**2.2 Novel ureas and thioureas & macrolide-sulfonamide conjugate**

Various 9a-carbamoyl and thiocarbamoyl derivatives **8** & **9** were prepared (Scheme 2) by reaction of intermediate **2** with corresponding isocyanates or isothiocyanates (Kujundžić et al., 1995). Reactions were usually conducted in toluene to achieve easily crystallisable *N*alkyl or *N*-aryl substituted ureas. Structures of the *N*-isopropyl– (**8a**) (Kujundžić et al., 1995) and *N*-(4-pyridyl)– (**8g**) (Sheldrick et al., 1995) derivatives were confirmed by single crystal X-ray analysis. In biological testing, only a few derivatives **8** & **9** showed moderate antibacterial activity. Additional halogen-aryl derivatives of **8** & **9** have been synthesized showing moderate activity against resistant strains (Marušić-Ištuk et al., 2000).

Introduction of novel interactive groups into the azalide backbone resulted in further improvements in activity. Strategy which involved macrolide conjugates incorporating antibacterial sulfonamides, such as sulfanilamide, sulfabenz, sulfapyridine and sulfamethoxazole, showed an increased affinity for the ribosome (Bukvić Krajačić et al., 2007). Significant activity against inducible resistant *S. pyogenes* strains was observed by modifications at position 9a of an azalide lacton ring, by the carbamoyl group linked sulfonamides (Scheme 3). Conjugates of 15-membered azalides and sulfonamides **10a**-**10d** were prepared by the reaction of **2** (Djokić et al. 1986; Djokić et al. 1988) with 4- (chlorosulfonyl)phenylisocyanate. The smoothly formed 9a-(4-chlorosulfonylpheyl) carbamoyl derivative was transformed without the isolation into the compounds **10a**-**10d**, by the reaction of ammonia, aniline, 2-aminopyridine and 5-methyl-3-aminoisoxasole, respectively.

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 MLSB 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 & 2007).

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

Various 9a-carbamoyl and thiocarbamoyl derivatives **8** & **9** were prepared (Scheme 2) by reaction of intermediate **2** with corresponding isocyanates or isothiocyanates (Kujundžić et al., 1995). Reactions were usually conducted in toluene to achieve easily crystallisable *N*alkyl or *N*-aryl substituted ureas. Structures of the *N*-isopropyl– (**8a**) (Kujundžić et al., 1995) and *N*-(4-pyridyl)– (**8g**) (Sheldrick et al., 1995) derivatives were confirmed by single crystal X-ray analysis. In biological testing, only a few derivatives **8** & **9** showed moderate antibacterial activity. Additional halogen-aryl derivatives of **8** & **9** have been synthesized

Introduction of novel interactive groups into the azalide backbone resulted in further improvements in activity. Strategy which involved macrolide conjugates incorporating antibacterial sulfonamides, such as sulfanilamide, sulfabenz, sulfapyridine and sulfamethoxazole, showed an increased affinity for the ribosome (Bukvić Krajačić et al., 2007). Significant activity against inducible resistant *S. pyogenes* strains was observed by modifications at position 9a of an azalide lacton ring, by the carbamoyl group linked sulfonamides (Scheme 3). Conjugates of 15-membered azalides and sulfonamides **10a**-**10d** were prepared by the reaction of **2** (Djokić et al. 1986; Djokić et al. 1988) with 4- (chlorosulfonyl)phenylisocyanate. The smoothly formed 9a-(4-chlorosulfonylpheyl) carbamoyl derivative was transformed without the isolation into the compounds **10a**-**10d**, by the reaction of ammonia, aniline, 2-aminopyridine and 5-methyl-3-aminoisoxasole,

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

 Further expanding the range of antimicrobial activity, especially against MLSB 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.,

observed in comparison with **10a** and **10b** analogs (Bukvić Krajačić et al., 2007).

introduction of a propyl linker and additional cyanoethyl side chain.

**2.2 Novel ureas and thioureas & macrolide-sulfonamide conjugate** 

showing moderate activity against resistant strains (Marušić-Ištuk et al., 2000).

respectively.

