Redefining the Structure-Activity Relationships of Benzazocines 4. Opioid Receptor Binding Properties of do Analogues of Cyclazocine and Ethylketocycalzocine

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Redefining the Structure-Activity Relationships of 2,6-Methano-3-benzazocines. 4. Opioid

Receptor Binding Properties of 8-[ N -(4′-phenyl)-phenethyl)carboxamido] Analogues of

Cyclazocine and Ethylketocycalzocine

Mark P. Wentland,*,† Melissa VanAlstine,† Robert Kucejko,† Rongliang Lou,† Dana J. Cohen,‡ Amy L. Parkhill,‡ andJean M. Bidlack ‡

Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, and Department of Pharmacology

and Physiology, School of Medicine and Dentistry, UniVersity of Rochester, Rochester, New York 14642

ReceiVed March 13, 2006

The synthesis and evaluation of a series of aryl-containing N-monosubstituted analogues of the lead compound8-carboxamidocyclazocine were performed to probe a putative hydrophobic binding pocket of opioid receptors.High binding affinity to µ, κ, and δ opioid receptors was observed for the 8-[ N -(4′-phenyl)-phenethyl)-carboxamido] analogue.

Introduction

8-Carboxamidocyclazocine (8-CAC;a 1) has high affinity for µ and κ opioid receptors (Figure 1).1 This result was unexpectedbased on the long-standing knowledge2 that a phenolic hydroxyl

group was required for the high affinity binding of many opioid-receptor interactive ligands (e.g., cyclazocine; 23). In in vivostudies, 8-CAC showed high antinociception activity and a muchlonger duration of action than cyclazocine (15 h vs 2 h) whenboth were dosed at 1 mg/kg ip in mice.4 Preliminary structure-activity relationship studies for 8-CAC revealed that monosub-stitution of the carboxamide nitrogen of 1 with methyl or phenylreduced binding affinity for guinea pig µ receptors 75- and 2313-fold, respectively, whereas dimethylation of the carboxamidegroup reduced binding affinity 9375-fold.1

The finding that monosubstitution of the carboxamide nitro-gen had such a detrimental effect was puzzling because of ourresults from another study indicating that opioid receptors couldaccommodate aryl groups on the 8-position of the cyclazocinecore structure. For example, the 8-phenylamino and 8-benzyl-amino derivatives, 3 and 4, respectively, of cyclazocine hadapproximately 7-fold higher affinity for µ than the correspondingunsubstituted (e.g., 8-NH2) amino variant 5.5

To further probe opioid receptor space for what we believecontains a putative hydrophobic pocket complementary to thearyl groups of 3 and 4, we now report the synthesis and opioidreceptor binding properties of a series of N-monosubstitutedcarboxamide analogues of 8-CAC (Table 1). Design of targetswas based on two factors, namely, the distance between thecarboxamide N and the aryl group as well as the nature of thearyl group itself. Specifically, we chose derivatives havingspacers of 0-3 methylene groups and those where the aryl group

was phenyl or 4-biphenyl. The 4-biphenyl group (4-C6H4C6H5)is known to be a privileged functional group for recognition toG protein-coupled receptors.6 After observing the 8-[ N -(4′-phenyl)-phenethyl)carboxamido] analogue of 8-CAC (15) toexhibit very high binding affinity to µ, κ, and δ opioid receptors,

we also prepared its enantiomers (-)-16 and (+)-17 to assessenantioselectivity of drug binding as well as the corresponding8-[ N -(4′-phenyl)-phenethyl)carboxamido] analogue (21) havingthe ethylketocyclazocine (EKC) core.

Chemistry

Using general Method A (Scheme 1), novel racemic targets10-12 and 15 were conveniently made from common inter-mediate 7 via acylation in pyridine of the appropriate com-mercially available amine in yields of 42-83%. The synthesisof 7 was performed using a method we previously described7

wherein the triflate ester 65 of cyclazocine (2) was treated withpalladium acetate, carbon monoxide, triethylamine, and N -hydroxysuccinamde in dimethyl sulfoxide using 1,1′-bis(diphe-nyl-phosphino)ferrocene or Xantphos as the palladium ligand.

Methods B and C (Scheme 2), both one-step procedures, wereused to prepare target compounds 13, 14, 16-19, and 21. For13, 14, 18, and 19, triflate 6 was treated with the appropriateamine and carbon monoxide/palladium acetate/1,1′-bis(diphenyl-phosphino)ferrocene/dimethyl sulfoxide (Method B) or carbonmonoxide/dichloro[1,1′-bis(diphenylphosphino)-ferrocene]pal-ladium (II) dichloromethane adduct/dimethylformamide (MethodC). Amines were commercially available except 3-(4-biphenyl)-propylamine used to make target 18; this amine was preparedby reducing p-phenyl-cinnamamide with lithium aluminum

* To whom correspondence should be addressed. Phone: 518-276-2234.Fax: 518-276-4887. E-mail: [email protected].

† Rensselaer Polytechnic Institute.‡ University of Rochester.a Abbreviations: 8-CAC, 8-carboxamidocyclazocine; DAMGO, [D-Ala2,N-

Me-Phe4,Gly-ol5]- enkephalin; DMF, N , N -dimethylformamide; DMSO,dimethyl sulfoxide; nor-BNI, norbinaltorphimine; ip, intraperitoneal.

Figure 1. Structures of lead compounds for this study.

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10.1021/jm060278n CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 08/10/2006



hydride/tetrahydrofuran (see Experimental Section). Method Cwas also used to convert the triflate ester of cyclazocineenantiomers5 and the racemic triflate ester 81 of EKC to novel8-carboxamido- N -(2-[1,1′-biphenyl]-4-ylethyl) derivatives, (-)-16, (+)-17, and 21, respectively.

Results and Discussion

Affinities of target compounds for human µ, δ, and κ opioidreceptors stably expressed in chinese hamster ovary (CHO) cellswere assessed by generating K i values using well-documentedreceptor binding assays.8 These data, compared to the bindingaffinities of 8-CAC (1), are summarized in Table 1. As statedpreviously, N-phenyl substitution on the carboxamide group of 8-CAC (compare 1 and 9) significantly reduced binding affinityfor µ, δ, and κ opioid receptors; for human receptors, thisdecrease was 2710-, 404-, and 4500-fold, respectively. Forcompound 10, an analogue of 9 where the N -phenyl and-carboxamide groups are separated by one methylene, binding

affinity is decreased 87-, 40-, and 600-fold for µ, δ, and κ,

respectively, relative to 1. However, relative to 9, affinity is

increased 31-, 10-, and 8-fold, for µ, δ, and κ, respectively. For

the analogues with 2 and 3 methylene spacers, 11 and 12,

respectively, binding affinities were similar to each and have

an order of magnitude higher affinity for µ and κ (ca. 4- foldfor δ) compared to the single methylene spacer analogue 10.

