Development of Nitric Oxide-Donating Netarsudil Derivatives as a Synergistic Therapy for Glaucoma with Reduced Ocular Irritation.

Based on the synergistic therapeutic effect of nitric oxide (NO) and Rho-associated protein kinase (ROCK) inhibitors on glaucoma, a series of NO-donating Netarsudil derivatives were designed, synthesized, and their activities in vitro and in vivo were evaluated. Among them, (S)-10e released an appropriate amount of NO in aqueous humor in vitro and displayed potent ROCK inhibition. Topical administration of (S)-10e significantly lowered intraocular pressure in an acute ocular hypertension rabbit model and protected retinal ganglion cells in a magnetic microbead occlusion mouse model. A metabolism investigation revealed that (S)-10e released 7a, a metabolite after NO releasing, and 13, an active metabolite of (S)-Netarsudil, in rabbit eyes. Notably, introducing an NO donor moiety attenuated ROCK inhibition-induced ocular irritation in an sGC-independent manner, suggesting that the attenuated conjunctival hyperemia effect of (S)-10e is related to the NO-induced protein S-nitrosation of phosphodiesterase 3A (PDE3A). Overall, (S)-10e is a promising candidate for glaucoma treatment.

Synthesis and Biological Studies on (KLAKLAK)2-NH2 Analog Containing Unnatural Amino Acid ß-Ala and Conjugates with Second Pharmacophore.

(1) Background: Peptides are good candidates for anticancer drugs due to their natural existence in the body and lack of secondary effects. (KLAKLAK)2 is an antimicrobial peptide that also shows good anticancer properties. (2) Methods: The Solid Phase Peptide Synthesis (Fmoc-strategy) was used for the synthesis of target molecules, analogs of (KLAKLAK)2-NH2. The purity of all compounds was monitored by HPLC, and their structures were proven using mass spectrometry. Cytotoxicity and antiproliferative effects were studied using 3T3 NRU and MTT tests, respectively. For determination of antimicrobial activity, the disc-diffusion method was used. Hydrolytic stability at three pH values, which mimic the physiological pH in the body, was investigated by means of the HPLC technique. (3) Results: A good selective index against MCF-7 tumor cell lines, combined with good cytotoxicity and antiproliferative properties, was revealed for conjugates NphtG-(KLAKLAK)2-NH2 and Caf-(KLAKLAK)2-NH2. The same compounds showed very good antifungal properties and complete hydrolytic stability for 72 h. The compound Caf-(KLß-AKLß-AK)2-NH2 containing ß-Ala in its structures exhibited good antimicrobial activity against Escherichia coli K12 407 and Bacillus subtilis 3562, in combination with very good antiproliferative and cytotoxic properties, as well as hydrolytic stability. (4) Conclusions: The obtained results reveal that all synthesized conjugates could be useful for medical practice as anticancer or antimicrobial agents.

New insight into nucleo α-amino acids - Synthesis and SAR studies on cytotoxic activity of ß-pyrimidine alanines.

Three series of the ß-pyrimidine alanines, including willardiine - a naturally occurring amino acid, were prepared from the l-serine-derived sulfamidates. Compounds 3b, 4a and 4b demonstrated antiproliferative activity toward the studied cancer cell lines, albeit the effect of these compounds on human brain astrocytoma MOG-G-CCM cells was more significant than on human neuroblastoma SK-N-AS cells. The cytosine analog of willardiine, compound 4b, reduced viability of MOG-G-CCM cells with EC50 = 36 ± 2 µM, more effectively than AMPA antagonist GYKI 52466. Willardiine showed possible capability of affecting invasiveness of glioblastoma U251 MG cells with no effect on their viability and morphology. Compound 3d, the ethyl ester of willardiine, featured activity toward binding domain hHS1S2I of the GluR2 receptor. Docking analysis revealed that the location mode of compound 3d at the S1S2 domain of hGluR2 (PDB ID: 3R7X) might differ from that of willardiine.

2,3-Diaminopropanols Obtained from d-Serine as Intermediates in the Synthesis of Protected 2,3-l-Diaminopropanoic Acid (l-Dap) Methyl Esters.

