An imidazole based H-Phe-Phe-NH2 peptidomimetic with anti-allodynic effect in spared nerve injury mice.

The dipeptide amide H-Phe-Phe-NH2 (1) that previously was identified as a ligand for the substance P 1-7 (SP1-7) binding site exerts intriguing results in animal models of neuropathic pain after central but not after peripheral administration. The dipeptide 1 is derived from stepwise modifications of the anti-nociceptive heptapeptide SP1-7 and the tetrapeptide endomorphin-2 that is also binding to the SP1-7 site. We herein report a strong anti-allodynic effect of a new H-Phe-Phe-NH2 peptidomimetic (4) comprising an imidazole ring as a bioisosteric element, in the spare nerve injury (SNI) mice model after peripheral administration. Peptidomimetic 4 was stable in plasma, displayed a fair membrane permeability and a favorable neurotoxic profile. Moreover, the effective dose (ED50) of 4 was superior as compared to gabapentin and morphine that are used in clinic.

Discovery of pyrazinone based compounds that potently inhibit the drug-resistant enzyme variant R155K of the hepatitis C virus NS3 protease.

Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wild-type and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

Aminocarbonylation of 4-iodo-1H-imidazoles with an amino acid amide nucleophile: synthesis of constrained H-Phe-Phe-NH2 analogues.

A simple and an expedient process to prepare 5-aryl-1-benzyl-1H-imidazole-4-carboxamides by the aminocarbonylation of 5-aryl-4-iodo-1H-imidazoles using ex situ generation of CO from Mo(CO)6 with an amino acid amide nucleophile is reported. Furthermore, a microwave-assisted protocol for the direct C-5 arylation of 1-benzyl-1H-imidazole and a regioselective C-4 iodination method to acquire starting material for our aminocarbonylation are presented. The method can be used to prepare imidazole based peptidomimetics, herein exemplified by the synthesis of constrained H-Phe-Phe-NH2 analogues.

Palladium-Catalyzed Aminocarbonylation in Solid-Phase Peptide Synthesis: A Method for Capping, Cyclization, and Isotope Labeling.

A new synthetic approach for introducing N-capping groups onto peptides attached to a solid support, combining aminocarbonylation under mild conditions using a palladacycle precatalyst and solid-phase peptide synthesis, is reported. The use of a silacarboxylic acid as an in situ CO-releasing molecule allowed the reaction to be performed in a single vial. The method also enables versatile substitution of side chains, side-chain-to-side-chain cyclizations, and selective [13C] acyl labeling of modified peptides.

Importance of N- and C-terminal residues of substance P 1-7 for alleviating allodynia in mice after peripheral administration.

The heptapeptide SP1-7 (1, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7) is the major bioactive metabolite formed after proteolytic processing of the neuropeptide substance P (SP, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2). The heptapeptide 1 frequently exhibits opposite effects to those induced by SP, such as exerting antinociception, or attenuating thermal hyperalgesia and mechanical allodynia. The heptapeptide SP1-7 amide (2, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-NH2) is often more efficacious than 1 in experimental pain models. We have now assessed the anti-allodynic outcome after systemic administration of 2 and a series of Ala-substituted and truncated analogues of 2, in the spared nerve injury (SNI) mice model and the results obtained were correlated with in vitro plasma stability and permeability measurements. It is herein demonstrated that an intact Arg1 in SP1-7 amide analogues is fundamental for retaining a potent in vivo effect, while Lys3 of 2 is less important. A displacement with Ala1 or truncation rendered the peptide analogues either inactive or with a significantly attenuated in vivo activity. Thus, the pentapeptide SP3-7 amide (7, t1/2=11.1 min) proven to be the major metabolite of 2, demonstrated an in vivo effect itself although considerably less significant than 2 in the SNI model. Intraperitoneal administration of 2 in a low dose furnished the most powerful anti-allodynic effect in the SNI model of all the analogous evaluated, despite a fast proteolysis of 2 in plasma (t1/2=6.4 min). It is concluded that not only the C-terminal residue, that we previously demonstrated, but also the N-terminal with its basic side chain, are important for achieving effective pain relief. This information is of value for the further design process aimed at identifying more drug-like SP1-7 amide related peptidomimetics with pronounced anti-allodynic effects.

Impact of N-methylation of the substance P 1-7 amide on anti-allodynic effect in mice after peripheral administration.

Substance P 1-7 (SP1-7, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7) is the major bioactive metabolite formed after proteolytic degradation of the tachykinin substance P (SP). This heptapeptide often opposes the effects of the mother peptide. Hence, SP1-7 is having anti-inflammatory, anti-nociceptive and anti-hyperalgesic effects in experimental models. Despite all encouraging properties of SP1-7 its exact mode of action has not yet been elucidated which has hampered further development of this heptapeptide in drug discovery. Contrary to SP that mediates its biological activity via the NK-1 receptor, the N-terminal fragment SP1-7 acts through an unknown target that is distinct from all known opioid and tachykinin receptors. The SP1-7 amide 1 (Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-NH2) was previously shown to be superior to the endogenous SP1-7 in all experimental pain models where the two compounds were compared. Herein, we report that N-methylation scan of the backbone of the SP1-7 amide (1) results in peptides that are significantly less prone to undergo proteolysis in plasma from both mouse and human. However, with the two exceptions of the [MeLys3]SP1-7 amide (3) and the [MeGln5]SP1-7 amide (4), the peptides with a methyl group attached to the backbone are devoid of significant anti-allodynic effects after peripheral administration in the spared nerve injury (SNI) mouse model of neuropathic pain. It is suggested that the N-methylation does not allow these peptides to form the accurate bioactive conformations or interactions required for efficient binding to the macromolecular target. The importance of intact N-terminal Arg1 and C-terminal Phe7, anticipated to serve as address and message residues, respectively, for achieving the anti-allodynic effect is emphasized. Notably, the three heptapeptides: the SP1-7 amide (1), the [MeLys3]SP1-7 amide (3) amide and the [MeGln5]SP1-7 amide (4) are all considerably more effective in the SNI mouse model than gabapentin that is widely used in the clinic for treatment of neuropathic pain.