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International Journal of Medicinal Chemistry
Volume 2012 (2012), Article ID 715123, 10 pages
http://dx.doi.org/10.1155/2012/715123
Review Article

Developmental Potential for Endomorphin Opioidmimetic Drugs

1Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Chuo-ku, Kobe 650-8586, Japan
2Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, 44100 Ferrara, Italy
3Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA

Received 18 January 2012; Accepted 27 March 2012

Academic Editor: O. Bruno

Copyright © 2012 Yoshio Okada et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Morphine, which is agonist for μ-opioid receptors, has been used as an anti-pain drug for millennia. The opiate antagonists, naloxone and naltrexone, derived from morphine, were employed for drug addiction and alcohol abuse. However, these exogenous agonists and antagonists exhibit numerous and unacceptable side effects. Of the endogenous opioid peptides, endomorphin(EM)-1 and endomorphin(EM)-2 with their high μ-receptor affinity and exceptionally high selectivity relative to δ- and κ-receptors in vitro and in vivo provided a sufficiently sequence-flexible entity in order to prepare opioid-based drugs. We took advantage of this unique feature of the endomorphins by exchanging the N-terminal residue Tyr1 with 2′,6′-dimethyl-L-tyrosine (Dmt) to increase their stability and the spectrum of bioactivity. We systematically altered specific residues of [Dmt1]EM-1 and [Dmt1]EM-2 to produce various analogues. Of these analogues, [N-allyl-Dmt1]EM-1 (47) and [N-allyl-Dmt1]EM-2 (48) exhibited potent and selective antagonism to μ-receptors: they completely inhibited naloxone- and naltrexone-induced withdrawal from following acute morphine dependency in mice and reversed the alcohol-induced changes observed in sIPSC in hippocampal slices. Overall, we developed novel and efficacious opioid drugs without deleterious side effects that were able to resist enzymatic degradation and were readily transported intact through epithelial membranes in the gastrointestinal tract and the blood-brain-barrier.

1. Introduction

Morphine, which represents the quintessential agonist for μ-opioid receptor, has been used as a pain-killing drug for millennia. Since natural occurring opioid antagonists are nonexistent, naloxone and naltrexone were derived from morphine and currently find use in drug addiction and alcohol cessation programs; however, these alkaloid-derived antagonists exhibit numerous deleterious side effects. In 1975, the endogenous opioid peptides enkephalins (H-Tyr-Gly-Gly-Phe-Met-OH/Leu-OH) were discovered [1], followed sequentially by the endorphins [2], dynorphins [3], and the endomorphins [4], all of which are involved in the modulation and attenuation of pain and regulation of homeostatic mechanisms.

Of the endogenous opioid peptides, endomorphin-1 (EM-1: H-Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (EM-2: H-Tyr-Pro-Phe-Phe-NH2) exhibited high μ-opioid receptor affinity (Ki = 0.36 and 0.69 nM, resp.) with high selectivity: 4,000- and 13,000-fold preference over the δ-opioid receptor and a similar 15,000- and 7,500-fold preference for μ-receptor relative to κ-opioid receptors [4]. These data underline the potential importance of these opioid ligands in all phases of human homeostatic mechanisms. Considering this premise, our research was directed toward the eventual development of endomorphin opioidmimetics, which would exhibit agonist and antagonist properties with potentially minimal side effects. We review the approach in this field, focusing basic research on key factors in the rational development of novel and highly efficacious opioid drugs able to resist enzymatic degradation and readily transported intact through epithelial membranes in the gastrointestinal tract and the blood-brain barrier.

2. Properties of Endomorphin Analogues

Opioid peptides and their G-protein-coupled receptors (δ, κ, and μ), which are distributed in the central nervous system and peripheral tissues, were initially classified on the basis of their functional pharmacological activity. However, despite a common mode of biological action as agonists, the structural differences among opioids permitted a division into two separate classes based on their N-terminal message domain: namely, H-Tyr-Gly-Gly-Phe-, a sequence that comprises the enkephalins, endorphins, and dynorphins, while H-Tyr-Pro-Trp/Phe- defines endomorphins-1 and -2. It is the unique sequence of the latter opioids that gave rise to their flexibility in the production of bioactive analogues.

2.1. Synthesis of Stereoisomeric Analogues of Endomorphin-2 and Their Activities

Initially, in order to gain insight on the interaction between opioid ligands with their receptors, we substituted D-amino acids into endomorphin-2 [5]. The rationale for the use of D-amino acids is their ability to generally affect biological activity due to a subtle change induced in peptide conformation that, if bioactive, can lead to enhanced stability against enzymatic degradation [6].

