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A. PROTOTYPES AND STRUCTURE–ACTIVITY RELATIONSHIPS

A. PROTOTYPES AND STRUCTURE–ACTIVITY RELATIONSHIPS

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Phe-NH2 (TIPP-NH2), was found to be a moderately potent A agonist in

the GPI assay and a potent y antagonist in the MVD assay (Table 3).

Replacement of Tyr1 in TIPP-NH2 with Dmt produced the compound

H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2), showing an increase in both Aagonist potency and y-antagonist activity by nearly two orders of magnitude [59,60]. In the receptor binding assays DIPP-NH2 displayed very high

y-receptor affinity and still some preference for y receptors over A receptors.

In comparison with DIPP-NH2, the corresponding analogue with a reduced peptide bond between the Tic2 and Phe3 residues, H-Dmt-TicC

[CH2-NH]Phe-Phe-NH2 (DIPP-NH2[C]), was about twice as potent as

agonist in the GPI assay and about half as potent as antagonist in the

MVD assay. Showing A- and y-receptor affinities that were both in the

subnanomolar range, DIPP-NH2[C] was essentially nonselective (K Ai /K yi

= 2.11; Table 3). Therefore, DIPP-NH2[C] represents the first known

opioid compound with balanced A-agonist/y-antagonist properties

[59,60]. In the rat tail flick test, DIPP-NH2[C] given intracerebroventricularly (ICV) produced a potent analgesic effect, being about three

times more potent than morphine. It produced less acute tolerance than

morphine, but still a certain level of chronic tolerance. Unlike morphine,

DIPP-NH2[C] produced no physical dependence upon chronic administration at high doses. Thus, DIPP-NH2[C] fulfilled to a large extent

the expectations based on the mixed A-agonist/y-antagonist concept [60].

Surprisingly, elimination of the C-terminal carboxylate function

of the tripeptide y antagonist TIP resulted in H-Tyr-Tic-NH-(CH2)2-Ph

(Ph = phenyl), a compound that was a moderately potent full y agonist

(Table 3) [61]. Interestingly, lengthening of the phenylethyl substituent by

insertion of an additional methylene group restored y antagonism, as

indicated by the finding that the dipeptide derivative H-Tyr-Tic-NH(CH2)3-Ph was a moderately potent y antagonist in the MVD assay and a

relatively weak partial A agonist in the GPI assay. This remarkable

dependence of y-agonist versus y-antagonist behavior on the length of

the phenylalkyl substituent may be due to conformational effects resulting

in different clustering of the three aromatic moieties present in these

molecules. The analogue H-Dmt-Tic-NH-(CH2)3-Ph showed very high

affinity for both A and y receptors and was a potent y antagonist in the

MVD assay (Ke = 1.69 nM). That this compound displayed relatively

modest agonist potency in the GPI assay suggests that it also may have

partial A-agonist properties and that it may represent a mixed partial A

agonist/y antagonist. In the case of high affinity A-receptor ligands, partial

agonism is not always directly apparent in the GPI assay because the



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Table 3 In Vitro Opioid Activities and Receptor Affinities of Mixed A Agonist/y Antagonists

GPI

Compound



IC50, nM



H-Tyr-Tic-Phe-Phe-NH2

H-Dmt-Tic-Phe-Phe-NH2

H-Dmt-TicC[CH2-NH]Phe-Phe-NH2

H-Tyr-Tic-NH-(CH2)2-Ph

H-Tyr-Tic-NH-(CH2)3-Ph

H-Dmt-Tic-NH-(CH2)3-Ph

H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-]

H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-]

H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-]



1700

18.2

7.71

3010

(42 %)c

102

2.14

384

7.88



Binding assaysa



MVD

IC50, (nM)



Ke (nM)b



K Ai (nM)



18.0

0.209

0.537



78.8

1.19

0.943

69.1

160

0.386

0.881

5.89

0.460



82.0

41.9

1.69

4.89

233

2.13



Displacement of [3H]DAMGO (A-selective) and [3H]DSLET (y-selective) from rat brain membrane binding sites.

