medicinal - ACS Publications - American Chemical Society

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JOURNAL OF

MEDICINAL CHEMISTRY Q Copyright 1993 by the American Chemical Society

Volume 36, Number 20

October 1, 1993

Articles Structure-Activity Relationships of trans-3,4-Dimethyl-4-(3-hydroxyphenyl)piperidineAntagonists for F - and K-Opioid Receptors Dennis M. Zimmerman,. J. David Leander, Buddy E. Cantrell, Jon K. Reel, John Snoddy, Laurane G. Mendelsohn, Bryan G. Johnson, and Charles H. Mitch' CNS Division, Lilly Research Laboratories, Indianapolis, Indiana 46285 Received February 18, 1993

A series of racemic N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines were evaluated for opioid agonist and antagonist activity at p and K receptors. Several highly potent p and K antagonists were discovered; however, no compounds with high selectivity for either the c or K receptor were identified. Importantly, no derivative was found to have significant opioid agonist activity. Two derivatives were resolved, and the activities of the enantiomers were investigated. Only a limited stereochemical effect on opioid receptor selectivities was observed. The structureactivity relationships described establish the existence of an important lipophilic binding site distal to the nitrogen for both p and K receptors and confirm the pure opioid antagonist pharmacophore nature of the trans-3,4-dimethyl-4-(3-hydroxypheny1)piperidine structure. Previously we described the discovery of opioid antagonist activity in a series of trans-3,4-dimethyl-4-(3hydroxyphenyl)piperidinesl$ (1, Figure 1). These 4-phenylpiperidine antagonists were structurally unique, since prior to their discovery, opioid antagonists were generally N-allyl or N-methylcyclopropyl analogs of morphine and morphine-like agonists derived from the morphine structure (e.g., in 4,5-epoxymorphinan-6-one,3morphinan,4 benzomorphan: and isoquinoline seriess). Unlike these polycyclic structures,the antagonist activity in the trans3,4-dimethyl-4-(3-hydroxyphenyl)piperidines was shown to be a consequence of substitution at the 3 position of the piperidine ring rather than substitution at the nitrogen. Compound 2 (the N-methyl derivative, Figure l), has antagonist potency comparableto that of nalorphine (the N-allyl analog of morphine), but is without opioid agonist effects. In contrast, the des-3-methyl analog 10 is a morphine-like agonist.'^^ Antagonist potency in the trans3,4-dimethyl-4-phenylpiperidines is not altered by N-allyl or N-methylcyclopropyl substitution (e.g., 3 and 4) but is increased in a stepwise manner from N-methyl to 8-phenethyl (51, 2-(phenylcarbony1)ethyl (6), and 3-hydroxy-3-

phenylpropyl (7, LY117413). Resolution of the 2-(phenylcarbony1)ethyl (6) provided only partial separation of activity as both are opiate antagonists with the (+)-3R,4Risomer 8 (Figure 2) being 2-6 times more potent than the (-)-3S,4S-isomer 9. As with 2, compounds 3-7, including both enantiomers of 6, have no measurableopioid agonist properties. Prior to the discovery of compounds 2-7, only two other pharmacologically pure opioid antagonists (antagonists devoid of any opioid agonists effects) had been well characterized, naloxone and naltrexone.3 Even today, there are still relatively few pure opioid antagonists known, and the structural requirements for such activity appear to be quite precise.8a In accordance with this, minor alterations of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine structure often impart opioid agonist activity to the molecule. The cis-3,4-dimethylanalog (11, Figure 1) has mixed agonist-antagonist properties. Substitution of n-propyl for the 4-methyl group of 1 and 11 increases opioid agonist activity and led to the discovery of the opioid analgesic, picenadol, which has mixed opioid agonist and antagonist properties.2Jo

0022-262319311836-2833$04.00/0 0 1993 American Chemical Society

2834 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 20

E' cn,

2

CH&HZPh

5

R

CH&HzcOPh

6 '

1

CHzCHzCHOHPh

Z

I

nou no

Zimmerman et al.

(LY117413)

no

LNI J

LNI J

k

&

ip II BWladQl Figure 1. Compounds 2-7, 11, and picenadol are racemic mixtures. Compound 7 is also a diasteriomeric mixture.

Since the originaldisclosure of opioid antagonist activity in the trans-3,4-dimethyl-4(3-hydroxyphenyl)piperidines, the understanding of opioid receptor mediated pharmacology has advanced considerably. Three opioid receptors have been characterized ( p , K , and 61,endogenous ligands for these receptors have been identified and the existence of opioid receptor subtypes has been postulated.11-17 These findings have led to the realization that opioid receptors are involved in a multitude of physiologicalprocesses and to suggestions of several new therapeutic uses for opioid receptor antagonists.18 These findings have also demonstrated the need for selectiveopioid antagonists to further study this complex receptor system. The 4-phenylpiperidine antagonists were originally identifiedas preceptor antagonists;however,K-antagonist effects were later identified within this series.le Compounds 2 and 5-7 were also shown to have significant affinity for the S receptor.20 It was further determined that the (+) and (-)-isomers of 6 had similar relative activity for p, K , and 6 receptors ( p > K = 6 compounds 8 and 9, Table I). In this manuscript, we report the further characterization of the structureactivity relationships (SAR) within this opioid antagonist series. Substituents bound to nitrogen were varied systematically, and the effects at p and K receptors, both in vitro and in vivo, were evaluated. Because of ease of synthesis and the limited stereochemicaleffect observed with the enantiomers of 6, SAR comparisonswere made on racemic mixtures and in a few cases on diasterameric mixtures. The intent was to further document the pure opioid antagonist nature of trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines and rapidly characterize effects which maximize activities for p and K receptors. Selected compoundswith high potency for either p and/or K receptors were to be studied further, including separation and evaluation of individual stereoisomers and evaluation for &receptor activitiesa21 Chemistry The synthesis of the 4-(&methoxyphenyl)piperidine16 has been recently described and is outlined in Scheme 1.22 The tetrahydropyridine 12,23was converted to the metalloenamine 13 and then alkylated regioselectively with methyl iodide giving 14. Metalloenaminessuch as 13have proven to be highly versatile intermediates in the synthesis

CH.