Compared to 1, however, compounds 11 and 12 had reduced

binding affinity. From these limited data, it is apparent that

contributions of the phenyl group to the receptor binding

affinities of these 8-carboxamidocyclazocine derivatives is

highly dependent on its spatial arrangement with regard to

interaction with a putative complimentary hydrophobic pocket

in the receptors. To further probe this receptor space, we

prepared analogue 15 where the phenyl group of 11 was replaced

with 4-biphenyl, a group known for its frequent high affinity

binding for G-protein coupled receptors (i.e., a privileged

structure).6 This design strategy proved to be quite effective in

Table 1. Comparative Opioid Receptor Binding Data for 2,6-Methano-3-benzazocine Derivatives

K i (nM ( SEM)a

compd R [3H]DAMGO ( µ) [3H]naltrindole (δ) [3H]U69,593 (κ)

1b H 0.31 ( 0.03 5.2 ( 0.36 0.06 ( 0.0019b C6H5 840 ( 64 2100 ( 112 270 ( 1410c CH2C6H5 27 ( 5.5 210 ( 55 36 ( 1.111c (CH2)2C6H5 3.5 ( 0.27 59 ( 6.6 1.7 ( 0.1812c (CH2)3C6H5 2.5 ( 0.27 47 ( 1.6 3.0 ( 0.3513c 4-C6H4C6H5 18 ( 1.6 110 ( 7.1 27 ( 0.8614c CH2(4-C6H4C6H5) 11 ( 0.40 170 ( 9.4 26 ( 2.015c (CH2)2(4-C6H4C6H5) 0.30 ( 0.036 0.74 ( 0.019 1.8 ( 0.19

(-)-16c

0.25 ( 0.031 0.24 ( 0.014 0.35 ( 0.009(+)-17c 6.4 ( 0.50 9.9 ( 0.44 8.5 ( 1.0718c (CH2)3(4-C6H4C6H5) 5.8 ( 0.31 72 ( 11 2.7 ( 0.2119c NHR ) N(CH3)(CH2)2(4-C6H4C6H5) 6.7 ( 1.7 12 ( 2.4 11 ( 0.4420b H 2.1 ( 0.30 23 ( 2.3 0.47 ( 0.02421c (CH2)2(4-C6H4C6H5) 3.1 ( 1.3 3.9 ( 1.4 1.3 ( 0.072

a See Experiemntal Section. The K d values for [3H]DAMGO, [3H]U69,593, and [3H]naltrindole were 0.56 nM, 0.34 nM, and 0.10 nM, respectively. Thesevalues were used to calculate the K i values. b See ref 1. c Proton NMR, IR, and MS were consistent with the assigned structures of all new compounds. C,H, and N elemental analyses were obtained for all new targets and most intermediates and were within (0.4% of theoretical values.

Scheme 1. Syntheses of Target Compounds via an Activated Ester Common Intermediate (Method A)a

a (i) Pd(OAc)2, dppf, CO, Et3N, NHS, DMSO, 70 °C; (ii) Pd(OAc)2, Xantphos, CO, Et3N, NHS, DMSO, 70 °C; (ii) RNH2, pyridine.

5636 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 Brief Articles



that binding affinities for 15 compared to 11, were 12-fold and80-fold greater for µ and δ, respectively, with sub-nanomolarK i values observed. For the κ opioid receptor, the K i values for11 and 15 were comparable. Compared to 1, compound 15 hadcomparable binding affinity for µ, 7-fold increased affinity forδ, and 30-fold lower affinity for κ. To assess the spatial

requirements of the 4-biphenyl group with respect to thecarboxamide moiety, we made three analogues, 13, 14, and 18,which had spacers of 0, 1, and 3 methylene groups, respectively.Not surprisingly, we found that compared to the two-methylenespacer analogue 15, the three compounds had significant loweraffinities for µ (20- to 60-fold) and δ (100- to 230-fold)receptors. For κ, however, 18 had comparable affinity as 15,while compounds 13 and 14 had 15-fold lower affinity than15. What was surprising however, was the observation that withone exception, affinities of 13, 14, and 18 for all three receptorswere about the same; that exception being that for κ compound18 had 10-fold greater binding affinity than compounds 13 and14. Relative to 1, compounds 13, 14, and 18 had significantlylower affinities for µ (20- to 60-fold), δ (15- to 33-fold), and κ

(45- to 450-fold) receptors. To assess the enantioselectivity of binding of 15 for opioid receptors, we evaluated its enantiomers(-)-16 and (+)-17. Consistent with what we observed for8-CAC enantiomers,1 a large enantiopreference for binding toall three receptors was observed (eudismic ratios 24-41) andthe active enantiomer (-)-16 had the expected (2 R,6 R,11 R)absolute configuration.

In our early 8-CAC structure-activity relationship (SAR)study,1 we found that dimethylation of the carboxamide es-sentially abolished binding affinity for opioid receptors sug-gesting the importance of an H-bond donating group at the8-position. To assess if this NH is a requirement in our N -(4′-phenyl)-phenethyl)carboxamido series, we evaluated the N -methyl analogue 19 of lead 15. While compound 19 has lower

affinity for µ, δ, and κ than 15, the difference in K i values (22-,16-, and 6-fold, respectively) does not come close to the muchlarger separation noted for the pair of 8-CAC analogues having

N -methyl and N,N -dimethyl substitutions (125-, >550-, 962-fold, respectively). These data strongly suggest the (4′-phenyl)-phenethyl) group of 15 or 19 sustains a specific hydrophobicinteraction with opioid receptors and this interaction helpsanchor these ligands into the active site lessening the importanceof the H-bond donor ability of the 8-CONHCH2CH2C6H4C6H5

group compared to that of the 8-CONHCH3 group.To determine the effects of N -(4′-phenyl)-phenethyl) substitu-

tion on other carboxamide-containing opiate core structures, wemade the corresponding analogue 21 of 8-carboxamidoEKC 20.1

A similar trend in the SAR was noted for the EKC-derived

compounds 20 and 21, compared to the cyclazocine-derived

derivatives, 1 and 15. Relative to the unsubstituted carboxamideanalogue 20, the N -(4′-phenyl)-phenethyl) derivative 21 hadcomparable affinity for µ, enhanced affinity for δ and reducedaffinity for the κ receptor.

Intrinsic opioid-receptor mediated activity for 15 and its activeenantiomer (-)-16 was determined using [35S]GTPγS bindingassays at µ, δ, and κ receptors; results compared to 8-CAC areshown in Table 2. Results from these assays indicate that at κand δ receptors, compounds 15 and 16 exhibited agonistproperties. Compound 1 was also an agonist at κ however,moderate affinity for δ receptors precluded generating functionalactivity data. At the µ receptor, all three compounds displaypartial agonist properties which may be a consequence of

Scheme 2. Syntheses of Target Compounds via Pd-Catalyzed Carboxamidation Procedures (methods B and C) a

a (i) RR′NH, Pd(OAc)2, dppf, CO, Et3N, DMSO (Method B); (ii) R′NH, PdCl2(dppf), CO, Et3N, DMF (Method C).

