Antibacterial Activity of Novel Sulfonylureas, Ureas and Thioureas of 15-Membered Azalides

One of the 20th century's significant achievements is a discovery of azithromycin (1) and its development to commercial product for effective treatment of various infective diseases. Owing to its exceptional therapeutic and biopharmaceutical properties, it has come to be one of the most successful antibiotics worldwide. For the discovery of azithromycin, in addition to receiving numerous awards, in the year 2000, PLIVA's scientists Slobodan Djokic and Gabrijela Kobrehel together with the representatives from the US-based Pfizer, Gene Michael Bright and Arthur E. Girard, (Anonymous, 2000) were granted the honourable titles of "Heroes of Chemistry" by the American Chemical Society (ACS), a non-profit association of American chemists and chemical engineers, and the largest association of scientists in the world. This prestigious award is taken to be also recognition of the achievement of PLIVA's entire team working on azithromycin. The success of azithromycin has positioned PLIVA among the few pharmaceutical companies in the world that have developed their own blockbuster drug, and has entitled Croatia to join a small group of nations that have developed a new antibiotic.


Introduction
One of the 20th century's significant achievements is a discovery of azithromycin (1) and its development to commercial product for effective treatment of various infective diseases. Owing to its exceptional therapeutic and biopharmaceutical properties, it has come to be one of the most successful antibiotics worldwide. For the discovery of azithromycin, in addition to receiving numerous awards, in the year 2000, PLIVA's scientists Slobodan Djokic and Gabrijela Kobrehel together with the representatives from the US-based Pfizer, Gene Michael Bright and Arthur E. Girard, (Anonymous, 2000) were granted the honourable titles of "Heroes of Chemistry" by the American Chemical Society (ACS), a non-profit association of American chemists and chemical engineers, and the largest association of scientists in the world. This prestigious award is taken to be also recognition of the achievement of PLIVA's entire team working on azithromycin. The success of azithromycin has positioned PLIVA among the few pharmaceutical companies in the world that have developed their own blockbuster drug, and has entitled Croatia to join a small group of nations that have developed a new antibiotic.
Nowadays, on the occasion of the 30 th anniversary of azithromycin's invention  an increasing prevalence of antibiotic-resistant pathogens suggests that we deeply entered into a "Post-Antimicrobial Era" (Cohen 1992;Travis 1994;Kirst 1996b). Investment in newer anti-infective platforms is essential and urgent in order to achieve a significant progress in our understanding of bacterial resistance and new approaches how to control it.
Macrolides as polyketide class of natural products have a long history as effective therapeutic agents for treating infectious diseases (Schönfeld & Kirst, 2002; G.T. T. Kaneko et al., 2006). The popularity of this class of antibiotics, inhibiting bacterial protein synthesis by interfering with ribosome function, is largely due to their spectrum of activity and their relative safety. They are still in the centre of interest of many research groups from academic institutions and pharmaceutical companies and much effort is directed toward the discovery of new macrolide antibiotics by chemical modification of the existing classes of natural derivatives (Sunazuka et al. 2002;Pal 2006). Antibacterial macrolides have attracted considerable attention for two main reasons: (a) the emergence of atypical and/or new pathogens and extensive clinical application of these antibiotics had resulted in an increasing emergence of bacterial resistance, especially among macrolideresistant Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus strains, and, therefore, the development of alternative antibacterial agents became essential; (b) macrolide derivatives, especially 14-and 15-membered classes, have also become interesting for treating important chronic diseases, that is, asthma, chronic sinusitis, diffuse panbronchiolitis, cystic fibrosis (Čulić, 2001;Labro, 2000;Labro, 2004), bronchiolitis obliterans syndrome (BOS) (Vanaudenaerde et al., 2008;Culic et al., 2006), etc. Some macrolides proved active in treatment of malaria Kuschner et al., 1994;Andersen et al., 1995;Ohrt et al., 2002;Sidhu et al., 2007) and cancer (Romano et al., 2004;Oyelere et al. 2009;Mwakwari et al. 2010;Bao et al., 2010), showed antiparasitic activity (Lee et al. 2011) or act as motilides, ie. macrolides with gastrointestinal motor stimulating activity (Takanashi et al. 2009).