A synthetic strategy for the preparation of two orthogonally protected methyl esters of the non-proteinogenic amino acid 2,3-l-diaminopropanoic acid (l-Dap) was developed. In these structures, the base-labile protecting group 9-fluorenylmethyloxycarbonyl (Fmoc) was paired to the p-toluensulfonyl (tosyl, Ts) or acid-labile tert-butyloxycarbonyl (Boc) moieties. The synthetic approach to protected l-Dap methyl esters uses appropriately masked 2,3-diaminopropanols, which are obtained via reductive amination of an aldehyde prepared from the commercial amino acid Nα-Fmoc-O-tert-butyl-d-serine, used as the starting material. Reductive amination is carried out with primary amines and sulfonamides, and the process is assisted by the Lewis acid Ti(OiPr)4. The required carboxyl group is installed by oxidizing the alcoholic function of 2,3-diaminopropanols bearing the tosyl or benzyl protecting group on the 3-NH2 site. The procedure can easily be applied using the crude product obtained after each step, minimizing the need for chromatographic purifications. Chirality of the carbon atom of the starting d-serine template is preserved throughout all synthetic steps.

Discovery of potent and orally bioavailable indazole-based glucagon receptor antagonists for the treatment of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is characterized by chronically elevated plasma glucose levels. The inhibition of glucagon-induced hepatic glucose output via antagonism of the glucagon receptor (GCGR) using a small-molecule antagonist is a promising mechanism for improving glycemic control in the diabetic state. The present work discloses the discovery of indazole-based ß-alanine derivatives as potent GCGR antagonists through an efficient enantioselective synthesis and structure-activity relationship (SAR) exploration and optimization. Compounds within this class exhibited excellent pharmacokinetic properties in multiple preclinical species. In an acute dog glucagon challenge test, compound 13K significantly inhibited glucagon-mediated blood glucose increase when dosed orally at 10 mg/kg.

Exploring Orthogonal Hydrogen Bonding towards Designing Organic-Salt-Based Supramolecular Gelators: Synthesis, Structures, and Anticancer Properties.

A series of primary ammonium monocarboxylate (PAM) salts derived from ß-alanine derivatives of pyrene and naphthalene acetic acid, along with the parent acids, were explored to probe the plausible role of orthogonal hydrogen bonding resulting from amide⋅⋅⋅amide and PAM synthons on gelation. Single-crystal X-ray diffraction (SXRD) studies were performed on two parent acids and five PAM salts in the series. The data revealed that orthogonal hydrogen bonding played an important role in gelation. Structure-property correlation based on SXRD and powder X-ray diffraction data also supported the working hypothesis upon which these gelators were designed. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell migration assay on a highly aggressive human breast cancer cell line, MDA-MB-231, revealed that one of the PAM salts in the series, namely, PAA.B2, displayed anticancer properties, and internalization of the gelator salt in the same cell line was confirmed by cell imaging.

Rationally Developed Organic Salts of Tolfenamic Acid and Its ß-Alanine Derivatives for Dual Purposes as an Anti-Inflammatory Topical Gel and Anticancer Agent.

A new series of primary ammonium monocarboxylate (PAM) salts of a nonsteroidal anti-inflammatory drug (NSAID), namely, tolfenamic acid (TA), and its ß-alanine derivatives were generated. Nearly 67 % of the salts in the series showed gelling abilities with various solvents, including water (biogenic solvent) and methyl salicylate (typically used for topical gel formulations). Gels were characterized by rheology, electron microscopy, and so forth. Structure-property correlations based on single-crystal and powder XRD data of several gelator and nongelator salts revealed intriguing insights. Studies (in vitro) on an aggressive human breast cancer cell line (MDA-MB-231) with the l-tyrosine methyl ester salt of TA (S7) revealed that the hydrogelator salt was more effective at killing cancer cells than the mother drug TA (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay); displayed better anti-inflammatory activity compared with that of TA (prostaglandin E2 assay); could be internalized within the cancer cells, as revealed by fluorescence microscopy; and inhibited effectively migration of the cancer cells. Thus, the easily accessible ambidextrous gelator salt S7 can be used for two purposes: as an anti-inflammatory topical gel and as an anticancer agent.

Synthesis of C-14 radiolabeled glucagon receptor antagonist and its use in a human mass balance study.

The synthesis of the radiolabeled glucagon receptor antagonist 1-[14 C] was accomplished based on decarboxylative iodination of acid 2 followed by "reattachment" of 14 C carboxylic function. The method allowed a significant reduction in the number of steps in preparation of the radiolabeled compound. Iodide 4, obtained by the halodecarboxylation, was converted to cyanide 5-[14 C], which was hydrolyzed to provide the radiolabeled acid 2-[14 C]. Coupling with ß-alanine fragment and hydrolysis of ester 6-[14 C] completed the synthesis of the target molecule 1-[14 C]. The resulting compound was utilized in a mass balance and metabolism study where hepatic oxidation followed by a trace amount of sulfate conjugation and elimination was the main clearance pathway for 1 in humans.

Effective killing of cancer cells and regression of tumor growth by K27 targeting sulfiredoxin.

