5'-C-ethyl-tetrazolyl-N 6-substituted adenosine and 2-chloro-adenosine derivatives as highly potent dual acting A1 adenosine receptor agonists and A3 adenosine receptor antagonists

A series of N(6)-substituted-5'-C-(2-ethyl-2H-tetrazol-5-yl)-adenosine and 2-chloro-adenosine derivatives was synthesized as novel, highly potent dual acting hA1AR agonists and hA3AR antagonists, potentially useful in the treatment of glaucoma and other diseases. The best affinity and selectivity profiles were achieved by N(6)-substitution with a 2-fluoro-4-chloro-phenyl- or a methyl- group. Through an in silico receptor-driven approach, the molecular bases of the hA1- and hA3AR recognition and activation of this series of 5'-C-ethyl-tetrazolyl derivatives were explained

Design, synthesis and biological evaluation of novel inhibitors of key enzymes in c-di-GMP metabolism as potential antibiofilm drugs

Bacterial biofilm is responsible for numerous chronic infections, which are characterized by persisting inflammation and tissue damage. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host’s immune defenses, antibiotic therapy and disinfectant chemicals. 3',5'-Cyclic diguanylic acid (c-di-GMP) is a widely conserved second-messenger signal that regulates a wide range of functions including motility, synthesis of virulence factors, adhesion and biofilm formation. In particular, high levels of intracellular cyclic di-GMP promote biofilm formation and sessility, whereas low levels induce biofilm dispersion and motility. Since the pathways involved in c-di-GMP signaling are not present in mammalians, they represent attractive targets for the development of anti-virulence drugs. Synthesis of c-di-GMP occurs via diguanylate cyclase (DGC) enzymes, encoding GGDEF domains, while degradation of c-di-GMP occurs via phosphodiesterase (PDE) enzymes, encoding either an EAL or a HD-GYP domain. The identification of compounds reducing c-di-GMP levels in bacteria would allow the development of new antibiofilm drugs.\ud In my PhD thesis, an array of c-di-GMP-based molecules has been synthesized and tested on both DGC and PDE enzymes, in particular RocR (PDE), WspR and YfiN (DGCs) from P. aeruginosa, and PleD (DGC) from C. crescentus. Three series of compounds have been rationally designed as linear c-di-GMP analogues using a molecular simplification strategy and a click chemistry method. In particular, a unique scaffold was identified, where two guanine bases and their derivatives were directly linked by a triazole moiety. The most potent inhibitor of the series resulted to inhibit PleD (89%, at 100 μM inhibitor) and RocR (59%, at 100 of μM inhibitor) with IC50 values of 17.5 +- 1.1 μM and 66.3 +- 1.3 μM, respectively. The novel inhibitors possess a number of properties that are attractive from a drug development point of view, compared with other c-di-GMP analogues. They are low-molecular-weight compounds, stable and easily synthesizable. More importantly, this study allowed us to identify the minimal structural determinants required for specifically targeting the I-site of DGCs. Although none of these compounds were able to inhibit biofilm formation in vivo, they could become potential tools for mechanicistic biochemical studies, in particular for hybrid protein which contains both the GGDEF and EAL domains.\ud During my research experience at the Institute of Organic Chemistry at the Department of Chemistry of University of Hamburg (Germany), I was involved in a project comprising the stereoselective synthesis of new iso-carbocyclic nucleoside analogues for the further study of their bioactivity in the context of anti-HIV and anti-HCV inhibition. It is widely known that carbocyclic nucleoside analogues possess important biological advantages compared to natural nucleosides and they have received much attention as potential antiviral drugs over the last few decades. Carbovir, abacavir and carba-dT are examples of these efforts, resulting in efficient inhibitors of HIV’s reverse transcriptase. A convergent approach was used for the synthesis, offering the formation of a variety of nucleoside analogues by the direct coupling of a chiral cyclopentanol with purine and pyrimidine nucleobases. These compounds are currently under investigation as potential antiviral drugs

