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

Mirjana Bukvić Krajačić1,2 and Miljenko Dumić<sup>3</sup> *1GlaxoSmithKline Research Centre Zagreb, Zagreb, 2Galapagos Research Center Ltd., Zagreb, 3Department of Biotechnology, University of Rijeka, Rijeka, Croatia* 

*Dedicated to all our colleagues engaged worldwide in discovery and development of azithromycin, on the occasion of the 30th anniversary of its invention (1981-2011)* 

### **1. Introduction**

84 Antimicrobial Agents

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23, pp. 1089-1094.

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 30th anniversary of azithromycin's invention (1981-2011) 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.

Antibacterial Activity of Novel Sulfonylureas, Ureas and Thioureas of 15-Membered Azalides 87

O

3"

4"

N

2' 3'

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

OH

Essential

Essential

Essential

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 (MLSB) 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

The discovery of highly potent representatives of the third-generation of macrolides**,** like ketolides (Agouridas 1998), acylides (Tanikawa et al. 2001; Tanikawa et al. 2003), anhydrolides (Elliott et al. 1998), etc., was a step forward to tackle the efflux problems (LeMahieu et al. 1974; Pestka & LeMahieu 1974a & 1974b, Pestka et al. 1974; Pestka et al. 1976; Allen. 1977; Van Bambeke et al. 2008)' to (Tanikawa et al., 2001; Tanikawa et al., 2003), anhydrolides (Elliott et al., 1998), etc., was a step forward to tackle the efflux problems (LeMahieu et al., 1974; Pestka & LeMahieu 1974a & 1974b, Pestka et al., 1974; Pestka et al.,

Fig. 1. Azithromycin **(1)** and its position subjected to derivatization

OH

9

8

3

**Azithromycin 1**

O

O

N

11

HO OH

12 6

13 5

2

9a

<sup>O</sup> <sup>O</sup>

O

HO

MeO

of the desirable features of the earlier generation of macrolides.

1976; Allen. 1977; Van Bambeke et al., 2008).

(Cunningham et al., 2000).

Macrolides as polyketide class of natural products have a long history as effective therapeutic agents for treating infectious diseases (Schönfeld & Kirst, 2002; G.T. Hansen et al., 2002; 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 (Andersen et al., 1994; 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).

Following this trend, the chemists from PLIVA Pharmaceuticals (Zagreb, Croatia) discovered in 1980 the famous azithromycin molecule, 1 (Fig. 1), characterized by unique 15-membered macrolide ring system, having a basic methylamino group inserted into the erythromycin aglycone (Kobrehel & Djokić 1982;. Kobrehel et al., 1982; Kobrehel & Djokić, 1985; Djokić et al. 1986; Djokić et al. 1987; Djokić et al. 1988). Soon after the publication of PLIVA's Belgian azithromycin patent, researchers at Pfizer (Groton, USA) prepared azithromycin independently, as the results of their own research program (Bright 1984).

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.

Macrolides as polyketide class of natural products have a long history as effective therapeutic agents for treating infectious diseases (Schönfeld & Kirst, 2002; G.T. Hansen et al., 2002; 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 (Andersen et al., 1994; 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

Following this trend, the chemists from PLIVA Pharmaceuticals (Zagreb, Croatia) discovered in 1980 the famous azithromycin molecule, 1 (Fig. 1), characterized by unique 15-membered macrolide ring system, having a basic methylamino group inserted into the erythromycin aglycone (Kobrehel & Djokić 1982;. Kobrehel et al., 1982; Kobrehel & Djokić, 1985; Djokić et al. 1986; Djokić et al. 1987; Djokić et al. 1988). Soon after the publication of PLIVA's Belgian azithromycin patent, researchers at Pfizer (Groton, USA) prepared azithromycin

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

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

independently, as the results of their own research program (Bright 1984).

stimulating activity (Takanashi et al. 2009).

antibiotics (Spaventi 2002).

compounds with advantageous pharmacokinetics.

Fig. 1. Azithromycin **(1)** and its position subjected to derivatization

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 (MLSB) 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.

The discovery of highly potent representatives of the third-generation of macrolides**,** like ketolides (Agouridas 1998), acylides (Tanikawa et al. 2001; Tanikawa et al. 2003), anhydrolides (Elliott et al. 1998), etc., was a step forward to tackle the efflux problems (LeMahieu et al. 1974; Pestka & LeMahieu 1974a & 1974b, Pestka et al. 1974; Pestka et al. 1976; Allen. 1977; Van Bambeke et al. 2008)' to (Tanikawa et al., 2001; Tanikawa et al., 2003), anhydrolides (Elliott et al., 1998), etc., was a step forward to tackle the efflux problems (LeMahieu et al., 1974; Pestka & LeMahieu 1974a & 1974b, Pestka et al., 1974; Pestka et al., 1976; Allen. 1977; Van Bambeke et al., 2008).

