FACT SHEET Ocfentanil

Science at the service of Public health, Food chain safety and Environment.

FACT SHEET

Ocfentanil

May 2017

For more information, please contact: Dr. P. Blanckaert Coordinator Belgian Early Warning System Drugs Scientific Institute of Public Health National Focal Point on Drugs Jyliette Wytsmanstraat 14 B-1050 Brussels, Belgium Tel : 02/642 5408 [email protected]

The information in this message is exclusively meant for the EWS-network, and was

sent to you, as a member of this network, in a confidential way. Therefore the information in this message may not be copied, transferred or made public without

the prior permission of the WIV-ISP. The WIV-ISP takes responsibility for the editing of a press release, if considered as necessary in the framework of its mission.

The information contained in this document is also available on the BEWSD-website (with corresponding pdf-files and analytical data).

This part of the website is not accessible for the general public. A login can be requested by contacting [email protected].

© Scientific Institute of Public Health, Brussels 2011

This report may not be reproduced, published or distributed without the consent of the ISP | WIV.

A. General information Recent death cases in Belgium Substance: Ocfentanil (mixture with caffeine and paracetamol) Discovered in vicinity of deceased victim Date of death: May 2017 Product type: powder Region: Ghent Substance: Ocfentanil (mixture with caffeine and paracetamol) Date of collection: March 2015 Date of analysis: April 2015 Product type: powder Color: brown Region: Dendermonde Created April 2015 Updated May 2017 Type Narcotic Drugs Group Opioids Name Ocfentanil (A-3217) Nature of substance Ocfentanil is a potent synthetic opioid substance structurally related to fentanyl. Compound with a methoxy group instead of a methylgroup, and a fluorine atom placed on the benzene ring. When studied as a supplement to general anaesthesia, researchers concluded that ocfentanil had a similar mode of action to fentanyl and 3 micrograms of ocfentanil was approximately equivalent to 5 micrograms of fentanyl. Systematic chemical name N-(2-fluorophenyl)-2-methoxy-N-[1-(2-phenylethyl)piperidin-4-yl]acetamide Other names /

B. Alerts Alerts Belgium: fatal intoxication linked to the consumption (sniffing) of Ocfentanil, March 2015. Reported by Eurofins forensic lab. A brown powder was found in the house of the victim, containing caffeine, paracetamol and Ocfentanil. Post-mortem blood analysis contained Ocfentanil. The sample was purchased with the use of bitcoins on the internet (darknet?). Reports to EMCDDA Belgium: On 8 April 2015, the Belgian NFP reported the analysis of this sample of Ocfentanil, found during the house-search of a victim of fatal intoxication. The Netherlands: On 24 October 2013, the Dutch FP reported a seizure of white powder seized by the police in September 2013. It was a component of a paracetamol caffeine mixture sold at the drug market as synthetic heroin. Estonia: On 3 March 2017 the Estonian FP reported a seizure of 1,01g white powder seized on 23.04.2016 by the Customs at Tallinn. Post delivery from Netherlands. Sweden: On 29 September 2016 the Swedish FP reported a seizure of 0,97 g and 1,18 g brown pieces (similar to haschish), seized on 14.01.2016 by the Customs at Stockholm. Containing also Paracetamol, coffeine. The substance has been analytically confirmed by GC-MS and TLC at The Swedish Customs Laboratory. France: On 24 June 2016 the French FP reported 3 intoxications that occurred on 31.10.2015. Denmark: On 23 June 2016 the Danish FP reported a seizure of 1g brown powder seized on 22.05.2016 by the Customs at Copenhagen International postoffice. Containing also Caffeine, paracetamol, mannitol. Sent from the Netherlands to Denmark. Packed in a zip bag between several layers of white paper and in a white envelope. Germany: On 29 January 2016 the German FP reported a death case that ocurred on 12.03.2015. Sweden: On 18 December 2015 the Swedish FP reported a seizure of 0.9 g brown powder seized on 2015-11-25 by the Customs at Stockholm. The substance was identified by the Swedish National Forensic Centre (NFC) using GC-MS, LC-HRMS and NMR. Spain: On 2 October 2015 the Spanish FP reported that on June 3rd , 2015, a few milligrams sample of OCFENTANYL was collected by Energy Control´s Drug Checking Service from a user in Madrid (Autonomous Community of Madrid). Sample consisted of a white-coloured powder. Product was sold as heroin on the Internet (160 €/gram). User bought it in March 2015.

C. Pictures Powder found in the 2015 case, Belgium.

Powder collected and analysed by Wedinos, UK

Seizure of brown pieces, Sweden, January 2016

D. Clinical information / Use & Risks Usage Ocfentanil (INN) is an opioid analgesic that is an analogue of fentanyl was developed in the early 1990s. It is similar to fentanyl in effects, producing strong analgesia and sedation, but is slightly more potent. Side effects of fentanyl analogues are similar to those of fentanyl, and include itching, nausea and potentially serious respiratory depression which can be life-threatening. Belgian case of 2015 reported administration through sniffing. Ocfentanil is a potent synthetic opioid structurally related to fentanyl. It was developed as one of a series of potent naloxone-reversible opioids in an attempt to obtain an opioid that had better therapeutic indices in terms of cardiovascular effects and respiratory depression as compared to fentanyl. Study of the analgesic activity of ocfentanil using the mouse hot plate test gave an ED50 of 0.007 mg/kg compared to 0.018 mg/kg for fentanyl; ocfentanil being approximately 2.5 times as potent as fentanyl in this test. In human volunteers ocfentanil induces effective analgesia at 1 μg/kg, while in doses up to 3 μg/kg, analgesia and respiratory depression occurred in a dose-dependent manner. While a further study suggests that ocfentanil may be as effective as morphine in post-operative relief. Ocfentanil was also studied as a supplement to general anaesthesia, in which the researchers concluded that it appears to be similar in action to fentanyl, with 3 μg/kg of ocfentanil approximately equivalent to 5 μg/kg of fentanyl. Modes and scope of the established or expected use Epidemiology What is the availability of ocfentanil on the drug market? It appears that ocfentanil is not being openly sold on the surface web by means of web pages advertising the substance. Reports to the EMCDDA suggest availability of ocfentanil on the dark net. The total amount of ocfentanil seized reported to the EMCDDA between 2014 – 2016 does not exceed 200 grams. The last reported seizure occurred in May 2016. The apparent low amounts of ocfentanil seized should be interpreted in the context of its potency as an analgesic. Fentanyl and its derivatives have been linked with the recent surge in overdose deaths in the USA and Canada. Misuse of fentanyl has also been reported in Estonia as one of the major causes of drug-related deaths. It should also be noted that ocfentanil has been controlled in China since October 2015. Who is using ocfentanil and why? The available data suggests that ocfentanil can be unknowingly used by those seeking heroin; data also suggests that those on opioid substitution programs (such as methadone clinics) and psychonauts may actively seek ocfentanil.

Health risks Pharmacology and toxicology The available data suggests that ocfentanil is a potent opioid similar in structure to fentanyl, the effects of which are reversed by naloxone. It is not specified in the literature which opioid receptors ocfentanil is active at, but it does result in typical fentanyl adverse effects with its ability to cause respiratory depression, bradycardia and hypotension. In mouse hot plate test, ocfentanil produced an ED50 of 7.7 µg/kg compared with fentanyl at 18 µg/kg and morphine at 10100 µg/kg in vivo. Based on the results of this test, ocfentanil is roughly 2.5 times as potent as fentanyl and 1,300 times more potent than morphine. A study by Fletcher and co-workers reported that a 3 µg/Kg dose of ocfentanil produced an equivalent level of analgesia to 5 µg/kg of fentanyl in humans. They do state however, that it was impossible to determine an exact potency relationship from their study and it is important to be aware that differences in the sensitivity and pharmacodynamics of opioids occur when comparing information from in vivo to humans. It has been reported that ocfentanil has a greater therapeutic index (TI) than fentanyl. The therapeutic index is a ratio that quantifies the ‘safety’ margin of a compound. A large therapeutic index results in a greater safety margin between the therapeutic dose and toxic effects. A small therapeutic index results in the opposite and a smaller window between the therapeutic dose and toxic effects. In the tail-flick test (conscious freely moving rat), the TI for respiratory depression of ocfentanil was 7.80 and 5.00 for fentanyl. Whilst in the hot plate test this was 5.90 for ocfentanil and 2.50 for fentanyl. This shows that in the hot plate test that ocfentanil has double the therapeutic index of fentanyl and 1.3 times greater in the tail-flick test. Leslie and co-workers have also reported that the therapeutic index of ocfentanil was consistently greater than that of fentanyl. Although the therapeutic index is higher for ocfentanil, it cannot be considered ‘safe’ due to its higher potency relative to morphine and fentanyl. Self-reported effects Limited data is available on the subjective effects of ocfentanil. Two users who submitted samples to drug checking services reported “distinct, short-acting opioid-like effects”. Other reported effects include: euphoria, relaxation, nausea and to a lesser extent paranoia, agitation and visual hallucinations. Respiratory depression and unconsciousness following consumption were also reported. Other uses No official use known.

E. Legal status Ocfentanil is a scheduled substance in Belgium. Furthermore, it has been scheduled as well in Czech Republic, Denmark, Estonia, Italy, Lithuania, Sweden, Turkey, China and Japan.

F. Chemistry Other chemical names and variants / Chemical Abstracts Service (CAS) registry number 101343-69-5 Molecular information Molecular structure:

Molecular formula: C22H27FN2O2

Molecular weight: 370.46 g/mol

Physical description: Powder; has been described in a brown (reported by Belgium) and a white powder (reported by The Netherlands) Identification and analytical profile can be found at the end of this fact sheet.

Mass spectrum kindly provided by the Dutch NFP.