Endomorphin-2 and D-amino acid containing stereoisomers were prepared by Fmoc solid-phase method using Fmoc (9-fluorenylmethyloxycarbonyl) amide resin as follows: solid support, Fmoc-D- or L-Tyr(But)-OH, Fmoc-D- or L-Pro-OH, Fmoc-D- or L-Phe-OH, and HBTU/HOBt/DMF, DIEPA/NMP were used. After each coupling reaction, the Fmoc group was removed by piperidine/NMP. For the final deblocking, dried protected peptide resin was suspended in TFA/H2O, and the reaction mixture was stirred at room temperature for 2 h. The material was filtered and ether added to filtrate to precipitate the peptides, which were collected by filtration and lyophilized from 1 M HCl to >98% purity. Receptor binding data are detailed in Table 1  (2–17) [5]. All D-amino acids containing analogues exhibited less binding affinities to the μ-opioid receptor (Ki = 24.3–2,755 nM), resulting in the loss of high selectivity over δ-opioid receptor (Kiδ/Kiμ = 2.6–177). Interestingly, although [D-Pro2]EM-2 (12) exhibited only low affinity towards the μ-receptor (Ki = 512.4 nM), it substantially exhibited more potent and longer activity in an in vivo tail flick test in mice compared to EM-2 [7]. These data clearly indicate an enhanced bioactivity most likely due to its resistance to proteolytic degradation, presumably by dipeptidyl peptidase IV [8].

tab1
Table 1: Opioid receptor affinities of opioid ligands 1–50.
2.2. Synthesis of [2′,6′-Dimethyl-L-tyrosine1 (Dmt1)]EM-2 Analogues: Structure-Activity Relationships

In order to develop potentially more potent analgesics, 2′,6′-dimethyl-L-tyrosine (Dmt) was substituted for Tyr as the N-terminal residue, since Dmt markedly increases the affinity and bioactivity of numerous opioid peptide agonists and antagonists [9, 1416]. Optically pure 2′,6′-dimethyl-L-tyrosine was prepared as previously described [17].

As summarized in Tables 1 and 3, substitution of Dmt1 in EM-1 and EM-2 and in C-terminal deletion analogues profoundly affected all the measured parameters. In each case, the affinity of [Dmt1]EM-1 (19) and [Dmt1]EM-2 (20) towards the μ-opioid receptor increased 6.6 and 4.6 times compared to the parent molecules (1, 2), respectively, and increased δ-opioid receptor affinity by 270- and 327-fold. The functional bioactivity of [Dmt1]EM-1 (Table 3, 19) increased μ-bioactivity by 15-fold over EM-1. Interestingly, [Dmt1]EM-1 (19) was transformed to potent mixed μ-agonist/δ-antagonist, while the bioactivity of [Dmt1]EM-2 (20) greatly increased both μ- and δ-agonist bioactivities by 98- and 184-fold greater than EM-2, respectively. Similarly, the deletion of C-terminal carboxyl group of [Dmt1]EM-2 to yield H-Dmt-Pro-Phe-NH-C2H4-Ph (22) also exhibited mixed μ-agonist/δ-antagonist properties, but with over an order of magnitude less activity than those observed for 19. The marked change in the Dmt-containing analogues relative to both receptor interaction and bioactivity could be a result of an alteration in the topography of the peptide. In fact, the 1H NMR spectra of EM-2 analogues revealed that the rotamers around the Dmt-Pro amide bond existed predominantly in the cis configuration [9].

2.3. Synthesis of C-Terminal-Modified [Dmt1]EM-2 Analogues (H-Dmt-Pro-Phe-NH-X)

Reports suggested that opiate tolerance and physical dependence could be blocked by δ-opioid receptor antagonists without compromising the antinociception produced by drug interaction at μ-opioid receptors [21]. From that point of view, H-Dmt-Pro-Phe-NH-C2H4-Ph (22) might be a candidate as an antinociceptive drug although its bioactivity was not exceptionally potent as a μ-agonist/δ-antagonist in vitro (GPI: IC50 = 5.03 nM, MVD: IC50 > 10,000 nM, pA2 = 7.05) [9]. Therefore, we substituted hydrophobic groups in lieu of the phenethyl group to develop the class of H-Dmt-Pro-Phe-NH-X ligands (Figure 1) [11]. In Tables 1 and 3, the [Dmt1]EM-2 analogues (22–33) demonstrated μ-opioid receptor affinity higher than EM-2 (Kiμ = 0.69 nM) with Ki = 0.11 to 0.52 nM, except for H-Dmt-Pro-Phe-NH-Ph (24) (Kiμ = 1.11 nM). In terms of their in vitro bioactivity, they were μ-opioid agonists based on a guinea pig ileum (GPI) bioassay (Table 3). It should be noted that 1-Nph (28), 5-Qln (30) and 5-Isq (33) exhibited potent μ-opioid receptor agonism (IC50 < 1 nM). In the mouse vas deferens (MVD) bioassay, they exhibited δ-opioid agonism (28, 30) with weak δ-opioid receptor antagonism (pA2 = 5.41–7.18). Compound 33, a modest μ-agonist/δ-antagonist in vitro, produced a dose-dependent antinociceptive effect after i.c.v. administration in mice that was antagonized completely by naltrexone, indicating that its antinociception occurred through μ-opioid receptors similar to that of morphine [11].

715123.fig.001
Figure 1: Structure of H-Dmt-Pro-Phe-NH-X.

These data substantiate that N-terminal Dmt-containing ligands permit development of novel bioactive opioidmimetics for potential therapeutic and clinical applications. The methyl groups on the tyramine ring of Dmt undoubtedly play a dominant role in the interaction within the opioid ligand-binding domains either by direct interaction with hydrophobic side chains of receptor residues or more interestingly by stabilization of favored cis conformer in solution prior to and during binding, or a combination of both mechanisms.

