The conjugates of 5'-deoxy-5-fluorocytidine and hydroxycinnamic acids - synthesis, anti-pancreatic cancer activity and molecular docking studies.

New amide conjugates 1-6 of hydroxycinnamic acids (HCA) and 5'-deoxy-5-fluorocytidine (5-dFCR), the prodrug of 5-fluorouracil (5-FU), were synthesized and tested in vitro against pancreatic cancer lines (PDAC). The compounds showed slightly higher efficacy against primary BxPC-3 cells (IC50 values of 14-45 µM) than against metastatic AsPC-1 (IC50 values of 37-133 µM), and similar to that of 5-FU for both PDAC lines. Compound 1, which has a para-(acetyloxy)coumaroyl substituent, was found to be the most potent (IC50 = 14 µM) with a selectivity index of approximately 7 to normal dermal fibroblasts (IC50 = 96 µM). The potential pharmacological profiles were discussed on the basis of the ADME data. Docking to the carboxylesterase CES2 showed that the synthesized compounds have the ability to bind via hydrogen bonding between a specific acetate group of the sugar moiety and Ser228, which belongs to the catalytic triad that causes hydrolysis. Docking to albumin, a major transport protein in the circulatory system, revealed a strong interaction of the conjugates at the binding site which is native to warfarin and responsible for its transport in the body.

The Conjugates of Indolo[2,3-b]quinoline as Anti-Pancreatic Cancer Agents: Design, Synthesis, Molecular Docking and Biological Evaluations.

New amide conjugates of hydroxycinnamic acids (HCAs) and the known antineoplastic 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ), an analog of the natural alkaloid neocryptolepine, were synthesized and tested in vitro for anticancer activity. The compound 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline (2), which contains the ortho-coumaric acid fragment, demonstrated dose-dependent effectiveness against both normal BxPC-3 and metastatic AsPC-1 pancreatic cancer cells. The IC50 values for AsPC-1 and BxPC-3 were 336.5 nM and 347.5 nM, respectively, with a selectivity index of approximately 5 for both pancreatic cancer cells compared to normal dermal fibroblasts. Conjugate 2 did not exhibit any hemolytic activity against human erythrocytes at the tested concentration. Computational studies were performed to predict the pharmacokinetic profile and potential mechanism of action of the synthesized conjugates. These studies focused on the ADME properties of the conjugates and their interactions with DNA, as well as DNA-topoisomerase alpha and beta complexes. All of the conjugates studied showed approximately one order of magnitude stronger binding to DNA compared to the reference DiMIQ, and approximately two orders of magnitude stronger binding to the topoisomerase II-DNA complex compared to DiMIQ. Conjugate 2 was predicted to have the strongest binding to the enzyme-DNA complex, with a Ki value of 2.8 nM.

Synthesis, Biological Activity, ADME and Molecular Docking Studies of Novel Ursolic Acid Derivatives as Potent Anticancer Agents.

A series of new ursolic acid (UA) derivatives substituted with various amino acids (AAs) or dipeptides (DP) at the C-3 position of the steroid skeleton was designed and synthesized. The compounds were obtained by the esterification of UA with the corresponding AAs. The cytotoxic activity of the synthesized conjugates was determined using the hormone-dependent breast cancer cell line MCF-7 and the triple-negative breast cancer cell line MDA. Three derivatives (l-seryloxy-, l-prolyloxy- and l-alanyl-l-isoleucyloxy-) showed micromolar IC50 values and reduced the concentrations of matrix metalloproteinases 2 and 9. Further studies revealed that for two compounds (l-seryloxy- and l-alanyl-l-isoleucyloxy-), a possible mechanism of their antiproliferative action is the activation of caspase-7 and the proapoptotic Bax protein in the apoptotic pathway. The third compound (l-prolyloxy- derivative) showed a different mechanism of action as it induced autophagy as measured by an increase in the concentrations of three autophagy markers: LC3A, LC3B, and beclin-1. This derivative also showed statistically significant inhibition of the proinflammatory cytokines TNF-α and IL-6. Finally, for all synthesized compounds, we computationally predicted their ADME properties as well as performed molecular docking to the estrogen receptor to assess their potential for further development as anticancer agents.

