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Bioorg Med Chem Lett. Author manuscript; available in PMC 2013 December 15.
Published in final edited form as:
PMCID: PMC3508289

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 8. High affinity ligands for opioid receptors in the picomolar Ki range: Oxygenated N-(2-[1,1′-biphenyl]-4-ylethyl) analogues of 8-CAC


N-[2-(4′-methoxy[1,1′-biphenyl]-4-yl)ethyl]-8-CAC (1) is a high-affinity (Ki = 0.084 nM) ligand for the μ opioid receptor and served as the lead compound for this study. Analogues of 1 were made in hopes of identifying an SAR within a series of oxygenated (distal) phenyl derivatives. A number of new analogues were made having single-digit pM affinity for the μ receptor. The most potent was the 3′,4′-methylenedioxy analogue 18 (Ki = 1.6 pM).

The broad goal of our research is to identify orally-available, long-acting modulators of μ and κ opioid G-protein coupled receptors as potential anticocaine medications. In 2001, we reported the synthesis and biological properties of 8-carboxamidocyclazocine (8-CAC; 2 in Figure 1), an opioid modulator having a novel carboxamido (CONH2) group at the 8-position of the 2,6-methano-3-benzazocine core in place of the prototypic (of opioids) phenolic-OH of cyclazocine (3).1 At that time, cyclazocine was undergoing clinical evaluation to treat cocaine addiction, however, it was beset by having a short duration of action in humans.2,3 8-CAC displays similar binding affinities to μ and κ receptors as cyclazocine and has a much longer duration of action in a mouse antinociception model,4 however; its utility as an anticocaine medication is questioned by having an undesirable side effect profile.5

While studying the effects of N-substitution of the carboxamido group of 8-CAC on binding affinity, we observed that affinity for opioid receptors significantly decreased upon mono-substitution (e.g., 8-CONHCH3, 8-CONHPh) or di-substitution (e.g., 8-CON(CH3)2).1 The discovery that an N-(2-[1,1′-biphenyl]-4-ylethyl) (a.k.a. N-BPE in this work) appendage (i.e., 4) gave comparable binding affinity to μ as the unsubstituted carboxamide was clearly an unexpected finding based on our previous experience.6 Preliminary SAR studies regarding the substitution of the distal phenyl ring of 4 subsequently revealed that a 4′-methoxy group was very beneficial (3.7-fold decrease in Ki value) for binding affinity to μ and it is this derivative 1 that served as the lead compound for the current study. Herein we report the syntheses and pharmacological characterizations of oxygenated (distal) phenyl analogues of 1 to probe the SAR of this exciting series of high affinity ligands.

As shown in the Scheme 1, novel target compounds 5-13 and 15-20 were made from the racemic 4-bromophenethyl intermediate 237 using standard Suzuki coupling conditions (Methods A or B) in yields of 14–61%. All reagents, including boronic acids or esters, were commercially available. The optically active enantiomer 14 of racemate 13, was made from the [(−)-[(2R,6R,11R)]]-triflate ester of cyclazocine8 via a procedure very similar to that used to make 13. The last coupling step was accomplished in 22% yield using Method B. Lastly, the methoxy-substituted naphthalene analogue 21 was prepared from the triflate ester (24) of cyclazocine8 and 6-methoxy-2-naphthaleneethanamine9 using the known conditions7 summarized in Scheme 2.

Target compounds were evaluated for their affinity and selectivity for human μ, δ and κ opioid receptors stably expressed in Chinese hamster ovary (CHO) cell membranes. Data are summarized in Table 1. For comparison purposes, literature opioid receptor binding affinity data for lead compound 1, 8-CAC (2), cyclazocine (3) and the unsubstituted N-BPE derivative 4 are included.7 High affinity binding to the μ receptor was observed for all new N-BPE target compounds 5-20. With one exception, Ki values were < 1.0 nM and five compounds had values that were single digit picomolar. New compounds 5-20 displayed higher selectivity for the μ receptor over δ and κ receptors (see selectivity ratios in Table 1). Analogue 18 exhibited the highest selectivity for μ having a μ:δ:κ Ki ratio of 1:625:456. There was little consistency in this group of compounds as far as selectivity between δ and κ.

Table 1
Comparative opioid receptor binding data for 2,6-methano-3-benzazocine derivatives.

In the first set of structural variations, we studied the effect of methoxy substitution at different sites on the distal phenyl ring on binding affinity at μ. The 3′-derivative 5 was 5-fold more potent than the 4′-substituted lead 1 while the 2′-methoxy derivative 6 and 1 were equipotent. We also studied the effects of other alkoxy substituents at the 4′-position. Of the variations studied, the methoxy derivative 1 was 7.6-, 2.7-, 21- and 3.6-fold more potent than those analogues with 4′ substituents being OCH2CH3 (7), OCH(CH3)2 (8), OCF3 (11), and OCHF2 (12), respectively. The only mono-4′-sustituted compound to show better potency than lead 1 was the des-alkyl derivative 13. The Ki value for this 4′-OH analogue 13 is a notable 0.0056 nM which represents a 15-fold increase in binding affinity at μ compared to 1.

We previously reported the eudismic ratio of the enantiomers of the original lead N-BPE compound 4 was 26 at the μ receptor with the (−)-[(2R,6R,11R)]-enantiomer being the eutomer.6 Since SAR in this series was tracked using racemates, we wanted to confirm in at least one example, that the bulk of the observed activity of a racemate resided in (2R,6R,11R)-enantiomer. To that end, we made and tested the (−)-[(2R,6R,11R)]-enantiomer 14 of the highly potent 4′-hydroxy racemic derivative 13 and found Ki values of the optically active partner slightly less than those of the racemate against all three opioid receptors. Within the variability of the assay where a 2-fold difference in Ki values is not considered significantly different, these data confirm that, in at least one example, the (2R,6R,11R)-enantiomer exhibits very high potency.

