ArzneimForschDrugRes Analgetika · Antiphlogistika · Antirheumatika · Entzündungshemmer

Analgesics · Anti-inflammatories · Antiphlogistics · Antirheumatic Drugs

Synthesis with Improved Yieldand Study on the Analgesic Effectof 2-HydroxyphencyclidineAbbas Ahmadia, Mahshid Shafiezadehb, Yaghoob Fathollahib, Mohammad Raouf Darvichb, Ali Mahmoudia,Manouchehr Bahmanic, and Batol Rahmatid

Department of Science, School of Chemistry, Islamic Azad University a, Karaj (Iran), Institute of Biochemistry andBiophysics (I. B. B.), University of Tehranb, Tehran (Iran), Department of Science, School of Chemistry, IslamicAzad University, North Branchc, Tehran (Iran), and Department of Physiology, Shahed University d, Tehran (Iran)

Key words

� CAS 956-90-1� 2-Hydroxyphencyclidine,

analgesic effect, rat, synthesis� Phencyclidine derivatives

Arzneim.-Forsch./Drug Res.55, No. 3, 172−176 (2005)

Summary

Phencyclidine (1-(1-phenylcyclohexyl)pi-peridine, CAS 956-90-1, PCP) has shownanalgesic effects. Some of its derivativeshave been synthesized and their biolog-ical properties were studied. Since a hy-droxyl group has been added to the posi-tion 2 of the cyclohexane ring of PCP,this compound would be more hydro-philic than PCP. This compound was syn-thesized using a different and improvedmethod with a higher yield. Its analgesiceffect was studied using the tail-flick teston rats and was compared with that ofketamine (CAS 1867-66-9).

Zusammenfassung

Synthese mit verbesserter Ausbeute undUntersuchung der analgetischen Wirkungvon 2-Hydroxyphencyclidin

Phencyclidin (1-(1-Phenylcyclohexyl)-piperidin, CAS 956-90-1, PCP) hat analge-tische Wirkung gezeigt. Einige Derivatevon PCP werden synthetisiert und ihrebiologischen Eigenschaften untersucht.Da eine Hydroxyl-Gruppe in der Position2 des Cyclohexan-Rings von PCP einge-führt wurde, ist diese Verbindung hydro-philer als PCP. Diese Verbindung wurdenach einer anderen und verbesserten Me-thode mit höherer Ausbeute syntheti-siert. Die analgetische Wirkung wurde imTail-Flick-Test an Ratten im Vergleich zuKetamin (CAS 1867-66-9) untersucht.

The results showed that 2-hydroxy-phencyclidine can increase tail-flick la-tencies as compared to the control groupand indicate that the maximum anal-gesic effect of this compound occurs 2−5min after its injection while the effect ofketamine is observed 10−25 min after in-jection.

Die Ergebnisse zeigen, daβ 2-Hydroxy-phencyclidin die Verzögerung beim Tail-Flick-Test im Vergleich zur Kontroll-gruppe erhöhen kann, wobei die maxi-male analgetische Wirkung 2 bis 5 minnach der Verabreichung beobachtetwurde, während der entsprechende Ef-fekt von Ketamin nach 10 bis 25 min auf-trat.

Arzneim.-Forsch./Drug Res. 55, No. 3, 172−176 (2005) ECV · Editio Cantor Verlag, Aulendorf (Germany)172 Ahmadi et al. − 2-Hydroxyphencyclidine

Analgesics · Anti-inflammatories · Antiphlogistics · Antirheumatic Drugs

1. IntroductionIt is well known that phencyclidine (1-(1-phenyl-cyclo-hexyl)piperidine, CAS 956-90-1, PCP), its derivativesand analogues have biological properties and affect thecentral nervous system. Changes in the phencyclidinemolecule can bring about changes in its properties.

PCP appears to represent a unique class of drugswhose spectrum of action is readily distinguishablefrom that of other classes of psychoactive drugs, includ-ing narcotics, LSD-like hallucinogens, amphetamine-like stimulants and cholinergic agonists. PCP binds tothe N-methyl-D-asparate (NMDA) receptor complexand blocks NMDA-mediated gating of the calciumchannel conductance [1]. The most powerful approachin characterizing PCP’s analgesic effect stems from re-cent pharmacodynamic studies that have identifiedspecific binding sites in the brain. PCP and similar com-pounds are classified as non-competitive, “open chan-nel blockers” of the NMDA receptor [2, 3]. PCP and ana-logues have many behavioral effects in common withother phencyclidine-like drugs, including anaesthetic,antinociceptive, psychomimetic, anticonvulsant, neur-oprotective and amnesic drugs [4]. PCP is a semi-rigidmolecule containing a cyclohexane ring with attachedaromatic and piperidine rings (see structure formulas).