Antioxidant activity of novel diosgenin derivatives: Synthesis, biological evaluation, and in silico ADME prediction.

A series of novel diosgenin (DSG) derivatives has been synthesized and tested in vitro for their antioxidant activity. Initially, four analogues have been evaluated for their cytotoxicity using normal human skin fibroblast (NHDF) as model cells. As a result, 84% of NHDF cells were still alive at 5 µM, so these compounds can be considered as innoxious to fibroblasts at this concentration. Then, hemolytic activity against human erythrocytes was studied in order to evaluate the potential impact of tested compounds against normal host cells. The result < 5% of hemolysis rates suggest no lytic activity for most compounds. After that, the main test - evaluation the antioxidant effect of DSG and its new derivatives against lipid peroxidation in the o/w emulsion model - was performed. The most promising compound (8) exhibited the significant antioxidant activity and the biocompatibility towards normal human dermal fibroblasts and red bloods cells. This p-aminobenzoic derivative revealed 61.6% blocking of induced lipid oxidation. Furthermore, eleven predicted ADME properties were predicted for all tested compounds and revealed that they are in compliance with drug-likeness criteria.

Mucin 1 as a Molecular Target of a Novel Diisoquinoline Derivative Combined with Anti-MUC1 Antibody in AGS Gastric Cancer Cells.

BACKGROUND: The aim of the study was to examine the molecular mechanism of the anticancer action of a monoclonal antibody against MUC1 and a diisoquinoline derivative (OM-86II) in human gastric cancer cells. METHODS: The cell viability was measured by the MTT assay. The disruption of mitochondrial membrane potential and activity of caspase-8 and caspase-9 was performed by flow cytometry. Fluorescent microscopy was used to confirm the proapoptotic effect of compounds. LC3A, LC3B and Beclin-1 concentrations were analyzed to check the influence of the compounds on induction of autophagy. ELISA assessments were performed to measure the concentration of mTOR, sICAM1, MMP-2, MMP-9 and pro-apoptotic Bax. RESULTS: The anti-MUC1 antibody with the diisoquinoline derivative (OM-86II) significantly reduced gastric cancer cells' viability. This was accompanied by an increase in caspase-8 and caspase-9 activity as well as high concentrations of pro-apoptotic Bax. We also proved that the anti-MUC1 antibody with OM-86II decreased the concentrations of MMP-9, sICAM1 and mTOR in gastric cancer cells. After 48 h of incubation with such a combination, we observed higher levels of the crucial component of autophagosomes (LC3) and Beclin-1. CONCLUSIONS: Our study proved that the anti-MUC1 antibody sensitizes human gastric cancer cells to the novel diisoquinoline derivative (OM-86II) via induction of apoptosis and autophagy, and inhibition of selected proteins such as mTOR, sICAM1 and MMP-9.

Unexpected Reaction Products of Uracil and Its Methyl Derivatives with Acetic Anhydride and Methylene Chloride.

New acetyl derivatives of uracil, 6-methyluracil, and thymine were obtained in the course of an unconventional synthesis in methylene chloride. It was shown that products with the acetyloxymethyl fragment are formed according to a mechanism different from that for products with the acetyloxyethyl group. In particular, for uracil it was proven that the reaction with Ac2O, TEA, and CH2Cl2 leads to 1-acetyloxymethyluracil, where the N1 substituent is composed of the -CH2- fragment that originated from CH2Cl2 and the 1-acetyloxy moiety from Ac2O. The reaction of uracil with Ac2O, TEA, CH2Cl2, and DMAP leads to an acetyloxyethyl derivative in which the -CH2-CH2- fragment originates from TEA and the 1-acetyloxy moiety from Ac2O. A possible mechanism for the formation of new compounds was suggested and supported by the density functional theory/B3LYP quantum mechanical calculations. New compounds (39 in total, including seven deuterated) were fully characterized by nuclear magnetic resonance and high-resolution mass spectrometry techniques.

Synthesis of Thiol Derivatives of Biological Active Compounds for Nanotechnology Application.

