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. Author manuscript; available in PMC: 2010 Jan 27.
Published in final edited form as: J Med Chem. 2008 Aug 5;51(16):5109–5117. doi: 10.1021/jm800587e

Further Studies on Lead Compounds Containing the Opioid Pharmacophore Dmt-Tic

Gianfranco Balboni †,‡,*, Stella Fiorini ‡, Anna Baldisserotto ‡, Claudio Trapella ‡, Yusuke Sasaki ^, Akihiro Ambo ^, Ewa D Marczak #, Lawrence H Lazarus #, Severo Salvadori ‡,*
PMCID: PMC2812024  NIHMSID: NIHMS166259  PMID: 18680274

Abstract

Opioids containing the Dmt-Tic pharmacophore, especially the δ agonists H-Dmt-Tic-Gly-NH-Ph 1 and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid 4 (UFP-512) were evaluated for the influence of the substitution of Gly with aspartic acid, its chirality, and the importance of the – NH-Ph and N1H-Bid hydrogens relative to δ agonism. The results provide the following conclusions: (i) Asp increases δ selectivity by lowering μ affinity; (ii) -NH-Ph and N1H-Bid nitrogen methylation transforms δ agonists into δ antagonists; (iii) substitution of Gly with L-Asp/D-Asp in the δ agonist H-Dmt-Tic-Gly-NH-Ph resulted in δ antagonists, while the same substitution in the δ agonist H-Dmt-Tic-NH-CH2-Bid yielded more selective δ agonists, H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid and H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid; (iv) L-Asp seems important only for functional bioactivity, not receptor affinity; (v) H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me) (10) revealed analgesia similar to 4, which was reversed by naltrindole only in the tail-flick test. Compounds 4 and 10 had opposite behaviours in mice: 4 caused agitation, while 10 gave sedation and convulsions.

Introduction

The prototype opioid pharmacophore Dmt-Tic,α, 1 which evolved from H-Tyr-Tic-OH2 as a simplified form of TIP(P),3 represents the minimum sequence that selectively interacts with δ opioid receptors as a potent δ antagonist. Extensive structure-activity studies on this prototype revealed that even minor chemical modifications changed its pharmacological profile, including: a wide range of properties such as: enhanced δ antagonism,4 appearance of mixed μ agonism/δ agonism5 as well as mixed μ agonism/δ antagonism,5 μ agonism,6 μ antagonism,6 δ inverse agonism,7 and δ agonism.5, 8–10 Among all synthesized analogues, some lead compounds were obtained; for example, the potent and selective δ antagonist N,N(Me)2-Dmt-Tic-OH11 and the δ inverse agonist N,N(Me)2-Dmt-Tic-NH2 12, 13 as useful tools for pharmacological studies. Some other lead compounds endowed with potential utility as therapeutic agents are represented by: the μ agonist/δ antagonist H-Dmt-Tic-Gly-NH-Bzl (2); the μ agonist/δ agonist H-Dmt-Tic-Gly-NH-Ph (1); and the potent δ agonists H-Dmt-Tic-NH-CH2-Bid 3 (UFP-502)5 and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid 4 (UFP-512)8. On the basis of different pharmacological studies, μ agonists/δ antagonists14 and μ agonists/δ agonists15 may provide new classes of analgesics with low propensity to induce tolerance, physical dependence, constipation and other side effects. δ Opioid receptor agonists are known to produce many pharmacological effects in rodents, including analgesia,16 antidepressant,17, 10 neuroprotection/neurogenesis18 and anti-Parkinson19 activities. Moreover, peripheral δ-opioid receptors seem to be involved in cancer,20 cardiovascular disease,21 gastrointestinal disorders,22 and newer paradigms for pain relief that use peripherally restricted opioids.23 Starting from our selected lead compounds, here we report some new attempts to gain a better understanding of their biological profiles, especially in the light of the fact that even minor modifications can change their pharmacological characteristics.24, 25 In particular, we focused our attention once again on the importance of the C-terminal Bid (1H-benzimidazole-2-yl) and the anilide function (considered as an open ring surrogate of Bid); in fact, both groups are important for δ agonist activity. From previous studies, hydrogen linked to the N1 of Bid24, 25 (and probably the corresponding hydrogen linked to the anilide function) seem to be important for the induction of δ agonism. To ascertain this hypothesis we synthesized some N1-Bid and N-anilide methylated analogues of our selected lead compounds. Moreover, to verify the importance of the negative charge in the induction of δ selectivity and to confirm the ineffectiveness of the C-terminal chiral center,8 we substituted Gly with L-Asp or D-Asp residues. Furthermore, this modification seems to be capable of influencing the blood-brain barrier (BBB) penetration of opioids; in fact, both 326, 27 and 410 show antidepressant activities when administered icv., but only 4 expresses these properties after intraperitoneal administration.

Chemistry

All peptides and pseudopeptides (5–13) were prepared stepwise in solution using conventional synthetic methods as outlined in Scheme 1 for the more representative compound (10). The intermediates containing Bid at their C-termini, Boc-NH-(S)CH(CH2-COOBzl)-Bid8 and Boc-NH-(R)CH(CH2-COOBzl)-Bid were prepared according to the procedure of Nestor et al.28 Briefly, mixed carbonic anhydride coupling of Boc-Asp(OBzl)-OH or Boc-D-Asp(OBzl)-OH with o-phenylendiamine gave the crude intermediate amides, which were converted without purification to the desired benzimidazole heterocycles (Bid) by cyclization / dehydration in acetic acid at 60 °C for 1 h. The corresponding Boc-protected intermediate anilides, or N(Me)-anilides, or benzylamides were prepared by condensation of Boc-Gly-OH, or Boc-Asp(OBzl)-OH, or Boc-D-Asp(OBzl)-OH with aniline, or N-methyl-aniline, or benzylamine, via mixed carbonic anhydride. Each intermediate, after Boc deprotection with TFA, was condensed with Boc-Tic-OH via WSC/HOBt. N1-Bid methylation was accomplished in DMF using K2CO3 as a base, followed after 1 h by the reaction with iodomethane.24 Subsequently, Boc deprotection (TFA) and the final condensation (WSC/HOBt) with Boc-Dmt-OH gave the fully protected compounds. Final deprotection of Asp or D-Asp side chains (by catalytic hydrogenation) and N-terminal amine functions (by TFA treatment) gave the crude final compounds (5–13), which were purified by preparative reverse phase HPLC.

Scheme 1.

Scheme 1

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Synthesis of Compound 10.

