Synthesis and Biological Study of 4-Aminopyridine-Peptide Derivatives Designed for the Treatment of Neurodegenerative Disorders.

BACKGROUND: Alzheimer's disease (AD) and Multiple sclerosis (MS) lead to neurodegenerative processes negatively affecting millions of people worldwide. Their treatment is still difficult and practically incomplete. One of the most commonly used drugs against these neurodegenerative diseases is 4-aminopyridine. However, its use is confined by the high toxicity. OBJECTIVES: The aim of this work is to obtain new peptide derivatives of 4-aminopyridine with decreased toxicity compared to 4-aminopyridine. METHODS: Synthesis was conducted in solution using a consecutive condensation approach. The new derivatives were characterized by melting points, NMR, and Mass spectra. Important ADME (absorption, distribution, metabolism, and excretion) properties have been studied in silico using ACD/Percepta v.2020.2.0 software. Acute toxicity was determined in mice according to a Standard protocol. All new derivatives were tested in vitro for cytotoxic activity in a panel of human (HEP-G2, BV-173) and murine (NEURO 2A) tumor cell lines via a standard MTT-based colorimetric method. ß-secretase inhibitory activity was determined by applying the fluorescent method. RESULTS: New derivatives of 4-aminopyridine containing analogues of the ß-secretase inhibitory peptide (Boc-Val-Asn-Leu-Ala-OH) were obtained. The in vivo toxicity of the tested compounds was found to be as high as 1500 mg/kg. Cell toxicity screening against tumor cell lines of different origins showed negligible growth-inhibitory effects of all investigated 4-aminopyridine analogues. CONCLUSION: Synthesis of new peptide derivatives of 4-aminopyridine is reported. Acute toxicity studies revealed a ca. 150 times lower toxicity of the new compounds as compared to 4-aminopyridine that may be ascribed to their peptide fragment.

Kinetic and structural studies on the inhibition of acetylcholinesterase and butyrylcholinesterase by a series of multitarget-directed galantamine-peptide derivatives.

Complex neurological disorders, including Alzheimer's disease, are one of the major therapeutic areas to which multitarget drug discovery strategies have been applied in the last twenty years. Due to the complex multifactorial etiopathogenesis of Alzheimer's disease, it has been proposed that to be successful the pharmaceutical agents should act on multiple targets in order to restore the complex disease network and to provide disease modifying effects. Here we report on the synthesis and the anticholinergic activity profiles of seven multitarget anti-Alzheimer compounds designed by combining galantamine, a well-known acetylcholinesterase inhibitor, with different peptide fragments endowed with inhibitory activity against BACE-1. A complementary approach based on molecular docking simulations of the galantamine-peptide derivatives in the active sites of acetylcholinesterase and of the related butyrylcholinesterase, as well as on inhibition kinetics, by global fitting of the reaction progress curves, allowed to gain insights into the enzyme-inhibitor mechanism of interaction. The resulting structure-activity relationships pave the way towards the design of more effective pharmacodynamic/pharmacokinetic multitarget inhibitors.

Synthesis and biological study of new galanthamine-peptide derivatives designed for prevention and treatment of Alzheimer's disease.

The Alzheimer's disease leads to neurodegenerative processes and affecting negatively million people worldwide. The treatment of the disease is still difficult and incomplete in practice. Galanthamine is one of the most commonly used drugs against the illness. The main aim of this work is design and synthesis of new derivatives of galanthamine comprising peptide moiety as well as study of their ß-secretase inhibitory activity and the anti-aggregating effect. All new derivatives of galanthamine containing analogues of Leu-Val-Phe-Phe (Aß17-Aß20) were synthesized in solution using fragment and consecutive condensation approaches. The new derivatives were characterized by melting points, NMR, and HPLC/MS. They were tested in vitro for ß-secretase inhibition activity by means of fluorescent method and were investigated in vitro for anti-aggregation activity on sheep platelet-rich plasma. Although the new compounds do not contain a structural element responsible for the ß-secretase inhibition, five of them show high or good ß-secretase inhibitory activity between 19.98 and 51.19% with IC50 between 1.95 and 5.26 nM. Four of the new molecules were able to inhibit platelet aggregation between 55.0 and 90.0% with IC50 between 0.69 and 1.36 µM. Four of the compounds were able to inhibit platelet aggregation and two of them have high anti-aggregating effects.