G. References Dussy FE, et al. An acute ocfentanil fatality: a case report with postmortem concentrations. J Anal Tox. 2016. http://dx.doi.org/10.1093/jat/bkw096 Cookman V. Ocfentanil overdose fatality in the recreational drug scene. Forensic Sci Int. 2016. DOI: http://dx.doi.org/10.1016/j.forsciint.2016.07.005 Quintana, P. et al., Ocfentanil: a novel fentanyl derivative detected as an adulterant of Heroin, 2016, Poster, Information from Energy control; https://energycontrol.org. Mounteney, J. et al., Fentanyls: are we missing the signs? Highly potent and on the rise in Europe. Int. J. Drug. Pol., 2014; 26 (7): 626-631. Bowdle, TA. Adverse effects of opioid agonists and agonist-antagonists in anaesthesia. Drug Saf. 1998; 19(3):173-89. Filer CN, et al. The synthesis of [fluorophenyl-3H(N)] ocfentanil and [fluorophenyl-3H(N)] brifentanil. Journal of Labelled Compounds and Radiopharmaceuticals. 1995;36(11):1019–27. Fletcher JE, Sebel PS, Murphy MR, Mick SA, Fein S. Comparison of ocfentanil and fentanyl as supplements to general anesthesia. Anesth Analg. 1991 Nov;73(5):622-6. PubMed PMID: 1952145 Ebrahim Z, et al. Multiple dose evaluation of the efficacy of ocfentanil hydrochloride (A3217) to produce postoperative analgesia. Anesth Analg. 1991;72:S63. Bagley J. R. et al., Evolution of the 4-Anilidopiperidine Class of Opioid Analgesics, Medicinal Research Reviews, Vol. 11, No. 4, 403436 (1991 Lalinde N, et al. Synthesis and pharmacological evaluation of a series of new 3-methyl-1,4-disubstituted-piperidine Analgesics. J Med Chem. 1990;33:2876–82 Leslie JB et al. Hemodynamic observations of ocfentanil (A-3217) in patients with ischemic heart disease. Anesth Analg. 1990;70:1-450. Weblinks: Warning: Fentanyl Being Sold As Heroin On DNMS, DeepDotWeb, 17 October 2015 https://www.deepdotweb.com/2015/10/17/warning-fentanyl-being-sold-as-heroin/ Héro coupée à l'ocfentanyl sur le deep web, Psych Activ, 16 October 2015 https://www.psychoactif.org/forum/t16265-p1-Hero-coupee-ocfentanyl-sur-deep-web.html "Warning" Ocfentanyl cutting heroin, Bluelight, 16 October 2015 http://www.bluelight.org/vb/threads/772519-quot-Warning-quot-Ocfentanyl-cutting-hero%C3%AFn

Heroin Batch Alerts 2015, Drugs Forum, 15 October 2015 https://drugs-forum.com/forum/showthread.php?p=1664313#post1664313 L'ocfentanil vendu pour de l'héro, PsychActif, 09.05.2015 https://www.psychoactif.org/forum/t14236-p1-L-ocfentanil-vendu-pour-hero.html Media reports from BE on ocfentanyl, April 2015 https://www.google.pt/search?hl=en&tbm=nws&q=Ocfentanyl+OR+ocfentanil&oq=Ocfentanyl+OR+ocfentanil&gs_l=news-cc.3..43j43i53.2922714.2938237.0.2940982.24.3.0.21.21.0.322.573.0j2j0j1.3.0...0.0...1ac.1.nNeDtBIb_cg&gws_rd=cr&ei=tOgnVbmIAsHlUpKtgYAP

Qualitative Analysis Report

Data Filename 2167.D Sample Name 2167

Sample Type Position 25

Column3 Column4 Column3 Column4

Instrument Name GCMSD User Name IFE

Acq Method EC LARGO.M Acquired Time 6/29/2015 2:32:32 PM

IRM Calibration Status Not Applicable DA Method CGL.m

Comment

D:\MassHunter\GCMS\1\5977

\

IFE

MassHunter GC/MS

Acquisition B.07.00

SP2.1654 29-Aug-2013

Copyright © 1989-2013

Agilent Technologies, Inc.

Expected Barcode

Dual Inj Vol

TunePath

OperatorName

Acquisition SW

Version

Compounds

atune.u

6.00.19

True

Sample Amount

TuneName

MSFirmwareVersion

RunCompletedFlag

1

Page 1 of 3 Printed at: 2:56 PM on: 10/1/2015




--- End Of Report ---


Forensic Science International 266 (2016) 469–473

Contents lists available at ScienceDirect

Forensic Science International

journal homepage: www.e lsev ier .com/ locate / forsc i in t

Ocfentanil overdose fatality in the recreational drug scene

Vera Coopman a, Jan Cordonnier a,*, Marc De Leeuw b,c, Vincent Cirimele d

a Eurofins Forensics Belgium, Lieven Bauwensstraat 6, 8200 Brugge, Belgiumb Emergency Department, Algemeen Stedelijk Ziekenhuis, Merestraat 80, 9300 Aalst, Belgiumc Department of Forensic Medicine, Ghent University, Campus UZ , De Pintelaan 185, 9000 Gent, Belgiumd ChemTox, Rue Gruninger 3, 67405 Illkirch, France

A R T I C L E I N F O

Article history:

Received 17 May 2016

Received in revised form 4 July 2016

Accepted 5 July 2016

Available online 17 July 2016

Keywords:

Ocfentanil

Fentanyl analogue

Tissue distribution

New psychoactive substances (NPS)

Ultra performance liquid chromatography

mass spectrometry (UPLC-MS)

A B S T R A C T

This paper describes the first reported death involving ocfentanil, a potent synthetic opioid and structure

analogue of fentanyl abused as a new psychoactive substance in the recreational drug scene. A 17-year-

old man with a history of illegal substance abuse was found dead in his home after snorting a brown

powder purchased over the internet with bitcoins. Acetaminophen, caffeine and ocfentanil were

identified in the powder by gas chromatography mass spectrometry and reversed-phase liquid

chromatography with diode array detector.

Quantitation of ocfentanil in biological samples was performed using a target analysis based on

liquid–liquid extraction and ultra performance liquid chromatography tandem mass spectrometry. In

the femoral blood taken at the external body examination, the following concentrations were measured:

ocfentanil 15.3 mg/L, acetaminophen 45 mg/L and caffeine 0.23 mg/L. Tissues sampled at autopsy were

analyzed to study the distribution of ocfentanil. The comprehensive systematic toxicological analysis on

the post-mortem blood and tissue samples was negative for other compounds.

Based on circumstantial evidence, autopsy findings and the results of the toxicological analysis, the

medical examiner concluded that the cause of death was an acute intoxication with ocfentanil. The

manner of death was assumed to be accidental after snorting the powder.

� 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Ocfentanil is a synthetic opioid and structure analogue offentanyl. Ocfentanil (also called A-3217) was developed inthe early 1990s in the attempt to obtain an analgesic opioid withless cardiovascular and respiratory effects. The activity ofocfentanil was studied in human volunteers, showing thatocfentanil is similar in action to fentanyl, given effectiveanalgesia at 1 mg/kg and being approximately 2 times as potentas fentanyl. Nausea, itching and dose-dependent potentially life-threatening respiratory depression are reported side effects offentanyl and analogues [1–5]. Ocfentanil is not approved formedical use.

To the author’s knowledge, this paper describes the firstreported death involving ocfentanil abused as a new psychoactivesubstance (NPS). The intoxication occurred in March 2015 inBelgium [6,7]. A target analysis on ocfentanil in the postmortem

* Corresponding author. Tel.: +32 50 31 02 52; fax: +32 50 31 02 54.

E-mail address: [email protected] (J. Cordonnier).

http://dx.doi.org/10.1016/j.forsciint.2016.07.005

0379-0738/� 2016 Elsevier Ireland Ltd. All rights reserved.

Downloaded from ClinicalKey.com at Inova FaFor personal use only. No other uses without permission. C

tissues was performed using liquid–liquid extraction and ultraperformance liquid chromatography tandem mass spectrometryoperating in multiple reaction monitoring mode.

2. Case history

A 17-year-old man was found dead in his home, seated andleaning forward on the toilet at 6:00 am. The victim was last seenalive at 22:30 pm when his parents went to sleep. No farewellletter was present. The parents stated that their son has left schoolat age 15 and never left the house. He stopped taken antidepres-sants 3 month before and was prescribed sleeping medication. Theyoung man had a history of illegal substance abuse and waspreviously hospitalized with an acute intoxication due to thecombined intake of cocaine and sleeping tablets. The abusedproducts were purchased over the internet with bitcoins. Drugparaphernalia were found in the proximity of the victim: a brownpowder (2.07 g) in a small zip-locked plastic bag which was lyingon a card with a straw. Residue of a brown powder, similar to thepowder in the bag, were present on the card.

irfax Hospital - JCon January 07, 2017.opyright ©2017. Elsevier Inc. All rights reserved.

http://crossmark.crossref.org/dialog/?doi=10.1016/j.forsciint.2016.07.005&domain=pdf

http://crossmark.crossref.org/dialog/?doi=10.1016/j.forsciint.2016.07.005&domain=pdf


mailto:[email protected]

http://www.sciencedirect.com/science/journal/03790738

www.elsevier.com/locate/forsciint


V. Coopman et al. / Forensic Science International 266 (2016) 469–473470

3. Materials and methods

3.1. Materials

Certified reference ocfentanil (N-(2-fluorophenyl)-2-methoxy-N-[1-(2-phenylethyl)-4-piperidyl]acetamide) was obtained fromViwit Pharmaceutical co., Ltd (Shanghai, China). The referencematerial fentanyl-d5 100 mg/mL in methanol was from Cerilliant(Round Rock, Texas). Standard compounds were diluted inmethanol (1 mg/mL) and stored at �18 8C. Methanol and acetoni-trile were obtained from Fisher Chemical (Fisher Bioblock,Belgium). Formic acid 98–100% was purchased from Merck(VWR, Leuven, Belgium). Water was purified by a Milli-Q systemobtained from Millipore. All solvents and inorganic chemicals wereof analytical grade. The potassium carbonate solution wasprepared by dissolving 13.6 g of anhydrous K2CO3 (VWR, Leuven,Belgium) into a 100 mL volumetric flask and made up to volumewith water.

3.2. Instrumentation

The UPLC-MS/MS analysis was performed using a Acquityseparations module coupled to the Acquity TQD mass detectorequipped with ES interface (Waters Milford, MA, USA). Chro-matographic separation was achieved using a Acquity UPLC HSSC18 column (150 mm length x 2.1 mm i.d., 1.8 mm particle size)with a HSS C18 Vanguard column (5 mm length x 2.1 mm i.d.,1.8 mm particle size) as guard column at 50 8C. The mobile phasesconsisted of 0.15% formic acid (A) and 0.15% formic acid inacetonitrile (B). The following gradient elution was used (runtime15.00 min), starting with 13% B held for 0.50 min., increased to 50%B in 9.50 min., changed to 95% B in 0.75 min and held for 1.50 min.,and finally changed back to initial conditions in 0.25 min and heldfor 2.50 min. The flow rate was 0.400 mL/min. The electrospraysource was operated in the positive ionisation mode (ES+). Productions were obtained by collision-induced dissociation whichallowed the MS/MS to be operated in the multiple reactionmonitoring (MRM) mode. The MRM transitions and conditions forthe measurement of ocfentanil (retentiontime: 5.24 min.); 371.00/188.00 (qualifier) and 371.00/105.00; cone voltage 28 V; collisionenergy 24 V and 32 V, respectively; fentanyl-d5 (retentiontime:6.26 min.): 342.45/188.25 (qualifier) and 342.45/105.10; conevoltage 40 V; collision energy 25 V and 38 V, respectively. Quantita-tions were carried out using the first transition (qualifier). Forconfirmation, the percent ratio of the second transition to thequalifier was calculated and monitored. The source temperature anddesolvation gas (nitrogen) temperature were set at 150 8C and 400 8C,respectively. The gas flow was delivered at a rate of 800 L/h. Thecapillary voltage was 3.00 kV. Waters Mass-lynx system softwareVersion 4.1 was used for instrument control and quantitation.