Results and Discussion

Receptor Affinity Analysis

Receptor binding data for δ- and μ-receptors and δ-selectivity (Kiμ/Kiδ) are reported in Table 1. All new compounds (5–13) had subnanomolar affinities for δ-opioid receptors (Kiδ = 0.036–0.186 nM) which is in good accordance with the reference compounds. As expected, the introduction of a negative charge, derived from the substitution of Gly with L- or D-Asp, increased δ-selectivity essentially by decreasing μ-affinity. In fact, compounds 6–13 with a Kiμ = 7.49–364.3 nM exhibited a δ-selectivity ranging from 101 to 5730 in comparison with the reference compounds and 5 lacking the negative charge (δ-selectivity = 4–14). The largest increase in δ-selectivity derived from the substitution of Gly with aspartic acid was seen in H-Dmt-Tic-Gly-NH-Bzl (2); in fact, its selectivity (Kiμ/Kiδ = 5) rose over 3 orders of magnitude to 5730. The same substitution gave lower increases in selectivity when applied to the reference compounds 1 and 3. On the basis of the C-terminal aromatic substituents, the highest selectivity appeared in the following series: –NH-Bzl > -N(Me)-Ph > -NH-Ph > -Bid ≥ -Bid(N1-Me). With regard to the aspartic acid chirality, no final conclusions can be drawn about its influence on receptor selectivity.

Table 1.

Receptor Binding Affinities and Functional Bioactivities of Compounds 1–13.

Structure Receptor affinityb (nM) Selectivity Functional bioactivity
Ref. Kiδ Kiμ Kiμ/Kiδ MVD pA2d MVD IC50c (nm) GPI I C50c (nM)
1 H-Dmt-Tic-Gly-NH-Phe 0.042 0.16 4 - 3.02 2.57
2 H-Dmt-Tic-Gly-NH-Bzle 0.031 0.16 5 9.25 - 2.69
3 H-Dmt-Tic-NH-CH2-Bide 0.035 0.50 14 - 0.13 26.92
4 H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bidf 0.443 53.9 122 - 0.12 1724
Cpd.
5 H-Dmt-Tic-Gly-N(Me)-Ph 0.143 ± 0.024 (4) 1.75 ± 0.067 (3) 12 8.80 - 1466 ± 629
6 H-Dmt-Tic-Asp-NH-Ph 0.036 ± 0.002 (3) 10.8 ± 1.2 (5) 300 9.40 - 265 ± 10.4
7 H-Dmt-Tic-D-Asp-NH-Ph 0.058 ± 0.003 (3) 7.49 ± 0.57 (4) 129 8.62 - 2655 ± 127.8
8 H-Dmt-Tic-Asp-N(Me)-Ph 0.186 ± 0.008 (3) 364.3 ± 37 (3) 1958 8.75 - >10000
9 H-Dmt-Tic-D-Asp-N(Me)-Ph 0.084 ± 0.008 (4) 45.0 ± 4.3 (5) 536 8.06 - >10000
10 H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me) 0.059 ± 0.008 (4) 5.93 ± 0.82 (5) 101 9.90 - 2886 ± 983
11 H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid(N1-Me) 0.070 ± 0.007 (4) 11.8 ± 1.4 (5) 169 9.65 - >10000
12 H-Dmt-Tic- NH-(R)CH(CH2-COOH)-Bid 0.067 ± 0.008 (5) 16.1 ± 1.9 (6) 240 - 1.21 179
13 H-Dmt-Tic-Asp-NH-Bzl 0.054 ± 0.006 (4) 309.4 ± 24 (3) 5730 8.53 - >10000
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b

The Ki values (nM) were determined according to Cheng and Prusoff.43 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.

c

Agonist activity was expressed as IC50 obtained from dose-response curves. These values represent the mean ± SE for at least four tissue samples. DPDPE and PL017 were the internal standards for MVD (δ-opioid receptor bioactivity) and GPI (μ-opioid receptor bioactivity) tissue preparation, respectively.

d

The pA2 values of opioid antagonists against the δ and μ agonists (deltorphin II and endomorphin-2, respectively) were determined by the method of Kosterlitz and Watt.49

e

Data taken from Balboni et al.5

f

Data taken from Balboni et al.8

Functional Bioactivity

Compounds 5–13 were tested in the electrically stimulated MVD and GPI pharmacological assays for intrinsic functional bioactivity (Table 1). As quite usually observed with compounds containing the Dmt-Tic pharmacophore, a close correlation between binding and functional bioactivity data is often lacking. Recently, a similar lack of correlation was observed by Hruby et al. with 4-anilidopiperidine analogues.29 As expected and partially demonstrated for 3,24, 25 all N-methylated analogues of anilides and N1-Bid (5, 8–11) revealed potent and selective δ-opioid antagonist activity (MVD, pA2 = 8.06–9.90), confirming the importance of the hydrogen of –NH-Ph and N1H-Bid on the induction of δ agonism. Surprisingly, the substitution of Gly with L-Asp (6) or D-Asp (7) in reference compound 1, gave two potent and quite selective δ antagonists (MVD, pA2 = 9.40 and 8.62, respectively) despite of the presence of the –NH-Ph hydrogen. Compound 12, the diastereoisomer containing the D-Asp side chain of δ agonist 4, indicated for the first time that better results can be obtained using L-amino acids in the synthesis of compounds containing a C-terminal Bid. In fact, it shows a δ agonist activity of one order of magnitude lower than 4, and a μ agonist activity of almost one order of magnitude higher. Interestingly, compound 10, the N1-Bid methylated analogue of 4, yielded the highest δ antagonism (pA2 = 9.90) in this series of compounds, and associated with a μ agonism 1.7 fold greater than 4. The substitution of Gly with Asp (13) in the μ agonist/δ antagonist 2 was detrimental in its activity profile; in fact, 13 had a selective δ antagonist activity (5-fold lower than 2) and associated with a very weak μ antagonist activity (GPI, pA2 = 6.26, not reported in Table 1). Finally, in the 3 pairs of compounds (6, 7; 8, 9; and 10, 11) and in the pair consisting of 4 and 12, the best δ activities were consistently seen with the analogues containing L-aspartic acid; however, this trend is not supported by the corresponding affinity data.

In Vivo Biological Activity

In recallling the data reported by Codd et al.,30 they demonstrated the in vivo biotrasformation of a δ opioid agonist into a μ agonist by N deethylation. A close look at our new compounds (5–13), a similar behaviour might be theoretically expected from all N-methylated analogues (5, 8–11). On the basis of this hypothesis,30 we chose a potent and selective δ antagonist (10) as a potential protodrug of the potent and selective δ agonist 4. However, preliminary enzymatic degradation studies (Supporting Information) failed to demonstrate and support this assumption; in fact, both compounds 4 and 10 appeared to be fully stable to enzymic degradation for 4 h and 2 h in plasma and brain homogenate, respectively. Notwithstanding the preceding negative results, we further tested 10 for in vivo analgesia in comparison with 4; a positive result might be tentatively considered as indirect evidence of the N-demethylation of 10 (δ antagonist) to the corresponding 4 (δ agonist) based on the analgesic effects of the tail-flick and hot-plate tests. Results reported in Figure 1 indicated a similar dose dependent analgesic effect for both compounds after intracerebroventricular injection: analgesia of both compounds was reversed by the δ selective antagonist naltrindole and the non-selective antagonist naloxone in the tail-flick test, but not in the hot-plate test (Figures 2 and 3). Interestingly, at the same dose 4 and 10 provided opposite behavioural effects; namely 4 caused excessive grooming and agitation (constant, fast moving in the cage, burrowing in the nesting material), while with 10 the mice appeared sedated, quiet, easily handled, and moving slowly if at all. Furthermore, 4 did not induce convulsions even at the highest doses, confirming our previous data on its antidepressant and anxiolytic studies,9, 10 which are in accord with observations about the higher convulsive effects of the nonpeptidic δ agonists in comparison to opioid peptides.26, 31, 32

Figure 1.