2.4. Synthesis of μ-Opioid Receptor Ligands Incorporating Unique Tyrosine Analogues

The enhancement of opioid activity upon inclusion of Dmt in the sequence of opioid peptides provided the impetus to develop further analogues with systematic modifications at the 2′ and 6′positions of the Tyr aromatic ring and investigate their impact on the activity of EM-2. In this study, six tyrosine analogues containing different alkyl groups were prepared, namely, 2′-monomethyltyrosine (Mmt), 2′,3′,6′-trimethyltyrosine (Tmt), 2′-ethyl-6′-methyltyrosine (Emt), 2′-isopropyl-6′-methyltyrosine (Imt), 2′,6′-diethyltyrosine (Det), and 2′,6′-diisopropyltyrosine (Dit). Opioid receptor affinities and in vitro functional bioactivity of the EM-2 analogues (34–39) are summarized in Tables 1 and 3, respectively [12]. Except for [Dit1]EM-2 (38: Kiμ = 2.29 nM), the [Xaa1]EM-2 analogues exhibited similar or higher μ-receptor affinity (Kiμ = 0.063–0.13 nM) to [Dmt1]EM-2. Evaluation of their in vitro bioactivities indicated that [Mmt1]- (34), [Emt1]- (35), [Det1]-(37), and [Tmt1]EM-2 (39) exhibited high GPI potencies (IC50 = 0.623–2.31 nM), although less than that of [Dmt1]EM-2 (20), while [Imt1]-(36) and [Dit1]EM-2 (38), which have bulky isopropyl groups, exhibited weak GPI potencies (IC50 = 10.6 and 299 nM, resp.). [Dit1]EM-2 (38) had unexpectedly low GPI and MVD potencies, although it retained high affinity toward both μ- and δ-opioid receptors, implying that this analogue may interact with the receptors but fail to trigger a bioactive response.

The in vivo biological activities of [Dmt1]EM-2 (20) and [Det1]EM-2 (37) were assessed by the induction of analgesia via the tail-flick test (spinally mediated mechanism) and hot-plate test (supraspinal effect) in comparison to both EM-2 (2) and morphine. The results revealed the following potency profile: [Dmt1]EM-2 (20) > [Det1]EM-2 (37) > EM-2 (2), which yielded activity ratios of 1.00 : 0.86 : 0.65 in the tail-lick tests and 1.00 : 0.47 : 0.30 in the hot-plate tests. These results indicated that the methyl side chain located at the 2′ and 6′ positions of Tyr represent the optimal alkyl groups for interaction with and activation of μ- and δ-opioid receptors. Interestingly, [Dmt1]EM-2 (20) was approximately 16% as effective as morphine.

2.5. [Dmt1]EM-2 Analogues Substituted at Position 3 with Alkylated Phe: Mixed μ-Agonist/δ-Antagonist and Dual μ-Agonist/δ-Agonist Opioid Ligands

The aromatic amino acid residue in position 3 is the defining structural determinant between EM-1 (Trp3) and EM-2 (Phe3). As shown, [Dmt1]EM-1 is a μ-agonist/δ-antagonist and [Dmt1]EM-2 is a μ-agonist/δ-agonist, further suggesting that the difference in chemical nature and the physical structure between Trp and Phe affected the properties of these opioid receptor ligands. Another alkylated Phe analogue, 2′,6′-dimethylphenylalanine (Dmp), was found to be an effective surrogate for phenylalanine in several opioid peptides [22, 23]. Interestingly, its replacement for Tyr1 in endomorphin [23] indicated that it was nearly as effective as the parental peptide, despite the absence of the important hydroxyl group on the tyramine ring, suggesting that alkylation of the aromatic ring enhances hydrophobicity and stability and/or limits rotational freedom. Therefore, we embarked on the synthesis of Phe analogues: 2′-methyl (Mmp), 3′5′-dimethyl (3,5Dmp), 2′,6′-dimethyl (Dmp), 2′,4′,6′-trimethyl (Tmp), 2′-ethyl-6′-methyl (Emp), and 2′-isopropyl-6′-methyl-phenylalanine (Imp) as reported [24] and their incorporation into H-Dmt-Pro-Xaa-Phe-NH2 [13].

As summarized in Table 1, the alkylated Phe3 analogues essentially enhanced the affinities for both μ- and δ-opioid receptors in these [Dmt1,Xaa3]EM-2 ligands (4046). Of these analogues, the highest μ-opioid selectivity occurred with [Dmt1,3]EM-2 (43) (Kiδ/Kiμ = 878). One analogue of considerable interest is [Dmt1,Tmp3]EM-2 (44) with a 44-fold enhancement toward δ-opioid receptors relative to [Dmt1,3]EM-2 (43). This suggested that the hydrogen donor capacity of the hydroxyl group of Dmt was apparently less effective in affecting receptor interaction when substituted within the sequence of the peptide than the hydrophobicity of a 4′ methyl group; that is, the hydroxyl group may contribute a negative influence when it occurred as an internal residue. κ-Opioid receptor affinities for Dmt derivatives (40–46) were quite weak relative to the interaction of these peptides to both μ- and δ-opioid receptors [13].

The functional bioactivities of [Dmt1,Xaa3]EM-2 analogues generally remained essentially unchanged (4046) relative to [Dmt1]EM-2 (20) (Table 3). Interestingly, the absence of a 4′ OH group (42) or its replacement by a methyl group yielded [Dmt1,Tmt3]EM-2 (44) and produced excellent ligands with mixed μ-agonist/δ-antagonist properties: δ-antagonism was 2 orders of magnitude greater than that obtained for Dmt3 (43). We have seen (supra vide) that [Dmt1]EM-1 (19) is a mixed μ-opioid agonist/δ-antagonist (GPI IC50 = 0.27 nM; MVD pA2 = 8.6), but [Dmt1,Tmp3]EM-2 (44) is obviously more potent (Table 3) [13].

