Analgetika Antiphlogistika Antirheumatika Entzndungshemmer

Synthesis with Improved Yield and Study on the Analgesic Effect of 2-Methoxyphencyclidine
Abbas Ahmadi and Ali Mahmoudi Department of Science, School of Chemistry, Islamic Azad University, Karaj (Iran)
Corresponding author: Prof. Dr. Abbas Ahmadi, Department of Science, School of Chemistry, Islamic Azad University, P. O. Box 31485-313, Karaj (Iran); e-mail: abbasahmady3957@yahoo.com

Summary
Phencyclidine (1-(1-phenylcyclohexyl)piperidine, CAS 956-90-1, PCP) has shown analgesic effects. Some of its derivatives have been synthesized and their biological properties were studied. Since a methoxy group has been added to the position 2 of the cyclohexane ring of PCP , the resulting compound is more polar than PCP This compound was synthe. sized using an improved method with a higher yield. Its analgesic effect was studied using the tail-flick test on rats and was compared with that of ketamine (CAS 1867-66-9). The results showed that 2-methoxyphencyclidine increased tail-flick latencies as compared to the control group. The maximum analgesic effect of the compound occurred 510 min after its injection, while the effect of ketamine was observed 1025 min after injection.

Zusammenfassung Key words

Analgesics CAS 956-90-1 2-Methoxyphencyclidine, analgesic effect, rat, synthesis Phencyclidine derivatives Arzneim.-Forsch./Drug Res. 56, No. 5, 346350 (2006)
Synthese mit verbesserter Ausbeute und Untersuchung der analgetischen Wirkung von 2-Methoxyphencyclidin Phencyclidin (1-(1-Phenylcyclohexyl)piperidine, CAS 956-90-1, PCP) besitzt analgetische Wirkung. Einige Derivate von PCP werden synthetisiert und ihre biologischen Eigenschaften untersucht. Da eine Methoxy-Gruppe in Position 2 des Cyclohexan-Rings von PCP eingefhrt wurde, besitzt die neue Verbindung grere Polaritt und Aktivitt als PCP . Diese Verbindung wurde nach einer verbesserten Methode mit hherer Ausbeute synthetisiert. Die analgetische Wirkung wurde im Tail-flick-Test an Ratten im Vergleich zu Ketamin (CAS 1867-66-9) untersucht. Die Ergebnisse zeigten, da 2-Methoxyphencyclidin die Verzgerung im Tailflick-Test im Vergleich zur Kontrollgruppe erhhte, wobei die maximale analgetische Wirkung 5 bis 10 min nach der Verabreichung beobachtet wurde, whrend der entsprechende Effekt von Ketamin nach 10 bis 25 min auftrat.

346

Ahmadi et al. 2-Methoxyphencyclidine

Arzneim.-Forsch./Drug Res. 56, No. 5, 346350 (2006) ECV Editio Cantor Verlag, Aulendorf (Germany)