2005 & 2007).

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

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

Antibacterial Activity of Novel Sulfonylureas, Ureas and Thioureas of 15-Membered Azalides 93

Introduction of unsaturated, sp2 hybridized, carbamoyl unit at 9a position placed nitrogen atom of **2** significantly change electronic properties and also steric environment in the 'upper part' of the macrolide, what resulted in increased antibacterial activity of the novel sulfonylureas **3,5,7** (Bukvić Krajačić et al., 2005), azalide-sulfonamide conjugates **10** (Bukvić Krajačić et al., 2007), and ureas and thioures **11-14** (Bukvić Krajačić et al., 2009). On the other hand, the selectively achieved cleavage of cladinose sugar, significantly changes the structural behaviour of the 'lower part' of 9a-carbamoyl 15-membered azalides, leading to the 3-*O*-decladinosyl-3-hydroxy ureas and thiouras lacking of any, as expected, antibacterial activity (Bukvić Krajačić et al., 2005; Bukvić Krajačić et al., 2007; Marušić Ištuk et al., 2007). However, there are some novel highly potent 3-*O*-decladinosyl derivatives of 14-membered macrolides, *e.g.* ketolides (Agouridas et al, 1998), acylides (Tanikawa et al., 2001; Tanikawa et al., 2003), anhydrolides (Elliott et al., 1998), etc. (Schönfeld & Kirst 2002; Pal 2006; Kaneko

**2.3 3-Decladinosyl-derivatives of sulfonylureas, ureas and thioureas** 

et al. , 2006; Mutak 2007) (Fig. 2), proved active against resistant bacterial strains.

O O

Q

**Acylides**

O O

Q

O O

O O

O

Q

**3-O-phenyl ethers** Q= Erythromycin scaffold

Q

**2,3-Anhydrolides** Q= Erythromycin scaffold

O O

N HO <sup>O</sup>

> O O

O O

O

<sup>O</sup> <sup>X</sup> Y

N HO

**3-Deoxy derivatives** Q= Azithromycin scaffold (R=H) Q= Erythromycin scaffold (R=Me)

<sup>R</sup> <sup>Q</sup>

O O

<sup>N</sup> HO <sup>O</sup>

O R' O

Q= Erythromycin scaffold (R=Me) Q= Azithromycin scaffold (R=H)

<sup>N</sup> HO <sup>O</sup>

R

HO

<sup>Q</sup> <sup>=</sup>

O O

Q

O O

**3,6-Ketals** Q= Azithromycin scaffold

Q

<sup>N</sup> R'

Q

**Ketolides**

Q= Azithromycin scaffold Q= Erythromycin scaffold

N HO O O

O O

O O

O

**3,6-Cyclic ethers** Q= Erythromycin scaffold

O O

N HO

O O

O

<sup>N</sup> HO <sup>O</sup>

OH

N HO

O O

N HO O O

O O

OH **3-Hydroxy-derivatives**

N HO OH

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

OH 9a

Azithromycin scaffold

Erythromycin scaffold

O 9

O O

**3,6-Bycicloids** Q= Erythromycin scaffold

Fig. 2. Novel classes of 3-*O*-decladinosyl derivatives of 14- and 15-membered macrolides

were improved in comparison with sulfonylureas **3a-3f** (Bukvić Krajačić et al., 2005) and azithromycin-sulfonamide conjugates **10a-10d** (Bukvić Krajačić et al., 2007). Ureas **11f, 11g** and **11h** demonstrated high activity against *Moraxella catarrhalis*. Naphthyl substituted ureas **11f, 11g** and **11h** showed better activity against Gram-negative pathogens involved in respiratory tract infections (RTI), *M. catarrhalis* (MIC 0.25 µg/ml) and *H. influenza* (MIC 1 µg/ml) than derivatives with phenyl ring on the alkyl side-chain **11b-11d**. In case of phenylethylsubstituents in **11d** and **12d** the presence of thiocarbamoyl moiety seemed to improve activity against *H. influenzae*. The urea **13** with cyanoethyl chain showed similar antibacterial activity in comparison to the urea **11**. The observed antibacterial activity of ureas and thioureas increased in the series **8** & **9** < **11** & **12** < **13** & **14**, by the introduction of a propyl linker and additional cyanoethyl side-chain (Bukvić Krajačić et al., 2009)**.**