Figure 1

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Dose dependent effect of icv injected 4 (A, B) and 10 (C, D) in the hot-plate (A, C) and tail-flick (B, D) tests. Each point represents the mean ± SEM (n = 5 mice). The asterisks denote AUC values that are significantly different from saline treated mice by Dunnett's test (*, p < 0.05; **, p < 0.01; ***, p < 0.001) following ANOVA (panel A: P < 0.0001: F = 71.49, d.f. 4; panel B: P < 0.0001: F = 251.7, d.f. 4; panel C: P < 0.001, F = 9.356, d.f. 3; panel D: p < 0.0001: F = 15.14, d.f. 3).

Figure 2.

Figure 2

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Effect of sc injected naltrindole (3 mg/kg) and naloxone (10 mg/kg) on 10 induced antinociception in hot-plate (A, C) and tail-flick (B, D) tests. Compound 10 was administered icv (10 μg/mouse) 20 min after administration of antagonists. (A, B) Time course, (C, D) Area Under the Curve (AUC). Each point represents the mean ± SEM (n = 5 mice). The asterisks denote AUC values that are significantly different from saline treated mice by Dunett's test (*, p < 0.05; ***, p < 0.001) following ANOVA (panel C: p = 0.2138: F = 1.759, d.f. 2; panel D: p <0.001: F = 13.61, d.f. 2).

Figure 3.

Figure 3

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Effect of sc injected naltrindole (3 mg/kg) and naloxone (10 mg/kg) on 4 induced antinociception in hot-plate (A, C) and tail-flick (B, D) tests. 4 was administered icv (10 μg/mouse) 20 min after administration of antagonists. (A, B) Time course, (C, D) Area Under the Curve (AUC). Each point represents the mean ± SEM (n = 5 mice). The asterisks denote AUC values that are significantly different from saline treated mice by Dunnett's test (**, p < 0.01; ***, p < 0.001) following ANOVA (panel C: p = 0.0136: F = 6.277, d.f. 2; panel D: p < 0.0001: F = 87.13, d.f. 2).

Conclusions

Starting from the assumption that even minor chemical modifications can change the pharmacological profile of opioids, such as peptides and pseudopeptides containing the Dmt-Tic pharmacophore or nonpeptide derivatives related to morphine,33 we selected some reference compounds, especially our known δ agonists 1 and 4, and evaluated the influence of aspartic acid and its chirality, and the importance of the –NH-Ph and N1H-Bid hydrogens in the induction of δ agonism. The results obtained confirmed two expectations based on prior experimental data: (i) Asp increased the δ selectivity by lowering μ affinity; and (ii) methylation of -NH-Ph and N1H- Bid nitrogen transformed potent δ agonists into potent δ antagonists. On the other hand, several observations were quite unexpected: (i) the substitution of Gly with L-Asp or D-Asp in the δ agonist 1 gave potent and selective δ antagonists, while the same substitution in the δ agonist 3 produced the more selective δ agonists 4 and 12; (ii) Asp chirality seems to be important only in terms of functional bioactivity, since analogues 4, 6, 8, and 10, containing L-Asp are more active than the corresponding diastereoisomers 12, 7, 9, and 11, but the same is not true for receptor affinity; (iii) finally and totally unexpected— and in our opinion of potential interest—the potent and selective δ antagonist 10 yielded an analgesic effect similar to 4 that was reversed by naltrindole only when it was tested by the tail-flick test, and not in the hot-plate test. Furthermore, 4 and 10 resulted in opposite behavioural effects in mice; namely, 4 caused grooming and agitation, while with 10 mice appeared nearly sedated; convulsions were observed only in animals treated with the δ antagonist at very high doses icv. Considering the novelty of the pharmacological profile of this compound, more detailed pharmacological studies are in progress both in vivo (as an analgesic for neuropatic and inflammatory pain, antidepressant and anxiolytic) and in vitro, considering also its potential interaction with different receptors,34 or with heterodimers involving δ receptors.35 Preliminary enzymatic degradation studies that failed to demonstrate the N-demethylation of 10 to the corresponding 4 necessitates a more detailed study involving the use of different methods36 and/or other tissue preparations, such as kidney and liver30 which are planned in collaboration with other laboratories. If enzymatic N-demethylation occurs under these conditions, 6 could be considered a new lead compound of potential pharmacological utility for in vivo studies relative to δ opioid receptors trafficking at the membrane level.37–39

Experimental Section

Chemistry

Boc-Gly-N(Me)-Ph

A solution of Boc-Gly-OH (1.00 g, 5.71 mmol) and NMM (0.62 mL, 5.71 mmol) in DMF (10 mL) was treated at −20 °C with IBCF (0.74 mL, 5.71 mmol). After 10 min. at −20 °C, N-methylaniline (0.62 mL, 5.71 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 citric acid (10% in H2O), NaHCO3 (5% in H2O), and brine and dried over Na2SO4. The solution was filtered, the solvent evaporated, and the residual oil was precipitated from Et2O/Pe (1:9, v/v): yield 1.39 g (92%); Rf(B) 0.52; HPLC K′ 6.01; mp 99–101 °C; m/z 265 (M+H)+; 1H-NMR (DMSO-d6) δ 1.40 (s, 9H), 2.78 (s, 3H), 3.88–3.92 (m, 2H), 7.10–7.31 (m, 5H).

TFȦH-Gly-N(Me)-Ph

Boc-Gly-N(Me)-Ph (1.36 g, 5.15 mmol) was treated with TFA (2.5 mL) for 0.5 h at room temperature. Et2O/Pe (1:1, v/v) were added to the solution until the product precipitated: yield 1.36 g (95%); Rf(A) 0.49; HPLC K′ 5.12; mp 115–117 °C; m/z 165 (M+H)+.

Boc-Tic-Gly-N(Me)-Ph

To a solution of Boc-Tic-OH (0.20 g, 0.72 mmol) and TFȦH-Gly- N(Me)-Ph (0.20 g, 0.72 mmol) in DMF (10 mL) at 0 °C, NMM (0.08 mL, 0.72 mmol), HOBt (0.12 g, 0.79 mmol), and WSC (0.15 g, 0.79 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 0.27 g (87%); Rf(B) 0.69; HPLC K′ 7.35; mp 133–135 °C; [α]20D - 30.1; m/z 425 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.78 (s, 3H), 2.92–3.17 (m, 2H), 4.14–4.27 (m, 4H), 4.92 (m, 1H), 6.96–7.31 (m, 9H).

