Novel cyclic biphalin analogue with improved antinociceptive properties.

Two novel opioid analogues have been designed by substituting the native d-Ala residues in position 2,2' of biphalin with two residues of d-penicillamine or l-penicillamine and by forming a disulfide bond between the thiol groups. The so-obtained compound 9 containing d-penicillamines showed excellent µ/δ mixed receptor affinities (K i (δ) = 5.2 nM; K i (µ) = 1.9 nM), together with an efficacious capacity to trigger the second messenger and a very good in vivo antinociceptive activity, whereas product 10 was scarcely active. An explanation of the two different pharmacological behaviors of products 9 and 10 was found by studying their conformational properties.

Potent biphalin analogs with µ/δ mixed opioid activity: in vivo and in vitro biological evaluation.

Biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2 ] is an octapeptide with mixed µ/δ opioid activity. Its structure is based on two identical enkephalin-like portions linked "tail-to-tail" by a hydrazine bridge. This study presents the synthesis and in vitro and in vivo bioassays of two biphalin analogs that do not present the toxicity connected with the presence of the hydrazine moiety and are able to elicit a higher antinociceptive effect than biphalin.

A Potent (R)-alpha-bis-lipoyl Derivative Containing 8-Hydroxyquinoline Scaffold: Synthesis and Biological Evaluation of Its Neuroprotective Capabilities in SH-SY5Y Human Neuroblastoma Cells.

A novel bis-lipoyl derivative containing 8-hydroxyquinoline scaffold (LA-HQ-LA, 5) was synthesized as a new multifunctional drug candidate with antioxidant, chelant, and neuroprotective properties for the treatment of neurodegenerative diseases. We have investigated the potential effectiveness of LA-HQ-LA against the cytotoxicity induced by 6-OHDA and H2O2 on human neuroblastoma SH-SY5Y cell line. Our outcomes showed that LA-HQ-LA resulted in significant neuroprotective and antioxidant effects against H2O2- and 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cells, as assessed by MTT assay. In particular, it showed potent neuroprotective effects against 6-OHDA in RA/PMA differentiated cells at all the tested concentrations.

A glutathione derivative with chelating and in vitro neuroprotective activities: synthesis, physicochemical properties, and biological evaluation.

Metal-ion dysregulation and oxidative stress have been linked to the progressive neurological decline associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Herein we report the synthesis and chelating, antioxidant, and in vitro neuroprotective activities of a novel derivative of glutathione, GS(HQ)H, endowed with an 8-hydroxyquinoline group as a metal-chelating moiety. In vitro results showed that GS(HQ)H may be stable enough to be absorbed unmodified and arrive intact to the blood-brain barrier, that it may be able to remove Cu(II) and Zn(II) from the Aß peptide without causing any copper or zinc depletion in vivo, and that it protects SHSY-5Y human neuroblastoma cells against H2 O2 - and 6-OHDA-induced damage. Together, these findings suggest that GS(HQ)H could be a potential neuroprotective agent for the treatment of neurodegenerative diseases in which a lack of metal homeostasis has been reported as a key factor.

Synthesis and neuromodulatory effects of TRH-related peptides: inhibitory activity on catecholamine release in vitro.

BACKGROUND: A detailed comprehension of central mechanisms underlying feeding behavior holds considerable promise for the treatment of alimentary disorders. METHODS: In order to elucidate the tight interrelationships occurring at the hypothalamic neuronal endings between aminergic neurotransmitters and co-localized appetite modulators, we initially studied the effects of two anorexigenic peptides structurally related to thyrotropin-releasing hormone (TRH, 1), namely cyclo(His-Pro) (CHP, 2) and pGlu-His-Gly-OH (3), on [(3)H]-norepinephrine and [(3)H]-dopamine release from perfused rat hypothalamic synaptosomes. Furthermore, a number of TRH and CHP analogues were synthesized and tested for their ability to influence neurotransmitter release in the selected neuronal model. RESULTS: Peptide 3 showed only a slight inhibitory activity on norepinephrine release, whereas no effect was observed for compound 2. TRH analogue 8, metabolically stabilized by the replacement of pyroglutamate with the pyrohomocysteic acid (pHcs), was found to be inactive. Conversely, a significant inhibitory effect on dopamine and norepinephrine release was observed for the CHP-related diketopiperazines cyclo(Leu-Pro) (11) and cyclo(His-Gly) (14). CONCLUSIONS: These results suggest a potential role for cyclo-dipeptides 11 and 14 in the hypothalamic modulation of appetite suppressant circuitry.