Since a hydroxy group has been added to position 2of the cyclohexane ring of PCP (2-OH-PCP), this com-pound is more hydrophilic than PCP and has previouslybeen synthesized with low yield [5].

The analgesic effect of ketamine (2-O-chlorophenyl-2-methylaminocyclohexan, CAS 1867-66-9; see struc-ture formula), another PCP analogue, was first de-scribed by Domino and collaborators in 1965. Ketaminein low sub-anaesthetic doses is reliable as an analgesicin acute pain [7]. In low sub-anaesthetic doses, ket-amine acts more selectively as a non-competitiveblocker of the NMDA receptor. It does so by binding tothe PCP recognition site in the NMDA receptor complex[8]. The analgesic effect of ketamine occurs at concen-trations within its PCP site occupancy range [9], but athigher concentrations, ketamine interacts with opioidreceptors, sigma sites, kapa and delta receptors [10].

2. Material and methods2.1. General

Cyclohexanone, 1,5-dibromopentane, sodium azide, LiAlH4

and all other chemicals were purchased from Merck (Darm-stadt, Germany). Ketamine was purchased from the Sigma-Ald-rich chemical company (Poole, Dorset, England). Meltingpoints (uncorrected) were determined using a Gallencamp ap-paratus (CAT.No.29/MF-370; Watford Herts WD1852, England)with a capillary tube. 1H- and 13C-NMR spectra were recordedon a Bruker 80 MHz, Ac-80 spectrometer (Spectrospin, Fael-landen, Switzerland) (internal reference TMS). IR spectra wererecorded on a Shimadzu FT-IR 4800 spectrophotometer (Kyoto,

NC6H5

1

PCP

NC6H5

OH

2

2-OH-PCP

3

C6H4Cl NHCH3O

Ketamine

Japan). Mass spectra were recorded on a Shimadzu QP-1000 EXspectrometer. Adult male NMRI rates, weighing about 250−300g, were used for the pharmacological tests.

2.2. Synthesis of compounds

2.2.1. 1-Phenylcyclohexanol 4

This compound was prepared in 62 % yield from phenyl mag-nesium bromide and cyclohexanone according to the knownprocedure [11]. The product was recrystallized from ether-pet-roleum benzene (1:1) (m. p. 63−63.5 °C).

2.2.2. 1-Phenylcyclohexene 5

This compound was prepared from 1-phenylcyclohexanol anda mixture of sulfuric and glacial acetic acid according to theknown procedure [12] (b. p. 110−111 °C, 5 mmHg).

2.2.3. cis-1-Phenylcyclohexane-1,2-diol 6

This compound was prepared from 1-phenylcyclohexen andKMnO4 at −5 °C according to the known procedure [13]. Theproduct was recrystallized from ethanol-petroleum benzene(1:1) (m. p. 94−96.5 °C).

2.2.4. cis-2-Azido-2-phenylcyclohexanol 7

This compound was prepared from cis-1-phenylcyclohexane-1,2-diol and sodium azide and a solution of perchloric acid70 % at 5 °C for 5 days [14] and was recrystalized from ether-petroleum benzene (6:1) (m. p. 61−63 °C).

2.2.5. cis-2-Amino-2-phenylcyclohexanol 8

This compound was prepared by refluxing cis-2-azido-2-phenylhexanol and LiAlH4 in dry ether for 48 h [15] (m. p.103−104 °C).

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

A solution containing 0.15 g (0.78 mmol) of amine with 0.2 ml(2.4 mmol) 1,5-dibromopentane in 5 ml of dry acetone wasrefluxed for 2 days. The reaction mixture was cooled, then0.18 g (1.3 mmol) of K2CO3 was added and refluxed for 5 days.The solvent was removed and ether was added. The residuewas treated with 5 % solution of HCl. The aqueous layer wasseparated and extracted with ether and neutralized with 10 %NaOH, extracted with ether, dried over Na2SO4, and 170 mg(83.7 %) of product was obtained (m. p. 135 °C ).

IR (KBr): 3420, 3050, 2970, 2920, 1600, 1496, 1442, 1159,1076, 987, 704 cm-1 1H-NMR (CDCl3) (ppm): 1.7-2 (19H, m),4.4−4.7 (1H, m), 7.1−7.4 (5H, m).

