Endomorphin analogues with balanced affinity for both μ- And δ-opioid receptors

Article abstract of DOI:10.1016/j.cclet.2011.01.035

Analogues of endomorphin and tripeptides modified at positions 4 and 3, respectively, with various phenylalanine analogues were synthesized and their affinities for opioid receptors were evaluated. Most of the peptides exhibited potent μ-receptor affinity and selectivity, among them, compound 7 (Dmt-Pro-Tmp-Tmp-NH₂) exhibited potent affinity for both μ- and δ-receptors (K_iμ = 0.47 nmol/L, K_iδ = 1.63 nmol/L).

Full text of DOI:10.1016/j.cclet.2011.01.035

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 907–910
www.elsevier.com/locate/cclet
Endomorphin analogues with balanced affinity
for both m- and d-opioid receptors
Liang Zhang a, Lei Chang a, Lei Lei Yu a, Jin Chun Liu a,
Jia Jia Chen a, Xiao Wen Li a, Lawrence H. Lazarus b, Ting You Li a,
a School of Pharmacy, Nanjing Medical University, Nanjing 210029, China
*
b Medicinal Chemistry Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA
Received 3 November 2010
Available online 18 May 2011
Abstract
Analogues of endomorphin and tripeptides modified at positions 4 and 3, respectively, with various phenylalanine analogues
were synthesized and their affinities for opioid receptors were evaluated. Most of the peptides exhibited potent m-receptor affinity
and selectivity, among them, compound 7 (Dmt-Pro-Tmp-Tmp-NH2) exhibited potent affinity for both m- and d-receptors
(Kim = 0.47 nmol/L, Kid = 1.63 nmol/L).
# 2011 Ting You Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Endomorphin; Opioid; Analgesics
Morphine, fentanyl, and pethidine are the most effective and most commonly prescribed analgesics among the
analgesics used in clinic, but their prolonged administration is greatly limited due to the side effects of tolerance,
dependence, respiratory inhibition, and constipation. Therefore, developing analgesics with less adverse action has
long been the target of medicine scientists [1]. The endomorphins (EM), endomorphin-1 (Tyr-Pro-Trp-Phe-NH2,
EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2) are putative endogenous opioids with high affinity and
remarkable selectivity for the m-opioid receptor [2]. Since EM exerts analgesic effect through action on the m-opioid
receptor and express similar pharmacological properties as morphine, they exhibit fewer side effects. Extensive
medicinal chemistry investigation has been performed in order to obtain compounds with improved affinity,
selectivity, bioactivity, metabolic stability, and to induce a new bioactive profile [3]. For example, we previously
reported that substitution of Tyr in the EM-2 sequence with 20,60-dimethyl-L-tyrosine (Dmt) greatly increased EM
affinity and bioactivity [4] and N-allyl-Dmt converted an agonist to a potent antagonist which inhibited alcohol
suppression of GABA mediated neurotransmission in the hippocampus [5]. The introduction of alkyl modified Phe
analogues, such as 20,60-dimethyl-L-phenylalanine (Dmp), 20,40,60-trimethyl-L-phenylalanine (Tmp), and Dmt at the
third position of EM-2 resulted in opioids with mixed m-agonist and d-antagonist properties, which are expected to
show low tolerance and dependence [6]. In keeping with our continuous interests on the investigation of the
* Corresponding author.
E-mail address: litingyou@yahoo.com.cn (T.Y. Li).
1001-8417/$ – see front matter # 2011 Ting You Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.01.035