For the 4′-isopropoxy and 4′-OH derivatives, 8 and 13, respectively, we also studied the attachment of the group at the 3′- and 2′-positions on the distal phenyl. In contrast to the trio of methoxy analogues 1, 5 and 6 where the 3′-derivative was the most potent, in the hydroxy case, the 3′-derivative 15 was least potent having a Ki value 4-fold higher than both the 4′-OH (13) and 2′-OH (16) regioisomers. For the isopropoxy substituent, the Ki value for the 4′-isomer 8 was within 2-fold of the values for the 3′- and 2′-isomers, 9 and 10, respectively.

We also studied the effects of 3′,4′- and 2′,4′-disubstitution on the distal phenyl ring. Addition of a methoxy group at the 3′-position of 1 to provide 17 resulted in a 12-fold increase in binding affinity at the μ receptor. An even greater enhancement of potency was realized when the 3′,4′-methylenedioxy group was introduced on the distal phenyl ring. The resulting compound, 18, exhibited a Ki = 0.0016 nM which represents a 53-fold increase in potency relative to 1. We also studied methyl substitution at the 3′- and 2′-positions of lead 1 giving targets 19 and 20, respectively. Compared to 1, binding affinity at μ for 19 was comparable and was slightly deceased (2.7-fold) for 20.

In our original report7 describing the discovery of lead compound 1, we also provided data for the highly potent N-[2-(2-naphthalenyl)ethyl]-analogue 22 (Ki = 0.18 nM Table 1). In the current study, we investigated the effect of methoxy substitution at the 6′-position on the naphthalene ring of 22 resulting in 21. Compared to 22, binding affinity of 21 for μ increased 2.8-fold.

A number of target compounds were also evaluated in [35S]GTPγS assays for functional activity at the μ, κ and δ opioid receptors (Table 2). Like lead compound 1, all new N-BPE derivatives tested (5, 8, 14, 16-19) were partial agonists at the μ receptor, however, potencies for receptor stimulation (EC50’s) were considerably more potent (4- to 200-fold) than 1. In contrast to the partial agonist activity observed for the N-BPE derivatives, the 6′-methoxy naphthalene derivative 21 was an antagonist of moderate potency (IC50 = 45 nM). Like lead compound 1, new compounds tested were agonists at the δ receptor with EC50 values in the 0.16 to 18 nM range. One exception is compound 17 which was a partial agonist at the δ receptor. Compound 17 had an Imax value of 32% in inhibiting agonist-stimulated [35S]GTPγS binding. When tested alone, compound 17 had an Emax value of 73%. These data demonstrate that compound 17 had both agonist and antagonist properties. A compound that produced both agonist and antagonist activity in our assay was regarded as a partial agonist. At the κ receptor, all compounds tested, included 1, were agonists with EC50 values in the 0.086 to 5.4 nM range.

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, δ and κ opioid receptors.a

Certain trends in the SAR emerged during the course of this study. For 4′-monosubstiuted N-BPE analogues, the rank order (from highest to lowest) of potency of these compounds (plus two from an earlier study7) is the following: OH (13) > OCH3 (1) > OCH(CH3)2 (8) ~ Cl7 ~ CH37 ~ OCHF2 (12) > OCH2CH3 (7) > OCF3 (11). While certainly not a perfect correlation, a trend is evident that electron donating groups are beneficial for binding to the μ receptor. This trend is corroborated by the observation that addition of an alkoxy group at the 3′-position of 1 significantly enhances binding affinity (i.e., 3′,4′-OCH2O- (18) > 3′,4′-OCH3 (17) [dbl greater-than sign] 4′-OCH3 (1)). Despite the fact that substituents are tolerated at the 2′- or 3′-positions of the N-BPE group, however, substitution of 1 with a mildly electron donating methyl group whether it is at the 3′-position (19) or 2′-position (20) provides no benefit. From this limited data set, there is a tendency that a single electron donating alkoxy or hydroxy group is beneficial at any position on the distal phenyl ring as evidenced by the following rank orders: when Y = OCH3 (3′ > 4′ ~ 2′); Y = OCH(CH3)2 (4′ ~ 2′ ~ 3′); and Y = OH (4′ ~ 2′ > 3″). Also, SAR appears to be additive regarding the benefit of a methoxy group on the distal part of the N-substituent as evidenced by 4′-OCH3 (1) > 4′-H (4) by 3.7-fold when N-BPE and 4′-OCH3 (21) > 4′-H (22) by 2.8-fold when N-2-naphthaleneethyl.

In conclusion, valuable insights into opioid SAR have been made by studying the opioid receptor binding properties of a series of 8-CAC analogues where the distal regions of the carboxamide N-substituent have been modified. In doing so, we have discovered a number of novel derivatives having single-digit pM Ki values for the μ receptor. We continue to explore the SAR of lead compound 1 to corroborate/refute our current hypothesis that the BPE group of 1 occupies previously unexplored receptor space in opioid receptors that is largely hydrophobic in nature and deep within this pocket lies the potential for polar non-covalent interactions (e.g., π-cation, H-bond acceptor/donor) responsible for enhancing binding affinity.


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).


Presented in part, as Bidlack, J. M.; Cohen, D. J.; Gargano, J.; Jia, X.; Jo, S.; VanAlstine, M. A.; Wentland, M. P. Abstracts of Papers, 240th National Meeting of the American Chemical Societ, Boston, MA, 2010; Abstract MEDI-110.

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References and notes

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