Further Studies on the Effect of Lysine at the C-Terminus of the Dmt-Tic Opioid Pharmacophore

Abstract

A wide range of bioactivities are induced by Lys when introduced at the C-terminus of the δ-opioid Dmt-Tic pharmacophore through the α-amine group, such as improved δ-antagonism, and presence of μ-agonism and μ-antagonism. We report the synthesis of a new series of compounds with the general formula H-Dmt-Tic-NH-(CH₂)₄-CH(R)-R’ (R = -NH₂, -NH-Ac, -NH-Z; R’ = CO-NH-Ph, -CO-NH-CH₂-Ph, -Bid) in which Lys is linked to Dmt-Tic through its amine group side chain. The compounds (1-9) displayed a potent and selective δ-antagonism (pA₂ = 7.81-8.27) independent of the functionalized α-amine and carboxylic groups of the C-terminal Lys. This suggests direct application as a prototype intermediate, such as Boc-Dmt-Tic-ε-Lys(Z)-OMe, which could be applied in the synthesis (after Z or methyl ester removal) of unique “designed multiple ligands” containing the pharmacophore of the quintessential δ-antagonist Dmt-Tic and another opioid or biologically active non-opioid ligand.

Introduction

Extensive structure-activity studies of the prototype δ-opioid receptor antagonist H-Dmt-Tic-OH ^a,¹ revealed that even minor chemical modifications changed its pharmacological profile.² This included enhanced δ-antagonism³ and reversal to δ-agonism,⁴ the appearance of dual μ-agonist/δ-agonist,⁵ as well as formation of mixed μ-agonist/δ-antagonist,⁵ and ligands with specific μ-agonism⁶ and μ-antagonism.⁶ Each pharmacological profile indicated interesting potential for therapeutic applications, such as production of analgesia with low tolerance and minimal dependence,⁵ antidepressant activity,^7,8 neuroprotection and neurogenesis,⁹ regulation of food intake ¹⁰ and in the treatment of alcoholism.¹¹

Recently, we demonstrated that the substitution of the C-terminal amino acid in tri- and tetrapeptides containing the Dmt-Tic pharmacophore with side chain protected Lys improved δ-antagonist potency.^12,13 On the basis of those results, we extended the substitution of the side chain protected or unprotected Lys to other biologically active compounds previously developed by us [H-Dmt-Tic-NH-CH(CH₂-COOH)-Bid (Bid = 1H-benzimidazole-2-yl) a δ-agonist; H-Dmt-Tic-Gly-NH-Ph a dual μ-agonist/δ-agonist; H-Dmt-Tic-Gly-NH-CH₂-Ph a mixed μ-agonist/δ-antagonist] with a quite surprising array of interesting results. Lysine, when introduced in place of the C-terminal amino acid in the above reference compounds did not produce a simple improvement in the original pharmacological activities, but provided opioid ligands which exhibited a plethora of pharmacological properties ranging from δ-antagonism and μ-agonism as well as μ-antagonism.⁶ Considering the variety of the biological effects induced by Lys in tripeptides and pseudotripeptides based on the general formula H-Dmt-Tic-Lys(R)-R’, the studies described herein extend our initial investigations on the synthesis and biological evaluation of a new series of constitutional isomers developed on the framework of H-Dmt-Tic-ε-Lys(R)-R’, where Lys is linked to the Dmt-Tic dipeptide through the ε amine group in order to further evaluate the important influence of Lys on opioid receptor interactions and functional bioactivities to produce opioid ligands for potential translation into human health initiatives.

Chemistry

Peptides (1-6) and pseudopeptides (7-9) were prepared stepwise by solution peptide synthetic methods, as outlined in Schemes 1 and 2, respectively. Boc-Tic-OH was condensed with commercially available Z-Lys-OMe or Ac-Lys-OMe via WSC/HOBt obtaining the corresponding Boc-Tic-ε-Lys(Z)-OMe or Boc-Tic-ε-Lys(Ac)-OMe. C-Terminal methyl ester protecting groups were removed by hydrolysis with 1N NaOH and then each pseudodipeptide was condensed with benzylamine or aniline via WSC/HOBt. N-terminal Boc-protected pseudodipeptide amides were treated with TFA and condensed with Boc-Dmt-OH via WSC/HOBt. Final N-terminal Boc deprotection with TFA gave compounds (1, 2, 4 and 5; Scheme 1). Catalytic hydrogenation (10% Pd/C) and TFA treatment of Boc-Dmt-Tic-ε-Lys(Z)-amides gave the final products 3 and 6 (Scheme 1). Pseudopeptides (7-9) containing C-terminal 1H-benzimidazol-2-yl (Bid) were synthesized in a similar manner (Scheme 2). Mixed carbonic anhydride coupling of Boc-Tic-ε-Lys(Z)-OH or Boc-Tic-ε-Lys(Ac)-OH with o-phenylendiamine gave the corresponding crude intermediate monoamides, which were converted without purification to the desired heteroaromatic derivatives by cyclization and dehydration in acetic acid. As detailed in Scheme 1, after N^α deprotection with TFA, each derivative was condensed with Boc-Dmt-OH via WSC/HOBt. Final N-terminal Boc deprotection with TFA gave compounds (7, 8, Scheme 2). Catalytic hydrogenation (10% Pd/C) and TFA treatment of Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid provided the product 9 (Scheme 2). Final compounds (1-9) were purified by preparative HPLC as described in Supporting Information.

