‘Synthesise, Characterise, Analyse (SCA)’: A mul disciplinary approach to tackling New Psychoac ve Substances (NPS)
Gavin McLaughlin, B.Sc., M.Sc., MRSC. PhD Candidate, Athlone Ins tute of Technology (AIT), Ireland Research Assistant, Trinity College Dublin (TCD), Ireland New Psychoac ve Substances (NPS)
• In recent years, there has been an unprecedented increase in the number, types and seizures of chemicals frequently referred to as new psychoac ve substances (NPS). • The constant release of NPS onto the recrea onal drug market con nues to create challenges for scien sts in the forensic, clinical and toxicology disciplines.
120 101 100 100 81 80 74
60 49 41 40 Number of Number of NPS Number of 24 NPS detected 20
0 2009 2010 2011 2012 2013 2014 2015 Year Categories New drugs encountered by 5F-MDMB-PINACA 5F-AKB-48 Pentylone Forensic Science Ireland in 2016 5F-PB-22 Dipentylone (FSI Annual Report 2016) Dibutylone Ethylhexedrone N-ethylpentedrone 3-Fluorophenmetrazine α-PVP Clephedrone MPHP PV8 (α-PHP) PV9 (α-POP) Chloro-PVP 4-chloro-α-PVP
Methyltryptamine Dimethyltryptamine 25I-NBOMe 5-MeO-MiPT
Phenazepam Nitrozolam Fentanyl E zolam Pentanoyl fentanyl Chlorodiazepam 2-fluorofentanyl Problems
• Main problems: – Absence of chemical and pharmacological informa on – Availability (online) – Availability of scien fic and patent literature • Forensic Science Laboratories tend to struggle when faced with the challenge of iden fying these NPS. • Most laboratories do not have access to the sophis cated analy cal techniques, such as NMR and HR-MS, required for the iden fica on of NPS. • Absence of chemical reference standards and searchable drug reference libraries. Current a empts for NPS iden fica on
• Examples: The RESPONSE project & WEDINOS
• These projects provide some important analy cal details about NPS.
• However, the aims of these projects are not to provide extensive chemical data or pharmacological data on NPS.
Importance of Organic Synthesis
• Facilitates the unambiguous identification of an unknown sample through targeted synthesis of all possible isomers. • Allows synthesis of compounds, which may not be commerically available. • Allows for the observation of synthesis related impurities or route specific by-products. • Extending the synthesis of a currently emerging psychoactive substance to a range of analogues and derivatives provides important proactive analytical data to the forensic, law enforcement and clinical communities. • Once synthesised, these compounds may be used for studies beyond analytical characterisation, such as exploration of pharmacological, metabolism or toxicological features. Characterisa on
Range of analy cal techniques u lised to facilitate the detec on, iden fica on and structural elucida on of drugs Importance of Pharmacological Evalua on
• Evalua on of the molecular mechanism of ac on – Technique used: In vitro Monoamine Transporter Assays in rat brain synaptosomes. – Informa on obtained: Informa on on the re-uptake and releasing proper es of a selected drug at the dopamine transporters, norepinephrine transporters and serotonin transporters as well as its selec vity for and potency at each transporter.
• Evalua on of the metabolite profile of NPS – Technique used: pooled Human Liver Microsomes and LC-MS/MS – Informa on obtained: Metabolite profile of a selected drug, which aids in the iden fica on of NPS in biological matrices.