Analgesics Anti-inflammatories Antiphlogistics Antirheumatic Drugs

1. Introduction
It is well known that, phencyclidine (1-(1-phenylcyclohexyl) piperidine, CAS 956-90-1, PCP), its derivatives and analogues have biological properties and affect the central nervous system. Changes in the phencyclidine molecule can bring about changes in its properties [1]. It is a highly potent and widely abused psychomimetic drug [2]. It displays analgesic, stimulant, depressant and hallucinogenic effects and is known to bind to receptors, but evidence is available suggesting that it has its own specific receptor [3]. PCP has been shown to exert its analgesic effect through opiate receptors. However, probably the most powerful approach in characterizing PCP analgesic effect stems from recent pharmacodynamic studies that have identified specific binding sites in the brain [4]. PCP also was originally introduced as a general anesthetic agent [57], but it was subsequently withdrawn from use in humans because of severe psychomimetic side effects [813]. The focus of research on PCP has shifted from its use as an anesthetic toward potential applications as a neuropharmaceutical [14, 15]. PCP binds to the N-methyl-D-asparate (NMDA) receptor complex and blocks NMDA-mediated gating of the calcium channel conductance [16].The most powerful approach in characterizing PCPs analgesic effect stems from recent pharmacodynamic studies that have identified specific binding sites in the brain. PCP and similar compounds are classified as non-competitive open channel blockers of the NMDA receptor [17, 18]. PCP and analogues have many behavioral effects in common with other phencyclidine-like drugs, including anaesthetic, antinociceptive, psychotomimetic, anticonvulsant, neuroprotective and amnesic drugs [19]. PCP is a semi-rigid molecule containing a cyclohexane ring with attached aromatic and piperidine rings (see structure formulas). The analgesic effect of ketamine (2-O-chlorophenyl2-methylaminocyclohexane, CAS 1867-66-9; see structure formula), another PCP analogue, was first described by Domino and collaborators in 1965. Ketamine in low sub-anaesthetic doses is reliable as an analgesic in acute pain [20]. In low sub-anaesthetic doses, ketamine acts more selectively as a non-competitive blocker of the NMDA receptor. It does so by binding to the PCP recognition site in the NMDA receptor complex [21]. At higher concentration, ketamine interacts with opioid receptors, sigma sites, kappa and delta receptors [22, 23]. Since a methoxy group has been added to position 2 of the cyclohexane ring of PCP (2-OCH3-PCP), this compound is more polar and active than PCP and has previously been synthesized with low yield [24, 25]. In this paper, we synthesized 2-methoxy phencyclidine with higher yield than the previous methods and tested its analgesic effect by the tail-flick test, comparing it with ketamine.
Arzneim.-Forsch./Drug Res. 56, No. 5, 346350 (2006) ECV Editio Cantor Verlag, Aulendorf (Germany)
C6H5 N C6H5 N OH 1 PCP 2-OH-PCP 2 Ketamine C6H4Cl NHCH3 O 3

C6H5

N OCH3 4

2-OCH 3-PCP

Structure formulas of PCP 2-OH-PCP, ketamine and 2-OCH3-PCP ,

2. Material and methods

2.1. General
Cyclohexanone, 1,5-dibromopentane, sodium azide, LiAlH4, tetrahydrofuran, sodium hydride, iodomethane and all other chemicals were purchase from Merck (Darmstadt, Germany). Ketamine was purchased from Sigma-Aldrich chemical company (Poole, Dorset, England). Melting points (uncorrected) were determined using a Gallencamp apparatus (CAT.No.29/ MF-370; Watford, Herts, England) with a capillary tube. 1H- and 13 C-NMR spectra were recorded on a Bruker 80 MHz, Ac-80 spectrometer (Spectrospin, Faellanden, Switzerland) (internal reference: TMS). IR spectra were recorded on a Shimadzu FTIR 4800 spectrophotometer (Kyoto, Japan). Mass spectra were recorded on a Shimadzu QP-1000 EX spectrometer. Spectroscopic data (IR, 1H- and 13C-NMR) confirmed the structures of all reported compounds; the melting points of known compounds could also confirm their identity. The purity of each compound was checked by TLC [dioxin:toluene:ethanol:concentrated ammonia, 50:40:5:5 (by volumes), as the mobile phase]. Adult male NMRI rats, weighting about 250300 g, were used for pharmacological testing. All procedures were carried out in accordance with institutional guidelines for animal care and use.

2.2. Synthesis of compounds 2.2.1. 1-Phenyl cyclohexanol 5

This compound was prepared in 62 % yield from phenyl magnesium bromide and cyclohexanone according to the known procedure [26]. The product was recrystallized from ether-petroleum benzene (1:1) (m.p. 6363.5 C).

2.2.2. 1-Phenyl cyclohexene 6

This compound was prepared from 1- phenyl cyclohexanol and a mixture of sulfuric and glacial acetic acid according to the known procedure [27] (b.p. 110111 C, 5mmHg).

2.2.3. cis-1-Phenylcyclohexane-1, 2-diol (7)

This compound was prepared from 1-phenylcyclohexen and KMnO4 at 5 C according to the known procedure [28]. The product was recrystallized from ethanol-petroleum benzene (1:1) (m.p: 9496.5 C).