Azithromycin was, beside clarithromycin, the leader of the second-generation of macrolides, the first representative of new series of macrolides termed "azalides" ( Schönfeld & Mutak 2002;Mutak 2007), and today the golden standard for macrolide antibiotics (Spaventi 2002).
Azithromycin has broad spectrum of activity against all relevant bacteria causing respiratory tract infections, including Haemophilus influenzae and Moraxella catarrhalis (Mutak, 2007). It also possesses excellent safety and tolerability profiles and is widely prescribed for the treatment of upper and lower respiratory tract infections (Kirst, 1996a;Girard et al., 1987;Schönwald et al, 1991;Retsema et al., 1986). The greatest advantages of azithromycin compared to other macrolide antibiotics are its unusual pharmacokinetics: high tissue distribution and metabolic stability. These properties have led in recent years to the widespread use of the azalide scaffold for synthesis of new antibacterial active compounds with advantageous pharmacokinetics. The growing resistance to antibiotics conferred by microorganisms commonly involved in respiratory tract infections has become a serious clinical problem (Prieto et al., 2002). The widespread use of macrolides has contributed to the increase of resistance within Streptoccoccus pyogenes and Streptoccoccus pneumoniae strains and its level varies worldwide, with an alarming upper rate of 25% in some European countries (Granizo et al., 2000;Szczypa et al., 2000;Nagai 2002 et al.,;Albrich et al., 2004). Gram-positive S. pyogenes and S. pneumonia is the most common bacterial strains implicated in acute pharyngitis, skin and soft tissue infections and also one of the most problematic respiratory pathogen (Cunningham et al., 2000).
It has been shown that the resistance to macrolide antibiotics in Gram-positive microorganisms can be attributed to two main mechanisms: target site modification and active efflux (Nakajima et al., 1999). It is known that macrolides exert their activity by binding to the large 50S ribosomal subunit. They inhibit bacteria protein synthesis at peptidyl transferase center by blocking the nascent peptide exit tunnel (Poehlsgarrd & Douthwaite , 2003). The modification of specific rRNA bases can prevent macrolides to bind. This may be due to the action of methylases encoded either by erm(B) or erm(A) genes (Weisblum, 1998). The methylases are responsible for developing macrolide, lincosamide and streptogramin B (MLS B ) resistance; inducible-(iMLS) or constitutive (cMLS). The active drug efflux is another common type of resistance developed by bacteria and is mediated by the membrane-associated pump encoded by the mef(A) gene (Sutcliffe et al. 1996). In order to overcome the resistance problems, lots of efforts have been made worldwide to search for novel and more potent agents with all of the desirable features of the earlier generation of macrolides.
In this paper, we present the short overview leading to the discovery of novel sulfonylureas, ureas and thioureas of 15-membered azalides as a new class of compounds and their antibacterial activity against some key erythromycin resistant pathogens. Structural features that guided design of novel macrolides included (1) a properly attached aryl/heteroarylcarbamoyl group for improving activity against MLS B resistance and (2) cleavage of cladinose sugar and ketolide backbone for improving potency and activity against efflux resistance. It was expected that introduction of unsaturated unit, that is, carbamoyl group, on nitrogen at position 9a of 1 ( Fig. 1) will significantly change electronic properties and also steric environment in the 'upper part' of the macrolide. It will also serve as an excellent linker for the attachment of various groups affording preparation of a library of compounds with the goal of identifying novel bacterial inhibitors.
Several N-derivatisation lines of azalides skeleton were started in PLIVA in the early 1990s (Schönfeld & Mutak 2002;Mutak 2007), and some of the synthesized compounds showed antibacterial activity. In that respect, the observed activity of initially prepared 9a-Ncarbamoyl and N-thiocarbamoyl derivatives of 2  encouraged us to extend our study in this direction.
