Toxicology, Medical Center – University Freiburg, Albertstr. 9, 79104 Freiburg, Germany *

Corresponding author, **manuscript prepared in equal parts with corresponding

author

Abstract In a seizure of German customauthorities two 3-([1,3]-thiazol-2-yl)indoles (N(2-methoxyethyl),N-iso-propyl-2-(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine (1) and N,N-diethyl-2-(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine Page 2 of 61

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(2)) and one benzimidazole (1-(cyclohexylmethyl)-2-[(4-ethoxyphenyl)methyl]N,N-diethyl-1H-benzimidazole-5-carboxamide

(6))

were

seized

as

pure

compounds. The compounds have been detected in Germany for the first time,

ip t

and no analytical data had been previously published. Mass spectrometric (MS),

cr

infrared (IR) spectroscopic, and nuclear magnetic resonance (NMR) spectros-

us

copic data are presented and the way of the structure elucidation of these rather

an

uncommon compounds is discussed.

Key words

M

3-([1,3]-thiazol-2-yl)indole, thiazolemethanamine, benzimidazole, cannabimi-

Ac ce p

te

d

metic, structure elucidation, GC-MS, LC-HRMS, NMR, IR

Page 3 of 61

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Two thiazolylindoles and a benzimidazole: novel compounds on the designer drug market with potential cannabinoid receptor activity

ip t

Abstract

cr

In a seizure of German customauthorities two 3-([1,3]-thiazol-2-yl)indoles (N-

us

(2-methoxyethyl),N-iso-propyl-2-(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine (1) and N,N-diethyl-2-(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine

an

(2)) and one benzimidazole (1-(cyclohexylmethyl)-2-[(4-ethoxyphenyl)methyl]N,N-diethyl-1H-benzimidazole-5-carboxamide

(6))

were

seized

as

pure

M

compounds. The compounds have been detected in Germany for the first time,

d

and no analytical data had been previously published. Mass spectrometric (MS),

te

infrared (IR) spectroscopic, and nuclear magnetic resonance (NMR) spectros-

Ac ce p

copic data are presented and the way of the structure elucidation of these rather uncommon compounds is discussed.

Key words

3-([1,3]-thiazol-2-yl)indole, thiazolemethanamine, benzimidazole, cannabimimetic, structure elucidation, GC-MS, LC-HRMS, NMR, IR

Introduction Cannabimimetics are the compound class with the largest structural diversity of new psychoactive substances (NPS) presently appearing on the market. Many of Page 4 of 61

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them show high binding activities to CB1 and/or CB2 receptors and are known to mimic the effects of Δ9-tetrahydrocannabinol (THC), the major psychotropic active ingredient of cannabis. The effects of THC are mediated by binding to

ip t

and activation of CB1 receptors. In recent years, a very large variety of

cr

cannabimimetics entered the illegal drug market [1-8]. Starting with naphthoyl-,

us

benzoyl-, phenacetyl-, adamantoyl-, and cyclopropoylindols now indolamides, indazoles, indazol(di)amides and indol(chinolinyl)esters have appeared,

an

occasionally combined with other rare or less common structures. With increasing frequency, even pharmaceuticals are entering the designer drug

M

market in large quantities not in typical pharmaceutical packaging but in pure

te

d

state for misuse purposes [9].

Ac ce p

Besides three already known cannabimimetics (JTE-907, A-834,735 and A836,339) two brown oily compounds (1 and 2, each of them about 10 grams) and a beige-colored powder (6, about 5 g) were seized from a mail package from China by German custom authorities. The three substances were not identifiable by comparison with IR- and MS-spectra of the available databases. Thus, for structure elucidation high resolution mass spectrometry (HRMS) and NMR spectroscopy were performed in addition to IR and GC-MS and GC-MS/MS analysis.

Page 5 of 61

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Methods Chemicals

ip t

Diethylether and ethanol were purchased from Merck (analytical grade, Darmstadt, Germany). CDCl3 was obtained from Euro-Isotope (Saint Aubin,

cr

France). Formic acid (Rotipuran® ≥ 98%, p.a.) and 2-propanol (Rotisolv®

us

≥ 99.95%, LC-MS grade) were purchased from Carl Roth (Karlsruhe, Germany). Sodium hydroxide (volumetric solution, 1 mol/L) was supplied by

an

Riedel-de Haën (Seelze, Germany), acetic acid (Emsure®, glacial, anhydrous,

M

100%) was from Merck (Darmstadt, Germany), and ammonium formate (99.995%) as well as methanol (LC-MS-grade) were purchased from Sigma

d

Aldrich (Steinheim, Germany). A cartridge deionizer (Memtech, Moorenweis,

te

Germany) was used for the preparation of deionized water.

Ac ce p

Gaschromatography mass spectrometry and product ion spectrometry (GC-MS and GC-MS/MS) Sample preparation:

For the analysis of the seized compounds 1 and 2 approximately 2 mg of the oils were dissolved in 1.5 mL ethanol. Approximately 2 mg of the powder (6) were dissolved in 2 mL of deionized water, alkalized with 5 % NaOH, and extracted with 2 mL diethylether. For analysis 1 µL of the ethanolic solution/the ethereal extract was injected into the GC-MS system.

Page 6 of 61

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Equipment: Electron ionization (EI) and chemical ionization (CI) mass spectra were obtained with a Finnigan TSQ 7000 triple stage quadrupole mass spectrometer

ip t

(Thermo Scientific, Dreieich, Germany) coupled to a gas chromatograph (Trace

cr

GC Ultra, Thermo Scientific, Dreieich, Germany) with an auto sampler CTC

us

CombiPAL (CTC Analytics, Switzerland).

an

GC parameters:

Samples were introduced via the gas chromatograph with splitless injection

M

using a fused silica capillary column DB-1 (30 m × 0.25 mm, film thickness

d

0.25 µm). The temperature program started with an initial temperature of 80 °C,

te

held for 2 min, followed by a ramp to 310 °C with 20 °C/min. The final

Ac ce p

temperature was held for 25 min. The injector temperature was 250 °C. The transfer line temperature was maintained at 300 °C. The carrier gas was helium in constant flow mode at a flow rate of 1.2 mL/min.

MS parameters:

The electron ionization (EI) energy was 70 eV with an emission current of 200 µA. The scan time was 1 s and the scan range was m/z 29-700. The ion source temperature was maintained at 175 °C.

Page 7 of 61

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The chemical ionization (CI) energy was 70 eV with an emission current of 200 µA and a source temperature of 175 °C. The reactant gas was methane and the source pressure was 1.5 mTorr (0.2 Pa). The scan time was 0.5 s and the

cr

ip t

scan range was m/z 50-600.

us

In the EI-MS/MS-product-ion-mode the ionization energy was 70 eV with an emission current of 200 µA and a source temperature of 175 °C. The scan time

an

was 1 s and the scan range was m/z 10 to 10 amu above the selected ion mass. The collision gas was argon. The collision energy was set to approximately

M

20 eV and the collision gas pressure to approximately 1.5 mTorr (0.2 Pa). The

d

exact target-thickness [10] was set using n-butyl benzene and adjusting intensity

te

ratios m/z 92/91 to 0.2 and m/z 65/91 to 0.02 by variation of collision energy and

Ac ce p

collision gas pressure [10]. This ensures the reproducibility of the product ion mass spectra and the use of a product ion mass spectra library for the identification of the structure of the product ions [11].

Retention indices (RI) are given as Kovats indices calculated after measurement of an n-alkane mixture analyzed with the above mentioned temperature program.

Page 8 of 61

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High resolution liquidchromatography spectrometry (LC-HRMS) Equipment: For HRMS analysis a maXis impact Q-TOF instrument (Bruker Daltonik,

ip t

Bremen, Germany) coupled with a Dionex UltiMate 3000 RSLC HPLC-system,

cr

consisting of a SRD-3600 solvent rack degasser, a HPG-3400RS binary pump

us

with solvent selection valve, a WPS-3000TRS thermostatted autosampler, and a TCC-3000RS thermostatted column compartment (Thermo Fisher Scientific,

an

Dreieich, Germany) was used.

M

LC parameters:

d

Chromatographic separation was performed on a Dionex Acclaim RSLC 120

te

C18 2.2µm 120Å 2.1 x 100mm column (Thermo Fisher Scientific, Dreieich, Germany) using H2O/MeOH 90/10 (v/v) with 5 mM ammonium formate and

Ac ce p

0.01 % formic acid (A) and MeOH with 5 mM ammonium formate and 0.01 % formic acid (B). Gradient elution was as follows: Starts with 1 % B at a flow rate of 0.2 mL/min, for 1 min, increases to 39 % B in 2 min, increases to 99.9 % B and a flow rate of 0.4 mL/min in 9 min, holds 99.9 % B for 2 min and increases flow rate to 0.48 mL/min. Initial mobile phase composition was restored within 0.1 min and the flow rate decreases to 0.2 mL/min after 3 min. Starting conditions were held for 0.9 min. The temperature of the column compartment and the autosampler were set to 30 °C and 5 °C, respectively. The injection volume was 1 µL. HyStar ver. 3.2 and DataAnalysis ver. 4.2 (including

Page 9 of 61

- 10-

the software tool SmartFormula) software (Bruker Daltonik, Bremen, Germany) were used for data acquisition and evaluation, respectively.

ip t

MS parameters:

cr

The instrument was operated in positive ionization mode with an m/z range from

us

30-1000. The dry gas temperature was 200 °C with a dry gas flow of 8.0 L/min. Nebulizer gas pressure was 2.0 bar. Full scan and broadband collision induced

an

dissociation (bbCID) data were acquired in one run. The collision energy applied for bbCID was 30 ± 6 eV. Nitrogen was used as collision gas. The

M

voltages for the capillary and end plate offset were set to 2500 and 500 V,

te

d

respectively.

Ac ce p

External mass calibration was performed per working day using sodium formate/acetate clusters and high precision calibration (HPC) mode. Internal mass calibration was automatically performed within every run using sodiumformate/acetate clusters and HPC mode. The calibration solution was prepared as follows: 750 µL acetic acid, 250 µL formic acid and 500 µL sodium hydroxide solution (1M) were added to 500 mL of a mixture of 2-propanol and H2O (50:50, v/v).