Effects of New Galantamine Derivatives in a Scopolamine Model of Dementia in Mice.

Alzheimer's disease (AD), a progressive neurodegenerative disorder characterized by memory loss and cognitive functions decline, is a leading cause for dementia and currently ranked as the sixth foremost cause of death. As of present, treatment of AD is symptomatic without convincing therapeutic benefits and new, effective, therapeutic agents are pursued. Due to massive loss of cholinergic neurons and decreased acetylcholine levels, cholinesterase inhibitors like galantamine, remain the backbone of pharmacological treatment of the disease. In the present study, using behavioral and biochemical methods, four newly synthesized galantamine derivatives, Gal 34, Gal 43, Gal 44, and Gal 46, were evaluated for a beneficial effect in a scopolamine model of dementia in mice. They were designed to have all the advantages of galantamine and additionally to inhibit ß-secretase and exert favorable effects on plasma lipids. Behavioral tests included step-through inhibitory avoidance, T-maze, and the hole-board test, whereas biochemical evaluations involved assessment of acetylcholinesterase activity, brain monoamines levels, lipid peroxidation, catalase, glutathione peroxidase, and superoxide dismutase activities along with measurement of total glutathione. Results show that Gal 43, Gal 44, and, in particular, Gal 46 are especially effective in improving both short- and long-term memory and in the case of Gal 46 having a significant effect on exploratory activity as well. Although Gal 34 did not show behavioral effects as convincing as those of the other three galantamine derivatives, it demonstrated persuasive antioxidant and restorative capacities, making all four galantamine derivatives promising AD treatment agents and prompting further research, especially that in many of our studies they performed better than galantamine.

New Galantamine Derivatives with Inhibitory Effect on Acetylcholinesterase Activity.

BACKGROUND: Inhibitors of acetylcholinesterase (AChE) are used to treat many disorders, among which are neurodegenerative upsets, like Alzheimer's disease (AD). One of the limited licensed AChE inhibitors (AChEIs) used as drugs is the natural compound galantamine (Gal). OBJECTIVE: As Gal is a toxic compound, here we expose data about its four derivatives in hybrid peptide-norgalantamine molecules, which have shown 100 times lower toxicity. METHODS: Four newly synthesized galantamine derivatives have been involved in docking analysis made by Molegro Virtual Docker. Biological assessments were performed on ICR male mice. The change in short and long-term memory performance was evaluated by passive avoidance test. AChE activity and levels of main oxidative stress parameters: lipid peroxidation, total glutathione (GSH), enzyme activities of catalase (CAT), superoxide dismutase, and glutathione peroxidase were measured in brain homogenates. RESULTS: Our experimental data revealed that the new hybrid molecules did not impair memory performance in healthy mice. Two of the compounds demonstrated better than Gal AChE inhibitory activity in the brain. None of them changed the level of lipid peroxidation products, one of the compounds increased GSH levels, and all of them increased CAT enzyme activity. CONCLUSION: The new galantamine-peptide hybrids demonstrated a potential for inhibition of AChE and antioxidant activity and deserve further attention.

Neuroprotective effect of newly synthesized 4-aminopyridine derivatives on cuprizone-induced demyelination in mice-a behavioral and immunohistochemical study.