2.2.4. cis-2-Azido-2-phenylcyclohexanol (8)

This compound was prepared from cis-1-phenylcyclohexane1,2-diol and sodium azide and a solution of perchloric acid

Ahmadi et al. 2-Methoxyphencyclidine

347

Analgetika Antiphlogistika Antirheumatika Entzndungshemmer

70 % at 5 C for 5 days [29] and was recrystalized from etherpetroleum benzene (6:1) (m.p. 6163 C).

2.2.5. cis-2-Amino-2-phenylcyclohexanol (9)

This compound was prepared by refluxing of cis-2-azido-2phenylhexanol and LiALH4 in dry ether for 48 h [30] (m.p. 103104 C).

tency in s was used as a parameter of pain sensitivity. The light source was adjusted to give an average baseline latency of about 34 s in untreated rats. Two trails of the tail flick test were run at 5-min intervals.

2.3.2. Effect of 2-methoxyphencyclidine and ketamine hydrochloride on analgesic activity

2-Methoxyphencyclidine (2-OCH3-PCP) was dissolved in dimethylsulfoxide (DMSO) and was given 15 min before the actual testing. In other groups, animals were given saline (vehicle) and ketamine in saline, respectively. The difference in the tail-flick latencies scores were evaluated using analysis of variance (ANOVA). The p< 0.05 level was considered to represent significant difference. 2-OCH3-PCP was injected in two doses (3 and 6 mg/ kg i.p) and the tail-flick latency was determined 2, 5, 10, 15, 20, 25 and 30 min after injection. Another analogue of PCP ket, amine hydrochloride was injected for comparison (at 0.5, 1, 6 mg/kg i.p.).

2.2.6. cis-2-Piperidino-2-phenylcyclohexanol (2)

This compound was prepared from refluxing of cis-2-amino-2phenylhexanol and 1,5-dibromopentane in dry acetone for 2 days [31] (m.p. 135 C ).

2.2.7. cis-1-Methoxy-2-piperidino-2phenylcyclohexane (4)

A solution containing 150 mg (0.58 mmol) of compound 2 with 127.5 mg (2.34 mmol) of 60 % NaH in 50 ml of dry THF was refluxed for 5 h. The reaction mixture was cooled, then 442 mg (2.32 mmol) ofCH3I was added and the mixture was refluxed again for 5 h. The reaction was quenched by the addition of ice-water. The solvent was removed and the residue was treated with 5 % solution of HCl. The aqueous layer was separated and extracted with ether and neutralized with 10 % NaOH, extracted with ether, dried over Na2SO4. 147.4 mg (93.2 %) of compound 4 was obtained (m.p. 9092 C). IR (KBr): 3050, 2979, 2925, 1600, 1490, 1465, 1371, 1271, 1168, 1097, 954.7, 756, 696 cm-1. 1 H-NMR (CDCl3) (ppm): 1.32.3 (18H, m), 3.25 (3H, m), 3.4 3.5 (1H, m), 77.2 (5H, m). 13 C-NMR (CDCl3) (p.p.m.): 20.3, 21.5, 25.1, 26.2, 31.4, 48.1, 52, 63.4, 84.4, 125.7, 126.7, 128.2, 144.8. MS: m/e (regulatory intensity): 273 (34).

3. Results
3.1. Chemistry 2-OCH3-PCP was synthesized as outlined in Scheme 1. This compound has been synthesized previously, but in this study, we synthesized it using another method with higher yield. We employed the known procedure for the synthesis of compounds 5 to 8 with the appropriate modifications described previously [2629]. 3.2. Pharmacology 3.2.1. The analgesic activity of 2-OCH3-PCP and ketamine hydrochloride Intraperitoneal injections of ketamine (0.5, 1, 6 mg/kg) and 2-OCH3-PCP (3 and 6 mg/kg) produced analgesic effects in the tail-flick test. The experiments showed that two doses of 2-OCH3-PCP (3 and 6 mg/kg) increased tail-flick latencies compared to the control group receiving DMSO, and the maximum analgesic effect was observed 510 min after its injection (Fig. 1). Ketamine produced analgesia in the tail-flick test in all injected doses (Fig. 2), and the maximum effect was observed 1015 min after its injection.