1 . C H 2 C H z ~

Figure 2. All compounds,unless otherwiseindicated, are racemic mixtures. Compounds 7, 48, and 49 are also diasteriomeric mixtures.

of opioid related str~ctures.6~2'~~ Treatment of 14 with dimethylamine and formaldehyde gave 16 in high yield28 which was reduced with high stereoselectivityto the trans3,4-dimethylarylpiperidine16. N-Demethylation with

Antagonists for p- a n d K-Opioid Receptors

J o u r n a l of Medicinal Chemistry, 1993, Vol. 36,No.20 2836

Scheme I"

Scheme I1

cn,o

cn,o

cn,o

b

a

I

CH,

I2

-

-

-

C

I

a, l.3

19

cH'o'o

cH1#462 I

cn,

\Ho

Scheme IIP Reagents: (a) n-BuLi, T H F (b) CHsI; (c) (CH&NH, CH20, (d) Hz, 5% Pt/C, EtOH; (e) C l C O C H 4 H 2 , proton sponge; (f) HC1, EtOH; (g) 48% HBr, HOAc; (h) RCOCl, Red-Al or LiAlH4; (i) R-X, NaCOs. a

vinyl chloroformatemfollowed by 0-demethylation with HBr in acetic acid afforded 18,while 0-demethylation of 16 gave 2. The trans configuration of the 3,4-dimethyl groupswas proven by analysis of lH and 13CNMR spectra and this assignment was confirmed by X-ray crystallography.30 Alkylation or acylation of 18 followed by reduction of the intermediate amide provided the N-substituted derivatives3-5,19-33,35-48, and 53 (SchemeI and Figure 2). Alkylation of 18 with a l-aryl-3-(dimethylamino)-lpropanone methiodide afforded 6 and 51 (Scheme11)which were subsequently reduced to give the 1-aryl-3-propanols 7 and 52. Alkylation of 18 with styrene oxide and 2,3epoxy-1-phenylpropanegave 49 and 50 while reaction with 4-vinylpiperidine gave 34 (Scheme I1 and Figure 2). The N-furanylmethyl derivatives 54 and 55 were synthesized through reduction of the intermediate Schiff bases derived from 17 and 2 or 3-furaldehyde and subsequent O-demethylation (Scheme I11 and Figure 2). The (+)-(38,48)- and (-)-(3S,4S)-isomers of 3,4-dimethyl-4-phenylpiperidine16 were resolved by fractional crystallizatiooof the dibenzoyl tartrate salts as previously describedaZ1Their absolute configurations have been established by synthesis using Sharpless asymmetric expoxidation.21*s0Compounds 8, 9, 42, and 43 were synthesized from 18 as described for 6 and 41. Biological Assays Affinity for the p-opioid receptor was determined by assaying a compound's ability to displace PHInaloxone

1L

W

CH, *R

a,.R 54 and 55

a

Reagents (a) 5% Pd/C; (b) n-PrSH, Kt-BuO.

( [3H]NAL)binding from rat brain homogenates. Affinity for the K receptor was determined by the ability of a compound to displace [3Hlethylketocyclazocine(PHIEKC) from guinea pig cortical tissue. In the K-receptor binding assay, fentanyl and D-Ala2-D-Leu6-enkephalin (DADL) were added to inhibit binding of PHIEKC to p and 6 receptors. Affinity for the b receptor was determined using PHIDADL with rat brain homogenates.sl Opioid antagonist activity was determined using the mouse writhing analgesic assay, measuring the test compound's ability to block morphine-induced (preceptormediated) and U50,488-induced (K-receptor-mediated) analgesia.

Rssults The results of this structure-activity relationship study are shown in Tables I and 11. In accord with previous findings, compounds 2-7 antagonize morphine-induced analgesia, and the antagonist A D m values obtained using the mouse writhing test correlate well to those obtained by other measures.l Comparison of the values in Tables I and I1show that there appears to be a correlation between the ability of compounds 2-7 to block morphine-induced analgesia and affinity for the p receptor. Compounds 2-7

2896 Journal of Medicinal Chemistry, 1993, Vol. 36, No.20 Table I. Affinities of the 4-Phenylpiperidine Antagonista for the p- and rr-Opioid Receptors % displacement [8HlNALa binding PHI EKCb binding assay (cc receptor): assay (K receptor) compd Ki(nM)d 1 0 n M 100nM Ki(nM)d 1 0 n M 100nM 2 80 833 313 3 16 21 36 4 1.5 52 6 208 5.4 6 12.5 7 0.96 163 8' 3.6 12 318 gr 18 52 19 44 78 4.3 20 9.6 0.29 21 57 87 0.62 22 57 82 65 89 23 15 0.69 24 22 70 2.0 26 55 83 3.5 26 54 82 0.30 27 90 100 7.5 28 35 82 83 100 29 61 13 95 52 30 26 58 50 90 31 60 93 3.2 32 72 34 5.3 33 20 51 54 99 34 22 77 1.1 35 53 80 0.95 36 8.9 0.62 37 73 2.8 49 38 63 19 89 100 39 10 0.26 40 6.1 0.56 41 3.3 0.20 42 1.8 13 43 1.4 14 44 41 78 63 100 45 72 20 23 46 69 1.3 57 47 61 1.0 48 34 70 49 48 97 42 77 88 100 60 15 1.5 61 11.7 0.50 62 26 60 9.3 29 53 82 100 63 90 54 71 5.8 55 66 3.7 naloxone naltrexone 0.56 3.9 ~~

a Naloxone.

Ethylketocyclazocine. Percent stereospecific displacement of either [SHINAL or [SHIEKC run in triplicata a t the concentration indicated. Kb were derived from six different concentrations each run in triplicate. Ki values (nM) for [SHI-D-AWD-bus-enkephalin (6 receptor) displacement for 8 and 9 are 93 and 246, respectively. @

also have affinity for the K receptor and are antagonists of K-induced analgesia and diuresis. These compounds exhibited varying degrees of selectivity for p vs K receptors, but in general, the effects of substitution at the nitrogen on potency are similar for both p and K receptors. Of these compounds, 7 (LY1174131, is the most potent p- and K-receptor antagonist. Its p- and K-receptor in uiuo antagonist activities are equivalent to those of naloxone. These early SAR findings supported the existence of an important lipophilic binding region for both p and K receptors in the proximity of the nitrogen binding site. Interaction with this region could be achieved with appropriate N-substitution and this accounts for the increase in antagonist potencies with compounds 2-7. In

Zimmerman et al. Table 11. Opioid Antagonist Effeds of the 4-Phenylpiperidine Antagon ista antagonism of opioid analgesia mouse writhing W h (mg/kg, ac)la receptor (morphine) 0.74 1.3 1.3 0.16 0.14 p

compd 2 3 4 5 6 7 8 9 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 36 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 naloxone naltrexone

0.050

receptor (U50,488)

K

>5.0

>10 6.1d 1.4 4.5 0.92

>1.25 0.37 0.052 0.21' 0.29 2.4C 0.28 0.31 0.44 0.19 0.27 >1.25

>1.25 0.60 0.11 >OW 0.45 5.3' >1.25C 0.51 0.38 0.33 0.95 >1.25

0.14 0.25 >1.25 0.08 0.18

0.84 0.25 >1.25 0.51 >0.64 0.09 >0.32 >1.25 0.24 0.30 0.25 0.65 0.26 0.91 0.43 >4oe >1.25C 11.8' 0.85

0.05 >1.25 0.76 0.12 0.22 0.03 0.24 0.08 0.25 0.16 0.14' >1.25' 2.4c 0.27 0.07

0.80 0.40 0.71 0.08

0.05

0.14 0.75 0.71 1.35 1.1 0.06

antagonism of K

diuresis:

ADSO(rnalka.

