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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Bioorg Med Chem Lett. Author manuscript; available in PMC Dec 1, 2008.
Published in final edited form as:
PMCID: PMC2137165
Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 5. Opioid receptor binding properties of N-((4′-phenyl)-phenethyl) analogues of 8-CAC
Melissa A. VanAlstine,a Mark P. Wentland,a* Dana J. Cohen,b and Jean M. Bidlackb
aDepartment of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
bDepartment of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA
*Corresponding author. Tel.: +1 518 276 2234; fax: +1 518 276 4887; e-mail: wentmp/at/
A series of aryl-containing N-monosubstituted analogues of the lead compound 8-[N-((4′-phenyl)-phenethyl)]-carboxamidocyclazocine were synthesized and evaluated to probe a putative hydrophobic binding pocket of opioid receptors. Very high binding affinity to the μ opioid receptor was achieved though the N-(2-(4′-methoxybiphenyl-4-yl)ethyl) analogue of 8-CAC. High binding affinity to μ and very high binding affinity to κ opioid receptors were observed for the N-(3-bromophenethyl) analogue of 8-CAC. High binding affinity to all three opioid receptors were observed for the N-(2-naphthylethyl) analogue of 8-CAC.
We recently reported the synthesis and opioid receptor binding properties of 1, an analogue of 8-carboxamidocyclazocine (8-CAC, 2), having an N-((4′-phenyl)-phenethyl) substitution.1 8-CAC is a long-acting2 analogue of cyclazocine (3)3 with high affinity for μ and κ opioid receptors4 having the potential to treat cocaine addiction in humans. Based on the long-standing knowledge that a phenolic hydroxyl group was required for the high affinity binding of many opioid-receptor interactive ligands,5 8-CAC's high affinity for μ and κ opioid receptors was unexpected. Until recently all substitution of the carboxamide nitrogen of 2 was detrimental towards opioid receptor binding except N-((4′-phenyl)-phenethyl) (1), which produced high binding affinity to μ and δ, and moderate affinity to κ.1
To further probe opioid receptor space for what we believe contains a putative hydrophobic pocket complementary to the aryl groups on the 8-position of 1, we now report the synthesis and opioid receptor binding properties of a series of N-mono-substituted carboxamide analogues of 8-CAC (Table 1). Design of targets was based on the substitution on either of the aryl rings, both in its nature and its placement with respect to the ethylene linker. Specifically, we chose derivatives with different attachment points, substituents and analogues where the aryl group was switched from biphenyl to the naphthyl or bromophenyl.
Table 1
Table 1
Comparative opioid receptor binding data for 2,6-methano-3-benzazocine derivatives.
Using a one-step procedure (Scheme 1), novel racemic targets 7-9, 11 and 14-19 were conveniently made by treatment of triflate 46 with the appropriate amine, dichloro[1,1′-bis(diphenylphosphino)-ferrocene] palladium (II) dichloromethane adduct, triethylamine, and carbon monoxide in dimethylsulfoxide. Amines were commercially available or made using known procedures.
Scheme 1
Scheme 1
Syntheses of target compounds via Pd-catalyzed carboxamidation procedures. Reagents and conditions: (i) RNH2, PdCl2(dppf), Et3N, DMSO, CO, 70°C
As shown in Scheme 2, targets 5 and 6 were prepared by treating 8 and 9, respectively, with phenylboronic acid, palladium acetate, triphenylphosphine, and sodium carbonate in toluene (microwaves) at 120 °C for 20 minutes. Targets 10, 12 and 13 were similarly prepared from 7 using 4-methoxyphenylboronic acid, 3,4-dichlorophenylboronic acid and 4-methylphenylboronic acid, respectively. Yields in these Suzuki couplings were in the 64-80 % range.
Scheme 2
Scheme 2
Syntheses of target compounds via Suzuki coupling. Reagents and condition: (i) Pd(OAc)2, PPh3, Na2CO3, tol, microwaves (20W), 20 min, 120 °C.