Scheme 1.

Synthesis of Compounds 1-6.

Scheme 2.

Synthesis of Compounds 7-9.

Results and Discussion

Receptor Affinity Analysis

Receptor binding and functional bioactivities are reported in Table 1. All the compounds (1-9) exhibited nanomolar affinity for δ-opioid receptors (K_i^δ = 0.21-2.64 nM). As expected, the lack of a free carboxylic function in molecules containing the Dmt-Tic pharmacophore induced a substantial increase in μ-opioid receptor affinity (K_i^μ = 0.60-3.43 nM).^12,4 Compounds (1, 2, 4, 5, 7, 8) containing a Lys residue protected at the α-amine function (Z, Ac) had weak δ-opioid receptor selectivity (K_i^μ/K_i^δ = 16.3-3.2); the acetyl protecting group conferred marginally better selectivity than the Z group. Removal of the α-amine protecting group of Lys (3, 6, 9) shifted the δ-opioid selectivity to very weak μ-selective ligands, which is essentially attributable to the presence of the additional positive charge.¹⁴ The same general behaviour was observed previously in the series of Dmt-Tic containing peptides containing Lys in the third position.⁶

Table 1.

Receptor Binding Affinities and Functional Bioactivities of Compounds 1-9

		receptor affinitya (nM)		selectivity	functional bioactivity
Comp.	Structure	Kiδ	Kiμ	Kiμ/Kiδ	MVD pA2c	GPI IC50 (nM)b
	H-Dmt-Tic-NH2d	1.22	277	227	7.2	>10000
1	H-Dmt-Tic-ε-Lys(Z)-NH-CH2-Ph	0.53±0.08 (4)	3.15±0.39 (4)	5.9	8.27	1451±200
2	H-Dmt-Tic-ε-Lys(Ac)-NH-CH2-Ph	0.21±0.01 (3)	3.43±0.54 (4)	16.3	8.07	1990±674
3	H-Dmt-Tic-ε-Lys-NH-CH2-Ph	1.00±0.02 (4)	0.60±0.11 (4)	1.7*	7.81	553±173
4	H-Dmt-Tic-ε-Lys(Z)-NH-Ph	0.47±0.04 (4)	2.67±0.41 (4)	5.7	8.23	711±194
5	H-Dmt-Tic-ε-Lys(Ac)-NH-Ph	0.22±0.005 (3)	2.57±0.34 (4)	11.7	8.18	486±63
6	H-Dmt-Tic-ε-Lys-NH-Ph	2.02±0.20 (4)	0.89±0.12 (4)	2.3*	7.92	515±73
7	H-Dmt-Tic-NH-(CH2)4-CH(NH-Z)-Bid	0.86±0.06 (3)	2.78±0.27 (4)	3.2	7.82	618±140
8	H-Dmt-Tic-NH-(CH2)4-CH(NH-Ac)-Bid	0.21±0.01 (3)	0.99±0.06 (4)	4.7	8.12	610±185
9	H-Dmt-Tic-NH-(CH2)4-CH(NH2)-Bid	2.64±0.18 (3)	1.02±0.08 (3)	2.6*	8.09	434±26

^aThe K_i values (nM) were determined according to Cheng and Prusoff.²⁴ The mean ± SE with n repetitions in parenthesis is based on independent duplicate binding assays with five to eight peptide doses using several different synaptosomal preparations.

^bAgonist activity was expressed as IC₅₀ obtained from dose-response curves. These values represent the mean ± SE for at least five to six fresh tissue samples. Deltorphin II and endomorphin-2 were the internal standards for MVD (δ-opioid receptor bioactivity) and GPI (μ-opioid receptor bioactivity) tissue preparation, respectively.

^cThe pA₂ values of opioid antagonists against the agonist deltorphin II were determined by the method of Kosterlitz and Watt.²⁵

^dData taken from Salvadori et al.¹

^*μ-opioid receptor selectivity K_iδ/K_iμ.