3.3. Samples

The brown powder in the small zip-locked plastic bag wassubmitted to the laboratory for analysis. Femoral blood, vitreoushumor and a swab of the mucous membrane of the nose weretaken at the external body examination by the medical examiner at11:00 am. The blood samples contained respectively sodiumfluoride and EDTA as preservative.

An autopsy was carried out 3 days later. Multiple samples weretaken for toxicological investigation: cardial blood (withoutpreservative and EDTA, respectively), urine, stomach content(40 mL), liver, kidney, brain tissue, bile and a hair sample from thescalp. The tissue samples were stored at �18 8C for 6 weeks untilarrival of the reference compound ocfentanil and before thedistribution study was performed.

Downloaded from ClinicalKey.com at InovaFor personal use only. No other uses without permission

4. Methods

4.1. Systematic toxicological analysis

The brown powder was subjected to the authors systematictoxicology identification scheme (ISO/IEC 17025:2005 accredited)based on analysis of freshly prepared methanolic sample solutionon reversed-phase liquid chromatography with diode arraydetector (HPLC/PDA) and gas chromatography mass spectrometry(GC/MS) as previously described [8].

A comprehensive systematic toxicological analysis was per-formed on the post-mortem tissue samples to investigate for illegaldrugs, medical drugs, alcohol, volatile substances and otherpoisons. Blood was analyzed for the presence of carboxyhemoglo-bin and cyanide. Screening for the presence of basic drugs in urineand stomach contents was performed by GC/MS and in blood byHPLC/PDA. Analysis for the presence of alcohol in blood, vitreoushumor and urine and of other volatile substances in blood wasperformed by gas chromatography and static headspace gaschromatography with flame ionisation detector. The screening forthe presence of illicit drugs of abuse and medical drugs (includingopiates, amphetamines, methadone and metabolite, cocaine andmetabolites, ketamine/norketamine, fentanyl/norfentanyl andmedical analogues, cannabinoids, benzodiazepines, narcoticanalgesics, antidepressant drugs and several new psychoactivesubstances) were investigated in blood and b-glucuronidasehydrolyzed urine using UPLC-MS/MS methods. Color spot testson urine and gastric content were used to detect salicylates,acetaminophen, phenothiazines and imipramines.

Quantitative determination of acetaminophen (matrix-matched standard calibration using acetaminophen-d4 asinternal standard) and caffeine (matrix-matched externalstandard calibration) in the blood taken at the external bodyexamination (with EDTA as preservative) were performed usingUPLC-MS/MS using the same liquid/liquid extraction and LC-conditions as applied for ocfentanil. The hair was analyzed on thepresence for amphetamine, methamphetamine, MDMA, MDA,MDEA, cocaine, benzoylecgonine, norcocaine, cocaethylene,morphine, 6-monoacetylmorphine, codeine, tetrahydrocannabinol,cannabidiol, cannabinol, ketamine and norketamine. Hair specimenwas decontaminated twice with methylene chloride. The proximal6 cm-long hair section was homogenized by cutting with scissorsinto small pieces (< 1 mm). Twenty mg of hair were incubatedovernight at 40 8C using 400 mL of methanol in the presence of thedeuterated analogues as internal standard. An aliquot of the mediawas evaporated to dryness and injected into the UPLC-MS/MS inMRM mode (ES+). Each compound was identified based on two MRMtransitions and quantified using a calibration curve (ISO/IEC17025:2005 accredited).

4.2. Sample preparation and extraction

The biological tissue samples were submitted to toxicologicalexamination. Liquid–liquid extraction was made after homogeni-zation. Kidney, liver, stomach content (semi-solid), bile and braintissue were homogenized in water at a ratio (m/m) of 1:2 or 1:5 bymeans of a Ultra Turrax1 (IKA T18 basis). The swab of the mucousmembrane of the nose was extracted in 0.5 mL ultrapure water.To a 0.5 mL aliquot of sample (blood, urine, vitreous humor, bile,extract of nose swab) or 0.5 g homogenized tissue sample, 5 mL ofinternal standard solution (fentanyl-d5 1 mg/mL) was added. Afteraddition of the internal standard solution (5 ng fentanyl-d5/sample), the samples were vortex mixed and allowed to equilibrate30 min. prior to extraction. Alkalinization was obtained byaddition of 1.0 mL potassium carbonate solution followed byagitation in a vortex mixer. Extraction was performed with 5 mL of

Fairfax Hospital - JCon January 07, 2017.. Copyright ©2017. Elsevier Inc. All rights reserved.

V. Coopman et al. / Forensic Science International 266 (2016) 469–473 471

a mixture of n-hexane: ethyl acetate (7:3, v/v). After vortex mixingduring 2 min. and centrifugation at 3000 rpm for 5 min, the upperorganic layer was evaporated to dryness under a slow stream ofnitrogen at 40 8C. The dried extracts were reconstituted in 0.5 mLof initial mobile phase. The reconstituted tissue extracts werecentrifuged at 14000 rpm for 5 min. A 10 mL aliquot was injectedinto the UPLC-MS/MS system. A blank was injected before everysample. All samples were analysed in duplicate or triplicate andmean values determined.

4.3. Calibration and validation

Calibrators and quality controls (low: 4.2 mg/L; medium:10.5 mg/L; high: 16.8 mg/L) were prepared by addition of standardsolutions to ocfentanil free pooled whole blood prior to extraction.Six point calibration curves were constructed within the concen-tration range 2.1–21.0 mg/L blood (see Table 1.). Calibrators andcontrols were different preparations of the same drug standard lot.Method validation was based on the document ‘Standard Practicesof Method Validation in Forensic Toxicology’ published by theScientific Working Group of Forensic Toxicology [9]. The followingvalidation parameters were assessed: calibration model, bias,precision, carry-over, interferences, ionization suppression/en-hancement, limit of detection (LOD) and limit of quantitation(LOQ).

The extraction recovery (%) was determined by comparing thepeak areas of an extracted aqueous quality control (medium) withthe peak areas of an unextracted solution with the sameconcentration (n = 6). The internal standard fentanyl-d5 was addedafter extraction and before reconstitution of the evaporated(extracted) quality control.

Table 1Validation parameters and validation data.

Validation parameter Validation data

Calibration model Unweighted linear curve fit, fentanyl-d5 as

internal standard

Mean correlation coefficient (r): 0.9993

Residual plots were evaluated, confirming that

the used calibration model was appropriate

Six point calibration curves with calibration

levels: 2.1 mg/L, 5.2 mg/L, 10.4 mg/L, 15.7 mg/L

and 20.9 mg/L

Bias Measured using three separate samples per

concentration over five different runs:

At concentration low (4.2 mg/L): �0.70%

At concentration medium (10.5 mg/L): 1.69%

At concentration high (16.8 mg/L): �0.10%

Precision Measured using three separate samples per

concentration over five different runs:

Within-run CV: 2.86% (low), 1.14% (medium),

1.79% (high)

Between-run CV: 3.78% (low), 2.24% (medium),

2.08% (high)

Carryover No carryover was observed after highest

calibrator (n = 5)

A blanc was run before every sample

Interference studies No interfering signal from matrix, internal

standard, common drugs abuse (including

medical fentanyl analogues) and prescription

medications

Ionization suppression/

enhancement

Post-extraction addition approach on 10

different postmortem whole blood sources

+7.4% (14.6% CV) at concentration low

+6.2% (7.3% CV) at concentration high

Limit of detection

(LOD)/limit of

quantitation (LOQ)

LOD and LOQ defined as the value of lowest

non-zero calibrator (2.1 mg/L)

Recovery 87.6% (4.6% CV)


5. Results

The initial analysis of the powder by HPLC/PDA revealed thepresence of caffeine, acetaminophen, benzoic acid and anunknown. In addition to these identified compounds, a hit forocfentanil was found on GC/MS by means of computer basedlibrary search of the SWGDRUG Mass Spectral Library (Version 2.2)installed on the Agilent Chemstation. After arrival of the referencecompound ocfentanil, identification was confirmed based onretention time and similarity index or mass spectrum. The massspectrum of the analysis of the brown powder and reference massspectrum of ocfentanil and the UV-spectrum of ocfentanil is shownin Figs. 1 and 2, respectively. Quantitative results were obtained byanalysis of different dilutions of 2 methanolic sample solutions onHPLC/PDA using a 5 point calibration curve: acetaminophen 58.4%(m/m), caffeine 13.7% (m/m) and ocfentanil 2.54% (m/m).

The comprehensive systematic toxicological analysis of thebiological samples, only showed the presence acetaminophen andcaffeine in blood, urine and stomach contents. The followingconcentrations were measured in the femoral blood taken at theexternal body examination: a high therapeutic concentration of45 mg/L acetaminophen and a toxicological irrelevant level of0.23 mg/L caffeine. In the 0–6 cm segment of the hair weredetected: cocaine (0.33 ng/mg), norcocaine (present at a concen-tration below the LOQ of 0.005 ng/mg), benzoylecgonine (0.14 ng/mg), 6-monoacetylmorphine (0.54 ng/mg), morphine (1.10 ng/mg)and codeine (0.11 ng/mg).

Quantitation of ocfentanil in the biological samples wasperformed using a target analysis based on liquid–liquid extractionand ultra performance liquid chromatography tandem massspectrometry. An overview of the assessed validation parametersand validation data is shown in Table 1. The distribution ofocfentanil in the post-mortem blood and tissue samples is shownin Table 2.

6. Discussion

Ocfentanil is a synthetic opioid and structure analogue offentanyl. The chemical structures of fentanyl (N-(1-phenethyl-4-piperidyl)-N-phenylpropanamide) and ocfentanil (N-(2-fluoro-phenyl)-2-methoxy-N-[1-(2-phenylethyl)-4-piperidyl]acetamide)are shown in Fig. 3. In accordance with findings described in opioidintoxications, pulmonary oedema and lung injury were observed atautopsy. The comprehensive systematic toxicological analysis onlyrevealed the presence of acetaminophen and caffeine. Therecognition and analysis of highly potent, new psychoactivesubstances in tissues are challenging in postmortem toxicology.Without the identification of ocfentanil in the powder and targetanalysis of postmortem samples with hyphenated techniques, thepresence of ocfentanil would be overlooked due to the low toxictissue concentrations. Based on the result of the nose swab and theparaphernalia found in the vicinity of the victim, it was concludedthat the brown powder was snorted.

Ocfentanil is a very potent opioid (approximately 90 times aspotent as morphine), making the abuse in the recreational drugscene exceptionally dangerous, in particular in opioid intolerantusers. The decedent had no known history of heroin usage. Nopunction marks were observed at autopsy. It was not clear what heintended to purchased on the internet. The hair was analyzed onthe presence for amphetamine, methamphetamine, MDMA, MDA,MDEA, cocaine, benzoylecgonine, norcocaine, cocaethylene, mor-phine, 6-monoacetylmorphine, codeine, tetrahydrocannabinol,cannabidiol, cannabinol, ketamine and norketamine to providean understanding of the historical pattern of drug use. Cocaine wasdetected in the 0–6 cm hair section at 0.33 ng/mg within twococaine metabolites, benzoylecgonine and norcocaine. Cocaine


[(Fig._1)TD$FIG]

Fig. 1. Mass spectrum of the analysis of the brown powder (upper) and reference mass spectrum of ocfentanil present in the SWGDRUG library (lower).