13C-NMR (CDCl3) (ppm): 21.08, 21.40, 25.17, 26.54, 27.67,47.14, 69.14, 75.42, 77.03, 78.62, 126.41 127.63, 127.93.

MS: m/e (regulatory intensity): 41 (37), 55 (14), 77 (18), 84(41), 91 (27), 103 (18), 104 (16), 117 (20), 174 (10), 186 (52), 200(100), 258 (34), 259 (23).

Arzneim.-Forsch./Drug Res. 55, No. 3, 172−176 (2005) ECV · Editio Cantor Verlag, Aulendorf (Germany) Ahmadi et al. − 2-Hydroxyphencyclidine 173

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Oa OH

C6H5

C6H5

b cOH

OH

C6H5

d

OH

C6H5

N3e

OH

NH2

C6H5

f

OH

N

654

2 8 7C6H5

Scheme 1: Synthesis of 2-OH-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.

2.3. Pharmacological methods

Adult male NMRI rats, bred at the Institute of Biochemistry andBiophysics at Tehran University, were maintained on a 12-hlight-dark cycle in a temperature-controlled room (25 ± 1 °C)at 50 % relative humidity. They were allowed free access to astandard laboratory rat chow (Pars Company, Tehran, Iran) andtap water ad libitum. The animals (n = 35, total) (250−300 gbody weight) were assigned randomly to various groups. Theywere brought into the experimental room for acclimatization24 h before the experiment. All experiments were performedbetween 8 h: 00 min and 16 h: 00 min under normal room lightand at a temperature of 25 °C.

All animals were injected by one investigator and evaluatedby another one.

2.3.1. Tail-flick test (TF test)

In this test a commercially available analgesia meter (Apelex,model DS 20, Socrel, Bagneux, France) was used. It is con-sidered as a modification of the method of D’Amour andSmith [16].

Various groups of rats (n = 5 animals/group) were used forthe test. The whole body of each rat except the tail was coveredwith a piece of thick cloth. A beam of light at a temperature of130 °C was focused onto the last 1−2 cm of the tail.

The latency time (in s) at which the animal withdrew its tailwas noted before treatment and 2, 5, 10, 15, 20, 25 and 30 minafter administration of the drug. A cut-off time of 10 s was usedfor each light exposure to avoid damage of the tail. The firsttail withdrawal latency in s was used as a parameter of painsensitivity. The light source was adjusted to give an averagebaseline latency of about 3−4 s in untreated rats. Two trails ofthe tail flick test were run at 5-min intervals.

2.3.2. Effect of 2-hydroxyphencyclidine and ketaminehydrochloride on analgesic activity

2-Hydroxyphencyclidine (2-OH-PCP) was dissolved in di-methylsulfoxide (DMSO) and was given 15 min before the ac-tual testing. In other groups, animals were given with saline(vehicle) and ketamine in saline. The difference in the tail-flicklatencies scores were evaluated using analysis of variance(ANOVA). The p < 0.05 level was considered to represent signifi-cant difference. 2-OH-PCP was injected in two doses (3 and 6mg/kg i. p.) and the tail-flick latency was determined 2, 5, 10,15, 20, 25 and 30 min after injection. For the comparison, an-other analogue of PCP, ketamine hydrochloride (2-O-chloro-phenyl-2-methylaminocyclohexane) was injected (0.5, 1, 6 mg/kg i. p.).

3. Results3.1. Chemistry

2-OH-PCP was synthesized as outlined in Scheme 1.This compound has been synthesized previously, but inthis study, we synthesized it using another method. Thepresent method includes two more steps than the previ-ous method but its yield is higher than that of the previ-ous one. We employed the known procedure for thesyntheses of compounds 4 to 8 with the appropriatemodifications described previously.

Spectroscopic data (IR, 1H- and 13C-NMR) confirmedthe structures of all reported compounds; the meltingpoints of known compounds could also confirm theidentity of them. The purity of each compound waschecked by TLC [dioxin:toluene:ethanol:concentratedammonia, 50:40:5:5 (by volumes) as the mobile phase].