Page 10 of 61

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Sample preparation: The samples were diluted with methanol, 100 µL were transferred to an LC-vial,

cr

Nuclear magnetic resonance spectroscopy (NMR)

ip t

evaporated and redissolved in 100 µL LC-eluent A : B (50 : 50, v/v).

us

Approximately10 mg of the compounds were dissolved in 0.6 mL deuterated

an

chloroform that contained tetramethylsilane as internal standard.

M

One- and two-dimensional NMR measurements were performed on a BRUKER DRX 500 MHz or Avance III 600 MHz NMR spectrometer with cryogenically

d

cooled triple resonance probehead at 300 K, employing the manufacturer’s pulse

13

C-, 1H-13C-HSQC-, 1H-13C-HMBC-, 1H,1H-gCOSY-

Ac ce p

dimensional 1H- and

te

programs and standard parameters. The following spectra were recorded: one

experiments. Further, for compounds 1, 2 and 6 concentrated solutions were prepared dissolving 76 mg (1), 78 mg (2),and 53 mg (6) in 0.6 mL CDCl3, respectively.

13

C-13C-INADEQUATE experiments were acquired in 24 to 36

hours, using 640 or 900 scans, a spectra width of 180 ppm and 16 data points in the direct 13C(F2) dimension, and a spectral width of 50 ppm and 64 total data points in the indirect, double quantum frequency (F1) dimension.

Page 11 of 61

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Infrared spectroscopy (IR) The spectrometer used was a Nicolet 380 FT-IR with Smart Golden Gate Diamond ATR. The wave length resolution was set to 4 cm-1. The IR spectrum

cr

ip t

was collected in a range of 650–4000 cm-1.

us

Results and discussion

The GC/MS spectra of the compounds 1 and 2 after electron ionization (EI) and

an

after chemical ionization (CI) with methane as reagent gas are shown in figures

Ac ce p

te

d

M

1 and 2.

Page 12 of 61

- 13T: + c EI Q3MS [ 29,00-700,00] 283

100

RI: 3172 (DB-1)

95 90 85 80 75 70

60 55 284 50

ip t

Relative Abundance

65

45 40 35 30

367

25 116

298 155

15

213

43

5 0

354

156

10

30

71 45

84

72

50

101

113

128 143 142

100

212 211

177

150

227 228

200

324

281

254 250

300

369

334

95 90

80 75 70 65 60

M

55 50 45 40 401,26

35 30

400

[M+H]+

398,24

367

368 401

[M+29]+

25 20 15

403,26

397,42

10

71

76

86

100

398

146

155

400

173

402 m/z

150

200

200

213

398 428

404,35

227

241

384 369

283

404

te

5

116 118 102 130

d

402,26 399,33

0

398

400

an

85

384

350

us

400,25

100

Relative Abundance

368

310

m/z

T: + c CI Q3MS [ 50,00-700,00]

cr

20

267

250 m/z

281

286

311

402

354 325

300

342 350

[M+41]+

429 403 400

441 450

Ac ce p

Fig. 1: EI-MS spectrum (top) and CI-MS spectrum of 1 (bottom)

Page 13 of 61

- 14T: + c EI Q3MS [ 29,00-700,00] 355

284

100 95

RI: 2930 (DB-1)

90 85 80 75 70

60 55 356

50

ip t

Relative Abundance

65

45 40 357

35

358

30 355

356 m/z

25

357

358

285

15

142

283

10

155

213 214 226

72 43

58

0

71

73

113

86

50

101 100

6656-05-CI #995 RT: 20,31 AV: 1 T: + c CI Q3MS [ 50,00-600,00]

156

128

170

150

SB: 2 19,94 , 21,24

212

185

200 m/z

NL: 1,67E7

95 90

254

287

326

298

300

356,23

80 75 70 65 60 55

M

50 45 40 35 30 25

[M+H]+

[M+29]+

72,11

10 58,08

102,13 114,12

384,25 340,19

213,14

155,08

188,13

te

0 100

d

74,12

5

350

284,15

20 15

356

341

an

85

281

250

100

355

286

227 241

us

5

Relative Abundance

cr

20

150

241,19

200

250

313,20 326,23

269,21

300

370,24 350

[M+41]+

396,25 400

m/z

Ac ce p

Fig. 2: EI-MS spectrum (top) and CI-MS spectrum of 2 (bottom)

Compound 1 has a molecular weight of 399 amu. The molecular radical cation of 1 is only very weak in the MS spectrum after EI. Compound 2 has a molecular weight of 355 amu. Both compounds show a base peak at m/z 283/284 and some similar fragments at m/z 115,155, 213 and 227. The GC-MS product ion spectra of the three fragments m/z 155, 213 and 227 of both compounds are each identical, so these fragments were assumed to have the same structure in 1 and 2. Therefore, it could be concluded that both compounds 1 and 2 have a common substructure element. Fragments at m/z 115, 143/144,

Page 14 of 61

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155 and 214 are typical – but not exclusive of course – for cannabimimetics with indole partial structure, N-pentyl substitution at the indole nitrogen, and a carbonyl group attached to the indole ring [12-15]. Compounds 1 and 2 both

ip t

show a fragment at m/z 213.This is one mass unit lower than 214, and thus

cr

suggested a substitution of O by NH in the carbonyl moiety of an N-pentylated

us

indoloylcation.

an

Both compounds show characteristic fragments that originate from immonium ions: the fragment of compound 1 at m/z 116 belongs to the oxygen-containing

M

immonium ion series with the molecular formula C6H14NO+, indicating an

d

alkoxy-substituted aminomethyl moiety. Compound 2 exhibits two fragments at

te

m/z 86 and m/z 72, belonging to the normal immonium series with molecular

Ac ce p

formulas C5H12N+ and C4H10N+, respectively. Product ion spectrometric examinations [11] showed that the fragment m/z 86 is caused by a N,N-diethylamino moiety. The fragment m/z 116 could not be identified by product ion spectrometry as our database did not contain any entry for this species.

The relatively large M+2 isotope signals (inserts in Figs. 1 and 2) suggested the presence of one sulfur atom in the molecule. The presence of sulfur along with a possible molecular formula was confirmed by LC-HRMS measurement for both compounds (Figs. 3 to 6).

Page 15 of 61

- 16Intens.

x105 4

+MS, 9.54min #1126

1+ 400.2435

Acquired spectrum

3 2

1+ 401.2463

ip t

1

1+ 402.2445

0 x105

2

cr

3

us

4

C 23 H 34 N 3 O 1 S 1 , 400.2417

1+ 400.2417

Simulated spectrum of C23H34N3OS ([M+H]+)

1+ 401.2447

1

399

400

Error [ppm] -4.6 1.7 -1.7 3.4 39.8

Error [mDa] -1.8 0.7 -0.7 1.4 15.9

402

mSigma 18.7 38.2 40.1 64.3 14.7

403

rdb 8.5 4.5 10.5 0.5 8.5

N-Rule ok ok ok ok ok

m/z

e¯-Config. even even even even even

te

d

Suggested molecular formulae for compound 1: Measured m/z # Ion Formula m/z 400.2435 1 C23H34N3OS 400.2417 2 C19H34N3O6 400.2442 3 C16H26N13 400.2429 4 C12H34N9O4S 400.2449 5 C23H34N3O3 400.2595

401

M

398

an

0

1+ 402.2420

Acquired and simulated LC-HRMS-spectrum of compound 1 with suggested molecular formulae (SmartFormula)

Ac ce p

Fig.3: Intens. x106

1.0

0.8

EIC C23H34N3OS ([M+H]+)

0.6

400.2417 ± 0.005 m/z

0.4

EIC C23H34N3O3 ([M+H]+) 0.2

400.2595 ± 0.005 m/z

0.0 8.0

Fig.4:

8.5

9.0

9.5

10.0

10.5

11.0

11.5

Time [min]

Extracted chromatogram traces of C23H34N3OS and C23H34N3O3, illustrating the presence of one sulfur atom instead of two oxygens

Page 16 of 61

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Intens. x105 5

1+

Acquired spectrum

+MS, 9.27min #1094

356.2166

4

1+ 357.2197

2

1+ 358.2162

1

4 3

C 21 H 30 N 3 S 1 , 356.2155

cr

5

1+ 356.2155

Simulated spectrum of C21H30N3S ([M+H]+)

us

0 x105

1+ 357.2185

2

an

1 0 355.5

356.0

356.5

Error [ppm] -3.2 3.8 -3.7 46.6

Error [mDa] -1.1 1.4 -1.3 16.6

358.0

mSigma 13.2 28.7 42.5 11.6

358.5

359.0

rdb 8.5 4.5 5.5 8.5

N-Rule ok ok ok ok

m/z

e¯-Config. even even even even

Acquired and simulated LC-HRMS-spectrum of compound 2 with suggested molecular formulae (SmartFormula)

Ac ce p

Fig. 5:

357.5

1+ 358.2115

te

d

Suggested molecular formulae for compound 2 Measured m/z # Ion Formula m/z 356.2166 1 C21H30N3S 356.2155 2 C17H30N3O5 356.2180 3 C13H26N9O3 356.2153 4 C21H30N3O2 356.2333

357.0

M

355.0

ip t

3

Intens. x106 1.25

1.00

EIC C21H30N3S ([M+H]+)

0.75

356.2155 ± 0.005 m/z

0.50

0.25

EIC C21H30N3O2 ([M+H]+) 356.2333 ± 0.005 m/z

0.00 2

Fig. 6:

4

6

8

10

12

Time [min]

Extracted chromatogram traces of C21H30N3S and C21H30N3O2, illustrating the presence of one sulfur atom instead of two oxygens

Page 17 of 61

- 18-

Based on these results, compound 1 has a molecular formula of C23H33N3OS and compound 2 a molecular formula of C21H29N3S. The isobaric molecular formulae C23H33N3O3 and C21H29N3O2 with two oxygen atoms instead of the

ip t

sulphur could be excluded as follows (Figs. 3/4 and 5/6, respectively): The only

cr

oxygen containing molecular formulae showed very large deviations in

us

measured and calculated mass (Figs. 3 and 5, respectively). Further, no extracted ion chromatogram for the molecular formulae only containing oxygen atoms

an

instead of the sulfur could be obtained (Figs. 4 and 6, respectively). All other

Ac ce p

te

d

with NMR results (see below).

