In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

Vladimir Sizoi, Tim Sobolevsky, Svetlana Appolonova, Grigory Rodchenkov

Prosidol: new piperidine-type narcotic

Moscow Anti-Doping Centre, Russia

Introduction Prosidol (1-(2-ethoxyethyl)-4-phenyl-4-propionyloxypiperidine) is a new synthetic developed in the Laboratory of Medicinal Compounds at the Kazakhstan Institute of Chemical Sciences in the 1990s and firstly synthesized in Novokuznetsk Chemical- Pharmaceutical Institute, Russia [1]. The drug has been patented in Russian Federation, Kazakhstan, Switzerland, Italy and Great Britain, and now it is used in medical practice. There is a wide spectrum of piperidine derivatives that possess analgesic activity (Fig. 1).

Ph COOEt COOEt Ph COOEt Ph

N N N COOEt Ph

O O N CH 3 NH2 O OH

furethidine carbetidine

Ph OC(O)Et

OC(O)Et OC(O)Et Ph Ph CH3 N H C CH 3 3 prosidol N N CH CH3 3 O promedol CH3

Fig. 1. Piperidine-type derivatives with opioid activity. They all bind to opioid µ-receptors and, according to Beckett-Casey hypothesis [2], share the same pharmacophore group (Fig. 2):

111 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

R N C

Fig. 2. Pharmacophore group for opioid according to Beckett-Casey (quaternary carbon atom with adjacent aromatic ring and tertiary nitrogen atom separated by ethylene bridge).

Prosidol is claimed to be as highly active analgesic as but less addictive and much more tolerable, although the side effects are typical for and include addiction, abstinence syndrome, respiratory depression, bradycardia etc. Prosidol can be administered intravenously, intramuscularly, subcutaneously (as 1% solution), orally (25 mg tablets) or buccally (10 and 20 mg tablets). Maximal therapeutic dose is 250 mg per day. The analgesic effect starts in 20 min and lasts for several hours [3]. The Prohibited List specifies only restricted number of narcotic analgesics, although there is a wide spectrum of compounds with such activity. Logically, application of any narcotic analgesic should constitute a doping violation. Therefore, it seems reasonable to monitor if the athletes abuse pharmaceuticals that are not formally prohibited but provide the same physiological effects as typical opioids like morphine. We have performed the excretion study of prosidol after buccal administration of single 20 mg tablet. The drug and two main metabolites identified as O-desethyl and O-desethyl-O’- despropionyl were found non-conjugated in urine. In addition, 2 minor conjugated metabolites, supposedly hydroxyprosidol and its desethyl analog, were detected by GC-MS EI as trimethylsilyl derivatives.

Experimental Urine samples were collected 2 and 8 hrs after buccal administration of 20 mg to a cancer patient who was receiving prosidol by medical prescription. No blank urine was available. A small part of prosidol tablet was dissolved in methanol (not quantitatively) and analyzed by GC-MS directly and after silylation with MSHFBA-TMSI-TMCS. Urine was subjected to standard screening procedures for volatile stimulants and total anabolic steroids [4]. Following the first procedure, to 5 ml of urine 30 µl of diphenylamine internal standard solution, 300 µl of 5M KOH, 5 ml of diethyl ether and 1 g of anhydrous

112 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

Na2SO4 were added followed by vortexing and centrifugation to separate layers. Ether layer was evaporated to dryness at 60°C and reconstituted in 50 µl of methyl tert-butyl ether. Two µl aliquot was injected into GC-NPD Agilent 6890N and GC-MS Agilent 6890N/5973inert in split mode 1:10 at injection port temperature of 250°C. Separation was performed on a HP-1 Ultra quartz capillary column 12 m × 0.2 mm × 0.33 µm, temperature program was as follows: 110°C (2 min), 15°C/min to 300°C (2 min). Helium carrier gas flow rate 1.1 ml/min (GC-NPD) and 0.8 ml/min (GC-MS). Following the second procedure, to 3 ml of urine 50 µl of methyltestosterone internal standard solution, 1 ml of 1M acetate buffer (pH 5.5) and 30 µl of β-glucuronidase from Helix Pomatia were added and enzymolysis was performed at 57°C for 3 hrs. After enzymolysis pH was adjusted to 9 by adding carbonate/bicarbonate buffer, and then ca. 2 g of anhydrous