>10 1.8 3.2 2.5

0.69 0.70 3.3 2.5 1.1 4.1 3.4 3.6

0.60 5.7 11 NA 1.4 3.8 14 >5.0

2.2 1.4 10 11 1.9 1.0

8.4 3.3 3.0 8.4 6.9 3.7 1.4 3.2 3.8 3.5 2.5

0 Dose required for 50% reduction in the analgesic response to either morphine (1.25 mg/kg, sc) or U50,488(2.5 mg/kg, sc). *Dose required to decrease the 5-h bremazocine-induced (0.08 mg/kg, SC) urination by 50 % c At higher dosee this compound inhibitedwrithii itaelf and the ADw had to be approximated.

.

this SAR study, different strategies were used to take advantage of this effect and increase opioid receptor affiiity. It was hoped that this strategy would also identify the means to achieve selectivity for a particular opioid receptor. The alkylphenyl analogs (5 and 23-25) with one to four carbon atom spacers were synthesizedto define the optimal distance between the nitrogen and a phenyl substituent. Affinity for the the p and K receptors is found to be maximized with the 3-phenylpropyl derivative (24); however, all four alkylphenyl analogs show significant affinity for both receptors and, in uiuo, are p- and K-receptor antagonists (Table 11). Unexpectedly, on the basis of its affinity for p and K receptors, 24 is only a weak antagonist of opioid mediated analgesia. Instead, 24 produces an

Antagonists for p- and K-Opioid Receptors

Journal of Medicinal Chemistry, 1993, Vol. 36, No.20 2837

antinociceptive response in the writhing test (EDm= 15 mg/kg, sc), and it is this effect which appears to mask its abilities to antagonize opioid-induced analgesia. The antinociceptiveactivity of 24 is not blocked by a high dose of the opioid antagonist naloxone (10 mg/kg, 8c) and thus appears to be a nonopioid effect. As noted in Table 11, a limited number of other compounds significantly inhibit mouse writhing at relatively high doses, but at doses which appear to affect their ADm values. In all cases these antinociceptive activities were judged to be nonopioid effects. Other compounds with various nitrogen substituents were synthesized to further increase opioid antagonist activities and to characterize structural requirements for maximum binding at p and K receptors. The phenoxyethyl (47)and phenoxypropyl (48)derivatives both have high affinity for the p receptor, but relatively weak affinity for the K receptor. Compound 47 is a potent antagonist of morphine-induced analgesia but has relatively weak nonopioid agonist effects in the writhing test (EDm= 6.8 mg/kg, sc). Substitution of a hydroxyl a to the phenyl of 24,giving 7, has little effect on affinity for either p or K receptors; however, 7 is devoid of the nonopioid agonist effects observed with 24. Several different lipophilic groups in addition to phenyl apparently bind tightly to the hydrophobic site distal to nitrogen including thiophene (33,40,41,51, and 52),furan (44,54,and 55), and n-alkyl (20-22). The 3-(2-furanyl)propyl(44),342- and3-thieny1)propyl (40and 411,and l-hydroxy-l-(2-thienyl)propanol3-yl (52)derivatives are highly potent p- and K-receptor antagonists. With N-alkyl substitution, replacement of methyl (2) with ethyl (19)leads to a loss in affinity for the p receptor while increasing affinity for the K receptor. Increasing the alkyl chain length to 5,6, and 7 carbons markedly increases opioid antagonist activities. The n-hexyl(21) and heptyl (22)derivatives are found to have high affinity for the p receptor. Compound 21 is a highly potent antagonist of p- and K-mediated analgesia and of K-induced diuresis. The n-pentyl derivative 20 is a potent antagonist of K-agonistinduced diuresis. The N-phenylethyl and N-phenylpropylderivatives (5 and 24) were chosen to explore the effects on opioid antagonist activity of substitutions on the distal phenyl ring. In general, methyl or chloro substitution produced relatively minor effects on p- and K-receptor activities. For 5, substitution of methyl in the ortho position (28) enhances affinity at the K receptor. In accord with this, 28 is a potent blocker of K diuresis. p-Methyl substitution of 5 (30) reduces activity at both p and K receptors. Addition of o-methyl to 24 gives compound 37 which has highly potent p - and K-receptor antagonist activities but is devoid of the nonopioid agonist effects found with 24 in the mouse writhing test. The 3-(2-thienyl)propylderivative 41 was resolved and the activities of the enantiomers (42and 43,Tables I and 11) were investigated. As with the 2-propiophenone derivative 6 only a limited stereochemical effect on I( and K activities was observed. The (+)-3R,U-isomer 44 has the highest affinity and antagonist activity for both I( and K receptors. Its potencies are approximately 8-9 (p receptor) and 2-4 ( K receptor) times that of the (-)-3S,4Sisomer.