Functional Bioactivity

Compounds 1-9 were tested in the electrically stimulated MVD and GPI assays for intrinsic functional bioactivity (Table 1). We and other investigators have previously discussed the discrepancy in the correlation between receptor binding affinities and functional bioactivity. Unfortunately, we have neither definitive nor comprehensive explanations for these observations.^12,6 In comparison to the Dmt-Tic peptides containing a protected or unprotected Lys residue at the C-terminus,⁶ all the analogues are inactive as δ-opioid agonists in the MVD assay (Table 1). Furthermore, they exhibited a weak or very weak μ-agonism in the GPI assay (IC₅₀ 434-1990 nM), which is in quite good agreement with the previous studies, except analogues containing Bid at the C-terminus.⁶ When Lys was linked to Dmt-Tic through the α-amine group and its carboxylic function transformed into Bid, the pseudopeptides predominately exhibited selective μ-agonism. On the other hand, when Lys was linked to Dmt-Tic through the ε-amine group and its carboxylic function once again transformed into Bid, no exceptional μ-agonism activity was observed. Interestingly, compounds (1-9) exhibited δ-antagonism to approximately the same order of magnitude (MVD, pA₂ 7.82-8.27), which was independent of the substitutions and modifications made on the α amine and carboxylic functions of Lys, but were in quite good agreement with the reference dipeptide H-Dmt-Tic-NH₂.¹

Conclusions

Considering the new derivatives (1-9) as analogues of the published reference compounds [H-Dmt-Tic-NH-CH₂-Bid (δ-agonist); H-Dmt-Tic-NH-CH(CH₂-COOH)-Bid (δ-agonist); H-Dmt-Tic-Gly-NH-Ph (dual μ-agonist/δ-agonist); H-Dmt-Tic-Gly-NH-CH₂-Ph (mixed μ-agonist/δ-antagonist)], the introduction of Lys (linked through its ε amine group to the Dmt-Tic pharmacophore) in place of the C-terminal amino acid failed to maintain the original pharmacological activity as previously reported for the corresponding isomers containing Lys linked through the α amine group.⁶ While isomers containing the C-terminal α-Lys revealed a variety of functional opioid bioactivities (δ-antagonism, μ-agonism, μ-antagonism),⁶ the isomers containing a C-terminal ε-Lys demonstrated δ-opioid antagonism to approximately the same order of magnitude and independent of the substituents linked to the α position.

Without taking into consideration the different behaviour of Lys when coupled to the Dmt-Tic pharmacophore through its α or ε amine group, it would be significantly beneficial to utilize these new isomers as potential precursors in the synthesis of “designed multiple ligands,” where one of the two pharmacophores is represented by the δ-selective antagonist dipeptide Dmt-Tic.^15,16 The four methylene groups of the Lys side chain can be considered as the spacer linking the first pharmacophore (Dmt-Tic) and whose length, as reported by Neumeyer et al., generally does not influence the biological activity of either pharmacophore.^17,18 The second pharmacophore, required to complete the potential “designed multiple ligands,” could be opioid compounds or other pharmacophores endowed with activity toward receptors exhibiting completely different (non-opioid) bioactivities.^16,19 More importantly, the second pharmacophore can be conveniently inserted using the deprotected α amino or carboxylic function depending on the required final bioactive product. In fact, very similar compounds were obtained when using fluorescent chromophores in place of the second pharmacophore.^20,21 Furthermore, Okada et al. reported the synthesis of similar substances with the general formula H-Dmt-Tic-NH-(CH₂)₆-NH-R (where R = Dmt, Phe, Tic and Tic-Dmt) in which all the opioids displayed increased δ antagonism, which as attributable to the additional aromatic amino acids.²² However, the same enhancement in δ-antagonist activity could be derived, at least in part, by the presence of the spacer as reported here and in preceding studies.^20,21

In summary, we suggest the possibility using a unique intermediate [for example, Boc-Dmt-Tic-ε-Lys(Z)-OMe] in the synthesis of “designed multiple ligands” containing the following constituents: (a) the δ-antagonist pharmacophore Dmt-Tic; (b) a spacer of defined length; or (c) two different protected functionalities (amine and carboxylic functions) for linkage to a variety of second pharmacophores. As a further exploration of this proposal, the synthesis of multiple ligands derived from the coupling of the selectively deprotected δ-antagonist intermediate Boc-Dmt-Tic-ε-Lys(Z)-OMe with salvinorin A^23a (κ-agonist), and the synthesis of H-Dmt-Tic-ε-Lys(4-fluorobenzoyl)-OH as a potential pharmacological tool for PET imaging of δ receptors^23b,c are currently in progress in our laboratory.

Experimental Section

Chemistry. General Methods

Crude peptides and pseudopeptides were purified by preparative reversed-phase HPLC [Waters Delta Prep 4000 system with Waters Prep LC 40 mm Assembly column C18 (30 cm × 4 cm, 15 μm particle)] and eluted at a flow rate of 25 mL/min with mobile phase solvent A (10% acetonitrile + 0.1% TFA in H₂O, v/v), and a linear gradient from 25 to 75% B (60%, acetonitrile + 0.1% TFA in H₂O, v/v) in 25 min. Analytical HPLC analyses were performed with a Beckman System Gold (Beckman ultrasphere ODS column, 250 mm × 4.6 mm, 5 μm particle). Analytical determinations and capacity factor (K’) of the products used HPLC in solvents A and B programmed at flow rate of 1 mL/min with linear gradients from 0 to 100% B in 25 min. Analogues had less than 1% impurities at 220 and 254 nm.

