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

94 Antimicrobial Agents

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**

O

O OH

2. NaH, DMF/THF, 0°C 3. MeOH, rt, 24h

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

 R' = H, R = isopropyl R' = H, R = 2,4-dichlorophenyl R' = CH3, R = isopropyl R' = CH3, R = 2,4-dichlorophenyl

1. Methylsulfonyl anhydride, pyridine, rt, 3h

*2,3-anhydrolides 3,6-cyclic ethers*

O O

N HO

O2N

 pivaloyl chloride, TEA, pyridine 2. MeOH, rt, 24h

1.

O

O

O

**25** R = isopropyl **26** R = 2,4-dichlorophenyl

OH HO

O

O

N

N H

R

*3-acylides*

O O

**29** R = isopropyl **30** R = 2,4-dichlorophenyl

O O

O O

N HO

O O

N HO

O

N HO

NO2

N

O

OH <sup>O</sup>

N H

**21** R = isopropyl **22** R = 2,4-dichlorophenyl

O

OH HO

O

O OH

O

N

N H

R

R

OH HO OH

N

O

RNCO/RNCS

N H

1. Aceticanhydride, TEA

3. MeOH, rt, 24h

R

*3-ketolides 3,6-hemiketals*

2. EDC, DMSO, pyridinium trifluoroacetate

OH HO OR'

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

O

O

**27** R = isopropyl **28** R = 2,4-dichlorophenyl

OH HO

O

O O

OMe

N HO

O

N

N H

O

O OH

**15** R' = H **16** R' = CH3

N H

OH HO OR'

R

OH HO

O

O O

**23** R = isopropyl **24** R = 2,4-dichlorophenyl

O O

O O

N HO

OMe

N HO

N

N H

R

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., 2001; Mutak, 2007).

Fig. 3. 3-Decladinosyl-3-hydroxy- azalides from the urea, thiorea and sulfonylurea series, lacking any significant antimicrobial activity

Antibacterial Activity of Novel Sulfonylureas, Ureas and Thioureas of 15-Membered Azalides 97

and 3-decladinosyl azithromycin-sulfonamide conjugates **32** (Bukvić Krajačić et al., 2007). Activity of (±)-**33a** and **35a** against *H. influenzae* is only one dilution lower than the corresponding MIC of azithromycin (MIC 2 µg/ml). Urea (±)-**33c** was more potent (MIC 8 µg/ml) than its 3-cladinosyl analogue (±)-**11h** (MIC 16 µg/ml) against *Enterococcus faecalis* and **33a** showed the same activity against *E. coli* in comparison to its cladinosyl analogue

Thus, it seems that appropriate linked urea or thiourea moiety at 9a-*N* of 3-decladinosyl-3 hydroxy- 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; Hansen et al., 2002), two bound conformations of telithromycin from *Deinococcus radiodurans*  (Berisio et al. 2003) and *Haloarcula marismortui (*Hansen et al., 2002) 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) (Hansen et al., 2002), ABT-773 (cyan) ( Auerbach et al., 2009), two conformations of telithromycin from *Deinococcus radiodurans* (magenta)( Berisio et al. 2003) and *Haloarcula marismortui* (yellow)( Hansen et al., 2002) complexes and most stable conformation for compound **35a** (red) (Bukvić Krajačić et

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; Hansen et al., 2002; Berisio et al., 2003), that high structural diversity is tolerated within the flexible macrolide-binding site of

**11f**. (Bukvić Krajačić et al., 2011a).

al., 2011a).

This is supported by recently published NMR binding studies (trNOESY and STD experiments) on 6-*O*-methyl-homoerythromycin derivatives, showing that the absence of cladinose sugar has been found to be the main cause of their inability to bind to their target ribosome (Novak et al., 2009). Stability study of the most active compounds **11f** and **13f** in the artificial gastric juice (Bukvić Krajačić et al., 2009) led to the formation of two decladinosyl derivatives **33a** and **34a** which were tested only against panel of *S. pneumoniae* strains. As was expected decladinosyl urea derivative **34a** did not show activity against tested strains. However, decladinosyl urea derivative **33a** showed significant activity against erythromycin susceptible *S. pneumoniae* strain (1 µg/ml), as well as efflux-mediated *S. pneumoniae* resistant strain (8 µg/ml) comparable to azithromycin. This finding initiated the synthesis of a small library of 3-decladinosyl-3-hydroxy ureas and thioureas of 15 membered azalides termed *"decladinosylides"* (Bukvić Krajačić et al., 2011a).

