Stereocontrolled synthesis of psi[CH = CH] dipeptide isosteres.

A new stereocontrolled synthesis of the psi[CH = CH] dipeptide isostere is described. They key step of the sequence relies on the stereospecific alpha-alkylation of delta-amino-gamma-mesyloxy-alpha,beta-unsaturated esters. The broad availability of nucleophilic alpha-side chains by this method allows the preparation of a wide variety of psi[CH = CH] isosteres with predictable stereochemistry.     

Endomorphin 1[psi] and endomorphin 2[psi], endomorphins analogues containing a reduced (CH2NH) amide bond between Tyr1 and Pro2, display partial agonist potency but significant antinociception

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are highly potent and selective mu-opioid receptor agonists and have significant antinociceptive action. In the mu-selective pocket of endomorphins (EMs), Pro2 residue is a spacer and directs the Tyr1 and Trp3/Phe3 side chains into the required orientation. The present work was designed to substitute the peptide bond between Tyr1 and Pro2 of EMs with a reduced (CH2NH) bond and study the agonist potency and antinociception of EM1[psi] (Tyr[psi(CH2NH)]Pro-Trp-Phe-NH2) and EM2[psi] (Tyr[psi(CH2NH)]Pro-Phe-Phe-NH2). Both EM1[psi] and EM2[psi] are partial mu opioid receptor agonists showing significant loss of agonist potency in GPI assay. However, EMs[psi] exhibited potent supraspinal antinociceptive action in vivo. In the mice tail-flick test, EMs[psi] (1, 5, 10 nmol/mouse, i.c.v.) produced potent and short-lasting antinociception in a dose-dependent and naloxone (1 mg/kg) reversed manner. At the highest dose of 10 nmol, the effect of EM2[psi] was prolonged and more significant than that of EM2. In the rat model of formalin injection induced inflammatory pain, EMs[psi] (0.1, 1, 10 nmol/rat, i.c.v.), like EMs, exerted transient but not dose-dependent antinociception. These results suggested that in the mu-selective pocket of EMs, the rigid conformation induced by the peptide bond between Tyr1 and Pro2 is essential to regulate their agonist properties at the mu opioid receptors. However, the increased conformational flexibility induced by the reduced (CH2NH) bond made less influence on their antinociception.     

Transition state isosteres of the gamma-glutamyl peptide bond hydrolysis: synthesis and characterization of the psi (CH2NH) pseudopeptide analogue of glutathione.

The fully deprotected glutathione analogue containing the aminomethylene unit as transition state isostere of the gamma-Glu-Cys peptide bond was synthesized for the first time and characterized in both the reduced and oxidized forms.     

A gastrin-releasing peptide antagonist containing A psi (CH2O) amide bond surrogate

The [Leu26-psi(CH2O)Leu27] derivative of N-Ac-GRP20-27-peptide amide was prepared and evaluated as a gastrin-releasing peptide antagonist. This psi(CH2O) derivative was found to be a more potent inhibitor of [3H-Phe15]GRP15-24NH2 binding and N-Ac-GRP20-27NH2 induced mitogenesis in Swiss 3T3 fibroblasts than the related nitrogen analog [Leu13-psi(CH2NH)Leu14] bombesin. Possible reasons for the improved activity of the (CH2O) insert relative to the (CH2NH) group include increased hydrophobicity and a reduced tendency of the oxygen derivative to form hydrogen bonds.     

Triazole-linked reduced amide isosteres: an approach for the fragment-based drug discovery of anti-Alzheimer's BACE1 inhibitors

In the course of a β-site APP-cleaving enzyme 1 (BACE1) inhibitor discovery project an in situ synthesis/screening protocol was employed to prepare 120 triazole-linked reduced amide isostere inhibitors. Among these compounds, four showed modest (single digit micromolar) BACE1 inhibition. Our ligand design was based on a potent reduced amide isostere 1, wherein the P₂ amide moiety was replaced with an anti-1,2,3-triazole unit. Unfortunately, this replacement resulted in a 1000-fold decrease in potency. Docking studies of triazole-linked reduced amide isostere A3Z10 and potent oxadiazole-linked tertiary carbinamine 2a with BACE1 suggests that the docking poses of A3Z10 and 2a in the active sites are quite similar, with one exception. In the docked structures the placement of the protonated amine that engages D228 differs considerably between 2a and A3Z10. This difference could account for the lower BACE1 inhibition potency of A3Z10 and related compounds relative to 2a.     