STEREOSELECTIVE SYNTHESIS OF ISO-CARBOCYCLIC NUCLEOSIDE ANALOGUES AS POTENTIAL ANTIVIRAL DRUGS

ABSTRACT A series of iso-carbocyclic nucleoside analogues were prepared starting from a chiral cyclopentanol, by using a convergent approach. In the key step, an enantiomerically pure cyclopentanol was condensed with N3- protected pyrimidine nucleobases using a modified Mitsunobu protocol. INTRODUCTION Over the last few decades, carbocyclic nucleosides represent an attractive approach for the development of new antiviral drugs. Due to their hydrolytic and enzymatic stability in comparison to the natural nucleosides they show important biological properties. Carbocyclic nucleoside analogues like carbovir and entecavir were found to be potent inhibitors of HIV´s reverse transcriptase and recently the carbocyclic 2’- deoxythymidine analogue carba-dT has shown high in vitro activity against HIV replication (Fig.1). This compound shows a unique mechanism of inhibition termed delayed chain termination, resulting in the blocking of DNA synthesis.[ 1] Moreover, the synthesis of carba-iso-dT was reported. This isomer showed a 20-fold decrease of the antiviral activity (EC50 = 10 M) compared to carba-dT, but surprisingly without exhibiting any cytotoxicity.[2]On the basis of these results, the interest is directed to the investigation of new carbocyclic nucleosides against the hepatitis C virus (HCV), a RNA virus that uses a RNAdependent RNA-polymerase (RdRp) for the viral replication. The current standard treatment for HCV-infections is a combination of subcutaneous interferon- with oral admission of the nucleoside drug ribavirin. Because of the low response rates as well as toxic side effects, these therapies are inadequate. Moreover, there is no established vaccineagainst HCV and there is an urgent need for improved therapeutic agents that effectively combat chronic HCV infection. RESULTS AND DISCUSSION We report on the synthesis of a series of iso-carbocyclic nucleoside analogues as potential inhibitors of RdRp (Fig.2). A convergent approach was used for the synthesis, offering the formation of a variety of nucleoside analogues by the direct coupling of a cyclopentanol derivative and different heterocyclic bases. We decided to start from enantiomerically pure (1R,2S)- 2-(benzyloxymethyl)cyclopent-3-enol, which was used for the preparation of a cyclopentanol derivate. The coupling with N3-protected pyrimidine nucleobases under standard Mitsunobu conditions presented a critical step; unfortunately, the nucleobases react as ambident nucleophiles, leading to a mixture of N1- and O2-isomers. In order to maximizethe reaction yields and to control the regioselective coupling, different reactions conditions were tested [3] and the results will be presented. Figure 2. Structures of the new iso-carbocyclic nucleosides. CONCLUSION We successfully synthesized new iso-carbocyclic nucleoside analogues. These will be evaluated as potential antiviral compounds. REFERENCES 1. Boyer, P.L., Vu, B.C., Ambrose Z., Julias, J.G., Warnecke, S., Liao, C., Meier, C., Marquez, V.E., Hughes, S.H. J. Med. Chem. 2009, 52, 5356-5364. 2. Ludek, O.L., Krämer, T., Balzarini, J., Meier, C. Synthesis 2006, 8, 1313-1324. 3. Radi M., Rao J.R., Jha A.K, Chu C.K. Nucl. Nucl. &Nucl. Acids 2009, 28, 504–518

Novel Inhibitors of Inosine Monophosphate Dehydrogenase as Potential Anti-Cancer Drugs: A Patent Review (2002-2014)

Inosine monophosphate dehydrogenase (IMPDH), an NAD-dependent enzyme that controls de novo synthesis of guanine nucleotides, has received considerable interest in recent years as an important target enzyme, not only for the discovery of anticancer drugs, but also for antiviral, antiparasitic, and immunosuppressive chemotherapy. The field of IMPDH inhibitor research is highly important for providing potential therapeutics against a validated target for disease intervention. This patent chapter examines the chemical structures and biological activities of recently reported IMPDH inhibitors. Patent databases SciFinder and Espacenet and Delphion were used to locate patent applications that were published between January 2002 and November 2013, claiming chemical structures for use as IMPDH inhibitors. From 2002 to 2014, around 64 primary patent applications have claimed IMPDH inhibitors, which we analyzed by target and applicant. The level of newly published patent applications covering IMPDH inhibitors remains high and a diverse range of scaffolds has been claimed

Design, synthesis and biological evaluation of novel inhibitors of NAD kinase

Nicotinamide adenine dinucleotide kinase (NADK) is an ubiquitous enzyme that catalyzes a magnesium-dependent phosphorylation of the 2’-hydroxyl group of the adenosine ribose moiety of NAD using ATP or inorganic polyphosphates as phosphoryl donors to produce NADP. To our knowledge this reaction represents the only route leading to de novo NADP biosynthesis, which is a crucial pathway for many cellular processes in living organism. Particularly it was demonstrated that NAD kinase is essential for the survival of Mycobacterium tuberculosis, becoming an appealing new target for the development of potential drugs against multi-drug resistant and extensively drug resistant tuberculosis (TB). With the recent discovery of NAD analogues as molecular probes, a number of nucleobase and sugar modified analogues have been synthesized, such as diadenosine disulfide (DTA) (1), and they were found to be moderate inhibitors of Mycobacterium tuberculosis and human NADKs. Locking the conformation of adenine moiety to syn by substitution with a bulky bromine atom at C8 in one or two adenine rings of DTA, the NADK inhibitory activity has been improved.1 On this basis, we report the synthesis and the inhibitory activity of NAD analogues modified in the sugar and adenine moiety, maintaining the short di-sulfur linker instead of the pyrophosphate linkage of NAD. The results of this study will be presented. 1Petrelli, R.; Cappellacci, L. et al. Bioorg. Med. Chem., 2009, 17(15), 5656-5664

