Characterization of the Thermal Degradation Product of UR‐144 Paul D. Kennedy, Gregory W. Endres Cayman Chemical Company, Ann Arbor, MI 48108 Anna Deakin United States Army Criminal Investigation Laboratory (USACIL), Gillem Enclave, Forest Park, GA 30297 David Eckre Marshall University Huntington, West Virginia 25755
Part 1: Data provided by Cayman Chemical Company, including: Introduction Figure 1: TIC of GC/MS(EI) of UR-144 with small degradation peak EI Spectra of UR-144 and UR-144 Degradant Figure 2: EI Spectrum of purified UR-144 Degradant Figure 3: EI Spectrum of UR-144 Figure 4:
1
Figure 5:
13
H NMR C NMR and
13
C- APT
Figure 6: APCI MS Figure 7: FTIR-ATR
Part 2: Data provided by the United States Army Criminal Investigation Laboratory (USACIL), including: Figure 8: GC/MS(EI) of UR-144 Degradant Figure 9: ESI MS Full Scan Figure 10: ESI MS/MS Product Ion Scan of m/z = 214 and 312 Figure 11: FTIR spectrum via GC-IR
References:
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UR‐144 Degradant Structure: CAS Registry No.: N/A Formal Name: 3,3,4‐trimethyl‐1‐(1‐pentyl‐1H‐indol‐3‐yl)pent‐4‐en‐1‐one Synonyms: UR‐144 Degradant, MF: C21H29NO FW: 311.46 Storage: ambient SMILES: CCCCCN1C2=CC=CC=C2C(C(CC(C)(C)C(C)=C)=O)=C1 InChI Key: NBJHWTCAQOYUND‐UHFFFAOYSA‐N InChI: InChI=1S/C21H29NO/c1‐6‐7‐10‐13‐22‐15‐18(17‐11‐8‐9‐12‐19(17)22)20(23)14‐21(4,5)16(2)3/h8‐9,11‐12,15H,2,6‐ 7,10,13‐14H2,1,3‐5H3
Background UR‐144 is a tetramethylcyclopropyl (TMCP) synthetic cannabinoid analog which has become a popular unregulated replacement for previous generation synthetic cannabinoids that have been recently regulated in the United States.1 Invented by Abbott Laboratories2 for pharmaceutical applications, UR‐144 shows higher affinity for the CB2 receptor over the CB1 receptor.3 GCMS spectral data has been presented for UR‐144 as well as other TMCP cannabinoid analogs such as XLR‐11 and A‐796,260.4 Common to all of these analyses is the presence of an impurity related to the parent TMCP compound. In all cases, the impurity possesses a similar spectrum to its parent TMCP compound with the exception that there is a prominent fragment ion 15amu higher than the parent compound’s Base Peak ion. Furthermore, published reports demonstrate that pyrolysis of UR‐144 results in the formation of the major impurity with the proposed structure in Scheme 1 (Compound 2).5 Based on the assumption that the TMCP cannabinoids likely undergo thermal decomposition, and further based on the mass spectrum showing a prominent fragment ion 15amu higher than the base peak, the working hypothesis was that the tetramethylcyclopropyl ring undergoes opening upon heating to produce a degradant having the same molecular weight as the parent compound, but with the ability to undergo a McLafferty rearrangement to produce the observed fragment ion in the GC‐MS. The mechanism of the rearrangement is unclear but it is possible that the mechanism is a heterolytic ring cleavage, possibly aided by the presence of trace nucleophiles such as water or solvents present in the neat sample, and further stabilized by the carbonyl. Rearrangement of UR‐144 (1) and the proposed structure of the UR‐144 degradant (2) is shown in Scheme 1. The GCMS data for UR‐144 and the UR‐144 degradant is shown in Figure 1 along with the proposed McLafferty rearrangement to yield the characteristic fragment ion. Scheme 1
Nu
O
O
H
N
N 1
2
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Figure 1: UR‐144 and UR‐144Degradant GC‐MS Data
UR‐144
UR‐144 Degradant
UR‐144 GC‐MS
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UR‐144 degradant in UR‐144
To confirm the structure of the UR‐144 Degradant, neat UR‐144 (Cayman Chemical, Item 11502) was heated under nitrogen atmosphere at 130 C overnight. The resulting crude mixture consisting of starting material and the ring‐opened degradant were separated by column chromatography. Purity was determined to be >98% by HPLC chromatography (90:10:0.1 methanol/water/acetic acid). As expected, the GC‐ MS matched the degradant detected in the UR‐144 sample (Figure 2). Furthermore, the purified UR‐144 Degradant was fully characterized by ESI‐MS, GC‐MS, FTIR, 1H NMR, 13C NMR, and 13C‐APT NMR (Figures 3‐7). All data was consistent with the proposed structure including notably the 1H NMR spectrum which shows the vinylic CH2 protons, and the 13C APT spectrum which clearly shows 12 quaternary/methylene carbons as well as 5 aromatic methine carbons and 3 methyl carbons (the gem‐dimethyl carbons are equivalent and appear as a single 13C peak at 27.6ppm). Figure 2. GCMS of Purified UR‐144 Degradant.
