Parallel Synthesis of Hexahydrodiimidazodiazepines Heterocyclic

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Parallel Synthesis of Hexahydro-Diimidazo Diazepines Heterocyclic Peptidomimetics and Their In Vitro and In Vivo activities Against µ- (MOR), #- (DOR), and #- (KOR) Opioid Receptors Shainnel O Eans, Michelle L Ganno-Sherwood, Eliza L Mizrachi, Richard A Houghten, Colette T. Dooley, Jay P. McLaughlin, and Adel Nefzi J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501637c • Publication Date (Web): 21 May 2015 Downloaded from http://pubs.acs.org on May 26, 2015

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Journal of Medicinal Chemistry

Parallel Synthesis of Hexahydro-Diimidazo Diazepines Heterocyclic Peptidomimetics and Their In Vitro and In Vivo activities Against µ- (MOR), δ- (DOR), and κ- (KOR) Opioid Receptors

Shainnel O. Eans, Michelle L. Ganno, Elisa Mizrachi, Richard A. Houghten, Colette T. Dooley, Jay P. McLaughlin, Adel Nefzi*

Torrey Pines Institute for Molecular Studies; 11350 SW Village Parkway; Port St. Lucie, FL 34987

KEYWORDS: heterocyclic peptidomimetics, parallel synthesis, combinatorial chemistry, solidphase synthesis, opioid receptors, delta-opioid receptor agonist, antinociception, peripherallyrestricted agonist.

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Abstract The development of analgesics with mixed-opioid agonist, peripherally-selective activity is expected to decrease side effects, minimizing respiratory depression and reinforcing properties generating significantly safer analgesic therapeutics. We synthesized diazaheterocyclics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for µ- (MOR), δ- (DOR), and κ- (KOR) opioid receptors across the series, with the diimidazo-diazepine 14 (2065-14) displaying good affinity for DOR and KOR. Central (i.c.v.), intraperitoneal (i.p.) or oral (p.o.) administration of 14 produced dose-dependent, opioidreceptor mediated antinociception in the mouse 55oC warm-water tail-withdrawal assay. Only trace amounts of compound 14 was found in brain up to 90 min later, suggesting poor BBB penetration and possible peripherally-restricted activity. Central administration of 14 did not produce locomotor effects, acute antinociceptive tolerance or conditioned-place preference or aversion. The data suggest these diazaheterocyclic mixed activity opioid receptor agonists may hold potential as new analgesics with fewer liabilities of use.


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INTRODUCTION

The modification of peptides to peptidomimetics has included the manipulation of peptide side chains, amino acid extensions, deletions and substitutions, and most recently backbone modification.1-8 The side chain groups of amino acid residues in polypeptide hormones, neurotransmitters, growth factors, substrates, antigens, and other bioactive peptides have been demonstrated to be extremely important pharmacophores for receptor binding and for signal transduction.1-8 Continuing with our work on the generation of heterocyclic compounds from modified peptides,7-8 we presently combined solid-phase and solution-phase synthesis to synthesize fused diimidazo-diazepines from reduced tripeptides, and then characterized their opioid activity with in vitro and in vivo assays. Nitrogen heterocycles of different ring sizes, with different substitution patterns and embedded in various molecular frameworks constitute important structure classes in the search for bioactivity. For example, the nitrogen-containing diazepine scaffold is an important pharmacophore that displays a wide range of biological activities 9including inhibitors of TNFalpha converting enzyme (TACE) and matrix metalloproteinase (MMP),10 antimicrobials,11 and inhibitors of human protein kinases and histamine H3 antagonists.12 Diazepam itself is used as an adjuvant analgesic to morphine for alleviating pain induced by the skeletal muscle spasms associated with painful vertebral metastases.13 To date, reported fused imidazo-diazepines display a wide range of biological activities including anti-hepatitis C virus (HCV) activity, inhibitory activity against the west Nile virus nucleoside triphosphatases (NTPase)/helicase,14 inhibition of adenosine deaminase,15 inhibition of guanase,16 and cannabinoid receptor modulators.17