TLC was performed on precoated plates of silica gel F254 (Merck, Darmstadt, Germany): (A) 1-butanol/AcOH/H₂O (3:1:1, v/v/v); (B) CH₂Cl₂/toluene/methanol (17:1:2). Ninhydrin (1% ethanol, Merck), fluorescamine (Hoffman-La Roche) and chlorine spray reagents. Melting points were determined on a Kofler apparatus and are uncorrected. Optical rotations were assessed at 10 mg/mL in methanol with a Perkin-Elmer 241 polarimeter in a 10 cm water-jacketed cell. Molecular weights of the compounds were determined by a MALDI-TOF analysis (Hewlett Packard G2025A LD-TOF system mass spectrometer) and α-cyano-4-hydroxycinnamic acid as a matrix. ¹H NMR (δ) spectra were measured, when not specified, in DMSO-d₆ solution using a Bruker AC-200 spectrometer, and peak positions are given in parts per million downfield from tetramethylsilane as internal standard.

Peptide Synthesis. Boc-Tic-ε-Lys(Z)-OMe

To a solution of Boc-Tic-OH (0.9 g, 3.24 mmol) and HCl^.Z-Lys-OMe (0.95 g, 3.24 mmol) in DMF (10 mL) at 0 °C, NMM (0.35 mL, 3.24 mmol), HOBt (0.54 g, 3.56 mmol) and WSC (0.68 g, 3.56 mmol) were added. The reaction mixture was stirred for 3 h at 0 °C and 24 h at room temperature. After DMF was evaporated, the residue was dissolved in EtOAc and washed with citric acid (10% in H₂O), NaHCO₃ (5% in H₂O), and brine. The organic phase was dried (Na₂SO₄) and evaporated to dryness. The residue was precipitated from Et₂O/Pe (1:9, v/v): yield 1.4 g (78%); R_f (B) 0.89; HPLC K’ 5.43; mp 101-103 °C; [α]²⁰_D -20.1; m/z 554 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.90 (m, 15H), 3.05-3.67 (m, 7H), 4.22-4.42 (m, 3H), 4.92-5.34 (m, 3H), 6.96-7.19 (m, 9H).

Boc-Tic-ε-Lys(Z)-OH

To a solution of Boc-Tic-ε-Lys(Z)-OMe (1.4 g, 2.53 mmol) in MeOH (10 mL) was added 1N NaOH (2.8 mL). The reaction mixture was stirred for 24 h at room temperature. After solvent evaporation, the residue was dissolved in EtOAc and washed with citric acid (10% in H₂O) and brine. The organic phase was dried (Na₂SO₄) and evaporated to dryness. The residue was precipitated from Et₂O/Pe (1:1, v/v): yield 1.1 g (81%); R_f (B) 0.42; HPLC K’ 3.71; mp 120-122 °C; [α]²⁰_D -21.2; m/z 540 (M+H)⁺.

Boc-Tic-ε-Lys(Z)-NH-CH₂-Ph

To a solution of Boc-Tic-ε-Lys(Z)-OH (0.52 g, 0.96 mmol) and benzylamine (0.1 mL, 0.96 mmol) in DMF (10 mL) at 0 °C, HOBt (0.16 g, 1.06 mmol) and WSC (0.2 g, 1.06 mmol) were added. The reaction mixture was stirred for 3 h at 0 °C and 24 h at room temperature. After DMF was evaporated, the residue was dissolved in EtOAc and washed with citric acid (10% in H₂O), NaHCO₃ (5% in H₂O), and brine. The organic phase was dried (Na₂SO₄) and evaporated to dryness. The residue was precipitated from Et₂O/Pe (1:9, v/v): yield 0.53 g (88%); R_f (B) 0.85; HPLC K’ 5.37; mp 102-104 °C; [α]²⁰_D -17.7; m/z 629 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.79 (m, 15H), 3.05-3.20 (m, 4H), 4.22-4.53 (m, 5H), 4.92-5.34 (m, 3H), 6.96-7.14 (m, 14H).

TFA^.H-Tic-ε-Lys(Z)-NH-CH₂-Ph

Boc-Tic-ε-Lys(Z)-NH-CH₂-Ph (0.47 g, 0.75 mmol) was treated with TFA (2 mL) for 0.5 h at room temperature. Et₂O/Pe (1:1, v/v) were added to the solution until the product precipitated: yield 0.34 g (87%); R_f (A) 0.51; HPLC K’ 4.3; mp 115-117 °C; [α]²⁰_D -19.8; m/z 529 (M+H)⁺.