M

hits for the calculated molecular formulae in Figs. 4 and 6 were inconsistent

Page 18 of 61

- 19Intens.

Intens.

x106

+bbCID MS, 30.0eV, 9.56min

x106

283.1276

1.25

Intens. x104

213.1393

1.0 0.8

1.00

1.0

0.6

227.0645

0.4

0.75

0.0 190

0.50

EIC 283.1276 ± 0.001 m/z

210

220

230 m/z

0.25 116.1072

0.4

100

150

EIC 116.1072 ± 0.002 m/z

200

250

300

350

us

0.2

400.2411

0.00 50

cr

0.6

200

ip t

0.2

0.8

400

m/z

EIC 400.2411 ± 0.002 m/z

9.0

9.5

10.0

10.5

11.0

Error [mDa] 0.6 -1.2 -0.7 -0.7 -0.2

mSigma 10.0 12.3 52.2 51.5 29.9

12.0

rdb 8.5 9.5 9.5 7.5 0.5

12.5

N-Rule ok ok ok ok ok

Time [min]

e- Config. even even even even even

bbCID spectrum and extracted chromatographic traces of compound 1 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 400.2411 are not annotated (non-parallel EICs). EICs of 213.1393 and 227.0645 are not illustrated due to their relatively low intensity

Ac ce p

Fig. 7:

11.5

te

d

M

Molecular formulae of compound 1 and its fragments Measured Error m/z Ion Formula m/z [ppm] 400.2411 C23H34N3OS 400.2417 1.6 283.1276 C17H19N2S 283.1263 -4.3 227.0645 C13H11N2S 227.0637 -3.2 213.1393 C14H17N2 213.1386 -3.1 116.1072 C6H14NO 116.1070 -1.9

an

0.0

Page 19 of 61

- 20Intens. x106

Intens.

1.25

1.25

+bbCID MS, 30.0eV, 9.30min

x106

283.1269

1.00

1.00

0.75

ip t

0.75

0.50

EIC 283.1263 ± 0.005 m/z

0.25

98.9843 213.1389 70.9952

EIC 213.1389 ± 0.005 m/z

EIC 227.0642 ± 0.005 m/z

9

10

11

12

Error [mDa] -1.2 -0.5 -0.5 -0.3

250

300

350

m/z

mSigma 18.5 7.1 9.7 30.8

13

rdb 8.5 9.5 9.5 7.5

14

N-Rule ok ok ok ok

Time [min]

e¯-Config. even even even even

d

M

Molecular formulae of compound 2 and its fragments Measured Error m/z [ppm] Ion Formula m/z 356.2167 C21H30N3S 356.2155 -3.4 283.1269 C17H19N2S 283.1263 -1.8 227.0642 C13H11N2S 227.0637 -2.1 213.1389 C14H17N2 213.1386 -1.3

bbCID spectrum and extracted chromatographic traces of compound 2 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 356.2167 are not annotated (non-parallel EICs)

Ac ce p

te

Fig. 8:

200

EIC 356.2155 ± 0.005 m/z

0.00 8

150

an

0.25

100

us

50

356.2167

227.0642

0.00

cr

0.50

The fragments observed in the LC-HRMS spectra parallel the fragments in the GC-CI and/or GC-EI mass spectra. With HRMS the molecular formula of the oxygen containing immonium ion m/z 116 could be confirmed (Fig. 7). Further, both compounds yield the same fragments at m/z 283, 227 and 213 after ESI with the molecular formulae of C17H19N2S+, C13H11N2S+ and C14H17N2+(Fig. 7 and 8), respectively. The calculated molecular formulae of fragments m/z 227 and 213 of compound 1 and 2 by LC-HRMS confirmed the same element composition of these fragments as they already have been predicted by the GCMS/MS product ion experiments. The mSigma-values are a measure for the Page 20 of 61

- 21-

consistency of the acquired isotopic peak pattern and the theoretically calculated pattern for the suggested formula (lower mSigma values imply a better fit). Some fragments showed elevated mSigma values due to weak intensities. The

ip t

fragmentation pathways for m/z 213, 227 and 283 will be discussed after

us

cr

presentation of the NMR results.

The IR spectra (Figs. 9 and 10) of 1 and 2 are both dominated by an intense

an

absorption at 737/738 cm-1. They also show common absorptions at 879, 1013, 1252, 1332/1333, 1347/1348, 1393, 1450, 1518/1519, 1545, 1557/1558, and

M

1613 cm-1 also proving their structural similarity. The absorptions in the keto

d

region at 1613 cm-1 are only very weak and could refer to C=N instead of C=O.

te

The intense absorption at 737/738 cm-1 could possibly be assigned to a sulfur

Ac ce p

containing thioamide fragment.

100

N-2-Methoxyethyl,N-i-propyl-2-(1-pentyl-indol-3-yl)-4-thiazolmethanamin

95 90 85 80 75 70 65 60 55 50 45

4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Page 21 of 61

- 22-

95 3, 3 1 6 1

4, 6 2 9

1, 9 1 5 1

85

75

9, 7 5 5 4, 1 5 4 5 1

70 %

8, 8 6 9

7, 1 5 2 1

80

6, 0 5 4 1

6, 7 7 0 1

6, 1 3 3 1

7, 0 6 3 1

2, 3 9 3 1

7, 8 5 0 1

5, 8 6 7

8, 8 7 8

1, 8 7 1 1 3, 6 1 1 1

60

6, 2 2 8

4, 3 1 0 1

1, 8 4 3 1

65

5, 6 5 8

9, 5 9 8

cr

55

ip t

90

50

us

45 40

1600

1400

1200

1000

-1

Wavenumber (cm )

800

100

M

an

Fig. 9: IR spectrum of 1 (top) and detail (bottom)

0, 8 3 7

N,N-Diethyl-2-(1-pentyl-indol-3-yl)-4-thiazolmethanamin

95 90

d

85

te

80 75 70

Ac ce p

65 60 55 50 45 40 35

3500

3000

2500

2000

1500

1000

Wavenumber (cm-1)

Page 22 of 61

- 23y

( p

y

y)

95 2, 3 1 6 1

85

1, 4 2 9

2, 8 1 5 1

4, 9 8 9

2, 1 5 2 1

80 75

8, 1 7 4 1

70

4, 7 5 4, 5 5 1 4 5 1

65

4, 1 7 3 1 4, 0 5 4 1

8, 2 9 3 1

5, 4 1 1 1 6, 3 6 1 1

4, 3 3 3 1

8, 5 3 1 1

0, 3 1 0 1

0, 0 0 1 1 6, 8 5 0 1

1, 1 8 1 1

2, 7 4 3 1

0, 5 6 9

55

0, 7 1 8

7, 7 6 7

cr

50

3, 6 0 8

2, 1 9 8

7, 8 7 8

60

8, 9 5 6

7, 8 5 8

ip t

90

45

35 30 1600

1400

1200

us

40

1000

-1

800

an

Wavenumber (cm )

3, 7 3 7

M

Fig. 10: IR spectrum of 2 (top) and detail (bottom)

With these first results in mind, NMR measurements were performed (the 1H13

C-spectra for compounds 1 and 2 can be found in the electronic

d

and

te

supplementary data). In large parts, the structures could be derived and

Ac ce p

confirmed in a straightforward fashion using standard correlation experiments. Owing to the presence of heteroatoms and its novel appearance in such drugs, the heterocycle was however challenging and required special verification. The presence of both compounds simplified this process somewhat, as it was also immediately obvious in the NMR data that these compounds are analogues differing in one side chain substituent only. The respective substituents were fully and unambiguously characterized by COSY and HMBC data. Also, the Npentylindole substructure was easily established, as it exhibited all expected correlations in the 2D experiments, and its data and chemical shifts compared well to previously characterized compounds. The carbon atoms C-5’, C-4’, and Page 23 of 61

- 24-

C-1’’ were connected by HMBC correlations, and the absence of a COSY cross peak defined the order of methylene, an olefinic quarternary carbon and an olefinic methine group, as well as the connection of the methylene group to the

ip t

other two amine substituents. The thioaza-heterocycle thus directly resulted

cr

from the molecular formula, remaining double bond equivalents, and a 3J

us

HMBC correlation between H-5’ and C-2’. The position of the methylene group (1’’) at this heterocycle, or in other words the relative position of the sulfur and

an

nitrogen could not be unambiguously established from correlations, and thus remained solely suggested on the basis of chemical shift estimates. Notably, a

M

crucial connectivity expected for these compounds, a HMBC correlation

d

between heterocyclic C-2’ and the H-2 of the indole moiety, was not observed in

te

neither compounds in several attempts. We thus aimed to establish this C-C

Ac ce p

bond in an alternative manner. Concentrated solutions of compounds 1 and 2 were prepared and several versions of 2D INADEQUATE experiments were acquired. In these, cross peaks established the expected C-C bond between C-3 and C-2’. 2D INADEQUATE data also confirmed all other carbon connections presented in the structural formulae proving the assignment of the compounds.

The structures of 1 and 2 were thus elucidated to N-(2-methoxyethyl),N-isopropyl-2-(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine 1 and N,N-diethyl-2(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine 2 (Figs. 11 and 12).

