A series of novel low-molecular weight bispecific radioligands were developed, which were able to target the prostate-specific membrane antigen (PSMA) and the gastrin releasing peptide receptor (GRPr), both expressed on prostate cancer cells. These bispecific radiotracers combined the peptidomimetic urea-based pseudo-irreversible inhibitor of PSMA: Glu-ureido-Lys with the bombesin (BN) analogue: H₂N-PEG₂-[D-Tyr⁶, β-Ala¹¹, Thi¹³, Nle¹⁴]BN(6–14), which binds to GRPr with high affinity and specificity. The two pharmacophores were linked together through the chelating agent HBED-CC and spacers made of positively charged His (H) and negatively charged Glu (E): -(HE)_n-, (n=0-3) amino acids. The positron emitter ⁶⁸Ga (t_1/2 = 68 min, β⁺ 88 %, E_β+ max. 1.9 MeV) was used for the radiolabelling of the bispecific radioligands and preliminary pharmacological data were collected from in vitro assays on prostate cancer cell lines (PC-3, AR42J, LNCaP) and in vivo experiments in normal and tumor bearing mice (biodistribution and small animal PET imaging studies). The new bispecific ligands in vitro showed binding affinities, which essentially matched the ones of the respective monomers, while in vivo they were able to target both PSMA (LNCaP) and GRPr (PC-3) positive tumors. In addition the charged -(HE)_n-, (n=1-3), linkers improved the tracer’s pharmacokinetics by significantly reducing the normal organ uptake (i.e. kidney and spleen) and by increasing the tumor to-background ratio. In conclusion, the bispecific (PSMA and GRPr) targeting ligands, developed in this study could be considered as novel radiotracer candidates for more sensitive PET/CT-imaging of prostate cancer (PCa) in future clinical application.

A series of 15 alkanediamide-linked bisbenzamidines and related analogs were synthesized and tested in vitro against two Trypanosoma brucei (Tb) strains: T. b. brucei (Tbb) and T. b. rhodesiense (Tbr), two Plasmodium falciparum (Pf) strains: a chloroquine-sensitive strain (NF54) and a chloroquine-resistant strain (K1), Trypanosoma cruzi (Tc), and Leishmania donovani (Ld). The in vitro cytotoxicity was determined against rat myoblast cells (L6). Seven compounds (5, 6, 10, 11, 12, 14, 15) showed high potency toward both strains of Tb and Pf with the inhibitory concentrations for 50% (IC₅₀) in the nanomolar range (IC₅₀ = 1-96 nM). None of the tested derivatives was significantly active against Tc or Ld. Three of the more potent compounds (5, 6, 11) were evaluated in vivo in mice infected with the drug-sensitive (Lab 110 EATRO and KETRI 2002) or drug-resistant (KETRI 2538 and KETRI 1992) clinical isolates of T. brucei. Compounds 5 and 6 were highly effective in curing 100% mice infected with the drug-sensitive strains, including a drug-resistant strain KETRI 2538, but were ineffective against KETRI 1992. Thermal melting of DNA and molecular modeling studies indicate AT-rich DNA sequences in the minor grove as possible binding sites for these compounds. Several of the tested compounds are excellent leads for the development of improved antiparasitic agents.

A series of new quinoline analogues was prepared in two steps. All the synthesized compounds were characterized by IR, NMR and mass spectral data. The anticancer activity was determined as per the standard protocol and LC50, TGI and GI50 were calculated. 1-(7-Hydroxy-4-methyl-2-oxoquinolin-1(2H)-yl)-3-(4-methoxylphenyl)urea (5j) showed maximum anticancer activity with GI50 of 35.1 µM against HeLa (Cervix Cancer Cell Line) and 60.4 µM against MDA-MB-435 (Breast Cancer Cell Line). A molecular docking study implying epidermal growth factor receptor tyrosine kinase (EGFR-TK) was carried out to observe the binding mode of the new quinoline analogues on the active site of EGFR-TK. The compound 5j showed maximum docking score among the series of compounds. The amino acid residues Met793 showed backbone H-bonding with the hydroxyl group, while Asp855 showed side chain H-bonding with aryl NH group.

Recently we have demonstrated that some bis(oxyphenylene)benzimidazoles constituted potential antiplasmodial candidates but they are characterized by a high lipophilic character. To circumvent that drawback, two series of structural analogs have been prepared. In the first series, oxygen atoms have been introduced in the linker separating both pharmacophores. In the second series, the heterocyclic skeletons have been replaced by imidazo[4,5-b]pyridine moieties. The antiplasmodial activity of the newly synthesized compounds has been evaluated against the chloroquine-sensitive strain NF-54.  Their cytotoxicity in the presence of L6 rat skeletal muscle cells has also been determined. Among the active derivatives, 2,2’-[propane-1,3-diylbis(oxy-1,4-phenylène)]bis-1H-imidazo[4,5-b]pyridine emerged as the most promising hit.