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* (IC50s ≥ 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 (Table 1) (Bukvić Krajačić et al., 2011b). All compounds demonstrated good *in vitro*, metabolic stability with t1/2 greater than 120 min (t1/2 = 103 min for compound **14d** in human liver microsomes). As was observed with azithromycin, and in line with the *in vitro* data, these analogs had a low systemic clearance, moderate to high volume of distribution and a very long half-life, however, the oral bioavailability was low (**12c**, **12e**) to moderate (Bukvić Krajačić et al., 2011b).


CL - blood clearance, Vd – apparent volume of distribution at the terminal phase based on drug concentration in blood, t1/2 - half life, a - IV parameters determined in one rat

Table 1. Pharmacokinetic parameters estimated in blood after intravenous (IV) and oral gavage (PO) administration to Sprague-Dawley rats (10 mg/kg IV and 30 mg/kg PO) (Bukvić Krajačić t al., 2011b).

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.

#### **2.3 3-Decladinosyl-derivatives of sulfonylureas, ureas and thioureas**

92 Antimicrobial Agents

were improved in comparison with sulfonylureas **3a-3f** (Bukvić Krajačić et al., 2005) and azithromycin-sulfonamide conjugates **10a-10d** (Bukvić Krajačić et al., 2007). Ureas **11f, 11g** and **11h** demonstrated high activity against *Moraxella catarrhalis*. Naphthyl substituted ureas **11f, 11g** and **11h** showed better activity against Gram-negative pathogens involved in respiratory tract infections (RTI), *M. catarrhalis* (MIC 0.25 µg/ml) and *H. influenza* (MIC 1 µg/ml) than derivatives with phenyl ring on the alkyl side-chain **11b-11d**. In case of phenylethylsubstituents in **11d** and **12d** the presence of thiocarbamoyl moiety seemed to improve activity against *H. influenzae*. The urea **13** with cyanoethyl chain showed similar antibacterial activity in comparison to the urea **11**. The observed antibacterial activity of ureas and thioureas increased in the series **8** & **9** < **11** & **12** < **13** & **14**, by the introduction of a propyl linker and additional

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* (IC50s ≥ 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 (Table 1) (Bukvić Krajačić et al., 2011b). All compounds demonstrated good *in vitro*, metabolic stability with t1/2 greater than 120 min (t1/2 = 103 min for compound **14d** in human liver microsomes). As was observed with azithromycin, and in line with the *in vitro* data, these analogs had a low systemic clearance, moderate to high volume of distribution and a very long half-life, however,

the oral bioavailability was low (**12c**, **12e**) to moderate (Bukvić Krajačić et al., 2011b).

concentration in blood, t1/2 - half life, a - IV parameters determined in one rat

Table 1. Pharmacokinetic parameters estimated in blood after intravenous (IV) and oral gavage (PO) administration to Sprague-Dawley rats (10 mg/kg IV and 30 mg/kg PO)

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

 CL (mL/min/kg) Vd (L/kg) t1/2 (hr) Oral F (%) **Azithromycin** 11.0 20.0 24.0 33.0 **12c** 4.0 10.4 30.0 3.4 **12e**<sup>a</sup> 2.3 2.6 13.4 1.3 **14e** 24.5 31.7 15.2 21 CL - blood clearance, Vd – apparent volume of distribution at the terminal phase based on drug

cyanoethyl side-chain (Bukvić Krajačić et al., 2009)**.**

(Bukvić Krajačić t al., 2011b).

novel antibacterial and antimalarial azalides.