Discussion Further explorationof the effectsof N-substitution with trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine has led to the discovery of several new, highly potent opioid antagonists. No antagonists with high selectivity for the p or even moderate selectivity for the K receptor were discovered and in general, the consequence of structural changes at the nitrogen is similar for p and K receptors, with all antagonists possessing somewhat higher affinity for the p receptor. The SAR presented here further establishes the existence of an important lipophilic binding site distal to the nitrogen for both p and K receptors to which a variety of different substituents can tightly bind. Our data suggest that this lipophilic pocket is either very large or is a domain which has considerable malleability. These conclusions have been derived principally from evaluation of racemic mixtures. While it is possible that further separations of p and K activities could be achieved through separation of individual stereoisomers, the very limited stereochemical effects observed with the isomers of 6 and 41 suggest that this is unlikely.M Significant opioid agonist activity (analgesia that is naloxone reversible) in the mouse writhing assay was not detected with any of the newly synthesized antagonists. This is particularlynoteworthy because the mouse writhing test is known to be highly sensitive for detecting analgesic activities of opioid partial agonists. The opioid agonist and antagonist effects of a representative series of 4-phenylpiperidines have been further assessed in the isolated MVD. Potent p-, K-, and &receptor antagonist activities were observed; however, no opioid agonist activities were detected with any N-substituted derivatives tested (M. L. Cohen, personal communication)." Consequently, the trans-3,4-dimethyl-4(3-hydroxyphenyl)pipridinestructure appears to be a pure opioid antagonist pharmacophore in that structural changes at nitrogen affect only the molecule's affinity for a particular opioid receptor. This feature differentiates these compounds from other series of opioid antagonists. We have used molecular modeling techniques in an attempt to identify the 3-dimensional structure of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines that binds to opioid receptors. Conformational energy analyses (Macromodel, MM2 force field) indicate that theee 4-phenylpiperidines can exist in either an axial-phenyl or equatorial-phenyl conformation. The energy difference between these conformations is relatively small with the equatorial-phenyl conformer being of lower energy (AG= 2.6 kcal/mol). Others using high-field NMR spectroscopy (IH, l3C) have noted a preference for the 1-methyl-trans3,4-dimethyl-4-(3-hydroxyphenyl)piperidineto exist in an equatorial phenyl conformation.a*w These data and SAR studies exploring the effect of 6-methyl substitution led us to conclude that the opioid antagonist activity is mediated through the equatorial-phenyl conformati~n.~~ We have compared equatorial-phenyl low-energy conformations of the phenylpiperidine antagonists with other opioid antagonists and agonists. These studies have led us to postulate that the 3-hydroxyphenyl substituent of the piperidine antagonists binds to the site occupied by the 3-hydroxyphenyl substituent of rigid, multicyclic opioids such as naloxone98 and WIN44,441.= We have also concluded that the lipophilic substituent distal to the nitrogen in the N-substituted 4-phenylpiperidine antagonists binds in the same region as does the cyclopentyl

2838 Journal of Medicinal Chemistry, 1993, Vol. 36, No.20

Zimmerman et al.

group, extended from the 5 position of the benzomorphan nucleus, of WIN44,441. A more thorough discussion of these 3-dimensional structural comparisons will be the subject of a future publication. The failure to discover highly selective antagonists for either the p or K receptor within this series of racemic trans-3,4-dimethyl-4-arylpiperidineswas disappointing; however, compounds with significant selectivity for the p receptor were identified and the degree of selectivity for p versus K receptors could be altered with N-substitution. This implies that additional SAR work could lead to the discovery of.receptor selective opioid antagonists. Most importantly, we were able to more firmly establish the antagonist pharmacophore nature of the trans-3,4-dimethyl-C(3-hydroxypheny1)piperidine nuc1eus.a Thus, there are now available for the first time a large number of highly potent pure opioid antagonists for pharmacological study.

over silica gel eluting with hexane/ethyl acetate (3:l) containing 0.5% triethylamine. Treatment with HCland trituration in ether gave the HCl salt: mp 155-157 "C. Anal. (CaoHuClNO) C, H,

N. (i)-l-Benzyl-3(R*),4(R*)-dimethyl-4-(3-hydroxyphenyl)piperidine (23) was prepared from 18 and benzoyl chloride. The crude amide was reduced with Red-Al and chromatographed over silica gel eluting with hexane/ethyl acetate (2.51.5). The HC1 salt was prepared and triturated in ether: mp 127-130 OC. Anal. (C&inClNO) C, H, N.