Target compounds were evaluated for their affinity and selectivity for μ, δ and κ opioid receptors stably expressed in Chinese hamster ovary (CHO) cell membranes.7 Data are summarized in Table 1. All compounds in Table 1 are racemic including cyclazocine. For comparison purposes, opioid binding affinity data for 8-[N-((4′-phenyl)-phenethyl)]-CAC (1),1 8-CAC (2),4 and cyclazocine (3)3 are included. As stated previously, N-((4′-phenyl)-phenethyl) substitution on the carboxamide group of 8-CAC had similar binding affinity for μ, slightly weaker for δ and 30-fold less affinity for κ opioid receptors, compared to 8-CAC (2). The optimal linker length between the carboxamide nitrogen and the biphenyl group has been shown to be a two-methylene spacer.1 To test the optimal orientation of the distal phenyl group, the N-((3′-phenyl)-phenethyl) and N-((2′-phenyl)-phenethyl) analogues 5 and 6, respectively, were synthesized. When the distal phenyl group was in the 3-position (5), binding affinity decreased only slightly for μ, δ, and κ (3-, 8- and 1.2-fold, respectively) compared to 1. However, when the distal phenyl group is in the 2-position (6) binding affinity is significantly decreased by 22-, 28-, and 1.3-fold for μ, δ, and κ receptors, respectively. These data suggest the receptors can accommodate the distal phenyl in the 2- and 3-positions; however, the 4-position is optimal.
To ascertain whether the role of the distal phenyl group in binding was mainly hydrophobics, pi-pi stacking or both, the corresponding bromophenyl analogues 7-9 were synthesized. When the distal phenyl group of 1 was replaced by bromide (7), binding affinity was reduced for μ and δ, 8- and 3-fold, respectively but increased 5-fold for κ. This was the first instance of subnanomolar potency for the κ opioid receptor in this series. The 2- and 3-bromophenethyl analogues of 8-CAC were synthesized as well. Compared to 1, the 3-bromophenethyl analogue 8 showed comparable affinity for μ, 5-fold decreased affinity for δ and 29-fold increased affinity for κ. With a binding affinity of 0.063 nM, compound 8 had had comparable affinity for the κ receptor as cyclazocine (3). The 2-bromophenethyl analogue 9 had decreased affinity for μ, δ, and κ receptors, 13-, 203-, and 10-fold, respectively, compared to 1.
The Topliss approach8 was applied to this series and the 4′-methoxy, 4′-chloro, 3′,4′-dichloro, and 4′-methyl analogues of 1 were synthesized. The 4′-methoxy analogue 10 had 4-fold increased binding affinity for μ, and δ opioid receptors compared to 1 while against κ receptors, similar affinity was observed. The 4′-chloro and 4′-methyl analogues, 11 and 13, respectively, had very similar binding to all three opioid receptors compared to 1. The 3′,4′-dichloro analogue 12 showed 3-fold lower affinity to both μ and δ opioid receptors and similar affinity to the κ opioid receptor compared to 1. From this activity pattern at the μ receptor, the probable operative parameter is deduced to be -π, implying that a more hydrophilic group would increase potency.
A series of naphthyl derivatives of 1 were made to further probe this putative hydrophobic interaction with the receptors. The 2-naphthylmethyl analogue 14 showed decreased binding affinity for μ, δ, and κ (4-, 42- and 11-fold, respectively). The 2-naphthylethyl analogue 15 showed similar binding affinity to μ and δ opioid receptors compared to 1 and affinity increased by 9-fold for the κ opioid receptor. The 2-naphthylpropyl analogue 16 had decreased affinity for μ and δ, 6- and 24-fold, respectively, but increased affinity (10-fold) for κ. All three 1-naphthyl analogues of 8-CAC, 1-naphthylmethyl (17), 1-naphthylethyl (18), and 1-naphthylpropyl (19) had decreased affinity for μ (8- to 14-fold), δ (24- to 70-fold), and κ (1- to 10-fold) opioid receptors compared to 1.