An efficient method of thiol group introduction to the structure of common natural products and synthetic active compounds with recognized biological efficacy such genistein (1), 5,11-dimethyl-5H-indolo[2,3-b]quinolin (2), capecitabine (3), diosgenin (4), tigogenin (5), flumethasone (6), fluticasone propionate (7), ursolic acid methyl ester (8), and ß-sitosterol (9) was developed. In most cases, the desired compounds were obtained easily via two-step processes involving esterification reaction employing S-trityl protected thioacetic acid and the corresponding hydoxy-derivative, followed by removal of the trityl-protecting group to obtain the final compounds. The results of our preliminary experiments forced us to change the strategy in the case of genistein (1), and the derivatization of diosgenin (4), tigogenin (5), and capecitabine (3) resulted in obtaining different compounds from those designed. Nevertheless, in all above cases we were able to obtain thiol-containing derivatives of selected biological active compounds. Moreover, a modelling study for the two-step thiolation of genistein and some of its derivatives was accomplished using the density functional theory (B3LP). A hypothesis on a possible reason for the unsuccessful deprotection of the thiolated genistein is also presented based on the semiempirical (PM7) calculations. The developed methodology gives access to new sulphur derivatives, which might find a potential therapeutic benefit.

Enigmatic Histamine Receptor H4 for Potential Treatment of Multiple Inflammatory, Autoimmune, and Related Diseases.

The histamine H4 receptor, belonging to the family of G-protein coupled receptors, is an increasingly attractive drug target. It plays an indispensable role in many cellular pathways, and numerous H4R ligands are being studied for the treatment of several inflammatory, allergic, and autoimmune disorders, including pulmonary fibrosis. Activation of H4R is involved in cytokine production and mediates mast cell activation and eosinophil chemotaxis. The importance of this receptor has also been shown in inflammatory models: peritonitis, respiratory tract inflammation, colitis, osteoarthritis, and rheumatoid arthritis. Recent studies suggest that H4R acts as a modulator in cancer, neuropathic pain, vestibular disorders, and type-2 diabetes, however, its role is still not fully understood.

A diversity of alkylation/acylation products of uracil and its derivatives: synthesis and a structural study.

tert-Butyl dicarbonate (Boc2O) and ethyl iodide (EtI) reactions with uracil (U), thymine (T) and 6-methyluracil (6-MU) were performed following routine procedures in pyridine/DMF solvents and with DMAP as the catalyst. Among 20 synthesized compounds, a derivative of 6-methyluracil substituted by the Boc-pyridine moiety at the C5 position appeared unexpectedly. The NMR spectra confirmed the molecular structure of all uracil derivatives. Parallel quantum mechanical DFT calculations supported the experimental findings.

Synthesis and antimicrobial activity of chiral quaternary N-spiro ammonium bromides with 3',4'-dihydro-1'H-spiro[isoindoline-2,2'-isoquinoline] skeleton.

A new class of highly functionalized tetrahydroisoquinolines with a quaternary carbon stereocenter was synthesized starting from an easily accessible L-tartaric acid. Nine strains of bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans, Streptococcus salivarius, Bacillus subtilis, Enterococcus faecalis, Moraxella catarrhalis, Escherichia coli, Campylobacter jejuni) were used for the determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of synthesized compounds. The influence of analyzed compounds on viability and induction of apoptosis in human skin fibroblasts was determined. A majority of the synthesized compounds showed the strongest antibacterial properties toward some gram-negative bacteria (M. catarrhalis and C. jejuni) with a high level of selectivity. High antibacterial compounds have bactericidal activity ratio MBC/MIC ≤4. Our studies also proved that the novel compounds do not possess cytotoxic and proapoptotic potential in normal cells.

Pemetrexed conjugated with gold nanoparticles - Synthesis, characterization and a study of noncovalent interactions.

Gold nanoparticles (AuNPs) have been widely used as nanocarriers in drug delivery application. However, the binding mechanism between AuNPs and drug bases still remains a puzzle. Our study included: (i) optimization of three synthesis of the AuNPs-pemetrexed (PE) nanocomposites formation which was monitored by UV-Vis spectroscopy, (ii) identification of PE in gold nanocomposites and mechanism of PE interaction with gold nanoparticles by electrochemistry, NMR and Raman measurements, (iii) characterization of the three nanocomposites by TEM, DSL, ESL, zeta potential, XRPD and TGA analysis. The obtained nanocomposites are homogeneously shaped and have a maximum diameter of around 14nm and 88nm, as measured by the TEM and DSL techniques, respectively. The zeta potential of the nanocomposites is -43mV and suggests a high stability of the nanoparticles and lower toxicity for the normal cells. Quantum chemical calculations were also performed on model systems to estimate the strength of the AuNPs-PE interaction. Taking into account the experimental and theoretical data a mechanism of the nanocomposites' formation has been proposed in which PE interacts with the gold surface by the COOH/COO- group.