(~)-1-(4-Phenylbutyl)-3(R*),4(~)-dimethyl-4-(3-hydrx-

ypheny1)piperidine (25) was prepared from 18 and 4-phenylbutyryl chloride. The crude amide was reduced with LiAlHd and chromatographed over silica gel eluting with hexane/ethyl acetate (2.5:1.5). Treatment with HCl and trituration in ether gave the HCl salt: mp 159-160 "C. Anal. (CBHs2ClNO) C, H, N. (&)-1-[2-(2-Chlorophenyl)ethy1]-3(R*),4(R*)-dimethyl-4(3-hydroxypheny1)piperidine(26) was prepared from 18 and (2-chloropheny1)acetylchloride. The crude amide was reduced with Red-A1 and chromatographed over silica gel eluting with hexane/ethyl acetate (41). Treatment with HCl and trituration in ether gave the HC1salt: mp 98-104.5 OC, with foaming. Anal. Experimental Section (CalHgC12NO) C, H, N. Melting points were determined for all solids on a Thomas(i)-1-[2-(3-Chlorophenyl)ethyl]-3(R*),4(R*)-dimethyl-4Hoover apparatus and are unconnected. NMR spectra were (3-hydroxypheny1)pipridine(27) was prepared from 18 and recorded on either a Varian T-60, brucker WM-270, or GE QE(3-chloropheny1)acetylchloride. The crude amide was reduced 300 spectrometer and were consistent with assigned structures. with Red-Al and chromatographed with silica gel eluting with Mass spectra and microanalyses were determined by the Struchexane/ethyl acetate (4:l). Treatment with HCl and trituration tural and Organic Chemistry Research Department of the Lilly in ether gave the HC1 salt: mp 179-185 OC. Anal. (C21Hd312Research Laboratories. Mass spectra were consistent with NO) C, H, N. assigned Structures for all compounds. All compounds were (i)-l-[2-(2-Methylphenyl)ethy1]-3(R*),4(R*)-dimethyl-4elementally analyzed within 0.4% of theoretical value unless (3-hydroxypheny1)piperidine(28) was prepared from 18 and othersise indicated. Column chromatography was performed by (2-methylpheny1)acetylchloride. The crude amide was reduced gravitational flow with use of Allied Fisher Silica (70-150 mesh). with Red-A1 and chromatographed over silica gel eluting with General Acylation/Reduction Procedures for Preparing ethyl acetate. The HCl salt was prepared and triturated in ethyl N-Substituted 3(R*),4(R*)-Dimethyl-4-(3-hydroxyphenyl)acetate: mp 127-132 OC with foaming. Anal. (C22HmClNO)C, piperidines (Method A). To a solution of 1.00 g (0.0049 mol) H, N. of 18 and 1.25 g (0.012 mol) of triethylamine in 70 mL of DMF (&)-1-[2-(3-Methylphenyl)ethy1]-3(~),4(R*)-dimethyl-4was added dropwise 0.012 mol of the appropriate acid chloride (3-hydroxypheny1)piperidine(29) was prepared from 18 and at room temperature under nitrogen. After the reaction mixture (3-methylpheny1)acetylchloride. The crude amide was reduced was heated for 2 h at 90 OC, the solution was cooled and poured with Red-Al and chromatographed over silica gel eluting with into 100 mL of water. The desired amide was extracted into hexane/ethyl acetate (1:l). The HCl salt was prepared and ether. The ether layer was washed two times with water, dried triturated in ether: mp 198-200 OC. Anal. (CaH&lNO) C, H, over KzCOs, and concentrated under vacuum. N. Red-A1Reduction. To 6 mL of Red-Al in 20 mL toluene was (&)-1-[2-(4-Methylpheny1)et hyl]-3(R*),4(R*)-dimet hyl-4added dropwise a solution of the crude amide dissolved in (3-hydroxypheny1)piperidine(30) was prepared from 18 and approximately 50 mL of toluene. The reaction was heated to (4methylphenyl)acetyl chloride. The crude amide was reduced 60-70 OC for 2 h and quenched by the addition of 400 mL of a with Red-Al and chromatographed over silica gel eluting with pH 10 buffer. The pH of the mixture was adjusted to approxhexane/ethyl acetate (1:l). The HCl salt was prepared and imately 9.8 with 1 N hydrochloric acid, and the mixture was triturated in ether: mp 185-187 "C. Anal. (CaHmClNO) C, H, extracted with toluene. The organic extracts were combined and N. dried over anhydrous sodium sulfate. The filtrate was concen(&)-1-[2-(3,4-Dichlorophenyl)ethyl]-3(~),4(~)-dimethtrated under vacuum, and the resulting residue was chromatoyl-4-(3-hydroxyphenyl)piperidine(31) was prepared from 18 graphed over silica gel. and (3,4-dichlorophenyl)acetylchloride. The crude amide was LiAlH, Reduction. The crude amide was dissolved into 75 reduced with Red-Al and chromatographed over silica gel eluting mL of anhydrous THF and added dropwise to 0.75 g of LiAlH4 with ethyl acetate. The HCl salt was prepared and triturated in dispersed in 50 mL of anhydrous THF. After a 4-h reflux, the ether: mp 107-114.5 OC. Anal. (C21HaCbNO) C, H, N. reaction mixture was cooled, and the excess LiAlH4 was neu(i)-1-[2-(3-Fluorophenyl)ethyl]-3(R.),4(R*)-dimethyl-4tralized by the careful addition of 10 mL of ethyl acetate with (3-hydroxypheny1)piperidine(32) waa prepared from 18 and ice cooling. Saturated N&Cl solution was then added to (3-fluoropheny1)acetylchloride. The crude amide was reduced precipitate the lithium salts. The solution containing the desired with Red-Al and chromatographed over silica gel eluting with product waa separated, evaporated to dryness, and worked up as ethyl acetate. The HCl salt was recrystallized from ethyl acetate/ above. Overall yields with either reducing agent were generally ethanoVisopropylether: mp 185-186 OC. Anal. (C21HnClFNO) 20-35%. C, H, N. (i)-1-(Cyclopropylmethyl)-3(~),4(R*)-dimethyl-4-(3-hy(~)-1-[2-(2-ThSenyl)ethyl]-3(R*),4(R*)-dimethyl-4-(3-hydroxypheny1)piperidine (4) was prepared from 18 and cyclodroxypheny1)piperidine (33) was prepared from 18 and propane carboxylic acid chloride. The crude amide was reduced 2-thienylacetyl chloride. The crude amide was reduced with with Red-AI and chromatographed over silica gel eluting with a LiAlHd and chromatographed over silica gel eluting with hexane/ solvent gradiant of ethyl acetate to ethyl acetate/methanol (1:l). ethyl acetate (3:l) containing 0.5% triethylamine. Treatment The HCl salt was prepared and recrystallized from isopropyl with HC1 and trituration in ether gave the HC1 salt: mp 98-100 ether/acetone/ethanol: mp 217.5-218.5 OC. Anal. (C1,H&lNO) OC. Anal. (C1oH&lNOS) C, H, N. C, H, N. (i)-l-[3-(2-Methoxyphenyl)propyl]-3(R*),4(AC)-dimeth(i)-l-n-Heptyl-3(~))P(AC)-dimethyl-4-(3-hy~~henyl)-yl-4-(3-hydroxyphenyl)piperidine(36) was prepared from 18 piperidine (22) was prepared from 18 and heptanoyl chloride. and 3-(2-methoxylphenyl)propionylchloride. The crude amide The crude amide was reduced with LiAlH, and chromatographed was reduced with Red-A1 and chromatographed over silica gel

Antagonists for H - and K-Opioid Receptors

Journal of Medicinal Chemistry, 1993, Vol. 36, No, 20 2839

eluting with hexane/ethyl acetate (1:l). The HC1 salt was prepared and triturated in ether: mp 94-97 "C. Anal. (CBHSSClNO2) C, H, N. (f)-1-[3-(2-Methylphenyl)propyl]-3(~),4(~)-dimethyl4-(3-hydroxyphenyl)piperidine(37) was prepared from 18and 3-(2-methylphenyl)propionylchloride. The crude amide was reduced with Red-Al and then chromatographed over silica gel eluting with ethyl acetate. The HC1 salt was prepared and triturated in ether: mp 91-95 "C. Anal. (CzsHs2ClNO) C, H, N. (f)-1-[3-(3-Methy lpheny1)propyl]-3( P ) , 4 (R*)-dim&hy 14-(3-hydroxyphenyl)piperidine(38) wasprepared from 18and 3-(3-methylphenyl)propionylchloride. The crude amide was reduced with Red-Al and then chromatographed over silica gel eluting with hexane/ethyl acetate (1:4). The HC1 salt was prepared and triturated in ether: mp 83-89 "C. Anal. (CuHszClNO) C, H, N. (~)-1-[3-(3-Chlorophenyl)propyl]-3(R*),4(~)-dimethyl4-(3-hydroxyphenyl)piperidine(39) was prepared from 18and 3-(3-~hlorophenyl)propionylchloride. The crude amide was reduced with Red-Al and then chromatographed over silica gel eluting with ethyl acetate. The HC1 salt was prepared and triturated in ether: mp 73-77 OC. Anal. (CaH&l2NO) C, H,