Table 2. EC50 and E max Values for the Stimulation of [35S]GTPγSBinding and IC50 and I max Values for the Inhibition of Agonist-Stimulated [35S]GTPγS Binding to the Human µ, κ, and δOpioid Receptorsa

compdEC50

(nM) E max

(% max. stim)IC50

(nM) I max

(% max. inhib)

Mu Opioid Receptor

DAMGO 55( 7 116 ( 4 NIc NI1 3.7b(1.2-7.6) 27b(15-37) 21 ( 3.7 79 ( 3.015 NAd 5.6 ( 3.4 150 ( 25 99 ( 1.316 3.0 ( 0.65 55 ( 3.3 34 ( 2.7 at 1 µM f

Kappa Opioid ReceptorU50,488 36( 5 77 ( 11 NI NI1 4.4 ( 0.73 87 ( 6.5 NI NI15 3.0 ( 0.50 76 ( 6.7 NI NI16 3.2 ( 0.16 74 ( 3.9 NI NI

Delta Opioid ReceptorSNC80 4.8 ( 0.60 120 ( 4.7 NI NI1 NTe NT NT NT15 3.0 ( 0.24 69 ( 8.6 NI NI16 6.9 ( 1.9 47 ( 5.0 NI NI

a See Experimental Section. Data are the mean E max and EC50 values (SEM from at least three separate experiments, performed in triplicate. Forcalculation of the E max values, the basal [35S]GTPγS binding was set at0%. b Data from three separate experiments, performed in triplicate, wereaveraged together, and are presented with 95% confidence limits. c NI )No inhibition. d NA ) Not applicable. e NT ) Not tested. f Compound 16at a concentration of 1 µM inhibited 34 ( 2.7% of DAMGO-stimulated[35S]GTPγS binding. An IC50 value was not reported because higherconcentrations could not be used without having the DMSO vehicle interferewith the assay. A concentration of 200 nM DAMGO, which gave 96 (

3.1% stimulation, was used to measure inhibition of DAMGO-stimulated[35S]GTPγS binding. A concentration of 100 nM U50,488, which gave 64( 1.9% stimulation, was used to measure inhibition of U50,488-stimulated[35S]GTPγS binding, and 10 nM SNC 80, which gave 66 ( 4.3%stimulation, was used to measure inhibition of [35S]GTPγS binding,mediated by the δ opioid receptor.

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overlapping concentration-response curves for agonist andantagonist effects.

Conclusions

Examination of opioid receptor binding data for this smallseries of N-monosubstituted carboxamide analogues of 8-CAChas yielded valuable insights into the SAR of the 8-substituentof 8-CAC and related compounds. Our observation that a (4′-phenyl)-phenethyl group situated on the carboxamide nitrogen

of 8-CAC is responsible for very high affinity to opioid receptorsrelative to other N-substituents leads us to conclude thathydrophobics play an important role in binding in this seriesand affinity for the receptors is highly dependent on the natureof the hydrophobic group and the distance between it and thecarboxamide. Since potency is much higher with the two-methylene spacer than with 0, 1, or 3 methylenes, conformationof the ligand also appears to play an important role. It isinteresting to speculate what amino acid residues on thereceptors are complementary to the hydrophobic biphenylethylgroup of 1. From the homology model of the µ receptor boundto nor-BNI,9 there are three phenylalanine (Phe152, Phe237,and Phe241) residues in the region of the 8-position of benzomorphans which may create a hydrophobic pocket comple-

mentary to the biphenylethyl group of 1.Our data suggest the N -(4′-phenyl)-phenethyl group to be such

an important part of the pharmacophore of 15 that even whenit is methylated to give 19, moderate affinity is still observed.This contrasts our earlier SAR study indicating that at least oneH on the carboxamide was a prerequisite for activity. Theputative hydrophobic binding pocket in the receptors that webelieve is complimentary to the N -(4′-phenyl)-phenethyl) groupof 15 has not been previously explored to any great extentbecause most opiate SAR studies lack the ability to probe thisreceptor space due to the lower (than nitrogen) valence of theoxygen of the phenolic-OH, the prototypic group of opiates.The SAR in this series appears to be additive in that the benefitof the N -(4′-phenyl)-phenethyl group of 15 crosses over to

another core structure, namely the 8-carboxamido-EKC. Inaddition to high affinity for opioid receptors, other attributesof N -(4′-phenyl)-phenethyl)-8-CAC derivatives are high enan-tiospecificity of binding with the (2 R,6 R,11 R)-isomer, (-)-16,being the active enantiomer and intrinsic activity as demon-strated in [35S]GTPγS assays.

It is our belief that the knowledge gained from this studywill assist in the design of new high affinity opioid receptor-interactive ligands modified at the phenolic-OH position of opiates. To further explore this novel SAR, the synthesis andevaluation of new targets related to 15 is ongoing in ourlaboratories. Target selection will include those analogues witha diverse array of spacer and (hetero)aryl groups on the8-position of 2,6-methano-3-benzazocines and the 3-position of morphinans and 4,5R-eopxymorphinans.

Experimental Section

(()-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -(2-[1,1′-biphenyl]-4-ylethyl)-2,6-methano-3-benzazo-cine-8-carboxamide (15). Method A. Conditions similar to thosepreviously reported were used.1 A solution of (()-7 (140 mg, 0.35mmol) and 2-(4-biphenylethylamine) (84 mg, 0.42 mmol) in 2.5mL of dry pyridine was stirred at room temperature for 48 h. Thesolvent was removed in vacuo, and the residue was taken up inmethylene chloride (40 mL) and washed once with saturated sodiumbicarbonate solution, water, and brine. The organic phase was driedover sodium sulfate, filtered, and concentrated to give a brownresidue, which was purified by flash chromatography (CH2Cl2:CH3-

OH:NH4OH 15:1:0.1) to give 15 as an off-white foam (110 mg,0.23 mmol, 66%).

(()-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -(2-phenylethyl)-2,6-methano-3-benzazocine-8-carbox-amide (11). This compound was prepared using Method A andphenethylamine. Off-white foam (93 mg, 0.231 mmol, 83%).

(()-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -(3-phenylpropyl)-2,6-methano-3-benzazocine-8-car-boxamide (12). This compound was prepared using Method A and3-phenyl-1-propylamine. Off-white foam (72 mg, 0.174 mmol,

68%).(()-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-

methyl- N -(phenylmethyl)-2,6-methano-3-benzazocine-8-carbox-amide (10). This compound was prepared using Method A andbenzylamine. Off-white oil (80 mg, 0.21 mmol, 42%).