Thus, a series of new sulfonylurea, urea and thiourea derivatives of 15-membered azalides were prepared in order to study whether antibacterial activity toward resistant strains would be achieved by introduction of aryl-sulfonylcarbamoyl/carbamoyl/thicarbamoyl group into the azalide molecule and how the activity would be affected by nature and position of the substituents in the phenyl ring (Bukvić Krajačić et al., 2005). Of particular interest was to study the influence of the linker between sulfonylcarbamoyl/carbamoyl/thicarbamoyl-group and aglycon moiety on the antibacterial activity. A special attention was paid to achieving the activity against S. pyogenes and S. pneumonia resistant strains. Scheme 1. Synthesis of sulfonylureas 3, 5 and 7.
Azalide-sulfonamide conjugates 10a and 10b possess two to three times better activity against iMLS resistant S. pyogenes strain (MIC 2 µg/ml) when compared to both azithromycin 1 (MIC 8 µg/ml) and starting cyclic amine 2 (MIC 16 µg/ml) (Bukvić Krajačić et al., 2007). These activities are comparable to those observed for azalide sulfonylureas 3a-3f (Bukvić Krajačić et al., 2005). New azithromycin-sulfonamide conjugates 10a and 10b exhibit somewhat lower activity than 1 against sensitive S. pneumonia and S. pyogenes strains. Furthermore, the 10c and 10d showed in general lower activity against most of the tested bacterial strains except for sensitive S. aureus and M. catarrhalis where better activity was observed in comparison with 10a and 10b analogs (Bukvić Krajačić et al., 2007).
Further expanding the range of antimicrobial activity, especially against MLS B and effluxmediated resistant S. pyogenes and S. pneumoniae strains was achieved by introduction of carbamoyl and thiocarbamoyl groups attached on propyl linker at the 9a position (Bukvić Krajačić et al., 2009). Novel N''-aryl substituted 9a-(N'-carbamoyl/thiocarbamoyl-γaminopropyl)-11, 12 and 9a-[N'-(β-cyanoethyl)-N'-(carbamoyl/thiocarbamoyl-γaminopropyl)]-13, 14 derivatives were obtained according to efficient procedure described for the preparation of the previous classes of compounds (Scheme 2) (Bukvić Krajačić et al., 2005.  On the basis of excellent in vitro antibacterial activity and their structural similarity, several  compounds 11f, 11g, 11h, 12c, 12d, 12e, 12f, 13h, 14c, 14d, 14e were screened for acid stability, cytotoxicity and preliminary pharmacokinetic parametars. In acidic conditions compounds exhibited azithromycin like stability (Bukvić Krajačić et al., 2009). In vitro cytotoxicity on Hep G2 and THP-1 cell lines measured for the selected set of compounds revealed that all compounds showed relatively low cytotoxicity in vitro (IC 50 s ≥ 4 μM) (Bukvić Krajačić et al., 2011b). These marked them as potent and selective compounds for further profiling (Steinmeyer, 2006). Metabolic stability of ureas and thioureas were screened in vitro using human and mouse liver microsomes and only a few were selected for in vivo rat pharmacokinetic studies in order to determine their pharmacokinetic profiles ( Preliminary in vitro microsomal stability data indicated that these compounds had good metabolic stability, as was confirmed by low clearances in vivo for the compounds tested. In comparison to azithromycin, known for its extensive tissue distribution, (Schönfeld & Mutak, 2002) these derivatives had a tendency toward higher volumes of distribution, in line with their increased lipophilic character (approx. 2-3 log units higher than azithromycin, according to calculated logP values, data not shown) due to the presence of strong lipophilic aromatic phenyl and naphtyl rings in the 9a-N substituent (Bukvić Krajačić et al., 2011b). Overall, with increased in vitro activity and promising pharmacokinetic properties, this series of molecules represents a good starting platform for the design of novel antibacterial and antimalarial azalides.