TFȦH-Tic-Gly-N(Me)-Ph

Boc-Tic-Gly-N(Me)-Ph (0.24 g, 0.57 mmol) was treated with TFA (1.5 mL) for 0.5 h at room temperature. Et2O/Pe (1:1, v/v) were added to the solution until the product precipitated: yield 0.24 g (95%); Rf(A) 0.39; HPLC K′ 6.22; mp 158–160 °C; [α]20D - 30.6; m/z 324 (M+H)+.

Boc-Dmt-Tic-Gly-N(Me)-Ph

To a solution of Boc-Dmt-OH (0.10 g, 0.32 mmol) and TFȦH-Tic-Gly-N(Me)-Ph (0.14 g, 0.32 mmol) in DMF (10 mL) at 0 °C, NMM (0.03 mL, 0.32 mmol), HOBt (0.05 g, 0.35 mmol), and WSC (0.07 g, 0.35 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%); Rf(B) 0.68; HPLC K′ 10.01; mp 141–143 °C; [α]20D −18.5; m/z 616 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.35 (s, 6H), 2.78 (s, 3H), 2.92–3.17 (m, 4H), 4.14–4.92 (m, 6H), 6.29 (s, 2H), 6.96–7.31 (m, 9H).

TFȦH-Dmt-Tic-Gly-N(Me)-Ph (5)

Boc-Dmt-Tic-Gly-N(Me)-Ph (0.14 g, 0.23 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 0.14 g (97%); Rf(A) 0.45; HPLC K′ 7.25; mp 150–152 °C; [α]20D −20.3; m/z 516 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.78 (s, 3H), 2.92–3.17 (m, 4H), 3.95–4.92 (m, 6H), 6.29 (s, 2H), 6.96–7.31 (m, 9H); Anal. C32H35F3N4O6: C; H; N.

Boc-Asp(OBzl)-NH-Ph

This intermediate was obtained by condensation of Boc-Asp(OBzl)- OH with aniline via mixed anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.57 g (93%); Rf (B) 0.73; HPLC K' 7.02; mp 129–132 °C; [α]20D +12.5; m/z 399 (M+H)+, 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.72–2.97 (d, 2H), 5.17–5.34 (m, 3H), 7.19–7.60 (m, 10H).

TFȦH-Asp(OBzl)-NH-Ph

Boc-Asp(OBzl)-NH-Ph was treated with TFA as reported for TFȦH-Gly-N(Me)-Ph: yield 0.54 g (96%); Rf(A) 0.75; HPLC K′ 5.02; mp 138–140 °C; [α]20D +15.2; m/z 299 (M+H)+.

Boc-Tic-Asp(OBzl)-NH-Ph

This intermediate was obtained by condensation of Boc-Tic-OH with TFȦH-Asp(OBzl)-NH-Ph via WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield 0.21 g (88%); Rf(B) 0.82; HPLC K′ 6.43; mp 147–149 °C; [α]20D +5.2; m/z 559 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.72–3.17 (m, 4H), 4.17–4.27 (m, 2H), 4.92–5.34 (m, 4H), 6.96–7.64 (m, 14H).

TFȦH-Tic-Asp(OBzl)-NH-Ph

Boc-Tic-Asp(OBzl)-NH-Ph was treated with TFA as reported for TFȦH-Tic-Gly-N(Me)-Ph: yield 0.18 g (96%); Rf(A) 0.73; HPLC K ′4.21; mp 145–147 °C; [α]20D +5.3; m/z 459 (M+H)+.

Boc-Dmt-Tic-Asp(OBzl)-NH-Ph

This intermediate was obtained by condensation of Boc-Dmt-OH with TFȦH-Tic-Asp(OBzl)-NH-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-Gly- N(Me)-Ph: yield 0.14 g (87%); Rf(B) 0.78; HPLC K′ 9.31; mp 165–167 °C; [α]20D −2.5; m/z 750 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.35 (s, 6H), 2.72–3.17 (m, 6H), 4.41–4.51 (m, 2H), 4.92–5.34 (m, 5H), 6.29 (s, 2H), 6.96–7.64 (m, 14H).

Boc-Dmt-Tic-Asp-NH-Ph

To a solution of Boc-Dmt-Tic-Asp(OBzl)-NH-Ph (0.11 g, 0.15 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 crystallized from Et2O/Pe (1:9, v/v): yield 0.09 g (95%); Rf(B) 0.67; HPLC K′ 7.19; mp 169–171 °C; [α]20D −6.4; m/z 660 (M+H)+.

TFȦH-Dmt-Tic-Asp-NH-Ph (6)

Boc-Dmt-Tic-Asp-NH-Ph was treated with TFA as reported for TFȦH-Dmt-Tic-Gly-N(Me)-Ph: yield 0.09 g (96%); Rf(A) 0.67; HPLC K′ 4.21; mp 158–160 °C; [α]20D −7.3; m/z 560 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.72–3.17 (m, 6H), 3.95–4.51 (m, 3H), 4.86–4.92 (m, 2H), 6.29 (s, 2H), 6.96–7.64 (m, 9H); Anal. C33H35F3N4O8: C; H; N.

Boc-D-Asp(OBzl)-NH-Ph

This intermediate was obtained by condensation of Boc-D-Asp(OBzl)-OH with aniline via mixed anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.38 g (92%); Rf (B) 0.73; HPLC K' 7.02; mp 129–132 °C; [α]20D −12.5; m/z 399 (M+H)+, 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.72–2.97 (d, 2H), 5.17–5.34 (m, 3H), 7.19–7.60 (m, 10H).

TFȦH-D-Asp(OBzl)-NH-Ph

Boc-D-Asp(OBzl)-NH-Ph was treated with TFA as reported for TFȦH-Gly-N(Me)-Ph: yield 0.25 g (96%); Rf(A) 0.75; HPLC K′ 5.02; mp 138–140 °C; [α]20D −15.2; m/z 299 (M+H)+.

Boc-Tic-D-Asp(OBzl)-NH-Ph

This intermediate was obtained by condensation of Boc-Tic-OH with TFȦH-D-Asp(OBzl)-NH-Ph via WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield 0.17 g (87%); Rf(B) 0.80; HPLC K′ 6.38; mp 147–149 °C; [α]20D +8.4; m/z 559 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.73–3.19 (m, 4H), 4.21–4.29 (m, 2H), 4.90–5.31 (m, 4H), 6.96–7.64 (m, 14H).

TFȦH-Tic-D-Asp(OBzl)-NH-Ph

Boc-Tic-D-Asp(OBzl)-NH-Ph was treated with TFA as reported for TFȦH-Tic-Gly-N(Me)-Ph: yield 0.15 g (97%); Rf(A) 0.71; HPLC K′ 4.17; mp 146–148 °C; [α]20D +10.1; m/z 459 (M+H)+.