Na2SO4 were added followed by liquid-liquid extraction with 5 ml of diethyl ether. After centrifugation ether layer was evaporated to dryness at 60°C and the residue was derivatized

with 50 µl of MSTFA / NH4I / dithiothreitol (1000:2:4 v/w/w) or MSHFBA / TMSI / TMCS (100:2:5 v/v/v) at 70°C for 20 min. After cooling and transferring to vial, 3 µl aliquot was injected in GC-MS Agilent 6890N/5973inert in split mode 1:10 at injection port temperature of 280°C. Separation was performed on a HP-1 Ultra quartz capillary column 12 m × 0.2 mm × 0.33 µm, temperature program was as follows: 190°C (0 min), 2°C/min to 234°C (0 min), 12°C/min to 300°C (4.5 min). Helium carrier gas head pressure was 10.1 psi.

Results and Discussion Tablet profile We analyzed a methanolic solution of prosidol tablet by GC-MS without derivatization. It was found that the pharmaceutical contains a number of impurities structurally related to prosidol, and some of them cannot be deduced from the synthesis layout presented by manufacturer. Based on mass spectral data, the following structures were attributed to three main tablet constituents: 1-(2-ethoxyethyl)-1,2,3,6-tetrahydropyridine (estimated content 21%, Fig. 3a), 1-(2-ethoxyethyl)-3-phenylpyrrolidine (9%, Fig. 3b), and prosidol (68%, Fig. 4). There were also detected 6 minor impurities including even brominated prosidol (!).

113 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

172 102

N 58 N

O

CH3 O

42 42 CH3

158 231 91 160 115 128 143 77 115 91 131 219 56 77 102 65 202186 216 144 204 Fig. 3a. Impurity in prosidol tablet Fig. 3b. Impurity in prosidol tablet

Prosidol gives characteristic electron ionization mass spectrum with prominent ion at m/z 172 by successive elimination of propionyl and ethoxy groups. This ion is common for all prosidol-related structures.

172

+ N 2CH

O

+ N O 2CH

CH3 246

OH

+ N 2CH 42 190 57 77 91 103115 143 160 129 231 305 260 276290 Fig. 4. Mass spectrum of prosidol (the molecular ion is at m/z 305).

It is worth mentioning that urine matrix has only slight interference with ion at m/z 172 and therefore it can be easily detected.

114 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

Prosidol metabolism Prosidol is readily metabolized to produce mostly desethyl (I) and desethyl despropionyl (IV) metabolites, which can be found in free fraction, and two minor hydroxy metabolites excreted as conjugates (desethyl hydroxyprosidol, II, and hydroxyprosidol, III, Fig. 5). Structure of the metabolites was proposed from electron ionization mass spectrometry data; no confirmatory synthesis was performed. Prosidol can be detected only in the initial period of a few hours after administration, and after 8 hrs it has been almost completely metabolized (Fig. 6). It is interesting to notice that metabolite II was not detected in the initial period of 2 hrs.

O O O

O O O

CH3 CH3 OH

N N N III

O OH OH

CH3

O

O OH

CH3

N III N IV

O OH

OH

Fig. 5. Metabolism of prosidol.

115 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

45 8 hrs 2 hrs 40 8 hrs

35

30 2 hrs

25 2 hrs 20 8 hrs 15 2 hrs

10 8 hrs 5 2 hrs 8 hrs 0 Metabolite IV Metabolite I Metabolite III Metabolite II Prosidol

Fig. 6. Metabolic distribution of prosidol and its metabolites (%) vs. time.

Detection of prosidol Prosidol as well as metabolites I and IV are stable and volatile enough to be detected by gas chromatography without derivatization. The mass spectra of these metabolites are given in Fig. 7a and 7b (the metabolites hereinafter are presented in their elution order). 190 172

246

42 172

190 56 77 203 91 105 42 86 115 131 143 162 221 74 97 115 129143157 214 277 Fig. 7a. Mass spectrum of IV (MW 221). Fig. 7b. Mass spectrum of I (MW 277).