The trans labilization of cis-[PtCl2(13CH3NH2)2] by glutathione can be monitored at physiological pH by [1H,13C] HSQC NMR

In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl₂(¹³CH₃NH₂)₂] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release of ¹³CH₃NH₂ but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds are formed and Pt–NH₃ bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C] HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best method for characterizing the different species in solution. However, the intensity of the peaks in the ¹⁵NH₃–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity, and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum complexes with sulfur-containing ligands.     

Supraspinal and spinal effects of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 on nociception in the rat

A new derivative of the neuropeptide nociceptin (NC) has recently been developed. This molecule, the pseudopeptide [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was found to antagonize NC inhibitory effects in peripheral smooth muscle preparations in vitro. However, contrasting results have appeared as regards its pharmacodynamic profile in the CNS. Here, we investigated the pseudopeptide effects, in vivo, on nociceptive responses in the rat. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) (alone or in combination with NC), and tail-flick latencies (TFL) to radiant heat were assessed. I.c.v. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 (1-10 nmol/rat) caused a short-lasting decrease (5 min) of TFL and did not antagonize the threshold lowering effect of i.c.v. NC (1 nmol/rat). At the spinal level, the i.t. administration (0.2-10 nmol/rat) of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 produced a dose-dependent and long-lasting antinociceptive effect that was not modified by the administration of a high dose (30 nmol/rat i.t.) of the opioid antagonist naloxone. The i.t. co-administration of the pseudopeptide (10 nmol/rat) did not block the antinociceptive effect of i.t. NC (10 nmol/rat). These data indicate that the pseudopeptide behaves as an NC agonist at supraspinal and spinal levels in the rat tail-flick test of nociception. These different profiles in the periphery and the CNS could suggest differences between central and peripheral NC receptor/s and provide a basis for further development of antagonist molecules suitable for their characterization.     

Effects of [Nphe1]nociceptin(1-13)NH2, [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2, and nocistatin on nociceptin inhibited constrictions of guinea pig isolated bronchus

Electric field stimulation (EFS) causes excitatory non adrenergic-non cholinergic (eNANC) and cholinergic constrictions in the guinea pig isolated bronchus, the activation of eNANC and cholinergic nerves respectively. We investigated the effects of [Nphe1]nociceptin(1-13)NH2 ([Nphe1]NC(1-13)NH2), [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2 ([F/G] NC(1-13)NH2), and nocistatin (NST) on nociceptin (NC) inhibited constrictions in isolated bronchus of guinea pig. The results show that NC (1 micromol/L) inhibited EFS-induced eNANC and cholinergic constrictions compared with the control, in which nociceptin was not applied. After pretreatment with [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, or NST, the inhibitions of NC were antagonized by [Nphe1]NC(1-13)NH2 and [F/G]NC(1-13)NH2 but not NST. However, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, and NST did not affect the inhibitions induced by morphine. Furthermore, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2 and NST did not cause any appreciable effects on EFS-induced eNANC and cholinergic constrictions in guinea pig bronchi. The results demonstrate that [Nphe1]NC(1-13)NH2 and [F/G]NC(1- 13)NH2 but not NST act as selective antagonists of the NC receptor and the effects of NC on EFS-induced constrictions of guinea pig isolated bronchus.     

Discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors

The design of amide and heteroaryl amide isosteres as replacements for the carbamate substructure in previously disclosed 2,6-disubstituted piperidine N-arylsulfonamides is described. In several cases, amides lessened CYP liabilities in this class of gamma-secretase inhibitors. Selected compounds showed significant reduction of Abeta levels upon oral dosing in a transgenic murine model of Alzheimer's disease.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Reaction of cis- and trans-[PtCl2(NH3)2] with reduced glutathione inside human red blood cells, studied by 1H and 15N-[1H] DEPT NMR