Boc-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph

To a solution of Boc-Dmt-OH (0.075 g, 0.24 mmol) and TFA^.H-Tic-ε-Lys(Z)-NH-CH₂-Ph (0.15 g, 0.24 mmol) in DMF (10 mL) at 0 °C, NMM (0.03 mL, 0.24 mmol), HOBt (0.04 g, 0.26 mmol) and WSC (0.05 g, 0.26 mmol) were added. The reaction mixture was stirred for 3 h at 0 °C and 24 h at room temperature. After DMF was evaporated, the residue was dissolved in EtOAc and washed with citric acid (10% in H₂O), NaHCO₃ (5% in H₂O), and brine. The organic phase was dried (Na₂SO₄) and evaporated to dryness. The residue was precipitated from Et₂O/Pe (1:9, v/v): yield 0.17 g (87%); R_f (B) 0.81; HPLC K’ 5.23; mp 129-131 °C; [α]²⁰_D -16.6; m/z 820 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.79 (m, 15H), 2.35 (s, 6H), 3.05-3.20 (m, 6H), 4.46-4.53 (m, 5H), 4.92-5.34 (m, 4H), 6.29 (s, 2H), 6.96-7.19 (m, 14H).

TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph (1)

Boc-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph (0.11 g, 0.13 mmol) was treated with TFA (1 mL) for 0.5 h at room temperature. Et₂O/Pe (1:1, v/v) were added to the solution until the product precipitated: yield 0.09 g (96%); R_f (A) 0.47; HPLC K’ 3.63; mp 125-127 °C; [α]²⁰_D -14.6; m/z 721 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.79 (m, 6H), 2.35 (s, 6H), 3.05-3.20 (m, 6H), 3.95-4.53 (m, 6H), 4.92-5.34 (m, 3H), 6.29 (s, 2H), 6.96-7.19 (m, 14H). Anal Calcd for C₄₄H₅₀F₃N₅O₈: C, 63.37; H, 6.04; N, 8.40. Found: C, 63.22; H, 5.98; N, 8.21.

Boc-Tic-ε-Lys(Ac)-OMe

This intermediate was obtained by condensation of Boc-Tic-OH with HCl^.Ac-Lys-OMe via WSC/HOBt, as reported for Boc-Tic-ε-Lys(Z)-OMe: yield 1.6 g (82%); R_f (B) 0.77; HPLC K’ 5.32; mp 127-129 °C; [α]²⁰_D -20.5; m/z 463 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.90 (m, 15H), 2.02 (s, 3H), 2.92-3.67 (m, 7H), 4.17-4.92 (m, 4H), 6.96-7.02 (m, 4H).

Boc-Tic-ε-Lys(Ac)-OH

This intermediate was obtained by hydrolysis of Boc-Tic-ε-Lys(Ac)-OMe as reported for Boc-Tic-ε-Lys(Z)-OH: yield 1.26 g (82%); R_f (B) 0.45; HPLC K’ 5.18; mp 135-137 °C; [α]²⁰_D -22.3; m/z 449 (M+H)⁺.

Boc-Tic-ε-Lys(Ac)-NH-CH₂-Ph

This intermediate was obtained by condensation of Boc-Tic-ε-Lys(Ac)-OH with benzylamine via WSC/HOBt as reported for Boc-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.33 g (81%); R_f (B) 0.79; HPLC K’ 5.32; mp 108-110 °C; [α]²⁰_D -18.6; m/z 537 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.79 (m, 15H), 2.02 (s, 3H), 2.92-3.20 (m, 4H), 4.17-4.92 (m, 6H), 6.96-7.14 (m, 9H).

TFA^.H-Tic-ε-Lys(Ac)-NH-CH₂-Ph

Boc-Tic-ε-Lys(Ac)-NH-CH₂-Ph was treated with TFA as reported for TFA^.H-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.21 g (97%); R_f (A) 0.48; HPLC K’ 3.92; mp 121-123 °C; [α]²⁰_D -20.7; m/z 437 (M+H)⁺.

Boc-Dmt-Tic-ε-Lys(Ac)-NH-CH₂-Ph

This intermediate was obtained by condensation of Boc-Dmt-OH with TFA^.H-Tic-ε-Lys(Ac)-NH-CH₂-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.14 g (83%); R_f (B) 0.75; HPLC K’ 4.92; mp 135-137 °C; [α]²⁰_D -17.5; m/z 729 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.79 (m, 15H), 2.02 (s, 3H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 4.41-4.92 (m, 7H), 6.29 (s, 2H), 6.96-7.14 (m, 9H).

TFA^.H-Dmt-Tic-ε-Lys(Ac)-NH-CH₂-Ph (2)

Boc-Dmt-Tic-ε-Lys(Ac)-NH-CH₂-Ph was treated with TFA as reported for TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.08 g (96%); R_f (A) 0.45; HPLC K’ 3.21; mp 131-133 °C; [α]²⁰_D -15.5; m/z 629 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.79 (m, 6H), 2.02 (s, 3H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.95-4.92 (m, 7H), 6.29 (s, 2H), 6.96-7.14 (m, 9H). Anal Calcd for C₃₈H₄₆F₃N₅O₇: C, 61.53; H, 6.25; N, 9.44. Found: C, 61.77; H, 6.39; N, 9.15.