908
L. Zhang et al. / Chinese Chemical Letters 22 (2011) 907–910
structure–activity relationship of [Dmt1]EM-2, we report herein the preliminary results of two series of endomorphin
analogues, one is modified at the fourth position of [Dmt1]EM-2 with various Phe analogues (Tmp, Dmp, Dmt, Trp,
1-Nal (1-naphthylalanine), and 2-Nal (2-naphthylalanine)), and the other one consists of the Dmt-Pro-Phe-NH2
analogues modified at the third position of Phe with the same modified Phe residues.
1. Results and discussion
The unnatural amino acids Dmt [4], Dmp and Tmp [7] were prepared as reported method through [Rh(1,5-
COD)(R,R-DIPAMP)]BF4 mediated asymmetric catalytic hydrogenation of the corresponding acetamidoacrylate. All
the peptides were synthesized by segment condensation method in solution. Peptides 1–6 were formed by coupling
Boc-Dmt-Pro-Phe-COOH with amides of corresponding amino acids using PyBop as coupling reagent. Peptides 8–12
were prepared from the coupling of Boc-Dmt-Pro-COOH with amides of corresponding amino acids, and peptide 7
was prepared by coupling Boc-Dmt-Pro-COOH with Tmp-Tmp-NH2. The final Boc protecting group was removed
with TFA in the presence of anisole, and the resulting peptide was purified by semipreparative RP-HPLC. The
identification of the final compounds was verified using MS, and the purity of the peptides was determined by
analytical HPLC. The final compounds exhibited greater than 98% purity were used for biological assay. Some
analytical data of the final compounds are summarized in Table 1.
The m- and d-opioid receptor affinities of the synthesized peptides were determined by competitive displacement
assay using brain P2 synaptosomal membranes prepared from Sprague–Dawley rats [5,6], [3H]DAMGO and
[3H]deltorphin-II were used for labeling m- and d-receptor, respectively (Table 2). EM-2 is one of the opioids isolated
from mammalian brain that shows a high affinity and selectivity for m-opioid receptor (Kim = 1.33 nmol/L,
Kid = 6085 nmol/L, Kid/Kim = 4575) [2]. While substitution of the first amino acid Tyr with Dmt, [Dmt1]EM-2,
improves the affinity of EM-2 for both m- and d-receptor, it greatly enhanced its affinity for d-receptor
(Kim = 0.26 nmol/L, Kid = 99.2 nmol/L, Kid/Kim = 382) [4]. Previously, we reported that introduction of alkyl
substituted Phe analogue Tmp and Dmp, and Dmt to the third position of [Dmt1]EM-2 to give Dmt-Pro-[Tmp/Dmp/
Dmt]-Phe-NH2 with slightly improved affinities for m-receptor and with profoundly enhanced affinity for d-receptor,
for example, [Dmt1,Tmp3]EM-2 has Kim = 0.18 nmol/L and Kid = 1.83 nmol/L [6]. In contrast, when the same amino
acids Tmp, Dmp, Dmt, and other aromatic amino acids, such as Trp, 1-Nal, and 2-Nal, were introduced to the fourth
position of [Dmt1]EM-2 resulted in compounds 1–6, respectively. Their affinities for the m-receptor, in general, were
nearly unchanged (1–5, Kim = 0.28–0.37 nmol/L) except 6 that decreased by threefold compared with the parent
compound [Dmt1]EM-2 (Kim = 0.28 nmol/L).
In regard to the d-receptor, increased lipophilicity at the forth position amino acid (1–6) enhanced opioid’s affinity
for d-receptor. Of Phe surrogates, Tmp (2), Trp (4), 2-Nal (6) had the most potent affinity (Kid = 23.2–25.4 nmol/L).
Table 1
Analytical data of endomorphin-2 analogues.
Compd.
Peptide
TLC
TOF mass [M+1]
Calcd.
HPLC (min)
Rfa
Found
tR
b
1
2
H-Dmt-Pro-Phe-Dmp-NH2
H-Dmt-Pro-Phe-Tmp-NH2
H-Dmt-Pro-Phe-Dmt-NH2
H-Dmt-Pro-Phe-Trp-NH2
H-Dmt-Pro-Phe-1-Nal-NH2
H-Dmt-Pro-Tmp-2-Nal-NH2
H-Dmt-Pro-Tmp-Tmp-NH2
H-Dmt-Pro-Dmp-NH2
0.83
0.85
0.81
0.78
0.82
0.82
0.85
0.76
0.74
0.72
0.75
0.75
627
641
643
638
649
649
683
480
494
491
502
502
628
642
644
639
650
650
684
481
495
492
503
503
11.01
11.68
9.57
3
4
12.67
13.72
13.74
12.75
9.50
5
6
7
8
9
H-Dmt-Pro-Tmp-NH2
10.05
9.24
10
11
12
H-Dmt-Pro-Trp-NH2
H-Dmt-Pro-1-Nal-NH2
H-Dmt-Pro-2-Nal-NH2
10.12
10.30
a
Solvent: n-BuOH/AcOH/H2O = 4:1:1.
b
HPLC elution on a Cosmosil C18 column (4.6 mm ꢀ 250 mm, 5 mm) using the solvent system of 0.05% (v/v) TFA in water (A) and 0.05% (v/v)
TFA in CH3CN (B) and a linear gradient of 10–90% solvent B over 20 min at a flow rate of 1.2 mL/min.