Introduction of unsaturated, sp2 hybridized, carbamoyl unit at 9a position placed nitrogen atom of **2** significantly change electronic properties and also steric environment in the 'upper part' of the macrolide, what resulted in increased antibacterial activity of the novel sulfonylureas **3,5,7** (Bukvić Krajačić et al., 2005), azalide-sulfonamide conjugates **10** (Bukvić Krajačić et al., 2007), and ureas and thioures **11-14** (Bukvić Krajačić et al., 2009). On the other hand, the selectively achieved cleavage of cladinose sugar, significantly changes the structural behaviour of the 'lower part' of 9a-carbamoyl 15-membered azalides, leading to the 3-*O*-decladinosyl-3-hydroxy ureas and thiouras lacking of any, as expected, antibacterial activity (Bukvić Krajačić et al., 2005; Bukvić Krajačić et al., 2007; Marušić Ištuk et al., 2007).

However, there are some novel highly potent 3-*O*-decladinosyl derivatives of 14-membered macrolides, *e.g.* ketolides (Agouridas et al, 1998), acylides (Tanikawa et al., 2001; Tanikawa et al., 2003), anhydrolides (Elliott et al., 1998), etc. (Schönfeld & Kirst 2002; Pal 2006; Kaneko et al. , 2006; Mutak 2007) (Fig. 2), proved active against resistant bacterial strains.

Erythromycin scaffold Azithromycin scaffold

Fig. 2. Novel classes of 3-*O*-decladinosyl derivatives of 14- and 15-membered macrolides

Antibacterial Activity of Novel Sulfonylureas, Ureas and Thioureas of 15-Membered Azalides 95

& **18**, and 6-methoxy-, **19** & **20** derivatives and subsequent base-promoted elimination led to the formation of different products. Whereas 6-hydroxy derivatives **17** & **18** produce 3,6 cyclic ethers **25** and **26**, 6-methoxy derivatives **19** & **20** afford 2,3-anhydro azalides **27** and **28**. Among already known 3-acylides of 14-membered macrolides, *3-O*-(4-nitrophenyl)acetyl derivative of clarithromycin (TEA-0777) showed the best antibacterial activity (Tanikawa et al., 2001). Accordingly, 9a-carbamoyl acylides having (4-nitrophenyl)acetyl- functionality attached to 3-O position, azalides **29** and **30,** were prepared, to test if antibacterial activity

3-Decladinosyl-6-hydroxy and 6-methoxy azalides **15** and **16** and 9a-carbamoyl/9athiocarbamoyl derivatives **17, 18, 19** & **20** proved antibacterially inactive against tested strains. Similar situation can be seen with 3,6-hemiketals **21** & **22** and 3,6-cyclic ethers **25** & **26**. However, anhydrolides **27** and **28** as well as ketolides **23** and **24** show good antibacterial activity against efflux resistant *S. pneumonia* but lower in comparison to erythromycin. 9a-Carbamoyl-3-*O*-(4-nitrophenyl)acetyl- acylides **29** and **30** showed the best antibacterial activity against efflux resistant *S. pneumoniae* (MIC 4 µg/ml), and better in comparison to erythromycin (MIC 8 µg/ml). Acylides **29** and **30** also show weak activity against

As expected, 3-decladinosyl-3-hydroxy- azalides **17** – **20** and **31** - **34** (Fig. 3) lacked any significant antimicrobial activity (Marušić Ištuk et al., 2007; Bukvić Krajačić et al., 2005; Bukvić Krajačić et al., 2007) being consistent with the role cladinose was found to play in antimicrobial activity (LeMahieu et.al., 1974; Kaneko et.al., 2006; Pal, 2006; Tanikawa et.al.,

OH

O

O

O O

N

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

N O N H

CN

O

OH

O

N

R'

O O

N

N H O

HO OH HO OH

SO2

N H

N

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

**32a-32d** 

O O

SO2

R''

O O

OH

O

could be enhanced upon attachment of favorite side-arm.

erythromycin-resistant *S. aureus* (Marušić Ištuk et al., 2007).