(()-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -[1,1′-biphenyl]-4-yl-2,6-methano-3-benzazocine-8-car-boxamide (13). Method B. Conditions similar to those previouslyreported were used.1 4-Aminobiphenyl (635 mg, 3.75 mmol),palladium acetate (17 mg, 0.075 mmol), and dppf (42 mg, 0.075mmol) were added to a two-neck flask charged with triflate 6 (300mg, 0.75 mmol). The reaction was equipped with a condenser andsealed with a septum and a balloon. The whole system wasvacuumed and backfilled with nitrogen for three cycles. DMSO (2mL) was added via syringe. Then it was vacuumed again and

backfilled with a mixture of carbon monoxide. The resulting mixturewas heated at 70 °C for 18 h. The cooled reaction mixture wasdiluted with ethyl acetate (30 mL) and washed with saturatedbicarbonate solution, water, and brine. The organic phase was driedover sodium sulfate, filtered, and concentrated to give a black oil,which was purified by flash chromatography (CH2Cl2:CH3OH:NH4-OH 25:1:0.1) to give 13 as a brown oil (191 mg, 0.42 mmol, 57%).

(()-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -([1,1′-biphenyl]-4-ylmethyl)-2,6-methano-3-benzazo-cine-8-carboxamide (14). This compound was prepared usingMethod B and 4-phenylbenzylamine. Off-white foam (275 mg, 0.59mmol, 68%).

(-)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -(2-[1,1′-biphenyl]-4-ylethyl)-2,6-methano-3-benzazo-cine-8-carboxamide [(-)-16]. Method C. Conditions similar to

those previously reported were used.2 2-(4-Biphenylethylamine) (85mg, 0.43 mmol) PdCl2(dppf) (16 mg, 0.02 mmol) were added to atwo-neck flask charged with triflate ester of (-)-cyclazocine5 (158mg, 0.39 mmol). The reaction was equipped with a condenser andsealed with a septum and a balloon. The whole system wasvacuumed and backfilled with nitrogen for three cycles. DMF (2mL) and triethylamine (0.09 mL, 0.62 mmol) were added viasyringe. Then it was vacuumed again and backfilled with a mixtureof carbon monoxide. The resulting mixture was heated at 70 °Cfor 18 h. The cooled reaction mixture was diluted with ethyl acetate(30 mL) and washed with saturated bicarbonate solution, water,and brine. The organic phase was dried over sodium sulfate, filtered,and concentrated to give a black oil, which was purified by flashchromatography (CH2Cl2:CH3OH:NH4OH 25:1:0.1) to give (-)-16 as an off-white foam (100 mg, 0.21 mmol, 53%).

(+)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -(2-[1,1′-biphenyl]-4-ylethyl)-2,6-methano-3-benzazo-cine-8-carboxamide [(+)-17]. This compound was prepared usingMethod C and triflate ester of (+)-cyclazocine.5 Off-white foam(90 mg, 0.19 mmol, 49%).

(()-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -(3-[1,1′-biphenyl]-4-ylpropyl)-2,6-methano-3-benzazo-cine-8-carboxamide (18). This compound was prepared usingMethod C and 3-[1,1′-biphenyl]-4-propylamine. Off-white foam(250 mg, 0.51 mmol, 63%).

(()-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-di-methyl- N -(2-[1,1′-biphenyl]-4-ylethyl)- N -methyl-2,6-methano-3-benzazocine-8-carboxamide (19). This compound was preparedusing Method C and N -methyl-[1,1′-biphenyl]-4-ethanamine. Off-white foam (190 mg, 0.39 mmol, 60%).

5638 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 Brief Articles



(()-3-(Cyclopropylmethyl)-6-ethyl-1,2,3,4,5,6-hexaahydro- cis-11-methyl- N -(2-[1,1′-biphenyl]-4-ylethyl)-1-oxo-2,6-methano-3-benzazocine-8-carboxamide (21). This compound was preparedusing Method C and the triflate ester 81 of EKC and 2-(4-biphenylethylamine). Off-white foam (200 mg, 0.39 mmol, 61%).

Acknowledgment. We gratefully acknowledge the contribu-tions of Rensselaer’s mass spectroscopist Dr. Dmitri Zagorevskiand the technical assistance provided by Brain I. Knapp of theUniversity of Rochester. Funding of this research was from

NIDA (DA12180, T32 DA07232, and KO5-DA00360).

Supporting Information Available: Experimental details,NMR data, and elemental analyses for all compounds. This materialis free of charge via the Internet at http://pubs/acs/org.

References

(1) Part 1: Wentland, M. P.; Lou, R.; Ye, Y.; Cohen, D. J.; Richardson,G. P.; Bidlack, J. M. 8-Carboxamidocyclazocine Analogues: Rede-fining the Structure-Activity Relationships of 2,6-Methano-3-benzazocines. Bioorg. Med. Chem. Lett. 2001, 11, 623-626.

(2) Aldrich, J. V.; Vigil-Cruz, S. C. In Burger’s Medicinal Chemistryand Drug DiscoVery, 6th ed.; Abraham, D. J., Ed.; John Wiley &Sons: New York, 2003; Vol. 6, pp 329-481.

(3) Archer, S.; Glick S. D.; Bidlack, J. M. Cyclazocine Revisited.

Neurochem. Res. 1996, 21, 1369-1373.

(4) Bidlack, J. M.; Cohen, D. J.; McLaughlin, J. P.; Lou, R.; Ye, Y.;

Wentland, M. P. 8-Carboxamidocyclazocine: A Long-Acting, Novel

Benzomorphan. J. Pharmacol. Exp. Ther. 2002, 302, 374-380.(5) Wentland, M. P.; Ye, Y.; Cioffi, C. L.; Lou, R.; Zhou, Q.; Xu, G.;

Duan, W.; Dehnhardt, C. M.; Sun, X.; Cohen, D. J.; Bidlack, J. M.

“Syntheses and Opioid Receptor Binding Affinities of 8-Amino-2,6-

methano-3-benzazocines.” J. Med. Chem. 2003, 46 , 838-849.(6) Hajduk, P. J.; Bures, M.; Praestgaard, J.; Fesik, S. E. Privileged

Molecules for Protein Binding Identified from NMR-Based Screen-

ing. J. Med. Chem. 2000, 43, 3443-3447.(7) Lou, R.; VanAlstine, M.; Sun, X.; Wentland, M. P. Preparation of

N-hydroxysuccinimido esters via palladium-catalyzed carbonylationof aryl triflates and halides. Tetrahedron Lett. 2003, 44, 2477-2480.

(8) Neumeyer, J. L.; Zhang, A.; Xiong, W.; Gu, X.; Hilbert, J. E.; Knapp,

B. I.; Negus, S. S.; Mello, N. K.; Bidlack, J. M. Design and Synthesis

of Novel Dimeric Morphinan Ligands for κ and µ Opioid Receptors.

J. Med. Chem. 2003, 46 , 5162.

(9) Fowler, C. B.; Pogozheva, I. D.; LeVine, H.; Mosberg, H. I.

Refinement of a Homology Model of the µ-Opioid Receptor UsingDistance Constraints from Intrinsic and Engineered Zinc-Binding

Sites. Biochemistry 2004, 43, 8700-8710.