Cancer cells have been suggested to be more susceptible to oxidative damages and highly dependent on antioxidant capacity in comparison with normal cells, and thus targeting antioxidant enzymes has been a strategy for effective cancer treatment. Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of sulfinylated peroxiredoxins and thereby reactivates them. In this study we developed a Srx inhibitor, K27 (N-[7-chloro-2-(4-fluorophenyl)-4-quinazolinyl]-N-(2-phenylethyl)-ß-alanine), and showed that it induces the accumulation of sulfinylated peroxiredoxins and oxidative stress, which leads to mitochondrial damage and apoptotic death of cancer cells. The effects of K27 were significantly reversed by ectopic expression of Srx or antioxidant N-acetyl cysteine. In addition, K27 led to preferential death of tumorigenic cells over non-tumorigenic cells, and suppressed the growth of xenograft tumor without acute toxicity. Our results suggest that targeting Srx might be an effective therapeutic strategy for cancer treatment through redox-mediated cell death.

Synthesis and biological activity of novel ester derivatives of N(3)-(4-metoxyfumaroyl)-(S)-2,3-diaminopropanoic acid containing amide and keto function as inhibitors of glucosamine-6-phosphate synthase.

A short series of novel ester derivatives of N(3)-4-metoxyfumaroyl-(S)-2,3-diaminopropanoic acid (FMDP) containing amide or keto functions have been designed and synthesized. Their antifungal activity and inhibitory properties toward fungal glucosamine-6-phosphate synthase has also been evaluated. The obtained compounds 11-13 and 15-17 demonstrated good antifungal activity against Candida albicans. Compounds 11-13 displayed also potent inhibitory activity against fungal glucosamine-6-phosphate synthase, comparable to that of FMDP.

The Cytolytic Amphipathic ß(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma.

In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.

Releasable Conjugation of Polymers to Proteins.

Many synthetic strategies are available for preparing well-defined conjugates of peptides/proteins and polymers. Most reports on this topic involve coupling methoxy poly(ethylene glycol) to therapeutic proteins, a process referred to as PEGylation, to increase their circulation lifetime and reduce their immunogenicity. Unfortunately, the major dissuading dogma of PEGylation is that, in many cases, polymer modification leads to significant (or total) loss of activity/function. One approach that is gaining momentum to address this challenge is to release the native protein from the polymer with time in the body (releasable PEGylation). This contribution will present the state-of-the-art of this rapidly evolving field, with emphasis on the chemistry behind the release of the peptide/protein and the means for altering the rate of release in biological fluids. Linkers discussed include those based on the following: substituted maleic anhydride and succinates, disulfides, 1,6-benzyl-elimination, host-guest interactions, bicin, ß-elimination, biodegradable polymers, E1cb elimination, ß-alanine, photoimmolation, coordination chemistry, zymogen activation, proteolysis, and thioesters.

Synthesis, anticandidal activity of N(3)-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic amide derivatives--novel inhibitors of glucosamine-6-phosphate synthase.

Novel FMDP amides 4-6 have been synthesized and tested against Candida strains. The anticandidal activity has been confined only to Candida albicans. Anticandidal activity of the tested amides has correlated with their inhibitory activity of glucosamine-6-phosphate synthase in cell free extract from C. albicans.

Monitoring penetratin interactions with lipid membranes and cell internalization using a new hydration-sensitive fluorescent probe.

A new fluorescent label N-[4'-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-ß-alanine (7AF) was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The 7AF label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms.

Synthesis, characterization and antioxidant activity of some new thiazolidin-4-one derivatives.

AIM: To design new thiazolidin-4-ones derivatives and to evaluate their potential antioxidant effects using in vitro methods. MATERIAL AND METHODS: New ethyl esters of the 2-(R-phenyl)-4-oxo-thiazolidin-3-yl propionic acid were synthesized using "one step reaction" between different aromatic aldehydes, thioglycolic acid and beta-alanine ethyl ester hydrochloride. The antioxidant potential of the synthesized compounds was evaluated using the DPPH radical scavenging assay and phosphomolybdenum method. RESULTS: Eight thiazolidine-4-one derivatives were obtained in good yields and high purity. The structure of the synthesized compounds was confirmed using IR spectroscopy. The evaluation of antioxidant activity showed that 2-[(4-NO2)-phenyl]-4-oxo-thiazolidin-3-yl propionic acid ethyl ester (compound 16) was the most active compound. For this derivative the DPPH radical scavenger activity (I% = 91.63% +/- 0.77) and the total antioxidant capacity (absorbance = 1.0691 +/- 0.0763) were similar with that of ascorbic acid used as standard antioxidant. CONCLUSIONS: The antioxidant activity of the thiazolidine-4-one derivatives depends on the nature of the phenyl ring substituents, the NO2 and OH radicals having the most significant influence.