Antibacterial Activity of Novel Sulfonylureas, Ureas and Thioureas of 15-Membered Azalides 89

aglycon moiety on the antibacterial activity. A special attention was paid to achieving the

O

O O O

O OH

> ' **R a** H **b** *p*-Me **c** *o*-Me

> > OCN SO2 toluene, 0-5°C, 1h

OCN SO2 toluene, 0-5°C, 1h

O O

**2** 

1eq acrylonitrile, methanol, reflux, 7h

> O O

addition of the substituted benzensulfonyl isocyanates to the intermediate **6**.

N

<sup>N</sup> HO HO OH OH

**5a-5f 7a-7f** 

**6** 

NH

NC

O

Intermediates **2** (Djokić et al., 1986; Djokić et al., 1988) and **4**, smoothly reacted with substituted benzensulfonyl isocyanates to form 9a-*N*-[*N*'-(aryl)sulfonylcarbamoyl] derivatives, **3a-3f** and **5a-5f** in high yields (Scheme 1). The key intermediate, 9a-*N*-(γaminopropyl) derivative **4** was prepared by standard Michael addition of acrylonitrile to the amine **2**, followed by catalytic hydrogenation of obtained 9a-*N*-(β-cyanoethyl) derivative with PtO2 as a catalyst (Bright et al., 1988). Derivatives **7a-7f**, were prepared by the selective cyanoethylation of amine **4** with equivalent amounts of acrylonitrile, followed by the

For the sulfonylureas directly linked to macrocyclic ring **3a-3f** it was observed that compounds with methyl group and chlorine in *p*- **3b** (MIC 1 µg/ml)**, 3d** (MIC 1 µg/ml) and *o*- **3c** (MIC 0.5 µg/ml)**, 3e** (MIC 2 µg/ml) positions and fluorine in *p*-position **3f** (MIC 2 µg/ml) showed significantly improved activity against iMLS resistant *S. pyogenes* strain when compared to azithromycin **1** (MIC 8 µg/ml) and starting amine **2** (MIC 16 µg/ml). Also, these compounds exhibited two level of dilution better activity than **2** (MIC 0.25 µg/ml) and similar activity to **1** (MIC <0,125 µg/ml) against sensitive *S. pneumonia* (Bukvić Krajačić et al. 2005). However, the activities against Gram-negative bacteria were all lower than those for **1** and **2**. Generally, it was observed that antibacterial activity of the novel

O O O

O OH

N H

9a

<sup>N</sup> HO HO OH OH

> O O

N

<sup>N</sup> HO HO OH OH

NC

O

O

O

O OH

O

N

R'

O O

' **R d** *p*-Cl **e** *o*-Cl **f** *p*-F

R'

N

N H O

SO2

HO HO OH OH

**3a-3f** 

O

N <sup>H</sup> SO2 <sup>N</sup> O

<sup>O</sup> <sup>O</sup>

O

R'

O OH

activity against *S. pyogenes* and *S. pneumonia* resistant strains.

1. acrylonitrile, 60°C, 12h 2. H2/ PtO2, ethanol, 20 bar,

O

OCN SO2 toluene, 0-5°C, 1h

O

O

O OH

O

O O

**4**

O O

N

<sup>N</sup> HO OHHO OH

O

N <sup>H</sup> SO2 <sup>N</sup> H

R'

Scheme 1. Synthesis of sulfonylureas **3, 5** and **7**.

O

O O

**2.1 Sulfonylureas** 

O

O OH

N

<sup>N</sup> HO OHHO OH

NH2

However, some serious drawbacks have been observed for those compound classes: the emergence of resistance developed shortly after their introduction and rare but serious side effects which lead to restrictions and withdrawal (Bambeke et al., 2008) as seen recently with telithromycin, approved by the United States (USA) Food and Drug Administration (FDA) approved in 2004 by for treatment of mild to moderate community-acquired bacterial pneumonia (CABP) (Cruzan, 2007; Farrell et al., 2010).

Recently, considering azithromycin's beneficial pharmacokinetic properties, our group have led the widespread modification of the azalide scaffold (Fig. 1) in a search for new, to resistant bacterial strains active azalides (Fajdetić et al., 2010; Fajdetić et al., 2011; Hutinec et al., 2010; Kapić et. al, 2010; Kapić et. al, 2011a; Kapić et. al., 2011b; Marušić Ištuk et al., 2011; Matanović Škugor et al., 2010; Palej Jakopović et al., 2010; Pavlović et al., 2010; Pavlović & Mutak, 2011; Štimac et al., 2010).

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 MLSB 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.