These data permitted us to conclude the following: (i) the bulky side chain of Trp in combination with Dmt1 caused either a steric hindrance in the conformation of the peptide or a shift in hydrophobicity to potentiate the induction of δ-opioid antagonism; (ii) [Dmt1,Emp3]EM-2 (45) and [Dmt1,Imp3]EM-2 (46) exhibited dual μ/δ-agonism similar to that seen for [Dmt1]EM-2 (20), while compounds 4044 had δ-opioid antagonism ranging from a weak pA2 = 6.59 to a potent pA2 = 9.05. Thus, these bifunctional molecules are targets in the design of new antinociceptive opioids that could potentially alleviate acute or chronic pain with a low degree of physical dependence and tolerance [25].

2.6. Transformation of [Dmt1]EM-1 and [Dmt1]EM-2 into Potent and μ-Selective Antagonists

The development of potent and selective opioid antagonists, especially μ-opioid receptor antagonists, is very important in order to delineate critical biochemical, pharmacological, and physiological roles played by these receptors and for their possible application as clinically and therapeutically relevant agents. Table 1 revealed that [N-allyl-Dmt1]EM-1 (47) exhibited better affinity compared to [N-allyl-Dmt1]EM-2 (48); however, in terms of their in vitro functional bioactivity (Table 3), [N-allyl-Dmt1]EM-2 (48) exhibited somewhat better μ-opioid antagonism with pA2 = 8.59 versus pA2 = 8.18 for [N-allyl-Dmt1]EM-1 (47) [10]. Furthermore, both antagonists are defined as neutral μ-antagonists due to their lack of inverse agonist properties determined by functional guanosine 5′-O-(3-[35S]thiotriphosphate) assays in vitro from membranes of cells grown in the presence of morphine or alcohol [26]. They also completely inhibited naloxone- and naltrexone-elicited withdrawal symptoms following acute morphine dependency in mice [26]. [N-Allyl-Dmt1]EM-2 (48) induced a dose-dependent suppression of an ethanol-induced increase of sIPSC frequency with full reversal at 300 nM that was several orders of magnitude more potent than naltrexone [27]. These results suggest a potential therapeutic application in the treatment of drug addiction and alcohol abuse without the adverse effects observed with inverse agonist alkaloid-derived compounds, such as naltrexone and naloxone that produce severe withdrawal symptoms.

3. Opioidmimetics

3.1. Agonists

The presence of Dmt in lieu of Tyr1 in opioid peptides enhanced affinities, bioactivity, and analgesia. In order to assess the possible effect of Dmt per se on opioid activities, H-Dmt-NH-CH3 was prepared and examined [28]. This compound had Kiμ = 7.45 nM and Kiδ = 460 nM values that were nearly equivalent to those of morphine. However, the in vitro bioactivity in a GPI assay was three orders of magnitude lower than that of EM-2 and [Dmt1]EM-2 and essentially inactive in the MVD assay. Its analgesic response relative to morphine was insignificant (0.64% in hot-plate test and 1.3% in tail-flick test). According to the message-address concept of opioid functionality [29], Dmt would be considered an important pharmacophore interacting within opioid receptors as an integral component of the message domain even though it had no intrinsic activity of its own. Thus, to test this hypothesis, we set out to construct ligands containing two message and address domains.

3.1.1. Development of Receptor Agonists by Dimerization of Dmt with Unbranched Alkyl Chains

The receptor affinities and in vitro bioactivities of the synthetic Dmt dimer analogues are summarized in Tables 2 and 4 [18]. The bis-Dmt-containing ligands 52 and 53 exhibited high μ-opioid receptor affinity (Ki = 0.04–0.05 nM) but modest receptor selectivity (δ/μ = 1302 and 870). The optimal distance between the Dmt residues for maximum μ-opioid receptor affinity appeared to be butyl (52) = hexyl (53) > octyl (54) > ethyl (51). Despite the relatively good μ-receptor agonism of 52 and 53 (IC50 = 5.3 and 3.1 nM, resp.), they had undetectable δ-agonism and very weak δ-antagonism (pA2 = 5.5–6.4). In terms of their in vivo bioactivity, 52 rapidly produced central mediated (i.c.v.) analgesia that was 1.5–2.2- fold greater than morphine and naloxone-reversible; the supraspinal nociceptive pathway revealed equivalent analgesia to morphine. Subcutaneous injection of 52 produced analgesia that was 10–20% as potent as morphine, indicating that 52 indeed crossed epithelial membranes and the blood-brain barrier [30].

tab2
Table 2: Opioid receptor affinities of opioid ligands 51–70.
tab3
Table 3: Functional bioactivities of opioid ligands 1, 2, and 19–50.
tab4
Table 4: Functional bioactivities of opioid ligands 51–70.
3.1.2. Development of Orally Available Opioidmimetic Analgesics by Dimerization of Dmt with Diaminoalkylpyrazinones