3.2. Pharmacology

3.2.1. The analgesic activity of 2-OH-PCP andketamine hydrochloride

Intraperitoneal injections of ketamine (0.5, 1, 6 mg/kg)and 2-OH-PCP (3 and 6 mg/kg) produced analgesic ef-fects in the tail-flick test. The experiments showed thattwo doses of 2-OH-PCP (3 and 6 mg/kg) increased thetail-flick latencies [F (2,23) = 077, p = 0.0083] comparedto the control group receiving DMSO, and the max-imum analgesic effect was observed 2−5 min after itsinjection (p < 0.01, Fig. 1). Ketamine produced analgesiain the tail-flick test [F(3,31) = 18.93, p < 0.0001] in allinjected doses (Fig. 2), and the maximum effect was ob-served 10−15 min after its injection. Meanwhile, the an-aesthetic effect was produced by injection of moredoses of 2-OH-PCP in rats.

4. DiscussionElectrophysiologic and binding studies revealed thatvarious antagonists of NMDA receptors, includingphencyclidine, ketamine and dizocilpine (MK-801),bind to the PCP-site mainly when the channels are inthe open or activated state [16, 17]. Previous studiessuggest that ketamine may interact with the NMDA re-

Arzneim.-Forsch./Drug Res. 55, No. 3, 172−176 (2005) ECV · Editio Cantor Verlag, Aulendorf (Germany)174 Ahmadi et al. − 2-Hydroxyphencyclidine

Analgesics · Anti-inflammatories · Antiphlogistics · Antirheumatic Drugs

**

***

3

4

5

6

7

8

2 5 10 15 20 25 30

Time (min)

Tai

l-fli

ck la

tenc

y

DMSO

2-OH-PCP(3 mg/kg)

2-OH-PCP(6 mg/kg)

Fig. 1: Mean tail-flick latencies in animals receiving 2-OH-PCP i.p. The tail-flick test was conducted 2, 5, 10, 15, 20, 25 and 30min after injection. Each point represents the mean ± S.E.M. of 5animals. ** p < 0.01, *** p < 0.001; significantly different from theDMSO group.

**

*

**** ***

* *****

*** ***

3

4

5

6

7

8

2 5 10 15 20 25 30

Time (min)

Tai

l-fli

ck la

tenc

y

Saline

Ketamine(6 mg/kg)

Ketamine(1 mg/kg)

Ketamine(0.5 mg/kg)

Fig. 2: The average latencies of heat-induced tail-flick responses2, 5, 10, 15, 20, 25 and 30 min after the i. p. administration ofsaline and ketamine (0.5, 1, 6 mg/kg). Each point represents themean ± S.E.M. of 5 animals. * p < 0.05, ** p < 0.01, *** p < 0.001;significantly different from the saline group.

ceptor at two potentially distinct sites: one site locatedwithin the channel pore and a second site associatedwith the hydrophobic domain of the protein. The bind-ing of the agonist to the receptor is assumed to modifythe binding of ketamine to both sites. Ketamine is for-mulated as a hydrochloride salt and highly water-sol-uble [18], but under physiological conditions, a largefraction of the drug exists in the lipid-soluble form.

Therefore, the concentration of ketamine in the lipidphase is several orders of magnitude greater than in theaqueous phase. Ketamine gained access to a blockersite associated with the lipid membrane of the lipidprotein interface [19]. On the other hand, the predom-inance of closed-channel blockade suggests that ketam-ine’s analgesic properties might result from closed-channel rather than open-channel blockade. But 2-OH-PCP has a hydrophilic structure, therefore, the concen-tration of this drug in the aqueous phase is greater thanin the lipid phase, and the analgesic properties of thisdrug might result from open-channel block impeding

ionic flow. This may explain why 2-OH-PCP has a rapidanalgesic effect (2−5 min after injection) on acute andphasic pain. The acute analgesic effect of ketamine isclosely associated with sensory and locomotor side ef-fects. These motor side effects are increased dependingon the dose, limiting the use of ketamine in chronicpain [20]. At the doses used in this study, 2-OH-PCP didnot show motor side effect.

In conclusion, the present study demonstrates thatthe test compound, synthesized by a different methodin comparison with previous methods [5] with higheryield and purity, attenuates acute pain in the tail-flicktest and may possibly exert its antinociceptive effectsthrough NMDA receptor blockade.