Hybrid peptides endomorphin-2/DAMGO: design, synthesis and biological evaluation.

Endomorphin-2 [Tyr-Pro-Phe-Phe-NH2] and DAMGO [Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol] are natural (EM2) and synthetic (DAMGO) opioid peptides both selective for µ opioid receptor with high analgesic activity. In this work we report synthesis, in vitro and in vivo biological evaluation of five new hybrid EM2/DAMGO analogues, with the aim to obtain compounds with high affinity at µ-opioid receptor, high activity in animal nociception tests (hot plate and tail flick) and improved enzymatic stability. Double N-methylation on both Phe residues and C-terminal ethanolamide led to analogue 6e, which possesses a good in vitro µ affinity (Kiµ=34 nM), combined with a remarkable in vivo antinociceptive activity.

Biological active analogues of the opioid peptide biphalin: mixed α/ß(3)-peptides.

Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo-ß(3) amino acids. The derivative 1 containing hß(3) Phe in place of Phe showed good µ- and δ-receptor affinities (Ki(δ) = 0.72 nM; Ki(µ) = 1.1 nM) and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma.

Structure-activity relationships of biphalin analogs and their biological evaluation on opioid receptors.

Biphalin (Tyr-D-Ala-Gly-Phe-NH-NH < -Phe < -Gly < -D-Ala < -Tyr) is an opioid octapeptide with a dimeric structure based on two identical pharmacophore portions, derived from enkephalins, joined "tail to tail" by a hydrazide bridge. This particular structure enhances the antinociceptive activity of the native enkephalins with an unknown mechanism, probably based on a cooperative binding and improved enzymatic stability. Biphalin has excellent binding affinity for µ and δ receptors and it is a highly potent analgesic, as potent as or more than ethorphine. A definitive explanation of the extraordinary in vivo potency shown by this compound, which has pronounced efficacy in pain modulation, is still not available; it has been suggested, however, that the high agonist activity may be related to its binding mode at both µ and δ opioid receptors. Biphalin has significantly higher potency than other analgesics with novel biological profiles; in particular, most recent data show that biphalin is unlikely to produce dependency in chronic use. In the past 20 years, there have been many attempts to modify its structure to obtain products unaffected by the action of enkephalinases, to enhance its antinociceptive activity and to modify the BBB penetration. In addition, structure-activity relationship studies (SAR) were performed in order to understand the elements responsible for biphalin's high activity. The aim of the studies reported in this review was to clarify: i) the role of the hydrazide bridge, ii) the role of residues in position 4, 4' and 3, 3', iii) the consequences of molecular simplifications (truncation, delection), iv) the consequences of cyclization through a disulfide bridge, v) conjugation with PEG and fluorescet residues, and vi) radiolabeling on Tyr.

Antinociceptive profile of potent opioid peptide AM94, a fluorinated analogue of biphalin with non-hydrazine linker.

AM94 is a fluorinated analog of biphalin with non-hydrazine linker that has an in vitro affinity for µ-opioid and δ-opioid receptors tenfold higher than biphalin. Furthermore, in vivo evaluation in rats showed that AM94 has in hot plate test - after both intracerebroventricular and intravenous administrations - a greater and more durable efficacy than biphalin. Here, the antinociceptive profile of AM94 is further evaluated by following two different administration routes, intrathecal and subcutaneous, and two different animal species, rats and mice. The analgesic potency of AM94 is compared with that of both the parent peptide biphalin and morphine. Results show that in rats (tail flick test) and in mice (formalin test), AM94 has a higher and more durable analgesic effect than biphalin after intrathecal and subcutaneous administrations. Conformational properties of biphalin and AM94 were also investigated by variable-temperature (1)H NMR and energy minimization.