Biological evaluation of octahydropyrazin[2,1-a:5,4-a']diisoquinoline derivatives as potent anticancer agents.

In this study, we evaluated the cytotoxic activity and antiproliferative potency of novel octahydropyrazin[2,1-a:5,4-a']diisoquinoline derivatives (1-7) in MCF-7 and MDA-MB-231 breast cancer cell lines. Annexin V binding assay and disruption of the mitochondrial potential were performed to determine apoptosis. The activity of caspases 3, 8, 9, and 10 was measured after 24 h of incubation with tested compounds to explain detailed molecular mechanism of induction of apoptosis. The results from experiments were compared with effects obtained after incubation in the presence of camptothecin and etoposide. Our study demonstrated that the most active compounds in both analyzed breast cancer cell lines were compounds 3 and 4. We also observed that all compounds induced apoptosis. We demonstrated the higher activity of caspases 3, 8, 9, and 10, which confirmed that induction of apoptosis is associated with external and internal cell death pathway. Our study revealed that the novel compounds in group of diisoquinoline derivatives are promising candidates in anticancer treatment by activation of both extrinsic and intrinsic apoptotic pathways.

New Polymorphic Forms of Pemetrexed Diacid and Their Use for the Preparation of Pharmaceutically Pure Amorphous and Hemipentahydrate Forms of Pemetrexed Disodium.

The preparation of stable amorphous pemetrexed disodium of pharmaceutical purity as well as the process optimization for the preparation of the hemipentahydrate form of pemetrexed disodium are described. Analytical methods for the polymorphic and chemical purity studies of pemetrexed disodium and pemetrexed diacid forms were developed. The physicochemical properties of the amorphous and hydrate forms of pemetrexed disodium, as well as new forms of pemetrexed diacid (a key synthetic intermediate) were studied by thermal analysis and powder X-ray diffraction. High-performance liquid chromatography and gas chromatography methods were used for the chemical purity and residual solvents determination. In order to study the polymorphic and chemical stability of the amorphous and hemipentahydrate forms, a hygroscopicity test (25 °C, 80% RH) was performed. Powder diffraction and high-performance liquid chromatography analyses revealed that the amorphous character and high chemical purity were preserved after the hygroscopicity test. The hemipentahydrate form transformed completely to the heptahydrate form of pemetrexed disodium. Both pemetrexed disodium forms were produced with high efficiency and pharmaceutical purity in a small commercial scale. Amorphous pemetrexed disodium was selected for further pharmaceutical development. Two new polymorphs (forms 1 and 2) of pemetrexed diacid were used for the preparation of high purity amorphous pemetrexed disodium.

Synthesis and physicochemical characterization of the impurities of pemetrexed disodium, an anticancer drug.

A physicochemical characterization of the process-related impurities associated with the synthesis of pemetrexed disodium was performed. The possibility of pemetrexed impurities forming has been mentioned in literature, but no study on their structure has been published yet. This paper describes the development of the synthesis methods for these compounds and discusses their structure elucidation on the basis of two-dimensional NMR experiments and MS data. The identification of these impurities should be useful for the quality control during the production of the pemetrexed disodium salt.

N-terminal guanidinylation of the cyclic 1,4-ureido-deltorphin analogues: the synthesis, receptor binding studies, and resistance to proteolytic digestion.