[(Fig._2)TD$FIG]

Fig. 2. UV-spectrum of ocfentanil (reference solution 50 mg/mL).

V. Coopman et al. / Forensic Science International 266 (2016) 469–473472

concentration is low when compared to the general population ofdrug addicts, suggesting that the donor of the hair specimen wasoccasionaly exposed to cocaine. Once in the body, heroin(diamorphine) is broken down in the body to yield 6-mono-acetylmorphine and morphine. The hair sample was positive for6-monoacetylmorphine in the 0–6 cm head hair section at a lowconcentration (0.54 ng/mg) in view of what can be observed inrecreational drug abusers, suggesting that the submitted hairbelongs to a subject that was occasionaly exposed to heroin overthe last 6 months period before death. The presence of codeine islikely to be the result of the presence of acetylcodeine in illicit

Table 2Distribution of ocfentanil in the postmortem biological samples.

Biological sample Concentration ocfentanil

(mg/L of mg/kg)

Taken at the external body examination

Femoral blood (EDTA) 15.3

Vitreous humor 12.5

Mucous membrane of the nose Concentration above highest

calibrator (calculated value in

undiluted sample extract:

2999 ng/swab)

Taken at autopsy

Cardial blood (EDTA) 23.3

Cardial blood (without preservative) 21.9

Urine 6.0

Stomach content 17.1

Liver 31.2

Kidney 51.2

Brain tissue 37.9

Bile 13.7

Downloaded from ClinicalKey.com at InovaFor personal use only. No other uses without permission

heroin. This is a common component and it is broken down withinthe body to codeine.

Ocfentanil in combination with caffeine and acetaminophen,was reported in one previous seizure in the Netherlands in 2013,where it was sold as ‘synthetic heroin’ [6]. Intoxications with otherfentanyl analogues and ‘heroin substitutes’ such as acetyl fentanyl,butyrfentanyl, 4-fluorobutyrfentanyl and ß-hydroxythiofentanylare reported in recent literature [10–16]. Fentanyl or analoguessuch as 3-methylfentanyl are known to be added in small amountsin normal heroin [17], making them more potent and profitable.Neither heroin nor other fentanyl analogues were detected in thepowder.

Internet searches carried out in July 2015 revealed a vendorreview posted on ‘DarkNetMarkets’ including results of presump-tive tests (Mecke, Marquis, Mandelin, Froehde, Liebermann andGallic Acid) and GC/MS analysis carried out on a powder marketedas ‘#40 and purchased as heroin. In the online review wasmentioned that a reference heroin was tested as control, that noheroin was detected in the sample ‘#40 and the major constituentsfound were in order of magnitude: acetaminophen, caffeine andocfentanil [18]. Widinos reported the presence of ocfentanil in 3white powders and 3 brown powders recieved in the period March2015–July 2015 [19]. The route of administration was stated‘sniffing/snorting’ for 2 powders, ‘smoking’ for 1 powder and‘unknown’ for the others. Three powders where profiled and foundto contain ocfentanil, caffeine and acetaminophen in varyingcombinations, one containing mannitol and methamphetaminerespectively.

[(Fig._3)TD$FIG]

Fig. 3. Chemical structure of fentanyl (A) and ocfentanil (B).

Fairfax Hospital - JCon January 07, 2017.. Copyright ©2017. Elsevier Inc. All rights reserved.

V. Coopman et al. / Forensic Science International 266 (2016) 469–473 473

The reported ocfentanil fatality was an isolated case. No clusterof intoxications were reported as seen in the past with NPSentering the recreational drug scene as street or club drugs [20].

7. Conclusions

Ocfentanil is a new fentanyl anologue abused as NPS. This is thefirst reported death involving this designer drug. Based oncircumstantial evidence, the autopsy findings and the results ofthe toxicological analysis, the medical examiner concluded thatdeath was caused by an acute accidental intoxication withocfentanil after snorting powder containing the substance.

References

[1] US Patent application 2002/176888 A1.[2] J. Bagley, Evolution of the 4-anilidopiperidine class of opioid analgesics, Med. Res.

Rev. 11 (4) (1991) 403–436.[3] J. Fletcher, P. Sebel, M. Murphy, S. Mick, S. Fein, Comparison of ocfentanil and fentanyl

as supplements to general anesthesia, Anesth. Analg. 73 (5) (1991) 622–626.[4] P. Glass, The analgesic efficacy of A3217, Anesthesiology 71 (71) (1989) A321.[5] Z. Z. Ebrahim, Multiple dose evaluation of the efficacy of ocfentanil hydrochloride

(A3217) to produce postoperative analgesia, Anesth. Analg. 72 (1991) S63.[6] P. Blanckaert, Fact Sheet Ocfentanil. Belgian Early Warning System Drugs Alert,

issued April 2015.[7] R. Christie, Death in Belgium associated with ocfentanil (N-(2-fluorophenyl)-2-

methoxy-N-[1-(2-phenylethyl)-4-piperidyl]acetamide). EU Early Warning Sys-tem Alert, EMCDDA issued 22/04/2015.

[8] V. Coopman, J. Cordonnier, Counterfeit drugs and pharmaceutical preparations seizedfrom the black market among bodybuilders, Ann. Toxicol. Anal. 24 (2) (2012) 73–80.


[9] Scientific Working Group for Forensic Toxicology (SWGTOX), Standard prac-tices for method validation in forensic toxicology, J. Anal. Toxicol. 37 (7)(2013) 452–474.

[10] I. Mc Intyre, A. Trochta, R. Gary, M. Malamatos, J. Lucas, An acute acetyl fentanylfatality, J. Anal. Toxicol. 39 (6) (2015) 490–494.

[11] J. Cole, J. Dunbar, S. McIntire, W. Regelmann, T. Slusher, Butyrfentanyl overdoseresulting in diffuse alveolar hemorrhage, Pediatrics 135 (March (3)) (2015)e740–e743.

[12] Centers for Disease Control and Prevention (CDC), Acetyl fentanyl overdosefatalities – Rhode Island, March–May 2013, MMWR Morb. Mortal. Wkly. Rep.62 (34) (2013) 703–704.

[13] M. Lozier, M. Boyd, C. Stanley, L. Ogilvie, E. King, C. Martin, L. Lewis, Acetylfentanyl, a novel fentanyl analog, causes 14 overdose deaths in Rhode Island,March–May 2013, J. Med. Toxicol. 11 (2015) 208–217.

[14] M. Blackberg, O. Beck, K. Jonsson, A. Helander, Opioid intoxications involvingbutyrfentanyl, 4-fluorobutyrfentanyl, and fentanyl from the Swedish STRIDAproject, Clin. Toxicol. 17 (1–9) (2015).

[15] J. Mounteney, I. Giraudon, G. Denissov, P. Griffiths, Fentanyls: are we missing thesigns? Highly potent and on the rise in Europe, Int. J. Drug Policy 26 (7) (2015)626–631.

[16] E. Shoff, The Real Heroin in South Florida: The Detection of a Fentanyl Analog inPostmortem Specimens Using Liquid Chromatography (LC)-Ion Trap TandemMass Spectrometry (MS/MS), http://aafs.org/sites/default/files/2016/abstracts/K67.pdf (accessed 01.04.16).

[17] European Monitoring Centre for Drugs and Drug Addition (EMCDDA). FentanylDrug Profile, http://www.emcdda.europa.eu/publications/drug-profiles/fentanyl(accessed 01.04.16).

[18] https://www.reddit.com/r/DNMAvengers/comments/3bp1kv/frenchconnection_nucleus_1g_heroin_4_gcms_marquis (accessed 28.07.15).

[19] Philtre Bulletin, Wedinos Quarterly Newsletter, Issue 6 April–June 2015, http://www.wedinos.org/resources/downloads/Philtre_Issue_6.pdf (accessed 21.02.16).

[20] E. De Letter, V. Coopman, J. Cordonnier, M. Piette, One fatal and seven non-fatalcases of 4-methylthioamphetamine (4-MTA) intoxication: clinico-pathologicalfindings, Int. J. Legal Med. 114 (6) (2001) 352–356.


http://refhub.elsevier.com/S0379-0738(16)30309-7/sbref0110





























http://aafs.org/sites/default/files/2016/abstracts/K67.pdf

http://aafs.org/sites/default/files/2016/abstracts/K67.pdf

http://www.emcdda.europa.eu/publications/drug-profiles/fentanyl

https://www.reddit.com/r/DNMAvengers/comments/3bp1kv/frenchconnection_nucleus_1g_heroin_4_gcms_marquis

https://www.reddit.com/r/DNMAvengers/comments/3bp1kv/frenchconnection_nucleus_1g_heroin_4_gcms_marquis

http://www.wedinos.org/resources/downloads/Philtre_Issue_6.pdf

http://www.wedinos.org/resources/downloads/Philtre_Issue_6.pdf




2876

Synthesis and Pharmacological Evaluation of a Series of New 3-Methyl-1,4-disubstituted-piperidine Analgesics

J. Med. Chem. 1990, 33, 2876-2802

Nhora Lalinde,*+ John Moliterni,' Denny Wright,!' H. Kenneth Spencer,l Michael H. Ossipov,t Theodore C. Spaulding,* and Frieda G. Rudos

Anaquest Pharmaceuticals, BOC Technical Center, 100 Mountain Ave., Murray Hill, New Jersey 07974, and University of Maryland, Baltimore College of Dental Surgery, 666 West Baltimore Street, Baltimore, Maryland 21201 -1586. Received November 27. 1989

The synthesis and intravenous analgesic activity of a series of 3-methyl-4-(N-phenyl amido)piperidines, entries 34-79, is described. The methoxyacetamide pharmacophore produced a series of compounds with optimal analgesic potency and short duration of action. cis-42 was 13036 times more potent than morphine and 29 times more potent than fentanyl; however, the corresponding diastereomer 43 was only 2778 and 6 times more potent, respectively. Compounds 40, 43, 47, and 57 are extremely short acting; all had durations of action of about 2 min, which was about of that of fentanyl in the mouse hot-plate test a t a dose equivalent to 2 times the EDw analgesic dose. Among the many compounds that displayed exceptional analgesic activity, duration of action was one of the main factors for choosing a candidate for further pharmacological investigation. At present, cis-1-[2-(4-ethy1-4,5-dihydro-5-0~0- lH-tetrazol-1-yl)ethyl]-3-methyl-4-[N-z 2-fluorophenyl)methoxyacetamido]piperidine hydrochloride (40) (Anaquest, A-3331.HC1, Brifentanil) is in clinical evaluation. Opiate analgesics that possess short duration of action are excellent candidates for short surgical procedures in an outpatient setting where a rapid recovery is required.