Page 24 of 61

- 25-

5

2 6

7a

5''

2' 3

3a

CH3

4''

N 3' 4

2''

N

1' S

O

1

6''

CH3

N1

7 1'''

3''' 2'''

CH3

5''' 4'''

H-2

7.77 ppm, s

H-6 H-5 H-4 H-5'

7.27 ppm, m 7.26 ppm, m 8.21 ppm, m 7.10 ppm, s

H-7 H-5'' H-6'' H-2'' H-1'' H-4'' H-1''' H-2''' H-3''' H-4''' H-3'' H-5'''

7.36 ppm, m 3.44 ppm, t 3.31 ppm, s 3.09 ppm, sept 3.88 ppm, s 2.79ppm, t 4.14 ppm, t 1.88 ppm, p 1.33 ppm, m 1.34 ppm, m 1.08 ppm, d 0.885 ppm, t

δC [ppm]

C-2' C-4' C-7a C-2 C-3a C-6 C-5 C-4 C-5' C-3 C-7 C-5'' C-6'' C-2'' C-1'' C-4'' C-1''' C-2''' C-3''' C-4''' C-3'' C-5'''

162.6 ppm 156.9 ppm 136.6 ppm 127.7 ppm 125.3 ppm 122.4 ppm 120.9 ppm 120.8 ppm 111.3 ppm 111.1 ppm 109.9 ppm 72.36 ppm 58.81 ppm 51.84 ppm 51.41 ppm 50.12 ppm 46.73 ppm 29.79 ppm 29.06 ppm 22.30 ppm 18.45 ppm 13.89 ppm

ip t

3''

Atom

us

4'

an

5'

M

CH3 1''

δH [ppm]

cr

Atom

Ac ce p

te

d

Fig. 11: NMR assignments of N-(2-methoxyethyl),N-iso-propyl-2-(1-pentyl-1H-indol-3-yl)4-thiazolemethanamine(1). s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, sept = septet, m = multiplet

Page 25 of 61

- 26-

4'

N

1' S

3''

N 3' 4

3a

2

2 6

7a

CH3

2' 3

5

CH3

2''

N1

H-2

7.77 ppm, s

H-6 H-5 H-4 H-5'

7.27 ppm, m 7.26 ppm, m 8.21 ppm, m 7.02 ppm, s

H-7 H-1'' H-2'' H-1''' H-2''' H-3''' H-4''' H-5''' H-3''

7.36 ppm, m 3.87 ppm, s 2.67 ppm, q 4.12 ppm, t 1.87 ppm, p 1.33 ppm, m 1.33 ppm, m 0.873 ppm, t 1.14 ppm, t

4'''

C-2' C-4' C-7a C-2 C-3a C-6 C-5 C-4 C-5' C-3 C-7 C-1'' C-2'' C-1''' C-2''' C-3''' C-4''' C-5''' C-3''

162.5 ppm 154.4 ppm 136.6 ppm 127.9 ppm 125.4 ppm 122.4 ppm 120.9 ppm 120.8 ppm 112.2 ppm 111.1 ppm 109.9 ppm 52.82 ppm 47.22ppm 46.73 ppm 29.79 ppm 29.05 ppm 22.30 ppm 13.90 ppm 12.00 ppm

M

2'''

CH3

5'''

an

3'''

δC [ppm]

us

7 1'''

Atom

ip t

1''

5'

δH [ppm]

cr

Atom

d

Fig. 12: NMR assignments of N,N-diethyl-2-(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine(2), abbreviations for multiplicitiesof1H signals see Fig. 11

te

With the elucidation of the structures of 1 and 2 by NMR the fragmentation

Ac ce p

pathway of m/z 213, 227 and 283 occurring after ESI can be discussed (Fig. 15): After protonation at the amino nitrogen of the side chain of 1 or 2 the corresponding amine could inductively been split off. The resulting cation – which is identical for 1 and 2 – may rearrange to a six membered, resonance stabilized thiazolane with m/z 283. This thiazolane can lose C4H8 forming m/z 227 (Fig. 13, way (a)) after double 1,3-H-shift or may split off C3H2S forming an aza-analogous carbonylcation with m/z 213 (Fig. 13, way (b)). These suggestions are well supported by the results of the HRMS measurements of the fragments m/z 213, 227 and 283 (Figs. 7 + 8).Therefore, m/z 213 could indeed be an aza-analogues indoloylcation as assumed above. Page 26 of 61

- 27-

NR'R'' N

N

S

H

CI/ESI

N r

- NHR'R''

N

cr

N

CH3

+

CH3

S

CH3

S

N

N

N

1,3-H, i

an

(a)

+N

S

N

1,3-H N

ip t

i

N

S

+

+ NR'R''

S

us

S

+N

(2)

-C4 H8

+N CH3

(1)

H CH3

i

C17 H19 N2 S

CH3

M

(b)

+

m/z 283

H

S

C13 H11 N2 S

+

m/z 227

H

+ N

te

+N

Ac ce p

i

N

CH3

d

H

H

-C3 H2 S

CH3

C14 H17 N2 N

+

m/z 213

CH3

Fig. 13: Possible fragmentation pathways for m/z 213, 227 and 283 after CI/ESI

To the best of our knowledge compounds 1 and 2 have not been described in literature yet and have appeared the first time on the designer drug market. However, compounds with 3-([1,3]-thiazol-2-yl]indole substructure have been investigated as agonists at the CB1 receptor [16 - 18] (Fig. 14).

Page 27 of 61

- 28-

R1 NR2

S

CH3 NR2

S

N

N

CH3

S

N

N

ip t

R1

OH

N

3

OMe

Cl

Cl

X

O

X = CH2 or O R = alkyl sidechains, partially O-containing 1

an

R = H, C2 H5 or Cl

5

us

X

N

4

cr

N

M

Fig. 14: 3-([1,3]-thiazol-2-yl)indoles with CB1 receptor activity according to [16-18]

d

Adam-Worall et al. mainly investigated 7-methoxy and 7-chloro substituted

te

indoles with different heterocyclic substituents in the 3-position of the indole ring: Besides thiazoles (Fig. 14) thiadiazoles, oxadiazoles, oxazoles, isoxazoles,

Ac ce p

isothiazoles, and triazoles have been investigated. The compounds beared exclusively (cyclohexyl)methyl or (tetrahydropyran-4-yl)methyl substituents at the indole nitrogen. One of the investigated compounds (5) exhibited a KihCB1 of 10 nM (hCB1 = human CB1 receptors, for comparison THC has a KihCB1 of 2.9 nM and a KihCB2 of 42 nM [19]), had a relatively long plasma half-life and an increased brain penetration with a strong analgesic effect and was chosen for subsequent clinical development and pharmacokinetic evaluation in a Phase 1 clinical trial [20]. None of the investigated compounds carried a n-pentyl sidechain at the indole nitrogen. Whether compound 1 and 2 show CB1receptor Page 28 of 61

- 29-

binding and intrinsic activities is not known and will be part of future examinations.

ip t

Figure 15 shows the MS spectra after EI and CI of the third seized compound 6

cr

with a completely different structure from compounds 1 and 2. Compound 6 has

us

a molecular weight of 447 amu and an intense molecular radical cation with loss of a hydrogen atom after EI. Compound 6 shows a very high retention index

an

compared to 1 and 2.From LC-HRMS experiments the molecular formula was

Ac ce p

te

d

M

calculated to C28H37N3O2 (Figs. 16 and 17).

Page 29 of 61

- 30T: + c EI Q3MS [ 29,00-700,00] 375

100 95

RI: 3836 (DB-1)

90 85 446

80 75 70

447

60 55 50

ip t

Relative Abundance

65

45 40 35

376

30 107

25

279 222

20

448

55

251

10

157

5

41

56

73

77

129

312

350

250

174 193

221

280 281

252

236

136

313

346

0 100

150

200

250 m/z

300

T: + c CI Q3MS [ 50,00-700,00] 100 95 90

377

378

402

418

448

449

432

400

450

[M+H]+

an

85 80 75 70 65 60 55

M

Relative Abundance

351

350

us

50

cr

135 15

50 45 40 35 30

449

[M+29]+

25 476

d

20 15 10 72

74 95

0 50

97 100

123

137

165 150

241

te

5

177

207

200

222

251

[M+41]+ 314 375 279

250

312 300

315

342

349

350

377

446 405 400

450

420

462

477 489

450

m/z

Ac ce p

Fig. 15: EI-MS spectrum (top) and CI-MS spectrum of 6 (bottom)

Page 30 of 61

- 31Intens.

1+ 449.2987

1

1+ 450.3017

0 3

2

1

C 28 H 38 N 3 O 2 , 448.2959

1+ 448.2959

Simulated spectrum of C28H38N3O2 Simulated +) ([M+H] spectrum of C28H38N3O2 ([M+H]+)

cr

x105

+MS, 11.50min #1362

ip t

2

1+ 448.2958

Acquired spectrum Acquired spectrum

1+ 449.2990

us

x105 3

0 444

446

Error [mDa] 0.0 0.6 -0.8

452

454

m/z

mSigma

rdb

N-Rule

e¯-Config.

14.8 55.6 68.6

11.5 4.5 -0.5

ok ok ok

even even even

Acquired and simulated spectrum of compound 6 after LC-HRMS with suggested molecular formulae (SmartFormula)

Ac ce p

te

Fig. 16:

Erorr [ppm] 0.1 1.2 -1.8

450

d

Suggested molecular formulae of compound 6 Measured # Ion Formula m/z m/z 448.2958 1 C28H38N3O2 448.2959 2 C13H34N15O3 448.2964 3 C12H38N11O7 448.2950

448

M

442

an

1+ 450.3020

Page 31 of 61

- 32Intens.

x105

Intens. +bbCID MS, 30.0eV, 11.49min #1361

448.2957

x105 2.5 2.0

4

1.5

0.5

3

352.2019

279.1127

0.0 50

100

150

200

250

300

EIC 448.2957 ± 0.001 m/z

EIC 279.1127 ± 0.001 m/z 0 11.5

12.0

12.5

13.0

Error [mDa] 0.2 0.0 0.8

mSigma 6.9 30.0 125.4

14.5

rdb 11.5 10.5 11.5

15.0

N-Rule ok ok ok

Time [min]

e¯-Config. even even even

bbCID spectrum and extracted chromatographic traces of compound 6 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 448.2957 are not annotated (non-parallel EICs)

Ac ce p

te

Fig. 17:

14.0

d

M

Molecular formulae of compound 6 and its fragments Error Measred m/z Ion Formula m/z [ppm] 448.2957 C28H38N3O2 448.2959 0.4 352.2019 C21H26N3O2 352.2020 0.0 279.1120 C17H15N2O2 279.1128 3.0

13.5

an

11.0

450 m/z

400

us

EIC 352.2019 ± 0.001 m/z

1

350

cr

2

ip t

1.0

Page 32 of 61

- 33100

y

y

y

[(

y

y)

y]

y

95

90

85

ip t

80

75

cr

70

65

us

60

55

50 4000

y

y

y

[(

y

3000

y)

y] ,

2500

2000

95

85

0, 1 0 4 1

70

65

60 6, 9 0 5 1

3, 0 4 4 1

3, 4 2 4 1

50

45

1, 6 1 6 1

1600

3, 4 2 3 1

9, 9 8 2 1

2, 5 0 3 1

Ac ce p

55

500

1400

4, 6 9 6

3, 3 6 9

6, 4 0 7 7, 3 2 9

4, 5 9 8

5, 2 8 0 1

5, 9 7 2 1

te

5, 8 5 4 1 5, 5 6 4 1

1, 5 9 0 1

8, 3 6 2 1

5, 9 7 3 7, 1 1 6 3 1

1, 1 9 4 1

d

75

9, 3 5 1 1

7, 7 4 3 1

9, 7 7 4 1

0, 4 1 0 1

M

90

80

1000

Wavenumber (cm-1)

y

8, 2 8 5 1

1500

an

3500

4, 6 1 1 1 3, 6 7 1 1

1, 8 4 0 1

7, 2 7 6

8, 8 3 7 8, 4 5 7

0, 1 3 8 4, 2 4 8 8, 2 5 8

4, 1 7 7

2, 1 8 7

6, 2 3 2 1

7, 6 0 8 5, 2 2 8

1200

1000

800

Wavenumber (cm-1)

Fig. 18: IR spectrum of 6 (top) and detail (bottom)

According to the IR spectrum (Fig. 18) the compound bears a C=O functional group with a strong absorption at 1616 cm-1 indicating an amide moiety in the molecule. Structure elucidation was achieved via NMR measurements (1H- and 13

C-NMR can be found in the electronic supplementary data).