JM060278N

Brief Articles Journal of Medicinal Chemistry, 2006, Vol. 49, No. 185639



S1

Supporting Information

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 4.

Opioid receptor binding properties of 8-[N-(4'-phenyl)-phenethyl)carboxamido] analogues

of cyclazocine and ethylketocyclazocine.

Mark P. Wentland,*,a

Melissa VanAlstine,a

Robert Kucejko,a

Rongliang Lou,a

Dana J. Cohen, b

Amy L. Parkhill b

and Jean M. Bidlack b

a Department of Chemistry and Chemical Biology

, Rensselaer Polytechnic Institute, Troy, NY

12180 and b Department of Pharmacology and Physiology, School of Medicine and Dentistry,

University of Rochester, Rochester, NY 14642

______________________________________________________________________________

Table of Contents

Experimental Section_____________________________________________________ S2

Materials and Methods___________________________________________ S2

NMR Data_____________________________________________________ S3

Combustion Data_______________________________________________ S9

Radiolabeled Ligand Binding Assays________________________________ S9

[35

S]GTPγS Binding Assays._______________________________________ S10

References_____________________________________________________ S11



S2

Experimental Section

Materials and Methods. Proton NMR spectra and in certain cases13

C NMR were obtained on a

Varian Unity-300 or 500 NMR spectrometer with tetramethylsilane as an internal reference for

samples dissolved in CDCl3. Samples dissolved in CD3OD and DMSO-d 6 were referenced to the

solvent. Proton NMR multiplicity data are denoted by s (singlet), d (doublet), t (triplet), q

(quartet), m (multiplet), dd (doublet of doublets), and br (broad). Coupling constants are in

hertz. Direct insertion probe chemical ionization mass spectral data were obtained on a

Shimadzu GC-17A GC-MS mass spectrometer. Direct infusion electrospray ionization (in

positively charged ion mode) mass spectral data were obtained on an Agilent 1100 series

LC/MSD system (Germany). Melting points were determined on a Meltemp capillary melting

point apparatus and were uncorrected. Infrared spectral data were obtained on a Perkin-Elmer

Paragon 1000 FT-IR spectrophotometer. Optical rotation data was obtained from a Perkin-Elmer

241 polarimeter. The assigned structure of all test compounds and intermediates were consistent

with the data. Carbon, hydrogen, and nitrogen elemental analyses for all novel targets were

performed by Quantitative Technologies Inc., Whitehouse, NJ, and were within ± 0.4% of

theoretical values except as noted; the presence of water or other solvents was confirmed by

proton NMR. Reactions were generally performed in an argon or nitrogen atmosphere.

Commercially purchased chemicals were used without purification unless otherwise noted. The

following reagents were purchased from Aldrich Chemical Company: N-hydroxysuccinimide,

phenethylamine, 3-phenyl-1-propylamine, 4-aminobiphenyl, palladium acetate, 4-

phenylbenzylamine and benzyl amine. The following reagent was purchased from Trans World

Chemicals: 2-(4-biphenyl ethylamine). The following reagents were purchased from Strem



S3

Chemicals, Incorporated: 1,1’-bis(diphenyl-phosphino)ferrocene (dppf) and dichloro[1,1’-

bis(diphenylphosphino)-ferrocene]palladium (II) dichloromethane adduct [PdCl2(dppf)].

Pyridine was distilled from KOH. DMF and DMSO were distilled over CaH2 under reduced

pressure. Silica gel (Bodman Industries, ICN SiliTech 2-63 D 60A, 230-400 Mesh) was used for

all flash chromatography.

(±)-1-[[[3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl-2,6-

methano-3-benzazocin-8-yl]carbonyl]oxy]-2,5-Pyrrolidinedione (7). To a flask charge with

triflate 65

(403 mg, 1.00 mmol), N-hydroxy succinimide (230 mg, 2.00 mmol) palladium acetate

(22.4 mg, 0.10 mmol) and dppf (55.4 mg, 0.10 mmol) was added triethyl amine (0.28 mL, 2.00

mmol). The reaction was equipped with a condenser and sealed with a septum and a balloon.

The whole system was vacuumed and backfilled with nitrogen for three cycles. DMSO (1 mL)

was added via syringe. Then it was vacuumed again and backfilled with a mixture of carbon

monoxide. The resulting mixture was heated at 70oC

for 8.5 h. The cooled reaction mixture was

diluted with ethyl acetate (30 mL), washed with water, and brine. The organic phase was dried

over sodium sulfate, filtered, and concentrated to give a brown oil, which was purified by flash

chromatography (Ethyl acetate:Acetone:Hexane:Et3 N 2:1:0.4:0.03) to give 7 as a white foam

(217 mg, 0.55 mmol, 55%):1H NMR (500 MHz, CDCl3) δ 7.96 (d, 1H, J = 1.5 Hz), 7.82 (dd,

1H, J 1 = 1.5 Hz, J 2= 8.1 Hz), 7.17 (d, 1H, J = 8.1 Hz), 3.19 (m, 1H), 2.97 (d, 1H, J = 19.5 Hz),

2.85 (s, 4H), 2.73 (m, 2H), 2.44 (dd, 1H, J 1 = 6.4 Hz, J 2 =12.7 Hz), 2.33 (dd, 1H, J 1 = 6.6 Hz, J 2

=12.4 Hz ), 1.93 (m, 1H), 1.84 (d, 2H, J

= 8.5 Hz), 1.35 (s, 3H), 1.27 (m, 1H), 0.83 (m, 1H), 0.79

(d, 3H, J = 7.1 Hz), 0.48 (m, 2H), 0.08 (m, 2H). MS (ESI) m/z 397 (M+H)+; Anal. Calcd. for

C23H28 N2O4·0.5H2O: C 68.20, H 7.20, N 6.90. Found: C 68.04, H 6.92, N 7.12.



S4

(±)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl- N -(2-[1,1'-

biphenyl]-4-ylethyl)-2,6-methano-3-benzazocine-8-carboxamide (15).1H NMR (500 MHz,

CDCl3) δ 7.66 (d, 1H, J = 1.5 Hz), 7.57 (dd, 2H, J 1 = 1.3 Hz, J 2= 7.5 Hz), 7.55 (d, 2H, J = 8.5

Hz), 7.43 (t, 2H, J = 7.75 Hz), 7.39 (dd, 1H, J 1 = 1.8 Hz, J 2 = 7.75 Hz), 7.34 (t, 1H, J = 7.5 Hz),

7.31 (d, 2H, J = 8 Hz), 7.08 (d, 1H, J = 8 Hz), 6.32 (bt, 1H, J = 5.75 Hz), 3.72 (q, 2H, J = 6.7

Hz), 3.14 (m, 1H), 2.97 (t, 2H, J = 1.5 Hz), 2.93 (d, 1H, J = 18.5 Hz), 2.70 (m, 2H), 2.45 (dd,

1H, J 1 = 6.3 Hz, J 2 =12.75 Hz), 2.34 (dd, 1H, J 1 = 6.75 Hz, J 2 =12.75 Hz ), 1.93 (m, 3H), 1.39 (s,

3H), 1.32 (d, 1H, J = 9.5), 0.87 (m, 1H), 0.81 (d, 3H, J = 7.0 Hz), 0.50 (dd, 2H, J 1 = 1.5 Hz, J 2

=8.0 Hz), 0.12 (m, 2H). MS (ESI) m/z 479 (M+H)

+

; Anal. Calcd. for C33H38 N2O·1.0H2O: C

79.80, H 8.12, N 5.64. Found: C 79.72, H 8.07, N 5.96.

