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. Author manuscript; available in PMC: 2008 Dec 8.
Published in final edited form as: J Med Chem. 2005 Dec 29;48(26):8112–8114. doi: 10.1021/jm058259l

Conversion of the Potent δ-Opioid Agonist H-Dmt-Tic-NH-CH2-Bid into δ-Opioid Antagonists by N1-Benzimidazole Alkylation1

Gianfranco Balboni *, Remo Guerrini †, Severo Salvadori †, Lucia Negri ‡, Elisa Giannini ‡, Sharon D Bryant §, Yunden Jinsmaa §, Lawrence H Lazarus §,
PMCID: PMC2597450  NIHMSID: NIHMS64884  PMID: 16366592

Abstract

N1-Alkylation of 1H-benzimidizole of the δ agonist H-Dmt-Tic-NH-CH2-Bid with hydrophobic, aromatic, olefinic, acid, ethyl ester or amide (1–6) became δ antagonists (pA2 = 8.52–10.14). δ- and μ-Opioid receptor affinities were high (Kiδ = 0.12–0.36 nM and Kiμ = 0.44–1.42 nM). Only δ antagonism (pA2 = 8.52–10.14) was observed; μ agonism (IC50 = 30–450 nM) was not correlated with changes in alkylating agent or δ antagonism and some compounds yielded mixed δ antagonism/μ agonism.

Numerous opioid peptides2 and non peptide opiates3–5 interact with opioid receptors. H-Dmt-Tic-OH,6 which evolved from H-Tyr-Tic-OH,7 as a simplified form of TIP(P),8 represents the minimal sequence that selectively interacts with δ-opioid receptors as a potent δ-antagonist. The dipeptide was transformed into a potent δ agonist by replacing the carboxylic function with an alkyl amide terminated with 1H-benzimidazole (H-Dmt-Tic-NH-CH2-Bid).9,10 To restore the δ-opioid receptor selectivity, an acidic moiety was introduced by alkylation of N1-benzimidazole, yielding H-Dmt-Tic-NH-CH2-Bid(CH2-COOH),10 and whose pharmacological behaviour highlighted the role of benzimidazole-N1H in δ-receptor interaction and activation. Similarly, the presence of a nitrogen was required in C-terminally modified endomorphin-2 with naphthyl or isoquinolyl groups resulting in mixed μ-/delta;-agonists.11 To investigate the role of the N1-benzimidazole on δ and μ bioactivity, alkylation with various groups was initiated. All compounds reverted to potent δ-antagonists, and in several cases, μ agonism increased.

Pseudopeptides were prepared stepwise by solution peptide synthetic methods9 described in detail in Supporting Information. In brief, mixed carbonic anhydride coupling of tert-butyloxycarbonyl-glycine (Boc-Gly-OH) with o-phenylendiamine gave intermediate monoamide, which was converted without purification to the desired 1H-benzimidazol-2-yl-methyl)-carbamic acid tert-butyl ester (Boc-NH-CH2-Bid) by cyclization and dehydration in acetic acid (AcOH) in the Scheme. After N-terminal Boc deprotection with TFA, H2N-CH2-Bid was condensed with Boc-Tic-OH via WSC/HOBt. Alkylation of N1-Bid was carried out by treatment of Boc-Tic-NH-CH2-Bid9 with K2CO3 and iodomethane, benzyl bromide, allyl bromide, cyclopropylmethyl bromide or ethyl bromoacetate.10 Boc-Tic-NH-CH2-Bid(R) (R = alkyl groups) was deprotected with TFA and condensed with Boc-Dmt-OH via WSC/HOBt. Compound (6) was obtained from Boc protected (5) after hydrolysis with NaOH 1N and reaction with NH3 via mixed anhydrides. Final compounds (1–6) were obtained after TFA treatment and purified by preparative HPLC.

Compounds (1–6) (Table) had subnanomolar affinity for δ-opioid receptors (Kiδ 0.12–0.36 nM); alkylation decreased affinity by approximately an order of magnitude relative to the reference compounds H-Dmt-Tic-NH-CH2-Bid (a) and H-Dmt-Tic-NH-CH2-Bid(CH2-COOH) (b). μ-Opioid receptor affinity was within the same order of magnitude as H-Dmt-Tic-NH-CH2-Bid and the lack of a carboxylic function caused a significant increase in μ-opioid receptor affinity.6,15,18 Thus, the analogues remained essentially neutral and non-selective, except 5 which was comparable to H-Dmt-Tic-NH-CH2-Bid (a), but considerably less selective than H-Dmt-Tic-NH-CH2-Bid(CH2-COOH) (b) (Table).

Table. Receptor affinity and functional bioactivity of 1–6.