Page 33 of 61

- 34-

The NMR spectra of this compound are of very good quality, but notably broad lines are present in both the 1H and

13

C-experiments. All sharp lines showed

good correlations in 2D experiments that were fully consistent with the proposed

ip t

structure. HMBC correlations particularly confirmed the three ring moieties and

cr

the substitution patterns. For example, the position of the cyclohexylmethyl

us

moiety at the nitrogen N-1 of the benzimidazole was established by 3J correlations of the methylene protons (H-1’) to C-2 and C-7a. The position of

an

the benzyl moiety was unambiguously established by a correlation in 2DINADEQUATE data, which was also performed with this compound. The

M

mentioned broad lines originated from the diethylcarboxamide substituent in

d

position 5 of the benzimidazole, broadened due to slow to intermediate time

te

scale rotation around the carbonylamide partial double bond. These resonances

Ac ce p

could however be sharpened into two sets of ethylgroup signals (E and Z) by sample cooling to 278 K (data not shown). Both sets then showed the expected and identical correlations in 2D data. According to the data compound 6 could unambiguously be identified as 1-(cyclohexylmethyl)-2-[(4-ethoxyphenyl)methyl]-N,N-diethyl-1H-benzimidazol-5-carboxamide (Fig. 19).

Page 34 of 61

- 35-

6''

H3 C

7''

O 5''

O

4''

2'''

N

4'''

H3C

3'''

1'''

N3

7a

N1

2''

5 2

6

3''

1''

7

5'''

1'

6

2' 5'

3'

7.13 ppm, d

H-6 H-4 H-4'' H-7 H-6'' H-1' H-2''' H-3''' H-2',4',5' H-1'' H-3'

7.30 ppm, AB 7.75 ppm, s 6.82 ppm, d 7.29 ppm, AB 3.99 ppm, q 3.80 ppm, d 3.56 ppm, br 3.38 ppm, br 1.69 ppm, m, 5H 4.26 ppm, s 1.55 ppm, d and 0.955 ppm, q 1.69 ppm, m, 5H 1.11 ppm, m, 2H 1.39 ppm, t 1.22 ppm, br 1.22 ppm, br

M

4'

H-3''

C-1''' C-5'' C-2 C-3a C-7a C-5 C-3'' C-2'' C-6 C-4 C-4'' C-7 C-6'' C-1' C-2''' C-3''' C-2' C-1'' C-3'

171.8 ppm 158.0 ppm 154.9 ppm 141.9 ppm 136.5 ppm 130.9 ppm 129.4 ppm 128.0 ppm 121.3 ppm 117.5 ppm 114.8 ppm 110.0 ppm 63.47 ppm 50.29 ppm 43.56 ppm 39.27 ppm 38.52 ppm 33.87 ppm 31.09 ppm

C-5' C-4' C-7'' C-4''' C-5'''

26.12 ppm 25.67 ppm 14.82 ppm 14.19 ppm 13.12 ppm

te

d

H-2',4',5' H-4',5' H-7'' H-4''',5''' H-4''',5'''

δC [ppm]

us

H3 C

3a

an

4

Atom

ip t

δH [ppm]

cr

Atom

Ac ce p

Fig. 19: NMR assignments of 1-(cyclohexylmethyl)-2-[(4-ethoxyphenyl)methyl]-N,Ndiethyl-1H-benzimidazol-5-carboxamide(6), AB = AB-system, br = broad, other abbreviations for multiplicities of1H signals see Fig. 11

Compound 6 has been described in the literature, but to our knowledge no spectroscopic data has been published. Compound 6 shows strong binding affinity to the hCB2 receptor (Ki = 3.7 nM) [19,21]. With a Ki(hCB1) of 110 nM it offers the strongest CB1-binding constant of all investigated benzimidazoles of this type. The CB1-binding constant of 6is nearly the same as in WIN 55212-2, but it has an approximately 5fold stronger binding affinity to CB2 receptor than WIN 55212-2. Due to the compounds high selectivity for the CB2 receptor it is at least questionable whether there is a psychotropic activity. Other Page 35 of 61

- 36-

benzimidazoles show binding constants down to Ki = 0.53 nM for the hCB1 receptor [22], resulting in a higher probability for psychotropic cannabimimetic

ip t

activity.

cr

Conclusions

us

Two 3-([1,3]-thiazol-2-yl)indoles (N-(2-methoxyethyl),N-iso-propyl-2-(1-pentyl1H-indol-3-yl)-4-thiazolemethanamine (1) and N,N-diethyl-2-(1-pentyl-1H-

an

indol-3-yl)-4-thiazolemethanamine (2)) as well as one benzimidazole (1(cyclohexylmethyl)-2-[(4-ethoxyphenyl)methyl]-N,N-diethyl-1H-benzimidazo-

M

le-5-carboxamide (6)) have been identified for the first time in seizures on the

d

designer drug market in Germany. Compared to already established

te

cannabmimetics, the structures of the here described compounds are rather

Ac ce p

uncommon. Their structure elucidation was relatively complex compared to other substances owing to their unusual/novel structural features. Whereas the CB1-/CB2-affinities and intrinsic activities of 1 and 2 are unknown and will be determined as a part of further examinations, compound 6 possesses a relative strong affinity for the CB2 receptor. If these substances act as psychoactive cannabimimetics and show sufficient potency has yet to be shown. This would be a prerequisite for becoming prevalent on the “legal high” market.

Page 36 of 61

- 37-

References 1 N. Uchiyama, S. Matsuda, M. Kawamura, Y. Shimokawa, R. Kikura-Hanajiri, K. Aritake,

ip t

Y. Urade, Y. Goda,Characterization of four new designer drugs, 5-chloro-NNEI, NNEI indazole analog, α-PHPP and α-POP, with 11 newly distributed designer drugs in illegal

cr

products. Forensic Sci. Int. 2014;243C:1-13.

2 S.Dresen, N. Ferreirós, M. Pütz, F. Westphal, R. Zimmermann, V. Auwärter, Monitoring

us

of herbal mixtures potentially containing synthetic cannabinoids as psychoactive compounds. J. Mass Spectrom. 2010;45(10):1186-1194.

an

3 N. Uchiyama, M. Kawamura, R. Kikura-Hanajiri,Y. Goda, URB-754: a new class of designer drug and 12 synthetic cannabinoids detected in illegal products. Forensic Sci. Int.

M

2013;227(1-3):21-32.

4 F. Westphal, U. Girreser, S. Knecht, Structure elucidation of a new open chain isomer of

d

the cannabimimetic cyclopropoylindole A-796,260. Forensic Sci. Int. 2014;234:139-148.

te

5 R. Kikura-Hanajiri, N.U. Kawamura, Y. Goda, Changes in the prevalence of new

Ac ce p

psychoactive substances before and after the introduction of the generic scheduling of synthetic cannabinoids in Japan. Drug Test Anal. 2014;6(7-8):832-839. 6 S. Kneisel, F. Westphal, P. Bisel, V. Brecht, S. Broecker, V. Auwärter, Identification and structural characterization of the synthetic cannabinoid 3-(1-adamantoyl)-1-pentylindole as an additive in 'herbal incense'. J. Mass Spectrom. 2012;47(2):195-200. 7 B. Moosmann, S. Kneisel, U. Girreser, V. Brecht, F. Westphal, V. Auwärter, Separation and structural characterization of the synthetic cannabinoids JWH-412 and 1-[(5fluoropentyl)-1H-indol-3yl]-(4-methylnaphthalen-1-yl)methanone using GC-MS, NMR analysis and a flash chromatography system. Forensic Sci. Int. 2012;220(1-3):e17-22. 8 N. Uchiyama, S. Matsuda, M. Kawamura, R. Kikura-Hanajiri, Y. Goda, Two new-type cannabimimetic quinolinyl carboxylates, QUPIC and QUCHIC, two new cannabimimetic

Page 37 of 61

- 38-

carboxamide derivatives, ADB-FUBINACA and ADBICA, and five synthetic cannabinoids detected with a thiophene derivative α-PVT and an opioid receptor agonist AH-7921 identified in illegal products. Forensic Toxicol.2013;31(2):223-240.

cr

Drugs and Drug Addiction (EMCDDA), Lisbon, May 2014:

ip t

9 European Drug Report 2014: Trends and developments, European Monitoring Centre for

http://www.emcdda.europa.eu/attachements.cfm/att_228272_DE_TDAT14001DEN.pdf

us

10 P.H. Dawson, W.F. Sun, A round robin on the reproducibility of standard operating

Mass Spectrom. Ion Proc. 1984;55:155-170.

an

conditions for the acquisition of library MS/MS spectra using triple quadrupols. Int. J.

11 For production spectrometry of immonium ions for structure elucidation see: P. Rösner, T.

M

Junge, Investigation of the alkylamino group of aliphatic an arylaliphatic amines by collision-induced dissociation mass spectra of C4H10N+ immonium ions. J. Mass

d

Spectrom. 1996;31:1047-1053. T. Junge, P. Rösner, F. Westphal, Product ion mass

te

spectra of important organic ions, a free printed version can be ordered from the authors.