The aim of this study was to assess the effect of newly synthesized derivatives of 4-aminopyridine (4-AP) on cuprizone-induced model of brain demyelination in mice. 4-AP is already approved for the treatment of walking difficulties in patients with multiple sclerosis. The model of demyelination was carried out by the administration of cuprizone to the drinking water of the experimental mice. Besides cuprizone, 4-AP derivatives and 4-AP were administered to the groups in order to assess their protective effect on the demyelination. We used immunohistochemistry for visualization of changes in corpus callosum. Memory storage processes were also assessed with the passive avoidance test on the last two days of the experiment. The experimental mice treated with compounds 4b and 4c increased significantly their latency time on the second day in comparison to the control group which indicated an improved memory process. The number of mature oligodendrocytes in the groups treated with compounds 4b, 4c and 4-AP is closer to those in the control group. The results of our studies showed that the newly synthesized compounds 4b and 4c reverse the effect of cuprizone. These groups also showed increased latency time in the passive avoidance test in comparison to the control group.

Synthesis of New Galanthamine-Peptide Derivatives Designed for Prevention and Treatment of Alzheimer's Disease.

BACKGROUND: Although no effective treatment for the Alzheimer's disease currently exist, some drugs acting as Acetylcholinesterase inhibitors, like galanthamine have positively affected such patients. ß- and/or γ-secretase inhibitors are another type of potential drugs. Here we report synthesis of new peptide-galanthamine derivatives, with expected inhibitory activity against both Acetylcholinesterase and ß-secretase. OBJECTIVES: The aim of this work is obtaining new peptide derivatives of galanthamine with decreased toxicity compared to galanthamine. METHODS: Syntheses were conducted in solution using fragment condensation approach. The new derivatives were characterized by melting points, angle of optical rotation, NMR and Mass spectra. Acute toxicity was determined on mice, according to a Standard protocol. All new compounds were tested in vitro for cytotoxic activity in a panel of human (HEP-G2, BV-173) and murine (Neuro-2a) tumor cell lines via a standard MTT-based colorimetric method. RESULTS: New derivatives of galanthamine containing shortened analogues of ß-secretase inhibitor (Boc- Asn-Leu-Ala-Val-OH) and either nicotinic or isonicotinic residue, both connected with a linker (L-Asp) to position 11 of galanthamine were obtained. In vivo toxicity of some new compounds was found up to 1000 mg/kg. Cell toxicity screening against the tumor cell lines showed negligible growth-inhibiting properties of the galanthamine derivatives. CONCLUSION: Synthesis of new galanthamine derivatives comprising peptide moiety and nicotinic acid or isonicotinic acid is reported. Acute toxicity studies reveal they are about 100 times less toxic than galanthamine. This effect is due to the peptide fragment. Cytotoxicity studies show good correlation with low toxicity results. These results are encouraging for the application of this class compounds as medicines.

Synthesis of New Peptide Derivatives of Galanthamine Designed for Prevention and Treatment of Alzheimer's Disease.

New derivatives of galanthamine containing peptide fragments with ß-secretase inhibitor activity were synthesized. In position 6 of the galanthamine new shortened analogues of ß-secretase inhibitor OM 99-2 (Boc-Val-Asn-Leu-Ala-OH and Boc-Val-Asn-Leu-Ala-Val-OH) were included. The new derivatives of the galanthamine in position 11 including Boc and norgalanthamine in P3 or P4 positions, Val in P2' position and benzylamin in P3'-position were also synthesized. All new peptides were investigated on mice for acute toxicity. The test compounds were administered to mice via intraperitoneal (i.p.) route. They have low toxicity (LD50>1000 mg/kg) after i.p. The compound 11-N-demethyl-11-N-N-[Boc-Asp(Asp-Leu-Ala-Val-NH-Bzl)]-Galanthamine was investigated by two way active avoidance method. The compound has good influence on the conditioned reflexes, which improved the processes of learning and memory. Inhibition activity of newly synthesized compounds was monitored against BuChE and IC50 values are determined. All compounds show activity in micromolar concentration. Compounds 5 and 6 have around 10 times higher activity than galanthamine. Compounds 4 and 9 also show good activity. All newly synthesized compounds show low acute toxicity.