Boc-Dmt-Tic-ε-Lys-NH-CH₂-Ph

To a solution of Boc-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph (0.1 g, 0.12 mmol) in methanol (30 mL) was added Pd/C (10%, 0.07 g), and H₂ was bubbled for 1 h at room temperature. After filtration, the solution was evaporated to dryness. The residue was precipitated from Et₂O/Pe (1:9, v/v): yield 0.07 g (85%); R_f (B) 0.58; HPLC K’ 4.98; mp 144-145 °C; [α]²⁰_D -18.7; m/z 687 (M+H)⁺.

2TFA^.H-Dmt-Tic-ε-Lys-NH-CH₂-Ph (3)

Boc-Dmt-Tic-ε-Lys-NH-CH₂-Ph was treated with TFA as reported for TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.07 g (95%); R_f (A) 0.39; HPLC K’ 3.32; mp 148-150 °C; [α]²⁰_D -16.2; m/z 587 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.79 (m, 6H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.56-4.92 (m, 7H), 6.29 (s, 2H), 6.96-7.14 (m, 9H). Anal Calcd for C₃₈H₄₅F₆N₅O₈: C, 56.08; H, 5.57; N, 8.61. Found: C, 56.30; H, 5.68; N, 8.70.

Boc-Tic-ε-Lys(Z)-NH-Ph

This intermediate was obtained by condensation of Boc-Tic-ε-Lys(Z)-OH with aniline via WSC/HOBt as reported for Boc-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.52 g (88%); R_f (B) 0.81; HPLC K’ 5.61; mp 94-96 °C; [α]²⁰_D -19.4; m/z 615 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.89 (m, 15H), 2.92-3.20 (m, 4H), 4.17-5.34 (m, 6H), 6.96-7.64 (m, 14H).

TFA^.H-Tic-ε-Lys(Z)-NH-Ph

Boc-Tic-ε-Lys(Z)-NH-Ph was treated with TFA as reported for TFA^.H-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.37 g (95%); R_f (A) 0.46; HPLC K’ 4.32; mp 111-113 °C; [α]²⁰_D -19.9; m/z 515 (M+H)⁺.

Boc-Dmt-Tic-ε-Lys(Z)-NH-Ph

This intermediate was obtained by condensation of Boc-Dmt-OH with TFA^.H-Tic-ε-Lys(Z)-NH-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.17 g (87%); R_f (B) 0.76; HPLC K’ 5.73; mp 124-126 °C; [α]²⁰_D -15.9; m/z 807 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.89 (m, 15H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 4.41-5.34 (m, 7H), 6.29 (s, 2H), 6.96-7.64 (m, 14H).

TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-Ph (4)

Boc-Dmt-Tic-ε-Lys(Z)-NH-Ph was treated with TFA as reported for TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.09 g (90%); R_f (A) 0.40; HPLC K’ 3.70; mp 133-135 °C; [α]²⁰_D -13.9; m/z 707 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.89 (m, 6H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.95-5.34 (m, 7H), 6.29 (s, 2H), 6.96-7.64 (m, 14H). Anal Calcd for C₄₃H₄₈F₃N₅O₈: C, 62.99; H, 5.90; N, 8.54. Found: C, 62.95; H, 5.76; N, 8.41.

Boc-Tic-ε-Lys(Ac)-NH-Ph

This intermediate was obtained by condensation of Boc-Tic-ε-Lys(Ac)-OH with aniline via WSC/HOBt as reported for Boc-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.32 g (81%); R_f (B) 0.74; HPLC K’ 4.21; mp 100-102 °C; [α]²⁰_D -19.9; m/z 524 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.89 (m, 15H), 2.02 (s, 3H), 2.92-3.20 (m, 4H), 4.17-4.92 (m, 4H), 6.96-7.64 (m, 9H).

TFA^.H-Tic-ε-Lys(Ac)-NH-Ph

Boc-Tic-ε-Lys(Ac)-NH-Ph was treated with TFA as reported for TFA^.H-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.27 g (96%); R_f (A) 0.43; HPLC K’ 3.47; mp 117-119 °C; [α]²⁰_D -20.8; m/z 424 (M+H)⁺.

Boc-Dmt-Tic-ε-Lys(Ac)-NH-Ph

This intermediate was obtained by condensation of Boc-Dmt-OH with TFA^.H-Tic-ε-Lys(Ac)-NH-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.15 g (88%); R_f (B) 0.71; HPLC K’ 5.21; mp 130-122 °C; [α]²⁰_D -16.8; m/z 715 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.89 (m, 15H), 2.02 (s, 3H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 4.41-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.64 (m, 9H).

TFA^.H-Dmt-Tic-ε-Lys(Ac)-NH-Ph (5)

Boc-Dmt-Tic-ε-Lys(Ac)-NH-Ph was treated with TFA as reported for TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.08 g (98%); R_f (A) 0.37; HPLC K’ 2.89; mp 127-129 °C; [α]²⁰_D -14.8; m/z 615 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.89 (m, 6H), 2.02 (s, 3H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.95-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.64 (m, 9H). Anal Calcd for C₃₇H₄₄F₃N₅O₇: C, 61.06; H, 6.09; N, 9.62. Found: C, 60.96; H, 6.03; N, 9.48.