Synthesis, characterisa on and monoamine transporter ac vity of the New Psychoac ve Substance (NPS) Mexedrone and differen a on from its N-methoxy posi onal isomer, N-methoxymephedrone Background
O O H N α NH2 β
Cathinone Mephedrone
O
N
α−PVP Generic Legisla on O
α NH2 β
Any substance (not being bupropion) structurally derived from 2-amino-1- phenyl-1-propanone by modifica on in any of the following ways: • By subs tu on in the phenyl ring to any extent with alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylenedioxy, haloalkyl or halo subs tuents, whether or further in the phenyl ring by one or more other univalent subs tuents. • By subs tu on at the 2- or 3- posi on of the propanone side chain with alkyl subs tuents • By subs tu on at the nitrogen atom with one or more alkyl or dialkyl groups, or by inclusion of nitrogen atom in a cyclic structure Replacement for Mephedrone
A A O O O OO O H H H H N N N NN N O O O O
MephedroneA Mexedrone N-Methoxymephedrone O O O MephedroneH HMexedrone N-Methoxymephedrone N N N O B O O O BBr CH3NHOCHO 3 HCl N O O Br CH3NHOCH3 HCl N Mephedrone Mexedrone N-Methoxymephedrone O DIPEA (a)(b) DIPEA B O (a)(O b) C Br CH NHOCH HCl N O O 3 3 O O NaOMe Br MeNHO H C 2 Br 2 N O DIPEA O O O MeOH (a)(NaOMeCH2Cl2 b) Br2 MeNH2 H Cl NaI O O BrO N (c)(d)(e)(f) MeOH CH2Cl2 C Cl NaI O O O O (c)(O d)(O e)(O f) NaOMe Br MeNH H 2 Br 2 N D O O O MeOH CH2Cl2 H Cl Br NaI2 O MeNH2 O O (c)(d)(Br e)(N f) D CH2Cl2 O O O Cl Cl Cl H (g) (h)(Br2 Br i) MeNH2 N D O CH Cl O O Cl 2 2 Cl H Cl Br MeNH2 (g) 2 Br (h)(N i) CH Cl Cl 2 2 Cl Cl (g) (h)(i) Synthesis of Mexedrone & N- methoxymephedrone
Mexedrone N-methoxymephedrone O O O O
Sodium methoxide Br N N,O-dimethylhydroxylamine. HCl O
Sodium iodide N,N-diisopropylethylamine Cl O
(3-chloro-1-(4-methyphenyl)propan-1-one) (3-Methoxy-1-(4-methylphenyl)propan-1-one) (alpha-bromo-4-methylpropiophenone) (N-Methoxymephedrone)
Br2
O O
H N Methanolic methylamine Br
O O
Mexedrone 2-Bromo-3-methoxy-1-(4-methyphenyl)propan-1-one A 88 280000 Mexedrone EI-MS 240000 6.57 min
200000
160000
120000
80000 56 40000 42 65 119 162 73 105 134 147 175 0 207 40 60 80 100 120 140 160 180 200 m/z B 88 3000000 N-Methoxymephedrone EI-MS 2600000 6.18 min
2200000
1800000
1400000
1000000 Characterisa on: GC-MS 56 600000 42 119 A 88 65 Mexedrone 200000 280000 77 105 133 148 160 174 192 207 EI-MS 40 60 80 100 120 140 160 180 200 m/z 240000 6.57 min C 200000 O O H H H O N 160000 N N N NH Mexedrone 120000 O O 80000 56 m/z 207 m/z 162 m/z 119 m/z 88 m/z 56 m/z 42 40000 M 42 65 119 162 73 105 134 147 175 0 207 40 60 80 100 120 140 160 180 200 m/z B 88 3000000 N-Methoxymephedrone N-Methoxymephedrone EI-MS O O O 2600000 6.18 min N N N N NH O O O 2200000
1800000 m/z 207 m/z 192 m/z 119 m/z 88 m/z 56 m/z 42 1400000 M
