Antimalarial activity of 9a-N substituted 15-membered azalides with improved in vitro and in vivo activity over azithromycin.

Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis.

Synthesis, structure-activity relationship, and antimalarial activity of ureas and thioureas of 15-membered azalides.

Azithromycin, a first member of the azalide family of macrolides, while having substantial antimalarial activity, failed as a single agent for malaria prophylaxis. In this paper we present the first analogue campaign to identify more potent compounds from this class. Ureas and thioureas of 15-membered azalides, N''-substituted 9a-(N'-carbamoyl-ß-aminoethyl), 9a-(N'-thiocarbamoyl-ß-aminoethyl), 9a-[N'-(ß-cyanoethyl)-N'-(carbamoyl-ß-aminoethyl)], 9a-[N'-(ß-cyanoethyl)-N'-(thiocarbamoyl-ß-aminoethyl)], 9a-{N'-[ß-(ethoxycarbonyl)ethyl]-N'(carbamoyl-ß-aminoethyl)}, and 9a-[N'-(ß-amidoethyl)-N'-(carbamoyl-ß-aminoethyl)] of 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A, were synthesized and their biological properties evaluated. The results obtained indicate a substantial improvement of the in vitro activity against P. falciparum (up to 88 times over azithromycin), particularly for compounds containing both sugars on the macrocyclic ring and aromatic moiety on 9a-position. The improved in vitro activity was not confirmed in the mouse model, likely due to an increase in lipophilicity of these analogues leading to a higher volume of distribution. Overall, with increased in vitro activity, promising PK properties, and modest in vivo efficacy, this series of molecules represents a good starting platform for the design of novel antimalarial azalides.

Discovery of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides active against efflux-mediated resistant Streptococcus pneumoniae.

A series of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides, were discovered, structurally characterized and biologically evaluated. They have shown good antibacterial activity against selected Gram-positive and Gram-negative bacterial strains. These include N″ substituted 9a-(N'-carbamoyl-γ-aminopropyl)- (6a,c), 9a-(N'-thiocarbamoyl-γ-aminopropyl)- (7a,e), 9a-[N'-(ß-cyanoethyl)-N'-(carbamoyl-γ-aminopropyl)]- (9a-c, 9g) 9a-[N'-(ß-cyanoethyl)-N'-(thiocarbamoyl-γ-aminopropyl)]-derivatives (10d-f) of 5-O-desosaminyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythronolide A (3). Among the synthesized compounds thiourea 7a and urea 9b have shown substantially improved activity comparable to azithromycin (1) and significantly better activity than the 3-decladinosyl-azithromycin (2) and the parent 3-cladinosyl analogues against efflux-mediated resistant S. pneumoniae.

Novel ureas and thioureas of 15-membered azalides with antibacterial activity against key respiratory pathogens.

The new ureas and thioureas of 15-membered azalides, N''-substituted 9a-(N'-carbamoyl-gamma-aminopropyl) (4), 9a-(N'-thiocarbamoyl-gamma-aminopropyl) (6), 9a-[N'-(beta-cyanoethyl)-N'-(carbamoyl-gamma-aminopropyl)] (8) and 9a-[N'-(beta-cyanoethyl)-N'-(thiocarbamoyl-gamma-aminopropyl)] (10) of 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2), were synthesized and structurally characterized by NMR and IR spectroscopic methods and mass spectrometry. The new compounds were evaluated in vitro against a panel of erythromycin susceptible and erythromycin-resistant gram-positive and gram-negative bacterial strains. These compounds displayed an excellent overall antibacterial in vitro activity against erythromycin sensitive gram-positive strains, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, and good against negative strains, Moraxella catarrhalis and Haemophilus influenzae. In addition, several ureas with naphthyl substituents (4f, 4g, 4h) showed better activity in comparison to azithromycin against inducible resistant S. pyogenes. Ureas with naphthyl substituents 4g, 4h and thiourea 8h displayed moderate activity against constitutively resistant S. pneumoniae.

Structural characterization of PEGylated rHuG-CSF and location of PEG attachment sites.

Mass spectrometry structural characterization is an essential tool in validating the quality of PEG-rHu-proteins. However, in either case top-down or bottom-up fashion, the interference of high intensity PEG signals on MS detection or detrimental influence of PEG on protein structure, leads to incomplete structural characterization. We propose here a method that permits complete and reliable structural characterization of PEGylated recombinant human granulocyte-colony stimulating factor (rHuG-CSF). The approach includes on-column 2-methoxy-4,5-dihydro-1H-imidazole derivatization of digested PEG rHuG-CSF and subsequent LC/MS investigation. By comparing the LC/MS retention of derivatized and underivatized digested PEG rHuG-CSF, location of the PEG attachment within rHuG-CSF could be deduced. Besides, the protein sequence coverage and position of the disulfide bridges was fully deducible from the MS data interpretation. Additionally, ultra performance liquid chromatography-mass spectrometry-to-the-E (UPLC-MS(E)) was introduced for analysis of label-free digested PEG rHuG-CSF here to enable high resolution and mass accuracy of MS detection and facilitate deep structural insights of peptides.

Azithromycin-sulfonamide conjugates as inhibitors of resistant Streptococcus pyogenes strains.

Novel hybrid compounds 6a-6d, conjugates of 15-membered azalides and sulfonamides, i.e. unsubstituted, 4-aryl- and 4-heteroaryl-aminosulfonyl derivatives of 9a-[N'-(phenylcarbamoyl)]-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A were synthesized and characterized by IR, one- and two-dimensional NMR spectroscopies and MALDI-TOF and MS/MS mass spectrometry. The new compounds were evaluated in vitro against a panel of sensitive and resistant Gram-positive and Gram-negative bacterial strains. 9a-{N'-[4-(Aminosulfonyl)phenyl]carbamoyl}--(6a) and 9a-{N'-[4-(phenylaminosulfonyl)phenyl]carbamoyl}--(6b) derivatives showed improvements in activity against inducible resistant Streptococcus pyogenes in comparison with macrolide antibiotic azithromycin and starting material 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2). In addition, the synthesized azithromycin-sulfonamide conjugates 6a-6d showed good antibacterial activity against sensitive S. pyogenes and Streptococcus pneumoniae strains. The kinetics of degradation in the artificial gastric juice showed that the most active compounds, 6a and 6b, exhibited azithromycin like stability. The cleavage of the cladinose sugar was found to be the main decomposition pathway leading to inactive 7a and 7b, prepared also as analytical standards by the alternative synthetic route together with 7c and 7d.