Ac ce p

12 F. Westphal, T. Junge, F. Sönnichsen, P. Rösner, J. Schäper, Ein neuer Wirkstoffin SPICE-artigen Kräutermischungen: Charakterisierung von JWH-250, seinen Methyl- und Trimethylsilyderivaten (A new active compound in SPICE-like products: characterization of JWH-250, it’s methyl- and trimethylsilyl derivatives). Toxichem Krimtech 2010 (77/1) 46-58.

13 L. Ernst, H.-M. Schiebel, C. Theuring, R. Lindigkeit, T. Beuerle, Identification and characterization of JWH-122 used as new ingredient in „Spice-like“ herbal incences. Forensic Sci. Int. 2011;208:e31-e35. 14 P. Jankovics, A. Váradi, L. Tölgyesi, S. Lohner, J. Németh-Palotás, Detection and identification of the new potential synthetic cannabinoids 1-pentyl-3-(2-

Page 38 of 61

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iodobenzoyl)indole and 1-pentyl-3-(1-adamantoyl)indole in seized bulk powders in Hungary. Forensic Sci. Int. 2012;214:27-32. 15 D.N. Harris, S. Hokanson, V. Miller, G.P. Jackson, Fragmentation differences in the EI

ip t

spectra of three synthetic cannabinoid positional isomers: JWH-250, JWH-302, and JWH-

cr

201. Int. J. Mass Spectrom. 2014;368:23-29.

16 A.J. Morrison, J.M. Adam, J.A. Baker, R.A. Campbell, J.K. Clark, J.E. Cottney,

us

M. Deehan, A.-M. Easson, R. Fields, S. Francis, F. Jeremiah, N. Keddie, T. Kiyoi, D.R. McArthur, K. Meyer, P.D. Ratcliffe, J. Schulz, G. Wishart, K. Yoshiizumi, Design,

an

synthesis, and structure-activity relationships of indole-3-heterocycles as agonists of the CB1 receptor. Bioorg. Med. Chem. Lett. 2011;21: 506-509.

M

17 J. Adam-Worrall, A.J. Morrison, G. Wishart, T. Kiyoi, R.D. McArthur, Patent WO 2005/089754 A1.

te

7,700,634 B3, 2010.

d

18 J. Adam-Worrall, A.J. Morrison, G. Wishart, T. Kiyoi, D.R. McArthur, Patent US

Ac ce p

19 D. Pagé, E. Balaux, L. Boisvert, Z. Liu, C. Milburn, M. Tremblay, Z. Wei, S. Woo, X. Luo, Y.-X. Cheng, H. Yang, S. Srivastava, F. Zhou, W. Brown, M. Tomaszewski, C. Walpole, L. Hodzic, S. St-Onge, C. Godbout, D. Salois, K. Payza, Novel benzimidazole derivatives as selective CB2 agonists. Bioorg. Med. Chem. Lett. 2008;18: 3695-3700. 20 P. Ratcliffe, J.M. Adam, J. Baker, R. Bursi, R. Campbell, J.K. Clarke, J.E. Cottney, M. Deehan, A.-M. Easson, D. Ecker, D. Edwards, O. Epemolu, L. Evans, R. Fields, S. Francis, P. Harradine, F. Jeremiah, T. Kiyoi, D. McArthur, A. Morrison, P. Passier, J. Pick, P.G. Schnabel, J. Schulz, H. Steinbrede, G. Walker, P. Westwood, G. Wishart, J. Udo de Haes, Design, synthesis and structure-activity relationships of (indo-3-yl) heterocyclic derivatives as agonists of the CB1 receptor. Discovery of a clinical candidate. Bioorg. Med. Chem. Lett. 2011;21: 2541-2546.

Page 39 of 61

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21 E. Cichero, S. Cesarini, L. Mosti, P. Fossa, CoMFA and CoMSIA analyses on 1,2,3,4tetrahydropyrrolo[3,4-b]indole and benzimidazole derivatives as selective CB2 receptor agonists. J. Molec. Modeling 2010;16(9): 1481-1498.

ip t

22 J.A. Mella-Raipán, C.F. Lagos, G. Recabarren-Gajardo, C, Espinosa-Bustos, J. Romero-

cr

Parra, H. Pessoa-Mahana, P. Iturriaga-Vásquez, C.D. Pessoa-Mahana, Design, synthesis,

binding and docking-based 3D-QSAR studies of 2-pyridylbenzimidazoles – a new family

Ac ce p

te

d

M

an

us

of high affinity CB1 cannabinoid ligands. Molecules 2013;18: 3972-4001.

Page 40 of 61

- 41Figures with legends

T: + c EI Q3MS [ 29,00-700,00] 283

100

RI: 3172 (DB-1)

ip t

95 90 85 80 75 70

cr

60 55 284 50 45 40

us

Relative Abundance

65

35 30 25 116

20

155

15

213 156 43

5 0

30

71 45

84

72

50

101

113

128 143 142

100

227

212 211

177

150

an

10

367

298

228

200

324

281

254

250

354 368

310

369

334

300

384

350

398

400

m/z

T: + c CI Q3MS [ 50,00-700,00]

400,25

100

400

[M+H]+

M

95 90 85 80 75 70

d

60 55 50

te

Relative Abundance

65

45 40

367

401,26

35 30

368 401

398,24

Ac ce p

25 20

[M+29]+

402,26

398

399,33

15

403,26

397,42

10

5 0

71

76

86

116 118 102 130

100

398

146

150

155

400

173

402 m/z

200 200

213

428

404,35

227

384 369

283

404

241

267

250 m/z

281

286

311

402

354 325

300

342 350

[M+41]+

429 403 400

441 450

Fig. 1: EI-MS spectrum (top) and CI-MS spectrum of 1 (bottom)

Page 41 of 61

- 42-

T: + c EI Q3MS [ 29,00-700,00] 355

284

ip t

100 95

RI: 2930 (DB-1)

90 85 80 75 70

cr

60 55 50

356

45 40 357

35

358

30 355

25

356 m/z

357

358

us

Relative Abundance

65

285

20 15

142 155

213 214 226

72 5 43

58

0

71

73

86

50

113 101

156

128

100

170

212

185

150

an

283

10

200 m/z

T: + c CI Q3MS [ 50,00-600,00]

281

254

250

95 90 85 80

355

286 287

326

298

356

341

300

350

356,23

M

100

227 241

[M+H]+

75

d

70

60 55

te

Relative Abundance

65

50 45 40 35

Ac ce p

30

284,15

[M+29]+

25 20

74,12

15

384,25

72,11

5

58,08

102,13

114,12

155,08

213,14 188,13

241,19

0

100

150

[M+41]+

340,19

10

200

250

313,20 326,23

269,21

300

370,24 350

396,25 400

m/z

Fig. 2: EI-MS spectrum (top) and CI-MS spectrum of 2 (bottom)

Page 42 of 61

- 43-

Intens.

4

+MS, 9.54min #1126

1+ 400.2435

Acquired spectrum

ip t

x105

2

cr

3 1+ 401.2463

1

3 2

1+ 401.2447

1 0 399

400

401

d

398

Ac ce p

te

Suggested molecular formulae for compound 1: Measured m/z # Ion Formula m/z 400.2435 1 C23H34N3OS 400.2417 2 C19H34N3O6 400.2442 3 C16H26N13 400.2429 4 C12H34N9O4S 400.2449 5 C23H34N3O3 400.2595

Fig. 3:

us an

4

C 23 H 34 N 3 O 1 S 1 , 400.2417

1+ 400.2417

Simulated spectrum of C23H34N3OS ([M+H]+)

M

0 x105

1+ 402.2445

Error [ppm] -4.6 1.7 -1.7 3.4 39.8

Error [mDa] -1.8 0.7 -0.7 1.4 15.9

1+ 402.2420 402

mSigma 18.7 38.2 40.1 64.3 14.7

403

rdb 8.5 4.5 10.5 0.5 8.5

N-Rule ok ok ok ok ok

m/z

e¯-Config. even even even even even

Acquired and simulated LC-HRMS-spectrum of compound 1with suggested molecular formulae (SmartFormula)

Page 43 of 61

- 44-

1.0

0.8

EIC C23H34N3OS ([M+H]+)

0.6

us

400.2417 ± 0.005 m/z

cr

ip t

Intens. x106

0.4

EIC C23H34N3O3 ([M+H]+) 0.2

0.0 9.0

9.5

10.0

10.5

11.0

11.5

Time [min]

Extracted chromatogram traces of C23H34N3OS and C23H34N3O3, illustrating the presence of one sulfur atom instead of two oxygens

Ac ce p

te

d

Fig. 4:

8.5

M

8.0

an

400.2595 ± 0.005 m/z

Page 44 of 61

- 45-

5

1+

Acquired spectrum

+MS, 9.27min #1094

356.2166

ip t

Intens. x105

4 3

cr

1+ 357.2197

2

1+ 358.2162

1

4 3

1+ 356.2155

C 21 H 30 N 3 S 1 , 356.2155

us

5

Simulated spectrum of C21H30N3S ([M+H]+)

an

0 x105

1+ 357.2185

2 1 0 355.5

356.0

356.5

Error [ppm] -3.2 3.8 -3.7 46.6

357.5

Error [mDa] -1.1 1.4 -1.3 16.6

358.0

mSigma 13.2 28.7 42.5 11.6

358.5

359.0

rdb 8.5 4.5 5.5 8.5

N-Rule ok ok ok ok

m/z

e¯-Config. even even even even

Ac ce p

te

d

Suggested molecular formulae for compound 2 Measured m/z # Ion Formula m/z 356.2166 1 C21H30N3S 356.2155 2 C17H30N3O5 356.2180 3 C13H26N9O3 356.2153 4 C21H30N3O2 356.2333

357.0

M

355.0

1+ 358.2115

Fig. 5:

Acquired and simulated LC-HRMS-spectrum of compound 2 with suggested molecular formulae (SmartFormula)

Page 45 of 61

- 46-

ip t

Intens. x106

cr

1.25

1.00

0.75

us

EIC C21H30N3S ([M+H]+) 356.2155 ± 0.005 m/z

0.25

EIC C21H30N3O2 ([M+H]+)

0.00 2

4

6

8

10

12

Time [min]

Extracted chromatogram traces of C21H30N3S and C21H30N3O2, illustrating the presence of one sulfur atom instead of two oxygens

Ac ce p

te

d

Fig. 6:

M

356.2333 ± 0.005 m/z

an

0.50

Page 46 of 61

- 47-

Intens.