1000000 56 600000 42 119 65 200000 77 105 133 148 160 174 192 207 40 60 80 100 120 140 160 180 200 m/z C O O H H H O N N N N NH Mexedrone O O
m/z 207 m/z 162 m/z 119 m/z 88 m/z 56 m/z 42 M
N-Methoxymephedrone O O O N N N N NH O O O
m/z 207 m/z 192 m/z 119 m/z 88 m/z 56 m/z 42 M [%] A LC-ESI-Q-MS 100 158.1 Characterisa on: LC-MS Mexedrone [%] 8.01 min A LC-ESI-Q-MS A O O 110 V 158.1 A H2O H H2 O H 100 Mexedrone N Loss 2of MeOH N 2 80 + N Loss of MeOH + N 8.01 min + + 110 V O 80 176.1 O Observed: 208.13374 Observed: 176.10712 60
119.1 + + 176.1 + C H NO : 208.13321 C H NO : 176.10699 + H 12 18Observed:2 208.13374 11 14 Observed: 176.10712 [M + H] O Δ = 2.58 ppm + Δ = 0.71 ppm + 60 H + C12H18NO2 : 208.13321 C11H14NO : 176.10699 119.1 N H + O Δ = 2.58 ppm Δ = 0.71 ppm 40 [M + H] H
208.1 N 149.1 O + + OH 40 H OH2 208.1 H 190.1 H 149.1 + N + [M + Na] O + N + N 20 117.1 148.2 190.1 + OH OH2 160.2 H [M + Na] O O H O + H
191.1 N 91.1 177.2 141.0 N N 105.0
20 163.1 120.1 195.9 117.1 148.2 84.9 230.2 88.1 160.2 191.1 91.1 177.2
141.0 Observed: 190.12279 105.0 163.1 120.1 195.9 84.9 230.2 88.1 O + 0 OC12H16NO : 190.12264 O 0 60 80 100 120 140 160 180 200 220 240 m/z Δ = 0.77 ppm 60 80 100 120 140 160 180 200 220 240 m/z H Observed: 190.12279 O Loss of H O + MeOHC12H16NO : 190.12264 N Loss of CO 10.63 min Δ = 0.77 ppm 160000 + 10.63 min 160000 OH N O Loss of H 140000 O MeOH N Observed: 119.04903 Loss of CO 140000 C H O+ : 119.04914 8 7 Observed: 158.09650 + Δ = -0.90 ppm C H N+ : 158.09643 120000 O 11 12 N 120000 Δ = 0.45 ppm 8.01 min B 8.01 min H Observed: 119.04903+ B 100000 O B 100000 + O + C8H7O : 119.04914 N Observed: 158.09650 Δ = -0.90 ppm O C H N+ : 158.09643 80000 11 12 80000 Δ = 0.45 ppm B 119.1 LC-ESI-Q-MSLC-ESI-Q-MS 119.1 + H 100100 60000 60000 H + N-MethoxymephedroneN-Methoxymephedrone O Observed: 208.13297O Observed:119.04901 + + + O 10.6310.63 min min N C12H18NO2 : 208.13321 N C8H7O : 119.04914 O = -1.15 ppm = -1.03 ppm 110110 V V 40000 40000 O 8080 20000 O O 20000 H H Loss of MeOH + N N H + O + 60 0 O Observed: 208.13297 Observed:119.04901 60 0 + + N C12H18NO2 : 208.13321 C8H7O : 119.04914 7.5 10 12.5 min O = -1.15 ppm = -1.03 ppm 7.5 10 12.5 min Observed: 176.10689 40 91.1 C H NO+ : 176.10699 91.1 11 14 40 = -0.60ppm + O H O [M + H] H + N Loss of MeOH N [M + H] + O + 20 105.1 140.9
20 208.2 158.1 120.1 149.0 176.0 105.1 140.9 85.0 195.9 208.2 161.1 158.1 120.1 149.0 176.0 85.0 195.9 0 161.1 Observed: 176.10689 60 80 100 120 140 160 180 200 220 240 m/z + 0 C11H14NO : 176.10699 60 80 100 120 140 160 180 200 220 240 m/z = -0.60ppm Monoamine Transporter Ac vity
Pharmacology summary: • N-Methoxymephedrone showed a transporter-meditated releasing profile comparable to mephedrone, although much lower in potency.
• By contrast, mexedrone was found to be a weak monoamine transporter uptake blocker and weak serotonin releasing agent – ‘hybrid’ ac vity.