Boc-Dmt-Tic-ε-Lys-NH-Ph

Boc-Dmt-Tic-ε-Lys(Z)-NH-Ph was dissolved in methanol and treated with Pd/C (10%) and H₂ as reported for Boc-Dmt-Tic-ε-Lys-NH-CH₂-Ph: yield 0.08 g (87%); R_f (B) 0.55; HPLC K’ 5.12; mp 146-148 °C; [α]²⁰_D -19.1; m/z 673 (M+H)⁺.

2TFA^.H-Dmt-Tic-ε-Lys-NH-Ph (6)

Boc-Dmt-Tic-ε-Lys-NH-Ph was treated with TFA as reported for TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.04 g (95%); R_f (A) 0.37; HPLC K’ 2.58; mp 147-149 °C; [α]²⁰_D -14.8; m/z 573 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.89 (m, 6H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.56-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.64 (m, 9H). Anal Calcd for C₃₇H₄₃F₆N₅O₈: C, 55.57; H, 5.42; N, 8.76. Found: C, 55.82; H, 5.53; N, 8.47.

Boc-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid

To a solution of Boc-Tic-ε-Lys(Z)-OH (0.5 g, 0.93 mmol) and NMM (2.1 mL, 0.93 mmol) in DMF(10 mL) was treated at -20 °C with IBCF (0.12 mL, 0.93 mmol). After 10 min. at -20 °C, o-phenylendiamine (0.1 g, 0.93 mmol) was added. The reaction mixture was allowed to stir while slowly warming to room temperature (1 h) and was then stirred for an additional 3 h. The solvent was evaporated and the residue was partitioned between EtOAc and H₂O. The EtOAc layer was washed with NaHCO₃ (5% in H₂O) and brine and dried over Na₂SO₄. The solution was filtered, the solvent evaporated, and the residual solid was dissolved in glacial acetic acid (10 mL). The solution was heated at 65 °C for 1 h. After the solvent was evaporated, the residue was precipitated from Et₂O/Pe (1:9, v/v): yield 0.47 g (82%); R_f (B) 0.66; HPLC K’ 4.92; mp 134-136 °C; [α]²⁰_D -12.8; m/z 613 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.84 (m, 15H), 2.92-3.20 (m, 4H), 4.17-5.34 (m, 6H), 6.96-7.70 (m, 13H).

2TFA^.H-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid

Boc-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid was treated with TFA as reported for TFA^.H-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.33 g (96%); R_f (A) 0.41; HPLC K’ 3.57; mp 137-139 °C; [α]²⁰_D -14.1; m/z 513 (M+H)⁺.

Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid

To a solution of Boc-Dmt-OH (0.075 g, 0.24 mmol) and 2TFA^.H-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid (0.18 g, 0.24 mmol) in DMF (10 mL) at 0 °C, NMM (0.05 mL, 0.48 mmol), HOBt (0.04 g, 0.26 mmol) and WSC (0.05 g, 0.26 mmol) were added. The reaction mixture was stirred for 3 h at 0 °C and 24 h at room temperature. After DMF was evaporated, the residue was dissolved in EtOAc and washed with NaHCO₃ (5% in H₂O), and brine. The organic phase was dried (Na₂SO₄)and evaporated to dryness. The residue was precipitated from Et₂O/Pe (1:9, v/v): yield 0.16 g (85%); R_f (B) 0.66; HPLC K’ 4.93; mp 137-139 °C; [α]²⁰_D -14.3; m/z 804 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.84 (m, 15H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 4.41-5.34 (m, 7H), 6.29 (s, 2H), 6.96-7.70 (m, 13H).

2TFA^.H-Dmt-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid (7)

Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid. was treated with TFA as reported for TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.05 g (94%); R_f (A) 0.33; HPLC K’ 2.95; mp 143-145 °C; [α]²⁰_D -17.8; m/z 704 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.84 (m, 6H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.95-5.34 (m, 7H), 6.29 (s, 2H), 6.96-7.70 (m, 13H). Anal Calcd for C₄₅H₄₈F₆N₆O₉: C, 58.06; H, 5.20; N, 9.06. Found: C, 57.97; H, 5.16; N, 8.89.

Boc-Tic-NH-(CH₂)₄-CH(NH-Ac)-Bid

This intermediate was obtained by condensation of Boc-Tic-ε-Lys(Ac)-OH with o-phenylendiamine via mixed anhydrides (IBCF) as reported for Boc-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid: yield 0.34 g (88%); R_f (B) 0.60; HPLC K’ 4.31; mp 140-142 °C; [α]²⁰_D -13.7; m/z 521 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.84 (m, 15H), 2.02 (s, 3H), 2.92-3.20 (m, 4H), 4.17-4.92 (m, 4H), 6.96-7.70 (m, 8H).