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File :Y:\Forensics\11928-0442099.D Operator : PDK Acquired : 14 Nov 2012 13:36 using AcqMethod CAY_DRUG_CATH.M Instrument : Instrument #1 Sample Name: UR-144 Degradant Misc Info : 30mx0.32mm, 0.5u Rtx-5MS, 50C-30C/min-300C Vial Number: 2 Abundance
TIC: 11928-1.D\data.ms 10.170
1100000 1000000 900000 800000 700000
Figure 3
Instrument: Agilent 6890 GC / 5973 MSD Column: 30mx0.32mm, 0.5um Rtx-5MS Carrier Gas: Helium Flow: 2mL/min Inlet temp: 300C 15:1 split Oven Program: 240C for 1 minute, ramp to 300C at 30C/min, Hold at 30C for 27minutes Transfer Line Temp: 300C
600000 500000 400000 300000 200000 100000 0 Time--> Abundance
4.50
5.00
5.50
6.00
6.50
7.00
130000
7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 Scan 1816 (10.167 min): 11928-1.D\data.ms (-1758) (-) 214
120000 229
110000 100000 90000
296
80000 70000
144
60000 50000 311 40000
130
41 55
30000
172
116 20000
187
83
67
268
200
254
102
10000
156 75
238
91
282
0 m/z-->
40
50
60
70
80
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 5 of 16
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\\sulfur\nmrdata\JW7-298-119-144_20121023_\JW7-298-119-144_20121023_01\JW7-298-119-144_20121023_001.fid\fid Oct 23 2012 1H 399.97 Nucleus Frequency (MHz) CHLOROFORM-d Number of Transients 16
1
10
03/21/13
9
8
8.49 8.48 8.47 8.46 8.46 8.45 8.45 8.44
0.92
2.06
7
7.33 7.29 7.28 7.27 7.27 7.26
7.68 1.03
6
5
H NMR (400 MHz, CHLOROFORM-d) 8.40-8.52 (m, 1H), 7.68 (s, 1H), 7.31-7.37 (m, 1H), 7.24-7.31 (m, 2H), 4.80 (s, 1H), 4.76-4.79 (m, 1H), 4.14 (t, J=7.14 Hz, 2H), 2.89 (s, 2H), 1.85-1.93 (m, 2H), 1.84 (s, 3H), 1.27-1.42 (m, 4H), 1.25 (s, 6H), 0.90 (t, J=6.96 Hz, 3H)
File Name Date Solvent
Lot #0442099
Item #11928
4.80 4.78 4.78 4.77 0.96
4.16 4.14 4.12
4
2.13
3
2.05
2.89
1.84 2.89
1.92 1.90 1.88
2
N
O
1.25 0.90
Chemical Shift (ppm)
6.00 2.78
1.37 1.36 1.34 1.34
6 of 16 0.91 0.88
UR-144 Degradant
Figure 4
0
0.05
0.10
0.15
0.20
0.25
0.30
0
4.77 4.78
4.80
0.96
4.5
8.45
8.44 8.43
8.47 8.46 8.46
8.49 8.48
8.5
2.13
4.0
3.5
8.0 2.89
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3.0
2.05
7.68 1.03
2.5
7.5
7.35 7.35 1.08 2.06
2.0
2.89
1.92 1.90 1.88
0.92
7.36
7.27 7.26 7.25
7.29 7.29 7.28 7.34 7.34 7.33 7.31 1.84
0.05
Chemical Shift (ppm) 1.25
2.78
Chemical Shift (ppm)
6.00
1.40 1.37 1.36 1.35 1.34 1.30 1.34
0.10
4.14
4.16 4.12
0.90 0.91 0.88
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1.25 1.27 1.28 1.29 1.30 1.31 1.32 1.32 1.33 1.34 1.34 1.35 1.36 1.37 1.39 1.39 1.40 1.41 1.84 1.86 1.88 1.90 1.92 2.89 4.12 4.14 4.16 4.77 4.78 4.78 4.80 7.25 7.26 7.27 7.27 7.28 7.29 7.29 7.31 7.32 7.32 7.33 7.34 7.34 7.35 7.35 7.35 7.36 7.68 8.43 8.44 8.45 8.45 8.46 8.46 8.46 8.47
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
8.49
0.91
3
8.48
0.90
2
62
0.88
1
61
(ppm)
No.