Synthesis and in vitro antitumor activity of new octapeptide analogs of somatostatin containing unnatural amino acids.

Some modified octapeptide analogs of somatostatin with the following structure D-Phe-c(Cys-Phe-D-Trp-Xxx-Yyy-Cys)-Thr-NH2, where Xxx is Lys or Orn and Yyy is Aib (α-aminoisobutyric acid), Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) have been synthesized. The peptides were prepared by standard Fmoc-solid phase peptide chemistry method. The direct disulphide bond formation has been employed on the solid phase by Tl(CF3CO2)3. The cytotoxic effects of the compounds were tested in vitro against a panel of tumor cell lines: HT-29 (human colorectal cancer cell line), MDA-MB-23 (human breast cancer cell line), Hep-G2 (human hepatocellular carcinoma cell line), HeLa (cervical cancer cell line) and normal human diploid cell line Lep-3. The new peptides exhibited different concentration-dependent antiproliferative effect against the tumor cell lines after 24 h treatment. The compounds were most effective to the HT-29 tumor cells. The compound 4C (Orn(5), Aib(6)) demonstrated the most pronounced antiproliferative effects on HT-29 cells with the IC50 = 0.0199 µM.

Synthesis and characterization of new galanthamine derivatives comprising peptide moiety.

New analogues of galanthamine containing peptide fragments either at 6 or 11 position, were synthesized by reaction between galanthamine molecule and dipeptides and tripeptide, derivatives of N-(3,4-dichlorophenyl)-D,L-Ala-OH. The best results according to yields, easily purification of the target products, and simplicity of the scheme realisation was achieved by using of cyanomethyl ester of Boc-Gly-OH as activated compound.

Synthesis of two peptide mimetics as markers for chemical changes of wool's keratin during skin unhairing process.

The sheep skins unhairing process with preliminary alkaline treatment of the wool leads to two unnatural dipeptide mimetics lysinoalanine (Lys(*) - Ala) and ornithinoalanine (Orn(*)- Ala) obtaining. They are result from the keratin hydrolysis process. The changes of wool keratin make it resistant to sulphide degradation. We synthesized and characterized these unnatural dipeptides under the experimental conditions. The structures and mechanism of Lys(*) - Ala and Orn(*)- Ala obtaining were elucidated. The using of newly synthesized products as markers for control of wool's keratin changes during skin unhairing process was demonstrated. The developments have also been the result of economic and environmental pressures to meet environmental regulations.

Investigation of kinetic parameters in vitro of serine proteinases included in the blood coagulation cascade.

During the last years the cases and death due to hemostatic violations exceed that of tumors. Enormous efforts have devoted to the prevention and treatment of some diseases such as arterial thrombosis. Antistasin, a 15 kDa anticoagulant protein isolated from salivary glands of the Mexican leech Haementeria officinalis, has been shown to be a potent inhibitor of Factor Xa in the blood coagulation cascade. Some short analogues which are hybrid structure between isoform 2 and 3 of antistasin and tripeptides inhibitors of serine proteinases were synthesized and reported in our previous work. Inhibitor constants, mechanism, and type of inhibition of some short analogues of antistasin are investigated. These analogs which show high anticoagulant activity in vitro in pure platelet human plasma.

Synthesis and structure-activity relationship of new analogues of antistasin.

Intensive investigation connected with the development of new anticoagulant agents for the treatment of cardiovascular diseases was carried out. Direct and specific inhibition of thrombin and Factor Xa-like serine proteases in the coagulation cascade has been the focus of many efforts to design novel anticoagulants over the past decade. This work reports the synthesis and biological activity of new anticoagulant peptide analogues of natural isoforms 2 and 3 of antistasin. In addition they include different tripeptide sequences in their molecules, which are highly active inhibitors of different serine proteases such as plasmin, trypsin, thrombin and Factor Xa.