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Known opioid analgesics such as morphine remain the gold standard in efficaciously treating many types of pain, but their clinical use is limited by side effects mediated primarily through centrally-located µ-opioid receptors (MOR),18 including respiratory depression, constipation and addiction. Attempting to retain opioid analgesia while limiting side effects, a number of agonists developed have selectively targeted δ- (DOR) and κ– (KOR) opioid receptors. Unfortunately, this strategy is limited by inherent side effects mediated by DOR (notably seizures) and KOR (notably aversion and psychomimetic effects) located in the CNS.19 In contrast, the development of efficacious compounds possessing mixed opioid agonist activity at some or all three of the opioid receptors has resulted in clinically useful medications,20-25

suggesting that new,

structurally diverse ligands with mixed opioid activity may prove to be effective new analgesics.18-25 Previous studies by our group have also shown the feasibility and utility of generating highly active compounds from peptidomimetics.7, 26-30 A number of low molecular weight compounds derived from modified peptides, have demonstrated good affinity for opioid receptors in radioligand competition binding assays.28-31 For instance, screening of permethylated chiral tetramines derived from resin-bound tripeptides identified permethylated YYF, a high affinity (0.5 nM), selective MOR antagonist.32 Likewise, the screening of a series of bicyclic guanidines derived from reduced tripeptides led to the identification of a bicyclic guanidine with good affinity and selectivity for the KOR.26 Recently, we reported the identification of two novel, potent, low-liability antinociceptive compounds, from the direct in vivo screening of a large mixture-based combinatorial library of pyrrolidine bis-cyclic guanidines derived from resin bound, proline containing acylated tetrapeptides,31 further suggesting the therapeutic potential of peptidomimetics. Finally, given that peptides generally cross the blood brain barrier and


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penetrate the CNS poorly after peripheral administration, it was theorized that peripherallyselective peptidomimetic opioid agonists would demonstrate analgesia without the liabilities associated with the activation of opioid receptors in the CNS.32 Continuing with our efforts toward the generation of heterocyclic peptidomimetics from modified resin bound peptides, we report the parallel synthesis of fused diimidazo-diazepines, from reduced tripeptides.

The diimidazo-diazepine compounds were subjected to initial

pharmacological screening in vitro for opioid receptor affinity with radioligand competition binding assays. The antinociceptive activity of a promising opioid ligand so identified, the diimidazo-diazepine 14 (2065-14), was then characterized in vivo with the mouse 55oC warmwater tail-withdrawal test after intracerebroventricular (i.c.v), intraperitoneal (i.p.) or oral (p.o.) administration, and opioid receptor agonist and antagonist profiles assessed. Bioavailability of the diimidazo-diazepine 14 in both blood and brain was evaluated up to 90 min after oral administration. Finally, the effect on acute antinociceptive tolerance, gastrointestinal transit, coordinated locomotor activity and place-conditioning preference was investigated to evaluate potential liabilities of use.

RESULTS AND DISCUSSION Design rationale and chemistry. Our approach toward the parallel synthesis of fused diimidazo-diazepines is outlined in Scheme 1. Starting from resin-bound acylated tripeptides, the amide bonds were exhaustively reduced in the presence of BH3-THF. Typical reaction conditions for the solid-phase reduction of polyamides consist of the treatment of resin-bound peptides with BH3-THF at 65°C for 72 hours.34,35 The generated resin-bound borane-amine complexes are then disproportionate



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following overnight treatment with neat piperidine at 65°C. As reported earlier by our group and others, the reduction of polyamides with borane is free of racemization.34-36 The resulting resin bound tetra-amines were cleaved from the solid-support in the presence of anhydrous HF, and then extracted with acetic acid and lyophilized to obtain solid white powder. The obtained crude tetra amine compounds were then separately treated with diisopropylethylamine (DIEA) and diethyl malonoimidate dihydrochloride in DMF at 85 oC for 48 hours and then purified to generate the desired diimidazo-diazepines compounds in good yield and high purity.