Reactions of cis- and trans-[PtCl2(NH3)2] with glutathione (GSH) inside intact red blood cells have been studied by 1H spin-echo nuclear magnetic resonance (NMR). Upon addition of trans-[PtCl2(NH3)2] to a suspension of red cells, there was a gradual decrease in the intensity of the resonances for free GSH, and new peaks were observed that were assignable to coordinated GSH protons in trans-[Pt(SG)Cl(NH3)2], trans-[Pt(SG)2(NH3)2], and possibly the S-bridged complex trans-[[NH3)2PtCl)2SG]+. Formation of trans-[Pt(SG)2(NH3)2] inside the cell was confirmed from the 1H NMR spectrum of hemolyzed cells, which were ultrafiltered to remove large protein molecules; the ABM multiplet of the coordinated GSH cys-beta CH2 protons was resolved using selective-decoupling experiments. Seventy percent of the total intracellular GSH was retained by the ultrafiltration membrane, suggesting that the mixed complex trans-[Pt(SG)(S-hemoglobin)(NH3)2] also is a major metabolite of trans-[PtCl2(NH3)2] inside red cells. The reaction of cis-[PtCl2(NH3)2] with intracellular GSH was slower; only 35% of the GSH had been complexed after a 4-hr incubation compared to 70% for the trans isomer. There was a gradual decrease in the intensity of the GSH 1H spin-echo NMR resonances, but no new peaks were resolved. This was interpreted as formation of high-molecular weight Pt:GSH and mixed GS-Pt-S(hemoglobin) polymers. By using a 15N-[1H] DEPT pulse sequence, we were able to study the reaction of cis-[PtCl2(15NH3)2] with red cells at concentrations as low as 1 mM. 15NH3 ligands were released, and no resonances assignable to Pt-15NH3 species were observed after a 12-hr incubation.     

Conformational studies on bombesin antagonists: CD and NMR characterization of [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14).

The conformational flexibility of the [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14) nonapeptide has been studied by CD and one- and two-dimensional (1D and 2D) nmr techniques. The CD and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for the parent bombesin (BN) and [D-Phe12, Leu14]BN. A preliminary investigation on spantide is also reported. In particular, the results obtained from CD measurements indicate that there is a shift from random coil structures, in aqueous solutions, toward folded structures in apolar media (2,2,2-trifluoroethanol) and in a membrane-mimetic environment (40 mM SDS) for all three peptides, namely BN, [D-Phe12, Leu14]BN, and [Thr6, Leu13 psi(CH2NH) Met14]BN (6-14). Spantide, which also possesses some inhibitory activity against BN but very little sequence similarity, even in water, shows an ordered conformation. Nuclear magnetic resonance parameters such as backbone NH-alpha CH coupling constant values, amidic temperature coefficients, and the presence of only sequential nuclear Overhauser effects have not provided, so far, any clear evidence for a preferential ordered structure in the peptides studied, and this may be due to rapid exchange among different conformers in the nmr time scale.     

Proline isosteres in a series of 2,4-disubstituted pyrrolo[1,2-f][1,2,4]triazine inhibitors of IGF-1R kinase and IR kinase

Pyrrolidine, pyrrolidinone, carbocyclic, and acyclic groups were used as isosteric proline replacements in a series of insulin-like growth factor I receptor kinase/insulin receptor kinase inhibitors. Examples that were similar in potency to proline-containing reference compounds were shown to project a key fluoropyridine amide into a common space, while less potent compounds were not able to do so for reasons of stereochemistry or structural rigidity.     

Cyclosporine inhibition of CH series murine B-cell lymphomas

To investigate the mechanisms by which cyclosporine (CsA) inhibits B-cell function, the effect of this agent on murine B-lymphoma cell lines of the CH series was tested. These lymphomas appear to be derived from a restricted B-cell population on the basis of their common expression of the Ly-1 cell surface marker and autoantibody products. Proliferation of each of the six cell lines tested was inhibited by CsA at doses without effect on the nonlymphoid HeLa cell line. The cell lines, however, differed from each other in their sensitivity to this agent. To correlate this sensitivity to other functional B-cell properties, the effect of lipopolysaccharide (LPS) on the proliferation of the CH cell lines was tested. Three of the lines showed enhanced proliferation to LPS; two were inhibited while one was unaffected. The cell lines that responded with increased proliferation to LPS were the most sensitive to CsA. These results indicate that sensitivity to CsA may be a common property of B cells of certain lineages, although the degree of sensitivity may be influenced by the activation properties of these cells.     