IDENTIFICATION OF MOLECULES THAT INHIBITS c-di-GMP SYNTHESIS TO TARGET BIOFILM FORMATION

Bacteria are able to organize in organized communities named biofilms, difficult to eradicate, highly resistant to antimicrobials and to the host immune system. The scientific challenge is thus to find new therapeutic options that specifically target bacteria growing in biofilms. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger and has a central role in modulation of several cellular processes, including the switch between planktonic and biofilm-related lifestyle. As this signalling system is found only in bacteria, targeting c-di-GMP metabolism represents thus an attractive strategy for the development of antibiofilm drugs. C-di-GMP levels are modulated by the opposite activity of diguanylate cyclase (DGCs), which catalyze its synthesis and phosphodiesterases (PDEs), which hydrolyze it. In order to provide a novel tool in treating drug-resistant bacterial infections, we are testing several compounds that target selectively c-di-GMP synthesis in vitro. We undertook two parallel and multidisciplinary approaches targeting active site by in silico screening of a small molecule database using known structure of DGC (PleD from C. crescents) and targeting inhibitory site by chemical synthesis of c-di-GMP analogues. Our results identify, by each approach, at least 2 compounds able to significatively decrease DGC activity. This promising results pave the way to test more compounds both in vitro end in vivo in human pathogens

Novel Inhibitors of Inosine Monophosphate Dehydrogenase in Patent Literature of the Last Decade

Inosine monophosphate dehydrogenase (IMPDH), an NAD-dependent enzyme that controls de novo synthesis of guanine nucleotides, has received considerable interest in recent years as an important target enzyme, not only for the discovery of anticancer drugs, but also for antiviral, antiparasitic, and immunosuppressive chemotherapy. The field of IMPDH inhibitor research is highly important for providing potential therapeutics against a validated target for disease intervention. This patent review examines the chemical structures and biological activities of recently reported IMPDH inhibitors. Patent databases SciFinder and Espacenet and Delphion were used to locate patent applications that were published between January 2002 and July 2012, claiming chemical structures for use as IMPDH inhibitors. From 2002 to 2012, around 47 primary patent applications have claimed IMPDH inhibitors, which we analyzed by target and applicant. The level of newly published patent applications covering IMPDH inhibitors remains high and a diverse range of scaffolds has been claimed

Synthesis and biological evaluation of (methylene)bisphosphonate derivatives of 2’-C-methyl-, and 3’-C-methyl-adenosine

P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2X receptors (seven types; P2X1-7) contain intrinsic pores that open by binding with ATP, and P2Y receptors (eight types; P2Y1,2,4,6,11-14) are activated by nucleotides and couple to intracellular second-messenger systems through heterotrimeric G-proteins. The P2Y1 and P2Y2 receptors are expressed in most human tissues including the heart, placenta, vascular endothelia, prostate, ovary, platelets and brain. They are attractive pharmaceutical targets due to their involvement in the modulation of various functions in many tissues and organs under both normal and pathophysiological conditions. P2Y1 agonists may have potential as antihypertensive or antidiabetic agent. P2Y2 receptor is a target in therapeutics of pulmonary diseases such as cystic fibrosis, and ophtalmic diseases. P2Y1 receptor is activated by ADP, while P2Y2 is activated equipotently by both ATP and UTP (1). Recently, we have synthesized the 2’- and 3’-C-methyl derivatives of ADP and evaluated their capacity to promote hP2Y1 and hP2Y2 receptor-mediated activation of PLC at recombinant human receptors expressed in atrocytoma cells. From the functional assay 2’-C-methyl-ADP resulted a full agonist at P2Y1 receptor, while 3’-Cmethyl- ADP acted as partial agonist at P2Y2 receptor. Having a nucleotide scaffold, these compounds suffer from limitations due their chemical and metabolic instability. One of the approaches to overcome the inherent instability of nucleotide-based drug candidates include the use of isoster-based non-hydrolyzable nucleotides. Based on these considerations, the (methylene)bisphosphonate derivatives of 2’-Cmethyl-, and 3’-C-methyl-adenosine were synthesized and evaluated for their capacity to promote hP2Y1 and hP2Y2 receptor-mediated activation of PLC at recombinant human receptors expressed in astrocytoma cells. The results of the functional assay will be discussed