2TFA^.H-Tic-NH-(CH₂)₄-CH(NH-Ac)-Bid

Boc-Tic-NH-(CH₂)₄-CH(NH-Ac)-Bid was treated with TFA as reported for TFA^.H-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.20 g (90%); R_f (A) 0.44; HPLC K’ 3.21; mp 143-145 °C; [α]²⁰_D -15.0; m/z 421 (M+H)⁺.

Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH-Ac)-Bid

This intermediate was obtained by condensation of Boc-Dmt-OH with 2TFA^.H-Tic-NH-(CH₂)₄-CH(NH-Ac)-Bid via WSC/HOBt as reported for Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid: yield 0.14 g (85%); R_f (B) 0.60; HPLC K’ 4.21; mp 132-134 °C; [α]²⁰_D -15.2; m/z 712 (M+H)⁺; ¹H NMR (DMSO-d₆) δ 1.29-1.84 (m, 15H), 2.02 (s, 3H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 4.41-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.70 (m, 8H).

2TFA^.H-Dmt-Tic-NH-(CH₂)₄-CH(NH-Ac)-Bid (8)

Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH-Ac)-Bid. was treated with TFA as reported for TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.08 g (98%); R_f (A) 0.30; HPLC K’ 2.63; mp 149-151 °C; [α]²⁰_D -18.7; m/z 612 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.84 (m, 6H), 2.02 (s, 3H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.95-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.70 (m, 8H). Anal Calcd for C₃₉H₄₄F₆N₆O₈: C, 55.84; H, 5.29; N, 10.02. Found: C, 56.01; H, 5.38; N, 10.12.

Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH₂)-Bid

Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH-Z)-Bid was dissolved in methanol and treated with Pd/C (10%) and H₂ as reported for Boc-Dmt-Tic-ε-Lys-NH-CH₂-Ph: yield 0.08 g (86%); R_f (B) 0.51; HPLC K’ 3.56; mp 140-142 °C; [α]²⁰_D -19.3; m/z 670 (M+H)⁺.

3TFA^.H-Dmt-Tic-NH-(CH₂)₄-CH(NH₂)-Bid (9)

Boc-Dmt-Tic-NH-(CH₂)₄-CH(NH₂)-Bid. was treated with TFA as reported for TFA^.H-Dmt-Tic-ε-Lys(Z)-NH-CH₂-Ph: yield 0.06 g (95%); R_f (A) 0.29; HPLC K’ 2.86; mp 154-156 °C; [α]²⁰_D -19.9; m/z 570 (M+H)⁺, ¹H NMR (DMSO-d₆) δ 1.29-1.84 (m, 6H), 2.35 (s, 6H), 2.92-3.20 (m, 6H), 3.90-4.92 (m, 5H), 6.29 (s, 2H), 6.96-7.70 (m, 8H). Anal Calcd for C₃₉H₄₃F₉N₆O₉: C, 51.43; H, 4.76; N, 9.23. Found: C, 51.32; H, 4.62; N, 9.12.

Pharmacology. Radioreceptor Binding Assays

Opioid receptor affinity were determined under equilibrium conditions [2.5 h at room temperature (23 °C)] in a competition assay using brain P₂ synaptosomal membranes prepared from Sprague-Dawley rats.^26,27 Synaptosomes were preincubated to remove endogenous opioid peptides and stored at -80 °C in buffered 20% glycerol.^26,28 Each analogue was analyzed in duplicate assays using five to eight dosages and three to five independent repetitions with different synaptosomal preparations (n values are listed in Table 1 in parenthesis and results are mean ± SE). Unlabeled peptide (2 μM) was used to determine non-specific binding in the presence of 1.9 nM [³H]deltorphin II (45.0 Ci/mmol, Perkin Elmer, Boston, MA; K_D = 1.4 nM) for δ-opioid receptors and 3.5 nM [³H]DAMGO (50.0 Ci/mmol), Amersham Bioscience, Buckinghamshire, U. K.; K_D = 1.5 nM) for μ-opioid receptors. Glass fibre filters (Whatman GFC) were soaked in 0.1% polyethylenimine in order to enhance the signal-to-noise ratio of the bound radiolabeled-synaptosome complex, and the filters were washed thrice in ice-cold buffered BSA.²⁶ The affinity constants (K_i) were calculated according to Cheng and Prusoff.²⁴

Biological Activity in Isolated Tissue Preparations

The myenteric plexus longitudinal muscle preparations (2-3 cm segments) from the small intestine of male Hartley strain guinea pigs (GPI) measured μ-opioid receptor agonism, and a single mouse vas deferens (MVD) was used to determine δ-opioid receptor agonism as described previously.^6,29 The isolated tissues were suspended in organ baths containing balanced salt solutions in a physiological buffer, pH 7.5. Agonists were tested for the inhibition of electrically evoked contraction and expressed as IC₅₀ (nM) obtained from the dose-response curves. The IC₅₀ values represent the mean ± SE of five or six separate assays. δ-antagonist potencies in the MVD assay were determined against the δ-agonist deltorphin-II and is expressed as pA₂ determined using the Schild Plot.³⁰