• This might explain why this substance received mixed reviews on
A D psychonaut forums. 120 120 DAT 80 Mephedrone 80 Mephedrone N-Methoxymephedrone N-Methoxymephedrone 40
H]DA uptake H]DA 40 Mexedrone
3 Mexedrone release H]MPP+ 3 0 % [ % -Chloromethylmephedrone -Chloromethylmephedrone
0 [ % -9 -7 -5 -3 -9 -7 -5 -3 log [dose] M log [dose] M B E 120 120 NET 80 80 Mephedrone Mephedrone N-Methoxymephedrone 40 N-Methoxymephedrone
H]NE uptake uptake H]NE 40 Mexedrone
3 Mexedrone H]MPP+ release H]MPP+ 3 0 -Chloromethylmephedrone % [ % -Chloromethylmephedrone 0 [ % -9 -7 -5 -3 -9 -7 -5 -3 log [dose] M C log [dose] M F 120 120 SERT 80 80 Mephedrone Mephedrone N-Methoxymephedrone 40 N-Methoxymephedrone
40 release H]5HT Mexedrone H]5HT uptake uptake H]5HT Mexedrone 3 3 -Chloromethylmephedrone 0 -Chloromethylmephedrone % [ % % [ % 0 -9 -7 -5 -3 -9 -7 -5 -3 log [dose] M log [dose] M Effects of Mexedone, N-methoxymephedrone and mephedrone on inhibi on of Schema c representa on of the release assay method uptake and s mula on of release at DAT, SERT and NET in rat brain synaptosomes. Mexedrone: Conclusion
• Chemical profile of mexedrone ascertained. • Pharmacological evalua on of mexedrone concluded it is much less potent than mephedrone. • Vendor sample found to contain mexedrone. • This was the first study on mexedrone, which reiterates the con nuous need to remain vigilant on the availability of newly emerging psychoac ve substances. Overall Conclusion
• This approach of combining chemistry with pharmacology allows for the genera on of essen al data and is an effec ve approach to tackling an increasingly complex area of inves ga on where growing demands are placed on inves gators in the field of NPS. Overall Conclusion
• This ‘SCA’ approach has been used for several other NPS including:
4,4-dimethylaminorex 3’,4’-methylenedioxy-4-methylaminorex Diphenidine 2-Methoxydiphenidine
NH2 NH2
O O N N N N O O O
AB-CHMFUPPYCA 3-Fluorophenmetrazine Mexedrone 4-Fluoromethylphenidate
O O F O H N NH2 F NH NH O O O O N N
F Acknowledgements
• Dr. Noreen Morris (Athlone Ins tute of Technology) • Dr. Pierce Kavanagh and his colleagues (Trinity College Dublin) • Dr. Simon Brandt (Liverpool John Moores University) • Mr. John Power (Forensic Science Ireland) • Dr. Geraldine Dowling (Dublin Ins tute of Technology) • Dr. Rachel Chris e (EMCDDA) • Dr. Michael H. Baumann and his colleagues (Na onal Ins tute of Drug Abuse) • Dr. Volker Aüwarter and his colleagues (University Medical Center Freiburg) Publica on list
1. Gavin McLaughlin, Noreen Morris, Pierce V. Kavanagh, John D. Power, Geraldine Dowling, Brendan Twamley, John O’Brien, Gary Hessman, Brian Murphy, Donna Walther, John S. Par lla, Michael H. Baumann, Simon D. Brandt. Analy cal characteriza on and pharmacological evalua on of the new psychoac ve substance 4-fluoromethylphenidate (4F-MPH) and differen a on between the (±)-threo and (±)-erythro diastereomers. Drug Tes ng and Analysis. 2017, 9, 347-357. DOI: 10.1002/dta.2167
2. John D. Power, Pierce V. Kavanagh, Gavin McLaughlin, Michael Barry, Geraldine Dowling, Simon D. Brandt. ‘APAAN in the neck’ – A reflec on on some novel impuri es found in seized materials containing amphetamine in Ireland during forensic analysis. Drug Tes ng and Analysis. 2017. DOI: 10.1002/dta.2194
3. Geraldine Dowling, Pierce V. Kavanagh, Brian Talbot, John O’Brien, Gary Hessman, Gavin McLaughlin, Brendan Twamley, Simon D. Brandt. Outsmarted by nootropics? An inves ga on into the thermal degrada on of modafinil, modafinic acid, adrafinil, CRL-40,940 and CRL-40,941 in the GC injector: forma on of 1,1,2,2-tetraphenylethane and its tetra fluoro analog. Drug Tes ng and Analysis. 2017, 9, 446-452. DOI: 10.1002/dta.2142
4. Gavin McLaughlin, Noreen Morris, Pierce V. Kavanagh, John D. Power, Geraldine Dowling, Brendan Twamley, John O’Brien, Brian Talbot, Donna Walther, John S. Par lla, Michael H. Baumann, Simon D. Brandt. Synthesis, characteriza on and monoamine transporter ac vity of the new psychoac ve substance mexedrone and its N-methoxy posi onal isomer, N-methoxymephedrone. Drug Tes ng and Analysis. 2017, 9, 358-368. DOI: 10.1002/dta.2053
5. Florian Franz, Verena Angerer, Simon D. Brandt, Gavin McLaughlin, Pierce V. Kavanagh, Bjoern Moosmann, Volker Auwarter. In vitro metabolism of the synthe c cannabinoid 3,5-AB-CHMFUPPYCA and its 5,3-regioisomer and inves ga on of their thermal stability. Drug Tes ng and Analysis. 2017, 9, 311-316. DOI: 10.1002/dta.1950.