(Hz)
3393.9
3389.8
3386.9
3385.4
3382.5
3381.8
3380.3
3378.1
3374.4
3371.9
3072.3
2945.6
2941.6
2939.4
2938.3
2936.8
2933.9
2932.8
2929.5
2925.9
2921.8
2917.1
2915.2
2911.6
2908.3
2906.1
2902.4
2899.1
1919.6
1911.9
1910.4
1909.3
1663.6
1656.3
1649.3
1156.1
766.5
758.8
751.4
744.5
737.2
564.3
560.7
557.4
554.8
547.8
542.0
540.9
537.2
535.4
533.6
529.9
527.7
524.4
520.4
517.1
511.6
509.8
498.4
365.5
358.9
351.6
-
-
-
-
-
-
-
-
-
-
7.68
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
*(ppm)
-
-
-
-
-
-
-
-
-
-
3072.23
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
*(Hz)
Height
0.0020
0.0071
0.0309
0.0251
0.0255
0.0234
0.0234
0.0297
0.0042
0.0005
0.1696
0.0044
0.0212
0.0214
0.0205
0.0223
0.0338
0.0468
0.0106
0.0052
0.0211
0.0628
0.0848
0.0720
0.0535
0.0370
0.0506
0.0035
0.0820
0.0580
0.0742
0.0437
0.0706
0.1256
0.0691
0.3173
0.0130
0.0344
0.0478
0.0338
0.2525
0.0031
0.0049
0.0088
0.0110
0.0243
0.0389
0.0363
0.0422
0.0501
0.0478
0.0400
0.0244
0.0290
0.0186
0.0139
0.0066
0.0094
1.0000
0.0793
0.1869
0.0892
-
-
-
-
-
-
-
-
-
-
0.1690
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
*Height
-
-
-
-
-
-
-
-
-
-
1.09
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
*FWHH
200
180
160
140
120
100
80
60
CHLOROFORM-d
\\sulfur\nmrdata\JW7-298-119-144_20121026_\JW7-298-119-144_20121026_01\JW7-298-119-144_20121026_002.fid\fid Oct 26 2012 13C 100.58 Nucleus Frequency (MHz) CHLOROFORM-d Number of Transients 512
Varian Innova 400MHz NMR with VNMRJ v2.2revD
File Name Date Solvent
152.48
Lot # 0442099
134.53 136.68
122.98 122.32 123.11 126.51
8 of 16 118.03
109.00 109.65
Item #11928
77.32 77.00 76.69
194.58
N
O
38.94
40
27.64
13.87
19.92
22.20
Chemical Shift (ppm)
28.95 29.52
UR-144 Degradant
Figure 5
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47.04
49.56
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7720.4
22.24
27.68
28.98
29.55
38.98
47.07
49.58
76.76
77.07
77.39
109.05
109.70
118.04
122.35
123.00
123.15
126.55
134.62
136.72
152.49
194.63
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
0.2541
-0.1169
-0.1390
-0.1600
0.4444
-0.1824
0.7059
0.5973
0.6977
-0.1595
0.7210
-0.7477
-0.3149
-0.3218
-0.2965
-0.4065
-0.5629
-0.1848
-0.5120
-0.5022
1.0000
-0.3269
\\sulfur\nmrdata\JW7-298_APT.fid\fid 100.58 Solvent
136.72
140
118.04
120
100
Nov 6 2012
76.76 77.07 77.39
80
60
13C Nucleus Number of Transients 256
40
38.98
03/21/13
160
Date
N
27.68
CH0 + CH2
CH + CH3
13.91 19.96
Chemical Shift (ppm)
28.98 29.55
180
CHLOROFORM-d
O
22.24
200
APT PARAMETERS Varian Innova 400MHz NMR with VNMRJ v2.2revD Relax delay 1s 1st pulse 90deg, 2nd pulse 45deg acq. time 1.303s, width 25125.6 Hz Observe C13 100MHz, Decouple H1 400MHz pwr 41dB on during acq WALTZ-16 modulated line broadening 0.5Hz
19576.6
15338.1
13751.4
13540.4
12728.3
12386.3
12371.1
12306.2
11873.0
11034.1
10968.0
7784.1
7752.0
4734.1
3920.9
2972.6
2914.7
2784.4
2236.6
2008.0
Height
0.4330
194.63
13C
4987.2
19.96
2
(Hz)
1
1398.8
(ppm)
13.91
No.