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.

Among them, most of the synthesized 3-decladinosyl-3-hydroxy derivatives showed moderate to high activity against efflux-mediated resistant *S. pneumoniae* and moderate activity against susceptible *S. pneumoniae* and *S. pyogenes* strains. Against efflux-mediated resistant *S. pneumoniae* compound **35a** (MIC 2 µg/ml) posses better activity compared to azithromycin (**1**) (MIC 8 µg/ml) (Bukvić Krajačić et al., 2011a) and their parent 3-cladinosyl analogues **11** & **13** (MIC 4 to 16 µg/ml) ( Bukvić Krajačić et al., 2009), and significantly better in comparison to the 3-decladinosyl-3-hydroxy azithromycin (**16**) (MIC >64 µg/ml).

Scheme 4. Synthesis of novel 3-decladinosyl ureas and thioureas of 15-membered azalides.

The racemic urea derivative **33c** showed the highest activity against both, susceptible *S. pneumoniae* and *S. pyogenes* strains, and the same activity as its 3-cladinosyl analogue (±)-**11h**  and azithromycin (MIC <0.125 µg/ml) (Bukvić Krajačić et al., 2009).

Interestingly, some of discovered 3-decladinosyl-3-hydroxy ureas **33** & **34**, and thioureas **35**  & **36**, maintain antibacterial activity against Gram-negative pathogens *H. influenzae* and *M. catarrhalis* (Bukvić Krajačić et al., 2011a) in comparison to their parent 3-cladinosyl derivatives **11, 12, 13** & **14**, (Bukvić Krajačić et al., 2009) and demonstrate large improvement in comparison to the inactive 3-decladinosyl sulfonylureas **31**( Bukvić Krajačić et al., 2005)

This is supported by recently published NMR binding studies (trNOESY and STD experiments) on 6-*O*-methyl-homoerythromycin derivatives, showing that the absence of cladinose sugar has been found to be the main cause of their inability to bind to their target ribosome (Novak et al., 2009). Stability study of the most active compounds **11f** and **13f** in the artificial gastric juice (Bukvić Krajačić et al., 2009) led to the formation of two decladinosyl derivatives **33a** and **34a** which were tested only against panel of *S. pneumoniae* strains. As was expected decladinosyl urea derivative **34a** did not show activity against tested strains. However, decladinosyl urea derivative **33a** showed significant activity against erythromycin susceptible *S. pneumoniae* strain (1 µg/ml), as well as efflux-mediated *S. pneumoniae* resistant strain (8 µg/ml) comparable to azithromycin. This finding initiated the synthesis of a small library of 3-decladinosyl-3-hydroxy ureas and thioureas of 15-

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

Among them, most of the synthesized 3-decladinosyl-3-hydroxy derivatives showed moderate to high activity against efflux-mediated resistant *S. pneumoniae* and moderate activity against susceptible *S. pneumoniae* and *S. pyogenes* strains. Against efflux-mediated resistant *S. pneumoniae* compound **35a** (MIC 2 µg/ml) posses better activity compared to azithromycin (**1**) (MIC 8 µg/ml) (Bukvić Krajačić et al., 2011a) and their parent 3-cladinosyl analogues **11** & **13** (MIC 4 to 16 µg/ml) ( Bukvić Krajačić et al., 2009), and significantly better

Cl

**f g**

**a b**

R'''

N X N H R'''

<sup>N</sup> HO OHHO OH

> **33 X = O, R1 = H 35 X = S, R1 = H 34 X = O, R1 = CH2CH2CN 36 X = S, R1 = CH2CH2CN**

O O

OH

N

O O

R

1

**<sup>d</sup> <sup>e</sup>**

Cl

**c**

in comparison to the 3-decladinosyl-3-hydroxy azithromycin (**16**) (MIC >64 µg/ml).

O O

R'''NCX

Scheme 4. Synthesis of novel 3-decladinosyl ureas and thioureas of 15-membered azalides.