Boc-Dmt-Tic-D-Asp(OBzl)-NH-Ph

This intermediate was obtained by condensation of Boc-Dmt-OH with TFȦH-Tic-D-Asp(OBzl)-NH-Ph via WSC/HOBt as reported for Boc-Dmt-Tic- Gly-N(Me)-Ph: yield 0.11 g (89%); Rf(B) 0.75; HPLC K′ 8.98; mp 160–162 °C; [α]20D +8.6; m/z 750 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.35 (s, 6H), 2.70–3.20 (m, 6H), 4.40–4.53 (m, 2H), 4.93–5.32 (m, 5H), 6.29 (s, 2H), 6.96–7.64 (m, 14H).

Boc-Dmt-Tic-D-Asp-NH-Ph

Boc-Dmt-Tic-D-Asp(OBzl)-NH-Ph was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.08 g (95%); Rf(B) 0.64; HPLC K′ 6.95; mp 164–176 °C; [α]20D +12.8; m/z 660 (M+H)+.

TFA.H-Dmt-Tic-D-Asp-NH-Ph (7)

Boc-Dmt-Tic-D-Asp-NH-Ph was treated with TFA as reported for TFA.H-Dmt-Tic-Gly-N(Me)-Ph: yield 0.04 g (93%); Rf(A) 0.64; HPLC K′ 4.15; mp 150–152 °C; [α]20D +12.5; m/z 560 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.70–3.19 (m, 6H), 3.96–4.53 (m, 3H), 4.85–4.90 (m, 2H), 6.29 (s, 2H), 6.96–7.66 (m, 9H); Anal. C33H35F3N4O8: C; H; N.

Boc-Asp(OBzl)-N(Me)-Ph

This intermediate was obtained by condensation of Boc-Asp(OBzl)-OH with N-methylaniline via mixed anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.43 g (90%); Rf (B) 0.75; HPLC K' 7.11; mp 126–128 °C; [α]20D +10.3; m/z 413 (M+H)+, 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.72–2.97 (m, 5H), 5.17–5.34 (m, 3H), 7.10–7.31 (m, 10H).

TFA.H-Asp(OBzl)-N(Me)-Ph

Boc-Asp(OBzl)-N(Me)-Ph was treated with TFA as reported for TFA.H-Gly-N(Me)-Ph: yield 0.40 g (97%); Rf(A) 0.77; HPLC K′ 5.11; mp 135–139 °C; [α]20D +14.7; m/z 313 (M+H)+.

Boc-Tic-Asp(OBzl)-N(Me)-Ph

This intermediate was obtained by condensation of Boc-Tic-OH with TFA.H-Asp(OBzl)-N(Me)-Ph via WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield 0.21 g (85%); Rf(B) 0.84; HPLC K′ 6.45; mp 144–146 °C; [α]20D +4.8; m/z 573 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.73–3.19 (m, 7H), 4.17–4.27 (m, 2H), 4.92–5.34 (m, 4H), 6.96–7.31 (m, 14H).

TFA.H-Tic-Asp(OBzl)-N(Me)-Ph

Boc-Tic-Asp(OBzl)-N(Me)-Ph was treated with TFA as reported for TFA.H-Tic-Gly-N(Me)-Ph: yield 0.18 g (97%); Rf(A) 0.75; HPLC K′ 4.25; mp 141–143 °C; [α]20D +4.6; m/z 473 (M+H)+.

Boc-Dmt-Tic-Asp(OBzl)-N(Me)-Ph

This intermediate was obtained by condensation of Boc-Dmt-OH with TFA.H-Tic-Asp(OBzl)-N(Me)-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-Gly-N(Me)-Ph: yield 0.13 g (88%); Rf(B) 0.81; HPLC K′ 9.36; mp 160–162 °C; [α]20D −3.5; m/z 764 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.35 (s, 6H), 2.70–3.17 (m, 9H), 4.43–4.51 (m, 2H), 4.92–5.34 (m, 5H), 6.29 (s, 2H), 6.96–7.31 (m, 14H).

Boc-Dmt-Tic-Asp-N(Me)-Ph

Boc-Dmt-Tic-Asp(OBzl)-N(Me)-Ph was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.08 g (94%); Rf(B) 0.69; HPLC K′ 7.23; mp 165–167 °C; [α]20D −7.3; m/z 674 (M+H)+.

TFA.H-Dmt-Tic-Asp-N(Me)-Ph (8)

Boc-Dmt-Tic-Asp-N(Me)-Ph was treated with TFA as reported for TFA.H-Dmt-Tic-Gly-N(Me)-Ph: yield 0.04 g (94%); Rf(A) 0.69; HPLC K′ 4.26; mp 152–154 °C; [α]20D −8.2; m/z 574 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.70–3.17 (m, 9H), 3.95–4.51 (m, 3H), 4.86–4.92 (m, 2H), 6.29 (s, 2H), 6.96–7.31 (m, 9H); Anal. C34H37F3N4O8: C; H; N.

Boc-D-Asp(OBzl)-N(Me)-Ph

This intermediate was obtained by condensation of Boc-D-Asp(OBzl)-OH with N-methylaniline via mixed anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.42 g (88%); Rf (B) 0.75; HPLC K' 7.11; mp 126–128 °C; [α]20D −10.3; m/z 413 (M+H)+, 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.72–2.97 (m, 5H), 5.17–5.34 (m, 3H), 7.10–7.31 (m, 10H).

TFA.H-D-Asp(OBzl)-N(Me)-Ph

Boc-D-Asp(OBzl)-N(Me)-Ph was treated with TFA as reported for TFA.H-Gly-N(Me)-Ph: yield 0.39 g (97%); Rf(A) 0.77; HPLC K′ 5.11; mp 135–139 °C; [α]20D −14.7; m/z 313 (M+H)+.

Boc-Tic-D-Asp(OBzl)-N(Me)-Ph

This intermediate was obtained by condensation of Boc-Tic-OH with TFA.H-D-Asp(OBzl)-N(Me)-Ph via WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield 0.19 g (87%); Rf(B) 0.83; HPLC K′ 6.44; mp 145–147 °C; [α]20D +7.8; m/z 573 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.73–3.19 (m, 7H), 4.21–4.29 (m, 2H), 4.90–5.31 (m, 4H), 6.96–7.35 (m, 14H).

TFA.H-Tic-D-Asp(OBzl)-N(Me)-Ph

Boc-Tic-D-Asp(OBzl)-N(Me)-Ph was treated with TFA as reported for TFA.H-Tic-Gly-N(Me)-Ph: yield 0.16 g (96%); Rf(A) 0.73; HPLC K′ 4.24; mp 142–144 °C; [α]20D +9.2; m/z 473 (M+H)+.