(±)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl- N -(2-

phenylethyl)-2,6-methano-3-benzazocine-8-carboxamide (11).1H NMR (500 MHz, CDCl3) δ

7.61 (d, 1H, J = 2.0 Hz), 7.35 (m, 3H), 7.26 (m, 3H), 7.08 (d, 1H, J = 8 Hz), 6.07 (bt, 1H, J = 5.0

Hz), 3.71 (q, 2H, J = 6.5 Hz), 3.16 (m, 1H), 2.94 (m, 3H), 2.70 (m, 2H), 2.47 (m, 1H), 2.32 (m,

1H), 1.93 (m, 3H), 1.40 (s, 3H), 1.33 (d, 1H, J = 11.5), 0.87 (m, 1H), 0.82 (d, 3H, J = 7.0 Hz),

0.52 (d, 2H, J =8.0 Hz), 0.11(m, 2H); MS (ESI) m/z 403 (M+H)+; Anal. Calcd. for

C27H34 N2O·0.5H2O: C 78.79, H 8.57, N 6.81. Found: C 78.90, H 8.55, N 6.86.

(±)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl- N -(3-

phenylpropyl)-2,6-methano-3-benzazocine-8-carboxamide (12).1H NMR (500 MHz, CDCl3)

δ 7.66 (d, 1H, J = 1.5 Hz), 7.30 (m, 3H), 7.21 (m, 3H), 7.09 (d, 1H, J = 8 Hz), 6.02 (bt, 1H, J =

5.5 Hz), 3.50 (q, 2H, J = 6.8 Hz), 3.15 (m, 1H), 2.95 (d, 1H, J = 19.0 Hz), 2.71 (m, 4H), 2.46 (m,

1H), 2.32 (m, 1H), 1.94 (m, 5H), 1.42 (s, 3H), 1.34 (d, 1H, J = 9.75), 0.87 (m, 1H), 0.82 (d, 3H, J



S5

= 7.0 Hz), 0.51 (d, 2H, J = 8.0 Hz), 0.11(m, 2H); MS (ESI) m/z 417 (M+H)+; Anal. Calcd. for


(±)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl- N -

(phenylmethyl)-2,6-methano-3-benzazocine-8-carboxamide (10).1H NMR (500 MHz,

CDCl3) δ 7.73 (d, 1H, J = 1.4 Hz), 7.45 (dd, 1H, J 1 = 1.4 Hz, J 2 =7.8 Hz), 7.36 (m, 5H), 7.10 (d,

1H, J = 8.1 Hz), 6.33 (bt, 1H), 4.65 (d, 2H, J = 5.7 Hz), 3.14 (m, 1H), 2.95 (d, 1H, J = 19.0 Hz),

2.70 (m, 2H), 2.46 (dd, 1H, J 1 = 6.6 Hz, J 2 =12.7 Hz), 2.31 (dd, 1H, J 1 = 6.6 Hz, J 2 =12.75 Hz),

1.91 (m, 3H), 1.42 (s, 3H), 1.35 (d, 1H, J = 9.3), 0.86 (m, 1H), 0.82 (d, 3H, J = 7.1 Hz), 0.52 (m,

2H), 0.10(m, 2H); MS (ESI) m/z 389 (M+H)

+

; Anal. Calcd. for C26H32 N2O·0.25H2O: C 79.45,

H 8.33, N 7.12. Found: C 79.16, H 8.15, N 7.05.

(±)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl- N -[1,1'-

biphenyl]-4-yl-2,6-methano-3-benzazocine-8-carboxamide (13).1H NMR (500 MHz,

CDCl3) δ 8.18 (s, 1H), 7.82 (d, 1H, J = 1.5 Hz), 7.73 (d, 2H, J = 8.5 Hz), 7.57 (m, 5H), 7.42 (t,

2H, J = 7.5 Hz), 7.32 (t, 1H, J = 7.25 Hz), 7.13 (d, 1H, J = 8.0 Hz), 3.16 (m, 1H), 2.97 (d, 1H, J

= 18.5 Hz), 2.71 (m, 2H), 2.46 (dd, 1H, J 1 = 6.5 Hz, J 2 =12.5 Hz), 2.32 (dd, 1H, J 1 = 6.75 Hz, J 2

=12.25 Hz), 1.93 (m, 3H), 1.42 (s, 3H), 1.34 (d, 1H, J = 9.5 Hz), 0.86 (m, 1H), 0.84 (d, 3H, J =

7.5 Hz), 0.51 (d, 2H, J = 8.25 Hz), 0.11 (m, 2H). MS (ESI) m/z 451 (M+H)+; Anal. Calcd. for


(±)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl- N -([1,1'-

biphenyl]-4-ylmethyl)-2,6-methano-3-benzazocine-8-carboxamide (14).

1

H NMR (300 MHz,

CDCl3) δ 7.76 (d, 1H, J = 1.2 Hz), 7.57 (d, 4H, J = 8.1 Hz), 7.45 (m, 5H), 7.35 (t, 1H. J = 6.9

Hz), 7.19 (d, 1H, J = 8.0 Hz), 6.53 (b, 1H), 3.17 (m, 1H), 2.96 (d, 2H, J = 6.0 Hz), 2.72 (m, 2H),

2.48 (dd, 1H, J 1 = 6.0 Hz, J 2 =12.0 Hz), 2.32 (dd, 1H, J 1 = 6.0 Hz, J 2 =12.0 Hz), 1.93 (m, 3H),



S6

1.43 (s, 3H), 1.3 (m, 1H), 0.88 (m, 1H), 0.83 (d, 3H, J = 6.9 Hz), 0.52 (d, 2H, J = 8.1 Hz), 0.12

(m, 2H). MS (ESI) m/z 465 (M+H)+; Anal. Calcd. for C32H36 N2O·0.5H2O: C 81.14, H 7.87, N

5.91. Found: C 80.91, H 7.77, N 5.81.

(-)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl- N -(2-[1,1'-

biphenyl]-4-ylethyl)-2,6-methano-3-benzazocine-8-carboxamide [(-)-16].1H NMR (300

MHz, CDCl3) δ 7.68 (s, 1H), 7.57 (m, 4H), 7.43 (m, 3H), 7.33 (m, 3H), 7.08 (d, 1H, J = 7.8 Hz),

6.34 (bt, 1H), 3.73 (q, 2H, J = 6.0 Hz), 3.16 (m, 1H), 2.94 (m, 3H), 2.71 (m, 2H), 2.48 (m, 1H),

2.31 (m, 1H), 1.93 (m, 3H), 1.40 (s, 3H), 1.32 (m, 1H), 0.87 (m, 1H), 0.82 (d, 3H, J = 7.2 Hz),

0.51 (d, 2H, J = 6.6 Hz), 0.11 (m, 2H). MS (ESI) m/z 479 (M+H)

+

; Anal. Calcd. for

C33H38 N2O·1.25H2O: C 79.08, H 8.14, N 5.59. Found: C 79.23, H 7.84, N 5.57. For (-)-16:

[α]25

D = -69.1o

(c = .75, acetone).