In [35S]GTPγS functional assays (Table 2),1 compound 1 showed antagonist properties at μ while the 4-bromo analogue 7 showed only agonist properties at μ receptor. Compounds 5, 6, 8, 10, 11 and 16 showed partial agonist properties as well as antagonist properties at μ receptor. All of the compounds were pure agonists at the κ opioid receptor. They had similar Emax values, ranging from 87 – 110% stimulation over control, which were slightly greater than the Emax value of 77% stimulation produced by the κ-selective agonist U50,488. There was not a strong correlation between the EC50 values for stimulating [35S]GTPγS binding mediated by the κ receptor and the Ki values for the inhibition of [3H]U69,593 binding to the κ receptor. For example, compounds 8 and 16 had the highest affinity for the κ receptor in the receptor binding assay. In the [35S]GTPγS binding assay, compounds 1, 8, 10, 11, and 16 had similar EC50 values. Due to their lower affinity for the δ receptor, compounds 6 and 16 were not tested in the [35S]GTPγS binding assay in CHO membranes expressing the δ opioid receptor. As observed with the κ receptor, compounds 1, 5, 7, 8, 10 and 11 were shown to be pure agonists at the δ opioid receptor. Compound 10, which had the highest binding affinity for the δ receptor within the series, also was the most potent at δ in the [35S]GTPγS assay. Other than this observation, there was little correlation at the δ opioid receptor between binding affinity and functional activity.
Table 2
Table 2
EC50 and Emax values for the stimulation of [35S]GTPγS binding and IC50 and Imax values for the inhibition of agonist-stimulated [35S]GTPγS binding to the human μ, κ and δ opioid receptors.a
Valuable insights into the SAR of the 8-position substituent of 8-CAC have been made by examination of the opioid receptor binding properties of a series of N-monosubstituted carboxamide analogues of 8-CAC. Our observation that the N-((3′-phenyl)-phenethyl) and N-((2′-phenyl)-phenethyl) groups were less potent then the N-((4′-phenyl)-phenethyl) analogue leads us to believe that the alkyl biaryl groups need to be in a near-linear arrangement. The loss in activity in the 4-bromophenylethyl analogue 7 compared to 1, suggests that molecular recognition may not be purely hydrophobic in nature but could also involve pi-pi stacking. Within this series of N-monosubstituted carboxamide analogues of 8-CAC, the 3-bromophenylethyl analogue 8 was the first in this series to show subnanomolar affinity to the κ receptor. Application of the Topliss approach showed the physicochemical parameter -π appears to be the most important physicochemical parameter for binding affinity. This information will guide future efforts. Evaluation of napthyl derivatives 14-19 showed that binding affinity was much higher when the naphthyl ring was attached to the linker via the 2-position and that the two carbon linker (ethylene) was optimal.
Results from this study will facilitate the design of new high affinity opioid receptor ligands. The synthesis and evaluation of new targets related to 1 is ongoing in our laboratories to further explore this novel SAR. New targets will include those analogues with a diverse array of (hetero) aryl groups on the 8-carboxamido group of 2,6-methano-3-benzazocines as well as the corresponding 3-carboxamido morphinans and 4,5α-epoxymorphinans.
Figure 1
Figure 1
Structures of lead compounds for this study.
We gratefully acknowledge the contributions of Rensselaer's mass spectroscopist Dr. Dmitri Zagorevski and the technical assistance provided by Brian I. Knapp of the University of Rochester. Funding of this research was from NIDA (DA12180 and KO5-DA00360) and the NSF (Agilent 1100 series LC/MSD system).
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References and notes
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3. Archer S, Glick SD, Bidlack JM. Neurochem Res. 1996;21:1369. [PubMed]
4. Wentland MP, Lou R, Ye Y, Cohen DJ, Richardson GP, Bidlack JM. Bioorgan Med Chem Lett. 2001;11:623.
5. Aldrich JV, Vigil-Cruz SC. In: Burger's Medicinal Chemistry and Drug Discovery. 6th. Abraham DJ, editor. Vol. 6. John Wiley & Sons; New York: 2003. pp. 329–481.
6. Wentland MP, Ye Y, Cioffi CL, Lou R, Zhou Q, Xu G, Duan W, Dehnhardt CM, Sun X, Cohen DJ, Bidlack JM. J Med Chem. 2003;46:838. [PubMed]
7. Neumeyer JL, Zhang A, Xiong W, Gu X, Hilbert JE, Knapp BI, Negus SS, Mello NK, Bidlack JM. J Med Chem. 2003;46:5162. [PubMed]
8. Topliss JG. J Med Chem. 1977;20:463. [PubMed]