File Name Frequency (MHz)
134.62
Lot # 0442099
123.15
Item #11928
122.35
UR-144 Degradant
Figure 5
49.58 47.07
109.70 109.05
126.55
152.49
Figure 6
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0
8
16
24
32
4000
3052.89 3090.5 3106.89
40
2931.39 2959.36
48
3800
3600
3400
3200
3000
2729.86
2871.61
2800
2600
2400
2200
2000
1730.87 1781.01
1800
1613.23
56
1483.05
64
1575.62
72
80
1526.93
1636.85
1600
O
N
1200
684.15 744.42
890.99
Wavenumber (cm-1)
957.04 1013.93 1059.26 1104.09 1134.47 1189.91 1217.88 1306.11 1339.38 1386.63
1465.7
1400
768.53 792.15 830.24
Nicolet iS10 FTIR with iTR Smart Diamond ATR 16 scans background subtracted ATR Corrected and Baseline corrected 4000 - 650 cm-1 4cm-1 resolution
Lot #0442099
Item #11928
UR-144 Degradant
%Transmittance
Figure 7
GC/MS(EI) Data Figure 8:
GC/MS(EI) of UR-144 Degradant
Sample preparation: Instrument: GC Parameters:
MS Parameters:
Extracted solid with methanol to yield a concentration of ~ 1 mg/mL Agilent 7890A gas chromatograph / 5975C mass spectrometer Column: HP-5MS; 30m x 0.250mm x 0.25 micron Carrier gas: He Oven program: 1) 220°C initial temperature for 2.0 minute 2) Ramp to 280°C at 5°C/minute 3) Hold at 280°C for 2 minutes Split ratio: 100:1, 1 microliter injected Flow: 1 mL/minute for 16.0 minutes Temperatures: Injector: 250° C Transfer line: 290° C Source: 230°C Quad: 150°C Mass scan range: 34-550 amu Threshold: 150 Tune file: atune.u
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LC-MS/MS(ESI) Data Experiment Parameters: Standard ~ 100 ug/mL in methanol, 1 uL injection INSTRUMENT: Thermo TSQ Quantum Access MAX in ESI Mode (HESI II Ion Source) equipped with an Accela Autosampler and Quaternary Pump. LC GRADIENT: Ammonium Acetate Buffer (15mM) (A) and Acetonitrile (B) Time %A %B Flow 0 50 50 300 uL/min 2 25 75 300 uL/min 4 25 75 300 uL/min 5 50 50 300 uL/min MS EXPERIMENTS FULL SCAN (ESI): 100 650 m/z PRODUCT SCANS: Ions 214 and 312 at varying Collision Energies (CE) and resulting product spectra (mass range 50 350) were collected. Both discreet energy levels and an energy ramp (RER) were employed. Argon was the collision gas used. INSTRUMENT SETTINGS Spray Voltage: Vaporizer Temp: Sheath Gas Pressure: Ion Sweep Gas Pressure: Aux Gas Pressure: Capillary Temperature: Tube Lens Offset: Skimmer Offset: Capillary Offset:
Figure 9:
3500 150 10 10 10 270 101 0 35
ESI MS (Full Scan)
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Figure 10:
ESI MS/MS Product Ion Scan of m/z = 214 and 312
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GC-IR Data Figure 11:
FTIR spectrum
Instrument: Parameters:
Spectra Analysis Discover IR-GC Transfer line: 250° C Disk speed: 3mm/minute Oven: 250° C FTIR Detector: 4000-650 cm-1 MCT; 4 cm-1 resolution Restrictor: 250° C -40° C Disk:
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References 1. Food and Drug Administration Safety and Innovation Act, Subtitle D‐Synthetic Drugs, Sections 1151‐1153. 2. WO application 2006069196, Pace JM, Tietje K, Dart MJ, Meyer MD, "3‐Cycloalkylcarbonyl indoles as cannabinoid receptor ligands", published 2006‐06‐29. 3. Frost, Jennifer M.; Dart, Michael J.; Tietje, Karin R.; Garrison, Tiffany R.; Grayson, George K.; Daza, Anthony V.; El‐Kouhen, Odile F.; Yao, Betty B. et al. (January 2010). "Indol‐3‐ylcycloalkyl ketones: effects of N1 substituted indole side chain variations on CB(2) cannabinoid receptor activity". J. Med. Chem. 53 (1): 295–315. 4. http://forendexforum.southernforensic.org/viewtopic.php?f=4&t=86&p=462&hilit=bp+311#p338 5. Kavanaugh, P.; Grigoryev, A.; Savcuck, S.; Mikhura, I.; Formanovsky, A. Drug Testing and Analysis, published online in Wiley Online Library Jan 2013.
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