Role of formyl peptide receptors (FPR) in abnormal inflammation responses involved in neurodegenerative diseases.

Neurodegenerative disorders, such as multiple sclerosis, prion diseases, Alzheimer's disease and Parkinson's disease are often associated with inflammatory process, which involves various components of the immune system in the central nervous system, in particular astrocytes and microglial cells. Inflammation mediators such as cytokines, leukotrienes, superoxide radicals, eicosanoids, the complement cascade, and FPR agonists (formyl peptides) may play a significant role in pro-inflammatory responses, in which infiltration of activated mononuclear phagocytes at the sites of lesion is a common feature. To prevent long-term inflammation damage, the central nervous system could be treated with antinflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDs), but only few drugs were found to be effective and their therapeutic benefits is limited by side effects. Accumulating evidences suggest that targeting glia-neuron system might be a therapeutic approach in the treatment of neurodegenerative disease progression, in particular of Alzheimer's disease. Aminopyridazine derivative discovered in unbiased cell-based screens for new synthetic compounds, have proved to be able to suppress selective glial activation responses via mechanisms distinct from NSAIDs. In this review, we report the potential involvement of FPR receptors in inflammatory responses and the potential use of their antagonists to modulate the inflammatory responses of the microglia. Recent results demonstrate that targeting of inflammatory glia cytokine pathways, can suppress Aß-induced neuroinflammation in vivo, resulting in the attenuation of neuronal damage.

(R)-α-lipoyl-glycyl-L-prolyl-L-glutamyl dimethyl ester codrug as a multifunctional agent with potential neuroprotective activities.

The (R)-α-lipoyl-glycyl-L-prolyl-L-glutamyl dimethyl ester codrug (LA-GPE, 1) was synthesized as a new multifunctional drug candidate with antioxidant and neuroprotective properties for the treatment of neurodegenerative diseases. Physicochemical properties, chemical and enzymatic stabilities were evaluated, along with the capacity of LA-GPE to penetrate the blood-brain barrier (BBB) according to an in vitro parallel artificial membrane permeability assay for the BBB. We also investigated the potential effectiveness of LA-GPE against the cytotoxicity induced by 6-hydroxydopamine (6-OHDA) and H2O2 on the human neuroblastoma cell line SH-SY5Y by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Our results show that codrug 1 is stable at both pH 1.3 and 7.4, exhibits good lipophilicity (log P=1.51) and a pH-dependent permeability profile. Furthermore, LA-GPE was demonstrated to be significantly neuroprotective and to act as an antioxidant against H2O2- and 6-OHDA-induced neurotoxicity in SH-SY5Y cells.

cis-4-amino-L-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues: part 2.

Recently, we reported synthesis and activity of a constrained cyclic analogue of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) and related linear models containing the cis-4-amino-L-proline (cAmp) in place of native Pro(2). In the present article, the adopted rationale is the possible modulation of the receptor affinity of the cAmp containing EM-2 analogues by assigning a different stereochemistry to the Phe(3) and Phe(4) residues present in the ring. Thus, eight more analogues with different absolute configuration at the chiral center of the aromatic residues in positions 3 and 4 have been synthesized and their opioid activity examined. The stereochemical change at the α-carbon atoms leads to a meaningful enhancement of the affinity and activity toward µ opioid receptors with respect to the prototype compound 9: e.g., 9a, K(i)(µ) = 63 nM, GPI (IC(50)) = 480 nM; 9b, K(i)(µ) = 38 nM, GPI (IC(50)) = 330 nM.

Synthesis of short cationic antimicrobial peptidomimetics containing arginine analogues.