The inability of opioid peptides to be transported through epithelial membranes in the gastrointestinal tract and pass the blood-brain barrier limits their effectiveness for oral application in an antinociceptive treatment regime. To overcome this limitation, we enhanced the hydrophobicity and maintained the aqueous solubility properties of ligands by employing two identical N-termini. This consisted of Dmt coupled to a pyrazinone ring platform by means of alkyl chains to yield the class of 3,6-bis-[Dmt-NH-(CH2)n]-5-methyl-2(1H)-pyrazinones (Figure 2) [19]. Their receptor affinities and in vitro bioactivities are summarized in Tables 2 and 4, respectively. The 3,6-bis-[Dmt-NH-(CH2)n]-5-methyl-2(1H)-pyrazinone compounds exhibited high affinity to both μ- (56–58: Kiμ = 0.04–0.12 nM) and δ-opioid receptors (55–58: Kiδ = 7.3–23.2 nM). Compound 57, 3,6-bis-[Dmt-NH-(CH2)3]-5-methyl-2(1H)-pyrazinone exhibited the highest affinity (Kiμ = 0.042 nM) that was ca. 3-fold greater than that of either 3,6-bis-[Dmt-NH-(CH2)2]-5-methyl-2(1H)-pyrazinone (56) or 3,6-bis-[Dmt-NH-(CH2)4]-5-methyl-2(1H)-pyrazinone (58) and nearly 30 times greater than that of 3,6-bis-[Dmt-NH-CH2]-5-methyl-2(1H)-pyrazinone (55). Thus, the length of the interposing alkyl chain determines the efficacy of receptor binding: propyl > ethyl, butyl ≫ methyl. Compounds 5558 were biologically active and generally reflected the values obtained for the affinity constants: 57 was the most active (GPI, IC50 = 1.33 nM) and more potent than the bis-[Dmt-NH]-alkyl compounds (51–54: GPI IC50 = 3.08–2,844 nM) [18] and was a μ-selective agonist without measurable δ-bioactivity. Compound 58, which exhibited 30% less μ-agonism than 57, had weak δ-agonism (MVD, IC50 = 41.5 nM). Similar to the bis-[Dmt-NH]-alkyl compounds (5154: pA2 = 5.5–6.5) [18], compounds 55 and 56 were weak δ-antagonists (pA2 = 6.47 and 6.56, resp.).

715123.fig.002
Figure 2: Structure of 3,6-bis [Dmt-NH-(CH2)n]-5-methyl-2(1H)-pyrazinones.

Compound 57 produced naloxone reversible analgesia by i.c.v., s.c. and oral (po) administration. While i.c.v. analgesia was 50- and 20-fold more potent than morphine in the tail-flick and hot-plate tests, respectively, both s.c. and p.o. were somewhat less active than morphine. These results demonstrated that compound 57 crossed epithelial membrane barriers in both the intestine and microcapillaries in mouse brain to interact with brain μ-opioid receptors. Similar conclusions were obtained by Igarashi et al. [31] and Koda et al. [32]. These results indicated that pyrazinone derivatives could be potential candidates for clinical and therapeutic applications in the treatment of pain arising from postoperative procedure or cancer, associated with birth, or act as possible veterinary drugs.

3.2. Development of μ- and δ-Opioid Receptor Antagonists by Dimerization of Dmt-Tic with Diaminoalkanes or Diaminoalkylpyrazinones

We expanded our studies with Dmt through the synthesis and analysis of the biological properties of unique series of dimeric H-Dmt-Tic (2′,6′-dimethyl-L-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) analogues linked either through diaminoalkanes of variable length (6668) or by symmetric or asymmetric 3,6-diaminoalkyl-5-methyl-2(1H)-pyrazinone derivatives (59–65). Salvadori et al. [14] first reported that H-Dmt-Tic-OH had not only δ high affinity (Kiδ = 0.022 nM) but also extraordinary selectivity for the δ-opioid receptor (Kiμ/Kiδ = 150,800) without interaction to κ-opioid receptors and exhibited δ-selective antagonism.

As summarized in Table 2, most of the compounds exhibited high subnanomolar affinities to δ-opioid receptors (Kiδ = 0.095–0.323 nM) independent of the spacer used, except compound 68 (Kiδ = 1.53 nM); μ-receptor affinities fell within the low nanomolar range of 1–5 nM [20]. Compared to H-Dmt-Tic-OH [14], our observed μ-affinities increased by several orders of magnitude.

In the series of dimeric H-Dmt-Tic-OH, ligands (5970) listed in Table 4 were devoid of δ-opioid receptor mediated agonism; all the compounds were exceptionally potent δ-antagonists with pA2 values ranging from 10.42 to 11.28, which represent orders of magnitude greater than that of both naltrindole (pA2 = 9.20) and H-Dmt-Tic-OH (pA2 = 8.48). In contrast to their μ-opioid receptor affinities (Table 2), the compounds exhibited very weak to nonexistent μ-agonism, especially, 69 and 70, which exhibited pure and potent δ- and μ-antagonism in the same molecule. In fact, the μ-opioid receptor antagonism of 69 and 70 exceeds that of other known peptidic [33] and nonpeptidic [34] antagonists.

The extraordinary dual δ/μ-antagonism of 69 and 70 qualifies these compounds as potential pharmacological tools for application in the clinical and therapeutic treatment of drug addiction and alcohol dependency. Considering that the bis-Dmt analogues containing alkylpyrazinone are orally active and pass through the blood-brain barrier [19, 31, 32], we would anticipate that 69 and 70 might show similar properties or may be even more potent due to their increased hydrophobicity [35].