     N-substituted benzisoselenazol-3(2H)-ones have been shown to have a broad spectrum of biological activities including anti-inflammatory and antioxidant activity and are believed to be a novel anticancer agents. Ebselen derivatives possess the ability to mimic the capacity of glutathione peroxidase (GPx), an antioxidant enzyme which removes the excess of reactive oxygen species and prevents from oxidative stress.

     The aim of the study was to test the antiproliferative and cytotoxic activity of benzisoselenazolone derivatives and to select those with antitumor activity.

     Prostate cancer cell lines with genetic background (PC-3, DU145) were treated with different concentrations of bensisoselenazolone analogs and corresponding diselenides. The cytotoxity and inhibition of cell proliferation were identified by Sulforhodamine B assay (SRB). The changes in level of Akt were evaluated using Western blot method.

     We observed that among twenty structurally different ebselen derivatives, four of them demonstrated antiproliferative activity at 40 µM concentration. Three of them were more cytotoxic to DU145 cell lines than to PC-3 and this data correlates with basal Akt activity, which is higher in PC-3 cells. On the other hand the cytotoxicity of N-butyl-1,2-benzisoselenazol-3(2H)-one was similar in both cell lines indicating different mode of action compared to other three selenocompounds. In conclusion, our initial data demonstarte the anticancer efficiency of benzisoselenazolones and corresponding diselenides.

Based on the data of compounds known from the literature to be active against various types of Ser/Thr kinases a general pharmachophore model for these types of kinases was developed. Search for the molecules fitting to this pharmacophore among ASINEX proprietary library revealed a number of compounds, which were tested and appeared to possess some activity against such Ser/Thr kinases as Aurora A, Aurora B and Haspin.

Our work on optimization of these molecules to Aurora A kinase allowed us to achieve several hits in 3-5 nM range of activity, with rather good selectivity and ADME properties.

Thus we showed the possibility of performing the fine-tuning of the general Ser/Thr pharmacophore designed to desired types of kinase to get active and selective compounds.

OBJECTIVE. Ebselen (1) is a multifunctional drug with a wide range of pharmacological effects that are predominantly due to its interaction with selenoenzymes, e.g. glutathione peroxidase, thioredoxin reductase [1,2]. Such enzymes play an important role in protecting biomembranes and other cellular components from oxidative stress [2]. Fluorescence-labeled probes containing ebselen can be suitable for further biological and medicinal studies, to profile enzyme activities, identify target enzymes and characterize their functions [3].

SYNTHESIS. The synthetic route starts with the procedure for synthesizing ebselen [4]. Anthranilic acid as the starting material was converted into a diazonium salt, which was treated with disodium diselenide to obtain the 2,2´-diselenobisbenzoate (2). After treating 2 with thionyl chloride, a reaction with an appropriate para-substituted aniline derivative with a protected primary aliphatic amine group was performed to give the first component, compound 3. To obtain the PEG linker for the connection of the two components, the amino group of 2-(2-aminoethoxy)ethanol was first Cbz-protected. Afterwards, the hydroxyl group was alkylated with tert.-butyl bromoacetate followed by a Cbz deprotection. The resulting primary aliphatic amine was coupled with the second component, the fluorescent coumarin 343 (4). This was synthesized by submitting 8-hydroxyjulolidine-9-carboxaldehyde to a Knoevenagel condensation. In the final steps, both the ebselen derivative 3 and the PEG-coumarin 343 building block were deprotected and the desired probe 5 was assembled.

APPLICATION. The new probe will be provided to biochemical and pharmacological studies.

Cysteine cathepsins are lysosomal cysteine proteases which play roles in many physiological processes. Cathepsin F is predominantly expressed in macrophages. Major histocompatibility complex class II molecules (MHC-II) are expressed by antigen-presenting cell types including macrophages, B cells, and dendritic cells. The cleavage of the invariant chain is the key event in the pathway of MHC-II complexes. Cathepsin S was described as the major processing enzyme of the invariant chain, but it was shown that cathepsin F can adopt its role in cathepsin S deficient mice.^[1] Low molecular weight inhibitors for cathepsin F have not been investigated so far. We have chosen the dipeptide nitrile^[2] chemotype to develop covalent-reversible inhibitors for this target.