Intens.

+bbCID MS, 30.0eV, 9.56min

x106

283.1276

1.25

Intens. x104

213.1393

1.0 0.8 0.6

227.0645

0.4

0.75

0.2 0.0 190

0.50

EIC 283.1276 ± 0.001 m/z

200

210

220

230 m/z

0.25

0.2

0.00 50

100

EIC 116.1072 ± 0.002 m/z

an

116.1072

0.4

us

0.8

0.6

cr

1.00

1.0

150

ip t

x106

200

250

300

400.2411 350

400

m/z

0.0 9.0

9.5

10.0

M

EIC 400.2411 ± 0.002 m/z

10.5

Ac ce p

te

d

Molecular formulae of compound 1 and its fragments Measured Error m/z Ion Formula m/z [ppm] 400.2411 C23H34N3OS 400.2417 1.6 283.1276 C17H19N2S 283.1263 -4.3 227.0645 C13H11N2S 227.0637 -3.2 213.1393 C14H17N2 213.1386 -3.1 116.1072 C6H14NO 116.1070 -1.9

Fig. 7:

11.0

Error [mDa] 0.6 -1.2 -0.7 -0.7 -0.2

11.5

mSigma 10.0 12.3 52.2 51.5 29.9

12.0

rdb 8.5 9.5 9.5 7.5 0.5

12.5

N-Rule ok ok ok ok ok

Time [min]

e- Config. Even Even Even Even Even

bbCID spectrum and extracted chromatographic traces of compound 1 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 400.2411 are not annotated (non-parallel EICs). EICs of 213.1393 and 227.0645 are not illustrated due to their relatively low intensity

Page 47 of 61

- 48-

Intens. x106

Intens.

1.25

1.25

+bbCID MS, 30.0eV, 9.30min

x106

ip t

283.1269

1.00

1.00

0.50

EIC 283.1263 ± 0.005 m/z

0.25

98.9843

us

0.75

cr

0.75

213.1389

70.9952

0.50

200

250

300

350

m/z

EIC 213.1389 ± 0.005 m/z

EIC 227.0642 ± 0.005 m/z

EIC 356.2155 ± 0.005 m/z

0.00 8

150

9

10

M

0.25

100

an

50

356.2167

227.0642

0.00

11

Error [mDa] -1.2 -0.5 -0.5 -0.3

mSigma 18.5 7.1 9.7 30.8

13

rdb 8.5 9.5 9.5 7.5

14

N-Rule ok ok ok ok

Time [min]

e¯-Config. even even even even

Ac ce p

te

d

Molecular formulae of compound 2 and its fragments Measured Error m/z [ppm] Ion Formula m/z 356.2167 C21H30N3S 356.2155 -3.4 283.1269 C17H19N2S 283.1263 -1.8 227.0642 C13H11N2S 227.0637 -2.1 213.1389 C14H17N2 213.1386 -1.3

12

Fig. 8:

bbCID spectrum and extracted chromatographic traces of compound 2 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 356.2167 are not annotated (non-parallel EICs)

Page 48 of 61

- 49-

N-2-Methoxyethyl,N-i-propyl-2-(1-pentyl-indol-3-yl)-4-thiazolmethanamin

ip t

100 95 90

cr

85 80

us

75 70 65

an

60 55 50

4000

3500

M

45

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

3, 3 1 6 1

1, 9 1 5 1

85 80 75 70

%

6, 0 5 4 1

2, 3 9 3 1

Ac ce p

9, 7 5 5 4, 1 5 4 5 1

te

90

d

95

4, 6 2 9 8, 8 6 9

7, 1 5 2 1 6, 7 7 0 1

6, 1 3 3 1

7, 0 6 3 1

7, 8 5 0 1

6, 2 2 8 5, 8 6 7

4, 3 1 0 1

1, 8 4 3 1

8, 8 7 8

1, 8 7 1 1

65

5, 6 5 8

9, 5 9 8

3, 6 1 1 1

60 55 50 45 40

1600

1400

0, 8 3 7

1200

1000

800

-1

Wavenumber (cm )

Fig. 9: IR spectrum of 1 (top) and detail (bottom)

Page 49 of 61

100

ip t

- 50-

N,N-Diethyl-2-(1-pentyl-indol-3-yl)-4-thiazolmethanamin

cr

95 90

us

85 80 75

an

70 65 60 55

M

50 45 40

3500

d

35 3000

2500

2000

1500

1000

-1

Wavenumber (cm )

( p

y

y)

te

y

95 90

2, 8 1 5 1

Ac ce p

85

2, 3 1 6 1

80 75

8, 1 7 4 1

70

4, 7 5 4, 5 5 1 4 5 1

65

4, 1 7 3 1

4, 0 5 4 1

8, 2 9 3 1

1, 4 2 9 4, 9 8 9

2, 1 5 2 1

5, 4 1 1 1 6, 3 6 1 1

4, 3 3 3 1

8, 5 3 1 1

1, 1 8 1 1

2, 7 4 3 1

0, 3 1 0 1

0, 0 0 1 1 6, 8 5 0 1

0, 5 6 9

8, 9 5 6

7, 8 5 8 2, 1 9 8

3, 6 0 8 0, 7 1 8

7, 7 6 7

7, 8 7 8

60 55 50 45 40 35

3, 7 3 7

30 1600

1400

1200

1000

800

Wavenumber (cm-1)

Fig. 10: IR spectrum of 2 (top) and detail (bottom)

Page 50 of 61

- 51-

N 3' 4 5

2 6

7a

5''

2' 3

3a

CH3

4''

O

1

6''

CH3

N1

7 1'''

3'''

4'''

H-2

7.77 ppm, s

H-6 H-5 H-4 H-5'

7.27 ppm, m 7.26 ppm, m 8.21 ppm, m 7.10 ppm, s

H-7 H-5'' H-6'' H-2'' H-1'' H-4'' H-1''' H-2''' H-3''' H-4''' H-3'' H-5'''

7.36 ppm, m 3.44 ppm, t 3.31 ppm, s 3.09 ppm, sept 3.88 ppm, s 2.79 ppm, t 4.14 ppm, t 1.88 ppm, p 1.33 ppm, m 1.34 ppm, m 1.08 ppm, d 0.885 ppm, t

C-2' C-4' C-7a C-2 C-3a C-6 C-5 C-4 C-5' C-3 C-7 C-5'' C-6'' C-2'' C-1'' C-4'' C-1''' C-2''' C-3''' C-4''' C-3'' C-5'''

162.6 ppm 156.9 ppm 136.6 ppm 127.7 ppm 125.3 ppm 122.4 ppm 120.9 ppm 120.8 ppm 111.3 ppm 111.1 ppm 109.9 ppm 72.36 ppm 58.81 ppm 51.84 ppm 51.41 ppm 50.12 ppm 46.73 ppm 29.79 ppm 29.06 ppm 22.30 ppm 18.45 ppm 13.89 ppm

te

d

2'''

CH3

5'''

δC [ppm]

cr

N

1' S

Atom

us

3'' 2''

an

4'

M

CH3 1''

5'

δH [ppm]

ip t

Atom

Ac ce p

Fig. 11: NMR assignments of N-(2-methoxyethyl),N-iso-propyl-2-(1-pentyl-1H-indol-3-yl)4-thiazolemethanamine(1). s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, sept = septet, m = multiplet

Page 51 of 61

N

1' S

3''

N 3' 4 5

2

2 6

7a

CH3

2' 3

3a

CH3

2''

N1

H-2

7.77 ppm, s

H-6 H-5 H-4 H-5'

7.27 ppm, m 7.26 ppm, m 8.21 ppm, m 7.02 ppm, s

H-7 H-1'' H-2'' H-1''' H-2''' H-3''' H-4''' H-5''' H-3''

7.36 ppm, m 3.87 ppm, s 2.67 ppm, q 4.12 ppm, t 1.87 ppm, p 1.33 ppm, m 1.33 ppm, m 0.873 ppm, t 1.14 ppm, t

1'''

3'''

CH3

5''' 4'''

δC [ppm]

162.5 ppm 154.4 ppm 136.6 ppm 127.9 ppm 125.4 ppm 122.4 ppm 120.9 ppm 120.8 ppm 112.2 ppm 111.1 ppm 109.9 ppm 52.82 ppm 47.22ppm 46.73 ppm 29.79 ppm 29.05 ppm 22.30 ppm 13.90 ppm 12.00 ppm

Ac ce p

te

d

2'''

M

7

C-2' C-4' C-7a C-2 C-3a C-6 C-5 C-4 C-5' C-3 C-7 C-1'' C-2'' C-1''' C-2''' C-3''' C-4''' C-5''' C-3''

us

4'

Atom

an

1''

5'

δH [ppm]

cr

Atom

ip t

- 52-

Fig. 12: NMR assignments of N,N-diethyl-2-(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine(2), abbreviations for multiplicities of 1H signals see fig. 11.