Boc-Dmt-Tic-D-Asp(OBzl)-N(Me)-Ph

This intermediate was obtained by condensation of Boc-Dmt-OH with TFA.H-Tic-D-Asp(OBzl)-N(Me)-Ph via WSC/HOBt as reported for Boc-Dmt-Tic-Gly-N(Me)-Ph: yield 0.12 g (88%); Rf(B) 0.79; HPLC K′ 9.08; mp 156–158 °C; [α]20D +7.8; m/z 764 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.35 (s, 6H), 2.70–3.20 (m, 9H), 4.40–4.53 (m, 2H), 4.93–5.32 (m, 5H), 6.29 (s, 2H), 6.96–7.35 (m, 14H).

Boc-Dmt-Tic-D-Asp-N(Me)-Ph

Boc-Dmt-Tic-D-Asp(OBzl)-N(Me)-Ph was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.08 g (93%); Rf(B) 0.68; HPLC K′ 7.03; mp 155–157 °C; [α]20D +11.3; m/z 674 (M+H)+

TFA.H-Dmt-Tic-D-Asp-N(Me)-Ph (9)

Boc-Dmt-Tic-D-Asp-N(Me)-Ph was treated with TFA as reported for TFA.H-Dmt-Tic-Gly-N(Me)-Ph: yield 0.04 g (96%); Rf(A) 0.68; HPLC K′ 4.23; mp 145–147 °C; [α]20D +11.7; m/z 574 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.70–3.19 (m, 9H), 3.96–4.53 (m, 3H), 4.85–4.90 (m, 2H), 6.29 (s, 2H), 6.96–7.35 (m, 9H); Anal. C34H37F3N4O8: C; H; N.

(S)-tert-butyl3-((S)-2-((benzyloxy)carbonyl)-1-(1-methyl-1H-benzo[d]imidazol-2-yl)ethylcarbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate [Boc-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me)]

To a solution of Boc-Tic-NH-(S)CH(CH2-COOBzl)-Bid8 (0.27 g; 0.40 mmol) in DMF (10 mL) at room temperature, K2CO3 (0.25 g; 1.8 mmol) and, after 1 h, iodomethane (0.03 mL; 0.42 mmol) were added. The reaction mixture was stirred for 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 0.20 g (90%); Rf(B) 0.81; HPLC K′ 6.15; mp 151–153 °C; [α]20D +9.7; m/z 570 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.80–3.17 (m, 4H), 3.63 (s, 3H), 4.17–4.27 (m, 2H), 4.92–5.51 (m, 4H), 6.96–7.70 (m, 13H).

2TFA.H-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me)

Boc-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me) was treated with TFA as reported for TFA.H-Tic-Gly-N(Me)-Ph: yield 0.21 g (93%); Rf(A) 0.71; HPLC K′ 5.52; mp 144–146 °C; [α]20D +10.1; m/z 470 (M+H)+

Boc-Dmt-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me)

To a solution of Boc-Dmt-OH (0.08 g, 0.26 mmol) and 2TFA.H-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me) (0.18 g, 0.26 mmol) in DMF (10 mL) at 0 °C, NMM (0.06 mL, 0.52 mmol), HOBt (0.04 g, 0.29 mmol), and WSC (0.05 g, 0.29 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 0.17 g (86%); Rf(B) 0.68; HPLC K′ 9.23; mp 165–167 °C; [α]20D +3.8; m/z 760 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.35 (s, 6H), 2.92–3.20 (m, 6H), 3.63 (s, 3H), 4.41–4.51 (m, 2H), 4.92–5.51 (m, 5H), 6.29 (s, 2H), 6.96–7.70 (m, 13H).

Boc-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me)

Boc-Dmt-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me) was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.11 g (94%); Rf(B) 0.55; HPLC K′ 7.26; mp 166–168 °C; [α]20D +4.6; m/z 671 (M+H)+

2TFA.H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me) (10)

Boc-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N1-Me) was treated with TFA as reported for TFA.H-Dmt-Tic-Gly-N(Me)-Ph: yield 0.05 g (96%); Rf(A) 0.69 HPLC K′ 4.56; mp 152–154 °C; [α]20D +7.3; m/z 571 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.92–3.20 (m, 6H), 3.63–3.95 (m, 4H), 4.41–4.51 (m, 2H), 4.92–5.20 (m, 2H), 6.29 (s, 2H), 6.96–7.70 (m, 8H); Anal. C36H37F6N5O9: C; H; N.

tert-butyl (R)-2-((benzyloxy)carbonyl)-1-(1H-benzo[d]imidazol-2-yl)ethylcarbamate [Boc-NH-(R)CH(CH2-COOBzl)-Bid]

A solution of Boc-D-Asp(OBzl)-OH (1 g, 3.1 mmol) and NMM (0.34 mL, 3.1 mmol) in DMF (10 mL) was treated at −20 °C with IBCF (0.4 mL, 3.1 mmol). After 10 min. at −20 °C, o-phenylendiamine (0.33 g, 3.1 mmol) was added. The reaction mixture was allowed to stir while slowly warming to room temperature (1h) and was then stirred for 3 h. The solvent was evaporated, and the residue was partitioned between EtOAc and H2O. The AcOEt layer was washed with NaHCO3 (5% in H2O) and brine and dried over Na2SO4. The solution was filtered, the solvent was evaporated, and the residual solid was dissolved in glacial acetic acid (10 mL). The solution was heated at 60 °C for 1h.. After the solvent was evaporated and the residue was precipitated from Et2O/Pe (1:9, v/v): yield 1 g (82%); Rf(B) 0.52; HPLC K′ 6.50; mp 136–138 °C; [α]20D −15.8; m/z 396 (M+H)+; 1H-NMR (DMSO-d6) δ 1.40 (s, 9H), 2.67–2.92 (m, 2H), 5.34–5.51 (m, 3H), 7.19–7.70 (m, 9H).

2TFA.H2N-(R)CH(CH2-COOBzl)-Bid

Boc-NH-(R)CH(CH2-COOBzl)-Bid was treated with TFA as reported for TFA.H-Gly-N(Me)-Ph: yield 0.51 g (96%); Rf(A) 0.78; HPLC K′ 4.20; mp 142–144 °C; [α]20D −17.3; m/z 296 (M+H)+.

Boc-Tic-NH-(R)CH(CH2-COOBzl)-Bid

To a solution of Boc-Tic-OH (0.28 g, 1 mmol) and 2TFA.H2N-(R)CH(CH2-COOBzl)-Bid (0.52 g, 1 mmol) in DMF (10 mL) at 0 °C, NMM (0.2 mL, 2 mmol), HOBt (0.17 g, 1.1 mmol), and WSC (0.21 g, 1.1 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 0.48 g (87%); Rf(B) 0.72; HPLC K′ 5.92; mp 155–157 °C; [α]20D −1.6; m/z 556 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.80–3.17 (m, 4H), 4.17–4.27 (m, 2H), 4.92–5.51 (m, 4H), 6.96–7.70 (m, 13H).