Synthesis,molecular modeling and BACE-1 inhibitory study of tetrahydrobenzo[b] pyran derivatives

β-Secretase (BACE1) has been broadly documented as one of the possible therapeutic targets for the treatment of Alzheimer’s disease. In this paper, we report the synthesis and the for β-secretase (BACE-1) inhibitory activity of new series of tetrahydrobenzo [b] pyran derivatives. One-pot synthesis of tetrahydrobenzo [b] pyrans was carried out by condensing aromatic aldehyde, malononitrile and 1,3-cyclohexanedione using ionic liquid 1-butyl-3-methyl imidazolium chloride ([bmIm]Cl⁻) in aqueous alcohol media. The addition of alcohol and water in the ratio of 1:2 keeps all the reactants in solution which facilitates the reaction and makes the product formation very easy. The synthesized compounds were subjected to BACE1 inhibition assay and six compounds, 4d, 4e, 4f, 4h, 4i, and 4p have shown significant IC₅₀ values at micromolar level. Among these six active compounds, 4e was a potential inhibitor with its IC₅₀ value in nanomolar range. All the synthesized compounds were docked onto the active site of β-Secretase enzyme.     

Achieving multi-isoform PI3K inhibition in a series of substituted 3,4-dihydro-2H-benzo[1,4]oxazines

The SAR and pharmacokinetic profiles of a series of multi-isoform PI3K inhibitors based on a 3,4-dihydro-2H-benzo[1,4]oxazine scaffold are disclosed.     

Leu9 psi(CH2NH)Leu10]-neurokinin A (4-10) (MDL 28,564) distinguishes tissue tachykinin peptide NK2 receptors

The neurokinin A analogue, MDL 28,564 (Asp-Ser-Phe-Val-Gly-Leu-CH2NH-Leu-NH2), inhibited 125I-NKA binding to hamster urinary bladder NK2 receptors with a KI of 130 nM. For rat submaxillary gland NK1 receptors and cerebral cortical NK3 receptors, the KI's for MDL 28,564 were greater than 250 microM and greater than 500 microM, respectively. MDL 28,564 did not relax dog carotid artery (NK1 tissue) or contract rat portal vein (NK3 tissue). In guinea-pig trachea tissues, MDL 28,564 stimulated phosphatidylinositol turnover and induced contraction with maximum effects similar to those of neurokinin A. In hamster urinary bladder tissue, MDL 28,564 stimulated phosphatidylinositol turnover with maximum effect only 10% of that of neurokinin A, did not produce sustained contraction itself and antagonized NKA-induced contraction. MDL 28,564 also produced full contraction in rabbit pulmonary artery (NK2 tissue) but was inactive in rat vas deferens (NK2 tissue). These data with MDL 28,564 are consistent with the NK2 receptors in guinea-pig trachea and rabbit pulmonary artery being different from those in hamster urinary bladder and rat vas deferens.     

BACE-1 inhibitors part 2: identification of hydroxy ethylamines (HEAs) with reduced peptidic character

This paper describes the discovery of non-peptidic, potent, and selective hydroxy ethylamine (HEA) inhibitors of BACE-1 by replacement of the prime side of a lead di-amide 2. Inhibitors with nanosmolar potency and high selectivity were identified. Depending on the nature of the P(1)(') and P(2)(') substituents, two different binding modes were observed in X-ray co-crystal structures.     

Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors may result in additional analgesia with fewer side effects. Here, we evaluated the pharmacological profile of our formerly developed mixed mu/kappa-opioid receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH₂ (C-36) and Dmt-c[D-Lys-Phe-p-CF₃-Phe-Asp]NH₂ (F-81). The ability of these peptides to cross the blood–brain barrier was tested in the parallel artificial membrane permeability (PAMPA) assay. On the basis of the hot-plate test in mice after central and peripheral administration, analog F-81 was selected for the anti-nociceptive and anti-inflammatory activity assessment after peripheral administration.