2.3. Pharmacological methods

Adult male NMRI rats, bred at the institute of Biochemistry and Biophysics at Tehran University, were maintained on a 12-h light-dark cycle in a temperature-controlled room (25 1 C) at 50 % relative humidity. They were allowed free access to a standard laboratory rat chow (Pars Company, Tehran, Iran) and tap water ad libitum. The animals (n = 35, total; 250300 g body weight) were randomly assigned to various groups. They were brought into the experimental room for acclimatization 24 h before the experiment. All experiments were performed between 8 h 0 min and 16 h 0 min under normal room light and at the temperature of 25 C. All of the animals were injected by one investigator and evaluated by another one.

2.3.1. Tail-flick test

A commercially available analgesia meter (Apelex, model DS 20, Socrel, Bagneux, France) was used. The test is a modification of the DAmour and Smith method [4]. Various groups of rats (n = 5 animals/group) were used for the test. The whole body of each rat except the tail was covered with a piece of thick cloth. A beam of light at a temperature of 130 C was focused onto the last 12 cm of the tail. The latency time (in s) at which the animal withdrew its tail was noted before treatment and 2, 5, 10, 15, 20, 25 and 30 min after administration of the drug. A cut-off time of 10 s was used for each light exposure to avoid damage of the tail. The first tail withdrawal laO

OH C6H5

C6H5

OH OH C6H5

5
OCH3
N

6 d
OH NH2 C6H5

7
OH N3 C6H5

OH

C6H5

C6H5

Scheme 1: Synthesis of 2-OCH3-PCP (a) C6H5MgBr, dry ether; (b) . H2SO4, ACOH; (c) KMnO4; (d) NaN3, HClO4; (e) LiAlH4, dry Et2O; (f ) 1,5-diboromo pentane, K2CO3, dry acetone; (g) CH3I, NaH, dry THF.

348

Ahmadi et al. 2-Methoxyphencyclidine

Arzneim.-Forsch./Drug Res. 56, No. 5, 346350 (2006) ECV Editio Cantor Verlag, Aulendorf (Germany)

Analgesics Anti-inflammatories Antiphlogistics Antirheumatic Drugs

DMSO

8
Tail-flick latency

2-OCH3-PCP (3 mg/kg) 2-OCH3-PCP (6 mg/kg)

7 6 5 4 3 2 5 10 15 20 25 30 Time (min)

Fig. 1: Mean tail flick latencies in animals that receiving 2-OCH3PCP i.p. The tail-flick test was conducted 2, 5, 10, 15, 20, 25 and 30 min after injection. Each point represents mean SEM of 5 animals.

Saline

8 7
Tail-flick latency

Ketamine (6 mg/kg) Ketamine (1 mg/kg) Ketamine (0.5 mg/kg)

form. Therefore, the concentration of ketamine in the lipid phase is several orders of magnitude greater than in the aqueous phase. Ketamine gained access to a blocker site associated with the lipid membrane of the lipid protein interface [35]. On the other hand, the predominance of closed-channel blockade suggests that ketamines analgesic properties might result from closed-channel rather than open-channel blockade. 2-OCH3-PCP has a polar structure; therefore, the concentration of this drug in the aqueous phase is greater than in the lipid phase, and its analgesic properties might result from open-channel block impeding ionic flow. This may explain why 2-OCH3-PCP has a rapid analgesic effect (5-10 min after injection) on acute and phasic pain. The acute analgesic effect of ketamine is closely associated with sensory and locomotor side effects. These motor side effects are increased depending on the dose, limiting the use of ketamine in chronic pain [34]. At the doses used in this study, 2OCH3-PCP did not show motor side effect. In conclusion, the present study demonstrates that the test compound, synthesized by different by a different method in comparison with previous methods [24, 25] with higher yield and purity, attenuates acute pain in the tail-flick test, possibly exerting its antinociceptive effects through NMDA receptor blockade. Acknowledgements
This work was done as a project at Tehran University, Department of Science and I.B.B. (Institute of Biochemistry and Biophysics). The author would like to thank Mrs. Mahshid Shafiezadeh and Mrs. T. Yousefifard for their assistance with the pharmacological tests.

6 5 4 3 2 5 10 15 20 25 30 Time (min)

Fig. 2: Average latencies of heat-induced tail-flick responses 2, 5, 10, 15, 20, 25 and 30 min after the i.p. administration of saline or ketamine (0.5, 1, 6 mg/kg). Each point represents the mean SEM of 5 animals [1].