(+)-3-(Cyclopropylmethyl)-1,2,3,4,5,6-hexahydro- cis-6,11-dimethyl- N -(2-[1,1'-

biphenyl]-4-ylethyl)-2,6-methano-3-benzazocine-8-carboxamide [(+)-17].1H NMR (500

MHz, CDCl3) δ 7.68 (s, 1H), 7.57 (d, 2H, J = 7.5 Hz), 7.55 (d, 2H, J = 7.5 Hz) 7.42 (m, 3H),

7.32 (m, 3H), 7.07 (d, 1H, J = 8.0 Hz), 6.40 (bt, 1H), 3.72 (q, 2H, J = 6.0 Hz), 3.13 (m, 1H), 2.94

(m, 3H), 2.69 (m, 2H), 2.45 (dd, 1H, J 1 = 6.5 Hz, J 2=13.0 Hz), 2.30 (dd, 1H, J 1 = 6.5 Hz, J 2=12.5

Hz), 1.93 (m, 3H), 1.39 (s, 3H), 1.32 (m, 1H), 0.87 (m, 1H), 0.81 (d, 3H, J = 7.0 Hz), 0.50 (d,

2H, J = 8.0 Hz), 0.11 (m, 2H). MS (ESI) m/z 479 (M+H)+; Anal. Calcd. for

C33H38 N2O·0.67H2O: C 80.78, H 8.07, N 5.71. Found: C 80.96, H 8.13, N 5.45. For (+)-17:

[α]25

D = +81.3o

(c = 1.02, acetone).

3-[1,1'-biphenyl]-4-propylamine. To a vigorously stirred solution of 4-

biphenylacrylamide (440 mg, 1.97 mmol) in 10 mL of THF under nitrogen atmosphere was

added 1.0 M lithium alumina hydride solution in THF (4.0 mL, 4.0 mmol). The resulting



S7

mixture was stirred for 2 h at reflux. The reaction was then cooled in an ice bath, quenched with

water, diluted with ethyl acetate and filtered. The filtrate was washed with saturated bicarbonate

solution, water, and brine. The organic phase was dried over magnesium sulfate, filtered, and

concentrated to give an oil, which was purified by flash chromatography

(CH2Cl2:CH3OH:NH4OH 10:1:0.1) to give 3-[1,1'-biphenyl]-4-propylamine as a clear oil (147

mg, 0.66 mmol, 34%):1H NMR (300 MHz, CDCl3) δ 7.59 (d, 2H, J = 7.5 Hz), 7.53 (d, 2H, J =

7.8 Hz), 7.44 (t, 2H, J = 7.65 Hz), 7.33 (m, 1H), 7.27 (d, 2H, J = 7.5 Hz), 2.77 (b, 2H), 2.71 (t,

2H, J = 7.65 Hz), 1.99 (b, 2H), 1.82 (m, 2H); MS (ESI) m/z 212 (M+H)+; Anal. Calcd. for

C15H17 N.


biphenyl]-4-ylpropyl)-2,6-methano-3-benzazocine-8-carboxamide (18).1H NMR (500 MHz,

CDCl3) δ 7.68 (s, 1H), 7.57 (d, 2H, J = 7.5 Hz), 7.52 (d, 2H, J = 7.5 Hz) 7.43 (t, 2H, J = 7.75

Hz), 7.32 (m, 4H), 7.05 (d, 1H, J = 7.5 Hz), 6.09 (bt, 1H), 3.52 (q, 2H, J = 6.7 Hz), 3.13 (m, 1H),

2.93 (d, 1H, J = 19 Hz), 2.77 (t, 2H, J = 7.75 Hz), 2.67 (m, 2H), 2.45 (dd, 1H, J 1 = 6.0 Hz, J 2

=12.5 Hz), 2.30 (dd, 1H, J 1 = 6.75 Hz, J 2 =12.25 Hz), 1.93 (m, 5H), 1.41 (s, 3H), 1.32 (m, 1H),

0.85 (m, 1H), 0.81 (d, 3H, J = 7.5 Hz), 0.51 (d, 2H, J = 8.0 Hz), 0.10 (m, 2H). MS (ESI) m/z

493 (M+H)+; Anal. Calcd. for C34H40 N2O·0.75H2O: C 80.67, H 8.26, N 5.53. Found: C 80.78,

H 8.12, N 5.51.


biphenyl]-4-ylethyl)- N

-methyl-2,6-methano-3-benzazocine-8-carboxamide (19).

1

H NMR

(500 MHz, CDCl3) δ 7.56 (m, 4H), 7.43 (m, 3H), 7.39 (m, 1H), 7.33 (t, 2H, J = 6.75 Hz), 7.22 (s,

1H), 7.05 (d, 1H, J = 7.5 Hz), 3.80 (b, 1H), 3.48 (b, 1H), 3.14 (b, 3H), 3.04 (b, 1H), 2.90 (m,

3H), 2.70 (m, 2H), 2.47 (m, 1H), 2.32 (m, 1H), 1.93 (m, 3H), 1.35 (s, 3H), 1.30 (d, 1H, J = 12.5),



S8

0.84 (m, 1H), 0.84 (d, 3H, J = 6.5 Hz), 0.51 (d, 2H, J = 7.5 Hz), 0.12 (m, 2H). MS (ESI) m/z 493

(M+H)+; Anal. Calcd. for C34H40 N2O·0.13H2O: C 82.51, H 8.20, N 5.66. Found: C 82.33, H

8.07, N 5.69.