Introduction In the search for the ideal analgesic devoid of the typical

side effects common to all morphinomimetic compounds, investigators have modified the fentanyl structure (1) in various ways. Fentanyl is a well-known analgesic, 80-100 times more potent than morphine, with a fast onset and short duration of action. However, as with earlier opioids it produces profound respiratory depression, muscle rigidity, postoperative nausae, and physical dependence. As a result of this ongoing effort to develop the ideal analgesic, there are today an interesting number of novel compounds of the fentanyl family, all exhibiting an array of analgesic profiles. Carfentanil and sufentanil are the safest and most potent fentanyl congeners.'S2 Afentanil is characterized by its short duration of action3 while lofentanil is long- acting. Respiratory depression after an oral, subcutaneous or intravenous dose of lofentanil (2) of 0.7 mg/kg can last up to 48 h.4

1

3

4

* Author to whom correspondence should be addressed.

t Department of Pharmacology, Anaquest. 8 Department of Pharmacology, University of Maryland. 11 401 Route 22, North Plainfield, NJ 07060.

Department of Chemistry, Anaquest.

Organon Inc., 375 Mt. Pleasant Ave., West Orange, NJ 07052.

As part of the continuing effort of our laboratories to develop therapeutically advantageous analgesics with rapid onset and short duration of action: we initiated a research program based on the synthesis of a series of racemic cis and trans stereoisomers of 3-methyl-4-anilid~piperidine~ (3). We wanted to evaluate the effect caused by changing the propionyl group to a methoxyacetyl, since this substitution is known to confer short duration of action in 4-anilidopiperidine analgesic^.^ It was foreseen that the highly lipophilic moieties would diffuse faster through the blood-brain barrier, while the more hydrophilic or ionized substituents would lead to analgesics with less lipid solu- bility (lower partition coefficients), little or no accumula- tion in fatty tissues, and rapid excretion. Opioid receptor binding affinities were determined for many of the compounds. These studies and secondary pharmacological results of selected compounds will be discussed herein.

Chemistry The synthesis is outlined in Schemes 1-111. All the

compounds in Table I were synthesized with l-benzyl-3- methylpiperidin-4-one (5) or with 1-phenethyl-3-methylpiperidin-4-one (26) as the starting material, which were prepared by the double Michael addition of N-benzyl- or

Van Bever, W. F. M.; Niemegeers, C. J. E.; Schellekens, K. H. L.; Janssen, P. A. J. Arzneim.-Forsch. (Drug Res.) 1976, 26, 1548. Niemegeers, C. J. E.; Schellekens, K. H. L.; Van Bever, W. F. M.; Janssen, P. A. J. Arzneim.-Forsch, (Drug Res.) 1976, 26, 1551. Niemergeers, C. J. E.; Janssen, P. A. J. Drug Deu. Res. 1981, 1,83. Nauta, J.; DeLange, S.; Koopman, D.; Spierdijk, J.; Van Kleef, J.; Stanley, T. H. Anesth. Analg. 1982, 61, 267. Leysen, J. E.; Laduron, P. M. Arch. Int. Pharmacodyn. 1978, 232, 343. We have defined a compound to be short acting (S) if the effect is less than 6 min and intermediate (I) if between 6 and 15 min, and long duration (L) is anytime greater than 15 min, at a dose equivalent to 2 times the EDw hot-plate analgesic dose. The term "cis" is applicable to those configurations in which the two functional groups in C-3 and C-4 lie on the same side of the plane of the ring and the term "trans" when the two groups lie on opposite sides of the plane. Janssen, P. A. J.; et al. US. Patent 3 907 813, 1975. Huang, B. S.; Terrell, R. C.; Deutsche, K. H.; Kudzma, L. V.; Lalinde, N. L. U S . Patent 4584303, 1986.

0022-2623/90/1833-2876$02.50/0 @ 1990 American Chemical Society

New 3-Methyl-1,4-disubstituted Piperidine Analgesics

Scheme Io

5 Ge:X=F b : X = H C: X = OCH3 d:X=CI

separation of momers

c I CH3 X CH3 X

(=y.=j-y& \ / H -Qr&& CO-R

>cis 8: tram

CH3 X CH3 X

L-N$& - H - . C S - & CO-R CO-R

11 cis 12 trans

a Reagents: (i) a-x-aniline, PhCH,, p-TsOH, A; (ii) NaBH,CN, MeOH; (iii) RCOCl, CHzCIz; (iv) 10% Pd(OH),/C, EtOH, Hz (50 psi); (v) LBr, acetonitrile, NaI, NazCO,.

N-phenethylamine, with methyl methacrylate and ethyl acrylate: followed by cyclization and decarboxylation. By the use of N-benzyl-3-methyl-4-piperidin-4-one (5) as the starting material, it was possible to couple various piperidines (i.e., 11-23) with different L substituents, such as 2-(chloroethyl)-2-thiophene and 1-(2-bromoethyl)-4- ethyl-1,4-dihydro-5H-tetrazol-5-one, as shown in Scheme I.

The synthesis of the cis isomers 36, 37, 45, 46, 51, 53, 61,62,70,76, and 77 and of trans congeners 52,58,71, and 73 is depicted in Scheme I. The condensation of the piperidone 5 with aniline or 1-substituted aniline, followed by reduction of the Schiff base with sodium borohydride, gave a 1 -benzyl-3-methyl-4-( pheny1amino)piperidine (6). Compounds 6 were produced in an approximate 7:3 (cis/trans) isomeric ratio. This was in agreement with previous literature The cis isomer was always

(8) (a) Beckett, A. H.; Casey, A. F.; Kick, G. J. Med. Chen. 1959, I, 31. (b) Ziering, A.; Motchane, A.; Lee, J. J . Med. Chem. 1957, 22, 1521. (c) Ganellin, C. R.; Spickett, R. G. J. Med. Chem. 1965, 8,619. (d) Carabateas, P. M.; Grumback, L. J. Med. Chem. 1962,5,913.

(9) (a) Van Bever, W. F. M.; Niemegeers, C. J. E.; Janssen, P. A. J. J. Med. Chem. 1974,17, 1047. (b) Assuming a chair con- formation for the piperidine ring, one would expect that the most predominant conformer would have an equatorial 4-N- (COCH,OCH,) group with an equatorial 3-Me group for the trans compound and an axial 3-Me group for the cis com-

This was confirmed by the splitting pattern of the 4-proton on the piperidine ring. Compound 40 showed a multiplet, centered at 6 4.53, consisting of a doublet (J = 10.5 Hz) of triplets (J = 5.2 Hz). On the other hand, the trans compound 41 showed a multiplet centered, at 6 4.82, consisting of a triplet (J = 12.53 Hz) of doublets (J = 2.64 Hz).

Journal of Medicinal Chemistry, 1990, Vol. 33, No. 10 2877

less polar than the trans isomer on silica, Rf (0.28) and ( O . l l ) , respectively (EtoAc/hexane, 1:4). Compounds 6 were separated by column chromatography into intermediates 7 and 8, respectively. Acylation of 7 and 8, with methoxyacetyl, furoyl, and methoxypropionyl chlorides, in methylene chloride, afforded intermediates 9 and 10, respectively. Subsequent catalytic debenzylation produced the secondary amines 11 and 12, which were N-alkylated in acetonitrile with appropriate L-X electrophiles.

In a different sequence of reactions (Scheme 11, path A), intermediates 6 were first separated into isomers 15 and 16, which in turn were treated with the corresponding acid chlorides, affording intermediates 17, and 18. The benzyl group of 17 and 18 was catalytically removed, giving rise to compounds 19 and 20, which in turn were alkylated with the corresponding L-X groups (L = phenethyl, thienylethyl, and tetrazolinonylethyl) to give compounds 67 and 69.

Compounds 21 were obtained by the acylation of the intermediates 6 (Scheme 11). The products 21 were catalytically debenzylated as described above to give the nor compounds 22. The intermediates 22 were utilized in two ways. Alkylation with appropriate L-X groups (path B) gave isomeric mixtures which were chromatographically separated to give compounds 25. Specifically, compounds 40,9b 42, and 72 are of the cis form, and 41,43, and 73 are their trans counterparts.

The final products, 56, and 57, were obtained by isomeric separation of the nor compound 22 (X = F) (Scheme 11, path C) into intermediates 23 and 24, followed by N-alkylation with 2-(chloroethyl)-2-thiophene and 142- bromoethyl)-l,4-dihydro-5H-tetrazol-5-one, respectively.

All the compounds where L is phenethyl were prepared as indicated in Scheme 111. The starting N-phenyethyl- 3-methyl-4-piperidone (26) was condensed with aniline or 1-substituted aniline and reduced in the usual manner with sodium borohydride, producing the amines 27. In one sequence of reactions, 27 was separated into compounds 28 and 29. The final cis products 34,38,47,49,55,59,63, 65, 74, and 78, were produced through acylation of the amine 28 with the corresponding acid chlorides in dry methylene chloride. Compounds 35,39,48,60,64,66,75, and 79 were the products of acylation of intermediate 29. Compound 44 was obtained by a small variation in the reaction sequence. The intermediate 27 (X = OCH,) was first acylated with the methoxyacetyl chloride, and then the resulting isomeric amides 30 were separated by column chromatography to afford 37. Pharmacology. Results and Discussion

The analgesic properties of these compounds were evaluated by the mouse 55 " C hot-plate assay. An initial dose of 1 mg/kg was administered, and if 100% analgesia was observed, then lower dosing was continued until an ED, was generated. If less than 100% analgesia was observed, then 5 mg/kg was administered. The duration of analgesia was also determined. Analgesics with relatively short duration of action, Le., less than 6 min, at 2 X EDm were again evaluated at doses equivalent to 8 times the ED,, analgesic dose, and those with durations of less than 9 min at 8 X ED, were evaluated again at 16 times the ED50 analgesic dose. Many compounds were screened in vitro for their ability to displace [3H]naloxone from its binding sites in rat brain membranes. Mouse EDm's and duration values are given in Table 11.

The analgesic activity of compound 40, along with that of fentanyl was evaluated in the mouse hot-plate (MHP), rat tail-flick (RTF), and rat hot-plate (RHP) tests (Table 111). In addition, their anesthetic profile was evaluated

2878 Journal of Medicinal Chemistry, 1990, Vol. 33, No. 10

Scheme 11"

6

w 21

\

Lalinde et al.

17 cis 10 trans

J ii

19 20 Cis trans

15 ds 16trans

25

I 22

path C 1 23 24 trans cis

L D

: i " i ! j 14 trans iil

D 13 cis iii

X = F, H, OCHj, CI R = rnethoxyacetyl, rnethoxypropionyl, furoyl L = tetrazdinonylethyl, thienylethyl

'Reagents: ( i ) RCOC1, CHzClz; (ii) 10% Pd(OH)p/C, EtOH, H2 (50 psi); (iii) LBr, 4-methyl-2-pentanone, NaI, Na2C03.