4. Conclusion

Based on the structure of endomorphins (H-Tyr-Pro-Trp/Phe-Phe-NH2), which exhibited very high selectivity toward μ-opioid receptors, we developed various analogues and examined their activities by alterations of a specific residue. From the studies on the stereoisomers of EM-2, [D-Pro2]EM-2 (12) exhibited more potent and prolonged analgesia [7] although it exhibited low μ-affinity [5], indicating an enhanced bioactivity due to a presumed resistance to enzymatic degradation by dipeptidyl peptidase IV [8]. Substitution of Tyr1 by Dmt yielded [Dmt1]EM-1 (19) and [Dmt1]EM-2 (20): the former, containing Trp3, had mixed μ-agonism/δ-antagonism properties, and the latter, with Phe3, exhibited dual μ/δ-agonism. The differences between bulkiness of Trp and Phe defined their biofunctional properties, suggesting the existence of fine differences in the stereo geometry of the ligand-binding site between μ- and δ-opioid receptors. These data provided us with methodology to design ligands with agonism or antagonism towards their respective receptors. Thus, we could develop various compounds with dual μ-/δ-agonism or mixed μ-agonism/δ-antagonism in the same molecule.

On the other hand, alkylation of the N-termini of [Dmt1]EM-1 and [Dmt1]EM-2 converted μ-agonists into neutral acting μ-antagonists: [N-allyl-Dmt1]EM-1 (47) and [N-allyl-Dmt1]EM-2 (48) exhibited potent and highly selective μ-antagonism without inverse agonism, suggesting a potential clinical application in the treatment of drug addiction and alcohol abuse without adverse effects [10, 26, 27, 35]. Similarly ligands with two Dmt residues separated by diaminoalkane or diaminoalkylpyrazinone produced orally available opioidmimetic analgesics. The compound 3,6-bis-[Dmt-NH-(CH2)3]-5-methyl-2(1H)-pyrazinone (57), μ-selective agonist, produced naloxone reversible analgesia following oral administration, with a potency that was 42% and 24% compared to morphine in tail-flick and hot-plate tests in mice, respectively. These results demonstrated that 57 passed through membranes in the gastrointestinal tract and the blood-brain barrier [19]. This observation paves the way for its clinical and therapeutic application in the treatment of pain. Dimerization of potent and δ-selective antagonist H-Dmt-Tic-OH [14] separated by diaminoalkane or 3,6-diaminoalkylpyrazinone produced the dual μ/δ-antagonists, bis-[N,N-dimethyl-Dmt-Tic-NH]hexane (69) and 3,6-bis-[N,N-dimethyl-Dmt-Tic-NH-(CH2)3]-5-methyl-2(1H)-pyrazinone (70) [20]. These extraordinary dual μ/δ-antagonists (69 and 70) also qualify as potential drugs with clinical and therapeutic applications.

Abbreviations

But:tert-Butyl
HBTU:O-Benzotriazole-1-yl-N,N, -tetramethyluronium hexafluorophosphate
DIPEA:Diisopropylethylamine
HOBt:1,2,3-Benzotriazole-1-ol
NMM:N-Methylmorpholine
NMP:N-Methylpyrrolidone
TFA:Trifluoroacetic acid
Ph:Phenyl
Bzl:Benzyl
4-Py:4-Pyridyl
3-Py:3-Pyridyl
2-Py:2-Pyridyl
1-Nph:1-Naphthyl
3-Qln:3-Quinolyl
5-Qln:5-Quinolyl
6-Qln:6-Quinolyl
8-Qln:8-Quinolyl
5-Isq:5-Isoquinolyl
Dmt:2′,6′-Dimethyl-L-tyrosine
Emt:2′-Ethyl-6′-methyl-L-tyrosine
Mmt:2′-Methyl-L-tyrosine
Imt:2′-Isopropyl-6′-methyl-L-tyrosine
Det:2′,6′-Diethyl-L-tyrosine
Dit:2′,6′-Diisopropyl-L-tyrosine
Tmt:2′,3′,6′-Trimethyl-L-tyrosine
Dmp:2′,6′-Dimethyl-L-phenylalanine
Mmp:2′-Methyl-L-tyrosine
3,5Dmp:3′,5′-Dimethyl-L-phenylalanine
Tmp:2′,4′,6′-Trimethyl-L-phenylalanine
Emp:2′-Ethyl-6′- methyl-L-phenylalanine
Imp:2′-Isopropyl-6′-methyl-L-phenylalanine
IPSC:Inhibitory postsynaptic currents.

Acknowledgments

The authors appreciate the critical review of this paper by Dr. Robert Langenbach. This work was supported in part by Kobe Gakuin University and in part by the Intramural Research Program of the NIH and NIEHS.