(f)1-(2-Phenylethyl)-3(R*),4(R*)-dimethyl-4-(3-hydroxypheny1)piperidine (5) was prepared from 18and 2-phenylethyl bromide and eluted from a silica gel column with a solventgradient of ethyl acetate to ethyl acetate/methanol (1:l). The HCl salt was prepared and dissolved in ethanol and precipitated with isopropyl ether: mp 125-128 "C. Anal. (C21H&lNO) C, H, N. (*)- 1-Ethyl-3(R*),4(R*)-dimet hyl-4-(3-hydroxyphenyl)piperidine (19) was prepared from 18and ethyl iodide and eluted from a silica gel column with a solvent gradient of ethyl acetate to ethyl acetate/methanol (1:l).The HC1 salt was prepared and triturated in ether: mp 150-152 "C. Anal. (ClJ-IuClNO) C, H, N. (*)-1-n-Pentyl-3(R*),4(R*)-dimethyl-4-(3-hydroxyphenyl)piperidine (20) was prepared from 18 and n-pentyl bromide and eluted from a silica gel column with ethyl acetate. The HCl salt was triturated in ether: mp 83-86 OC. Anal. (C&&lNO) C, H. N. (~)-l-m-Hexyl-3(~),4(~)-dimethyl-4-(3-hydroxyphenyl)piperidine (21) was prepared from 18 and n-hexyl iodide and eluted from a silica gel column with ethyl acetate. The HCl salt was prepared and triturated in ether: mp 140-143 OC with decomposition. Anal. (ClsHs2ClNO) C, H, N. (*)1-(3-Phenylpropyl)-3(R*),4( R*)-dimet hyl-4-(3-hydmxypheny1)piperidine (24) was prepared from 18 and 3-phenylpropyl bromide and eluted from a silica gel column with ethyl acetate. The HC1 salt was prepared and triturated in ether: mp 103-107 "C. Anal. (CaH&lNO) C, H, N. (A)-1-(2-Naphthalen-1-y1)ethyl)-3(R*) ,4(R* )-dimet hy l-4(3-hydroxypheny1)piperidine(35) was prepared from 18 and 2-naphthalen-1-ylethyl bromide. The crude free base was recrystallizedfromethylacetate: mp 201-202 "C. Anal. ( C a w NO) C, H, N. (~)-1-(4-Phenylpentyl)-3(R*),4(R*)-dimethyl-4-(3-hydrxypheny1)piperidine (45) was prepared from 18 and 4-phenylpentyl bromide and eluted from silica with ethyl acetate. The HCl salt was prepared and triturated in ether: mp 187-189 OC. Anal. (CuH&lNO) C, H, N.

N.

(f)-l-[3-(3-Thienyl)propyl]-3(R*),4(R*)-dimethyl-4-(3hydroxypheny1)piperidine (40)was prepared from 18and 343thieny1)propionyl chloride. The crude amide was reduced with Red-Al and chromatographed over silica gel eluting with hexane/ ethyl acetate (2:l). Treatment with HC1and trituration in ether gave the HCl salt: mp decomposed. Anal. (C&&lNOS) C, H, N. (f)-1-[3-(2-Thienyl)propyl]-3(~),4(R*)-dimethyl-4-(3hydroxypheny1)piperidine (41) was prepared from 18and3-(2thieny1)propionyl chloride. The crude amide was reduced with LiAlH, and was chromatographed over silica gel eluding with hexane/ethyl acetate (31) containing 0.5% triethylamine. Treatment with HC1 and trituration in ether gave the HCl salt: mp 101-103 'C. Anal. (C&z,aClNOS) C, H, N. (+)-1-[ 3-(2-Thienyl)propyl]-3(R*),4(R)-dimethyl-4-(3-hy(f)-1-(2-Phenoxyet~1)-3(~),4(R*)-~t~l~-(3-hydmxdroxypheny1)piperidine (42) was prepared from (+)-3(R),4ypheny1)piperidine (47) was prepared from 18 and 2-phenox(R)-18 and as described for 41. Treatment with HC1 and yethyl bromide and eluted from a silica gel column with a solvent trituration in ether gave the HCl salt: mp 110-112 "C; [a]'% = gradient of ethyl acetate to ethyl acetate/methanol (1:l). The +54.9 (c = 1.0, MeOH). Anal. (C&=ClNOS), C, H, N. HC1 salt was prepared, dissolved in ethanol, and precipitated with isopropyl ether: mp 105-106 "C. Anal. (C21H&lN02) C, (-)-1-[3-(2-Thienyl)propyl]-3(9),4(S)-dimethyl-4-(3-hyH, N. droxypheny1)piperidine (43) was prepared from (-)-3(S),4(S)-l8 and as described 42. Treatment with HC1 and trituration (f)-l-(3-Phenoxypropyl)-3(R*),4(~)-dimethyl-4-(3-hyin ether gave the HCl salt: mp 110-112 "C; [ a ] m=~-55.2 (c = droxypheny1)piperidine (48) was prepared from 18 and 1.0, MeOH). Anal. (C&&lNOS), C, H, N. 3-phenoxypropyl bromide and eluted from a silica gel column (f)-1-[3-(2-Furanyl)propyl]-3(R*),4(~)-dimethyl-4-(3- with ethyl acetate. The HC1 salt was prepared and triturated in ether: mp 78-81 "C. Anal. ( C ~ H W C ~ N O C,ZH, ) N. hydroxypheny1)piperidine (44) was prepared from 18and 342furany1)propionylchloride. The crude amide was reduced with (f)-l-(Tetrahydro-2(R,~-furanylmethyl]-3(R*),4(R*)Red-A1and chromatographed over silica gel eluting with hexane/ dimethyl-4-(3-hydroxyphenyl)piperidine(53) was prepared ethyl acetate (1:l). The HC1 salt was prepared and triturated from 18and tetrahydrofuranyl bromide and eluted from a silica in ether: mp 78-81 "C. Anal. (CmH&lNOz) C, H, N. gel column with hexane/ethyl acetate (1:7). The HCl salt was (~)-1-[4-(2-Thienyl)butyl]-3(~),4(R*)-dimethyl-4-(3-hy- prepared and triturated in ethanol/isopropyl ether: mp 177-179 "C. Anal. (C&9ClN02)C, H, N. droxypheny1)piperidine (46) was prepared from 18 and 442thieny1)butylchloride. The crude amide was reduced with LiAW (*)-l-( l-Phenyl-l-oxoprop-3-yl)-3(R*),4(R*)-dimethyl-4and chromatographed over silica gel eluting with hexane/ethyl (3-hydroxypheny1)piperidine(6). A mixture of 3.70 g (0.018 acetate (3:l) containing 0.5 % triethylamine. Treatment with mol) of 18,3.82 g (0.036 mol) of NaZCOs, and 6.41 g (0.020 mol) HC1 and trituration in ether gave the HC1 salt: mp 148-150 "C. methiodide in 37 of l-phenyl-3-(dimethylamino)-l-propanone Anal. (CzlH&lNOS) C, H, N. mL DMF was stirred at room temperature for 4 h with nitrogen continuously bubbling through. The mixture was poured into General Alkylation Procedure for Preparing N-Subetituted 3(R*),4(R*)-Dimethyl-4-(3-hydm~phenyl)pi~n~n~ 200 mL of water and extracted with ether. The ether layer was washed, dried over KlCOs, and concentrated under vacuum. The (Method B). Compound 18 (500 mg) was refluxed for 1h in 35 maleic acid salt was prepared and recrystallized from ethyl acetate mL DMF',with 1.1equiv of the appropriate alkyl halide and 1.1 to give 6.8 g (86%): mp 70-71 "C. Anal. (CaeHslNO6)C, H, N. equiv of NaHCOa. The mixture was cooled and poured into 100 mL of water. The pH was adjusted to 9.8, and the product was (+)-1-( l-Phenyl-l-oxoprop-3-yl)-3(R),4(R)-dimethyl-4-(9extracted into ether. The ether layer was washed, dried over hydroxypheny1)piperidine (8). A mixture of 2.540 (0.012 mol) K2CO8, and concentrated under vacuum. The producta were of (+)-3(R),4(R)-l8, 2.63 g (0.025) of NasCOs, and 4.35 g (0.014 purified using silica gel column chromatography. The HC1salta methiodide in mol) of l-phenyl-3-(dimethylamino)-l-propanone were prepared and further purified by recrystallization or 45 mL of DMF was stirred at room temperature for 4 h with trituration. Yields of purified products were generally 35-80%. nitrogen continuously bubbling through. The ether layer was washed, dried over KaCOa, and concentrated under vacuum. The (A)-1-Al y l-3(R*),4(RI )-dimethy l-4- (3-hydroxypheny1)piHCl salt was prepared and recrystallized from 2-ieopropylalcohoV peridine (3) was prepared from 18 and allyl bromide. The HCI isopropyl ether: mp 184-186"c; [aIasD = +48.7 (c = 1.0, MeOH). salt was recrystallized from ethanol/isopropyl ether: mp 200.5Anal. (CaH&lNO2) C, H, N. 203 "C. Anal. (Cl&&lNO), C, H, N.