(S)-tert-b u t y l 3-((R)-2-((benzyloxy)carbonyl)-1-(1-methyl-1H-benzo[d]imidazol-2-yl)ethylcarbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate [Boc-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me)]

This intermediate was obtained by alkylation of Boc-Tic-NH-(R)CH(CH2-COOBzl)-Bid with K2CO3 and iodomethane as reported for Boc-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me): yield 0.22 g (88%); Rf(B) 0.77; HPLC K′ 6.07; mp 154–156 °C; [α]20D +4.3; m/z 570 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.82–3.20 (m, 4H), 3.63 (s, 3H), 4.15–4.29 (m, 2H), 4.90–5.49 (m, 4H), 6.96–7.70 (m, 13H).

2TFȦH-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me)

Boc-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me) was treated with TFA as reported for TFȦH-Tic-Gly-N(Me)-Ph: yield 0.21 g (92%); Rf(A) 0.68; HPLC K′ 5.39; mp 148–150 °C; [α]20D +5.4; m/z 470 (M+H)+.

Boc-Dmt-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me)

This intermediate was obtained by condensation of Boc-Dmt-OH with 2TFȦH-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me) via WSC/HOBt as reported for Boc-Dmt-Tic-NH-(S)CH(CH2-COOBzl)-Bid(N1-Me): yield 0.17 g (86%); Rf(B) 0.64; HPLC K′ 8.28; mp 169–171 °C; [α]20D −2.7; m/z 760 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.35 (s, 6H), 2.94–3.22 (m, 6H), 3.63 (s, 3H), 4.40–4.52 (m, 2H), 4.92–5.48 (m, 5H), 6.29 (s, 2H), 6.96–7.70 (m, 13H).

Boc-Dmt-Tic-NH-(R)CH(CH-COOH)-Bid(N1-Me)

Boc-Dmt-Tic-NH-(R)CH(CH2-COOBzl)-Bid(N1-Me) was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.10 g (92%); Rf(B) 0.50; HPLC K′ 6.98; mp 172–174 °C; [α]20 −1.9; m/z 671 (M+H)+D −1.9; m/z 671 (M+H)+.

2TFȦH-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid(N1-Me) (11)

Boc-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid(N1-Me) was treated with TFA as reported for TFȦH-Dmt-Tic-Gly- N(Me)-Ph: yield 0.07 g (96%); Rf(A) 0.58 HPLC K′ 4.02; mp 161–163 °C; [α]20D +2.2; m/z 571 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.90–3.22 (m, 6H), 3.61–3.93 (m, 4H), 4.42–4.53 (m, 2H), 4.90–5.18 (m, 2H), 6.29 (s, 2H), 6.96–7.70 (m, 8H); Anal. C36H37F6N5O9: C; H; N.

2TFȦH-Tic-NH-(R)CH(CH2-COOBzl)-Bid

Boc-Tic-NH-(R)CH(CH2-COOBzl)-Bid was treated with TFA as reported for TFȦH-Tic-Gly-N(Me)-Ph: yield 0.18 g (93%); Rf(A) 0.66; HPLC K′ 5.35; mp 149–151 °C; [α]20D +6.3; m/z 456 (M+H)+.

Boc-Dmt-Tic-NH-(R)CH(CH2-COOBzl)-Bid

This intermediate was obtained by condensation of Boc-Dmt-OH with 2TFȦH-Tic-H2N-(R)CH(CH2-COOBzl)-Bid via WSC/HOBt as reported for Boc-Dmt-Tic-NH-(S)CH(CH -COOBzl)-Bid(N1-Me): yield 0.15 g (87%); Rf(B) 0.61; HPLC K′ 8.01; mp 174–176 °C; [α]20D −3.5; m/z 747 (M+H)+; 1H-NMR (DMSO-d6) δ 1.36–1.40 (d, 9H), 2.35 (s, 6H), 2.63–3.17 (m, 6H), 4.42–4.53 (m, 2H), 4.92–5.51 (m, 5H), 6.29 (s, 2H), 6.96–7.70 (m, 13H).

Boc-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid

Boc-Dmt-Tic-NH-(R)CH(CH2-COOBzl)-Bid was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.09 g (93%); Rf(B) 0.46; HPLC K′ 6.84; mp 176–178 °C; [α]20D −2.8; m/z 657 (M+H)+.

2TFẠH-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid (12)

Boc-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid was treated with TFA as reported for TFȦH-Dmt-Tic-Gly-N(Me)-Ph: yield 0.06 g (96%); Rf(A) 0.52; HPLC K′ 3.92; mp 166–168 °C; [α]20D +3.5; m/z 557 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.90–3.17 (m, 6H), 3.95–4.52 (m, 3H), 4.90–5.21 (m, 2H), 6.29 (s, 2H), 6.96–7.70 (m, 8H); Anal. C35H35F6N5O9: C; H; N.

Boc-Asp(OBzl)-NH-Bzl

This intermediate was obtained by condensation of Boc- Asp(OBzl)-OH with benzylamine via mixed anhydride as reported for Boc-Gly-N(Me)-Ph: yield 0.45 g (90%); R (B) 0.77; HPLC K' 7.18; mp 126–128 °C; [α]20D −11.2; m/z 413 (M+H)+, 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 2.63–2.88 (d, 2H), 4.46 (s, 2H), 5.17–5.34 (m, 3H), 7.06–7.19 (m, 10H).

TFȦH-Asp(OBzl)-NH-Bzl

Boc-Asp(OBzl)-NH-Bzl was treated with TFA as reported for TFȦH-Gly-N(Me)-Ph: yield 0.42 g (96%); Rf(A) 0.78; HPLC K′ 5.18; mp 135–147 °C; [α]20D +14.3; m/z 313 (M+H)+.

Boc-Tic-Asp(OBzl)-NH-Bzl

This intermediate was obtained by condensation of Boc-Tic-OH with TFȦH-Asp(OBzl)-NH-Bzl via WSC/HOBt as reported for Boc-Tic-Gly-N(Me)-Ph: yield 0.22 g (87%); Rf(B) 0.86; HPLC K′ 6.51; mp 144–146 °C; [α]20D +4.7; m/z 573 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.63–3.17 (m, 4H), 4.17–4.46 (m, 4H), 4.92–5.34 (m, 4H), 6.96–7.19 (m, 14H).

TFȦH-Tic-Asp(OBzl)-NH-Bzl

Boc-Tic-Asp(OBzl)-NH-Bzl was treated with TFA as reported for TFȦH-Tic-Gly-N(Me)-Ph: yield 0.18 g (97%); Rf(A) 0.77; HPLC K′ 4.29; mp 140–142 °C; [α]20D +4.7; m/z 473 (M+H)+.

Boc-Dmt-Tic-Asp(OBzl)-NH-Bzl

This intermediate was obtained by condensation of Boc-Dmt-OH with TFȦH-Tic-Asp(OBzl)-NH-Bzl via WSC/HOBt as reported for Boc-Dmt-Tic-Gly- N(Me)-Ph: yield 0.13 g (88%); Rf(B) 0.82; HPLC K′ 9.38; mp 160–162 °C; [α]20D −1.9; m/z 764 (M+H)+; 1H-NMR (DMSO-d6) δ 1.38–1.42 (d, 9H), 2.35 (s, 6H), 2.63–3.17 (m, 6H), 4.41–4.51 (m, 4H), 4.92–5.34 (m, 5H), 6.29 (s, 2H), 6.96–7.19 (m, 14H).