Scheme 111"

26

i, ii J

H 27

b e p a r a t i o n ot Borers

CO-R 30

H 28 cis 29 trans

i 'I'

I---------- 31 cis 32 trans

'Reagents: (i) 2-X-aniline, PhCH3, p-TsOH, A; ( i i ) NaBH,, MeOH; ( i i i ) RCOCl, CH2C12.

in the rat loss of righting test (Table IV). The methoxyacetamide pharmacophore produced a se-

ries of compounds with optimal analgesic potency and short duration of action. Compound 42 was 13 OOO times more potent than morphine and 29 times more potent than fentanyl1° in the mouse hot-plate test, while 43 was only

2778 and 6 times more potent, respectively (Table 11). The furoyl analogues displayed potencies similar to that of fentanyl, and the methoxypropionyl moiety rendered compounds with significant diminution of analgesic activity (e.g., 55, 57, 58, 59, 60, 62, and 63).

Within each series of compounds, the order of potency versus change of the ortho substituent on the anilido phenyl ring was observed to decrease in the order 2-F L 2-H > 2-C1> 2-OCH3 (i.e., 42, 37,47,46). Although, the quantitative effect of the ortho fluorine on the analgesic potency was not as profound as reported by Huang7 and Kudzma,ll in most cases, it produced congeners with increased agonism, Le., entries 35 and 39. The duration of action was unaffected by those variations. In general, one could say that in an isomeric pair the greatest agonism was displayed by the cis isomer. Although in the instances where the ortho substituent was chlorine, the trans isomers possessed always higher analgesic potencies (e.g., 48 and 78, 79).

Closer examination of the series of compounds with an o-fluoro substituent in the anilido moiety reveals that the order of potency was directly dependent on the N-1 substituent, i.e., thienylethyl > phenylethyl > 4-ethyl- tetrazolinone (e.g., 42,38, and 40). This trend was not as rigorous for analogues with different ortho substituents, but in general, the introduction of a 2-phenylethyl or 2- thienylethyl group rendered compounds with appreciable agonist activity (e.g., 34 and 42) vs the 4-ethyl- tetrazolinones analogues 40 and 41.12

(10) Lalinde, N.; Spencer, K. H.; Wright, D. Abstracts of Papers, 193rd National Meeting of the American Chemical Society, Denver, CO, 1987; American Chemical Society: Washington, DC, 1987.

(11) Kudzma, L. V.; Severnak, S. A.; Benvenga, M.; Ezell, E. F.; Ossipov, M. H.; Knight, V. V.; Rudo, F. G.; Spencer, H. K.; Spaulding, T. H. J. Med. Chem. 1989, 32, 2534.

New 3-Methyl-1,4-disubstituted Piperidine Analgesics Journal of Medicinal Chemistry, 1990, Vol. 33, No. 10 2879

34 cis 35 trans 36 cis 37 cis 38 cis 39 trans 40 cis 41 trans 42 cis 43 trans 44 cis 45 cis 46 cis 47 cis 48 trans 49 cis 50 trans 51 cis 52 trans 53 cis 54 trans 55 cis 56 trans 57 cis 58 trans 59 cis 60 trans 61 cis 62 cis 63 cis 64 trans 65 cis 66 trans 67 cis 68 cis 69 cis 70 cis 71 trans 72 cis 73 trans 74 cis 75 trans 76 cis 77 cis 78 cis 79 trans

phenylethyl phenylethyl tetrazolylethyl thienylethyl phenylethyl phenylethy tetrazolylethyl tetrazolylethyl thienylethyl thienylethyl phenylethyl tetrazolylethyl thienylethyl phenylethyl phenylethyl phenylethyl phenylethyl tetrazolylethyl tetrazolylethyl thienylethyl thienylethyl phenylethyl tetrazolylethyl thienylethyl thienylethyl phenylethyl phenylethyl tetrazolylethyl thienylethyl phenylethyl phenylethyl phenylethyl phenylethyl tetrazolylethyl thienylethyl phenylethyl tetrazolylethyl tetrazolylethyl thienylethyl thienylethyl phenylethyl phenylethyl tetrazolylethyl thienylethyl phenylethyl ohenvlethvl

CHzOCH3 CHQOCH, CH~OCH, CHZOCH, CH20CH3 CHZOCH, CHzOCH, CH20CH3 CHZOCH, CH20CH3 CHZOCH, CHZOCH, CHZOCH, CHZOCH, CH20CH3 CH(CH3)OCHS CH(CHS)OCH3 CH(CH3)0CH3 CH(CH,)OCH, CH(CHJOCH3 CH(CHJOCH3 CH(CHJOCH3 CH(CH3)OCH:, CH(CH.JOCH3 CH(CHJOCH3 CH(CH3)0CH3 CH(CH3)OCHS CH(CH3)OCHS CH(CH3)OCH:, CH(CH3)OCHS CH(CH,)OCH3 furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furovl

H H H H F F F F F F OCH3 OCH, OCH3 c1 c1 H H H H H H F F F F OCH, OCH, OCH3 OCH, Cl c1 H H H H F F F F F OCH3 OCH3 OCH, OCH, c1 c1

135-136 190-191 145-146 161-163 168-169 180-181 151-153 148-150 185-186 155-156 175-176 137-138 194-195 171-175 171-172 2 0 4 - 2 0 5 167-169 147-148 164-165 2 17-2 18 180-181 179-180 206-209 204-205 2 0 3 - 2 0 4 159- 161 223-224 220-222 172-173 2 12-2 13 138-140 177-178 169-170 124-1 25 201-204 208-212 154-155 174-175 191-192 168-169 202-204 227-229 150-151 195-196 201-202 187-189

Table I. Chemical Properties of N - [ 1-(Substituted alkyl)-3-methyl-4-piperidinyl]-N-phenylalkanamides

L-N &lR x&

entry L R X mp, OC formulaa

C23Hd202'C2H204 C23Hd202'C2H204

C20H30N603'C2H204 C21H28N202*C2H204 C23H29N204F'C2H204

C23H29N20'2F'C2H204 C,H,FN603*C2H204 C&2BN6F'C2H204

C21H27FN202S*C2H204 C21H,FN202S*C2H2Od C24H32N203.HCl C21H32N604'CZH204 C22HNN20,S.HCl C23H29N202'C2H204 C2,H,N202Cl.C2H204 C24H32N202'CZH204

C24H32N202*C2H204

C21H32N603'C2H204 CZ1H32N603'C2H204

C22H30N202S'C2H204 C24H31N202F'C2H204 C21H3,FN603*HCl C22HBFN202SC2H204 CzzHZsFN202SCC2H204 CEHSNzO3.HC1 C25H34N204'C2H204

CZZH34N6O4*CZH204 C23H32NZ03S'C2H204 C~H31N~O~Cl.C2H~04 C24H31N202C1~C2H204

C25H28N202'C2H204 C25H28N202'C2H204 CZZ%N603*C2HZO4 C23H26N202S'C2H204 C26H2A7N202FC2HZO CZZH27FN603'C2H204

C22H~FN603'C2H204 C23HsFN2SCC2H204 C23H25FN202S'C2H204 C26H30N203'CZH204

C26H30N203'C2H204

C23H30N604'C2H204 C24H28N2O3S'C2H204 C25H27N202C1'C2H204

. * < C ~ ~ H ~ N ~ O ~ C l C ~ H ~ 0 4

C22H30N2S'C2H204

Analytical results were within f0.4% of the theoretical values. Recrystallized from methanol except where indicated. The compounds are either oxalates or hydrochloride salts.

The duration of action of 40 in the mouse hot-plate test, a t the 2 X EDW dose was 1.98 min, which was similar to that of compounds 42,57, and 55. However, superiority was clearly demonstrated at 8X and 16X MHP ED,, analgesic doses. At 8X MHP ED,, 40 had a duration of action of only 9.00 min, and at the 16X MHP EDW the duration was approximately and was comparable to that of fentanyl and sulfentanil at their 2X MHP EDw analgesic doses (Table 11). The onset of the antinociceptive activity of 40 in the mouse hot plate (MHP) occurred within 1 min after iv injection. From the series of compounds in Table I1 40 was selected for further pharmacological investigation because it satisfied our criteria for short duration of action.

In the rat tail-flick experiments, a t 2X ED, 40 had a duration of action of 8.4 min, which was comparable to that

(12) Colapret, J. A.; Diamantidis, G.; Spencer, H. K.; Spaulding, T. C.; Rudo, F. G . J . Med. Chem. 1989,32,968.

of fentanyl (7.6 min), but at 8X RTF EDW its duration of action was significantly shorter (20.8 min) and that of fentanyl (34 min). In the rat hot-plate test the duration of action of 40 at 2X RTF EDw was 2.8 min and of fentanyl 11.62 min. At 8X RHP EDW fentanyl had a duration of action of 32.35 min, which was 4.1 times longer than that of 40, 7.9 min (Table 111).

Due to its overall superior analgesic profile, the pharmacology of 40 was further scrutinized. The anesthetic activity of 40 and fentanyl was evaluated in the loss of righting test (LOR) in rats (Table IV). The ED, for LOR in rats administered iv with 40 and fentanyl were calculated (percent responding vs dose) to be 0.152 and 0.0175 mg/kg, respectively. However at equi-efficacious doses for LOR (i.e., 100% responding), the duration of 40 was 1.7 min compared to 8.8 min for fentanyl. In addition, 40 appeared to produce less muscular rigidity and have a better behavioral syndrome, rated by the severity of pri-

2880 Journal of Medicinal Chemistry, 1990, Vol. 33, No. 10 Lalinde et al.

Table 11. N - [ 1-(Substituted alkyl)-3-methyl-4-piperidinyl]-N-phenylalkanamides Preliminary Pharmacology

durationbsc entry L R x w / k g 2n 8n 16n Ki,dnM

0.0016 (0.001023-0.0024) 9.16 -- -- 0.33 34 cis phenylethyl 35 trans 36 cis 37 cis 38 cis 39 trans 40 cis 41 trans 42 cis 43 trans 44 cis 45 cis 46 cis 47 cis 48 trans 49 cis 50 trans 51 cis 52 trans 53 cis 54 trans 55 cis 56 trans 57 cis 58 trans 59 cis 63 trans 61 cis 62 cis 63 cis 64 trans 65 cis 66 trans 67 cis 68 cis 69 cis 70 cis 71 trans 72 cis 73 trans 74 cis 75 trans 76 cis 77 cis 78 cis 79 trans morphine alfentanil fentanyl sufentanyl

. .

phenylethyl tetrazolylethyl thien ylethyl phenylethyl phenylethyl tetrazolylethyl tetrazolylethyl thienylethyl thienylethyl phenylethyl tetrazolylethyl thienylethyl phenylethyl phenylethyl phenylethyl phenylethyl tetrazolylethyl tetrazol y let h yl thien ylethyl thienylethyl phenylethyl tetrazolylethyl thienylethyl thienylethyl phenylethyl phenylethyl tetrazolylethy 1 thienylethyl phenylethyl phenylethyl phenylethyl phenylethyl tetrazolylethyl thienylethyl phenylethyl tetrazolylethyl tetrazolylethy 1 thienylethyl thienylethyl phenylethyl phenylethyl tetrazolylethyl thienylethyl phenylethyl phenylethyl