2840 Journal of Medicinal Chemistry, 1993, Vol. 36,No. 20

Zimmerman et al.

(-)-l-( l-Phenyl-l-oxoprop-3-yl)-3(9),4(~-dimethyl-4-(3- toasolutionof n-propanethiol(2.0mL,0.0022mol),1.25g (0.0011 mol) of potassium tert-butoxide and 25 mL of DMF at room hydroxypheny1)piperidine(9) was prepared as described for temperature. The solution was then heated at 145 "C for 5 h. 8 from (-)-3(S),4(&18: mp 184-185.5 OC; = -50.1 (c = The volatile8 were removed under vacuum at 75 "C. To the 1.0, MeOH). Anal. (CaH&lNOz) C, H, N. (*)-l-[R,&( l-Hydroxy-l-phenylprop-3-yl)]-3(RC),4(RC~- residue was added 50 mL of water and the pH was adjusted to 9.8 by the addition of 1N HC1. The product was extracted into dimethyl-4-(3-hydroxyphenyl)piperidine(7). Compound 6 toluene/l-butanol (3:1), dried over NaCVNaaSO4, and concen(3.50 g, 0.010 mol) in 30 mL of benzene was added dropwise to trated under vacuum. The crude product was chromatographed 8.0 mL of Red-Al, and the mixture was reflexued for 5 h. The over silica gel eluting with hexane/ethyl acetate (3:l) containing reaction was cooled and carefully added to 100 mL water, and 0.1 % triethylamine. The HClsalt was prepared and recrystallized the pH was adjusted to 9.8. The product was extracted into from isopropyl alcohollisopropylether: mp 217-219 OC. Anal. ether, dried over KaCOa, and concentrated under vacuum. The (CieHuClNOz) C, H, N. HClsalt was prepared and triturated in ether to give 2.8 g (74%1: (*)-(2-Furanylmethyl)-3(A()Po-dimethyE mp 86-89 "C with decomposition. Anal. (C=H&lNOz) C, H, ypheny1)piperidine (55). This compound was prepared from N. 17 and 2-furaldehyde as described for the synthesis of 54. The (+)-1-[2-( 4-Pyridinyl)ethyl]-3(R*),4(RC)-dimethyl-4-(3HC1 salt was prepared and titurated in methylene chloride/ hydroxypheny1)piperidine (34). A mixture of 18 as the isopropylether: mp 197-199 OC. Anal. (C&#ClNO1) C, H, N. hydrochloridesalt (1.17 g, 0.0049 mol),4-vinylpyridine(1.05 mL, Mouse Writhing Analgesic and Opioid Antagonist Aasay. 0.0097 mol), and 0.37 mL of water was heated at 100 OC for 4.5 Five CF-1 male mice (Charles River Portage, MI), weighing h. After cooling the mixture was diluted with 1 N HC1 poured approximately 20 g after being fasted overnight, were observed into water and the pH adjusted to 9.8. The product was extracted simultaneouslyfor the writhingresponse. The writhingresponse into ether, dried over KaCOa, and concentrated under vacuum. was induced by the intraperitoneal administration of 0.6 7% acetic The crude product was chromatographed over silica gel eluting acid in a volume of 1mL/100 g of body weight. The observation with ethyl acetate containing 1% methanol. The product period was 10 min in duration, beginning 5 min after injection crystallized as the free base on standing and was triturated with of acetic acid. The percent inhibition of writhing was calculated hexane to give 0.16 g (9%): mp 170-172 OC. Anal. (CZOHZ&O) from the average number of writhes in the control (nondrug) C, H, N. group. Each data point is the mean (+ standard error) for five (*)-l-[&S-( l-Hydroxy-l-phenyleth-3-y1)]-3(R*),4(R*)mice. Dose-response effects were measured by varying the (49). To a solution dimethyl-4-(3-hydroyphenyl)piperidine antagonist 2-fold, until minimum and maximum doses were of 0.50 g (0.0025 mol) of 18 in 50 mL of ethanol was added 0.46 determined. A minimum of five different doses were used. The g (0.0038 mol) of styrene oxide in 1.5 mL of methylene chloride. response for each dose had a standard error < 25 % The ADSO The reaction mixture was heated at 50 OC for 7 h, cooled to room was defiied as the dose of antagonist that reduced the inhibition temperature, and concentrated under vacuum. The resulting of writhing produced by a standard dose of the agonist (1.25 residue was chromatographed over silica gel eluting with ethyl mg/kg for morphine or 2.5 mg/kg for U-50,488H) to 50%. Each acetate/methanol (1:l).The HCl salt was prepared and triturated mouse was used only once. All drugs were administered in acetone/ethyl ether to give 0.25 g (28%): mp 120 OC with subcutaneously (1mL/100 g of body weight) 20 rnin before the decomposition. Anal. (C21HaClN02) C, H, N. (+)-1-[~(l-Hy~rl-(phenylmethyl)~h-3-y1)]-3(RC~,4- injection of acetic acid. The drugs used and the forms in which the doses were calculated are as follows: morphine sulfate, and (Rc)-dimethyl-4-(3-hydroxyphenyl)piperidine (50) was preU-50,488Hmethane sulfonate (The Upjohn Co., Kalamazoo, MI). pared by the procedure described for 49. The HCl salt was Antagonism Of K (Bremamine-Induced) Diumsis in Rats. prepared and triturated in acetone/ethyl ether: mp 90 "C with The animals used were a pool of 50male Long-Evans hooded rata decomposition. Anal. (C=H&lNO2) C, H, N. (f)-1-[ l-Oxo-l-(2-thienyl)prop3-yl)-3(R*),4(R*)-dimeth- (Charles River, Portage, MI) weighing 350-500 g. They were individuallyhoused in a colony room (23OC) illuminated between yl-4-(3-hydroxyphenyI)piperidine(51) was prepared by the 0600and 1800h. Rodent chow and tap water were