Worldwide efforts are underway to develop new antimicrobial agents against bacterial resistance. To identify new compounds with a good antimicrobial profile, we designed and synthesized two series of small cationic antimicrobial peptidomimetics (1-8) containing unusual arginine mimetics (to introduce cationic charges) and several aromatic amino acids (bulky moieties to improve lipophilicity). Both series were screened for in vitro antibacterial activity against a representative panel of Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacterial strains, and Candida albicans. The biological screening showed that peptidomimetics containing tryptophan residues are endowed with the best antimicrobial activity against S. aureus and S. epidermidis in respect to the other synthesized derivatives (MIC values range 7.5-50 µg/ml). Moreover, small antimicrobial peptidomimetics derivatives 2 and 5 showed an appreciable activity against the tested Gram-negative bacteria and C. albicans. The most active compounds (1-2 and 5-6) have been tested against Gram-positive established biofilm, too. Results showed that the biofilm inhibitory concentration values of these compounds were never up to 200 µg/ml. The replacement of tryptophan with phenylalanine or tyrosine resulted in considerable loss of the antibacterial action (compounds 3-4 and 7-8) against both Gram-positive and Gram-negative bacterial strains. Furthermore, by evaluating hemolytic activity, the synthesized compounds did not reveal cytotoxic activities, except for compound 5.

(Acyloxy)alkoxy moiety as amino acids protecting group for the synthesis of (R,R)-2,7 diaminosuberic acid via RCM.

A new synthetic pathway is described to prepare asymmetrically protected 2,7-diaminosuberic acid. This strategy exploits (acyloxy)alkoxy promoiety as protecting group and RCM reaction using second generation Grubbs catalyst and provides the trans isomer of (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-(tert-butoxycarbonylamino)-8- methoxy-8-oxooct-4-enoic acid, which was in turn reduced to obtain (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)- 2-(tert-butoxycarbonylamino)-8-methoxy-8-oxooctanoic acid.

Recent advances in the treatment of neurodegenerative diseases based on GSH delivery systems.

Neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease(AD), are a group of pathologies characterized by a progressive and specific loss of certain brain cell populations. Oxidative stress, mitochondrial dysfunction, and apoptosis play interrelated roles in these disorders. It is well documented that free radical oxidative damage, particularly on neuronal lipids, proteins, DNA, and RNA, is extensive in PD and AD brains. Moreover, alterations of glutathione (GSH) metabolism in brain have been implicated in oxidative stress and neurodegenerative diseases. As a consequence, the reduced GSH levels observed in these pathologies have stimulated a number of researchers to find new potential approaches for maintaining or restoring GSH levels. Unfortunately, GSH delivery to the central nervous system (CNS) is limited due to a poor stability and low bioavailability. Medicinal-chemistry- and technology-based approaches are commonly used to improve physicochemical, biopharmaceutical, and drug delivery properties of therapeutic agents. This paper will focus primarily on these approaches used in order to replenish intracellular GSH levels, which are reduced in neurodegenerative diseases. Here, we discuss the beneficial properties of these approaches and their potential implications for the future treatment of patients suffering from neurodegenerative diseases, and more specifically from PD and AD.

Synthesis and bioactivity of secondary metabolites from marine sponges containing dibrominated indolic systems.

Marine sponges. (e.g., Hyrtios sp., Dragmacidin sp., Aglophenia pleuma, Aplidium cyaneum, Aplidium meridianum.) produce bioactive secondary metabolites involved in their defence mechanisms. Recently it was demonstrated that several of those compounds show a large variety of biological activities against different human diseases with possible applications in medicinal chemistry and in pharmaceutical fields, especially related to the new drug development process. Researchers have focused their attention principally on secondary metabolites with anti-cancer and cytotoxic activities. A common target for these molecules is the cytoskeleton, which has a central role in cellular proliferation, motility, and profusion involved in the metastatic process associate with tumors. In particular, many substances containing brominated indolic rings such as 5,6-dibromotryptamine, 5,6-dibromo-N-methyltryptamine, 5,6-dibromo-N-methyltryptophan (dibromoabrine), 5,6-dibromo-N,N-dimethyltryptamine and 5,6-dibromo-L-hypaphorine isolated from different marine sources, have shown anti-cancer activity, as well as antibiotic and anti-inflammatory properties. Considering the structural correlation between endogenous monoamine serotonin with marine indolic alkaloids 5,6-dibromoabrine and 5,6-dibromotryptamine, a potential use of some dibrominated indolic metabolites in the treatment of depression-related pathologies has also been hypothesized. Due to the potential applications in the treatment of various diseases and the increasing demand of these compounds for biological assays and the difficult of their isolation from marine sources, we report in this review a series of recent syntheses of marine dibrominated indole-containing products.