The synthesis of a series of N-guanidinylated cyclic ureidopeptides, analogues of 1,4-ureido-deltorphin/dermorphine tetrapeptide is described. The δ- and µ-opioid receptor affinity of new guanidinylated analogues and their non-guanidinylated precursors was determined by the displacement radioligand binding experiments. Our results indicate that the guanidinylation of cyclic 1,4-ureidodeltorphin peptide analogues does not exhibit a uniform influence on the opioid receptor binding properties, similarly as reported earlier for some linear peptides. All analogues were also tested for their in vitro resistance to proteolysis during incubation with large excess of chymotrypsin, pepsin, and papain by means of mass spectroscopy. Guanidinylated ureidopeptides 1G-4G showed mixed µ agonist/δ agonist properties and high enzymatic stability indicating their potential as therapeutic agents for treatment of pain.

Antinociceptive effect of D-Lys(2), Dab(4)N-(ureidoethyl)amide, a new cyclic 1-4 dermorphin/deltorphin analog.

BACKGROUND: A preliminary evaluation of antinociceptive activity of a new cyclic dermorphin/deltorphin tetrapeptide analog restricted via a urea bridge and containing C-terminal ureidoethylamid {[H-Tyr-d-Lys(&(1))-Phe-Dab(&(2))-CH2CH2NHCONH2][&(1)CO&(2)]} (cUP-1) revealed a significant and long-lasting increase of pain threshold to thermal stimulation after systemic application. The current studies were aimed at further evaluation of cUP-1 activity in animal models of somatic and visceral pain. The influence of cUP-1 on motor functions was also investigated. METHODS: The influence of cUP-1 (0.5-2mgkg(-1), iv) on nociceptive threshold to mechanical pressure and analgesic efficacy in formalin and acetic acid-induced writhing tests were estimated. The antinociceptive effect of cUP-1 was compared to that of morphine (MF). The influence of cUP-1 (1, 4 and 8mgkg(-1), iv) on locomotor activity, motor coordination and muscle strength was estimated using open field and rota-rod tests and a grip strength measurement. RESULTS: Administration of cUP-1 in doses of 1 and 2mgkg(-1) elicited a significant increase of nociceptive threshold to mechanical pressure. MF applied in the same doses induced an antinociceptive effect only at the higher dose (2mgkg(-1)). There were no marked differences between the effect of cUP-1 and MF at each dose, at relative time points. In the writhing test and both phases of the formalin test, cUP-1 showed a significant, dose-dependent antinociceptive effect which did not markedly differ from that of MF. cUP-1 did not significantly affect motor functions of mice. CONCLUSIONS: Systemic application of cUP-1 elicited a dose-dependent antinociceptive effect. The analgesic efficacy of cUP-1 on mechanical nociception, visceral and formalin-induced pain was comparable to that of MF. cUP-1 did not impair motor functions of mice.

Synthesis, biological activity and resistance to proteolytic digestion of new cyclic dermorphin/deltorphin analogues.

A series of novel cyclic ureidopeptides, analogues of dermorphine/deltorphine tetrapeptide, were synthesized by solid phase peptide synthesis and/or in solution. The antinociceptive activity of N-substituted amides 1-10 was evaluated using hot-plate and tail-flick tests. Analogue 1 showed significant, stronger than morphine, antinociceptive effect after systemic applications. All analogues were also tested for their in vitro resistance to proteolysis by means of mass spectroscopy and it was found that all substituted amides 1-10 showed full stability during incubation with large excess of chymotrypsin and pepsin. Compound 1 is a lead molecule for further evaluation.

Analogues of acyclic nucleosides derived from tris-(hydroxymethyl)phosphine oxide or bis-(hydroxymethyl)phosphinic acid coupled to DNA nucleobases.

A series of novel acyclic nucleoside analogues containing bis-(hydroxymethyl)phosphinic acid (BHPA) or tris(hydroxymethyl)phosphine oxide (THPO) coupled with DNA nucleobases or with 5-fluorouracil were prepared and their antiviral activity was studied against cytomegalovirus (CMV), varicella-zoster virus (VZV), parainfluenza-virus type 3, reovirus-type 1, sindbis, coxsackie B4, punta toro, vesicular stomatitis and respiratory syncytial virus, herpes simplex virus-type 1 (KOS) and type 2 (G), vaccinia virus and herpes simplex virus-1 (TK- KOS ACVr). No specific antiviral effects were noted for any of test compounds against viruses evaluated, except thymine, cytosine and adenine derivatives of BHPA exerting borderline activity against respiratory syncytial virus at the 80 mg/ml concentration.