References
[1] Ahmadi, A., Shafiezadeh, M., Fathollahi, Y., Synthesis with improved yield and study on analgesic effect of 2-hydroxyphencyclidine. Arzneim-Forsch./Drug Res. 55, 172 (2005) [2] Peterson, R. C., Stillman, R. C., An overview in phencyclidine abuse, pp. 117, National Institute on Drug Abuse, Washington, DC (1978) [3] Casy, A. F., Dewar, G. H., Al-deeb, O. A. A., Opioid properties of some isomeric derivatives of phencyclidine. J. Pharm. Pharmacol. 44, 19 (1992) [4] Al-deeb, O. A. A., Synthesis and analgesic activity of new phencyclidine derivatives. Arzneim-Forsch./Drug Res. 44, 1141 (1994) [5] Luby, E. D., Gottlieb, J. S., Rosenbaum, G. et al., Model psychoses and schizophrenia. Am. J. Psychiatry. 119, 61 (1962) [6] Rainey, J. M., Crowder, M. K., Prolonged psychoses attributed to phencyclidine: report of three cases. Am. J. Psychiatry 132, 1076 (1975) [7] Fauman, B., Baker, F., Coppleson, L. et al., J. Am. Coll. Emerg. Physicians 4, 223 (1975) [8] Luisada, P., Brown, B. I., Clinical management of phencyclidine psychosis. Clin. Toxicol. 9, 539 (1976) [9] Balster, R, L., Chait, L. D., The behavioral pharmacology of phencyclidine. Clin. Toxicol. 9, 513 (1976)

4. Discussion
Electrophysiologic and binding studies revealed that various antagonists of NMDA receptors, including phencyclidine, ketamine and dizocilpine (MK-801), bind to the PCP site mainly when the channels are in the open or activated state [3, 33]. Previous studies suggest that ketamine may interact with the NMDA receptor at two potentially distinct sites: one site located within the channel pore and a second site associated with the hydrophobic domain of the protein. The binding of the agonist to the receptor is assumed to modify the binding of ketamine to both sites. Ketamine is formulated as a hydrochloride salt and thus is highly water-soluble [34], but under physiological conditions, a large fraction of the drug exists in the lipid-soluble
Arzneim.-Forsch./Drug Res. 56, No. 5, 346350 (2006) ECV Editio Cantor Verlag, Aulendorf (Germany)

Ahmadi et al. 2-Methoxyphencyclidine

349

Analgetika Antiphlogistika Antirheumatika Entzndungshemmer

[10] Johnson, K. M., Jones, S. M., Neuropharmacology of phencyclidine: Basic mechanisms and therapeutic potential. Annu. Rev. Pharmacol. Toxicol. 39, 707 (1990) [11] Contreras, P. C., Monohan, J. B., Lanthorn, T. H. et al., Phencyclidine: physiological actions, interactions with excitatory amino acids and endogenous ligands. Mol. Neurobiol. 1, 191 (1987) [12] Chen, G., Bohner, B., Anticonvulsant properties of (1(1-phenylcyclohexyl) piperidine.HCl and certain other drugs. Proc. Soc. Exp. Biol. Med. 106, 632 (1961) [13] Leander, J. D., Rathbun, R. C., Zimmeman, D. M., Anticonvulsant effects of phencyclidine-like drugs: relation to Nmethyl-D-aspartic acid antagonism. Brain Res. 454, 368 (1988) [14] Sagratella, S, Niglio, T., Scotti de Carolis, A., An investigation on the mechanism of anticonvulsant action of ketamine and phencyclidine on convulsions due to cortical application of penicillin in rabbits. Pharmacol. Res. Commun. 17, 773 (1989) [15] Hayes, B. A., Balster, R. L., Anticonvulsant properties of phencyclidine-like drugs in mice. Eur. J. Pharmacol. 117, 121 (1985) [16] Honey, C. R., Miljkovic, Z., McDonald, J. F., Ketamine and phencyclidine cause a voltage-dependent block of responses to L-aspartic acid. Neurosci. Lett. 61, 135 (1985) [17] Kemp, J. A., Foster, A. C., Wong, E. H. F., Non-competitive antagonists of excitatory amino acid receptors. Trends Neurosci. 10, 294 (1987) [18] Anis, N. A., Berry, S. C., Burtan, M. et al., The dissociative anesthetics ketamine and phencyclidine selectively reduce excitation of control mammalian neurons by N-methyl-asparate. J. Pharmacol. 79, 565 (1983) [19] Shimoyama, N., Shimoyama, M., Inturrisi, C. E. et al., Ketamine attenuates reverses morphine tolerance in rodents. Anesthesiology 85, 1357 (1996) [20] Stubhaug, A., Breivik, H., Long-term treatment of chronic neuropathic pain with the NMDA (N-methyl-D-asparate) receptor antagonist ketamine. Acta. Anaesthesiol. Scand. 41, 329 (1997) [21] Oye, I., Paulsen, O., Maurset, A., Effects of ketamine on sensory perception: evidence for a role of N-methyl-D-asparate receptors. J. Pharmacol. Exp. Ther. 260, 1209 (1992)