CHZOCH, CH20CH3

CH20CH3 CH20CH3 CHzOCH3 CH2OCH3 CHzOCH3 CHzOCH3 CH20CH3 CHZOCH, CHZOCH, CHZOCH, CHZOCH, CHZOCH, CH(CH,)OCH, CH(CHJOCH, CH(CHJOCH3 CH(CHJOCH3

CHzOCH3

CH(CH3)OCH3 CH(CH,)OCH, CH(CH3)OCH3 CH(CH3)OCH3 CH(CH3)0CH3 CH(CH3)OCH3

CH(CH,)OCH,

CH(CH3)0CH3

furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl furoyl

CH(CH3)OCH,

CH(CHJOCH3

CH(CHJOCH3 CH(CH3)OCH,

H H H H F F F F F F OCH3 OCH, OCH, e1 C1 H H H H H H F F F F OCH, OCH, OCHS OCH, OCH, C1 H H H H F F F F F OCH, OCH, OCH, OCH, c1 c1

0.041 (0.029-0.06) inactive 0.0021 (0.00186-0.002508)

0.00069 (0.000535-0.00091)

inactive 0.00056 (0.000453-0.000694) 0.0027 (0.001085-0.00587) 0.1125 (0.1016-0.1485) inactive 0.547

0.00486 (0.00226-0.010463)

0.0041 (0.002977-0.00551)

0.0980 (0.091 -0.156)

0.078 (0.0724-0.12644)

0.035 (0.028406-0.044195) 0.575 (0.004-82.72) inactive inactive 0.0119 (0.006573-0.032742) 0.0244 (0.0154-0.0388) 0.410 (0.286-0.589)

0.0057 (0.00496-0.009692) 0.0244 (0.0154-0.0388) inactive 0.651 (0.477-0.889) inactive 2.5 0.669 (0.52-0.291) 0.188 (0.122-0.291) 0.005 (0.003-0.007027) 0.082 (0.000896-0.6987) 0.638 (0.47-8.66) 0.0054 (0.0039-0.0073) 0.041 (0.001-1.3) 0.305 (0.298-0.451)

0.004 (0.0008-0.018) 0.025 (0.01845-0.034407) 0.217 (0.156-0.303) 0.118 (0.11013-0.18712) inactive 0.568 (0.434-0.743) 1.96 0.247 (0.14948-0.40843) 7.3 0.047 (0.034-0.065) 0.018 (0.014-0.023)

inactive

inactive

0.0029

3.24

5.32 5.13 25.40 2.00

16.00 2.33 8.60

23.00 1.41 4.85 7.70 6.10

_-

--

_ _

_ _ _ _ 2.0 10.56 11.54

1.73 10.56

10.20

_ _

_ _

_ _ _ _ 6.88 12.55 7.5 7.8 6.46 15.60 11.55 4.57

16.80 4.76 7.20 6.16

12.00

26 >60.0 4.1 11.70 12.52

- _

_.

"_

0.86 NDe ND 0.88 0.19 2.00 >iod ND ND ND ND ND 1.10 ND 4.20 32.2 ND ND ND ND 8.10 ND 2.16 ND ND 7.62 ND 11.2 ND 2.80 0.30 0.40 ND 0.15 ND ND ND ND ND ND 0.78 > 100 2.00 ND 0.13 2.1 8.21 2.16 0.22

"ED, for mouse hot plate with 95% confidence limits in parentheses; inactive refers to doses up to 5 mg/kg. *Duration in minutes at indicated multiples of the ED, dose. Analgesics with relatively short duration of action, less than 6 min at 2 times ED, were evaluated at doses equivalent to 8 times the ED, analgesic dose, and those with durations of less than 9 min, at 8 times ED, were evaluated again at 16 times the ED, analgesic dose. K i denotes the ability to displace [3H]naloxone from the p opioid receptor isolated from rat brain membranes. 'ND = not determined. f A greater than sign (>) denotes no displacement of [3H]naloxone at the concentration indicated.

mary overt effects (POE; e.g. ataxia, tremors, myotactic reflex, vascular tone, salivation), which was indicated by a lower behavioral or POE score (0.53 and 0.98 for 40 and fentanyl, respectively).

Many compounds were screened in vitro for their ability to displace [SH]naloxone from its binding sites in rat brain membranes. Compound 40 exhibited a receptor-binding affinity (Ki, nmol) of 2.0, which was comparable to that of morphine (2.1) and fentanyl (2.161, though 40 was 70 times more potent than morphine and 6 times less potent than fentanyl. The variance in in vivo agonism with the

in vitro data suggested that pharmacokinetic, rather than pharmacodynamic, factors are paramount.

The ability of iv naloxone to reverse the antinociceptive action of 40 was determined in the rat tail-flick test. Administration of the Aw dose, i.e., dose calculated to produce 99% of the maximal possible effect (99% MPE), of 40 (0.11 mg/kg, iv) followed by one injection of saline 1 min later, produced 81% maximum possible effect (MPE). The effect was significantly (p < 0.05) reduced to 25% and 3% MPE, following injections of 0.01 and 0.1 mg/kg of naloxone, respectively. These data suggest that

New 3-Methyl-1,4-disubstituted Piperidine Analgesics Journal of Medicinal Chemistry, 1990, Vol. 33, No. 10 2881

Table 111. Potencv and Duration of Action of 40, Fentanyl, and Morphine in Various Tests 40 fentanyl morphine

durationsc durationsC durations' EDmasb 2n 8n EDmavb 2n 8n 16n EDmapb 2n 8n 16n

mouse hot plate 0.098 1.98 9.0 12.6 0.018 7.9 27.1 41.07 4.68 66.3 217.8 (0.091-0.156) (0.015-0.022) (1.77 -12.39)

rat tail flick 0.059 8.4 20.8 -- 0.0043 7.6 34.0 -- 1.11 18.5 102.2 (0.046-0.076) (0.003-0.0061) (0.67-1.82)

rat hot plate 0.075 2.8 7.9 -- 0.0086 11.62 32.35 -- 2.77 37.0 115.3 (0.049-0.1141 (0.0065-0.010) (2.32-3.30)

a Milligram/kilogram. b95% confidence limits in parentheses. 'Minutes, at the given multiple of the EDW

Table IV. Equiefficacious Dose and Duration of Action for the Lost of Righting in Rata following Intravenous 40 and Fentanyl

40 fentanyl POE POE

dose0vb durationc score dosensb durationc score 0.152 1.7 0.53 0.0209 8.8 0.98 (0.139-0.165) (0.0169-0.0249) a Milligrams/kilogram. bLOR EDw with 95% confidence limits in

parentheses. cMinutes, at a given dose. 100% responding.

40 acts at the opioid receptor and that the effects of an overdose of 40 may be readily reversed by naloxone in the clinical setting.

In conclusion, our efforts in the search for a better analgesic agent have proven to be fruitful. Compound 40 (Anaquest, A-3331.HC1, Brifentanil, structure 4) was chosen as a development compound because it met the criteria we had set forth a t the beginning of the project. The short duration of analgesia and minimal opioid side effects gave this compound a unique position within the class of clinically useful opioid analgesics.

Experimental Section General Information. Melting points were recorded on a

Thomas-Hoover melting point apparatus and are uncorrected. Elemental analyses were obtained from the Analytical Services Division, BOC Technical Center, Murray Hill, NJ, and from Galbraith Laboratories, Knoxville, TN. 'H NMR spectra were recorded on a Varian E 360 (60 MHz) spectrometer. The NMR data for the assignment of the cis and trans isomer was acquired on the JEOL GSX-270 and transformed by using an exponential broadening factor of 0.2 Hz and utilizing a trapezoidal window for resolution enhancement. IR spectra were recorded on a Perkin-Elmer 197 spectrophotometer. Conventional chromatography was performed with fine silica (EM Science, 230-400 mesh). Reaction progress and purity of products were checked by analytical TLC using Analtech (GHLF) silica-coated glass plates. Spots were visualized with UVzM light or iodine. For pharmacological screening, oxalate salts were prepared by stirring a solution of the base, in 2-propanol, with excess oxalic acid a t room temperature and recrystallizing the solids to analytical purity. Starting Materials. l-Phenethyl-3-methyl-4-piperidone (5)

and 1-benzyl-3-methyl-4-piperidone (26) were prepared by standard procedures.8 The acid chlorides were commercially available from Aldrich, Milwaukee, WI, and most did not require further purification. 3-Methyl-4-phenyl-4-anilinopiperidines. The procedure

described is for the preparation of 40 (Scheme 11, path B). Nevertheless it is representative of those depicted in Schemes I and I1 (path A) and Scheme 111.

l-Benzyl-3-methyl-4-(2-fluoroanilino)piperidine (6). A mixture of 5 (25 g, 132 mmol), 2-fluormiline (23.25 g, 209 mmol), a few crystals of p-toluenesulfonic acid, and toluene (350 mL) was stirred under reflux overnight. This was the time required for collection of the theoretical quantity of water byproduct (2.2 mL) in a Dean-Stark trap. The reaction mixture was cooled and concentrated in vacuo to give a brownish oil which exhibited a strong C=N absorption band a t 1665 cm-' by IR analysis. The crude Schiff base was dissolved in methanol (250 mL) and then NaBH4 (19 g) was added in small portions. The reaction mixture

was stirred at room temperature overnight and concentrated in vacuo. Water (200 mL) was added, followed by extraction with toluene (450 mL). The organic layer was dried over MgSO,. Concentration in vacuo left a brown oil; this was purified by vacuum distillation (0.1 mmHg, 140-170 "C), yielding 21.6 g (53%) of 6 as a pale yellow oil: 'H NMR (CDCL3) 6 1.05 (d, J = 6 Hz, 6 H), 1.67-2.75 (complex, 7 H), 3.58 (s, 2 H), 3.75-4.2 (br, 1 H), 6.58-7.29 (complex, 3 H), 7.39 (s, 5 H). Elemental analysis for Cl9HZ3FN2. Calcd: C, 76.48; H, 7.77; N, 9.39. Found: C, 76.79; H, 7.83; N, 9.51.

l-Benzyl-3-methyl-4-[N-(2-fluorophenyl)methoxyacet- amidolpiperidine (21). A solution of 6 (10 g, 33 mmol), methoxyacetyl chloride (4.0 g, 37 mmol), and anhydrous THF (50 mL) was stirred a t room temperature for 2 days. The acidic mixture was carefully alkalinized with 10% NaOH. The phases were separated, and the liberated free base was extracted with CH2Clz (100 mL). The organic extract was dried over MgSOI. Concen- tration in vacuo yielded 21 as a pale yellow oil (12.33 g, 99%): 'H NMR (CDC13) 6 1.29 (d, J = 6.5 Hz, 6 H), 2.45-3.21 (complex, 5 H), 3.42 (s, 5 H), 3.76 (2, 2 H), 4.3-4.75 (complex, 1 H), 7.1S7.4 (complex, 8 H). Elemental analysis for C22HnFN20. Calcd: C, 71.33; H, 7.35; N, 7.56. Found: C, 70.69; H, 7.27; N, 7.91. 3-Methyl-4-[N-(2-fluorophenyl)met hoxyacetamidol-

piperidine (22). A mixture of 21 (12 g, 32 mmol) and palladium on carbon (0.5 g, 10%) in ethanol (50 mL) was shaken under a hydrogen atmosphere (50-60 psi) for 18 h. TLC (EtOAc, silica gel) indicated complete consumption of the starting material, and the slurry was filtered. The filtrate was concentrated in vacuo to afford a yellowish oil which was purified by column chromatography (methanol/NH40H 1:0.5 v:v) to yield 3.2 g (45%) of 22

(complex, 5 H), 3.2 (s, 3 H), 3.62 (s, 2 H), 4.12-4.74 (complex, 1 H), 7.0-7.4 (complex, 4 H). Elemental analysis for C1SH21FN20. Calcd: C, 64.27; H, 7.55; N, 9.99. Found: C, 63.94; H, 7.19; N, 9.71.