High reactivity of secondary and primary amino groups of 3-decladinosyl-derivatives 37 and 38 toward isocyanates and isothiocyanates assured highly site-selective introduction of carbamoyl and thiocarbamoyl groups and preparation of ureas 33 & 34 and thioureas 35 & 36 in high yield (Scheme 4). They were found to posses good antibacterial activities against key respiratory Gram-positive and Gram-negative pathogens including efflux-mediated resistant strains.
Thus, it seems that appropriate linked urea or thiourea moiety at 9a-N of 3-decladinosyl-3hydroxy-azalides might interact with particular ribosome binding sites and "substitute" the cladinose sugar interaction. In order to gain more information about that conformational analysis of a compound 35a was carried out by using systematic conformational search around flexible propyl linker. Analysis of NOE cross peaks in the NOESY spectrum indicated that there is no strong interaction between macrolactone ring and the substituent at 9a-position of 35a, pointing to the stretched conformations that were also found to be most stable ones in the conformational analysis (Bukvić Krajačić et al., 2011a). Superposed xray conformations of ABT-773, (Auerbach et al., 2009), azithromycin (Schlunzen et al., 2003;, two bound conformations of telithromycin from Deinococcus radiodurans (Berisio et al. 2003) and Haloarcula marismortui  and the lowest conformation for compound 35a were shown in Fig. 4 (Bukvić Krajačić et al., 2011a). Fig. 4. Superposed x-ray conformations for azithromycin (green) , ABT-773 (cyan) ( Auerbach et al., 2009), two conformations of telithromycin from Deinococcus radiodurans (magenta) ( Berisio et al. 2003) and Haloarcula marismortui (yellow)  complexes and most stable conformation for compound 35a (red) (Bukvić Krajačić et al., 2011a).
It is clear that substituents at different positions have different spatial arrangements with respect to macrolactone. Until now there is a number of evidence including here mentioned ketolides (Auerbach et al., 2009;Schlunzen et al., 2003;Berisio et al., 2003), that high structural diversity is tolerated within the flexible macrolide-binding site of ribosome. In spite of the knowledge gained so far on macrolide binding, (Novak et al., 2006;Novak et al., 2009;Auerbach et al., 2009;Schlunzen et al., 2003;Berisio et al., 2003) an understanding of the mode of their interactions with ribosome still remain incomplete with many issues unresolved. Therefore, it can only be speculated about the possible binding mode of the compound 35a but it is likely that the additional interaction involving 1-naphthyl-propyl-side-chain, attached at the 9a position, might lead to a further stabilization of a complex with ribosome (Bukvić Krajačić et al., 2011a).

Concluding remarks
In summary, the coupling of a arylsulfonyl and benzenesulfonamido moiety to the 9a position of 15-membered azalide scaffold via carbamoyl linker has indicated improvement in antibacterial activity of novel azalides.  Hence, newly synthesised sulfonyl ureas of azalides 3b-3f, and azalide-sulfonamide conjugates 10a and 10b displayed significantly improved activity against inducible resistant S. pyogenes strain when compared to azithromycin.
In addition, the introduction of carbamoyl and thiocarbamoyl group at the 9a position of azithromycin like azalide skeleton via propyl linker proved to be promising method to tackle the resistance problems.
As a result of a preliminary optimization of an alkyl/aryl moiety attached at the carbamoyl and thiocarbamoyl group all prepared and tested compounds had high in vitro activity against erythromycin susceptible Gram-positive aerobes and Gram-negative microorganisms and especially resistant S. pyogenes and S. pneumoniae strains. It was also, shown here that urea and thiourea derivatives of 3-decladinosyl-3-hydroxy azalides, although lacking a cladinose sugar, showed noticeable antibacterial activity.

Acknowledgements
The Ministry of Science, Education and Sports of the Republic of Croatia is acknowledged for the financial support (Project No. 006-0000000-3216).