[22] Rabben, T., Skjelbred, P Oye, I., Prolonged analgesic ., effect of ketamine, an N-methyl-D-asparate receptor inhibitor, in patients with chronic pain. J. Pharmacol. Exp. Ther. 289, 1060 (1999) [23] Meller, S. T., Ketamine relief from chronic pain through actions at the NMDA receptors. Pain 68, 435 (1996) [24] Kamenka, J. M., Geneste, P., Constantes hydrophobes en serie de la phencyclidine au moyen de la chromatographie liquide. Eur. J. Med. Chem. Ther. 16, 213 (1981) [25] Kamenka, J. M., Ung, M. S. N., Herrmann, P. et al., Determination conformationnelle de derives de la phencyclidine en vue dune correlation acture-active. Eur. J. Med. Chem. Ther. 14, 301 (1979) [26] Treppmann, A., Zur Kenntnis ungesttigter hydroaromatischer Kohlenwasserstoffe. Chem. Ber. 48, 1216 (1915) [27] Karabinos, P., The dehydration of cis- and trans-2phenylcyclohexanols. J. Am. Chem. Soc. 62, 1160 (1940) [28] Mangoni, L., Adinolfi, M., Barone, G. et al., A convenient procedure for the cis-hydroxylation of olefins. Tetrahedron Lett. 45, 4485 (1973) [29] Yoshito, T., Hidekazu, I., Tsuchiya, Y., Homochiral ligands derived from cis-1-phenylcyclohexane-1, 2-diol and cis2-azido-phenylcyclohexanol. Tetrahedron Asymmetry 22, 3735 (1997) ` [30] Hedayatullah, M., Guy, A., Synthese et reduction dazidosulfates daryle. Tetrahedron Lett. 29, 2455 (1975) [31] Ahmadi, A., Mahmoudi, A., Synthesis and Biological Properties of 2-Hydroxy-1-(1-phenyltetralyl)piperidine and some of its intermediates as derivatives of phencyclidine. Arzneim.-Forsch./Drug Res. 55, 528 (2005) [32] Parsons, C. G., Gibbens, H., Magnago, T. S. L. et al., At which sigma site are the spinal actions of ketamine mediated? Neurosci. Lett. 85, 322 (1988) [33] Qian, J., Brown, S. D., Carlton, S. M., Systemic ketamine attenuates nociceptive behaviors in a rat model of peripheral neuropathy. Brain Res. 715, 51 (1996) [34] Shimoyama, M., Shimoyama, N., Gorman, A. L. et al., Oral ketamine is antinociceptive in the rat formalin test: role of the metabolite, nor ketamine. Pain 81, 85 (1999) [35] Farance, C. P., Snyder, A. M., Woods, J. H., Analgesic effects of phencyclidine-like drugs in rhesus monkeys. J. Pharmacol. Exp. Ther. 250, 197 (1989)

350

Ahmadi et al. 2-Methoxyphencyclidine

Arzneim.-Forsch./Drug Res. 56, No. 5, 346350 (2006) ECV Editio Cantor Verlag, Aulendorf (Germany)