Synthesis and biological evaluation of new active For-Met-Leu-Phe-OMe analogues containing para-substituted Phe residues.

In the present study, we report synthesis and biological evaluation of the N-Boc-protected tripeptides 4a-l and N-For protected tripeptides 5a-l as new For-Met-Leu-Phe-OMe (fMLF-OMe) analogues. All the new ligands are characterized by the C-terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a-l and the antagonism of 4a-l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF-OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with -CH(3) and -C(CH(3))(3), respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i, containing -F and -I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed.

The cis-4-amino-L-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues.

Endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) is an endogenous tetrapeptide that combines potency and efficacy with high affinity and selectivity toward the µ opioid receptor, the most responsible for analgesic effects in the central nervous system. The presence of the Pro(2) represents a crucial factor for the ligand structural and conformational properties. Proline is in fact an efficient stereochemical spacer, capable of inducing favorable spatial orientation of aromatic rings, a key factor for ligand recognition and interaction with receptors. Here the Pro(2) has been replaced by 4(S)-NH(2)-2(S)-proline (cAmp), a proline/GABA cis-chimera residue. This bivalent amino acid maintains the capacity to influenc the tetrapeptide conformation and offers the opportunity to generate new linear models and unusually constrained cyclic analogues characterized by an N-terminal Tyr bearing a free α-amino group. The results indicate that the new analogues do not show affinity for both δ and κ opioid receptors and bind only poorly to the µ receptors (for cyclopeptide 9: K(i)(µ) = 660 nM; GPI (IC(50)) = 1.4% at 1 µM; for linear tetrapeptide acid 13: K(i)(µ) = 2000 nM; GPI (IC(50)) = 0% at 1 µM; for linear tetrapeptide amide 15: K(i)(µ) = 310 nM; GPI (IC(50)) = 894 nM).

Delivery methods of camptothecin and its hydrosoluble analogue irinotecan for treatment of colorectal cancer.

Camptothecins are a family of alkaloids originally extracted from the Chinese tree Camptotheca acuminata, Nyssaceae, exhibiting a strong activity against colorectal cancer (CRC). CRC is a common malignancy worlwide. Despite significant developments in the treatment of this disease, it still causes considerable morbidity and mortality. Recent advances include both newer cytotoxic chemotherapies and novel biological agents including the more hydrosoluble camptothecin derivative, namely irinotecan. Camptothecin and irinotecan are selective human topoisomerase I inhibitors but their application for curing CRC is compromised by their intrinsic high toxicity, insolubility and instability. Furthermore, pharmacology studies have determined that continuously and prolonged schedules of administration are required. The aim of this work is to review the state of the art of camptothecin and its derivative irinotecan's delivery methods.

CNS delivery of L-dopa by a new hybrid glutathione-methionine peptidomimetic prodrug.

Parkinson's disease (PD) is a neurodegenerative disorder associated primarily with loss of dopamine (DA) neurons in the nigrostriatal system. With the aim of increasing the bioavailability of L: -dopa (LD) after oral administration and of overcoming the pro-oxidant effect associated with LD therapy, we designed a peptidomimetic LD prodrug (1) able to release the active agent by enzyme catalyzed hydrolysis. The physicochemical properties, as well as the chemical and enzymatic stabilities of the new compound, were evaluated in order to check both its stability in aqueous medium and its sensitivity towards enzymatic cleavage, providing the parent LD drug, in rat and human plasma. The radical scavenging activities of prodrug 1 was tested by using both the DPPH-HPLC and the DMSO competition methods. The results indicate that the replacement of cysteine GSH portion by methionine confers resistance to oxidative degradation in gastric fluid. Prodrug 1 demonstrated to induce sustained delivery of DA in rat striatal tissue with respect to equimolar LD dosages. These results are of significance for prospective therapeutic application of prodrug 1 in pathological events associated with free radical damage and decreasing DA concentration in the brain.