Discovery Of Benzhydrylpiperazine Opioid Receptor Ligands

In the late 1970s, the molecular biology and pharmacology of opioid receptors were under investigation in the laboratories of Kwen-Jen Chang at Burroughs Wellcome. Early research into delta receptor biology relied on peptidic ligands, such as the enkephalins, as molecular tools. The metabolic instability, formulation difficulties, and in vivo absorption and distribution characteristics made these suboptimal tools for in-depth in vivo studies. The availability of rat brain membranes expressing the delta receptor to evaluate ligand binding, and the use of mouse vas deferens tissue to identify the functional response of delta-receptor ligands made it possible to search for structurally novel opioids. However, the ability to screen large numbers of compounds against a receptor did not exist as it does today. Careful selection of compounds for in vitro evaluation was necessary.

The Burroughs Wellcome compound collection was carefully studied in search of structures that might have opioid activity. Medicinal chemists considered the structures of the morphinoids as well as the enkephalins in choosing novel compounds to test for delta receptor activity. Among the small set of molecules selected for testing were benzhydrylpiperazines. Structures reminiscent of morphinoids would include a phenol and a basic amine (a tyramine-like pharmacophore) with significant structural constraint. The initial benzhydrylpiperazines sent for testing preserved this motif. Additionally, Leu- and Met-enkephalins (Tyr-Gly-Gly-Phe-Leu and Tyr-Gly-Gly-Phe-Met, respectively), delta receptor agonists that were known at that time, have a second phenyl ring (part of a phenylalanine residue), as do the benzhydrylpiperazines. Compounds were evaluated for opioid receptor affinity via displacement of radiolabeled ligands from rat brain membranes (RBM) [17a,b]. Function activity was evaluated via inhibition of electrically stimulated twitch in mouse vas deferens (MVD) and guinea pig ileum (GPI) tissue preparations for the delta opioid and mu opioid receptors, respectively [18]. This initial testing identified a relatively simple benzhydrylpiperazine, compound 4a (Fig. 3), as a selective delta opioid receptor antagonist (mu receptor IC50 = 7000 nM, delta receptor IC50 = 200 nM, pA2 from mouse vas deferens = 5.7). Simple manipulation of the substitution on the piperazine nitrogen distal to the 4'-chloro-3-benzhydrol provided interesting structure-activity data. Removal of the methyl group as well as replacement with an ethyl group (compounds 4b and 4c, respectively) provided similar delta opioid receptor antagonists. Lengthening the chain to «-propyl and «-butyl incrementally reduced delta opioid receptor affinity (4d and 4e, respectively), with the «-butyl compound having a delta receptor IC50 of 1.5 |aM. The N-allylpiperazine analogue, compound 4f, provided an exciting result. Delta opioid receptor affinity had increased (IC50 = 50 nM), and the mouse vas deferens assay revealed this compound to be a relatively weak but effective delta receptor agonist (MVD ED50 = 2 ||M). The N-allylpiperazine present in this first agonist would prove to be a key structural feature, present in many of the potent benzhydrylpiperazine opioid agonists produced at Burroughs Wellcome. After the identification of this first

Compounds 4a-f Figure 3 Early benzhydrylpiperazine opioids.

benzhydrylpiperazine delta receptor agonist, medicinal chemistry research focused on developing the SAR to discover highly potent, selective delta receptor agonists.

2.1 Early SAR of Benzhydrylpiperazine Opioids

Early medicinal chemistry efforts in the benzhydrylpiperazine series focused on structural modifications to explore opioid receptor potency and selectivity, with a goal of identifying potent, selective delta opioid receptor agonists for potential therapeutic use in the treatment of severe pain. Potent antagonists were also desirable as valuable pharmacological tools. With compound 4f in hand and initial SAR indicating the value of the allyl group for agonist activity, much of the early structural exploration focused on two areas: 1) additional small alkyl substitution on the piperazine ring; 2) substitution on and heterocyclic replacement of the nonphenolic aryl ring. Based on the precedent of the morphinoids and enkephalins, the phenolic —OH was hypothesized to be valuable for optimal opioid potency and was therefore a common feature of most of the early benzhydrylpiperazine analogues. While the most potent compounds to date retain the phenolic —OH, we now know that it is not critical for obtaining potent and selective delta-opioid receptor agonists.

With the allyl group on the piperazine providing delta opioid receptor agonism, substitution on the carbons of the piperazine ring was explored. For example, N-allyldimethylpiperazine analogues were prepared, and the placement and relative stereochemistry (early in the program, mixtures of stereo-isomers were often tested) of the two methyl groups affected delta opioid receptor potency and selectivity (see Table 1). Interesting analogues include a direct analog of 4f containing a czs-3,5-dimethyl-N-allylpiperazine substituent that was a modestly potent, selective delta receptor antagonist (5a). The analogue featuring a czs-2,5-dimethyl substitution pattern (5b) had slightly weaker delta opioid receptor binding affinity and less delta selectivity, and was not evaluated for functional activity. The czs-2,3-dimethyl analogue (5c) exhibited potent, selective affinity for the delta opioid receptor (IC50 = 10 nM, 300-fold selective vs. the mu receptor), but displayed weak agonist activity in the mouse vas deferens assay (ED50 = 20 ||M). The trans-2,3-dimethyl analogue (5d) possessed much more delta opioid agonism, with an ED50 of ~ 0.4 |aM. A breakthrough was realized with the trans-2,5-dimethyl analogue, which provided a more potent, selective delta opioid agonist. The two pairs of enantiomers were separated for this compound, and the more potent pair (5e) displayed delta opioid agonist activity with an ED50 of 40 nM in the MVD assay, with 20-fold tissue selectivity. While preferred absolute stereochemistry was addressed in a later series of analogs, these results sug-

TABLE 1 Delta and Mu Opioid Activity of Dimethylpiperazine Analogues3

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