Scheme 1

Based on previous studies from our laboratory and other groups,37-39 tyrosine and aromatic residues were universally found in the wide family of opioid peptide and peptidomimetic ligands. Thus, we selected Val and Phe, for the first position of diversity (R1), Phe and Tyr for the second position of diversity (R2), Leu and Tyr for the third position of diversity (R3), and phenylacetic acid and H (no acylation) for the fourth and final position of diversity (R4). Using tea-bag technology,65 we performed the parallel synthesis of 16 diimidazo-diazepines (Table 1; see also Figure 1). Compounds were purified by RP-HPLC and purities of the tested compounds were determined to be at least 95% for each compound.

Table 1.


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Evaluation of opioid receptor affinity and selectivity by the diimidazo-diazepine compounds. All synthesized compounds were tested in vitro with radioligand competition binding assays to measure their affinities for MOR, DOR and KOR by determining the IC50 values for the inhibition of [3H]DAMGO, [3H]DPDPE and [3H]U69,593, respectively, from their rat brain membrane binding sites. Calculated inhibitory constants (Ki) of the analogues are listed underneath their structures in Figure 1. A number of compounds displayed affinity for DOR, with compounds 5, 6, 21 and 22 demonstrating affinities of less than 70 nM for DOR (Figure 1). Of interest, incorporation of a phenylethyl group at the R4 position decreased DOR-selectivity by increasing affinity for the KOR (Figure 1). Additionally compound 14 demonstrated equivalent good affinity (>35 nM) for both kappa and delta opioid receptors. Accordingly, for further functional characterization, we selected those analogs having an aliphatic amino acid (Val; compound 14) or aromatic amino acid (Phe; compound 6) side chain at R1, and the tripeptide analog matching compound 5, but lacking the phenylethyl at the R4 position (compound 5). Evaluation of functional delta opioid receptor activity for diimidazo-diazepine compounds in vitro. The functional activity of three diimidazo-diazepinecompounds found to have good (>60 nM) affinity for DOR were assessed in Chinese hamster ovary cells stably expressing the DOR (CHO-DOR) using a cAMP inhibition assay. The DOR agonist DPDPE was tested as a positive control. A dose response curve for DOR-mediated activity was determined for each compound (Figure 2), with EC50 values (mean ± SEM) of 366±170 nM, 1639±530 nM and 6365±2580 nM for compounds 14, 6 and 5, respectively. Although the DOR agonist DPDPE proved more potent (EC50 value = 18 ± 10 nM), these data prove that each diimidazo-diazepine analog tested produced full agonist efficacy.



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Figure 1

Figure 2

In Vivo characterization of diimidazo-diazepine 14 in mouse assays: On the basis of the in vitro data, compound 14 was chosen for characterization in vivo. The antinociceptive potency of 14 was determined with the 55°C warm-water tail-withdrawal assay. The opioid agonist morphine and KOR-selective agonist

(±)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]

benzeneacetamide (U50,488) were used as positive controls.40 Morphine, U50,488 and 14 each demonstrated antinociceptive activity after either i.c.v. (Figure 3A, circles), i.p. (Figure 3A, triangles) or p.o. administration (Figure 3A, squares). After i.c.v. administration, morphine and 14 exhibited equivalent dose-dependent antinociceptive potency, with ED50 (and 95% confidence interval) values of 2.35 (1.13–5.03) nmol, and 5.37 (3.84–8.00) nmol, respectively, whereas U50,488 proved modestly (but significantly) less potent (8.62 (5.74-11.9) nmol; F(1,106)=19.3, P

Parallel Synthesis of Hexahydrodiimidazodiazepines Heterocyclic

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