6. Gavin McLaughlin, Noreen Morris, Pierce V. Kavanagh, Geraldine Dowling, John D. Power, Brendan Twamley, John O'Brien, Brian Talbot, Harald H. Si e, Simon D. Brandt. Test purchase, synthesis and characteriza on of 3- fluorophenmetrazine (3-FPM) and differen a on from its ortho- and para-subs tuted isomers. Drug Tes ng and Analysis. 2017, 9, 369-377. DOI: 10.1002/dta.1945.
Publica on list
7. John D. Power, Pierce V. Kavanagh, Gavin McLaughlin, Geraldine Dowling, Michael Barry, John O'Brien, Brian Talbot, Brendan Twamley, Simon D. Brandt. Forensic analysis of P2P derived amphetamine synthesis impuri es: iden fica on and characteriza on of indene by-products. Drug Tes ng and Analysis. 2017, 9, 446-452. DOI: 10.1002/dta.1944.
8. Gavin McLaughlin, Noreen Morris, Pierce V. Kavanagh, John D. Power, Brendan Twamley, John O’Brien, Brian Talbot, Geraldine Dowling, Simon D. Brandt. The synthesis and characterisa on of the ‘research chemical’ N-(1-amino-3-methyl-1- oxobutan-2-yl)-1-(cyclohexylmethyl)-3-(4-fluorophenyl)-1H-pyrazole-5-carboxamide (3,5-AB-CHMFUPPYCA) and differen a on from its 5,3-regioisomer. Drug Tes ng and Analysis. 2016, 8, 920-929. DOI: 10.1002/dta.1864.
9. Gavin McLaughlin, Noreen Morris, Pierce V. Kavanagh, John D. Power, John O’Brien, Brian Talbot, Simon P. Ellio , Jason Wallach, Hamilton Morris, Simon D. Brandt. Test purchase, synthesis and characteriza on of 2-methoxydiphenidine (MXP) and differen a on from its meta- and para-subs tuted isomers. Drug Tes ng and Analysis. 2015, 8, 98-109. DOI: 10.1002/dta. 1800.
10. John D. Power, Pierce Kavanagh, Gavin McLaughlin, John O’Brien, Brian Talbot, Michael Barry, Brendan Twamley, Geraldine Dowling, Simon D. Brandt. Iden fica on and characteriza on of an imidazolium by-product formed during the synthesis of 4- methylmethcathinone (mephedrone). Drug Tes ng and Analysis. 2015, 7, 894-902. DOI: 10.1002/dta.1789.
11. Gavin McLaughlin, Noreen Morris, Pierce V. Kavanagh, John D. Power, Brendan Twamley, John O’Brien, Brian Talbot, Geraldine Dowling, Olivia Mahony, Simon D. Brandt, Julian Patrick, Roland P. Archer, John S. Par lla, Michael H. Baumann. Synthesis, characteriza on and monoamine transporter ac vity of the new psychoac ve substance 3’,4’-methylenedioxy-4- methylaminorex (MDMAR). Drug Tes ng and Analysis. 2014, 7, 555-564. DOI: 10.1002/dta.1732.
12. Jason Wallach, Pierce V. Kavanagh, Gavin McLaughlin, Noreen Morris, John D. Power, Simon P. Ellio , Marion S. Mercier, David Lodge, Hamilton Morris, Nicola M. Dempster, Simon D. Brandt. Prepara on and characteriza on of the ‘research chemical’ diphenidine, its pyrrolidine analogue, and their 2,2-diphenylethyl isomers. Drug Tes ng and Analysis. 2014, 7, 358-367. DOI: 10.1002/dta.1689