**2.3.2 3-Decladinosyl-3-hydroxy derivatives** 

OH

**18** R' = H, R = 2,4-dichlorophenyl **19** R' = CH3, R = isopropyl **20** R' = CH3, R = 2,4-dichlorophenyl

O

O

**31a-31f 17** R' = H, R = isopropyl

O O

lacking any significant antimicrobial activity

N

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

N H O N H

O

OH

O

**33a 34a** Fig. 3. 3-Decladinosyl-3-hydroxy- azalides from the urea, thiorea and sulfonylurea series,

N

2001; Mutak, 2007).

O O

N

N H X

HO OR' HO OH

R

#### **2.3.1 3-Decladinosyl-3-O-substituted derivatives**

Isopropyl- and 2,4-dichlorophenyl- derivatives of 9a-carbamoyl-6-hydroxy (**17** & **18**) and 9acarbamoyl-6-methoxy azalides (**19** & **20**) lacking any antibacterial activity, were selected to study the effects of the 'lower part' of azalide skeleton modifications *via* chemical transformations of hydroxyl group at C-3 position (Scheme 3) (Marušić Ištuk et al., 2007). They afforded formation of the new ketolides **23** and **24**, anhydrolides **27** and **28**, hemiketals **21** and **22**, cyclic ethers **25** and **26**, and acylides **29** and **30** (Scheme 4). In order to perform chemical transformations on the hydroxyl group at position 3, 2'-hydroxyl group which is the most reactive one, was suitably protected. Consequently, reaction of 3-decladinosyl-3 hydroxy- azalides **17**, **18**, **19**, and **20** with acetic anhydride in the presence of a base smoothly afforded 2'-*O*-acetyl-3-decladinosyl-3-hydroxy-6-hydroxy azalides, that under conditions of Pfitzner–Moffat 3-OH group oxidation, followed by subsequent methanolysis of 2'-*O*-acetyl intermediate produces internal 3,6-hemiketal structures **21** and **22**. Under the same reaction conditions 2'-*O*-acetyl-3-decladinosyl-3-hydroxy-6-methoxy derivatives afford 3-keto azalides **23** and **24** (Scheme 3). Introduction of mesyl group at position C-3 of 6-hydroxy-, **17**

Scheme 3. Synthesis of 3-decladinosyl-3-*O*-substituted azalides

& **18**, and 6-methoxy-, **19** & **20** derivatives and subsequent base-promoted elimination led to the formation of different products. Whereas 6-hydroxy derivatives **17** & **18** produce 3,6 cyclic ethers **25** and **26**, 6-methoxy derivatives **19** & **20** afford 2,3-anhydro azalides **27** and **28**.

Among already known 3-acylides of 14-membered macrolides, *3-O*-(4-nitrophenyl)acetyl derivative of clarithromycin (TEA-0777) showed the best antibacterial activity (Tanikawa et al., 2001). Accordingly, 9a-carbamoyl acylides having (4-nitrophenyl)acetyl- functionality attached to 3-O position, azalides **29** and **30,** were prepared, to test if antibacterial activity could be enhanced upon attachment of favorite side-arm.

3-Decladinosyl-6-hydroxy and 6-methoxy azalides **15** and **16** and 9a-carbamoyl/9athiocarbamoyl derivatives **17, 18, 19** & **20** proved antibacterially inactive against tested strains. Similar situation can be seen with 3,6-hemiketals **21** & **22** and 3,6-cyclic ethers **25** & **26**. However, anhydrolides **27** and **28** as well as ketolides **23** and **24** show good antibacterial activity against efflux resistant *S. pneumonia* but lower in comparison to erythromycin. 9a-Carbamoyl-3-*O*-(4-nitrophenyl)acetyl- acylides **29** and **30** showed the best antibacterial activity against efflux resistant *S. pneumoniae* (MIC 4 µg/ml), and better in comparison to erythromycin (MIC 8 µg/ml). Acylides **29** and **30** also show weak activity against erythromycin-resistant *S. aureus* (Marušić Ištuk et al., 2007).
