Original articleSynthesis and in vitro opioid activity profiles of DALDA analogues
Introduction
Centrally acting μ opioid agonists (e.g. morphine) are still the most frequently used analgesics for the relief of severe pain, but their usefulness is limited by a number of well known side-effects, including tolerance, physical dependence, respiratory depression, adverse gastrointestinal effects, etc. Therefore, current efforts to develop centrally acting opioid analgesics are aimed at developing compounds with different opioid activity profiles (e.g. δ agonists or mixed μ agonist/δ antagonists (for a review, see ref. [1]). However, very hydrophilic μ opioid agonists that are unable to cross certain biological barriers still have considerable therapeutic potential for some analgesic applications. For example, μ opioid compounds that cannot penetrate the blood–brain barrier (BBB) may elicit peripheral analgesic effects without producing centrally mediated side effects. Also, some μ opioid agonists with polar character may not be able to cross the placental barrier (PB) and, therefore, may have potential for use in obstetrical analgesia.
In general, opioid peptides are more hydrophilic than non-peptide opiates. Opioid peptides with high μ receptor binding affinity and high μ receptor selectivity include several enkephalin analogues as well as morphiceptin and its analogues (for a review, see ref. [2]). The putative endogenous μ opioid receptor ligands endomorphin-1 and endomorphin-2 are also potent and very selective μ agonists [3], but are subject to enzymatic degradation [4], [5]. Among various analogues of the N-terminal tetrapeptide segment of dermorphin, the compounds TAPS (H-Tyr-d-Arg-Phe-Ser-OH) [6] and DALDA (H-Tyr-d-Arg-Phe-Lys-NH2) [7] were reported to be potent and selective μ agonists.
Among these selective μ agonist peptides, DALDA has the most polar character because it carries a net positive charge of 3+ at physiological pH. DALDA was shown to be metabolically stable and to have very limited distribution across the BBB [8]. The results of a recent study indicated that DALDA did not cross the PB to a significant extent in the pregnant sheep model [9]. In agreement with this finding, DALDA had no effect on fetal cardiorespiratory parameters when given to pregnant sheep [10]. Furthermore, DALDA produced only a transient, minor increase in blood pressure and did not affect the maternal respiratory pattern. Taken together, these results suggest that DALDA or DALDA analogues may have potential for use in obstetrical analgesia or peripheral analgesia.
In the present paper we describe a series of DALDA analogues that were designed with the goal to develop highly polar μ opioid agonists with increased potency. The design was mainly based on structural modification of the Tyr1 residue. Tyr1 was replaced with 2′,6′-dimethyltyrosine (Dmt), N,2′,6′-trimethyltyrosine (Tmt), 2′-methyltyrosine (Mmt) or 2′-hydroxy,6′-methyltyrosine (Hmt). Analogues containing an α,ω-diamino acid with a shorter side chain (ornithine [Orn] or α,γ-diaminobutyric acid [A2bu]) in place of Lys4 were also prepared.
Section snippets
Chemistry
Peptides were synthesized by the solid-phase method using tert-butyloxycarbonyl (Boc)-protected amino acids and 1,3-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) as coupling agents. Boc-Tmt was prepared as described elsewhere [11]. Mmt was first synthesized by hydrogenolysis of 7-hydroxy,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic(OH)] using Pd-black as catalyst and a reaction temperature of 90 °C [12]. Under these conditions extensive racemization occurred. Van
In vitro bioassays and opioid receptor binding assays
For the determination of their in vitro opioid activities, analogues were tested in bioassays based on inhibition of electrically evoked contractions of the guinea pig ileum (GPI) and the mouse vas deferens (MVD). The GPI assay is usually considered as being representative for μ opioid receptor interactions, even though the ileum does also contain κ opioid receptors. In the MVD assay opioid effects are primarily mediated by δ opioid receptors but μ and κ receptors also exist in this tissue.
Results and discussion
DALDA (1) is a full μ agonist in the μ receptor representative GPI assay (table II). It has high μ receptor binding affinity and very weak binding affinities for δ and κ receptors (table III). Therefore, DALDA has very high selectivity for μ receptors vs. δ and κ receptors with selectivity ratios of Kiδ/Kiμ=11400 and Kiκ/Kiμ=2500.
Replacement of Tyr1 in DALDA with Dmt produced a 180-fold potency enhancement in the GPI assay and a somewhat less pronounced (34-fold) potency increase in the MVD
Conclusions
Most of the prepared DALDA analogues displayed higher μ receptor binding affinity and higher μ agonist potency than the DALDA parent peptide and they were all μ receptor-selective. The most potent μ agonist was [Dmt1]DALDA (2), which also showed highest μ receptor binding affinity, highest μ vs. δ receptor selectivity and substantial preference for μ receptors over κ receptors. This compound was 5 times more potent in the GPI assay and had 14 times higher μ receptor binding affinity than
General methods
Precoated plates (silica gel 60 F254, 250 μm; Merck, Darmstadt, Germany) were used for ascending TLC in the following systems (all v/v): (I) CHCl3/AcOH/MeOH (85:10:15); (II) n-BuOH/AcOH/H2O (4:1:1); (III) n-BuOH/pyridine/AcOH/H2O (15:10:3:12). A Varian VISTA 5500 liquid chromatograph was used for the purification and the purity control of the peptides. Preparative reversed-phase HPLC was performed on a Vydac 218-TP column (22×250 mm) with a linear gradient of 10–40% MeOH in 0.1% TFA at a flow
Acknowledgements
This work was supported by a Multicenter Consortium Grant (PO1-DA08924) from the National Institute on Drug Abuse, NIH, USA.
References (23)
- et al.
Brain Res.
(1999) - et al.
Jpn. J. Pharmacol.
(1999) - et al.
Am. J. Obstet. Gynecol.
(1998) - et al.
Biochem. Biophys. Res. Commun.
(1978) - et al.
Eur. J. Pharmacol.
(1979) - et al.
Biopolymers (Peptide Science)
(1999) - et al.
Nature
(1997) - et al.
J. Pharmacol. Exp. Ther.
(1987) - et al.
J. Med. Chem.
(1989)
Am. J. Physiol.
Cited by (150)
Gram-scale preparation of DAMGO by typical solid phase synthesis
2022, Tetrahedron LettersDevelopment of disulfide-functionalized peptides covalently binding G protein-coupled receptors
2022, Bioorganic and Medicinal ChemistryDesign, synthesis and evaluation of [ <sup>111</sup> In]-labeled, DOTA-conjugated tetrapeptides having high affinity and selectivity for mu opioid receptors
2019, Nuclear Medicine and BiologyCitation Excerpt :Crude material was triturated with diethyl ether, and then isolated by reversed-phase (RP) HPLC. The same strategy was used for H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA, 6; Fig. 1), and resembles the original preparation of Schiller and colleagues [29]. Analytical RP-HPLC data, including characterizations by electrospray ionization mass spectrometry (ESI-MS), are given in Table 1.