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.

The synthesis of indolo[2,3-b]quinoline derivatives with a guanidine group: highly selective cytotoxic agents.

The synthesis of indolo[2,3-b]quinoline derivatives containing guanidine, amino acid or guanylamino acid substituents as well as their in vitro evaluation for the cytotoxic and antifungal activity are reported. The influence of the guanidine group on the selective cytotoxic and hemolytic properties of indolo[2,3-b]quinoline was investigated. Most of the compounds displayed a high cytotoxic activity in vitro and two of the most promising compounds (3 and 12) exhibited a high selectivity between normal and cancer cell-lines. The cytotoxic activity of compound 3 was about 600-fold lower against normal fibroblasts than against A549 and MCF-7 cancer cell lines. Novel entities acted as the DNA-intercalators when tested using a DNA-methyl green assay but demonstrated zero or low hemolytic activity in comparison to their unsubstituted analogs. The mechanism of action was studied for guanidine derivatives 3 and 12 and both compounds were found to be very effective inducers of apoptosis.

A study on the stereochemical purity of trandolapril and octahydro-1H-indole-2-carboxylic acid by HPLC method.

HPLC conditions for the identification of stereoisomers and stereochemical purity of the key intermediate in Trandolapril synthesis, octahydro-1H-indole-2-carboxylic acid, and final drug were elaborated. The chemical and stereochemical purity of synthetic Trandolapril was proved to be as high as 99.3-99.8%, on both non chiral and chiral RP-columns.

99mTc-labeled vasopressin peptide as a radiopharmaceutical for small-cell lung cancer (SCLC) diagnosis.

The 99mTc-labeled conjugates of the vasopressin (AVP) peptide and of its analogue d(CH2)5[D-Tyr(Et2)-Ile4-Eda9]AVP (AVP(an)) have been synthesized using the technetium complexes with tetradentate tripodal chelator (the tris(2-mercaptoethyl)amine (NS3)) and the monodentate isocyanide ligand (CN-peptide). The conjugates exhibit high stability in the presence of 100 times the molar excess of standard amino acids cysteine or histidine and also satisfactory stability in human serum. The 99mTc(NS3)(CN-AVP) and 99mTc(NS3)(CN-AVP(an)) ability of binding to small-cell lung cancer (SCLC) cell line H69 was studied in vitro. The results suggest that the novel vasopressin conjugate 99mTc(NS3)(CN-AVP(an)) is a desirable compound for imaging oncogene receptors overexpressed in SCLC cells and can be an important basis for further consideration the conjugate as a potential diagnostic radiopharmaceutical for patients suffering from small-cell lung cancer.

Synthesis, physicochemical and biological evaluation of technetium-99m labeled lapatinib as a novel potential tumor imaging agent of Her-2 positive breast cancer.

Tumors that are Her-2-positive tend to grow and spread more quickly than other types of breast cancer. Overexpression of Her-2 can be a predictive biomarker for stratification of patients for therapy with Herceptin (containing humanized IgG1 monoclonal antibody trastuzumab) or Tykerb (containing lapatinib di-p-toluenesulfonate) drug. Usually, Her-2 status is determined by immunohistochemical (IHC) as well as fluorescent or chromogenic in situ hybridisation (FISH or CISH) analysis of biopsy material. The objective of the present work was to standardize the conjugation of anti-cancer drug lapatinib (which recognizes selectively the Her-2 extracellular domain) with technetium-99m complex, of type '4+1', to obtain (99m)Tc(NS3)(CN-lapatinib) conjugate for use as in vivo tracer of the Her-2 expression in breast cancer. The conjugate (99m)Tc(NS3)(CN-lapatinib) was formed with high yield, high radiochemical purity and specific activity within the range 25-30 GBq/µmol. The biological in vitro and in vivo studies of the conjugate showed its high affinity to Her-2 receptor (Kd = 3.5 ± 0.4 nM, Ki = 2.9 ± 0.5 nM, Bmax = 2.4 ± 0.3 nM, approximate number of 2.4 × 10(6) binding sites per cell, IC50 = 41.2 ± 0.4 nM) and also pointed out to the clearance through the hepatic and renal route in comparable degree. Basing on these results one can conclude that (99m)Tc(NS3)(CN-lapatinib) conjugate could be a promising radiopharmaceutical for in vivo diagnosis of the Her-2 status in breast with impact on treatment planning.

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.

Physicochemical characteristics of sunitinib malate and its process-related impurities.

In this article, details of crystal and molecular structures of sunitinib malate (SUM), an anticancer therapeutic, and its key synthetic intermediate are presented. Both these compounds were also characterized spectroscopically and thermally. SUM crystallizes in the monoclinic P2(1) space group with two molecules in the asymmetric part of the unit cell, whereas the intermediate crystallises in the triclinic P-1 space group with four independent molecules in the asymmetric unit. The three-dimensional structure of SUM consists of two different layers of molecules. The first one is built of the malic anions, whereas the other layer consists of more hydrophobic sunitinib molecules. This layer is formed by two types of molecules creating a herring bond-like pattern with pairs of neighboring cations forming the V-shape arrangement of molecules. The V-shaped pairs of molecules form ribbons by fitting into two neighboring V shapes. The characteristic V-shape assemble of the moieties is hold together with three C-H…F weak interactions. Besides, process-related impurities of SUM were identified and their structures confirmed by separate synthesis. These impurities were fully characterized by spectroscopic and crystallographic methods as well as by differential scanning calorimetry and thermogravimetric analysis. The H-, (13)C-, and (15)N-nuclear magnetic resonance signals were fully assigned structurally and the resulting structures were confirmed by Fourier-transformed infrared spectroscopy. It was the herein elaborated synthesis of impurity-free SUM that allowed for growing of its single crystals suitable for X-ray crystallographic studies. Comparison of the powder X-ray diffraction pattern for SUM with that derived from single-crystal X-ray crystallographic analysis indicated that SUM formed the polymorph I crystal phase, which is also encountered in its pharmaceutical formulation.

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.