Conversion of the potent δ-opioid agonist H-Dmt-Tic-NH-CH 2-Bid into δ-opioid antagonists by N1-benzimidazole alkylation

Article abstract of DOI:10.1021/jm058259l

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 w

Full text of DOI:10.1021/jm058259l

8112
J. Med. Chem. 2005, 48, 8112-8114
1H-benzimidazol-2-yl-methyl)-carbamic acid tert-butyl
ester (Boc-NH-CH2-Bid) by cyclization and dehydration
in acetic acid (AcOH) in 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 bromoac-
etate.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 1 N NaOH and
reaction with NH3 via mixed anhydrides. Final com-
pounds 1-6 were obtained after TFA treatment and
purified by preparative HPLC.
Compounds 1-6 (Table 1) had subnanomolar affinity
for δ-opioid receptors (Kiδ ) 0.12-0.36 nM); alkylation
decreased affinity by approximately 1 order of magni-
tude 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 nonselec-
tive, 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 1).
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,§ and
Lawrence H. Lazarus§,*
Department of Toxicology, University of Cagliari, I-09124,
Cagliari, Italy, Department of Pharmaceutical Sciences and
Biotechnology Center, University of Ferrara, I-44100
Ferrara, Italy, Department of Human Physiology and
Pharmacology “Vittorio Erspamer,” University La Sapienza,
I-00185 Rome, Italy, and Medicinal Chemistry Group,
Laboratory of Pharmacology and Chemistry, National
Institute of Environmental Health Sciences, Research
Triangle Park, North Carolina 27709
Received August 8, 2005
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 antago-
nism/µ agonism.
Numerous opioid peptides2 and nonpeptide 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-antago-
nist. 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 alkyla-
tion of N1-benzimidazole, yielding H-Dmt-Tic-NH-CH2-
Bid(CH2-COOH),10 and whose pharmacological behavior
highlighted the role of benzimidazole-N1H in δ-receptor
interaction and activation. Similarly, the presence of a
nitrogen was required in C-terminally modified endo-
morphin-2 with naphthyl or isoquinolyl groups resulting
in mixed µ/δ 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.
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 con-
verted 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 impli-
cated in δ-receptor interactions. Compounds 1-6 had
improved µ-opioid receptor affinity and agonism com-
pared to H-Dmt-Tic-NH-CH2-Bid(CH2-COOH) (b), sup-
porting evidence that the carboxylic function prevents
µ-opioid receptor activation.2a,6 Alkylation of N1H-benz-
imidazole 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.
Pseudopeptides were prepared stepwise by solution
peptide synthetic methods9 described in detail in Sup-
porting 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
In summary, the alkyl groups (hydrophobic, aromatic,
olefinic, acid, ethyl ester, amide) modify δ-opioid recep-
tor activation which suggests the importance of N1H-
benzimidazole in these events. The allyl and cycloprop-
ylmethyl (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
* To whom correspondence should be addressed. Tel.: +1-919-541-
3238; Fax: +1-919-541-0696. E-mail: lazarus@niehs.nih.gov.
| University of Cagliari.
† University of Ferrara.
‡ University La Sapienza.
§ National Institute of Environmental Health Sciences.
10.1021/jm058259l CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/23/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8113
Table 1. Receptor Affinity and Functional Bioactivity of 1-6
functional bioactivity
receptor affinity (nM)
MVD
GPI
compd no.
Ki(δ)
Ki(µ)
µ/δ
IC50 (nM)
0.035 ( 0.003
pA2
IC50 (nM)
a
0.035 ( 0.006 (3)
0.021 ( 0.0025 (4)
0.16 ( 0.03 (3)
0.20 ( 0.06 (4)
0.13 ( 0.02 (4)
0.36 ( 0.05 (4)
0.12 ( 0.02 (3)
0.16 ( 0.03 (4)
0.24 ( 0.06 (6)
178.6 ( 18 (15)
0.50 ( 0.054 (3)
6.92 ( 0.25 (4)
0.83 ( 0.07 (5)
1.02 ( 0.19 (4)
0.44 ( 0.04 (3)
0.52 ( 0.08 (4)
1.42 ( 0.08 (3)
0.49 ( 0.02 (3)
272 ( 50 (11)
1.22 ( 0.13 (22)
14
330
-
40.7 ( 5
3193 ( 402
450 ( 51
245 ( 35
72 ( 6
e
b
-
9.57
10.14
8.52
9.34
9.47
9.77
9.26
-
1
5
5
3
1
12
3
-
2
-
3
-
4
-
64 ( 5
5
-
30 ( 4
6
-
77 ( 5
DELc
DERd
1133
0.17 ( 0.02
15.2 ( 2
1300 ( 150
1.9 ( 0.3
0.0068
-
a (H-Dmt-Tic-NH-CH2-Bid), Balboni et al.9 b [H-Dmt-Tic-NH-CH2-Bid(CH2-COOH), Balboni et al.10 c DEL (deltorphin C) Lazarus et
al.19 d DER (dermorphin) Melchiorri and Negri.20 e -, No activity. The number of independent repetitions (n) is listed for the radioreceptor
assays conducted in duplicate; bioassays represent means ( SE for at least six different tissue samples.
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; TIP(P), H.-Tyr-
Tic-Phe-(Phe)-OH.; TLC, thin-layer chromatography; WSC, 1-eth-
yl-3-[3′-dimethyl)aminopropyl]-carbodiimide hydrochloride; Z,
lower degree of tolerance as seen with analgesics of the
µ-selective opiates.14
Binding assays were conducted as described else-
where using rat brain P2 synaptosomes preincubated
to remove endogenous opioids,6,15 and labeled with 2.1
nM [3H]deltorphin II (45.0 Ci/mmol, Amersham, Buck-
inghamshire, 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 An-
tagonism 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.
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Acknowledgment. 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 ser-
vices of the library staff of the NIEHS.
Supporting Information Available: Additional experi-
mental details. This material is available free of charge via
the Internet at http://pubs.acs.org.
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