**2. Novel sulfonylureas, ureas and thioureas of 15-membered azalides**

The first discovered representative of the 15-membered azalides, cyclic amine **2** (Scheme 1) named 9-deoxo-9a-aza-9a-homoerythromycin A, permitted a derivatisation line at the 9anitrogen atom (Scheme 1). Its first derivatization was methylation at 9a position and synthesis of 9-deoxo-9a-aza-9a-methyl-9a-homoerythromycin A, azithromycin **(1)**(Kobrehel & Djokić, 1982).

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-*N*carbamoyl and *N*-thiocarbamoyl derivatives of **2** (Kujundžić et al., 1995) 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**.

#### **2.1 Sulfonylureas**

88 Antimicrobial Agents

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

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 &

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

**2. Novel sulfonylureas, ureas and thioureas of 15-membered azalides** 

The first discovered representative of the 15-membered azalides, cyclic amine **2** (Scheme 1) named 9-deoxo-9a-aza-9a-homoerythromycin A, permitted a derivatisation line at the 9anitrogen atom (Scheme 1). Its first derivatization was methylation at 9a position and synthesis of 9-deoxo-9a-aza-9a-methyl-9a-homoerythromycin A, azithromycin **(1)**(Kobrehel

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-*N*carbamoyl and *N*-thiocarbamoyl derivatives of **2** (Kujundžić et al., 1995) encouraged us to

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

pneumonia (CABP) (Cruzan, 2007; Farrell et al., 2010).

with the goal of identifying novel bacterial inhibitors.

Mutak, 2011; Štimac et al., 2010).

& Djokić, 1982).

extend our study in this direction.

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

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

Antibacterial Activity of Novel Sulfonylureas, Ureas and Thioureas of 15-Membered Azalides 91

O

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

O

O OH

> O O

O O

**R'''**

N

O

O

N

**f g**

sulfonamide conjugates

**d e**

**<sup>a</sup> <sup>b</sup> <sup>c</sup>**

**9 X = S**

O

O

O OH

O

N

O O

O N

**R'**

N

N H X

HO OH HO OH

R'

<sup>N</sup> HO OHHO OH

> **11 X = O 12 X = S**

N H X N H R'''

1. acrylonitrile, 60°C 2. H2/ PtO2, ethanol

toluene, 0-5°C R'NCX

O

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

O

O O

N H

<sup>N</sup> HO OHHO OH

3. R'''NCX 4. R'''NCX

**10a-d 8 X = O**

**2** 

O OH

**<sup>a</sup> <sup>b</sup> <sup>c</sup> <sup>d</sup>**

**<sup>e</sup> <sup>f</sup> <sup>g</sup> <sup>h</sup>**

Scheme 2. Synthesis of novel ureas and thioureas of 15-membered azalides and azalide-

Ureas **11** and **13** and thioureas **12** and **14** (Bukvić Krajačić et al., 2009) showed a significant improvement in antibacterial activity against all tested macrolide-susceptible and resistant bacteria in comparison with carbamoyl/thiocarbamoyl derivatives **8** & **9** (Kujundžić et al. 1995), sulfonylcarbamoyl derivatives **3a-3f** (Bukvić Krajačić et al., 2005) and azithromycinsulfonamide conjugates **10a-10d** (Bukvić Krajačić et al., 2007). Also, these compounds exhibited a substantially improved *in vitro* antimalarial activity against *P. falciparum (*Bukvić Krajačić et al., 2011b; Hutinec et al., 2011)*.* Several ureas bearing naphthyl supstituents (**11f, 11g, 11h**) were superior *in vitro* to the azithromycin against inducible resistant *S. pyogenes* (MIC 2 µg/ml). Ureas **11f, 11g** and thioureas **12c, 12d, 12e, 12f** possesses good activity against effluxmediated resistant *S. pyogenes* (MIC 4 µg/ml), comparable to azithromycin (MIC 4 µg/ml).

In general, all tested compounds had high *in vitro* activity against erythromycin susceptible Gram-positive aerobes, *S. pneumoniae* and *S. pyogenes* (MIC < 0.125 μg/ml) (Bukvić Krajačić et al., 2009). Ureas **11** and **13** and thioureas **12** and **14** exhibited excellent activity against susceptible *S. aureus* (MIC 0.25-1 µg/ml), but lacked activity against resistant *S. aureus* strains. Ureas **11f, 11g** and thiourea **12f** also showed *in vitro* activity against efflux-mediated resistant *S. pneumoniae* with MICs 4 μg/ml and their activities were comparable with those observed for azithromycin (MIC 8 µg/ml). Ureas **11g, 11h** and **13h** showed moderate activity against cMLS *S. pneumonia* (MIC 16 µg/ml) (Bukvić Krajačić et al., 2009). *In vitro* activities of ureas **11** and **13**, thioureas **12** and **14** against key community-acquired Gram-negative respiratory pathogens

N

<sup>N</sup> HO OHHO OH

> **13 X = O 14 X = S**

N X N H R'''

1. acrylonitrile, 60°C 2. H2/ PtO2, ethanol 3. 1eq acrylonitrile, MeOH

CN

OCN SO2Cl

toluene, 0-5°C, 1h

2 R''H

O

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

O

N H O

N

<sup>N</sup> HO OHHO OH

> N H

 -NH2 **a b**

**R''**

O O

SO2

R''

O O

O OH

N H

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

**c d**

N O

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

O OH

arylsulfonylcarbamoyl derivatives **3a-3f**, **5a-5f** and **7a-7f** against all the tested erythromycin susceptible (Ery-S) Gram-positive strains decreased in the series **3a-3f** > **5a-5f** > **7a-7f** by the introduction of a propyl linker and additional cyanoethyl side chain.