SINTESI E VALUTAZIONE BIOLOGICA DI INIBITORI DI ENZIMI CHIAVE NEL METABOLISMO DI BIOFILM BATTERICI

I biofilm sono comunità strutturate di cellule batteriche racchiuse in una matrice polimerica autoprodotta, contenente polisaccaridi (EPS), proteine e DNA ed adesa ad una superficie inerte o vivente. I biofilm differiscono significativamente dalle cellule planctoniche per la loro fisiologia, l’espressione genica e persino la morfologia e risultano 1000 volte più resistenti agli antimicrobici convenzionali [1]. I biofilm batterici sono responsabili di molte infezioni persistenti e croniche, come endocardite, otite media, polmonite associata a fibrosi cistica (CF) e della contaminazione di dispositivi medici, costituendo un problema cruciale in ambito medico, ambientale e tecnologico. Pertanto, l’obiettivo principale della ricerca è quello di identificare nuovi composti mirati a contrastare la formazione del biofilm oppure a promuoverne la dispersione. L’acido bis(3',5')-diguanilico ciclico (c-di-GMP) è un’importante molecola signaling, presente esclusivamente nei batteri, che controlla un'ampia gamma di processi cellulari coinvolti nella formazione del biofilm, come la motilità, l'adesione cellulare ed il differenziamento [2]. Il c-di-GMP intracellulare promuove la formazione del biofilm e sopprime la motilità ad elevate concentrazioni, mentre il c-di-GMP extracellulare ad alte concentrazioni sopprime la formazione del biofilm. Il c-di-GMP è sintetizzato dalle diguanilato ciclasi (DGC) a partire da due molecole di GTP e degradato da fosfodiesterasi specifiche (PDE), che producono 5'pGpG o GMP [2]. Dato il ruolo chiave del c-di-GMP nella formazione del biofilm batterico e dato che la maggior parte delle DGC sono soggette all’inibizione allosterica da prodotto, la sintesi di composti strutturalmente correlati al c-di-GMP rappresenta una strategia interessante per lo sviluppo di nuovi farmaci anti-biofilm [3]. In questo lavoro di ricerca sono stati sintetizzati analoghi dinucleosidici aciclici (seco) del c-di-GMP, nei quali il ponte fosfato è stato sostituito da un linker 1,2,3-triazolico come gruppo non ionico, conformazionalmente rigido ed isostere del linker fosfato. I composti sintetizzati sono stati testati nei confronti delle proteine bersaglio DGC e PDE da Pseudomonas aeruginosa, uno degli agenti principali delle infezioni ospedaliere mediate da biofilm nell’uomo ed i risultati di tale studio verranno discussi

NOVEL 2-SUBSTITUTED 2’/3’-C-METHYL-ADENOSINE DERIVATIVES: SYNTHESIS AND BIOLOGICAL EVALUATION AGAINST TRYPANOSOMA BRUCEI

Human African trypanosomiasis (HAT), which is also known as sleeping sickness, is a devastating parasitic disease that affects more than 300000 people of sub-Saharan Africa each year. The causative agent of this affliction is the protozoan Trypanosoma brucei, which is introduced in the mammalian host by the tsetse fly. T. brucei attacks the central nervous system leading to dementia, epileptic attacks, coma, and, if left untreated, death. Current treatment for this disease, including suramin, pentamidine, melarsoprol, and difluoromethylornithine (DFMO), is often antiquated, highly toxic and frequently ineffective. Therefore, new highly effective and not toxic drugs are needed. Like most obligate intracellular parasites, T. brucei has lost the capacity to synthesize purines de novo and depends on the salvage pathway of nucleosides from the body fluids of the host. Bloodstream T. brucei can take up different types of purines and interconverts them into essential cellular nucleotides. Cordycepin (3’-deoxyadenosine) is an adenosine derivatives able to cure mice inoculated with the human pathogenic T. brucei even after parasites have penetrated into the brain, but requires co-administration with the adenosine deaminase (ADA) inhibitor coformycin to prevent deamination. However, the toxicity of coformycin has stimulated the search of adenosine analogues active against the parasite, but resistant to ADA. In our previous work, we found that the introduction of a methyl group in position 2’- or 3’- of the sugar moiety of adenosine (2’-C-methyladenosine and 3’-C-methyladenosine, respectively) confers a certain grade of resistance to ADA.(1) In fact, 3’-MeAdo is resistant to ADA, while 2’-MeAdo is deaminated by ADA even though the rate of deamination was 1/25 that observed with adenosine. Moreover, some 2, N6-disubstituted adenosine analogs have been reported to show antitrypanosomal activity.(2) Based on these findings a new series of 2-substituted-2’-C-methyl-, and 3’-C-methyl-adenosine derivatives were synthesized and tested for their antiprotozoal activity. The results of this study will be discussed. (1) Franchetti, P, et al. J Med Chem 1998, 41, 1708-1715; Franchetti, P, et al. J Med Chem 2005, 48, 4983-4989; Cappellacci, L, et al. J Med Chem 2008, 51, 4260-4269. (2) Rodenko, B, et al. Antimicrob. Agents Chemother. 2007, 51, 3796-3802