References

  1. J. Hughes, T. W. Smith, H. W. Kosterlitz, L. A. Forthergill, B. A. Morgan, and H. R. Morris, “Identification of two related pentapeptides from the brain potent opiate agonist activity,” Nature, vol. 258, pp. 577–580, 1975.
  2. C. H. Li and D. Chung, “Isolation and structure of an untriakontapeptide with opiate activity from camel pituitary glands,” Proceedings of the National Academy of Sciences of the United States of America, vol. 73, no. 4, pp. 1145–1148, 1976. View at Scopus
  3. A. Goldstein, S. Tachibana, L. I. Lowney, M. Hunkapiller, and L. Hood, “Dynorphin(1-13), an extraordinarily potent opioid peptide,” Proceedings of the National Academy of Sciences of the United States of America, vol. 76, no. 12, pp. 6666–6670, 1979. View at Scopus
  4. J. E. Zadina, L. Hackler, L. J. Gee, and A. J. Kastin, “A potent and selective endogenous agonist for the μ-opiate receptor,” Nature, vol. 386, no. 6624, pp. 499–502, 1997. View at Publisher · View at Google Scholar · View at Scopus
  5. Y. Okada, A. Fukumizu, M. Takahashi et al., “Synthesis of stereoisomeric analogues of endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2, and examination of their opioid receptor binding activities and solution conformation,” Biochemical and Biophysical Research Communications, vol. 276, no. 1, pp. 7–11, 2000. View at Publisher · View at Google Scholar · View at Scopus
  6. L. Terenius, A. Wahlstrome, G. Lindeberg, S. Karlsson, and U. Ragnarson, “Opiate receptor affinity of peptides related to Leu-enkephalin,” Biochemical and Biophysical Research Communications, vol. 71, no. 1, pp. 175–179, 1976.
  7. R. Shane, S. Wilk, and R. J. Bodnar, “Modulation of endomorphin-2-induced analgesia by dipeptidyl peptidase IV,” Brain Research, vol. 815, no. 2, pp. 278–286, 1999. View at Publisher · View at Google Scholar · View at Scopus
  8. T. Kato, T. Nagatsu, T. Kimura, and S. Sakakibara, “Studies on substrate specificity of X-prolyl dipeptidyl-aminopeptidase using new chromogenic substrates, X-Y-p-nitroanilides,” Experientia, vol. 34, no. 3, pp. 319–320, 1978. View at Scopus
  9. Y. Okada, Y. Fujita, T. Motoyama et al., “Structural studies of [2′,6′-dimethyl-l-tyrosine1]endomorphin-2 analogues: enhanced activity and cis orientation of the Dmt-Pro amide bond,” Bioorganic and Medicinal Chemistry, vol. 11, no. 9, pp. 1983–1994, 2003. View at Publisher · View at Google Scholar · View at Scopus
  10. T. Li, Y. Jinsmaa, M. Nedachi et al., “Transformation of μ-opioid receptor agonists into biologically potent μ-opioid receptor antagonists,” Bioorganic and Medicinal Chemistry, vol. 15, no. 3, pp. 1237–1251, 2007. View at Publisher · View at Google Scholar · View at Scopus
  11. Y. Fujita, Y. Tsuda, T. Li et al., “Development of potent bifunctional endomorphin-2 analogues with mixed μ-/δ-opioid agonist and δ-opioid antagonist properties,” Journal of Medicinal Chemistry, vol. 47, no. 14, pp. 3591–3599, 2004. View at Publisher · View at Google Scholar · View at Scopus
  12. T. Li, Y. Fujita, Y. Tsuda et al., “Development of potent μ-opioid receptor ligands using unique tyrosine analogues of endomorphin-2,” Journal of Medicinal Chemistry, vol. 48, no. 2, pp. 586–592, 2005. View at Publisher · View at Google Scholar · View at Scopus
  13. T. Li, K. Shiotani, A. Miyazaki et al., “Bifunctional [2′,6′-dimethyl-l-tyrosine1] endomorphin-2 analogues substituted at position 3 with alkylated phenylalanine derivatives yield potent mixed μ-agonist/δ-antagonist and dual μ-agonist/δ-agonist opioid ligands,” Journal of Medicinal Chemistry, vol. 50, no. 12, pp. 2753–2766, 2007. View at Publisher · View at Google Scholar · View at Scopus
  14. S. Salvadori, M. Attila, G. Balboni et al., “δ opioidmimetic antagonists: prototypes for designing a new generation of ultraselective opioid peptides,” Molecular medicine, vol. 1, no. 6, pp. 678–689, 1995. View at Scopus
  15. S. D. Bryant, S. Salvadori, P. S. Cooper, and L. H. Lazarus, “New δ-opioid antagonists as pharmacological probes,” Trends in Pharmacological Sciences, vol. 19, no. 2, pp. 42–46, 1998. View at Publisher · View at Google Scholar · View at Scopus
  16. L. H. Lazarus, S. D. Bryant, P. S. Cooper, R. Guerrini, G. Balboni, and S. Salvadori, “Design of δ-opioid peptide antagonists for emerging drug applications,” Drug Discovery Today, vol. 3, no. 6, pp. 284–294, 1998. View at Publisher · View at Google Scholar · View at Scopus
  17. J. H. Dygos, E. E. Yonan, M. G. Scaros et al., “A convenient asymmetric synthesis of the unnatural amino acid 2,6-dimethyl-L-tyrosine,” Synthesis, no. 8, pp. 741–743, 1992. View at Scopus
  18. Y. Okada, Y. Tsuda, Y. Fujita et al., “Unique high-affinity synthetic μ-opioid receptor agonists with central- and systemic-mediated analgesia,” Journal of Medicinal Chemistry, vol. 46, no. 15, pp. 3201–3209, 2003. View at Publisher · View at Google Scholar · View at Scopus