One-pot efficient synthesis of N(α)-urethane-protected ß- and γ-amino acids.

1-[(4-Methylphenyl)oxy]pyrrolidine-2,5-dione and 1-[(4-methylphenyl)oxy]piperidine-2,6-dione react in a Lossen-type reaction with primary alcohols in the presence of triethylamine to furnish corresponding N(α)-urethane-protected ß-alanine and γ-aminopropionic acid (GABA), respectively, with excellent yields and purities, in an essentially "one-pot" procedure.

Transition metal complexes bearing flexible N3 or N3O donor ligands: reactivity toward superoxide radical anion and hydrogen peroxide.

Mononuclear complexes of N-methylpropanoate-N,N-bis-(2-pyridylmethyl)amine (MPBMPA) and N-propanoate-N,N-bis-(2-pyridylmethyl)amine (HPBMPA) with first row transition metals from Mn to Cu were synthesized and characterized by spectroscopy (infrared, UV-visible), electrochemistry (cyclic voltammetry), microanalysis and in four cases X-ray crystallography. Structure of the complexes revealed high flexibility of these ligands that can adopt facial (Fe) and meridional (Cu) geometry. Activity in the degradation of reactive oxygen species (superoxide radical anion: superoxide dismutase (SOD)-like activity and hydrogen peroxide: catalase-like activity) was tested throughout the complex series in aqueous solutions. In connection with the catalytic dismutation of H(2)O(2), bleaching tests with morin were also conducted in water. Comparison of the two ligands helped in elucidating the possible role of the carboxylate moiety in the different catalytic reactions. Although no general trends could be revealed between reactivity and constitution of the first coordination sphere, plausible explanations for differences are discussed individually for SOD like, catalase-like and bleaching activity.

Designing hybrid foldamers: the effect on the peptide conformational bias of ß- versus α- and γ-linear residues in alternation with (1R,2S)-2-aminocyclobutane-1-carboxylic acid.

Several oligomers constructed with (1R,2S)-2-aminocyclobutane-1-carboxylic acid and glycine, ß-alanine, and γ-amino butyric acid (GABA), respectively, joined in alternation have been synthesized and studied by means of NMR and CD experiments as well as with computational calculations. Results account for the spacer length effect on folding and show that conformational preference for these hybrid peptides can be tuned from ß-sheet-like folding for those containing a C(2) or C(4) linear segment to a helical folding for those with a C(3) spacer between cyclobutane residues. The introduction of cyclic spacers between these residues does not modify the extended ribbon-type structure previously manifested in poly(cis-cyclobutane) ß-oligomers.

Inactivation of glucosamine-6-phosphate synthase by N3-oxoacyl derivatives of L-2,3-diaminopropanoic acid.

N(3)-Oxoacyl derivatives of L-2,3-diaminopropanoic acid 1-4, containing either an epoxide group or a conjugated double bond system, inactivate Saccharomyces cerevisiae glucosamine-6-phosphate (GlcN-6-P) synthase in a time- and concentration dependent manner. The results of kinetics studies on inactivation suggested a biphasic course, with formation of the enzyme-ligand complex preceding irreversible modification of the enzyme. The examined compounds differed markedly in their affinity to the enzyme active site. Inhibitors containing a phenyl ketone moiety bound much more strongly than their methyl ketone counterparts. The molecular mechanism of enzyme inactivation by phenyl ketone compounds 1 and 3 was elucidated by using a stepwise approach with 2D NMR, MS and UV-visible spectroscopy. A substituted thiazine derivative was identified as the final product of a model reaction between an epoxide compound, 1, and L-cysteine ethyl ester (CEE); and the respective cyclic product, found as a result of reaction between 1 and CGIF tetrapeptide, was identical to the N-terminal fragment of GlcN-6-P synthase. On the other hand, the reaction of a double-bond-containing compound, 3, with CEE, CGIF and GlcN-6-P synthase led to the formation of a C-S bond, without any further conversion or rearrangement. Molecular mechanisms of the reactions studied are proposed.

Enantiospecific synthesis of genetically encodable fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid.

Fluorescent unnatural amino acids (UAAs), when genetically incorporated into proteins, can provide unique advantages for imaging biological processes in vivo. Synthesis of optically pure L-enantiomer of fluorescent UAAs is crucial for their effective application in live cells. An efficient six-step synthesis of L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap), a genetically encodable and polarity-sensitive fluorescent UAA, has been developed. The synthesis takes advantage of a high-yield and enantiospecific Fukuyama-Mitsunobu reaction as the key transformation.