To detect the other prosidol metabolites, urine should be subjected to enzymolysis followed by extraction and silylation. Interestingly, when the mixture of MSHFBA / TMSI / TMCS is used, prosidol and all its metabolites may be detected (for mass spectra see Fig. 8a –

116 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

8d, respective mass chromatogram plotted for m/z 172 is presented in Fig. 9). On the

contrary, using the common in doping analysis derivatization mixture of MSTFA / NH4I / dithiothreitol that promotes enolization, the parent molecule and its metabolites, except metabolite IV (desethyl despropionyl prosidol), are converted to prosidol artifact, namely, desethyl desoxyprosidol. Therefore, only two peaks can be detected in the chromatogram (same urine, Fig. 10): desethyl desoxyprosidol (structure and mass spectrum as TMS derivative are given in Fig. 11, retention time relatively to methyltestosterone – 0.1677) that reflects contribution of all metabolites except metabolite IV, and metabolite IV which is detected as is (RRT 0.2243). 262 172

172

246 73

103 73 350 57 130 190 130 158 91 59 115 158 260275 334 144 191 218 276 365 144 349 Fig. 8a. Mass spectrum of IV as bis- Fig. 8b. Mass spectrum of I as TMS TMS (MW 365). (MW 349). 172 172

334

73 246

130 289 91 73 56 147 144 190 158 57 91 115 128 191 217 257 317 274 Fig. 8c. Mass spectrum of III as TMS Fig. 8d. Mass spectrum of II as bis-TMS (MW 393). (MW 437).

117 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

160000 160000 IV 150000 IV 150000 140000 140000 I 130000 130000 PA 120000 120000

110000 110000

100000 100000

90000 90000

80000 80000

70000 70000

60000 60000 50000 III 50000 40000 40000

30000 30000 20000 PA II 20000 10000 P 10000 0 0 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00

Fig. 9. Derivatization with MSHFBA / TMSI Fig. 10. Derivatization with MSTFA / NH4I / / TMCS (PA – prosidol artifact, P – prosidol, dithiothreitol (PA – prosidol artifact, IV – roman numerals correspond to respective metabolite IV). metabolites).

172

N

158

73 H 130 O 115 143 275 59 91102 202 260 Fig. 10. Mass spectrum and structure of prosidol artifact (MW 275 as TMS).

Conclusion Metabolism of a new piperidine-type narcotic analgesic prosidol was studied. In humans prosidol is readily metabolized to produce four metabolites, two of which (desethyl and desethyl despropionyl prosidol) are excreted free and volatile enough to be detected by GC as is, and other two (hydroxy prosidol and desethyl hydroxy prosidol) are conjugated. As to doping control analysis, prosidol may be easily determined in urine either in screening for volatile stimulants or anabolic steroids. For confirmation purposes enzymolysis followed by extraction and silylation with MSHFBA / TMSI / TMCS is preferable as it would enable detecting all the metabolites of prosidol.

118 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

References [1] V.K. Yu, A.D. Nagimova, K.D. Praliev, S.N. Shin and N. Kempe. Synthesis, Antibacterial and Analgesic Activity of 1-(2-ethoxyethyl)-4-hydroxy(acyloxy)-piperidine-4-carboxylic Acids // Pharm. Chem. J. 2002, 36(7), 382-384. [2] A.H. Beckett, A.F. Casey. Synthetic Analgesics: Stereochemical Considerations // J. Pharm. Pharmacol. 1954, 6, 986-1001. [3] N.A. Osipova, G.A. Novikov, M.S. Vetsheva, B.M. Prokhorov, V.A. Beresnev, N.A. Loseva, S.I. Zemskaia, T.A. Smolina. First Experience in the Use of a New Russian Narcotic Analgesic Prosidol in Oncology // Anesteziol. Reanimatol, 1994, 4, 53-57 (in Russian). [4] M. Donike, H. Geyer, A. Gotzmann, M. Kraft, F. Mandel, E. Nolteernsting, G. Opfermann, G. Sigmund, W. Schanzer, J. Zimmermann. Dope Analysis // Proceedings of I.A.F. World Symposium on Doping in Sport, Florence, May 10-12, 1987.

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