(±)-3-(Cyclopropylmethyl)-6-ethyl-1,2,3,4,5,6-hexaahydro- cis-11-methyl- N -(2-[1,1'-

biphenyl]-4-ylethyl)-1-oxo-2,6-methano-3-benzazocine-8-carboxamide (21).1H NMR (500

MHz, CDCl3) δ 8.00 (d, 1H, J = 8.0 Hz), 7.82 (s, 1H), 7.58 (m, 4H), 7.51 (d, 2H, J = 8.0 Hz)

7.44 (t, 2H, J = 7.5 Hz), 7.33 (m, 3H), 6.19 (bt, 1H), 3.77 (q, 2H, J = 6.5 Hz), 3.32 (d, 1H, J =

8.0 Hz), 3.00 (t, 2H, J = 6.75 Hz) 2.92 (dd, 1H, J 1 = 3.75 Hz, J 2 =12.25 Hz), 2.65 (dd, 2H, J 1 =

5.75 Hz, J 2 =8.25 Hz), 2.36 (m, 1H), 2.29 (m, 1H), 2.10 (m, 1H), 1.97 (dd, 1H, J 1 = 7 .5 Hz, J 2 =

13.0 Hz), 1.90 (m, 1H), 1.82 (m, 1H), 1.24 (d, 1H, J = 12.0 Hz), 1.05 (t, 3H, J = 7.75 Hz), 0.87

(m, 1H), 0.79 (d, 3H, J = 7.0 Hz), 0.48 (m, 2H), 0.26 (m, 1H), 0.01 (m, 1H). MS (ESI) m/z 507

(M+H)+; Anal. Calcd. for C34H38 N2O2·1.35H2O: C 76.91, H 7.73, N 5.28. Found: C 76.89, H

7.48, N 4.89.



S9

COMBUSTION DATA

calculated values found

compound formula C H N C H N

7 C23H28 N2O4 .0.50 H2O 68.20 7.20 6.90 68.04 6.92 7.12

10 C26H32 N2O.0.25 H2O 79.45 8.33 7.12 79.16 8.15 7.05

11 C27H34 N2O.0.50 H2O 78.79 8.57 6.81 78.90 8.55 6.86

12 C28H36 N2O.0.33 H2O 79.58 8.75 6.63 79.71 8.75 6.66

13 C31H34 N2O.1.25 H2O 78.70 7.78 5.92 78.85 7.56 5.73

14 C32H36 N2O

.

0.50 H2O 81.14 7.87 5.91 80.91 7.77 5.81

15 C33H38 N2O.1.00 H2O 79.80 8.12 5.64 79.72 8.07 5.96

(-)-16 C33H38 N2O.1.25 H2O 79.08 8.14 5.59 79.23 7.84 5.57

(+)-17 C33H38 N2O.0.67 H2O 80.78 8.07 5.71 80.96 8.13 5.45

18 C34H40 N2O.0.75 H2O 80.67 8.26 5.53 80.78 8.12 5.51

19 C34H40 N2O.0.25 H2O 82.13 8.21 5.63 82.33 8.07 5.69

21 C34H38 N2O2 . 1.35 H2O 76.91 7.73 5.28 76.89 7.48 4.89

Radiolabeled Ligand Binding Assays. Binding assays used to screen compounds are similar to

those previously reported.8

Membrane protein from CHO cells that stably expressed one type of

the human opioid receptor were incubated with 12 different concentrations of the compound in

the presence of either 1 nM [3H]U69,593

10(κ ), 0.25 nM [

3H]DAMGO

11(μ) or 0.2 nM

[3H]naltrindole

12(δ) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25

oC. Incubation

times of 60 min were used for [3H]U69,593 and [

3H]DAMGO. Because of a slower association

of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples



S10

incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl

fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was

terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using

a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of

cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For

[3H]naltrindole and [

3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for

at least 60 min before use. IC50 values were calculated by least squares fit to a logarithm-probit

analysis. K i values of unlabeled compounds were calculated from the equation K i = (IC50)/1+S

where S = (concentration of radioligand)/(K d of radioligand).

13

Data are the mean ± SEM from

at least three experiments performed in triplicate.

[35

S]GTPγS Binding Assays. In a final volume of 0.5 mL, 12 different concentrations of each

test compound were incubated with 15 μg (κ ), 10 μg (δ) or 7.5 μg (μ) of CHO cell membranes

that stably expressed either the human κ , δ or μ opioid receptor. The assay buffer consisted of 50

mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 μM GDP, and 100 mM NaCl. The final

concentration of [35

S]GTPγS was 0.080 nM. Nonspecific binding was measured by inclusion of

10 μM GTPγS. Binding was initiated by the addition of the membranes. After an incubation of

60 min at 30oC, the samples were filtered through Schleicher & Schuell No. 32 glass fiber filters.

The filters were washed three times with cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL

of Ecoscint scintillation fluid. Data are the mean Emax and EC50 values ± S.E.M. from at least

three separate experiments, performed in triplicate. For calculation of the Emax values, the basal

[35

S]GTPγS binding was set at 0%. To determine antagonist activity of a compound at the μ

opioid receptors, CHO membranes expressing the μ opioid receptor, were incubated with 12



S11

different concentrations of the compound in the presence of 200 nM of the μ agonist DAMGO.

To determine antagonist activity of a compound at the κ opioid receptors, CHO membranes

expressing the κ opioid receptor, were incubated with the compound in the presence of 100 nM

of the κ agonist U50,488. To determine if a compound was an antagonist at δ receptors, CHO

membranes expressing the δ receptor were incubated with 12 different concentrations of the test

compound in the presence of 10 nM of the δ -selective agonist SNC 80.

References:

1. Part 1: Wentland, M. P.; Lou, R.; Ye, Y.; Cohen, D. J.; Richardson, G. P.; Bidlack, J. M.

8-

Carboxamidocyclazocine Analogues: Redefining the Structure-Activity Relationships of

2,6-Methano-3-benzazocines. Bioorgan. Med. Chem. Lett. 2001, 11, 623-626.

2. Aldrich, J. V.; Vigil-Cruz, S. C. In Burger’s Medicinal Chemistry and Drug Discovery, 6th

ed.; Abraham, D. J., Ed.; John Wiley & Sons: New York, 2003; Vol. 6, pp 329-481.

3. Archer, S.; Glick S. D.; Bidlack, J. M. Cyclazocine Revisited. Neurochem. Res. 1996, 21,

1369-1373.

4. Bidlack, J. M.; Cohen, D. J.; McLaughlin, J. P.; Lou, R.; Ye, Y.; Wentland, M. P. 8-

Carboxamidocyclazocine: A Long-Acting, Novel Benzomorphan. J. Pharmacol. Exp.

Ther. 2002, 302, 374-380.

5. Wentland, M. P.; Ye, Y.; Cioffi, C. L.; Lou, R.; Zhou, Q.; Xu, G.; Duan, W.; Dehnhardt,

C. M.; Sun, X.; Cohen, D. J.; Bidlack, J. M. "Syntheses and Opioid Receptor Binding

Affinities of 8-Amino-2,6-methano-3-benzazocines." J. Med. Chem. 2003, 46 , 838-849.

6. Hajduk, P.J.; Bures, M.; Praestgaard, J.; Fesik, S.E. "Privileged Molecules for Protein

Binding Identified from NMR-Based Screening." J. Med. Chem. 2000, 43, 3443-3447.



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7. Lou, R.; VanAlstine, M.; Sun, X.; Wentland, M. P. Preparation of N-hydroxysuccinimido

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