Boc-Dmt-Tic-Asp-NH-Bzl

Boc-Dmt-Tic-Asp(OBzl)-NH-Bzl was treated with Pd/C and H2 as reported for Boc-Dmt-Tic-Asp-NH-Ph: yield 0.09 g (96%); Rf(B) 0.71; HPLC K′ 7.24; mp 164–166 °C; [α]20D −5.2; m/z 674 (M+H)+.

TFȦH-Dmt-Tic-Asp-NH-Bzl (13)

Boc-Dmt-Tic-Asp-NH-Bzl was treated with TFA as reported for TFȦH-Dmt-Tic-Gly-N(Me)-Ph: yield 0.05 g (96%); Rf(A) 0.69; HPLC K′ 4.29; mp 154–166 °C; [α]20D −6.7; m/z 574 (M+H)+; 1H-NMR (DMSO-d6) δ 2.35 (s, 6H), 2.63–3.17 (m, 6H), 3.95–4.51 (m, 5H), 4.86–4.92 (m, 2H), 6.29 (s, 2H), 6.96–7.14 (m, 9H); Anal. C34H37F3N4O8:C; H; N.

Pharmacology

Competitive Binding Assays

Opioid receptor affinities were determined under equilibrium conditions [2.5 h room temperature (23 °C)] in competition assays using brain P2 synaptosomal membranes prepared from Sprague-Dawley rats.40, 41 Synaptosomes were preincubated to remove endogenous opioid peptides and stored at −80 °C in buffered 20% glycerol.40, 42 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; KD = 1.4 nM) for δ-opioid receptors and 3.5 nM [3H]DAMGO (50.0 Ci/mmol), Amersham Bioscience, Buckinghamshire, U. K.; KD = 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 icecold buffered BSA,40 and the affinity constants (Ki) were calculated according to Cheng and Prusoff..43

Biological Activity in Isolate Tissue Preparations

The myenteric plexus longitudinal muscle preparations (2–3 cm segments) from the small intestine of male Hartley strain of 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.44 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, and the δ-antagonist potencies in the MVD assay were determined against the δ agonist deltorphin-II, while μ antagonism (GPI assay) used the μ agonist endomorphin-2. Antagonism is expressed as pA2 determined using the Schild Plot.45

Animals for in vitro and in vivo studies

Laboratory animals were used under protocols approved and governed by the Animal Care and Use Committees of Tohoku Pharmaceutical University, University of Ferrara and the National Institute of Environmental Health Sciences.

In vivo Assessment of Opioid Bioactivity

Male Swiss-Webster mice (20–25 g, Taconic, Germantown, NY) were housed on a 12-h light/dark cycle with free access to food and water and experimental protocol approved by the NIEHS Animal Care and Use Committee. Intracerebroventricular injection (icv) used a Hamilton microsyringe with disposable 26-gauge needle that was inserted 2.3–3 mm into the ventricular sinus at the bregma as described;46 the total volume administered was 5 μL (peptide in saline). Upon completion of the study, mice were sacrificed according ACUC protocols and those animals having needle tract 2 mm lateral from the midline were counted as being valid. Hot-plate test consisted of animals placed on an electrically heated plate (55 ± 0.1 °C, IITC MODEL 39D Hot Plate analgesia meter, IITC Inc, Woodland Hills, CA) 10 min after icv administration. Hot-plate latency (HPL) is the interval between placement of mice onto the hot plate and observing movement consisting of either jumping, licking or shaking their hind paws; a baseline latency of 5–10 sec (pre-response time) and maximal cut off time of 30 sec. Measurements were repeated every 10 min and testing was terminated when the HPL was close to the pre-response time.

Spinal effects used of a tail-flick instrument (Columbus Instruments, Columbus, OH). Radiant heat was applied on the dorsal surface of the tail and the latency before removal of the tail from the onset of the radiant heat is defined as the tail-flick latency. The baseline was to 2–3 sec (pre-response time) and a cut off time was set at 8 sec to avoid external heat-related damage. The time sequence was the same as described for the HPL test47, 48

Statistical Analysis

Statistical significance of the data was estimated by one-way analysis of variance (ANOVA) followed by Dunnett's test using the computer software program JMP (SAS Institute Inc, Cary, NC) and considered significant at p < 0.05. Minimum Effective Dose (MED) is the minimum dose of compound showing statistically significant antinociceptive effect expressed as the area under the time-response curve (AUC) value compared to a saline-treated group. The AUC was obtained by plotting the response time (sec) on the ordinate and time (min) on the abscissa after administration of the compounds. The percent maximum possible effect (% MPE) was calculated as follows: ([post-drug response latency — pre-drug response latency]/[cutoff time (30 s) — pre-drug response latency]) x100.

Acknowledgments

This study was supported in part by the University of Cagliari (GB), the University of Ferrara (SS) and in part by the Intramural Research Program of the NIH and NIEHS (LHL and EDM).

Abbreviations

In addition to the IUPAC-IUB Commission on Biochemical Nomenclature (J. Biol. Chem. 1985, 260, 1 4–42), this paper uses the following additional symbols and abbreviations:

AcOEt

ethyl acetate

AcOH

acetic acid

Bid

1H-benzimidazole-2-yl

Boc

tert-butyloxycarbonyl

DAMGO

[D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin

DEL C

deltorphin II (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2)

DMF

N,N-dimethylformamide

DMSO-d6

hexadeuteriodimethyl sulfoxide

Dmt

2′6′-dimethyl-L-tyrosine

DPDPE

(D-Pen2,D-Pen5)-enkephalin

endomorphin-2

H-Tyr-Pro-Phe-Phe-NH2

Et2O

diethyl ether

GPI

guinea-pig ileum

HOBt

1-hydroxybenzotriazole

HPLC

high performance liquid chromatography

MALDI-TOF

matrix assisted laser desorption ionization time-of-flight

MVD

mouse vas deferens

NMM

4-methylmorpholine

NLX

naloxone

NTI

naltrindole

pA2

negative log of the molar concentration required to double the agonist concentration to achieve the original response

Pe

petroleum ether

PL017

H-Tyr-Pro-(N-Me)Phe-D-Pro-NH2

TEA

triethylamine

TFA

trifluoroacetic acid

Tic

1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

TLC

thin-layer chromatography

WSC

1-ethyl-3-[3′-dimethyl)aminopropyl]-carbodiimide hydrochloride

Z

benzyloxycarbonyl

Footnotes

Supporting Information Available: Chemistry general methods, enzymatic degradation general methods, elemental analysis, MS and HPLC data. This material is available free of charge via the Internet at http://pubs.acs.org.

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