5. References[1] Honey, C. R., Miljkovic, Z., McDonald, J. F., Ketamine andphencyclidine cause a voltage-dependent block of responses toL-aspartic acid. Neurosci. Lett. 61, 135 (1985)

[2] Kemp, J. A., Foster, A. C., Wong, E. H. F., Non-competitiveantagonists of excitatory amino acid receptors. Trends Neuro-sci. 10, 294 (1987)

[3] Anis, N. A., Berry, S. C., Burtan, M. et al., The dissociativeanesthetics ketamine and phencyclidine, selectively reduce ex-citation of control mammalian neurons by N-methyl-asparate.J. Pharmacol. 79, 565 (1983)

[4] Shimoyama, N., Shimoyama, M., Inturrisi, C. E., et al.,Ketamine attenuates reverses morphine tolerance in rodents.Anesthesiology 85, 1357 (1996)

[5] Kmenka, J. M., Michaud, M., Genest, P., Recherche dedifferences conformation nelles of biochimiques entre phency-clidine et ketamine. Eur. J. Med. Chem. Chim. Ther. 20, 419(1985)

[6] Kelly, K., The Little Book of Ketamine, p. 96. Ronin Pub-lishing, Portland, OR, USA (1999)

[7] Stubhaug, A., Breivik, H., Long-term treatment of chronicneuropathic pain with the NMDA (N-methyl-D-asparate) re-ceptor antagonist ketamine. Acta Anaesthesiol. Scand. 41, 329(1997)

[8] Oye, I., Paulsen, O., Maurset, A., Effects of ketamine onsensory perception: evidence for a role of N-methyl-D-asparatereceptors. J. Pharmacol. Exp. Ther. 260, 1209 (1992)

[9] Rabben, T., Skjelbred, P., Oye, I., Prolonged analgesic ef-fect of ketamine, an N-methyl-D-asparate receptor inhibitor, inpatients with chronic pain. J. Pharmacol. Exp. Ther. 289, 1060(1999)

[10] Meller, S. T., Ketamine relief from chronic pain throughactions at the NMDA receptors. Pain 68, 435 (1996)

[11] Treppmann, A., Zur Kenntnis Ungesättigter hydroarom-atischer Kohlenwasserstoffe. Chem. Ber. 48, 1216 (1915)

[12] Karabinos, P., The dehydration of cis-and trans-2-phenylcyclohexanols. J. Am. Chem. Soc. 62, 1160 (1940)

[13] Mangoni, L., Adinolfi, M., Barone, G. et al., A conveni-ent procedure for the cis-hydroxylation of olefins. TetrahedronLett. 45, 4485 (1973)

[14] Yoshito, T., Hidekazu, I., Tsuchiya, Y., Homochiral li-gands derived from cis-1-phenylcyclohexane-1,2-diol and cis-2-azido-2-phenylcyclohexanol. Tetrahedron Asymmetry 22,3735 (1997)

Arzneim.-Forsch./Drug Res. 55, No. 3, 172−176 (2005) ECV · Editio Cantor Verlag, Aulendorf (Germany) Ahmadi et al. − 2-Hydroxyphencyclidine 175

Analgetika · Antiphlogistika · Antirheumatika · Entzündungshemmer

[15] Hedayatullah, M., Guy, A., Synthese et reduction d’azi-dosulfates d’aryle. Tetrahedron Lett. 29, 2455 (1975)

[16] Al-Deeb, O. A., Synthesis and analgesic activity of newphencyclidine derivatives. Arzneim-Forsch./Drug Res. 44 (II),1141 (1994)

[17] Parsons, C. G., Gibbens, H., Magnago, T. S. L. et al., Atwhich ’sigma’ site are the spinal actions of ketamine mediated?Neurosci. Lett. 85, 322 (1988)

[18] Qian, J., Brown, S. D., Carlton, S. M., Systemic ketamineattenuates nociceptive behaviors in a rat model of peripheralneuropathy. Brain Res. 715, 51 (1996)

[19] Shimoyama, M., Shimoyama, N., Gorman, A. L. et al.,Oral ketamine is antinociceptive in the rat formalin test: roleof the metabolite, norketamine. Pain 81, 85 (1999)

[20] Farance, C. P., Snyder, A. M., Woods, J. H., Analgesiceffects of phencyclidine-like drugs in rhesus monkeys. J. Phar-macol. Exp. Ther. 250, 197 (1989)

Acknowledgements

This work was done as M. S. project at Tehran University, De-partment of Science and I.B.B. Center. The author would liketo thank Mrs. T. Yosefifard for her assistance with the pharma-cological tests.

Correspondence:Dr. Abbas Ahmadi, Department of Science,School of Chemistry, Islamic Azad University,P. O. Box 31485-313, Karaj (Iran)E-mail: abbas−ahmady−3957@yahoo.com

Arzneim.-Forsch./Drug Res. 55, No. 3, 172−176 (2005) ECV · Editio Cantor Verlag, Aulendorf (Germany)176 Ahmadi et al. − 2-Hydroxyphencyclidine