  19. Y. Jinsmaa, A. Miyazaki, Y. Fujita et al., “Oral bioavailability of a new class of μ-opioid receptor agonists containing 3,6-bis[Dmt-NH(CH2)n]-2(1H)-pyrazinone with central-mediated analgesia,” Journal of Medicinal Chemistry, vol. 47, no. 10, pp. 2599–2610, 2004. View at Publisher · View at Google Scholar · View at Scopus
  20. T. Li, Y. Fujita, K. Shiotani et al., “Potent Dmt-Tic pharmacophoric δ- and μ-opioid receptor antagonists,” Journal of Medicinal Chemistry, vol. 48, no. 25, pp. 8035–8044, 2005. View at Publisher · View at Google Scholar · View at Scopus
  21. E. E. Abdelhamid, M. Sultana, P. S. Portoghese, and A. E. Takemori, “Selective blockage of delta opioid receptors prevents the development of morphine tolerance and dependence in mice,” Journal of Pharmacology and Experimental Therapeutics, vol. 258, no. 1, pp. 299–303, 1991. View at Scopus
  22. Y. Sasaki, M. Hirabuki, A. Ambo, H. Ouchi, and Y. Yamamoto, “Enkephalin analogues with 2′,6′,-dimethylphenylalanine replacing phenylalanine in position 4,” Bioorganic and Medicinal Chemistry Letters, vol. 11, no. 3, pp. 327–329, 2001. View at Publisher · View at Google Scholar · View at Scopus
  23. Y. Sasaki, A. Sasaki, H. Niizuma, H. Goto, and A. Ambo, “Endomorphin-2 analogues containing Dmp residue as an aromatic amino acid surrogate with high μ-opioid receptor affinity and selectivity,” Bioorganic and Medicinal Chemistry, vol. 11, no. 5, pp. 675–678, 2003. View at Publisher · View at Google Scholar · View at Scopus
  24. T. Li, Y. Tsuda, K. Minoura et al., “Enantioselective synthesis of a phenylalanine library containing alkyl groups on the aromatic moiety: confirmation of stereostructure by X-ray analysis,” Chemical and Pharmaceutical Bulletin, vol. 54, no. 6, pp. 873–877, 2006. View at Publisher · View at Google Scholar · View at Scopus
  25. M. E. Fundytus, P. W. Schiller, M. Shapiro, G. Weltrowska, and T. J. Coderre, “Attenuation of morphine tolerance and dependence with the highly selective δ-opioid receptor antagonist TIPP[ψ],” European Journal of Pharmacology, vol. 286, no. 1, pp. 105–108, 1995. View at Publisher · View at Google Scholar · View at Scopus
  26. E. D. Marczak, Y. Jinsmaa, T. Li et al., “[N-Allyl-Dmt1]-endomorphins are μ-opioid receptor antagonists lacking inverse agonist properties,” Journal of Pharmacology and Experimental Therapeutics, vol. 323, no. 1, pp. 374–380, 2007. View at Publisher · View at Google Scholar · View at Scopus
  27. Q. Li, Y. Okada, E. Marczak, W. A. Wilson, L. H. Lazarus, and H. S. Swartzwelder, “The novel μ-opioid receptor antagonist, [N-Allyl-Dmt1] endomorphin-2, attenuates the enhancement of GABAergic neurotransmission by ethanol,” Alcohol and Alcoholism, vol. 44, no. 1, pp. 13–19, 2009. View at Publisher · View at Google Scholar · View at Scopus
  28. Y. Fujita, Y. Tsuda, T. Motoyama et al., “Studies on the structure-activity relationship of 2′,6′-dimethyl-l-tyrosine (Dmt) derivatives: bioactivity profile of H-Dmt-NH-CH3,” Bioorganic and Medicinal Chemistry Letters, vol. 15, no. 3, pp. 599–602, 2005. View at Publisher · View at Google Scholar · View at Scopus
  29. R. Schwyzer, “Molecular mechanism of opioid receptor selection,” Biochemistry, vol. 25, no. 20, pp. 6335–6342, 1986. View at Scopus
  30. Y. Okada, Y. Tsuda, T. Yokoi, S. D. Bryant, and L. H. Lazarus, New opioid derivative, Patent WO 03/064375 A1, 2003.
  31. K. Igarashi, Y. Murabayashi, K. Hotta, et al., “Application of liquid chromatography-tandem mass spectrometry for determination of opioimimetics in the brain dialysates from rat treated with opioidmimetics intraperitoreally,” Journal of Chromatography B, vol. 806, pp. 53–57, 2004.
  32. Y. Koda, K. Shiotani, I. Toth, Y. Tsuda, Y. Okada, and J. T. Blanchfield, “Comparison of the in vitro apparent permeability and stability of opioid mimetic compounds with that of the native peptide,” Bioorganic and Medicinal Chemistry Letters, vol. 17, no. 7, pp. 2043–2046, 2007. View at Publisher · View at Google Scholar · View at Scopus
  33. A. Ambo, T. Terashima, and Y. Sasaki, “Novel [d-Arg2]dermorphin(1-4) analogs with μ-opioid receptor antagonist activity,” Chemical and Pharmaceutical Bulletin, vol. 50, no. 10, pp. 1401–1403, 2002. View at Publisher · View at Google Scholar · View at Scopus
  34. S. J. Ward, P. S. Portoghese, and A. E. Takemori, “Pharmacological characterization in vivo of the novel opiate, β-funaltrexamine,” Journal of Pharmacology and Experimental Therapeutics, vol. 220, no. 3, pp. 494–498, 1982. View at Scopus
  35. L. H. Lazarus, Y. Okada, T. Li, et al., Dmt-derivative compounds and related compositions and method of use. Patent WO2007027628A, 2011.