The racemic urea derivative **33c** showed the highest activity against both, susceptible *S. pneumoniae* and *S. pyogenes* strains, and the same activity as its 3-cladinosyl analogue (±)-**11h** 

Interestingly, some of discovered 3-decladinosyl-3-hydroxy ureas **33** & **34**, and thioureas **35**  & **36**, maintain antibacterial activity against Gram-negative pathogens *H. influenzae* and *M. catarrhalis* (Bukvić Krajačić et al., 2011a) in comparison to their parent 3-cladinosyl derivatives **11, 12, 13** & **14**, (Bukvić Krajačić et al., 2009) and demonstrate large improvement in comparison to the inactive 3-decladinosyl sulfonylureas **31**( Bukvić Krajačić et al., 2005)

OH

**38 R1 = CH2CH2CN**

and azithromycin (MIC <0.125 µg/ml) (Bukvić Krajačić et al., 2009).

NH

1

R

N

<sup>N</sup> HO OHHO OH

> O O

membered azalides termed *"decladinosylides"* (Bukvić Krajačić et al., 2011a).

resistant strains.

O O

N H

<sup>N</sup> HO OHHO OH

O

OH

O

1. acrylonitrile 2. H2/ PtO2 3. 1eq acrylonitrile

**15 37 R1 = H**

and 3-decladinosyl azithromycin-sulfonamide conjugates **32** (Bukvić Krajačić et al., 2007). Activity of (±)-**33a** and **35a** against *H. influenzae* is only one dilution lower than the corresponding MIC of azithromycin (MIC 2 µg/ml). Urea (±)-**33c** was more potent (MIC 8 µg/ml) than its 3-cladinosyl analogue (±)-**11h** (MIC 16 µg/ml) against *Enterococcus faecalis* and **33a** showed the same activity against *E. coli* in comparison to its cladinosyl analogue **11f**. (Bukvić Krajačić et al., 2011a).

Thus, it seems that appropriate linked urea or thiourea moiety at 9a-*N* of 3-decladinosyl-3 hydroxy- 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; Hansen et al., 2002), two bound conformations of telithromycin from *Deinococcus radiodurans*  (Berisio et al. 2003) and *Haloarcula marismortui (*Hansen et al., 2002) 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) (Hansen et al., 2002), ABT-773 (cyan) ( Auerbach et al., 2009), two conformations of telithromycin from *Deinococcus radiodurans* (magenta)( Berisio et al. 2003) and *Haloarcula marismortui* (yellow)( Hansen et al., 2002) 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; Hansen et al., 2002; Berisio et al., 2003), that high structural diversity is tolerated within the flexible macrolide-binding site of

Antibacterial Activity of Novel Sulfonylureas, Ureas and Thioureas of 15-Membered Azalides 99

Hence, newly synthesised sulfonyl ureas of azalides **3b**-**3f**, and azalide-sulfonamide conjugates **10a** and **10b** displayed significantly improved activity against inducible resistant

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

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,

• The observed increase of antibacterial activity in the series of ureas and thioureas **11, 12, 13** and **14** (Bukvić Krajačić et al., 2009) in comparison with those of their analogues **8** and **9**  (Kujundžić et al., 1995), was opposite to the results obtained for the sulfonylcarbamoyl derivatives **3, 5** and **7** (Bukvić Krajačić et al., 2005) where a decrease of activity was found

> NT NT

*S. pyogenes*-iMLS

16 8 4 2 1 0.5 0.25

when sulfonylcarbamoyl moiety was further away from the azalide ring (Fig. 6)

Fig. 6. Antibacterial activity of selected novel sulfonylureas, ureas and thioureas of 15 membered azalides on *S. Pneumoniae efflux-mediated* (Bukvić Krajačić et al., 2009) and *S. pyogenes* iMLS (Bukvić Krajačić et al., 2005) resistant strains in comparison to azithromycin

**Azi 3b 3c 3e 10a 10b 11b 11c 11f 11g 12c 12d 12e 12f 33a 34b**

NT NT NT NT NT

NT = Not tested

MIC (μg/m) MIC (μg/mL)

• Several novel sulfonylureas (Bukvić Krajačić et al., 2005), ureas and thioureas (Bukvić Krajačić et al. 2009) of 15-membered azalides showed same or significantly better activity

although lacking a cladinose sugar, showed noticeable antibacterial activity.

Overall mutual comparison of obtained results can be summarized in three items:

*S. pyogenes* strain when compared to azithromycin.

*S. pneumoniae-*M

1 2 4 8 16 32

tackle the resistance problems.

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; Hansen et al., 2002; 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).