See text for summary and pertinent references. Parent compounds: a(H-Dmt-Tic-NH-CH2-Bid), Balboni et al.9 and b[H-Dmt-Tic-NH-CH2-Bid(CH2-COOH), Balboni et al.10. Standard compounds: cDEL (deltorphin C) Lazarus et al.19 and dDER (dermorphin) Melchiorri and Negri.20 –, No activity. The number of independent repetitions (n) is listed for the radioreceptor assays conducted in duplicate; bioassays represent means ± SE for at least 6 different tissue samples.

Functional bioactivity
Cmpd no. Receptor affinity (nM) MVD GPI
Ki(δ) Ki(μ) μ/δ IC50 (nM) pA2 IC50 (nM)
a 0.035 ± 0.006 (3) 0.50 ± 0.054 (3) 14 0.035 ± 0.003 - 40.7 ± 5
b 0.021 ± 0.0025 (4) 6.92 ± 0.25 (4) 330 - 9.57 3193 ± 402
1 0.16 ± 0.03 (3) 0.83 ± 0.07 (5) 5 - 10.14 450 ± 51
2 0.20 ± 0.06 (4) 1.02 ± 0.19 (4) 5 - 8.52 245 ± 35
3 0.13 ± 0.02 (4) 0.44 ± 0.04 (3) 3 - 9.34 72 ± 6
4 0.36 ± 0.05 (4) 0.52 ± 0.08 (4) 1 - 9.47 64 ± 5
5 0.12 ± 0.02 (3) 1.42 ± 0.08 (3) 12 - 9.77 30 ± 4
6 0.16 ± 0.03 (4) 0.49 ± 0.02 (3) 3 - 9.26 77 ± 5
DELc 0.24 ± 0.06 (6) 272 ± 50 (11) 1133 0.17 ± 0.02 - 1300 ±150
DERd 178.6 ± 18 (15) 1.22 ± 0.13 (22) 0.0068 15.2 ± 2 - 1.9 ± 0.3
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Alkylation transformed the δ agonist H-Dmt-Tic-NH-CH2-Bid (IC50 = 0.035 nM, MVD) (a) into δ antagonists (1–6) without effect on μ-opioid receptors (GPI). The analogues demonstrated high δ antagonism (pA2 = 8.52 to 10.14) without μ antagonism; a 15-fold difference in μ-opioid agonism occurred among 1–6. Although the alkylating agent per se does not appear important, methyl (1) improved δ antagonism slightly more than the bulky substituents (2–4), particularly the aromatic benzyl group (2). Interestingly, a single methyl converted naltrindole, an opiate δ antagonist, into a μ agonist.12

Modification of the carboxylic function into an ester (5) or amide (6) did not change δ antagonism, suggesting these functional groups are weakly implicated in δ-receptor interactions. Compounds (1–6) had improved μ-opioid receptor affinity and agonism compared to H-Dmt-Tic-NH-CH2-Bid(CH2-COOH) (b), supporting evidence that the carboxylic function prevents μ-opioid receptor activation.2a,6 Alkylation of N1H-benzimidazole did not modify the pharmacological activity toward μ-opioid receptors indicating that this nitrogen is not implicated in μ-opioid receptor activation. Thus, 1–6 had a pattern of pharmacological activities as mixed μ agonists/δ antagonists.

In summary, the alkyl groups (hydrophobic, aromatic, olefinic, acid, ethylester, amide) modify δ-opioid receptor activation which suggests the importance of N1H-benzimidazole in these events. The allyl and cyclopropylmethyl (3,4) substituents induce antagonism when present at the amino function of alkaloid opiates.13 The δ-antagonism/μ-agonism profile of 1–6 is similar to the bioactivity of opioids that elicit analgesia and display a lower degree of tolerance as seen with analgesics of the μ-selective opiates.14

Binding assays were conducted as described elsewhere using rat brain P2 synaptosomes preincubated to remove endogenous opioids,6,15 and labelled with 2.1 nM [3H]deltorphin II (45.0 Ci/mmol, Amersham, Buckinghamshire, UK; KD = 1.4 nM) for δ-opioid receptors, and 3.5 nM [3H]DAMGO (50.0 Ci/mmol, Amersham, Buckinghamshire, UK; KD = 1.5 nM) for μ-opioid receptors; the affinity constants (Ki) were calculated.17

In vitro activity utilized guinea-pig ileum (μ) and mouse vas deferens (δ) in competitive bioassays.6 Antagonism was the shift of deltorphin C (MVD) and dermorphin (GPI) log(concentration)-response curve to the right; pA2 values were determined using the Schild Plot.18 Agonism was the inhibition of the electrically-evoked twitch; the IC50 values (nM) represent the mean ± SE of not less than six tissue samples.

Supplementary Material

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Acknowledgments

This research was supported in part in part by the University of Ferrara and in part by the Intramural Research Program of the NIH, and NIEHS. The authors appreciate the professional services of the library staff of the NIEHS.

References

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