An active site mapping with a library of 52 nitrile-based cathepsin inhibitors was performed at human cathepsin F to draw structure-activity relationships. With the kinetic data in hand, new compounds with optimized residues in P1, P2 and P3 position were synthesized and evaluated. With all dipeptide nitriles including the newly synthesized derivatives, a 3D activity landscape was generated to visualize similarity-activity relationships of this series of cathepsin F inhibitors.

 

References

[1] Shi GP, Bryant RA, Riese R, Verhelst S, Driessen C, Li Z, Brömme D, Ploegh HL, Chapman HA. J. Exp. Med. 2000, 191, 1177-1186.

[2] Frizler M, Stirnberg M, Sisay MT, Gütschow M. Curr. Top. Med. Chem. 2010, 10, 294-322.

Study of the antiviral action of the abnormal nucleoside analogue 2-(beta-D-ribofuranosyl)-5-amino-1,2,4-triazine-3(2H)-one was performed with Epstein-Barr virus (EBV) positive human B-cells (Raji) superinfected by EBV, as an acute infection model, and EBV-producing tamarin (Saguinus oedipus) B-cell line (B95-8), as a chronic infection model. The antiviral activity of the compound was determined by RT-PCR. It was shown that the IC₅₀ values (concentration of compound that inhibits the accumulation of the viral genome at 50 %) for 2-(beta-D-ribofuranosyl-5-amino-1,2,4-triazine-3(2H)-one were 0.5 ug/ml for the acute infection of EBV infected Raji cells and 3.8 ug/ml for the model of chronic infection. The anti-EBV action of Ganciclovir (the official anti-herpetic drug) was studied for comparison. IC₅₀ values for Ganciclovir were 1.0 ug/ml for the acute model and 6.2 ug/ml for the chronic model of EBV infections. Thus abnormal nucleoside 2-(beta-D-ribofuranosyl)-5-amino-1,2,4-triazine-3(2H)-one is a promising anti-EBV compound deserving further research.

Hepatocellular carcinoma (HCC), is the second most common cause of death from cancer worldwide (745 000 deaths). Since 2008, HCC is the cancer with the highest mortality rate (0.95).^[1] Nowadays, the only systemic treatment that has demonstrated a real benefit in advanced HCC is Sorafenib, but it remains associated with many side effects and this therapy is still very expensive. So, it is desirable to offer a treatment more efficient, and cheaper.

Many studies showed the overexpression in tumor tissue, and especially in HCC cases, of somatostatin receptors in contrast to healthy cells. ^{[2, 3]} That’s why, these kind of receptors are interesting for tumor targeting, either for imaging or for therapy. Indeed, selective localization or destruction of cancer cells by means of such radiolabelled bioconjugates is a simple and attractive concept, based on the use of the recognition properties of biomolecules towards tumour cells (magic bullet concept).^[4] The challenge is to develop radiotracers, so-called radiopharmaceuticals, which consist in a three-parts system including a biomolecule, a bifunctional chelating agent (BCA) and a radioactive isotope which delivers γ or β^- emission. To be efficient, this system must be stable in vivo in order to image and/or irradiate selectively the targeted tumour mass. Consequently, our challenge is to develop a targeting radiopharmaceutical with Rhenium-188 as destructive part, a tripodal N₂O bifunctional chelator and as biovector, a synthetic somatostatin analogue.

 

In this communication, we reported our first results related to the development of a targeting radiopharmaceutical including: (i) the synthesis of original tripodal N₂O BCAs based on a triazolyl moiety, these chelators being synthesised via a click chemistry approach ^[5], (ii) a complete structural study of corresponding non-radioactive tricarbonylrhenium complexes (iii) the first trials of coupling and of ¹⁸⁸Re-labelling of the tripodal ligand (proof of concept).

 

 

 

 

 

References:

^[1]            J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Int. J. Cancer, 2014, 136, E359–E386

^[2]               J. C. Reubi, A. Zimmermann, S. Jonas, B. Waser, P. Neuhaus, U. Läderach, B. Widenmann, Gut, 1999, 45, 766-774

^[3]            H. Reynaert, K. Rombouts, A. Vandermonde, D. Urbain, U. Kumar, P. Bioulac-Sage, M. Pinzani, J. Rosenbaum, A. Geerts, Gut, 2004, 53, 1180-1189

^[4]            C.F. Ramogida, C. Orvig, Chem. Commun., 2013, 49, 4720-4739

^[5]            S. Guizani, N. Malek-Saied, C. Picard, E. Benoist, M. Saidi, J. Label. Compd Radiopharm., 2014, 57, 158-163.