Determined against DPDPE.

c

Maximal inhibition of the contractions at 10 AM.

a

b



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



K yi (nM)

3.00

0.118

0.447

5.22

3.01

0.0871

13.2

17.2

0.457



K Ai /K yi

26.3

10.1

2.11

13.2

53.2

4.43

0.0667

0.342

1.01



ileum has a very high A-receptor reserve. Substituted Tyr-Tic-dipeptide

amides with mixed A-agonist/y-antagonist properties are of interest because

their small molecular size and lipophilic character may facilitate their

passage across the blood–brain barrier (BBB). Further efforts aimed at

strengthening the A-agonist component of this class of compounds may

be required.

Another prototype of a mixed A agonist/y antagonist is the cyclic hcasomorphin analogue H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-] [62]. This

compound turned out to be a fairly potent A agonist in the GPI assay

and showed relatively modest y-antagonist potency in the MVD assay

(Table 3). The 2-naphthylalanine (2-Nal) residue in this compound is a

key structural determinant for its y-antagonist behavior, since the corresponding Phe3 analogue, H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-] was found to

be a full y agonist in the MVD assay [62]. As expected, an analogue

containing Dmt in place of Tyr1, H-Dmt-c-[-D-Orn-2-Nal-D-Pro-Gly-],

showed greatly increased A-agonist and y-antagonist potency [63]. This

pentapeptide displayed almost equal affinities for A and y receptors in the

subnanomolar range and, thus, represents another example of a balanced

A agonist/y antagonist. In comparison with DIPP-NH2[C], H-Dmt-c[-DOrn-2-Nal-D-Pro-Gly-] has the same A-agonist potency in the GPI assay

and is about four times less potent as a y antagonist in the MVD assay.

The various compounds described in this section represent the only

known mixed A-agonist/y-antagonist substances reported to date. Analgesic testing of all these prototypes will reveal which type of compound

has the greatest potential for the development of viable analgesics.

Further analogues may have to be prepared and examined to determine

the ratio between A-agonist and y-antagonist potency required for

optimal attenuation of tolerance and dependence development. Additional structural modifications may be necessary to increase analgesic

potency and bioavailability.



B. Conformational Study of H-Tyr-c[-D-Orn-2-Nal-DPro-Gly-]

The conformation of the mixed A agonist/y antagonist H-Tyr-c[-D-Orn-2Nal-D-Pro-Gly-] in comparison to that of H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-]

was studied in DMSO-d6 by NMR spectroscopy and by molecular mechanics calculations [62,64]. Neither peptide showed nuclear Overhauser

effects between CaH protons or chemical exchange cross peaks in spectra

obtained by total correlation and rotating frame Overhauser enhance-



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



ment spectroscopy (TOCSY, ROESY). These results indicated that the

average preferred solution conformation of both peptides was characterized by all-trans peptide bonds. The results of temperature-dependence

studies of the amide proton chemical shifts in conjunction with those of

the molecular mechanics studies indicated that the two analogues had

backbone conformations that were both stabilized by Tyr1-COÁÁÁHNPhe3 (or 2-Nal3) and D-Orn2-COÁÁÁHNy-D-Orn2 hydrogen bonds. Furthermore, ROESY experiments revealed a close proximity between the

aromatic moiety of the 3-position residue and the pyrrolidine ring of the

4

D-Pro residue in these two compounds. The comparison of all calculated

low-energy conformations with the various proton NMR parameters led

to proposals for the solution conformation of these two peptides (Fig. 4).

Inspection of the structures reveals that the Phe3- and 2-Nal3analogues have similar backbone conformations and the same side chain

orientation at the 3 position. These results suggest that the y-antagonist



Figure 4 Proposed solution conformations of H-Tyr-c-[-D-Orn-2-Nal-D-ProGly-] (left panel) and H-Tyr-c-[D-Orn-Phe-D-Pro-Gly-] (right panel).



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



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