freely available procedure describedfor 6 except the crude product was extracted except during the measurement of urinary output. The animals into ethyl acetate and purified by flash chromatography over were used repeatedly, but no more frequently than twice silica gel eluting with hexane/ethyl acetate (1:l)containing 0.5 % (separated by two days) during a week. To measure urinary triethylamine. The HC1 salt was prepared and triturated in output, the animals were removed from their home cages at about ether: mp 118-120 OC. Anal. (C&-I&lNOzS) C, H, N. (+)-1-[ l-Hydroxy-1-(2-thienyl)prop-3-y1)-3(~),4(R*)- lo00 h, weighed, injected, and placed individuallyin metabolism cages for the next 5 h. Excreted urine was funneled into graduated dimethyl-4-(3-hydroxyphenyl)piperidine(52). NaBH4 (0.25 cylinders, and the volume was recorded at 2 h and 5 h after g, 0.0066 mol) was added to a solution of 51 (2.00 g, 0.0058 mL) injection. Bremazocine hydrochloride (Sandoz Ltd., Basle, in 50 mL isopropyl alcohol at 0 OC. After 30 min of stirring, a Switzerland) was used as the K agonist to induce urination. second 0.25 g (0.0066 mol) portion of NaBH4 was added. The Bremazocine HCl was injected subcutaneously in a dose of 0.08 mixture was stirred for an additional 30 min following which 50 mg/kg, and without delay doses of the potential antagonists were mL of 1 N HCl was carefully added. The mixture was conceninjected subcutaneously on the opposite side of the rat. For each trated under vacuum, and the residue was dissolved into 50 mL test compound three rata per dose were used. The response for of water. The pH was adjusted to 9.8 and extracted with ethyl each dose had a standard error < 25 % . Test doses were varied acetate. The extra& were washed with NaCl solution, dried 2-fold until minimum and maximum effect doses were identified. over NaCl/Na&04 and concentrated under vacuum. The crude Opioid Receptor Binding Assays. The affiities of test product was chromatographedover silica gel eluting with hexane/ compounds of p- and r-opioid receptors were determined by a ethyl acetate (1:l)containing 0.6 % triethylamine. The HC1 salt modification of previously published rnethods.'l To perform was prepared and triturated in ether: mp 90-92 OC. Anal. preceptor binding,washed crude synaptosomalmembranesfrom (C&=ClNO&) C, H. N. (+)-l-(bFuranylmethyl)-3(R,),4(~)-dimethyl-4-(3-hy- rat whole brain tissue (minus cerebellum) were prepared and stored at -125 OC until use. Samples were incubated for 20 droxypheny1)piperidine(54). A solution of 2.40 g (0.011 mol) minutes at 37 OC in the presence of tissue, [SHlnaloxone (0.5 of 17.1.6 g (0.017 mol) of 3-furaldehyde,and 0.030 g of 5% Pd/C nM), and the test compound. The incubation buffer combted in 100 mL of ethanol was adjusted to pH 6 with ethanolic HCl of a modified Krebs-HEPES buffer (NaCl 118.2 mM; KCl4.6 and hydrogenated at 45 psi for 1 h at room temperature. The m M CaCl2 1.6 mm: MgS04 1.2 mM; KHzPO4 1.2 m M glucose catalyst was filtered, and the solvent was concentrated under 10 m M and HEPES 25 mM pH 7.4). Nonspecific binding was vacuum. The residue was partitioned between 1N NaOH and determined in the presence of lo00 nM of unlabeled naloxone. ether. The ether extracta were washed with aqueous NaCl, dried Bound radioactivity was separated from free ligand by fitration over NaCYNaaOd,and concentratedunder vacuum. The residue through Whatman GF-C glass fiber filters. Filters were further was chromatographed over silica gel using hexane/ethyl acetate washed with 2 x 6 mL of ice-cold buffer. Bound radioactivity (31) containing 0.1% triethylamine, giving 1.10 g (0.0011 mol) of (+)-1-(3-furanylmethyl)-3(R+),4(R+)-dimethyl-4-(3-methox- on the filter was quantitated using liquid scintillation spectrometry. ypheny1)piperidine. This was dissolved in 25 mLDMF and added

.

Antagonists for

p-

Journal of Medicinal Chemistry, 1993, Vol. 33, No.20 2841

and K-Opioid Receptors

For K-receptor binding, washed crude synaptosomal membranes were prepared from guinea pig cortical tiseue and stored at -126 O C until we. Tissue was suspended in modified KrebsHEPES buffer and incubated at 37 “C for 46 min with 1.0 nM [*Hlethylketocyclazocineand the test compound. Fentanyl and D-Ala2-DLeu6-enkephalinwere also added to the samples at 100 nM concentration to inhibit binding to and 6 receptors, respectively. Nonspecificbinding was determiued in the presence of lo00nM unlabeled ethylketocyclazocine. Bound radioactivity was separated and quantitated as described above for p binding. Methods for 6 receptor binding are deacribed in the accompanying paper. For all opioid receptor binding assays Ki values were derived from at least six different concentrations each run in triplicate. The standard errors from the extrapolated Ki values from the regression lines were less than 50% of the Ki. The correlation coefficient for calculating the Ki values were >0.9 and the triplicate values generally different by less than 10%

.

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