1-[ 2-(4-Ethyl-4,5-dihydro-1H-tetrazolyl)ethyl]-3-methyl- 4-[N-(2-fluorophenyl)methoxyacetamido]piperidine (40). l-(2-Chloroethyl)-4-ethyl-1,4-dihydro-5Htetrazol-5-0ne~~ (1.68 g, 9.5 mmol) was added to a stirring solution of 22 (2.42 g, 8.6 mmol), powdered anhyrous potassium carbonate (11.9 g, 86 mmol), and a few crystals of potassium iodide in acetonitrile (60 mL). The reaction mixture was heated at reflux for 18 h, cooled, and filtered. The filtrate was concentrated in vacuo, affording the intermediate 25 as a yellowish oil. Column chromatography with EtOAc (100%) afforded 1.2 g of 13, upper spot (R, - 0.3), and 0.75 g of 14, lower spot (Rf - 0.24). The combined total yield was 56%. Intermediate 13 was crystallized as the oxalate salt (mp 151-153 "C), affording 1.25 g of 40. Note: The compounds where the N-1 substituent is phenethyl were prepared with 1- phenethyl-3-methyl-4-piperidone (26) as the starting material, which, in turn, was prepared according to ref 8.

cis -l-[2-(2-Thienyl)ethyl]-3-methyl-4-[N-(2-fluorophenyl)-2-methoxyacetamido]piperidinium oxalate (42): IH NMR (CDC13) 6 1.28 (d, J = 6 Hz, 6 H), 1.78-3.2 (complex, 8 H), 3.45 (s, 3 H), 3.78 (s, 2 H), 4.3-4.85 (complex, 1 H), 6.8-7.6 (complex, 7 H).

cis - 1-[ 2- (2-Thieny1)et hyl1-3-met hyl-4-[ N-( 2-met hoxy- phenyl)-2-methoxypropionamido]piperidinium oxalate (62): 'H NMR (CDC13) 6 0.89-1.32 (complex, 6 H), 1.8-3.02 (complex,

(R10.2): 'H NMR (CDC13) 6 1.0 (d, J = 4.6 Hz, 6 H), 2.22-2.8

(13) Jansaen, F.; Torremans, J.; Jansaen, P. A. J. Med. Chern. 1986, 29, 2290.

2882 Journal of Medicinal Chemistry, 1990, Vol. 33, No. 10 Lalinde et al.

Naloxone Reversibility Test. Male Sprague-Dawley rats (15&250 g), housed with free access to food and water, were identified, weighed, and randomly assigned to a treatment group ( N = 6). After the animals acclimated to the laboratory environment (1 h), pretreatment talil-flick latencies were determined for each animal. Drugs were dissolved in 0.9% saline or appropriate vehicle and administered in a volume of 1 mL/kg. All groups were administered the A , dose of agonist followed 1 min later by either saline or naloxone. The animals posttreatment tail-flick latency was determined 1 min after naloxone (or saline) administration.

The percent maximal effect (% MPE) was determined for each animal by the equation

(posttreatment) - (control)

(cuboff)- (control) % MPE = x 100

The mean % MPE was then determined for each treatment group. The T test for unpaired data was then used to determine whether the % MPE obtained after naloxone administration was significantly different from that obtained after saline administration. Treatment groups having a signficant difference (p = 0.05) were considered as naloxone reversible.

Inhibition of [3H]Naloxone Binding. Compounds were studied for their ability to displace [3H]naloxone from membrane binding sites. This assay is a good estimate of rank orders of potency for opiate agonists. It is based upon the method reported by Pasternak and his colleagues,18 who used membrane fractions prepared from homogenates of rat brains. The membranes were incubated in Tris buffer in the presence of 1.0 nM [3H]naloxone (50-60 Ci/mmol) and varying concentrations of either 40 or drug standards. After incubation at 25 "C for 30 min, the membranes with radioactivity bound to them were separated from free or unbound radioactivity by filtering the incubation mixtures over glass-fiber fdters. After they were washed, the filters with adherent membranes and bound radioactivity were put into scintillation vials. Scintillation cocktail was added and radioactivity was estimated by liquid scintillation spectrometry. The results were plotted, and from the plots an ICw for displacement was calculated. The Ki for inhibition was calculated by Cheng and Prusoff's method.lg

Screen for Pr imary Overt Effects for Hypnotics/An- esthetics in the Rat. Six SpragueDawley rats (23G290 g) were used. Immediately following iv drug administration, the animal was removed from the restrainer and observed along with control (i.e., the next test animal) for 20 min. Righting is lost when the animal remains supine for at least 30 s. The EDw for anesthesia was determined by linear regression. The confidence limits were determined as previously described.20 Core temperature is obtained rectally prior to and during the 5 min postdrug period and the difference is recorded. Other primary overt effects are scored from 0 to plus or minus 3 (0 = normal, 1 = mild, 2 = moderate, 3 = marked) and recorded a t 5-min intervals for 30 min.

Acknowledgment. T h e technical assistance of Mark Benvenga, John Capacchione, Steve Waters, Sarah Harris, (Anaquest Pharmacology Department, Murray Hill, NJ, and of t he Dental School of the University of Maryland at Baltimore) is gratefully acknowledged. We also thank the discussions and valuable insight of Dr. Bao-Shan Huang and Dr. Jerome Bagley (Anaquest Chemistry De- partment) during the course of this investigation and the preparation of this manuscript.

9 H), 3.25 ( s , 3 H), 3.32-3.9 (complex, 4 H), 4.1-4.6 (complex, 1 H), 6.7-7.3 (complex, 7 H).

cis - l-(2-Phenylethyl)-~henylethyl)3-methyl-4-(N-phenyl-N-2-furoyl- amino)piperidinium oxalate (65): 'H NMR (CDC13) 6 1.3 (d, J = 6 Hz, 3 H), 1.7-3.2 (complex, 10 H), 4.1-4.7 (complex, 2 H), 5.45-6.65 (3 m, 3 H), 7.3-7.6 (complex, 10 H).

cis -1-[2-(2-Thienyl)ethyl]-3-methyl-4-(N-phenyl-~-2- furoy1amino)piperidinium oxalate (68): 'H NMR (CDC1,) b 1.2 (d, J = 6.5 Hz, 3 H), 1.6-3.2 (complex, 10 H), 3.8-4.9 (complex, 2 H), 5.5 (d, J = 4 Hz, 1 H), 6.15 (complex, 1 H), 6.7-7.7 (complex, 9 H).

Pharmacological Methods. In Vivo. Analgesic. A. 55 "C Mouse Hot Plate.14 The hot-plate assay utilized nonfasted male mice (Swiss-Webster) weighing between 18 and 22 g. The surface of the hot-plate apparatus was maintained a t 55 f 0.5 'C. To determine the percentage of maximum pharmacological effect (MPE) using the mouse hot-plate (MHP) assay, vehicle (saline) or drug solution (10 mL/kg) was injected into the lateral tail vein of groups of 10 mice and placed on the hot plate after 1 min. An initial dose of 1 mg/kg of compound was administered. If 100% MPE was observed, then lower dosing was continued until an EDSo was generated. If less than 100% MPE was observed, then 5 mg/ kg was administered. In addition to analgesia, side effects were noted. These were chiefly categorized as rigidity, sedation, respiratory depression, tremors, convulsions, and cyanosis. For each experiment, control latency times were determined in 10 mice and treatment latency times determined in additional groups after each dose of compound. The response latency was the time between the initial contact on the hot surface and the fiit paw-lick response. Animals were removed from the hot plate immediately after a response or until the cut-off time 30 s was reached. An- tinociceptive effect was defined as a doubling of the latency time to paw-lick over control times.

test time - control time x 100 30 s -control time

?& MPE =

The EDw and 95% confidence limits were calculated by using a standard computer program of the method of Litchfield and W i l c o ~ i n ~ ~ fitted to a minioomputer.16 Calculation of the EDw (95% confidence limits) was corrected for base content of the salts.

Duration of Analgesia. Two times the ED, was administered to 10 mice and the hot-plate latencies were determined at various times after injection in the lateral tail vein. The mean MPE was calculated for each time period and a time-effect curve was generated. A test compound was defined to be short acting if the duration of action to 50% MPE was less than 6 min, intermediate duration was 6.1-15 min, and long acting was a duration greater than 15.1 min.

B. 55 "C Rat Hot Plate. This assay was performed siilarly to the above with six male Sprague-Dawley rats weighing between 300 and 400 g. C. Rat Tail Flick Median Effective Dose (ED,) Deter-

mination." Rat tail flick latencies were determined for 40 and drug standards as a measure of antinociceptive activity. Two pretreatment latencies were determined for each rat. They were then restrained gently and their tails were placed under a focused light that produced radiant heat. When the animals flicked their tail aside, a photocell was uncovered and the instrument's circuitry stopped a timer. Postinjection tail flick latencies were determined after iv injection. The quantal ED& were calculated by Litchfield and Wilcoxin's method.ls

Fifer, E. K.; Davis, W. M.; Borne, R. F. Eur. J . Med. Chem.- Chim. Ther. 1984,6,519. Wynn, R. L.; Ford, R. D.; McCourt, P. J.; Ramkumar, V.; Bergman, S. A.; Rudo, F. G . Drug Deu. Res. 1986, 9, 233. Litchfield, J. T.; Wilcoxin, F. J. J . Pharmacol. Exp. Ther. 1949, 96,99. Tallarida, R. J.; Murray, R. B. Manual of Pharmacological Calculations with Computer Programs; Springer-Verlag: New York, 1981; p 85. D'Armoour, F. E.; Smith, D. L. J. Pharrnacol. Exp. Ther. 1941, 72, 74.

(18) Pasternak, G. W.; Wilson, H. A.; Snyder, S. H. Differential effects of protein-modifying reagents on receptor binding of opioid agonists and antagonists. Mol. Pharmacol. 1975, 11, 340.

(19) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (Ki) and the concentration of inhibitor that causes a 50% inhibition of an enzymatic reaction. Biochem. Pharma- col. 1973, 22, 3099.

(20) Kushner, H. W.; Demaio, G. Basic Statistics; Holden-Day: San Francisco, 1980.

Documents

FACT SHEET Ocfentanil