Page 52 of 61

N

N

CH3

CH3

S N

H

te

m/z 283 H

H

Ac ce p

S

CH3

+

i

N

CH3

(2)

-C4 H8

+N CH3

(1)

CH3 C13 H11 N2 S

+

m/z 227

H

+ N

+N

-C3 H2 S

+N

H

d

C17 H19 N2 S

N

1,3-H, i

M

(a)

+N

S

N

1,3-H

CH3

CH3

S

N

N

N

an

+

r

- NHR'R''

N

i

N

i

N

(b)

S

H

CI/ESI

S

+

+ NR'R''

S

cr

NR'R''

us

S

ip t

- 53-

C14 H17 N2 N

+

m/z 213

CH3

Fig. 13: Possible fragmentation pathways for m/z 213, 227 and 283 after CI/ESI

Page 53 of 61

- 54-

R1 NR2

NR2

S

N

N

S

N

N

CH3

cr

S

CH3

ip t

R1

OH

N

3 Cl

X

5

Cl

X

O

X = CH2 or O R = alkyl sidechains, partially O-containing 1

M

R = H, C2 H5 or Cl

an

OMe

N

4

us

N

Ac ce p

te

d

Fig. 14: 3-([1,3]-thiazol-2-yl)indoles with CB1 receptor activity according to [16-18]

Page 54 of 61

- 55-

T: + c EI Q3MS [ 29,00-700,00] 375

95 90 85

446

80 75 70

cr

65 60 55 50 45

447

us

Relative Abundance

RI: 3836 (DB-1)

ip t

100

40 35

376

30 107

25

279 222

20 135 55

251

10

157

5

41

56

73

77

129

250

174 193

221

236

136

0 50

100

150

200

280 281

252

250 m/z

T: + c CI Q3MS [ 50,00-700,00]

95 90 85 80

448

350

313

300

346

351

377 378

350

402

418

449

432

400

450

448

M

100

312

an

15

[M+H]+

75

d

70

60 55

te

Relative Abundance

65

50 45 40 35

Ac ce p

30

449

[M+29]+

25

476

20

[M+41]+

15

314

10

5

72

74

95

0

50

97

100

123

137

165

150

177

207

200

375 222

241

251

279

250

312 300

315

342

349

350

377

446 405 400

450

420

462

477 489

450

m/z

Fig. 15: EI-MS spectrum (top) and CI-MS spectrum of 6 (bottom)

Page 55 of 61

- 56-

Intens.

1+ 450.3017

0

1

an

2

1+ 449.2990

0 444

446

448

d

442

Ac ce p

te

Suggested molecular formulae of compound 6 Measured # Ion Formula m/z m/z 448.2958 1 C28H38N3O2 448.2959 2 C13H34N15O3 448.2964 3 C12H38N11O7 448.2950

Fig. 16:

C 28 H 38 N 3 O 2 , 448.2959

1+ 448.2959

Simulated spectrum of C28H38N3O2 Simulated +) ([M+H] spectrum of C28H38N3O2 ([M+H]+)

M

3

ip t

1+ 449.2987

1

x105

+MS, 11.50min #1362

cr

2

1+ 448.2958

Acquired spectrum Acquired spectrum

us

x105 3

Erorr [ppm] 0.1 1.2 -1.8

1+ 450.3020 450

Error [mDa] 0.0 0.6 -0.8

452

454

m/z

mSigma

rdb

N-Rule

e¯-Config.

14.8 55.6 68.6

11.5 4.5 -0.5

ok ok ok

even even even

Acquired and simulated spectrum of compound 6 after LC-HRMS with suggested molecular formulae (SmartFormula)

Page 56 of 61

- 57-

Intens.

x105

Intens. +bbCID MS, 30.0eV, 11.49min #1361

448.2957

x105

ip t

2.5 2.0

4

1.5

0.5

3

279.1127

0.0 100

150

200

250

2 EIC 448.2957 ± 0.001 m/z

350

400

450 m/z

an

EIC 352.2019 ± 0.001 m/z

300

352.2019

us

50

cr

1.0

1

EIC 279.1127 ± 0.001 m/z

11.0

11.5

12.0

12.5

M

0

13.5

Error [mDa] 0.2 0.0 0.8

14.0

mSigma 6.9 30.0 125.4

14.5

rdb 11.5 10.5 11.5

15.0

N-Rule ok ok ok

Time [min]

e¯-Config. even even even

bbCID spectrum and extracted chromatographic traces of compound 6 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 448.2957 are not annotated (non-parallel EICs)

Ac ce p

Fig. 17:

te

d

Molecular formulae of compound 6 and its fragments Error Measred m/z Ion Formula m/z [ppm] 448.2957 C28H38N3O2 448.2959 0.4 352.2019 C21H26N3O2 352.2020 0.0 279.1120 C17H15N2O2 279.1128 3.0

13.0

Page 57 of 61

100

y

y

y

[(

y

y)

y] ,

ip t

- 58-

y

cr

95

90

us

85

80

75

an

70

65

M

60

55

50 4000

3500

y

y

[(

y

y)

y] ,

2500

te

90

85

Ac ce p

8, 2 8 5 1

9, 7 7 4 1 1, 1 9 4 1

75

0, 1 0 4 1

5, 8 5 4 1

5, 5 6 4 1

60

6, 9 0 5 1

55

3, 0 4 4 1

3, 4 2 4 1

50

45

1, 6 1 6 1

1600

1000

500

4, 6 9 6 0, 4 1 0 1

9, 3 5 1 1

7, 7 4 3 1

1, 5 9 0 1

8, 3 6 2 1

5, 9 7 3 7, 1 1 6 3 1

70

65

1500

Wavenumber (cm-1)

y

95

80

2000

d

y

3000

5, 2 8 0 1

5, 9 7 2 1

3, 4 2 3 1

4, 6 1 1 1

9, 9 8 2 1

3, 6 7 1 1

2, 5 0 3 1

1, 8 4 0 1

3, 3 6 9

6, 4 0 7 7, 3 2 9

4, 5 9 8

7, 2 7 6

8, 8 3 7 8, 4 5 7

0, 1 3 8 4, 2 4 8 8, 2 5 8

4, 1 7 7

2, 1 8 7

6, 2 3 2 1

7, 6 0 8 5, 2 2 8

1400

1200

1000

800

Wavenumber (cm-1)

Fig. 18: IR spectrum of 6 (top) and detail (bottom)

Page 58 of 61

δH [ppm]

6''

H3 C

7''

O

4''

H3 C

2'''

N

4'''

H3C

3'''

1'''

3a

N3

7a

N1

2

6

3''

2''

5 1''

7

5'''

1'

6

2' 5'

3'

7.13 ppm, d

H-6 H-4 H-4'' H-7 H-6'' H-1' H-2''' H-3''' H-2',4',5' H-1'' H-3'

7.30 ppm, AB 7.75 ppm, s 6.82 ppm, d 7.29 ppm, AB 3.99 ppm, q 3.80 ppm, d 3.56 ppm, br 3.38 ppm, br 1.69 ppm, m, 5H 4.26 ppm, s 1.55 ppm, d and 0.955 ppm, q 1.69 ppm, m, 5H 1.11 ppm, m, 2H 1.39 ppm, t 1.22 ppm, br 1.22 ppm, br

Ac ce p

te

d

M

4'

H-3''

an

4

δC [ppm]

C-1''' C-5'' C-2 C-3a C-7a C-5 C-3'' C-2'' C-6 C-4 C-4'' C-7 C-6'' C-1' C-2''' C-3''' C-2' C-1'' C-3'

171.8 ppm 158.0 ppm 154.9 ppm 141.9 ppm 136.5 ppm 130.9 ppm 129.4 ppm 128.0 ppm 121.3 ppm 117.5 ppm 114.8 ppm 110.0 ppm 63.47 ppm 50.29 ppm 43.56 ppm 39.27 ppm 38.52 ppm 33.87 ppm 31.09 ppm

C-5' C-4' C-7'' C-4''' C-5'''

26.12 ppm 25.67 ppm 14.82 ppm 14.19 ppm 13.12 ppm

us

5''

O

Atom

cr

Atom

ip t

- 59-

H-2',4',5' H-4',5' H-7'' H-4''',5''' H-4''',5'''

Fig. 19: NMR assignments of 1-(cyclohexylmethyl)-2-[(4-ethoxyphenyl)methyl]-N,Ndiethyl-1H-benzimidazol-5-carboxamide(6), AB = AB-system, br = broad, other abbreviations for multiplicities of 1H signals see fig. 11

Page 59 of 61

- 60Legends

cr

Acquired and simulated LC-HRMS-spectrum of compound 1with suggested molecular formulae (SmartFormula)

an

Fig. 3:

us

Fig. 2: EI-MS spectrum (top) and CI-MS spectrum of 2 (bottom)

ip t

Fig. 1: EI-MS spectrum (top) and CI-MS spectrum of 1 (bottom)

Acquired and simulated LC-HRMS-spectrum of compound 2 with suggested molecular formulae (SmartFormula)

d

Fig. 5:

M

Fig. 4: Extracted chromatogram traces of C23H34N3OS and C23H34N3O3, illustrating the presence of one sulfur atom instead of two oxygens

te

Fig. 6: Extracted chromatogram traces of C21H30N3S and C21H30N3O2, illustrating the presence of one sulfur atom instead of two oxygens

Ac ce p

Fig. 7: bbCID spectrum and extracted chromatographic traces of compound 1 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 400.2411 are not annotated (non-parallel EICs). EICs of 213.1393 and 227.0645 are not illustrated due to their relatively low intensity Fig. 8: bbCID spectrum and extracted chromatographic traces of compound 2 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 356.2167 are not annotated (non-parallel EICs) Fig. 9: IR spectrum of 1 (top) and detail (bottom)

Fig. 10: IR spectrum of 2 (top) and detail (bottom)

Page 60 of 61

- 61Fig. 11: NMR assignments of N-(2-methoxyethyl),N-iso-propyl-2-(1-pentyl-1H-indol-3-yl)4-thiazolemethanamine(1). s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, sept = septet, m = multiplet

ip t

Fig. 12: NMR assignments of N,N-diethyl-2-(1-pentyl-1H-indol-3-yl)-4-thiazolemethanamine(2), abbreviations for multiplicities of 1H signals see fig. 11

us

cr

Fig. 13: Possible fragmentation pathways for m/z 213, 227 and 283 after CI/ESI

an

Fig. 14: 3-([1,3]-thiazol-2-yl)indoles with CB1 receptor activity according to [16-18]

Acquired and simulated spectrum of compound 6 after LC-HRMS with suggested molecular formulae (SmartFormula)

d

Fig. 16:

M

Fig. 15: EI-MS spectrum (top) and CI-MS spectrum of 6 (bottom)

Ac ce p

te

Fig. 17: bbCID spectrum and extracted chromatographic traces of compound 6 and its fragments with suggested molecular formulae (SmartFormula). Signals not deriving from the precursor 448.2957 are not annotated (non-parallel EICs) Fig. 18: IR spectrum of 6 (top) and detail (bottom)

Fig. 19: NMR assignments of 1-(cyclohexylmethyl)-2-[(4-ethoxyphenyl)methyl]-N,Ndiethyl-1H-benzimidazol-5-carboxamide(6), AB = AB-system, br = broad, other abbreviations for multiplicities of 1H signals see fig. 11

Page 61 of 61