L. Zhang et al. / Chinese Chemical Letters 22 (2011) 907–910
909
Table 2
Receptor affinity of endomorphin-2 analogues.
Compd.
Peptide
Kim nmol/L ꢁ SE (n)a
Kid nmol/L ꢁ SE (n)b
Kim/Kid
1
2
3
4
5
6
7
H-Dmt-Pro-Phe-Dmp-NH2
H-Dmt-Pro-Phe-Tmp-NH2
H-Dmt-Pro-Phe-Dmt-NH2
H-Dmt-Pro-Phe-Trp-NH2
0.31 ꢁ 0.07 (3)
0.37 ꢁ 0.02 (3)
0.28 ꢁ 0.02 (3)
0.34 ꢁ 0.03 (3)
0.28 ꢁ 0.02 (3)
0.81 ꢁ 0.15 (4)
0.47 ꢁ 0.07 (3)
1.33
40.0 ꢁ 4.4 (4)
24.5 ꢁ 2.3 (3)
87.2 ꢁ 6.7 (4)
25.4 ꢁ 3.2 (3)
51.7 ꢁ 6.3 (4)
23.2 ꢁ 2.4 (3)
1.63 ꢁ 0.2 (4)
6085
129
66
311
59
H-Dmt-Pro-Phe-1-Nal-NH2
H-Dmt-Pro-Phe-2-Nal-NH2
H-Dmt-Pro-Tmp-Tmp-NH2
H-Tyr-Pro-Phe-Phe-NH2 (EM-2)c
H-Dmt-Pro-Phe-Phe-NH2 ([Dmt1]EM-2)c
H-Dmt-Pro-Tmp-Phe-NH2 ([Dmt1,Tmp3]EM-2)c
H-Dmt-Pro-Dmp-NH2
185
29
3.5
4575
382
10
0.26
99.2
0.18
1.83
8
9
0.15 ꢁ 0.03 (4)
0.13 ꢁ 0.03 (4)
0.21 ꢁ 0.02 (3)
0.70 ꢁ 0.08 (4)
16.9 ꢁ 3.1 (4)
0.12
33.0 ꢁ 7.1 (4)
13.1 ꢁ 1.7 (4)
66.4 ꢁ 7.1 (3)
120.8 ꢁ 10.2 (4)
68.3 ꢁ 9.9 (4)
53.2
220
101
316
173
4
H-Dmt-Pro-Tmp-NH2
10
11
12
H-Dmt-Pro-Trp-NH2
H-Dmt-Pro-1-Nal-NH2
H-Dmt-Pro-2-Nal-NH2
d
H-Dmt-Pro-Phe-NH2
170
a
Versus [3H]DAMGO.
b
c
d
Versus [3H]deltorphin-II. n is the number of independent repetitions conducted for each analogue using five to eight doses of peptide.
Data are taken from Ref. [6].
Data are taken from Ref. [7], the Kid versus [3H]DPDPE.
Compared with [Dmt1, Dmp4]EM-2 (1) and [Dmt1, Tmp4]EM-2 (2), the additional hydroxyl group at the side chain of
the fourth amino acid of 3 ([Dmt1, Dmt4]EM-2) was detrimental to binding, indicating that decreased lipophilicity at
this position is unfavorable to binding to d-receptor. Compound 6 showed onefold greater d-receptor affinity than
compound 5, indicating that the orientation of the aromatic group also affects peptide binding to d-receptor. On the
whole, modification at position 4 slightly decreases affinity for m-receptor and slightly improves affinity for d-receptor.
Compound 7 containing two Tmp residues at 3 and 4 positions exhibited the synergic effects of the substitution
position 3 on d-receptor activity; the change at position 4 (compound 7) revealed high affinity for both m- and d-
receptor (Kim = 0.47 nmol/L, Kid = 1.63 nmol/L, Kid/Kim = 3.5), with a more balanced receptor selectivity than
[Dmt1]EM-2 (Kid/Kim = 10).
Research on the structure of endomorphins indicated that the Tyr1 and either Phe3/Trp3 or Phe4 in opioid peptides
are important topological elements that interact with opioid receptors [8]. Research also showed that deletion of the
forth position’s amino acid of [Dmt1]EM-2 to give Dmt-Pro-Phe-NH2 maintained high binding affinity for both m- and
d-receptors [9]. Substitution of the Phe3 of the Dmt-Pro-Phe-NH2 with Tmp, Dmp, Trp, 1-Nal, and 2-Nal resulted in
peptides 8–12, respectively. The binding assay indicated that all the new opioids except 12 retained high affinity for m-
receptor. Of these analogues, compound 9 had the highest affinity (Kim = 0.13 nmol/L). Relative to the d-receptor, 8
and 9 showed enhanced affinity, and in agreement with the first series, Tmp containing compound 9 contained the most
potent affinity for the d-receptor. The results also indicated that 1-Nal (9) is unfavorable to adopt the binding pocket of
d-receptor and showed decreased affinity.
In conclusion, both position 4 of [Dmt1]EM-2 and position 3 of Dmt-Pro-Phe-NH2 were well tolerated by
substitution with various aromatic amino acids, of which Tmp is the best for both positions in its interaction with
the m- and d-binding pockets. Pharmacological and biochemical evidence reveals that m- and d-opioid receptors
readily form heterodimers [10], and either d-receptor agonists or antagonists can facilitate internalization of
morphine-binding m-receptor, thereby decreasing morphine-induced tolerance and dependence [11]. This,
therefore, becomes the rationale in opioid research focused on the development of compounds with mixed and
dual opioid receptor bioactivities [1]. Among the newly synthesized peptides, Tmp at positions 3 and 4
(compound 7) exhibited potent and balanced affinities for both m- and d-receptors, and preliminary functional
assay indicated that compound 7 is a potent m-receptor agonist, therefore, this compound is of special interesting
in screening analgesics with low tolerance and dependence. Further detailed functional and pharmacological
evaluations are undergoing.

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L. Zhang et al. / Chinese Chemical Letters 22 (2011) 907–910
Acknowledgments
This work was supported by grants 08KJB350002 and 08NMUZ028, and in part by the Intramural Research
Program of the NIH and NIEHS.
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